Diversity of anaerobic microbes in spacecraft assembly clean rooms

Abstract

Although the cultivable and noncultivable microbial diversity of spacecraft assembly clean rooms has been previously documented using conventional and state-of-the-art molecular techniques, the occurrence of obligate anaerobes within these clean rooms is still uncertain. Therefore, anaerobic bacterial communities of three clean-room facilities were analyzed during assembly of the Mars Science Laboratory rover. Anaerobic bacteria were cultured on several media, and DNA was extracted from suitable anaerobic enrichments and examined with conventional 16S rRNA gene clone library, as well as high-density phylogenetic 16S rRNA gene microarray (PhyloChip) technologies. The culture-dependent analyses predominantly showed the presence of clostridial and propionibacterial strains. The 16S rRNA gene sequences retrieved from clone libraries revealed distinct microbial populations associated with each clean-room facility, clustered exclusively within gram-positive organisms. PhyloChip analysis detected a greater microbial diversity, spanning many phyla of bacteria, and provided a deeper insight into the microbial community structure of the clean-room facilities. This study presents an integrated approach for assessing the anaerobic microbial population within clean-room facilities, using both molecular and cultivation-based analyses. The results reveal that highly diverse anaerobic bacterial populations persist in the clean rooms even after the imposition of rigorous maintenance programs and will pose a challenge to planetary protection implementation activities.

FIG. 1.

Rarefaction analysis of anaerobic bacterial diversity from several spacecraft assembly facilities. The coverage values (C) for each clone library are given in parentheses. Rarefaction curves of standard and gradient PCR samples are depicted with asterisks and in italics, respectively.

FIG. 2.

Principal coordinate analyses showing the relationships between samples.

FIG. 3.

Overview of phyla retrieved from the DNA microarray analysis. Numbers in parentheses on the x axis after each sampling event are the number of OTU counts retrieved. Families having a minimum of 10 OTU counts are given in parentheses. Underlined families were also detected in conventional clone libraries of related samplings. The “others” category denotes the phyla having fewer than 10 OTU counts as well as unclassified members.