Peroxidase catalyzed co-polymerization of pentachlorophenol anda potential humic precursor

Kengo Morimoto, Kenji Tatsumi*, Ken-Ichi Kuroda

Hydrospheric environmental protection department, national institute for resources and environment, 16-3 Onogawa, Tsukuba, Ibaraki 3058569,

Japan

Accepted 24 January 2000

Abstract

The transformation of xenobiotics in the soil was studied in a model experimental system using pentachlorophenol (PCP), anenzyme, and a potential humic precursor. It has been shown that potential humic precursors such as p-coumaric acid greatly

accelerated the peroxidase-catalyzed reaction of PCP (Morimoto K. and Tatsumi K, 1997. E�ect of humic substances on theenzymatic formation of OCDD from PCP. Chemosphere 34, 1277±1283). About 90% of the PCP reacted but almost notransformation products were detected by GC/MS analysis. Most of the transformed PCP was detected in the polymericproducts by the determination of the adsorbable organic halide (AOX) in gel permeation chromatography (GPC) fractions.

Pyrolysis gas chromatography/mass spectrometry (py-GC/MS) of the polymers showed that tetrachlorophenol (TeCP) was themajor pyrolysis product. This accords with the ion chromatography observation that about 20% of the PCP chlorine wasreleased as chloride ion. However, nitrobenzene oxidation produced only a small amount of PCP and no TeCP. Based on the

selectivity of the two degradation methods, it is concluded that TeCP is bound to the polymer by a diphenylether linkage, andPCP by an ether linkage to the aliphatic side chain. Thus, it was concluded that the major part of the PCP was incorporatedinto the polymer from p-coumaric acid by the release of a chlorine atom from the PCP molecule to form a diphenylether linkage

with the p-hydroxyl group of the p-coumaric acid polymer. 7 2000 Elsevier Science Ltd. All rights reserved.

Keywords: PCP; p-Coumaric acid; Humic substance; Peroxidase; Pyrolysis-GC/MS; Alkaline nitrobenzene oxidation

1. Introduction

Large amounts of toxic chemicals have been appliedas agricultural chemicals or discharged as industriale�uent into the environment. Their fate and the possi-bility of their bioremediation have been an active con-cern.

The transformation of phenolic substances by whiterot fungi may provide a solution for part of this pro-blem. The fungi excrete extracellular phenol-oxidizingenzymes such as laccase or peroxidase, which bringabout the transformation. Peroxidase is a commonextracellular enzyme of white rot fungi but also occurs

elsewhere, e.g. in lignin synthesis, in plants or in thereduction of superoxide in various organisms (Everseet al., 1991). It is known to produce phenoxy radicalsby dehydrogenation of phenolic substances in the pre-sence of hydrogen peroxide, leading to coupling reac-tions which form phenolic polymers such as lignin.

This enzyme also has an important role in humi®ca-tion reactions in soils (Ladd and Butler, 1975; Sul®taand Bollag, 1981), a complex process including bothdegradation and synthesis. The ability of the enzymeto cause phenolic coupling is also likely to allow theimmobilization of toxic phenolic substances such aschlorophenols (Bollag et al., 1987; Ruttimann-Johnsonand Lamar, 1997). In any study of such immobiliz-ation, it is easier to use the isolated enzyme ratherthan fungal cultures, with their attendant growthrestrictions. If the process is to become an instrument

Soil Biology & Biochemistry 32 (2000) 1071±1077

0038-0717/00/$ - see front matter 7 2000 Elsevier Science Ltd. All rights reserved.

PII: S0038-0717(00 )00016 -X

www.elsevier.com/locate/soilbio

* Corresponding author.

E-mail address: tatsumi@nire.go.jp (K. Tatsumi).

for bioremediation, it will also be safer to use the iso-lated enzyme rather than whole micro-organisms.

