Application Of Bacillus Coagulans In Chinese Fir Biorefinery And The Study Of Its Stress Resistance Mechanism

As the main fast-growing forest resource in China,there are large amount of Chinese fir(CF)processing residues not effectively utilized every year.The utilization of CF residues belongs to the research field of forestry engineering.Through chemical conversion and biological conversion,three major components of cellulose,hemicellulose,and lignin contained in CF can be converted to platform chemicals such as monosaccharides,lactic acid,and phenols,which can reduce environmental pressure.However,as one of the typical representatives of softwood,CF has strong biological antagonism.The biorefining of CF faces two major problems:one is the low pretreatment efficiency and saccharification yield of CF,and the other one is the inhibitors produced by harsh pretreatment condition seriously affects the fermentation.These problems limit the application of CF residues in industry.In this paper,we aim to overcome the recalcitrance of CF and the total utilization of carbohydrates and lignin of CF.Through the study on the component removal of lignocellulose depolymerization process,this work used the combined pretreatment to overcome the high recalcitrance of CF,and realized the cascade separation of the three major components.Meanwhile,a high hydrolysate tolerance strain of B.coagulans was obtained through adapted domestication and successfully used to efficiently convert hemicellulose and cellulose to lactic acid,and the application potential of oxidized lignin was also studied.In addition,the molecular biological mechanism and key gene of B.coagulans response for stress resistance were analyzed and successfully constructed highly resistant B.coagulans using overexpression.The main research results are summarized as follows:(1)Single pretreatment could not achieve the simultaneous removal of hemicellulose and lignin of CF.There was an interaction between the removal yield of hemicellulose and lignin,and the presence of hemicellulose limited the removal of lignin.In this study,a combined pretreatment of dilute sulfuric acid followed by sodium chlorite(DSASCP)was established and realized the cascade separation of hemicellulose,lignin,and cellulose.The hemicellulose was mainly selectively dissolved in step 1 and the removal yield was 92.3%.The lignin component was mainly separated in the second step and the dissolution yield was 93.2%.Cellulose was mainly recovered as solid form and the glucan content in the solid was 79.3%.(2)The most important limiting factor of CF saccharification was lignin removal yield and hemicellulose removal yield was the second important.Based on removal yields,a reliable model(R~2=0.9482)that could accurately evaluate the effectiveness of pretreatment was established.The sample treated by DSASCP exhibited excellent enzymatic hydrolysis performance and fermentability.Through the fed-batch enzymatic hydrolysis,a hydrolysate with a high glucose concentration of 138.8 g/L under 160 g/L substrate content,and the enzymatic yield was 77.7%.After fermentation using S.cerevisiae NL22,the hydrolysate produced 64.6 g/L of ethanol,which was a 93.2%of theoretical conversion rate.(3)A high hydrolysate tolerance strain of B.coagulans CC17A was obtained through adapted domestication in real dilute acid hydrolysate.By avoiding solid-liquid separation and detoxification,B.coagulans CC17A could integrate in-suit online detoxification,xylose fermentation and simultaneous cellulose saccharification and fermentation.Finally,80 g of wheat straw could produce 35.5 g of lactic acid,which is 70.9%of the theoretical carbohydrate production of lactic acid.(4)The stress resistance of B.coagulans was analyzed from the metabolism and transcription.B.coagulans CC17A not only has furan aldehyde and phenol aldehyde reduction ability,but also has the ability of phenolic acid decarboxylation.Transcriptome analysis showed that a total of 331 genes of B.coagulans CC17A were significantly differentially expressed under environmental stress,mainly involving transporters and transmembrane transporters,required cofactor proteins,redox processes,and membrane components.The stress resistance mechanisms of B.coagulans CC17A include ABC transmembrane transport protein,increased expression of cytochrome c oxidase and hemoglobin synthesis-related genes in the oxidative respiratory chain,and overall up-regulation of sulfur transport and degradation metabolic pathways in stress high-salt environments.(5)The up-regulated oxidoreductase genes and phenolic acid decarboxylase gene were successfully overexpressed in B.coagulans DSM1,respectively.RS25280,as a phenolic acid decarboxylase,played a key role in phenolic acid stress resistance.Overexpression of RS25280could not only promote the ability of strain degrade phenolic acid in a non-oxidative state,but also the first discovery of the presence of phenolic acid decarboxylase was beneficial to the conversion of vanillin and promotes the conversion of vanillin into low-toxic substances.The overexpression of RS25275 could promote the conversion of syringaldehyde obviously,and it plays a leading role in phenolic stress resistance.The non-oxidative decarboxylation and oxidoreductase reduction processes of B.coagulans had an interactive effect on the degradation of phenolic acid and phenolic aldehyde.(6)An integrated biorefining technology for CF was established using combined pretreatment and high stress resistance strain.The reused hemicellulose hydrolysate technology could not only save 54%of acid usage but also increased the concentration of fermentation sugar in hydrolysate by 2.8 fold.Then,B.coagulans CC17A was applied for whole utilize the separated hemicellulose and cellulose component.The reused hemicellulose hydrolysate could produce 28.6 g/L lactic acid without detoxification,and the cellulose component could produce128.8 g/L lactic acid by using fed-batch SSCF.Through the mass balance,after resuing hydrolysate DSASCP pretreatment,3.0 kg CF could finally produce 1.1 kg lactic acid,which was the 59.8%of theoretical conversion of whole carbohydrates.Meanwhile,a total of 0.49 kg lignin component was recovered through selective precipitation.The structure of O-lignin was highly depolymerized and the 2D-HSQC results showed that not only theβ-O-4 ether bond was greatly destroyed,but theβ–βandβ-1 linkages between benzene rings were also opened.However,the O-lignin still retained the benzene ring structure and mainly was guaiacol structure.These results showed that the O-lignin has the potential of thermal depolymerization to guaiacol products.