Study On The Mechanism Of Lignin Bio-depolymerization Improving The Saccharification Of Bamboo

Lignocellulose is one of the most abundant renewable resources,which is an alternative to fossil resources for acquiring biomass energy.Efficient saccharification of lignocellulose is the key step for producing the liquid fuel.However,the lignin structure barrier severely limits the efficient saccharification of lignocellulose.In nature,white rot fungi were considered as the best lignin degrader,which could completely mineralize lignin into water and carbon dioxide through ligninolytic enzymes system.However,it is still unclear how white rot fungi use lignin enzymes to destroy lignin macromolecular structures to obtain carbon nutrition from ligninocellulose.Based on this,a highly efficient lignin-degrading white rot fungus Physisporinus vitreus was used as our research object.Firstly,we characterized the key modification in the structure of bamboo lignin during P.vitreus pretreatment,then the transcriptome was analyzed to identify the ligninolytic enzyme-related genes expressed in P.vitreus.The major extracellular ligninolytic enzymes were isolated and purified.We further studied the ligninolytic enzymes degradation of lignin model compounds and natural lignin,as well as its effect on the hydrolysis of moso bamboo.Therefore,the mechanism by which the white rot fungus using its ligninolytic enzymes to depolymerize lignin and to improve the enzymatic hydrolysis and saccharification of bamboo was illuminated.The main results of the research are as follows:(1)Lignin depolymerization by white rot fungus P.vitreus improving the saccharification of bambooThe white rot fungus P.vitreus can selectively degrade lignin in lignocellulose and significantly improve the efficiency of enzymatic hydrolysis of bamboo,resulting in increasing the maximum glucose yield of bamboo by 207.08%.2D-NMR characterization of lignin structure showed that the white rot fungus P.vitreus could effectively break the linkages between lignin units,especially the hard-degradable C-C bonds,which decreased the total content of lignin linkages by 20.42%.The relative content of hard-degradable C-C bonds decreased,and the relative content of easily degradable ?-O-4' bonds increased.The decreasement in the ratio of C-C bonds/?-O-4' bonds reduce the lignin condensation by 51.97%,which led to lignin depolymerization and the enhancement of bamboo saccharification.(2)Isolation and purification of Ligninolytic enzymes from P.vitreus,and the degradation of Lignin Model CompoundsBased on the extracellular ligninolytic enzymes activity and transcriptome expression profile data of white rot fungus P.vitreus cultured in bamboo system,the main ligninolytic enzymes of lignin depolymerized by white rot fungi P.vitreus are type II peroxidase(including Versatile peroxidases and manganese peroxidases)and laccases.Two kinds of ligninolytic enzymes were obtained from the extracellular fluid of P.vitreus by fractional salting-out,hydrophobic interaction chromatography and ion-exchange chromatography.They were versatile peroxides and laccase and were named as Pv-Lac and Pv-VP respectively.The lignin degradation ability of Pv-Lac and Pv-VP was studied using lignin model compounds monomers and dimers.The results showed that Pv-Lac could only degrade phenolic lignin model compounds,while Pv-VP could oxidize both phenolic and non-phenolic lignin model compounds.The product generated by the two enzymes were analyzed by LC-ESI-MS.We found that Pv-Lac only oxidized the side chain of the 5-5' dimer,whereas Pv-VP could really break the ?-O-4' and 5-5' linkages of dimers and produce monomers.(3)Lignin depolymerization and enhancement of bamboo saccharification was mainly depended on Pv-VP and supplemented by Pv-LacThe degradation of natural lignin by Pv-Lac and Pv-VP were studied using MMW as a substrate.Like the white rot fungi P.vitreus,Pv-Lac and Pv-VP reduced the total content of lignin linkages.Furthermore,the reduction of the C-C bond/?-O-4' bond ratio reduced the lignin condensation.Individual Pv-Lac,Pv-VP and the combination of two enzymes reduced the total bond content of lignin by 3.25%,4.66% and 6.63%,while the condensation degree was decreased by 33.07%,42.52% and 47.24%.Pv-VP depolymerized natural lignin more efficiently than Pv-Lac.In the enzymatic hydrolysis system of bamboo,Pv-VP improved the saccharification of bamboo more effectively than Pv-Lac.Moreover,when supplemented with both Pv-Lac and Pv-VP,the saccharification of bamboo was similar to that with Pv-VP supplementation alone It was revealed that lignin depolymerization and the enhancement of bamboo saccharification was mainly depended on Pv-VP and supplemented by Pv-Lac,and this approach was also universal in corn stover substrate.In summary,through studying of the lignin degradation process by white rot fungi and its ligninolytic enzymes,we uncover that white rot fungi mainly secrete certain ligninolytic enzymes to break the hard-to-degrade bond between lignin units,which reduces the lignin condensation to depolymerize the lignin macromolecules and increases efficiency of bamboo saccharification.This study complements and improves the theoretical basis for the lignocellulose biotransformation.