Biological Sciences in Space, Vol.25 No.2-4, 83-92,Takahashi, 2011 Y. et al. Original Paper Survivability of and Fungal irradiated for 30 min. On the other hand, a colony did not develop if the spores were Spores in Tests Simulating taken off the beads and irradiated for 10 min. Conditions of the ISS Outer Wall This indicates that UV does not penetrate to the other side of the beads, and so the spores on Yuichi Takahashi1*, Hirofumi Hashimoto2, that side can be protected from UV radiation. 3 1 ©2011 Jpn. Soc. Biol. Sci. Space; Article ID: Takuo Nakagawa and Shinpei Shibata 112502011 1Department of Astrophysics, Interactive Symbio- sphere Science, Yamagata University Graduate Key words; fungi, moss, , survivability, thermal cycle treatment, UV irradiation School of Science and Engineering, Kojirakawa- machi 1-4-12, Yamagata-City, Yamagata 990-8560, Introduction Japan 2 Institute of Space and Astronautical Science, There are many factors that can influence the survivability JAXA, Yoshinodai 3-1-1 Chuo-ku, Sagamihara- of terrestrial (TL) in interstellar and interplanetary space, City, Kanagawa 252-5210, Japan for example full-spectrum electromagnetic radiation from 3Inspection room, Kojirakawa-Shiseidoh Hospital, the sun, temperature fluctuations, cosmic particle radiation, Higashihara-machi 1-12-26, Yamagata-City, Ya- vacuum, and microgravity. However, space conditions can magata 990-0034, Japan only be partially simulated in ground experiments. Recently, several experiments have investigated the possibility of TL Abstract survival in space, for example EXPOSE-E (ESA Human Space Flight (2010) Expose, http:// smsc.cnes.fr/EXPOSE/), BIORISK (Baranow et al., 2009; Novikova et al., 2010) and To investigate whether terrestrial life (TL) can BIOPAN (De la Torre Noetzel et al., 2007; Devara et al., survive in interstellar and interplanetary space, 2010), while another one, TANPOPO, will take place in the an experiment was performed to simulate near future. In the BIORISK experiment, 31 kinds of TL were conditions in the Earth’s orbit. There are many examined such as higher plants, insects, crustaceans, fungi factors which influence survivability. Among and . A high survival rate was observed in some them, the effects of temperature fluctuations samples of plant seeds (barley, rice) and chironomid larvae, and UV irradiations were addressed in this whereas a low survival rate was observed in samples of paper. Six of moss spores and 3 bacteria and fungi. The plastic dishes of bacteria and fungi species of fungal spores were selected as melted during the 2nd experiment (Baranow et al., 2009; target TL. Temperature was fluctuated between Novikova et al., 2010), and after the experiment, the melting 80˚C and -80˚C once every 90 min, whereas UV 2 temperature of the plastic dishes was determined to be at 254nm was irradiated for 10 min (471mJ/cm ) 2 95˚C. So they were unable to reach a reliable conclusion and 30 min (1,413mJ/cm ). The moss spores of about the obtained results. Funaria hygrometrica (exp 1) and Pogonatum In this paper, a thermal cycle experiment and an inflexum (exp 2) were the most tolerable species ultraviolet ray (UV) irradiation experiment were carried to the thermal cycle treatment, with germination out as a ground experiment under defined conditions rates of 3.3±1.2% (n=5) and 7.9±3.2% (n=6), simulating the Earth’s orbit. Our purpose is to seek a respectively, after 3 weeks of treatment. candidate species for the TANPOPO and other space Germination occurred even after the spores had experiment such as BIORISK, and hopefully to participate been UV irradiated for 30 min (0.7% to 23.5%). in the space experiment itself. Furthermore, we aim Only a slight difference in the germination rate to test whether cellular tissues of living organisms are was observed using different culture media. damaged physically and their functions stopped due The treated spores were transferred to to the repetition of short-time temperature fluctuations where they grew into , then through thermal cycle treatment and UV irradiation. , and finally formed new capsules This paper describes a pilot study using moss and after 7-8 months. Two species of fungal spores fungal spores as potential candidates for the TANPOPO were allowed to adsorb onto beads before the project (Yamagishi et al., 2008), which proposes to beads were directly irradiated for 10 min and analyse the interplanetary migration of microbes and 30 min, respectively. Colonies developed the organic compounds and survivability of microbes at the International Space Station (ISS), located ca. 400 km Received: May 2, 2011; Accepted: November 8, 2011 above the Earth’s surface. As a part of this project, they *To whom correspondence should be addressed: plan to test the survivability of the microbes in space using Tel: +81-(0)23-628-4552; Fax: +81-(0)23-628-4567 ; a direct exposure experiment, which will take place in the E-mail: [email protected] Japanese exposure unit “Kibo” on board the ISS. «Can life migrate between planets?» is a question that many Abbreviations TL: terrestrial life, exp 1: experiment 1, exp 2: experiment 2 people have an interest in. Nicholson (2006) reviewed the potential interplanetary transport of microbes and

