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Lignocellulosic biomass is an abundant byproduct from cereal crops that can potentially be valorized as a feedstock to produce biomaterials. Zea mays CINNAMYL ALCOHOL DEHYDROGENASE 2 (ZmCAD2) is involved in lignification, and is a promising target to improve the cellulose–to–glucose conversion of maize stover. Here, we analyzed a field–grown zmcad2 Mutator transposon insertional mutant. Zmcad2 mutant plants had an 18% lower Klason lignin content, whereas their cellulose content was similar to that of control lines. The lignin in zmcad2 mutants contained increased levels of hydroxycinnamaldehydes, i.e. the substrates of ZmCAD2, ferulic acid and tricin. Ferulates decorating hemicelluloses were not altered. Phenolic profiling further revealed that hydroxycinnamaldehydes are partly converted into (dihydro)ferulic acid and sinapic acid and their derivatives in zmcad2 mutants. Syringyl lactic acid hexoside, a metabolic sink in CAD–deficient dicot trees, appeared not to be a sink in zmcad2 maize. The enzymatic cellulose–to–glucose conversion efficiency was determined after 10 different thermochemical pre–treatments. Zmcad2 yielded significantly higher conversions compared with controls for almost every pre–treatment. However, the relative increase in glucose yields after alkaline pre–treatment was not higher than the relative increase when no pre–treatment was applied, suggesting that the positive effect of the incorporation of hydroxycinnamaldehydes was leveled off by the negative effect of reduced p–coumarate levels in the cell wall. Here, taken together, our results reveal how phenolic metabolism is affected in CAD–deficient maize, and further support mutating CAD genes in cereal crops as a promising strategy to improve lignocellulosic biomass for sugar–platform biorefineries.
Abstract Background Pretreatments are commonly used to facilitate the deconstruction of lignocellulosic biomass to its component sugars and aromatics. Previously, we showed that iron ions can be used as co-catalysts to reduce the severity of dilute acid pretreatment of biomass. Transgenic iron-accumulating Arabidopsis and rice plants exhibited higher iron content in grains, increased biomass yield, and importantly, enhanced sugar release from the biomass. Results In this study, we used intracellular ferritin (FerIN) alone and in combination with an improved version of cell wall-bound carbohydrate-binding module fused iron-binding peptide (IBPex) specifically targeting switchgrass, a bioenergy crop species. The FerIN switchgrass improved by 15% in height and 65% in yield, whereas the FerIN/IBPex transgenics showed enhancement up to 30% in height and 115% in yield. The FerIN and FerIN/IBPex switchgrass had 27% and 51% higher in planta iron accumulation than the empty vector (EV) control, respectively, under normal growth conditions. Improved pretreatability was observed in FerIN switchgrass (~ 14% more glucose release than the EV), and the FerIN/IBPex plants showed further enhancement in glucose release up to 24%. Conclusions We conclude that this iron-accumulating strategy can be transferred from model plants and applied to bioenergy crops, such as switchgrass. The intra- and extra-cellular iron incorporation approach improves biomass pretreatability and digestibility, providing upgraded feedstocks for the production of biofuels and bioproducts.
Increasing global populations, finite arable land, and the anthropogenic release of carbon dioxide into the atmosphere are driving the search for bio-based alternatives to the petroleum-derived fuels and chemicals that underpin the global economy. With rapid growth rates, a propensity for coppicing, and a wide geographic range across Canada, native shrub willows (Salix spp.) are an attractive source of low-input, high-volume biomass. To date, most willow research has focused on increasing yields and improving cropping systems, while comparatively little work has been done to assess the intrinsic diversity in cell wall traits and bioenergy potential. In this study, we characterized the cell wall composition and wood ultrastructure of 338 xylem samples from two Canadian willow species, Salix famelica and Salix eriocephala, harvested from a common garden experimental plot. Lignin content ranged from 17.5–25.1% in S. famelica and 18.6–24.3% in S. eriocephala. Following alkali pretreatment with 62.5 mM NaOH at 90 °C for 3 h and a 70-h enzymatic digestion with Accellerase 1000, glucose release ranged from 23.0–38.9 wt% in S. famelica and from 20.5–37.7 wt% in S. eriocephala, while xylose release ranged from 9.4–14.9% in S. famelica and from 9.5–15.2% in S. eriocephala. Here, partial least squares regression modelling showed that lignin content and composition were important negative regulators of glucose release. Overall, this work highlights the innate variability in cell wall traits of native willows and identifies potential genotypes that should be considered in future breeding and deployment strategies for Canadian bioenergy production.
