Impact of freeze-thaw cycle on metagenomics in subsurface wastewater infiltration systems: Ecological implications for greenhouse gas emissions.

Nitrous oxide (N2O) is a potent greenhouse gas, with a global warming potential 273 times that of carbon dioxide (CO2) and is a significant byproduct of wastewater treatment. Subsurface wastewater infiltration systems (SWIS) effectively treat nitrate-rich wastewater but can also contribute to N2O emissions, particularly during freeze-thaw cycles. This study used metagenomics and 15N isotope tracing to investigate the impacts of freeze-thaw on microbial ecology and nitrogen transformation in SWIS. Results show that freeze-thaw significantly increased abundances of denitrifying bacteria (Bradyrhizobium, Streptomyces and Nocardioides), on average, by 16-63 %. Denitrification genes (nirK and norB) were also increased by 40 ± 16 % and 22 ± 5 %, while the N2O reductase gene (nosZ) decreased by 19 ± 0.46 %. These impacts collectively increased N2O emissions by more than 20 %. During freezing, about one-third of the added 15NO3--N was recovered as gas (25 % as N2O and 13 % as N2), increasing to 43 % during thawing (29 % N2O and 15 % N2). This study underscores the need for targeted strategies N2O emission in SWIS, particularly under freeze-thaw conditions, to maximize their sustainability in wastewater treatment.