Non-productive cellulase adsorption onto lignin has always been deemed to negatively affect the enzymatic hydrolysis of lignocellulosic feedstocks. Therefore, understanding enzyme-lignin interactions is essential for the development of enzyme mixtures, the processes of lignocellulose hydrolysis, and the genetic modification of lignocellulosic biomass and enzymes. In this work, we examined the properties of six lignins from diverse types of lignocellulosic biomass (aspen, pine, corn stover, kenaf, and two Arabidopsis lines, wild-type and SALK mutant of fah1) to determine the mechanism of differences in their adsorption of enzymes.

In the bioconversion of lignocellulosic substrates, the adsorption behavior of cellulase onto lignin has a negative effect on enzymatic hydrolysis of cellulose, decreasing glucose production during enzymatic hydrolysis, thus decreasing the yield of fermentation and the production of useful products. Understanding the interaction between lignin and cellulase is necessary to optimize the components of cellulase mixture, genetically engineer high-efficiency cellulase, and reduce cost of bioconversion. Most lignin is not removed during liquid hot water (LHW) pretreatment, and the characteristics of lignin in solid substrate are also changed. To understand the interactions between cellulase and lignin, this study investigated the change in the characteristics of lignin obtained from corn stover, as well as the behavior of cellulase adsorption onto lignin, under various severities of LHW pretreatment. LHW pretreatment removed most hemicellulose and some lignin in corn stover, as well as improved enzymatic digestibility of corn stover. After LHW pretreatment, the molecular weight of lignin obviously increased, whereas its polydispersity decreased and became more negative. The hydrophobicity and functional groups in lignin also changed. Adsorption of cellulase from Penicillium oxalicum onto lignin isolated from corn stover was enhanced after LHW pretreatment, and increased under increasing pretreatment severity. Different adsorption behaviors were observed in different lignin samples and components of cellulase mixtures, even in different cellobiohydrolases (CBHs), endo-beta-1, 4-glucanases (EGs). The greatest reduction in enzyme activity caused by lignin was observed in CBH, followed by that in xylanase and then in EG and β-Glucosidase (BGL). The adsorption behavior exerted different effects on subsequent enzymatic hydrolysis of various biomass substrates. Hydrophobic and electrostatic interactions may be important factors affecting different adsorption behaviors between lignin and cellulase. LHW pretreatment changed the characteristics of the remaining lignin in corn stover, thus affected the adsorption behavior of lignin toward cellulase. For different protein components in cellulase solution from P. oxalicum, electrostatic action was a main factor influencing the adsorption of EG and xylanase onto lignin in corn stover, while hydrophobicity affected the adsorption of CBH and BGL onto lignin.

Reed is a widespread-growing, inexpensive, and readily available lignocellulosic material source in northeast China. The objective of this study is to evaluate the liquid hot water (LHW) pretreatment efficiency of reed based on the enzymatic digestibility and ethanol fermentability of water-insoluble solids (WISs) from reed after the LHW pretreatment. Several variables in the LHW pretreatment and enzymatic hydrolysis process were optimized. The conversion of glucan to glucose and glucose concentrations are considered as response variables in different conditions. The optimum conditions for the LHW pretreatment of reed area temperature of 180°C for 20min and a solid-to-liquid ratio of 1 : 10. These optimum conditions for the LHW pretreatment of reed resulted in a cellulose conversion rate of 82.59% in the subsequent enzymatic hydrolysis at 50°C for 72 h with a cellulase loading of 30 filter paper unit per gram of oven-dried WIS. Increasing the pretreatment temperature resulted in a higher enzymatic digestibility of the WIS from reed. Separate hydrolysis and fermentation of WIS showed that the conversion of glucan to ethanol reached 99.5% of the theoretical yield. The LHW pretreatment of reed is a suitable method to acquire a high recovery of fermentable sugars and high ethanol conversion yield.

