Enzymatic Hydrolysis Of Lignocellulose Research Articles

Synthesis of temperature and pH responsive lignin-grafted sulfobetaine for efficiently recycling cellulase

Recycling cellulase can reduce the cost of lignocellulosic enzymatic hydrolysis. Here, a lignin-grafted sulfobetaine (LSB) was first synthesized by grafting sulfobetaine (SB) on enzymatic hydrolysis lignin (EHL). LSB had a sensitive response of pH and temperature. LSB dissolved under the conditions of lignocellulosic enzymatic hydrolysis (pH 5.0, 50 °C). After hydrolysis, LSB co-precipitated with cellulase when lowering pH of the hydrolysate to 4.0 and cooling to 25 °C. When 3.0 g/L LSB-100 was added to the hydrolysis system of corncob residue (CCR), 70 % of amount of cellulase was saved. LSB had a remarkable response and stronger cellulase recovery capacity. This was attributed that carboxylate radical in LSB was protonated, and positive and negative ions of SB associated to form salt at 25 °C. This work provides a new idea for reducing the cost for preparing fermentable sugars from lignocellulose, and increasing the added value of EHL.

Биоконверсия целлюлозосодержащего сырья. Ферментативный гидролиз целлюлозы (обзор литературы)

Lignocellulose is the most abundant biopolymer on earth. The hydrolysis of lignocellulose to fermentable sugars is a prerequisite for its successful use as a substrate for the large-scale production of value-added industrial products. Enzymatic hydrolysis of lignocellulose is carried out by cellulases, hemicellulases, and ligninases in synergy or individually. The review describes the enzymatic hydrolysis of cellulose, hemicellulose, and lignin. Information on solid-phase and submerged microorganisms fermentation used to obtain cellulases was given. Methods for increasing the level of production and activity of cellulases were characterized. The industrial application of cellulases, including for the production of bioethanol, was described.

Enzymatic formulation strategies unlock highly-efficient saccharification of distinct pretreated corncobs

Optimization of the composition of enzyme cocktail is an effective way to improve the hydrolytic efficiency and reduce the cost during enzymatic hydrolysis of lignocellulose. Herein, the optimal enzymes ratios of enzyme cocktails for efficient hydrolysis of corncobs with distinct pretreatments were quantitatively formulated. As the importance of pretreatment type on xylan extraction, the optimized cellulase/hemicellulase ratios for NaClO2, H2SO4 and steam explosion treated corncob were 1:1.5, 1:0.15 and 1:0.75, respectively. Meanwhile, different requirements for types of cellulases are also determined: endoglucanase and cellobiohydrolase were key enzymes in the hydrolysis of NaClO2 and H2SO4 treated corncobs, respectively, while the synergy of three kinds of cellulases was preferred for steam-exploded corncob. Furthermore, based on the optimized cellulase/hemicellulase ratios, cellulases proportions for hydrolyzing the corncobs pretreated with distinctive methods were determined. Notably, by the enzymatic formulation strategy, 100% yields of glucose and xylose were obtained simultaneously from NaClO2 treated corncob, which was up to 70.1% and 80.1% at high solid loading (20%), respectively. Without detoxification of hydrolysate, high isopropanol yield of 0.45 g/g (of dry matter) was achieved. Hence, this study demonstrated a low cost and high efficient conversion strategy for prospective high-value utilization of lignocellulose.

