Phylogenetic Characterization and Description of Novel-tolerant Bacillus Species Isolated from Spacecraft Assembly Facility

Microbial biofilms representing both Gram-positive and Gram-negative bacteria were artificially coated onto aluminum metal surfaces that are commonly used in building spacecraft. Changes in bacterial numbers were monitored over time on metal test sections by conventional spread plate technique and polymerase chain reaction (PCR). Conventional standard plating technique did not recover desiccated bacterial biomass from either glass or aluminum metal surfaces even with a density of 10⁶ cells per coupon. However, whole-cell PCR (without extracting DNA) showed a positive signal for a density of 10⁵ dehydrated microbial cells. Electron and epifluorescent microscopic examinations revealed that bacteria entered a non-cultivable state once the water activity was reduced to minimum. However, a simple DNA extraction protocol combined with PCR amplification of a specific 16S rDNA fragment improved the recovery of non-viable desiccated microbial biofilm from the metal test sections. In ongoing investigations to map and archive the microbial footprints in various components of spacecraft and its accessories, we examined the microbial populations of the JPL-Spacecraft Assembly Facility (SAF). We exposed witness plates that are made up of spacecraft materials and/or painted with spacecraft-quality paints for ~7 to 9 months. In the initial studies reported here, we examined the total cultivable aerobic heterotrophs. The results showed that the witness plates coated with spacecraft quality paints attracted more dust particles than the non-coated stainless steel witness plates. Among four paints tested, witness plates coated with NS43G (an off-white conductive paint [silicate binder]) accumulated the highest number of particles, hence attracting more cultivable microbes and spore-formers. The microbiological examination revealed that the SAF High Bay-1 harbors mainly Gram-positive microbes, the majority being spore-forming Bacillus species. The phylogenetic relationships among these heat-tolerant microbes were examined using a battery of morphological, physiological, molecular and chemotaxonomic characterizations. Using phenotypic characterization alone, only 9 strains were identified. By 16S rDNA analysis, the isolates fell into seven clades: Bacillus licheniformis, B. pumilus, B. cereus, B. circulans, Staphylococcus capitis, Planococcus sp. and Micrococcus lylae.