Pentachlorophenol (PCP) has commonly been usedas a herbicide and it is still used for wood preservationin some countries (Tanjore and Viraraghavan, 1994).It is well known that peroxidase catalyzes the trans-formation of PCP in the presence of hydrogen per-oxide. This reaction is thought to involve thepolymerization of PCP but also yields octachlorodi-benzo-p-dioxin (OCDD) as a by-product (Oberg et al.1990; Oberg and Rappe, 1992). However, this resultwas obtained in an experiment where no humic sub-stances were present. To assess the fate of PCP in soil,it is necessary to consider the participation of humicsubstances in any transformation process (Tatsumi etal., 1994a, 1994b; Roper et al. 1995; Ruttimann-John-son and Lamar, 1996). Model experiments (Morimotoand Tatsumi, 1997) have shown that the oxidation ofPCP in the presence of potential humic precursorssuch as p-coumaric acid or p-hydroxybenzoic acid canremove up to 90% of the PCP. This reaction also sig-ni®cantly reduced the yields of chlorinated quinones ordimers, such as chlorinated dioxins or phenoxyphenols.However, the details of the PCP reaction pathway arenot clearly understood.

We studied the dechlorination and co-polymeriz-ation of PCP with p-coumaric acid, and the resultingchlorine balance. From analyses using alkaline nitro-benzene oxidation and pyrolysis GC/MS, the chemicalstructure of the polymeric product is discussed.

2. Materials and methods

2.1. Materials

Pentachlorophenol and p-coumaric acid were pur-chased from Nacalai Tesque (Kyoto, Japan). Nitro-benzene was obtained from Aldrich Chemical.(Milwaukee, WI, USA.). Horseradish peroxidase(HRP) was obtained from Wako Pure Chemical Indus-try (Osaka, Japan).

2.2. Reaction conditions

To observe any PCP decrease and dechlorination,the reaction solution contained 50 mM PCP, 200 mMp-coumaric acid, 1 unit HRP mlÿ1, 0.5 mM H2O2. ThepH was adjusted to 5, and the mixture was reacted at288C under aerobic conditions using a water bath sha-ker. PCP and chloride ion concentrations in the sol-ution were monitored during the reaction.

To obtain su�cient polymeric product for structureanalysis, the concentrations of the reactants wereraised in a second experiment to 150 mM PCP, 300mM p-coumaric acid, 6 units HRP mlÿ1, 0.9 mM

H2O2. To dissolve this quantity of PCP, acetone wasadded (25% v/v) to the solution.

2.3. Sample preparation

The reaction solution was fractionated by gel per-meation chromatography (GPC) after 0, 5 and 30 minincubation. Fractions (15 ml) were collected and theadsorbable organic halide (AOX, see below) of eachfraction was measured as an index of the total organicchloride.

In a further reaction experiment (500 ml volume), 50ml samples were taken at ®me points 0, 5, 15, 30,60,120 and 240 min. The pH of each sample was raisedto 9 with NaOH to stop the reaction and to dissolvethe transformation products. Then 50 ml samples fromthese solutions were used to determine AOX, and toobtain the polymeric products and the remainder wasdialyzed against water in cellulose tubing (mean porediameter 2.4 nm). The AOX of the dialyzed reactionsolution was measured to evaluate the quantity of PCPthat was incorporated into the enzymatic polymeriz-ation, and the remaining solution was taken to drynessand analyzed by pyrolysis-GC/MS (Meier and Faix,1992). A further 2 l of solution was similarly reactedto obtain 20 mg of polymer products for alkalinenitrobenzene oxidation analysis.

2.4. Analytical methods

PCP in the reaction solution was monitored byHPLC using a Jasco PU-980 (Japan Spectroscopic,Tokyo, Japan) provided with a UV detector and anintegrator. A reverse phase column, Cosmosil 5C18-SL(4.6 mm i.d. � 25 cm, Nacalai tesque, Kyoto,Japan), was used with a 10:90 methanol/0.08% phos-phate bu�er mobile phase (¯ow rate 1.0 ml minÿ1).

