Reactivation of Microbial Strains and Synthetic Communities After a Spaceflight to the International Space Station: Corroborating the Feasibility of Essential Conversions in the MELiSSA Loop

Abstract

To sustain human deep space exploration or extra-terrestrial settlements where no resupply from the Earth or other planets is possible, technologies for in situ food production, water, air, and waste recovery need to be developed. The Micro-Ecological Life Support System Alternative (MELiSSA) is such a Regenerative Life Support System (RLSS) and it builds on several bacterial bioprocesses. However, alterations in gravity, temperature, and radiation associated with the space environment can affect survival and functionality of the microorganisms. In this study, representative strains of different carbon and nitrogen metabolisms with application in the MELiSSA were selected for launch and Low Earth Orbit (LEO) exposure. An edible photoautotrophic strain (Arthrospira sp. PCC 8005), a photoheterotrophic strain (Rhodospirillum rubrum S1H), a ureolytic heterotrophic strain (Cupriavidus pinatubonensis 1245), and combinations of C. pinatubonensis 1245 and autotrophic ammonia and nitrite oxidizing strains (Nitrosomonas europaea ATCC19718, Nitrosomonas ureae Nm10, and Nitrobacter winogradskyi Nb255) were sent to the International Space Station (ISS) for 7 days. There, the samples were exposed to 2.8 mGy, a dose 140 times higher than on the Earth, and a temperature of 22°C ± 1°C. On return to the Earth, the cultures were reactivated and their growth and activity were compared with terrestrial controls stored under refrigerated (5°C ± 2°C) or room temperature (22°C ± 1°C and 21°C ± 0°C) conditions. Overall, no difference was observed between terrestrial and ISS samples. Most cultures presented lower cell viability after the test, regardless of the type of exposure, indicating a harsher effect of the storage and sample preparation than the spaceflight itself. Postmission analysis revealed the successful survival and proliferation of all cultures except for Arthrospira, which suffered from the premission depressurization test. These observations validate the possibility of launching, storing, and reactivating bacteria with essential functionalities for microbial bioprocesses in RLSS.

1. Introduction

Human space exploration missions of long or permanent duration require autonomy from terrestrial resupply. Hence, extensive regeneration of consumables and waste to provide food, water, and breathable air is necessary (Barta and Henninger, 1994). A Regenerative Life Support System (RLSS) enables recycling the waste in a simplified ecosystem that can incorporate physicochemical and biological processes. The use of microbial bioprocesses has specific advantages that can improve the self-sufficiency of the system and allow a higher diversity of sustainable conversions, due to the diversification in the microbial metabolism (National Research Council, 1997). The conversion of organic compounds into CO₂ can be performed aerobically or anaerobically by heterotrophic bacteria, and it can find use in the treatment of carbon-enriched waste streams, such as fecal material, food waste, and urine (Wheeler, 2003). Autotrophic bacteria can be utilized for the fixation of CO₂ into edible biomass or the oxidation of inorganic elements, such as ammonium into nitrite and nitrate. Phototrophic bacteria utilize light as an energy source for biomass production that is rich in proteins and valuable nutrients. Their presence in an RLSS can provide dietary supplement for the astronauts (Hendrickx et al., 2006; Hendrickx and Mergeay, 2007; Escobar and Nabity, 2017).

