New approach to improve degradation of recalcitrant azo dyes by Streptomyces spp. and Phanerochaete chrysosporium

New approach to improve degradation of recalcitrant azo dyes by Streptomyces spp. and Phanerochaete

chrysospormm

A. Paszczynski, M. B. Pasti, S. Goszczynski, D. L. Crawford and R. L. Crawford D e p a r t m e n t o f Bacteriology a n d Biochemistry, Colh, ge o f Agricttlture a n d C e n t e r fi~r H a z a r d o u s W a s t e R e m e d i a t i o n R e s e a r c h , University o f Idaho, M o s c o w , I d a h o

Three azo dyes, the commercially available aeid yellow 9(4-amino-l, 1 '-azobenzene-3,4'-disulfonie acid), and two synthesized dyes, azo dye 1 [4-(3-methoxy-4-hydroxyphenylazo)-azobenzene-3,4'-disulfonie acid] and azo dye 2 (3-methoxy-4-hydroxy-azobenzene-4'-sulfonic acid), plus sulfanilic acid attd vanillic acid, were tested as substrates for degradation by 12 Streptomyces spp. and the white-rot fttngus Phanerochaete chrysosporium. None of the Streptomyccs spp. degraded acid yellow 9 or suIjanilie acid. The linkage of a guaiacol molecule onto acid yellow 9 or sulfanilie acid via azo-linkages resulted in dyes that were decolorized by 5 o f the 12 Streptomyces strains. These Streptomyces were those that could also attack uanillic acid, which has the same ring substitution pattern (4-hydroxy-3-methoxy) as guaiaeol. While P. chrysosporium tran,~fi~rmed both acid yellow 9 and sulfanilic acid, the two guaiaeof substituted azo dyes were deeolorized more readily by P. chrysosporium than the corresponding ansubstitated nzoleeuh's. Ligninase and manganese peroxidase preparations fi'om the P. chrysosporium culture were inuolued in the degradation. The diseouery that one may enhanc'e the degradahility of azo dyes by linking selected, readily degradabh" substitaents into the dyes' c'hemical structure, is a novel observation. This approach shouhl be considered for use in commercial production of azo dyes or other reeah'itrant compounds as a way o f producing eompoands that are more easily degraded in the environment.

Keywords: Azo dyes; Phanerochaete chcvsosporium; Streptomyces; ligninase; manganese peroxidase; biodegradation; decolorization

Introduction The importance of dyes to human civilization, both ancient and c o n t e m p o r a r y , is well documented. Recent history has shown a switch from natural to synthesized dyes. Our knowledge about the synthesis of azo dyes is well established, and each year new azo dyes are being developed, primarily for industrial use. ~Dyestuff and textile industries are major producers and, respcclively, users of azo dyes, and effluents from these in-

Address reprint requests to Dr. Crawford at the Department of Bacteriology and Biochemistry, College of Agriculture and Center for Hazardous Waste Remediation Research, University of Idaho, Moscow. Idaho 83843, USA Paper No. 90517 of the Idaho Agricultural Experiment Station. Received 24 July 199(I; revised 10 October 1990

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dustrial processes are usually resistant to biological treatment. Costly physical-chemical decontamination processes arc often the only treatment alternatives available for such wastewaters. -~Azo linkages and aromatic sulfo groups are generally not synthesized by living organisms, and our knowledge about biodegradalion of these c o m p o u n d s in nature is limited. An interest in understanding the biodegradation of environmentally hazardous c o m p o u n d s has sparked efforts to isolate microorganisms that are able to utilize azo dyes as carbon sources. Efforts to isolate such microorganisms have been largely unsuccessful; however, azo dye-degrading bacteria, specifically P s e u d o m o n a s strains, have recently been isolated from chemostat cultures. 3 The degradation mechanism describcd tbr thc P s e u d o m o n a s involved an oxygen-insensitive azoreductase which catalysed the reductive cleavage of the azo group using NAD(P)H as an elec-

