Lignocellulose Saccharification Research Articles

Construction of an enzymatic shuttling compartment based on reverse micellar for bamboo biomass hydrolysis in ionic liquids

The enzymatic saccharification of regenerated lignocellulose must occur separately due to the toxicity of ionic liquids to cellulase. Therefore, it is important to develop a biocompatible IL-cellulase system which effectively achieves activation and saccharification of lignocellulose. For this purpose, a dual-phase “enzyme-shuttling compartment” was constructed in this study. Tween 80 was found to form reverse micelles in the isooctane-IL two-liquid phase, acting as a microenvironment that maintains the energetic conformation of the reactive cellulase. The activated bamboo biomass was enzymatically hydrolyzed in 20% (w/v) 1-ethyl-3-methylimidazolium dimethyl phosphate and 50 mM citrate buffer at 50 °C, achieving a high total reducing sugar yield of 71.2% and maintaining an enzymatic activity of 91.2% after 24 h. Thus, an efficient system with the simultaneous activation and saccharification of natural biomass was successfully developed in a one-pot procedure at low temperatures, ensuring large-scale biomass conversion into biofuels and biological products.

Dissolved xylan inhibits cellulosome-based saccharification by binding to the key cellulosomal component of Clostridium thermocellum

Polysaccharides derived from lignocellulose are promising sustainable carbon sources. Cellulosome is a supramolecular machine integrating multi-function enzymes for effective lignocellulose bio-saccharification. However, how various non-cellulose components of lignocellulose affect the cellulosomal saccharification is hitherto unclear. This study first investigated the stability and oxygen sensitivity of the cellulosome from Clostridium thermocellum during long-term saccharification process. Then, the differential inhibitory effects of non-cellulose components, including lignin, xylan, and arabinoxylan, on the cellulosome-based saccharification were determined. The results showed that lignin played inhibitory roles by non-productively adsorbing extracellular proteins of C. thermocellum. Differently, arabinoxylan preferred to bind with the cellulosomal components. Almost no adsorption of cellulosomal proteins on solid xylan was detected. Instead, xylan in water-dissolved form interacted with the cellulosomal proteins, especially the key exoglucanase Cel48S, leading to the xylan inhibitory effect. Compared to xylan, xylooligosaccharides influenced the cellulosome activity slightly. Hence, this work demonstrates that the timely hydrolysis or removal of dissolved xylan is important for cellulosome-based lignocellulose saccharification.

Fungal pretreatments of Napier grass and sugarcane leaves for high recovery of lignocellulosic enzymes and methane production

Effective biological pretreatment is a sought-after approach to solving the dilemma between environmental protection versus sustainable and economical production of bio-based products. In many countries, grasses are among the most attractive and widely cultivated crops for bioenergy. Here, Napier grass, including Alafal, Pakchong 1, and Merkeron varieties, and sugarcane were investigated for their potential applications in lignocellulosic enzyme and biomethane production. This was accomplished through biological pretreatments using a wood-rotting fungal Xylaria species under solid-state fermentation (SoSF) at ambient temperatures. Merkeron produced an enzymatic cocktail of xylanase and cellulase at activities of 103.1 ± 2.5 or 42.4 ± 1.61 U/g, respectively, on day 14. In contrast, using yeast Pichia pastoris expressing engineered Bacillus firmus xylanases, the highest xylanase activities were achieved between 58 and 61 U/g using Pakchong 1 or sugarcane leaves under submerged fermentation (SmF) after 2–4 days. Preferential fermentation mode between SoSF and SmF, utilized by fungi or yeast was observed during lignocellulosic saccharification. The Highest cellulase and xylanase productivity was achieved at 0.24–0.51 U/g/h, using the yeast-pretreatment via SmF. Alternatively, Xylaria-pretreated Napier grass mixed with cow manure effectively increased the daily biogas yield up to 212.8 ± 2.3 cm3/gVS with methane content over 80% and methane productivity of 2.31%/day, respectively. Thus, this study highlights potential uses of fungi in eco-friendly pretreatment of abundant feedstocks for optimal production of lignocellulosic enzymes and green energy biomethane.

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.

