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Article history:Received 8 August 2013Received in revised form12 March 2014

this production was released to the Strzsky canal (maximal con-centration 5 g of PCBs per kilogram of sediment) and Laborec Riverand resulted in serious contamination of soil, sediments, surfaceand ground waters (Langer et al., 2012). The contamination wasdeposited in sediments and accumulated in the aquatic organisms

f PCBs in Slovakiants still representsconcentrations ofnska Srava wateriller et al., 2010).and cost-effectiveich et al., 2011). Itulation strategiesg bacterial strainsn of nutrients and

oxygen (Mrozik and Piotrowska-Seget, 2010). To stimulate thedegradation abilities of bacterial strains, several inducers have beenproposed. Biphenyl is structurally related to PCB congeners andhence is commonly used as a growth substrate and inducer(Harkness et al., 1993; Luo et al., 2007, 2008). Glucose is an easilyutilized carbon source for most microorganisms. Several publica-tions observed that glucose may inhibit PCB biodegradation (Waliaet al., 1990). The use of biphenyl as carbon source in degradationexperiments was questioned (Billingsley et al., 1997). Our previous

* Corresponding author. Tel.: 421 02 59325710.

Contents lists availab

International Biodeterior

.e

International Biodeterioration & Biodegradation 91 (2014) 52e59E-mail address: katarina.dercova@stuba.sk (K. Dercov).1. Introduction

Decades of industrial production of polychlorinated biphenyls(PCBs) as well as their improper disposal has resulted in contami-nation of many areas. Due to the massive and uncontrolled use ofthese compounds in the industry, PCBs became ubiquitous con-taminants worldwide (Brzov et al., 2012). The former producer(Chemko Strzske) of the commercial PCB mixture DELOR 103 inSlovakia, manufactured altogether approximately 21,500 tons ofthis product (Dercov et al., 2008). A huge amount of waste from

(Brzov et al., 2012). Although the production owas banned in 1984, the pollution of bed sedimea source of continuous contamination. The highPCBs in shes from Laborec River and Zemplreservoir represent a threat for human health (H

Bioremediation is considered as an efcientprocess for the decontamination of PCBs (Tandlmay involve bioaugmentation and/or biostimwhich mean the introduction of PCB-degradinindividually or as a consortium and introductioAccepted 12 March 2014Available online

Keywords:BacteriaBiodegradationBiphenylPolychlorinated biphenylshttp://dx.doi.org/10.1016/j.ibiod.2014.03.0110964-8305/ 2014 Elsevier Ltd. All rights reserved.a b s t r a c t

We investigated the PCB-degrading abilities of four bacterial strains isolated from long-term PCB-contaminated soil (Alcaligenes xylosoxidans and Pseudomonas stutzeri) and sediments (Ochrobactrumanthropi and Pseudomonas veronii) that were co-metabolically grown on glucose plus biphenyl which isan inducer of the PCB catabolic pathway. The aim of study was to determine the respective contributionof biomass increase and expression of degrading enzymes on the PCB degrading abilities of each isolate.Growth on 5 g l1 glucose alone resulted in the highest stimulation of the growth of bacterial strains,whereas grown on 10 mg l1, 100 mg l1, 1 g l1, or 5 g l1 biphenyl did not effected the bacterial growth.None of the strains used in this study was able to grow on PCBs as the sole carbon source. Cells grown onglucose exhibited enhanced degradation ability due to an increased biomass. Addition of biphenyl atconcentrations of 1 or 5 g l1 did not increase total PCB degradation, but stimulated the degradation ofhighly chlorinated congeners for some of the strains. The degradation of di- and tri-chlorobiphenyls wassignicantly lower for cells grown on 5 g l1 biphenyl independently on glucose addition. The highestdegradation of the PCBs was obtained for A. xylosoxidans grown in the presence of glucose. ThusA. xylosoxidans appears to be the most promising among the four bacterial isolates for the purpose ofbioremediation.

2014 Elsevier Ltd. All rights reserved.Water Research Institute, Nbrezie arm. gen. L. Svobodu 5, 812 49 Bratislava, SlovakiaDegradation of polychlorinated biphenyisolates obtained from the PCB-contamiPCB-contaminated sediment

