Appl Microbiol Biotechnol(1993) 38:808-814

Applied Microbiology Biotechnology ©, Springer-Verlag 1993

Dechlorination of Fenclor 54 and of a synthetic mixture of polychlorinated biphenyls by anaerobic microorganisms Fabio Fava, Sergio Cinti,Leonardo Marchetti Department of Applied Chemistryand Material Science, EngineeringFaculty, Universityof Bologna, Viale Risorgimento2, 40136 Bologna, Italy Received: 12 June 1992/Accepted: 8 October 1992

Abstract. Microorganisms obtained from a contaminated experimental soil were found to reductively dechlorinate the polychlorobiphenyls (PCBs) of ex-commercial Fenclor 54 and of a synthetic mixture of single congeners, under laboratory anaerobic conditions. The dechlorination rate and extent tended to increase as the chlorination degree of F 54 congeners increased. Several penta:clorinated congeners temporarily accumulated during the final period of incubation. Dechlorination occurred primarily from the rneta and para positions while ortho-sustituted congeners accumulated in the medium during incubation. The dechlorination pattern observed with these unacclimated microorganisms in both PCB mixtures could be only partially compared to patterns reported in the literature. The low product yield deriving from reductive dechlorination of PCBs, i.e. diand tri-chlorinated biphenyls, :and the slow rate of PCB biotransformation can be attributed to a lower dehalogenation capability of artificially contaminated soil microorganisms and, perhaps, also to the inadequacy of the adopted anaerobic medium.

Introduction Fenclor is the commercial name of the complex mixtures of polychlorinated biphenyls (PCBs) manufactured in Italy by Caffaro up to 1965 for a variety of industrial purposes. Fenclor 42 (F 42), which is very similar to Aroclor 1242, contains about 42% of chlorine by weight, whereas Fenclor 54 (F 54), like Aroclor 1254, contains about 54% of chlorine by weight. Several lowchlorine PCB congeners of F 42 were easily degraded by one pure and two mixed aerobic cultures under growingcell conditions: the co-metabolism of F 42 PCBs, however, was limited to congeners with four or fewer chlorines (Fava et al. 1991). More interesting degrading abilities of aerobic cultures on other complex PCB mixtures

Correspondence to: F. Fava

(Aroclors, Kanachlors) have been demonstrated (Furukawa et al. 1983; Bedard et al. 1986, 1987a, b; Kohler et al. 1988. Microbial aerobic populations have also been shown to mineralize some PCB congeners to chlorine ions and unhalogenated simple organic compounds (Sylvestre et al. 1985; Adriaens et al. 1989; Pettigrew et al. 1990; Fava and Marchetti 1991). However, aerobic degradation of complex mixtures of PCBs is generally limited to low chlorinated congeners, produces chlorobenzoates as final stable products, and always requires an additional growth substrate (i.e., biphenyl or 4-chlorobiphenyl) (Bedard 1989). The microbiologically mediated reductive dechlorination of PCBs is therefore of great interest to the development of a biotreatment system for these compounds (Bedard 1989). Evidence for dechlorination of PCBs in many anaerobic sediments and in other spill sites was preliminarily reported by Brown et al. (1984, 1987a, b). Following on from these initial findings, many laboratory studies confirmed that bacterial communities obtained from PCB-contaminated sediments were responsible for reductive dechlorination of PCBs: differences in congener specificity were detected in different consortia (Bedard 1989). The removal of meta and para chlorine atoms (Quensen et al. 1988, 1990; Nies and Vogel 1990), or ortho and meta chlorines (Van Dort and Bedard 1991), although not decreasing the molar concentration of PCBs, can be expected to decrease the mammalian toxicity of PCB residues and to make them more readily degradable by aerobic bacteria (Quensen et al. 1988, 1990). The chemical mechanism of microorganism-mediated reductive dechlorination of PCBs has been also studied (Nies and Vogel 1991; Assaf-Anid et al. 1992). The aim of this work was to characterize the dechlorination activity of microorganisms obtained from an artificially contaminated soil using both an ex-commercial complex mixture (F 54) and a simple synthetic mixture of PCBs.

