The International Space Station Environment Triggers Molecular Responses in Aspergillus niger

Due to immense phenotypic plasticity and adaptability, Aspergillus niger is a cosmopolitan fungus that thrives in versatile environments, including the International Space Station (ISS). This is the first report of genomic, proteomic, and metabolomic alterations observed in A. niger strain JSC-093350089 grown in a controlled experiment aboard the ISS. Whole-genome sequencing (WGS) revealed that ISS conditions, including microgravity and enhanced irradiation, triggered non-synonymous point mutations in specific regions, chromosomes VIII and XII of the JSC-093350089 genome when compared to the ground-grown control. Proteome analysis showed altered abundance of proteins involved in carbohydrate metabolism, stress response, and cellular amino acid and protein catabolic processes following growth aboard the ISS. Metabolome analysis further confirmed that space conditions altered molecular suite of ISS-grown A. niger JSC-093350089. After regrowing both strains on Earth, production of antioxidant—Pyranonigrin A was significantly induced in the ISS-flown, but not the ground control strain. In summary, the microgravity and enhanced irradiation triggered unique molecular responses in the A. niger JSC-093350089 suggesting adaptive responses.

Filamentous fungi are producers of a myriad of bioactive compounds or secondary metabolites (SMs). These SMs often confer environmental advantage, which facilitate survival in hostile niches despite not being directly essential for survival (Keller et al., 2005;Fox and Howlett, 2008;Brakhage and Schroeckh, 2011;Rohlfs and Churchill, 2011;Brakhage, 2013). SMs span from potent bioactive molecules used in the drug discovery processes (Borel et al., 1995;Elander, 2003;Mulder et al., 2015) or other branches of the industry (Vandenberghe et al., 2000;Rodríguez Couto and Toca Herrera, 2006;Piscitelli et al., 2010;Dhillon et al., 2011) to health hazardous toxins (Barnes, 1970;Shephard, 2008;Hof and Kupfahl, 2009;Eaton and Groopman, 2013). Altered production of various SMs is one potential mechanism of fungal adaptation to extreme environments. For example, increased production of melanin, a pigment with UV protective properties, was observed in fungi isolated from Chernobyl nuclear power plant (Dadachova et al., 2008) and "Evolution Canyon" (Singaravelan et al., 2008). One such highly melanized fungal species is Aspergillus niger.
Industrially important A. niger (Schuster et al., 2002) has been isolated from various ecological niches, including decaying leaves (Nikolcheva et al., 2003), common households (Adams et al., 2013;Barberán et al., 2015), and the ISS (Checinska et al., 2015). The A. niger strain JSC-093350089 isolated from the surface of the US compartment of the ISS was previously characterized using multi-omics techniques. Performed analyses revealed genetic variance typical for the A. niger clade, increased abundance of proteins involved in starvation response, oxidative stress, and cell wall modulation (Romsdahl et al., 2018), and alteration in SM production levels when compared to wellstudied A. niger ATCC 1015 strain (Romsdahl et al., 2019). However, definite ascribing of observed molecular alterations to the ISS environment was not possible, since the strains were not grown in microgravity using a controlled experiment with ground counterparts. Nevertheless, in-depth characterization of ISS-isolated JSC-093350089 A. niger provided insight into potential space-induced molecular phenotypes.
This study is the first report of the multi-omics characterization of A. niger JSC-093350089 grown aboard the ISS and compared to ground controls. To study the impact of the enhanced irradiation and microgravity on JSC-093350089, the strain was transported to and grown aboard the ISS. Upon return to Earth, ISS-grown samples, along with ground controls, were immediately processed for metabolomic, proteomic, and genomic analyses with the aim of obtaining important insights into the adaptive responses of A. niger to space conditions. In addition, ISS-grown samples were regrown on Earth to identify any conserved molecular alterations.

Isolation and Identification of Aspergillus niger
Procedures to isolate and identify A. niger collected from the ISS were described previously (Romsdahl et al., 2018). In brief, sterile swabs soaked in saline solution were used to sample the ISS surface and transported to Earth. Particles retrieved from the swab were spread into potato dextrose agar (PDA) plates and any growing colonies were purified, collected, and further analyzed. One of the collected isolates was identified as A. niger via ITS region sequencing, which was subsequently confirmed via whole-genome sequencing (WGS).

