Adsorption and removal of pentachlorophenol by white rot fungi in batch culture

Pergamon

0043-1354(93)E0018-N

Wat. Res. Vol. 28, No. 7, pp. 1533--1538,1994 Copyright© 1994ElsevierScienceLtd Printed in Great Britain.All rights reserved 0043-1354/94 $7.00+ 0.00

ADSORPTION A N D REMOVAL OF PENTACHLOROPHENOL BY WHITE ROT FUNGI IN BATCH CULTURE BRUCE E. LOGANI*~, BRUCE C. ALLEMAN2, GARY L. AMY30 and ROBERT L. GILBERTSON4 IDepartment of Chemical and Environmental Engineering, University of Arizona, Tucson, AZ 85721, 2Research Scientist, Environmental Technology Department, Battelle, Columbus, OH 43201, 3Civil, Architectural and Environmental Engineering Department, University of Colorado, Boulder, CO 80309 and 4Department of Plant Pathology, University of Arizona, Tucson, AZ 85721, U.S.A. (First received May 1993; accepted in revised form December 1993)

Abstract--The adsorption and removal of pentachlorophenol (PCP) by 12 species of white rot fungi is a function of species and culture conditions. In general, PCP adsorption to mycelia was very low, ranging from 0.01 to 0.05 g PCP g mycelium-i (dry wt basis) at 40 mg PCP l -I, with no apparent correlation between species. After long pre-incubation periods (8-20 d), all species of fungi reduced PCP by >50% within 12 days of PCP addition. Several species of fungi, including Phanerochaete chrysosporium, Trametes versicolor, and all four Ganoderma sp. removed > 50% of the PCP within 24 h, although the largest overall reduction of PCP (96%) was achieved by Inonotus rickii. PCP removal in 250ml flasks by shallow (10 ml) cultures was greater than removal by deep (50 ml) cultures indicating that the ratio of surface area to volume of liquid media is an important factor in the extent of PCP removal by white rot fungi. Key words--adsorption, biodegradation, white rot fungi, pentachlorophenol, Phanerochaete chrysosporium, suspended cultures

INTRODUCTION Many polycyclic aromatic pollutants are aerobically degraded by white rot fungi. During lignolytic metabolism, lignin peroxidase and manganesedependent peroxidases are secreted by white rot fungi to extracellularly degrade lignin in wood (Bumpus et al., 1985; Bumpus and Aust, 1987a; Kersten and Kirk, 1987; Farrell et al., 1989). These enzymes have been implicated in the initial degradative reactions of chlorinated aromatic compounds. Of the more than 1600 species of wood decaying species (Gilbertson, 1980), only a few have been investigated for their potential to mineralize pollutants in engineered systems. The most intensively studied fungus for bioremediation and wastewater treatment is Phanerochaete chrysosporium since it has a high growth rate, exhibits a high lignin degrading capacity, and readily forms conidia (spores) in laboratory culture. Fungi differ from bacteria in that they have complicated growth cycles and grow by hyphal extension and not by binary fission. Some species such as P. chrysosporium preferentially grow at the air-water interface, and in mechanically aerated cultures can *Author to whom all correspondence should be addressed.

fail to secrete lignolytic enzymes and lose their ability to degrade chlorinated aromatic compounds (Kirk et al., 1978; Jiiger et al., 1985; Alleman, 1991). In order to plan for new reactor designs that can accomplish chemical degradation using fungal cultures, factors that enhance chemical degradation by fungi must be better known. The purpose of this investigation was to examine the extent of adsorption and removal of a model aromatic pollutant, pentachiorophenol (PCP), by several different species of white rot fungi. Most of the information reported in the literature is based on the properties of a single species of white rot fungus (P. chrysosporium). For this study, 12 species of fungi were selected based on their exceptional lignin degrading capacity and known responses to PCP toxicity (Lamar et al., 1990; Alleman et al., 1992). Using 10 of these species it is shown that PCP adsorption is relatively unimportant compared to other variables such as fungai species, growth stage, and culture conditions such as volume of liquid media. Although nitrogen concentrations have been found to be important in PCP degradation by P. chrysosporium, it is shown for three other fungal species that nitrogen concentrations are relatively unimportant over the time period necessary for complete removal of PCP from cultures.

