Degradation by white-rot fungi of high concentrations of PCB extracted from a contaminated soil

Degradation by white-rot fungi of high concentrations of PCB extracted from a contaminated soil

Advances in Environmental Research 6 Ž2002. 559᎐568 Degradation by white-rot fungi of high concentrations of PCB extracted from a contaminated soil G...

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Advances in Environmental Research 6 Ž2002. 559᎐568

Degradation by white-rot fungi of high concentrations of PCB extracted from a contaminated soil Graciela M.L. Ruiz-Aguilar, Jose ´ M. Fernandez-Sanchez, ´ ´ U Refugio Rodrıguez-Vazquez , Hector Poggi-Varaldo ´ ´ ´ Departamento de Biotecnologıa Nacional 2508, ´ y Bioingenierıa, ´ CINVESTAV-IPN, A¨ . Instituto Politecnico ´ San Pedro Zacatenco, Deleg. Gusta¨ o A. Madero, 07360, Mexico, D.F., Mexico ´ Accepted 24 June 2001

Abstract White-rot fungi are known to degrade a wide variety of recalcitrant pollutants. In this work, three white-rot fungi were used to degrade a mixture of PCBs at high initial concentrations from 600 to 3000 mgrl, in the presence of a non-ionic surfactant ŽTween 80.. The PCBs were extracted from a historically PCB-contaminated soil. Preliminary experiments showed that Tween 80 exhibited the highest emulsification index of the three surfactants tested ŽTergitol NP-10, Triton X-100 and Tween 80.. Tween 80 had no inhibitory effect on fungal radial growth, whereas the other surfactants inhibited the growth rate by 75᎐95%. Three initial PCB concentrations Ž600, 1800 and 3000 mgrl. were assayed with three fungi for the PCB degradation tests. The extent of PCB modification was found to depend on PCB concentration Ž P- 0.001. and fungal species Ž P- 0.001.. PCB degradation ranged from 29 to 70%, 34 to 73% and 0 to 33% for Trametes ¨ ersicolor, Phanerochaete chrysosporium and Lentinus edodes, respectively, in 10-day incubation tests. The highest PCB transformation Ž70%. was obtained with T. ¨ ersicolor at an initial PCB concentration of 1800 mgrl, whereas P. chrysosporium could modify 73% at 600 mgrl. Interestingly, P. chrysosporium was the most effective for PCB metabolization at an initial concentration of 3000 mgrl, and it reduced up to 34% of the PCB mixture. As an overall effect, an increase in the initial PCB concentration led to a decrease in the pollutant degradation, from 57% to 21%. P. chrysosporium and L. edodes accumulated low chlorinated congeners. In contrast, T. ¨ ersicolor removed both low and high-chlorinated congeners of PCBs. 䊚 2002 Elsevier Science Ltd. All rights reserved. Keywords: Degradation; Fungus; PCBs; Surfactant

1. Introduction Polychlorinated biphenyls ŽPCBs. are pollutants of concern found in soil and sediments. PCBs have been

U

Corresponding author. Tel.: q52-5-747-7000, ext. 4351; fax: q52-5-747-7002. E-mail address: [email protected] ŽR. Rodrıguez´ .. Vazquez ´

widely used in a variety of industrial applications such as hydraulic and dielectric fluids. The widespread uses of PCBs coupled with improper disposal practices have led to their environmental ubiquity. PCBs are highly stable in the environment and are readily transported from localized or regional contaminated sites ŽSafe et al., 1987.. In situ bioremediation is an attractive alternative for the treatment of PCB-contaminated soils and sediments. Nevertheless, the rates of PCB bioremediation

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Ruiz-Aguilar Graciela M.L. et al. r Ad¨ ances in En¨ ironmental Research 6 (2002) 559᎐568

