Efficient Hydrolysis Of Cellulose Research Articles

A novel GH3-β-glucosidase from soda lake metagenomic libraries with desirable properties for biomass degradation.

Beta-glucosidases catalyze the hydrolysis of the glycosidic bonds of cellobiose, producing glucose, which is a rate-limiting step in cellulose biomass degradation. In industrial processes, β-glucosidases that are tolerant to glucose and stable under harsh industrial reaction conditions are required for efficient cellulose hydrolysis. In this study, we report the molecular cloning, Escherichia coli expression, and functional characterization of a β-glucosidase from the gene, CelGH3_f17, identified from metagenomics libraries of an Ethiopian soda lake. The CelGH3_f17 gene sequence contains a glycoside hydrolase family 3 catalytic domain (GH3). The heterologous expressed and purified enzyme exhibited optimal activity at 50°C and pH 8.5. In addition, supplementation of 1M salt and 300mM glucose enhanced the β-glucosidase activity. Most of the metal ions and organic solvents tested did not affect the β-glucosidase activity. However, Cu2+ and Mn2+ ions, Mercaptoethanol and Triton X-100 reduce the activity of the enzyme. The studied β-glucosidase enzyme has multiple industrially desirable properties including thermostability, andalkaline, salt, and glucose tolerance.

Efficient hydrolysis of cellulose into sugars in aqueous systems using fibrous core-shell structure FS@C/HPW

Phosphotungstic acid hydrate (HPW)-doped hydroxyl-rich hydrothermal carbon-coated fibrous silica catalyst (FS@C/HPW) is fabricated to induce the highly efficient hydrolysis of cellulose to sugar in an ultra-low acid aqueous system. The obtained FS90@C/HPW12 owns good thermal stability under 200 °C and the highest specific surface area at 28 m2/g. Owing to its special fibrous microstructures, FS90@C/HPW12 can cause linear damage and structural fragmentation on the surface of cellulose during hydrolysis. Accordingly, rather highly efficient hydrolysis with the cellulose conversion of 100 %, total reducing sugar and glucose yields at 92.2 % and over 85 % is achieved in the aqueous system with FS90@C/HPW12 and 5 mmol/L H2SO4. FS90@C/HPW12 also exhibits good reusability in multiple hydrolysis. This research provides a particular way for the design and synthesis of solid catalysts to induce the highly efficient hydrolysis of cellulose to sugar by chemical catalysis in a full aqueous system.

Tailoring whole slurry bioprocessing for sugary stovers to augment sugar production by integrating soluble and insoluble carbohydrates

Obtaining sufficient concentrations of fermentable sugars from the polysaccharides of lignocellulosic materials represents a long-term challenge in achieving sustainable biorefineries. This work aimed to enhance sugar titers by employing high sugar-containing stovers (HSS) and combining its intrinsic soluble sugars with the two insoluble carbohydrates, namely cellulose and hemicellulose. Whole-slurry hydrolysis (WSH) was performed under low-to-high solid conditions using non-detoxified pretreated slurry, which exhibited a decrease in cellulose hydrolysis efficiency (CHE) from 89.08% to 42.1% as the solid loadings increased from 7% to 20%. A further WSH with detoxified slurry showed a significant increase in CHE from 42.1% to 59.52% at 20% solid loading, indicating that inhibitors present in the pretreated slurry had negative effects on CHE. Lower CHE with non-detoxified slurry accompanied by higher losses of cellulase and β-glucosidase activities, particularly at higher solid loadings. Therefore, systematic modification of WSH was performed by incorporating 2% syringic acid during pretreatment and 0.4% polyethylene glycol during saccharification, reformulating enzyme cocktails, and implementing a fed-batch experiment. These modifications resulted in a notable improvement in sugar production under high-solid loading (20%) and non-detoxified conditions, reaching 142.87 g/L total sugars with a CHE of 90.7%. These findings have revealed the potential of integrating soluble sugars with holocellulose for obtaining high-titer fermentable sugars in lignocellulosic biorefineries.