One example of microbial utilization in an RLSS is the Micro-Ecological Life Support System Alternative (MELiSSA) designed for the European Space Agency (Hendrickx et al., 2006). At first, anaerobic thermophilic conversion of organic waste into volatile fatty acids, hydrogen gas, CO₂, and ammonium (NH₄ ⁺) is performed in the first compartment (CI) of the MELiSSA loop (Lissens et al., 2004). The removal of the soluble organic compounds from the filtrate of CI is carried out in the compartment CII via anoxygenic photosynthesis by the edible purple nonsulfur bacterium Rhodospirillum rubrum S1H (Mastroleo et al., 2009a). The conversion of ammonium into nitrate, which is the preferred form of nitrogen for the hydroponic plant compartment (CIVb), takes place in the nitrification compartment (CIII) with autotrophic nitrifiers such as, for example, Nitrosomonas europaea ATCC19718 and Nitrobacter winogradskyi Nb255 (Clauwaert et al., 2017). With a richer community, urine can be treated in CIII (De Paepe et al., 2018). In the case of composing the community synthetically, a specific ureolytic autotroph (e.g., Nitrosomonas ureae Nm10) to convert the urea into ammonium or heterotroph (e.g., Cupriavidus pinatubonensis 1245) to convert the urea into ammonium and to oxidize organic compounds in urine will be necessary. The production of edible biomass and O₂ is obtained in compartment CIVa with the photosynthetic cyanobacterium Arthrospira sp. PCC8005 and in CIVb, the plant compartment (Olsson-Francis and Cockell, 2010).

The ultimate goal of RLSS technologies is its utilization in space. This will expose the microorganisms to an extreme environment in which altered gravity, possibly temperature fluctuations, and higher levels of radiation can significantly affect microorganisms (Horneck et al., 2010). However, the effect of space on microorganisms that are suitable for RLSS conversion has been limited to some functionalities, tested at different conditions. Several phototrophs were tested for outer space conditions, and both algae (Chlorella and Rosenvingiella) and cyanobacteria (Gloeocapsa) survived 548 days outside the International Space Station (ISS) (Cockell et al., 2011). The CIVb Arthrospira sp. PCC8005 has not yet been included in the spaceflights experiment. The CII R. rubrum grown on agar plates was sent twice to the ISS, which demonstrated its survival and indicated that radiation has a higher impact than microgravity (Mastroleo et al., 2009b). The ureolytic heterotroph C. pinatubonensis and key nitrogen cycle bacteria also survived a 44-day space flight on board of the unmanned satellite FOTON M-5 (Lindeboom et al., 2018). N. urea and its synthetic communities have never been exposed to Low Earth Orbit (LEO) conditions.

The aim of this study is to elucidate the reactivation of metabolically diverse microorganisms for potential use in the MELiSSA Life Support System, after exposure to the same LEO environment, launch, and storage conditions.

An edible autotrophically grown cyanobacteria (Arthrospira sp. PCC 8005), a heterotrophically grown purple nonsulfur bacterium (R. rubrum S1H), a ureolytic heterotrophic strain (C. pinatubonensis 1245), and combinations of C. pinatubonensis 1245 and autotrophic ammonia and nitrite oxidizing strains (N. europaea ATCC19718, N. ureae Nm10, N. winogradskyi Nb255) were selected. The cultures were sent to the ISS for 7 days, where they were exposed to 2.8 mGy, a dose 140 times higher than on the Earth at an average temperature of 22°C ± 1°C. To maximize the resolution of the experiment and identify the effect of LEO exposure, the transfer times between the laboratories at the launch base were optimized compared with previous experiments, which presented a storage and travel time amounting to 2/3 of the total experimental time (Lindeboom et al., 2018), and control cultures were also transferred to the launch site.

On return on the Earth, the culture's reactivation was tested for cell viability (cell number or ATP quantification) and ability to proliferate (growth or activity preservation). LEO-exposed cultures were compared with terrestrial control cultures, exposed to 0.02 mGy background radiation dose and various test temperatures, namely refrigerated controls (5°C ± 2°C); controls were stored at room temperature (21°C ± 0°C); and spare samples were preserved at the launch site in Baikonur (22°C ± 1°C). Prestorage values were also compared with the poststorage, elucidating for the first time the role of storage over LEO exposure.

