Whole cell biosensor for PCB analysis based on optical
detection
P Gavlasova1,3,*, G Kuncova1, L Kochánkova2 and M Mackova3
1
Institute of Chemical Process Fundamentals, Czech Academy of Sciences,
Rozvojová 135, Prague 6, 165 02, Czech Republic
2
Department of Environmental Chemistry, Faculty of Environmental
Technology, ICT, Technická 5, Prague 6 , 166 28, Czech Republic
3
Department of Biochemistry and Microbiology, Faculty of Food and
Biochemical Technology, ICT, Technická 5, Prague 6 , 166 28, Czech
Republic
*author for correspondence: email: gavlasova@icpf.cas.cz
Abstract. Whole cell optical sensor of polychlorinated biphenyls (PCBs) based on
production of coloured intermediates by silica entrapped cells is described. The
reusable sensor (WCB) was prepared by co-immobilization of cells Pseudomonas
species 2 with biphenyl in the silica matrix. The sensors were exposed to 13 individual
PCB congeners, the commercial PCB mixture Delor 103, PAHs (anthracene, pyrene,
phenanthrene), solvents (toluene, xylene), naphthalene, 1-methylnaphthalene and
dibenzofuran. The results show that silica entrapped cells produce yellow
intermediates (absorbance λmax = 398 nm) selectively in the presence of two PCB
congeners: 2,4,4´-trichlorobiphenyl (2,4,4´-CB) and 2,4´,5-trichlorobiphenyl (2,4´,5-CB)
and Delor 103. The PCBs detection was not influenced by PAHs. Naphthalenes and
solvents decreased production of yellow intermediates. In the presence of
dibenzorufane, PCBs detection was interfered with development of orange
metabolites. The WCB´s detection limit was 1.2 ± 0.4 mg.L-1 (Delor103) and
0.2 ± 0.08 mg.L-1 (2,4,4´CB), response time 3 hours, reproducibility ± 5%, reusability 3
times and ≥ 4 weeks storage stability was demonstrated.
1. Introduction
Polychlorinated biphenyls are highly persistent toxic compounds; which accumulate in the
food chain and can negatively affect the health of different organisms. In Czechoslovakia
PCB mixtures, called Delor, had been produced. Despite, Delor production was banned in
1984, their residues can be still found in the environment together with other aromatics.
Standard methods of PCBs determination are gas chromatography and high performance
liquid chromatography (GC-MS/ECD, HPLC). For screening tests, sensors having wide
variety of sensitivity and selectivity were developed. Commercially available imunosensors
are based on indirect competitive type of analysis with covalent bounded anti-PCB antibodies
on paramagnetic particles. They were proposed for semiquantitative analysis of polar
samples as soil, water or milk [1]. Among whole cells biosensors, bioluminescent reporter
strain, Ralstonia eutropha ENV307 (pUTK60), was constructed for PCBs detection. The
minimum detection limits were ranged from 0.15 mg.L-1 for 4-chlorobiphenyl to 1.5 mg.L-1 for
Aroclor 1242 [2].
PCBs contaminating the natural environment are co-metabolically transformed to
chlorobenzoic acids by aerobic bacteria through the biphenyl catabolic pathway [3]. During
the third step of the so called upper PCB metabolic pathway the yellow meta ring-fission
product 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid (HOPDA) is formed and its formation
was used for selection of microorganism with PCB degradative enzymes [4, 5].
An increase in light absorption at λ = 400 nm was used for detection of commercial PCB
mixture D103 in water and oils [6]. Production of yellow HOPDA was caused by
biodegradation of chlorinated derivates by bacteria selected from contaminated soil,
Pseudomonas species 2 [7, 8] physically adsorbed on glass carrier in an aerobic bioreactor.
The disadvantage of this method is the time-consuming immobilization and bioreactor set-up.
An important consideration in the development of biosensor lies in identifying
immobilization method. Silica matrices are relatively inexpensive to synthesize and have
several desirable properties including chemical inertness, optical transparency, enhanced
thermo stability, biocompatibility, and resistance to microbial attack. Viability and longevity of
cells entrapped in silica were shown in optical sensors and bioreactors [9].
The aim of this work was to develop a whole cell screening method for the detection of
PCBs, ease of handling and usage, which did not utilize GMO. Pseudomonas species 2 were
entrapped in silica to form homogeneous and optically translucent films. A film fabrication
and sensing manual were work out to prepare PCB bioassay (WCB). WCBs´ films were
exposed to individual PCB congeners, commercial PCB mixture Delor 103 (D103) and to
estimate interferences of D103 detection to PAHs, naphthalenes and solvents.
