Selection of the microorganism composition to exposure in outer space. At the stage of the ground preparatory phase, studies were conducted to select the composition of microorganisms that are most resistant to high doses of UV radiation. Low-pressure gas-discharge amalgam lamps with a UV radiation power of 256 ± 28 W at a wavelength of 253.7 nm were used to irradiate microbial cultures. The UV irradiation lamp was placed in a Class 5 microbiological safety laminar box. Test samples were placed in Petri dishes. The distance from the lamp to the surface of the samples in Petri dishes was 50 cm.
To obtain bacterial spores of the species Bacillus pumilus and Bacillus subtilis, the technique described by Newcombe D.A. et al. was used. in 2005 with modifications [38]. Bacterial cells were cultured on a nutrient medium - potato agar without glucose. The crops were incubated for 24 hours at 37 0C, and then 4–5 days at 20–22 0C in natural light. Subsequently, a control microscopic examination of test cultures was carried out (the number of spore forms was at least 99%), then a suspension of spores of each test culture of bacteria was prepared in a phosphate buffer using the McFarland 2 turbidity reference standard (BioMerieux, France), which corresponds to the number of microbial cells 6x108/ml, and heated for 15 minutes at 80 oS for inactivation of vegetative forms. Then, by a series of successive dilutions in a sterile phosphate buffer, the concentration of the suspension of each test microorganism was achieved equal to 105–106 cells in 1 ml.
The spores of the fungus A. pullulans were obtained for 7 days during surface cultivation on a nutrient medium - potato dextrose agar (KDA) (HiMedia).
To study the effect of UV radiation on test cultures, suspensions of bacterial and fungal spores were prepared at a concentration of at least 1.0 x 106 CFU/ml. Then 0.1 ml of suspension of each culture was applied to the surface of a sterile Petri dish with a triple repetition. After exposure to UV radiation, 25 ml of cooled (up to 40 0C) medium R2A (HiMedia) was poured into cups with cultures, and then cups with bacterial and fungal crops were incubated at 30 0C and 28 0C, respectively.
Preparation to exposure in a vacuum near the ISS. To determine the duration of the survival of microorganisms in the conditions of open space, the device "Test" was developed (Fig. 1, Fig. 4), intended for the transportation of selected test cultures of microorganisms from the earth and their exposure. The device consists of a transport unit 1 (Fig. 4), an exposure module 2 (Fig. 4) in which rods 3 (Fig. 4) are fixed with cotton wool contaminated with microorganisms for conducting experiments. There are threaded holes in the housing of the transport unit 1, into which rods with cotton wool with applied test cultures are screwed. The design of this part of the device made it possible to maintain the tightness of the delivered microorganisms during transportation. During the spacewalk, the astronaut unscrewed the rod 3 from the transport unit and fixed it in a special free hole of the exposure module, as shown in Fig. 4.
Preparation and carrying out of the exposure on the outside near the ISS. After selecting the cultures for the experiment on exposure on the outside of the ISS, the following works were carried out:
- cultures were grown on the surface of appropriate nutrient media to obtain isolated colonies;
- spores were selected from one colony of a spore-forming bacterium and a fungus to increase biomass by surface cultivation of a continuous lawn;
- a suspension of test culture spores was prepared in the highest possible concentration of at least 1x10 9–10 CFU/ml of bacteria and 1x107-8 CFU/ml of fungus;
- preparation of the methane-forming archaea was carried out according to a specific technique [16];
- under aseptic conditions in an ISO Class 5 laminar box, using sterile disposable syringes, the suspension of each culture was applied to the surface of a separate rod (each culture had its own rod with cotton wool) and dried in the box at room temperature for the time necessary for the cotton wool to dry. As a control, test samples were used, which were stored in the refrigerator in frozen form (at a temperature of -80 0C) during the entire exposure time.
Suspensions of cells of each of the selected test cultures were applied one by one to the surface of cotton wool of the individual rod of the "Test" device, for exposure for 12 and 24 months, to ensure a two-fold repetition. During the spacewalk, the astronaut placed devices with bio-objects on the outside of the Russian ISS module (Fig. 5). The installation locations of the "Test" devices are indicated by red arrows.
The process of installing the "Test" device by an astronaut is shown in Fig. 6.
