When compared with H-2, mixed electron donors (MED), comprising S2-, S2O3 (2-), and NO2 (-), could generally improve the CO2-fixing efficiency of nonphotosynthetic microbial communities (NPMCs). However, a large amount of abiotic precipitates combined with bacteria produced during culture may be unfavorable for the recycling and reuse of bacteria. The main component of the abiotic precipitates is S-0, which influences the enrichment and reuse of bacteria but is not conducive for CO2 fixation in the subsequent step. In this study, a two-step culture method (TSCM), employing H-2 and MED, respectively, was verified to be feasible for improving the CO2-fixing efficiency of NPMCs in the second step. In the TSCM, the net-fixed CO2 increased to 854 mg/L and abiotic precipitates were not produced in the medium. Sequence analysis of 16 s rDNA from NPMC indicated the presence of microbial symbioses in the NPMC, supporting the possible applications of TSCM.