Fermentative Hydrogen Producing Bacteria And The Population Ecology Of Mixed Culture Systems

Many researches based on ethanol type fermentative H2-producing technique and theory have been developed of late years for increasing H2-producing efficiency, improving engineering controlling methods and realizing industrial H2- production. Several bacteria of Ethanoligenens and a lot of engineering datas had been achieved.But the engineering controlling countermeasures and H2-producing efficiency still need to be improved and increased . Particularly the ecology mechanism of ethanol type fermentative H2-producing systems have not been disclosed, and monitoring technique still need to be improved. In this paper, ethanol type fermentative H2-producing system was the focus. Several bacteria procucing ethanol and H2 were identified. And population ecology of mixed culture fermentative H2-producing systems were studied deeply by improved FISH technique.First, two fermentative hydrogen producing bacteria C12 and C3 were isolated from an active sludge sample. Their characteristics of morphology, physiology, biochemistry and H2-production influencing factors were compared with Ethanoligenens harbinense B49 by methods of biochemistry, electronic microscope and molecular biology. The full-length 16S rDNA sequences of them and phylogenesis were studied. And their resemblances of 16S-23S rDNA intergenic spacer region(ISR) were analyzed. Results indicated that these two bacteria all belong to Ethanoligenens the same as B49. Based on those results, searching for 16S rRNA oligonucleotide probe of Ethanoligenens for FISH experiment was tried to do. It was proved that the oligonucleotide probe of Ethanoligenens couldn't be achieved in 16S rRNA gene sequences. It should be resereched in 23S rRNA gene sequences or 16S-23S rRNA intergenic spacer regions(ISR).Second, normal FISH techniques using in environmental microbe were improved. Three oligonucleotide probes Chis150,Ent183 and HGC suitable for monitoring fermentative H2-producing system and the best hybridization conditions of them were determined. A FISH monitoring system with Clostridium, Ethanoligenens and Enterobacteriaceae as targets was built up primarily. A lot of FISH experiments were compared and analyzed. According to pre-experiment results, the processes were simplified, the experiment conditions were improved and the cost was decreased. So that the improving FISH was more suitable for monitoring mixed culture fermentation H2-producing system quickly.Third, the variations of goal populations in CSTR fermentative hydrogen producing reactor were studied by improving FISH during the starting period, the running courses and the changing course of ethanol and butyric acid type fermentations including the richness and variarions of goal populations in ABR fermentative hydrogen producing system. Effects of bacterial population structures on MLVSS, fermentative products and hydrogen producing rates were analyzed, and the relations between population ecology and fermentation types were discussed. Important fountions of Clostridium, Ethanoligenens and Enterobacteriaceae on deciding fermentation types were revealed for the first time. The dominant population of ethanol type fermentation were Clostridium and Ethanoligenens, and the dominant population of butyric acid type fermentation was Enterobacteriaceae .Furthermore, COD and pH affected the fermentation types of reactors during the starting period together. But the pH was the main controlling factor during running courses. Also several advices for engineering controlling were suggested.