Effects of Nitrogen Supplements on Degradation of Aspen Wood Lignin and Carbohydrate Components by Phanerochaete chrysosporium

A supplement of KH₂PO₄, MgSO₄, CaCl₂, trace elements, and thiamine accelerated the initial rate of aspen wood decay by Phanerochaete chrysosporium but did not increase the extent of lignin degradation. Asparagine, casein hydrolysate, and urea supplements (1% added N) strongly inhibited lignin degradation and weight loss. The complex nitrogen sources peptone and yeast extract stimulated lignin degradation and weight loss. Albumen and NH₄Cl had intermediate effects. Conversion of [¹⁴C]lignin to ¹⁴CO₂ and water-soluble materials underestimated lignin degradation in the presence of the complex N sources. The highest ratio of lignin degradation to total weight loss and the largest increase in cellulase digestibility occurred during the decay of unsupplemented wood. Rotting of aspen wood by P. chrysosporium gives smaller digestibility increases than have been found with some other white-rot fungi.     

Elicitor-Induced Spruce Stress Lignin (Structural Similarity to Early Developmental Lignins)

Suspension cultures of Picea abies (L.) Karst released polymeric material into the culture medium when treated with an elicitor preparation from the spruce needle pathogen Rhizosphaera kalkhoffii. The presence of lignin (about 35%, w/w) was demonstrated by phloroglucinol/HCI reactivity and quantitation with thioglycolic acid. Carbohydrate (about 14%, w/w) and protein (about 32%, w/w) were also detected. Amino acid analysis revealed that hydroxyproline and proline predominated. Thioacidolysis and subsequent Raney nickel desulfurization allowed the analysis of lignin-building units and interunit bonds. Compared with spruce wood lignin, an approximately 20-fold higher relative amount of p-hydroxyphenyl units was determined. A high content of p-hydroxyphenyl units is typical for certain developmental lignins, such as conifer compression wood and middle lamella lignins, as well as all induced cell culture lignins so far analyzed. Cross-linkages of the pinoresinol type ([beta]-[beta]) in the excreted cell culture lignin were markedly increased, whereas [beta]-1 interunit linkages were decreased relative to spruce wood lignin. The amount and nature of cross-linkages were shown to be intermediate between those in wood lignin and in enzymatically prepared lignins. In summary, the elicitor-induced stress lignin was excreted as a lignin-extensin complex that closely resembled early developmental lignins.     

Wood Degradation by White Rot Fungi: Cytochemical Studies Using Lignin Peroxidase-Immunoglobulin-Gold Complexes

Using an anti-lignin peroxidase antiserum-protein A-gold complex, we found lignin peroxidase mainly intracellularly in several white rot fungi colonizing sawdust under laboratory conditions. This enzyme was also present in fungi found in naturally decayed wood. However, in all cases, lignin peroxidase was located mainly inside the fungal cells. Labeled lignin peroxidase did not bind to the lignocellulosic samples tested, with the exception of poplar milled-wood lignin. These results are discussed in relation to the role of lignin peroxidase during wood degradation.     

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.     

Wood stimulates the demethoxylation of [O14CH3]-labeled lignin model compounds by the white-rot fungi Phanerochaete chrysosporium and Phlebia radiata

Mineralization of polymeric wood lignin and its substructures is a result of complex reactions involving oxidizing and reducing enzymes and radicals. The degradation of methoxyl groups is an essential part of this process. The presence of wood greatly stimulates the demethoxylation of a non-phenolic lignin model compound (a [O¹⁴CH₃]-labeled β-O-4 dimer) by the lignin-degrading white-rot fungi Phlebia radiata and Phanerochaete chrysosporium. When grown on wood, both fungi produced up to 47 and 40% ¹⁴CO₂ of the applied ¹⁴C activity, respectively, under air and oxygen in 8 weeks. Without wood, the demethoxylation of the dimer by both fungi was lower, varying between 0.5 and 35%. Addition of nutrient nitrogen together with glucose decreased demethoxylation when the fungi were grown on spruce wood under air. Because the evolution of ¹⁴CO₂ in the absence of wood was poor, the fungi may have preferably used wood as a carbon and nitrogen source. The amount of fungal mycelium, as determined by the ergosterol assay, did not show connection to demethoxylation. P. radiata also showed a high demethoxylation of [O¹⁴CH₃]-labeled vanillic acid in the presence of birch wood. The degradation of lignin and lignin-related substances should be studied in the presence of wood, the natural substrate for white-rot fungi.     

