Biodegradation of pentachlorophenol by the white rot fungus Phanerochaete chrysosporium.

Extensive biodegradation of pentachlorophenol (PCP) by the white rot fungus Phanerochaete chrysosporium was demonstrated by the disappearance and mineralization of [14C]PCP in nutrient nitrogen-limited culture. Mass balance analyses demonstrated the formation of water-soluble metabolites of [14C]PCP during degradation. Involvement of the lignin-degrading system of this fungus was suggested by the fact the time of onset, time course, and eventual decline in the rate of PCP mineralization were similar to those observed for [14C]lignin degradation. Also, a purified ligninase was shown to be able to catalyze the initial oxidation of PCP. Although biodegradation of PCP was decreased in nutrient nitrogen-sufficient (i.e., nonligninolytic) cultures of P. chrysosporium, substantial biodegradation of PCP did occur, suggesting that in addition to the lignin-degrading system, another degradation system may also be responsible for some of the PCP degradation observed. Toxicity studies showed that PCP concentrations above 4 mg/liter (15 microM) prevented growth when fungal cultures were initiated by inoculation with spores. The lethal effects of PCP could, however, be circumvented by allowing the fungus to establish a mycelial mat before adding PCP. With this procedure, the fungus was able to grow and mineralize [14C]PCP at concentrations as high as 500 mg/liter (1.9 mM).     

Biodegradation of pentachlorophenol by the white rot fungus Phanerochaete chrysosporium

Extensive biodegradation of pentachlorophenol (PCP) by the white rot fungus Phanerochaete chrysosporium was demonstrated by the disappearance and mineralization of [14C]PCP in nutrient nitrogen-limited culture. Mass balance analyses demonstrated the formation of water-soluble metabolites of [14C]PCP during degradation. Involvement of the lignin-degrading system of this fungus was suggested by the fact the time of onset, time course, and eventual decline in the rate of PCP mineralization were similar to those observed for [14C]lignin degradation. Also, a purified ligninase was shown to be able to catalyze the initial oxidation of PCP. Although biodegradation of PCP was decreased in nutrient nitrogen-sufficient (i.e., nonligninolytic) cultures of P. chrysosporium, substantial biodegradation of PCP did occur, suggesting that in addition to the lignin-degrading system, another degradation system may also be responsible for some of the PCP degradation observed. Toxicity studies showed that PCP concentrations above 4 mg/liter (15 microM) prevented growth when fungal cultures were initiated by inoculation with spores. The lethal effects of PCP could, however, be circumvented by allowing the fungus to establish a mycelial mat before adding PCP. With this procedure, the fungus was able to grow and mineralize [14C]PCP at concentrations as high as 500 mg/liter (1.9 mM).     

Effects of xylan and starch on secretome of the basidiomycete Phanerochaete chrysosporium grown on cellulose

Lignocellulosic biomass contains cellulose and xylan as major structural components, and starch as a storage polysaccharide. In the present study, we have used comparative secretomic analysis to examine the effects of xylan and starch on the expression level of proteins secreted by the basidiomycete Phanerochaete chrysosporium grown on cellulose,. Forty-seven spots of extracellular proteins expressed by P. chrysosporium separated by two-dimensional electrophoresis were identified by liquid chromatography-tandem mass spectrometry analysis. Addition of starch to the cellulolytic culture did not affect fungal growth significantly, but did decrease the production of total extracellular enzymes, including cellulases and xylanases. In contrast, addition of xylan increased mycelial volume and the production of extracellular proteins. Xylan increased synthesis of several glycoside hydrolase (GH) family 10 putative endoxylanases and a putative glucuronoyl esterase belonging to carbohydrate esterase family 15, for which plant cell wall xylan may be a substrate. Moreover, cellobiose dehydrogenase and GH family 61 proteins, which are known to promote cellulose degradation, were also increased in the presence of xylan. These enzymes may contribute to degradation by the fungus of not only cellulose but also complex carbohydrate components of the plant cell wall.     

