ISOLATION AND CHARCTERIZATION OF
HYDROCARBON DEGRADING BACTERIA
ISOLATED FROM SOIL CONTAMINATED
WITH ENGINE OIL.
SUBMITTED TO
VEER NARMAD SOUTH GUJARAT UNIVERSITY
GUIDED BY :
Miss. Priya Bande
Miss. Neha Vora
MITCON BIOPHARMA CENTER,
PUNE-411005,
MAHARASTRA,
INDIA.
SUBMITTED BY:
Hinal Desai
M.Sc. Biotechnology,
Department of Biotechnology,
Veer Narmad South Gujarat University,
Surat-395007.
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1. INTRODUCTION
Petroleum-based products are the major source of energy for industry and daily
life. Leaks and accidental spills occur regularly during the exploration, production,
refining, transport, and storage of petroleum and petroleum products. Release of
hydrocarbons into the environment whether accidentally or due to human activities is a
main cause of water and soil pollution. Soil contamination with hydrocarbons causes
extensive damage of local system since accumulation of pollutants in animals and plant
tissue may cause death or mutations. The technology commonly used for the soil
remediation includes mechanical, burying, evaporation, dispersion, and washing.
However, these technologies are expensive and can lead to incomplete decomposition of
contaminants. The process of bioremediation, defined as the use of microorganisms to
detoxify or remove pollutants due to their diverse metabolic capabilities is an evolving
method for removal and degradation of many environmental pollutants including the
products of petroleum industry. In addition, bioremediation technology is believed to be
noninvasive and relatively cost-effective. Biodegradation by natural populations of
microorganisms represents one of the primary mechanisms by which petroleum and other
hydrocarbon pollutants can be removed from the environment and is cheaper than other
remediation technologies.
The success of oil spill bioremediation depends on one’s ability to establish
and maintain conditions that favor enhanced oil biodegradation rates in the contaminated
environment. One important requirement is the presence of microorganisms with the
appropriate metabolic capabilities. If these microorganisms are present, then optimal rates
of growth and hydrocarbon biodegradation can be sustained by ensuring that adequate
concentrations of nutrients and oxygen are present and that the pH is between 6 and 9.
The physical and chemical characteristics of the oil and oil surface area are also important
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determinants of bioremediation success.
[Fig.1.1 A bird covered in the oil spill]
There are the two main approaches to oil spill bioremediation:
(a) bioaugmentation, in which known oil-degrading bacteria are added supplement the
existing microbial population, and
(b) biostimulation, in which the growth of indigenous oil degraders is stimulated by the
addition of nutrients or other growth-limiting cosubstrates.
Most existing studies have concentrated on evaluating the factors affecting oil
bioremediation or testing favored products and methods through laboratory studies.
Only limited numbers of pilot scale and field trials have provided the most convincing
demonstrations of this technology. The scope of current understanding of oil
bioremediation is also limited because the emphasis of most of these field studies has
been given on the evaluation of bioremediation technology for dealing with large-scale oil
spills on marine shorelines.
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1.1 WHAT ARE HYDROCARBONS????
A hydrocarbon is an organic compound consisting entirely of hydrogen and
carbon. They can be straight-chain, branched chain or cyclic molecules. Hydrocarbon
derivatives are formed when there is a substitution of a fuctional group at one or more
positions. An almost unlimited number of carbon compounds can be formed by the
addition of a functional group to hydrocarbon.
[Fig 1.2 Derivatives of hydrocarbon]
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1.2
TYPES OF HYDROCARBON
There are four types of hydrocarbon:
1.
2.
3.
4.
Saturated hydrocarbons
Unsaturated hydrocarbons
Cycloalkanes and
Aromatic hydrocarbons
1. Saturated hydrocarbons (alkanes) are the simplest of the hydrocarbon
species and are composed entirely of single bonds and are saturated with
hydrogen. The general formula for saturated hydrocarbons is
CnH2n+2. Saturated hydrocarbons are the basis of petroleum fuels and are
found as either linear or branched species. Branched hydrocarbons can
be chiral. Chiral saturated hydrocarbons constitute the side chains of
biomolecules such as chlorophyll and tocopherol. Hydrocarbons with
the same molecular formula but different structural formulae are called
structural isomers.
