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ORIGINAL PAPER
Characterization of multiple novel aerobic polychlorinated
biphenyl (PCB)-utilizing bacterial strains indigenous to
contaminated tropical African soils
Sunday A. Adebusoye ÆFlynn W. Picardal Æ
Matthew O. Ilori ÆOlukayode O. Amund Æ
Clay Fuqua
Received: 29 November 2006 / Accepted: 10 April 2007 / Published online: 30 May 2007
Springer Science+Business Media B.V. 2007
Abstract Contaminated sites in Lagos, Nigeria were
screened for the presence of chlorobiphenyl-degrading
bacteria. The technique of continual enrichment on
Askarel fluid yielded bacterial isolates able to utilize
dichlorobiphenyls (diCBs) as growth substrates and six
were selected for further studies. Phenotypic typing and
16S rDNA analysisclassified these organisms as species
of Enterobacter,Ralstonia and Pseudomonas. All the
strains readily utilized a broad spectrum of xenobiotics
as sole sources of carbon and energy. Growth was
observed on all monochlorobiphenyls (CBs), 2,20-, 2,3-,
2,40-, 3,30- and 3,5-diCB as well as di- and trichloro-
benzenes Growth was also sustainable on Askarel
electrical transformer fluid and Aroclor 1221. Time-
course studies using 100 ppm of 2-, 3- or 4-CB resulted
in rapid exponential increases in cell numbers and CB
transformation to respective chlorobenzoates (CBAs)
within 70 h. Significant amounts of chloride were
recovered in culture media of cells incubated with 2-CB
and 3-CB, suggesting susceptibilities of both 2- and 3-
chlorophenyl rings to attack, while the 4-CB was
stoichiometrically transformed to 4-CBA. Extensive
degradation of most of the congeners in Aroclor 1221
was observed when isolates were cultivated with the
mixture as a sole carbon source. Aroclor 1221 was
depleted by a minimum of 51% and maximum of 71%.
Substantial amounts of chloride eliminated from the
mixture ranged between 15 and 43%. These results
suggest that some contaminated soils in the tropics may
contain exotic micro-organisms whose abilities and
potentialsare previously unknown. An understanding of
these novel strains therefore, may help answer questions
about the microbial degradation of polychlorinated
biphenyls (PCBs) in natural systems and enhance the
potential use of bioremediation as an effective tool for
cleanup of PCB-contaminated soils.
Keywords Aerobic biodegradation
Bioremediation Chlorobenzene PCBs
Polychlorinated biphenyls
Introduction
The manufacture, use and disposal of polychlorinated
biphenyls (PCBs) have been subject to strict govern-
mental regulations for more than three decades owing
S. A. Adebusoye (&)M. O. Ilori O. O. Amund
Department of Botany and Microbiology,
Faculty of Science, University of Lagos,
Akoka, Yaba, Lagos, Nigeria
e-mail: sadebusoye@yahoo.com
S. A. Adebusoye F. W. Picardal
Environmental Science Research Center, School of Public
and Environmental Affairs, Indiana University,
Bloomington, IN 47405, USA
C. Fuqua
Department of Biological Sciences, College of Art and
Sciences, Indiana University, Bloomington, IN 47405,
USA
123
Biodegradation (2008) 19:145–159
DOI 10.1007/s10532-007-9122-x
to their environmental and potential carcinogenic
effects. Although many developing countries in Africa
include PCBs and PCB-waste oils on the list of
hazardous substances, the discharge of these pollutants
into the environment is often not monitored and
regulated. Pollution, when it occurs is never reported
or investigated. As a result, data on importation, use,
transportation and environmental fate of PCBs are
usually unavailable. Leakage from transformers and
indiscriminate discharge of the spent PCB insulating
fluid (Askarel) are regular occurrences. The safe and
economical degradation of PCBs, therefore, is one of
the major environmental challenges facing many
African countries today.
In spite of the superhydrophobicity and chemical
stability, it is amazing that some bacterial strains that
can metabolize specific PCB congeners have been
described. The isolation of two bacterial strains
capable of aerobic degradation of 2-, 3-, 4-mono-
chlorobiphenyl (CB) and 2,20- and 4,40-dichlorobi-
phenyl (diCB) as co-cultures in 1973 (Ahmed and
Focht 1973) changed our perception of PCBs as
immutable chemicals. Since then, a large number of
micro-organisms that can degrade PCBs were iso-
lated from contaminated soils and sludges (Furukawa
et al. 1979; Bopp 1986; Bedard et al. 1987a; Kim and
Picardal 2000) and studied in greater detail, culmi-
nating in elucidation of the PCB biodegradation
pathway. One similarity among PCB degrading
micro-organisms is that they are all biphenyl utilizers
which metabolize PCBs with the same suite of
enzymes employed in biphenyl catabolism (Ahmed
and Focht 1973; Furukawa et al. 1979; Abramowicz
1990). Aerobic catabolism of PCB usually begins
with the attack of biphenyl dioxygenase on an
unsubstituted 2,3 position of a non-chlorinated or
less chlorinated ring. This is followed by meta-
cleavage, producing chlorobenzoic acids (CBAs) and
a 5-carbon aliphatic acid (2-hydroxy 2,4-pentadienoic
acid). None of these steps is linked to energy
conservation. If an organism is to grow on the PCB
substrate, it must get energy from subsequent
metabolism of either the CBA or more commonly,
the 5-C fragment, with the resultant accumulation of
the former in the culture fluid (Bevinakatti and
Ninnekar 1993; Kim and Picardal 2000). Micro-
organisms that can utilize PCBs as growth substrates
must either utilize non-chlorinated rings, producing
CBAs as products, or produce enzymes capable of
carbon–chlorine bond cleavage. Ability of micro-
organisms to grow on PCBs is generally limited to
monochlorophenyls, while congeners containing 2 or
more chlorines are usually aerobically degraded via
co-metabolism (Furukawa et al. 1979; Bedard et al.
1987b; Abramowicz 1990), which requires biphenyl
as a growth substrate and inducer of the requisite
enzymes. Biphenyl, however, is toxic, often subject
to regulatory restrictions and not easily dispersed in
contaminated soils or sludges. Hence, isolation of
organisms that grow on environmental PCB mixtures
without the need of biphenyl as a primary substrate
might obviate this problem.