can be limited, among other factors, by the low concentration of microorganisms that are able to remove these compounds. Thus, the addition of exogenous microorganisms and nutrients could be beneficial for their modification. The rate of microbial degradation of PCBs can also be limited by the low bioavailability of these hydrophobic compounds ŽProvidenti et al., 1993; Robinson and Lenn, 1994; Verstraete and Devliegher, 1996.. White-rot fungi can rapidly oxidize and mineralize a broad spectrum of diverse aromatic compounds, including PCBs ŽBumpus et al., 1985a,b.; however, PCB transformation by white-rot fungi can also be limited by its bioavailability. In this regard, surfactants might promote microbial degradation of hydrophobic compounds by enhancing their apparent solubilization and increasing their availability ŽAronstein et al., 1991.. Aerobic biodegradation of PCBs has been extensively studied, mainly with bacteria ŽFiebeg et al., 1993; Quensen et al., 1990; Rojas-Avelizapa, et al., 1999.. It is reported that PCBs are attacked by fungi ŽAsther et al., 1987; Beaudette et al., 1998; Bumpus et al., 1985a,b; Eaton, 1985; Novotny et al., 1997; Sasek et al., 1993; Thomas et al., 1992; Vyas et al., 1994.. It is known that some basidiomycetes, like P. chrysosporium, Pleurotus florida ŽArisoy, 1998., T. ¨ ersicolor ŽJohansson and Nyman, 1993., L. tigrinus ŽHomolka et al., 1995. and Grifola frondosa ŽSeto et al., 1999. produce lignin degrading enzymes under nitrogen-limiting conditions. Yet, some ligninolytic species of fungi were found to produce these enzymes under non-limiting conditions ŽJackson et al., 1999. depending on the strain, culture system, etc. ŽCameron et al., 2000; Hattaka, 1994; Orth et al., 1993.. Usually, Phanerochaete chrysosporium has been used as a model in studies of PCB degradation by fungi. Still, other white-rot fungi have been examined for the aerobic transformation of chloroaromatic compounds, including Pleurotus ostreatus and Trametes ¨ ersicolor ŽSasek et al., 1993.. P. chrysosporium was reported to degrade 75% of the congeners in Aroclor 1260 Ža complex mixture of biphenyl congeners averaging five chlorine atoms. at low initial concentration of 0.90 mgrl over a 3-week period ŽAsther et al., 1987.. This result is comparable with the activity of the aerobic bacterium Pseudomonas strain LB400, which removed 68% of the congeners in Aroclor 1242 at an initial concentration of 50 mgrl within 24 h. Such studies used well-defined mixtures of PCBs and did not address the ability of fungi to degrade effectively complex PCB mixtures at environmentally relevant concentrations. More extensive studies are necessary to evaluate the potential of white-rot fungi for enhancing PCB bioremediation and elucidating the effects of pollutant concentration and culture conditions. To address these issues, we carried out a study of the PCB biodegradation with three white-rot

fungi in the presence of a non-ionic surfactant at different PCB concentrations.

2. Materials and methods 2.1. Microorganisms Phanerochaete chrysosporium strain ŽH-298 CDBB500., Trametes ¨ ersicolor strain ŽH-1051 CDBB-500. and Lentinus edodes strain ŽH-925 CDBB-500. were obtained from the Culture Collection of the CINVESTAV-IPN, Mexico. Fungal strains were maintained on malt extract agar slops ŽMerck᎐Mexico, S.A...

2.2. Media A medium containing yeast᎐peptone᎐glucose ŽYPG. was used for inoculum production. The composition of the medium was Žfor 1 l of distilled water.: 20 g malt extract, 30 g glucose, 20 g peptone, and 1.5 g yeast extract. A nitrogen-limited mineral medium ŽMorgan et al., 1991; Kirk et al., 1978. was used for the PCB degradation studies. The medium formulation was modified by substituting glucose for a PCB-contaminated soil extract as the carbon source.

2.3. Inoculum production Inocula were prepared by transfering 750 mg of mycelium Ždry weight. grown on malt extract agar ŽMerck᎐Mexico, S.A.. plates to Erlenmeyer flasks containing 270 ml of the YPG medium. The P. chrysosporium cultures were grown at 39⬚C for 2 days and T. ¨ ersicolor and L. edodes at 28⬚C for 4 days. All cultures were mixed at 150 rev.rmin.

2.4. Extract from a PCB contaminated soil The PCB contaminated soil was obtained from an abandoned chemical company in the northern zone of Mexico City. PCBs were extracted from the contaminated soil according to Fernandez-Sanchez et al. Ž1999.. ´ ´ Briefly, a 30-g sample of PCB contaminated soil was Soxhlet-extracted for 9 h with 100 ml of nhexaneracetone Ž1:1, vrv. mixture. The extract volume was reduced to 5 ml in a rotatory evaporator, the organic matter was removed from the extract with concentrated H 2 SO4 and the extract was further cleaned by solidrliquid chromatography. The extract was filtered through a 30 = 1.5 cm column packed with 60᎐100 mesh Florisil, eluted once with 100 ml of hexane and the resulting eluants Ž50 ml. were concen-

Ruiz-Aguilar Graciela M.L. et al. r Ad¨ ances in En¨ ironmental Research 6 (2002) 559᎐568

trated to 5 ml in a rotatory evaporator. The resulting concentrate was used for all the experiments reported in this study. PCB analysis showed a mixture of bithrough nonachlorobiphenyls, similar to Aroclor 1260.

