Nitrate sources and sinks in oligotrophic groundwater

Despite the high relevance of karstic aquifers as drinking water reservoirs, nitrate pollution of groundwater is posing an increasing threat on a global scale. Microbially mediated processes such as nitrification, denitrification, or anaerobic ammonia oxidation (anammox) play a key role in the formation of nitrate or nitrogen loss as N₂ gas, respectively. We aimed to disentangle different sources and sinks of nitrate and key microbial players involved in nitrogen transformation processes in oligotrophic limestone aquifers of the Hainich Critical Zone Exploratory (CZE; Germany). Assessment of process rates using ¹⁵N-labeling techniques revealed a variance of nitrification rates by two orders of magnitude (1 – 125 nmol NO_x^- L^-1 d^-1) across four oxic groundwater wells. Strong discrepancy between the potential for nitrate formation and the nitrate concentrations present in the groundwater pointed to surface inputs rather than in situ production for wells with nitrate concentrations higher than 300 µmol L^-1. Anammox and denitrification as potential nitrate or nitrite sinks varied from 1 to 5 nmol N₂ L⁻¹ d⁻¹ across anoxic wells with anammox contributing about 84% to nitrogen loss from the groundwater. Molecular surveys of groundwater microbial communities of nitrifiers, denitrifiers, and anammox bacteria over several years revealed consistent spatial patterns, pointing to the existence of distinct “hotspots” of nitrification and anammox across the heterogeneous aquifer assemblages. Overall, our findings suggest that spatial patterns of nitrogen cycling processes in these karstic aquifers are driven by heterogeneity of hydrochemical conditions, groundwater residence times, and surface connectivity. Knowledge of these processes and their controls is crucial to assess the groundwater’s capacity to buffer elevated nitrate inputs.