Christopher G. Hunt

Laccase and Its Role in Production of Extracellular Reactive Oxygen Species during Wood Decay by the Brown Rot Basidiomycete Postia placenta

ABSTRACT 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 Fe3+, thus generating perhydroxyl radicals and Fe2+, which subsequently react further to produce biodegradative hydroxyl radicals. However, many brown rot fungi also secrete high levels of oxalate, which chelates Fe3+ 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 104 M−1 s−1, and measurements of the H2O2 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.

A biopulping mechanism: Creation of acid groups on fiber

Abstract We investigated how biopulping modifies chemical and physical properties of wood and how these changes affect the properties of the resulting fiber. Mechanical and chemical testing revealed wood cell changes during 2 weeks of colonization by Ceriporiopsis subvermispora. Typical mechanical properties, such as modulus of elasticity and maximum load, tracked reductions in energy needed for mechanical refining to pulp. The data indicate the fiber saturation point of spruce increased from 29% to 42% during biopulping. At the same time, titratable acid groups increased up to 62%. Chemical analysis showed that oxalic acid esters were produced in the wood during biopulping in sufficient amounts to account for the increase in acid groups. The benefits of biopulping—energy savings and increased handsheet strength—as well as other physical property changes are consistent with the mechanism we propose: biopulping increases the acid group content of wood.

REGULATION OF GENE EXPRESSION DURING THE ONSET OF LIGNINOLYTIC OXIDATION BY PHANEROCHAETE CHRYSOSPORIUM ON SPRUCE WOOD

ABSTRACT 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 Fe3+ 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.

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.

The influence of lathe check depth and orientation on the bond quality of phenol-formaldehyde – bonded birch plywood

Abstract During the rotary peeling of veneer for plywood or the laminated veneer lumber manufacture, checks are formed in the veneer that are as deep as 70–80% of the veneer thickness. The results of this study show that, during adhesive bond testing, deep lathe checks in birch (Betula pendula Roth.) veneer significantly reduce the shear strength and the percent wood failure of phenol-formaldehyde (PF) – bonded plywood. The results also show that specimens tested with the checks pulled open or closed can fail by different mechanisms. Dried rotary peeled birch veneers were sanded to create uniform surfaces with lathe check depths varying from 30% to 90% of veneer thickness. Then, 7-ply plywood was manufactured with a commercial PF resin. After the preparation of the test specimens, the check depth of each specimen was measured microscopically. Subsequently, bond quality was measured according to EN 314. The results show that veneer checking alone can bring EN 314 specimens to the brink of failure even with an excellent adhesive. These findings stress the importance of measuring the depth of lathe checks and considering the orientations of checks during the testing to get a better understanding of bond quality in veneer-based products.

The influence of felling season and log-soaking temperature on the wetting and phenol formaldehyde adhesive bonding characteristics of birch veneer

Abstract Most adhesive studies employing wood veneer as the substrate assume that it is a relatively uniform material if wood species and veneer thickness are constant. In the present study, veneers from rotary cut birch (Betula pendula Roth) were produced from logs harvested in spring, autumn and winter, and soaked at 20°C and 70°C prior to peeling. Firstly, veneers produced from logs felled in autumn were dried at 103°C for 24 h and subsequently half of these veneers were heat-treated at 180°C for 3 h. In addition, veneers produced from logs felled in all three seasons were dried at 160°C for 3.5 min to simulate industrial drying. The wettability of veneers was evaluated goniometrically, and bonding strength was evaluated with an automated bonding evaluation system (ABES). The results show that soaking birch logs at 70°C rather than at 20°C before peeling, or harvesting trees in the spring rather than in the autumn or winter, gives rise to veneers with enhanced wettability and higher bond strengths with a phenol-formaldehyde adhesive. Changes in the preparation and history of a veneer surface may have a profound effect on the development of adhesive bonds.

High temperature performance of soy-based adhesives

We studied the high temperature performance of soy meal processed to different protein concentrations (flour, concentrate, and isolate), as well as formulated soy-based adhesives, and commercial nonsoy adhesives for comparison. No thermal transitions were seen in phenol-resorcinol-formaldehyde (PRF) or soy-phenol-formaldehyde (SoyPF) or in as-received soy flour adhesive during differential scanning calorimetry scans heating at 10 °C/min between 35 and 235 °C. Heat flow rates decreased in the order soy flour (as received) > SoyPF > PRF > emulsion polymer isocyanate (EPI). In thermogravimetric analysis (TGA) scans from 110 to 300 °C at 2 °C/min, total weight loss decreased in the order soy flour (as-received)>SoyPF > PRF > casein > maple > EPI. For bio-based materials, the total weight loss (TGA) decreased in the order soy flour (as-received) > concentrate, casein > isolate. Dynamic mechanical analysis from 35 to 235 °C at 5 °C/min of two veneers bonded by cured adhesive showed 30–40% decline in storage modulus for maple compared to 45–55% for the adhesive made from soy flour in water (Soy Flour) and 70–80% for a commercial poly(vinyl acetate) modified for heat resistance. DMA on glass fiber mats showed thermal softening temperatures increasing in the order Soy Flour < casein < isolate < concentrate. We suggest that the low molecular weight carbohydrates plasticize the flour product. When soy-based adhesives were tested in real bondlines in DMA and creep tests in shear, they showed less decrease in storage modulus than the glass fiber-supported specimens. This suggests that interaction with the wood substrate improved the heat resistance property of the adhesive. Average hot shear strengths (ASTM D7247) were 4.6 and 3.1 MPa for SoyPF and Soy Flour compared to 4.7 and 0.8 MPa for PRF and EPI and 4.7 for solid maple. As a whole, these data suggest that despite indications of heat sensitivity when tested neat, soy-based adhesives are likely to pass the heat resistance criterion required for structural adhesives.

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.