Research Article
Open access
Published Online: 9 December 2021

Investigation of Nostoc sp. HK-01, Cell Survival over Three Years during the Tanpopo Mission

Publication: Astrobiology
Volume 21, Issue Number 12

Abstract

The survival of the terrestrial cyanobacterium Nostoc sp. HK-01 was tested as part of the Tanpopo mission experiment, which was conducted both outside and inside the International Space Station (ISS). The selection of Nostoc sp. HK-01 was based on the results of on-ground experiments that demonstrated that the cyanobacterium can survive simulated space environments. This study verified cell survival after exposure to the outside environment in low Earth orbit (LEO). We examined the cellular tolerance of Nostoc sp. HK-01 simultaneously outside and inside of the ISS over a 3-year period. After the experiments were conducted, we confirmed cell viability by fluorescein diacetate (FDA). Cell growth abilities for 3 years without sunlight in space-vacuum-exposed cells were not significantly different from those of cells kept in the dark of control cells in the ISS and on the ground. Though a few light-exposed cells in space vacuum survived outside the ISS after 3 years as judged by FDA staining assay, the survival could not be verified by testing the growth ability due to an insufficient number of cells.
To the best of our knowledge, this is the first pure strain of Nostoc sp. HK-01 that survived in a space environment on the inside and outside of the ISS with and without sunlight for more than 3 years (1126 days).

1. Introduction

Cyanobacteria are photosynthetic organisms that contributed to the atmospheric oxidation of ancient Earth; therefore, their use in a space environment for oxygen production is often discussed (Arai et al., 2008; Brown et al., 2008; Billi et al., 2019; Tomita-Yokotani et al., 2020). In our previous study, we reported that a strain of the terrestrial cyanobacterium Nostoc sp. HK-01 demonstrated a high tolerance to high- and low-temperature cycles under vacuum, helium-ion beam radiation, vacuum ultraviolet (172 nm) radiation, ultraviolet (254 nm) radiation, and gamma rays (Tomita-Yokotani et al., 2020). Owing to their high tolerance against several environmental factors, the Nostoc sp. HK-01 cells were selected for use as biological material for the Tanpopo mission, a space experiment outside the Japanese Experiment Module (JEM) of the International Space Station (ISS) (Kawaguchi et al., 2013, 2016; Tomita-Yokotani et al., 2020). The Japanese word “Tanpopo” refers to the dandelion in English, the seeds of which are spread via wind dispersal. Considering the meaning of the term, the dandelion seeds elicit an image of the interplanetary migration of microbes and organic compounds. The Tanpopo mission experiment was conducted in the Exposed Facility (EF) of the JEM (also known as “Kibo”) at the ISS for over 3 years (Yamagishi et al., 2018). In this study for the Tanpopo mission, there were two scientific aims and six subthemes, as described previously (Kawaguchi et al., 2013; Sasaki et al., 2019). An experiment using a cyanobacterium was included in Subtheme 2: Exposure of microbes in space. The name of our research group is “Cyanobacterial Inception & Operation” (CINO), and the CINO research group focused on providing evidence that a terrestrial cyanobacterium, Nostoc sp. HK-01, has a high tolerance to space environments. Evidence to date suggests that the cells have the potential to survive transfer within interplanetary space; therefore, we took the first step to test this hypothesis and examined cell survival of this organism after exposure to the space environment in low Earth orbit (LEO).
We reported previously that cyanobacteria would be suitable as a food source and fertilizer in martian environments (Arai et al., 2008; Kimura et al., 2015, 2016). Recycling of oxygen and carbon dioxide by photosynthesis in extraterrestrial habitats, such as on Mars (e.g., Verseux et al., 2016), would contribute to life support for human exploration. To date, several research groups have focused on the study of cyanobacteria as a useful organism for future space habitats (Arai et al., 2008; Kimura et al., 2015; Verseux et al., 2016; Billi et al., 2017; Tomita-Yokotani et al., 2020).
Previous studies that have reported the use of photosynthetic organisms for exposure experiments in space have reported promising results (Mancinelli et al., 1998; Olsson-Francis et al., 2009, 2010; Eitner and Augustin, 2017; Billi et al., 2019; de Vera et al., 2019). Haloarcula and Synechococcus were used as the main study materials, and viability was tested over a period of 15 days (Mancinelli et al., 1998). It has been reported that Nostoc commune (PCC7524) can survive vacuum and low temperature but not high UV radiation based on a space exposure experiment of 548 days' duration (Cockell et al., 2011). Billi et al. (2019) reported space exposure experiments using the cyanobacterium Chroococcidiopsis sp., which was selected because of its high tolerance to space environments (Billi et al., 2019). The experiment was performed for 16 months outside the ISS (Billi et al., 2019). In the experiment, the tested cells were set in a compartment mixed with or without martian soil simulants, together with a Mars-like atmosphere (Billi et al., 2019). After the exposure experiment, it was reported that some cells within the mixed soil survived.
The CINO group selected Nostoc sp. HK-01 as a material for this mission. The strain's genomic information was previously investigated, and the strain inhabits environments globally (Katoh et al., 2003; Kimura et al., 2017a). Many related species have also undergone genomic analysis, and their genomic information is also known (Katoh, personal communication). Furthermore, this strain is a photosynthetic organism with a distinct life cycle, and it is a purified strain among the Nostoc species (Fig. 1). The distinct life cycle makes this organism a candidate for future space studies, such as for studies of dormancy or the functional substances related to their differentiation (Kimura et al., 2020).
FIG. 1. Cell cycle of Nostoc sp. HK-01. Akinete cells (shown in the upper left outlined in green) have high tolerances to any severe environments as described in the text. Black scale bar is 10 μm.
For the Tanpopo mission, the CINO group focused on demonstrating the feasibility of transferring cyanobacteria between the ISS and Earth over a 3-year period.