Combination of microwave with acid deep eutectic solvent pretreatment for reed (Phragmites australis) fractionation

The cost-effective and low-carbon fractionation of lignocellulosic biomass will enhance the economic viability of bio-refining. To achieve efficient reed fractionation under favorable conditions, microwave-assisted choline chloride and p-toluene sulfonic acid (M-ChCl/p-TsOH) pretreatment was employed. The fractionation of reed components demonstrated higher effectiveness with M-ChCl/p-TsOH pretreatment compared to conventional DES pretreatment. Under M-ChCl/p-TsOH conditions, a high hemicellulose removal rate (∼90%) and delignification rate (∼65%) were achieved while achieving a saccharification rate of 88% during cellulase hydrolysis. Microwave assistance not only significantly reduced reaction time to less than 60 s but also enhanced the pretreatment efficacy. Furthermore, the obtained lignin products consisted of low-polymerization lignin (MW < 800 g/mol) and acid-soluble lignin, providing a solid foundation for subsequent high-value utilization of lignin. This study provides a promising strategy for the low carbon fractionation of reed using DES, aiming to simultaneously achieve efficient cellulose hydrolysis and obtain tractable lignin.

Cloning of AA9 Polysaccharide Monooxygenase gene AN3860 into pEX2B for expression in Aspergillus oryzae

Polysaccharide monooxygenases (PMOs) catalyze oxidative degradation of recalcitrant carbohydrate chains in cellulose, starch, and chitin. In biofuel industry, the conversion of rich lignocellulose source to fermentable sugars by hydrolytic cellulases can be synergistically boosted by cellulose-active PMOs (AA9 PMOs) found in a vast number of fungi that grow on biomass. Aspergillus nidulans, a filamentous fungus, possess a dozen of PMO-encoding genes, but only the AN3860 is expressed at a high level when cultured with wheat straw as the sole carbon source. Bioinformatic analysis indicates that AN3860 belongs to type 3 AA9 PMO subfamily that is capable of hydroxylating both C1 and C4 of the glycosidic linkages. Therefore, AN3860 may be a potential enzyme to improve cellulose hydrolysis efficiency, which has not been characterized. In this study, we describe the AN3860 cloning into Aspergillus oryzae AUT1-PlD. To facilitate the purification of AN3860, we added a CBM20 tag to its C-terminal. The recombinant vector was designed and constructed successfully. Simultaneously, we have obtained the clone of A. oryzae carrying the target gene by the ATMT method. Further expression optimization and characterization of AN3860 by both activity assays and spectroscopic techniques are underway.

Acetic acid as a protic solvent for reducing sulphuric acid concentrations in the production of cellulose nanocrystals alongside transition metal co-catalysts

Cellulose nanocrystals (CNC) conventionally involve highly concentrated sulphuric acid, which typically resulted in the formation of undesirable by-products. Although less corrosive mineral acids have been explored as alternatives, high concentrations are still required. In this study, CNC was successfully isolated from Leucaena leucocephala wood using mild sulphuric acid with acetic acid as protic solvent, and it was further studied with the addition of Lewis acids in the form of multivalent transition metal salts as co-catalyst. Selected divalent and trivalent transition metal salts including (Cr(NO3)3, Fe(NO3)3, Co(NO3)2, and Ni(NO3)2) were investigated. The morphology, chemical structure, particle size, and physicochemical properties of the CNCs were determined. Controlled depolymerization of cellulose was observed using transmission electron microscopy (TEM). Rod-like morphology for all CNCs was obtained during the hydrolysis process with the smallest CNC particles found at an average length of 278.1 ± 35.1 nm and a diameter of 13.4 ± 3.0 nm. The results showed that higher valence state metal ions resulted in better cellulose hydrolysis efficiency. In addition, the use of transition metal salt as a co-catalyst improved production efficiency and minimised carbonization of CNC while maintaining desired crystallinity and thermal properties.

In situ bifunctional solid acids bearing B–OH and –COOH groups for efficient hydrolysis of cellulose to sugar in a pure aqueous phase

Pyruvaldehyde as a carbon source is synthesized together with boric acid to produce a carbon-based solid catalyst with in situ bifunctional groups, which is utilized to induce the hydrolysis of cellulose to sugar in a pure aqueous phase.

