Enzymatic Hydrolysis Of Lignocellulose Research Articles

Corn-derived Expansin synergistically promotes enzymatic hydrolysis of corn cob

The enzymatic hydrolysis of lignocellulose is hindered by challenges such as high enzyme usage and associated costs. It is essential to explore effective approaches to improve the efficiency of enzymatic hydrolysis while reducing costs. Expansins are non-enzymatic proteins that can interact with lignocellulose and facilitate the loosening of plant cell walls. Given their natural affinity to plant cell walls, we hypothesized that a corn Expansin could enhance the enzymatic hydrolysis of corn lignocellulose. In this study, we expressed a corn (Zea mays) Expansin, EXPA17, in yeast cells and explored the synergistic effect between EXPA17 and commercial cellulase, and found that EXPA17 exhibited a pronounced synergistic effect on the enzymatic hydrolysis of corn cobs. The addition of 0.015 mg/mL EXPA17 resulted in a 14.00 % increase in glucose yield. Fourier-transform infrared (FTIR) spectroscopy and scanning electron microscopy (SEM) analysis revealed that EXPA17 proteins disrupted hydrogen bonds in the amorphous regions of corn cobs, leading to a more porous and looser structure, thereby enhancing cellulose accessibility. Our work leveraged the synergistic effect between Expansin and lignocellulose from the same source of corn, providing a novel strategy to improve the efficiency of enzymatic hydrolysis of corn lignocellulose while potentially reducing the associated costs.

Constructing of ultrahigh pH-responsive polycarboxylic copolymers for enhanced recycling cellulase from enzymatic hydrolyzate of corncob resides

The high cost of the enzymatic hydrolysis of lignocellulose is a major problem for the industrialization of lignocellulose-based sugar platform technology. Cellulase recovery is a vital approach to reduce the enzymatic hydrolysis cost. In this study, ultrahigh pH-responsive copolymers (PAMAs) were obtained from methacrylamide, sodium methacrylate and [2-(methacryloyloxy)ethyl]-trimethylammonium chloride. A PAMA addition method coupled with substrate reabsorption was used to efficiently recover cellulase in a narrow pH range. The enzymatic hydrolysis system of corncob resides (pH = 5.0) was supplemented with 1.0 g/L PAMA-1. After hydrolysis, the PAMAs coprecipitate with cellulase when adjusting the pH value of the hydrolysate to 4.4. When coupled with substrate reabsorption, 50 % of the cellulase was recovered. This method therefore represents a feasible solution to reduce the high enzymatic hydrolysis cost of the industrialization of lignocellulose-based sugar platform technology.

Relation between structural feature and non-ionic surfactant improving enzymatic hydrolysis of lignocellulose

Relation between structural feature and non-ionic surfactant improving enzymatic hydrolysis of lignocellulose

Valorization of residual lignin from corncob residues into thermosensitive lignin-based “molecular glues” for recycling cellulase

The cost of enzymolysis is a major bottleneck for the industrialisation of lignocellulosic enzymatic hydrolysis technology, and recycling cellulase can reduce this cost. Herein, a sulfobetaine prepolymer (CPS) with terminal chlorine was grafted onto enzymatic hydrolysis residual lignin (EHL) from corncob to construct thermosensitive lignin-based “molecular glues” (lignin-based sulfobetaine polymers, L-CPS) that were used to recover and recycle cellulase. L-CPS2 (1.0 g/L) was added to the corncob residue (CCR) enzymolysis system (50 °C, pH 4.5). After hydrolysis, L-CPS2 co-precipitated with cellulase through hydrophobic binding when cooling to 25 °C. This co-precipitation decreased the amount of cellulase by 40 %. In summary, a thermally responsive lignin-based molecular glue was constructed for green recycling of cellulase, providing a new approach to decreasing the cost of lignocellulosic enzymolysis and high value utilisation of industrial lignin.

