| Lignocellulose content in crop stover is approximately 80%,and it is an important biomass resource,while its comprehensive utilization level needs to be improved urgently.How to effectively transform and utilize straw resources has attracted extensive attention of researchers.Bio-refining technology based on sugar platform is an effective approach to utilize lignocellulose raw materials.Lignin in biomass is a natural barrier to cellulase hydrolysis,which is one of the factors that affect the efficiency of enzymatic reactions.How to safely,effectively and maximally remove lignin has become a hot topic of researches.Microbial method has become an ideal method of straw degradation in industrial production with their strong lignin degradation ability and green environmental protection advantages.In this process,screening strains with good lignin degradation ability and high enzyme production activity is the key to the development and utilization of straw resources.In this study,corn stover was used as research materials to screen out strains with good degradability to lignin from nature.Corn stover was pretreated with screened fungi to reduce enzymatic resistance barrier of lignocellulose.The degradation effect of the screened fungus on corn stover was evaluated systematically.The lignin degradation ability was improved by breeding mutations and optimizing fermentation conditions.The functions of key enzyme genes related to lignin degradation were explored by high-throughput sequencing and bioinformatics analysis.The expression pattern of enzyme-producing genes in related microorganisms was revealed.The mechanism of the synergistic effect of the screened strain on enzymatic hydrolysis of corn stover was elucidated.This research provides the theoretical basis for the industrialization of microbial transformation of corn stover and promotes the comprehensive utilization efficiency of corn stover.The main results are summarized as follows:?1?Alkali lignin was used as the sole carbon source for quantitative screening of lignin-degrading bacteria.Sixty-three strains which can grow well in alkaline lignin medium were screened out from nature.Compared with the model strain,Phanerochaete chrysosporium,it was found that the alkaline lignin removal ability and the enzyme production capacity in corn stover culture medium of strain No.61 were higher than the model strain.The activities of laccase?Lac?,lignin peroxidase?LiP?and manganese peroxidase?MnP?secreted by strain No.61 reached 6.61 U/g,0.78 U/g and 1.30 U/g biomass,respectively.Comprehensive data showed that strain No.61 has the ability to degrade lignin,and its degradation ability is superior to the model strain.Strain No.61 is Myrothecium verrucaria by biology.?2?The content of lignocellulose in corn stover before and after fermentation was determined according to National Energy Laboratory Standard?NREL?Method.Microstructure changes of corn stover after fermentation by Myrothecium verrucaria was determined by scanning electron microscopy?SEM?,Brunauer-Emmet-Teller?BET?,X-ray diffraction?XRD?,Fourier Transform Infrared Spectroscopy?FTIR?and Gas Chromatography-Mass Spectrometer?GC-MS?.The degradation of corn stover by Myrothecium verrucaria was evaluated systematically.Results showed that the lignin content of corn stover was decreased by 42.30%after the fermentation of Myrothecium verrucaria with little cellulose and hemicellulose losses in comparison with that of untreated corn stover.Meanwhile,the surface structure of fiber bundles in corn stover was destroyed,the porosity was increased,and the crystallinity of corn stover was decreased after Myrothecium verrucaria fermentation.Small molecules of benzene ring in the degradation products were detected,too.These results indicated that lignin in corn stover could be effectively degraded.?3?The ARTP mutagenesis was used to mutagenize Myrothecium verrucaria,and the mutant library of Myrothecium verrucaria was established successfully.The positive mutant T0701 with higher lignin removal ability than wild-type strain and with stable genetics was screened out.Under the same fermentation conditions,the lignin removal rate of corn stover after fermentation with mutant T0701 was up to 44.79%,which was 5.89%higher than that of the wild-type strain.Response surface methodology was used to optimize the lignin degradation ability of mutant strain T0701,indicating that fermentation time,liquid-to-solid ratio and Mn2+addition have significant effects on lignin degradation,and the optimum condition was determined.After optimization,the lignin degradation rate of corn stover was up to 49.76%,11.09%higher than the degradation rate before optimization,and 17.64%higher than that of wide-type fungus pretreatment.?4?The corn stover was enzymatically hydrolyzed after fermentation by Myrothecium verrucaria,and the conversion rate of cellulose reached 56.81%,which was 123.84%higher than that of corn stover without pretreatment.Compared with the pretreatment effect of ultrasonic method,dilute acid?H2SO4?method and dilute alkali?NaOH?method,the pretreatment effect of Myrothecium verrucaria was much higher than that of ultrasonic pretreatment,similar to dilute acid pretreatment and slightly lower than dilute alkali pretreatment,indicating that Myrothecium verrucaria could be used for microbial pretreatment of corn stover.To further improve the effect of pretreatment,the second step pretreatment was adopted with dilute acid directly after the pretreatment of Myrothecium verrucaria to remove the hemicellulose and replace the corn stover regeneration process.The effects of different acids on the two-stage pretreatment were evaluated.The results showed that the removal rates of hemicellulose and lignin in corn stover reached 51.15%and 52.44%,respectively after two-stage pretreatment with Myrothecium verrucaria combined with solid acid.Conversion rate of two-stage pretreatment was up to 81.54%,which was 221.28%higher than that of the untreated corn stover,indicating that the two-stage pretreatment is an effective method to improve the utilization rate of cellulose in corn stover.SEM,XRD and FTIR results also confirmed that the two-stage pretreatment had a significant effect on the microstructure of corn stover.?5?Transcriptome sequencing of the wild-type strain and the mutant strain T0701 in three growth periods was performed.And the sequencing results were analyzed by bioinformatics.A total of 50159 Unigenes were obtained,26,052 Unigenes which distributed in 50 GO terms were annotated in GO.348?340 and 592 differentially expressed genes were obtained by differential expression analysis.A total of 105,73,183 genes were up-regulated and 0,74,383genes were down-regulated after pearson correlation analysis.Pathway enrichment analysis of differentially expressed genes was subsequently carried out based on KEGG database.Most genes were involved in Metabolism pathway.A large number of genes involved in lignin degradation through RNA sequencing were found.It contained many genes encoding laccase,protocatechuate-2,3-dioxygenase and catechol-2,3-dioxygenase.Multiple differential genes map to aromatic degradation pathways associated with lignin degradation,such as glyoxylate and dicarboxylic acid metabolic pathways,carbon metabolism pathways,ether ester metabolic pathways,and fatty acid metabolism pathways.The molecular mechanism of the degradation of lignin by M.verrucaria was preliminarily explained at molecular level.?6?Previous studies have found that Myrothecium verrucaria has a high laccase activity,the application of this advantage in production practice was investigated.The mutant strain M-6with high yield of laccase and stable genetics was screened from the previously constructed mutant library,and the laccase activity reached 2.62 U/mL,which was 75.51%higer than that of the wild-type strain.The conditions of laccase production by mutant M-6 were optimized by one-factor-at-a-time experiments and orthogonal experiments.After optimization,the laccase activity reached 30.08 U/mL,which was 11.48 folds higher than that of basal fermentation condition.The degradations of 2-CP with different laccase fermentation broth?wide stype fungus in basal fermentation condition,mutant M-6 in basal fermentation condition and mutant M-6 in optimal fermentation condition?were compared.The results showed that mutant M-6had higher degradation ability and faster degradation rate,and the degradation rate of 2-CP could reach 97.13%,indicating that the degradation of 2-CP by crude Myrothecium verrucaria fermentation broth is an effective way to reduce the content of 2-CP in pollutants,and laccase activity is an important parameter of 2-CP degradation rate. |
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