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International Biodeterioration & Biodegradation 62 (2008) 219–225

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International Biodeterioration & Biodegradation

journal homepage: www.elsevier .com/locate/ ib iod

Isolation and identification of PCB-degrading microorganisms fromcontaminated sediments

Katarına Dercova a,*, Jana �Cicmanova a, Petra Lovecka b, Katerina Demnerova b, Martina Mackova b,Pavel Hucko c, Patrik Kusnır c

a Slovak University of Technology, Faculty of Chemical and Food Technology, Institute of Biotechnology and Food Science, Department of Biochemical Technology,Radlinskeho 9, 812 37 Bratislava, Slovakiab ICT Prague, Faculty of Food and Biochemical Technology, Department of Biochemistry and Microbiology, Technicka 5, 166 28 Praha 6, Czech Republicc Water Research Institute, Nabr. arm. gen. L. Svobodu 5, 812 49 Bratislava, Slovakia

a r t i c l e i n f o

Article history:Received 19 July 2007Received in revised form 24 January 2008Accepted 24 January 2008Available online 30 June 2008

Keywords:BiodegradationGenotoxicityPCBsSedimentsToxicity

* Corresponding author. Tel.: þ421 2 5932 5710; faE-mail address: katarina.dercova@stuba.sk (K. Der

0964-8305/$ – see front matter � 2008 Elsevier Ltd.doi:10.1016/j.ibiod.2008.01.016

a b s t r a c t

PCBs represent a serious ecological problem due to their low degradability, high toxicity, and strongbioaccumulation. The goal of this study was to analyze the PCB-contaminated sediments from Stra�zskycanal and Zemplınska sırava water reservoir from several points of view. The study of ecotoxicity con-firmed that both sediments were toxic for various tested organisms. The genotoxicity test has not provedthe mutagenic effect. The subsequent step included microbiological analysis of the contaminated sedi-ments and isolation of pure bacterial cultures capable of degrading PCBs. In order to determine thegenetic potential for their biodegradability, the gene bphA1 encoding the enzyme biphenyldioxygenase,responsible for the first step of PCB aerobic degradation, was identified using a PCR technique. The ul-timate goal of the work was to perform aerobic biodegradation of PCBs in the sediments. The bacteriapresent in both sediments are able to degrade certain low chlorinated congeners. The issue of bio-diversity is still open and has to be studied in more detail to reveal the factual interactions betweenbacteria.

� 2008 Elsevier Ltd. All rights reserved.

1. Introduction

Polychlorinated biphenyls (PCBs) are highly persistent pollut-ants that have been produced on the large scale in the past decades.The production was stopped after they had been proved to be toxicto humans. Once in the environment, PCBs tend to accumulate indifferent stages of the food chain and along with being toxic, theyare also carcinogenic (Safe, 1992). Environmental and economicreasons have urged the development of bioremediation technolo-gies for the PCB removal from the contaminated sites. A crucial stepis the isolation or genetic construction of microbial strains withbiodegradation potential. Several aerobic microorganisms are ableto biodegrade certain, usually less chlorinated, PCB congeners(Bedard et al., 1987; Kohler et al., 1988; Sylvestre and Fateux, 1982;Furukawa et al., 1978). Although the complete PCB degradation isthe ultimate goal, often only partial solutions of this complexproblem can be reached resulting in elimination of the mostharmful properties. For PCBs, the top priority is to lower theirbioaccumulation potential or, equivalently, their lipophilicity. In the

x: þ421 2 5296 7085.cova).

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aerobic biodegradation process, dioxygenases introduce hydroxylgroups into the PCB molecule, decreasing its lipophilicity and mak-ing the further cleavage easier (Sondossi et al., 1991). The initialoxidation and subsequent ring cleavage seem to be the rate-limitingsteps of the process. In our previous publications, a simple apparatusfor effective monitoring of the PCB evaporation kinetics in batchbiodegradation experiments was described (Vrana et al., 1995,1996a,b) together with a simple mathematical model (Dercova et al.,1996) that takes evaporation and biodegradation into account.

