Journal of Environmental Protection, 2011, 2, 243-254
doi:10.4236/jep.2011.23028 Published Online May 2011 (http://www.scirp.org/journal/jep)
Copyright © 2011 SciRes. JEP
243
Characterization of Aromatic Hydrocarbon
Degrading Bacteria from Petroleum
Contaminated Sites
Swarnakaran Hemalatha, Panchanathan Veeramanikandan
Department of Biotechnology, Vels University, Chennai, India.
Email: hemas81@gmail.com
Received November 18th, 2010; revised January 13th, 2011; accepted February 10th, 2011.
ABSTRACT
Aromatic hydrocarbons such as benzene, hexane, toluene, naphthalene and xylene degrading bacteria such as Flavo-
bacterium spp.1 & 2 and Pseudomonas spp.1 & 2 were isolated from petroleum contaminated soil samples. They were
resistant to heavy metals such as lead, iron, zinc, cobalt and mercury. The optimum pH for hydrocarbon degradation by
Flavobacterium spp.1 was 9. Flavobacterium spp.2, Pseudomonas sp p.1 & 2 have shown optimum pH 7 for th eir deg-
radation. The optimum temperature for hydrocarbon degradation by Flavobacterium spp.1 & 2 and Pseudomonas
spp.1 & 2 were at 40˚C & 45˚C.
Keywords: Aromatic Hydrocarbon, Flavobacterium, Pseudomonas, Heavy Metals, Temperature
1. Introduction
Aromatic hydrocarbons are common environmental pol-
lutants with toxic, genotoxic, mutagenic and carcinogenic
properties [1]. They mainly occur in petroleum industry
activities [2]. Oil spills because of pipeline breakages,
tanks leakages or storage and transportation accidents
can be considered as the most frequent causes of hydro-
carbon release, included PAHs into soils [3]. BTEX
compounds are components of gasoline and aviation fu-
els that are carcinogenic and neurotoxic to most organ-
isms [4].
Bacteria play a major role in hydrocarbon degrad ation.
The reason for petroleum biodegradation is the ability of
microorganisms to utilize hydrocarbons to satisfy their
cell growth and energy needs. Low molecular weight
alkanes are degraded most rapidly whereas mixed cul-
tures carry out more extensive biodegradation of petro-
leum through pure cultures [5,6]. Therefore, biodegrada-
tion using microorganisms is usually preferred. They
play major role in PAHs removal from contaminated
environments because of some advantages such as cost
effectiveness and more complete cleanup [7].
Toluene degrading bacteria putid a PaW1, P. putid a F1,
P. mendocina KRI, Burkholderia cepacia G4, B. cepacia
and Ralstonia p ickettii PKO1 were characterized [8]. The
biodegradation of naphthalene has been well studied at
the molecular level and thus it serves as one of the prin-
cipal models for understanding the mechanisms of bacte-
rial benzene ring metabolism. Isolates CMBLHC1-
CMBLHC8 have potential use to clean up the environ-
ment containing hydrocarbons such as salicylate, phe-
nanthrene, SDS and naphthalene. Eight bacterial strains
(CMBLHC1-CMBLHC8) were isolated from petrol
pumps soil samples and their tolerance was also checked
on other hydrocarbons such as 0.01 M salicylate, 0.008 M
phenanthrene, 0.01 M SDS [sodium dodecyl sulfate) and
naphthalene vapours [9]. CMBLHC2 and CMBLHC6
show maximum tolerance up to 0.5 M benzoic acid and
CMBLHC2, CMBLHC4, CMBLHC7 showed maximum
tolerance up to 0.5 M salicylate. Optimum pH ranged
from 6.0 to 7.0. The optimum temperature for all strains
was 37˚C. Isolated bacteria have also shown resistance
against heavy metals and antibiotics.
