Decomposition of 14C-labelled lignin and phenols by a Nocardia sp.

A Gram-positive bacterium which was isolated from a Finnish soil and identified as a Nocardia sp., was able to decompose lignin and to assimilate lignin degradation products as a carbon source. It could release ¹⁴CO₂ from ¹⁴C-labelled methoxyl groups, side chains or ring carbons of coniferyl alcohol dehydropolymers (DHP) and from specifically ¹⁴C-labelled lignin of plant material. Furthermore, it could release ¹⁴CO₂ from phenolcarboxylic and cinnamic acids and alcohols labelled in the OCH₃, COOH groups, side chain or aromatic ring carbons.Non-Common Abbreviations Used DHP dehydropolymers of coniferyl alcohol     

Screening for lignin degrading bacteria by means of 14C-labelled lignins

Several Nocardia and Pseudomonas spp., as well as some unidentified bacteria, isolated from lake water containing high loads of waste lignin, were tested for their capacity to release ¹⁴CO₂ from specifically ¹⁴C-labelled dehydropolymer of coniferyl alcohol (DHP) or corn stalk lignins. The bacteria were selected according to their ability to degrade phenolic compounds. However, only some of them could release significant amounts of ¹⁴CO₂ from the labelled lignin. The tested Nocardia spp. were more active than the Pseudomonas spp. and the unidentified bacteria. The most active strains belonged to N. autotrophica. These strains released CO₂ significantly from the methoxyl group and transformed the other carbons from the phenylpropane skeleton of lignin also into CO₂. Other less demethylating strains also released little CO₂ from the other carbons of the lignin molecule. From corn stalk materials which were specifically labelled in the lignin part only small amounts of labelled CO₂ were released.Non-Common-Abbreviation Used DHP dehydropolymers of coniferyl alcohol     

Degradation of lignin and lignin related compounds by protoplasts isolated from Fomes annosus

Protoplasts from a lignolytic fungus Fomes annosus were prepared through enzymatic hydrolysis of mycelium utilizing Novozym, a wall lytic enzyme preparation. Isolated protoplasts and living mycelium were compared in their ability to degrade ¹⁴C-labelled lignin related phenols and dehydropolymers of labelled coniferyl alcohol (synthetic lignin). The amounts of ¹⁴CO₂ released from O¹⁴CH₃-groups, ¹⁴C-2-side chains and ¹⁴C-rings by protoplasts was in the same range as those for intact mycelium. The methoxyl groups of synthetic lignin were more rapidly metabolized by protoplasts than by mycelium. When calculated in dpm of released ¹⁴CO₂ per mg protein the decomposition of ¹⁴C-labelled synthetic lignin and lignin-related monomers in a hyphae-free system of protoplasts was considerable higher than that obtained by the intact mycelium. The presence of intact hyphae is thus not necessary for lignin degradation to occur.Non-common-abbreviations used DHP Dehydropolymer of coniferyl alcohol - LS lignosulfonates prepared from DHP     

New form of lignolytically active mycelium generated by immobilization of protoplasts isolated from the white rot fungi Heterobasidion annosum and Polyporus pinsitus

Summary Immobilized mycelia regenerated from immobilized protoplasts isolated from lignin-degrading Basiodiomycetes have been shown to be able to decompose specifically ¹⁴C-labelled dehydropolymers of coniferylalcohol (DHP-lignin) and monomeric lignin-related compounds more intensively than native mycelium, by decarboxylation, demethylation, ring and side chain cleavage. Protoplasts of two white rot fungi were immobilized by entrapment in Na- alginate gel and remained intact after the immobilization procedure. Within the first 3 days of incubation in culture medium, regeneration of hyphal cells occurred. Since hyphal cells regenerated from protoplasts within gel beads were hindered from stretching by the matrix, the microbial immobilized cells differed from native mycelium in terms of their morphology. The time course and extent of lignin degradation by native mycelium and regenerated mycelium of the examined white rot fungi also differed, a sign that there may also be differences between them in terms of the physiology of lignin degradation.     

