Significant Achievements in Space Bioscience 1958-1964 Part 3

s.p.a.cECRAFT STERILIZATION

The search for extraterrestrial life with unmanned s.p.a.ce 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-s.p.a.ce conditions (low temperature, high ultraviolet flux, and low dose levels of ionizing radiation) indicate that these conditions will not sterilize contaminated s.p.a.cecraft (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 compet.i.tion 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 s.p.a.ce 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 s.p.a.cecraft. 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 a.s.says 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 s.p.a.cecraft, 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 s.p.a.cecraft 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 s.p.a.cecraft 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 s.p.a.cecraft 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 s.p.a.cecraft. Without careful microbiological monitoring of manufacture and a.s.sembly procedures, many bacteria could be trapped in parts and suba.s.semblies. To permit sterilization at the lowest temperature-time regimen that will insure kill of all organisms, the microbiological load inside all parts and suba.s.semblies must be held to a minimum.

The role of industrial clean rooms in reducing the biological load on s.p.a.cecraft 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 s.p.a.cecraft 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 s.p.a.cecraft. 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 s.p.a.cecraft (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 s.p.a.cecraft 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 s.p.a.cecraft 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' alt.i.tude.

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 s.p.a.ce.

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 acc.u.mulate 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 const.i.tuents, 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 110?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 s.p.a.cecraft.

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 a.s.sumed 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 a.s.sist 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 s.p.a.cE RADIATION

This section includes part of the Summary of the Panel on Radiation Biology of the Environmental Biology Committee s.p.a.ce Science Board, NAS/NRC (1963), and results of research by the Bioscience Programs, NASA.

Radiation sources in s.p.a.ce 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 s.p.a.ce 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 att.i.tude that all radiobiological experiments need be repeated in the s.p.a.ce environment should be resolutely rejected. Since fundamental radiobiology cannot be performed easily in s.p.a.ce, it has been recommended that, wherever possible, these investigations be carried out in ground laboratories in preference to flying laboratories.

s.p.a.ce 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 s.p.a.ce. 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 s.p.a.ce environment: e.g., zero g.

Previous s.p.a.ce radiobiological studies have depended solely on very low and inaccurately measured doses of ambient s.p.a.ce 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 s.p.a.ce radiobiology.

Controlled Radiobiological Experiments in s.p.a.ce

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 s.p.a.ce 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 quant.i.ties and qualities of radiation; (4) the use of adequate controls.

High-alt.i.tude 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 s.p.a.ce-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 alt.i.tudes up to 200 nautical miles.

Most biological materials flown so far have been for the express purpose of investigating s.p.a.ce-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 s.p.a.ce 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: