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Microorganisms in groundwater play an important role in aquifer biogeochemical cycles and water quality. However, the mechanisms linking the functional diversity of microbial populations and the groundwater physicochemistry are still not well understood due to the complexity of interactions between surface and subsurface. Here, we used phospholipid fatty acids (PLFAs) relative abundances to link specific biochemical markers within the microbial communities to the spatio-temporal changes of the groundwater physicochemistry. PLFAs were isolated from groundwater of two physicochemically distinct aquifer assemblages in central Germany (Thuringia). The functional diversities of the microbial communities were mainly correlated with groundwater chemistry, including dissolved O2, Fet and NH4+ concentrations. Abundances of PLFAs derived from eukaryotes and potential nitrite oxidizing bacteria (11MeC16:0 as biomarker for Nitrospira moscoviensis) were high at sites with elevated O2 concentration where groundwater recharge supplies both bioavailable organic substrates and NH4+ needed to sustain heterotrophic growth and nitrification processes. In anoxic groundwaters more rich in Fet, PLFAs abundant in sulphate reducing bacteria (SRB), iron-reducing bacteria and fungi increased with Fet and HCO3− concentrations suggesting the occurrence of active iron-reduction and the possible role of fungi in meditating iron solubilisation and transport in those aquifer domains. In NH4+ richer anoxic groundwaters, anammox bacteria and SRB- derived PLFAs increased with NH4+ concentration further evidencing the dependence of the anammox process on ammonium concentration and potential links between SRB and anammox bacteria. Additional support of the PLFA-based bacterial communities was found in DNA and RNA-based Illumina MiSeq amplicon sequencing of bacterial 16S rRNA genes, which evidenced high predominance of nitrite-oxidizing bacteria Nitrospira e.g. Nitrospira moscoviensis in oxic zones of the aquifers and of anammox bacteria in NH4+ richer anoxic groundwater. Higher relative abundances of sequence reads in the RNA-based data sets affiliated with iron-reducing bacteria in Fet richer groundwater supported the occurrence of active dissimilatory iron-reduction. The functional diversity of the microbial communities in these biogeochemically distinct groundwater assemblages can be largely attributed to the redox conditions linked to changes in bioavailable substrates and input of substrates with the seepage. Our results demonstrate the power of complementary information derived from PLFA-based and sequencing-based approaches

The terrestrial subsurface contains nearly all of Earth’s freshwater reserves and harbours the majority of our planet’s total prokaryotic biomass. Although genetic surveys suggest these organisms rely on in situ carbon fixation, rather than the photosynthetically derived organic carbon transported from surface environments, direct measurements of carbon fixation in the subsurface are absent. Using an ultra-low level 14C-labelling technique, we estimate in situ carbon fixation rates in a carbonate aquifer. We find these rates are similar to those measured in oligotrophic marine surface waters and up to six-fold greater than those observed in the lower euphotic zone. Our empirical carbon fixation rates agree with nitrification rate data. Metagenomic analyses reveal abundant putative chemolithoautotrophic members of an uncharacterized order of Nitrospiria that may be behind the carbon fixation. On the basis of our determined carbon fixation rates, we conservatively extrapolate global primary production in carbonate groundwaters (10% of global reserves) to be 0.11 Pg carbon per year. These rates fall within the range found for oligotrophic marine surface waters, indicating a substantial contribution of in situ primary production to subsurface ecosystem processes. We further suggest that, just as phototrophs are for marine biogeochemical cycling, such subsurface carbon fixation is potentially foundational to subsurface trophic webs.

Despite the high relevance of anaerobic ammonium oxidation (anammox) for nitrogen loss from marine systems, its relative importance compared to denitrification has less been studied in freshwater ecosystems, and our knowledge is especially scarce for groundwater. Surprisingly, phospholipid fatty acids (PLFA)-based studies identified zones with potentially active anammox bacteria within two superimposed pristine limestone aquifer assemblages of the Hainich Critical Zone Exploratory (CZE; Germany). We found anammox to contribute an estimated 83% to total nitrogen loss in suboxic groundwaters of these aquifer assemblages at rates of 3.5–4.7 nmol L−1 d−1, presumably favored over denitrification by low organic carbon availability. Transcript abundances of hzsA genes encoding hydrazine synthase exceeded nirS and nirK transcript abundances encoding denitrifier nitrite reductase by up to two orders of magnitude, providing further support of a predominance of anammox. Anammox bacteria, dominated by groups closely related to Cand. Brocadia fulgida, constituted up to 10.6% of the groundwater microbial community and were ubiquitously present across the two aquifer assemblages with indication of active anammox bacteria even in the presence of 103 μmol L−1 oxygen. Co-occurrence of hzsA and amoA gene transcripts encoding ammonia mono-oxygenase suggested coupling between aerobic and anaerobic ammonium oxidation under suboxic conditions. These results clearly demonstrate the relevance of anammox as a key process driving nitrogen loss from oligotrophic groundwater environments, which might further be enhanced through coupling with incomplete nitrification.