Study of lignin biotransformation by Aspergillus fumigatus and white-rot fungi using (14)C-labeled and unlabeled kraft lignins

The biodegradation of lignin by fungi was studied in shake flasks using (14)C-labeled kraft lignin and in a deep-tank fermentor using unlabeled kraft lignin. Among the fungi screened, A. fumigatus-isolated in our laboratories-was most potent in lignin biotransformation. Dialysis-type fermentation, designed to study possible accumulation of low MW lignin-derived products, showed no such accumulation. Recalcitrant carbohydrates like mi-crocrystalline cellulose supported higher lignolytic activity than easily metabolized carbohydrates like cellobiose. An assay developed to distinguish between CO(2) evolved from lignin and carbohydrate substrates demonstrated no stoichiometric correlation between the metabolism of the two cosubstrates. The submerged fermentations with unlabeled lignin are difficult to monitor since chemical assays do not give accurate and true results. Lignolytic efficiencies that allowed monitoring of such fermentations were defined. Degraded lignins were analyzed for structural modifications. A. fumigatus was clearly superior to C. versicolor in all aspects of lignin degradation; A. fumigatus brought about substantial demethoxylation and dehydroxylation, whereas C. versicolor degraded lignins closely resembled undegraded kraft lignin. There was a good agreement among the different indices of lignin degradation, namely, (14)CO evolution, OCH(3) loss, OH loss, and monomer and dimer yield after permanganate oxidation.     

Degradation of lignin and lignin derivatives by Acinetobacter sp.

An Acinetobacter sp. utilized 2-methoxy-4-formylphenoxyacetic acid, dehydrodivanillyl alcohol, dehydrodiisoeugenol and conidendrin as sole carbon source. It also degraded ¹⁴C-labelled DHP lignin and teakwood lignin. Vanillic acid, protocatechuic acid and catechol were separated from 2-methoxy-4-formylphenoxyacetic acid grown cultures. Both protocatechuic acid and catechol were formed from dehydrodivanillyl alcohol, dehydrodiisoeugenol and conidendrin. On the dimeric lignin model substances this Acinetobacter sp. produced protocatechuate 3,4-dioxygenase and catechol 1,2-dioxygenase.     

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.     

14C-[lignin]-lignocellulose biodegradation by bacteria isolated from polluted soil

Four bacterial species [Branhamella catarrhalis (gram -ve), Brochothrix species (gram -ve), Micrococcus luteus (gram +ve) and Bacillus firmus (gram +ve)], isolated from the soil polluted with cane sugar factory effluents, were found capable of growing on solid media supplemented with indulin AT (a polymeric industrial lignin) as sole C source. All the four species could metabolize cinnamic acid (a non-hydroxylated phenylpropanoid) as sole carbon source with significant suppression on addition of readily metabolizable carbon source (glucose). However, Br. catarrhalis and Brochothrix sp. were capable of metabolizing ferulic acid, but could not do so on addition of glucose. Of the four species, Br. catarrhalis could evolve significant amount of 14CO2 from U-14C (lignin)-lignocellulose prepared from rice stalks (ca. 10% of the added radioactivity in 3 weeks), in addition to solubilization of another 11.7% radioactivity in culture filtrate. The other three species could not significantly evolve 14CO2, though a significant fraction of added 14C-lignin (6.1 to 11.2%) could be solubilized into culture filtrate, suggesting lack of ring-cleavage or other CO2 evolving mechanisms in these species.     

Enzymatic co-polymerization of lignin with low-molecular mass compounds

The oxidoreductive enzyme laccase (E.C.1.10.3.2.) isolated from a culture medium of white-rot fungus Trametes versicolor transformed lignin preparations solubilized in a dioxane-H₂O (7:3) mixture. The obvious net result of lignin transformation was an increase in molecular mass. A superoxide radical was found in the reaction mixture during lignin incubation with laccase. It appeared that a change in the reaction medium or in the lignin molecule instigated by laccase could lead to polymerization after the lignin molecules had crossed a dialysis membrane and were separated from the enzyme. Two possible mechanisms are suggested, either diffusion of an activated oxygen species or diffusion of primed lignin molecules. Laccase was able to co-polymerize lignin with low-molecular-mass compounds of different origins, particularly with aromatics containing either carboxyl or isocyanate groups, as well as acrylamide — an aliphatic monomer containing a vinyl group. Correspondence to: O. Milstein     

Intermediates and products of synthetic lignin (dehydrogenative polymerizate) degradation by Phlebia tremellosa.