2-Naphthol modification alleviated the inhibition of ethanol organosolv lignin on enzymatic hydrolysis

It has been reported that the lignins deposited on surfaces of pretreated biomass directly affected the enzyme adsorption, thus impeding enzymatic hydrolysis of lignocellulose. Alleviating the surface lignin inhibition is urgently needed for establishing an efficient lignocellulose biorefinery process. In this study, ethanol organosolv lignins (EOLs) from masson pine and poplar were employed as representatives of surface lignins. Aiming to mitigate the EOLs inhibition on enzymatic hydrolysis, 2-naphthol as a carbocation scavenger was added in the organosolv pretreatment. The function of 2-naphthol for suppressing lignin condensation was verified by investigating the lignin structural changes with NMR analysis. Moreover, it was verified that 2-naphthol modification remarkably eliminated the negative effects of EOLs from both two wood species, leading to a 6.8–34.4% increase in hydrolysis yields. The effects of 2-napthol modified lignins were even reversed to slightly promote the enzymatic hydrolysis of pure cellulose. The mechanism of 2-naphthol modification for relieving lignin inhibition was revealed by evaluating the relationship between lignin structural features and hydrolysis performance with Pearson's correlation heatmap. Results enrich our understanding of surface lignin inhibition, thereby providing ideas for promoting enzymatic hydrolysis by alleviating the surface lignin inhibition.

Improving enzymatic saccharification of corn stover via thioglycolic acid-mediated Fenton pretreatment

Fenton pretreatment is known as a green and mild approach, but it is usually time-consuming and inefficient in promoting lignocellulose enzymatic hydrolysis. Here we demonstrate that thioglycolic acid (TA) significantly accelerates Fenton pretreatment (FP) of corn stover (CS) and improves enzymatic hydrolysis of cellulose and xylan. The results showed that the reducing sugar yield of CS reached 77.0% with TA-assisted FP in only 16 h, while it was only 34.1% and 38.0% with FP alone in 16 h and 72 h, respectively. Via examination of the changes of composition, physical and chemical characteristics of CS before and after pretreatment, it was found that TA-assisted FP greatly improved the removal of hemicellulose and lignin, resulting in increment of the pore volume, pore size and cellulose exposure of CS substrate, which together promoted the enzymatic saccharification of CS.

Ammonia fiber expansion (AFEX) combined with white-rot fungi pretreatment to improve enzymatic hydrolysis of lignocellulose

Ammonia fiber expansion (AFEX) combined with white-rot fungi pretreatment to improve enzymatic hydrolysis of lignocellulose

Recombinant Family 1 Carbohydrate-Binding Modules Derived From Fungal Cellulase Enhance Enzymatic Degradation of Lignocellulose as Novel Effective Accessory Protein.

Fungal cellulases usually contain a family 1 carbohydrate-binding module (CBM1), and its role was considered to recognize the substrate specifically. This study testified that the CBM1s derived from cellobiohydrolase I of Trichoderma reesei, Penicillium oxalicum, and Penicillium funiculosum could be used as an effective accessory protein in cellulase cocktails to enhance the saccharification of lignocellulose, and its enhancement effect was significantly superior to some reported accessory proteins, such as bovine serum albumin (BSA). The promoting effects of the CBM1s were related to not only the CBM1 sources and protein dosages, but also the substrate characteristics and solid consistency during enzymatic hydrolysis. The adsorption capacity of the CBM1s, the adsorption kinetic of TrCBM from T. reesei and cellobiohydrolase, endoglucanase, and β-glucosidase from P. oxalicum, and the effect of adding TrCBM on enzyme activities of free cellulases in the hydrolysis system were investigated, and the binding conformations and affinities of CBM1s to cellulose and lignin were predicted by molecular docking. It was speculated that the higher affinity of the CBM1s to lignin than cellulases could potentially enable the CBM1s to displace cellulase adsorbed on lignin or to preferentially adsorb onto lignin to avoid ineffective adsorption of cellulase onto lignin, which enhanced cellulase system efficiency during enzymatic hydrolysis of lignocellulose.

Roles of Surfactants in Oriented Immobilization of Cellulase on Nanocarriers and Multiphase Hydrolysis System.