The concentration of chloride ions released fromPCP was measured in a DIONEX series 4000i ionchromatograph using a HPIC AS12A column with 0.3mM NaHCO3/2.7 mM Na2CO3 as eluant.

GPC was carried out on Biogel P-30 polyacrylamidegel (Bio-Rad, California, USA) packed in a 2.5 � 50cm column using 50 mM NaOH/50 mM Na2B4O7 aseluant.

To evaluate the quantity of total organic chlorine,the reaction solution was injected into a 3 � 40 mmcolumn of activated carbon powder (40 mg). Afterwashing the column repeatedly with KNO3 to removeinorganic halide ion, the total chloride concentrationof this column was measured by a TOX-10S instru-ment (Mitsubishi Kasei, Tokyo, Japan). The halideconcentration measured as above is called adsorbableorganic halide (AOX) that is used as an index of totalorganic halide.

Following dialysis, the polymeric reaction product

K. Morimoto et al. / Soil Biology & Biochemistry 32 (2000) 1071±10771072

structure was analysed by pyrolysis-GC/MS and bynitrobenzene oxidation, using syringaldehyde as an in-ternal standard. About 100 mg of the material was pyr-olyzed at 5008C for 5 s in a JHP-3 curie pointpyrolyzer (Japan Analytical Industry Co. Ltd.,Mizuho, Tokyo, Japan). The pyrolyzate was directlyinjected into an HP5972 / HP5890 series II GC/MS(Hewlett Packard, Palo Alto, CA, USA) equipped witha HP-1 fused silica capillary column (0.2 mm � 25 m).The temperature of the injector and the detector trans-fer line was kept at 3008C. The column oven tempera-ture was held at 508C for 5 min and then raised to3008C at a rate of 88C minÿ1.

The polymer (20 mg) was also oxidised in alkalinenitrobenzene at 1708C for 2.5 h in an oil bath shaker(Chen, 1992). The resultant mixture was ®ltered andthe acidi®ed ®ltrate was extracted with diethylether.The ether-soluble products were analyzed by GC/MSas described above.

3. Results and discussion

3.1. Incubation analysis

Morimoto and Tatsumi (1997) have shown thathorseradish peroxidase can e�ectively catalyze thetransformation of PCP when reacted with p-coumaricacid. Almost no chlorinated monomeric or dimericproducts were detected. This suggests that the PCPwas incorporated into polymeric structures or furtherdegraded by dechlorination and ring cleavage. Toquantify the degradation of PCP, its concentrationduring the reaction was determined, along with thechloride ion released (Fig. 1). This chloride wasexpressed as a percentage of the total chlorine con-tained in the PCP in the initial solution. On this basis,more than 80% of the PCP disappeared within the®rst 5 min of reaction and more than 90% within 20min. Along with the PCP decrease, 18% of the chlor-ine initially contained in PCP was released as chloride

ion in the ®rst 5 min, but thereafter the process slowedwith the maximum being near 20%, corresponding tothe release of one chlorine atom from each PCP mol-ecule. However, about 70% of the chlorine in the in-itial PCP was not detected in the reaction solution. Itsincorporation in the polymeric products is the onlyremaining explanation for its fate.

3.2. Molecular weight distribution of the chlorinatedreaction products

Dialysis and GPC were used to detect co-polymeriz-ation of PCP as a possible fate of the unknown 70%chlorine. To obtain more products for fractionation,the concentration of PCP in the reaction solution wasraised three-fold. This decreased the transformationyield and 15% of the initial PCP was left unchangedfor 30 min after the reaction started.

