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Lignin degradation and humus formation in alluvial soils and sediments.

The contribution of lignin to the formation of humic compounds was examined in different environments of the terrestrial-aquatic interface in the Garonne River valley in southwestern France. Alluvial soils and submerged or nonsubmerged river and pond sediments containing alder, poplar, or willow [C-lignin]ligno-celluloses were incubated. After a 49-day incubation period, 10 to 15% of labeled lignins in alluvial soils was recovered as evolved CO(2). In nonsubmerged sediments, 10% of the applied activity was released as CO(2), and in submerged sediments, only 5% was released after 60 days of incubation. In the different alluvial soils and sediments, the bulk of residual activity (70 to 85%) remained in the two coarsest-grain fractions (2,000 to 100 and 100 to 50 mum). Only 2 to 6% of the residual activity of these two coarse fractions was recovered as humic and fulvic acids, except in the case of alder [C-lignin]lignocellulose, which had decomposed in a soil collected beneath alders. In this one 55% of the residual activity was extracted as humic substances from the 2,000- to 100-mum fraction. Humic and fulvic acids represented from 6 to 50% of the residual activity in the finest-grain fractions (50 to 20 and 20 to 0 mum). The highest percentages were obtained in soil collected beneath alders and in submerged pond sediment. The contribution of different groups of microorganisms, as well as nutrients and clay content, may influence humic-substance formation in such environments. Physical stability also may be an important factor for complex microbial activity involved in this process.

Journal Article↗

Physiological Aspects of Biosynthesis of Lignin Peroxidases by Phanerochaete chrysosporium.

Methods based on UV-visible diffuse reflectance spectroscopy were used to study the physiological aspects of lignin-peroxidase biosynthesis by Phanerochaete chrysosporium. Here we introduce the use of cytochrome aa(3) as an indicator of active fungal biomass and of its redox state to calculate the oxygen mass transport coefficient between the growth medium and the fungal cell interior. When lignin peroxidase biosynthesis was enhanced by the addition of Tween 80 or Tween 20 to the growth medium, a higher proportion of reduced cytochrome aa(3) and a higher oxygen diffusion barrier were observed compared with control cultures. In cultures supplemented with Tween 80 or Tween 20, a higher oxygen mass transport coefficient between the growth medium and the interior of the fungal cell was also found. The beginning of the lignin peroxidase activity in these cultures was found to coincide with a temporary cessation in the dry biomass increase and a reduction in the relative active-biomass concentration. During the lignin peroxidase activity, a decrease in the intracellular pH and an increase in the growth medium pH were determined in cultures supplemented with Tween 80.

Journal Article↗

Synthetic Lignin Mineralization by Ceriporiopsis subvermispora Is Inhibited by an Increase in the pH of the Cultures Resulting from Fungal Growth.

(sup14)C-synthetic lignin mineralization by the basidiomycete Ceriporiopsis subvermispora occurs at the highest rate (about 30% after 29 days) in liquid cultures containing 1% glucose and a growth-limiting amount (1 mM) of ammonium tartrate. The titers of manganese peroxidase (MnP) and laccase are lower in these cultures than in cultures containing 1% glucose and 10 mM ammonium tartrate, where the extent of lignin mineralization in the same period is only about 15%. The inverse correlation between enzyme activity and lignin mineralization is also observed when ammonium tartrate is replaced by ammonium chloride or Casamino Acids as the source of nitrogen. This phenomenon can be explained by a gradual increase in the pH of the medium that takes place only in the cultures with high nitrogen concentrations. Supporting this finding, when cultures with 1 mM ammonium tartrate were grown at different pHs, (sup14)CO(inf2) evolved more rapidly from those with pH values near the optimum for MnP activity. On the other hand, (sup14)CO(inf2) evolution from cultures containing 1% glucose supplemented with 1 mM ammonium tartrate plus 9 mM sodium tartrate was as low as that from cultures with a high ammonium tartrate concentration. Since the changes in the pH of these cultures were not as pronounced as those in cultures containing high nitrogen concentrations, tartrate itself may also be contributing to limit the extent of lignin mineralization. Considering that pH instability seems to constitute a common feature of fungal cultures, precautions must be taken to avoid underestimation of their ligninolytic efficiencies.