Surfactants, especially non-ionic surfactants, play an important role in the preparation of nanocarriers and can also promote the enzymatic hydrolysis of lignocellulose. A broad overview of the current status of surfactants on the immobilization of cellulase is provided in this review. In addition, the restricting factors in cellulase immobilization in the complex multiphase hydrolysis system are discussed, including the carrier structure characteristics, solid-solid contact obstacles, external diffusion resistance, limited recycling frequency, and nonproductive combination of enzyme active centers. Furthermore, promising prospects of cellulase-oriented immobilization are proposed, including the hydrophilic-hydrophobic interaction of surfactants and cellulase in the oil-water reaction system, the reversed micelle system of surfactants, and the possible oriented immobilization mechanism.

Structural Changes of Alkali Lignin under Ozone Treatment and Effect of Ozone-Oxidized Alkali Lignin on Cellulose Digestibility

In this study, the structural changes of alkali lignin induced by ozonation were investigated, and the effect of ozone-treated alkali lignin and its mechanism on Avicel enzymatic hydrolysis was examined. The physicochemical properties of alkali lignin were analyzed by FTIR, 1H-13C HSQC NMR, and GPC. It was revealed that ozone pretreatment increased the content of carboxyl and/or aldehyde groups and the negative zeta potential of alkali lignin, which enhanced the electrostatic repulsion between alkali lignin and cellulase; The S/G ratio was reduced, indicating the hydrophobic interaction was diminished. The Langmuir adsorption isotherm showed that the cellulase binding strength of ozone pretreated alkali lignin (OL-pH3, OL-pH7, and OL-pH12 were 16.67, 13.87, and 44.05 mL/g, respectively) was significantly lower than that of alkali lignin (161.29 mL/g). The 72 h hydrolysis yields of Avicel added with OL-pH3, OL-pH7, and OL-pH12 were 55.4%, 58.6%, and 54.9% respectively, which were 2.6–6.3% higher than that of Avicel added with AL (52.3%). This research aimed to reduce the non-productive adsorption between cellulase and lignin by investigating the structural changes of lignin caused by ozone treatment. For the first time, we discovered that ozone-treated alkali lignin has a further promotion effect on the enzymatic digestion of cellulose, providing a green and feasible pretreatment process for the enzymatic hydrolysis of lignocellulose and aiding in the more efficient utilization of biomass.

Understanding the toxicity of lignin-derived phenolics towards enzymatic saccharification of lignocellulose for rationally developing effective in-situ mitigation strategies to maximize sugar production from lignocellulosic biorefinery

The lignin-derived phenolics are highly inhibitory toward lignocellulose enzymatic hydrolysis, while the relationship between phenolic structure and inhibitory effect is still not fully understood. In this study, the compositions of phenolics from dilute acid pretreated wheat straw were analyzed and their impact on cellulose hydrolysis was studied. With increase of pretreatment severity, more toxic phenolics were produced from lignin degradation reactions, which were the major contributor to the increased inhibitory effect of pretreatment hydrolysate towards cellulases. Through analyzing the relationship of phenolic structure and their inhibitory effect, a useful model was developed to predict the phenolics-caused inhibition by combining the indexes of electrophilicity and hydrophobicity. Further, through understanding the interactions between phenolics and cellulases, a novel biocomponent alleviator was rationally designed to block the phenolics-cellulase interactions, the degree of improvement of enzymatic hydrolysis reached as high as 135.8%. This study provides directions for developing more effective pretreatment and detoxification methods.

Integrating mild chemical pretreatments with endogenous protein supplement for complete biomass saccharification to maximize bioethanol production by enhancing cellulases adsorption in novel bioenergy Amaranthus

