Studies On Biohydrogen Production By A Two-step Process Of Sequential Dark- And Photo-fermentation And Related Biotechnologies

Biological hydrogen production,which is carried out at normal temperature,normal pressure and low energy-consuming as well as environmental-friendly,has been attracting the common attentions.Among the several main biological hydrogen-production pathways (photolysis of water,photo-fermentation of small molecular organic compounds,and light-independent anaerobic fermentation from relatively large molecular organic compounds),light-independent anaerobic(dark-) fermentation and photo-fermentation are currently the closest to application,owing to thier higher hydrogen production rate.Many recent investigations demonstrated that photo-fermetnation further converted the metabolites from dark-fermentation to hydrogen and the two-step process of sequential dark- and photo-fermentation could not only improve the hydrogen yield but also remove the most COD in the dark-fermentational effluent.It provides a new promising approach to produce biohydrogen from waste biomass.To further improve the hydrogen producing efficiency of the two-step process of sequential dark- and photo-fermentation,the succession of microbial composition during the hydrogen producing process of darkfermentation was investigated,the bacterial strains with high hydrogen producing efficiency were screened and the conditions for photo-fermentation were optimized in this study.The dissertation includes the following five parts:1.Hydrogen production from different substrates by the sequential dark- and photo-fermnetation.One of the main problems on developing bioenergy is to seek suitable feedstock.A Feasible study of sequential dark- and photo-fermentation hydrogen production from cassava and food waste was testified.In dark-fermentation,the average yield of hydrogen was approximately 212 mL H₂/g-cassava and 226 mL H₂/g-food waste. In subsequent photo-fermentation,the average yield of hydrogen from the effluent of dark-fermentation was approximately 611±11 mL H₂/g-cassava and 451±20 mL H₂/g-food waste.The process of sequential dark-fermentation and photo-fermentation could reach two-four fold hydrogen yields that dark-fermentation did.And meanwhile,the COD decreased greatly with a removal efficiency of 84.3%in cassava batch and 80.2%in food waste batch.These results demonstrate that cassava and food waste could be ideal substrates for biohydrogen production.And a two-step process of sequential dark-fermentation and photo-fermentation was highly efficient on both preventing the environmental issue and improving biohydrogen production capacity.2.The succession of dominant bacteria during the hydrogen producing process by dark-fermentation in a 10 L continuous stirred tank reactor was investigated.To elucidate the bacterial community structure-function relationships of 10 L continuous stirred tank reactor(CSTR) system possessing relatively high hydrogen producing potential,molecular marker of 16S-rRNA and hydA gene,denaturing gradient gel electrophoresis(DGGE), qrt-PCR and hydA gene-based DNA and cDNA clone library were applied.The results of DGGE described a general profile of bacterial population dynamics:Bacillus spp.grew in the lag phase;Clostridium perfringens and Clostridium beijerinckii became dominant when hydrogen production was initiated;other Clostridium spp.began to appear when hydrogen production stopped and hydrogen uptake was observed.Deatailed dynamic profiles about categories and abundances of hydrogen producers in different stage were acquired with respect to hydA gene-based DNA and cDNA clone library.Expression level of hydA mRNA in different stages were studied by qrt-PCR,the results were that total hydA mRNA expression level in the culture reached the peak at stationary phase and is positively related to the instant hydrogen production rate,but relative hydA mRNA expression level of one hydrogen-producer reached the peak during exponential phase.The above results demonstrated that predominant bacterial communities and specific profile of succession can be monitored by molecular ecology technologies adopted.3.Isolation of the dominant hydrogen producers in the dark-fermentation system.The strain 08-2 was isolated from the 8h-sample of 10L CSTR,and identified as Bacillus cereus according to16S rRNA gene sequences.14 strains of hydrogen producers were isolated from 16h-sample of 10L CSTR,and then were merged as 3 different strains quickly by ERIC-PCR.Among them,the strain 08-1 was identified as Clostridium beijerinckii based on its phenotype and 16S rRNA gene.Sucrose,biomass and wastes which components complicated