Adding tetrahydrofuran to dilute acid pretreatment provides new insights into substrate changes that greatly enhance biomass deconstruction by Clostridium thermocellum and fungal enzymes

BackgroundLiquid hot water (LHW) pretreatment has been considered as one of the most industrially viable and environment-friendly methods for facilitating the transformation of lignocelluloses into biofuels through biological conversion. However, lignin fragments in pretreatment hydrolysates are preferential to condense with each other and then deposit back onto cellulose surface under severe conditions. Particularly, lignin tends to relocate or redistribute under high-temperature LHW pretreatment conditions. The lignin residues on the cellulose surface would result in significant nonproductive binding of cellulolytic enzymes, and therefore negatively affect the enzymatic conversion (EC) of glucan in pretreated substrates. Although additives such as bovine serum albumin (BSA) and Tween series have been used to reduce nonproductive binding of enzymes through blocking the lignin, the high cost or non-biocompatibility of these additives limits their potential in industrial applications.ResultsHere, we firstly report that a soluble soy protein (SP) extracted from inexpensive defatted soy powder (DSP) showed excellent performance in promoting the EC of glucan in LHW-pretreated lignocellulosic substrates. The addition of the SP (80 mg/g glucan) could readily reduce the cellulase (Celluclast 1.5 L®) loading by 8 times from 96.7 to 12.1 mg protein/g glucan and achieve a glucan EC of 80% at a hydrolysis time of 72 h. With the same cellulase (Celluclast 1.5 L®) loading (24.2 mg protein/g glucan), the ECs of glucan in LHW-pretreated bamboo, eucalyptus, and Masson pine substrates increased from 57%, 54% and 45% (without SP) to 87%, 94% and 86% (with 80 mg SP/g glucan), respectively. Similar effects were also observed when Cellic CTec2, a newer-generation cellulase preparation, was used. Mechanistic studies indicated that the adsorption of soluble SP onto the surface of lignin residues could reduce the nonproductive binding of cellulolytic enzymes to lignin. The cost of the SP required for effective promotion would be equivalent to the cost of 2.9 mg cellulase (Celluclast 1.5 L®) protein (or 1.2 FPU/g glucan), if a proposed semi-simultaneous saccharification and fermentation (semi-SSF) model was used.ConclusionsNear-complete saccharification of glucan in LHW-pretreated lignocellulosic substrates could be achieved with the addition of the inexpensive and biocompatible SP additive extracted from DSP. This simple but remarkably effective technique could readily contribute to improving the economics of the cellulosic biorefinery industry.Electronic supplementary materialThe online version of this article (10.1186/s13068-019-1387-x) contains supplementary material, which is available to authorized users.

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Promoting enzymatic hydrolysis of lignocellulosic biomass by inexpensive soy protein

Luo

Liu

Zheng

et al. 2019

“…Clostridium thermocellum is one of the most attractive candidates for efficient cellulose solubilization because it produces the cellulosome, a highly organized multiprotein supermolecular complex containing both enzymatic subunits and non-catalyzing scaffoldins [21]. Using the cellulosome, C. thermocellum has been determined a robust and effective biocatalyst that outperforms commercial fungal cellulase cocktails in lignocellulose solubilization [22–25]. However, industrial-scale application of CBP remains challenging so far because of the low lignocellulose saccharification efficiency [26].…”

Section: Introductionmentioning

confidence: 99%

Construction of consolidated bio-saccharification biocatalyst and process optimization for highly efficient lignocellulose solubilization

Liu

Feng

et al. 2019

Background The industrial conversion of biomass to high-value biofuels and biochemical is mainly restricted by lignocellulose solubilization. Consolidated bio-saccharification (CBS) is considered a promising process for lignocellulose solubilization depending on whole-cell biocatalysts that simultaneously perform effective cellulase production and hydrolysis. However, it usually takes a long time to reach a high saccharification level using the current CBS biocatalyst and process. Results To promote the saccharification efficiency and reduce the cost, a Clostridium thermocellum recombinant strain ∆pyrF ::KBm was constructed as a new CBS biocatalyst in this study. The key CBS factors, including the medium, inoculum size and cultivation, and substrate load, were investigated and optimized. The saccharification process was also stimulated by adding free hemicellulases, suggesting the need to further enhance hemicellulase activity of the whole-cell catalyst. Under the optimal conditions, the CBS process was shortened by 50% with pretreated wheat straw as the substrate. The sugar yield reached 0.795 g/g and the saccharification level was 89.3%. Conclusions This work provided a new biocatalyst and an optimized process of CBS and confirmed that CBS is a feasible strategy for cost-efficient solubilization of lignocellulose, which will greatly promote the industrial utilization of lignocellulosic biomass. Electronic supplementary material The online version of this article (10.1186/s13068-019-1374-2) contains supplementary material, which is available to authorized users.

“…The miscible mixture of tetrahydrofuran (THF) with water and dilute acid used for CELF has been demonstrated to preferentially solvate lignin, thus, allowing for its facile removal from cellulose and preventing lignin self-aggregation and redeposition [23,24]. The resulting pretreated solids are highly digestible, achieving nearly theoretical glucose yields at a very low enzyme loadings [25,26]. Although the substantial removal of hemicellulose and lignin has been shown to play a major role in higher glucan conversions for CELFpretreated solids [24,27], the role of residual lignin and its impact on enzyme activity on cellulase activity is not clearly understood.…”

mentioning

confidence: 99%

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

Patri

Mohan

et al. 2021

Self Cite

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.