Slavomra Murnov a,b, Katarna Dercov a,*, Hanaa Slovak University of Technology, Faculty of Chemical and Food Technology, Institute ofTechnology, Radlinskho 9, 812 37 Bratislava, Slovakiab

journal homepage: www(PCBs) by four bacterialted soil and

dsov a

technology and Food Science, Department of Biochemical

le at ScienceDirect

ation & Biodegradation

lsevier .com/locate/ ibiod

rioradata indicated that addition of biphenyl did not affect toxicity ofPCBs toward the tested bacteria. On the other hand, addition ofglucose together with PCBs increased cell survival ability anddecreased adaptation responses of the bacteria (Zordov et al.,2011; Zordov-Murnov et al., 2012). The aim of the currentresearch was thus to examine the effect of growing PCB-degradersco-metabolically on glucose and biphenyl on their PCB-biodegradation abilities. We compared the PCB-degrading abili-ties of four bacterial isolates obtained from long-term PCB-contaminated soil and sediments in order to nd out how theydiffer in their PCB-degrading abilities when they are grown oneither one of these substrates alone or co-metabolically on both ofthem. Our intention was to determine which of the biphenyl in-duction of the biphenyl degrading enzymes or of the growthstimulation of glucose is most appropriate to enhance PCB degra-dation and to verify if there would be a synergic effect of bothsubstrates.

2. Materials and methods

2.1. Bacterial strains and chemicals

Bacterial strains used in the study were isolated from long-termcontaminated area surrounding the former producer of PCBsChemko Strzske. Alcaligenes xylosoxidans and Pseudomonas stut-zeri were obtained from PCBs contaminated soil by enrichment inmineral medium with biphenyl according to Dercov et al. (1996).Ochrobactrum anthropi and Pseudomonas veronii were recentlyisolated from contaminated sediment sampled from Strzsky canalaccording methods described by Dudsov et al. (2014). All fourisolates were identied and maintained at the Czech Collection ofMicroorganisms (Masaryk University, Brno, Czech Republic).

PCB degradation was determined using the commercial DELOR103 (Chemko Strzske, Slovakia) mixture which is equivalent toAROCLOR 1242. Biphenyl and n-hexane (Merck, Germany), glucose(Mikrochem, Slovakia), SILIPOR (55e105 mm), and other chemicalsfor minimal mineral medium (MMmedium) (Lachema Brno, CzechRepublic) were used in the degradation experiments.

2.2. Design of degradation experiments

Bacterial inocula were prepared by growing the cultures ac-cording to Dudsov et al. (2012) on a rotary shaker 180 rpm for48 h at 28 C. Themedium contained 60mg of biphenyl to stimulatePCBs degradation ability (Chong et al., 2012). Biomass was har-vested by centrifugation, washed two times with saline solutionand added to 50 ml of MM medium at a nal concentration of1 g l1. The composition of MM medium has been described byDudsov et al. (2012). DELOR 103 was added to each ask as so-lution in DMSO at a nal concentration of 100 mg l1. Two differentsets of experiments were carried out in parallel. The rst settingconsisted of cultures grown in absence of glucose and presence ofvarious concentrations of biphenyl (10 mg l1, 100 mg l1, 1 g l1,and 5 g l1). The second experimental setting consisted of culturescontaining 5 g l1 glucose plus 10 mg l1, 100 mg l1, 1 g l1, or5 g l1 biphenyl. Three parallel sets for each experiment were runfor a statistical assessment. Concentration of glucose decreased tozero after 72 h of cultivation.

Biodegradation of PCBs by bacterial strains was carried out in500 ml Erlenmeyer asks equipped with glass columns lled withSILIPOR C18 sorbent (Vrana et al., 1995, 1996) and closed with acotton wool stopper. The apparatus has been described previously(Dercov et al., 1996). Flasks were incubated in the dark at 28 C ona rotary shaker for seven days. Abiotic control without biomass was

S. Murnov et al. / International Biodeterun in parallel. Biodegradation was calculated as the initial amountminus the amount in the abiotic control (physico-chemical changesincluding evaporation) at the end of incubation. Elimination of PCBcongeners was evaluated and expressed as percentage of the nalamount minus the abiotic depletion and evaporation according tothe equation: P A - X + Y=A$100%, where P stands for thebiodegradation of the particular PCB congener (%), A is the abioticamount detected at the end of cultivation (mg), X is the total amountof the individual PCB congener in cultivation medium (mg) and Y isthe total amount of PCB congener evaporated during the experi-ment (mg).

2.3. PCB extraction

After seven days of cultivation, 5 ml of n-hexane were added toeach ask. Flasks were kept in ultrasonic bath for 10 min for celldisruption and release of the bound PCBs. The content was thentransferred into separatory funnel and vigorously shaken for 2 min.To reduce the foam originated from biomass proportional amountof anhydrous K2CO3 was added to the mixture which was shakenfor 15 s. The n-hexane layer was collected and the aqueous layerwas extracted again with 15 ml of n-hexane. Both n-hexane layerswere dried with anhydrous Na2SO4, combined in a 25 ml volu-metric ask, lled to 25 ml volume with n-hexane and subse-quently analyzed by gas chromatography (GC). The effectiveness ofPCB extractionwas determined by the addition of PCB209 congeneras internal standard into MM medium prior to ultrasonic treat-ment. Standard deviation (SD) was calculated for statisticalevaluation.

