Biodegradation of kerosene by Aspergillus ochraceus NCIM-1146

400

Journal of Basic Microbiology 2007, 47, 400 – 405

www.jbm-journal.com

Research Paper

Biodegradation of kerosene by Aspergillus ochraceus
NCIM-1146

Ganesh Saratale, Satish Kalme, Sanjyot Bhosale and Sanjay Govindwar

Department of Biochemistry, Shivaji University, Kolhapur, India

The filamentous fungus Aspergillus ochraceus NCIM-1146 was found to degrade kerosene, when
previously grown mycelium (96 h) was incubated in the broth containing kerosene. Higher
levels of NADPH-DCIP reductase, aminopyrine N-demethylase and kerosene biodegradation
activities were found to be present after the growth in potato dextrose broth for 96 h, when
compared with the activities at different time intervals during the growth phase. NADPH was
the preferred cofactor for enzyme activity, which was inhibited by CO, indicating cytochrome
P450 mediated reactions. A significant increase in all the enzyme activities was observed when
mycelium incubated for 18 h in mineral salts medium, containing cholesterol, camphor,
naphthalene, 1,2-dimethoxybenzene, phenobarbital, n-hexane, kerosene or saffola oil as
inducers. Acetaldehyde produced by alcohol dehydrogenase could be used as an indicator for
the kerosene biodegradation.

Keywords: Aspergillus ochraceus / Biodegradation / Amino N-demethylase / Kerosene

Received: February 12, 2007; returned for modification: March 20, 2007; accepted: April 10, 2007

DOI 10.1002/jobm.200710337

Introduction

Pollution by petroleum and its byproducts due to the
accidental spillage is a problem in the environment. It
is known that the main microorganisms that can con-
sume petroleum hydrocarbons are bacteria and fungi.
In the literature, the potential of microorganisms
pointed out as degrading agents of several compounds
indicates biological treatment as being the most prom-
ising alternative for reducing the environmental impact
of oil spills (Facundo et al. 2001, Robert et al. 2003). Fil-
amentous fungi play an important role in degrading
diesel and kerosene by producing capable enzymes.
Because of their aggressive growth, greater biomass
production and extensive hyphal growth in soil, fungi
offer potential for biodegradation technology (Kenneth
1995, Saadoun 2002).
Mixed-function oxidase system is of great catalytic
versatility and widely distributed in nature, playing an

Correspondence: Prof. S. P. Govindwar, Department of Biochemistry,
Shivaji University, Kolhapur- 416 004, India
E-mail: spg_biochem@unishivaji.ac.in
Tel: +91-231-2609152
Fax: +91-231-2692333

important role in the biotransformation of a wide vari-
ety of both exogenous and endogenous organic com-
pounds (Bonaventura and Johnson 1997).

The list of

monooxygenation reactions that are catalyzed includes,
aliphatic hydroxylation, desaturation, heteroatom oxy-
genation and dealkylation, epoxidation, oxidative group
migration and various modes of mechanism based inac-
tivation. Biodegradation of n-alkanes takes place by
common hydroxylation pathways, such as terminal,
subterminal or diterminal

γ oxidation pathway (May

and Katoposis 1990). The bacterial degradation of aro-
matics normally involves the formation of a diol, fol-
lowed by ring cleavage and formation of dicarboxylic
acid. Fungi and other eukaryotes normally oxidize
aromatic compounds using mono-oxygenase, forming a
trans-diol (Leahy et al. 2002).
The major constituents of kerosene are alkanes and
cycloalkanes (65 – 70%), benzene and substituted ben-
zene (10 – 15%), naphthalene and substituted naphtha-
lene (ASTM 2001). Kerosene exhibits moderate to highly
acute toxicity to biota, with product-specific toxicity
related to the type and concentration of aromatic com-
pounds (Song and Bartha 1990). Kerosene spills have
the potential for causing acute toxicity in some forms
of aquatic life.

