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.

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.

Background: Liquid hot water (LHW) pretreatment is an effective and environmentally friendly method to produce bioethanol with lignocellulosic materials. In our previous study, high ethanol concentration and ethanol yield were obtained from water-insoluble solids (WIS) of reed straw and corn stover pretreated with LHW by using fed-batch semi-simultaneous saccharification and fermentation (S-SSF). However, high cellulase loading and the large amount of wash water possibly limit the practical application of LHW pretreatment. To decrease cellulase loading and the amount of wash water, we performed Tween 40 pretreatment before WIS was subjected to bioethanol fermentation. Results: Results showed that the optimum conditions of Tween 40 pretreatment were as follows: Tween 40 concentration of 1.5%, WIS-to-Tween 40 ratio of 1:10 (w/v), and pretreatment time of 1 hour at ambient temperature. After Tween 40 pretreatment, cellulase loading could be greatly reduced. After Tween 40 pretreatment, the residual liquid could be recycled for utilization but slightly affected ethanol concentration and yield. The unwashed WIS could obtain a high ethanol concentration of 56.28 g/L (reed straw) and 52.26 g/L (corn stover) by Tween 40 pretreatment using fed-batch S-SSF. Ethanol yield reached a maximum of 69.1% (reed straw) and 71.1% (corn stover). Conclusions: Tween 40 pretreatment was a very effective and less costly method with unwashed WIS. This pretreatment could greatly reduce cellulase loading and save wash water. Higher ethanol concentration was obtained almost without reducing ethanol yield.