Growth Conditions
JSC-093350089 was cultivated on glucose minimal medium (GMM) agar plates (6 g/l NaNO 3 , 0.52 g/l KCl, 0.52 g/l MgSO 4 ·7H 2 O, 1.52 g/l KH 2 PO 4 , 10 g/l D-glucose, and 15 g/l agar supplemented with 1 ml/l of Hutner's trace elements) covered with a cellophane membrane. Each of 10 prepared Petri plates (D = 10 cm) was inoculated with 1 × 10 7 conidia/ plate. Subsequently, plates were sealed with 3 M™ Micropore™ Surgical Tape (VWR International, Radnor, PA, United States) and placed in four Biological Research in Canister (BRIC) systems (three and two plates/BRIC). BRICs were divided into two groups, which were exact mimics, and transferred to 4°C. The exact timeline of the experiment is presented in Supplementary Figure 1. The whole experiment lasted 42 days from preparing the payload by a science team prior to launch till the handout of the ISS-grown samples back to the science team after the flight. The first group of BRICs was sent to the ISS and continuously kept at 4°C (1-20 days) prior to being transferred to ambient temperature for the active growth phase ~22°C for 12 days (21-32 days). After that time BRICs were stored at 4°C before returning to Earth (33-42 days). Upon arrival to Earth, BRICs were turned over to the science team for the downstream analyses, which commenced immediately. The second group of BRICs, treated as controls, was kept on Earth at Kennedy Space Center (KSC) and mimicked the ISS experiment timeline with roughly 2 h of delay. BRICs containing control samples were shipped from KSC to research team along with the ISS-grown samples. Lastly, for an additional secondary metabolite analysis, 1 × 10 7 conidia/plate of the ISS-and ground-grown JSC-093350089 were grown on GMM medium at 28°C for 5 days.
Fluorometer and used for paired-end library preparation with TruSeq Nano DNA Library Preparation Kit (Illumina, San Diego, California, United States) followed by WGS at the Duke Center for Genomic and Computational Biology. Samples were sequenced using a HiSeq 4,000 Illumina Sequencer generating 101 base long reads.

Protein Extraction
Mycelia and conidia from GMM agar plates were collected and stored at -80°C prior to protein extraction. Proteins were extracted with the lysis buffer consisting of 100 mm triethylammonium bicarbonate (TEAB) with 1:100 Halt Protease Inhibitor Cocktail (Thermo Scientific, Rockford, IL) and 200 μg/ ml phenylmethylsulfonyl fluoride (Sigma-Aldrich, St. Louis, MO, United States). Mycelia and conidia were homogenized by bead beating using Precellys 24 homogenizer (Bertin, Rockville, MD). The lysed fungal material was centrifuged at 17,000 g for 15 min and the protein concentration in the supernatants was measured by the Bradford assay (Bio-Rad Laboratories, Inc. Hercules, CA, United States).

LC-MS/MS Analysis
The samples were analyzed on an Orbitrap Fusion Tribrid mass spectrometer with an EASY-nLC 1,000 Liquid Chromatograph, a 75 μm × 2 cm Acclaim PepMap100 C18 trapping column, and a 75 μm × 25 cm PepMap RSLC C18 analytical column, and an Easy-Spray ion source (Thermo Scientific, Waltham, MA, United States). The peptides were eluted at 45°C with a flow rate of 300 nl/min over a 110 min gradient, from 3 to 30% solvent B (100 min), 30-50% solvent B (3 min), 50-90% solvent B (2 min), and 90% solvent B (2 min). The solvent A was 0.1% formic acid in water and the solvent B was 0.1% formic acid in acetonitrile.
The full MS survey scan (m/z 400-1,500) was acquired at a resolution of 120,000 and an automatic gain control (AGC) target of 2 × 10 5 in the Orbitrap with the 50 ms maximum injection time for MS scans. Monoisotopic precursor ions were selected with charge states 2-7, a ± 10 ppm mass window, and 70 s dynamic exclusion. The MS 2 scan (m/z 400-2000) was performed using the linear ion trap with the 35% collisioninduced dissociation (CID) energy. The ion trap scan rate was set to "rapid, " with an AGC target of 4 × 10 3 , and a 150 ms maximum injection time. Ten fragment ions from each MS 2 experiment were subsequently selected for an MS 3 experiment. The MS 3 scan (m/z 100-500) was performed to generate the TMT reporter ions in the linear ion trap using higher-energy collisional dissociation (HCD) at a 55% collision energy, a rapid scan rate and an AGC target of 5 × 10 3 , and a maximum injection time of 250 ms.

