World Journal of Microbiology & Biotechnology 15: 269±276, 1999. Ó 1999 Kluwer Academic Publishers. Printed in the Netherlands.

269

Degradation of polychlorinated biphenyls by extracellular enzymes of Phanerochaete chrysosporium produced in a perforated plate bioreactor Pavel KrcÏmaÂrÏ 1,*, Alena KubaÂtovaÂ2, Jaroslav Votruba2, Pavla ErbanovaÂ2, CÏeneÏk NovotnyÂ2 and VaÂclav SÏasÏ ek2 1 Veterinary Research Institute, 621 32 Brno, Czech Republic 2 Institute of Microbiology, Academy of Sciences of the Czech Republic, VõÂdenÏska 1083, 142 20 Prague 4, Czech Republic *Author for correspondence: E-mail: [email protected] Received in revised form 4 January 1999; accepted 9 January 1999

Keywords: Degradation, ligninolytic enzymes, polychlorinated biphenyls, white rot fungi

Summary The white rot fungus Phanerochaete chrysosporium was cultivated in a perforated plate bioreactor and the expression of activities of manganese-dependent peroxidase (MnP) and lignin peroxidase (LiP) was measured. Peak activities of the two enzymes were reached close to day 11 and therefore the cultivation was terminated on that day. Extracellular proteins were concentrated and both peroxidases separated by isoelectric focusing. Degradation of technical PCB mixtures containing low and highly chlorinated congeners (Delor 103 and Delor 106 as equivalents of Aroclor 1242 and Aroclor 1260, respectively) was performed using intact mycelium, crude extracellular liquid and enriched MnP and LiP. A decrease in PCB concentration caused by a 44-h treatment with mycelium (74% w/w for Delor 103 and 73% for Delor 106) or crude extracellular liquid (62% for Delor 103 and 58% for Delor 106) was observed. The degradation was not substrate-speci®c, because no signi®cant di€erences between the respective degradation rates were observed with di-, tri-, tetra-, penta-, hexa-, hepta-, and octachlorinated congeners. In contrast, MnP and LiP isolated from the above-mentioned extracellular liquid did not catalyse any degradation.

Introduction Polychlorinated biphenyls (PCBs) have been shown to contaminate various environmental matrices worldwide. Due to their toxicity, mutagenicity and bioaccumulation their biodegradation is of environmental interest (Safe 1994). Most biodegrading organisms studied so far have been bacteria (Abramowicz 1990). However, within the group of fungi that in nature cause white rot of wood (white rot fungi), the ability to degrade PCBs, (as well as other aromatic organopollutants) has been described (Bumpus et al. 1985; Paszczynski & Crawford 1995). Phanerochaete

chrysosporium Burds. has been the white rot fungus most often studied and its degrading ability was attributed to its ligninolytic system (Eaton 1985; Bumpus & Aust 1986). Biodegradation and even mineralization of PCBs under ligninolytic, i.e., nutrient-limited, conditions were described (Eaton 1985; Vyas et al. 1994; Dietrich et al. 1995), however, a direct correlation between the biodegradative ability and activities of ligninolytic enzymes was not proven (Thomas et al. 1992; Novotny et al. 1997). Yadav et al. (1995) described a similar PCB degradative ability in P. chrysosporium under both ligninolytic and non-ligninolytic conditions. KrcÏmaÂrÏ & Ulrich (1998) even

270 observed degradation of a PCB technical mixtures under non-ligninolytic conditions, and no degradation under ligninolytic conditions. The purpose of the present paper is to elucidate this unresolved problem using mycelium, fungal extracellular ¯uid and isolated ligninolytic enzymes. Materials and Methods Chemicals Commercial PCB mixtures Delor 103 (an analogue of Aroclor 1242), containing 40±42% bound chlorine, and Delor 106 (an analogue of Aroclor 1260), containing 60% bound chlorine were both ex-products of Chemko (StraÂzÏskeÂ, Slovakia). Standard solutions of Delor 103 (1 ml, conc. 24.72 mg/ml) and Delor106 (1 ml, conc. 25.08 mg/ ml) were combined, evaporated to dryness and dissolved in 5 ml of acetone. The diluted standards were dispensed into test tubes in which degradation was measured. The ®nal concentrations of Delor 103 and Delor 106 in the test tubes were 250 ppm. Culture conditions Phanerochaete chrysosporium, strain ATCC 24 725 was cultivated in an aerated, perforated plate bioreactor. A glass/stainless steel vessel of 7 l MBR Bioreactor (Sulzer, Switzerland) equipped with a ring sparger was modi®ed to a novel bioreactor function. The magnetically driven agitator body was removed from the bioreactor. Three perforated polyurethane plates were horizontally ®xed on ba‚es. The diameter of each plate was 125 mm, its thickness was 12 mm. The plates were perforated by circular holes of 11 mm in diameter. The distance between the holes was about 25 mm, and the distance between the plates was 30 mm. The perforated plate bioreactor that was ®lled with water was twice sterilized for 30 min at 120 °C before cultivation. The empty sterile reactor was ®lled with 4 l of an N-limited mineral medium (Tien & Kirk 1988) and the contents of the bioreactor were sterilized in a similar way. To prevent the medium becoming brown, the concentrated glucose solution was sterilized separately and added aseptically to the bioreactor together with