The cost of sugar production remains a key challenge in future lignocellulosic biorefineries. We demonstrate that ZnBr 2 , an inexpensive inorganic salt, provides nearly theoretical yields of glucose and xylose in one-step from poplar wood at 85 °C and short reaction times at molten salt hydrate (MSH) conditions without an acid. Catalytic depolymerization of the isolated MSH lignin, using a CoS 2 catalyst, yields 17% phenol-like monomers compared to only 1% produced from the acidified MSH lignin. Reductive catalytic fractionation of MSH lignin over Ru/C resulted in two times higher total monomer yield compared to the AMSH lignin. Both the lignin samples were characterized using 2D HSQC NMR and the thioacidolysis method. Thioacidolysis studies reveal 8.4% and 1.8% of β-O-4 linkages in MSH and acidified MSH lignin, respectively. Thermodynamic modeling and 13 C NMR spectroscopy indicate that the effectiveness of this catalyst arises from the strong interaction of the Lewis acidic zinc cation (Zn 2+ ) with the coordinated water molecules resulting in hydrolysis of the metal aquo complex and to the salt-driven increase in the H + activity coefficient. Techno-economic analysis demonstrates that despite being slower, the ZnBr 2 MSH media has cost advantages, compared to conventional hydrolysis and even to the LiBr and ZnBr 2 AMSH, due to the higher quality of lignin.
A method of converting a lignocellulosic biomass to monosaccharides, and optionally further converting the monosaccharides to one or both of furfural and HMF, includes contacting the biomass with a reactive liquid phase comprising LiBr, H 2 SO 4 , and water, wherein H 2 SO 4 preferably constitutes at most 1.0 wt %, more preferably at most 0.5 wt %, and most preferably at most 0.1 wt % of the reactive liquid phase, and water preferably constitutes at most at most 60 wt %, more preferably at most 50 wt %, and most preferably at most 30 wt % of the reactive liquid phase.
The present invention provides for a method of fermenting or saccharifying a biomass comprising: (a) (i) contacting a biomass comprising a polysaccharide, and an ionic liquid (IL) to form a first solution, or (ii) providing the first solution comprising the biomass and the IL, (b) contacting the first solution and carbon dioxide such that the first solution results in a lower pH, (c) introducing (i) an enzyme capable of enzymatically to breakdown at least one bond in the polysaccharide or a breakdown product of the polysaccharide, and/or (ii) a microorganism that capable of producing the enzyme and/or fermenting the polysaccharide or a breakdown product of the polysaccharide, such that the polysaccharide is at least partially broken down and the first solution is transformed into a second solution.
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The present invention provides for a method of fermenting or saccharifying a biomass comprising: (a) (i) contacting a biomass comprising a polysaccharide, and an ionic liquid (IL) to form a first solution, or (ii) providing the first solution comprising the biomass and the IL, (b) contacting the first solution and carbon dioxide such that the first solution results in a lower pH, (c) introducing (i) an enzyme capable of enzymatically to breakdown at least one bond in the polysaccharide or a breakdown product of the polysaccharide, and/or (ii) a microorganism that capable of producing the enzyme and/or fermenting the polysaccharide or a breakdown product of the polysaccharide, such that the polysaccharide is at least partially broken down and the first solution is transformed into a second solution.
Previous lignin microspheres (LMS) preparation needs multiple steps with very low yield and high cost. Herein, we developed a high-solid deep eutectic solvent (DES) pretreatment for an effective lignin fractionation and enzymatic saccharification of moso bamboo under mild temperature (110 °C) with high-solid loading. Lignin was significantly removed from the plant cell wall, and cellulose properties (e.g., crystallinity and degree of polymerization) were also altered during the pretreatment. As a result, the enzymatic digestibility of the pretreated bamboo was dramatically increased. Uniform micro-spherical lignin was directly produced from the pretreatment system, and its particle size could be regulated by controlling the solid loadings and pretreatment temperatures. The lignin microspheres formation mechanism was investigated by analyzing the lignin's size distribution, molecular weight distribution, chemical structure, and hydrophobicity. The DES showed excellent recyclability, and the recycled DES could still remove 42.78% lignin even after 7th circulation associated with 100% glucan saccharification. Further, the mass balance based on 1000 g biomass showed that 196.28 g LMS was directly recovered, which exhibited a high RhB adsorption. Besides, 404.91 g glucose and 36.67 g xylose were obtained after the enzymatic saccharification process. Specifically, GAPI analysis exhibited a near total green and yellow portions of the pictogram, indicating our DES process was green enough to make this biorefinery sustainable. Overall, the proposed DES generated synergistic productions of digestible solid and LMS which could contribute to establish a green and sustainable biorefinery sequence with diverse outputs in one pot.
