The hydrogen production yield from glucose by an isolate was investigated and compared to that by microflora. The isolate, Thermoanaerobacterium thermosaccharolyticum KU001, from the microflora demonstrated approximately 2.4 mol/mol-glucose of hydrogen production with acetate/butyrate formation in an artificial medium. The fermentation pattern was similar to that observed for the hydrogen fermentation of wastewater by the microflora. A PCR-DGGE analysis of the bacterial 16S rDNA detected T. thermosaccharolyticum in the microflora with strong intensity of the characteristic 16S rDNA band, although the microflora was enriched from an artificial medium. These results imply that T thermosaccharolyticum could be a predominant species of the microflora that is involved in hydrogen-producing acetate/butyrate fermentation. The nitrogen source in the medium affected the carbohydrate metabolism of KU001, and caused a change in hydrogen yield.