Pyrene-degradation potentials of Pseudomonas species isolated from polluted tropical soils

Abstract

Three Pseudomonas species isolated from oil polluted soils in Lagos, Nigeria were studied for their pyrene degradation potentials. These isolates
exhibited broad substrate specificities for hydrocarbon substrates including polycyclic aromatic hydrocarbons, petroleum fractions
and chlorobenzoates. All three isolates tolerated salt concentrations of more than 3%. They resisted ampicillin, cenfuroxime,
but susceptible to ofloxacin and ciprofloxacin. Pseudomonas sp. strain LP1 exhibited growth rates and pyrene degradation rates of 0.018h−1 and 0.111mgl−1h−1 respectively, while P.aeruginosa strains LP5 and LP6 had corresponding values of 0.024, 0.082 and 0.017, 0.067 respectively. The overall respective percentage
removal of pyrene obtained for strains LP1, LP5 and LP6 after a 30-day incubation period were 67.79, 66.61 and 47.09. Resting
cell assay revealed that strain LP1 had the highest uptake rate. Strains LP1, LP5, and LP6 also used the ortho-cleavage pathway. Enzyme study confirmed activity of catechol 1,2-dioxygenase in all with values 0.6823, 0.9199, and 0.8344μmolmin−1mg−1 respectively for LP1, LP3 and LP6. To the best of our knowledge, ours is the first report of pyrene-degraders from the sub-Saharan
African environment.

Full text

ORIGINAL PAPER
Pyrene-degradation potentials of Pseudomonas species isolated
from polluted tropical soils
Oluwafemi S. Obayori Æ Matthew O. Ilori Æ
Sunday A. Adebusoye Æ Ganiyu O. Oyetibo Æ
Olukayode O. Amund
Received: 29 February 2008 / Accepted: 2 June 2008 / Published online: 4 July 2008
Ó Springer Science+Business Media B.V. 2008
Abstract Three Pseudomonas species isolated from oil
polluted soils in Lagos, Nigeria were studied for their
pyrene degradation potentials. These isolates exhibited
broad substrate specificities for hydrocarbon substrates
including polycyclic aromatic hydrocarbons, petroleum
fractions and chlorobenzoates. All three isolates tolerated
salt concentrations of more than 3%. They resisted ampi-
cillin, cenfuroxime, but susceptible to ofloxacin and
ciprofloxacin. Pseudomonas sp. strain LP1 exhibited
growth rates and pyrene degradation rates of 0.018 h
-1
and
0.111 mg l
-1
h
-1
respectively, while P. aeruginosa strains
LP5 and LP6 had corresponding values of 0.024, 0.082 and
0.017, 0.067 respectively. The overall respective percent-
age removal of pyrene obtained for strains LP1, LP5 and
LP6 after a 30-day incubation period were 67.79, 66.61 and
47.09. Resting cell assay revealed that strain LP1 had the
highest uptake rate. Strains LP1, LP5, and LP6 also used
the ortho-cleavage pathway. Enzyme study confirmed
activity of catechol 1,2-dioxygenase in all with values
0.6823, 0.9199, and 0.8344 lmol min
-1
mg
-1
respectively
for LP1, LP3 and LP6. To the best of our knowledge, ours
is the first report of pyrene-degraders from the sub-Saharan
African environment.
Keywords Biodegradation 
Polycyclic aromatic hydrocarbons  Pollution 
Pseudomonas  Pyrene
Introduction
Petroleum is a major source of energy in the world today
and Nigeria is one of the major oil producing countries
(Amund et al. 1987). Polycyclic aromatic hydrocarbons
(PAHs) are present as natural constituents of fossil fuels
and are formed anthropogenically as a result of incomplete
combustion of organic materials. They are, therefore,
present in relatively high concentrations in refined products
of fossil fuel and in every environment where fossil fuels
and other organic materials are combusted. This, in com-
bination with global transport phenomenon, results in their
worldwide distribution (Kanaly and Harayama 2000).
Pyrene is a pericondensed, four-ring polycyclic aromatic
hydrocarbon (PAH). It is a regulated contaminant at sites
polluted with petroleum (Kazunga and Aitken 2000).