Mathematical and statistical modeling of glucose permeation through ultrafiltration system

Membrane bioreactors (MBRs) have been recently proposed for enhancing enzymatic hydrolysis of lignocelluloses by simultaneously and selectively removing the produced sugars from the reaction system. An inverted dead-end MBR with polyethersulfone membrane was used to investigate glucose permeation. The effects of glucose concentration, water flowrate, and membrane molecular weight cut-offs (MWCO), were investigated. The developed diffusion-convective model predicted glucose permeation with R2 value of 0.96. The statistical analysis showed that the effects of glucose concentration and water flowrate were significant, with P-value less than 0.05 for both, whereas that of the MWCO was insignificant (P-value of 0.66). This is the first attempt to mathematically describe the behavior of glucose across ultrafiltration membrane, while taking into consideration both molecular diffusion and convective flow. The findings of this work are essential for understanding the behavior and enhancing the performance of solute permeation through ultrafiltration membrane, which is the heart of many processes.

Self-Buffering Effect of Solids During High-Solid Enzymatic Hydrolysis of Lignocellulose

Self-Buffering Effect of Solids During High-Solid Enzymatic Hydrolysis of Lignocellulose

Upgrading pectin methylation for consistently enhanced biomass enzymatic saccharification and cadmium phytoremediation in rice Ospmes site-mutants

Crop straws provide enormous biomass residues applicable for biofuel production and trace metal phytoremediation. However, as lignocellulose recalcitrance determines a costly process with potential secondary waste liberation, genetic modification of plant cell walls is deemed as a promising solution. Although pectin methylation plays an important role for plant cell wall construction and integrity, little is known about its regulation roles on lignocellulose hydrolysis and trace metal elimination. In this study, we initially performed a typical CRISPR/Cas9 gene-editing for site mutations of OsPME31, OsPME34 and OsPME79 in rice, and then determined significantly upgraded pectin methylation degrees in the young seedlings of three distinct site-mutants compared to their wild type. We then examined distinctively improved lignocellulose recalcitrance in three mutants including reduced cellulose levels, crystallinity and polymerization or raised hemicellulose deposition and cellulose accessibility, which led to specifically enlarged biomass porosity either for consistently enhanced biomass enzymatic saccharification under mild alkali pretreatments or for cadmium (Cd) accumulation up to 2.4-fold. Therefore, this study proposed a novel model to elucidate how pectin methylation could play a unique enhancement role for both lignocellulose enzymatic hydrolysis and Cd phytoremediation, providing insights into precise pectin modification for effective biomass utilization and efficient trace metal exclusion.

Deep eutectic solvent cocktail enhanced the pretreatment efficiency of lignocellulose

Pretreatment of lignocellulosic biomass by breaking down its complex structure is an essential step to facilitate enzymatic hydrolysis of lignocellulose and subsequent bioethanol fermentation. Herein, an eco-friendly pretreatment strategy was developed by applying deep eutectic solvent (DES) cocktail. Attributing to the synergistic effect of two DESs (e.g., betaine-glycerol and choline chloride-oxalic acid (or choline chloride-acetamide)), an impressive 89.51% digestibility for glucan and 75.43% for xylan were achieved. Characterization of pretreated corn stover showed that DES cocktail changed substrate surface structure and enlarged structure pores, which could improve the accessibility of cellulase. The pretreated corn stover was also employed in ethanol fermentation and achieved the yield of 73.35%. Recycling experiments demonstrated the potential reusability of DES cocktail in a sustainable manner towards various biomass sources. These findings highlight the effectiveness and versatility of the developed DES cocktail pretreatment method, paving the way for sustainable bioethanol production from lignocellulosic feedstocks.