Isolates, which can utilize 4-CB as a growth
substrate are common (Barton and Crawford 1988;
Mondello 1989; Ahmad et al. 1990; Arensdorf and
Focht 1995). Reports of isolates, which utilize 2- and
3-CB are less common (Potrawfke et al. 1998;
Bedard et al. 1987a; Hickey et al. 1992; Kim and
Picardal 2000) and, with the exception of the isolates
described by Arensdorf and Focht (1994) and Kim
and Picardal (2000), no organism has been reported
to grow on all 3 monochlorobiphenyl isomers. In
addition, most of the reported isolates possess narrow
substrate spectra. Furthermore, there is paucity of
information on the biodegradation of PCBs by
organisms isolated from tropical African environ-
ment. In fact, almost, if not all of the micro-
organisms characterized to date were obtained from
contaminated temperate soils. It is unlikely, however,
that identical xenobiotic-degrading bacteria are uni-
formly distributed around the globe due to differing
ambient environmental conditions, soil composition,
organic carbon soil inputs and many other factors. In
view of the wider geochemical and physical varia-
tions in both environments, tropical microbes may
exhibit a fascinating metabolic diversity and possess
novel catabolic properties for real time degradation of
PCBs. In the current study, we isolated a number of
organisms from contaminated tropical soils that
exponentially grow on all CBs and some diCBs as
sole carbon and energy sources under aerobic condi-
tions. These organisms were also able to extensively
transform several congeners in commercial mixture
of Aroclor 1221 with evidence of chloride elimina-
tion. To the best of our knowledge, this is the first
report demonstrating the occurrence of competent
multiple PCB-degrading micro-organisms in African-
contaminated systems.
146 Biodegradation (2008) 19:145–159
123
Materials and methods
Chemicals
Polychlorinated biphenyl congeners (> 99% purity),
PCB commercial mixture (Aroclor 1221; > 98.6–100%
purity) and chlorobenzene isomers (> 98.6% purity)
were purchased from AccuStandard Inc. (New Haven,
CT, USA). Since none of the single PCB congeners
contained biphenyl as an impurity, they were utilized
as received from the manufacturer without further
purification. PCB analytical standards were obtained
from Ultra Scientific (North Kingston, RI, USA).
Biphenyl, naphthalene (99+%), chloroacetic acid
(98% purity) and mono- and dichlorobenzoic acids
(98% purity) were acquired from Sigma-Aldrich Corp.
(St Louis, MD, USA). Sodium benzoate (99+% purity),
2,2,4,4,6,8,8-heptamethylnonane (HMN), and all other
organic solvents (HPLC grade) were obtained from
Fisher Scientific Co. (Springfield, NJ, USA). Askarel
oil (a blend of PCBs and chlorobenzenes) was gener-
ously supplied by the NEPA Transformer Workshop
(Ijora, Lagos, Nigeria).
Stock solutions and media
The chloride-free mineral salts (MS) medium used in
this study was formulated according to Kim and
Picardal (2000). Due to limited aqueous solubility,
stock solutions of each PCB and chlorobenzene
congener were prepared in HMN, a non-degradable
carrier, to provide an initial concentration of 100 ppm.
The concentration given represents the total mass in
both the aqueous and HMN phases, divided by the
aqueous volume. The appropriate stock solution was
added in 20 ml aliquots to provide a test compound
concentration of 100 ppm in the finished medium.
Solid MS medium was made by the addition of 1.6%
Bacto-agar (Difco Laboratories, Detroit, MI, USA).
MS medium was supplemented with test compounds to
achieve an experiment-dependent concentration of
either 100 ppm or 2.5 mM. Unless otherwise stated,
cultures were incubated at 25 8C on a shaker table to
improve mass transfer from the HMN-phase into the
aqueous phase. In order to establish that the HMN
carrier was not a growth substrate for our isolates,
preliminary growth studies were performed in MS
medium supplemented with HMN as the sole carbon
and energy source.
Enrichment of microbial communities
Soil samples were collected randomly from six
contaminated sites in Lagos. Three of these sites,
namely Ojota; National Electric Power Authority
(NEPA) Transformer Workshop, Ijora; and NEPA
Thermal Station, Surulere have been heavily polluted
for decades, mainly with PCB- and chlorobenzene-
containing, electrical transformer fluid (Askarel).
Chlorobiphenyl-degrading bacteria were initially iso-
lated by traditional enrichment culture methods: Soil
samples (1 g) were used to inoculate MS medium
contained in a conical flask (500 ml). The medium
was supplemented with Askarel fluid (0.1% v/v) as
the primary carbon source. Enrichment cultures were
incubated on a gyratory shaker incubator (New
Brunswick Scientific Co., Edison NJ, USA) at
120 rpm for 30 days. Subsequent transfers from
these enrichments were made (every month) by using
the same methods and conditions. A parallel enrich-
ment was also set up in 160-ml serum bottles
containing MS medium (40 ml) supplemented with
100 ppm Aroclor 1221. Bottles were inoculated with
soil sample (5 g), crimp-sealed with Teflon-coated
stoppers and incubated horizontally on a shaker table
(Labline Instruments Inc., Melrose Park, IL, USA).
After 1 month, enrichment cultures were transferred
to fresh medium using a 10% inoculum and continued
cultivation under the same conditions. Subsequent
transfers were carried out using 1% inoculum and the
procedure repeated for six successive times.
Isolation, purification and characterization of pure
cultures
Pure cultures from Askarel-enriched media were
isolated by directly plating out appropriate aliquots
(0.1 ml) of highly enriched cultures onto nutrient
agar. Since we desired to use these same enrich-
ments to isolate bacteria growing on chlorobenzenes
in the Askarel (Adebusoye et al. 2007), we used
nutrient agar to obtain pure cultures rather than MS
medium supplemented with biphenyl or benzoate
which may have selected against chlorobenzene-
degraders unable to utilize those substrates. Pure
colonies selected from the nutrient agar plates were
subsequently screened for their ability to utilize
selected chlorobiphenyls in MS medium as de-
scribed below.
Biodegradation (2008) 19:145–159 147
123
Organisms in Aroclor 1221 enriched cultures were
obtained by a spray plate technique. Desired dilutions
(0.1 ml) were spread on MS agar. Immediately after,
an ethereal solution of biphenyl was uniformly
sprayed onto the surface of the agar. The plates were
sealed with paraffin film and incubated for 4–6 weeks.