Use of coimmobilized biological systems to degrade toxic organic compounds

The concept of coimmobilizing cell mass (and/or enzyme) and adsorbent in a hydrogel matrix for biodegradation of toxic organic chemicals was introduced. Under defined experimental conditions, the coimmobilized system using activated carbon and Phanerochaete chrysosporium was compared with nonimmobilized systems for the degradation of pentachlorophenol (PCP). It was demonstrated that the coimmobilized system degraded PCP more effectively than the nonimmobilized system. A solid substrate included in the coimmobilized system could support the biodegradation. Isolation of the degrading agents from a model interrupting microorganism by the coimmobilized capsule membrane reduced the interference on the biodegradation. In simulated contaminated soil extract and sand, the coimmobilized system also exhibited higher degradative ability and stability than the nonimmobilized systems.     

Adsorption capacity as a key parameter for enzyme induction and pentachlorophenol degradation in Mycobacterium chlorophenolicum PCP-1.

Adsorption of pentachlorophenol (PCP) on induced cells of Mycobacterium chlorophenolicum PCP-1 and its influence on enzyme induction and PCP degradation of this strain were studied. Compared to non-induced cells, induced degrading cells had a lower adsorption capacity (q(ads)), particularly at prolonged induction and low PCP concentration. Unlike the effects of pH and biomass concentration previously reported for non-induced cells, the variation of q(ads) of induced cells was associated with changes of both the capacity and intensity constants of the Freundlich equation which was used to describe PCP adsorption on M. chlorophenolicum PCP-1. This indicated changes of cell surface properties during enzyme induction and PCP degradation. The latter was shown in turn to be affected by several parameters such as PCP concentration, pH value and induction time. Interestingly, irrespective of the pH and PCP concentration, the specific PCP degradation rate (q(t)(PCP)) at a given induction time was found to be solely a function of q(ads), revealing that adsorption capacity is an inherent key parameter for enzyme induction and PCP degradation. Based on this knowledge, a kinetic model was developed for q(t)(PCP) which used only q(ads) and induction time as variables. The model considered inhibition of PCP on both enzyme induction and enzyme activity and described the experimental data at different PCP concentrations and pH values well. q(ads) also turned out to be a useful criterion for choosing optimum induction concentration of PCP. Irrespective of pH and biomass concentration, an initial adsorption capacity of 2-3 micromol PCP/g cells was found to be optimum for enzyme induction in M. chlorophenolicum PCP-1.     

Mineralization of biphenyl and PCBs by the white rot fungus Phanerochaete chrysosporium

The white rot fungus Phanerochaete chrysosporium is unique in its ability to totally degrade a wide variety of recalcitrant pollutants. We have investigated the degradation of biphenyl and two model chlorinated biphenyls, 2,2',4,4'-tetrachlorobiphenyl and 2-chlorobiphenyl by suspended cultures of P. chrysosporium grown under conditions that maximize the synthesis of lignin-oxidizing enzymes. Radiolabeled biphenyl and 2'-chlorobiphenyl added to cultures at concentrations in the range 260 nM to 8.8 muM were degraded extensively to CO(2) within 30 days. In addition, from 40% to 60% of the recovered radioactivity was found in water-soluble compounds. A correlation between the rate of degradation and the synthesis of ligninases or Mn-dependent peroxidases could not be observed, indicating that yet unknown enzymatic system may be resonsible for the initial oxidation of PCBs. The more heavily chlorinated PCB congener, 2,2',4,4'-tetrachlorobiphenyl was converted to CO(2) less readily; approximately 9% and 0.9% mineralization was observed in cultures incubated with 40 nM and 5.3 muM, respectively. Overall, our results indicate that P. chrysosporium is a promising organism for the treatment of wastes contaminatd with lightly and moderately chlorinated PCBs. (c) 1992 John Wiley & Sons, Inc.     

