International Biodeterioration & Biodegradation 91 (2014) 52e59

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International Biodeterioration & Biodegradation

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Degradation of polychlorinated biphenyls (PCBs) by four bacterial

isolates obtained from the PCB-contaminated soil and
PCB-contaminated sediment
Slavomra Murnov a, b, Katarna Dercov a, *, Hana Dudsov a
a

Slovak University of Technology, Faculty of Chemical and Food Technology, Institute of Biotechnology and Food Science, Department of Biochemical
Technology, Radlinskho 9, 812 37 Bratislava, Slovakia
Water Research Institute, Nbrezie arm. gen. L. Svobodu 5, 812 49 Bratislava, Slovakia

a r t i c l e i n f o

a b s t r a c t

Article history:
Received 8 August 2013
Received in revised form
12 March 2014
Accepted 12 March 2014
Available online

We investigated the PCB-degrading abilities of four bacterial strains isolated from long-term PCBcontaminated soil (Alcaligenes xylosoxidans and Pseudomonas stutzeri) and sediments (Ochrobactrum
anthropi and Pseudomonas veronii) that were co-metabolically grown on glucose plus biphenyl which is
an inducer of the PCB catabolic pathway. The aim of study was to determine the respective contribution
of biomass increase and expression of degrading enzymes on the PCB degrading abilities of each isolate.
Growth on 5 g l
1 glucose alone resulted in the highest stimulation of the growth of bacterial strains,
whereas grown on 10 mg l
1, 100 mg l
1, 1 g l
1, or 5 g l
1 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 on
glucose exhibited enhanced degradation ability due to an increased biomass. Addition of biphenyl at
concentrations of 1 or 5 g l
1 did not increase total PCB degradation, but stimulated the degradation of
highly chlorinated congeners for some of the strains. The degradation of di- and tri-chlorobiphenyls was
signicantly lower for cells grown on 5 g l
1 biphenyl independently on glucose addition. The highest
degradation of the PCBs was obtained for A. xylosoxidans grown in the presence of glucose. Thus
A. xylosoxidans appears to be the most promising among the four bacterial isolates for the purpose of
bioremediation.
2014 Elsevier Ltd. All rights reserved.

Keywords:
Bacteria
Biodegradation
Biphenyl
Polychlorinated biphenyls

1. Introduction
Decades of industrial production of polychlorinated biphenyls
(PCBs) as well as their improper disposal has resulted in contamination of many areas. Due to the massive and uncontrolled use of
these compounds in the industry, PCBs became ubiquitous contaminants worldwide (Brzov et al., 2012). The former producer
(Chemko Str
zske) of the commercial PCB mixture DELOR 103 in
Slovakia, manufactured altogether approximately 21,500 tons of
this product (Dercov et al., 2008). A huge amount of waste from
this production was released to the Str
zsky canal (maximal concentration 5 g of PCBs per kilogram of sediment) and Laborec River
and resulted in serious contamination of soil, sediments, surface
and ground waters (Langer et al., 2012). The contamination was
deposited in sediments and accumulated in the aquatic organisms

* Corresponding author. Tel.: 421 02 59325710.

E-mail address: katarina.dercova@stuba.sk (K. Dercov).
http://dx.doi.org/10.1016/j.ibiod.2014.03.011
0964-8305/ 2014 Elsevier Ltd. All rights reserved.

(Brzov et al., 2012). Although the production of PCBs in Slovakia

was banned in 1984, the pollution of bed sediments still represents
a source of continuous contamination. The high concentrations of

PCBs in shes from Laborec River and Zemplnska Srava
water
reservoir represent a threat for human health (Hiller et al., 2010).
Bioremediation is considered as an efcient and cost-effective
process for the decontamination of PCBs (Tandlich et al., 2011). It
may involve bioaugmentation and/or biostimulation strategies
which mean the introduction of PCB-degrading bacterial strains
individually or as a consortium and introduction of nutrients and
oxygen (Mrozik and Piotrowska-Seget, 2010). To stimulate the
degradation abilities of bacterial strains, several inducers have been
proposed. Biphenyl is structurally related to PCB congeners and
hence is commonly used as a growth substrate and inducer
(Harkness et al., 1993; Luo et al., 2007, 2008). Glucose is an easily
utilized carbon source for most microorganisms. Several publications observed that glucose may inhibit PCB biodegradation (Walia
et al., 1990). The use of biphenyl as carbon source in degradation
experiments was questioned (Billingsley et al., 1997). Our previous