Biphenyl-degrading micro-organisms were identified
by compound-cleared zones surrounding individual
colony. Such colonies were purified on MS agar
containing 2.5 mM benzoate.
Organisms capable of growing on PCB congeners as
a sole source of carbon and energy were classified
using standard cultural and morphological techniques.
The API 20 E test kits (bioMerieux Vitek, Hazelwood,
MO, USA) were also employed. Identification of the
micro-organisms was achieved on the basis of 16S
rRNA gene analysis. Genomic DNA was isolated from
overnight cultures of isolates on 2.5 mM benzoate
using an ultra clean DNA prep kit (MoBio Laborato-
ries, Solana Beach, CA, USA). Two Eubacterial PCR
primers; forward primer 63f and reverse primer 1387r,
were used to amplify *1,300 bp of the 16S rRNA
gene. The resulting PCR-generated fragments were
gel-purified and cloned into the PCR-TOPO 1 plasmid
(Invitrogen Life Technologies, Carlsbard, CA, USA)
vector. Recombinant plasmids with correct rDNA
inserts were digested with EcoRI and then sequenced
(ABI 3700 sequencer; AP Biotech, Buck, UK), and
were probed against the GenBank database with the
BLAST algorithm. Organism identities so obtained
were subsequently verified using the Ribosomal Data-
base Project (Cole et al. 2007) and identical results
were obtained.
Growth on different carbon sources and
determination of PCB transformation potential
Pure cultures were tested for their ability to grow on a
variety of defined carbon sources. The tests were
performed in MS medium supplemented with the test
compound as the sole carbon source. Substrates were
added to 6 ml MS medium in Balch tubes at a
concentration of 100 ppm and inoculated with 10
5
–
10
6
cells/ml of phosphate buffer (pH 7.2)-washed cells
pre-grown with 2.5 mM benzoate. Tubes were crimp-
sealed with Teflon-coated stoppers to prevent abiotic
losses and incubated horizontally on an orbital shaker
at 100 rpm. Substrates tested included all mono- and
dichlorobenzoic acid congeners, chloroacetic acid,
biphenyl, naphthalene and various congeners of chlo-
robenzenes and chlorobiphenyls. Stock solutions of all
substrates were autoclaved prior to use. Growth was
evaluated by microscopy and visual monitoring of
turbidity in conjunction with periodic GC and HPLC
analyses to measure test compound disappearance or
appearance of products. In these substrate-screening
studies, growth was scored as positive if turbidity was
notably greater than in controls lacking the test
compound, microscopic examination revealed an
increase in cell numbers, and GC or HPLC analysis
showed loss of the test compound. Growth tests were
conducted in triplicate for each substrate.
In time course experiments, three replicate tubes
were sacrificed at each time point. Reactions were
stopped by adding 10 ml of hexane, vortexing for 1–
2 min and thereafter, mixing continuously on a tube
rotator for 12–24 h. The aqueous phase was centrifuged
(Hermle Z 180M Labnet) at 13,000 ·g, and filtered.
The hexane extracts and aqueous phases were sepa-
rately collected for analysis. Growth of the cultures
was monitored by a direct counting method using
acridine orange staining (Kepner et al. 1994).
Transformation of Aroclor 1221
Experiments to study degradation of the commercial
PCB mixture, Aroclor 1221, were similarly con-
ducted in Balch tubes. All tubes were inoculated with
respective bacterial cultures, crimp-sealed and incu-
bated horizontally with shaking at room temperature.
Transformation reactions were stopped after 12 days
by the addition of 10 ml hexane. To assess the
dynamics of degradation and release of chloride, the
entire contents of triplicate tubes were sacrificed and
analysed. Percent degradation was calculated as the
decrease in total summation of all ECD (electron
capture detector) area counts. The percentage of
chloride released from the Aroclor mixture was
determined based on percent composition of chlorine
in the mixture. For instance, Aroclor 1221 was
assumed to contain a chlorine content of 21%.
Analytical methods
GC analysis
Hexane extracts were analysed on an HP 5890 series
II gas chromatograph (GC) (Hewlett Packard Co.,
148 Biodegradation (2008) 19:145–159
123
Palo Alto, CA, USA) fitted with an HP 3396 series II
Integrator, and an ECD. Hexane extracts (1 ml)
injected using a 10-ml Hamilton syringe was carried
through a 30 m DB-5 megabore fused-silica capillary
column (J & W Scientific, Folsom, CA, USA;
0.53 mm id, 2.5 mm film thickness) coated with 5%
phenyl substituted methylpoly siloxane stationary
phase to the ECD. The carrier gas was helium, with a
linear velocity of 30 cm/s at 100 8C. The injector and
detector temperatures were 200 and 275 8C, respec-
tively. The GC was programmed at an initial
temperature of 70 8C; this was held for 1 min, then
ramped at 30 8C/min to 160 8C, ramped to 200 8Cat
28C/min for 1 min, and then held for 14 min.
Analytical standards of PCBs were prepared in
hexane at concentration range of 0.05–1 mM. Typical
coefficients of correlation for standard curves were
0.95–0.99.
HPLC analysis of CBAs and chloride
Chlorobenzoate metabolites in the aqueous phase
were analysed by HPLC (Waters Corp., Milford, MA,
USA) equipped with a UV, dual absorbance detector
(Model 2487). Separation was performed on a YMC-
Pack ODS-AQ reversed-phase column (YMC Co.
Ltd., Kyoto, Japan) at 308C by isocratic elution with a
mobile phase consisting of a mixture of 25% phos-
phate buffer (50 mM, pH 2.5), 25 of 50% methanol,
and 50% acetonitrile at a flow rate of 0.7 ml/min.
Injection volume was 10 ml; chlorobenzoates were
monitored by UV detection (k
238 nm
) and identified
with reference standards by retention time.
Chloride was measured using the above HPLC
equipped with a conductivity detector (Model 432).
Separation was performed on an IonPac AS17
analytical column (4 ·250 mm; Dionex) which
was preceded by an AG17 guard column at a flow
rate of 1.2 ml/min. Eluent composition was 100%
5 mM NaOH. Injection volume was 10 ml. A
calibrated check standard and blank were run with
each sample test.
