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diff --git a/40268-0.txt b/40268-0.txt index 721d3d1..476de38 100644 --- a/40268-0.txt +++ b/40268-0.txt @@ -1,37 +1,4 @@ -The Project Gutenberg EBook of Significant Achievements in Space -Bioscience 1958-1964, by National Aeronautics and Space Administration - -This eBook is for the use of anyone anywhere at no cost and with -almost no restrictions whatsoever. You may copy it, give it away or -re-use it under the terms of the Project Gutenberg License included -with this eBook or online at www.gutenberg.org - - -Title: Significant Achievements in Space Bioscience 1958-1964 - -Author: National Aeronautics and Space Administration - -Release Date: July 17, 2012 [EBook #40268] - -Language: English - -Character set encoding: UTF-8 - -*** START OF THIS PROJECT GUTENBERG EBOOK ACHIEVEMENTS IN SPACE BIOSCIENCE *** - - - - -Produced by K.D. 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You may copy it, give it away or -re-use it under the terms of the Project Gutenberg License included -with this eBook or online at www.gutenberg.org - - -Title: Significant Achievements in Space Bioscience 1958-1964 - -Author: National Aeronautics and Space Administration - -Release Date: July 17, 2012 [EBook #40268] - -Language: English - -Character set encoding: ISO-8859-1 - -*** START OF THIS PROJECT GUTENBERG EBOOK ACHIEVEMENTS IN SPACE BIOSCIENCE *** - - - - -Produced by K.D. Thornton, Enrico Segre and the Online -Distributed Proofreading Team at http://www.pgdp.net - - - - - - - - - - NASA SP-92 - - - Significant Achievements in - - - Space Bioscience - 1958-1964 - - - - - _Scientific and Technical Information Division_ 1966 - - NATIONAL AERONAUTICS AND SPACE ADMINISTRATION - - _Washington, D.C._ - - - - - For sale by the Superintendent of Documents, - U.S. Government Printing Office - - Washington, D.C., 20402--Price 55 cents - - - - -_Foreword_ - - -This volume is one of a series which summarize the progress made during -the period 1958 through 1964 in discipline areas covered by the Space -Science and Applications Program of the United States. In this way, the -contribution made by the National Aeronautics and Space Administration -is highlighted against the background of overall progress in each -discipline. Succeeding issues will document the results from later -years. - -The initial issue of this series appears in 10 volumes (NASA Special -Publications 91 to 100) which describe the achievements in the following -areas: Astronomy, Bioscience, Communications and Navigation, Geodesy, -Ionospheres and Radio Physics, Meteorology, Particles and Fields, -Planetary Atmospheres, Planetology, and Solar Physics. - -Although we do not here attempt to name those who have contributed to -our program during these first 6 years, both in the experimental and -theoretical research and in the analysis, compilation, and reporting of -results, nevertheless we wish to acknowledge all the contributions to a -very fruitful program in which this country may take justifiable pride. - - _Homer E. Newell_ - _Associate Administrator for_ - _Space Science and Applications, NASA_ - - - - -_Preface_ - - -This summary of certain aspects of the space biology program of the -National Aeronautics and Space Administration brings together some -results of NASA research and NASA-sponsored research under grants and -contracts from 1958 through 1964. Closely related research even though -not sponsored by NASA is also included. - -The space biology program has had a late start in comparison with the -space physics program, and only a token program existed before 1962. -Much of the present research involves preparation of space-flight -experiments and obtainment of adequate baseline information. Perhaps -half the research results reported are derived from the NASA program. -Additional information is included from many other sources, especially -the U.S. Air Force with its long history of work in aviation and -aerospace medicine. - -Relatively few biological space-flight experiments have been undertaken. -These have been to test life-support systems and to demonstrate, before -manned space flight, an animal's capability to survive. Few critical -biological experiments have been placed in orbit by NASA, but a -biosatellite program will soon make a detailed study of the fundamental -biological effects of weightlessness, biorhythms, and radiation. - -The search for extraterrestrial life has been limited to ground-based -research and planning for planetary and lunar landings. Life-detection -experiments have been developed and tested, and an important and -exciting program is being planned to detect and study extraterrestrial -life, if it exists. - -Interest in space biology has been slow in developing, and there has -been some caution and controversy in the scientific community. However, -increased interest is starting to push forward the frontier of this new -and important scientific field, and future outlook appears to be -optimistic. - -This summary was written and compiled by the members of the Bioscience -Programs Division of the Office of Space Science and Applications. The -report was edited and chapters 1, 3, 6, and 7 were written by Dale W. -Jenkins, Chief, Environmental Biology; chapter 2, by Gregg Mamikunian, -Staff Scientist, Exobiology; chapters 4 and 8, by Richard E. Belleville, -Chief, Behavioral Biology; and chapter 5, by George J. Jacobs, Chief, -Physical Biology. - - - - -_Contents_ - - - page - 1 BACKGROUND ................................................... 1 - 2 EXOBIOLOGY ................................................... 5 - 3 ENVIRONMENTAL BIOLOGY ........................................ 23 - 4 BEHAVIORAL BIOLOGY ........................................... 43 - 5 MOLECULAR BIOLOGY AND BIOINSTRUMENTATION ..................... 57 - 6 FLIGHT PROGRAMS .............................................. 65 - 7 MANNED SPACE FLIGHT .......................................... 77 - 8 SIGNIFICANCE OF THE ACHIEVEMENTS ............................. 111 - REFERENCES ..................................................... 119 - - - - - chapter 1 - -_Background_ - - -The biological program of the National Aeronautics and Space -Administration had a late start. A small life sciences group, organized -in 1958, was concerned with life support and use of primates for system -and vehicle testing for the Mercury program. Three small suborbital -flights of biological materials were flown in space. - -The Bioscience Program Office of the Office of Space Science and -Applications was organized in 1962. The goals of the Bioscience Program -are: (1) to determine if extraterrestrial life exists anywhere in the -solar system and to study its origin, nature, and level of development, -if it is present; (2) to determine the effects of space and planetary -environments on Earth organisms, including man; (3) to conduct -biological research to develop life support and protective measures for -extended manned space flight; and (4) to develop fundamental theories in -biology relative to origin, development, and relationship to -environment. Research and development has been carried out to design -life-detection experiments and instruments for future flights to Mars -and to develop experiments to study the effects of the space environment -on living organisms. A biosatellite program, started in 1963, has the -first of six flights scheduled for 1966. - -Space exploration has demanded a rigorous development, especially in the -biosciences area. Investigation of the solar system for exotic life -forms, the environmental extremes to which Earth organisms (including -man) are being exposed, the possibilities for modification of planetary -environments by biological techniques yet to be developed, and the -problems of communication in biosystems are areas which have required -refinement of the theoretical framework of biology before progress could -be made rapidly enough to keep pace with technological advances in -transportation. - -Of all the sciences, biology alone has not yet benefited from -comparisons with the universe beyond Earth. It is reasonable to suppose -that breakthroughs might be made in biology on the basis of comparisons -with life from other worlds. Organisms elsewhere may have found -alternatives to processes we think of as basic characteristics of life. - -In contrast, physical science has advanced sufficiently to provide a -great body of laws which may be expressed in mathematical terms, and by -which phenomena may be predicted with complete accuracy. A well-known -characteristic of biological phenomena is variability. The Darwinian -concept of evolution is perhaps the only pervading generalization in -biology. This concept has been supported by evidence of a hereditary -mechanism in the discovery of genes and gene mutations. - -Space bioscience represents the convergence of main disciplines with a -single orientation, whose direction is determined by the problems of -manned space travel which have, in turn, created a host of -bioengineering problems concerned with supporting man in space. - -Foremost among these questions is the possibility of the existence of -extraterrestrial life. The field which is concerned with the search for -extraterrestrial life has come to be called "exobiology." In addition to -the challenge of great technological problems which must be solved, -exobiology is so closely related to the central scientific questions in -biological science that it is considered by some to be the most -significant pursuit in all of science. - -One of the major opportunities already presented by the advances in -propulsion systems is the ability to escape from the influence of the -Earth, which has made possible the study of organism-environment -relationships, particularly the role that environmental stimuli play in -the establishment and maintenance of normal organization in living -systems. - -Transcending even these formidable objectives of space bioscience is an -objective shared by all life sciences, the discovery of nature's scheme -for coding the messages contained in biological molecules. -Extraterrestrial biology seeks to find not only evidence of life now -present, but the vestigial chemicals of its previous existence. The ways -and means have already been made available to study molecules on whose -long, recorded messages is written the autobiography of evolution--the -history of living organisms extending back to the beginnings of life. On -this same basis, it is now within the realm of science to foresee the -means of predicting the development of life from primordial, nonliving -chemical systems. Closely allied to the search for extraterrestrial life -is research which seeks to identify the materials and the conditions -which are the prerequisites of life. - -Space bioscience research is now extending human knowledge of -fundamental biological phenomena, both in space and on Earth, just as -the physical sciences explore other aspects of the universe. The -accomplishment of bioscience objectives is totally dependent upon -advances in the technology of space flight. A highly developed -launch-vehicle capability is essential to accomplish the long-duration -missions required in the search for extraterrestrial life. - -Life on other planets in the solar system (with emphasis on Mars) will -be investigated by full exploitation of space technology which will -allow both remote (orbiter) and direct (lander) observations of the -planetary atmosphere, surface, and subsurface. Certain characteristics -of terrestrial life, such as growth and reproduction, provide a basis -for relatively simple experiments which may be used on early missions to -detect the existence of life on Mars. Later missions will provide -extensive automatic laboratory capabilities for analyzing many samples -taken from various depths and locations. Because of the hypothetical -nature of current experiment designs, it is likely that visual -observations of the planet will be required. Many technical problems are -involved in storing and transmitting the large amounts of data over -planetary distances. Such visual observations might very well be crucial -in interpreting results from other experiments. Critical to all -exploration of the Moon and planets are the requirements to: (1) prevent -contamination of the environment with Earth organisms and preserve the -existing conditions of the planet for biological exploration; (2) -provide strict quarantine for anything returned to Earth from the Moon -and planets. - -The biological exploration of Mars is a scientific undertaking of the -greatest significance. Its realization will be a major milestone in the -history of human achievement. The characterization of life, if present, -and study of the evolutionary processes involved and their relationship -to the evolution of terrestrial life would have a great scientific and -philosophical impact. What is at stake is nothing less than knowledge of -our place in nature. - -Extended Earth orbital flights with subhuman specimens will be used to -determine the effects on Earth organisms of prolonged weightlessness, -radiation, and removal from the influence of the Earth's rotation. Such -flights of biosatellites and other suitable spacecraft are expected to: -(1) establish biological specifications for extending the duration of -manned space flight; (2) provide a flexible means of testing unforeseen -contingencies, thus providing an effective biological backup for manned -missions; (3) yield experimental data more rapidly by virtue of the -greater number and expendability of subjects; (4) anticipate possible -delayed effects appearing in later life or in subsequent generations, -through use of animal subjects with more rapid development and aging; -(5) develop and test new physiological instrumentation techniques, -surgical preparations, prophylactic techniques, and therapeutic -procedures which are not possible on human subjects; and (6) provide a -broad background of experience and data which will permit more accurate -interpretations of observed effects of space flight on living organisms, -including man. - - - - - chapter 2 - -_Exobiology_ - - -The possibility of discovering an independent life form on a planet -other than Earth presents an unequaled challenge in the history of -scientific search. Therefore, the detection of life within the solar -system is a major objective of space research in the foreseeable future. - -The scientific data presently available concerning the possible -existence of a Martian life form and the chemical constitution of the -surface of Mars are disappointingly few. In fact, it is impossible to -make a statement about any of the many surface features, other than the -polar caps, with any degree of certainty. The observational results have -been accounted for by many conflicting hypotheses which can only be -resolved by the accumulation of new evidence. - -The arguments supporting the existence of Martian life ([ref.1]) are -based on the following observations: - - (1) The various colors, including green, exhibited by the dark areas - (2) The seasonal changes in the visual albedo and polarization of the - dark areas - (3) The ability of the dark areas to regenerate after an extensive - "duststorm" - (4) The presence of absorption bands at 3.3µ-3.7µ, attributed to - organic molecules - -Conflicting interpretations of the above observations have been -advanced. The argument based on the colors is inconclusive, and several -workers have suggested that the color is a contrast effect with the -bright-reddish continents. The meager quantitative data have been -discussed by Öpik ([ref.2]) who has reduced Kozyrev's photometric -observations of the very dark area of Syrtis Major to intrinsic -reflectivities by allowing for the estimated atmospheric attenuation and -reflectivity. Kuiper ([ref.3]) similarly demonstrated the absence of the -near-infrared reflection maximum, which is characteristic of most green -plants, indicating that chlorophyll was not responsible for the color. - -The second and third arguments remain the most cogent. However, serious -limitations are imposed on the second if the severity of the Martian -climate is considered. Föcas ([ref.4]) has photometrically measured the -seasonal changes in the fine structure of the dark areas of Mars and -concludes that-- - - (1) The dark areas of Mars show periodic variation of intensity - following the cycle of the darkening element - (2) The average intensity of the dark area, not including the action - of the darkening waves, increases from the poles toward the - equator - (3) The action of each of the darkening waves decreases from the poles - toward the equator. This decrease is balanced in the equatorial - zone by the combined action of the two darkening waves alternately - originating at the two poles. The mechanism of the - darkness-generating element seems to be constant for all latitudes - during the Martian year. - -The variation in intensity has been explained recently by nonlife -mechanisms for Depressio Hellespontica (an area showing one of the -greatest seasonal changes) ([ref.2]). Similar nonlife mechanisms may be -applicable to the other dark regions, and, thus, the "darkening" can be -used only as circumstantial evidence in support of a Martian life form. - -If inorganic interpretations of the seasonal albedo variation are -accepted, then an inorganic interpretation must also be advanced for the -polarization variation. Two possibilities can be suggested: - - (1) A change in surface texture, caused by varying absorption of - atmospheric constituents, causing both the albedo and polarization - to change in the manner observed - (2) A change in surface texture, in which the surface material becomes - rougher, which also explains the observed polarization data - ([ref.5]) - -The third argument against the regenerative feature of the dark areas -being a life process has been advanced by Kuiper ([ref.6]). It is based -on atmospheric circulation causing dust, presumably lava, to be blown on -the dark areas of Mars during the late summer, autumn, and winter, and -then removed during the spring. Mamikunian and Moore have recently -advanced the similar explanation that carbonaceous chondrites or -asteroidal matter may induce the observed phenomenon if they are -abundant on the planet's surface. The pulverized chondritic material -will exhibit a high degree of opacity due to localization and, hence, a -change in polarization characteristics and a decrease in polarization -following mixing of the chondritic material with indigenous surface -minerals. - -The fourth observational argument, the Sinton bands ([ref.7]), has been -shown to be at least doubtful. Rea, Belsky, and Calvin ([ref.8]) -recorded infrared reflection spectra for a large number of inorganic and -organic samples, including minerals and biological specimens, for the -purpose of interpreting the 3µ-to-1µ spectrum of Mars. These authors -state that a previous suggestion that the Martian "bands" be attributed -solely to carbohydrates is not a required conclusion. At the same time -they fail to present a satisfactory alternate explanation, and the -problem remains unsolved. More recently, Rea et al. ([ref.9]) noted the -similarity between the 3.58µ and 3.69µ minima in the Martian infrared -spectra and those of D2O-HDO-H2O mixtures and, particularly, of HDO. - -With all this marked disagreement in interpreting the observational data -concerning Mars, it becomes clearly evident that an experimental -approach to the detection of life on Mars should provide the maximum -positive information possible. Some life-detection experiments developed -with NASA support have been summarized by Quimby ([ref.10]). - -The schema of the biological exploration of a planet is to conduct a -series of complementary experiments proceeding from general to specific. -The general experiments will examine gross characteristics of the -planet's environment and surface for determining the probability of an -active biota (life). Data from the general experiments will be -significant in-- - - (1) Defining the nature of specific experiments in which life - detection is the major objective; and - (2) Providing a high degree of confidence in undertaking specific - experiments, since indications from the gross characterization of - the planet in question will influence the choice and design of the - specific experiments. - -The biological exploration of planets is then to be defined as the -search for those parameters relevant to the origin, development, -sustenance, and degradation of life in a planetary environment. This -definition will give rise to a critical question for each progressively -specific and complex experiment to determine-- - - (1) The existence of life on the planet - (2) The degree of similarity or dissimilarity (structure and function) - with respect to terrestrial life - (3) The origin of this planetary life - -The immediate objective of the biological explorations of the planet is -to define the state of the planetary surface, which may exhibit the -following properties: - - (1) A prebiota (defined as the absence of life) - (2) An active biota (defined as the presence of life) - (3) An extinct biota (defined as evidence of former life) - -The identification and the detailed characterization of each of the -above stages of planetary development constitute the subject matter of -the biological exploration of the planets and, specifically, Mars. - - -THE EXPERIMENTAL INVESTIGATION OF CHEMICAL EVOLUTION - -Attempts have been made to simulate and approximate models of primitive -Earth conditions for abiogenic synthesis, and successful synthesis of -essential biochemical constituents necessary for maintaining life has -been partly accomplished. - -Urey ([ref.11]) has clearly pointed out the possible role of a reducing -atmosphere in the synthesis of prebiological organic molecules. Miller -([ref.12]) synthesized a variety of amino acids in a reducing atmosphere -by means of an electrical discharge. A variety of organic compounds have -been synthesized by the action of various energy sources upon reducing -atmospheres, and several investigators have extended the -Urey-Miller-type reactions to synthesize nucleic acid components -([ref.13]), adenosine triphosphate ([ref.14]), and a host of -biologically essential organic compounds. - -It is likely that in the synthesis of organic moieties, simple and -specific molecules were first produced when the planets had a reducing -atmosphere. Further complexity or degradation of the organic compounds -produced varied, depending on the geochemical changes of the planet's -surface, the atmospheric constituents, the degree of interaction between -surface and atmosphere, and the rate of the organic synthesis. Oparin -([ref.15]) presented the most detailed mechanisms for the spontaneous -generation of the first living organism arising in a sea of organic -compounds synthesized in a reducing atmosphere on Earth. - -It is generally accepted that, under favorable conditions, life can -arise by spontaneous generation. A primary requirement for this -initiation is that there be abundant organic compounds concentrated in -one or more specific zones. These simple organic molecules would undergo -modification to develop a greater structural complexity and specificity, -finally giving rise to a "living" organism. Therefore, because of the -ease with which organic compounds can be synthesized under reducing -conditions, planetary surfaces may contain an abundant source of similar -organic matter. However, difficulties arise in postulating steps for -further organization or modification of the above synthesized organic -matter into a living state. Most of the original organic matter produced -in the primary reducing atmospheres of the various planets may have been -quite similar. However, major variations between planets, in chemical -evolution beyond the prebiotic stage, must have been the rule rather -than the exception. - -The primary interest in this area of research has been the realization -of the possible existence of organic molecules on planetary surfaces -and, particularly, Mars. Pertinent synthesis may be either biological or -abiological. Research conducted in the simulation of cosmochemical -synthesis has used most of the available solar spectrum. Simulation -experiments devised to study the effects of these energies on the -assumed early atmosphere of the Earth have yielded products that play a -dominant role in molecular and biochemical organization of the cell. - -Calvin ([ref.16]) irradiated water and carbon dioxide in a cyclotron, -obtaining formaldehyde and formic acid. Miller ([ref.17]) found that -when methane, ammonia, water, and hydrogen were subjected to a -high-frequency electrical discharge, several amino acids were produced -along with a variety of other organic compounds. - -Corroborating experiments established that the synthesis of amino acids -occurred readily. The apparent mechanism for the production of amino -acids is as follows: aldehydes and hydrogen cyanide are synthesized in -the gas phase by the electrical discharge. These substances react -together and also together with ammonia in the water phase of the system -to give hydroxy and amino nitriles, which are then hydrolyzed to hydroxy -and amino acids. Among the major constituents were aspartic acid, -glutamic acid, glycine, [alpha]-alanine, and [beta]-alanine. - -The "Miller-Urey" reaction mixture has been extended and several -modifications introduced. Oró ([ref.18]) introduced hydrogen cyanide -into the system as the primary gas component. Adenine was obtained when -Oró heated a concentrated solution of hydrogen cyanide in aqueous -ammonia for several days at temperatures up to 100° C. Adenine is an -essential component of nucleic acids and of several important coenzymes. -Guanine and urea were the two other products identified in the hydrogen -cyanide reaction. Oró further obtained guanine and uracil as products of -nonenzymatic reactions by using certain purine intermediates as starting -materials. - -Ponnamperuma ([ref.19]) also obtained adenine upon irradiation of -methane, ammonia, hydrogen, and water, using a high-energy electron beam -as the source of energy of irradiation. These results indicate that -adenine is very readily synthesized under abiotic conditions. Adenine, -among the biologically important purines and pyrimidines, has the -greatest resonance energy, thus making its synthesis more likely and -imparting greater radiation stability to the molecule. - -The formation of adenine and guanine, the purines in RNA and DNA, by a -relatively simple abiological process lends further support to the -hypothesis that essential biochemical constituents of life may have -originated on Earth by a gradual chemical evolution and selection. In -this respect, the examination of planetary surfaces--specifically -Mars--presents practical implications for current research on the -problem of chemical evolution. - -When Ponnamperuma et al. ([ref.14]) exposed adenine and ribose to -ultraviolet light in the presence of phosphate, adenosine was produced. -When the adenine and ribose were similarly exposed in the presence of -the ethyl ester of polyphosphoric acid, adenosine diphosphate (ADP) and -adenosine triphosphate (ATP) were produced. The abiological formation of -ATP was a major stride along the path of chemical evolution, since ATP -is the principal free energy source of living organisms. - -Oparin ([ref.15]) postulated that [alpha]-amino acids could have been -formed nonbiologically from hydrocarbons, ammonia, and hydrogen cyanide -at a time when the Earth's atmosphere contained these substances in high -concentrations. Oparin's hypothesis has received strong experimental -support, as evidenced by the work of Miller ([ref.12]). Bernal -([ref.20]) has emphasized the role played by ultraviolet light in the -formation of organic compounds at a certain stage of the Earth's -evolution. - -Generally it has been believed that the first proteins or foreprotein -were nonbiologically formed by the polycondensation of preformed free -amino acids ([ref.21]). Akabori ([ref.22]) proposed a hypothesis for the -origin of the foreprotein and speculated that it must have been produced -through reactions consisting of the following three steps. - -The first step is the formation of aminoacetonitrile from formaldehyde, -ammonia, and hydrogen cyanide. - - CH2O + NH3 + HCN --------> H2N--CH2--CN + H2O - -The second is the polymerization of aminoacetonitrile on a solid -surface, probably absorbed on clay, followed by the hydrolysis of the -polymer to polyglycine and ammonia. - - x H2N--CH2--CN --------> (--NH--CH2--C--)x - || - || - NH - | - | + x H2O - | - V - (--NH--CH2--CO--)x + x NH3 - -The third step is the introduction of side chains into polyglycine by -the reaction with aldehydes or with unsaturated hydrocarbons. Akabori -has demonstrated experimentally the formation of cystinyl and cysteinyl -residue in his above-postulated mechanism. - -Fox's theory of thermal copolymerization ([ref.23]) suggests that -proteins or like molecular units could have been formed in the Earth's -crust, under geothermal conditions. The accumulated amino acids were -heat polymerized and transported into the primary oceans for further -modifications. Fox has obtained polymers consisting of all 18 amino -acids usually present in proteins. The polymerization is generally done -at 160° C to 200° C, although in the presence of polyphosphoric acid it -can be accomplished at temperatures below 100° C. Molecular weights -increased from 3600 in a proteinoid made at 160° C to 8600 in one made -at 190° C. - -Fox showed that when hot saturated solutions of thermal copolymers -containing the 18 common amino acids were allowed to cool, large numbers -of uniform, relatively firm, and elastic spherules separate. These range -from 0.2µ to 60µ in diameter and are quite uniform within each -preparation. Various chemical observations suggest the presence of -peptide bonds in the structural organization of these proteinoids. -Continuing observations of these microspheres have established further -characteristics that point to the possibility of their interpretation as -a kind of primitive protein macromolecule with self-organizing -properties, such that a primitive form of cell, with boundary and other -properties, might form. - -In laboratory experiments the behavior of gram-negative and -gram-positive microspheres in dilute alkali parallels that of -gram-negative and gram-positive bacteria ([ref.23]). Furthermore, -time-lapse studies indicate that the proteinoid microspheres undergo a -septate kind of fission, mimicking cell division as shown in figure 1. -Cytochemical studies show that the microsphere's boundary is -membranelike in having a primitive selectivity. Electron micrographs of -sections of stained microspheres also indicate the presence of a -boundary. - -Oparin ([ref.15]) states that the type of organization peculiar to life -could only result from the evolution of a multimolecular organic system -separated from its environment by a distinct boundary but constantly -interacting with this environment. In his concept of coacervates as -precell models, Oparin ([ref.24]) indicates that present-day protoplasm -possesses a number of features similar to coacervate structure. These -coacervates could represent the starting point for evolution leading to -the origin of life. Moreover, in the course of their evolution the -initial systems may gradually become more complex. Oparin also showed -([ref.15]) that mixing solutions of different proteins and other -substances of high molecular weight produced these coacervate droplets. -These droplets are characterized by the formation of a surface layer -with altered structure and mechanical properties, thus providing a -somewhat selective barrier in which to house a molecular system capable -of replication. However, these coacervates are unstable structurally. - -[Illustration: Figure 1.--_Protenoid microspheres undergoing septate -fission. Small microspheres and filamentous associations thereof are -also shown ([ref.25])._] - -The NASA program has further provided considerable impetus for -continuing research with respect to the chemical evolution of life, -since its life-detection experiments may encounter prebiological -molecules in their search for extraterrestrial life on other planetary -surfaces. - -In the area of exobiological research, the significant accomplishments -to date have been-- - - (1) The reconstruction of some of the pathways which may have led to - the origin of life, by means of laboratory simulation of processes - yielding prebiological organic molecules - (2) The developments in experimental and theoretical biology; - specifically, the role of nucleic acid-protein interactions in - storage and transmission of information both within living cells - and from generation to generation of cells - (3) The suspected role of DNA in information storage and the - development of new concepts of the coding mechanism in DNA that - may lead to a universal biological theory embracing evolutionary, - as well as homeostatic, adaptation to environment and learned - behavioral systems - -With the essential biochemical constituents of life and the mechanism of -replication beginning to be understood, the challenge for the synthesis -of living matter by abiogenic experimental techniques has become to many -scientists the ultimate goal of the scientific era. - -NASA has established an exobiology laboratory at Ames Research Center in -addition to the sizable support of research at various academic centers -of excellence for the continuation of abiogenic synthesis. - -Although research on organochemical evolution is in its infancy, the -data from relatively few experiments have already created an immense -enthusiasm for knowledge of the biochemical pathways of evolution. This -kind of research will ultimately elucidate the terrestrial evolution of -life and, perhaps, the nature of life on other planetary bodies and the -distribution of life in our galaxy. - -This program, with its vast demands on the scientific community at -large, is coordinated with related endeavors of a number of Federal -agencies. It is allied with certain biochemical studies at the National -Institutes of Health for the eventual elucidation of the dynamic -pathways in cosmochemical synthesis of life's essential biochemical -constituents. - - - METEORITES AND ORGANIC GEOCHEMISTRY - -Meteorites - -A significant area of exobiological research is the investigation of a -special class of stony meteorites known as "carbonaceous chondrites." It -is increasingly apparent that almost all life-detection concepts rely on -the eventual analysis of the solid materials that may be available on -Mars and other planetary surfaces. Cosmic dust and meteorites are two -classes of material bodies that reach the Earth from outer space. The -carbonaceous chondrites are the only extraterrestrial materials known to -contain organic carbon. - -The study of meteorites has generated an astonishing diversity of -hypotheses. There is agreement at only one point: that meteorites are -preserved chunks of very ancient, perhaps primordial, planetary matter -and that when we are able to understand the curious structures and -chemical and isotopic variations in the meteorites, we will also know a -great deal about early planetary (and perhaps preplanetary) history. - -Meteorites provide a more representative sample of average planetary -matter than the highly differentiated crust of the Earth. Although it is -known that the meteorite parent bodies ceased to be geochemically active -shortly after their formation, some 4½ billion years ago, there is no -consensus on the nature of the meteorite parent bodies, not even on such -basic properties as size, location, and multiplicity. This is not -surprising because the meteorite samples commonly available for study -represent only about 10^-23 to 10^-26 of the parent body. - - -Carbonaceous Meteorites - -Analysis and characterization of the chemical constituents (organic) of -carbonaceous chondrites, including the possible mechanism of their -formation, may be expected to improve methods of analyzing samples from -the Moon and planets and of interpreting remote automated biological -analyses on the planets' surfaces. - -Carbon has been detected in all meteorites analyzed; however, both the -amount and forms present vary considerably. Among the forms of meteorite -carbon are diamond, graphite, cohenite (Fe,Ni,Co)3 C, moissanite SiC, -calcite CaCO3, dolomite (Ca,Mg)CO3, bruennerite (Mg,Fe)CO3. A summary of -the results of carbon analyses on large numbers of meteorites is given -in table I ([ref.26]). - - - Table I.--_Meteorite Carbon_ - - ----------------------------------------------------------------- - Meteorite group Number Mean carbon content, - analyzed percent by weight - ----------------------------------------------------------------- - Pallasites 10 0.08 - ----------------------------------------------------------------- - Ureilites 2 .69 - ----------------------------------------------------------------- - Bronzite chondrites 12 .05 - ----------------------------------------------------------------- - Hypersthene chondrites 8 .04 - ----------------------------------------------------------------- - Enstatite chondrites 8 .29 - ----------------------------------------------------------------- - Carbonaceous chondrites 16 2.04 - ----------------------------------------------------------------- - - -Most meteorites possess only traces of carbon, and studies of this -carbon indicate that it is composed largely of graphite, cohenite, and -moissanite, with some diamond. However, studies of the carbon in the -carbonaceous chondrites have failed to detect any of these forms. Some -carbonates are present in a minority of the carbonaceous group, but -account for only a small percentage of the total carbon (perhaps about -10 percent of the total C in type I only). - -The carbonaceous chondrites contain organic carbon. The word "organic" -is not used in a biological sense, merely as a chemical term to describe -compounds of carbon other than carbonates, bicarbonates, and carbides. -No evidence has been found of any form of carbon other than organic, -except for traces of carbonates. - -Various studies have demonstrated possible methods of estimating the -total amount of organic matter present in meteorites. Wiik ([ref.27]) -has suggested that organics can be estimated by measuring the loss of -weight on ignition. Unfortunately, this method has several disadvantages -and gives very low values. Corrections must be made for weight gains due -to oxidation of reduced constituents, such as FeO, Fe, Ni, and Co, and -for weight losses due to H2O, S, etc. The water loss is exceedingly -difficult to estimate, as part comes from the combustion of organic -hydrogen and part comes from the loss of mineral-bound water. The carbon -also proves difficult to combust completely, and high temperatures (over -1000° C) are required for efficient conversion to CO2. - -In one study the major fraction of organic matter removed proved to have -a carbon content of about 47 percent ([ref.28]). Thus, if all the -meteorite carbon is present as organic matter of approximately this -composition, total organics must be approximately double the carbon -content; that is, 2 percent by weight carbon indicates 4 percent by -weight organic matter. This estimate may be too low, for Mueller -([ref.29]) has extracted a major organic fraction containing only 24 -percent carbon; however, this work has not been confirmed for other -meteorites. - -Briggs and Mamikunian ([ref.26]) have pointed out that only 25 percent -of the organic matter has been extracted, and only about 5 percent of -this has been chemically characterized. Most of this 5 percent is a -complex mixture of hydroxylated aromatic acids together with -hydrocarbons of the aliphatic, napalicyclic, and aromatic series. Small -amounts of amino acids, sugars, and fatty acids are also present. - -Thus far, these chemical analyses point to an abiogenic origin for the -organic matter, and no conclusive evidence exists of biological activity -on the meteorite parent body. Microbiological investigations of samples -of the carbonaceous chondrites have yielded only inconclusive evidence -on the problem of "organized elements." - -Several of these microstructures from different carbonaceous chondrites -are illustrated in a paper by Mamikunian and Briggs ([ref.30]). It has -been difficult to identify the organized structures, and most do not -have morphologies identical to known terrestrial micro-organisms. -However, they may prove to be a variety of mineral grains, droplets of -organic matter and sulfur, as well as a small amount of contaminating -terrestrial debris. - -A comparison between the photographs of the organized elements observed -in the Orgueil and Ivuna meteorites and the synthetic proteinoid -microspheres observed by Fox ([ref.25]) point to similarities between -the two. One inference from this finding is that the organized elements -in carbonaceous chondrites were never alive but, rather, should be -considered as natural experiments in molecular evolution. Also, these -similarities strengthen the belief that the laboratory experiments are -similar to the natural experiments in space. - -In cooperation with the Smithsonian Astrophysical Observatory, NASA has -a network to track meteors in the Midwest (South Dakota, Nebraska, -Kansas, Oklahoma, Iowa, Missouri, and Illinois). Photographs of meteor -trails are used for scientific study, and attempts are made to track and -recover meteorites for examination for traces of organic material of -extraterrestrial origin. - -Fundamental research in terrestrial organic geochemistry has shown that -ancient sediments and drill core samples subjected to organic analysis -contain certain stable biochemical components of past life. This -preserved record is significant not only in studies of early-life -chemical pathways but also in studies of the interaction of organic -matter with the geological factors. Since life on any planetary body -will interact with the soil, or surface material, it is of interest to -understand the relationship. - - - CONCEPTS FOR DETECTION OF EXTRATERRESTRIAL LIFE - -It is not possible to present completely convincing evidence for the -existence of extraterrestrial life. The problem often reduces to -probabilities and to estimates of observational reliability. In almost -all cases the evidence is optimistically considered strongly suggestive -of--or, at the worst, not inconsistent with--the existence of -extraterrestrial life. Alternatively, there is a pessimistic view that -the evidence advanced for extraterrestrial life is unconvincing, -irrelevant, or has another, nonbiological explanation. - -In studies of the laboratory synthesis of life-related compounds and its -significance concerning the origin of life, several results seem to -suggest that organochemical synthesis is a general process, occurring -perhaps on all planets which retain a reducing atmosphere. The -temperature ranges must be such that precursors and reaction products -are not thermally dissociated. The reaction rates for the synthesis of -more complex organic molecules diminish to a negligible value when the -temperature range is below 100° C. - -Besides the planetary parameter of temperature, an even more fundamental -necessity for a living state exists--a liquid solvent system. For -terrestrial life forms, water serves this purpose. Water has this and -other properties of biological significance because of hydrogen bonding -between adjacent molecules in the liquid state. - -Ultraviolet radiation could serve as an extraterrestrial energy source -for organic synthesis. Research shows that, while an atmosphere is -important, living systems can survive a wide range of ambient pressures -and are little affected by a wide range of magnetic field strengths. - -Oxygen is not a prerequisite for all living systems. While it is -sometimes concluded that free oxygen is needed for all but the simplest -organisms, less efficient metabolic processes coupled with higher food -collection efficiency--or a more sluggish metabolism--would seem to do -just as well. Earth is the only planet in the solar system on which -molecular oxygen is known to be present in large amounts. Since plant -photosynthesis is the primary source of atmospheric oxygen, it seems -safe to infer that no other planet has large-scale plant photosynthesis -accompanied by the production of oxygen. - -The possibility of the existence of extraterrestrial life raises the -important question of man's being able to detect it. Research on -extraterrestrial life detection is predicated on the ability to develop -ways to detect it even when the living systems are based on principles -entirely different from those on Earth. - -The substitution of various molecules for those of known biological -significance to living organisms as we know them has been investigated; -the substitution of NH2 for OH in ammonia-rich environments leads to a -diverse, and biologically very promising, chemistry. The hypothesis that -silicon may replace carbon does not support the construction of -extraterrestrial genetics based on silicon compounds. (Silicon compounds -participate in redistribution reactions which tend to maximize the -randomness of silicon bonding, and the stable retention of genetic -information over long time periods is thus very improbable.) - -Evidence relevant to life on Mars has been summarized by Sagan (ch. 1 of -[ref.10]): - - _The Origin of Life_ - - In the past decade, considerable advances have been made in our - knowledge of the probable processes leading to the origin of - life on Earth. A succession of laboratory experiments has shown - that essentially all the organic building blocks of contemporary - terrestrial organisms can be synthesized by supplying energy to - a mixture of the hydrogen-rich gases of the primitive - terrestrial atmosphere. It now seems likely that the laboratory - synthesis of a self-replicating molecular system is only a short - time away from realization. The syntheses of similar systems in - the primitive terrestrial oceans must have occurred--collections - of molecules which were so constructed that, by the laws of - physics and chemistry, they forced the production of identical - copies of themselves out of the building blocks in the - surrounding medium. Such a system satisfies many of the criteria - for Darwinian natural selection, and the long evolutionary path - from molecule to advanced organism can then be understood. Since - nothing except very general primitive atmospheric conditions and - energy sources are required for such syntheses, it is possible - that similar events occurred in the early history of Mars and - that life may have come into being on that planet several - billions of years ago. Its subsequent evolution, in response to - the changing Martian environment, would have produced organisms - quite different from those which now inhabit Earth. - - _Simulation Experiments_ - - Experiments have been performed in which terrestrial - micro-organisms have been introduced into simulated Martian - environments, with atmospheres composed of nitrogen and carbon - dioxide, no oxygen, very little water, a daily temperature - variation from +20° to -60° C, and high ultraviolet fluxes. It - was found that in every sample of terrestrial soil used there - were a few varieties of micro-organisms which easily survived on - "Mars." When the local abundance of water was increased, - terrestrial micro-organisms were able to grow. Indigenous - Martian organisms may be even more efficient in coping with the - apparent rigors of their environment. These findings underscore - the necessity for sterilizing Mars entry vehicles so as not to - perform accidental biological contamination of that planet and - obscure the subsequent search for extraterrestrial life. - - _Direct Searches for Life on Mars_ - - The early evidence for life on Mars--namely, reports of vivid - green coloration and the so-called "canals"--are now known to be - largely illusory. There are three major areas of contemporary - investigation: visual, polarimetric, and spectrographic. - - As the Martian polar ice cap recedes each spring, a wave of - darkening propagates through the Martian dark areas, sharpening - their outlines and increasing their contrast with the - surrounding deserts. These changes occur during periods of - relatively high humidity and relatively high daytime - temperatures. A related dark collar, not due to simple dampening - of the soil, follows the edge of the polar cap in its - regression. Occasional nonseasonal changes in the form of the - Martian dark regions have been observed and sometimes cover vast - areas of surface. - - Observations of the polarization of sunlight reflected from the - Martian dark areas indicate that the small particles covering - the dark areas change their size distribution in the spring, - while the particles covering the bright areas _do_ not show any - analogous changes. - - Finally, infrared spectroscopic observations of the Martian dark - areas show three spectral features which, to date, seem to be - interpretable only in terms of organic matter, the particular - molecules giving rise to the absorptions being hydrocarbons and - aldehydes. [However, see p. 7 and Rea et al. ([ref.9]).] - - Taken together, these observations suggest, but do not - conclusively prove, that the Martian dark areas are covered with - small organisms composed of familiar types of organic matter, - which change their size and darkness in response to the moisture - and heat of the Martian spring. We have no evidence either for - or against the existence of more advanced life forms. There is - much more information which _can_ be garnered from the ground, - balloons, Earth satellites, Mars flybys, and Mars orbiters, but - the critical tests for life on Mars can only be made from - landing vehicles equipped with experimental packages.... - -Results of Kaplan et al. ([ref.31]) indicate that Mars has no detectable -oxygen, but does contain small amounts of water vapor, more abundant -carbon dioxide, possibly a large surface flux of solar ultraviolet -radiation, and estimated daily temperature variations of 100° C at many -latitudes. Studies have shown that terrestrial micro-organisms can -survive these extremely harsh environments. Furthermore, a variety of -physiological and ecological adaptations might enable the biota to -survive the low nighttime temperatures and intracellular ice -crystallization. - -Less evidence is available to support the possibility of -extraterrestrial life on other planets. The Moon has no atmosphere, and -extremes of temperature characterize its surface. However, the Moon -could have a layer of subsurface permafrost beneath which liquid water -might be trapped. The temperatures of these strata might be biologically -moderate. - -Studies by Davis and Libby ([ref.32]) on the atmosphere of Jupiter -support the possibility of the production of organic matter in its -atmosphere in a manner analogous to the processes which may have led to -the synthesis of organic molecules in the Earth's early history. It is -difficult to assess the possibility that life has evolved on Jupiter -during the 4- or 5-billion-year period in which the planet has retained -a reducing atmosphere. - -The question of extraterrestrial life and of the origin of life is -interwoven. Discovery of the first and analysis of its nature may very -well elucidate the second. - -The oldest form of fossil known today is that of a microscopic plant -similar in form to common algae found in ponds and lakes. Scientists -know that similar organisms flourished in the ancient seas over 2 -billion years ago. However, since algae are a relatively complex form of -life, life in some simpler form could have originated much earlier. -Organic material similar to that found in modern organisms can be -detected in these ancient deposits as well as in much older Precambrian -rocks. - -Although the planets now have differing atmospheres, in their early -stages the atmospheres of all the planets may have been essentially the -same. The most widely held theory of the origin of the solar system -states that the planets were formed from vast clouds of material -containing the elements in their cosmic distribution. - -It is believed that the synthesis of organic compounds preceding the -origin of life on Earth occurred before its atmosphere was transformed -from hydrogen and hydrides to oxygen and nitrogen. This theory is -supported by laboratory experiments of Calvin ([ref.16]), Miller -([ref.33]), and Oró ([ref.34]). - -The Earth's present atmosphere consists of nitrogen and oxygen in -addition to relatively small amounts of other gases; most of the oxygen -is of biological origin. Some of the atmospheric gases, in spite of -their low amounts, are crucial for life. The ultraviolet-absorbing ozone -in the upper atmosphere and carbon dioxide are examples of such gases. - -Significant in the search for extraterrestrial life are the data (e.g., -planet's temperature) transmitted by Mariner II, which was launched from -Cape Canaveral on August 27, 1962, and flew past Venus on December 14, -1962. Mariner II's measurements showed temperatures on the surface of -Venus of the order of 800° F, too hot for life as known on Earth. - -The question "Is life limited to this planet?" can be considered on a -statistical basis. Although the size of the sample (one planet) is -small, the statistical argument for life elsewhere is believed by many -to be very strong. While Mars is generally considered the only other -likely habitat of life in our solar system, Shapley ([ref.35]) has -calculated that more than 100 million stars have planets sufficiently -similar in composition and environment to Earth to support life. Of -course, yet unknown factors may significantly reduce or even eliminate -this probability. - - - SPACECRAFT STERILIZATION - -The search for extraterrestrial life with unmanned space probes requires -the total sterilization of the landing capsule and its contents. -Scientists agree that terrestrial organisms released on other planets -would interfere with exobiological explorations (refs. -[ref.36]-[ref.43]). Any flight that infects a planet with terrestrial -life will compromise a scientific opportunity of almost unequaled -proportions. Studies on microbiological survival in simulated deep-space -conditions (low temperature, high ultraviolet flux, and low dose levels -of ionizing radiation) indicate that these conditions will not sterilize -contaminated spacecraft (refs. [ref.44]-[ref.48]). Furthermore, many -terrestrial sporeformers and some vegetative bacteria, especially those -with anaerobic growth capabilities, readily survive in simulated Martian -environments (refs. [ref.49]-[ref.54]). It has been estimated that a -single micro-organism with a replication time of 30 days could, in 8 -years of such replication, equal in number the bacterial population of -the Earth. This potential could result not only in competition with any -Martian life, but in drastic changes in the geochemical and atmospheric -characteristics of the planet. To avoid such a disaster, certainly the -first, and probably many succeeding landers on Mars, must be -sterile--devoid of terrestrial life ([ref.55]). Since the space -environment will not in itself kill all life aboard, the lander must -leave the Earth in a sterile condition. - -The sterility of an object implies the complete absence of life. The -presence of life or the lack of sterility may be proven; but the absence -of life or sterility cannot be proven, for the one viable organism that -negates sterility may remain undetected. Many industrial products which -must be guaranteed as sterile cannot be tested for sterility in a -nondestructive manner. A similar situation exists in determining the -sterility of a spacecraft. Certification of sterility--based on -experience with the sterilizing process used, knowledge of the kinetics -of the death of micro-organisms, and computation of the probability of a -survivor from assays for sterility--is the only accurate approach to -defining the sterility of such treated items. - -Macroscopic life can be readily detected and kept from or removed from -the spacecraft, but the detection and removal of microscopic and -submicroscopic life is an extremely difficult task. The destruction of -micro-organisms can be achieved by various chemical and physical -procedures. Sterilizing agents have been evaluated not only for their -ability to kill microbial life on surfaces and sealed inside components, -but also for the agents' effects on spacecraft reliability as well -(refs. [ref.56]-[ref.59]). Of the available agents, only heat and -radiation will penetrate solid materials. Radiation is expensive, -hazardous, difficult to control, and apparently damages more materials -than does heat. Heat, therefore, has been selected as the primary method -of spacecraft sterilization and will be used, except in specific -instances where radiation may prove to be less detrimental to the -reliability of critical parts ([ref.60]). - -The sterilization of spacecraft is a difficult problem if flight -reliability is not to be impaired. The development of heat-resistant -parts will enable the design and manufacture of a heat-sterilizable -spacecraft. Without careful microbiological monitoring of manufacture -and assembly procedures, many bacteria could be trapped in parts and -subassemblies. To permit sterilization at the lowest temperature-time -regimen that will insure kill of all organisms, the microbiological load -inside all parts and subassemblies must be held to a minimum. - -The role of industrial clean rooms in reducing the biological load on -spacecraft is currently being defined. NASA-supported studies indicate -that biological contamination in industrial clean rooms for extended -time periods is about 1 logarithm less (tenfold reduction), compared -with conditions in a well-operated microbiological laboratory -([ref.61]). With the use of clean-room techniques and periodic -decontamination by low heat cycles or ethylene oxide treatment, it -should be possible to bring a spacecraft to the point of sterilization -with about 10^6 organisms on board ([ref.60]). - -The sterilization goal established for Mars landers is a probability of -less than 1 in 10 000 (10^-4) that a single viable organism will be -present on the spacecraft. Laboratory studies of the kinetics of -dry-heat kill of resistant organisms show that at 135° C the number of -bacterial spores can be reduced 1 logarithm (90 percent) for every 2 -hours of exposure (refs. [ref.58] and [ref.62]). The reduction in -microbial count needed is the logarithm of the maximum number on the -spacecraft (10^6) plus the logarithm of the reciprocal of the -probability of a survivor (10^4), or a total of 10 logarithms of -reduction in microbial count. Thus, with an additional 2 logarithms -added as a safety factor, a total of 12 logarithms of reduction in count -has been accepted as a safe value which can be achieved by a dry-heat -treatment of 135° C for 24 hours. This is the heat cycle that is -currently under study and being developed for use in spacecraft -sterilization ([ref.60]). However, other heat treatments at temperatures -as low as 105° C for periods of 300 hours or longer are under study -([ref.63]). - -Based on results to date, it is reasonable to believe that a full -complement of heat-sterilizable hardware will be available when needed -for planetary exploration. Every effort is being made to improve the -state of the art to a point where spacecraft can not only withstand -sterilization temperatures, but will be even more reliable than the -present state-of-the-art hardware that is not heated. - - - - - chapter 3 - -_Environmental Biology_ - - - BIOLOGICAL EFFECTS OF WEIGHTLESSNESS AND ZERO GRAVITY - -High priority has been given to studies of weightlessness. Gravity is -one of the most fundamental forces that acts on living organisms, and -all life on Earth except the smallest appears to be oriented with -respect to gravity, although certain organisms are more responsive to it -than others. The gravity force on Earth is 1 g, but this force may be -experimentally varied from zero g, or weightlessness, to many thousands -of g's. - -Zero gravity or decreased gravity occurs during freefall, in parabolic -trajectory, or during orbit around the Earth. Gravitational force -decreases by the square of the distance away from the Earth's center. It -is reduced about 5 percent at about 200 nautical miles' altitude. -Gravitational force greater than 1 g can be obtained by acceleration, -deceleration, or impact. It also can be increased by using a centrifuge -which adds a radial acceleration vector to the 1 g of Earth. - -On the ground, the biological effects of gravity have been studied at 1 -g, and experimentally, forces of many g have been produced. In addition, -modifications of the effects of the 1-g force have been induced by -suspension of the organism in water or by horizontal immobilization of -an erect animal such as man. The biological effects of such modification -have been of significant value in understanding some of the possible -consequences of human exposure to the zero-g environment of space. - -Weightlessness in an Earth-orbiting satellite occurs when the continuous -acceleration of Earth's gravity is exactly counterbalanced by the -continuous radial acceleration of the satellite. In such a weightless -state, organisms are liberated from their natural and continuous -exertion against 1 g, but this liberation may carry with it certain -serious physical penalties. - -Some of the physical processes which probably have the greatest -biological effects are (1) convective flow of fluid, e.g., protoplasmic -streaming, transport of nutrient materials, oxygen, waste products, and -CO2 from the immediate environment of the cell, and (2) sedimentation -occurring within cells; substances of higher density sediment in a -gravitational field, and those of lighter density rise. A separation of -particles of different densities probably occurs. The removal of gravity -would change a distribution of particles like mitochondria by 10 percent -([ref.64]). - -Gravity has effects on the physical processes involved in mitosis and -meiosis. Study under weightlessness might contribute to our -understanding of the general cellular information-relay process. - -A gravitational effect is known in the embryonic development of the frog -_Rana sylvatica_. After fertilization, the eggs rotate in the -gravitational field so that the black animal hemisphere is uppermost. -Development becomes abnormal if this position is disturbed. If the egg -is inverted following the first cleavage and held in this position, two -abnormal animals result, united like Siamese twins. This phenomenon -appears to be related to the gravitational separation of low- and -high-density components of the egg. The size of the egg is about 1 to 2 -mm and is suspended in water of about the same density. This system is -very sensitive to gravity; and, under weightlessness, the separation of -different density components might be irregular, leading to aberrant -development. When certain aquatic insect eggs are inverted, subsequent -development results in shortened abnormal larvae. - -The directional growth of plant shoots and plant roots is probably due -to this sedimentation phenomenon, particularly the effect on movement of -auxins ([ref.65]). - -Free convection flow is a major transport process, and under its -influence the mixing of substances is much more effective than when -diffusion operates alone. Free convection flow is a macroscopic -phenomenon which increases not only with g, but varies also -approximately with the five-fourths power of the bulk concentration -involved. Whether or not convection is important at the microscopic -level remains an experimentally unsolved question. The Grashoff number -limits free convection to the macroscopic domain. It would appear in -weightlessness that the contribution of free convective flow would be -small and that only diffusion should occur. This phenomenon would cause -equilibration to occur much more slowly than that occurring with free -convection and diffusion. The absence of convective transfer raises a -problem as to how nutrients may be obtained and waste products removed -in living cells during weightlessness. In a liquid substrate, nutrients -and oxygen would be depleted, and waste products would accumulate around -the cell. - -Absence of gravity may have far-reaching consequences in the homeostatic -aspects of cell physiology. The outstanding characteristics of living -cells which are most likely to be influenced by the absence of gravity -are the ability of the cell to maintain its cytoplasmic membrane in a -functional state, the capacity of the cell to perform its normal -functions during the mitotic cycle, and the capacity of the cytoplasm to -maintain the constant reversibility of its sol-gel system ([ref.66]). - -Two-phase systems, e.g., air-in-water and air-in-oil, possess entirely -different characteristics at zero g than at 1 g. These physical -differences in phase interaction could well be suspected of interfering -with the orientation and flow pattern of cell constituents, thus -hindering the cellular processes involved in the movement, metabolism, -and storage of nutrients and waste. - -On the basis of theoretical calculations, weightlessness can be expected -to have some effect even on one individual cell if its size exceeds 10 -microns in diameter ([ref.64]). Cell colonies might be affected. In -larger cells there may be a redistribution of enzyme-forming systems -which give rise to polarization. The low surface tension of the cell -membrane lends itself to hydrostatic stress distortion, implying an -alteration in permeability and thus an almost certain alteration of cell -properties under low gravity conditions. - -Another aspect of gravity that affects the growth and development of -living organisms is the directionality of the gravitational field. In -fact, some plants are so sensitive that they are able to direct their -growth with as little stimulus as a 1×10^-6 gravitational field. -Investigations of plant growth in altered gravitational fields are -underway at Argonne National Laboratory and Dartmouth College. - -The Argonne Laboratory has designed and developed a 4-pi, or -omnidirectional, clinostat. By rotating a plant so that the force of -gravity is distributed evenly over all possible directions, the -directional effects of gravity are eliminated, simulating some aspects -of the zero-g state. It was shown that certain plants grew more slowly -and had fewer and smaller leaves, while others had about 25 percent -greater replication of fronds and had greater elongation of certain -plant parts. It will be extremely interesting to compare these effects -under zero-g conditions in orbiting spacecraft. - -The effect of gravity in transporting growth hormones in plants has been -demonstrated at Dartmouth College using radiocarbon-labeled growth -hormones. Plant geotropisms and growth movements have been studied and -biosatellite experiments developed. - -Anatomy is considered a derivative adaptation to gravity ([ref.67]). A -large background of plant research exists on the effect of orientation -on plant responses. Information from clinostat experiments is considered -susceptible of extrapolation to low gravity conditions because the -threshold period for gravitational triggering is relatively long. - -Once over critical minimum dimensions, the major effects of low gravity -would be assumed to occur in those heterocellular organisms that develop -in more or less fixed orientation with respect to terrestrial gravity -and which respond to changes in orientation with relatively long -induction periods; these are the higher plant orders. On the other -extreme are the complex primates which respond rapidly, but whose -multiplicity of organs and correlative mechanisms are susceptible to -malfunction and disorganization. It may be suggested that the -heterocellular lower plants and invertebrates will be less affected. -Perturbations of the environment to which the experimental organism is -exposed must be limited or controlled to reduce uncertainties in -interpretation of the results. At the same time, the introduction of -known perturbations may assist in isolating the effects due solely to -gravity. Study of _de novo_ differentiation and other phenomena -immediately after syngamy may be of particular importance. Study of -anatomical changes after exposure of the organism to low gravity is -important. - - - BIOLOGICAL EFFECTS OF SPACE RADIATION[1] - - [1] This section includes part of the Summary of the Panel on Radiation - Biology of the Environmental Biology Committee Space Science Board, - NAS/NRC (1963), and results of research by the Bioscience Programs, - NASA. - - -Radiation sources in space are of three types: galactic cosmic -radiation, Van Allen belts, and solar flares with an intense proton -flux. Cosmic radiation has higher energy levels than radiation produced -by manmade accelerators. - -The Panel on Radiation Biology, while recognizing the need for -radiobiological studies of an applied nature with reference to manned -flight programs, stated that it would be shortsighted for the United -States to confine its efforts to the solution of immediate problems -since, in the long run, successful exploration of space will be aided by -the contributions of basic research. Both the immediate biological -research program and the continuing program for basic studies should be -built upon the large body of existing knowledge of radiation effects. -The attitude that all radiobiological experiments need be repeated in -the space environment should be resolutely rejected. Since fundamental -radiobiology cannot be performed easily in space, it has been -recommended that, wherever possible, these investigations be carried out -in ground laboratories in preference to flying laboratories. - -Space environment does vary from the terrestrial environment, but the -variations are not so great as to lead to the expectation of strikingly -different biological effects of radiation in space. However, it is -conceivable that radiations whose effects are well known under -terrestrial conditions may have some unsuspected biological effects when -combined with unusual features of the space environment: e.g., zero g. -Previous space radiobiological studies have depended solely on very low -and inaccurately measured doses of ambient space radiation. It has been -difficult to distinguish between the observed response levels and the -random noise; thus, experiments have been inconclusive. - - -Biological Effects of Heavy Ions and Mesons - -The biological effects of heavy ions (especially Z>2) and mesons are of -specific interest to space radiobiology. - - -Controlled Radiobiological Experiments in Space - -There is the remote possibility that the radiobiological response may be -modified by factors as yet unknown and perhaps not susceptible to -terrestrial study. Experiments have been designed to settle this matter -including the exposure of biological materials during space flight which -meet the following criteria of reliability: (1) the use of well-known -biological systems, e.g., mutation induction or chromosome breakage; (2) -the use of a sufficient number of individuals in the experiment to -guarantee statistical precision on the results; (3) the exposure of the -system to known quantities and qualities of radiation; (4) the use of -adequate controls. - -High-altitude balloon ascents of the 1930's initiated study of the -biological effects of cosmic rays. They were limited to the exploration -of secondary cosmic radiation effects. After World War II, the research -extended to the use of V-2 rockets fired from the White Sands Proving -Ground. Interest returned to balloons and a significant program was -underway by 1950, first using mice and then hamsters, fruit flies, cats, -and dogs. These flights gave no evidence of radiation damage. However, -it was realized that the flights were too far south to obtain a -significant exposure, and more northerly flights began in 1953. Mice and -guinea pigs were flown on these later flights. Chase ([ref.68]) showed -the most unequivocal results to that time, a statistically significant -increase in light hairs on black animals and the streaks of white hair -up to 10 times wider than expected. Brain lesions were detected in the -guinea pigs flown on Man High in 1957. Many other types of biological -material were sent aloft in an effort to further corroborate existing -information and to investigate genetic and developmental effects of -cosmic radiation. - -From the earlier V-2 rocket flights to the Jupiter missile launchings of -the monkeys Able and Baker, cosmic-ray research was continued, but the -short flight durations of these vehicles did not provide substantial -information. The USAF Discoverer satellite program has given impetus to -cosmic-ray research and provided for longer "staytimes." - -It has been difficult to separate radiation effects from other -space-flight factors: therefore, some of the alterations observed are -still subject to debate. Vibration, acceleration, and weightlessness -appear to be the three most important additional parameters. -Measurements of radiation dosage have been made by chemical and -photographic dosimetry, ion chambers, and biological dosimetry. All -evidence to date indicates that radiation exposure levels are not -hazardous to man at present orbital altitudes up to 200 nautical miles. -Most biological materials flown so far have been for the express purpose -of investigating space-radiation levels and effects. The biological -materials have ranged from tissue cultures to entire organisms and from -phage and bacterial cells to man. The studies have required much of the -space and weight resources allotted biology by the U.S.S.R. and the -United States. They have been accompanied by ground-based controls. - -The Vostok series provided the following data: - - (1) A small, but statistically significant, increase was observed in - the percentage of chromosome aberrations in the rootlet cells of - air-dried wheat and pea seeds after germination. In this case - only, the increase did not depend on flight duration. - (2) Lysogenic bacteria exhibited an increase of genetic alterations - and increased phage production. Length of flight was associated - with increased bacteriophage production by the lysogenic bacteria. - There was an increase of recessive lethals coupled with - nonconvergence of chromosomes (sex linked) in the fruit fly. A - stimulation of cell division in wheat and pea seeds was observed. - Cultures of human cells exposed to space-flight factors did not - differ significantly from terrestrial controls with respect to - such indicators as proliferation rate, percentage of mortality and - morphological, antigenic, and cultural properties. Repeated - flights of the identical HeLa cells revealed that there was a - longer latent period for restoration of growth capacity than in - cells carried into space once or not flown at all. - (3) The most definite radiation effects observed were only revealed in - genetic tests. No harmful influence on those characteristics - affecting the viability of the organism has been discovered. - -The Air Force Discoverer series launched from the west coast had a few -successful flights incorporating organisms. With severe environmental -stress and long recovery times, data on radiation exposure were -equivocal up to Discoverer XVII and XVIII when cultures of human tissue -were flown, recovered, and assessed for radiation exposure effects. -Comparison with ground-based controls revealed no measurable -differences. - -Radiation dosimetry from the Mercury series established that minimal -exposures were encountered at those orbital altitudes. A typical example -is the MA-8 flight of W. M. Schirra, Jr., during which the body surface -dosage was less than 30 millirads. - -NASA has supported fundamental radiation studies at the Oak Ridge -National Laboratory and the Lawrence Radiation Laboratory. Emphasis has -been placed on the biological effects of high-energy proton radiation -and particulate radiation from accelerators. - -At the NASA Ames Research Center extensive fundamental studies are being -carried out on the effects of radiation, especially in the nervous -system. It has been demonstrated that deposits accumulate in the brain -following exposure to large doses of ionizing particle radiation as well -as after X-irradiation. These deposits, referred to as a "chemical -lesion," result from an accumulation of glycogen. The formation of these -deposits during exposure to large doses of X-irradiation was not -increased in environments of 99.5 percent oxygen and increased -atmospheric pressure. - - - SIMULATION OF PLANETARY (MARTIAN) ENVIRONMENTS - -Attempts have been made to simulate to some degree the various -parameters of the Martian environment, such as atmospheric composition, -pressure, radiation flux, temperatures, and the day-night as well as -seasonal cycles. Certain factors for Mars cannot yet be simulated, such -as soil composition, gravitational field, magnetic field, and electrical -field. - -Caution is required in interpreting all simulation experiments. How -Earth organisms respond to simulated Martian environments probably has -nothing to do with life on Mars, but these experiments may show whether -or not anything in the environment of Mars makes life as we know it -impossible. We must expect that on Mars, life will have evolved and have -adapted over long periods of time under conditions which are quite -different from conditions on Earth. The simulation experiments also -provide some information about the possibility of contaminating the -planet Mars, or any planet, with organisms from Earth. In addition, they -give us some clues about the possibilities of adaptation and evolution -of life under these conditions. - -From an evolutionary point of view, if life has developed on Mars, we -expect it to have evolved at least to a microbial stage. On Earth, -micro-organisms are the most ubiquitous and numerous forms of life. This -fact should be considered in studying extraterrestrial bodies. - -Micro-organisms have been selected as the best test organisms, and -bacteria and fungi have been used because they are durable and easy to -grow. Also, because of their rapid growth, many generations can be -studied in a relatively short period of time. The organisms include -chemoautotrophic bacteria, which are able to synthesize their cell -constituents from carbon dioxide by energy derived from inorganic -reactions; anaerobic bacteria, which grow only in the absence of -molecular oxygen; photoautotrophic plants such as algae, lichens, and -more complex seed plants; and small terrestrial animals. - -Organisms have been collected from tundra, desert, hot springs, alpine, -and saline habitats to obtain species with specialized capabilities to -conserve water, balance osmotic discrepancies, store gases, accommodate -to temperature extremes, and otherwise meet stresses. An attempt is made -in these simulation experiments to extend these processes across the -possible overlapping microenvironments which Earth and Mars may share. - -Scientists have developed various special environmental simulators, -including "Mars jars" and "Marsariums." These have made possible -controlled temperatures, atmospheres, pressures, water activities, and -soil conditions for duplicating assumed Martian surface. A complex -simulator, developed by Young et al. ([ref.52]), reproduces the -formation of a permafrost layer with some water tied up in the form of -ice beneath the soil surface. This simulator serves as a model to study -the wave of darkening, thus supporting the hypothesis that the -pole-to-equator wave of darkening is correlated with the availability of -subsurface water. The simulator is a heavily insulated 2-cu-ft capacity -chamber with an internal pressure of 0.1 atm. The chamber contains a -soil mixture of limonite and sand and an atmosphere of carbon dioxide -and nitrogen. With the use of a liquid nitrogen heat exchanger at one -end and an external battery of infrared lamps at the other end, the -temperature simulates that of Mars from pole to equator. Thermocouples -throughout the soil monitor the temperatures in the chamber. - -Zhukova and Kondratyev ([ref.69]) designed a structure measuring -100×150×180 cm. Micro-organisms were placed at the surface of a copper -bar made in a special groove separated by glass cloth. Copper was -selected as one of the best heat-conduction materials permitting a rapid -change of temperature. The lower end of the bar was immersed into a -mixture of dry ice and ethyl alcohol, which made it possible to create a -temperature of -60° C. Heating was performed by an incandescent spiral. - -As the knowledge concerning the Martian environment becomes more -refined, scientists can more accurately simulate this environment under -controlled conditions in the laboratory. Determination of the effects of -the Martian environment on Earth organisms will permit better -theorization on the forms of life we might find on Mars and will permit -us to estimate the potential survival of Earth contaminants on Mars. - -However, until the environmental conditions of Mars are defined more -accurately, the experiments must be changed continually to fit newly -determined conditions. Therefore, existing simulation data are made less -valid for comparison. The data resulting from the simulation experiments -for Mars have been compiled in table II, and the experiments are -summarized below. - -The earliest simulation studies were carried out by the Air Force, and -the studies during the past 6 years have been supported by NASA. -Recently, these studies have received less support or have been -terminated in favor of critical studies on the effects of biologically -important environmental extreme factors on Earth organisms. These -critical studies permit establishing the extreme environmental factor -parameters in which Earth life can grow or survive. These data will have -valuable application to the consideration of life on any planet, to the -design of life-detection instruments, to the sterilization of space -vehicles, and to the problem of contamination of planets. - -Some exploratory experimental studies are in progress to study the -capabilities of organisms to grow under the assumed conditions on -Jupiter. These include studies at high pressure with liquid ammonia, -methane, and other reducing compounds. - -Early experiments simulating Martian conditions using soil bacteria were -carried out by Davis and Fulton ([ref.70]) at the Air Force School of -Aviation Medicine, San Antonio, Tex. Mixed populations of soil bacteria -were put in "Mars jars" with the following conditions: 65-mm Hg -pressure, 1 percent water or less, nitrogen atmosphere, sandstone-lava -soil, and a temperature day-night cycle of +25° to -25° C. The moisture -was controlled by desiccating the soil and adding a given amount of -water. Experiments, conducted up to 10 months, demonstrated that -obligate aerobes died quickly. The anaerobes and sporeformers survived. -Although a small increase in the total number of organisms indicated -growth, the increases in the number of bacteria may have been due to -breaking up clumps of dirt. - -Roberts and Irvine ([ref.71]) reported that, in a simulated Martian -environment, colony counts of a sporeforming bacterium, _Bacillus -cereus_, increased when 8 percent moisture was added. Moisture was -considered more important than temperature or atmospheric gases inasmuch -as a simulated Martian microenvironment containing 8 percent moisture -permitted germination and growth of endospores of _Clostridium -sporogenes_. Increases in colony counts of _Bacillus cereus_ appeared to -be influenced by temperature cycling ([ref.72]). - - - Table II.