Bacterial catabolism of acetovanillone, a lignin-derived compound.

Bacterial catabolic pathways have considerable potential as industrial biocatalysts for the valorization of lignin, a major component of plant-derived biomass. Here, we describe a pathway responsible for the catabolism of acetovanillone, a major component of several industrial lignin streams. Rhodococcus rhodochrous GD02 was previously isolated for growth on acetovanillone. A high-quality genome sequence of GD02 was generated. Transcriptomic analyses revealed a cluster of eight genes up-regulated during growth on acetovanillone and 4-hydroxyacetophenone, as well as a two-gene cluster up-regulated during growth on acetophenone. Bioinformatic analyses predicted that the hydroxyphenylethanone (Hpe) pathway proceeds via phosphorylation and carboxylation, before ß-elimination yields vanillate from acetovanillone or 4-hydroxybenzoate from 4-hydroxyacetophenone. Consistent with this prediction, the kinase, HpeHI, phosphorylated acetovanillone and 4-hydroxyacetophenone. Furthermore, HpeCBA, a biotin-dependent enzyme, catalyzed the ATP-dependent carboxylation of 4-phospho-acetovanillone but not acetovanillone. The carboxylase's specificity for 4-phospho-acetophenone (kcat/KM = 34 ± 2 mM-1 s-1) was approximately an order of magnitude higher than for 4-phospho-acetovanillone. HpeD catalyzed the efficient dephosphorylation of the carboxylated products. GD02 grew on a preparation of pine lignin produced by oxidative catalytic fractionation, depleting all of the acetovanillone, vanillin, and vanillate. Genomic and metagenomic searches indicated that the Hpe pathway occurs in a relatively small number of bacteria. This study facilitates the design of bacterial strains for biocatalytic applications by identifying a pathway for the degradation of acetovanillone.

Functional Lignin Building Blocks: Reactive Vinyl Esters with Acrylic Acid.

Introducing vinyl groups onto the backbone of technical lignin provides an opportunity to create highly reactive renewable polymers suitable for radical polymerization. In this work, the chemical modification of softwood kraft lignin was pursued with etherification, followed by direct esterification with acrylic acid (AA). In the first step, phenolic hydroxyl and carboxylic acid groups were derivatized into aliphatic hydroxyl groups using ethylene carbonate and an alkaline catalyst. The lignin was subsequently fractionated using a downward precipitation method to recover lignin of defined molar mass and solubility. After recovery, the resulting material was then esterified with AA, resulting in lignin with vinyl functional groups. The first step resulted in approximately 90% of phenolic hydroxyl groups being converted into aliphatic hydroxyls, while the downward fractionation resulted in three samples of lignin with defined molar masses. For the esterification reaction, the weight ratio of reagents, reaction temperature, and reaction time were evaluated as factors that would influence the modification efficacy. 13C NMR spectroscopy analysis of lignin samples before and after esterification showed that the optimized reaction conditions could reach approximately 40% substitution of aliphatic hydroxyl groups. Both steps only used lignin and the modifying reagent (no solvent), with the possibility of recovery and reuse of the reagent by dilution and distillation. An additional second esterification step of the resulting lignin sample with acetic acid or propionic acid converted 90% of remaining hydroxyl groups into short-chain carbon aliphatic esters, making a hydrophobic material suitable for further copolymerization with synthetic hydrophobic monomers.

Revisiting the Molar Mass and Conformation of Derivatized Fractionated Softwood Kraft Lignin.

The limited utilization of reliable tools and standards for determination of the softwood kraft lignin molar mass and the corresponding molecular conformation hampers elucidation of the structure-property relationships of lignin. At issue, conventional size exclusion chromatography (SEC) is unable to robustly measure the molar mass because of a lack of calibration standards with a similar structure to lignin. In the present work, the determination of the absolute molar mass of acetylated technical lignin was revisited utilizing SEC combined with multi-angle light scattering with a band pass filter to suppress the fluorescence. Fractionated lignin isolated using sequential techniques of solvent and membrane methods was used to enhance the clarity of light-scattering profiles by narrowing the molar mass distribution of lignin fractions. Further information on the molecular conformation of derivatized samples was studied utilizing a differential viscometer, and chemical structures were identified by NMR spectroscopy analysis. Through the help of fractionation, intrinsic viscosity values were determined for the different fractions as a function of molecular weight cut-off membranes. The derivatized acetone-soluble lignin was found to possess a lower molecular weight and an extremely compact structure relative to the derivatized acetone-insoluble fraction based on a significantly lower "α" value in the Mark-Houwink-Sakurada plot (0.15 acetone-soluble vs 0.33 acetone-insoluble). The differences in geometry were supported by the linkage analysis from NMR showing the acetone-soluble part containing fewer native linkages. In both of these examples, kraft lignin behaved like a solid sphere, limiting the ability to provide entanglements between molecular chains. From this standpoint, macroscopic properties of lignin are justified with this knowledge of a dense and extremely compact structure.