Proteome Data Analysis
All MS data (MS 1 , MS 2 , and MS 3 ) were searched using the Proteome Discoverer (version 2.2.0.388, Thermo Scientific) with the Sequest-HT searching engines against an Aspergillus niger CBS 513.88 database containing 10,549 sequences (NCBI). The searches were performed with the following parameters: 2 maximum missed cleavage sites, 6 minimum peptide length, 5 ppm tolerance for precursor ion masses, and 0.6 Dalton tolerance for fragment ion masses. The static modification settings included carbamidomethyl of cysteine residues, and Frontiers in Microbiology | www.frontiersin.org dynamic modifications included oxidation of methionine, TMT6plex modification of lysine ε-amino groups and peptide N-termini, and acetyl modification of protein N-terminus. A false discovery rate (FDR) of 1% was set using a target-decoy database search. The reporter ions integration tolerance was 0.5 Da and the co-isolation threshold was 75%. The average signal-to-noise threshold of all reporter peaks was bigger than 10. The total intensity of a reporter ion for a protein was calculated based on the sum of all detected reporter ions of associated peptides from that protein. The ratios between an abundance of a reporter and the average abundance of all reporters were used to estimate the abundance ratio of each protein.
For statistical analysis, the sum of reporter ion intensities for each protein was Log2 transformed and the biological duplicate and technical triplicate measurement for each protein was averaged. Only the proteins that were identified and quantified in both biological and technical replicates were used for the analysis. Student t-test was performed to identify proteins with changed abundance. Proteins with value of p <0.05 were further evaluated for increased and decreased abundance using a cut-off value of ≥ ± 1 fold (Log2) change.

Secondary Metabolite Extraction and Analysis
Upon return to Earth single plates from both ground-and ISS-kept BRICs were used to collect agar plugs in triplicate for secondary metabolite (SM) extraction. Plugs were extracted with 3 ml of methanol and 1:1 methanol/dichloromethane each followed by 1 h sonication. Crude extract was evaporated in vacuo to yield a residue that was then suspended in 1 ml of 20% dimethyl sulfoxide/methanol and 10 μl was examined by high-performance liquid chromatography-photodiode array detection-mass spectrometry (HPLC-DAD-MS) analysis. HPLC-MS was carried out using ThermoFinnigan LCQ Advantage ion trap mass spectrometer with an RP C18 column (Alltech Prevail C18 3 mm 2.1 × 100 mm) at a flow rate 125 μl/min. The solvent gradient for LC-MS was 95% acetonitrile/H2O (solvent B) in 5% acetonitrile/H2O (solvent A) both containing 0.05% formic acid, as follows: 0% solvent B from 0 min to 5 min, 0 to 100% solvent B from 5 min to 35 min, 100% solvent B from 35 min to 40 min, 100 to 0% solvent B from 40 min to 45 min, and re-equilibration with 0% solvent B from 45 min to 50 min. The SM profiles of ISS-and ground-grown JSC-093350089, which were regrown at 28°C were obtained following the procedure described above.

SM Statistical Analysis
To compare the yields of produced SMs in ISS-grown, groundgrown, and regrown samples, the area under the electrospray ionization curve (ESI) was integrated for each compound. SM data collected from three independent biological replicates of ISS-and ground-grown, and regrown JSC-093350089 were used for testing statistical significance of production yields of identified SMs by Welch's corrected t-test. The data are presented as column charts with corresponding error bars. Data analysis was conducted using GraphPad Prism version 7.

