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International Biodeterioration & Biodegradation 60 (2007) 1624 www.elsevier.com/locate/ibiod

PCB-degrading potential of aerobic bacteria enriched from marine sediments

Ana Begonja Kolara,, Dubravka Hrs aka, Sanja Finglerb, Helena Cetkovicc, Ines Petrica, a Nikolina Udikovic Kolic
a

kovic Institute, POB 180, HR 10002 Zagreb, Croatia Division for Marine and Environmental Research, RuXer Bos b Institute for Medical Research and Occupational Health, Zagreb, POB 291, HR 10001 Zagreb, Croatia c kovic Institute, Zagreb, POB 180, HR 10002 Zagreb, Croatia Division of Molecular Biology, RuXer Bos Received 16 October 2006; received in revised form 14 November 2006; accepted 14 November 2006 Available online 5 January 2007

Abstract The main objective of this work was to study catabolic potential of marine sediment bacteria in aerobic degradation of polychlorinated biphenyls (PCBs). Marine sediment samples were collected at urban areas of the Croatian Adriatic coast, and microcosm enrichment experiments were performed in seawater mineral salts (SMS) medium with the addition of biphenyl as the only carbon source. After two to four subcultures, all enriched mixed cultures demonstrated the capability to use biphenyl, indicating that biphenyl-utilizing bacteria are widespread in coastal marine sediments. PCB-degrading activity of the enrichment cultures as well as that of their biphenyl-utilizing members were further studied in SMS medium with the addition of PCB mixtures containing di- to heptachlorinated congeners. GCMS analyses of the extracted cultures suggested that, although they differed in PCB-degrading capabilities, all of the enrichment cultures expressed activity toward at least some of the lower chlorinated congeners (di- to tetrachlorobiphenyls). Biphenyl-utilizing bacteria isolated from the most active PCB-degrading mixed cultures showed little taxonomic diversity (six out of seven isolates belonged to the genus Rhodococcus and one to the genus Sphingomonas). All isolated Rhodococcus strains (R. erythropolis and R. ruber) showed substantial PCB-degrading activity, suggesting that these bacteria might play an important role in aerobic PCB degradation in polluted marine sediment. r 2006 Elsevier Ltd. All rights reserved.
Keywords: Marine sediment; PCB; PCB degradation; Rhodococcus

1. Introduction Polychlorinated biphenyls (PCBs) are synthetic chemicals that have been used for several decades in a wide range of industrial applications such as oil in transformers, dielectrics in capacitors, hydraulic uids in hydraulic tools, lubricants for turbines and pumps, in the formulations of cutting oils for metal treatment and surface coatings. A large quantity of PCBs was released in the environment during their early usage. Since PCBs are highly recalcitrant to biodegradation, they persist in the environment for a long time (Borja et al., 2005). They are hydrophobic chemicals and their strong adsorption to organic matter
Corresponding author. Tel.: +38514680944; fax: +38514680242.

E-mail address: begonja@irb.hr (A. Begonja Kolar). 0964-8305/$ - see front matter r 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.ibiod.2006.11.004

leads to the accumulation in soil and aquatic sediments. It is generally believed that estuarine and marine sediments are the ultimate sinks for worldwide accumulation of PCBs; high PCB levels were found especially in harbours and coastal sediments under the impact of industrial activity (Kuo et al., 1999; Wiegel and Wu, 2000; Frignani et al., 2001; Fava et al., 2003). Despite their recalcitrance, microbial-mediated degradation is considered one of the main processes in alleviation of PCB pollution from the contaminated environment. Numerous PCB-degrading bacteria which transform PCBs through the biphenyl pathway have been isolated from soil and freshwater sediments (Furukawa et al., 1979; Bedard et al., 1986; Seto et al., 1995; Sakai et al., 2002). The enzymes involved in this catabolic pathway have broad substrate specicity, allowing the bacteria to

