Deposition of Extreme-Tolerant Bacterial Strains Isolated during Different Phases of Phoenix Spacecraft Assembly in a Public Culture Collection

1. Deposition of Bacterial Strains in Public Culture Collection

A team of Jet Propulsion Laboratory (JPL) scientists isolated 262 strains from the Phoenix spacecraft assembly clean room environments. Eighty-two of the bacterial strains exhibited the capacity to withstand multiple extreme physiological conditions (hereafter called extreme-tolerant) and were consigned to the Agricultural Research Station (ARS) Culture Collection. The ARS Culture Collection is the world's largest publicly funded culture collection, with more than 100,000 strains of bacteria and fungi in its open (nonpatent) holdings. At ARS, the JPL-isolated strains were archived both as lyophilized pellets stored at 4°C and as liquid cultures in medium with 20% glycerol, frozen in liquid nitrogen. The ARS Culture Collection will make these strains available to professional (nonprofit) and academic organizations for their research purposes.

2. Background of the Cultures Deposited

The microbial burden of the Phoenix spacecraft assembly environment was assessed by using several cultivation-based techniques, and a detailed report was published (Ghosh et al., 2010). Sample sets were collected from the Payload Hazardous Servicing Facility (PHSF) during three assembly phases of the Phoenix spacecraft. The assembly phases took place (1) prior to the arrival of the Phoenix spacecraft (1P; April 25, 2007), (2) during the assembly and testing operations of the Phoenix spacecraft but prior to launch (2P; June 27, 2007), and (3) after removal of the spacecraft to the launch pad (3P; August 1, 2007). Extreme-tolerant bacteria that could potentially survive conditions experienced en route to Mars or on the planet's surface were isolated by using a series of cultivation-based assays. Wet surface sampling of PHSF floor and wall locations was performed with Biological Sampling Kits (BiSKits, QuickSilver Analytics, Abingdon, MD) as previously described (La Duc et al., 2009). The cultivation conditions of various kinds of bacteria were published elsewhere (La Duc et al., 2007; Ghosh et al., 2010). The bacteria are mesophilic heterotrophic bacteria (growth at 25°C on R2A plates), thermophiles (growth at 65°C on R2A plates), psychrophiles (growth at 4°C on R2A plates), alkaliphiles (growth at pH ∼11 on R2A plates), acidophiles (growth at pH ∼3 on R2A plates), halophiles (25% NaCl on R2A plates), spore-formers (heat shock at 80°C for 15 min with subsequent growth at 32°C on TSA plates), UV- or hydrogen peroxide–resistant bacteria, and anaerobes. UV-resistant bacteria were grown on R2A medium after a portion of samples was exposed to 1000 J/m² of UVC radiation, while hydrogen peroxide–resistant bacteria were isolated from samples by exposing aliquots to 5% hydrogen peroxide for 1 h before the samples were inoculated onto R2A. Anaerobes were cultured on thioglycolate medium in a GasPak anaerobic chamber. Detailed growth conditions for all cultivable bacteria have been described previously (La Duc et al., 2007). Detailed polyphasic taxonomic analyses of 262 isolates were carried out, and results have already been documented (Ghosh et al., 2010). Isolation, identification, and diversity profiles are given in an additional document (see Supplementary File S1, available online at www.liebertonline.com/ast). Among 262 strains isolated, 82 representative strains were deposited in the ARS Culture Collection for permanent archiving and distribution. Care was taken to avoid depositing multiple strains of the same taxonomic identity in the ARS Culture Collection, with the exception of B. pumilus strains, phenotypic diversity of which is known to be important from a planetary protection perspective. The diversity, taxonomic positions, and accession numbers (GenBank and NRRL) of these 82 isolates are shown in Supplementary Table S1.

3. Phylogenetic Diversity

A total of 67 bacterial species was isolated, 27 of which were found to be potentially novel by 16S rRNA gene sequence analysis (Stackebrandt and Ebers, 2006). In general, a more diverse community of bacteria was detected before the Phoenix spacecraft was brought into the clean room (1P) compared to what was detected in the locations sampled during spacecraft assembly (2P) and after the spacecraft left the PHSF (3P). Furthermore, the clean room locations that were sampled before Phoenix spacecraft arrival (1P) harbored a greater number of previously unknown bacteria. When sampling was conducted in PHSF locations before Phoenix arrived (1P), 11 novel bacterial species were discovered, whereas only 6 new species were found during the time the spacecraft was assembled (2P). No novel species were encountered from those same locations after the spacecraft left the facility (3P). A distinct shift in bacterial populations was observed in samples collected from the PHSF before Phoenix arrived (1P) when compared to the samples collected during (2P) and after the spacecraft left the facility (3P). Isolates collected from the pre-Phoenix period (1P) included representatives from the Proteobacteria (9 species), low GC gram-positive organisms (Firmicutes, 17 species), high GC gram-positive organisms (Actinobacteria, 17 species), and one each of Bacteroidetes and Deinococcus groups. Strains collected during Phoenix (2P) belonged to members of the Proteobacteria, Firmicutes, and Actinobacteria, while only Proteobacteria and Firmicutes members were detected from post-Phoenix samples (3P). The markedly reduced diversity during 2P and 3P coincided with the increased prevalence of Acinetobacter johnsonii and Brevundimonas diminuta (Ghosh et al., 2010), indicating that these two species were better adapted to the clean room conditions. Analysis of overlapping cultivable bacterial communities from all 3 sampling events revealed that 31 of the 43 species present during 1P were no longer detected during 2P and 3P. Only two species, B. pumilus and B. diminuta, were detected during all three sampling events.

