Biomass Enzymatic Saccharification Research Articles

Upgrading pectin methylation for consistently enhanced biomass enzymatic saccharification and cadmium phytoremediation in rice Ospmes site-mutants

Crop straws provide enormous biomass residues applicable for biofuel production and trace metal phytoremediation. However, as lignocellulose recalcitrance determines a costly process with potential secondary waste liberation, genetic modification of plant cell walls is deemed as a promising solution. Although pectin methylation plays an important role for plant cell wall construction and integrity, little is known about its regulation roles on lignocellulose hydrolysis and trace metal elimination. In this study, we initially performed a typical CRISPR/Cas9 gene-editing for site mutations of OsPME31, OsPME34 and OsPME79 in rice, and then determined significantly upgraded pectin methylation degrees in the young seedlings of three distinct site-mutants compared to their wild type. We then examined distinctively improved lignocellulose recalcitrance in three mutants including reduced cellulose levels, crystallinity and polymerization or raised hemicellulose deposition and cellulose accessibility, which led to specifically enlarged biomass porosity either for consistently enhanced biomass enzymatic saccharification under mild alkali pretreatments or for cadmium (Cd) accumulation up to 2.4-fold. Therefore, this study proposed a novel model to elucidate how pectin methylation could play a unique enhancement role for both lignocellulose enzymatic hydrolysis and Cd phytoremediation, providing insights into precise pectin modification for effective biomass utilization and efficient trace metal exclusion.

Synergistic β-glucosidases for improving cellulases recyclability and biomass enzymatic saccharification in wheat straw

Cellulases required for the conversion of biomass to sugars contribute to a significant part of the final bioethanol cost in the overall lignocellulosic biorefinery. It has been proven that recycling cellulases for saccharification is a viable method to lower the cost of the enzymatic hydrolysis process. Any method for recycling enzymes must begin with a stable cellulase cocktail that can withstand operational recycling conditions. The majority of commercial cellulase preparations contain a small quantity of β-glucosidases (BG), which lowers overall recyclability and causes low hydrolysis yields. This study examines the use of synergistic β-glucosidases (AtuBG3II and PfuBG1 from Aspergillus tubingensis and Pyrococcus furiosus, respectively) to enhance the recyclability of cellulases during the hydrolysis process. It was able to recycle a significant amount of cellulases by recycling the insoluble solid fraction and liquid fraction following enzymatic hydrolysis of two-step treated wheat straw (ts-WS). In addition, the in-house cellulase cocktail (C-Mix) retained β-glucosidase activity and demonstrated glucose productivity of 18.9 g/L/h at 5% loading and enzyme efficiency (0.41 g of glucose per mg of protein) as AtuBG3II and PfuBG1 did not adsorb significantly to the biomass. This resulted in a 3.5-fold improvement in the recyclability and a 33% increase in the hydrolysis yield during the study. Therefore, all these results signify better cellulase recovery using two-step treated lignocellulose and stable cellulase cocktails, demonstrating great potential for enzymatic recycling.

BsEXLX of engineered Trichoderma reesei strain as dual-active expansin to boost cellulases secretion for synergistic enhancement of biomass enzymatic saccharification in corn and Miscanthus straws

In this study, bacterial BsEXLE1 gene was overexpressed into T. reesei (Rut-C30) to generate a desirable engineered TrEXLX10 strain. While incubated with alkali-pretreated Miscanthus straw as carbon source, the TrEXLX10 secreted the β-glucosidases, cellobiohydrolases and xylanses with activities raised by 34%, 82% and 159% compared to the Rut-C30. Supplying EXLX10-secreted crude enzymes and commercial mixed-cellulases for two-step lignocellulose hydrolyses of corn and Miscanthus straws after mild alkali pretreatments, this work measured consistently higher hexoses yields released by the EXLX10-secreted enzymes for synergistic enhancements of biomass saccharification in all parallel experiments examined. Meanwhile, this study detected that the expansin, purified from EXLX10-secreted solution, was of exceptionally high binding activities with wall polymers, and further determined its independent enhancement for cellulose hydrolysis. Therefore, this study raised a mechanism model to highlight EXLX/expansin dual-activation roles for both secretion of stable biomass-degradation enzymes at high activity and biomass enzymatic saccharification in bioenergy crops.