Table 1 Statistical three level factorial design Ž3 2 . used in the PCB degradation of an extract obtained from a contaminated soil Factor

2.6. Effect of surfactants on fungal growth The three fungal strains were grown on 2% malta agar plates. Surfactants were added to autoclaved agar medium at 75 mgrl for Triton X-100, 74 mgrl for Tergitol NP-10 and 302 mgrl for Tween 80. These concentrations resulted from the emulsification assays. The culture conditions were: for P. chrysosporium, 39⬚C, pH 4.5; and for T. ¨ ersicolor and L. edodes, 28⬚C, pH 5.0. The growth rate was estimated by measuring their radial growth several times until the mycelia covered the agar surface completely ŽTrinci, 1971.. As a control, each fungus was grown on agar plates without surfactant.

2.7. PCB degradation studies Degradation studies were performed following a three-level factorial design with two factors Ž3 2 , Table 1.. Then, nine treatments were performed with three replicates, giving a total of 27 experimental units ŽMontgomery, 1991; Myers and Montgomery, 1995.. Factors were the fungus species Žthree levels: P. chrysosporium, T. ¨ ersicolor and L. edodes. and the PCB initial concentration Žthree levels: 600, 1800 and 3000 mgrl.. Each species of fungus was exposed to each level of PCB concentration. The percentage of PCB degradation and fungal growth were measured as response variables. Three controls were run for each fungus Žthree replicates each.: Ž1. fungus without PCBs; Ž2. inactive fungus with PCBs Žabiotic control.; and Ž3.

Level Ž0.

2.5. Emulsification assays The PCB-contaminated soil extract was emulsified by non-ionic surfactants, as indicated by Ghurye et al. Ž1994.. Three non-ionic surfactants were tested to establish their ability to emulsify a PCB-contaminated soil extract: Triton X-100, Tergitol NP-10 and Tween 80, supplied by Baker ŽUSA, Cat. No. X198-05., Sigma ŽUSA, Cat. No. NP-10., and Merck ŽGermany, Cat. No. 822187., respectively. Each surfactant was added at different concentrations Žfrom 6 to 604 mgrl. to 5 ml of nitrogen-limited mineral medium and 0.5 ml of extract in a 10-ml test tube Ž100 = 10 mm.. The mixture was vortex-shaken for 1 min after each addition and allowed to equilibrate for 2 min. The thickness of the emulsified layer was measured in millimeters. The surfactant concentration selected for further study was the one that was able to maintain the emulsification.

561

White-rot fungus PCB extract from contaminated soil Žmgrl.

a

Le 600

Ž1. a

Tv 1800

Ž2. Pcb 3000

Le s Lentinus edodes, Tv s Trametes ¨ ersicolor, Pc s Phanerochaete chrysosporium. Conditions: 150 rev.rmin, 302 mg Tween 80rl, 10 days of treatment. a 28⬚C and pH 5.0. b 39⬚C and pH 4.5.

PCBs without fungus. The abiotic control was prepared by autoclaving the cultures at 121⬚C for 60 min at 2 kgrcm2. Liquid cultures were carried out in 150-ml flasks with 42-ml of nitrogen-limiting medium, as described in Section 2.2, plus fungal biomass from cultures, as described in Section 2.3 in a 10% Žwrv. proportion. Experimental units were aerobically incubated at 150 rev.rmin for 10 days. Samples were taken at the beginning and at the end of a 10-day incubation period.