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classified the following stages; surviving the launch from well as one kind used in exp 1 were exposed for 21 days. a donor planet, surviving the transit through space, and As for fungi, spores were adsorbed onto the surface surviving atmospheric entry and landing onto a recipient of MicrobankTM beads (Pro-Lab Diagnostics, 20 Mural planet. Thus, TANPOPO’s proposed experiment will test Street, Unit #4, Richmond Hill, ON and L4B1K3 Canada). Nicholson’s second stage (i.e., surviving space transit). The MicrobankTM system consists of sterile vials containing The exposure experiment of microbes in space has beads which act as carriers to support the fungi for a long been performed mainly using the spore-forming bacterium time. Each bead is 3.5 mm in outer diameter, 1.0 mm in Bacillus sp. (Nicholson et al., 2000). In TANPOPO, inner diameter and 2.5 mm in thickness. Each bead can various microbial cells including Deinococcus radiodurans support 103 to 104 microbes (Chandler, 1994). Aspergillus and the newly isolated Deinococcus sp. from high- oryzae spores were exposed for 26 days, Cladosporium sp. altitude (ca. 10 km) will be used for exposure experiments spores for 14 days and Aspergillus niger spores for 21 days. (Yamagishi et al., 2008). Deinococcus radiodurans is UV irradiation known to show extreme tolerance to UV-light and gamma A UVS-14 EL series UV lamp with an Hg-law lamp radiation. Some isolates of Deinococcus sp. showed was used for the UV irradiation treatment. The amount of higher tolerance to UV-light than D. radiodurans (Yang et UV at 254nm irradiated from 7.6cm distance is 0.785mW/ al., 2008; Yang et al., 2009). In addition to the microbes, 2 cm . The expected amount of UV was calculated to be we are examining candidate species of spores belonging 2 2 471mJ/cm for 10 min and 1,413 mJ/cm for 30 min. to , which are expected to be tolerant to the UV treatment of moss spores was performed after space environment. Our candidate moss spores can they were taken out from the capsules and placed into the tolerate being in a vacuum (Saegusa et al., 2008). Since culture medium. The spore concentration was adjusted they are autotrophic, if they can be brought to Mars, 6 to 1×10 spores/ml using a haemocytometer. Twenty μl they would be expected to contribute to future space of spores were put into each well of a 96-well microplate agriculture (http://surc.isas.ac.jp/space_agriculture/ (Nunc company, Roskilde, Denmark, non-treated; flat; Archive/PDF/Space_Agri_ 2008-a.pdf). Considering the well volume 0.4ml) and irradiated for 10 or 30 min. A flat Earth’s history, were one of the first plants to and small size well (7mm in diameter) was used in order inhabit terrestrial (Kenrick, 2003; Wellman et to irradiate the UV light uniformly. UV treated moss spores al., 2003), and have been playing an important role in preparing the soil for vascular plant growth. Fungal spores are found in spaceships such as ISS (Novikova, 2010; Vesper, 2008), where they can cause trouble because they are difficult to get rid of once the fungi propagates and some of them cause allergies. The survivability of spores was obtained from germination rates in the case of moss spores and from colony formation in the case of fungal spores. Detection of fungal spores in the air is especially suitable for us, because we have developed an immunochemical technique for detecting fungi such as Cladosporium sp. in the air (Suzuki et al., 2008). It is the right target species for the examination of the change in allergenicity when it is carried out in interstellar and interplanetary space.

