Size-controlled synthesis of xylan micro / nanoparticles by self-assembly of alkali-extracted xylan.

Valorization of underutilized biobased feedstocks like hetero-polysaccharides is critical for the development of the biorefinery concept. Towards this goal, highly uniform xylan micro/nanoparticles with a particle size ranging from 400 nm to 2.5 µm in diameter were synthesized by a facile self-assembly method in aqueous solutions. Initial concentration of the insoluble xylan suspension was utilized to control the particle size. The method utilized supersaturated aqueous suspensions formed at standard autoclaving conditions without any other chemical treatments to create the resulting particles as solutions cooled to room temperature. Processing parameters of the xylan micro/nanoparticles were systematically studied and correlated with both the morphology and size of xylan particles. By adjusting the crowding of the supersaturated solutions, highly uniform dispersions of xylan particles were synthesized of defined size. The xylan micro/nanoparticles prepared by self-assembly have a quasi-hexagonal shape, like a tile, and depending upon solution concentrations xylan nanoparticles with a thickness of <100 nm were achieved at high concentrations. Based on the usefulness of polysaccharide nanoparticles, like cellulose nanocrystals, these particles have potential for unique structures for hydrogels, aerogels, drug delivery, and photonic materials. This study highlights the formation of a diffraction grating film for visible light with these size-controlled particles.

The production of lactic acid from chemi-thermomechanical pulps using a chemo-catalytic approach.

Previous work has shown that sulfonation and oxidation of chemi-thermomechanical pulps (CTMPs) significantly enhanced enzyme accessibility to cellulose while recovering the majority of carbohydrates in the water-insoluble component. In the work reported here, modified (sulfonated and oxidized) CTMPs derived from hard-and-softwoods were used to produce a DL-mix of lactic acid via a chemo-catalytic approach using lanthanide triflate (Ln (OTf)3) catalysts (Ln = La, Nd, Er, and Yb). It was apparent that sulfonation and oxidation of chemi-thermomechanical pulps (CTMPs) also enhanced Ln(OTf)3 catalyst accessibility to the carbohydrate components of the pulps, with the Er(OTf)3 catalysts resulting in significant lactic acid production. Under optimum conditions (250 °C, 60 min, 0.5 mmol catalyst g-1 biomass), 72% and 67% of the respective total carbohydrate present in the hard-and-softwood CTMPs could be converted to lactic acid compared to the respective 59% and 51% yields obtained after energy-intensive ball milling.

Enhancing Enzyme-Mediated Cellulose Hydrolysis by Incorporating Acid Groups Onto the Lignin During Biomass Pretreatment.

Lignin is known to limit the enzyme-mediated hydrolysis of biomass by both restricting substrate swelling and binding to the enzymes. Pretreated mechanical pulp (MP) made from Aspen wood chips was incubated with either 16% sodium sulfite or 32% sodium percarbonate to incorporate similar amounts of sulfonic and carboxylic acid groups onto the lignin (60 mmol/kg substrate) present in the pulp without resulting in significant delignification. When Simon's stain was used to assess potential enzyme accessibility to the cellulose, it was apparent that both post-treatments enhanced accessibility and cellulose hydrolysis. To further elucidate how acid group addition might influence potential enzyme binding to lignin, Protease Treated Lignin (PTL) was isolated from the original and modified mechanical pulps and added to a cellulose rich, delignified Kraft pulp. As anticipated, the PTLs from both the oxidized and sulfonated substrates proved less inhibitory and adsorbed less enzymes than did the PTL derived from the original pulp. Subsequent analyses indicated that both the sulfonated and oxidized lignin samples contained less phenolic hydroxyl groups, resulting in enhanced hydrophilicity and a more negative charge which decreased the non-productive binding of the cellulase enzymes to the lignin.

Non-productive celluase binding onto deep eutectic solvent (DES) extracted lignin from willow and corn stover with inhibitory effects on enzymatic hydrolysis of cellulose.