Evaporated PCB congeners were captured into SILIPOR C18sorbent placed in glass columns. Sorbent was extractedwith 8ml ofn-hexane and analyzed by GC with electron capture detector (ECD).

2.4. Analysis of PCBs

All samples were analyzed by Agilent Technologies 7890A GCSystem (Santa Clara, CA, USA) equipped with a split/splitlessinjector (in splitless mode, 1 min at 250 C) and a micro electroncapture detector (300 C, nitrogen make up gas 25 ml min1). Theanalytes were separated on an HP-5 capillary column(30 m 0.32 mm 0.25 mm lm thickness) from Agilent Tech-nologies using helium as a carrier gas at constant ow of1.8 ml min1. The column was kept at 80 C for 1 min, then tem-perature was raised to 160 C at 30 C min1, kept for 1 min, andthen raised to 260 C at 4 C min1, kept for 3 min. Peak identi-cation and calibration was carried out using a standard mixture ofPCB congeners (PCB4, PCB8, PCB15, PCB18, PCB28, PCB52, PCB101,PCB118, PCB138, PCB153, PCB180, and PCB203) (Dr. Ehrenstorfer,Germany). Individual PCB congeners with the IUPAC numbers,chlorine substitution patterns and retention time are listed inTable 1.

2.5. Enumeration of bacterial cells

The number of viable bacteria in experimental sets was moni-tored using the drop plate method. Solid MM medium supple-mented with 15 g l1 Nobel agar (Difco) was used for bacterialgrowth. 1 ml of media at the beginning and end of experiment wasdiluted serially with sterile MMmedium and applied to Petri plates.Plates were incubated at 28 C for 48 h in the dark with subsequentcolonies counting.

2.6. Statistic evaluation

All experiments were performed in triplicate. Data were pro-

tion & Biodegradation 91 (2014) 52e59 53cessed using standard software packages Microsoft Excel and

3. Results and discussion

The purpose of this study was to compare the PCB-degrading

rioration & Biodegradation 91 (2014) 52e59Table 1Description of PCB congeners determined at the end of degradation experiment.

S. Murnov et al. / International Biodete54KruskaleWallis One Way Analysis of Variance on Ranks (SigmaPlot) for statistical evaluation. The differences in the mean valuesamong the treatment groups are greater thanwould be expected bychance (there is a statistically signicant difference), if the p< 0.05.

abilities of four bacterial isolates that originated from the long-term contaminated soil and sediments. The investigation aimedprincipally at examining the effect on their PCB-degrading abilitywhen these strains were grown co-metabolically on glucose plusvarious concentrations of biphenyl. Data allowed determiningwhich of biomass increase or of the biphenyl pathway inductionaffected most their PCB degrading abilities.

3.1. Bacterial growth

Two different experimental settings were run in parallel. Therst setting consisted of monitoring PCB degradation during cellgrowth on various concentration of biphenyl. The second settingconsisted of monitoring PCB degradation when the cells were

IUPAC No.(Millset al., 2007)

Retentiontime (min)

Systematic name Molecule structure

4 8.2 2,20-dichlorobiphenyl

8 9.4 2,40-dichlorobiphenyl

15 10.8 4,40-dichlorobiphenyl

18 10.7 2,20 ,5-trichlorobiphenyl

28 12.3 2,4,40-trichlorobiphenyl

52 13.6 2,20 ,5,50-tetrachlorobiphenyl

101 17.1 2,20 ,4,5,50-pentachlorobiphenyl

118 19.6 2,30 ,4,40 ,5-pentachlorobiphenyl

138 21.6 2,20 ,3,4,40 ,50-hexachlorobiphenyl

153 20.6 2,20 ,4,40 ,5,50-hexachlorobiphenyl

180 24.4 2,20 ,3,4,40 ,5,50-heptachlorobiphenyl

203 26.2 2,20 ,3,4,40 ,5,50 ,6-heptachlorobiphenylgrown co-metabolically on glucose (5 g l1) plus biphenyl. As ex-pected, when the biomass was cultivated without any additive butPCBs, no growth was observed after 7 days of incubation (Table 2).Similar results were observed for cultures containing 10 mg l1

biphenyl or 10mg l1 biphenyl plus PCBs (Table 2). None of the fourstrains was able to grow on DELOR 103. The viable count of cellsgrown in the presence of the PCBs alone was signicantly lower atthe seventh day of growth than at the beginning (p < 0.05) sug-gesting cell death during incubation.

