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.     

Role of veratryl alcohol in lignin degradation by Phanerochaete chrysosporium

Several aromatic compounds increased initial lignin degradation rates in cultures of Phanerochaete chrysosporium. This activation was connected to increased H₂O₂ production and glucose oxidation rates. Veratryl alcohol, a natural secondary metabolite of P. chrysosporium, also activated the lignin-degrading system. In the presence of added veratryl alcohol the ligninolytic system appeared 6–8 h earlier than in reference cultures. This effect was only seen when lignin was added after the primary growth was completed because lignin itself also caused earlier appearance of the degradative system. In cultures which received no added lignin or veratryl alcohol the ligninolytic activity only appeared once the alcohol started to accumulate. The degradation patterns of veratryl alcohol and lignin were similar. The activity levels of lignin degradation and glucose oxidation could be regulated by veratryl alcohol concentration. It is suggested that either veratryl alcohol itself or a metabolite derived from it is actually responsible for the low levels of ligninolytic activity in glucose grown cultures.     

Factors Involved in the Regulation of a Ligninase Activity in Phanerochaete chrysosporium

The regulation of an H₂O₂-dependent ligninolytic activity was examined in the wood decay fungus Phanerochaete chrysosporium. The ligninase appears in cultures upon limitation for nitrogen or carbohydrate and is suppressed by excess nutrients, by cycloheximide, or by culture agitation. Activity is increased by idiophasic exposure of cultures to 100% O₂. Elevated levels of ligninase and, in some cases, of extracellular H₂O₂ production are detected after brief incubation of cultures with lignins or lignin substructure models, with the secondary metabolite veratryl alcohol, or with other related compounds. It is concluded that lignin degradation (lignin → CO₂) by this organism is regulated in part at the level of the ligninase, which is apparently inducible by its substrates or their degradation products.     

Relationship Between Lignin Degradation and Production of Reduced Oxygen Species by Phanerochaete chrysosporium

The relationship between the production of reduced oxygen species, hydrogen peroxide (H₂O₂), superoxide (O₂), and hydroxyl radical (·OH), and the oxidation of synthetic lignin to CO₂ was studied in whole cultures of the white-rot fungus Phanerochaete chrysosporium Burds. The kinetics of the synthesis of H₂O₂ coincided with the appearance of the ligninolytic system; also, H₂O₂ production was markedly enhanced by growth under 100% O₂, mimicking the increase in ligninolytic activity characteristic of cultures grown under elevated oxygen tension. Lignin degradation by whole cultures was inhibited by a specific H₂O₂ scavenger, catalase, implying a role for H₂O₂ in the degradative process. Superoxide dismutase also inhibited lignin degradation, suggesting that O₂ is also involved in the breakdown of lignin. The production of ·OH was assayed in whole cultures by a benzoate decarboxylation assay. Neither the kinetics of ·OH synthesis nor the final activity of its producing system obtained under 100% O₂ correlated with that of the lignin-degrading system. However, lignin degradation was inhibited by compounds which react with ·OH. It is concluded that H₂O₂, and perhaps O₂, are involved in lignin degradation; because these species are relatively unreactive per se, their role must be indirect. Conclusions about a role for ·OH in ligninolysis could not be reached.     

Involvement of singlet oxygen in the fungal degradation of lignin

The possible involvement of singlet oxygen (¹O₂) in the degradation of lignin by $\text{Phanerochaete}\text{chrysosporium}$ was examined. Ligninolytic cultures and photochemically generated ¹O₂ gave the same oxidation products from the lignin substructure model compound 1,2-bis(3-methoxy-4-alkoxyphenyl)propan-1,3-diol. Fluorescence and near UV absorbance of the specific ¹O₂ trapping agent anthracene-9,10-bisethanesulfonic acid (AES) disappeared in ligninolytic cultures, indicating that ¹O₂ was produced. AES strongly inhibited oxidation of ¹⁴C-lignin, but not ¹⁴C-glucose, to ¹⁴CO₂ in cultures, and also strongly suppressed oxidation of the model compound. These results indicate the ¹O₂ plays an integral role in lignin biodegradation.     

