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.

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.

The use of carbohydrate binding modules (CBMs) to monitor changes in fragmentation and cellulose fiber surface morphology during cellulase- and Swollenin-induced deconstruction of lignocellulosic substrates.

Although the actions of many of the hydrolytic enzymes involved in cellulose hydrolysis are relatively well understood, the contributions that amorphogenesis-inducing proteins might contribute to cellulose deconstruction are still relatively undefined. Earlier work has shown that disruptive proteins, such as the non-hydrolytic non-oxidative protein Swollenin, can open up and disaggregate the less-ordered regions of lignocellulosic substrates. Within the cellulosic fraction, relatively disordered, amorphous regions known as dislocations are known to occur along the length of the fibers. It was postulated that Swollenin might act synergistically with hydrolytic enzymes to initiate biomass deconstruction within these dislocation regions. Carbohydrate binding modules (CBMs) that preferentially bind to cellulosic substructures were fluorescently labeled. They were imaged, using confocal microscopy, to assess the distribution of crystalline and amorphous cellulose at the fiber surface, as well as to track changes in surface morphology over the course of enzymatic hydrolysis and fiber fragmentation. Swollenin was shown to promote targeted disruption of the cellulosic structure at fiber dislocations.

Enhancing the enzymatic hydrolysis of lignocellulosic biomass by increasing the carboxylic acid content of the associated lignin.

To assess the effects that the physical and chemical properties of lignin might have on the enzymatic hydrolysis of pretreated lignocellulosic substrates, protease treated lignin (PTL) and cellulolytic enzyme lignin (CEL) fractions, isolated from steam and organosolv pretreated corn stover, poplar, and lodgepole pine, were prepared and characterized. The adsorption of cellulases to the isolated lignin preparations corresponded to a Langmuir adsorption isotherm. It was apparent that, rather than the physical properties of the isolated lignin, the carboxylic acid functionality of the isolated lignin, as determined by FTIR and NMR spectroscopy, had much more of an influence when lignin was added to typical hydrolysis of pure cellulose (Avicel). An increase in the carboxylic content of the lignin preparation resulted in an increased hydrolysis yield. These results suggested that the carboxylic acids within the lignin partially alleviate non-productive binding of cellulases to lignin. To try to confirm this possible mechanism, dehydrogenative polymers (DHP) of monolignols were synthesized from coniferyl alcohol (CA) and ferulic acid (FA), and these model compounds were added to a typical enzymatic hydrolysis of Avicel. The DHP from FA, which was enriched in carboxylic acid groups compared with the DHP from CA, adsorbed a lower mount of cellulases and did not decrease hydrolysis yields when compared to the DHP from CA, which decreased the hydrolysis of Avicel by 8.4%. Thus, increasing the carboxylic acid content of the lignin seemed to significantly decrease the non-productive binding of cellulases and consequently increased the enzymatic hydrolysis of the cellulose.

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.

The effect of isolated lignins, obtained from a range of pretreated lignocellulosic substrates, on enzymatic hydrolysis.

The influence of the residual lignin remaining in the cellulosic rich component of pretreated lignocellulosic substrates on subsequent enzymatic hydrolysis was assessed. Twelve lignin preparations were isolated by two isolation methods (protease treated lignin (PTL) and cellulolytic enzymatic lignin (CEL)) from three types of biomass (corn stover, poplar, and lodgepole pine) that had been pretreated by two processes (steam and organosolv pretreatments). Comparative analysis of the isolated lignin showed that the CEL contained lower amounts of carbohydrates and protein than did the PTL and that the isolated lignin from corn stover contained more carbohydrates than did the lignin derived from the poplar and lodgepole pine. The lower yields of acid insoluble lignin (AIL) obtained from the corn stover when using the PTL method indicated that the lignin from the corn stover had a higher hydrophilicity than did the lignin from the poplar and lodgepole pine. The isolated lignin preparations were added to the reaction mixture containing crystalline cellulose (Avicel) and their possible effects on enzymatic hydrolysis were assessed. It was apparent that the lignin isolated from lodgepole pine and steam pretreated poplar decreased the hydrolysis yields of Avicel, whereas the other isolated lignins did not appear to decrease the hydrolysis yields significantly. The hydrolysis yields of the pretreated lignocellulose and those of Avicel containing the PTL showed good correlation, indicating that the nature of the residual lignin obtained after pretreatment significantly influenced hydrolysis.