809

Materials and methods Source o f anaerobic microorganisms. Aroclors-contaminated soil was collected from an experimental agricultural field of the Missouri region. This soil, contaminated 10 and 15 years ago with high concentrations of Aroclor 1242, 1254 and 1260, was collected anaerobically up to a depth of about 1 m and transported, in a filled and tightly sealed vessel, to the laboratory where it was stored at 4 ° C until use. Ten litres of non-PCB-contaminated sludge featuring 20-25 days of retention time and about 2°70 of total organic matter was collected from a primary anaerobic digestor (municipal digestor of Reggio Emilia, Italy). The sludge was distributed as a thin layer in a container and allowed to evaporate slowly under normal atmospheric conditions. The dry matter (about 30 g residue/1 of treated sludge) was well ground in a mortar, sieved and stored in an anaerobic glove box (Coy Laboratory Products, USA) until use. This apparatus, used for all operations requiring anaerobic conditions, was fed with Oz-free Nz and COz (80:20).

Preparation o f test tubes. Degradation assays were carried out in 25 ml Pyrex serum tubes with fitted teflon screw caps. Samples were PrePared according to Quensen et al. (1990) in an anaerobic glove box. "Thirty-six test tubes were prepared for each mixture of PCBs tested, 18 of which were used as controls. Each tube was added with about 1 g of dried PCB-free sludge, 2 ml of reduced anaerobic mineral medium (RAMM medium following Shelton and Tiedje (1984) with 1 mg/1 of Resazurin) containing anaerobic microorganisms eluted from dried slugde and 2 gl of ethanol. Another four test tubes (called "indicator test tubes") were prepared as reported above in tubes equipped with screw caps containing butyl rubber septa. All test tubes, tightly capped under anaerobic conditions, were incubated at 37 ° C for 8-9 days, i.e. until methane was detected in the headspace of each indicator test tubes. Gas composition in the headspace of these tubes was determined by gas chromatography (GC) according to Biavati et al. (1988). The tubes were then sterilized by autoclaving at 121 ° C for 3 h.

Inoculation and incubation o f cultures. The inoculum was prepared from contaminated soil as reported by Quensen et al. (1990). All sterile assay tubes were inoculated in the anaerobic chamber with 5 ml RAMM suspension of microorganisms. Controls were then autoclaved twice at 121°C for 1 h (the second time 24 h after the first) before addition of the PCB mixture. A 100 g/1 solution (7.5 ~tl) of F 54 in acetone were added to a first group of 36 test tubes (750 gg/g on a sludge dry weight basis) while 25 Ixl of an acetone solution of PCB synthetic mixture (10 g / 1 of di-chlorobiphenyls and 5 g/1 of tetra- and penta-chlorobiphenyls corresponding to 250 gg/g and 125 gg/g on a sludge dry weight basis, respectively) were added to the remaining 36 tubes; this operation was carried out in the anaerobic chamber under aseptic conditions. Test tubes were tightly recapped, shaken thoroughly to ensure the distribution of PCBs, wrapped in aluminum foil and then incubated statically in the dark at 25°C for several weeks. Samples of each type of PCB mixture and related controls were drawn in duplicate from the incubator at pre-established times and frozen until extraction.

Preparation o f additional anaerobic cultures. Four additional sterfie assay tubes, prepared as reported above, were inoculated with 5 ml RAMM suspension containing 1 g contaminated soil. No exogenous PCBs (such as a synthetic mixture of PCBs or F 54) were added to these tubes; after sterilization of the two control samples, all the tubes were incubated at 25 ° C for 20 weeks as reported for the other cultures.

Extraction o f PCBs. In order to study the percentage of recovery, before the addition of extraction solvents 100 ~tl of a 0.5 g/1 acetonic solution of 2,4,4',6-tetrachlorobiphenyl (congener no. 75, which was absent in the original F 54) was added to each culture

containing F 54, and 125 gl of acetone solution of 2,4,5,2',5'-pentachlorobiphenyl (congener no. 101, 1 g/l) was added to each culture containing the synthetic mixture of PCBs. Acetone (10 ml) was then added to each tube, which was closed hermetically and shaken thoroughly. The content of the tube was carefully transferred to a 100-ml teflon screw capped silanized bottle; another 20 ml acetone was put into the same tube; after shaking, they were combined with the acetonic fraction previously transferred into the bottle. The entire contents of the test tubes were then extracted in the bottles using an ultrasonic bath. The extractions were carried out according to Quensen et al. (1990) using three times more solvent volume and quantities of chemicals. The sample eluted with hexane in a Florisil-copper powder column was added with 200 gl of a hexane solution of lindane (70 rag/l) and 700 I.tl of a hexane solution of octachloronaphthalene (OCN, 100 rag/l) and, finally, with hexane to obtain a final volume of 100 ml.

