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, α-alanine, and β-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 α-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)₃C, 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 10⁶ 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 (10⁶) plus the logarithm of the reciprocal of the probability
of a survivor (10⁴), 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 RADIATION¹

   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 10⁹
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 10⁶ 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, 40
                110 000 g      _Escherichia coli_    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   10⁸ erg/cm²,   Bean embryos          Suppressed growth
                2537 Å
  ----------------------------------------------------------------------
  X-ray         2×10⁶ rad      Bacteria              Growth
  ----------------------------------------------------------------------
  Gamma ray     2.45×10⁶ 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 (P³² 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.


                            NEUROPHYSIOLOGY²

   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 C¹⁴ from HC¹⁴O₃—. 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 10⁷ gauss-cm², which is equivalent to detecting a
change in specific susceptibility of 1 in 10⁹ 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 PROGRAM³

   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 (Sr⁸⁵) 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 man⁴        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 ATMOSPHERES⁹

   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 SPACE¹⁰

  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. Before manned
voyages for extended periods into deep space will be possible, solutions
must be found for problems such as the development of bioregenerative
life-support systems, communication with nonhuman species, and the
development of new methods for transferring knowledge to the human
brain.

The problems are all of the type that could perhaps be solved by truly
great advances in biological theory, and probably not by any other
avenue.




                                                            _References_


   [ref.1] _Rea, D. G.:_ The Evidence for Life on Mars. Nature, vol. 200,
           1963, p. 114.

   [ref.2] _Öpik, E. J.:_ Spectroscopic Evidence of Vegetation on Mars.
           Irish Astron. J., vol. 5, 1958, pp. 12-13.

   [ref.3] _Kuiper, G. P.:_ Visual Observations of Mars, 1956. Astrophys.
           J., vol. 125, 1957, p. 307.

   [ref.4] _Föcas, J. H.:_ Seasonal Evolution of the Fine Structure of
           the Dark Areas of Mars. Planetary Space Sci., vol. 9, 1962, p.
           371.

   [ref.5] _Dollfus, A.:_ Interpretation of the Polarization of Light
           Reflected by the Different Regions of the Surface of Mars.
           Compt. Rend., vol. 233, 1951, p. 467.

   [ref.6] _Kuiper, G. P.:_ On the Martian Surface Features. Publ.
           Astron. Soc. Pacific, vol. 67, 1955, p. 271.

   [ref.7] _Sinton, W. M.:_ Spectroscopic Evidence for Vegetation on
           Mars. Astrophys. J., vol. 126, 1957, p. 231.

   [ref.8] _Rea, D. G.; Belsky, T.; and Calvin, M.:_ Interpretation of
           the 3- to 4-Micron Infrared Spectrum of Mars. Science, vol.
           141, 1963, p. 923.

   [ref.9] _Rea, D. G.; O’Leary, B. T.; and Sinton, W.:_ Mars and the
           Origin of the 3.58- and 3.69-Micron Minima in the Infrared
           Spectra. Science, vol. 147, 1965, p. 1286.

  [ref.10] _Quimby, F. H.:_ Concepts for Detection of Extraterrestrial
           Life. NASA SP-56, 1964.

  [ref.11] _Urey, H. C.:_ The Planets. Their Origin and Development. Yale
           Univ. Press (New Haven), 1952.

  [ref.12] _Miller, S. L.:_ Mechanism of Synthesis of Amino Acids by
           Electric Discharge. Biochim. Biophys. Acta, vol. 23, 1957, p.
           480.

  [ref.13] _Oró, J.:_ Synthesis of Adenine From Ammonium Cyanide.
           Biochem. Biophys. Res. Commun., vol. 2, 1960, p. 407.

  [ref.14] _Ponnamperuma, C.; Sagan, C.; and Mariner, R.:_ Synthesis of
           Adenosine Triphosphate Under Possible Primitive Earth
           Conditions. Nature, vol. 199, 1963, p. 222.

  [ref.15] _Oparin, A. I.:_ The Origin of Life. Macmillan Co. (New York),
           1938.

  [ref.16] _Calvin, M.:_ Reduction of Carbon Dioxide in Aqueous Solutions
           by Ionizing Radiation. Science, vol. 114, 1951, p. 416.

  [ref.17] _Miller, S. L.:_ A Production of Amino Acids Under Possible
           Primitive Earth Conditions. Science, vol. 117, 1953, pp.
           528-529.

  [ref.18] _Oró, J.:_ Studies in Experimental Cosmochemistry. Ann. N. Y.
           Acad. Sci., vol. 108, 1963, pp. 464-481.

  [ref.19] _Ponnamperuma, C.:_ Chemical Evolution and the Origin of Life.
           Nature, vol. 201, 1964, pp. 337-340.

  [ref.20] _Bernal, J. D.:_ The Physical Basis of Life. Routledge &
           Kegan-Paul (London), 1951.

  [ref.21] _Fox, S.:_ Anhydrocopolymerization of Amino Acids Under the
           Influence of Hypothetically Primitive Terrestrial Conditions.
           Federation Proc., vol. 13, 1954, p. 211.

  [ref.22] _Akabori, S.:_ On the Origin of the Fore-Protein. _In_ The
           Origin of Life on the Earth. A. I. Oparin et al., eds.,
           Pergamon Press (New York), 1955, p. 189.

  [ref.23] _Harada, K.; and Fox, S. W.:_ The Thermal Synthesis of Amino
           Acids From a Hypothetically Primitive Terrestrial Atmosphere.
           _In_ The Origin of Prebiological Systems and Their Molecular
           Matrices. Academic Press (New York and London), 1965.

  [ref.24] _Oparin, A. I.:_ Biochemical Processes in the Simplest
           Structures. _In_ The Origin of Life on the Earth. Pergamon
           Press (New York), 1959.

  [ref.25] _Fox, S. W.:_ Simulated Natural Experiments in Spontaneous
           Organization of Morphological Units for Proteinoid. _In_ The
           Origin of Prebiological Systems and of their Molecular
           Matrices. Academic Press (New York), 1965.

