Habitat heterogeneity shapes chemoautotrophic microbial diversity, ecological interactions, and carbon fixation function in an oligotrophic karst cave

Karst caves represent important yet understudied components of the global carbon cycle, where microbial dark carbon fixation contributes substantially to subterranean carbon sequestration. However, how habitat heterogeneity regulates autotrophic microbial communities and how CO₂ concentration impacts on their carbon fixation performance remains poorly understood. Here, we investigated autotrophic microbiomes across weathered rocks of cave wall and sediment along the cave passage, as well as the overlying-soil, in Luohandu Cave, a typical karst ecosystem in southwestern China, by integrating community profiling, ecological network analysis, and microcosm validation experiments. Functional prediction based on 16S rRNA gene sequencing identified six carbon fixation pathways, among which the Calvin-Benson-Bassham (CBB) cycle, Wood-Ljungdahl pathway, and 3-hydroxypropionate bicycle exhibited marked variation across the three habitats. High-throughput sequencing of the cbbL gene further revealed pronounced habitat-specific community structures: overlying soils exhibited the highest diversity and enrichments of Mesorhizobium, Variovorax, and Bradyrhizobium, whereas sediments were dominated by Pseudonocardia. cbbL communities were significantly influenced by total organic carbon, total nitrogen, pH, Ca²⁺, and Mg²⁺. Co-occurrence network analysis revealed highly modular interaction structures with predominant positive links among nodes and a limited number of keystone taxa. Microcosm incubations under a CO₂ gradient further demonstrated a nonlinear response of microbial carbon fixation, with both the microbial inorganic carbon incorporation rate (1.29 mg C kg^-1·d^-1) and cbbL abundance (1.77 × 10⁶ copies g^-1) peaking at 2000 ppm CO₂. Collectively, these findings establish an ecological framework that links habitat heterogeneity, autotrophic community structure, microbial interactions, and carbon fixation function, thereby providing mechanistic insights into microbial contributions to carbon sequestration in karst cave ecosystems.