Hirofumi Hashimoto

To investigate microbial viability and DNA damage, dried cell pellets of the radiation-resistant bacterium Deinococcus radiodurans were exposed to various space environmental conditions at the Exposure Facility of the International Space Station (ISS) as part of the Tanpopo mission. Mutation analysis was done by sequencing the rpoB gene encoding RNA polymerase β-subunit of the rifampicin-resistant mutants. Samples included bacteria exposed to the space environment with and without exposure to UV radiation as well as control samples held in the ISS cabin and at ground. The mutation sites of the rpoB gene obtained from the space-exposed and ISS/ground control samples were similar to the rpoB mutation sites previously reported in D. radiodurans. Most mutations were found at or near the rifampicin binding site in the RNA polymerase β-subunit. Mutation sites found in UV-exposed samples were mostly shared with non-exposed and ISS/ground control samples. These results suggest that most mutations found in our experiments were induced during procedures that were applied across all treatments: preparation, transfer from our laboratory to the ISS, return from the ISS, and storage before analysis. Some mutations may be enhanced by specific factors in the space experiments, but the mutations were also found in the spontaneous and control samples. Our experiment suggests that the dried cells of the microorganism D. radiodurans can travel without space-specific deterioration that may induce excess mutations relative to travel at Earth's surface. However, upon arrival at a recipient location, they must still be able to survive and repair the general damage induced during travel.

The Tanpopo experiment was the first Japanese astrobiology mission on board the International Space Station. It included exposure experiments of microbes and organic compounds as well as a capture experiment of hypervelocity impacting microparticles. We deployed three Exposure Panels, each consisting of 20 Exposure Units that contained microbes, organic compounds, an alanine UV dosimeter or an ionizing radiation dosimeter. The three Exposure Panels were situated on the zenith face of the Exposed Experiment Handrail Attachment Mechanism (ExHAM) that was pointing in zenith direction toward space, which was attached on a handrail of the Japanese Experiment Module (Kibo) Exposed Facility (JEM-EF) outside the International Space Station. The three Exposure Panels were one by one retrieved and returned to the ground after approximately 1, 2, and 3 years of exposure to the space environment. Capture Panels, each of which contained one or two blocks of amorphous silica aerogel, were exposed to collect hypervelocity impact microparticles. Possible captured particles may include micrometeoroids, human-made orbital debris, and natural terrestrial particles. Each year, Capture Panels containing from 11 to 12 aerogel blocks were attached to the three faces of the ExHAM (pointing to zenith, ram, and port); they remained in place for about 1 year and were then returned to the laboratory. This process was repeated three times, in total, during 2015-2018. Additional exposure of a Capture Panel facing ram was conducted between 2018 and 2019. Once the aerogel blocks were returned to the laboratory, they were encapsulated in dedicated transparent plastic cases and optically inspected by a specially designed microscopic system. Once located and recorded, hypervelocity impact signatures were excavated one by one and distributed for further detailed analyses. The apparatus, operation, and environmental factors of all the Tanpopo experiments are summarized in this article.

Radiation dosimetry was carried out at the exposure facility (EF) and the pressurized module (PM) of the Japanese Kibo module installed in the International Space Station as one study on environmental monitoring for the Tanpopo mission. Three exposure panels and three references including biological and organic samples and luminescence dosimeters were launched to obtain data for different exposure durations during 3 years from May 2015 to July 2018. The dosimeters were equipped with additional shielding materials (0.55, 2.95, and 6.23 g/cm2 mass thickness). The relative dose variation, as a function of shielding mass thickness, was observed and compared with Monte Carlo simulations with respect to galactic cosmic rays (GCRs) and typical solar energetic particles (SEPs). The mean annual dose rates were DEF = 231 ± 5 mGy/year at the EF and DPM = 82 ± 1 mGy/year at the PM during the 3 years. The PM is well shielded, and the GCR simulation indicated that the measured mean dose reduction ratio inside the module (DPM/DEF = 0.35) required ∼26 g/cm2 additional shielding mass thickness. Observed points of the dose reduction tendency could be explained by the energy ranges of protons (10-100 MeV), where the protons passed through, or were absorbed in, the shielding materials of different mass thickness that surrounded dosimeters.