1533

1534

BRUCE E. LOGAN et al. Table I. Selected species for PCP degradation and adsorption experiments Species Isolate Phanerochaete chrysosporium Trametes versicolor Ganoderma colossum Ganoderma zonatum Ganoderma Iobatum Ganoderma oregonense Inonotus rickii lnonotus dryophilus Perenniporiafraxinophila Perenniporia ohiensis Perenniporia medulla-panis Perenniporia phloiophila

ME 446 MAD 697 NHC 2959 JPL 1847 RLG 16384 RLG 16381 RLG 16385 RLG 16297 JF 17898 JF 17894 JEA 832 M B 2405

METHODS

Cultures

Twelve species of white-rot fungi were selected for testing based on their known high lignin degrading capacity (Table 1). Phanerochaete chrysosporium ME-446 and Trametes versicolor 697 were obtained from the U.S. Forest Product Laboratory, Madison, Wis. All other species were obtained from the University of Arizona Mycological Collection, Dept of Plant Pathology, University of Arizona, Tucson. Cultures were maintained at room temperature by aseptically cutting and transferring a piece of mycelium growing on agar (2% malt extract) from a mature slant with a dissecting needle to fresh media every 3 0 ~ 0 days. Liquid media contained (per liter of distilled water): 20 g glucose, 2.5g sodium citrate, 5.0g KH2PO 4, 1.78g NH4NO 3, 0.2 g MgSO4.7H20, 0.068 g CaCO 3, and 1 mg thiamine (Kirk et al., 1978) adjusted to a pH of 6 (unless indicated otherwise). PCP remot,al

Fungi were grown in static (non-agitated) 250ml Erlenmeyer flasks at room temperature (23°C) unless indicated otherwise. Rubber stoppers used to seal the flasks were fitted with three-way valves to allow either gas exchange through 0.2/~ m filters or sampling through stainless steel (18 gauge) syringe needles. Flasks containing either 10ml ("shallow" cultures) or 50ml ("deep" cultures) of liquid media were aseptically inoculated (in triplicate) by transferring a 5ram plug cut from the outer edge of an actively growing culture on an agar plate. Liquid cultures were incubated for 8 14d (as indicated) prior to addition of PCP at final concentrations of 5~10mg PCP1 -t. Liquid subsamples (1.5 ml) were withdrawn using a syringe, centrifuged (16,600×g) for 10min, and analysed for PCP. Reported PCP concentrations are averages ( ~< 10% coefficient of variation) of three separate samples unless indicated otherwise. Since others (Mileski et al., 1988) have shown using radiolabeled PCP that removal is correlated to degradation, only removal was quantified in this study, PCP adsorption isotherms

The extent that chemical adsorption to mycelia contributed to PCP removal from solution was examined using adsorption isotherms for 11 species of fungi. Cultures used in adsorption experiments were grown as described above, except that larger culture volumes (100ml) and longer incubation periods were necessary to produce sufficient biomass for adsorption experiments. Most fungi were grown for 20-27 d, but longer incubations were necessary for Ganoderma lobatum and Perenniporia fraxinophila (36 d), G. colossum and Perenniporia phloiophila (43 d), and Perenniporia ohiensis (53d). All cultures used in adsorption experiments were killed by heating the culture in an incubation at 5YC for 24 h. This heating procedure was developed to measure assimilable organic carbon in drinking

