Enzymatic Hydrolysis Of Cellulose Research Articles

Specific features of enzymatic hydrolysis of cellulose

In this article, the features of enzymatic hydrolysis of cellulose samples with different structural characteristics were studied. It was discovered that outer paracrystalline layers of crystallites are accessible and susceptible to hydrolysis along with the poorly ordered amorphous domains of cellulose. The thermodynamic analysis showed that the hydrolysis process of cellulose samples at 298 K is exothermic. Since reaction enthalpy is negative and the temperature-entropy factor is positive, the Gibbs potential of this process becomes negative, which contributes to the implementation of enzymatic hydrolysis of cellulose. Moderate enhancement in temperature to optimal value, 323 K, increases the negative value of Gibbs potential and thereby promotes the hydrolysis of cellulose substrates. The most negative Gibbs potential was observed for the hydrolysis of the most accessible amorphous cellulose. Thus, amorphization of cellulose substrates facilitates enzymatic hydrolysis. On the other hand, the enzymatic hydrolysis of high-ordered crystalline domains of cellulose cannot be performed even at optimal temperature since the Gibbs potential of this process is close to zero.

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.

An experimental and modeling approach to describe the deactivation of cellulases at the air-liquid interface.

Understanding the reaction mechanisms involved in the enzymatic hydrolysis of cellulose is important because it is kinetically the most limiting step of the bioethanol production process. The present work focuses on the enzymatic deactivation at the air-liquid interface, which is one of the aspects contributing to this global deactivation. This phenomenon has already been experimentally proven, but this is the first time that a model has been proposed to describe it. Experiments were performed by incubating Celluclast cocktail solutions on an orbital stirring system at different enzyme concentrations and different surface-to-volume ratios. A 5-day follow-up was carried out by measuring the global FPase activity of cellulases for each condition tested. The activity loss was proven to depend on both the air-liquid surface area and the enzyme concentration. Both observations suggest that the loss of activity takes place at the air-liquid surface, the total amount of enzymes varying with volume or enzyme concentration. Furthermore, tests performed using five individual enzymes purified from aTrichoderma reesei cocktail showed that the only cellulase that is deactivated at the air-liquid interface is cellobiohydrolaseII. From the experimental data collected by varying the initial enzyme concentration and the ratio surface to volume, it was possible to develop, for the first time, a model that describes the loss of activity at the air-liquid interface for this configuration.

Enzymatic hydrolysis of cellulose banana stem (alkaline microwave-assisted pre-treatment)

The banana stem waste holds immense promise as a readily available and abundant source of lignocellulosic biomass, making it a compelling alternative for biofuel and biochemical applications. Therefore, this study focuses on investigating the impact of both time and substrate loading on the enzymatic hydrolysis of banana stem cellulose that has undergone alkaline microwave-assisted pre-treatment. The pre-treatment method involves subjecting the biomass to 5% KOH for 30 min, followed by microwave exposure at 300 W for 5 min, a process aimed at enhancing the accessibility of cellulose. Enzymatic hydrolysis experiments were carried out utilizing cellulase enzymes derived from Aspergillus niger, with variations in hydrolysis times (ranging from 5 to 45 h) and enzyme-to-substrate ratios (ranging from 1:1 to 1:10). The results of this investigation revealed a substantial improvement in hydrolysis efficiency, owing to the synergistic effects of alkaline microwave-assisted pre-treatment, signifying enhanced cellulose accessibility. Notably, the highest concentration of reducing sugars (1.3 mg mL−1) was achieved at a substrate-to-enzyme ratio of 1:1 and a hydrolysis duration of 45 h. These findings provide valuable insights into the conversion of lignocellulosic biomass, emphasizing the potential of integrated pre-treatment strategies for sustainable biorefinery applications. This research contributes to advancing our understanding of lignocellulosic biomass utilization, offering a promising avenue for biofuel and biochemical production from banana stem waste.

Transcriptional regulation of cellobiose utilization by PRD-domain containing Sigma54-dependent transcriptional activator (CelR) and catabolite control protein A (CcpA) in Bacillus thuringiensis.