Twenty four bacteria capable of utilizing naphthalene
as their sole source of carbon and energy for growth from
three different soils in Nsukula, Nigeria were isolated
and characterized [10]. The usage of petroleum hydro-
carbon products has been increased. Therefore, the soil
contamination with diesel and engine oil is becoming one
of the major environmental problems. Various microbial
species are effective degraders of hydrocarbons in natural
environment. Microorganisms such as bacteria, fungi,
Characterization of Aromatic Hydrocarbon Degrading Bacteria from Petroleum Contaminated Sites
244
yeast and microalgae can degrade petroleum hydrocar-
bons [11,12] .
Flavobacterium, Acnetobacterium and Pseudomonas
isolates are capable of utilizing used engine oil as a car-
bon source. The potential of Flavobacterium, Acneto-
bacterium and Pseudomonas for oil bioremediation in
situ and ex situ were demonstrated [13]. Three bacteria
such as Pseudomonas sp, Flavobacterium sp and Rhodo-
coccus sp were isolated and considered as efficient gaso-
line degrading bacteria. The optimal growth conditions
of three bacteria including pH, temperature and the con-
centration of gasoline were similar [14]. Bacterial and
fungal species such as Pseudomonas, Bacillus, Micro-
coccus, Aspergillus, Rhizopus, Muc or and Penicillium
degrade hydrocarbon from the oil spilled area [15].
As the usage of petroleum hydrocarbons products in-
creased, soil contamination with diesel and engine oils
has become one of the major environmental problems.
Uncontrolled and catastrophic releases of petroleum pose
ecological and environmental repercussions, as a lot of
hydrocarbon components are toxic and persistent in ter-
restrial and aquatic environments. Several physico-
chemical methods of decontaminating the environment
have been established and employed. Biological degra-
dation, a safe, effective and an economic alternative
method, is a process of decay initiated by biological
agents, specifically in this case by microorganisms.
Therefore the counter measure to remediate soils con-
taminated with oils is by bioremediation. Bioremediation
provide an effective and efficient strategy to speed up the
clean up process. Therefore, the present study deals with
the isolation and characterization of aromatic hydrocar-
bons degrading bacteria from oil contaminated sites.
2. Materials and Methods
2.1. Sample Collection
About 5 g of soil samples were aseptically collected from
different petroleum contaminated sites in and around
Chennai City, Tamil Nadu, India. Immediately they were
brought to the laboratory. All samples were placed in to
sterile polythene bags and stored at 4˚C.
2.2. Hydrocarbon Substrates
Hydrocarbon substrates such as Toluene, Benzene, Hex-
ane, Xylene and Naphthalene were selected for study.
2.3. Isolation of Bacteria
Bacteria were isolated from soil samples using an en-
richment medium containing petrol. Petrol was added to
the medium after autoclaving. 1.0 g of soil sample was
inoculated into the medium and incubated at 170 rpm at
30˚C in an orbital shaker for one week. After one week,
1.0 ml of the sample was taken from each culture and
transferred into fresh enrichment medium, followed by
incubation as described above for one week. At the end
of second week, the bacterial contents were serially di-
luted and 10–2 dilution was placed in Bushnell Haas me-
dium containing 15.0 g/L of pure agar. Inoculated plates
were purified repeatedly by sub culturing. Pure culture
was sto red in nutrient agar slants and stored at 4˚C.
2.4. Identification of Bacteria
The bacteria isolated were identified based on physical
characterization and the biochemical tests outlined in
Bergey’s Manual of determinative Bacteriology [16].
2.5. Characterization of Hydrocarbon Degrading
Bacteria
2.5.1. Effect of Aromatic Hydrocarbon
A loopful of isolated bacteria was inoculated in a freshly
prepared and autoclaved BH medium in presence of five
different aromatic hydrocarbons such as Benzene, Hex-
ane, Toluene, Naphthalene and Xylene, whose concen-
tration is 0.02%. After 24 hours of incubation at 30˚C,
the utilization of the aromatic hydrocarbons as sole car-
bon source was assayed for O.D at 600 nm in U-V spec-
trophotometer at an interval of 30 minutes. The Bushnell
Haas medium (BH) devoid of aromatic hydrocarbon
served as con trol.