Interest in the biodegradation of PAHs such as pyrene is
spurred in part by its mutagenicity and shared structure
with carcinogenic PAH such as benzo(a)pyrene (Cerniglia
1992; Kanaly and Harayama 2000; Cheung and Kinkle
2001). Bacteria capable of degrading a wide range of
hydrocarbons abound in the soil environment. Whereas
microorganisms capable of utilising two- and three-ring
PAHs as sole carbon and energy sources have been well-
documented in the literature, until the late 1980s there was
no literature on microorganisms capable of using four and
five ring PAHs as sole carbon and energy sources (Heitk-
amp et al. 1988). Pyrene is mainly degraded by
actinomycetes such as Mycobacterium and Rhodococcus
(Grosser et al. 1991; Walter et al. 1991; Kastner et al.
O. S. Obayori (&)
Faculty of Science, Department of Microbiology, Lagos State
University, Ojo, Lagos, Nigeria
e-mail: femiobayori@yahoo.com
O. S. Obayori  M. O. Ilori  S. A. Adebusoye 
G. O. Oyetibo  O. O. Amund
Department of Botany and Microbiology, University of Lagos,
Akoka, Lagos, Nigeria
123
World J Microbiol Biotechnol (2008) 24:2639–2646
DOI 10.1007/s11274-008-9790-7

1994; Dean-Rose and Cerniglia 1996; Schneider et al.
1996; Kanaly and Harayama 2000; Derz et al. 2004).
However, there have also been reports of few non-actino-
mycete degraders of pyrene, particularly the pseudomonads
(Caldini et al. 1995; Bouchez et al. 1995). Sarma et al.
(2004) isolated a strain of the enteric bacterium Leclercia
adecarboxylata capable of degrading pyrene from oily
sludge contaminated soil. Das and Mukherjee (2007)
reported the pyrene-induced production of biosurfactant by
a Bacillus and two Pseudomonas species.
Because of the ubiquitous nature and recalcitrance of
pyrene, and the significance of this for bioremediation,
there is a growing interest in the ability of pyrene-
degrading strains to also degrade other related PAHs and
other components present in petroleum (Churchill et al.
1999). The role of pyrene-degrading organisms in the fate
of mixture of PAHs in the environment has also been the
subject of investigation by various workers (Trzesicka-
Mlynarz and Ward 1995; Juhasz et al. 1997). Vila et al.
(2001) identified a novel metabolite 6,6
0
-dihydroxy-2,2
0
-
biphenyl dicarboxylic acid resulting from the ortho cleav-
age of both central rings of pyrene. While Krivobok et al.
(2003) identified Pyrene-induced proteins in Mycobacte-
rium sp. strain 6PY1, thus suggesting the presence of two
ring-hydroxylating dioxygenases. It has been suggested
that products derived from pyrene transformation, partic-
ularly by pseudomonads and Bacillus species have the
potential to accumulate in PAH-contaminated sites and that
such products may significantly interfere with the removal
of other PAHs (Kazunga and Aitken 2000).
In spite of the existing body of knowledge on pyrene
metabolism, there is virtually no report of study of pyrene
degradation in the tropical African environment and in Nigeria
especially where gas flaring and unabated release by auto-
mobiles are loading the environment with cocktails of PAHs.
Improving on the available bank of microbial resources (iso-
lates) and information is crucial to the proper management of
petroleum-polluted sites. In this paper we report the isolation
and characterisation of three strains of Pseudomonas able to
grow on pyrene as sole source of carbon and energy.
Materials and methods
Sample sites
Soil samples were collected from three contaminated sites
MVOA, DGED and LAS. MVOA samples were collected
from a mechanic village at Egbeda, Lagos, with a long
history of contamination with spent engine oil, diesel,
gasoline and transformer fluid. DGED samples were col-
lected from a diesel generator exhaust-contaminated soil, at
Dopemu-Agege, Lagos. LAS is a diesel generator site soil,
dark and thick with soot. The samples were collected at a
depth of 10–12 cm with sterile trowel after clearing debris
from the soil surface.