Fig. 2 shows the AOX change in the reaction sol-ution and the dialysis residue along with the residualPCP concentration. The AOX of the reaction solutionwas about 20±30% less than the initial concentrationof PCP, corresponding to a dechlorination of nearly20%. The AOX of the dialysis residue was slightlymore than 50%. Since this fraction cannot passthrough the 2.4 nm pore-sized dialysis tubing, thesemolecules should contain at least 5 benzene rings. Thedi�erence in AOX between the reaction solution andthe dialysis residue should correspond to the amountof organic chlorine (including that of residual PCP) inproducts smaller than 2.4 nm.

Dialysis showed most of the transformed PCP wascoupled with p-coumaric acid to form oligomeric orpolymeric products. GPC was used to determine themolecular size distribution of these products in thereaction solution and the AOX of each GPC fractionis shown in Fig. 3. Most of the organic chlorine wasdetected in two peaks, corresponding to the exclusionlimit and low molecular weight peaks. The latter peakwas found to be residual PCP by comparing AOXwith the PCP concentration of the same fractions

Fig. 2. Adsorbable organic halide (AOX) change of the reaction sol-

ution and the dialyzed reaction solution during incubation.

Fig. 1. The disappearance of PCP and the production of chloride ion

during enzymatic transformation of PCP and p-coumaric acid.

K. Morimoto et al. / Soil Biology & Biochemistry 32 (2000) 1071±1077 1073

measured by HPLC, using the AOX content of 150mM PCP solution as a standard. Another peak ofalmost the same AOX content appeared at the exclu-sion volume, which corresponds to a 40,000 Da pro-tein standard. Allowing for the physical di�erencesbetween phenolic polymers and protein molecules, thispeak suggests that the polymer contained more than200 monomer units, and the detection of AOX fromthis fraction indicates the incorporation of PCP intothat polymer, since PCP was the only chlorine sourcein the solution mixture.

HPLC analysis showed the total absence of p-cou-maric acid after 2 min of reaction, at the point wherePCP just started to decrease. In addition, preliminaryexperiments showed that when PCP was reacted withthe polymer resulting from a 10 min previous reactionof p-coumaric acid, the PCP reduction rate was almostthe same or even a little faster than with the p-couma-ric acid monomer. These results suggest that p-couma-ric acid ®rst polymerized and then the PCP was boundto that polymer, a mechanism that also explains theabsence of dimeric or trimeric products includingchlorine. No oligomers or dimers were present even inthe very early stages of the reaction, despite severalattempts to obtain dimeric or trimeric product.

3.3. Characterization of the polymerized product

Chromatograms of the nitrobenzene oxidation pro-ducts from the 30 min-reaction product are shown inFig. 4(a) and the pyrogram is shown in Fig. 4(b). Theonly chlorinated substance produced by nitrobenzeneoxidation was PCP and the yield was 2% of the initialsolution. In contrast, pyrolysis produced a consider-able amount of tetrachlorophenol (TeCP) and also asmall amount of trichlorophenol (TrCP) and PCP, thePCP is almost the same yield as in the nitrobenzeneoxidation. The detection of TeCP means one chlorine

atom of PCP was lost at the binding site where PCPforms a linkage with the polymer.

Fig. 5 shows the variation of each chlorinated pyrol-ysis product in the dialyzed reaction solution whileFig. 2 shows the AOX variation in the same incu-bation. The total chlorophenol recovery at 120 minwas about 18% of the initial solution and 35% of thedialysis residue AOX. This yield is appropriate consid-ering that the recovery of phenolic substances from lig-nin in general is about 30% (Lam et al., 1990; Kurodaet al., 1994).

TeCP was the major organohalogen detected, corre-sponding to the observation that nearly 20% of thechlorine was released as chloride ion from the trans-formed PCP. The concentration of pyrolysis productsincreased for the ®rst 2 h of reaction, but decreasedafterwards. However the AOX of the dialysis residueshowed no such decline (Fig. 2). One possible expla-nation is that the polymer became more resistant topyrolysis as the reaction proceeded, indicating a con-tinuous condensation within the polymer structureeven after most of the monomers were consumed.

