A. B. Regberg1, C. L. Castro2, H. C. Connoly Jr.3, R. E. Davis4, J. P. Dworkin5, D

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Microbial Ecology of the OSIRIS-REx Assembly Test and Launch Environment Abstract #1113

A. B. Regberg1, C. L. Castro2, H. C. Connoly Jr.3, R. E. Davis4, J. P. Dworkin5, D. S. Lauretta6, S. R. Messenger1, F.M. McCubbin1, K. Righter1, S. E. Stahl2, S. L. Wallace7 1Astromaterials Research and Exploration Science Division, NASA Johnson Space Center, 2101 NASA Park-way, Houston TX 77058,2 JES Tech, 16870 Royal Crest, Houston, TX 77058, 3Rowan University, Glassboro, NJ 08028, 4 Jacobs@NASA/Johnson Space Center, Houston, TX 77058, 5Astrochemistry Laboratory, Goddard Space Flight Center, Greenbelt, MD 20771, 6Lunar and Planetary Laboratory, University of Arizona, 1629 E University Blvd Tucson AZ 85721-0092, 7Biomedical Research and Environmental Sciences Division, Johnson Space Center, 2101 NASA Parkway, Houston TX 77058. Email: [email protected]

Introduction Results

• Our goal is to characterize the microbes present during diﬀerent phases of 384,526 the OSIRIS–REx ATLO (Assembly Test Launch Operations) Range of Bacterial and Archaeal 93,435 • These data can inform future planetary protection controlled missions help OTU’s identiﬁed from JPL Space Craft Assembly Facility to understand the relationship between organic an biological cleanliness (Minich et al., 2018)

• We extracted DNA from 17 witness foils and two blanks originally intended 64 for amino acid characterization Bacteria and Archaea 12 12 • We successfully ampliﬁed and sequenced DNA from bacteria, archaea, and Fungi fungi. • We ampliﬁed the 16S ribosomal rRNA gene to identify bacteria and ar-

chaea facility assembly JPL Space craft from of 16S reads Range al., 2018) (Minich et • We amplified the ITS region to identify fungi • Bacterial and Archaeal diversity is relatively low compared to a similar aerospace clean room • We were not able to sequence DNA from any other Eukaryotes. • Fungal diversity appears to increase slightly with time • The DNA amplification and sequencing techniques used had a detection • Sample 4 had the highest 16S diversity, Sample 15 and the fairing sample had the highest fungal diversity limit of ~7 bacterial cells. • Only one 16S sample had too few sequences for further analysis Fungi • 3 fungal samples had 0 reads • It is common for the blanks and control samples to have relatively large numbers of DNA sequences Methods • The number of bacterial reads appears to increase with time • The number of fungal reads is more variable • Sample 11 (TVAC lid opened) and sample 12 (launch container) have highest number of 16S and fungal DNA Extraction and Tag reads respectively Sample Information Sequencing Bacteria Table 1: Sample Information Firmicutes Exposure Exposure Sample start stop Location ATLO Processes OR-CKP-01-1-A,0 3/11/2015 4/14/2015 LM/Denver SRC assem. & funct. test; TAGSAM assem. w/ clean qual head OR-CKP-02-1-A,0 4/14/2015 5/11/2015 LM/Denver TAGSAM funct. Develop. OR-CKP-03-1-A,0 5/11/2015 6/10/2015 LM/Denver avionics box/SRC funct. Post-vibe; OR-CKP-04-1-A,0 6/12/2015 7/14/2015 LM/Denver SARA TVAC/launch container/ TAGSAM deploy. funct. Post-vibe/OVIRS/OTES OR-CKP-05-1-A,0 7/14/2015 8/19/2015 LM/Denver TAGSAM install, deploym. OR-CKP-06-1-A,0 8/19/2015 9/18/2015 LM/Denver OCAMS install/SARA deploym. OR-CKP-07-1-A,0 9/18/2015 11/4/2015 LM/Denver SARA deploym./move to RAL OR-CKP-08-1-A,0 11/4/2015 12/9/2015 LM/Denver RAL sine-vibe/move to SSB OR-CKP-09-1-A,0 12/9/2015 1/7/2016 LM/Denver REXIS OLA install/SC moved/shipping container OR-CKP-10-1-A,0 1/8/2016 2/5/2016 LM/Denver Flt. TAGSAM/SC to RAL/ EMI-EMC/TVAC pre-cert. OR-CKP-11-1-A,0 2/5/2016 3/16/2016 LM/Denver TVAC lid opened/SC to SSL/TVAC pumpdown/lid opened 3/10/move to SSB OR-CKP-12-1-A,0 3/6/2016 4/26/2016 LM/Denver launch container/SARA Actinobacteria OR-CKP-13-1-A,0 4/26/2016 4/27/2016 LM/Denver TAGSAM flight install OR-CKP-14-1-A,0 4/27/2016 6/17/2016 KSC SARA deploym./Flight head test/KSC OR-CKP-15-1-A,0 6/17/2016 7/14/2016 KSC SRC battery enable/TAGSAM cleaning OR-CKP-16-1-A,0 7/14/2016 8/26/2016 KSC He load/prop tests/TAGSAM bottle loads/fuel sampling/cleaning&inspection/pack&ship preps Basidiomycota Ascomycota OR-GCKP-17-1-A1 JSC/Goddard Shipping Blank ORX Faring Blank – FB - 18 KSC Fairing Blank ORX Fairing CK Fck – 19 KSC Fairing CK (Dworkin et al., 2018) Swab Blank – Sb 20 JSC Kit Blank – Kb -21 JSC

