Multiplex CRISPR editing of wood for sustainable fiber production.

The domestication of forest trees for a more sustainable fiber bioeconomy has long been hindered by the complexity and plasticity of lignin, a biopolymer in wood that is recalcitrant to chemical and enzymatic degradation. Here, we show that multiplex CRISPR editing enables precise woody feedstock design for combinatorial improvement of lignin composition and wood properties. By assessing every possible combination of 69,123 multigenic editing strategies for 21 lignin biosynthesis genes, we deduced seven different genome editing strategies targeting the concurrent alteration of up to six genes and produced 174 edited poplar variants. CRISPR editing increased the wood carbohydrate-to-lignin ratio up to 228% that of wild type, leading to more-efficient fiber pulping. The edited wood alleviates a major fiber-production bottleneck regardless of changes in tree growth rate and could bring unprecedented operational efficiencies, bioeconomic opportunities, and environmental benefits.

Tracing Sweetgum Lignin's Molecular Properties through Biorefinery Processing.

Changes to the molecular properties of lignin over the course of biorefinery processing were investigated by using sweetgum as a feedstock. Hydrothermal pretreatment has been used because it is an economically attractive, green process. Three representative biorefinery lignin preparations were obtained, with about 70 % yield based on raw lignin. The three fractions included soluble lignin adsorbed on resin (XADL), solvent-extracted lignin (HTCELp), and an additional ball-milled residual lignin (HTRELp). By comparing the raw and biorefinery lignin preparations, it can be concluded that lignin undergoes both degradation and condensation throughout the various stages of the hydrothermal-based biorefinery process. The two fractions made soluble by biorefinery processing, XADL and HTCELp, were found to be low-molecular-weight degradation products enriched with free phenolic hydroxyl groups. In addition, about 15 % of noncondensed phenolic units were involved in condensation reactions. Quantitative NMR spectroscopy analysis revealed that at least about 28 % of ß-O-4' substructures were cleaved. Hibbert's ketones were identified in XADL and HTRELp, which provided evidence of lignin undergoing acidolysis. The contents of ß-5' and ß-ß' did not change significantly upon biorefinery processing. Finally, episyringaresinol was detected in XADL and HTCELp. It is hoped that these findings will help to further demonstrate the specific effects of biorefinery processing on lignin in hardwood and facilitate its utilization to improve biorefinery economics.

Cleavage of aryl-ether bonds in lignin model compounds using a Co-Zn-beta catalyst.

Efficient cleavage of aryl-ether linkages is a key strategy for generating aromatic chemicals and fuels from lignin. Currently, a popular method to depolymerize native/technical lignin employs a combination of Lewis acid and hydrogenation metal. However, a clear mechanistic understanding of the process is lacking. Thus, a more thorough understanding of the mechanism of lignin depolymerization in this system is essential. Herein, we propose a detailed mechanistic study conducted with lignin model compounds (LMC) via a synergistic Co-Zn/Off-Al H-beta catalyst that mirrors the hydrogenolysis process of lignin. The results suggest that the main reaction paths for the phenolic dimers exhibiting α-O-4 and ß-O-4 ether linkages are the cleavage of aryl-ether linkages. Particularly, the conversion was readily completed using a Co-Zn/Off-Al H-beta catalyst, but 40% of α-O-4 was converted and ß-O-4 did not react in the absence of a catalyst under the same conditions. In addition, it was found that the presence of hydroxyl groups on the side chain, commonly found in native lignin, greatly promotes the cleavage of aryl-ether linkages activated by Zn Lewis acid, which was attributed to the adsorption between Zn and the hydroxyl group. Followed by the cobalt catalyzed hydrogenation reaction, the phenolic dimers are degraded into monomers that maintain aromaticity.

Production of liquefied fuel from depolymerization of kraft lignin over a novel modified nickel/H-beta catalyst.