Recent advances in studies of the mechanisms of microbial degradation of lignins

Major advances in our understanding of the biochemical and enzymological mechanisms of lignin biodegradation have been made in the past three years. Research has principally involved two ligninolytic microorganisms, the white rot fungus Phanerochaete chrysosporium and the actinomycete Streptomyces viridosporus. Research has been centred on attempts to identify the microbial catalysts that mediate lignin decay in these two microbes. Emphasis has been on studies concerned with isolating specific lignin catabolic enzymes and/or reduced forms of oxygen involved in attacking the lignin polymer. The possibility that lignin degradation might be non-enzymatic and mediated by extracellular reduced oxygen species such as hydrogen peroxide (H₂O₂), superoxide (O₂∪c-|_.), hydroxyl radical (·OH) or singlet oxygen (¹O₂) has been investigated with both microorganisms. Using methods which have not always been unequivocal, the question of involvement of reduced oxygen species in lignin degradation by P. chrysosporium has been examined exhaustively. Evidence for the involvement of H₂O₂ is conclusive. However, there is little evidence to support the involvement of other extracellular reduced oxygen species, including ·OH, directly in the process of lignin degradation. Scavenger studies have been inconclusive because of questions of their specificity. If activated oxygen species are involved, the activated oxygen is probably held within the active site of an enzyme molecule. With S. viridosporus, scavenger studies also strongly indicate that extracellular reduced oxygen species are not involved in lignin degradation since scavengers generally do not significantly affect the ligninolytic system. The involvement of specific enzymes in lignin degradation by both P. chrysosporium and S. viridosporus has now been confirmed. With P. chrysosporium, ligninolytic enzymes recently discovered include extracellular non-specific peroxidases and oxygenases. They show numerous activities including dehydrogenative, peroxidatic, oxygenative and C_α?C_β cleavages of lignin side chains. At least one P. chrysosporium enzyme, a unique H₂O₂-requiring oxygenase, has been purified to homogeneity. Evidence has been presented to show that S. viridosporus also produces a ligninolytic enzyme complex involved in demethylation of lignin's aromatic rings and in the oxidation of lignin side chains and cleavage of β-tether linkages within the polymer. The combined activites of these enzymes generate water-soluble polymeric modified lignin fragments, which are then slowly degraded further by S. viridosporus. The β-ether cleaving enzyme complex is probably membrane associated, but it is not extracellular. These first isolations of ligninolytic enzymes have changed the course of basic research on lignin biodegradation. New research priorities are already emerging and include enzyme purifications, kinetic studies, enzyme reaction mechanism studies and screenings for more enzymes. In addition, genetic studies are being carried out with both P. chrysosporium and S. viridosporus. Genetic manipulations include not only classical mutagenesis techniques, but also recombinant DNA techniques such as protoplast fusion. This latter technique has already been used to generate overproducers of the ligninolytic enzyme complex in S. viridosporus and it has been successfully used to recombine mutant strains of P. chrysosporium.     

Incorporation of 18O2 and absence of stereospecificity in primary product formation during fungal metabolism of a lignin model compound

The metabolism of a lignin substructure model compound, 1,2-bis(3-methoxy-4-ethoxyphenyl)propane-1,3-diol (Ia) in ligninolytic cultures of Phanerochaete chrysosporium was studied to help elucidate the biochemical mechanism of lignin degradation. The primary reaction was cleavage of the model compound between C₁ and C₂ of the propane moiety to produce 1-(3-methoxy-4-ethoxyphenyl)ethane-1,2-diol and a C₆-C₁ product (probably 3-methoxy-4-ethoxybenzaldehyde). Other identified products arose secondarily; all were further metabolized. Even though the model compound was a mixture of four stereoisomers, no stereoselectivity was observed in its metabolism. In cultures under ¹⁸O₂, the initial cleavage produced the diol product with ≈70% enrichment by ¹⁸O in the benzyl alcohol group. The diol was a mixture of the two possible enantiomers, and the O₂-derived hydroxyl was incorporated at the asymmetric (benzyl) carbon. (Limited optical activity in the diol was traced to selective further metabolism of the D form.) These results show that the primary cleavage reaction lacked stereospecificity and was primarily oxygenative, implicating a nonspecific oxygenase or a nonenzymatic reaction involving activated oxygen. Preliminary experiments demonstrated no cell homogenate activity against Ia.     