2.8. PCBs extraction and analysis The mycelium was filtered and washed twice with 18 ml of acetone and 18 ml of the hexane᎐acetone mixture Ž9:1, vrv.. The washings were combined with the medium and shaken for 20 min at 150 rev.rmin. The whole extracts were then re-extracted three times with hexane in a separation funnel and the resulting extracts were combined. The mixture was then extracted twice with 36 ml of 2% of NaCl and once with 7 ml of concentrated H 2 SO4 . The PCB extract was evaporated down to 5 ml by rotatory evaporation filtered through a Florosil-packed chromatographic column and analyzed by high-performance liquid chromatography ŽHPLC. according to Fernandez-Sanchez et al. Ž1999.. The ´ ´ HPLC system consisted of a pump ŽVarian 9012. and a UV detector ŽVarian 9050. operated at 254 nm. The column was a C18 ŽChromanetics, 150 = 4.6 mm, and particle diameter 5 ␮m.. The solvents were acetonitrile-deionizated water Ž80:20, vrv. chromatographic grade with an isocratic flow rate at 1.0 mlrmin. The peak areas of the chromatograms were integrated using a Star Integrator software package Žversion 4.51, Varian Associates, Inc... PCB degradation was calculated as the difference of the initial and final sum of the peak areas of the experimental unit; the corresponding areas of the abiotic control were discounted, and then this difference was divided by the initial sum of peak area times 100 ŽFava et al., 1996; Joshi and Walia,

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1995; Mondello, 1989; Rojas-Avelizapa et al., 1999.. In this way, the degradation here defined gives a good approximation of the biological degradation of PCBs ŽBrock et al., 1984.. The recovery of PCBs was 88.8" 1.1%, using Aroclor 1260 as standard.

3. Results and discussion 3.1. Emulsification assays The effect of the non-ionic surfactants on the emulsification of PCB extract obtained from the contaminated soil plus nitrogen-limited mineral medium is shown in Fig. 1. For Tergitol NP-10 and Triton X-100, the emulsification capacity remained almost constant Žaverage 10 mm. in the range of 10᎐604 mg surfactantrl. On the other hand, the emulsification capacity of Tween 80 was poor Ž- 7 mm. in the range from 0 to 280 mgrl. At that point, an increase in the concentration of Tween 80 resulted in a sharp increase of the emulsification capacity Žfrom 7 to 25 mm in the range of 280᎐305 mgrl.. Further increase in Tween 80 concentration beyond 305 mgrl did not lead to a significant increase in the emulsification capacity Ž27 " 2.5 mm maximum thickness, for 604 mgrl.. To determine whether the surfactants had an effect on fungal growth, a common emulsification index of 10 mm was selected. Practical concentrations were chosen for Tergitol NP-10 and Triton X-100 Ž74 and 75 mgrl, respectively. which showed a 10-mm thickness of the selected emulsification. Incidentally, the Triton X-100 concentration tested was similar to that used by Beaudette et al. Ž2000. to dissolve six PCB congeners in a low-nitrogen medium. Concerning Tween 80, a 302mgrl concentration was selected, which was associated with an emulsification index of approximately 10 mm.

3.2. Effect of surfactants on fungal growth

Surfactants have the ability to increase aqueous concentrations of poorly soluble compounds enhancing their availability to microorganisms. However, surfactants have been reported to inhibit the biodegradation of organic compounds ŽLaha and Luthy, 1992; Providenti et al., 1993; Robinson and Lenn, 1994; Shiau et al., 1995.. Thus, it was necessary to establish if the surfactants used in this work would inhibit fungal growth and, consequently, decrease the PCB degradation. Fig. 2 and Table 2 show the results of fungal growth in the presence of the three surfactants: Triton X-100 Ž75 mgrl., Tergitol NP-10 Ž74 mgrl. and Tween 80 Ž302 mgrl.. Tween 80 appeared to have no effect on the fungal growth as compared to the control without the addition of surfactant. Indeed, the growth rates were 1.21, 0.34 and 0.23 mmrh and the corresponding control rates were 1.67, 0.34 and 0.24 mmrh for P. chrysosporium, T. ¨ ersicolor and L. edodes, respectively. Our results seem to agree with Kotterman et al. Ž1998. who reported that fungal growth was not affected, when Tween 80 was present in the medium Žrange tested from 250 to 10 000 mgrl.. Also, Asther et al. Ž1987. found no negative effect of Tween 80 on the growth of P. chrysosporium. For all species, Tergitol NP-10 and Triton X-100 decreased the growth rate by 75% and 95%, respectively, when compared to the control ŽTable 2.. For example, T. ¨ ersicolor showed a growth rate of 0.34 mmrh without surfactant, but this rate decreased considerably when Tergitol NP-10 and Triton X-100 were used Ž0.06 and 0.16 mmrh, respectively.. According to Aronstein et al. Ž1991., Triton X-100 and Tergitol NP-10 have also been used for emulsification at low concentration. Inhibitory effects of Triton X-100 on other

Fig. 1. Emulsification of the PCB extract from contaminated soil with three surfactants in nitrogen-limited mineral medium. Error bars represent the standard deviation ŽS.D.. from the mean of three replicates. ' Tergitol NP-10; I Triton X-100; 䢇 Tween 80.

