Journal of Environmental Protection, 2010, 1, 420-425
doi:10.4236/jep.2010.14048 Published Online December 2010 (http://www.SciRP.org/journal/jep)
Biodegradation of Tritium Labeled Polychlorinated
Biphenyls (PCBs) by Local Salt Tolerant Mesophylic
Bacillus Strains
Rasulov Bakhtiyor Abdughafurovich1, Kim Andrey Andreevich2, Lorenz Adrian3,
Yadgarov Khojiakbar Tashpulatovich1
1Institute of Genetics and Plant Experimental Biology, Uzbekistan Academy of Sciences, Tashkent, Uzbekistan; 2Institute of Nuclear
Physics, Uzbekistan Academy of Sciences, Tashkent, Uzbekistan; 3Department Isotope Biogeochemistry, Helmholtz Centre for
Environmental Research, Leipzig, Germany.
Received August 13th, 2010; revised September 8th, 2010; accepted September 13th, 2010.
ABSTRACT
Salt-resistant Bacillus strains, isolated from agricultural soils in Uzbekistan, were tested for degradation activity to-
wards a mixture of polychlorinated biphenyls (PCBs) under aerobic conditions. The study employed the use of tritium
labeled PCB congeners and traced the tritium label in cultures with high salt content. The experiments show that most
of the selected strains were able to adsorb a part of the radioactivity, indicating transformation of the added PCBs. Gas
chromatography demonstrated transformation of PCBs. The radioactive label was removed from several cultures by up
to 91%, indicating also mineralization of PCBs. The study suggests that the isolated strains might be good candidates
for the bioremediation of contaminated high-salt soils in Uzbekistan and other Central-Asian countries.
Keywords: Bacillus, Biodegradation, PCBs, Salinity, Tritium Label
1. Introduction
Excessive use of fertilisers and pesticides in agriculture,
primarily in the cotton and wheat plantations over the
past decades, has resulted in significant contamination of
soils not only in Uzbekistan, but also in a number of
other Central Asian countries. Huge former cotton and
wheat plantations now contain many different types of
these chemicals.
One important example for these chemicals is the
polychlorinated biphenyls (PCBs that have been widely
used for numerous open and closed industrial applica-
tions. Commercial PCB mixtures were sold under the
names Aroclor (Monsanto, USA), Phenoclor (Caffaro,
Italy), Clophen (Bayer, Germany), Kanechlor (Kanega-
fuchi, Japan), and Sovol (former USSR). Due to their
widespread use and their thermal and chemical stability
they have also found their way to agricultural soil. PCBs
are organic chemicals belonging to a class of hydrocar-
bons that are among the most hazardous environmental
contaminants, as their physical and chemical properties
especially their low water solubility and high lipophilic-
ity lead to a high persistence in the environment and to
bioaccumulation in the food chains [1,2].
Several bacteria have been described to degrade PCBs
under aerobic conditions. Most of these bacteria also
degrade biphenyls as a growth substrate. Such PCB de-
graders are mainly obligate aerobes, motile, gram nega-
tive rods. They can be enriched by cultivation with bi-
phenyl or PCBs as a growth substrate on agar plates and
are identified on agar plates by detecting clearing of bi-
phenyl or PCB films around colonies. Biphenyl is mostly
degraded by the initial action of a 2, 3-dioxygenase at-
tack on the 2, 3-carbons and meta-cleavage, producing
benzoate. Similarly, there are many bacterial strains from
the genera Pseudomonas [3], Arthrobacter [4], Vibrio,
Aeromonas, Micrococcus, Acinetobacter, Bacillus, Rho-
dococcus [5], and Streptomyces that degrade mono-, di-,
tri-, and several tetrachlorinated PCBs by meta-cleavage
of non-chlorinated 2,3-carbons.
