Anammox and DNRA dominate anaerobic nitrogen transformation in carbonate-rock and sandstone aquifers

Microbially driven anaerobic nitrogen transformation processes play an important role in groundwater biogeochemical cycling. However, the contribution of processes other than denitrification, such as anaerobic ammonia oxidation (anammox) or dissimilatory nitrate reduction to ammonium (DNRA), and the overall influence of geological settings, have received little attention so far. We hypothesize that groundwaters from two contrasting geological settings – carbonate rock (Hainich Critical Zone Exploratory) and sandstone aquifers (Saale-Elster-Sandstone Observatory) in Thuringia (Germany) – differ in the dominant anaerobic nitrogen transformation pathways, the preferred electron donors for nitrate reduction, and the respective microbial communities. ¹⁵N-based approaches revealed that anammox contributed about 80% to total nitrogen loss in suboxic carbonate-rock groundwater with maximum rates of 9.8 nmol N₂ L^-1 d, while DNRA was the dominant nitrate reducing process in sandstone groundwater (12.8 nmol N L^-1 d^-1). Despite a high genetic potential for denitrification, measured activities remained below 0.6 nmol N₂ L^-1 d^-1 across all groundwater wells of the two locations. Microcosm experiments conducted over 70 days showed that nitrate reducing communities from the sandstone groundwater responded more rapidly to the addition of organic carbon compounds or hydrogen than those from carbonate-rock groundwater. In sandstone groundwater microcosms, incubation with both types of electron donors resulted in an increase in nirS and nrfA genes encoding NO-forming nitrite reductase (denitrification) and ammonia-forming nitrite reductase (DNRA), respectively, by two orders of magnitude. In contrast, microorganisms with the genetic potential for DNRA were not stimulated in carbonate-rock groundwater microcosms. Our findings suggest that the geological setting strongly influences preferential nitrogen transformation pathways and electron donor usage in groundwater. DNRA could play an important role in nitrate reduction in groundwater from the sandstone aquifers, potentially supported by hydrogen and organic carbon compounds.