2. Materials and Methods

2.1. Materials

An axenic culture of Nostoc sp. HK-01 that has been maintained in our laboratory (University of Tsukuba) for over 10 years was used for all experiments. These cultures are maintained at 25°C in BG-11 medium (Rippka et al., 1979; Arai et al., 2008) and are routinely subcultured. This strain develops small colonies during growth in liquid medium that include four types of cells, the different morphotypes of which are shown in Fig. 1. Each cell type can be routinely distinguished by microscopic observation. Cell preparation for flight was carried out as follows: A small colony of cells of Nostoc sp. HK-01 was incubated in BG-11 at 25°C for 2 months under room light (20.2 ± 0.9 μmol·m−2·s−1) in 45 mL of medium in a 100 mL volume Sakaguchi flask with a 3 cm amplitude for 75 cycles·min−1 on an SR-1 reciprocating shaker (As One, Osaka, Japan). Small portions of colonies of cells in liquid medium, including akinete and other types of cells, were extracted and placed into a small tube and shaken. A cell suspension was obtained, and the percentage of akinete cells was determined because only the akinete cells survive the air-drying process, a protocol for spaceflight.

2.2. Preparation of materials for the Tanpopo mission

To prepare the cells for flight, nine aliquots of cells in 500 μL of BG-11 were used for each year of exposure in space. The cell culture was determined to contain 5.5 × 107 ± 1.0 × 107 cells, of which 10% were akinete cells. Each 500 μL volume of homogenized, suspended cell extracts was placed on each of the nine pieces of aluminum foil (φ = 17.8 mm and φ = 11.8 mm) and left to air dry on a clean bench at room temperature. Thereafter, the dried akinete cell suspensions (5.5 × 106 ± 1.0 × 106 akinete cells) on the aluminum foil were placed into the exposure unit together with the exposure sample plate in the exposure unit base. Figure 2A shows a cross-section of the exposure unit. Three pieces of aluminum foil with air-dried cells attached (5.5 × 106 ± 1.0 × 106 akinete cells per piece of aluminum foil) were placed in an exposure base for each year of the experiment. One piece of aluminum foil (φ = 17.8 mm) was placed at the bottom of the exposure unit and labeled “Dark,” where it would remain in the dark and be exposed to the vacuum of space until retrieval. A second piece of aluminum foil (φ = 11.8 mm) with cells, labeled “Space,” was placed underneath the MgF2 window of the exposure unit, where it was exposed to the vacuum of space and sunlight. A third smaller piece of the aluminum foil (φ = 11.8 mm), which was also placed in the exposure unit though it remained in the dark, was exposed to the vacuum of space and is referred to as the “Dark Reserve” sample. The other 6 pieces of aluminum foil were split equally between the dark control that remained in the ISS (labeled “ISS”) and the dark laboratory control that remained on the ground (labeled “Ground”). The ISS and Ground samples were both kept at 1 atm pressure and ambient temperature for the duration of the experiment.
FIG. 2. Diagram of the base of exposure unit. Cross-section of exposure unit (A) and the internal parts of the exposure unit (B).
Three exposure units were flown during the 3-year mission, and one exposure unit was removed at the end of each year. A diagram of the exposure unit and the position of the samples is provided in Fig. 2. The internal part of the exposure units was produced by the Research Facility Center for Science and Technology, University of Tsukuba, Japan.

2.3. Timeline for the Tanpopo mission

The timeline for the Tanpopo mission is shown in Table 1. Materials were launched on Space-X CRS-6 on April 15, 2015, and the exposure experiments began on May 26, 2015. The end of the first, second, and third periods was June 13, 2016; July 19, 2017; and July 20, 2018, respectively (Table 1).
Table 1. Timeline of the Tanpopo Mission for Dried Colonies of the Terrestrial Cyanobacterium Nostoc sp. HK-01
YearMonthDayEventsDays after first launch
20150309Delivered to Tsukuba Space Center 
20150313Arrival at Lyndon B. Johnson Space Center; JSC 
20150415Launch on Space-X CRS-6 
20150514Attachment of samples to ExHAM 
20150526Initiation of the exposure experiments 
20160613End of the first year exposure384
20160617Removal of exposure panel (EP) for the first year 
20160827Return to ground by Dragon SpX-9 
20160913Arrival at Tsukuba Space Center 
20160920Delivery to JAXA at Sagamihara 
20161003Removal of a part of EPs 
20170719End of the second year exposure769
20170721Removal of exposure panel (EP) for year two 
20170917Return to ground by Dragon SpX-12 
20171003Arrival at Tsukuba Space Center 
20171006Delivery to JAXA at Sagamihara 
20171012Removal of a part of EPs 
20180720End of the third year exposure1126
20180723Removal of a part of EPs 
20180804Return to ground by Dragon SpX-15 
20180822Arrival at Tsukuba Space Center 
20180824Delivery to JAXA at Sagamihara 
20180903Removal of a part of EPs 

2.4. Apparatus for the Tanpopo mission

The exposure units were secured in an exposure panel (EP) developed for the Tanpopo mission for the exposure of microbes and organic materials (Yamagishi et al., 2018). The dried Nostoc sp. HK-01 cells outside the ISS in the EP were exposed at the same time as samples from other investigations conducted during the mission. The position of the experimental units on the EP is shown in Fig. 3 (green squares).
FIG. 3. Photographs of exposure panels (100 × 100 × 20 mm): outside exposure panel (A) and inside panel of the ISS (B). Cyanobacterial cells were set within the green square.

2.5. Outside and inside environments of the ISS

According to data obtained from the thermometer on board the Kibo exposure facility during the Tanpopo mission, the maximum and minimum temperatures were 24°C ± 5°C and -21°C ± 5°C, respectively (Hashimoto et al., 2016; Yamagishi et al., 2018). The UV energy was measured using an alanine dosimeter (Yamagishi et al., 2018). Kawaguchi et al. (2020) reported that the mean UV fluence was estimated at 124 MJ m−2 yr−1, and 177 MJ m−2 yr−1 on the side of the MgF2 window. Ionizing radiation in space was 232 ± 5 mGy yr−1 on the exterior and 83 ± 1 mGy yr−1 in the interior of the Kibo module during flight (Kawaguchi et al., 2020).