--_Survival and Growth of Organisms in Simulated Planetary - (Martian) Environments_ - - ------------------------------------------------------------------ - Species Survival, Moisture Temperature, - months °C - - ------------------------------------------------------------------ - Conditions on Mars: 14µ±7µ -70 to +30 - ------------------------------------------------------------------ - Anaerobic 6 Low, -60 to +20 - sporeformers (CaSO4) - _Clostridia_, - _Bacillus - planosarcina_ - ------------------------------------------------------------------ - Anaerobic 6 Low, -60 to +20 - nonsporeformers (CaSO4) - _Pseudomonas_, - _Rhodopseudomonas_ - ------------------------------------------------------------------ - Anaerobes Growth Very wet -75 to +25 - _Aerobacter - aerogenes_, - _Pseudomonas sp._ - ------------------------------------------------------------------ - _Clostridium_, 10 1 -25 to +25 - _Corynebacteria_ percent - "Thin short rod" or less - ------------------------------------------------------------------ - _Bacillus cereus_ 2 0.5 -25 to +25 - percent - soil - ------------------------------------------------------------------ - _Clostridium sporogenes_ 1 8.4 -25 to +25 - (growth) percent - ------------------------------------------------------------------ - _Clostridium botulinum_ 10 Lyophilized -25 to +25 - - ------------------------------------------------------------------ - _Klebsiella pneumoniae_ 6 Lyophilized -25 to +25 - - ------------------------------------------------------------------ - _Bacillus subtilis_ var. 4 2 percent -25 to +25 - _globigii_ - ------------------------------------------------------------------ - _Sarcina aurantiaca_ 4 0.5 percent -25 to +25 - - ------------------------------------------------------------------ - _Clostridium tetani_ 2 or less 1 percent -60 to +25 - ------------------------------------------------------------------ - _Aspergillus niger_ Over 6 hr Very dry -60 to +25 - - - - ------------------------------------------------------------------ - _Aspergillus oryzae_ Over 6 hr Very dry -60 to +25 - ------------------------------------------------------------------ - _Mucor plumbeus_ Over 6 hr Very dry -60 to +25 - ------------------------------------------------------------------ - _Rhodotorula rubra_ Over 6 hr Very dry -60 to +25 - ------------------------------------------------------------------ - Pea, bean, tomato, rye, 0.3 Moist +25 - sorghum, rice. - ------------------------------------------------------------------ - Winter rye 0.6 Moist -10 to +23 - ------------------------------------------------------------------ - - - Table II.--_Survival and Growth of Organisms in Simulated Planetary - (Martian) Environments_ - - ---------------------------------------------------------------------- - Species Atmospheric N2, CO2, Substrate - pressure, percent percent - mm Hg - ---------------------------------------------------------------------- - Conditions on Mars: 85, 3 to 30 - 25±15, 11 - ---------------------------------------------------------------------- - Anaerobic 76 95 5 Air-dried - sporeformers soil - _Clostridia_, - _Bacillus - planosarcina_ - ---------------------------------------------------------------------- - Anaerobic 76 95 5 Air-dried - nonsporeformers soil - _Pseudomonas_, - _Rhodopseudomonas_ - ---------------------------------------------------------------------- - Anaerobes 760 100 (?) Difco - _Aerobacter infusion - aerogenes_, broth - _Pseudomonas sp._ - ---------------------------------------------------------------------- - _Clostridium_, 65 100 (?) Soil - _Corynebacteria_ - "Thin short rod" - ---------------------------------------------------------------------- - _Bacillus cereus_ 65 94 2.21 Sandstone - soil - - ---------------------------------------------------------------------- - _Clostridium sporogenes_ 65 94 2 Enriched - soil - ---------------------------------------------------------------------- - _Clostridium botulinum_ 65 95 0 to Lava soil - 0.5 - ---------------------------------------------------------------------- - _Klebsiella pneumoniae_ 65 95 0 to Lava soil - 0.5 - ---------------------------------------------------------------------- - _Bacillus subtilis_ var. 85 95 0.3 Media - _globigii_ - ---------------------------------------------------------------------- - _Sarcina aurantiaca_ 85 95 0.3 Desert - soil - ---------------------------------------------------------------------- - _Clostridium tetani_ 85 95 0.3 Soil - ---------------------------------------------------------------------- - _Aspergillus niger_ 76 95.5 0.25 Glass - cloth on - copper - bar - ---------------------------------------------------------------------- - _Aspergillus oryzae_ 76 95.5 0.25 Do. - ---------------------------------------------------------------------- - _Mucor plumbeus_ 76 95.5 0.25 Do. - ---------------------------------------------------------------------- - _Rhodotorula rubra_ 76 95.5 0.25 Do. - ---------------------------------------------------------------------- - Pea, bean, tomato, rye, 75 100 0 Filter - sorghum, rice. paper - ---------------------------------------------------------------------- - Winter rye 76 98 0.24 Soil - ---------------------------------------------------------------------- - -Studies of the effects of simulated Martian environments on sporeforming -anaerobic bacteria were carried out by Hawrylewicz et al. ([ref.49]). -They showed that the encapsulated facultative anaerobe, _Klebsiella -pneumoniae_, survived under simulated Martian atmosphere for 6 to 8 -months, but were less virulent than the freshly isolated organisms. -Spores of the anaerobe _Clostridium botulinum_ survived 10 months in the -simulator. Hagen et al. ([ref.53]) found that the addition of moisture -to dry-simulated Martian soil did not improve the survival of _Bacillus -subtilis_ or _Pseudomonas aeruginosa_. _Bacillus cereus_ spores -survived, with added organic medium plus moisture, but no germination of -the spores resulted. - -Hawrylewicz et al. ([ref.49]) put rocks from Antarctica bearing various -lichens in simulated Martian conditions in a large desiccator. They -found that the algal portion of a lichen, _Trebouxia erici_, showed only -slight resistance to the Martian environment. They also pointed out the -effect moisture had on the physical condition of lichens. The -undersurface of a lichen has great water-absorbing capability, and the -slightest amount of moisture on a rock surface is absorbed by the lichen -which can turn green in 15 minutes. - -Scher et al. ([ref.51]) exposed desert soils to simulated environmental -conditions and diurnal cycles of Mars. The atmosphere consisted of 95 -percent nitrogen and 5 percent carbon dioxide (no oxygen) and was dried, -using calcium sulfate as a desiccant. The total atmospheric pressure was -0.1 atm. The temperature ranged from -60° to +20° C in 24-hour cycles. -One hour was spent at the maximum and at the minimum temperatures. The -chambers were irradiated with ultraviolet, 2537 Å, with a dose of 10^9 -ergs/cm², which is comparable to a daily dose found on Mars, and easily -exceeds the mean lethal dose for unprotected bacteria. Soil aliquots -were removed weekly and incubated at 30° C. The scoring was done both -aerobically and anaerobically. Sporeforming obligate and facultative -anaerobes, including _Clostridium_, _Bacillus_, and _Planosarcina_, and -nonsporeforming facultative anaerobes, including _Pseudomonas_ and -_Rhodopseudomonas_, were found. The experimental chambers were frozen -and thawed cyclically up to 6 months. Organisms that were able to -survive the first freeze-thaw cycle were able to survive the entire -experiment. The ultraviolet irradiation did not kill subsurface -organisms, and a thin layer of soil served as an ultraviolet shield. All -of the samples showed survivors. - -Young et al. ([ref.52]) assumed that water is present on Mars, at least -in microenvironments, and that nutrients would be available. The primary -objective of their experiments was to determine the likelihood of -contaminating Mars with Earth organisms should a space probe from Earth -encounter an optimum microenvironment in terms of water and nutrients. -The experiments used bacteria in liquid nutrient media. The environment -consisted of a carbon dioxide-nitrogen atmosphere, and the temperature -cycling was -70° to +25° C, with a maximum time above freezing of 4½ -hours. _Aerobacter aerogenes_ and _Pseudomonas sp._ grew in nutrient -medium under Martian freezing and thawing cycles. Atmospheric pressure -was not a significant factor in the growth of bacteria under these -conditions. - -Silverman et al. ([ref.47]) studied bacteria and a fungus under -extreme--but not "Martian"--conditions. Spores of five test organisms -(_B. subtilis_ var. _niger_, _B. megaterium_, _B. stearothermophilus_, -_Clostridium sporogenes,_ and _Aspergillus niger_) and soils were -exposed while under ultrahigh vacuum to temperatures of from -190° to -+170° C for 4 to 5 days. Up to 25° C there was no loss in viability; at -higher temperatures, differences in resistivity were observed. At 88° C, -only _B. subtilis_ and _A. niger_ survived in appreciable numbers; at -107° C, only _A. niger_ spores survived; none were recoverable after -exposure to 120° C. _B. subtilis_ survived at atmospheric pressure and -90° C for 5 days, but none of the other spores were viable alter 2 days. -Four groups of soil organisms (mesophilic, aerobic, and anaerobic -bacteria, molds, and actinomycetes) were similarly tested in the vacuum -chamber. From one sample only actinomycetes survived 120° C, while one -other soil sample yielded viable bacteria after exposure to 170° C. -Several organisms resisted 120° C in ultrahigh vacuum for 4 to 5 days. -When irradiated with gamma rays from a cobalt 60 source, differences -were observed between vacuum-dried spores irradiated while under vacuum -and those exposed to air immediately before irradiation. A reduction of -from one-third to one-ninth of the viability of spores irradiated in -vacuum occurred with vacuum-treated spores irradiated in air. - -Siegel et al. ([ref.73]), in approximate simulations of Martian -environments, studied tolerances of certain seed plants, such as -cucumbers, corn, and winter rye, to low temperatures and lowered oxygen -tensions. Lowered oxygen tensions enhanced the resistance of seedlings, -particularly cucumber and rye to freezing, and lowered the minimum -temperature required for germination. Germination of seeds in the -absence of liquid water has also been studied. In this case, seeds of -xerophytes have been suspended in air at 75-mm Hg pressure above water. -The air was thus saturated. Germination was slow but did occur. - -Siegel et al. (refs. [ref.73] and [ref.74]) found that the growth rate -of several higher plants was enhanced by certain gases usually thought -to be toxic, such as N2O. This finding is significant inasmuch as the -presence of nitrogen oxides in the Martian atmosphere has been cited as -evidence for the nonexistence of plants on that planet by Kiess et al. -([ref.75]). Exploratory survival tests showed that various mature -plants, as well as the larvae, pupae, and adult specimens of a -coleopteran insect, were undamaged when exposed to at least 40 hours of -an atmosphere containing 96.5 percent N2O, 0.7 percent O2, and 2.8 -percent N2. - -Lichens are of interest because of their ability to survive and thrive -under extreme environmental conditions on Earth. Biological activity of -slow-growing lichens was detected by metabolic gas exchange, CO2 -detection being especially convenient. Siegel points out that this -method is sensitive and nondestructive, to be preferred to staining -techniques, which at present are limited because they are only -semiquantitative, subjective, and destructive of the lichen. - -A Russian study of simulated planetary environments has been performed -with good simulation but for periods of only 2 to 6 hours. Comments on -simulation experiments made by Zhukova and Kondratyev ([ref.69]) are -presented as follows: - - On the basis of modern conceptions on Martian conditions it is - difficult to imagine that higher forms of animals or plants - exist on the planet. A Martian change of seasons similar to that - of our planet empowers us to think that there is a circulation - of an organic substance on Mars, which cannot exist without - participation of microbic forms of life. Microorganisms are the - most probable inhabitants of Mars although the possibility is - not excluded that their physiological features will be very - specific. That is why the solution of the problem concerning the - character of life on Mars is of exceptional interest. But still - the answer to this question can be verified only by simulating - Martian conditions, taking into account the information obtained - from astrophysicists. - - Experiments aimed at creating artificial Martian climatic - conditions have been started quite recently; their number is not - large since they cannot be combined with the results of numerous - experiments investigating the effect of extreme factors on - microorganisms. The result of the effect of such physicochemical - parameters of the medium as pressure, sharp temperature changes, - the absence of oxygen and insolation, depends on their - combination and simultaneity. These examples convincingly show - that while simulating Martian conditions one should strive to - the most comprehensive complex of simultaneously acting factors. - The creation of individual climatic parameters acting - successively leads to absolutely different, often opposite - results. It should be mentioned also that refusal to imitate - insolation and the performance of experiments with specimens of - soil which itself has protective effect on cells of - microorganisms, but not with pure culture of bacteria, are usual - shortcomings in the bulk of studies on this problem. - -It appears that organisms from Earth might survive in large numbers when -introduced to Martian environment. Whether these organisms will be -capable of growth and explosive contamination of the planet in a -biological sense or not is highly questionable. The likelihood of an -organism from Earth finding ideal conditions for growth on Mars seems -extremely low. However, the likelihood of an organism from Earth serving -as a contaminant for any life-detection device flown to Mars for the -purpose of searching out carbon-based life is considerably higher. The -chance that life has originated and evolved on Mars is a completely -separate question and much more difficult to answer. - -It would be interesting to attempt to determine possible evolutionary -trends which might occur on a planet by means of selection of organisms -in a simulated planetary environment. Rapid genetic selection combined -with radiation and chemicals to speed up mutation rate under these -conditions should reveal possible evolutionary trends under the -planetary environmental conditions. This could be attempted after the -planetary environments are more accurately defined. - - - EXTREME AND LIMITING ENVIRONMENTAL PARAMETERS OF LIFE - -The question of the existence of extraterrestrial life is one of the -most important and interesting biological questions facing mankind and -has been the subject of much controversial discussion and conjecture. -Many of the quantitative, and even qualitative, environmental -constituents of the planets also are still subjects of controversy and -speculation. Best guesses about a relatively unknown planetary -environment, combined with lack of information about the capabilities of -Earth life to grow in extreme environments, do not provide the basis for -making informed scientific estimates. - -Life on Earth is usually considered to be relatively limited in its -ability to grow, reproduce, or survive in extreme environmental -conditions. While many common plants and animals (including man) are -quite sensitive to, or incapable of, surviving severe chemical and -physical changes or extremes of environment, a large number of -micro-organisms are highly adapted and flourish in environments usually -considered lethal. Certain chemoautotrophic bacteria require high -concentrations of ammonia, methane, or other chemicals to grow. -Anaerobic bacteria grow only in the absence of oxygen. - -Besides adapting to the extremes of environments on Earth, life is also -capable of growing and reproducing under extreme environmental -conditions not normally encountered: e.g., from a few rad of radiation -in normal habitats to 10^6 or more rad from artificial sources, from 0.5 -gauss of Earth magnetism to 167 000 gauss in manmade magnetic fields, -and from 1-g force of gravity to 110 000 g. The extreme ranges of -physical and chemical environmental factors for growth, reproduction, -and survival for Earth micro-organisms are phenomenally large. - -Life is ubiquitous on Earth and is found in almost every possible -environment, including the most severe habitats, from the bottom of the -ocean to the highest mountain tops and from cold Arctic habitats to hot -springs, as well as in volcanic craters, deep wells, salt flats, and -mountain snowfields. Earth life has become adapted to, and has invaded, -nearly every habitat, no matter how severe. The physiological and -morphological adaptations of life are exceedingly diverse and complex. - -Surprisingly, the extreme parameters or ranges of the physical and -chemical environmental factors permitting growth, reproduction, and -other physiological processes of Earth organisms have not been -critically compiled. A partial compilation of certain selected -environmental factors has been made by Vallentyne ([ref.76]). A -compilation of available published data on certain environmental -extremes, particularly from recent NASA-supported research (compiled by -Dale W. Jenkins, in press), is presented in tables III to VI. These data -can serve as a starting point for a more intensive literature review by -specialists, critical evaluation, standardization of end points, and -especially to point out areas where critical experimentation is urgently -needed. - -This critical compilation involves a review of a very broad and complex -range of subjects involved in many different disciplines with widely -scattered literature. Since the effects of many of the specific -environmental factors are harmful, it is difficult to select a point on -a scale from no effect to death and use some criteria to say that normal -or even minimal growth and reproduction are occurring. The effects of -environmental factors are dependent on (1) the specific factor, times, -(2) the concentration or energy, times, (3) the time of exposure or -application of the factor. Many reports, especially older ones, do not -give all of the necessary data to permit proper evaluation. A -complicating factor is that the effect of each factor depends on the -other factors before, during, and after its application. The condition -of the organism itself is a great variable. Proper evaluation requires -the critical review by a variety of biological specialists, physicists, -and chemists. - -To determine the potential of Earth organisms to survive or grow under -other planetary environmental conditions, a number of experiments have -been carried out attempting to simulate planetary environments, -especially of Mars, as reviewed previously. While the results are of -real interest, they do not provide much basic information. Further, as -the Martian environment is more accurately defined, the experimental -conditions are changed. In addition, some experimenters have altered -certain factors, such as water content, to allow for potential -microhabitats or for areas which might contain more water at certain -times. - - - Table III.--_Extreme Physical Environmental Factors_ - - ----------------------------------------------------------------- - Physical Minimum Organism - factors - ----------------------------------------------------------------- - Temperature -30° C Algae (photosynthesis), - pink yeast (growth) - ----------------------------------------------------------------- - Magnetism 0-50 gamma (=×10^-5 Human - gauss) - - ----------------------------------------------------------------- - Gravity 0 g Human, plants, animals - - ----------------------------------------------------------------- - Pressure 10^-9 mm Hg (5 days) _Mycobacterium_ - _smegmatis_ - ----------------------------------------------------------------- - Microwave 0 W/cm² - - - ----------------------------------------------------------------- - Visible 0 ft-c Animals, fungi, - bacteria - - ----------------------------------------------------------------- - Ultraviolet 0 erg/cm² - - ----------------------------------------------------------------- - X-ray 0 rad - ----------------------------------------------------------------- - Gamma ray 0 rad - - - ----------------------------------------------------------------- - Acoustic 0 dyne/cm² - - - - - ----------------------------------------------------------------- - - - Table III.--_Extreme Physical Environmental Factors_ - - ---------------------------------------------------------------------- - Physical Maximum Organism Activity - factors - ---------------------------------------------------------------------- - Temperature 104° C (1000 _Desulfovibrio Grows and reduces - atm) desulfuricans_ sulfate - ---------------------------------------------------------------------- - Magnetism 167 000 _Neurospora_ 1 hr--no effect, - gauss _Arbacia_ _Arbacia_ - _Drosophila_ development delayed - ---------------------------------------------------------------------- - Gravity 400 000 g _Ascaris_ eggs 1 hr--eggs hatch, - 110 000 g _Escherichia coli_ 40 days' growth - ---------------------------------------------------------------------- - Pressure 1400 atm Marine organisms Growth - - ---------------------------------------------------------------------- - Microwave 2450 Mc/sec _Drosophila_ 68 hr, growth not - 0.3 to 1 affected - W/cm² - ---------------------------------------------------------------------- - Visible 50 000 ft-c _Chlorella_, Seconds, - 17 000 ft-c higher plants recurrently - continuous - ---------------------------------------------------------------------- - Ultraviolet 10^8 erg/cm², Bean embryos Suppressed growth - 2537 Å - ---------------------------------------------------------------------- - X-ray 2×10^6 rad Bacteria Growth - ---------------------------------------------------------------------- - Gamma ray 2.45×10^6 rad _Microcoleus_ Continued growth - _Phormidium_ - _Synechococcus_ - ---------------------------------------------------------------------- - Acoustic 140 db or Man Threshold of pain - 6500 - dyne/cm² at - 0.02 to 4.8 - kcs/sec - ---------------------------------------------------------------------- - - - Table IV.--_Extreme Low and High Temperature Effects Permitting - Life Processes_ - - ----------------------------------------------------------------- - Minimum Organism Activity or condition - temperature, - °C - ----------------------------------------------------------------- - -11 Bacteria Growth (on fish) - ----------------------------------------------------------------- - -12 Bacteria Growth - ----------------------------------------------------------------- - -12 Molds Growth - ----------------------------------------------------------------- - -15 _Pyramidomonas_ Swimming - ----------------------------------------------------------------- - -15 _Dunaliella salina_ Swimming - ----------------------------------------------------------------- - -18 Mold Growth - ----------------------------------------------------------------- - -18 Yeast Growth - ----------------------------------------------------------------- - -18 _Aspergillus Growth (in glycerol) - glaucus_ - ----------------------------------------------------------------- - -18 to -20 Mold Growth (in fruit juice) - ----------------------------------------------------------------- - -18 to -20 _Pseudomonads_ Growth (in fruit juice) - ----------------------------------------------------------------- - -20 Bacteria Growth - ----------------------------------------------------------------- - -20 Bacteria Growth - ----------------------------------------------------------------- - -20 Bacteria Luminescence development - accelerated - ----------------------------------------------------------------- - -20 to -24 Insect eggs - (diapause) - ----------------------------------------------------------------- - -30 Algae Photosynthesis - ----------------------------------------------------------------- - -30 Pink yeast Growth (on oysters) - ----------------------------------------------------------------- - -30 Lichens Photosynthesis - ----------------------------------------------------------------- - -20 to -40 Lichens and conifers Photosynthesis - ----------------------------------------------------------------- - -44 Mold spores Sporulation and germination - ----------------------------------------------------------------- - - - Table IV.--_Extreme Low and High Temperature Effects Permitting - Life Processes_ - - ------------------------------------------------------------------- - Maximum Organism Activity or condition - temperature, - °C - ------------------------------------------------------------------- - 73 Thermophilic organisms Growth (P^32 metabolism) - ------------------------------------------------------------------- - 73 _Phormidium_ (alga) Acclimatized - ------------------------------------------------------------------- - 70 to 73 _Bacillus calidus_ Growth and spore - germination - ------------------------------------------------------------------- - 70 to 74 _Bacillus cylindricus_ Growth and spore - germination - ------------------------------------------------------------------- - 70 to 75 _Bacillus tostatus_ Growth and spore - germination - ------------------------------------------------------------------- - 80 _Bacillus Cultured in laboratory - stearothermophilus_ - ------------------------------------------------------------------- - 83 Sulfate-reducing Found in a well - bacteria - ------------------------------------------------------------------- - 89 Sulfate-reducing Found in oil waters - bacteria - ------------------------------------------------------------------- - 65 to 85 Sulfate-reducing Cultured in laboratory - bacteria - ------------------------------------------------------------------- - 89 Micro-organisms Found in hot springs - ------------------------------------------------------------------- - 95 _Bacillus coagulans_ In 80 min. sporulation - activation - ------------------------------------------------------------------- - 110 _Bacillus coagulans_ In 6 min, sporulation - activation - ------------------------------------------------------------------- - 104 _Desulfovibrio Grow and reduce sulfate - desulfuricans_ at 1000 atm - ------------------------------------------------------------------- - - - Table V.--_Extreme Temperature Limits of Survival_ - - -------------------------------------------------- - Minimum Organism - temperature - °C - -------------------------------------------------- - -190 Yeast bacteria, 10 species - -------------------------------------------------- - -197 _Trebouxia erici_ from lichens - -------------------------------------------------- - -197 Protozoa, _Anguillula_ - -------------------------------------------------- - -252 Yeasts, molds, bacteria, 10 species - -------------------------------------------------- - -253 Black currant, birch - -------------------------------------------------- - -273 Bacteria, many species - -------------------------------------------------- - -273 Bacteria, many species - -------------------------------------------------- - -272 Desiccated rotifers - -------------------------------------------------- - -269 Human spermatozoa - -------------------------------------------------- - - - Table V.--_Extreme Temperature Limits of Survival_ - - ------------------------------------------------------------------ - Maximum Organism Time of exposure - temperature - °C - ------------------------------------------------------------------ - 140 Bacterial spores 5-hr immersion - ------------------------------------------------------------------ - 170-200 Desiccated rotifers 5 min - ------------------------------------------------------------------ - 151 Desiccated rotifers 35 min - ------------------------------------------------------------------ - 150 _Clostridium tetani_ 180 min - ------------------------------------------------------------------ - 170 Aerobic bacteria, molds. 5 days at - actinomycetes 6×10^-9mm Hg - ------------------------------------------------------------------ - 127 (dry) Bacteria (in activated charcoal) 60 min - ------------------------------------------------------------------ - 110 (wet) _Bacillus subtilis_ var. _niger_ 400 min - ------------------------------------------------------------------ - 120 _Bacillus subtilis_ var. _niger_ 400 min - ------------------------------------------------------------------ - 141 _Bacillus subtilis_ var. _niger_ 70 min - ------------------------------------------------------------------ - 160 _Bacillus subtilis_ var. _niger_ 15 min - ------------------------------------------------------------------ - 180 _Bacillus subtilis_ var. _niger_ 2 min - ------------------------------------------------------------------ - 188 _Bacillus subtilis_ var. _niger_ 1 min - ------------------------------------------------------------------ - 120 (wet) _Bacillus stearothermophilus_ 25 min - ------------------------------------------------------------------ - 120 (dry) _Bacillus stearothermophilus_ 100 min - ------------------------------------------------------------------ - 141 _Bacillus stearothermophilus_ 12 min - ------------------------------------------------------------------ - 160 _Bacillus stearothermophilus_ 2 min - ------------------------------------------------------------------ - 166 _Bacillus stearothermophilus_ 1 min - ------------------------------------------------------------------ - - - Table VI.--_Extremes of Chemical Environmental Factors - Permitting Growth or Activity_ - - -------------------------------------------------------- - Chemical Minimum Organism - factor - -------------------------------------------------------- - O2 0% HeLa cells, _Cephalobus_, - anaerobic bacteria - -------------------------------------------------------- - O3 (ozone) 0% - - - -------------------------------------------------------- - H2 0% - - -------------------------------------------------------- - H2O Aw 0.48 _Pleurococcus vulgaris_ - ------------------------------------------ - Aw 0.5 _Xenopsylla cheopis_ - (prepupae) - -------------------------------------------------------- - H2O2 0% - - -------------------------------------------------------- - He 0% - - -------------------------------------------------------- - CO 0% - - - -------------------------------------------------------- - CO2 0% - - -------------------------------------------------------- - CH4 0% - -------------------------------------------------------- - CH2O 0% - -------------------------------------------------------- - CH3OH 0% - -------------------------------------------------------- - N2 0% - - -------------------------------------------------------- - NO 0% - - -------------------------------------------------------- - NO2 0% - - -------------------------------------------------------- - N2O 0% - - - - - - -------------------------------------------------------- - Ar 0% - -------------------------------------------------------- - NaCl, - Na2SO4, - NaHCO3 - -------------------------------------------------------- - H2S 0% - - -------------------------------------------------------- - H2SO4 0% - - - - - -------------------------------------------------------- - Cu^++ - - -------------------------------------------------------- - Zn^++ - - -------------------------------------------------------- - pH 0 _Acontium velatum_ - _Thiobacillus thioodixans_ - - - - - -------------------------------------------------------- - Eh -450 mV Sulfate-reducing bacteria - at pH 9.5 - -------------------------------------------------------- - - - - Table VI.--_Extremes of Chemical Environmental Factors - Permitting Growth or Activity_ - - ---------------------------------------------------------------------- - Chemical Maximum Pressure, Time, Organism Activity - factor atm days - ---------------------------------------------------------------------- - O2 100% 1 Plants, Growth - animals - ---------------------------------------------------------------------- - O3 100 ppm 5 _Armillaria Growth - (ozone) --------------------------- mellea_ ----------------- - 500 ppm 5 Light emission - ---------------------------------------------------------------------- - H2 100% Various Germination - plants - ---------------------------------------------------------------------- - H2O Aw 1.0 1 Various Growth - aquatic - organisms - - ---------------------------------------------------------------------- - H2O2 0.34% Rye Germination - enhanced - ---------------------------------------------------------------------- - He 100% Wheat, rye, Germination - rice - ---------------------------------------------------------------------- - CO 100% Rye Germination - -------------------------------------------------------------- - 80% 1.1 4 _Hydrogenomonas_ Growth - ---------------------------------------------------------------------- - CO2 100% 1.1 4 Rye Growth and - germination - ---------------------------------------------------------------------- - CH4 100% 1.1 4 Rye Germination - ---------------------------------------------------------------------- - CH2O 50% Rye Germination - ---------------------------------------------------------------------- - CH3OH 50% Rye Germination - ---------------------------------------------------------------------- - N2 100% .1 10 Various plants Germination and - root growth - ---------------------------------------------------------------------- - NO 18% .018 10 Sorghum, rice Germination and - root growth - ---------------------------------------------------------------------- - NO2 18% .018 10 Rye, rice Germination and - root growth - ---------------------------------------------------------------------- - N2O 100% 1.2 4 Rye Germination - -------------------------------------------------------------- - 96.5% 1.7 Rye Germination - ------------------------------------ - _Tenebrio Survival - molitor_ - ---------------------------------------------------------------------- - Ar 100% 1.2 2 Rye Germination - ---------------------------------------------------------------------- - NaCl, 67% Photosynthetic Growth - Na2SO4, bacteria - NaHCO3 - ---------------------------------------------------------------------- - H2S 0.96 _Desulfovibrio Growth - g/liter desulfuricans_ - ---------------------------------------------------------------------- - H2SO4 7% _Acontium Growth - velatum_ - ------------------------------- - Thiobacilli Growth, - reproduction - ---------------------------------------------------------------------- - Cu^++ 12 _Thiobacillus Growth - g/liter ferrooxidans_ - ---------------------------------------------------------------------- - Zn^++ 17 _Thiobacillus Growth - g/liter ferrooxidans_ - ---------------------------------------------------------------------- - pH 13 _Plectonema Growth - nostocorum_ - ------------------------------- - _Nitrobacter_ Growth - ------------------------------- - _Nitrosomonas_ Growth - ---------------------------------------------------------------------- - Eh 850 mV Iron bacteria Growth - at pH 3 - ---------------------------------------------------------------------- - - - - - chapter 4 - -_Behavioral Biology_ - - - EFFECTS OF THE SPACE ENVIRONMENT ON BEHAVIOR - -NASA was established in 1958, shortly after the Russian launching of the -second Earth satellite Sputnik II, the first vehicle to carry life into -orbit around the Earth. This accomplishment was preceded by the -pioneering work of Henry et al. ([ref.77]), in which animals were -exposed briefly to low-gravity states in Aerobee rockets. A -motion-picture camera photographed the behavior of two white mice in -rotating drums during this series of flights, which marked the first -time that simple psychological tests were made on animals in the -weightless condition. While this behavioral experiment was relatively -simple, it provided the basic concepts for recent studies which involved -rotation of animals during the weightless state. Subsequent flights such -as Project MIA (Mouse-in-Able) reflected a preoccupation with -physiologic measures (refs. [ref.78] and [ref.79]), although the flights -of Baker and Able included preflight and postflight performance studies -([ref.80]). Able's behavior was recorded in detail on in-flight film, -but none of the behavior was programed or under experimental control. - -The first flights in which behavior or performance was explicitly -programed were those of Sam and Miss Sam in flights of the Little Joe -rocket with the Mercury capsule, launched from Wallops Island in 1959 -and 1960 ([ref.81]). The first major space achievement in the behavioral -sciences was the successful in-flight measurement of the behavior of the -chimpanzee Ham in early 1961, in which the pretrained animal performed -throughout the flight. The second achievement along these lines was in -1962 when the chimpanzee Enos made several orbits around Earth and -performed continuously on a complex behavioral task. The tasks which the -animals performed during these flights have been described in detail by -Belleville et al. ([ref.82]), and the results of the in-flight -performance have been presented by Henry and Mosely ([ref.83]). These -early flights provided much of the technological framework on which -current biological experiments on organisms during flights of extended -duration are based. Due largely to the efforts of Grunzke (refs. -[ref.84] and [ref.85]), the apparatus needed to sustain animals during -space flight, such as zero-g watering and feeding devices, are now -commonplace ([ref.86]). Advanced systems of programing stimulus -presentations and recording responses, developed for Project Mercury, -may now be seen in many basic research laboratories throughout the -country. - -Several other noteworthy advances have been made as an outgrowth of the -Mercury animal flights. Immediately before the orbital flight MA-5, in -which the chimpanzee Enos was employed, it was unexpectedly found that -this 5-year-old animal was hypertensive. Subsequent centrifuge studies -showed that its vascular responses exceeded those of a control group. -Consideration of the animal's preflight experience led to speculation -concerning the origin of this hypertension. An explanation of the -high-blood-pressure responses detected in Enos has been pursued by -Meehan et al. ([ref.87]). Persistent hypertension has been produced in -other laboratory chimpanzees restrained in the same manner as those -participating in space flight and exposed to demanding performance -tasks, a demonstration which has important implications for prolonged -manned space flight and for cardiovascular medicine in general. - -Studies more directly concerned with behavior and performance have been -extended from those of Project Mercury. These extensions have been in -the following directions: (1) the establishment and maintenance of -complex behavioral repertoires under conditions of full environmental -control, (2) the refinement of behavioral techniques for assessing -sensory and motor processes, and (3) the maintenance of sustained -performance under conditions of long-term isolation and confinement and -preliminary extension of such experimental analysis to man. - -Numerous studies with primate subjects, including several at Ames -Research Center, have been devoted to developing methods for maintaining -optimum performance in environments with limited sources of stimulation. -Monkeys, baboons, and chimpanzees, for example, have been isolated for -periods of longer than 2 years with no decrement in performance on -complicated behavioral tasks ([ref.88]). The behavioral techniques used -in these studies are closely related to those employed on human subjects -under NASA sponsorship at the University of Maryland ([ref.89]). The -essence of these techniques is in the proper programing of environmental -stimuli ([ref.90]). It is not sufficient to provide the subject with his -physiological requirements for survival, but he must be given the -psychological motivation for using these provisions. This statement, of -course, is an oversimplification of the problem, but it serves to -illustrate the essence of these experimental programs. - -Gravity has long been known as one of the major factors influencing -various life processes and the orientation of both plants and animals. -One of the most challenging problems of space research has been to -define this influence more precisely. Related to the effect of gravity -on living processes is the problem of the effects of weightlessness. Of -particular interest to psychologists are the possible modifications an -altered gravitational environment might produce in behavioral patterns -basic to the animal's maintenance and survival, such as eating, sensory -and discriminative processes, development and maturation, and learning -capacity ([ref.91]). - -One prominent method of studying gravitational effects is to simulate an -increase in gravity by centrifugation. Smith et al. ([ref.92]) and -Winget et al. ([ref.93]) have investigated the effects of long-term -acceleration on birds, primarily chickens, while Wunder (refs. [ref.94] -and [ref.95]) and his coworkers (refs. [ref.96]-[ref.99]) have used -fruit flies, mice, rats, hamsters, and turtles. The general findings are -that, when animals are subjected to a prolonged period of acceleration -of moderate intensity, they exhibit decreased growth, delayed -maturation, and an increase in the size of certain muscles and organs, -dependent on the species. With regard to the decreased growth effect, -the data of these investigators show some exceptions. When the -gravitational increase is kept below a certain limit, growth was greater -than that of controls in the fruit fly, turtle, mouse, and chicken. The -limit below which enhancement of growth was observed varied with the -species studied. - -The data on food intake do not present a consistent picture. Wunder -([ref.94]) found that food intake in accelerated mice was markedly -reduced from that of nonaccelerated control animals. Smith, however, -found that in chickens, food intake increased up to 36 percent over -controls and has derived an exponential relation between food intake and -acceleration. After six generations of selective breeding, Smith has -produced a strain of chickens better adapted to prolonged exposure to -high g. - -A very relevant finding of their research with birds was that exposure -to chronic acceleration in some way appears to interfere with -habituation to rotatory stimulation. Chickens who were being subjected -to chronic acceleration were given repeated rotatory stimulation tests -to estimate their labyrinthine sensitivity. This study revealed that -centrifuged animals showed a marked reduction in labyrinthine -sensitivity. This result appeared to persist after the acceleration was -terminated. In animals who developed gait or postural difficulties as a -result of acceleration, there was no evidence of a postnystagmus in -response to the rotatory stimulation test, which the investigators point -out may be evidence of a lesion in the labyrinth or its neural pathways. - -Smith has implicated social factors as interfering with acceleration -effects. His subjects were typically accelerated four or six to a cage. -When groups were mixed midway through the experiment, they exhibited a -higher mortality rate and incidence of acceleration symptoms than did -groups whose constituency remained unchanged. - -At the U.S. Naval School of Aerospace Medicine, numerous studies have -been conducted on the effects of slow rotation on the behavior and -physiology of humans and animals ([ref.100]). Rotation initially -produces decrements in performance, but adaptation to a rotating -environment ensues quite rapidly (refs. [ref.101]-[ref.103]). Perceptual -distortion, nystagmus, nausea, and other signs of discomfort are common -responses to slow rotation. These symptoms are generally reduced with -continued exposure (adaptation). Interestingly, however, adaptation is -delayed when the subjects are exposed to a fixed reference outside their -rotating environment. - -At NASA-Ames, rodents have been used in experiments by Weissman and -Seldeen to delimit the stimulus effects of rotation. In these -experiments the subjects must discriminate between different speeds of -rotation in order to obtain food reinforcement. The results thus far -provide evidence that these animals are capable of discriminating -between the different speeds at which they are being rotated. The range -of speeds studied was 0-25 rpm, with tests of discrimination being made -at intervals of less than 5 rpm. Experiments such as these will lead to -the development of techniques for measuring rotational sensitivity in -many species, including man. - -The optimum configuration of manned spacecraft will depend, in part, -upon biomedical considerations. A voluminous literature now exists on -the possible hazards to man of prolonged exposure to zero-g conditions. -Should prolonged weightlessness prove to be a serious detriment to -health, consideration must be given to design concepts which provide -artificial gravity. - -No data exist on the minimum gravity requirements necessary to sustain -basic biological functions for extended periods. A limit of 0.2 g has -been given as the lower level at which man can walk unaided ([ref.104]). -It has also been recommended that angular velocity be maintained -at the lowest possible level in order to minimize the occurrence of -vestibular disturbances. These recommendations are based on human-factor -requirements, rather than upon biological considerations, which may -significantly modify these values. In recent studies, a technique has -been devised which promises to provide reliable criteria for biological -acceptability, since it is based on fundamental biological and -behavioral principles. - -As animals progress up the evolutionary stale, their survival depends -less and less upon stereotyped physiological reactions which occur in -reflex fashion, in response to environmental stimulation. In higher -organisms, survival depends more upon the capacity of organisms to -modify their behavior. At the highest levels of functional efficiency, -the ultimate form of adaptation is seen--the manipulation of the -environment by the organism. Developments in behavioral science now -permit us to utilize the adaptive behavior of animals to investigate -many problems of biological interest. Recent studies on the -self-selection of gravity levels represent a further attempt to exploit -the adaptive capacities of animals, in order to provide information -relevant to problems of space exploration. - -One such project allows animals to select their own gravity environment -in an apparatus designed to create g-forces through centrifugal action -by rotation at 60 rpm ([ref.105]). The surface of this centrifuge is -parabolic, so that the resultant of the centrifugal g and the Earth's -gravity is always normal to the surface. When the animal moves away from -the center, increasing the radius of rotation, it is exposed to -increasing gravity. Motion toward the center reduces the gravity level. -By this means, an animal is free to select its own gravity environment. - -When the animal moves toward or away from the center, he is moving from -one tangential velocity to another. He is therefore acted upon by a -third force--due to Coriolis acceleration. The effects of Coriolis -forces are a major problem difficult to eliminate in studies such as -these, but they must be taken into account in the design of spacecraft -which produce artificial gravity by rotation. Motion of the head in any -direction not parallel to the centrifugal force vector would result in -bizarre stimulation of the semicircular canals and consequent motion -sickness. This effect is likely to become even more pronounced if the -sensitivity of these organs is increased by prolonged exposure to -reduced gravity. Methods such as these are currently being developed for -conducting a refined psychophysical analysis of gravity, including -studies by Lange and Broderson on the perception of angular, linear, and -Coriolis acceleration. - -The results of animal studies such as these will be of great value in -arriving at a decisive judgment concerning the need for artificial -gravity in a manned orbiting space station, or other vehicles designed -for long-term occupancy. - -To aid in the interpretation of in-flight data, other studies are -underway to determine the functions of the vestibular system, as a -principal brain center related to orientation in space and to the -physiology of posture and movement, as well as with the influences of -acceleration, rotation, and weightlessness. Experiments are presently -being conducted on monkeys and cats in order to trace these complex -neurological connections and to determine their functional organization. - - - BIOLOGICAL INFORMATION SYSTEMS - -The nature of memory has been the subject of considerable speculation in -the past. It has long been felt intuitively that retention of -information in the central nervous system involves either an alteration -of preexisting material or structure, or, alternatively, synthesis of -materials not present previously. The cellular site of operational -alteration was unknown but, again intuitively, was felt to be closely -associated with the synapses. The problems faced by early investigators -were great; but nevertheless much information relevant to the question -of biological information storage was obtained. With the relatively -recent advent of more refined tools and methodologies, there has been -rapid progress. - -A significant amount of the work which has been conducted in the area of -biological information and communication systems is easily classified as -"basic research" (refs. [ref.106]-[ref.109]). This discussion will be -limited to those aspects closely related to the fields of molecular -biology and experimental psychology, which seem to have universal -application to all known animal life forms. Studies involving the basic -principles of acquisition, processing, storage, and retrieval of -information in living systems are emphasized. - - -Early Work - -Early speculations on the operational nature of memory have been based -upon relatively little experimental evidence. Charles Darwin observed -that domestic rabbits had smaller brains than their wild counterparts, -and attributed this to lack of exercise of their intellect, senses, and -voluntary movements. Unfortunately, subsequent studies of the brains of -men with greatly differing intellectual capability did not substantiate -the hypothesis. Idiots sometimes had larger brains than geniuses. Later, -an idea proposed by Ramon y Cajal came into favor. Since brain cells did -not increase in number after birth, he proposed that memory involved the -establishment of new and more extended intercortical connections. -Unfortunately, methods were not available to test this hypothesis -adequately and it has remained until quite recently in the realm of -conjecture. - -Another major hypothesis was that there were two or more stages in the -information storage process. The final form the information took in the -brain was called a brain engram, or memory trace. However, prior to the -formation of the engram, a transitory process denoted as -"reverberational memory" was postulated to exist for a relatively short -time (minutes to hours) (refs. [ref.106] and [ref.107]). This hypothesis -was used by Pauling to explain why an elderly chairman of a board could -brilliantly summarize a complex 8-hour meeting and yet, after its -conclusion and his return to his office, not even remember having -attended the meeting. Thus, this individual's reverberational memory -functioned well, but advanced years had seriously impaired his brain's -ability to form a permanent engram. Similar, although less dramatic, -observations in other situations are not uncommon. A wide variety of -experiments have been conducted to study this aspect of memory and to -relate it to the process whereby the information is transformed to a -more stable form (refs. [ref.110]-[ref.112]). - -More recently, the concept of a specific biochemical activity during the -process of long-term storage of information has gained considerable -favor. Initially, neither the site nor the nature of the change was well -defined. Quite recent studies by Krech et al. (refs. [ref.113] and -[ref.114]), Bennett et al. ([ref.115]), Rosenzweig et al. (refs. -[ref.116] and [ref.117]) support the view that alteration of the levels -of acetylcholinesterase at cortical synapses play an important role in -information storage. These studies will be discussed in a later section. -However, these authors do not claim that the changes observed are -unambiguously related to the storage of memory. It may well be that the -alterations observed are in some way related to this process but are -still secondary to some other, more basic, process. - -An alternative hypothesis is that the information resides in its -ultimate form in some more central structure of the neurone than the -synapse. (It has even been postulated that the basic information is -stored in nonneuronocortical material.) Perhaps Halstead was the first -to postulate the involvement of nucleoprotein in this process -([ref.107]). From the biochemist's point of view, this is an extremely -attractive hypothesis. Both proteins and nucleic acids possess -sufficient possible permutations of structure to permit storage of a -lifetime's accumulation of information in an organ the size of the -brain. From the previously known ability of the nucleic acids to code -genetic information, they are the prime suspects. However, from the -known regulatory ability of nucleic acids in specific protein synthesis, -it is possible that the final repository is protein. - - -Recent Biochemical Studies - -Among the foremost investigators of the chemistry and biochemistry of -the central nervous system is Holger Hyden at the University of -Göteborg, Sweden. He and others (refs. [ref.118]-[ref.120]) have for -many years performed elegant microanalytical studies of single nerve -cells. The evidence which Hyden has obtained is consistent with the -hypothesis that the initial electrical reverberations in the brain -induce a change in the molecular structure of the ribonucleic acid (RNA) -of the neurones which, in turn, leads to a subsequent deposition of -specific proteins. It is well known from other investigations that a -major role of RNA in any type of cell is to specify and mediate -synthesis of the protein enzymes of the cells. Thus, in this hypothesis, -it is only necessary to postulate the modification of brain RNA by the -activities associated with reverberational memory. Particularly -pertinent to this hypothesis are observations that-- - - (1) Large nerve cells have a very high rate of metabolism of RNA and - proteins, and, of the somatic cells, are the largest producers of - RNA. - (2) Vestibular stimulation by passive means leads to an increase in - the RNA content of the Deiters nerve cells of rabbits ([ref.121]). - The protein content of these cells is also increased. - (3) Changes in the RNA composition of neurones and glia of the - brainstem occur during a learning situation. Animals were trained - over a period of 4 to 5 days to climb a steeply inclined wire to - obtain food. The big nerve cells and the glia of their lateral - vestibular apparatus were analyzed, since the Deiters neurones - present in this structure are directly connected to the middle - ear. The amount of RNA was found to be increased in the nerve - cells; and, more significantly, the adenine-to-uracil ratio of - both the nuclear RNA of nerve cells and glia cells became - significantly increased ([ref.119]). A variety of control - experiments were conducted. Although there was an increase in RNA - content of these cells in animals exposed to passive stimulation, - there was no change in the ratio of adenine to uracil. Nerve cells - from the reticular formation, another portion of the brain, had - only an increased content of RNA with no base-ratio change. - Animals subjected to a stress experiment involving the vestibular - nucleus showed only an increase in content of RNA. Littermates - living in cages on the same diet as learning animals showed no - change in content of RNA. Thus, it would appear that the change in - the base ratio of the RNA synthesized is not due to increased - neurone function per se, but is more directly related to the - learning process. The fact that this was nuclear RNA implies that - it was immediately related to chromosomal DNA. - (4) Neuronal RNA with changed cytosine-guanine ratios synthesized - during a short period of induced protein synthesis could be - blocked by actinomycin D. It was concluded, therefore, that the - RNA was immediately DNA dependent and directly related to the - genetic apparatus. - -Rats which were normally right handed were forced to modify their -handedness in order to obtain food. The RNA of nerve cells in that part -of the cortex, whose destruction destroys the ability to transfer -handedness, was analyzed. A significant increase in RNA of nerve cells -of the fifth to sixth cortical layers on the right side of the brain was -observed. The corresponding nerve cells on the opposite side of the same -brain served as controls. There was an increase in RNA and a significant -increase in the purine bases relative to the pyrimidine bases in the -learning side of the cortex. When the animals were not forced to learn a -new procedure, only an increase of RNA was observed, with no change in -base ratio. - -Frank Morrell, head of the Neurology Department at Stanford Medical -School, has also been active in this field during the past 6 years. He -has found that if a primary epileptic lesion is induced on one side of -the cortex, a secondary mirror lesion eventually develops in the -contralateral homologous cortex. This secondary lesion, which showed -self-sustaining epileptiform discharge, could be isolated, whereupon the -epileptiform discharge disappeared. This was interpreted as learned -behavior of the secondary lesion. From changes in the staining -properties of the secondary lesion, Morrell concluded that changes in -RNA had occurred in the cell. Changes in the composition of the RNA -could not be shown by these techniques. - -At the University of California at Berkeley, Drs. Rosenzweig, Bennett, -and Krech have conducted extensive studies related to this topic. These -investigators have directed their efforts toward demonstrating -alterations in the cerebral cortex of animals exposed to continuing -learning situations or continuously deprived of sensory stimulation. In -a recent publication ([ref.116]), which also summarizes a considerable -amount of previous work, they report studies which demonstrate the -following: - - (1) Rats given enriched experience develop, in comparison with their - restricted littermates, greater weight and thickness of cortical - tissue and an associated proportional increase in total - acetylcholinesterase activity of the cortex. - (2) The gain in weight of cortical tissue is relatively larger than - the increase in enzymatic activity. Acetylcholinesterase activity - increases in other portions of the brain even though tissue weight - decreases. - (3) The changes appear in a variety of lines of rats, although - differing in amount between strains. - (4) The changes are observed in both the young and adult animals. - -The previous studies were comparisons between experience-enriched -animals and animals maintained in isolation. Animals which were housed -in colonies, but given no special treatment, showed intermediate effects -in those situations studied. - -The Berkeley group emphasized that the finding of changes in the brain -subsequent to experience does not prove that the changes have anything -to do with memory storage, but do establish the fact that the brain can -respond to environmental pressure. However, the results are compatible -with the hypothesis that long-term memory storage involves the formation -of new somatic connections among neurones. Calculations of the amount of -additional material required to permit this to exist are compatible with -the increases observed. - -A number of investigators have studied the effects of antimetabolites -and drugs on the learning process. Since their specific metabolic -effects are known in other tissues, the rationale is that if these -materials do interfere with memory, then specific types of metabolic -activities may be implicated in the deposition of the engram. - -One of the initial studies of this type was conducted by Dingman and -Sporn ([ref.122]), presently at the National Institute of Mental Health. -They showed that 8-azaguanine, a purine antagonist, injected -intra-cisternally was incorporated into the RNA of the brains of rats. -Associated with this incorporation was an impairment of the -maze-learning ability of the animals. These findings have been -confirmed. - -Flexner and his associates injected puromycin, an inhibitor of protein -synthesis, into the brains of mice, which were then trained to perform -in a maze. Losses of short-term or long-term memory were obtained, -depending upon the site of the injection. The results indicate that the -hippocampal region is the site of recent memory. - -The hippocampal region is of interest in connection with memory -processes for a number of other reasons. Adey et al. ([ref.123]) and his -group observed a transient fall in electrical impedance in this region -when cats learned to perform in a T-maze in response to a visual cue. It -was supposed that the electrodes were situated within glial cells of the -dendritic zone of the hippocampal pyramidal cell layer. Extinction of -the learned habit abolished the briefly evoked impedance changes, which -subsequently reappeared with retraining. - -A number of other studies more or less indirectly implicate RNA in the -learning processes. For instance, in retinal cells of rabbits raised in -darkness, there was virtually no ribonucleoprotein as compared with -normal amounts in the cells of animals raised in light ([ref.124]). -Further, maintenance of normal electrical activity of isolated perfused -cat brains is highly dependent upon the presence of the ribonucleic acid -precursors, uridine and cytidine, in the perfusate ([ref.125]), and -severe derangements occur if any of a variety of pyrimidine antagonists -are added ([ref.126]). Brief electrical stimulation of cat cortical -tissue causes an increase in nucleic acid cytidine and adenine, thus -indicating a synthesis of altered polynucleotides. Finally, injections -of RNA in animals have shown interesting effects. When given at a dose -of 116 mg/kg daily for 1 month, rats showed an enhanced response and -greater resistance to extinction in a shock-motivated behavioral -response. It has been shown by another group that injections of RNA -enhance the ability of young animals to learn various tasks. - -Planaria have been used in a variety of studies which seem to bear on -the problem of memory. Quite recent evidence by Bennett, Calvin, and -their associates has cast somewhat of a pall over the studies; -nevertheless, the work may have some validity. Interest in the use of -flatworms, particularly planaria, for study of memory began with a -demonstration by McConnell that these simple animals could undergo -conditioning ([ref.127]). Subsequently, it was found that some -conditioning was retained when the animal was transected and allowed to -regenerate. The retention of training was found in both new animals, -although the very simple brain, really only two ganglia, was in the head -section ([ref.128]). - -Apparently, some diffusely distributed component of the animal was -responsible for retention of learning. Evidence has accumulated to -indicate that this material is RNA. Among this evidence is the -following: - - (1) The two halves of a trained planaria were allowed to regenerate in - a solution containing RNA-destroying enzymes. Whereas the head - ends retained some training, no retention was observed in the - animals derived from the tail end ([ref.129]). - (2) When pieces of trained planaria were fed to untrained animals, the - untrained cannibal required a shorter time to become trained to a - criterion. It would appear that the digestive system of planaria - is so simple that the material responsible for the transfer of the - information was not broken down. - (3) When RNA, obtained from trained planaria, is injected into the - digestive tract of untrained animals, there is a transfer of - information. - - - NEUROPHYSIOLOGY[2] - - [2] Excerpt from [ref.130]. - - -Neurophysiological studies concern the functions of the nervous -system--in particular the central nervous system (CNS)--under normal, -simulated, and actual flight conditions. Of paramount importance is the -maintenance of equilibrium and orientation in three-dimensional space. -The ability of man and his close relatives among the vertebrates to -maintain these functions depends on an integrated sensory input from the -vestibular organ; the eyes; the interoceptors of the muscles, tendons, -joints, and viscera; and the exteroceptors of the skin. - -Certain parameters of the environmental and space-flight conditions -drastically affect man's ability to maintain equilibrium and spatial -orientation. Centrifugal forces modify or reverse the directional vector -of gravity. Linear acceleration may increase enormously, as may angular -stimulation. The sensory organs listed above are unreliable under such -conditions. The very organ which is designed specifically to furnish -information on spatial orientation may malfunction in man while he is in -flight. Thus, with respect to sensory orientation, these labyrinthine -organs are by no means precision instruments. - -The use of classical histological methods and the observation of -equilibrium disturbances resulting from operative interference with the -internal ear have in the past been the two principal sources of -knowledge concerning the structure and function of the labyrinth, but -the answers given to various questions vary considerably in their value. -The development of electrophysiological techniques and the refinement in -recent years of the ultrastructural analysis by means of the electron -microscope may allow more precise experimental studies of the -correlation of function and structure. - -Before considering vestibular impulses in their bulbar and descending -spinal pathways, a recent study concerning the generation of impulses in -the labyrinth must be mentioned. Von Bekesy's finding ([ref.131]) of the -direct current potentials in the cochlea aroused speculation about the -existence of similar labyrinthine potentials. Such dc potentials were -also detected in the semicircular canal of the guinea pig by Trincker -([ref.132]), who measured the potential changes in the endolymph, -surface of the cupula, or side of the crista during cupular deflection. -It seems likely, however, that the effects do not represent the -physicochemical changes in the cupula but the electrical potentials in -the nerve and nerve endings of the crista. Attempts at differentiating -these effects have failed so far. Great expectations are brought by the -advances of microchemistry, microphysiology, and physical chemistry with -regard to the excitatory processes, the generation of the nerve impulse. -Quite apart from a need to understand vestibular nerve discharges and -patterns more adequately in such terms, the analysis of the vestibular -system has in the past revealed general biological principles which were -not readily discernible through the examination of other tissues -([ref.133]). - -The neural connections of the vestibular organ consist of numerous -chains of neurons, reciprocally linked in many ways and having their -synapses in various anatomical nuclei. All the chains work in intimate -collaboration, and the final pattern of reflex responses is attributable -largely to the highly complex integrating activity of the center. The -labyrinthine function is automatic, carried out in a reflex fashion: in -other words, mostly below the level of consciousness. The brain centers -through which the labyrinth elicits the various appropriate muscular -reactions of the head, body, limbs, and eyes--the righting, the -postural, and the ocular reflexes--represent an intricate mechanism. -Before we can hope for a satisfactory understanding of their functional -organization, we will have to know their anatomy in more detail. Thus, -we are confronted with a fruitful field for the exploration of basic -mechanisms of neuronal activity. Major advances dining the last years -have provided us with new information about the neuroanatomy of the -vestibular system (refs. [ref.134]-[ref.137]). - -Vestibular impulses entering the brainstem ascend and descend the -neuroaxis and cross the midline. It was previously believed that the -vestibular apparatus had only subcortical projections. Recently, -however, it has been established by means of electrophysiological -methods that the organ is represented by a projection area in the -cerebral cortex of some animals (refs. [ref.138]-[ref.141]). The use of -brief electrical stimulation of the vestibular nerve in order to elicit -a cortical response has been of great value for the mapping of these -areas. - -Among a great variety of sensory receptors, the vestibular ones are -capable of evoking the most widespread somatovisceral effects throughout -the body. Moreover, vestibular effects seem to be imperious and less -dependent upon the state of readiness of the nervous system. As a -consequence of the extensive distribution of vestibular effects, there -are many opportunities for central integration. Proprioceptive and -vestibular systems are both known to be active in posture and -locomotion; streams of impulses arising from the receptors in each of -these systems must converge to influence the activity of the final -common path. The state of the motor centers of the spinal cord, as -affected by vestibular stimulation, has been tested by dorsal root and -other sensory input interventions. These experiments have provided us -with insight into the mechanisms concerned with the vestibular control -of spinal reflexes (refs. [ref.142]-[ref.146]). - -It has long been known that the vestibular apparatus is essential for -the development of motion sickness. Commonplace subjective experience of -nausea relates to visceral changes mediated through autonomic efferent -pathways and may ultimately involve rhythmic somatic nerve discharges to -skeletal muscles responsible for retching and vomiting. However, very -little is known about the central nervous mechanisms responsible for -elaboration of the whole syndrome. Since the maintenance of vestibular -bombardment for some length of time seems essential for the development -of motion sickness, one would presume this to be an instance of slow -temporal summation. Experimental findings demonstrate a powerful effect -of temporal summation upon somatic motor outflow during vestibular -stimulation ([ref.147]), and not upon parasympathetic outflow. - -The practical implication of these studies is closely related to -physiological effects of weightlessness. Based on experimental evidence -from short weightless periods obtained in aircraft, it was concluded -that "when the exposure becomes longer, there may develop minor -physiologic disturbances which, if cumulative or irritating, may cause -or enhance psychiatric symptoms" ([ref.148]). Although the zero-g -condition, per se, does not cause spatial disorientation if visual cues -are provided, the astronauts reported a temporary loss of orientation -during the orbital flight while they were engaged in activities which -diverted their attention. However, no disturbing sensory inputs were -observed during the weightless period. Violent head maneuvers within the -limited mobility of the helmet were performed in every direction without -illusions or vertigo. The subjective sensations of "tumbling forward" -after sustainer engine cutoff reported by the Mercury astronauts, and -Titov's motion sickness attacks, which were particularly dismaying -during head movements, were well within the entire range of -psychosomatic experiences already obtained during aerodynamic -trajectories ([ref.149]). Interestingly enough it now appears that the -otolithic output in mammals including man is the differential of linear -acceleration, and therefore unaffected by zero g. - -Of interest in this connection are the problems which may be encountered -during and following long-term exposure to weightlessness. Although -there is no evidence of adverse effects on operative behavior, the -possibility of biological disturbances on a cellular or subcellular -level, which may cause a deterioration of the somatic basis, has been -repeatedly stressed. Whether effects of this sort will occur or whether -the organism will be able to adapt is still an open question. Since -motion sensitivity based on vestibular stimulation differs widely among -individuals, the selection of astronauts may solve the problem of zero-g -vestibular disturbance. Reports from the MA-8 (Sigma 7) and Vostok III -and IV flights seem to support this assumption. Moreover, experiments -are being made in the slow rotation room at the Naval School of Aviation -Medicine to study the Coriolis effects which arise when "artificial -gravity" is produced by angular acceleration. Since man can adapt to -wave motion on shipboard within a few days, a similar process may be -expected to occur in the case of long-term weightlessness ([ref.150]). - - - - - chapter 5 - -_Molecular Biology and Bioinstrumentation_ - - -To support biological investigations in space and to accumulate baseline -data needed for manned space flight, NASA has conducted a program in -laboratory research and theory. A multidisciplinary approach has -included such fields as ecology, physiology, organic and biological -chemistry, engineering, electronics, and optics. Emphasis in this -program has been placed on qualitative and theoretical rather than -purely descriptive research, and the investigation of fundamental -biological phenomena at all levels, from the molecular to the total life -form. - - - MOLECULAR BIOLOGY - -Research in molecular biology has included chemical, physical, -biological, and theoretical investigations of prebiological conditions -on Earth and, possibly, on other planets; studies of cellular -inclusions; genetic material (DNA and RNA) and coding; as well as energy -transfer in biological systems. - -The understanding of prebiological conditions on Earth, and possible -conditions on other planets, depends upon the nature of the complex -chemical species which might be encountered. Scientists have shown that -biologically important compounds, such as amino acids, can be generated -by applying an electrical discharge, ultraviolet radiation, or heat to a -gaseous mixture. Biologically interesting compounds can be removed from -such a system by condensation or absorption; however, in the limited -time and space available in such experiments, many compounds are not -produced in sufficient quantity to be measured. - -The National Biomedical Research Foundation (NBRF) and the National -Bureau of Standards (NBS) are conducting an investigation on equilibria -in multielement systems. The distribution of molecular species at -equilibrium is independent of the way equilibrium was reached and is -dependent only on pressure, temperature, and elemental composition. Many -of the conditions which might have arisen naturally can be approximated -by thermodynamic equilibrium. Compounds which can be formed at -equilibrium need no special mechanism to explain their presence. -However, special mechanisms have to be sought for those compounds which -could not be so produced and which would have been required for the -structure and nutrition of the first living organisms. - -In the absence of precise knowledge of the composition of the primitive -planetary atmospheres, equilibrium concentrations with a wide range of -temperatures, pressures, and elemental compositions are being -investigated by NBRF and NBS. These investigators have postulated that -the maximum atmospheric pressure may have approached 100 atm if the -primitive Earth was sufficiently hot and if an appreciable portion of -the water on Earth's surface today was present on primitive Earth. (If -the present oceans were to evaporate, the surface pressure would be -approximately 300 atm.) Low pressures of 10^-6 atm and temperatures -between 500° and 1000° K are being used. - -A large range of N, O, C, and H compositions are being investigated for -interesting and plausible combinations of factors. In these calculations -an IBM 7090 computer is being used to obtain data on a very large number -of combinations of chemicals. Other chemical species will be added as -the research continues. Some results of this study give an insight into -the variety of biologically significant chemicals which might have -existed during Earth's primitive prebiological condition or may now -exist on the surfaces and in the atmospheres of other planets (refs. -[ref.151]-[ref.153]). The general method described by White et al. -([ref.152]), minimizing the free energy of the system, was used. The -solution was approached by an iterative process, starting with an -initial guess of concentrations of the compounds. At each step, _M_+1 -linear equations are solved where _M_ is the number of elements in the -system. - -In addition to listing of the concentrations of all compounds included -in each problem, the results of three-element problems have been -expressed on a triangular composition diagram for convenience. A coarse -grid of 60 points is used to survey all elemental compositions, with -finer grids being used in regions of particular interest. The calculated -concentrations of the compounds at each composition are stored, and -finally a series of triangular diagrams is printed out, each showing the -concentrations of as many as four compounds at the grid points. - -Figure 2 shows the results obtained in the C, H, and O systems. Organic -compounds in concentrations greater than 10^-20 mole fraction are found -everywhere except where free O2, is present. Solid carbon theoretically -becomes stable along the lower dashed line at 500° K. However, reactions -producing it are very slow. The supersaturated region beyond the line of -potential carbon formation was also investigated. A threshold was found -where polynuclear aromatic compounds are sufficiently concentrated to -form a liquid phase. These conditions may have been involved in the -primordial formation of asphaltic petroleum. - -[Illustration: Figure 2.