Addition of biphenyl at any concentration together with glucoseled to an enhanced growth of all four strains (p < 0.05). Oppositeresults were observed when biphenyl was the sole substrate. Whencells were grown on biphenyl alone at concentrations of 10 mg l1,100 mg l1, 1 g l1, or 5 g l1, the biomass did not increasedsignicantly. The viable counts of O. anthropi and P. veronii cultures(both originated from sediment) grown on glucose plus biphenylwere lower for cultures containing the highest concentration ofbiphenyl (1 and 5 g l1), but in case of A. xylosoxidans and P. stutzeribiphenyl concentrations did not appear to have any effect on cellgrowth. The differences in the behavior of the individual bacterialstrains toward biphenyl may be explained by different environ-mental origins of the strains.

In previous reports, biphenyl has been demonstrated to stimu-late bacterial growth and their PCB degrading abilities (Dercovet al., 2008; Luo et al., 2008). However, in this work, the results ofthe experiments with biphenyl added at various concentrationstogether with PCBs indicate that not all concentrations of biphenyllead to the stimulation of the bacterial growth of all tested bacterialstrains.

Table 2Biomass expressed in CFUml1 of each bacterial strain after seven days cultivation ineach experimental settings described in the rst column.

Experimental seta A. xylosoxidans O. anthropi P. stutzeri P. veronii

CFU 108 ml1

Initial number 17.76 0.76 15.62 1.12 16.67 0.93 14.98 0.91Control (without

PCBs or glc)13.64 0.74 5.75 0.13 5.25 0.43 3.65 0.29

Bip 10 12.66 0.63 6.02 0.46 5.81 0.24 4.68 0.74PCBs 14.75 0.31 11.04 0.62 9.30 0.53 7.72 0.51PCBs bip10 16.18 0.83 16.05 1.18 8.10 0.61 5.37 0.63PCBs bip100 18.62 0.94 13.44 0.53 17.18 1.16 9.60 0.31PCBs bip1 17.85 1.66 9.41 0.72 16.92 0.47 11.10 0.24PCBs bip5 19.20 1.89 8.64 0.52 17.08 0.72 7.76 0.42PCBs glc 56.48 3.32 42.56 2.29 26.94 1.42 31.62 2.30PCBs glc bip10 44.52 4.85 40.60 3.24 36.60 3.93 32.88 1.67PCBs glc bip100 38.78 2.93 47.96 4.23 32.90 1.35 32.20 1.99PCBs glc bip1 40.64 2.28 30.36 4.85 32.36 3.32 29.56 2.17PCBs glc bip5 39.12 3.23 27.72 3.34 30.36 2.65 23.18 3.86

a glc, glucose, bip, biphenyl, numbers refer to concentration in mg l1.

3.2. Biodegradation of DELOR 103

The extraction efciencywas determined relatively to the valuesobtained with PCB209 as internal standard. The recovery of sampleextraction was 85.3% with 4.7% accuracy.

Fig. 1A shows the total degradation of DELOR 103 by each of thefour bacterial isolates. A. xylosoxidans exhibited the highest PCB-degrading ability exhibiting 55% PCB depletion for the glucose-grown cultures and 46% depletion for cultures grown on10 mg l1 biphenyl. The effect of biphenyl on total PCB degradationby A. xylosoxidans depended on the concentration of addedbiphenyl. For example, PCB depletionwas higher for cells grown on10 mg l1 biphenyl than those grown on 100 mg l1. Moreover, athigher biphenyl concentrations (1 or 5 g l1) total PCB depletionwas even lower. It is also noteworthy that for A. xylosoxidans, grownco-metabolically on glucose plus various concentrations ofbiphenyl, PCB depletion decreasedwith increasing concentration ofbiphenyl. A similar observation was made for O. anthropi whichshowed the highest PCB degradation for cells grown on glucosealone (33%), whereas in co-metabolically grown cultures, a pro-nounced decrease of the PCB degradation was observed for thehighest biphenyl concentrations 1e5 g l1 (p < 0.05). PCB degra-dation by abovementioned strain in the absence of glucose was thehighest when biphenyl was added at 10 mg l1. P. stutzeri demon-strated the highest degradation of DELOR 103 when grown onglucose (5 g l1) plus biphenyl at 10 mg l1 (27%) (p< 0.05) and thelowest degradation was observed for cultures grown on glucoseplus 5 g l1 (p < 0.05). P. veronii analogously to P. stutzeri exhibitedthe highest degradation of DELOR 103 (40%) when grown onglucose plus 10 mg l1 biphenyl, whereas 30% degradation wasobserved when glucose was added together with 100 mg l1