2.6. Survival analysis of cells by cell staining test

Randomly selected subsamples from the experimental units, 1/16 of each piece of aluminum foil with Nostoc sp. HK-01 cells, were used for survival analysis. After the exposure experiments, exposed and unexposed cells were stored in water for 2 days. The surviving cells were stained with fluorescein diacetate (FDA), as previously described (Tomita-Yokotani et al., 2020). The stained cells were observed under a fluorescence microscope (BX50, Olympus, Japan). Cells that were stored inside the ISS and on the ground, both of which were maintained under dark conditions, were used as controls. The survival cell rate (%) was calculated as the number of fluorescent akinetes (dormant cells) divided by the total number of akinete cells, multiplied by 100. For each of the randomly selected fields of view selected for microscopic observation, the cell survival rate was calculated, and an average survival cell rate and standard error were calculated. Cell staining tests were performed within 6 months after return to Earth. Cell staining tests were also performed to check the survival rate (%) when several other experiments were performed. All of the cells that were part of the experiments were stored in a desiccator at room temperature, 25°C, in the dark in the laboratory.

2.7. Measurement of thickness of cell layer

The thickness of each cell layer was analyzed using a scanning 3D profilometer (NanoMap LS). Three pieces of aluminum foil (numbered No. 1, 2, and 3, Fig. 8) with the dried suspensions of Nostoc sp. HK-01 from the ground control were used for the analysis of the thickness of each cell layer. A small piece of aluminum foil on which cells were placed was fixed directly on a slide. The dried cells were scratched along a line by a toothpick to separate the dried cell suspension and enable us to measure the thickness of the cell layer. The exact thickness of the cell layer and aluminum foil was measured three times using the NanoMap Contact software, and the value of the aluminum thickness was subtracted after the measurements.

2.8. Separation and preparation of materials for cell growth tests

A subsample (1/16) of the aluminum foil with Nostoc sp. HK-01 cells that was used in the space experiments was placed into a small sterile culture tube. BG-11 (400 μL) was added to the test tube, and the cells and aluminum foil were separated by agitating the fluid with the pipette multiple times. The cells were then incubated at 37°C for 30 min in the dark. Subsequently, 100 μL of cells together with 1000 μL BG-11 liquid medium were incubated in a 24-well microplate (P24F01S, As One, Japan) for 3 weeks under a light (14.0 ± 0.8 μmol m−2 s−1) / dark cycle of 12/12 hours with a 2.3 cm amplitude for 30 cycles·min−1 on See-Saw Shaker NA-101 (Nissinrika, Tokyo, Japan). After 3 weeks of incubation, the cells were observed visually and photographed.
The number of akinete cells in the initial state was (1.3 ± 0.3) × 105 cells mL−1, determined by microscopic analysis (BX50, Olympus, Japan). The concentration of akinetes was calculated by using a hemocytometer (Nippon Rinsho Kikai Kogyo Co. Ltd., Japan). The cell growth rates of samples that remained in the EP for 3 years was also determined by optical microscopy.

2.9. Time course of cell germination rate in the early stage of re-incubation after 3 years of LEO exposure

The recovered cells after 3 years of LEO exposure were dried and re-incubated with BG-11 liquid medium. The methods of observation and calculation of germinated cells by microscopy were the same as those described above. A germinated cell rate (%) was calculated for each randomly selected field of view in the microscope and the average germinated cell rate and standard error determined. The tests were performed several times within a year after the third-year cells were returned to Earth.