Journal of Basic Microbiology 2007, 47, 400 – 405

Biodegradation of kerosene by A. ochraceus 401

www.jbm-journal.com

The present report describes a study of kerosene
biodegradation by Aspergillus ochraceus NCIM 1146. Ear-
lier investigations show the ability of bacterial strains
to degrade diesel fuel by transforming the diesel fuel to
alcohol, Saadoun (2002) has modified a method pre-
viously employed by Jacobs et al. (1983). The present
study deals with the ability of A. ochraceus to oxidize the
alkanes, a simple and rapid assay procedure for kero-
sene biodegradation by modifying the earlier method.
This method can be used in the screening of fungi for
their potential to transform kerosene to alcohol.

Materials and methods

Organism and culture conditions
A. ochraceus NCIM-1146 was obtained from National
Collection of Industrial Microorganisms, National
Chemical Laboratory, India. Stock cultures were main-
tained on the potato dextrose agar slants, subcultured
periodically and stored at 4

°C. Mycelium cut from agar

slant was used to inoculate 100 ml potato dextrose
broth (PDB) in 250 ml Erlenmeyer flasks, and grown
aerobically for 96 h at 30

°C which was then used for

kerosene degradation experiments. Mineral salts me-
dium containing (g l

–1

): K

HPO

1.71; KH

1.32;

Cl, 1.26; MgCl

, 0.011; and CaCl

0.02 was used for

the induction experiments. All media were autoclaved
at 120

°C for 20 min.

Kerosene degradation study
Media containing 50 mM phosphate buffer (phosphate
medium) or 0.1 g l

–1

yeast extract (YE medium) were

inoculated with PDB-grown mycelium (96 h, 3.4 g dry
weight) which was harvested by centrifugation at
6000

× g for 20 min. The mycelium was divided into

small portions with sterile seizers approximately into
1 cm pieces, which were transferred into 100 ml phos-
phate and YE medium in 500 ml Erlenmeyer flasks in
triplicate, which contained 10% kerosene (v/v, density
0.798 g ml

–1

) and were incubated at 30

°C on a rotary

shaker at 120 rpm. One uninoculated flask was kept as
a control to measure abiotic loss. Percentage removal of
total petroleum hydrocarbon (TPH) was observed at
4-day intervals up to 20 days (100 ml media with de-
graded petroleum hydrocarbon (TPH) was extracted
with 100 ml benzene, twice and dried in rotary evapo-
rator under vacuum). Fungal growth was determined at
each sampling time by measuring the dry weight of the
centrifuged mycelium in a pre-weighed aluminium pan
after oven drying at 70

°C, overnight. The pH change

was also reported at each extraction time. Biodegrada-

tion progress was monitored by Fourier transform in-
frared (FTIR) spectroscopic analysis of the residual
kerosene.

Extraction and analysis of TPH from kerosene
The culture broth was centrifuged at 10,000

× g for

30 min and TPH was extracted in benzene (1 : 1, v/v)
from the supernatant, and the mycelium was washed
twice to extract any bound kerosene. TPH samples were
dehydrated by addition of granular anhydrous sodium
sulfate. After drying to the constant weight, total non-
volatile hydrocarbon was measured gravimetrically and
compared with the uninoculated control flasks. The
degraded kerosene was characterized by FTIR spectros-
copy using Perkin-Elmer, USA, Spectrum One equip-
ment in the mid-IR region (400 – 4000 cm

–1

) at 16 scan

speed. The samples were kept within spectroscopically
pure KBr pellets.

Preparation of cell free extract and enzyme assays
Mycelium was removed after the growth for 96 h and
cut into small pieces (approximately into 1 cm

pieces),

and blotted dried. Five g (fresh weight) of mycelium
was homogenized at 4

°C with 10 ml of 50 mM potas-

sium phosphate buffer (pH 7.4) in a Potter-Elvehjem
type homogenizer. This crude homogenate was used as
an enzyme source without centrifugation. The protein
content was estimated by the Biuret method. Amino-
pyrine N-demethylase and NADPH-DCIP reductase ac-
tivities were assayed according to the procedures re-
ported earlier by Bhosale et al. (2006) and Salokhe and
Govindwar (1999).