2. Unsaturated hydrocarbons have one or more double or triple bonds
Between carbon atoms. Those with double bonds are called alkenes with
formula CnH2n. Those containing triple bonds are called alkynes, with
general formula CnH2n-2.
3. Cycloalkanes are hydrocarbons containing one or more carbon rings to
which hydrogen atoms are attached.
4. Aromatic hydrocarbons, also known as arenes, are hydrocarbons that
have at least one aromatic ring.
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[Fig 1.3 Types of Hydrocarbon]
Hydrocarbon can be gases (e.g. methane and propane), liquids (e.g. hexane and benzene),
waxes or low melting solids (e.g. paraffin wax and naphthalene) or polymer (e.g.
polyethylene, polypropylene and polystyrene).
[ Fig 1.4 Hydrocarbon Methane]
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1.3 GENERAL PROPERTIES OF HYDROCARBONS
Because of differences in molecular structure, the empirical formula remains different
between hydrocarbons, the amount of bonded hydrogen lessens in alkenes and alkynes
due to the "self-bonding" or catenation of carbon preventing entire saturation of the
hydrocarbon by the formation of double or triple bonds. This inherent ability of
hydrocarbons to bond to themselves is referred to as catenation and allows hydrocarbon
to form more complex molecules, such as cyclohexane,and in rarer cases, arenes such
as benzene. This ability comes from the fact that bond character between carbon atoms is
entirely non-polar, in that the distribution of electrons between the two elements is
somewhat even due to the same electronegativity values of the elements, and does
not result in the formation of an electrophile.
Hydrocarbons are hydrophobic and are lipids.
Some hydrocarbons also are abundant in the solar system. Lakes of liquid methane and
ethane have been found on Titan, Saturn's largest moon. Hydrocarbons are also abundant
in nebulae forming polycyclic aromatic hydrocarbons - PAH compounds.
1.4 USES OF HYDROCARBONS
Hydrocarbons are one of the Earth's most important energy resources. The predominant
use of hydrocarbons is as a combustible fuel source. In their solid form, hydrocarbons
take the form of asphalt. Mixtures of volatile hydrocarbons are now used in preference to
the chlorofluorocarbons as a propellant for aerosol sprays, due to chlorofluorocarbon's
impact on the ozone layer. Methane [1C] and ethane [2C] are gaseous at ambient
temperatures and cannot be readily liquefied by pressure alone. Propane [3C] is however
easily liquefied, and exists in 'propane bottles' mostly as a liquid.Butane [4C] is so easily
liquefied that it provides a safe, volatile fuel for small pocket lighters. Pentane [5C] is a
clear liquid at room temperature, commonly used in chemistry and industry as a powerful
nearly odorless solvent of waxes and high molecular weight organic compounds,
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including greases. Hexane [6C] is also a widely used non-polar, non-aromatic solvent, as
well as a significant fraction of common gasoline. The [6C] through [10C] alkanes,
alkenes and isomeric cycloalkanes are the top components of gasoline, naphtha,
jetfuel and specialized industrial solvent mixtures. With the progressive addition of
carbon units, the simple non-ring structured hydrocarbons have higher viscosities,
lubricating indices, boiling points, solidification temperatures, and deeper color. At the
opposite extreme from [1C] methane lie the heavy tars that remain as the lowest
fraction in a crude oil refining retort. They are collected and widely utilized as roofing
compounds, pavement composition, wood preservatives and as extremely high viscosity
sheer-resisting liquids.
