Isolation, H₂-producing Characterization Of A Photosynthetical Bacterium PB-a And Study On Microbial Population Of H₂ Producing System

This thesis deals with the preliminary theoretical studies on the photosynthetic hydrogen production in the laboratory scale.The main results obtained from this study are as follows:1.A highly efficient hydrogen producing photosynthetic bacterium,named PB-a,was isolated from the fermentation sullage with pig dung.According to the《Bergey's Manual of Systemaic Bacteriology》,strain PB-a was identified to be Rhodopseudomonas based on morphology, physio-biochemicial and almost complete 16S rDNA sequences.The 16S rDNA sequences of PB-a were 99%similar with representatives of genus Rhodopseudomonas palustris.Phylogenetic analysis using neighbour-jioning treeing algorithms and bootstrap evaluation with the software of Clustalxl[1].83 showed PB- a was most closely related to the type strain of Rhodopseudomonas palustris S55.2.The medium components(trace element,carbon source,nitrogen source and the other mineral elements)and the culturing conditions(light intensity,pH and temperature)were optimized for the best growth and hydrogen production of strain PB-a.The optimized medium was composed of(g/L):sodium acetate 40 mmol/L,sodium glutamate 15 mmol/L,MgSO₄ 0.3, NaCl 0.4,KH₂PO₄ 0.2,K₂HPO₄ 0.6,NaHCO₃ 0.756,and 1 ml trace elements solution.The trace element solution contained(g/L):FeSO₄·7H₂O 2,ZnCl₂ 0.1,MnCl₂·4H₂O 0.1,H₃BO₄ 0.1, CoCl₂·6H₂O 0.1,NiCl₂·6H₂O 0.02,Na₂MoO₄·2H₂O 0.75,CuCl₂·2H₂O 0.01.The results showed that,four trace elements(Fe,Mo,Ni and Mn)had a significant impact on photosynthetic hydrogen production,with order of Fe＞Mo＞Mn＞Ni.The optimum conditions were light intensity 4000 lux,pH 7 and temperature at 30℃.Under the optimized conditions, strain PB-a could accumulate the maximum hydrogen production gas of 140.33 ml,reach the maximum average 20.9 ml/h/g(cdw)and the maximum special rate of hydrogen production of 30 ml/h/g(cdw)in the 100 ml volume.3.The relation of nitrogenase,uptake-hydeogenase and hydrogen release activity in the hydrogen producing process was analyzed.The results showed that nitrogenase activity and hydrogen release activity had a consistent trend,both increased rapidly in the3-4th day.Then,the activity declined slowly.Meanwhile,the activity of uptake-hydrogen had been raised with the time.Integrating of the change of their activities,it was consistent with the change of total hydrogen gas.The whole cell protein SDS-PAGE map of PB-a in the hydrogen producing process displayed that its composition and quantity in the expression had no significantly different.The character of whole cell protein change in the culture medium lacking different trace elements was analyzed due to the importance of the trace elements Fe,Mo and Ni.The result revealed that there existed significant differences in protein components and quantity expressed under the different culture conditions.All of the conclusions provided a basic foundation for the future study of hydrogen production.4.A PCR-DGGE(denaturing gradient gel electrophoresis of polymerase chain reaction) protocol was used for monitoring the dynamic changes of the microbial population during photo-hydrogen producing period.Total DNA was extracted directly from the mixed bacterial community in the reactor and subjected to PCR with V3-16S rDNA and pufM gene primers,and then the amplifications were analyzed by DGGE.The DGGE profiles demonstrated the dynamics of community structure and the shift of microbial diversity,which corresponded to different running periods of the reactor.The optimal hydrogen producing community formed on the 10th day.Using DGGE analysis with the pufM gene fragments was superior to V3-16S rDNA region genes for detecting the dynamic variations of photosynthetic bacteria population during hydrogen production.The DGGE profile with pufM gene fragments displayed more multiple clones than the DGGE profile with V3-16S rDNA region.The comparative sequence analysis of excised DGGE bands showed the relationship between specific population structures and system performance.It could be presumed that Rhodopseudomonas palustris was one of the dominant community members for hydrogen production in the reactor.From the two DGGE profiles,we found that different periods of hydrogen production resulted under different niche conditions,and formed different microbial community structures.Moreover,the sequence analysis provided the information for enriching and selecting the target dominant populations.We could design an updated reactor with the highly efficient hydrogen producing photosynthetic bacteria to enhance photo-hydrogen producing efficiency and substrate conversion in further study.