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simultaneously cometabolize PCBs. Degradation begins with the attack of biphenyl dioxygenase on an unsubstituted 2,3 position of biphenyl ring and thus formed dihydroxy metabolites are transformed through metacleavage products to chlorobenzoic acids (Hofer et al., 1994; Sakai et al., 2002). The bacteria that have been isolated and examined in aerobic PCB degradation studies are Gram-negative bacteria belonging to the genera Burkholderia, Acinetobacter, Ralstonia and Pseudomonas (Furukawa et al., 1979; Bedard et al., 1986; Gibson et al., 1993; Hofer et al., 1994; Novakova et al., 2002), as well as Gram-positive bacteria, often Rhodococcus species (Asturias and Timmis, 1993; Chung et al., 1994; Seto et al., 1995). It is important to note that numerous studies on aerobic PCB degradation have been conducted by using the bacteria isolated from the soil and freshwater environment; however, there is no information about aerobic PCB degradation activity of the bacteria originating from marine sediments. Therefore, our main research interest is to investigate whether bacteria-mediated aerobic PCB-degradation occurs in marine environment. In this work, we describe the enrichment of biphenyl-utilizing bacteria from coastal marine sediments, taxonomic and physiological characteristics of these bacteria, focusing primarily on their metabolic activity in aerobic PCB-degradation.
2. Materials and methods 2.1. Sediment samples
Samples of the upper layer (010 cm) of marine sediment were collected at sea depths from 5 to 34 m in the vicinity of three urban centres at the Croatian Adriatic coast (three samples were collected in Rijeka harbour, three samples in Zadar harbour and two samples in the Dubrovnik area). PCB concentrations in marine sediments ranged from 0.2 to 1.66 mg kg1 (dry weight).

Biphenyl was obtained from Merck, Hohenbrunn, Germany and dihydroxybiphenyl from Fluka, Sigma-Aldrich, Deisenhofen, Germany. Acetone and n-hexane were purchased from Merck, Darmstadt, Germany. All other chemicals (p.a. purity) were the products of Kemika, Zagreb, Croatia.

2.4. Enrichment and isolation of biphenyl-utilizing bacteria

Marine sediments (10 g) were added in Erlenmeyer asks (300 ml) containing SMS medium (100 ml). Biphenyl (1 g l1) was added and asks were shaken on a rotary shaker (150 rpm) at 30 1C. After ten to fourteen days, the cultures were allowed to settle, inoculated into fresh SMS medium (10%), and incubated under the same conditions. After two to four subcultures, intensive yellow colour was observed in all asks, indicating the presence of 2,3-dihydroxybiphenyl dioxygenase and metacleavage of 2,3-dihydroxybiphenyl in all enriched cultures. During enrichment experiments, the number of heterotrophs and biphenyl-utilizing bacteria in mixed cultures was determined using plate count method (Brown, 2005). Serial dilutions of the cultures were plated in triplicates onto Marine agar plates for heterotrophs and SMS agar plates with biphenyl in the vapour phase for biphenyl-utilizing bacteria. The heterotrophs were counted after a ve day incubation and biphenylutilizers after ten to fteen day incubation at 30 1C. Colonies grown on SMS agar plates were further assayed for 2,3-dihydroxybiphenyl dioxygenase activity by spraying with an aqueous solution containing 0.1% dihydroxybiphenyl (wt./vol) and 10% (vol/vol) acetone. Appearance of yellow colour within several minutes suggested the presence of 2,3dihydroxybiphenyl dioxygenase (Wagner-Dobler et al., 1998). Morpholo gically different colony types were isolated and puried after several subcultures on SMS agar plates under the biphenyl atmosphere. Table 1 PCB congeners assignment to analysed peaks in the GSMS chromatogram of PCB50 mixture Peak IUPAC number number 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
a b

Chemical structure Di-CBb Di-CB Tri-CB 2,20 ,5; 4,40 ; 2,20 ,4 2,30 ,6 2,20 ,3; 2,40 ,6 2,30 ,5; 2,30 ,4 2,4,40 20 ,3,4; 2,20 ,5,60 ; 2,3,30 ; 2,3,4 Tetra-CB 2,3,40 ; 2,20 ,4,60 2,20 ,3,6 2,20 ,5,50 2,20 ,4,50 ; 2,20 ,3,5 2,20 ,4,40 ; 2,20 ,4,5; 2,4,40 ,6 2,20 ,3,50 ; 3,4,40 2,20 ,3,40 ; 2,3,30 ,6 2,3,40 ,6; 2,30 ,40 ,6; 2,20 ,3,4 2,20 ,3,30 2,30 ,4,5; 2,20 ,4,40 ,6; 2,3,30 ,5; 2,20 ,4,50 ,6 2,3,40 ,5 2,4,40 ,5; 2,20 ,3,5,60 2,30 ,40 ,5 2,30 ,4,40 ; 20 ,3,4,5; 2,20 ,3,50 ,6; 2,20 ,4,5,60 ; 2,20 ,3,5,6 2,20 ,3,40 ,6; 2,3,30 ,4 2,3,4,40 ; 2,3,30 ,40 2,20 ,4,5,50 ; 2,20 ,3,40 ,5