Soils in Uzbekistan vary considerably in their chemi-
cal composition and their salinity. The average soil salin-
ity exceeds 40-60 mM [6]. However, in some provinces,
particularly in Karakalpakstan, and Khorezm, Syrdarya
provinces the degree of salinity is several times higher
than this. Overall climate and specific weather patterns

Biodegradation of Tritium Labeled Polychlorinated Biphenyls (PCBs) by Local Salt Tolerant Mesophylic Bacillus Strains 421
play a major role in the impacts that soil salinity will
have with regard to the different regional soils of Uz-
bekistan. Microbial biodegradation is the major process
leading to transformation of organic chemicals in soil,
and hence bioremediation is viewed as the most attrac-
tive approach to the ultimate destruction of exogenous
agricultural chemicals added to cultivated soils. However,
the efficiency of bioremediation of agrochemicals in soils
depends on the soil conditions. Therefore, under the giv-
en climate and soil conditions in Uzbekistan, it appears
necessary to use salt tolerant strains for bioaugmentation
approaches. The application of microorganisms encoding
contaminant-specific biodegradative pathways may pro-
vide the most cost-effective technology for the remedia-
tion of pollutant-contaminated sites. This type of tech-
nology may also be the most acceptable for the public
because it represents a natural process that is essentially
taking advantage of nature’s recycling abilities. The most
effective and acceptable bioremediation systems strive
for complete mineralization of pollutants to inorganic
products.
The aim of our research was to investigate the biodeg-
radation of PCBs in the presence of sodium chloride –
the main source of salinity in the soil. Therefore, we used
salt tolerant bacterial strains of the genus Bacillus from
our strain collection and tested them for PCB degradation
capability.
2. Materials and Methods
2.1. Strains
We used a bacterial strain collection that was set up in a
number of state projects on biodegradation of hazardous
organic pesticides, such as PCBs, HCCH (hexachlorecy-
clohexane), DDD (1,1-di(4’-chlorophenyl)-2,2 dichloro-
ethane) and DDT (1,1-di(4’-chlorophenyl)-2,2,2 trichlo-
roethane).
2.2. Culture Medium
The strains were cultured in synthetic mineral medium
with the following additions (g/l): NaCl – 0.5; KH2PO4 –
0.7; (NH4)2HPO4 – 1.5; MgSO4·7 H2O – 0.5; pH 7.3. As
the sole source of carbon and energy, PCBs were added
in a concentration of 20 μg/ml. Incubation was carried
out on a shaker at 28℃ for 60 days.
2.3. Labeling of PCBs
Before labeling, the labeling system and the capture was
washed with nitrogen and a mixture of hexane:acetone
(1:1). PCBs were labeled by thermal activation of tritium
with the help of a labeling tool developed in the labora-
tory of biological radiochemistry [7]. A solution of non-
labeled PCBs was inserted to the surface of the reaction
retort, cooled with liquid nitrogen and vacuumized to 5 ×
10-3 mm mercury. Labeling was carried out in five cycles,
with five times filling the system with gaseous tritium up
to a pressure of 8 × 10-3 mm mercury. Tritium was acti-
vated with a heated tungsten spiral three times with a 20
minute time interval between heating the spiral. The
mixture of PCBs was analysed before and after the la-
beling via HPLC. The labeled PCBs were purified on
silica gel in hexane (Rf = 0.78).
2.4. GC Analyses
GC analysis was done using an electron capture detector.
A Chromaton N-Super column (2 m × 4 mm) (Chemapol,
Czech Republic) was used, with a corn size of 0.125-0.16
mm, column temperature 200℃, injector temperature
230℃, detector temperature 260℃; carrier gas 30 ml/
min, 5% DS-200 (Chemapol, Czech Republic).
2.5. Analysis of Accumulation of PCBs by the
Strains
Strains cultured on synthetic agar-solidified medium were
transferred to liquid medium. After one day of incubation
300 µl of culture (108 cells/ml) were transferred to a test
tube containing 1 ml synthetic medium and 20 µl of the
prepared solution of tritium-labeled PCBs in hexane. The
tritium-labeled PCB was the sole carbon and energy
source. The strains were incubated from several days up
to two months at 28℃ on a shaker. After incubation bac-
terial cells were separated by centrifugation at 6500 rpm
for 30 min. Cells and supernatant were dried and PCBs
were extracted with hexane.
