Fiber fractionation to understand the effect of mechanical refining on fiber structure and resulting enzymatic digestibility of biomass.

Mechanical refining results in fiber deconstruction and modifications that enhance enzyme accessibility to carbohydrates. Further understanding of the morphological changes occurring to biomass during mechanical refining and the impacts of these changes on enzymatic digestibility is necessary to maximize yields and reduce energy consumption. Although the degree of fiber length reduction relative to fibrillation/delamination can be impacted by manipulating refining variables, mechanical refining of any type (PFI, disk, and valley beater) typically results in both phenomena. Separating the two is not straightforward. In this study, fiber fractionation based on particle size performed after mechanical refining of high-lignin pulp was utilized to successfully elucidate the relative impact of fibrillation/delamination and fiber cutting phenomena during mechanical refining. Compositional analysis showed that fines contain significantly more lignin than larger size fractions. Enzymatic hydrolysis results indicated that within fractions of uniform fiber length, fibrillation/delamination due to mechanical refining increased enzymatic conversion by 20-30 percentage points. Changes in fiber length had little effect on digestibility for fibers longer than ~0.5 mm. However, the digestibility of the fines fractions was high for all levels of refining even with the high-lignin content.

High-Strength Antibacterial Chitosan-Cellulose Nanocrystal Composite Tissue Paper.

A heightened need to control the spread of infectious diseases prompted the current work in which functionalized and innovative antimicrobial tissue paper was developed with a hydrophobic spray-coating of chitosan (Ch) and cellulose nanocrystals (CNCs) composite. It was hypothesized that the hydrophobic nature of chitosan could be counterbalanced by the addition of CNC to maintain fiber formation and water absorbency. Light-weight tissue handsheets were prepared, spray-coated with Ch, CNC, and their composite coating (ChCNC), and tested for antimicrobial activity against Gram-negative bacteria Escherichia coli and a microbial sample from a human hand after using the rest room. Water absorption and strength properties were also analyzed. To activate the surface of cationized tissue paper, an oxygen/helium gas atmospheric plasma treatment was employed on the best performing antimicrobial tissue papers. The highest bactericidal activity was observed with ChCNC-coated tissue paper, inhibiting up to 98% microbial growth. Plasma treatment further improved the antimicrobial activity of the coatings. Water absorption properties were reduced with Ch but increased with CNC. This "self-disinfecting" bactericidal tissue has the potential to be one of the most innovative products for the hygiene industry because it can dry, clean, and resist the infection of surfaces simultaneously, providing significant societal benefits.

Interactions between Cellulolytic Enzymes with Native, Autohydrolysis, and Technical Lignins and the Effect of a Polysorbate Amphiphile in Reducing Nonproductive Binding.

Understanding enzyme-substrate interactions is critical in designing strategies for bioconversion of lignocellulosic biomass. In this study we monitored molecular events, in situ and in real time, including the adsorption and desorption of cellulolytic enzymes on lignins and cellulose, by using quartz crystal microgravimetry and surface plasmon resonance. The effect of a nonionic surface active molecule was also elucidated. Three lignin substrates relevant to the sugar platform in biorefinery efforts were considered, namely, hardwood autohydrolysis cellulolytic (HWAH), hardwood native cellulolytic (MPCEL), and nonwood native cellulolytic (WSCEL) lignin. In addition, Kraft lignins derived from softwoods (SWK) and hardwoods (HWK) were used as references. The results indicated a high affinity between the lignins with both, monocomponent and multicomponent enzymes. More importantly, the addition of nonionic surfactants at concentrations above their critical micelle concentration reduced remarkably (by over 90%) the nonproductive interactions between the cellulolytic enzymes and the lignins. This effect was hypothesized to be a consequence of the balance of hydrophobic and hydrogen bonding interactions. Moreover, the reduction of surface roughness and increased wettability of lignin surfaces upon surfactant treatment contributed to a lower affinity with the enzymes. Conformational changes of cellulases were observed upon their adsorption on lignin carrying preadsorbed surfactant. Weak electrostatic interactions were determined in aqueous media at pH between 4.8 and 5.5 for the native cellulolytic lignins (MPCEL and WSCEL), whereby a ∼20% reduction in the enzyme affinity was observed. This was mainly explained by electrostatic interactions (osmotic pressure effects) between charged lignins and cellulases. Noteworthy, adsorption of nonionic surfactants onto cellulose, in the form cellulose nanofibrils, did not affect its hydrolytic conversion. Overall, our results highlight the benefit of nonionic surfactant pretreatment to reduce nonproductive enzyme binding while maintaining the reactivity of the cellulosic substrate.