P. KrcÏmaÂrÏ et al. a spore inoculum of 400 ml spore suspension (5 ´ 106 spore/ml). After inoculation, the temperature in the bioreactor was adjusted to 32 °C. During the ®rst three days of cultivation, the medium was aerated with pure oxygen at a ¯ow rate of 0.2 l/min. After addition of veratryl alcohol (to a ®nal concentration of 2 mM; cf. Cancel et al. 1993) on the fourth day of cultivation, oxygen was replaced by sterile air at a ¯ow rate of 0.5 l/min. Fifteen ml samples of culture broth were collected from the bioreactor in 24 h intervals starting after 3 days of cultivation to estimate activities of MnP and LiP and total protein content. Enzyme activities Manganese-dependent peroxidase (MnP, EC 1.11.1.13) activity was measured via oxidation of 2,2¢-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS). The reaction mixture (0.7 ml) contained 25 ll sample and in addition 425 ll 0.1 M succinic acid and 0.1 M lactic acid as bu€er (pH 4.5), 0.5 mM ABTS and 50 lM H2O2. The reaction was initiated by addition of 35 ll 2 mM MnSO4 and absorbance was measured at 415 nm after 1 min (spectrophotometer Perkin Elmer lambda 11). One unit of the enzyme activity was de®ned as the amount of the enzyme converting 1 lmol of the substrate in 1 min (Glenn & Gold 1985). Laccase (Lac; EC 1.10.3.2) activity was measured as described for MnP except that MnSO4 and H2O2 were omitted. Lignin peroxidase (LiP; EC 1.11.1.14) activity was measured via oxidation of veratryl alcohol according by Tien & Kirk (1988) except that the concentration of veratryl alcohol was 4 mM (Linko & Haapala 1993). One unit of the enzyme activity was de®ned as the amount of the enzyme converting 1 lmol of the substrate in 1 min. All measurements of enzyme activities were done at 20±22 °C. Protein concentration Total extracellular proteins were determined by the Bradford method. Microtitre plates were used and each well contained 40 ll sample and the same volume of Coomassie Brilliant Blue G-250. Absorbance was read at 620 nm (Labsystems EMS

Biodegradation of PCBs Reader MF, software Genesis) after 2 min. Bovine serum albumin was used as standard. Puri®cation of LiP and MnP Part of the extracellular ¯uid harvested from the bioreactor (2 l, MnP activity 163 U/l, LiP 31 U/l) was concentrated by ultra®ltration (YM 10 membrane, Amicon USA) to a volume of 73 ml (MnP 2530 U/l, LiP 1227 U/l). The enzymes were puri®ed by isoelectric focusing using the Rotofor technique (Bio Rad). Ampholyte 4/6 was diluted with the sample to a ®nal concentration of 0.5%. Fifty ®ve ml of this sample was focused using a standard cell for 2.5 h. In all twenty fractions the pH values were measured (Beckman F 110 ISFET pH Meter with F Smart ISFET Micro Probe, Beckman, USA). Fractions 9±14 (pH 3.11±6.26) were combined, and 17 ml of this solution was applied to a mini cell and focused for 2 h. Fractions were harvested, their pH values as well as MnP and LiP activities measured. Then SDSPAGE was performed and the samples lyophilized. SDS-PAGE SDS-PAGE was performed with Mini-Protean Electrophoresis system (Bio Rad, USA) according to the Bio Rad Manual. The Low Molecular Weight calibration set (Pharmacia, Sweden) was used as standard. Proteins were silver stained. In vitro degradation of PCBs The reduction of PCB congener concentration by exposure to intact mycelium, crude extracellular liquid, and the isolated LiP and MnP was measured and compared with the e€ect of heat-killed (autoclaved) controls. All measurements were done in triplicate in 5 ml-glass stoppered test tubes. Degradation with the mycelium: 1 g of wet mycelium was mixed with 50 ll of a Delor mixture and left standing for 44 h at 20±22 °C in the dark. Degradation with extracellular ¯uid: 950 ll of crude extracellular liquid was mixed with 50 ll of the Delor mixture. At 8 h intervals 20 ll of 2.5 mM H2O2 was added to reach a total conc. of 50 lM, and the mixture was left standing at 20±22 °C in the dark for 44 h.

