Recurrent Isolation of Extremotolerant Bacteria from the Clean Room Where Phoenix Spacecraft Components Were Assembled

Growth of Bacillus subtilis cells, normally adapted at Earth-normal atmospheric pressure (ϳ101.3 kPa), was progressively inhibited by lowering of pressure in liquid LB medium until growth essentially ceased at 2.5 kPa. Growth inhibition was immediately reversible upon return to 101.3 kPa, albeit at a slower rate. A population of B. subtilis cells was cultivated at the near-inhibitory pressure of 5 kPa for 1,000 generations, where a stepwise increase in growth was observed, as measured by the turbidity of 24-h cultures. An isolate from the 1,000-generation population was obtained that showed an increase in fitness at 5 kPa when compared to the ancestral strain or a strain obtained from a parallel population that evolved for 1,000 generations at 101.3 kPa. The results from this preliminary study have implications for understanding the ability of terrestrial microbes to grow in low-pressure environments such as Mars.Microorganisms grow optimally within a characteristic range of fundamental physical parameters such as temperature, osmolarity, pH, and pressure. Cells that can grow at the extreme limits of these parameters are known collectively as extremophiles (reviewed in reference 8). Thus, in extreme environments on Earth there exist halophiles in hypersaline niches, psychrophiles and thermophiles in extreme cold and hot environments, and acidophiles or alkaliphiles in environments of extreme acidic or basic pH. Regarding extremes of pressure, piezophiles (barophiles) have been isolated from high-pressure (hyperbaric) submarine environments and have been studied rather extensively; in addition, high pressure has been shown to exert lethal and inhibitory effects on various microbial systems not normally adapted to high pressure (reviewed in reference 19). On the opposite extreme, there has been very little investigation concerning the survival and growth of microbes under conditions of extremely low atmospheric pressure (hypobaria), primarily because such environments do not exist in nature on the Earth's surface. However, some investigators in the emerging field of astrobiology have become concerned with microbial survival and/or growth at low pressures because (i) the closest potential life-bearing planet, Mars, contains a low-pressure atmosphere, and (ii) the possibility exists that terrestrial microbes could be transferred from Earth to Mars as a result of natural impacts (23) or human spaceflight activities (24). Most of the experiments conducted have concentrated on testing the ability of terrestrial microbes merely to survive in the Mars surface environment (reviewed in references 6, 23, and 28); relatively few experiments have tested the ability of specific Earth microbes to grow or metabolize at reduced pressure (12,14). We previously reported that growth of at least 37 different microorganisms on semisolid agar medium was inhibited at pressures approaching ϳ2.5 to 3.5 kPa (3,4,27,30). (Note that atmospheric pressures at the surfaces of Earth and Mars average ϳ101.3 kPa and ϳ0.7 kPa, respectively [26].) T...

Section: Resultsmentioning

confidence: 99%

Exploring the Low-Pressure Growth Limit: Evolution of Bacillus subtilis in the Laboratory to Enhanced Growth at 5 Kilopascals

Nicholson¹,

Fajardo-Cavazos²,

Fedenko³

et al. 2010

“…Previous studies evaluating the effectiveness of sampling devices for the recovery of microorganisms from clean-room surfaces have used purified bacterial spores (8,9,11,12,29,32,34,39) or pure cultures of vegetative cells (7) as positive controls. However, such controls do not accurately represent the highly diverse microbial populations which can be expected in nearly all environments, including clean rooms (10,15,30,35). Additionally, whenever samples are collected for the purpose of molecular-analysis-based microbial monitoring, results will be affected not only by the presence of living cells but also by dead and decaying cells and spores, cellular debris, and indigenous biomolecules (e.g., nucleic acids, lipids, proteins, carbohydrates).…”

mentioning

confidence: 99%

Evaluation of Procedures for the Collection, Processing, and Analysis of Biomolecules from Low-Biomass Surfaces

Kwan

Cooper

Duc

et al. 2011

To comprehensively assess microbial diversity and abundance via molecular-analysis-based methods, procedures for sample collection, processing, and analysis were evaluated in depth. A model microbial community (MMC) of known composition, representative of a typical low-biomass surface sample, was used to examine the effects of variables in sampling matrices, target cell density/molecule concentration, and cryogenic storage on the overall efficacy of the sampling regimen. The MMC used in this study comprised 11 distinct species of bacterial, archaeal, and fungal lineages associated with either spacecraft or clean-room surfaces. A known cellular density of MMC was deposited onto stainless steel coupons, and after drying, a variety of sampling devices were used to recover cells and biomolecules. The biomolecules and cells/spores recovered from each collection device were assessed by cultivable and microscopic enumeration, and quantitative and speciesspecific PCR assays. rRNA gene-based quantitative PCR analysis showed that cotton swabs were superior to nylon-flocked swabs for sampling of small surface areas, and for larger surfaces, biological sampling kits significantly outperformed polyester wipes. Species-specific PCR revealed differential recovery of certain species dependent upon the sampling device employed. The results of this study empower current and future molecular-analysis-based microbial sampling and processing methodologies.

“…Within category A, rRNA gene abundances remained at consistent levels (Յ10 5 16S rRNA gene copies/m 2 ), with the exception of two samples (GI-15 and GI-17; Table 1) which also gave the highest diversity. Previous studies have demonstrated that whenever spacecraft-associated surface samples yield fewer than ϳ1 ϫ 10 5 16S rRNA gene copies/m 2 , they can be expected to give rise to very few, if any, cultivable cells or spores (13,18). The results of this study support this observation.…”

Section: Resultsmentioning

confidence: 99%

Comparison of Innovative Molecular Approaches and Standard Spore Assays for Assessment of Surface Cleanliness

Cooper

Duc

Probst

et al. 2011

Self Cite

A bacterial spore assay and a molecular DNA microarray method were compared for their ability to assess relative cleanliness in the context of bacterial abundance and diversity on spacecraft surfaces. Colony counts derived from the NASA standard spore assay were extremely low for spacecraft surfaces. However, the PhyloChip generation 3 (G3) DNA microarray resolved the genetic signatures of a highly diverse suite of microorganisms in the very same sample set. Samples completely devoid of cultivable spores were shown to harbor the DNA of more than 100 distinct microbial phylotypes. Furthermore, samples with higher numbers of cultivable spores did not necessarily give rise to a greater microbial diversity upon analysis with the DNA microarray. The findings of this study clearly demonstrated that there is not a statistically significant correlation between the cultivable spore counts obtained from a sample and the degree of bacterial diversity present. Based on these results, it can be stated that validated state-of-the-art molecular techniques, such as DNA microarrays, can be utilized in parallel with classical culture-based methods to further describe the cleanliness of spacecraft surfaces.