| Economic growth and our lifestyle in the last few decades have been strongly dependent on fossil fuels as sources of energy. However, the excessive dependence on fossil fuels has caused severe problems to human beings due to continual rising of their cost, insecurity in their sustainability, as well as their impacts on global warming and environmental pollution. Hydrogen is a clean and efficient fuel, widely recognized as a potential energy substitute for fossil fuels. Photo-hydrogen production process can combine treatment of organic wastewater, utilization of solar energy and production of clean energy source, so it is considered the most environment-friendly hydrogen production method. Rhodobacter sphaeroides ZX-5, a recently isolated purple non-sulfur (PNS) bacterial strain, has displayed higher hydrogen-producing capabilities. In this study, the physiological and metabolic characteristics of R. sphaeroides ZX-5 were investigated systematically. Meanwhile, the process of photo-fermentation by R. sphaeroides ZX-5 with RCVBN medium was optimized, and the efficient photobioreactors for photo-hydrogen production were designed. The main contents of the paper are as follow:The optimum conditions for the cell growth and hydrogen production by R. sphaeroides ZX-5 in 38-ml anaerobic tubes were determined as follow:temperature 30-35℃, pH 7.0, inoculum size:3.3%, medium volume:34 ml medium/38 ml anaerobic tube and inoculum age: 18-24 h. Moreover, the optimal concentration of carbon source and nitrogen source used for hydrogen production were 30-mM DL-malic acid and 7-mM L-glutamic acid. In this study, a noteworthy pH self-adjustment phenomenon was found, that is, all final pH values remained stable at 7.15±0.1 during batch hydrogen production process, even when the initial pH values varied within the range of 6.0-10.0. The effects of vitamins (nicotinic acid, vitamin Bi and biotin) on the growth and hydrogen production of R. sphaeroides ZX-5 were investigated. The results showed that nicotinic acid, as a precursor of NAD+/NADH, plays a crucial role in effectively enhancing the phototrophic hydrogen synthesis. Lack of nicotinic acid in hydrogen production medium resulted in the failure of photo-hydrogen production. In addition, though vitamin B1 and biotin do not have direct impact on photo-hydrogen production, they are still essential and must exist in either growth medium or hydrogen production medium. Without either of them, photo-hydrogen production decreased seriously, regardless of the existence of nicotinic acid.The light attenuation kinetic model, I=Ioexp[-(0.4762+0.32660D660)·L],well described the relationship among optical path (L), cell density (OD660) and light intensity (I). Based on this kinetic equation, a novel cultural method of photosynthetic bacteria was originated, i.e., shaking and extra-light supplementation (SELS), which greatly increased the rate and substrate conversion efficiency of photo-hydrogen production. Under shaking and elevated illumination (7000-8000 lux), the culture was effective in promoting photo-H2 production, resulting in a 59% and 56% increase of the maximum and average hydrogen production rate, respectively, in comparison with the culture under standing and 4000-5000 lux conditions. Moreover, substrate conversion efficiencies to hydrogen of both cultures were of no significant difference (ca.89%). To our knowledge, the hydrogen-producing rate of 165.9 ml H2/l·h under the application of SELS approach is currently the highest hydrogen production rate of non-immobilized PNS bacteria. This optimal performance of photo-H2 production using SELS approach is a favorable choice of sustainable and economically feasible strategy to improve phototrophic H2 production efficiency. In addition, the optimum illumination condition for shaking-culture by strain ZX-5 increased to 7000-8000 lux, markedly higher than that for standing-culture (4000-5000 lux).A new outer-cycle flat-panel photobioreactor equipped with oxidation-reduction potential (ORP) control unit was designed. In order to obtain the high hydrogen yield, photo-hydrogen production by fed-batch culture with on-line ORP feedback control was developed. Once ORP reached the threshold value of -400 mV, the concentrated medium would be added to culture system automatically right away. The results indicated that the optimum feeding concentration of malic acid was 50 g/l. Besides, due to the intrinsic pH self-adjustment feature of R. sphaeroides ZX-5, the pH of fermentation broth during fed-batch process did not need to be controlled. The photo-fermentation process with three times feeding consumed a total of 160 hours. The average substrate conversion efficiency was improved to 66.43% throughout the entire repeated fed-batch photo-fermentation. The maximum substrate conversion efficiency of 73.03% was observed in the first fed-batch process, much higher than that obtained from batch culture process (59.81%). In addition, compared to the batch culture, a significantly higher maximum hydrogen production rate (102.33 ml H2/l·h) was achieved during fed-batch culture. The results demonstrated that photo-hydrogen production using fed-batch operation based on ORP feedback control appears to be capable of attaining high-rate and high-yield phototrophic H2 production at the same time.In order to develop high-effective and low-cost photobioreactor for biohydrogen production, it is necessary to determine and evaluate the distinct effects of as many parameters as possible. The artificial light sources (e.g. incandescent lamp) which emit light in the red-infrared region are suitable for illumination of the photobioreactor for hydrogen production. The results from light/dark cycle experiment set a solid base for operating photobioreactors under outdoor conditions which would be exposed to the diurnal cycle. The other factor evaluated was hydrogen partial pressure in the culture system. The substrate conversion efficiency increased from 86.07% to 95.56% along with the decrease of the total pressure in the photobioreactor from 1.082×105 Pa to 0.944×105 Pa, which indicated that reduction of hydrogen partial pressure by regulating the total pressure in the headspace of photobioreactor substantially improved hydrogen production in an anaerobic, photo-fermentation process. Additionally, the shaking velocity of 120 rpm was the optimum condition for hydrogen production by R. sphaeroides ZX-5. Meanwhile, shaking during hydrogen production phase (i.e., cell growth stationary phase) of photo-fermentation played a crucial role on effectively enhancing the phototrophic hydrogen production, rather than that during cell exponential growth phase.Based on the understanding of physiological and metabolic characteristics of R. sphaeroides ZX-5 and the analysis of key factors for photobioreactor design, three different kinds of bioreactors for photo-H2 production were developed. The maximum hydrogen production rate of 141.65 ml J2/l·h and the total hydrogen production of 3056 ml H2/l medium were obtained using the side-stirred flat-panel photobioreactor, which were markedly higher than that obtained using the other two kinds of photobioreactors. The results demonstrated that side stirring pattern is in favor of the good mixing of cells and substrate, thus speed up H2 production.Computational fluid dynamics (CFD) technology was applied to simulate mass transfer process of hydrogen from the fermentation broth (liquid phase) to the bubble (gas phase) in tube-photobioreactor, and the facilitating function of SELS approach in the photo-hydrogen production was described from the microscopic view. In addition, the distribution and characteristics of flow field in different kinds of photobioreactors was simulated with CFD technology. Also, the influences of the flow field on the growth and hydrogen production of R. sphaeroides ZX-5 were investigated systematically, which would provide a theoretical basis for photobioreactors design and scale-up in the future. |
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