Enrichment and isolation of pyrene-degrading bacteria
Bacteria able to degrade pyrene were isolated on pyrene
minimal salt medium (MSM) by continual enrichment
method (Churchill et al. 1999). The mineral medium
described by Kastner et al. (1994) was used. The medium
contains per litre Na
2
HPO
4
, 2.13 g; KH
2
PO
4
, 1.30 g;
NH
4
Cl, 0.50 g and MgSO
4
 7H
2
O, 0.20 g. The pH of the
medium for bacteria was adjusted to 7.2 and fortified with
nystatin at 50 lg/ml to suppress fungal growth. Trace
elements solution (1 ml per litre) described by Bauchop
and Elsden (1960) was sterilised separately and added
aseptically to the medium. Contaminated soil sample
(5.0 g) was added to 45 ml MSM containing 50 ppm of
pyrene. Enrichment was carried out with shaking
(175 rpm) for 4–5 weeks in the dark until there was tur-
bidity. After 5 consecutive transfers, pyrene degraders were
isolated by plating out dilutions from the final flask on
Luria–Bertani (LB) agar. Several of the colonies that
appeared were further purified by subculturing once onto
LB agar. Ability to degrade pyrene was confirmed by
inoculating washed LB broth grown culture (18 h) into
fresh MSM flask supplemented with 50 ppm pyrene as a
sole carbon source and observing (OD
500
of 0.2 was con-
sidered significant). Pure isolates were maintained at -
20°C in glycerol: LB broth medium (50:50).
Identification and characterisation of isolates
Pure cultures of bacterial isolates were identified on the basis
of their colonial morphology, cellular morphology and bio-
chemical characteristics according to the scheme of Cowan
and Steel (Barrow and Feltham 1995). This was comple-
mented by using the ID32 E system for the identification of
Enterobacteriaceae and other non-fastidious Gram-negative
rods. The ID32 E kit was used according to the manufac-
turer’s specifications. (Biomerieux Inc., Durham, NC, USA).
Antibiotic sensitivities of isolates were carried out using
multidiscs. Salt tolerance was tested in LB broth containing
NaCl ranging from 1 to 10% (w/v). Incubation was carried out
at room temperature (27 ± 2.0°C) for 2 weeks with shaking
and daily observation for growth as indicated by turbidity.
Substrate specificity
The ability of the isolates to grow on pure hydrocarbon
substrates was evaluated in liquid media amended with
50 ppm of respective hydrocarbons as a sole carbon and
energy source. Incubation was carried out under similar
2640 World J Microbiol Biotechnol (2008) 24:2639–2646
123

conditions as described above. Degradation was monitored
by cell increases and visual observation for turbidity. The
hydrocarbons tested include naphthalene anthracene,
phenanthrene, benzene, phenol, biphenyl and dibenzo-
thiophene. Isolates were also tested for growth on crude oil
and petroleum products. Liquid hydrocarbons were auto-
claved and separately added to sterile MSM at 0.1% (v/v).
Incubation was carried out for 21 days.
Evaluation of pyrene biodegradation
Pyrene degradation was carried out by inoculating replicate
250-ml flasks containing 50 ml of MSM already supple-
mented with pyrene as a sole carbon and energy source at
concentration of 100 ppm. Flasks were inoculated with
0.5 ml of MSM-washed 18 h LB agar-grown cells and
subsequently incubated with shaking at 150 rpm for
30 days at room temperature.
At each time point, entire replicate flasks were sacri-
ficed. Metabolic reactions were stopped with the addition
of hexane. Previous experiments in our laboratory have
shown that hexane extraction lyses cells and that identical
substrate recoveries are obtained using killed-cell controls
(Adebusoye et al. 2007a, b).The total viable counts (TVCs)
in each flask were determined after plating out aliquots of
appropriate dilutions onto nutrient agar.