Phenol oxidizing enzymes such as peroxidase pro-duce phenoxy radicals through dehydrogenation of thehydroxyl group (Courteix and Bergel, 1995a, 1995b).This radical is resonance-stabilized as shown in Fig. 6for p-coumaric acid. Here radical coupling occurs atthe p-hydroxyl group, the m-position of the benzenering and the b-carbon of the side-chain.

Fig. 7 shows the probable linkage in an analogouslignin structure and the possibility of their cleavage bynitrobenzene oxidation and pyrolysis. L2 and L4 areC±C linkages, presumed to form enzymatically,between PCP and p-coumaric acid. These linkages arenot cleaved either by nitrobenzene oxidation or by py-rolysis. Among the other probable linkages (L1, L3and L5), nitrobenzene oxidation would cleave etherlinkages between aliphatic and aromatic structures(L1) but not diphenylether bonds (L3 and L5 Ð Villeret al., 1997). Thus, the PCP in both the nitrobenzeneoxidation and the pyrolysis products would have origi-nated from the aliphatic side-chain. As stated earliermost of the p-coumaric acid might have been polymer-ized before the incorporation of the PCP. In this case,the side-chain of p-coumaric acid would have beentransformed beforehand. However, some aliphatic car-bons in the side-chains or in bridges between benzenerings would still be capable of radical coupling withPCP as exempli®ed by the L1 linkage.

On the other hand, would TeCPs detected only bypyrolysis were probably derived from a diphenyletherlinkage, because if they were on the side-chain theymust also be cleaved and detected by nitrobenzene oxi-dation. Pyrolysis of the diphenylether linkage isthought to be di�cult and has not been studied indetail. However, when pyrolyzed, some lignin dipheny-

Fig. 3. AOX of gel chromatography fractions (Bio-Gel P-30) from

reaction solutions.

K. Morimoto et al. / Soil Biology & Biochemistry 32 (2000) 1071±10771074

lethers were cleavable to a remarkable extent. In ourhands, 52% of 2-(4-carboxy-2-methoxyphenoxy)-4,5-dimethoxybenzoic acid was cleaved at 5008C, andaround 32% of 3-(4-carboxy-2-methoxyphenoxy)-4,5-dimethoxybenzoic acid. Diphenylethers including apolychlorinated phenol such as PCP may cleave readily

due to the reduction of the ring aromaticity by thechlorine substituents.

The oxygen of the ether linkage connecting TeCP tothe polymer must have come from p-coumaric acid orits polymer, because any linkage through the hydroxylgroup of PCP will cleave to form PCP and not TeCP.

Fig. 4. GC/MS chromatogram of the oxidation products from the polymer produced by the peroxidase catalyzed transformation of PCP and p-

coumaric acid. The degradation methods were (a) nitrobenzene oxidation, and (b) pyrolysis.

K. Morimoto et al. / Soil Biology & Biochemistry 32 (2000) 1071±1077 1075

The phenolic p-hydroxyl group (L5) is the only pointwhere p-coumaric acid or its polymer can supply thephenoxy radical to form a diphenylether. This corre-sponds with the observation (Morimoto and Tatsumi,1997) that the enzymatic transformation of PCP washighly a�ected by a methoxyl group at the meta pos-ition of the humic precursors. This can be explainedby the steric hindrance between the m-methoxyl groupsand any incoming PCP molecule approaching the p-hydroxyl group.

Thus, we can conclude that the major part of thePCP enzymatically transformed with p-coumaric acidwas bound to the polymer through diphenylether lin-kages at the p-hydroxyl groups of p-coumaryl subunitsand the remaining PCP was bound to their aliphaticside chains by an ether linkage.