Agitated, nitrogen-limited cultures of Phlebia tremellosa caused substantial changes in the distribution of 14C-labelled synthetic lignin (dehydrogenative polymerizate [DHP]) between water-soluble, dioxane-soluble, alkali-soluble, and insoluble fractions before much lignin carbon was metabolized to CO2. First, the insoluble form increased at the expense of the dioxane-soluble form. Later, the amounts of alkali-soluble and water-soluble 14C increased, and release of 14CO2 began. The molecular weight distribution of the dioxane-soluble lignin remained constant during degradation, but that of the water-soluble fraction changed to higher molecular weights. Culture agitation accelerated the attachment of suspended DHP to the mycelia and stimulated production of water-soluble 14C and 14CO2. The nonionic detergent Tween 80 also hastened release of 14CO2 and increased the early conversion of dioxane-soluble DHP to the alkali-soluble and insoluble forms. Oxidative polymerization is suggested as the first step in degradation of DHP by P. tremellosa.     

Intermediates and products of synthetic lignin (dehydrogenative polymerizate) degradation by Phlebia tremellosa.

Agitated, nitrogen-limited cultures of Phlebia tremellosa caused substantial changes in the distribution of 14C-labelled synthetic lignin (dehydrogenative polymerizate [DHP]) between water-soluble, dioxane-soluble, alkali-soluble, and insoluble fractions before much lignin carbon was metabolized to CO2. First, the insoluble form increased at the expense of the dioxane-soluble form. Later, the amounts of alkali-soluble and water-soluble 14C increased, and release of 14CO2 began. The molecular weight distribution of the dioxane-soluble lignin remained constant during degradation, but that of the water-soluble fraction changed to higher molecular weights. Culture agitation accelerated the attachment of suspended DHP to the mycelia and stimulated production of water-soluble 14C and 14CO2. The nonionic detergent Tween 80 also hastened release of 14CO2 and increased the early conversion of dioxane-soluble DHP to the alkali-soluble and insoluble forms. Oxidative polymerization is suggested as the first step in degradation of DHP by P. tremellosa.     

Degradation of labelled lignins and veratrylglycerol-beta-guaiacyl ether by Acinetobacter sp

Acinetobacter sp. evolved 14CO2 from 14C-(ring)DHP lignin and 14C-teakwood lignin. Veratrylglycerol-beta-guaiacyl ether, a lignin model compound with beta-o-4 linkage was cleaved by Acinetobacter sp. Veratrylglycerol-beta-guaiacyl ether into 2(o-methoxyphenoxy) ethanol and veratrylalcohol 2(o-methoxyphenoxy) ethanol was degraded to guaiacol and then to catechol whereas veratrylalcohol was converted to veratraldehyde, veratric acid, vanillic acid, protocatechuic acid and catechol. Both catechol 1,2-dioxygenase and protocatechuate 3,4-dioxygenase were detected in veratrylglycerol-beta-guaiacyl ether grown cultures.     

The involvement of hydroxyl radical derived from hydrogen peroxide in lignin degradation by the white rot fungus Phanerochaete chrysosporium

The possible involvement of hydrogen peroxide (H2O2)-derived hydroxyl radical (.OH) in lignin degradation ([14C]lignin leads to 14CO2) by Phanerochaete chrysosporium was investigated. When P. chrysosporium was grown in low nitrogen medium (2.4 mM N), an increase in the specific activity for H2O2 production in cell extracts was observed to coincide with the appearance of ligninolytic activity and both activities appeared after the culture entered stationary phase. The production of .OH in ligninolytic cultures of P. chrysosporium was demonstrated by alpha-keto-gamma-methiolbutyric acid-dependent formation of ethylene. Hydrogen peroxide-dependent .OH formation was also shown in cell extracts of ligninolytic cultures. The radical species was demonstrated to be .OH by the .OH-dependent hydroxylation of p-hydroxybenzoic acid to form protocatechuic acid and by using 5,5-dimethyl-1-pyrroline-N-oxide and detecting the production of the nitroxide radical of 5,5-dimethyl-1-pyrroline-N-oxide by EPR. These reactions were inhibited by .OH-scavenging agents and were stimulated when azide was added to inhibit endogenous catalase. Lignin degradation by P. chrysosporium was markedly suppressed in the presence of the .OH-scavenging agents mannitol, benzoate, and the nonspecific radical scavenging agent butylated hydroxytoluene. The above results indicate that .OH derived from H2O2 is involved in lignin biodegradation by P. chrysosporium.     