An extracellular H2O2-requiring enzyme preparation involved in lignin biodegradation by the white rot basidiomycete Phanerochaete chrysosporium

An H₂O₂-requiring enzyme system was found in the extracellular medium of ligninolytic cultures of Phanerochaete chrysosporium. The enzyme system generated ethylene from 2-keto-4-thiomethyl butyric acid (KTBA), and oxidized a variety of lignin model compounds including the diarylpropane 1-(4′-ethoxy-3′-methoxyphenyl) 1,3-dihydroxy-2-(4″-methoxyphenyl)propane (I), a β-ether dimer 1-(4′-ethoxy-3′-methoxyphenyl)glycerol-β-guaiacyl ether (IV) and an olefin 1-(4′-ethoxy-3′-methoxy-phenyl)1,2-propene (VI). The products found were equivalent to the metabolic products previously isolated from intact ligninolytic cultures. In addition, the enzyme system partially degraded ¹⁴C-ring labeled lignin. The enzyme was not found in high nitrogen (N) cultures, nor in cultures of a ligninolytic mutant strain which is incapable of metabolizing lignin.     

Ultrastructural Localization of Hydrogen Peroxide Production in Ligninolytic Phanerochaete chrysosporium Cells

Previous studies have shown that the hydroxyl radical derived from hydrogen peroxide (H₂O₂) is involved in lignin degradation by Phanerochaete chrysosporium. In the present study, the ultrastructural sites of H₂O₂ production in ligninolytic cells of P. chrysosporium were demonstrated by cytochemically staining cells with 3,3′-diaminobenzidine (DAB). Hydrogen peroxide production, as evidenced by the presence of oxidized DAB deposits, appeared to be localized in the periplasmic space of cells from ligninolytic cultures grown for 14 days in nitrogen-limited medium. When identical cells were treated with DAB in the presence of aminotriazole, periplasmic deposits of oxidized DAB were not observed, suggesting that the deposits resulted from the H₂O₂-dependent peroxidatic oxidation of DAB by catalase. Cells from cultures grown for 3 or 6 days in nitrogen-limited medium or for 14 days in nitrogen-sufficient medium had little ligninolytic activity and low specific activity for H₂O₂ production and did not contain periplasmic oxidized DAB deposits. The results suggest that in cultures grown in nitrogen-limited medium, there is a positive correlation between the occurrence of oxidized DAB deposits, the specific activity for H₂O₂ production in cell extracts, and ligninolytic activity.     

Methanol formation during lignin degradation by Phanerochaete chrysosporium

Summary Methanol formation during the degradation of synthetic lignin (DHP), spruce and birch milled wood lignin (MWL) by Phanerochaete chrysosporium Burds. was studied under different culture conditions. When 100-ml flasks with 15–20 ml volumes of culture media containing high glucose and low nitrogen concentrations were used the metabolism of methanol to formaldehyde, formic acid and CO₂ was repressed thereby facilitating methanol determination. In standing cultures with oxygen flushing the fungus converted up to 25% of the DHP-methoxyl groups to methanol and 0.5–1.5% to ¹⁴CO₂ within 22–24 h. Methanol formation from methoxyl-labelled DHP was strongly repressed by high nitrogen in the medium, by addition of glutamic acid and by culture agitation. These results indicate that methanol is formed only under ligninolytic conditions and during secondary metabolism. Methanol is most likely released both from the lignin polymer itself and from lignin degradation products. Methanol was also formed from MWL preparations with higher percentage yields produced from birch as compared to spruce MWL.Small amounts of methanol detected in cultures without lignin probably emanated from demethoxylation of veratryl alcohol synthesized de novo from glucose by the fungus during secondary metabolism. Catalase or superoxide dismutase added to the fungal culture prior to addition of lignin, did not decrease methanol formation. Horseradish peroxidase plus H₂O₂ in vitro caused 5–7% demethoxylation of O¹⁴CH₃-DHP in 22 h, while laccase gave smaller amounts of methanol (1.8%). Since addition of H₂O₂ gave similar results as peroxidase plus H₂O₂, it seems likely that the main effect of peroxidase demethoxylation emanates from the hydrogen peroxide.     

Decolorization of Several Polymeric Dyes by the Lignin-Degrading Basidiomycete Phanerochaete chrysosporium

The polymeric dyes Poly B-411, Poly R-481, and Poly Y-606 were examined as possible alternatives to the radiolabeled lignin previously used as a substrate in lignin biodegradation assays. Like lignin degradation, the decolorization of these dyes by the white rot basidiomycete Phanerochaete chrysosporium occurred during secondary metabolism, was suppressed in cultures grown in the presence of high levels of nitrogen, and was strongly dependent on the oxygen concentration in the cultures. A variety of inhibitors of lignin degradation, including thiourea, azide, and 4′-O-methylisoeugenol, also inhibited dye decolorization. A pleiotropic mutant of P. chrysosporium, 104-2, lacking phenol oxidase and ligninolytic activity was also not able to decolorize the polymeric dyes, whereas a phenotypic revertant strain, 424-2, regained this capacity. All of these results suggest that the ligninolytic degradation activity of the fungus was responsible for the decolorization of these dyes.     