Full-Chain FeCl3 Catalyzation Is Sufficient to Boost Cellulase Secretion and Cellulosic Ethanol along with Valorized Supercapacitor and Biosorbent Using Desirable Corn Stalk

Cellulosic ethanol is regarded as a perfect additive for petrol fuels for global carbon neutralization. As bioethanol conversion requires strong biomass pretreatment and overpriced enzymatic hydrolysis, it is increasingly considered in the exploration of biomass processes with fewer chemicals for cost-effective biofuels and value-added bioproducts. In this study, we performed optimal liquid-hot-water pretreatment (190 °C for 10 min) co-supplied with 4% FeCl3 to achieve the near-complete biomass enzymatic saccharification of desirable corn stalk for high bioethanol production, and all the enzyme-undigestible lignocellulose residues were then examined as active biosorbents for high Cd adsorption. Furthermore, by incubating Trichoderma reesei with the desired corn stalk co-supplied with 0.05% FeCl3 for the secretion of lignocellulose-degradation enzymes in vivo, we examined five secreted enzyme activities elevated by 1.3-3.0-fold in vitro, compared to the control without FeCl3 supplementation. After further supplying 1:2 (w/w) FeCl3 into the T. reesei-undigested lignocellulose residue for the thermal-carbonization process, we generated highly porous carbon with specific electroconductivity raised by 3-12-fold for the supercapacitor. Therefore, this work demonstrates that FeCl3 can act as a universal catalyst for the full-chain enhancement of biological, biochemical, and chemical conversions of lignocellulose substrates, providing a green-like strategy for low-cost biofuels and high-value bioproducts.

Large-scale screening of diverse barely lignocelluloses for simultaneously upgrading biomass enzymatic saccharification and plant lodging resistance coupled with near-infrared spectroscopic assay

Barley is an important economical crop for food and beer industry with abundant lignocellulose residues convertible for biofuels and biochemicals. Although lignocellulose recalcitrance provides mechanic strength to maintain plant lodging resistance for biomass production, it also leads to a costly process for biofuel production. By collecting total 94 barely samples with diverse genetic backgrounds, this study examined large variations of lignocellulose levels, sugars yields and lodging index values, and further identified that the cellulose level was a crucial factor either positively accounting for biomass enzymatic saccharification or negatively affecting plant lodging resistance. Despite lignin level showed slight impact on lodging resistance, it could negatively affect biomass saccharification in barley samples examined. Regarding normal distributions of barley samples, this study generated optimal near-infrared spectroscopy (NIRS) equations applicable for rapid assessment of lignocellulose levels, sugars yields and lodging-related parameters with high determination coefficients for external validation (0.71–0.97), cross-validation (0.89–0.95) and calibration (0.89–0.95), and acceptable ratio performance deviation (2.85–4.35). Notably, the established NIRS models could be employed for selection of the desirable barley samples that are simultaneously of better lodging resistance and higher biomass saccharification. Therefore, this study has established a powerful approach applicable for both rapidly screening lignocellulose-related traits in crop breeding and precisely selecting the ideal lignocellulose substrates for green-like biomass process into cost-effective biofuels and value-added bioproducts.

Overexpression of PtoMYB115 improves lignocellulose recalcitrance to enhance biomass digestibility and bioethanol yield by specifically regulating lignin biosynthesis in transgenic poplar

BackgroundWoody plants provide the most abundant biomass resource that is convertible for biofuels. Since lignin is a crucial recalcitrant factor against lignocellulose hydrolysis, genetic engineering of lignin biosynthesis is considered as a promising solution. Many MYB transcription factors have been identified to involve in the regulation of cell wall formation or phenylpropanoid pathway. In a previous study, we identified that PtoMYB115 contributes to the regulation of proanthocyanidin pathway, however, little is known about its role in lignocellulose biosynthesis and biomass saccharification in poplar.ResultsHere, we detected the changes of cell wall features and examined biomass enzymatic saccharification for bioethanol production under various chemical pretreatments in PtoMYB115 transgenic plants. We reported that PtoMYB115 might specifically regulate lignin biosynthesis to affect xylem development. Overexpression of PtoMYB115 altered lignin biosynthetic gene expression, resulting in reduced lignin deposition, raised S/G and beta-O-4 linkage, resulting in a significant reduction in cellulase adsorption with lignin and an increment in cellulose accessibility. These alterations consequently improved lignocellulose recalcitrance for significantly enhanced biomass saccharification and bioethanol yield in the PtoMYB115-OE transgenic lines. In contrast, the knockout of PtoMYB115 by CRISPR/Cas9 showed reduced woody utilization under various chemical pretreatments.ConclusionsThis study shows that PtoMYB115 plays an important role in specifically regulating lignin biosynthesis and improving lignocellulose features. The enhanced biomass saccharification and bioethanol yield in the PtoMYB115-OE lines suggests that PtoMYB115 is a candidate gene for genetic modification to facilitate the utilization of biomass.