(c 1991 Butterworth-Heinemann

Degradation of recalcitrant azo dyes." A. Paszczynski et aL tron donor 4. Various anaerobic bacteria have been reported to degrade azo dyesS6: however, tinder aerobic conditions these dyes have been considered to bc essentially nonbiodegradablc. 27 Rccently, however, Cripps et al. ~ found that the fungus Phanero('haete chrysosporium aerobically degrades polycyclic hydrocarbons containing azo and sulfo groups. These authors showed that the ligninolytic system of this fungus is in some way involved in the degradation of three azo dyes: Tropaeolin O, Congo Red, and Orangc 11. They described several unidentified mctabolites of these compounds after incubation with crude ligninasc preparations, but the possible mechanism of degradation was not explained. It also has bccn shown that P. chrvsosporium is able to mineralize chloroaniline/lignin conjugates9 and xenobiotic molecules bound to humic acids. ~0 In the present study, we have examined a new approach to increase the susceptibility of azo dyes to degradation by aerobic microorganisms, specifically soil Streptomyces spp. and Phanerochaete ('ho'sosporium. In this approach, an azo dye is made more dcgradable by introduction of a metabolizable subslitucnt into the dye's chemical structure. This approach to biodegradation studies with azo dyes also revealed information on the enzymatic mechanisms ofaxo dye degradation by filamentous microorganisms.

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Materials and methods

Substrates Sulfanilic acid, guaiacol, sodium nitrite, and 4-hydroxy-3-methoxybenzoic acid (vanillic acid) were purchased from Aldrich Chemical Co. The azo dyc 4amino-I,l'-azobenzene-3,4'-disulfonic acid (acid yellow 9) was purchased from Sigma Chemical Co. Two additional azo dyes wcrc synthesized by attaching guaiacol through an azo linkage to acid yellow 9, forming 4-(3- met hoxy-4- hydroxyphenylazo)-azobcnzcne-3,4' disulfonic acid (azo dye 1), or to sulfanilic acid, forming 3-methoxy-4-hydroxy-azobenzene-4'-sulfonic acid (azo dye 2). Structures and MS fragmentations of the dyes are shown in Figure la and b. Both synthesized dyes were also chcckcd for purity by TLC and HPLC analysis. No significant impurities were detected. From HPLC integration data, the purity of the synthesized dyes was approximately 98% for azo dye 1 and 97% for azo dye 2.

Synthesis o f azo dye I 4-Amino-azobenzcne-3,4'-disulfonic acid sodium salt (0.76 g) was dissolved in 5% sodium hydroxide (8 ml), and a solution of sodium nitrite (0.14 g in 0.5 ml of water) was added. Crushed ice (10 g) and concentrated HCI (i.8 ml) were introduced to the solution, which was then vigorously stirred for 15 min. To the cooled guaiacol solution (0.25 g dissolved in 3.2 ml 5% sodium hydroxide) the diazotised yellow 9 solution was added portionwise within 15 min under mechanical stirring. Saturated sodium chloride solution was added (15 ml),

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and the mixture was left fi)r crystallization overnight at 5°C. The cryslallinc product was filtered, washed with acetone and ethcr, and dried in air. Dark brown crystals (0.98 g) were collected (86.6% of theoretical yield).

Synthesis o f azo dye 2 Sulfanilic acid (I .73 g) was suspcndcd in 23 ml of water, and 8 ml of 5% NaOH was addcd. The mixture was stirred until the acid dissolvcd and then sodium nitrite solution (0.7 g in 2 ml H:O) was added. The solution was poured on a crushed ice (25 g) and concentrated HCI (2 ml) mixture and mixed until copious precipitation took place (KJ starch test was positive). The diazotized sulfanilic acid was added portionwise to the cooled guaiacol solution (I.24 g in 20 ml 5% sodium hydroxide) with stirring. NaCI (20 g) was added and stirring was continued for 30 min at room temperature. The crystalline deep orange precipitate was filtered off

Enzyme Microb. Technol., 1991,vol. 13, May 379

Papers and washed with ethanol and ether; 2.62 g of the product was obtained (64.4% of theoretical yield).

plates. The medium also contained 120 mg 1- ~ of specific azo dye.