3. Results

3.1. Visual observation of the surface of dried Nostoc sp. HK-01 cells

The visual appearance of dried colonies of Nostoc sp. HK-01 after exposure for 3 years differed significantly depending upon whether the samples were exposed to sunlight or kept in the dark. As shown in Fig. 4, colonies exposed to space vacuum beneath the MgF2 window that were exposed to sunlight appeared brown in color, whereas those exposed to space vacuum but kept in the dark maintained a green color for the entire 3-year duration of the experiment. The green color of the dried colonies kept in the dark was the same as that of the initial preparation and each year's ground control of dried colonies (Fig. 4).
FIG. 4. Photographs of an exposure unit and initial state of Nostoc sp. HK-01 cells deposited on aluminum foil (A) and after exposure in exposure units for 1–3 years during the Tanpopo mission. (B) These cells were exposed to LEO; upper panel shows cells after exposure on the window side; lower panel shows cells after exposure on the dark side of the exposure unit after each exposure period from 1 to 3 years. The “Ground” panel shows each year's ground control of dried colonies.

3.2. FDA cell viability assay and cell growth test

The viability of cells was tested using the FDA staining technique. The FDA staining test was repeated three times using cells on 1/16th of the aluminum foil for each type of sample (Space, Dark, etc.), and measurements were made at the end of each year. Microscopic analysis (n > 100) revealed that there were no significant differences in the viability of cells kept in the dark, regardless of whether they were exposed to space vacuum, kept on plates inside the ISS, or kept on the ground in the laboratory, though the survival cell rate (%) over the 3 years decreased with time (Fig. 5). Nearly all of the viable cells exposed to space vacuum and sunlight in LEO perished prior to the end of year 1.
FIG. 5. Survival rates (%) of Nostoc sp. HK-01 cells according to the FDA cell staining test of ground control cells (Ground), cells kept inside the ISS (ISS), cells exposed on the dark side and kept in the vacuum of space (Dark), and cells exposed on the window side in space (Space). Data are represented as the mean ± standard error.
Shown in Fig. 6 are optical contrast and fluorescent light microscope photographs of the stained control cell samples (Ground and ISS) and the cell samples exposed to the space environment (Dark, which was kept on the dark side of the exposure unit, and Space, which was kept on the window side of the exposure unit). There were no detectable differences between the ground control and the space-exposed cells except for those cells exposed to sunlight on the window side of the exposure unit. During the search for viable cells in the Space sample exposed to space vacuum and LEO sunlight for 3 years, we observed several areas with fluorescent cells (Extra box in Fig. 6). It was not possible to express this finding as a statistically valid percentage of cell survivors, though the microscopic observation of cells stained by FDA led us to conclude that a residual number of cells survived after 3 years of the experiment (Extra box in Fig. 6).
FIG. 6. Microscopic images of Nostoc sp. HK-01 cell control samples (Ground and ISS) and cells exposed in space (Dark side and Space sample on the window side of the exposure unit). NL, normal light; FL, fluorescent light. Extra box shows the area with fluorescent cells.
For cell growth, cells in space on the dark side of the exposure unit (Dark), cells kept in the dark inside the ISS (ISS), and cells in the dark on the ground (Ground) were incubated for 3 weeks. The concentration of incubated cells in liquid medium was 1.3 ± 0.3 × 105 cells mL−1. In these tests, cells exposed on the window side to space vacuum (Space) were not examined because, as noted, the number of surviving cells was insufficient. As shown in Fig. 7A, cell propagation of the cells tested for growth from the Dark, ISS, and Ground foils were clearly recognizable visibly. The cells from the Dark, ISS, and Ground foils after 3 years of exposure were re-incubated with liquid medium of BG-11 after re-drying (Fig. 7B). As shown in Fig. 7B, the germination rate and growth of the flight samples after several days of incubation did not differ significantly from that of the cell growth rate of the ground control. Indeed, during the early incubation period, such as on day two, the rate of germinated cells in the ISS sample was higher than that of the cells that had been kept in the dark in space and higher than those in the ground control (Fig. 7B).
FIG. 7. Growth of Nostoc sp. HK-01 cells (A) and germination of re-dried and re-incubated cells (B) in liquid medium. (A) Results of the survival cell growth test for the control cells (ISS cell sample kept in the dark in the space station and Ground cell sample exposed to space but kept in the dark on the exposure unit) and space-vacuum-exposed cells kept in the dark (Dark). Cells of all samples were incubated with liquid BG-11 for 3 weeks. (B) Cells recovered after 3 years of the Tanpopo experiment were dried and re-incubated with liquid BG-11 for 5 days. Bars indicate average ± standard error.