--_Equilibrium diagram for the system C-H-O._] - -Jukes and associates ([ref.154]) at the University of California at -Berkeley have been investigating the code for amino acids in protein -synthesis, the key for translating the sequence of bases in DNA into the -sequence of amino acids in proteins. The amino acid code was solely a -matter of theory until Nirenberg and Matthaei ([ref.155]) at the -National Institutes of Health carried out a crucial experiment. This -experiment bridged the last remaining gap separating theoretical -genetics and test-tube biochemistry. It now became experimentally -possible to search for codes for all 20 amino acids concerned in the -synthesis of proteins. - -The amino acid bases of DNA are: A, adenine; C, cytosine; G, guanine; T, -thymine; and U, uracil, which replaces thymine in RNA. There are only 16 -ways of arranging A, C, G, and T in pairs. For this and other reasons it -is thought that a triplet of three consecutive bases is needed to code -for each amino acid. The sequences of bases in a strand of DNA are known -to be unrestricted with respect to the order in which they occur; -apparently any one of the four bases can be next to any of the other -four, although, of course, each base must be paired with the -corresponding complementary base in the adjacent strand. Since the same -freedom is true of the amino acid sequences in the polypeptide chains of -proteins, any one of the 20 amino acids can occur next to any other. -Moreover, the sequences in DNA are subject to mutational changes in -which one base replaces another, or bases are added to or deleted from -the DNA. Such rearrangements plus the possibility of lengthening of DNA -molecules are numerous enough to account for all the genetics of living -forms since the first appearance of life on Earth. - -Most of our knowledge is based on experiments with synthetic RNA carried -out with extracts of _E. coli_. The majority of the work has been at -Nirenberg's laboratory at the National Institutes of Health and at -Ochoa's laboratory at New York University ([ref.155]). Various -combinations of A, C, G, and U were used in preparing the synthetic RNA -molecules that are used in experiments to explore the code. These -molecules are made by incubating a mixture of ribonucleoside -diphosphates with a specific enzyme, polynucleotide phosphorylase. An -important property of this enzyme is that it condenses the nucleoside -diphosphates into polynucleotide strands containing random sequences -depending on the proportion of each base. For example, if the enzyme -were furnished with a mixture of 5 parts of A and 1 part of C, it would -make strands containing, on the average, 25 sequences of AAA, 5 of AAC, -5 of ACA, 5 of CAA, and 1 each of ACC, CAC, and CCA. The proportion of -triplets within the strands of a polynucleotide is reflected in the -proportion of amino acids in polypeptides that are obtained in the -cell-free system. Most of the present knowledge of the amino acid code -is based on this concept. All the proposed codes have been discovered by -this experimental approach where synthetic RNA molecules are used as -"artificial" messenger RNA. - -Representative of another class of activities in molecular biology is -the examination of passive ion flux across axon membranes. This work is -being done by Goldman at the National Naval Medical Center. The question -of stimulus transmission by nerve tissue is far from simple, and the ion -concentrations associated with nerve membranes is a significant part of -the answer. Because the space environment may very well produce -alterations in these ion potentials, an investigation of their natures -and significance becomes extremely important. A working theory is now -being developed as a result of this study. - -Vital cell processes, chemical transformations, and mechanisms that -provide energy for cell maintenance and activity have been studied by -Kiesow (refs. [ref.157] and [ref.158]) at the Naval Medical Research -Institute. The common objective of all phases of this project is the -elucidation of reaction steps in which energy and matter are transformed -in living systems. Compared with _photo_synthetic organisms, -_chemo_synthetic bacteria offer distinct advantages for the study of -energy assimilation. These studies have led to the following -experimental findings. - -With the energy from oxidation of nitrite, NO2-- to nitrate, NO3-- as an -_inorganic_ source, and with added _organic_ chemical energy from the -hydrolysis of adenosinetriphosphate (ATP) to adenosinediphosphate (ADP) -and inorganic phosphate, chemosynthetic bacteria are capable of reducing -diphosphopyridinenucleotide (DPN^+) to DPNH, in a coupled -oxidoreduction-dephosphorylation. Thus, in the crucial step of -chemosynthesis, _ATP is consumed, not produced_. However, in -simultaneously proceeding cell respiration, the energy donor, DPNH, is -oxidized and generates more ATP than is required for DPN^+ reduction. -This "breeder cycle" for DPNH--with different ratios of cell respiration -and biosynthesis--results in a net production of either DPNH, or ATP, or -both. Production of DPNH in the cycle leads immediately to the -assimilation of C^14 from HC^14 O3--. These observations explain the -bacteria's energy source without the classical hypotheses of either -direct phosphorylation or direct CO2 reduction by inorganic chemical or -electromagnetic energy. The cycle transforms the free energy of nitrite -oxidation into the free energy of the organic compounds. Cell -respiration and elementary biosynthesis proceed through structure-bound -enzyme systems in the same fraction of subcellular particles. Three -components, two cytochromes and one flavoprotein, have been identified. -A thermodynamic analysis of the DPNH "breeder cycle" appears to be -attainable by measurements of redox potentials and calorimetric -determinations of heats of reaction. - -Studies are also being conducted by Pollard and associates at -Pennsylvania State University in an attempt to formulate a theoretical -basis for the description of the processes of synthesis, growth, -division, and differentiation of the living cell. Such a theory would be -basic to an understanding of very primitive life forms or prebiological -material which might be found elsewhere in the universe. For these -purposes, studies are being undertaken in macromolecular reproduction -which differ from the studies involving cellular genetic material. -Theories concerning the problem of replication of cellular structures -and information storage in two-dimensional systems are being developed. -Theories are also being developed about the mechanisms which control and -regulate receptor and enzymatic activities within the cell. - -One study involved the rate of mutation in cells and disposed of the -suggestion that the process of mutation consists of a "tunneling" of -proton from one base to another in DNA. Such a suggestion can no longer -be advanced as a major explanation of mutations. - -Work is also being conducted on the centrifugation of cells of _E. -coli_. It has been shown that cells exposed to as little as 100 g have a -modification in their function. This has been looked at from the point -of view of thymine uptake, which would be concerned with the formation -of DNA, and also from the point of view of the induction of an enzyme, -which would correspond to the transcription of the DNA. Preliminary -experiments in the latter case indicate considerable centrifugation -effect. The thymine uptake is affected, but not nearly as much as -formerly thought. Further work is in progress in this area. - -Important work has been completed on the cells of _E. coli_ grown on -maltose, which can be induced to produce betagalactosidase by the -addition of thiomethyl galactoside. If cells are irradiated shortly -after induction, the transcription of the DNA ceases and the enzyme -produced by the messenger RNA is observed to reach a maximum. This -enables the calculation of the half-life of unstable messenger RNA. The -half-life for this decay is readily measurable, and values are given -over a temperature range of 17° C (5.2 minimum) to 45° C (0.56 minimum). -These agree very well with half-lives measured by others by inducing for -short times and measuring the course of enzyme formation. The rate of -transcription is involved in the kinetics of cessation of enzyme -induction, and the rate of transcription can be measured. Arrhenius -plots for this rate and the rate of decay are given, and the activation -energies measured are about 16 000 cal/mole. The cessation of -transcription is linked to the degradation, possibly of only one strand, -of DNA. - -Pollard has suggested that one important action of ionizing radiation is -concerned with the transcription of the genetic message into RNA. -Clayton and Adler ([ref.159]) showed that induced catalase synthesis in -_Rhodopseudomonas spheroides_ is inhibited by low doses of X-rays, -giving experimental support to the idea. Pollard and Vogler ([ref.160]), -using cells in which the process of induction involved permease, showed -that there is some sensitivity to gamma radiation. Novelli et al. -([ref.161]) found a reduced sensitivity as compared with colony -formation, but it is still a considerable sensitivity. - -The process of induction of an enzyme indicates that the transcription -of the genetic message is repressed by something which can be acted on -by a small molecule, the inducer, to remove repression and permit the -formation of messenger RNA, which then acts to make the enzyme. The -messenger RNA undergoes decay through a process which is still not -clear. Very elegant measurements by Kepes ([ref.162]) show that for the -messenger RNA for betagalactosidase, the half-life is 1.02 min at 37° C -and 2.05 min at 25° C. The time of onset of enzyme formation after -induction was found to be about 3 minutes. - -If the process of transcription is indeed sensitive to ionizing -radiation, then the irradiation of cells which have just been induced -should show formation of the enzyme to the extent of formation of new -messenger RNA within a few minutes, plus the formation of the enzyme -while the messenger RNA is decaying. This pattern was found by Clayton -and Adler. The experiments conducted by Pollard and associates amplify -and extend their work and also agree with the work of Kepes ([ref.162]). - - - BIOINSTRUMENTATION - -Fernandez-Moran (refs. [ref.163]-[ref.165]), at the University of -Chicago, has devised a new multielectrode electrostatic lens which he -has incorporated into an electron microscope. This necessitated the -development of a novel high-voltage power source and voltage regulator -of extreme stability and accuracy. Some promising work has now been done -on superconducting lenses. In a series of experiments with a simple -electron microscope without pole pieces, using high-field -superconducting niobium-zirconium solenoid lenses in an open air core, -liquid helium Dewar, electron microscopic images of test specimens have -been recorded while operating at 32 200 gauss in a persistent current -mode, with regulated accelerating potentials of 4 to 8 kilovolts. These -preliminary experiments have demonstrated the exceptional stability of -the images (both short term and long term) over a period of 4 to 8 hours -and the relatively high quality of the images. - -Progress has been made on the viscosimeter for high intrinsic -viscosities. This is now working, and the viscosity of DNA preparations -has been measured. It is hoped to use the viscosimeter to study the -variation in DNA viscosity as a function of the cell cycle. - -An instrument is under development by Wald at the University of -Pittsburgh to automatically analyze cytogenetic material and, thus, -extend cytogenetic methodology both for research and as a biological -monitoring procedure, using automatic electronic scanning and computer -analysis of chromosomes. Chromosomal aberrations can thus be monitored -under unusual and abnormal conditions such as weightlessness and -radiation, since chromosomes are very sensitive to stress situations. In -this device a sample will be prepared and automatically inserted under a -microscope lens. The device will then scan, identify, and photograph on -35-mm film a predetermined number of mitotic cells and process the film. -The data will be recorded under the direct control of a digital -computer. The computer will perform a detailed quantitative analysis of -the pictorial data. - -Significant effort has been expended in the development of -instrumentation for measuring and recording electrophysiological -information. One such instrument, developed by the Franklin Institute, -Philadelphia, Pa., is a temperature-sensing microprobe. This microprobe -is an implantable and remote broadcasting instrument. These developments -are associated, in part, with training programs so that competent -individuals may be trained not only in electronics but also in the -biological uses of the devices they construct. - -A project of interest, conducted at the Stanford Research Institute, is -the investigation of the uses of an extremely sensitive method for -measuring magnetic susceptibility having the possibility of detecting -macroscopic quantum effects in macromolecules of biological interest. -Good progress has been made in the first 15 months of a project devoted -to the development and initial use of equipment specifically designed -for this purpose. A new superconducting circuit, together with -superconducting magnetic shields, has been constructed. This apparatus -can measure the magnetic susceptibility of small organic samples at -temperatures between 1° and 300° K in fields up to 40 000 gauss. It can -detect flux changes of 10^7 gauss-cm², which is equivalent to detecting -a change in specific susceptibility of 1 in 10^9 in a 100-mg sample -under an applied field of 10 000 gauss. - -Several hundred preliminary measurements were made on samples of -coronene. The most reliable of these were in agreement with published -values of the magnetic susceptibility of coronene. Experience during -these measurements led to changes which have resulted in an apparatus -well suited to the measurements on macromolecules. An improved version -of the superconducting circuit now available shows promise of a further -improvement in sensitivity by a factor of more than a thousand -([ref.166]). - -Living organisms possess many unique processes and systems which are -complex and poorly understood. The new theoretical approaches, combined -with laboratory studies, are expected to result in advances which will -expand both our scientific and technological horizons. - - - - - chapter 6 - -_Flight Programs_ - - - BALLOONS - -Biological and medical experiments carried out on balloon flights, both -manned and unmanned, antedate the establishment of NASA. Aside from the -early use of balloons in flights that could be called simply -flight-survival studies, balloons have made important contributions to -our present knowledge of the effects of cosmic radiation and to various -aspects of space travel. - -The achievements of the Strato-Lab and Man High series by the U.S. Navy -and Air Force include a wealth of information on balloon travel and on -the survival of man at altitudes close to and above 100 000 feet. -Generally, balloon launches of animals, which reached a maximum in 1953 -when 23 balloons were released, have established the feasibility of a -program of extended manned balloon flights to high altitudes. - -Atmospheric life studies outside the area of cosmic radiation effects -have been comparatively few. Results from two manned flights, Strato-Lab -I and II, indicate that the flights did produce pronounced changes in -white blood cell count; however, the data suggest that these changes -were due to psychological rather than physical stress. Exposure to -altitudes above 90 000 feet for a total of 62 hours did not produce any -general behavioral change in two Java monkeys, according to other -balloon flights. Many of these flights were effective in testing -equipment, telemetering devices, and in pointing the way for other -flights. - -Stratoscope I and II, originally undertaken by the Office of Naval -Research (ONR), are projects involving various astronomical observations -with the aid of a balloon-borne telescope and television and camera -systems. NASA cooperated with ONR on Stratoscope II (36-inch telescope -compared with Stratoscope I's 12-inch telescope) which has already -resulted in significant discoveries about the nature of the planets and -stars. Water vapor has been identified in the atmosphere of cool red -stars and an analysis of the Martian spectra showed a greater abundance -of carbon dioxide than had previously been believed. Since the -balloon-borne telescope was carried beyond Earth's obscuring atmosphere, -the Stratoscope projects have yielded valuable photographs of the Sun, -stars, and various planets. - - - ROCKETS AND SATELLITES - -Historically, biological experiments aboard rockets and satellites have -been limited to a "piggyback" and "noninterference" basis on military -rockets. For the past few years, however, as the effort toward manned -space flight leading to lunar and Martian landings increased, more -attention was devoted to experiments designed to show the effects of the -space environment on living systems. As in the balloon flight programs, -the U.S. Army, Navy, and Air Force played an important role, reaching -what might be considered a high point with the successful launch and -recovery of a ballistic rocket experiment with monkeys Able and Baker. -Aerobee rockets as well as Thor IRBM's carried biological payloads -consisting of mice and monkeys on six launches, contributing to our -knowledge of the effects of weightlessness and radiation on higher -animals. - -Van der Wal and Young ([ref.78]) used Thor-Able combinations to serve as -boosters for lifting a 20-pound biocapsule to a peak altitude of 1400 -miles and over a distance of about 5300 miles from Cape Canaveral to the -west coast of Africa. Weightlessness was attained for a period of almost -40 minutes. During reentry into the atmosphere, a peak deceleration of -about 60 g was reached. Each of the three capsules flown carried one -mouse (Mouse-in-Able); two of the mice were instrumented for heart-rate -telemetry. Although all three mice were lost, the two experiments with -Laska and Benji yielded physiological results. - -The experimenters designed effective instrumentation for registering the -electrical activity of the mouse's heart through a single commutated -telemetry channel. Records were obtained for both animals during various -portions of the flight. The results indicate that both animals were -alive when the nose cones hit the water. - -Two South American squirrel monkeys (Gordo and Baker) and a rhesus -monkey (Able) were launched into space from Cape Canaveral in 1958 and -1959 by U.S. Army Jupiter missiles. The vehicles reached speeds of -approximately 10 000 mph and altitudes of 300 miles on flights which -lasted about 15 min. - -Time courses of cardiac and respiratory rates ([ref.80]) of the two -squirrel monkeys showed that the noise of the engine at liftoff -immediately produced an increase in their heart rates. Respiration also -increased temporarily, but slowed later with increasing acceleration. -Heart rates fluctuated considerably during launch acceleration, which -reached about 15 g at cutoff. - -The period of free flight and weightlessness was characterized by -pronounced fluctuations of heart activity in the postacceleration phase. -Thereafter, the heart rate of Baker remained relatively constant, -whereas the cardiac activity of Gordo fluctuated markedly and decreased -slowly almost to the end of his flight. Slight changes, which were -transient and not pathological in nature, were also noted in the -electrocardiogram. Gordo's respiration was very shallow during maximum -launch acceleration, when Baker's reached its highest value, only to be -approximated again during reentry when forces of about 35 g were -encountered. - -Able's cardiac and respiratory rates indicated that, after an initial -startle reaction, the heart rate dropped transiently and then increased -steeply, reaching a maximum of 259 during the 10-second interval at peak -acceleration. Respiration increased only slightly throughout the -launching phase. There was a period of tachycardia during -postacceleration weightlessness, after which the heart rate declined -steadily and was disturbed only by several startling missile events. At -the end of the subgravity phase, Able's cardiac rate was slightly below -normal. - -Although the periods of high g force and free flight were short, the -extremes were considerable, and the changes from one state to the next -were rapid. In spite of this, the cardiovascular, hemodynamic, and -electrocardiographic phenomena were remarkably well maintained. -Apparently the animals were not in serious plight at any time. That -psychological factors entered into the observed phenomena is clearly -evident from the increase in cardiac rate associated with the noise of -the engine prior to liftoff and also from the cinematographic record of -facial expressions. Nevertheless, the integrated responses indicated -that the animals' physiological states remained sufficiently normal to -insure a safe flight. - - - LITTLE JOE FLIGHTS - -The first step in an attempt at animal verification of the adequacy of -the Mercury flight program was the development of two tests by NASA in -collaboration with the U.S. Air Force School of Aviation Medicine in -which there would be a biomedical evaluation of the accelerations -experienced during the abort of a Mercury flight at and shortly after -liftoff. These flights were launched at the NASA Wallops Station with a -Little Joe solid-fuel launch vehicle. - -Two Little Joe launches were made with activation of the escape rockets -during the boost phase to secure maximum acceleration; only a brief -period of weightlessness was attained. The first launch was on December -4, 1959, and the other on January 21, 1960. A 36 by 18-inch sealed, -125-pound, cylindrical capsule containing the subject, an 8-pound -_Macaca mulatta_, the necessary life-support system, and associated -instrumentation was flown in a "boilerplate" model of the Mercury -spacecraft. The rhesus monkeys were named "Sam" and "Miss Sam." - -The flight profile included maximum accelerations of about 10 to 12 g -and periods of about 3 minutes at 0±0.02 g. The peak altitude obtained -in the last ballistic flight was about 280 000 feet. The experimental -capsule was pressurized at 1 atmosphere with 100 percent oxygen at the -start of the experiment and fell to just below a half atmosphere of -oxygen due to breathing during flight. The capsule temperature was kept -between 10° and 20° C in both flights. - -The measurements taken from the rhesus monkeys were the -electrocardiogram, respiration, body temperature, eye movements, and bar -pressing, but only partial results were obtained in the first flight. -Oxygen tension, total pressure, capsule temperature, and relative -humidity were recorded. Both animals were recovered alive and did not -show pathologic alterations in their physiologic and psychological -reactions. - - - MERCURY ANIMAL TEST FLIGHTS - -In the Mercury animal test program a Redstone missile carried the -chimpanzee Ham on a ballistic flight to a height of 155 miles to provide -animal verification of the success with which the Mercury system could -be applied to manned flight. The male chimpanzee was trained to perform -a two-phased reaction task during the 16 minutes of flight. The -chimpanzee Enos was put into orbit for 3 hours and 20 minutes. Results -of the two flights gave the following information: - - (1) Pulse and respiration rates during both the ballistic (MR-2) and - the orbital (MA-5) flights remained within normal limits - throughout the weightless state. Effectiveness of heart action, as - evaluated from the electrocardiograms and pressure records, was - also unaffected by the flights. - (2) Blood pressures, both arterial and venous, were not significantly - changed from preflight values during 3 hours of the weightless - state. - (3) The performance of a series of tasks involving continuous and - discrete avoidance, fixed ratio responses for food reward, delayed - response for a fluid reward, and solution of a simple oddity - problem was unaffected by the weightless state. - (4) Animals trained in the laboratory to perform during simulated - acceleration, noise, and vibration of launch and reentry were able - to maintain performance throughout an actual flight. - -From the results of the MR-2 and MA-5 flights, the following conclusions -were drawn: - - (1) The numerous objectives of the Mercury animal test program were - met. The MR-2 and MA-5 tests preceded the first ballistic and - orbital manned flights, respectively, and provided valuable - training in countdown procedures and range monitoring and recovery - techniques. The bioinstrumentation was effectively tested and the - adequacy of the environmental control system was demonstrated. - (2) A 7-minute (MR-2) and a 3-hour (MA-5) exposure to the weightless - state were experienced by the subjects in an experimental design - which left visual and tactile references unimpaired. There was no - significant change in the physiological state or performance of - the animals as measured during a series of tasks of graded - motivation and difficulty. - (3) Questions were answered concerning the physical and mental demands - that the astronauts would encounter during space flight, and it - was shown that these demands would not be excessive. - (4) It was also demonstrated that the young chimpanzee can be trained - to be a highly reliable subject for space-flight studies. - -The suborbital ballistic flight of Ham on January 31, 1961, was the -prelude to Alan R. Shepard's suborbital space flight, while the orbital -flight of Enos on November 29, 1961, preceded the orbital flight of John -H. Glenn. - -The fact that we now categorize these events as belonging to the rather -distant past, although they occurred only about 4 years ago, serves to -emphasize the pace of development in the exploration of space. While the -chimpanzee program may pale in the light of subsequent successes, its -scientific and technological contribution should not be overlooked. - -The significance of this project can be fully appreciated, and its -contribution judged, only by considering the lack of knowledge existing -at the time of its conception. In addition to its essential training -function, this project verified the feasibility of manned space flight -through operational tests of the Mercury life-support system. It -demonstrated that complex behavioral processes and basic physiological -functions remained essentially unperturbed during brief exposures to -space flight. The Mercury chimpanzee program marked the first time that -physiological and behavioral assessment techniques were combined for -evaluating the functional efficiency of the total organism in space. - -Perhaps the ultimate contribution of this program was in providing the -framework of knowledge upon which future scientific experiments on -biological organisms, exposed to flights of extended durations, must be -based. Biosatellite experiments designed to seek more subtle and elusive -effects of prolonged space flight on biological functioning will require -even more refined and difficult techniques, but will depend heavily on -the groundwork laid in these early steps of Project Mercury. - -A summary of the more important animal suborbital and orbital flights -during the period 1957 to 1964 is presented in table VII. - -In another NASA-supported flight, _NERV_ 1, various experiments were -carried in a suborbital flight of 20 minutes. _Neurospora_ molds showed -a surprisingly high level of mutation, but the control molds also had -high rates. - -The Discoverer XVII and XVIII flights, to which the Air Force -contributed, resulted in many interesting findings relative to the -responses of living systems to space flight. On the Discoverer XVII -flight, samples of human gamma globulin and rabbit antiserum specific -for human gamma globulin showed an increase in reactivity, and samples -of synovial and conjunctival cells showed no changes in their -cytological characteristics. - -Discoverer XVIII was launched during a massive solar flare which lasted -for the first 13 hours of the 48-orbit, 3-day flight. _Neurospora -conidia_, nerve tissue, algae, human bone marrow, eyelid tissue, gamma -globulin, and cancer cells were put in orbit. The results indicated that -biological specimens may be able to withstand radiation from solar -flares with a minimum of shielding and that aluminum shielding may be -better than lead. - -In 1949, the U.S.S.R. began a systematic, uninterrupted research program -in biological space experimentation. They have studied the effects of -physical stress, immune reactions, psychobiology and behavior, genetics, -and responses to environmental factors such as spacecraft dynamics and -ambient radiation. The organisms and biological materials included -tobacco mosaic and influenza viruses; T2 and T4 bacteriophage; _Bacillus -aerogenes_; lysogenic bacteria; _Clostridium butyricum_; _Escherichia -coli_; actinomycetes; yeasts; _Chlorella pyrenoidosa_; seeds of fir, -pine, onion, corn, lettuce, wheat, cabbage, carrot, buckwheat, cucumber, -beet, _Euonymus_, fennel, mustard, pea, broad bean, tomato, and nutmeg; -_Tradescantia paludosa_; _Ascaris_ eggs; snail spawn; _Drosophila -melanogaster_; loach roe; frog eggs and sperm; guinea pigs; mice; rats; -hamsters; rabbits; dogs; monkeys; human and rabbit skin; HeLa tissue -cultures and other tissues (refs. [ref.167] and [ref.168]). - - - Table VII.--_Orbital and Suborbital Animal Flights for 1957-64_ - - ---------------------------------------------------------------- - Year Animal subject Flight profile - ---------------------------------------------------------------- - United States - ---------------------------------------------------------------- - 1958 Mice _Wickie_, 1400 miles. None of the three - _Laska_, and flights were recovered. - _Benji_ - ---------------------------------------------------------------- - 1958 Squirrel monkey 300-mile maximum altitude over a - _Old Reliable_ 1300-mile distance via a Jupiter - rocket. Not recovered. - ---------------------------------------------------------------- - 1959 Rhesus monkeys 300-mile maximum altitude over a - _Able_ and _Baker_ 1500-mile distance via a Jupiter - rocket. Recovered. - ---------------------------------------------------------------- - 1959 Black mice 500 seconds of weightlessness in - Discoverer III via a Thor-Able - rocket. The Discoverer vehicle did - not go into orbit and the animals - were lost. - ---------------------------------------------------------------- - 1959 Rhesus monkey 53-mile altitude in Little Joe. - _Sam_ Recovered. - ---------------------------------------------------------------- - 1960 Rhesus monkey 9-mile altitude in Little Joe. - _Miss Sam_ Recovered. - ---------------------------------------------------------------- - 1960 C-57 black mice 650-mile altitude over a 5000-mile - distance via Atlas RVX-2A. - Recovered. - ---------------------------------------------------------------- - 1961 Chimpanzee _Ham_ 156-mile altitude over a 414-mile - distance via a Redstone booster, - Mercury capsule. Recovered. - ---------------------------------------------------------------- - 1961 Chimpanzee _Enos_ 2 Earth orbits. 183 minutes of - weightlessnessat an apogee of 146 - miles anda perigee of 99 miles. - Atlas booster, Mercury capsule. - Recovered. - ---------------------------------------------------------------- - Soviet Union - ---------------------------------------------------------------- - 1958 Dogs _Belyanka_ 280-mile altitude in hermetically - and _Pestraya_ sealed cabin. Recovered. - ---------------------------------------------------------------- - 1959 Dog _Otyazhnaya_ Over 100-mile altitude. Recovered. - and a rabbit - ---------------------------------------------------------------- - 1960 Dogs _Belka_ and 16 Earth orbits (24 hours) via - _Strelka_, 21 Sputnik V. First successful recovery - black and 21 white of living creature from orbital - mice flight. - ---------------------------------------------------------------- - 1960 Dogs _Pchelka_ and 16 Earth orbits (24 hours). - _Mushka_ Spacecraft destroyed during reentry. - ---------------------------------------------------------------- - 1961 1 dog, mice, 1 Earth orbit at an apogee of 155 - guinea pigs, and miles and a perigee of 114 miles. - frogs Recovered. - ---------------------------------------------------------------- - 1961 Dog _Laetzpochka_ 1 Earth orbit. Recovered. - ---------------------------------------------------------------- - France - ---------------------------------------------------------------- - 1961 Rat _Hector_ 95-mile attitude in a capsule - boosted by a Veronique rocket. - Recovered. - ---------------------------------------------------------------- - 1963 Cat Felicette 95-mile altitude in a capsule - boosted by a Veronique rocket. Over - 5 min of weightlessness. Recovered. - ---------------------------------------------------------------- - - - THE NASA BIOSATELLITE PROGRAM[3] - - [3] From [ref.169]. - -The space environment offers a unique opportunity to study the basic -properties of living Earth organisms with new tools and opens up new -areas of research for which biological theory fails to provide adequate -predictions. Unique components of the space environment of biological -importance are weightlessness or greatly decreased gravity, the -imposition of an environment disconnected from Earth's 24-hour rotation -(particularly its effect on biorhythms), and cosmic radiation with -energies and particle sizes unmatched by anything produced artificially -on Earth ([ref.169]). - -As progress is made in the manned exploration of space, the biological -effects of its unique environmental factors become of greater -importance. It is essential to determine the effects of space -environment on man's ability to perform physical and mental tasks. In -addition, it is necessary to develop those systems required for his -survival and for his physiological and psychological well-being, both in -space and in his subsequent resumption of normal life patterns. Despite -nearly a century of research and development in environmental -physiology, a number of phenomena will be encountered in long-term space -flight with which we have had neither the experience that would enable -us to predict the effects nor to develop the necessary protective or -remedial measures ([ref.170]). Many of the experimental programs in -bioscience are being carried out or planned so that the deleterious -effects of these phenomena may be determined, predicted, or avoided -before they are encountered in manned flight. - -Biological experimentation has been carried out in orbiting spacecraft -by Soviet and American scientists preparatory to manned space flight. -These first-generation exploratory experiments had the following -objectives: - - (1) To discover whether complex organisms could survive space - conditions and to test life-support systems - (2) To determine whether complex organisms (dogs and primates) could - survive launch, orbital space flight, reentry, and recovery - (3) To determine the effects of space radiation and any obvious - effects of weightlessness on biological organisms - -These biological studies indicate that manned space flight was -practicable, and the various cosmonaut and astronaut flights have proven -the validity of the results. - -The National Academy of Sciences' Space Science Board summer study -([ref.171]) recommended that-- - - NASA should exploit special features of the space environment as - unique situations for the general analysis of the - organism-environment relationships including, especially, the - role environmental inputs play in the establishment and - maintenance of normal organization in the living system. NASA - should support studies in ground-based and in orbiting - laboratories [biosatellites] on the biological effects of - gravity fields both above and below normal. This should be - considered a major responsibility of NASA in the area of - environmental opportunities. NASA should support studies of - biological rhythms in plants and animals including man as part - of its effort in environmental biology. Investigate by - observation of rhythms in organisms in space in (_a_) polar and - equatorial low orbits; (_b_) orbits less than, equal to and - greater than 22,000 miles. Properly designed experiments should - be conducted to explore the effects of different environmental - factors when these impinge simultaneously on test organisms. - -The Panel on Gravity of the Space Science Board ([ref.67]) stated that -the major effects of low gravity would be expected in heterocellular -organisms that develop in more or less fixed orientation with respect to -terrestrial gravity and which respond to changes in orientation with -relatively long induction periods, including the higher plants. On the -other extreme are the complex primates which respond rapidly, but whose -multiplicity of organs and correlative mechanisms make the occurrence of -malfunction and disorganization probable, but not certain. The Panel -recommended emphasis on early embryogenesis and histogenesis, -particularly of plants during exposure to low gravity, and anatomical -studies after low gravity. They stated that perturbations of the -environment to which the experimental organism is exposed must be -limited or controlled to reduce uncertainties in interpretation of -results. At the same time, the introduction of known perturbations may -assist in isolating the effects due solely to gravity. Ground-based -clinostats and centrifuges should be used in conjunction with the -experiments, and an attempt should be made to extrapolate effects of low -gravity with the clinostat. - -The study of the effects of unique or unknown space environmental -factors will probably yield unexpected results which may drastically -modify future technical approaches. The results from these biosatellite -studies will have broad application to longer term, manned space flight, -including manned space stations and lunar and planetary exploration. - -The biosatellite program is a second-generation series of carefully -planned and selected experiments, including some highly sophisticated -experiments which have required several years of baseline study and -equipment development. These orbiting recoverable biosatellites will -provide opportunities for critical testing of major biological -hypotheses in the areas of genetics, evolution, and physiology. - -The scientific community showed great interest in the biosatellite -program, and scientists from universities, industry, and Government have -submitted 185 flight experiments involving primates and other mammals, -vertebrate and invertebrate animals, micro-organisms, and plants. - -The selected biosatellite experiments include studies at the cellular, -tissue, and organism levels, including embryological development and -growth experiments at the tissue level and physiological, behavioral, -reproductive, and genetic studies at the organism level. The experiments -are divided into six categories: - - (1) Primates - (2) Mammals (nonprimate) - (3) Animal, cellular, and egg - (4) Plant morphogenesis, photosynthesis, and growth - (5) Biorhythm - (6) Radiation - -Twenty experiments have been selected for flight to study the effects of -weightlessness and decreased gravity during 3- to 30-day orbital -periods. The experiments include a wide variety of plants and animals -from single-celled organisms to higher plants and animals. The effects -of weightlessness on the primate will be studied, especially the central -nervous, the cardiovascular, and the skeletal systems during 30-day -orbits. - -Experiments have been selected to study the genetic and somatic effects -of weightlessness combined with a known source of radiation (Sr^85) to -determine if there are any antagonistic or synergistic effects -([ref.172]). Experiments are also included for studying the effects of -the unique environment of the Earth-orbiting satellite and removal from -the Earth's rotation in relation to biological rhythms of plants and -animals. - -Six biosatellites are included in the presently approved program, with -the first flight in 1966. They will be launched from Cape Kennedy by the -improved two-stage, thrust-augmented Thor-Delta into a nearly equatorial -circular orbit at an altitude of 180-200 miles for periods up to 30 -days. Recovery will be by Air Force airplane during capsule/parachute -descent. The spacecraft weigh 1000-1200 pounds, have a 280-pound -recoverable capsule and, while in orbit, will not experience greater -than 1/10 000 g of acceleration. The life-support system will provide an -environment at sea-level pressure of 80 percent nitrogen, 20 percent -oxygen, and no more than 0.5 percent carbon dioxide with a temperature -of 75° F ±5° F. - -All experiments are in various stages of development or testing and -flight test hardware has been and is being constructed. The experiments -and hardware are being subjected to preflight tests simulating launch -and recovery stresses. Rhesus, pigtail, and squirrel monkeys have been -subjected to the dynamic forces of the simulated flight under conditions -of complete, partial, and no restraint. Three types of centrifuges have -been used to simulate the flight profile. Primates were fully -instrumented with deep brain electrode implants, implanted catheters, -and other implanted sensors. During centrifugation, motion pictures were -taken. These primates were semirestrained in form-fitted couches which -allowed movement of the body while facing the accelerative force in a -ventrodorsal position (eyeballs in). In this series of tests, all -primates were normal following the tests and exhibited no unusual -behavior or effects. X-rays showed that implanted catheters and -electrodes remained in place, and there were no movements causing tissue -damage. However, when the primates were placed with their backs toward -the accelerative force, dorsoventral (eyeballs out), the animals -suffered visible damage. At 6 g there was no visible stress, but at 8 g -swelling of the lower eyelids was noticeable. At 11 g both eyelids were -swollen shut. In the biosatellite program, primates will be placed in -the semirestraint couches in a position facing accelerative forces, -ventrodorsal (eyeballs in), to prevent these effects. - - - - - chapter 7 - -_Manned Space Flight_ - - - BIOREGENERATIVE LIFE-SUPPORT SYSTEMS - -Placing a man in space requires a complete life-support system capable -of supplying sufficient oxygen, food, and water and removing excess -carbon dioxide, water vapor, and human body wastes. In addition, the -oxygen, carbon dioxide, and pressure must be maintained at a suitable -level. Any accumulated toxic products and noxious odors must be removed. - -In the spacecraft the human is confined in a restricted environment in -which it is necessary to establish a balanced microcosm or closed -ecological system. This is an enormous biological and bioengineering -problem. Weight, size, simplicity of operation, and reliability -particularly are important factors. - -For relatively short missions involving one or several astronauts, food, -oxygen, and water can be stored and made available as required, and the -various waste products can be stored. On longer missions, particularly -those involving more than one astronaut, efficient chemical or -biological regenerative systems will be required. Any regenerative -system introduces a fixed cost in weight of processing equipment and -energy requirements. - -Chemical, or partially regenerative, methods for providing breathing -oxygen by the regeneration of metabolic products such as water vapor and -carbon dioxide include the thermal decomposition of water and CO2, -photolysis and radiolysis of water, electrolysis of fused carbonates and -aqueous solutions, and the chemical reduction of CO2 with H2, followed -by electrolysis of the water formed. Chemical regenerative systems have -been developed to remove excess carbon dioxide and water vapor from the -atmosphere. Nonbiological regenerative systems are time limited by the -amount of food, water, and oxygen that can be carried or recovered. -These physical-chemical processes show great potential, but they also -present many difficulties, including requirements for extremely high -temperatures and considerable amounts of power, the formation of highly -toxic materials, and high susceptibility to inactivation. None of the -presently studied nonbiological processes can function as completely as -a bioregenerative system. All these nonbiological systems have -unrealistic supply requirements and produce unusable wastes. -Consequently, for long planetary missions the bioregenerative systems, -though also beset with problems, are potentially far superior to their -physical and chemical counterparts. - -Table VIII shows average daily metabolic data for a 70-kg astronaut. A -man breathes about 10 cubic feet of air per minute, or 400 000 liters, -daily. The expired air contains about 4 percent carbon dioxide. Man -normally breathes air containing 0.03 percent CO2, but can withstand -comfortably about 1.5 percent CO2. Anything in excess of 1.5 percent -will produce labored breathing, headaches, and, if greatly exceeded, -death. A man exhales about 1.1 pounds of water per day and this, in -addition to water from perspiration and other sources, must be removed -from the air. - - - Table VIII.--_Average Daily Metabolic Data for a 70-kg, - 25-Year-Old Astronaut With Normal Spacecrew Activity_ - [From [ref.173]] - - ----------------------------------------------------------------- - O2 input, kg 0.862 - ----------------------------------------------------------------- - CO2 output, kg 1.056 - ----------------------------------------------------------------- - Drinking water, liters 2.5 - ----------------------------------------------------------------- - Food rehydrating water, liters 1 - ----------------------------------------------------------------- - Caloric value of food, kcal 3000 - ----------------------------------------------------------------- - Water output: - ----------------------------------------------------------------- - Urine, liters 1.6 - ----------------------------------------------------------------- - Respiration and perspiration, liters 2.13 - ----------------------------------------------------------------- - Feces, kg 0.09 - ----------------------------------------------------------------- - Total heat output, Btu 11 100 - ----------------------------------------------------------------- - - -Two types of biological regenerative systems have been proposed. The -photosynthetic closed ecological system was proposed as early as 1951. -This involves the use of single-celled algae or higher plants, including -floating aquatic and terrestrial plants, and requires the interaction of -light energy with CO2 and H2O to produce O2 and plant cells. Another -system, proposed in 1961, involves electrolysis of water into oxygen and -hydrogen, and the concurrent use of _Hydrogenomonas_ bacteria which take -up hydrogen, some oxygen, carbon dioxide, and urine yielding water and -bacterial cells. - - - Table IX.--_Requirements for Regenerative Life-Support Systems_ - --------------------------------------------------------------------- - Requirements / Requirements / - 1 man[4] 3 men (270 man-day - System mission)[5] - --------------------------------------- - Weight, Power, Weight, Power, - kg kW kg kW - --------------------------------------------------------------------- - Partial chemoregenerative [7]332 1.75 - --------------------------------------------------------------------- - LiOH 125 1.40 - --------------------------------------------------------------------- - NaOH 155 7.68 - --------------------------------------------------------------------- - CO2-H2 34 .36 - --------------------------------------------------------------------- - Full bioregenerative--algae: - --------------------------------------------------------------------- - Artificial illumination 116 [6]10.40 591 25.00 - --------------------------------------------------------------------- - Solar illumination 103 1.70 356 .60 - --------------------------------------------------------------------- - Electrolysis-_hydrogenomonas_ 55 .25 129 2.60 - --------------------------------------------------------------------- - - [4] From [ref.174]. - - [5] From [ref.175]. - - [6] From [ref.176]. - - [7] Includes instrumentation and food storage. - - -The values given in table IX indicate relative weights and powers -required by various systems to provide the gaseous environment for -manned space cabins. If one considers operating temperatures and -hazards, other systems may offer advantages which offset the weight and -power advantages of the hydrogen reduction of LiOH systems. - -Research is being conducted by NASA on life-support-system technology -applicable to missions planned for 20 years in the future. Life-support -systems include the requirements for supplying breathing gases, control -of contaminants in the cabin atmosphere, water reclamation, food supply, -and personal hygiene. The disciplines involved in such systems include -biology and microbiology, cryogenic fluid handling at zero g, heat -transfer, and thermal integration with other systems, such as power. The -physiological, psychological, and sociological problems of the crew are -also being considered. - - -Photosynthetic System - -Green plants contain chlorophyll which captures light energy -thermodynamically required to convert carbon dioxide and water into -carbohydrate which can subsequently be transformed into other foods such -as protein and fat. During this process, carbon dioxide is consumed, and -an approximately equal amount of oxygen gas is liberated. As a first -approximation, photosynthesis is the reverse of the oxidative metabolism -of animal life: - - Oxidation - C6H12O6 + 6O2 --------------> 6CO2 + 6H2O + heat - - Photosynthesis - 6CO2 + 6H2O + light --------------> C6H12O6 + 6O2 - -The photosynthetic process in plants and respiration during -photosynthesis have been studied intensively, and several metabolic -pathways have been elucidated. Mechanisms are being studied to explain -the inhibitory effect of strong visible light on this process. This -program may lead to the use of chloroplasts or chlorophyll without cells -in future photosynthetic bioregenerative systems for long-term space -travel. - -One of the prime considerations of a closed ecological system is that -the environmental gases shall remain physiologically tolerable to all of -the ecologic components. Ideally, a photosynthetic gas exchange organism -should possess a high ratio of gas exchange to total mass (considering -all equipment and material incidental to growth, harvesting, processing, -and utilization); and a controllable assimilation rate to maintain -steady-state gas composition. It should also be (1) amenable to -confining quarters which may be imposed by inflexibility of rocket or -space station design; (2) genetically and physiologically stable and -highly resistant to anticipated stresses; (3) edible and capable of -supplying most or all human nutritional requirements; (4) capable of -utilizing raw or appropriately treated organic wastes; and (5) amenable -to water recycling as demanded by other components of the ecosystem. - - -Higher Plants - -Efforts to utilize multicellular plants as photosynthetic gas exchangers -have been somewhat neglected, since it has been assumed by many that -algae would be more efficient. The family _Lemnaceae_ (duckweeds) are -small primitive aquatic plants with a minimum of tissue differentiation. -Practically all of the cells of the plant contain chlorophyll and are -capable of photosynthetic activity. They reproduce principally by -asexual budding of parent leaflike fronds. They can be grown readily on -moist surfaces ([ref.177]) on almost any medium suitable for the growth -of autotrophic plants. With duckweeds the problems of gaseous exchange -and harvesting are simplified and the volume of medium can be greatly -decreased as compared with algae. - -Ney ([ref.177]) obtained a very high gas exchange rate with duckweeds. -Using small cultures under controlled optimal conditions of temperature, -light (600-1000 ft-c), and CO2, concentration, he estimated that 2.3 m² -of frondal surface of duckweed, at a gas exchange rate of 10.8 liters -m²/hr would provide sufficient gas exchange for one man. This would -produce about 25 grams of dry plant material per hour. - -A few nutritional studies have been carried out with duckweeds. Nakamura -([ref.178]) considered _Wolffia_ as a possible source of food for space -travel and found that it contained carbohydrate 25-60 percent, protein -8-10 percent, fat 18-20 percent, minerals 6-8 percent (all dry weights), -and vitamins B2, B6, and C, with C the most abundant. - -One of the desirable features of a duckweed system is that the gas -exchange is direct between the atmosphere and the plant and does not -require dissolving the respiratory gases in a bulky fluid system which -introduces special engineering difficulties in zero- or low-gravity -conditions. - -In the design of equipment for photosynthetic studies, careful -consideration should be given to the material used in the construction -of the unit. Most plastic materials are subject to photo-oxidative -degradation, with CO as one of the products. When air is recirculated -through plastic tubing and transparent rigid plastics in the presence of -light, considerable quantities of CO are given off. With high-intensity -illumination such as sunlight, a CO buildup of several hundred parts per -million is not uncommon. Also, plant pigments such as the carotenoids -and chlorophylls will react similarly when exposed to light of high -intensity. If the plants die, then CO is released quite rapidly. - -At Colorado State University the responses of plants to high-intensity -radiation (ultraviolet to infrared) are being studied. Plants from high -mountaintops that are exposed to greater ultraviolet light are being -studied for specialized adaptations. The effect of temperature on -photosynthesis is being explored. Various plants are also being studied -under germ-free conditions. - -Screening of higher plants for possible use in bioregenerative systems -at Connecticut Agriculture Experiment Station resulted in the selection -of corn, sugarcane, and sunflower. Under optimal conditions it has been -shown that 100 to 130 ft² of leaf surface are required to support an -astronaut. - -Plants considered as possible food sources include soybeans, peanuts, -rice, and tomatoes, which can be combined with algae to give a -well-balanced and reasonably varied diet. Hydroponic systems use large -quantities of water, but progress is being made in reducing this. - -The possibility of using animals in the closed ecological system is open -to question, particularly in the absence of gravity, and much work -remains to be done on using plant materials as animal food and on the -disposal of wastes. Animals which have been considered are crustaceans, -fish, chickens, rabbits, and goats. - - -Algae - -Algae have the fastest growth rate and are among the most efficient -plants for oxygen and food production. It has been amply demonstrated by -Myers ([ref.179]) and other workers that _Chlorella_ can be used in a -closed ecological system to maintain animals such as mice and a monkey. -The use of algae for supplying O2 and food, and for removing CO2 and -odors has been considered by many authors for use in spacecraft, space -platforms, and for establishing bases on the Moon or Mars. - -Estimates of total efficiency are based on extrapolated laboratory data -and vary widely, since many different types of data have been used as a -basis for these estimates. - -The respired air containing about 4-5 percent CO2 is bubbled into the -_Chlorella_ culture, at either atmospheric or increased pressure. Air -containing a high percentage of oxygen and saturated with moisture is -released from the algal system. - -The use of algae for several purposes might require from one to three -separate algal systems. For food production, _Chlorella_ produces 50 -percent protein and 50 percent lipids in high-nitrogen media. In -low-nitrogen media, it produces 85 percent lipids. Proper choice of -_Chlorella_ strains and media will produce not only the necessary -calories but also the necessary specific nutrients required. Certain -strains are more effective in O2 production, and others in the use of -urine and other wastes. - -Some of the early estimates, using _Chlorella_ grown at 25° C, for -supplying these requirements for a single man in space include the -following: 168 kg of algal suspension ([ref.179]), 200 kg of algal -suspension and 50 kg of equipment including pumps (refs. [ref.180] and -[ref.181]), and 100 kg of algal suspension and 50 cubic feet for -equipment and gas exchange ([ref.182]). Using the blue-green alga -_Synechocystis_, 600 kg of algal suspension would be required, according -to Gafford and Craft. These estimates are based on preliminary studies, -are quite high, and are not of real practical value. - -Other studies have indicated an extremely efficient algal system which -offers a real potential for a practical and effective gas exchanger -([ref.183]). A thermophilic strain of _Chlorella_ with an optimum growth -temperature of 39° C and an optimum temperature for photosynthesis of -about 40° C can increase its cell mass 10 000-fold per day. When -operating at one-half maximum efficiency, this alga produces 100 times -its cell volume of oxygen per hour. Burk et al. ([ref.183]) state: -"Future engineering development should lead to a space requirement, per -adult person, of no more than 3 to 5 cubic feet of algal culture, -equipment, and instrumentation for adequate purification of air." The -requirements of this system would require additional energy in the form -of light and of small amounts of nitrogenous and mineral material for -the algae. The light source used by Burk et al. ([ref.183]) is a -tungsten filament quartz lamp the size of a pencil, which has a long -life, produces a luminous flux 5-10 times greater than sunlight on -Earth, and operates at a 10-12 percent light efficiency. - -Research is being carried out on algal regenerative systems by about 40 -or 50 laboratories in the United States. NASA is supporting several -basic studies on photosynthesis, the physiology of algae, and -engineering pilot-plant development. Much of the research on algae is -being supported by the Air Force. - -Most algal studies have been carried out in small units and the data -obtained have been used as a basis for extrapolating logistic values for -the use of these organisms in manned space vehicles. Myers ([ref.179]) -has shown that the quantity of algae necessary to support a man (with an -assumed O2 requirement of 625 liters per day) would yield about 600-700 -grams dry weight of new cells per day. If algal growth in mass cultures -could be maintained in a steady-state concentration of 2.5 gram dry -weight per liter with such a growth rate as to yield 10 grams weight per -liter per day, the volume of algal culture would be 60-70 liters and the -total mass of the system would approximate 200-250 pounds. - -Using an 8-liter system, Ward et al. ([ref.176]) have produced algal -concentrations of 5-7 grams of dry algae per liter with a -high-temperature algal strain. The maximum growth rate observed with the -culture was 0.375 gram dry weight per liter per hour, or 9 grams dry -weight per liter per day. This was accomplished by using 1-centimeter -layers of culture and a light intensity of 8000 foot-candles. The -culture system consisted of a rectangular plastic chamber having an area -of 0.5 square meter and illuminated on each side to an intensity of 4000 -foot-candles (cool-white). To produce 25 liters of oxygen per hour, an -area of 8.3 square meters (85 square feet) would be required. - -The major problem in large-scale production of algae is that of -illumination. Conversion of electricity to light has an efficiency of -only 10 to 20 percent. In addition, the maximum efficiency of light -utilization by _Chlorella_ algae lies in the range of 18-22 percent. -This results in a maximum efficiency of only 4 percent for -photosynthetic systems. Another problem involved in conversion of -electricity to light is the production of heat which has to be removed -even with thermophilic algae. With a human demand of 600 liters of -oxygen per day, the minimum electrical requirement becomes 4 kW. No -large-scale culture has yet been managed at anything close to this -minimum figure. - -Another problem is the poor penetration of light into concentrated -cultures of algae. This necessitates construction of large tanks of only -about ¼-inch thickness. This results frequently in fouling of the -surfaces of the tank by algae and makes the removal of the excess algae -difficult. Production of 1 liter of oxygen results in the production of -1 gram dry weight of algae. Although a small amount of CO is produced by -some algae, it can probably be removed by catalytic oxidation. Other -problems include mutation and genetic drift of the algae and the -necessity for maintaining bacteria-free cultures. There are also -difficulties in maintaining a sterile culture if urine is to be used as -a nitrogen source. While there is a potential for using algae as food, -more research is required before it can be determined what quantity and -methods of processing can be used. Research and development on algae is -much greater than on both the higher plants and the -electrolysis-_Hydrogenomonas_ systems together. - -The difference between the photosynthetic and -electrolysis-chemosynthetic systems is the way electrical energy is made -available to the organisms. In the photosynthetic system, electrical -energy is converted to light which the algae or plants transform into -chemical energy. In the chemosynthetic process, electrical energy is -transformed into the chemical energy of hydrogen gas which is used by -the bacteria. Both organisms use the chemical energy available to them -to synthesize cell material with similar degrees of efficiency. The -problem is to make the conversion of electricity to available chemical -energy as efficient as possible. - -In photosynthetic systems much energy is lost in the conversion of -electricity to light, a process only 10-20 percent efficient at best. -When this is combined with the loss from the inefficient use of light by -plants, an overall efficiency of about 4 percent is obtained. In the -electrolysis-_Hydrogenomonas_ system, the two steps are very efficient. -Electrolysis cells can operate at up to 85 percent efficiency and the -overall efficiency can be up to seven times that of a photosynthetic -system. - - - ELECTROLYSIS-_HYDROGENOMONAS_ SYSTEM - -Electrolysis is carried out in a closed unit containing an electrolyte -(KOH solution) with an anode and a cathode. These cells produce a -maximum yield (60-80 percent or more) in gas production per unit of -power consumption. According to Dole and Tamplin ([ref.184]), a unit -capable of producing enough oxygen to sustain one man would be highly -reliable, weigh approximately 18 kg, and require a power input of 0.25 -kW. - -One approach to zero-gravity operation is to rotate the electrolysis -cell as described by Clifford and McCallum ([ref.185]) and Clifford and -Faust ([ref.186]). The smallest known electrolysis cell under -development uses this artificial gravity to separate oxygen from the -anode and electrolyte, while the dry hydrogen gas permeates through the -foil cathode, fabricated from palladium-silver alloy. This electrolysis -cell, which would provide breathing oxygen for three men, has a volume -of 1.4 liters, weighs 4.5 kg, and requires 0.67 kW, excluding auxiliary -equipment, and has an efficiency of 84 percent. - -The chemosynthetic conversion is carried out by the hydrogen bacteria. -By the oxidation of molecular hydrogen, supplied from the electrolysis -of water, energy is made available for biosynthesis. The generation of -this "biological energy" is mediated by the stable enzyme hydrogenase -which is present in the bacteria. On the average, the oxidation of 4 -moles of H2 is required for the conversion of 1 mole of CO2 (the hourly -production of a man). The removal of this amount of CO2 would thus -require the cleavage of 4 moles of water. In addition, to supply oxygen -for human respiration (at a rate of 1 mole of O2 per hour) the cleavage -of two additional moles of water is required. Therefore, the -chemosynthetic regeneration and human respiration together would -require, on the average, the splitting of 6 moles of water per hour. - -The material balance for electrolysis, biosynthesis, and human -metabolism, with gram molecular weights in parentheses, are shown in -equations (1) to (3), respectively: - - 6H2O --------------> 3O2 + 6H2 - (108) --------------> (96) + (12) (1) - -The bacterial synthesis requires 6 moles of H2, 2 moles of O2, and 1 -mole of CO2 (from the astronaut), as shown in equation 2: - - 6H2 + 2O2 + CO2 --------------> CH2O + 5H2O - (12) + (64) + (44) --------------> (30) + (90) (2) - -The respiration of the astronaut requires 1 "food" mole (CH2O) -representing about 120 kcal, and 1 mole of O2, as shown in equation 3: - - CH2O + O2 --------------> CO2 + H2O - (30) + (32) --------------> (44) + (18) (3) - -The metabolic data in table VIII show that the CO2 of the astronaut and -the bacteria must balance at about 1.056 kg per day. - -The water relations are not completely balanced, but are fairly close. -About 2.6 liters per day of water are split by electrolysis. The -astronaut has an intake of 3.5 liters of water per day, 2.5 liters for -drinking and 1 liter for preparing dehydrated food. The output is about -1.6 liters of urine and 2.1 liters of water of respiration and -perspiration per day, or a total output of 3.7 liters, with the -0.2-liter excess due mainly to water of metabolism. The -bacteria-produced water, amounting to 2.2 liters per day, and the excess -from the astronaut would supply 2.4 liters toward balancing the 2.6 -liters of water electrolyzed. - - -Bacterial Culture - -Hydrogen bacteria are characterized by their ability to metabolize and -multiply in a strictly inorganic medium, when supplied with H2, CO2 and -O2 in required amounts. They can be grown in batch culture or in -continuous culture using different methods of supplying entire medium or -components on a demand feed system. - -A medium was developed for batch culture of _Hydrogenomonas eutropha_ by -Repaske ([ref.187]) with quantitation of a number of components -including trace minerals. Experiments by Bongers ([ref.188]) showed that -a simplified medium, using laboratory-grade chemicals, could be used. A -definite requirement was found for magnesium and ferrous iron (Fe^++). -The optimal growth requirements observed for _Hydrogenomonas eutropha_ -are shown in table X. - - - Table X.