Active dark carbon fixation evidenced by ¹⁴C isotope assimilation and metagenomic data across the estuarine-coastal continuum

Estuaries, as important land-ocean transitional zones across the Earth's surface, are hotspots of microbially driven dark carbon fixation (DCF), yet understanding of DCF process remains limited across the estuarine-coastal continuum. This study explored DCF activities and associated chemoautotrophs along the estuarine and coastal environmental gradients, using radiocarbon labelling and molecular techniques. Significantly higher DCF rates were observed at middle- and high-salinity regions (0.65–2.31 and 0.66–2.82 mmol C m⁻² d⁻¹, respectively), compared to low-salinity zone (0.07–0.19 mmol C m⁻² d⁻¹). Metagenomic analysis revealed relatively stable DCF pathways along the estuarine-coastal continuum, primarily dominated by Calvin-Benson-Bassham (CBB) cycle and Wood-Ljungdahl (WL) pathway. Nevertheless, chemoautotrophic communities driving DCF exhibited significant spatial variations. It is worth noting that although CBB cycle played an important role in DCF in estuarine sediments, WL pathway might play a more significant role, which has not been previously recognized. Overall, this study highlights that DCF activities coincide with the genetic potential of chemoautotrophy and the availability of reductive substrates across the estuarine-coastal continuum, and provides an important scientific basis for accurate quantitative assessment of global estuarine carbon sink.