Indian Journal of Experimental Biology

Vol. 47, September 2009, pp. 760-765

Isolation of hydrocarbon degrading bacteria from soils contaminated with

crude oil spills

Anupama Mittal* & Padma Singh

Department of Microbiology, Kanya Gurukul Mahavidyalaya (Gurukul Kangri University),
Jwalapur, Haridwar 249 407, India
Received 24 December 2008; revised 19 May 2009

A feasibility study was conducted to evaluate the capability of bacterial strains to degrade crude oil under in vitro
conditions. Pseudomonas strain PS-I could degrade alkanes (70.69%) and aromatics (45.37%). Alkanes and aromatic
fractions separated by column chromatography were analyzed by gas chromatography. In case of Pseudomonas strain PS-I,
nC17/Pr, nC18/Ph ratios decreased from 2.5100 to 0.1232 and from 7.2886 to 0.3853, respectively. It was concluded that out
of the isolated strains, Pseudomonas strain PS-I, PS-II and PS-III were comparatively better and potent hydrocarbon
degraders. Pseudomonas strain PS-I was almost comparable with standard strain of Acinetobacter calcoaceticus in crude oil
biodegradation potency.

Keywords: Acinetobacter calcoaceticus, Bioremediation, Crude Oil, Pseudomonas

Oil spills have become a global problem particularly
in industrialized and developing countries. Attention
has been focused on the marine environment, because
oceans and estuaries have generally been the sites of
the largest and most dramatic spills1. Apparently
inevitable spillages, which occur during routine
operations of crude oil production, refining,
distribution and as a consequence of acute accidents,
have generated continuous research interest in this
field2. The parameters typically measured in
laboratory tests of bioremediation efficacy include
enumeration of microbial populations3-5,
determination or fate of hydrocarbon degradation
(disappearance of individual hydrocarbons and/or
total hydrocarbons)6. Undoubtedly, the most direct
measure of bioremediation efficacy is the monitoring
of hydrocarbon disappearance rates7. Generally type
and identity of fresh to biodegraded oils and
petroleum products can be readily revealed their GC-
FID traces especially where the biodegradation of
spilled oil or petroleum product is heavy and
background hydrocarbon levels are low in an
impacted environment. In addition to measuring TPH
(Total petroleum hydrocarbons) in samples, GC-FID
_____________________
*Address for correspondence: Department of Microbiology,
Kanya Gurukul Mahavidylaya, Gurukul Kangri University,
Haridwar 249407, India
Telephone: (91) 9999882926 (Mobile)
chromatograms provide a distribution pattern of
petroleum hydrocarbons {e.g., carbon range and
profile of UCM (Unresolved complex mixture)},
fingerprints of the major oil components (e.g.,
individual resolved n-alkanes and major isoprenoids),
and information on the biodegradation extent of the
spilled oil. Comparing biodegradation indicators
(such as nC17/Pristane nC18/Phytane) for the spilled oil
to the source oil can also be used to monitor the effect
of microbial degradation on the loss of hydrocarbons
at the spill site8,9.
The objective of the study is to assess the crude oil
biodegradation potential of selected five bacterial
strains under in vitro conditions. Degradation studies
to be carried out with different isolates at varying
interval of time will help to find out the most potent
hydrocarbon degrading strains, which can be used for
any bioaugmentation studies during bioremediation.
Materials and Methods
Soil samplesSamples (500g) contaminated with
oil since 1986 (when oil production began in Lingala
oil field, India) used for isolation of hydrocarbon
utilizing microorganisms, were collected from oil
production site of ONGC (oil wells from Lingala Oil
field Project, India). Subsurface soil sample
contaminated with refined product of crude oil viz.
diesel and lubricating oil was collected from local
 MITTAL & SINGH: ISOLATION OF HYDROCARBON DEGRADING BACTERIA
area, Haridwar, India. The soil samples were collected
in pre-sterilized sample bottles and also in whirl pack
bags following aseptic conditions. The samples duly
labeled were stored at - 4C for further analysis.
Crude oil contentsPhysico-chemical properties
of crude oil of Lingala well # X were - depth (m),
1893.5-95; Formation, Sand stone; Type, Oil; API
Gravity, 35.6; Saturates, 67.3%; Aromatic, 21.2%;
NSO (Nitrogen, Sulphur, Oxygen containing
compounds), 11.2%; Asphaltenes, 0.3%; Sat/Aro,
3.17; HC/NSO, 7.7; Pr/Ph, 4.3; Pr/nC17, 1.693;
Ph/nC18, 0.266; Pr+nC17/ Ph+nC18, 1.434; nC21+nC22/
nC28+nC29, 1.219.