In this work, deep eutectic solvent (DES) was prepared by mixing choline chloride (ChCl) with lactic acid (LA), and effects of cellulase non-productive binding onto DES-extracted lignin from willow and corn stover on enzymatic hydrolysis of cellulose was investigated. The correlation between hydrolysis yield of cellulose and chemical features of lignin was evaluated, and a potential inhibitory mechanism was proposed. Condensation of lignin was observed during DES treatment, and these condensed aromatic structures had an increased tendency to adsorb enzymes through hydrophobic interactions. As well as hydrophobic interactions mediated by lignin condensation, an increase in phenolic hydroxyl groups resulted in a greater amount of hydrogen bonds between cellulases and lignin that appeared to inhibit enzymatic hydrolysis yields of cellulose (39.96-42.86 % to 31.96-32.68 %). Although large amounts of COOHs were generated, the elevated electrostatic repulsion as a result of ionic groups was insufficient to decrease non-productive adsorption.

Enzyme-Mediated Lignocellulose Liquefaction Is Highly Substrate-Specific and Influenced by the Substrate Concentration or Rheological Regime.

The high viscosities/yield stresses of lignocellulose slurries makes their industrial processing a significant challenge. However, little is known regarding the degree to which liquefaction and its enzymatic requirements are specific to a substrate's physicochemical and rheological properties. In the work reported here, the substrate- and rheological regime-specificities of liquefaction of various substrates were assessed using real-time in-rheometer viscometry and offline oscillatory rheometry when hydrolyzed by combinations of cellobiohydrolase (Trichoderma reesei Cel7A), endoglucanase (Humicola insolens Cel45A), glycoside hydrolase (GH) family 10 xylanase, and GH family 11 xylanase. In contrast to previous work that has suggested that endoglucanase activity dominates enzymatic liquefaction, all of the enzymes were shown to have at least some liquefaction capacity depending on the substrate and reaction conditions. The contribution of individual enzymes was found to be influenced by the rheological regime; in the concentrated regime, the cellobiohydrolase outperformed the endoglucanase, achieving 2.4-fold higher yield stress reduction over the same timeframe, whereas the endoglucanase performed best in the semi-dilute regime. It was apparent that the significant differences in rheology and liquefaction mechanisms made it difficult to predict the liquefaction capacity of an enzyme or enzyme cocktail at different substrate concentrations.

Acidic deep eutectic solvent assisted isolation of lignin containing nanocellulose from thermomechanical pulp.

Nanocellulose is a promising material but its isolation generally requires unrecyclable hazardous chemicals and high energy consumption and its overall yield is low due to the use of high purity cellulose as precursor. In order to overcome these shortcomings, in this study, thermomechanical pulp (TMP) was investigated as a precursor for isolating lignin containing nanocellulose (LNC) using an environmentally friendly acidic deep eutectic solvent (DES) pre-treatment. Flat "ribbon" like LNCs (around 7.1 nm wide, 3.7 nm thick) with uniformly distributed lignin nanoparticles of 20-50 nm in diameter were successfully obtained at 57 % yield under optimum pre-treatment conditions (90 °C, 6 h, 1:1 oxalic acid dihydrate to choline chloride ratio). The LNCs exhibit cellulose Iß structure, high lignin content (32.6 %), and high thermal stability (Tmax of 358 °C). In general, green acidic DES pre-treatment has shown high efficiency in converting high lignin content biomass into value-added LNC, which benefits both lignocellulose utilization and environmental protection.

Elucidation of Changes in Cellulose Ultrastructure and Accessibility in Hardwood Fractionation Processes with Carbohydrate Binding Modules.

We have recently presented a sequential treatment method, in which steam explosion (STEX) was followed by hydrotropic extraction (HEX), to selectively fractionate cellulose, hemicellulose, and lignin in hardwood into separate process streams. However, above a treatment severity threshold, the structural alterations in the cellulose-enriched fraction appeared to restrict the enzymatic hydrolyzability and delignification efficiency. To better understand the ultrastructural changes in the cellulose, hardwood chips were treated by single (STEX or HEX) and combined treatments (STEX and HEX), and the cellulose accessibility quantified with carbohydrate-binding modules (CBMs) that bind preferentially to crystalline (CBM2a) and paracrystalline cellulose (CBM17). Fluorescent-tagged versions of the CBMs were used to map the spatial distribution of cellulose substructures with confocal laser scanning microscopy. With increasing severities, STEX increased the apparent crystallinity (CBM2a/CBM17-ratio) and overall accessibility (CBM2aH6 + CBM17) of the cellulose, whereas HEX demonstrated the opposite trend. The respective effects could also be discerned in the combined treatments where increasing severities further resulted in higher hemicellulose dissolution and, although initially beneficial, in stagnating accessibility and hydrolyzability. This study suggests that balancing the severities in the two treatments is required to maximize the fractionation and simultaneously achieve a reactive and accessible cellulose that is readily hydrolyzable.

