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In this thesis, two kinds of hydrogen-producing microorganism were mixed cultured for hydrogen production under illumination. After optimization of Rhodobacter sphaeroides growing and hydrogen production conditions, R. sphaeroides was mixed cultured with Halobacterium salinarum in different forms (packed cells, purple membrane fragments, fixed-based packed cells, fixed-type purple membrane fragments), and understood the roles in hydrogen production of different forms of H. salinarum. H. salinarum was domesticated by decreasing the NaCl concentration, then the domesticated strain was mixed cultured with R. sphaeroides for hydrogen production. Conclusions were summarized as follows:①Select four major environmental impact factors: different carbon source, temperature, light intension and the initial pH value, and carried out three level- orthogonal test, initial defined the the best growth conditions for R. sphaeroides. Sodium pyruvate and sodium succinate is the suitable substrate for growth; sodium acetate is not the best carbon source for R. sphaeroides. The best growth results of R. sphaeroides was under the mode of pH 6.5~7.0, the light intensity for the 5000 ~ 6000 lx, temperature for 30 ~ 35℃and anaerobic-illumination conditions. R. sphaeroides was cultured in Biolog carbon metabolism plate under aerobic and anaerobic environment. It was observed that, R. sphaeroides hours had a high metabolic activity under aerobic illumination within 48h, used a large number of carbon sources; it had a high activity anaerobic light between 48 ~ 72h, the used carbon sources were more than them in aerobic training.②A preliminary study R. sphaeroides on batch culture, the dynamics of hydrogen producing, as well as the different carbon source, light intensity, temperature and light /dark alternation on the impact of hydrogen production. With the increase in biomass, the total hydrogen production was improved. Sodium succinate, sodium pyruvate and glucose were good hydrogen donors for hydrogen production on R. sphaeroides, the maximum hydrogen production rate was 10.3 ~ 22.1ml/h/L, and the substrate conversion efficiency was 26.2% ~ 40.5%. Sewage as a single substrate, there was no hydrogen produced. In the light intensity range was 5000 lx ~ 9000 lx, the hydrogen production capacity increases by the light intensity; but decreased to some certain level. In 30℃~ 35℃range, R. sphaeroides was higher hydrogen production activity; When the temperature is greater than 38℃, the hydrogen production capacity was declined. In the light / dark alternation environment, the total hydrogen production increased slightly, but there is no hydrogen production in the dark phase.③R. sphaeroides was mix-cultured with suspended packed cells(PC) and purple membeane fragment (PM). Total gas production was increased from 124 to 217 ml with addition of 80 nmol of BR, and hydrogen production rate was increased from 9.6 to 17.9 ml/h/L of culture. In a certain extent, hydrogen production capacity was increased with the addition of BR concentration. The addition of BR in the form of PM fragments had no significant effect on hydrogen production. The enhancement of hydrogen production by R. sphaeroides by PC of H. salinarum could be ascribed to the additional protons coming from the light induced proton pumping of BR. The provided protons were readily not only used by nitrogenase of R. sphaeroides, but also adjusted the pH of the nutrient solution.④The pacted cells and purple membeane were immobilized on sodium alginate. R. sphaeroides was mixed cultured with SA-PC、SA-PM, respectively. SA-PC can enhance the stability of packed cells, decrease the impact of the low ionic concentration, increase the total hydrogen production by R. sphaeroides. When BR was 40nmol, the maximum hydrogen production rate was 16.3 ml / h /L, the total hydrogen production was 1165 mL /L culture. PM did not contain any cellular components, therefore PM on SA had long term stability and strong resistance on low ionic concentration. SA-PM could enhance hydrogen production of R. sphaeroides. Photoactivity of SA-PM is stronger than SA-PC. When BR was 20nmol, the hydrogen production rate of 14.6 mL / h / L, the total hydrogen production was 1085 mL /L of culture When R. sphaeroides and SA-PC (BR was160nmol) were semi-connected cultured, the protons coming from the light induced proton pumping of BR transfer through the proton exchange membrane, enhanced the hydrogen production by R. sphaeroides. Containing the same amount of BR, different forms of H. salinarum enhanced the hydrogen production of R. sphaeroides as followed: SA-PM> SA-PC> PC> PM.⑤H. salinarum was domesticated through decreasing the NaCl concentration gradually, and the strain could be grown well in 14% NaCl environment. PC and PM were separated from the domesticated strain. PC (BR was 160nmol) could promote the R. sphaeroides hydrogen production, the total hydrogen production increased to 234ml; there is no obvious role with addition PM. Compared to initial strain, BR in the form of PC, PM photosensitivity has not been a substantial increase. While in the 14% of the environment, the biomass and purple membrane of H. salinarum can be very good, but the low-ionic concentration of medium could weaken the photosensitive of BR, and affect the hydrogen production of mixed System.
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