were favorite carbon sources for growth and hydrogen production by the strain 08-1.The optimum temperature was 40℃and pH was 5.5 for hydrogen production of strain 08-1.Under batch fermentative hydrogen production conditions:concentration of sucrose 20 g/L,temperature 40℃,pH 5.5 controlled and stirred rate 100 rpm,the maximal hydrogen yield and volumetric hydrogen production rate for strain 08-1 were 3.09 mol H₂/(mol-sucrose) and 245 mL/L.h,respectively.The results demonstrate that the strain possesses potential application in biohydrogen production from wastes.The strain 08-11 was a facultative anaerobic hydrogen producer and was judged as a new species based on its 16S rRNA gene.Clostridium perfringens were counted and isolated from 24 h-samples of 10L CSTR by selective medium.Moreover,128 strains of hydrogen producers isolated from other hydrogen production system,and among them,5 strains of Clostridium butyricum,3 strains of Enterobacterium sp.,1 strain of Clostridium perfringens,Clostridium beijerinckii,Bacillus licheniformis and condensate bacillus. These important functional strains acquired from 10L CSTR further demonstrate validity on forecasting hydrogen production community structure by molecular marker-based monitoring system.Furthermore,ERIC-PCR method was successfully applied to quick identifying and classifying dark-fermentation hydrogen production strains for the first time.4.Screening photosynthetic dacteriat strains to produce hydrogen from wastewater of biodiesel manufacture.13 strains of photosynthetic bacterial isolated from different water bodies.Their maximum hydrogen production rates and substrate conversion efficiency from 30mM malate were 47-103 mL/l·h and 27.98-72.00%,respectively.Photo-hydrogen production was carried out from acetate and butyrate by 4 selected photosynthetic bacterial with higher biohydrogen efficiency from malate.The strain HL-1 can utilize both acetate and butyrate for growth and hydrogen production.Strain SC-6 and SC-7 can grow but can't produce hydrogen from acetate and butyrate.The strain HL-5 can can grow but can't produce hydrogen from acetate,and utilize butyrate for growth and hydrogen production. These strains would be excellent material for investigating different acetate and butyrate metablic pathway.A strain(designated as DB803) producing hydrogen efficiently from glycerol as the carbon source was screened from 37 photosynthetic bacterial strains.This strain was identified as a new strain of Rhodobacter sphaeroides based on its phenotype, physiological and biochemical characteristics,the sequence of its 16S rRNA gene and fingerprinting of ERIC-PCR.Its hydrogen yields from biodiesel wastewater at different dilution times were investigated under the anaerobic condition of 30℃,4000lux of illumination.The average hydrogen-producing rate of 38 mL/L·h was observed at log phase when the initial COD value of wastewater was diluted to 11.5 g/L.Meanwhile,the COD decreased greatly with a removal efficiency of 91.2%at batch test.The results demonstrate that the strain possesses potential application in hydrogen production from wastewater of biodiesel manufacture.And moreover,DB803 would be material for further studing metabolic pathway of glycerol for hydrogen production by photosynthetic bacterial. 5.The optimal conditions for photo-fermentation and investigation on hydrogen production of photo-fermentation under sunlight.The effects of different inoculum,culture volume and gas types on photohydrogen production were investigated.Hydrogen production potential inoculated by inoculum under photo and anaerobic conditions was superior to the inoculum under dark and aerobic conditions.Substrate conversion efficiency was high with smaller headspacein bioreactor.Nitrogen gas might promote the metabolic activities of photosynthetic bacterium.Moreover,Production of hydrogen by photosynthetic bacteria under sunlight was studied.In the experiment on hydrogen production by natural sunlight during daytime and supplement with artificial light during night,the hydrogen production rate by the photosynthetic bacteria depended on the sunlight intensity;the average hydrogen production rate during daytime and night was 12.4 mL/L·h and 9.0 mL/L·h,respectively.And the substrate conversion efficiency was relatively high,72.4%.Another experiment on hydrogen production by natural sunlight during daytime and not supplement with artificial light during night was also conducted,its substrate conversion efficiency was 45.02%.The cause of time prolonged in amplified photobioreactor would be contributed to smaller specific surface area of cylindrical photobioreactor with bigger diameter height ratio.