Amaranthus is a fast-growing perennial plant containing large amounts of extractable green proteins and lignocellulose residues. However, it remains to explore optimal Amaranthus biomass process technology for biofuel production. In this study, we examined that mild alkali and acid pretreatments (1% NaOH, 2% H2SO4) were sufficient for near-complete biomass enzymatic saccharification, while 8% Amaranthus endogenous proteins were supplemented into the enzymatic hydrolyses. By performing classic yeast fermentation with all carbon sources from directly-extractable sugars and starch and lignocellulose enzymatic hydrolysis, this work achieved the bioethanol yield up to 23.5% (% dry matter). Notably, with respect to extremely high yield of Amaranthus straw, the maximum bioethanol yield was estimated at 14.46 t/ha/year, which was much higher than those of other major bioenergy crops as previously reported. Furthermore, this work proposed a hypothetic model to elucidate how the complete biomass saccharification was achieved to maximize bioethanol production at large scale, based on the effective wall polymer extraction and distinct lignocellulose feature modification for remarkably enhanced biomass porosity and cellulases adsorption. Hence, this work has demonstrated that Amaranthus could be applied as a novel and leading bioenergy crop, providing a powerful strategy for optimal biomass processing towards high bioethanol production.

Ammonia Fiber Expansion (Afex) Pretreatment Combined with White Rot Fungi Treatment to Improve Enzymatic Hydrolysis of Lignocellulose

Ammonia Fiber Expansion (Afex) Pretreatment Combined with White Rot Fungi Treatment to Improve Enzymatic Hydrolysis of Lignocellulose

Using poly(N-Vinylcaprolactam) to Improve the Enzymatic Hydrolysis Efficiency of Phenylsulfonic Acid-Pretreated Bamboo.

Chemical pretreatment followed by enzymatic hydrolysis has been regarded as a viable way to produce fermentable sugars. Phenylsulfonic acid (PSA) pretreatment could efficiently fractionate the non-cellulosic components (hemicelluloses and lignin) from bamboo and result in increased cellulose accessibility that was 10 times that of untreated bamboo. However, deposited lignin could trigger non-productive adsorption to enzymes, which therefore significantly decreased the enzymatic hydrolysis efficiency of PSA-pretreated bamboo substrates. Herein, poly(N-vinylcaprolactam) (PNVCL), a non-ionic surfactant, was developed as a novel additive for overcoming the non-productive adsorption of lignin during enzymatic hydrolysis. PNVCL was found to be not only more effective than those of commonly used lignosulfonate and polyvinyl alcohol for overcoming the negative effect of lignin, but also comparable to the robust Tween 20 and bovine serum albumin additives. A PNVCL loading at 1.2 g/L during enzymatic hydrolysis of PSA pretreated bamboo substrate could achieve an 80% cellulosic enzymatic conversion and meanwhile reduce the cellulase loading by three times as compared to that without additive. Mechanistic investigations indicated that PNVCL could block lignin residues through hydrophobic interactions and the resultant PNVCL coating resisted the adsorption of cellulase via electrostatic repulsion and/or hydration. This practical method can improve the lignocellulosic enzymatic hydrolysis efficiency and thereby increase the productivity and profitability of biorefinery.

Identifying the negative cooperation between major inhibitors of cellulase activity and minimizing their inhibitory potential during hydrolysis of acid-pretreated corn stover

Soluble compounds produced during the enzymatic hydrolysis of lignocelluloses hampers cellulose conversion. Cellobiose and vanillin most severely inhibited the effect of cellobiohydrolase I. A concentration-dependent negative cooperative effect was found between cellobiose and vanillin. The combined inhibitory effect was about 83.5% of the cellobiose and 88.1% of the vanillin when their concentration was 20 mg/ml. However, the negative synergy could be eliminated by excessive enzyme loading. Differences in their binding sites on the catalytic domain of cellobiohydrolase I lead to negative synergistic inhibition, which should be considered in devising strategies to alleviate this effect. Combined β-glucosidase and PEG addition at an appropriate dose was feasible to balance cost and hydrolytic efficiency. To achieve efficient hydrolysis, especially at high solid concentrations, it is important to understand the synergistic inhibition between these inhibitors.