Recent advances in the processing of Napier grass (Pennisetum purpureum Schumach) as a potential bioenergy crop for bioethanol production

Pennisetum purpureum is an easily cultivated fast-growing perennial grass with great potential as a bioenergy crop due to its high biomass production. Recent advancements in the processing of P. purpureum for the generation of bioethanol are highlighted in this review paper. Various pretreatment methods, namely, enzymatic, acid, and alkaline pretreatment have been investigated to enhance the efficiency of cellulose hydrolysis, which is a crucial step in bioethanol production. Moreover, genetic engineering approaches for enhancing the biomass yield and reducing the lignin are being discussed in this review. The production of bioethanol can be increased by several fermentation processes, including saccharification, separated hydrolysis, simultaneous saccharification, and fermentation. This comprehensive review explores recent advancements in bioethanol production, encompassing technological innovations, diversification of feedstock sources, genetic engineering methodologies, inventive pretreatment techniques, refinements in enzymatic hydrolysis, and enhancements in fermentation, ultimately highlighting the potential of P. purpureum as a sustainable bioethanol source while addressing the challenges and opportunities in its commercial-scale production.

Robust fabrication of sulfonated graphene oxide/poly (ether sulfone) catalytic membrane reactor for efficient cellulose hydrolysis and product separation

The efficient conversion of cellulose to high value-added products is important for the utilization of cellulose biomass. Achieving efficient cellulose hydrolysis and timely products separation is the essential target. Herein, a modified sulfonated graphene oxide/polydopamine deposited polyethersulfone (mGO(SO3H)-PDA/PES) membrane reactor, combining in the same unit a conversion effect and a separation effect, was prepared by suction filtration and subsequent polymerization and adhesion. The structure of PES membrane and deposition of PDA was regulated to sure that small molecules can pass through the membrane, while cellulose could not. As a result, the mGO(SO3H)-PDA/PES membrane realized the efficient cellulose hydrolysis and timely products separation under cross-flow circulation mode at 0.1 MPa, avoiding the further degradation of reducing sugar products. The yields of total reducing sugar (TRS) and glucose in separated hydrolysate reached 93.2 % and 85.5 %, respectively. This strategy provides potential guidance for efficient conversion of cellulose.

A robust mesoporous sulfated zirconium‐doped KIT‐6 solid acid catalyst for the hydrolysis of microcrystalline cellulose

AbstractBACKGROUNDThe exploration of a robust solid acid catalyst for the efficient hydrolysis of cellulose is a research hotspot due to the need for clean production and sustainable development. KIT‐6 silica offers abundant three‐dimensionally interconnected mesopores, which are expected to be superior to mesoporous structures with one‐ or two‐dimensional channels due to faster diffusion of reactants and products during reaction. The catalytic conversion of microcrystalline cellulose is examined in detail over a functionalized KIT‐6 catalyst prepared by the method of Zr addition and subsequent sulfation.RESULTSIn terms of cellulose conversion and product yield, the activity of the catalysts followed the order of sulfated Zr‐KIT‐6 &gt; Zr‐KIT‐6 &gt; SO42−‐KIT‐6 &gt; KIT‐6. Over the sulfated Zr‐KIT‐6 catalyst with a Si/Zr molar ratio of 7:1, a maximum cellulose conversion of 95.0% was achieved with glucose yield of 23.4%, 5‐hydroxymethylfufural yield of 4.9% and levulinic acid yield of 11.7% under the optimized reaction conditions: reaction temperature of 200 °C, reaction time of 2 h, catalyst amount of 0.3 g, cellulose‐to‐water mass ratio of 1:1.CONCLUSIONThe sulfated Zr‐KIT‐6 catalyst possessed the strongest and richest Brønsted acid sites, which readily provided protons for the cleavage of β‐1,4 glycosidic bonds in cellulose, thus conferring superior catalytic activity in cellulose hydrolysis. A plausible pathway at the initial stage of cellulose hydrolysis over the catalyst was proposed. © 2023 Society of Chemical Industry (SCI).