water while preventing dissolved organic carbon release from bacteria (van der Kooij et aL, 1982) and was used here to kill the fungi and minimize cellular disruption compared to typical autoclaving procedures used by others (Bell and Tsezos, 1982). Mycelia used in adsorption experiments were separated from culture media by vacuum filtration at 60kPa (450ram Hg) with glass fiber filters (GF/C, Whatman Corp.) and rinsed (4 times) with 200 ml of ultrapure water (Millipore Corp.). Portions of washed mycelial biomass were weighed and added to bottles. Wet weights were converted to dry weights by drying portions of each sample at 105'C for 24 h, cooling in a desiccator, and reweighing until a constant weight (_+ 5%) was obtained. Adsorption experiments were conducted (in triplicate) in 35-ml amber bottles sealed with Teflon-lined screw caps. Each bottle was filled with 25 ml of liquid media (without glucose) containing 80mg PCPI -~, placed on a shaker table for 48h, and then analysed for PCP concentration. PCP adsorption during removal was further examined R~r four species of fungi at four different PCP concentrations. PCP was added to flasks as described above to shallow (10ml of liquid media) cultures after a pre-incubation period of 10d at concentrations of 5, 10, 20 and 40mg PCPI ~. Flasks were incubated for an additional 8 days. and then sacrificed and analysed for PCP concentrations. The percent of adsorbed PCP was calculated from adsorption isotherm data using the residual PCP concentration and the total dry weight of mycelia in the sample. F
Low ratios of nitrogen to carbon in growth media have been shown to enhance the extent of compound mineralization by P. chrysosporium (Kirk et al., 1978). The media used in the experiments described above had an excess of nitrogen, referred to as nitrogen-sufficient media. The effect of nitrogen-limited, or nitrogen-deficient, media on PCP removal was examined for three species of fungi, T. tersicolor, L dryophilus, and G. oregonense. Nitrogen-deficient media was prepared by reducing the concentration of NH4NO 3 to 0.178 g l ~. Cultures were grown (in triplicate) in 10ml of either nitrogen-sufficient or -deficient media for 10 days, dosed with 10 mg PCPI ~, and examined for PCP concentration for 10 days, Analytical methods

PCP was measured at a level of detection of 0.1 mg 1 to within + 5% (coefficient of variation) using high performance liquid chromatography (HPLC, Beckman) equipped with a C-18 reverse phase column (4.6 × 250mm, 5#mEconosphere C-18, Alltech Assoc.). PCP was eluted with a 75:25:0.125 mixture of acetronitrile:water:acetic acid, monitored at 238 nm, and concentrations determined by comparing resulting peak area counts to a standard calibration curve (Mileski et al., 1988). PCP, acetonitrile, and methanol were obtained from Aldrich Chemical Company (Milwaukee, WI). RESL I,TS Ten species o f fungi g r o w n in 5 0 m l o f m e d i a for 1 4 d a n d d o s e d with 8 r a g P C P I ~ r e d u c e d the c o n c e n t r a t i o n o f P C P by 50% 4 days after P C P a d d i t i o n (Fig. 1). Several species o f fungi, including P. chrysosporium, T. t,ersicolor, and all four G a n o d e r m a sp. r e m o v e d m o r e than 50% o f the P C P within I day. T h e largest overall r e d u c t i o n o f P C P (96%) was achieved by 1. rickii. In c o n t r a s t , only 67% o f the P C P was r e m o v e d by the m o r e c o m m o n l y studied species o f white rot fungus, P. chrysosporium.

PCP degradation by fungi

1535

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Fig. 1. Degradation of PCP by 10 species of white rot fungi over a 10 day period. Fungi were grown for 14 d prior to PCP addition in deep (50 ml) cultures. (A) Control (no fungi), Phanerochaete and Trametes sp., (B) Inonotus sp., (C) Ganoderma sp., and (D) Perenniporia sp. (Control has no mycelia. PCP concentrations made on a sample prepared by combining 3 separate flasks.)

