Screening And Identification Of A Strain With High-Yield Bacterial Cellulase Activity And Optimization Of Fermentation Conditions

The aim of this study was to isolate cellulase-producing bacteria microorganisms from termite hindgut and bovine rumen,congo-red staining method was used to isolated18bacteria strains of cellulose-decomposing.According to the detection of cellulase activity,one bacteria strain with high-yield bacterial cellulose activity were isolated.16S rDNA sequence analysis were used for the strain25-2,and aphylogenetic tree was constructed by comparing the16S rDNA sequence with other relative bacteria species in the GenBank database.The results showed that the complete16S rDNA gene sequence was1511bp,with Bacillus amyloliquefaciens subsp have100%similarity value.The strain25-2was used as parent strain and mutagenized by ultraviolet rays(UV),congo-red staining method was used to isolated11bacteria strains with the lager ratio of the diameter between hydrolysis cycle and the colonies on cong-red medium.Secondary screening with flask-shaking fermentation method,and determina-tion of cellulase activity.One mutant strain Y-9with high cellulase-producing capacity was obtained through compare enzyme activity size and analyse genetic stability,which CMCase activity were16.108U/mL,24.01%higher than cellulase produced by parent strain.In order to enhance the cellulase production of mutant strain Y-9, response surface methodology was applied to optimize the fermentation conditions. Firstly, two level factorial design of Plackett-Burman was used to evaluate the influence of six related factors.The results showed that the effects of culture time(X1),fermentation temperature(X2) and initial pH value(X3) on the activities of cellulase were significant among the six factors.Secondly,the path of steepest ascent was taken to approach the optimal region of fermentation conditions,Box-Behnken design and response surface methodology (RSM) analysis were applied to analyze the relationships between these factors and to find out the optimal values of the critical factors.And establish the second-orderregression equation model,the optimal conditions of the three factors were assessed by the RSA as follows:21.7h of culture time,46℃of fermentation temperature and initial pH value4.8.Under the optimum conditions, the activity of cellulase increased nearly1.704times to27.907U/mL and the experimental values argreed with the predicted values.