Vol. 57, No. 10APPLIED AND ENVIRONMENTAL MICROBIOLOGY, OCt. 1991, p. 2858-28630099-2240/91/102858-06$02.00/0Copyright © 1991, American Society for Microbiology

Degradation of Organochlorine Compounds in Spent SulfiteBleach Plant Effluents by Actinomycetes

BRUNO WINTER,* ARMIN FIECHTER, AND WOLFGANG ZIMMERMANN

Institute of Biotechnology, ETH-Honggerberg, CH-8093 Zurich, Switzerland

Received 19 February 1991/Accepted 10 June 1991

Actinomycetes isolated from different soil samples were tested for their abilities to utilize spent sulfite bleacheffluents from a paper mill. Degradation and dechlorination of the chlorinated compounds in the effluents of thefirst, two bleaching stages, i.e., chlorination stage [(C + D)red.] and alkaline extraction stage (E10), weremonitored by determining total organic carbon (TOC) and activated-carbon-adsorbable organic-boundhalogen (AOX). The isolates showed increased degradation rates after repeated incubations in the effluent-containing medium. Separation of the culture supernatants by ultrafiltration into three fractions of differentmolecular weights revealed substantial AOX and TOC reductions in the low-molecular-weight fraction. TheAOX values of the higher-molecular-weight fractions were also reduced. Extracellular peroxidase and cellwall-bound catalase activities were produced during growth of the microorganisms on bleach effluents.

-During cellulose pulp production, lignin is removed in thecooking stage and in subsequent bleaching stages, where inconventional bleaching, chlorolignins as well as low-molec-ular-weight chlorinated-aromatic and aliphatic compoundsare formed. Most of the chlorinated compounds are pro-duced in the first chlorination stage, (C+D)red, and in thesubsequent alkaline extraction stage, E10, of the bleachingsequence (19, 22). Many of the low-molecular-weight com-pounds, especially chlorinated guaiacols and phenols, aretoxic to the aquatic environment (2, 10, 15). These com-pounds can be partially removed from the effluents in thewastewater treatment plant by a coupled anaerobic-aerobicprocess (18). However, under aerobic conditions, chlori-nated guaiacols and phenols can be 0-methylated to thecorresponding anisoles and veratroles, which are more re-sistant to biodegradation and have a higher potential forbioaccumulation in fish (7, 20, 21). The medium- and high-molecular-weight compounds are only slightly modified asthey pass through the wastewater treatment plant into thereceiving river, where they release toxic and lipophiliclow-molecular-weight compounds during their slow decay(7). 2,4,6-Trichlorophenol, 3,4,5-trichloroguaiacol, and tet-rachloroguaiacol have been shown to accumulate in fish (15).The amounts of organochlorine compounds (measured asactivated-carbon-adsorbable organic-bound halogen [AOX])and dissolved organic carbon (DOC) from five pulp millsdischarging into the River Rhine have been calculated to be2,100 tonnes of AOX and 33,000 metric tons of DOC peryear. This would account for 90% of the total AOX load and10% of the DOC load of the Rhine (25a). Worldwide, anestimated 250,000 metric tons ofAOX are produced per year(14). A reduction in the amount of chlorinated compounds,especially those of higher molecular weights, in bleach planteffluents is highly desirable and demands an improved waste-water treatment procedure.

In nature, actinomycetes are involved in the degradationof lignocellulose (26, 27). Their ability to produce lignin-degrading enzymes has been demonstrated (26). Other re-search work has shown their ability to degrade chlorinatedaromatic compounds and has elucidated the degradation

* Corresponding author.

pathways of some model compounds (6, 9, 12, 13, 20, 25).Actinomycetes could therefore be candidates for the biolog-ical treatment of spent bleach plant effluents.

In the present investigation, the microbial conversion ofchlorinated bleach plant effluents by actinomycete isolateswas studied to obtain information on changes in the chloro-lignin fractions and to identify extracellular enzymes pro-duced during growth on spent sulfite bleach plant effluents.

