Enzymatic Hydrolysis Efficiency Research Articles

Blocking miR396 activity by overexpression MIM396 improved switchgrass tiller number and biomass yield.

MicroRNA396 (miR396) plays an important role in the regulation of plant growth and development by repressing the expression level of its target growth-regulating factor (GRF) family genes. In our previous study, we found that overexpression of miR396 negatively regulated both tillering and biomass yield in switchgrass (Panicum virgatum L.). We, therefore, speculated that blocking the expression of miR396 could enhance switchgrass tillering and biomass yield. Here, we produced transgenic switchgrass plants overexpressing a target mimicry form of miR396 (MIM396) in wild type (WT) and Os-MIR319b overexpressing switchgrass plant (with higher enzymatic hydrolysis efficiency, but reduced tillering), in which the expression of miR396 was blocked. The phenotype and biological yields of these plants were analyzed. Blocking miR396 to improve its target PvGRFs expression in switchgrass improved the tiller number and dry weight of transgenic plants. Further morphological analysis revealed that MIM396 plants increased the number of aerial branches and basal tillers compared to those of wild-type plants. The enzymatic efficiency of MIM396 plants was reduced; however, the total sugar production per plant was still significantly higher than that of wild-type plants due to the increase in biomass. In addition, blocking miR396 in a transgenic switchgrass plant overexpressing Os-MIR319b (TG21-Ms) significantly increased the PvGRF1/3/5 expression level and tiller number and biomass yield. The miR156-target gene PvSPL4, playing a negative role in aerial and basal buds outgrowth, showed significant downregulated in MIM396 and TG21-Ms. Those results indicate that miR396-PvGRFs, through disrupting the PvSPL4 expression, are involved in miR319-PvPCFs in regulating tiller number, at least partly. MIM396 could be used as a molecular tool to improving tiller number and biomass yield in switchgrass wild type and miR319b transgenic plants. This finding may be applied to other graminaceous plants to regulate plant biological yield.

A novel additive promoting lignocellulose alkali pretreatment for its “two birds with one stone” role

Alkali pretreatment is one of the most commonly used pretreatment methods in lignocellulose bioprocessing. However, there are still some “pseudo-lignin” that hinder enzymatic hydrolysis and seriously affect the industrialization process of biorefining.Excellent pretreatment additives can effectively cover these shortages. However, most additives have disadvantages like high operating temperature, low effective utilization efficiency, or poor performance. To address this issue, we proposed a novel additive, dicyandiamide (DICY), by means of quantum chemical prediction, and verified its ability on assisting alkali to remove lignin by experiments. The results indicate that the lignin removal by KOH-DICY pretreatment is about 15% higher than KOH pretreatment, thereby increasing the enzymatic efficiency to 97%, while the reaction temperature was lower 20℃ than common alkali pretreatment. These are due to the “two birds with one stone” role of DICY on lignocellulose alkali-DICY pretreatment. DICY not only dissolves small amounts of lignin, but also assists alkali to remove lignin by reducing the diffusion resistance of KOH in lignin and blocking the generation of “pseudo-lignin”. Thus, DICY effectively improved the lignin removal and enzymatic hydrolysis efficiency of alkali pretreated lignocellulose, and performed better than urea. Moreover, the waste liquid pretreated by DICY combined with KOH has the potential on preparation of agricultural fertilizers. In summary, DICY is a new, environmentally friendly, and energy conservation additive for lignocellulose alkali pretreatment.

Selective Removal of Hemicellulose by Diluted Sulfuric Acid Assisted by Aluminum Sulfate.

Hemicellulose can be selectively removed by acid pretreatment. In this study, selective removal of hemicellulose was achieved using dilute sulfuric acid assisted by aluminum sulfate pretreatment. The optimal pretreatment conditions were 160 °C, 1.5 wt% aluminum sulfate, 0.7 wt% dilute sulfuric acid, and 40 min. A component analysis showed that the removal rate of hemicellulose and lignin reached 98.05% and 9.01%, respectively, which indicated that hemicellulose was removed with high selectivity by dilute sulfuric acid assisted by aluminum sulfate pretreatment. Structural characterizations (SEM, FTIR, BET, TGA, and XRD) showed that pretreatment changed the roughness, crystallinity, pore size, and functional groups of corn straw, which was beneficial to improve the efficiency of enzymatic hydrolysis. This study provides a new approach for the high-selectivity separation of hemicellulose, thereby offering novel insights for its subsequent high-value utilization.