© Copyright © 2020 Kawaguchi, Shibuya, Kinoshita, Yatabe, Narumi, Shibata, Hayashi, Fujiwara, Murano, Hashimoto, Imai, Kodaira, Uchihori, Nakagawa, Mita, Yokobori and Yamagishi. The hypothesis called “panspermia” proposes an interplanetary transfer of life. Experiments have exposed extremophilic organisms to outer space to test microbe survivability and the panspermia hypothesis. Microbes inside shielding material with sufficient thickness to protect them from UV-irradiation can survive in space. This process has been called “lithopanspermia,” meaning rocky panspermia. We previously proposed sub-millimeter cell pellets (aggregates) could survive in the harsh space environment based on an on-ground laboratory experiment. To test our hypothesis, we placed dried cell pellets of the radioresistant bacteria Deinococcus spp. in aluminum plate wells in exposure panels attached to the outside of the International Space Station (ISS). We exposed microbial cell pellets with different thickness to space environments. The results indicated the importance of the aggregated form of cells for surviving in harsh space environment. We also analyzed the samples exposed to space from 1 to 3 years. The experimental design enabled us to get and extrapolate the survival time course to predict the survival time of Deinococcus radiodurans. Dried deinococcal cell pellets of 500 μm thickness were alive after 3 years of space exposure and repaired DNA damage at cultivation. Thus, cell pellets 1 mm in diameter have sufficient protection from UV and are estimated to endure the space environment for 2–8 years, extrapolating the survival curve and considering the illumination efficiency of the space experiment. Comparison of the survival of different DNA repair-deficient mutants suggested that cell aggregates exposed in space for 3 years suffered DNA damage, which is most efficiently repaired by the uvrA gene and uvdE gene products, which are responsible for nucleotide excision repair and UV-damage excision repair. Collectively, these results support the possibility of microbial cell aggregates (pellets) as an ark for interplanetary transfer of microbes within several years.

Cyanobacteria are photosynthetic organisms. This study aimed to examine the cellular tolerance of a terrestrial cyanobacterium, Nostoc sp. HK-01 to cosmic rays to assess its potential application in studies on extraterrestrial environments and the possibility of interplanetary transformation. This strain is a candidate organism for introduction in extraterrestrial environments such as those on Mars owing to its high tolerance to heat, dryness, and vacuum conditions. Dried cells were exposed to extreme environments and survival germinated cell rates were conformed after the tests. Dried cells of Nostoc sp. HK-01 colonies were exposed to a temperature cycle of 80 degrees C and - 80 degrees C for 90 min, vacuum, a helium-ion beam (He), ultraviolet (VUV) rays (172 nm), UV rays (254 nm), and gamma rays (5 kGy). Live cells were confirmed in all experiments. The D-10 values of a helium-ion beam, VUV, UV and a gamma rays (5 kGy) tests were 2.9 kGy, 11.5 kJ cm(-2), 1.2 kJ cm(-2) and 19.9 kGy, respectively. The present results indicate that cyanobacteria can be potentially used for future manned space missions and it was confirmed that these cells could be transported to extraterrestrial environments.

The Tanpopo mission has two objectives: (1) test the panspermia hypothesis and (2) test whether organic compounds may have been transferred to Earth before the origin of life. We developed an exposure panel (EP) designed to expose microbes and organic compounds to the space environment and a capture panel designed to capture high-velocity particles on the International Space Station (ISS) using aerogel contained in an aluminum container. The panels returned after 1 year of exposure at the Exposure Facility of the Japan Experimental Module, ISS. In this communication, we report the measurements of temperature, radiation dosimeter and vacuum ultraviolet dosimeter in the EP, and survival data of Deinococcus aetherius. The environmental data are consistent with survival data of microbes and organic compounds, which will be presented elsewhere in detail.

Plants play an important role in bio-regenerative life support systems (BLSSs) for long-term manned space missions. During parabolic airplane flights, we investigated stem sap flow without forced air movement and water vapor conductance with forced air movement using sweetpotato plants. Stem sap flow was promoted under microgravity, but only when forced air movement was applied to the plants. The water vapor conductance of the plant leaves increased under microgravity at an air velocity of 0.25 m.s(-1). Leaf temperatures also increased under microgravity at an air velocity of 0.02 m.s(-1). This suggests that forced air movement is important in maintaining long-term, healthy plant growth in BLSSs.

