Demethylation Of Lignin Research Articles

Enzymatic Kraft lignin demethylation and fungal O-demethylases like vanillate-O-demethylase and syringate O-demethylase catalyzed catechol-Fe3+ complexation method

The ability of enzymatic Kraft Lignin (KL) demethylation was determined using catechol and ferric ion coordination (catechol-Fe3+ complexes) by reduction of Fe3+ to Fe2+ and formation of mono, bis- and/or tris-catechol-Fe3+ complexes has been investigated to identify enzyme that can strip-off O-methyl groups from lignin such as O-demethylase. To detect fungal demethylation and release of catechol-like structures, these were demonstrated using catechol, gallic acid and caffeic acid as standard model compounds to forms mono, bis- and/or tris-catechol-Fe3+ complexes. The catechol-Fe3+ complexes formation controlled by pH via the deprotonation of the catechol hydroxyls was investigated at pH 2.5, 8.0 and 10.0 and demonstrated that catechol formed mono, bis- and/or tris-catechol-Fe3+ complexes, and showed maximum absorbance at 547 nm. Lignin demethylation (O-demethylase) and formation of pyrocatecholic structures was detected using Aspergillus sp. and Galerina autumnalis culture filtrates as the enzyme source. The produced aromatic vicinal diol groups in lignin model compounds (LMCs) and KL were determined using different catecholic-binding reagents with the influence of H2O2, along with 4-antiaminopyrine reagent, was analyzed by the following: i) Fe3+-catechol complexation method, ii) HNO2 method, iii) FAS (Ferric Ammonium-Sulfate) method, iv) Ti(III)-NTA (Titanium (III)- Nitrilotriacetate) method for hydrolytic zone formation. Among the tested methods showing lytic zone formation was Fe3+-catechol complexation. The LMCs and KL treated using Aspergillus sp. culture filtrate showed maximum Fe3+-catechol complexes with 3-methoxy catechol (91 μmol/mL), o-vanillin (44 μmol/mL) and KL (100 μmol/mL). In addition, Galerina autumnalis culture filtrate showed demethylation of vanillin (48 μmol/mL), 3-methoxy catechol (82 μmol/mL), o-vanillin, (33 μmol/mL), 3 4-dimethoxybenzyl alcohol (49 μmol/mL) and KL (41 μmol/mL). The results suggest that lignin demethylation (O-demethylases) activity that strip-off methyl groups in LMCs and KL and produced vicinal diols that covalently bind with Fe3+ to form Fe3+-catechol complexes. The new Fe3+-catechol complexation method has the ability to characterize pyrocatechol and galloyl structures in chemically or biologically modified lignins and to detect O-demethylase activity.

Demethylation and other modifications of industrial softwood kraft lignin by laccase-mediators

AbstractSubstitution of phenol in phenol-formaldehyde (PF) resin preparations by technical lignins is hindered by the inherently lower reactivity of lignin compared to phenol. Demethylation of an industrial softwood kraft lignin (SKL) to improve its reactivity is the focus of this paper. To this purpose, kraft lignin (KL) was treated with two commercial laccases, NS51002 (L1) and NS51003 (L2), for 24 h in combination with three mediators, 2,2′-azinobis-(3-ethyl-benzothiazoline-6-sulfonic acid) diammonium salt (ABTS), 1-hydroxybenzotriazole (HBT) and 2,2,6,6-tetramethylpiperidin-1-oxyl (TEMPO). The characterizations of the reaction solution and the resultant KL showed that methanol was released as a result of the methoxy group splitting from the aromatic rings, while such demethylation was dependent on the laccase-mediator system (LMS). The catechol structures formed, which were further oxidized to a quinone structures prone to polymerization, led to molecular mass increment. Also this reaction was LMS dependent. The same is true to the cleavage of β-O-4′ linkages, which resulted in depolymerization. The L1-ABTS, L1-TEMPO and L2-HBT combinations are the most efficient and the resulting modified lignin would be suitable to phenol substitution. Challenging is the lignin polymerization following the demethylation, especially in case of L1-ABTS, which might inhibit the reactivity of the treated lignin.

Imidazolium-Based Ionic Liquids as Efficient Reagents for the C-O Bond Cleavage of Lignin.