Online prediction of the enzymatic hydrolysis efficiency of crop straw

The extent of removal of lignin and hemicellulose are crucial indicators for evaluating the efficiency of enzymatic hydrolysis of crop straw. Numerous factors influence these two indices. Establishing a quantitative model that correlates these factors with hydrolysis efficiency is essential, as it can guide efficient hydrolysis. In this study, a predictive method for enzymatic hydrolysis efficiency in crop straw was proposed using Grey relational analysis (GRA), Kernel principal component analysis (KPCA), and a least squares support vector machine (LSSVM). The authors collected a dataset from actual production data and developed an efficiency predictive model using GRA for variable selection, KPCA for dimensionality reduction, and LSSVM for model training. This model allows for the direct estimation of the final enzymatic hydrolysis efficiency based on production condition variables, which can include enzyme amount, temperatures, pH, time, agitation, and straw dimensions. Extensive experimental testing validated the effectiveness of the proposed method, resulting in minimal errors, a high degree of fit, and exceptional performance. The methodology described in this study can serve as a foundation for optimising the design of efficient enzymatic hydrolysis production processes for crop straw. Additionally, it offers valuable soft measurements to support efficient control of the enzymatic hydrolysis process.

Lignin's complex role in lignocellulosic biomass Recalcitrance: A case study on bamboo

Lignin is widely recognized as a key factor influencing the recalcitrance of lignocellulosic biomass, yet the specifics of this relationship remain complex and not fully understood due to the complex interplay between lignin content variation and cell wall structure. Bamboo, which exhibits greater recalcitrance compared to wood in biorefinery processing, was chosen as the focus of investigation in this study. Three variants of Dendrocalamus farinosus with high, middle and low lignin contents were systematically analyzed and compared, in terms of molecular structure of lignin, hemicelluloses and cellulose, cell wall polymer spatial orientation, lignin-carbohydrate complex (LCC) linkages, cell wall geometrical size, as well as deconstruction efficiency, including delignification, alkaline extraction, and cellulose enzymatic hydrolysis efficiency. Findings reveal that an increase in lignin content generally lowers bamboo's deconstruction efficiency, but the mechanisms are more nuanced than previously thought. While lignin content variations do not significantly affect the molecular structure of xylan, cellulose and the chemical linkage type of LCC linkages, they do impact cellulose crystallinity, xylan orientation and notably, the thickness of fiber cell wall in the free fiber stands of bamboo. These findings highlight that lignin's role extends beyond mere physical protection of the cell wall, but also induces subtle variations in polymer structure and cell wall morphologies, which collectively influence the deconstruction efficiency of bamboo cell walls.

Development of an LC-MS/MS method for the determination of five psychoactive drugs in postmortem urine by optimization of enzymatic hydrolysis of glucuronide conjugates.

Toxicological analyses of biological samples play important roles in forensic and clinical investigations. Ingested drugs are excreted in urine as conjugates with endogenous substances such as glucuronic acid; hydrolyzing these conjugates improves the determination of target drugs by liquid chromatography-tandem mass spectrometry (LC-MS/MS). In this study, we sought to improve the enzymatic hydrolysis of glucuronide conjugates of five psychoactive drugs (11-nor-9-carboxy-Δ9-tetrahydrocannabinol, oxazepam, lorazepam, temazepam, and amitriptyline). The efficiency of enzymatic hydrolysis of glucuronide conjugates in urine was optimized by varying temperature, enzyme volume, and reaction time. The hydrolysis was performed directly on extraction columns. This analysis method using LC-MS/MS was applied to forensic autopsy samples after thorough validation. We found that the recombinant β-glucuronidase B-One® quantitatively hydrolyzed these conjugates within 3min at room temperature directly on extraction columns. This on-column method saved time and eliminated the loss of valuable samples during transfer to the extraction column. LC-MS/MS-based calibration curves processed with this method showed good linearity, with r2 values exceeding 0.998. The intra- and inter-day accuracies and precisions of the method were 93.0-109.7% and 0.8-8.8%, respectively. The recovery efficiencies were in the range of 56.1-104.5%. Matrix effects were between 78.9 and 126.9%. We have established an LC-MS/MS method for five psychoactive drugs in urine after enzymatic hydrolysis of glucuronide conjugates directly on extraction columns. The method was successfully applied to forensic autopsy samples. The established method will have broad applications, including forensic and clinical toxicological investigations.

