Warming often accelerates methane (CH₄) emissions from seagrass meadows, while aerobic methanotrophs act as a crucial biological sink. However, responses of active methanotrophic communities to warming remain poorly understood. Here, seagrass sediments were incubated with methane under three temperature treatments: W0 (CK, 28℃), W1 (+2.7℃, 30.7℃) and W2 (+4.4℃, 32.4℃), representing IPCC moderate and extreme warming scenarios. DNA-based stable isotope probing (DNA-SIP) technology was used to investigate the methanotrophic community structure and metabolic pathways of active taxa. Results showed that warming significantly increased aerobic methane oxidation (AMO) potential, with a higher temperature sensitivity (Q10) observed at +2.7℃ (3.67) than at +4.4℃ (1.92). Warming significantly affected the active microbial communities during methane incubation (P < 0.05). DNA-SIP identified type I genera Methylomarinum, Methylobacter, Methylomonas, Methyloprofundus and the type II genus Methyloceanibacter as the primary active methanotrophs. Under W1, type I methanotrophs using the RuMP pathway were more abundant, whereas under W2, the abundance of microorganisms associated with the serine pathway, including Methyloceanibacter and Hyphomicrobium, was higher. Correspondingly, the RuMP pathway proportion increased under W1 but was lowest under W2, whereas the serine pathway proportion increased significantly with rising temperature (P < 0.05). The RuMP pathway has higher carbon assimilation efficiency than the serine pathway. Consistently, methane assimilation rate was highest at W1 and lowest at W2 in our study. These results indicate that warming alters methane assimilation efficiency by reshaping active methanotrophic communities and shifting carbon assimilation pathways, providing new insight into methane oxidation regulation in seagrass ecosystems under climate change.

 

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