Cara Magnabosco

A metagenomic window into carbon metabolism at 3 km depth in Precambrian continental crust.

Subsurface microbial communities comprise a significant fraction of the global prokaryotic biomass; however, the carbon metabolisms that support the deep biosphere have been relatively unexplored. In order to determine the predominant carbon metabolisms within a 3-km deep fracture fluid system accessed via the Tau Tona gold mine (Witwatersrand Basin, South Africa), metagenomic and thermodynamic analyses were combined. Within our system of study, the energy-conserving reductive acetyl-CoA (Wood-Ljungdahl) pathway was found to be the most abundant carbon fixation pathway identified in the metagenome. Carbon monoxide dehydrogenase genes that have the potential to participate in (1) both autotrophic and heterotrophic metabolisms through the reversible oxidization of CO and subsequent transfer of electrons for sulfate reduction, (2) direct utilization of H2 and (3) methanogenesis were identified. The most abundant members of the metagenome belonged to Euryarchaeota (22%) and Firmicutes (57%)—by far, the highest relative abundance of Euryarchaeota yet reported from deep fracture fluids in South Africa and one of only five Firmicutes-dominated deep fracture fluids identified in the region. Importantly, by combining the metagenomics data and thermodynamic modeling of this study with previously published isotopic and community composition data from the South African subsurface, we are able to demonstrate that Firmicutes-dominated communities are associated with a particular hydrogeologic environment, specifically the older, more saline and more reducing waters.

Comparisons of the composition and biogeographic distribution of the bacterial communities occupying South African thermal springs with those inhabiting deep subsurface fracture water.

South Africa has numerous thermal springs that represent topographically driven meteoric water migrating along major fracture zones. The temperature (40–70°C) and pH (8–9) of the thermal springs in the Limpopo Province are very similar to those of the low salinity fracture water encountered in the South African mines at depths ranging from 1.0 to 3.1 km. The major cation and anion composition of these thermal springs are very similar to that of the deep fracture water with the exception of the dissolved inorganic carbon and dissolved O2, both of which are typically higher in the springs than in the deep fracture water. The in situ biological relatedness of such thermal springs and the subsurface fracture fluids that feed them has not previously been evaluated. In this study, we evaluated the microbial diversity of six thermal spring and six subsurface sites in South Africa using high-throughput sequencing of 16S rRNA gene hypervariable regions. Proteobacteria were identified as the dominant phylum within both subsurface and thermal spring environments, but only one genera, Rheinheimera, was identified among all samples. Using Morisita similarity indices as a metric for pairwise comparisons between sites, we found that the communities of thermal springs are highly distinct from subsurface datasets. Although the Limpopo thermal springs do not appear to provide a new window for viewing subsurface bacterial communities, we report that the taxonomic compositions of the subsurface sites studied are more similar than previous results would indicate and provide evidence that the microbial communities sampled at depth are more correlated to subsurface conditions than geographical distance.

Phylogeny and phylogeography of functional genes shared among seven terrestrial subsurface metagenomes reveal N-cycling and microbial evolutionary relationships

Comparative studies on community phylogenetics and phylogeography of microorganisms living in extreme environments are rare. Terrestrial subsurface habitats are valuable for studying microbial biogeographical patterns due to their isolation and the restricted dispersal mechanisms. Since the taxonomic identity of a microorganism does not always correspond well with its functional role in a particular community, the use of taxonomic assignments or patterns may give limited inference on how microbial functions are affected by historical, geographical and environmental factors. With seven metagenomic libraries generated from fracture water samples collected from five South African mines, this study was carried out to (1) screen for ubiquitous functions or pathways of biogeochemical cycling of CH4, S, and N; (2) to characterize the biodiversity represented by the common functional genes; (3) to investigate the subsurface biogeography as revealed by this subset of genes; and (4) to explore the possibility of using metagenomic data for evolutionary study. The ubiquitous functional genes are NarV, NPD, PAPS reductase, NifH, NifD, NifK, NifE, and NifN genes. Although these eight common functional genes were taxonomically and phylogenetically diverse and distinct from each other, the dissimilarity between samples did not correlate strongly with geographical or environmental parameters or residence time of the water. Por genes homologous to those of Thermodesulfovibrio yellowstonii detected in all metagenomes were deep lineages of Nitrospirae, suggesting that subsurface habitats have preserved ancestral genetic signatures that inform the study of the origin and evolution of prokaryotes.

An oligotrophic deep-subsurface community dependent on syntrophy is dominated by sulfur-driven autotrophic denitrifiers

Significance Microorganisms are known to live in the deep subsurface, kilometers below the photic zone, but the community-wide metabolic networks and trophic structures (the organization of their energy and nutritional hierarchy) remain poorly understood. We show that an active subsurface lithoautotrophic microbial ecosystem (SLiME) under oligotrophic condition exists. Taxonomically and metabolically diverse microorganisms are supported, with sulfur-driven autotrophic denitrifiers predominating in the community. Denitrification is a highly active process in the deep subsurface that evaded recognition in the past. This study highlights the critical role of metabolic cooperation, via syntrophy between subsurface microbial groups, for the survival of the whole community under the oligotrophic conditions that dominate in the subsurface. Subsurface lithoautotrophic microbial ecosystems (SLiMEs) under oligotrophic conditions are typically supported by H2. Methanogens and sulfate reducers, and the respective energy processes, are thought to be the dominant players and have been the research foci. Recent investigations showed that, in some deep, fluid-filled fractures in the Witwatersrand Basin, South Africa, methanogens contribute <5% of the total DNA and appear to produce sufficient CH4 to support the rest of the diverse community. This paradoxical situation reflects our lack of knowledge about the in situ metabolic diversity and the overall ecological trophic structure of SLiMEs. Here, we show the active metabolic processes and interactions in one of these communities by combining metatranscriptomic assemblies, metaproteomic and stable isotopic data, and thermodynamic modeling. Dominating the active community are four autotrophic β-proteobacterial genera that are capable of oxidizing sulfur by denitrification, a process that was previously unnoticed in the deep subsurface. They co-occur with sulfate reducers, anaerobic methane oxidizers, and methanogens, which each comprise <5% of the total community. Syntrophic interactions between these microbial groups remove thermodynamic bottlenecks and enable diverse metabolic reactions to occur under the oligotrophic conditions that dominate in the subsurface. The dominance of sulfur oxidizers is explained by the availability of electron donors and acceptors to these microorganisms and the ability of sulfur-oxidizing denitrifiers to gain energy through concomitant S and H2 oxidation. We demonstrate that SLiMEs support taxonomically and metabolically diverse microorganisms, which, through developing syntrophic partnerships, overcome thermodynamic barriers imposed by the environmental conditions in the deep subsurface.

