The fuel shortage is now the subject of debate in all countries of the world, so far as the years pass, the less the existence of so-called energy from fossil fuels, or non-renewable energy. For this reason, it has been great investment in alternative energies, which are renewable and environmentally sustainable. The current research specifies a conversion of biomass to bioethanol of 30 to 50% only however novel processes increase the conversion yield to about 92% of the theoretical yield. Bacillus subtilis was isolated from three different samples soil, bark of the tree and cow dung and the preferred sample was evaluated for cellulase enzyme activity. The crude enzyme produced on cellulose basal broth by these potent isolates was partially purified by 80-85% ammonium sulphate precipitation. Saccharification of substrate into reducing sugars was estimated by DNS reagent. To detect 16S rRNA of the Bacillus subtilis polymerase chain reaction was performed which showed band exactly in 595 bp. Sample S3 obtained from soil was found to be most potent with 19.76% cellulose conversion rate from CMC and 5.20% cellulose conversion rate from filter paper. The synergistic action of samples S4, D2 and B2 (3 out of 4 isolated) was found to be the most effective with 25.80% cellulose conversion from CMC and 5.90% cellulose conversion from filter paper. 1.75% alcohol solution was obtained  from NaOH pre-treated rice straw and 2.43% alcohol solution was obtained when CMC was used as substrate by using the mixed culture of S4,D2 and B2.



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