Growth of 48 Built Environment Bacterial Isolates on Board the International Space Station (ISS)
Background: While significant attention has been paid to the potential risk of pathogenic microbes aboard crewed spacecraft, the non-pathogenic microbes in these habitats have received less consideration. Preliminary work has demonstrated that the interior of the International Space Station (ISS) has a microbial community resembling those of built environments on earth. Here we report results of sending 48 bacterial strains, collected from built environments on earth, for a growth experiment on the ISS. This project was a component of Project MERCCURI (Microbial Ecology Research Combining Citizen and University Researchers on ISS).
Results: Of the 48 strains sent to the ISS, 45 of them showed similar growth in space and on earth using a relative growth measurement adapted for microgravity. The vast majority of species tested in this experiment have also been found in culture-independent surveys of the ISS. Only one bacterial strain showed significantly different growth in space. Bacillus safensis JPL-MERTA-8-2 grew 60% better in space than on earth.
Conclusions: The majority of bacteria tested were not affected by conditions aboard the ISS in this experiment (e.g., microgravity, cosmic radiation). Further work on Bacillus safensis could lead to interesting insights on why this strain grew so much better in space.
From 2012-2014, we conducted a nationwide citizen science project, Project MERCCURI http://spacemicrobes.org/, aimed at raising public awareness of microbiology and research on board the International Space Station (ISS). Project MERCCURI (Microbial Ecology Research Combining Citizen and University Researchers on the ISS) was a collaborative effort involving the "microbiology of the Built Environment network" (microBEnet) group, Science Cheerleader, NanoRacks, Space Florida, and SciStarter. One of the goals of Project MERCCURI was to examine how a number of non-pathogenic bacteria associated with the built environment would grow on board the ISS compared to on earth.
Most previous work growing bacteria in space has focused on species known to contain pathogenic strains (e.g. Escherichia coli (Klaus 1997) (Brown 2002) and Pseudomonas aeruginosa (Crabbé 2011) (Kim 2013)), and much less attention has been paid to the non-pathogenic microbes that surround us. An understandable bias towards pathogens and pathogenic pathways is highlighted by work on topics such as biofilm formation ((Kim 2013a), (McLean 2001)), antibiotic resistance/production ((Benoit 2006), (Juergensmeyer 1999), (Lam 2002) reviewed in (Klaus 2006)), and virulence ((Nickerson 2000), (Hammond 2013)).
Although concern about pathogens in spacecraft is certainly warranted, it should be emphasized that the ability of a pathogen to survive outside a host and the ability to infect a host are both, at least in part, dependent on the existing community of non-pathogenic microbes in those locations. For example, the infectivity of some pathogens has been shown to be very dependent on the host microbiome (e.g. (Schuijt 2015), (Ichinohe 2011), (van 2015) (Reeves 2011)). Therefore, it is important to understand the entire microbial ecosystem of spacecraft. Indeed, in recent years, several culture-independent studies have examined the microbiome of the ISS ((Castro 2004), (Venkateswaran 2014), (Moissl 2007)), including another part of Project MERCCURI (Lang Submitted). These studies have shown, not surprisingly, that the microbiome of the ISS bears a strong resemblance to the microbiome of human-associated built environments on earth. Therefore, it is of interest to see how microbes from human-associated environments behave in space.
For this study, samples from human-associated surfaces (e.g. toilets, doorknobs, railings, floors, etc.) were collected at a variety of locations around the United States, usually in collaboration with the public. A wide variety of bacteria were cultured from these samples, and 48 non-pathogenic strains were selected for a growth assay comparing growth in microgravity on the ISS and on earth.
Materials and Methods
Samples were collected from built environment surfaces throughout the United States on cotton swabs (Puritan 25-806 2PC) and mailed (usually overnight) to the University of California Davis where they were transferred to lysogeny broth (LB) plates. Colonies were chosen for further examination based on maximizing morphological variation. Each chosen colony was double-dilution streaked (two rounds of streak plates) and then the identity determined by direct PCR and Sanger sequencing using the 27F (5'-AGAGTTTGATCMTGGCTCAG-3') and 1391R (5'-GACGGGCGGTGTGTRCA-3') primers (see (Dunitz 2015) for details). Sanger sequences were trimmed and aligned using Geneious (Kearse 2012). The resulting consensus sequence was identified through a combination of BLAST (Altschul 1990) and building phylogenetic trees using the Ribosomal Database Project (RDP) website (Cole 2014). The 48 candidates for spaceflight were chosen on the basis of biosafety level (BSL-1 only), taxonomic variety, and human interest. In the absence of international standards, the biosafety level of each organism was determined by searching the American Biological Safety Association (ABSA) risk group database, the American Tissue Culture Collection (ATCC), the Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ), and other public databases. An organism was removed from consideration if it was listed as BSL-2 or higher in any country or collection in the world. Human interest was an arbitrary set of criteria such as unusual physiology, catchy name, or a memorable original isolation source.
