Selective Delignification of Aspen Wood Blocks In Vitro by Three White Rot Basidiomycetes

Aspen wood blocks were selectively delignified in the laboratory by Ischnoderma resinosum, Poria medulla-panis, and Xylobolus frustulatus. After 8 weeks only the outer surfaces of wood blocks were selectively delignified. The percentages of weight loss obtained after 4, 8, and 12 weeks showed that decay occurred at a relatively constant rate. Selectively delignified wood could be identified by using scanning electron microscopy only when lignin had been extensively removed from cell walls. X. frustulatus was able to form pockets of delignified wood throughout blocks after 12 weeks.     

Poplar Lignin Decomposition by Gram-Negative Aerobic Bacteria

Eleven gram-negative aerobic bacteria (Pseudomonadaceae and Neisseriaceae) out of 122 soil isolates were selected for their ability to assimilate poplar dioxane lignin without a cosubstrate. Dioxane lignin and milled wood lignin degradation rates ranged between 20 and 40% of initial content after 7 days in mineral medium, as determined by a loss of absorbance at 280 nm; 10 strains could degrade in situ lignin, as evidenced by the decrease of the acetyl bromide lignin content of microtome wood sections. No degradation of wood polysaccharides was detected. Lignin biodegradation by Pseudomonas 106 was confirmed by ¹⁴CO₂ release from labeled poplar wood, although in lower yields compared with results obtained through chemical analysis based on acetyl bromide residual lignin determination.     

Thermal behavior of biodegraded lime wood

The effects of the soft-rot fungus Trichoderma viride Pers., on the thermal behavior of lime wood (Tillia cordata Mill.) were investigated. The lime wood pieces were inoculated with the fungus over a 12-week period. At pre-established time intervals two samples were withdrawn from the medium and analyzed by thermogravimetry and differential calorimetry, and the results were correlated with mass loss. Fungal activity was indicated by continuous decrease of sample mass.Modification of the wood because of the presence of the fungus was evidenced by structural changes that affected its thermal properties, both in respect to the hydrophilicity of the wood (evidenced mainly in desorption process) and in its decomposition behavior. The shape of DTG curves depends on the exposure time of wood to the action of microorganisms. The peak temperature assigned to the decomposition of wood components increases, while the global kinetic parameters for the main peak decrease with increasing exposure time of the wood to the attack by microorganisms.The increased characteristic temperatures of water desorption and cellulose decomposition processes and lower thermal stability could be explained by newly formed structures, mainly the oxidized ones.     

Enhanced delignification of mixed wood pulp by <Emphasis Type="Italic">Aspergillus fumigatus</Emphasis> laccase mediator system

An environmentally sound biobleaching to get high quality paper pulp from mixed wood pulp was attempted employing laccase from Aspergillus fumigatus VkJ2.4.5 for lignin removal. Laccase treatment was performed in the presence of a mediator N-hydroxybenzotriazole (HBT, 1.5% w/w), resulting into notably higher level of delignification of the pulp. Enzyme at 10 Ug⁻¹ of pulp at 50°C, pH 6.0, for 2 h with a pulp consistency of 10% was found suitable for enabling maximum decrease in the kappa number. The kappa number and yellowness decreased by 14 and 4% whereas ISO brightness improved by 7%. The presence of a characteristic peak at 280 nm indicated the presence of lignin in the effluent during biobleaching. Analysis of FTIR spectra of residual lignin revealed characteristic modifications following enzymatic bleaching by laccase mediator system (LMS). Variations in morphology and crystallinity of pulp were evaluated by scanning electron microscopy and X-ray diffraction analysis.     

Impact of Dilute Sulfuric Acid,Ammonium Hydroxide,and Ionic Liquid Pretreatments on the Fractionation and Characterization of Engineered Switchgrass

Improving plant characteristics for better environmental resilience and more cost-effective transformation to fuels and chemicals is one of the focus areas in biomass feedstock development. In order to bridge lignin engineering and conversion technologies, this study aimed to fractionate and characterize lignin streams from wild-type and engineered switchgrass using three different pretreatment methods, i.e., dilute sulfuric acid (DA), ammonium hydroxide (AH), and aqueous ionic liquid (IL). Results demonstrate the low lignin content and high S/G ratio switchgrass mutant (4CL) was more susceptible to pretreatment and subsequently more digestible by enzymes as compared to wild-type switchgrass and AtLOV1 mutant. In addition, when compared to DA and AH pretreatment, aqueous IL (cholinium lysinate) was demostrated to be an efficient lignin solvent, as indicated by the high (> 80%) lignin solubility and reduced lignin molecular weight. FTIR and differential scanning calorimetry measurements suggest that pretreatment chemistry greatly influenced the structural and compositional changes and thermal properties of the pretreated switchgrass and recovered lignin-rich streams. The comparative data obtained from this work deepen our understanding of how lignin modification impacts the fractionation and properties of biomass feedstocks.     