Materials and Method Thermal cycle treatment The thermal cycle test device of the Institute of Space and Astronautical Science, Japan Aerospace Exploration Agency (JAXA) was used for the treatment (Fig.1). This Fig. 1. Thermal cycle test device, (a) sample containers and (b) the plate which controls the temperature of the container. device is designed to reproduce a repeating change in The sample containers (a) were accommodated on the plate (b) temperature at short-time intervals. We use temperature shown in this figure. fluctuations between 80˚C and -80˚C once every 90 min, Capsules were put into a hole in the sample containers (the and the vacuum level of the device is less than 1Pa. The areas surrounded by black squares). actual atmospheric pressure at the altitude of the ISS Five to ten capsules* were put into a hole in the sample -4 -6 container depending on the capsule size and were subjected to (400 km) is 10 to 10 Pa, though. A typical result of the the thermal cycle treatment for a defined period. temperature fluctuation obtained from the device is shown *The capsule size is greatly different. C. purpureus and B. in Fig.2. argenteum have small capsules and P. inflexum has a large one. Capsules were used for the moss experiment, which F. hygrometricas has a medium size one. The beads onto which the fungal spores were absorbed (c) were was performed twice. In experiment 1 (exp 1), capsules put on the plate directly. of four kinds of moss were exposed for 7 days, 14 days Temperature was monitored with a sensor attached to the inner or 24 days. In experiment 2 (exp 2), another two kinds as side of the plate.

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100.0 and P. inflexum were collected from the roadside of a mountain path. All four species have a characteristic

) cooling capsule and can be easily discriminated from each 50.0 other and other species of moss. They were selected partly from a large cluster where contamination by other 0.0 species was not recognized. Capsules of each moss heating were collected and used in the experiment. The capsules

mperature (˚C were dried on a glass plate and then transferred to a 1.5ml

Te -50.0 Eppendorf tube and kept at 4˚C until needed. Cultivation of moss spores -100.0 The thermal cycle treated capsules were rehydrated 0102030405060708090 100 in a culture medium and the spores were taken out of Time (min) the medium. Murashige and Skooge Modified Basal Medium (MS culture medium: Phyto Technology Lab. Fig. 2. Temperature fluctuation of the plate of the thermal cycle Shawnee mission, USA) was used in exp 1. To ascertain test device. A complete cycle consists of 20 min cooling, 25 min machine whether the difference in culture media may influence stop, 20 min heating and 25 min machine stop. the germination rate, two kinds of culture medium were examined in exp 2 under the same experimental conditions. Hyponica culture medium (HP culture were adjusted to 1×105 spores/ml with culture medium medium: Kyowa Co. Ltd. Takatsuki, Japan) was used as and cultivated for 2 weeks at 25˚C. The germination rates the other culture medium. The spore concentration was 5 were examined after the cultivation. adjusted to 1×10 spores/ml in each medium, transferred Fungal spores were irradiated using two methods; to a flat-bottom cell culture plate with 6 holes, and then one by irradiating the Microbank TM beads directly with cultivated for 14 days at a 14 h light / 10 h dark cycle at UV, the other by irradiating the solution of spores (taken 25˚C. Illumination for plant culture was achieved using a off the Microbank TM beads in advance) with UV. plant raising light with red and blue LED (LED MY PLANT, Wako Electric lab, Yao City, Japan). The moss spores Sampling and cultivation of moss spores, and were illuminated from a distance of 20cm (1,000 lux, ca. 2 calculation of the germination rate 1.5w/m at 555nm). Sampling the moss spores Calculation of the germination rate and subsequent Artrichum undulatum and Bryum argenteum were re-cultivation After cultivation, the spore solutions were adjusted to purchased from Moss Farm (Oomiya, Shizuoka City, 5 Japan). Funaria hygrometrica, Ceratodon purpureus, a suitable concentration (ca. 2.5×10 spores/ml ) in the Brachythecium populeum and Pogonatum inflexum were culture medium (i.e., either concentrated or diluted) and collected from the natural environment. F. hygrometrica were mixed thoroughly until the solution became uniform. was collected from a place where an expressway will be After mixing a part of the sample was immediately constructed in the near future and other plants had not deposited onto a slide glass. The spores were observed grown there in abundance. Ceratodon purpureus was with a light microscope at 100× magnification. Spores collected from a thatched roof. Brachythecium populeum with buds that had obviously come out were counted