Effect of various aromatic compounds with different functional groups on enzymatic hydrolysis of microcrystalline cellulose and alkaline pretreated wheat straw

Low molecular aromatic compounds are detrimental to the enzymatic hydrolysis of lignocellulose. However, the specific role of their functional groups remains unclear. Here, a series of nine aromatic compounds as additives were tested to understand their effect on the hydrolysis yield of microcrystalline cellulose (MCC) and alkaline pretreated wheat straw. Based on the results, the inhibition of aldehyde groups on MCC was greater than that of carboxyl groups, whereas for the alkaline pretreated wheat straw case, the inhibitory effect of aldehyde groups was lower than that of carboxyl groups. Increased methoxyl groups of aromatic compounds reduced the inhibitory effect on enzymatic hydrolysis of both substrates. Stronger inhibition of aromatic compounds on MCC hydrolysis was detected in comparison with the alkaline pretreated wheat straw, indicating that the substrate lignin can offset the inhibition to a certain extent. Among all aromatic compounds, syringaldehyde with one aldehyde group and two methoxyl groups improved the glucan conversion of the alkaline pretreated wheat straw.

Recycling enzymatic hydrolysis lignin residues saved cellulase in enzymatic hydrolysis of lignocellulose: An insight from cellulase adsorption mechanism

Enzymatic hydrolysis lignin residues (EHLRs) of lignocellulose usually adsorbs cellulase, which can be recycled and used to replace parts of cellulase in the hydrolysis process. To understand this phenomenon during enzymatic hydrolysis (EH) of sugarcane bagasse (SCB) treated with sulfite (SPORL) and dilute acid (DA), the adsorption characteristics between lignin and cellulase in EHLRs were investigated, focusing on interaction force at molecular level and enzymatic activity. The results revealed that SPORL-EHLR adsorbed more β-glucosidases (β-GLs) through the stronger electrostatic attraction and hydrogen bond force, causing higher cellulase adsorption amount compared to DA-EHLR. Further exploration demonstrated that the cellobiose’s catalytic and binding sites on β-GLs were separated from the binding site of SPORL-EHLR on β-GLs, resulting in minimal inhibition of β-GLs activity when bound to SPORL-EHLR. Furthermore, adding SPORL-EHLR in SCB hydrolysis saved 40% cellulase. This study deepens the understanding of the adsorption behavior between lignin and cellulase.

Steam pre-treatment of sugarcane bagasse and wheat straw as a cleaner feedstock for black soldier fly larvae rearing

Abstract With the pressing need for alternative waste management strategies that are friendly to the environment, black soldier fly larvae (BSFL) are being cultivated as exceptional insects for the bioconversion of organic waste into larval biomass rich in protein and fat content. Agricultural residues, such as wheat straw and sugarcane bagasse are recognised as important renewable biomass sources, with potential to replace insufficient amount of suitable organic wastes available for BSFL feeding. This study evaluated the steam pre-treatment from 140 to 215 °C and enzymatic hydrolysis of lignocelluloses as BSFL feed supplement prior to blending in an equal ratio (50:50) with the standard feed for BSFL rearing. Key findings illustrated that steam pre-treatment and enzymatic hydrolysis are vital in liberating the sugar monomers of the lignocellulose biomass for BSFL utilisation, with an optimum steam pre-treatment temperature of 185 °C. With a lignocellulosic feed prepared at this temperature plus supplemented standard feed, a BSFL dry-weight bioconversion of 16% was achieved in 8 to 11 days of rearing. The inhibitory by-products formed by degradation of lignocelluloses during pre-treatment had a significantly negative effect on the rearing of the BSFL, particularly a furan concentration of 0.2 g/L, which should be investigated for efficient BSFL rearing. Finally, the crude protein content in the BSFL reared on the standard feed was higher than the feed substrate supplemented with lignocellulose. This study demonstrated the need for process optimisation and component supplements in the BSFL feed substrates that contain lignocellulosic feedstocks.

Boosting enzymatic hydrolysis of steam-pretreated softwood by laccase and endo-β-mannanase enzymes from Streptomyces ipomoeae CECT 3341

Ligninases and hemicellulases are crucial as accessory enzymes to increase the enzymatic hydrolysis of lignocellulose, boosting sugars production from which biofuels and bioproducts could be obtained. In order to find new sources of these accessory enzymes, this study evaluates the potential of laccase and mannanase enzymes from Streptomyces ipomoeae for improving the conventional hydrolysis with commercial cellulases of steam-pretreated softwood. For that, different laccase treatment and mannanase supplementation strategies were performed. S. ipomoeae laccase increased both glucose and xylose production (17.8% and 9.3%, respectively), which was attributed to a removal of phenols of 29%. Moreover, the combination of laccase and alkaline extraction produced a lignin reduction of 16.2%, improving the glucose and xylose production by almost 41.3% and 44.9%, respectively. On the other hand, the supplementation of S. ipomoeae mannanase to the hydrolysis 24 h before the addition of cellulases increased the glucose (18.4%), xylose (12.3%), and mannose (47.2%) production.

