Screening And Identification Of Efficient Cellulase-producing Fungus And Optimization Of The Fermentation Condition

Lignocellulose is the most abundant renewable resource in nature, accounting for more than90%of plant cell dry weight. In general, lignocellulose is composed of45%cellulose,25%hemicellulose and30%lignin. Cellulose as the predominent component can be degradated into reducing sugars, which could be used for ethanol fermentation and other purposes, by cellulase without adverse environmental or energy effects. However, the utilization of lignocellulosic resources is limited by cellulase activity that at present is not high enough. Therefore, screening for microbial strains that produce cellulase efficiently is a key need to successful bioconversion of lignocellulose.An efficient cellulase-producing fungal strain B-5was isolated from the primary forest soil in different regions of the protected areas in Shennongjia, Hubei, China, using the methods of CMC-Na culture medium, Congo red staining, disintegration of filter paper and measurement of extracellular enzyme activity. The strain was identified as Fusarium sp by colony morphology, microscopic characteristics and ITS sequence homology analysis. The ITS sequence of B-5has a99%similarity with Fusarium Equiseti.Filter paper disintegration assay showed that Fusarium B-5had strong capacity of filter paper digestion in72h and the weight loss rate of filter paper reached37.9%. With CMC-Na as the carbon source, peptone as nitrogen source, and fermentation for4days at28℃and180r/min, its initial activity of FPAase was2.09IU/mL and CMCase was5.20IU/mL. The optimal fermentation conditions were:rice straw as the sole carbon source,80mL fermentation liquid in250mL triangular flask, initial pH6.1, nitrogen concentration1.0%, carbon concentration2.5%,0.1%rhamnolipid addition, fermentation for120hours under the temperature of28℃and the rotation speed of180r/min. The ultimate activity of FPAase reached2.504IU/mL.We also explored suitable environmental conditions for cellulose hydrolysis by cellolase produced by Fusarium B-5. Centrifugal condition was4000r/min for10 minutes to recover the enzyme. The hydrolysis reaction time was standardized at60minutes, temperature at55℃and the initial pH was adjusted to5.0. Different metal ions in concentration of10mmol/mL had variable effects on the cellulase enzyme activity. Co2+, Mn2+, Mg2+and Fe2+had a stimulative effect, Na+, K+and Ca2+had no apparent effect, and Cu2+and Zn2+had an inhibitive effect, although Cu2+was a stronger inhibitor than Zn2+.