The importance of phenol oxidase activity in lignin degradation by the white-rot fungus Sporotrichum pulverulentum

The lignin degradation abilities of wildtype, a phenol oxidase-less mutant and a phenol oxidase-positive revertant of Sporotrichum pulverulentum were compared to determine if phenol oxidase activity is necessary for lignin degradation by white-rot fungi. The phenol oxidase-less mutant was unable to degrade kraft lignin or wood. The phenol oxidase-positive revertant, however, regained the ability of the wildtype to degrade kraft lignin and all of the major components of wood. It was found that kraft lignin and lignin-related phenols decreased cellulase and xylanase production by the phenol oxidase-less mutant. Addition of highly purified laccase increased the production of endo-1,4--glucanase in the phenol oxidase-less mutant in the presence of vanillic acid and kraft lignin. After addition of laccase to kraft lignin agar plates, the phenol oxidase-less mutant could degrade kraft lignin.It is proposed that phenol oxidase function in regulating the production of both lignin-and polysaccharide-degrading enzymes by oxidation of lignin and lignin-related phenols when S. pulverulentum is growing on wood.Abbreviation WT wildtype Sporotrichum pulverulentum Research supported by a grant from Stiftelsen Nils and Dorthi Troëdssons forskningsfond     

Absence of microbial mineralization of lignin in anaerobic enrichment cultures

The existence of anaerobic biodegradation of lignin was examined in mixed microflora. Egyptian soil samples, in which rapid mineralization of organic matter takes place in the presence of an important anaerobic microflora, were used to obtain the anaerobic enrichment cultures for this study. Specifically, 14CO2 or [14C]lignin wood was used to investigate the release of labeled gaseous or soluble degradation products of lignin in microbial cultures. No conversion of 14C-labeled lignin to 14CO2 or 14CH4 was observed after 6 months of incubation at 30 degrees C in anaerobic conditions with or without NO3-. A small increase in soluble radioactivity was observed in certain cultures, but it could not be related to the release of catabolic products during the anaerobic biodegradation of lignin.     

Absence of microbial mineralization of lignin in anaerobic enrichment cultures.

The existence of anaerobic biodegradation of lignin was examined in mixed microflora. Egyptian soil samples, in which rapid mineralization of organic matter takes place in the presence of an important anaerobic microflora, were used to obtain the anaerobic enrichment cultures for this study. Specifically, 14CO2 or [14C]lignin wood was used to investigate the release of labeled gaseous or soluble degradation products of lignin in microbial cultures. No conversion of 14C-labeled lignin to 14CO2 or 14CH4 was observed after 6 months of incubation at 30 degrees C in anaerobic conditions with or without NO3-. A small increase in soluble radioactivity was observed in certain cultures, but it could not be related to the release of catabolic products during the anaerobic biodegradation of lignin.     

Selective Degradation of Wood Components by White-Rot Fungi

In order to find naturally occurring white-rot fungi which preferentially degrade lignin. 25 different species of such fungi were cultivated on pine wood blocks and on kraft lignin agar plates with and without cellulose. Due to differences in phenol oxidase reactions on the kraft lignin agar plates, the 25 fungi could be divided into two groups, 1 and 2, which also differed in other properties. The three Group I fungi Sporotrichum pulverulentum, Phanerochaete sp. L1 and Polyporus dichrous produced high levels of endo-l,4-β-glucanase and cellobiose:quinone oxidoreductase in shaking cellulose flasks and a low level of phenol oxidase in standing wood meal flasks, The four fungi Merulius tremellosus, Phlebia radiata, Pycuoporus cinnabarinus and Pleurotus ostreatus from Group 2, on the other hand, produced low levels of endo-1,4-β-glucanase and cellobiose:.quinone oxidoreductase in the cellulose. flasks and a high level of phenol oxidase in the wood meal flasks. Analyses of pine wood blocks degraded by the above-mentioned fungi in the presence of either malt extract, asparagine or NH₄H₂PO₄ revealed that malt extract gave good lignin degradation. In the presence of this nutrient source. P. cinnabarinus, at 3.4% weight loss, even degraded 12.5% lignin without loss of cellulose or mannan. No common degradation pattern was, however, obtained using mall extract, asparagine or NH₄H₂PO₄, It is suggested that while-rot fungi, which preferentially degrade lignin, may be found among Group 2 fungi producing large amounts of phenol oxidases.     