2. Materials and Methods

2.1. Sample preparations and hardware

Overview of the cultures origin and cultivation is presented in section S1 of Supplementary Data. All cultures were prepared at Belgian Nuclear Research Center (SCK•CEN) on August 25, 2015. The nitrifying cultures were prepared by mixing equal volumes of each strain. The culture of C. pinatubonensis intended for the nitrifying cultures was pelleted and resuspended in PBS beforehand. An overview of the cultures is presented in Table 1, whereas the final concentration of the nitrogen species present in the media is reported in Supplementary Fig. S1. Aliquots of the cultures were then divided into quadruplicates in polypropylene cryotubes, with a silicone washer seal and an internal thread (VWR International, Radnor). Each cryotube contained 5 mL of liquid culture. The cryotubes were then stored in the dark in a 1.5 L polypropylene plastic Biojar (Air Sea Containers, Birkenhead, United Kingdom) (Fig. 1a). The lid and the bottom of the Biojar were equipped with 1 track-etch detector each (Harzlas TD-1; Fukuvi Chemical Industry CO. Ltd., Fukui-city, Japan) (Fig. 1b), and 32 cryotube lids were equipped with 144 thermoluminescent detectors (MTS-6, MTS-7, MCP-6, and MCP-7; Radcard, Kraków, Poland) (Fig. 1c). The track-etch and thermoluminescent detectors were provided and analyzed, respectively, by the radiation dosimetry departments of the Nuclear Physics Institute of the Czech Academy of Sciences in Prague (Czech Republic) and SCK•CEN. In addition, stand-alone temperature loggers (iButton; HQMatics, Dokkum, the Netherlands) were added to the payload to monitor the temperature over the full mission.

FIG. 1.

Pictures of (a) the Biojar, (b) iButton temperature loggers inside the container, and (c) cryotubes containing the samples. The blue cap indicates the presence of thermoluminescent detectors.

Table 1.

Overview of the Cultures, the Nitrogen-Related Metabolic Activities, and OD Recorded on Sample Preparation

Culture	Code	Phototrophic production of edible biomass		Production of nitrate in urine (CIII)			OD ISS	OD G
Culture	Code	Autotrophic/oxygenic photosynthesis (CIVa)	Heterotrophic/anoxygenic photosynthesis (CII)	Ureolysis	Ammonia oxidation	Nitrite oxidation	OD ISS	OD G
Arthrospira sp. PCC8005	A	X	—	—	—	—	1.06	1.09
Rhodospirillum rubrum S1H	R	—	X	—	—	—	0.92	0.92
Cupriavidus pinatubonensis strain “1245”	C	—	—	X	—	—	1.85	1.85
Nitrosomonas europaea strain ‘Winogradsky (ATCC19718)^a	E	—	—	—	X	—	0.10	0.10
Nitrosomonas ureae strain Nm10^a	U	—	—	X	X	—	0.05	0.05
Nitrobacter winogradskyi strain “Nb255”	W	—	—	—	—	X	0.07	0.14
N. europaea+N. winogradskyi ^a	EW	—	—	—	X	X	0.07	0.07
C. pinatubonensis ^a +N. europaea+N. winogradskyi+	CEW	—	—	X	X	X	0.14	0.14
N. ureae+N. winogradskyi ^a	UW	—	—	X	X	X	0.05	0.05
C. pinatubonensis ^a +N. ureae+N. winogradskyi+	CUW	—	—	X	X	X	0.10	0.10

Column headers refer to the relevant MELiSSA compartment number (CII, CIII, and CIVa) for every conversion.

Contamination was observed at the end of the tests in the cultures.

ISS, International Space Station; MELiSSA, Micro-Ecological Life Support System Alternative; OD, Optical Density.

To ensure the tightness of the cryotubes, the samples intended for LEO exposure (ISS) and spare samples preserved at the launch site in Baikonur (G) were subjected to a depressurization test on the day of their preparation (August 25, 2015), as reported in section S1.2 of Supplementary Data (see https://www.liebertpub.com/doi/suppl/10.1089/ast.2018.1973).