CEF3 is involved in membrane trafficking and essential for secondary cell wall biosynthesis and its mutation enhanced biomass enzymatic saccharification in rice

BackgroundAs one of the most important staple food crops, rice produces large of agronomic biomass residues that contain lots of secondary cell walls (SCWs). Membrane trafficking plays key roles in SCWs biosynthesis, but information association membrane trafficking and SCWs formation in plants is limited.ResultsIn this study, we report the function characterization of a rice mutant, culm easily fragile 3 (cef3), that exhibits growth retardation and fragile culm phenotype with significantly altered cell wall composition and reduced secondary wall thickness. Map-based cloning revealed that CEF3 encodes a homologous protein of Arabidopsis STOMATAL CYTOKINESIS DEFECTIVE2 (SCD2). The saccharification assays revealed that CEF3 mutation can improve biomass enzymatic saccharification. Expression pattern analysis indicated that CEF3 is ubiquitously expressed in many organs at different developmental stages. Subcellular localization revealed that CEF3 is a Golgi-localized protein. The FM4-64 uptake assay revealed CEF3 is involved in endocytosis. Furthermore, mutation of CEF3 not only affected cellulose synthesis-related genes expression, but also altered the abundance of cellulose synthase catalytic subunit 9 (OsCESA9) in the PM and in the endomembrane systems.ConclusionsThis study has demonstrated that CEF3 participates in the membrane trafficking that is essential for normal cellulose and other polysaccharides biosynthesis of the secondary cell wall, thereby manipulation of CEF3 could alter cellulose content and enhance biomass enzymatic saccharification in rice plants. Therefore, the study of the function of CEF3 can provide a strategy for genetic modification of SCWs in bioenergy crops.

Specific lignin and cellulose depolymerization of sugarcane bagasse for maximum bioethanol production under optimal chemical fertilizer pretreatment with hemicellulose retention and liquid recycling

By screening out total nine sugarcane cultivars that contain directly-fermentable sugars, this study examined largely varied hexoses yields from direct enzymatic hydrolysis of bagasse residues. Using two representative sugarcane bagasse substrates, this study sorted out optimal K3PO4 pretreatments by performing classic response surface methodology, which respectively extracted 58% and 63% lignin and significantly reduced cellulose CrI and DP values in two pretreated bagasse residues. These consequently led to remarkably raised cellulose accessibility by 1.4- and 3.6 folds for near-complete biomass enzymatic saccharification with hexoses yields close to 100% (% cellulose). Notably, unlike physical (hot water, steam explosion) and chemical (acid, alkali) pretreatments as previously examined, this optimal pretreatment (6.4% K3PO4, 144.0 °C, 1 h) could not significantly extract arabinose-rich hemicellulose, suggesting that almost all xylose of hemicelluloses was applicable for engineered yeast co-fermentation to achieve maximum bioethanol at 29% (% dry matter) without the necessity of solid-liquid separation, water washing, and detoxification process. Furthermore, this study collected all K3PO4 liquid waste as chemical fertilizer into rice culture for much enhanced plant growth and biomass production up to 1.5 folds. Therefore, this work has proposed a mechanism model to highlight a cost-effective and green-like strategy for high yield of bioethanol with potential value-added bioproducts by using sugarcane bagasse and other lignocellulose residues.

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.