The demethylation of lignin in ionic liquids (ILs) was investigated by using pure lignin model monomers and dimers together with dioxane-isolated lignins from poplar, miscanthus, and maize. Different methylimidazolium ILs were compared and the samples were treated with two different heating processes: microwave irradiation and conventional heating in a sealed tube. The conversion yield and influence of the treatment on the lignin structure were assessed by 31 P NMR spectroscopy, size-exclusion chromatography, and thioacidolysis. The acidic methylimidazolium IL [HMIM]Br was shown to be an effective combination of solvent and reagent for the demethylation and depolymerization of lignin. The relatively mild reaction conditions, the clean work-up, and the ability to reuse the IL makes the described procedure an attractive and new green method for the conversion of lignin to produce phenol-rich lignin oligomers.

Fast Curing Bio-Based Phenolic Resins via Lignin Demethylated under Mild Reaction Condition.

Demethylation technique has been used to enhance lignin reactivity for preparation of phenolic resins. However, the demethylation efficiency and the demethylated lignin (DL) reactivity were still unsatisfactory. To improve the demethylation efficiency, alkali lignin was demethylated under different mild conditions using sodium sulfite as a catalyst. Lignin and DL were characterized by 1H-NMR (nuclear magnetic resonance) and Fourier transform infrared (FT-IR) spectroscopy to determine the demethylation mechanism. With the demethylation of lignin, the methoxyl group content decreased from 1.93 m mol/g to 1.09 m mol/g, and the phenolic hydroxyl group content increased from 0.56 m mol/g to 0.82 m mol/g. These results revealed that methoxyl groups were attacked by SO32−, and some methoxyl groups were converted to phenolic hydroxyl groups by a nucleophilic substitution reaction, generating DL with high reactivity. The chemical properties of lignin-based phenolic resins were studied by 13C-NMR and FT-IR spectroscopy, and their physical properties were also investigated. The results indicated that lignin-based phenolic resins exhibited faster curing rate and shorter gel time. In addition, the bonding strength increased from 0.92 MPa to 1.07 MPa, and the formaldehyde emission decreased from 0.58 mg/L to 0.22 mg/L after lignin demethylated at the optimum condition.

Catalytic demethylation of organosolv lignin in aqueous medium using indium triflate under microwave irradiation

The anticipated shortage and price increase of fossil resources rendered material applications of renewable resources especially lignocellulosics highly attractive. When using lignin for polymeric materials, the macromolecular nature of lignin can be retained to reduce synthesis efforts. However, the effective integration of lignin into polymers is often lacking a sufficient number of reactive sites. Here, we present a method for the demethylation of lignin using the water-tolerant Lewis acid indium triflate, and compared conventional heating to a microwave-assisted approach. By appropriate dissolution of the organosolv lignin, char formation could be eliminated, and the reaction could be improved using microwave irradiation. The products were characterized by 1H, 13C and 31P NMR spectroscopy as well as size exclusion chromatography. An increase in the number of aromatic hydroxyl groups from 2.1 to 4.4mmolg−1 was obtained pointing towards enhanced suitability in application as polymeric precursor for materials such as phenolic and epoxy resins as well as polyurethanes.

Demethylation of Wheat Straw Alkali Lignin for Application in Phenol Formaldehyde Adhesives.

Lignin is a natural biopolymer with a complex three-dimensional network. It is the second most abundant natural polymer on earth. Commercially, lignin is largely obtained from the waste liquors of pulping and bioethanol productions. In this study, wheat straw alkali lignin (WSAL) was demethylated by using an in-situ generated Lewis acid under an optimized demethylation process. The demethylation process was monitored by a semi-quantitative Fourier Transform Infrared Spectroscopy (FTIR) method. The demethylated wheat straw alkali lignin (D-WSAL) was further characterized by Proton Nuclear Magnetic Resonance (1H NMR), Gel Permeation Chromatography (GPC), and titration methods. After the demethylation process, it was found that the relative value of the methoxy group decreased significantly from 0.82 to 0.17 and the phenolic hydroxyl group increased from 5.2% to 16.0%. Meanwhile, the hydroxyl content increased from 6.6% to 10.3%. GPC results suggested that the weighted averaged molecular weight of D-WSAL was lower than that of WSAL with a smaller polydispersity index. The D-WSAL was then used to replace 60 wt % of phenol to prepare lignin-based phenol formaldehyde adhesives (D-LPF). It was found that both the free formaldehyde content and the free phenol content in D-LPF were less than those of the lignin-based phenol formaldehyde adhesives without lignin demethylation (LPF). Gel time of D-LPF was shortened. Furthermore, the wet and dry bonding strengths of lap shear wood samples bonded using D-LPF were higher than those of the samples bonded using LPF. Therefore, D-WSAL has shown good potential for application in phenol formaldehyde adhesives.