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.     

Mechanism of aromatic ring cleavage of β-O-4 lignin substructure models by lignin peroxidase

This investigation examined the aromatic ring cleavage of β-O-4 lignin substructure model compounds by lignin peroxidase of Phanerochaete chrysosporium. Based on tracer experiments using H₂¹⁸O and ¹⁸O₂, mechanisms of the aromatic ring cleavage of the β-O-4 lignin models were proposed. The mechanisms involve one-electron oxidation of the β-O-4 lignin models by the enzyme followed by attack of nucleophiles and radical coupling with O₂.     

Delignification of an unbleached hardwood kraft pulp by Phanerochaete chrysosporium

Summary Culture conditions affecting lignin degradation of an unbleached hardwood kraft pulp by Phanerochaete chrysosporium have been examined. Optimum pH and temperature for lignin degradation (about 33%) were 3.5 and 38°C, respectively. Optimum fungal growth was at a pH of 4.5 and a temperature of around 32°C. Addition of exogeneous glucose to the cultures lessened the degradation of pulp carbohydrates. Lignin degradation was stimulated by oxygen atmosphere and non-agitated cultures. Increased surface to volume ratio (decreased culture depth) enhanced lignin degradation (about 56% at a depth of 1.2 cm). Finally, the correlations: pulp yield vs. residual glucose, ligninase activity vs. mycelium, and extent of delignification vs. residual extracellular H₂O₂ were discussed in light of recent findings of ligninases responsible for ligninolysis.     

Influence of culture parameters on lignin metabolism byPhanerochaete chrysosporium

Culture parameters influencing metabolism of synthetic¹⁴C-lignins to¹⁴CO₂ in defined media have been studied in shallow batch cultures of the ligninolytic wood-destroying HymenomycetePhanerochaete chrysosporium Burds. Study of the effect of O₂ concentration in the gas phase above non-agitated cultures indicated essentially complete absence of attack on the lignin polymer at 5% O₂ in N₂, and a 2- to 3-fold enhancement by 100% O₂ as compared to air (21% O₂). Agitation of the cultures resulting in the formation of mycelial pellets greatly suppressed lignin decomposition. The optimum culture pH for lignin decomposition was 4 to 4.5, with marked suppression above 5.5 and below 3.5. The source of nutrient nitrogen (NO ₃ ^– , NH ₄ ⁺ , amino acids) had little influence on lignin decomposition, but the concentration of nitrogen was critical; decomposition at 24 mM was only 25–35% of that at 2.4 mM N. Thiamine was the only vitamin required for growth and lignin decomposition. Under the optimum conditions developed, decomposition of 5 mg of synthetic lignin was accompanied by utilization of approximately 100 mg of glucose. The influence of the various culture parameters was analogous for metabolism of synthetic lignin labeled in the ring-,side chain-, and methoxyl carbon atoms.     

Polysaccharide synthesis by Phanerochaete chrysosporium during degradation of kraft lignin

Summary Phanerochaete chrysosporium (Sporotrichum pulverulentum) produced an extracellular glucan type polysaccharide when grown in a chemostat under nitrogen limitation. When cells were transferred to a standing mode of cultivation in the presence of excess glucose (6 gl^–1), the amount of non-glucose total carbohydrates in the culture increased from 0.58 gl^–1 to 1.76 gl^–1 during 15 day experiments. The change in total carbohydrates was due to an increase in extracellular and cell-bound glucan type polysaccharide. This increase occured simultaneously with formation of mycelial mats and appearance of ligninolytic activity. When the cultures were agitated under atmospheric oxygen rather than 100% O₂, their non-glucose total carbohydrate content increased to 2.15 gl^–1 in 4 days. The excess polysaccharide formation had an inhibitory effect on lignin degradation as more lignin was degraded by cells with lower polysaccharide content. The lignin that was associated with cells after the degradation had stopped could be further degraded by new active cells.     

