Microbial assembly and metabolic potential in the Subsurface Critical Zone (SCZ) are substantially impacted by subsurface geochemistry and hydrogeology, selecting for microbes distinct from those in surficial soils. In this study, we integrated metagenomics and geochemistry to elucidate how microbial composition and metabolic potential are shaped and impacted by vertical variations in geochemistry and hydrogeology in terrestrial subsurface sediment. A sediment core from an uncontaminated, pristine well at Oak Ridge Field Research Center in Oak Ridge, Tennessee, including the shallow subsurface, vadose zone, capillary fringe, and saturated zone, was used in this study. Our results showed that subsurface microbes were highly localized, and communities were rarely inter-connected. Microbial community composition as well as metabolic potential in carbon and nitrogen cycling varied even over very short vertical distances. Further analyses indicated strong depth-related co-variation of community composition with a subset of 12 environmental variables. Analysis of dissolved organic carbon (DOC) quality via ultrahigh resolution mass spectrometry suggested that the SCZ was generally a low-carbon environment with the relative portion of labile DOC decreasing and that of recalcitrant DOC increasing along the depth, selecting microbes from copiotrophs to oligotrophs and also impacting the microbial metabolic potential in C cycle. Our study demonstrates that sediment geochemistry and hydrogeology are vital in selecting distinct microbial population and metabolism in the SCZ.
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