| Cellulase is a compound enzyme,which is sufficient for effective conversion of cellulose to glucose.In light of their wide spectrum of catalytic substrates,cellulases have many potential applications,for example,paper industry,textile industry,alcohol fermentation,food processing and animal feed production.Recently,the ascomycete fungi such as Trichoderma,Aspergillus,Penicillium have been used in various industries for cellulase production,due to their capability to synthesize all the three essential components of cellulase system.Currently,expensive cellulase production process,poor stability and low efficiency of cellulolytic enzymes still present a major obstacle in commercial and industrial applications.Thus,explorations of highly efficient cellulases is extremely pivotal for bioconversion of lignocellulosic biomass into value-added products.The objective of this dissertation was to determine various process parameters for the production of cellulolytic enzyme cocktails using I.obliquus（Hymenochaetaceae,Basidiomycetes）under solid state fermentation（SSF）.The enzyme cocktail obtained under the optimal conditions was characterized in terms of its optimum pH and temperature,and thermal stability.The application of the enzyme cocktails obtained on different time points in hydrolysis of lignocellulosic biomass was conducted.Below are the main conclusions from our research.（1）Optimization of cellulase production under SSF.The cellulase production by the fungus I.obliquus was optimized following one factor at a time（OFAT）approach.The effect of various factors such as different carbon sources,initial pH（3.5-9.0）,substrate to moisture ratio（1:1-1:4）and inoculum size（v/w）were investigated.The results indicated that,the highest activities of CMCase and FPase（27.15±1.17 IU/g and 3.16±0.06 IU/g,respectively）achieved on Day 10,and the maximumβ-glucosidase activity（2.53±0.01 IU/g）was observed on Day 12 using wheat bran at40（4）（v/w）inoculum level,initial pH of 6.0 and substrate-moisture ratio of 1:2.5[the mineral salt solution containing（g/L）:（NH4）2SO4 1.7,K2HPO4 2.0,CaCl2 0.3,peptone1.0,MgSO4 0.3,FeSO4 0.005,MnSO4 0.002,ZnSO4.7H2O 0.0016,and CoCl2 0.0014,and 1 m L Tween-80].The CMCase,FPase andβ-Glucosidase production was found to be increased by 1.68-fold,1.46-fold and 1.43-fold with optimized media as compared to unoptimized basal media,respectively.（2）Characterization of cellulolytic enzyme cocktail.The cellulase cocktail produced on Day 10 by I.obliquus under the optimal conditions was characterized.The temperature and pH had a great influence on the biocatalyst activity of cellulases.The enzyme cocktail exhibited promising properties in terms of high catalytic activity at 40-60°C and at pH 3.0-4.5,indicating that the cellulolytic enzymes represent thermophilic and acidophilic characteristics.The CMCase,FPase andβ-Glucosidase from I.obliquus showed half-lives of 12h、1.5h and 1h at 50°C,respectively.Theβ-glucosidase from I.obliquus retained less than 50%of its original activities after incubation for 0.5 h at55°C;the CMCase lost 49.2%activity when kept at 55°C for 2 h;the CMCase component of the cocktail produced by I.obliquus revealed a half-life of 2 h at 55°C.The results indicated that the enzymes produced by I.obliquus exhibited relatively excellent thermal stability.（3）Effect of Tween 80 on lignocellulolytic enzyme production and wheat straw degradation.Different time in fermentation processing,addition of 1g/L Tween 80exerted different influence on CMCase and FPase production by I.obliquus.However,the addition of Tween 80 under submerged fermentation had suppression effect on theβ-glucosidase production（P<0.05）.Different from the case in the cellulolytic enzymes,Tween 80 presented in medium synergistically induced MnP,LiP and Lac production by more than 1200%,125%and 39.9%greater than the control medium,respectively.After 12 day of fermentation,presence of Tween 80 showed a strong stimulatory effect on the degradation rate of cellulose,hemicellulose and lignin with a significant（P<0.05）increase of 49.48%,9.26%and 12.61%compared to the control,respectively.（4）The saccharification efficiency is associated with the cellulolytic and ligninolytic enzyme components.The time course of enzymatic saccharification revealed that the enzyme cocktail sampled on Day 12 achieved the highest release of reducing sugar from the raw rice straw（125.36 mg/g of substrate）and wheat straw（130.24 mg/g of substrate）after 48h of hydrolysis.Interestingly,hydrolysis efficiency of the cellulase cocktail sampled on Day 6 was higher than the enzyme cocktails sampled on Day 8 and 10.These results were probably caused by the action of high-activity-level MnP-LiP andβ-glucosidase.The MnP-LiP played an important part in the process of biodegradation of lignin.The enzymes removed or disrupted lignin and liberated cellulose from lignin,and reduced the cellulase adsorption onto lignin,which facilitated the process of saccharification for the reducing sugar production.Theβ-glucosidase is the rate-limiting enzyme of the final step of enzymatic saccharification,sufficiently converting cellobiose into glucose.The above-mentioned results showed that the hydrolysis efficiency of raw rice straw and wheat straw was dependent on both the cellulolytic and ligninolytic enzyme components and activities in the cellulase cocktails.（5）The NaOH-pretreatment sharply decreased lignin content and reduced the cellulase adsorption onto lignin,which facilitated the process of saccharification for the reducing sugar production.The conversion yields in the hydrolysis of NaOH-treated sugarcane bagasse and H2O2-treated sugarcane bagasse by the cellulolytic enzyme cocktails sampled on Day 12 from I.obliquus were higher than the untreated sugarcane bagasse,with a 52.05%and 31.94%increase compared to the control.Through the saccharification processing after 48 h of hydrolysis using mixed crude cellulases obtained on Day 6 and 12,the higher yield of the reducing sugar from sugarcane bagasse pretreated with diluted NaOH or H2O2 increase by 35.70%and 29.23%compared with the control while using the cellulolytic enzyme cocktails sampled on Day 6 from I.obliquus were higher than the untreated sugarcane bagasse,with a 32.51%and 29.06%increase compared to the control,respectively.This results indicated that NaOH-pretreatment effectively improved the enzymatic hydrolysis efficiency of bagasse cellulose.In conclusion,the white rot fungus I.obliquus was an efficient and effective producer in terms of high-activity-level cellulolytic and ligninolytic enzyme production under solid state fermentation.The cellulase cocktail exhibited excellent thermal stability and great tolerance toward acidic pH.Interestingly,saccharification efficiency of raw rice straw and wheat straw was dependent on both the cellulolytic and ligninolytic enzyme components and activities in the cellulase cocktails.This study provided an approach to solve the problem of highly efficient both lignolytic and cellulase production for bioconversion of lignocellulosic biomass into biofeuls. |