Analytical methods
Residual pyrene was determined by a method modified
from Das and Mukherjee (2007). Residual pyrene was
extracted from culture (20 ml) with equal volume of hex-
ane. After vortexing hexane-culture mixture for 2 min, it
was centrifuged at 10,000g for 10 min to remove cell
debris and separate aqueous and organic phases. Hexane
extract was concentrated and re-constituted in 20 ml of
hexane. Pyrene concentration was determined on a 6305
UV/Vis Spectrophotometer at 335 nm and the amount of
pyrene read on a standard curve. Control tubes inoculated
with heat-killed cells were similarly treated.
Alternatively, undegraded pyrene was quantified using
gas chromatographic (GC) analysis in order to validate the
results obtained from spectrophotometric method described
above. Residual pyrene was extracted according to the
method described by Sarma et al. (2004). Briefly, culture
(20 ml) was extracted once with an equal volume of tolu-
ene and then twice with an equal volume of chloroform.
After the solvents were vented off, the residual pyrene was
dissolved in acetone, and concentrated to 2 ml. Pyrene
concentrations in the acetone were determined using a
Hewlett Packard 5890 Series II gas chromatograph equip-
ped with flame ionization detector (FID) and 30 m long
HP-5 column (internal diameter, 0.25 mm; film thickness,
0.25 lm). The injector and detector temperatures were
maintained at 300 and 320°C respectively. The column
temperature was programmed to rise from 60 to 500°C for
27 min. The GC was programmed at an initial temperature
of 60°C; this was held for 2 min, then ramped at 12°C/min
to 205°C and held for 16 min.
Resting cell assay
Pyrene uptake by resting cells was measured spectropho-
tometrically as described by Stringfellow and Aitkens
(1995). Pyrene-grown cells were harvested by centrifuga-
tion and washed twice in 50 mM KH
2
PO
4
buffer (pH 7.2).
Cells were resuspended in buffer (20 mM phosphate buffer
containing 150 M NaCl, pH 7.0) to a final volume of
3.0 ml and O.D
600
of 1.0 in a 3.5 ml quartz curvette in a
UV/Vis spectrophotometer (Jenway 6305). About 60 ng of
pyrene (in 10 ll of acetone) was injected into the curvette.
Decrease in A
273
was measured from 0.5 to 30 min post-
addition of pyrene. Acetone without pyrene served as
control. A second control was set up for heat-killed cells to
check decreases in pyrene concentration resulting from
adsorption. Decrease in pyrene content was calculated from
a standard curve of pyrene and result expressed as nano-
gram of pyrene uptake by 2.0 9 10
7
bacterial cells.
Detection of ring-fission enzymes
The methods of Stanier et al. (1966) and Rothera (1908)
were used thus: bacterial strains were grown on pyrene (0.1
w/v, MSM) for 15 days. Cells were harvested from 30 ml
broth culture by centrifugation (10,000g for 10 min, 4°C).
Cells were resuspended in 4 ml Tris–HCl buffer (0.02 M
pH 8.0) in a test tube. Catechol solution (4 ml, pH 8.0,
0.01 M) was then added to the mixture and shaken for
5 min. The appearance of a yellow colour within a few
minutes as a result of formation of 2-hydroxymuconic
semi-aldehyde indicated meta-fission. When yellow colour
failed to appear the mixture was shaken for additional 2 h
at 30°C and tested for b-keto-adipic acid by the method of
Rothera (1908) thus: a small quantity of Rothera’s reagent
was added to the bottom of a test tube. A few drops of the
test liquid were added to the powder with a Pasteur’s
pipette just to moisten it. The mixture was left for 1 min for
the colour to develop. Appearance of a purple colour
within 1 min indicated ortho-cleavage. The test tube was
held against a sheet of white paper so that any colour
change could be quickly noticed.
Preparation of cell extracts and enzyme assay
Cell extract was prepared according to the method of
Phillips et al. (2001). About 2 ml of bacterial culture was
World J Microbiol Biotechnol (2008) 24:2639–2646 2641
123

centrifuged and the pellet was resuspended in 1 ml of basal
medium. Cells were lysed by the addition of 10 ll toluene.
Cell debris and unbroken cells were removed by centrifu-
gation (16g, 30 s) and the supernatant was immediately
used for the experiment or kept on ice for not more than
10 min. Deactivated cell extracts were made by boiling the
extract for 15 to 30 min.