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.

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.

Comparison of methods to assess the enzyme accessibility and hydrolysis of pretreated lignocellulosic substrates.

Fiber size analysis, water retention value, and Simons' stain measurements were assessed for their potential to predict the susceptibility of a given substrate to enzymatic hydrolysis. Slight modifications were made to the fiber size analysis and water retention protocols to adapt these measurements to evaluate substrates for cellulolytic hydrolysis rather than pulps for papermaking. Lodgepole pine was pretreated by the steam and ethanol-organosolv processes under varying conditions. The Simons' stain procedure proved to be an effective method for indicating the potential ease of enzymatic hydrolysis of substrates pretreated by either process or when the pretreatment conditions were altered.

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.

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.

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.

Evaluating the distribution of cellulases and the recycling of free cellulases during the hydrolysis of lignocellulosic substrates.

The recycling of cellulase enzymes is one potential strategy for reducing the cost of the enzymatic hydrolysis step during the bioconversion of lignocellulosics to ethanol. To determine the influence of lignin on the post-hydrolysis distribution of cellulase enzymes between the liquid and solid phases, the hydrolysis of Avicel was compared to an organosolv-pretreated Douglas fir substrate with a lignin content of 3.0%. After a 12 h hydrolysis reaction on Avicel, 90% of the added cellulases (including beta-glucosidases) remained "free" in the liquid phase compared to only 65% in the case of the hydrolysis of the organosolv-pretreated Douglas fir substrate. The readsorption of free cellulases by supplementing the hydrolysis reaction with fresh substrate was explored as a potential means of recovering the free cellulases that remain in the liquid phase after hydrolysis. The Langmuir adsorption isotherm was used to develop a model predicting that 82% of the free cellulases could be recovered via readsorption onto fresh substrates during the hydrolysis of an ethanol-pretreated mixed softwood substrate with a lignin content of 6%. Recoverable free cellulase values of 85% and 88% based on cellulase activity and protein content, respectively, were obtained after experimental verification of the model. The readsorption of free cellulases onto fresh lignocellulosic substrates was shown to be an effective method for free enzyme recovery.

Organosolv ethanol lignin from hybrid poplar as a radical scavenger: relationship between lignin structure, extraction conditions, and antioxidant activity.

Twenty-one organosolv ethanol lignin samples were prepared from hybrid poplar (Populus nigra xP. maximowiczii) under varied conditions with an experimental matrix designed using response surface methodology (RSM). The lignin preparations were evaluated as potential antioxidants. Results indicated that the lignins with more phenolic hydroxyl groups, less aliphatic hydroxyl groups, low molecular weight, and narrow polydispersity showed high antioxidant activity. Processing conditions affected the functional groups and molecular weight of the extracted organosolv ethanol lignins, and consequently influenced the antioxidant activity of the lignins. In general, the lignins prepared at elevated temperature, longer reaction time, increased catalyst, and diluted ethanol showed high antioxidant activity. Regression models were developed to enable the quantitative prediction of lignin characteristics and antioxidant activity based on the processing conditions.

Strategies to enhance the enzymatic hydrolysis of pretreated softwood with high residual lignin content.