MATERIALS AND METHODS

Microorganisms and culture maintenance. Actinonmyceteswere isolated by selective isolation procedures (16, 17) from16 soil samples obtained from various places in SoutheastAsia and southern Europe. Stock cultures of the isolateswere maintained at 4°C after growth and sporulation at 30 or37°C on MS agar (20 g of mannitol, 20 g of soya, and 20 g ofagar per liter of deionized water), DSM (German Collectionof Microorganisms, Braunschweig) medium 53 corynebacte-rium agar (10 g of casein peptone [tryptic digest], 5 g of yeastextract, 5 g of glucose, 5 g of NaCl, and 15 g of agar [adjustedto pH 7.2 to 7.4] per liter of deionized water), no. 65streptomyces medium (4 g of glucose, 4 g of yeast extract, 10g of malt extract, and 12 g of agar [adjusted to pH 7.2] perliter of deionized water), or no. 83 Czapek peptone agar (30g of saccharose, 2 g of KNO3, 1 g of K2HPO4, 0.5 g ofMgSO4 7H20, 0.5 g of KCl, 0.01 g of FeSO4. 7H20, 2 g ofyeast extract, 5 g of peptone, and 15 g of agar [adjusted to pH7.3] per liter of deionized water). Liquid cultures consistedof 0.3 g of NaNO3, 0.66 g of K2HPO4- 3H20, 0.01 g ofCaCl2, 5 g of glucose, 5 g of D-mannitol, 2 g of yeast extract,1 ml of trace element solution (11), and 8.75 ml of 0.5 Mphosphate buffer (pH 6.0) per liter of deionized water.Arthrobacter picolinophilus (DSM 20665) was obtained

from the German Collection of Microorganisms and main-tained at 4°C after growth at 30°C on DSM medium 53.

Screening procedure. Experiments were carried out withpure cultures. Sampling and substrate additions were madeunder aseptic conditions, and appropriate noninoculatedcontrols were included. Screening for growth on sulfite spentbleach plant effluents from the first two stages of a conven-tional bleaching sequence was performed on agar platescontaining a mixture of 166 ml of E10-stage effluent, 333 ml

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TABLE 1. AOX, TOC, and CU contents of fractions of E1O-and (C+D),ed.-stage bleach effluents"

Mol wt Contents at stage:fractions E,O (C+D)redAOXb 120.9 98.6L 15.2 21.2M 49.9 40.2H 45.2 31.1

TOC' 1,295 653L 470 438M 405 125H 420 90

CU 6,194 3,152L 470 432M 1,163 784H 4,561 1,936

"After adjustment to pH 7.0 and filtration through 8-,um-pore-size cellulosenitrate filters, the filtrate was separated into three molecular weight fractions,i.e., L (<1,000), M (1,000 to 10,000), and H (>10,000), by ultrafiltrationthrough Amicon membranes PM 10 and YM 2.bIn mg of Cl liter-.In mg of C liter-

of (C+D),ed -stage bleach effluent, 500 ml of double-distilledwater, and 20 g of agar per liter of medium. Isolates growingon this medium were subcultured on the same medium toobtain spores to be used as inocula for liquid cultures. Theliquid medium for degradation experiments contained 14.8 gof Na2HPO4 2H20, 2.3 g of KH2PO4, 5 g of yeast extract,2 g of (NH4)2SO4, 0.05 g of Ca(NO3)2, 1 ml of trace elementsolution (11), 333 ml of (C+D),ed.-stage bleach effluent, 166ml of E10-stage bleach effluent, and 500 ml of double-distilled water per liter. The E1O/C+D ratio of the mediumreflects the conditions found in the effluents of the papermill. Liquid cultures for degradation experiments and pro-duction of extracellular enzymes were performed in 500-mlshaking flasks with 180 ml of medium at 30 or 37°C, respec-tively, and 150 rpm. The inoculum size for isolates growingas single cells gave a final cell density of about 107/ml ofmedium. The inoculum sizes of isolates forming mycelialpellets could not be determined. Autoclaved media wereused for adaptation and maintenance of the isolates. Cul-tures used for measuring AOX, total organic carbon (TOC),and DOC reductions and extracellular peroxidase activitiescontained nonautoclaved bleach effluents. The media had apH ofpH 7.2, which was readjusted with 3 N HNO3 every 24to 36 h.

