Spatial heterogeneity plays a vital role in shaping the structure and function of estuarine carbon-fixing bacterial communities.

Carbon-fixing bacterial communities are essential drivers of carbon fixation in estuarine ecosystems that critically affect the global carbon cycle. This study compared the abundances of the Calvin cycle functional genes cbbL and cbbM and Reductive tricarboxylic acid cycle gene aclB, as well as compared carbon-fixing bacterial community features in the two estuaries, predicted potential ecological functions of carbon-fixation bacteria, and analyzed their symbiosis strategies in two estuaries having different geographical distributions. Gammaproteobacteria was the dominant carbon-fixing bacterial community in the two estuaries. However, a higher number of Alphaproteobacteria were noted in the Liaohe Estuary, and a higher number of Betaproteobacteria were found in the Yalujiang Estuary. The carbon-fixing functional gene levels exhibited the order of aclB > cbbL > cbbM, and significant effects of Cu, Pb, and petroleum were observed (p < 0.05). Nitrogen-associated nutrient levels are major environmental factors that affect carbon-fixing bacterial community distribution patterns. Spatial factors significantly affected cbbL carbon-fixing functional bacterial community structure more than environmental factors. With the increase in offshore distance, the microbial-led processes of methylotrophy and nitrogen fixation gradually weakened, but a gradual strengthening of methanotrophy and nitrification was observed. Symbiotic network analysis of the microorganisms mediating these ecological processes revealed that the carbon-fixing bacterial community in these two estuaries had a non-random symbiotic pattern, and microbial communities from the same module were strongly linked among the carbon, nitrogen, and sulfur cycle. These findings could advance the understanding of carbon fixation in estuarine ecosystems.