2.6. Analysis of PCBs Degradation by the
Selected Strains
Bacterial cells, cultured on the agar-solidified mineral
medium, were transferred to liquid medium. After one
day, 300 μl (108 cells/ml) of this culture were transferred
into test tubes with 5 ml liquid medium containing pep-
tone (1%) and 100 μg of PCBs. In the controls, cells
were killed by autoclaving for 30 min. The strains were
incubated in a thermostat at 28℃ for 60 days. Then, cells
were harvested by centrifugation. Pellets and super-
natants were dried at 60℃ and PCBs were extracted with
a mixture of hexane and acetone (1:1, vol/vol). The ex-
tracts were then analyzed by GC as described above.
2.7. Analysis of Biodegradation of PCBs in the
Soil
Autoclaved soil samples (15 g each) were mixed with
100 μg of PCBs in 5 ml of hexane. Then, soil samples
were dried to remove hexane from the samples. Each soil
sample was inoculated with 300 μl from one of the liq-

Biodegradation of Tritium Labeled Polychlorinated Biphenyls (PCBs) by Local Salt Tolerant Mesophylic Bacillus Strains 422
uid-medium cultures (108 cells/ml). The control consisted
of a PCBs containing soil sample without bacteria. Dur-
ing the two month incubation period all samples were
kept humid by the addition of sterile water. After incuba-
tion, samples were dried at 60℃ and PCBs were ex-
tracted with a mixture of hexane and acetone (1:1,
vol/vol). The extracts were analyzed by GC as described
above.
3. Results
We investigated the capability of selected pure Bacillus
strains (denominated №№ 20, 22, 23, 24, 27, 28, 29, 30,
31, and 32) to utilize tritium-labeled PCBs in liquid cul-
ture and soil. The strains were isolated from soils in Uz-
bekistan that were heavily polluted since more than 40
years. We added 0.1 mg tritium-labeled PCBs solved in 5
ml of hexane to the bottom of the tubes. PCBs were then
dried by complete evaporation of hexane. The applied
radioactivity inserted with 0.1 mg of tritium labeled
PCBs amounted to 13,647 imp/10 seconds. Finally, the
tubes were inoculated with 5 ml of bacterial cultures. The
control sample consisted of sterilized medium. The tubes,
containing the culture liquids of the strains, were incu-
bated in the thermostat at 28℃ for 10 days. After incuba-
tion the cultures were transferred to centrifuge tubes, and
bacterial cell were pelleted at 8000 rpm for 30 min. The
supernatant was then transferred to fresh culture tubes.
The pellets, containing bacterial cells were dried and
weighed. Supernatants (culture liquid) were also dried at
80℃ until they were completely dry. From the dried
samples of bacterial strains and culture liquid PCBs were
extracted using a mixture of hexane and aceton (1:1,
vol/vol) three times. The combined PCB extracts were
again dried, dissolved in 100 µl of hexane and analyzed
for radioactivity. Radioactivity was measured by liq-
uid-scintillation counting in РЖС-05 (Liquid Scintilla-
tion Radiometer) in a toluene scintillate cocktail (1 l to-
luene containing 4 g of РРО and 0.05 g of РОРОР). For
standardization a volume of 25 µl of a control solution
was taken. Obtained data were then recalculated to ac-
count for the whole volume. The results are given in the
Figure 1.
It was observed that a large part of the tritium label
was incorporated into cell mass, showing that the PCBs
were transformed and used for cell growth. Only a minor
part was still found in the medium as non-evaporable
substances. Another part of the tritium label was lost
from the tubes either by the production of volatile trit-
ium-labeled intermediates or by the transfer of tritium
from the PCBs into water and successive evaporation
during the analysis procedure. In the strains B. mycoides
0
2000
4000
6000
8000
10000
12000
14000
16000
2022 23242728 293031 32Control
Bacterial strain number
Radioactivity in 100 µl (imp/10 sec)
Culture medium
Pelleted bacteria
Figure 1. Radioactivity originating from tritium-labeled
PCBs detected associated to the bacteria and the culture
medium after incubation for 60 days.
