Screening And Identification Of Thermostable Cellulose-degrading Bacterial Consortium And Studies On Its Enzymes Activity

The present energy structure based on the fossil fuel which lead to the exhaustionof fossil energy and the dramatic deterioration of ecological environment. Change theconsumption structure of energy and utilization of renewable biomass as organicmaterial to produce energy get more and more attention. Lignocellulosic biomass isconsidered as the most abundant, cheap and clean renewable organic material in theearth. It can be transformed to the fermentable syrup mixture, composedpolysaccharides and monosaccharides, that can be fermented to the clean bio-ethanol.However, the low efficiency of cellulose degradation and the high cost of cellulaseproduction are the main bottlenecks of the development of biomass energy industry.In order to produce efficient cellulase and improve the efficiency of cellulosedegradation, a thermostable cellulase producing bacterial consortium XM70wasscreened, identified and its enzyme producing conditions were studied. Theextracellular cellulase from the XM70was purified and its enzyme activity wascharacterized. Carbon source control mechanism of cellulase production waspreliminarily investigated. The results are as follows:1.A novel thermophilic cellulose-degrading bacterial consortium, named XM70,was obtained from a hot spring in Xiamen (Fujian, China) with pretreated bagasse asthe sole carbon source at70°C. Molecular community structure of the XM70wasdetermined by phylogenetic analysis of16S rDNA gene sequences and it mainlyincluded70%Geobacillus sp.,10%Desulfotomaculum sp.,5%Bacillus sp.,5%Exiguobacterium sp.,5%Paenibacillus sp. and5%Enterobacter sp.. Three majorcomponents of the cellulases [carboxymethyl cellulase (CMCase), filter papercellulase and β-glucosidase] were produced with maximal activities (0.14,0.06and0.08U/mL), and the yield of reducing sugars peaked0.11g.g-1dry feed stock. Thebacterial consortium XM70can utilize lignocellulosic biomass bagasse to producecellulases and reducing sugar, which has good research and application value.2.The extracellular cellulase from the XM70was purified, and its relativemolecular mass was approximately31.0kDa. Optimal temperature and pH for theCMCase activity of the purified enzyme were recorded as70°C and7.0respectively.Maximum CMCase exhibited37.7U/mg specific activity and it also retained about90%and70%after incubation at70°C and80°C for60min. This study could providetheoretical and technical support for purification of thermostable cellulase from cellulose-degrading bacterial consortium.3.12strains of bacteria were isolated from XM70. They belonged to3generaincluding Geobacillus sp., Bacillus sp. and Enterobaceter sp. by16S rDNA geneanalysis. In the12strains bacteria, XM70R7has the highest enzyme activity which is0.12U/mL. The carbon source regulation mechanism of cellulase production ofXM70R7was investigated by SDS-PAGE protein electrophoresis technology, usingbagasse, JunCao, starch, microcrystalline cellulose and sodium carboxy methylcellulose as five different carbon sources. After fermentation of five kinds of carbonsources, there were six differences in the strips of protein electrophoresis, the fifthdifferent strip with about23kD was analyzed by mass spectrometry. The resultshowed that they are superoxide dismutase and translaldolase. The study on carbonsource regulation mechanism could provide theoretical basis and technical support forutilizing lignocellulosic biomass such as bagasse and JunCao to produce efficientcellulase and fuel ethanol.