  [ref.26] _Briggs, M. H.; and Mamikunian, G.:_ Organic Constituents of
           the Carbonaceous Chondrites. Space Sci. Rev., vol. 1, 1963, p.
           647.

  [ref.27] _Wilk, H. B.:_ Composition of Some Stony Meteorites. Geochim.
           Cosmochim. Acta, vol. 9, 1956, p. 279.

  [ref.28] _Briggs, M. H.:_ Organic Extracts of Some Carbonaceous
           Meteorites. Life Sciences, vol. 2, 1963, p. 1.

  [ref.29] _Mueller, G.:_ Interpretation of the Microstructures of
           Carbonaceous Chondrites. Nature, vol. 196, 1962, p. 929.

  [ref.30] _Mamikunian, G.; and Briggs, M. H.:_ Some Microstructures of
           Complex Morphology Observed in Preparations of Carbonaceous
           Choridrites Made Under Sterile Conditions. Nature, vol. 197,
           1963, p. 1245.

  [ref.31] _Kaplan, L. D.; Munch, G.; and Spinrad, H.:_ An Analysis of
           the Spectrum of Mars. Astrophys. J., vol. 139, 1964, p. 1.

  [ref.32] _Davis. D. R.; and Libby, W. F.:_ Positive Ion Chemistry.
           Science, vol. 144, 1961, p. 991.

  [ref.33] _Miller, S. L.:_ Production of Some Organic Compounds Under
           Possible Primitive Earth Conditions. J. Am. Chem. Soc., vol.
           77, 1955, p. 235.

  [ref.34] _Oró, J.:_ Experimental Cosmochemistry. _In_ Current Aspects
           of Exobiology. G. Mamikunian and M. H. Briggs, eds., Pergamon
           Press (New York), 1965.

  [ref.35] _Shapley, H.:_ Of Stars and Men. Beacon Press (Boston), 1958,
           pp. 11 and 157.

  [ref.36] _Lederberg, J.; and Cowie, D. B.:_ Moondust. Science, vol.
           127, 1958, pp. 1473-1475.

  [ref.37] CETEX: Development of International Efforts To Avoid
           Contamination by Extraterrestrial Exploration. Science, vol.
           128, 1958, pp. 887-889.

  [ref.38] CETEX: Contamination by Extraterrestrial Exploration. Nature,
           vol. 183, 1959, pp. 925-928.

  [ref.39] _Sagan, C.:_ Biological Contamination of the Moon. Proc. Natl.
           Acad. Sci. U.S., vol. 46, 1960, pp. 393-401.

  [ref.40] _Davies, R. W.; and Communizis, M. G.:_ Sterilization of Space
           Vehicles To Prevent Extraterrestrial Biological Contamination.
           Proc. 10th Intern. Astro. Congr., Springer-Verlag (Vienna),
           1960, pp. 495-504.

  [ref.41] _Lederberg, J.:_ Exobiology: Approaches to Life Beyond the
           Earth. Science, vol. 132, 1960, pp. 393-400.

  [ref.42] _Brown, A. H.:_ Report of the Working Subgroup on Space Probe
           Sterilization. A Review of Space Research. Natl. Acad.
           Sci.-Natl. Res. Council, Publ. 1079, 1962, ch. 10.

  [ref.43] _Imshenetskii, A. A.:_ Prospects of the Development of
           Exobiology. _In_ Space Research III, W. Priester, ed.,
           North-Holland Publ. Co. (Amsterdam), 1963.

  [ref.44] _Portner, D. M.; Spiner, D. R.; Hoffman, R. K.; and Phillips,
           C. R.:_ Effect of Ultrahigh Vacuum on Viability of
           Microorganisms. Science, vol. 134, 1961, p. 2047.

  [ref.45] _Morelli, F. A.; Fehlner, F. P.; and Stembridge, C. H.:_
           Effects of Ultra-High Vacuum on _Bacillus subtilis_ var.
           _niger_. Nature, vol. 196, 1962, pp. 106-107.

  [ref.46] _Davis, N. S.; Silverman, G. J.; and Keller, W. H.:_ Combined
           Effects of Ultrahigh Vacuum on the Viability of Some Spores
           and Soil Organisms. Appl. Microbiol., vol. II, 1963, pp.
           202-211.

  [ref.47] _Silverman, G. J.; Davis, N. S.; and Keller, W. H.:_ Exposure
           of Microorganisms to Simulated Extraterrestrial Space Ecology.
           _In_ Life Sciences and Space Research II, M. Florkin and A.
           Dollfus, eds., North-Holland Publ. Co. (Amsterdam), 1964, pp.
           372-384.

  [ref.48] _Imshenetskii, A. A.; Bogrov, N.; and Lysenko, S.:_ Resistance
           of Microorganisms to High Vacuum. Doklady Akad. Nauk SSSR,
           vol. 154, 1964, pp. 1188-1190.

  [ref.49] _Hawrylewicz, E. J.; Gowdy, B.; and Ehrlich, R.:_
           Microorganisms Under a Simulated Martian Environment. Nature,
           vol. 193, 1962, p. 497.

  [ref.50] _Packer, E.; Scher, S.; and Sagan, C.:_ Biological
           Contamination of Mars. Part II. Cold and Aridity as
           Constraints on the Survival of Terrestrial Microorganisms in
           Simulated Martian Environments. Icarus, vol. 2, 1963, pp.
           292-316.

  [ref.51] _Scher, S.; Packer, E.; and Sagan, C.:_ Biological
           Contamination of Mars: I. Survival of Terrestrial
           Microorganisms in Simulated Martian Environments. _In_ Space
           Research IV. P. Muller, ed., North-Holland Publ. Co.
           (Amsterdam), 1964.

  [ref.52] _Young, R. S.: Deal, P. H.; Bell, J.; and Allen, J. L.:_
           Bacteria Under Simulated Martian Conditions. _In_ Space
           Research IV, P. Muller, ed., North-Holland Publ. Co.
           (Amsterdam), 1963.