Developing an efficient deconstruction step of woody biomass for biorefinery has been drawing considerable attention since its xylem cell walls display highly recalcitrance nature. Here, we explored transcriptional factors (TFs) that reduce wood recalcitrance and improve saccharification efficiency in Populus species. First, 33 TF genes up-regulated during poplar wood formation were selected as potential regulators of xylem cell wall structure. The transgenic hybrid aspens (Populus tremula × Populus tremuloides) overexpressing each selected TF gene were screened for in vitro enzymatic saccharification. Of these, four transgenic seedlings overexpressing previously uncharacterized TF genes increased total glucan hydrolysis on average compared to control. The best performing lines overexpressing Pt × tERF123 and Pt × tZHD14 were further grown to form mature xylem in the greenhouse. Notably, the xylem cell walls exhibited significantly increased total xylan hydrolysis as well as initial hydrolysis rates of glucan. The increased saccharification of Pt × tERF123-overexpressing lines could reflect the improved balance of cell wall components, i.e., high cellulose and low xylan and lignin content, which could be caused by upregulation of cellulose synthase genes upon the expression of Pt × tERF123. Overall, we successfully identified Pt × tERF123 and Pt × tZHD14 as effective targets for reducing cell wall recalcitrance and improving the enzymatic degradation of woody plant biomass.
Switchgrass ( Panicum virgatum L.) is a promising perennial bioenergy crop that achieves high yields with relatively low nutrient and energy inputs. Modification of cell wall composition for reduced recalcitrance can lower the costs of deconstructing biomass to fermentable sugars and other intermediates. We have engineered overexpression of OsAT10 , encoding a rice BAHD acyltransferase and QsuB , encoding dehydroshikimate dehydratase from Corynebacterium glutamicum , to enhance saccharification efficiency in switchgrass. These engineering strategies demonstrated low lignin content, low ferulic acid esters, and increased saccharification yield during greenhouse studies in switchgrass and other plant species. In this work, transgenic switchgrass plants overexpressing either OsAT10 or QsuB were tested in the field in Davis, California, USA for three growing seasons. No significant differences in the content of lignin and cell wall-bound p -coumaric acid or ferulic acid were detected in transgenic OsAT10 lines compared with the untransformed Alamo control variety. However, the transgenic overexpressing QsuB lines had increased biomass yield and slightly increased biomass saccharification properties compared to the control plants. This work demonstrates good performance of engineered plants in the field, and also shows that the cell wall changes in the greenhouse were not replicated in the field, emphasizing the need to validate engineered plants under relevant field conditions.
This project will conduct biomass deconstruction (pretreatment and enzymatic saccharification) on two representative biomass feedstocks and four different pretreatment processes, including three high temperature steam/chemical pretreatments and one low temperature chemical/mechanical pretreatment. A third biomass feedstock will undergo biomass deconstruction with three different pretreatment processes, including two high temperature chemical pretreatments and one low temperature chemical/mechanical pretreatment. Several pretreatment conditions will be performed in a screening study using NLR pilot-scale pretreatment equipment to generate a range of pretreated biomass substrates. A selected number of these substrates will be chosen for enzymatic saccharification evaluation, based on standard compositional analysis of the pretreated substrates as a primary indicator of pretreatment efficacy. Resulting enzymatic hydrolysis slurries will be analyzed to determine overall biomass sugar yields. Additional compositional analysis will be performed to determine oligomeric sugar composition and structure, to analyze structural characteristics of solids fractions on native, pretreated, and enzymatically saccharified biomass residues for one of the biomass feedstocks, corn stover. The enzymatically saccharified materials will undergo 2,3-butanediol fermentation in a shaker flask as bench scale. Using relevant process performance data collected in these various conversion steps, technoeconomic analysis activities will be performed to compare the economic potential of the various biomass feedstock and pretreatment processes and to identify key economic drivers and sustainability metrics.
