The effect of substrate to inoculum (So/Xo) 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 So/Xo ratio of 1.0 gCOD/gVSS achieved maximal hydrogen yield (HY) of 79.2 ± 11.9 mL H2/gCOD removed. Supplementation of anaerobes with 100 mg/L MGO promoted HY up to 112.4 ± 10.5 mL H2/gCOD 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/gVSS, respectively. However, propionate (HPr) concentration dropped from 35.9 ± 2.7 to 15 ± 1.3 mg/gVSS. Hydrogenase enzyme activity increased 9-folds and the anaerobes elongated from ca. 1.8-2.9 to ca. 2.5-5.1 ?m with MGO addition. Moreover, Proteobacteria, Firmicutes, Clostridia and Bacilli were detected with the batches supplemented with MGO. © 2016 Elsevier Ltd.