2.5.2. Effect of pH
Enrichment BH medium with varying pH such as 5, 6, 7,
8, 9 were prepared and autoclaved. A loopful of isolated
bacteria was inoculated into it. Th e utilization of suitable
hydrocarbon at different pH was read at 600 nm in U-V
spectrophotometer at an interval of 30 minutes. The me-
dium devoid of hydrocarbons served as control.
2.5.3. Effect of Temperature
A loopful of isolated bacteria was inoculated in enrich-
ment BH medium. It was maintained at different tem-
perature such as 30˚C - 45˚C. The utilization of suitable
hydrocarbon at different temperature was read at 600 nm
in UV spectrophotometer at an interval of 30 minutes.
The medium devoid of hydrocarbons served as control.
2.6. Heavy Metal Tolerance Spectrum of
Hydrocarbon Degrading Bacteria
The tolerance of bacterial isolates to various heavy met-
als such as Cobalt (Cobalt Chloride), Iron (Ferrous Sul-
phate), Mercury (Mercury Sulphate), Zinc (Zinc Sulphate)
and Lead (Lead acetate) was studied by inoculating
loopful of overnight grown cultures on Nutrient agar
plates amended with 1, 3 and 5 mM concentration of
heavy metals and incubated at 37˚C. After 24 hours of
incubation, the p lates were observed for growth. Nutr ient
Copyright © 2011 SciRes. JEP
Characterization of Aromatic Hydrocarbon Degrading Bacteria from Petroleum Contaminated Sites 245
Agar plates without heavy metals served as control.
2.7. Estimation of Protein in Hydrocarbon
Degrading Bacteria
To 1ml of the bacterial supernatant, 1ml of 20% TCA
was added and kept for half hour incubation. It was then
centrifuged at 8000 rpm for 20 minutes. The pellet was
washed with acetone twice and again centrifuged it. The
supernatant was discarded and the pellet was dissolved in
0.2 m phosphate buffer. The sample was stored at 4˚C for
further study. Protein concentrations were determined by
Lowry’s method using BSA (bovine serum albumin) as
standard.
3. Results and Discussion
Polycyclic aromatic hydrocarbons are ubiquitous con-
taminants of terrestrial ecosystems whose presence is
attributable to a number of petrogenic and pyrogenic
sources, which had increased since the end of the II
world war. Environments contaminated with PAHs are
considered hazardous to humans exposed to them.
Therefore, removal of pollutants from the environment is
essential. Varieties of physical, chemical and biological
ways were adopted already. But bacterial bior emediation
technique offers new possibilities to accelerate the pollu-
tion degradation because they are widely present in the
environment itself.
In the present study, four bacterial isolates have been
isolated from different petroleum contaminated soil sam-
ples by crowded plate technique. With reference to
physical and biochemical tests as outlined in Bergey’s
manual of determinative bacteriology, the bacteria were
identified as Flavobacterium species and Pseudomonas
species (Table 1). Their potential to degrade PAH was
studied. The isolate which have shown the highest Opti-
cal Density (OD) value in different hydrocarbon was
considered as the suitable hydrocarbon source for their
degradation. The presence of local soil microbial popula-
tion adapted to hydrocarbons form the basis of microbial
methods.
It is also believed that the isolated organisms should
posses catabolic enzymes for the specific biodegradation
in the presence of different PAHs. The catabolic path-
ways, which encode the different aromatic hydrocarbon
degradation routes, are frequently located on plasmids.
Although degradation genes can be located on either
chromosome or plasmid. Phenanthrene degrading Pseu-
domonas from crude oil contaminated soil samples col-
lected in a petroleum refining area were isolated and
characterized [17]. They concluded that hydrocarbon deg-
radation routes are frequently located on plasmid.