A tailored deep eutectic solvent for high-yield conversion of poplar residues to bio-based building blocks at mild conditions

The generation of condensed compounds in a deep eutectic solvent (DES) pretreatment has become a paramount factor that inhibits the further conversion of pretreated cellulose-rich substrate. This study proposed a novel three-constituent DES system for the value-added utilization of poplar residues by efficiently suppressing hemicellulose derived condensation reactions. The results showed that adding ethanol into the DES system could effectively deconstruct the recalcitrant structure of poplar residues, simultaneously hindering the condensed composition formation. Under optimum ethanol addition (30%), the maximum xylan and lignin removal of 87.94% and 90.99% could be achieved, with more than 90.47% of glucan being preserved. With this, the glucan enzymatic hydrolysis efficiency was remarkably improved to 100% under the pretreatment conditions of 100 °C for 60 min, which was 84.69 times higher than the regular DES-pretreated poplar residues (1.17%). The working mechanism of relieving condensation reactions was characterized by Py-GC–MS, NMR, SEM, FTIR, and others. The results demonstrated that the addition of ethanol could regulate the degradation/conversion pathway of hemicellulose and lignin precipitation, thus inhibit the formation of condensed compounds during the pretreatment.

Unraveling interplay between lignocellulosic structures caused by chemical pretreatments in enhancing enzymatic hydrolysis

To investigate the interplay between substrate structure and enzymatic hydrolysis (EH) efficiency, poplar was pretreated with acidic sodium-chlorite (ASC), 3 % sodium-hydroxide (3-SH), and 3 % sulfuric acid (3-SA), resulting in different glucose yields of 94.10 %, 74.35 %, and 24.51 %, respectively, of pretreated residues. Residues were fractionated into cellulose, lignin and unhydrolyzed residue after EH (for lignin-carbohydrate complex (LCC) analysis) and analyzed using HPLC, FTIR, XPS, CP MAS 13C NMR and 2D-NMR (Lignin and LCC analysis). After delignification, holocellulose exhibited a dramatic increase in glucose yield (74.35 % to 90.82 % for 3-SH and 24.51 % to 80.0 % for 3-SA). Structural analysis of holocellulose suggested the synergistic interplay among cellulose allomorphs to limit glucose yield. Residual lignin analysis from un/pretreated residues indicated that higher β-β' contents and S/G ratios were favorable to the inhibitory effect but unfavourable to the holocellulose digestibility and followed the trend in the following order: 3-SA (L3) > 3-SH (L2) > native-lignin (L1). Analysis of enzymatically unhydrolyzed pretreated residues revealed the presence of benzyl ether (BE1,2) LCC and phenyl glycoside (PG) bond linking to xylose (X) and mannose (M), which yielded a xylan-lignin-glucomannan network. The stability, steric hindrance and hydrophobicity of this network may play a central role in defining poplar recalcitrance.

Low-temperature and low-concentration sodium hydroxide pretreatment for enhanced enzyme hydrolysis rate from Quercus variabilis Blume

