Metabolomic differential display analysis of the white-rot basidiomycete Phanerochaete chrysosporium grown under air and 100% oxygen

When the cultural atmosphere of the white-rot basidiomycete, Phanerochaete chrysosporium, was changed from air to 100% oxygen, the lyophilized mycelial weight increased and thickening of extracellular glucan layer was observed in 2-3 days. To better understand the oxygen-stress responsive mechanism of P. chrysosporium, the metabolomic differential display analysis was performed using metabolites isolated from fungal cells grown under either air or 100% O(2) atmosphere. In the GC-MS total ion chromatogram of methanol-extracts from fungal cells, at least 183 peaks were detected and 53 compounds were identified. Among them, veratryl alcohol (VA), threonate, and erythronate were identified as oxygen-stress responsive metabolites. The intracellular concentration of VA increased dramatically within 1 h after an oxygen purge, indicating that VA production is sensitive to the oxygen stress in P. chrysosporium.     

Lignin-modifying enzymes from selected white-rot fungi: production and role from in lignin degradation

Abstract: White-rot fungi produce extracellular lignin-modifying enzymes, the best characterized of which are laccase (EC 1.10.3.2), lignin peroxidases (EC 1.11.1.7) and manganese peroxidases (EC 1.11.1.7). Lignin biodegradation studies have been carried out mostly using the white-rot fungus Phanerochaete chrysosporium which produces multiple isoenzymes of lignin peroxidase and manganese peroxidase but does not produce laccase. Many other white-rot fungi produce laccase in addition to lignin and manganese peroxidases and in varying combinations. Based on the enzyme production patterns of an array of white-rot fungi, three categories of fungi are suggested: (i) lignin-manganese peroxidase group (e.g. P. chrysosporium and Phlebia radiata ), (ii) manganese peroxidase-laccase group (e.g. Dichomitus squalens and Rigidoporus lignosus ), and (iii) lignin peroxidase-laccase group (e.g. Phlebia ochraceofulva and Junghuhnia separabilima ). The most efficient lignin degraders, estimated by ¹⁴CO₂ evolution from ¹⁴C-[Ring]-labelled synthetic lignin (DHP), belong to the first group, whereas many of the most selective lignin-degrading fungi belong to the second, although only moderate to good [¹⁴C]DHP mineralization is obtained using fungi from this group. The lignin peroxidase-laccase fungi only poorly degrade [¹⁴C]DHP.     

The effects of co-fungal cultures and supplementation with carbohydrate adjuncts on lignin biodegradation and substrate digestibility

The ability of three fungal strains (Pleurotus sajor-caju, Phanerochaete chrysosporium, Trametes versicolor) to decrease the lignin content and to enhance in vitro rumen digestibility of lignified spruce sawdust was assessed. In monoculture solid substrate fermentation (SSF) studies, a considerable length of time (6 weeks) elapsed before 4 to 14% lignin was degraded. In contrast, paired or multiple cultures of these fungi caused an 8 to 16% loss of native lignin within three weeks of incubation. There were also synergistic effects on total polysaccharide/hemicellulose degraded by mixed cultures. A similar observation was made for in vitro digestibility of fungal fermented samples: Total solubles (carbohydrate products) which accumulated in cultures were significantly higher in mixed cultures than in respective monocultures. In contrast, mixtures of cell free enzyme extracts of these fungi did not cause any marked reduction in lignin or cellulose content. Supplementation of wood sawdust with carbohydrate adjuncts prior to fungal treatment also led to substantial reduction in lignin content and increased substrate digestibility.F.O. Asiegbu is with the Department of Forest Mycology & Pathology, Swedish University of Agricultural Sciences, P.O. Box 7026, S-750 07 Uppsala, Sweden; A. Paterson and J.E. Smith are with the Department of Bioscience and Biotechnology, University of Strathclyde, Glasgow, G1 1XW, UK.     

