In situ treatment of nitrate polluted groundwater by methane-dependent denitrification: meso‑scale flume proof-of-concept.

Nitrate concentrations in groundwater frequently exceed the EU drinking water limit of 50 mgL⁻¹, threatening drinking water quality. This study evaluates a novel in situ nitrate removal strategy based on methane injection to stimulate autochthonous denitrifiers. Methane was injected into a meso‑scale artificial aquifer equipped with horizontal injection wells and a comprehensive monitoring system. We hypothesized that methane injection promotes methanotrophic denitrification and enhances microbial nitrate removal in groundwater. Following methane injection over four months, nitrate concentrations declined from ∼55 mg L⁻¹ (0.89 mM) to 36 mg L⁻¹ (0.58 mM). Concurrent isotopic shifts of up to 11‰ in both δ¹⁵N of dissolved nitrate and δ¹³C of dissolved methane provided strong evidence for enhanced microbial nitrate reduction coupled to methane oxidation. Spatio-temporal analyses of sediment microbiomes revealed successive enrichment of canonical aerobic methano- and methylotrophs (Methylomonandaceae and Methylophilaceae). While only the first hosted metagenomic methane oxidation capacities, the second was associated with complete denitrification. Likely, they thus interacted synergistically under oxygen-limited conditions, suggesting an indirect coupling between oxygen limited methane oxidation and denitrification. Only towards the distal, anoxic end of the flume, true anaerobic methanotrophs affiliated with the Methylomirabilaceae also were enriched. Spatial analyses indicated that sediment heterogeneity influenced methane distribution and, therefore, microbial nitrate removal in the flume. Overall, methane injection effectively stimulated microbial nitrate degradation, providing meso‑scale proof of concept for future pilot-scale remediation of nitrate-contaminated groundwater.