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

data indicated that addition of biphenyl did not affect toxicity of

PCBs toward the tested bacteria. On the other hand, addition of
glucose together with PCBs increased cell survival ability and
decreased adaptation responses of the bacteria (Zordov et al.,
2011; Zordov-Murnov et al., 2012). The aim of the current
research was thus to examine the effect of growing PCB-degraders
co-metabolically on glucose and biphenyl on their PCBbiodegradation abilities. We compared the PCB-degrading abilities of four bacterial isolates obtained from long-term PCBcontaminated soil and sediments in order to nd out how they
differ in their PCB-degrading abilities when they are grown on
either one of these substrates alone or co-metabolically on both of
them. Our intention was to determine which of the biphenyl induction of the biphenyl degrading enzymes or of the growth
stimulation of glucose is most appropriate to enhance PCB degradation and to verify if there would be a synergic effect of both
substrates.
2. Materials and methods
2.1. Bacterial strains and chemicals
Bacterial strains used in the study were isolated from long-term
contaminated area surrounding the former producer of PCBs
Chemko Str
zske. Alcaligenes xylosoxidans and Pseudomonas stutzeri were obtained from PCBs contaminated soil by enrichment in
mineral medium with biphenyl according to Dercov et al. (1996).
Ochrobactrum anthropi and Pseudomonas veronii were recently
isolated from contaminated sediment sampled from Str
zsky canal
according methods described by Dudsov et al. (2014). All four
isolates were identied and maintained at the Czech Collection of
Microorganisms (Masaryk University, Brno, Czech Republic).
PCB degradation was determined using the commercial DELOR
103 (Chemko Str
zske, Slovakia) mixture which is equivalent to
AROCLOR 1242. Biphenyl and n-hexane (Merck, Germany), glucose
(Mikrochem, Slovakia), SILIPOR (55e105 mm), and other chemicals
for minimal mineral medium (MM medium) (Lachema Brno, Czech
Republic) were used in the degradation experiments.
2.2. Design of degradation experiments
Bacterial inocula were prepared by growing the cultures according to Dudsov et al. (2012) on a rotary shaker 180 rpm for
48 h at 28  C. The medium contained 60 mg of biphenyl to stimulate
PCBs degradation ability (Chong et al., 2012). Biomass was harvested by centrifugation, washed two times with saline solution
and added to 50 ml of MM medium at a nal concentration of
1 g l
1. The composition of MM medium has been described by
Dudsov et al. (2012). DELOR 103 was added to each ask as solution in DMSO at a nal concentration of 100 mg l
1. Two different
sets of experiments were carried out in parallel. The rst setting
consisted of cultures grown in absence of glucose and presence of
various concentrations of biphenyl (10 mg l
1, 100 mg l
1, 1 g l
1,
and 5 g l
1). The second experimental setting consisted of cultures
containing 5 g l
1 glucose plus 10 mg l
1, 100 mg l
1, 1 g l
1, or
5 g l
1 biphenyl. Three parallel sets for each experiment were run
for a statistical assessment. Concentration of glucose decreased to
zero after 72 h of cultivation.
Biodegradation of PCBs by bacterial strains was carried out in
500 ml Erlenmeyer asks equipped with glass columns lled with
SILIPOR C18 sorbent (Vrana et al., 1995, 1996) and closed with a
cotton wool stopper. The apparatus has been described previously
(Dercov et al., 1996). Flasks were incubated in the dark at 28  C on
a rotary shaker for seven days. Abiotic control without biomass was
run in parallel. Biodegradation was calculated as the initial amount