Former Czechoslovakia (nowadays Slovakia and Czech Republic)belonged to the eight largest world producers of commercialmixtures of PCBs. Through Regional Centre of UNDP (UnitedNations Development Programme), Slovakia has obtained a GEF(Global Environment Facility) grant to support implementation ofthe Stockholm Convention commitments and to elaborate theNational Implementation Plan within the project ‘‘Initial assistanceto Slovakia to meet the obligations under the Stockholm Conven-tion on Persistent Organic Pollutants (POPs)’’. The total productionof PCBs was approximately 21,500 tons. About 46% of the PCBproduction was exported. The rest (11,600 tons) was used in Slo-vakia (5600 tons) and Czech Republic (6000 tons) in dielectricfluids for transformers and power capacitors, as heat exchanger andhydraulic fluids, and as paint additives and lubricants. PCBs from

K. Dercova et al. / International Biodeterioration & Biodegradation 62 (2008) 219–225220

damaged devices can easily enter the environment. At present, theuse of PCBs in open systems is forbidden, however, in closed sys-tems such as transformers and capacitors, they still can be used.Basing on extensive inventories, which were carried out in theyears 2000–2002, current existence of about 3500 tons of PCBs maybe assumed in the territory of Slovakia (Kocan et al., 1999).

Contamination at the factory Chemko Stra�zske surroundingsbelongs to the so-called ‘‘old environmental burdens’’. Contami-nated areas are found inside the factory area as well as in widersurroundings. The contamination is primarily spread through sur-face water by a gradual release from the contaminated sediments ofan open effluent Stra�zsky canal that leads from the factory toLaborec River, and subsequently through the filling canal contam-inates Zemplınska sırava water reservoir. The contaminationresulted in the increased PCB content in the monitored componentsof environment as well as in the population of this district, com-paring with the other parts of Slovakia. PCB concentrations inindustrial canal fluctuate from grams to tens of grams of PCBs perkg of sediment dry weight, in some places in Laborec at hundreds ofmilligrams, and in the sediment of Zemplınska sırava in milligrams.Initial approximation assumes that at least 40,000 tons of PCB-contaminated sediments are still present in the effluent canal,Laborec River, and Zemplınska sırava water reservoir. Thesesediments are an abundant PCB source causing the long-termcontamination of the waters of Eastern Slovakia. As expected, thehighest value of PCBs was found in a muddy part of the effluentcanal flowing from the Chemko factory containing about 3–5 kgPCBs in 1 ton of dry mud. It is doubtless that the polluted effluentcanal flowing into the Laborec River has caused the contamination.It should be stressed that, in general, PCB levels in Slovak humanpopulation are higher than those in other European countries(Langer et al., 2006a,b; Zhiwei et al., 2007). Sediments from the areaof former PCB production sites in Slovakia show high values even21 years after the termination of the production – up to 4.1 mg kg�1

(on average, 0.3 mg kg�1). A possible solution involves the detailedexamination of an extent of the pollution of the water reservoir, theremoval of the contaminated sediment, and the decomposition ofPCBs by a suitable technology, e.g. thermal desorption, chemicaldehalogenation, solvent extraction, or bioremediation.

The goals of the present work were to study ecotoxicity andgenotoxicity of PCB-contaminated sediments from Stra�zsky canal andZemplınska sırava water reservoir, to isolate the microorganismswith degradative ability, and to perform biodegradation of PCBs innatural and bioaugmented sediments under laboratory conditions.

2. Materials and methods

2.1. Chemicals

The following reagents were used: agarose (BioRad, USA), bacteriological agar(Oxoid, UK), biphenyl (Sigma-Aldrich, USA), dibenzofuran (Sigma-Aldrich, USA),diethyl pyrocarbonate (Sigma-Aldrich, USA), DNA markers (Promega, USA), 100 bpladder, 1 kb ladder DNA polymerase (Finzymes, FI), ethidium bromide (Fluka, Ger-many), n-hexane (Chromservis, Czech Republic), IRS solution (Mo Bio Laboratories,USA), Luria–Bertani (LB) medium (Oxoid, UK), Plate Count Agar (PCA) (Oxoid, UK),primers F350 and R674, F38GC and R518 (Biotech, Czech Republic), sodium pyro-phosphate (Sigma-Aldrich, USA), reaction buffer solution for DNA polymerase(Finzymes, FI), Trisma base (Lachema, Czech Republic), mixture of nucleotides(Promega, USA).

Table 1Criteria for evaluation of ecotoxicity using assessment of an inhibition effect on thebioluminescence of the standard bacteria Vibrio fischeri

Relative inhibition H (%) Ecotoxicity

0–5 Non-toxic sample5–20 Potentially toxic sample>20 Toxic sample

2.2. Biological material and the environmental samples

Bacterial strains used were Burkholderia xenovorans LB400 (bph operon) and theisolates from sediments. Histidine dependent strains of Salmonella typhimurium TA98 (3811) and TA 100 (3812) were received from the Czech Collection of Microor-ganisms, Brno, Czech Republic. Sediment sampling protocol was in agreement withthe Slovak technical norm, ISO 5667-12:2001. (Water Quality. Sampling. Part 12:Guidance on sampling of bottom sediments) (757051).