Molecular Orientation and Organization of Technical Lignin-Based Composite Nanofibers and Films.

Natural materials are highly anisotropic, maximizing performance of the polymeric structures while conserving mass and enhancing function. In synthetic materials, nanoscale fibers produced by electrospinning often contain molecular alignment of polymers along the fiber axis achieving some similarity to natural fibers. In this study, isolated softwood kraft lignin (SKL) was electrospun into aligned fibers utilizing a special collector. The molecular organization of lignin within the aligned nanofibers was investigated by polarized light optical microscopy. Furthermore, the functional groups that had preferred alignment along the fiber axis were identified with polarized Fourier transform infrared (FTIR) spectroscopy based on dichroism measurements. In addition, nanocrystalline cellulose (NCC) was added to the lignin solutions in order to create composite nanofibers. Both the orientation of NCC within the nanoscale fibers and the impact this component had on the degree of orientation of SKL within the aligned nanofibers were revealed by utilizing polarized FTIR. Finally, solvent cast lignin films were analyzed for their anisotropic polarizability, demonstrating birefringence with and without nanocrystalline cellulose. The work provided unique insight into both preferred orientation (fibers) and assembly (films) for technical lignin due to processing.

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.

Current characterization methods for cellulose nanomaterials.

A new family of materials comprised of cellulose, cellulose nanomaterials (CNMs), having properties and functionalities distinct from molecular cellulose and wood pulp, is being developed for applications that were once thought impossible for cellulosic materials. Commercialization, paralleled by research in this field, is fueled by the unique combination of characteristics, such as high on-axis stiffness, sustainability, scalability, and mechanical reinforcement of a wide variety of materials, leading to their utility across a broad spectrum of high-performance material applications. However, with this exponential growth in interest/activity, the development of measurement protocols necessary for consistent, reliable and accurate materials characterization has been outpaced. These protocols, developed in the broader research community, are critical for the advancement in understanding, process optimization, and utilization of CNMs in materials development. This review establishes detailed best practices, methods and techniques for characterizing CNM particle morphology, surface chemistry, surface charge, purity, crystallinity, rheological properties, mechanical properties, and toxicity for two distinct forms of CNMs: cellulose nanocrystals and cellulose nanofibrils.

Preparation and evaluation of nanocellulose-gold nanoparticle nanocomposites for SERS applications.

Nanocellulose is of research interest due to its extraordinary optical, thermal, and mechanical properties. The incorporation of guest nanoparticles into nanocellulose substrates enables production of novel nanocomposites with a broad range of applications. In this study, gold nanoparticle/bacterial cellulose (AuNP/BC) nanocomposites were prepared and evaluated for their applicability as surface-enhanced Raman scattering (SERS) substrates. The nanocomposites were prepared by citrate mediated in situ reduction of Au(3+) in the presence of a BC hydrogel at 303 K. Both the size and morphology of the AuNPs were functions of the HAuCl4 and citrate concentrations. At high HAuCl4 concentrations, Au nanoplates form within the nanocomposites and are responsible for high SERS enhancements. At lower HAuCl4 concentrations, uniform nanospheres form and the SERS enhancement is dependent on the nanosphere size. The time-resolved increase in the SERS signal was probed as a function of drying time with SERS 'hot-spots' primarily forming in the final minutes of nanocomposite drying. The application of the AuNP/BC nanocomposites for detection of the SERS active dyes MGITC and R6G as well as the environmental contaminant atrazine is illustrated as is its use under low and high pH conditions. The results indicate the broad applicability of this nanocomposite for analyte detection.

Assembly of debranched xylan from solution and on nanocellulosic surfaces.

