Study On Cellulase Production By Mixed Fermentation

Cellulase is a complex enzyme composed by cellobiohydrolase, endoglucanase and β-glucosidase.Cellulase, which was widely used in the textile industry, animal feeds, food process, and bioenergy refinery. In order to develop and applicate cellulase, a multi-species mixed fermentation in liquid-state or solid-state fermentation to produce cellulose has been researched in this paper. The main contents and results were as follows:(1) The determination of the mixed strains.20cellulase-producing strains preserved in laboratory of Nanchang University. were detected. Based on the capacity of cellobiohydrolase, endoglucanase and β-glucosidase production, these strains with higher activity were selected to mixed fermentation test. The results showed that the White-rot fungi NS75and Aspergillus niger NS83were a good combination, which’s mixed fermentation cellulase activity of CMC, β-G and FPA reached170.4U/mL,252.7U/mL and52.0U/mL respectively.(2) Research on mixed liquid fermentation. White-rot fungi NS75and Aspergillus niger NS83were mixed to liquid-state fermention, and the conditions of enzyme-producing was optimized. The results showed, under the medium composition with bran25g/L, peptone15g/L, Tween800.3%and inoculation volume ratio(v/v)3:1, initial pH5.0,30℃,200r/min culture5d, the enzyme activity produced of CMC, β-G and FPA had up to252.3U/mL,335.7U/mL and64.3U/mL. respectively.(3) Cellulases production by mixed strains fermentation in straw powder. Straw powder was used as the raw material, the solid fermentation conditions of cellulases production by mixed strains of White-rot fungi NS75and Aspergillus niger NS83had been investigated. The results of comparative experiments between single strain solid-state fermentation and mixed strains solid-state fermentation showed that, the cellulose enzyme activity produced by mixed strains had increased obviously than that produced in single strain solid-state fermentation, in which, the P-G enzyme activity had increased120.9%than that produced by White-rot fungi NS75single fermentation, and the CMC enzyme activity had increased140.8%than that produce by Aspergillus niger NS83single fermentation..In the mixed strains fermentation, the effects of straw powder content, water content ratio, inoculation volume ratio of White-rot fung NS75and Aspergillus niger NS83, and the stirring mode on cellulases production have been researched with single factor and orthogonal experiment modes. According to the experiment results the optimum fermentation conditions were determined as follows:straw powder content90%, feed-water ratio1:2, inoculation volume ratio of White-rot fung NS77and Aspergillus niger NS831:1(v/v), start stirring on the third day and once a day,30℃culture5days. Under this conditions the enzyme activity of CMC enzyme,β-G enzyme and FPA enzyme produced have reached respectively16650U/g,16108U/g and3164U/g.(4) The factors that affect the activity and stability of cellulase, such as reaction temperature, pH, glucose, cellobiose, metal ions(Zn2+, Ca2+, K+, Co2+, Fe2+and Mn2+) were investigated. The results indicated tha the crude supernatant of mixed bacteria fermentation presented the strongest cellulase activity under temperature60℃, pH5.0. Glucose presented the inhibition effect for CMC enzymes and β-G enzymes, and cellobiose possessed the inhibition for CMC enzymes. However, a small amount of cellobiose presented the promotion effect for β-G enzymes. Moreover, ions Ca2+, K+Co2+and Fe2+were activators for the activity of CMC enzymes and β-G enzymes. But ion Zn2+was a strong inhibitor.