Kinetics of pentachlorophenol degradation by a Flavobacterium species

The kinetics of pentachlorophenol (PCP) degradation by a Flavobacterium sp. ATCC 39723 has been investigated. Sodium glutamate was supplied as an additional carbon source to increase the rate of cell growth and PCP degradation. A kinetic model including PCP toxicity for cell growth and PCP inhibition of its own degradation was developed. This model was also applied to 2,4-dichlorophenol (DCP) degradation by the same organism. Although PCP and DCP are degraded by different pathways, the model describes these two degradation processes very well.     

Degradation of phenanthrene by Phanerochaete chrysosporium occurs under ligninolytic as well as nonligninolytic conditions.

In order to delineate the roles of lignin and manganese peroxidases in the degradation of polycyclic aromatic hydrocarbons by Phanerochaete chrysosporium, the biodegradation of phenanthrene (chosen as a model for polycyclic aromatic hydrocarbons) was investigated. The disappearance of phenanthrene from the extracellular medium and mycelia was determined by using gas chromatography. The disappearance of phenanthrene from cultures of wild-type strains BKM-F1767 (ATCC 24725) and ME446 (ATCC 34541) under ligninolytic (low-nitrogen) as well as nonligninolytic (high-nitrogen) conditions was observed. The study was extended to two homokaryotic (basidiospore-derived) isolates of strain ME446. Both homokaryotic isolates, ME446-B19 (which produces lignin and manganese peroxidases only in low-nitrogen medium) and ME446-B5 (which totally lacks lignin and manganese peroxidase activities), caused the disappearance of phenanthrene when grown in low- as well as high-nitrogen media. Moreover, lignin and manganese peroxidase activities were not detected in any of the cultures incubated in the presence of phenanthrene. Additionally, the mineralization of phenanthrene was observed even under nonligninolytic conditions. The results collectively indicate that lignin and manganese peroxidases are not essential for the degradation of phenanthrene by P. chrysosporium. The observation that phenanthrene degradation occurs under nonligninolytic conditions suggests that the potential of P. chrysosporium for degradation of certain environmental pollutants is not limited to nutrient starvation conditions.     

Degradation of phenanthrene by Phanerochaete chrysosporium occurs under ligninolytic as well as nonligninolytic conditions.

In order to delineate the roles of lignin and manganese peroxidases in the degradation of polycyclic aromatic hydrocarbons by Phanerochaete chrysosporium, the biodegradation of phenanthrene (chosen as a model for polycyclic aromatic hydrocarbons) was investigated. The disappearance of phenanthrene from the extracellular medium and mycelia was determined by using gas chromatography. The disappearance of phenanthrene from cultures of wild-type strains BKM-F1767 (ATCC 24725) and ME446 (ATCC 34541) under ligninolytic (low-nitrogen) as well as nonligninolytic (high-nitrogen) conditions was observed. The study was extended to two homokaryotic (basidiospore-derived) isolates of strain ME446. Both homokaryotic isolates, ME446-B19 (which produces lignin and manganese peroxidases only in low-nitrogen medium) and ME446-B5 (which totally lacks lignin and manganese peroxidase activities), caused the disappearance of phenanthrene when grown in low- as well as high-nitrogen media. Moreover, lignin and manganese peroxidase activities were not detected in any of the cultures incubated in the presence of phenanthrene. Additionally, the mineralization of phenanthrene was observed even under nonligninolytic conditions. The results collectively indicate that lignin and manganese peroxidases are not essential for the degradation of phenanthrene by P. chrysosporium. The observation that phenanthrene degradation occurs under nonligninolytic conditions suggests that the potential of P. chrysosporium for degradation of certain environmental pollutants is not limited to nutrient starvation conditions.     

