Screening Of Cellulase Producing Strains And Synergistic Degradation Of Corn Straw With Ionic Liquids

Corn straw is a kind of renewable biomass resource which is rich in resources and obtain easily.Unreasonable treatment methods lead to serious waste of resources and environmental pollution.Cellulose is the main component of corn straw biomass.Thus it is significance to find an efficient and feasible straw pretreatment method.It not only to reduce the fermentation cost of bioethanol,but also to the rational utilization of resources and environmental protection.According to the research status at home and abroad in recent years,the low cellulase activity is one of the main factors limiting the utilization of biomass resources such as straw.Therefore,the screening of high cellulase producing strains has become a key is sue.In this experiment,Congo red staining and iodine staining were used to isolate and screen two high-yield cellulase strains from corn straw soil in Daxinganling area,Heilongjiang,China and Laiwu,Shandong,China.According to colony morphology observation and its sequence homology analysis,Penicillium janthinellum FHH1 and Penicillium oxalicum FLY4.Through the optimization of enzyme production conditions,P.janthinellum FHH1 was cultured with corn straw as carbon source,beef extract as nitrogen source,initial p H 9.0,and fermentation for 9 days at 30?and 150 r/min,the CMCase,?-gluase and FPase enzyme activities were 0.383 U/m L,0.421 U/m L,0.111 U/m L,respectively.P.oxalicum FLY4 was cultured with corn straw as carbon source,peptone as nitrogen source,initial p H 5.0,and fermentation for 7 days at 30?and 150 r/min.The enzyme activities of CMCase,?-gluase and FPase were 0.430 U/m L,0.484 U/m L and 0.047 U/m L,respectively.When the strain was cultured under different concentrations of salt and ionic liquid,it was found that two strains were not tolerant to salt and ionic liquids.The results showed that the optimal conditions of P.janthinellum FHH1 cellulase hydrolysis were:p H 4.0,hydrolysis temperature60?,the CMCase,?-gluase and FPase activities were 0.561 U/m L,1.335 U/m L,0.062 U/m L,respectively.The optimal conditions of P.oxalicum FLY4 cellulase hydrolysis were:p H 4.0,hydrolysis temperature 35?,the CMCase,?-gluase and FPase activities were 0.727 U/m L,0.194U/m L,0.144 U/m L,respectively.Studies on the Na Cl and ionic liquids stability of cellulase showed that when the concentration of Na Cl was less than 15%,the cellulose of P.janthinellum FHH1,CMCase hardly changed.When the Na Cl concentration was 15%,the maximum activity of enzyme was 1.039 U/m L.The CMCase enzyme activity of P.oxalicum FLY4 could still retain more than 55%of the maximum enzyme activity when the concentration of Na Cl in buffer was higher than 40%.However,ionic liquid had a great effect on cellulase.The cellulase of P.janthinellum FHH1 and P.oxalicum FLY4 didn't have the stability of ionic liquid.In addition,due to the complex composition of lignocellulose,it has high crystallinity and polymerization degree,which affects the cellulase to effectively hydrolyze cellulose into available reducing sugar.As a green organic solvent,ionic liquid has good properties of dissolving cellulose and other organic compounds.Compared with other organic solvents,ionic liquid is non-toxic,non-volatile,and environmentally friendly.In the study,[?PYR?C₄Cl][PF₆],[?PYR?C₅Cl][PF₆],[PF₆][?PYR?C₄?MIM?][Cl],[PF₆][?PYR?C₄?PYR?][Cl],[PF₆][?PYR?C₅?MIM?][Cl],[PF₆][?PYR?C₅?PYR?][Cl],[?MIM?C₂COOH][PF₆]and[?PYR?C₂COOH][PF₆]were synthesized and characterize-ed by means of ¹HNMR.The properties of TGA,melting point and solubility were determined.[PF₆][?PYR?C₄?MIM?][Cl]?[PF₆][?PYR?C₄?PYR?][Cl]?[?MIM?C₂COOH][PF₆]and[?PYR?C₂COOH][PF₆]were selected for single or combined pretreatment of corn straw.When treated with[?PYR?C₂COOH][PF₆]and[PF₆][?PYR?C₄?PYR?][Cl],the degradation of corn stalk was the best,and the yield of reducing sugar was 20.75%when pretreatment was 8 h.In this paper,ionic liquid and reducing sugar was determined in the process of recycling.The recoveries of ionic liquid[?MIM?C₂COOH][PF₆]and[PF₆][?PYR?C₄?MIM?][Cl]in three cycles were 94.91%,86.76%and93.5%,respectively.And the yields of reducing sugar were 100%,81.91%and 80.2%,respectively.The recoveries of ionic liquid[?PYR?C₂COOH][PF₆]and[PF₆][?PYR?C₄?PYR?][Cl]in three cycles were 81.17%,80.73%and 85.27%,respectively,and the yields of reducing sugar were 100%,75.1%and 79.0 5%,respectively.Repeated experiments showed that the recovered ionic liquid can be reused several times and degradation efficiency.The cellulase of P.janthinellum FHH1 and P.oxialicum FLY4 was used to hydrolyze corn straw,and the best results were achieved at 96 h,18%and 33.75%,respectively.Corn straw pretreated with ionic liquid was hydrolyzed by strains P.janthinellum FHH1 and P.oxialicum FLY4,and the yield of reducing sugar was determined.The strain P.janthinellum FHH1 showed the maximum value of enzymatic hydrolysis at 96 h,and the yield of reducing sugar was 30.93%.P.oxialicum FLY4 showed the maximum value of enzymatic hydrolysis at 72 h,and the yield of reducing sugar was 52.37%.To sum up,two cellulase-producing fungi were screened,the enzyme producing conditions were optimized,the tolerance of the strains and cellulase to Na Cl and ionic liquid was tested,and a series of ionic liquids were synthesized and applied to the pretreatment of corn straw.The residue of corn straw was hydrolyzed by cellulase and pretreated by ionic liquid,and the yield of reducing sugar was high.Therefore,the screened strains and the synthesized ionic liquids have important significance and good prospective application in the degradation of corn straw cellulose.