2.3. Cultivation media

LB medium: 25 g LB and 15 g bacteriological agar in 1 l distilled water.Minimal liquid medium (MM): 1 g (NH4)2SO4; 2.7 g KH2PO4; 10.95 g

Na2HPO4 � 12 H2O filled with distilled water to 1 l. After sterilization of the solution,salts containing particular trace elements sterilized by filtration were added: 250 mlFeSO4 � 7 H2O (2 g l�1); 250 ml, Ca (NO3)2 � 4 H2O (6 g l�1); and 250 mlMgSO4 � 7 H2O (40 g l�1), each in 50 ml of the prepared medium.

Minimal solid medium: 5.37 g Na2HPO4 � 12 H2O; 1.30 g KH2PO4; 0.50 g NH4Cl;and 0.20 g MgSO4 � 7 H2O filled with distilled water to 1 l. To congelate the medium,15 g l�1 agar was added (Nobel Agar, Difco, UK).

Plate Count Agar (PCA): 23.5 g PCA in 1 l of distilled water (the guide of OxoidCompany, UK).

Phosphate buffer (pH 7.4): Solution A: 13.8 g NaH2PO4 � H2O filled with distilledwater to 500 ml; solution B: 14.2 g Na2HPO4 filled with distilled water to 500 ml. Atotal of 60 ml of solution A and 440 ml of solution B were mixed; TBE buffer (pH 8.0):stock solution – 10� concentrated: 54 g Tris, 27.5 g boric acid; 3.72 g EDTA, filledwith distilled water to 1 l.

Solution for Ames test: (Mortelmans and Zeiger, 2000): Solution A (10�): 105 g(37.5 g) K2HPO4 (K3PO4), 45 g KH2PO4, 10 g (NH4)2, 5.5 g sodium citrate pentahy-drate filled with distilled water to 500 ml.

Solution B: 30 g l�1 Noble Agar; solution C (10�): 20% MgSO4 � 7H2O; solutionD: 5% glucose. After sterilization, a mixture of 100 ml A, 2 ml C, and 20 ml D solutionswas filled to 1000 ml by distilled water and mixed with 1000 ml of solution B.

Top agar: 6 g Noble Agar and 5 g NaCl filled with distilled water to 1000 ml.

2.4. Determination of the inhibition effect

Specific inhibitory effects of PCBs on the bioluminiscence of intact cells of thestandard bacteria Vibrio fischeri NRRL-B-11177 were measured applying a LumisToxmethod by using a luminometer Biocounter LUMAC 1500 (Perstorp, Netherlands).Toxicity was expressed as ID50 value after 15 min incubation of bacteria in thepresence of toxicant. To evaluate ecotoxicity, the criteria listed in Table 1 were used.The isopropyl alcohol and water extracts of sediments were prepared from 10 g ofsediments and 100 ml of 2% isopropyl alcohol or water, respectively. Bacterial con-sortia were extracted 24 h at 28 �C on a rotary shaker (120 rpm). After extractionsediments were centrifuged (5 min, 4000 rpm).

The Salmonella/microsome reversion assay was conducted using the plate in-corporation procedure described by Maron and Ames (1983). The Ames test wasperformed utilizing two different bacterial strains of Salmonella typhimurium: strainTA 97 for frame-shift mutations and strain TA 100 for base-pair substitution. Alltester strains were maintained and stored according to the standard procedures(Mortelmans and Zeiger, 2000). All samples were tested without metabolic activa-tion (S9) to detect direct mutagenic compounds. The test was performed with plateincorporation method in duplicate at the increasing concentrations. Sodium azidefor TA 100 and 9-aminoacridine for TA 97 were used as positive controls and run inparallel with the test samples. Sterile distilled water and DMSO were used as neg-ative control (number of spontaneous revertants without metabolic activation: TA97 – 100–200 revertants and TA 100 – 142 revertants). The plates were incubated at37 �C for 72 h in the dark. The number of hisþ revertants was estimated using cellcounter. Results were expressed as mutation ratios (F) calculated as the number ofcolonies on test plates/number of colonies on solvent control plates. A compound isconsidered mutagenic when F is more than 2 and a dose-response effect is evident(U.S. EPA, 1983).