Polystyrene sulfonate is effective for enhancing biomass enzymatic saccharification under green liquor pretreatment in bioenergy poplar

BackgroundWater-soluble lignin (particularly lignosulfonate, LS) has been well documented for its significance on enzymatic saccharification of lignocellulose, though the promotion mechanism has not been fully understood. Much attention has been paid to natural lignin or its derivatives. The disadvantage of using natural lignin-based polymers as promoting agents lies in the difficulty in tailor-incorporating functional groups due to their complex 3D structures. To further improve our understanding on the promotion mechanism of water-soluble lignin in the bioconversion of lignocellulose and to pursue better alternatives with different skeleton structures other than natural lignin, herein we reported a synthetic soluble linear aromatic polymer, sodium polystyrene sulfonate (PSS), to mimic LS for enhancing the efficiency of enzymatic saccharification.ResultsThe role of PSS in enzymatic saccharification of pure cellulose and green liquor-pretreated poplar (GL-P) was explored by analyzing substrate enzymatic digestibility (SED) under different addition dosages and various pH media, along with LS for comparison. At the cellulase loading of 13.3 FPU/g-glucan, the glucose yield of GL-P increased from 53% for the control to 81.5% with PSS addition of 0.1 g/g-substrate. It outperformed LS with the addition of 0.2 g/g-substrate by 6.3%. In the pH range from 4.5 to 6, PSS showed a positive effect on lignocellulose saccharification with the optimum pH at 4.8, where the most pronounced SED of GL-P was achieved. The underlying mechanism was unveiled by measuring zeta potential and using Quartz Crystal Microbalance (QCM) and Multi-parametric Surface Plasmon Resonance (MP-SPR). The results confirmed that the complexes of cellulase and PSS were conjugated and the negatively supercharged complexes reduced non-productive binding effectively along with the improved saccharification efficiency. The thickness of PSS required to block the binding sites of cellulase film was less than half of that of LS, and the PSS adlayer on cellulase film is also more hydrated and with a much lower shear modulus than LS adlayer.ConclusionsPSS as LS analogue is effective for enhancing the biomass enzymatic saccharification of GL-pretreated poplar. PSS exhibited a severer inhibition on the enzymatic saccharification of pure cellulose, while a more positive effect on bioconversion of lignocellulose (GL-P) than LS. In addition, a much lower dosage is required by PSS. The dynamic enzymatic hydrolysis indicated PSS could prolong the processive activity of cellulase. The valid data stemmed from QCM and SPR expressed that PSS bound to cellulases and the as-formed complexes reduced the non-productive adsorption of cellulase onto substrate lignin more efficiently than LS due to its flexible skeleton and highly hydrated structure. Therefore, PSS is a promising alternative promoting agent for lignocellulose saccharification. From another perspective, the synthetic lignin mimics with controllable structures enable us to reach an in-depth understanding of the promotion mechanism of soluble lignins on enzymatic saccharification.

Multistep Structural and Chemical Evaluation of Sugarcane Baggase, Pretreated With Alkali for Enhancing the Enzymatic Saccharification by Cellulase and Xylanase of the Pseudomonas sp. CVB-10 (MK443365) and Bacillus paramycoides T4 (MN370035) Mix-Culture System

The enzymatic saccharification of sugarcane bagasse was significantly increased by alkali pretreatment under mild conditions. The effectiveness of different concentrations of alkali and acid pretreatment of sugarcane bagasse for improving the enzymatic saccharification of lignocellulose has been evaluated. The sugarcane bagasse was characterized to contain 39.52% celluloses, 25.63% hemicelluloses, and 30.36% lignin. After that, sugarcane bagasse was pretreated with 5 and 10% of H2SO4 and NaOH at 121°C for 60 min. FTIR, XRD, and SEM analyses also showed significant molecular and surface structure changes of the sugarcane bagasse with 10% NaOH. Maximum saccharification was 489.5 mg/g from 10% NaOH pretreatment followed by 322.75, 301.25, and 276.6 mg/g from 10% H2SO4, 5% NaOH, and 5% H2SO4, respectively, which were 55.1, 32.0, 27.1, and 20.6 times higher than the that of the control. Cellulase and xylanase produced by Pseudomonas sp. CVB-10 (MK443365) and Bacillus paramycoides T4 (MN370035) were used to hydrolyze the pretreated bagasse, and the optimal condition was determined to be 30 h of the enzymatic reaction with the 3:1 ratio of enzymes under the temperature of 55°C, pH 5.0, and substrate concentration of 3%, leading to celluloses and hemicelluloses conversion in the enzymatic hydrolysis/saccharification that is more proficient.