--_Optimum Growth Requirements of_ - Hydrogenomonas eutropha - - --------------------------------------------------------- - Culture parameter Optimum value - --------------------------------------------------------- - Cell density, g (dry weight)/liter 10 - --------------------------------------------------------- - Temperature, °C 35 - --------------------------------------------------------- - Pressure, atm 1 - --------------------------------------------------------- - pH (phosphate buffer) 6.8 (6.4-8.0) - --------------------------------------------------------- - H2, percent 75 - --------------------------------------------------------- - O2, percent 15 - --------------------------------------------------------- - CO2, percent 10 - --------------------------------------------------------- - Urea CO(NH2)2, g/liter 1 - --------------------------------------------------------- - MgSO4·7H2O, g/liter 0.1 - --------------------------------------------------------- - Fe(NH4)2 (SO4)2, g/liter 0.008 - --------------------------------------------------------- - - -The effects of temperatures ranging from 20° to 42.5° C on the growth -rates of _Hydrogenomonas eutropha_ were studied by Bongers ([ref.189]), -and the optimal temperature was found to be about 35° C. Experiments at -25° and 35° C indicated that the efficiency of energy conversion was -essentially identical at both temperatures. _Hydrogenomonas_ requires, -as part of its substrate, a mixture of three gases: hydrogen, oxygen, -and carbon dioxide. Experiments were performed by Bongers ([ref.189]) to -determine the toleration limits of the three gases. Growth rates were -found to be identical when hydrogen varied from 5 to 80 percent. Nearly -identical growth was obtained when CO2 partial pressures were 5 to 60 -percent, being slightly lower at higher partial pressures. The organism -was highly sensitive to oxygen concentration. Dissolved oxygen -concentrations above 0.13 mM were found to inhibit cell division; energy -utilization was also affected by oxygen concentration. At 0.2 mM oxygen -concentration, the efficiency of energy conversion was approximately -half the value observed with 0.05 mM. - -Another parameter of importance is the total volume of suspension which -would be required to balance the metabolic needs of one man. The volume -of suspension is determined by the conversion capacity of a unit volume. -This capacity is a function of the cell concentration; hence, the more -cells that can be packed in a unit volume of suspension (and adequately -provided with H2, O2, and CO2), the less the volume of suspension -required. - -Results of experiments by Bongers (refs. [ref.190] and [ref.191]) on -conversion capacity-density relationships show that the rate of CO2 -conversion obtained with suspensions up to approximately 10 grams (dry -weight) per liter is linear with relation to density. This indicates -that the supply of H2, O2, and CO2 is adequate. Upon a further increase -in cell concentration, the conversion rate still increases but not -linearly. The highest amount of CO2 taken up per liter of suspension was -approximately 2 liters per hour. At these very high cell concentrations, -the relationship between rate of conversion and density is no longer -linear. This is demonstrated when the conversion rate is calculated per -unit cell weight instead of per unit suspension volume. The rate per -gram dry weight per liter decreases from 146 to 68 ml of CO2 per hour. -With a suspension at a density of approximately 10 grams, the conversion -of 1.1 liters of CO2 per liter per hour is obtained. At a CO2 output of -22 liters per man per hour, 20 liters of suspension would be sufficient -to balance the gas exchange needs of one man. - -At higher cell concentrations, less volume of suspension would suffice -if gas equilibration could be maintained at the higher consumption rates -to avoid anaerobic conditions which could lead to a shift in metabolism. -In the final analysis, the technical problem of gas transfer from the -gas to the liquid phase determines the optimal cell concentration and, -therefore, the required suspension volume. - -From data presently available, it can be concluded that, using the -slow-growing _H. facilis_, the volume of suspension required to support -one man is about 500 liters. Using _H. eutropha_, Schlegel ([ref.192]) -calculated a suspension volume of 66 liters with 1 gram dry weight of -bacteria per liter. - -In recent NASA-supported research, the amount of culture medium has been -estimated using improved cultivation methods and conditions. For batch -culture, the data show that from 10 to 66 liters would be required per -man, with a best practical estimate of 20 liters at 9 to 10 grams dry -weight of bacteria per liter ([ref.191]). For continuous culture using -the turbidostat, the present data indicate a demand for some 30 liters -of suspension, and a volume of 20 liters (at approximately 10 grams dry -weight of bacteria per liter) as a realistic goal. - -In the foregoing section, the material balance for gases and water was -discussed. It was shown that a close match could be obtained with these -components of the closed environment. - -Less abundant, though no less important, are the nonwater components of -urine and feces. The urine is important for the content of fixed -nitrogen and other products of man's metabolism and serves as a very -effective substrate for cultivation of hydrogen bacteria. Maximum -closure of the system necessitates utilization of the urea in urine as a -nitrogen source. - -The average man produces 1.2 to 1.6 liters of urine per 24-hour period. -This contains about 0.00005 gram per liter of iron, 0.113 gram per liter -of magnesium, and 24.5 grams per liter of urea ([ref.193]). As shown in -table X, each liter of bacterial medium requires 0.008 gram per liter of -Fe(NH4)2 (SO4)2, about 0.1 gram of MgSO4·7H2O, and 1.0 gram per liter of -urea. In comparing the daily urine output with the estimated required -ingredients of a bacterial medium, a relatively close balance is -observed, with the exception of iron. - -For the fixation of 24 moles of CO2 (288 grams of C) produced per man -per day, the production of about 640 grams dry bacterial mass is -required. At an average N-content of 12 percent, the nitrogen -requirement would be some 100 grams. A comparison of daily output -(urine) and daily requirement by the bacterial suspension reveals that -only 10 to 33 percent of this amount could be recovered from average -urine. To obtain a material balance, either the man must be fed a -protein-rich diet or the bacterial suspension must be grown under -conditions which lead to the production of a cell mass relatively low in -protein content. Experiments have indicated that nitrogen starvation of -the bacterial culture might be a promising solution. Culture "staging" -(cultivation under nitrogen-rich conditions, followed by cultivation in -the absence of substrate nitrogen and subsequent harvesting for food -processing) will probably be the most promising means of nitrogen -economy in the closed environment. As discussed in a following section, -a biomass of relatively high lipid content can be obtained under -conditions of nitrogen starvation. - - -Continuous Culture of _Hydrogenomonas_ Bacteria - -Growth of hydrogen bacteria in a batch culture, after an initial period -of adjustment, becomes steady and rapid during the exponential growth -phase. This steady state of growth is temporary and ceases when nutrient -substrate or gas concentrations drop to limiting values. For long -periods a continual supply of nutrients must be provided. Growth then -occurs under steady-state conditions for prolonged periods, and such -factors as pH, concentration of nutrient, oxygen, and metabolic products -(which change during batch culture) are all maintained constant in -continuous culture. - -Two methods can be used for control of continuous cultures: the -turbidostat and the chemostat. In the turbidostat, regulation of medium -input and cell concentration is controlled by optically sensing the -turbidity of the culture. - -The dilution rate varies with the population density of the culture and -maintains the density within a narrow range. Organisms grow at the -maximum rate characteristic of the organism and the conditions. The -growth rate can be changed by modifying the nutrient medium, gas -concentration, or incubation temperature. A disadvantage of the -turbidostat is that all nutrient concentrations in the culture chamber -are necessarily higher than the minimum, resulting in inefficient -utilization of nutrients. - -The turbidostat system for continuous culture of _Hydrogenomonas_ -bacteria, developed by Battelle Memorial Institute ([ref.194]), includes -electrolysis of water in a separate unit. Hydrogen and oxygen are fed -separately up to the point of injection into the culture vessel, and the -mixed volume is kept very small to minimize am possibility of explosion. -However, the two gases may be injected simultaneously if there is a -demand for both. - -In the chemostat, growth of the organisms is limited by maintaining one -essential nutrient concentration below optimum. A constant feed of -medium, with one nutrient in limiting concentration and with constant -removal of culture at the same rate, is used to achieve the steady -state. The dilution rate is set at an arbitrary value, and the microbial -population is allowed to find its own level. By appropriate setting of -the dilution rate, the growth rate may be held at any desired value from -slightly below the maximum possible to nearly zero. This constitutes a -self-regulating system and allows selection of a desired growth rate. - -A combined electrolysis-chemostat method, developed by Magna Corp., -maintained the hydrogen-producing electrode of an electrolysis cell in -the bacterial culture. Resting cells of _Hydrogenomonas eutropha_ -consumed hydrogen produced at the cathode of an electrolysis cell built -into a specially constructed Warburg flask. Attempts to immobilize -_Hydrogenomonas_ cells on a porous conductor were partially successful. -This system could lower the volume requirements compared with those for -the isolated subsystems. Disadvantages of this integrated system include -electrolysis of the bacterial medium, possibly resulting in toxic -breakdown products, and the possible effects of electric power and the -KOH electrolyte on the bacteria. The main disadvantage of an integrated -system would be the disparity between optimal conditions for efficient -electrolysis and efficient bacterial conversion, particularly -temperature and pH, with the combination possibly resulting in -considerably higher power and weight demands. - -Both continuous-culture approaches are being studied with NASA support. -The turbidostat offers the greatest potential efficiency in weight and -volume, but uses nutrient materials less efficiently and is more -complex. The chemostat is less efficient in weight and volume, but has -greater simplicity and reliability. - -_Hydrogenomonas eutropha_ has been grown in 15-liter batch cultures and -in 2.1-liter continuous cultures. A 20-liter continuous culture, -sufficient to balance the requirements of a man, is under development. - -The potential problem areas in large-scale continuous production of the -bacteria include assuring genetic stability, preventing or controlling -bacteriophage and foreign bacterial contamination, and preventing -heterotrophic growth caused by exposure to organic material from the -urine. Genetics of hydrogen bacteria and phage infection have been -studied by DeCicco. Research on these problems indicates that they are -not of major importance, but cause significant effects and must be -eliminated or controlled. - - -Bacterial Composition and Nutrition - -_Hydrogenomonas_ bacteria can be used for at least part of the -astronauts' diet. The washed bacteria have a mild taste and are being -studied for their total energy content, protein and lipid digestibility, -and vitamin content. Carbon and nitrogen balances, and respiratory -quotient are to be determined in animals fed the bacteria as their sole -food source. No toxic constituents have been discovered. Sonicated and -cooked bacteria, when fed to white rats as 12 percent of the solids of a -nutritionally balanced diet, were eaten readily and produced no ill -effects. Net utilization of the protein appears to be somewhat lower -than casein and about the same as legume proteins. - -The composition of _Hydrogenomonas eutropha_ is shown in table XI. The -composition of the bacteria varies with the age and growth phase of the -cells and with the medium and gas available. It is possible to modify -the growth conditions to grow the type of bacteria desired for nutritive -purposes. - -_Hydrogenomonas_ cells contain about 75 percent water. Of the dry -weight, about 74 percent is protein, calculated as 6.25 times the -nitrogen content. Table XI shows the amino acid composition to be -comparable with other bacterial proteins, except for higher tryptophan -and methionine values. - - - Table XI--_Analysis of_ Hydrogenomonas eutropha _Cells Grown in - Continuous Culture_ [From [ref.194]] - - ----------------------------------------------------------------- - Constituent Percent by weight - ----------------------------------------------------------------- - Moisture 74.55 - ----------------------------------------------------------------- - Fat .44 - ----------------------------------------------------------------- - Ash 1.73 - ----------------------------------------------------------------- - Nitrogen 3.02 (wet) - ------------------- - 11.87 (dry) - ----------------------------------------------------------------- - Protein (N × 6.25) 18.90 (wet) - ------------------- - 74.26 (dry) - ----------------------------------------------------------------- - Amino acids (dry weight)[8] - ----------------------------------------------------------------- - Alanine 4.47 - ----------------------------------------------------------------- - Arginine 3.41 - ----------------------------------------------------------------- - Aspartic acid 4.32 - ----------------------------------------------------------------- - Cystine .08 - ----------------------------------------------------------------- - Glutamic acid 7.67 - ----------------------------------------------------------------- - Glycine 2.76 - ----------------------------------------------------------------- - Histidine .95 - ----------------------------------------------------------------- - Isoleucine 2.17 - ----------------------------------------------------------------- - Leucine 4.04 - ----------------------------------------------------------------- - Lysine 2.65 - ----------------------------------------------------------------- - Methionine 1.14 - ----------------------------------------------------------------- - Phenylalanine 2.20 - ----------------------------------------------------------------- - Proline 2.06 - ----------------------------------------------------------------- - Serine 1.80 - ----------------------------------------------------------------- - Threonine 2.15 - ----------------------------------------------------------------- - Tryptophan .78 - ----------------------------------------------------------------- - Tyrosine 1.79 - ----------------------------------------------------------------- - Valine 3.03 - ----------------------------------------------------------------- - - [8] Trace amounts of the following were also found: methionine - sulfoxide, citrulline, alpha-amino-n-butyric acid, homocitrulline, - glucosamine, galactosamine, methionine sulfoximine, ethionine, and - ethanolamine. - - -The lipid content of rapidly growing cells is normally quite low (0.45 -to 2.3 percent crude ether extractable lipids). The most important lipid -is poly-beta-hydroxybutyric acid, which is stored under the growing -conditions of insufficient nitrogen or oxygen supply (refs. [ref.187] -and [ref.191]). Under these conditions, this unusual polymer constitutes -up to 80 percent of the dry weight. While the monomer itself, -beta-hydroxybutyric acid, is rapidly and efficiently used in cell -metabolism, the nutritive value of the polymer is yet to be determined. -The fatty acids found include lauric, myristic, palmitic, palmitoleic, -heptadecaenoic, C17 saturated(?), stearic, linoleic, and linolenic(?) -([ref.195]). - - -Application to Spacecraft System - -A bioregenerative life-support system will be required in long manned -space flight, especially with several astronauts such as would be -required for a manned mission to Mars in the 1980 time period. While -almost 15 years is a long leadtime, the biological research and -engineering problems are formidable, and a system would have to be -developed at least 5 years before the mission. - -The power and weight requirements for both chemical and biological -regenerative life-support systems were presented in table VIII. These -should be considered tentative best estimates based on present data. - -The use of bioregenerative systems in spacecraft systems has been -studied by Bongers and Kok ([ref.175]) who put the -electrolysis-_Hydrogenomonas_ system in proper perspective with the -following statement: - - The bioregenerative systems are more or less in a transitory - phase between research and development. The power data can be - considered fairly accurate, at least within ±20 percent. The - postulated weight data, however, represent approximations, - particularly with respect to auxiliary equipment and - construction materials. Also omitted are the weight penalties - most probably involved in the processing of the solid output of - the exchangers, elegantly defined as potential food. Further - research is required in this area to evaluate the regenerative - systems, especially the bacteria, with respect to this - potential. Furthermore, as yet there is no experimental proof - that the growth rates of the heavy bacterial suspensions can be - realized in a large design, determined on a relatively small - scale with fairly precise control of physiological conditions - and gas exchange. This aspect may affect considerably the weight - involved in a chemosynthetic balanced system. Nevertheless, at - present, this approach still seems most promising. - - - CABIN ATMOSPHERES[9] - - [9] Includes part of [ref.196]. - -In the first U.S. manned space flight program, Project Mercury, and in -the face of very severe weight limitations, a cabin atmosphere of pure -oxygen at one-third atmospheric pressure was adopted. This choice -probably represented the greatest simplification which could be achieved -readily and, at the same time, provide protection against some of the -risks of rapid decompression. Although breathing pure oxygen at higher -pressures was known to be attended by some undesirable physiological -effects, the short duration of the flights to be undertaken, and the low -pressure employed, suggested that no harmful results would result in -this case. That these expectations were generally borne out is now -history. Preparations for space flights of longer duration--many weeks -or months--present similar problems and require special attention to -phenomena which may be either undetectable or of trivial significance on -a time scale of a few days. - - -Physiological Criteria in the Choice of Cabin Atmosphere - -If maintenance of normal respiratory function were the only -consideration, a cabin atmosphere of about sea-level composition and -pressure might be an ideal and straightforward choice for manned -spacecraft. In fact, this atmosphere has been used in the manned space -flights conducted by the U.S.S.R. No other atmosphere has been shown to -be more satisfactory from the physiological point of view, and the -tedious respiratory studies which should accompany the use of other -atmospheres can be avoided. Nevertheless, the formidable problems of -spacecraft design and the necessary precautions for safeguarding the -crew from accident require that other atmospheric compositions and -pressures be considered. For example, if a cabin at 1-atm pressure were -decompressed to space suit pressure (0.3 atm), the occupants would -develop decompression sickness; i.e., "bends." - -Several engineering considerations argue for low cabin pressures and -pure oxygen composition. Among these are structural design, weight of -atmospheric gas storage and control equipment, and the difficulty of -contriving pressure suits which allow operation at pressures near one -atmosphere. Such departures from the normal human gaseous environment, -however, require the demonstration of an acceptable level of safety and -physiological performance. - -The limits of the composition and pressure of acceptable cabin -atmospheres are then set by-- - - (1) A pure oxygen atmosphere at a pressure which will provide an - alveolar oxygen partial pressure equal to that provided by air at - sea level - (2) A mixed gas (oxygen and inert gas) atmosphere having a pressure - and composition that will allow decompression to the highest - acceptable suit pressure without the risk of bends - -A numerical value for the lower limit (1) is approximately 0.2 atm of -pure oxygen. The upper limit (2) is determined by the operating pressure -and composition of the space-suit atmosphere and may be of the order of -0.5 atm for a cabin atmosphere of 50 percent oxygen. It is necessary to -determine the astronaut's ability to survive and perform his duties in -any atmosphere selected. - - -Atelectasis and Pulmonary Edema - -Localized or diffuse collapse of alveoli in the lungs may, if the -condition persists, lead to arterial hypoxia which may be extremely -undesirable under the stresses of space flight. The alveoli are probably -unstable when pure oxygen is breathed; they tend to collapse if there is -blockage of the airways, especially at low pressures. This collapse -occurs because each of the gases present in the alveoli (oxygen, water -vapor, and carbon dioxide) is subject to prompt and complete absorption -from the alveoli by the blood. - -The alveoli are normally stabilized against collapse by the presence of -inert and relatively insoluble gas (nitrogen) and an internal coating of -lipoprotein substances with low surface tension. - -Theoretical and experimental results strongly suggest the desirability -of using oxygen-inert gas atmospheres for long missions to avoid -atelectasis and other gas absorption phenomena, such as retraction of -the eardrum. However, further experimental evidence is required both to -confirm this point and to establish its upper limit of suitability of -pure oxygen atmospheres. - -At Ohio State University in 1962, scientists studied the effect on young -rats exposed for 27 days to 100 percent oxygen (with no nitrogen), at a -reduced barometric pressure equivalent to 33 000 feet altitude. The rats -showed no difference in growth rate, oxygen consumption, food and water -intake, or behavior from control rats in air at 1 atm. - - -Oxygen Toxicity - -It has long been known that breathing pure oxygen at normal atmospheric -pressure often produces pulmonary irritation and other toxic effects -both in man and animals. This knowledge has occasioned concern over the -use of pure oxygen atmospheres in spacecraft. - -The effect of 100 percent oxygen at a simulated altitude of 26 000 feet -for 6 weeks was studied using white rats at Oklahoma City University -under a NASA grant. Radioactive carbon techniques revealed a 15-percent -reduction of metabolism in the 100-percent oxygen-exposed rats, compared -with rats in air at 1 atmosphere. There was a 20-percent decrease in -lipid metabolism in the liver compared with controls, but no decrease in -heart metabolism. There was no gross change in body weight. - -The White Leghorn chick between 2 and 7 weeks old is markedly resistant -to the toxic effects of 1 atm of O2. Continuous exposure (Ohio State -University) for as long as 4 weeks did not cause deaths, obvious -morbidity, or any signs of pulmonary damage on gross autopsy. -Nevertheless, the hyperoxia had some adverse effects, primarily reducing -the growth rate to between three-fourths to one-fourth of normal; -reducing feed intake per unit body weight to three-fourths of normal; -slowing respiratory rate by 30 percent; decreasing erythrocytes, -hemoglobin, and hematocrit by 9 to 12 percent; and causing reversible -histological changes in the lungs. Arterial O2 tensions were elevated -over 300-mm Hg, but arterial pCO2 and blood pH were unaffected. No -residual effects were noted upon return to air breathing. It is possible -that the anatomical peculiarities of the avian lung play some role in -the chicks' resistance to hyperoxia, but it is also possible that this -resistance is a function of age, similar to the tolerance shown by the -young rat but not the adult. - - -Carbon Dioxide Tolerance - -Studies of CO2 tolerance in submarine crews indicate that no loss of -performance is involved if the concentration in air at normal pressure -does not exceed 1.5 percent with exposures of 30 to 40 days. However, -biochemical adaptive changes were observed at this concentration. - - -Inert-Gas Components - -If other investigations establish the need for an inert gas in manned -spacecraft atmospheres, gases other than nitrogen may be considered. -Compared with nitrogen, the physical properties or helium and neon offer -advantages with respect to solubility in body fluids, storage weight, -and thermal properties. - -Studies at Ohio State University in 1964, under a NASA grant, showed -that helium substituted for nitrogen in a closed container causes humans -to feel "cold" at a normally comfortable temperature. Studies with -animals have shown that in a helium atmosphere there is greater heat -loss due to the increased conducting capacity and probably greater -evaporative capacity. In 6 days at 21 percent oxygen and 79 percent -helium at 1-atmosphere pressure, young rats grew at the same rate as -controls, but drank more water, excreted more urine, and had a higher -rate of food and oxygen consumption than controls in air at 1 -atmosphere. Men are being tested on a bicycle ergometer in saturated and -low relative humidity helium atmospheres to study heat balance. - -Mice were exposed to 80 percent argon and 20 percent oxygen continuously -at 1-atmosphere pressure for 35 days at Oklahoma City University. Carbon -14 studies of metabolism showed a slight slowing and a twofold to -threefold increase in fat deposition. - - -Bends - -Decompression, whether accidental (due to damage of the spacecraft) or -intentional (as in the use of the pressure suit outside the capsule), -carries the risk of bends if the inert gases dissolved in the tissues -and body fluids come out of solution. The magnitude of this risk is -determined to a very considerable extent by-- - - (1) Individual susceptibility - (2) The extent to which the nitrogen (or other inert gas) - concentrations of tissues and body fluids have been reduced - (3) The magnitude and rate of the inert-gas, partial pressure change - on decompression - -The probability of getting bends is reduced by-- - - (1) Selection of bends-resistant individuals - (2) Thorough denitrogenation before flight - (3) Limitation of decompressive pressure changes by appropriate choice - of cabin atmosphere pressure and composition - (4) Space-suit pressure setting - -In some cases, further improvements might be obtained by using, in the -cabin atmosphere, an inert-gas component which has a lower solubility in -tissue and body fluids or less tendency than nitrogen to form bubbles. - - -Fire Hazard - -Experience indicates that fires in pure oxygen atmospheres, even at low -pressures (e.g., 1/3 atm), are extremely difficult to extinguish. While -this phenomenon has nothing to do with respiratory physiology, the risk -on flights of long duration may be so serious as to demand special -measures. Unless effective countermeasures can be devised, this risk may -argue very strongly against the use of such atmospheres in the future. -Further experimental investigation is required. - - -Acceleration Effects on the Lungs and Pulmonary Circulation - -Forces produced by high acceleration overdistend one part and compress -another part of the lungs. Blood flow diminishes in some parts of the -lungs and increases in others. Fluid leaks from the blood into the -tissues and into the air sacs in parts of the lungs. These effects cause -difficulty in breathing, low arterial oxygen saturation, and impaired -consciousness during high sustained acceleration and, to a lesser -extent, after its cessation. They must be considered when selecting the -best gas to be breathed, since a high partial pressure of oxygen is -favorable for consciousness, but a low inert-gas concentration during -acceleration is unfavorable for rapid lung recovery afterward. - - - PHYSIOLOGICAL PROBLEMS - -A study of the manned space flights and laboratory observations to date -suggests that during long periods of weightlessness, some physiological -difficulties may arise which may produce serious effects on human -performance. Although recent experience gives no grounds for expecting -insuperable difficulties, neither the quantity nor quality of the -available observations permits the conclusion that long-term exposure to -weightlessness will _not_ have serious consequences. The critical role -to be played by the astronaut demands that every effort be made to -identify in advance those phenomena which may affect performance, and to -study their qualitative and quantitative relationships so that proper -precautions can be taken. - -Lawton ([ref.197]), in reviewing the literature on prolonged -weightlessness, found few instances in which physiological function was -truly gravity dependent. He stated that the physiological systems likely -to be most affected by weightlessness were the musculoskeletal system, -the cardiovascular system, and the equilibrium senses. Subsequent -experience proved this to be the case. McCally and Lawton ([ref.198]) -analyzed the data from experiments since 1961 and concluded that much -more basic laboratory work is necessary. Studies using immobilization, -immersion, and cabin-confinement techniques were recommended approaches -toward simulating weightlessness. - -Much of the difficulty in obtaining precise information of anticipated -problems arises from a lack of knowledge of normal mammalian physiology. -Many of these deficiencies can be remedied in the laboratory. In -space-flight development, however, two distinct investigational -approaches can be adopted. The first of these may be characterized as -empirical and incremental; that is, the capabilities of the astronaut -are explored in successive flights involving relatively modest increases -in difficulty or severity of the environmental conditions. In this way -it is hoped to ascertain the human limitations without running too great -a risk. The second approach can be described as fundamental: determining -by a series of controlled experiments the effects of exposure to -space-flight conditions upon comparative mammalian physiology, with -emphasis on man. A fundamental understanding of the observed effects -would be sought so that predictions for new situations and possible ways -to control them could be made with confidence. - -It is not possible now to predict for flights of 30 days or more-- - - (1) The effects of sudden reimposition of reentry accelerations and - terrestrial gravity - (2) Changes in body fluid distribution and composition - (3) The effects of violent physical effort on respiratory and - cardiovascular systems in prolonged weightlessness - (4) Central nervous system functions, especially coordination, skilled - motor performance, judgment, and sleep-wakefulness cycles - -NASA has emphasized that planning for manned space programs involves a -systematic extension from physiological observations in animals to man, -and finally the establishment of man as part of the man-vehicle system -design. Moreover, these studies require the evaluation of central -nervous, cardiovascular, respiratory, gastrointestinal, and other -systems as a matrix in mutual interdependence. There is particular -interest in the effects of weightlessness on flights exceeding 30 days. - -Mammalian flights of about 30 days also merit attention, including the -development of the life-support systems which must precede such a -program. Development of facilities for biological experiments may well -be an important requirement for studies in anticipation of manned -flights of longer duration than Apollo. Unless the biological satellite -programs of the type mentioned above are successful in providing the -necessary data, a manned orbiting laboratory may also be important in -studies of shorter range. - - -General Studies of Biological Rhythmicity - -The effects of weightlessness on the organism as a whole may be -manifested by important changes in certain integrated behavioral -patterns having an inherently rhythmic character. Modifications in basic -behavioral patterns and performance may occur as disruptions of rhythmic -physiological phenomena, which are themselves the end product of -interrelated functional activity in a number of physiological systems, -such as the neuroendocrine, cardiovascular, and central nervous systems. - -Measurements of interdependent components of biological rhythmicity are -beginning to be analyzed by methods well established in -physics--including correlation and spectral analyses, and phase -modulation and variance in rhythmic processes. A wide variety of -physiological functions can be treated as periodic variables in the -analysis, including rhythmicities in cardiac output and blood pressure, -respiration, brain waves, and the slower tides of appetite, and -sleep-wakefulness. The importance of such investigations argues for -their inclusion in forthcoming flight programs. Their experimental -simplicity is an additional advantage. Biorhythms have been discussed in -more detail in the section on "Environmental Biology." - - -Effects of Weightlessness on the Cardiovascular System - -Earlobe oximetry, indirect measurements of blood flow and of blood -pressure by finger plethysmography or impedance plethysmography, and -ballistocardiographic techniques have potential application to manned -space flight. - -Adaptation to prolonged exposure to weightlessness or to lunar gravity -may cause difficulties when the astronaut is exposed again to reentry -forces and terrestrial gravity. It is possible that these adaptive -changes may thus produce unacceptable effects on performance or cause -risk to life. It is important to obtain experimental evidence on this -subject. - -It is common knowledge that following a stay in bed, dizziness, -faintness, and weakness characterize arising, and that a feeling of -general weakness may persist for several days. The phenomenon has been -investigated in a number of laboratories. One approach has been to put -healthy young subjects to bed, and even in extensive casts for periods -of 2 or 3 weeks or more. Two major findings have emerged from these -studies. First, a substantial adjustment in the blood circulatory system -occurs, which is termed the "hypodynamic state." Second, there is a -large decrease in the skeletal and muscle mass of the body. - -There are two kinds of evidence for the hypodynamic state: measurement -of parameters of circulatory function, and measurement of the response -of the individuals to a quantitatively imposed mild gravitational load. -After 3 weeks in bed, otherwise healthy persons exhibit an increase of -more than 20 percent in heart rate; a reduction of 10 to 20 percent in -total blood volume, primarily as a result of reduction of plasma volume; -and a decrease in heart size of about 8 percent. Coupled with these -cardiovascular changes is a reduction of 10 percent in the basal -metabolic rate. It appears as though the circulation and metabolism are -reset to a lower functional level commensurate with the reduced demands -placed on the whole organism. - -After 3 weeks of bed rest, all of the subjects tested showed pronounced -orthostatic hypotension. After tilting, the average heart rate increased -by 37 beats per minute, the systolic blood pressure fell some 12-mm Hg, -and some of the subjects fainted. The measurements were continued for 16 -days after the bed-rest period, and it was round that recovery was not -quite complete when the experiment was terminated. - -There is little question that in prolonged exposures to the weightless -state, there is a fair probability of extensive circulatory adjustments, -the seriousness of which cannot yet be foretold. While it is likely that -the astronauts will adapt successfully to long periods of weightlessness -at some new circulatory functional level, the remote possibility exists -that the circulatory changes may be progressive to the point of ultimate -failure. - - -Metabolic Effects of Weightlessness - -Without metabolic information, accurate planning of environmental -systems for long flights is difficult. Importance is also attached to -early evaluation of weightlessness effects on body-fluid equilibria. The -results of Earth orbital flights and of terrestrial water-immersion -experiments suggest the occurrence of undesirable changes, although no -effects leading to operational incapacity have yet arisen. - -In both recumbency and immersion, a similar redistribution of body -fluids occurs. It has been suggested that recumbency may affect an -extracellular fluid-volume receptor mechanism which by decreasing -aldosterone secretion by the adrenal gland, would decrease sodium -reabsorption by the renal tubules. Aldosterone excretion decreases -during recumbency and during standing in water, but increases while -standing in air. There is also evidence for cardiac atrial volume -receptor mechanisms which respond to increased filling of the left -atrium with reflex inhibition of release of pituitary antidiuretic -hormone (ADH), resulting in diuresis (Henry-Gauer reflex). - -Altered fluid equilibrium in buoyant states is accompanied by shifts in -intracellular and extracellular electrolyte distribution, especially -sodium and potassium. Evidence from recumbency studies indicates a -strong correlation between loss of erect posture or weight bearing and -excretion of calcium stores in bone. - -A bone X-ray densitometry method has been developed by Mack, at Texas -Woman's University, for accurately determining the loss of bone mineral -(±2 percent accuracy) in humans and animals. The heel bone and spine are -X-rayed using a calibrated aluminum wedge as a standard. This technique -will be used for preflight and postflight analysis of the primate being -flown in the 30-day biosatellite. Comparative appraisal of bone mineral -behavior in astronauts participating in the Gemini and Apollo programs -will be invaluable for future flight missions. - -Bed rest and immobilization studies by Mack have shown loss of skeletal -mineral and increased calcium in the urine and excreta. Four bed-rest -studies, each extending for 2 weeks, compared different levels of -calcium intake. Four men were used in each study and served as their own -controls during extended ambulatory periods. During 2-week periods, up -to 10 percent of calcium mineral was lost from the heel bone. Calcium -was also determined in the urine and feces. In other studies, isometric -exercises reduced loss of bone mineral during bed rest. - -Excretion of calcium in the urine is accompanied by risk of its -deposition as calculi or "kidney stones" in the urinary tract. -Currently, changes in calcium metabolism resulting from weightlessness -over periods up to 2 weeks is not considered a hazard requiring -precautionary measures. - -Flights in excess of 2 weeks, however, constitute a problem serious -enough to warrant study on the 11-day orbital flights and the 30-day -biosatellite primate mission. Therapeutic immobilization, -post-poliomyelitis immobility, and experimental restraint in normal -subjects lead to a negative calcium balance, with hypercalciuria. - - -Central Nervous System Functions in Weightlessness - -The wide range of individual tolerances to the disturbing effects of -vestibular stimulation has emphasized the importance of this factor in -astronaut selection. At the same time, vestibular functions must be -considered jointly with visual task performance, since both have special -significance for such maneuvers as vehicle docking. Vestibular function -in the weightless state remains almost completely unknown. Limited -evidence from animal and manned space flights suggests that head -turning, resulting from vestibular stimulation, may seriously interfere -with visuomotor performance, but that susceptibility to these -disturbances is significantly different between individuals and that -partial adaptation occurs relatively quickly. - -NASA is currently collecting extensive baseline electroencephalogram -data under controlled conditions in a form suitable for mathematical -analysis. Data are being taken from about 200 subjects in major national -and overseas centers. It is intended that this study will assist in -astronaut selection and monitoring in space. - -Studies on many effects of weightlessness on nervous functions require -monitoring of the autonomic nervous system, including such autonomic -effects as gastrointestinal activity, secretion, lacrimation, -salivation, sweating, and the central control of respiration. Urinary -estimations of catecholamines and 5-hydroxyindoleacetic acid would -provide important data on autonomic system activity if collected in -flight and compared with preflight and postflight controls. - -Major areas have been outlined in which prolonged weightlessness may be -expected to interfere with performance, judgment, and, ultimately, -chances of survival. These include cardiovascular, metabolic, central -nervous, psychophysiological, and biorhythmic effects. They have been -dealt with separately and in sequence, but have not been intended to be -viewed as hierarchic. The relative scarcity of data necessarily -precludes such an evaluation. - -Soviet experience with zero gravity and weightlessness has increased -their emphasis on this space-flight factor and was an important topic at -the May 1964 COSPAR meeting. Discussion of the postflight medical status -of Bykovsky (5-day flight) and Tereshkova (3-day flight) revealed a -concern for the significance of prolonged weightlessness and the -presence of postflight physical debility and fatigue following Vostok -flights 3 through 6. These changes persisted for several days. Among the -physiological conditions singled out for mention were-- - - (1) _Body fluids_-- Cosmonauts have shown a postflight weight loss of - 1.9 to 2.4 kg apparently resulting from a redistribution of body - fluid in response to elimination of the hydrostatic pressure - gradients caused by Earth gravity. There is the suggestion that - this redistribution is complete within the first 24 hours of - flight. Titov is reported to have been dehydrated alter his flight - with early hemoconcentration. These findings directly support - predictions made from ground-based research. - (2) _Cardiovascular_-- Postflight orthostatic tachycardia is reported - for Titov as long as 23 hours after landing; at 48 hours there was - significant residual intolerance to the upright posture. - Cosmonauts have demonstrated a 20- to 35-percent increase in - oxygen consumption during the standard postflight exercise test. - -In both of these areas there was a return to normal within the -postflight period of study. The Soviets have continued their biological -experiments in space with the Vostok/Voshkod series. Fixing of -histologic specimens in flight by Bykovsky demonstrated a critical role -for man and made possible an expanded experimental program. Biopackages -have become more complex with each succeeding flight. - -With the exception of postflight orthostatic intolerance after the third -and fourth Mercury flights, changes as a result of exposure to a -zero-gravity environment have not been noted by U.S. investigations in -space. Ground-based research proceeds here at an advanced pace and is -supported in large measure by both the USAF and NASA. A study of the -relationships among renal and systemic hemodynamics, neurohumoral -cardiovascular regulation, and renal excretory function in differently -positioned subjects is underway, as are studies of acceleration -tolerance. - - - DEPRESSED METABOLISM - -In anticipation of prolonged manned space flights, NASA has sponsored -research related to metabolism depression. The daily food requirements, -for example, of astronauts during a voyage of several months can -constitute a major portion of the weight and storage capacity of the -spacecraft. A somewhat promising and fundamental approach to this -problem is the reduction of the astronauts' daily metabolic -requirements. It has been suggested that astronauts on prolonged space -missions be put in a state of suspended animation until their -destination is reached. Though this sounds fantastic, 10 years ago no -cell had been frozen to cryogenic temperatures and survived. Today it is -commonplace for tissues to be frozen, stored at low temperatures, and -thawed and then to maintain their viability and function. - -Animal metabolism may be depressed by reducing body temperature, as in -hibernation and hypothermia. Other means by which metabolism can be -lowered include drugs and electronarcosis. Hibernation is a nonstressful -state and results in a great decrease in metabolism. However, human -beings are not hibernators, and much research is needed before the -mechanism of hibernation is understood, and the possibility of inducing -it in humans evaluated. Hypothermia is the direct cooling of the body to -temperatures where metabolism is substantially depressed. Extracorporeal -circulation systems combined with cooling are in routine use in most -medical centers throughout the world. Hypothermia is not an ideal -solution, however, since general body hypothermia is a stressful -condition. Pharmacologic induction of hypothermia can be accomplished by -such drugs as chlorpromazine and harbamil. Other drugs can be used to -depress metabolism, but all have some disadvantage. - -In recent years there has been a growing interest in electronarcosis, -the induction of sleep by an electric current. Although potentially -valuable, this method is far from routine application. - -Outstanding advances have been made in metabolism suppression. Recent -progress in the biochemistry and physiology of hibernation and -hypothermia have shown that the oxygen requirements of individual -mammals, organs, and tissues can be reduced. When the chemical -composition of the blood and the cardiac output are sufficient to meet -cellular requirements, regulatory mechanisms remain effective and animal -survival is assured. In contrast, when oxygen transport is interrupted, -a reduction in cellular activity occurs and regulation is impaired. In -induced hypothermia, the low temperature slows the rates of all -processes and modifies the action of metabolites and other substances. -This in itself is not harmful, as shown by the true hibernating animal -(e.g., ground squirrel), but will become disastrous as soon as anoxia -and chemical imbalance begin to develop. - -The phenomenon of natural hibernation is being investigated in the -laboratory in the hope that the unusual tolerance of hibernating animals -to reduced metabolism and low body temperature may some day be produced -artificially in ordinary laboratory animals and man. Experiments with -the ground squirrel, a typical hibernator, show that the artificially -cooled ground squirrel does not tolerate such long periods of low body -temperature as does a naturally hibernating animal. - -Other studies of the brown adipose tissue (fat), which is present in -most hibernating mammals, show it to be essential to hibernation. -Indications that brown fat has a thermogenic role in rats exposed to low -temperatures suggest that this may be the case in true hibernators -([ref.199]). Arousal of the hibernating animal by cold is triggered by -sympathetically activated thermogenesis in areas of brown fat so -located, relative to the vasculature, that the heat is transferred to -areas of the body concerned with normal metabolic and nervous activity. - -Soviet work comparing various depressed metabolic states and resistances -to acceleration shows deep winter hibernation to be most effective, -followed by deep hypothermia, and drug narcosis as the least effective. - -Experimental evidence is being accumulated to show that hibernation and -hypothermia somewhat protect animals against radiation. Clinical studies -on irradiation of cancer patients indicate that lowering the body -temperature reduces cellular metabolism and thus decreases tissue -sensitivity to gamma radiation ([ref.200]). - -The use of prolonged hypothermia, hibernation, drugs, and -electronarcosis appears to hold some potential for reducing astronauts' -metabolic requirements. If one or mote of these methods become -practical, human requirements for food and oxygen could be drastically -reduced. Simultaneously, these methods may afford radiation protection -and acceleration tolerance. - - - NUTRITION IN SPACE[10] - - [10] Includes part of [ref.201]. See also [ref.202]. - -The human body can use food stores so that the nutritional requirements -can be reduced for a short time. This will vary widely among individuals -and each individual may exhibit characteristic patterns of nutritional -behavior. During reduced food intake, muscular efficiency may not change -significantly over a period of 4 to 6 days; unfortunately, however, -mental activity begins to decline after 24 hours. Feeding requirements -can be divided into two categories: short term (for missions of less -than 21 days) and long term. Since dehydration can occur in a matter of -hours under adverse conditions, water requirements must be considered as -a special case. - - -Water Requirements - -Water requirements are extremely critical and the amount supplied should -not under any circumstances be kept to a minimum. Rather, a large margin -of safety should be allowed. - -Present data on water requirements show a very strong dependence upon -suit inlet temperatures. In the absence of an accurately controlled suit -temperature, water requirements can easily double. If this should occur, -the mission would probably have to be aborted, since it is doubtful if -electrolyte balance would be maintained at such high rates of water -loss. Normal or even extreme conditions of the terrestrial environment -usually include diurnal variation in temperature which may modify water -needs. These conditions will not be obtained in the spacecraft. - -In addition to ground-based experiments, measurements of water intake -should be made under actual flight conditions. Data from short-term -flights should be used for extrapolation to longer missions. - - -Formula Diets - -The tacit assumption which now prevails, "Astronauts even on short-term -missions require a diet of great variety," is apparently not well -supported. In many parts of the world, people live on a monotonous diet -consisting of only a few types of food with no apparent ill effects, -provided their nutritional requirements are satisfied. Experimental -evidence from many sources (e.g., the Army Medical Research and -Nutrition Laboratory) shows that individuals can be kept on a single -disagreeable formula diet for as long as 60 to 90 days without harm. -Since highly motivated individuals are chosen for space flights, it is -unlikely that they would object to the monotony of a formula diet and -would probably prefer its simplicity. Also, there are definite -possibilities of developing a much more acceptable formula than present -types. There is no reason to anticipate adverse effects from the use of -formula diets in short-term flights. - -Formula diets would be extremely desirable for short-term flights. A -formula diet (a rehydrated liquid formula could be used) would -considerably reduce the number of manipulations and the time required -for in-flight preparation, compared to a varied diet. These two -improvements could contribute materially to the safety of a flight, -since the astronauts would not be preoccupied with food preparation for -so long a period, and the food could be dispensed without removing suit -components, such as gloves. Storage requirements could be simplified -with this type of diet. Weight, however, would not be lowered without -the development of more refined formulas than those now available. -Formula diets could readily be adapted to the determined metabolic -requirements of the individual astronaut. Packaging problems will be -simplified by using formula diets, which can easily be given a variety -of flavors and colors. - - -Waste - -The problem of waste production is intimately related to nutrition and -can be solved or simplified by dietary changes. Any diet should be -adjusted for the minimum production of feces, before and during even -short flights. Water will be sequestered by accumulation in the feces, -and the net loss, under normal conditions, would be approximately 40 to -60 grams per man per day. Flatus can be a serious problem, since -considerable concentrations of toxic gases may accumulate. The -purification system for the recirculated atmosphere must be able to -remove these, although the diet should be planned to minimize the -problem. The collection of urine and its storage is of importance, -particularly on short-term flights, and individual packaging and -labeling of urine specimens will be necessary for the analyses. - - -Metabolism - -An accurately measured intake of nutrients, calories, and water is -necessary for determining metabolic demands imposed in any space flight. -There is insufficient knowledge to predict total metabolic requirements -under the numerous stresses which can be anticipated. Simulator studies -are of great importance even for short-duration flights. - -The two most important variables to be considered in establishing the -minimal diet are protein and energy requirements. NASA is supporting -research at the University of California (Berkeley) to determine these -requirements and to estimate individual variation in healthy young men. -The possibility of minimizing need through biological adaptation is -being explored. - -It is difficult to estimate the minimum protein requirement of an adult -man. The generally accepted criterion of minimum adequate protein -nutrition in the adult is the maintenance of nitrogen balance at minimum -intake. The minimum protein requirements depend on endogenous nitrogen -loss. Analysis of the little data available indicates a best estimate of -2 mg of nitrogen per kilocalorie of basal energy expenditure. However, -this figure is higher than that noted in experiments in some human -subjects. - -After minimum nitrogen requirements and minimum amino acid requirements -have been established, studies will be directed toward investigating -caloric restriction and adaptation to restriction of calories. It has -been suggested that caloric restriction in animals and man results in -apparent decreased energy need for the same activity. This apparent -paradox has never been explained. It has been shown that there is -adaptation to repeated episodes of caloric restriction both in animals -and man, so that subsequent periods of caloric restriction result in -decreased rate of weight loss, nitrogen loss, and longer survival. - -Additional experiments are urgently required to determine the metabolic -demands for minerals--in particular, the metabolic balance of calcium, -potassium, sodium, and phosphorus. Under conditions of high water -consumption, large mineral losses are to be expected. Failure to replace -these can cause an imbalance which could impair the efficiency of the -individual to the extent of endangering the flight. - -Analysis of samples taken in flight, both of urine and feces, should be -made. Respiratory quotients can be determined in flight, blood samples -should be taken before and immediately after flight for analyzing -selected components (in simulator studies these could be taken -periodically), and nutritional intakes (which would be facilitated by -formula diets) must be measured and analyzed. - - -Short-Range Technology - -There are many practical difficulties in providing for food storage and -accessibility in spacecraft. The packaging of food materials, both -dehydrated and liquid, has proceeded satisfactorily under the -supervision of the Food and Container Institute. If packaging materials -are to be made to withstand very high relative humidities and large -variations in temperature, additional investigations are required, since -such containers are not yet available. In packaging, serious -consideration must be given to the ease with which the food may be -reached and eaten. - -If dehydrated formula foods are to be fed on short-term missions, -additional work is required on the rehydration of such formulas. Present -methods of water measurement under weightless conditions are not -satisfactory, and better methods will have to be contrived. - - -Long-Term Nutritional Problems - -There is a dearth of metabolic information, even for short-duration -flights, without which changes in metabolic patterns to longer flights -cannot be extrapolated. However, using scattered information, certain -changes which may be encountered can be hypothesized. Decalcification of -bone and changes in water-holding capacity of the body may be -anticipated. It is also possible that changes in proportion of fat to -lean body mass could be experienced and should be considered in -nutritional planning. Nutritional requirements depend on size, -particularly lean body mass, sex, physiological state, and individual -metabolic rates. Therefore, individuals for space flight should be -screened with these factors in mind if it is desirable to minimize food -intake in long flights. The factors which influence the total -nutritional requirements of the individual also influence his mental and -physical responses to stress. - - -Synthetic Foods - -The development of food materials other than those derived directly from -animal or vegetable origin is of interest. Advantages of such diets may -be low residue, ease of storage, rehydration, and manipulation. -Experiments with chemically defined synthetic diet for humans have been -carried out by Medical Sciences Research Foundation, San Mateo, Calif. -The complete liquid diet is composed of required amino acids, fat, -carbohydrate, vitamins, and minerals. A cubic foot of the diet (50 -percent solids in H2O) supplies 2500 calories per day for 1 month, and -has been given a variety of artificial flavors. - -This synthetic diet has been fed to human volunteers for 6 months in a -pilot study at the California Medical Facility, Vacaville, Calif., and -the results are being reviewed. Schwarz Bioresearch, Inc., is studying -the storage, stability, and packaging of chemically defined synthetic -diets for human and animal flights. - - -Food Production in Space - -Long-term feeding in space depends upon a payload of stored food unless -food is produced during flight. If sufficient propulsive energy is -available, the duration of missions using stored food may be quite long. -However, in emergencies in which a mission lasts longer than planned, -survival may depend on the ability to produce food extraterrestrially. -Eventually it will be desirable or necessary to produce food beyond the -confines of Earth. - -The nutritional requirements of the crew will be influenced by such -factors as activity, physical and psychological stress, individual size -of the members, and individual metabolic rates. The food intake will -have to be adjusted to meet these requirements. It is necessary to know -the nutritional requirements of each astronaut and the way in which -these are altered by the conditions of space flight in order to estimate -needs on long missions. Without this information, the food supplies for -the longer flights may be too much, too little, or improperly balanced. -Where dependence would not be on stored food alone, but on food produced -en route, more exact information on requirements is needed to determine -the capacity of food production units. - -In the discussion of bioregenerative systems, it was suggested that food -materials could be produced by photosynthetic organisms (e.g., algae, -duckweed, and other higher plants) or by nonphotosynthetic organisms -(e.g., _Hydrogenomonas_). In contrast to the use of living organisms, -reprocessing waste materials by chemical treatment or the actual -synthesis of high-energy compounds has been suggested. No chemical -system has yet been demonstrated as workable for the economical -production of food in space, and the systems considered produce -materials which may be converted to food, but are not food as such. - -Algal cultures have had the most extensive investigation as food in -space, but the technical problems of using this material as a food -source have not yet been solved. It is apparent from the investigations -to date that algae will require treatment before they can be used as -food. In limited trials, difficulties have been experienced with amino -acid deficiencies, digestibility, high residues, and gastric distress. -Processing methods which would be applicable in space travel and the -possibility of secondary conversion by other animals or plants should be -systematically investigated. - - - - - chapter 8 - -_Significance of the Achievements_ - - - SIGNIFICANCE TO SCIENCE - -One of the most critical research areas of the space program is -bioscience. Of both practical and philosophical significance in -exploring the origins of life and the possibilities of life on other -planets, bioscience also promises much in medical aspects. Space offers -biologists completely new environmental factors, such as the effects of -zero gravity and of removal from Earth's rotation. These effects have -been studied in attempts to advance understanding of basic mechanisms of -physiology and biological rhythms. These studies can be of great value -in dealing with problems of disease and metabolic disorders. - -Biological research is fundamental to the problem of successfully -protecting and sustaining man in the peculiar and hostile space -environment. Understanding human requirements and variations in their -response to various environmental factors offers value in medical -research for human survival and comfort. The many technological -discoveries and advancements in electronic and engineering equipment -greatly enhance medical diagnosis, treatment of disease, and the -extension of human life. - -The life sciences, biology and medicine, are fundamental to the success -of manned exploration of space, which marks a unique and significant -development in the long history of man's conquest of new frontiers. -Those who pioneered other frontiers on land and sea and in the air were -not forced to await biological and medical research. Even the pioneers -of aerial flight began their efforts without first seeking biomedical -data. The search for such data followed flight experience and, indeed, -was made only after problems arose. - -Project Mercury, NASA's first program for manned space flight, -stimulated immediate and extensive studies in the life sciences to -sustain man in space. Before a vehicle could be designed to carry an -astronaut into space, anticipated biomedical problems associated with -space flight were studied. Life-support systems were designed to offer -adequate protection from environmental stresses peculiar to space, such -as zero gravity, removal from Earth's rotation, and high-energy cosmic -radiation. These life-support systems used knowledge already gained from -research for manned space flight by the U.S. Air Force. - -Our entry into space has put us at the threshold of fundamental and -far-reaching discoveries in the biological realm which have profound -implications for other areas of human thought and endeavor. As man goes -farther into space, the hazards increase; but past accomplishments -indicate that the road ahead holds more promise than peril and that the -vistas of knowledge that may be foreseen are as vast as space itself. - -Almost everything which now can be said about the effects of -extraterrestrial environments and about life on the Moon or the planets -lies in the realm of pure speculation. There is one prediction, however, -that can be made with considerable certainty by reason of historical -precedent--the opportunity to investigate a totally new area, such as is -offered by space exploration, is certain to produce a burst of -scientific interest as soon as the path is charted by a few pioneers. -Over the next few decades a progressively larger proportion of -biological interest will turn to space. We may well expect that the -discoveries made here will revolutionize some of our concepts of -biology. - -It should be fully realized that the accumulation and dissemination of -biological and other scientific information is not only of great value -to science and humanity but is of tremendous import to the prestige of -the Nation. - - - SIGNIFICANCE FOR PRACTICAL APPLICATIONS - -It can be predicted as confidently for space biology as for other space -sciences that the economic costs will be amply repaid in the long run by -applications of space-oriented biotechnology to other fields of biology -and medicine. There are inevitable substantial, though indirect, -contributions of NASA's continuing efforts in space biology. - -NASA-supported biological research has many practical applications and -"spinoffs" which contribute to the fields of health and medicine, food -and agriculture, and industry and manufacturing. Some of these are -presented to show the range and value of applications which have -resulted from basic and applied biological research. In addition to -those listed are many others from the biosatellite program, particularly -in the fields of bioengineering and miniaturization. - - -Health and Medicine - -Solar cells, which have powered space systems, are now being used as a -power source in studies on brain function. A miniaturized solar cell -developed by General Electric provides enough power, under ordinary -house lights, to stimulate an animal's brain and to telemeter -respiratory, cardiovascular, and brain-wave data while the animal is -allowed to move about freely. Such a system is now used by the National -Institute of Mental Health Laboratory at Rethesda, Md. - -Scientists at the Ames Research Center have devised a new technique for -studying organic compounds, whether synthesized in the laboratory or -produced by a living system. This technique is based on a property of -matter called optical activity. Previous methods of measuring optical -activity have been plagued by low sensitivity. The new method is many -tunes more sensitive and represents a real contribution to modern -analytical instrumentation. - -Studies on calcium metabolism and bed rest simulating weightlessness are -adding knowledge on the prevention of demineralization of the skeleton; -treatment of Paget's disease and osteoporosis prevention of muscular -atrophy; the cause and treatment of renal calculi (kidney stones); -optimal calcium for the human diet; and the factors influencing calcium -absorption, metabolism, and excretion. The results will have great -importance in bone healing and repair, care and treatment of fracture -cases, treatment of paraplegics, and treatment of polio patients and -similar cases. These grant studies at Texas Woman's University have also -proven that the X-ray bone densitometry method can accurately detect -changes in the skeleton. - -A primary objective of the planetary exploration program is the -detection of possible extraterrestrial life. The study of the -fundamental properties of living things on Earth is restricted to the -type of life which has evolved and survived here. Life which has been -exposed to totally different environmental conditions may have markedly -different physiological characteristics. The impact of the new -information obtainable from the study of extraterrestrial life upon the -sciences of medicine and biology will unquestionably be of fundamental -and far-reaching importance. Advancement in the treatment of disease and -the problems of aging are among the many possible consequences. - -New developments in such techniques as ultraviolet spectrophotometry, -polarimetry, and gas chromatography will find use in the detection of -biochemicals and other compounds in hospitals and in toxicology and -pathology laboratories. They will also be useful in studies of -atmospheric pollutants such as smog. - -Studies of the chemistry of living systems, molecular biology, and -biophysics of cellular processes will create a better understanding of -the basic mechanisms of life, leading to an understanding of both -inherited and acquired disease, especially neoplastic conditions and -chemical disturbances incident to mental disease. - -The University of Pittsburgh is conducting a study to increase the -availability of cytological technique in research and as a monitoring -procedure by developing an automatic electronic scanning device using -computer analysis for recording, counting, and sorting chromosomes. -Structural changes in blood cell chromosomes can indicate the degree of -radiation damage as well as damage resulting from various environmental -stresses. Accordingly, this instrument, when developed, can be used as a -radiation dosimeter in civil defense by swiftly detecting the degree and -type of chromosomal aberrations in blood cells. Thus, casualties in -nuclear attack could be quickly detected and treated. This system would -also be useful for nuclear industrial plants and for military maneuvers. -In medicine, various disease trends could be monitored. (Chromosomes -exhibit anomalies in leukemia and mental retardation as well as in other -states.) In space exploration and experimentation, the device can spot -monitor radiation dose levels as well as changes resulting from any of -the environmental stresses experienced in space. This apparatus can be -modified for use as an extraterrestrial-life-detecting instrument by -scanning the growth of cells (or cellular inclusions), computing rates, -and telemetering changes to the researcher. - -Investigations of rhythmic phenomena of various physiological systems -can result in knowledge of the utmost importance to medicine. Rhythmic -phenomena are found in the cardiovascular system of normal humans. -Changes in these rhythms have the potential of foretelling abnormalities -(heart disease, arteriosclerosis) before outward signs are manifested, -allowing for earlier diagnosis, treatment, and control or cure. - -The spacecraft sterilization program requires the use of rooms having -the lowest attainable level of bacterial contamination. The rate of -dissemination of bacteria from the humans in the room is basic to the -problem. Data on this matter are being obtained through support of the -Communicable Disease Center of the U.S. Public Health Service. The -findings are affecting the measures used in surgical practice to lower -infection rates. - -Studies on the physiology of hibernation in mammals are important to -understand temperature regulation and the mechanism of survival at low -body temperatures. The purpose of this type of research is to understand -and use reduced metabolic activity in astronauts on future extended -space flight. Other applications involve studies of the mechanisms of -injury and freezing biological organisms, for improving techniques in -hypothermic surgery, pathology, and preservation of tissue for human -grafting. - - -Food and Agriculture - -Gathering agricultural information by remote sensing of Earth's surface -from aircraft, balloons, and satellites has a potential application in -research and development. Current needs for data gathered in this way -include crop and livestock surveys for marketing planning; soil mapping; -crop disease, insect, and weed surveys; soil conservation management and -research; and crop acreage control programs. As population and world -trade increase, the needs will become even more intense for regularly -scheduled synoptic surveys of the world's agricultural lands for crop -plantings and harvests; determining the condition of crops as affected -by drought, disease, or insect outbreaks; and studies of the lands -suitable for agricultural development in underdeveloped countries. The -only way that worldwide synoptic surveys can be made is by using -orbiting platforms. - -The NASA nutrition program for developing diets for prolonged manned and -animal space flight lends itself to civil defense purposes; military -maneuvers where space and weight are prime considerations; polar and -desert exploration; reducing hunger in underdeveloped countries; and -detecting metabolic diseases as well as diseases of infancy and old age. -For space research such a diet can be used on prolonged manned space -flights, animal experiments in space, manned orbiting laboratories, and -space and planetary stations. Studies on the packaging and stability of -foods under various conditions of humidity, temperature, and radiation -will lead to better processing and storage. - -Learning how microbial spores are transported by air is important to -biology, agriculture, and medicine. Besides spreading crop destruction, -microbial spores produce allergic responses in some human beings. To -obtain the facts, not only the biology of micro-organisms but also the -weather factors that induce the flight of mature spores must be known. -Thus, both biological and meteorological problems are involved. Data -obtained under a NASA contract with the General Mills Electronic -Division (now part of Litton Industries, Inc.) indicate that spores of -fungi are present in low numbers in the stratosphere. A reservoir of -spores exists which cannot be brought down by the normal scrubbing -mechanisms of rainfall and other meteorological disturbances in the -troposphere. This finding has important implications for reducing the -spread of agricultural crop diseases and for protecting persons -suffering from allergies. This project has indicated the necessity for -designing novel biological samplers for use in the stratosphere. Such -samplers will aid in determining various pollutants of the atmosphere. - -The NASA program for developing sterile spacecraft for the biological -exploration of Mars will contribute improved methods of sterilization -that can be applied to the canning industry. Studies on sterilization at -low temperatures for long periods of time are being supported by NASA at -the Massachusetts Institute of Technology and the Communicable Disease -Center and the Sanitary Engineering Center of the Public Health Service. -The developing capability is making possible the heat sterilization of -products that never before could be thoroughly sterilized. - -In preparing for missions to search for extraterrestrial life, research -on the psychrophilic or cold bacteria, on halophytic or salt bacteria, -and on specialized bacteria and other organisms growing in extreme -environments is defining the extremes under which life can exist. -Increased knowledge about organisms that can grow in or on refrigerated, -dried, or salted foods and other materials should have practical -applications for food storage and preservation. Research on -psychrophilic bacteria is being conducted by Whirlpool Corp. and the -NASA Ames Research Center. - -Theoretical studies of Martian life involve investigations of plant and -bacterial spores. Many of these forms are spoilage organisms and some -produce lethal toxins. This work has potential importance for food -processing and for obtaining more precise knowledge of how wounds become -infected. The program for investigating possible forms of life on Mars -includes a thorough study of anaerobic micro-organisms. This research -has led to the discovery of new types of nitrogen-fixing bacteria other -than the familiar types found in the root nodules of leguminous plants. -Thus, it may be possible to use these microorganisms, or the principles -involved, in the incorporation of vital atmospheric nitrogen into -terrestrial soils which are now unproductive. - - -Industry and Manufacturing - -Batteries that have been developed in the space program to endure high -sterilization temperatures for extended times will have greatly -increased shelf life at normal storage temperatures and will be -serviceable after many hours of baking at high temperatures. - -Currently, the highest quality tape recorders are subject to imperfect -reproduction because the tapes are heat labile; i.e., they soften and -stretch when warm. The development of high-quality magnetic tapes for -space-data recorders is an outgrowth of the materials developed to meet -spacecraft sterilization requirements. These improved tapes will be -useful for all types of recording--industry, automation controls, home, -and studio. - - - OUTLOOK FOR BIOSCIENCE--MAJOR PROBLEMS - -The problems undertaken are among the most challenging, if not _the_ -most challenging, man faces on the space frontier. These include the -quest for the origin of life, the explanation of life and life -processes, the elucidation of the environment's role in establishing and -maintaining normal organization in living organisms, the possibility of -extraterrestrial life on other planets--the concern of exobiology. The -greatest promise for their solution lies in advances in biological -theory rather than other avenues of research; therefore, it is fortunate -that the need to solve them has come at a time when developments in -experimental biology are at a high level. In addition, technological -developments in electronics and engineering are providing new and -wonderful instruments for this great exploration into the sources of -life. Many of these have had practical application that has made -possible important advances in medical diagnosis and treatment. - -The broad national space goals initially charted by NASA have gone -beyond space flight in near-Earth orbit to lunar and interplanetary -exploration by man and machine. For such missions, more intensive and -comprehensive research in the life sciences is needed. 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K.;_ - - *p. 124, l. -10:* _Rosenszweig_ ----> _Rosenzweig_ - - *p. 128, l. 29:* AMRL Tech. Doc. Rept. ----> AMRL-Tech. Doc. Rept. - -Variant spelling: Both forms _microorganism_ and _micro-organism_ have -been retained, as quoted from different sources or bibliographic -reference titles. - -Tables: Where necessary, the widths of columns have been adjusted, and -some tables have been split to accommodate the width restrictions on -this text format. Split tables have had blank lines inserted in order to -maintain the alignment between the two parts should they be re-joined at -a future date. In the original text, Table VI was split over two pages -but has been rejoined in this version. - -References: For ease of searching, references in the text, as well as -those in the list of references, have been enclosed in square brackets, -e.g. [ref.3]. - -Representation of UTF-8 characters: In this version of the text, certain -characters cannot be represented directly. 