Catechol 1,2-dioxygenase and catechol 2,3 dioxygenase
activities in the crude cell extracts were evaluated by the
methods of Ka-Leung et al. (1990) and Kataeva and Go-
lovleva (1990) respectively. The protein contents of cell
extracts were determined with the method of Bradford
(1976). The concentration of protein was read off a stan-
dard curve prepared with bovine serum albumin (0–10 lg).
Results
Isolation and characterisation of pyrene degraders
Pyrene-degrading strains namely, LP1 and LP5 were
obtained from MVOA and DGED sites respectively while,
the third strain, LP6 originated from the soil obtained from
LAS. The colony strain LP1 measured 2–3 mm with
irregular edges on LB agar and it spread on the line of
streaking on blood, chocolate and MacConkey agar. It was
Gram-negative motile rods, oxidase- and catalase-positive.
It failed to ferment almost all sugars tested and was indole,
urease and citrate negative, attributes suggestive of Pseu-
domonas sp. It produced no pigment, thus clearly showing
that it is not Pseudomonas aeruginosa. Cultural morphol-
ogies of LP5 showed circular, raised and smooth edge
translucent colony (3–4 mm in diameter) while colonies of
LP6 measured about 10 mm and were cream with a round,
and smooth edge. Both strains were Gram-negative, motile
rods that shared a lot of biochemical characteristics with
LP1, however, they grew at 42°C on nutrient agar. Addi-
tionally, strain LP5 was urease- and malonate-positive, and
was also positive for the pigment pyoverdin. On the other
hand, LP6 utilized citrate and was positive for the pigment
pyorubrin. These characteristics indicate that both isolates
were strains of Pseudomonas aeruginosa. All three isolates
tolerated salt concentrations of more than 3%. They all
resisted ampicillin, cenfuroxime, but were susceptible to
ofloxacin and ciprofloxacin. Only LP6 was resistant to both
gentamycin and terramycin (Table 1).
Substrate susceptibility of isolates
Results summarized in Table 2 show that all the isolates
utilized pyrene but not phenol and hexane. Strain LP1 grew
very well on naphthalene, phenanthrene, anthracene,
dibenzothiophene, biphenyl and some congeners of
chlorobenzoate (CBA) as sole carbon and energy sources
while, these pollutants were weakly utilized by LP6.
Interestingly, LP5 failed to grow on naphthalene, phenan-
threne, anthracene and biphenyl. Additionally strain LP1
grew excellently well on crude oil, diesel and engine oil,
but only fairly well on kerosene. LP5, on the other hand,
Table 1 Antibiotic resistance patterns of pyrene-degrading isolates
Antibiotic Isolate
LP1 LP5 LP6
Norfloaxacin (10 lg) S R R
AX (20 lg) R R R
Ofloxacin (5 lg) S S R
Chloramphenicol (10 lg) S ND R
Cenfuroxime (30 lg) R R R
Ampicillin (25 lg) R R R
Gentamycin (10 lg) S S R
Nitrofurantion (100 lg) R ND R
Ciprofloxacin (50 lg) S S S
Terramycin (50 lg) S S R
R: resistant; S: susceptible; ND: not determined
Table 2 Substrate specificity of pyrene degrading isolates
Substrate Isolate
LP1 LP5 LP6
Naphthalene + – ++
Phenanthrene ++ – ++
Anthracene ++ – ++
Pyrene +++ +++ ++
Dibenzothiophene ++ ++ ++
Biphenyl +++ – +
Benzene ++++
Catechol +++ +++ ++
Phenol – – –
Toluene +++
Hexane – – –
Crude oil +++ +++ ++
Diesel ++ +++ ++
Engine oil +++ +++ +
Kerosene +++
Benzoate +++ ++ ++
2-Chlorobenzoate ++ +++ +
3-Chlorobenzoate +++ – ++
4-Chlorobenzoate +++ ––
1,4-Dichlorobenzene +++ + +
2,4-Dichlorobenzoate +++ ++ +
2,5-Dichlorobenzoate + +++ +
2,6-Dichlorobenzoate ++ +++ +
+++: Luxuriant growth; ++: Growth; +: Poor growth; –: No growth
2642 World J Microbiol Biotechnol (2008) 24:2639–2646
123

only grew poorly on crude oil and kerosene, fairly well on
diesel, but failed to grow at all on engine oil. LP6 grew
slightly on all the petroleum cuts tested (Table 2).