Efficient saccharification of wheat straw pretreated by solid particle-assisted ball milling with waste washing liquor recycling

Wheat straw was pretreated using ball milling (BM) promoted by solid particles (NaOH, NaCl, citric acid). NaOH showed the best synergistic interaction effect, due to the breakage of β-1,4-glycosidic bonds among cellulose molecules by the alkali solid particles induced by BM. NaOH-BM pretreatment decreased the straw crystallinity from 46% to 21.4% and its average particle size from 398.3 to 50.6 μm in 1 h. After 4 h milling, the reducing-end concentration of cellulose increased by 3.8 times from 12.5 to 60.2 μM, with glucose yield increased by 2.1 times from 26.6% to 82.4% for 72 h enzymatic hydrolysis at cellulase loading of 15 FPU/g dry substrate. The pretreatment washing liquor was recycled for the re-treatment of partially pretreated biomass at 121 °C for 30 min, resulting in 99.4% glucose yield by enzymatic hydrolysis. BM assisted with alkali particles was an effective approach for improving biomass enzymatic saccharification.

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.

Down-regulation of OsMYB103L distinctively alters beta-1,4-glucan polymerization and cellulose microfibers assembly for enhanced biomass enzymatic saccharification in rice

BackgroundAs a major component of plant cell walls, cellulose provides the most abundant biomass resource convertible for biofuels. Since cellulose crystallinity and polymerization have been characterized as two major features accounting for lignocellulose recalcitrance against biomass enzymatic saccharification, genetic engineering of cellulose biosynthesis is increasingly considered as a promising solution in bioenergy crops. Although several transcription factors have been identified to regulate cellulose biosynthesis and plant cell wall formation, much remains unknown about its potential roles for genetic improvement of lignocellulose recalcitrance.ResultsIn this study, we identified a novel rice mutant (Osfc9/myb103) encoded a R2R3-MYB transcription factor, and meanwhile generated OsMYB103L-RNAi-silenced transgenic lines. We determined significantly reduced cellulose levels with other major wall polymers (hemicellulose, lignin) slightly altered in mature rice straws of the myb103 mutant and RNAi line, compared to their wild type (NPB). Notably, the rice mutant and RNAi line were of significantly reduced cellulose features (crystalline index/CrI, degree of polymerization/DP) and distinct cellulose nanofibers assembly. These alterations consequently improved lignocellulose recalcitrance for significantly enhanced biomass enzymatic saccharification by 10–28% at p < 0.01 levels (n = 3) after liquid hot water and chemical (1% H2SO4, 1% NaOH) pretreatments with mature rice straws. In addition, integrated RNA sequencing with DNA affinity purification sequencing (DAP-seq) analyses revealed that the OsMYB103L might specifically mediate cellulose biosynthesis and deposition by regulating OsCesAs and other genes associated with microfibril assembly.ConclusionsThis study has demonstrated that down-regulation of OsMYB103L could specifically improve cellulose features and cellulose nanofibers assembly to significantly enhance biomass enzymatic saccharification under green-like and mild chemical pretreatments in rice. It has not only indicated a powerful strategy for genetic modification of plant cell walls in bioenergy crops, but also provided insights into transcriptional regulation of cellulose biosynthesis in plants.

Integrated NIRS and QTL assays reveal minor mannose and galactose as contrast lignocellulose factors for biomass enzymatic saccharification in rice

BackgroundIdentifying lignocellulose recalcitrant factors and exploring their genetic properties are essential for enhanced biomass enzymatic saccharification in bioenergy crops. Despite genetic modification of major wall polymers has been implemented for reduced recalcitrance in engineered crops, it could most cause a penalty of plant growth and biomass yield. Alternatively, it is increasingly considered to improve minor wall components, but an applicable approach is required for efficient assay of large population of biomass samples. Hence, this study collected total of 100 rice straw samples and characterized all minor wall monosaccharides and biomass enzymatic saccharification by integrating NIRS modeling and QTL profiling.ResultsBy performing classic chemical analyses and establishing optimal NIRS equations, this study examined four minor wall monosaccharides and major wall polymers (acid-soluble lignin/ASL, acid-insoluble lignin/AIL, three lignin monomers, crystalline cellulose), which led to largely varied hexoses yields achieved from enzymatic hydrolyses after two alkali pretreatments were conducted with large population of rice straws. Correlation analyses indicated that mannose and galactose can play a contrast role for biomass enzymatic saccharification at P < 0.0 l level (n = 100). Meanwhile, we found that the QTLs controlling mannose, galactose, lignin-related traits, and biomass saccharification were co-located. By combining NIRS assay with QTLs maps, this study further interpreted that the mannose-rich hemicellulose may assist AIL disassociation for enhanced biomass enzymatic saccharification, whereas the galactose-rich polysaccharides should be effectively extracted with ASL from the alkali pretreatment for condensed AIL association with cellulose microfibrils.ConclusionsBy integrating NIRS assay with QTL profiling for large population of rice straw samples, this study has identified that the mannose content of wall polysaccharides could positively affect biomass enzymatic saccharification, while the galactose had a significantly negative impact. It has also sorted out that two minor monosaccharides could distinctively associate with lignin deposition for wall network construction. Hence, this study demonstrates an applicable approach for fast assessments of minor lignocellulose recalcitrant factors and biomass enzymatic saccharification in rice, providing a potential strategy for bioenergy crop breeding and biomass processing.

