Nitrogen Loss from Pristine Carbonate-Rock Aquifers of the Hainich Critical Zone Exploratory (Germany) Is Primarily Driven by Chemolithoautotrophic Anammox Processes

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.