Cellulose and lignin biosynthesis is altered by ozone in wood of hybrid poplar (Populus tremula × alba)

Wood formation in trees is a dynamic process that is strongly affected by environmental factors. However, the impact of ozone on wood is poorly documented. The objective of this study was to assess the effects of ozone on wood formation by focusing on the two major wood components, cellulose and lignin, and analysing any anatomical modifications. Young hybrid poplars (Populus tremula × alba) were cultivated under different ozone concentrations (50, 100, 200, and 300 l l(-1)). As upright poplars usually develop tension wood in a non-set pattern, the trees were bent in order to induce tension wood formation on the upper side of the stem and normal or opposite wood on the lower side. Biosynthesis of cellulose and lignin (enzymes and RNA levels), together with cambial growth, decreased in response to ozone exposure. The cellulose to lignin ratio was reduced, suggesting that cellulose biosynthesis was more affected than that of lignin. Tension wood was generally more altered than opposite wood, especially at the anatomical level. Tension wood may be more susceptible to reduced carbon allocation to the stems under ozone exposure. These results suggested a coordinated regulation of cellulose and lignin deposition to sustain mechanical strength under ozone. The modifications of the cellulose to lignin ratio and wood anatomy could allow the tree to maintain radial growth while minimizing carbon cost.     

Modification of lignin for the production of new compounded materials

The cell walls of woody plants are compounded materials made by in situ polymerization of a polyphenolic matrix (lignin) into a web of fibers (cellulose), a process that is catalysed by polyphenoloxidases (laccases) or peroxidases. The first attempt to transform the basic strategy of this natural process for use in human craftsmanship was the ancient lacquer method. The sap of the lacquer tree (Rhus verniciflua) contains large amounts of a phenol (urushiol), a polysaccharide and the enzyme laccase. This oil-in-water emulsion solidifies in the presence of oxygen. The Chinese began using this phenomenon for the production of highly creative artwork more than 6,000 years ago. It was the first example of an isolated enzyme being used as a catalyst to create an artificial plastic compound. In order to apply this process to the production of products on an industrial scale, an inexpensive phenol must be used, which is transferred by an enzyme to active radicals that react with different components to form a compounded material. At present, the following approaches have been studied: (1) In situ polymerization of lignin for the production of particle boards. Adhesive cure is based on the oxidative polymerization of lignin using phenoloxidases (laccase) as radical donors. This lignin-based bio-adhesive can be applied under conventional pressing conditions. The resulting particle boards meet German performance standards. By this process, 80% of the petrochemical binders in the wood-composite industry can be replaced by materials from renewable resources. (2) Enzymatic copolymerization of lignin and alkenes. In the presence of organic hydroperoxides, laccase catalyses the reaction between lignin and olefins. Detailed studies on the reaction between lignin and acrylate monomers showed that chemo-enzymatic copolymerization offers the possibility to produce defined lignin-acrylate copolymers. The system allows control of the molecular weights of the products in a way that has not been possible with chemical catalysts. This is a novel attempt to enzymatically induce grafting of polymeric side chains onto the lignin backbone, and it enables the utilization of lignin as part of new engineering materials. (3) Enzymatic activation of the middle-lamella lignin of wood fibers for the production of wood composites. The incubation of wood fibers with a phenol oxidizing enzyme results in oxidative activation of the lignin crust on the fiber surface. When such fibers are pressed together, boards are obtained which meet the German standards for medium-density fiber boards (MDF). The fibers are bound together in a way that comes close to that by which wood fibers are bound together in naturally grown wood. This process will, for the first time, yield wood composites that are produced solely from naturally grown products without any addition of resins.     

