On different sensitivities of microorganisms to lowered gravitation.

The influence of lowered gravitation on biomass and CO2 production in B. megaterium, a xerophyte, and Spirillum azotocolligens, an aqueous spirillum, in liquid nutrient medium on a horizontal clinostat at 0.1 g has been studied. As controls we considered: 1) growth under stationary conditions of cultivation with test tubes oriented horizontally; 2) growth on a synchronously revolving centrifuge; and 3) growth on a swing with stirring. A horizontal clinostat at 0.1 g stimulates biomass production and CO2 release in B. megaterium as compared with the controls. Spirillum azotocolligens growth is reduced as a result of clinostating. The best development and CO2 production are observed under stationary conditions. The results do not support the assumption that microorganisms living in water are more resistant to lowered gravitation than those living in soil.

Biological studies of Martian soil analogues.

Results of the study of the influence of Martian soil analogues, both as described by American scientists and as prepared by us, and of hydrogen peroxide on the viability of microorganisms are presented. The experiments were carried out using mixtures of soil analogues with desert soil and black earth (chernozem) samples, and pure cultures of microorganism. Microorganisms capable of withstanding a concentration of hydrogen peroxide in the medium as high as 1.5-2.0% were isolated. None of the 40 strains of microorganisms studied, all belonging to different systematic and physiological groups, exhibited growth inhibition on solid media in the presence of Martian soil analogues. In view of the fact that Martian soil cannot contain microorganisms in great quantities, we suggest using electroadsorption for their concentration, to make detection reliable. A device was designed for this purpose, using the principle of electroadsorption on a polarisable carrier (sterile cotton wool or cheesecloth). The concentrated suspension of microorganisms thus obtained was then characterized by various physicochemical methods.

Microorganisms of the upper layer of the atmosphere and the protective role of their cell pigments.

Of the six species of microorganisms isolated from the mesosphere, five contained pigments and were more resistant to UV radiation compared with their pigment-free mutants. The black pigment isolated from the conidia of Aspergillus niger considerably increased the UV resistance of the unpigmented mutant conidia of Penicillium notatum, the spore Circinella muscae and the vegetative cells of Micrococcus albus. From the data it is possible to conclude that in the upper layers of the Earth's atmosphere the predominant proportion of pigmented microorganisms is the consequence of natural selection by UV radiation.

Upper boundary of the biosphere.

By using meterological rockets fitted with specially designed analyzers, samples for microbiological investigation have been taken. The analyzer design prevented extraneous microorganisms from penetrating into the analyzer. Before being used, the analyzers were sterilized with high gamma-ray doses. For the first time microorganisms have been detected in the mesosphere at an altitude of 48 to 77 km. The microorganisms are microscopic fungi having black conidia or spores (Circinella muscae, Aspergillus niger, Papulaspora anomala) and one species forming green conidia (Penicillium notatum). Colonies of Mycobacterium luteum and Micrococcus albus have also grown. Five of the six species have synthesized pigments. The presence of pigmented microbial forms leads us to believe that natural selection is occurring in the mesosphere because cells possessing chromogenous pigments (carotenoids, melanins) are more resistant to ultraviolet-ray action. A greater number of microorganisms have been registered in the mesosphere during dust storms than in the absence of strong winds.

Resistance of stratospheric and mesospheric micro-organisms to extreme factors.

Studies of the stratosphere and mesosphere, by means of special analysers installed on meteorological rockets, have thrown more light on our knowledge of the upper boundary of the biosphere. The presence of the following micro-organisms was registered at heights of 49-77 km: Aspergillus niger, Penicillium notatum, Circinella muscae, Papulaspora anomala, Mycobacterium luteum and Micrococcus albus. The isolated micro-organisms were subjected to the action of gamma-irradiation, high vacuum and UV radiation in order to evaluate the quality of sterilization by gamma-rays (3.2-3.5 Mrad) prior to sampling and the resistance of these micro-organisms to physical factors of the stratosphere and mesosphere. No species with high radio-resistance were detected among the isolated cultures. The D10 index for fungal spores and bacterial vegetative cells, freeze-dried or suspended in a physiological solution, did not exceed 290 krad. These data confirm that sterilization of the analyser with gamma-rays assured the purity of biological experiments during sampling. The isolated micro-organisms were found to be very resistant to high vacuum (10(-9) mmHg) and UV radiation, with the exception of the pigmentless Micrococcus albus. This evidence shows that pigmented micro-organisms can survive in the earth's atmosphere at high altitudes.

Physiology of xerophytic micro-organisms growing under Martian conditions.

The main factor limiting growth of terrestrial organisms under Martian conditions is the low humidity; therefore the biology of xerophytic micro-organisms has been studied. We found earlier that Bacillus megaterium var. halotolerant and Mycococcus ruber could survive and grow under Martian conditions. In this work, we studied the zone of tolerance of these and some other micro-organisms towards various levels of water activity, and the toxicity of substances which were used to control this activity. A technique for quantitative evaluation of the xerophytic nature of micro-organisms is proposed. The technique is based on quantitative evaluation by gas chromatography of carbon dioxide evolved in the course of growth of micro-organisms on media with different values of water activity.

