Metabolic diversity and aero-tolerance in anammox bacteria from geochemically distinct aquifers

Abstract Background Anaerobic ammonium oxidation (anammox) is important for converting bioavailable nitrogen into dinitrogen gas, particularly in carbon poor environments. Yet, the diversity and prevalence of anammox bacteria in the terrestrial subsurface – a typically oligotrophic environment – is little understood across different geochemical conditions. To determine the distribution and activity of anammox bacteria across a range of aquifer lithologies and physicochemistries, we analysed 16S rRNA genes, metagenomes and metatranscriptomes, and quantified hydrazine synthase genes and transcripts sampled from 59 groundwater wells distributed over 1 240 km 2 . Results Data indicate that anammox-associated bacteria (class Brocadiae ) and the anammox process are prevalent in aquifers (identified in aquifers with sandy-gravel, sand-silt and volcanic lithologies). While Brocadiae diversity decreased with increasing DO, Brocadiae 16S rRNA genes and hydrazine synthase genes and transcripts (hydrazine synthase, hzsB ) were detected across a wide range of bulk groundwater dissolved oxygen (DO) concentrations (0 – 10 mg/L). Anammox genes and transcripts ( hzsB ) correlated significantly with those involved in bacterial and archaeal ammonia oxidation (ammonia monooxygenase, amoA ), which could represent a major source of nitrite for anammox. Differences in anammox community composition were strongly associated with DO and bore depth (and to a lesser extent pH and phosphate), revealing niche differentiation among anammox bacteria in groundwater that was largely driven by water oxygen contents, and not ammonium/nitrite. Eight Brocadiae genomes (63-95% estimated completeness) reconstructed from a subset of groundwater sites belong to 2 uncharacterized families and 6 novel species (based on average nucleotide identity). Distinct groups of these genomes dominated the anammox-associated community at dysoxic and oxic sites, further reflecting the influence of DO on Brocadiae composition. Six of the genomes (dominating dysoxic or oxic sites) have genes characteristic of anammox (hydrazine synthase and/or dehydrogenase). These genes, in addition to aerotolerance genes, belonging to four Brocadiae genomes, were transcriptionally active, although transcript numbers clearly highest in dyoxic groundwater. Conclusions Our findings indicate anammox bacteria contribute to loss of fixed N across diverse anoxic-to-oxic aquifer conditions, and that this is likely supported by nitrite from aerobic ammonia oxidation. Results provide an insight into the distribution and activity of anammox bacteria across distinct aquifer physicochemisties.