Methane fermentation of Japanese cedar wood pretreated with a white rot fungus, Ceriporiopsis subvermispora

Methane fermentation of Japanese cedar wood was carried out after pretreatment with four strains of white rot fungi, Ceriporiopsis subvermispora ATCC 90467, CZ-3, CBS 347.63 and Pleurocybella porrigens K-2855. These fungi were cultivated on wood chip media with and without wheat bran for 4-8 weeks. The pretreated wood chip was fermented anaerobically with sludge from a sewage treatment plant. Pretreatments with C. subvermispora ATCC 90467, CZ-3 and CBS 347.63 in the presence of wheat bran for 8 weeks decreased 74-76% of beta-O-4 aryl ether linkages in the lignin to accelerate production of methane. After fungal treatments with C. subvermispora ATCC 90467 and subsequent 30-days methane fermentation, the methane yield reached 35 and 25% of the theoretical yield based on the holocellulose contents of the decayed and original wood, respectively. In contrast, treatment with the three strains of C. subvermispora without wheat bran cleaved 15-26% of the linkage and produced 6-9% of methane. There were no significant accelerating effects in wood chips treated with P. porrigens which has a lower ability to decompose the lignin. Thus, it was found that C. subvermispora, with a high ability to decompose aryl ether bonds of lignin, promoted methane fermentation of softwood in the presence of wheat bran.     

Cellobiose dehydrogenase (cellobiose oxidase) from Phanerochaete chrysosporium as a wood-degrading enzyme. Studies on cellulose,xylan and synthetic lignin

Degradation of carboxymethylcellulose (CMC), xylan and synthetic lignin was studied in a cellobiose dehydrogenase system, that reduced Fe(III) to Fe(II) with cellobiose as electron donor, which in the presence of hydrogen peroxide degraded all the above representatives of the main wood components, probably by forming Fenton's reagent. The production of hydroxyl radicals was shown by benzoate decarboxylation. For the CMC and xylan studies viscometry was used, while lignin degradation was studied by measuring the passage of ¹⁴C-labelled synthetic lignin (DHP) through membranes of different molecular-mass cut-off. The possible participation of cellobiose dehydrogenase, Fe(III) and hydrogen peroxide in wood degradation by white-rot and brown-rot fungi is discussed.     

Ratio of erythro and threo forms of beta-O-4 structures in tension wood lignin

The ratio of erythro and threo forms of beta-O-4 structures in tension wood lignin was investigated by ozonation analysis of wood meal taken from various positions in the stem of yellow poplar (Liriodendron tulipifera). The proportion of the erythro form was higher in tension wood than in opposite wood, and the methoxyl group content showed a similar trend. The proportion of the erythro form and the methoxyl group content in the 7 positions in the stem lignin was correlated (correlation coefficient R=0.98), suggesting that the type of aromatic ring, syringyl or guaiacyl, is one of the factors which stereochemically controls the ratio of erythro and threo forms of beta-O-4 structures during lignin formation.     

Biological Pretreatment of Lignocellulosic Substrates for Enhanced Delignification and Enzymatic Digestibility

Sheer enormity of lignocellulosics makes them potential feedstock for biofuel production but, their conversion into fermentable sugars is a major hurdle. They have to be pretreated physically, chemically, or biologically to be used by fermenting organisms for production of ethanol. Each lignocellulosic substrate is a complex mix of cellulose, hemicellulose and lignin, bound in a matrix. While cellulose and hemicellulose yield fermentable sugars, lignin is the most recalcitrant polymer, consisting of phenyl-propanoid units. Many microorganisms in nature are able to attack and degrade lignin, thus making access to cellulose easy. Such organisms are abundantly found in forest leaf litter/composts and especially include the wood rotting fungi, actinomycetes and bacteria. These microorganisms possess enzyme systems to attack, depolymerize and degrade the polymers in lignocellulosic substrates. Current pretreatment research is targeted towards developing processes which are mild, economical and environment friendly facilitating subsequent saccharification of cellulose and its fermentation to ethanol. Besides being the critical step, pretreatment is also cost intensive. Biological treatments with white rot fungi and Streptomyces have been studied for delignification of pulp, increasing digestibility of lignocellulosics for animal feed and for bioremediation of paper mill effluents. Such lignocellulolytic organisms can prove extremely useful in production of bioethanol when used for removal of lignin from lignocellulosic substrate and also for cellulase production. Our studies on treatment of hardwood and softwood residues with Streptomyces griseus isolated from leaf litter showed that it enhanced the mild alkaline solubilisation of lignins and also produced high levels of the cellulase complex when growing on wood substrates. Lignin loss (Klason lignin) observed was 10.5 and 23.5% in case of soft wood and hard wood, respectively. Thus, biological pretreatment process for lignocellulosic substrate using lignolytic organisms such as actinomycetes and white rot fungi can be developed for facilitating efficient enzymatic digestibility of cellulose.     