2.5. Isolation of microorganisms from the sediments

A mixture of 10 g sediment, 250 mg biphenyl, and 100 ml liquid mineral mediawas cultivated at 28 �C for 7 days. To determine the total number of microorganisms,solid LB medium was used. For isolation of the potential degraders of biphenyl,mineral medium with Nobel agar was used. Microbial strains were cultivated in thepresence of 250 mg biphenyl placed into the lid of a Petri dish.

2.6. Screening of phenotypes exhibiting 2,3-dioxygenase activity

Microorganisms isolated from sediment were inoculated into solid minimalmedium. Biphenyl and dibenzofuran were sprinkled into the lid of a Petri dish. Di-benzofuran, a substance structurally similar to PCB, is split by the enzyme dioxy-genase to yield a so-called meta-cleavage product having yellow color. In this way, it

Table 2Concentration of the selected PCB congeners in the sediments from Stra�zsky canal(Sk) and Zemplınska sırava (Zs) before degradation

PCB congener Sk (mg kg�1) Zs (mg l�1)

8 81.1 208.328 124.5 187.852 40 26.3101 55.3 29.1118 58.1 44.3138 40.6 19.2153 50.2 22.5180 209 <2.5203 15.1 6.9

K. Dercova et al. / International Biodeterioration & Biodegradation 62 (2008) 219–225 221

is possible to detect bacteria producing dioxygenase, which indicates their potentialto degrade PCBs (Becher et al., 2000).

2.7. Classification of microorganisms using Nefermtest 24

The commercial set Nefermtest 24 is assigned for identification of Gram-nega-tive non-fermentative bacteria. A bacterial culture cultivated on PCA for 24 h at28 oC was used. The procedure followed the guide for the use of the commercial setNefermtest 24. The principle of this method is in detection of the biochemicalreactions of the tested strains with different substrates or identification of particularenzyme activities. A suspension of the tested strain was added to a specifieddehydrated substrate and after 48 h individual reactions were evaluated on the basisof a color reaction, and the particular strain was identified using key manual.

2.8. Isolation of the bacterial DNA

Bacterial DNA of Gram-negative bacteria was isolated by thermolysis. For iso-lation, a culture from solid media was used and incubated in 50 ml sterile distilledwater for 10 min at 95 oC in a thermoblock. After thermolysis, the mixture wasstirred and centrifuged at 1300 rpm. The obtained sediment contained the bacterialDNA. QIAamp DNA Mini Kit – Qiagen, USA (a set for isolation of the total DNA frompure culture) and UltraClean Soil DNA Isolation Kit – Mo Bio Laboratories, USA (a setfor isolation of microbial DNA from soil) were used.

2.9. Amplification of the gene bphA1 in isolated DNA using PCR

For detection and amplification of the genes of bacterial DNA, a PCR methodwith specific primers was used. Composition of the mixture was: 34.5 ml sterile de-ionized water, 5 ml of polymerase buffer Finzymes, 1 ml dNTP (10 mM), 2 � 1 ml ofprimers for detection of gene bphA1 (primers F350 and R674 at a concentration of10 pmol ml�1) (Ryslava et al., 2003), 2 ml BSA, 0.5 ml of polymerase Finzymes, 5 ml ofDNA sample.

2.10. Detection of the PCR products by gel electrophoresis

A suitable concentration of the agarose gel was used: from 1% (for fragmentswith number of bp higher than 100) up to 1.5% (for fragments with number of bpbelow 100). To determine fragment size, a marker of 100 bp DNA standard was used(Sambrook et al., 1989).

2.11. Biodegradation of PCBs in the natural sediments

To assess aerobic biodegradation of PCBs present in contaminated sedimentsfrom Stra�zsky canal and Zemplınska sırava without additional bioaugmentation ofnatural isolated microorganisms, 10 g sediments, 250 mg biphenyl, and 50 ml liquidminimal medium were added. Biodegradation was carried out for 14 days at130 rpm and 28 �C. Negative control was represented by a chemically sterilizedsediment with 10 ml of diethylpyrocarbonate in 10 ml sample.