All the four isolates degraded benzene, toluene, hex-
ane, xylene and naphthalene. Flavobacterium sp. 1 have
utilised xylene as carbon source and the highest OD of
0.37 was recorded (Figure 1). Flavobacterium sp. 2 have
utilized toluene as carbon source and the highest OD of
0.372 was recorded (Figure 2). Pseudomonas sp. 1 and
Pseudomonas sp. 2 have shown good growth in Toluene
containing medium and the highest OD of 0.314 was
recorded for both the organisms (Figures 3 & 4).
The suitable hydrocarbon alone is not sufficient for
bioremediation of environmental pollutants. Other physi-
cal parameters such as pH and Temperature are also es-
sential because physio-chemical influence cause the mi-
crobial degradation of hydrocarbon. The optimum pH
and temperature for degradation by four isolates were
determined. The isolate, which have shown the highest
OD value in varying pH, was considered as the suitable
pH for their degradation and the results are represented in
Figures 5-8. The optimum pH for Flavobacterium sp. 1
was recorded as pH 9. It shows the maximum peak value
of 0.292 (Figure 5). The optimum pH for the Flavobac-
terium sp. 2, Pseudomonas sp. 1 and Pseudomonas sp. 2
were recorded as pH 7 (Figures 6, 7 & 8). Moreover
these isolates illustrate maximum peak value of 0.404,
0.207 and 0. 28 8 respect i vel y .
The pH range that is most suitable for soil microor-
ganism is below 6 and 8, with an optimum pH of about
seven for most species. The optimum pH for Flavobacte-
rium sp. 2, Pseudomonas sp. 1 and Pseudomonas sp. 2
was 7 in the presence of the hydrocarbon, which suppor ts
the investigation done by [18] who studied the effects of
aerobic bacterial mixtures pH, temperature and carbon
sources on polyaromatic hydrocarbons. The optimal con-
ditions for PAH biodegradation were determined as 30˚C
and pH 7. The isolate, which have shown the highest OD
value in varying temperature, was considered as the
suitable temperature for their degradation. The optimum
temperature for degradation by Flavobacterium sp. 1and
2 was at 40˚C. They have shown optimum OD value of
0.353 & 0.464 respectively (Figures 9 & 10). The opti-
mum temperature for degradation by Pseudomonas spp1
was also at 40˚C. They have shown optimum OD value
of 0.304. (Figure 11). But Pseudomonas sp. 2 degraded
maximum hydrocarbon at 45˚C with OD of 0.345. (Fig-
ure 12). The optimum petroleum degradation rate by
aerobic bacteria occurs at temperature below 15 and
30˚C [19]. Since the selected organism have shown their
maximum degradation at 40˚C and 45˚C, these organism
can be used for bioremediation to be carried out in sum-
mer months. Increases in optimum temperature increases
the solubility of hydrocarbons to o.
The biodegradation of aliphatic and aromatic hydro-
carbons at high temperatures were investigated [20]. The
solubility of naphthalene is increased by a factor of ap-
proximately ten if the temperatur e is increased from 20 to
Copyright © 2011 SciRes. JEP
Characterization of Aromatic Hydrocarbon Degrading Bacteria from Petroleum Contaminated Sites
Copyright © 2011 SciRes. JEP
246
75˚C, which increases the availability of sparingly solu-
ble hydrocarbons.
The heavy metal tolerance spectrum of Flavobacte-
rium spp. 1, Flavobacterium spp. 2, Pseudomonas spp. 1
and Pseudomonas spp. 2 were studied and the results are
presented in Table 2. All the four isolates were tolerant
to iron, zinc and lead upto 5 mM concentration. But they
were sensitive to cobalt and mercury even at 1 mM con-
centration. Being sensitive no growth was observed in
their plates. These results revealed that the presence of
heavy metals such as cobalt and mercury even in 1 mM
concentration was highly toxic to the bacteria isolated
from oil contaminated sites and they will pose serious
threat to their metabolism in the environment. But they
have shown resistance towards iron, zinc and lead. This
could be attributed due to the presence of plasmid in their
genetic makeup. The transferable plasmid encodes resis-
tance to various heavy metals [21]. The protein content
of hydrocarbon degrading bacteria was determined. It has
increased to a smaller extent after utilizing hydrocarbon
as substrate. But Pseudomonas spp. 2 has showed de-
crease protein content than control sample. The protein
content of hydrocarbon degrading bacteria has varied
between 1.4 - 1. 8 mg/100 ml (Table 3).