Biodegradation of pyrene
The growth kinetics of the LP strains on pyrene are illus-
trated in Fig. 1 and Table 3. The organisms exhibited slight
lag phases, especially strains LP1 and LP5. Growth was
gradual with concomitant decreases in pyrene concentra-
tion. LP1 grew from an initial density of 13.3 9 10
6
cfu/ml
to peak at 2.0 9 10
8
cfu/ml resulting in over 15-fold
increase. Similar growth dynamics was obtained with LP5
though with over 20-fold cell increase at a growth rate of
0.024 h
-1
which was the highest observed. In the case of
strain LP6, there was a rapid cell increase between days 5
and 6 compared to other time points. By day 12, sharp drop
in cell density was obtained which peaked and remained
relatively stationary for more than 6 days before assuming
a decreasing trend.
The rates of pyrene utilization observed as quantified by
spectrophotometric technique were 0.111, 0.082 and
0.067 mg l
-1
h
-1
respectively for LP1, LP5 and LP6. In
the control tubes, no apparent decrease of the substrate was
observed. This indicates that the depletion of the pyrene
from the basal media was due to biodegradation rather than
to non-specific losses such as compound volatility or
adsorption to the glass tubes or mere accumulation of the
PAH substrate in cells. Although strain LP5 grew with the
least doubling time (28.4 h), the best pyrene degrader was
LP1 with nearly 68% degradation. The overall percentage
removal of pyrene observed for other strains were 67 (LP5)
and 47 (LP6) after a 30-day incubation period. It is note-
worthy that in spite of decline in growth observed from day
21, degradation of the pyrene substrate lasted the experi-
mental period. It is noteworthy that these results were
comparable with those obtained during GC analysis (GC
fingerprints not shown).
Rate of pyrene uptake
Figure 2 shows the rate of uptake of pyrene by the isolates.
The organisms exhibited divergent pyrene uptake rate. The
uptake rate peaked for LP1 within 10 min at 14 ng/
2.0 9 10
7
bacteria cells; LP5 peaked within 6 min at
7.5 ng/2.0 9 10
7
bacteria cells, while LP6 recorded a
value of 9 ng/2.0 9 10
7
bacteria cells within 10 min.
Enzyme activities
Cell suspensions of the three isolates failed to show
appearance of yellow colouration in the presence of cate-
chol, indicating the absence of meta-cleavage of catechol
by these organisms. The appearance of purple colour upon
testing for b-keto-adipic acid showed the presence of the
ortho-cleavage pathway in the three isolates. Furthermore,
analysis of the cell-free extracts confirmed the presence of
ortho-cleavage enzymes for the metabolism of catechol.
The respective specific activities of catechol 1,2-dioxy-
genase obtained for LP1, LP5 and LP6 were 0.6823,
0.9199, and 0.8344 lmol min
-1
mg of protein
-1
(Table 4).
Fig. 1 Growth dynamics of LP1 (a), LP5 (b) and LP6 (c) strains in
minimal medium supplemented with 100 mg l
-1
pyrene showing
total viable count, TVC (d) and residual pyrene (j). Data points
represent the means of three replicate tubes and were determined with
reference to pyrene recovered from heat-killed controls
World J Microbiol Biotechnol (2008) 24:2639–2646 2643
123

Discussion
Degradation of pyrene and other PAHs in the environment
occurs predominantly by microbial processes (Das and
Mukherjee 2007). The recovery of only one species from
each of the soils MVOA and DGEG is in tandem with
earlier reports and may be attributed to the fact that
enrichment medium imposes stress on the microbial com-
munity in order to select the required phenotype leading to
reduction of species diversity and dominance of a single
species (Stach and Burns 2002). Most of the high molec-
ular weight-PAH degrading bacteria so far described are
actinomycetes and the knowledge about biodegradation of
pyrene by non-actinomycetes is scanty. However, a variety
of non-actinobacteria, particularly pseudomonads, have
also been reported to mineralize pyrene (Thibault et al.