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Table 2 Effect of the surfactant on the growth rate of three white-rot fungi Treatment

With surfactant Tergitol NP-10 Triton X-100 Tween 80 Without surfactant Control

Concentration tested Žmgrl.

Radial growth rate Žmmrh. Fungus P. chrysosporiuma H-298 CDBB-500

T. ¨ ersicolorb H-1051 CDBB-500

L. edodesb H-925 CDBB-500

74 75 302

0.10 0.08 1.21

0.06 0.16 0.34

0.06 0.10 0.23

1.67

0.34

0.24

In all cases R G 0.99. Conditions: 8᎐30 days of treatment. a 39⬚C and pH 4.5. b 28⬚C and pH 5.0. 2

fungi have also been reported elsewhere ŽPardo, 1996; Yazdi et al., 1990.. Tween 80 was thus selected for PCB emulsification because it did not have a deleterious effect on fungal growth and simultaneously provided a

Fig. 2. Effect of surfactants on fungal growth Ža. P. chrysosporium H-298 CDBB-500; Žb. T. ¨ ersicolor H-1051 CDBB-500; and Žc. L. edodes H-985 CDBB-500 on plate. Conditions: T. ¨ ersicolor and L. edodes, 28⬚C and pH 5.0, P. chrysosporium, 39⬚C and pH 4.5. ᎐⽧᎐ Tergitol NP-10 Ž74 mgrl.; --I-- Triton X-100 Ž75 mgrl.; ᎐'᎐ Tween 80 Ž302 mgrl.; --`-- Control Žwithout surfactant ..

reasonable emulsification of the extract from a PCB contaminated soil.

3.3. PCB degradation results The effect of three PCB concentrations on the degradation ability of three white-rot fungi in the presence of Tween 80 was examined. Each fungus was exposed to the three levels of PCBs as described in Table 1. The extent of PCB degradation was found to depend on PCB concentration Ž P- 0.001. and fungal species Ž P- 0.001; see Figs. 3 and 4.. PCB degradation ranged from 29 to 70%, 34 to 73% and 0 to 33% for T. ¨ ersicolor, P. chrysosporium and L. edodes, respectively, in 10-day incubation tests. As it is the practice in the analysis of experimental designs ŽMontgomery, 1991; Myers and Montgomery, 1995., the inspection of the overall degradation averages per species pointed out to a higher effectiveness of T. ¨ ersicolor cultures Ž55%., followed by P. chrysosporium Ž48%., and last L. edodes Ž21%.. The general trend of PCB transformation as function of the initial PCB concentration indicated an impairment of degradation at increasing concentration Ž57, 46 and 21% at 600, 1800 and 3000 mgrl, respectively.. The quantity of PCB absorbed onto glass was negligible. When T. ¨ ersicolor was tested, a greater extent of degradation was found at concentrations of 1800 mgrl Ž70%, the highest among species at this level. and 600 mgrl Ž67%.. PCBs transformation also occurred at 3000 mgrl, but to a lesser extent Ž29%.. P. chrysosporium also presented an important modification of PCBs at 600 mgrl Ž73%, the highest among species at this level., however, its degradation ability was half of that at 1800 and 3000 mgrl ŽFig. 3.. Interestingly, P. chrysosporium was the most effective at an initial concentration of 3000 mgrl, and could reduce up to 34% of PCBs. L. edodes was able to degrade 32% of the

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Ruiz-Aguilar Graciela M.L. et al. r Ad¨ ances in En¨ ironmental Research 6 (2002) 559᎐568

Fig. 3. Degradation of PCB in an extract from a contaminated soil by white-rot fungi. I Lentinus edodes H-925 CDBB-500; B Trametes ¨ ersicolor H-1051 CDBB-500; B Phanerochaete chrysosporium H-298 CDBB-500. The asterisk ŽU . on the top of the bars indicates reductive transformation evidenced by an increase in the concentration of lesser-chlorinated PCB congeners.