Fungal demethylation of Kraft lignin

Demethylation of industrial lignin has been for long coveted as a pathway to the production of an abundant natural substitute for fossil-oil derived phenol. In an attempt to possibly identify a novel Kraft lignin-demethylating enzyme, we surveyed a collection of fungi by using selected ion flow tube-mass spectrometry (SIFT-MS). This method readily identifies methanol resulting from lignin demethylation activity. Absidia cylindrospora, and unidentified Cylindrocladium sp. and Aspergillus sp. were shown to metabolize lignin via different pathways, based on the HPLC analysis of lignin fragments. Of these three, Cylindrocladium and Aspergillus were shown to retain most of the lignin intact after 3 weeks in culture, while removing about 40% of the available methoxy groups. Our results demonstrate that after optimization of culture and lignin recovery methods, biological modification of Kraft lignin may be a feasible pathway to obtaining demethylated lignin for further industrial use.

Adaptation of Ti(III)-NTA colorimetric assay for use in detecting microbial demethylation of lignin and lignin derived compounds in aerobic conditions

An anaerobic colorimetric assay for quantifying microbial demethylation activity was adapted for aerobic use in studying lignin and lignin-derived compounds. Standard curves of 0–500μM pyrocatechol with and without 0.3% lignin demonstrated the use in either case. This method detects demethylation products up to 500μM without using additional dilutions.

Colocalizing incipient reactions in wood degraded by the brown rot fungus Postia placenta

Brown rot fungi are theorized to use both free radicals and enzymes to degrade wood. If these incompatible agents are employed in sequence (enzymatic after oxidative) in order to avoid interaction, this should be resolvable spatially in rotting wood. To assess this, we used thin spruce wafers as substrates, with the largest face the transverse plane. Propped wafers were colonized from the bottom (tangential to grain) by Postia placenta, using wood cell orientation and gravity to slow fungal egress and accentuate spatial gradients. After brief colonization, wafers were cut into 1-mm strips progressing up the wafer, and subsectioned for complementary analyses. Analyses included fungal growth, pH, cellulase activity, and wood modifications attributable to non-enzymatic mechanisms. Hyphae were imaged using confocal microscopy of fluorophore-tagged chitin. Dilute alkali solubility and lignin demethylation were measured as proxies (consequences) of carbohydrate depolymerization and lignin oxidation, respectively. Because P. placenta lacks genes for cellobiohydrolases, endoglucanase (EG) activity was measured. In composites of reassembled sections, hyphal fronts and apparent depolymerization preceded EG and lignin demethylation fronts by more than 6 mm. Although detection limits are a caveat when implicating novel fungal metabolites, results encourage and provide methodology for targeting this interesting leading edge of cellulolysis.

A New Method for Demethylation of Lignin from Woody Biomass using Biophysical Methods

Demethylation of softwood kraft lignin from woody biomass to improve the hydroxyl number in modified demethylated lignin and to produce lignin-based polyols was investigated using several biophysical techniques. Lignin is a tremendously under-developed natural polymer co-generated through papermaking and biomass fractionation. Molecular weights of lignins were analyzed by high-performance size-exclusion chromatography (HPSEC). Molecular weights of softwood kraft lignin (SKL) and demethylated lignin (DL) were determined as 1071 and 891 gmol-1, respectively. For demethylation, iodocyclohexane in dimethyl formamide (DMF) was used under reflux conditions, with a resulting yield of 87%. Fourier transform infrared spectroscopy (FTIR) was used to determine characteristic absorption peaks of softwood kraft lignin and demethylated lignin. Significant spectral differences were noticed between the two types of lignin due to changes in chemical structure. Total hydroxyl numbers were determined by titration. Phosphorus nuclear magnetic resonance spectrometry (31P-NMR) was employed to analyze the structure of lignin and different types of phenolic hydroxyl units. Nevertheless, further chemical and biological modifications within the lignin molecule are needed for various industrial applications to synthesize polyurethane foam by using chemically modified lignin-based polyols.