In this study, a novel modified nickel/H-beta (Ni/DeAl-beta) catalyst, which has active acidic sites and hydrogen binding sites, was prepared and used to produce liquefied fuel from lignin. The bifunctional Ni/DeAl-beta catalyst efficiently converted kraft lignin into liquefied fuel due to the synergistic effect of aluminum Lewis acid sites and nickel hydrogen binding sites. At a nickel content of 0.6 mmol/gzeolite, the Ni/DeAl-beta catalyst gave a high liquid product yield of 88.6% at 300 °C for 36 h. Most of the liquid product was dissolved in petroleum ether (73% of 88.6%), which was mainly composed of monomeric and dimeric degradation products. Under these conditions, the higher heating values (HHV) increased from 24.9 MJ/kg for kraft lignin to 32.0 MJ/kg for the liquid product. These results demonstrated the bifunctional Ni/DeAl-beta catalyst could be an efficient catalyst for lignin to liquefied fuel conversion.

Improving wood properties for wood utilization through multi-omics integration in lignin biosynthesis.

A multi-omics quantitative integrative analysis of lignin biosynthesis can advance the strategic engineering of wood for timber, pulp, and biofuels. Lignin is polymerized from three monomers (monolignols) produced by a grid-like pathway. The pathway in wood formation of Populus trichocarpa has at least 21 genes, encoding enzymes that mediate 37 reactions on 24 metabolites, leading to lignin and affecting wood properties. We perturb these 21 pathway genes and integrate transcriptomic, proteomic, fluxomic and phenomic data from 221 lines selected from ~2000 transgenics (6-month-old). The integrative analysis estimates how changing expression of pathway gene or gene combination affects protein abundance, metabolic-flux, metabolite concentrations, and 25 wood traits, including lignin, tree-growth, density, strength, and saccharification. The analysis then predicts improvements in any of these 25 traits individually or in combinations, through engineering expression of specific monolignol genes. The analysis may lead to greater understanding of other pathways for improved growth and adaptation.

Furfural production from biomass pretreatment hydrolysate using vapor-releasing reactor system.

Biomass hydrolysate from autohydrolysis pretreatment was used for furfural production considering it is in rich of xylose, xylo-oligomers, and other decomposition products from hemicellulose structure. By using the vapor-releasing reactor system, furfural was protected from degradation by separating it from the reaction media. The maximum furfural yield of 73% was achieved at 200 °C for biomass hydrolysate without the use of the catalyst. This is because the presence of organic acids such as acetic acid in hydrolysate functioned as a catalyst. According to the results in this study, biomass hydrolysate with a vapor-releasing system proves to be efficient for furfural production. The biorefinery process which allows the separation of xylose-rich autohydrolysate from other parts from biomass feedstock also improves the overall application of the biomass.

Comparison of One-Stage Batch and Fed-Batch Enzymatic Hydrolysis of Pretreated Hardwood for the Production of Biosugar.

Fed-batch method has shown a great promise in debottlenecking the high-solid enzymatic hydrolysis for the commercialization of cellulosic biosugar conversion for biofuel/biochemical production. To further improve enzymatic hydrolysis efficiency at high solid loading, fed-batch methods of green liquor-pretreated hardwood were performed to evaluate their effects on sugar recovery by comparing with one-stage batch method in this study. Among all the explored conditions, the fed-batch at 15% consistency gave higher sugar recovery on green liquor-pretreated hardwood compared to that of one-stage batch. By using general linear model analysis, the percentage of enzymatic sugar recovery in fed-batch consistency method (increasing consistency from the initial 10.7 to 15% at intervals of 24 and 48 h) was higher than that of batch hydrolysis at higher density of 15% consistency. Under that best fed-batch condition, the total sugar recovery of pretreated hardwood in enzymatic hydrolysate reached approximately 48.41% at Cellic® enzyme loading of 5 filter-paper unit (FPU)/g and 58.83% at Cellic® enzyme loading of 10 FPU/g with a hydrolysis time of 96 h.

Enhanced furfural production from raw corn stover employing a novel heterogeneous acid catalyst.