  [ref.53] _Hagen, C. A.; Hawrylewicz, E. J.; and Ehrlich, R.:_ Survival
           of Microorganisms in a Simulated Martian Environment. Appl.
           Microbiol., vol. 12, 1964, pp. 215-218.

  [ref.54] _Hawrylewicz, E. J.; Hagen, C. A.; and Ehrlich, R.:_ Response
           of Microorganisms to a Simulated Martian Environment. _In_
           Life Sciences and Space Research III, M. Florkin, ed.,
           North-Holland Publ. Co. (Amsterdam), 1965.

  [ref.55] _Nicks, O. W.; and Reynolds, O. E.:_ Decontamination and
           Sterilization of Lunar and Planetary Spacecraft. Science, vol.
           142, 1963, pp. 539-540.

  [ref.56] _Phillips, C. R.; and Hoffman, R. K.:_ Sterilization of
           Interplanetary Vehicles. Science, vol. 132, 1960, pp. 991-995.

  [ref.57] _Hobby, G.:_ Review of NASA-JPL Spacecraft Sterilization
           Program. A Review of Space Research. Natl. Acad. Sci.-Nat.
           Res. Council, Publ. 1079, 1962, ch. 10. App. III.

  [ref.58] _Bruch, C. W.; Koesterer, M. G.; and Bruch, M. K.:_ Dry-Heat
           Sterilization: Its Development and Application to Components
           of Exobiological Space Probes. Develop. Ind. Microbiol., vol.
           4, 1963, pp. 334-342.

  [ref.59] _Koesterer, M. G.:_ Thermal Death Studies on Microbial Spores
           and Some Considerations for the Sterilization of Spacecraft
           Components. Develop. Ind. Microbiol., vol. 6, 1964, pp.
           268-276.

  [ref.60] _Hall, L. B.; and Bruch, C. W.:_ Procedures Necessary for the
           Prevention of Planetary Contamination. _In_ Life Sciences and
           Space Research III, M. Florkin, ed., North-Holland Publ. Co.
           (Amsterdam), 1965.

  [ref.61] _Portner, D. M.; Hoffman, R. K.; Decker, H. M.; and Phillips,
           C. R.:_ The Level of Microbial Contamination in a Clean Room
           During a One-Year Period. Rept. 11-65 on NASA Interagency
           Agreement R-35, 1964.

  [ref.62] _Bruch, C. W.:_ Some Biological and Physical Factors in
           Dry-Heat Sterilization: A General Review. _In_ Life Sciences
           and Space Research II. M. Florkin and A. Dollfus, eds.,
           North-Holland Publ. Co. (Amsterdam), 1964.

  [ref.63] _Bruch, C. W.:_ Dry-Heat Sterilization for Planetary-Impacting
           Spacecraft. Tech. Rept. on Spacecraft Sterilization. Jet
           Propulsion Lab., 1965.

  [ref.64] _Pollard, E.:_ Pilot Theoretical Study of the Effect of
           Weightlessness and Densely Ionizing Radiation on Single Cells.
           Progr. Rept. on Contract NsG-182-62. NASA, 1962.

  [ref.65] _Lyon, C. J.:_ Auxin Transport in Leaf Epinasty. Plant
           Physiol., vol. 38, 1963, pp. 567-574.

  [ref.66] _McKinney, R.; Montgomery, P. O’B.; and Gell, C. F.:_ A Study
           of the Effects of Zero Gravity on Cell Physiology. _In_
           Physical and Biological Phenomena in a Weightless State.
           Second AAS Symp. Phys. Biol. Phenomena Under Zero Gravity
           Conditions. E. T. Benedikt and R. W. Halliburton, eds., Advan.
           Astronaut. Sci., vol. 14, 1963, pp. 291-306.

  [ref.67] Committee on Environmental Biology: Report of Panel on
           Gravity. Space Science Board, Natl. Acad. Sci.-Natl. Res.
           Council, 1964.

  [ref.68] _Chase, H. B.:_ Cutaneous Effects of Primary Cosmic Radiation.
           J. Aviation Med., vol. 25, 1954, p. 388.

  [ref.69] _Zhukova, A. I.; and Kondratyev, I. I.:_ On Artificial Martian
           Conditions Reproduced for Microbiological Investigations. _In_
           Life Sciences and Space Research III, M. Florkin, ed.,
           North-Holland Publ. Co. (Amsterdam), 1965, pp. 120-126.

  [ref.70] _Davis, I.; and Fulton, J. D.:_ Microbiological Studies on
           Ecological Considerations of the Martian Environment. USAF SAM
           Review, vol. 2, 1959, p. 60.

  [ref.71] _Roberts, T. S.; and Irvine, L. A.:_ Studies With a Simulated
           Martian Environment. Germination and Growth of Bacterial
           Spores. Tech. Doc. Rept. 63-75. USAF School Aerospace Med.,
           1963.

  [ref.72] _Roberts, T. L.; and Wynn, E. S.:_ Studies With a Simulated
           Martian Environment. Bacterial Survival and Soil Moisture
           Content. Tech. Doc. Rept. 62-121. USAF School Aerospace Med.,
           1962.

  [ref.73] _Siegel, S. M.; Giumarro, C.; and Latterall, R.:_ Behavior of
           Plants Under Extraterrestrial Conditions: Seed Germination in
           Atmospheres Containing Nitrogen Oxides. Proc. Natl. Acad. Sci.
           U.S., vol. 52, 1961, pp. 11-13.

  [ref.74] _Siegel, S. M.; Halpern, L. A.; Giumarro, C.; Renwick, G.; and
           Davis, G.:_ Martian Biology: The Experimentalist’s Approach.
           Nature, vol. 193, 1963, pp. 329-331.