Increased biooxidation of lignosulfonates by Sporotrichum pulverulentum in the presence of polyacrylic acid

Summary The superiority of polyacrylic acid used as a buffer at 0.25% (w/v) during biodegradation of high molecular weight ¹⁴C-labeled lignosulfonates (LS) by the white rot fungus Sporotrichum pulverulentum is demonstrated. Compared to 2,2-dimethylsuccinate (DMS) the release of ¹⁴CO₂ from the LS occurs earlier, does not show the levelling-off symptom and reaches higher levels. Changes in pH values of the medium cannot be correlated with the stimulating effects of polyacrylic acid on the ligninolytic activity of the fungus. It seems that interaction between LS and also of dehydropolymers of coniferyl alcohol (DHP) with the polymeric buffer increases the accessibility to the fungus.     

Bacterial degradation of dehydropolymers of coniferyl alcohol

A bacterial isolate identified as Xanthomonas sp. proved to be ligninolytic due to its ability to degrade ¹⁴C-labeled dehydropolymers of coniferyl alcohol (DHP) and [14C]lignocellulose complexes from corn plants (Zea mays). Several parameters of ligninolysis were evaluated and it was shown that resting cells degrade DHP as sole carbon source. Enhancement of DHP degradation in the presence of ferulic acid or water-soluble fractions of DHP or of dioxane lignin from wheat was demonstrated. It is shown that a dissociation of DHP takes place during incubation in the absence of the bacteria which is reflected in a shift of DHP to lower molecular weight fractions. Bacterial degradation of [14C] DHP results in the release of ¹⁴CO₂ and in the incorporation of the ¹⁴C-label into the biomass of the bacteria, as shown by chemical and biological methods.Abbreviations Bq Becquerel, measure for radioactivity according to SI nomenclature - DHP dehydropolymers of coniferyl alcohol - DMF dimethylformamide - DMSO dimethyl sulfoxide - HPLC high performance liquid chromatography - TCA trichloroacetic acid - THF tetrahydrofuran     

Lignocellulosic polysaccharides and lignin degradation by wood decay fungi: the relevance of nonenzymatic Fenton-based reactions

The brown rot fungus Wolfiporia cocos and the selective white rot fungus Perenniporia medulla-panis produce peptides and phenolate-derivative compounds as low molecular weight Fe³⁺-reductants. Phenolates were the major compounds with Fe³⁺-reducing activity in both fungi and displayed Fe³⁺-reducing activity at pH 2.0 and 4.5 in the absence and presence of oxalic acid. The chemical structures of these compounds were identified. Together with Fe³⁺ and H₂O₂ (mediated Fenton reaction) they produced oxygen radicals that oxidized lignocellulosic polysaccharides and lignin extensively in vitro under conditions similar to those found in vivo. These results indicate that, in addition to the extensively studied Gloeophyllum trabeum—a model brown rot fungus—other brown rot fungi as well as selective white rot fungi, possess the means to promote Fenton chemistry to degrade cellulose and hemicellulose, and to modify lignin. Moreover, new information is provided, particularly regarding how lignin is attacked, and either repolymerized or solubilized depending on the type of fungal attack, and suggests a new pathway for selective white rot degradation of wood. The importance of Fenton reactions mediated by phenolates operating separately or synergistically with carbohydrate-degrading enzymes in brown rot fungi, and lignin-modifying enzymes in white rot fungi is discussed. This research improves our understanding of natural processes in carbon cycling in the environment, which may enable the exploration of novel methods for bioconversion of lignocellulose in the production of biofuels or polymers, in addition to the development of new and better ways to protect wood from degradation by microorganisms.     

Comparison of pyrene biodegradation by white rot fungi

Six strains of white rot fungi, isolated from soil in Korea, were evaluated as to their ability to biodegrade the 4-ring polycyclic aromatic hydrocarbon pyrene. While growing in a complex fungal medium, Irpex lacteus, Trametes versicolor KR11W, and Phanerochaete chrysosporium mineralized 15.6, 12.7 and 7.0% of the added 0.84 nmol of radioactive pyrene, respectively. In these cultures, 33–46% of the added pyrene was converted to water-soluble polar metabolites, and 22–40% was incorporated into fungal biomass. Pleurotus ostreatus mineralized only 2.5% of the added pyrene, while T. versicolor KR65W and Microporus vernicipes failed to evolve ¹⁴CO₂ from pyrene. The information obtained aids in strain selection for clean-up of polycyclic aromatic hydrocarbon contamination.     