Lack of lignin degradation by glucose oxidase-negative mutants of Phanerochaete chrysosporium

Glucose oxidase-negative (gox-) mutants of Phanerochaete chrysosporium were isolated after exposing conidia to UV irradiation. The gox- mutants exhibited little or no ability to degrade lignin (2-[14C]-synthetic lignin to 14CO2); however, they retained other secondary metabolic features such as the ability to conidiate and produce veratryl alcohol, suggesting that they are not pleiotropic for secondary metabolism. Lignin degradation activity was restored in gox+ revertants. These results, in support of earlier evidence, indicate that glucose oxidase activity plays an important role in lignin degradation by P. chrysosporium.     

Microbial degradation of lignocellulose: the lignin component

A new procedure was developed for the study of lignin biodegradation by pure or mixed cultures of microorganisms. Natural lignocelluloses were prepared containing C in primarily their lignin components by feeding plants l-[U-C]phenylalanine through their cut stems. Lignin degradation was observed in numerous soils by monitoring evolution of CO(2) from [C]lignin-labeled oak (Quercus albus), maple (Acer rubrum), and cattail (Typha latifola). An organism (Thermonospora fusca ATCC 27730) that is known to degrade cellulose but not lignin was shown to grow on lignocellulose in the presence of [C]lignocelluloses without evolution of CO(2). A known lignin degrader (a white-rot fungus, Polyporus versicolor) was shown to readily evolve CO(2) from damp C-labeled cattail and C-labeled maple.     

Demethoxylation of [O14CH3]-labelled lignin model compounds by the brown-rot fungi Gloeophyllum trabeum and Poria (Postia) placenta

Demethoxylation reactions in the cultures of the brown-rot fungi Gloeophyllum trabeum and Poria placenta were studied by determining the evolution of (14)CO(2) from a non-phenolic lignin model, beta-O-4 dimer, [O(14)CH(3)]-labelled at position 4 in the A ring (model I), and from [O(14)CH(3)]-labelled vanillic acid (model II). The fungi were grown under oxygen or air atmosphere on an agar medium with or without spruce sapwood blocks. The dimeric model (I) was impregnated onto agar or wood block in cultures to clarify the possible effect of wood as growth substrate. In the case of vanillic acid (model II), birch wood was used. The effect of supplemented nutrient nitrogen (2 mM N) and glucose (0.1 or 1.0% w/v) on demethoxylation was also studied. G. trabeum enhanced the production of (14)CO(2) from the dimer in the presence of spruce wood blocks. It released (14)CO(2) from the methoxyl groups giving 30-60% of the applied activity in 8 weeks. P. placenta produced almost 30% (14)CO(2 )from vanillic acid (model II) in 9 weeks under oxygen, but from the methoxyl group of the dimer only 3% of (14)CO(2) was evolved in 4 weeks. The biomasses determined as ergosterol assay showed variation from 14 to 226 microg g(-1) dry weight of agar, and 2 to 9 microg g(-1 )of wood, but they did not correlate with the production of (14)CO(2). The results indicate that these brown-rot fungi possess different mechanisms for demethoxylation.     

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     

Inhibition of the lignin peroxidase of Phanerochaete chrysosporium by hydroxylamino-dinitrotoluene, an early intermediate in the degradation of 2,4,6-trinitrotoluene.

The ability of the white rot fungus Phanerochaete chrysosporium to mineralize 2,4,6-trinitrotoluene (TNT) was studied in the concentration range of 0.36 to 20.36 mg/liter. The initial rate of 14CO2 formation was 30% in 4 days at 0.36 mg of [14C]TNT per liter and decreased to 5% in 4 days at 20.36 mg of [14C]TNT per liter. Such a pronounced inhibition was not observed when a mixture of [14C]2-amino-4,6-dinitrotoluene and [14C]4-amino-2,6-dinitrotoluene was used as a substrate. 2-Hydroxylamino-4,6-dinitrotoluene and its isomer 4-hydroxylamino-2,6-dinitrotoluene were identified as the first detectable degradation products of TNT. Their transient accumulation correlated with the inhibition of TNT degradation and of the veratryl alcohol oxidase activity of lignin peroxidase. With purified lignin peroxidase H8, it could be shown that the two isomers of hydroxylamino-dinitrotoluene were oxidized by lignin peroxidase. The corresponding nitroso-dinitrotoluenes apparently were formed, as indicated by the formation of azoxy-tetranitrotoluenes.     