1.5 MICROBIAL DEGRADATION OF PETROLEUM
HYDROCARBON
Biodegradation of petroleum hydrocarbons is a complex process that depends on the
nature and on the amount of the hydrocarbons present. Petroleum hydrocarbons can be
divided into four classes: the saturates, the aromatics, the asphaltenes (phenols, fatty
acids, ketones, esters, and porphyrins), and the resins (pyridines, quinolines, carbazoles,
sulfoxides, and amides). Different factors influence hydrocarbon degradation. One of the
important factors that limit biodegradation of oil pollutants in the environment is their
limited availability to microorganisms. Petroleum hydrocarbon compounds bind to soil
components, and they are difficult to be removed or degraded. Hydrocarbons differ in
their susceptibility to microbial attack. The susceptibility of hydrocarbons to microbial
degradation can be generally ranked as follows: linear alkanes > branched alkanes > small
aromatics > cyclic alkanes. Some compounds, such as the high molecular weight
polycyclic aromatic hydrocarbons (PAHs), may not be degraded at all. Microbial
degradation is the major and ultimate natural mechanism by which one can cleanup
the petroleum hydrocarbon pollutants from the environment. Hydrocarbons in the
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environment are biodegraded primarily by bacteria, yeast, and fungi. The reported
efficiency of biodegradation ranged from 6% to 82% for soil fungi, 0.13% to 50% for
soil bacteria, and 0.003% to 100% for marine bacteria. Many scientists reported that
mixed populations with overall broad enzymatic capacities are required to degrade
complex mixtures of hydrocarbons such as crude oil in soil, fresh water, and marine
environments. Bacteria are the most active agents in petroleum degradation, and they
work as primary degraders of spilled oil in environment . Several bacteria are even known
to feed exclusively on hydrocarbons. In earlier days, the extent to which bacteria, yeast,
and filamentous fungi participate in the biodegradation of petroleum hydrocarbons was
the subject of limited study, but appeared to be a function of the ecosystem and local
environmental conditions. Though algae and protozoa are the important members of the
microbial community in both aquatic and terrestrial ecosystems, reports are scanty
regarding their involvement in hydrocarbon biodegradation. brown alga, and two diatoms
could oxidize naphthalene.
1.6 LIST OF MICROORGANISMS INVOLVED IN
HYDROCARBON DEGRADATION
 BACTERIA











Arthrobacter
Burkholderia
Mycobacterium
Sphingomonas
Pseudomonas fluorescens
Pseudomonas aeruginosa
Pseudomonas alcaligens
Staphylococcus sp.
Bacillus subtilis
Bacillus sp.
Alcaligenes sp.
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




Flavobacterrium sp.
Acinetobacterium sp.
Micrococcus roseus
Corynebacterium sp.
Xanthomonas sp.
 Fungi







Amorphoteca sp.
Aspergillus
Cephalosporium
Penicillium
Neosartorya sp.
Talaromyces sp.
Graphium sp.
 Yeast




Candida sp.
Yorrowia sp.
Pichiya sp.
Geotrichum sp.
1.7 MECHANISM OF PETROLEUM HYDROCARBON
DEGRADATION
The most rapid and complete degradation of the majority of organic pollutants is brought
about under aerobic conditions. Figure 1.5 shows the main principle of aerobic
degradation of hydrocarbons. The initial intracellular attack of organic pollutants is an
oxidative process and the activation as well as incorporation of oxygen is the enzymatic
key reaction catalyzed by oxygenases and peroxidases. Peripheral degradation pathways
convert organic pollutants step by step into intermediates of the central intermediary
metabolism, for example, the tricarboxylic acid cycle. Biosynthesis of cell biomass occurs
from the central precursor metabolites, for example, acetyl-CoA, succinate, pyruvate.
Sugars required for various biosyntheses and growth are synthesized by gluconeogenesis.