271 Degradation with MnP: Lyophilized fractions 12 and 14 (original volumes of 961 and 544 ll, respectively) were each dissolved in 450 ll water and combined. The reaction mixture (1 ml) contained bu€er (0.1 M succinic acid and 0.1 M lactic acid, pH 4.5), 50 lM H2O2, 0.1 mM MnSO4, 50 ll of the Delor mixture and 100 ll MnP 238 U/l. At 8 h intervals 20 ll of 2.5 mM H2O2 was added to reach a total conc. of 50 lM and left standing at 20±22 °C in the dark for 44 h. Degradation with LiP: Lyophilized fractions 3, 4 and 5 (original volumes of 902, 268 and 734 ll, respectively) were each dissolved in 300 ll water and combined. The reaction mixture (1 ml) contained bu€er (50 mM sodium tartrate, pH 3.0), 4 mM veratryl alcohol, 0.4 mM H2O2, 50 ll of the Delor mixture, and 100 ll LiP 24 U/l. At 8 h intervals 20 ll of 2.5 mM H2O2 was added to reach a total conc. of 50 lM and the mixture was left standing at 20±22 °C in the dark for 44 h. Extraction and determination of residual PCBs After incubation, the residual PCBs were extracted 3 times with 3 ml of a n-hexane-acetone mixture (9:1) using sonication (15 min) and Vortex mixing (1 min). The extraction method was based on the procedures described by Yadav et al. (1995) and ZacharÏ et al. (1996). The extraction eciencies can be expressed on the bases of amounts of PCBs found in heat-killed controls. The recoveries from the crude extracelluar liquid and the enzyme media were 44±61%. The recoveries from the intact mycelia were 16% and these values were increased by adding another extraction step employing Soxhlet apparatus to 48% (KubaÂtova 1997). The low recoveries (about 50%) were mentioned in other works and they are dependent on the sample matrix and PCBs studied (ZacharÏ et al. 1996; Beaudette et al. 1998). The extracts were dehydrated (anhydrous Na2SO4), combined, the volume adjusted to 10 ml and applied to GC. A congenerspeci®c analysis was performed using a gas chromatograph Hewlett-Packard 5890 Series II with 63 NiECD (Hewlett-Packard, USA), PC datastation with a software HP GC ChemStation Rev. A 04.02. The capillary column DB-5 (J & W Scienti®c), 30 m ´ 0.25 mm I.D., with a ®lm thickness of 0.25 lm, was used. Helium was employed as a carrier gas under a constant pressure of 90 kPa.

272

P. KrcÏmaÂrÏ et al.

The temperature program initiated at 50 °C for 1.5 min (splitless mode), followed by a temperature gradient 25 °C/min up to 140 °C and then 3 °C/min up to 290 °C; and ®nally hold isothermally for 10 min. The temperature of injector and detector was 290 °C and 300 °C, respectively. The PCB mixture composition was determined using linear regression analysis (KubaÂtova et al. 1996).

Results The fungus was cultivated in a bioreactor to produce a sucient amount of enzyme necessary for in vitro biodegradation experiments. The activity of MnP was detectable after day 6 and was close to its maximum (163 U/l) on day 11 of cultivation. Activity of LiP appeared on day 9 and reached a maximum (31 U/l) also on day 11, when the cultivation was terminated. No laccase activity was detected in the medium during the cultivation. Extracellular protein concentration was rather constant (5.4±5.9 lg/ml), did not ¯uctuate during the experiment with the exception of decrease on day 6 (4.7 lg/ml) and increase on day 7 (7.7 lg/ ml) (Figure 1). A simple isoelectric focusing technique used for isolation of MnP and LiP was sucient for enzyme separation (see Figures 2 and 3). Two peaks of MnP (with isoelectric points of 5.38 and 5.95, and molecular mass of about 45 kDa) and one peak of LiP (with an isoelectric point of 3.37 and molecular mass of about 40 kDa) were obtained. In vitro degradation was carried out with combined enzyme fractions ± fractions 12 and 14 (MnP) and fractions 3, 4 and 5 (LiP), respectively.