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.

Combined steam explosion and optimized green-liquor pretreatments are effective for complete saccharification to maximize bioethanol production by reducing lignocellulose recalcitrance in one-year-old bamboo

Bamboo is a fast-growing perennial plant rich at lignocellulose convertible for biofuels and biochemical production. Despite various physical and chemical pretreatments have been implemented for bamboo biomass utilization, it becomes essential to explore optimal technology for complete biomass saccharification to maximize bioethanol production in the desirable bamboo substrates. In this study, the steam explosion followed with optimized green-liquor pretreatments were conducted in different-year-old bamboo samples using response surface methodology. Compared to the older samples, the one-year-old bamboo (PhY1) showed a complete biomass enzymatic saccharification with hexoses yield of 100.0% (% cellulose), leading to the highest bioethanol yield of 20.3% (% dry biomass) achieved among all previously-reported bamboo processes. Notably, those combined pretreatments could not only cause an effective co-extraction of hemicellulose-lignin complexes, but also distinctively modify major wall polymer features (cellulose DP and accessibility, hemicellulosic Xyl/Ara and lignin S/G) for significantly reduced lignocellulose recalcitrance, which should lead to an integrated enhancement to biomass enzymatic saccharification in the PhY1 bamboo sample. Therefore, this study has demonstrated a powerful strategy for a green-like biomass process, providing an applicable technology to achieve maximum bioethanol production in bamboo and other lignin-rich bioenergy crops.

Recyclable cascading of arsenic phytoremediation and lead removal coupled with high bioethanol production using desirable rice straws

Crop straws provide enormous lignocellulose residues convertible to biofuels, whereas Arsenic (As) and lead (Pb) pollute agricultural soil and water source. Hence, it becomes important to explore a compelling biomass process technology for both bioethanol production and trace metal remediation. In this study, we selected classic rice cultivars that distinctively accumulated As from 10.25 to 16.18 μg/g in their mature straws from the As-polluted farming soils, being up to 10-fold higher than those of the previously-reported rice straws and other crops. By performing various chemical pretreatments with the As-accumulated rice straws, this study sorted out that the optimal two-step 2%H2SO4 and 2%NaOH pretreatments could cause almost complete biomass enzymatic saccharification for high bioethanol production in all rice straws examined, along with a full As release for As recycling as potential high-value crystallized chemical. Furthermore, all remaining lignin-rich solid resides were finally examined to act as active biosorbent for Pb adsorption, with the optimal Pb adsorption conditions established (1 g/L adsorbent dose at pH6.0 and room temperature). Hence, this study has not only demonstrated a cascading-like strategy for efficient As and Pb remediation in agricultural soil and water source, but it has also provided a value-added approach for complete utilization of crop straws towards high bioethanol production.

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.

Ecologically adaptable Populus simonii is specific for recalcitrance‐reduced lignocellulose and largely enhanced enzymatic saccharification among woody plants