Substrates and chemicals. Bleach effluents were from apaper mill (Cellulose Attisholz AG, Luterbach, Switzer-land). (C+D)red.-stage bleach effluent contained spruce andbeech chlorolignins, and E10-stage bleach effluent containedbeech chlorolignin only. The distribution of AOX, TOC, andcolor unit (CU) values in the molecular weight fractions ofthe two bleach effluents is given in Table 1. The pulp millused a bleaching sequence of (C+D)red, E10, (H+ D), E,and then D. All other chemicals used were from Aldrich,Merck, or Sigma and were analytical grade.Enzyme assays. (i) Peroxidase and phenol oxidase. After

being separated from the cells by centrifugation and pHadjustment to pH 7.0, culture supernatants were routinelyassayed for peroxidase and phenol oxidase activities. Allassays were performed at 40°C. Final volumes of 1.0 ml ofthe reaction mixtures contained (i) 100 mM phosphate buffer

(pH 7.0), 1 mM L-3,4-dihydroxyphenylalanine (L-DOPA),and 4 mM H202 (23); (ii) 100 mM acetic acid-acetate buffer(pH 5.5), 48 p.M o-dianisidine, and 4 mM H202 (3); (iii) 50mM morpholineethanesulfonic acid (MES)-NaOH buffer(pH 5.5), 320 p.M syringaldazine (dissolved in ethanol; thefinal ethanol concentration in the assay was 10%), and 4 mMH02 (4); (iv) 100 mM sodium succinate buffer (pH 5.5), 82mM 4-aminoantipyrine, 1 mM 2,4-dichlorophenol, and 4 mMH202 (23) and (as part of all assays) 750 pl of enzymesolution. The reaction was initiated by the addition ofhydrogen peroxide solution for peroxidase assays, and theincreases in A470, A460, A525, and A510, respectively, weremonitored for 5 min, starting 30 s after the addition ofhydrogen peroxide. Phenol oxidase assays were started bythe addition of enzyme solution. One unit of enzyme activitywas expressed as the conversion of 1 pLmol of substrate permin. The following extinction coefficients were used:L-DOPA r470, 3,088 M-1 cm-'; o-dianisidine r460, 24,039M-1 cm-'; syringaldazine r525, 31,659 M-1 cm-1; and2,4-dichlorophenol M510, 21,647 M-1 cm-'.

(ii) Catalase. Catalase activity in culture supernatants andcell suspensions was measured with a Rank Brothers (Bot-tisham, England) Clark-type oxygen electrode. Appropriatevolumes of enzyme preparation or cell suspension werebrought to 50°C and degassed by being purged with nitrogen.After dilution with 50 mM phosphate buffer (pH 7.0) to afinal volume of 2 ml in a temperature-controlled measuringcell, the reaction was initiated by the addition of 20 pu1 of 0.1M sodium perborate solution. Reading of the electrodecurrent started 15 s after the addition of sodium perborate.Calibration of the oxygen electrode was done with catalase(from beef liver; catalog no. 106810; Boehringer MannheimGmbH) and various amounts of sodium perborate (8, 24).Cells were treated for 30 s by ultrasonification at 50 W(Branson Sonifier B-12; Branson, Danbury, Conn.) in a 50mM phosphate buffer (pH 7.0) containing 0.3% CHAPS[3-(3-cholamidopropyl)dimethylammonio-1-propane sulfonate;Serva, Heidelberg, Germany] to release cell wall-boundcatalase.Chemical analysis. Determination of AOX in the culture

supernatant was done after removal of the cells by centrifu-gation. Organochlorine compounds associated with cells ormycelium during the growth in bleach plant effluent-con-taining medium were determined by direct application ofwashed cells to the AOX analyzer. Analysis was performed(according to DIN 38409, part 14 [la]) with an AOX analyzer(Euroglas, Delft, The Netherlands).

Determinations of TOC and DOC were performed simul-taneously with a Hydromat (Westhoff GmbH Me,Btechnik,Bochum, Germany). In contrast to the standard TOC andDOC analysis methods, filtration of the sample through0.45-p.m-pore-size membranes was omitted because of ad-sorption of AOX material on the membrane.

Culture supernatants were separated by molecular weightinto three fractions by using ultrafiltration in a 400-mlAmicon cell (W. R. Grace & Co., Amicon Div., Danvers,Mass.) with Amicon membranes PM 10 (10,000 cutoff) andYM 2 (1,000 cutoff). Samples were adjusted to pH 7.0 andfiltered through 8-pm-pore-size cellulose nitrate membranes(Sartorius, Gottingen, Germany) prior to ultrafiltration. Themedium was cencentrated 10-fold and washed four times byrefilling the cell with water purified with a NANOpuresystem (Barnstead Thermoline Corp., Dubuque, Iowa) to itsoriginal volume and then concentrated. The efficiency of thewashing steps was controlled by measuring the conductivityof the filtrate.

