Co-occurring aquatic acidification and hypoxia promote methane emissions from estuarine ecosystems

Estuaries worldwide are experiencing intensifying acidification and hypoxia, driven synergistically by anthropogenic activities and global climate change. Nevertheless, their combined impact on the emissions of the potent greenhouse gas methane (CH₄) and its underlying regulatory mechanisms remains poorly understood, undermining our ability to project climate feedbacks. Here, we integrated ¹³C stable isotope tracing, DNA/mRNA-based qPCR, and amplicon/metagenomic sequencing to unravel how acidification-hypoxia interactions regulate the complex balance between CH₄ production and consumption in estuarine sediments. Results showed that aquatic acidification and hypoxia combined to significantly increase CH₄ emissions from estuarine sediments (P < 0.05), in a non-additive (antagonistic) manner where oxygen availability was the dominant factor governing this response. Notably, acidification increased CH₄ emissions by suppressing methanotrophy more strongly than methanogenesis, whereas hypoxia preferentially stimulated methanogenic activity over CH₄ oxidation. These response patterns were further demonstrated by metagenomic sequencing and mRNA-based quantitative PCR analyses, which revealed coordinated shifts in both the relative abundance and transcriptional activity of key functional genes. These findings uncover a previously overlooked mechanism whereby the worldwide co-occurrence of acidification and hypoxia in estuarine ecosystems jointly promote CH₄ emissions, providing a scientific basis for improving predictive models of the global CH₄ cycle and its climate feedbacks under combined anthropogenic and climatic stressors.