Kerosene biodegradation assay
The test conducted for biodegradation of kerosene by a
monooxygenase pathway was examined by the method
of Jacobs et al. (1983) and Kalme et al. (2007). Acetalde-
hyde, the reaction product of alcohol dehydrogenase
activity was estimated using Nash reagent (1953). The
test is based on the following reactions:

Ethanol + nicotinamide adenine dinucleotide
phosphate (NADP

)

Alcohol dehydrogenase

Acetaldehyde + NADP

+ H

Acetaldehyde + Nash reagent

58 °C, 20 min

Diacetyldihydrocollidine (yellow coloured compound,
λ max 388 nm)

402 G.

Saratale

et al.

Journal of Basic Microbiology 2007, 47, 400 – 405

www.jbm-journal.com

The assay mixture for kerosene biodegradation was
0.5 ml 50 mM HEPES [N-(2-hydroxyethyl) piperazine-N

′-

(2-ethanesulfonic acid] buffer (pH 7.8), 0.2 ml NADPH-
generating system (NADP, 2.6

mM: glucose 6-phos-

phate, 12.5 mM; glucose 6-phosphate dehydrogenase,
4 units), 0.5 ml cell free extract and 0.4 ml kerosene.
After incubation at 37 °C for 30 min with constant ro-
tary shaking at 100 rpm, the reaction was terminated
by adding 1 ml ice cold 20% trichloroacetic acid solu-
tion. After centrifugation at 3000

× g, 1 ml Nash re-

agent was added to the clear supernatant and test tubes
were incubated at 58 °C for 20 min. The diacetyldihy-
drocollidine, obtained from the amount of acetalde-
hyde liberated and Nash reagent, was determined at
388 nm (Nash 1953). The cofactor requirement (NADP

and NAD

), the synergistic effect of both cofactors and

the inhibition of enzyme activities in presence of
carbon monoxide were investigated for aminopyrine
N-demethylase and kerosene biodegradation activity.
The status of three enzymes was assessed in PDB within
the growth period from 48 to 144 h.

Enzyme induction study
The mycelium

of A. ochraceus grown in 100 ml PDB for

96 h, was harvested by centrifugation at 8,000

× g for

20 min and transferred (approximately 1 cm

pieces)

into mineral salts medium containing one of the series
of organic compounds present as a carbon source at the
concentration indicated in Table 2. After incubation for
18 h with constant rotary shaking at 120 rpm at 30

°C,

the mycelium was again harvested. A mycelium-free
extract was prepared as described in section 2.5. This
extract was used as an enzyme source for investigation
of the induction of NADPH-DCIP reductase, amino-
pyrine N-demethylase and kerosene degradation activ-
ity. A flask incubated with the individual inducer for
2 min was used as control.

Statistical data analyses
The

data was analyzed by one-way analysis of variance

(ANOVA) with the Tukey-Kramer multiple comparison
test.

Results

Biodegradation of kerosene
Aerobic biodegradation of kerosene in phosphate me-
dium and YE medium showed an increase in the utili-
zation of petroleum hydrocarbons with increased dry
weight. Degradation of TPH in kerosene in phosphate
medium was 77.5% and in YE medium 83.8%. Dry

Time (days)

)

ight

)

Figure 1. Growth profile of A. ochraceus in phosphate and YE
medium containing 10% kerosene showing percentage TPH
degraded (phosphate

䊏

, YE

䊐

), growth as change in dry weight

(phosphate

䉱

, YE

) and pH change (phosphate

䊉

, YE

䊊

weight of the mycelium after incubation for 20 days
with kerosene in these two media was almost the same,
50 – 51 mg ml

–1

medium. The pH decreased from 7.4 to

6.7 in phosphate medium and from 7.2 to 6.6 in YE
medium (Fig. 1). The emulsion formed in both the
media was stable (data not shown). Measuring abiotic
loss in combination with biodegradation studies helped
to avoid overestimation of biodegradation activity

and underestimation of other possible disappearance
mechanisms. Abiotic loss measured for uninoculated
flasks at the time of last sampling, showed a loss of
20 – 21% in both the media.