53

minus the amount in the abiotic control (physico-chemical changes

including evaporation) at the end of incubation. Elimination of PCB
congeners was evaluated and expressed as percentage of the nal
amount minus the abiotic depletion and evaporation according to
the equation: P A - X + Y=A$100%, where P stands for the
biodegradation of the particular PCB congener (%), A is the abiotic
amount detected at the end of cultivation (mg), X is the total amount
of the individual PCB congener in cultivation medium (mg) and Y is
the total amount of PCB congener evaporated during the experiment (mg).
2.3. PCB extraction
After seven days of cultivation, 5 ml of n-hexane were added to
each ask. Flasks were kept in ultrasonic bath for 10 min for cell
disruption and release of the bound PCBs. The content was then
transferred into separatory funnel and vigorously shaken for 2 min.
To reduce the foam originated from biomass proportional amount
of anhydrous K2CO3 was added to the mixture which was shaken
for 15 s. The n-hexane layer was collected and the aqueous layer
was extracted again with 15 ml of n-hexane. Both n-hexane layers
were dried with anhydrous Na2SO4, combined in a 25 ml volumetric ask, lled to 25 ml volume with n-hexane and subsequently analyzed by gas chromatography (GC). The effectiveness of
PCB extraction was determined by the addition of PCB209 congener
as internal standard into MM medium prior to ultrasonic treatment. Standard deviation (SD) was calculated for statistical
evaluation.
Evaporated PCB congeners were captured into SILIPOR C18
sorbent placed in glass columns. Sorbent was extracted with 8 ml of
n-hexane and analyzed by GC with electron capture detector (ECD).
2.4. Analysis of PCBs
All samples were analyzed by Agilent Technologies 7890A GC
System (Santa Clara, CA, USA) equipped with a split/splitless
injector (in splitless mode, 1 min at 250  C) and a micro electron
capture detector (300  C, nitrogen make up gas 25 ml min
1). The
analytes were separated on an HP-5 capillary column
(30 m  0.32 mm  0.25 mm lm thickness) from Agilent Technologies using helium as a carrier gas at constant ow of
1.8 ml min
1. The column was kept at 80  C for 1 min, then temperature was raised to 160  C at 30  C min
1, kept for 1 min, and
then raised to 260  C at 4  C min
1, kept for 3 min. Peak identication and calibration was carried out using a standard mixture of
PCB 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 in
Table 1.
2.5. Enumeration of bacterial cells
The number of viable bacteria in experimental sets was monitored using the drop plate method. Solid MM medium supplemented with 15 g l
1 Nobel agar (Difco) was used for bacterial
growth. 1 ml of media at the beginning and end of experiment was
diluted serially with sterile MM medium and applied to Petri plates.
Plates were incubated at 28  C for 48 h in the dark with subsequent
colonies counting.
2.6. Statistic evaluation
All experiments were performed in triplicate. Data were processed using standard software packages Microsoft Excel and

54

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

Table 1
Description of PCB congeners determined at the end of degradation experiment.
IUPAC No.
(Mills
et al., 2007)

Retention
time (min)

Systematic name

8.2

2,20 -dichlorobiphenyl

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

Molecule structure

3. Results and discussion

The purpose of this study was to compare the PCB-degrading
abilities of four bacterial isolates that originated from the longterm contaminated soil and sediments. The investigation aimed
principally at examining the effect on their PCB-degrading ability
when these strains were grown co-metabolically on glucose plus
various concentrations of biphenyl. Data allowed determining
which of biomass increase or of the biphenyl pathway induction
affected most their PCB degrading abilities.
3.1. Bacterial growth
Two different experimental settings were run in parallel. The
rst setting consisted of monitoring PCB degradation during cell
growth on various concentration of biphenyl. The second setting
consisted of monitoring PCB degradation when the cells were
grown co-metabolically on glucose (5 g l
1) plus biphenyl. As expected, when the biomass was cultivated without any additive but
PCBs, no growth was observed after 7 days of incubation (Table 2).
Similar results were observed for cultures containing 10 mg l
1
biphenyl or 10 mg l
1 biphenyl plus PCBs (Table 2). None of the four
strains was able to grow on DELOR 103. The viable count of cells
grown in the presence of the PCBs alone was signicantly lower at
the seventh day of growth than at the beginning (p < 0.05) suggesting cell death during incubation.
Addition of biphenyl at any concentration together with glucose
led to an enhanced growth of all four strains (p < 0.05). Opposite
results were observed when biphenyl was the sole substrate. When
cells were grown on biphenyl alone at concentrations of 10 mg l
1,
100 mg l
1, 1 g l
1, or 5 g l
1, the biomass did not increased
signicantly. The viable counts of O. anthropi and P. veronii cultures
(both originated from sediment) grown on glucose plus biphenyl
were lower for cultures containing the highest concentration of
biphenyl (1 and 5 g l
1), but in case of A. xylosoxidans and P. stutzeri
biphenyl concentrations did not appear to have any effect on cell
growth. The differences in the behavior of the individual bacterial
strains toward biphenyl may be explained by different environmental origins of the strains.
In previous reports, biphenyl has been demonstrated to stimulate bacterial growth and their PCB degrading abilities (Dercov
et al., 2008; Luo et al., 2008). However, in this work, the results of
the experiments with biphenyl added at various concentrations
together with PCBs indicate that not all concentrations of biphenyl
lead to the stimulation of the bacterial growth of all tested bacterial
strains.
Table 2
Biomass expressed in CFU ml
1 of each bacterial strain after seven days cultivation in
each experimental settings described in the rst column.
Experimental seta