Development of novel enzyme assay procedures to determine demethylation of kraft lignin by wood-decay Fungi: Modifying lignin as substitute for phenol in formaldehyde-based polymers

L is a highly methylated natural aromatic biopolymer found in the cell walls of plants. Biological demethylation bywooddecay fungiremoves O-methyl groups from lignin increasing its phenolic content. The objective was to demonstrate a new enzyme associated with lignin demethylation (lignin demethylase, LDM) that cleaves O-methyl groups from benzyl rings comprising lignin making the latter more phenolic in structure toserve as phenol substitutes in formaldehyde-based polymers. Assays for LDM activity were developedthat specifically measured (i) methanol liberated as a consequence of demethylation, followed by (ii) an increase in pyrocatecholic structure content of modified lignin. Wood-decay fungi isolated from the Boreal Forest (31 isolates growing solely on Kraft lignin, KL) were screened for enzymes that modify lignin.An assay for LDM consisted of coupling an enzymatic oxidation step using alcohol oxidase to convert methanol released to formaldehyde and hydrogen peroxide, and then employing specific reagents to yield colored complexes measured spectrophotometrically. Fivebiochemical methods were developed to determine LDM using KL andlignin-like model compounds (LMC; guaiacol, veratryl, syringyl alcohols). Formaldehyde was measured using (i) pentan-2,4-dione/ammonium acetate. (ii) Hydrogen peroxide was assayed against ABTS in the presence of horseradish peroxidase. The increase in pyrocatecholic content of modified LMC’s and KLas consequence of demethylation, was determined spectrophotometrically by either (iii)complexation with Ti(III)-nitrilotriacetic acid, or (iv) reaction with4-aminoantipyrine. Biologically modified KL structures were determined by FT-IR. SIFT-MS(v), a highly sensitive procedure that measuresvolatile organic compounds, was also used to measure methanol,and demonstrated unequivocally that methanol was released from LMC’s and KL examined. The potential of ligninolytic enzymes to modify KL and LMCs was performed on 6 ligninolytic fungal isolates, and the results obtained are discussed.Supported by NSERC-CRD.

Methods to improve lignin’s reactivity as a phenol substitute and as replacement for other phenolic compounds: A brief review

Lignin is readily available as a by-product from the pulp and paper industry. It is considered to be a promising substitute for phenol in phenol-formaldehyde (PF) resin synthesis, given the increasing concerns of the shortage of fossil resources and the environmental impact from petroleum-based products. One hurdle that prevents the commercial utilization of lignin is its low reactivity due to its chemical structure. Many efforts have been made to improve its reactivity by modification and/or depolymerization of lignin molecules. Methylolation and phenolation are the two most studied modification approaches aimed at introducing reactive functional groups to lignin molecules. Modified lignin from these two methods could partially replace phenol in PF resin synthesis. Demethylation of lignin could effectively increase the reactivity of lignin by forming catechol moieties in the lignin macromolecule. Other methods, including reduction, oxidation, and hydrolysis, have also been studied to improve the reactivity of lignin as well as to produce phenolic compounds from lignin. Most current methods of lignin modification are not economically attractive. One can expect that efforts will be continued, aimed at improving the utilization of lignin for value-added products.

Methods to improve lignin’s reactivity as a phenol substitute and as replacement for other phenolic compounds: A brief review

Lignin is readily available as a by-product from the pulp and paper industry. It is considered to be a promising substitute for phenol in phenol-formaldehyde (PF) resin synthesis, given the increasing concerns of the shortage of fossil resources and the environmental impact from petroleum-based products. One hurdle that prevents the commercial utilization of lignin is its low reactivity due to its chemical structure. Many efforts have been made to improve its reactivity by modification and/or depolymerization of lignin molecules. Methylolation and phenolation are the two most studied modification approaches aimed at introducing reactive functional groups to lignin molecules. Modified lignin from these two methods could partially replace phenol in PF resin synthesis. Demethylation of lignin could effectively increase the reactivity of lignin by forming catechol moieties in the lignin macromolecule. Other methods, including reduction, oxidation, and hydrolysis, have also been studied to improve the reactivity of lignin as well as to produce phenolic compounds from lignin. Most current methods of lignin modification are not economically attractive. One can expect that efforts will be continued, aimed at improving the utilization of lignin for value-added products.

