Single-cell analysis reveals an active and heterotrophic microbiome in the Guaymas Basin deep subsurface with significant heterotrophic inorganic carbon fixation

Abstract

The marine subsurface is a long-term sink of atmospheric carbon dioxide with significant implications for climate on geologic timescales. Subsurface microbial cells can either enhance or reduce the potential for the subsurface to sequester carbon, depending on their metabolic activity. However, the activity of subsurface microbes is rarely measured, leaving their role in biogeochemical cycling poorly characterized. Here, we used nanoscale secondary ion mass spectrometry to quantify anabolic activity in 3,203 individual cells from the thermally altered deep subsurface in the Guaymas Basin, Mexico (3-75 m below the seafloor, 0-14 C). We observed that a large majority of cells were active (83-100%), although rates of biomass generation were low, suggesting cellular maintenance rather than doubling. Mean single-cell activity decreased with increasing sediment depth and temperature, and was most strongly correlated with porewater sulfate concentrations. Intracommunity heterogeneity in cell-specific activity decreased with increasing sediment depth and age. Using a dual-isotope labelling approach we determined that all active cells analyzed at all depths were heterotrophic. We detected and quantified inorganic carbon assimilation by heterotrophs and found that it contributes on average at least 5% of total heterotrophic biomass carbon in this community. Our results therefore suggest that the deep marine biosphere at Guaymas Basin is largely active and contributes to subsurface carbon cycling primarily by assimilating organic carbon but also by mediating heterotrophic inorganic carbon fixation. Heterotrophic assimilation of inorganic carbon may be a small yet significant and widespread underappreciated source of labile carbon in the global subsurface. ImportanceThe global subsurface is the largest reservoir of microbial life on the planet yet remains poorly characterized. The activity of life in this realm has implications for long-term elemental cycling, particularly of carbon, as well as how life survives in extreme environments. Here, we recovered cells from the deep subsurface of the Guaymas Basin and investigated the level and distribution of activity, the physicochemical drivers of activity, and the relative significance of organic versus inorganic carbon to subsurface biomass. Using a sensitive single-cell assay we find that the majority of cells are active, that activity is likely driven by availability of energy, and that while organic carbon supplies most cellular carbon, inorganic carbon also contributes. We additionally find that the inorganic carbon assimilation observed was mediated by heterotrophs, not autotrophs, highlighting the importance of this often overlooked mode of carbon assimilation in the subsurface and beyond.