A. xylosoxidans O. anthropi
8

180

203

24.4

26.2

2,20 ,3,4,40 ,5,

50 -heptachlorobiphenyl

2,20 ,3,4,40 ,5,50 ,

6-heptachlorobiphenyl

KruskaleWallis One Way Analysis of Variance on Ranks (Sigma

Plot) for statistical evaluation. The differences in the mean values
among the treatment groups are greater than would be expected by
chance (there is a statistically signicant difference), if the p < 0.05.

P. stutzeri

P. veronii

1

CFU  10 ml
Initial number
Control (without
PCBs or glc)
Bip 10
PCBs
PCBs bip10
PCBs bip100
PCBs bip1
PCBs bip5
PCBs glc
PCBs glc bip10
PCBs glc bip100
PCBs glc bip1
PCBs glc bip5
a

17.76  0.76
13.64  0.74
12.66
14.75
16.18
18.62
17.85
19.20
56.48
44.52
38.78
40.64
39.12













0.63
0.31
0.83
0.94
1.66
1.89
3.32
4.85
2.93
2.28
3.23

15.62  1.12 16.67  0.93 14.98  0.91

5.75  0.13 5.25  0.43 3.65  0.29
6.02
11.04
16.05
13.44
9.41
8.64
42.56
40.60
47.96
30.36
27.72













0.46
0.62
1.18
0.53
0.72
0.52
2.29
3.24
4.23
4.85
3.34

5.81
9.30
8.10
17.18
16.92
17.08
26.94
36.60
32.90
32.36
30.36













0.24
0.53
0.61
1.16
0.47
0.72
1.42
3.93
1.35
3.32
2.65

4.68
7.72
5.37
9.60
11.10
7.76
31.62
32.88
32.20
29.56
23.18

glc, glucose, bip, biphenyl, numbers refer to concentration in mg l
1.













0.74
0.51
0.63
0.31
0.24
0.42
2.30
1.67
1.99
2.17
3.86

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

3.2. Biodegradation of DELOR 103

The extraction efciency was determined relatively to the values
obtained with PCB209 as internal standard. The recovery of sample
extraction was 85.3% with 4.7% accuracy.
Fig. 1A shows the total degradation of DELOR 103 by each of the
four bacterial isolates. A. xylosoxidans exhibited the highest PCBdegrading ability exhibiting 55% PCB depletion for the glucosegrown cultures and 46% depletion for cultures grown on
10 mg l
1 biphenyl. The effect of biphenyl on total PCB degradation
by A. xylosoxidans depended on the concentration of added
biphenyl. For example, PCB depletion was higher for cells grown on
10 mg l
1 biphenyl than those grown on 100 mg l
1. Moreover, at
higher biphenyl concentrations (1 or 5 g l
1) total PCB depletion
was even lower. It is also noteworthy that for A. xylosoxidans, grown
co-metabolically on glucose plus various concentrations of
biphenyl, PCB depletion decreased with increasing concentration of
biphenyl. A similar observation was made for O. anthropi which
showed the highest PCB degradation for cells grown on glucose
alone (33%), whereas in co-metabolically grown cultures, a pronounced decrease of the PCB degradation was observed for the
highest biphenyl concentrations 1e5 g l
1 (p < 0.05). PCB degradation by abovementioned strain in the absence of glucose was the
highest when biphenyl was added at 10 mg l
1. P. stutzeri demonstrated the highest degradation of DELOR 103 when grown on
glucose (5 g l
1) plus biphenyl at 10 mg l
1 (27%) (p < 0.05) and the
lowest degradation was observed for cultures grown on glucose
plus 5 g l
1 (p < 0.05). P. veronii analogously to P. stutzeri exhibited
the highest degradation of DELOR 103 (40%) when grown on
glucose plus 10 mg l
1 biphenyl, whereas 30% degradation was
observed when glucose was added together with 100 mg l
1