This study focused on the assembly characteristics of debranched xylan onto cellulose surfaces. A rye arabinoxylan polymer with an initial arabinose/xylose ratio of 0.53 was debranched with an oxalic acid treatment as a function of time. The resulting samples contained reduced arabinose/xylose ratios significantly affecting the molecular architecture and solution behavior of the biopolymer. With this treatment, an almost linear xylan with arabinose DS of only 0.04 was obtained. The removal of arabinose units resulted in the self-assembly of the debranched polymer in water into stable nanoparticle aggregates with a size around 300 nm with a gradual increase in crystallinity of the isolated xylan. Using quartz crystal microbalance with dissipation monitoring, the adsorption of xylan onto model cellulose surfaces was quantified. Compared to the nonmodified xylan, the adsorption of debranched xylan increased from 0.6 to 5.5 mg m(-2). Additionally, adsorption kinetics suggest that the nanoparticles rapidly adsorbed to the cellulose surfaces compared to the arabinoxylan. In summary, a control of the molecular structure of xylan influences its ability to form a new class of polysaccharide nanoparticles in aqueous suspensions and its interaction with nanocellulose surfaces.

Chemical shifts of phenolic monomers in solution and implications for addition and self-condensation.

Alkali metal counter-cations alter the electron density of phenolates in solution by electrostatic interactions. This change in electron density affects their reactivity toward formaldehyde, hydroxymethylphenols, and isocyanates during polymerization. The electronic perturbation of phenolic model compounds in the presence of alkali metal hydroxides was investigated with (13)C and (1)H nuclear magnetic resonance in polar solvents relative to non-ionic controls, altering the chemical shifts of the model compounds, thus indicating changes in electron density using the chemical shift as a proxy. These shifts were attributed to Coulombic electrostatic interactions of the counter-cation with the phenolate anion that correlated to hydrated ionic radius and solvent dielectric constants. The predicted relative reaction rates for formaldehyde addition based on electron density ranking from (13)C nuclear magnetic resonance of the phenolic models was compared with the literature values. Predictions for condensation reactions of 2- and 4-hydroxymethylphenol from chemical shifts were consistent with published results. The results permit predictions for the reaction of phenolic compounds for the formation of thermosetting polymeric materials.

Exploring the impact of water on the morphology and crystallinity of xylan hydrate nanotiles.

There has been a resurgence of studies on xylan particles describing various properties and exploring new applications. The aim of this study was to analyze xylan hydrate crystals in the wet state and after air-drying using state-of-art imaging techniques in order to assess the impact of water on both crystallinity and particle morphology. Xylan from esparto grass (Stipa tenacissima) was crystallized and formed convex platelets, termed 'nanotiles'. Fully hydrated xylan crystals were examined in a layer of vitreous ice by cryogenic electron microscopy. Selected area electron diffraction of the xylan hydrate crystals revealed an oriented crystalline core, unlike the dried crystals that showed no orientation. The surface topographies and thickness of wet and air-dried xylan nanotiles were observed using atomic force microscopy imaging in both liquid and in air. X-ray diffraction was used to assess the crystallinity of xylan nanotiles after drying to varying levels. Air-dried crystals gave diffraction maxima corresponding to xylan hydrate, while wet crystals gave diffraction maxima corresponding to xylan dihydrate. This study offers new insight into xylan hydrate particles, focusing on the role of water on their crystallinity, ultrastructure, and orientation of the crystalline layers.

Reduced cellulose accessibility slows down enzyme-mediated hydrolysis of cellulose.

Enzyme-mediated hydrolysis of cellulose always starts with an initial rapid phase, which gradually slows down, sometimes resulting in incomplete cellulose hydrolysis even after prolonged incubation. Although mechanisms such as end-product inhibition are known to play a role, the predominant mechanism appears to be reduced cellulose accessibility to the enzymes. When using Simon's stain to quantify accessibility, the accessibility of mechanically disintegrated and phosphoric acid-swollen cellulose substrates decreased as hydrolysis proceeded. In contrast, the poor initial accessibility of Avicel remained low throughout hydrolysis. However, washing the residual cellulose increased cellulose accessibility, likely due to the removal of tightly bound but non-productive enzymes which blocked access to more active enzymes in solution. Atomic force microscopy (AFM) analysis of the initial and residual cellulose collected when the hydrolysis plateaued, showed an increase in the roughness of the cellulose surface, possibly resulting in the tighter binding of less active cellulases.

Solventless Amination of Lignin and Natural Phenolics using 2-Oxazolidinone.