Pretreatment of Douglas-fir by steam explosion produces a substrate containing approx 43% lignin. Two strategies were investigated for reducing the effect of this residual lignin on enzymatic hydrolysis of cellulose: mild alkali extraction and protein addition. Extraction with cold 1% NaOH reduced the lignin content by only approx 7%, but cellulose to glucose conversion was enhanced by about 30%. Before alkali extraction, addition of exogenous protein resulted in a significant improvement in cellulose hydrolysis, but this protein effect was substantially diminished after alkali treatment. Lignin appears to reduce cellulose hydrolysis by two distinct mechanisms: by forming a physical barrier that prevents enzyme access and by non-productively binding cellulolytic enzymes. Cold alkali appears to selectively remove a fraction of lignin from steam-exploded Douglas-fir with high affinity for protein. Corresponding data for mixed softwood pretreated by organosolv extraction indicates that the relative importance of the two mechanisms by which residual lignin affects hydrolysis is different according to the pre- and post-treatment method used.

A NaBH4 Coupled Ninhydrin-Based Assay for the Quantification of Protein/Enzymes During the Enzymatic Hydrolysis of Pretreated Lignocellulosic Biomass.

Accurate protein quantification is necessary in many of the steps during the enzymatic hydrolysis of pretreated lignocellulosic biomass, from the fundamental determination of enzyme kinetics to techno-economic assessments, such as the use of enzyme recycling strategies, evaluation of enzyme costs, and the optimization of various process steps. In the work described here, a modified, more rapid ninhydrin-based protein quantification assay was developed to better quantify enzyme levels in the presence of lignocellulosic biomass derived compounds. The addition of sodium borohydride followed by acid hydrolysis at 130 °C greatly reduced interference from monosaccharides and oligosaccharides and decreased the assay time 6-fold. The modified ninhydrin assay was shown to be more accurate as compared to various traditional colorimetric protein assays when commercial cellulase enzyme mixtures were quantified under typical pretreated lignocellulosic biomass enzymatic hydrolysis conditions. The relatively short assay time and microplate-reading capability of the modified assay indicated that the method could likely be used for high-throughput protein determination.

Accessory enzymes influence cellulase hydrolysis of the model substrate and the realistic lignocellulosic biomass.

The potential of cellulase enzymes in the developing and ongoing "biorefinery" industry has provided a great motivation to develop an efficient cellulase mixture. Recent work has shown how important the role that the so-called accessory enzymes can play in an effective enzymatic hydrolysis. In this study, three newest Novozymes Cellic CTec cellulase preparations (CTec 1/2/3) were compared to hydrolyze steam pretreated lignocellulosic substrates and model substances at an identical FPA loading. These cellulase preparations were found to display significantly different hydrolytic performances irrelevant with the FPA. And this difference was even observed on the filter paper itself when the FPA based assay was revisited. The analysis of specific enzyme activity in cellulase preparations demonstrated that different accessory enzymes were mainly responsible for the discrepancy of enzymatic hydrolysis between diversified substrates and various cellulases. Such the active role of accessory enzymes present in cellulase preparations was finally verified by supplementation with ß-glucosidase, xylanase and lytic polysaccharide monooxygenases AA9. This paper provides new insights into the role of accessory enzymes, which can further provide a useful reference for the rational customization of cellulase cocktails in order to realize an efficient conversion of natural lignocellulosic substrates.

The accessible cellulose surface influences cellulase synergism during the hydrolysis of lignocellulosic substrates.

Effective enzymatic hydrolysis of insoluble cellulose requires the synergistic action of a suite of cellulase components. Most previous studies have only assessed cellulase synergism on model cellulosic substrates. When the actions of individual and combinations of cellulases (Cel7A, Cel6A, Cel7B, Cel5A) were assessed on various pretreated lignocellulosic substrates, Cel7A was shown to be the major contributor to overall cellulose hydrolysis, with the other enzymes synergistically enhancing its hydrolytic efficiency, at least partially, by facilitating Cel7A desorption (assessed by a double-sandwich enzyme-linked immunosorbent assay). When the influences of various substrate physicochemical characteristics on the effectiveness of enzyme synergism were assessed, a strong relationship was observed between cellulose accessibility (as determined by the cellulose binding module technique) and the degree of synergism, with greater synergy observed on the more disorganized/accessible cellulose surface.