0.197 0.197 0.1970.1970.197
0.193
0.249
0.353 0.37
0.198
0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
0.4
HEXANE BENZENETOLUENE XYLENENAPHTHALENE
O D at 600 nm
Substrates Control Sample
Figure 1. Average optical density obtained from Flavobacterium spp. 1 using different hydrocarbon.
0.191 0.191 0.191 0.191 0.1910.191
0.24
0.372
0.291
0.214
0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
0.4
HEXANE BENZENETOLUENE XYLENENAPHTHALENE
O D a t 600 nm
Substrates Control Sample
Figure 2. Average optical density obtained from Flavobacterium spp. 2 using different hydrocarbons.
Characterization of Aromatic Hydrocarbon Degrading Bacteria from Petroleum Contaminated Sites 247
0.1820.182 0.1820.1820.182
0.204
0.256
0.314
0.273
0.185
0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
HEXANE BENZENETOLUENE XYLENENAPHTHALEN
E
OD at 600 nm
Sub strates
Control Sample
Figure 3. Average optical density obtained from Pseudomonas spp. 1 using different hydrocarbons.
0.182 0.182 0.182 0.182 0.182
0.204
0.256
0.314
0.273
0.185
0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
HexaneBenzeneTolueneXylene Naphthalene
o. d a t 600 n m
substrates
Control Sample
Figure 4. Average optical density obtained from Pseudomonas spp 2 using different hydrocarbons.
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Characterization of Aromatic Hydrocarbon Degrading Bacteria from Petroleum Contaminated Sites
248
0.1920.208
0.2450.234
0.211
0.281
0.238
0.278
0.226
0.292
0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
56789
O D a t 600 nm
pH Control Sample
Figure 5. Average optical density of Flavobacterium spp. 1 grown in Bushnell haas medium using different pH.
0.152
0.219
0.182 0.179
0.122
0.32
0.364
0.404
0.357
0.27
0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
0.4
0.45
56789
O D a t 6 00 nm
pH Control Sample
Figure 6. Average optical density of Flavobacterium spp. 2 grown in Bushnell haas medium using different pH.
Copyright © 2011 SciRes. JEP
Characterization of Aromatic Hydrocarbon Degrading Bacteria from Petroleum Contaminated Sites 249
0.224 0.22 0.21
0.231
0.212
0.184 0.177
0.207
0.159
0.205
0
0.05
0.1
0.15
0.2
0.25
56789
O D a t 600 nm
pH
ControlSample
Figure 7. Average optical density of Pseudomonas spp. 1 grown in Bushnell haas medium using different pH.
0.232
0.205 0.2 0.213
0.287
0.18
0.262
0.288
0.198
0.16
0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
56789
OD at 600 nm
pH
Control Sampl
e
Figure 8. Average optical density of Pseudomonas spp. 2 grown in Bushnell haas medium using different pH.
Copyright © 2011 SciRes. JEP
Characterization of Aromatic Hydrocarbon Degrading Bacteria from Petroleum Contaminated Sites
250
0.137 0.153
0.108 0.102
0.188
0.145
0.353
0.218
0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
0.4
30°C35°C40°C45°C
OD at 600 nm
Temperature
CONTROL SAMPLE
Figure 9. Average optical density of Flavobacterium spp. 1 grown in Bushnell haas medium at different te mperature.
