Optimization Of Combinatorial Fungi Producing Lignin-degrading Enzymes And Promotion Of Anaerobic Fermentation Of Corn Straw

At present,the use of corn stalks for anaerobic fermentation is the main way of energy utilization,and the large amount of lignin in straw can lead to a series of questions,such as incomplete hydrolysis of anaerobic fermentation,low gas production rate,long start-up time and easy rancidity.Therefore,reducing the lignin content in corn stover is an effective way to improve the utilization rate of the substrate.Due to low cost of microbial pretreatment,no pollution and green cleaning,it has has attracted wide attention all of the word.This study selected three white rot fungi with lignin degradation ability: Phanerochaete chrysosporium,Trametes versicolor and Pleurotus ostreatus.After excluding the antagonism of the strains by the antagonistic test to construct the combined fungi of white rot fungi,and sing corn straw culture medium to induce the production of lignin-degrading enzymes,analysed their enzyme-producing ability.The optimal culture temperature and rotation speed of the lignin-degrading enzyme produced by the combined fungi were determined by single factor test.The superior p H,inoculum volume and solid-liquid ratio of the medium were determined by the orthogonal rotation center combination test.Selecting three kinds of the laccase,manganese peroxidase and lignin peroxidase as indicators,to optimize the best enzyme production culture conditions of the combined fungi,apply it to the anaerobic fermentation process of corn straw pretreatment.The specific results are as follows:(1)Three white rot fungi were able to produce lignin-degrading enzymes,Lac,Mn P and Li P activities of Phanerochaete chrysosporium were: 190.36 U/L,296.35 U/L and 1346.23 U/L,Lac,Mn P and Li P activities of Trametes versicolor were: 480.54 U/L,278.65 U/L and 1030.60 U/L,and Lac,Mn P and Li P activities of Pleurotus ostreatus were 356.28 U/L,435.26 U/,and 850.29U/L,respectively.Therefore,the different strains have different expressions for lignin-degrading enzymes.Among the four combinatorial fungi constructed by the antagonistic test,the combined of the three strains greatly improved the comprehensive activity,and the Lac,Mn P and Li P activities of that were: 498.00 U/L,525.56 U/L and 2329.30 U/L;compared with the maximum value of a single strain increased by 3.63%,20.75% and 73.02%,respectively.(2)The results of single factor test showed that the optimal culture temperature is 34 °C,the optimal rotation speed was 165 r/min;the superior p H value was 4.0~5.0,the solid-liquid ratio was10~18%,and the inoculum volume was 12 ~18%,when cultured the combined fugus.Using Lac,Mn P and Li P activities as indicators,the orthogonal rotation center combination testdetermined the optimal enzyme production conditions : pH value was 4.6,solid-liquid ratio was15.60%,and inoculum size was 15.65%.Under this condition,the Lac activity was 1086.25U/L,the Mn P activity was 915.50 U/L,and the Li P activity was 2790.90 U/L.Under the optimal conditions of enzyme production,the degradation rates of corn stalk lignin,cellulose and hemicellulose were 38.65%,28.50% and 44.23%,respectively.The results of transmission electron microscopy showed that the combined fungi could invade corn stover cells and destroy the dense network structure of lignocellulose.The absorption peak of infrared spectrum confirmed the degradation and transformation of lignin macromolecules in corn stover.(3)Applying the combined fungi to the anaerobic fermentation pretreatment process of corn stover : It showed that the anaerobic fermentation process of T3(corn stalk was pretreated by combined fungi for 9 days)started faster,and the maximum volatile fatty acid content was 14.20g/L,which was higher than CK group.56.56%;the maximum daily methane production was 48.00 m L/g,and the VS methanogenic rate was 288.26 mL/g VS,which was 30.25% higher than that of the CK group.The result of the microbial community indicated that the combined fungi pretreatment could accelerate the anaerobic fermentation process,and improve the richness and diversity of microbial communities.Firmicutes,Bacteroidetes and Clostridia are dominant bacterias.The relative abundance of Firmicutes and Clostridia increased by pretreatment of combined fungi,and the relative abundance of Spirochaetia,Chloroflexi,Euryarchaeota and Methanomicrobia were changed.About the relative abundance of microorganism is also a certain impact on the anaerobic fermentation process.The lignin-degrading enzyme activity of the combined fungi is higher than that of a single strains,and its enzyme-producing ability and stability are both better than single strains.Appling the combined fungi to the anaerobic fermentation pretreatment process of corn stover,which can accelerate the hydrolysis rate and increase the methane production.It is of great significance for the anaerobic fermentation of agricultural waste.