2.2. LEO flight and ground storage conditions

The samples were hand-carried in phase change material boxes (series 22 PCM) to RSC-Energia (Moscow, Russia) on August 27, 2015, and they were subsequently sent to the launch site (Baikonour, Kazakhstan). Here, samples destined for the ISS were placed inside the capsule and launched on September 2, 2015, whereas the controls for terrestrial refrigerated (G-5) and room (G-21) temperature exposure were sent back from Baikonur to SCK•CEN, where they arrived on September 4, 2015. Spare samples (G) were preserved at the launch site and returned together with the ISS samples from September 2, 2015 to September 12, 2015. The ISS samples were launched on September 2 and installed in the Columbus module of the ISS on September 5. The ISS samples were discharged on September 11 and landed back on the Earth on September 12. Total ISS permanence was 9.84 days. Sample analysis was performed 1 day after the recovery of ISS samples in the lab, on September 13, 2015. An overview of the temperature and radiation profile is presented in Fig. 2.

FIG. 2.

Temperature profile (°C) from the iButton (a) and radiation absorbed dose in water measured with MTS-6, MTS-7, MCP-6, and MCP-7 detectors for the ISS and G samples (b). ISS, International Space Station.

The ground control G (22°C ± 1°C) was selected as the reference for the nitrification tests of the ISS samples (22°C ± 1°C), unless otherwise mentioned. The terrestrial storages in a temperature-controlled environment experienced a refrigerated temperature of 5°C ± 2°C (G-5) and a room temperature of 21°C ± 0°C (G-21) (Fig. 2a).

The thermoluminescent detectors showed that, during the 9.84 days mission, the ISS samples were exposed to a total radiation absorbed dose in water of 2.4 mGy (Fig. 2b). Next, 0.4 mGy was added to the overall dose, due to the radiation with high ionization density (heavy nuclei and high-energy neutrons) measured by the track-etch detectors (Fig. 2c). Hence, the total absorbed dose in water received by the ISS samples is 2.8 mGy, more or less 140 times higher than the 0.02 mGy measured for the terrestrial controls G, G-5, and G-21.

2.3. Postmission characterization and reactivation

On retrieval, the nitrogen composition of the storage media and the purity of the nitrifying cultures were assessed as described in sections S1.3 and S1.4 of Supplementary Data. Despite the culture preparation in sterile conditions, the development of heterotrophic populations was observed for all the nitrifying cultures, but not for the Arthrospira sp. and R. rubrum and C. pinatubonensis cultures. An overview of the operational taxonomic units (OTUs) detected is presented in Supplementary Fig. S2 and Supplementary Table S1.

Postmission cultures were characterized evaluating the cell viability and ability to proliferate.

Arthrospira sp. and R. rubrum and C. pinatubonensis's cell viability was assessed with flow cytometric photosynthetic potential evaluation (FL3/FL4 ratio), LIVE/DEAD ratio, and plate count quantification, respectively. Their ability to proliferate was checked monitoring the growth after subculture in liquid medium. Pulse Amplitude Modulated analysis (PAM) was additionally performed on Arthrospira sp.'s cultures. Detailed materials and methods are presented in section S1.5 of Supplementary Data.

Due to the slow growth of autotrophic bacteria, cell viability for the nitrifying cultures was assessed measuring intracellular ATP (adenosine triphosphate). The ability to proliferate was checked evaluating the presence of nitrifying activity, monitoring over time the concentration of ammonia, nitrite, and nitrate. An overview of the materials and methods utilized is described in section S1.6 of Supplementary Data.

3. Results

3.1. Photoautotrophic stain Arthrospira sp. PCC 8005 was irreversibly affected by a preflight depressurization test

The pH of Arthrospira sp. media before the test was 10.3. Poststorage pH measurements on ISS, G, G-5, and G-21 revealed a similar endpoint pH, on average 10.2 ± 0.0.

