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Co-substrate utilization of various wastes with complementary characteristics can provide a complete medium for higher hydrogen production. This study evaluated potential of apple pomace hydrolysate (APH) co-fermented with crude glycerol (CG) for increased H 2 production and decreased by-products formation. The central composite design (CCD) along with response surface methodology (RSM) was used as tool for optimization and 15 g/L of CG, 5 g/L of APH and 15% (v/v) inoculum were found to be optimum to produce as high as 26.07 ± 1.57 mmol H 2 /L of medium. The p -value of 0.0017 indicated that APH at lower concentration had a significant effect on H 2 production. By using CG as sole carbon source, reductive pathway of glycerol metabolism was favored with 19.46 mmol H 2 /L. However, with APH, oxidative pathway was favored with higher H 2 production (26.07 ± 1.57 mmol/L) and decrease in reduced by-products (1,3-propanediol and ethanol) formation. APH inclusion enhanced H 2 production, and decreased substrate inhibition. [ABSTRACT FROM AUTHOR]