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     

Biodegradation of radiolabelled synthetic lignin (14C-DHP) and mechanical pulp in a compost environment

Mineralization of radioactive synthetic lignin (14C-DHP) was studied in a compost environment at 35, 50 and 58 degrees C. Compost samples were successively extracted with water, dioxane and alkali, and the molecular weight distribution of some extracts was determined by gel permeation chromatography (GPC). Biodegradation of lignin-containing spruce groundwood (SGW) and pine sawdust was concurrently determined in controlled composting tests by measuring evolved CO2. The temperatures were the same as in the 14C-DHP mineralization experiment and bleached kraft paper, with a lignin content of 0.2%, was used as a reference. The mineralization of 14C-DHP was relatively high (23-24%) at 35 degrees C and 50 degrees C, although the mixed population of compost obviously lacks the most effective lignin degraders. At 58 degrees C the mineralization of 14C-DHP, as well as the biodegradation of SGW and sawdust, was very low, indicating that the lignin-degrading organisms of compost were inactivated at this temperature. SGW was poorly biodegradable (<40%) in controlled composting tests compared with kraft paper (77-86%) at all temperatures, which means that lignin inhibits the degradation of carbohydrates. During the incubation, water-soluble degradation products, mainly monomers and dimers, and the original 14C-DHP were either mineralized or bound to humic substances. A substantial fraction of 14C-DHP was incorporated into humin or other insolubles.     

Biochemistry of the oxidation of lignin by Phanerochaete chrysosporium

The objective of this research was to identify the biochemical agents responsible for the oxidative degradation of lignin by the white-rot fungus Phanerochaete chrysosporium. We examined the hypothesis that activated oxygen species are involved, and we also sought the agent in ligninolytic cultures responsible for a specific oxidative degradative reaction in substructure model compounds. Results of studies of the production of activated oxygen species by cultures, of the effect of their removal on ligninolytic activity, and of their action on substructure model compounds support a role for hydrogen peroxide (H(2)O(2)) and possibly superoxide (O(2)(*)(-)) in lignin degradation. Involvement of hydroxyl radical (*OH) or singlet oxygen (1O(2)) is not supported by our data. The actual biochemical agent responsible for one important oxidative C-C bond cleavage reaction in non-phenolic lignin substructure model compounds, and in lignin itself, was found to be an enzyme. The enzyme is extracellular, has a molecular weight of 42,000 daltons, is azide-sensitive, and requires H(2)O(2) for activity.     

Enhanced degradation of lignin by the strain fusion of Pseudomonas putida and Gordonia sp

The protoplast fusion technique was applied to construct a more efficient engineering microbial strain to degrade lignin by fusing two strains, Pseudomonas putida and Gordonia sp. At an initial lignin concentration of 900?mg/L, COD, BOD, TOC removal efficiencies increased from 69–76%, 69–72%, and 70–72% by the parent stains to 83%, 83%, and 83% of the fused strain, respectively. IR and HPLC analyses of the treated solution suggested that the fused strains were more capable of breaking the C_α–C_β bonds of the benzene ring in lignin compared to its parent strains, yielding syringyls as the main product. GC–MS analysis was used to identify the release of three-types of lower molecular intermediates: ring-opening, monomer, and dipolymer products. The phenolic hydroxyl group in lignin was oxidized to carbonyls, followed by further degradation to acids and esters. The carboxyl group on the ether linkage that maintains the macromolecular structure of lignin was oxidized to acyls, which further led to depolymerization and the opening of benzene ring.     

Factors affecting lignin degradation in lignocellulose by Phanerochaete chrysosporium

Cultural conditions affecting lignin degradation by Phanerochaete chrysosporium in various lignocellulosic materials were studied in comparison to an isolated lignin preparation. With shallow mycelial cultures, the degradation of lignin in wood proceeded more slowly in a 100% O₂-atmosphere than in an air atmosphere, indicating that pure oxygen was toxic to the fungus. The organism was able to degrade lignin efficiently even under 30% CO₂ and 10% O₂ concentrations. Evolution of ¹⁴CO₂ from labelled lignocellulosic materials was shown not to be representative of total lignin degradation. Addition of glucose to the culture did not affect lignin degradation measured by ¹⁴CO₂ evolution, whereas lignin degradation measured by Klason lignin method stopped completely (poplar) or slowed considerably (straw). Due to partial depolymerization of lignin to soluble products, measuring only the evolution of ¹⁴CO₂ results in an underestimation of the total amount of lignin bioaltered. The soluble products from all of the tested lignocellulosic materials and from the isolated lignin had an average molecular weight of about 1,000 and the products could be further fractionated by ion exchange chromatography. The relative amount of these products could be varied from 15 to 45% from the original lignin.