Flow cytometry count on the Arthrospira sp. mother culture gave an FL3/FL4 ratio around 0.5, indicating a healthy physiological state of the cells before the experiment. A similar ratio was observed in the G-5 samples. G-21 showed a ratio of 1, sign of a partially impaired photosynthetic system, whereas ISS and G presented a ratio higher than 3, indicating a fully damaged system (Fig. 3a). Pulse Amplitude Modulated analysis (PAM) measurements of the photosynthetic efficiency of G-5 and G-21 showed Fv/Fm values close to 0.6, namely 0.521, indicating a healthy culture. Fv/Fm values could not be determined for ISS and G samples. Arthrospira sp. samples ISS and G could not resume growth, whereas G-21 and G-5 reached an OD₇₅₀ after 15 days of cultivation (Fig. 3b).

FIG. 3.

Overview of the cell viability for PM, LEO-exposed samples (ISS), spare terrestrial samples preserved at the launch site in Baikonur (G), terrestrial refrigerated samples (G-5), and samples stored at room temperature (G-21), evaluated with FL3/FL4 ratio (a) and growth curve (b) for Arthrospira sp. LIVE/DEAD cell count (c) and growth curve (d) for Rhodospirillum rubrum and CFU counts (e) and growth curves (f) for Cupriavidus pinatubonensis. LEO, Low Earth Orbit; PM, premission,

Since Arthrospira sp. PCC8005 is highly resistant to ionizing radiation (Badri et al., 2015), it is suspected that the effects observed on the flight and ground control samples are not related to the space experiment but rather to preflight sample preparation. Indeed, the samples that did not resume the growth (ISS and G) were the ones subjected to the depressurization test. Hence, the same treatment was performed on freshly grown cultures of Arthrospira sp. Their ability to propagate was further evaluated.

Before the depressurization procedure, the pH of the culture (split in 20 tubes) was set at 10.3. Immediately after the depressurization, the pH increased to 11.5 and then stabilized to pH 10.6 after about 1 min, when reaching the equilibrium with ambient air. After 20 days of storage, the estimated theoretical time between sample preparation on the Earth and activating experiment on ISS, of these samples in the dark, the pH was 10.4 and the OD₇₅₀ was 0.984. The subculture of the samples after 20 days yielded an OD₇₅₀ of 0.294 at a pH of 9.9 after 4 weeks of cultivation, indicating a lack of growth.

3.1.1. Rhodospirillum rubrum

The pH measurements of R. rubrum after the storage revealed a mild acidification of the media in all the samples, with no difference among conditions (ISS, G, G-5, G-21), from a pH of 7.8 to an average value of 7.0 ± 0.1. The mother culture of R. rubrum presented a fraction of live cells of ca. 20%. After the storage, a value 10 times lower was observed in all the samples (Fig. 3c).

No difference among samples (ISS, G, G-5, G-21) was observed during growth, and all the cultures reached stationary phase after 3 days (Fig. 3d).

3.1.2. Cupriavidus pinatubonensis

The storage medium of C. pinatubonensis was adjusted on sample preparation to a pH of 6.8. After the test, no variation was detected among the ISS, G, and G-21 cultures, which presented an average pH of 7.0 ± 0.1, 6.9 ± 0.0, and 6.9 ± 0.0, respectively. A mild acidification to a pH of 6.7 ± 0.1 was observed in the G-5 cultures.

C. pinatubonensis's viable cell number was similar among samples, with a logCFU mL⁻¹ of 9, irrespective of the treatment (Fig. 3e). The Illumina analysis confirmed that for both the ISS and the G sample, no other strains were present (Supplementary Fig. S2). The growth observed after the storage was similar among ISS, G, G-21, and G-5, as all samples reached the stationary phase 1 day after the subculture (Fig. 3f).

3.2. Ureolytic communities

Even though the reactivation and heterotrophic of C. pinatubonensis (C) was demonstrated in the previous section, its ureolytic activity was also evaluated, for comparison with the cultures including the autotrophic ureolytic strain N. ureae (U). An overview of the rates is presented in Supplementary Table S2. Since urease is a widely distributed enzyme, the heterotrophic contaminant identified in the samples U, CEW, and CUW could possibly contribute to ureolysis. Nonetheless, C showed the highest activity (1.77 ± 0.06 and 1.72 ± 0.04 mg-N L⁻¹ h⁻¹ for ISS and G, respectively) and the ureolytic rate in samples U and the ureolytic rate in samples U, where no Cupriavidus was detected, was on average 7–11 times lower than C for ISS and G, respectively. C showed the highest activity (42.48 ± 1.44 and 41.28 ± 0.96 mg-N L⁻¹ d⁻¹ for ISS and G, respectively), and the ureolytic rate in samples U was on average 7–11 times lower than C for ISS and G, respectively. Ureolysis was observed also in all the samples where C. pinatubonensis was mixed with other bacteria (Fig. 4a).