AB C D 100 ) 10

1

0.1 Germination rate (%

0.01 0714 24 0714 24 0714 24 0714 24 Exposure period (days)

Fig. 3. Effects of temperature fluctuation under low pressure on moss germination (exp1). The horizontal axis represents the time taken and the vertical axis the germination rate (percentage) of each moss spore on a logarithmic scale. (A) A. undulatum (B) F. hygrometrica (C) B. argenteum (D) C. purpureus

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Murashige-Skooge Hyponica EFGEFG 100 100 ) 10 10

1 1

0.1 0.1 Germination rate (%

0.01 0.01 0 21 0 21 0 21 0 21 0 21 0 21 Exposure period (days)

Fig. 4. Effects of temperature fluctuation under low pressure and the examination of the influence of culture media on moss germination (exp 2). The horizontal axis represents the time taken and the vertical axis the germination rate (percentage) of each moss spore on a logarithmic scale. (E) F. hygrometrica (F) B. populeum (G) P. inflexum

A as germinated spores. About a hundred to two hundred spores were counted in each sample and 4-6 samples were counted for each condition. The spore germination rate in each well was calculated from the number of germinated spores to the total number of spores. Further cultivation in soil was performed using Jiffy seven after 2 weeks of cultivation in liquid medium. The soil was purchased from Jiffy-Garden Series, Sakata-no-Tane Co., Ltd., Yokohama, Japan.

Source, cultivation and preservation of fungal spores, and calculation of the colony formation rate Thermal cycle treated beads non treated control beads Source, cultivation and preservation (incubated for 7 days) (incubated for 7 days) Aspergillus oryzae was separated from malt bought at the market, whereas spores of B Cladosporium sp. and A. niger were isolated from the air. Colonies were extracted, placed into isolation culture and cultivated on Sabouraud dextrose agar plates. The fungal spores were adsorbed onto Microbank TM beads and kept at -80˚C until needed. At the time of the treatment they were thawed and a sufficient number of beads were taken out for thermal cycle treatment or UV treatment. Calculation of the colony formation rate The treated beads were put on Sabouraud dextrose agar plates and rolled all over the plate. The plates were then incubated at 25˚C for 2 days. Cultivation was continued if colonies were not Thermal cycle treated beads non treated control beads observed. The colony formation rate was calculated (incubated for 7 days) (incubated for 2 days) from the number of colonies on the treated plate to Fig. 5. Colony formation of (A) A. niger after a 21-day thermal cycle that on the non-treated control plate. treatment and (B) Cladosporium sp. after a 14-day thermal cycle treatment. MicrobankTM beads were put on a Sabouraud dextrose agar Statistics plate and rolled all over the plate and then incubated at 25˚C for 7 days. State Mate ver 4.01 was used for the calculation Blue grains on the figure are the beads. We think spore growth would not be influenced even if the beads were left on the plate.

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Table 1. Germination rates (%) of moss spores after thermal cycle treatment The number of data for each condition is indicated in parenthesis. The number is the mean value and standard deviation for each condition. (exp 1) moss name control 1 week 2 weeks 3 weeks A. undulatum 71.9±5.5 (6) 2.3±0.6 (6) 0.8±0.3 (4) 0.05±0.03 (4) F. hygrometrica 82.1±4.1 (6) 47.6±15.3 (6) 16.8±2.9 (6) 3.3±1.2 (5) B. argenteum 35.1±7.0 (6) 5.3±1.2 (6) 0.5±0.3 (4) 0.02±0.01 (6) C. purpureus 74.5±3.7 (6) 8.2±3.5 (6) 0.5±0.3 (4) 0.02±0.01 (4)

(exp 2) moss name control 3 weeks B. populeum 71.6±5.4 (6) 1.7±1.0 (4) P. inflexum 57.4±13.2 (6) 7.9±3.0 (6) F. hygrometrica 63.2±11.0 (4) 2.0±0.9 (5)