GC analysis. The samples were analysed with a gas chromatograph (5890 II series Hewlett-Packard, Palo Alto, Calif., USA), equipped with a Supelchem PTE-5 capillary column (30m by 0.25 mm, SE-54 equivalent) and an electron capture detector (ECD). All runs were conducted under the following conditions: initial temperature, 60 ° C; isothermal for 1 rain; first temperature rate, 20 ° C/min; final temperature, 180°C; isothermal for 2 min; second temperature rate, 4°C/rain; final temperature, 225°C; third temperature rate, 5° C/min; final temperature, 275 ° C; isothermal for 8 min; injector (splitless mode), 240 ° C; ECD, 320 ° C; carrier gas flow rate (N2), 6 ml/min; sample volume, 0.5 gl. The identification of the individual PCB congeners of F 54 was performed by using available pure congeners and/or by comparing the data reported in previous studies (Ballschmiter and Zell 1980; Capel et al. 1985) and the retention times relative to OCN (RRT), calculated by Mullin et al. (1984). The 33 highest peaks of F 54, numbered in order of elution from the gas chromatographic column, were studied; the corresponding congener assignments with the chlorine substitution pattern and the numbers designated by International Union of Pure and Applied Chemistry (IUPAC) as tabulated by Ballschmiter and Zell (1980) are reported in Table 1. Possible co-eluting isomers were also identified. Quantification (mg/l) for each PCB isomer identified was performed by using a Hewlett-Packard 3396 A integrator, and four-point calibration curves were plotted using F 54 and the synthetic PCB mixture as standards, and lindane plus OCN as internal standards. Fourpoint calibration curves were also plotted for individual congeners (nos. 75 and 101), which were used as "standards of recovery". The percentage of dechlorination of each PCB (i.e. the decrease of each chromatographic peak), was calculated with the following equation: Average dechlorination percentage = ~ - g ~ - - x 100, where Ci t ° and C~ represent the average concentrations (mg/1) of the congener i, in the controls and in the active cultures, respectively, at a given incubation time. t ° and C~ were determined from the i PCB peak area and corrected on the basis of the recovery by the internal standard quantification method. This dechlorination parameter was determined as reported above for the culture and controls which were taken from the incubator at weeks 4, 6, 8, 10, 12, 14, 16, 18 and 20 of incubation. Possible co-eluting congeners indicated in Table 1 were not considered in terms of extent and rate of decrease.

Chemicals. F 54 (ex-commercial sample) was provided by Caffaro (Brescia, Italy). All mineral medium, chemicals were obtained from Merck (FRG), pesticide-free hexane and acetone were obtained from J. T. Baker (Holland), and PCB pure congeners, lindane and OCN were purchased from Ultra Scientific (North Kingstown, R.I., USA).

810 Table 1. Assignment of congeners to chro-

matographic peak of F 54 and their retention time relative to octachloronaphthalene (RRT)

Peak no.

RRT

IUPAC no.

Substitution pattern

Lindane 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 27 28 29 30 31 32 33

0.4080 0.5084 0.5137 0.5334 0.5485 0.5768 0.5811 0.5868 0.5947 0.6075 0.6145 0.6213 0.6414 0.6475 0.6529 0.6596 0.6746 0.6800 0.6889 0.7104 0.7175 0.7215 0.7327 0.7439 0.7501 0.7600 0.7699 0.7758 0.7823 0.7969 0.8031 0.8086 0.8278 0.8610

52 49 44 72(41-64-71) 74 70(76) 95(66-102) 91 60(56) 101(90) 99 97(86) 87(115) 85 110(77) 151 135(144) 149(118) 122(131) 146 153 141 137 138(163) 158 178 175 187(182) 183 167 185 180 170