20, B. subtilis 28 and Bacillus sp. 30 the percentage of
the remaining radioactivity was between 53 and 75% (in
the strain of Bacillus sp. 23 up to 91%). In these strains
(B. mycoides 20, Bacillus sp. 23, B. subtilis 28 and Ba-
cillus sp. 30) between 86 and 95% of the remaining trit-
ium was incorporated into the bacteria. In other strains
the remaining radioactivity was 16-30%, while the rest
was lost. In the control sample no decrease of the radio-
activity was observed. Therefore, we can exclude the
possibility that there was an isotope exchange of tritium
fixed in the added PCBs with hydrogen from the water
phase.
The tritium-labeled PCBs remaining in the medium
were analyzed by GC (Figure 2). It was observed that
the spectra of PCB fractions changed during the incuba-
tion when bacteria were present. This proves active
transformation of PCBs by the strains in the presence of
NaCl. The chromatogram of Sovol was also compared
with the standard chromatograms of Aroclor (Aroclor
1016/1260 Standard, Aroclor 1221 Standard, Aroclor
1232 Standard, Aroclor 1242 Standard, Aroclor 1248
Standard, Aroclor 1254 Standard), available at the stan-
dard library NIST02. The chlorination spectrum of Sovol
considerably differs from that of the various Aroclors,
which were used for calibration of the mass-selective
detector Hewlett-Packard HP5972. That is why we de-
veloped quantitative estimation of PCBs content in the
tested samples. The obtained chromatograms from our
incubations were reprocessed with MSD ChemStation
Agilent Technologies in the overlay chromatogram re-
gime. Chromatographic analysis of soil samples showed
that they were rich in low chlorinated biphenyls.
The amount of tritium-labeled PCBs that was trans-
formed and incorporated into the bacteria was recalcu-
lated from the incorporated radioactivity and the dry
weight of bacterial cells. The results of these calculations
are given in the Table 1. Here, the high capacity of
strains 20 and 23 to specifically transform PCBs be

Biodegradation of Tritium Labeled Polychlorinated Biphenyls (PCBs) by Local Salt Tolerant Mesophylic Bacillus Strains 423
Figure 2. Chromatogram of extracted PCBs from Sovol-amended soils. Upper panel: control microcosm without bacteria;
lower panel: PCB content after incubation with Bacillus sp. 23.
comes evident by values of above 50 µg incorporated
PCBs per mg of dry mass.
According to the literature monochlorinated biphenyls
are more volatile than PCB isomers with 8-9 chlorine
atoms. Also, the evaporation of the isomers depends on
temperature [8,9]. It is necessary to point out that, under
the hot climate conditions of Central Asia the tempera-
ture is one of the basic factors in the removal of PCBs
from soil. In this climate the volatility of low chlorinate
biphenyls are high. Therefore, even without the complete
biodegradation of the lower chlorinated biphenyls, the
toxicity of technical mixture of PCBs is greatly de-
creased, since low chlorinated (less than three chlorine
atoms) are less toxic.
4. Discussion
Biodegradation of polychlorinated biphenyls has been
shown and the activity of the products of pcb genes in

Biodegradation of Tritium Labeled Polychlorinated Biphenyls (PCBs) by Local Salt Tolerant Mesophylic Bacillus Strains 424
Table 1. Determination of tritium-labeled PCBs bound to the bacteria.
Strain Radioactivity bound to the
bacteria, imp/10 sec
Dry weight of bacterial
cells
Bound PCBs
(% out of exposed radioactivity)
Bound PCBs per mg dry
weight of bacterial cells
B. mycoides 20 8,853 1.2 mg 64.87 µg (64.87%) 54.06 µg
Bacillus sp. 22 984 2.3 mg 7.21 µg (7.21%) 3.13 µg
sBacillus sp. 23 11,064 1.2 mg 81.07 µg (81.07%) 67.56 µg
B. megaterium 24 1,208 1.1 mg 8.85 µg (8.85%) 8.05 µg
B. subtilis 27 2,148 4.6 mg 15.74 µg (15.74%) 3.42 µg
B. subtilis 28 7,768 10.3 mg 56.92 µg (56.92%) 5.54 µg
B. megaterium 29 640 1.5 mg 4.69 µg (4.69%) 3.13 µg
Bacillus sp. 30 6,216 3.8 mg 45.55 µg (45.55%) 11.99 µg
B. megaterium 31 3,728 2.7 mg 27.32 µg (27.32%) 10.12 µg
B. subtilis 32 2,396 20.9 mg 17.56 µg (17.56%) 0.84 µg
different extreme environments has been documented.