Über O-Demethylierung und Decarboxylierung von Benzoesäuren in pflanzlichen Zellsuspensionskulturen

Summary Various benzoic acids ¹⁴C-labelled in para and meta methoxyl groups as well as (O-methyl-¹⁴C) p-methoxy cinnamic acid were tested for O-demethylation in cell suspension cultures of Phaseolus aureus Roxb. and Glycine max Merr. On the basis of ¹⁴CO₂-formation and product analyses the O-demethylation reactions were shown to be specific for para methoxyl groups. A vanillate-O-demethylase known from microbial sources seemed to be absent in the plant cell cultures.In this and in an earlier publication (Berlin et al., 1971) some twenty ¹⁴C-labelled aromatic compounds were tested for catabolic reactions in the cell cultures, and here we report on the product analyses and the general pattern of distribution of radioactivity. Finally, some indications for compartmentalisation in connection with catabolic studies of aromatic compounds in plant cell cultures are discussed.Decarboxylation of substituted benzoic acids in the cell cultures is restricted to aromatic acids possessing a hydroxyl group in the para position. Only trace amounts of labelled CO₂ were released from (carboxyl-¹⁴C)-anisic acid. This acid, however, was nearly quantitatively demethylated to p-hydroxybenzoic acid, which itself was decarboxylated to a considerable extent after being fed to cell suspension cultures. Similar differences in respect to decarboxylation were observed with syringic acid produced by demethylation of trimethoxybenzoic acid and syringic acid applied directly to the cell cultures.     

Recent Developments in Using Advanced Sequencing Technologies for the Genomic Studies of Lignin and Cellulose Degrading Microorganisms

Lignin is a complex polyphenyl aromatic compound which exists in tight associations with cellulose and hemicellulose to form plant primary and secondary cell wall. Lignocellulose is an abundant renewable biomaterial present on the earth. It has gained much attention in the scientific community in recent years because of its potential applications in bio-based industries. Microbial degradation of lignocellulose polymers was well studied in wood decaying fungi. Based on the plant materials they degrade these fungi were classified as white rot, brown rot and soft rot. However, some groups of bacteria belonging to the actinomycetes, α-proteobacteria and β-proteobacteria were also found to be efficient in degrading lignocellulosic biomass but not well understood unlike the fungi. In this review we focus on recent advancements deployed for finding and understanding the lignocellulose degradation by microorganisms. Conventional molecular methods like sequencing 16s rRNA and Inter Transcribed Spacer (ITS) regions were used for identification and classification of microbes. Recent progression in genomics mainly next generation sequencing technologies made the whole genome sequencing of microbes possible in a great ease. The whole genome sequence studies reveals high quality information about genes and canonical pathways involved in the lignin and other cell wall components degradation.     

