This study is aiming at investigating the metabolic characteristics of Ethanoligenens Harbinense YUAN-3. The effects of yeast extract powder and supplemental vitamins on the growth medium were assessed. With yeast powder as the sole nitrogen source, the biomass yield and hydrogen-producing capacity of YUAN-3 from glucose fermentation significantly increased. Adding vitamin in yeast powder can enhance hydrogen yield compared with no adding. Therefore vitamin supplement can be considered as an effective growth factor in this study. Batch culture also produced larger yields of hydrogen.