Genome Variation in the ISS-Grown JSC-093350089 Aspergillus niger
The genomes of ISS-flown and ground-grown JSC-093350089 were compared to identify occurring genetic variations. Obtained reads were aligned to the CBS 513.88 reference genome and single-nucleotide polymorphisms (SNPs) present in the ground control were filtered. This revealed presence of 375 SNPs and 620 INDELs that occurred because of the exposure to conditions aboard the ISS ( Table 1). All identified genetic variations are summarized in Supplementary Tables 1 and 2, presenting SNPs and INDELs, respectively. Distribution of non-synonymous point mutations among genes is presented in Table 2. Interestingly, about 80% of these mutations occurred in chromosome VIII and 13% occurred in chromosome XII, while the remaining 7% were distributed among other chromosomes (Supplementary Figure 2A). The majority of missense point mutations (75%) were observed within genes of unknown function. However, several characterized genes containing missense SNPs have DNA-binding activity (An06g01180, An08g11890, and An12g00840), DNA polymerase activity (An08g11520), protein kinase and transferase activity (An08g12110), phospholipase activity (An08g12250), and chromosome anchoring RacA protein binding activity (An12g06420 ; Table 2). Additional, mutations like one stop lost, one start gained and one 5 prime untranslated region (UTR) mutation were observed. Most of the observed SNPs (~55%) and INDELs (71%) were located in intergenic regions (Table 1). Interestingly, unlike SNPs, INDELs were distributed throughout all chromosomes. However, similarly to SNPs, the highest number of INDELS was found in chromosomes VIII and XII. Among observed INDELS 109 caused frameshift, 14 lead to disruptive inframe deletion, and a few caused start lost and stop gained (Supplementary Table 2  Proteomic Characterization of ISS-Grown JSC-093350089 Aspergillus niger Differentially expressed proteins in ISS-grown JSC-093350089 strain were investigated following the extraction of total protein from two biological replicates of ISS-grown and ground control counterpart strains. Due to the low yields of extracted proteins, biological replicates were combined and divided into two parts that were then TMT labeled and subtracted to analysis via LC-MS/MS followed by spectrum/sequence matching using A. niger CBS 513.88 protein database (NCBI). Protein abundance ratios in ISS-grown JSC-093350089 were normalized to Earthgrown counterparts, which enabled identification of 70 up-and 142 downregulated proteins (fold change (FC) > |2|, p < 0.05) in response to space conditions (Supplementary Tables 3 and  4, respectively). AspGD Gene Ontology (GO) Slim terms (Cerqueira et al., 2014) were used to study the distribution of differentially expressed proteins in ISS-grown JSC-093350089 when compared to ground controls (Figure 1). Among differentially expressed proteins, 29 were involved in carbohydrate metabolism, 18 in stress responses, 21 in amino acid metabolism, and 15 in protein catabolic processes (Figure 1). Protein GO term enrichment analysis was conducted using FungiDB (Stajich et al., 2012), which revealed that significantly over-represented upregulated biological processes included carbohydrate metabolic processes (28% of all upregulated proteins) and stress response (10%), whereas significantly over-represented downregulated processes included cellular amino acid metabolic processes (13%), proteasomal ubiquitin-independent (10%) and dependent processes (10%), and proteasomal protein catabolic processes (10%; Supplementary Table 5).

Metabolomic Characterization of ISS-Grown JSC-093350089 Aspergillus niger
Alterations in SM production in response to ISS conditions were assessed by extracting organic compounds from three biological replicates of ISS-and ground-grown JSC-093350089. The extracts were analyzed using HPLC-DAD-MS. Compounds were identified based on mass, UV absorption, and retention time, which were in agreement with literature (Chiang et al., 2011; Figure 2). Assessment of SM production yields revealed slight decrease in production of bicoumanigrin A, fonsecinones B and C, aurasperones C and B, and kotanin in ISS-grown isolate. Production levels of pestalamide B, nigerazine B, and nigragillin exhibited a reduction of approximately 50% when compared to control strains.
Both ISS-and ground-grown JSC-093350089 were regrown at 28°C and SMs production was evaluated. Due to the difference in growth temperature, obtained SM profiles of regrown isolates differed from those grown on the ISS (~22°C; Figure 3), as the trend in the production of SMs was the opposite of the one observed immediately after growth on the ISS. The majority of identified compounds showed slight increase in the production levels. Further, statistically significant increase in the production of the antioxidant pyranonigrin A (Miyake et al., 2007) and kotanin was observed when compared to ground controls.

