Characterization of Aromatic Hydrocarbon Rading Bacteria from Petroleum Contaminated Sites

doi:10.4236/jep.2011.23028

Paper Menu >>

Journal Menu >>

Journal of Environmental Protection, 2011, 2, 243-254

doi:10.4236/jep.2011.23028 Published Online May 2011 (http://www.scirp.org/journal/jep)

243

Characterization of Aromatic Hydrocarbon

Degrading Bacteria from Petroleum

Contaminated Sites

Swarnakaran Hemalatha, Panchanathan Veeramanikandan

Department of Biotechnology, Vels University, Chennai, India.

Email: [email protected]

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

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

Characterization of Aromatic Hydrocarbon Degrading Bacteria from Petroleum Contaminated Sites

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

0.05

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

0.05

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

0.05

0.1

0.15

0.2

0.25

0.3

0.35

HEXANE BENZENETOLUENE XYLENENAPHTHALEN

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

0.05

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.

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

0.05

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

0.05

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.

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

0.05

0.1

0.15

0.2

0.25

56789

O D a t 600 nm

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

0.05

0.1

0.15

0.2

0.25

0.3

0.35

56789

OD at 600 nm

Control Sampl

Figure 8. Average optical density of Pseudomonas spp. 2 grown in Bushnell haas medium using different pH.

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

0.05

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

0.05

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.

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

0.05

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

0.05

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.

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

Characterization of Aromatic Hydrocarbon Degrading Bacteria from Petroleum Contaminated Sites

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.

REFERENCES

[1] G. Mastrangela, E. Fadda and V. Marzia, “Polycyclic

Aromatic Hydrocarbons and Cancer in Man,” Environ-

ment Health Perspect, Vol. 104, No. 11, 1997, pp. 1166-

1170. doi:10.1289/ehp.961041166

[2] M. Blumer, “Polycyclic Aromatic Hydrocarbons in Na-

ture,” Scientific Americian, Vol. 234, No. 1, 1976, pp.

34-44. doi:10.1038/scientificamerican0376-34

[3] M. Bossert, W. Kachel and R. Bartha, “Fate of Hydro-

carbons during Oily Sludge Disposal in Soil,” Applied

Environment Microbiology, Vol. 47, No. 4, 1984, pp.

763-767.

[4] L. A. Levin and V. A. Forchiassin, “Degradation of Or-

ganic Pollutants by the White Rot Basidiomycete Tram-

etes Trogii,” International Biodeterioration & Biodegra-

dation, Vol. 52, 2003, pp. 1-5.

doi:10.1016/S0964-8305(02)00091-4

[5] M. F. Ghazali, N. R. Zakha, R. N. Abdul, A. B. Salleh

and M. Basri, “Biodegradation of Hydrocarbons in Soil

by Microbial Consortium,” International Biodeterioration

and Biodegradation, Vol. 54, No. 1, 2004, pp. 61-67.

doi:10.1016/j.ibiod.2004.02.002

[6] P. V. O. Trindada, L. G. Sobral, A. C. L. Rizzo, S. G. F.

Leitic and A. U. Soriano, “Bioremediation of a Weath-

ered and Recently Oil Contaminated Soils from Bra-

zil—A Comparison Study,” Chemosphere, Vol. 58, No. 4,

2004, pp. 515-522.

doi:10.1016/j.chemosphere.2004.09.021

[7] J. V. Pothuluri and C. E. Cerniglia, “Microbial Metabo-

lism of Polycyclic Aromatic Hydrocarbons,” In: G. R.

Chaudry, Ed., Biological Degradation and Bioremedia-

tion Toxic Chemicals, Chapman and Hall, London, 1994,

pp. 92-124.