B. populeum 72.0±4.5 (5) 1.9±0.9 (6) P. inflexum 61.3±8.3 (6) 2.6±0.8(4) F. hygrometrica 68.7±12.8 (6) 2.8±0.8(5) of the correlation coefficient and the analysis of the 2). The germination rate of F. hygrometrica in exp 1 was simple linear regression. 3.3 ± 1.2% (n=5) and that of P. inflexum was 7.9 ± 3.2% (n=6) after 3 weeks of treatment. Results Fungi Thermal cycle treatment Colonies of A. oryzae and A. niger were not formed when cultivated on Sabouraud dextrose agar plates for Moss 7 days, after the beads were treated for 26 days and 21 The results of the thermal cycle treated moss spores days (Fig.5 (A)), respectively. On the other hand, colonies are shown in Fig.3 (exp 1), Fig.4 (exp 2) and Table 1 (exp1 were formed with the non-treated control cultivated at the and exp2). As the treatment periods became longer, same time. In the case of Cladosporium sp., a colony was the germination rates decreased. The germination rates formed by 14-day treated spores. The size of the colony of the control (without treatments) were 82%-35 %. It was, however, smaller than that of the control. Cultivation declined to 7.9% - 0.02% after 3 weeks of treatment. The was continued for a further 7 days for verification (Fig.5 highest rate was seen in F. hygrometrica in MS culture (B)). Approximately 100 colonies were found in the non- medium (exp 1) and P. inflexum in HP culture medium (exp treated control and one colony was formed in the treated

AB CD 100 100 100 100 ) Germination rate (%

10 10 10 10 0 10 30 0 10 30 0 10 30 0 10 30

Irradiation time (min)

Fig. 6. Effects of UV irradiation on moss germination (exp 1). The horizontal axis represents the irradiation time and the vertical axis the germination rate (percentage) of each moss spore on a logarithmic scale. (A) A. undulatum (B) F. hygrometrica (C) B. argenteum (D) C. purpureus

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Murashige-Skooge Hyponica

BG HBGH

) 100 100

10 10

Germination rate (% 1 1 10 20 10 20 30 10 20 30 10 20 30 Irradiation time (min)

Fig. 7. Effects of UV irradiation on moss germination and examination of the influence of the culture media (exp 2) The horizontal axis represents the irradiation time and the vertical axis the germination rate (percentage) of each moss spore on a logarithmic scale. (B) F. hygrometrica (G) B. populeum (H) P. inflexum

spores. Thus, the colony formation rate of Cladosporium dilution in the case of non-irradiated A. oryzae (control) sp. was ca. 1%. (Fig.9) and Cladosporium sp. (control). UV irradiation Germination, cultivation and the extrapolation of the Moss survivability of treated mosses The result of the UV irradiation is shown in Fig.6, The germinated spores increased their volume and Fig.7 and Table 2. Some spores can germinate after 30 size and many green bodies (chloroplasts) were seen in min irradiation (1,413 mJ/cm2) though the germination the cytoplasm of the spores. The shape of the germinated rate decreased to 0.7-23.5%. spores of 6 kinds of moss after 14 days of cultivation is shown in Fig.10. The thermal cycle treated spores of F. Fungi hygrometrica under three different conditions (7 days, 14 Colonies of Cladosporium sp. (Fig.8) and A. oryzae days and 21 days) and non-treated control were put on were recognized after being irradiated directly on the Jiffy seven cultivation soil. The treated spores developed beads for 10 min or 30 min, but colonies were not seen in into gametophytes, then sporophytes and capsules after spore solutions taken off the beads and irradiated directly 7-8 months (Fig.11). So, the spores which were treated for 10 min or 30 min. Colonies were seen at a 32-fold

Table 2 Germination rates (%) of moss spores after UV irradiation. The number of data for each condition is indicated in parenthesis. The number is the mean value and standard deviation for each condition. moss name control 10 min 30 min (exp 1) A. undulatum 91.8±2.0 (6) 41.3±4.9 (6) 18.9±7.5 (6) F. hygrometrica 78.9±7.2 (6) 50.6±3.0 (6) 17.4±2.6 (6) B. argenteum 48.1±6.3 (6) 35.3±3.8 (6) 14.8±1.3 (6) C. purpureus 65.3±3.8 (6) 43.0±2.3 (6) 20.3±1.4 (6)