Binary additives for in-situ mitigating the inhibitory effect of lignin-derived phenolics on enzymatic hydrolysis of lignocellulose: Enhanced performance and synergistic mechanism

Separation-free sugar-platform biorefinery from lignocellulose has drawn increasing attention due to its feasibility. However, the presence of lignin-derived phenolics within the whole slurry after pretreatment, is highly inhibitory toward enzymatic hydrolysis of lignocellulose especially at high solid loadings. Developing effective methods to mitigate the phenolics-caused enzyme inhibition in-situ can further improve the cellulose hydrolysis, while the relevant studies are limited. In this study, the effects of different kinds of additives on mitigating the phenolic-caused inhibition was determined and the synergy among the additives were systematically evaluated. Based on the analysis, we developed novel binary additives to alleviate the phenolic-caused inhibition of cellulose hydrolysis in-situ. In addition to surfactants, a reducing agent (l-cysteine) was introduced to enhance the enzymatic hydrolysis of cellulose and the strong synergism between the reducing agent and the surfactant was firstly demonstrated. Further, the mechanism study indicated that these additives could prevent the strong interactions between phenolics and cellulase and improve the major cellulase activities. Finally, with the addition of the binary additives, the cellulose hydrolysis for acid/alkaline pretreated corn stover were significantly improved with low enzyme loading without detoxification. This study provides directions for designing novel additives to improve the enzymatic hydrolysis of pretreated lignocellulosic biomass.

Unveiling the role of long-range and short-range forces in the non-productive adsorption between lignin and cellulases at different temperatures

Quantitatively understanding of interaction mechanism between lignin and cellulases is essential for the efficient improvement of lignocellulose enzymatic hydrolysis. However, the individual contribution of multiple forces between lignin and cellulases to the non-productive adsorption of enzymes still remains deeply ambiguous, especially in situations of near enzymatic hydrolysis temperatures. Herein, atomic force microscopy (AFM) and computational simulations were utilized to quantitatively analyze the intermolecular forces between lignin and enzyme at 25 °C and 40 °C. Our results unveiled that an increase in temperature obviously improved adsorption capacity and total intermolecular forces between lignin and cellulases. This positive relationship mainly comes from the increase in the decay length of hydrophobic forces for lignin-cellulases when temperature increases. Different from the hydrophobic interaction which provides long-range part of attractions, van der Waals forces dominate the intermolecular force only at approaches < 2 nm. On the other hand, electrostatic forces exhibited repulsive effects, and its intensity and distance were limited due to the low surface potential of cellulases. Short-range forces including hydrogen bonding (main) and π-π stacking (minor) stabilize the non-specific binding of enzymes to lignin, but increasing temperature reduces hydrogen bond number. Therefore, the relative contribution of long-range forces increased markedly at higher temperatures, which benefits protein capture and brings lignin and cellulase close together. Finally, the structure–activity relationships between lignin physicochemical properties and its inhibitory effect to enzymes indicated that hydrophobic interactions, hydrogen bonding, and steric effects drive the final adsorption capacity and glucose yields. This work provides quantitative and basic insights into the mechanism of lignin-cellulase interfacial interactions and guides design of saccharification enhancement approaches.