  [ref.75] _Kiess, C. C.; Corliss, C. H.; and Kiess, H.:_ Evidence for
           Oxides of Nitrogen in the Atmosphere of Mars. Science, vol.
           131, 1960, p. 1319.

  [ref.76] _Vallentyne, J. R.:_ Environmental Biophysics and Microbial
           Ubiquity. Ann. N.Y. Acad. Sci., vol. 108, 1963, pp. 342-352.

  [ref.77] _Henry, J. P.; Ballinger, E. R.; Maher, P. J.; and Simons, D.
           G.:_ Animal Studies of the Subgravity State During Rocket
           Flight. J. Aviation Med., vol. 23, 1952, p. 421.

  [ref.78] _Van der Wal., F. L.; and Young, W. D.:_ A Preliminary
           Experiment With Recoverable Biological Payloads in Ballistic
           Rockets. Project MIA. Rept. 715-58, Am. Rocket Soc., 1958.

  [ref.79] _Van der Wal, F. L.; and Young, W. D.:_ Project MIA
           (Mouse-in-Able) Experiment on Physiological Response to Space
           Flight. ARS J., vol. 29, 1954, p. 716.

  [ref.80] _Graybiel, A.; Holmes, R. A.; Beischer, D. E.; Champlin, G.
           E.; Pedigo, G. P.; Hixson, W. C.; Davis, T. R. A.; Barr, N.
           L.; Kistler, W. G.; Niven, J. T.; Wilbarger, E.; Stullken, D.
           E,; Augerson, W. S.; Clark, R.; and Berrian, J. H.:_ An
           Account of Experiments in Which Two Monkeys Were Recovered
           Unharmed After Ballistic Space Flight. Aerospace Med., vol.
           30, 1959, p. 871.

  [ref.81] _Green, C. D.; Welch, B. E.; Brown, W. L.; Lamb, L. E.; Tang,
           P. C.; Gisler, D. B.; and Blodgett, H. C.:_ Studies of Escape
           From Ballistic Space Vehicles. Rept. 61-29, School Aviation
           Med., sec. 1, pp. 1-24; sec. 2, pp. 1-16, 1961.

  [ref.82] _Belleville, R. E.; Rohles, F. H.; Grunzke, M. E.; and Clark,
           F. C.:_ Development of a Complex Multiple Schedule in the
           Chimpanzee. J. Exp. Anal. Behav., vol. 6, 1963, pp. 549-556.

  [ref.83] _Henry, J. P.; and Mosely, J. D.:_ Results of the Project
           Mercury Ballistic and Orbital Chimpanzee Flights. NASA SP-39,
           1963.

  [ref.84] _Grunzke, M. E.:_ A Liquid Dispenser for Primates. J. Exp.
           Anal. Behav., vol. 4, 1961, p. 326.

  [ref.85] _Grunzke, M. E.:_ Feeding Devices for Use With Primates in
           Space Flight. Tech. Doc. Rept. 61-35, USAF Missile Develop.
           Center, 1961.

  [ref.86] _Gilbert, G. A.:_ A Zero Gravity Pellet Dispenser for Use With
           Primates in Long-Term Space Flights. ARL Tech. Rept. 64-15,
           1964.

  [ref.87] _Meehan, J. P.; Fineg, J.; and Mosely, J. D.:_ The Effect of
           Restraint and Training on the Arterial Pressure of the
           Immature Chimpanzee. Federation Proc., vol. 23, 1964.

  [ref.88] _Findley, J.; and Weissman, N.:_ "Counting" in a Baboon. Paper
           presented at Psychonomic Soc. Meeting (New York), 1961.

  [ref.89] _Findley, J.; and Brady, J. V.:_ Exposure to Total and
           Continuous Environmental Control With a Single Human Organism.
           Paper presented at Symp. at 7th Annual Meeting, Human Factors
           Soc. (Palo Alto, Calif.), 1963.

  [ref.90] _Findley, J.:_ An Experimental Outline for Building and
           Exploring Multioperant Behavior Repertories. J. Exp. Anal.
           Behav., vol. 5, 1962, pp. 113-116.

  [ref.91] _Beasley, J.; and Seldeen, B.:_ The Effect of Prolonged
           Acceleration on Eating and Performance. NASA TM X-54:077,
           1964.

  [ref.92] _Smith, A. H.; Winget, C. M.; and Kelly, C. F.:_ Growth and
           Survival of Birds Under Chronic Acceleration. Growth, vol. 23,
           1959, p. 97.

  [ref.93] _Winget, C. M.; Smith, A. H.; and Kelly, C. F.:_ Effects of
           Chronic Acceleration on Induced Nystagmus in the Fowl. J.
           Appl. Physiol., vol. 17, 1962, p. 709.

  [ref.94] _Wunder, C. C.:_ Food Consumption of Mice During Continual
           Centrifugation. Proc. Iowa Acad. Sci., vol. 68, 1961, pp.
           616-624.

  [ref.95] _Wunder, C. C.:_ Survival of Mice During Chronic
           Centrifugation. Aerospace Med., vol. 33, 1962, pp. 866-870.

  [ref.96] _Wunder, C. C.; Lutherer, L. C.; and Dodge, C. H.:_ Survival
           and Growth of Organisms During Life-Long Exposure to High
           Gravity. Aerospace Med., vol. 34, 1963, pp. 5-11.

  [ref.97] _Dodge, C. H.; and Wunder, C. C.:_ Growth of Turtles During
           Continual Centrifugation. Proc. Iowa Acad. Sci., vol. 69,
           1962, pp. 594-599.

  [ref.98] _Oyama, J.; and Platt, W.:_ Effects of Deceleration on Rats
           Exposed to Prolonged Centrifugation. Nature, vol. 203, 1964,
           pp. 766-767.

  [ref.99] _Matthews, B. H. C.:_ Adaptation to Centrifugal Acceleration.
           J. Physiol., vol. 122, 1953, p. 31.