Bioprospecting of xylanase producing fungal strains: Multilocus phylogenetic analysis and enzyme activity profiling.

The study aims to explore potential xylanase-producing indigenous fungi isolated from soil and vegetable wastes containing plant degraded matter, reporting multilocus phylogenetic analysis and xylanase enzyme activity from selective strains. Four potential xylanolytic fungi were identified through distinct primary and secondary screening of 294 isolates obtained from the samples. Morphological characterization and multigene analysis (ITS rDNA, 18S rDNA, LSU rDNA, β-tubulin, and actin gene) confirmed them as Aspergillus sp. AUMS56, Aspergillus tubingensis AUMS60 and AUMS64, and Aspergillus fumigatus AUKEMS24; achieving crude xylanase activities (through submerged fermentation using corn cobs) of 18.9, 32.29, 30.68, and 15.82 U ml-1 , respectively. AUMS60 and AUMS64 (forming lineage with A. tubingensis and Aspergillusniger in the same phylogroup with 100% Bayesian posterior probability support) secreted single xylanase (Xyn60; 36 kDa) and multiple xylanases (Xyn64A and Xyn64B; 33.4 and 19.8 kDa) respectively, having pH optima of 6.0 and exhibiting maximal activity at 60°C. These enzymes were highly stable at 40°C (120 h) and retained more than 70% activity at 50°C and at pH 5-6 (upon 72 h incubation). Our analysis suggested these enzymes to be endoxylanases demonstrating substrate hydrolysis within 15 min of reaction and maximum efficiency of xylanases from AUMS60 and AUMS64 achieving 51.1% (13 h) and 52.2% (24 h) saccharification, respectively. They also showed enhanced catalytic activity with various cations. Based on our investigation on xylan hydrolysis, we believe that these xylanases may find significant industrial applications as they have a real potential of working as a bio-catalytic cocktail (patent file number: IN E1/38213/2020-DEL) for the enhanced saccharification of lignocelluloses.

Improved saccharification of pretreated lignocellulose by Clostridium thermocellum with the addition of surfactant, low loading of cellulose

Saccharification of lignocellulose is prerequisite for the biorefinery. Clostridium thermocellum is a promising candidate for the hydrolysis of lignocellulose. A study on the saccharification by Clostridium thermocellum combined with surfactant and cellulase addition was investigated. The reducing sugars content increased with increasing substrate loading in the fermentation of C. thermocllum with isooctyl alcohol polyoxyethylene ether (IAPE) addition. Based on IAPE addition, the glucose content of two-step saccharification was higher than that of the batch saccharification. Besides, the addition of IAPE and Triton X-100 had the same effect on saccharification. The saccharification performance was related to the substrate, especially the lignin content. For the fed-batch hydrolysis of 20 % (w/v) rice straw pretreated by hydrogen peroxide-acetic acid (HPAC), 66.9 g/L glucose and 19.5 g/L xylose were obtained by using C. thermocellum DSM 1313 and 1 FPU/g substrate cellulase with 0.1 % (w/v) IAPE addition, and the glucose and xylose yield reached 63.2 % and 37.4 % respectively.

Saccharification Potential of Transgenic Greenhouse- and Field-Grown Aspen Engineered for Reduced Xylan Acetylation.