Journal Article↗

Manganese-Mediated Lignin Degradation by Pleurotus pulmonarius.

Pleurotus pulmonarius produced the strongest degradation of lignin during solid-state fermentation of [(sup14)C]lignin wheat straw with different fungi. A manganese-oxidizing peroxidase seemed to be involved in lignin attack, since the addition of Mn(sup2+) to the culture increased lignin mineralization by ca. 125%. This enzyme was purified and characterized from both solid-state fermentation and liquid cultures.

Journal Article↗

Evidence That Ceriporiopsis subvermispora Degrades Nonphenolic Lignin Structures by a One-Electron-Oxidation Mechanism.

The white-rot fungus Ceriporiopsis subvermispora is able to degrade nonphenolic lignin structures but appears to lack lignin peroxidase (LiP), which is generally thought to be responsible for these reactions. It is well established that LiP-producing fungi such as Phanerochaete chrysosporium degrade nonphenolic lignin via one-electron oxidation of its aromatic moieties, but little is known about ligninolytic mechanisms in apparent nonproducers of LiP such as C. subvermispora. To address this question, C. subvermispora and P. chrysosporium were grown on cellulose blocks and given two high-molecular-weight, polyethylene glycol-linked model compounds that represent the major nonphenolic arylglycerol-(beta)-aryl ether structure of lignin. The model compounds were designed so that their cleavage via one-electron oxidation would leave diagnostic fragments attached to the polyethylene glycol. One model compound was labeled with (sup13)C at C(inf(alpha)) of its propyl side chain and carried ring alkoxyl substituents that favor C(inf(alpha))-C(inf(beta)) cleavage after one-electron oxidation. The other model compound was labeled with (sup13)C at C(inf(beta)) of its propyl side chain and carried ring alkoxyl substituents that favor C(inf(beta))-O-aryl cleavage after one-electron oxidation. To assess fungal degradation of the models, the high-molecular-weight metabolites derived from them were recovered from the cultures and analyzed by (sup13)C nuclear magnetic resonance spectrometry. The results showed that both C. subvermispora and P. chrysosporium degraded the models by routes indicative of one-electron oxidation. Therefore, the ligninolytic mechanisms of these two fungi are similar. C. subvermispora might use a cryptic LiP to catalyze these C(inf(alpha))-C(inf(beta)) and C(inf(beta))-O-aryl cleavage reactions, but the data are also consistent with the involvement of some other one-electron oxidant.

Journal Article↗

A small-scale method for screening of lignin-degrading microorganisms.

A new method to facilitate rapid screening of lignin-degrading microorganisms was developed. Fungal strains are cultivated in tissue culture plates containing C-ring-labeled dehydrogenation polymerizate (DHP) (synthetic lignin). Evolved CO(2) is trapped in barium-saturated filter paper and is detected by exposing the paper to X-ray film. Analysis of the autoradiograms, carried out by density measurement with an image analysis program, allows for a semiquantitative estimation of the amount of CO(2) evolved. The method is especially useful for screening for new, powerful lignin-degrading strains in both man-made and natural environments. It eliminates the need for special equipment for their cultivation and trapping of CO(2) as well as laborious sample analysis. The method has in this study been used to test three novel fungal isolates and a laccaseless mutant of the basidiomycete Pycnoporus cinnabarinus. Their ligninolytic capacities were compared with those of the potent lignin degrader Ceriporiopsis subvermispora.