PCBs present in the medium at 600 and 1800 mgrl. However, its degradation ability was practically suppressed at 3000 mgrl of PCB. P. chrysosporium and L. edodes accumulated low chlorinated congeners whereas T. ¨ ersicolor removed both low and high-chlorinated congeners. Fig. 4 shows typical chromatograms of T. ¨ ersicolor and L. edodes units. A decrease in the peak area of chromatograms was apparent when T. ¨ ersicolor was used ŽFig. 4a vs. b.. Notably, a decrease in some peak areas was found

in the range 8᎐40 min. In contrast, an increase in the peak area corresponding to low chlorinated congeners Žretention times lower than 8 min. was observed when L. edodes was grown in the presence of PCBs at 1800 mgrl ŽFig. 4c vs. d.. For example, if we focus on peaks a and b Žbelonging to the range of di- and trichlorobiphenyls; Fig. 4c,d., their areas increased almost twofold after treatment Ž133 000 and 289 000 for peaks a and b, respectively, before treatment, and 214 000 and 429 000 after treatment .. Some reduction in the peak areas at retention times higher than 10 min was obtained after treatment with this fungus, but this is not so evident. It is known that aerobic microorganisms may reductively dechlorinate compounds into less chlorinated intermediates, which could explain the formation of low-chlorinated congeners. Degradation may be followed by ring cleavage, and, in some cases, a complete mineralization of compounds ŽAbramowicz, 1990; Bedard and Quensen, 1995.. For instance, other fungal species Ž P. chrysosporium and others. were reported to convert highly chlorinated congeners to CO 2 and water-soluble products with little accumulation of low-chlorinated congeners ŽEaton, 1985; Bumpus et al., 1985a,b; Thomas et al., 1992.. In general, initial PCB concentration affected fungal growth ŽFig. 5.. An exception was P. chrysosporium, which was able to tolerate 3000 mgrl Žwith only a 30% inhibition; Fig. 5c.. Growth of L. edodes and T. ¨ ersicolor were the most negatively affected, particularly at 3000 mgrl initial PCB concentration ŽFig. 5a,b.. Murado et al. Ž1976. reported a progressive reduction in

Fig. 4. Typical chromatogram profiles of PCB transformation. Ža. Start and Žb. end of treatment Ž10 days. using T. ¨ ersicolor H-1051 CDBB-500; Žc. start and Žd. end of treatment Ž10 days. using L. edodes H-925 CDBB-500. Peak chapters a and b represent low-chlorinated congeners, as discussed in the text.

Ruiz-Aguilar Graciela M.L. et al. r Ad¨ ances in En¨ ironmental Research 6 (2002) 559᎐568

growth for Aspergillus fla¨ us when exposed to higher Aroclor 1254 concentrations Žin the range 5᎐50 mgrl.. Although fungal growth was affected by the initial PCB concentration used in our study, the fungi were still able to degrade congeners of extract. Support of fungal metabolism is possible whether structural changes from higher to lower chlorinated congeners occur ŽEaton, 1985. or to another compound more easily metabolizable ŽVyas et al., 1994.. These compounds may be used as a carbon source. In addition, Tween 80 could be a ligninase inducer in agitated cultures ŽAsther et al., 1987. that protects the enzymes from denaturing ŽMoyson and Verachtert, 1993.. It is possible that high PCB concentrations Ždespite their inhibition of fungal growth. would not be entirely inhibitory to the enzyme activities responsible for PCB degradation or for cellular metabolism in general, thus resulting in good levels of degradation. On the other hand, white-rot fungi are known to produce a variety of ligninolityc enzymes that can be expressed depending on culture conditions and the strain used ŽCameron et al., 2000; Hattaka, 1994; Orth et al., 1993., adding the possibility of different alternative pathways for PCB degradation among the tested strains. We found that at concentrations above 1800 mgrl, white-rot fungi could degrade PCBs to the same extent as bacteria did but at lower PCBs concentrations. Bacterial consortia could degrade 50% of initial 100 mgrl PCBs ŽAroclor 1242. present in the medium ŽGuilbeault et al., 1994.. Mixed bacterial cultures could remove 75% of initial PCBs emulsified with Triton X-100 in liquid media ŽRojas-Avelizapa et al., 1999.. In this study, higher PCB degradation was obtained than that reported by Novotny et al. Ž1997., Sasek et al. Ž1993. and Vyas et al. Ž1994.; see Table 3. Moreover, it should emphasized that we used high PCB concentrations extracted from a historically contaminated soil as opposed to the off-the-shelf low PCB concentrations used in those studies. In our work, PCB degradation efficiencies were higher than those obtained for the same PCB-contaminated soil bioaugmented with P. chrysosporium, in solid culture ŽFernandez-Sanchez et ´ ´ al., 1999.. To our best knowledge, this is the first reported case of PCB degradation by white-rot fungi at high PCB concentrations in liquid culture.