FTIR analysis of degraded kerosene
FTIR of non-degraded kerosene just after the addition
to phosphate medium (uninoculated) revealed two
prominent peaks representing saturated hydrocarbons
due to the > CH

symmetric (2855 cm

–1

) and asymmetric

stretch (2925 cm

–1

). A – CH

symmetric and asymmetric

bend for an aliphatic hydrocarbon chain and for either
a linear aliphatic hydrocarbon chain or a methyl

benzene derivative was observed at 1460, 1377 cm

–1

. A

ring vibration at 1022 and 1607 cm

–1

represented alkyl

cycloalkanes and aromatic hydrocarbons, and peaks
at 698, 722, 740, and 810 represented mono-, tri- and
tetra-substituted benzene derivatives. TPH extracted
after incubation for 4 days showed bands at 2749
and 2729

–1

and seven sharp bands between

794 – 1402 cm

–1

indicated the formation of aliphatic

and aromatic aldehydes. Increased transmittance at
1604 cm

–1

, with bands at 475 – 810 cm

–1

, represented

mono-, meta- and para-substituted benzene (Fig. 2).

Journal of Basic Microbiology 2007, 47, 400 – 405

Biodegradation of kerosene by A. ochraceus 403

www.jbm-journal.com

4000.0

3000

2000

1500

1000

450.0

-7.6

83.7

cm-1

2925.60

2855.46

2729.88

1607.36

1460.00

1377.67

1022.06

810.38

740.11

722.76

698.73

2924.65

2729.58

1604.24

1460.02

1377.37

1305.23

1168.20

1033.86

964.22

871.14

810.92

745.93

722.12

675.08

537.20

475.60

Figure 2. FTIR comparison of TPH extracted from control (a) and culture of A. ochraceus grown in presence of kerosene (b; 4th day
sample).

Enzyme assays and induction
The fungus showed NADPH-DCIP reductase, amino-
pyrine N-demethylase and kerosene biodegradation
activities. Mycelium-free extracts of autoclaved myce-
lium did not show any activity of enzymatic nature.
A statistically significant decrease in aminopyrine

N-demethylase (61%) and kerosene biodegradation ac-
tivity (44%) was recorded after purging the reaction

Table 1. Cofactor requirement and criteria for cytochrome
P450-mediated reactions for aminopyrine N-demethylase and
kerosene biodegradation activity in A. ochraceus NCIM-1146.

Assay conditions

Aminopyrine
N-demethylase

Kerosene
biodegradation
activity

Without cofactor

No activity

NADPH generating
system

4.39

± 0.09

7.89

± 0.14

NADH

2.30*

± 0.09

4.96*

± 0.07

NADPH + NADH

5.02*

± 0.01

8.58*

± 0.05

Autoclaved mycelia

No activity

Activity in presence of
carbon monoxide (CO)

2.68*

± 0.07

4.46*

± 0.07

nmol of formaldehyde liberated min

–1

(mg protein)

–1

µmol

of acetaldehyde liberated min

–1

(mg protein)

–1

, Values are

mean of three experiments

± SEM Significantly different

from control (assay containing NADPH generating system) at
*

P < 0.001 by one-way ANOVA and Tukey-Kramer multiple

comparisons test.

mixture with carbon monoxide. NADPH was the pre-
ferred cofactor for both the activities. A synergistic
effect in activity was observed in the presence of both
NADH and NADPH (Table 1). In PDB, maximum ac-
tivities of NADPH-DCIP reductase, aminopyrine-N-de-
methylase and kerosene biodegradation were recorded
at 96 h growth (Fig. 3).

