| Recent studies indicated that co-cultured methanogens could increase the lignocellulosic digestibility of anaerobic fungi.However,there are few reports on further improvement of the straw digestibility of anaerobic fungi co-cultured with methanogens.This study,based on the latest reported methods bioaugmentation and steam explosion,investigated their effects on the straw digestibility of an anaerobic fungus co-cultured with methanogen.Meanwhile,a feruloyl esterase from the anaerobic fungus was expressed and its biological characteristics was analyzed.This study was divided into three parts:1 Effect of bioaugmentation on the straw degradation of a co-culture of anaerobic fungus and methanogenThis experiment used a co-culture of anaerobic fungus and methanogen(Pecoramyces sp.F1+Methanobrevibacter thaueri)and the mono-culture of anaerobic fungus(Pecoramyces sp.F1)as inocula,studying the effects of bioaugmentating with different volumes of anaerobic fungal culture(0,10,and 20 mL)on the straw degradation,lignocellulase activities,and fermentation end-products of a co-culture of anaerobic fungus and methanogen.The weight of 1.0 g wheat straw was used as substrate.The volume of 10 mL inocula was added and the bottles were incubated at 39℃ for 96 hours without shaking.The gas,methane,and hydrogen production were determined every 6 hours.At the end of fermentation,the digestibilities of different components of straw,lignocellulase activities,and the concentrations of domain metabolites were determined.The results showed that no significant difference was observed on the total gas production between group bioaugmented with 10 mL anaerobic fungal culture and control group(P>0.05).The total gas production of group bioaugmented with 20 mL anaerobic fungal culture was significantly lower than that of the other two groups(P<0.05).There was no significant difference on the methane production among all the three groups(P>0.05).The hydrogen production of group bioaugmented with 20 mL anaerobic fungal culture was significantly higher than that of the other two groups(P<0.05),and its digestibilities of dry matter,NDF,ADF,cellulose,and hemicellulose were significantly lower than those of the other two groups(P<0.05).There was no significant difference on the digestibilities of dry matter,NDF,ADF,cellulose,and hemicellulose of the other two groups(P>0.05).The xylanase activity of control group was significantly higher than that of the other two groups(P<0.05),but there was no significant difference on the carboxymethyl cellulase activity among all the three groups(P>0.05).The concentration of formate of group bioaugmented with 10 mL anaerobic fungal culture was significantly higher than that of the other two groups(P<0.05).The concentration of ethanol of group bioaugmented with 10 mL anaerobic fungal culture was significantly higher than that of the other two groups(P<0.05).There was no significant difference in the pH and concentration of lactate and acetate among these three groups(P>0.05).The results indicated that bioaugmentation with anaerobic fungus can accelerate the process of degradation of straw by the co-culture of anaerobic fungus and methanogen,but could not improve its digestibility.Exorbitant bioaugmentation amount of anaerobic fungus could restrain the degradation of straw by the co-culture of anaerobic fungus and methanogen.2 Effect of steam explosion on the stover degradation of a co-culture of anaerobic fungus and methanogenThis experiment used a co-culture of anaerobic fungus and methanogen(Pecoramyces sp.F1+Methanobrevibacter thaueri)as inoculum,studying the effects of steam explosion treatment on the stover degradation.lignocellulase activities,and metabolites of the co-culture.This study contained untreated corn stover group and steam-exploded corn stover group.using 1.0 g untreated corn stover and steam-exploded corn stover as substrates,respectively.The 90 mL medium was inoculated with 10 mL co-culture of anaerobic fungus and methanogen,and all bottles were incubated at 39℃ for 72 hours without shaking.The concentrations of reducing sugar,xylose,and glucose in the supernatant of each group and the contents of different chemical components in untreated and steam-exploded corn stover were determined before fermentation.During fermentation,total gas,methane,and hydrogen production were measured at specific time point.At the end of fermentation,the digestibilities of different components of straw,lignocellulase activities,and the concentrations of domain metabolites were determined.The results demonstrated that steam explosion treatment reduced the contents of neutral detergent fiber and hemicellulose of corn stover significantly(P<0.05),increased the contents of reducing sugar,xylose,acid detergent fiber,neutral detergent solute,and lignin significantly(P<0.05),but did not affect the contents of cellulose and glucose(P>0.05).Steam explosion treatment improved the total gas production,hydrogen production,the digestibility of neutral detergent solute,the concentrations of formate and lactate significantly(P<0.05),had no significant effects on the methane production,the digestibilities of dry matter and cellulose,the pH value,carboxymethyl cellulase activity,concentrations of acetate and ethanol in the supernatant(P>0.05),but decreased the xylanase activity,the digestibilities of neutral detergent fiber,acid detergent fiber,and hemicellulose significantly(P<0.05).In conclusion,steam explosion treatment could convert the hemicellulose of corn stover into neutral detergent solute,but could not improve the digestibility of corn stover by the co-culture of anaerobic fungus and methanogen,which implied that the co-culture of anaerobic fungus and methanogen can efficiently degrade stover without any pretreatments.3 The expression and biological characterization of a feruloyl esterase from anaerobic fungus Pecoramyces sp.F1This study was aimed at expressing a feruloyl esterase gene from anaerobic fungus Pecoramyces sp.F1,analyzing the biological characteristics and paving the way for its application.Screening the feruloyl esterase gene sequences in the fungal genome,which had high activity through genomic technology and expressing it successfully in E.coli(DE3).Ni-affinity chromatography,ionic exchange chromatography,and gel filtration were used to purify the zymoprotein.According to the gene sequence and structure of the feruloyl esterase,three mutants were obtained by mutating the active amino acids of esterase(EST),glycoside hydrolase 11(GH11),and EST+GH11,respectively.The biological characteristics of wild feruloyl esterase and mutants was analyzed.The results showed that the length of feruloyl esterase gene is 1878 bp,and the protein encoded by this gene contains 625 amino acids.Isoelectric point is 5.93.and instability coefficient is 15.36.The optimum temperature and pH were 37℃ and 7.5.respectively.The molecular weights of wild feruloyl esterase(WT),feruloyl esterase mutated EST(S126A),feruloyl esterase mutated GH11(E510A),and double mutant(S126A/E510A)were 67.80 kDa,67.78 kDa,67.74 kDa,and 67.72 kDa,respectively.For degradation of polysaccharide,the specific activity of S126A was significantly higher than that of WT(P<0.05),E510A and S126A/E510A both showed very low specific activity.The result of degrading esters and feruloyl oligosaccharides showed that the degrading capacity of WT was significantly higher than that of the three mutants(P<0.05),and three mutants all appeared extremely low esterase activities.This study expressed a feruloyl esterase and its mutants from anaerobic fungus Pecoramyces sp.F1 successfully.The results showed that the glycoside hydrolase 11 in this enzyme assisted the esterase to degrade esters,which has the function of preserving and improving the activity of esterase. |
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