Reactive amine compounds are critical for a vast array of useful chemicals in society, yet a limited number of them are derived from renewable resources. This study developed an efficient route to obtain aminated building blocks from phenolic resources derived from nature, such as lignin and tannic acid, for enhancing their utility in applications such as epoxy resins, nylons, polyurethanes, and other polymeric materials. The reaction utilized a carbon storage compound, 2-oxazolidinone as a solvent and as a reagent circumventing the need of hazardous chemistry of conventional amination routes such as those involving formaldehyde. Both free acids and hindered phenolics were readily converted into aminoethyl derivatives resulting in aromatics with primary amine functionality. The aminated compounds, with the potential for enhanced reactivity, can pave the way toward more advanced renewable building blocks.

The pre-addition of "blocking" proteins decreases subsequent cellulase adsorption to lignin and enhances cellulose hydrolysis.

The pre-adsorption of non-catalytic/blocking proteins onto the lignin component of pretreated biomass has been shown to significantly increase the effectiveness of subsequent enzyme-mediated hydrolysis of the cellulose by limiting non-productive enzyme adsorption. Layer-by-layer adsorption of non-catalytic proteins and enzymes onto lignin was monitored using Quartz Crystal Micro balancing combined with Dissipation monitoring (QCM-D) and conventional protein adsorption. These methods were used to assess the interaction between soft/hardwood lignins, cellulases and the three non-catalytic proteins BSA, lysozyme and ovalbumin. The QCM-D analysis showed higher adsorption rates for all of the non-catalytic proteins onto the lignin films as compared to cellulases. This suggested that the "blocking" proteins would preferentially adsorb to the lignin rather than the enzymes. Pre-incubation of the lignin films with blocking proteins resulted in reduced adsorption of cellulases onto the lignin, significantly enhancing cellulose hydrolysis.

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.

Discovery of lignin-transforming bacteria and enzymes in thermophilic environments using stable isotope probing.

Characterizing microorganisms and enzymes involved in lignin biodegradation in thermal ecosystems can identify thermostable biocatalysts. We integrated stable isotope probing (SIP), genome-resolved metagenomics, and enzyme characterization to investigate the degradation of high-molecular weight, 13C-ring-labeled synthetic lignin by microbial communities from moderately thermophilic hot spring sediment (52 °C) and a woody "hog fuel" pile (53 and 62 °C zones). 13C-Lignin degradation was monitored using IR-GCMS of 13CO2, and isotopic enrichment of DNA was measured with UHLPC-MS/MS. Assembly of 42 metagenomic libraries (72 Gb) yielded 344 contig bins, from which 125 draft genomes were produced. Fourteen genomes were significantly enriched with 13C from lignin, including genomes of Actinomycetes (Thermoleophilaceae, Solirubrobacteraceae, Rubrobacter sp.), Firmicutes (Kyrpidia sp., Alicyclobacillus sp.) and Gammaproteobacteria (Steroidobacteraceae). We employed multiple approaches to screen genomes for genes encoding putative ligninases and pathways for aromatic compound degradation. Our analysis identified several novel laccase-like multi-copper oxidase (LMCO) genes in 13C-enriched genomes. One of these LMCOs was heterologously expressed and shown to oxidize lignin model compounds and minimally transformed lignin. This study elucidated bacterial lignin depolymerization and mineralization in thermal ecosystems, establishing new possibilities for the efficient valorization of lignin at elevated temperature.

Supramolecular structure characterization of molecularly thin cellulose I nanoparticles.

Unusual fractions of cellulose microfibrils from woody material with dimensions of hundreds of nanometers in length and single digit angstrom thickness were obtained by intensive sonication of TEMPO-oxidized cellulose fibers. These cellulose microfibril fragments, composed of many mono- and bilayer molecular sheets, were analyzed with scattering and spectroscopy techniques to understand the structural changes at the supramolecular level. XRD data indicated that sonication breaks the cellulose microfibrils along its (200) planes, yet some form of the Iß crystalline structure is still retained with reduced crystallinity. The Raman and FTIR analysis indicated structural changes to the cellulose microfibrils do not occur until after sonication; furthermore, AFM observation indicates that the structural changes began to occur within 5 min of sonication. An altered supramolecular structure is evident after sonication: major features from cellulose I are preserved, although certain spectral features similar to mercerized and ball milled cellulose appeared in its FTIR and Raman spectra. These spectral differences are traced to changes in the methine environment, hydroxymethyl conformations, and skeletal vibrations. By integrating the present findings and previous research, a cellulose molecular sheet delamination scheme is proposed to describe this microfibril fragmentation along its (200) plane.

Genomics and metatranscriptomics of biogeochemical cycling and degradation of lignin-derived aromatic compounds in thermal swamp sediment.