Cellulase immobilization on zeolitic imidazolate frameworks for boosting cellulose hydrolysis at high solids loading

This study investigates the cellulase immobilization onto zeolitic imidazolate frameworks (ZIFs) to enable efficient cellulose hydrolysis at high solids loading. Different ZIF carriers (ZIF-8, ZIF-8-NH2, Fe3O4@ZIF-8, and Fe3O4@ZIF-8-NH2) were prepared and characterized. The resulting ZIF-immobilized cellulases were subsequently analyzed for their loading capacity and enzyme activity, as well as their biochemical properties such as temperature stability, pH stability, storage stability, enzyme leaching, and enzyme kinetics. Results revealed that the cellulase immobilized on Fe3O4@ZIF-8-NH2 exhibited an exceptional loading capacity of 359.89 mg/g and a relative enzyme activity of 69.39%. Remarkably, at a solid loading of 15% (w/w) and an enzyme dosage of 5 FPU/g cellulose, the Fe3O4@ZIF-8-NH2 immobilized cellulase demonstrated a 20.44% higher hydrolysis yield compared to the free cellulase. Furthermore, this immobilized enzyme system displayed robust reusability, retaining 71.03% of its initial activity even after 10 cycles of reuse. A proposed mechanism of enhanced enzymatic hydrolysis by ZIF-immobilized cellulases was also presented. These findings have significant implications for the future application of ZIF-immobilized cellulases in efficient and cost-effective lignocellulosic bioconversion.

Efficient hydrolysis of cellulose to reducing sugars over peanut shell‐derived carbon‐based solid acid with a large surface area

AbstractA carbon‐based solid acid with a large surface area (CSALA; SBET = 237.9 m2/g) was prepared from peanut shells. Compared with a CSA with a small specific surface area (SBET = 5.6 m2/g), the CSALA enabled greater cellulose hydrolysis. Under optimized conditions, the yield of reducing sugars over 2.5 h at 110°C was 60.5% using the CSALA. When a CSA was used, the yield of reducing sugar only reached 58.6% after 4 h at the same temperature. The results showed that the CSALA had higher activity because of its larger specific surface area and more numerous surface acid centers. However, it was unstable and was deactivated after one use. The loss of some of the sulfonic groups on the CSALA surface, and the conversion of some of those groups to sulfate groups under the action of 1‐butyl‐3‐methylimidazolium chloride ionic liquid were the main reasons for the deactivation of the CSALA. In addition, most of the residual sulfonic groups were located in the micropores of CSALA and could not participate in the cellulose hydrolysis reaction.

Pretreatment with ChOH/Urea under mild conditions for the efficient deconstruction of poplar cell wall

In this study, DES consisting of Choline hydroxide (ChOH) and Urea (Ur) was used to pretreat poplar under mild conditions (60 ℃). The 72 h saccharification efficiency of the pretreated samples for 10 min was significantly improved (69.97 %), which amounted to a 2.4-fold increase in sugar yield over the raw material (29.89 %). The remarkable enhancement could be correlated to the topochemical and morphological changes occurring in poplar cell walls, including the removal of lignin and hemicelluloses, decreased crystallinity and microstructural disruption. Noteworthy, X-ray diffraction analysis showed that cellulose I underwent a crystallographic transformation from cellulose I to cellulose II during DES pretreatment. Our study demonstrated that ChOH/Ur pretreatment was quite effective in deconstructing poplar cell walls and increasing the enzymatic hydrolysis efficiency of cellulose under mild conditions.

Improving the cellobiose hydrolysis activity of glucose-stimulating β-glucosidase Bgl2A

β-Glucosidases with high catalytic activity and glucose tolerant properties possess promising applications in lignocellulose-based industries. To obtain enzymes possessing these properties, a semi-rational strategy was employed to engineer the glucose-stimulating β-glucosidase Bgl2A for high cellobiose hydrolysis activity. A total of 18 mutants were constructed. A22S, V224D, and A22S/V224D exhibited high specific activities of 272.06, 237.60, and 239.29 U/mg toward cellobiose, which were 2.5- to 2.8-fold of Bgl2A. A22S, V224D, and A22S/V224D exhibited increased kcat values, which were 2.7- to 3.1-fold of Bgl2A. A22S and V224D maintained glucose-stimulating property, whereas A22S/V224D lost it. Using 150 g/L cellobiose as the substrate, the amount of glucose produced by A22S was the highest, yielding 129.70 g/L glucose after 3 h reaction at 35 °C. The synergistic effects of the engineered enzymes with commercial cellulase on hydrolyzing cellulose were investigated. Supplemented with the commercial cellulase and A22S, the highest glucose amount of 23.30 g/L was yielded from cellulose with hydrolysis rate of 21.02 %. Given its high cellobiose hydrolysis activity and glucose-stimulating properties, A22S can be used as a component of enzyme cocktail to match mesophilic cellulases for efficient cellulose hydrolysis.

