Optimization Of Cellulase Production By Solid Fermentation Of Aspergillus Niger And Study On Straw Saccharification

The excessive use of non-renewable fossil energy brings a lot of environmental problems to the earth.More and more countries attach importance to the biomass energy such as lignocellulose.Cellulase is a kind of enzyme that can hydrolyze cellulose to produce glucose and one of the key factors for efficient utilization of lignocellulose such as straw.In this paper,aspergillus Niger was used as the research object to optimize the process conditions of producing filter paper enzyme and endoglucanase by solid-state fermentation,and to study the enzymatic properties of producing cellulase,the conditions of enzymatic saccharification of straw and the effects of additives on the efficiency of enzymatic saccharification.The main findings are as follows:In this study,a strain of aspergillus Niger with high activity cellulase was selected from the preserved strains in the laboratory and applied to straw fermentation to produce cellulase to degrade cellulose.Firstly,through the initial screening of the strain on the plate with sodium carboxymethyl cellulose as the only carbon source,14 strains of the pure strain were obtained after multiple dilution coating and plate marking,and then the pure strain was stained with Congo red.Taking the ratio of the diameter of the hydrolysis circle to the diameter of the strain as the index,the strains with high enzyme activity of no.7,no.10 and no.14 were obtained.Then,based on the filter paper enzymatic activity and endoglucanase activity of the strain producing enzyme in the medium of wheat bran,corn straw and straw,the number 7 strain with enzymatic activity of 0.64 IU/g and 7.96 IU/g were screened.It was observed morphologically in medium and under light microscope.The bacteria grew slowly in the first 2 days,and became beige on the third day.There are distinct traces between the colonies.Radial furrows and outwards concentric circles,colonies rounded and flat with neat edges.On day 4-5,the plate was covered with spores and the back was slightly yellow in the middle.Under 40-fold optical microscope,hyphae are transparent,apical capsule is large spherical,peduncle is double-layered,conidia is spherical,black,conidium is produced by thick hyphae.It conforms to the basic morphology of aspergillus.The technological conditions of aspergillus Niger solid-state fermentation were explored,and the basic medium of aspergillus Niger solid-state fermentation was obtained: bran was the carbon source,ammonium sulfate was the nitrogen source,and potassium dihydrogen phosphate was the phosphorus.First,single factor experimentand Plackett-Burman(PBD)experiment design were carried out on the culture conditions,and three significant factors including initial pH,potassium dihydrogen phosphate concentration and ammonium sulfate concentration were selected from the7 factors.Then the Box-Behnken(BBD)experimental design and response surface analysis to optimize,the solid-state fermentation of aspergillus Niger produces the best culture conditions of cellulose enzyme: solid substrate 1.5 g,bran,maize straw,rice straw = 3:2:1,ammonium sulfate content of 5.2 g/L,optimum adding amount of potassium dihydrogen phosphate as 7.0 g/L,magnesium sulfate optimum adding amount was 5.0 g/L,pH 4.3,10% inoculated quantity,in 30 ? constant temperature incubator culture 4 days.After optimization,the activity of filter paper and endoglucanase were 1.88 times and 1.54 times higher than before.Research on enzymatic properties: the enzymatic reaction temperature has a greater effect on the filter paper enzyme activity of aspergillus Niger crude enzyme solution,and a smaller effect on the endoglucanase activity.The optimum enzymatic reaction temperature is 50?-55?.The optimum crude enzyme reaction pH is 4.It shows that the enzyme system is a kind of thermophilic and acidophilic cellulase.And aspergillus Niger cellulase under 50? insulation 1h,maintain about 85% of the vitality.After 4h,the enzyme activity of filter paper and endoglucanase decreased to70% and 50% respectively.For enzymatic hydrolysis of rice straw and corn straw through 200 mesh sieve,the substrate was 1.0g,the enzymatic hydrolysis time was 24 h and the enzyme dosage was 5 FPU/g,and the glucose yield and saccharification rate were the best.Tween 80,PEG6000,bovine serum protein and soybean isolate protein four different additives after straw pretreatment,soybean protein effect is the best.Moreover,after 18%soybean isolate protein pretreatment,the glucose yield and saccharification rate of cellulose in the enzymatic hydrolysis of straw straw and corn straw were 268.89 mg/g and 44.59%,224.55 mg/g and 38.58%,respectively.