Statistical analysis
All statistical tests were performed using the Prism
2.01 computer software programme (GraphPad Soft-
ware, San Diego, CA, USA).
Nucletotide sequence accession numbers
The 16S rRNA gene sequences determined in this
study have been deposited in the GenBank database
under accession numbers DQ854840 through
DQ854845 as shown in Table 1.
Results
Taxonomic characteristics of PCB-utilizing
isolates
A total of 150 different microbial colonies were
selected from nutrient agar plates following initial
enrichment on Askarel oil and Aroclor 1221. Upon
screening individual isolates for growth on PCBs or
CBAs, we obtained six isolates for further study.
Initial partial characterization was carried out with
API 20 E test kit. A positive identification was
established for each isolate genotypically by cloning
Table 1 Genotypic identities of PCB-degrading bacterial isolates obtained from cloned sequences of 16S rDNA fragment of
genomic DNA
Bacterial
strain
Tentative identity Origin Closest relative Bacteria
subdivision
%
ID
1
GenBank
accession no.
Length
(nt)
2
SA-1 Pseudomonas
aeruginosa
Ijora Pseudomonas aeruginosa strain
SCD-13
c-Proteobacteria 99 DQ854840 1,304
SA-2 Enterobacter sp. Ijora Enterobacter aerogenes c-Proteobacteria 98 DQ854842 1,376
SA-3 Ralstonia sp. Ojota Ralstonia sp. AV5BG b-Proteobacteria 99 DQ854843 1,322
SA-4 Ralstonia sp. Ijora Ralstonia sp. BPC3 b-Proteobacteria 98 DQ854844 1,364
SA-5 Ralstonia sp. Ijora Ralstonia sp. BPC3 b-Proteobacteria 99 DQ854845 1,364
SA-6 Pseudomonas sp. Ojota Pseudomonas sp. Bu34 c-Proteobacteria 99 DQ854841 1,363
1
ID identity,
2
nt nucleotides
Biodegradation (2008) 19:145–159 149
123
and sequencing PCR-amplified 16S rRNA gene
fragments. A significant proportion of sequences in
the libraries were highly similar to those of the
bacterial genus b-proteobacteria of Ralstonia while
others were c-proteobacteria of the genera Enterob-
acter or Pseudomonas (Table 1). The strains were all
Gram-negative, motile short to medium rods, non-
H
2
S producers, b-galactosidase-, lysine decarboxyl-
ase-, VP- and oxidase-negative (except SA-1 and SA-
6) and were non-sugar fermenters with the exception
of SA-1 and SA-6 that fermented arabinose. Isolate
SA-1 had a clone sequence type similar to the 16S
rRNA gene sequence of species of Pseudomonas
aeruginosa strain BPC3. In addition, the colonies
were flat, spreading and had green colour pigmenta-
tion that diffused relatively quickly. Isolate SA-6, to a
large extent, showed similar morphological and
phenotypic traits as strain SA-1. Unlike SA-1, the
cells utilized glucose in addition to arabinose. It also
differed from SA-1 in that citrate utilization was
positive. Dynamics of nitrate reduction varied mark-
edly. SA-6 reduced nitrate to gaseous nitrogen while
SA-1 reduced same to nitrate. SA-2 was 98%
homologous to Enterobacter aerogenes 16S rRNA
gene sequence. The three isolates, SA-3, SA-4 and
SA-5 were identical in their cultural (medium,
circular, smooth and elevated colonies) and cellular
morphologies, but differ in their 16S rRNA gene
sequences. Whereas, SA-3 hydrolysed gelatine and
utilized citrate, the other two did not. Nitrate
reduction differed among the three strains; SA-3
and SA-5 reduced nitrate to nitrite, while only SA-4
reduced it to gaseous nitrogen.
Substrate diversity of bacterial strains
The growth of these isolates on PCB congeners and
other environmental pollutants as sole carbon sources
is summarized in Table 2. All the strains showed
visible signs of growth on non-chlorinated organic
compounds, e.g. benzoate, within 12 h of incubation.
In addition, they were able to utilize all CB isomers
quite rapidly, though growth patterns varied for
different isolates. Interestingly, growth was observed
on some diCBs including 2,3-, 2,20-, 2,40-, 3,30-
and .3,5-diCB. In some instances, the bacterial
growth was accompanied by the production of yellow
metabolites. Production of this metabolite was occa-
sional and/or persisted in some strains, while in
others, it disappeared with time suggesting that the
yellow product was further subjected to metabolism.
The yellow–green colour observed could be an
indication of meta-cleavage product—hydroxyl 6-
oxo-6-penta 2,4-dienoic acid (HOPDA) in the case of
PCBs and 2-hydroxymuconic semialdehyde in the
case of benzoate. All di- and trichlorobenzenes tested
were good growth substrates while tetrachloroben-
zene isomers supported no growth. Similarly, the
strains failed to utilize all mono- and dichlorobenzo-
ate congeners tested. Furthermore, the six strains
grew heavily on Askarel fluid producing intense
turbidity of the culture media. Similar results were
also observed when Aroclor 1221 was supplied as the
sole source of carbon and energy.
Growth of bacterial strains on CBs
Since PCBs and other sparingly soluble substrates
were dissolved in HMN, there was the need to
establish that any observed growth was due to the
presence of the test substrate rather than the HMN.
When HMN alone was added as the only carbon
source in preliminary experiments (data not shown),
there was a slight to moderate increase in the cell
number of some isolates. Over the course of the
experiments, this increase was relatively small for
SA-4 and SA-6, while no appreciable growth was
observed for SA-3 and SA-5. In the case of SA-2,
however, an almost tenfold increase in cell numbers
was observed at one time-point, possibly due to
continued cell division by the inoculum, continued
utilization of endogenous substrates, or a transient
artefact of our counting method. This increase was
obtained only after incubating the cells for more than
120 h, was not observed at the next sampling period,
and the overall results indicated no major cell
increase on HMN in the absence of CBs. In all cases
where growth occurred on the test substrate, cell
numbers increased by at least 1.5 orders-of-magni-
tude more than in tubes containing the HMN carrier
alone, clearly demonstrating growth on the test
compound.