The use of fluorescent protein-tagged carbohydrate-binding modules to evaluate the influence of drying on cellulose accessibility and enzymatic hydrolysis.

The influence of drying on cellulose accessibility and enzymatic hydrolysis was assessed. Dissolving pulp was differentially dried by freeze-, air- and oven-drying at 50 °C and subsequently hydrolyzed using the commercial CTec 3 cellulase preparation. It was apparent that drying reduced the ease of enzymatic hydrolysis of all of the substrates with a pronounced reduction (48%) exhibited by the oven-dried pulp. To assess if the ease of hydrolysis was due to enzyme accessibility to the substrate, microscopy (SEM), FTIR spectroscopy, water retention value (WRV), fiber aspect ratio analysis, Simons' stain and the selective binding of Fluorescent Protein-tagged Carbohydrate Binding Modules (FP-CBMs): CBM3a (crystalline cellulose) and CBM17 (amorphous cellulose) in combination with confocal laser scanning microscopy (CLSM) were used. The combined methods indicated that, if the gross characteristics of the substrate limited enzyme accessibility, the cellulases, as represented by the FP-CBMs, could not in turn access the finer structural components of the cellulosic substrates.

Potential of Xylanases to Reduce the Viscosity of Micro/Nanofibrillated Bleached Kraft Pulp.

The generally high viscosity of micro/nanofibrillated cellulose limits its applications in cream and fluid products. A bleached softwood Kraft (BSK) pulp was refined with increasing energy (500-2500 kWh t-1) to produce micro/nanofibrillated cellulose (MNBSK). Subsequent xylanase treatment was shown to influence the viscosity, gel point, aspect ratio, and fiber surface morphology of the MNBSK. It was apparent that the accessibility to xylanases was increased even at low refining energies (500 kWh t-1). Depending on the initial degree of cellulose fibrillation, xylanase treatment decreased the viscosity of the MNBSK from 4190-2030 to 681-243 Pa·s at a shear rate of 0.01 s-1, corresponding to the reduction in the aspect ratio from 183-296 to 163-194. It was likely that the xylanases were predominantly acting on the xylan present on the fiber surfaces, reducing the cross-linking points on the cellulose fibers and consequently resulting in the reduction in MNBSK viscosity.

Laccase-mediated hydrophilization of lignin decreases unproductive enzyme binding but limits subsequent enzymatic hydrolysis at high substrate concentrations.

One of the predominant mechanisms by which lignin restricts effective enzymatic deconstruction of lignocellulosic materials is the unproductive adsorption of enzymes. Although this inhibition can be partially mitigated through hydrophilization of lignin during thermochemical pretreatment, these types of treatments could potentially worsen slurry rheology, consequently making it more difficult to process the material at high substrate concentrations. In the work reported here, laccases were used to specifically modify lignin hydrophilicity within steam-pretreated substrate via in situ phenolic compound grafting. While lignin hydrophilization reduced unproductive enzyme adsorption, high-solids hydrolysis efficiency decreased significantly due to mass transfer limitations. It was apparent that low-solids hydrolysis experiments were a poor predictor of substrate digestibility at high-solids conditions and that substrate-water interactions impacted both substrate digestibility and slurry rheology.

Functionalizing Cellulose Nanocrystals with Click Modifiable Carbohydrate-Binding Modules.

Functionalized cellulose nanocrystals (CNC) have unique properties that make them attractive in various applications such as drug delivery, hydrogels, and emulsions. However, the predominant chemical methods currently used to functionalize cellulose nanocrystals have a large environmental footprint. Although greener methods are desirable, the relatively inert nature of cellulose crystals presents a major challenge to their potential modification in aqueous media. In the work reported here, carbohydrate binding modules (CBMs) were used to introduce new functionality to cellulose surfaces. CBM2a, which has a strong affinity for crystalline cellulose, was functionalized with an alkyne at the terminal amine position. The alkyne group, which was introduced onto the cellulose surface with CBM2a, underwent a Click reaction with polyethylene glycol (PEG) to modify CNC surfaces. This provided a strong, non-covalent modification of cellulose surfaces that was carried out in a one-pot reaction in aqueous media. The CBM-PEG modification of cellulose surfaces increased CNC redispersion after drying and improved suspension stability based on steric interactions. It was apparent that hybrid polysaccharide-protein, self-assembled nanoparticles could be effectively produced, with potential for nanomedicine, immunoassay, and drug delivery applications.

