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 eight groundwater wells and one karstic spring in the Ammer River catchment (SW Germany). Comparing groundwater hydrochemistry with microbial community composition, relative abundances of 16S rRNA and N-cycling gene copies (nirK, nirS, amoA) showed that declining O2 and dissolved organic carbon along the aquifer corresponded to lower bacterial and archaeal 16S rRNA gene copy numbers. Despite oxic conditions in the recharge zone, NO3⁻ can be reduced heterotrophically and autotrophically in anoxic microniches. In the confined anoxic zone, taxa such as Aquabacterium, Acidovorax and Gallionella may couple NO3⁻ reduction to pyrite-derived Fe(II) oxidation. Microorganisms such as Rhodoferax, Sediminibacterium and Sulfurifustis may oxidize pyrite-derived reduced sulfur compounds. Hydrogen-oxidizing (e.g. Hydrogenophaga) and CH4-oxidizing microorganisms (e.g. Candidatus Methylomirabilis) may also contribute to NO3⁻ turnover. These findings suggest that NO3⁻ turnover in the carbonate aquifer shifts from predominantly organic matter-supported pathways in recharge groundwater towards increasing reliance on pyrite-derived electron donors under anoxic conditions.