 [ref.100] _Riccio, D.:_ The Effects of Vestibular Stimulation on
           Performance Under Several Schedules of Reinforcement. Paper
           presented at Eastern Psych. Assoc. Meeting (Atlantic City,
           N.J.), 1965.

 [ref.101] _Guedry, F. E.; and Biberman, N.:_ Apparent Adaptation Effects
           in Vestibular Reactions. Rept. 293, USA Med. Res. Lab., 1957.

 [ref.102] _Guedry, F. E.; Cramer, R. L.; and Koella, W. P.:_ Experiments
           on the Rate of Development and Rate of Recovery of Apparent
           Adaptation Effects in the Vestibular System. Rept. 338. USA
           Med. Res. Lab., 1958.

 [ref.103] _Guedry, F. E.; and Graybiel, A.:_ The Appearance of
           Compensatory Nystagmus in Human Subjects as a Conditional
           Response During Adaptation to a Continuously Rotating
           Environment. Rept. 531, USA Med. Res. Lab., 1962.

 [ref.104] _Loret, B. J.:_ Optimization of Manned Orbital Satellite
           Vehicle Design With Respect to Artificial Gravity. ASD Tech.
           Rept. 61-688, Wright-Patterson AFB, Ohio, 1961.

 [ref.105] _Belleville, R. E.; Clark. F. C.; and Lange, K. O.:_ The
           Behavior of Small Animals Under the Accelerative Conditions
           Found in Space Travel. NASA TT F-9080, 1964.

 [ref.106] _Hebb, D. O.:_ The Organization of Behavior. John Wiley &
           Sons, Inc., 1949.

 [ref.107] _Jeffress, L. A.:_ Cerebral Mechanisms in Behavior. John Wiley
           & Sons, Inc., 1951.

 [ref.108] _Konorsky, J.:_ Mechanisms in Animal Behavior. Symp. Soc. Exp.
           Biol., vol. 4, Academic Press, 1950.

 [ref.109] _Eccles, J. C.:_ The Neurophysiological Basis of Mind. Oxford
           Univ. Press (London), 1953.

 [ref.110] _Thorpe, W. H.:_ Learning and Instinct in Animals. Methuen
           (London), 1956.

 [ref.111] _Sperry, R. W.:_ On the Neural Basis of the Conditioned
           Response. Brit. J. Anim. Behav., vol. 3, 1955, p. 41.

 [ref.112] _Burns, B. D.:_ The Mammalian Cerebral Cortex. Monographs
           Physiol. Soc. (London), vol. 5, 1958.

 [ref.113] _Krech, D.; Rosenzweig, M. R.; and Bennett, E. L.:_ Dimensions
           of Discrimination and Level of Cholinesterase Activity in
           Cerebral Cortex of the Rat. J. Comp. Physiol. Psychol., vol.
           49, 1956, p. 261.

 [ref.114] _Krech, D.; Rosenzweig, M. R.; and Bennett, E. L.:_
           Correlation Between Brain Cholinesterase and Brain Weight
           Within Two Strains of Rats. Am. J. Physiol., vol. 196, 1959,
           pp. 31-32.

 [ref.115] _Bennett, E. L.; Rosenzweig, M. R.; Krech, D.; Karlsson, H.;
           Dye, N.; and Ohlander, A.:_ Individual, Strain, and Age
           Differences in Cholinesterase Activity of the Rat Brain. J.
           Neurochem., vol. 3, 1958, p. 153.

 [ref.116] _Rosenzweig, M. R.; Krech, D.; and Bennett, E. L.:_ Effect of
           Pentobarbital Sodium on Adaptive Behavior in the Rat. Science,
           vol. 123, 1956, pp. 371-372.

 [ref.117] _Rosenzweig, M. R.; Krech, D.; and Bennett, E. L.:_ A Search
           for Relations Between Brain Chemistry and Behavior. Psychol.
           Bull., vol. 57, 1960, p. 476.

 [ref.118] _Hyden, H.:_ Biochemistry of the Central Nervous System.
           Pergamon Press (New York), 1959.

 [ref.119] _Hyden, H.; and Egyhazi, E.:_ Nuclear RNA Changes of Nerve
           Cells During a Learning Experiment in Rats. Proc. Natl. Acad.
           Sci. U.S., vol. 48, 1962, p. 1366.

 [ref.120] _Brachet, J.; and Mirsky, A. E.:_ The Cell: Biochemistry,
           Physiology, Morphology. Vol. IV, ch. 5. Academic Press (New
           York), 1960.

 [ref.121] _Tower, D. B.; and Schade, J. P.:_ Structure and Function of
           the Cerebral Cortex. Elsevier (New York), 1960.

 [ref.122] _Dingman, W.; and Sporn, M. B.:_ The Incorporation of
           8-Azaguanine Into Rat Brain RNA and Its Effect on Maze
           Learning by the Rat. An Inquiry Into the Biochemical Basis of
           Memory. J. Psychiatric Res., vol. 1, 1961, pp. 1-11.

 [ref.123] _Adey, W. R.; Kado, R. T.: Didio, J.; and Schindler, W. J.:_
           Impedance Changes in Cerebral Tissue Accompanying a Learned
           Discriminative Performance in the Cat. Exp. Neurol., vol. 7,
           1963, pp. 282-293.

 [ref.124] _Brattgard, S. O.:_ Acta Radiol. Suppl., vol. 96, 1952, p. 1.

 [ref.125] _Geiger, A.:_ Correlation of Brain Metabolism and Function by
           the Use of a Brain Perfusion Method _In Situ_. Physiol. Rev.,
           vol. 38, 1958, pp. 1-20.

 [ref.126] _Geiger, A.:_ Metabolism of the Nervous System. D. Richter,
           ed., Pergamon Press (New York), 1957.

 [ref.127] _Thompson, R.; and McConnell, J.:_ Classical Conditioning in
           the Planarian _Dugesia dorotocephala_. J. Comp. Physiol.
           Psychol., vol. 48, 1955, p. 65.