[8] J. G. Leahy, K. D. Tracy, M. H. Eley, “Degradation of

Mixtures of Aromatic and Aliphatic Hydrocarbons by

Aromatic Hydrocarbon-Degrading Bacteria,” FEMS-Mi-

crobiology-Ecology, Vol. 43, No. 2, 2003, pp. 271-276.

doi:10.1111/j.1574-6941.2003.tb01067.x

[9] R. Shakoori and B. Zahra, “Hydrocarbon Degrading Bac-

teria Isolated from Soil Samples and Industrial Effluents

and Their Potential Use in Environmental Cleanup,”

Proceedings of Pakistan Congress of Zoology, Vol. 19,

1999, pp. 309-338.

[10] I. Nnamchi, A. N. Obeta and L. I. Ezeogu, “Isolation and

Characterization of Polycyclic Aromatic Hydrocarbon

Degrading Bacteria from Nsukka Soils in Nigeria,” In-

ternational Journal of Environmental Science and Tech-

nology, Vol. 3, No. 2, 2006, pp. 181-190.

[11] E. R. Roberts, “Bioremediation of Petroleum Contami-

nated Sites,” RC Press InC., Boca Raton, 1992.

[12] J. G. Bundy, G. I. Paton and C. D. Campbell, “Combined

microbial Community Level and Single Species Biosen-

sor Responses to Monitor Recovery of Oil Polluted Soil,”

Soil Biology and Biochemistry, Vol. 36, 2004, pp. 1149-

1159. doi:10.1016/j.soilbio.2004.02.025

[13] T. Mandri and J. Lin, “Isolation and Characterization of

Engine Oil Degrading Indigenous Micro Organisms in

Kwazulu Natal, South Arica,” African Journal of Bio-

technology, Vol. 6, No. 1, 2007, pp. 23-27.

[14] S. J. Lu, H. Q. Wang and Z. H. Yao, “Isolation and Char-

acterization of Gasoline Degrading Bacteria from Gas

Station Leaking Contaminated Soil,” Journal of Envi-

ronmental Sciences, Vol. 8, 2006, pp. 969-972.

doi:10.1016/S1001-0742(06)60023-5

[15] Sivapriya and R. Nirmala, “Biodegradation of Cyclohex-

onal by Bacteria Isolated from Oil Spilled Soil Around

Vandavasi,” Journal of Ecotoxicology and Environmental

Monitoring, Vol. 13, 2003, pp. 185-190.

Characterization of Aromatic Hydrocarbon Degrading Bacteria from Petroleum Contaminated Sites

254

[16] J. G. Holt, “Bergy’s Manual of Determinative Bacteriol-

ogy,” 9th Edition, Williams and Wilkins, Philadelphia,

1994.

[17] G. Coral and S. Karagoz, “Isolation and Characterization

of Phenanthrene Degrading Bacteria from Petroleum Re-

finery Soil,” Annals of Microbiology, Vol. 55, No. 4,

2005, pp. 255-259.

[18] S. Y. Yuan, S. W. Chang, B. V. Chang, “Biodegradation

of Polycyclic Aromatic Hydrocarbons in Sludge,” Bulle-

tin of Environmental Contamination and Toxicology, Vol.

7, No. 3, 2003, pp. 625-632.

doi:10.1007/s00128-003-8841-x

[19] N. Englert, G. Harms and F. Widdel, “Aerobic Petroleum

Degradation Rate of Aromatic Hydrocarbon,” Achieves of

Microbiology, Vol.172, 1993, pp. 303-312.

[20] H. Feitkenhauer and H. Mark, “Biodegradation of Ali-

phatic and Aromatic Hydrocarbons at High Tempera-

ture,” Water Science and Technology, Vol. 47, No. 10,

2003, pp. 123-130.

[21] A. S. R. Ghosh and V. Singh, “Characterization of Large

Plasmid Encoding Resistance to Toxic Heavy Metals in

Salmonella Abortus Equi,” Biochemical and Biophysical

Research Communications, Vol. 272, No. 1, 2000, pp.

6-11. doi:10.1006/bbrc.2000.2727