Metabolic Diversity and Aero-Tolerance in Anammox Bacteria from Geochemically Distinct Aquifers

Anaerobic ammonium oxidation (anammox) is important for converting bioavailable nitrogen into dinitrogen gas, particularly in carbon-poor environments. However, the diversity and prevalence of anammox bacteria in the terrestrial subsurface—a typically oligotrophic environment—are little understood. To determine the distribution and activity of anammox bacteria across a range of aquifer lithologies and physicochemistries, we analyzed 16S rRNA genes and quantified hydrazine synthase genes and transcripts sampled from 59 groundwater wells and metagenomes and metatranscriptomes from an oxic-to-dysoxic subset. Data indicate that anammox and anammox-associated bacteria (class “Candidatus Brocadiae”) are prevalent in the aquifers studied, and that anammox community composition is strongly differentiated by dissolved oxygen (DO), but not ammonia/nitrite. While “Candidatus Brocadiae” diversity decreased with increasing DO, “Candidatus Brocadiae” 16S rRNA genes and hydrazine synthase (hzsB) genes and transcripts were detected across a wide range of bulk groundwater DO concentrations (0 to 10 mg/L). Anammox genes and transcripts correlated significantly with those involved in aerobic ammonia oxidation (amoA), potentially representing a major source of nitrite for anammox. Eight “Candidatus Brocadiae” genomes (63 to 95% complete), representing 2 uncharacterized families and 6 novel species, were reconstructed. Six genomes have genes characteristic of anammox, all for chemolithoautotrophy. Anammox and aerotolerance genes of up to four “Candidatus Brocadiae” genomes were transcriptionally active under oxic and dysoxic conditions, although activity was highest in dysoxic groundwater. The coexpression of nrfAH nitrite reductase genes by “Candidatus Brocadiae” suggests active regeneration of ammonia for anammox. Our findings indicate that anammox bacteria contribute to loss of fixed N across diverse anoxic-to-oxic aquifer conditions, which is likely supported by nitrite from aerobic ammonia oxidation. IMPORTANCE Anammox is increasingly shown to play a major role in the aquatic nitrogen cycle and can outcompete heterotrophic denitrification in environments low in organic carbon. Given that aquifers are characteristically oligotrophic, anammox may represent a major route for the removal of fixed nitrogen in these environments, including agricultural nitrogen, a common groundwater contaminant. Our research confirms that anammox bacteria and the anammox process are prevalent in aquifers and occur across diverse lithologies (e.g., sandy gravel, sand-silt, and volcanic) and groundwater physicochemistries (e.g., various oxygen, carbon, nitrate, and ammonium concentrations). Results reveal niche differentiation among anammox bacteria largely driven by groundwater oxygen contents and provide evidence that anammox is supported by proximity to oxic niches and handoffs from aerobic ammonia oxidizers. We further show that this process, while anaerobic, is active in groundwater characterized as oxic, likely due to the availability of anoxic niches.