3.3. Measurement of thickness of cell layer

The measurement of cell thickness was done by the profilometer using the Ground samples, which contained the cells on the aluminum foil that remained on the ground. The cell thickness results are shown in Fig. 8. Three round pieces of aluminum foil that were the same size as the pieces of foil used in the exposure tests were analyzed. As shown in Fig. 8, the number of areas measured on the foils were as follows: No. 1 (n = 24), No. 2 (n = 30), and No. 3 (n = 37). The average thicknesses were No. 1, 35.1 ± 3.9 μm; No. 2, 30.8 ± 4.5 μm; and No. 3, 20.0 ± 6.1 μm. The total average thickness was 27.5 ± 2.2 μm. Figure 8 shows the distribution of those results. According to the results of the cell thickness, there was an area in which the cell thickness was over 100 μm.
FIG. 8. Thickness of the cells on the aluminum foil kept in the laboratory in the dark on Earth. Cell thickness was analyzed using a profilometer. The number of measurement areas was as follows: No. 1 (n = 24), No. 2 (n = 30), and No. 3 (n = 37).

4. Discussion

4.1. Visual observation of the surface of dried cells of Nostoc sp. HK-01

Pigments that absorb photons are crucial for photosynthesis (Blankenship, 2014). The role of chlorophyll in oxygenic photosynthetic organisms is to transfer light energy to photosystems (Kumar et al., 2014; Batista-Silva et al., 2020). The green color of the cells in cyanobacteria indicates the presence of chlorophyll a (Yasuda et al., 2019). The cells on the window side of the experimental units appeared brown in color for all 3 years of the experiment (Fig. 4). There is a possibility that the pigments in the thin layer of dried cells on the window side were degenerated by sunlight. One of the reasons for a change in color from green to brown is the decomposition of the remaining chlorophyll a in the vegetative cells. The absorption spectrum of chlorophyll shifts to the long-wavelength side during the decomposition of chlorophyll a (Yasuda et al., 2019). However, confirmation of this hypothesis will require detailed analyses of the specific changes in the cell components including a comparison to dark control experiments exposed to the vacuum in space and on the ground. Furthermore, it would be necessary to analyze the other pigments related to photosynthesis in the cells in addition to chlorophyll during such long exposure experiments.