With the aim to enhance the direct conversion of raw corn stover into furfural, a promising approach was proposed employing a novel heterogeneous strong acid catalyst (SC-CaCt-700) in different solvents. The novel catalyst was characterized by elemental analysis, N2 adsorption-desorption, FT-IR, XPS, TEM and SEM. The developed catalytic system demonstrated superior efficacy for furfural production from raw corn stover. The effects of reaction temperature, residence time, catalyst loading, substrate concentration and solvent were investigated and optimized. 93% furfural yield was obtained from 150mg corn stover at 200°C in 100min using 45mg catalyst in γ-valerolactone (GVL). In comparison, 51.5% furfural yield was achieved in aqueous media under the same conditions (200°C, 5h, and 45mg catalyst), which is of great industrial interest. Furfural was obtained from both hemicelluloses and cellulose in corn stover, which demonstrated a promising routine to make the full use of biomass.

Liquefaction of kraft lignin by hydrocracking with simultaneous use of a novel dual acid-base catalyst and a hydrogenation catalyst.

In this study, a novel catalyst, S2O82--KNO3/TiO2, which has active acidic and basic sites, was prepared and used in lignin hydrocracking with a co-catalyst, Ru/C. Ru/C is an efficient hydrogenation catalyst and S2O82--KNO3/TiO2 is a dual catalyst, which could efficiently degrade lignin. This catalytic hydrogenation system can reduce solid products to less than 1%, while giving a high liquid product yield of 93%. Catalytic hydrocracking of kraft lignin at 320°C for 6h gave 93% liquid product with 0.5% solid product. Most of this liquid product was soluble in petroleum ether (60% of 93%), which is a clear liquid and comprises mainly of monomeric and dimeric degradation products. These results demonstrated that the combination of the two catalysts is an efficient catalyst for liquefaction of lignin, with little char formation (∼1%). This concept has the potential to produce valuable chemicals and fuels from lignin under moderate conditions.

Lignocentric analysis of a carbohydrate-producing lignocellulosic biorefinery process.

A biologically-based lignocellulosic biorefinery process for obtaining carbohydrates from raw biomass was investigated across six diverse biomasses (three hardwoods & three nonwoods) for the purpose of decoding lignin's influence on sugar production. Acknowledging that lignin could positively alter the economics of an entire process if valorized appropriately, we sought to correlate the chemical properties of lignin within the process to the traditional metrics associated with carbohydrate production-cellulolytic digestibility and total sugar recovery. Based on raw carbohydrate, enzymatic recovery ranged from 40 to 64% w/w and total recovery ranged from 70 to 87% w/w. Using nitrobenzene oxidation to quantify non-condensed lignin structures, it was found that raw hardwoods bearing increasing non-condensed S/V ratios (2.5-5.1) render increasing total carbohydrate recovery from hardwood biomasses. This finding indicates that the chemical structure of hardwood lignin influences the investigated biorefinery process' ability to generate carbohydrates from a given raw hardwood feedstock.

A two-stage pretreatment process using dilute hydrochloric acid followed by Fenton oxidation to improve sugar recovery from corn stover.

A two-stage pretreatment process is proposed in this research in order to improve sugar recovery from corn stover. In the proposed process, corn stover is hydrolyzed by dilute hydrochloric acid to recover xylose, which is followed by a Fenton reagent oxidation to remove lignin. 0.7wt% dilute hydrochloric acid is applied in the first stage pretreatment at 120°C for 40min, resulting in 81.0% xylose removal. Fenton reagent oxidation (1g/L FeSO4·7H2O and 30g/L H2O2) is performed at room temperature (about 20°C) for 12 has a second stage which resulted in 32.9% lignin removal. The glucose yield in the subsequent enzymatic hydrolysis was 71.3% with a very low cellulase dosage (3FPU/g). This two-stage pretreatment is effective due to the hydrolysis of hemicelluloses in the first stage and the removal of lignin in the second stage, resulting in a very high sugar recovery with a low enzyme loading.

Novel process for the coproduction of xylo-oligosaccharides, fermentable sugars, and lignosulfonates from hardwood.

Many biorefineries have not been commercialized due to poor economic returns from final products. In this work, a novel process has been developed to coproduce valuable sugars, xylo-oligosaccharides, and lignosulfonates from hardwood. The modified process includes a mild autohydrolysis pretreatment, which enables for the recovery of the xylo-oligosaccharides in auto-hydrolysate. Following enzymatic hydrolysis, the residue is sulfomethylated to produce lignosulfonates. Recycling the sulfomethylation residues increased both the glucan recovery and lignosulfonate production. The glucose recovery was increased from 81.7% to 87.9%. Steady state simulation using 100g of hardwood produced 46.7g sugars, 5.9g xylo-oligosaccharides, and 25.7g lignosulfonates, which were significantly higher than that produced from the no-recycling process with 39.1g sugars, 5.9g xylo-oligosaccharides, and 15.0g lignosulfonates. The results indicate that this novel biorefinery process can improve the production of fermentable sugars and lignosulfonate from hardwood as compared to a conventional biorefinery process.