Enhancing cellulosic digestibility of wheat straw by adding sodium lignosulfonate and sodium hydroxide to hydrothermal pretreatment

Surfactant-assisted pretreatment has been widely reported to improve the enzymatic hydrolysis of lignocellulose by promoting removal of xylan and lignin. Hence, this work innovatively proposed the use of sodium lignosulfonate (SL) as an additive of alkaline pretreatment (AP), and evaluated its influence on the cellulosic digestibility of wheat straw (WS). The results displayed that the maximum of 72-h cellulosic digestibility could reach 83.5% as 15 g/L SL was introduced to the AP process (SAP), while the cellulosic digestibility of hydrothermal and alkaline pretreated WS was only 63.6% and 70.2%, respectively. These increments were subsequently attributed to the improvement of 6.5% xylan and 26.8% lignin accelerated by SAP, resulting in positive changes in structural characteristics such as accessibility, specific surface area, and cellulosic crystalline structure. The utilization of lignin-based surfactants in pretreatment has realized the economic feasibility of lignocellulosic biorefining and broadened the application prospect of surfactants.

The Role of Lignin Structure on Cellulase Adsorption and Enzymatic Hydrolysis

Lignin is one of the important components of lignocellulosic cell walls, which endows plant cell walls with rigidity and strength and protects them from microbial invasion. The presence of lignin is thought to hinder the conversion of biomass to bioenergy, so understanding enzyme-lignin interactions is very important in order to reduce the inhibition of lignin and improve the hydrolysis yields. Conversion of lignocellulosic raw materials into bioethanol is divided into pretreatment, enzymatic hydrolysis, and fermentation. In this paper, both pretreatment and enzymatic hydrolysis of lignocellulose are described in detail. Finally, the reasons why lignin hinders enzymatic hydrolysis efficiency, mainly from forming spatial barriers and interacting with cellulase, are discussed, and the influencing factors and mechanisms of action of cellulase hydrolysis are explored with a view to targeted regulation of lignin structure to improve lignocellulosic saccharification.

Lignin-grafted quaternary ammonium phosphate with temperature and pH responsive behavior for improved enzymatic hydrolysis and cellulase recovery

The cost of lignocellulosic enzymatic hydrolysis was reduced by enhancing enzymatic hydrolysis and recycling cellulase. Lignin-grafted quaternary ammonium phosphate (LQAP) with sensitive temperature and pH response, was obtained by grafting quaternary ammonium phosphate (QAP) onto enzymatic hydrolysis lignin (EHL). LQAP dissolved under the hydrolysis condition (pH 5.0, 50 °C) and enhanced the hydrolysis. After hydrolysis, LQAP and cellulase co-precipitated by the hydrophobic binding and electrostatic attraction, when lowering pH to 3.2, and cooling to 25 °C. LQAP had significant performances of pH-UCST response, enzymatic hydrolysis enhancement and cellulase recovery at the same time. When 3.0 g/L LQAP-100 was added to the system of corncob residue, SED@48 h increased from 62.6 % to 84.4 %, and 50 % of amount of cellulase was saved. Precipitation of LQAP at low temperature was mainly attributed to the salt formation of positive and negative ions in QAP; LQAP enhanced the hydrolysis for its ability to decrease the ineffective adsorption of cellulase by forming a hydration film on lignin and through the electrostatic repulsion. In this work, a lignin amphoteric surfactant with temperature response, was used to enhance hydrolysis and recover cellulase. This work will provide a new idea for reducing the cost of lignocellulose-based sugar platform technology, and high-value utilization of industrial lignin.

Synthesis of Recyclable UCST-Type Copolymer PPSP and Its Application in Enhancing Lignocellulosic Enzymatic Hydrolysis and Recycling Cellulase

The cost of lignocellulosic enzymatic hydrolysis can be reduced by adding additives with upper critical solution temperature (UCST) response. A recyclable UCST-type copolymer PPSP was synthesized, for enhancing lignocellulosic enzymatic hydrolysis and the cellulase recovery effect of substrate reabsorption. For the hydrolysis system of corncob residue without adding PPSP, only 50% of cellulase amount was saved at a room temperature of 25 °C by adding the fresh corncob residue. The substrate enzymatic digestion efficiency for 72 h was increased by 1.3 times with only adding 0.5 g/L PPSP10-3. After the hydrolysis, 70% of cellulase amount was saved at 25 °C by using the above method. Both lignocellulosic hydrolysis and recovery of cellulase performances were improved by adding PPSP. This may be attributed to the fact that PPSP preferentially adsorbed on lignin to form a hydration membrane to reduce the no-productive adsorption of cellulase and enhanced lignocellulosic enzymatic hydrolysis, while increasing the concentration of free cellulase in the system and strengthening recovery of cellulase. This work will provide one way to reduce the cost of lignocellulosic enzymatic hydrolysis.

