Phototrophic Hydrogen Production Using Mixed Culture Biotechnology (MCB)

Fossil fuel depletion and pollutant emission led to serious energy crisis and environmental problems. Hydrogen gas was considered as an ideal alternative energy because of high energy yield and nonpolluting. Compared with conventional hydrogen production methods, biohydrogen production has received considerable attention in recent years owing to the advantage of renewable, high efficiency and clean. And phototrophic biohydrogen production technology combined solar energy utilization, organic wastes treatment and energy production together, which will be the most competitive power technology of hydrogen production. Research on phototrophic hydrogen production was very important.Mixed culture is a kind of microorganism resource which deserves pay more attention and carefully studied. Mixed culture biotechnology (MCB) could become an attractive addition or alternative to traditional pure culture based biotechnology for the production of bioenergy. Compared with pure culture based industrial biotechnology, specific advantages of MCB include: no sterilization requirements, adaptive capacity owing to microbial diversity, the capacity to use mixed substrates, and the possibility of a continuous process.In this paper, phototrophic hydrogen production was carried out in batch experiment using mixed cultures extracted from active sludge of marsh gas tank, and the contrastive test was using pure culture Rh.Palustris Z02. The modified Gompertz model was used for kinetic analysis of hydrogen production. The experimental result showed that hydrogen yield of mixed cultures was higher than that of pure culture Rh.Palustris Z02, especially using sugar and soluble starch as substrate. Mixed cultures utilized these two carbon sources more sufficiently; hydrogen yield was 3.47mol H₂/mol sugar and 6.68mmol H₂/g soluble starch respectively, hydrogen content was 83.30% and 76.06% respectively, and no methane gas was detected. Moreover, effects of various parameters, initial pH, temperature, inoculums concentration, light intensity etc, were examined with respect to maximum hydrogen yield. The conditions of hydrogen production using mixed cultures are not strict as using pure cultures. Temperature affected the hydrogen production rate according to the Arrhenius equation.The effects of three influencing factors (gas phase, ammonia and iron group ion) on phototropic hydrogen production using MCB were studied. The results showed that a large amount of O₂ in gas phase was toxic for nitrogenase and hydrogenase, which resulted in no hydrogen gas produced. However, a small quantity of O₂ can promote hydrogen production. When Argon gas and a small quantity of O₂ mixed together, the highest hydrogen yield of 2.38mol/mol acetate was achieved, and the lag phase was shortest. Furthermore, the effects of various gas phases on hydrogen production of mixed cultures were fewer than that of pure culture. Contrast test of pH controlled and uncontrolled was carried out. The result showed that the inhibitory effect of NH₄⁺ was obvious when pH was uncontrolled. And appropriate low NH₄⁺ concentration could enhance hydrogen yield and hydrogen production rate when pH was controlled at 7.0. The depletion of NH₄⁺ triggered hydrogen production whenever pH controlled or uncontrolled. The effect of NH₄⁺ concentration on growth and hydrogen production of mixed cultures were lesser than those of pure culture Rh.Palustris Z02. Fe²⁺, Co²⁺ and Ni²⁺ as the prosthetic group of metalloenzyme are important for phototrophic bacterial growth and hydrogen production. The experimental results indicated that appropriate concentration of Fe²⁺, Co²⁺ and Ni²⁺ promoted hydrogen production and increased cell growth. The optimal concentration of Fe²⁺, Co²⁺ and Ni²⁺ for hydrogen production was 9μmol/L, 0.45μmol/L and 0.1μmol/L, respectively. The effect on hydrogen production of iron group ion followed the order of Fe²⁺ > Ni²⁺ > Co²⁺. For cell growth, Co²⁺ was the most important ion than the others two. The effect of Fe²⁺ concentrations on hydrogen production at various pH values was investigated. The result showed that the effect of Fe²⁺ concentration on hydrogen production was conspicuous when pH was controlled at 7.0. As pH value diverged from 7.0, the effect of Fe²⁺ concentration on hydrogen production was decreased gradually.The classes and amount of carbon source and nitrogen source are the important factors for hydrogen production. The effects of carbon and nitrogen on hydrogen phototropic production were examined by mixed cultures. Appropriate carbon source and nitrogen source of hydrogen production and the optimum C/N mass ratio of hydrogen production were determined. The experiments of phototrophic hydrogen production from simulated starch wastewater by mixed cultures were conducted. The results showed that starch concentration was the main influencing factor. The cumulative hydrogen production increased and hydrogen yield decreased with the starch concentration increasing. Therefore, the starch concentration, hydrogen production rate and lag time should be considered carefully. The results indicated that it is feasible for phototrophic hydrogen production from starch wastewater by mixed cultures. Finally, PCR-DGGE method was applied to determine the relative genetic complexity of microbial communities in mixed cultures. Analysis of DGGE profile showed that the quantity and brightness of bands were different under light and dark condition during hydrogen production from starch, predominant microbial population of dark condition was more in quantity.