2.2. Culture media

Enrichment experiments were performed in seawater mineral salts (SMS) medium, supplemented with biphenyl as the sole carbon source. SMS medium was prepared with lter sterilized seawater and mineral salts medium according to Bedard et al., 1986 (1:1). Salinity of SMS medium was 18.5.

2.3. Chemicals
Commercial PCB mixtures, PCB50 and Aroclor 1248, were used in biodegradation experiments. PCB50 mixture, containing di- to heptachlorinated congeners, was obtained by courtesy of the Environmental Protection Institute, Maribor, Slovenia, origin unknown. The composition of PCB50 mixture was characterized by GCMS analyses and the selected congeners discussed in this work are presented in Table 1. The PCB congeners in the chromatogram of PCB50 mixture were identied by comparison with the chromatograms of commercially available standard Aroclor PCB mixtures published in the literature (Frame et al., 1996) as well as by comparison with the chromatogram of a standard mixture of twenty individual PCB congeners (IUPAC numbers: 28, 52, 60, 74, 77, 101, 105, 114, 118, 123, 126, 138, 153, 156, 157, 167, 169, 170, 180 and 189). Aroclor 1248 was purchased from Supelco, Bellefonte, USA.

NDa ND ND 18,15,17 27 16,32 26,25 28 33,53,20,21 ND 22,51 45 52 49, 43 47,48,75 44,37 42,59 64,71,41 40 67,100,57,103 63 74, 94 70 66,76,95,102,93 91,55 60,56 101,90 ND, not determined. CB, chlorobiphenyl.

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18 A. Begonja Kolar et al. / International Biodeterioration & Biodegradation 60 (2007) 1624 followed by 35 cycles of 94 1C for 1 min, 55 1C for 1 min and 72 1C for 2 min, with a nal extension of 72 1C for 10 min. PCR products were separated in 0.8% agarose gel and puried with a Qiagen QIAquick gel extraction kit according to the manufacturers directions. Sequence analyses were performed with automatic sequence analyser ABI PRISMs 3100-Avant Genetic Analyser (Applied Biosystem, USA) using the primers mentioned above and the internal primes 38f, 338f and 518r. 16S rDNA sequences were compared to those available in the GenBank NCBI (National Center for Biotechnology Information databases) using the BLAST server. Multiple sequence alignments and construction of the phylogenetic tree were performed using Clustal X programme (Thompson et al., 1997). Program TreeView, version 1.6.6. (http://www.taxonomy. zoology.gla.ac.uk/rod/treeview.html), was used for graphic presentation of the results.