1996; Juhasz et al. 1997; Sarma et al. 2004; Das and
Mukherjee 2007). The three bacteria isolated in this study
are members of the genus Pseudomonas. The fact that the
three organisms are species of Pseudomonas is not sur-
prising because they are a group of organisms with broad
substrate specificity for dioxygenases, a factor which
accounts for their metabolic diversity. The metabolic ver-
satility of the pseudomonads isolated in the present study
makes them unique and quite different from previously
characterized strains.
The ability of these organisms to grow at salt concen-
trations higher than 3% is noteworthy. This may be
important in their consideration for bioremediation purpose
upon further study as salinity has been found by Kastner
et al. (1998) to be a very important factor determining the
survival of bacterial inoculum in soil during bioaugmen-
tation. Antibiotics are produced by many soil
microorganisms for competitive edge. Their persistence,
after production, depends on many factors including cli-
mate, type of soil and antibiotic type. Many antibiotics are
biodegradable in soil and many also have a long half life.
Since antibiotics have both qualitative and quantitative
effects on terrestrial microbial communities (Chander et al.
2005). For survival in soils, it is important and required to
know the sensitivity and resistance patterns of microor-
ganisms with potentials for use as seeds for bioremediation.
Shared resistance to ampicillin and cenfuroxime by all the
isolates could be attributed to the fact that these antibiotics
are common in environmental compartments prompting
evolution of resistance by autochthonous strains and hence,
a survival strategy that allowed them to proliferate ahead of
other members of the community. In any case, the meta-
bolic versatility is an indication of the occurrence of
multifunctional dioxygenases in these strains particularly
LP1.
The broad spectrum of LP1 on PAHs may be attributed
to the more diverse nature of petroleum products at the site
from which it was recovered. This may also account for its
better performance on crude and most of the petroleum
products tested. LP1 also performed better on chloro-
benzoates tested than LP5 and LP6. One reason for this
may be the fact that in the mechanic village where this
organism was isolated, apart from oil products, other pol-
lutants such as coils, capacitor, and transformer oil
containing polychlorinated biphenyls (PCBs) abound and
the selective pressure exerted by these pollutants led to
acquisition of such special ability (Wackett and Hersh-
berger 2001; Adebusoye et al. 2007a).
Our isolates grew on pyrene with growth rates of 0.017–
0.024 h
-1
lower than those previously reported for many
actinomycetous pyrene-degraders, these values are better
than previously reported for some pseudomonads. Thus,
while Boldrin et al. (1993) reported 0.056 h
-1
for
Table 3 Growth kinetics of pyrene degrading isolate
Isolate
LP1 LP5 LP6
Growth rate (h
-1
) 0.018 0.024 0.017
Doubling time (h) 39.4 28.4 41.7
Rate of degradation (mg l
-1
h
-1
) 0.111 0.082 0.067
Fig. 2 Pyrene uptake by LP1 (d), LP5 (j) and LP6 (m) strains. No
significant pyrene uptake was observed in heat-killed control tubes as
well as uninoculated tubes
Table 4 Specific enzyme activity of pyrene degrading isolate
Isolate Enzyme activity (lmol min
-1
mg
-1
)
Catechol
1,2-dioxygenase
Catechol
2,3-dioxygenase
Pathway
LP1 0.6823 0.0 ortho
LP5 0.9199 0.0 ortho
LP6 0.8344 0.0 ortho
2644 World J Microbiol Biotechnol (2008) 24:2639–2646
123

Mycobacterium BB1, Thibault et al. (1996) reported 0.014
and 0.013 h
-1
for Pseudomonas sp. K-12 and B-24
respectively. Walter et al. (1991) also reported 0.023 h
-1
for their Rhodococcus UW1. Compared to previous reports,
the pyrene utilisation rates of our organisms are low.