Microorganisms and culture maintenance

Spectrophotometric assay

Twelve wild-type aclinomycetcs were selected from 20 strains isolated from higher termites in Kenya. ~ All strains have been identified as Strt7~tomyces. a'- based on the key of Williams et al. t3 Streptomyces uiridosporus T7A (ATCC 39115) was isolated from soil by D.I,. Sinden (M.S. thesis. University of Idaho, Moscow, 1979). Streptomyces badius 252 (ATCC 39117) was isolated from soil by Phelan et al. J4 and Streptomyees SR-10 is a protoplast fusion recombinant derived from a cross between S. uirhtosporus T7A and S. setonii 75Vi2. ~ Stock cultures of the Kenyan isolates were maintained at 4°C, after growth and sporulation at 37°C on the following medium (g I ~ of dcionized water): NH4NO ~, 1: KH,P() 4, 0.4: yeast nitrogen base (Difco), 0.67; yeast extract (Difco). 0.2; lactose, 15; bacto-agar (Difco), 18. S. uiridosporus T7A, S. badius 252, and Streptomyces SR-10 were maintained at 4°C, after growth and sporulation at 37°C on yeast extractmalt extract dextrose agar. ~' Stock cultures wcre subcultured every 2 to 10 weeks, and distilled water suspensions of sporulated growth were used as initial inocula in all experiments. Phanerochaete cho'sosporium BKM-|:-1767 (ATCC 24725) was received from The Forest Products l,aboratory, Madison, WI. The fungus was maintained and spore inocula were prepared as previously described. 17

A I-ml sample of actinomycete culture medium was centrifuged and then diluted 2.5-fold with water, or 1.0 ml of fungal supernatant was centrifuged and diluted fivefoid with 10 mM sodium 2,2-dimethylsuccinate buffer (DMS) of pH 4.5. Azo dye substrate present was then measured spectrophotometrically (HewlettPackard 8452 diode array spectrophotometer operated by PC Vectra computer equipped with HP's MS rMDOS/UV-VIS software). In order to be sure that changes in substrate spectra were not due to pH variations, the effects of pH on the visible absorption o f each compound were also assayed within physiological pH range in the culture media. While the spectra of sulfanilic acid (Max abs at 250 nm), vanillic acid (Max abs at 252 and 286 nm), and acid yellow 9 (Max abs at 386 nm) were unaffected by pH over the tested pH range, the spectra of the two novel azo dyes were changed (shifts of their Absm, 0. Thus, the spectrophotometric assays for these dyes were carried out at their isosbestic points. These were, respectively, 450 nm for azo dye 1 and 400 nm for azo dye 2.

Culture conditions Each Streptomyces sp. was grown in a cotton-plugged 250-ml flask containing 25 ml of the following medium: 0.2 M Tris buffer (pH 7.6), I00 ml; vitamin-free Casamino acids (Difco), 1.0 g: thiamine, 100/xg: biotin, 100 /zg; o-glucose, 2 g; deionized water, 900 ml. Thiamine. biotin, and o-glucose were filter sterilized and added to the autoclaved medium. ~ The dyes were filter sterilized and added at 0.005% (w/v) to the autoclaved basal medium. Three replicates of every culture were incubated, and each strain was grown in media supplemented individually with every substratc. Replicate sterile controls also wcrc run in each experiment. Cultures were incubated at 37°C for 14 days with shaking (2(X) rev min ~). Three replicates for each strain grown in only the basal medium were incubated as well. P. eho'sosporium was grown in a cotton-plugged 500-ml flask containing 250 ml defined medium, j'j with the addition of 75 mg adenine (6-aminopurine) and 27 mg L-phenylalanine per liter. This addition accelerated the growth of the fungus without inhibiting ligninase activity. Four substrates were tested: sulfanilic acid, acid yellow 9, and the two synthesized azo dyes; each was separately added at a concentration of about 0.02% (w/v). Cultures were incubated at 37°C for 7 to 15 days with shaking (250 rev min J). Solid agar media were also employed. The medium was 3.0% (w/v) malt extract (Difco l,aboratorics) agar dispensed in pctri

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lligh petformam'e liquid chromatography (llPLC) analysis Degradation of the dyes and aromatic compounds was confirmed by HPI,C. A Hewlett-Packard HP I090 Liquid Chromatograph equipped with a HP 40 diode array UV-VIS detector and automatic injector was used. The chromatograph was controlled by an HP 9000 series 300 computer which used HP 7995 A ChemStation software. A reverse phase column from Phenomenex (Rancho Palos Verdes, CA, type Spherex 5 C 18 size 250 x 2.0 mm, s/no PP/6474A) was used. Each 15-rain analysis used a solvent gradient of acetonitrile (solvent A) and 10 mM DMS buffer pH 4.5 (solvent B), with the following conditions: 0-5 rain, 100% A; 5-12 min, 25% A, 75?4, B: 12-15 rain, 100% B: post time 2 rain, injection volume 10/zl. Absorption was measured at 250, 325,350,400, and 450 nm, and spectra were collected automatically by the peak controller.