0.123 0.117
0.091 0.103
0.251 0.236
0.464
0.245
0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
0.4
0.45
0.5
30°C35°C40°C45°C
O D a t 600 nm
Temperature CONTROL SAMPLE
Figure 10. Average optical density of Flavobacterium spp. 2 grown in Bushnell haas medium at different temperature.
Copyright © 2011 SciRes. JEP
Characterization of Aromatic Hydrocarbon Degrading Bacteria from Petroleum Contaminated Sites 251
0.123 0.117 0.091 0.103
0.251 0.236
0.464
0.245
0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
0.4
0.45
0.5
30°C35°C40°C45°C
O D a t 600 nm
Temperature CONTROL SAMPLE
Figure 11. Average optical density of Pseudomonas spp.1 grown in Bushnell haas medium at different temperature.
0.109 0.123
0.096 0.112
0.246
0.165
0.244
0.345
0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
0.4
30°C35°C40°C45°C
O D a t 6 00 n m
Temperature CONTROL SAMPLE
Figure 12. Average optical density of Pseudomonas spp.2 grown in Bushnell haas medium at different temperature.
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Characterization of Aromatic Hydrocarbon Degrading Bacteria from Petroleum Contaminated Sites
252
Table 1. Bio chemical characterization of bacterial isolates from oil contaminated sites.
Bacterial Isolates
S.No Cultural CharacteristicsFlavo bacterium spp.1Flavo bacterium spp. 2Pseudomonas spp.1 Pseudomonas spp.2
1. Colony Morphology Rhizoid Yellow
pigented colonies. Rhizoid Yellow
pigmented colonies.
Small, Pigmented
Circular, Flat, Entire,
dry colonies
Small, Pigmented
Circular, Flat, Entire,
dry colonies
2. Gram’s Staining Gram negative Gram negative Gram negative Gram negative
3. Motility Gliding Motility Gliding Motility Active Motile Active Motile
4. Catalase + + + +
5. Oxidase + + +
6. Indole + + +
7. Methyl red
8. VP Test
9. Citrate Test +
10. Nitrate Test +
11. H2S production + + + +
+ Positive Negativ e
Table 2. Heavy metal tolerance spectrum of hydrocarbon degrading bacteria.
Heavy Metals Concentration
(g/ml) Flavo bacterium spp. 1Flavo bacterium spp. 2Pseudomonas spp.1 Pseudomonas spp.2
1 – – – –
3 – – – –
Cobalt (Cobalt
Chloride)
5 – – – –
1 + + + +
3 + + + +
Iron
(Ferrous Sulphate)
5 + + + +
1 – – – –
3 – – – –
Mercury (Mercury
Sulphate)
5 – – –
1 + + + +
3 + + + +
Lead (Lead
Acetate)
5 + + + +
1 + + + +
3 + + + +
Zinc (Zinc
Sulphate)
5 + + + +
…………. Sensitive +………… Resistant
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Characterization of Aromatic Hydrocarbon Degrading Bacteria from Petroleum Contaminated Sites
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253
Table 3. The protein conte nt of hydrocarbon degr ading bacteria.
Isolates Protein Content mg/100 ml
Control 1.7
Flavo bacterium spp. 1 Sample(X) 1.8
Control 1.5
Flavo bacterium spp. 2 Sample(X) 1.7
Control 1.7
Pseudomonas spp. 1 Sample (T) 1.7
Control 1.6
Pseudomonas spp.2 Sample (T) 1.4
X ------------ Xylene T ---------- -- Toluene
4. Conclusions
Hydrocarbon degrading bacteria from oil-contaminated
site have utilized aromatic hydrocarbons at wide range of
temperature and pH. Therefore, Flavobacterium spp and
Pseudomonas spp are recommended for bioremediating
oil contaminated sites.
5. Acknowledgements
The authors are thankful to the Management Vael’s
Educational Trust, Pallavaram, Chennai, India for pro-
viding all the facilities to carry out this research work.
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