2.12. Biodegradation of PCBs in the sediments amended with the isolated microbialconsortium

Microbial consortium isolated from the sediment was inoculated in 20 ml liquidminimal medium with biphenyl as a structure analog of PCBs and incubated 48 h at28 �C. To evaluate aerobic biodegradation of PCBs present in contaminated sedi-ments from Stra�zsky canal and Zemplınska sırava with additional bioaugmentationof natural isolated microorganisms, 10 g sediments, 250 mg biphenyl, and 5 mlmicrobial consortium of 48 h inoculum were used in the 45 ml liquid minimalmedium. Biodegradation was carried out for 14 days at 130 rpm and 28 �C.

2.13. Determination of PCBs

Aliquots (1 g) of overnight air-dried and sieved (1 mm mesh) sediment sampleshomogenized in liquid nitrogen, were extracted with hexane for 4 h. The extractswere concentrated to 1 ml by a nitrogen flow, purified on a Florisil column, anddiluted with hexane to the same volume as was used for the extraction. These ex-tracts were analyzed using a Hewlett-Packard 5890 gas chromatograph with anelectron capture detector and fused silica capillary column (30 m, 0.2 mm innerdiameter) coated with 0.25 mm immobilized phase SE-54 with nitrogen as carriergas (flow rate 1 ml min�1). The temperature program was 50 �C for 1 min, followedby an increase at a rate of 25 �C min�1 until the temperature was 280 �C and thenkept at this temperature. The injection volume was 2 ml. Results were calculatedfrom the residual amounts of congener peaks present in each sample. For theevaluation of the experiments, the US EPA method 8089/8081 for expressing of thetotal content of PCBs as a sum of recommended indicator congeners (PCB 77, 101, 118,138, 153, and 180) was applied.

3. Results

3.1. Determination of the inhibition effect of the sediments

The sediments from Zemplınska sırava water reservoir andStra�zsky canal were sampled according to the protocol. Thedetected PCB concentrations are presented in Table 2. Toxicity ofwater and isopropyl alcohol extracts of sediments was determinedusing bacterial strain Vibrio fischeri. As can be seen from Fig. 1, ex-tracts of both sediments inhibited bioluminescence. The toxicity ofisopropyl alcohol extracts was higher than those obtained by waterextraction. It was caused probably by the compounds soluble ina less polar solvent, which include also PCBs.

3.2. Determination of mutagenicity of the sediment extracts byAmes test using bacterial strain Salmonella typhimurium his–

Mutagenicity is the property of POPs that may complicate theuse of biological systems for environment decontamination due totheir potential danger for biota. Therefore, extracts of the PCB-contaminated sediments were tested for the mutagenic effectassessed by the Ames test using standard strains of Salmonellatyphimurium TA 100 and TA 97. The number of spontaneous re-vertant colonies was higher than that of the induced revertantcolonies. In the case of the mutagenic effect, ratio of Rt/Rc should beequal or higher than 2, however it has not been confirmed in ourexperiments (Table 3).

The genotoxicity test has not proved the mutagenic effect ofPCB-contaminated sediments for the tested microorganisms Sal-monella typhimurium TA 100 and TA 97.

3.3. Visualization of the presence of biphenyldioxygenase in themicrobial consortium isolated from both studied sediments

Microorganisms that grow on the minimal medium with bi-phenyl as the sole carbon source were isolated from the samples ofthe sediments. Biphenyl is an inducer of the biphenyl operon and itspresence causes induction of the so-called PCB ‘‘upper’’ degrada-tion pathway. Microorganisms isolated by this procedure are po-tential degraders of PCBs. Along with detection of the bacterialgrowth on biphenyl, there are many other screening methods,which enable to confirm the presence of biphenyldioxygenase, thefirst and one of the most important enzymes of the biodegradationpathway of PCBs. One of these pathways is the splitting of di-benzofuran. Dibenzofuran, a substance structurally similar to PCB,is split by the enzyme dioxygenase to yield a so-called meta-cleavage product having yellow color. In this way, we detectedbacteria producing dioxygenase, which indicates their potential todegrade PCBs. Using this method for a rapid screening of dioxy-genase, it was confirmed that the isolated strains that possess thisenzyme in their outfit produced yellow coloration in the presenceof dibenzofuran. This visual proof of dibenzofuran cleavage with

line 1 2 3 4 5 6

line1 2 3 4 5 6 7 8 9 10

bphA1 gen

bphA1 gen- 300 bp

- 100 bp

- 300 bp

- 100 bp

Fig. 2. Gel electrophoretic proof (1.5% gel) of the presence of bphA1 gene: (a) in thetotal DNA isolated separately from both sediments (Sk and Zs).