Preparation o f enzymes and enzyme assays Streptomyces spp. peroxidascs were prepared and assayed using 2,4-dichlorophenol (2,4-DCP) (Sigma) as substralc, as previously described. 2" P. chrvsosporium BKM-F-1767 was grown in a 21)-I carboy containing I 1 o f nitrogen-limited defined medium (BlI-medium), as described earlier. 2~ Preparation and assay of ligninase and manganese peroxidase from these P. cho'sosporittm cultures were carried out as previously reported. 22 Results

Biotran,~formation Table I shows the substrate utilization pattern of the Streptomyces spp. after a growth period of 14 days.

Degradation of recalcitrant azo dyes: A. Paszczynski et aL Table 1 Substrate r e m o v e d (%) by cultures of Streptomyces spp. and Phanerochaete chrysosporium during a g r o w t h period of, respectively, 14 days and 7 days. Growth condition described in Materials and m e t h o d s Sulfanilic acid

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Only six strains, A4, AI0, A l l , AI2, AI3, and AI4, significantly degraded vanillic acid, while none degraded sulfanilic acid or acid yellow 9 to a detectable extent. This confirms that the compounds characterized by aromatic sulfo group and azo linkages are quite recalcitrant. However, five strains, AI0, A l l , A12, AI3, and AI4, significantly decolorized the two new azo dyes. Moreover, azo dye 2 was decolorized by these strains to a large extent than azo dye 1. l"i~,,ure 2 shows the patterns of removal from the culture supernatant of each compound by strain S. chromoJics¢'us A I 1 versus time, as a typical example. P. cho'sosporium transformed sulfanilic acid and decolorized acid

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yellow 9, but only to a limited extent. The fungus almost completely decolorized (90%) azo dyes I and 2 alter a growth period of 7 days (/qA,ure 3). There was a characteristic lag of 80-90 h prior to decolorization of any of the compounds by the fungus, due to its slower growth rate than the , ~ ' I r e p l o m y ( ' e s . O n solid medium, P. chrvsosporium behaved similarly, leaving some undegraded color after 2 weeks of growth. In all cases, the mycelium remained colorless, indicating that no dye absorption to the mycelium had occurred.

Oxidation by enzyme preparations We were unable to show detectable decolorization of the substrates by extracellular enzyme preparations of the Streptomyces species.

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TIME (days) T r a n s f o r m a t i o n of vanillic acid (.- I), sulfanilic acid (V), acid y e l l o w 9 (O), and azo dye 1 (@) and 2 (T), by Streptomyces A l l g r o w n in agitated flasks at 37°C f o r a period of 14 days. M e d i u m contained 0.2 M Tris buffer (pH 7.6), 100 ml; vitaminfree Casamino acids, 1.0 g; thiamine, 100/~g; biotin, 100/xg; Dglucose, 2 g; and deionized water, 900 ml. Starting substrate concentrations w e r e 50 ppm

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Enzyme Microb. Technol., 1991, vol. 13, May

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Oxidation of azo dyc i by manganese pcroxidasc resuited in new peak formation at 355 nm. After I h incubation of azo dye 1 with manganese pcroxidase. the dye was almost completely degraded as compared to thc control chromatogram (l"Tg,re 5A and B). We detected no dccolorization of acid yellow 9 or oxidation of sulfanilic acid by manganese pcroxidase.

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Aromatic compound-utilizing actinomycetes have been reported in the past 23 and recently examined in more detail. 24-2~ Though not studied previously with actinomycetes, it has been observed with some microorganisms that the presence of substitutions on the aromatic ring can allow the degradation of an otherwise recalcitrant original substrate/° However, attempts to demonstrate the enzymes involved have been tmsuccessful. ~t Here, we have shown that the linkage of a guaiacol moiety into azo dye yellow 9 allowed Streptomy('t,s spp. capable of utilizing vanillic acid to dccolorizc an azo dye that they otherwise

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Figure 4 Oxidation of azo dyes by ligninase and manganese peroxidase. (A) Oxidation of the acid y e l l o w 9 by ligninase. Reaction conditions: 0.2 mM hydrogen peroxide, 50 mM sodium tartrate buffer pH 3, 10 #g of dye, 0.6 units of enzyme (20 #1) in total v o l u m e 1 ml. Cycle time 30 s, last measure after 15 min. (B) Oxidation of sulfanilic acid by ligninase. For reaction conditions see A. (C) Oxidation of azo dye 1 by manganese peroxidase. Reaction conditions: 0.2 mM hydrogen peroxide, 50 mM sodium tartrate buffer pH 5, 10 #g of dye, 1 unit of enzyme, 1 mM MnSO4 in total v o l u m e 1 ml. Cycle time 10 s, last measure after 3 rain of incubation. (D) Oxidation of azo dye 2 by manganase peroxidase. For reaction conditions see C