Medium used and maintenance of
microorganismsSoil sample (1 g) from each source
was suspended and vortexed with distilled water (10
mL). The suspension was allowed to settle down and
supernatant (5 mL) was used as inoculum, in 100 mL
of Luria Bertani broth containing crude oil (1%) in an
Erlenmeyer flask. The flask was incubated for 48 h at
37C on a rotary shaker at 100 rpm. Three successive
subculturing were done on the same medium
containing crude oil (1%). After three subculturing
steps broth was centrifuged at 5,000 rpm for 10 min
and the cell pellets were obtained. The cell pellets
were washed with 0.1M phosphate buffer solution
(pH-6.8) twice, after suspending in mineral salt
medium, cell pellets were inoculated in 100 mL of
mineral salt medium in an Erlenmeyer flask,
containing 1% crude oil as a single source of carbon
and energy. The mineral salt medium10 contained (g):
KNO3, 1.0; MgSO4.7H2O, 1.0; CaCl2.6H2O, 0.1;
FeSO4, 0.05; trace element sol, 250 mL; phosphate
buffer (1M; pH-6.8), 20 mL; and distilled water, 980
mL. Trace element solution11comprised(g) - SnCl2,
0.05; KI, 0.05; LiCl, 0.05; MnSO4.4H2O, 0.08; HBO3,
0.50; ZnSO4.7H2O, 0.10; CoCl2.6H2O, 0.10;
NiSO4.6H2O, 0.10; BaCl2, 0.05; Ammonium
molybdate, 0.05; and distilled water, 1000 mL (All
salts were dissolved in defined sequence only). Pure
hydrocarbon degrading strains were isolated on
petroleum agarose plates. In the preparation of
petroleum agarose plates 1-2 drops of sterile crude oil
was evenly spread with glass spreader, so that a film
of crude oil got absorb over the entire agarose surface
of mineral medium in the petriplates and then
inoculum was spread with spreader on the medium.
The plates were incubated at 37C for one week in an
incubator. Pure and representative colonies were
transferred to slants for preservation.
761
Preservation and subculture of the strainsThe
Isolated strains were preserved in 25% v/v glycerol
solution at -70C. For day-to-day experimentation
strains were maintained on nutrient agar slants at 4C
in refrigerator and sub-cultured at an interval of 30
days.
Biodegradation studiesLaboratory biodegrada-
ation studies were carried out under optimized
conditions as described elsewhere12 for assessing the
hydrocarbon degradation potential of isolated strains.
Mineral salt medium (200mL) in each, was prepared
in three sets of 24 Erlenmeyer flasks (1000mL
capacity) and autoclaved. These 24 Erlenmeyer flasks
were grouped in sets of six flasks and subjected to
treatment for 15, 30, 45, and 60 days Sterile crude oil
(2.4 g, sterilized by filtration, Millipore size, 0.25
mm) of Lingala well # X was added in each flask.
One mL inoculum (Bacterial number was adjusted to
give initial cell number 1 108 CFU ml-1) of each of
hydrocarbon degraders, standard strain (Acinetobacter
calcoaceticus) and test isolates (Pseudomonas strains
PS-I, PS-II, PS-III, PS-IV and Bacillus) was added in
sets of six flasks, respectively. Flasks were incubated
in incubator shaker at 30C, at 100 rpm. After desired
interval of time, the flasks were taken out and
bacterial activities were stopped by adding 1% 1N-
HCl. For extraction of crude oil, 50 mL of culture
broth was mixed with 50 mL petroleum ether :
acetone (1:1) in a separating funnel and was shaken
vigorously to get a single emulsified layer. Acetone
was then added to it and shaken gently to break the
emulsification, which resulted in three layers. Top
layer was a mixture of petroleum ether, crude oil and
acetone; clumping cells make the middle layer and the
bottom aqueous layer contains acetone, water and
biosurfactant in soluble form. The lower two layers
were separated out while top layer containing
petroleum ether mixed with crude oil and acetone was
taken out in a clean beaker. The extracted oil was
passed through anhydrous sodium sulphate to remove
moisture. The petroleum ether and acetone was
evaporated on a water bath. The gravimetric
estimation of residual oil left after biodegradation was
made by weighing the quantity of oil in a tared vial.
The biodegraded crude oils were further fractioned for
their gross and molecular composition.
Column chromatography Saturates, aromatics
and NSO fractions were fractionated quantitatively by
column chromatography13. Silica gel (60-120 mesh)
and alumina neutral were activated at 150C for 24 h
762 INDIAN J EXP BIOL, SEPTEMBER 2009
and cooled in desiccators. The glass column (Internal
diameter 1.1 cm, length-65 cm and reservoir capacity
60-100 mL) was packed by placing a thin cotton plug
at the bottom. The slurry of 20 g activated alumina was