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X 1000

c: 80:EC)0E 604)

40

20

0

x 00C4

cm0

0 80

60

40

BW42 108 154 171 184 212 A.pic.FIG. 1. Percentage of remaining AOX in the bleach effluent

medium incubated with BW 42, BW 108, BW 154, BW 171, BW 184,BW 212, and A. picolinophilus (A. pic.) at the end of the first (C1) andthird (U) 21-day incubations. All strains were incubated at 30°C.AOX was measured in the cell-free culture supernatant.

CU were measured according to the Canadian Pulp &Paper Association standard method (lb).

0

1 00 Cm0ou0CDcmcc

C)

80 000

60 e)

40

40

0 j 6 9 12 15 1 8 21Time [days]

FIG. 2. Time course of AOX, TOC, and DOC biodegradation inthe bleach effluent medium inoculated with isolates BW 22, BW 93,BW 184, BW 214, and A. picolinophilus. All strains were grown at30°C. AOX values are shown for isolates BW 22 (0), BW 93 (A),BW 184 (O) and BW 214 (V) and for A. picolinophilus (O), and thecorresponding TOC (V) and DOC (0) values are shown for isolateBW 214. AOX, TOC, and DOC values were determined in thesupematant after the cells were removed by centrifugation.

RESULTS

Isolation of actinomycetes. Isolation of actinomycetes from16 different soil samples resulted in 217 cultures, mainlystreptomycetes, growing at 30 and 37°C.

Screening and adaptation on bleach plant effluents. A totalof 208 actinomycete isolates and A. picolinophilus weretested for growth on agar plates containing bleach planteffluents. Of these isolates, 66 did not grow on the agarplates. A. picolinophilus and 105 of the isolates which grewwell on the agar plates were selected for further tests inliquid medium. Measurement of the remaining AOX con-tents at the end of the first and third incubation periods of 21days each revealed for isolates BW 42, BW 108, BW 154,BW 171, BW 184, and BW 212 and for A. picolinophilus anincrease in degradation rates (Fig. 1). Release of organicallybound chlorine increased during the adaptation period from12 to 45%, corresponding to reductions of 7.4 to 25 mg of Clliter-', respectively. Isolate BW 184 showed the highestreduction (27.2%, or 14.4 mg of Cl liter-') in AOX contentduring the first incubation period. Uninoculated controlsshowed a 10% decrease in AOX content due to autohydrol-ysis within 21 days.Time course of AOX, TOC, and DOC reductions. Figure 2

shows the decrease ofAOX in the culture supernatants ofA.picolinophilus and isolates BW 22, BW 93, BW 184, and BW214 and the corresponding TOC and DOC values of isolateBW 214. Most of the microbial AOX degradation, about 18to 28%, corresponding to 9 to 13.8 mg of Cl liter-', occurredduring the first 24 h. Further reductions of 12% (9 mg of Clliter-') could be measured during the following 48 to 72 h. Aslight increase of 2 to 3 mg of Cl liter-1 in the culturesupernatants was observed at the end of the incubationperiod. Figure 2 includes TOC and DOC values for isolateBW 214. Major reductions took place during the first 24 h,when TOC values decreased to 35.5% of the initial value(from 2,250 to 800 mg of C liter-l) and DOC decreased to36.8% of the initial value (from 5,950 to 2,190 mg of 02

liter-'). AOX, TOC, and DOC values behaved similarlyover the whole incubation period. The same pattern wasobserved for the other isolates (data not shown).Some cultures were inoculated with a spore suspension

(AOX-free inoculum) to estimate the amount of loss of AOXin the supernatant due to association with the cells byadsorption, undegraded intracellular metabolites, or entrap-ment in the microbial mycelium. The results indicated thatonly 3.5 to 4.9% of the initial AOX content becomes asso-ciated with the cells.Changes in AOX, TOC, and CU contents of three bleach