All three CBs were readily degraded by the six
strains and respective CBAs were recovered as
primary metabolites. Results are summarized in
Table 3. The growth profiles of the strains on CBs
showed a logarithmic population increase without
exhibiting any lag period. This was of significance, in
150 Biodegradation (2008) 19:145–159
123
that organisms were not pre-cultivated on biphenyl
that would have induced requisite enzymes of the
degradative pathways. All organisms exhibited sim-
ilar growth dynamics on 2-CB, resulting in 100-fold-
cell increase during the 66-h degradation studies
while the mean generation times ranged insignifi-
cantly (P< 0.05) from 8 to 10 h (Table 3). Although
nearly all 2-CB was utilized within the incubation
period, recovery of 2-CBA was non-stoichiometric
(Table 3, Fig. 1). Interestingly, some chloride was
dissociated from the ortho-chlorinated ring. Between
0.1 and 0.2 mM chloride were released into culture
fluids. However, carbon–chlorine cleavage was not
detected in cultures of SA-6 growing on 2-CB until
after 30 h of incubation (Fig. 1c). Moreover, a
reduction in 2-CBA observed in strains SA-2, SA-4
and SA-6 (see Fig. 1) at 50 h suggest that 2-CBA may
not be the terminal product of 2-CB metabolism.
Metabolism of 3-CB followed a similar trend as
2-CB. Population densities increased several
Table 2 Substrate utilization spectrum of PCB-degraders
Substrate SA-1 SA-2 SA-3 SA-4 SA-5 SA-6
Biphenyl
2
2
Benzoate
1
1
1
2
2
2
Naphthalene
2
Aroclor 1221
1
1
1
1
Aroclor 1242
Askarel fluid
2-CB
1
1
1
2
3-CB
1
1
4-CB
1
2
1
2
2,3-diCB NT
2,4-diCB –––––
2,6-diCB –––––
3,5-diCB NT –
3
–
2,2’-diCB
2
2,3’-diCB –––––
2,4’-diCB
3
3
3,3’-diCB –
4,4’-diCB ––––––
All monochlorobenzoates ––––––
All dichlorobenzoates ––––––
1,2-dichlorobenzene
1,3 –dichlorobenzene
1,4-dichlorobenzene
1,2,3 -trichlorobenzene
1,2,4-trichlorobenzene
1,3,5 -trichlorobenzene
1,2,3,4-tetrachlorobenzene NT –––––
1,2,3,5 -tetrachlorobenzene NT –––––
1,2,4,5 -tetrachlorobenzene NT –––––
Chloroacetate ––––––
, Growth; –, no growth; NT, not tested. Culture supernatant fluid turned a yellow colour that was;
1
permanent;
2
disappeared with
time and;
3
occasional
All compounds were supplied as sole carbon sources in MS medium at 100 ppm except benzoate presented at a concentration of
2.5 mM. All compounds with the exception of benzoate and chloroacetate were supplied in a HMN carrier.
Biodegradation (2008) 19:145–159 151
123
Table 3 Degradation of monochlorobiphenyl isomers and production of metabolites by tropical bacterial strains
Bacterial
strain
2-CB 3-CB 4-CB
Tg
(h)
%
Degradation
2-CBA
recovered (%)
Chloride
released (%)
Tg
(h)
Degradation
(%)
3-CBA
recovered (%)
Chloride
released (%)
Tg
(h)
Degradation
(%)
4-CBA
recovered (%)
Chloride
released (%)
SA-2 8 96 ± 5 34 ± 2.7 27 ± 13.6 7 91 ± 7.5 30 ± 8.2 0 9 88 ± 9.1 98 ± 5.3 0
SA-3 8 99 ± 1.3 49 ± 2.4 43 ± 6.8 9 98 ± 3.6 11 ± 3.8 11 ± 2.9 9 90 ± 2.1 96 ± 36 0
SA-4 10 99 ± 1.2 34 ± 5.7 39 ± 2.3 6 97 ± 1.3 19 ± 2.6 28.6 ± 5.7 9 97 ± 1.31 73 ± 13 0
SA-5 8 88 ± 16 22 ± 16 14 ± 6.8 8 86 ± 9.2 64 ± 19 23 ± 14.3 9 98 ± 1.4 92 ± 52 0
SA-6 10 94 ± 0.99 20 ± 8.8 50 ± 22.7 7 93 ± 7.7 6.4 ± 2 14 ± 22.779 10 90 ± 6.3 85 ± 13 0
Tg, mean generation time. All values are means ± SD for triplicate cultures. Percent degradation values have been calculated with reference to the amount recovered from
uninoculated control tubes. 2-CB, 3-CB and 4-CB were supplied at concentrations of 0.44, 0.35 and 0.66 mM, respectively
Fig. 1 Time course for 2-CB metabolism and production of
benzoate and chloride by strains SA-2 (a), SA-4 (b) and SA-6
(c). j, 2-CB concentration in experimental tubes; h,CB
concentration in non-inoculated controls; m, 2-CBA; open
triangular, Chloride; d, log cell number. In the controls
without cells, CBs were not metabolized and minimal abiotic
loss occurred. Data represent the averages and SD of triplicate
determinations. Large error bars were due to differential
response of cells to substrate in replicate tubes
152 Biodegradation (2008) 19:145–159
123
orders-of-magnitude (Fig. 2) with a doubling time in
the range of 6 and 9 h (Table 3). More than 90% of
the 0.35 mM 3-CB was removed by all cultures after
60 h exposure though, as was observed with 2-CB,
non-stoichiometric amounts of 3-CBA (in the range
of 6.4–64% of the expected CBA) were produced as
the main metabolite. Elimination of minor amounts of
chloride was observed in all the strains with the
exception of SA-2 (Table 3, Fig. 2). Since the sum of
chloride and chlorobenzoate products do not account
for all of the 3-CB consumed, this indicated that
unmeasured chlorinated metabolites were produced.
In the case of SA-5 chloride release became
noticeable only after 36 h (Fig. 2b), suggesting slow
metabolism of chlorinated intermediates. Neverthe-
less, partial elimination of chloride presents the
possibility that both rings can potentially be
metabolized.