Journal Article↗

Production of manganese peroxidase and organic acids and mineralization of 14C-labelled lignin (14C-DHP) during solid-state fermentation of wheat straw with the white rot fungus nematoloma frowardii

The basidiomycetous fungus Nematoloma frowardii produced manganese peroxidase (MnP) as the predominant ligninolytic enzyme during solid-state fermentation (SSF) of wheat straw. The purified enzyme had a molecular mass of 50 kDa and an isoelectric point of 3.2. In addition to MnP, low levels of laccase and lignin peroxidase were detected. Synthetic 14C-ring-labelled lignin (14C-DHP) was efficiently degraded during SSF. Approximately 75% of the initial radioactivity was released as 14CO2, while only 6% was associated with the residual straw material, including the well-developed fungal biomass. On the basis of this finding we concluded that at least partial extracellular mineralization of lignin may have occurred. This conclusion was supported by the fact that we detected high levels of organic acids in the fermented straw (the maximum concentrations in the water phases of the straw cultures were 45 mM malate, 3.5 mM fumarate, and 10 mM oxalate), which rendered MnP effective and therefore made partial direct mineralization of lignin possible. Experiments performed in a cell-free system, which simulated the conditions in the straw cultures, revealed that MnP in fact converted part of the 14C-DHP to 14CO2 (which accounted for up to 8% of the initial radioactivity added) and 14C-labelled water-soluble products (which accounted for 43% of the initial radioactivity) in the presence of natural levels of organic acids (30 mM malate, 5 mM fumarate).

Journal Article↗

The nature of lignin from steam explosion/ enzymatic hydrolysis of softwood: structural features and possible uses: scientific note.

Effective utilization of the lignin by-product is a prerequisite to the commercial viability of ethanol production from softwood wastes using a steam explosion (SE)/enzymatic hydrolysis (EH)/fermentation process. Changes in the chemical composition of Douglas fir wood on SO2-catalyzed SE followed by EH were assessed using conventional analytical methods and new halogen-probe techniques. A significant solubilization of hemicelluloses was observed in the SE stage, the severity of which affected subsequent fermentation of cellulose and sorption of enzymes. SE of softwood resulted in dramatic changes in the chemical structure of lignin in the residual material involving chemical reactions via the benzyl cation. This leads to a more condensed lignin with partly blocked alpha-reaction centres. Possible uses for this lignin are discussed.

Journal Article↗

[Effectiveness of lignin as a protective factor in exposure to polychlorinated biphenyls].

The effect of lignin on some biochemical and morphological parameters was studied in rats after short-term treatment with the industrial mixture of polychlorinated biphenyls (PCB) sovol at a daily dose of 5 mg/kg, 5 times a week for a fortnight. Dietary supplementation of 5% lignin as the drug polyphenane did not protect the induction of the rat hepatic P-450 cytochrome monooxygenase system, which is typical of the action of PCB, but slightly modified the inducing effect of sovol. In the polyphepane-fed animals, there was a decrease in impairments of the liver structure and its more rapid normalization than in control rats. The lower hepatotoxic effect of the PCB mixture when lignin was added to the ration was likely to be associated with the action of lignin as a nonspecific polyvalent enterosorbent. It is concluded that polyphepane is a promising protective agent on exposure to PCB.

Animals↗

Fungal laccase: properties and activity on lignin.

The sources of ligninocellulose that occur in various forms in nature are so vast that they can only be compared to those of water. The results of several, more recent experiments showed that laccase probably possesses the big ability for "lignin-barrier" breakdown of ligninocellulose. The degradation of this compound is currently understood as an enzymatic process mediated by small molecules, therefore, this review will focus on the role of these mediators and radicals working in concert with enzymes. The fungi having a versatile machinery of enzymes are able to attack directly the "lignin-barrier" or can use a multienzyme system including "feed-back" type enzymes allowing for simultaneous transformation of lignin and carbohydrate compounds.

Basidiomycota↗

BSA treatment to enhance enzymatic hydrolysis of cellulose in lignin containing substrates.