High acetylation of xylan in hardwoods decreases their value as biorefinery feedstocks. To counter this problem, we have constitutively suppressed RWA genes encoding acetyl-CoA transporters using the 35S promoter, or constitutively and wood-specifically (using the WP promoter) expressed fungal acetyl xylan esterases of families CE1 (AnAXE1) and CE5 (HjAXE), to reduce acetylation in hybrid aspen. All these transformations improved the saccharification of wood from greenhouse-grown trees. Here, we describe the chemical properties and saccharification potential of the resulting lines grown in a five-year field trial, and one type of them (WP:AnAXE1) in greenhouse conditions. Chemically, the lignocellulose of the field- and greenhouse-field-grown plants slightly differed, but the reductions in acetylation and saccharification improvement of engineered trees were largely maintained in the field. The main novel phenotypic observation in the field was higher lignification in lines with the WP promoter than those with the 35S promoter. Following growth in the field, saccharification glucose yields were higher from most transformed lines than from wild-type (WT) plants with no pretreatment, but there was no improvement in saccharification with acid pretreatment. Thus, acid pretreatment removes most recalcitrance caused by acetylation. We found a complex relationship between acetylation and glucose yields in saccharification without pretreatment, suggesting that other variables, for example, the acetylation pattern, affect recalcitrance. Bigger gains in glucose yields were observed in lines with the 35S promoter than in those with the WP promoter, possibly due to their lower lignin content. However, better lignocellulose saccharification of these lines was offset by a growth penalty and their glucose yield per tree was lower. In a comparison of the best lines with each construct, WP:AnAXE1 provided the highest glucose yield per tree from saccharification, with and without pretreatment, WP:HjAXE yields were similar to those of WT plants, and yields of lines with other constructs were lower. These results show that lignocellulose properties of field-grown trees can be improved by reducing cell wall acetylation using various approaches, but some affect productivity in the field. Thus, better understanding of molecular and physiological consequences of deacetylation is needed to obtain quantitatively better results.

Treating maize plants with benzohydrazide increases saccharification of lignocellulose: A non-transgenic approach to improve cellulosic ethanol production

Treating maize plants with benzohydrazide increases saccharification of lignocellulose: A non-transgenic approach to improve cellulosic ethanol production

Diverse Banana Pseudostems and Rachis Are Distinctive for Edible Carbohydrates and Lignocellulose Saccharification towards High Bioethanol Production under Chemical and Liquid Hot Water Pretreatments

Banana is a major fruit crop throughout the world with abundant lignocellulose in the pseudostem and rachis residues for biofuel production. In this study, we collected a total of 11 pseudostems and rachis samples that were originally derived from different genetic types and ecological locations of banana crops and then examined largely varied edible carbohydrates (soluble sugars, starch) and lignocellulose compositions. By performing chemical (H2SO4, NaOH) and liquid hot water (LHW) pretreatments, we also found a remarkable variation in biomass enzymatic saccharification and bioethanol production among all banana samples examined. Consequently, this study identified a desirable banana (Refen1, subgroup Pisang Awak) crop containing large amounts of edible carbohydrates and completely digestible lignocellulose, which could be combined to achieve the highest bioethanol yields of 31–38% (% dry matter), compared with previously reported ones in other bioenergy crops. Chemical analysis further indicated that the cellulose CrI and lignin G-monomer should be two major recalcitrant factors affecting biomass enzymatic saccharification in banana pseudostems and rachis. Therefore, this study not only examined rich edible carbohydrates for food in the banana pseudostems but also detected digestible lignocellulose for bioethanol production in rachis tissue, providing a strategy applicable for genetic breeding and biomass processing in banana crops.

Coordinated β-glucosidase activity with the cellulosome is effective for enhanced lignocellulose saccharification

Consolidated bio-saccharification (CBS) technology employs cellulosome-producing bacterial cells, rather than fungal cellulases, as biocatalysts for cost-effective production of lignocellulosic sugars. Extracellular β-glucosidase (BGL) expression in the whole-cell arsenal is indispensable, due to severe cellobiose inhibition of the cellulosome. However, high-level BGL expression in Clostridium thermocellum is challenging, and the optimal BGL production level for efficient cellulose saccharification is currently unknown. Herein, we obtained new CBS biocatalysts by transforming BGL-expressing plasmids into C. thermocellum, which produced abundant BGL proteins and hydrolyzed cellulose effectively. The optimal ratio of extracellular BGL-to-cellulosome activity was determined to be in a range of 5.5 to 21.6. Despite the critical impact of BGL, both excessive BGL expression and its assembly on the cellulosome via type I cohesin-dockerin interaction led to reduced cellulosomal activity, which further confirmed the importance of coordinated BGL expression with the cellulosome. This study will further promote industrial CBS application in lignocellulose conversion.