Fig. 1. Percent depletion of DELOR 103 (A) and degradation efciency per biomass unit
(1 g l
1) (B) by four bacterial strains. All asks contained 100 mg l
1 of DELOR 103.
PCBs bip10 contained 10 mg l
1 of biphenyl, PCBs bip100 contained 100 mg l
1
biphenyls, PCB bip1 contained 1 g l
1 of biphenyl, PCBs bip5 contained 5 g l
1 of
biphenyl. Columns with glc (or g) label, experiments ran with the addition of 5 g l
1 of
glucose.

55

biphenyl. Growth on biphenyl without glucose did not increase the

degradation of PCBs by P. veronii. An inverse proportionality between the amount of biphenyl and the extent of degradation was
also observed for this strain.
We measured the effects of growing cells on glucose and
biphenyl on the degradation efciency per unit cells which should
reect the level of induction of the biphenyl catabolic enzymes in
each individual cell. Data are shown in Fig. 1B. The most efcient
stimulation of the degradation enzymes of A. xylosoxidans and
P. veronii was observed for cells grown on biphenyl (10 mg l
1) and
absence of glucose (amount of biomass decreased compared to an
inoculated amount, but the degradation increased). Biphenyl in the
concentration range between 100 mg l
1 and 5 g l
1 decreased the
activity of PCB-degrading enzymes of A. xylosoxidans and
O. anthropi independently of the addition of glucose. On the contrary, biphenyl present at all applied concentrations stimulated the
activity of the degradation enzymes of P. stutzeri in the absence of
glucose (p < 0.05). Glucose did not hamper the induction effect of
10 mg l
1 biphenyl on the PCB degradation ability of P. stutzeri and
P. veronii. However, for both Pseudomonas species the degradation
efciency was signicantly lower (p < 0.05) when cells were grown
on glucose plus 5 g l
1 biphenyl. The PCB degradation efciency by
A. xylosoxidans grown on PCB alone without any other additive was
observed to be 50%, while the degradation efciency of cells grown
on glucose alone reached 36%. A. xylosoxidans cells grown on
10 mg l
1 biphenyl without glucose demonstrated a degradation
efciency higher than 75%. Generally, addition of biphenyl at the
highest used concentration (5 g l
1) led to a decrease of the
degradation efciency independently of the presence of glucose
(except for P. stutzeri). Biphenyl added at the lowest concentration
(10 mg l
1) stimulated PCB-degradation of three strains to the
highest extent, except for P. stutzeri where the stimulatory effect of
biphenyl at all concentrations was similar.
The percentage of PCBs degradation by A. xylosoxidans and
O. anthropi cells grown on glucose alone was higher compared to
cells grown co-metabolically on glucose plus biphenyl. However,
for both Pseudomonas species the highest percentage of PCB
degradation was observed for cells grown on glucose plus the
lowest concentration of biphenyl (10 mg l
1). This fact implies that
each strain respond differently with respect to their PCB-degrading
abilities when they are grown on biphenyl alone or cometabolically with glucose. Therefore, PCB degradation depended
not only on the type of carbon source, but was also conditioned by
the bacterial strain genetic background.
Growth on glucose alone or together with biphenyl at the lowest
concentration (10 mg l
1) stimulated bacterial growth resulting in
an increased PCB removal. However, this observation did not
necessarily imply stimulation of the degradation enzymes. More
likely, the enhanced degradation ability had been achieved due to
the increased bacterial biomass. Moreover, the degradation efciency decreased when glucose was present. Our results suggest
that addition of glucose or biphenyl may stimulate PCB degradation
probably via the three mechanisms: i) growth stimulation; ii) induction of catabolic enzymes required for the degradation of PCBs;
iii) and both, stimulation of growth and induction of catabolic enzymes. Our results could imply that glucose stimulated PCB
degradation only due to the stimulation of bacterial growth, while
it may inhibit the induction effect of biphenyl. These results are in
agreement with other studies (Sylvestre, 1995; Luo et al., 2007,
2008). Biphenyl enhanced the degradation ability only via the
second mechanism. Moreover, the induction effect disappeared
when biphenyl was added at the highest used concentration
(5 g l
1).
Parnell et al. (2010) have demonstrated that PCB degradation
starts at the stationary phase of bacterial growth and is absent at