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[Fig. 1.5 Main principle of aerobic degradation of hydrocarbon by
microorganisms]
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1.8 ENZYMES PARTICIPATING IN HYDROCARBON
DEGRADATION
Cytochrome P450 alkane hydroxylases constitute a super family of ubiquitous Hemethiolate Monooxygenases which play an important role in the microbial degradation of
oil, chlorinated hydrocarbons, fuel additives, and many other compounds. Depending on
the chain length, enzyme systems are required to introduce oxygen in the substrate to
initiate biodegradation [Table 1]. Higher eukaryotes generally contain several different
P450 families that consist of large number of individual P450 forms that may contribute
as an ensemble of isoforms to the metabolic conversion of given substrate. In
microorganisms such P450 multiplicity can only be found in few species. Cytochrome
P450 enzyme systems was found to be involved in biodegradation of petroleum
hydrocarbons. The capability of several yeast species to use n-alkanes and other aliphatic
hydrocarbons as a sole source of carbon and energy is mediated by the existence of
multiple microsomal Cytochrome P450 forms. These cytochrome P450 enzymes had
been isolated from yeast species such as Candida maltose, Candida tropicalis,
and Candida apicola. The diversity of alkaneoxygenase systems in prokaryotes and
eukaryotes that are actively participating in the degradation of alkanes under aerobic
conditions like Cytochrome P450 enzymes, integral membrane di-iron alkane
hydroxylases (e.g., alkB), soluble di-iron methane monooxygenases, and membranebound copper containing methane monooxygenases have also been studied.
12 | P a g e
[Table 1 Enzymes involved in petroleum hydrocarbon degradation]
1.9 FACTORS INFLUENCING PETROLEUM
HYDROCARBON DEGRADATION
A number of limiting factors have been recognized to affect the biodegradation of
petroleum hydrocarbons. The composition and inherent biodegradability of the petroleum
hydrocarbon pollutant is the first and foremost important consideration when the
suitability of a remediation approach is to be assessed. Among physical factors,
temperature plays an important role in biodegradation of hydrocarbons by directly
affecting the chemistry of the pollutants as well as affecting the physiology and diversity
of the microbial flora. At low temperatures, the viscosity of the oil increased, while the
volatility of the toxic low molecular weight hydrocarbons were reduced, this cause delay
of biodegradation. Temperature also affects the solubility of hydrocarbons . Although
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hydrocarbon biodegradation can occur over a wide range of temperatures, the rate of
biodegradation generally decreases with the decreasing temperature. Figure 1.6 shows
that highest degradation rates that generally occur in the range 30–40∘C in soil
environments, 20–30∘C in some freshwater environments and 15–20∘C in marine
environments. Ambient temperature of the environment affect both, the properties of
spilled oil and the activity of the microorganisms. Significant biodegradation of
hydrocarbons have been reported in psychrophilic environments in temperate regions.
[Fig. 1.6 Hydrocarbon degradation rate in soil, freshwater and marine
environment]
Nutrients are very important ingredients for successful biodegradation of hydrocarbon
pollutants especially nitrogen, phosphorus, and in some cases iron. Some of these
nutrients could become limiting factor thus affecting the biodegradation processes. When
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a major oil spill occurred in marine and freshwater environments, the supply of carbon
significantly increases and the availability of nitrogen and phosphorus generally become
the limiting factor for oil degradation. In marine environments, it was found to be more
pronounced due to low levels of nitrogen and phosphorous in seawater. Freshwater
wetlands are typically considered to be nutrient deficient due to heavy demands of
nutrients by the plants. Therefore, additions of nutrients were necessary to enhance the
biodegradation of oil pollutant. On the other hand, excessive nutrient concentrations can
also inhibit the biodegradation activity. Use of poultry manure as organic fertilizer in
contaminated soil was also reported, and biodegradation was found to be enhanced in the
presence of poultry manure alone. Photo-oxidation also increases the biodegradability of
petroleum hydrocarbon by increasing its bioavailability and thus enhancing microbial
activities.
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2. OBJECTIVE
 Isolation of hydrocarbon degrading bacteria from the soil of automobile
workshop.
 Identification of bacteria by their morphological and colony characteristics and
biochemical tests.
 To
check
ability of bacteria to utilize different hydrocarbon sources like
Benzene, Petrol, Engine oil, Diesel, Toluene as sole carbon source.