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You may copy it, give it away or -re-use it under the terms of the Project Gutenberg License included -with this eBook or online at www.gutenberg.org - - -Title: Significant Achievements in Space Bioscience 1958-1964 - -Author: National Aeronautics and Space Administration - -Release Date: July 17, 2012 [EBook #40268] - -Language: English - -Character set encoding: ASCII - -*** START OF THIS PROJECT GUTENBERG EBOOK ACHIEVEMENTS IN SPACE BIOSCIENCE *** - - - - -Produced by K.D. Thornton, Enrico Segre and the Online -Distributed Proofreading Team at http://www.pgdp.net - - - - - - - - - - NASA SP-92 - - - Significant Achievements in - - - Space Bioscience - 1958-1964 - - - - - _Scientific and Technical Information Division_ 1966 - - NATIONAL AERONAUTICS AND SPACE ADMINISTRATION - - _Washington, D.C._ - - - - - For sale by the Superintendent of Documents, - U.S. Government Printing Office - - Washington, D.C., 20402--Price 55 cents - - - - -_Foreword_ - - -This volume is one of a series which summarize the progress made during -the period 1958 through 1964 in discipline areas covered by the Space -Science and Applications Program of the United States. In this way, the -contribution made by the National Aeronautics and Space Administration -is highlighted against the background of overall progress in each -discipline. Succeeding issues will document the results from later -years. - -The initial issue of this series appears in 10 volumes (NASA Special -Publications 91 to 100) which describe the achievements in the following -areas: Astronomy, Bioscience, Communications and Navigation, Geodesy, -Ionospheres and Radio Physics, Meteorology, Particles and Fields, -Planetary Atmospheres, Planetology, and Solar Physics. - -Although we do not here attempt to name those who have contributed to -our program during these first 6 years, both in the experimental and -theoretical research and in the analysis, compilation, and reporting of -results, nevertheless we wish to acknowledge all the contributions to a -very fruitful program in which this country may take justifiable pride. - - _Homer E. Newell_ - _Associate Administrator for_ - _Space Science and Applications, NASA_ - - - - -_Preface_ - - -This summary of certain aspects of the space biology program of the -National Aeronautics and Space Administration brings together some -results of NASA research and NASA-sponsored research under grants and -contracts from 1958 through 1964. Closely related research even though -not sponsored by NASA is also included. - -The space biology program has had a late start in comparison with the -space physics program, and only a token program existed before 1962. -Much of the present research involves preparation of space-flight -experiments and obtainment of adequate baseline information. Perhaps -half the research results reported are derived from the NASA program. -Additional information is included from many other sources, especially -the U.S. Air Force with its long history of work in aviation and -aerospace medicine. - -Relatively few biological space-flight experiments have been undertaken. -These have been to test life-support systems and to demonstrate, before -manned space flight, an animal's capability to survive. Few critical -biological experiments have been placed in orbit by NASA, but a -biosatellite program will soon make a detailed study of the fundamental -biological effects of weightlessness, biorhythms, and radiation. - -The search for extraterrestrial life has been limited to ground-based -research and planning for planetary and lunar landings. Life-detection -experiments have been developed and tested, and an important and -exciting program is being planned to detect and study extraterrestrial -life, if it exists. - -Interest in space biology has been slow in developing, and there has -been some caution and controversy in the scientific community. However, -increased interest is starting to push forward the frontier of this new -and important scientific field, and future outlook appears to be -optimistic. - -This summary was written and compiled by the members of the Bioscience -Programs Division of the Office of Space Science and Applications. The -report was edited and chapters 1, 3, 6, and 7 were written by Dale W. -Jenkins, Chief, Environmental Biology; chapter 2, by Gregg Mamikunian, -Staff Scientist, Exobiology; chapters 4 and 8, by Richard E. Belleville, -Chief, Behavioral Biology; and chapter 5, by George J. Jacobs, Chief, -Physical Biology. - - - - -_Contents_ - - - page - 1 BACKGROUND ................................................... 1 - 2 EXOBIOLOGY ................................................... 5 - 3 ENVIRONMENTAL BIOLOGY ........................................ 23 - 4 BEHAVIORAL BIOLOGY ........................................... 43 - 5 MOLECULAR BIOLOGY AND BIOINSTRUMENTATION ..................... 57 - 6 FLIGHT PROGRAMS .............................................. 65 - 7 MANNED SPACE FLIGHT .......................................... 77 - 8 SIGNIFICANCE OF THE ACHIEVEMENTS ............................. 111 - REFERENCES ..................................................... 119 - - - - - chapter 1 - -_Background_ - - -The biological program of the National Aeronautics and Space -Administration had a late start. A small life sciences group, organized -in 1958, was concerned with life support and use of primates for system -and vehicle testing for the Mercury program. Three small suborbital -flights of biological materials were flown in space. - -The Bioscience Program Office of the Office of Space Science and -Applications was organized in 1962. The goals of the Bioscience Program -are: (1) to determine if extraterrestrial life exists anywhere in the -solar system and to study its origin, nature, and level of development, -if it is present; (2) to determine the effects of space and planetary -environments on Earth organisms, including man; (3) to conduct -biological research to develop life support and protective measures for -extended manned space flight; and (4) to develop fundamental theories in -biology relative to origin, development, and relationship to -environment. Research and development has been carried out to design -life-detection experiments and instruments for future flights to Mars -and to develop experiments to study the effects of the space environment -on living organisms. A biosatellite program, started in 1963, has the -first of six flights scheduled for 1966. - -Space exploration has demanded a rigorous development, especially in the -biosciences area. Investigation of the solar system for exotic life -forms, the environmental extremes to which Earth organisms (including -man) are being exposed, the possibilities for modification of planetary -environments by biological techniques yet to be developed, and the -problems of communication in biosystems are areas which have required -refinement of the theoretical framework of biology before progress could -be made rapidly enough to keep pace with technological advances in -transportation. - -Of all the sciences, biology alone has not yet benefited from -comparisons with the universe beyond Earth. It is reasonable to suppose -that breakthroughs might be made in biology on the basis of comparisons -with life from other worlds. Organisms elsewhere may have found -alternatives to processes we think of as basic characteristics of life. - -In contrast, physical science has advanced sufficiently to provide a -great body of laws which may be expressed in mathematical terms, and by -which phenomena may be predicted with complete accuracy. A well-known -characteristic of biological phenomena is variability. The Darwinian -concept of evolution is perhaps the only pervading generalization in -biology. This concept has been supported by evidence of a hereditary -mechanism in the discovery of genes and gene mutations. - -Space bioscience represents the convergence of main disciplines with a -single orientation, whose direction is determined by the problems of -manned space travel which have, in turn, created a host of -bioengineering problems concerned with supporting man in space. - -Foremost among these questions is the possibility of the existence of -extraterrestrial life. The field which is concerned with the search for -extraterrestrial life has come to be called "exobiology." In addition to -the challenge of great technological problems which must be solved, -exobiology is so closely related to the central scientific questions in -biological science that it is considered by some to be the most -significant pursuit in all of science. - -One of the major opportunities already presented by the advances in -propulsion systems is the ability to escape from the influence of the -Earth, which has made possible the study of organism-environment -relationships, particularly the role that environmental stimuli play in -the establishment and maintenance of normal organization in living -systems. - -Transcending even these formidable objectives of space bioscience is an -objective shared by all life sciences, the discovery of nature's scheme -for coding the messages contained in biological molecules. -Extraterrestrial biology seeks to find not only evidence of life now -present, but the vestigial chemicals of its previous existence. The ways -and means have already been made available to study molecules on whose -long, recorded messages is written the autobiography of evolution--the -history of living organisms extending back to the beginnings of life. On -this same basis, it is now within the realm of science to foresee the -means of predicting the development of life from primordial, nonliving -chemical systems. Closely allied to the search for extraterrestrial life -is research which seeks to identify the materials and the conditions -which are the prerequisites of life. - -Space bioscience research is now extending human knowledge of -fundamental biological phenomena, both in space and on Earth, just as -the physical sciences explore other aspects of the universe. The -accomplishment of bioscience objectives is totally dependent upon -advances in the technology of space flight. A highly developed -launch-vehicle capability is essential to accomplish the long-duration -missions required in the search for extraterrestrial life. - -Life on other planets in the solar system (with emphasis on Mars) will -be investigated by full exploitation of space technology which will -allow both remote (orbiter) and direct (lander) observations of the -planetary atmosphere, surface, and subsurface. Certain characteristics -of terrestrial life, such as growth and reproduction, provide a basis -for relatively simple experiments which may be used on early missions to -detect the existence of life on Mars. Later missions will provide -extensive automatic laboratory capabilities for analyzing many samples -taken from various depths and locations. Because of the hypothetical -nature of current experiment designs, it is likely that visual -observations of the planet will be required. Many technical problems are -involved in storing and transmitting the large amounts of data over -planetary distances. Such visual observations might very well be crucial -in interpreting results from other experiments. Critical to all -exploration of the Moon and planets are the requirements to: (1) prevent -contamination of the environment with Earth organisms and preserve the -existing conditions of the planet for biological exploration; (2) -provide strict quarantine for anything returned to Earth from the Moon -and planets. - -The biological exploration of Mars is a scientific undertaking of the -greatest significance. Its realization will be a major milestone in the -history of human achievement. The characterization of life, if present, -and study of the evolutionary processes involved and their relationship -to the evolution of terrestrial life would have a great scientific and -philosophical impact. What is at stake is nothing less than knowledge of -our place in nature. - -Extended Earth orbital flights with subhuman specimens will be used to -determine the effects on Earth organisms of prolonged weightlessness, -radiation, and removal from the influence of the Earth's rotation. Such -flights of biosatellites and other suitable spacecraft are expected to: -(1) establish biological specifications for extending the duration of -manned space flight; (2) provide a flexible means of testing unforeseen -contingencies, thus providing an effective biological backup for manned -missions; (3) yield experimental data more rapidly by virtue of the -greater number and expendability of subjects; (4) anticipate possible -delayed effects appearing in later life or in subsequent generations, -through use of animal subjects with more rapid development and aging; -(5) develop and test new physiological instrumentation techniques, -surgical preparations, prophylactic techniques, and therapeutic -procedures which are not possible on human subjects; and (6) provide a -broad background of experience and data which will permit more accurate -interpretations of observed effects of space flight on living organisms, -including man. - - - - - chapter 2 - -_Exobiology_ - - -The possibility of discovering an independent life form on a planet -other than Earth presents an unequaled challenge in the history of -scientific search. Therefore, the detection of life within the solar -system is a major objective of space research in the foreseeable future. - -The scientific data presently available concerning the possible -existence of a Martian life form and the chemical constitution of the -surface of Mars are disappointingly few. In fact, it is impossible to -make a statement about any of the many surface features, other than the -polar caps, with any degree of certainty. The observational results have -been accounted for by many conflicting hypotheses which can only be -resolved by the accumulation of new evidence. - -The arguments supporting the existence of Martian life ([ref.1]) are -based on the following observations: - - (1) The various colors, including green, exhibited by the dark areas - (2) The seasonal changes in the visual albedo and polarization of the - dark areas - (3) The ability of the dark areas to regenerate after an extensive - "duststorm" - (4) The presence of absorption bands at 3.3 mu - 3.7 mu, attributed to - organic molecules - -Conflicting interpretations of the above observations have been -advanced. The argument based on the colors is inconclusive, and several -workers have suggested that the color is a contrast effect with the -bright-reddish continents. The meager quantitative data have been -discussed by Oepik ([ref.2]) who has reduced Kozyrev's photometric -observations of the very dark area of Syrtis Major to intrinsic -reflectivities by allowing for the estimated atmospheric attenuation and -reflectivity. Kuiper ([ref.3]) similarly demonstrated the absence of the -near-infrared reflection maximum, which is characteristic of most green -plants, indicating that chlorophyll was not responsible for the color. - -The second and third arguments remain the most cogent. However, serious -limitations are imposed on the second if the severity of the Martian -climate is considered. Foecas ([ref.4]) has photometrically measured the -seasonal changes in the fine structure of the dark areas of Mars and -concludes that-- - - (1) The dark areas of Mars show periodic variation of intensity - following the cycle of the darkening element - (2) The average intensity of the dark area, not including the action - of the darkening waves, increases from the poles toward the - equator - (3) The action of each of the darkening waves decreases from the poles - toward the equator. This decrease is balanced in the equatorial - zone by the combined action of the two darkening waves alternately - originating at the two poles. The mechanism of the - darkness-generating element seems to be constant for all latitudes - during the Martian year. - -The variation in intensity has been explained recently by nonlife -mechanisms for Depressio Hellespontica (an area showing one of the -greatest seasonal changes) ([ref.2]). Similar nonlife mechanisms may be -applicable to the other dark regions, and, thus, the "darkening" can be -used only as circumstantial evidence in support of a Martian life form. - -If inorganic interpretations of the seasonal albedo variation are -accepted, then an inorganic interpretation must also be advanced for the -polarization variation. Two possibilities can be suggested: - - (1) A change in surface texture, caused by varying absorption of - atmospheric constituents, causing both the albedo and polarization - to change in the manner observed - (2) A change in surface texture, in which the surface material becomes - rougher, which also explains the observed polarization data - ([ref.5]) - -The third argument against the regenerative feature of the dark areas -being a life process has been advanced by Kuiper ([ref.6]). It is based -on atmospheric circulation causing dust, presumably lava, to be blown on -the dark areas of Mars during the late summer, autumn, and winter, and -then removed during the spring. Mamikunian and Moore have recently -advanced the similar explanation that carbonaceous chondrites or -asteroidal matter may induce the observed phenomenon if they are -abundant on the planet's surface. The pulverized chondritic material -will exhibit a high degree of opacity due to localization and, hence, a -change in polarization characteristics and a decrease in polarization -following mixing of the chondritic material with indigenous surface -minerals. - -The fourth observational argument, the Sinton bands ([ref.7]), has been -shown to be at least doubtful. Rea, Belsky, and Calvin ([ref.8]) -recorded infrared reflection spectra for a large number of inorganic and -organic samples, including minerals and biological specimens, for the -purpose of interpreting the 3 mu-to-1 mu spectrum of Mars. These authors -state that a previous suggestion that the Martian "bands" be attributed -solely to carbohydrates is not a required conclusion. At the same time -they fail to present a satisfactory alternate explanation, and the -problem remains unsolved. More recently, Rea et al. ([ref.9]) noted the -similarity between the 3.58 mu and 3.69 mu minima in the Martian -infrared spectra and those of D2O-HDO-H2O mixtures and, particularly, of -HDO. - -With all this marked disagreement in interpreting the observational data -concerning Mars, it becomes clearly evident that an experimental -approach to the detection of life on Mars should provide the maximum -positive information possible. Some life-detection experiments developed -with NASA support have been summarized by Quimby ([ref.10]). - -The schema of the biological exploration of a planet is to conduct a -series of complementary experiments proceeding from general to specific. -The general experiments will examine gross characteristics of the -planet's environment and surface for determining the probability of an -active biota (life). Data from the general experiments will be -significant in-- - - (1) Defining the nature of specific experiments in which life - detection is the major objective; and - (2) Providing a high degree of confidence in undertaking specific - experiments, since indications from the gross characterization of - the planet in question will influence the choice and design of the - specific experiments. - -The biological exploration of planets is then to be defined as the -search for those parameters relevant to the origin, development, -sustenance, and degradation of life in a planetary environment. This -definition will give rise to a critical question for each progressively -specific and complex experiment to determine-- - - (1) The existence of life on the planet - (2) The degree of similarity or dissimilarity (structure and function) - with respect to terrestrial life - (3) The origin of this planetary life - -The immediate objective of the biological explorations of the planet is -to define the state of the planetary surface, which may exhibit the -following properties: - - (1) A prebiota (defined as the absence of life) - (2) An active biota (defined as the presence of life) - (3) An extinct biota (defined as evidence of former life) - -The identification and the detailed characterization of each of the -above stages of planetary development constitute the subject matter of -the biological exploration of the planets and, specifically, Mars. - - -THE EXPERIMENTAL INVESTIGATION OF CHEMICAL EVOLUTION - -Attempts have been made to simulate and approximate models of primitive -Earth conditions for abiogenic synthesis, and successful synthesis of -essential biochemical constituents necessary for maintaining life has -been partly accomplished. - -Urey ([ref.11]) has clearly pointed out the possible role of a reducing -atmosphere in the synthesis of prebiological organic molecules. Miller -([ref.12]) synthesized a variety of amino acids in a reducing atmosphere -by means of an electrical discharge. A variety of organic compounds have -been synthesized by the action of various energy sources upon reducing -atmospheres, and several investigators have extended the -Urey-Miller-type reactions to synthesize nucleic acid components -([ref.13]), adenosine triphosphate ([ref.14]), and a host of -biologically essential organic compounds. - -It is likely that in the synthesis of organic moieties, simple and -specific molecules were first produced when the planets had a reducing -atmosphere. Further complexity or degradation of the organic compounds -produced varied, depending on the geochemical changes of the planet's -surface, the atmospheric constituents, the degree of interaction between -surface and atmosphere, and the rate of the organic synthesis. Oparin -([ref.15]) presented the most detailed mechanisms for the spontaneous -generation of the first living organism arising in a sea of organic -compounds synthesized in a reducing atmosphere on Earth. - -It is generally accepted that, under favorable conditions, life can -arise by spontaneous generation. A primary requirement for this -initiation is that there be abundant organic compounds concentrated in -one or more specific zones. These simple organic molecules would undergo -modification to develop a greater structural complexity and specificity, -finally giving rise to a "living" organism. Therefore, because of the -ease with which organic compounds can be synthesized under reducing -conditions, planetary surfaces may contain an abundant source of similar -organic matter. However, difficulties arise in postulating steps for -further organization or modification of the above synthesized organic -matter into a living state. Most of the original organic matter produced -in the primary reducing atmospheres of the various planets may have been -quite similar. However, major variations between planets, in chemical -evolution beyond the prebiotic stage, must have been the rule rather -than the exception. - -The primary interest in this area of research has been the realization -of the possible existence of organic molecules on planetary surfaces -and, particularly, Mars. Pertinent synthesis may be either biological or -abiological. Research conducted in the simulation of cosmochemical -synthesis has used most of the available solar spectrum. Simulation -experiments devised to study the effects of these energies on the -assumed early atmosphere of the Earth have yielded products that play a -dominant role in molecular and biochemical organization of the cell. - -Calvin ([ref.16]) irradiated water and carbon dioxide in a cyclotron, -obtaining formaldehyde and formic acid. Miller ([ref.17]) found that -when methane, ammonia, water, and hydrogen were subjected to a -high-frequency electrical discharge, several amino acids were produced -along with a variety of other organic compounds. - -Corroborating experiments established that the synthesis of amino acids -occurred readily. The apparent mechanism for the production of amino -acids is as follows: aldehydes and hydrogen cyanide are synthesized in -the gas phase by the electrical discharge. These substances react -together and also together with ammonia in the water phase of the system -to give hydroxy and amino nitriles, which are then hydrolyzed to hydroxy -and amino acids. Among the major constituents were aspartic acid, -glutamic acid, glycine, [alpha]-alanine, and [beta]-alanine. - -The "Miller-Urey" reaction mixture has been extended and several -modifications introduced. Oro ([ref.18]) introduced hydrogen cyanide -into the system as the primary gas component. Adenine was obtained when -Oro heated a concentrated solution of hydrogen cyanide in aqueous -ammonia for several days at temperatures up to 100 deg. C. Adenine is an -essential component of nucleic acids and of several important coenzymes. -Guanine and urea were the two other products identified in the hydrogen -cyanide reaction. Oro further obtained guanine and uracil as products of -nonenzymatic reactions by using certain purine intermediates as starting -materials. - -Ponnamperuma ([ref.19]) also obtained adenine upon irradiation of -methane, ammonia, hydrogen, and water, using a high-energy electron beam -as the source of energy of irradiation. These results indicate that -adenine is very readily synthesized under abiotic conditions. Adenine, -among the biologically important purines and pyrimidines, has the -greatest resonance energy, thus making its synthesis more likely and -imparting greater radiation stability to the molecule. - -The formation of adenine and guanine, the purines in RNA and DNA, by a -relatively simple abiological process lends further support to the -hypothesis that essential biochemical constituents of life may have -originated on Earth by a gradual chemical evolution and selection. In -this respect, the examination of planetary surfaces--specifically -Mars--presents practical implications for current research on the -problem of chemical evolution. - -When Ponnamperuma et al. ([ref.14]) exposed adenine and ribose to -ultraviolet light in the presence of phosphate, adenosine was produced. -When the adenine and ribose were similarly exposed in the presence of -the ethyl ester of polyphosphoric acid, adenosine diphosphate (ADP) and -adenosine triphosphate (ATP) were produced. The abiological formation of -ATP was a major stride along the path of chemical evolution, since ATP -is the principal free energy source of living organisms. - -Oparin ([ref.15]) postulated that [alpha]-amino acids could have been -formed nonbiologically from hydrocarbons, ammonia, and hydrogen cyanide -at a time when the Earth's atmosphere contained these substances in high -concentrations. Oparin's hypothesis has received strong experimental -support, as evidenced by the work of Miller ([ref.12]). Bernal -([ref.20]) has emphasized the role played by ultraviolet light in the -formation of organic compounds at a certain stage of the Earth's -evolution. - -Generally it has been believed that the first proteins or foreprotein -were nonbiologically formed by the polycondensation of preformed free -amino acids ([ref.21]). Akabori ([ref.22]) proposed a hypothesis for the -origin of the foreprotein and speculated that it must have been produced -through reactions consisting of the following three steps. - -The first step is the formation of aminoacetonitrile from formaldehyde, -ammonia, and hydrogen cyanide. - - CH2O + NH3 + HCN --------> H2N--CH2--CN + H2O - -The second is the polymerization of aminoacetonitrile on a solid -surface, probably absorbed on clay, followed by the hydrolysis of the -polymer to polyglycine and ammonia. - - x H2N--CH2--CN --------> (--NH--CH2--C--)x - || - || - NH - | - | + x H2O - | - V - (--NH--CH2--CO--)x + x NH3 - -The third step is the introduction of side chains into polyglycine by -the reaction with aldehydes or with unsaturated hydrocarbons. Akabori -has demonstrated experimentally the formation of cystinyl and cysteinyl -residue in his above-postulated mechanism. - -Fox's theory of thermal copolymerization ([ref.23]) suggests that -proteins or like molecular units could have been formed in the Earth's -crust, under geothermal conditions. The accumulated amino acids were -heat polymerized and transported into the primary oceans for further -modifications. Fox has obtained polymers consisting of all 18 amino -acids usually present in proteins. The polymerization is generally done -at 160 deg. C to 200 deg. C, although in the presence of polyphosphoric -acid it can be accomplished at temperatures below 100 deg. C. Molecular -weights increased from 3600 in a proteinoid made at 160 deg. C to 8600 -in one made at 190 deg. C. - -Fox showed that when hot saturated solutions of thermal copolymers -containing the 18 common amino acids were allowed to cool, large numbers -of uniform, relatively firm, and elastic spherules separate. These range -from 0.2 mu to 60 mu in diameter and are quite uniform within each -preparation. Various chemical observations suggest the presence of -peptide bonds in the structural organization of these proteinoids. -Continuing observations of these microspheres have established further -characteristics that point to the possibility of their interpretation as -a kind of primitive protein macromolecule with self-organizing -properties, such that a primitive form of cell, with boundary and other -properties, might form. - -In laboratory experiments the behavior of gram-negative and -gram-positive microspheres in dilute alkali parallels that of -gram-negative and gram-positive bacteria ([ref.23]). Furthermore, -time-lapse studies indicate that the proteinoid microspheres undergo a -septate kind of fission, mimicking cell division as shown in figure 1. -Cytochemical studies show that the microsphere's boundary is -membranelike in having a primitive selectivity. Electron micrographs of -sections of stained microspheres also indicate the presence of a -boundary. - -Oparin ([ref.15]) states that the type of organization peculiar to life -could only result from the evolution of a multimolecular organic system -separated from its environment by a distinct boundary but constantly -interacting with this environment. In his concept of coacervates as -precell models, Oparin ([ref.24]) indicates that present-day protoplasm -possesses a number of features similar to coacervate structure. These -coacervates could represent the starting point for evolution leading to -the origin of life. Moreover, in the course of their evolution the -initial systems may gradually become more complex. Oparin also showed -([ref.15]) that mixing solutions of different proteins and other -substances of high molecular weight produced these coacervate droplets. -These droplets are characterized by the formation of a surface layer -with altered structure and mechanical properties, thus providing a -somewhat selective barrier in which to house a molecular system capable -of replication. However, these coacervates are unstable structurally. - -[Illustration: Figure 1.--_Protenoid microspheres undergoing septate -fission. Small microspheres and filamentous associations thereof are -also shown ([ref.25])._] - -The NASA program has further provided considerable impetus for -continuing research with respect to the chemical evolution of life, -since its life-detection experiments may encounter prebiological -molecules in their search for extraterrestrial life on other planetary -surfaces. - -In the area of exobiological research, the significant accomplishments -to date have been-- - - (1) The reconstruction of some of the pathways which may have led to - the origin of life, by means of laboratory simulation of processes - yielding prebiological organic molecules - (2) The developments in experimental and theoretical biology; - specifically, the role of nucleic acid-protein interactions in - storage and transmission of information both within living cells - and from generation to generation of cells - (3) The suspected role of DNA in information storage and the - development of new concepts of the coding mechanism in DNA that - may lead to a universal biological theory embracing evolutionary, - as well as homeostatic, adaptation to environment and learned - behavioral systems - -With the essential biochemical constituents of life and the mechanism of -replication beginning to be understood, the challenge for the synthesis -of living matter by abiogenic experimental techniques has become to many -scientists the ultimate goal of the scientific era. - -NASA has established an exobiology laboratory at Ames Research Center in -addition to the sizable support of research at various academic centers -of excellence for the continuation of abiogenic synthesis. - -Although research on organochemical evolution is in its infancy, the -data from relatively few experiments have already created an immense -enthusiasm for knowledge of the biochemical pathways of evolution. This -kind of research will ultimately elucidate the terrestrial evolution of -life and, perhaps, the nature of life on other planetary bodies and the -distribution of life in our galaxy. - -This program, with its vast demands on the scientific community at -large, is coordinated with related endeavors of a number of Federal -agencies. It is allied with certain biochemical studies at the National -Institutes of Health for the eventual elucidation of the dynamic -pathways in cosmochemical synthesis of life's essential biochemical -constituents. - - - METEORITES AND ORGANIC GEOCHEMISTRY - -Meteorites - -A significant area of exobiological research is the investigation of a -special class of stony meteorites known as "carbonaceous chondrites." It -is increasingly apparent that almost all life-detection concepts rely on -the eventual analysis of the solid materials that may be available on -Mars and other planetary surfaces. Cosmic dust and meteorites are two -classes of material bodies that reach the Earth from outer space. The -carbonaceous chondrites are the only extraterrestrial materials known to -contain organic carbon. - -The study of meteorites has generated an astonishing diversity of -hypotheses. There is agreement at only one point: that meteorites are -preserved chunks of very ancient, perhaps primordial, planetary matter -and that when we are able to understand the curious structures and -chemical and isotopic variations in the meteorites, we will also know a -great deal about early planetary (and perhaps preplanetary) history. - -Meteorites provide a more representative sample of average planetary -matter than the highly differentiated crust of the Earth. Although it is -known that the meteorite parent bodies ceased to be geochemically active -shortly after their formation, some 4-1/2 billion years ago, there is no -consensus on the nature of the meteorite parent bodies, not even on such -basic properties as size, location, and multiplicity. This is not -surprising because the meteorite samples commonly available for study -represent only about 10^-23 to 10^-26 of the parent body. - - -Carbonaceous Meteorites - -Analysis and characterization of the chemical constituents (organic) of -carbonaceous chondrites, including the possible mechanism of their -formation, may be expected to improve methods of analyzing samples from -the Moon and planets and of interpreting remote automated biological -analyses on the planets' surfaces. - -Carbon has been detected in all meteorites analyzed; however, both the -amount and forms present vary considerably. Among the forms of meteorite -carbon are diamond, graphite, cohenite (Fe,Ni,Co)3 C, moissanite SiC, -calcite CaCO3, dolomite (Ca,Mg)CO3, bruennerite (Mg,Fe)CO3. A summary of -the results of carbon analyses on large numbers of meteorites is given -in table I ([ref.26]). - - - Table I.--_Meteorite Carbon_ - - ----------------------------------------------------------------- - Meteorite group Number Mean carbon content, - analyzed percent by weight - ----------------------------------------------------------------- - Pallasites 10 0.08 - ----------------------------------------------------------------- - Ureilites 2 .69 - ----------------------------------------------------------------- - Bronzite chondrites 12 .05 - ----------------------------------------------------------------- - Hypersthene chondrites 8 .04 - ----------------------------------------------------------------- - Enstatite chondrites 8 .29 - ----------------------------------------------------------------- - Carbonaceous chondrites 16 2.04 - ----------------------------------------------------------------- - - -Most meteorites possess only traces of carbon, and studies of this -carbon indicate that it is composed largely of graphite, cohenite, and -moissanite, with some diamond. However, studies of the carbon in the -carbonaceous chondrites have failed to detect any of these forms. Some -carbonates are present in a minority of the carbonaceous group, but -account for only a small percentage of the total carbon (perhaps about -10 percent of the total C in type I only). - -The carbonaceous chondrites contain organic carbon. The word "organic" -is not used in a biological sense, merely as a chemical term to describe -compounds of carbon other than carbonates, bicarbonates, and carbides. -No evidence has been found of any form of carbon other than organic, -except for traces of carbonates. - -Various studies have demonstrated possible methods of estimating the -total amount of organic matter present in meteorites. Wiik ([ref.27]) -has suggested that organics can be estimated by measuring the loss of -weight on ignition. Unfortunately, this method has several disadvantages -and gives very low values. Corrections must be made for weight gains due -to oxidation of reduced constituents, such as FeO, Fe, Ni, and Co, and -for weight losses due to H2O, S, etc. The water loss is exceedingly -difficult to estimate, as part comes from the combustion of organic -hydrogen and part comes from the loss of mineral-bound water. The carbon -also proves difficult to combust completely, and high temperatures (over -1000 deg. C) are required for efficient conversion to CO2. - -In one study the major fraction of organic matter removed proved to have -a carbon content of about 47 percent ([ref.28]). Thus, if all the -meteorite carbon is present as organic matter of approximately this -composition, total organics must be approximately double the carbon -content; that is, 2 percent by weight carbon indicates 4 percent by -weight organic matter. This estimate may be too low, for Mueller -([ref.29]) has extracted a major organic fraction containing only 24 -percent carbon; however, this work has not been confirmed for other -meteorites. - -Briggs and Mamikunian ([ref.26]) have pointed out that only 25 percent -of the organic matter has been extracted, and only about 5 percent of -this has been chemically characterized. Most of this 5 percent is a -complex mixture of hydroxylated aromatic acids together with -hydrocarbons of the aliphatic, napalicyclic, and aromatic series. Small -amounts of amino acids, sugars, and fatty acids are also present. - -Thus far, these chemical analyses point to an abiogenic origin for the -organic matter, and no conclusive evidence exists of biological activity -on the meteorite parent body. Microbiological investigations of samples -of the carbonaceous chondrites have yielded only inconclusive evidence -on the problem of "organized elements." - -Several of these microstructures from different carbonaceous chondrites -are illustrated in a paper by Mamikunian and Briggs ([ref.30]). It has -been difficult to identify the organized structures, and most do not -have morphologies identical to known terrestrial micro-organisms. -However, they may prove to be a variety of mineral grains, droplets of -organic matter and sulfur, as well as a small amount of contaminating -terrestrial debris. - -A comparison between the photographs of the organized elements observed -in the Orgueil and Ivuna meteorites and the synthetic proteinoid -microspheres observed by Fox ([ref.25]) point to similarities between -the two. One inference from this finding is that the organized elements -in carbonaceous chondrites were never alive but, rather, should be -considered as natural experiments in molecular evolution. Also, these -similarities strengthen the belief that the laboratory experiments are -similar to the natural experiments in space. - -In cooperation with the Smithsonian Astrophysical Observatory, NASA has -a network to track meteors in the Midwest (South Dakota, Nebraska, -Kansas, Oklahoma, Iowa, Missouri, and Illinois). Photographs of meteor -trails are used for scientific study, and attempts are made to track and -recover meteorites for examination for traces of organic material of -extraterrestrial origin. - -Fundamental research in terrestrial organic geochemistry has shown that -ancient sediments and drill core samples subjected to organic analysis -contain certain stable biochemical components of past life. This -preserved record is significant not only in studies of early-life -chemical pathways but also in studies of the interaction of organic -matter with the geological factors. Since life on any planetary body -will interact with the soil, or surface material, it is of interest to -understand the relationship. - - - CONCEPTS FOR DETECTION OF EXTRATERRESTRIAL LIFE - -It is not possible to present completely convincing evidence for the -existence of extraterrestrial life. The problem often reduces to -probabilities and to estimates of observational reliability. In almost -all cases the evidence is optimistically considered strongly suggestive -of--or, at the worst, not inconsistent with--the existence of -extraterrestrial life. Alternatively, there is a pessimistic view that -the evidence advanced for extraterrestrial life is unconvincing, -irrelevant, or has another, nonbiological explanation. - -In studies of the laboratory synthesis of life-related compounds and its -significance concerning the origin of life, several results seem to -suggest that organochemical synthesis is a general process, occurring -perhaps on all planets which retain a reducing atmosphere. The -temperature ranges must be such that precursors and reaction products -are not thermally dissociated. The reaction rates for the synthesis of -more complex organic molecules diminish to a negligible value when the -temperature range is below 100 deg. C. - -Besides the planetary parameter of temperature, an even more fundamental -necessity for a living state exists--a liquid solvent system. For -terrestrial life forms, water serves this purpose. Water has this and -other properties of biological significance because of hydrogen bonding -between adjacent molecules in the liquid state. - -Ultraviolet radiation could serve as an extraterrestrial energy source -for organic synthesis. Research shows that, while an atmosphere is -important, living systems can survive a wide range of ambient pressures -and are little affected by a wide range of magnetic field strengths. - -Oxygen is not a prerequisite for all living systems. While it is -sometimes concluded that free oxygen is needed for all but the simplest -organisms, less efficient metabolic processes coupled with higher food -collection efficiency--or a more sluggish metabolism--would seem to do -just as well. Earth is the only planet in the solar system on which -molecular oxygen is known to be present in large amounts. Since plant -photosynthesis is the primary source of atmospheric oxygen, it seems -safe to infer that no other planet has large-scale plant photosynthesis -accompanied by the production of oxygen. - -The possibility of the existence of extraterrestrial life raises the -important question of man's being able to detect it. Research on -extraterrestrial life detection is predicated on the ability to develop -ways to detect it even when the living systems are based on principles -entirely different from those on Earth. - -The substitution of various molecules for those of known biological -significance to living organisms as we know them has been investigated; -the substitution of NH2 for OH in ammonia-rich environments leads to a -diverse, and biologically very promising, chemistry. The hypothesis that -silicon may replace carbon does not support the construction of -extraterrestrial genetics based on silicon compounds. (Silicon compounds -participate in redistribution reactions which tend to maximize the -randomness of silicon bonding, and the stable retention of genetic -information over long time periods is thus very improbable.) - -Evidence relevant to life on Mars has been summarized by Sagan (ch. 1 of -[ref.10]): - - _The Origin of Life_ - - In the past decade, considerable advances have been made in our - knowledge of the probable processes leading to the origin of - life on Earth. A succession of laboratory experiments has shown - that essentially all the organic building blocks of contemporary - terrestrial organisms can be synthesized by supplying energy to - a mixture of the hydrogen-rich gases of the primitive - terrestrial atmosphere. It now seems likely that the laboratory - synthesis of a self-replicating molecular system is only a short - time away from realization. The syntheses of similar systems in - the primitive terrestrial oceans must have occurred--collections - of molecules which were so constructed that, by the laws of - physics and chemistry, they forced the production of identical - copies of themselves out of the building blocks in the - surrounding medium. Such a system satisfies many of the criteria - for Darwinian natural selection, and the long evolutionary path - from molecule to advanced organism can then be understood. Since - nothing except very general primitive atmospheric conditions and - energy sources are required for such syntheses, it is possible - that similar events occurred in the early history of Mars and - that life may have come into being on that planet several - billions of years ago. Its subsequent evolution, in response to - the changing Martian environment, would have produced organisms - quite different from those which now inhabit Earth. - - _Simulation Experiments_ - - Experiments have been performed in which terrestrial - micro-organisms have been introduced into simulated Martian - environments, with atmospheres composed of nitrogen and carbon - dioxide, no oxygen, very little water, a daily temperature - variation from +20 deg. to -60 deg. C, and high ultraviolet fluxes. - It was found that in every sample of terrestrial soil used there - were a few varieties of micro-organisms which easily survived on - "Mars." When the local abundance of water was increased, - terrestrial micro-organisms were able to grow. Indigenous - Martian organisms may be even more efficient in coping with the - apparent rigors of their environment. These findings underscore - the necessity for sterilizing Mars entry vehicles so as not to - perform accidental biological contamination of that planet and - obscure the subsequent search for extraterrestrial life. - - _Direct Searches for Life on Mars_ - - The early evidence for life on Mars--namely, reports of vivid - green coloration and the so-called "canals"--are now known to be - largely illusory. There are three major areas of contemporary - investigation: visual, polarimetric, and spectrographic. - - As the Martian polar ice cap recedes each spring, a wave of - darkening propagates through the Martian dark areas, sharpening - their outlines and increasing their contrast with the - surrounding deserts. These changes occur during periods of - relatively high humidity and relatively high daytime - temperatures. A related dark collar, not due to simple dampening - of the soil, follows the edge of the polar cap in its - regression. Occasional nonseasonal changes in the form of the - Martian dark regions have been observed and sometimes cover vast - areas of surface. - - Observations of the polarization of sunlight reflected from the - Martian dark areas indicate that the small particles covering - the dark areas change their size distribution in the spring, - while the particles covering the bright areas _do_ not show any - analogous changes. - - Finally, infrared spectroscopic observations of the Martian dark - areas show three spectral features which, to date, seem to be - interpretable only in terms of organic matter, the particular - molecules giving rise to the absorptions being hydrocarbons and - aldehydes. [However, see p. 7 and Rea et al. ([ref.9]).] - - Taken together, these observations suggest, but do not - conclusively prove, that the Martian dark areas are covered with - small organisms composed of familiar types of organic matter, - which change their size and darkness in response to the moisture - and heat of the Martian spring. We have no evidence either for - or against the existence of more advanced life forms. There is - much more information which _can_ be garnered from the ground, - balloons, Earth satellites, Mars flybys, and Mars orbiters, but - the critical tests for life on Mars can only be made from - landing vehicles equipped with experimental packages.... - -Results of Kaplan et al. ([ref.31]) indicate that Mars has no detectable -oxygen, but does contain small amounts of water vapor, more abundant -carbon dioxide, possibly a large surface flux of solar ultraviolet -radiation, and estimated daily temperature variations of 100 deg. C at -many latitudes. Studies have shown that terrestrial micro-organisms can -survive these extremely harsh environments. Furthermore, a variety of -physiological and ecological adaptations might enable the biota to -survive the low nighttime temperatures and intracellular ice -crystallization. - -Less evidence is available to support the possibility of -extraterrestrial life on other planets. The Moon has no atmosphere, and -extremes of temperature characterize its surface. However, the Moon -could have a layer of subsurface permafrost beneath which liquid water -might be trapped. The temperatures of these strata might be biologically -moderate. - -Studies by Davis and Libby ([ref.32]) on the atmosphere of Jupiter -support the possibility of the production of organic matter in its -atmosphere in a manner analogous to the processes which may have led to -the synthesis of organic molecules in the Earth's early history. It is -difficult to assess the possibility that life has evolved on Jupiter -during the 4- or 5-billion-year period in which the planet has retained -a reducing atmosphere. - -The question of extraterrestrial life and of the origin of life is -interwoven. Discovery of the first and analysis of its nature may very -well elucidate the second. - -The oldest form of fossil known today is that of a microscopic plant -similar in form to common algae found in ponds and lakes. Scientists -know that similar organisms flourished in the ancient seas over 2 -billion years ago. However, since algae are a relatively complex form of -life, life in some simpler form could have originated much earlier. -Organic material similar to that found in modern organisms can be -detected in these ancient deposits as well as in much older Precambrian -rocks. - -Although the planets now have differing atmospheres, in their early -stages the atmospheres of all the planets may have been essentially the -same. The most widely held theory of the origin of the solar system -states that the planets were formed from vast clouds of material -containing the elements in their cosmic distribution. - -It is believed that the synthesis of organic compounds preceding the -origin of life on Earth occurred before its atmosphere was transformed -from hydrogen and hydrides to oxygen and nitrogen. This theory is -supported by laboratory experiments of Calvin ([ref.16]), Miller -([ref.33]), and Oro ([ref.34]). - -The Earth's present atmosphere consists of nitrogen and oxygen in -addition to relatively small amounts of other gases; most of the oxygen -is of biological origin. Some of the atmospheric gases, in spite of -their low amounts, are crucial for life. The ultraviolet-absorbing ozone -in the upper atmosphere and carbon dioxide are examples of such gases. - -Significant in the search for extraterrestrial life are the data (e.g., -planet's temperature) transmitted by Mariner II, which was launched from -Cape Canaveral on August 27, 1962, and flew past Venus on December 14, -1962. Mariner II's measurements showed temperatures on the surface of -Venus of the order of 800 deg. F, too hot for life as known on Earth. - -The question "Is life limited to this planet?" can be considered on a -statistical basis. Although the size of the sample (one planet) is -small, the statistical argument for life elsewhere is believed by many -to be very strong. While Mars is generally considered the only other -likely habitat of life in our solar system, Shapley ([ref.35]) has -calculated that more than 100 million stars have planets sufficiently -similar in composition and environment to Earth to support life. Of -course, yet unknown factors may significantly reduce or even eliminate -this probability. - - - SPACECRAFT STERILIZATION - -The search for extraterrestrial life with unmanned space probes requires -the total sterilization of the landing capsule and its contents. -Scientists agree that terrestrial organisms released on other planets -would interfere with exobiological explorations (refs. -[ref.36]-[ref.43]). Any flight that infects a planet with terrestrial -life will compromise a scientific opportunity of almost unequaled -proportions. Studies on microbiological survival in simulated deep-space -conditions (low temperature, high ultraviolet flux, and low dose levels -of ionizing radiation) indicate that these conditions will not sterilize -contaminated spacecraft (refs. [ref.44]-[ref.48]). Furthermore, many -terrestrial sporeformers and some vegetative bacteria, especially those -with anaerobic growth capabilities, readily survive in simulated Martian -environments (refs. [ref.49]-[ref.54]). It has been estimated that a -single micro-organism with a replication time of 30 days could, in 8 -years of such replication, equal in number the bacterial population of -the Earth. This potential could result not only in competition with any -Martian life, but in drastic changes in the geochemical and atmospheric -characteristics of the planet. To avoid such a disaster, certainly the -first, and probably many succeeding landers on Mars, must be -sterile--devoid of terrestrial life ([ref.55]). Since the space -environment will not in itself kill all life aboard, the lander must -leave the Earth in a sterile condition. - -The sterility of an object implies the complete absence of life. The -presence of life or the lack of sterility may be proven; but the absence -of life or sterility cannot be proven, for the one viable organism that -negates sterility may remain undetected. Many industrial products which -must be guaranteed as sterile cannot be tested for sterility in a -nondestructive manner. A similar situation exists in determining the -sterility of a spacecraft. Certification of sterility--based on -experience with the sterilizing process used, knowledge of the kinetics -of the death of micro-organisms, and computation of the probability of a -survivor from assays for sterility--is the only accurate approach to -defining the sterility of such treated items. - -Macroscopic life can be readily detected and kept from or removed from -the spacecraft, but the detection and removal of microscopic and -submicroscopic life is an extremely difficult task. The destruction of -micro-organisms can be achieved by various chemical and physical -procedures. Sterilizing agents have been evaluated not only for their -ability to kill microbial life on surfaces and sealed inside components, -but also for the agents' effects on spacecraft reliability as well -(refs. [ref.56]-[ref.59]). Of the available agents, only heat and -radiation will penetrate solid materials. Radiation is expensive, -hazardous, difficult to control, and apparently damages more materials -than does heat. Heat, therefore, has been selected as the primary method -of spacecraft sterilization and will be used, except in specific -instances where radiation may prove to be less detrimental to the -reliability of critical parts ([ref.60]). - -The sterilization of spacecraft is a difficult problem if flight -reliability is not to be impaired. The development of heat-resistant -parts will enable the design and manufacture of a heat-sterilizable -spacecraft. Without careful microbiological monitoring of manufacture -and assembly procedures, many bacteria could be trapped in parts and -subassemblies. To permit sterilization at the lowest temperature-time -regimen that will insure kill of all organisms, the microbiological load -inside all parts and subassemblies must be held to a minimum. - -The role of industrial clean rooms in reducing the biological load on -spacecraft is currently being defined. NASA-supported studies indicate -that biological contamination in industrial clean rooms for extended -time periods is about 1 logarithm less (tenfold reduction), compared -with conditions in a well-operated microbiological laboratory -([ref.61]). With the use of clean-room techniques and periodic -decontamination by low heat cycles or ethylene oxide treatment, it -should be possible to bring a spacecraft to the point of sterilization -with about 10^6 organisms on board ([ref.60]). - -The sterilization goal established for Mars landers is a probability of -less than 1 in 10 000 (10^-4) that a single viable organism will be -present on the spacecraft. Laboratory studies of the kinetics of -dry-heat kill of resistant organisms show that at 135 deg. C the number -of bacterial spores can be reduced 1 logarithm (90 percent) for every 2 -hours of exposure (refs. [ref.58] and [ref.62]). The reduction in -microbial count needed is the logarithm of the maximum number on the -spacecraft (10^6) plus the logarithm of the reciprocal of the -probability of a survivor (10^4), or a total of 10 logarithms of -reduction in microbial count. Thus, with an additional 2 logarithms -added as a safety factor, a total of 12 logarithms of reduction in count -has been accepted as a safe value which can be achieved by a dry-heat -treatment of 135 deg. C for 24 hours. This is the heat cycle that is -currently under study and being developed for use in spacecraft -sterilization ([ref.60]). However, other heat treatments at temperatures -as low as 105 deg. C for periods of 300 hours or longer are under study -([ref.63]). - -Based on results to date, it is reasonable to believe that a full -complement of heat-sterilizable hardware will be available when needed -for planetary exploration. Every effort is being made to improve the -state of the art to a point where spacecraft can not only withstand -sterilization temperatures, but will be even more reliable than the -present state-of-the-art hardware that is not heated. - - - - - chapter 3 - -_Environmental Biology_ - - - BIOLOGICAL EFFECTS OF WEIGHTLESSNESS AND ZERO GRAVITY - -High priority has been given to studies of weightlessness. Gravity is -one of the most fundamental forces that acts on living organisms, and -all life on Earth except the smallest appears to be oriented with -respect to gravity, although certain organisms are more responsive to it -than others. The gravity force on Earth is 1 g, but this force may be -experimentally varied from zero g, or weightlessness, to many thousands -of g's. - -Zero gravity or decreased gravity occurs during freefall, in parabolic -trajectory, or during orbit around the Earth. Gravitational force -decreases by the square of the distance away from the Earth's center. It -is reduced about 5 percent at about 200 nautical miles' altitude. -Gravitational force greater than 1 g can be obtained by acceleration, -deceleration, or impact. It also can be increased by using a centrifuge -which adds a radial acceleration vector to the 1 g of Earth. - -On the ground, the biological effects of gravity have been studied at 1 -g, and experimentally, forces of many g have been produced. In addition, -modifications of the effects of the 1-g force have been induced by -suspension of the organism in water or by horizontal immobilization of -an erect animal such as man. The biological effects of such modification -have been of significant value in understanding some of the possible -consequences of human exposure to the zero-g environment of space. - -Weightlessness in an Earth-orbiting satellite occurs when the continuous -acceleration of Earth's gravity is exactly counterbalanced by the -continuous radial acceleration of the satellite. In such a weightless -state, organisms are liberated from their natural and continuous -exertion against 1 g, but this liberation may carry with it certain -serious physical penalties. - -Some of the physical processes which probably have the greatest -biological effects are (1) convective flow of fluid, e.g., protoplasmic -streaming, transport of nutrient materials, oxygen, waste products, and -CO2 from the immediate environment of the cell, and (2) sedimentation -occurring within cells; substances of higher density sediment in a -gravitational field, and those of lighter density rise. A separation of -particles of different densities probably occurs. The removal of gravity -would change a distribution of particles like mitochondria by 10 percent -([ref.64]). - -Gravity has effects on the physical processes involved in mitosis and -meiosis. Study under weightlessness might contribute to our -understanding of the general cellular information-relay process. - -A gravitational effect is known in the embryonic development of the frog -_Rana sylvatica_. After fertilization, the eggs rotate in the -gravitational field so that the black animal hemisphere is uppermost. -Development becomes abnormal if this position is disturbed. If the egg -is inverted following the first cleavage and held in this position, two -abnormal animals result, united like Siamese twins. This phenomenon -appears to be related to the gravitational separation of low- and -high-density components of the egg. The size of the egg is about 1 to 2 -mm and is suspended in water of about the same density. This system is -very sensitive to gravity; and, under weightlessness, the separation of -different density components might be irregular, leading to aberrant -development. When certain aquatic insect eggs are inverted, subsequent -development results in shortened abnormal larvae. - -The directional growth of plant shoots and plant roots is probably due -to this sedimentation phenomenon, particularly the effect on movement of -auxins ([ref.65]). - -Free convection flow is a major transport process, and under its -influence the mixing of substances is much more effective than when -diffusion operates alone. Free convection flow is a macroscopic -phenomenon which increases not only with g, but varies also -approximately with the five-fourths power of the bulk concentration -involved. Whether or not convection is important at the microscopic -level remains an experimentally unsolved question. The Grashoff number -limits free convection to the macroscopic domain. It would appear in -weightlessness that the contribution of free convective flow would be -small and that only diffusion should occur. This phenomenon would cause -equilibration to occur much more slowly than that occurring with free -convection and diffusion. The absence of convective transfer raises a -problem as to how nutrients may be obtained and waste products removed -in living cells during weightlessness. In a liquid substrate, nutrients -and oxygen would be depleted, and waste products would accumulate around -the cell. - -Absence of gravity may have far-reaching consequences in the homeostatic -aspects of cell physiology. The outstanding characteristics of living -cells which are most likely to be influenced by the absence of gravity -are the ability of the cell to maintain its cytoplasmic membrane in a -functional state, the capacity of the cell to perform its normal -functions during the mitotic cycle, and the capacity of the cytoplasm to -maintain the constant reversibility of its sol-gel system ([ref.66]). - -Two-phase systems, e.g., air-in-water and air-in-oil, possess entirely -different characteristics at zero g than at 1 g. These physical -differences in phase interaction could well be suspected of interfering -with the orientation and flow pattern of cell constituents, thus -hindering the cellular processes involved in the movement, metabolism, -and storage of nutrients and waste. - -On the basis of theoretical calculations, weightlessness can be expected -to have some effect even on one individual cell if its size exceeds 10 -microns in diameter ([ref.64]). Cell colonies might be affected. In -larger cells there may be a redistribution of enzyme-forming systems -which give rise to polarization. The low surface tension of the cell -membrane lends itself to hydrostatic stress distortion, implying an -alteration in permeability and thus an almost certain alteration of cell -properties under low gravity conditions. - -Another aspect of gravity that affects the growth and development of -living organisms is the directionality of the gravitational field. In -fact, some plants are so sensitive that they are able to direct their -growth with as little stimulus as a 1x10^-6 gravitational field. -Investigations of plant growth in altered gravitational fields are -underway at Argonne National Laboratory and Dartmouth College. - -The Argonne Laboratory has designed and developed a 4-pi, or -omnidirectional, clinostat. By rotating a plant so that the force of -gravity is distributed evenly over all possible directions, the -directional effects of gravity are eliminated, simulating some aspects -of the zero-g state. It was shown that certain plants grew more slowly -and had fewer and smaller leaves, while others had about 25 percent -greater replication of fronds and had greater elongation of certain -plant parts. It will be extremely interesting to compare these effects -under zero-g conditions in orbiting spacecraft. - -The effect of gravity in transporting growth hormones in plants has been -demonstrated at Dartmouth College using radiocarbon-labeled growth -hormones. Plant geotropisms and growth movements have been studied and -biosatellite experiments developed. - -Anatomy is considered a derivative adaptation to gravity ([ref.67]). A -large background of plant research exists on the effect of orientation -on plant responses. Information from clinostat experiments is considered -susceptible of extrapolation to low gravity conditions because the -threshold period for gravitational triggering is relatively long. - -Once over critical minimum dimensions, the major effects of low gravity -would be assumed to occur in those heterocellular organisms that develop -in more or less fixed orientation with respect to terrestrial gravity -and which respond to changes in orientation with relatively long -induction periods; these are the higher plant orders. On the other -extreme are the complex primates which respond rapidly, but whose -multiplicity of organs and correlative mechanisms are susceptible to -malfunction and disorganization. It may be suggested that the -heterocellular lower plants and invertebrates will be less affected. -Perturbations of the environment to which the experimental organism is -exposed must be limited or controlled to reduce uncertainties in -interpretation of the results. At the same time, the introduction of -known perturbations may assist in isolating the effects due solely to -gravity. Study of _de novo_ differentiation and other phenomena -immediately after syngamy may be of particular importance. Study of -anatomical changes after exposure of the organism to low gravity is -important. - - - BIOLOGICAL EFFECTS OF SPACE RADIATION[1] - - [1] This section includes part of the Summary of the Panel on Radiation - Biology of the Environmental Biology Committee Space Science Board, - NAS/NRC (1963), and results of research by the Bioscience Programs, - NASA. - - -Radiation sources in space are of three types: galactic cosmic -radiation, Van Allen belts, and solar flares with an intense proton -flux. Cosmic radiation has higher energy levels than radiation produced -by manmade accelerators. - -The Panel on Radiation Biology, while recognizing the need for -radiobiological studies of an applied nature with reference to manned -flight programs, stated that it would be shortsighted for the United -States to confine its efforts to the solution of immediate problems -since, in the long run, successful exploration of space will be aided by -the contributions of basic research. Both the immediate biological -research program and the continuing program for basic studies should be -built upon the large body of existing knowledge of radiation effects. -The attitude that all radiobiological experiments need be repeated in -the space environment should be resolutely rejected. Since fundamental -radiobiology cannot be performed easily in space, it has been -recommended that, wherever possible, these investigations be carried out -in ground laboratories in preference to flying laboratories. - -Space environment does vary from the terrestrial environment, but the -variations are not so great as to lead to the expectation of strikingly -different biological effects of radiation in space. However, it is -conceivable that radiations whose effects are well known under -terrestrial conditions may have some unsuspected biological effects when -combined with unusual features of the space environment: e.g., zero g. -Previous space radiobiological studies have depended solely on very low -and inaccurately measured doses of ambient space radiation. It has been -difficult to distinguish between the observed response levels and the -random noise; thus, experiments have been inconclusive. - - -Biological Effects of Heavy Ions and Mesons - -The biological effects of heavy ions (especially Z>2) and mesons are of -specific interest to space radiobiology. - - -Controlled Radiobiological Experiments in Space - -There is the remote possibility that the radiobiological response may be -modified by factors as yet unknown and perhaps not susceptible to -terrestrial study. Experiments have been designed to settle this matter -including the exposure of biological materials during space flight which -meet the following criteria of reliability: (1) the use of well-known -biological systems, e.g., mutation induction or chromosome breakage; (2) -the use of a sufficient number of individuals in the experiment to -guarantee statistical precision on the results; (3) the exposure of the -system to known quantities and qualities of radiation; (4) the use of -adequate controls. - -High-altitude balloon ascents of the 1930's initiated study of the -biological effects of cosmic rays. They were limited to the exploration -of secondary cosmic radiation effects. After World War II, the research -extended to the use of V-2 rockets fired from the White Sands Proving -Ground. Interest returned to balloons and a significant program was -underway by 1950, first using mice and then hamsters, fruit flies, cats, -and dogs. These flights gave no evidence of radiation damage. However, -it was realized that the flights were too far south to obtain a -significant exposure, and more northerly flights began in 1953. Mice and -guinea pigs were flown on these later flights. Chase ([ref.68]) showed -the most unequivocal results to that time, a statistically significant -increase in light hairs on black animals and the streaks of white hair -up to 10 times wider than expected. Brain lesions were detected in the -guinea pigs flown on Man High in 1957. Many other types of biological -material were sent aloft in an effort to further corroborate existing -information and to investigate genetic and developmental effects of -cosmic radiation. - -From the earlier V-2 rocket flights to the Jupiter missile launchings of -the monkeys Able and Baker, cosmic-ray research was continued, but the -short flight durations of these vehicles did not provide substantial -information. The USAF Discoverer satellite program has given impetus to -cosmic-ray research and provided for longer "staytimes." - -It has been difficult to separate radiation effects from other -space-flight factors: therefore, some of the alterations observed are -still subject to debate. Vibration, acceleration, and weightlessness -appear to be the three most important additional parameters. -Measurements of radiation dosage have been made by chemical and -photographic dosimetry, ion chambers, and biological dosimetry. All -evidence to date indicates that radiation exposure levels are not -hazardous to man at present orbital altitudes up to 200 nautical miles. -Most biological materials flown so far have been for the express purpose -of investigating space-radiation levels and effects. The biological -materials have ranged from tissue cultures to entire organisms and from -phage and bacterial cells to man. The studies have required much of the -space and weight resources allotted biology by the U.S.S.R. and the -United States. They have been accompanied by ground-based controls. - -The Vostok series provided the following data: - - (1) A small, but statistically significant, increase was observed in - the percentage of chromosome aberrations in the rootlet cells of - air-dried wheat and pea seeds after germination. In this case - only, the increase did not depend on flight duration. - (2) Lysogenic bacteria exhibited an increase of genetic alterations - and increased phage production. Length of flight was associated - with increased bacteriophage production by the lysogenic bacteria. - There was an increase of recessive lethals coupled with - nonconvergence of chromosomes (sex linked) in the fruit fly. A - stimulation of cell division in wheat and pea seeds was observed. - Cultures of human cells exposed to space-flight factors did not - differ significantly from terrestrial controls with respect to - such indicators as proliferation rate, percentage of mortality and - morphological, antigenic, and cultural properties. Repeated - flights of the identical HeLa cells revealed that there was a - longer latent period for restoration of growth capacity than in - cells carried into space once or not flown at all. - (3) The most definite radiation effects observed were only revealed in - genetic tests. No harmful influence on those characteristics - affecting the viability of the organism has been discovered. - -The Air Force Discoverer series launched from the west coast had a few -successful flights incorporating organisms. With severe environmental -stress and long recovery times, data on radiation exposure were -equivocal up to Discoverer XVII and XVIII when cultures of human tissue -were flown, recovered, and assessed for radiation exposure effects. -Comparison with ground-based controls revealed no measurable -differences. - -Radiation dosimetry from the Mercury series established that minimal -exposures were encountered at those orbital altitudes. A typical example -is the MA-8 flight of W. M. Schirra, Jr., during which the body surface -dosage was less than 30 millirads. - -NASA has supported fundamental radiation studies at the Oak Ridge -National Laboratory and the Lawrence Radiation Laboratory. Emphasis has -been placed on the biological effects of high-energy proton radiation -and particulate radiation from accelerators. - -At the NASA Ames Research Center extensive fundamental studies are being -carried out on the effects of radiation, especially in the nervous -system. It has been demonstrated that deposits accumulate in the brain -following exposure to large doses of ionizing particle radiation as well -as after X-irradiation. These deposits, referred to as a "chemical -lesion," result from an accumulation of glycogen. The formation of these -deposits during exposure to large doses of X-irradiation was not -increased in environments of 99.5 percent oxygen and increased -atmospheric pressure. - - - SIMULATION OF PLANETARY (MARTIAN) ENVIRONMENTS - -Attempts have been made to simulate to some degree the various -parameters of the Martian environment, such as atmospheric composition, -pressure, radiation flux, temperatures, and the day-night as well as -seasonal cycles. Certain factors for Mars cannot yet be simulated, such -as soil composition, gravitational field, magnetic field, and electrical -field. - -Caution is required in interpreting all simulation experiments. How -Earth organisms respond to simulated Martian environments probably has -nothing to do with life on Mars, but these experiments may show whether -or not anything in the environment of Mars makes life as we know it -impossible. We must expect that on Mars, life will have evolved and have -adapted over long periods of time under conditions which are quite -different from conditions on Earth. The simulation experiments also -provide some information about the possibility of contaminating the -planet Mars, or any planet, with organisms from Earth. In addition, they -give us some clues about the possibilities of adaptation and evolution -of life under these conditions. - -From an evolutionary point of view, if life has developed on Mars, we -expect it to have evolved at least to a microbial stage. On Earth, -micro-organisms are the most ubiquitous and numerous forms of life. This -fact should be considered in studying extraterrestrial bodies. - -Micro-organisms have been selected as the best test organisms, and -bacteria and fungi have been used because they are durable and easy to -grow. Also, because of their rapid growth, many generations can be -studied in a relatively short period of time. The organisms include -chemoautotrophic bacteria, which are able to synthesize their cell -constituents from carbon dioxide by energy derived from inorganic -reactions; anaerobic bacteria, which grow only in the absence of -molecular oxygen; photoautotrophic plants such as algae, lichens, and -more complex seed plants; and small terrestrial animals. - -Organisms have been collected from tundra, desert, hot springs, alpine, -and saline habitats to obtain species with specialized capabilities to -conserve water, balance osmotic discrepancies, store gases, accommodate -to temperature extremes, and otherwise meet stresses. An attempt is made -in these simulation experiments to extend these processes across the -possible overlapping microenvironments which Earth and Mars may share. - -Scientists have developed various special environmental simulators, -including "Mars jars" and "Marsariums." These have made possible -controlled temperatures, atmospheres, pressures, water activities, and -soil conditions for duplicating assumed Martian surface. A complex -simulator, developed by Young et al. ([ref.52]), reproduces the -formation of a permafrost layer with some water tied up in the form of -ice beneath the soil surface. This simulator serves as a model to study -the wave of darkening, thus supporting the hypothesis that the -pole-to-equator wave of darkening is correlated with the availability of -subsurface water. The simulator is a heavily insulated 2-cu-ft capacity -chamber with an internal pressure of 0.1 atm. The chamber contains a -soil mixture of limonite and sand and an atmosphere of carbon dioxide -and nitrogen. With the use of a liquid nitrogen heat exchanger at one -end and an external battery of infrared lamps at the other end, the -temperature simulates that of Mars from pole to equator. Thermocouples -throughout the soil monitor the temperatures in the chamber. - -Zhukova and Kondratyev ([ref.69]) designed a structure measuring -100x150x180 cm. Micro-organisms were placed at the surface of a copper -bar made in a special groove separated by glass cloth. Copper was -selected as one of the best heat-conduction materials permitting a rapid -change of temperature. The lower end of the bar was immersed into a -mixture of dry ice and ethyl alcohol, which made it possible to create a -temperature of -60 deg. C. Heating was performed by an incandescent -spiral. - -As the knowledge concerning the Martian environment becomes more -refined, scientists can more accurately simulate this environment under -controlled conditions in the laboratory. Determination of the effects of -the Martian environment on Earth organisms will permit better -theorization on the forms of life we might find on Mars and will permit -us to estimate the potential survival of Earth contaminants on Mars. - -However, until the environmental conditions of Mars are defined more -accurately, the experiments must be changed continually to fit newly -determined conditions. Therefore, existing simulation data are made less -valid for comparison. The data resulting from the simulation experiments -for Mars have been compiled in table II, and the experiments are -summarized below. - -The earliest simulation studies were carried out by the Air Force, and -the studies during the past 6 years have been supported by NASA. -Recently, these studies have received less support or have been -terminated in favor of critical studies on the effects of biologically -important environmental extreme factors on Earth organisms. These -critical studies permit establishing the extreme environmental factor -parameters in which Earth life can grow or survive. These data will have -valuable application to the consideration of life on any planet, to the -design of life-detection instruments, to the sterilization of space -vehicles, and to the problem of contamination of planets. - -Some exploratory experimental studies are in progress to study the -capabilities of organisms to grow under the assumed conditions on -Jupiter. These include studies at high pressure with liquid ammonia, -methane, and other reducing compounds. - -Early experiments simulating Martian conditions using soil bacteria were -carried out by Davis and Fulton ([ref.70]) at the Air Force School of -Aviation Medicine, San Antonio, Tex. Mixed populations of soil bacteria -were put in "Mars jars" with the following conditions: 65-mm Hg -pressure, 1 percent water or less, nitrogen atmosphere, sandstone-lava -soil, and a temperature day-night cycle of +25 deg. to -25 deg. C. The -moisture was controlled by desiccating the soil and adding a given -amount of water. Experiments, conducted up to 10 months, demonstrated -that obligate aerobes died quickly. The anaerobes and sporeformers -survived. Although a small increase in the total number of organisms -indicated growth, the increases in the number of bacteria may have been -due to breaking up clumps of dirt. - -Roberts and Irvine ([ref.71]) reported that, in a simulated Martian -environment, colony counts of a sporeforming bacterium, _Bacillus -cereus_, increased when 8 percent moisture was added. Moisture was -considered more important than temperature or atmospheric gases inasmuch -as a simulated Martian microenvironment containing 8 percent moisture -permitted germination and growth of endospores of _Clostridium -sporogenes_. Increases in colony counts of _Bacillus cereus_ appeared to -be influenced by temperature cycling ([ref.72]). - - - Table II.