Impact Of Microbial Synergism On Second Generation Production Of Bioethanol From Fruit Peels Wastes

Fruit peels annually accumulate in huge amounts, fruit wastes are rich in lignocellulo-sic component which can be recovered into mono simple carbohydrates able to utilize for bioethanol production. The enzymatic hydrol-ysis of lignocelluloses is known to be a key to the second-generation biofuel, the challenge is the still expensive enzymes involved in the saccharification process, loss of the most hem-icellulose pentose sugars which were non-fer-mentable as base, beside the presence of ham-pers lignin thus require to resolve its problem.As part of study, followed the cost-effective means for bioethanol producing from four fruit peels (Banana, Orange, Mango and Water-melon), yielding of enzymes from co-fungal cultivation carried by Aspergillus niger and Phanerochaete chrysosporium, followed by co-fermentation of the saccharified sugars us-ing yeast belong to Saccharomyces cerevisiae and Kluyveromyces marxianus which contrib-uted for bioethanol production in 8 L lab-scale reactor. The fermented sugars recovering was reached to 27.77 g.l-1 from banana peels which were found to be good exploited as potential raw source, co-fungal enzymatic hydrolysis followed by yeast co-fermentation led to sub-stantial yield by 10.74 g.l-1, the adding of calcium oxide increased the purity which leads finally to 97.5 wt % of pure bioethanol. Thrust towards fossil fuels replacement with renewa-ble clean fuels such as bioethanol by using fruit peels residuals which are considered renewa-ble energy source may also help in CO2 miti-gation.

Interdependence between lignocellulosic biomasses, enzymatic hydrolysis and yeast cell factories in biorefineries.

SummaryBiorefineries have a pivotal role in the bioeconomy scenario for the transition from fossil‐based processes towards more sustainable ones relying on renewable resources. Lignocellulose is a prominent feedstock since its abundance and relatively low cost. Microorganisms are often protagonists of biorefineries, as they contribute both to the enzymatic degradation of lignocellulose complex polymers and to the fermentative conversion of the hydrolyzed biomasses into fine and bulk chemicals. Enzymes have therefore become crucial for the development of sustainable biorefineries, being able to provide nutrients to cells from lignocellulose. Enzymatic hydrolysis can be performed by a portfolio of natural enzymes that degrade lignocellulose, often combined into cocktails. As enzymes can be deployed in different operative settings, such as separate hydrolysis and fermentation (SHF) or simultaneous saccharification and fermentation (SSF), their characteristics need to be combined with microbial ones to maximize the process. We therefore reviewed how the optimization of lignocellulose enzymatic hydrolysis can ameliorate bioethanol production when Saccharomyces cerevisiae is used as cell factory. Expanding beyond biofuels, enzymatic cocktail optimization can also be pivotal to unlock the potential of non‐Saccharomyces yeasts, which, thanks to broader substrate utilization, inhibitor resistance and peculiar metabolism, can widen the array of feedstocks and products of biorefineries.

Cellulose induced protein 1 (Cip1) from Trichoderma reesei enhances the enzymatic hydrolysis of pretreated lignocellulose

BackgroundTrichoderma reesei is currently the main strain for the commercial production of cellulase. Cellulose induced protein 1 (Cip1) is one of the most abundant proteins in extracellular proteins of T. reesei. Reported literatures about Cip1 mainly focused on the regulation of Cip1 and its possible enzyme activities, but the effect of Cip1 on the enzymatic hydrolysis of lignocellulose and possible mechanism have not still been reported.ResultsIn this study, Cip1 from T. reesei was cloned, expressed and purified, and its effects on enzymatic hydrolysis of several different pretreated lignocellulose were investigated. It was found that Cip1 could promote the enzymatic hydrolysis of pretreated lignocellulose, and the promoting effect was significantly better than that of bovine serum albumin (BSA). And especially for the lignocellulosic substrate with high lignin content such as liquid hot water pretreated corn stover and corncob residue, the promoting effect of Cip1 was even better than that of the commercial cellulase when adding equal amount protein. It was also showed that the metal ions Zn2+ and Cu2+ influenced the promoting effect on enzymatic hydrolysis. The Cip1 protein had no lyase activity, but it could destroy the crystal structure of cellulose and reduce the non-productive adsorption of cellulase on lignin, which partly interpreted the promoting effect of Cip1 on enzymatic hydrolysis of lignocellulose.ConclusionThe Cip1 from T. reesei could significantly promote the enzymatic hydrolysis of pretreated lignocellulose, and the promotion of Cip1 was even higher than that of commercial cellulase in the enzymatic hydrolysis of the substrates with high lignin content. This study will help us to better optimize cellulase to improve its ability to degrade lignocellulose, thereby reducing the cost of enzymes required for enzymatic hydrolysis.

