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Biohythane production from sugarcane bagasse by two-stage thermophilic fermentation process consisting of hydrogen production as first stage and methane production as second stage was investigated. Maximum hydrogen and methane productions from two-stage thermophilic fermentation process obtaining at 15 % w/v sugarcane bagasse were 4.87 L H2/L-waste and 28.54 L CH4/L-waste, respectively. Whereas, single stage gave maximum methane production of 10.35 L CH4/L-waste at 20 % w/v sugarcane bagasse. Pretreatment of solid fraction after hydrogen production by 1 % (v/v) H2SO4, steam explosion and microwave with subsequent methane production by two-stage thermophilic fermentation process improved methane production in second stage of 30, 160 and 200 % comparing with that of without pretreatment. Pretreatment of solid fraction after hydrogen production by 5 % NaOH for 24 hours gave maximum methane production of 7.28 L CH4/L-waste at 10% w/v sugarcane bagasse. Pretreatment of solid fraction after hydrogen production by 1 % (v/v) H2SO4 for 24 hours gave maximum methane production of 17.10 L CH4/L-waste at 15 % w/v sugarcane bagasse. Pretreatment of solid fraction after hydrogen production by steam explosion at 121 °C for 1 hour gave maximum methane production of 35.75 L CH4/L-waste at 15 % w/v sugarcane bagasse. Pretreatment of solid fraction after hydrogen production by 700-watt microwave for 3 min gave maximum methane production of 39.28 L CH4/L-waste at 30 % w/v sugarcane bagasse. Hydrogen and methane productions from sugarcane bagasse at 15 % by two-stage thermophilic fermentation process gave 33.41 L of mixed biogas that was a suitable composition (H2 = 6.0 %, CO2 = 35 % and CH4 = 58.9 %) as biohythane fuels with total energy of 1,384 KJ/L.
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