Enzymatic activity, osmotic stress and degradation of pesticide mixtures in soil extract liquid broth inoculated with Phanerochaete chrysosporium and Trametes versicolor

In this study we examined the extracellular enzymatic activity of two white rot fungi (Phanerochaete chrysosporium and Trametes versicolor) in a soil extract broth in relation to differential degradation of a mixture of different concentrations (0-30 p.p.m.) of simazine, dieldrin and trifluralin under different osmotic stress (-0.7 and -2.8 MPa) and quantified enzyme production, relevant to P and N release (phosphomonoesterase, protease), carbon cycling (beta-glucosidase, cellulase) and laccase activity, involved in lignin degradation. Our results suggest that T. versicolor and P. chrysosporium have the ability to degrade different groups of pesticides, supported by the capacity for expression of a range of extracellular enzymes at both -0.7 and -2.8 MPa water potential. Phanerochaete chrysosporium was able to degrade this mixture of pesticides independently of laccase activity. In soil extract, T. versicolor was able to produce the same range of enzymes as P. chrysoporium plus laccase, even in the presence of 30 p.p.m. of the pesticide mixture. Complete degradation of dieldrin and trifluralin was observed, while about 80% of the simazine was degraded regardless of osmotic stress treatment in a nutritionally poor soil extract broth. The capacity of tolerance and degradation of high concentrations of mixtures of pesticides and production of a range of enzymes, even under osmotic stress, suggest potential bioremediation applications.     

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.     

Heterologous expression of lignin peroxidase of Phanerochaete chrysosporium in Pichia methanolica

The cDNA encoding for lignin peroxidase of Phanerochaete chrysosporium was expressed in the Pichia methanolica under the control of the alcohol oxidase (AUG1) promoter which was followed by either the lignin peroxidase leader peptide of Phanerochaete chrysosporium or the Saccharomyces cerevisiae alpha-factor signal peptide. Both peptides efficiently directed the secretion of lignin peroxidase from the recombinant yeast cell. The extracellular lignin peroxidase activity in two recombinants was 932 U l(-1) and 1933 U l(-1). The purity of the recombinant product was confirmed by SDS-PAGE.     

Transformation of 2,4,6-trinitrotoluene (TNT) by immobilized Phanerochaete chrysosporium under fed-batch and continuous TNT feeding conditions

The cometabolic transformation of 2,4,6-trinitrotoluene (TNT) by an immobilized Phanerochaete chrysosporium culture was investigated under different TNT and/or glycerol feeding conditions in a 5-L reactor. In the fed-batch feeding mode, as a result of four spiking events at an average feeding rate of 20 mg TNT L(-1) d(-1) and 250 mg glycerol L(-1) d(-1), the initial TNT transformation rate and the glycerol uptake rate of the 7-day-old immobilized cell culture were 2.41 mg L(-1) h(-1) and 16.6 mg L(-1) h(-1), respectively. Thereafter, the TNT fed into the reactor depicted a negative effect on the cell physiology of P. chrysosporium, i.e., both rates decreased constantly. At 32 mg TNT L(-1) d(-1) feeding rate, also in the presence of glycerol (200 mg L(-1) d(-1)), this effect on the fungal cell metabolism was even more significant. When TNT was fed alone at 3.7 mg L(-1) d(-1), it showed an initial 0.75 mg L(-1) h(-1) rate of TNT transformation, i.e., one-third the initial level observed in the presence of glycerol. In contrast, in the continuous feeding mode (dilution rate, D = 0.11 d(-1)), at 5.5 mg TNT L(-1) d(-1) and 220 mg glycerol L(-1) d(-1), the immobilized cell culture exhibited a constant TNT transformation rate for cultivation periods of 50 and 61 days, under uncontrolled and controlled pH conditions, respectively. Thereafter, during the latter experiment, 100% TNT biotransformation was achieved at 1,100 mg L(-1) d(-1) glycerol feeding rate. Immobilized cells (115-day-old), sampled from a continuous TNT feeding experiment, mineralized [(14)C]-TNT to a level of 15.3% following a 41-day incubation period in a microcosm.     

Factors influencing fungal degradation of lignin in a representative lignocellulosic,thermomechanical pulp

This research examined culture parameters influencing the rate of degradation of lignin in lignocellulosic substrates by the Basidiomycete Phanerochaete chrysosporium. Thermomechanical pulps prepared from western hemlock (Tsuga heterophylla) and red alder (Alnus rubra) were chosen as model substrates. Degradation of lignin in shallow, liquid-phase, stationary cultures was 10 times as rapid as in agitated cultures. Lignin degradation was at least 50% more rapid in cultures under 100% O₂ than in those under air. Addition of 0.12% nutrient N (dry pulp basis) increased the rate of lignin degradation two- to fivefold; 1.2% added N at first suppressed, then stimulated, lignin degradation. Lignin in the alder pulp was degraded over five times as rapidly as in the hemlock pulp. Addition of glucose (35% of dry pulp) to the pulps containing 0.12% added N completely suppressed polysaccharide depletion during two weeks, but did not influence lignin degradation. The maximum rate of lignin degradation was 3%/day over a two-week incubation, or approximately 2.9 mg/mg fungal cell protein/day. The influence of the examined parameters was in complete accord with those found earlier for synthetic ¹⁴C-lignin metabolism by P. chrysosporium.     