Evaluation of the selectivity of white rot isolates using near infrared spectroscopic techniques

Seventeen isolates from white rotted beech wood and six strains from a local culture collection were evaluated for their capability to delignify beech and spruce wood selectively. Six peroxidase-positive isolates were found using a colorimetric agar plate test (Poly R-478), and genetically identified by their internal transcribed spacer (ITS1) or 28S rDNA sequences. Colonised on beech and spruce wood veneers, some of the peroxidase-positive isolates caused selective white rot on both wood species. Weight loss and lignin content of the degraded veneers were estimated from FT-NIR spectra with established linear regression models and multivariate models based on partial least squares regression (PLSR). Weight loss of the samples was also determined gravimetrically. A measure for the relative selectivity of the strains for lignin degradation was formulated and the values were calculated. Two strains that were identified as Oxyporus latemarginatus and Trametes cervina exhibited high selectivity on spruce wood, but the lignin content of the decayed wood was higher than that degraded by the reference strain Ceriporiopsis subvermispora. One strain – identified as Phlebia tremellosa – led to a lower lignin content of beech wood but caused also comparably high weight loss and thus exhibited an overall lower selectivity. The NIR spectroscopic method proved to be convenient for the quick screening of selective white rot fungi. Furthermore, the results revealed that high selectivity for lignin degradation is much more pronounced in early degradation stages.     

Fungal resistance of rubber wood modified by fatty acid chlorides

Decay resistance of Rubber wood (Hevea brasiliensis) esterified with three fatty acid chlorides (hexanoyl chloride (C6), decanoyl chloride (C10) and tetra-decanoyl chloride (C14)) was evaluated. Unmodified and modified wood samples were exposed to a brown rot (Polyporus meliae) and a white rot (Coriolus versicolor) fungus for 12 weeks. Unmodified rubber wood was severely decayed by P. meliae and C. versicolor, which was indicated by significant weight loss. The rate of decay by brown rot was higher than white rot. Modified wood samples exhibited very good resistant to brown and white-rot fungi. The degree of protection increased with increase in degree of modification. P. meliae, a brown rot fungus, removed structural carbohydrate component in unmodified wood selectively whereas, C. vesicolor showed preference to lignin. The FTIR spectra of modified wood exposed to fungi show no significant changes in relative peak intensities of lignin/carbohydrates indicating effectiveness of chemically modified wood in restricting chemical degradation. Chemical modification occurred more efficiently at carbohydrate portion of the wood. Therefore, it is more effective in retarding decay due to P. meliae.     

Linoleic acid peroxidation initiated by Fe-reducing compounds recovered from Eucalyptus grandis biotreated with Ceriporiopsis subvermispora

This work evaluates linoleic acid peroxidation reactions initiated by Fe³⁺-reducing compounds recovered from Eucalyptus grandis, biotreated with the biopulping fungus Ceriporiopsis subvermispora. The aqueous extracts from biotreated wood had the ability to reduce Fe³⁺ ions from freshly prepared solutions. The compounds responsible for the Fe³⁺-reducing activity corresponded to UV-absorbing substances with apparent molar masses from 3 kDa to 5 kDa. Linoleic acid peroxidation reactions conducted in the presence of Fe³⁺ ions and the Fe³⁺-reducing compounds showed that the rate of O₂ consumption during peroxidation was proportional to the Fe³⁺-reducing activity present in each extract obtained from biotreated wood. This peroxidation reaction was coupled with in-vitro treatment of ball-milled E. grandis wood. Ultraviolet data showed that the reaction system released lignin fragments from the milled wood. Size exclusion chromatography data indicated that the solubilized material contained a minor fraction representing high-molar-mass molecules excluded by the column and a main low-molar-mass peak. Overall evaluation of the data suggested that the Fe³⁺-reducing compounds formed during wood biodegradation by C. subvermispora can mediate lignin degradation through linoleic acid peroxidation.     