A set of bacterial plates were created for each aspect of the study: growth in microgravity on the ISS (space plates), or growth on earth (ground plates). The plates were created using clear agar to facilitate optical density (OD) measurements. 1.5 g of Gelzan™ CM agar (Sigma-Aldrich) was added to 1 liter of lysogeny broth (LB). Each well of a flat-bottomed 96-well plate (Costar) was plated with 200 μl of agar. The plates were flamed to remove bubbles and incubated for 48-72 hours at room temperature (~20 °C) to ensure sterility before adding bacteria. Fresh overnights of each bacterial isolate were diluted to .01 OD600 and made into 8% glycerol stocks. For plating, 10 μl of each thawed stock dilution was added to 2 wells per 96 well plate. 6 replicate plates were made. The bacteria were placed into different locations on each plate in order to account for drying at the edges or any other positional effects on the plates. The plates were then sealed with adhesive polypropylene film (VWR #60941-072), into which a grid of micron-diameter holes were cut with a laser to allow for airflow. The ground plates were stored at -80 °C at UC Davis, and the space plates were mailed on dry ice to the National Aeronautics and Space Administration (NASA) Johnson Space Center in Houston, TX before transfer (at -80 °C) to Cape Canaveral, FL for launch.
This payload was flown on the CRS-3 launch of the Space Exploration Technologies (SpaceX) Dragon spacecraft, on a Falcon 9 v1.1 rocket which successfully launched April 18, 2014. After six days, the space plates were removed from the MELFI (Minus Eighty Lab Freezer For ISS) and partially thawed. However, technical problems arose and the space plates were placed back into the MELFI until December 8, 2014. At that time, all three plates were thawed and the OD600 of each well (3x3 grid) was measured at time 0 (60 minutes after removal from the freezer) and then every 24 hours for 4 days. Measurements were performed in a Molecular Devices SpectraMax M5e plate reader which was modified for integration onto the ISS. On these same days, equivalent measurements of the ground plates were taken in a Molecular Devices SpectraMax M5e plate reader at UC Davis. The exception to this was the initial partial thawing, which was not replicated with the ground plates since the amount of thaw was not reported by the astronauts. After the experiment, the ground plates were placed back at -80 °C and the space plates were placed back into the MELFI. In February 2015, the space plates were transferred to a -95 °C freezer on board a Dragon spacecraft. The vehicle splashed down in the Pacific Ocean on Feb 10, 2015. The space plates were then mailed to UC Davis on dry ice and were transferred to -80 °C when received.
Once the plates arrived, we thawed all six plates and performed a high-density measurement in a Tecan M200 plate reader. OD600 readings were taken in a 5x5 grid covering the entire well, these 25 measurements were then averaged within each well.
For each sample, the averages of the six space replicates and six ground replicates were compared using a Student's t-test. To correct for multiple hypothesis testing, the p-values were adjusted using the False Discovery Rate (FDR) method (Benjamini 1995). All raw data, analyses and scripts can be found at https://zenodo.org/record/44661.
In order to confirm that the observed results were not due to contamination of the wells, each of the 12 replicates (six space, six ground) for the three bacteria showing statistically different growth between the ISS and earth were cultured after the experiment. Bacteria were struck from the wells onto LB-agar plates, then single colonies were grown into overnight cultures. DNA was extracted using a Wizard Genomic DNA Purification kit (Promega) from each of the 36 cultures (3 bacteria X 12 replicates) and the identity was confirmed by BLAST of the Sanger sequenced PCR product using the 27F and 1391R primers as described above.
Comparison to ISS swab data
The bacterial community on the ISS was recently surveyed by PCR amplification and sequencing of 16S rRNA genes from swabs (Lang Submitted). We compared the 16S sequence of each of our bacterial isolates to the "representative sequence" from each operational taxonomic unit (OTU) generated from the survey data. A BLASTN search was performed locally and a match was considered to be present in the data when there was 97% identity over at least 250 bp of the rRNA sequence (the amplified fragment is 253 bp).