Crop rotation and organic fertilizer maintains diversity and activity of cbbL-carrying CO2-fixing bacteria in reclaimed coal mining soils

CO₂-fixing bacteria are an important factor in restoring soil health in coal mining areas. The impact of crop rotation and fertilization on CO₂-fixing bacteria in reclaimed mining soils remains unclear. To narrow this knowledge gap, in this study, maize (Zea mays L.) monoculture (M) and maize-soybean (Glycine max) rotation (R) cropping systems were set up in a coal-mining reclamation area with four fertilization treatments in each, namely, CK (without fertilization), inorganic fertilizer (F), organic fertilizer (O), and combined organic and inorganic fertilizer (OF). The abundance, diversity, community composition and RubisCO activity of CO₂-fixing bacteria in topsoil under those treatments were investigated respectively using quantitative PCR, high-throughput sequencing based on the cbbL gene [that encodes ribulose-1,5-biphosphate carboxylase/oxygenase (RubisCO)] and enzyme-linked immunosorbent assay (ELISA). The results showed that R_O significantly increased easily oxidized organic carbon (EOC), total nitrogen (TN), and available nitrogen (AN) (P < 0.05). It also significantly increased the biomass of CO₂-fixing bacteria (P < 0.05) and altered the CO₂-fixing bacterial community. The CO₂-fixing bacteria in R_OF, R_O and M_O exhibited comparable community structures and harbored a greater co-occurrence network complexity than other treatments. Several CO₂-fixing bacteria associated with nitrogen cycling, such as Devosia, Nitrobacter, Hyphomicrobiales and Nitrosospira, were significantly enriched under the maize-soybean rotation system (P < 0.05). This study implied that crop rotation and organic fertilizer application could synergistically foster soil quality restoration in coal mining area by elevating soil nutrients and maintaining biomass, diversity and community structure of cbbL-carrying CO₂-fixing bacteria, establishing a theoretical foundation for optimizing carbon sequestration strategies in post-mining ecological rehabilitation.