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Bacteria produce hydrogen during anaerobic dark digestion of carbon rich natural resources including renewable cellulosic materials. The purpose of this work was to study the impact of maize silage pretreatment with Trichoderma fungi, bioaugmentation with defined bacterial inocula and/ or biostimulation with humic acids and an additional inorganic nitrogen source on the fermentative hydrogen production in laboratory batch assay. Experiments were carried out with and without Trichoderma asperellum pretreated silage. The selected bacterial inocula consisted of Clostridium, Enterobacter and Tissierella species, with or without Bacillus mycoides. Headspace gas composition, the amount of dry particulate matter, chemical oxygen demand and concentration of volatile fatty acids in liquid were determined. Bacterial communities were studied with fluorescence in situ hibridization. The predominant cultivable microbial species were isolated and identified. The study demonstrated a significant increase of hydrogen production from maize silage by indigenous bacteria after pretreatment with Trichoderma in comparison with silage untreated with Trichoderma. From tested factors, pretreatment, biostimulation with additional nutrients (ammonium nitrate and/ or humic acids) and bioaugmentation with defined bacterial inocula, pretreatment demonstrated significant improvement of hydrogen production from maize silage. Thereby, aerobic treatment with Trichoderma could be recommended for the pretreatment of silage for the purpose of fermentative production of hydrogen.