Biosynthesis of the secondary metabolite veratryl alcohol in relation to lignin degradation in Phanerochaete chrysosporium

The lignin-degrading basidiomycete Phanerochaete chrysosporium synthesizes veratryl alcohol (3,4-dimethoxybenzyl alcohol) via phenylalanine, 3,4-dimethoxycinnamyl alcohol and veratrylglycerol. Study of the conversion of 3,4-dimethoxycinnamyl alcohol to veratrylglycerol and veratryl alcohol showed is to be (a) catalyzed by a secondary metabolic system, (b) markedly suppressed by culture agitation, and (c) strongly inhibited by l-glutamate. The amount of veratryl alcohol synthesized de novo was positively correlated with the O₂ concentration after primary growth. Other work has shown that the cinnamyl alcohol terminal residue in a lignin substructure model compound is degraded via arylglycerol and benzyl alcohol structures in ligninolytic cultures of P. chrysosporium, and that the ligninolytic system exhibits traits (a)-(c) above. Ligninolytic activity is also strongly and positively correlated with O₂ concentration. The results here suggest, therefore, that the actual biosynthetic secondary metabolic product is 3,4-dimethoxycinnamyl alcohol, but that this is degraded by the ligninolytic system to veratryl alcohol via veratrylglycerol. Veratryl alcohol is only slowly metabolized by the fungus, and accumulates.Non-standard abbreviation tlc thin layer chromatography     

Induction, Isolation, and Characterization of Two Laccases from the White Rot Basidiomycete Coriolopsis rigida

Previous work has shown that the white rot fungus Coriolopsis rigida degraded wheat straw lignin and both the aliphatic and aromatic fractions of crude oil from contaminated soils. To better understand these processes, we studied the enzymatic composition of the ligninolytic system of this fungus. Since laccase was the sole ligninolytic enzyme found, we paid attention to the oxidative capabilities of this enzyme that would allow its participation in the mentioned degradative processes. We purified two laccase isoenzymes to electrophoretic homogeneity from copper-induced cultures. Both enzymes are monomeric proteins, with the same molecular mass (66 kDa), isoelectric point (3.9), N-linked carbohydrate content (9%), pH optima of 3.0 on 2,6-dimethoxyphenol (DMP) and 2.5 on 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), absorption spectrum, and N-terminal amino acid sequence. They oxidized 4-anisidine and numerous phenolic compounds, including methoxyphenols, hydroquinones, and lignin-derived aldehydes and acids. Phenol red, an unusual substrate of laccase due to its high redox potential, was also oxidized. The highest enzyme affinity and efficiency were obtained with ABTS and, among phenolic compounds, with 2,6-dimethoxyhydroquinone (DBQH₂). The presence of ABTS in the laccase reaction expanded the substrate range of C. rigida laccases to nonphenolic compounds and that of MBQH₂ extended the reactions catalyzed by these enzymes to the production of H₂O₂, the oxidation of Mn²⁺, the reduction of Fe³⁺, and the generation of hydroxyl radicals. These results confirm the participation of laccase in the production of oxygen free radicals, suggesting novel uses of this enzyme in degradative processes.     

The ligninolytic activities of Lentinus edodes and Phanerochaete chrysosporium

Summary Two important lignin-degrading fungi with existing or potential applications in the production of food, feed and/or fiber products from wood are Lentinus edodes (Berk.; Sing.=Lentinula edodes [Pegler]) and Phanerochaete chrysosporium (Burds). This study discusses their relative ability to degrade lignin and the factors controlling their ligninolytic activity (synthetic ¹⁴C-lignin¹⁴CO₂). Ligninolytic activity in P. chrysosporium is known to develop after the fungus ceases vegetative growth, and to require both O₂ and an exogenous carbon source such as glucose. It has an extracellular ligninase in high titer which is assayed by the oxidation of veratryl alcohol to veratraldehyde. Here, P. chrysosporium was found to have a high capacity for lignin degradation (it was not easily saturated with lignin). Certain inorganic elements, including Fe²⁺, Ca²⁺ and Mo⁶⁺, were found to stimulate its ligninolytic activity. Calcium addition was required, with 40 ppm Ca²⁺ giving the highest activity. As in P. chrysosporium, ligninolytic activity in L. edodes was found to require both O₂ and an exogenous carbon source. However, in contrast to P. chrysosporium, L. edodes was only moderately ligninolytic, had a lower capacity for lignin degradation (was more easily saturated with lignin), and showed maximal activity only during the vegetative growth period. Also in contrast to P. chrysosporium, ligninolytic activity in L. edodes was not stimulated by Ca²⁺. Instead, manganese was required, with 10 ppm Mn²⁺ giving optimal activity. An extracellular ligninase capable of oxidizing veratryl alcohol to veratraldehyde was not detected in L. edodes.     

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.