2,5,2',5' 2,4,2',5' 2,3,2',5' 2,5,3',5'(2,3,4,2'-2,3,6,4'-2,6,3',4') 2,4,5,4' 2,5,Y,4'(3,4,5,2') 2,3,6,2',5'(2;4,3',4'-2,4,5,2',6') 2,3,6,2',4' 2,3,4,4'(2,3,Y,4') 2,4,5,2',5'(2,3,5,2',4') 2,4,5,2',4' 2,4,5,2',3'(2,3,4,5,2') 2,3,4,2',5'(2,3,4,6,4') 2,3,4,2',4' 2,3,6,3',4'(3,4,Y,4') 2,3,5,6,2',5' 2,3,5,2',3',6'(2,3,4,6,2',5') 2,3,6,2',4',5'(2,4,5,3',4') 3,4,5,2',3'(2,3,4,6,2',3') 2,3,5,2',4',5' 2,4,5,2',4',5' 2,3,4,5,2',5' 2,3,4,5,2',4' 2,3,4,2',4',5'(2,3,5,6,3',4') 2,3,4,6,3',4' 2,3,5,6,2',3',5' 2,3,4,6,2',Y,5' 2,3,5,6,2',4',5'(2,3,4,5,2',4',6') 2,3,4,6,2',4',5' 2,4,5,3',4',5' 2,3,4,5,6,2',5' 2,3,4,5,2',4',5' 2,3,4,5,2',3',4'

IUPAC, International Union of Pure and Applied Chemistry

Results

PCB dechlorination activities of anaerobic cultures: preliminary observations The culture prepared using contaminated soil and PCBfree dried sludge showed, on the basis of simple visual inspection of the chromatograms reported in Fig. 1, a marked variation in the gas chromatographic peak distribution with respect to the corresponding control after 20 weeks of incubation. Neither PCB composition of contaminated sediment nor extent and pattern of endogenous PCB dechlorination have been studied here.

Dechlorination o f F 54 by contaminated soil microorgan&ms F 54 showed a certain dechlorination only after 10 weeks of incubation. The average percentage of decrease of each chosen peak of F 54 after 20 weeks of incubation of the cultures is given in Table 2. Several highly chlorinated congeners (nos. 170, 180, 167, 183,175, 178)exhibited a strong decrease with respect to most other peaks in F 54, whereas some penta-chlorinated congeners (nos. 97, 87, 85, 110) showed marked increases.

Hepta-chlorinated biphenyls with three chlorines in the ortho positions (nos. 185, 183, 187, 178, 175) showed, after the same incubation time, a lower degradation percentage than mono- or di-ortho-chlorinated ones (nos. 180, 167). The progressive dechlorination of tetra-, penta-, hexa- and hepta-chlorinated biphenyls of F 54 in living cultures is reported in Fig. 2. Hepta-, hexa- and tetrachlorobiphenyls were slowly dechlorinated after the first 8-10 weeks of incubation, while the concentration of penta-chlorobiphenyls slowly increased during the same period. The 2-chlorobiphenyl (RRT = 0.2947), 2,2'-dichlorobiphenyl (or 2,6-dichlorobiphenyl or both, RRT = 0.3375), 2,5-dichlorobiphenyl (or 2,4-dichlorobiphenyl or both, RRT--0.3698) and a PCB with RRT -- 0.4001, tentatively identified as 2,6,2'-trichlorobiphenyl, were detected in small concentrations only in the active cultures, f r o m week 16 to 20 of incubation. The presence of other early eluting PCBs was indicated by ambiguous peaks, which were found only in some chromatographic profiles related to the oldest living cultures.