Fungal secretomes—nature’s toolbox for white biotechnology

Adapting their metabolism to varying carbon and nitrogen sources, saprophytic fungi produce an arsenal of extracellular enzymes, the secretome, which allows for an efficient degradation of lignocelluloses and further biopolymers. Based on fundamental advances in electrophoretic, chromatographic, and mass spectrometric techniques on the one hand and the availability of annotated fungal genomes and sophisticated bioinformatic software tools on the other hand, a detailed analysis of fungal secretomes has become feasible. While a number of reports on ascomycetous secretomes of, e.g., Aspergillus, Trichoderma, and Fusarium species are already available, studies on basidiomycetes have been mainly focused on the two model organisms Phanerochaete chrysosporium and Coprinopsis cinerea so far. Though an impressive number and diversity of fungal biocatalysts has been revealed by secretome analyses, the identity and function of many extracellular proteins still remains to be elucidated. A comprehensive understanding of the qualitative and quantitative composition of fungal secretomes, together with their synergistic actions and kinetic expression profiles, will allow for the development of optimized enzyme cocktails for white biotechnology.     

Degradation of chlorinated phenols by a pentachlorophenol-degrading bacterium

A pentachlorophenol (PCP)-degrading Flavobacterium sp. was tested for its ability to dechlorinate other chlorinated phenols by using resting cells that had been grown in the presence or absence of PCP. Phenols with chlorine atoms at positions 2 and 6 of the phenol ring were dechlorinated completely by PCP-induced cells. Other chlorinated phenols were not significantly mineralized. When PCP was added to a culture growing on L-glutamate, there was a lag period before the start of PCP degradation. When similar cells were treated with chloramphenicol prior to the addition of PCP, they did not degrade added PCP, even after prolonged incubations. Thus, the enzymes necessary for PCP degradation appeared to be inducible. Suspensions of cells grown in the presence of 2,4,6-trichlorophenol or 2,3,5,6-tetrachlorophenol did not show a lag period for mineralization of PCP, 2,4,6-trichlorophenol, or 2,3,5,6-tetrachlorophenol, indicating that one enzyme system probably was induced for the biodegradation of all three compounds. Nondegradable chlorophenols were toxic toward the Flavobacterium sp., probably acting as uncouplers of oxidative phosphorylation.     

Degradation of chlorinated phenols by a pentachlorophenol-degrading bacterium.

A pentachlorophenol (PCP)-degrading Flavobacterium sp. was tested for its ability to dechlorinate other chlorinated phenols by using resting cells that had been grown in the presence or absence of PCP. Phenols with chlorine atoms at positions 2 and 6 of the phenol ring were dechlorinated completely by PCP-induced cells. Other chlorinated phenols were not significantly mineralized. When PCP was added to a culture growing on L-glutamate, there was a lag period before the start of PCP degradation. When similar cells were treated with chloramphenicol prior to the addition of PCP, they did not degrade added PCP, even after prolonged incubations. Thus, the enzymes necessary for PCP degradation appeared to be inducible. Suspensions of cells grown in the presence of 2,4,6-trichlorophenol or 2,3,5,6-tetrachlorophenol did not show a lag period for mineralization of PCP, 2,4,6-trichlorophenol, or 2,3,5,6-tetrachlorophenol, indicating that one enzyme system probably was induced for the biodegradation of all three compounds. Nondegradable chlorophenols were toxic toward the Flavobacterium sp., probably acting as uncouplers of oxidative phosphorylation.     

Effects of p-cresol and chlorophenols on pentachlorophenol biodegradation

The effects of three aromatic compounds, p-cresol, 2,4-dichlorophenol (DCP), and 2,4,6-trichlorophenol (TCP), on cell growth and pentachlorophenol (PCP) degradation bya Flavobacterium species were investigated. While p-cresol was not degraded by this bacterium, DCP and TCP were simultaneously degraded with PCP. Both DCP and TCP lowered cell growth and PCP degradation rate. Cell growth was modeled by cell death, because p-cresol, DCP, and TCP were toxic to the organism. A new model was used to predict cell death rate in a mixture of two toxic compounds from the cell death kinetics for each individual compound. PCP degradation rates were modeled by conventional inhibition models, but only over a small concentration range for the secondary toxic compound. However, a new empirical model described PCP degradation over a wider concentration range of the secondary toxic compound. (c) 1995 John Wiley & Sons Inc.     

