Project 6

Over the past decade there has been a profound shift in our understanding of the distribution of microbial life on Earth. Up to 90% of all bacteria and
archaea live in the subsurface based on chemoautotrophic metabolisms; gaining energy through redox reactions and carbon from inorganic trace
gasses. Up to an order of magnitude more of these cells are physically attached to their rock substrates as compared to ‘free-living’ in subsurface
aquifers making them difficult to capture using traditional extraction techniques. Our increased understanding of subsurface biospheres and
litho(auto)trophic microbial communities has facilitated a paradigm shift in our approach for search for life and biosignatures on Mars. While previous and current missions focus on defining habitability and searching for signs of either a present or potential past surficial biosphere, the Martian subsurface remains the longest-lived and largest potential habitable environment on Mars. A better understanding of terrestrial subsurface microbial communities will help inform future astrobiology missions targeting rock-hosted biosignatures on Mars. One the greatest challenges in assessing terrestrial subsurface and rock-hosted microbial communities is low biomass and, consequently, accurately capturing microbial diversity. Terrestrial subsurface communities in Mars analog environments are often characterized by 1) low biomass characterized by rock-associated cells and difficult to lyse (burst such that their DNA can be extracted); 2) a large diversity of rare taxa; and 3) a large proportion of unknown or unclassified taxa. Current methods of extracting nucleic acid are not well optimized for soils or rocks (hosting litho(auto)trophs) and the results are biased towards abundant, easy to lyse, and well-known taxa. To better characterize the in situ diversity of these subsurface communities we need to develop and benchmark extraction techniques that will work across bacterial and archaeal phyla, including recalcitrant, or difficult to lyse, cells. There are several published methods developed for extracting nucleic acid from soil samples, however, many of these studies are ranked by nucleic acid yield rather than recovered diversity and may be biased against rare and recalcitrant microbial taxa. In this project undergraduate students will develop experimental matrices to systematically evaluate several parameters of extraction protocols to develop a starting protocol optimized to maximize the recovered microbial diversity from hypersaline cold spring samples collected from Mars analog permafrost springs on Axel Heigberg Island. Developing these methods helps to both develop protocols and procedures for processing returned samples, but also, genetic diversity studies of terrestrial microbial communities in Mars analogue environments that determine diversity and metabolic potential to inform our understanding of putative extinct or extant subsurface biospheres on Mars.

This project will be broken into two main phases: Phase 1; evaluate pretreatment of soils; and Phase 2; evaluate optimum conditions for enzymatic cell
lysis. Each phase is designed to lead to a specific deliverable that will inform a major step of the nucleic acid extraction protocol. As such, completion
both phases is not required for success and students will learn to develop experimental matrices, develop sterile technique for working with low biomass,
and learn to extract nucleic acid from Mars analog samples within each phase. In all phases samples will be run in triplicate and with positive (using a
mock microbial community supplied by Zymo) and negative (using nucleic acid free beads) controls. Phase 1: Evaluate pretreatment of soils. Microbial
strains isolated from the AHI springs are known to entomb themselves in carbonate minerals, being hypersaline springs, these samples also have
inherently high salt concentrations. These characteristics are challenging for downstream nucleic acid extraction. Cells entombed in carbonate may not
be lysed and therefore their DNA will not be extracted and they will be missing from the final diversity evaluation, ie, extraction will be biased against cells entombed in carbonate. Pretreatment with acid digest will dissolve carbonate minerals, exposing these cells to downstream extraction. High salt
concentrations (and now acidic conditions) interfere with downstream enzymatic digests and DNA binding to extraction columns. To mitigate this, sample washes with phosphate buffered saline (PBS) will dilute salts and neutralize acid. Students will evaluate the effect of different concentrations of dilute HCl and different osmotic strengths of PBS in sequential sample pretreatment steps prior to following the established protocol for Qiagen PowerLyzer Power Soil DNA extraction kits on the recovered microbial diversity following sequencing. Phase 2: evaluate optimum conditions for enzymatic cell lysis. In order to capture recalcitrant cells, multiple methods of cell digestion and lyses are required. In phase 1, students evaluate the efficacy of acid or chemical digestion. In this phase, students will evaluate enzymatic digestion. There are two formulations of enzymatic solutions optimized for low biomass, litho(auto)trophic communities): Metapolyzyme, which contains a collections of enzymes that digest cell walls and Exopolyzyme, which contains a collection of enzymes that digest extrapolymeric substances, or the material that attaches cells to their physical rock substrates. Each specific enzyme in both mixes has a maximum efficiency at a specific optimum pH and temperature combination. Published literature protocols use an average temperature and pH, and different sample types may benefit from optimizing these parameters. Again, using AHI spring samples and the established protocol for Qiagen PowerLyzer Power Soil DNA extraction kits, students will develop experimental matrices to evaluate the effect different temperature and pH combination, optimized for individual enzymes within each mix, has on the recovered microbial diversity This work is expected to take approximately 8-10 hours/week on average.

After the completion of each phase the students will write a formal protocol with sufficient mentorship such that it can be published on Protocols.io. In
addition, the students will prepare a poster presentation of their results (phase 1 effect of acid digest and PBS washing; phase 2 effect of pH and
temperature optimization on enzymatic digest mixes) to present at Winter 27/ Fall 27/ Winter 28 symposium opportunities. The opportunities include:
NAU Biological Symposium, Flagstaff Astronomy Symposium, Arizona Astrobiology Symposium, or the Arizona/Southwest Nevada Regional American
Society for Microbiology (ASM) Branch Conference depending on project progress and student availability. If both phases are successfully completed it is
expected that the students will contribute to a published manuscript to be submitted to one of the following academic journals: Geomicrobiology, Applied
and Environmental Microbiology, Frontiers in Microbiology, or Astrobiology. In total, if both phases are successfully completed the expected products
would be: 2 protocols published on protocols.io, 2 student-led poster presentations at regional symposia, 3 student contribution to an academic journal
article detailing the role of extraction optimization on the recovered diversity of lithoautotrophic terrestrial subsurface microbial communitas. Additionally, there are no systematic studies currently published on the efficacy of Exopolyzyme, depending on results, this may comprise a 2nd publication.