Figure 1. Time dependence of the activity of MnP (m) and LiP (h), and extracellular proteins (s) of Phanerochaete chrysosporium.

Figure 2. Separation of MnP (m) and LiP (h) from Phanerochaete chrysosporium by isoelectric focusing, run II.

Figure 3. SDS-PAGE of fractions from isoelectric focusation. Line 1: molecular size marker proteins, line 2: sample before ultra®ltration, line 3: sample after ultra®ltration, lines 4±23: fractions 1±20 from isoelectric focusation. Molecular masses of marker proteins are indicated in kilodaltons on the left.

Biodegradation of PCBs

273

Degradation of Delor 103 and Delor 106 by mycelium, crude extracellular liquid and partially puri®ed MnP and LiP from P. chrysosporium were studied (Table 1). Forty-four hours after addition

of PCBs to the mycelium, a decrease in PCBs was measured of 74% w/w for Delor 103 and 73% for Delor 106. A comparable decrease of PCBs was observed if the PCB mixtures were added to the

Table 1. Degradation by P. chrysosporium of selected Delor 103 and 106 congeners. The nomenclature of congeners recommended by Ballschmiter & Zell (1980) was used. Peak no.

IUPAC no.

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26

4 8/5 19 18/17 27/24 16/32 26 25 28/31 33 22 45 52 49 48/47 44 42 41/64/71/72 40 74 70/76 66/95 56/60 101/90 87 110

Delor 103

Average

27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42

151 135/144 149/118 153/132 141 138/163/164 182/187 183 128 174 177 180 170/190 196/203 195 194

Delor 106

Average

Structure

Degradation of congeners (%) Mycelium

Liquid

2,2¢ 2,4¢/2,3 2,2¢,6 2,2¢,5/2,2¢,4 2,3¢,6/2,3,6 2,2¢,3/2,4¢,6 2,3¢,5 2,3¢,4 2,4,4¢/2,4¢,5 2¢,3,4 2,3,4¢ 2,2¢,3,6 2,2¢,5,5¢ 2,2¢,4,5¢ 2,2¢,4,5/2,2¢,4,4¢ 2,2¢,3,5¢ 2,2¢,3,4¢ 2,2¢3,4/2,3,4¢,6/2,3¢,4¢,6/2,3¢,5,5¢ 2,2¢,3,3¢ 2,4,4¢,5 2,3¢,4¢,5/2¢,3,4,5 2,3¢,4,4¢/2,2¢,3,5¢,6 2,3,3¢,4¢/2,3,4,4¢ 2,2¢,4,5,5¢/2,2¢,3,4¢,5 2,2¢,3,4,5¢ 2,3,3¢,4¢,6

78 77 81 74 73 78 75 78 74 76 67 79 56 76 79 73 70 75 75 74 75 75 72 70 73 73

65 62 68 58 61 62 60 65 62 62 59 65 57 58 65 58 57 61 62 68 65 64 65 54 60 60

74

62

2,2¢,3,5,5¢,6 2,2¢,3,3¢,5,6¢/2,2¢,3,4,5¢,6 2,2¢,3,4¢,5¢,6/2,3¢,4,4¢,5 2,2¢,4,4¢,5,5¢/2,2¢,3,3¢,4,6¢ 2,2¢,3,4,5,5¢ 2,2¢,3,4,4¢,5¢/2,3,3¢,4¢,5,6/2,3,3¢,4¢,5¢,6 2,2¢,3,4,4¢,5,6¢/2,2¢,3,4¢,5,5¢,6 2,2¢,3,4,4¢,5¢,6 2,2¢,3,3¢,4,4¢ 2,2¢,3,3¢,4,5,6¢ 2,2¢,3,3¢,4¢,5,6 2,2¢,3,4,4¢,5,5¢ 2,2¢,3,3¢,4,4¢,5/2,3,3¢,4,4¢,5,6 2,2¢,3,3¢,4,4¢,5¢,6/2,2¢,3,4,4¢,5,5¢,6 2,2¢,3,3¢,4,4¢,5,6 2,2¢,3,3¢,4,4¢,5,5¢