A surface response design was employed to develop a sodium hydroxide (NaOH) pretreatment method for Quercus variabilis Blume using low NaOH concentration at low temperature. Nevertheless, the persistent issues associated with alkaline pretreatment of lignocellulose, namely high-water consumption and wastewater generation, remain prevalent in this pretreatment process. To address these challenges, this study aimed to conduct enzymatic hydrolysis of NaOH-treated Q. variabilis Blume without the intermediary washing steps. The results revealed that, following pretreatment and solid-liquid separation, NaOH-treated Q. variabilis Blume could be directly subjected to cellulase-mediated hydrolysis with pH adjustment, eliminating the need for washing steps. The maximum enzymatic hydrolysis efficiency reached 95.9% under specific conditions (1.2% NaOH, 8.9 °C, 32.1 h). This approach offers a promising avenue to enhance the enzyme hydrolysis rate of NaOH-treated lignocellulose. Notably, the low-temperature and low-concentration NaOH treatment effectively removed a substantial portion of lignin and hemicelluloses, resulting in a higher crystallinity index of the cellulose-rich residue compared to substrates treated solely with steam explosion. The integration of direct pretreatment and alkaline treatment emerges as an environmentally friendly and economically viable method for producing glucose and high-purity lignin. The obtained lignin can be further transformed into high-value products within the biorefinery framework.

A reaction–diffusion coupled kinetic model for hydrothermal acid pretreatment of wheat straw

Hydrothermal acid pretreatment is a promising method to overcome the recalcitrance of lignocellulosic biomass for subsequent efficient enzymatic hydrolysis and biofuel production. However, little is known about heterogeneous catalysis reactions involving the proton diffusion in the pore of lignocellulosic biomass coupled with the hydrolysis of hemicellulose and cellulose catalyzed by proton. In this study, a reaction–diffusion coupled kinetic model for hydrothermal acid pretreatment of wheat straw was developed based on the visualization of particle geometry and pore features. The increase rate of average proton concentration in the pores is higher at a higher temperature or a smaller particle size. A mathematical model was successfully developed to describe the time courses of average proton concentration dictated by initial acid concentration and an unsteady diffusion fitting parameter. The kinetic parameters of each reaction step for hydrothermal acid pretreatment of wheat straw were obtained by fitting experimental data. The activation energies for the hydrolysis of slow-hydrolyzing cellulose, fast-hydrolyzing cellulose, and hemicellulose were determined of 937.8 kJ·mol−1, 90.7 kJ·mol−1, and 39.8 kJ·mol−1, respectively, indicating the temperature dependence of slow-hydrolyzing cellulose with a high crystalline index is strongest. The prediction accuracy of this model can be further improved at higher stirring speeds and initial acid concentrations. Additionally, the model demonstrates high prediction accuracy across three different particle sizes. Generally, this model can gratifyingly correlate data of formation and degradation of monosaccharides and furfural in the hydrothermal acid pretreatment of wheat straw with fair accuracy.

Numerical simulation of enzymatic hydrolysis in a bionic intestinal segmentation reactor

Enzymatic hydrolysis plays a critical role in the microbial conversion of lignocellulosic biomass, which has been widely performed in continuous stirred-tank reactors (CSTRs). However, low hydrolysis efficiency in CSTRs hinders the industrial application of lignocellulose to produce biofuels. A bionic intestinal segmentation reactor (BISR) was proposed for efficient enzymatic hydrolysis. In BISR, the dynamic reactor's wall with enzyme secretion led to a 2.0-fold improvement in the conversion of cellobiose as a model substrate compared to the control with a static one. Meanwhile, the reaction rate reached 0.89 mmol/l/s, which was increased by 6.7-fold. Flow fields further illustrated that secondary flow during the segmentation facilitated radial mass transfer between cellobiose and enzyme. Dimensionless secondary flow intensity Sem increased from 0.93 to 2.35 with amplitude, while decreasing from 2.86 to 2.26 with period. More importantly, the analysis of dimensionless Péclet number (Pe) and Damköhler number (Da) indicated that secondary flow could significantly enhance the hydrolysis process controlled by mass transfer.

The promoting effect of electron beam irradiation on enzymatic saccharification and alcohol fermentation of sorghum meal: Related mechanisms

In this study, sorghum meal underwent 2–10 kGy electron beam irradiation (EBI) to investigate alterations in its elemental composition, relevant physicochemical characteristics, and enzymatic hydrolysis fermentation properties. The results indicated a decrease in amylose content and an increase in reducing sugar content in sorghum meal after EBI, along with severe damage to the internal structure of starch granules, leading to increased solubility in water. Furthermore, irradiation enhanced the shear resistance and gelatinization stability of sorghum meal paste. Most importantly, EBI-induced degradation of sorghum starch molecules enhanced their susceptibility to interact with α-amylase, significantly improving the enzymatic hydrolysis and alcohol fermentation efficiency of sorghum meal. At an irradiation dose of 8 kGy, raw and cooked sorghum meal exhibited a 43.40% and 33.33% increase in ethanol concentration and a 43.44% and 30.29% increase in ethanol yield, respectively. This finding indicated that irradiation is an effective pretreatment method for starch-based ethanol fermentation.