S. Murnov et al. / International BiodeterioraFig. 1. Percent depletion of DELOR 103 (A) and degradation efciency per biomass unit(1 g l1) (B) by four bacterial strains. All asks contained 100 mg l1 of DELOR 103.PCBs bip10 contained 10 mg l1 of biphenyl, PCBs bip100 contained 100 mg l1biphenyls, PCB bip1 contained 1 g l1 of biphenyl, PCBs bip5 contained 5 g l1 of

1biphenyl. Columns with glc (or g) label, experiments ran with the addition of 5 g l ofglucose.biphenyl. Growth on biphenyl without glucose did not increase thedegradation of PCBs by P. veronii. An inverse proportionality be-tween the amount of biphenyl and the extent of degradation wasalso observed for this strain.

We measured the effects of growing cells on glucose andbiphenyl on the degradation efciency per unit cells which shouldreect the level of induction of the biphenyl catabolic enzymes ineach individual cell. Data are shown in Fig. 1B. The most efcientstimulation of the degradation enzymes of A. xylosoxidans andP. veronii was observed for cells grown on biphenyl (10 mg l1) andabsence of glucose (amount of biomass decreased compared to aninoculated amount, but the degradation increased). Biphenyl in theconcentration range between 100 mg l1 and 5 g l1 decreased theactivity of PCB-degrading enzymes of A. xylosoxidans andO. anthropi independently of the addition of glucose. On the con-trary, biphenyl present at all applied concentrations stimulated theactivity of the degradation enzymes of P. stutzeri in the absence ofglucose (p < 0.05). Glucose did not hamper the induction effect of10 mg l1 biphenyl on the PCB degradation ability of P. stutzeri andP. veronii. However, for both Pseudomonas species the degradationefciency was signicantly lower (p< 0.05) when cells were grownon glucose plus 5 g l1 biphenyl. The PCB degradation efciency byA. xylosoxidans grown on PCB alone without any other additive wasobserved to be 50%, while the degradation efciency of cells grownon glucose alone reached 36%. A. xylosoxidans cells grown on10 mg l1 biphenyl without glucose demonstrated a degradationefciency higher than 75%. Generally, addition of biphenyl at thehighest used concentration (5 g l1) led to a decrease of thedegradation efciency independently of the presence of glucose(except for P. stutzeri). Biphenyl added at the lowest concentration(10 mg l1) stimulated PCB-degradation of three strains to thehighest extent, except for P. stutzeri where the stimulatory effect ofbiphenyl at all concentrations was similar.

The percentage of PCBs degradation by A. xylosoxidans andO. anthropi cells grown on glucose alone was higher compared tocells grown co-metabolically on glucose plus biphenyl. However,for both Pseudomonas species the highest percentage of PCBdegradation was observed for cells grown on glucose plus thelowest concentration of biphenyl (10 mg l1). This fact implies thateach strain respond differently with respect to their PCB-degradingabilities when they are grown on biphenyl alone or co-metabolically with glucose. Therefore, PCB degradation dependednot only on the type of carbon source, but was also conditioned bythe bacterial strain genetic background.

Growth on glucose alone or together with biphenyl at the lowestconcentration (10 mg l1) stimulated bacterial growth resulting inan increased PCB removal. However, this observation did notnecessarily imply stimulation of the degradation enzymes. Morelikely, the enhanced degradation ability had been achieved due tothe increased bacterial biomass. Moreover, the degradation ef-ciency decreased when glucose was present. Our results suggestthat addition of glucose or biphenyl may stimulate PCB degradationprobably via the three mechanisms: i) growth stimulation; ii) in-duction of catabolic enzymes required for the degradation of PCBs;iii) and both, stimulation of growth and induction of catabolic en-zymes. Our results could imply that glucose stimulated PCBdegradation only due to the stimulation of bacterial growth, whileit may inhibit the induction effect of biphenyl. These results are inagreement with other studies (Sylvestre, 1995; Luo et al., 2007,2008). Biphenyl enhanced the degradation ability only via thesecond mechanism. Moreover, the induction effect disappearedwhen biphenyl was added at the highest used concentration(5 g l1).

