Versatile microbial community responsible for nitrate turnover in a carbonate aquifer in southwest Germany

Denitrifiers contribute to the remediation of agriculturally impacted aquifers by using NO3⁻ as an electron acceptor. However, the effects of local hydrogeochemical factors (e.g. O2, electron donors, carbon sources) on the abundance of denitrifiers remain poorly understood. To address this, we sampled planktonic (0.2-0.4 µm, 0.4-8.0 µm) and particle-associated (>8 µm) biomass from nine groundwater wells and one karstic spring in the Ammer River catchment (SW Germany). Comparing groundwater hydrochemistry with microbial community composition and the relative abundance of 16S rRNA and N-cycling genes (nirK, nirS, amoA) revealed a correlation between declining O2 and dissolved organic carbon levels in the aquifer and lower bacterial and archaeal 16S rRNA gene copy numbers. Despite oxic conditions in the recharge zone, NO3⁻ could be reduced heterotrophically and autotrophically in anoxic microniches. In the confined anoxic zone, taxa such as Aquabacterium, Acidovorax, and Gallionella could couple NO3⁻ reduction to pyrite-derived Fe(II) oxidation. Microorganisms such as Rhodoferax, Sediminibacterium, and Sulfurifustis could oxidize pyrite-derived reduced sulfur compounds. Hydrogen-oxidizing (e.g. Hydrogenophaga) and CH4-oxidizing microorganisms (e.g. Candidatus Methylomirabilis) could also contribute to NO3⁻ turnover. These findings suggest that the turnover of NO3- in the carbonate aquifer shifts from pathways predominantly supported by organic matter in recharge groundwater towards an increasing reliance on pyrite-derived electron donors under anoxic conditions.