Significant Achievements in Space Bioscience 1958-1964 Part 4

(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 a.s.sociated with increased bacteriophage production by the lysogenic bacteria.

There was an increase of recessive lethals coupled with nonconvergence of chromosomes (s.e.x linked) in the fruit fly. A stimulation of cell division in wheat and pea seeds was observed.

Cultures of human cells exposed to s.p.a.ce-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 s.p.a.ce 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 a.s.sessed 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 alt.i.tudes. 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 acc.u.mulate 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 acc.u.mulation 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 const.i.tuents 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 a.s.sumed 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 100150180 cm. Micro-organisms were placed at the surface of a copper bar made in a special groove separated by gla.s.s 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 s.p.a.ce 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 a.s.sumed 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: 147 -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 2515, 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 Gla.s.s 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 , with a dose of 10?

ergs/cm, 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]) a.s.sumed 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 s.p.a.ce 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 4 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 cuc.u.mbers, corn, and winter rye, to low temperatures and lowered oxygen tensions. Lowered oxygen tensions enhanced the resistance of seedlings, particularly cuc.u.mber 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.