signature=7be306a303cf7d5bf08da1e092a3a5e9,Development of a model community to evaluate efficient re...

Abstract

Despite advances in the specificity and sensitivity of molecular biological technologies, the ef-ficient recovery of DNA from low-biomass samples remains extremely challenging. Optimal methods to extract these biomolecules should 1) achieve the greatest total yield; 2) reflect comprehensive microbial diversity of the sampled environment; and 3) assert reproducible re-sults. For an in-depth assessment of the wide spectrum of microorganisms present in the low-biomass spacecraft assembly clean room environment, technologies facilitating efficient col-lection, sample processing, and analysis are needed. To this end, a homogenous mixture of equal concentrations of 11 distinct microbial lineages having significant relevance to planetary protection (bacteria, archaea, and fungi; aerobes and anaerobes; cells and spores; rods and cocci) was prepared. Suitable aliquots of this "model" community were then characterized us-ing a parallel set of downstream molecular analyses which revealed the level of microbial DNA, extracellular DNA, dissolved organic matter, and particulate non-microbial substances present in the community. Appropriate subsamples of this model community were dried on stainless steel metal surfaces, and procedures targeting the efficient removal and recovery of community member DNAs were evaluated. The collection and release of genetic materials from cotton and flocked nylon swabs were compared. Several automated nucleic acid extraction methods were assessed for both total DNA yield and conservation of microbial community structure. Uni-versal small subunit rrn Q-PCR, species-specific Q-PCR, and DNA microarray methodologies were used in concert to estimate the recovery of both individual members, and the community as a whole. Results of this study will enable consideration of future planetary protection policy amendments based on modern molecular methods.