811 300

A

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200 -

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t 100 L

il,J , l l lt/,l

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0

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_ i

300

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mV

chlorobiphenyl, which has two ortho chlorine atoms in addition to the 4,4'-substitution, was found to be more refractory to dechlorination than 4,4'-dichlorobiphenyl. Congener no. 105, a coplanar PCB characterized by a greater mammalian toxicity (Parkinson et al. 1983; Safe et al. 1985), was also found to be significantly degraded at week 20 of incubation (Table 3). Small accumulations of 2,6- and/or 2,2'-dichlorobiphenyl, 2-chlorobiphenyl (RRT=0.2935), 4-chlorobiphenyl (RRT= 0.3225) and a PCB with RRT=0.5875, identified as 2,4,3',4'-tetrachlorobiphenyl, were detected in the medium at the end of incubation. No other PCBs were unequivocally identified as intermediates or terminal products of the dechlorination process. A comparison of the undegraded percentage of each congener of the synthetic mixture of PCBs as a function of time is shown in Fig. 3. No dechlorination activity was observed until the weeks 8-10 of incubation, when it became significant: the dechlorination process continued at a low rate throughout all of the last 10 weeks.

Discussion

250Z

O

200 -

m

150 -

100 ~

50-

0 10.00

I

!iittl

1

{

t

i

15.00

22.50

30.00

37.50 rain 45.00

Fig. 1A, B. Reductive dechlorination of endogenous polychlorinated biphenyl (PCBs) of contaminated soil after 20 weeks of incubation: OCN, octachloronaphthalene. A Autoclaved control. B Active culture

Dechlorination o f a synthetic mixture o f P C B s

The assay mixture included six PCB congeners referring to various structural classes such as: 1. Congeners chlorinated only in the ortho position (nos. 10, 54). 2. Congener chlorinated in the meta position (no. 80). 3. Congeners chlorinated in the para position (nos. 15, 75). 4. Co-planar congener preferentially chlorinated in the meta and para positions (no. 105). Both meta- and para-chlorinated congeners were significantly degraded at week 20 of incubation, whereas the heaviest ortho-chlorinated congener (no. 54) showed a degree of degradation close to zero. 2,4,4',6-tetra-

Several endogenous PCBs of the contaminated soil used as a source of microorganisms in our experiment considerably decreased after 20 weeks of culture incubation: the more chlorinated congeners were degraded to the less chlorinated PCBs only in the living cultures (Fig. 1). This change in endogenous PCBs isomer and congener distribution indicated the occurrence of PCBs reductive dechlorination mediated by artificially contaminated soil microorganisms. Several congeners of F 54 ranging from tetra- to hep= ta-chlorobiphenyls were significantly dechlorinated by the same consortium of unacclimated microorganisms after 20 weeks of incubation (Table 2). Whereas hexaand hepta-chlorinated congeners of F 54 were extensively and progressively degraded after week 10 of incubation, several penta-chlorinated isomers slowly accumulated in the active cultures during the same incubation time (Fig. 2). A thorough evaluation of the substitution pattern of the biotransformed congeners (Table 2), suggested that the accumulated penta-chlorobiphenyls were the preliminary derivatives of the meta and para dechlorination of one or more heavier F 54 chlorinated biphenyls. The slow and apparently reversible accumulation of penta-chlorobiphenyls can be considered, therefore, to be the result of a high rate of dechlorination of the heavier F 54 chlorinated biphenyls present in the culture (Quensen et al. 1990), but also, probably, of a low ability of the same microorganisms to degrade these PCBs by the same mechanism. Also, Brown et al. (1984) observed that several penta-chlorobiphenyls, such as congeners nos. 110 and 91, present in F 54 and significantly accumulated in our cultures, were very slowly removed in different sediment layers. Dechlorination of tetrachlorinated biphenyls was less extensive than that observed on hexa- and hepta-chlorinated biphenyls (Table 2). These observations suggested that the dechlorination activity of the unacclimated microorganisms obtained

812 Table 2. Average percentage decrease of each chosen peak of F 54 after 20 weeks of incubation in living cultures

IUPAC no.

Substitution pattern

Degradation (°70)

52 49 44 72(41-64-72) 74 70(76) 95(66-102) 91 60(56) 101(90) 99 97(86) 87(115) 85 110(77) 151 135(144) 149(118) 122(131) 146 153 141 137 138(163) 158 178 175 187(182) 183 167 185 180 170