Parnell et al. (2010) have demonstrated that PCB degradation

tion & Biodegradation 91 (2014) 52e59 55starts at the stationary phase of bacterial growth and is absent at

the exponential phase. Our results corroborate these ndings. Alltested strains in the presence of glucose did not degrade PCBswithin the rst 3 days due to utilization of the primary substrate(exponential growth phase). On the other hand, the amount ofbiomass increased rapidly during this period in comparison withthe cultures grown on biphenyl alone. Hence, when cells reachedthe stationary phase and started to degrade PCBs, higher percent-ages of PCB degradation were observed at the end of experiment(compared to cells grown on biphenyl alone). Addition of biphenyldid not stimulate bacterial growth at all. Therefore, lower biomassamount contributed to the degradation process.

The aim of our efforts was to evaluate whether the effect ofglucose addition on bacterial growth was stronger than the in-duction effect of biphenyl toward the degradation enzymes. Ourresults showed that higher PCB degradation was achieved forA. xylosoxidans and O. anthropi cells grown on glucose alone or forP. stutzeri and P. veronii cells co-metabolically grown on glucoseplus low concentrations of biphenyl. These results may imply thatstimulation of the PCB degradation with biphenyl alone is notstrong enough compared to the effect of glucose or a synergisticeffect of both glucose and biphenyl. Easily utilized carbon sourcessuch as glucose (lactate, pyruvate or succinate, all more cost-effective ones for practice) could therefore be used to enhancePCB degradation during the bioremediation process. For somestrains, it is possible to use both biphenyl as the inducer of degra-dation and glucose as the primary carbon source and growthstimulator.

A very important observation was that biphenyl at the highconcentrations present together with glucose may inhibit PCBdegradation. The highest stimulatory effect of biphenyl was ach-ieved when its amount did not exceed the amount of PCBs

3.3. Degradation of PCB congeners

The percentage of degradation of twelve individual PCB conge-ners was monitored according to a mixture of standards (Section2.4). The percent evaporation of each individual PCB congener wasdetermined separately and this value was subtracted from thatobtained for total depletion. Evaporation of the higher chlorinatedcongeners was smaller than for the lower chlorinated ones (datanot shown). Evaporation represented about 1e3% of initially addedamount of PCBs.

Figs. 2 and 3 show the percent degradation of each PCBcongener. Fig. 2 shows the degradation of PCB congeners by cellsgrown on various concentration of biphenyl without glucose.A. xylosoxidans performed the best, which was in agreement withthe results obtained from the assessment of total PCB degradation(Fig. 1A). The highest degradation of lower chlorinated congeners(PCB8, PCB15, PCB18, PCB28, and PCB52) by this strain wasobserved in the presence of 10 mg l1 of biphenyl (p< 0.05). On thecontrary, the percent degradation of these congeners was lower forcells grown on 5 g l1 of biphenyl (Fig. 2A). However, the degra-dation of highly chlorinated biphenyls was enhanced when cellswere grown in the presence of 1 g l1 and 5 g l1 biphenyl. PCBdegradation by O. anthropi was less inuenced by biphenyl thanA. xylosoxidans for biphenyl concentrations of 100 mg l1, 1 g l1,and 5g l1 (Fig. 2B). However, in the case of the lower chlorinatedcongeners (PCB8, PCB15, PCB18, and PCB28), a slightly better levelof degradation was observed when O. anthropi was cultivated onPCBs alone or in the presence of 10 mg l1 biphenyl (p < 0.05). Thedegradation of di-chlorinated congeners (PCB4, PCB8) by P. stutzeriwas the highest for cells cultivated on PCBs alone or on 10 mg l1

biphenyl (Fig. 2C). Degradation of tri-chlorinated congeners (PCB18,

by

S. Murnov et al. / International Biodeterioration & Biodegradation 91 (2014) 52e5956(100 mg l1).

Fig. 2. Degradation of individual PCB congeners of DELOR 103 in the absence of glucose1 1100 mg l of PCBs. PCBs bip10 contained 10 mg l of biphenyl, PCBs bip100 contain

contained 5 g l1 of biphenyl.PCB28) by the same strain was similar at all used concentrations of

A. xylosoxidans (A), O. anthropi (B), P. stutzeri (C), and P. veronii (D). All asks contained1 1ed 100 mg l biphenyls, PCB bip1 contained 1 g l of biphenyl, and PCBs bip5

(A),bip1

rioraFig. 3. Degradation of PCB congeners in the presence of glucose (glc) by A. xylosoxidansand 5 g l1 of glucose. PCBs glc bip10 contained 10 mg l1 of biphenyl, PCBs glc PCBs glc bip5 contained 5 g l1 of biphenyl.