4.2. Cell viability assay using the FDA staining test and cell growth test

Fluorescein diacetate (FDA) staining is often used to identify living cells by measuring esterase activity in cells. The exposed cells were tested by hydration in the laboratory after they were returned to the ground. In the FDA staining test, the survival rate in the third-year cells, even for the ground control cells, was decreased; but this strain, Nostoc sp. HK-01, can survive on martian regolith simulant for over 8 years (Kimura et al., 2015). Recently, we confirmed the survival rate using the cells stored in our laboratory for 8 years, which was around 30% (detailed data not shown here). The decreased survival rate is often affected by subtle environmental conditions that we were not aware of during storage. This may be related to humidity, although cells were stored with a desiccant, which will need to be examined in the future. Therefore, in this study, the survival cell rate of this space experiment was compared to that of the ground control for each year.
In the first year, the results of this experiment seemed reasonable in that the surviving cell rate in the dark control of the ISS sample was higher than that of samples kept in the dark though exposed to space, presumably because the environment inside the ISS was more benign than that outside in the vacuum of space. However, such a hypothesis would predict that the cell survival rate for cells kept in the dark on the ground would be higher than for the cells kept in the dark on the ISS in space. The observed phenomena, that is, that the survival cell rate in the ISS was higher than that in the ground control, might be related to the radiation hormesis effect (Shibamoto and Nakamura, 2018). The proposed effect should be clarified by ground-based experiments with this organism. We have previously reported similar results in helium-ion beam exposure experiments (Tomita-Yokotani et al., 2020). Interestingly, the surviving cell rate in the third year in the ISS dark control was lower than that for the cells kept in the dark in space (Fig. 5). These results suggest that the conditions for the tested cells in the dark in space were more benign than those in the ISS. The humidity of the ISS pressurized area was 45–55% based on data from Kawaguchi et al. (2020), while there is no humidity on the exterior of the ISS. The environment in vacuum may be more advantageous than the environment with air to protect the samples from oxidation. Similar results were observed with the samples in the EXPOSE-R2 mission experiment when using a Mars-like atmosphere, and it was suggested that the samples would often sustain more damage in air than in vacuum (Billi et al., 2019).
For the cells exposed to space on the window side of the experimental unit, cell survivors after 1 and 2 years were not observed; however, a few surviving cells were observed after the third year by microscopic observation and a cell staining test. According to the cell profile thickness measurements, the average layer thickness of cells that remained on the ground was 25–35 μm, and there were few areas where the thickness was approximately over 100 μm (Fig. 8). It is possible that an area in which the cell thickness was over 100 μm occurred, although this would have been due to technical problems during the drying of the cells. This may be the reason why some surviving cells were observed on the window side of the third year: the upper cells may have protected lower cells in the exposed layers from the UV or harsh environments due to the thickness of the cell layer and protective functional substances. Compatible components, such as sucrose, glycine, betaine, and glucosylglycerol, may contribute to protection from UV and high temperature (Kimura et al., 2017a, 2017b). We recommend future experiments on long-duration LEO flight missions that repeat our experiments as well as carry new experiments designed to test several of the hypotheses derived from the observations made in our experiments.
