Different types of sludge pretreatments were tested, with thermal shock at 90 degrees C to 95 degrees C for 60 minutes plus a 6-hour rest period achieving the best results for inhibition of methanogen microorganisms and inoculum enrichment with H-2-producing bacteria, which produced a H-2-rich biogas (up to 65% mol/mol) without the presence of CH4. Wastewater from biodiesel production (WBP), containing mainly methanol (128 g/L) and glycerol (4 g/L), was evaluated as a potential substrate to produce H-2 through dark fermentation. Both methanol-based solutions and methanol-rich wastewater were not suitable for hydrogen production; however, these effluents showed a strong potential for CH4-rich biogas production. A fractional factorial design was employed to evaluate the effect of six substrate-related variables (glycerol content, 25% and 75%; COD content, 4 and 50 g/L, COD:VSS ratio, 1:1 and 5:1; COD:N:P ratio, 350:0:0 and 350:5:1; NaCl content, 0.5 and 12.0 g/L; and pH, 4.0 and 5.5) on the H-2 production from glycerol-methanol-based synthetic solutions (synthetic WBP). Some substrate-related variables had a crucial impact on the hydrogen production potential from WBP, which was significantly affected by the COD and salinity content in the substrate. WBP containing high glycerol (representing until 75% of the COD) and salinity (up to 12 g/L as NaCl) content could be turned into a potential substrate for H-2 production through dark fermentation as long as specific fermentation conditions are maintained, such as pH 5.5 to 5.7 and a substrate COD content up to 50 g/L. Using this condition, glycerol conversion, H-2 productivity, and H-2 yield of 81.3 +/- 8.9%, 102.8 +/- 18.2 mL H-2/L.d, and 24.5 +/- 4.4 mL H-2/g CODapplied, respectively, were obtained.