effluent molecular weight fractions. Ultrafiltration of the twobleach effluent fractions and the medium resulted in threemolecular weight fractions: L (<1,000), M (1,000 to 10,000),and H (>10,000). Table 2 lists the AOX, TOC, and CUcontents of the culture supernatant molecular weight frac-tions at the end of a 21-day incubation. AOX values de-creased in all three molecular weight fractions. AOX valuesin the L fractions decreased as much as 35 to 59% (4.7 to 7.9mg of Cl liter-'), and the AOX for A. picolinophilus wasbelow the detection limit. The corresponding TOC valuesdecreased by 68.8 to 94.4% (1,035 to 1,421 mg of C liter-').Expressed as a percentage, this decrease is much higher thanthat for the corresponding AOX values. In the M fractions,AOX values decreased by 28 to 70% (4.6 to 9.2 mg of Clliter-'). The corresponding TOC values decreased similarly,by 61.3 to 82.7% (230 to 310 mg of C liter-'). AOX values inthe H fractions decreased by 10 to 30% (1.7 to 6.6 mg of Clliter-'), whereas the TOC values increased up to 228.2%(from 195 to 445 mg of C liter-') of the initial value. All TOCvalues for H fractions at the end of the 21-day incubationwere above the initial value.The total CU in the culture supernatants of all isolates

increased during the incubation period. The increases intotal CU varied between 790 and 2,260 CU. L- and M-frac-

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TABLE 2. AOX, TOC, and CU contents of molecular weight fractions of bleach effluent medium at the end of 21-day incubation

Mol wt Incubation witha:fraction Control' A. picolinophilus BW 42 BW 93 BW 154 BW 171 BW 184 BW 212

AOXcL 13.5 ND 6.8 6.1 6.1 5.6 8.8 7.3M 16.0 4.8 11.2 10.3 10.9 11.2 8.2 11.4H 19.3 15.3 16.4 12.7 12.9 13.6 12.6 17.6

TOCdL 1,505 113 140 98 104 95 144 121M 375 75 100 85 90 90 65 110H 195 445 215 285 315 335 245 265

CUTotal 4,179 4,967 5,063 5,711 6,281 5,147 6,436 6,012L 1,134 418 431 446 362 361 704 625M 1,731 849 1,182 996 1,030 1,011 916 1,186H 1,314 4,746 3,356 3,620 4,318 4,909 3,526 4,625

a All strains and the contol were incubated at 30°C. ND, not detected.b Uninoculated medium.c In mg of Cl liter-.d In mg of C liter-'.

tion CU decreased by 430 to 1,000 CU, but H-fraction CUincreased by 2,042 to 3,595 CU.

Extracellular enzyme activities. Decreases in AOX andTOC values during growth on bleach effluents for com-

pounds with molecular weights above 1,000 were expectedto involve extracellular or cell wall-bound enzymes. Isolatesgrowing on bleach plant effluents were analyzed for extra-cellular peroxidase and phenol oxidase activities, withL-DOPA, syringaldazine, o-dianisidine, and 2,4-dichlorophe-nol used as substrates. Of 106 organisms, 47 showed perox-

idase activities in the culture supernatants with L-DOPA,reaching activity levels ofup to 24 mU ml-'. Only a few alsoexhibited low activities with 2,4-dichlorophenol, o-dianisi-dine, or syringaldazine as substrate, with activity levels of0.5 to 5 mU ml-'. Figure 3 shows the time courses ofperoxidase activities with L-DOPA as substrate in culturesupernatants of isolates BW 1, BW 9, BW 42, and BW 206

1 8

E 16

v 0122 64 0728 6182

0 8

C')

x

'IO4

0)

0 12 24 36 48 60 72 84 96 108 120

Time [hours]

FIG. 3. Time course of peroxidase activities in the culture super-natants of isolates BW 1 (0), BW 9 (A), BW 42 (O), and BW 206 (0)grown in the bleach effluent-containing medium. Isolates BW 1 andBW 9 were grown at 37°C, and BW 42 and BW 206 were grown at30°C. Peroxidase activity was measured with L-DOPA as substrate.

grown on the bleach effluents. Maximum L-DOPA-peroxi-dase activities were reached during the exponential growthphase after 24 to 48 h of incubation. The appearance of thisperoxidase activity and the reductions in AOX and TOCvalues coincided. The peroxidase activity measured with2,4-dichlorophenol as substrate reached its maximum duringthe late exponential growth phase about 96 h after inocula-tion. Most of the isolates showed extracellular phenol oxi-dase activity with 2,4-dichlorophenol as substrate, reachingactivity levels of up to 3 mU ml-'. Only a few also exhibitedphenol oxidase activities when L-DOPA or syringaldazinewas used as substrate. Phenol oxidase activity reached itspeak levels at 3 to 7 days after inoculation during the lateexponential and stationary growth phases.

Catalase activities could also be observed in culture su-pernatants. Maximum activity of 1.5 U ml-' was measuredin the culture supernatant of BW 42 grown on bleacheffluent-containing medium. Assays with cell suspensionsshowed that catalase activities were also associated with thecells. Only a small proportion of this catalase activity couldbe released from the cells by ultrasonic treatment in phos-phate buffer containing 0.3% CHAPS.

