The contributions of dark microbial CO2 fixation to soil organic carbon along a tropical secondary forest chronosequence on Hainan Island, China

Nonphototrophic CO₂ fixation by microorganisms can reduce soil pore CO₂ to organic matter in the dark. Tropical forest restoration changes soil microbial community structure and organic carbon (SOC) storage. However, whether the capacity for dark CO₂ fixation is altered and contributes to SOC accumulation during tropical forest restoration remains unclear. Here, in the topsoil and deep soil of a tropical forest restoration chronosequence, we investigated chemoautotrophic and heterotrophic microbes and pathways involved in dark CO₂ fixation using a metagenome and quantified CO₂ fixation rates with a ¹³C–CO₂ labelling experiment. Tropical forest restoration altered the autotrophic CO₂ fixation pathway abundance in the Ah horizon, which showed an increasing trend. Heterotrophic carboxylase gene abundance was influenced by soil layer and was more abundant in the B horizon. The main microbes involved in CO₂ fixation belong to Acidobacteria, Proteobacteria, and Actinobacteria. ¹³C–CO₂ labelling showed that the CO₂ fixation rates across the restoration chronosequence ranged from 0.035 to 0.155 μg C/g soil d⁻¹, and the middle- and late-stage secondary forests exhibited higher rates compared to other stages. The microbial assimilation of CO₂ into mineral-associated organic carbon was also observed and the rate exhibited a similar trend to that into SOC, indicating that dark CO₂ fixation contributes to stable carbon formation. Tropical forest restoration influenced the CO₂ fixation rate indirectly by changing microbial CO₂ fixation gene abundance. Specifically, autotrophic pathways (Calvin, reductive citrate, and Wood−Ljungdahl cycles) and heterotrophic carboxylase genes (phosphoenolpyruvate and pyruvate carboxylases) were vital for CO₂ fixation in the Ah and B horizons, respectively. Our results suggest that SOC formed by microbial CO₂ assimilation contributes to the long-term soil carbon sequestration, especially in secondary forests, which have recovered to middle- and late-stages. The study highlights the importance of dark microbial CO₂ fixation in soil carbon sequestration and provides a new understanding of tropical forest soil carbon processes.