FIG. 4.

Overview of the ureolysis (a), ammonia oxidation (b), and nitrite oxidation (c) rates as mg-N L⁻¹ d⁻¹ for the PM, LEO-exposed samples (ISS), spare terrestrial samples preserved at the launch site in Baikonur (G), terrestrial refrigerated samples (G-5), and samples stored at room temperature (G-21). The rates of the cultures not present in the graphs were not determined. The cultures marked with “*” present a significant difference between ISS and G samples.

The combinations CEW and CUW, where the relative abundance of C. pinatubonensis was between 50–55% and 61–63%, according to the Illumina results (Supplementary Fig. S2), showed G and ISS ureolytic rates of 547.2 ± 115.2 and 417.6 ± 52.8 mg-N L⁻¹ d⁻¹ for CEW, and of 168.0 ± 24.0 and 273.6 ± 91.2 mg-N L⁻¹ d⁻¹ for CUW. The presence of N. ureae in the latter community did not seem to have a beneficial effect on the total ureolysis.

Overall, the comparison between ISS and G samples did not show a significant impact of LEO conditions in any of the cultures tested (p > 0.05).

3.3. Nitrifying communities

An overview of the pH and nitrogen species after the test in the ISS and G nitrifying cultures is presented in Supplementary Fig. S1. A general acidification of the medium from the initial pH of 7.5 to 6.7–5.8 was observed in all the cultures containing ammonia oxidizing bacteria (AOB). The samples with N. europaea (E) presented the lowest average pH (5.8 ± 0.3), which could indicate active ammonia oxidation during the transportation and on flight.

An overview of the ATP concentration and equivalent active cells (EAC), assuming 10⁻¹⁸ mol ATP equals 1 EAC, is presented in Supplementary Fig. S3. Absolute ATP measurements showed that all the mother cultures presented EAC concentration in the range between 10⁷ and 10⁸ cells mL⁻¹. Despite the proliferation of heterotrophic contaminant, the comparison of the EAC value measured for the mother cultures shows a decrease in EAC after the experiment in all the cultures, except for EW. The comparison between ISS and G revealed mostly equal EAC levels, with the exception of E, whose ISS samples displayed 1.7 times higher ATP concentration. Comparisons of the samples stored at different temperature (G-5 and G-21) revealed mostly similar ATP levels. Samples that present higher EAC levels at G-5 are W and EW with 1.6 and 2 times higher values in G-5, whereas higher ATP levels in the G-21 cultures were observed only for CEW.

The ammonia and nitrite oxidation rates are presented in Supplementary Table S2. The OTUs table presented in Supplementary Fig. S1 shows that the only AOB present belongs to the Nitrosomonas genus, suggesting no contamination of ancillary AOB. The cultures in which ammonia oxidation is performed by N. europaea displayed a significantly lower activity (p < 0.05) in G samples compared with ISS and high variability among replicates during the activity test. Hence, the back-up cultures stored under refrigerated (G-5) and temperature-controlled (G-21) conditions were also analyzed for E, EW, and CEW. These cultures could all be reactivated in the first week of the test. Overall, the ammonia oxidation activity of the cultures with N. europaea as key AOB displayed no significant difference between ISS, G-5, and G-21 (p > 0.05) (Fig. 4b). Compared with the mother culture, E showed a five-fold decrease in activity, whereas EWC and EW presented comparable rates. This indicates that neither the relative abundance of the AOB nor the presence of heterotrophic contaminant is responsible for the decrease in activity of the sample E compared with the mother culture.