µµµ

120

144

Time (h)

DCI

reduc

(

-1

ycel

-1

)

For

ehyde

acet

dehy

leased

-1

min

-1

)

Figure 3. Change with time in activity of mixed function oxidase
enzymes and kerosene biodegradation activity in potato dextrose
broth; growth as dry weight (

䊏

), NADPH-DCIP reductase activity

(

䊐

), aminopyrine N demethylase activity (

䉱

), Kerosene biodegrada-

tion activity (

䉭

404 G.

Saratale

et al.

Journal of Basic Microbiology 2007, 47, 400 – 405

www.jbm-journal.com

Table 2. Effect of carbon sources on NADPH-DCIP reductase, aminopyrine N-demethylase, and kerosene biodegradation activities
in A. ochraceus (NCIM-1146). Mycelium incubated with carbon sources for 2 min in control and for 18 h in tests.

Carbon source

NADPH-DCIP reductase

Aminopyrine
N-demethylase

Kerosene biodegradation
activity

Substance Concentration

(g l

–1

)

Control Test

Control

Test

Control Test

Cholesterol

2.5 12.03

± 0.34 13.85** ± 0.45 2.12 ± 0.12 2.86** ± 0.03 0.94 ± 0.07 1.06* ± 0.03

Camphor

2.5 13.31

± 1.21 14.48 ± 1.23

4.34

± 0.07 5.30** ± 0.12 4.11 ± 0.03 5.13** ± 0.04

Naphthalene

7.84

± 0.57 10.28* ± 0.54 2.67 ± 0.16 3.40 ± 0.29

1.94

± 0.08 3.00** ± 0.11

1,2-dimethoxybenzene

†

19.80

± 1.07 25.87* ± 1.05 5.28 ± 0.03 7.28** ± 0.10 1.57 ± 0.14 2.19**

± 0.10

Phenobarbital

2.5

15.33

± 0.60 19.22** ± 0.52 4.32 ± 0.04 5.19** ± 0.05 2.44 ± 0.20 4.39** ± 0.32

n-Hexane

†

12.86

± 0.27 34.06** ± 1.70 4.43 ± 0.57 14.05** ± 1.52 0.72 ± 0.07 1.15** ± 0.12

Kerosene

†

13.42

± 0.76 22.99** ± 1.20 1.95 ± 0.06 4.01** ± 0.20 0.57 ± 0.03 2.19* ± 0.01

Saffola oil

†

11.47

± 1.21 19.71** ± 2.08 2.61 ± 0.07 4.61 ± 0.14

1.68

± 0.08 2.46** ± 0.13

†

ml l

-1

µg DCIP reduced min

–

(mg protein)

–1

nmol of formaldehyde liberated min

–1

(mg protein)

–1

µmol of acetaldehyde

liberated min

–1

(mg protein)

–1

Significantly different from control at * P < 0.05, ** P < 0.001 by One way analysis of variance and Tukey-Kramer multiple
comparisons test.

In order to investigate the inducibility of these activi-
ties, 96 h mycelium was incubated for 18 h in mineral
salts medium containing an inducer. All compounds
listed in Table 2 gave statistically significant increases
in NADPH-DCIP reductase, aminopyrine N-demethylase
and kerosene biodegradation activities.

Discussion

The results obtained in these investigations indicate
that A. ochraceus utilized kerosene when it was the sole
source of carbon and energy. When a major oil spill
occurs in marine and freshwater environments, the
supply of carbon is dramatically increased, and the
availability of nitrogen and phosphorus generally be-
comes a limiting factor for oil degradation (Prince et al.
2003). It is therefore of importance to note that for
A. ochraceus YE medium gave greater biodegradation of
kerosene than phosphorous medium (Fig. 1). Recently,
Wemedo et al. (2002) recorded the genera of fungi such
as  Penicillium, Aspergillus, Fusarium, Rhizopus and  Mucor
which are associated with kerosene-polluted soil. The
decrease in pH observed in present study might be due
to the formation of aldehyde and carboxylic acids dur-
ing biodegradation of kerosene, which is supported
by the FTIR data. The detection of aminopyrine

