Bio Remediation of Pentachlorophenol

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Land Contamination & Reclamation, 7 (4), 1999 1999 EPP Publications

Bioremediation of Pentachlorophenol

Pollution by the Fungus CoriolusVersicolor

Millie A. Ullah and Christine S. Evans

Abstract

Pentachlorophenol (PCP) is a persistent pollutant arising from its use as a wood pre-

servative and pesticide. Ligninolytic enzymes secreted by wood rotting fungi effect PCPremoval. Coriolus versicolor, a white-rot species, is one of the most effective

lignin-degraders mainly through secretion of the polyphenol oxidase, laccase and man-

ganese-dependent peroxidase. Purified laccase removed 100% of low concentrations

(25 ppm) of PCP and 40% of 200 ppm PCP in 24 hours.

Purified manganese peroxidase also removed PCP but less effectively than laccase,

with 15% removal of 50 ppm PCP and 10% removal of 100 ppm in 48 hours. Laccase

and manganese peroxidase combined did not enhance the removal rate for PCP. Cul-

tures ofC. versicolor in which laccase activity was the predominant activity were there-

fore investigated for use in solid state fermentations for PCP removal. Maximum activity

of laccase was produced after four weeks growth on wheat husks, which resulted in

100% removal of 200 ppm PCP. Some PCP bound to the wheat husks and the fungal

mycelium. PCP polymers formed by the action of laccase, in combination with its bind-

ing to the solid substrate permitted complete removal of PCP from solution with solidsubstrate cultures ofC. versicolor.

Key words: Coriolus versicolor; pentachlorophenol; laccase; bioremediation

INTRODUCTION

Pentachlorophenol (PCP) is one of the most recalci-

trant chemicals polluting the environment, introduced

by man as a pesticide and wood preservative (Bollag

1992). It is resistant to removal by abiotic degradation

with only a few biotic organisms having the potential

for its degradation. These organisms include the

wood-rotting basidiomycetes responsible for white-rot

decay, with Coriolus versicolorbeing one of the most

effective wood degraders. The ligninolytic ability of

this fungus is due to the production of laccases and

manganese-dependent peroxidases that produce highly

reactive free radical species that can attack aromatic

molecules bearing some similarity to lignin (Kirk and

Farrell 1987; Lamar 1992).

Laccase is a copper-containing polyphenol oxidase

that occurs in several forms, including blue laccases

type I and II produced in liquid cultures (Fahraeus and

Reinhammar 1967; Evans 1985) and yellow-brown

isomers produced during growth on solid substrates

(Iimura et al. 1995; Leontievsky et al. 1997). Previous

studies of laccase with PCP have produced conflicting

conclusions; Konishi and Inoue (1972) reporting ben-

zoquinones as breakdown products of reaction and Ric-cota et al. (1996) concluding that laccase did not play

an integral role in PCP mineralisation, and that cou-

pling of reaction products occurred to produce poly-

meric products. Using a purified laccase from C.

versicolor, the predominant end product from reaction

with PCP was a high molecular mass polymer (Ullah et

al. 1999).

Less chlorinated phenolics than PCP are more read-

ily degraded by laccase. The transformation efficiency

of chloride release has shown that mono- and di-chlo-

rinated phenols are more readily dechlorinated than tri-

and penta-chlorophenols (Roy-Arcand and Archibald1991; Kadhim et al. 1999).

Received September 1999; accepted September 1999

Authors

Millie A. Ullah and Christine S. Evans, Fungal BiotechnologyGroup, University of Westminster, London W1M 8JS


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Land Contamination & Reclamation / Volume 7 / Number 4 / 1999

White-rot species, particularly Phanerochaete

chrysosporium, have been used for decontamination of

PCP-polluted soils and aqueous effluents (Lamar and

Dietrich 1990; Alleman et al. 1995). The predominant

ligninolytic enzymes from P. chrysosporium are lignin

peroxidase and manganese-dependent peroxidases.

When white rot fungi are used for bioremediation of

soils contaminated with PCP, a dramatic decrease in

extractable PCP occurs. A large proportion of unde-

graded PCP is converted into soil-bound transforma-

tion products, which are not extractable with organic

solvents (Ruttiman-Johnson and Lamar 1996, 1997).

When fungi grown on solid substrates are used for

bioremediation of soils and water effluents, it is impor-

tant to understand the extent of physico-chemical bind-

ing to solid particles, to assess removal of pollutants

such as PCP.In this study we have used wheat husks as solid sub-

strate for growth ofC. versicolorto immobilise the fun-

gus and stimulate laccase production, for optimisation

of bioremediation of PCP in aqueous effluents.

MATERIALS AND METHODS

Culture conditions

Stock cultures ofCoriolus versicolor(FPRL-28A) sup-

plied by CABI, Egham, Surrey, UK, were maintained

on 3% malt2% agar slopes stored at 4C. Plate cul-tures of the same medium were incubated for seven

days at 26C.

Enzyme assays

Laccase activity was measured according to Evans and

Palmer (1983) using catechol as substrate, with one

unit of activity defined as a change in absorbance at

440 nm of 1.0 min-1 ml-1 at 25C. Manganese peroxi-

dase activity was measured by transformation of 1 mM

guaiacol in 40 mM phosphate buffer pH 5.5, with 20

mM citric acid, 0.1 mM MnSO4 and 50 M H2O2. Oxi-

dation of guaiacol was measured at 465 nm, with 1 unitof activity causing a change in absorbance of 1.0 min-1

ml-1 at 25C (Martinez et al. 1996).

Analysis of PCP

Quantification of PCP was by HPLC, using a reversed

phase C18 column (25 x 0.46 cm) with a mobile phase

of acetonitrile: water: acetic acid (75: 25: 0.125).