Selection of white-rot fungi for biopulping

Different rates of wood decay and ligninolytic activity were found in wood decayed by various white-rot fungi. Chemical and ultrastructural analyses showed wood decayed by Coriolus versicolor consisted of a nonselective attack on all cell wall components. Lignin degradation was restricted to the cell wall adjacent to hyphae or around the circumference of cell lumina. Decay by Phellinus pini, Phlebia tremellosus, Poria medullapanis and Scytinostroma galactinum was selective for lignin degradation. Secondary walls were void of lignin and middle lamellae were extensively degraded. A diffuse attack on lignin occurred throughout all cell wall layers. Variation in ligninolytic activity was found among strains of Phanerochaete chrysosporium. Differences in weight loss as well as lignin and polysaccharide degradation were also found when wood of different coniferous and deciduous tree species was decayed by various white-rot fungi.     

Production of New Unsaturated Lipids during Wood Decay by Ligninolytic Basidiomycetes

Lipids were analyzed by gas chromatography-mass spectrometry for a 7-week in vitro decay of eucalypt wood by four ligninolytic basidiomycetes. The sound wood contained up to 75 mg of lipophilic compounds per 100 g of wood. Hydrolysis of sterol esters, which represented 38% of total wood lipids, occurred during the fungal decay. The initial increase of linoleic and other free unsaturated fatty acids paralleled the decrease of sterol esters. Moreover, new lipid compounds were found at advanced stages of wood decay that were identified from their mass spectra as unsaturated dicarboxylic acids consisting of a long aliphatic chain attached to the C-3 position of itaconic acid. These dicarboxylic acids were especially abundant in the wood treated with Ceriporiopsis subvermispora (up to 24 mg per 100 g of wood) but also were produced by Phlebia radiata, Pleurotus pulmonarius, and Bjerkandera adusta. We hypothesize that three main alkylitaconic acids (tetradecylitaconic, cis-7-hexadecenylitaconic, and hexadecylitaconic acids) are synthesized by fungi in condensation reactions involving palmitic, oleic, and stearic acids. We suggest that both wood unsaturated fatty acids (present in free form or released from esters during natural decay) and unsaturated metabolites synthesized by fungi could serve as a source for peroxidizable lipids in lignin degradation by white rot basidiomycetes.     

Linoleic acid peroxidation and lignin degradation by enzymes produced by Ceriporiopsis subvermispora grown on wood or in submerged liquid cultures

Ceriporiopsis subvermispora is a white-rot fungus used in biopulping processes and seems to use the fatty acid peroxidation reactions initiated by manganese-peroxidase (MnP) to start lignin degradation. The present work shows that C. subvermispora was able to peroxidize unsaturated fatty acids during wood biotreatment under biopulping conditions. In vitro assays showed that the extent of linoleic acid peroxidation was positively correlated with the level of MnP recovered from the biotreated wood chips. Milled wood was treated in vitro by partially purified MnP and linoleic acid. UV spectroscopy and size exclusion chromatography (SEC) showed that soluble compounds similar to lignin were released from the milled wood. SEC data showed a broad elution profile compatible with low molar mass lignin fractions. MnP-treated milled wood was analyzed by thioacidolysis. The yield of thioacidolysis monomers recovered from guaiacyl and syringyl units decreased by 33% and 20% in MnP-treated milled wood, respectively. This has suggested that lignin depolymerization reactions have occurred during the MnP/linoleic acid treatment.     

Purification and characterization of two forms of the homologously expressed lytic polysaccharide monooxygenase (PvLPMO9A) from Penicillium verruculosum