AbstractWoody plants provide enormous biomass resource convertible for biofuels and bioproducts, but they are of typical lignified secondary cell walls with strong recalcitrance against biomass degradation. It thus becomes critical to find out the desirable woody plant enabled for efficient biomass enzymatic saccharification. In this study, we collected totally seven biomass samples from three major hardwood species showing worldwide geographic distributions and diverse cell wall compositions. Under acid (H2SO4) and alkali (NaOH, CaO) pretreatments, all biomass samples showed remarkably enhanced enzymatic saccharification, but the Populus simonii species had the highest hexoses yields from all pretreatments performed. In particular, the mild and green‐like pretreatment (10% CaO, 50°C) could lead to more than 70% cellulose degradation into fermentable hexoses in the P. simonii species, but only 24%–38% cellulose digestions were examined in the other six samples. Importantly, the P. simonii species is of the lowest ratios of lignin S/G and hemicellulose Xyl/Ara among seven samples, being two major factors accountable for much improved lignocellulose recalcitrance. These consequently caused the most reduced cellulose DP in the pretreated P. simonii residues for enhanced biomass saccharification. Furthermore, this study performed a genome‐wide profiling of gene expression to confirm distinct wall polymer biosynthesis and biomass metabolism in the P. simonii species, consistent with its significantly improved lignocellulose recalcitrance. Therefore, this study has found out the desirable model of woody plants for efficient biomass enzymatic saccharification under cost‐effective and green‐like pretreatments, providing a powerful strategy for genetic lignocellulose modification in woody plants and beyond.

Distinctively altered lignin biosynthesis by site‐modification of OsCAD2 for enhanced biomass saccharification in rice

AbstractCrop straws represent enormous biomass resource convertible for biofuels and bioproducts, but lignocellulose recalcitrance restricts its saccharification for commercial utility. Despite genetic modification of lignin biosynthesis being attempted to reduce recalcitrance in bioenergy crops, it remains challenging to optimize lignin deposition without an unacceptable yield penalty. Based on gene expression analysis and phylogenetic tree profiling, a cinnamyl alcohol dehydrogenase gene (OsCAD2) as the target for genetic engineering of lignin biosynthesis in rice was selected in this study. Using CRISPR/Cas9 technology, independent homozygous transgenic lines with precise site mutation of OsCAD2, which showed slightly reduced lignin levels but markedly decreased p‐hydroxyphenyl (H) contents in lignin by 34% and increased guaiacyl (G) contents by 16%, compared to the wild type were generated in this study. Under mild alkali pretreatment (1% NaOH, 50°C), the OsCAD2 site‐modified lines showed effective lignin extraction up to 70% (of total lignin) from mature rice straws, which caused either significantly increased biomass porosity and cellulose accessibility or remarkably reduced cellulase adsorption to lignin in pretreated lignocellulose residues. These consequently led to almost complete biomass enzymatic saccharification with increased hexoses yields by 61%–72% in the modified lines, being much higher than those of the lignin‐altered lines reported in previous studies. Hence, this study has demonstrated a novel genetic engineering strategy to reduce lignocellulose recalcitrance with minimized biomass loss for cost‐effective biomass conversion to bioethanol in rice and bioenergy crops.

LACCASE14 is required for the deposition of guaiacyl lignin and affects cell wall digestibility in poplar

BackgroundThe recalcitrance of lignocellulosic biomass provided technical and economic challenges in the current biomass conversion processes. Lignin is considered as a crucial recalcitrance component in biomass utilization. An in-depth understanding of lignin biosynthesis can provide clues to overcoming the recalcitrance. Laccases are believed to play a role in the oxidation of lignin monomers, leading to the formation of higher-order lignin. In plants, functions of only a few laccases have been evaluated, so little is known about the effect of laccases on cell wall structure and biomass saccharification.ResultsIn this study, we screened a gain-of-function mutant with a significant increase in lignin content from Arabidopsis mutant lines overexpressing a full-length poplar cDNA library. Further analysis confirmed that a Chinese white poplar (Populus tomentosa) laccase gene PtoLAC14 was inserted into the mutant, and PtoLAC14 could functionally complement the Arabidopsis lac4 mutant. Overexpression of PtoLAC14 promoted the lignification of poplar and reduced the proportion of syringyl/guaiacyl. In contrast, the CRISPR/Cas9-generated mutation of PtLAC14 results in increased the syringyl/guaiacyl ratios, which led to integrated enhancement on biomass enzymatic saccharification. Notably, the recombinant PtoLAC14 protein showed higher oxidized efficiency to coniferyl alcohol (precursor of guaiacyl unit) in vitro.ConclusionsThis study shows that PtoLAC14 plays an important role in the oxidation of guaiacyl deposition on cell wall. The reduced recalcitrance of the PtoLAC14-KO lines suggests that PtoLAC14 is an elite target for cell wall engineering, and genetic manipulation of this gene will facilitate the utilization of lignocellulose.