Pretreatment of corn stover for sugar production using a two-stage dilute acid followed by wet-milling pretreatment process.

A two-stage process was evaluated to increase sugar recovery. Firstly, corn stover was treated with dilute hydrochloric acid to recover the xylose, and then the residue was subjected to a wet-milling pretreatment. Dilute hydrochloric acid showed a high xylose recovery during the first stage. The optimal condition was 120°C and 40min for 0.7wt% dilute hydrochloric acid pretreatment followed by wet-milling pretreatment for 15min. The xylose and glucose yield were 81.0% and 64.0%, respectively, with a cellulase dosage at 3FPU/g of substrate. This two-stage process was effective on account of the removal of hemicelluloses in the first stage and the delamination of cell wall in the second stage, increasing the possibility of adsorption of cellulose to enzymes, and resulting in a high sugar recovery with a very low enzyme loading.

Catalytic conversion of xylose and corn stalk into furfural over carbon solid acid catalyst in γ-valerolactone.

A novel carbon solid acid catalyst was synthesized by the sulfonation of carbonaceous material which was prepared by carbonization of sucrose using 4-BDS as a sulfonating agent. TEM, N2 adsorption-desorption, elemental analysis, XPS and FT-IR were used to characterize the catalyst. Then, the catalyst was applied for the conversion of xylose and corn stalk into furfural in GVL. The influence of the reaction time, temperature and dosage of catalyst on xylose dehydration were also investigated. The Brønsted acid catalyst exhibited high activity in the dehydration of xylose, with a high furfural yield of 78.5% at 170°C in 30min. What's more, a 60.6% furfural yield from corn stalk was achieved in 100min at 200°C. The recyclability of the sulfonated carbon catalyst was perfect, and it could be reused for 5times without the loss of furfural yields.

Biomass pretreatments capable of enabling lignin valorization in a biorefinery process.

Recent techno-economic studies of proposed lignocellulosic biorefineries have concluded that creating value from lignin will assist realization of biomass utilization into valuable fuels, chemicals, and materials due to co-valorization and the new revenues beyond carbohydrates. The pretreatment step within a biorefinery process is essential for recovering carbohydrates, but different techniques and intensities have a variety of effects on lignin. Acidic and alkaline pretreatments have been shown to produce diverse lignins based on delignification chemistry. The valorization potential of pretreated lignin is affected by its chemical structure, which is known to degrade, including inter-lignin condensation under high-severity pretreatment. Co-valorization of lignin and carbohydrates will require dampening of pretreatment intensities to avoid such effects, in spite of tradeoffs in carbohydrate production.

Conversion of corn stalk into furfural using a novel heterogeneous strong acid catalyst in γ-valerolactone.

A novel solid acid catalyst was prepared by the copolymerization of p-toluenesulfonic acid and paraformaldehyde and then characterized by FT-IR, TG/DTG, HRTEM and N2-BET. Furfural was successfully produced by the dehydration of xylose and xylan using the novel catalyst in γ-valerolactone. This investigation focused on effects of various reaction conditions including solvent, acid catalyst, reaction temperature, residence time, water concentration, xylose loading and catalyst dosage on the dehydration of xylose to furfural. It was found that the solid catalyst displayed extremely high activity for furfural production. 80.4% furfural yield with 98.8% xylose conversion was achieved at 170°C for 10 min. The catalyst could be recycled at least five times without significant loss of activity. Furthermore, 83.5% furfural yield and 19.5% HMF yield were obtained from raw corn stalk under more severe conditions (190°C for 100 min).

Production of fermentable sugars from sugarcane bagasse by enzymatic hydrolysis after autohydrolysis and mechanical refining.