Vanillic acid metabolism by the white-rot fungus Sporotrichum pulverulentum

Vanillic acid metabolism was studied in wild-type Sporotrichum pulverulentum and three different mutants. Vanillic acid was found to be oxidatively decarboxylated to methoxyhydroquinone (MHQ) and simultaneously reduced to vanillin and vanillyl alcohol to different degrees depending upon the cultivation conditions. The reducing pathway cannot be utilized unless the fungus has access to an easily metabolized carbon source such as glucose or cellobiose, while decarboxylation takes place in cultures with only vanillic acid present. Polymerization reactions also occurred in the culture solutions. Some evidence for reoxidation of vanillin and vanillyl alcohol was obtained in vivo, and in vitro experiments using horseradish peroxidase.Using vanillic acids labelled in the carboxyl, methoxyl and the aromatic ring it was shown that decarboxylation occures before ring-cleavage, which in turn takes place earlier than the release of ¹⁴CO₂ from O¹⁴CH₃-vanillate. The ¹⁴CO₂ evolution from the methoxyl group is repressed by 1% cellobiose as compared to 0.25% cellobiose, but is stimulated by 26 mM nitrogen (as asparagine plus NH₄NO₃) compared to 2.6 mM nitrogen. Since S. pulverulentum appears to require three hydroxyl groups attached to the benzene ring before ring-cleavage can occur, preparation for ring-cleavage is apparently achieved by hydroxylation rather than by demethylation.A scheme for metabolism of vanillic acid by S. pulverulentum based upon these results is proposed.Non-Standard Abbreviations WT wild type Sporotrichum pulverulentum - MHQ methoxyhydroquinone - MQ methoxyquinone - NKM Norkrans medium - DMS dimethylsuccinate - DHP dehydropolymer of coniferyl alcohol     

Short communication: Isolation of a bacterium capable of limited degradation of industrial and labelled,natural and synthetic lignins

Pseudomonas putida, isolated from decomposing plant materials, degraded several lignin-related aromatic compounds. After 30 days of incubation in media containing polymeric Kraft-lignin (PKL), the amount of Klason lignin had decreased by about 13%. When ¹⁴C-labelled dehydropolymers of coniferyl alcohol (DHP) lignins and ¹⁴C-lignin-lignocelluloses were used as substrates, mineralization to ¹⁴CO₂ by the P. putida strain ranged from 1.4% to 2.1%.     

Bioremediation of Polycyclic Aromatic Hydrocarbons in Creosote-Contaminated Soil by Peniophora incarnata KUC8836

ABSTRACT Polycyclic aromatic hydrocarbons (PAHs) are present in products made from creosote, coal tar, and asphalt. When wood pile treated with creosote is placed in soil, PAHs can contaminate it. Creosote has been used for wood preservation in the past and is composed of approximately 85% PAHs and 15% phenolic compounds. PAHs cause harmful effects to humans and the environment because of their carcinogenic and mutagenic properties. White rot fungi can degrade not only lignin, but also recalcitrant organic compounds such as PAHs. Among numerous white rot fungi used in previous studies, four species were selected to degrade PAHs in a liquid medium. From this evaluation of the degradation of PAHs by the four fungal isolates, two species were ultimately selected for the highest rates of removal. Following 2 weeks of incubation with Peniophora incarnata KUC8836, the degradation rates of phenanthrene, fluoranthene, and pyrene were 86.5%, 77.4%, and 82.6%, respectively. Mycoaciella bispora KUC8201 showed the highest degradation rate for anthracene (61.8%). Hence, bioremediation of creosote-contaminated soil with an initial concentration of 229.49 mg kg^?1 PAHs was carried out using the two selected fungi because they could simultaneously degrade 13 more PAHs than the comparison species. More importantly, isolates of P. incarnata KUC8836 were discovered as powerful degraders of PAHs by producing laccase and manganese-dependent peroxidase (MnP), with 1.7- and 1.1-fold higher than the comparison species, respectively. Therefore, the white rot fungus may be proposed for the removal of PAHs and xenobiotic compounds in contaminated environments.     

Formation and Action of Lignin-Modifying Enzymes in Cultures of Phlebia radiata Supplemented with Veratric Acid