2.5. PCB degradation experiments

The same shake ask technique as described for the culture enrichment by using SMS medium (45 ml) supplemented with biphenyl (0.7 g l1) was applied in PCB degradation experiments. Three-day-old bacterial cultures grown in SMS medium with biphenyl were used as inocula. The PCB50 mixture was used in the experiments with mixed cultures and Aroclor 1248 was used in the experiments with isolated strains. In both experiments, PCB mixture was added to the cell suspensions as concentrated acetone solutions until the nal concentration of 50 mg l1. Control experiments with bacterial cells inactivated by autoclaving (121 1C for 15 min) were performed as well. The asks were incubated on a rotary shaker (150 rpm) at 30 1C. All experiments were performed in triplicates. After two weeks of incubation, cultures were sonicated in ultrasonic bath for 15 min. Subsequently, 20 ml of n-hexane was added to the asks, cultures were sonicated for additional 15 min and shaken vigorously (280 rpm for 2 h at 18 1C). After extraction, 5 g of anhydrous sodium sulphate was added to prevent the formation of stable emulsion. The phases were separated by centrifugation (4000g for 1 min) and the hexane layer was collected for GCMS analyses. GCMS analyses were performed by using Varian Saturn II GCMS system (Varian, Walnut Creek, CA, USA), consisting of a Varian 3400 gas chromatograph and ion trap detector (ITD). Separation of PCB congeners was performed on a Rtx-5MS fused silica column, dimensions 60 m 0.25 mm (i.d.), lm thickness 0.25 mm (Restek, Bellefonte, PA, USA). The column temperature was programmed from 60 1C (1 min hold) to 200 1C at 40 1C min1, then to 240 1C at 2 1C min1 and to 260 1C at 40 1C min1 (20 min hold). The injector temperature was programmed from 100 1C (0.1 min hold) to 270 1C at 200 1C min1 (3 min hold). The carrier gas was helium. Column head pressure was 103 Pa. The GCMS (ITD) was operating in the electron impact ionization mode (70 eV) at the lament emission current of 20 mA. Transfer line and manifold temperatures were 240 and 220 1C, respectively. The spectra of PCB congeners were recorded in the full scan acquisition mode (mass range 45500 m/z) at the scan rate of 1 scan/s. PCB-degrading activity was evaluated based on the reduction of the peak areas in GCMS chromatograms of the culture extracts compared to the peak areas in the chromatograms of the control sample extracts (with autoclaved cultures). In this way potential abiotic PCB elimination (adsorption to the bacterial cells and to the incubation ask walls) was taken in consideration. In the experiments with enrichment cultures, performed with PCB50 mixture, results were expressed as the percentage of the reduction of individual peak areas while in the experiments with isolated strains, performed with Aroclor 1248, the results were expressed as the percentage of the reduction of total peak areas.

2.8. Nucleotide sequence accession numbers

The 16S rDNA sequences of the isolates R1, R2, Z56, Z57, D3-1, D3-2 and D14 determined in this study have been deposited in GenBank (NCBI) database under the accession numbers DQ858957, DQ858958, DQ858959, DQ858960, DQ858961, DQ858962 and DQ858963, respectively.

3. Results 3.1. Spreading of biphenyl-utilizing bacteria in marine sediments Microcosm experiments described in this work, which were preformed in shake culture using seawater mineral salts (SMS) medium supplemented with biphenyl, resulted in fast enrichment of biphenyl-utilizing bacteria from all marine sediments collected along the Croatian Adriatic coast. This was initially indicated by the appearance of intensive yellow colour already upon two to four subcultures in fresh medium. Consistently with the nding of some previous studies (Wagner-Dobler et al., 1998; Leight et al., 2006) the characteristic yellow colour of the cultures suggested meta-cleavage of 2,3-dihydroxybiphenyl and the formation of 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoate. The enrichment of biphenyl-utilizing bacteria was also conrmed by the growth of these bacteria on solid SMS medium in the presence of biphenyl as the only carbon and energy source (one or two morphotypes were observed on the plates of each mixed cultures). Further characterization of the colonies grown on SMS agar plates indicated that these bacteria expressed 2,3-dihydroxybiphenyl dioxygenase activity (colonies turned yellow after spraying with 2,3dihydroxybiphenyl). Apart from the nding that biphenylutilizing bacteria were present in all enriched cultures, the analysis of the colonies grown on Marine agar suggested that the enrichment cultures were more or less complex associations (from three to six morphologically different colony types were observed on the plates of the mixed cultures). The evaluation of the colonies grown on both, Marine and SMS agar plates, suggested that biphenylutilizing bacteria were dominant members of the total culturable populations. The biphenyl-utilizing bacteria that were isolated and some of them further characterized in this work are presented in Table 2.

2.6. Phenotypic characterizations of biphenyl-utilizing bacteria

Colony morphology of the isolated biphenyl-utilizing bacteria was assessed by monitoring their growth on SMS and Marine agar plates. Cellular morphology was examined by light microscopy and transmission electron microscopy using a FEI Morgagni 268D microscope (FEI, Oregon, USA). Preparation techniques applied for electron microscopy were negative staining with 2% phosphowolfram acid, shadowing with paladium and ultrathin sectioning of bacterial cells (Hashimoto and Naylor, 1958).