Cellulase and bovine serum albumin (BSA) were added to Avicel cellulose and solids containing 56% cellulose and 28% lignin from dilute sulfuric acid pretreatment of corn stover. Little BSA was adsorbed on Avicel cellulose, while pretreated corn stover solids adsorbed considerable amounts of this protein. On the other hand, cellulase was highly adsorbed on both substrates. Adding a 1% concentration of BSA to dilute acid pretreated corn stover prior to enzyme addition at 15 FPU/g cellulose enhanced filter paper activity in solution by about a factor of 2 and beta-glucosidase activity in solution by about a factor of 14. Overall, these results suggested that BSA treatment reduced adsorption of cellulase and particularly beta-glucosidase on lignin. Of particular note, BSA treatment of pretreated corn stover solids prior to enzymatic hydrolysis increased 72 h glucose yields from about 82% to about 92% at a cellulase loading of 15 FPU/g cellulose or achieved about the same yield at a loading of 7.5 FPU/g cellulose. Similar improvements were also observed for enzymatic hydrolysis of ammonia fiber explosion (AFEX) pretreated corn stover and Douglas fir treated by SO(2) steam explosion and for simultaneous saccharification and fermentation (SSF) of BSA pretreated corn stover. In addition, BSA treatment prior to hydrolysis reduced the need for beta-glucosidase supplementation of SSF. The results are consistent with non-specific competitive, irreversible adsorption of BSA on lignin and identify promising strategies to reduce enzyme requirements for cellulose hydrolysis.

Adsorption↗

On the mechanism of the laccase-mediator system in the oxidation of lignin.

In an effort to elucidate the role of phenolic and non-phenolic lignin subunits in a laccase mediator (LM) system, vanillyl alcohol was oxidized with laccase in the presence and absence of the mediator 1-hydroxybenzotriazol (HBT). Furthermore, the role of phenolic, aliphatic hydroxyl, and carboxylic acid moieties in lignin degradation was elucidated by selectively blocking them. The modified samples were then subjected to laccase and laccase-HBT treatments. On the basis of this data it was possible to establish the role of this mediator. HBT mediates the oxidation of lignin by inducing side-chain oxidation and oxygen-addition products rather than oxidative coupling reactions.

Acetylation↗

Oxidative coupling during lignin polymerization is determined by unpaired electron delocalization within parent phenylpropanoid radicals.

The high degree of selectivity observed in the incorporation of phenylpropanoids into lignin may be a consequence of the influence exerted by methoxyl substituents on the ambident radicals generated during biosynthesis. Since unpaired electron distribution may be regarded as an important factor in determining positional selectivity during oxidative coupling, electron spin resonance spectroscopy and Austin Model 1 molecular computation were used to study the effects of methoxyl substitution on unpaired electron distribution in lignin precursor radicals. The data obtained were used to predict the effect of substitution on coupling and were compared with the linkage types observed in complementary dehydrogenation polymerization studies employing each of the lignin precursors. We report that methoxyl substitution increases unpaired electron density on the phenolic oxygen of the precursor phenylpropanoid radicals and that this subsequently determines the nature of the bond formation during polymerization.

Computer Simulation↗

Electrophoretic separation, characterization, and quantification of biologically active lignin-derived macromolecules.

Degraded macromolecular lignin, which was isolated from the effluents of commercial pulp processing and known to inhibit early development in marine organisms, was separated and characterized using several polyacrylamide gel electrophoresis (PAGE) techniques. This lignin-derived macromolecule (LDM), when subjected to native PAGE and stained with alcian blue, appeared as a single band. On sodium dodecyl sulfate (SDS)-PAGE, LDM appeared to consist of two subcomponents with apparent molecular weights of 11 and < 1 kDa. When subjected to isoelectrofocusing--PAGE of pH 3-9, LDM consisted of two major bands in the basic region of the gel, with less distinct banding in the more acidic region. Two-dimensional PAGE of LDM indicated that the higher molecular weight subcomponent corresponded to the more basic constituents, while the lower molecular weight subcomponent corresponded to acidic constituents. When the two subcomponents of LDM were isolated from SDS gels by electroelution and assessed for their effects on successful fertilization and early development, the higher molecular weight subcomponent possessed most of the inhibitory activity. This is the first report of the application of a variety of electrophoretic techniques to both structurally and biologically characterize lignin-derived macromolecules.