From the direct measurement of chemicals in soil, itis known that the major part of PCP applied to farm-land is retained in the soil and does not easily leachout (Weiss et al., 1982). However the mechanism ofretention is not known but adsorption to the soil or-ganic matter is thought to be one of the major causes(Christodoulatos et al., 1994; Christodoulatos andMohiuddin, 1996). The enzymatic binding of xeno-biotics into humic substances has also been highlighted(Bollag et al., 1992), but in the case of PCP, enzymecatalyzed association with ferulic or syringic acid wasquantitatively not so e�ective. Ruttimann-Johnson and

Lamar (1996) reported on the ability of manganeseperoxidase to polymerize PCP with ferulic acid, but lit-tle attention was paid to p-coumaric acid although it isthe most abundant humic precursor in various kindsof soils and sediments (Shindo et al., 1978; Matsumotoand Hanya, 1980). Our research shows that peroxidasecan e�ectively catalyze the linkage of PCP to p-couma-ric acid polymer, principally through a diphenyletherlinkage between the p-hydroxyl group of p-coumaricacid and the benzene ring of PCP by dechlorination atthe point of linkage. These results reinforce the im-portance of enzymatic coupling of PCP with humicsubstances in soil as an immobilization process.

Acknowledgements

We thank Dr Hiroshi Oi (Tsukuba University) forhis comments about the interpretation of chromato-

Fig. 5. The amounts of pyrolysis products from the dialyzed reaction

solution and their change with incubation.

Fig. 7. Structural analysis of the polymeric product from p-coumaric

acid and PCP. PCP detected by nitrobenzene oxidation and pyrolysis

GC/MS; TeCP detected only by pyrolysis GC/MS.

Fig. 6. Resonance stabilization of the dehydrogenation product from p-coumaric acid.

K. Morimoto et al. / Soil Biology & Biochemistry 32 (2000) 1071±10771076

graphic results and Dr Hamish Anderson of theMacanley Land Use Research Institute, Aberdeen forhis generous help with the preparation of the ®nal ver-sion of our paper.

References

Bollag, J.M., Chen, C.M., Sarkar, J.M, Michael, J.L, 1987.

Extraction and puri®cation of a peroxidase from soil. Soil

Biology & Biochemistry 19, 61±67.

Bollag, J.M., Myers, C.J, Minard, R.D, 1992. Biological and chemi-

cal interactions of pesticides with soil organic matter. Science of

the Total Environment 123/124, 205±217.

Chen, C.L, 1992. Nitrobenzene and cupric-oxide oxidations. In:

Stephen, Y.Lin, Carlton, W.Dence (Eds.), Methods in Lignin

Chemistry. Springer Verlag, Berlin, pp. 301±321.

Christodoulatos, C., Kor®atis, G.P., Talimcioglu, N.M, Mohiuddin,

M, 1994. Adsorption of pentachlorophenol by natural soils.

Journal of Environmental Science and Health. Part A:

Environmental Science and Engineering 29, 883.

Christodoulatos, C, Mohiuddin, M, 1996. Generalized models for

prediction of pentachlorophenol adsorption by natural soils.

Water Environment Research 68, 370±378.

Courteix, A, Bergel, A, 1995a. Horseradish peroxidase catalyzed hy-

droxylation of phenol. Part 1: Thermodynamic analysis. Enzyme

and Microbial Technology 17, 1087±1093.

Courteix, A, Bergel, A, 1995b. Horseradish peroxidase - catalyzed

hydroxylation of phenol. Part 2: Kinetic Model. Enzyme and

Microbial Technology 17, 1094±1100.

Everse, J., Everse, K.E., Grisham, M.B. (Eds.), 1991. Peroxidases in

Chemistry and Biology, vols. I and II. CRC Press, Boca Raton.

Kuroda, K., Yamaguchi, A, Sakai, K, 1994. Analysis of Sugi wood

and its lignin preparations by pyrolysis-gas chromatography.

Mokuzai Gakkaishi 40, 987±995.

Ladd, J.N, Butler, J.H.A, 1975. Humus-enzyme systems and syn-

thetic organic polymer-enzyme analogs. In: Paul, E.A., McLaren,

A.D. (Eds.), Soil Biochemistry, vol. 4. Marcel Dekker, New

York, pp. 143±194.