56

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

the exponential phase. Our results corroborate these ndings. All

tested strains in the presence of glucose did not degrade PCBs
within the rst 3 days due to utilization of the primary substrate
(exponential growth phase). On the other hand, the amount of
biomass increased rapidly during this period in comparison with
the cultures grown on biphenyl alone. Hence, when cells reached
the stationary phase and started to degrade PCBs, higher percentages of PCB degradation were observed at the end of experiment
(compared to cells grown on biphenyl alone). Addition of biphenyl
did not stimulate bacterial growth at all. Therefore, lower biomass
amount contributed to the degradation process.
The aim of our efforts was to evaluate whether the effect of
glucose addition on bacterial growth was stronger than the induction effect of biphenyl toward the degradation enzymes. Our
results showed that higher PCB degradation was achieved for
A. xylosoxidans and O. anthropi cells grown on glucose alone or for
P. stutzeri and P. veronii cells co-metabolically grown on glucose
plus low concentrations of biphenyl. These results may imply that
stimulation of the PCB degradation with biphenyl alone is not
strong enough compared to the effect of glucose or a synergistic
effect of both glucose and biphenyl. Easily utilized carbon sources
such as glucose (lactate, pyruvate or succinate, all more costeffective ones for practice) could therefore be used to enhance
PCB degradation during the bioremediation process. For some
strains, it is possible to use both biphenyl as the inducer of degradation and glucose as the primary carbon source and growth
stimulator.
A very important observation was that biphenyl at the high
concentrations present together with glucose may inhibit PCB
degradation. The highest stimulatory effect of biphenyl was achieved when its amount did not exceed the amount of PCBs
(100 mg l
1).

3.3. Degradation of PCB congeners

The percentage of degradation of twelve individual PCB congeners was monitored according to a mixture of standards (Section
2.4). The percent evaporation of each individual PCB congener was
determined separately and this value was subtracted from that
obtained for total depletion. Evaporation of the higher chlorinated
congeners was smaller than for the lower chlorinated ones (data
not shown). Evaporation represented about 1e3% of initially added
amount of PCBs.
Figs. 2 and 3 show the percent degradation of each PCB
congener. Fig. 2 shows the degradation of PCB congeners by cells
grown on various concentration of biphenyl without glucose.
A. xylosoxidans performed the best, which was in agreement with
the 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 was
observed in the presence of 10 mg l
1 of biphenyl (p < 0.05). On the
contrary, the percent degradation of these congeners was lower for
cells grown on 5 g l
1 of biphenyl (Fig. 2A). However, the degradation of highly chlorinated biphenyls was enhanced when cells
were grown in the presence of 1 g l
1 and 5 g l
1 biphenyl. PCB
degradation by O. anthropi was less inuenced by biphenyl than
A. xylosoxidans for biphenyl concentrations of 100 mg l
1, 1 g l
1,
and 5g l
1 (Fig. 2B). However, in the case of the lower chlorinated
congeners (PCB8, PCB15, PCB18, and PCB28), a slightly better level
of degradation was observed when O. anthropi was cultivated on
PCBs alone or in the presence of 10 mg l
1 biphenyl (p < 0.05). The
degradation of di-chlorinated congeners (PCB4, PCB8) by P. stutzeri
was the highest for cells cultivated on PCBs alone or on 10 mg l
1
biphenyl (Fig. 2C). Degradation of tri-chlorinated congeners (PCB18,
PCB28) by the same strain was similar at all used concentrations of

Fig. 2. Degradation of individual PCB congeners of DELOR 103 in the absence of glucose by A. xylosoxidans (A), O. anthropi (B), P. stutzeri (C), and P. veronii (D). All asks contained
100 mg l
1 of PCBs. PCBs bip10 contained 10 mg l
1 of biphenyl, PCBs bip100 contained 100 mg l
1 biphenyls, PCB bip1 contained 1 g l
1 of biphenyl, and PCBs bip5
contained 5 g l
1 of biphenyl.

