Effect of Protein Surface Hydrophobicity and Surface Amines on Soy Adhesive Strength.

Soy is considered one of the most promising natural materials for manufacturing wood adhesives due to its low cost, high protein content, and ready availability. However, more cost-effective ways of improving its wet shear strength are needed to achieve wider market acceptance. Protein adhesive wet strength depends on the use of (typically expensive) crosslinking additives as well as the processing/denaturation of the protein. It has been commonly stated in the literature that protein denaturation leads to higher bond strength by activating the surface and exposing the reactive groups. Therefore, we investigated how differences in surface reactive groups (surface hydrophobicity and reactive amine groups) brought on with different denaturation treatments relate to bonding performance. Fourteen soy protein isolates (SPIs) with different denaturation histories were investigated. Characterization of the SPIs included surface hydrophobicity, surface amine content, extent of protein hydrolysis, and bond strength (wet and dry, with and without polyamidoamine epichlorohydrin (PAE) crosslinking agent) by ASTM D7998. The molecular weight patterns showed that proteins denatured by extensive hydrolysis had very low bond strengths. Adding the crosslinker, PAE, improved all the shear strength values. We found that the number of water-accessible reactive amine groups on protein surfaces had no impact on the adhesive strength, even with the amine-reactive crosslinker, PAE. Conversely, increased surface hydrophobicity was beneficial to adhesive strength in all cases, though this correlation was only statistically significant for wet strength without PAE. While, in general, denatured proteins are typically thought to form better bonds than native state proteins, this work suggests that it matters how proteins are denatured, and what surfaces become exposed. Denaturation by hydrolysis did not improve bond strength, and extensive hydrolysis seemed highly detrimental. Moreover, exposing hydrophobic surface groups was beneficial, but exposing covalent bond-forming reactive amine groups was not.

Metabolism of Scenedesmus obliquus cultivated with raw plant substrates.

The potential benefits of adding raw, non-food, lignocellulosic plant material as a carbon source for mixotrophic growth of microalgae have previously been demonstrated. This approach has advantages over using traditional carbon sources like glucose or acetate due to wide-spread plant biomass availability and substrate recalcitrance to bacterial contamination. Here, we report the overall growth characteristics and explore the metabolic patterns of Scenedesmus obliquus cultured in the presence raw plant substrate. An initial screen of plant substrate candidates showed an increase in specific growth rate and biomass accumulation when S. obliquus was cultured in the presence of switchgrass or yard waste compared to media alone. We observed a near doubling of microalgal dry weight when S. obliquus was grown with 0.2% (w/v) switchgrass under ambient CO2. Scanning electron microscopy (SEM) of corn stem after S. obliquus cultivation exhibited substantial phloem degradation. Transcriptomic analyses of S. obliquus during mid- and late-log phase growth revealed a dynamic metabolic landscape within many KEGG pathways. Notably, differential expression was observed for several potential glycosyl hydrolases. We also investigated the influence of switchgrass on the growth of S. obliquus at 50 L volume in mini raceway ponds to determine the scalability of this approach.

The characteristics of insoluble softwood substrates affect fungal morphology, secretome composition, and hydrolytic efficiency of enzymes produced by Trichoderma reesei.