Comparison of Low-Temperature Alkali/Urea Pretreatments for Ethanol Production from Wheat Straw

NaOH/urea (NU) pretreatment at lower than 0 °C has been frequently applied for improving bio-conversion of lignocellulose, but the wastewater generated from the pretreatment process is hard to dispose. KOH/urea (KU) pretreatment for enhancing bioconversion of lignocellulose has recently attracted researchers’ attention due to the recycling of wastewater for facilitating crops’ growth. This study compared the effects of NU and KU pretreatments at cold conditions on the enzymatic hydrolysis and bioethanol yield from wheat straw (WS). By using response surface methodology an optimal pretreatment with an equal ratio of alkali/urea (4% w/v) at −20 °C for 3 h was established. The enzymatic hydrolysis of KU-treated WS was 81.17%, which was similar to that of NU-treated WS (83.72%) under the same condition. It means that KU pretreatment has equal ability to NU pretreatment to improve enzymatic saccharification of lignocellulose. KU pretreatment has the promising potential to replace NU pretreatment for facilitating bioconversion of lignocellulose in cold conditions due to the clean way to recycle its wastewater as fertilizer for crop growth. Hence, KU pretreatment combined with enzymatic hydrolysis and fermentation could be a promising green way to cellulosic ethanol production with zero waste emission.

Precise high-throughput online near-infrared spectroscopy assay to determine key cell wall features associated with sugarcane bagasse digestibility

BackgroundSugarcane is one of the most crucial energy crops that produces high yields of sugar and lignocellulose. The cellulose crystallinity index (CrI) and lignin are the two kinds of key cell wall features that account for lignocellulose saccharification. Therefore, high-throughput screening of sugarcane germplasm with excellent cell wall features is considered a promising strategy to enhance bagasse digestibility. Recently, there has been research to explore near-infrared spectroscopy (NIRS) assays for the characterization of the corresponding wall features. However, due to the technical barriers of the offline strategy, it is difficult to apply for high-throughput real-time analyses. This study was therefore initiated to develop a high-throughput online NIRS assay to rapidly detect cellulose crystallinity, lignin content, and their related proportions in sugarcane, aiming to provide an efficient and feasible method for sugarcane cell wall feature evaluation.ResultsA total of 838 different sugarcane genotypes were collected at different growth stages during 2018 and 2019. A continuous variation distribution of the near-infrared spectrum was observed among these collections. Due to the very large diversity of CrI and lignin contents detected in the collected sugarcane samples, seven high-quality calibration models were developed through online NIRS calibration. All of the generated equations displayed coefficient of determination (R2) values greater than 0.8 and high ratio performance deviation (RPD) values of over 2.0 in calibration, internal cross-validation, and external validation. Remarkably, the equations for CrI and total lignin content exhibited RPD values as high as 2.56 and 2.55, respectively, indicating their excellent prediction capacity. An offline NIRS assay was also performed. Comparable calibration was observed between the offline and online NIRS analyses, suggesting that both strategies would be applicable to estimate cell wall characteristics. Nevertheless, as online NIRS assays offer tremendous advantages for large-scale real-time screening applications, it could be implied that they are a better option for high-throughput cell wall feature prediction.ConclusionsThis study, as an initial attempt, explored an online NIRS assay for the high-throughput assessment of key cell wall features in terms of CrI, lignin content, and their proportion in sugarcane. Consistent and precise calibration results were obtained with NIRS modeling, insinuating this strategy as a reliable approach for the large-scale screening of promising sugarcane germplasm for cell wall structure improvement and beyond.

The characteristics of insoluble softwood substrates affect fungal morphology, secretome composition, and hydrolytic efficiency of enzymes produced by Trichoderma reesei