 [ref.128] _McConnell, J. V.; Jacobson, A. L.; and Kimble, D. P.:_
           Effects of Regeneration Upon Retention of a Conditioned
           Response in the Planarian. J. Comp. Physiol. Psychol., vol.
           52, 1959, p. 1.

 [ref.129] _Corning, W. C.; and John, E. E.:_ Effects of Ribonuclease on
           Retention of a Conditioned Response in Regenerating
           Planarians. Science, vol. 134, 1961, p. 1363.

 [ref.130] _Gerathewohl, S. J.; and Gernandt, B. E.:_ Physiological and
           Behavioral Sciences, Vol. 1, NASA SP-11, 1962, pp. 399-413.

 [ref.131] _Von Bekesy, G. J.:_ DC Potentials and Energy Balance of the
           Cochlear Partition. Acoust. Soc. Am., vol. 23, 1951, p. 576.

 [ref.132] _Trincker, D.:_ Bestandspotentiale in Bogengangssystem des
           Meerschweinchens und ihre Anderungen bei Experimentellen
           Cupula-Ablenkunges. Arch. Ges. Physiol., Bd. 264, 1957, p.
           351.

 [ref.133] _Livingston, R. B.:_ Neural Mechanisms of the Auditory and
           Vestibular Systems. Ch. 25, G. L. Rasmussen and W. F. Windle,
           eds., Charles C Thomas (Springfield, Ill.), 1960.

 [ref.134] _Brodal, A.:_ Neural Mechanisms of the Auditory and Vestibular
           Systems. G. L. Rasmussen and W. F. Windle, eds., Charles C
           Thomas (Springfield, Ill.), 1960.

 [ref.135] _Brodal, A.; Pompeiano, O.; and Walberg, F.:_ The Vestibular
           Nuclei and Their Connections, Anatomy, and Functional
           Correlations. Charles C Thomas (Springfield, Ill.), 1960.

 [ref.136] _Carpenter, M. B.:_ Neural Mechanisms of the Auditory and
           Vestibular Systems. Ch. 22, G. L. Rasmussen and W. F. Windle,
           eds., Charles C Thomas (Springfield, Ill.), 1960.

 [ref.137] _Dow, R. S.; and Moruzzi, G.:_ The Physiology and Pathology of
           the Cerebellum. Univ. Minn. Press (Minneapolis), 1958.

 [ref.138] _Kempinsky, W. H.:_ Cortical Projection of Vestibular and
           Facial Nerves in the Cat. J. Neurophysiol., vol. 14, 1951, p.
           203.

 [ref.139] _Mickle, W. A.; and Ades, H. W.:_ A Composite Sensory
           Projection Area in the Cerebral Cortex of the Cat. Am. J.
           Physiol., vol. 170, 1952, p. 682.

 [ref.140] _Walzl, E. M.; and Mountcastle, V.:_ Projection of Vestibular
           Nerve to Cerebral Cortex of the Cat. Am. J. Physiol., vol.
           159, 1949, p. 595.

 [ref.141] _Anderson, S.; and Gernandt, B. E.:_ Acta Oto-Laryngol.
           Suppl., vol. 116, 1954, p. 10.

 [ref.142] _Gernandt, B. E.; and Thulin, C. A.:_ Vestibular Mechanisms of
           Facilitation and Inhibition of Cord Reflexes. Am. J. Physiol.,
           vol. 172, 1953, pp. 653-660.

 [ref.143] _Gernandt, B. E.; and Terzuolo, C. A.:_ Effect of Vestibular
           Stimulation on Strychnine-Induced Activity of the Spinal Cord.
           Am. J. Physiol., vol. 183, 1955, pp. 1-8.

 [ref.144] _Gernandt, B. E.; Katsuki, Y.; and Livingston, R. B.:_
           Functional Organization of Descending Vestibular Influences.
           J. Neurophysiol., vol. 20, 1957, pp. 453-469.

 [ref.145] _Gernandt, B. E.; and Gilman, S.:_ Vestibular and
           Propriospinal Interactions and Protracted Spinal Inhibition by
           Brain Stem Activation. J. Neurophysiol., vol. 23, 1960, pp.
           269-287.

 [ref.146] _Gernandt, B. E.; and Gilman, S.:_ Interactions Between
           Vestibular, Pyramidal and Cortically Evoked Extrapyramidal
           Activities. J. Neurophysiol., vol. 23, 1960, pp. 516-533.

 [ref.147] _Akert, K.; and Gernandt, B. E.:_ Electroencephalog. Clin.
           Neurophysiol., vol. 14, 1962, p. 383.

 [ref.148] _Gerathewohl, S. J.; and Ward, J. E.:_ The Physics and
           Medicine of the Upper Atmosphere and Space. O. O. Benson and
           H. Strughold, eds., John Wiley & Sons, Inc., 1960, ch. 26.

 [ref.149] _Gerathewohl, S. J.:_ Personal Experiences During Short
           Periods of Weightlessness Reported by Sixteen Subjects.
           Astronaut. Acta, vol. 2, 1956, pp. 2O5-217.

 [ref.150] _Henry, J. P.; Augerson, W. S.; Belleville, R. E.; Douglas, W.
           K.; Grunzke, M. K.; Johnston, R. S.; Laughlin, P. C.; Mosely,
           J. D.; Rohles, F. H.; Voas, R. B.; and White, S. C.:_ Effects
           of Weightlessness in Ballistic and Orbital Flight. Aerospace
           Med., vol. 33, 1962, pp. 1056-1068.

 [ref.151] _Dayhoff, M. O.; Lippincott, E. R.; and Eck, R. V.:_
           Thermodynamic Equilibria in Prebiological Atmospheres.
           Science, vol. 146, 1964, p. 1461.

 [ref.152] _Armstrong, G. T.; Furukawa, G. T.; and Hilsenrath, J.:_ A
           Survey of Thermodynamic Properties of the Compounds of the
           Elements CHNOPS. NBS Rept. 8521, U.S. Dept. Com., 1964.