--_Survival and Growth of Organisms in Simulated Planetary - (Martian) Environments_ - - ------------------------------------------------------------------ - Species Survival, Moisture Temperature, - months deg. C - - ------------------------------------------------------------------ - Conditions on Mars: 14 mu +-7 mu -70 to +30 - ------------------------------------------------------------------ - Anaerobic 6 Low, -60 to +20 - sporeformers (CaSO4) - _Clostridia_, - _Bacillus - planosarcina_ - ------------------------------------------------------------------ - Anaerobic 6 Low, -60 to +20 - nonsporeformers (CaSO4) - _Pseudomonas_, - _Rhodopseudomonas_ - ------------------------------------------------------------------ - Anaerobes Growth Very wet -75 to +25 - _Aerobacter - aerogenes_, - _Pseudomonas sp._ - ------------------------------------------------------------------ - _Clostridium_, 10 1 -25 to +25 - _Corynebacteria_ percent - "Thin short rod" or less - ------------------------------------------------------------------ - _Bacillus cereus_ 2 0.5 -25 to +25 - percent - soil - ------------------------------------------------------------------ - _Clostridium sporogenes_ 1 8.4 -25 to +25 - (growth) percent - ------------------------------------------------------------------ - _Clostridium botulinum_ 10 Lyophilized -25 to +25 - - ------------------------------------------------------------------ - _Klebsiella pneumoniae_ 6 Lyophilized -25 to +25 - - ------------------------------------------------------------------ - _Bacillus subtilis_ var. 4 2 percent -25 to +25 - _globigii_ - ------------------------------------------------------------------ - _Sarcina aurantiaca_ 4 0.5 percent -25 to +25 - - ------------------------------------------------------------------ - _Clostridium tetani_ 2 or less 1 percent -60 to +25 - ------------------------------------------------------------------ - _Aspergillus niger_ Over 6 hr Very dry -60 to +25 - - - - ------------------------------------------------------------------ - _Aspergillus oryzae_ Over 6 hr Very dry -60 to +25 - ------------------------------------------------------------------ - _Mucor plumbeus_ Over 6 hr Very dry -60 to +25 - ------------------------------------------------------------------ - _Rhodotorula rubra_ Over 6 hr Very dry -60 to +25 - ------------------------------------------------------------------ - Pea, bean, tomato, rye, 0.3 Moist +25 - sorghum, rice. - ------------------------------------------------------------------ - Winter rye 0.6 Moist -10 to +23 - ------------------------------------------------------------------ - - - Table II.--_Survival and Growth of Organisms in Simulated Planetary - (Martian) Environments_ - - ---------------------------------------------------------------------- - Species Atmospheric N2, CO2, Substrate - pressure, percent percent - mm Hg - ---------------------------------------------------------------------- - Conditions on Mars: 85, 3 to 30 - 25+-15, 11 - ---------------------------------------------------------------------- - Anaerobic 76 95 5 Air-dried - sporeformers soil - _Clostridia_, - _Bacillus - planosarcina_ - ---------------------------------------------------------------------- - Anaerobic 76 95 5 Air-dried - nonsporeformers soil - _Pseudomonas_, - _Rhodopseudomonas_ - ---------------------------------------------------------------------- - Anaerobes 760 100 (?) Difco - _Aerobacter infusion - aerogenes_, broth - _Pseudomonas sp._ - ---------------------------------------------------------------------- - _Clostridium_, 65 100 (?) Soil - _Corynebacteria_ - "Thin short rod" - ---------------------------------------------------------------------- - _Bacillus cereus_ 65 94 2.21 Sandstone - soil - - ---------------------------------------------------------------------- - _Clostridium sporogenes_ 65 94 2 Enriched - soil - ---------------------------------------------------------------------- - _Clostridium botulinum_ 65 95 0 to Lava soil - 0.5 - ---------------------------------------------------------------------- - _Klebsiella pneumoniae_ 65 95 0 to Lava soil - 0.5 - ---------------------------------------------------------------------- - _Bacillus subtilis_ var. 85 95 0.3 Media - _globigii_ - ---------------------------------------------------------------------- - _Sarcina aurantiaca_ 85 95 0.3 Desert - soil - ---------------------------------------------------------------------- - _Clostridium tetani_ 85 95 0.3 Soil - ---------------------------------------------------------------------- - _Aspergillus niger_ 76 95.5 0.25 Glass - cloth on - copper - bar - ---------------------------------------------------------------------- - _Aspergillus oryzae_ 76 95.5 0.25 Do. - ---------------------------------------------------------------------- - _Mucor plumbeus_ 76 95.5 0.25 Do. - ---------------------------------------------------------------------- - _Rhodotorula rubra_ 76 95.5 0.25 Do. - ---------------------------------------------------------------------- - Pea, bean, tomato, rye, 75 100 0 Filter - sorghum, rice. paper - ---------------------------------------------------------------------- - Winter rye 76 98 0.24 Soil - ---------------------------------------------------------------------- - -Studies of the effects of simulated Martian environments on sporeforming -anaerobic bacteria were carried out by Hawrylewicz et al. ([ref.49]). -They showed that the encapsulated facultative anaerobe, _Klebsiella -pneumoniae_, survived under simulated Martian atmosphere for 6 to 8 -months, but were less virulent than the freshly isolated organisms. -Spores of the anaerobe _Clostridium botulinum_ survived 10 months in the -simulator. Hagen et al. ([ref.53]) found that the addition of moisture -to dry-simulated Martian soil did not improve the survival of _Bacillus -subtilis_ or _Pseudomonas aeruginosa_. _Bacillus cereus_ spores -survived, with added organic medium plus moisture, but no germination of -the spores resulted. - -Hawrylewicz et al. ([ref.49]) put rocks from Antarctica bearing various -lichens in simulated Martian conditions in a large desiccator. They -found that the algal portion of a lichen, _Trebouxia erici_, showed only -slight resistance to the Martian environment. They also pointed out the -effect moisture had on the physical condition of lichens. The -undersurface of a lichen has great water-absorbing capability, and the -slightest amount of moisture on a rock surface is absorbed by the lichen -which can turn green in 15 minutes. - -Scher et al. ([ref.51]) exposed desert soils to simulated environmental -conditions and diurnal cycles of Mars. The atmosphere consisted of 95 -percent nitrogen and 5 percent carbon dioxide (no oxygen) and was dried, -using calcium sulfate as a desiccant. The total atmospheric pressure was -0.1 atm. The temperature ranged from -60 deg. to +20 deg. C in 24-hour -cycles. One hour was spent at the maximum and at the minimum -temperatures. The chambers were irradiated with ultraviolet, 2537 A, -with a dose of 10^9 ergs/cm squared, which is comparable to a daily dose -found on Mars, and easily exceeds the mean lethal dose for unprotected -bacteria. Soil aliquots were removed weekly and incubated at 30 deg. C. -The scoring was done both aerobically and anaerobically. Sporeforming -obligate and facultative anaerobes, including _Clostridium_, _Bacillus_, -and _Planosarcina_, and nonsporeforming facultative anaerobes, including -_Pseudomonas_ and _Rhodopseudomonas_, were found. The experimental -chambers were frozen and thawed cyclically up to 6 months. Organisms -that were able to survive the first freeze-thaw cycle were able to -survive the entire experiment. The ultraviolet irradiation did not kill -subsurface organisms, and a thin layer of soil served as an ultraviolet -shield. All of the samples showed survivors. - -Young et al. ([ref.52]) assumed that water is present on Mars, at least -in microenvironments, and that nutrients would be available. The primary -objective of their experiments was to determine the likelihood of -contaminating Mars with Earth organisms should a space probe from Earth -encounter an optimum microenvironment in terms of water and nutrients. -The experiments used bacteria in liquid nutrient media. The environment -consisted of a carbon dioxide-nitrogen atmosphere, and the temperature -cycling was -70 deg. to +25 deg. C, with a maximum time above freezing -of 4-1/2 hours. _Aerobacter aerogenes_ and _Pseudomonas sp._ grew in -nutrient medium under Martian freezing and thawing cycles. Atmospheric -pressure was not a significant factor in the growth of bacteria under -these conditions. - -Silverman et al. ([ref.47]) studied bacteria and a fungus under -extreme--but not "Martian"--conditions. Spores of five test organisms -(_B. subtilis_ var. _niger_, _B. megaterium_, _B. stearothermophilus_, -_Clostridium sporogenes,_ and _Aspergillus niger_) and soils were -exposed while under ultrahigh vacuum to temperatures of from -190 deg. -to +170 deg. C for 4 to 5 days. Up to 25 deg. C there was no loss in -viability; at higher temperatures, differences in resistivity were -observed. At 88 deg. C, only _B. subtilis_ and _A. niger_ survived in -appreciable numbers; at 107 deg. C, only _A. niger_ spores survived; -none were recoverable after exposure to 120 deg. C. _B. subtilis_ -survived at atmospheric pressure and 90 deg. C for 5 days, but none of -the other spores were viable alter 2 days. Four groups of soil organisms -(mesophilic, aerobic, and anaerobic bacteria, molds, and actinomycetes) -were similarly tested in the vacuum chamber. From one sample only -actinomycetes survived 120 deg. C, while one other soil sample yielded -viable bacteria after exposure to 170 deg. C. Several organisms resisted -120 deg. C in ultrahigh vacuum for 4 to 5 days. When irradiated with -gamma rays from a cobalt 60 source, differences were observed between -vacuum-dried spores irradiated while under vacuum and those exposed to -air immediately before irradiation. A reduction of from one-third to -one-ninth of the viability of spores irradiated in vacuum occurred with -vacuum-treated spores irradiated in air. - -Siegel et al. ([ref.73]), in approximate simulations of Martian -environments, studied tolerances of certain seed plants, such as -cucumbers, corn, and winter rye, to low temperatures and lowered oxygen -tensions. Lowered oxygen tensions enhanced the resistance of seedlings, -particularly cucumber and rye to freezing, and lowered the minimum -temperature required for germination. Germination of seeds in the -absence of liquid water has also been studied. In this case, seeds of -xerophytes have been suspended in air at 75-mm Hg pressure above water. -The air was thus saturated. Germination was slow but did occur. - -Siegel et al. (refs. [ref.73] and [ref.74]) found that the growth rate -of several higher plants was enhanced by certain gases usually thought -to be toxic, such as N2O. This finding is significant inasmuch as the -presence of nitrogen oxides in the Martian atmosphere has been cited as -evidence for the nonexistence of plants on that planet by Kiess et al. -([ref.75]). Exploratory survival tests showed that various mature -plants, as well as the larvae, pupae, and adult specimens of a -coleopteran insect, were undamaged when exposed to at least 40 hours of -an atmosphere containing 96.5 percent N2O, 0.7 percent O2, and 2.8 -percent N2. - -Lichens are of interest because of their ability to survive and thrive -under extreme environmental conditions on Earth. Biological activity of -slow-growing lichens was detected by metabolic gas exchange, CO2 -detection being especially convenient. Siegel points out that this -method is sensitive and nondestructive, to be preferred to staining -techniques, which at present are limited because they are only -semiquantitative, subjective, and destructive of the lichen. - -A Russian study of simulated planetary environments has been performed -with good simulation but for periods of only 2 to 6 hours. Comments on -simulation experiments made by Zhukova and Kondratyev ([ref.69]) are -presented as follows: - - On the basis of modern conceptions on Martian conditions it is - difficult to imagine that higher forms of animals or plants - exist on the planet. A Martian change of seasons similar to that - of our planet empowers us to think that there is a circulation - of an organic substance on Mars, which cannot exist without - participation of microbic forms of life. Microorganisms are the - most probable inhabitants of Mars although the possibility is - not excluded that their physiological features will be very - specific. That is why the solution of the problem concerning the - character of life on Mars is of exceptional interest. But still - the answer to this question can be verified only by simulating - Martian conditions, taking into account the information obtained - from astrophysicists. - - Experiments aimed at creating artificial Martian climatic - conditions have been started quite recently; their number is not - large since they cannot be combined with the results of numerous - experiments investigating the effect of extreme factors on - microorganisms. The result of the effect of such physicochemical - parameters of the medium as pressure, sharp temperature changes, - the absence of oxygen and insolation, depends on their - combination and simultaneity. These examples convincingly show - that while simulating Martian conditions one should strive to - the most comprehensive complex of simultaneously acting factors. - The creation of individual climatic parameters acting - successively leads to absolutely different, often opposite - results. It should be mentioned also that refusal to imitate - insolation and the performance of experiments with specimens of - soil which itself has protective effect on cells of - microorganisms, but not with pure culture of bacteria, are usual - shortcomings in the bulk of studies on this problem. - -It appears that organisms from Earth might survive in large numbers when -introduced to Martian environment. Whether these organisms will be -capable of growth and explosive contamination of the planet in a -biological sense or not is highly questionable. The likelihood of an -organism from Earth finding ideal conditions for growth on Mars seems -extremely low. However, the likelihood of an organism from Earth serving -as a contaminant for any life-detection device flown to Mars for the -purpose of searching out carbon-based life is considerably higher. The -chance that life has originated and evolved on Mars is a completely -separate question and much more difficult to answer. - -It would be interesting to attempt to determine possible evolutionary -trends which might occur on a planet by means of selection of organisms -in a simulated planetary environment. Rapid genetic selection combined -with radiation and chemicals to speed up mutation rate under these -conditions should reveal possible evolutionary trends under the -planetary environmental conditions. This could be attempted after the -planetary environments are more accurately defined. - - - EXTREME AND LIMITING ENVIRONMENTAL PARAMETERS OF LIFE - -The question of the existence of extraterrestrial life is one of the -most important and interesting biological questions facing mankind and -has been the subject of much controversial discussion and conjecture. -Many of the quantitative, and even qualitative, environmental -constituents of the planets also are still subjects of controversy and -speculation. Best guesses about a relatively unknown planetary -environment, combined with lack of information about the capabilities of -Earth life to grow in extreme environments, do not provide the basis for -making informed scientific estimates. - -Life on Earth is usually considered to be relatively limited in its -ability to grow, reproduce, or survive in extreme environmental -conditions. While many common plants and animals (including man) are -quite sensitive to, or incapable of, surviving severe chemical and -physical changes or extremes of environment, a large number of -micro-organisms are highly adapted and flourish in environments usually -considered lethal. Certain chemoautotrophic bacteria require high -concentrations of ammonia, methane, or other chemicals to grow. -Anaerobic bacteria grow only in the absence of oxygen. - -Besides adapting to the extremes of environments on Earth, life is also -capable of growing and reproducing under extreme environmental -conditions not normally encountered: e.g., from a few rad of radiation -in normal habitats to 10^6 or more rad from artificial sources, from 0.5 -gauss of Earth magnetism to 167 000 gauss in manmade magnetic fields, -and from 1-g force of gravity to 110 000 g. The extreme ranges of -physical and chemical environmental factors for growth, reproduction, -and survival for Earth micro-organisms are phenomenally large. - -Life is ubiquitous on Earth and is found in almost every possible -environment, including the most severe habitats, from the bottom of the -ocean to the highest mountain tops and from cold Arctic habitats to hot -springs, as well as in volcanic craters, deep wells, salt flats, and -mountain snowfields. Earth life has become adapted to, and has invaded, -nearly every habitat, no matter how severe. The physiological and -morphological adaptations of life are exceedingly diverse and complex. - -Surprisingly, the extreme parameters or ranges of the physical and -chemical environmental factors permitting growth, reproduction, and -other physiological processes of Earth organisms have not been -critically compiled. A partial compilation of certain selected -environmental factors has been made by Vallentyne ([ref.76]). A -compilation of available published data on certain environmental -extremes, particularly from recent NASA-supported research (compiled by -Dale W. Jenkins, in press), is presented in tables III to VI. These data -can serve as a starting point for a more intensive literature review by -specialists, critical evaluation, standardization of end points, and -especially to point out areas where critical experimentation is urgently -needed. - -This critical compilation involves a review of a very broad and complex -range of subjects involved in many different disciplines with widely -scattered literature. Since the effects of many of the specific -environmental factors are harmful, it is difficult to select a point on -a scale from no effect to death and use some criteria to say that normal -or even minimal growth and reproduction are occurring. The effects of -environmental factors are dependent on (1) the specific factor, times, -(2) the concentration or energy, times, (3) the time of exposure or -application of the factor. Many reports, especially older ones, do not -give all of the necessary data to permit proper evaluation. A -complicating factor is that the effect of each factor depends on the -other factors before, during, and after its application. The condition -of the organism itself is a great variable. Proper evaluation requires -the critical review by a variety of biological specialists, physicists, -and chemists. - -To determine the potential of Earth organisms to survive or grow under -other planetary environmental conditions, a number of experiments have -been carried out attempting to simulate planetary environments, -especially of Mars, as reviewed previously. While the results are of -real interest, they do not provide much basic information. Further, as -the Martian environment is more accurately defined, the experimental -conditions are changed. In addition, some experimenters have altered -certain factors, such as water content, to allow for potential -microhabitats or for areas which might contain more water at certain -times. - - - Table III.--_Extreme Physical Environmental Factors_ - - ----------------------------------------------------------------- - Physical Minimum Organism - factors - ----------------------------------------------------------------- - Temperature -30 deg. C Algae (photosynthesis), - pink yeast (growth) - ----------------------------------------------------------------- - Magnetism 0-50 gamma (=x10^-5 Human - gauss) - - ----------------------------------------------------------------- - Gravity 0 g Human, plants, animals - - ----------------------------------------------------------------- - Pressure 10^-9 mm Hg (5 days) _Mycobacterium_ - _smegmatis_ - ----------------------------------------------------------------- - Microwave 0 W/cm squared - - - ----------------------------------------------------------------- - Visible 0 ft-c Animals, fungi, - bacteria - - ----------------------------------------------------------------- - Ultraviolet 0 erg/cm squared - - ----------------------------------------------------------------- - X-ray 0 rad - ----------------------------------------------------------------- - Gamma ray 0 rad - - - ----------------------------------------------------------------- - Acoustic 0 dyne/cm squared - - - - - ----------------------------------------------------------------- - - - Table III.--_Extreme Physical Environmental Factors_ - - ---------------------------------------------------------------------- - Physical Maximum Organism Activity - factors - ---------------------------------------------------------------------- - Temperature 104 deg. C _Desulfovibrio Grows and reduces - (1000 atm) desulfuricans_ sulfate - ---------------------------------------------------------------------- - Magnetism 167 000 _Neurospora_ 1 hr--no effect, - gauss _Arbacia_ _Arbacia_ - _Drosophila_ development delayed - ---------------------------------------------------------------------- - Gravity 400 000 g _Ascaris_ eggs 1 hr--eggs hatch, - 110 000 g _Escherichia coli_ 40 days' growth - ---------------------------------------------------------------------- - Pressure 1400 atm Marine organisms Growth - - ---------------------------------------------------------------------- - Microwave 2450 Mc/sec _Drosophila_ 68 hr, growth not - 0.3 to 1 affected - W/cm squared - ---------------------------------------------------------------------- - Visible 50 000 ft-c _Chlorella_, Seconds, - 17 000 ft-c higher plants recurrently - continuous - ---------------------------------------------------------------------- - Ultraviolet 10^8 erg/cm Bean embryos Suppressed growth - squared, 2537 A - ---------------------------------------------------------------------- - X-ray 2x10^6 rad Bacteria Growth - ---------------------------------------------------------------------- - Gamma ray 2.45x10^6 rad _Microcoleus_ Continued growth - _Phormidium_ - _Synechococcus_ - ---------------------------------------------------------------------- - Acoustic 140 db or Man Threshold of pain - 6500 - dyne/cm squared - at 0.02 to 4.8 - kcs/sec - ---------------------------------------------------------------------- - - - Table IV.--_Extreme Low and High Temperature Effects Permitting - Life Processes_ - - ----------------------------------------------------------------- - Minimum Organism Activity or condition - temperature, - deg. C - ----------------------------------------------------------------- - -11 Bacteria Growth (on fish) - ----------------------------------------------------------------- - -12 Bacteria Growth - ----------------------------------------------------------------- - -12 Molds Growth - ----------------------------------------------------------------- - -15 _Pyramidomonas_ Swimming - ----------------------------------------------------------------- - -15 _Dunaliella salina_ Swimming - ----------------------------------------------------------------- - -18 Mold Growth - ----------------------------------------------------------------- - -18 Yeast Growth - ----------------------------------------------------------------- - -18 _Aspergillus Growth (in glycerol) - glaucus_ - ----------------------------------------------------------------- - -18 to -20 Mold Growth (in fruit juice) - ----------------------------------------------------------------- - -18 to -20 _Pseudomonads_ Growth (in fruit juice) - ----------------------------------------------------------------- - -20 Bacteria Growth - ----------------------------------------------------------------- - -20 Bacteria Growth - ----------------------------------------------------------------- - -20 Bacteria Luminescence development - accelerated - ----------------------------------------------------------------- - -20 to -24 Insect eggs - (diapause) - ----------------------------------------------------------------- - -30 Algae Photosynthesis - ----------------------------------------------------------------- - -30 Pink yeast Growth (on oysters) - ----------------------------------------------------------------- - -30 Lichens Photosynthesis - ----------------------------------------------------------------- - -20 to -40 Lichens and conifers Photosynthesis - ----------------------------------------------------------------- - -44 Mold spores Sporulation and germination - ----------------------------------------------------------------- - - - Table IV.--_Extreme Low and High Temperature Effects Permitting - Life Processes_ - - ------------------------------------------------------------------- - Maximum Organism Activity or condition - temperature, - deg. C - ------------------------------------------------------------------- - 73 Thermophilic organisms Growth (P^32 metabolism) - ------------------------------------------------------------------- - 73 _Phormidium_ (alga) Acclimatized - ------------------------------------------------------------------- - 70 to 73 _Bacillus calidus_ Growth and spore - germination - ------------------------------------------------------------------- - 70 to 74 _Bacillus cylindricus_ Growth and spore - germination - ------------------------------------------------------------------- - 70 to 75 _Bacillus tostatus_ Growth and spore - germination - ------------------------------------------------------------------- - 80 _Bacillus Cultured in laboratory - stearothermophilus_ - ------------------------------------------------------------------- - 83 Sulfate-reducing Found in a well - bacteria - ------------------------------------------------------------------- - 89 Sulfate-reducing Found in oil waters - bacteria - ------------------------------------------------------------------- - 65 to 85 Sulfate-reducing Cultured in laboratory - bacteria - ------------------------------------------------------------------- - 89 Micro-organisms Found in hot springs - ------------------------------------------------------------------- - 95 _Bacillus coagulans_ In 80 min. sporulation - activation - ------------------------------------------------------------------- - 110 _Bacillus coagulans_ In 6 min, sporulation - activation - ------------------------------------------------------------------- - 104 _Desulfovibrio Grow and reduce sulfate - desulfuricans_ at 1000 atm - ------------------------------------------------------------------- - - - Table V.--_Extreme Temperature Limits of Survival_ - - -------------------------------------------------- - Minimum Organism - temperature - deg. C - -------------------------------------------------- - -190 Yeast bacteria, 10 species - -------------------------------------------------- - -197 _Trebouxia erici_ from lichens - -------------------------------------------------- - -197 Protozoa, _Anguillula_ - -------------------------------------------------- - -252 Yeasts, molds, bacteria, 10 species - -------------------------------------------------- - -253 Black currant, birch - -------------------------------------------------- - -273 Bacteria, many species - -------------------------------------------------- - -273 Bacteria, many species - -------------------------------------------------- - -272 Desiccated rotifers - -------------------------------------------------- - -269 Human spermatozoa - -------------------------------------------------- - - - Table V.--_Extreme Temperature Limits of Survival_ - - ------------------------------------------------------------------ - Maximum Organism Time of exposure - temperature - deg. C - ------------------------------------------------------------------ - 140 Bacterial spores 5-hr immersion - ------------------------------------------------------------------ - 170-200 Desiccated rotifers 5 min - ------------------------------------------------------------------ - 151 Desiccated rotifers 35 min - ------------------------------------------------------------------ - 150 _Clostridium tetani_ 180 min - ------------------------------------------------------------------ - 170 Aerobic bacteria, molds. 5 days at - actinomycetes 6x10^-9mm Hg - ------------------------------------------------------------------ - 127 (dry) Bacteria (in activated charcoal) 60 min - ------------------------------------------------------------------ - 110 (wet) _Bacillus subtilis_ var. _niger_ 400 min - ------------------------------------------------------------------ - 120 _Bacillus subtilis_ var. _niger_ 400 min - ------------------------------------------------------------------ - 141 _Bacillus subtilis_ var. _niger_ 70 min - ------------------------------------------------------------------ - 160 _Bacillus subtilis_ var. _niger_ 15 min - ------------------------------------------------------------------ - 180 _Bacillus subtilis_ var. _niger_ 2 min - ------------------------------------------------------------------ - 188 _Bacillus subtilis_ var. _niger_ 1 min - ------------------------------------------------------------------ - 120 (wet) _Bacillus stearothermophilus_ 25 min - ------------------------------------------------------------------ - 120 (dry) _Bacillus stearothermophilus_ 100 min - ------------------------------------------------------------------ - 141 _Bacillus stearothermophilus_ 12 min - ------------------------------------------------------------------ - 160 _Bacillus stearothermophilus_ 2 min - ------------------------------------------------------------------ - 166 _Bacillus stearothermophilus_ 1 min - ------------------------------------------------------------------ - - - Table VI.--_Extremes of Chemical Environmental Factors - Permitting Growth or Activity_ - - -------------------------------------------------------- - Chemical Minimum Organism - factor - -------------------------------------------------------- - O2 0% HeLa cells, _Cephalobus_, - anaerobic bacteria - -------------------------------------------------------- - O3 (ozone) 0% - - - -------------------------------------------------------- - H2 0% - - -------------------------------------------------------- - H2O Aw 0.48 _Pleurococcus vulgaris_ - ------------------------------------------ - Aw 0.5 _Xenopsylla cheopis_ - (prepupae) - -------------------------------------------------------- - H2O2 0% - - -------------------------------------------------------- - He 0% - - -------------------------------------------------------- - CO 0% - - - -------------------------------------------------------- - CO2 0% - - -------------------------------------------------------- - CH4 0% - -------------------------------------------------------- - CH2O 0% - -------------------------------------------------------- - CH3OH 0% - -------------------------------------------------------- - N2 0% - - -------------------------------------------------------- - NO 0% - - -------------------------------------------------------- - NO2 0% - - -------------------------------------------------------- - N2O 0% - - - - - - -------------------------------------------------------- - Ar 0% - -------------------------------------------------------- - NaCl, - Na2SO4, - NaHCO3 - -------------------------------------------------------- - H2S 0% - - -------------------------------------------------------- - H2SO4 0% - - - - - -------------------------------------------------------- - Cu^++ - - -------------------------------------------------------- - Zn^++ - - -------------------------------------------------------- - pH 0 _Acontium velatum_ - _Thiobacillus thioodixans_ - - - - - -------------------------------------------------------- - Eh -450 mV Sulfate-reducing bacteria - at pH 9.5 - -------------------------------------------------------- - - - - Table VI.--_Extremes of Chemical Environmental Factors - Permitting Growth or Activity_ - - ---------------------------------------------------------------------- - Chemical Maximum Pressure, Time, Organism Activity - factor atm days - ---------------------------------------------------------------------- - O2 100% 1 Plants, Growth - animals - ---------------------------------------------------------------------- - O3 100 ppm 5 _Armillaria Growth - (ozone) --------------------------- mellea_ ----------------- - 500 ppm 5 Light emission - ---------------------------------------------------------------------- - H2 100% Various Germination - plants - ---------------------------------------------------------------------- - H2O Aw 1.0 1 Various Growth - aquatic - organisms - - ---------------------------------------------------------------------- - H2O2 0.34% Rye Germination - enhanced - ---------------------------------------------------------------------- - He 100% Wheat, rye, Germination - rice - ---------------------------------------------------------------------- - CO 100% Rye Germination - -------------------------------------------------------------- - 80% 1.1 4 _Hydrogenomonas_ Growth - ---------------------------------------------------------------------- - CO2 100% 1.1 4 Rye Growth and - germination - ---------------------------------------------------------------------- - CH4 100% 1.1 4 Rye Germination - ---------------------------------------------------------------------- - CH2O 50% Rye Germination - ---------------------------------------------------------------------- - CH3OH 50% Rye Germination - ---------------------------------------------------------------------- - N2 100% .1 10 Various plants Germination and - root growth - ---------------------------------------------------------------------- - NO 18% .018 10 Sorghum, rice Germination and - root growth - ---------------------------------------------------------------------- - NO2 18% .018 10 Rye, rice Germination and - root growth - ---------------------------------------------------------------------- - N2O 100% 1.2 4 Rye Germination - -------------------------------------------------------------- - 96.5% 1.7 Rye Germination - ------------------------------------ - _Tenebrio Survival - molitor_ - ---------------------------------------------------------------------- - Ar 100% 1.2 2 Rye Germination - ---------------------------------------------------------------------- - NaCl, 67% Photosynthetic Growth - Na2SO4, bacteria - NaHCO3 - ---------------------------------------------------------------------- - H2S 0.96 _Desulfovibrio Growth - g/liter desulfuricans_ - ---------------------------------------------------------------------- - H2SO4 7% _Acontium Growth - velatum_ - ------------------------------- - Thiobacilli Growth, - reproduction - ---------------------------------------------------------------------- - Cu^++ 12 _Thiobacillus Growth - g/liter ferrooxidans_ - ---------------------------------------------------------------------- - Zn^++ 17 _Thiobacillus Growth - g/liter ferrooxidans_ - ---------------------------------------------------------------------- - pH 13 _Plectonema Growth - nostocorum_ - ------------------------------- - _Nitrobacter_ Growth - ------------------------------- - _Nitrosomonas_ Growth - ---------------------------------------------------------------------- - Eh 850 mV Iron bacteria Growth - at pH 3 - ---------------------------------------------------------------------- - - - - - chapter 4 - -_Behavioral Biology_ - - - EFFECTS OF THE SPACE ENVIRONMENT ON BEHAVIOR - -NASA was established in 1958, shortly after the Russian launching of the -second Earth satellite Sputnik II, the first vehicle to carry life into -orbit around the Earth. This accomplishment was preceded by the -pioneering work of Henry et al. ([ref.77]), in which animals were -exposed briefly to low-gravity states in Aerobee rockets. A -motion-picture camera photographed the behavior of two white mice in -rotating drums during this series of flights, which marked the first -time that simple psychological tests were made on animals in the -weightless condition. While this behavioral experiment was relatively -simple, it provided the basic concepts for recent studies which involved -rotation of animals during the weightless state. Subsequent flights such -as Project MIA (Mouse-in-Able) reflected a preoccupation with -physiologic measures (refs. [ref.78] and [ref.79]), although the flights -of Baker and Able included preflight and postflight performance studies -([ref.80]). Able's behavior was recorded in detail on in-flight film, -but none of the behavior was programed or under experimental control. - -The first flights in which behavior or performance was explicitly -programed were those of Sam and Miss Sam in flights of the Little Joe -rocket with the Mercury capsule, launched from Wallops Island in 1959 -and 1960 ([ref.81]). The first major space achievement in the behavioral -sciences was the successful in-flight measurement of the behavior of the -chimpanzee Ham in early 1961, in which the pretrained animal performed -throughout the flight. The second achievement along these lines was in -1962 when the chimpanzee Enos made several orbits around Earth and -performed continuously on a complex behavioral task. The tasks which the -animals performed during these flights have been described in detail by -Belleville et al. ([ref.82]), and the results of the in-flight -performance have been presented by Henry and Mosely ([ref.83]). These -early flights provided much of the technological framework on which -current biological experiments on organisms during flights of extended -duration are based. Due largely to the efforts of Grunzke (refs. -[ref.84] and [ref.85]), the apparatus needed to sustain animals during -space flight, such as zero-g watering and feeding devices, are now -commonplace ([ref.86]). Advanced systems of programing stimulus -presentations and recording responses, developed for Project Mercury, -may now be seen in many basic research laboratories throughout the -country. - -Several other noteworthy advances have been made as an outgrowth of the -Mercury animal flights. Immediately before the orbital flight MA-5, in -which the chimpanzee Enos was employed, it was unexpectedly found that -this 5-year-old animal was hypertensive. Subsequent centrifuge studies -showed that its vascular responses exceeded those of a control group. -Consideration of the animal's preflight experience led to speculation -concerning the origin of this hypertension. An explanation of the -high-blood-pressure responses detected in Enos has been pursued by -Meehan et al. ([ref.87]). Persistent hypertension has been produced in -other laboratory chimpanzees restrained in the same manner as those -participating in space flight and exposed to demanding performance -tasks, a demonstration which has important implications for prolonged -manned space flight and for cardiovascular medicine in general. - -Studies more directly concerned with behavior and performance have been -extended from those of Project Mercury. These extensions have been in -the following directions: (1) the establishment and maintenance of -complex behavioral repertoires under conditions of full environmental -control, (2) the refinement of behavioral techniques for assessing -sensory and motor processes, and (3) the maintenance of sustained -performance under conditions of long-term isolation and confinement and -preliminary extension of such experimental analysis to man. - -Numerous studies with primate subjects, including several at Ames -Research Center, have been devoted to developing methods for maintaining -optimum performance in environments with limited sources of stimulation. -Monkeys, baboons, and chimpanzees, for example, have been isolated for -periods of longer than 2 years with no decrement in performance on -complicated behavioral tasks ([ref.88]). The behavioral techniques used -in these studies are closely related to those employed on human subjects -under NASA sponsorship at the University of Maryland ([ref.89]). The -essence of these techniques is in the proper programing of environmental -stimuli ([ref.90]). It is not sufficient to provide the subject with his -physiological requirements for survival, but he must be given the -psychological motivation for using these provisions. This statement, of -course, is an oversimplification of the problem, but it serves to -illustrate the essence of these experimental programs. - -Gravity has long been known as one of the major factors influencing -various life processes and the orientation of both plants and animals. -One of the most challenging problems of space research has been to -define this influence more precisely. Related to the effect of gravity -on living processes is the problem of the effects of weightlessness. Of -particular interest to psychologists are the possible modifications an -altered gravitational environment might produce in behavioral patterns -basic to the animal's maintenance and survival, such as eating, sensory -and discriminative processes, development and maturation, and learning -capacity ([ref.91]). - -One prominent method of studying gravitational effects is to simulate an -increase in gravity by centrifugation. Smith et al. ([ref.92]) and -Winget et al. ([ref.93]) have investigated the effects of long-term -acceleration on birds, primarily chickens, while Wunder (refs. [ref.94] -and [ref.95]) and his coworkers (refs. [ref.96]-[ref.99]) have used -fruit flies, mice, rats, hamsters, and turtles. The general findings are -that, when animals are subjected to a prolonged period of acceleration -of moderate intensity, they exhibit decreased growth, delayed -maturation, and an increase in the size of certain muscles and organs, -dependent on the species. With regard to the decreased growth effect, -the data of these investigators show some exceptions. When the -gravitational increase is kept below a certain limit, growth was greater -than that of controls in the fruit fly, turtle, mouse, and chicken. The -limit below which enhancement of growth was observed varied with the -species studied. - -The data on food intake do not present a consistent picture. Wunder -([ref.94]) found that food intake in accelerated mice was markedly -reduced from that of nonaccelerated control animals. Smith, however, -found that in chickens, food intake increased up to 36 percent over -controls and has derived an exponential relation between food intake and -acceleration. After six generations of selective breeding, Smith has -produced a strain of chickens better adapted to prolonged exposure to -high g. - -A very relevant finding of their research with birds was that exposure -to chronic acceleration in some way appears to interfere with -habituation to rotatory stimulation. Chickens who were being subjected -to chronic acceleration were given repeated rotatory stimulation tests -to estimate their labyrinthine sensitivity. This study revealed that -centrifuged animals showed a marked reduction in labyrinthine -sensitivity. This result appeared to persist after the acceleration was -terminated. In animals who developed gait or postural difficulties as a -result of acceleration, there was no evidence of a postnystagmus in -response to the rotatory stimulation test, which the investigators point -out may be evidence of a lesion in the labyrinth or its neural pathways. - -Smith has implicated social factors as interfering with acceleration -effects. His subjects were typically accelerated four or six to a cage. -When groups were mixed midway through the experiment, they exhibited a -higher mortality rate and incidence of acceleration symptoms than did -groups whose constituency remained unchanged. - -At the U.S. Naval School of Aerospace Medicine, numerous studies have -been conducted on the effects of slow rotation on the behavior and -physiology of humans and animals ([ref.100]). Rotation initially -produces decrements in performance, but adaptation to a rotating -environment ensues quite rapidly (refs. [ref.101]-[ref.103]). Perceptual -distortion, nystagmus, nausea, and other signs of discomfort are common -responses to slow rotation. These symptoms are generally reduced with -continued exposure (adaptation). Interestingly, however, adaptation is -delayed when the subjects are exposed to a fixed reference outside their -rotating environment. - -At NASA-Ames, rodents have been used in experiments by Weissman and -Seldeen to delimit the stimulus effects of rotation. In these -experiments the subjects must discriminate between different speeds of -rotation in order to obtain food reinforcement. The results thus far -provide evidence that these animals are capable of discriminating -between the different speeds at which they are being rotated. The range -of speeds studied was 0-25 rpm, with tests of discrimination being made -at intervals of less than 5 rpm. Experiments such as these will lead to -the development of techniques for measuring rotational sensitivity in -many species, including man. - -The optimum configuration of manned spacecraft will depend, in part, -upon biomedical considerations. A voluminous literature now exists on -the possible hazards to man of prolonged exposure to zero-g conditions. -Should prolonged weightlessness prove to be a serious detriment to -health, consideration must be given to design concepts which provide -artificial gravity. - -No data exist on the minimum gravity requirements necessary to sustain -basic biological functions for extended periods. A limit of 0.2 g has -been given as the lower level at which man can walk unaided ([ref.104]). -It has also been recommended that angular velocity be maintained -at the lowest possible level in order to minimize the occurrence of -vestibular disturbances. These recommendations are based on human-factor -requirements, rather than upon biological considerations, which may -significantly modify these values. In recent studies, a technique has -been devised which promises to provide reliable criteria for biological -acceptability, since it is based on fundamental biological and -behavioral principles. - -As animals progress up the evolutionary stale, their survival depends -less and less upon stereotyped physiological reactions which occur in -reflex fashion, in response to environmental stimulation. In higher -organisms, survival depends more upon the capacity of organisms to -modify their behavior. At the highest levels of functional efficiency, -the ultimate form of adaptation is seen--the manipulation of the -environment by the organism. Developments in behavioral science now -permit us to utilize the adaptive behavior of animals to investigate -many problems of biological interest. Recent studies on the -self-selection of gravity levels represent a further attempt to exploit -the adaptive capacities of animals, in order to provide information -relevant to problems of space exploration. - -One such project allows animals to select their own gravity environment -in an apparatus designed to create g-forces through centrifugal action -by rotation at 60 rpm ([ref.105]). The surface of this centrifuge is -parabolic, so that the resultant of the centrifugal g and the Earth's -gravity is always normal to the surface. When the animal moves away from -the center, increasing the radius of rotation, it is exposed to -increasing gravity. Motion toward the center reduces the gravity level. -By this means, an animal is free to select its own gravity environment. - -When the animal moves toward or away from the center, he is moving from -one tangential velocity to another. He is therefore acted upon by a -third force--due to Coriolis acceleration. The effects of Coriolis -forces are a major problem difficult to eliminate in studies such as -these, but they must be taken into account in the design of spacecraft -which produce artificial gravity by rotation. Motion of the head in any -direction not parallel to the centrifugal force vector would result in -bizarre stimulation of the semicircular canals and consequent motion -sickness. This effect is likely to become even more pronounced if the -sensitivity of these organs is increased by prolonged exposure to -reduced gravity. Methods such as these are currently being developed for -conducting a refined psychophysical analysis of gravity, including -studies by Lange and Broderson on the perception of angular, linear, and -Coriolis acceleration. - -The results of animal studies such as these will be of great value in -arriving at a decisive judgment concerning the need for artificial -gravity in a manned orbiting space station, or other vehicles designed -for long-term occupancy. - -To aid in the interpretation of in-flight data, other studies are -underway to determine the functions of the vestibular system, as a -principal brain center related to orientation in space and to the -physiology of posture and movement, as well as with the influences of -acceleration, rotation, and weightlessness. Experiments are presently -being conducted on monkeys and cats in order to trace these complex -neurological connections and to determine their functional organization. - - - BIOLOGICAL INFORMATION SYSTEMS - -The nature of memory has been the subject of considerable speculation in -the past. It has long been felt intuitively that retention of -information in the central nervous system involves either an alteration -of preexisting material or structure, or, alternatively, synthesis of -materials not present previously. The cellular site of operational -alteration was unknown but, again intuitively, was felt to be closely -associated with the synapses. The problems faced by early investigators -were great; but nevertheless much information relevant to the question -of biological information storage was obtained. With the relatively -recent advent of more refined tools and methodologies, there has been -rapid progress. - -A significant amount of the work which has been conducted in the area of -biological information and communication systems is easily classified as -"basic research" (refs. [ref.106]-[ref.109]). This discussion will be -limited to those aspects closely related to the fields of molecular -biology and experimental psychology, which seem to have universal -application to all known animal life forms. Studies involving the basic -principles of acquisition, processing, storage, and retrieval of -information in living systems are emphasized. - - -Early Work - -Early speculations on the operational nature of memory have been based -upon relatively little experimental evidence. Charles Darwin observed -that domestic rabbits had smaller brains than their wild counterparts, -and attributed this to lack of exercise of their intellect, senses, and -voluntary movements. Unfortunately, subsequent studies of the brains of -men with greatly differing intellectual capability did not substantiate -the hypothesis. Idiots sometimes had larger brains than geniuses. Later, -an idea proposed by Ramon y Cajal came into favor. Since brain cells did -not increase in number after birth, he proposed that memory involved the -establishment of new and more extended intercortical connections. -Unfortunately, methods were not available to test this hypothesis -adequately and it has remained until quite recently in the realm of -conjecture. - -Another major hypothesis was that there were two or more stages in the -information storage process. The final form the information took in the -brain was called a brain engram, or memory trace. However, prior to the -formation of the engram, a transitory process denoted as -"reverberational memory" was postulated to exist for a relatively short -time (minutes to hours) (refs. [ref.106] and [ref.107]). This hypothesis -was used by Pauling to explain why an elderly chairman of a board could -brilliantly summarize a complex 8-hour meeting and yet, after its -conclusion and his return to his office, not even remember having -attended the meeting. Thus, this individual's reverberational memory -functioned well, but advanced years had seriously impaired his brain's -ability to form a permanent engram. Similar, although less dramatic, -observations in other situations are not uncommon. A wide variety of -experiments have been conducted to study this aspect of memory and to -relate it to the process whereby the information is transformed to a -more stable form (refs. [ref.110]-[ref.112]). - -More recently, the concept of a specific biochemical activity during the -process of long-term storage of information has gained considerable -favor. Initially, neither the site nor the nature of the change was well -defined. Quite recent studies by Krech et al. (refs. [ref.113] and -[ref.114]), Bennett et al. ([ref.115]), Rosenzweig et al. (refs. -[ref.116] and [ref.117]) support the view that alteration of the levels -of acetylcholinesterase at cortical synapses play an important role in -information storage. These studies will be discussed in a later section. -However, these authors do not claim that the changes observed are -unambiguously related to the storage of memory. It may well be that the -alterations observed are in some way related to this process but are -still secondary to some other, more basic, process. - -An alternative hypothesis is that the information resides in its -ultimate form in some more central structure of the neurone than the -synapse. (It has even been postulated that the basic information is -stored in nonneuronocortical material.) Perhaps Halstead was the first -to postulate the involvement of nucleoprotein in this process -([ref.107]). From the biochemist's point of view, this is an extremely -attractive hypothesis. Both proteins and nucleic acids possess -sufficient possible permutations of structure to permit storage of a -lifetime's accumulation of information in an organ the size of the -brain. From the previously known ability of the nucleic acids to code -genetic information, they are the prime suspects. However, from the -known regulatory ability of nucleic acids in specific protein synthesis, -it is possible that the final repository is protein. - - -Recent Biochemical Studies - -Among the foremost investigators of the chemistry and biochemistry of -the central nervous system is Holger Hyden at the University of -Goeteborg, Sweden. He and others (refs. [ref.118]-[ref.120]) have for -many years performed elegant microanalytical studies of single nerve -cells. The evidence which Hyden has obtained is consistent with the -hypothesis that the initial electrical reverberations in the brain -induce a change in the molecular structure of the ribonucleic acid (RNA) -of the neurones which, in turn, leads to a subsequent deposition of -specific proteins. It is well known from other investigations that a -major role of RNA in any type of cell is to specify and mediate -synthesis of the protein enzymes of the cells. Thus, in this hypothesis, -it is only necessary to postulate the modification of brain RNA by the -activities associated with reverberational memory. Particularly -pertinent to this hypothesis are observations that-- - - (1) Large nerve cells have a very high rate of metabolism of RNA and - proteins, and, of the somatic cells, are the largest producers of - RNA. - (2) Vestibular stimulation by passive means leads to an increase in - the RNA content of the Deiters nerve cells of rabbits ([ref.121]). - The protein content of these cells is also increased. - (3) Changes in the RNA composition of neurones and glia of the - brainstem occur during a learning situation. Animals were trained - over a period of 4 to 5 days to climb a steeply inclined wire to - obtain food. The big nerve cells and the glia of their lateral - vestibular apparatus were analyzed, since the Deiters neurones - present in this structure are directly connected to the middle - ear. The amount of RNA was found to be increased in the nerve - cells; and, more significantly, the adenine-to-uracil ratio of - both the nuclear RNA of nerve cells and glia cells became - significantly increased ([ref.119]). A variety of control - experiments were conducted. Although there was an increase in RNA - content of these cells in animals exposed to passive stimulation, - there was no change in the ratio of adenine to uracil. Nerve cells - from the reticular formation, another portion of the brain, had - only an increased content of RNA with no base-ratio change. - Animals subjected to a stress experiment involving the vestibular - nucleus showed only an increase in content of RNA. Littermates - living in cages on the same diet as learning animals showed no - change in content of RNA. Thus, it would appear that the change in - the base ratio of the RNA synthesized is not due to increased - neurone function per se, but is more directly related to the - learning process. The fact that this was nuclear RNA implies that - it was immediately related to chromosomal DNA. - (4) Neuronal RNA with changed cytosine-guanine ratios synthesized - during a short period of induced protein synthesis could be - blocked by actinomycin D. It was concluded, therefore, that the - RNA was immediately DNA dependent and directly related to the - genetic apparatus. - -Rats which were normally right handed were forced to modify their -handedness in order to obtain food. The RNA of nerve cells in that part -of the cortex, whose destruction destroys the ability to transfer -handedness, was analyzed. A significant increase in RNA of nerve cells -of the fifth to sixth cortical layers on the right side of the brain was -observed. The corresponding nerve cells on the opposite side of the same -brain served as controls. There was an increase in RNA and a significant -increase in the purine bases relative to the pyrimidine bases in the -learning side of the cortex. When the animals were not forced to learn a -new procedure, only an increase of RNA was observed, with no change in -base ratio. - -Frank Morrell, head of the Neurology Department at Stanford Medical -School, has also been active in this field during the past 6 years. He -has found that if a primary epileptic lesion is induced on one side of -the cortex, a secondary mirror lesion eventually develops in the -contralateral homologous cortex. This secondary lesion, which showed -self-sustaining epileptiform discharge, could be isolated, whereupon the -epileptiform discharge disappeared. This was interpreted as learned -behavior of the secondary lesion. From changes in the staining -properties of the secondary lesion, Morrell concluded that changes in -RNA had occurred in the cell. Changes in the composition of the RNA -could not be shown by these techniques. - -At the University of California at Berkeley, Drs. Rosenzweig, Bennett, -and Krech have conducted extensive studies related to this topic. These -investigators have directed their efforts toward demonstrating -alterations in the cerebral cortex of animals exposed to continuing -learning situations or continuously deprived of sensory stimulation. In -a recent publication ([ref.116]), which also summarizes a considerable -amount of previous work, they report studies which demonstrate the -following: - - (1) Rats given enriched experience develop, in comparison with their - restricted littermates, greater weight and thickness of cortical - tissue and an associated proportional increase in total - acetylcholinesterase activity of the cortex. - (2) The gain in weight of cortical tissue is relatively larger than - the increase in enzymatic activity. Acetylcholinesterase activity - increases in other portions of the brain even though tissue weight - decreases. - (3) The changes appear in a variety of lines of rats, although - differing in amount between strains. - (4) The changes are observed in both the young and adult animals. - -The previous studies were comparisons between experience-enriched -animals and animals maintained in isolation. Animals which were housed -in colonies, but given no special treatment, showed intermediate effects -in those situations studied. - -The Berkeley group emphasized that the finding of changes in the brain -subsequent to experience does not prove that the changes have anything -to do with memory storage, but do establish the fact that the brain can -respond to environmental pressure. However, the results are compatible -with the hypothesis that long-term memory storage involves the formation -of new somatic connections among neurones. Calculations of the amount of -additional material required to permit this to exist are compatible with -the increases observed. - -A number of investigators have studied the effects of antimetabolites -and drugs on the learning process. Since their specific metabolic -effects are known in other tissues, the rationale is that if these -materials do interfere with memory, then specific types of metabolic -activities may be implicated in the deposition of the engram. - -One of the initial studies of this type was conducted by Dingman and -Sporn ([ref.122]), presently at the National Institute of Mental Health. -They showed that 8-azaguanine, a purine antagonist, injected -intra-cisternally was incorporated into the RNA of the brains of rats. -Associated with this incorporation was an impairment of the -maze-learning ability of the animals. These findings have been -confirmed. - -Flexner and his associates injected puromycin, an inhibitor of protein -synthesis, into the brains of mice, which were then trained to perform -in a maze. Losses of short-term or long-term memory were obtained, -depending upon the site of the injection. The results indicate that the -hippocampal region is the site of recent memory. - -The hippocampal region is of interest in connection with memory -processes for a number of other reasons. Adey et al. ([ref.123]) and his -group observed a transient fall in electrical impedance in this region -when cats learned to perform in a T-maze in response to a visual cue. It -was supposed that the electrodes were situated within glial cells of the -dendritic zone of the hippocampal pyramidal cell layer. Extinction of -the learned habit abolished the briefly evoked impedance changes, which -subsequently reappeared with retraining. - -A number of other studies more or less indirectly implicate RNA in the -learning processes. For instance, in retinal cells of rabbits raised in -darkness, there was virtually no ribonucleoprotein as compared with -normal amounts in the cells of animals raised in light ([ref.124]). -Further, maintenance of normal electrical activity of isolated perfused -cat brains is highly dependent upon the presence of the ribonucleic acid -precursors, uridine and cytidine, in the perfusate ([ref.125]), and -severe derangements occur if any of a variety of pyrimidine antagonists -are added ([ref.126]). Brief electrical stimulation of cat cortical -tissue causes an increase in nucleic acid cytidine and adenine, thus -indicating a synthesis of altered polynucleotides. Finally, injections -of RNA in animals have shown interesting effects. When given at a dose -of 116 mg/kg daily for 1 month, rats showed an enhanced response and -greater resistance to extinction in a shock-motivated behavioral -response. It has been shown by another group that injections of RNA -enhance the ability of young animals to learn various tasks. - -Planaria have been used in a variety of studies which seem to bear on -the problem of memory. Quite recent evidence by Bennett, Calvin, and -their associates has cast somewhat of a pall over the studies; -nevertheless, the work may have some validity. Interest in the use of -flatworms, particularly planaria, for study of memory began with a -demonstration by McConnell that these simple animals could undergo -conditioning ([ref.127]). Subsequently, it was found that some -conditioning was retained when the animal was transected and allowed to -regenerate. The retention of training was found in both new animals, -although the very simple brain, really only two ganglia, was in the head -section ([ref.128]). - -Apparently, some diffusely distributed component of the animal was -responsible for retention of learning. Evidence has accumulated to -indicate that this material is RNA. Among this evidence is the -following: - - (1) The two halves of a trained planaria were allowed to regenerate in - a solution containing RNA-destroying enzymes. Whereas the head - ends retained some training, no retention was observed in the - animals derived from the tail end ([ref.129]). - (2) When pieces of trained planaria were fed to untrained animals, the - untrained cannibal required a shorter time to become trained to a - criterion. It would appear that the digestive system of planaria - is so simple that the material responsible for the transfer of the - information was not broken down. - (3) When RNA, obtained from trained planaria, is injected into the - digestive tract of untrained animals, there is a transfer of - information. - - - NEUROPHYSIOLOGY[2] - - [2] Excerpt from [ref.130]. - - -Neurophysiological studies concern the functions of the nervous -system--in particular the central nervous system (CNS)--under normal, -simulated, and actual flight conditions. Of paramount importance is the -maintenance of equilibrium and orientation in three-dimensional space. -The ability of man and his close relatives among the vertebrates to -maintain these functions depends on an integrated sensory input from the -vestibular organ; the eyes; the interoceptors of the muscles, tendons, -joints, and viscera; and the exteroceptors of the skin. - -Certain parameters of the environmental and space-flight conditions -drastically affect man's ability to maintain equilibrium and spatial -orientation. Centrifugal forces modify or reverse the directional vector -of gravity. Linear acceleration may increase enormously, as may angular -stimulation. The sensory organs listed above are unreliable under such -conditions. The very organ which is designed specifically to furnish -information on spatial orientation may malfunction in man while he is in -flight. Thus, with respect to sensory orientation, these labyrinthine -organs are by no means precision instruments. - -The use of classical histological methods and the observation of -equilibrium disturbances resulting from operative interference with the -internal ear have in the past been the two principal sources of -knowledge concerning the structure and function of the labyrinth, but -the answers given to various questions vary considerably in their value. -The development of electrophysiological techniques and the refinement in -recent years of the ultrastructural analysis by means of the electron -microscope may allow more precise experimental studies of the -correlation of function and structure. - -Before considering vestibular impulses in their bulbar and descending -spinal pathways, a recent study concerning the generation of impulses in -the labyrinth must be mentioned. Von Bekesy's finding ([ref.131]) of the -direct current potentials in the cochlea aroused speculation about the -existence of similar labyrinthine potentials. Such dc potentials were -also detected in the semicircular canal of the guinea pig by Trincker -([ref.132]), who measured the potential changes in the endolymph, -surface of the cupula, or side of the crista during cupular deflection. -It seems likely, however, that the effects do not represent the -physicochemical changes in the cupula but the electrical potentials in -the nerve and nerve endings of the crista. Attempts at differentiating -these effects have failed so far. Great expectations are brought by the -advances of microchemistry, microphysiology, and physical chemistry with -regard to the excitatory processes, the generation of the nerve impulse. -Quite apart from a need to understand vestibular nerve discharges and -patterns more adequately in such terms, the analysis of the vestibular -system has in the past revealed general biological principles which were -not readily discernible through the examination of other tissues -([ref.133]). - -The neural connections of the vestibular organ consist of numerous -chains of neurons, reciprocally linked in many ways and having their -synapses in various anatomical nuclei. All the chains work in intimate -collaboration, and the final pattern of reflex responses is attributable -largely to the highly complex integrating activity of the center. The -labyrinthine function is automatic, carried out in a reflex fashion: in -other words, mostly below the level of consciousness. The brain centers -through which the labyrinth elicits the various appropriate muscular -reactions of the head, body, limbs, and eyes--the righting, the -postural, and the ocular reflexes--represent an intricate mechanism. -Before we can hope for a satisfactory understanding of their functional -organization, we will have to know their anatomy in more detail. Thus, -we are confronted with a fruitful field for the exploration of basic -mechanisms of neuronal activity. Major advances dining the last years -have provided us with new information about the neuroanatomy of the -vestibular system (refs. [ref.134]-[ref.137]). - -Vestibular impulses entering the brainstem ascend and descend the -neuroaxis and cross the midline. It was previously believed that the -vestibular apparatus had only subcortical projections. Recently, -however, it has been established by means of electrophysiological -methods that the organ is represented by a projection area in the -cerebral cortex of some animals (refs. [ref.138]-[ref.141]). The use of -brief electrical stimulation of the vestibular nerve in order to elicit -a cortical response has been of great value for the mapping of these -areas. - -Among a great variety of sensory receptors, the vestibular ones are -capable of evoking the most widespread somatovisceral effects throughout -the body. Moreover, vestibular effects seem to be imperious and less -dependent upon the state of readiness of the nervous system. As a -consequence of the extensive distribution of vestibular effects, there -are many opportunities for central integration. Proprioceptive and -vestibular systems are both known to be active in posture and -locomotion; streams of impulses arising from the receptors in each of -these systems must converge to influence the activity of the final -common path. The state of the motor centers of the spinal cord, as -affected by vestibular stimulation, has been tested by dorsal root and -other sensory input interventions. These experiments have provided us -with insight into the mechanisms concerned with the vestibular control -of spinal reflexes (refs. [ref.142]-[ref.146]). - -It has long been known that the vestibular apparatus is essential for -the development of motion sickness. Commonplace subjective experience of -nausea relates to visceral changes mediated through autonomic efferent -pathways and may ultimately involve rhythmic somatic nerve discharges to -skeletal muscles responsible for retching and vomiting. However, very -little is known about the central nervous mechanisms responsible for -elaboration of the whole syndrome. Since the maintenance of vestibular -bombardment for some length of time seems essential for the development -of motion sickness, one would presume this to be an instance of slow -temporal summation. Experimental findings demonstrate a powerful effect -of temporal summation upon somatic motor outflow during vestibular -stimulation ([ref.147]), and not upon parasympathetic outflow. - -The practical implication of these studies is closely related to -physiological effects of weightlessness. Based on experimental evidence -from short weightless periods obtained in aircraft, it was concluded -that "when the exposure becomes longer, there may develop minor -physiologic disturbances which, if cumulative or irritating, may cause -or enhance psychiatric symptoms" ([ref.148]). Although the zero-g -condition, per se, does not cause spatial disorientation if visual cues -are provided, the astronauts reported a temporary loss of orientation -during the orbital flight while they were engaged in activities which -diverted their attention. However, no disturbing sensory inputs were -observed during the weightless period. Violent head maneuvers within the -limited mobility of the helmet were performed in every direction without -illusions or vertigo. The subjective sensations of "tumbling forward" -after sustainer engine cutoff reported by the Mercury astronauts, and -Titov's motion sickness attacks, which were particularly dismaying -during head movements, were well within the entire range of -psychosomatic experiences already obtained during aerodynamic -trajectories ([ref.149]). Interestingly enough it now appears that the -otolithic output in mammals including man is the differential of linear -acceleration, and therefore unaffected by zero g. - -Of interest in this connection are the problems which may be encountered -during and following long-term exposure to weightlessness. Although -there is no evidence of adverse effects on operative behavior, the -possibility of biological disturbances on a cellular or subcellular -level, which may cause a deterioration of the somatic basis, has been -repeatedly stressed. Whether effects of this sort will occur or whether -the organism will be able to adapt is still an open question. Since -motion sensitivity based on vestibular stimulation differs widely among -individuals, the selection of astronauts may solve the problem of zero-g -vestibular disturbance. Reports from the MA-8 (Sigma 7) and Vostok III -and IV flights seem to support this assumption. Moreover, experiments -are being made in the slow rotation room at the Naval School of Aviation -Medicine to study the Coriolis effects which arise when "artificial -gravity" is produced by angular acceleration. Since man can adapt to -wave motion on shipboard within a few days, a similar process may be -expected to occur in the case of long-term weightlessness ([ref.150]). - - - - - chapter 5 - -_Molecular Biology and Bioinstrumentation_ - - -To support biological investigations in space and to accumulate baseline -data needed for manned space flight, NASA has conducted a program in -laboratory research and theory. A multidisciplinary approach has -included such fields as ecology, physiology, organic and biological -chemistry, engineering, electronics, and optics. Emphasis in this -program has been placed on qualitative and theoretical rather than -purely descriptive research, and the investigation of fundamental -biological phenomena at all levels, from the molecular to the total life -form. - - - MOLECULAR BIOLOGY - -Research in molecular biology has included chemical, physical, -biological, and theoretical investigations of prebiological conditions -on Earth and, possibly, on other planets; studies of cellular -inclusions; genetic material (DNA and RNA) and coding; as well as energy -transfer in biological systems. - -The understanding of prebiological conditions on Earth, and possible -conditions on other planets, depends upon the nature of the complex -chemical species which might be encountered. Scientists have shown that -biologically important compounds, such as amino acids, can be generated -by applying an electrical discharge, ultraviolet radiation, or heat to a -gaseous mixture. Biologically interesting compounds can be removed from -such a system by condensation or absorption; however, in the limited -time and space available in such experiments, many compounds are not -produced in sufficient quantity to be measured. - -The National Biomedical Research Foundation (NBRF) and the National -Bureau of Standards (NBS) are conducting an investigation on equilibria -in multielement systems. The distribution of molecular species at -equilibrium is independent of the way equilibrium was reached and is -dependent only on pressure, temperature, and elemental composition. Many -of the conditions which might have arisen naturally can be approximated -by thermodynamic equilibrium. Compounds which can be formed at -equilibrium need no special mechanism to explain their presence. -However, special mechanisms have to be sought for those compounds which -could not be so produced and which would have been required for the -structure and nutrition of the first living organisms. - -In the absence of precise knowledge of the composition of the primitive -planetary atmospheres, equilibrium concentrations with a wide range of -temperatures, pressures, and elemental compositions are being -investigated by NBRF and NBS. These investigators have postulated that -the maximum atmospheric pressure may have approached 100 atm if the -primitive Earth was sufficiently hot and if an appreciable portion of -the water on Earth's surface today was present on primitive Earth. (If -the present oceans were to evaporate, the surface pressure would be -approximately 300 atm.) Low pressures of 10^-6 atm and temperatures -between 500 deg. and 1000 deg. K are being used. - -A large range of N, O, C, and H compositions are being investigated for -interesting and plausible combinations of factors. In these calculations -an IBM 7090 computer is being used to obtain data on a very large number -of combinations of chemicals. Other chemical species will be added as -the research continues. Some results of this study give an insight into -the variety of biologically significant chemicals which might have -existed during Earth's primitive prebiological condition or may now -exist on the surfaces and in the atmospheres of other planets (refs. -[ref.151]-[ref.153]). The general method described by White et al. -([ref.152]), minimizing the free energy of the system, was used. The -solution was approached by an iterative process, starting with an -initial guess of concentrations of the compounds. At each step, _M_+1 -linear equations are solved where _M_ is the number of elements in the -system. - -In addition to listing of the concentrations of all compounds included -in each problem, the results of three-element problems have been -expressed on a triangular composition diagram for convenience. A coarse -grid of 60 points is used to survey all elemental compositions, with -finer grids being used in regions of particular interest. The calculated -concentrations of the compounds at each composition are stored, and -finally a series of triangular diagrams is printed out, each showing the -concentrations of as many as four compounds at the grid points. - -Figure 2 shows the results obtained in the C, H, and O systems. Organic -compounds in concentrations greater than 10^-20 mole fraction are found -everywhere except where free O2, is present. Solid carbon theoretically -becomes stable along the lower dashed line at 500 deg. K. However, -reactions producing it are very slow. The supersaturated region beyond -the line of potential carbon formation was also investigated. A -threshold was found where polynuclear aromatic compounds are -sufficiently concentrated to form a liquid phase. These conditions may -have been involved in the primordial formation of asphaltic petroleum. - -[Illustration: Figure 2.