DISCUSSION

The data presented in this paper demonstrate the ability ofactinomycetes to degrade and partly dehalogenate chlori-nated compounds in bleach effluents. The strains wereisolated under nonselective conditions and had not previ-ously been in contact with bleach effluents. After repeatedincubations of several of the strains in bleach effluent-containing medium, increased AOX reduction rates werefound, indicating the ability of the organisms to adapt to theconditions prevailing in the medium. This AOX reduction inthe supernatants is not due to adsorption to the cells orautohydrolysis of unstable organochlorine bonds. The char-acterization of the degraded bleach effluents which wereseparated into three fractions by molecular weight revealeddifferent patterns of AOX and TOC reductions. Majorchanges occurred in the L and M fractions, in which theAOX, TOC, and CU values were reduced during the incu-bation period. The bleach effluents and the yeast extract,

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which serves as an additional C source, contribute to theTOC value measured in the medium. Therefore, the initialmolar TOC/AOX ratio, an indicator of the average degree ofhalogenation, was calculated from the TOC values of thecorresponding bleach effluent molecular weight fractions.Assuming a total consumption of yeast extract in the L andM fractions during the incubation period, the TOC/AOXratios increased in the L fractions in cultures of A. picolino-philus and isolate BW 42 and in all of the M fractions. Thisindicates a lower average degree of halogenation of theremaining compounds in the medium after treatment withthe organisms. The ratio stayed constant in the L fractionsfor all other isolates. With the exception of that of A.picolinophilus, no L fractions showed total AOX reductions.0-Methylation of phenolic compounds, which can be con-stitutively performed by actinomycetes, leads to the forma-tion of chloroveratroles and chloroanisoles (1, 20, 21). Theseconversions could have occurred in the medium and contrib-uted to the remaining AOX in the L and M fractions. In theH fractions, AOX reductions could be observed, but TOCvalues were increased. This indicates that the TOC reduc-tions detected in the other fractions could be partly due tocondensation reactions. Polymerization leading to com-pounds of higher molecular weights has not yet been re-ported to occur by treatment of bleach effluents with actino-mycetes. Other groups (1, 7, 20) have reported formation ofchloroguaniacols and its 0-methylation products by treat-ment of bleach effluent fractions above 1,000 molecularweight (H+M) with Arthrobacter spp. and other actino-mycete species. Eriksson et al. (7) reported only minor TOCreductions in H+M chlorolignin fractions during a 40-dayincubation. Another indication for modifications in the Hfractions and/or condensation of low-molecular-weight com-pounds is the observed increases in CU. Replacement ofchlorine substituents by hydroxyl groups and formation ofquinones increases the CU of the chlorolignin fractions.Chromophore contents in the M and L fractions weresubstantially reduced despite a strong increase in overallCU. Compounds in the H fractions are most likely attackedby extracellular or cell wall-bound enzymes. These modifi-cations also alter the solubility and hydrophobicity of thecompounds. The different degradation patterns found in thethree molecular weight fractions could indicate the involve-ment of enzymes in the degradation and dehalogenation ofbleach effluents.Four phenol oxidase-peroxidase substrates were used for

the determination of extracellular enzyme activities in theculture supernatants of organisms grown on bleach effluents.Mainly peroxidases were found when L-DOPA was used assubstrate, and low levels of peroxidases and phenol oxidasescould be detected when 2,4-dichlorophenol was used assubstrate. The isolates grown on chlorolignin showed peaksof peroxidase activity during their growth phase after 24 to48 h of incubation when L-DOPA was used as substrate.Major AOX and TOC reductions occurred during the sameperiod. The 2,4-dichlorophenol-peroxidase and 2,4-dichlo-rophenol-phenol oxidase activities appeared during the lateexponential and stationary growth phases, during which onlyminor AOX reductions occurred, indicating no major role forthese phenol oxidases and peroxidases in the degradation ofthe chlorinated compounds in bleach effluents under comet-abolic conditions. Horseradish peroxidase and a laccasefrom streptomycetes are known to polymerize chlorophe-nols with the release of chloride (5).

ACKNOWLEDGMENTS

We thank Urs Gage, Philip Lang, and Eugen Krebser, CelluloseAttisholz AG, for supplying the effluents and for measuring AOXand TOC.

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