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.

Quantifying cellulose accessibility during enzyme-mediated deconstruction using 2 fluorescence-tagged carbohydrate-binding modules.

Two fluorescence-tagged carbohydrate-binding modules (CBMs), which specifically bind to crystalline (CBM2a-RRedX) and paracrystalline (CBM17-FITC) cellulose, were used to differentiate the supramolecular cellulose structures in bleached softwood Kraft fibers during enzyme-mediated hydrolysis. Differences in CBM adsorption were elucidated using confocal laser scanning microscopy (CLSM), and the structural changes occurring during enzyme-mediated deconstruction were quantified via the relative fluorescence intensities of the respective probes. It was apparent that a high degree of order (i.e., crystalline cellulose) occurred at the cellulose fiber surface, which was interspersed by zones of lower structural organization and increased cellulose accessibility. Quantitative image analysis, supported by 13C NMR, scanning electron microscopy (SEM) imaging, and fiber length distribution analysis, showed that enzymatic degradation predominates at these zones during the initial phase of the reaction, resulting in rapid fiber fragmentation and an increase in cellulose surface crystallinity. By applying this method to elucidate the differences in the enzyme-mediated deconstruction mechanisms, this work further demonstrated that drying decreased the accessibility of enzymes to these disorganized zones, resulting in a delayed onset of degradation and fragmentation. The use of fluorescence-tagged CBMs with specific recognition sites provided a quantitative way to elucidate supramolecular substructures of cellulose and their impact on enzyme accessibility. By designing a quantitative method to analyze the cellulose ultrastructure and accessibility, this study gives insights into the degradation mechanism of cellulosic substrates.

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.

N-Heteropolycyclic compounds from the formal intramolecular (4 + 1)-cycloaddition of chromium aminocarbenes.

Chromium aminocarbenes tethered to dienes of all three electronic natures undergo an efficient intramolecular (4 + 1)-cycloaddition to give N-heteropolycyclic compounds. Ligands on chromium had a profound effect on the course of the reaction.