2.7. 16S rDNA sequencing and phylogenetic analysis

Sequence anayses of 16S rDNA were performed on biphenyl-utilizing isolates by amplifying the 16S rRNA genes using PCR with the eubacterial primers 27f and 1492r (Brousius et al., 1978). The PCR mixtures (50 ml) contained 10 mM Tris-HCl (pH 8), 50 mM KCl, 1.5 mM MgCl2, 0.2 mM of dNTPs, 0.5 pmol of each primer, 1.25 U of Taq polymerase (SigmaAldrich, Germany), and $10 ng of DNA template. PCR amplications were performed in Tgradient thermal cycler (Biometra, Goettingen, Germany) programmed as follows: 5 min of denaturation at 95 1C,

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3.2. PCB-degrading activity of enrichment cultures Since all enrichment cultures demonstrated the capability to use biphenyl as the sole carbon and energy source, they were further examined for their potential PCBdegrading activity by using PCB50 mixture (containing di- to heptachlorinated congeners). These experiments were performed in SMS medium at the initial PCB concentration of 50 mg l1. GCMS analyses of culture extracts after two-week experiments by using enrichment cultures originating from Rijeka marine sediments suggested that all
Table 2 Origin of the isolated biphenyl-utilizing bacteria enriched from marine sediments collected along the Croatian Adriatic coast in the vicinity of urban centres Location PCB mass fraction in sediment (mg kg1 dry wt.)a 0.58 0.50 1.66 Zadar harbour 0.20 0.22 0.54 0.15 0.21
a

Enrichment culture designation

Isolated biphenylutilizing bacteria

cultures showed PCB-degrading activity; however, one culture (RMC2) was more efcient than the other two cultures (RMC1 and RMC3), expressing considerably broader congener specicity. This is evident from Fig. 1, where it is shown that the culture RMC2 degraded at least twenty congeners with two to four chlorine atoms on the biphenyl ring, while the cultures RMC1 and RMC3 degraded at least six and ve congeners, respectively. In the experiments with the enriched cultures from Zadar marine sediments (Fig. 2), the cultures ZMC56 and ZMC57 showed similar congener specicity, degrading at least twenty PCB congeners, while the culture ZMC55 was signicantly less efcient in PCB degradation (degraded at least three congeners). The enriched cultures DMC3 and DMC14 from Dubrovnik marine sediments were efcient in the degradation of at least thirteen and nineteen PCB congeners, respectively (Fig. 3).

3.3. Growth characteristics and the identity of biphenylutilizing culture members

RMC1 RMC2 RMC3 ZMC55 ZMC56 ZMC57 DMC3 DMC14 R1 (Sphingomonas sp.) R1-2b R2 (R. erythropolis) R3b Z55b Z56 (R. ruber) Z57 (R. ruber) D3-1 (R. erythropolis) D3-2 (R. erythropolis) D14 (R. erythropolis) D14-2b

Rijeka harbour

Dubrovnik area

Determined against Aroclor 1248+1254. Not identied yet.

After conrming PCB-degrading activity, individual culture members expressing 2,3-dihydroxybiphenyl dioxygenase activity that were dominant in the enrichment cultures, were isolated and puried. All isolates showed good growth on Marine agar and all of them, except for the strain R1, showed good growth on SMS agar plates as well as in liquid SMS medium with the addition of biphenyl as the only carbon source. The lack of growth of the strain R1, although it expressed substantial 2,3-dihydroxybiphenyl dioxygenase activity (intensive yellow colour appeared within a few minutes after subculturing in the fresh medium), can be explained by the fact that, unlike other
RMC 1 RMC 2 RMC 3

100 90 80 Reduction (%) 70 60 50 40 30 20 10 0 27 45 52 28 40 63 di-CB 49,43 44,37 42,59

70

91,55

tri-CB

74,94

60,56

16,32

26,25

22,51

18,15,17

47,48,75

33,53,20,21

64,71,41

67,100,57,103

PCB congeners
Fig. 1. PCB-degrading activity of the mixed cultures RMC1, RMC2 and RMC3 enriched from Rijeka harbour sediments. Reduction of peak areas in GSMS chromatograms of culture extracts were determined after two week cultivation in seawater mineral salts medium supplemented with biphenyl. Peak reduction less than 20% was not considered signicant and is not reported. Reported values are the mean (7SD) of three independent experiments. Congener assignments for each peak are presented in Table 1.

