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Fluids circulating through oceanic crust play important roles in global biogeochemical cycling mediated by their microbial inhabitants, but studying these sites is challenged by sampling logistics and low biomass. Borehole observatories installed at the North Pond study site on the western flank of the Mid-Atlantic Ridge have enabled investigation of the microbial biosphere in cold, oxygenated basaltic oceanic crust. Here we test a methodology that applies redox-sensitive fluorescent molecules for flow cytometric sorting of cells for single cell genomic sequencing from small volumes of low biomass (approximately 10 3  cells ml –1 ) crustal fluid. We compare the resulting genomic data to a recently published paired metagenomic and metatranscriptomic analysis from the same site. Even with low coverage genome sequencing, sorting cells from less than one milliliter of crustal fluid results in similar interpretation of dominant taxa and functional profiles as compared to ‘omics analysis that typically filter orders of magnitude more fluid volume. The diverse community dominated by Gammaproteobacteria, Bacteroidetes, Desulfobacterota, Alphaproteobacteria, and Zetaproteobacteria, had evidence of autotrophy and heterotrophy, a variety of nitrogen and sulfur cycling metabolisms, and motility. Together, results indicate fluorescence activated cell sorting methodology is a powerful addition to the toolbox for the study of low biomass systems or at sites where only small sample volumes are available for analysis.

Oceanic Crustal Fluid Single Cell Genomics Complements Metagenomic and Metatranscriptomic Surveys With Orders of Magnitude Less Sample Volume