Proteobacteria • Only sample 13 had archaeal DNA

16S rRNA Barcode Gene for Bacteria and Archaea • Firmicutes is the dominant bacterial phyla Witness foil Samples v4 region, 515F-806R, 27Fmod-519Rmod (Caporaso et al. 2012; Walters et al. 2015) • This includes common clean room and human associated or- • Fungal DNA is relatively distinct for each sample ganisms like Bacillus, Lactobacillus, Listeria and Staphylococcus • Sample 11 is dominated by Micrococcus a type of human associ- DNA ampli cation with PCR • Sample 12 (launch container) is dominated by Cladosporium delicatum, a saprophytic fungi (Polymerase Chain Reaction) ated actinobacteria • Sample 16 is dominated by Zymoseptoria a genus containing plant pathogens Bacillus OTU matches several species Bacillus nakamurai strain NRRL B-41091 16S ribosomal RNA, partial sequence • Sample 19 (Fairing) appears to be different, dominated by mito- Bacillus pumilus strain SBMP2 16S ribosomal RNA gene, partial sequ... • Sample 13 (TAGSAM flight install) is dominated by the an unidentified Meruleacieae the family responsible for lignin degra- Bacillus safensis strain NBRC 100820 16S ribosomal RNA gene, part... chondrial DNA from an Oomecyte (water mold) Bacillus velezensis strain FZB42 16S ribosomal RNA, complete sequence dation Bacillus subtilis subsp. subtilis strain 168 16S ribosomal RNA, complete sequence Bacillus axarquiensis strain LMG 22476 16S ribosomal RNA gene, partial sequence Bacillus mojavensis strain ifo 15718 16S ribosomal RNA gene, partial sequence • Unique organisms from the faring Bacillus mojavensis strain NRRL B-14698 16S ribosomal RNA gene, partial sequence DNA extracted with a QIAamp Bacillus mojavensis strain NBRC 15718 16S ribosomal RNA gene, partial sequence Bacillus halotolerans strain DSM 8802 16S ribosomal RNA, partial sequence BiOstic Bacteremia kit ITS1 region for Fungi Bacillus axarquiensis strain CR-119 16S ribosomal RNA gene, partial sequence • Udenimyces pyricola (yeast) Bacillus malacitensis strain CR-95 16S ribosomal RNA gene, partial sequence 0.002 ITS1f-ITS2 lcl|Query_55793 • Itersonilia pannonica (plant associated fungi) Bacillus mojavensis strain IFO15718 16S ribosomal RNA gene, partial sequence DNA Sequencing Illumina MiSeq • Articulospora proliferate (aquatic fungi) • Phaeosphaeria caricola (plant fungi) Conclusions