Thermal swamps are unique ecosystems where geothermally warmed waters mix with decomposing woody biomass, hosting novel biogeochemical-cycling and lignin-degrading microbial consortia. Assembly of shotgun metagenome libraries resolved 351 distinct genomes from hot-spring (30-45 °C) and mesophilic (17 °C) sediments. Annotation of 39 refined draft genomes revealed metabolism consistent with oligotrophy, including pathways for degradation of aromatic compounds, such as syringate, vanillate, p-hydroxybenzoate, and phenol. Thermotolerant Burkholderiales, including Rubrivivax ssp., were implicated in diverse biogeochemical and aromatic transformations, highlighting their broad metabolic capacity. Lignin catabolism was further investigated using metatranscriptomics of sediment incubated with milled or Kraft lignin at 45 °C. Aromatic compounds were depleted from lignin-amended sediment over 148 h. The metatranscriptomic data revealed upregulation of des/lig genes predicted to specify the catabolism of syringate, vanillate, and phenolic oligomers in the sphingomonads Altererythrobacter ssp. and Novosphingobium ssp., as well as in the Burkholderiales genus, Rubrivivax. This study demonstrates how temperature structures biogeochemical cycling populations in a unique ecosystem, and combines community-level metagenomics with targeted metatranscriptomics to identify pathways with potential for bio-refinement of lignin-derived aromatic compounds. In addition, the diverse aromatic catabolic pathways of Altererythrobacter ssp. may serve as a source of thermotolerant enzymes for lignin valorization.

Tailoring renewable materials via plant biotechnology.

Plants inherently display a rich diversity in cell wall chemistry, as they synthesize an array of polysaccharides along with lignin, a polyphenolic that can vary dramatically in subunit composition and interunit linkage complexity. These same cell wall chemical constituents play essential roles in our society, having been isolated by a variety of evolving industrial processes and employed in the production of an array of commodity products to which humans are reliant. However, these polymers are inherently synthesized and intricately packaged into complex structures that facilitate plant survival and adaptation to local biogeoclimatic regions and stresses, not for ease of deconstruction and commercial product development. Herein, we describe evolving techniques and strategies for altering the metabolic pathways related to plant cell wall biosynthesis, and highlight the resulting impact on chemistry, architecture, and polymer interactions. Furthermore, this review illustrates how these unique targeted cell wall modifications could significantly extend the number, diversity, and value of products generated in existing and emerging biorefineries. These modifications can further target the ability for processing of engineered wood into advanced high performance materials. In doing so, we attempt to illuminate the complex connection on how polymer chemistry and structure can be tailored to advance renewable material applications, using all the chemical constituents of plant-derived biopolymers, including pectins, hemicelluloses, cellulose, and lignins.

n-p Heterojunction of TiO2-NiO core-shell structure for efficient hydrogen generation and lignin photoreforming.

Hydrogen evolution from biomass photoreforming has been widely recognized as a promising strategy for relieving the pressure from energy crisis and environmental pollution, as it could generate sustainable H2 and value-added bioproducts simultaneously. Combining p-type semiconductors with n-type semiconductors to form n-p heterojunction is an effective strategy to improve the photocatalytic quantum efficiency by enhancing the separation of photogenerated electrons and holes, which could greatly facilitate the realization of such biomass photorefinery concept. However, the incompact contact between the n-type and p-type semiconductors often induces the aggregation of photogenerated electrons and holes. In this work, we design and synthesize an ultrafine n-p heterojunction TiO2-NiO core-shell structure to overcome the incompact contact in the n-p interface. When the n-p heterojunction photocatalysts are evaluated for photocatalytic water splitting and biomass lignin photoreforming respectively, the as-fabricated TiO2-NiO nanocomposite with 3.25% NiO demonstrates the highest hydrogen generation of 23.5 mmol h-1 g-1 from water splitting and H2 (0.45 mmol h-1 g-1) and CH4 (0.03 mmol h-1 g-1) cogeneration with reasonable amount of fatty acids (palmitic acid and stearic acid) production from lignin photoreforming. The excellent photocatalytic activity is ascribed to the synergistic effects of high crystallinity of TiO2 ultrafine nanoparticles, core-shell structure and n-p heterojunction with NiO nanoclusters. This present work demonstrates a simple and efficient method to fabricate ultrafine n-p heterojunction core-shell structure for noble-metal free catalyst for both water splitting and biomass photoreforming.