Effects of sulfamethazine on microbially-mediated denitrifying anaerobic methane oxidation in estuarine wetlands

Nitrite/nitrate-dependent anaerobic methane oxidation (n-DAMO) is an important methane (CH₄) consumption and nitrogen (N) removal pathway in estuarine and coastal wetlands. Antibiotic contamination is known to affect microbially mediated processes; however, its influences on n-DAMO and the underlying molecular mechanisms remain poorly understood. In the present study, using ¹³CH₄ tracer method combined with molecular techniques, we investigated the responses of n-DAMO microbial abundance, activity, and the associated microbial community composition to sulfamethazine (SMT, a sulfonamide antibiotic, with exposure concentrations of 0.05, 0.5, 5, 20, 50, and 100 µg L^-1). Results showed that the effect of SMT exposure on n-DAMO activity was dose-dependent. Exposure to SMT at concentrations of up to 5 µg L^-1 inhibited the potential n-DAMO rates (the average rates of nitrite- and nitrate-DAMO decreased by 92.9 % and 79.2 % relative to the control, respectively). In contrast, n-DAMO rates tended to be promoted by SMT when its concentration increased to 20-100 µg L^-1 (the average rates of nitrite- and nitrate-DAMO increased by 724.1 % and 630.1 % relative to the low-doses, respectively). Notably, low-doses of SMT suppressed nitrite-DAMO to a greater extent than nitrate-DAMO, indicating that nitrite-DAMO was more sensitive to SMT than nitrate-DAMO. Molecular analyses suggest that the increased n-DAMO activity under high-doses SMT exposure may be driven by changes in microbial communities, especially because of the promotion of methanogens that provide more CH₄ to n-DAMO microbes. Moreover, the abundances of n-DAMO microbes at high SMT exposure (20 and 50 µg L^-1) were significantly higher than that at low SMT exposure (0.05-5 µg L^-1). These results advance our understanding of the ecological effects of SMT on carbon (C) and N interactions in estuarine and coastal wetlands.