Gel description bphA1 gene

Lane 1 marker100 bpLane 2 Zs sediment YesLane 3 Sk sediment YesLane 4 Sk sediment YesLane 5 LB 400 (positive control) YesLane 6 Negative control No

(b) in the microbial DNA isolated from individual strains from the sediments Sk and Zs.

Gel description bphA1 gene

Lane 1 Marker 100 bpLane 2 Zs 5a YesLane 3 Sk 2b NoLane 4 Sk 6 YesLane 5 Zs 4 YesLane 6 Zs 12a YesLane 7 Zs 10 YesLane 8 LB400 (positive control) YesLane 9 Negative control NoLane 10 Marker 100 bp

0

25

50

75

100

Sk Zšsediments

Relative in

hib

itio

n

of b

io

lu

min

escen

ce (%

)

Fig. 1. Inhibition effect of the sediments from Zemplınska sırava (Zs) and Stra�zskycanal (Sk) on bioluminiscence of Vibrio fischeri. , water extract isopropyl alcoholextract. The experiments were performed in triplets.

K. Dercova et al. / International Biodeterioration & Biodegradation 62 (2008) 219–225222

dioxygenase was manifested during the growth of colonies in Petridishes with solid minimal medium as well as in liquid mediumthroughout degradation (not shown). On the basis of the performedpositive screening, we have continued in the isolation of the purestrains, which were subsequently submitted to genetic analysis forthe presence of the bphA1 gene.

3.4. PCR amplification of bphA1 gene

A method of identification of the presence of a particular gene inthe microbial cell is based on the use of the polymerase chain re-action and the knowledge of the sequence of the primers, shortsequences of DNA, which enable replication of a particular gene andits amplification. We have based our experiments on the fact thatbiphenyldioxygenase determines the specificity of PCB cleavageand that it is the first enzyme of the biphenyl pathway. At ampli-fication, the primers F350 and R674 that were previously definedand verified were used (Ryslava et al., 2003). The results of am-plification and identification of the bphA1 gene are presented in thefigures illustrating the gel electrophoretic data. In Fig. 2, a gene forthe total DNA is shown, which was isolated from the both sedi-ments (Zs and Sk). The results indicate that in the microflora of thesediments, bacterial strains are present, which may express thisgene. Fig. 2 also depicts the detected bands of bphA1 gene for se-lected strains isolated from the both sediments. The used markerserved for the assessment of the number of the base-pairs of thedetected product of PCR.

Table 3Ratio of revertants F ¼ Rt/Rc for the diluted extracts of the PCB-contaminatedsediments from Stra�zsky kanal (Sk) and Zemplınska sırava (Zs)

Extract Number of revertants F ¼ Rt/Rc

TA 100 TA 97 TA 100 TA 97

Skwater 146 87 0.94 1.22Skisopropyl alcohol 124 86 0.79 1.20Zswater 172 74 1.11 1.04Zsisopropyl alcohol 138 44 0.88 0.61Skwater 1:1 126 43 0.81 0.61Zswater 1:1 102 67 0.66 0.94Skwater 1:2 143 73 0.92 1.02Zsisopropyl alcohol 1:2 153 0 0.98 0.00Skisopropyl alcohol 1:1 134 51 0.86 0.72Zsisopropyl alcohol 1:1 136 41 0.87 0.58Skisopropyl alcohol1:2 146 77 0.94 1.08Zsisopropyl alcohol 1:2 143 61 0.92 0.86Control 155 719-aminoacridine – 852Sodium azide 923 –

3.5. Classification of the bacterial strains with the identified bphA1 gene

The bacterial strains with the identified bphA1 gene weredetected using a Gram staining method. All isolates except one(Zs5a) were Gram-negative. The characterization of the strains wascontinued using the commercial Nefermtest 24 for detection ofnon-fermentative bacteria. Strain Zs5a was Gram-positive, so it wasexcluded from Nefermtest 24, which is applicable only on Gram-negative bacteria. It is feasible that PCB-degrading bacteria (mainlyG�) occur in this group of microorganisms. According to theobtained results, we identified the following microorganisms listedin Table 4: Pseudomonas fluorescens, Ochrobactrum anthropi, Pseu-domonas aeruginosa, and Agrobacterium radiobacter.