S. Murnov et al. / International Biodetebiphenyl. However, the degradation of penta-, hexa-, hepta-, andoctachlorobiphenyls (PCB101, PCB118, PCB138, PCB153, PCB180,and PCB203) reached the highest values when biphenyl concen-tration was in the range 1e5 g l1. Moreover, the lowest degrada-tion of the same congeners was observed in the absence ofbiphenyl. Similar degradation abilities were observed for P. veronii(Fig. 2D). The highest degradation of congeners PCB4, PCB8, PCB15,PCB18, and PCB28 (di- and tri-chlorobiphenyls) by P. veronii wasachieved for cells cultivated on 10 mg l1 biphenyl or without thebiphenyl (p< 0.05). Growth on 5 g l1 biphenyl caused a signicantdecrease of degradation of di- and tri-chlorobiphenyls.

The highest degradation of di-chlorinated congeners (PCB4,PCB8) in the cultures containing only PCBs was observed withPseudomonas strains. However, the degradation of tri-, tetra-,penta-, and hexa-chlorinated biphenyls was the highest withA. xylosoxidans (p < 0.05). Degradation of PCB180 and PCB203 wassimilar when A. xylosoxidans, O. anthropi and P. veronii were used.Similar results were observed in experiments with 10 mg l1 and100 mg l1 biphenyl. Addition of 5 g l1 biphenyl created betterconditions for degradation of di-, tri, and tetra-chlorinated conge-ners (PCB4, PCB8, PCB15, PCB18, PCB28, and PCB52) by O. anthropiand P. stutzeri compared to A. xylosoxidans and P. veronii. P. veroniishowed the lowest degradation of all analyzed congeners in thisexperiment.

Fig. 3 shows the total percentage of degradation of each PCBcongener for cultures grown in the presence of glucose (5 g l1) plusvarious concentrations of biphenyl. The highest degradation abilitywas observed with A. xylosoxidans (Fig. 3A). This strain showed themost efcient degradation of highly chlorinated congeners (PCB52,PCB101, PCB118, PCB138, PCB153, and PCB180) in the presence of10mg l1 or 100mg l1 biphenyl, or without biphenyl addition. Theaddition of 1 g l1 biphenyl led to signicantly lower degradation ofall congeners. Furthermore, addition of 5 g l1 biphenylO. anthropi (B), P. stutzeri (C), and P. veronii (D). All asks contained 100 mg l1 of PCBs00 contained 100 mg l1 biphenyls, PCB glc bip1 contained 1 g l1 of biphenyl, and

tion & Biodegradation 91 (2014) 52e59 57signicantly decreased the congeners degradation compared withthat observed in the presence of 1 g l1 biphenyl (p< 0.05). PCB153,PCB138, PCB180, and PCB203 were not degraded under theseconditions. The results indicate that A. xylosoxidans grown onglucose alone without biphenyl performed the best for PCBremoval. Glucose increased the degradation of PCB congeners mostlikely as a result of increased biomass. O. anthropi respondedsimilarly where PCB degradation was highest for cells grown inabsence or at low concentration of biphenyl (Fig. 3B). Addition of5 g l1 biphenyl inhibited the degradation of PCB4, PCB8, PCB15,PCB18, PCB28, and PCB52. On the contrary, at high biphenyl con-centration the degradation of PCB180 and PCB203 was stimulated.The results obtained from experiments with both Pseudomonasspecies were similar to those obtained from the experiments withO. anthropi. Degradation of highly chlorinated biphenyls by Pseu-domonas strains increased with the addition of 1 g l1 and 5 g l1

biphenyl, but the degradation of lower chlorinated congeners (di-and tri-chlorobiphenyls) was very low with P. stutzeri (Fig. 3C) ornon-detectable with P. veronii (Fig. 3D).

The cultures containing only glucose (without biphenyl) showedthe highest degradation of all analyzed congeners byA. xylosoxidans. An increase of biphenyl addition to 100 mg l1 ledto the most pronounced degradation of congeners PCB18, PCB28,PCB52, PCB101, PCB118, PCB138, PCB153, and PCB180 by strainsA. xylosoxidans and P. veronii. O. anthropi and P. stutzeri showedsignicantly lower degradation abilities toward these congenersthan the previously mentioned strains under the same conditions(p < 0.05). The presence of 1 g l1 or 5 g l1 biphenyl together with5 g l1 glucose decreased the degradation of lower chlorinatedcongeners (PCB4, PCB8, PCB15, PCB18, PCB28, and PCB52) by allfour strains. The degradation of penta-, hexa- and hepta-chlor-obiphenyls in the presence of glucose and 1 g l1 biphenyl washigher when O. anthropi and P. veronii were used (compared to