 [ref.153] _White, W. B.; Johnson, S. M.; and Dantzig, G. B.:_ J. Chem.
           Phys., vol. 28, 1958, p. 751.

 [ref.154] _Jukes, T. H.:_ Present Status of the Amino Acid Code. J. Am.
           Dietet. Assoc., vol. 45, 1964, p. 517.

 [ref.155] _Nirenberg, M. W.; and Matthaei, J. H.:_ The Dependence of
           Cell-Free Protein Synthesis in E. _coli_ upon Naturally
           Occurring or Synthetic Polyribonucleotides. Proc. Natl. Acad.
           Sci. U.S., vol. 47, 1961, p. 1588.

 [ref.156] _Ochoa, S.:_ Chemical Basis of Heredity, the Genetic Code.
           Experientia, vol. 20, 1964, p. 57.

 [ref.157] _Kiesow, L.:_ The Energy-Transforming Step in
           Nitrobacter-Chemosynthesis. Biochem. Z., Bd. 338, 1963, p.
           400.

 [ref.158] _Kiesow, L.:_ On the Assimilation of Energy From Inorganic
           Sources in Autotrophic Forms of Life. Proc. Natl. Acad. Sci.
           U.S., vol. 52, 1964, p. 980.

 [ref.159] _Clayton, R. K.; and Adler, H. I.:_ Protein Synthesis and
           Viability in X-Irradiated _Rhodopseudonomonas spheroides_.
           Biochim. Biophys. Acta, vol. 56, 1962, p. 257.

 [ref.160] _Pollard, E.; and Vogler, C.:_ Radiation Action on Some
           Metabolic Processes in E. _coli_. Radiation Res., vol. 15,
           1961, p. 109.

 [ref.161] _Novelli, G. D.; Kameyama, T.; and Eisenstadt, J. M.:_ Cold
           Spring Harbor Symp. Quant. Biol., vol. 26, 1961, p. 133.

 [ref.162] _Kepes, A.:_ Kinetics of Induced Enzyme Synthesis.
           Determination of the Mean Life of a Galactosidase-Specific
           Messenger RNA. Biochim. Biophys. Acta, vol. 76, 1963, p. 293.

 [ref.163] _Fernandez-Moran, H.:_ New Approaches in Correlative Studies
           of Biological Ultrastructure by High-Resolution Electron
           Microscopy. J. Roy. Microscop. Soc., vol. 83, 1964, p. 183.

 [ref.164] _Fernandez Moran, H.:_ Electron Microscope-Medicines Research:
           Tool of Unfulfilled Promise. J. Am. Med. Assoc. 189, 1964,
           p.31.

 [ref.165] _Fernandez Moran, H.:_ Electron Microscope With High Field
           Superconducting Solenoid Lenses. Proc. Natl. Acad. Sci. U.S.,
           vol.53, 1965, p. 445.

 [ref.166] _Deaver, B. S. Jr.; Swedlund, J. B.; and Bradley, H. Jr.:_
           Magnetic Properties of Some Macromolecules of Biological
           Interest. Final Rept. PHU 4644. Stanford Res. Inst., 1964.

 [ref.167] _Sisakyan, N. M.:_ Problems of Space Biology. Vol. I, USSR
           Acad. Sci. Publ. House (Moscow) 1962.

 [ref.168] _Sisakyan, N. M. and Yazdovskiy V. I._,: Problems of Space
           Biology. Vol. III. USSR Acad. Sci. Publ. House (Moscow),
           1964.

 [ref.169] _Jenkins D. W._,: The NASA Biosatellite Program. _In_ Life
           Sciences and Space Research III, M. Florkin, ed,.
           North-Holland Publ. Co. (Amsterdam), 1965, pp. 230-240.

 [ref.170] _Pack N.:_ The Effects of Weightlessness on Mammals. Space
           Biology: Proc. 24th Annual Biol. Colloq., Oregon St. Univ.,
           1963, pp. 65-74.

 [ref.171] _Anon.:_ A Review of Space Research. Ch. 9, Natl. Acad.
           Sci.-Natl. Res. Council. Publ. 1079, 1962.

 [ref.172] Committee on Environmental Biology: Report of Panel on
           Radiation Biology. Space Science Board, Natl. Acad. Sci.-Natl.
           Res. Council, 1963.

 [ref.173] _Taleot, J. M.:_ Life Support in Space Operations. Air Univ.
           Rev., vol. 16, 1965, pp. 42-52

 [ref.174] _Del Duca, M. G.; Konecci, E. B.; and Ingelfinger A. L.:_ Life
           Support-the Next Generation. Space Aeronautics, vol. 41, 1964,
           pp. 84-91.

 [ref.175] _Bongers. L.; and Kok B.:_ Life Support for Space Missions.
           Develop. Ind. Micro-biol., vol. 5, 1964, pp. 183-195.

 [ref.176] _Ward, C. H.; Wilks. S. S.; and Craft, H. L.;_ Use of Algae
           and Other Plants in the Development of Life Support Systems.
           Am. Biol. Teacher, vol. 25, 1963, pp. 512-521.

 [ref.177] _Ney L. F.:_ Gas Exchange by the Duckweed Family. ONR Contract
           No. Nonr-2887(00), Stanford Res. Inst., 1960.

 [ref.178] _Nakamura, H.:_ A Study of Wolffia as a New Food. Rept.
           Microalgal Res. Inst. Japan., vol. 1, 1960, pp. 7-13.

 [ref.179] _Myers, J.:_ Basic Remarks on the Use of Plants as Biological
           Gas Exchangers in a Closed System. _In_ Epitome of Space
           Medicine. J. Aviation Med., vol. 25, 1954, pp. 407-411.

 [ref.180] _Bowman, N. J.:_ The Food and Atmosphere Control Problems on
           Space Vessels. J. Brit. Interplanet. Soc, vol. 12, 1953, pp.
           118-123.

