Bacteria Community Succession In The Process Of Maize Straw Natural Fermation And Research Of BS-DX4 Cellulose Degrading Bacteria

Corn stalk,contains a lot of cellulose,is the most abundant and high application value of renewable resources in the world,but it wasn't reasonable exploited for a lon g time.In our country,most of corn stalk,about 265 million ton of each year,were b urned except a few used in feed.That caused the waste of resources and new atmosph eric pollution.Every two days,using DNS and phenol-sulfuric method to determine the contents of reducing sugar and total sugar,respectively.And the paper fully demonstrated the bacterial community diversity in natural fermentable maize straw by high-throughput sequencing method.As fermentation time goes by,the contents of total sugar and reducing sugar even presented trend like down-up-down.A total of80575 effective sequences and 3307 OTUs were obtained,which were classified in to194 genera from 18 phyla.The result of diversity analysis showed that bacterial diversity and richness of the fermantable samples changed significantly.The analysis of bacterial composition indicated,in the middle and late periods,Bacteroidetes phyla increased significantly,Proteobacteria decreased obviously,Firmicutes showed down-up trend.Additionally,the number of bacterial genus in tail stage increased by69.3% relative to initial stage.It proved extremely rich bacterial diversity in fermentation maize straw,and in the different periods,appeared different dominant bacterial community.The bacteria were screened from fermentation maize straw by using the Congo red dye test,gained a cellulose-producing Bacillus subtilis DX4,named BS-DX4.The results showed that the filter paper activity(FPA),endoglucanase activity(CMCase activity),exoglucanase activity(Cex activity)of were 256.051 U/ml,358.276 U/ml,5.536 U/ml,respectively.The strain grew well at 40 ? and medium with light concentration salt.Strain BS-DX4 was a cellulase-producing strain with broad development potential.First,from single factor test,temperature,time and pH were found to be significant factors influencing the production of endo-?-1,4-glucanase.Then using response surface methodology(RSM),and a quadric regression equation for predicting the endo-?-1,4-glucanase.The results showed optimal fermentationconditions were bran 10g/L,soybean powder 10g/L,31 ?,123 h,pH 7.0.Shaking culture under the optimal conditions,CMCase enzyme activity was 523.59U/mL,which is well matched with the predictive activity 546.53 U/mL.The production of enzyme activity was increased by 46.14 % compared with that using the original culture medium.Based on the method of conservative sequence cloning,primer pairs were designed and the ?-1,4-endoglucanase gene(bEDG)of BS-DX4 was cloned by PCR.The results showed that the bEDG,which length was 1564 bp,contained a complete ORF of 1527 bp.It encoded the 508 amino acid polypeptide with a molecular weight of 56.53 KD and the isoelectric point of 8.93.The protein contained trans-membrane signal area at the loci of 13~38,,a N-terminal signal peptide,and it was a secretory protein.In addition,it included two conserved domains,the GH5 family and the CBM-3 superfamily.By Congo red staining after SDS-PAGE preliminary analyzed cellulose enzyme special bands.