2. Whole cell biosensor of polychlorinated biphenyls (WCB): preparation and
measurements
2.1 Fabrication of WCB
2.1.1. Pre-polymerization of tetramethoxysilan (TMOS)
The mixture of TMOS:H2O:HCl = 1:4:10-4 (molar ratio) was stirred to form a clear solution
and left to pre-polymerize for 24 hours at 10°C. Biphenyl (B) was added as a 2% TMOS
solution (0.1 ml) to cold clear sol (6 ml) shortly before cell immobilization to final
concentration 0.1 g.L-1.
2.1.2. Cell immobilization Pseudomonas species 2 were pre-incubated in mineral medium
with biphenyl (3 g.L-1) as sole carbon source for one day. Pre-polymerized TMOS with
biphenyl (0.15 ml) was mixed with 0.05 M NaOH (0.15 ml) and with cell suspension (0.5 ml,
concentration of 6.108cells.ml-1). The mixture was poured into Petri dishes Ø 3.5 cm. The film
containing approximately 4.108 cells.ggel-1, thickness of 1.3 mm and weight 0,75 g (±1%)
gelled within 5 minutes at 25°C.
2.2. Application of WCB
2.2.1. WCB test Mineral media (2 ml) containing D103 at concentrations of 1 to 20 mg.L -1,
individual PCB congeners (10 mg.L-1), PAHs, naphthalenes, solvents or dibenzofuran (2.5
g.L-1) were added to WCB. Dishes were incubated for 3 hours at 30°C. Seven replicates of
each sample were used for the statistical interpretation. Petri dishes were stored at 10°C
between experiments, testing the reproducibility of D103 detection, for up to 41 days.
2.2.2. UV-VIS spectra and data evaluation. After WCB incubation, 0.6 ml of media was
drawn off from WCB and the absorption spectra were measured by UV-VIS
spectrophotometer HP-8452 in 3 cm (optical length) glass cell. The absorbance values of
yellow HOPDA products were obtained by subtraction of the reference absorbance at 600
nm and HOPDA absorption maxima 398 nm. The absorption of minimal detected Delor103
and 2,4,4´CB concentrations were calculated as a values on axis x corresponding to y value
calculated as triplicate of standard deviation (s0) of blank media samples (Figure 2).
3. Results of measurements with WCB
PCB congener
3.1. Congener selectivity
Among 13 tested individual PCB congeners the highest amounts of the yellow meta ringfission product (HOPDA) were produced by 2,4,4´CB and 2,5,4´CB (figure 1). These two
congeners are the most abundant congeners in commercial mixture D103 (21.58 % w/w)
[10]. The generation of stable yellow colour meta ring-fission product from the degradation of
2, 4,4´CB and 2,5,4´CB by various bacteria were reported [5, 11]. Authors concluded that the
bulk of the degraded PCB was blocked at earlier stages of metabolism, including the meta
ring-fission product. This metabolite formed from each of these congeners was generated
from attack on the 4-chlorophenyl ring. It could be supposed, that chlorine in the ortho
position on one of the PCB ring and chlorine in para position on the second ring is the
chlorination pattern needed for the production and accumulation of yellow meta ring-fission
product.
2,2´,4,5,5´CB
3,3´,4,4´CB
2,4,6,3´CB
2,2´,5,5´CB
2,2´,3,3´CB
3,4,2´CB
2,5,4´CB
2,4,4´CB
2,5,3´CB
2,3,3´CB
2,5,2´CB
4,4´CB
2,2´CB
B
0
0.5
1
1.5
2
2.5
3
3.5
Absorbance (398-600 nm) AU
Figure 1 The effect of chlorine substitution pattern on the production of HOPDA.
Individual PCB congeners (10 mg.L-1) were tested. Samples for
detection of PCB content were taken after 3 hours of WCB incubation
at 30°C.