 [ref.181] _Bowman, N. J.:_ The Food and Atmosphere Control Problems on
           Space Vessels. Part II. The Use of Algae for Food and
           Atmosphere Control. J. Brit. Interplanet. Soc. Vol. 12, 1953,
           pp. 159-166.

 [ref.182] _Bassham, J. A.:_ Experiments With Photosynthetic Gas
           Exchangers. Proc. World Symp. Appl. Solar Energy, Phoenix,
           Ariz., 1955, pp. 35-50.

 [ref.183] _Burk D.; Hobby, G.; and Gaucher, T.:_ Closed-Cycle Air
           Purification With Algae. Proc. First Intern. Symp. Submarine
           Space Med. Macmillan Co. (New York), 1958.

 [ref.184] _Dole, S. H.; and Tamplin, A. R.:_ The Sabatier Reaction for
           Inorganic Recovery of Oxygen in Manned Space Capsules. _In_
           Closed Circuit Respiratory Systems Symp. Tech. Rept. 60-574,
           Wright Air Develop. Div., 1960, pp. 239-287.

 [ref.185] _Clifford, J. E.; and McCallum, J.:_ Research on the
           Electrolysis of Water Under Weightless Conditions. AMRL-Tech.
           Doc. Rept. 62-44, 1962.

 [ref.186] _Clifford, J.; and Faust, C.:_ Research on Electrolysis of
           Water With a Hydrogen Diffusion Cathode To Be Used in a
           Rotating Cell. AMRL-Tech. Doc. Rept. 62-94, 1962.

 [ref.187] _Rupaske, R.:_ Nutritional Requirements for _Hydrogenomonas
           eutropha_. J. Bacteriol, vol. 83, 1962, pp. 418-422

 [ref.188] _Bongers, L.:_ Chemosynthetic Gas Exchanger. Quarterly
           Progress Rept. No. 4 on NASA Contract NASw-971, 1965, 23 pp.

 [ref.189] _Bongers, L.:_ Chemosynthetic Gas Exchanger. Ann. Rept. on
           NASA Contract NASw-713, 1964, 44 pp.

 [ref.190] _Bongers, L.:_ Chemosynthetic Gas Exchanger. Rept. RM
           153-1963. Martin-Marietta Space Systems Div. (Baltimore, Md.),
           1963

 [ref.191] _Bongers, L.:_ Sustaining Life in Space—A New Approach.
           Aerospace Med., vol. 35, 1964, pp. 139-144.

 [ref.192] _Schlegel, H.G.:_ Die Verwendung von H₂-oxydierenden Bakterien
           zur Regenerierung der Atemluft. Raumfahrtforschung, Bd. 8. Nr.
           2, 1964, pp. 65-67.

 [ref.193] _Webb. P., ed.:_ Bioastronautics Data Book. NASA SP-3006,
           1964.

 [ref.194] _Foster. J. F.; and Litchfield, J. H.:_ A Continuous Culture
           Apparatus for the Microbial Utilization of Hydrogen Produced
           by Electrolysis of Water in Closed-Cycle Space Systems.
           Biotech. Bioeng., vol. 6, 1964, pp. 441-456.

 [ref.195] _Callaway. D.; and Margen, S.:_ Nutritional Properties of
           _Hydrogenomonas eutropha_. Semiann. Progr. Rept., NASA Grant
           NGR-05-003-(089), 1965.

 [ref.196] Working Group on Gaseous Environment for Manned Spacecraft:
           Summary Report. Space Science Board. NAS/NRC, 1963.

 [ref.197] _Lawton, R. W.:_ Physiological Considerations Relevant to the
           Problem of Prolonged Weightlessness. Astronaut. Set. Rev.,
           vol. 4, 1962, pp. 1-16.

 [ref.198] _McCally, M.; and Lawton. R. W.:_ The Pathophysiology of
           Disuse and the Problem of Prolonged Weightlessness: A Review.
           AMRL-Tech. Doc. Rept. 63-3, Aerospace Med. Div.
           (Wright-Patterson AFB, Ohio), 1963.

 [ref.199] _Smith, R. E.; and Hock, R. J.:_ Brown Fat: Thermogenic
           Effector of Arousal in Hibernators. Science, vol. 140, 1963,
           p. 199.

 [ref.200] _Storer. J. B.; and Hempelman L. H.:_ Hypothermia and
           Increased Survival Rate of Infant Mice Irradiated With X-rays.
           Am. J. Physiol., vol. 171, 1952, pp. 341-348.

 [ref.201] Working Group on Nutrition and Feeding Problems: Summary
           Report. Space Science Board. Man-in-Space Comm., NAS/NRC,
           1963.

 [ref.202] _Anon.:_ Conference on Nutrition in Space and Related Waste
           Problems. NASA SP-70, 1964.

                    * U.S. GOVERNMENT PRINTING OFFICE 1966 - O - 798 520




                                                      Transcription note


The following typographic errors have been corrected:

  - *p. 2, l. 16:* to be called ‘"exobiology." ——> to be called
    "exobiology."
  - *Table III.- Ultraviolet/Maximum:* 10⁸ erg/cm², 2537° Å ——> 10⁸
    erg/cm², 2537 Å
  - *p. 58, l. 14-15:* approximately 300 atm) ——> approximately 300 atm.)
  - *p. 80, l. -2:* (600-1000 ft=c) ——> (600-1000 ft-c)
  - *p. 87, l. 1:* 0.2-mM ——> 0.2 mM
  - *p. 99, l. 1-2:* faintsess ——> faintness
  - *p. 104, l. 2:* hiberation ——> hibernation
  - *p. 116, l. 19:* processsing ——> processing
  - *p. 121, l. 1:* _Hoffman, R. K.,_ ——> _Hoffman, R. 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 rejoined 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].

UTF-8 characters: This version contains UTF-8 characters to represent
Greek letters, subscripts, superscripts and some punctuation. If these
characters do not appear correctly, you may need to select a different
font. In order for the table columns to line up correctly, this will
need to be a monospaced, or fixed-width, font.