3.6. Aerobic degradation of PCBs in the sediments

The mixture used for the assessment of aerobic degradation ofPCBs present in the sediments comprised 10 g of the contaminated

Table 4Microorganisms isolated from the sediments of Zemplınska sırava and Stra�zskycanal

Sediment Microorganism (Nefermtest 24)

Zemplınska sırava Pseudomonas fluorescens (Zs10)Ochrobactrum anthropi (Zs4)Pseudomonas aeruginosa (Zs12a)

Stra�zsky canal Agrobacterium radiobacter (Sk6)

0

20

40

60

80

100

28 52 101 138 153 180PCB congeners

deg

rad

atio

n (%

)

Fig. 4. Comparison of the results of 14-day biodegradation of the indicator PCB con-geners in sediments sampled from Stra�zsky canal and Zemplınska sırava withoutamendment with the isolated natural microbial consortium. Stra�zsky canal , Zem-plınska sırava -.

K. Dercova et al. / International Biodeterioration & Biodegradation 62 (2008) 219–225 223

sediment, 50 ml minimal mineral medium, and 250 mg biphenyl asthe inducer of biphenyl and PCB pathways. To evaluate the exper-iments, an indicator standard (a mixture of six PCB congeners: 28,52, 101, 138, 153, and 180) was used (Burkhard et al., 1997). A de-crease in the individual PCB congeners content after 14 days ofaerobic biodegradation is illustrated in Fig. 3. The results of thebiodegradation processes were compared to the control (sterilesediment without microorganisms), which represented abiotictransformation of PCB congeners. Fig. 4 shows that in the sedimentsfrom Stra�zsky canal, a more pronounced decrease of the PCB con-geners content was observed than in the sediments from Zem-plınska sırava. In the sediments from Stra�zsky canal, the PCBcongeners 28 (40%), 138 (31%) and 180 (30%) were the most de-graded congeners, while in the sediments from Zemplınska sıravathere were the PCB congeners 28 (14%) and 153 (13%).

0

10

20

30

40

50

60

28 52 101 138 153 180PCB congeners

mg

P

CB

/kg

o

f sed

im

en

t

0

0.002

0.004

0.006

0.008

0.01

28 52 101 138 153 180PCB congeners

mg

P

CB

/kg

o

f sed

im

en

t

Sk

Zš

A

B

Fig. 3. Comparison of PCB concentrations in the sediments from: (a) Zemplınska sıravabefore and after 14 days , of degradation; (b) Stra�zsky canal before and after ,

14 days of degradation. The experiments were performed in triplets.

3.7. Aerobic degradation of PCBs in the sediments bioaugmentedwith the isolated microbial consortia

In this experiment, a 48 h inoculum of the microbial consortiumobtained from the particular sediment was added to the bio-degradation system. The 14-day biodegradation of PCBs in thesediment with addition of the microbial consortium resulted onlyin a decrease of the amount of the PCB congener 28 from 20.38 to2.41 mg kg�1 in the sediment from Stra�zsky canal and from 3.18 to2.72 mg kg�1 in the sediment from Zemplınska sırava. The amountof all other studied PCB congeners has increased. It is possible thatthe presence of the higher amount of microorganisms caused in-creased production of the biosurfactants and subsequently the re-lease of sequestered PCBs from sediment structures. This probablycaused the increase of PCB content in comparison with their initialamount. This fact requires an additional detailed study.

4. Discussion

The goal of the present study was to analyze the real samples ofcontaminated sediments from Stra�zsky canal and Zemplınska sır-ava water reservoir from several points of view. Both sediments arecontaminated with PCBs and were therefore chosen for aerobicdegradation by an indigenous microbial consortium and were alsoused as a source for isolation of bacterial strains with a potential todegrade PCBs.

Toxicity of sediments depends on their physicochemical prop-erties (dilution in water, evaporation, polarity). These propertiesinfluence adsorption of PCBs on sediment particles or their ar-rangement in the particular phase (water, solid matrix, and air).The sensitivity of tests in water solution is very low especially fororganic compounds with low solubility in water. At the beginning,the ecotoxicity and genotoxicity of water and isopropyl alcoholextracts of the sediments were studied. The results confirmed thatboth sediments, mainly their isopropyl alcohol extracts were toxicfor various tested organisms. Chemical analysis alone is not suf-ficient for determination of the environmental risk. Sometimes,the byproducts are more toxic than the initial compound. Loveckaet al. (2004) and Vrana et al. (1996a,b) proved higher toxicity ofchlorobenzoic acids in case of several bioindicator models, whichare bacterial degradation products of PCBs produced by the ‘‘up-per’’ degradation pathway. Tests with water solutions did notrender unambiguous results due to the hydrophobic character ofsome toxic compounds, e.g. PCBs (Borja et al., 2005). The testsperformed with bacteria Vibrio fischeri demonstrated different