Cellobiose, a β-1,4-linked glucose dimer, is a major cellodextrin resulting from the enzymatic hydrolysis of cellulose. It is a major source of carbon for soil bacteria. In bacteria, the phosphoenolpyruvate (PEP): carbohydrate phosphotransferase system (PTS), encoded by the cel operon, is responsible for the transport and utilization of cellobiose. In this study, we analyzed the transcription and regulation of the cel operon in Bacillus thuringiensis (Bt). The cel operon is composed of five genes forming one transcription unit. β-Galactosidase assays revealed that cel operon transcription is induced by cellobiose, controlled by Sigma54, and positively regulated by CelR. The HTH-AAA+ domain of CelR recognized and specifically bound to three possible binding sites in the celA promoter region. CelR contains two PTS regulation domains (PRD1 and PRD2), which are separated by two PTS-like domains-the mannose transporter enzyme IIA component domain (EIIAMan) and the galactitol transporter enzyme IIB component domain (EIIBGat). Mutations of His-546 on the EIIAMan domain and Cys-682 on the EIIBGat domain resulted in decreased transcription of the cel operon, and mutations of His-839 on PRD2 increased transcription of the cel operon. Glucose repressed the transcription of the cel operon and catabolite control protein A (CcpA) positively regulated this process by binding the cel promoter. In the celABCDE and celR mutants, PTS activities were decreased, and cellobiose utilization was abolished, suggesting that the cel operon is essential for cellobiose utilization. Bt has been widely used as a biological pesticide. The metabolic properties of Bt are critical for fermentation. Nutrient utilization is also essential for the environmental adaptation of Bt. Glucose is the preferred energy source for many bacteria, and the presence of the phosphotransferase system allows bacteria to utilize other sugars in addition to glucose. Cellobiose utilization pathways have been of particular interest owing to their potential for developing alternative energy sources for bacteria. The data presented in this study improve our understanding of the transcription patterns of cel gene clusters. This will further help us to better understand how cellobiose is utilized for bacterial growth.

Integrating ball milling assisted enzymatic hydrolysis of bamboo cellulose for controllable production of xylo-oligosaccharides, monosaccharides and cellulose nanofibrils

Green bioconversion technology has been regarded as a fundamental solution to energy challenges and environmental issues. During bioconversion, a combination of mechanical and enzymatic treatment on lignocellulosic biomass can not only conformity with environmental concepts but also enable efficient conversion. In this study, a ball milling assisted enzymatic treatment was applied for full utilization of bamboo cellulose. In addition to the production of soluble saccharides, the residues that remained after enzymatic hydrolysis were collected for producing high-quality nanocellulose. Ball milling efficiently broke the hydrogen bonding interactions between glucan chains thus promoting the following xylanase and cellulase conversion. The highest Xylo-oligosaccharides (XOS) yield reached 21.6%. Meanwhile, xylanase facilitated cellulase hydrolysis by separation of hemicellulose on cellulose. With a low commercial cellulase loading of 5 FPU/g glucan, a maximum yield of 76.6% glucose and 53.3% xylose was reached. Finally, nanocellulose was extracted from the enzymatic hydrolyzate residue with a minimum width of 3.5 nm and an average size of 294.8 nm. The present study proposed an efficient combination of mechanical treatment assisted enzymatic digestion, which is not only environmentally friendly but also allows for complete bioconversion of bamboo cellulose.

Enzymatic hydrolysis of microcrystalline cellulose for efficient and economic production of astragalin in metabolic engineering of Escherichia coli

Cellulose is the most abundant biomass in nature, and the development of high-value utilization technologies for cellulose is of great significance. In this work, a recombinant strain was engineered by introducing Arabidopsis thaliana glycosyltransferase (AtUGT78D2) and the cellobiose phosphorolysis route to produce astragalin from kaempferol. By optimizing the transformation conditions, the production of astragalin increased from 1053 to 3031 mg/L with the addition of cellobiose. Subsequently, the cellulose enzymatic solution was used to replace cellobiose as the carbon source and UDP-glucose precursor for producing astragalin. By optimizing enzymatic conditions and adding additives, the concentration of cellobiose significantly increased, resulting in an increase in the production of astragalin to 2279 mg/L. The adsorption strategy was employed to modulate the composition and activity of cellulase, and the ratio of cellobiose to glucose in the cellulose enzymatic solution increased from 0.73 to 1.62, with the yield of cellobiose reaching up to 5.9 g/L. Finally, the highest production of astragalin in the recombinant strain reached 2654 mg/L when the optimal ratio of cellobiose to glucose was used as the carbon source. This study provides a novel method for producing astragalin by using the enzymatic hydrolysis of microcrystalline cellulose.