Variability of polyploid strains of Candida scottii in accumulation of riboflavin in the medium.

Polyploid strains of Candida scottii show a higher spontaneous and UV-induced variability in accumulation of riboflavin in the medium than the original haploid strain. UV irradiation affects the formation of variants and induces those which accumulate more riboflavin than any of the most productive variants resulting from spontaneous variability. In the polyploid strains the frequency of plus-variants increases. Therefore, polyploid strains of C. scottii are advantageous for selection processes.

On methods of detection of extraterrestrial life.

New methods have been developed for detecting microbial growth from the microflora of desert soils. The first is a polarimetric method in which the fall in optical activity due to assimilation of D-glucose is followed. Detectable changes with desert soils were seen in a few hours, and the method can be employed with small amounts of material. The second method is the release of heat from metabolizable substrates as measured by a microcalorimeter. In the presence of glucose a characteristic response from desert soils was found within 24 hours.

On micro-organisms of the stratosphere.

The lower parts of the biosphere are well studied since various live beings are found in oceans and at the bottom of large hollows. Contrary to this, we have no data about the upper boundaries of the biosphere. Samples were obtained with the help of specially constructed analysers which were installed in meteorological rockets and reached an altitude of 100 km. With the help of methods completely excluding the possibility of contamination of analysers with outside microflora it became possible to prove that earth microbes carried by air currents are present in the stratosphere. At an altitude of 48-77 km Circinella muscae, Asp. niger, Penicillium notatum were found as well as mycobacterium and micrococcus. The correlation of these cultures with external factors is studied and the weight of one conidium or one cell in isolated micro-organisms is estimated. These investigations will continue.

On the multiplication of xerophilic micro-organisms under simulated Martian conditions.

The environmental conditions prevailing on Mars would supposedly favour the existence there of micro-organisms belonging to xerophiles, anaerobes, or micro-aerophiles, oligonitrophiles, which are able to grow in wide temperature intervals. From soil samples taken in deserts and tundra, antarctic halophilic bacteria, able to grow in liquid media containing 20-25% of sodium chloride, were isolated. Some of these cultures appeared to be also osmophilic (growth on media with 50% glucose); they grew at temperatures from 5 degrees C to 50 degrees C, and developed on media without a nitrogen source (oligonitrophiles). Of special interest was the halophilic and osmophilic form of Bacillus megaterium isolated from the Nubian desert. In experiments with this bacterium the following technique was used. A thin film of potato extract agar was prepared on a glass slide and dried over a saturated K2SO4 solution in a closed container up to the level of maximal hygroscopic moisture. The cell suspension was then sprayed on to the agar surface, the film dried again at 45 degrees C, and the glass slide put in the test tube over the saturated solution of K2SO4. The test tube was evacuated, flushed three times with a gas mixture containing 80% CO2 plus 20% Ar, and sealed. Under these conditions the water content of the agar film was equal to the maximal hygroscopic moisture; only the xerophilic form of bacteria are able to develop at this moisture level. This halophilic strain of Bac. megaterium grew satisfactorily under these conditions, as did a halophilic and osmophilic strain of Mycococcus ruber isolated in Antarctica. Both the halophilic strain of Bac. megaterium and that of M. ruber were able to grow under simulated Martian conditions. Xerophily and halophily may be linked. This assumption was supported by relatively high incidence of xerophilic forms among halophilic bacteria isolated from different soils of both high and low salt content as well as from salty muds.

Multiplication of certain soil micro-organisms under simulated Martian conditions.

According to earlier observations, severe UV irradiation kills all micro-organisms in a chamber with simulated Martian conditions. However, even a thin soil layer protects buried micro-organisms from UV irradiation. The chief limiting factor for microbial multiplication under simulated Martian conditions seems to be soil humidity. Several micro-organisms were isolated from harsh environments (e.g., from Arctic, Antarctic desert and high-mountain soil samples). A strain of an oligonitrophilic mycococcus, isolated from Dixon Island, proved to be most resistant to low humidity. It multiplied in a mixture of limonite (maximal hygroscopical humidity 3.8%) + 2% (w/w) garden soil kept in a chamber simulating Martian conditions. Total cell count increased 7.6-fold and, in some experiments, 26-fold in 14 days. The oligonitrophilic mycococcus was able to grow even at a humidity level of 2.5%, that is less than maximal hygroscopical (3.8%). Under these conditions cell count increased 10-fold in 36 days. Thus, it was shown that even in Earth soils there are xerophytic micro-organisms which are able to multiply in limonite of low humidity. These data might correct our current concepts concerning microbial water requirements. One might speculate that Martian micro-organisms belong to xerophytic species.