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toxic effects of the water and isopropyl alcohol solutions of PCBson the bioluminescence of the tested organism. PCBs are hydro-phobic compounds and it is obvious that toxicity was expressedmore markedly in the case of the less polar solvent. Solubility ofPCBs in isopropyl alcohol is higher than in water and this fact maycause higher bioavailability and increase of solution toxicity. Thegenotoxicity assay of the water and isopropyl alcohol extract didnot prove any mutagenic effects. It has been confirmed that PCB-contaminated sediments represent a source of the adverse effectson live functions of the microorganisms. The next step was a mi-crobiological analysis of the contaminated sediments identifica-tion of bphA gene in total DNA isolated from sediments andadditional isolation of pure bacterial cultures able to degrade PCBsin the environment. In order to determine the genetic potential forbiodegradability of the isolated strains, the gene bphA1 wasidentified using PCR technique applied to the total DNA withspecific primers designed for conservative regions of the bphA1gene (Ryslava et al., 2003). Gene bphA1 codes a small subunit ofbiphenyldioxygenase, a specific enzyme of the ‘‘upper’’ degrada-tion pathway. The length of fragment was up to 324 bp. As a pos-itive control Burkholderia xenovorans LB400 strain was used. Firsttotal DNA from sediments was isolated and tested for presence ofbphA gene to detect possible degradative potential of bacterialconsortia. After confirmation of its presence, we continued theisolation of the pure strains, which were positive in dibenzofurantest. At the end, one strain from the sediments Stra�zsky canal andfour strains from the Zemplınska sırava water reservoir wereidentified as expressing bphA1 gene. All strains were subjected toother identification procedures such as Gram-test and Nefermtest24. Using the method for a rapid screening of dioxygenase, it wasconfirmed that the isolated strains that possess this enzyme intheir outfit produced yellow coloration in the presence of diben-zofurane. This visual proof of cleavage with dioxygenase wasmanifested during the growth of colonies in Petri dishes with solidminimal medium as well as in liquid medium throughout degra-dation. It is also possible to assess other biochemical characteris-tics that could describe an other important properties of theisolated bacteria. Identification using 16srDNA will be performedin the future.

The ultimate goal of the work was to carry out aerobic bio-degradation of PCBs in the contaminated sediments, as well as toperform aerobic biodegradation with the bioaugmentation bynatural microorganisms isolated from these sediments. PCB bio-degradation produced positive results. The microorganisms pres-ent in both sediments were capable of degrading certain lowchlorinated congeners. The issue of biodiversity is still open andmust be studied in more detail to reveal the factual cooperationbetween the degrading bacteria. Biodegradation of PCBs by intactsediments, i.e. without an amendment of additional microbialinoculum was significantly higher in the sediments from Stra�zskycanal in comparison with those of the Zemplınska sırava waterreservoir. We assumed that in the course of aerobic degradation,bioaugmented sediments should degrade PCBs faster and moreeffectively in comparison with the non-bioaugmented sediments.This fact was confirmed only in the case of PCB congener 28(2,40,4-trichlorobiphenyl), which content was markedly reducedby both sediments. This phenomenon can be explained by theeffect of amendment of sediment with a fresh microbial consor-tium and an increase of biosurfactants production, which mayrelease sequestered PCBs from the soil particles, which in the endresulted in the increased concentration of PCBs. It is also possiblethat higher chlorinated PCB congeners were dechlorinated to thelow chlorinated ones. Last but not least, it is necessary to take intoaccount a possible competition of microorganisms in the sub-strate. It might cause inhibition of the growth of microorganismsresponsible for degradation of the mentioned congeners.

Therefore, it is indispensable to determine microbial diversity andcoexistence of the bacteria present in the consortium. The bacte-rial strains isolated from Zemplınska sırava water reservoir pro-duced more positive results in comparison with those isolatedfrom Stra�zsky canal, where the initial concentration of PCBs wasone order higher.

Acknowledgement

This work was performed in the framework of the ProgramSocrates/Erasmus. Financial support from Scientific Grant Agencyof Ministry of Education of Slovak Republic and Slovak Academy ofSciences (Grants 1/1309/04 and 1/4357/07) and Grant Agency ofMinistry of Education of Czech Republic (Grants MSMT No NPVII2B06156 and MSM 6046137305) are gratefully acknowledged.

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