Hydrolysis of cellulose to glucose in aqueous phase with phosphate group modified hydroxy-rich carbon-based catalyst

By catalytically converting cellulose using a phosphate-modified hydroxyl-rich carbon-based catalyst (P@HRCBC), which was made by hydrothermal carbonization, glucose was successfully hydrolyzed from cellulose in an aqueous phase system (HTC). The glycosidic bond breaking catalyzed by the -PO3H2 and the cellulose matrix lamellar exfoliation are both enhanced by the excellent adsorption capacity of the exceptionally rich –OH. The activity of cellulase is perfectly imitated. Within 120 min, the glucose output and the conversion of cellulose achieved 96.3% and 47.1%, respectively. The numerous ways that P@HRCBC adsorbs cellulose and cellobiose alter its catalytic ability and make separating glucose easier. After numerous cycles, the conversion of cellulose was maintained at 90.3%, thanks to the remarkable stability of –OH and -PO3H2. P@HRCBC shows an actual "enzymatic" catalytic function that provides a new solution for the efficient green hydrolysis of cellulose into glucose.

Bismuth-Decorated Beta Zeolites Catalysts for Highly Selective Catalytic Oxidation of Cellulose to Biomass-Derived Glycolic Acid

Catalytic conversion of cellulose to liquid fuel and highly valuable platform chemicals remains a critical and challenging process. Here, bismuth-decorated β zeolite catalysts (Bi/β) were exploited for highly efficient hydrolysis and selective oxidation of cellulose to biomass-derived glycolic acid in an O2 atmosphere, which exhibited an exceptionally catalytic activity and high selectivity as well as excellent reusability. It was interestingly found that as high as 75.6% yield of glycolic acid over 2.3 wt% Bi/β was achieved from cellulose at 180 °C for 16 h, which was superior to previously reported catalysts. Experimental results combined with characterization revealed that the synergetic effect between oxidation active sites from Bi species and surface acidity on H-β together with appropriate total surface acidity significantly facilitated the chemoselectivity towards the production of glycolic acid in the direct, one-pot conversion of cellulose. This study will shed light on rationally designing Bi-based heterogeneous catalysts for sustainably generating glycolic acid from renewable biomass resources in the future.

Production of Enzymatic Extract with High Cellulolytic and Oxidative Activities by Co-Culture of Trichoderma reesei and Panus lecomtei

This work aimed to produce enzymatic fungi extracts with hydrolytic and oxidative activities to hydrolyze lignocellulosic biomasses efficiently. For this, the fungi Trichoderma reesei and Panus lecomtei were co-cultured using the vegetable biomasses oil palm decanter cake, wheat bran, and cottonseed cake as substrates in submerged fermentation. T. reesei and P. lecomtei showed partially compatible positive interaction on plates. The co-cultures respond positively to variations of temperature and inoculum interval, generating extracts responsible for higher hydrolysis yield when grown at 25 °C, and P. lecomtei is inoculated 24 h after T. reesei. The enzymatic extract production of co-cultures was also improved by modifying the components of the initial media and evaluating enzymatic activities, hydrolysis of sugarcane bagasse pretreated by autohydrolysis and ethanol production as a response. Five culture media were evaluated with variations in the composition of nutritional elements, minerals and substrates. The best extract showed a maximum cellulose hydrolysis efficiency of 68.7% compared with 44.8% of the initial medium. The ethanolic fermentation of hydrolysates obtained by co-culture extracts showed higher ethanol yields than monocultures. This work demonstrates the use of fungi co-cultures to produce enzymatic extracts composed of cellulolytic, hemicellulolytic, and ligninolytic enzymes complexes, which allow hydrolyzing pretreated lignocellulosic biomass with high efficiency, generating hydrolysates that are easier fermented by yeast.

Comparison of various delignification/desilication pre-treatments and indigenous fungal cellulase for improved hydrolysis of paddy straw.