3.2. Concentration dependence
WCB was calibrated for 2,4,4´CB and commercial mixture D103 (figure 2). Seven replicates
of 2,4,4´CB and D103 was tested. Concentration dependence of congener 2,4,4´CB (figure 2
a) follows Michaelis-Menten type curve up to 15 mg.L-1. At higher 2,4,4´CB concentrations,
lower HOPDA production might be ascribed to toxic effect of 2,4,4´CB rather than HOPDA
degradation. Linearity in the range 0.5-5 mg.L-1 was confirmed for two WCB batches that
slightly differ in cell concentrations (4.6 and 3.9.108cells/g). Therefore in this range, WCB
might be used for measurements of 2,4,4´CB concentrations and also pollutants rich of this
congener. These include commercial mixtures, which were formerly used as transformer
fillings: D103, Pyralen 3100, Clophen 30, Aroclor 1242, TCB. Linearity of concentration
dependence was confirmed for D103 (figure 1b).
1
Absorbance (398 - 600 nm) AU
Absorbance (398-600 nm) AU
3
2
1
y = 0.3433x + 0.1035
R2 =0.9868
0.8
0.6
0.4
y = 0.1146x + 0.0508
R2 =0.9864
0.2
0
0
0
10
20
2,4,4´CB (mg.L-1)
(a)
30
0
10
20
30
D103 (mgL-1)
(b)
Figure 2 The dependence of absorbance on the concentration of 2,4,4’CB (a) and
D103 (b).
3.3. Selective detection in presence of other contaminants
The influence of persistent soil contaminants, that often accompany PCBs (polyaromatic
hydrocarbons, naphthalenes, and aromatic solvents), on WCB’s detection is on figure 3. An
occurrence of phenanthrene, anthracene and pyrene did not interfere the detection of D103
but, toluene, xylene naphtalenes and dibenzofuran increased the measured absorbance
without D103 and lowered that in its presence.
Naphthalene and toluene are more advantageous substrates as PCBs thus their
metabolites appeared in absorption spectra meanwhile yellow HOPDA faded due to blocking
PCBs’ metabolisation. In contrast to that toluene and xylene impaired bacterial metabolism,
which we deduced from drop of coloration and scatter of results (see error bars in figure 3).
As many microbial cells incubated with dibenzofuran, after biphenyl pre-cultivation, WCB
showed the characteristic orange coloration of the medium indicating dibenzofuran meta–
ring-fission [12]. Orange hue is visible so WCB will not give false positive results of PCBs
occurrence but in presence of both contaminants, PCBs will not be detected.
dibenzofuran
toluene
xylene
4 mg D103/L
0 mg D103 /L
compound (2.5 g/L)
1methylnaphthalene
naphthalene
pyrene
anthracene
1-methylnaphthalene
phenanthrene
control
0
0.2
0.4
0.6
0.8
Absorbance (398-600 nm) AU
Figure 3 Effect of aromatics on HOPDA production by WCB in the
presence or absence of D103 (4 mg.L-1) in tested media.
Samples for detection of PCB’s content were taken after 3
hours of WCB incubation at 30°C. Control samples did not
contain any aromatics.
0 mg.L-1
1 mg.L-1
10 mg.L-1
15 mg.L-1
Figure 4 WCBs incubated 3 hours with 2,4,4´CB (0, 1, 10, 15 mg.L-1). WCBs were reused
after 41 days storage at 10°C.
3. Conclusions
Reusable bio-assay, WCB, is the silica film thickness ~1 mm containing immobilized
Pseudomonas species 2 (~108 cells.g-1) and co-immobilized biphenyl (0.1 g.L-1). A response
time of this assay was 3 hours with detection limit ~1 mgD103.L-1, reproducibility ± 5%,
reusability 3 times and storage stability ≥ 4 weeks (Figure 4). The detection was not
interfered with phenanthrene, anthracene and pyrene. Toluene, xylene and naphtalenes
increased the measured absorbancies without D103 and lowered that in its presence. D103
was not detected in presence of dibenzofuran.
Among PCB bioassays, WCB fills the niche between immunosensors, which are sensitive to
toxic congeners with 4 and more chlorine atoms in molecule [13] and whole cell
bioluminescent bioreporter [2], which produces luminescence consuming congeners with 1 or
2 chlorine atoms. PCBs with 3 chlorine atoms are not extremely toxic but its degradation to
final metabolite is slow.
The advantages of WCB are: facile preparation and measurement, low cost of bioassay and
electro-optical instrumentation.
Acknowledgment
The authors thank the Grant Agency of the Czech Republic for funding this work in the
projets No 104/05/2637, 203/06/1244 and Ministry of Education, Youth and Sports of the
Czech Republic project No OC121 1041/2006-32.
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