The increase of glycerol production due to the high demand for biodiesel has made this byproduct attractive for research. It can be used as a substrate in bioprocesses and when associated with the yeast extract, it can reduce the costs with culture mediums. Biohydrogen is an important bioproduct from fermentative metabolism; industrially important as a fuel and chemical compound. The objective of this paper was to evaluate hydrogen production from crude glycerol by Enterobacter sp strain 9 under different nutritional conditions. The comparison was performed in seven nutrient media at different concentrations of crude glycerol (15, 21, 33, 40 and 55 g/L). The highest yield of hydrogen was obtained without the addition of a complex mineral media by applying a buffer solution with pretreated lysed yeast cells.