BACKGROUND: On-site enzyme production using Trichoderma reesei can improve yields and lower the overall cost of lignocellulose saccharification by exploiting the fungal gene regulatory mechanism that enables it to continuously adapt enzyme secretion to the substrate used for cultivation. To harness this, the interrelation between substrate characteristics and fungal response must be understood. However, fungal morphology or gene expression studies often lack structural and chemical substrate characterization. Here, T. reesei QM6a was cultivated on three softwood substrates: northern bleached softwood Kraft pulp (NBSK) and lodgepole pine pretreated either by dilute-acid-catalyzed steam pretreatment (LP-STEX) or mild alkaline oxidation (LP-ALKOX). With different pretreatments of similar starting materials, we presented the fungus with systematically modified substrates. This allowed the elucidation of substrate-induced changes in the fungal response and the testing of the secreted enzymes' hydrolytic strength towards the same substrates. RESULTS: Enzyme activity time courses correlated with hemicellulose content and cellulose accessibility. Specifically, increased amounts of side-chain-cleaving hemicellulolytic enzymes in the protein produced on the complex substrates (LP-STEX; LP-ALKOX) was observed by secretome analysis. Confocal laser scanning micrographs showed that fungal micromorphology responded to changes in cellulose accessibility and initial culture viscosity. The latter was caused by surface charge and fiber dimensions, and likely restricted mass transfer, resulting in morphologies of fungi in stress. Supplementing a basic cellulolytic enzyme mixture with concentrated T. reesei supernatant improved saccharification efficiencies of the three substrates, where cellulose, xylan, and mannan conversion was increased by up to 27, 45, and 2800%, respectively. The improvement was most pronounced for proteins produced on LP-STEX and LP-ALKOX on those same substrates, and in the best case, efficiencies reached those of a state-of-the-art commercial enzyme preparation. CONCLUSION: Cultivation of T. reesei on LP-STEX and LP-ALKOX produced a protein mixture that increased the hydrolytic strength of a basic cellulase mixture to state-of-the-art performance on softwood substrates. This suggests that the fungal adaptation mechanism can be exploited to achieve enhanced performance in enzymatic hydrolysis without a priori knowledge of specific substrate requirements.

Functionality of Surface Mycelium Interfaces in Wood Bonding.

Filamentous fungi have been considered as candidates to replace petroleum-based adhesives and plastics in novel composite material production, particularly those containing lignocellulosic materials. However, the nature of the role of surface mycelium in the adhesion between lignocellulosic composite components is not well-known. The current study investigated the functionality of surface mycelium for wood bonding by incubating Trametes versicolor on yellow birch veneers and compared the lap-shear strengths after hot-pressing to evaluate if the presence of surface mycelium can improve the interface between two wood layers and consequently improve bonding. We found that the lap-shear strength of the samples was enhanced by the increase of surface mycelium coverage up to 8 days of incubation (up to 1.74 MPa) without a significant wood weight loss. We provide evidence that the bottom surface of the mycelium layer is more hydrophilic, contains more small-scale filamentous structure and contains more functional groups, resulting in better bonding with wood than the top surface. These observations confirm and highlight the functionality of the surface mycelium layer for wood bonding and provide useful information for future developments in fully biobased composites manufacturing.

Direct fluorescence imaging of lignocellulosic and suberized cell walls in roots and stems.

Investigating plant structure is fundamental in botanical science and provides crucial knowledge for the theories of plant evolution, ecophysiology and for the biotechnological practices. Modern plant anatomy often targets the formation, localization and characterization of cellulosic, lignified or suberized cell walls. While classical methods developed in the 1960s are still popular, recent innovations in tissue preparation, fluorescence staining and microscopy equipment offer advantages to the traditional practices for investigation of the complex lignocellulosic walls. Our goal is to enhance the productivity and quality of microscopy work by focusing on quick and cost-effective preparation of thick sections or plant specimen surfaces and efficient use of direct fluorescent stains. We discuss popular histochemical microscopy techniques for visualization of cell walls, such as autofluorescence or staining with calcofluor, Congo red (CR), fluorol yellow (FY) and safranin, and provide detailed descriptions of our own approaches and protocols. Autofluorescence of lignin in combination with CR and FY staining can clearly differentiate between lignified, suberized and unlignified cell walls in root and stem tissues. Glycerol can serve as an effective clearing medium as well as the carrier of FY for staining of suberin and lipids allowing for observation of thick histological preparations. Three-dimensional (3D) imaging of all cell types together with chemical information by wide-field fluorescence or confocal laser scanning microscopy (CLSM) was achieved.

Measurement of moisture-dependent ion diffusion constants in wood cell wall layers using time-lapse micro X-ray fluorescence microscopy.