The effect of high vacuum on oxidative reactions in bacteria and the activity of certain enzymes.

Among the physical factors which might influence micro-organisms one of the most potentially interesting is high vacuum. The effect of high vacuum is less studied as compared with other physical factors. It is impossible to achieve, under laboratory conditions, a vacuum of the order 10(-16) mm Hg which is probably characteristic of space. Earlier, the effect of high vacuum was studied on different bacteria, yeasts, molds and algae. It appeared that spores and fungal conidia were not killed by high vacuum. Later, the effect of high vacuum on physiological processes in micro-organisms was studied. The ability to oxidize glucose or ethanol was studied with Sarcina flava and Bacillus simplex cells after they were subjected for 72 hr to vacuum (10(-8) to 10(-9) mm Hg). The oxidation rate was followed polarographically. The oxidative ability of S. flava cells diminished [correction of dimished] after their subjection to vacuum, while B. simplex spores were unchanged in that respect. The following crystalline enzymes were subjected for 72 hr to the same vacuum: alpha-amylase, catalase, ribonuclease, trypsine and urease. Then the activity of the above enzymes was tested on corresponding substrates. Not a single enzyme was totally inactivated. About 50% of activity was lost with alpha-amylase; 25--35% of activity with catalase, ribonuclease and urease. Trypsine retained its total activity. Thus, high vacuum cannot be listed among factors rapidly inactivating enzymes of micro-organisms.

Exobiology and the effect of physical factors on micro-organisms.

A study of the action of different physical factors on micro-organisms is necessary for a further development of exobiology. The action of temperature on crystalline preparations of catalase and peroxidase was studied by means of oscillographic polarography. A determination of the height of polarographic waves at the decrease of temperature from 20 degrees C to 0 degrees C has shown that structural elements of the peroxidase molecule connected with the enzymatic activity are more stable with the decrease of temperature cf. catalase. A relative resistance of the dehydrogenase activity in Az. vinelandii cells to high vacuum was found. Incubation of azotobacter cells under vacuum of 10(-9) mm Hg during 72 hr did not decrease the activity of alcohol and succinic dehydrogenase. Bac. cereus spores can be preserved from bactericidal UV action by thin films of chrome. The thickness of chrome film being 200-670 angstroms, spores are killed by a dose of 7.8 x 10(7) erg/cm2 at 253.7 microns wave length. Spores covered by chrome film thicker than 800 angstroms remain alive after this treatment. Investigations carried out with an 'Artificial Mars' camera led to the following results. The growth of Bac. megaterium on liquid growth media in this camera ceases as a result of UV rays killing all cells after 3 weeks. Untreated bacteria grow in the camera for a long time. Spore-forming bacteria isolated from the sand of the Kara-Kum Desert grow in ground limonite (with the addition of 2% garden soil) having maximum hygroscopic humidity (3.8%). Freezing and thawing (from -60 degrees C to +25 degrees C) corresponding to day temperature deviations on Mars, low pressure (P=10 mm Hg) and the composition of the atmosphere (CO2-50%, N2-40%, Ar-10%) do not influence the growth of xerophylic bacteria under study. Humidity is the main factor limiting the growth of micro-organisms under 'Artificial Mars' conditions. According to the further development of the microbiological meteorite analysis methods, samples of rocks and stone meteorites were sterilized, incubated in the desert or on a snow surface in the Arctic and after different times (from 100 days to 7 months), investigated. In all cases, microbes were found only on the sample surfaces, whereas 1 cm from the surface and in the central parts micro-organism were completely absent. Hence, microbiological analysis of central parts of meteorites fallen in the Arctic or during dry periods of the year in the desert can give reliable results.

The possibility of life in outer space.

Experiments were carried out dealing with the effect of extreme factors on microorganisms. Methods for microbiological analysis of meteorites were developed. The effect of temperature, from -20 degrees C to +15 degrees C, on trypsine activity was studied. It was shown that at about -3 degrees C a definite intramolecular reorganization occurs in the enzyme, leading to decreased activation energy. Previously reported resistance of microorganisms to high vacuum was confirmed. New species were tested at l0(-8)-10(-9) mm Hg. It was shown that very thin metal and metal oxide films, as well as films from other substances, may fully protect microorganisms from the hazardous effect of UV rays. By using an artificial climate, camera forms of microorganisms were selected which have the highest resistance to the whole complex of Martian climatic factors. Pigmented and spore-forming microorganisms resist UV up to a dose of 4.04 x 10(8) erg/cm2. There are microorganisms on Earth which show some activity in soil at a relative humidity of 0.096-0.196%. Preliminary data permit one to assume the possibility of existence of Earth-like forms on Mars. The majority of meteorites are accessible to Earth microorganisms. While choosing a meteorite for microbiological analysis it is necessary to consider the soil and climatic factors of the country where the latter has been found.