Global warming caused by excessive emission of CO_2 is a major environmental problem currently. CO_2 fixing by microbial communities is a very important part of CO_2 resource utilization. Therefore, it is necessary to isolate highly efficient CO_2 fixing microorganisms that do not require light or hydrogen to enable a wider range of applications. Non-photosynthetic bacteria had done from some of the world's ocean by premier experiments. This research was based on the premier experiments and took the non-photosynthetic microbial communities separated from seawater as object to construct the optimal mix carbon source system to promote growth of microbacteria under the condition of different electron donors. When the optimal carbon source components were 374.24 mgC/L Na_2CO_3, 54.76 mgC/L NaHCO_3 and 0 mgC/L CO_2 with NH4+ as electron donor, the predicted max TOC was 3.06 mg/L. The value of the predicted max TOC was lower than that of using the Na_2CO_3 as single carbon source, but higher than that of using the CO_2 or NaHCO_3 as single carbon source respectively. When the optimal carbon source components was 0.26 mg/L Na_2CO_3, 0.59 mg/L NaHCO_3 and 71.48 mL/L CO_2 with H_2 as electron donor, the carbon fixation efficiency by non-photosynthetic microbial communities was up to 27.62 mg/L, which increased by about 35% comparing with CO_2 as the sole carbon source. The results suggested that hydroxypropionate cycle might be the main carbon fixing cycle and all the six carbon fixing cycles could be used by non-photosynthetic microbial communities with H_2.