Different responses of nitrite- and nitrate-dependent anaerobic methanotrophs to increasing nitrogen loading in a freshwater reservoir

Nitrite (NO₂⁻)- and nitrate (NO₃⁻)-dependent anaerobic oxidation of methane (AOM) are two new additions in microbial methane cycle, which potentially act as important methane sinks in freshwater aquatic systems. Here, we investigated spatial variations of community composition, abundance and potential activity of NO₂⁻- and NO₃⁻-dependent anaerobic methanotrophs in the sediment of Jiulonghu Reservoir (Zhejiang Province, China), a freshwater reservoir having a gradient of increasing nitrogen loading from upstream to downstream regions. High-throughput sequencing of total bacterial and archaeal 16S rRNA genes showed the cooccurrence of Candidatus Methylomirabilis oxyfera (M. oxyfera)-like and Candidatus Methanoperedens nitroreducens (M. nitroreducens)-like anaerobic methanotrophs in the examined reservoir sediments. The community structures of these methanotrophs differed substantially between the sediments of upstream and downstream regions. Quantitative PCR suggested higher M. oxyfera-like bacterial abundance in the downstream (8.6 × 10⁷ to 2.8 × 10⁸ copies g⁻¹ dry sediment) than upstream sediments (2.4 × 10⁷ to 3.5 × 10⁷ copies g⁻¹ dry sediment), but there was no obvious difference in M. nitroreducens-like archaeal abundance between these sediments (3.7 × 10⁵ to 4.8 × 10⁵ copies g⁻¹ dry sediment). The ¹³CH₄ tracer experiments suggested the occurrence of NO₂⁻- and NO₃⁻-dependent AOM activities, and their rates were 4.7–14.1 and 0.8–2.6 nmol CO₂ g⁻¹ (dry sediment) d⁻¹, respectively. Further, the rates of NO₂⁻-dependent AOM in downstream sediment were significantly higher than those in upstream sediment. The NO₃⁻ concentration was the key factor affecting the spatial variations of abundance and activity of NO₂⁻-dependent anaerobic methanotrophs. Overall, our results showed different responses of NO₂⁻- and NO₃⁻-dependent anaerobic methanotrophs to increasing nitrogen loading in a freshwater reservoir.