In contrast to what was observed on 2-CB and
3-CB, time-course degradation of 4-CB yielded near
stoichiometric production of 4-CBA and mirrored the
disappearance of substrate in the medium (Table 3,
Fig. 3). In the case of SA-3, SA-4, SA-5 and SA-6, an
early indication of 4-CB metabolism was a transient
change in the colour of the culture media to bright
yellow. The lack of chloride production together with
near stoichiometric CBA recovery indicates that the
extensive growth observed occurred solely on the
non-chlorinated ring.
Although we observed differences in patterns of
congener degradation and product formation by the
various bacteria, all isolates were clearly capable of
CB degradation on similar time scales. Analyses of
variance showed no significant difference at P< 0.05
level of significance in the degradation capabilities of
the test organisms.
Degradation of PCB Aroclor 1221
Degradation of Aroclor 1221 was evaluated using
washed, benzoate-grown cells. No carbon sources
other than the PCB commercial mixture were
provided. Growth on this mixture was evidence by
intense turbidity of the culture media and significant
reduction in concentration of the PCB substrate.
Values for net reduction (percent reduction in total
ECD area counts) in total PCB content were 51, 53,
54, 68, 71 and 66%, respectively, for SA-1, SA-2,
SA-3, SA-4, SA-5 and SA-6 (Fig. 4). Since Aroclor
Fig. 2 Time course for 3-CB metabolism and production of
benzoate and chloride by strains SA-2 (a), SA-5 (b) and SA-6
(c). j, 3-CB concentration in experimental tubes; h,CB
concentration in non-inoculated controls; m, 3-CBA; 4,
Chloride; d, log cell number. In the controls without cells,
CBs were not metabolized and minimal abiotic loss occurred.
Data represent the averages and standard deviations of
triplicate determinations. Large error bars were due to differ-
ential response of cells to substrate in replicate tubes
Biodegradation (2008) 19:145–159 153
123
1221 contains a significant amount of biphenyl
(*15% by weight), some of the observed disappear-
ance may have resulted from cometabolic degrada-
tion by cells growing on biphenyl. Our isolates,
however, were able to grow on all three CBs and
some di-CBs as a sole source of carbon and energy
and it is more likely that degradation occurred during
growth on the high percentage of CBs and di-CBs
(> 80%) present in Aroclor 1221. This inference is
further corroborated by preliminary studies that
showed extensive depletion of some congeners in
Aroclor 1242 (which contains only trace amounts of
biphenyl) without the addition of biphenyl (data not
shown) and the ability to utilize a broad and unusual
spectrum of PCB and chlorobenzenes congeners as
growth substrate (Table 2). Chloride produced during
Aroclor 1221 degradation ranged from 0.10 to
0.25 mM representing 15–43% of the chloride in
Aroclor 1221. There was a colour change from
colourless to yellow observed in culture media of
SA-1, SA-2, SA-3 and SA-5. It is noteworthy that this
meta-cleavage product persisted throughout the
incubation period (12 days).
The extent of degradation of individual PCB
congeners in Aroclor 1221 is tabulated in Table 4.
Although unambiguous identification of the individual
peaks with the exception of monochlorobiphenyls was
Fig. 3 Time course for 4-CB metabolism and production of
benzoate by strains SA-2 (a), SA-4 (b) and SA-6 (c). j, 4-CB
concentration in experimental tubes; h, CB concentration in
non-inoculated controls; m, 4-CBA; d, log cell number. In the
controls without cells, CBs were not metabolized and minimal
abiotic loss occurred. Data represent the averages and standard
deviations of triplicate determinations. Large error bars were
due to differential response of cells to substrate in replicate
tubes
Fig. 4 Transformation of Aroclor 1221, hand chloride
eliminated, jPercent degradation represents the net decrease
(in ECD area counts) in experimental cultures, compared with
that of the non-inoculated controls. No chloride was detected in
control tubes. Values presented are means ± SD for triplicate
cultures. The PCB mixture was supplied at a concentration of
100 ppm and incubated with each organism for 12 days
154 Biodegradation (2008) 19:145–159
123
not possible with available instrumentation due to
co-elution of some congeners, the degree of chlorine
substitution was assigned based on the relative reten-
tion times of eluting peaks and comparison with
retention times of selected mono- through hexachlo-
robipenyl standards. Congeners including those
assigned to more than three chlorine substituents were
extensively attacked. Degradation generally did not
follow a particular fashion. One would have expected
complete disappearance of congeners containing
between one and two chlorine atoms, but quite
surprisingly, extensive transformation was accom-
plished for congeners with more than three chlorine
substituents with degradation values higher than lesser
chlorinated congeners in some cases. In most cases,
strains SA-4, SA-5 and SA-6 depleted most congeners
displayed in Table 4by a minimum of 64.4% and a
maximum of 100%.
Discussion
One of the best ways to achieve the isolation of
micro-organisms with specific metabolic capabilities
is enrichment with substrates contaminated with the
target compound. For this approach to be particularly
useful, the target compound must serve as a potential
carbon source. Acclimation or prior exposure to the
compound may enhance the organism’s metabolic
capabilities. In the present study, microbial commu-
nities from contaminated soils were enriched on
Askarel as well as Aroclor 1221. In the case of
Askarel enrichment, more than 90% of the organisms
obtained could not grow on CBs, which likely
suggests that the bulk of the isolates may only be
marginally effective in stimulating the cometabolism
of chlorobiphenyls.
