In Situ Real-Time Monitoring for Aseptic Drilling: Lessons Learned from the Atacama Rover Astrobiology Drilling Studies Contamination Control Strategy and Implementation and Application to the Icebreaker Mars Life Detection Mission.

In 2019, the Atacama Rover Astrobiology Drilling Studies (ARADS) project field-tested an autonomous rover-mounted robotic drill prototype for a 6-Sol life detection mission to Mars (Icebreaker). ARADS drilled Mars-like materials in the Atacama Desert (Chile), one of the most life-diminished regions on Earth, where mitigating contamination transfer into life-detection instruments becomes critical. Our Contamination Control Strategy and Implementation (CCSI) for the Sample Handling and Transfer System (SHTS) hardware (drill, scoop and funnels) included out-of-simulation protocol testing (out-of-sim) for hardware decontamination and verification during the 6-Sol simulation (in-sim). The most effective five-step decontamination combined safer-to-use sterilants (3%_hydrogen-peroxide-activated 5%_sodium-hypochlorite), and in situ real-time verification by adenosine triphosphate (ATP) and Signs of Life Detector (SOLID) Fluorescence Immunoassay for characterization hardware bioburden and airborne contaminants. The 20- to 40-min protocol enabled a 4-log bioburden reduction down to <0.1 fmoles ATP detection limit (funnels and drill) to 0.2-0.7 fmoles (scoop) of total ATP. The (post-cleaning) hardware background was 0.3 to 1-2 attomoles ATP/cm2 (cleanliness benchmark background values) equivalent to ca. 1-10 colony forming unit (CFU)/cm2. Further, 60-100% of the in-sim hardware background was ≤3-4 bacterial cells/cm2, the threshold limit for Class <7 aseptic operations. Across the six Sols, the flux of airborne contaminants to the drill sites was ∼5 and ∼22 amoles ATP/(cm2·day), accounting for an unexpectedly high Fluorescence Intensity (FI) signal (FI: ∼6000) against aquatic cyanobacteria, but negligible anthropogenic contribution. The SOLID immunoassay also detected microorganisms from multiple habitats across the Atacama Desert (anoxic, alkaline/acidic microenvironments in halite fields, playas, and alluvial fans) in both airborne and post-cleaning hardware background. Finally, the hardware ATP background was 40-250 times lower than the ATP in cores. Similarly, the FI peaks (FImax) against the microbial taxa and molecular biomarkers detected in the post-cleaned hardware (FI: ∼1500-1600) were 5-10 times lower than biomarkers in drilled sediments, excluding significant interference with putative biomarker found in cores. Similar protocols enable the acquisition of contamination-free materials for ultra-sensitive instruments analysis and the integrity of scientific results. Their application can augment our scientific knowledge of the distribution of cryptic life on Mars-like grounds and support life-detection robotic and human-operated missions to Mars.

Life Detection and Microbial Biomarker Profiling with Signs of Life Detector-Life Detector Chip During a Mars Drilling Simulation Campaign in the Hyperarid Core of the Atacama Desert.

The low organic matter content in the hyperarid core of the Atacama Desert, together with abrupt temperature shifts and high ultraviolet radiation at its surface, makes this region one of the best terrestrial analogs of Mars and one of the best scenarios for testing instrumentation devoted to in situ planetary exploration. We have operated remotely and autonomously the SOLID-LDChip (Signs of Life Detector-Life Detector Chip), an antibody microarray-based sensor instrument, as part of a rover payload during the 2019 NASA Atacama Rover Astrobiology Drilling Studies (ARADS) Mars drilling simulation campaign. A robotic arm collected drilled cuttings down to 80 cm depth and loaded SOLID to process and assay them with LDChip for searching for molecular biomarkers. A remote science team received and analyzed telemetry data and LDChip results. The data revealed the presence of microbial markers from Proteobacteria, Acidobacteria, Bacteroidetes, Actinobacteria, Firmicutes, and Cyanobacteria to be relatively more abundant in the middle layer (40-50 cm). In addition, the detection of several proteins from nitrogen metabolism indicates a pivotal role in the system. These findings were corroborated and complemented on "returned samples" to the lab by a comprehensive analysis that included DNA sequencing, metaproteomics, and a metabolic reconstruction of the sampled area. Altogether, the results describe a relatively complex microbial community with members capable of nitrogen fixation and denitrification, sulfur oxidation and reduction, or triggering oxidative stress responses, among other traits. This remote operation demonstrated the high maturity of SOLID-LDChip as a powerful tool for remote in situ life detection for future missions in the Solar System.

Assessment of Automated Nucleic Acid Extraction Systems in Combination with MinION Sequencing As Potential Tools for the Detection of Microbial Biosignatures.

The utilization of nanopore technologies for the detection of organic biogenic compounds has garnered significant focus in recent years. Oxford Nanopore Technologies' (ONT) MinION instrument, which can detect and sequence nucleic acids (NAs), is one such example. These technologies have much promise for unambiguous life detection but require significant development in terms of methods for extraction and preparation of NAs for biosignature detection and their feasibility for use in astrobiology-focused field missions. In this study, we tested pre-existing, automated, or semiautomated NA extraction technologies, coupled with automated ONT VolTRAX NA sample preparation, and verification with Nanopore MinION sequencing. All of the extraction systems tested (SuperFastPrep2, ClaremontX1, and SOLID-Sample Preparation Unit) showed potential for extracting DNA from Canadian High Arctic environments analogous to Mars, Europa, and Enceladus, which could subsequently be detected and sequenced with the MinION. However, they differed with regard to efficacy, yield, purity, and sequencing and annotation quality. Overall, bead beating-based systems performed the best for these parameters. In addition, we showed that the MinION could sequence unpurified DNA contained in crude cell lysates. This is valuable from an astrobiology perspective because purification steps are time-consuming and complicate the requirements for an automated extraction and life detection system. Our results indicate that semiautomated NA extraction and preparation technologies hold much promise, and with increased optimization and automation could be coupled to a larger platform incorporating nanopore detection and sequencing of NAs for life detection applications.