Enhancing bacterial cellulose production via adding mesoporous halloysite nanotubes in the culture medium.

Although bacterial cellulose (BC) is a fascinating, highly pure cellulose material for various downstream applications, production has been challenged by its low productivity. This work reported a facile route to significantly enhance BC yield without compromising its structural advantages via adding mesoporous halloysite nanotubes (HNTs) in the culture medium at static cultivations. The BC productivity of Gluconacetobacter xylinus was increased from 2.2 to 5.9 g L-1 after 15 days of cultivation when 2 wt% of HNTs was added into the standard fructose medium. It appeared that the dual functionality of cell immobilization and oxygen release of the HNTs were responsible for enhancing the BC productivity. Moreover, the HNTs-resulted BC pellicle exhibited negligible content of HNTs contamination (∼2 wt%), higher degree of crystallinity (87.7%) and porosity (assessed by water holding capacity, 12.7 g g-1), and showed promising applications especially in the bio-adsorption field.

Minimizing cellulase inhibition of whole slurry biomass hydrolysis through the addition of carbocation scavengers during acid-catalyzed pretreatment.

The aim of this work was to study how to minimize cellulase inhibition of whole slurry biomass hydrolysis through addition of carbocation scavengers during acid-catalyzed pretreatment. Various potential carbocation scavengers were compared and their inhibition mitigating effects towards the hydrolytic performance of cellulase enzymes was assessed. The results indicated that the addition of carbocation scavengers during the pretreatment process could not only alleviate the inhibitory effect of the phenolics on the enzymatic hydrolysis but also increase the accessibility of cellulases to the pretreated substrates. It appeared that lignin-derived compounds such as 4-hydroxybenzoic acid, vanillic acid, syringic acid could all serve as efficient scavengers to alleviate the inhibitory effect of phenolics on cellulose hydrolysis where the syringic acid showed the best mitigating effect. By combining the carbocation scavengers in the pretreatment process, an improved cellulose hydrolysis of the pretreated whole slurry could be achieved without any post detoxification step.

The inhibition of hemicellulosic sugars on cellulose hydrolysis are highly dependant on the cellulase productive binding, processivity, and substrate surface charges.

In this study, the influence of major hemicellulosic sugars (mannose and xylose) on cellulose hydrolysis and major enzyme activities were evaluated by using both commercial enzyme cocktail and purified cellulase monocomponents over a "library" of cellulosic substrates. Surprisingly, the results showed that unlike glucose, mannose/xylose did not inhibit individual cellulase activities but significantly decreased their hydrolytic performance on cellulose substrates. When various enzyme-substrate interactions (e.g. adsorption/desorption, productive binding, and processive moving) were evaluated, it appeared that these hemicellulosic sugars significantly reduced the productive binding and processivity of Cel7A, which in turn limited cellulase hydrolytic efficacy. Among a range of major cellulose characteristics (e.g. crystallinity, degree of polymerization, accessibility, and surface charges), the acid group content of the cellulosic substrates seemed to be the main driver that determined the extent of hemicellulosic sugar inhibition. Our results provided new insights for better understanding the sugar inhibition mechanisms of cellulose hydrolysis.

Enzyme mediated nanofibrillation of cellulose by the synergistic actions of an endoglucanase, lytic polysaccharide monooxygenase (LPMO) and xylanase.