Selective lignin and polysaccharide removal in natural fungal decay of wood as evidenced by in situ structural analyses

Selective modification/degradation of the main plant polymers (cellulose, hemicelluloses and lignin) was investigated in a hardwood after white and brown-rot fungal decay under environmental conditions. The chemical changes produced in the plant cell wall were analysed in situ, by nuclear magnetic resonance (NMR) at the gel state, and analytical pyrolysis. Two-dimensional (2D) NMR of the white-rotted wood showed only cellulose and (deacetylated) hemicellulose, and the complete removal of lignin. On the other hand, the brown-rotted wood showed the nearly complete absence of polysaccharides, while the main features of lignin structure, as revealed by 2D-NMR, could be observed. These included well-resolved aromatic and side-chain cross-signals, although the intensity of the latter signals was lowered indicating a reduction in the number of side-chain linkages (β-O-4' and β-β') per aromatic unit (their relative abundances remaining unchanged). These results contrast with a recent study concluding that the aromatic polymer after brown-rot decay is not longer recognized as lignin. Some oxidative alteration of lignin during brown-rot decay was evidenced and, more interesting, several compounds with 3-methoxycatechol skeleton were released upon pyrolysis. Lignin demethylation is consistent with recent brown-rot transcriptomic/secretomic studies showing overexpression of methanol oxidase, which could use lignin-derived methanol to generate the peroxide required for cellulose depolymerization via Fenton chemistry.

Blue laccase from Galerina sp.: Properties and potential for Kraft lignin demethylation

We purified a laccase isoenzyme, Lac1 from Galerina sp. HC1 using a combination of anion exchange- and hydrophobic interaction chromatography. Lac1 has a molecular mass of 64 kDa, an isoelectric point of 4, and 3.35 copper atoms/enzyme molecule. The enzyme has features typical of fungal blue laccases. The sequences of two internal peptides were highly similar to reported laccase sequences from other fungi such as Trametes sp. Lac1 exhibited optimal activity on substrate 2,2′-azino-bis (3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) at pH 3 and 60 °C. It had high stability at ≤20 °C and at high pH. Lac1 exhibited high substrate affinity and the highest catalytic efficiency reported among laccases for ABTS, and among the lowest for syringaldazine. The most potent inhibitors of Lac1 were sodium azide, sodium cyanide, disulfide reducing agents, and metal ions in the order Li + > Sn 2+ > Hg 2+. The laccase efficiently catalyzed demethylation of eucalyptus hard wood Kraft lignin.

Characteristics of Gloeophyllum trabeum alcohol oxidase, an extracellular source of H2O2 in brown rot decay of wood.

A novel alcohol oxidase (AOX) has been purified from mycelial pellets of the wood-degrading basidiomycete Gloeophyllum trabeum and characterized as a homooctameric nonglycosylated protein with native and subunit molecular masses of 628 and 72.4 kDa, containing noncovalently bonded flavin adenine dinucleotide. The isolated AOX cDNA contained an open reading frame of 1,953 bp translating into a polypeptide of 651 amino acids displaying 51 to 53% identity with other published fungal AOX amino acid sequences. The enzyme catalyzed the oxidation of short-chain primary aliphatic alcohols with a preference for methanol (K(m) = 2.3 mM, k(cat) = 15.6 s(-1)). Using polyclonal antibodies and immunofluorescence staining, AOX was localized on liquid culture hyphae and extracellular slime in sections from degraded wood and on cotton fibers. Transmission electron microscopy immunogold labeling localized the enzyme in the hyphal periplasmic space and wall and on extracellular tripartite membranes and slime, while there was no labeling of hyphal peroxisomes. AOX was further shown to be associated with membranous or slime structures secreted by hyphae in wood fiber lumina and within the secondary cell walls of degraded wood fibers. The differences in AOX targeting compared to the known yeast peroxisomal localization were traced to a unique C-terminal sequence of the G. trabeum oxidase, which is apparently responsible for the protein's different translocation. The extracellular distribution and the enzyme's abundance and preference for methanol, potentially available from the demethylation of lignin, all point to a possible role for AOX as a major source of H(2)O(2), a component of Fenton's reagent implicated in the generally accepted mechanisms for brown rot through the production of highly destructive hydroxyl radicals.

Molecular Characterization of Biodegradable Dissolved Organic Matter Using Bioreactors and [12C/13C] Tetramethylammonium Hydroxide Thermochemolysis GC−MS

Little is known about the molecular composition of the biodegradable fraction of dissolved organic matter (BDOM) in stream ecosystems. We combined plug-flow biofilm reactors, tetramethylammonium hydroxide (TMAH) thermochemolysis GC-MS, and 13C-labeled TMAH thermochemolysis GC-MS to study the molecular composition of BDOM from two stream ecosystems. TMAH products derived from fatty acids, lignin, and other aromatic molecules were quantified using an internal standard approach. We applied the 13C-TMAH thermochemolysis procedure to differentiate between compounds in dissolved organic matter (DOM) that had natural methoxyl groups from those that acquired methoxyl groups during the TMAH reaction. In Rio Tempisquito, a stream draining a tropical evergreen forest, and White Clay Creek, a stream draining a temperate deciduous woodlands, carbohydrates, fatty acids, and lignin contributed to the DOM and BDOM molecular composition. We observed 97 different peaks in the chromatograms of streamwater, with 57% of the peaks common to both streams. The DOM and BDOM pools from each site also contained a unique suite of compounds. Our combined use of TMAH and 13C-TMAH thermochemolysis revealed that heterotrophic bacteria can selectively degrade and demethylate different types of compounds in the lignin residues of DOM. This demonstration of bacterial demethylation of lignin, an abundant and refractory plant molecule, has potential implications for global carbon cycling.