Biostimulation and Bioaugmentation of Anaerobic Pentachlorophenol Degradation in Contaminated Soils

The effects of bioaugmentation with a pentachlorophenol (PCP)-adapted consortium and biostimulation with glucose as a carbon source on anaerobic bioremediation of PCP-contaminated soil were investigated in terms of the initial PCP removal rate and the extent of PCP dechlorination and mineralization. Samples from two PCP-contaminated sites were prepared, put into a series of Hungate tubes, inoculated, and fed under different conditions. Chlorophenols in the tubes were monitored over a 4-month period to measure PCP transformation in the soil. In less contaminated soil (10 mg PCP/kg soil), it was found that biostimulation with glucose at 1 g/kg soil or bioaugmentation at 0.14 g volatile suspended solids (VSS)/kg soil could greatly improve PCP degradation. The best PCP degradation was obtained when both bioaugmentation and biostimulation were applied, but higher levels of glucose (2 g/kg soil) or inoculum (0.56 g VSS/kg soil) had little additional effect. The highest initial PCP-removal rate reached 8.1 μmol/kg soil-d, which is almost 20 times greater than in the unamended controls. PCP was dechlorinated to lesser chlorinated phenols with 0.6 chlorine remaining on average, and the extent of mineralization approached 70% in 4 months. In highly PCP-contaminated soil (90 mg PCP/kg soil), PCP degradation was partially inhibited, but the relative effects of augmentation, stimulation, and combined treatments were the same as in the less contaminated soil.     

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.     

Accelerated Mineralization of Pentachlorophenol in Soil upon Inoculation with Mycobacterium chlorophenolicum PCP1 and Sphingomonas chlorophenolica RA2

Mineralization of pentachlorophenol (PCP) was studied in nonsterile soil from a PCP-contaminated site upon inoculation with two PCP-degrading bacterial strains. At spiked [(sup14)C]PCP concentrations of 30 and 100 mg/kg, the effects of organism type, different inoculation techniques, including structural amendment with sawdust and cell attachment to polyurethane (PU), as well as the effect of different inoculum sizes of 10(sup4) to 10(sup8) cells per g (dry weight) of soil were compared with PCP mineralization by indigenous bacteria. Gas chromatographic analysis was used to monitor PCP disappearance and to check mass balances. The survival and activity of the released bacteria were examined by immunofluorescence microscopy and respiking experiments. Noninoculated soil completely mineralized 30 mg of PCP per kg within 7 months but showed no or only low degradation activity at 100 mg/kg in the same period. Structural amendment with PU or sawdust initiated slow mineralization after half a year. Soil inoculation with Sphingomonas chlorophenolica RA2 shortened the mineralization time drastically to 1 month at 30 mg of PCP per kg using 10(sup8) cells per g, with approximately 80% of the added radioactivity being converted to CO(inf2). The inoculated cells disappeared rapidly, with a count of 2 x 10(sup6) cells per g after 2.3 months and nondetectability after 7 months. At 100 mg/kg, mineralization was slower because of PCP toxicity but approached completion within 7.5 months. The inhibition could be overcome by addition of sawdust (1 g/kg of soil), resulting in a mineralization rate of 3 to 4 mg/kg(middot)d. PU had the opposite effect. Lower inoculum densities resulted in prolonged lag phases and lower rates, although mineralization was still enhanced over the background level. At 30 mg of PCP per kg, inoculation with Mycobacterium chlorophenolicum PCP1 increased mineralization slightly over the indigenous bacterial activity, regardless of inoculum size, but only when the organisms were attached to PU. At 100 mg of PCP per kg, only 27% were mineralized within 7.5 months. After 7 months, the original strain PCP1 inoculum of 10(sup8) cells per g was recovered at 5 x 10(sup6) to 3 x 10(sup7) cells per g, depending on the PCP concentration, but independent of PU amendment. Amendment with sawdust had no effect on the performance of this organism. Possible reasons for the poor performance of this strain include its sensitivity to PCP and its preference for slightly acidic soil conditions.     

Photoelectrocatalytic treatment of pentachlorophenol in aqueous solution using a rutile nanotube-like TiO2/Ti electrode.

