The photobiological hydrogen production by anaerobic photosynthetic bacteria, Rhodospirillum rubrum, from synthesis gas was studied. The influence of initial acetate concentration as the substrate for the microbial growth was investigated in a batch system. Series of experiments were conducted in the serum bottles as bioreactor at ambient temperature and atmospheric pressure. The agitation rate and light intensity were adjusted at 200rpm and 1000 lux, respectively. The concentration of acetate, as carbon source, was varied from 0.5 to 3.0 g/l. In this study, the synthesis gas consists of carbon dioxide, argon, hydrogen and carbon monoxide, 10, 15, 20 and 55%, respectively. It was observed that an increase in concentration of the carbon source from 2.5 to 3 g/l resulted in a decrease of the microorganism growth, R. rubrum, and also decrease in hydrogen production rate. Furthermore, the experimental results indicated that the optimum acetate concentrations range were from 1 to 2 g/l. The resulted data were also shown that in 1 to 2 g/l acetate, higher hydrogen formation and cell concentration were obtained. A biologically-based water-gas shift reaction provided an attractive alternative improvement of renewable resources to achieve higher hydrogen production. Addition of excess amount of acetate to the initial culture medium was inhibited the growth of R. rubrum. An inverse relationship between the acetate concentration and the initial cell growth was observed. This article presents a method of calculations involved in process parameters for gaseous substrates (CO) fermentation using gas and liquid system. The procedure is explained by experimental data for bioconversion of carbon monoxide to carbon dioxide, while simultaneously water is reduced to hydrogen.