Introduction

1. Results of studies on long term exposition of dormant forms of various organisms in outer space environmentN.D. Novikova, N.A. Polikarpov, E.A. Deshevaya, V. N. Sychev,M.A. Levinskikh, A.I. GrigorievEstablishment of the Russian Academy of Sciences RF SRC – Institute of Biomedical Problems, RAS, Moscow, Russia

2. Introduction Risk of contamination of Mars and other planets with terrestrial microorganisms delivered by spacecrafts is the one of the most discussed problems of exobiology and planetary protection. As a result of these discussions it has become apparent, that developing of joint position on the given issue is impossible without conducting of detailed research focused on survival rate of microorganisms from Earth on materials, used in space techniques in space environment. One can assume, that on the external side of both unmanned and manned space crafts there can be millions of microbial cells, many of which form spores and have an extremely high stability towards space flight factors.

3. Experiment«Biorisk» Hardware and a research program have been developed at the Institute for Biomedical Problems with the goal of carrying out a space experiment “Biorisk”. The first stage of this experiment provided a unique opportunity to study the most interesting species of bacteria and fungi, exposed to the open space for a long period of time and to evaluate adaptation and variability limits of these organisms. The purpose of the present "Biorisk" experiment is to obtain new data on possible limits of phenotypic adaptation and genotypic changes in bacterial and fungal associations, shaping a standard microbiota of construction materials, used in space techniques. Task of the experiment – is definition of basic capability of preservation of life capacity of test-cultures of microorganisms during the long (comparable with time of flight from Earth to Mars) modelling in space the "microorganisms - construction materials" systems.

4. Space experiment «Biorisk» Petri dish with sistem «microorganisms - structural materials» Equipment for the “Biorisk” experiment

5. Microorganisms used in the first stage of “Biorisk” experiment BACTERIA Bacillus pumilus Bacillus licheniformis (2 strains) Bacillus subtilis FUNGI Aspergillus versicolor Penicillium expansum Cladosporium cladosporioides

6. Materials used in the “Biorisk” experiment Materialsusedintheconstructionofexternalspacecraftsurfacessuchas: • steel, • aluminiumalloy, • heat-insulatingcoating werechosenastestsamplesfortheexperiment. The surface of each material sample was 16 cm 2.

7. Biorisk Disposition of ’’Biorisk” containersIn January 2005 three containers with materials and test microorganismswere placed on the external side of the Russiansegmentof the ISS

8. “Biorisk” containers Holder caser Russian segment Dismantling of ”Biorisk” containers on Russian segment of ISS

9. Results Unique data have been accumulated after a 7, 12 and 18 months exposure on the external side of the ISS, which have proved that representatives of bacteria and fungi possess a potential for long-term survival under the conditions of outer space. Extreme environment of outer space was found to have less influence on the survivability of bacterial spores comparing to fungal spores.

10. The contents of microorganisms on the materials(CFU in % to background) fungi bacteria

11. Cells of Bacillus subtilis After flight Before flight

12. Hyphae of Aspergillus versicolorThe cell wall of hyphae changed After flight Before flight

13. Resistance to antibiotics After exposingin to outer space test microorganisms have preserved high bioactivity. Also after the flight increase of the bacteria resistance to antimicrobial medications, in particular to antibiotics was observed.

14. Intensification of fungi's acid formation The comparative analysis of fungi samples acid formation before and after exposure on the external covering of ISS shows that the extreme conditions of the outer space can cause intensification of fungi's acid formation. Dynamics of P.expansum acid formation

15. Main result of theexperiment«Biorisk» Thus, the fact of that long-term survival of terrestrial microorganisms in the outer space environment have been established for the first time, which suggests the existing possibility of interplanetary transfer of microorganisms on external spacecraft surfaces.

16. The second stage of the experiment “Biorisk” On the 6-th of June 2007 three containers was placed on the external side of the ISS. You can see them on the following photo, which was taken by Russian cosmonaut during extravehicular activity after containers installation on outer surface of Russian segment ISS.

17. The second stage of the experiment “Biorisk” ”Biorisk” instruments photo 06.06.2007

18. Biological objectsusedin the 2-nd stage of“Biorisk” experiment

19. Package with biological samples in "Biorisk" experiment(Packages made of polymer film)

20. Package with biological samples in "Biorisk" experiment (Bags made of cotton)

21. The results of the second stage of the experiment “Biorisk” • Three containers were obtained and analyzed after 13, 18 and 31 months of exposure to outer space environment. • Most of the biological objects utilized in the second stage of the experiment have survived. • There were only a few of died biological objects: majority of the fungi, tomato seeds and killifish eggs. • Considerable part of survived biological objects have maintained reproduction ability.