In plant cell walls, lignin, cellulose, and the hemicelluloses form intricate three-dimensional structures. Owing to its complexity, lignin often acts as a bottleneck for the efficient utilization of polysaccharide components as biochemicals and functional materials. A promising approach to mitigate and/or overcome lignin recalcitrance is the qualitative and quantitative modification of lignin by genetic engineering. Feruloyl-CoA 6'-hydroxylase (F6'H1) is a 2-oxoglutarate-dependent dioxygenase that catalyzes the conversion of feruloyl-CoA, one of the intermediates of the lignin biosynthetic pathway, into 6'-hydroxyferuloyl-CoA, the precursor of scopoletin (7-hydroxy-6-methoxycoumarin). In a previous study with Arabidopsis thaliana, we demonstrated that overexpression of F6'H1 under a xylem-preferential promoter led to scopoletin incorporation into the cell wall. This altered the chemical structure of lignin without affecting lignin content or saccharification efficiency. In the present study, the same F6'H1 construct was introduced into hybrid aspen (Populus tremula × tremuloides T89), a model woody plant, and its effects on plant morphology, lignin chemical structure, global gene expression, and phenolic metabolism were examined. The transgenic plants successfully overproduced scopoletin while exhibiting severe growth retardation, a phenotype not previously observed in Arabidopsis. Scopoletin accumulation was most pronounced in the secondary walls of tracheary elements and the compound middle lamella, with low levels in the fiber cell walls. Overexpression of F6'H1 also affected the metabolism of aromatics, including lignin precursors. Heteronuclear single-quantum coherence (HSQC) NMR spectroscopy revealed that scopoletin in cell walls was bound to lignin, leading to a reduction in lignin content and changes in its monomeric composition and molar mass distribution. Furthermore, the enzymatic saccharification efficiency of the transgenic cell walls was more than three times higher than that of the wild-type plants, even without pretreatment. Although addressing growth inhibition remains a priority, incorporating scopoletin into lignin demonstrates significant potential for improving woody biomass utilization.
Bioconversion of renewable lignocellulosics to produce liquid fuels and chemicals is one of the most effective ways to solve the problem of fossil resource shortage, energy security, and environmental challenges. Among the many biorefinery pathways, hydrolysis of lignocellulosics to fermentable monosaccharides by cellulase is arguably the most critical step of lignocellulose bioconversion. In the process of enzymatic hydrolysis, the direct physical contact between enzymes and cellulose is an essential prerequisite for the hydrolysis to occur. However, lignin is considered one of the most recalcitrant factors hindering the accessibility of cellulose by binding to cellulase unproductively, which reduces the saccharification rate and yield of sugars. This results in high costs for the saccharification of carbohydrates. The various interactions between enzymes and lignin have been explored from different perspectives in literature, and a basic lignin inhibition mechanism has been proposed. However, the exact interaction between lignin and enzyme as well as the recently reported promotion of some types of lignin on enzymatic hydrolysis is still unclear at the molecular level. Multiple analytical techniques have been developed, and fully unlocking the secret of lignin-enzyme interactions would require a continuous improvement of the currently available analytical techniques. This review summarizes the current commonly used advanced research analytical techniques for investigating the interaction between lignin and enzyme, including quartz crystal microbalance with dissipation (QCM-D), surface plasmon resonance (SPR), attenuated total reflectance-Fourier transform infrared (ATR-FTIR) spectroscopy, atomic force microscopy (AFM), nuclear magnetic resonance (NMR) spectroscopy, fluorescence spectroscopy (FLS), and molecular dynamics (MD) simulations. Interdisciplinary integration of these analytical methods is pursued to provide new insight into the interactions between lignin and enzymes. Finally, this review will serve as a resource for future research seeking to develop new methodologies for a better understanding of the basic mechanism of lignin-enzyme binding during the critical hydrolysis process.
