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Ethanol as a renewable biofule is an appropriate and viable alternative to the challenging fossil fuels. Bacillus subtilis, a gram positive bacterium, seems to be a promising choice since it has many useful features. For example B.subtilis ferments broad range of sugars derived from lignocellulosic hydrolysis. Transformation of this cellulytic bacterium to an ethanologenic one was accomplished via metabolic engineering techniques and Ethanol production operon of Z.mobilis was introduced to the B.subtilis. SR1 and SR21 strains expressed plasmid-borne ethanologenic genes of Z.mobilis but the genes had been integrated into the SR22 genomic DNA. Also lactate dehydrogenase gene had been knocked-out in SR21 and SR22 strains. Defect of cell growth in SR21 and SR22, suggests that NAD+ oxidation by lactate dehydrogenase is important for anaerobic growth. Considering the impact of Fe2+ ion on alcohol dehydrogenase II activity, in further experiments Fe2+ was added to the culture media and improvement in growth rates was seen. Final yield of ethanol production of SR1, SR21, and SR22 strains were 53.8%, 86.7%, and 83.9% respectively.

Keywords: "Bacillus subtilis", "Ethanol", "Biofuel", "pyruvate decarboxylase", "alcohol dehydrogenase"

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