Taking pentachlorophenol (PCP) as a reference, we investigated the photoelectrocatalytic degradation of organic pollutants using a rutile nanotube-like TiO(2)/Ti film electrode. The nanotube-like TiO2 electrode was prepared by first oxidizing the surface of a titanium sheet to form rutile TiO2 and then treating it to form the tubular structure in NaOH aqueous solution. The occurrence of PCP degradation was indicated by the decrease in pH, concentration of PCP and TOC, and by the formation of chloride ions. The photoelectrochemical (PEC) efficiency of the nanotube-like TiO2/Ti electrode has been determined in terms of degradation of PCP and the incident photo-to-current conversion efficiency (IPCE). The experimental results showed that PCP could be degraded more efficiently by a photoelectrocatalytic process than by a photocatalytic or electrochemical oxidation alone. A significant photoelectrochemical synergetic effect was observed. The kinetic constant of PEC degradation of PCP using a nanotube-like TiO2 electrode was over 60% higher than that using a TiO2 film electrode. It is noted that chloride ion and hydrogen ion concentration increased with irradiation time in the PEC degradation of PCP; PCP was gradually mineralized and the complete minimization of PCP took more time than its degradation.     

Degradation of polychlorinated biphenyls by extracellular enzymes of Phanerochaete chrysosporium produced in a perforated plate bioreactor

The white rot fungus Phanerochaete chrysosporium was cultivated in a perforated plate bioreactor and the expression of activities of manganese-dependent peroxidase (MnP) and lignin peroxidase (LiP) was measured. Peak activities of the two enzymes were reached close to day 11 and therefore the cultivation was terminated on that day. Extracellular proteins were concentrated and both peroxidases separated by isoelectric focusing. Degradation of technical PCB mixtures containing low and highly chlorinated congeners (Delor 103 and Delor 106 as equivalents of Aroclor 1242 and Aroclor 1260, respectively) was performed using intact mycelium, crude extracellular liquid and enriched MnP and LiP. A decrease in PCB concentration caused by a 44-h treatment with mycelium (74% w/w for Delor 103 and 73% for Delor 106) or crude extracellular liquid (62% for Delor 103 and 58% for Delor 106) was observed. The degradation was not substrate-specific, because no significant differences between the respective degradation rates were observed with di-, tri-, tetra-, penta-, hexa-, hepta-, and octachlorinated congeners. In contrast, MnP and LiP isolated from the above-mentioned extracellular liquid did not catalyse any degradation.     

Manganese regulation of manganese peroxidase expression and lignin degradation by the white rot fungus Dichomitus squalens.

Extracellular manganese peroxidase and laccase activities were detected in cultures of Dichomitus squalens (Polyporus anceps) under conditions favoring lignin degradation. In contrast, neither extracellular lignin peroxidase nor aryl alcohol oxidase activity was detected in cultures grown under a wide variety of conditions. The mineralization of 14C-ring-, -side chain-, and -methoxy-labeled synthetic guaiacyl lignins by D. squalens and the expression of extracellular manganese peroxidase were dependent on the presence of Mn(II), suggesting that manganese peroxidase is an important component of this organism's lignin degradation system. The expression of laccase activity was independent of manganese. In contrast to previous findings with Phanerochaete chrysosporium, lignin degradation by D. squalens proceeded in the cultures containing excess carbon and nitrogen.     

Extracellular oxidative systems of the lignin-degrading Basidiomycete Phanerochaete chrysosporium

The US Department of Energy has assembled a high quality draft genome of Phanerochaete chrysosporium, a white rot Basidiomycete capable of completely degrading all major components of plant cell walls including cellulose, hemicellulose and lignin. Hundreds of sequences are predicted to encode extracellular enzymes including an impressive number of oxidative enzymes potentially involved in lignocellulose degradation. Herein, we summarize the number, organization, and expression of genes encoding peroxidases, copper radical oxidases, FAD-dependent oxidases, and multicopper oxidases. Possibly relevant to extracellular oxidative systems are genes involved in posttranslational processes and a large number of hypothetical proteins.