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.

Evaluation of the factors affecting avicel reactivity using multi-stage enzymatic hydrolysis.

Multi-stage and single-stage enzymatic hydrolysis of cellulose (Avicel PH-101) were conducted to investigate individual factors that affect the rate-reducing kinetics of enzymatic hydrolysis. Understanding factors affecting enzymatic hydrolysis of Avicel will help improve hydrolysis of various biomasses. Product inhibition, enzyme deactivation, and the changes of substrate are potential factors that can affect the hydrolysis efficiency of Avicel. Multi-stage enzymatic hydrolysis resulted in 36.9% and 25.4% higher carbohydrate conversion as compared to a single-stage enzymatic hydrolysis with an enzyme loading of 5 and 20 FPU/g in a 96 h reaction. However, a decline in carbohydrate conversion of 1.6% and 2.6% was observed through each stage with 5 and 20 FPU/g, respectively. This indicated that the substrate became more recalcitrant as hydrolysis progressed. The decreased reactivity was not due to crystallinity because no significant change in crystallinity was detected by X-ray diffraction. Product inhibition was significant at low enzyme loading, while it was marginal at high enzyme loading. Therefore, product inhibition can only partially explain this decreased conversion. Another important factor, enzyme deactivation, contributed to 20.3% and 25.4% decrease in the total carbohydrate conversion of 96 h hydrolysis with 5 and 20 FPU/g, respectively. This work shows that an important reason for the decreased Avicel digestibility is the effect of enzyme blockage, which refers to the enzymes that irreversibly adsorb on accessible sites of substrate. About 45.3% and 63.2% of the total decreased conversion at the end of the 8th stage with 5 and 20 FPU/g, respectively, was due to the presence of irreversibly adsorbed enzymes. This blockage of active sites by enzymes has been speculated by other researchers, but this article shows further evidence of this effect.

A systematic examination of the dynamics of water-cellulose interactions on capillary force-induced fiber collapse.

Cellulosic fiber collapse is a phenomenon of fundamental importance for many technologies that include tissue/hygiene to packaging because it governs their essential materials properties such as tensile strength, softness, and water absorption; therefore, we elaborate cellulose fiber collapse from water interactions. This is the first attempt to directly correlate fiber collapse and entrapped or hard-to-remove (HR) water content through DSC, TGA and SEM. Freeze-drying and oven drying were individually investigated for influence on collapse. SEM of the fibers at different moisture contents show that irreversible collapsing begins as entrapped water departs the fiber surface. The removal of HR water pulls cell walls closer due to strong capillary action which overwhelms the elastic force of the fiber lumen which results in partially or fully irreversible collapse. The initial moisture content and refining intensity were found to regulate HR water content and consequently played a vital role in fiber collapsing.

Quantification of lignin-carbohydrate linkages with high-resolution NMR spectroscopy.

A quantitative approach to characterize lignin-carbohydrate complex (LCC) linkages using a combination of quantitative ¹³C NMR and HSQC 2D NMR techniques has been developed. Crude milled wood lignin (MWLc), LCC extracted from MWLc with acetic acid (LCC-AcOH) and cellulolytic enzyme lignin (CEL) preparations were isolated from loblolly pine (Pinus taeda) and white birch (Betula pendula) woods and characterized using this methodology on a routine 300 MHz NMR spectrometer and on a 950 MHz spectrometer equipped with a cryogenic probe. Structural variations in the pine and birch LCC preparations of different types (MWL, CEL and LCC-AcOH) were elucidated. The use of the high field NMR spectrometer equipped with the cryogenic probe resulted in a remarkable improvement in the resolution of the LCC signals and, therefore, is of primary importance for an accurate quantification of LCC linkages. The preparations investigated showed the presence of different amounts of benzyl ether, γ-ester and phenyl glycoside LCC bonds. Benzyl ester moieties were not detected. Pine LCC-AcOH and birch MWLc preparations were preferable for the analysis of phenyl glycoside and ester LCC linkages in pine and birch, correspondingly, whereas CEL preparations were the best to study benzyl ether LCC structures. The data obtained indicate that pinewood contains higher amounts of benzyl ether LCC linkages, but lower amounts of phenyl glycoside and γ-ester LCC moieties as compared to birch wood.

Micro- and nanofibrillated cellulose from virgin and recycled fibers: A comparative study of its effects on the properties of hygiene tissue paper.

