THF co-solvent pretreatment prevents lignin redeposition from interfering with enzymes yielding prolonged cellulase activity

Abhishek S Patri,Chang G Yoo,David Kisailus,Arthur J Ragauskas,Rajeev Kumar,Charles E Wyman,Ramya Mohan,Yunqiao Pu,Charles M Cai

doi:10.1186/s13068-021-01904-2

Abstract

BackgroundConventional aqueous dilute sulfuric acid (DSA) pretreatment of lignocellulosic biomass facilitates hemicellulose solubilization and can improve subsequent enzymatic digestibility of cellulose to fermentable glucose. However, much of the lignin after DSA pretreatment either remains intact within the cell wall or readily redeposits back onto the biomass surface. This redeposited lignin has been shown to reduce enzyme activity and contribute to rapid enzyme deactivation, thus, necessitating significantly higher enzyme loadings than deemed economical for biofuel production from biomass.ResultsIn this study, we demonstrate how detrimental lignin redeposition on biomass surface after pretreatment can be prevented by employing Co-solvent Enhanced Lignocellulosic Fractionation (CELF) pretreatment that uses THF–water co-solvents with dilute sulfuric acid to solubilize lignin and overcome limitations of DSA pretreatment. We first find that enzymatic hydrolysis of CELF-pretreated switchgrass can sustain a high enzyme activity over incubation periods as long as 5 weeks with enzyme doses as low as 2 mg protein/g glucan to achieve 90% yield to glucose. A modified Ninhydrin-based protein assay revealed that the free-enzyme concentration in the hydrolysate liquor, related to enzyme activity, remained unchanged over long hydrolysis times. DSA-pretreated switchgrass, by contrast, had a 40% drop in free enzymes in solution during incubation, providing evidence of enzyme deactivation. Furthermore, measurements of enzyme adsorption per gram of lignin suggested that CELF prevented lignin redeposition onto the biomass surface, and the little lignin left in the solids was mostly integral to the original lignin–carbohydrate complex (LCC). Scanning electron micrographs and NMR characterization of lignin supported this observation.ConclusionsEnzymatic hydrolysis of solids from CELF pretreatment of switchgrass at low enzyme loadings was sustained for considerably longer times and reached higher conversions than for DSA solids. Analysis of solids following pretreatment and enzymatic hydrolysis showed that prolonged cellulase activity could be attributed to the limited lignin redeposition on the biomass surface making more enzymes available for hydrolysis of more accessible glucan.

Highlights

Conventional aqueous dilute sulfuric acid (DSA) pretreatment of lignocellulosic biomass facilitates hemicellulose solubilization and can improve subsequent enzymatic digestibility of cellulose to fermentable glucose
The mass of components in solids produced by application of the maximum sugar recovery pretreatment conditions for both DSA and Co-solvent Enhanced Lignocellulosic Fractionation (CELF) pretreatments were adjusted to a basis of 100 g of unpretreated switchgrass (Additional file 1: Figure S1)
The data show that residual lignin in THF-washed DSA switchgrass contained a higher percentage of β-O-4 linkages and lignin recovered in the THF wash liquid contained a lower percentage of β-O-4 linkages when compared to lignin found in DSA-pretreated switchgrass. β-O-4 linkages are one of the major interunit linkages in unpretreated switchgrass [31]. These results provide further evidence that lignin in THF-washed DSA switchgrass is likely lignin remaining in the lignin–carbohydrate complex (LCC), unlike the likely depolymerized and recondensed lignin recovered from the THF wash liquid, and binds more enzyme during enzymatic hydrolysis

Summary

Introduction

Conventional aqueous dilute sulfuric acid (DSA) pretreatment of lignocellulosic biomass facilitates hemicellulose solubilization and can improve subsequent enzymatic digestibility of cellulose to fermentable glucose. Much of the lignin after DSA pretreatment either remains intact within the cell wall or readily redeposits back onto the biomass surface This redeposited lignin has been shown to reduce enzyme activity and contribute to rapid enzyme deactivation, necessitating significantly higher enzyme loadings than deemed economical for biofuel production from biomass. Achieving sufficiently high sugar yields during enzymatic hydrolysis still requires uneconomically high enzyme loadings, largely due to the presence of lignin which remains attached to the solid fraction after pretreatment [10, 11]. An effective pretreatment should have high sugar yields during enzymatic hydrolysis at low enough enzyme loadings to reduce overall costs of producing 2nd-generation fuels from biomass

Objectives

Results

Conclusion