Transformation of veratric (3,4-dimethoxybenzoic) acid by the white rot fungus Phlebia radiata was studied to elucidate the role of ligninolytic, reductive, and demeth(ox)ylating enzymes. Under both air and a 100% O₂ atmosphere, with nitrogen limitation and glucose as a carbon source, reducing activity resulted in the accumulation of veratryl alcohol in the medium. When the fungus was cultivated under air, veratric acid caused a rapid increase in laccase (benzenediol:oxygen oxidoreductase; EC 1.10.3.2) production, which indicated that veratric acid was first demethylated, thus providing phenolic compounds for laccase. After a rapid decline in laccase activity, elevated lignin peroxidase (ligninase) activity and manganese-dependent peroxidase production were detected simultaneously with extracellular release of methanol. This indicated apparent demethoxylation. When the fungus was cultivated under a continuous 100% O₂ flow and in the presence of veratric acid, laccase production was markedly repressed, whereas production of lignin peroxidase and degradation of veratryl compounds were clearly enhanced. In all cultures, the increases in lignin peroxidase titers were directly related to veratryl alcohol accumulation. Evolution of ¹⁴CO₂ from 3-O¹⁴CH₃-and 4-O¹⁴CH₃-labeled veratric acids showed that the position of the methoxyl substituent in the aromatic ring only slightly affected demeth(ox)ylation activity. In both cases, more than 60% of the total ¹⁴C was converted to ¹⁴CO₂ under air in 4 weeks, and oxygen flux increased the degradation rate of the ¹⁴C-labeled veratric acids just as it did with unlabeled cultures.     

Mn(II) Regulation of Lignin Peroxidases and Manganese-Dependent Peroxidases from Lignin-Degrading White Rot Fungi

Two families of peroxidases—lignin peroxidase (LiP) and manganese-dependent lignin peroxidase (MnP)—are formed by the lignin-degrading white rot basidiomycete Phanerochaete chrysosporium and other white rot fungi. Isoenzymes of these enzyme families carry out reactions important to the biodegradation of lignin. This research investigated the regulation of LiP and MnP production by Mn(II). In liquid culture, LiP titers varied as an inverse function of and MnP titers varied as a direct function of the Mn(II) concentration. The extracellular isoenzyme profiles differed radically at low and high Mn(II) levels, whereas other fermentation parameters, including extracellular protein concentrations, the glucose consumption rate, and the accumulation of cell dry weight, did not change significantly with the Mn(II) concentration. In the absence of Mn(II), extracellular LiP isoenzymes predominated, whereas in the presence of Mn(II), MnP isoenzymes were dominant. The release of ¹⁴CO₂ from ¹⁴C-labeled dehydrogenative polymerizate lignin was likewise affected by Mn(II). The rate of ¹⁴CO₂ release increased at low Mn(II) and decreased at high Mn(II) concentrations. This regulatory effect of Mn(II) occurred with five strains of P. chrysosporium, two other species of Phanerochaete, three species of Phlebia, Lentinula edodes, and Phellinus pini.     

Lignin Peroxidase Activity Is Not Important in Biological Bleaching and Delignification of Unbleached Kraft Pulp by Trametes versicolor

The discovery in 1983 of fungal lignin peroxidases able to catalyze the oxidation of nonphenolic aromatic lignin model compounds and release some CO₂ from lignin has been seen as a major advance in understanding how fungi degrade lignin. Recently, the fungus Trametes versicolor was shown to be capable of substantial decolorization and delignification of unbleached industrial kraft pulps over 2 to 5 days. The role, if any, of lignin peroxidase in this biobleaching was therefore examined. Several different assays indicated that T. versicolor can produce and secrete peroxidase proteins, but only under certain culture conditions. However, work employing a new lignin peroxidase inhibitor (metavanadate ions) and a new lignin peroxidase assay using the dye azure B indicated that secreted lignin peroxidases do not play a role in the T. versicolor pulp-bleaching system. Oxidative activity capable of degrading 2-keto-4-methiolbutyric acid (KMB) appeared unique to ligninolytic fungi and always accompanied pulp biobleaching.     

Decolorizing Reactive Textile Dyes with White‐Rot Fungi by Temporary Immersion Cultivation

The decontamination of effluents from textile industries is problematic due to the fact that textile dyes are resistant to degradation in the environment. Enzymes from white rot fungi, especially laccase, are able to degrade various complex aromatic structures, and are therefore able to decolorize textile dyes. The white‐rot fungi Trametes versicolor and Phanerochaete chrysosporium were immobilized, separately, on both pine wood chips and palm oil fiber, and cultivated in the temporary immersion RITA® (Récipient à Immersion Temporaire Automatique) System, which was adapted to serve as a fungal bioreactor in a series of four experiments to determine optimal conditions for decolorizing the textile dyes Levafix Blue and Remazol Brilliant Red. The maximum rate of decolorization of both dyes occurred within 24 h of incubation, and laccase was detected in the system.     

