Microbial interactions in nitrogen cycling in oligotrophic groundwater

Understanding the links between anaerobic and aerobic processes in the nitrogen cycle is crucial for studying nitrogen dynamics in natural environments. Coupling between anaerobic and aerobic nitrogen transformation processes was previously shown to support nitrogen loss in the marine water column. However, the importance of such interactions for N dynamics in groundwater environments is poorly understood. Previous findings demonstrated a high relevance of anaerobic ammonia oxidation (anammox) for nitrogen loss from carbonate-rock aquifers in the Hainich Critical Zone Exploratory (Germany). Here, we hypothesize that (i) anammox in oligotrophic groundwater is supported by tight coupling with dissimilatory nitrate reduction to ammonium (DNRA) and aerobic ammonia oxidation, providing ammonium and nitrite, respectively, for the anammox reaction, and (ii) that this coupling is reflected by spatial co-localization of the respective microbial groups in the aquifer system. Comparative investigations of two suboxic groundwater wells (0.3 to 2.3 µmol L^-1 dissolved oxygen) pointed to overall favorable conditions for anammox (12-25 µmol L^-1 NO₃^-, 29-40 µmol L^-1 NH₄⁺) along with anammox activity (H52: 0.9 nmol N₂ L^-1 d^-1, H53: 2.5-9.7 nmol N₂ L^-1 d^-1). Metagenomic analysis indicated the genetic potential for DNRA by members of the class Thermodesulfovibrionales (Nitrospirota), which were found to consistently co-occur with anammox bacteria in those two wells, together accounting for 13-25% of the bacterial community. Although oxygen is limited, aerobic nitrifiers, especially the genus Nitrospira (Nitrospirota) constituted 1-15% across both wells which may support anammox activity. Ongoing experiments utilize CARD-FISH to detect anammox bacteria along with members of the Nitrospirota in attached aquifer communities. Potential coupling between anammox, DNRA, and aerobic ammonia oxidation is being tested using ¹⁵N-based approaches. Our findings suggest that differences in anammox activity in the studied aquifer system could primarily be linked to the presence of oxygen and the intensity of coupling with aerobic N cycling processes.