Polymerisation of lignins by ligninases from Phanerochaete chrysosporium

Abstract Four major hemoproteins were purified by isoelectric focusing from an extracellular crude enzyme preparation, produced by the white rot fungus Phanerochaete chrysosporium under carbon-limited conditions. Both the crude enzyme and the purified proteins oxidised milled wood lignin, HCl-dioxane-extracted straw lignin and alkali straw lignin in the presence of hydrogen peroxide. The oxidation resulted mainly in further polymerisation of the lignins and was enhanced by addition of veratryl alcohol to the reaction mixture. Alkali straw lignin was also polymerised by horseradish peroxidase, although veratryl alcohol had no influence on this reaction.     

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.     

Biodegradation of Lignin by White Rot Fungi

A review is presented related to the biochemistry of lignocellulose transformation. The biodegradation of wood constituents is currently understood as a multienzymatic process with the mediation of small molecules; therefore, this review will focus on the roles of these small molecular compounds and radicals working in concert with enzymes. Wood rotting basidiomycetous fungi penetrate wood and lead to more easily metabolized, carbohydrate constituents of the complex. Having a versatile machinery of enzymes, the white rot fungi are able to attack directly the "lignin barrier." They also use a multienzyme system including so-called "feed back" type enzymes, allowing for simultaneous transformation of both lignin and cellulose. These enzymes may function separately or cooperatively.     

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.     

Analysis of lignin–carbohydrate and lignin–lignin linkages after hydrolase treatment of xylan–lignin,glucomannan–lignin and glucan–lignin complexes from spruce wood

Xylan–lignin (XL), glucomannan–lignin (GML) and glucan–lignin (GL) complexes were isolated from spruce wood, hydrolyzed with xylanase or endoglucanase/β-glucosidase, and analyzed by analytical pyrolysis and 2D-NMR. The enzymatic hydrolysis removed most of the polysaccharide moieties in the complexes, and the lignin content and relative abundance of lignin–carbohydrate linkages increased. Analytical pyrolysis confirmed the action of the enzymatic hydrolysis, with strong decreases of levoglucosane and other carbohydrate-derived products. Unexpectedly it also revealed that the hydrolase treatment alters the pattern of lignin breakdown products, resulting in higher amounts of coniferyl alcohol. From the anomeric carbohydrate signals in the 2D-NMR spectra, phenyl glycoside linkages (undetectable in the original complexes) could be identified in the hydrolyzed GML complex. Lower amounts of glucuronosyl and benzyl ether linkages were also observed after the hydrolysis. From the 2D-NMR spectra of the hydrolyzed complexes, it was concluded that the lignin in GML is less condensed than in XL due to its higher content in β-O-4′ ether substructures (62 % of side chains in GML vs 53 % in XL) accompanied by more coniferyl alcohol end units (16 vs 13 %). In contrast, the XL lignin has more pinoresinols (11 vs 6 %) and dibenzodioxocins (9 vs 2 %) than the GML (and both have ~13 % phenylcoumarans and 1 % spirodienones). Direct 2D-NMR analysis of the hydrolyzed GL complex was not possible due to its low solubility. However, after sample acetylation, an even less condensed lignin than in the GML complex was found (with up to 72 % β-O-4′ substructures and only 1 % pinoresinols). The study provides evidence for the existence of structurally different lignins associated to hemicelluloses (xylan and glucomannan) and cellulose in spruce wood and, at the same time, offers information on some of the chemical linkages between the above polymers.