The Tanpopo mission will address fundamental questions on the origin of terrestrial life. The main goal is to test the panspermia hypothesis. Panspermia is a long-standing hypothesis suggesting the interplanetary transport of microbes. Another goal is to test the possible origin of organic compounds carried from space by micrometeorites before the terrestrial origin of life. To investigate the panspermia hypothesis and the possible space origin of organic compounds, we performed space experiments at the Exposed Facility (EF) of the Japanese Experiment Module (JEM) of the International Space Station (ISS). The mission was named Tanpopo, which in Japanese means dandelion. We capture any orbiting microparticles, such as micrometeorites, space debris, and terrestrial particles carrying microbes as bioaerosols, by using blocks of silica aerogel. We also test the survival of microbial species and organic compounds in the space environment for up to 3 years. The goal of this review is to introduce an overview of the Tanpopo mission with particular emphasis on the investigation of the interplanetary transfer of microbes. The Exposed Experiment Handrail Attachment Mechanism with aluminum Capture Panels (CPs) and Exposure Panels (EPs) was exposed on the EF-JEM on May 26, 2015. The first CPs and EPs will be returned to the ground in mid-2016. Possible escape of terrestrial microbes from Earth to space will be evaluated by investigating the upper limit of terrestrial microbes by the capture experiment. Possible mechanisms for transfer of microbes over the stratosphere and an investigation of the effect of the microbial cell-aggregate size on survivability in space will also be discussed.

The fabrication of an ultralow-density hydrophobic silica aerogel for the intact capture cosmic dust during the Tanpopo mission is described. The Tanpopo experiment performed on the International Space Station orbiting the Earth includes the collection of terrestrial and interplanetary dust samples on a silica aerogel capture medium exposed to space for later ground-based biological and chemical analyses. The key to the mission's success is the development of high-performance capture media, and the major challenge is to satisfy the mechanical requirements as a spacecraft payload while maximizing the performance for intact capture. To this end, an ultralow-density (0.01 g cm(-3)) soft aerogel was employed in combination with a relatively robust 0.03 g cm(-3) aerogel. A procedure was also established for the mass production of double-layer aerogel tiles formed with a 0.01 g cm(-3) surface layer and a 0.03 g cm(-3) open-topped, box-shaped base layer, and 60 aerogel tiles were manufactured. The fabricated aerogel tiles have been demonstrated to be suitable as flight hardware with respect to both scientific and safety requirements. [GRAPHICS] .

In this paper, we report the progress in developing a silica-aerogel-based cosmic dust capture panel for use in the Tanpopo experiment on the International Space Station (ISS). Previous studies revealed that ultralow-density silica aerogel tiles, comprising two layers with densities of 0.01 and 0.03 g/cm(3) developed using our production technique, were suitable for achieving the scientific objectives of the astrobiological mission. A special density configuration (i.e., box framing) aerogel with a holder was designed to construct the capture panels. Qualification tests for an engineering model of the capture panel as an instrument aboard the ISS were successful. Sixty box-framing aerogel tiles were manufactured in a contamination-controlled environment.

We have proposed an experiment (the Tanpopo mission) to capture microbes on the Japan Experimental Module of the International Space Station. An ultra low-density silica aerogel will be exposed to space for more than 1 year. After retrieving the aerogel, particle tracks and particles found in it will be visualized by fluorescence microscopy after staining it with a DNA-specific fluorescence dye. In preparation for this study, we simulated particle trapping in an aerogel so that methods could be developed to visualize the particles and their tracks. During the Tanpopo mission, particles that have an orbital velocity of similar to 8 km/s are expected to collide with the aerogel. To simulate these collisions, we shot Deinococcus radiodurans-containing Lucentite particles into the aerogel from a two-stage light-gas gun (acceleration 4.2 km/s). The shapes of the captured particles, and their tracks and entrance holes were recorded with a microscope/camera system for further analysis. The size distribution of the captured particles was smaller than the original distribution, suggesting that the particles had fragmented. We were able to distinguish between microbial DNA and inorganic compounds after staining the aerogel with the DNA-specific fluorescence dye SYBR green I as the fluorescence of the stained DNA and the autofluorescence of the inorganic particles decay at different rates. The developed methods are suitable to determine if microbes exist at the International Space Station altitude.