Novel ferrous disulfide loaded palygorskite composites as additives in lignocellulosic waste composting for improving humification

Improving the composting efficiency of lignocellulose wastes has been the focus in the field of aerobic fermentation. In this study, FeS2 was modified on the surface of palygorskite to regulate the straw composting. Material characterizations showed that FeS2/Pal composite exhibited glutathione oxidase and catalase activities, attributing to the fact that nano FeS2 filled the aggregation space of the original palygorskite, exposing more active sulfur vacancy defects. During the composting process, the addition of FeS2/Pal could adjust pH value and increase electrical conductivity, which was conducive to improving the efficiency of biological redox process. The total degradation ratio of cellulose was 52.54% with FeS2/Pal added. The repolymerization of small molecule intermediates was strengthened, and the final polymerization degree and maturity of composting products was improved. Since the sequence abundance of aromatic amino acids biosynthesis and carbon fixation pathways was higher revealed by PICRUSt. High-throughput sequencing results showed that bacterial community was more dominant than fungi, with community richness index increased by 2.3–3.7 times. The pathogenic bacteria such as Atopostipes and Corynebacterium were disappeared, and heat-resistant bacteria were dominant. The key bacteria and fungi in the group added with FeS2/Pal were Limnochordaceae and Melanocarpus genus, which were related to the cellulose enzymatic hydrolysis and humification degree.

Understanding the Influence of Water-Soluble Compounds from Unpretreated Corn Stover Pellets on Enzymatic Hydrolysis of Cellulose

Understanding the Influence of Water-Soluble Compounds from Unpretreated Corn Stover Pellets on Enzymatic Hydrolysis of Cellulose

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.

Controlled Semi-Batch Process to Enhance Glucose Concentration in an Enzymatic Hydrolysis Stirred Tank Reactor

In this work, it is explored a controlled semi-batch operation for a stirred tank reactor in which an enzymatic hydrolysis of cellulose is carried out to enhance the concentration of reducing sugar. A high concentration of reducing sugar impacts fermentation processes in a biorefinery. Cellulose and enzyme feedings are driven by controllers to maintain the highest cellulose concentration allowed by a physical constraint in the process; in consequence, more cellulose is fed than in a typical batch operation and a greater glucose concentration is obtained. The construction of controllers is systematic, and performance is illustrated by simulation.

A waste-free biorefinery pathway to the valorisation of Chinese hickory shell through alkaline hydrogen peroxide pretreatment

Efficiently harnessing the intrinsic value of lignocellulose requires the integration of multifarious capacities within a zero-waste biorefinery. Chinese hickory shell, a food industry byproduct, remains largely untapped. Unleashing their full potential as a valuable biomass resource has become an urgent challenge. This study utilises the alkaline hydrogen peroxide pretreatment for Chinese hickory shell, exploring the effect of H2O2 dosage, alkali concentration, and temperature on lignocellulosic fractionation. Under optimal conditions, nearly complete removal of lignin and hemicelluloses is achieved, with cellulose enzymatic hydrolysis up to 98.1%, surpassing the original material by over 15-fold. Simultaneously, hemicelluloses transformed into high-value organic acids, yielding 33.5%. Notably, fractionated lignin is abundant in aryl ether linkages after AHP, facilitating subsequent transformation into aromatic chemicals. Through AHP, efficient fractionation of Chinese hickory shell is achieved, yielding fermentable glucose, organic acids, and lignin enriched with high aryl ether content. This research emphasises the pivotal role of comprehensive Chinese hickory shell utilisation in elevating the revenue of zero-waste biorefinery, sparking the production of valuable products from biomass waste streams, and further contributing to establishing resource-efficient biorefineries.

Changes in various lignocellulose biomasses structure after microwave-assisted hydrotropic pretreatment

Effective pretreatment of lignocellulosic biomass combined with delignification is a condition for effective enzymatic hydrolysis of cellulose, which is of key significance for the efficiency of the process of production of cellulosic ethanol. Becoming aware of the full range of changes in the structure of lignocellulose as a result of biomass pretreatment is important in view of the necessity to optimise the process of technological production of bioethanol. Our research was aimed at a comprehensive analysis of changes in structure of pine chip (softwood), beech chip (hardwood) and wheat straw (non-wood) biomasses resulting from a newly developed pretreatment method combining the use of hydrotrope in the form of sodium cumene sulfonate and microwave radiation. The performed analyses of the biomass composition indicate a high effectiveness of the proposed pretreatment method in the area of biomass delignification and hemicellulose removal. An effective removal of amorphous substances, i.e. lignin and hemicellulose, led to an increase in the crystallinity of pine chip biomass to 55%, and beech chips and wheat straw biomasses to 61%.