Our future bioeconomy depends on increased utilization of renewable lignocellulosic biomass. Controlling the diffusion of chemicals, such as inorganic ions, within secondary plant cell walls is central to many biomass applications. However, insufficient understanding of intra-cell-wall diffusion within secondary plant cell walls is hindering the advancement of many lignocellulosic biomass applications. In this work, X-ray fluorescence microscopy was used to measure diffusion constants of K+, Cu2+, and Cl- diffusing through loblolly pine (Pinus taeda) cell wall layers under 70%, 75%, or 80% relative humidity (RH). Results revealed that diffusion constants increased with RH, the larger Cu2+ diffused more slowly than the K+, and the Cl- diffusion constant was the same as that for the counter cation, indicating cations and anions diffused together to maintain charge neutrality. Comparison with electrical conductivity measurements showed that conductivity is being controlled by ion mobility over these RH. The results further support that intra-cell-wall diffusion of inorganic ions is a Fickian diffusion process occurring through rubbery amorphous polysaccharides, which contradicts previous assertions that intra-cell-wall diffusion is an aqueous process occurring through water pathways. Researchers can now utilize polymer science approaches to engineer the molecular architecture of lignocellulosic biomass to optimize properties for specific end uses.

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.

Fully Bio-Based Hybrid Composites Made of Wood, Fungal Mycelium and Cellulose Nanofibrils.

Novel hybrid panel composites based on wood, fungal mycelium, and cellulose nanofibrils (CNF) were developed and investigated in the present study. In one set of experiments, mycelium was grown on softwood particles to produce mycelium-modified wood which was then hybridized with various levels of CNF as binder. The other set of experiments were conducted on unmodified wood particles mixed with CNF and pure mycelium tissue. It was found that the composites made of mycelium-modified wood and CNF resulted in enhanced physical and mechanical properties compared to the ones made by physically mixing wood, mycelium, and CNF. Scanning electron microscopy (SEM) images showed that mycelium modification covered wood particles with a network of fungal hyphae whereas CNF formed a uniform mycelial film over wood particles. Mycelium modification had a significant effect on reducing water absorption and thickness swelling of the hybrid composites and CNF increased the modulus of rupture and modulus of elasticity, optimally at 2.5% addition. We also present results and analysis pertaining to the development of unique lightweight composite systems with physical and mechanical properties optimized at 5% CNF addition with potential to be used in packaging and furniture applications.

Fungal lignin peroxidase does not produce the veratryl alcohol cation radical as a diffusible ligninolytic oxidant.

Peroxidases are considered essential agents of lignin degradation by white-rot basidiomycetes. However, low-molecular-weight oxidants likely have a primary role in lignin breakdown because many of these fungi delignify wood before its porosity has sufficiently increased for enzymes to infiltrate. It has been proposed that lignin peroxidases (LPs, EC 1.11.1.14) fulfill this role by oxidizing the secreted fungal metabolite veratryl alcohol (VA) to its aryl cation radical (VA+•), releasing it to act as a one-electron lignin oxidant within woody plant cell walls. Here, we attached the fluorescent oxidant sensor BODIPY 581/591 throughout beads with a nominal porosity of 6 kDa and assessed whether peroxidase-generated aryl cation radical systems could oxidize the beads. As positive control, we used the 1,2,4,5-tetramethoxybenzene (TMB) cation radical, generated from TMB by horseradish peroxidase. This control oxidized the beads to depths that increased with the amount of oxidant supplied, ultimately resulting in completely oxidized beads. A reaction-diffusion computer model yielded oxidation profiles that were within the 95% confidence intervals for the data. By contrast, bead oxidation caused by VA and the LPA isozyme of Phanerochaete chrysosporium was confined to a shallow shell of LP-accessible volume at the bead surface, regardless of how much oxidant was supplied. This finding contrasted with the modeling results, which showed that if the LP/VA system were to release VA+•, it would oxidize the bead interiors. We conclude that LPA releases insignificant quantities of VA+• and that a different mechanism produces small ligninolytic oxidants during white rot.

Acridine Orange Indicates Early Oxidation of Wood Cell Walls by Fungi.

Colonization of wood blocks by brown and white rot fungi rapidly resulted in detectable wood oxidation, as shown by a reduced phloroglucinol response, a loss of autofluorescence, and acridine orange (AO) staining. This last approach is shown to provide a novel method for identifying wood oxidation. When lignin was mildly oxidized, the association between AO and lignin was reduced such that stained wood sections emitted less green light during fluorescence microscopy. This change was detectable after less than a week, an interval that past work has shown to be too short for significant delignification of wood. Although fungal hyphae were observed in only a few wood lumina, oxidation was widespread, appearing relatively uniform over regions several hundred micrometers from the hyphae. This observation suggests that both classes of fungi release low molecular weight mild oxidants during the first few days of colonization.