However, a strong effect of temperature on PCBs degra-
dation has been found [10,11], and biphenyl dioxygenase
was induced in psychorotolerant, polychlorinated biphe-
nyl (PCBs) degrading Pseudomonas spp. [10,11]. How-
ever, biodegradation of polychlorinated biphenyls in salt
affected environments still needs to be better clarified.
There is no detailed data on salinity influence on biodeg-
radation activity of catabolic strains, expression of pcb
genes, or synthesis and functioning of biphenyl dioxy-
genase(s). Therefore, in the present study we examined
the biodegradative activity of catabolic strains, isolated
from salt-rich, pesticide-polluted soils in Uzbekistan.
Former pollutants included DDT, and HCCH. The final
concentration of NaCl in our study was 4%, in which the
tested strains survived in previous experiments. We ob-
served degradation of the PCBs by several catabolic
strains within 4 months of incubation, whereas other re-
searchers incubated for 1 year in the temperature range
from 4 to 66℃ for microbial dechlorination of 2,3,4,6-
tetrachlorobiphenyl [8].
In all tested samples with salt-tolerant catabolic strains,
PCB removal (up to 85-90%) was much higher than with
salt-intolerant catabolic strains of the same genus. That
again proved normal catabolic functioning of the tested
strains. It is important to note, that despite all strains be-
longed to the same genus, the products of biodegradation
slightly varied in respect to the number of chlorine atoms
and their positions in the ring. Especially, it was specific
to the intermediates with penta- and hexachlorinated
rings in the early stages of incubation. The chroma-
tographic analysis of a technical mixture of PCBs show-
ed abundant presence of highly chlorinated biphenyls,
such as heptachlorobiphenyls (2,2’,3,4,4’,5’,6-, 2,2’,
3,3’,4,- 6,6’-, and 2,2’,3,4’,5,5’,6-heptachlorbiphenyl),
hexachlorobiphenyls (2,2’,3,4,4’,5’-, 2,2’4,4’,5,5’-, and
2,2’,3,-4,4’,5’- hexachlorobiphenyls), and 2,2’,4,5,5’-
pentachlorobi-phenyl. Also tri- and tetrachlorobiphenyls
were detected in the mixture. After two and four months
of incubation the amount of highly chlorinated biphenyls
was strongly reduced in all inoculated samples. The
chromatographic analysis of two month samples revealed
that the amount of heptachlorobiphenyls and hexachlore-
biphenyls was sharply reduced.
It is known that some bacterial strains can loose their
biodegradation ability after introducing them into soil.
Therefore, we performed additional tests on the func-
tioning of the tested strains in soil. The strains were
grown under model conditions in the soil with tritium
labeled PCBs as the sole electron and carbon source. We
found that most strains effectively mineralized PCBs
resulting in decreasing level of tritium label in the soil
with time (Figure 1).
Based on the obtained data we can conclude that the
tested strains effectively utilize tritium-labeled PCBs and
different PCBs metabolism intermediates in the presence
of high concentrations of sodium chloride, which is the
main contributor to salinity. In the NaCl containing mi-
crocosms the strains Bacillus sp. 23, B. mycoides 20, B.
subtilis 28 and Bacillus sp. 30 transformed PCBs mainly
to cell mass. Other strains most probably produced vola-
tile compounds with low numbers of chlorine atoms,
which evaporated during incubation and the analysis
procedure. Herewith, we can suppose that practically all
tritium labeled PCBs and their intermediates are incor-
porated into the strains, and the degree of biodegradation
under model conditions in the soil was lower than that of
cultures without soil.