(exp 2) B. populeum 69.0±14.6 (6) 8.9±2.3 (5) 3.8±2.2 (4) P. inflexum 83.7±5.3 (6) 38.9±3.3 (4) 7.7±4.8 (6) F. hygrometrica 77.9±7.2 (6) 59.2±16.5 (4) 23.5±6.0 (6) B. populeum 68.0±13.8 (6) 5.4±2.8(4) 0.7±0.2 (4) P. inflexum 82.7±4.7 (6) 37.1±12.2 (6) 17.5±5.7 (6) F. hygrometrica 78.1±7.2 (6) 32.3±8.4 (6) 17.0±1.7 (4)

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10min UV treat (471mJ/cm2)

control

Fig. 8. Colony formation of Cladosporium sp. on the beads after 30 min of UV irradiation Microbank TM beads were put on a Sabouraud dextrose agar plate and rolled all over the plate and Fig. 9. UV irradiated solutions of A. oryzae spores after being then incubated at 25 ˚C for 14 days. taken off the MicrobankTM beads. UV at 254nm was irradiated for 10 min. The total amount of UV irradiation is 471mJ/cm2. Colonies were recognized until the 32-fold dilution in the control for 3 weeks with the thermal cycle treatment could solution without UV irradiation. Colonies were not seen on the UV treated plate. complete their whole life history safely. The germination rates of the spores of F. hygrometrica after 1 month, 2 months, 3 months and 1 year of treatment 167 min (C. purpureus), 128 min (P. inflexum) and 40 min were extrapolated using the following formula which was (B. populeum). obtained from the results of linear regression in graph b of Fig.3. y=−0.0596x+2.003, where y designates the Discussion logarithm (common logarithm) value of the germination rate and x designates the duration of the treatment (day). Russian Soyuz TMA-02M (27S) was launched in early Presuming that extrapolation from the linear regression June 2011, and fortunately, we could participate in the equation is logical, the extrapolated results after 1 month, experiment. Samples for the BIORISK experiment were 2 months and 1 year were calculated to be 1.66%, 0.03% carried on the rocket, and were installed on the wall of the and the order of 10-20 respectively. ISS in July, 2011. The BIORISK experiment included our To estimate how many capsules are needed for the experiment, we counted the number of spores in each capsule. The mean number ABCD of spores in a capsule of F. hygrometrica was 2.4×105 ± 0.9×105 grains (n=8). The number of germinating spores in each capsule was estimated to be 103 after 1 month, 10 after 2 months, 1 out of 10 capsules after 3 months and 1 out of 1015 capsules after 1 year of treatment from the result of exp 1. In the case of UV irradiation, the germination rate decreases with time to EF 0.1%, as extrapolated from the results of linear regression. The following formulae were used for the estimation. A. undulatum: y=− 114.5x+223.3, F. hygrometrica: y=− 45.3x+86.4, B. argenteum: y = −57.5x+97.7, C. purpureus: y=−59.4x+107.5, P. inflexum : y=−44.3x+83.2, B. populeum: y=−14.9x+25.3, where x is the irradiation time (min) and y the logarithm (log10) value of the Fig. 10. Microscopic images of the germinated spores. germination rate. A value of 0.1 % survivability (A) A. undulatum, (B) F. hygrometrica, (C) B. argenteum, (D) C. purpreus, to UV irradiation of 0.785mW/cm2 was (E) B. populeum, (F) P. inflexum extrapolated to 338 min (A. undulatum), 135 Many green bodies were seen in the germinated spores and some of them came out and moved around the circumference. min (F. hygrometrica), 155 min (B. argenteum),