DNA ampli cation with PCR (Polymerase Chain Reaction) • We successfully sequenced bacterial and fungal DNA from clean room wit- Bioinformatics ness coupons demonstrating that these types of samples can be useful for biological contamination knowledge Quality Control and OTU clustering • Witness coupons from the TVAC testing and from the rocket fairing had Taxonomy • DNA sequences were quality filtered, trimmed analyzed using the QIIME software package. distinct microbial communities • We utilized the Deblur pipeline to identify 100% unique OTU’s (Operational Taxonomic Units) P-value:0.0119474 • The abundance of fungal sequences may correlate to the amount of • OTU’s were identified using the Silva v132 database for bacteria and archaea and the UNITE database for fungi. P-value:0.0034466 Equation: ln(Fungal Swab Sequences) = 5.64776*% Fe + 6.88946 Equation: ln(Fungal Swab Sequences) = -6.09474*% C + 10.518 carbon and iron bearing particles from replicate witness plates. Known concentrations of Bacillus subtilus sequenced in order to deter- • Further data analysis is ongoing mine the detection limit for this sequencing run • Dedicated biological contamination knowledge samples should be routine- ly collected for missions where biology could affect mission requirements • There are weak relationships between the % of carbon particles and iron e.g. Mars2020, Europa Clipper particles identified on witness plates and the number of fungal sequences

Detection Limit = 5,611 reads • There are no clear relationships between the sequencing data and the Detection Limit = 6.759 cells References measured amino acid concentrations

Amir, Amnon et al. 2017. “Deblur Rapidly Resolves Single-Nucleotide Community Sequence Patterns.” MSystems 2(2):e00191-16. Auchtung, Jennifer M. et al. 2015. “Cultivation of Stable, Reproducible Microbial Communities from Different Fecal Donors Using Minibioreactor Arrays (MBRAs).” Microbiome 3(1):42. Blattner, Frederick R. et al. 1997. “The Complete Genome Sequence of Escherichia Coli K-12.” Science 277(5331):1453–62. Caporaso, J. Gregory et al. 2010. “QIIME Allows Analysis of High-Throughput Community Sequencing Data.” Nature Methods 7(5):335–36. Caporaso, J. Gregory et al. 2012. “Ultra-High-Throughput Microbial Community Analysis on the Illumina HiSeq and MiSeq Platforms.” The ISME Journal 6(8):1621–24. Dworkin, J. P. et al. 2018. “OSIRIS-REx Contamination Control Strategy and Implementation.” Space Sci Rev 214(9). Minich, Jeremiah J. et al. 2018. “KatharoSeq Enables High-Throughput Microbiome Analysis from Low-Biomass Samples” edited by M. J. McFall-Ngai. MSystems OSIRIS-REX | LUNAR AND PLANETARY SCIENCE CONFERENCE | MARCH 2019 3(3):e00218-17. Assuming an average of 5.14 fg of DNA per cell (Blattner et al. 1997), this is Nilsson, Rolf Henrik et al. 2018. “The UNITE Database for Molecular Identification of Fungi: Handling Dark Taxa and Parallel Taxonomic Classifications.” Nucleic Acids Research. a detection limit of approximately 34 fg of DNA Quast, Christian et al. 2012. “The SILVA Ribosomal RNA Gene Database Project: Improved Data Processing and Web-Based Tools.” Nucleic Acids Research 41(D1):D590–96. .