The Isolation And Identification Of Facultative Anaerobic Cellulose Decomposing Bacteria Of Rhizomys Intestine And And Research On Its Enzyme Production Activity

Straw stalk crops contain high cellulose,which is a potential natural resource. Today,in the global resource crisis, making the most of them to develop the animal husbandry vigorously can not only avoid the waste of resource,but also avoid the straw burning polluting the ecological environment. Using cellulase to decompose cellulose is one of the effective methods for the use of cellulose resource, and the strains which can produce cellulase most isolated from the rumen fluid of ruminant,compost, soil,etc,and the strains had high ability of cellulose-degradation were due to strict anaerobic strains, on account of the strict culture conditions, the application in the production was limited. The aim of this study was to select the excellent facultative anaerobic cellulase-producing microorganism from Chinese rhizomys intestine, in order to lay a foundation for the development and utilization of cellulose resource to the uttermost, and promote the rapid development of new energy, food, animal husbandry and other related industries so on. This research mainly includes the following four parts:Experiment1:Through the strict anaerobic combined with aerobic cultivation, using sodium carboxymethyl cellulose as the sole carbon source, initially isolated cellulose decomposing strains from Chinese rhizomys intestinal contents by spread plate cultivation, and then screened the desired strains with congored staining and enzyme assay. The test results showed that:17strains were obtained from the ileum, cecum, colon of rhizomys, from which7strains of suspected cellulose decomposing bacteria were screened according to the morphology of strains,they were respectively ileal strain H-1, H-2; strain M-1,M-2, M-3of caecum, strain J-1,J-2of colon; are combined with the transparent circle and colony diameter ratio by congo red staining, after determined the enzyme activity of strains fermented liquid,strain M-2, M-3showed higher enzyme activity, were0.5029U/mL,0.4874U/mL respectively.Experiment2:Strain M-2, M-3from the cecum as objective strain would be identified to the bacterial species or genus by the morphological observation, Gram staining, the strains physiological and biochemica test, and molecular biological identification. Test results show that:the morphological characteristics of strain M-2, M-3combined with the physiological and biochemical experiment results, and PCR amplification the16SrDNA sequence,then cloning, sequencing, BLASTin Gene bankdatabase showsthat, strain M-2was belong to the Serratia of Enterobacteriaceae spp., strain M-3was identified as Klebsiella pneumoniae.Experiment3:Study the enzyme production nature of caecal strain M-2which had higher enzyme activity through testing the influnce of different fermentation time, carbon source, nitrogen source, inoculation amount, temperature, and initial pH value on enzyme production activity of strain M-2. The test results show that:All of the6factors had significant effects on these strains’enzyme producing, and the optimum culture conditions about enzyme producing was that:strain M-2could show the maximum enzyme activity with glucose as carbon source, yeast powder as nitrogen source, initial pH7,40℃shake flask culture24h, the optimum inoculum4%.