Impacts of temperature on hydrogenotrophic denitrifying aquifer microbiota

ABSTRACT Groundwater is a globally relevant drinking water resource, yet nitrate pollution from agriculture increasingly threatens its quality, especially in shallow, oxygen-rich aquifers where natural attenuation is limited. This study examined whether gaseous hydrogen can stimulate autochthonous chemolithoautotrophic denitrification in nitrate-polluted, gravel aquifer sediments, and whether this process remains effective across temperatures relevant for laboratory and field conditions. We hypothesized that both microbial community composition and the kinetics of nitrate reduction will change under distinct temperature regimes. Microcosm incubations of sediments from a nitrate-polluted aquifer successfully enriched native hydrogenotrophic denitrifiers. Denitrification activity was quantified at 12°C, 15°C, 20°C, and 25°C, with maximum rates ranging from ~1 to ~1.8 µmol g⁻¹ sediment day⁻¹, and average rates generally following the expected kinetic temperature responses. Higher temperatures supported greater microbial biomass and community diversity, including a broader range of denitrifiers. Nevertheless, a single population, a novel strain of Acidovorax defluvii , dominated the community across all temperatures. Unlike the heterotrophic type strain, it possessed a conserved, likely transferable gene cluster for hydrogen oxidation, hydrogen sensing, and CO₂ fixation, as revealed by metagenomics. Although complete denitrifiers with hydrogen-oxidation capacity dominated at all temperatures, nitrite accumulated transiently below 15°C. The presence of aerobic respiration genes in all enriched denitrifiers indicates their ability to switch to oxygen as an electron acceptor if available. Our study demonstrates a notable presence of an autochthonous hydrogenotrophic denitrification potential in the studied aquifer and its effective stimulation by hydrogen injection alone. Furthermore, it identifies mass transfer constraints and redox control as targets for future process optimization. IMPORTANCE Nitrate contamination of groundwater remains a widespread challenge in agricultural regions, and effective in situ remediation strategies are urgently needed. Hydrogen-stimulated chemolithoautotrophic denitrification represents a promising, low-carbon approach, yet its robustness under environmentally relevant temperature conditions has remained unclear. Our findings show that indigenous aquifer microorganisms can rapidly and consistently perform hydrogenotrophic denitrification across a broad temperature range (12–25°C), driven by a dominant Acidovorax species with a specialized hydrogen-oxidizing gene cluster. Identification of a conserved, potentially transferable gene cluster conferring chemolithoautotrophic lifestyle is not only biotechnologically interesting but also contributes to debate about genomic and ecological definitions of microbial species. This demonstrates that hydrogen injection can reliably activate existing microbial potential without requiring bioaugmentation and that temperature-driven shifts in community composition do not compromise nitrate removal. These insights advance the development of hydrogen-based bioremediation strategies for nitrate-impacted aquifers.