A wide variety of organic compounds have been found in space, and their relevance to the origin of life is discussed. Interplanetary dust particles (IDPs) are most promising carriers of extraterrestrial organic compounds, but presence of bioorganic compounds are controversial since they are so small and were collected in the terrestrial biosphere. In addition, IDPs are directly exposed to cosmic and solar radiation. Thus, it is important to evaluate the stability of organics in IDPs in space environment. We are planning a novel astrobiology mission named Tanpopo by utilizing the Exposed Facility of Japan Experimental Module (JEM/EF) of the International Space Station (ISS). Two types of experiments will be done: Capture experiments and exposure experiments. In the exposure experiments, organics and microbes will be exposed to the space environments to examine possible alteration of organic compounds and survivability of microbes. Selected targets for the exposure experiments of organic compounds are as follows: Amino acids (glycine and isovaline), their possible precursors (hydantoin and 5-ethyl-5-methyl hydantoin) and complex precursors "CAW" synthesized from a mixture of carbon monoxide, ammonia and water by proton irradiation. In addition to them, powder of the Murchison meteorite will be exposed to examine possible alteration of meteoritic organics in space. We will show the results of preparatory experiments on ground by using a UV lamp, a ⁶⁰Co source, synchrotron facilities, and a heavy ion irradiation facility.

To investigate the possible interplanetary transfer of life, numerous exposure experiments have been carried out on various microbes in space since the 1960s. In the Tanpopo mission, we have proposed to carry out experiments on capture and space exposure of microbes at the Exposure Facility of the Japanese Experimental Module of the International Space Station (ISS). Microbial candidates for the exposure experiments in space include Deinococcus spp.: Deinococcus radiodurans, D. aerius and D. aetherius. In this paper, we have examined the survivability of Deinococcus spp. under the environmental conditions in ISS in orbit (i.e., long exposure to heavy-ion beams, temperature cycles, vacuum and UV irradiation). A One-year dose of heavy-ion beam irradiation did not affect the viability of Deinococcus spp. within the detection limit. Vacuum (10(-1) Pa) also had little effect on the cell viability. Experiments to test the effects of changes in temperature from 80 °C to -80 °C in 90 min (± 80 °C/90 min cycle) or from 60 °C to -60 °C in 90 min (± 60 °C/90 min cycle) on cell viability revealed that the survival rate decreased severely by the ± 80 °C/90 min temperature cycle. Exposure of various thicknesses of deinococcal cell aggregates to UV radiation (172 nm and 254 nm, respectively) revealed that a few hundred micrometer thick aggregate of deinococcal cells would be able to withstand the solar UV radiation on ISS for 1 year. We concluded that aggregated deinococcal cells will survive the yearlong exposure experiments. We propose that microbial cells can aggregate as an ark for the interplanetary transfer of microbes, and we named it 'massapanspermia'.

Tardigrades are tiny (less than 1 mm in length) invertebrate animals that have the potential to survive travel to other planets because of their tolerance to extreme environmental conditions by means of a dry ametabolic state called anhydrobiosis. While the tolerance of adult tardigrades to extreme environments has been reported, there are few reports on the tolerance of their eggs. We examined the ability of hydrated and anhydrobiotic eggs of the tardigrade Ramazzottius varieornatus to hatch after exposure to ionizing irradiation (helium ions), extremely low and high temperatures, and high vacuum. We previously reported that there was a similar pattern of tolerance against ionizing radiation between hydrated and anhydrobiotic adults. In contrast, anhydrobiotic eggs (50% lethal dose; 1690 Gy) were substantially more radioresistant than hydrated ones (50% lethal dose; 509 Gy). Anhydrobiotic eggs also have a broader temperature resistance compared with hydrated ones. Over 70% of the anhydrobiotic eggs treated at either -196 degrees C or +50 degrees C hatched successfully, but all the hydrated eggs failed to hatch. After exposure to high-vacuum conditions (5.3 x 10(-4) Pa to 6.2 x 10(-5) Pa), the hatchability of the anhydrobiotic eggs was comparable to that of untreated control eggs.