Physiochemical methods have generally been used to "open-up" biomass substrates/pulps and have been the main method used to fibrillate cellulose. However, recent work has shown that canonical cellulase enzymes such as endoglucanases, in combination with "amorphogenesis inducing" proteins such as lytic polysaccharide monooxygenases (LPMO), swollenin and hemicellulases, are able to increase cellulose accessibility. In the work reported here different combinations of endoglucanase, LPMO and xylanase were applied to Kraft pulps to assess their potential to induce fibrillation at low enzyme loading over a short time period. Although gross fiber properties (fiber length, width and morphology) were relatively unchanged, over a short period of time, the intrinsic physicochemical characteristics of the pulp fibers (e.g. cellulose accessibility/DP/crystallinity/charge) were positively enhanced by the synergistic cooperation of the enzymes. LPMO addition resulted in the oxidative cleavage of the pulps, increasing the negative charge (~100 mmol kg-1) on the cellulose fibers. This improved cellulose nanofibrilliation while stabilizing the nanofibril suspension (zeta potential ζ = ~60 mV), without sacrificing nanocellulose thermostability. The combination of endoglucanase, LPMO and xylanases was shown to facilitate nanofibrillation, potentially reducing the need for mechanical refining while resulting in a pulp with a more uniform nanofibril composition.

Lignin Sulfonation and SO2 Addition Enhance the Hydrolyzability of Deacetylated and Then Steam-Pretreated Poplar with Reduced Inhibitor Formation.

The merit of deacetylation of corn stover prior to pretreatment is decreasing the formation of inhibitors and improving enzyme hydrolysis, proved in dilute acid pretreatment. However, few studies are done on how deacetylation would affect bioconversion process containing steam explosion. In this study, the effect of deacetylation on steam explosion was conducted using poplar as substrate. About 57 to 90% of acetyl group in poplar, depending on alkaline types and concentration, was removed by dilute alkaline deacetylation in 6 h. Deacetylation eliminated over 85% of inhibitor formation during downstream steam explosion. However, deacetylation prior to steam explosion decreased the dissolution of hemicellulose, thus reducing the cellulose accessibility of pretreated poplar, finally resulting in 5-20% decrease in glucose yield and 20-35% decrease in xylose yield. The addition of 5% SO2 during steam explosion significantly improved the hydrolysis of deacetylated and pretreated poplar without significantly increasing the concentration of inhibitors. Incorporating 45 mmol/kg sulfoacid group in lignin fraction of deacetylated and then pretreated poplar dramatically improved the xylose yield to about 100% and increased the glucose yield by 30%.

The potential of tailoring the conditions of steam explosion to produce xylo-oligosaccharides from sugarcane bagasse.

In this study, the potential of the steam explosion (SE) method to produce high levels XOS from sugarcane bagasse, a xylan-rich hemicellulosic feedstock, was assessed. The effect of different operating conditions on XOS production yield and selectivity were investigated using a mini-pilot scale SE unit. The results show that even under a non-optimized condition (190 °C, 5 min and 0.5% H2SO4 as catalyst), SE led to about 40% xylan recovery as XOS, which was comparable to the well-known, multi-step, enzymatic production of XOS from alkaline-extracted xylan, and other commonly employed chemical methods. In addition, the XOS-rich hydrolysate from SE constituted of greater diversity in the degree of polymerization, which has been shown to be desirable for prebiotic application.

Limitation of cellulose accessibility and unproductive binding of cellulases by pretreated sugarcane bagasse lignin.

BACKGROUND: The effectiveness of the enzymatic hydrolysis of cellulose in plant cell wall is strongly influenced by the access of enzymes to cellulose, which is at least in part limited by the presence of lignin. Although physicochemical treatments preceding the enzymatic catalysis significantly overcome this recalcitrance, the residual lignin can still play a role in the process. Lignin is suggested to act as a barrier, hindering cellulose and limiting the access of the enzymes. It can also unspecifically bind cellulases, reducing the amount of enzymes available to act on cellulose. However, the limiting role of the lignin present in pretreated sugarcane bagasses has not been fully understood yet. RESULTS: A set of sugarcane bagasses pretreated by five leading pretreatment technologies was created and used to assess their accessibility and the unproductive binding capacity of the resulting lignins. Steam explosion and alkaline sulfite pretreatments resulted in more accessible substrates, with approximately 90% of the cellulose hydrolyzed using high enzyme loadings. Enzymatic hydrolysis of alkaline-treated (NaOH) and steam-exploded sugarcane bagasses were strongly affected by unproductive binding at the lowest enzyme loading tested. Analysis of the extracted lignins confirmed the superior binding capacity of these lignins. Sulfite-based pretreatments (alkaline sulfite and acid sulfite) resulted in lignins with lower binding capacities compared to the analogue pretreatments without sulfite (alkaline and acidic). Strong acid groups present in sulfite-based pretreated substrates, attributed to sulfonated lignins, corroborated the lower binding capacities of the lignin present in these substrates. A more advanced enzyme preparation (Cellic CTec3) was shown to be less affected by unproductive binding at low enzyme loading. CONCLUSIONS: Pretreatments that increase the accessibility and modify the lignin are necessary in order to decrease the protein binding capacity. The search for the called weak lignin-binding enzymes is of major importance if hydrolysis with low enzyme loadings is the goal for economically viable processes.