An integrated pretreatment strategy for enhancing enzymatic hydrolysis efficiency of poplar: Hydrothermal treatment followed by a twin-screw extrusion

To reduce the dependence on chemical inputs during biomass pretreatment, an integrated process on the basis of hydrothermal pretreatment followed by twin-screw extrusion for poplar was proposed. A comparative study was conducted on the pore structure, crystallinity, relative surface lignin concentration, cellulose accessibility, and enzymatic hydrolysis efficiency of the pretreated materials. Results suggested that the relative surface lignin concentration of fibers pretreated by hydrothermal treatment (conducted at 170 ℃ for 90 min) coupled with extrusion (HE) treatment was decreased compared to the sole hydrothermal (H) process, which had a positive impact on the subsequent enzymatic hydrolysis. Additionally, the HE process resulted in an increase in pore size by 34.4%, pore volume by 447.5%, and specific surface area by 93.5%, which enhanced the penetration and interaction of water into the fibers and thus increased cellulose accessibility by 143.2%. SEM and TEM observations revealed that fibers were separated and finely fibrillated, large amounts of fragmentary structure and cracks appeared on the fiber. In addition, the cell wall structure was deformed and damaged, when pretreated by HE process. Overall, this work showed that hydrothermal treatment followed by twin-screw extrusion was an achievable strategy for high fermentable sugar production of hardwood.

Enhanced Enzymatic Degradability of Livestock Blood Pretreated with Ultrasonic Technique

Livestock blood, a major organic waste generated by the livestock industry, poses a risk of pollution due to its rapid decomposition. However, it is a potential protein source for agricultural purposes due to its protein-rich organic matter. In this study, we conducted studies on an eco-friendly, scalable, and effective protein degradation process using livestock blood to reduce waste and produce an amino acid liquid fertilizer that can be recycled for agricultural use. Ultrasonic technology was applied as a pretreatment method to improve the enzymatic hydrolysis efficiency of proteins in livestock blood, and the optimal conditions that led to 95.91% solubilization rate of hemoglobin were ultrasound duration for 30 min at an ultrasound density of 0.5 W/mL. As a result of hydrolyzing ultrasonically pretreated blood by mixing exo- and endo-type proteolytic enzymes, the optimal combination was a mixture of Savinase® 1% and Flavourzyme® 1%. After 4 h of reaction, the protein concentration was 27.8 mg/mL and the amino acid concentration was confirmed to be 54.6 mg/mL. This is about 4.2 times higher than the amino acid concentration of blood without ultrasound pretreatment, 13.1 mg/mL, and it was confirmed that sonication has a significant effect on improving protein degradation efficiency. As protein degradation increased, the viscosity of blood gradually decreased, suggesting that the physical force applied to the agitator torque diminished during the enzyme reaction; a significant correlation between protein and amino acid concentrations (biological factors) and torque (mechanical factor) was observed. Measuring torque during an enzyme reaction can confirm the extent of the enzyme reaction, so it can be used as an indicator of reaction progress when scaling up the process in the future.

Hydrodynamic studies of innovative membrane reactor for enzymatic hydrolysis of lignocellulosic waste.

This paper presents the study concerning the impact of the basic operational parameters on the performance of an innovative microfiltration membrane reactor applied for enzymatic hydrolysis of lignocellulosic biomass. The concept and basic hydrodynamics of the reactor with tubular ceramic membranes and a propeller agitator were shown. Besides, the efficiency of enzymatic hydrolysis of corn straw was studied to check reactor functionality. It has been proven that the proposed reactor construction can improve the microfiltration of lignocellulosic suspension by reducing the cake layer on the membrane surface. Increasing the rotational speed of the propeller agitator also improved the filtration efficiency. The permeate flux during the microfiltration experiments was lower for smaller lignocellulose biomass fraction (D<425 μm) when compared to the less fragmented corn straw (425<D<900 μm). For larger solid fractions, a stirring speed increase enhanced the separation efficiency regardless of the differences in biomass concentration. In contrast, this trend for the finer biomass fraction was only noticeable for the highest used biomass concentration (C=2.0%). Considering the enzymatic hydrolysis of corn straw, membrane separation of reaction products positively influenced the process yield, and the results depended on the applied operational parameters.