Tailoring the expression of Xyr1 leads to efficient production of lignocellulolytic enzymes in Trichoderma reesei for improved saccharification of corncob residues

BackgroundThe filamentous fungus Trichoderma reesei is extensively used for the industrial-scale cellulase production. It has been well known that the transcription factor Xyr1 plays an important role in the regulatory network controlling cellulase gene expression. However, the role of Xyr1 in the regulation of cellulase expression has not been comprehensively elucidated, which hinders further improvement of lignocellulolytic enzyme production.ResultsHere, the expression dosage of xyr1 was tailored in T. reesei by differentially overexpressing the xyr1 gene under the control of three strong promoters (Pegl2, Pcbh1, and Pcdna1), and the transcript abundance of xyr1 was elevated 5.8-, 12.6-, and 47.2-fold, respectively. We found expression of cellulase genes was significantly increased in the Pegl2-driven xyr1 overexpression strain QE2X, whereas relatively low in the Pcbh1- and Pcdna1-driven overexpression strains. We also found that the Pegl2-driven overexpression of xyr1 caused a more significant opening of chromatin in the core promoter region of the prominent cellulase genes. Furthermore, the cellulase activity showed a 3.2-fold increase in the strain QE2X, while insignificant improvement in the Pcbh1- and Pcdna1-driven strains. Finally, the saccharification efficiency toward acid-pretreated corncob residues containing high-content lignin by the crude enzyme from QE2X was increased by 57.2% compared to that from the parental strain. Moreover, LC–MS/MS and RT-qPCR analysis revealed that expression of accessory proteins (Cip1, Cip2, Swo1, and LPMOs) was greatly improved in QE2X, which partly explained the promoting effect of the Pegl2-driven overexpression on enzymatic hydrolysis of lignocellulose biomass.ConclusionsOur results underpin that the precise tailoring expression of xyr1 is essential for highly efficient cellulase synthesis, which provide new insights into the role of Xyr1 in regulating cellulase expression in T. reesei. Moreover, these results also provides a prospective strategy for strain improvement to enhance the lignocellulolytic enzyme production for use in biorefinery applications.

Synergistic Enhancement Effect of Compound Additive of Organic Alcohols and Biosurfactant on Enzymatic Hydrolysis of Lignocellulose

The insufficient of lignocellulose degradation enzymes, such as cellulase and hemicellulase, is the major obstacle that hinders the bioconversion of lignocellulosic biomass to monosaccharides, especially during the woody biomass hydrolysis process. The addition of additives has received significant attention due to their enhancement of the enzymatic degradation efficiency of lignocellulose. In the present study, a combination of organic alcohols and a biosurfactant could synergistically enhance the saccharification of the cellulose substrate of Avicel, as well as that of pretreated poplar. Results showed that compound additives can greatly improve the conversion rate of enzymatic hydrolysis. The combination of 0.1% (v/v) n-decanol and 1% (v/v) sophorolipid dramatically increased the poplar enzymatic conversion rate from 17.9% to 85%, improving it by 67.1%. Enzyme-rich Hypocrea sp. W63 was fermented to obtain beta-glucosidase (BGL) and xylanase (XYL), which were used as auxiliary enzymes during enzymatic hydrolysis. It was found that the effects of such a combination of additives improved the filter paper activity, stability, and longevity, helping in the recovery of the cellulase cocktail. The compound additives associated with the commercial cellulase and Hypocrea sp. W63 enzyme solution formed an excellent formula for improving the stability of BGL and XYL. The results provide insight into compound additives and the use of a cellulase and auxiliary enzyme cocktail to improve enzymatic hydrolysis for lignocellulose conversion into biofuels.