Results of this investigation have confirmed that
cultures isolated from contaminated sites in Nigeria
exhibited both the chlorobiphenyl and chlorobenzene
biodegradation phenotypes. The HMN used as a
carrier did not support significant or sustained
increases in cell numbers in the absence of CBs or
other added substrates. Since we don’t expect induc-
tion of aromatic ring oxygenases by this highly
branched alkane, we believe that HMN functioned as
intended, i.e. to facilitate mass transfer of CBs into
the aqueous phase. We cannot completely discount,
however, the possibility that HMN influenced our
results in an unknown manner. As summarized in
Table 2, all the bacterial strains isolated possess
unique ability to assimilate a diverse range of lightly
chlorinated PCB and chlorobenzene congeners when
Table 4 Analysis of the transformation of Aroclor 1221 by microbial strains
Retention
time (min)
Peak
number
Number of
chlorines
Percent degradation
SA-1 SA-2 SA-3 SA-4 SA-5 SA-6
7.65 1 1 100 (0) 100 (0) 78.3 (8.5) 96.3 (0.7) 91.9 (0.9) 81.6 (13.4)
8.66 2 1 72.2 (3.5) 61.7 (54.1) 60.0 (23.5) 100 (0) 100 (0) 100 (0)
8.78 3 1 61.8 (10.2) 47.0 (24.0) 66.5 (19.4) 98.3 (2.4) 94.5 (2.1) 80.1 (10.0)
9.48 5 2 59.0 (14.7) 60.9 (9.8) 74.1 (13.5) 77.9 (21.9) 83.1 (6.9) 77.1 (7.7)
10.38 6 2 60.9 (12.6) 72.3 (19.3) 74.9 (13.6) 73.6 (26.9) 85.5 (8.8) 80.1 (12.0)
10.8 8 2 62.0 (11.1) 73.0 (19.4) 76.2 (25.3) 94.2 (5.4) 85.7 (9.3) 80.0 (12.0)
11.05 9 2 59.3 (11.8) 71.8 (19.7) 63.6 (17.6) 95.6 (5.7) 95.8 (0.6) 95.6 (5.7)
13.1 15 2,3 40.2 (10.0) 60.9 (27.4) – 76.0 (19.5) 74.4 (12.1) 64.4 (7.4)
13.96 18 3 59.8 (6.2) 63.4 (11.8) 54.5 (27.1) 80.4 (19.6) 81.8 (10.0) 80.2 (8.6)
15.48 23 3,4 56.2 (10.6) 63.3 (11.0) 67.7 (21.0) 79.9 (17.7) 78.6 10.0) 79.7 (8.2)
16.04 24 4 73.3 (6.2) 73.7 (6.3) 43.5 (17.8) 87.0 (10.5) 94.5 (6.8) 92.8 (7.9)
16.49 25 >3 56.9 (11.1) 74.2 (15.4) 64.0 (22.3) 72.8 (16.0) 83.3 (5.7) 81.1 (7.1)
Percent degradation represents the net decrease (in ECD area counts) in experimental cultures, compared with that of the non-
inoculated controls. Values presented are means of triplicate samples, while those in parentheses represent percent standard
deviations. The PCB commercial mixture was supplied at a concentration of 100 ppm and incubated with each organism for 12 days.
Chlorine number assignment was based on retention time on a DB-5 megabore capillary column. Blanks are where results could not
be ascertained
Biodegradation (2008) 19:145–159 155
123
supplied as sole carbon and energy sources. The
ability of our isolates to utilize both chlorobiphenyls
and chlorobenzenes is quite rare and unusual. Since
Askarel was the enrichment substrate for isolation
(with the exception of SA-1 that was enriched using
Aroclor 1221), the technique may have selected for
those organisms with capacity for both PCB and
chlorobenzene phenotypes. In addition, it has been
noted that enzymes of PCB pathways may not only
transform PCBs and their metabolites, but also other
related compounds, such as monocyclic aromatics
(Pellizari et al. 1996; Suenaga et al. 2001) and vice
versa. Suenaga et al. (2001) engineered the bphA1
gene of Pseudomonas pseudoalcaligenes KF707 and
obtained some novel biphenyl dioxygenase that
exhibited multifunctional oxygenase activity not only
for PCBs but also for several xenobiotics including
polyaromatic hydrocarbons (PAHs). In an earlier
study, Pellizari et al. (1996) reported that several
bacterial species isolated by naphthalene enrichment
have PCB-metabolizing ability and concluded that
the former (and perhaps other aromatic co-contam-
inants) may be marginally effective in stimulating the
cometabolism of PCBs. Generally, this substrate
overlap means that other pollutants in a site
may act as co-substrates that can influence the
competitiveness and activity of PCB-metabolizing
communities.
Of significance is the substrate diversity of Ente-
robacter sp. strain SA-2. The ability of an enteric
organism to grow with either PCBs or chlorobenz-
enes or both is an uncommon phenomenon, even
though dechlorination of DDT in pure systems of E.
aerogenes,E. cloacae and Escherichia coli has been
described (Aislabie et al. 1997; Juhasz and Naidu
2000). The well characterized and widely studied
organisms, Alcaligenes eutrophus H850, Pseudomo-
nas testosteroni H430, Corynebacterium sp. MB1 and
Burkholderia sp.LB400,havebeenreportedto
possess an exceptional ability to degrade even a
larger range of congeners including penta- and
hexachlorobiphenyl co-metabolically (Bopp 1986;
Bedard et al. 1987a,b; Commandeur et al. 1996;
Potrawfke et al. 1998). These other micro-organisms,
however, have not been shown to utilize the range of
xenobiotics reported for SA-2 (or any of the SA
strains) in this investigation for growth as sole carbon
sources (McCullar et al. 1994; Kim and Picardal
2001; Rodrigues et al. 2006).
It is possible that CBA-degrading bacteria are rare
to all the six contaminated soils examined, since no
growth was possible on all CBAs tested. This may
have been a result of the fact that we did not utilize
CBAs as enrichment substrates. It may also reflect an
observation by Knackmuss (1984) that the frequency
of CBA-utilizers to benzoate-utilizers was about one
per million. In another report, only three out of 42
bacterial strains isolated by enrichment on biphenyl
could grow on 3-CBA (Hernandez et al. 1995). In
addition, the investigators detected no CBA-utilizers
in all three soils analysed by direct-plating of diluted
soil samples onto MS agar supplemented with
monochlorobenzoates. In spite the fact that CBA-
degrading micro-organisms have been well-studied,
they may be relatively scarce in the environment. It is
interesting to note that the 3-CBA degrader reported
by Dorn et al. (1974) required several months of
adaptation to the substrate. Similarly, Marks et al.
(1984) documented an adaptive period of 4 months
before a 4-CBA-degrader could be isolated.
Unlike LB400 and H850 (Bedard et al. 1987a;
Potrawfke et al. 1998), our data clearly established
that all three CBs, 2-, 3- and 4-CB were rapidly
degraded by all the isolates on the basis of growth
studies and disappearance of substrates and appear-
ance of respective metabolites. The layout of growth
patterns indicated exponential increase in cell density
(Figs. 1,2,3) that was similar to previous report by
Kim and Picardal (2000). Having pre-grown the
organisms on benzoate rather than biphenyl, the
observation suggests constitutive expression of the
enzymes of the upper biphenyl degradative pathway.