In the cell growth test, we confirmed that a statistically valid number of cells survived 3 years of exposure to space when kept in the dark (Fig. 7). In this experiment, though we could not quantify a statistically significant number of cells that survived exposed to LEO sunlight on the window side in the third year, we did observe that some cells survived. In general, organisms often depend on cell density, and there are cases where it is difficult to perform general incubation under cell low density (Pande et al., 2020). We will have to establish the evolutionary incubation method for which even a low density of cells, such as the two or three cells observed on the window side of the experimental units and flown in space, can survive. The evolutionary analysis of these species is currently in progress. In the re-incubation test, the survival rate in the third-year cells flown inside the ISS was recovered, which is an interesting phenomenon (Fig. 7B). There is a possibility that some cells affected by low radiation effects support cell germination in the early stages of the flight. However, some of these matters remain undetermined. The third-year cells were affected by some damage though living cells should have high recovery abilities.

4.3. Studies in space environments of Nostoc sp. HK-01 and future experiments

In this study, dried Nostoc sp. HK-01 was exposed to a space environment that is severe for most Earth-bound organisms (UNSCEAR, 1993; Goka et al., 2011). Though dried cells of this strain have a high tolerance to severe environments, such as the dark conditions in the LEO, several unknown phenomena still remain. Additional experiments to determine how these cells can survive in space environments such as outside the ISS are needed. We aim in future experiments to identify functional substances related to their viability functions. Kawaguchi et al. (2020) reported the exact number of required cell layers for protection of upper cell layers. We confirmed that a small number of cells did survive on the window sunlight-exposed side of the experimental unit after 3 years and suggest this was possible due to variation in the thickness of the cell layer. We will investigate this potential protective effect in the next space exposure experiment.
We also detected a low radiation effect on cyanobacterial germination. This strain has a distinctive life cycle that involves morphological and physiological differentiation (Fig. 1). Information gleaned about this and similar strains in the future will be relevant to the new results obtained in our space survival study and will advance this work, as well as future space-bound experimental studies of these cyanobacteria and other space-relevant organisms. Additional research focused on investigations into the role of functional substances that may enhance survival during extraterrestrial exposure, such as the role of compatible solutes in the protection of important proteins during cell drying (Kimura et al., 2017a, 2017b), will also advance our findings.
Chroococcidiopsis cells have also been exposed to simulated extraterrestrial environments like that of Mars, and such cells have been mixed with martian soil simulant (Billi et al., 2019). These exposure experiments demonstrate that these cells can survive when exposed to the atmospheric conditions on Mars, though such experimental environments cannot simulate all of the salient conditions on Mars.
Our future efforts to study the survival capability of this strain, Nostoc sp. HK-01, to extraterrestrial environments will also include a focus on some plant growth regulators, and we consider the present study a step forward in our study of the potential for crop production on Mars (Kimura et al., 2020).
Given that the ISS is currently flying in a low Earth orbit within the Van Allen belt, our results compel us to consider the potential for cyanobacteria to have survived travel in the past within LEO. Our findings indicate that Nostoc sp. HK-01 may persist for at least 3 years (1126 days) under such conditions, which is the longest time reported for a photosynthetic microorganism in a space exposure experiment.