The cultures with N. ureae had a more stable response to reactivation among replicates already during the first ammonia oxidation activity test. The rates achieved by U and CUW were comparable to the ones of the mother cultures. Similar to the cultures with N. europaea, the ISS and G rates were not affected by the storage conditions for N. ureae.

Nitrite oxidation after the ISS and ground storage could be observed in all cultures tested (Fig. 4c). All the sequences associated with nitrite oxidizing bacteria (NOB) belonged to Bradyrhizobiaceae, suggesting no contamination of ancillary NOB (Supplementary Fig. S1). The consortium W, which did not present any AOB, showed the highest activity loss compared with the mother culture, showing six and three times lower rates than ISS and G, respectively. W represented also the only culture in which the G rates were two times higher than the ISS rates (p < 0.05). The samples that presented Nitrosomonas sp., instead, did not display any significant difference among ISS and G cultures (p > 0.05).

4. Discussion

4.1. Storage exposed the samples to stable conditions

Results from the characterization of the storage conditions reveal that the samples were exposed to very stable conditions throughout the experiment and especially during the flight itself (Fig. 1), which is not always feasible during LEO flight experiments (Cockell et al., 2011; Byloos et al., 2017; Lindeboom et al., 2018). The higher standard deviation measured for the ISS samples originates from the lower temperature experienced during the launch and landing activities, whereas the one registered for the G-5 samples was caused by the 5 h needed to cool down from room temperature, as shown in Fig. 1a. Hence, the results observed were not caused by thermic fluctuations. This allows the analysis of the storage and radiation independently from the effect of temperature.

Overall, the radiation experienced by the cultures showed no significant difference in dose between the different tube positions. All samples were exposed to a higher dose than the terrestrially preserved controls (ca. 140 times), which could have impacted the performance of the ISS samples. A systematic difference between the different detector types, associated with the different sensitivity toward radiation with high ionization density (MCP detectors are commonly less sensitive than MTS), low energy neutrons (measured only by the six-type detector), or heavy ions and high energy neutrons (measured by the track-etch detectors), was also observed. This expected variability indicates that the use of the different detector types can provide a better insight in the quality of the radiation field.

4.2. Photoautotrophic strain Arthrospira sp. PCC 8005 was irreversibly affected by a preflight depressurization test

This mission represents the first spaceflight for Arthrospira sp. PCC8005, although it was already exposed to high doses of ionizing radiations (i.e., more than 6000 Gy) in lab-scale simulations, without significant consequences (Badri et al., 2015). The disruption of the photosynthetic system and the inability to propagate after the mission was observed in both the ISS and G samples. This is, therefore, not a result of the higher radiation dose experienced, but rather of the preflight assembly procedures. This hypothesis finds confirmation in the results of the follow-up depressurization test, which had a dramatic impact on the growth of terrestrially preserved Arthrospira sp. The off-gassing of CO₂ probably induced a harmful or deadly increase in pH, from which the culture was not able to recover. The results obtained highlight the need to transition from standardized sample preparation procedures, which do not take into account the microbial response, toward individualized procedures based on the type of microorganism. In addition, the media selected can promote either microbial growth (if substrate is available) or inhibition (generated by pH fluctuations), further indicating the need for case-specific choices.

4.3. R. rubrum resumed growth after storage despite postmission biomass deterioration

R. rubrum showed a 10-fold decrease in live cells in all the cultures after 13 days of storage. A mild acidification of the media was also observed after the experiment, possibly contributing to the poor culture health observed for R. rubrum. A longer storage time will be required for cultures intended for distant human outposts or as back-up, which can possibly affect permanently their ability to recover. The development of optimized storage conditions and media (i.e., higher buffer capacity) will be crucial for real-life application. The utilization of different techniques, such as freezing with proper cryoprotectants, will allow the preservation of heterotrophic strains for a longer period (Kerckhof et al., 2014). Nonetheless, it could grow after sub-culture in fresh media and reached a steady state after few days of cultivation. This was the third spaceflight for MELiSSA CII compartment bacterium, R. rubrum, after MESSAGE-2 and BASE-A spaceflight experiments, but the first including R. rubrum in liquid culture, confirming its survival in different culture conditions (Mastroleo et al., 2009b).