4. Functional Diversity

Strains that could grow under several physiological conditions and were isolated in other exposure conditions included alkalitolerant bacteria (26 strains; 21 species with 8 novel species), facultative anaerobes (7 strains; 6 species; 1 novel), mesophiles (12 species; 4 novel), psychrotolerant bacteria (14 species; 6 novel), spore-formers (19 strains; 13 species; 8 novel), and UV₂₅₄-resistant bacteria (4 species). Direct cultivation in high-alkaline medium yielded a total of 21 species, 8 of which were likely novel. All 6 facultative anaerobic species, including a novel species of the genus Serratia, were also able to grow aerobically. A total of 12 species were isolated when grown in mesophilic conditions, 3 of which were likely novel species, and one of these has been fully described (Vaishampayan et al., 2013). Growth under low-temperature conditions resulted in the isolation of 13 species, 7 of which were novel. Spore-formers demonstrated diverse metabolic capabilities: 11 species were detected from the spore-favoring heat shock assay, 7 were obtained from high-alkaline media, 7 were cultivated under anaerobic conditions, 5 were isolated on neutral pH R2A under mesophilic conditions, 2 were isolated under psychrophilic conditions, and 4 survived high UV exposure. Of the 4 UV-resistant bacterial species analyzed, 3 belonged to Firmicutes and were the members of previously described species. It should be noted that a single species, B. pumilus, was found to tolerate nearly all experimental stresses, including growth at high temperature (∼65°C) and exposure to UV₂₅₄ conditions, but was not isolated under low-temperature conditions (4–8°C). Thus, in total 13 B. pumilus strains were archived at the ARS Culture Collection that might be useful for future researchers for elucidating the metabolic plasticity present in this “problematic” bacterial spore-forming species (Newcombe et al., 2005; Vaishampayan et al., 2012).

5. Novel Bacterial Diversity

Among the 27 species deemed novel based on 16S rRNA gene sequence analysis (Stackebrandt and Goebel, 1994), 8 alkalitolerant, 8 spore-forming, 6 psychrotolerant, 4 mesophilic heterotrophs, and 1 facultative anaerobic bacteria were identified. Among these novel bacteria, one novel genus (Tersicoccus; Vaishampayan et al., 2013), two novel species (B. horneckiae; Vaishampayan et al., 2010), and Paenibacillus phoenicis (Benardini et al., 2011) were described by our group. Polytaxonomic characterization of two additional species, Acinetobacter phoenicis and Deinococcus phoenicis, has been ongoing with the intent to delineate novel species status (Vaishampayan et al., unpublished data).

Several of these novel strains exhibited tolerance to multiple physiological conditions. For example, some of the psychro- and alkalitolerant strains were also spore-formers under low nutrient conditions. The strains isolated as UV-resistant all belonged to species that have already been described. Except for one each of Bacillus and Georgenia species, novel species were not found in more than a single sampling event; however, several isolates of the same novel species were often found in various locations of that sampling event. One novel alkalitolerant species, identified as B. horneckiae, was found in at least five locations during the 1P sampling event and has recently been more fully described by using a variety of metabolic and phylogenetic analyses (Vaishampayan et al., 2010).

6. Importance and Implications of Spacecraft-Associated Microbes in Public Culture Collection

This collection of extreme-tolerant bacteria from a NASA mission such as Phoenix and deposition into a publicly accessible microbial culture collection is the first of its kind in the United States. Thus, it is now possible for nonprofit organizations to have access to these extreme-tolerant strains and explore their potential for research and biotechnological applications. Recently, an ESA-funded project collected several extreme-tolerant microbes and placed them in the German Culture Collection (DSMZ; Moissl-Eichinger et al., 2012). Such efforts to isolate, identify, and characterize extremophilic microorganisms will also inform NASA and other national space agencies in the setting of appropriate planetary protection standards for future missions based on an enhanced understanding of relevant extremophilic microbiota. Finally, appropriate cleaning and bioreduction regimes can be developed for outbound spacecraft, based on the limits of survival of extremophiles actually present on spacecraft and associated surfaces. The present study reports the isolation of the radiation-tolerant Deinococcus phoenicis from the Phoenix spacecraft-associated environments (Vaishampayan et al., unpublished data), and such an isolate will be a highly valuable resource for estimating the extent of radiation resistance of organisms on spacecraft. In addition, such extreme-tolerant bacteria will be useful in multiple NASA programs for the development of tools to predict the effect of microorganisms on extraterrestrial environments during potential future human exploration efforts.