The decolorization of acid yellow 9 azo dye and the oxidation of sulfanilic acid by a lignin peroxidasc preparation of Phanerochaete ('ht3,sosporium arc shown in Figure 4o and h, respectively. During a period of 15 min the ligninase exhibited a stable activity which decolorized about 3/xg out of 10/xg of acid yellow 9 in the reaction mixture (Figure 4A). Sulfanilic acid was transformed slowly by ligninasc, with an increase in absorbancc at about 480 nm (Figure 4B). We detected 382

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Degradation of recalcitrant azo dyes: A. Paszczynski et aL could not transform (Table I). S. rochei A4 was the only vanillic acid degrader that could not attack either of the synthesized dyes. Possibly, this strain catabolizes vanillic acid by attacking its carboxylic group, a substituent absent in the guaiacol moiety. Microbial degradation of vanillatc by nonoxidative decarboxylation has been reported for Bacillus megaterium, 32 Streptomyces strain 179,~'- and S. setonii strain 75Vi2. ~-9Strains AI0. AI I, AI2, AI3, and AI4, on the other hand, may utilize vanillic acid by way of oxidative dcmethylation of the mcthoxyl group, which was present in the two synthesized azo dyes. This is a common pathway for microbial catabolism of vanillate. 3~-3~' Our conclusion is that utilization ot" the two dyes starts at the guaiacol substituent. Though a mechanism of "'bacterial" azo dyes degradation has been previously described, ~7 the pathway used by these Streptomyces spp. remains to bc elucidated. Attempts to demonstrate a behavior similar to P. chrysosporium, with an involvement of extraccllular pcroxidases in the utilization of the two synthesized dyes, have thus far bccn unsuccessful. P. cht3,sosporittm degrades a large variety of aromatic compounds, ~" including polycyclic aromatic hydrocarbons, dibenzolp]-dioxins) '~ polychlorinated phenols, 4° and trinitrotoluenc. 4j Only recently, it has also been shown to degrade azo dyes, using its ligninolytic enzymes." In this report, we used several times higher concentrations of azo dyes than reported earlier and tested the ability of this organism to oxidize sulfonated azo aromatic compounds (Figure 3). The maximum rate of decolorization occurred on the fourth day of growth in the Bll medium for all of the compounds. However, in the cultures with yellow 9 or sulfanilic acid, as assayed by spectrophotometric and HPLC analysis, some undegraded dye remained in the medium after decolorization ended. Yet using HPLC, we were not able to detect any residual substrate in the culture broth after I week of growth, cvcn though color was still present in the culture tiltratcs. One explanation is the finding of Kulla et al.,~7 who found that in cultures of Pseudomonas which wcrc actively degrading azo dyes, secondary oxidative coupling occurred between sulfonated and nonsulfonatcd phenols, giving dead-end polymers resistant to further degradation. In testing which, if either, of the ligninolytic pcroxidases of P. chrysosporium was involved in the degradation of these azo compounds, wc tbund that ligninasc oxidized yellow 9 and sulfanilic acid (Figure 4A and B), while manganese peroxidase oxidized azo dyes 1 and 2 (bTgure 4C and D). Thc HPLC analysis of the reaction mixture after incubation of azo dye I with manganese pcroxidasc revealed polymorphic reaction products (t:igure 5B). Oxidation of sulfanilic acid by ligninasc produced a purple unstable product, which upon exposure to air precipitated. During a 15-min incubation period, we did not detect oxidation of yellow 9 or sulfanilic acid by manganese pcroxidase, or the oxidation of azo dye I or 2 by ligninase. Thus, it is possible that ligninases may cooperate in

the degradation of azo dyes I and 2. Further studies are needed to elucidate their exact role. The fact that shaking cultures decolorized the dyes is evidence that a nonligninolytic system could bc involved, since shaking suppresses expression of the system in P. cho'sosporium BKM-F-1767. -~l Azo dyes I and 2 were decolorized to a greater extent by P. chrysosporium than was acid yellow 9 (l~bh, 1). This difference was even more striking when decolorization was lollowed during the growth of S. c h r o m o f l t S ( ' t t s All (Figure 2). These results suggest that linkage of a guaiacol molecule into the dye structure increased its susceptibility to degradation. Azo dye structures are typically conjugated multiunsaturated systems. This makes it possible to change only one fragment of the molecule and yet have the entire conjugated system become accessible to enzymatic attack, particularly when microorganisms use oxidative enzymes that generate cation radicals. This finding may have general application for strategies to synthesize more easily biodegradable azo dyes and other recalcitrant compounds.