22. The results of the second stage of the experiment “Biorisk” Germinated «space» seeds of barley Hordeum vulgare cv.Haruna Nijo and plants of radish Raphanus sativus strain «Cherry Bomb», planted from “space” seeds Biorisk-MSN canisters and their location on the PIRS Docking compartment Growth of Bacillus subtilis # 24after 31 months of exposure to outer space on the outer side of ISS Sleeping chironomid larvae “Nemuri-usurika” rehydrated after 31 months in outer space Reactivation of “space” and control Artemia

23. Experiment «EXPOSE-R»(10.03.2009 – 21.01.2011) The main factor, which can influences on survival of microorganisms and other biologics objects in outer space is ultraviolet. Equipment of “Biorisks’” experiment doesn’t allow to evaluate this factor. The experiment “Expose-R” proposed by European Space Agency and we tooke part in it. Originality of this experiment consists in about that part of samples was discover for ultraviolet emanation.

24. Experiment EXPOSE-R To expose dormant forms of various organisms on the external surface of the ISS, which include not only bacterial and fungal spores but also seeds and dormant embryos of lower crustaceans, as well as multi-cellular organism in the cryptobiotic state.

25. Location of biological samples in the trays of EXPOSE-R Pressurized teflon packages 77 mm and 1010 mmwith biological samples (spores of fungi, dried embryos of lower crustaceans, dried chironomid larvae, seeds of plant) Trays with fixed teflon packages containing biological samples

26. Experiment “Expose-R” ((10.03.2009 – 21.01.2011) BACTERIA Group 18, subgroup Bacillaceae, genera Bacillus, species: Bacillus subtilis-2335/105 ; Bacillus subtilis-20 ; Bacillus pumilus-25 ; Bacillus licheniformis-24. Gram-positive rods, 0,5-2,5 x 1,2-10 mkm, spore - 6 mkm MOLDS Taxon - Mitosporic fungi, genera Aspergillus, Penicillium,Geomyces species: Aspergillussydowi- 9-6; Geomycespannorum– 2241; Aspergillusversicolor-4-3-4; Penicilliumaurantiogresium –9-9; Penicilliumexpansum– 4-3-3. Spore 3-10 mkm ANIMALS Dried encysted embryos (diameter of a single egg is 200 micron) Daphnia magna belongs to family Daphniidae, order Daphniiformes, class Crustacea. Dried encysted embryos (diameter of a single egg is 160 micron)Streptocephalustorvicornis belongs to family Streptocephalidae, order Anostraca, class Crustacea. Dried encysted embryos(diameter of a single egg is 180-200 micron)Artemiasalina belongs to family Artemiidae, order Anostraca, class Branchiopoda, subphylum: Crustacea. Dried encysted embryos(diameter of a single egg is 180-200 micron)Eucypris species belongs to family Cyprididae, order Ostracoda, class Crustacea. Cryptobiotic dried larvae (length 5 mm  0,8 mm) of Polypedilumvanderplankibelongs to family Chironomidae, order Diptera, classInsecta. PLANTS Seeds ofLycopersicumesculentum(Tomato Micro-Tom) Seeds ofArabidopsis thalianaline WT–2.

27. Main result For the first time, selective effect of space ultraviolet (>200nm) on resting stages of different biological objects was analyzed in "Expose-R" experiment, conducted on the outer side of Russian Segment of ISS. In contrast to UV-shielded conditions where most of the species survived, spore of microorganisms, seeds of higher plants, dry embryos of crustaceans died under effect of the ultraviolet. At the same time, spores of microscopic fungi (Aspergillus sydowii, Aspergillus versicolor) and anhydrobiotic larvae of the sleeping chironomid (Polypedilum vanderplankii, Diptera) survived even under such harsh conditions. These data are very important both for fundamental space life science and for applications targeted on developing of planetary safety protocols for future interplanetary spaceflights.

28. The third stage of the experiment “Biorisk” On the 3-th of August 2011 three containers, including 59 biological objects was placed on the external side of the ISS.

29. Thank you for your attention!