Lignin demethylation and polysaccharide decomposition in spruce sapwood degraded by brown rot fungi

The organic residues produced in the brown-rot (BR) of wood by many basidiomycetes fungi are ubiquitous on most coniferous forest floors. This degraded wood tissue is characterized by low levels of polysaccharides and a high proportion of demethylated lignin with minor glycerol side chain oxidation. Because of the selective enrichment in an aromatic dihydroxy-rich lignin residue, the chemical and biological reactivity of BR degraded wood will be distinctly different from white rot, the other primary class of fungal wood decay, which typically produces oxidized, lignin-depleted residues. The biochemical mechanism by which BR fungi perform this distinctive degradative chemistry is only starting to become known, and molecular studies which examine the chemical changes imparted to lignin over the long-term decay process are lacking. Using 13C-labeled tetramethylammonium hydroxide thermochemolysis (13C-TMAH) and solid state 13C NMR, we investigated the relationship between lignin oxidation/demethylation and polysaccharide metabolism in a 32-week time series study of spruce sapwood inoculated with either of two BR fungi (Postia placenta and Gloeophyllum trabeum). Our findings demonstrate a close relationship between lignin demethylation and polysaccharide loss and suggest demethylation may play a mechanistic role in polysaccharide loss, possibly by assisting in Fenton reactions where catechol/quinone oxidation and cycling aids in iron reduction. The residue remaining after 16 weeks of decay is devoid of polysaccharides, in contrast to the 68% polysaccharide carbon present in the initial spruce, and exhibits an increased aromatic dihydroxy content (resulting from demethylation of the 3-methoxyl carbon) of up to 22% of the lignin, as determined by 13C-TMAH thermochemolysis. In a typical soil or porewater environment these chemical changes would make BR residues highly reactive toward redox sensitive polyvalent metals (e.g. ferric iron) and likely to adsorb to metal hydroxide surfaces.

Recalcitrance as a catalyst for new developments

Humic substances are very recalcitrant in anaerobic environments as substrates, mostly due to their non-hydrolyzable high molecular weight structure, which is incompatible for cell uptake. The recalcitrant nature can be applied towards immobilizing pollutants into non-toxic humic substances. Certain anaerobic bioconversions such as demethylation of lignin and reduction of nitro- or azo groups can activate pollutants for humification processes upon subsequent exposure to air. Although humic substances provide no carbon or energy for microorganisms, the evidence presented here indicates that they are very active as terminal electron acceptors and redox mediators promoting the anaerobic degradation and biotransformation of priority pollutants.

Twenty-second annual meeting of the Japan Neuroscience Society

Brown rot fungi are theorized to use both free radicals and enzymes to degrade wood. If these incompatible agents are employed in sequence (enzymatic after oxidative) in order to avoid interaction, this should be resolvable spatially in rotting wood. To assess this, we used thin spruce wafers as substrates, with the largest face the transverse plane. Propped wafers were colonized from the bottom (tangential to grain) by Postia placenta, using wood cell orientation and gravity to slow fungal egress and accentuate spatial gradients. After brief colonization, wafers were cut into 1-mm strips progressing up the wafer, and subsectioned for complementary analyses. Analyses included fungal growth, pH, cellulase activity, and wood modifications attributable to non-enzymatic mechanisms. Hyphae were imaged using confocal microscopy of fluorophore-tagged chitin. Dilute alkali solubility and lignin demethylation were measured as proxies (consequences) of carbohydrate depolymerization and lignin oxidation, respectively. Because P. placenta lacks genes for cellobiohydrolases, endoglucanase (EG) activity was measured. In composites of reassembled sections, hyphal fronts and apparent depolymerization preceded EG and lignin demethylation fronts by more than 6 mm. Although detection limits are a caveat when implicating novel fungal metabolites, results encourage and provide methodology for targeting this interesting leading edge of cellulolysis.