The extent of PCP adsorption varied as a function of fungal species and PCP concentration (Fig. 2). In general, there was very little PCP adsorption to the mycelia. All species adsorbed between 0.01 and 0.05 g PCP g mycelium -~ at a PCP concentration of 40 mg 1 J. Some adsorption isotherms (e.g. P. chrysosporium, T. versicolor) were nearly linear while others were curved. The extent of chemical adsorption during PCP removal is shown in Fig. 3 for four species of fungi sacrificed 8 days after PCP addition. At low PCP concentrations (5 and 10mgl - l ) less than 5% of the PCP added to the cultures of P. chrysosporium, L dryophilus, and G. oregonense was adsorbed to the mycelia. PCP adsorption was only a substantial fraction of the total PCP dose at low PCP concentrations for T. versicolor. At the highest PCP concentration of 4 0 m g l -~, the fraction of PCP adsorbed was the lowest for L dryophilus and the highest for G. oregonense. The overall removals of PCP decreased in the order: T. versicolor (84%), P. chrysosporiurn (78%), I. dryophilus (75%), and G. oregonense (57%). These results indicate that adsorption can contribute to overall removal, but that the extent of adsorption and

degradation is a function of chemical dose and species. The use of nitrogen-deficient media did not enhance the total amount or rate of PCP removal by any of three species of fungi. As shown in Fig. 4, PCP removal in nitrogen-deficient media generally lagged behind PCP removal in nitrogen-sufficient media. However, after 10 days all three species of fungi had removed PCP to the level of detection (0.1 mg l-l). Nitrogen-deficient conditions could be considered to improve PCP if removal is expressed on a mass to mass basis (mg PCP removed per mg mycelia). Due to lower nitrogen availability, the nitrogendeficient cultures typically produced less than 50% of the total biomass than the nitrogen-sufficient cultures (Table 2). The ratio of biomass concentrations (mg l - l ) in nitrogen-deficient to nitrogensufficient cultures after 10 days of growth (after complete PCP removal) was 60:25 for I. dryophilus and 50:25 for T. versicolor. Since less mass was necessary to achieve the same PCP removal by day 10, on a mass basis the nitrogen-deficient cultures appear more efficient at PCP removal than the nitrogen-sufficient cultures.

BRUCEE. LOGANet al.

1536

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DISCUSSION All species of fungi examined were able to degrade PCP although the extent of PCP removal was a function of species, adsorption to mycelia, time of 2.5

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Fig. 3. An examination of PCP adsorption during PCP removal for four different fungi: P = P. chrysosporium, I = 1. drysophilus, T = T. versicolor, G = G. oregonense. Measurements were made 8 d after PCP additions to 10 d old (50-ml) cultures. Adsorption calculated from residual PCP concentrations in solution and adsorption isotherms in Fig. 2.

exposure to PCP, carbon to nitrogen ratios in culture media, culture age, and depth of fluid media. For the base conditions of PCP addition after 2 weeks of growth in 50ml of media, the largest observed percent of PCP removal (97%) occurred using L dryophilus, and not the more commonly studied species P. c h r y s o s p o r i u m (Fig. I). Biosorption of chemicals by bacteria is known to be a function of the microorganism and chemical (Bell and Tsezos, 1988). Thus, adsorption isotherms need to be examined on a case by case basis. Of the 11 species examined, the lowest amount of PCP was adsorbed by Perenniporia phloiophila and the highest by P. chrvsosporium. These results indicate that although PCP removal can occur through adsorption the extent of adsorption is small but generally species dependent. The selection of a specific species of fungi for a bioreactor would therefore depend on whether the system is designed for overall chemical removal, or to minimize chemical adsorption to biomass that will ultimately require disposal. The extent of PCP degradation is a function of two time variables: the time of incubation prior to addition of PCP, and the time of exposure after PCP addition. The rate of PCP removal for most species

PCP degradation by fungi 12"

Table 2. Productionof mycelialbiomassby two species of white rot fungi during exposure to 10mgl ~of PCP Biomass (rag 1-i ) Species Nitrogen status Initial Final

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N-sufficientand -deficientrefer to nitrogenconcentrationsin culture media of 1.78 and 0.178g 1-I of NH4NO3. The fungi were dosed with PCP 8 days after inoculation (initial), and analysedon day 10 (final) for biomass.

