Substantial dark carbon fixation contributes to carbon sink in human-impacted estuarine and coastal waters

Dark carbon fixation (DCF) is an integral component of the global carbon sink. However, quantitative data on its contribution to total carbon fixation in estuaries are scarce, and factors driving DCF remain under exploration. In this study, radio-¹⁴C labeling and molecular techniques were employed to investigate the temporal-spatial distribution of DCF and photosynthesis rates in the Yangtze Estuary and its adjacent coastal areas (YEA), along with the potential microorganisms involved. DCF rates ranged from 0.17 to 3.79 μmol C L⁻¹ h⁻¹ in the YEA, accounting for 15.4–97.7% of integrated total daily carbon fixation, suggesting the large variability in both DCF rates and their contributions to the carbon sink. An estimate based on ¹⁵NH₄Cl labeling experiments revealed that the contribution of DCF by ammonia-oxidizing microorganisms was low (< 3.97%) in surface waters of the YEA. Bacteria bearing the cbbL-IA&IC gene were the potential essential contributors to DCF, while eukaryotic phytoplankton harboring the cbbL-ID gene may also contribute to DCF through light-independent β-carboxylation. DCF rates were mainly influenced by nutrients, particulate organic carbon, and salinity, which affect microbial abundance. Our findings underscore the importance and variability of the DCF process in human-impacted estuarine and coastal waters, contributing to a better understanding of microbial carbon fixation processes and their potential mechanisms.