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A B C

Fig. 11. Images of thermal cycle treated spores of F. hygrometrica cultured on Jiffy seven. Arrows indicate newly formed capsules. (A) 7 day treated spores after 7 months of cultivation, (B) 14 day treated spores after 7 months of cultivation, (C) 21 day treated spores after 8 months of cultivation. samples of spores of the moss F. hygrometrica and the to acquire baseline data on the repeating short-time Cladosporium sp. If on schedule, these samples temperature fluctuations in our search for a candidate TL will be recovered 8 months, 1.5 years and 2.5 years after which can survive in the Earth’s orbit. exposure on the ISS. The results mentioned here will It could be confirmed, this time, that some of the become the control experiment for the space environment 3-week treated moss spores can germinate and develop study. to sporophytes and form capsules. But, we could not Although there may be interest in the survivability of confirm whether the newly formed spores can normally TL in the Earth’s orbit, there is no way to collect samples differentiate and finally form capsules. These are future once dispersed in interplanetary space. Therefore, subjects. According to the preceding research using the samples must be fixed onto a space ship such as the same thermal cycle device, the germination rate of 3 kinds outer wall of the ISS. There are various problems in of higher plant seeds (Jute, Komatsuna and Cucumber) carrying out an experiment simulating the conditions of after 6 days of treatments was 50-100%, and that after 60 the ISS outer wall. The outer wall and the samples are days was 10-20% (Hashimoto, 2010). Cucumber was the subject to harsh conditions such as high temperature, most tolerant seed among them. The seeds of this higher because many pieces of equipment are designed to plant also seem to be tolerant to temperature fluctuations. reflect heat. For example, a machine nominally designed But, they were not a candidate for “TANPOPO”. The for temperatures as high as 60˚C sometimes reaches spores of mosses and fungi examined this time also were 120˚C. In addition to this, the temperature is not always not candidates for “TANPOPO”. From the viewpoint of uniform even in the same place, due to the orbit and the literature, higher cell viability (120%) (survivability) posture of the ISS. The β angle to the sun gradually than the untreated control was reported after a changes day by day. The ISS rotates around the Earth −20˚C/20˚C thermal cycle treatment of antarctic black every 90 min. ISS’s own solar battery panel causes fungi (Cryomyces minteri) for 2 weeks. It was reported shadows over various parts of the hull, and so also to be 120% compared to the untreated control (Onofri et contributes to the temperature fluctuation. Reflection al., 2008) and ca. 40% survivability at UV-C (254nm) is heat and radiation heat from the surrounding machines 1kJ/m2. Deinococcus sp. is reported to be the strongest are another cause. Thermal model calculations by ESA species against UV and gamma radiation so far reported. suggested that the temperature during the space fight Yang et al reported that 10% survivability at UV-C (254nm) may reach as high as 90˚C without any covering. The of Deinococcus aerius sp. (type strain, TR0125T ) is 1.2kJ/ fluctuation of the temperature (−80˚C to 80˚C) was set m2 (Yang, et al., 2008, Yang, et al., 2009). It is reported based on the above-mentioned facts, though the ISS has that 0.1% survivability of a species of A. niger at UV-C different temperature readings in various locations. Onofri, (254nm) is 417mJ/cm2 (4.2kJ/m2) (Yoshino, 2009). This et al. (2008) employed a temperature fluctuation between irradiation value is slightly smaller than our 10 minute −20˚C and 20˚C (maintenance at −20˚C and 20˚C for 1 h) irradiation (471mJ/cm2). Colonies of A. niger were not and used 2 h heating, 2.5 h cooling for their thermal cycle formed in our experiment. UV-C irradiation of 417mJ/cm2 experiment to simulate the change in temperature on the (4.2kJ/m2) therefore may be the limit. surface of Mars. Our defined conditions seem to be more It was found that F. hygrometrica was the most severe than that those encountered in the actual Earth’s tolerant species to the thermal cycle treatment among the orbit, especially regarding our high temperature setting. 6 kinds of moss tested this time in MS culture medium (exp Our purpose is not to reproduce the exact temperature 1 and exp 2). P. inflexum was another tolerant species fluctuations for the exposure part of the ISS. We want in a different culture medium (HP culture medium) in exp 2. In the case of F. hygrometrica, 1 spore out of