Two forms of C1/C4-oxidizing lytic polysaccharide monooxygenase (PvLPMO9A) from Penicillium verruculosum (Talaromyces verruculosus) homologously expressed in P. verruculosum B1-537 auxotrophic strain were isolated in a homogeneous state using two-stage chromatography. The PvLPMO9A-hm form represented a full-size enzyme encoded by the intact lpmo1 gene, while the PvLPMO9A-lm was a truncated enzyme variant consisting of a conserved catalytic core of AA9 family LPMOs and lacking a C-terminal extra peptide sequence that is present in PvLPMO9A-hm. The N-terminal histidine was partially methylated in both enzymes. Most of properties of PvLPMO9A-hm and PvLPMO9A-lm, such as specific activities determined using the 2,6-dimethoxyphenol/H₂O₂ assay, pH-optima of activity observed at pH 7.5, synergistic effects exhibited with purified cellobiohydrolase I (Cel7A) and/or endoglucanase II (Cel5A) from P. verruculosum in hydrolysis of Avicel and milled aspen wood, were also very similar, except for the higher PvLPMO9A-hm thermostability studied using differential scanning calorimetry (DSC). The DSC profile for the PvLPMO9A-hm holoenzyme demonstrated two overlapping peaks (with maxima at 56.3 and 59.6 °C) due to the presence of two unfolding protein domains, while the PvLPMO9A-lm DSC profile represented one peak with maximum at 48.1 °C. After removing the active site copper with EDTA, the PvLPMO9A-hm and PvLPMO9A-lm melting temperatures decreased by ~10–11 and ~1 °C, respectively. These data show that both active site copper and C-terminal domain present in the PvLPMO9A-hm protect the enzyme from thermal unfolding, while the stabilizing effect of metal is much less pronounced in the truncated PvLPMO9A-lm form.     

Quantification of compression wood severity in tracheids of Pinus radiata D. Don using confocal fluorescence imaging and spectral deconvolution

Confocal fluorescence microscopy was used to examine the spectral characteristics of lignin autofluorescence in secondary cell walls of normal and compression wood from Pinus radiata. Using UV excitation, fluorescence spectra of normal and compression wood sections showed significant differences, especially in the outer secondary cell wall of tracheids, with a shift in maxima from violet to blue wavelengths between normal and compression wood. A comparison of normal wood, mild and severe compression wood, showed that the wavelength shift was intermediate in the mild compression wood compared to the severe compression wood, thus offering the possibility of quantifying the severity by measuring ratios of fluorescence at violet and blue wavelengths. Fluorescence induced by blue light, rather than UV, was less well differentiated amongst wood types. Spectral deconvolution indicated the presence of a minimum of five discrete lignin fluorophores in the cell walls of both normal and compression wood tracheids. Comparison with lignin model compounds suggest that the wavelength shift may correspond in part to increased levels of p-hydroxy type lignin in the compression wood samples. The combination of confocal fluorescence imaging and related spectral deconvolution therefore offers a novel technique for characterising cell wall lignin in situ.     

Ultramicroscopic composition and supramolecular structure of wood matrix

The ultramicroscopic composition and supramolecular structure of wood matrix were investigated by methods of electron and atomic force microscopy (AFM). New data on specific features of the composition and cell wall structure of Juniper wood (Juniperus communis L.) were obtained. Native lignin was found to be water soluble. It was also shown that lignin does not constitute a continuous matrix between cellulose fibrils, but is deposited as spherical particles.     

Biodegradation of lignin by <Emphasis Type="Italic">Pseudomonas</Emphasis> sp. Q18 and the characterization of a novel bacterial DyP-type peroxidase

Lignin valorization can be obtained through cleavage of selected bonds by microbial enzymes, in which lignin is segregated from cellulose and hemicellulose and abundant phenolic compounds can be provided. In this study, Pseudomonas sp. Q18, previously isolated from rotten wood in China, was used to degrade alkali lignin and raw lignocellulosic material. Gel-permeation chromatography, field-emission scanning electron microscope, and GC–MS were combined to investigate the degradation process. The GC–MS results revealed that the quantities of aromatic compounds with phenol ring from lignin increased significantly after incubation with Pseudomonas sp. Q18, which indicated the degradation of lignin. According to the lignin-derived metabolite analysis, it was proposed that a DyP-type peroxidase (PmDyP) might exist in strain Q18. Thereafter, the gene of PmDyP was cloned and expressed, after which the recombinant PmDyP was purified and the enzymatic kinetics of PmDyP were assayed. According to results, PmDyP showed promising characteristics for lignocellulosic biodegradation in biorefinery.     

