The purpose of this study was to optimize the conditions of continuous mixed culture of C.butyricum and R. spaeroides K-7, which were able to produce hydrogen using biomass-dreived substrate. To investigate the possibility of continuous culture, semi-continuous culture was carried out for 20 days. In semi-continuous culture using the reactor system, the replacement rate of fresh medium was 30% of total medium volume for the highest hydrogen evolution. In continuous culture, the optimum dilution rate was determined to be 0.05$h^{-1}$. The continuous culture produced 3.1 times as compared with the hydrogen on batch culture. On the other hand, the continuous mixed culture produced 1.3~2.1 times as much as hydrogen of the continuous monoculture of C. butyricum. When 10g of glucose in the media (1l) was supplied as a carbon source on continuous culture, mixed culture of C. butyricum and R. sphaeroides K-7 increased hydrogen evolution rate. Because considerable amount of glutamate was contained in waste water of glutamate fermentation, utilization of glutamate was examined in mixed culture. As a result of examination, production of hydorgen was slightly inhibited by high concentration of glutamate, more than 20mM, on continuous monoculture of R. sphaeroides K-7. On the other hand, both on continuous monoculture of C. butyricum and on mixed culture of C. butyricum and R. sphaeroides K-7, production of hydrogen was not inhibited by high concentration of glutamate such as 100mM. Hence this suggests that high concentration of waste water can be used as good substrate for hydrogen production on monoculture of C. butyricum and mixed culture of C. butyricum and R. sphaeroides K-7.