Biodegradation of Tritium Labeled Polychlorinated Biphenyls (PCBs) by Local Salt Tolerant Mesophylic Bacillus Strains 425
Based on our results we can recommend the use of all
our investigated Ba cillus strains to different extends as
new biopreparations for the biodegradation of PCBs in
the salt-affected soils in the Republic of Uzbekistan, and
other Central Asian countries, including Kazakhstan,
Turkmenistan, Tajikistan, and Kyrgyzstan, where tech-
nical PCBs were heavily used in industrial and agricul-
tural processes.
REFERENCES
[1] J. F. Quensen, J. M. Tiedje and S. A. Boyd, “Reductive
Dechlorination of Polychlorinated Biphenyls by Anaero-
bic Microorganisms from Sediment,” Science, Vol. 242,
No. 4879, 1988, pp. 752-754.
[2] V. A. McFarland and J. U. Clarke, “Environmental Oc-
currence, Abundance, and Potential Toxicity of Poly-
chlorinated Biphenyl Congeners: Considerations for a
Congener-Specific Analysis,” Environment Health Per-
spect, Vol. 81, 1989, pp. 225-239.
[3] B. D. Erickson and F. J. Mondello, “Enhanced Biodegra-
dation of Polychlorinated Biphenyls after Site-Directed
Mutagenesis of a Biphenyl Dioxygenase Gene,” Applied
and Environmental Microbiology, Vol. 59, No. 11, 1993,
pp. 3858-3862.
[4] E. S. Gilbert and D. E. Crowley, “Plant Compounds that
Induce Polychlorinated Biphenyl Biodegradation by Ar-
throbacter sp. Strain B1B,” Applied and Environmental
Microbiology, Vol. 63, No. 5, 1997, pp. 1933-1938.
[5] A. W. Boyle, C. J. Silvin, J. P. Hassett, J. P. Nakas and
S.W. Tanenbaum, “Bacterial PCB Biodegradation,” Bio-
degradation, Vol. 3, No. 2-3, 1992, pp. 285-298.
[6] A. Abdullayev, “Azospirillum in Saline Soils of Uzbeki-
stan,” Ph.D Dissertation, Institute of Microbiology of
Uzbekistan Academy of Sciences, Tashkent, 2006.
[7] A. A Kim, G. T. Djuraeva, P. V. Zinovev, I. I. Sadikov, A.
A. Rylov, “A New Technique for Tritium Labeling of
Complex Technical Mixture of PCB Congeners,” Journal
of Radioanalytical and Nuclear Chemistry, Vol. 272, No.
3, 2007, pp. 483-489.
[8] E. R. Master and W. W. Mohn, “Induction of bpha, En-
coding Biphenyl Dioxygenase, in Two Polychlorinated
Biphenyl-Degrading Bacteria, Psychrotolerant Pseudo-
monas Strain Cam-1 and Mesophilic Burkholderia Strain
lb400,” Applied and Environmental Microbiology, Vol.
67, No. 6, 2001, pp. 2669-2676.
[9] E. R. Master, N. Y. R. A. Leticia Gómez-Gil, J. B. Pow-
lowski, W. W. Mohn and L. D. Eltis, “Biphenyl Dioxy-
genase from an Arctic Isolate is not Cold Adapted,” Ap-
plied and Environmental Microbiology, Vol. 74, No. 12,
2008, pp. 3908-3911.
[10] Q. Wu, D. L. Bedard and J. Wiegel, “Effect of Incubation
Temperature on the Route of Microbial Reductive Dech-
lorination of 2,3,4,6-Tetrachlorobiphenyl in Polychlori-
nated Biphenyl (PCB)-Contaminated and PCB-Free Fre-
shwater Sediments,” Applied and Environmental Micro-
biology, Vol. 63, No. 7, 1997, pp. 2836-2843.
[11] E. R. Master and W. W. Mohn, “Psychrotolerant Bacteria
Isolated from Arctic Soil that Degrade Polychlorinated
Biphenyls at Low Temperatures,” Applied and Environ-
mental Microbiology, Vol. 64, No. 12, 1998, pp. 4823-
-4829.