This study investigated a high-solid diol deep eutectic solvent (DES) pretreatment using a wet substrate as the starting material. This pretreatment led to a remarkable glucan saccharification of 94.8% with efficient lignin and xylan removal (as high as 63.1% and 73.0%, respectively). Here the chemical structures of the substrates were analyzed comprehensively to reveal the impact of the pretreatment. In addition, over 90% of the removed lignin was recovered from the pretreated liquid, which exhibited a well-preserved β-O-4 structure (46–56/100Ar). The protection mechanism of our DES was investigated by 2D HSQC NMR, GPC, and 31 P NMR analysis. This study emphasized that diol-based DES pretreatment of undried lignocellulosic biomass at a high-solid loading can significantly utilize both carbohydrates and lignin fractions with high saccharification yields and high-quality lignin as a co-product.
Abstract Plant lignocellulosic biomass, i.e. secondary cell walls of plants, is a vital alternative source for bioenergy. However, the acetylation of xylan in secondary cell walls impedes the conversion of biomass to biofuels. Previous studies have shown that REDUCED WALL ACETYLATION (RWA) proteins are directly involved in the acetylation of xylan but the regulatory mechanism of RWAs is not fully understood. In this study, we demonstrate that overexpression of a Populus trichocarpa PtRWA-C gene increases the level of xylan acetylation and increases the lignin content and S/G ratio, ultimately yielding poplar woody biomass with reduced saccharification efficiency. Furthermore, through gene coexpression network and expression quantitative trait loci (eQTL) analysis, we found that PtRWA-C was regulated not only by the secondary cell wall hierarchical regulatory network but also by an AP2 family transcription factor HARDY (HRD). Specifically, HRD activates PtRWA-C expression by directly binding to the PtRWA-C promoter, which is also the cis-eQTL for PtRWA-C. Taken together, our findings provide insights into the functional roles of PtRWA-C in xylan acetylation and consequently saccharification and shed light on synthetic biology approaches to manipulate this gene and alter cell wall properties. These findings have substantial implications for genetic engineering of woody species, which could be used as a sustainable source of biofuels, valuable biochemicals, and biomaterials.
Abstract Background The development of bioenergy crops with reduced recalcitrance to enzymatic degradation represents an important challenge to enable the sustainable production of advanced biofuels and bioproducts. Biomass recalcitrance is partly attributed to the complex structure of plant cell walls inside which cellulose microfibrils are protected by a network of hemicellulosic xylan chains that crosslink with each other or with lignin via ferulate (FA) bridges. Overexpression of the rice acyltransferase OsAT10 is an effective bioengineering strategy to lower the amount of FA involved in the formation of cell wall crosslinks and thereby reduce cell wall recalcitrance. The annual crop sorghum represents an attractive feedstock for bioenergy purposes considering its high biomass yields and low input requirements. Although we previously validated the OsAT10 engineering approach in the perennial bioenergy crop switchgrass, the effect of OsAT10 expression on biomass composition and digestibility in sorghum remains to be explored. Results We obtained eight independent sorghum ( Sorghum bicolor (L.) Moench) transgenic lines with a single copy of a construct designed for OsAT10 expression. Consistent with the proposed role of OsAT10 in acylating arabinosyl residues on xylan with p -coumarate ( p CA), a higher amount of p -coumaroyl-arabinose was released from the cell walls of these lines upon hydrolysis with trifluoroacetic acid. However, no major changes were observed regarding the total amount of p CA or FA esters released from cell walls upon mild alkaline hydrolysis. Certain diferulate (diFA) isomers identified in alkaline hydrolysates were increased in some transgenic lines. The amount of the main cell wall monosaccharides glucose, xylose, and arabinose was unaffected. The transgenic lines showed reduced lignin content and their biomass released higher yields of sugars after ionic liquid pretreatment followed by enzymatic saccharification. Conclusions Expression of OsAT10 in sorghum leads to an increase of xylan-bound p CA without reducing the overall content of cell wall FA esters. Nevertheless, the amount of total cell wall p CA remains unchanged indicating that most p CA is ester-linked to lignin. Unlike other engineered plants overexpressing OsAT10 or a phylogenetically related acyltransferase with similar putative function, the improvements of biomass saccharification efficiency in sorghum OsAT10 lines are likely the result of lignin reductions rather than reductions of cell wall-bound FA. These results also suggest a relationship between xylan-bound p CA and lignification in cell walls.