Anaerobic degradation of methoxylated aromatic compounds by Clostridium methoxybenzovorans and a nitrate-reducing bacterium Thauera sp. strain Cin3,4

The coupling of growth of the o-demethylating bacterium, Clostridium methoxybenzovorans SR3, with a nitrate-reducing bacterium able to degrade aromatic compounds, Thauera sp. Cin3,4, allowed complete mineralization of poorly oxidizable methoxylated aromatic compounds such as vanillate, isovanillate, vanilline, anisate, ferulate and veratrate. C. methoxybenzovorans o-demethylated these aromatic compounds to their corresponding hydroxylated derivatives and fermented the side chains to acetate and butyrate. The hydroxylated compounds and the fermentation end-products in the C. methoxybenzovorans spent growth medium were then completely metabolized to CO₂ on inoculation with the Thauera strain. Kinetic studies with veratrate indicated that C. methoxybenzovorans initially o-demethylated the substrate to vanillate and then further to protocatechuate together with the production of acetate and butyrate from the demethylated side chains. Protocatechuate, acetate and butyrate were then utilized as a carbon source by the Thauera strain aerobically or anaerobically in the presence of nitrate. The results therefore suggest that mono- or dimethoxylated aromatic compounds can be completely mineralized by coupling the growth of a fermentative bacterium with a nitrate-reducing bacterium, and a metabolic pathway for this is proposed.     

Vanillic acid metabolism by selected soft-rot,brown-rot,and white-rot fungi

Metabolism of vanillic acid, a product of lignin degradation, has been studied in selected representatives of soft-rot, brown-rot and white-rot fungi. All of the brown-and white-rot species examined decarboxylated vanillate to methoxyhydroquinone oxidatively. Mycelium extracts of all these fungi, except Pleurotus ostreatus contained high levels of an NAD(P)H-dependent vanillate hydroxylase. P. ostreatus also released ¹⁴CO₂ from ¹⁴COOH-vanillate but by a different mechanism possibly involving phenoloxidases. Most of these fungi also contained a dioxygenase which catalysed the intra-diol cleavage of hydroxyquinol (1,2,4-trihydroxybenzene) to form maleylacetate. No 3-O-demethylase activity was detected, and data indicate that in some of the fungi examined cleavage of the aromatic ring occurs without prior removal of the methoxyl group. None of the soft-rot fungi tested contained vanillate hydroxylase or hydroxyquinol 1,2-dioxygenase, but very low levels of protocatechuate 3,4-dioxygenase were detected in mycelium extracts. Vanillate catabolism among members of this group occurs via a different route which may involve ring demethylation although no 3-O-demethylase activity was detected in this study. The enzyme NAD(P)H-quinone oxidoreductase was demonstrated to exist in all the studied groups of fungi.     

Wood-decay type and fungal guild dominance across a North American log transplant experiment

We incubated 196 large-diameter aspen (Populus tremuloides), birch (Betula papyrifera), and pine (Pinus taeda) logs on the FACE Wood Decomposition Experiment encompassing eight climatically-distinct forest sites in the United States. We sampled dead wood from these large-diameter logs after 2 to 6 y of decomposition and determined wood rot type as a continuous variable using the lignin loss/density loss ratio (L/D) and assessed wood-rotting fungal guilds using high-throughput amplicon sequencing (HTAS) of the ITS-2 marker. We found L/D values in line with a white rot dominance in all three tree species, with pine having lower L/D values than aspen and birch. Based on HTAS data, white rot fungi were the most abundant and diverse wood-rotting fungal guild, and soft rot fungi were more abundant and diverse than brown rot fungi in logs with low L/D values. For aspen and birch logs, decay type was related to the wood density at sampling. For the pine logs, decay type was associated with the balance between white and brown/soft rot fungi abundance and OTU richness. Our results demonstrate that decay type is governed by biotic and abiotic factors, which vary by tree species.