Abbreviations Used

CINO
Cyanobacterial Inception & Operation
EP
exposure panel
FDA
fluorescein diacetate
ISS
International Space Station
JEM
Japanese Experiment Module
LEO
low Earth orbit

Acknowledgments

The authors thank Professor Akihiko Yamagishi, PI of the Tanpopo mission, and all members of the Tanpopo mission team for their helpful suggestions. We also thank all the staff related to our research for their cooperation at JAXA and NASA. We also thank the members for their cooperation at the Research Facility Center for Science and Technology at the University of Tsukuba. We deeply thank the reviewers and editors of Astrobiology for their contributions to strengthen the reporting of these Tanpopo mission results.
This work was supported by the Astrobiology Center Program of the National Institutes of Natural Sciences (NINS) (AB282002, AB292002, AB302005, AB312006, and AB022002).

Declaration of Interests

The authors declare no competing interests.

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Associate Editor: Petra Rettberg

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cover image Astrobiology
Astrobiology
Volume 21Issue Number 12December 2021
Pages: 1505 - 1514
PubMed: 34889664

History

Published online: 9 December 2021
Published in print: December 2021
Accepted: 11 November 2021
Received: 15 September 2021

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Kaori Tomita-Yokotani [email protected]
Faculty of Life and Environmental Sciences, University of Tsukuba, Tsukuba, Ibaraki, Japan.
Shunta Kimura
Faculty of Life and Environmental Sciences, University of Tsukuba, Tsukuba, Ibaraki, Japan.
Present address: ISAS/JAXA, Sagamihara, Kanagawa, Japan.
Midori Ong
Graduate School of Life and Environmental Sciences, University of Tsukuba, Tsukuba, Ibaraki, Japan.
Miku Tokita
Graduate School of Life and Environmental Sciences, University of Tsukuba, Tsukuba, Ibaraki, Japan.
Hiroshi Katoh
Division of Plant Functional Genomics, Advanced Science Research Promotion Center, Organization for the Promotion of Regional Innovation, Mie University, Tsu, Mie, Japan.
Tomoko Abe
School of Science and Engineering, Tokyo Denki University, Ishizaka, Hatoyama, Hiki-gun, Saitama, Japan.
Hirofumi Hashimoto
Institute of Space and Astronautical Sciences, Japan Aerospace Exploration Agency (ISAS/JAXA), Sagamihara, Kanagawa, Japan.
Kintake Sonoike
Faculty of Education and Integrated Arts and Sciences, Waseda University, Shinjuku-ku, Tokyo, Japan.
Masayuki Ohmori
The University of Tokyo, Komaba, Graduate School of Arts and Sciences, Meguro-ku, Tokyo, Japan.

Notes

Address correspondence to: Kaori Tomita-Yokotani, Faculty of Life and Environmental Sciences, University of Tsukuba, Tsukuba, Ibaraki 305-8572, Japan [email protected]

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