Carboxylated and quaternized lignin enhanced enzymatic hydrolysis of lignocellulose treated by p-toluenesulfonic acid due to improving enzyme activity

Modificated lignins can affect enzymatic hydrolysis efficiency (EHE) because of changing physicochemical properties of lignin. In this study, carboxylated and quaternized lignin (CQL) and hydroxymethylated lignin (HML) were prepared to explore the effect of lignin modification on cellulase adsorption and EHE of p-toluenesulfonic acid treated corn stover (PCS). The results showed that CQL enhanced EHE of PCS due to the higher β-glucosidase (β-GL) activity, resulting from the formation of CQL-β-GL complexes with a lower binding free energy and the improvement of β-GL conformation made by the binding of CQL and β-GL. However, the drop in EHE due to the addition of HML was consequent on β-GL deactivation that was because the binding site of HML and β-GL overlapped with the carbohydrate binding domain of β-GL, causing the decrease in β-GL activity compared with CQL. This study would help deeply elucidate the effect of modified lignins on EHE and cellulase adsorption.

Bioreactor Design For Solid State Lignocellulosic Hydrolysis: A Review

Enzymatic hydrolysis of lignocellulose to produce reducing sugars to be used as bioethanol raw material has recently attracted many scientists’ attention to investigate. It needs to be performed on a big scale, so it is essential to select an appropriate bioreactor. This work discusses enzymatic hydrolysis in solid-state. Solid hydrolysis is suitable for the growth of filamentous fungi to produce hydrolytic enzymes. A bioreactor is the heart of the process since the chemical reactions take place in it. In biochemical reactions, bioreactor plays two complementary functions, i.e., the reaction holder and the place for microorganism to grow. Therefore, to design a good bioreactor, these two functions should be considered carefully. Besides, we also developed a bioreactor with a continuous air supply to provide oxygen for hydrolytic fungi.

Impact of Alkaline Pretreatment Condition on Enzymatic Hydrolysis of Sugarcane Bagasse and Pretreatment Cost.

A combined severity factor (RCSF) which is usually used to evaluate the effectiveness of hydrothermal pretreatment at above 100 °C had been developed to assess the influence of temperature, time, and alkali loading on pretreatment and enzymatic hydrolysis of lignocellulose. It is not suitable for evaluating alkaline pretreatment effectiveness at lower than 100 °C. According to the reported deducing process, this study modified the expression of [Formula: see text] as [Formula: see text] which is easier and more reasonable to assess the effectiveness of alkaline pretreatment. It showed that RCSF exhibited linear trend with lignin removal, and quadratic curve relation with enzymatic hydrolysis efficiency (EHE) at the same temperature. The EHE of alkali-treated SCB could attain the maximum value at lower RCSF, which indicated that it was not necessary to continuously enhance strength of alkaline pretreatment for improving EHE. Within a certain temperature range, the alkali loading was more important than temperature and time to influence pretreatment effectiveness and EHE. Furthermore, the contribution of temperature, time, and alkali loading to pretreatment cost which was seldom concerned was investigated in this work. The alkali loading contributed more than 70% to the pretreatment cost. This study laid the foundation of further optimizing alkaline pretreatment to reduce cost for its practical application.