Biobased lignin-blockers enable efficient production of glucose from lignocelluloses

Enzymatic hydrolysis of cellulose in lignocelluloses exhibits low efficiency due to low cellulose accessibility and adverse lignin effects. By using phenylsulfonic acid pretreatment to improve cellulose accessibility, we have developed several new plant proteins such as corn germ and green rapeseed proteins that extracted from inexpensive defatted meals as lignin-blockers. For pretreated lignocellulosic substrates with high cellulose accessibility, such plant proteins could effectively overcome negative lignin effects and achieve robust cellulose enzymatic conversion (88–97 %) at a low cellulase loading of 5 FPU/g glucan. To reach the same level of cellulose enzymatic conversion, adding corn germ protein could save cellulase loading by 3.6 times as compared to those without lignin-blockers. But these plant proteins showed insignificant promotion effects on substrates with low cellulose accessibility. Overall, this study coupled high cellulose-accessible substrates and efficient lignin-blockers to synergistically improve the profitability of lignocellulosic biorefinery.

Novel supramolecular deep eutectic solvent-enabled in-situ lignin protection for full valorization of all components of wheat straw

Lignin is usually deemed as an inhibitor to enzymatic hydrolysis of cellulose due to its physical barrier, non-productive adsorption, and steric hindrance. Herein, a novel supramolecular deep eutectic solvent (SUPRADES), comprising ethylene glycol and citric acid in 5:1 M ratio, and β-cyclodextrin (β-CD) in a concentration of 3.5% (w/w), was developed to be efficient for pretreating wheat straw. The delignification rate, cellulose enzymatic digestibility, and hemicellulose removal reached 90.45%, 97.36% and 87.24%, respectively, which may be attributed to the introduction of β-CD with superior ability of both adsorption and in-situ lignin protection to efficiently remove lignin with intact structure from cellulose surface. The mechanisms of high-efficiency lignin extraction/protection were systematically illustrated by adsorption kinetics. Moreover, Trichosporon cutaneum grown on the hemicellulose and cellulose fractions after pretreatment afforded 8.8 g total lipids from 100 g wheat straw. The green SUPARDES pretreatment strategy offers a new avenue for upgrading lignocellulose to biofuels.

A novel mineral-acid free biphasic deep eutectic solvent/γ-valerolactone system for furfural production and boosting the enzymatic hydrolysis of lignocellulosic biomass

The failure of hemicellulose valorization in a deep eutectic solvent (DES) pretreatment has become a bottleneck that challenges its further development. To address this issue, this study developed a DES/GVL (γ-valerolactone) biphasic system for effective hemicellulose-furfural conversion, enhanced cellulose saccharification and lignin isolation. The results indicated that the biphasic system could significantly improve the lignin removal (as high as 89.1%), 86.0% higher than the monophasic DES, accompanied by ∼100% hemicellulose degradation. Notably, the GVL in the biphasic solvent restricted the condensation of hemicellulose degradation products, which as a result generated large amount of furfural in the pretreatment liquid with a yield of 68.6%. With the removal of hemicellulose and lignin, cellulose enzymatic hydrolysis yield was boosted and reached near 100%. This study highlighted that the novel DES/GVL is capable of fractionating the biomass and benefiting their individual utilization, which could provide a new biorefinery configuration for a DES pretreatment.

An aggregated understanding of the influence of aqueous ammonia pretreatment on the physical deconstruction of cell walls in sugar beet pulp.

The underlying interplay between physicochemical property and enzymatic hydrolysis of cellulose still remains unclear. The impacts of matrix glycan composition of sugar beet pulp (SBP) on physical structure and saccharification efficiency were emphasized. The results showed that aqueous ammonia (AA) pretreatment could remove the non-cellulosic polysaccharides and destroy the linkage between the pectin and lignin. The cellulose supramolecule was changed significantly after AA pretreatment, in terms of the decline in hardness, gumminess, springiness, thickness and degree of polymerization. Furthermore, vascular cell was exposed and degraded. The highest reducing sugar yield of 355.06mg/g was obtained from the pretreated SBP (80°C) with enzyme loading of 30 U/g, which was 1.01 times higher than that of the untreated SBP. This research also supported the idea that recognizing and precisely removing the primary epitopes in cell walls might be an ideal strategy to accomplish the improved enzymatic hydrolysis through mild pretreatment.