Degradation of lignin in pulp mill wastewaters by white-rot fungi on biofilm

An investigation was conducted to explore the lignin-degrading capacity of attached-growth white-rot fungi. Five white-rot fungi, Phanerochaete chrysosporium, Pleurotus ostreatus, Lentinus edodes, Trametes versicolor and S22, grown on a porous plastic media, were individually used to treat black liquor from a pulp and paper mill. Over 71% of lignin and 48% of chemical oxygen demand (COD) were removed from the wastewater. Several factors, including pH, concentrations of carbon, nitrogen and trace elements in wastewater, all had significant effects on the degradation of lignin and the removal of COD. Three white-rot fungi, P. chrysosporium, P. ostreatus and S22, showed high capacity for lignin degradation at pH 9.0-11.0. The addition of 1 g l-1 glucose and 0.2 g l-1 ammonium tartrate was beneficial for the degradation of lignin by the white-rot fungi studied.     

Oxidative degradation of phenanthrene by the ligninolytic fungus Phanerochaete chrysosporium.

The ligninolytic fungus Phanerochaete chrysosporium oxidized phenanthrene and phenanthrene-9,10-quinone (PQ) at their C-9 and C-10 positions to give a ring-fission product, 2,2'-diphenic acid (DPA), which was identified in chromatographic and isotope dilution experiments. DPA formation from phenanthrene was somewhat greater in low-nitrogen (ligninolytic) cultures than in high-nitrogen (nonligninolytic) cultures and did not occur in uninoculated cultures. The oxidation of PQ to DPA involved both fungal and abiotic mechanisms, was unaffected by the level of nitrogen added, and was significantly faster than the cleavage of phenanthrene to DPA. Phenanthrene-trans-9,10-dihydrodiol, which was previously shown to be the principal phenanthrene metabolite in nonligninolytic P. chrysosporium cultures, was not formed in the ligninolytic cultures employed here. These results suggest that phenanthrene degradation by ligninolytic P. chrysosporium proceeds in order from phenanthrene----PQ----DPA, involves both ligninolytic and nonligninolytic enzymes, and is not initiated by a classical microsomal cytochrome P-450. The extracellular lignin peroxidases of P. chrysosporium were not able to oxidize phenanthrene in vitro and therefore are also unlikely to catalyze the first step of phenanthrene degradation in vivo. Both phenanthrene and PQ were mineralized to similar extents by the fungus, which supports the intermediacy of PQ in phenanthrene degradation, but both compounds were mineralized significantly less than the structurally related lignin peroxidase substrate pyrene was.     

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.     

Mineralization of mono- and dichlorobenzenes and simultaneous degradation of chloro- and methyl-substituted benzenes by the white rot fungus Phanerochaete chrysosporium.

Phanerochaete chrysosporium extensively degraded and mineralized chlorobenzene and o-, m-, and p-dichlorobenzenes. The rate of degradation was in the following order: monochlorobenzene > m-dichlorobenzene > o-dichlorobenzene > p-dichlorobenzene. Net level of degradation was generally higher than mineralization. Maximal degradation and mineralization of chlorobenzenes were observed in malt extract cultures in which the lignin peroxidases and manganese peroxidases are not known to be produced. The fungus degraded both chlorobenzene and toluene when presented as a mixture, indicating its ability to simultaneously degrade chloro-substituted and methyl-substituted benzenes.     

Free hydroxyl radical is not involved in an important reaction of lignin degradation by Phanerochaete chrysosporium Burds.

Hydroxyl radical (HO.) has been implicated in the degradation of lignin by Phanerochaete chrysosporium. This study assessed the possible involvement of HO. in degradation of lignin substructural models by intact cultures and by an extracellular ligninase isolated from the cultures. Two non-phenolic lignin model compounds [aryl-C(alpha)HOH-C(beta)HR-C(gamma)H2OH, in which R = aryl (beta-1) or R = O-aryl (beta-O-4)] were degraded by cultures, by the purified ligninase, and by Fenton's reagent (H2O2 + Fe2+), which generates HO.. The ligninase and the cultures formed similar products, derived via an initial cleavage between C(alpha) and C(beta) (known to be an important biodegradative reaction), indicating that the ligninase is responsible for model degradation in cultures. Products from the Fenton degradation were mainly polar phenolics that exhibited little similarity to those from the biological systems. Mass-spectral analysis, however, revealed traces of the same products in the Fenton reaction as seen in the biological reactions; even so, an 18O2-incorporation study showed that the mechanism of formation differed. E.s.r. spectroscopy with a spin-trapping agent readily detected HO. in the Fenton system, but indicated that no HO. is formed during ligninase catalysis. We conclude, therefore that HO. is not involved in fungal C(alpha)-C(beta) cleavage in the beta-1 and beta-O-4 models and, by extension, in the same reaction in lignin.     

