Screening,Identification And Optimization Of High-Yield Lignase Producing Fungus

With the increasing exhausted of fossil energy and serious environmental pollution,exploring the environmentally friendly,sustainable and adequate alternative energy becomes extremely important.Bioconversion of lignocellulosic biomass into biofuels and other biomaterials by fermentation received more and more attention.However,as the complex structure of lignocellulosic biomass formed by lignin chemically interacting with cellulose and hemicellulose through covalent and non-covalent bonds,it is dificult to degrade cellulose and hemicellulose by enzymes.Therefore,delignification has become one of the important steps before lignocellulosic biomass biodegradation.In this study,isolation,screening and identification of fungus with high activity of lignin-degrading enzymes were firstly performed.Then,the lignin degradation characteristics of the obtained strains were explored.Furthermore,the conditions for the strains producing lignin-degrading enzymes were optimized.The main findings are as follows:(1)Isolation,screening and identification of strains with high activity of lignin-degrading enzymes.The media contains aniline blue or guaiacol were used for screen.Among the 28 isolated strains,strains labelled as Z-1 and X-13 showed the better effects than other strains on the decolorization of aniline blue and guaiacol.The decolorizing circle diameter of the aniline blue and guaiacol containing media by Z-1 reached 65.5 cm and 33.4 cm,respectively,and 73.3 cm and 34.1 cm is for that of X-13,respectively.According to the analysis of phylogenetic of ITS sequence and morphological characteristics,Z-1 and X-13 were identified as genus of Trametes versicolor and Trametes hirsute,respectively.Both the two strains have been deposited in China General Microbial Culture Collection Management Center(CGMCC).(2)The lignin degradation properties of T.versicolor Z-1 and T.hirsute X-13.During the process of cultivation on the medium contained lignin as the sole carbon resource,The maximum activities of laccase(Lac)and manganese peroxidase(MnP)by strain T.versicolor Z-1 observed on day 7,with the values of 40.67 IU/mL and 30.85 IU/mL respectively.While the maximum rates of lignin degradation and decolorization by strain T.versicolor Z-1 were detected on day 13,with the values of 13.54%and 26.43%respectively.The maximum activities of laccase(Lac)and manganese peroxidase(MnP)by strain T.hirsuta X-13 observed on day 5 and 7,with the values of 9.68 IU/mL and 23.08 IU/mL respectively.While the maximum rates of lignin degradation and decolorization by strain T.hirsuta X-13 were detected on day 13,with the values of 21.88%and 52.43%respectively.The results suggested that both strains T.hirsuta X-13 and T.versicolor Z-1 could efficiently degrade lignin,which exhibited great potential for the pretreatment of lignocellulose by delignification.(3)Optimization of medium compositions for lignin-dergading enzymes production.A single factor experiment combined with response surface analysis were conducted.The optimal medium for lignin-dergading enzymes production by T.versicolor Z-1 contained:wheat bran 18.3 g/L,ammonium sulfate 4.55 g/L,PEG-40000.045 g/L.Cultivating on this medium,the activity values of Lac and MnP produced by strain T.versicolor Z-1 reached 137.29 ± 2.35 IU/mL and 119.48±2.07 IU/mL,respectively;The optimal medium for lignin-dergading enzymes production by T.hirsuta X-13 contained:corncob powder 25.75 g/L,ammonium tartrate 3.29 g/L,Tween-80 0.0454 g/L.Cultivating on this medium,the activity values of Lac and MnP produced by strain T.versicolor Z-1 reached 77.29±2.15 IU/mL and 86.47±2.43 IU/mL,respectively.The activity values of Lac and MnP for strain T.versicolor Z-1 increased by 2.4 times and 2.8 times relative to unoptimized medium,while there were 7.0 times and 2.7 times for strain T.hirsuta X-13,respectively.The results indicated that the activities of Lac and MnP for both two strains were improved by media compositions optimization.(4)Optimization of fermentation conditions for for lignin-dergading enzymes production.A single factor experiment combined with response surface analysis were conducted.The optimal medium for lignin-dergading enzymes production by T.versicolor Z-1 contained:inoculation volume 8.04%(V/V%),initial pH value 5.28,culture temperature 31.63℃.Cultivating on this medium,the activity values of Lac and MnP produced by strain T.versicolor Z-1 reached 150.36 ± 3.15 IU/mL and 133.05 ±2.451 IU/mL,respectively;The optimal medium for lignin-dergading enzymes production by T.hirsuta X-13 contained:inoculation volume 8.42%(V/V),initial pH value 4.60,culture temperature 28.18 ℃.Cultivating on this medium,the activity values of Lac and MnP produced by strain T.hirsuta X-13 reached 94.42 ±1.85 IU/mL and 99.93±2.13 IU/mL,respectively.The results suggested that the activities of Lac and MnP for both two strains were greatly enhanced after optimizations of medium compositions and fermentation conditionsIn conclusion,two strains T.hirsute X-13 and T versicolor Z-1 with high activity of lignin-degrading enzymes were isolated.Both two strains exhibited strong capacity for lignin degradation.Furthermore,the activities of Lac and MnP for both two strains were greatly improved after optimization.This work suggested that the newly isolated strains X-13 and T.versicolor Z-1 might be promising candidates for lignocellulosic biomass pretreatment by delignification.Meanwhile it laid a theoretical foundation for industrial application with this two strains.