vated in a medium containing no glucose or biphenyl or in a me-

rioradium containing 10 mg l1 biphenyl without glucose. Thepreferential degradation of the higher chlorinated congeners wasobserved by all strains when theywere cultivated in the presence ofhigh concentration of biphenyl. It seems that biphenyl added at aconcentration above that of PCBs stimulated the degradation ofpenta-, hexa-, hepta-, and octa-chlorobiphenyls. Other reports havedescribed the ability of aerobic bacteria to degrade higher chlori-nated congeners (Commandeur et al., 1995; Dercov et al., 1996; Lizet al., 2009; Dudsov et al., 2012), however, they did not examinethe effect of the inducer concentration on their preferential PCBcongener degradation. The ability to degrade highly chlorinatedPCB congeners of all tested strains may be explained by the ob-servations made by Komancov et al. (2003). In this paper theauthors showed that 2,3-dioxygenase was able to oxidize the car-bon bound to chlorine atomwhich was subsequently released. Thisallowed the degradation of highly chlorinated congeners. Similarmechanism was described for chlorobenzoates (Fava et al., 1993).

A. xylosoxidans exhibited highest PCB degradation abilitiesamong of all four bacterial strains. This strain seems to be the mostpromising one for further bioaugmentation strategy. This strainwas isolated from a long-term PCB-contaminated soil and degradesmostly tetra-, penta-, and hexa-chlorobiphenyls. This nding is inaccordance with the observations of Luo et al. (2008) whodescribed higher removal of especially higher chlorinated conge-ners by soil microorganisms.

4. Conclusions

Addition of glucose as substrate stimulated the degradationabilities of PCB-degrading bacterial isolates via growth stimulation.Biphenyl stimulated degradation ability just via induction of thebiphenyl catabolic enzymes. Synergic effect of the addition ofglucose and biphenyl together stimulated degradation ability ofPseudomonas species via both abovementioned mechanisms onlywhen biphenyl was added at 10 mg l1. According to our results itseems that stimulation of PCB degradation with just biphenyl ismuch less efcient than the synergic effect of both glucose andbiphenyl. Moreover, at signicantly high concentration, biphenylmay inhibit the PCB degradation abilities of some bacterial strainsand for others it may promote the degradation of the higherchlorinated biphenyls. This observation indicates that other similarcarbon sources but more cost-effective could be used to improvebiodegradation of PCBs in bioremediation technologies.A. xylosoxidans exhibited the highest PCB degradation abilitiesA. xylosoxidans and P. stutzeri). The presence of 5 g l1 biphenyl plus5 g l1 glucose led to a more signicant degradation of highlychlorinated congeners by O. anthropi and P. stutzeri.

Many investigators described Pseudomonas species as the mosteffective PCB degraders (Hickey et al., 1992; Gibson et al., 1993;Novkov et al., 2002; Tandlich et al., 2011). In this investigationthe Pseudomonas strains showed lower degradation potential to-ward PCB congeners than A. xylosoxidans. It is in agreement withour previous publication, in which we found that the Pseudomonasstrains had lower adaptation ability to PCBs (Zordov-Murnovet al., 2012). Findings of Tandlich et al. (2001) regarding the inhi-bition of PCB degradation by glucose were not conrmed. Severalreports have brought evidences that under aerobic conditions, thelower chlorinated congeners are more easily degraded than thehigher chlorinated ones (Sondossi et al., 1992;Mondello et al., 1997;Potrawfke et al., 1998; Field and Sierra-Alvarez, 2008; Luo et al.,2008). In our experiments the higher removal of lower chlorinatedcongeners was only observed for Pseudomonas species cells culti-

S. Murnov et al. / International Biodete58among of all four bacterial strains. The strain seems to be anappropriate inoculum for further remediation experiments due toits degradation ability.

Acknowledgment

Authors gratefully acknowledge the nancial support from theSlovak Grant Agency (grant No.1/0734/12) of Ministry of Education,Science, and Sports of Slovak Republic. This work was also sup-ported by the Slovak Research and Development Agency under thecontract No. APVV-0656-12.

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S. Murnov et al. / International Biodeterioration & Biodegradation 91 (2014) 52e59 59

Degradation of polychlorinated biphenyls (PCBs) by four bacterial isolates obtained from the PCB-contaminated soil and PCB- ...1 Introduction2 Materials and methods2.1 Bacterial strains and chemicals2.2 Design of degradation experiments2.3 PCB extraction2.4 Analysis of PCBs2.5 Enumeration of bacterial cells2.6 Statistic evaluation

3 Results and discussion3.1 Bacterial growth3.2 Biodegradation of DELOR 1033.3 Degradation of PCB congeners

4 ConclusionsAcknowledgmentReferences