Southern blot screening for lignin peroxidase and aryl-alcohol oxidase genes in 30 fungal species

Screening to detect genes encoding lignin peroxidase (LiP) and aryl-alcohol oxidase (AAO) has been carried out with 30 fungal strain using DNA probes from genes lpo of Phanerochaete chrysosporium (encoding LiP isoenzyme H8) and aao of Pleurotus eryngii. Evidence for the presence of genes closely related to lpo was found in Bjerkandera adusta, Fomes fomentarius, Ganoderma applanatum, Ganoderma australe, Lentinula degener, Peniophora gigantea, P. chrysosporium, Phanerochaete flavido-alba and Trametes tersicolor, whereas the gene aao was detected in Pleurotus species and B. adusta. The presence of both genes was only detected in B. adusta. These results suggest that different enzymatic system, formed by enzymes encoded by different genes, are responsible for lignin degradation by white-rot fungi.     

Diuron degradation by Phanerochaete chrysosporium BKM- F-1767 in synthetic and natural media

When incubated in synthetic (N-limited) medium and on ashwood chips, Phanerochaete chrysosporium BKM-F-1767 degraded 14 and 10 mg/l diuron, respectively. The wood chips were used as support and sole nutrient source for the fungus. A higher degradation efficiency was found in ashwood culture as compared to the liquid culture, probably as a result of the synergetic effect of attached fungal growth, presence of limiting-substrate conditions and the microenvironment provided by ashwood, all favorable for production of high extracellular enzyme titres. Diuron degradation occured during the idiophasic growth, in the presence of manganese peroxidase, detected as dominant enzyme in both cultures.     

Methanogenic bacteria as a key factor involved in changes of town gas stored in an underground reservoir

Growth of Phanerochaete chrysosporium in a nitrogen-limited medium buffered with sodium acetate, instead of the commonly used 2,2-dimethylsuccinate (DMS), resulted in quantitative and qualitative differences in the production of various extracellular lignin peroxidases (LIPs) and manganese-dependent peroxidases (MNPs) involved in lignin degradation. The results indicate that production of LIPs and MNPs can be selectively enhanced by manipulation of culture conditions. Partial N-terminal analyses of the major LIPs and MNPs have made it possible to assign a specific protein to the specific genes and cDNAs that have been reported recently. The LIPs and MNPs differed widely in their ability to decolorize various dyes that are known to be degraded by the lignin degrading enzyme system of P. chrysosporium.     

Influence of 2,4,6-trinitrotoluene (TNT) concentration on the degradation of TNT in explosive-contaminated soils by the white rot fungus Phanerochaete chrysosporium.

The ability of Phanerochaete chrysosporium to bioremediate TNT (2,4,6-trinitrotoluene) in a soil containing 12,000 ppm of TNT and the explosives RDX (hexahydro-1,3,5-trinitro-1,3,5- triazine; 3,000 ppm) and HMX (octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine; 300 ppm) was investigated. The fungus did not grow in malt extract broth containing more than 0.02% (wt/vol; 24 ppm of TNT) soil. Pure TNT or explosives extracted from the soil were degraded by P. chrysosporium spore-inoculated cultures at TNT concentrations of up to 20 ppm. Mycelium-inoculated cultures degraded 100 ppm of TNT, but further growth was inhibited above 20 ppm. In malt extract broth, spore-inoculated cultures mineralized 10% of added [14C]TNT (5 ppm) in 27 days at 37 degrees C. No mineralization occurred during [14C]TNT biotransformation by mycelium-inoculated cultures, although the TNT was transformed.     