BackgroundOn-site enzyme production using Trichoderma reesei can improve yields and lower the overall cost of lignocellulose saccharification by exploiting the fungal gene regulatory mechanism that enables it to continuously adapt enzyme secretion to the substrate used for cultivation. To harness this, the interrelation between substrate characteristics and fungal response must be understood. However, fungal morphology or gene expression studies often lack structural and chemical substrate characterization. Here, T. reesei QM6a was cultivated on three softwood substrates: northern bleached softwood Kraft pulp (NBSK) and lodgepole pine pretreated either by dilute-acid-catalyzed steam pretreatment (LP-STEX) or mild alkaline oxidation (LP-ALKOX). With different pretreatments of similar starting materials, we presented the fungus with systematically modified substrates. This allowed the elucidation of substrate-induced changes in the fungal response and the testing of the secreted enzymes’ hydrolytic strength towards the same substrates.ResultsEnzyme activity time courses correlated with hemicellulose content and cellulose accessibility. Specifically, increased amounts of side-chain-cleaving hemicellulolytic enzymes in the protein produced on the complex substrates (LP-STEX; LP-ALKOX) was observed by secretome analysis. Confocal laser scanning micrographs showed that fungal micromorphology responded to changes in cellulose accessibility and initial culture viscosity. The latter was caused by surface charge and fiber dimensions, and likely restricted mass transfer, resulting in morphologies of fungi in stress. Supplementing a basic cellulolytic enzyme mixture with concentrated T. reesei supernatant improved saccharification efficiencies of the three substrates, where cellulose, xylan, and mannan conversion was increased by up to 27, 45, and 2800%, respectively. The improvement was most pronounced for proteins produced on LP-STEX and LP-ALKOX on those same substrates, and in the best case, efficiencies reached those of a state-of-the-art commercial enzyme preparation.ConclusionCultivation of T. reesei on LP-STEX and LP-ALKOX produced a protein mixture that increased the hydrolytic strength of a basic cellulase mixture to state-of-the-art performance on softwood substrates. This suggests that the fungal adaptation mechanism can be exploited to achieve enhanced performance in enzymatic hydrolysis without a priori knowledge of specific substrate requirements.

Cloning, expression, biochemical characterization, and molecular docking studies of a novel glucose tolerant β-glucosidase from Saccharomonospora sp. NB11

Most of the presently known β-glucosidases are sensitive to end-product inhibition by glucose, restricting their potential use in many industrial applications. Identification of novel glucose tolerant β-glucosidase can prove a pivotal solution to eliminate end-product inhibition and enhance the overall lignocellulosic saccharification process. In this study, a novel gene encoding β-glucosidase BglNB11 of 1405bp was identified in the genome of Saccharomonospora sp. NB11 and was successfully cloned and heterologously expressed in E. coli BL21 (DE3).The presence of conserved amino acids; NEPW and TENG indicated that BglNB11 belonged to GH1 β-glucosidases. The recombinant enzyme was purified using a Ni–NTA column, with the molecular mass of 51 kDa, using SDS‐PAGE analysis. BglNB11 showed optimum activity at 40 °C and pH 7 and did not require any tested co‐factors for activation. The kinetic values, Km, Vmax, kcat, and kcat/Km of purified enzyme were 0.4037 mM, 5735.8 μmol/min/mg, 5042.16 s−1 and 12487.71 s−1 mM−1, respectively. The enzyme was not inhibited by glucose to a concentration of 4 M but was slightly stimulated in the presence of glucose. Molecular docking of BglNB11 with glucose suggested that the relative binding position of glucose in the active site channel might be responsible for modulating end product tolerance and stimulation. β-glucosidase from BglNB11 is an excellent enzyme with high catalytic efficiency and enhanced glucose tolerance compared to many known glucose tolerant β-glucosidases. These unique properties of BglNB11 make it a prime candidate to be utilized in many biotechnological applications.

Alkaline incubation improves the saccharification of poplar after sodium chlorite pretreatment with ultra-low cellulase loading

The limitation of xylan hydrolysis is a characteristic problem of sodium chlorite (SC) pretreatment, which greatly restricted the saccharification of SC-pretreated lignocelluloses. In this work, the effect of alkali incubation on the xylan hydrolysis of SC-pretreated poplar was investigated. SC pretreatment of poplar removed 84.7% lignin, which yielded 71.2% glucose and 60.0% xylose under a cellulase loading of 20 filter paper units (FPU)/g dry matter (DM). The subsequent alkali incubation reduced the acetyl group, surface lignin, and surface chlorine contents of SC-pretreated poplar. Glucose and xylose yields were increased to 92.4% and 86.3%, respectively, by incubating SC-pretreated samples with 1.0% NaOH at 50 °C for 1 h using a cellulase loading of 2 FPU/g DM. Both Tween 80 and xylanase improved the enzymatic hydrolysis yield of SC-pretreated poplar. Alkali incubation showed a 9-fold decrease in cellulase loading and increased the xylose yield of SC-pretreated poplar by 119.8%. This work overcame the limitation of xylan hydrolysis in sodium chlorite-pretreated lignocelluloses and represented a novel method to efficiently saccharification of poplar with ultra-low cellulase loading.