--_Equilibrium diagram for the system C-H-O._] - -Jukes and associates ([ref.154]) at the University of California at -Berkeley have been investigating the code for amino acids in protein -synthesis, the key for translating the sequence of bases in DNA into the -sequence of amino acids in proteins. The amino acid code was solely a -matter of theory until Nirenberg and Matthaei ([ref.155]) at the -National Institutes of Health carried out a crucial experiment. This -experiment bridged the last remaining gap separating theoretical -genetics and test-tube biochemistry. It now became experimentally -possible to search for codes for all 20 amino acids concerned in the -synthesis of proteins. - -The amino acid bases of DNA are: A, adenine; C, cytosine; G, guanine; T, -thymine; and U, uracil, which replaces thymine in RNA. There are only 16 -ways of arranging A, C, G, and T in pairs. For this and other reasons it -is thought that a triplet of three consecutive bases is needed to code -for each amino acid. The sequences of bases in a strand of DNA are known -to be unrestricted with respect to the order in which they occur; -apparently any one of the four bases can be next to any of the other -four, although, of course, each base must be paired with the -corresponding complementary base in the adjacent strand. Since the same -freedom is true of the amino acid sequences in the polypeptide chains of -proteins, any one of the 20 amino acids can occur next to any other. -Moreover, the sequences in DNA are subject to mutational changes in -which one base replaces another, or bases are added to or deleted from -the DNA. Such rearrangements plus the possibility of lengthening of DNA -molecules are numerous enough to account for all the genetics of living -forms since the first appearance of life on Earth. - -Most of our knowledge is based on experiments with synthetic RNA carried -out with extracts of _E. coli_. The majority of the work has been at -Nirenberg's laboratory at the National Institutes of Health and at -Ochoa's laboratory at New York University ([ref.155]). Various -combinations of A, C, G, and U were used in preparing the synthetic RNA -molecules that are used in experiments to explore the code. These -molecules are made by incubating a mixture of ribonucleoside -diphosphates with a specific enzyme, polynucleotide phosphorylase. An -important property of this enzyme is that it condenses the nucleoside -diphosphates into polynucleotide strands containing random sequences -depending on the proportion of each base. For example, if the enzyme -were furnished with a mixture of 5 parts of A and 1 part of C, it would -make strands containing, on the average, 25 sequences of AAA, 5 of AAC, -5 of ACA, 5 of CAA, and 1 each of ACC, CAC, and CCA. The proportion of -triplets within the strands of a polynucleotide is reflected in the -proportion of amino acids in polypeptides that are obtained in the -cell-free system. Most of the present knowledge of the amino acid code -is based on this concept. All the proposed codes have been discovered by -this experimental approach where synthetic RNA molecules are used as -"artificial" messenger RNA. - -Representative of another class of activities in molecular biology is -the examination of passive ion flux across axon membranes. This work is -being done by Goldman at the National Naval Medical Center. The question -of stimulus transmission by nerve tissue is far from simple, and the ion -concentrations associated with nerve membranes is a significant part of -the answer. Because the space environment may very well produce -alterations in these ion potentials, an investigation of their natures -and significance becomes extremely important. A working theory is now -being developed as a result of this study. - -Vital cell processes, chemical transformations, and mechanisms that -provide energy for cell maintenance and activity have been studied by -Kiesow (refs. [ref.157] and [ref.158]) at the Naval Medical Research -Institute. The common objective of all phases of this project is the -elucidation of reaction steps in which energy and matter are transformed -in living systems. Compared with _photo_synthetic organisms, -_chemo_synthetic bacteria offer distinct advantages for the study of -energy assimilation. These studies have led to the following -experimental findings. - -With the energy from oxidation of nitrite, NO2-- to nitrate, NO3-- as an -_inorganic_ source, and with added _organic_ chemical energy from the -hydrolysis of adenosinetriphosphate (ATP) to adenosinediphosphate (ADP) -and inorganic phosphate, chemosynthetic bacteria are capable of reducing -diphosphopyridinenucleotide (DPN^+) to DPNH, in a coupled -oxidoreduction-dephosphorylation. Thus, in the crucial step of -chemosynthesis, _ATP is consumed, not produced_. However, in -simultaneously proceeding cell respiration, the energy donor, DPNH, is -oxidized and generates more ATP than is required for DPN^+ reduction. -This "breeder cycle" for DPNH--with different ratios of cell respiration -and biosynthesis--results in a net production of either DPNH, or ATP, or -both. Production of DPNH in the cycle leads immediately to the -assimilation of C^14 from HC^14 O3--. These observations explain the -bacteria's energy source without the classical hypotheses of either -direct phosphorylation or direct CO2 reduction by inorganic chemical or -electromagnetic energy. The cycle transforms the free energy of nitrite -oxidation into the free energy of the organic compounds. Cell -respiration and elementary biosynthesis proceed through structure-bound -enzyme systems in the same fraction of subcellular particles. Three -components, two cytochromes and one flavoprotein, have been identified. -A thermodynamic analysis of the DPNH "breeder cycle" appears to be -attainable by measurements of redox potentials and calorimetric -determinations of heats of reaction. - -Studies are also being conducted by Pollard and associates at -Pennsylvania State University in an attempt to formulate a theoretical -basis for the description of the processes of synthesis, growth, -division, and differentiation of the living cell. Such a theory would be -basic to an understanding of very primitive life forms or prebiological -material which might be found elsewhere in the universe. For these -purposes, studies are being undertaken in macromolecular reproduction -which differ from the studies involving cellular genetic material. -Theories concerning the problem of replication of cellular structures -and information storage in two-dimensional systems are being developed. -Theories are also being developed about the mechanisms which control and -regulate receptor and enzymatic activities within the cell. - -One study involved the rate of mutation in cells and disposed of the -suggestion that the process of mutation consists of a "tunneling" of -proton from one base to another in DNA. Such a suggestion can no longer -be advanced as a major explanation of mutations. - -Work is also being conducted on the centrifugation of cells of _E. -coli_. It has been shown that cells exposed to as little as 100 g have a -modification in their function. This has been looked at from the point -of view of thymine uptake, which would be concerned with the formation -of DNA, and also from the point of view of the induction of an enzyme, -which would correspond to the transcription of the DNA. Preliminary -experiments in the latter case indicate considerable centrifugation -effect. The thymine uptake is affected, but not nearly as much as -formerly thought. Further work is in progress in this area. - -Important work has been completed on the cells of _E. coli_ grown on -maltose, which can be induced to produce betagalactosidase by the -addition of thiomethyl galactoside. If cells are irradiated shortly -after induction, the transcription of the DNA ceases and the enzyme -produced by the messenger RNA is observed to reach a maximum. This -enables the calculation of the half-life of unstable messenger RNA. The -half-life for this decay is readily measurable, and values are given -over a temperature range of 17 deg. C (5.2 minimum) to 45 deg. C (0.56 -minimum). These agree very well with half-lives measured by others by -inducing for short times and measuring the course of enzyme formation. -The rate of transcription is involved in the kinetics of cessation of -enzyme induction, and the rate of transcription can be measured. -Arrhenius plots for this rate and the rate of decay are given, and the -activation energies measured are about 16 000 cal/mole. The cessation of -transcription is linked to the degradation, possibly of only one strand, -of DNA. - -Pollard has suggested that one important action of ionizing radiation is -concerned with the transcription of the genetic message into RNA. -Clayton and Adler ([ref.159]) showed that induced catalase synthesis in -_Rhodopseudomonas spheroides_ is inhibited by low doses of X-rays, -giving experimental support to the idea. Pollard and Vogler ([ref.160]), -using cells in which the process of induction involved permease, showed -that there is some sensitivity to gamma radiation. Novelli et al. -([ref.161]) found a reduced sensitivity as compared with colony -formation, but it is still a considerable sensitivity. - -The process of induction of an enzyme indicates that the transcription -of the genetic message is repressed by something which can be acted on -by a small molecule, the inducer, to remove repression and permit the -formation of messenger RNA, which then acts to make the enzyme. The -messenger RNA undergoes decay through a process which is still not -clear. Very elegant measurements by Kepes ([ref.162]) show that for the -messenger RNA for betagalactosidase, the half-life is 1.02 min at 37 -deg. C and 2.05 min at 25 deg. C. The time of onset of enzyme formation -after induction was found to be about 3 minutes. - -If the process of transcription is indeed sensitive to ionizing -radiation, then the irradiation of cells which have just been induced -should show formation of the enzyme to the extent of formation of new -messenger RNA within a few minutes, plus the formation of the enzyme -while the messenger RNA is decaying. This pattern was found by Clayton -and Adler. The experiments conducted by Pollard and associates amplify -and extend their work and also agree with the work of Kepes ([ref.162]). - - - BIOINSTRUMENTATION - -Fernandez-Moran (refs. [ref.163]-[ref.165]), at the University of -Chicago, has devised a new multielectrode electrostatic lens which he -has incorporated into an electron microscope. This necessitated the -development of a novel high-voltage power source and voltage regulator -of extreme stability and accuracy. Some promising work has now been done -on superconducting lenses. In a series of experiments with a simple -electron microscope without pole pieces, using high-field -superconducting niobium-zirconium solenoid lenses in an open air core, -liquid helium Dewar, electron microscopic images of test specimens have -been recorded while operating at 32 200 gauss in a persistent current -mode, with regulated accelerating potentials of 4 to 8 kilovolts. These -preliminary experiments have demonstrated the exceptional stability of -the images (both short term and long term) over a period of 4 to 8 hours -and the relatively high quality of the images. - -Progress has been made on the viscosimeter for high intrinsic -viscosities. This is now working, and the viscosity of DNA preparations -has been measured. It is hoped to use the viscosimeter to study the -variation in DNA viscosity as a function of the cell cycle. - -An instrument is under development by Wald at the University of -Pittsburgh to automatically analyze cytogenetic material and, thus, -extend cytogenetic methodology both for research and as a biological -monitoring procedure, using automatic electronic scanning and computer -analysis of chromosomes. Chromosomal aberrations can thus be monitored -under unusual and abnormal conditions such as weightlessness and -radiation, since chromosomes are very sensitive to stress situations. In -this device a sample will be prepared and automatically inserted under a -microscope lens. The device will then scan, identify, and photograph on -35-mm film a predetermined number of mitotic cells and process the film. -The data will be recorded under the direct control of a digital -computer. The computer will perform a detailed quantitative analysis of -the pictorial data. - -Significant effort has been expended in the development of -instrumentation for measuring and recording electrophysiological -information. One such instrument, developed by the Franklin Institute, -Philadelphia, Pa., is a temperature-sensing microprobe. This microprobe -is an implantable and remote broadcasting instrument. These developments -are associated, in part, with training programs so that competent -individuals may be trained not only in electronics but also in the -biological uses of the devices they construct. - -A project of interest, conducted at the Stanford Research Institute, is -the investigation of the uses of an extremely sensitive method for -measuring magnetic susceptibility having the possibility of detecting -macroscopic quantum effects in macromolecules of biological interest. -Good progress has been made in the first 15 months of a project devoted -to the development and initial use of equipment specifically designed -for this purpose. A new superconducting circuit, together with -superconducting magnetic shields, has been constructed. This apparatus -can measure the magnetic susceptibility of small organic samples at -temperatures between 1 deg. and 300 deg. K in fields up to 40 000 gauss. -It can detect flux changes of 10^7 gauss-cm squared, which is equivalent -to detecting a change in specific susceptibility of 1 in 10^9 in a -100-mg sample under an applied field of 10 000 gauss. - -Several hundred preliminary measurements were made on samples of -coronene. The most reliable of these were in agreement with published -values of the magnetic susceptibility of coronene. Experience during -these measurements led to changes which have resulted in an apparatus -well suited to the measurements on macromolecules. An improved version -of the superconducting circuit now available shows promise of a further -improvement in sensitivity by a factor of more than a thousand -([ref.166]). - -Living organisms possess many unique processes and systems which are -complex and poorly understood. The new theoretical approaches, combined -with laboratory studies, are expected to result in advances which will -expand both our scientific and technological horizons. - - - - - chapter 6 - -_Flight Programs_ - - - BALLOONS - -Biological and medical experiments carried out on balloon flights, both -manned and unmanned, antedate the establishment of NASA. Aside from the -early use of balloons in flights that could be called simply -flight-survival studies, balloons have made important contributions to -our present knowledge of the effects of cosmic radiation and to various -aspects of space travel. - -The achievements of the Strato-Lab and Man High series by the U.S. Navy -and Air Force include a wealth of information on balloon travel and on -the survival of man at altitudes close to and above 100 000 feet. -Generally, balloon launches of animals, which reached a maximum in 1953 -when 23 balloons were released, have established the feasibility of a -program of extended manned balloon flights to high altitudes. - -Atmospheric life studies outside the area of cosmic radiation effects -have been comparatively few. Results from two manned flights, Strato-Lab -I and II, indicate that the flights did produce pronounced changes in -white blood cell count; however, the data suggest that these changes -were due to psychological rather than physical stress. Exposure to -altitudes above 90 000 feet for a total of 62 hours did not produce any -general behavioral change in two Java monkeys, according to other -balloon flights. Many of these flights were effective in testing -equipment, telemetering devices, and in pointing the way for other -flights. - -Stratoscope I and II, originally undertaken by the Office of Naval -Research (ONR), are projects involving various astronomical observations -with the aid of a balloon-borne telescope and television and camera -systems. NASA cooperated with ONR on Stratoscope II (36-inch telescope -compared with Stratoscope I's 12-inch telescope) which has already -resulted in significant discoveries about the nature of the planets and -stars. Water vapor has been identified in the atmosphere of cool red -stars and an analysis of the Martian spectra showed a greater abundance -of carbon dioxide than had previously been believed. Since the -balloon-borne telescope was carried beyond Earth's obscuring atmosphere, -the Stratoscope projects have yielded valuable photographs of the Sun, -stars, and various planets. - - - ROCKETS AND SATELLITES - -Historically, biological experiments aboard rockets and satellites have -been limited to a "piggyback" and "noninterference" basis on military -rockets. For the past few years, however, as the effort toward manned -space flight leading to lunar and Martian landings increased, more -attention was devoted to experiments designed to show the effects of the -space environment on living systems. As in the balloon flight programs, -the U.S. Army, Navy, and Air Force played an important role, reaching -what might be considered a high point with the successful launch and -recovery of a ballistic rocket experiment with monkeys Able and Baker. -Aerobee rockets as well as Thor IRBM's carried biological payloads -consisting of mice and monkeys on six launches, contributing to our -knowledge of the effects of weightlessness and radiation on higher -animals. - -Van der Wal and Young ([ref.78]) used Thor-Able combinations to serve as -boosters for lifting a 20-pound biocapsule to a peak altitude of 1400 -miles and over a distance of about 5300 miles from Cape Canaveral to the -west coast of Africa. Weightlessness was attained for a period of almost -40 minutes. During reentry into the atmosphere, a peak deceleration of -about 60 g was reached. Each of the three capsules flown carried one -mouse (Mouse-in-Able); two of the mice were instrumented for heart-rate -telemetry. Although all three mice were lost, the two experiments with -Laska and Benji yielded physiological results. - -The experimenters designed effective instrumentation for registering the -electrical activity of the mouse's heart through a single commutated -telemetry channel. Records were obtained for both animals during various -portions of the flight. The results indicate that both animals were -alive when the nose cones hit the water. - -Two South American squirrel monkeys (Gordo and Baker) and a rhesus -monkey (Able) were launched into space from Cape Canaveral in 1958 and -1959 by U.S. Army Jupiter missiles. The vehicles reached speeds of -approximately 10 000 mph and altitudes of 300 miles on flights which -lasted about 15 min. - -Time courses of cardiac and respiratory rates ([ref.80]) of the two -squirrel monkeys showed that the noise of the engine at liftoff -immediately produced an increase in their heart rates. Respiration also -increased temporarily, but slowed later with increasing acceleration. -Heart rates fluctuated considerably during launch acceleration, which -reached about 15 g at cutoff. - -The period of free flight and weightlessness was characterized by -pronounced fluctuations of heart activity in the postacceleration phase. -Thereafter, the heart rate of Baker remained relatively constant, -whereas the cardiac activity of Gordo fluctuated markedly and decreased -slowly almost to the end of his flight. Slight changes, which were -transient and not pathological in nature, were also noted in the -electrocardiogram. Gordo's respiration was very shallow during maximum -launch acceleration, when Baker's reached its highest value, only to be -approximated again during reentry when forces of about 35 g were -encountered. - -Able's cardiac and respiratory rates indicated that, after an initial -startle reaction, the heart rate dropped transiently and then increased -steeply, reaching a maximum of 259 during the 10-second interval at peak -acceleration. Respiration increased only slightly throughout the -launching phase. There was a period of tachycardia during -postacceleration weightlessness, after which the heart rate declined -steadily and was disturbed only by several startling missile events. At -the end of the subgravity phase, Able's cardiac rate was slightly below -normal. - -Although the periods of high g force and free flight were short, the -extremes were considerable, and the changes from one state to the next -were rapid. In spite of this, the cardiovascular, hemodynamic, and -electrocardiographic phenomena were remarkably well maintained. -Apparently the animals were not in serious plight at any time. That -psychological factors entered into the observed phenomena is clearly -evident from the increase in cardiac rate associated with the noise of -the engine prior to liftoff and also from the cinematographic record of -facial expressions. Nevertheless, the integrated responses indicated -that the animals' physiological states remained sufficiently normal to -insure a safe flight. - - - LITTLE JOE FLIGHTS - -The first step in an attempt at animal verification of the adequacy of -the Mercury flight program was the development of two tests by NASA in -collaboration with the U.S. Air Force School of Aviation Medicine in -which there would be a biomedical evaluation of the accelerations -experienced during the abort of a Mercury flight at and shortly after -liftoff. These flights were launched at the NASA Wallops Station with a -Little Joe solid-fuel launch vehicle. - -Two Little Joe launches were made with activation of the escape rockets -during the boost phase to secure maximum acceleration; only a brief -period of weightlessness was attained. The first launch was on December -4, 1959, and the other on January 21, 1960. A 36 by 18-inch sealed, -125-pound, cylindrical capsule containing the subject, an 8-pound -_Macaca mulatta_, the necessary life-support system, and associated -instrumentation was flown in a "boilerplate" model of the Mercury -spacecraft. The rhesus monkeys were named "Sam" and "Miss Sam." - -The flight profile included maximum accelerations of about 10 to 12 g -and periods of about 3 minutes at 0+-0.02 g. The peak altitude obtained -in the last ballistic flight was about 280 000 feet. The experimental -capsule was pressurized at 1 atmosphere with 100 percent oxygen at the -start of the experiment and fell to just below a half atmosphere of -oxygen due to breathing during flight. The capsule temperature was kept -between 10 deg. and 20 deg. C in both flights. - -The measurements taken from the rhesus monkeys were the -electrocardiogram, respiration, body temperature, eye movements, and bar -pressing, but only partial results were obtained in the first flight. -Oxygen tension, total pressure, capsule temperature, and relative -humidity were recorded. Both animals were recovered alive and did not -show pathologic alterations in their physiologic and psychological -reactions. - - - MERCURY ANIMAL TEST FLIGHTS - -In the Mercury animal test program a Redstone missile carried the -chimpanzee Ham on a ballistic flight to a height of 155 miles to provide -animal verification of the success with which the Mercury system could -be applied to manned flight. The male chimpanzee was trained to perform -a two-phased reaction task during the 16 minutes of flight. The -chimpanzee Enos was put into orbit for 3 hours and 20 minutes. Results -of the two flights gave the following information: - - (1) Pulse and respiration rates during both the ballistic (MR-2) and - the orbital (MA-5) flights remained within normal limits - throughout the weightless state. Effectiveness of heart action, as - evaluated from the electrocardiograms and pressure records, was - also unaffected by the flights. - (2) Blood pressures, both arterial and venous, were not significantly - changed from preflight values during 3 hours of the weightless - state. - (3) The performance of a series of tasks involving continuous and - discrete avoidance, fixed ratio responses for food reward, delayed - response for a fluid reward, and solution of a simple oddity - problem was unaffected by the weightless state. - (4) Animals trained in the laboratory to perform during simulated - acceleration, noise, and vibration of launch and reentry were able - to maintain performance throughout an actual flight. - -From the results of the MR-2 and MA-5 flights, the following conclusions -were drawn: - - (1) The numerous objectives of the Mercury animal test program were - met. The MR-2 and MA-5 tests preceded the first ballistic and - orbital manned flights, respectively, and provided valuable - training in countdown procedures and range monitoring and recovery - techniques. The bioinstrumentation was effectively tested and the - adequacy of the environmental control system was demonstrated. - (2) A 7-minute (MR-2) and a 3-hour (MA-5) exposure to the weightless - state were experienced by the subjects in an experimental design - which left visual and tactile references unimpaired. There was no - significant change in the physiological state or performance of - the animals as measured during a series of tasks of graded - motivation and difficulty. - (3) Questions were answered concerning the physical and mental demands - that the astronauts would encounter during space flight, and it - was shown that these demands would not be excessive. - (4) It was also demonstrated that the young chimpanzee can be trained - to be a highly reliable subject for space-flight studies. - -The suborbital ballistic flight of Ham on January 31, 1961, was the -prelude to Alan R. Shepard's suborbital space flight, while the orbital -flight of Enos on November 29, 1961, preceded the orbital flight of John -H. Glenn. - -The fact that we now categorize these events as belonging to the rather -distant past, although they occurred only about 4 years ago, serves to -emphasize the pace of development in the exploration of space. While the -chimpanzee program may pale in the light of subsequent successes, its -scientific and technological contribution should not be overlooked. - -The significance of this project can be fully appreciated, and its -contribution judged, only by considering the lack of knowledge existing -at the time of its conception. In addition to its essential training -function, this project verified the feasibility of manned space flight -through operational tests of the Mercury life-support system. It -demonstrated that complex behavioral processes and basic physiological -functions remained essentially unperturbed during brief exposures to -space flight. The Mercury chimpanzee program marked the first time that -physiological and behavioral assessment techniques were combined for -evaluating the functional efficiency of the total organism in space. - -Perhaps the ultimate contribution of this program was in providing the -framework of knowledge upon which future scientific experiments on -biological organisms, exposed to flights of extended durations, must be -based. Biosatellite experiments designed to seek more subtle and elusive -effects of prolonged space flight on biological functioning will require -even more refined and difficult techniques, but will depend heavily on -the groundwork laid in these early steps of Project Mercury. - -A summary of the more important animal suborbital and orbital flights -during the period 1957 to 1964 is presented in table VII. - -In another NASA-supported flight, _NERV_ 1, various experiments were -carried in a suborbital flight of 20 minutes. _Neurospora_ molds showed -a surprisingly high level of mutation, but the control molds also had -high rates. - -The Discoverer XVII and XVIII flights, to which the Air Force -contributed, resulted in many interesting findings relative to the -responses of living systems to space flight. On the Discoverer XVII -flight, samples of human gamma globulin and rabbit antiserum specific -for human gamma globulin showed an increase in reactivity, and samples -of synovial and conjunctival cells showed no changes in their -cytological characteristics. - -Discoverer XVIII was launched during a massive solar flare which lasted -for the first 13 hours of the 48-orbit, 3-day flight. _Neurospora -conidia_, nerve tissue, algae, human bone marrow, eyelid tissue, gamma -globulin, and cancer cells were put in orbit. The results indicated that -biological specimens may be able to withstand radiation from solar -flares with a minimum of shielding and that aluminum shielding may be -better than lead. - -In 1949, the U.S.S.R. began a systematic, uninterrupted research program -in biological space experimentation. They have studied the effects of -physical stress, immune reactions, psychobiology and behavior, genetics, -and responses to environmental factors such as spacecraft dynamics and -ambient radiation. The organisms and biological materials included -tobacco mosaic and influenza viruses; T2 and T4 bacteriophage; _Bacillus -aerogenes_; lysogenic bacteria; _Clostridium butyricum_; _Escherichia -coli_; actinomycetes; yeasts; _Chlorella pyrenoidosa_; seeds of fir, -pine, onion, corn, lettuce, wheat, cabbage, carrot, buckwheat, cucumber, -beet, _Euonymus_, fennel, mustard, pea, broad bean, tomato, and nutmeg; -_Tradescantia paludosa_; _Ascaris_ eggs; snail spawn; _Drosophila -melanogaster_; loach roe; frog eggs and sperm; guinea pigs; mice; rats; -hamsters; rabbits; dogs; monkeys; human and rabbit skin; HeLa tissue -cultures and other tissues (refs. [ref.167] and [ref.168]). - - - Table VII.--_Orbital and Suborbital Animal Flights for 1957-64_ - - ---------------------------------------------------------------- - Year Animal subject Flight profile - ---------------------------------------------------------------- - United States - ---------------------------------------------------------------- - 1958 Mice _Wickie_, 1400 miles. None of the three - _Laska_, and flights were recovered. - _Benji_ - ---------------------------------------------------------------- - 1958 Squirrel monkey 300-mile maximum altitude over a - _Old Reliable_ 1300-mile distance via a Jupiter - rocket. Not recovered. - ---------------------------------------------------------------- - 1959 Rhesus monkeys 300-mile maximum altitude over a - _Able_ and _Baker_ 1500-mile distance via a Jupiter - rocket. Recovered. - ---------------------------------------------------------------- - 1959 Black mice 500 seconds of weightlessness in - Discoverer III via a Thor-Able - rocket. The Discoverer vehicle did - not go into orbit and the animals - were lost. - ---------------------------------------------------------------- - 1959 Rhesus monkey 53-mile altitude in Little Joe. - _Sam_ Recovered. - ---------------------------------------------------------------- - 1960 Rhesus monkey 9-mile altitude in Little Joe. - _Miss Sam_ Recovered. - ---------------------------------------------------------------- - 1960 C-57 black mice 650-mile altitude over a 5000-mile - distance via Atlas RVX-2A. - Recovered. - ---------------------------------------------------------------- - 1961 Chimpanzee _Ham_ 156-mile altitude over a 414-mile - distance via a Redstone booster, - Mercury capsule. Recovered. - ---------------------------------------------------------------- - 1961 Chimpanzee _Enos_ 2 Earth orbits. 183 minutes of - weightlessnessat an apogee of 146 - miles anda perigee of 99 miles. - Atlas booster, Mercury capsule. - Recovered. - ---------------------------------------------------------------- - Soviet Union - ---------------------------------------------------------------- - 1958 Dogs _Belyanka_ 280-mile altitude in hermetically - and _Pestraya_ sealed cabin. Recovered. - ---------------------------------------------------------------- - 1959 Dog _Otyazhnaya_ Over 100-mile altitude. Recovered. - and a rabbit - ---------------------------------------------------------------- - 1960 Dogs _Belka_ and 16 Earth orbits (24 hours) via - _Strelka_, 21 Sputnik V. First successful recovery - black and 21 white of living creature from orbital - mice flight. - ---------------------------------------------------------------- - 1960 Dogs _Pchelka_ and 16 Earth orbits (24 hours). - _Mushka_ Spacecraft destroyed during reentry. - ---------------------------------------------------------------- - 1961 1 dog, mice, 1 Earth orbit at an apogee of 155 - guinea pigs, and miles and a perigee of 114 miles. - frogs Recovered. - ---------------------------------------------------------------- - 1961 Dog _Laetzpochka_ 1 Earth orbit. Recovered. - ---------------------------------------------------------------- - France - ---------------------------------------------------------------- - 1961 Rat _Hector_ 95-mile attitude in a capsule - boosted by a Veronique rocket. - Recovered. - ---------------------------------------------------------------- - 1963 Cat Felicette 95-mile altitude in a capsule - boosted by a Veronique rocket. Over - 5 min of weightlessness. Recovered. - ---------------------------------------------------------------- - - - THE NASA BIOSATELLITE PROGRAM[3] - - [3] From [ref.169]. - -The space environment offers a unique opportunity to study the basic -properties of living Earth organisms with new tools and opens up new -areas of research for which biological theory fails to provide adequate -predictions. Unique components of the space environment of biological -importance are weightlessness or greatly decreased gravity, the -imposition of an environment disconnected from Earth's 24-hour rotation -(particularly its effect on biorhythms), and cosmic radiation with -energies and particle sizes unmatched by anything produced artificially -on Earth ([ref.169]). - -As progress is made in the manned exploration of space, the biological -effects of its unique environmental factors become of greater -importance. It is essential to determine the effects of space -environment on man's ability to perform physical and mental tasks. In -addition, it is necessary to develop those systems required for his -survival and for his physiological and psychological well-being, both in -space and in his subsequent resumption of normal life patterns. Despite -nearly a century of research and development in environmental -physiology, a number of phenomena will be encountered in long-term space -flight with which we have had neither the experience that would enable -us to predict the effects nor to develop the necessary protective or -remedial measures ([ref.170]). Many of the experimental programs in -bioscience are being carried out or planned so that the deleterious -effects of these phenomena may be determined, predicted, or avoided -before they are encountered in manned flight. - -Biological experimentation has been carried out in orbiting spacecraft -by Soviet and American scientists preparatory to manned space flight. -These first-generation exploratory experiments had the following -objectives: - - (1) To discover whether complex organisms could survive space - conditions and to test life-support systems - (2) To determine whether complex organisms (dogs and primates) could - survive launch, orbital space flight, reentry, and recovery - (3) To determine the effects of space radiation and any obvious - effects of weightlessness on biological organisms - -These biological studies indicate that manned space flight was -practicable, and the various cosmonaut and astronaut flights have proven -the validity of the results. - -The National Academy of Sciences' Space Science Board summer study -([ref.171]) recommended that-- - - NASA should exploit special features of the space environment as - unique situations for the general analysis of the - organism-environment relationships including, especially, the - role environmental inputs play in the establishment and - maintenance of normal organization in the living system. NASA - should support studies in ground-based and in orbiting - laboratories [biosatellites] on the biological effects of - gravity fields both above and below normal. This should be - considered a major responsibility of NASA in the area of - environmental opportunities. NASA should support studies of - biological rhythms in plants and animals including man as part - of its effort in environmental biology. Investigate by - observation of rhythms in organisms in space in (_a_) polar and - equatorial low orbits; (_b_) orbits less than, equal to and - greater than 22,000 miles. Properly designed experiments should - be conducted to explore the effects of different environmental - factors when these impinge simultaneously on test organisms. - -The Panel on Gravity of the Space Science Board ([ref.67]) stated that -the major effects of low gravity would be expected in heterocellular -organisms that develop in more or less fixed orientation with respect to -terrestrial gravity and which respond to changes in orientation with -relatively long induction periods, including the higher plants. On the -other extreme are the complex primates which respond rapidly, but whose -multiplicity of organs and correlative mechanisms make the occurrence of -malfunction and disorganization probable, but not certain. The Panel -recommended emphasis on early embryogenesis and histogenesis, -particularly of plants during exposure to low gravity, and anatomical -studies after low gravity. They stated that perturbations of the -environment to which the experimental organism is exposed must be -limited or controlled to reduce uncertainties in interpretation of -results. At the same time, the introduction of known perturbations may -assist in isolating the effects due solely to gravity. Ground-based -clinostats and centrifuges should be used in conjunction with the -experiments, and an attempt should be made to extrapolate effects of low -gravity with the clinostat. - -The study of the effects of unique or unknown space environmental -factors will probably yield unexpected results which may drastically -modify future technical approaches. The results from these biosatellite -studies will have broad application to longer term, manned space flight, -including manned space stations and lunar and planetary exploration. - -The biosatellite program is a second-generation series of carefully -planned and selected experiments, including some highly sophisticated -experiments which have required several years of baseline study and -equipment development. These orbiting recoverable biosatellites will -provide opportunities for critical testing of major biological -hypotheses in the areas of genetics, evolution, and physiology. - -The scientific community showed great interest in the biosatellite -program, and scientists from universities, industry, and Government have -submitted 185 flight experiments involving primates and other mammals, -vertebrate and invertebrate animals, micro-organisms, and plants. - -The selected biosatellite experiments include studies at the cellular, -tissue, and organism levels, including embryological development and -growth experiments at the tissue level and physiological, behavioral, -reproductive, and genetic studies at the organism level. The experiments -are divided into six categories: - - (1) Primates - (2) Mammals (nonprimate) - (3) Animal, cellular, and egg - (4) Plant morphogenesis, photosynthesis, and growth - (5) Biorhythm - (6) Radiation - -Twenty experiments have been selected for flight to study the effects of -weightlessness and decreased gravity during 3- to 30-day orbital -periods. The experiments include a wide variety of plants and animals -from single-celled organisms to higher plants and animals. The effects -of weightlessness on the primate will be studied, especially the central -nervous, the cardiovascular, and the skeletal systems during 30-day -orbits. - -Experiments have been selected to study the genetic and somatic effects -of weightlessness combined with a known source of radiation (Sr^85) to -determine if there are any antagonistic or synergistic effects -([ref.172]). Experiments are also included for studying the effects of -the unique environment of the Earth-orbiting satellite and removal from -the Earth's rotation in relation to biological rhythms of plants and -animals. - -Six biosatellites are included in the presently approved program, with -the first flight in 1966. They will be launched from Cape Kennedy by the -improved two-stage, thrust-augmented Thor-Delta into a nearly equatorial -circular orbit at an altitude of 180-200 miles for periods up to 30 -days. Recovery will be by Air Force airplane during capsule/parachute -descent. The spacecraft weigh 1000-1200 pounds, have a 280-pound -recoverable capsule and, while in orbit, will not experience greater -than 1/10 000 g of acceleration. The life-support system will provide an -environment at sea-level pressure of 80 percent nitrogen, 20 percent -oxygen, and no more than 0.5 percent carbon dioxide with a temperature -of 75 deg. F +-5 deg. F. - -All experiments are in various stages of development or testing and -flight test hardware has been and is being constructed. The experiments -and hardware are being subjected to preflight tests simulating launch -and recovery stresses. Rhesus, pigtail, and squirrel monkeys have been -subjected to the dynamic forces of the simulated flight under conditions -of complete, partial, and no restraint. Three types of centrifuges have -been used to simulate the flight profile. Primates were fully -instrumented with deep brain electrode implants, implanted catheters, -and other implanted sensors. During centrifugation, motion pictures were -taken. These primates were semirestrained in form-fitted couches which -allowed movement of the body while facing the accelerative force in a -ventrodorsal position (eyeballs in). In this series of tests, all -primates were normal following the tests and exhibited no unusual -behavior or effects. X-rays showed that implanted catheters and -electrodes remained in place, and there were no movements causing tissue -damage. However, when the primates were placed with their backs toward -the accelerative force, dorsoventral (eyeballs out), the animals -suffered visible damage. At 6 g there was no visible stress, but at 8 g -swelling of the lower eyelids was noticeable. At 11 g both eyelids were -swollen shut. In the biosatellite program, primates will be placed in -the semirestraint couches in a position facing accelerative forces, -ventrodorsal (eyeballs in), to prevent these effects. - - - - - chapter 7 - -_Manned Space Flight_ - - - BIOREGENERATIVE LIFE-SUPPORT SYSTEMS - -Placing a man in space requires a complete life-support system capable -of supplying sufficient oxygen, food, and water and removing excess -carbon dioxide, water vapor, and human body wastes. In addition, the -oxygen, carbon dioxide, and pressure must be maintained at a suitable -level. Any accumulated toxic products and noxious odors must be removed. - -In the spacecraft the human is confined in a restricted environment in -which it is necessary to establish a balanced microcosm or closed -ecological system. This is an enormous biological and bioengineering -problem. Weight, size, simplicity of operation, and reliability -particularly are important factors. - -For relatively short missions involving one or several astronauts, food, -oxygen, and water can be stored and made available as required, and the -various waste products can be stored. On longer missions, particularly -those involving more than one astronaut, efficient chemical or -biological regenerative systems will be required. Any regenerative -system introduces a fixed cost in weight of processing equipment and -energy requirements. - -Chemical, or partially regenerative, methods for providing breathing -oxygen by the regeneration of metabolic products such as water vapor and -carbon dioxide include the thermal decomposition of water and CO2, -photolysis and radiolysis of water, electrolysis of fused carbonates and -aqueous solutions, and the chemical reduction of CO2 with H2, followed -by electrolysis of the water formed. Chemical regenerative systems have -been developed to remove excess carbon dioxide and water vapor from the -atmosphere. Nonbiological regenerative systems are time limited by the -amount of food, water, and oxygen that can be carried or recovered. -These physical-chemical processes show great potential, but they also -present many difficulties, including requirements for extremely high -temperatures and considerable amounts of power, the formation of highly -toxic materials, and high susceptibility to inactivation. None of the -presently studied nonbiological processes can function as completely as -a bioregenerative system. All these nonbiological systems have -unrealistic supply requirements and produce unusable wastes. -Consequently, for long planetary missions the bioregenerative systems, -though also beset with problems, are potentially far superior to their -physical and chemical counterparts. - -Table VIII shows average daily metabolic data for a 70-kg astronaut. A -man breathes about 10 cubic feet of air per minute, or 400 000 liters, -daily. The expired air contains about 4 percent carbon dioxide. Man -normally breathes air containing 0.03 percent CO2, but can withstand -comfortably about 1.5 percent CO2. Anything in excess of 1.5 percent -will produce labored breathing, headaches, and, if greatly exceeded, -death. A man exhales about 1.1 pounds of water per day and this, in -addition to water from perspiration and other sources, must be removed -from the air. - - - Table VIII.--_Average Daily Metabolic Data for a 70-kg, - 25-Year-Old Astronaut With Normal Spacecrew Activity_ - [From [ref.173]] - - ----------------------------------------------------------------- - O2 input, kg 0.862 - ----------------------------------------------------------------- - CO2 output, kg 1.056 - ----------------------------------------------------------------- - Drinking water, liters 2.5 - ----------------------------------------------------------------- - Food rehydrating water, liters 1 - ----------------------------------------------------------------- - Caloric value of food, kcal 3000 - ----------------------------------------------------------------- - Water output: - ----------------------------------------------------------------- - Urine, liters 1.6 - ----------------------------------------------------------------- - Respiration and perspiration, liters 2.13 - ----------------------------------------------------------------- - Feces, kg 0.09 - ----------------------------------------------------------------- - Total heat output, Btu 11 100 - ----------------------------------------------------------------- - - -Two types of biological regenerative systems have been proposed. The -photosynthetic closed ecological system was proposed as early as 1951. -This involves the use of single-celled algae or higher plants, including -floating aquatic and terrestrial plants, and requires the interaction of -light energy with CO2 and H2O to produce O2 and plant cells. Another -system, proposed in 1961, involves electrolysis of water into oxygen and -hydrogen, and the concurrent use of _Hydrogenomonas_ bacteria which take -up hydrogen, some oxygen, carbon dioxide, and urine yielding water and -bacterial cells. - - - Table IX.--_Requirements for Regenerative Life-Support Systems_ - --------------------------------------------------------------------- - Requirements / Requirements / - 1 man[4] 3 men (270 man-day - System mission)[5] - --------------------------------------- - Weight, Power, Weight, Power, - kg kW kg kW - --------------------------------------------------------------------- - Partial chemoregenerative [7]332 1.75 - --------------------------------------------------------------------- - LiOH 125 1.40 - --------------------------------------------------------------------- - NaOH 155 7.68 - --------------------------------------------------------------------- - CO2-H2 34 .36 - --------------------------------------------------------------------- - Full bioregenerative--algae: - --------------------------------------------------------------------- - Artificial illumination 116 [6]10.40 591 25.00 - --------------------------------------------------------------------- - Solar illumination 103 1.70 356 .60 - --------------------------------------------------------------------- - Electrolysis-_hydrogenomonas_ 55 .25 129 2.60 - --------------------------------------------------------------------- - - [4] From [ref.174]. - - [5] From [ref.175]. - - [6] From [ref.176]. - - [7] Includes instrumentation and food storage. - - -The values given in table IX indicate relative weights and powers -required by various systems to provide the gaseous environment for -manned space cabins. If one considers operating temperatures and -hazards, other systems may offer advantages which offset the weight and -power advantages of the hydrogen reduction of LiOH systems. - -Research is being conducted by NASA on life-support-system technology -applicable to missions planned for 20 years in the future. Life-support -systems include the requirements for supplying breathing gases, control -of contaminants in the cabin atmosphere, water reclamation, food supply, -and personal hygiene. The disciplines involved in such systems include -biology and microbiology, cryogenic fluid handling at zero g, heat -transfer, and thermal integration with other systems, such as power. The -physiological, psychological, and sociological problems of the crew are -also being considered. - - -Photosynthetic System - -Green plants contain chlorophyll which captures light energy -thermodynamically required to convert carbon dioxide and water into -carbohydrate which can subsequently be transformed into other foods such -as protein and fat. During this process, carbon dioxide is consumed, and -an approximately equal amount of oxygen gas is liberated. As a first -approximation, photosynthesis is the reverse of the oxidative metabolism -of animal life: - - Oxidation - C6H12O6 + 6O2 --------------> 6CO2 + 6H2O + heat - - Photosynthesis - 6CO2 + 6H2O + light --------------> C6H12O6 + 6O2 - -The photosynthetic process in plants and respiration during -photosynthesis have been studied intensively, and several metabolic -pathways have been elucidated. Mechanisms are being studied to explain -the inhibitory effect of strong visible light on this process. This -program may lead to the use of chloroplasts or chlorophyll without cells -in future photosynthetic bioregenerative systems for long-term space -travel. - -One of the prime considerations of a closed ecological system is that -the environmental gases shall remain physiologically tolerable to all of -the ecologic components. Ideally, a photosynthetic gas exchange organism -should possess a high ratio of gas exchange to total mass (considering -all equipment and material incidental to growth, harvesting, processing, -and utilization); and a controllable assimilation rate to maintain -steady-state gas composition. It should also be (1) amenable to -confining quarters which may be imposed by inflexibility of rocket or -space station design; (2) genetically and physiologically stable and -highly resistant to anticipated stresses; (3) edible and capable of -supplying most or all human nutritional requirements; (4) capable of -utilizing raw or appropriately treated organic wastes; and (5) amenable -to water recycling as demanded by other components of the ecosystem. - - -Higher Plants - -Efforts to utilize multicellular plants as photosynthetic gas exchangers -have been somewhat neglected, since it has been assumed by many that -algae would be more efficient. The family _Lemnaceae_ (duckweeds) are -small primitive aquatic plants with a minimum of tissue differentiation. -Practically all of the cells of the plant contain chlorophyll and are -capable of photosynthetic activity. They reproduce principally by -asexual budding of parent leaflike fronds. They can be grown readily on -moist surfaces ([ref.177]) on almost any medium suitable for the growth -of autotrophic plants. With duckweeds the problems of gaseous exchange -and harvesting are simplified and the volume of medium can be greatly -decreased as compared with algae. - -Ney ([ref.177]) obtained a very high gas exchange rate with duckweeds. -Using small cultures under controlled optimal conditions of temperature, -light (600-1000 ft-c), and CO2, concentration, he estimated that 2.3 m -squared of frondal surface of duckweed, at a gas exchange rate of 10.8 -liters m squared/hr would provide sufficient gas exchange for one man. -This would produce about 25 grams of dry plant material per hour. - -A few nutritional studies have been carried out with duckweeds. Nakamura -([ref.178]) considered _Wolffia_ as a possible source of food for space -travel and found that it contained carbohydrate 25-60 percent, protein -8-10 percent, fat 18-20 percent, minerals 6-8 percent (all dry weights), -and vitamins B2, B6, and C, with C the most abundant. - -One of the desirable features of a duckweed system is that the gas -exchange is direct between the atmosphere and the plant and does not -require dissolving the respiratory gases in a bulky fluid system which -introduces special engineering difficulties in zero- or low-gravity -conditions. - -In the design of equipment for photosynthetic studies, careful -consideration should be given to the material used in the construction -of the unit. Most plastic materials are subject to photo-oxidative -degradation, with CO as one of the products. When air is recirculated -through plastic tubing and transparent rigid plastics in the presence of -light, considerable quantities of CO are given off. With high-intensity -illumination such as sunlight, a CO buildup of several hundred parts per -million is not uncommon. Also, plant pigments such as the carotenoids -and chlorophylls will react similarly when exposed to light of high -intensity. If the plants die, then CO is released quite rapidly. - -At Colorado State University the responses of plants to high-intensity -radiation (ultraviolet to infrared) are being studied. Plants from high -mountaintops that are exposed to greater ultraviolet light are being -studied for specialized adaptations. The effect of temperature on -photosynthesis is being explored. Various plants are also being studied -under germ-free conditions. - -Screening of higher plants for possible use in bioregenerative systems -at Connecticut Agriculture Experiment Station resulted in the selection -of corn, sugarcane, and sunflower. Under optimal conditions it has been -shown that 100 to 130 ft squared of leaf surface are required to support -an astronaut. - -Plants considered as possible food sources include soybeans, peanuts, -rice, and tomatoes, which can be combined with algae to give a -well-balanced and reasonably varied diet. Hydroponic systems use large -quantities of water, but progress is being made in reducing this. - -The possibility of using animals in the closed ecological system is open -to question, particularly in the absence of gravity, and much work -remains to be done on using plant materials as animal food and on the -disposal of wastes. Animals which have been considered are crustaceans, -fish, chickens, rabbits, and goats. - - -Algae - -Algae have the fastest growth rate and are among the most efficient -plants for oxygen and food production. It has been amply demonstrated by -Myers ([ref.179]) and other workers that _Chlorella_ can be used in a -closed ecological system to maintain animals such as mice and a monkey. -The use of algae for supplying O2 and food, and for removing CO2 and -odors has been considered by many authors for use in spacecraft, space -platforms, and for establishing bases on the Moon or Mars. - -Estimates of total efficiency are based on extrapolated laboratory data -and vary widely, since many different types of data have been used as a -basis for these estimates. - -The respired air containing about 4-5 percent CO2 is bubbled into the -_Chlorella_ culture, at either atmospheric or increased pressure. Air -containing a high percentage of oxygen and saturated with moisture is -released from the algal system. - -The use of algae for several purposes might require from one to three -separate algal systems. For food production, _Chlorella_ produces 50 -percent protein and 50 percent lipids in high-nitrogen media. In -low-nitrogen media, it produces 85 percent lipids. Proper choice of -_Chlorella_ strains and media will produce not only the necessary -calories but also the necessary specific nutrients required. Certain -strains are more effective in O2 production, and others in the use of -urine and other wastes. - -Some of the early estimates, using _Chlorella_ grown at 25 deg. C, for -supplying these requirements for a single man in space include the -following: 168 kg of algal suspension ([ref.179]), 200 kg of algal -suspension and 50 kg of equipment including pumps (refs. [ref.180] and -[ref.181]), and 100 kg of algal suspension and 50 cubic feet for -equipment and gas exchange ([ref.182]). Using the blue-green alga -_Synechocystis_, 600 kg of algal suspension would be required, according -to Gafford and Craft. These estimates are based on preliminary studies, -are quite high, and are not of real practical value. - -Other studies have indicated an extremely efficient algal system which -offers a real potential for a practical and effective gas exchanger -([ref.183]). A thermophilic strain of _Chlorella_ with an optimum growth -temperature of 39 deg. C and an optimum temperature for photosynthesis -of about 40 deg. C can increase its cell mass 10 000-fold per day. When -operating at one-half maximum efficiency, this alga produces 100 times -its cell volume of oxygen per hour. Burk et al. ([ref.183]) state: -"Future engineering development should lead to a space requirement, per -adult person, of no more than 3 to 5 cubic feet of algal culture, -equipment, and instrumentation for adequate purification of air." The -requirements of this system would require additional energy in the form -of light and of small amounts of nitrogenous and mineral material for -the algae. The light source used by Burk et al. ([ref.183]) is a -tungsten filament quartz lamp the size of a pencil, which has a long -life, produces a luminous flux 5-10 times greater than sunlight on -Earth, and operates at a 10-12 percent light efficiency. - -Research is being carried out on algal regenerative systems by about 40 -or 50 laboratories in the United States. NASA is supporting several -basic studies on photosynthesis, the physiology of algae, and -engineering pilot-plant development. Much of the research on algae is -being supported by the Air Force. - -Most algal studies have been carried out in small units and the data -obtained have been used as a basis for extrapolating logistic values for -the use of these organisms in manned space vehicles. Myers ([ref.179]) -has shown that the quantity of algae necessary to support a man (with an -assumed O2 requirement of 625 liters per day) would yield about 600-700 -grams dry weight of new cells per day. If algal growth in mass cultures -could be maintained in a steady-state concentration of 2.5 gram dry -weight per liter with such a growth rate as to yield 10 grams weight per -liter per day, the volume of algal culture would be 60-70 liters and the -total mass of the system would approximate 200-250 pounds. - -Using an 8-liter system, Ward et al. ([ref.176]) have produced algal -concentrations of 5-7 grams of dry algae per liter with a -high-temperature algal strain. The maximum growth rate observed with the -culture was 0.375 gram dry weight per liter per hour, or 9 grams dry -weight per liter per day. This was accomplished by using 1-centimeter -layers of culture and a light intensity of 8000 foot-candles. The -culture system consisted of a rectangular plastic chamber having an area -of 0.5 square meter and illuminated on each side to an intensity of 4000 -foot-candles (cool-white). To produce 25 liters of oxygen per hour, an -area of 8.3 square meters (85 square feet) would be required. - -The major problem in large-scale production of algae is that of -illumination. Conversion of electricity to light has an efficiency of -only 10 to 20 percent. In addition, the maximum efficiency of light -utilization by _Chlorella_ algae lies in the range of 18-22 percent. -This results in a maximum efficiency of only 4 percent for -photosynthetic systems. Another problem involved in conversion of -electricity to light is the production of heat which has to be removed -even with thermophilic algae. With a human demand of 600 liters of -oxygen per day, the minimum electrical requirement becomes 4 kW. No -large-scale culture has yet been managed at anything close to this -minimum figure. - -Another problem is the poor penetration of light into concentrated -cultures of algae. This necessitates construction of large tanks of only -about 1/4-inch thickness. This results frequently in fouling of the -surfaces of the tank by algae and makes the removal of the excess algae -difficult. Production of 1 liter of oxygen results in the production of -1 gram dry weight of algae. Although a small amount of CO is produced by -some algae, it can probably be removed by catalytic oxidation. Other -problems include mutation and genetic drift of the algae and the -necessity for maintaining bacteria-free cultures. There are also -difficulties in maintaining a sterile culture if urine is to be used as -a nitrogen source. While there is a potential for using algae as food, -more research is required before it can be determined what quantity and -methods of processing can be used. Research and development on algae is -much greater than on both the higher plants and the -electrolysis-_Hydrogenomonas_ systems together. - -The difference between the photosynthetic and -electrolysis-chemosynthetic systems is the way electrical energy is made -available to the organisms. In the photosynthetic system, electrical -energy is converted to light which the algae or plants transform into -chemical energy. In the chemosynthetic process, electrical energy is -transformed into the chemical energy of hydrogen gas which is used by -the bacteria. Both organisms use the chemical energy available to them -to synthesize cell material with similar degrees of efficiency. The -problem is to make the conversion of electricity to available chemical -energy as efficient as possible. - -In photosynthetic systems much energy is lost in the conversion of -electricity to light, a process only 10-20 percent efficient at best. -When this is combined with the loss from the inefficient use of light by -plants, an overall efficiency of about 4 percent is obtained. In the -electrolysis-_Hydrogenomonas_ system, the two steps are very efficient. -Electrolysis cells can operate at up to 85 percent efficiency and the -overall efficiency can be up to seven times that of a photosynthetic -system. - - - ELECTROLYSIS-_HYDROGENOMONAS_ SYSTEM - -Electrolysis is carried out in a closed unit containing an electrolyte -(KOH solution) with an anode and a cathode. These cells produce a -maximum yield (60-80 percent or more) in gas production per unit of -power consumption. According to Dole and Tamplin ([ref.184]), a unit -capable of producing enough oxygen to sustain one man would be highly -reliable, weigh approximately 18 kg, and require a power input of 0.25 -kW. - -One approach to zero-gravity operation is to rotate the electrolysis -cell as described by Clifford and McCallum ([ref.185]) and Clifford and -Faust ([ref.186]). The smallest known electrolysis cell under -development uses this artificial gravity to separate oxygen from the -anode and electrolyte, while the dry hydrogen gas permeates through the -foil cathode, fabricated from palladium-silver alloy. This electrolysis -cell, which would provide breathing oxygen for three men, has a volume -of 1.4 liters, weighs 4.5 kg, and requires 0.67 kW, excluding auxiliary -equipment, and has an efficiency of 84 percent. - -The chemosynthetic conversion is carried out by the hydrogen bacteria. -By the oxidation of molecular hydrogen, supplied from the electrolysis -of water, energy is made available for biosynthesis. The generation of -this "biological energy" is mediated by the stable enzyme hydrogenase -which is present in the bacteria. On the average, the oxidation of 4 -moles of H2 is required for the conversion of 1 mole of CO2 (the hourly -production of a man). The removal of this amount of CO2 would thus -require the cleavage of 4 moles of water. In addition, to supply oxygen -for human respiration (at a rate of 1 mole of O2 per hour) the cleavage -of two additional moles of water is required. Therefore, the -chemosynthetic regeneration and human respiration together would -require, on the average, the splitting of 6 moles of water per hour. - -The material balance for electrolysis, biosynthesis, and human -metabolism, with gram molecular weights in parentheses, are shown in -equations (1) to (3), respectively: - - 6H2O --------------> 3O2 + 6H2 - (108) --------------> (96) + (12) (1) - -The bacterial synthesis requires 6 moles of H2, 2 moles of O2, and 1 -mole of CO2 (from the astronaut), as shown in equation 2: - - 6H2 + 2O2 + CO2 --------------> CH2O + 5H2O - (12) + (64) + (44) --------------> (30) + (90) (2) - -The respiration of the astronaut requires 1 "food" mole (CH2O) -representing about 120 kcal, and 1 mole of O2, as shown in equation 3: - - CH2O + O2 --------------> CO2 + H2O - (30) + (32) --------------> (44) + (18) (3) - -The metabolic data in table VIII show that the CO2 of the astronaut and -the bacteria must balance at about 1.056 kg per day. - -The water relations are not completely balanced, but are fairly close. -About 2.6 liters per day of water are split by electrolysis. The -astronaut has an intake of 3.5 liters of water per day, 2.5 liters for -drinking and 1 liter for preparing dehydrated food. The output is about -1.6 liters of urine and 2.1 liters of water of respiration and -perspiration per day, or a total output of 3.7 liters, with the -0.2-liter excess due mainly to water of metabolism. The -bacteria-produced water, amounting to 2.2 liters per day, and the excess -from the astronaut would supply 2.4 liters toward balancing the 2.6 -liters of water electrolyzed. - - -Bacterial Culture - -Hydrogen bacteria are characterized by their ability to metabolize and -multiply in a strictly inorganic medium, when supplied with H2, CO2 and -O2 in required amounts. They can be grown in batch culture or in -continuous culture using different methods of supplying entire medium or -components on a demand feed system. - -A medium was developed for batch culture of _Hydrogenomonas eutropha_ by -Repaske ([ref.187]) with quantitation of a number of components -including trace minerals. Experiments by Bongers ([ref.188]) showed that -a simplified medium, using laboratory-grade chemicals, could be used. A -definite requirement was found for magnesium and ferrous iron (Fe^++). -The optimal growth requirements observed for _Hydrogenomonas eutropha_ -are shown in table X. - - - Table X.