Regulation of Gene Expression during the Onset of Ligninolytic Oxidation by Phanerochaete chrysosporium on Spruce Wood.

Since uncertainty remains about how white rot fungi oxidize and degrade lignin in wood, it would be useful to monitor changes in fungal gene expression during the onset of ligninolysis on a natural substrate. We grew Phanerochaete chrysosporium on solid spruce wood and included oxidant-sensing beads bearing the fluorometric dye BODIPY 581/591 in the cultures. Confocal fluorescence microscopy of the beads showed that extracellular oxidation commenced 2 to 3 days after inoculation, coincident with cessation of fungal growth. Whole transcriptome shotgun sequencing (RNA-seq) analyses based on the v.2.2 P. chrysosporium genome identified 356 genes whose transcripts accumulated to relatively high levels at 96 h and were at least four times the levels found at 40 h. Transcripts encoding some lignin peroxidases, manganese peroxidases, and auxiliary enzymes thought to support their activity showed marked apparent upregulation. The data were also consistent with the production of ligninolytic extracellular reactive oxygen species by the action of manganese peroxidase-catalyzed lipid peroxidation, cellobiose dehydrogenase-catalyzed Fe(3+) reduction, and oxidase-catalyzed H2O2 production, but the data do not support a role for iron-chelating glycopeptides. In addition, transcripts encoding a variety of proteins with possible roles in lignin fragment uptake and processing, including 27 likely transporters and 18 cytochrome P450s, became more abundant after the onset of extracellular oxidation. Genes encoding cellulases showed little apparent upregulation and thus may be expressed constitutively. Transcripts corresponding to 165 genes of unknown function accumulated more than 4-fold after oxidation commenced, and some of them may merit investigation as possible contributors to ligninolysis.

Synchrotron-based X-ray fluorescence microscopy in conjunction with nanoindentation to study molecular-scale interactions of phenol-formaldehyde in wood cell walls.

Understanding and controlling molecular-scale interactions between adhesives and wood polymers are critical to accelerate the development of improved adhesives for advanced wood-based materials. The submicrometer resolution of synchrotron-based X-ray fluorescence microscopy (XFM) was found capable of mapping and quantifying infiltration of Br-labeled phenol-formaldehyde (BrPF) into wood cell walls. Cell wall infiltration of five BrPF adhesives with different average molecular weights (MWs) was mapped. Nanoindentation on the same cell walls was performed to assess the effects of BrPF infiltration on cell wall hygromechanical properties. For the same amount of weight uptake, lower MW BrPF adhesives were found to be more effective at decreasing moisture-induced mechanical softening. This greater effectiveness of lower MW phenolic adhesives likely resulted from their ability to more intimately associate with water sorption sites in the wood polymers. Evidence also suggests that a BrPF interpenetrating polymer network (IPN) formed within the wood polymers, which might also decrease moisture sorption by mechanically restraining wood polymers during swelling.

Spatial mapping of extracellular oxidant production by a white rot basidiomycete on wood reveals details of ligninolytic mechanism.

Oxidative cleavage of the recalcitrant plant polymer lignin is a crucial step in global carbon cycling, and is accomplished most efficiently by fungi that cause white rot of wood. These basidiomycetes secrete many enzymes and metabolites with proposed ligninolytic roles, and it is not clear whether all of these agents are physiologically important during attack on natural lignocellulosic substrates. One new approach to this problem is to infer properties of ligninolytic oxidants from their spatial distribution relative to the fungus on the lignocellulose. We grew Phanerochaete chrysosporium on wood sections in the presence of oxidant-sensing beads based on the ratiometric fluorescent dye BODIPY 581/591. The beads, having fixed locations relative to the fungal hyphae, enabled spatial mapping of cumulative extracellular oxidant distributions by confocal fluorescence microscopy. The results showed that oxidation gradients occurred around the hyphae, and data analysis using a mathematical reaction-diffusion model indicated that the dominant oxidant during incipient white rot had a half-life under 0.1 s. The best available hypothesis is that this oxidant is the cation radical of the secreted P. chrysosporium metabolite veratryl alcohol.

Laccase and its role in production of extracellular reactive oxygen species during wood decay by the brown rot basidiomycete Postia placenta.