4.4. C. pinatubonensis was not affected by LEO exposure

C. pinatubonensis proves to be unaffected by the experimental conditions tested. It displayed similar cell count as before the space mission (PM samples), was able to resume growth in fresh medium, and also showed the highest urea hydrolysis rate. This might indicate that, at least for some conversions, the use of C. pinatubonensis might be preferred due to its robustness. Nonetheless, conversion of nitrogen to undesired by-products should be carefully evaluated as heterotrophic strains might perform them with higher speed. This was, for example, observed evaluating the nitrate removal under anoxic conditions (Supplementary Fig. S4). In the culture of C. pinatubonensis, complete depletion of nitrate was observed within the 24 h of experiment in both ISS and ground controls, whereas all the other cultures still presented residual NO₃ ⁻ in the media.

Autotrophic nitrogen conversions were not affected by LEO exposure but showed biomass deterioration after storage.

The cultures associated with nitrogen metabolism mostly showed lower EAC values after the space mission and terrestrial storage, indicating a general decay of the biomass, despite the development of heterotrophic contaminations. Overall, the temperature (G-5 vs. G21) did not seem to influence the EAC values, whereas ISS samples yielded mostly similar EAC concentrations than G storage. This suggests that the presence of contaminants that most likely originated before the cultures were split for the different storage conditions, which seems to find confirmation in the similar sequencing profile of the G and ISS samples (Supplementary Fig. S2). Although autotrophic bacteria can release in the media small organic compounds (soluble microbial products), which can function as organic substrate for heterotrophic growth, the EAC results indicate that, in minimal medium without added organic substrates, the proliferation of heterotroph contaminants was limited and generally the biomass decayed. AOB and NOB were never outcompeted by the heterotrophic populations and were active in all the samples analyzed. This co-existence points toward the use of microbial synthetic communities combining multiple metabolisms. Nitrifying communities have been previously exposed to LEO conditions, and all metabolic functions could be recovered. However, the previously reported experimental setup foresaw longer storage and travel time amounting to 2/3 of the total experimental time, with 1 month passing from the landing of the sample to the activity tests (Lindeboom et al., 2018). Hence, the effect of long storage time and spaceflight could not be completely differentiated, as, for instance, AOB activity has been recovered after 5 months of terrestrial storage at 4°C and 20°C (Vlaeminck et al., 2007).

5. Conclusions

In conclusion, this study presents for the first time the results on key bacteria and synthetic communities for RLSS exposed to the same LEO environment. All cultures except for Arthrospira could be successfully prepared and exposed to LEO conditions, proving that they could be efficiently preserved and reactivated to an extent comparable to terrestrially stored cultures. Further, the results validate the storage in liquid medium for space mission.

The confirmation that microorganisms involved in RLSS can withstand space conditions for a mission lasting longer than the time required to reach the Moon eliminates one of the main uncertainties regarding its applicability in space, and it promotes the development of additional studies and technologies.

Footnotes

Acknowledgments

The authors gratefully thank (i) the Belgian Science Policy Office (BELSPO) for their support in the framework of the PRODEX program, (ii) MELiSSA (www.esa.int/Our_Activities/Space_Engineering_Technology/Melissa), ESA's life support system R&D program, for scientific and logistic support, and (iii) Tim Lacoere, for performing DNA extractions.

Author Disclosure Statement

No competing financial interests exist.

Supplementary Material

Supplementary Data

Supplementary Table S1

Supplementary Table S2

Supplementary Figure S1

Supplementary Figure S2

Supplementary Figure S3

Supplementary Figure S4

Associate Editor: Petra Rettberg

Abbreviations Used

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