Growth of isolates on 2-CB and 3-CB resulted in non-
stoichiometric production of 2-CBA and 3-CBA,
respectively, concomitant with substantial release of
chloride from the substituted rings of these isomers.
On the contrary, the bacterial strains stoichiometri-
cally transformed 4-CB to 4-CBA, with no detectable
chloride (Table 3). The production of 4-CBA indi-
cates that the bacterial dioxygenase attacked the
unsubstituted aromatic ring. Early indication of
utilization of the PCB substrates was a change in
colour of the culture media to yellow (Table 2),
suggesting the operation of a meta-cleavage pathway
for the degradation of CBs similar to other PCB-
degrading organisms. Similar results have been
reported by Commandeur and Parsons (1990) and
Bevinakatti and Ninnekar (1993). In the case of
156 Biodegradation (2008) 19:145–159
123
4-CB, the yellow metabolite was apparently produced
by the meta-cleavage of 2,3-dihydroxy 4-chlorobi-
phenyl which was further transformed into 4-CBA
and 5-C fragment by enzymatic hydrolysis (Kobay-
ashi et al. 1996; Omori et al. 1986; Ahmed and Focht
1973). Since the strains could neither grow on nor
oxidize 4-CBA, therefore, the growth on 4-CB
seemed to depend solely on utilizing the 5-C
fragment as the carbon source. Furthermore, the
absence of chloride in the reaction tube strongly
suggests that the failure of these strains to mineralize
4-CB may be due to the lack of or inhibition of
enzymes effecting dehalogenation.
Several workers have shown that isolates able to
grow on CBs as sole carbon and energy sources
usually metabolise the non-chlorinated ring to pro-
duce a CBA or other chlorinated compound as a
product (Furukawa and Chakrabarty 1982; Barton
and Crawford 1988; Kobayashi et al. 1996). The
exceptions to this rule were the reports of Potrawfke
et al. (1998), Kim and Picardal (2000) and the results
obtained in the present study during growth on 2-CB
and 3-CB. Besides significant quantities of chloride
released during metabolism of these isomers, espe-
cially during growth of SA-6 on 2-CB (Table 3), the
incomplete recovery of 2-CBA and 3-CBA observed
in all strains implied susceptibilities of both 2- and
3-chlorophenyl rings to attack. CBAs may, after all,
not be the final product of chlorobiphenyl metabolism
by the organisms.
Since the sum of chloride and chlorobenzoate
products do not account for all of the 2-CB and 3-CB
consumed, our data suggests accumulation of un-
known, chlorinated metabolites. Indeed, incomplete
degradation of PCBs is frequently observed with the
formation and accumulation of different intermedi-
ates besides CBAs (Bruhlmann and Chen 1999;
Arensdorf and Focht 1994; Fava and Marchetti 1991).
It is also possible that the CBAs were transformed to
other chlorinated compounds such as chlorocatechol,
thus escaping detection. This inference is further
supported by the fact that limited production of
CBAs, e.g. production of 3-CBA in Fig. 2, did not
always yield a corresponding increase in chloride
concentration. Conversion of 3-CB to 3-chlorocate-
chol (3-CC) and 4-CC was previously reported by
Fava and Marchetti (1991). It is possible that
chlorocatechols are able to reduce the catechol
dioxygenase activity resulting in suicide inactivation
of the entire pathway. In fact, other reports show that
the formation of chlorocatechol in the medium can be
the cause of the incomplete degradation of 3-CBA
(Haller and Finn 1979). Arensdorf and Focht (1994)
also documented that 3-CC interfered with the
utilization of CBs as a carbon by Pseudomonas
cepacia P166 by arresting biphenyl transformation.
The authors proposed a likely mechanism for this
phenomenon as the inactivation by 3-CC of 2,3-
dihydroxybiphenyl 1,2-dioxygenase.
Data from transformation of Aroclor 1221 further
illustrate the notable catabolic properties displayed
by our isolates. Since congeners assigned to more
than 3 substituents were sometimes degraded more
extensively than those assigned to 1 or 2 chlorine
substituents, it is possible that the extent of chlorine
substitution, by itself, may not determine the effec-
tiveness of oxidative attack by the bacterial enzyme
system. Further work, however, is necessary to
determine the relative importance of chlorination
extent and ring position on CB degradation by our
isolates. More importantly, degradation of Aroclor
1221 and components by Enterobacter sp. strain SA-
2, an enteric organism is novel. To our knowledge,
this is the first substantial report of degradation of
PCB mixture by this organism unknown to have
xenobiotic degradative capability. Generally, there is
paucity of information on the release of chloride
during metabolic transformation of PCB mixtures by
biphenyl-oxidizing bacteria, presumably because
they are unable to dehalogenate the chlorinated
products. In our own case, we were able to recover
significant amounts of chloride. This would mean that
some congeners were either completely mineralized
or transformed to CBAs or other products with
concomitant chloride release.
In summary, results of our investigation have
shown the occurrence of multiple bacterial strains
with PCB catabolic competence in Nigerian contam-
inated soils. This conclusion is supported by several
lines of evidence. First, isolates were capable of
growth on CBs and diCBs, particularly ortho-substi-
tuted congeners known for their recalcitrance to
aerobic and anaerobic biodegradation. Second, Aroc-
lor 1221 was extensively degraded and possibly
partially mineralized. It is noteworthy that organisms
were pre-grown on benzoate prior to inoculation into
PCB supplemented MS medium and that CB degra-
dation occurred without a notable lag period, thus
Biodegradation (2008) 19:145–159 157
123
suggesting either that the enzymes of the upper
biphenyl/PCB catabolic route are constitutively
expressed or that the biphenyl pathway is co-induced
with the benzoate pathway. The catabolic properties
of these strains with wider substrate spectrum could
be an important step in studying the specificity of
aromatic oxygenases, constructing new hybrids with
unique abilities, and developing effective PCB bio-
remediation strategies.
Acknowledgements Authors would like to acknowledge the
financial support of the ICSC-World Laboratory, Lausanne,
Switzerland and the School of Public and Environmental
Affairs, Indiana University, Bloomington, IN, USA. We thank
Nathan Grindle for assistance with 16S rRNA gene sequence
analysis and the anonymous reviewers for helpful comments
that improved the manuscript.
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