filled, followed by 20 g silica gel. The column was
washed with petroleum ether. The oil sample (approx.
100 mg/known weight) was dissolved in chloroform,
adsorbed on silica gel and evaporated the excess
chloroform at 80C. The adsorbed sample was charged
at the top and eluted saturates with 60 mL of petroleum
ether (40-60C), aromatics with 90 mL benzene and
NSO with 60 mL methanol, respectively. The solvents
were evaporated in rotary vacuum evaporator at 60C.
Each fraction was transferred in tared sample vials,
dried and weighed.
Gas chromatographic analysisChemito-1000 gas
chromatograph was used for gas chromatographic
analysis of whole oils, saturate and aromatic fractions on
a FID detector under the conditions mentioned below.
Conditions for saturate (alkanes)Column, BP-5
(30 m 0.25 mm); Initial temperature, 80C; final
temperature, 300C; ramp rate, 4C per min; carrier
gas, nitrogen (40 mL/min); injection temperature,
310C.
Conditions for aromatic analysisColumn, BP-5
(60 m X 0.25 mm); Initial temperature, 80C; final
temperature, 300C; ramp rate, 3C per min; carrier
gas, nitrogen (30 mL/min); injection temperature,
310C.
Identification of isolated potent hydrocarbon
degraderAfter examining the chromatograms, a
greater decrease in front-end boiling range and
residual carbon of the oil samples degraded by
Pseudomonas strain PS-I suggested that this strain
was responsible for the breakdown of higher
molecular weight constituents of the oil compared to
two Pseudomonas strain PS-II, PS-III. Therefore,
identification of Pseudomonas strain PS-I (the potent
strain) was carried out by biochemical test as well as
by 16Sr-DNA genes sequencing from The Energy and
Resources Institute (TERI), New Delhi, India.
16S rDNA sequencing of PS-Ifor molecular
characterizationBacterial strain PS-I was identified
by 16S rDNA sequence structure. The 500 bases
sequence of 16S rDNA was obtained by sequencing.
The electrophoregram of partial sequence was aligned
with blast search of NCBI, RDP and microseqTM
databases. The sequence aligned gave 97% similarity
with Pseudomonas putida. Out of 474 bases matched
with databases, 468 bases gave homology with
Pseudomonas putida. Therefore, bacterial strain PS-I
was identified as Pseudomonas putida.
Results and Discussion
Test microorganismsTwenty microbial strains
capable of using hydrocarbon as sole source of
carbon, were isolated of which 11 (A1-A11) were
recorded from the soil sample of oil production site of
Lingala oil field and other 9 (B1-B9) from oil
contaminated soil of a nearby garage of Hardwar.
Similarly, in preliminary study 57 oil degrading
bacteria were isolated from oil impregnated soil in
search of potent hydrocarbon degrader14. These
isolates were purified from soil samples on the basis
of their colony morphology, texture, growth,
temperature and hydrocarbon utilization ability.
Preliminary screening and laboratory
biodegradation studyThe pure hydrocarbon
utilizing bacterial strains were isolated first in mineral
salt broth containing 1% crude oil (Lingala well # X)
and later on petroleum agarose plates. The isolated
strains were examined further for substrate utilization.
Over the year, substrate specificity in newly isolated
organisms has been a routine diagnostic test for
speciation and identification15. Microbes isolated on
petroleum agarose plate have been tested for its
ability to utilize the major classes of hydrocarbons
viz., gaseous alkanes, n-paraffins, aromatics etc.,
which has been described elsewhere12. After substrate
utilization study, it was concluded that out of 20, only
five strains (A2, A7, B1, A9, B5) were potent
hydrocarbon degraders and capable of degrading
practically most of the representative fractions of
petroleum. After morphological identification and
number of biochemical tests, these isolates (A2, A7,
B1, A9, and B5) were identified as Pseudomonas
strains PS-I, PS-II, PS-III, PS-IV and Bacillus,
respectively. Pseudomonas spp. comprised the first
most numerous group than other taxa among
petroleum degraders isolated from an oil-
contaminated site in colgate creek in Baltimore
Harbor. It was 27% of the total isolates identified16.
Pseudomonas is most frequently reported and, so far,
most studied of hydrocarbon degradation genus17-19.
Laboratory biodegradation studies of crude oil (whole
oil) have been conducted up to a period of 60 days,
under optimized conditions of pH, temperature and
crude oil concentration on Lingala well # X crude oil
by deploying standard strain of Acinetobacter
 MITTAL & SINGH: ISOLATION OF HYDROCARBON DEGRADING BACTERIA 763