This study aims to understand the effect of micro- and nanofibrillated cellulose (MNFC) on the tensile index, softness, and water absorbency of tissue paper. MNFC was produced from four different fiber sources. The results show that MNFC acts as an effective strength enhancer at the expense of a reduced water absorbency and softness. The impact of the fiber source on MNFC manufacturing cost and the trade-off with performance was also investigated. MNFCs produced from southern bleached hardwood kraft, northern bleached softwood kraft, and deinked pulp exhibited similar performance trends with the MNFC from the deinked pulp having a significantly lower cost. This suggests that MNFCs with similar degrees of fibrillation may be used interchangeably regardless of the fiber source, revealing the possibility to minimize MNFC manufacturing costs based on fiber selection. MNFC produced from bleached Eucalyptus kraft showed the lowest degree of fibrillation and the lowest strength improvements among the MNFCs evaluated.

Two-stage autohydrolysis and mechanical treatment to maximize sugar recovery from sweet sorghum bagasse.

Modified autohydrolysis combined with mechanical refining has been suggested to recover free sugars from sweet sorghum bagasse and facilitates enzyme access to cellulose in bagasse for enhancing its conversion to fermentable sugars. The amount of total available sugars in sweet sorghum bagasse was found to be 76.1% and this value was used to evaluate the efficiency of the process suggested. Total sugar recovery was achieved up to 68.1% through the single-stage autohydrolysis at 170 °C for 60 min, followed by mechanical refining and enzymatic hydrolysis; however, the sugar recovery through partial degradation of free sugars induced by high-temperature autohydrolysis was lower than expected. A modified two-stage autohydrolysis was suggested to prevent sugar degradation and the total sugar recovery using this process reached 83.9% of total available sugars in sweet sorghum bagasse.

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.

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.

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.

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.

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.

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).

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.

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%.

Elucidation of lignin structure through degradative methods: comparison of modified DFRC and thioacidolysis.

Milled wood and milled wood lignin (MWL) samples were subjected to DFRC and thioacidolysis. Despite the fact that both methods selectively cleave aryl ether bonds, substantial differences in results were obtained. Lignin thioacidolysis gave total molar yields of degradation monomer products in the range of 3.5-7 mol % higher than DFRC. GPC analysis showed that the thioacidolysis-treated lignin was degraded to a lower average molecular weight than that treated by DFRC. Contrary to results reported for lignin model compounds, these results indicate that the DFRC method does not completely or efficiently degrade the lignin polymer. In fact, the DFRC-degraded lignin retained much of the characteristics of the original MWL. Elemental analysis revealed the presence of bromine in the DFRC-treated lignin, and two-dimensional (1)H-(13)C HMQC NMR spectroscopy showed the presence of beta-O-4 linkages in the DFRC-treated lignin. No beta-O-4 interunit linkages were detected in the thioacidolysis-treated lignin. These results are consistent with the lower monomer yields and the higher average molecular weight of the DFRC-treated lignin and indicate inefficiency in the chemistry of the method, probably due to steric constraints of the polymeric nature of lignin.

Studies on the effect of ball milling on lignin structure using a modified DFRC method.

The structures of milled wood lignin (MWL), cellulolytic enzyme lignin (CEL), and residual lignin (REL) from a loblolly pine were analyzed using a modified derivatization followed by reductive cleavage (DFRC) method developed to allow the quantitative determination of three different structural monomeric products originating in lignin: phenolic beta-O-4, alpha-O-4, and etherified beta-O-4 structures. Results show that MWL and CEL are structurally identical, with an increased phenolic beta-O-4 content compared to that of the original Wiley milled wood. These results indicate that the portion of lignin linked to carbohydrates and that not linked to carbohydrates are structurally the same. Modified DFRC analysis of the effect of ball milling on the structure of lignin in wood, MWL, CEL, and REL indicate that vibratory ball milling does not change the lignin structure provided certain precautions are taken. Specifically, dry vibratory ball milling under a nitrogen atmosphere causes substantial structural changes including condensation, whereas vibratory ball milling in toluene had little effect on the lignin structure. This indicates that the structural differences observed in MWL and CEL arise because of the extraction procedure, which preferentially extracts phenolic lignin structures. MWL and CEL are representative of the total lignin in wood; however, due primarily to the solvent extraction process, higher phenolic hydroxyl contents are observed. Nitrobenzene oxidation showed structural results similar to those from the modified DFRC method.