66,76,95,102,93

101,90

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100 90 80 Reduction (%) 70 60 50 40 30 20 10 0 40 63 27 28 45 52 16,32 26,25 22,51 49,43 44,37 42,59 74,94 70

ZMC 55 ZMC 56 ZMC 57

91,55
91,55

tri-CB

60,56
60,56

di-CB

47,48,75

18,15,17

33,53,20,21

64,71,41

67,100,57,103

PCB congeners
Fig. 2. PCB-degrading activity of the mixed cultures ZMC55, ZMC56 and ZMC57 enriched from Zadar harbour sediments. Further description as in Fig. 1.

100 90 80 Reduction (%) 70 60 50 40 30 20 10 0 66,76,95,102,93 67,100,57,103 33,53,20,21 18,15,17 47,48,75 64,71,41

66,76,95,102,93

DMC 3 DMC 14

PCB congeners
Fig. 3. PCB-degrading activity of the mixed cultures DMC3 and DMC14 enriched from coastal sediments in Dubrovnik area. Further description as in Fig. 1.

isolates, the strain R1 was not capable to use benzoic acid as the carbon source. Cell morphology examination showed that the isolate R1 was a Gram-negative, motile bacterium, while other isolates were Gram-positive, nonmotile bacteria. The isolates R2, D3-1, D3-2 and D14 were short rods that elongated with the prolonged cultivation and formed laments, whereas the isolates Z56 and Z57 were coccoid bacteria. Apart from polyphosphate granules found in all isolates, a large number of sphere-shaped lipid inclusions were also observed in D3-1, Z56 and Z57 cells. After characterization on the basis of their morphological and phenotypic properties, bacterial isolates were

further identied by 16S rDNA sequencing. In 16S rDNA sequence comparisons with the entries in GenBank, the isolate R1 showed the highest sequence similarity to the genus Sphingomonas (98% to Sphingomonas sp. MBIC1965 and 96% to Sphingomonas sp. KA1). The other isolates showed the highest similarity to the genus Rhodococcus. The relationships between these isolates and Rhodococcus strains available from the GenBank (NCBI), detected or isolated from the environment, are presented in a phylogenetic tree (Fig. 4). The isolates R2, D3-1, D3-2 and D14 revealed the highest sequence similarity (99%) to Rhodococcus erythropolis DCL14 isolated from a freshwater sediment (van der Werf et al., 1999), to Rhodococcus

101,90

tri-CB

di-CB

16,32

26,25

22,51

49,43

44,37

42,59

74,94

27

28

45

52

40

63

70

101,90

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erythropolis OUCZ211 isolated from a PCB contaminated soil (Leight et al., 2006) and to Rhodococcus sp. X309 isolated from marine sediment (Denis-Larose et al., 1997). On the other hand, the isolates Z56 and Z57 showed highest sequence similarity (99% and 98%, respectively) to R. ruber M2 isolated from an industrial wastewater (Daye et al., 2003) and were aligned with R. ruber strains in a separated branch. The bacteria we isolated from marine sediments were not aligned in the same branch with Rhodococcus sp. RHA1 and Rhodococcus sp. P6, the most investigated Gram-positive PCB-degrading bacteria isolated from the soil (Asturias and Timmis, 1993; Seto et al., 1995). 3.4. PCB-degrading activity of biphenyl-utilizing culture members In order to better elucidate PCB-degrading capacity of the enrichment cultures, all seven identied biphenylutilizing isolates were further examined for their potential catabolic activity in aerobic PCB degradation. These experiments were performed by employing the same shake culture method as applied for the mixed cultures and by using commercial mixture Aroclor 1248. The results of GC-MS analyses of the culture extracts showed that only the isolate R1, identied as Sphingomonas sp., did not show signicant PCB-degrading activity, while all other isolates, identied as Rhodococcus sp., were efcient in the degradation of Aroclor 1248 (Table 3). Furthermore, although they were isolated from different enrichment cultures, the isolates exhibited similar PCB-degrading activity, except for isolate Z56 which showed slightly lower activity. 4. Discussion It is generally believed that estuarine and marine sediments are the ultimate repository of polychlorinated biphenyls. Their high levels were especially found within industrial and urban areas where increased human activities take place (Kuo et al., 1999; Frignani et al., 2001; Fava et al., 2003). Therefore, it was not surprising that PCBs were detected in all tested marine sediments collected in the vicinity of urban centres at the Croatian Adriatic coast. Furthermore, it was assumed that indigenous bacteria present in these contaminated sediments have developed catabolic pathway for biphenyl degradation and could have catabolic potential for aerobic PCB degradation. Our investigation started with the enrichment of biphenyl-utilizing bacteria from eight marine sediment samples by employing microcosm enrichment approach and using seawater mineral salts medium. Biphenyl was added as the only carbon and energy source. Good growth under these conditions in all enrichments, and the detection of 2,3-dihydroxybiphenyl dioxygenase activity suggested