calcoaceticus as well as isolated Pseudomonas strains
PS-I, PS-II, PS-III, PS-IV and Bacillus. The
biodegradation effect was seen after every 15 days.
The concentration of oil before and after
biodegradation has been estimated gravimetrically.
The six different strains under study have shown
varying biodegradation potential (Table 1). Out of
four Pseudomonas strains, PS-I proved to be the most
potent hydrocarbon degrader and crude oil
degradation gradually increased from 29.77% in 15
days to 57.13% in 60 days. The study indicates that
this strain was more or less as potent hydrocarbon
degrader the standard strain. Gas chromatographic
(GC) analysis of the crude oil and its fractions
(saturate and aromatic) has been used to determine the
biodegradability of crude oil20.
Effect of biodegradation on fractions of crude
oilEffect of biodegradation on alkane, aromatics
and NSO + asphaltene fractions by standard strain and
various isolated strains has been studied for 60 days.
Effect was seen at time interval of 15 days (Table 2).
Alkanes and aromatic fractions separated by column
chromatography were analyzed by gas
chromatography. Gas chromatogram of alkane
fraction of crude oil, biodegraded by standard strain
Acinetobacter calcoaceticus revealed extensive
biodegradation in 60 days. Most of the lighter alkane
fractions were metabolized. The most commonly used
biodegradation parameters i.e. nC17/Pr, nC18/Ph and
Pr/Ph were calculated from the gas chromatograms
(Table 3). The ratios of Pr/Ph represent isoprenoidal
alkanes and are considered as recalcitrant and
therefore, are also referred to as biomarker
parameter9,21. The visual examination of gas
chromatogram of alkane fraction of crude oil
biodegraded by isolated Pseudomonas strain PS-I
indicated extensive biodegradation in 60 days, which
was almost comparable with standard strain,
Acinetobacter calcoaceticus. The results clearly
revealed the extent of biodegradation as nC17/Pr ratio
decreased from 2.5100 to 0.1232 in Pseudomonas
strain PS-I in 60 days. Similarly, nC18/Ph ratio also
decreased from 7.2886 to 0.3853. The ratio of Pr/Ph
indicated meager degradation of pristane.
Index used to monitor the progress of
biodegradation is the rate of decrease in the ratios of
nC17/Pristane and nC18/Phytane. The decreasing rate
in the ratios of nC17/Pristane and nC18/Phytane has
been used to monitor the progress of biodegradation
during the EPA Alaska oil spill bioremediation
project22. The application of this concept was based
on the principle that during biodegradation, decrease
of total oil residues could occur because of other non-
biological processes. Thus, changes in hydrocarbon
composition that are indicative of biodegradation
must be measured accurately and this is done
historically by examining the weight ratio between
hydrocarbons known to be readily biodegradable,
such as the C17 and C18 alkanes, and those that
biodegrade slowly, such as the branched alkanes
(Pristane and Phytane), but with very close
chromatographic behavior6. The two isolates
(Acinetobacter calcoaceticus and Pseudomonas strain
PS-I) showed a similar trend in the decreasing ratios
of nC17/Pristane and nC18/Phytane up till the stage that
the recalcitrant Pristane and Phytane were degraded.
Gas chromatogram of alkane fraction of crude oil
biodegraded by isolated Pseudomonas strain PS-II,
Pseudomonas strain PS-III indicated moderate
biodegradation and Pseudomonas strain PS-IV,
Bacillus showed less biodegradation in 60 days.
Nevertheless, other parameters indicated progressive