2,5,2',5' 2,4,2',5' 2,3,2',5' 2,5,3',5'(2,3,4,2'-2,3,6,4'-2,6,3',4') 2,4,5,4' 2,5,3',4'(3,4,5,2') 2,3,6,2',5'(2,4,Y,4'-2,4,5,2',6') 2,3,6,2',4' 2,3,4,4'(2,3,3',4') 2,4,5,2',5'(2,3,5,2',4') 2,4,5,2',4' 2,4,5,2',Y(2,3,4,5,2') 2,3,4,2',5'(2,3,4,6,4') 2,3,4,2',4' 2,3,6,Y,4'(3,4,3',4') 2,3,5,6,2',5' 2,3,5,2',Y,6'(2,3,4,6,2',5') 2,3,6,2',4',5'(2,4,5,3',4') 3,4,5,2',3'(2,3,4,6,2',3') 2,3,5,2',4',5' 2,4,5,2',4',5' 2,3,4,5,2',5' 2,3,4,5,2',4' 2,3,4,2',4',5'(2,3,5,6,3',4') 2,3,4,6,3',4' 2,3,5,6,2',3',5' 2,3,4,6,2',3',5' 2,3,5,6,2',4',5'(2,3,4,5,2',4',6') 2,3,4,6,2',4',5' 2,4,5,3',4',5' 2,3,4,5,6,2',5' 2,3,4,5,2',4',5' 2,3,4,5,2',3',4'

10 Not resolved 28 64 53 42 10 +(11) 27 + (5) + (7) + (9) + (11) + (21) + (15) 43 40 13 7 20 17 23 47 10 10 45 41 5 49 75 35 70 55

The + sign stands for polychlorinated biphenyls (PCBs) accumulated in the medium

Table 3. Average percentage decrease of each PCB of the synthetic mixture after 20 weeks of incubation in living cultures

110 U p-,

90 ~ 80 i ~I~

c

~~ ~ ~ p~rrA.CHLOR1NATED ~ ~RA-CHLOR~NATED

7O

IUPAC no.

RRT

Substitution pattern

Degradation (°7o)

10 15 54 75 80 105

0.3384 0.4211 0.4537 0.5175 0.5835 0.7228

2,6 4,4' 2,6,2',6' 2,4,6,4' 3,5,3',5' 2,3,4,3',4'

+ (1.5) 37 3 21 39 33

The + sign stands for PCB accumulated in the medium ~

60

>

50

0

10

20

30

Incubation time (weeks} Fig. 2. Comparison of undegraded average percentage of total tetra-, penta-, hexa- and hepta-chlorinated biphenyls of F 54 versus time in the living cultures

f r o m artificially c o n t a m i n a t e d soil d e p e n d e d on the degree o f c h l o r i n a t i o n o f P C B s . I n fact, the rate a n d the extent o f reductive d e c h l o r i n a t i o n t e n d e d to increase as the degree o f c h l o r i n a t i o n o f P C B s increased. T h e d e c h l o r i n a t i o n o f the i d e n t i f i e d P C B s o f F 54 occ u r r e d p r i m a r i l y f r o m the meta a n d para p o s i t i o n s (Table 2): the presence o f m o r e t h a n one chlorine a t o m in the ortho p o s i t i o n was the m a i n cause o f the decrease in b i o c o n v e r s i o n o f h e p t a - a n d h e x a - c h l o r i n a t e d congeners a n d o f the recalcitrance o f congeners nos. 4, 10, 19 a n d o f 2 - c h l o r o b i p h e n y l . This o b s e r v a t i o n was c o n f i r m e d b y the results o b t a i n e d f r o m the d e c h l o r i n a t i o n o f the syn-

813 mllO ,~1o0 >

No. 10

o 9o

8

No. 15 No. 54 No, 75 No. 80 No. 105

,-~ 70

I

~ 50 0

10

~

I

20

30

I n c u b a t i o n time (weeks) Fig. 3. Comparison of undegraded average percentage of congeners of a synthetic mixture of PCBs versus time in the living cultures

