Screening Of Photo-fermentation Bacteria And The Combination Of Dark And Photo- Fermentation Bacteria For Hydrogen Production

At present, energy substitute for fossil fuels need urgently because the lack of fossil energy source and the increase of international petrolic price. Hydrogen is to replace fossil fuels in the future, will become world's"clean energy choice", it has to be produced renewably and in large scale, through environmentally benign processes. Among bio-hydrogen production processes, photo-fermentation hydrogen production can combining solar energy, wastewater treatment and cleaning energy source production, the cost was decreased, considered as a potential hydrogen production technology in engineering application. The combination of dark and photo-fermentation can improve hydrogen yield and substrate conversion efficiency, and it is significant for scale bio-hydrogen production in future.In this study, a photo-fermentation bacterium strain RLD-53 was isolated from freshwater pond sludge. According to their morphology, physiological and biochemical characteristics, blast of the sequences of 16S rDNA and analysis of phylogenetic tree, we characterized it as a new strain within the species R. faecalis and designated as R. faecalis strain RLD-53. Optimal culture and H₂ production conditions of strain RLD-53 were determined in batch tests. Strain RLD-53 can convert acetate into H₂ with high efficiency. The hydrogen yields were 2.64-2.84 mol H₂/mol acetate and the maximum hydrogen production rate was 32.62 ml-H₂/l/h and average hydrogen content was about 81%。R. faecalis RLD-53 can not utilize butyrate and ethanol as sole carbon source for hydrogen production under anaerobic light conditions. But, butyrate and ethanol are main byproducts in butyrate and ethanol fermentation, respectively. The ratio of butyrate and ethanol to acetate was key factors for hydrogen production of the combination of dark and photo-fermentation bacteria. Therefore, the capacity of hydrogen production was studied when dual carbon sources were used as substrate. Results indicate that butyrate added in acetate medium significantly affected on photo-hydrogen production and butyrate of high concentration evidently inhibited on the growth and hydrogen production of RLD-53, and the optimal ratio of butyrate to acetate for H₂ production was determined. When the ratio of butyrate to acetate at 1:2, the hydrogen yields and maximum hydrogen production rate were 3.43 mol H₂/mol acetate and 32.53 ml H₂/l/h, respectively, and average hydrogen content reached about 84.27%. Effect of ethanol on hydrogen production was slightly. Effects of Ni²⁺, Fe²⁺ and Mg²⁺ on photo-hydrogen production and cell growth were obviously. In an appropriate range, Ni²⁺ and Fe²⁺ can increase the hydrogen production and cell growth with increasing their concentration. A low or high Ni²⁺ and Fe²⁺ concentration will inhibit photo-hydrogen production. Mg²⁺ concentration did not obviously affect hydrogen production by R. faecalis RLD-53. However, Mg²⁺ of different concentration in culture medium just promoted cell growth.Different gases as gas phase evidently influenced on photo-hydrogen production. Observation indicates that CO2 of high concentration have inhibited effect on cell growth and H₂ production. Therefore, we proposed a method of separation of CO2 from reaction system and it successfully stimulated photo-H₂ production in the entire H₂ production process.In fed-batch culture hydrogen production process, hydrogen production was improved by the control of feeding acetate concentration and pH. The average hydrogen yield of 3.17 mol H₂/mol acetate in this study was substantially higher than that in other study by pure cultures of bacteria. So experiments demonstrated the repeated fed-batch mode compared with batch culture obtained higher efficiency for hydrogen production.In order to increase acid-resistant capacity of RLD-53 and efficiency of substrate conversion into hydrogen, immobilized cells were used. The effects of diameter of agar granule, inoculant age, agar concentration, biomass in agar and light intensty on immobilized RLD-53 were investigated and at pH 5.0 hydrogen was also produced.The combination of dissociate dark-fermentation bacteria and immobilized photo-fermentation bacteria for hydrogen production was adopted first time in this study. To important, phosphate buffer was used to maintain different pH of culture system and the change of component of metabolites from dark-fermentation, and the content of acetate was successfully increased. Results showed that the dilution ratio of metabolites from dark-fermentation play an important role in two step hydrogen production and the control of initial pH and the ratio of dark and photo-bacteria were key factors in mixed culture hydrogen production. At initial pH 7.5, pH of entire process at 6.5-7.5, and properly increased the concentration of strain RLD-53, these were favorable to dark and photo-fermentation bacteria. The mechanism and key factors of hydrogen production by the combination of dark and photo-fermentation was analyzed preliminary.