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

57

Fig. 3. Degradation of PCB congeners in the presence of glucose (glc) by A. xylosoxidans (A), O. anthropi (B), P. stutzeri (C), and P. veronii (D). All asks contained 100 mg l
1 of PCBs
and 5 g l
1 of glucose. PCBs glc bip10 contained 10 mg l
1 of biphenyl, PCBs glc bip100 contained 100 mg l
1 biphenyls, PCB glc bip1 contained 1 g l
1 of biphenyl, and
PCBs glc bip5 contained 5 g l
1 of biphenyl.

biphenyl. However, the degradation of penta-, hexa-, hepta-, and

octachlorobiphenyls (PCB101, PCB118, PCB138, PCB153, PCB180,
and PCB203) reached the highest values when biphenyl concentration was in the range 1e5 g l
1. Moreover, the lowest degradation of the same congeners was observed in the absence of
biphenyl. 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 was
achieved for cells cultivated on 10 mg l
1 biphenyl or without the
biphenyl (p < 0.05). Growth on 5 g l
1 biphenyl caused a signicant
decrease of degradation of di- and tri-chlorobiphenyls.
The highest degradation of di-chlorinated congeners (PCB4,
PCB8) in the cultures containing only PCBs was observed with
Pseudomonas strains. However, the degradation of tri-, tetra-,
penta-, and hexa-chlorinated biphenyls was the highest with
A. xylosoxidans (p < 0.05). Degradation of PCB180 and PCB203 was
similar when A. xylosoxidans, O. anthropi and P. veronii were used.
Similar results were observed in experiments with 10 mg l
1 and
100 mg l
1 biphenyl. Addition of 5 g l
1 biphenyl created better
conditions for degradation of di-, tri, and tetra-chlorinated congeners (PCB4, PCB8, PCB15, PCB18, PCB28, and PCB52) by O. anthropi
and P. stutzeri compared to A. xylosoxidans and P. veronii. P. veronii
showed the lowest degradation of all analyzed congeners in this
experiment.
Fig. 3 shows the total percentage of degradation of each PCB
congener for cultures grown in the presence of glucose (5 g l
1) plus
various concentrations of biphenyl. The highest degradation ability
was observed with A. xylosoxidans (Fig. 3A). This strain showed the
most efcient degradation of highly chlorinated congeners (PCB52,
PCB101, PCB118, PCB138, PCB153, and PCB180) in the presence of
10 mg l
1 or 100 mg l
1 biphenyl, or without biphenyl addition. The
addition of 1 g l
1 biphenyl led to signicantly lower degradation of
all congeners. Furthermore, addition of 5 g l
1 biphenyl

signicantly decreased the congeners degradation compared with

that observed in the presence of 1 g l
1 biphenyl (p < 0.05). PCB153,
PCB138, PCB180, and PCB203 were not degraded under these
conditions. The results indicate that A. xylosoxidans grown on
glucose alone without biphenyl performed the best for PCB
removal. Glucose increased the degradation of PCB congeners most
likely as a result of increased biomass. O. anthropi responded
similarly where PCB degradation was highest for cells grown in
absence or at low concentration of biphenyl (Fig. 3B). Addition of
5 g l
1 biphenyl inhibited the degradation of PCB4, PCB8, PCB15,
PCB18, PCB28, and PCB52. On the contrary, at high biphenyl concentration the degradation of PCB180 and PCB203 was stimulated.
The results obtained from experiments with both Pseudomonas
species were similar to those obtained from the experiments with
O. anthropi. Degradation of highly chlorinated biphenyls by Pseudomonas strains increased with the addition of 1 g l
1 and 5 g l
1
biphenyl, but the degradation of lower chlorinated congeners (diand tri-chlorobiphenyls) was very low with P. stutzeri (Fig. 3C) or
non-detectable with P. veronii (Fig. 3D).
The cultures containing only glucose (without biphenyl) showed
the highest degradation of all analyzed congeners by
A. xylosoxidans. An increase of biphenyl addition to 100 mg l
1 led
to the most pronounced degradation of congeners PCB18, PCB28,
PCB52, PCB101, PCB118, PCB138, PCB153, and PCB180 by strains
A. xylosoxidans and P. veronii. O. anthropi and P. stutzeri showed
signicantly lower degradation abilities toward these congeners
than the previously mentioned strains under the same conditions
(p < 0.05). The presence of 1 g l
1 or 5 g l
1 biphenyl together with
5 g l
1 glucose decreased the degradation of lower chlorinated
congeners (PCB4, PCB8, PCB15, PCB18, PCB28, and PCB52) by all
four strains. The degradation of penta-, hexa- and hepta-chlorobiphenyls in the presence of glucose and 1 g l
1 biphenyl was
higher when O. anthropi and P. veronii were used (compared to