2l 0

1537

indicating that older cultures had greater potential to remove PCP from solution. An effect of culture age has been observed in other studies using P. chrysosporium (Leisola et al., 1987). The time of production, the concentration, and the types of lignolytic and peroxidase enzymes in solution are a function of the age of the culture (Bumpus and Aust, 1987b; Leisola et al., 1987; Farrell et al., 1989). It may be necessary, therefore, to use cultures at different growth stages and ages in order to maximize chemical removal and degradation. The depth of fluid media is an important factor in chemical degradation by fungi. When fungi were grown in deep (50-ml) liquid suspensions in 250-ml flasks, only one of the 10 species (L rickii) achieved nearly complete PCP removal (Fig. 1). However, all four species grown in shallow (10-ml) suspensions completely removed PCP within 10 d (Fig. 4). It is not clear what the exact reason is for the enhanced growth of P. chrysosporium and T. versicolor in the shallow (10-ml) versus deep (50-ml) cultures (Figs 1 and 4), but oxygen transfer is probably an important factor. Leisola et al. (1983) found that mat formation limited oxygen transfer into the fluid of non-agitated cultures and inhibited lignin degradation by P. chrysosporium. The importance of oxygenated media during PCP removal is u n k n o w n and could not be separated from other experimental

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Fig. 4. PCP degradation by nitrogen-sufficient and nitrogendeficient shallow (10 ml) cultures: (A) L dryophilus, (B) T. versicolor, and (C) G. oregonense. Nitrogen-sufficient (Fq) and nitrogen-deficient ( + ) controls (no fungi); nitrogensufficient ( 1 ) and nitrogen-deficient (&) cultures.

generally decreased with time (Fig. 1). Although culture age was not tested as a variable for species examined in this study, the age of the culture prior to the addition of PCP can be an important factor in PCP removal by white rot fungi. Shown in Fig. 5 are the PCP concentrations measured in P. chrysosporium cultures (50 ml) incubated for 10, 15 and 20 days prior to PCP addition. The a m o u n t of PCP degraded after 60-70 h increased with culture age,

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Fig. 5. PCP degradation by P. chrysosporium cultures as a function of culture age prior to PCP addition (8 mg 1- ~) to deep (50-ml) cultures in nitrogen-deficient media at 37°C. (Control has no mycelia.)

1538

BRUCEE. LOGANet al.

variables in the current study since some species of fungi are adversely affected by fluid agitation. For example, P. chrysosporium grown in mechanically agitated submerged cultures was unable to degrade PCP (Alleman, 1991). Oxygen transfer in the static cultures occurred either by direct utilization by mycelia extending out of the fluid or by diffusion through the surface of the liquid media to submerged mycelia. Increasing the oxygen concentration from 0.2 to 0.8 arm has been found by others to increase lignin-oxidizing activities in fungal cultures (Bar-Lev and Kirk, 1981). However, in experiments with P. ehrysosporium Alleman (1991) tbund that using a pure atmosphere of oxygen increased the initial rate of PCP degradation, but did not alter the final percent of PCP removal after 70 h. Alteration of the total volume of liquid media in the flask also produces different biomass yields per fixed time interval. Since the toxicity and mass of PCP degraded is a function of the dose, expressed as g PCP g mycelia ~ (Alleman et al., 1992), biomass yield is an additional factor influencing the extent of PCP removal by shallow and deep static cultures. These findings suggest that some generalizations can be made concerning factors important for conceptual design of fungal bioreactors. First, long detention times (10-40d from inoculation to complete PCP removal) may be necessary to completely remove chemicals from solution. Detention times can be reduced, however, by decreasing the chemical dose. Second, chemical adsorption should be examined on a case-by-case basis. In this study PCP adsorption was very low, but it was variable with fungal species. Third, the design of systems with nitrogen-limitations in the bioreactor feed may not be useful for long-term operation of bioreactors. Although nitrogen limitations may be useful to stimulate lignolytic enzyme induction, we found for three species that growth in nitrogen-limited media decreased both the initial rate of PCP degradation and the rate of biomass production. Finally, systems should be designed to produce shallow fluid interfaces since shallow culture conditions were shown here to increase the rate of PCP degradation. The reasons for enhanced degradation by shallow cultures is not well understood, however, and needs to be further investigated. Acknowledgements This research was supported by Mclntire-Stennis funds for projects in the University of Arizona Agriculture Experiment Station, a grant from ITT Rayonier Inc., and a Fulbright Grant (to BEL).