Detection of Lignin Peroxidase and Xylanase by Immunocytochemical Labeling in Wood Decayed by Basidiomycetes

The white rot fungi used in this study caused two different forms of degradation. Phanerochaete chrysosporium, strain BKM-F-1767, and Phellinus pini caused a preferential removal of lignin from birch wood, whereas Trametes (Coriolus) versicolor caused a nonselective attack of all cell wall components. Use of polyclonal antisera to H8 lignin peroxidase and monoclonal antisera to H2 lignin peroxidase followed by immunogold labeling with protein A-gold or protein G-gold, respectively, showed lignin peroxidase extra-and intracellularly to fungal hyphae and within the delignified cell walls after 12 weeks of laboratory decay. Lignin peroxidase was localized at sites within the cell wall where electron-dense areas of the lignified cell wall layers remained. In wood decayed by Trametes versicolor, lignin peroxidase was located primarily along the surface of eroded cell walls. No lignin peroxidase was evident in brown-rotted wood, but slight labeling occurred within hyphal cells. Use of polyclonal antisera to xylanase followed by immunogold labeling showed intense labeling on fungal hyphae and surrounding slime layers and within the woody cell wall, where evidence of degradation was apparent. Colloidal-gold-labeled xylanase was prevalent in wood decayed by all fungi used in this study. Areas of the wood with early stages of cell wall decay had the greatest concentration of gold particles, while little labeling occurred in cells in advanced stages of decay by brown or white rot fungi.     

Acid-Free Ethanol-Water Pretreatment with Low Ethanol Concentration for Robust Enzymatic Saccharification of Cellulose in Bamboo

Lignin deposition phenomenon during the liquid-hot-water (LHW) pretreatment negatively affects substrate enzymatic digestibility (SED). To overcome this limitation of LHW, acid-free ethanol-water (EW) pretreatment with low ethanol concentration was developed. With less cellulose loss and similar hemicellulose removal, adding 10% (v/v) ethanol into water (EW10 pretreatment) resulted in a lignin removal of 5.8% higher than that of LHW pretreatment conducted at the same conditions (such as 200 °C for 40 min). Although the lignin removal did not increase significantly, differential scanning calorimetry (DSC) and X-ray photoelectron spectroscopy (XPS) characterizations indicated that LHW pretreatment-induced lignin condensation was alleviated by EW10 pretreatment, leading less lignin condensates deposited on the corresponding surface of solid substrate. Moreover, compared with lignin separated from LHW-pretreated substrate, the non-productive adsorption between EW10 pretreatment-induced lignin and cellulase was significantly weakened. As a result, the SED of EW10 pretreatment was improved to 91.7%, which was higher than LHW pretreatment by 19.5%. Due to the advantages of suppressing the deposition of lignin condensates and employing ethanol at low concentration, EW10 pretreatment shows practical significance for producing fermentable sugar from abundant non-woody biomass (bamboo) in China.     

Differential Scanning Calorimetrie Study of the Thermal Denaturation of Almond β-Glucosidase

The thermal denaturation of almond β-glucosidase [EC 3.2.1.21] was studied by differential scanning calorimetry. The shape of the DSC trace was highly dependent on pH; two peaks were observed between pH 6–8, but only one peak between pH 4–5. All of the DSC curves were resolved into three components according to the model of independent two-state processes, and the thermodynamic parameters for the denaturation were evaluated. The dependence of the shape of DSC curves was accounted for mainly by the rapid changes of denaturation enthalpy and denaturation temperature of the third component in the acidic pH region.     

Amino-Acid Composition of Wood Proteins

The ammo-acid compositions of proteins present in the sapwoodand heartwood from three species of Eucalyptus, in normal, reaction,and opposite wood from Pinus radiata seedlings, and in reactionand opposite wood from Eucalyptus goniocalyx, are listed. Thequestion of the location of protein within wood cell walls isdiscussed. The amounts of tyrosine found in the woods from P.radiata seedlings are incompatible both with the belief thatit determines the intensity of staining of reaction wood cellwalls with Millon's reagent, and that it is the source of thep-hydroxybenzaldehyde obtained when milled wood lignin is oxidizedwith nitrobenzene. Loss of amino acids in the presence of milledwood lignin, -cellulose, hemicellulose, glucose, mannose, galactose,xylose, arabinose, glucuronic, and galacturonic acids is demonstrated.The presence of numerous peaks which are not indentifiable withknown amino acids in the chromatograms of wood hydrolysatesis believed to be due largely to the products of interactionof amino acids and sugars. Some retention of nitrogen by theresidues of wood hydrolysates occurs. The significance of theresults obtained with the wood samples listed above is discussedagainst this background.