Fig. 4. Phylogenetic tree representing the relationships of 16S rDNA gene sequences of isolated biphenyl-degrading bacteria and Rhodococcus sequences from GenBank (NCBI). The tree was constructed using a neighbour-joining analysis based on the examination of 1400 16S rDNA gene nucleotide positions. The numbers indicate the present bootstrap support based on a neighbour-joining analysis of 1000 resampled datasets. The scale bar indicates 0.01 nucleotide substitution per position. The sequences of strains denoted in bold characters were determined in this study. Sequence accession numbers used for phylogenetic analysis are shown in parentheses following the species name.

Table 3 PCB-degrading activity of biphenyl-utilizing bacteria isolated from the enrichment culture Enrichment culture RMC1 RMC2 ZMC56 ZMC57 DMC3 DMC14
a

Isolated strain

Aroclor 1248 degradation (%)a 5b 61 40 59 57 50 58

R1 (Sphingomonas sp.) R2 (R. erythropolis) Z56 (R. ruber) Z57 (R. ruber) D3-1 (R. erythropolis) D3-2 (R. erythropolis) D14 (R. erythropolis)

Reduction of total peak areas in GCMS chromatograms of the culture extracts after two week incubation (see Section 2). b Degradation of total PCBs less than 10% was not considered signicant.

the presence of biphenyl-utilizing bacteria in all examined marine sediments. The analysis of the composition of the enrichment cultures suggested that they were stable associations consisting of three to six morphologically different

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22 A. Begonja Kolar et al. / International Biodeterioration & Biodegradation 60 (2007) 1624 Table 4 Comparison of PCB-degrading activitya of enriched mixed cultures originating from different marine sediments PCB congeners Degradation (%) Biphenyl-degrading mixed cultures RMC2 2,30 ,6-CB 2,4,40 -CB 2,20 ,3,6-CB 2,20 ,5,50 -CB 2,20 ,3,30 -CB 2,3,40 ,5-CB 61 98 23 25 27 49 ZMC56 100 50 32 25 28 18 ZMC57 97 50 23 32 31 17 DMC3 37 85 21 25 15 30 DMC14 52 96 29 23 26 42

bacteria, with one or two dominant biphenyl-utilizing strains. These results undoubtedly demonstrated that biphenyl-utilizing bacteria were widespread in coastal marine sediments along the Croatian Adriatic coast. The results of biodegradation studies, in which the complex PCB50 mixture containing di- to heptachlorobiphenyls was used, suggested that all enrichment cultures exhibited PCB-degrading activity during incubation in seawater mineral salts medium supplemented with biphenyl. However, the cultures differed in their PCB-degrading capabilities and congener specicities. Thus, all enrichment cultures degraded dichlorobiphenyls, while their capabilities to degrade congeners with three and four substituted chlorines varied greatly. Four enrichment cultures (RMC2, DMC14, ZMC56 and ZMC57) were capable of degrading some pentachlorobiphenyls, while the degradation of hexaand heptachlorobiphenyls was not observed. Further analyses of their PCB-degrading activity showed that ve from eight enriched cultures exhibited broad congener specicity, while the remaining cultures degraded considerably smaller number of di- to tetrachlorobiphenyls. The broadest congener specicity was exhibited by four cultures: one culture originating from Rijeka harbour sediment (RMC2), two cultures from Zadar harbour sediment (ZMC56 and ZMC57) and one culture from Dubrovnik marine sediment (DMC14). These cultures also showed almost the same extent in total PCB degradation of complex PCB50 mixture. Although the results of the PCB degradation experiments by using complex PCB mixture did not allow more detailed discussion about congener specicity of the enrichment cultures, the analysis of six peaks (5, 8, 12, 13, 19 and 21) in GCMS chromatograms representing single PCB congeners suggested some differences in their congener preferences (Table 4). Thus, the cultures comprising R. erythropolis strains were more efcient in the degradation of double para-substituted congener 2,4,40 trichlorobiphenyl (2,4,40 -CB), while the cultures containing R. ruber strains were more efcient in the degradation of double ortho-substituted congener (2,30 ,6-CB). Additionally, the cultures containing R. erythropolis strains were more efcient in the degradation of 2,3,40 ,5-CB than those containing R. ruber strains. Furthermore, the fact that all enrichment cultures were capable of degrading the congener with the only free 2,3position (2,4,40 -CB) and the congeners with free both 2,3and 3,4-positions (2,30 ,6-CB; 2,20 ,3,6-CB) as well as the congener with the only free 3,4-position (2,20 ,5,50 -CB), suggested the presence of bacteria exhibiting both 2,3 and 3,4-dioxygenase activities. It is also important to note that the enrichment cultures were catabolically active toward ortho-substituted congeners (2,30 ,6-CB; 2,20 ,3,6-CB; 2,20 ,5,50 -CB; 2,20 ,3,30 -CB), which are expected to be present in marine sediments as a result of reductive dechlorination of highly chlorinated PCBs (Wu et al., 2000; Fava et al., 2003). Therefore, enrichment of biphenyl-utilizing bacteria from marine sediments exhibiting the capability to degrade