Brown rot basidiomycetes initiate wood decay by producing extracellular reactive oxygen species that depolymerize the structural polysaccharides of lignocellulose. Secreted fungal hydroquinones are considered one contributor because they have been shown to reduce Fe(3+), thus generating perhydroxyl radicals and Fe(2+), which subsequently react further to produce biodegradative hydroxyl radicals. However, many brown rot fungi also secrete high levels of oxalate, which chelates Fe(3+) tightly, making it unreactive with hydroquinones. For hydroquinone-driven hydroxyl radical production to contribute in this environment, an alternative mechanism to oxidize hydroquinones is required. We show here that aspen wood undergoing decay by the oxalate producer Postia placenta contained both 2,5-dimethoxyhydroquinone and laccase activity. Mass spectrometric analysis of proteins extracted from the wood identified a putative laccase (Joint Genome Institute P. placenta protein identification number 111314), and heterologous expression of the corresponding gene confirmed this assignment. Ultrafiltration experiments with liquid pressed from the biodegrading wood showed that a high-molecular-weight component was required for it to oxidize 2,5-dimethoxyhydroquinone rapidly and that this component was replaceable by P. placenta laccase. The purified laccase oxidized 2,5-dimethoxyhydroquinone with a second-order rate constant near 10(4) M(-1) s(-1), and measurements of the H(2)O(2) produced indicated that approximately one perhydroxyl radical was generated per hydroquinone supplied. Using these values and a previously developed computer model, we estimate that the quantity of reactive oxygen species produced by P. placenta laccase in wood is large enough that it likely contributes to incipient decay.

Involvement of reactive oxygen species in the electrochemical inhibition of barnacle (Amphibalanus amphitrite) settlement.

The role of reactive oxygen species (ROS) in electrochemical biofouling inhibition was investigated using a series of abiotic tests and settlement experiments with larvae of the barnacle Amphibalanus amphitrite, a cosmopolitan fouler. Larval settlement, a measure of biofouling potential, was reduced from 43% +/- 14% to 5% +/- 6% upon the application of pulsed electric signals. The application of ROS scavengers such as glutathione and catalase counteracted the inhibitory effects of the electric signals, allowing settlement, and thus indicating that ROS are antifouling agents. Based on the experimental evidence, the proposed mechanism for ROS-based fouling prevention with interdigitated electrodes involved the electrochemical generation of hydrogen peroxide by oxygen reduction, and its likely reduction to hydroxyl radicals. Either hydroxyl radicals or products of hydroxyl radical reactions appeared to be the main deterrents of larval settlement.

Fungal hydroquinones contribute to brown rot of wood.

The fungi that cause brown rot of wood initiate lignocellulose breakdown with an extracellular Fenton system in which Fe(2+) and H(2)O(2) react to produce hydroxyl radicals (.OH), which then oxidize and cleave the wood holocellulose. One such fungus, Gloeophyllum trabeum, drives Fenton chemistry on defined media by reducing Fe(3+) and O(2) with two extracellular hydroquinones, 2,5-dimethoxyhydroquinone (2,5-DMHQ) and 4,5-dimethoxycatechol (4,5-DMC). However, it has never been shown that the hydroquinones contribute to brown rot of wood. We grew G. trabeum on spruce blocks and found that 2,5-DMHQ and 4,5-DMC were each present in the aqueous phase at concentrations near 20 microM after 1 week. We determined rate constants for the reactions of 2,5-DMHQ and 4,5-DMC with the Fe(3+)-oxalate complexes that predominate in wood undergoing brown rot, finding them to be 43 l mol(-1) s(-1) and 65 l mol(-1) s(-1) respectively. Using these values, we estimated that the average amount of hydroquinone-driven .OH production during the first week of decay was 11.5 micromol g(-1) dry weight of wood. Viscometry of the degraded wood holocellulose coupled with computer modelling showed that a number of the same general magnitude, 41.2 micromol oxidations per gram, was required to account for the depolymerization that occurred in the first week. Moreover, the decrease in holocellulose viscosity was correlated with the measured concentrations of hydroquinones. Therefore, hydroquinone-driven Fenton chemistry is one component of the biodegradative arsenal that G. trabeum expresses on wood.