N-demethylase and NADPH-DCIP reductase, together
with decrease in the activity of these enzymes in pres-
ence of carbon monoxide (Table 1), can be taken as an
indicator for the presence of cytochrome P450. Serratia
marcescens NCIM 5115 also showed the presence of these
biotransformation enzymes, which were inducible by
some polycyclic aromatic hydrocarbons examined by

Salokhe and Govindwar (1999). The study of induction
in A. ochraceus NCIM 1146 (Table 2) demonstrated statis-
tically significant increase in all three activities in
the presence of carbon sources studied. These organic
compounds (camphor, n-hexane, naphthalene, 1,2-di-
methoxybenzene) also showed induction of kerosene
degradation activity in P. desmolyticum NCIM

2112

(Kalme et al. 2007). These inducers might have increased
the amount of soluble cytochrome P450 in the

A. ochraceus. In Rhodococcus sp., compounds that induced
emulsifying ability simultaneously induced the cyto-
chrome P-450 containing alkane oxidizing system
(Bredholt et al. 2002).
  In the present study, emulsification was stable in
both the medium up to 20 days. This emulsification
action overcomes the interfacial area limitation and
also permits effective contact between cells and hydro-
carbons (Breuil and Kushner 1980, Rittman and John-
son 1989). Saffola oil in presence of kerosene showed
more emulsification compared to the other carbon
sources. Saffola oil might have resulted in statistical
significant induction because of increased emulsifica-
tion with enhanced bioavailability of kerosene to the
mycelium. In Pseudomonas sp. F21, isolated from a min-
eral medium containing Arabian crude oil, degrades all
the n-alkanes and branched alkanes of low molecular
weight, and also selective depletes methylated naphtha-
lenes, phenanthrenes, chrysenes and pyrenes (Bayona
et al. 1986, Cho et al. 1997). Such strains, as these are
having a mixed-function oxidase system inducible by
n-alkanes as well as PAH, have great potential for the
biodegradation of crude oil and kerosene.
  As alkanes comprise one of the major components of
kerosene, the detection of acetaldehyde production as a

Journal of Basic Microbiology 2007, 47, 400 – 405

Biodegradation of kerosene by A. ochraceus 405

www.jbm-journal.com

result of alkane oxidation would offer an approach to
detect the microbial activity against kerosene. As a
rapid and suitable method of the screening for micro-
organisms that degrade diesel, Saadoun (2002) modified
the method of Jacobs et al. (1983) used to determine the
ability of different bacterial strains in degrading diesel
fuel by transforming the diesel fuel to alcohol. Saadoun
used the diesel as a source of alkane, from which alco-
hol dehydrogenase produces acetaldehyde. NADH gen-
erated in this reaction was used as the reducing equiva-
lent for DCPIP. In present study, the detection of
acetaldehyde in the test sample can be used as a rapid
assay in screening fungi, for their ability to degrade
kerosene (Kalme et al. 2007). The liberated acetaldehyde
is detected by using Nash reagent in assaying the kero-
sene biodegradation, where the reaction product of
acetaldehyde and Nash reagent, diacetyldihydrocol-
lidine, shows maximum absorption at 388 nm. FTIR
analysis and induction studies carried out supports a
possible role for A. ochraceus NCIM-1146 in catabolism of
variable chain length alkanes and PAHs. Since it de-
grades alkanes as well as PAHs, this strain could be
used effectively to reduce the hydrocarbon load.
Although

A. ochraceus isolates most commonly pro-

duce ochratoxin A, which displays nephrotoxic, hepa-
totoxic, teratogenic and immunosuppressive properties
(O’Callaghan et al. 2006), however the production of
ochratoxin A is not above the level of detection
(0.01

µg ml

–1

) (Bayman et al. 2002

)

. Study of toxic effects

of liberated products is a matter of further investigation.

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