Detection of PCP was at 254 nm, with calibration linear

in the range 20175 g ml-1 (Ullah et al. 1999).

Reactions of laccase and MnP with PCP

Stock solutions of PCP were prepared in 33% ethanol.For activity measurements, PCP replaced catechol and

guaiacol in the laccase and MnP assays respectively.

Control reaction mixtures included either boiled

enzymes or were prepared without enzymes.

Bioreactor studies

Solid-substrate cultures, grown on wheat husks for four

weeks, were added as inoculum to 1 litre shaken flask

cultures containing 400 ml of water and PCP in 1 ml of

ethanol (50200 ppm). Control fermentations were

inoculated with uninfected wheat husks or autoclaved

wheat husk cultures. The size (510% w/v) and age (0

6 weeks) of inoculum were evaluated. Scale-up of

shaken flask cultures was to a 5 litre stirred tank reactor

containing 4 litres of water and PCP in 10 ml of ethanol

(200 ppm) by fed batch operation.

RESULTS AND DISCUSSION

To determine the action of laccase on PCP, etha-

nol-water was used as solvent for PCP as its solubility

in water is only 0.014 ppm at 25C (Crosby 1981). Eth-

anol affected laccase activity, with increasing concen-

trations of ethanol in the assay mixture with catechol,

reducing enzyme activity (Figure 1). To solubilise up to

200 ppm PCP, 33% ethanol was required, which gave

approximately 50% reduction in laccase activity. With

100 ppm PCP and 20% ethanol in the reaction mixture

with 100 units of laccase, over 95% of PCP wasremoved over 48 hours. Increasing ethanol to 50%

reduced the reaction rate, with ~10% PCP removed

from the reaction over 48 hours, while at 33% ethanol

50% PCP was removed. With 100 units of laccase,

reaction with PCP could be measured over a range of

substrate concentrations of 25200 ppm. Previous

studies have shown that the major reaction product is a

high molecular mass polymer, with trace amounts of

benzoquinones (Ullah et al. 1999).

Reactions of PCP with MnP showed that less PCP

was removed from reaction mixtures than had occurredwith laccase. From 50 ppm PCP, only 15% was

Figure 1. Effect of ethanol concentration on laccase

oxidation of catechol at 440 nm


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Bioremediation of Pentachlorophenol Pollution by the Fungus Coriolus Versicolor

degraded by 45 units of MnP in 48 hours and


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a total of 800 ppm PCP from solution over twelve days.

Other reactor designs such as the bubble column reac-

tor by Smith and Valsaraj (1997), achieved 94%

removal of pentachlorophenol. Katagiri et al. (1995)

used a SSF system for treatment of KRAFT pulp by C.

versicolorand P. chrysosporium. Alleman et al. (1995)

described a novel bioreactor design with rotating tubes

of fungi immobilised in steel tubes. C. versicolorhas

been known to have the greatest resistance to PCP tox-

icity, and the greatest degree of PCP dehalogenation,

indicated by the production of chloride. Bioreactorscan be manipulated by batch, fed batch or continuous

operational strategies that enable maximum removal of

contamination to be achieved.

Successful PCP biodegradation has been achieved

by continuous operation of fluidised bed enrichment

cultures (Melin et al. 1997). Continuous operation can

be advantageous, as the need to stop and start the biore-

actor could be reduced or even eliminated. However,

the most important feature is the maintenance of a via-

ble biofilm culture at high contaminant concentrations.

Biofilm processes are more stable in treatment of inhib-

itory substrates, and high loading rates can be achieved.One of the best examples of this are fluidised bed reac-

tors (FBR). FBRs achieve higher loading rates and

removal efficiencies than other types of biofilm (Melin

et al. 1997, 1998). Another method for maintaining a

microbial population is by recycling biomass

(Konopka et al. 1996). Laugero et al. (1997) compared

the degradation of radioactive PCP and its metabolite

pentachloroanisole (PCA) by static and agitated immo-

bilised cultures ofP. chrysosporium. It was found that

greater PCA accumulated in static cultures compared

to agitated cultures, due to the metabolism not allowing

any storage of intermediate metabolites. Although suchprocesses are extremely useful for decontamination of

effluent streams, overloading can lead to process fail-

ure and recovery is often very slow. Discontinuous

addition of reactants allowed increased turnover time,

by maintaining viability of the culture, as shown in the

bioreactor studies described in this paper. Aqueous fer-mentations ofP. chrysosporium have been employed

for modelling of the mechanisms involved in PCP

removal by cell mass and enzyme enhancement cul-

tures (Lin et al. 1990).

SSF are applicable to the clean-up of contaminated

soil by in situ treatment, in a manner that can be both

cost-effective and environmentally friendly. If the con-

tamination is resistant to attack by indigenous organ-

isms it has been demonstrated that white rot fungi can

be inoculated into soil to initiate microbial attack

(Bumpus and Aust 1986; Field et al. 1993). To main-

tain microbial cultures it is necessary to supplement thesoil with plant derived carbon sources, such as cereal

Figure 4. Effect of increasing age of inoculum on the

removal of PCP by 20g and 10g of wheat husk

Figure 5. Removal of PCP at increasing concentrations by

wheat husk inoculated withC. versicolor 50 ppm;

100 ppm; 150 ppm; and 200 ppm

Figure 6. Continuous bioreactor for removal of PCP [4 x 200

ppm] by inoculated wheat husk. inoculated PCP;

uninoculated PCP; laccase activity

0

20

40

60

80

100

0 55 110 165 220 275 330 385

Time [hrs]

PCPRemoval%

0

0.02

0.04

0.06

0.08

0.1

0.12

LaccaseActivityUnits

P C P

P C PP C PP C P


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Bio Remediation of Pentachlorophenol