Pyranose Oxidase, a Major Source of H(2)O(2) during Wood Degradation by Phanerochaete chrysosporium, Trametes versicolor, and Oudemansiella mucida

The production of the H(2)O(2)-generating enzyme pyranose oxidase (POD) (EC 1.1.3.10) (synonym, glucose 2-oxidase), two ligninolytic peroxidases, and laccase in wood decayed by three white rot fungi was investigated by correlated biochemical, immunological, and transmission electron microscopic techniques. Enzyme activities were assayed in extracts from decayed birch wood blocks obtained by a novel extraction procedure. With the coupled peroxidase-chromogen (3-dimethylaminobenzoic acid plus 3-methyl-2-benzothiazolinone hydrazone hydrochloride) spectrophotometric assay, the highest POD activities were detected in wood blocks degraded for 4 months and were for Phanerochaete chrysosporium (149 mU g [dry weight] of decayed wood), Trametes versicolor (45 mU g), and Oudemansiella mucida (1.2 mU g), corresponding to wood dry weight losses of 74, 58, and 13%, respectively. Mn-dependent peroxidase activities in the same extracts were comparable to those of POD, while lignin peroxidase activity was below the detection limit for all fungi with the veratryl alcohol assay. Laccase activity was high with T. versicolor (422 mU g after 4 months), in trace levels with O. mucida, and undetectable in P. chrysosporium extracts. Evidence for C-2 specificity of POD was shown by thin-layer chromatography detection of 2-keto-d-glucose as the reaction product. By transmission electron microscopy-immunocytochemistry, POD was found to be preferentially localized in the hyphal periplasmic space of P. chrysosporium and O. mucida and associated with membranous materials in hyphae growing within the cell lumina or cell walls of partially and highly degraded birch fibers. An extracellular distribution of POD associated with slime coating wood cell walls was also noted. The periplasmic distribution in hyphae and extracellular location of POD are consistent with the reported ultrastructural distribution of H(2)O(2)-dependent Mn-dependent peroxidases. This fact and the dominant presence of POD and Mn-dependent peroxidase in extracts from degraded wood suggest a cooperative role of the two enzymes during white rot decay by the test fungi.     

Influence of 2,4,6-trinitrotoluene (TNT) concentration on the degradation of TNT in explosive-contaminated soils by the white rot fungus Phanerochaete chrysosporium.

The ability of Phanerochaete chrysosporium to bioremediate TNT (2,4,6-trinitrotoluene) in a soil containing 12,000 ppm of TNT and the explosives RDX (hexahydro-1,3,5-trinitro-1,3,5- triazine; 3,000 ppm) and HMX (octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine; 300 ppm) was investigated. The fungus did not grow in malt extract broth containing more than 0.02% (wt/vol; 24 ppm of TNT) soil. Pure TNT or explosives extracted from the soil were degraded by P. chrysosporium spore-inoculated cultures at TNT concentrations of up to 20 ppm. Mycelium-inoculated cultures degraded 100 ppm of TNT, but further growth was inhibited above 20 ppm. In malt extract broth, spore-inoculated cultures mineralized 10% of added [14C]TNT (5 ppm) in 27 days at 37 degrees C. No mineralization occurred during [14C]TNT biotransformation by mycelium-inoculated cultures, although the TNT was transformed.     

Fungal pretreatment of lignocellulose by Phanerochaete chrysosporium to produce ethanol from rice straw

Phanerochaete chrysosporium is a wood‐rot fungus that is capable of degrading lignin via its lignolytic system. In this study, an environmentally friendly fungal pretreatment process that produces less inhibitory substances than conventional methods was developed using P. chrysosporium and then evaluated by various analytical methods. To maximize the production of manganese peroxidase, which is the primary lignin‐degrading enzyme, culture medium was optimized using response surface methodologies including the Plackett–Burman design and the Box–Behnken design. Fermentation of 100 g of rice straw feedstock containing 35.7 g of glucan (mainly in the form of cellulose) by cultivation with P. chrysosporium for 15 days in the media optimized by response surface methodology was resulted in a yield of 29.0 g of glucan that had an enzymatic digestibility of 64.9% of the theoretical maximum glucose yield. In addition, scanning electronic microscopy, confocal laser scanning microscopy, and X‐ray diffractometry revealed significant microstructural changes, fungal growth, and a reduction of the crystallinity index in the pretreated rice straw, respectively. When the fungal‐pretreated rice straw was used as a substrate for ethanol production in simultaneous saccharification and fermentation (SSF) for 24 h, the ethanol concentration, production yield and the productivity were 9.49 g/L, 58.2% of the theoretical maximum, and 0.40 g/L/h, respectively. Based on these experimental data, if 100 g of rice straw are subjected to fungal pretreatment and SSF, 9.9 g of ethanol can be produced after 96 h, which is 62.7% of the theoretical maximum ethanol yield. Biotechnol. Bioeng. 2009; 104: 471–482 © 2009 Wiley Periodicals, Inc.