Acknowledgements This research was supported by the Idaho Agricultural Experiment Station, by Competitive Research grants 87-FSTY-9-0255 and 88-37233-4037 from the US Department of Agriculture, and by grant BCS-8807000 from the National Science Foundation.

References I

2 3

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7 8 9 10 II 12

13

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Phelan, M. B., Crawford, D. L. and Pometto II1, A. L. Can. J. Microhiol. 1979.27, 636-638 Pettey, T. M. and Crawford, D. 1+. Appl. Environ. Microbiol. 1984, 47, 439-440 Pridham, T. G. and Gottlieb, l). J. Bacteriol. 1948.56, 107-114 Huynh, V. B. and Crawford. R. I,. FEMS Microhi~d. l,ett. 1985.28, 119-123 McCarthy, A. amt Broda, P. J. Gen. Microbiol. 1984, 130, 29{)5- 2913 Jeffries, T. W., Choi, S. and Kirk. T. K. Appl. Entfiron. Microbiol. 1981, 42, 290-296 Ramachandra, M., Crawford, D. 1,. and Hertel, G. Appl. Em,,iton. Microbiol. 1988, 54, 3057-3063 Paszczynski, A., H u y n h , V. B. and (,rawford. R. L. Arch. Biochem. Biophy,~. 1986, 44, 750-765 Paszczynski, A.. ('rawford, R. L. and H u y n h , V. B. Methods En:.vmol. 1988. 161,264-270 Fuhs, G. W. Arch. Microbiol. 1961, 39, 374-422 ('rawford. R. L., Crawford, 1). 1,. and Dizikes, G. J. Arch. Microhiol. 1981. 129, 204-209 Sutherland, J. B.. Cra~,l'ord, D. L. and Pometto Ill. A. L. Appl. Environ. Microhiol. 1981, 4 1 , 4 4 2 - 4 4 8 Sutherland, J. B., Crawfi~rd, 1). L. and Pometto 111, A. I+. Can. J. Microhiol. 1983, 29, 1253-1257 Antai, S. P. and Crawford, D. 1,. Can. J. Micr,hiol. 1983, 29, 142-143 Pometto II1. A. L. and Crawford, l). 1+. Appl. Enuiron. Microbiol. 1985, 49, 727-729

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Pometto 111, A. L., Sutherland, J. B. and Crawfi)rd, D. L. Can. J. Microbiol. 1981, 27, 636-638 Traxler, R. W. and Flannery. W. L. in Biodeterioration o f Materials (Waiters, A. II. and Elphick, J. J., eds.) Elsevier, A m s t e r d a m , 1968, pp. 44-54 Donoghue. N. A.. Griffin, M.. Norris, D. B. and Trudgill, P. W. in Proceedinl,,s of+the lhird International Biodegradation Symposium (Sharpley, J. M. and Kaplan, A. M., eds.) Applied Science Publishers, London, 1976, pp. 43-56 Crawford, R. I,. and Perkins OIson, P. Appl. Environ. Microbiol. 1978, 36, 539-543 Cartwright, N. J. and Buswell, J. A. Bioctu'm. J. 1967, 105, 767-770 ('artwright, N. J. and Smith. A. R. W. Biochem. J. 1967, 102, 826-84 I Crawford, R. I,., Kirk. T. K. and McCoy, E. Can. J. Microbiol. 1975, 2 1 , 5 7 7 - 5 7 9 T o m s , A. and Wt~.)d, J. M. Biochemisto" 1969, 9, 337-343 Kulla, H. G., Klausner, F., Meyer, U., l,udeke, B. and Lesinger. T. Arch. Microhiol. 1983. 135, I-7 B u m p u s , J. A. and Aust, S. l). BioEv,says 1986, 6, 166170 H a m m e l , K. E.. K a l y a n a n l m a n , B. and Kirk, T. K. J. Biol. Chem. 1986. 261, 16948-16952 Hammcl. K. E. and Tardone, P. J. Bio(hemi.~tO" 1988, 27, 6563-6568 Fernando, T., B u m p u s . J. A. and Aust. S. 1). Appl. Enuiron. Microhiol. 199{), 56, 1666-1671