thetic mixture of PCBs: also in this case, only the congeners chlorinated in meta and para positions showed an important dechlorination, and the presence of chlorine atoms in the ortho positions was the cause of recalcitrance to reductive dechlorination (Table 3), as well as of the accumulation of some mono- and di-chlorinated congeners. The production in living cultures of 2,4,3',4'tetrachlorobiphenyl, which is considered to be the major meta-dechlorination product of the congener no. 105 (Abramowicz et al. 1989), was a further confirmation of the congener dehalogenation specificity of our microorganisms. These assessments, which suggest a microbial dechlorination selectivity similar to that already reported in the literature (Quensen et al. 1988, 1990; Abramowicz et al. 1989; Nies and Vogel 1990), allowed us to exclude the presence of an ortho-dechlorination activity in our microorganisms (originally suggested by Brown et al. (1987a) and recently demonstrated under laboratory conditions (Van Dort and Bedard 1991)). The dechlorination of F 54 and of the synthetic mixture of PCBs apparently followed the C dechlorination pattern described by Brown et al. (1984, 1987a, b) as the most commonly encountered in the Hudson River region. In fact, the dechlorination process of F 54 apparently converted, presumably stepwise, most of the mono-ortho-substituted congeners to 2-mono-, 2,4-diand 2,5-di-chlorobiphenyls, most of the di-ortho-substituted congeners to 2,2'-di- a n d / o r 2,6-di-chlorobiphenyls, and most of tri-ortho-substituted congeners to 2,6,2'-trichlorobiphenyl. The detection of other chlorinated biphenyls (congeners nos. 2 and 66) in the cultures with the PCB-synthetic mixture, the accumulation of Several penta-chlorinated biphenyls in the F 54 cultures and the generally low yield of less chlorinated products of reductive dehalogenation, however, made it quite difficult to unambiguously compare our results with the dehalogenation patterns

previously described in the literature (Brown et al. 1984, 1987a, b; Quensen et al. 1988, 1990; Nies and Vogel 1990; Van Dort and Bedard 1991). The low yield in the formation of di- and tri-chlorihated biphenyls suggested that dechlorination occurred quite slowly in our cultures. Several other experimental findings, such as a longer lag phase, a stronger accumulation of penta-chlorinated congeners and a lower degradation percentage of PCBs with respect to those observed by Quensen et al. (1990) on Aroclor 1254 at comparable incubation times, confirmed this hypothesis. A lower reductive dehalogenation activity of our microorganisms may be the cause of the slow rate of the process. Our microorganism consortium was obtained from an artificially contaminated soil, i.e. from a site different from those naturally contaminated by PCBs normally used as a source of microorganisms (Quensen et al. 1988, 1990; Abramowicz.et al. 1989; Nies and Vogel 1990, 1991). Since the rate (as well as the pattern of dechlorination) is generally dependent on the contaminated sediment used for the inoculum (Quensen et al. 1990), the low rate of dechlorination observed in our experiments may therefore be attributed to the peculiar origin of our microorganisms. The slow rate of dechlorination may also be attributed to the medium chosen for our experiments which included, besides PCB-free sediments (Quensen et al. 1988, 1990), an elective methanogenic substrate, quite rich in organic matter. A higher concentration of complex organic compounds, which generally corresponds to a significant availability of electron acceptors and, therefore, to good microbial growth, may reduce the tendency of the microorganisms to use PCBs as terminal electron acceptors (Brown.et al. 1987a, b; Quensen et al. 1988). The limited capacity of our microorganisms to express PCB dechlorination may therefore be also attributed to the peculiar composition of the culture medium. Microscopic investigations showed that our methanogenic consortia were made up of four or more species or strains of anaerobic microorganisms. So far no studies have been conducted in order to characterize these microorganisms. We can therefore conclude that also the indigenous microorganisms selected from anaerobic artificially contaminated soils are responsible for the extensive dechlorination of PCBs in simple and complex mixtures (F 54). Since a great part of the mammalian toxicity of PCBs is generally linked to the meta and para chlorine atoms (Parkinson et al. 1983; Safe et al. 1985), the anaerobic treatment described should be able to reduce the intrinsic toxicity of F 54. The data reported underscore the importance of microbially catalysed dechlorination of PCBs in the environmental detoxification of contaminated sites. Acknowledgments. We are greatly indebted to Dr. Andrea Tilche of ENEA (Department of Environmental Engineering, ENEA of Bologna) for helpful suggestions and for critically commenting on the results. We thank Mr. P. Luigi Tonetti of Caffaro, Italy, who kindly supplied us with an old laboratory sample of Fenclor 54 and Mr. W. Tumiatti of Sea Marconi Technologies, Italy, for the anaerobic PCB-contaminated soil.

814

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