Whereas we report here values of 0.111, 0.082 and
0.067 mg l
-1
h
-1
), Dean-Ross and Cerniglia (1996)
reported 0.56 mg l
-1
h
-1
for Mycobacterium flavescens.It
is noteworthy however that growth rates on substrate and
rate of utilisation are not intrinsic properties of isolates
independently of culture conditions. For instance, Boldrin
et al. (1993) had shown that pyrene crystal sizes greatly
influence pyrene degradation rates by Mycobacterium
species. The authors found that large crystals were utilised
at the rate of 1.2 while smaller ones were utilised at the rate
of 5.6 lgml
-1
h
-1
. It has also been shown that variations
in physico-chemical parameter such as pH may also sig-
nificantly influence mineralization rates (Grosser et al.
1991; Kim et al. 2005).
The overall percentage utilisation of pyrene exhibited by
strains LP1 and LP5 are well within the ranges previously
reported for many pyrene-degraders, including actinomy-
cetous: Mycobacterium sp. PYR1––52.4% (Heitkamp et al.
1988) Rhodococcus sp. UW1––72% (Walter et al. 1991)
Leclercia adecarboxylata––61.5% (Sarma et al. 2004). The
lower degradation recorded for LP6 (40.09%) is in tandem
with its generally poor performance on most hydrocarbon
substrates tested (Table 3). This may be due to possible
effects of some of some of the metabolites which inhibit
further degradation. This argument is buttressed by its early
attainment of stationary phase, in spite of early similarities
in utilisation of pyrene it shared with LP5.
Since bacteria initiate PAH degradation by the action of
intracellular dioxygenase, the PAHs must be taken up by
the cells before degradation can take place (Johnsen et al.
2005). Thus uptake rate is an important parameter amidst
the gamut of factors worthy of consideration when
assessing the biodegradation abilities of potential candi-
dates for bioaugmentation or biostimulation. Recent report
by Das and Mukherjee (2007) on the role of biosurfactant
in the uptake of pyrene by Bacillus and Pseudomonas spp.
has further underscored this. Our organisms demonstrated
higher uptake rates than the non-biosurfactant enhanced
resting cell of the two Pseudomonas strains reported by
these authors. It is noteworthy that preliminary studies
(data not shown) already showed that ours are poor bio-
surfactant producers with pyrene, suggesting therefore
other mechanisms of uptake than biosurfactant production
in the growth medium. The higher rate of uptake of pyrene
recorded for LP1 (Fig. 2) is in consonance with its higher
rate of utilisation. The same also applies to LP5, which had
the lowest total uptake, but peaked only within 6 min. The
rapidity of uptake may account for the relatively higher
growth rate of this organism. The inconsistencies between
growth rate and amount of pyrene utilised can be attributed
to difference in the efficiency of conversion of the carbon
source to biomass by the different strains.
Pathway of degradation of aromatic compounds usually
involves the incorporation of two atoms of oxygen into the
aromatic ring and subsequent cleavage of the dihydroxy-
lated compound. Such cleavage could either be ortho
(between the two hydroxylated carbons) or meta (between
a hydroxylated and non-hydroxylated carbon; Cerniglia
1992). The three organisms in this study degraded catechol
via the ortho pathway, but more interestingly, LP1 with the
highest rate of degradation of pyrene showed the least
enzyme activity. This is not unlikely however as the
dioxygenase enzymes are highly specific and the ones
involved in the cleavage of pyrene ring itself in this case
are actually more crucial than those of the lower pathway.
The rate of degradation is ultimately a function of inter-
action among a number of factors including rate of uptake
and cellular physico-chemistry.
We have described for the first time organisms exhibiting
pyrene catabolic phenotypes from Nigerian contaminated
systems. Our results have shown that pyrene-degraders may,
as well, act on a number of hydrocarbon environmental
mixtures including aliphatic and aromatic compounds.
Pyrene degraders are not so common in the environment;
however, such organisms are needed for use in developing
models and strategies for removing PAH pollutants.
Obtaining them require careful and painstaking search.
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