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10 capsules is expected to germinate after a 3-month will occur frequently once they become exposed to the thermal cycle treatment. It is realistically easy to provide space environment, and eventually more virulent species several tens of capsules for space experiments and the will appear in the space environment. number is realistic of that thrown out into the universe by Aspergillus sp., Cladosporium sp. and Penicillium a meteorite collision etc. A three month experiment with F. sp. are the major fungal taxa found in the cabin of the hygrometrica should be planned to confirm that the result ISS (Navikoba et al., 2006; Vesper et al., 2008). It is from linear regression is correct and to get conclusive important to estimate how long they survive after leakage evidence in the near future. out into interplanetary space. The long-term existence of Another unsolved problem is the maximum time these fungi in space seems improbable from the results it takes for thermal cycle treated mosses and fungi to obtained here. We will continue to search for other complete their whole biological life cycle. It is found, candidates, such as lichens that have symbiotic relations until now, that at least 21-day thermal cycle treated F. with fungi and (Pogoda de la Vega et al., 2007) hygrometrica spores can complete the whole biological ,(De la Torre Noetzel et al., 2007),(Sancho et al., 2007), life cycle safely. and examine other conditions for simulating planetary We need to estimate how long the moss spores can environments with the same technique. exist under UV irradiation in the Earth’s orbit. The energy Our data indicated that the germination rate changed of the UV lamp used in this study is 785μW/cm2 and the according to the culture medium used. We applied liquid solar irradiance belonging to the biologically damaging culture medium instead of an agar culture plate, because UV range is 9520μW/cm2 (Rabbow et al., 2009). The discrimination of the germinating spores was difficult on intensity of the UV lamp is about 12 times weaker than the agar plate. The volume and length of the germinated that of the Sun. However, the former is the UV light in spores increases remarkably and they overlapped each the wavelength of 254nm and the latter is the light in the other. On the other hand, discrimination is easy in the wide range spectrum through the UV field. A comparison case of liquid culture, because they can be diluted or was made presuming that the damaging effect by the two concentrated freely to the desirable concentration. A lights is the same, but in reality they may be different. difference in cultivation method and culture media may In the Earth’s orbit, the time of survivability without influence the result. Further examination is necessary to covering decreases to 0.1% as extrapolated using linear assess the differences in cultivation method and culture regression and equates to 27.9 min in A. undulatum, 11.2 media. min in F. hygrometrica, 12.8 min in B. argenteum, 13.8 min in C. purpureus, 10.6 min in P. inflexum and 3.3 min Acknowledgements in B. populeum. Next, we will examine the effect of UV irradiation This work was accomplished under the TANPOPO TM with some coverings. Microbank beads were used in meeting. We appreciate valuable comments and this fungal experiment. They are made of ceramics and arguments from the meeting member. Especially, we can have the same effect on sheltering of UV as clay. extend our thanks to Prof. Dr. Akihiko Yamagishi (in It is known that UV light will not reach several tens to charge of the meeting) and Dr. Shinichi Yokobori of hundreds of μm in depth (Yamagishi, et al. 2008). The Department of Molecular , Tokyo University thickness of the beads is 2.5mm. So UVC does not of Pharmacy and Life Sciences. Prof. Dr. Masamichi penetrate to the other side of the beads and as a result Yamashita of Institute of Space and Astronautical some surviving spores remain. Protection against UV Science, JAXA and Prof. Dr. Norimi Hasegawa of Faculty radiation can be easily achieved with some covering. of Science, Yamagata University for various meaningful Examination of the survivability of TL in interstellar comments and advices, and to Prof. Dr. Richard W. and interplanetary space is related to the problem of Jordan of Faculty of Science, Yamagata University whether TL can move through the universe. A TL is for precious revision. Dr. Tomomichi Fujita of Faculty sometimes thrown out into space in natural processes of Science, Hokkaido University for profitable advices such as a huge meteorite drop, volcanic eruption and regarding cultivation of moss spores. so on (lithopanspermia)(Nicholson, 2006). Except for these natural processes, numerous microbes are brought References to the ISS together with the astronauts. Until now only astronauts have been staying at Russia Mir space station, Baranov, V.M., Novikova, N.D., Polikarpov, N.A., Sychev, the ISS, but the general public will go into interplanetary V.N., Levinskikh, M.A., Alekseev, V.R., Okuda, T., space and visit planets such as Mars in the future. We Sugimoto, M., Gusev, O.A. and Grigor, A.I. (2009) think, nowadays, that the possibility that microbes may The Biorisk experiment: 13-month exposure of resting reach other planets by space probes and human space forms of organism on the outer side of the Russia flight missions, either unconsciously or intentionally, segment of the International Space Station. Doklady may be higher than by the natural processes (the topics Biol. Sci., 426, 267-270. mentioned above). The existence in space of microbes Chandler, D. (1994) Cryopreservation of fungal spores with symbiotic relations with mankind will be more using porous beads. Mycol. Rev., 98, 525-526. important as human beings advance further into space. De la Torre Noetzel, R., Sancho, L.G., Pintado, A., Such microbes are unwanted in space, since Rettberg, P., Rabbow, E., Panitz, C., Deutschmann, U.,

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