The efficient removal of lignin and silica from paddy straw is essential for its volarization into biofuels and other value-added products. In this work, different chemical pre-treatments viz. acid/alkali, organosolv and deep eutectic solvents were carried out to assess the extent of delignification and desilication of paddy straw. Maximum lignin and silica removal of 96.08 and 95.51% was observed with two step acid (0.5% sulphuric acid) followed by alkali (4% sodium hydroxide) pre-treatment with significantly low total lignin (2.30%) and silica content (0.80%) of the treated straw residue. The treated straw residue contained significantly high holocellulose (91.65%), cellulose (75.01%) and hemicellulose content (16.64%). Among the four indigenous fungal isolates, Penicillium mallochii (JS17) cellulase showed better accessibility for the treated straw residue with maximum release of 504.18mgg-1 of reducing sugars and saccharification efficiency of 56.90%. The two-step acid/alkali pre-treatment of paddy straw was highly effective for removing lignin and silica from paddy straw, thereby, resulting in enhanced enzymatic accessibility of the substrate and more efficient hydrolysis of cellulose into fermentable sugars.

Гидролиз целлюлозы с использованием газовоздушных смесей хлористого водорода

Abstract. Microcrystalline cellulose (MCC) is a common product used in pharmaceutical, food and other industries. MCC is obtained by liquid-phase hydrolysis of cotton or wood bleached pulp with diluted 0.5–10.0 % mineral acids at a temperature of 100–140 °C. This process requires significant consumption of acid, water, and heat energy. Production of MCC in general is very expensive, which determines its high cost and the need to find alternative methods of cellulose hydrolysis. It is proposed to hydrolyze cellulose with concentrated hydrochloric acid produced by absorption of hydrogen chloride. We studied the processes of hydrogen chloride adsorption by bleached wood pulp with 8–18 % humidity. It is shown that adsorption of hydrogen chloride is determined by pulp humidity and is 3–5 % of the dry pulp mass. The sorption of hydrogen chloride leads to the formation of hydrochloric acid with a concentration of 25–40 % in the raw material moisture, significant heating of the mass and rapid hydrolysis of the amorphous cellulose fraction. It has been found that the use of pure hydrogen chloride for saturation causes strong darkening and humification of pulp. We recommend the use of hydrogen chloride gas-air mixtures to saturate the pulp, which will significantly reduce the sorption temperature and eliminate the strong darkening of the pulp during hydrolysis. The adsorption of hydrogen chloride by cellulose proceeds at an extremely high rate and is accompanied by the formation of a clearly visible sorption front at a temperature of 45–60 °C. Hydrolysis occurs for 15–30 min at 40–60 °C until the amorphous cellulose fraction is completely decomposed. A very small amount of monosaccharides is formed (4 % of dry pulp). The yield of MCC is high, more than 95 %. These circumstances are probably related to the recrystallization of a part of the amorphous fragments of cellulose macromolecules, which is a characteristic of hydrolysis with concentrated acids. The product obtained by cellulose hydrolysis is identical to MCC according to the data of IR spectroscopy, X-ray diffraction and viscometry. The article shows the high efficiency of cellulose hydrolysis with hydrogen chloride gas-air mixtures compared to traditional methods of MCC production.

A Combination Method of Liquid Hot Water and Phosphotungstic Acid Pretreatment for Improving the Enzymatic Saccharification Efficiency of Rice Straw

Chemical pretreatment can significantly improve the enzymatic hydrolysis efficiency of lignocellulosic biomass, thereby improving the yield of sugar materials for the production of cellulosic ethanol, but commonly used acid–base catalysts are difficult to recover and reuse. In this work, a combination method of liquid hot water (LHW) and phosphotungstic acid (PTA) pretreatment was performed to improve the saccharification efficiency of rice straw, and we attempted to evaluate the reuse effect of PTA catalysts. The rice straw was first treated with LHW at 180 °C for 90 min, and then treated with 20 mM PTA at 130 °C for 60 min. After pretreatment, the cellulose hydrolysis efficiency and glucose recovery of the rice straw increased by 201.85% and 164.25%, respectively. Glucose accounted for 96.8% of the total reducing sugar in the final enzymatic hydrolysate. After each PTA pretreatment, approximately 70.8–73.2% of the PTA catalyst could be recycled. Moreover, the catalytic activity of the PTA catalyst that had been used five times did not decrease. The improved enzymatic saccharification efficiency was attributed to the removal of 89.24% hemicellulose and 21.33% lignin from the lignocellulosic substrate. The two-step LHW-PTA pretreatment could pretreat biomass in the field of cellulosic ethanol production.