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Not AvailableLignocellulosic wastes such as rice straw gain importance in ensuring sustainable production of biofuels, being one of the most abundant agricultural residues worldwide. This study points the need for characterization and selection of substrate, optimizing the pretreatment method, as well as employing suitable fermenting organism, which can be of immense help in the path towards a more efficient ethanol production process. Microwave assisted alkali, acid, or water pretreated rice straw of five cultivars (PRH-10, Pusa Basmati 1121, Pusa 44, Taraori basmati and IR-36) were subjected to enzymatic hydrolysis, followed by fermentation with Pichia stipitis 3498. Saccharified hydrolysate of pretreated rice straw gave 4–5 times higher sugar recovery compared to untreated straw. Total sugar recovery from different cultivars after pretreatment and saccharification ranged between 20 and 59%. Among the cultivars, Taraori basmati gave significantly higher sugar recovery and ethanol yield of 39.65%±3.70 and 5.16% respectively, through microwave/alkali pretreatment which was found best. Kinetic studies done in biofermentor were able to produce the maximum yield of 6.63% (v/v) ethanol, equivalent to 92.9% of the theoretical yield, with an ethanol yield coefficient of 0.47gg⁠-1. This study confirmed the importance of considering varietal differences of substrate, highlighted the higher efficiency of microwave/alkali pretreatment in enhancing straw digestibility and also the potential of the strain P. stipitis 3498 for ethanol production from lignocellulosic biomass.Not Availabl