Empty fruit bunch was considered as substrate for second generation of bioethanol because it consists of lignin, cellulose, and hemicelluloses. For lignocelluloses materials, it usually needs pretreatment and hydrolysis to convert cellulose into glucose. Two methods of enzymatic hydrolysis, Separated Hydrolysis and Fermentation (SHF) and Simultaneous Saccharification and Fermentation (SSF) were carried out in this study. The performance of both SHF and SSF was concerned to evaluate the effect of hydrolysis methods and enzyme concentration for producing ethanol. Pretreatment was conducted in a reactor using 10% sodium hydroxide at temperature 150°C during 30 minutes. Two kinds of enzyme, Cellic® CTec2 and Cellic® HTec2 from novozyme were added in 15% (gr/ml) of pretreated EFB at pH 4.8. Four concentration of enzyme Cellic® CTec2, 10, 20, 30, 40 FPU per gram biomass were performed in SHF and SSF processes respectively, while Cellic® HTec2 was added 20% from Cellic® CTec2. Contents of glucose, xylose, and ethanol were recorded every 24 hours. Using 40 FPU of concentration enzyme, it could be produce 4.74% of ethanol in 72 hours fermentation by SHF process and 6.05% of ethanol in 24 hours by SSF process. From this study, the SSF method was considered as a better process than SHF due to rapidly ethanol production and the highest concentration of produced ethanol.

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