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Hydrogen is known as a clean energy resource. The recent surge in global H2 production research promises to make this clean fuel a replacement energy utilization of fossil fuels. The biological production of hydrogen by using wastewater and other biomass as raw materials has been attracting attention due to the characteristic of conversion of waste materials to energy resources. An anaerobically fermentative continuous culture system using condensed molasses fermentation solubles (CMS), which comes from a monosodium glutamate factory, as nutrient substrate was used to study hydrogen production. Previous results of reverse transcription polymerase chain reaction (RT-PCR) indicated that Clostridium spp. existed in the system. In fact, one Clostridium tyrobutyricum-like and one C. pasteurianum-like clostridia (F4 and F40) were substantially isolated from CMS by using PYG agar plate (glucose as sole carbon source). One C. sporosphaeroides-like clostridia (F52) were also isolated from CMS by using VR agar plate (monosodium glutamate as sole carbon source). C. sporosphaeroides was reported that they could produce hydrogen when using monosodium glutamate as sole carbon source. Primers were synthesized according to the consensus sequence of different clostridial hydrogenase genes. The results of PCR and RT-PCR indicated that those clostridial hydrogenase genes actively expressed under HRT 8 h and HRT 4 h were similar to the hydrogenase genes of C. acetobutylicum. Phylogenetic analysis of all the hydrogenase amino acid sequences obtained from RT-PCR, and PCR indicated that clostridial hydrogenase genes actively expressed under HRT 8 h and HRT 4 h were similar to hydrogenase genes of Clostridium sp. existed in CMS. The result reveal that the major hydrogen-producing clostridia may come from CMS. The hydrogen-producing rate of this system varied with different hydraulic retention time (HRT), probably due to the variation of microbial composition in this system. Indeed, this variation was clearly shown in the denaturing gradient gel electrophoresis (DGGE) of 16S rDNA fragment that were produced by PCR and RT-PCR from total cellular DNA and RNA at HRT 8 h and HRT 4 h, respectively. When those 16S rDNA fragments that significantly appeared on the DGGE gel were further cloned and sequenced, the results indicated that Clostridium tyrobutyricum and C. butyricum were probably the major hydrogen-producing microbes under HRT 8 h and HRT 4 h. Because CMS contain carbohydrate and monosodium glutamate, it was speculated that two strains of bacteria using glucose and monosodium glutamate, respectively, might efficiently use CMS to produce hydrogen. Hence co-culture of F4、F40、F52 using CMS medium, reveal that co-culture of F40 and F52 can have the highest H2 production up to 96 ml .

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