Prokaryotic Diversity and Aqueous Geochemistry of Subsurface Environments of the Death Valley Regional Flow System

Joshua David Sackett


This dissertation summarizes over four years of effort towards the completion of a Ph.D. in Biological Sciences. The work presented in this document covers a broad range of topics, but a central unifying theme is prokaryotic life in the continental subsurface. The work presented in each chapter relied heavily on cultivation-independent methods for assessing prokaryotic communities, including prokaryotic community structure reconstruction from high-throughput sequencing of 16S rRNA gene libraries and single-cell genome analysis of novel uncultivated bacteria.

Chapter 2 examines the aqueous geochemistry and prokaryotic diversity of Devils Hole, a cavernous limnocrene and sole natural habitat for the critically endangered Devils Hole pupfish (Cyprinodon diabolis), and of the Ash Meadows Fish Conservation Facility (AMFCF), a replica of the Devils Hole environment constructed to aid in Devils Hole pupfish propagation and conservation. Comparative analysis of the microbiology and geochemistry of these two locations revealed marked differences: Devils Hole planktonic and sediment samples were dominated by the cyanobacterium Oscillatoria, which proved virtually undetectable at AMFCF. Conversely, predicted heterotrophic organisms in the Verrucomicrobiaceae family dominated AMFCF communities, but were comparatively rare in Devils Hole. Furthermore, bioavailable nitrogen (primarily nitrate) was 5x lower in AMFCF. We propose that the paucity of bioavailable nitrogen in AMFCF is reflected in the prokaryotic community disparity and that the lack of cyanobacteria in AMFCF may ultimately impact survivorship and recruitment of refuge populations of the Devils Hole pupfish.

Chapter 3 examines the spatiotemporal dynamics of surface-colonized prokaryotic communities throughout an 883.5 meter deep borehole near Death Valley, California, USA. 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 high-volume pumping and discrete samples from the static water column) and in situ synthetic and natural sponge colonization experiments by employing aqueous geochemical characterization, thermodynamic modeling, and analysis of 16S rRNA gene libraries. Physical measurements collected at 752 meters depth were indicative of hot, anoxic, circumneutral, and highly reducing conditions. Thermodynamic calculations revealed an energetically austere environment. Hydrogenotrophic sulfate reduction was the most exergonic metabolic reaction modeled. Furthermore, prokaryotic communities of planktonic and attached samples were dominated by yet-to-be cultivated, phylogenetically deeply branching taxa, including Acetothermia and Aminicenantes, Candidatus Desulforudis, Hadesarchaea, and novel lineages in the Nitrospirae family. Our observations suggest that the deep fractured rock ecosystem of the Death Valley Regional Flow System discharge zone is a repository of novel biodiversity and that the sampling of water samples from subsurface environments result in an underestimation of biodiversity.

Chapter 4 examines the metabolic potential of novel uncultured Kiritimatiellae inhabiting deep subsurface fluids of the Death Valley Regional Flow System using a single-cell genomics approach. Genome assemblies from four Kiritimatiellae cells collectively encoded an atypical Embden-Meyerhof-Parnas pathway lacking pyruvate kinase, a nearly complete pentose phosphate pathway, and several of the genes involved in the tricarboxylic acid cycle. Genomes also encoded genes involved in nitrite reduction to ammonia (NrfAH), thiosulfate oxidation to tetrathionate (DoxD), and cytochrome bd ubiquinol oxidase (CydAB), although cytochrome bd ubiquinol oxidase likely serves as a mechanism for removal of dioxygen as has been observed in some obligate anaerobes. Putative geochemical assessment coupled with analysis of genes encoded in genome assemblies suggest the potential for a saccharolytic, fermentative lifestyle with glucose, mannose, and N-acetylglucosamine as likely carbon substrates.

Chapter 5 examines the prokaryotic biogeochemistry of hyporheic zone fluids collected from six samples associated with the McCarran Ranch channel bar (MRCB) in the Truckee River, Nevada, USA. We characterized the prokaryotic community structure, metabolic potential, and aqueous chemistries of flowing river surface water and porewater. The concentrations of potential respiratory electron acceptors were highest in the surface water and riverbed porewater samples and were sequentially depleted (O2, then NO3-, then SO42-) in porewater from the MRCB. Likewise, we observed a shift in metabolic strategy, from aerobic heterotrophy in river surface water, to chemolithotrophy along the inferred hyporheic flow path, as evidenced by shifts in putative metabolic strategies of dominant members of the prokaryotic communities. These data suggest that prokaryotic communities within the MRCB are phylogenetically and metabolically diverse and contribute to biogeochemical cycling in this common yet relatively understudied habitat.