58

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

A. xylosoxidans and P. stutzeri). The presence of 5 g l
1 biphenyl plus

5 g l
1 glucose led to a more signicant degradation of highly
chlorinated congeners by O. anthropi and P. stutzeri.
Many investigators described Pseudomonas species as the most
effective PCB degraders (Hickey et al., 1992; Gibson et al., 1993;
Novkov et al., 2002; Tandlich et al., 2011). In this investigation
the Pseudomonas strains showed lower degradation potential toward PCB congeners than A. xylosoxidans. It is in agreement with
our previous publication, in which we found that the Pseudomonas
strains had lower adaptation ability to PCBs (Zordov-Murnov
et al., 2012). Findings of Tandlich et al. (2001) regarding the inhibition of PCB degradation by glucose were not conrmed. Several
reports have brought evidences that under aerobic conditions, the
lower chlorinated congeners are more easily degraded than the
higher 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 chlorinated
congeners was only observed for Pseudomonas species cells cultivated in a medium containing no glucose or biphenyl or in a medium containing 10 mg l
1 biphenyl without glucose. The
preferential degradation of the higher chlorinated congeners was
observed by all strains when they were cultivated in the presence of
high concentration of biphenyl. It seems that biphenyl added at a
concentration above that of PCBs stimulated the degradation of
penta-, hexa-, hepta-, and octa-chlorobiphenyls. Other reports have
described the ability of aerobic bacteria to degrade higher chlorinated congeners (Commandeur et al., 1995; Dercov et al., 1996; Liz
et al., 2009; Dudsov et al., 2012), however, they did not examine
the effect of the inducer concentration on their preferential PCB
congener degradation. The ability to degrade highly chlorinated
PCB congeners of all tested strains may be explained by the observations made by Komancov et al. (2003). In this paper the
authors showed that 2,3-dioxygenase was able to oxidize the carbon bound to chlorine atom which was subsequently released. This
allowed the degradation of highly chlorinated congeners. Similar
mechanism was described for chlorobenzoates (Fava et al., 1993).
A. xylosoxidans exhibited highest PCB degradation abilities
among of all four bacterial strains. This strain seems to be the most
promising one for further bioaugmentation strategy. This strain
was isolated from a long-term PCB-contaminated soil and degrades
mostly tetra-, penta-, and hexa-chlorobiphenyls. This nding is in
accordance with the observations of Luo et al. (2008) who
described higher removal of especially higher chlorinated congeners by soil microorganisms.

4. Conclusions
Addition of glucose as substrate stimulated the degradation
abilities of PCB-degrading bacterial isolates via growth stimulation.
Biphenyl stimulated degradation ability just via induction of the
biphenyl catabolic enzymes. Synergic effect of the addition of
glucose and biphenyl together stimulated degradation ability of
Pseudomonas species via both abovementioned mechanisms only
when biphenyl was added at 10 mg l
1. According to our results it
seems that stimulation of PCB degradation with just biphenyl is
much less efcient than the synergic effect of both glucose and
biphenyl. Moreover, at signicantly high concentration, biphenyl
may inhibit the PCB degradation abilities of some bacterial strains
and for others it may promote the degradation of the higher
chlorinated biphenyls. This observation indicates that other similar
carbon sources but more cost-effective could be used to improve
biodegradation of PCBs in bioremediation technologies.
A. xylosoxidans exhibited the highest PCB degradation abilities
among of all four bacterial strains. The strain seems to be an

appropriate inoculum for further remediation experiments due to

its degradation ability.
Acknowledgment
Authors gratefully acknowledge the nancial support from the
Slovak Grant Agency (grant No. 1/0734/12) of Ministry of Education,
Science, and Sports of Slovak Republic. This work was also supported by the Slovak Research and Development Agency under the
contract No. APVV-0656-12.
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