4. Summary and conclusions White-rot fungi were able to degrade PCBs extracted from a historically contaminated soil. Preliminary tests demonstrated that Tween 80, at 302 mgrl, could emulsify the PCB extract and it does not have an effect on fungal growth rate, whereas Triton X-100 and Tergitol NP-10 severely inhibited the fungal growth.

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Fig. 5. Fungal growth in the presence of a PCB-contaminated soil extract at the end of treatment Ž10 days.. Ža. L. edodes H-925 CDBB-500; Žb. T. ¨ ersicolor H-1051 CDBB-500; Žc. P. chrysosporium H-298 CDBB-500. Error bars represent the S.D. from the mean of three replicates.

From the three initial PCB concentrations Ž600, 1800 and 3000 mgrl. assayed and the three fungi used for the PCB degradation, in the presence of Tween 80 at 302 mgrl, it was found that PCB transformation depended on PCB concentration Ž P- 0.001. and fungal species Ž P- 0.001.. PCB degradation ranged from 29 to 70%, 34 to 73% and 0 to 33% for T. ¨ ersicolor, P. chrysosporium and L. edodes, respectively in 10-day incubation tests. Moreover, the highest PCB transformation Ž70%. was obtained with T. ¨ ersicolor at an initial PCB concentration of 1800 mgrl, whereas P. chrysosporium could transform 73% at 600 mgrl. In addition, it was found that P. chrysosporium was the most effective for PCB degradation at an initial concentration of 3000 mgrl, and it reduced up to 34% of the PCB extract. In general, an increase in the initial

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Table 3 Comparison of PCB degradation in different studies using white-rot fungi PCB used

PCB concentration Žmgrl.

Aroclor 1260a

2.61

No. 77b

8.77= 10y4

Declor 106a Žequivalent at Aroclor 1260. PCB extract from contaminated soil

0.90

3000 1800

Fungus

PCB degradation Ž%.

Time of treatment Žweeks.

Reference

Pc Le Po Pc Tv Co Pc Tv Po Pc Tv Le

10.81 23.72 28.95 1.39c 0.40c 0.02c 75.00 50.00 0.00 72.97 70.37 32.63

6.0

Sasek et al., 1993

4.0

Vyas et al., 1994

3.0

Asther et al., 1987

1.4

This work

a

s Liquid culture; b s Solid culture; c s Mineralised. Pc s P. chrysosporium; Tvs T. ¨ ersicolor; Le s L. edodes; Po s Pleurotus ostreatus; Cos Coriolopsis polysona.

PCB concentration led to a decrease in the pollutant transformation, from 57% down to 21%. Low chlorinated congeners were accumulated when P. chrysosporium and L. edodes were employed. In contrast, both low and high-chlorinated congeners of PCBs were transformed by T. ¨ ersicolor. The ability of white-rot fungi to degrade high PCB concentrations supports their use for the treatment of PCB-contaminated soils.

5. Nomenclature CDBB Le Pc Tv

Coleccion ´ del CINVESTAV-IPN Lentinus edodes Phanerochaete chrysosporium Trametes ¨ ersicolor

Acknowledgements We are grateful to Ms. Blanca Patricia Arroyo-Arista, Ms. Dolores Dıaz-Cervantes and Mr. Alfredo Medina´ Davila for their excellent technical assistance. We wish ´ to thank Professor Elvira Rıos-Leal for her exceptional ´ help in establishing the HPLC methodology. We also express our appreciation to C. Ph. D. Sondra Miller at the University of Iowa for her comments on the manuscript. This work was supported by Fondo de apoyo al desarrollo de proyectos de investigacion ´ basica ´ y tecnologica en colaboracion ´ ´ con las Instituciones de Educacion ´ Superior ŽFIES 95-106-VI., IMP, Mexico. ´

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