Table 1Laboratory biodegradation studies of crude oil (whole oil)

[Values are the mean SE of 3 replications]
Strains Acinetobacter Pseudomonas Bacillus
calcoaceticus
Strain-I Strain-II Strain-III Strain-IV
Incub.Period(d) F D F D F D F D F D F D
0d 2.463 - 2.489 - 2.474 - 2.467 - 2.49 - 2.454 -
[Initial conc. (g)]
15d 1.6260.54 33.98 1.7480.80 29.77 1.9560.75 20.94 2.0740.75 15.93 2.3480.75 5.7 2.2890.75 6.72
30d 1.1620.67 52.82 1.3130.56 47.25 1.7430.45 29.56 1.7990.69 27.07 2.2010.59 11.61 2.0880.76 14.91
45d 1.0630.89 56.84 1.2020.66 51.71 1.5660.74 36.7 1.6870.37 31.62 1.9640.23 21.24 2.0050.69 18.3
60d 0.9660.32 60.78 1.0670.78 57.13 1.3830.23 44.1 1.4730.49 40.29 1.8140.53 27.15 1.9300.82 21.35
F-Final Conc. (g); Ddegradation (%); d-days
764 INDIAN J EXP BIOL, SEPTEMBER 2009

Table 2Effect of biodegradation on various fractions of crude oil

[Values are the mean SE of 3 replications]
Strain Treatment period Alkane Aromatic NSO+Asphalt
(days) R D R D R
Acinetobacter 0 1.6580.34 - 0.5220.45 - 0.2830.65
calcoaceticus 15 0.8970.45 45.89 0.4400.56 15.7 0.2890.35
30 0.4940.23 70.21 0.3760.34 27.96 0.2920.30
45 0.4530.12 72.68 0.3380.28 35.24 0.2720.17
60 0.4130.09 75.09 0.2730.22 47.7 0.2800.18
Pseudomonas strain I 0 1.6750.78 - 0.5280.34 - 0.2860.12
15 0.9850.48 41.19 0.4710.14 10.80 0.2920.07
30 0.6240.33 62.75 0.4140.09 21.59 0.2750.11
45 0.5390.17 67.82 0.3650.12 30.87 0.2980.13
60 0.4910.20 70.69 0.2890.04 45.37 0.2870.06
Pseudomonas strain II 0 1.6750.67 - 0.5240.24 - 0.2850.07
15 1.1890.84 28.59 0.4910.15 6.30 0.2760.03
30 1.0130.53 38.16 0.4410.19 15.84 0.2890.14
45 0.8960.08 46.19 0.3960.05 24.43 0.2740.03
60 0.7520.30 54.83 0.3420.08 34.73 0.2890.11
Pseudomonas strain III 0 1.6600.96 - 0.5230.15 - 0.2840.14
15 1.2850.73 22.59 0.4920.23 5.93 0.2790.07
30 1.0730.74 35.36 0.4510.08 13.77 0.2750.05
45 0.9770.56 41.14 0.4120.05 21.22 0.2980.11
60 0.8380.48 49.52 0.3660.04 30.02 0.2690.03
Pseudomonas strain IV 0 1.6760.77 - 0.5280.25 - 0.2860.25
15 1.5610.56 6.86 0.4980.28 5.68 0.2890.18
30 1.4190.62 15.33 0.4850.18 8.14 0.2970.02
45 1.2290.45 26.67 0.4610.13 12.69 0.2740.09
60 1.1030.38 34.19 0.4300.05 18.56 0.2810.07
Bacillus 0 1.6520.92 - 0.5200.32 - 0.2820.07
15 1.4890.43 9.87 0.5040.16 3.08 0.2960.11
30 1.3170.33 20.28 0.5940.13 5.00 0.2700.07
45 1.2470.52 24.52 0.4740.21 8.85 0.2840.04
60 1.2040.21 27.12 0.4480.16 13.85 0.2780.08
Rremaining Conc. (g); Ddegradation (%)
Table 3Biodegradation indices
Acinetobacter Pseudomonas
Bacillus
calc. PS-I PS-II PS-III PS-IV
Parameters Initial (0d) 30d 60d 30d 60d 30d 60d 30d 60d 30d 60d 30d 60d
nC17/Pr 2.510 0.140 0.120 0.143 0.123 0.182 0.143 0.185 0.151 0.272 0.199 0.363 0.262
nC18/Ph 7.289 0.433 0.350 0.424 0.385 0.665 0.427 0.706 0.436 0.881 0.493 1.932 1.185
Pr/Ph 2.891 2.885 2.620 2.860 2.694 2.809 2.722 2.802 2.763 2.883 2.871 2.891 2.890

bacterial attack upon the crude oil. The ratios of
isoprenoids, pristane and phytane increase to
North West suggesting depletion of normal
paraffins 23.
Gas chromatograms of standard strain and
isolated Pseudomonas strains PS-I, PS-II, PS-III
revealed that the lower aromatic fractions were
removed over a period of time and higher aromatic
hydrocarbon abundance was seen. This clearly
indicated that Pseudomonas strains PS-I, PS-II and
PS-III were comparatively better and potent
aromatic hydrocarbon degraders. Out of these
Pseudomonas strains PS-I was almost comparable
with standard strain of Acinetobacter calcoaceticus.
 MITTAL & SINGH: ISOLATION OF HYDROCARBON DEGRADING BACTERIA
Acknowledgement
Authors are thankful to Dr Rajeev Sharma, ONGC,
Dehradun for their constant help during this investigation.
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