Spatiotemporal microbial community dynamics of in situ sponge colonization experiments in a deep continental borehole. Spatiotemporal microbial community dynamics of in situ sponge colonization experiments in a deep continental borehole

BLM1 is an 883.5-m-deep monitoring borehole near Death Valley, California, USA, that intercepts fault-associated paleometeoric water from the discharge zone of the Death Valley Regional Flow System (DVRFS): a fractured carbonate rock aquifer that underlies much of Southern Nevada and Eastern California. The borehole is cased in low-carbon steel to a depth of 750 m, below which limited uncased completion is currently accessible, providing a unique window into the DVRFS. The aim of this study was to develop a better understanding of fracture-associated microbial communities in BLM1 through a combination of both planktonic characterizations (from hydrologic pumping tests and discrete samples) and in situ synthetic and natural sponge colonization experiments by employing aqueous geochemical characterization, thermodynamic modeling, and Illumina sequencing of 16S rRNA gene libraries. Physical measurements collected along the vertical profile of BLM1 show a transition from cool (25°C), suboxic (11.9% saturation), basic (pH 9.3), and oxidizing (58 mV) conditions at 30 meters depth to hot (57°C), anoxic, circumneutral (pH 6.92), and highly reducing (-242 mV) conditions at 752 meters depth. Thermodynamic calculations revealed many exergonic metabolic reactions involving the reduction of SO42-, NO32-, and O2, although when normalized per mole of limiting reactant and per kg of fluid (energy density), the austerity of energy availability in the subsurface became clear. Our analysis of planktonic and surface-colonized microorganisms throughout BLM1, a window into the deep terrestrial biosphere, has revealed microbial communities dominated by yet-to-be cultivated, phylogenetically deeply branching taxa, including candidate division Acetothermia, candidate division Aminicenantes, Candidatus Desulforudis, Hadesarchaea, and novel lineages in the Nitrospirae family. Colonized sponge and planktonic communities from discrete water samples were more diverse and taxonomically distinct when compared with pumped samples. Hierarchical clustering of abundance-weighted UniFrac distances revealed clustering by sample type (e.g.: pumped, bailed, synthetic sponge, and natural sponge communities clustered independently of one another). Mantel tests and non-metric multidimensional scaling (NMDS) ordinations of UniFrac distances revealed that sponge-colonized microbial communities were structured spatially (by depth). NMDS ordination also revealed similarity between sponge-colonized and planktonic microbial communities obtained via discrete sampler. Natural sea sponges and co-located synthetic sponges were colonized primarily by Aminicenantes and Thermotogae, which were comparatively rare in all other samples, suggesting that deep subsurface microbial communities are active and respond to inputs of fixed carbon and nitrogen. Our observations suggest that the deep terrestrial biosphere is a repository of novel diversity and that water samples from subsurface environments represent just a fraction of the diversity harbored in subsurface biofilms.