--_Optimum Growth Requirements of_ - Hydrogenomonas eutropha - - --------------------------------------------------------- - Culture parameter Optimum value - --------------------------------------------------------- - Cell density, g (dry weight)/liter 10 - --------------------------------------------------------- - Temperature, deg. C 35 - --------------------------------------------------------- - Pressure, atm 1 - --------------------------------------------------------- - pH (phosphate buffer) 6.8 (6.4-8.0) - --------------------------------------------------------- - H2, percent 75 - --------------------------------------------------------- - O2, percent 15 - --------------------------------------------------------- - CO2, percent 10 - --------------------------------------------------------- - Urea CO(NH2)2, g/liter 1 - --------------------------------------------------------- - MgSO4.7H2O, g/liter 0.1 - --------------------------------------------------------- - Fe(NH4)2 (SO4)2, g/liter 0.008 - --------------------------------------------------------- - - -The effects of temperatures ranging from 20 deg. to 42.5 deg. C on the -growth rates of _Hydrogenomonas eutropha_ were studied by Bongers -([ref.189]), and the optimal temperature was found to be about 35 deg. -C. Experiments at 25 deg. and 35 deg. C indicated that the efficiency of -energy conversion was essentially identical at both temperatures. -_Hydrogenomonas_ requires, as part of its substrate, a mixture of three -gases: hydrogen, oxygen, and carbon dioxide. Experiments were performed -by Bongers ([ref.189]) to determine the toleration limits of the three -gases. Growth rates were found to be identical when hydrogen varied from -5 to 80 percent. Nearly identical growth was obtained when CO2 partial -pressures were 5 to 60 percent, being slightly lower at higher partial -pressures. The organism was highly sensitive to oxygen concentration. -Dissolved oxygen concentrations above 0.13 mM were found to inhibit cell -division; energy utilization was also affected by oxygen concentration. -At 0.2 mM oxygen concentration, the efficiency of energy conversion was -approximately half the value observed with 0.05 mM. - -Another parameter of importance is the total volume of suspension which -would be required to balance the metabolic needs of one man. The volume -of suspension is determined by the conversion capacity of a unit volume. -This capacity is a function of the cell concentration; hence, the more -cells that can be packed in a unit volume of suspension (and adequately -provided with H2, O2, and CO2), the less the volume of suspension -required. - -Results of experiments by Bongers (refs. [ref.190] and [ref.191]) on -conversion capacity-density relationships show that the rate of CO2 -conversion obtained with suspensions up to approximately 10 grams (dry -weight) per liter is linear with relation to density. This indicates -that the supply of H2, O2, and CO2 is adequate. Upon a further increase -in cell concentration, the conversion rate still increases but not -linearly. The highest amount of CO2 taken up per liter of suspension was -approximately 2 liters per hour. At these very high cell concentrations, -the relationship between rate of conversion and density is no longer -linear. This is demonstrated when the conversion rate is calculated per -unit cell weight instead of per unit suspension volume. The rate per -gram dry weight per liter decreases from 146 to 68 ml of CO2 per hour. -With a suspension at a density of approximately 10 grams, the conversion -of 1.1 liters of CO2 per liter per hour is obtained. At a CO2 output of -22 liters per man per hour, 20 liters of suspension would be sufficient -to balance the gas exchange needs of one man. - -At higher cell concentrations, less volume of suspension would suffice -if gas equilibration could be maintained at the higher consumption rates -to avoid anaerobic conditions which could lead to a shift in metabolism. -In the final analysis, the technical problem of gas transfer from the -gas to the liquid phase determines the optimal cell concentration and, -therefore, the required suspension volume. - -From data presently available, it can be concluded that, using the -slow-growing _H. facilis_, the volume of suspension required to support -one man is about 500 liters. Using _H. eutropha_, Schlegel ([ref.192]) -calculated a suspension volume of 66 liters with 1 gram dry weight of -bacteria per liter. - -In recent NASA-supported research, the amount of culture medium has been -estimated using improved cultivation methods and conditions. For batch -culture, the data show that from 10 to 66 liters would be required per -man, with a best practical estimate of 20 liters at 9 to 10 grams dry -weight of bacteria per liter ([ref.191]). For continuous culture using -the turbidostat, the present data indicate a demand for some 30 liters -of suspension, and a volume of 20 liters (at approximately 10 grams dry -weight of bacteria per liter) as a realistic goal. - -In the foregoing section, the material balance for gases and water was -discussed. It was shown that a close match could be obtained with these -components of the closed environment. - -Less abundant, though no less important, are the nonwater components of -urine and feces. The urine is important for the content of fixed -nitrogen and other products of man's metabolism and serves as a very -effective substrate for cultivation of hydrogen bacteria. Maximum -closure of the system necessitates utilization of the urea in urine as a -nitrogen source. - -The average man produces 1.2 to 1.6 liters of urine per 24-hour period. -This contains about 0.00005 gram per liter of iron, 0.113 gram per liter -of magnesium, and 24.5 grams per liter of urea ([ref.193]). As shown in -table X, each liter of bacterial medium requires 0.008 gram per liter of -Fe(NH4)2 (SO4)2, about 0.1 gram of MgSO4.7H2O, and 1.0 gram per liter of -urea. In comparing the daily urine output with the estimated required -ingredients of a bacterial medium, a relatively close balance is -observed, with the exception of iron. - -For the fixation of 24 moles of CO2 (288 grams of C) produced per man -per day, the production of about 640 grams dry bacterial mass is -required. At an average N-content of 12 percent, the nitrogen -requirement would be some 100 grams. A comparison of daily output -(urine) and daily requirement by the bacterial suspension reveals that -only 10 to 33 percent of this amount could be recovered from average -urine. To obtain a material balance, either the man must be fed a -protein-rich diet or the bacterial suspension must be grown under -conditions which lead to the production of a cell mass relatively low in -protein content. Experiments have indicated that nitrogen starvation of -the bacterial culture might be a promising solution. Culture "staging" -(cultivation under nitrogen-rich conditions, followed by cultivation in -the absence of substrate nitrogen and subsequent harvesting for food -processing) will probably be the most promising means of nitrogen -economy in the closed environment. As discussed in a following section, -a biomass of relatively high lipid content can be obtained under -conditions of nitrogen starvation. - - -Continuous Culture of _Hydrogenomonas_ Bacteria - -Growth of hydrogen bacteria in a batch culture, after an initial period -of adjustment, becomes steady and rapid during the exponential growth -phase. This steady state of growth is temporary and ceases when nutrient -substrate or gas concentrations drop to limiting values. For long -periods a continual supply of nutrients must be provided. Growth then -occurs under steady-state conditions for prolonged periods, and such -factors as pH, concentration of nutrient, oxygen, and metabolic products -(which change during batch culture) are all maintained constant in -continuous culture. - -Two methods can be used for control of continuous cultures: the -turbidostat and the chemostat. In the turbidostat, regulation of medium -input and cell concentration is controlled by optically sensing the -turbidity of the culture. - -The dilution rate varies with the population density of the culture and -maintains the density within a narrow range. Organisms grow at the -maximum rate characteristic of the organism and the conditions. The -growth rate can be changed by modifying the nutrient medium, gas -concentration, or incubation temperature. A disadvantage of the -turbidostat is that all nutrient concentrations in the culture chamber -are necessarily higher than the minimum, resulting in inefficient -utilization of nutrients. - -The turbidostat system for continuous culture of _Hydrogenomonas_ -bacteria, developed by Battelle Memorial Institute ([ref.194]), includes -electrolysis of water in a separate unit. Hydrogen and oxygen are fed -separately up to the point of injection into the culture vessel, and the -mixed volume is kept very small to minimize am possibility of explosion. -However, the two gases may be injected simultaneously if there is a -demand for both. - -In the chemostat, growth of the organisms is limited by maintaining one -essential nutrient concentration below optimum. A constant feed of -medium, with one nutrient in limiting concentration and with constant -removal of culture at the same rate, is used to achieve the steady -state. The dilution rate is set at an arbitrary value, and the microbial -population is allowed to find its own level. By appropriate setting of -the dilution rate, the growth rate may be held at any desired value from -slightly below the maximum possible to nearly zero. This constitutes a -self-regulating system and allows selection of a desired growth rate. - -A combined electrolysis-chemostat method, developed by Magna Corp., -maintained the hydrogen-producing electrode of an electrolysis cell in -the bacterial culture. Resting cells of _Hydrogenomonas eutropha_ -consumed hydrogen produced at the cathode of an electrolysis cell built -into a specially constructed Warburg flask. Attempts to immobilize -_Hydrogenomonas_ cells on a porous conductor were partially successful. -This system could lower the volume requirements compared with those for -the isolated subsystems. Disadvantages of this integrated system include -electrolysis of the bacterial medium, possibly resulting in toxic -breakdown products, and the possible effects of electric power and the -KOH electrolyte on the bacteria. The main disadvantage of an integrated -system would be the disparity between optimal conditions for efficient -electrolysis and efficient bacterial conversion, particularly -temperature and pH, with the combination possibly resulting in -considerably higher power and weight demands. - -Both continuous-culture approaches are being studied with NASA support. -The turbidostat offers the greatest potential efficiency in weight and -volume, but uses nutrient materials less efficiently and is more -complex. The chemostat is less efficient in weight and volume, but has -greater simplicity and reliability. - -_Hydrogenomonas eutropha_ has been grown in 15-liter batch cultures and -in 2.1-liter continuous cultures. A 20-liter continuous culture, -sufficient to balance the requirements of a man, is under development. - -The potential problem areas in large-scale continuous production of the -bacteria include assuring genetic stability, preventing or controlling -bacteriophage and foreign bacterial contamination, and preventing -heterotrophic growth caused by exposure to organic material from the -urine. Genetics of hydrogen bacteria and phage infection have been -studied by DeCicco. Research on these problems indicates that they are -not of major importance, but cause significant effects and must be -eliminated or controlled. - - -Bacterial Composition and Nutrition - -_Hydrogenomonas_ bacteria can be used for at least part of the -astronauts' diet. The washed bacteria have a mild taste and are being -studied for their total energy content, protein and lipid digestibility, -and vitamin content. Carbon and nitrogen balances, and respiratory -quotient are to be determined in animals fed the bacteria as their sole -food source. No toxic constituents have been discovered. Sonicated and -cooked bacteria, when fed to white rats as 12 percent of the solids of a -nutritionally balanced diet, were eaten readily and produced no ill -effects. Net utilization of the protein appears to be somewhat lower -than casein and about the same as legume proteins. - -The composition of _Hydrogenomonas eutropha_ is shown in table XI. The -composition of the bacteria varies with the age and growth phase of the -cells and with the medium and gas available. It is possible to modify -the growth conditions to grow the type of bacteria desired for nutritive -purposes. - -_Hydrogenomonas_ cells contain about 75 percent water. Of the dry -weight, about 74 percent is protein, calculated as 6.25 times the -nitrogen content. Table XI shows the amino acid composition to be -comparable with other bacterial proteins, except for higher tryptophan -and methionine values. - - - Table XI--_Analysis of_ Hydrogenomonas eutropha _Cells Grown in - Continuous Culture_ [From [ref.194]] - - ----------------------------------------------------------------- - Constituent Percent by weight - ----------------------------------------------------------------- - Moisture 74.55 - ----------------------------------------------------------------- - Fat .44 - ----------------------------------------------------------------- - Ash 1.73 - ----------------------------------------------------------------- - Nitrogen 3.02 (wet) - ------------------- - 11.87 (dry) - ----------------------------------------------------------------- - Protein (N x 6.25) 18.90 (wet) - ------------------- - 74.26 (dry) - ----------------------------------------------------------------- - Amino acids (dry weight)[8] - ----------------------------------------------------------------- - Alanine 4.47 - ----------------------------------------------------------------- - Arginine 3.41 - ----------------------------------------------------------------- - Aspartic acid 4.32 - ----------------------------------------------------------------- - Cystine .08 - ----------------------------------------------------------------- - Glutamic acid 7.67 - ----------------------------------------------------------------- - Glycine 2.76 - ----------------------------------------------------------------- - Histidine .95 - ----------------------------------------------------------------- - Isoleucine 2.17 - ----------------------------------------------------------------- - Leucine 4.04 - ----------------------------------------------------------------- - Lysine 2.65 - ----------------------------------------------------------------- - Methionine 1.14 - ----------------------------------------------------------------- - Phenylalanine 2.20 - ----------------------------------------------------------------- - Proline 2.06 - ----------------------------------------------------------------- - Serine 1.80 - ----------------------------------------------------------------- - Threonine 2.15 - ----------------------------------------------------------------- - Tryptophan .78 - ----------------------------------------------------------------- - Tyrosine 1.79 - ----------------------------------------------------------------- - Valine 3.03 - ----------------------------------------------------------------- - - [8] Trace amounts of the following were also found: methionine - sulfoxide, citrulline, alpha-amino-n-butyric acid, homocitrulline, - glucosamine, galactosamine, methionine sulfoximine, ethionine, and - ethanolamine. - - -The lipid content of rapidly growing cells is normally quite low (0.45 -to 2.3 percent crude ether extractable lipids). The most important lipid -is poly-beta-hydroxybutyric acid, which is stored under the growing -conditions of insufficient nitrogen or oxygen supply (refs. [ref.187] -and [ref.191]). Under these conditions, this unusual polymer constitutes -up to 80 percent of the dry weight. While the monomer itself, -beta-hydroxybutyric acid, is rapidly and efficiently used in cell -metabolism, the nutritive value of the polymer is yet to be determined. -The fatty acids found include lauric, myristic, palmitic, palmitoleic, -heptadecaenoic, C17 saturated(?), stearic, linoleic, and linolenic(?) -([ref.195]). - - -Application to Spacecraft System - -A bioregenerative life-support system will be required in long manned -space flight, especially with several astronauts such as would be -required for a manned mission to Mars in the 1980 time period. While -almost 15 years is a long leadtime, the biological research and -engineering problems are formidable, and a system would have to be -developed at least 5 years before the mission. - -The power and weight requirements for both chemical and biological -regenerative life-support systems were presented in table VIII. These -should be considered tentative best estimates based on present data. - -The use of bioregenerative systems in spacecraft systems has been -studied by Bongers and Kok ([ref.175]) who put the -electrolysis-_Hydrogenomonas_ system in proper perspective with the -following statement: - - The bioregenerative systems are more or less in a transitory - phase between research and development. The power data can be - considered fairly accurate, at least within +-20 percent. The - postulated weight data, however, represent approximations, - particularly with respect to auxiliary equipment and - construction materials. Also omitted are the weight penalties - most probably involved in the processing of the solid output of - the exchangers, elegantly defined as potential food. Further - research is required in this area to evaluate the regenerative - systems, especially the bacteria, with respect to this - potential. Furthermore, as yet there is no experimental proof - that the growth rates of the heavy bacterial suspensions can be - realized in a large design, determined on a relatively small - scale with fairly precise control of physiological conditions - and gas exchange. This aspect may affect considerably the weight - involved in a chemosynthetic balanced system. Nevertheless, at - present, this approach still seems most promising. - - - CABIN ATMOSPHERES[9] - - [9] Includes part of [ref.196]. - -In the first U.S. manned space flight program, Project Mercury, and in -the face of very severe weight limitations, a cabin atmosphere of pure -oxygen at one-third atmospheric pressure was adopted. This choice -probably represented the greatest simplification which could be achieved -readily and, at the same time, provide protection against some of the -risks of rapid decompression. Although breathing pure oxygen at higher -pressures was known to be attended by some undesirable physiological -effects, the short duration of the flights to be undertaken, and the low -pressure employed, suggested that no harmful results would result in -this case. That these expectations were generally borne out is now -history. Preparations for space flights of longer duration--many weeks -or months--present similar problems and require special attention to -phenomena which may be either undetectable or of trivial significance on -a time scale of a few days. - - -Physiological Criteria in the Choice of Cabin Atmosphere - -If maintenance of normal respiratory function were the only -consideration, a cabin atmosphere of about sea-level composition and -pressure might be an ideal and straightforward choice for manned -spacecraft. In fact, this atmosphere has been used in the manned space -flights conducted by the U.S.S.R. No other atmosphere has been shown to -be more satisfactory from the physiological point of view, and the -tedious respiratory studies which should accompany the use of other -atmospheres can be avoided. Nevertheless, the formidable problems of -spacecraft design and the necessary precautions for safeguarding the -crew from accident require that other atmospheric compositions and -pressures be considered. For example, if a cabin at 1-atm pressure were -decompressed to space suit pressure (0.3 atm), the occupants would -develop decompression sickness; i.e., "bends." - -Several engineering considerations argue for low cabin pressures and -pure oxygen composition. Among these are structural design, weight of -atmospheric gas storage and control equipment, and the difficulty of -contriving pressure suits which allow operation at pressures near one -atmosphere. Such departures from the normal human gaseous environment, -however, require the demonstration of an acceptable level of safety and -physiological performance. - -The limits of the composition and pressure of acceptable cabin -atmospheres are then set by-- - - (1) A pure oxygen atmosphere at a pressure which will provide an - alveolar oxygen partial pressure equal to that provided by air at - sea level - (2) A mixed gas (oxygen and inert gas) atmosphere having a pressure - and composition that will allow decompression to the highest - acceptable suit pressure without the risk of bends - -A numerical value for the lower limit (1) is approximately 0.2 atm of -pure oxygen. The upper limit (2) is determined by the operating pressure -and composition of the space-suit atmosphere and may be of the order of -0.5 atm for a cabin atmosphere of 50 percent oxygen. It is necessary to -determine the astronaut's ability to survive and perform his duties in -any atmosphere selected. - - -Atelectasis and Pulmonary Edema - -Localized or diffuse collapse of alveoli in the lungs may, if the -condition persists, lead to arterial hypoxia which may be extremely -undesirable under the stresses of space flight. The alveoli are probably -unstable when pure oxygen is breathed; they tend to collapse if there is -blockage of the airways, especially at low pressures. This collapse -occurs because each of the gases present in the alveoli (oxygen, water -vapor, and carbon dioxide) is subject to prompt and complete absorption -from the alveoli by the blood. - -The alveoli are normally stabilized against collapse by the presence of -inert and relatively insoluble gas (nitrogen) and an internal coating of -lipoprotein substances with low surface tension. - -Theoretical and experimental results strongly suggest the desirability -of using oxygen-inert gas atmospheres for long missions to avoid -atelectasis and other gas absorption phenomena, such as retraction of -the eardrum. However, further experimental evidence is required both to -confirm this point and to establish its upper limit of suitability of -pure oxygen atmospheres. - -At Ohio State University in 1962, scientists studied the effect on young -rats exposed for 27 days to 100 percent oxygen (with no nitrogen), at a -reduced barometric pressure equivalent to 33 000 feet altitude. The rats -showed no difference in growth rate, oxygen consumption, food and water -intake, or behavior from control rats in air at 1 atm. - - -Oxygen Toxicity - -It has long been known that breathing pure oxygen at normal atmospheric -pressure often produces pulmonary irritation and other toxic effects -both in man and animals. This knowledge has occasioned concern over the -use of pure oxygen atmospheres in spacecraft. - -The effect of 100 percent oxygen at a simulated altitude of 26 000 feet -for 6 weeks was studied using white rats at Oklahoma City University -under a NASA grant. Radioactive carbon techniques revealed a 15-percent -reduction of metabolism in the 100-percent oxygen-exposed rats, compared -with rats in air at 1 atmosphere. There was a 20-percent decrease in -lipid metabolism in the liver compared with controls, but no decrease in -heart metabolism. There was no gross change in body weight. - -The White Leghorn chick between 2 and 7 weeks old is markedly resistant -to the toxic effects of 1 atm of O2. Continuous exposure (Ohio State -University) for as long as 4 weeks did not cause deaths, obvious -morbidity, or any signs of pulmonary damage on gross autopsy. -Nevertheless, the hyperoxia had some adverse effects, primarily reducing -the growth rate to between three-fourths to one-fourth of normal; -reducing feed intake per unit body weight to three-fourths of normal; -slowing respiratory rate by 30 percent; decreasing erythrocytes, -hemoglobin, and hematocrit by 9 to 12 percent; and causing reversible -histological changes in the lungs. Arterial O2 tensions were elevated -over 300-mm Hg, but arterial pCO2 and blood pH were unaffected. No -residual effects were noted upon return to air breathing. It is possible -that the anatomical peculiarities of the avian lung play some role in -the chicks' resistance to hyperoxia, but it is also possible that this -resistance is a function of age, similar to the tolerance shown by the -young rat but not the adult. - - -Carbon Dioxide Tolerance - -Studies of CO2 tolerance in submarine crews indicate that no loss of -performance is involved if the concentration in air at normal pressure -does not exceed 1.5 percent with exposures of 30 to 40 days. However, -biochemical adaptive changes were observed at this concentration. - - -Inert-Gas Components - -If other investigations establish the need for an inert gas in manned -spacecraft atmospheres, gases other than nitrogen may be considered. -Compared with nitrogen, the physical properties or helium and neon offer -advantages with respect to solubility in body fluids, storage weight, -and thermal properties. - -Studies at Ohio State University in 1964, under a NASA grant, showed -that helium substituted for nitrogen in a closed container causes humans -to feel "cold" at a normally comfortable temperature. Studies with -animals have shown that in a helium atmosphere there is greater heat -loss due to the increased conducting capacity and probably greater -evaporative capacity. In 6 days at 21 percent oxygen and 79 percent -helium at 1-atmosphere pressure, young rats grew at the same rate as -controls, but drank more water, excreted more urine, and had a higher -rate of food and oxygen consumption than controls in air at 1 -atmosphere. Men are being tested on a bicycle ergometer in saturated and -low relative humidity helium atmospheres to study heat balance. - -Mice were exposed to 80 percent argon and 20 percent oxygen continuously -at 1-atmosphere pressure for 35 days at Oklahoma City University. Carbon -14 studies of metabolism showed a slight slowing and a twofold to -threefold increase in fat deposition. - - -Bends - -Decompression, whether accidental (due to damage of the spacecraft) or -intentional (as in the use of the pressure suit outside the capsule), -carries the risk of bends if the inert gases dissolved in the tissues -and body fluids come out of solution. The magnitude of this risk is -determined to a very considerable extent by-- - - (1) Individual susceptibility - (2) The extent to which the nitrogen (or other inert gas) - concentrations of tissues and body fluids have been reduced - (3) The magnitude and rate of the inert-gas, partial pressure change - on decompression - -The probability of getting bends is reduced by-- - - (1) Selection of bends-resistant individuals - (2) Thorough denitrogenation before flight - (3) Limitation of decompressive pressure changes by appropriate choice - of cabin atmosphere pressure and composition - (4) Space-suit pressure setting - -In some cases, further improvements might be obtained by using, in the -cabin atmosphere, an inert-gas component which has a lower solubility in -tissue and body fluids or less tendency than nitrogen to form bubbles. - - -Fire Hazard - -Experience indicates that fires in pure oxygen atmospheres, even at low -pressures (e.g., 1/3 atm), are extremely difficult to extinguish. While -this phenomenon has nothing to do with respiratory physiology, the risk -on flights of long duration may be so serious as to demand special -measures. Unless effective countermeasures can be devised, this risk may -argue very strongly against the use of such atmospheres in the future. -Further experimental investigation is required. - - -Acceleration Effects on the Lungs and Pulmonary Circulation - -Forces produced by high acceleration overdistend one part and compress -another part of the lungs. Blood flow diminishes in some parts of the -lungs and increases in others. Fluid leaks from the blood into the -tissues and into the air sacs in parts of the lungs. These effects cause -difficulty in breathing, low arterial oxygen saturation, and impaired -consciousness during high sustained acceleration and, to a lesser -extent, after its cessation. They must be considered when selecting the -best gas to be breathed, since a high partial pressure of oxygen is -favorable for consciousness, but a low inert-gas concentration during -acceleration is unfavorable for rapid lung recovery afterward. - - - PHYSIOLOGICAL PROBLEMS - -A study of the manned space flights and laboratory observations to date -suggests that during long periods of weightlessness, some physiological -difficulties may arise which may produce serious effects on human -performance. Although recent experience gives no grounds for expecting -insuperable difficulties, neither the quantity nor quality of the -available observations permits the conclusion that long-term exposure to -weightlessness will _not_ have serious consequences. The critical role -to be played by the astronaut demands that every effort be made to -identify in advance those phenomena which may affect performance, and to -study their qualitative and quantitative relationships so that proper -precautions can be taken. - -Lawton ([ref.197]), in reviewing the literature on prolonged -weightlessness, found few instances in which physiological function was -truly gravity dependent. He stated that the physiological systems likely -to be most affected by weightlessness were the musculoskeletal system, -the cardiovascular system, and the equilibrium senses. Subsequent -experience proved this to be the case. McCally and Lawton ([ref.198]) -analyzed the data from experiments since 1961 and concluded that much -more basic laboratory work is necessary. Studies using immobilization, -immersion, and cabin-confinement techniques were recommended approaches -toward simulating weightlessness. - -Much of the difficulty in obtaining precise information of anticipated -problems arises from a lack of knowledge of normal mammalian physiology. -Many of these deficiencies can be remedied in the laboratory. In -space-flight development, however, two distinct investigational -approaches can be adopted. The first of these may be characterized as -empirical and incremental; that is, the capabilities of the astronaut -are explored in successive flights involving relatively modest increases -in difficulty or severity of the environmental conditions. In this way -it is hoped to ascertain the human limitations without running too great -a risk. The second approach can be described as fundamental: determining -by a series of controlled experiments the effects of exposure to -space-flight conditions upon comparative mammalian physiology, with -emphasis on man. A fundamental understanding of the observed effects -would be sought so that predictions for new situations and possible ways -to control them could be made with confidence. - -It is not possible now to predict for flights of 30 days or more-- - - (1) The effects of sudden reimposition of reentry accelerations and - terrestrial gravity - (2) Changes in body fluid distribution and composition - (3) The effects of violent physical effort on respiratory and - cardiovascular systems in prolonged weightlessness - (4) Central nervous system functions, especially coordination, skilled - motor performance, judgment, and sleep-wakefulness cycles - -NASA has emphasized that planning for manned space programs involves a -systematic extension from physiological observations in animals to man, -and finally the establishment of man as part of the man-vehicle system -design. Moreover, these studies require the evaluation of central -nervous, cardiovascular, respiratory, gastrointestinal, and other -systems as a matrix in mutual interdependence. There is particular -interest in the effects of weightlessness on flights exceeding 30 days. - -Mammalian flights of about 30 days also merit attention, including the -development of the life-support systems which must precede such a -program. Development of facilities for biological experiments may well -be an important requirement for studies in anticipation of manned -flights of longer duration than Apollo. Unless the biological satellite -programs of the type mentioned above are successful in providing the -necessary data, a manned orbiting laboratory may also be important in -studies of shorter range. - - -General Studies of Biological Rhythmicity - -The effects of weightlessness on the organism as a whole may be -manifested by important changes in certain integrated behavioral -patterns having an inherently rhythmic character. Modifications in basic -behavioral patterns and performance may occur as disruptions of rhythmic -physiological phenomena, which are themselves the end product of -interrelated functional activity in a number of physiological systems, -such as the neuroendocrine, cardiovascular, and central nervous systems. - -Measurements of interdependent components of biological rhythmicity are -beginning to be analyzed by methods well established in -physics--including correlation and spectral analyses, and phase -modulation and variance in rhythmic processes. A wide variety of -physiological functions can be treated as periodic variables in the -analysis, including rhythmicities in cardiac output and blood pressure, -respiration, brain waves, and the slower tides of appetite, and -sleep-wakefulness. The importance of such investigations argues for -their inclusion in forthcoming flight programs. Their experimental -simplicity is an additional advantage. Biorhythms have been discussed in -more detail in the section on "Environmental Biology." - - -Effects of Weightlessness on the Cardiovascular System - -Earlobe oximetry, indirect measurements of blood flow and of blood -pressure by finger plethysmography or impedance plethysmography, and -ballistocardiographic techniques have potential application to manned -space flight. - -Adaptation to prolonged exposure to weightlessness or to lunar gravity -may cause difficulties when the astronaut is exposed again to reentry -forces and terrestrial gravity. It is possible that these adaptive -changes may thus produce unacceptable effects on performance or cause -risk to life. It is important to obtain experimental evidence on this -subject. - -It is common knowledge that following a stay in bed, dizziness, -faintness, and weakness characterize arising, and that a feeling of -general weakness may persist for several days. The phenomenon has been -investigated in a number of laboratories. One approach has been to put -healthy young subjects to bed, and even in extensive casts for periods -of 2 or 3 weeks or more. Two major findings have emerged from these -studies. First, a substantial adjustment in the blood circulatory system -occurs, which is termed the "hypodynamic state." Second, there is a -large decrease in the skeletal and muscle mass of the body. - -There are two kinds of evidence for the hypodynamic state: measurement -of parameters of circulatory function, and measurement of the response -of the individuals to a quantitatively imposed mild gravitational load. -After 3 weeks in bed, otherwise healthy persons exhibit an increase of -more than 20 percent in heart rate; a reduction of 10 to 20 percent in -total blood volume, primarily as a result of reduction of plasma volume; -and a decrease in heart size of about 8 percent. Coupled with these -cardiovascular changes is a reduction of 10 percent in the basal -metabolic rate. It appears as though the circulation and metabolism are -reset to a lower functional level commensurate with the reduced demands -placed on the whole organism. - -After 3 weeks of bed rest, all of the subjects tested showed pronounced -orthostatic hypotension. After tilting, the average heart rate increased -by 37 beats per minute, the systolic blood pressure fell some 12-mm Hg, -and some of the subjects fainted. The measurements were continued for 16 -days after the bed-rest period, and it was round that recovery was not -quite complete when the experiment was terminated. - -There is little question that in prolonged exposures to the weightless -state, there is a fair probability of extensive circulatory adjustments, -the seriousness of which cannot yet be foretold. While it is likely that -the astronauts will adapt successfully to long periods of weightlessness -at some new circulatory functional level, the remote possibility exists -that the circulatory changes may be progressive to the point of ultimate -failure. - - -Metabolic Effects of Weightlessness - -Without metabolic information, accurate planning of environmental -systems for long flights is difficult. Importance is also attached to -early evaluation of weightlessness effects on body-fluid equilibria. The -results of Earth orbital flights and of terrestrial water-immersion -experiments suggest the occurrence of undesirable changes, although no -effects leading to operational incapacity have yet arisen. - -In both recumbency and immersion, a similar redistribution of body -fluids occurs. It has been suggested that recumbency may affect an -extracellular fluid-volume receptor mechanism which by decreasing -aldosterone secretion by the adrenal gland, would decrease sodium -reabsorption by the renal tubules. Aldosterone excretion decreases -during recumbency and during standing in water, but increases while -standing in air. There is also evidence for cardiac atrial volume -receptor mechanisms which respond to increased filling of the left -atrium with reflex inhibition of release of pituitary antidiuretic -hormone (ADH), resulting in diuresis (Henry-Gauer reflex). - -Altered fluid equilibrium in buoyant states is accompanied by shifts in -intracellular and extracellular electrolyte distribution, especially -sodium and potassium. Evidence from recumbency studies indicates a -strong correlation between loss of erect posture or weight bearing and -excretion of calcium stores in bone. - -A bone X-ray densitometry method has been developed by Mack, at Texas -Woman's University, for accurately determining the loss of bone mineral -(+-2 percent accuracy) in humans and animals. The heel bone and spine -are X-rayed using a calibrated aluminum wedge as a standard. This -technique will be used for preflight and postflight analysis of the -primate being flown in the 30-day biosatellite. Comparative appraisal of -bone mineral behavior in astronauts participating in the Gemini and -Apollo programs will be invaluable for future flight missions. - -Bed rest and immobilization studies by Mack have shown loss of skeletal -mineral and increased calcium in the urine and excreta. Four bed-rest -studies, each extending for 2 weeks, compared different levels of -calcium intake. Four men were used in each study and served as their own -controls during extended ambulatory periods. During 2-week periods, up -to 10 percent of calcium mineral was lost from the heel bone. Calcium -was also determined in the urine and feces. In other studies, isometric -exercises reduced loss of bone mineral during bed rest. - -Excretion of calcium in the urine is accompanied by risk of its -deposition as calculi or "kidney stones" in the urinary tract. -Currently, changes in calcium metabolism resulting from weightlessness -over periods up to 2 weeks is not considered a hazard requiring -precautionary measures. - -Flights in excess of 2 weeks, however, constitute a problem serious -enough to warrant study on the 11-day orbital flights and the 30-day -biosatellite primate mission. Therapeutic immobilization, -post-poliomyelitis immobility, and experimental restraint in normal -subjects lead to a negative calcium balance, with hypercalciuria. - - -Central Nervous System Functions in Weightlessness - -The wide range of individual tolerances to the disturbing effects of -vestibular stimulation has emphasized the importance of this factor in -astronaut selection. At the same time, vestibular functions must be -considered jointly with visual task performance, since both have special -significance for such maneuvers as vehicle docking. Vestibular function -in the weightless state remains almost completely unknown. Limited -evidence from animal and manned space flights suggests that head -turning, resulting from vestibular stimulation, may seriously interfere -with visuomotor performance, but that susceptibility to these -disturbances is significantly different between individuals and that -partial adaptation occurs relatively quickly. - -NASA is currently collecting extensive baseline electroencephalogram -data under controlled conditions in a form suitable for mathematical -analysis. Data are being taken from about 200 subjects in major national -and overseas centers. It is intended that this study will assist in -astronaut selection and monitoring in space. - -Studies on many effects of weightlessness on nervous functions require -monitoring of the autonomic nervous system, including such autonomic -effects as gastrointestinal activity, secretion, lacrimation, -salivation, sweating, and the central control of respiration. Urinary -estimations of catecholamines and 5-hydroxyindoleacetic acid would -provide important data on autonomic system activity if collected in -flight and compared with preflight and postflight controls. - -Major areas have been outlined in which prolonged weightlessness may be -expected to interfere with performance, judgment, and, ultimately, -chances of survival. These include cardiovascular, metabolic, central -nervous, psychophysiological, and biorhythmic effects. They have been -dealt with separately and in sequence, but have not been intended to be -viewed as hierarchic. The relative scarcity of data necessarily -precludes such an evaluation. - -Soviet experience with zero gravity and weightlessness has increased -their emphasis on this space-flight factor and was an important topic at -the May 1964 COSPAR meeting. Discussion of the postflight medical status -of Bykovsky (5-day flight) and Tereshkova (3-day flight) revealed a -concern for the significance of prolonged weightlessness and the -presence of postflight physical debility and fatigue following Vostok -flights 3 through 6. These changes persisted for several days. Among the -physiological conditions singled out for mention were-- - - (1) _Body fluids_-- Cosmonauts have shown a postflight weight loss of - 1.9 to 2.4 kg apparently resulting from a redistribution of body - fluid in response to elimination of the hydrostatic pressure - gradients caused by Earth gravity. There is the suggestion that - this redistribution is complete within the first 24 hours of - flight. Titov is reported to have been dehydrated alter his flight - with early hemoconcentration. These findings directly support - predictions made from ground-based research. - (2) _Cardiovascular_-- Postflight orthostatic tachycardia is reported - for Titov as long as 23 hours after landing; at 48 hours there was - significant residual intolerance to the upright posture. - Cosmonauts have demonstrated a 20- to 35-percent increase in - oxygen consumption during the standard postflight exercise test. - -In both of these areas there was a return to normal within the -postflight period of study. The Soviets have continued their biological -experiments in space with the Vostok/Voshkod series. Fixing of -histologic specimens in flight by Bykovsky demonstrated a critical role -for man and made possible an expanded experimental program. Biopackages -have become more complex with each succeeding flight. - -With the exception of postflight orthostatic intolerance after the third -and fourth Mercury flights, changes as a result of exposure to a -zero-gravity environment have not been noted by U.S. investigations in -space. Ground-based research proceeds here at an advanced pace and is -supported in large measure by both the USAF and NASA. A study of the -relationships among renal and systemic hemodynamics, neurohumoral -cardiovascular regulation, and renal excretory function in differently -positioned subjects is underway, as are studies of acceleration -tolerance. - - - DEPRESSED METABOLISM - -In anticipation of prolonged manned space flights, NASA has sponsored -research related to metabolism depression. The daily food requirements, -for example, of astronauts during a voyage of several months can -constitute a major portion of the weight and storage capacity of the -spacecraft. A somewhat promising and fundamental approach to this -problem is the reduction of the astronauts' daily metabolic -requirements. It has been suggested that astronauts on prolonged space -missions be put in a state of suspended animation until their -destination is reached. Though this sounds fantastic, 10 years ago no -cell had been frozen to cryogenic temperatures and survived. Today it is -commonplace for tissues to be frozen, stored at low temperatures, and -thawed and then to maintain their viability and function. - -Animal metabolism may be depressed by reducing body temperature, as in -hibernation and hypothermia. Other means by which metabolism can be -lowered include drugs and electronarcosis. Hibernation is a nonstressful -state and results in a great decrease in metabolism. However, human -beings are not hibernators, and much research is needed before the -mechanism of hibernation is understood, and the possibility of inducing -it in humans evaluated. Hypothermia is the direct cooling of the body to -temperatures where metabolism is substantially depressed. Extracorporeal -circulation systems combined with cooling are in routine use in most -medical centers throughout the world. Hypothermia is not an ideal -solution, however, since general body hypothermia is a stressful -condition. Pharmacologic induction of hypothermia can be accomplished by -such drugs as chlorpromazine and harbamil. Other drugs can be used to -depress metabolism, but all have some disadvantage. - -In recent years there has been a growing interest in electronarcosis, -the induction of sleep by an electric current. Although potentially -valuable, this method is far from routine application. - -Outstanding advances have been made in metabolism suppression. Recent -progress in the biochemistry and physiology of hibernation and -hypothermia have shown that the oxygen requirements of individual -mammals, organs, and tissues can be reduced. When the chemical -composition of the blood and the cardiac output are sufficient to meet -cellular requirements, regulatory mechanisms remain effective and animal -survival is assured. In contrast, when oxygen transport is interrupted, -a reduction in cellular activity occurs and regulation is impaired. In -induced hypothermia, the low temperature slows the rates of all -processes and modifies the action of metabolites and other substances. -This in itself is not harmful, as shown by the true hibernating animal -(e.g., ground squirrel), but will become disastrous as soon as anoxia -and chemical imbalance begin to develop. - -The phenomenon of natural hibernation is being investigated in the -laboratory in the hope that the unusual tolerance of hibernating animals -to reduced metabolism and low body temperature may some day be produced -artificially in ordinary laboratory animals and man. Experiments with -the ground squirrel, a typical hibernator, show that the artificially -cooled ground squirrel does not tolerate such long periods of low body -temperature as does a naturally hibernating animal. - -Other studies of the brown adipose tissue (fat), which is present in -most hibernating mammals, show it to be essential to hibernation. -Indications that brown fat has a thermogenic role in rats exposed to low -temperatures suggest that this may be the case in true hibernators -([ref.199]). Arousal of the hibernating animal by cold is triggered by -sympathetically activated thermogenesis in areas of brown fat so -located, relative to the vasculature, that the heat is transferred to -areas of the body concerned with normal metabolic and nervous activity. - -Soviet work comparing various depressed metabolic states and resistances -to acceleration shows deep winter hibernation to be most effective, -followed by deep hypothermia, and drug narcosis as the least effective. - -Experimental evidence is being accumulated to show that hibernation and -hypothermia somewhat protect animals against radiation. Clinical studies -on irradiation of cancer patients indicate that lowering the body -temperature reduces cellular metabolism and thus decreases tissue -sensitivity to gamma radiation ([ref.200]). - -The use of prolonged hypothermia, hibernation, drugs, and -electronarcosis appears to hold some potential for reducing astronauts' -metabolic requirements. If one or mote of these methods become -practical, human requirements for food and oxygen could be drastically -reduced. Simultaneously, these methods may afford radiation protection -and acceleration tolerance. - - - NUTRITION IN SPACE[10] - - [10] Includes part of [ref.201]. See also [ref.202]. - -The human body can use food stores so that the nutritional requirements -can be reduced for a short time. This will vary widely among individuals -and each individual may exhibit characteristic patterns of nutritional -behavior. During reduced food intake, muscular efficiency may not change -significantly over a period of 4 to 6 days; unfortunately, however, -mental activity begins to decline after 24 hours. Feeding requirements -can be divided into two categories: short term (for missions of less -than 21 days) and long term. Since dehydration can occur in a matter of -hours under adverse conditions, water requirements must be considered as -a special case. - - -Water Requirements - -Water requirements are extremely critical and the amount supplied should -not under any circumstances be kept to a minimum. Rather, a large margin -of safety should be allowed. - -Present data on water requirements show a very strong dependence upon -suit inlet temperatures. In the absence of an accurately controlled suit -temperature, water requirements can easily double. If this should occur, -the mission would probably have to be aborted, since it is doubtful if -electrolyte balance would be maintained at such high rates of water -loss. Normal or even extreme conditions of the terrestrial environment -usually include diurnal variation in temperature which may modify water -needs. These conditions will not be obtained in the spacecraft. - -In addition to ground-based experiments, measurements of water intake -should be made under actual flight conditions. Data from short-term -flights should be used for extrapolation to longer missions. - - -Formula Diets - -The tacit assumption which now prevails, "Astronauts even on short-term -missions require a diet of great variety," is apparently not well -supported. In many parts of the world, people live on a monotonous diet -consisting of only a few types of food with no apparent ill effects, -provided their nutritional requirements are satisfied. Experimental -evidence from many sources (e.g., the Army Medical Research and -Nutrition Laboratory) shows that individuals can be kept on a single -disagreeable formula diet for as long as 60 to 90 days without harm. -Since highly motivated individuals are chosen for space flights, it is -unlikely that they would object to the monotony of a formula diet and -would probably prefer its simplicity. Also, there are definite -possibilities of developing a much more acceptable formula than present -types. There is no reason to anticipate adverse effects from the use of -formula diets in short-term flights. - -Formula diets would be extremely desirable for short-term flights. A -formula diet (a rehydrated liquid formula could be used) would -considerably reduce the number of manipulations and the time required -for in-flight preparation, compared to a varied diet. These two -improvements could contribute materially to the safety of a flight, -since the astronauts would not be preoccupied with food preparation for -so long a period, and the food could be dispensed without removing suit -components, such as gloves. Storage requirements could be simplified -with this type of diet. Weight, however, would not be lowered without -the development of more refined formulas than those now available. -Formula diets could readily be adapted to the determined metabolic -requirements of the individual astronaut. Packaging problems will be -simplified by using formula diets, which can easily be given a variety -of flavors and colors. - - -Waste - -The problem of waste production is intimately related to nutrition and -can be solved or simplified by dietary changes. Any diet should be -adjusted for the minimum production of feces, before and during even -short flights. Water will be sequestered by accumulation in the feces, -and the net loss, under normal conditions, would be approximately 40 to -60 grams per man per day. Flatus can be a serious problem, since -considerable concentrations of toxic gases may accumulate. The -purification system for the recirculated atmosphere must be able to -remove these, although the diet should be planned to minimize the -problem. The collection of urine and its storage is of importance, -particularly on short-term flights, and individual packaging and -labeling of urine specimens will be necessary for the analyses. - - -Metabolism - -An accurately measured intake of nutrients, calories, and water is -necessary for determining metabolic demands imposed in any space flight. -There is insufficient knowledge to predict total metabolic requirements -under the numerous stresses which can be anticipated. Simulator studies -are of great importance even for short-duration flights. - -The two most important variables to be considered in establishing the -minimal diet are protein and energy requirements. NASA is supporting -research at the University of California (Berkeley) to determine these -requirements and to estimate individual variation in healthy young men. -The possibility of minimizing need through biological adaptation is -being explored. - -It is difficult to estimate the minimum protein requirement of an adult -man. The generally accepted criterion of minimum adequate protein -nutrition in the adult is the maintenance of nitrogen balance at minimum -intake. The minimum protein requirements depend on endogenous nitrogen -loss. Analysis of the little data available indicates a best estimate of -2 mg of nitrogen per kilocalorie of basal energy expenditure. However, -this figure is higher than that noted in experiments in some human -subjects. - -After minimum nitrogen requirements and minimum amino acid requirements -have been established, studies will be directed toward investigating -caloric restriction and adaptation to restriction of calories. It has -been suggested that caloric restriction in animals and man results in -apparent decreased energy need for the same activity. This apparent -paradox has never been explained. It has been shown that there is -adaptation to repeated episodes of caloric restriction both in animals -and man, so that subsequent periods of caloric restriction result in -decreased rate of weight loss, nitrogen loss, and longer survival. - -Additional experiments are urgently required to determine the metabolic -demands for minerals--in particular, the metabolic balance of calcium, -potassium, sodium, and phosphorus. Under conditions of high water -consumption, large mineral losses are to be expected. Failure to replace -these can cause an imbalance which could impair the efficiency of the -individual to the extent of endangering the flight. - -Analysis of samples taken in flight, both of urine and feces, should be -made. Respiratory quotients can be determined in flight, blood samples -should be taken before and immediately after flight for analyzing -selected components (in simulator studies these could be taken -periodically), and nutritional intakes (which would be facilitated by -formula diets) must be measured and analyzed. - - -Short-Range Technology - -There are many practical difficulties in providing for food storage and -accessibility in spacecraft. The packaging of food materials, both -dehydrated and liquid, has proceeded satisfactorily under the -supervision of the Food and Container Institute. If packaging materials -are to be made to withstand very high relative humidities and large -variations in temperature, additional investigations are required, since -such containers are not yet available. In packaging, serious -consideration must be given to the ease with which the food may be -reached and eaten. - -If dehydrated formula foods are to be fed on short-term missions, -additional work is required on the rehydration of such formulas. Present -methods of water measurement under weightless conditions are not -satisfactory, and better methods will have to be contrived. - - -Long-Term Nutritional Problems - -There is a dearth of metabolic information, even for short-duration -flights, without which changes in metabolic patterns to longer flights -cannot be extrapolated. However, using scattered information, certain -changes which may be encountered can be hypothesized. Decalcification of -bone and changes in water-holding capacity of the body may be -anticipated. It is also possible that changes in proportion of fat to -lean body mass could be experienced and should be considered in -nutritional planning. Nutritional requirements depend on size, -particularly lean body mass, sex, physiological state, and individual -metabolic rates. Therefore, individuals for space flight should be -screened with these factors in mind if it is desirable to minimize food -intake in long flights. The factors which influence the total -nutritional requirements of the individual also influence his mental and -physical responses to stress. - - -Synthetic Foods - -The development of food materials other than those derived directly from -animal or vegetable origin is of interest. Advantages of such diets may -be low residue, ease of storage, rehydration, and manipulation. -Experiments with chemically defined synthetic diet for humans have been -carried out by Medical Sciences Research Foundation, San Mateo, Calif. -The complete liquid diet is composed of required amino acids, fat, -carbohydrate, vitamins, and minerals. A cubic foot of the diet (50 -percent solids in H2O) supplies 2500 calories per day for 1 month, and -has been given a variety of artificial flavors. - -This synthetic diet has been fed to human volunteers for 6 months in a -pilot study at the California Medical Facility, Vacaville, Calif., and -the results are being reviewed. Schwarz Bioresearch, Inc., is studying -the storage, stability, and packaging of chemically defined synthetic -diets for human and animal flights. - - -Food Production in Space - -Long-term feeding in space depends upon a payload of stored food unless -food is produced during flight. If sufficient propulsive energy is -available, the duration of missions using stored food may be quite long. -However, in emergencies in which a mission lasts longer than planned, -survival may depend on the ability to produce food extraterrestrially. -Eventually it will be desirable or necessary to produce food beyond the -confines of Earth. - -The nutritional requirements of the crew will be influenced by such -factors as activity, physical and psychological stress, individual size -of the members, and individual metabolic rates. The food intake will -have to be adjusted to meet these requirements. It is necessary to know -the nutritional requirements of each astronaut and the way in which -these are altered by the conditions of space flight in order to estimate -needs on long missions. Without this information, the food supplies for -the longer flights may be too much, too little, or improperly balanced. -Where dependence would not be on stored food alone, but on food produced -en route, more exact information on requirements is needed to determine -the capacity of food production units. - -In the discussion of bioregenerative systems, it was suggested that food -materials could be produced by photosynthetic organisms (e.g., algae, -duckweed, and other higher plants) or by nonphotosynthetic organisms -(e.g., _Hydrogenomonas_). In contrast to the use of living organisms, -reprocessing waste materials by chemical treatment or the actual -synthesis of high-energy compounds has been suggested. No chemical -system has yet been demonstrated as workable for the economical -production of food in space, and the systems considered produce -materials which may be converted to food, but are not food as such. - -Algal cultures have had the most extensive investigation as food in -space, but the technical problems of using this material as a food -source have not yet been solved. It is apparent from the investigations -to date that algae will require treatment before they can be used as -food. In limited trials, difficulties have been experienced with amino -acid deficiencies, digestibility, high residues, and gastric distress. -Processing methods which would be applicable in space travel and the -possibility of secondary conversion by other animals or plants should be -systematically investigated. - - - - - chapter 8 - -_Significance of the Achievements_ - - - SIGNIFICANCE TO SCIENCE - -One of the most critical research areas of the space program is -bioscience. Of both practical and philosophical significance in -exploring the origins of life and the possibilities of life on other -planets, bioscience also promises much in medical aspects. Space offers -biologists completely new environmental factors, such as the effects of -zero gravity and of removal from Earth's rotation. These effects have -been studied in attempts to advance understanding of basic mechanisms of -physiology and biological rhythms. These studies can be of great value -in dealing with problems of disease and metabolic disorders. - -Biological research is fundamental to the problem of successfully -protecting and sustaining man in the peculiar and hostile space -environment. Understanding human requirements and variations in their -response to various environmental factors offers value in medical -research for human survival and comfort. The many technological -discoveries and advancements in electronic and engineering equipment -greatly enhance medical diagnosis, treatment of disease, and the -extension of human life. - -The life sciences, biology and medicine, are fundamental to the success -of manned exploration of space, which marks a unique and significant -development in the long history of man's conquest of new frontiers. -Those who pioneered other frontiers on land and sea and in the air were -not forced to await biological and medical research. Even the pioneers -of aerial flight began their efforts without first seeking biomedical -data. The search for such data followed flight experience and, indeed, -was made only after problems arose. - -Project Mercury, NASA's first program for manned space flight, -stimulated immediate and extensive studies in the life sciences to -sustain man in space. Before a vehicle could be designed to carry an -astronaut into space, anticipated biomedical problems associated with -space flight were studied. Life-support systems were designed to offer -adequate protection from environmental stresses peculiar to space, such -as zero gravity, removal from Earth's rotation, and high-energy cosmic -radiation. These life-support systems used knowledge already gained from -research for manned space flight by the U.S. Air Force. - -Our entry into space has put us at the threshold of fundamental and -far-reaching discoveries in the biological realm which have profound -implications for other areas of human thought and endeavor. As man goes -farther into space, the hazards increase; but past accomplishments -indicate that the road ahead holds more promise than peril and that the -vistas of knowledge that may be foreseen are as vast as space itself. - -Almost everything which now can be said about the effects of -extraterrestrial environments and about life on the Moon or the planets -lies in the realm of pure speculation. There is one prediction, however, -that can be made with considerable certainty by reason of historical -precedent--the opportunity to investigate a totally new area, such as is -offered by space exploration, is certain to produce a burst of -scientific interest as soon as the path is charted by a few pioneers. -Over the next few decades a progressively larger proportion of -biological interest will turn to space. We may well expect that the -discoveries made here will revolutionize some of our concepts of -biology. - -It should be fully realized that the accumulation and dissemination of -biological and other scientific information is not only of great value -to science and humanity but is of tremendous import to the prestige of -the Nation. - - - SIGNIFICANCE FOR PRACTICAL APPLICATIONS - -It can be predicted as confidently for space biology as for other space -sciences that the economic costs will be amply repaid in the long run by -applications of space-oriented biotechnology to other fields of biology -and medicine. There are inevitable substantial, though indirect, -contributions of NASA's continuing efforts in space biology. - -NASA-supported biological research has many practical applications and -"spinoffs" which contribute to the fields of health and medicine, food -and agriculture, and industry and manufacturing. Some of these are -presented to show the range and value of applications which have -resulted from basic and applied biological research. In addition to -those listed are many others from the biosatellite program, particularly -in the fields of bioengineering and miniaturization. - - -Health and Medicine - -Solar cells, which have powered space systems, are now being used as a -power source in studies on brain function. A miniaturized solar cell -developed by General Electric provides enough power, under ordinary -house lights, to stimulate an animal's brain and to telemeter -respiratory, cardiovascular, and brain-wave data while the animal is -allowed to move about freely. Such a system is now used by the National -Institute of Mental Health Laboratory at Rethesda, Md. - -Scientists at the Ames Research Center have devised a new technique for -studying organic compounds, whether synthesized in the laboratory or -produced by a living system. This technique is based on a property of -matter called optical activity. Previous methods of measuring optical -activity have been plagued by low sensitivity. The new method is many -tunes more sensitive and represents a real contribution to modern -analytical instrumentation. - -Studies on calcium metabolism and bed rest simulating weightlessness are -adding knowledge on the prevention of demineralization of the skeleton; -treatment of Paget's disease and osteoporosis prevention of muscular -atrophy; the cause and treatment of renal calculi (kidney stones); -optimal calcium for the human diet; and the factors influencing calcium -absorption, metabolism, and excretion. The results will have great -importance in bone healing and repair, care and treatment of fracture -cases, treatment of paraplegics, and treatment of polio patients and -similar cases. These grant studies at Texas Woman's University have also -proven that the X-ray bone densitometry method can accurately detect -changes in the skeleton. - -A primary objective of the planetary exploration program is the -detection of possible extraterrestrial life. The study of the -fundamental properties of living things on Earth is restricted to the -type of life which has evolved and survived here. Life which has been -exposed to totally different environmental conditions may have markedly -different physiological characteristics. The impact of the new -information obtainable from the study of extraterrestrial life upon the -sciences of medicine and biology will unquestionably be of fundamental -and far-reaching importance. Advancement in the treatment of disease and -the problems of aging are among the many possible consequences. - -New developments in such techniques as ultraviolet spectrophotometry, -polarimetry, and gas chromatography will find use in the detection of -biochemicals and other compounds in hospitals and in toxicology and -pathology laboratories. They will also be useful in studies of -atmospheric pollutants such as smog. - -Studies of the chemistry of living systems, molecular biology, and -biophysics of cellular processes will create a better understanding of -the basic mechanisms of life, leading to an understanding of both -inherited and acquired disease, especially neoplastic conditions and -chemical disturbances incident to mental disease. - -The University of Pittsburgh is conducting a study to increase the -availability of cytological technique in research and as a monitoring -procedure by developing an automatic electronic scanning device using -computer analysis for recording, counting, and sorting chromosomes. -Structural changes in blood cell chromosomes can indicate the degree of -radiation damage as well as damage resulting from various environmental -stresses. Accordingly, this instrument, when developed, can be used as a -radiation dosimeter in civil defense by swiftly detecting the degree and -type of chromosomal aberrations in blood cells. Thus, casualties in -nuclear attack could be quickly detected and treated. This system would -also be useful for nuclear industrial plants and for military maneuvers. -In medicine, various disease trends could be monitored. (Chromosomes -exhibit anomalies in leukemia and mental retardation as well as in other -states.) In space exploration and experimentation, the device can spot -monitor radiation dose levels as well as changes resulting from any of -the environmental stresses experienced in space. This apparatus can be -modified for use as an extraterrestrial-life-detecting instrument by -scanning the growth of cells (or cellular inclusions), computing rates, -and telemetering changes to the researcher. - -Investigations of rhythmic phenomena of various physiological systems -can result in knowledge of the utmost importance to medicine. Rhythmic -phenomena are found in the cardiovascular system of normal humans. -Changes in these rhythms have the potential of foretelling abnormalities -(heart disease, arteriosclerosis) before outward signs are manifested, -allowing for earlier diagnosis, treatment, and control or cure. - -The spacecraft sterilization program requires the use of rooms having -the lowest attainable level of bacterial contamination. The rate of -dissemination of bacteria from the humans in the room is basic to the -problem. Data on this matter are being obtained through support of the -Communicable Disease Center of the U.S. Public Health Service. The -findings are affecting the measures used in surgical practice to lower -infection rates. - -Studies on the physiology of hibernation in mammals are important to -understand temperature regulation and the mechanism of survival at low -body temperatures. The purpose of this type of research is to understand -and use reduced metabolic activity in astronauts on future extended -space flight. Other applications involve studies of the mechanisms of -injury and freezing biological organisms, for improving techniques in -hypothermic surgery, pathology, and preservation of tissue for human -grafting. - - -Food and Agriculture - -Gathering agricultural information by remote sensing of Earth's surface -from aircraft, balloons, and satellites has a potential application in -research and development. Current needs for data gathered in this way -include crop and livestock surveys for marketing planning; soil mapping; -crop disease, insect, and weed surveys; soil conservation management and -research; and crop acreage control programs. As population and world -trade increase, the needs will become even more intense for regularly -scheduled synoptic surveys of the world's agricultural lands for crop -plantings and harvests; determining the condition of crops as affected -by drought, disease, or insect outbreaks; and studies of the lands -suitable for agricultural development in underdeveloped countries. The -only way that worldwide synoptic surveys can be made is by using -orbiting platforms. - -The NASA nutrition program for developing diets for prolonged manned and -animal space flight lends itself to civil defense purposes; military -maneuvers where space and weight are prime considerations; polar and -desert exploration; reducing hunger in underdeveloped countries; and -detecting metabolic diseases as well as diseases of infancy and old age. -For space research such a diet can be used on prolonged manned space -flights, animal experiments in space, manned orbiting laboratories, and -space and planetary stations. Studies on the packaging and stability of -foods under various conditions of humidity, temperature, and radiation -will lead to better processing and storage. - -Learning how microbial spores are transported by air is important to -biology, agriculture, and medicine. Besides spreading crop destruction, -microbial spores produce allergic responses in some human beings. To -obtain the facts, not only the biology of micro-organisms but also the -weather factors that induce the flight of mature spores must be known. -Thus, both biological and meteorological problems are involved. Data -obtained under a NASA contract with the General Mills Electronic -Division (now part of Litton Industries, Inc.) indicate that spores of -fungi are present in low numbers in the stratosphere. A reservoir of -spores exists which cannot be brought down by the normal scrubbing -mechanisms of rainfall and other meteorological disturbances in the -troposphere. This finding has important implications for reducing the -spread of agricultural crop diseases and for protecting persons -suffering from allergies. This project has indicated the necessity for -designing novel biological samplers for use in the stratosphere. Such -samplers will aid in determining various pollutants of the atmosphere. - -The NASA program for developing sterile spacecraft for the biological -exploration of Mars will contribute improved methods of sterilization -that can be applied to the canning industry. Studies on sterilization at -low temperatures for long periods of time are being supported by NASA at -the Massachusetts Institute of Technology and the Communicable Disease -Center and the Sanitary Engineering Center of the Public Health Service. -The developing capability is making possible the heat sterilization of -products that never before could be thoroughly sterilized. - -In preparing for missions to search for extraterrestrial life, research -on the psychrophilic or cold bacteria, on halophytic or salt bacteria, -and on specialized bacteria and other organisms growing in extreme -environments is defining the extremes under which life can exist. -Increased knowledge about organisms that can grow in or on refrigerated, -dried, or salted foods and other materials should have practical -applications for food storage and preservation. Research on -psychrophilic bacteria is being conducted by Whirlpool Corp. and the -NASA Ames Research Center. - -Theoretical studies of Martian life involve investigations of plant and -bacterial spores. Many of these forms are spoilage organisms and some -produce lethal toxins. This work has potential importance for food -processing and for obtaining more precise knowledge of how wounds become -infected. The program for investigating possible forms of life on Mars -includes a thorough study of anaerobic micro-organisms. This research -has led to the discovery of new types of nitrogen-fixing bacteria other -than the familiar types found in the root nodules of leguminous plants. -Thus, it may be possible to use these microorganisms, or the principles -involved, in the incorporation of vital atmospheric nitrogen into -terrestrial soils which are now unproductive. - - -Industry and Manufacturing - -Batteries that have been developed in the space program to endure high -sterilization temperatures for extended times will have greatly -increased shelf life at normal storage temperatures and will be -serviceable after many hours of baking at high temperatures. - -Currently, the highest quality tape recorders are subject to imperfect -reproduction because the tapes are heat labile; i.e., they soften and -stretch when warm. The development of high-quality magnetic tapes for -space-data recorders is an outgrowth of the materials developed to meet -spacecraft sterilization requirements. These improved tapes will be -useful for all types of recording--industry, automation controls, home, -and studio. - - - OUTLOOK FOR BIOSCIENCE--MAJOR PROBLEMS - -The problems undertaken are among the most challenging, if not _the_ -most challenging, man faces on the space frontier. These include the -quest for the origin of life, the explanation of life and life -processes, the elucidation of the environment's role in establishing and -maintaining normal organization in living organisms, the possibility of -extraterrestrial life on other planets--the concern of exobiology. The -greatest promise for their solution lies in advances in biological -theory rather than other avenues of research; therefore, it is fortunate -that the need to solve them has come at a time when developments in -experimental biology are at a high level. In addition, technological -developments in electronics and engineering are providing new and -wonderful instruments for this great exploration into the sources of -life. Many of these have had practical application that has made -possible important advances in medical diagnosis and treatment. - -The broad national space goals initially charted by NASA have gone -beyond space flight in near-Earth orbit to lunar and interplanetary -exploration by man and machine. For such missions, more intensive and -comprehensive research in the life sciences is needed. 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K.;_ - - *p. 124, l. -10:* _Rosenszweig_ ----> _Rosenzweig_ - - *p. 128, l. 29:* AMRL Tech. Doc. Rept. ----> AMRL-Tech. Doc. Rept. - -Variant spelling: Both forms _microorganism_ and _micro-organism_ have -been retained, as quoted from different sources or bibliographic -reference titles. - -Tables: Where necessary, the widths of columns have been adjusted, and -some tables have been split to accommodate the width restrictions on -this text format. Split tables have had blank lines inserted in order to -maintain the alignment between the two parts should they be re-joined at -a future date. In the original text, Table VI was split over two pages -but has been rejoined in this version. - -References: For ease of searching, references in the text, as well as -those in the list of references, have been enclosed in square brackets, -e.g. [ref.3]. - -Representation of non-ascii characters: In this version of the text, -certain characters cannot be represented directly. 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