DISCUSSION
It is critical to study molecular changes occurring in living organisms to understand the adaptation mechanisms allowing for surviving in extreme environments. One of such scientifically intriguing environments is the ISS, which is characterized by the presence of enhanced irradiation and microgravity. Due to its uniqueness, the ISS is under constant microbial monitoring, which allows for the isolation of wide array of microorganisms that often-become subjects of scientific investigations (Checinska et al., 2015;Knox et al., 2016;Checinska Sielaff et al., 2017;Venkateswaran et al., 2017;Bijlani et al., 2021). However, like in the case of the A. niger strain JSC-093350089 (Romsdahl et al., 2018(Romsdahl et al., , 2020, these investigations are more of a descriptive nature as definitive ascribing of observed molecular changes requires precisely controlled experiments. Therefore, to further investigate the differences in JSC-093350089 that were observed when compared to a "terrestrial" strain, the isolate was sent to the ISS in a planned experiment. Genomic, proteomic, and metabolomic alterations occurring in ISS-grown samples were analyzed following sample return and compared to groundgrown counterparts. Genome analysis of ISS-grown JSC-093350089 revealed the introduction of SNPs and INDELs in response to space conditions. Interestingly, the majority of observed non-synonymous SNPs and INDELS were located within chromosomes VIII and XII, which suggests that only selected regions of the genome undergo positive selection to confer selective advantage while adapting to the space environment. This is in agreement with previous reports of space-induced genetic variations, as ISS-grown Aspergillus nidulans (Romsdahl et al., 2019) and spaceflightgrown Staphylococcus aureus (Guo et al., 2015) both exhibited genetic mutations that occurred in specific clustered regions of the genome. Although the functions of many genes containing non-synonymous SNPs were unknown, several of these genes possessed transposable element and DNA-binding activity. One such gene, An08g11520, was an analogue of transposon I factor and has RNA-directed DNA polymerase activity, which is consistent with genetic changes observed in transposable element genes in both A. nidulans (Romsdahl et al., 2019) and S. aureus (Guo et al., 2015). Alterations in transposable element genes likely influence their activity and lead to the introduction of variations within the genome in response to environmental stress (Capy et al., 2000;Muszewska et al., 2017). The results from this study further underscore the significant role of transposable elements in adaptation to the spacecraft environment. Future studies should investigate the functions of uncharacterized genes containing non-synonymous SNPs, as such knowledge may provide key information on how fungi adapt to space conditions. Noteworthy, when radioadapted strain of Exophiala dermatitidis and the non-radioadapted control strain were exposed to Polonium-210, a mostly transcriptomic rather than genomic response to radiation was observed in radioadapted strain. This suggests that strains previously exposed to irradiation respond to subsequent exposures in a unique way (Malo et al., 2021). Based on the observations reported for E. dermatitidis, it is plausible that A. niger   contain high-level developmental and metabolic transcriptional regulators (Noble and Andrianopoulos, 2013;Lim et al., 2021). Therefore, the high occurrence of SNPs and INDELS in the intergenic regions may contribute to developing the space environment-induced phenotype in A. niger JSC-093350089. Investigation into the proteome of ISS-and ground-grown JSC-093350089 revealed that space conditions induce changes in protein expression when compared to ground controls. Observed, alterations in proteins involved in carbohydrate metabolism upon exposure to space conditions stay in agreement with previous studies of A. niger JSC-093350089 (Romsdahl et al., 2018), and other filamentous fungi, including Aspergillus fumigatus (Blachowicz et al., 2019a,b) and A. nidulans (Romsdahl et al., 2019). Similarly, a black fungus Knufia chersonesos exposed to low-shear simulated microgravity (LSSMG) also exhibited differential expression of proteins involved in carbohydrate metabolism (Tesei et al., 2021). On the other hand, a yeast, Candida albicans, exposed to spaceflight environment on the "SJ-10" satellite for 12 days showed decreased abundance of proteins involved in carbon metabolism, suggesting that yeast and filamentous fungi differ in adaptive responses to space environment . Additionally, ISS-grown JSC-093350089 exhibited upregulation of starvation-induced glycoside hydrolases, including AamA, and CbhB. This further confirms that changes in carbohydrate metabolism, may be a result of adaptation to scarce nutrient availability on ISS surfaces, due to the maintained cleaning regimen. Further, differential expression of XlnR-regulated proteins appears to be a key adaptive response to microgravity and enhanced irradiation, as both this and the previous study characterizing the space-induced molecular phenotype of JSC-093350089 (Romsdahl et al., 2018) revealed differential expression of various XlnR-regulated proteins. Activation of XlnR-regulated metabolic pathways leads to use of versatile carbon sources (Hasper et al., 2000), which may facilitate survival in hostile space environments. A few proteins upregulated in ISS-grown JSC-093350089 were involved in stress response, including the cell wall organization protein EcmA and flavohemoglobin, whose analogue in Aspergillus flavus has been reported to have a function correlated with hyphal growth phenotype (te Biesebeke et al., 2010). These observations further suggest that modulation of fungal growth and cell organization is an essential response to space conditions.