The pretreatment and enzymatic hydrolysis behaviors of lignocellulosic biomass (oak and larch) based on structural changes in its lignin-carbohydrate complex

This study investigated the structural changes in oak and larch lignin-carbohydrate complexes (LCCs) during hydrothermal treatment and enzymatic hydrolysis. Hydrothermal treatment caused hemicellulose degradation (degradation rate at 170 ℃: larch 19.40%; oak, 22.26%). The LCC1 (glucan-lignin) fraction was the highest in the raw material of both biomasses. The LCC3 (xylan-lignin) and LCC2 (glucomannan-lignin) fractions were high in oak and larch, respectively; these fractions decreased as the hydrothermal treatment temperature increased (owing to hemicellulose degradation). Both biomasses had phenyl glycoside, benzyl ether (BE), and γ-ester (Est) linkages. The BE and Est linkages decreased as the hydrothermal treatment temperature increased; the Est linkages decrease was more significantly in oak than in larch. The efficiency of enzymatic hydrolysis was higher in oak than that in larch and increased as the hydrothermal treatment temperature increased. Furthermore, enzyme adsorption on the LCC1 sensor was higher in oak than in larch. The chemical compositions and LCC linkages of the biomass were changed by the hydrothermal treatment, which affected the efficiency of enzymatic hydrolysis. Moreover, the efficiency of enzymatic hydrolysis was higher for oak than for larch because of the difference hemicellulose and cellulose contents and LCC linkages.

An eco-friendly process for low-degree polymerization xylo-oligosaccharides production from corncob via enzyme adsorption-hydrolysis integrated approach by reconstructing fusion protein

Xylo-oligosaccharides (XOS) have gained significant attention for their extensive health-boosting characteristics. However, the tedious procedures involved in lignocellulose pretreatment and XOS purification, coupled with the low efficiency of enzymatic hydrolysis, constrain the broader commercialization of XOS. In this study, the effects of fusion order and linker composition on enzyme activity and thermostability of fusion proteins consisting of a GH11 xylanase mutant (XYNm) and an expansin (EXLX) were investigated. The results indicated that the reconstructed protein XYNm-R2-EXLX exhibited 2.6-fold activity higher than EXLX-R2-XYNm. Furthermore, the denaturation thermodynamics evaluation demonstrated a significant enhancement in the thermostability of XYNm-R2-EXLX. Additionally, the reconstructed fusion protein showed a notable affinity adsorption efficiency on corncob, reaching a remarkable 95.5%. Subsequently, the fusion enzyme-corncob complex underwent direct hydrolysis, and a yield of 58.7 g XOS/kg corncob was obtained by employing RSM optimization techniques. Finally, a membrane-assisted refining process was implemented, resulting in the recovery of 68.2% (w/w) of the initial XOS (X2-X4) with a purity of 96.2%. Here, a chemical free and eco-friendly bioprocess for production of low-degree polymerization XOS was developed, eliminating the need for xylan separation from corncob and simplifying the process for product purification. These findings exhibited the great potential in XOS (X2-X4) production.

Pretreatment and enzymatic hydrolysis optimization of lignocellulosic biomass for ethanol, xylitol, and phenylacetylcarbinol co-production using Candida magnoliae.