a Determined based on the reduction of individual PCB congeners in PCB50 mixture.

ortho-substituted congeners is a promising indication that, after the initial reductive dechlorination step, bacteriamediated aerobic degradation of PCBs could proceed in marine environment. Our preliminary PCB degradation studies by using pure biphenyl-utilizing culture isolated from the most active enriched PCB-degrading cultures, which are sill in progress, conrmed that six isolates belonging to the genus Rhodococcus exhibited substantial PCB-degrading activity. This further conrmed that the isolated rhodococci were active members of the enrichment cultures, responsible for PCB degradation. In contrast, Sphingomonas sp., the only Gram-negative strain isolated from marine sediments, did not show signicant PCB-degrading activity, suggesting that although a dominant biphenyl-transforming member of the culture RMC1, this isolate did not contribute to PCB degradation. Therefore, it was assumed that the other biphenyl-metabolizing member which was detected in the culture RMC1 (not further examined) or some nonculturable strains were involved in PCB degradation. Rhodococci capable of degrading PCBs have already been isolated from different PCB-contaminated environments. However, this is the rst report about PCBdegrading Rhodococcus strains isolated from marine sediments. The fact that the strains exhibiting substantial PCB degradation and broad congener specicities were isolated from most of the examined marine sediments collected at distant sites indicated that these bacteria are widely distributed along the Croatian Adriatic coast. Phylogenetic studies showed that the isolated Rhodococcus strains were closely related to the PCB-degrading strains isolated from the soil (Leight et al., 2006) and further investigations are necessary to elucidate potential similarities/differences in catabolic activities among those bacteria of different origin. It has been well documented that rhodococci are one of the most diverse and abundant group of microorganisms present in different environments (Bell et al., 1998). Apart from freshwater environment they have also been isolated from coastal marine sediments as well as from the deep-sea

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sediments where the terrestrial inuence is negligible (Heald et al., 2001; Brandao et al., 2002). Since our PCB degrading bacteria were isolated from coastal marine sediments, it remains unclear whether these bacteria are indigenous marine microora or have been washed from the shore into the sea. Regardless of their indigenous origin (marine or terrestrial), the results of this study demonstrated good growth and PCB-degrading activity of the isolated rhodococci in seawater mineral salts medium indicating that they represent metabolically active members of culturable marine sediment communities. In conclusion, this work reports that biphenyl-utilizing bacteria with catabolic potential for aerobic PCB degradation were isolated from coastal marine sediments. Most of these bacteria belonged to the genus Rhodococcus and showed extensive degradation of lower ortho-chlorinated congeners (di- to tetrachlorobiphenyls) that could be found in marine sediments as a result of contamination or reductive dechlorination of highly chlorinated congeners. These ndings are a promising indication that rhodococci that have been recognized as PCB-degraders in freshwater and terrestrial environment may also play an important role in the biotransformation processes of polychlorinated biphenyls in marine environment. Acknowledgements This work was supported by the Croatian Ministry of Science, Education and Sports. We are grateful to Dr. Nikola Ljubes ic for his valuable cooperation in cell morphology of isolated bacteria by electron microscopy. References
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