Conducting ground-based research after exposure. After the end of the exposure period, the astronauts dismantled the Test devices, and then they were taken to the laboratory, where the number of surviving test cultures was determined by transferring cotton wool infected with strains from the rod into vials with saline solution for spore bacteria and fungus. Next, bacteria were seeded on the surface of the nutrient medium of tripkase-soy agar (TSA), and the fungus was seeded on the surface of the CD. The bacterial crops were incubated at 30 0C for 3 days, and the fungus was incubated for 7 days at 28 0C. The number of surviving crops was determined by the method of marginal dilutions. Then subcultures were isolated from individual colonies of cultures to create a collection and conduct subsequent experiments. The number of surviving archaea was determined by a specific technique consisting of dividing the cotton wool material into parts: one was flushed into an anaerobic medium for microscopy of cells, and the rest was seeded into nutrient media [16].
Determination of the radiation dose received by the samples during exposure. The absorbed radiation dose was calculated according to the method described in [39]. The calculation took into account the total impact:
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- electrons and protons of the Earth's radiation belts;
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- protons, neutrons and heavy charged particles (from alpha particles to nickel ions) of solar cosmic rays that occur during solar flares and are partially cut off by the Earth's magnetosphere in the ISS orbit;
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- protons and nuclei (from alpha particles to uranium nuclei) of galactic cosmic rays (GCL). GCLs penetrate the ISS orbit almost unhindered and constantly affect it.
Determination of radiation sensitivity of fungal strains and subcultures. When determining the radiation sensitivity of fungi that survived exposure for two years on the outside of the ISS, the method of survival of microorganisms under the influence of different doses of gamma radiation was used. For this purpose, 30 ml of suspension of each mushroom was prepared at a concentration of 106 spores / ml. Then the suspension of each strain of the fungus was poured into 6 sterile tubes of 5 ml, of which one tube was left in the laboratory to determine the initial concentration of spores. One tube of each strain (cultures of VKM F-1116 and the initial strain) and each subculture were exposed to radiation exposure of gamma radiation of the isotope CO60 at doses of 1 kGr, 2.5 kGr, 5 kGr, 10 kGr and 15 kGr at the GU-200M installation (with an average absorbed dose rate of 2.5 kGr/h). To control the absorbed dose, dosimeters were placed near the irradiation objects. After radiation exposure, the number of surviving viable units of each strain and subculture of the fungus was determined by sowing marginal dilutions of their suspension on the surface of the nutrient medium KDA. The level of the maximum suppressive dose of ionizing gamma radiation was judged by the growth of cultures or its absence. These studies were carried out in sevenfold repetition.
Determination of the sensitivity of strains and subcultures of the fungus Aureobasidium pullulans to the effects of extreme factors. After receiving data on the resistance of strains and subcultures of fungi to gamma radiation, it was decided to study their reaction to the action of other stress factors, such as increased temperature for fungi, exposure to different concentrations of hydrogen peroxide and a solution of 1% chlorine (the calculation of the concentration of chlorine in the solution was carried out according to the manufacturer's instructions) of the disinfectant Chlorapine (CJSC "Petrospirt" St. Petersburg).
These studies were carried out using 4 strains of the Aureobasidium pullulans fungus: VKM F-1116, the initial strain and 2 subcultures isolated from sowing after 2 years of exposure on the outside of the ISS: subculture No. 2.1 and subculture No. 2.6. For these studies, suspensions of spores of strains and subcultures of fungi were prepared in approximately equal concentrations are about 106 CFU/ml. The reaction of A. pullulans strains and subcultures to the effects of different temperatures was determined by heating each suspension for 15 minutes at temperatures of 37 0C, 45 0C, 65 0C and 85 0C. The survival of fungi after this procedure was assessed by the presence of growth of 1 ml of suspension of each strain and subculture deposited in the wells of cups with KDA nutrient medium. The qualitative reaction of action on strains and subcultures of different concentrations of hydrogen peroxide and 1% chlorine was determined by the zone of fungal growth retardation when applying discs impregnated with solutions of 1% and 3% H2O2 and 1% Sl solution to the lawn surface of each strain or subculture. Then the cups with the crops were incubated at 28 0C for five days. All studies were carried out in three-fold repetition. The reliability was determined using single-factor analysis of variance [40].