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

Abstract

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.

Biogeochemical Regimes in Shallow Aquifers Reflect the Metabolic Coupling of the Elements Nitrogen, Sulfur, and Carbon

“…1A). Clusters 4 and 5, both belonging to the HTU (Hainich transect upper aquifer assemblage) complex, are anoxic and feature lower sulfate levels in comparison and increased ammonium concentrations and are commonly associated with sulfate reduction and anaerobic ammonium oxidation (anammox), respectively (15)(16)(17). Phospholipid-derived fatty acid (PLFA) patterns (17) and marker gene studies (18) used to assess microbial community composition and functional clades partially confirmed the hydrochemical clustering.…”

“…A strong involvement of "Candidatus Brocadiales" in nitrogen cycling for well H5-2 was recently shown with available metaproteome data sets (15). Recent work determined anammox rates in the HTU to range from 3.5 to 4.7 nmol N 2 liter Ϫ1 day Ϫ1 (16).…”

Phylogenetic and metabolic diversity have contrasting effects on the ecological functioning of bacterial communities

“…The twenty-seven bacterial strains used in this study (Table S1) were isolated from pristine groundwater (Geesink et al 2018), obtained from the Hainich Critical Zone Exploratory located in Thuringia, Germany (Küsel et al 2016). The isolates were selected from a pool of more than 340 groundwater isolates as representatives of the most abundant taxonomic families within the total groundwater bacterial population, based on 16S rRNA gene MiSeq amplicon sequencing data (Schwab et al 2017, Kumar et al 2017), throughout the taxonomic classes of Actinobacteria , Bacilli , Flavobacteriia , Sphingobacteria , Alpha - and Gammaproteobacteria . The isolates were maintained on S2P medium agar (Geesink et al 2018).…”

14C‐Free Carbon Is a Major Contributor to Cellular Biomass in Geochemically Distinct Groundwater of Shallow Sedimentary Bedrock Aquifers

“…Recharge waters slowly percolate within the bedrock allowing for O 2 consumption, lowering of the redox potential and allowing chemical interaction with the bedrock to affect groundwater chemical composition (Kohlhepp et al, ; Küsel et al, ). Groundwaters sampled from both wells are anoxic (dissolved O 2 concentration < 0.02 mg/L = limit of detection; Figure ) but have distinct biogeochemistry indicative of the importance of iron reduction in H4.3 (which we refer to here as H4.3‐IR to emphasize iron reduction as a key biogeochemical process in this aquifer zone) and sulfate reduction/anammox metabolism in H5.2 (referred to below as H5.2‐SR/An; Küsel et al, ; Kumar et al, ; Schwab et al, ). DIC radiocarbon values in these wells reflect not only carbonate dissolution, but also the addition of C from a 14 C‐free source that has a 13 C‐content consistent with a C 3 organic matter source (Nowak et al, ).…”