Isolation And Identification Of Anaerobic Ethanol-producing Cellulolytic Bacterial Consortia And Studies On Its Fermentation Characteristics

By enrichment with cellulosic substrates as the sole carbon source, the diversity of cellulose-decomposing anaerobic bacteria in the water column and sediment of Great Basin Hot Spring in Nevada were surveyed. Two samples were resulted in two cellulose-decomposing bacterial consortia. SV79, one of the two bacterial consortia, which performed a better cellulose-decomposing ability, was passed 15 times with achievement of a purified culture of bacterial consortia. Studying on the optimal culturing conditions and the optimal cellulose-decomposing condition, the results showed that the optimum pH , temperature, cellulose concentration, incubation concentration, culture volume and time were 8.0, 42℃, 1 g/100 mL cellulose concentration, 1% incubation concentration, 100 mL culture volumes, and 72 h cultivation respectively. The degradation rate of cellulose reached 51.3% - 68.2%. The crude enzyme liquid was prepared for the determination of CMCase activity; filter paper enzyme activity andβ-glycosidase activity, the results showed the PFase activity was 9.125 U/mL; CMCase activity was 9.89 U/mL andβ-glycosidase activity was 7.70 U/mL. Fermentation supernatant of SV79 was deposited by ammonium sulphate and the CMCase was purified by using Sepharose CL 4B column and its molecular weight by SDS-PAGE was about 42 kDa. This purified CMCase activity was 13.69 U/mL. The optimum pH for CMCase activity was 8.0, and the optimum temperature for CMCase activity was 52℃. Products of the cellulose fermentation by SV79 was also analyzed, this bacterial consortia provided a variety sugars by using filter paper, CMC, fungi substance scrap and straw, and Ethanol yields were 1.97 g/L for filter paper, 1.19 g/L for CMC, 0.74 g/L for fungi substance scrap and 1.14 g/L for straw by Gas Chromatography Analysis.By the method of 16S rDNA PCR, we got 16 positive clones. The 16S rDNA sequences of these clones were analyzed by NCBI BLAST, quite diversity in bacterial population in SV79 was revealed, including Ruminococcus sp. Clostridium sp. and Sporomusa sp. It proved that Clostridium sp. play the most important role in bacterial consortia SV79, which account for 75% of the whole bacterial consortia, Ruminococcus sp. account for 20% of SV79 bacterial consortia and Sporomusa sp. account for 5% of SV79 bacterial consortia.Anaerobic bacteria had been considered as catalyst for ethanol production from renewable cellulosic substrates. These organism produced very active cellulase complex and ferments cellulose to ethanol. In situ production of cellulase was the distinguishing feature of thermophilic bacteria which hold the greatest potential for positive economic impact relative to a yeast-based system for ethanol production from cellulose.Ordinary, many cellulose-decomposing strains were isolated base on cultivation and pure culture. The traditional technique had its major limitation to the study of microbial ecology, because of the fact that most of the microbial species in nature can not be cultured. Besides, symbiosis between strains were widely exist in nature, especially anaerobic cellulose-decomposing strains. In this study, a cellulose-decomposing bacterial consortia, which could utilize cellulose as the sole carbon and energy source, was screened from nature. To realize the efficient cellulose-decomposing ability and ethnol-producing ability, the characteristics of SV79 were studied. The study of SV79 also provided as a guide for construction of manpower bacterial consortia, which would perform a better ethanol-producing ability.