Lam, T.B.T., Iiyama, K, Stone, B.A, 1990. Lignin in wheat inte-

nodes. Part 2: Alkaline nitrobenzene oxidation by wheat straw

lignin and its fractions. Journal of the Science of Food and

Agriculture 51, 493±506.

Matsumoto, G., Hanya, T., 1980. Gas chromatographic-mass spec-

trometric identi®cation of phenolic acids in recent sediments.

Journal of Chromatography 93, 89±94.

Meier, D, Faix, O, 1992. Pyrolysis-gas chromatography-mass spec-

trometry. In: Stephan, Y.L., Carlton, W.D (Eds.), Methods in

Lignin Chemistry, vol. 34. Springer Verlag, Berlin, pp. 177±199.

Morimoto, K, Tatsumi, K, 1997. E�ect of humic substances on the

enzymatic formation of OCDD from PCP. Chemosphere 34,

1277±1283.

Oberg, L.G., Glas, B., Swanson, S.E., Rappe, C, Paul, K.G, 1990.

Peroxidase-catalyzed oxidation of chlorophenols to polychlori-

nated dibenzo-p-dioxins and dibenzofurans. Archives of

Environmental Contamination and Toxicology 19, 930±938.

Oberg, L.G, Rappe, C, 1992. Biochemical formation of PCDD/Fs

from chlorophenols. Chemosphere 25, 49±52.

Roper, J.C., Sarkar, J.M., Dec, J, Bollag, J.M, 1995. Enhanced enzy-

matic removal of chlorophenols in the presence of co-substrates.

Water Research 29, 2720±2724.

Ruttimann-Johnson, C, Lamar, R.T, 1996. Polymerization of penta-

chlorophenol and ferulic acid by fungal extracellular lignin-

degrading enzymes. Applied and Environmental Microbiology 62,

3890±3893.

Ruttimann-Johnson, C, Lamar, R.T, 1997. Binding of pentachloro-

phenol to humic substances in soil by the action of white rot

fungi. Soil Biology & Biochemistry 29, 1143±1148.

Shindo, H., Ohta, S, Kuwatsuka, S, 1978. Behavior of phenolic sub-

stances in the decaying process of plants. IX Distribution of phe-

nolic acids in soils of paddy ®elds and forests. Soil Science and

Plant Nutrition 24, 223±243.

Sul®ta, J.M, Bollag, .M, 1981. Polymerization of phenolic com-

pounds by a soil-enzyme complex. Soil Science Society of

America Journal 45, 297±302.

Tanjore, S, Viraraghavan, T, 1994. Pentachlorophenol-water pol-

lution impacts and removal technologies. International Journal of

Environmental Studies 45, 155±164.

Tatsumi, K., Freyer, A., Minard, R.D, Bollag, J.M, 1994a. Enzyme-

mediated coupling of 3,4-dichloroanilline and ferulic acid: a

model for pollutant binding to humic materials. Environmental

Science and Technology 28, 210±215.

Tatsumi, K., Freyer, A., Minard, R.D, Bollag, J.M, 1994b.

Enzymatic coupling of chloroanilines with syringic acid, vanillic

acid and protocatechuic acid. Soil Biology & Biochemistry 26,

735±742.

Viller, J.C., Caperos, A, Garcia-Ochoa, F, 1997. Oxidation of hard-

wood kraft-lignin to phenolic derivatives. Nitrobenzene and cop-

per oxide as oxidants. Journal of Wood Chemistry and

Technology 17, 259±285.

Weiss, U.M., Scheunert, I., Klein, W, Korte, F, 1982. Fate of

pentachlorophenol-14C in soil under controlled conditions.

Journal of Agricultural and Food Chemistry 30, 1191±1194.

K. Morimoto et al. / Soil Biology & Biochemistry 32 (2000) 1071±1077 1077