Corn stover is a promising feedstock for bioethanol production because of its abundant availability in China. To obtain higher ethanol concentration and higher ethanol yield, liquid hot water (LHW) pretreatment and fed-batch semi-simultaneous saccharification and fermentation (S-SSF) were used to enhance the enzymatic digestibility of corn stover and improve bioconversion of cellulose to ethanol. The results show that solid residues from LHW pretreatment of corn stover can be effectively converted into ethanol at severity factors ranging from 3.95 to 4.54, and the highest amount of xylan removed was approximately 89%. The ethanol concentrations of 38.4 g/L and 39.4 g/L as well as ethanol yields of 78.6% and 79.7% at severity factors of 3.95 and 4.54, respectively, were obtained by fed-batch S-SSF in an optimum conditions (initial substrate consistency of 10%, and 6.1% solid residues added into system at the prehydrolysis time of 6 h). The changes in surface morphological structure, specific surface area, pore volume and diameter of corn stover subjected to LHW process were also analyzed for interpreting the possible improvement mechanism.

Cellulolytic enzyme hydrolysis of lignocellulose biomass to release fermentable sugars is one of the key steps in biofuel refining. Gene expression of fungal cellulolytic enzymes is tightly controlled at the transcriptional level. Key transcription factors such as activator ClrB/CLR2 and XlnR/XYR1, as well as repressor CreA/CRE1 play crucial roles in this process. The putative protein methyltransferase LaeA/LAE1 has also been reported to regulate the gene expression of the cellulolytic enzyme. The formation and gene expression of the cellulolytic enzyme was compared among Penicillium oxalicum wild type (WT) and seven mutants, including ΔlaeA (deletion of laeA), OEclrB (clrB overexpression), OEclrBΔlaeA (clrB overexpression with deletion of laeA), OExlnR (xlnR overexpression), OExlnRΔlaeA (xlnR overexpression with deletion of laeA), ΔcreA (deletion of creA), and ΔcreAΔlaeA (double deletion of creA and laeA). Results revealed that LaeA extensively affected the expression of glycoside hydrolase genes. The expression of genes that encoded the top 10 glycoside hydrolases assayed in secretome was remarkably downregulated especially in later phases of prolonged batch cultures by the deletion of laeA. Cellulase synthesis of four mutants ΔlaeA, OEclrBΔlaeA, OExlnRΔlaeA, and ΔcreAΔlaeA was repressed remarkably compared with their parent strains WT, OEclrB, OExlnR, and ΔcreA, respectively. The overexpression of clrB or xlnR could not rescue the impairment of cellulolytic enzyme gene expression and cellulase synthesis when LaeA was absent, suggesting that LaeA was necessary for the expression of cellulolytic enzyme gene activated by ClrB or XlnR. In contrast to LaeA positive roles in regulating prominent cellulase and hemicellulase, the extracellular β-xylosidase formation was negatively regulated by LaeA. The extracellular β-xylosidase activities improved over 5-fold in the OExlnRΔlaeA mutant compared with that of WT, and the expression of prominent β-xylosidase gene xyl3A was activated remarkably. The cumulative effect of LaeA and transcription factor XlnR has potential applications in the production of more β-xylosidase.

There is a rising interest in bioethanol production from lignocellulose such as corn stover to decrease the need for fossil fuels, but most research mainly focuses on how to improve ethanol yield and pays less attention to the biorefinery of corn stover. To realize the utilization of different components of corn stover in this study, different pretreatment strategies were used to fractionate corn stover while enhancing enzymatic digestibility and cellulosic ethanol production. It was found that the pretreatment process combining dilute acid (DA) and alkaline sodium sulfite (ASS) could effectively fractionate the three main components of corn stover, i.e., cellulose, hemicellulose, and lignin, that xylose recovery reached 93.0%, and that removal rate of lignin was 85.0%. After the joint pretreatment of DA and ASS, the conversion of cellulose at 72 h of enzymatic hydrolysis reached 85.4%, and ethanol concentration reached 48.5 g/L through fed-batch semi-simultaneous saccharification and fermentation (S-SSF) process when the final concentration of substrate was 18% (w/v). Pretreatment with ammonium sulfite resulted in 83.8% of lignin removal, and the conversion of cellulose and ethanol concentration reached 86.6% and 50 g/L after enzymatic hydrolysis of 72 h and fed-batch S-SSF, respectively. The results provided a reference for effectively separating hemicellulose and lignin from corn stover and producing cellulosic ethanol for the biorefinery of corn stover.