Lignin valorization: lignin nanoparticles as high-value bio-additive for multifunctional nanocomposites.

BACKGROUND: Although conversion of low value but high-volume lignin by-product to its usable form is one of the determinant factors for building an economically feasible integrated lignocellulose biorefinery, it has been challenged by its structural complexity and inhomogeneity. We and others have shown that uniform lignin nanoparticles can be produced from a wide range of technical lignins, despite the varied lignocellulosic biomass and the pretreatment methods/conditions applied. This value-added nanostructure lignin enriched with multifunctional groups can be a promising versatile material platform for various downstream utilizations especially in the emerging nanocomposite fields. RESULTS: Inspired by the story of successful production and application of nanocellulose biopolymer, two types of uniform lignin nanoparticles (LNPs) were prepared through self-assembling of deep eutectic solvent (DES) and ethanol-organosolv extracted technical lignins derived from a two-stage fractionation pretreatment approach, respectively. Both LPNs exhibited sphere morphology with unique core-shell nanostructure, where the DES-LNPs showed a more uniform particle size distribution. When incorporated into the traditional polymeric matrix such as poly(vinyl alcohol), these LPN products displayed great potential to formulate a transparent nanocomposite film with additional UV-shielding efficacy (reached ~80% at 400 nm with 4 wt% of LNPs) and antioxidant functionalities (reached ~160 µm mol Trolox g-1 with 4 wt% of LNPs). At the same time, the abundant phenolic hydroxyl groups on the shell of LNPs also provided good interfacial adhesion with PVA matrix through the formation of hydrogen bonding network, which further improved the mechanical and thermal performances of the fabricated LNPs/PVA nanocomposite films. CONCLUSIONS: Both LNPs are excellent candidates for producing multifunctional polymer nanocomposites using facile technical route. The prepared transparent and flexible LNPs/PVA composite films with high UV-shielding efficacy, antioxidant activity, and biocompatibility are promising in the advanced packaging field, which potentially provides an additional high-value lignin product stream to the lignocellulose biorefinery. This study could open the door for the production and application of novel LNPs in the nascent bioeconomy.Graphical abstractLignin nanoparticle for transparent nanocomposite film with UV-shielding efficacy.

Mechanistic insights into the liquefaction stage of enzyme-mediated biomass deconstruction.

Effective enzyme-mediated viscosity reduction, disaggregation, or "liquefaction," is required to overcome the rheological challenges resulting from the fibrous, hygroscopic nature of lignocellulosic biomass, particularly at the high solids loadings that will be required for an economically viable process. However, the actual mechanisms involved in enzyme-mediated liquefaction, as determined by viscosity or yield stress reduction, have yet to be fully resolved. Particle fragmentation, interparticle interaction, material dilution, and water-retention capacity were compared for their ability to quantify enzyme-mediated liquefaction of model and more realistic pretreated biomass substrates. It was apparent that material dilution and particle fragmentation occurred simultaneously and that both mechanisms contributed to viscosity/yield stress reduction. However, their relative importance was dependent on the nature of the biomass substrate. Interparticle interaction and enzyme-mediated changes to these interactions was shown to have a significant effect on slurry rheology. Liquefaction was shown to result from the combined action of material dilution, particle fragmentation, and alteration of interactions at particle surfaces. However, the observed changes in water retention capacity did not correlate with yield stress reduction. The relative importance of each mechanism was significantly influenced by the nature of the biomass substrate and its physicochemical properties. An ongoing challenge is that mechanisms, such as refining, which enhance enzyme accessibility to the cellulosic component of the substrate, are detrimental to slurry rheology and will likely impede enzyme-mediated liquefaction when high substrate concentrations are used.