Animals↗

Secretion of TNF-alpha from macrophages following induction with a lignin derivative.

Macrophages derived from rat bone marrow were treated with macrophage colony stimulating factor (M-CSF) to obtain a sufficient number of cells for the tumor necrosis factor (TNF-alpha) assay. The present study has been designed to investigate whether the production of TNF-alpha, which induces multinucleated giant cell formation, is regulated by polyanions such as lignin derivatives. ELISA for TNF-alpha showed that the polyanion induced TNF-alpha production by macrophages. The secretion of TNF-alpha from the cells reached a maximum at 3-6 h, and then showed a slight decline. Northern blotting of TNF-alpha mRNA showed that the amount of TNF-alpha reached a maximum within 1 h of macrophage culture in the presence of a lignin derivative. On the other hand, TNF-alpha mRNA was undetectable in the control cells. It was concluded that stimuli such as that provided by lignin derivatives increases the amount of TNF-alpha mRNA, which is then followed by translation of TNF-alpha.

Animals↗

The crystal structure of lignin peroxidase at 1.70 A resolution reveals a hydroxy group on the cbeta of tryptophan 171: a novel radical site formed during the redox cycle.

The crystal structure of lignin peroxidase (LiP) from the white rot fungus Phanerochaete chrysosporium was refined to an R-factor of 16.2 % utilizing synchrotron data in the resolution range from 10 to 1.7 A. The final model comprises all 343 amino acid residues, 370 water molecules, the heme, four carbohydrates, and two calcium ions. Lignin peroxidase shows the typical peroxidase fold and the heme has a close environment as found in other peroxidases. During refinement of the LiP model an unprecedented modification of an amino acid was recognized. The surface residue tryptophan 171 in LiP is stereospecifically hydroxylated at the Cbeta atom due to an autocatalytic process. We propose that during the catalytic cycle of LiP a transient radical at Trp171 occurs that is different from those previously assumed for this type of peroxidase. Recently, the existence of a second substrate-binding site centered at Trp171 has been reported, by us which is different from the "classical heme edge" site found in other peroxidases. Here, we report evidence for a radical formation at Trp171 using spin trapping, which supports the concept of Trp171 being a redox active amino acid and being involved in the oxidation of veratryl alcohol. On the basis of our current model, an electron pathway from Trp171 to the heme is envisaged, relevant for the oxidation of veratryl alcohol and possibly lignin. Beside the opening leading to the heme edge, which can accommodate small aromatic substrate molecules, a smaller channel giving access to the distal heme pocket was identified that is large enough for molecules such as hydrogen peroxide. Furthermore, it was found that in LiP the bond between the heme iron and the Nepsilon2 atom of the proximal histidine residue is significantly longer than in cytochrome c peroxidase (CcP). The weaker Fe-N bond in LiP renders the heme more electron deficient and destabilizes high oxidation states, which could explain the higher redox potential of LiP as compared to CcP.

Benzyl Alcohols↗

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.

Carbon Dioxide↗

Decolorization of synthetic textile dyes by lignin peroxidase of Phanerochaete chrysosporium.

Neem hull waste (containing a high amount of lignin and other phenolic compounds) was used for lignin peroxidase production by Phanerochaete chrysosporum under solid-state fermentation conditions. Maximum decolorization achieved by partially purified lignin peroxidase was 80% for Porocion Brilliant Blue HGR, 83 for Ranocid Fast Blue, 70 for Acid Red 119 and 61 for Navidol Fast Black MSRL. The effects of different concentrations of veratryl alcohol, hydrogen peroxide, enzyme and dye on the efficiency of decolorization have been investigated. Maximum decolorization efficiency was observed at 0.2 and 0.4 mmol/L hydrogen peroxide, 2.5 mmol/L veratryl alcohol and pH 5.0 after a 1-h reaction, using 50 ppm of dyes and 9.96 mkat/L of enzyme.

Azadirachta↗