Fluctuations in populations of subsurface methane oxidizers in coordination with changes in electron acceptor availability

ABSTRACT The concentrations of electron donors and acceptors in the terrestrial subsurface biosphere fluctuate due to migration and mixing of subsurface fluids, but the mechanisms and rates at which microbial communities respond to these changes are largely unknown. Subsurface microbial communities exhibit long cellular turnover times and are often considered relatively static—generating just enough ATP for cellular maintenance. Here, we investigated how subsurface populations of CH4 oxidizers respond to changes in electron acceptor availability by monitoring the biological and geochemical composition in a 1339 m‐below‐land‐surface (mbls) fluid‐filled fracture over the course of both longer (2.5 year) and shorter (2‐week) time scales. Using a combination of metagenomic, metatranscriptomic, and metaproteomic analyses, we observe that the CH4 oxidizers within the subsurface microbial community change in coordination with electron acceptor availability over time. We then validate these findings through a series of 13C‐CH4 laboratory incubation experiments, highlighting a connection between composition of subsurface CH4 oxidizing communities and electron acceptor availability.

The case for a dynamical subsurface ecosystem

The introduction and concentration of electron donors and acceptors in the subsurface biosphere is controlled by the mixing of subsurface fluids, but the mechanisms and rates at which microbial communities respond to changes induced by fluid mixing and transport are relatively unknown. Subsurface microbial ecosystems whose estimated doubling times range from 3,000 years are often considered to be relatively static. Despite marked changes in geochemistry over a 1-year period, the bacterial community inhabiting a 1339 m below land surface (mbls) fracture (Be326) remained largely unchanged and exhibited PLFA isotopic signatures consistent with the accumulation of 13C-DIC impacted by the microbial oxidation of CH4. These CH4 oxidizing (MO) bacteria and archaea are an essential link between the Be326 subsurface carbon cycle and microbial community and were hypothesized to contain members of the community that are most sensitive to environmental change. To evaluate this hypothesis, we used a combination of high throughput sequence analysis methods (DNA, RNA, and protein) and geochemical monitoring of Be326s in situ fracture fluids over the course of both longer (2.5 year) and shorter (2-week) timescales and validated our findings through a series of 13C-CH4 laboratory enrichment experiments. We show that Be326s MO organisms responded to changes in electron donor and acceptor availability in their natural subsurface habitat and under laboratory conditions over extended periods of time. These results provide the most definitive evidence to date that, like the marine subsurface, CH4 oxidation occurs and is an integral component of the deep terrestrial subsurface carbon cycle. Further, the responsiveness of this component of the microbial community to changes in geochemistry illustrates a more dynamic subsurface ecosystem than previously understood.The concentrations of electron donors and acceptors in the terrestrial subsurface biosphere fluctuate due to migration and mixing of subsurface fluids, but the mechanisms and rates at which microbial communities respond to these changes are largely unknown. Subsurface microbial communities exhibit long cellular turnover times and are often considered relatively static—generating just enough ATP for cellular maintenance. Here, we investigated how subsurface populations of CH4 oxidizers respond to changes in electron acceptor availability by monitoring the biological and geochemical composition in a 1,339 meters-below-land-surface (mbls) fluid-filled fracture over the course of both longer (2.5 year) and shorter (2-week) time scales. Using a combination of metagenomic, metatranscriptomic, and metaproteomic analyses, we observe that the CH4 oxidizers within the subsurface microbial community change in coordination with electron acceptor availability over time. We then validate these findings through a series of 13C-CH4 laboratory incubation experiments, highlighting a connection between composition of subsurface CH4 oxidizing communities and electron acceptor availability.

The biomass and biodiversity of the continental subsurface

Despite accounting for a significant portion of the Earth’s prokaryotic biomass, controls on the abundance and biodiversity of microorganisms residing in the continental subsurface are poorly understood. To redress this, we compiled cell concentration and microbial diversity data from continental subsurface localities around the globe. Based on considerations of global heat flow, surface temperature, depth and lithology, we estimated that the continental subsurface hosts 2 to 6 × 1029 cells and found that other variables such as total organic carbon and groundwater cellular abundances do not appear to be predictive of cell concentrations in the continental subsurface. Although we were unable to identify a reliable predictor of species richness in the continental subsurface, we found that bacteria are more abundant than archaea and that their community composition was correlated to sample lithology. Using our updated continental subsurface cellular estimate and existing literature, we estimate that the total global prokaryotic biomass is approximately 23 to 31 Pg of carbon C (PgC), roughly 4 to 10 times less than previous estimates.The abundance of microorganisms in the continental subsurface may have been overestimated, according to a review compilation of data from subsurface localities around the globe.