Cellulosic bioethanol production generally has a higher operating cost due to relatively expensive pretreatment strategies and low efficiency of enzymatic hydrolysis. The production of other high-value chemicals such as xylitol and phenylacetylcarbinol (PAC) is, thus, necessary to offset the cost and promote economic viability. The optimal conditions of diluted sulfuric acid pretreatment under boiling water at 95°C and subsequent enzymatic hydrolysis steps for sugarcane bagasse (SCB), rice straw (RS), and corn cob (CC) were optimized using the response surface methodology via a central composite design to simplify the process on the large-scale production. The optimal pretreatment conditions (diluted sulfuric acid concentration (% w/v), treatment time (min)) for SCB (3.36, 113), RS (3.77, 109), and CC (3.89, 112) and the optimal enzymatic hydrolysis conditions (pretreated solid concentration (% w/v), hydrolysis time (h)) for SCB (12.1, 93), RS (10.9, 61), and CC (12.0, 90) were achieved. CC xylose-rich and CC glucose-rich hydrolysates obtained from the respective optimal condition of pretreatment and enzymatic hydrolysis steps were used for xylitol and ethanol production. The statistically significant highest (p ≤ 0.05) xylitol and ethanol yields were 65% ± 1% and 86% ± 2% using Candida magnoliae TISTR 5664. C. magnoliae could statistically significantly degrade (p ≤ 0.05) the inhibitors previously formed during the pretreatment step, including up to 97% w/w hydroxymethylfurfural, 76% w/w furfural, and completely degraded acetic acid during the xylitol production. This study was the first report using the mixed whole cells harvested from xylitol and ethanol production as a biocatalyst in PAC biotransformation under a two-phase emulsion system (vegetable oil/1M phosphate (Pi) buffer). PAC concentration could be improved by 2-fold compared to a single-phase emulsion system using only 1M Pi buffer.

Elevating enzymatic hydrolysis efficiency and ethanol yield of poplar by hydrogen peroxide assisted p-toluenesulfonic acid pretreatment

Utilizing hydrogen peroxide (H2O2) and acid medium for oxidation-reduction reaction to facilitate the efficient biorefining of lignocellulose is the ultimate goal of oxidative pretreatment. Here, the p-toluenesulfonic acid (TsOH) was employed in the H2O2 pretreatment of poplar, which has achieved excellent depolymerizing effect of lignin and xylan. The results displayed that 99.9% xylan and 68.4% lignin were dramatically removed when 2% H2O2 and 40 g/L TsOH were introduced to the pretreatment (HTP) at 160 °C. After enzymatic hydrolysis and simultaneous saccharification and fermentation, the enzymatic hydrolysis efficiency and ethanol yield of poplar were advanced by 59.0% and 59.4%, respectively. The structural characterization indicated that the incremental efficiency was attributed to the advantageous accessibility, surface area, crystallinity, and crystal size of poplar by HTP’s augmentative removal of non-cellulosic components. This work expanded the application of acidic H2O2 pretreatment and suggested that HTP was a synergistic and feasible way in biorefinery.

Double in-situ lignin modification in surfactant-assisted glycerol organosolv pretreatment of sugarcane bagasse towards efficient enzymatic hydrolysis

Organosolv pretreatment effectively fractionates lignocellulosic biomass for efficient enzymatic hydrolysis, yielding fermentable sugars. However, substantial lignin redeposition on the substrate surface and residual lignin content exacerbate lignin’s inhibitory effects on subsequent enzymatic hydrolysis. An in-situ lignin modification is proposed to address this challenge by incorporating polyethylene glycol (PEG) series into glycerol organosolv (GO) pretreatment. According to the results obtained, PEG-assisted GO pretreated substrate exhibited significantly increased sugar yield during enzymatic hydrolysis, particularly with PEG 4000, showing 35.4% higher glucose yield over 72 h. The improved glucose yield with PEG could be attributed to changes in the physicochemical structure and properties of residual lignin, mitigating its non-productive interaction with cellulase enzymes. PEG's presence in GO pretreatment also increased β-O-4 linkages preservation by 55% and reduced phenolic hydroxyl groups by 28% in lignin fragments versus no surfactants. This outcome resulted from introducing glycerol and PEG hydroxyl tails into the lignin structure, forming α-etherified lignin and rendering it more hydrophilic. Simulations confirmed strong Van Der Waals and hydrogen bonding interactions between lignin units and PEG, hindering lignin fragments repolymerization. Incorporating PEG in the organosolv pretreatment proves highly beneficial for cellulase-mediated lignocellulosic biorefinery, resembling a lignin-first strategy. The optimized process enables efficient biomass utilization for sustainable fuels and chemicals production.