Metabolomic analysis of ISS-grown JSC-09335008 showed alterations in secondary metabolite production in response to the space environment. This observation was not surprising, as fungi often respond to versatile environmental conditions by altering the type and yield of produced SMs (Brakhage, 2013). Interestingly, ISS-grown JSC-09335008 showed decreased production of all SMs, which is the opposite production pattern observed during the initial characterization of the metabolome of JSC-09335008 when compared to a "terrestrial" strain (Romsdahl et al., 2020). This discrepancy may be related to the fact that metabolomic profile of ISS-isolated JSC-09335008 was compared to the well-studied ATCC 1015 strain, rather than a "proper" JSC-09335008 ground control. Further investigation of the space environment-induced SM profile of the JSC-09335008 strain should be conducted to confirm whether decreased production of pestalamide B, nigerazine B (alkaloid), and nigragillin (alkaloid) are important biological adaptations. Given that sending experiments to the ISS is not readily available, it will be critical to use more easily accessible microgravity simulators, like High Aspect Ratio Vessels-HARV, or random positioning machines-RPMs to gain more insights in the space-induced phenotype of A. niger JSC-09335008 strain.
Finally, to gain insight into observed differences in acquired SMs profiles between current and previous study (Romsdahl et al., 2020) further experiment was conducted. Both ISS-and ground-grown JSC-09335008 were regrown in the same conditions (28°C for 5 days) as used in the previous study, which resulted in observing similar trends in SM production. After regrowing at 28°C, the ISS-grown JSC-09335008 produced higher yields of all SMs when compared to the regrown ground control, including approximately 60% increased production of the antioxidant pyranonigrin A. Pyranonigrin A was previously proposed to have a radioprotective nature, as pyranonigrin A-deficient JSC-09335008 strain was more sensitive to UVC exposure than the wild type JSC-09335008 strain (Romsdahl et al., 2020). Interestingly, pyranonigrin A production was not detected in the ISS-and ground-grown samples following the  Frontiers in Microbiology | www.frontiersin.org experiment on the ISS where growth temperature was about at 22°C. Due to this temperature-dependent discrepancy in observed SM profiles, the most important question to address is whether pyranonigrin A truly provides A. niger protection while in space. Such protection could potentially have various biotechnological applications for use of pyranonigrin A, including within human space programs and cancer therapies. Therefore, studies confirming pyranonigrin A potential as a radioprotective agent should be warranted. Finally, future studies should examine production of pyranonigrin A under various temperatures aboard the ISS, as well as its protective nature within the space environment to definitively answer this question. This study is the first report of the multi-omics response of A. niger to space conditions during a controlled experiment, which enhances our understanding of its space-induced phenotype. Such understanding may be translated to development of protective measures for both astronauts and the spacecraft during future manned space explorations, as A. niger is ubiquitous fungus present in many human-occupied closed habitats (Checinska et al., 2015;Blachowicz et al., 2017). Lastly, a thorough understanding of the space-induced secondary metabolomic alterations of industrially important A. niger may result in creating a potent producer of compounds of interest during space voyages.

DATA AVAILABILITY STATEMENT
Raw WGS reads for JSC-093350089 ISS-and ground-grown are available in the NCBI SRA, under accession numbers SAMN25997338 and SAMN25997339 and BioProject accession number PRJNA807647. Proteomics data is accessible through Massive with the dataset identifier MSV000088986.

AUTHOR CONTRIBUTIONS
AB drafted the manuscript, contributed to sample processing, and conducted data analysis and interpretation. JR contributed to sample processing and data interpretation. AC and MK conducted protein sample processing, LC-MS analyses, and data processing. SM and JS contributed to genome analysis. TT designed the study and drafted the manuscript. KV and CW conceptualized the project, coordinated the flight experiment, designed the study, interpreted the data, and drafted the manuscript. All authors contributed to the article and approved the submitted version. design, data collection and interpretation, the writing of the manuscript, or the decision to submit the work for publication.

ACKNOWLEDGMENTS
Part of the research described in this publication was carried out at the Jet Propulsion Laboratory, California Institute of Technology, under a contract with NASA. We would like to thank astronaut Jeff Williams for handling the samples aboard the ISS and CASIS Team for coordinating this effort.