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

Abstract

Background Conventional 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. Results In 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. Conclusions Enzymatic 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.

Toward a Fundamental Understanding of the Role of Lignin in the Biorefinery Process

“…prevent lignin redeposition onto the biomass surface and prolong cellulases activity (Patri et al, 2021). Microscopic analysis showed that after autohydrolysis pretreatment, lignin from Eucalyptus globulus migrates out of the cell wall and redeposits in certain regions of the fibers to form droplet-like structures.…”

Organosolv pretreatment assisted by carbocation scavenger to mitigate surface barrier effect of lignin for improving biomass saccharification and utilization

“…3 ). Interestingly, a recent work on lignocellulosic fractionation pretreatment using tetrahydrofuran–water co-solvent revealed that the lignin globules deposited on the cellulose surface did not adsorb as much enzyme as lignin in the lignin–carbohydrate complex [ 9 , 39 ]. In other words, the deposited lignin merely provided an exterior physical obstacle on fiber surface, rather than strong adsorbents to cellulase enzymes [ 40 ], which allowed access of enzymes to cellulose with high-efficiency hydrolytic performance [ 41 ].…”

“…Results confirmed the aggravated exterior lignin coverage caused by 2-naphthol addition in acid EOS treatment, which in turn proved the greater fluidity of the less repolymerized lignin that modified by 2-naphthol. As discussed above, the lignin deposition on fiber surface probably cancelled out the negative effects of residual bulk lignin in the lignin–carbohydrate complex by reducing the unproductive binding, which turned out to be beneficial to enzymatic hydrolysis [ 9 , 40 ].

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“…However, lignin is not fully removed during acidic pretreatment, which would redeposit back onto fiber surface and restrict the cellulose accessibility to cellulase enzymes [ 8 ]. Besides, lignin also reduces the availability and activity of enzymes during hydrolysis through unproductive binding [ 9 ]. It is suggested that ethanol organosolv (EOS) pretreatment, in particular with acid catalyst, can effectively remove lignin and hemicellulose together through the cleaving of the lignin–carbohydrate complex, yielding cellulose-enriched solids with improved ease of enzymatic hydrolysis [ 1 , 3 ].…”

A Critical Review on the Effect of Lignin Redeposition on Biomass in Controlling the Process of Enzymatic Hydrolysis

“…Though the removal of hemicellulose and lignin plays a significant role in obtaining higher conversion of carbohydrates from CELF-pretreated solids, the actual impact of residual lignin on the action of enzyme is yet to be explored. In view of that, Patri et al [62] developed co-solvent-enhanced lignocellulosic fractionation (CELF) method in order to solubilize lignin and to decrease the limitations associated with acid pretreatment. It was observed that with an incubation period of 5 weeks and enzyme doses of 2 mg protein/g, an enzymatic hydrolysis of CELF-pretreated switchgrass at 7.5% (w/w) of solid loadings attributes towards glucan to glucose yield of more than 90%.…”