The known unknowns in lignin biosynthesis and its engineering to improve lignocellulosic saccharification efficiency

Biofuels produced from lignocellulosic biomass provide energy security and reduce greenhouse gas emissions with positive impacts on sustainability. Lignocellulose byproducts are expected to impact the production of cellulosic ethanol. However, lignin is a major factor imposing biomass recalcitrance to saccharification. Although structural phenolics can be removed from lignocellulosic biomass, physical and chemical pretreatments are expensive. In turn, the structure and composition of lignin is very flexible. Up- and downregulation of genes and, thereby, enzymes involved in core and non-core lignin biosynthesis can reduce lignin content and improve biomass saccharification. Alternatively, insertion of heterologous genes to the phenylpropanoid pathway may lead to the incorporation of alternative monomers into lignin, creating desirable properties and preserving the biological roles of lignin with a positive effect on saccharification. Studies reporting the effect of fine regulation on lignin structures are mapping how plants can be transformed to enhance saccharification while preserving productivity. Based on these findings and events, this review updates the state-of-art changes in lignin biosynthesis to improve saccharification efficiency. The main findings reported over the last decade were summarized to permit researchers an overview of this relevant scientific subject.

Novel β-glucanases along with xylanase identified in Thermomyces lanuginosus secretome for enhanced saccharification of different lignocellulosics

The LC/MS-based exo-proteome analysis of thermophilic fungus Thermomyces lanuginosus showed 22.59% (40 proteins) of the total identified proteins (177) as CAZymes (carbohydrate-active enzymes). The CAZymes were primarily represented by glycosyl hydrolases (72.5%) belonging to 21 different GH families. Xylanase (GH11) was found to be the major protein (24.3%) in addition to the β-glucanase and another complex polysaccharide-degrading (carbohydrate-active enzymes) CAZymes in the secretome. FPLC-based fractionation of secretome was employed to identify proteins responsible for enhancing the catalytic efficacy of Cellic Ctec2 during hydrolysis of acid pre-treated lignocellulosics and eventually xylanase (GH11) and β-glucanases (GH64 and GH81) were identified and purified. The purified xylanase when supplemented with Cellic Ctec2 resulted in 2.05-, 1.79-, and 1.60-fold increase in the release of glucose from acid-treated bagasse at 10, 15, and 20% substrate loading rate, respectively when compared with the control. Similarly, enhanced hydrolysis of acid pre-treated corn residue and rice straw was observed upon supplementation with xylanase. Spiking benchmark cellulase with purified GH64 and GH81 also resulted in 1.18- and 1.23-fold enhanced hydrolysis of acid pre-treated sugarcane bagasse. The supplementation of xylanase (1000 units/g substrate) was found to reduce the Cellic Ctec2 loading rate from 36 mg/g substrate by 2.70-, 2.88-, and 2.57-fold required for hydrolysis of acid treated bagasse, corn residue, and rice straw, respectively, at high substrate loading rate (20%) and thus, it is an important candidate for economizing the 2G ethanol process.

Natural surfactant-aided dilute sulfuric acid pretreatment of waste wheat straw to enhance enzymatic hydrolysis efficiency

Traditional surfactants have been reported to enhance enzymatic saccharification of lignocellulose, however, it is important to transfer these findings to a system that uses a high-efficiency and low-toxicity natural surfactant instead. In this work, a novel hybrid method involving use of the natural surfactant (humic acid, HA) during mild acid (H2SO4) pretreatment was developed for waste wheat straw (WWS) biorefinery. The HA was found to help remove lignin up to 40.6%, and hemicellulose up to 96.2%. As a result of these changes, the enzymatic hydrolysis efficiency reached as high as 92.9%. The success of enzymatic digestion was partly attributed to the improved accessibility of cellulose to cellulase and changes in lignocellulose structures. We anticipate that these findings will be used to further evaluate HA as a beneficial surfactant in biorefinery pretreatment processes, and perhaps spur others to identify other natural surfactants that may prove even more effective.