What lives in regolith simulants and why it matters for biological in‐situ resource utilization research

Regolith simulants are an essential tool for research in space biology, but criteria and protocols are limited for selecting and utilizing simulants in biological research. One critical issue regarding this is a poor understanding of existing simulant microbiomes. Here, we present an initial evaluation of the microbiome size and composition of lunar and martian regolith simulants, the effectiveness of and alterations caused by sterilization, and organic removal and test methods for improving DNA extraction from simulant materials. Numerous culturable non‐fastidious and mesophilic bacteria and fungi were detected in all simulants. No viable colony‐forming units (CFUs) were detected after the sterilization of small simulant volumes (500 mL). When autoclaved in large volumes (5 L), 1 × 10 3 to 6 × 10 4 CFU were still detected in the center of most autoclaved samples. Both autoclave and dry heat resulted in hydration or dehydration of salts. Higher furnace temperatures altered the structural properties of clays. DNA extractions with the addition of concentrated phosphate buffer solution had the highest concentrations and quality of extracted DNA from the simulants. The results of these initial evaluations demonstrate that simulants are not sterile materials, that autoclaving may not effectively sterilize simulants at bulk volumes, that specific simulant components show alterations detectable by X‐ray diffraction when autoclaved (120°C) or furnace‐heated (400°C), and that DNA extraction techniques can be adapted to better evaluate simulant microbiomes. This provides a starting point for the further evaluation of simulant microbiomes and the much‐needed establishment of guidelines for the selection, storage, and handling of simulant materials applied to biological research.