Gravity resistance is a principal graviresponse in plants. In resistance to hypergravity, the gravity signal may be perceived by the mechanoreceptors located on the plasma membrane, and then transformed and transduced via the structural continuum or physiological continuity of cortical microtubules-plasma membrane-cell wall, leading to an increase in the cell wall rigidity as the final response. The Resist Tubule experiment, which will be conducted in the Kibo Module on the International Space Station, aims to confirm that this hypothesis is applicable to resistance to 1 G gravity. There are two major objectives in the Resist Tubule experiment. One is to quantify the contributions of cortical microtubules to gravity resistance using Arabidopsis tubulin mutants with different degrees of defects. Another objective is to analyze the modifications to dynamics of cortical microtubules and membrane rafts under microgravity conditions on-site by observing green fluorescent protein (GFP)-expressing Arabidopsis lines with the fluorescence microscope in the Kibo. We have selected suitable mutants, developed necessary hardware, and fixed operation procedure for the experiment.

JAXAが検討している火星探査において，生命探査を行う意義について述べ，具体的方法を提案した。

微生物の惑星間移動および地球外有機物の地球への伝搬を検証するための宇宙実験「たんぽぽ」について，その目的と準備状況について述べた。

The treatment of sodium, Na, and potassium, K, presents a challenge in space agriculture material recycling, as humans require Na and plants cannot grow at high Na concentrations. Hence, we are proposing the use of marine macro-algae to harvest K and other minerals from composted human waste. Ulva was selected for this feasibility study, since it tolerates a wide range of salinity levels. Growth capability of Ulva was examined under various total salinity levels and proportions of Na and K in the incubation medium. A homeo-static feature of Ulva was found in its intra-cellular concentration of Na and K, and in the intra-cellular ratio between Na and K (at 0.58 +/- 0.30, lower than that of human metabolic waste). Intracellular concentration of K in Ulva is 20 times higher than seawater. Because of these characteristics, Ulva is a good candidate species for space agriculture. (C) 2009 COSPAR. Published by Elsevier Ltd. All rights reserved.

Overview of Space Agriculture on Mars: Mars is the second target of our manned space flight next to the Moon, and possibly the most distant extraterrestrial body to which we could travel, land and explore within the next half century. The requirements and design of life support for a Mars mission are quite different from those being operated on near Earth orbit or prepared for a lunar mission, because of the long mission duration. A Mars mission must include at least 2.5 years for round trip travel, and a restricted opening of the launch window, both for forward and return flights once every two years. Precursor manned mission to Mars might be conducted with a small number of crew and a conservative life support system on the space ship. Once the scale of the manned mission is enlarged, an advanced bio-regenerative life support system provides an economical advantage over the open loop life support, based on cost comparison between the cumulative sum of consumables with the open loop system versus the initial investment for a recycling system. We further propose use of on-site resources to supplement loss of component materials in the recycling process. Reproducing recycling materials on an expanded scale is another advantage of the use of on-site resources for space agriculture. © 2009 Springer-Verlag Berlin Heidelberg.

The Twenty-fifth Space Utilization Symposium (January 14-15, 2009: ISAS/JAXA Sagamihara, Japan)One of major problems in astrobiology is intense radiation, theoretical and experimental studies on biological protection and tolerance to radiation are needed. Air dried-microbial cells were irradiated with high-dose X-ray for 0 to 66,500 Gy and high-energy Fe ions for 0 to 2,000 Gy to examine their survivability based on the biological "energy currency", i.e., adenosine 5'-triphosphate(ATP), and the Most probably number (MPN). The well known radiation-resistant bacteria such as genus Deinococcus and Rubrobacter radiotolerans, highly stress-resistant spore former family Bacillaceae and radiation-sensitive Escherichia coli strains were used for this experiment. Irradiation of synchrotron X-ray (0.2 nm) at a high dose rate of 4.4 Gy/s (500 R/s) was done at KEK Photon Factory. All species showed exponential decrease in MPN against total dose. However, celluar ATP showed little decrease at low dose and linear (or exponential) decrease at high dose. Genus Deinococcus and Rubrobacter radiotolerans showing survival at 67 kGy, was more than 60 times resistant compared with normal E. coli. Irradiation of 500 MeV Fe ion at a dose rate of 0.172 Gy/s (2.5 x 10(exp 8) particles/s) was done at NIRS HIMAC. Generally similar results to X-ray irradiation were observed, but some genus Deinococcus and Rubrobacter radiotolerans showed no decrease at total dose 2000 Gy. And celluar ATP showed no decrease against to the dose. More biomacromolecule should be used in future study.