Dark fermentative hydrogen gas production from cheese whey powder solution was realized at 55 degrees C. Experiments were performed at different initial biomass concentrations varying between 0.48 and 2.86 g L-1 with a constant initial substrate concentration of 26 +/- 2 g total sugar (TS) per liter. The highest cumulative hydrogen evolution (633 mL, 30 degrees C), hydrogen yield (1.56 mol H2 mol-1 glucose), and H2 formation rate (3.45 mL h-1) were obtained with 1.92 g L-1 biomass concentration. The specific H2 production rate decreased with increasing biomasss concentration from the highest value (47.7 mL g-1 h-1) at 0.48 g L-1 biomass concentration. Total volatile fatty acid concentration varied beetween 10 and 14 g L-1 with the highest level of 14.2 g L-1 at biomass concentration of 0.48 g L-1 and initial TS content of 28.4 g L-1. The experimental data were correlated with the Gompertz equation and the constants were determined. The most suitable initial biomass to substrate ratio yielding the highest H2 yield and formation rate was 0.082 g biomass per gram of TS. (c) 2012 American Institute of Chemical Engineers Biotechnol. Prog., 28: 931936, 2012