| Lignocellulosic biomass has become a potential green resource due to its abundant resources,environmental friendliness and sustainability,but its complex structure is a bottleneck restricting the efficient utilization of this resource.Lignin is a kind of aromatic heteropolymer,which acts as a natural barrier against degradation of biomass by embedding into the gap between cellulose and hemicellulose structure to form matrix.It is of great significance to explore microorganisms that degrade lignin efficiently and further study the degradation mechanism of lignin.White rot fungus is the only fungus that can mineralize lignin into carbon dioxide and water,which has great application prospect in biorefinery.Due to the complex structure of natural lignin,alkali extraction lignin is widely used in lignin research.In this paper,a white rot fungus Trametes hirsuta X-13,which can degrade lignin efficiently,isolated and screened in the previous research,was taken as the research object.The characteristics and degradation mechanism of lignin degradation by this fungus were deeply studied,and the following research results were obtained:1.Degradation characteristics of lignin by T.hirsuta X-13In the medium with alkali lignin as the only carbon source,the mycelial biomass,lignin degradation rate and decolorization rate and enzyme activity of Trametes hirsuta X-13 kept increasing in the early stage of degradation.The degradation rate and decolorization rate of lignin were 39.88%and 52.43%respectively on the 11th day of culture.Lignin was degraded by laccase and manganese peroxidase,and the enzyme activity peaked at 9.68 IU/mL and 23.08 IU/mL on the 7th day,respectively.The results of Fourier transform infrared spectroscopy and thermal decomposition GC-MS analysis showed that the aromatic group structure of lignin residue was seriously damaged.The results of scanning electron microscopy showed that the particle size of lignin residue decreased and its surface became rough.These characterization results indicated that T.hirsuta X-13 could degrade lignin efficiently.The results of 2D-HSQC NMR analysis of the lignin structure before and after the degradation of T.hirsuta X-13 showed that the percentage of H,G and S type units in the lignin residue after the degradation of T.hirsuta X-13 decreased.This indicated that the bacteria could degrade these three units of lignin.The S type unit structure was more easily utilized by T.hirsuta X-13 in the early stage of culture,while the bacteria preferred to degrade G type units in the late stage of culture.In addition,the condensation degree of lignin(β-β/β-O-4),resin-alcohol(β-β),β-aryl ether,cinnamyl alcohol and phenylcoumarin decreased with the increase of degradation time,suggesting that β-5 and β-β cleavage pathways may also exist in the degradation process of lignin.2.Quantitative secretory Proteomics study on lignin degradation by T.hirsuta X-13The results of extracellular proteomics analysis showed that laccase,lignin peroxidase,manganese peroxidase and multifunctional peroxidase could be secreted during the degradation process.On the third day of degradation,the expression levels of peroxidase,laccase,glutathione S-transferase and GMC oxidation-reductase were higher than those on the seventh and eleventh days,while the expression levels of lignin degradation auxiliary enzymes were higher than those on the third day.These results indicate that the high expression of lignin oxidase in X-13 contributes to the depolymerization of lignin macromolecules in the early stage of lignin degradation,while the high expression of lignin degradation auxiliary enzymes in the late stage of degradation mainly participates in the modification and further transformation of aromatic compounds produced by lignin depolymerization.3.Extracellular metabolomics study of lignin degradation by T.hirsuta X-13Metabolomics analysis showed that the degradation of alkali lignin by laccase produced aromatic monomers and small molecular acids.Laccase cleaved β-β bond to produce vanillic acid.The downstream metabolite of vanillic acid was 4-hydroxybenzoic acid,and the downstream product of 4-hydroxybenzoic acid was benzoic acid.Or the body produces protocatechuic acid after loop opening by dioxygenase to 2-ketoadipic acid,and phenylacetic acid production was also detected.Based on the above results,it is speculated that the degradation of alkali lignin by T.hirsuta X-13 firstly results in oxidative decomposition of its β-β and β-5 bonds,and the formation of monomer aromatic ring compounds,vanillic acid,4-hydroxybenzoic acid,phthalic acid,terephthalic acid,phenylpyruvate,hypervanillic acid and caffeic acid,all of which belong to the metabolic pathway of benzoic acid.The downstream products of 4-hydroxybenzoic acid,β-ketoadipic acid,citric acid,pyruvate and fumaric acid were detected in the β-KPA pathway,while phenylacetic acid and succinic acid were detected in the phenylacetic acid pathway,indicating that the benzoic acid,β-KPA and phenylacetic acid metabolic pathways were further degraded into small molecular acids,respectively.In this paper,the degradation characteristics and mechanism of T.hirsuta X-13 lignin were investigated.The results showed that T.hirsuta X-13 could efficiently degrade lignin,and secreted abundant lignin-degrading enzymes during the degradation process,and could degrade lignin through β-5 and β-β cleavage pathways.The metabolic pathways of degraded aromatic compounds include benzoic acid pathway,β-KPA pathway and phenylacetic acid pathway.The results of this study indicate that T.hirsuta X-13 is a fungus resource with potential application in biomass pretreatment industry,which is of great significance to promote the development of biomass refining industry,and provides a theoretical basis for exploring the degradation process of natural lignocellulose by fungi. |