73 77 70 75 73 73 75 77 71 71 74 74 72 75 72 73

56 61 55 59 58 59 57 60 60 56 57 59 58 57 57 56

73

58

274 crude extracellular liquid together with H2O2 (62% for Delor 103 and 58% for Delor 106). The degradation was nonspeci®c since no signi®cant di€erences in the degradation were found among di-, tri-, tetra-, penta-, hexa-, hepta-, and octachlorinated congeners. However, a complete separation of all congeners present in the sample was not possible under the GC/ECD conditions used. Some of the peaks may result from a co-elution of more than one PCB congeners (Larsen et al. 1992). In contrast, MnP and LiP isolated from the above mentioned extracellular liquid with H2O2 added, did not show any degradation of PCBs. Discussion Thin mycelial mats of surface liquid cultures have generally been used for the study of ligninolytic enzymes in Phanerochaete chrysosporium, a model representative of white rot fungi (Kirk et al. 1978; Faison & Kirk 1985; Leisola et al. 1987). The scale-up using shaken cultures by growing the fungus in a fermentor has been hampered by the fact that the activities of ligninolytic enzymes were sensitive to aeration and agitation (Janshekar & Fiechter 1988; Michel et al. 1990). In order to overcome such problems, many e€orts have been put in to achieve a scalable reactor con®guration giving acceptable performance. Mechanical stirring was replaced by pneumatic agitation (Bonnarme et al. 1991), and immobilization of mycelium on various support materials gave good results both in agitated and air-lift bioreactors (Willershausen et al. 1987; Schmidt et al. 1990; Bonnarme et al. 1991; Bosco et al. 1996; Darah & Ibrahim 1998). In the present work, the operational parameters have not been optimized in order to assure high enzyme activities and, therefore, the peak activities obtained are not comparable with those under optimized conditions (cf. Bosco et al. 1996). The goal of our work was to get a reasonable production of ligninolytic enzymes in a simple, inexpensive and reliable way. Therefore, we used a standard bioreactor with a simple modi®cation; the magnetically driven agitation body was replaced with plates of pressed polyurethane (that is normally used as ®lling material to protect instruments during transport). The bioreactor was aerated

P. KrcÏmaÂrÏ et al. with pure oxygen only during the ®rst 3 days of cultivation, to start the synthesis of the ligninolytic enzymes. The inoculum of low spore concentration caused a slow culture development and a later enzyme expression, so that, the increase of activities was moderate enough to catch the maximum for enzyme harvesting. White rot fungi have been found to degrade many aromatic organopollutants, including chlorinated phenols, PCBs, DDT, dioxins, polycyclic aromatic hydrocarbons, nitrotoluenes, synthetic dyes, etc. (for review see Aust 1990; Field et al. 1993; Hammel 1995). Many of these compounds were degraded by isolated ligninolytic enzymes, namely LiP, MnP and laccase (see the list summarized by Field et al. (1993) and references therein). However, none of these enzymes has so far been implicated in PCB degradation. Our results demonstrating the extracellular nature of PCB-biodegrading agent (different from the intracellular systems such as cytochrome P-450 monooxygenases) and simultaneously eliminating the role of LiP or MnP, support conclusions of Field et al. (1993) that ``other unidenti®ed enzymes are implicated.'' Our results thus support ®ndings published by KoÈhler et al. (1988), Thomas et al. (1992), Yadav & Reddy (1993), Yadav et al. (1995), Novotny et al. (1997) holding in doubt a direct correlation between the activity of ligninolytic enzymes and degradation of xenobiotic aromatics. In our experiments, PCB-congener-speci®c analysis of the Delor mixtures degraded in vitro by both mycelium and separated culture medium of P. chysosporium showed a similar pattern to that obtained by Yadav et al. (1995) with Aroclors incubated for 30 days with whole cultures of the same fungal species. To our best knowledge, our results demonstrate for the ®rst time the extracellular nature of the degrading agent and that it cannot be attributed to LiP or MnP. Acknowledgements This work was supported by grant No. A 6301501 of the Grant Agency of the Academy of Sciences of the Czech Republic and grant No TA-MOU-95C15-190, U.S.-Israel Cooperative Development Research Program, Oce of the Science Advisor, U.S. Agency for International Development.

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