REFERENCES

Alleman B. C. (1991) Degradation of Pentachlorophenol by Selected Species of White Rot Fungi. Ph.D., dissertation, Dept of Civil Engin. & Engin. Mechanics, University of Arizona, Tucson. Alleman B. C., Logan B. g. and Gilbertson R. L. (1992t Toxicity of pentachlorophenol to six species of white rot fungi as a function of chemical dose. Appl. era'it. Microbiol. 58, 4048 4050 Bar-Lev S. S. and Kirk T K. (1981) Effects of molecular oxygen on lignin degradation by Phanerochaete chrysosporium, Biochem. Biophys. Res. Commun. 99, 373 378. Bell J. P. and Tsezos M. (1988) The selectivity of biosorption of hazardous organics by microbial biomass. H,~,. Re.s. 22, I245 1251. Bumpus J. A. and Aust S I). 11987a~ Biodegradation of DDT [l,l,l-trichloro-2,2-bis(4-chlorophenyl)ethane] by the white-rot fungus Phanerochaete chrysosporium. Appl. envir. Microbiol. 53, 2001 2(108. Bumpus J. A. and Aust S. D. (1987b) Biodegradation of environmental pollutants by the white rot fungus Phanerochaete chryso,~porium: involvement of the lignin degrading system. BioEssays 6, 166 170. Bumpus J. A., Tien M., Wright D. and Aust S. D. (19851 Oxidation of persistent environmental pollutants by a white rot fungus. Science 228, 1434 1436. Farrell R. L., Murtagh K. E., Tien M., Mozuch M. D. and Kirk T. K. (1989) Physical and enzymatic properties of lignin peroxidase isoenzymes from Phanerochaete chrysosporium. Enzyme Microhiol. Te¢hnol. 11, 322 328. Gilbertson R. L. (1980) Wood-rotting fungi of North America. Mycologia 72, 1 49. J/iger A., Croan S. and Kirk T. K. il985) Production of ligninases and degradation of lignin in agitated submerged cultures of Phanerochaete chrysosporium, Appl. envir. Microbiol. 50, 1274 1278. Kersten P. J. and Kirk T. K. (1987) lnvovlement of a new enzyme, glyoxal oxidase, in extracellular H20~ production by Phanerochaete ~hr~wosporium..L Bact. 169, 2195 2201. Kirk T. K., Schultz E., Conners W. J., Lorenz L. I~. and Zeikus J. G. (1978) Influence of cultural parameters on lignin metabilism by Phanerochaete chrysosporium. Arch. Microbiol. 117, 227 285 van der Kooij D, Vissel A. and Hijnen W. A M. (1982) Determining the concentration of easily assimilable organic carbon in drinking water. J. A W W A 74, 540 545. Lamar R T., Larsen M. ,I. and Kirk "[. K. (1990) Sensitivity to and degradation ot pentachlorophenol by Phanerochaete sp. Appl. envir. Microbiol. 56, 3519 3523. Leisola M., Ulmer D. and Fiechter A, (1983) Problem of oxygen transfer during degradation of lignin by Phanerochaete chryso,~porium. Eur ,1 Appl. Mierobiol. BiotechnoL 17, 113 116. Leisola M. S. A., Kozulic B., Meussdoerffer F. and Fiechter A. (1987) Homology among multiple extracellular peroxidases from Phanerochaete chrysosporium. J. biol. Chem. 262, 419424. Mileski G. J., Bumpus ,1. A., Jurek M. A. and Aust S. D. (1988) Biodegradation of pentachlorophenol by the white-rot fungus Phanerochaete chryso.q3orium. Appl. em, ir. Microbiol. 54, 28852889.