Combining heterogeneous photocatalysis and enzymatic catalysis via membrane: Conversion of biomass for H2 production from water

Solar reforming cellulosic biomass into hydrogen is an attractive research topic for sustainable biomass waste utilization and renewable energy development. To exert the advantages of enzymatic catalysis and heterogeneous photocatalysis, a glass fiber membrane integrated process that combines enzymatic hydrolysis of cellulose with sacrificial photocatalytic H2 production from water under mild condition has been proposed as an example of integrating enzymatic catalysis and heterogeneous photocatalysis. Specifically, a low−cost Cu0.5Ni0.5−TiO2 photocatalyst has been developed with presenting remarkable H2 production performance even comparable with Pt−TiO2 under UV light irradiation. The synergistic effect of Cu and Ni co−deposition onto TiO2 has been found to improve the photocatalytic H2 production. The condition for enzymatic hydrolysis of cellulose to generate glucose has been optimized in terms of reaction temperature, solution pH, types of cellulase and inorganic ions in order to obtain higher yields of glucose. To integrate the enzymatic catalysis and photocatalysis together, the glass fiber membrane with superior glucose penetration capability has been screened out. Lastly, the coupling of photocatalytic H2 production from water based on the Cu0.5Ni0.5 −TiO2 photocatalyst and enzymatic hydrolysis of cellulose has been quantitively evaluated in both mixed and membrane−separation systems. The membrane−separation system can avoid the depletion of cellulase activity induced by photocatalytic oxidation, and thus presents higher H2 production efficiencies with apparent quantum efficiency of 3.07 % at 365 ± 10 nm irradiation in initial 5 h. This work demonstrates that inorganic membrane integrated enzymatic catalysis and photocatalysis can be a powerful tool for different potential applications.

Differentiated Fractionation of Various Biomass Resources by p-Toluenesulfonic Acid at Mild Conditions.

Biomass is the ideal substitute for petrochemical resources because of its renewable and abundant sources. p-Toluenesulfonic acid (p-TsOH) can effectively separate lignin from biomass under mild conditions, so it is highly expected in biomass fractionation to improve the utilization efficiency. In this study, we investigated the effect of p-TsOH differentiated fractionation of poplar sawdust, eucalyptus sawdust, and rice straw below 100 °C. According to the experimental results, upon pretreatment by p-TsOH of the three kinds of raw biomass, most of the lignin and hemicellulose of poplar sawdust and eucalyptus sawdust were removed, whereas the cellulose was retained, but most of the hemicellulose and cellulose of rice straw were kept, whereas the lignin was removed at similar conditions. The structures and compositions of pretreatment residues, lignin, and hemicellulose extracted from raw biomass were characterized by XRD, FTIR, HSQC-NMR, XPS, and SEM. The differentiated fractionation mechanism of biomass was analyzed. A better recognition and understanding of the factors affecting biomatrix opening and fractionation will allow for the identification of new pretreatment strategies that improve biomass utilization and permit the rational enzymatic hydrolysis of cellulose.

Integrated pretreatment of poplar biomass employing p-toluenesulfonic acid catalyzed liquid hot water and short-time ball milling for complete conversion to xylooligosaccharides, glucose, and native-like lignin

This work aimed to study an integrated pretreatment technology employing p-toluenesulfonic acid (TsOH)-catalyzed liquid hot water (LHW) and short-time ball milling for the complete conversion of poplar biomass to xylooligosaccharides (XOS), glucose, and native-like lignin. The optimized TsOH-catalyzed LHW pretreatment solubilized 98.5% of hemicellulose at 160 °C for 40 min, releasing 49.8% XOS. Moreover, subsequent ball milling (20 min) maximized the enzymatic hydrolysis of cellulose from 65.8% to 96.5%, owing to the reduced particle sizes and cellulose crystallinity index. The combined pretreatment reduced the crystallinity by 70.9% while enlarging the average pore size and pore volume of the substrate by 29.5% and 52.4%, respectively. The residual lignin after enzymatic hydrolysis was rich in β-O-4 linkages (55.7/100 Ar) with less condensed structures. This lignin exhibited excellent antioxidant activity (RSI of 66.22) and ultraviolet absorbance. Thus, this research suggested a sustainable waste-free biorefinery for the holistic valorization of biomass through two-step biomass fractionation.