The autohydrolysis process has been considered a simple, low-cost and environmental friendly technology for generation of sugars from biomass. In order to improve accessibility of enzymes during enzymatic hydrolysis as well as to allow the recovery of hemicellulose in the filtrate, the sugarcane bagasse was pretreated using autohydrolysis followed by a mechanical refining process. The autohydrolysis was carried out in three different conditions. Autohydrolysis at 190°C for 10min provided the highest overall sugar (19.2/100g raw bagasse) in prehydrolyzate. The enzymatic hydrolysis step was performed for all the post-treated solids with and without refining at enzyme loadings of 5 and 10FPU/g for 96h. A total of 84.4% of sugar can be recovered from sugarcane bagasse at 180°C for 20min with 5 FPU/g enzyme charge. The economic analysis for the proposed method showed that the bioethanol production can have a financial return larger than 12%.

Autohydrolysis pretreatment of waste wheat straw for cellulosic ethanol production in a co-located straw pulp mill.

Waste wheat straw (WWS) is the waste product from feedstock preparation process in a straw pulp mill. It has a significant annual production rate and no commercial value has been explored on this material. In this study, waste wheat straw was pretreated using an autohydrolysis process followed by mechanical refining, and the pretreated materials were further enzymatically hydrolyzed to evaluate the total sugar recovery for bioethanol production. Results show that autohydrolysis at 170 °C for 40 min followed by 6000 revolution PFI refining provided the best result in this study, where a total sugar recovery (total sugars in autohydrolysis filtrate and enzymatic hydrolyzate over total carbohydrates on raw WWS) of 70 % at 4 filter paper unit per oven dry gram (FPU/OD g) substrate enzyme charge could be obtained. The economic evaluation of this biorefinery process indicates that cellulosic ethanol production from autohydrolysis of WWS is a very profitable business, with 28.4 % of internal rate of return can be achieved based on current ethanol wholesale price in China.

Effect of lignin chemistry on the enzymatic hydrolysis of woody biomass.

The impact of lignin-derived inhibition on enzymatic hydrolysis is investigated by using lignins isolated from untreated woods and pretreated wood pulps. A new method, biomass reconstruction, for which isolated lignins are precipitated onto bleached pulps to mimic lignocellulosic biomass, is introduced, for the first time, to decouple the lignin distribution issue from lignin chemistry. Isolated lignins are physically mixed and reconstructed with bleached pulps. Lignins obtained from pretreated woods adsorb two to six times more cellulase than lignins obtained from untreated woods. The higher adsorption of enzymes on lignin correlates with decreased carbohydrate conversion in enzymatic hydrolysis. In addition, the reconstructed softwood substrate has a lower carbohydrate conversion than the reconstructed hardwood substrate. The degree of condensation of lignin increases significantly after pretreatment, especially with softwood lignins. In this study, the degree of condensation of lignin (0.02 to 0.64) and total OH groups in lignin (1.7 to 1.1) have a critical impact on cellulase adsorption (9 to 70%) and enzymatic hydrolysis (83.2 to 58.2%); this may provide insights into the more recalcitrant nature of softwood substrates.

Comparison of sodium carbonate-oxygen and sodium hydroxide-oxygen pretreatments on the chemical composition and enzymatic saccharification of wheat straw.

Pretreatment of wheat straw with a combination of sodium carbonate (Na2CO3) or sodium hydroxide (NaOH) with oxygen (O2) 0.5MPa was evaluated for its delignification ability at relatively low temperature 110°C and for its effect on enzymatic hydrolysis efficiency. In the pretreatment, the increase of alkali charge (as Na2O) up to 12% for Na2CO3 and 6% for NaOH, respectively, resulted in enhancement of lignin removal, but did not significantly degrade cellulose and hemicellulose. When the pretreated solid was hydrolyzed with a mixture of cellulases and hemicellulases, the sugar yield increased rapidly with the lignin removal during the pretreatment. A total sugar yield based on dry matter of raw material, 63.8% for Na2CO3-O2 and 71.9% for NaOH-O2 was achieved under a cellulase loading of 20FPU/g-cellulose. The delignification efficiency and total sugar yield from enzymatic hydrolysis were comparable to the previously reported results at much higher temperature without oxygen.