The International Space Station Microbial Observatory Experiment

Postdoctoral Fellow: Aleksandra Checinska (352N) Principal Investigator: Kasthuri Venkateswawaran (352N)

Poster no. P-17

National Aeronautics and Space Administration

Jet Propulsion Laboratory California Institute of Technology Pasadena, California www.nasa.gov Copyright 2015. All rights reserved.

National Aeronautics and Space Administration

Objectives •  The safety of the International Space Station (ISS) crewmembers and maintenance of hardware are the

primary rationale for monitoring microorganisms in this closed habitat. •  National Research Council (NRC) recommended to utilize ISS –a closed habitat– and observe changes

encountered due to the microgravity. Subsequently, NASA Space Biology program funded JPL to catalogue microbial diversity of ISS surfaces and atmosphere under NASA – Microbial Observatory Program.

•  Molecular techniques were used to measure microbial burden and diversity associated with these samples that were previously.

•  This study provides the insight into microbial diversity of ISS using the state-of-the-art molecular techniques applied by JPL-352N group for the MARS program.

Sample Collection

Conclusions

•  Two sampling campaigns revealed presence of diverse microbial population with some microbial species dominating in ISS. The ongoing 16S rDNA Illumina sequencing will provide data on microbial diversity over time (subsequent months of sampling).

•  The long-term goal of this project is aimed to develop practices for better cleaning and maintenance of the ISS, cataloguing and preserving beneficial microorganisms for future applications, and the general knowledge on microbial ecology of closed, environmentally controlled built systems.

•  The microbial diversity study on the ISS will help to implement better practices for future robotic and human missions.

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. This research was funded by a 2012 Space Biology NNH12ZTT001N grant # 19-12829-26 under Task Order NNN13D111T award to K. Venkateswaran. Government sponsorship acknowledged.

Sample collection from 8 ISS locations using

polyester wipes, contact slides, opsite tape and air sampling device.

Sample processing upon the arrival to JPL; the samples returned on Soyuz TM-14M and

SpaceX CRS-6)

Sample collection from 8 Dragon capsule

locations (SpaceX) using polyester wipes, contact slides, opsite tape and air sampling device.

Sample Processing

Sample recovery and concentration

Microbial Bioburden Assessment measured

by Adenosine Triphosphate (ATP)

Assay

Cultivable population (sample was split for incubation on R2A,

PDA and Blood Agar)

Viable population and diversity (qPCR, Illumina based Next Generation Sequencing and

Metagenomics)

Propidium monoazide (PMA) assay and DNA extraction

Cultivable, Viable, Bacterial Community

Cultivable Microbial Diversity

A

B

Fig. 1. Microbial population from ISS location no. 4 (dining table) on PDA plate (fungi promoting media) (A). The contact slide used for sampling the ISS location no. 4 (dining table) (B). Contact slides cover a surfaces of 25 cm2 while a polyester wipes 1 m2. Phylogenetic tree for the isolates collected from Blood Agar plates (potential pathogens); IF, IIF– first and second sampling on the ISS, respectively.

DNA Illumina next generation sequencing for 16S rRNA (bacteria and archaea) and 18S rRNA (fungi) – microbial diversity of the ISS environmental surfaces

Metagenomics study for functional analysis: metabolism, defense/survival mechanisms, antibiotic resistance, pathogenicity

Whole genome sequencing –

novel species studies

Data storage/accessibility: 1. ISS-MO – project database

2. GeneLab - NASA database for all genetic data 3. NCBI – worldwide database for biological data

Molecular Microbial Community Diversity Analysis

Fig. 2 ATP assay measure total microbial burden (bacteria, fungi, archaea). The assay has been applied by JPL-352N for the estimation of microbial bioburden for the environmental surfaces of cleanrooms.

C

1.00E+00

1.00E+01

1.00E+02

1.00E+03

1.00E+04

1.00E+05

1.00E+06

1.00E+07

1.00E+08

1.00E+09

1.00E+10

1.00E+11

IIFCSW IIF1SW IIF2SW IIF3SW IIF4SW IIF5SW IIF6SW IIF7SW IIF8SW IIF2*SW

Cultivable (R2A)

Internal ATP

rrn

IFSW-P1B

IF7SW-P1A

Staphylococcus epidermidis ATCC 14990(T)

IF4SW-P4

IIF4SW-P1

Staphylococcus warneri ATCC 27836(T)

Staphylococcus aureus subsp. anaerobius ATCC 35844

IIFSW-P5

IIF2SW-P3

IF7SW-P3

Staphylococcus haemolyticus ATCC 29970(T)

IF4SW-P3

IIF2SW-P4(2)

IF4SW-P5

IF6SW-P3B

IF6SW-P1B

Staphylococcus saprophyticus subsp. saprophyticus ATCC 15305(T)

IIF2SW-P2

IIF3SW-P3

IF1SW-P2

IIF4SW-P2

Bacillus cereus ATCC 14579(T)

IIF4SW-P4

IIF4SW-P5

IIF4SW-P3

Paenibacillus jamilae CECT 5266(T)

IIF8SW-P4

IIF2SW-P4

Paenibacillus polymyxa ATCC 842(T)

IF8SW-P1

Micrococcus yunnanensis YIM 65004(T)

IIF1SW-P1

Acinetobacter pittii CIP 70.29(T)

IIF1SW-P3

IIF1SW-P4

IIF1SW-P5

IIF1SW-P2

IF1SW-P4

IF1SW-P3

IF2SW-P1

IF3SW-P1

Klebsiella quasipneumoniae subsp. similipneumoniae 07A044(T)

IF3SW-P2

IF2SW-P3

IF2SW-P2

Enterobacter ludwigii DSM 16688(T)

IF5SW-P2

Pantoea conspicua LMG 24534(T)

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