The effect of substrate to inoculum (S-o/X-o) ratio and supplementation of magnetite/graphene oxide (MGO) nano-composite material on hydrogen production from gelatinaceous wastewater via dark fermentation process was investigated. Results demonstrated that optimum S-o/X-o ratio of 1.0 g(COD)/g(VSS) achieved maximal hydrogen yield (HY) of 79.2 +/- 11.9 mL H-2/g(COD removed). Supplementation of anaerobes with 100 mg/L MGO promoted HY up to 112.4 +/- 10.5 mL H-2/g(COD removed). Moreover, the degradation efficiency of carbohydrates, proteins and lipids was improved to 80.8 +/- 7.6, 34.4 +/- 2.3 and 31.4 +/- 2.2%, respectively. Acetate (HAc) and butyrate (HBu) concentrations increased from 102 +/- 6.8 to 125.3 +/- 6.3 and from 31.1 +/- 1.5 to 48.8 +/- 3.5 mg/g(VSS), respectively. However, propionate (HPr) concentration dropped from 35.9 +/- 2.7 to 15 +/- 1.3 mg/g(VSS). Hydrogenase enzyme activity increased 9-folds and the anaerobes elongated from ca. 1.8-2.9 to ca. 2.5-5.1 mu m with MGO addition. Moreover, Proteobacteria, Firmicutes, Clostridia and Bacilli were detected with the batches supplemented with MGO. (C) 2016 Elsevier Ltd. All rights reserved.