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3. MATERIALS AND METHODS
3.1
MATERIALS












Soil contaminated with diesel
Luria Bertani (LB) Broth
Luria Bertani Agar Plate
Nutrient Agar Plate
Nutrient Broth
Psuedomonas Agar Plate
Mineral salt medium
0.1 M phosphate buffer
Media for Biochemical Tests
Reagents for Biochemical Tests
Reagents for gram’s staining
- Crystal violet stain
- Gram’s iodine
- 95% ethanol
- Safranin stain
Different carbon sources (Petrol, Diesel, Engine oil, Toluene,
Benzene)
All the instruments which were used are as following:











Weighing Balance
pH meter
Autoclave
Oven
Laminar Air Flow
Incubator
Shaker Water Bath
Orbital shaker
Microscope
Centrifuge
Spectrophotometer
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 Media and reagents for biochemical tests
Test
Medium
Reagent
Carbohydrate
fermentation test
Glucose, maltose,
Sucrose, Lactose,
Mannitol, Xylose
Phenol red
Red to Yellow
(gas production
in Durham’s
tube)
Urea utilization
test
Urea broth,
Phenol red
Pinkish red color
H2S Production
test
2% Peptone
Lead acetate
paper strip
Blackening of
paper
Gelatin hydrolysis
test
Nutrient gelatin
broth
–
Liquefaction at
4°C
Citrate utilization
test
Simmons Citrate
agar Slant
Bromothymole
blue
Green to Blue
Nitrate reduction
test
Peptone nitrate
broth
Sulphanilic acid
+
a-Naphthalamine
Red color
Oxidase test
Nutrient Agar Slant
Oxidase strip
Violet color
Catalase test
Nutrient Agar Slant
3% H2O2
Formation of
bubbles
M-R test
Glucose Phosphate
broth
Methyl red
Red color
V-P test
Glucose Phosphate
broth
40% KOH
+
a- Naphthol
Kovac`s reagent
Pink color
Iodole production
test
1% Tryptone
Positive
Results
Red ring
production
TSI slant
Triple Sugar iron
agar Slant
–
–
Macconkey`s Agar
plate
Macconkeys agar
plate
–
–
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3.2
METHOD
3.2.1 Collection of soil :
Oil contaminated soil sample was collected from automobile work shop
from Pune (Sample 1) and Surat (Sample 2). Soil samples were used to isolate the
Bacteria. Samples were collected at a depth within 5cm from the surface of the
soil. They were collected in sterile polythene bags and tightly packed.
3.2.2 Culture media

For Enrichment the culture LB broth and Mineral Salt Medium were used.

Isolation was carried out on LB agar plate and Mineral Salt agar medium
containing filtered engine oil.
Prepared media in D/W
Bring vol. 1 lit. & Autoclaving 15 psi, 121°C
Pour into sterile Petriplate
Allow to cool to room temp.
Invert Petri-plate
Spread 0.2 ml of hydrocarbon source on plate
3.2.3 Procedure for inoculum development :
1 gm soil sample (contaminated with diesel)
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Vortex with 10ml distilled water in testtube
Allow to settle
Use supernatent as inoculum
3.2.4 Procedure for Growth and Isolation of bacteria
100ml LB broth containing 1% Engine oil in flask
Add 10ml previously prepared supernatant into flask containing LB broth
.
Incubate flask at 37 c on shaker at 100rpm for 48hrs
Three successive subculture on same medium containing Engine oil
At every subculture, streak a loopfull of medium containing growth of
bacteria onto LB agar plate contaning Engine oil by four flame method
Incubate plate at 37.c in incubator for 48hrs
After 48hrs, observe plate for growth of bacteria
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After three subculturing, centrifuge broth at 5000rpm for 10 min, collect
cell pellets
Wash cell pellets with 0.1 M phosphate buffer (pH 6.8) twice
Transfer pellets into 100ml Mineral salt medium in flask containing 1%
Engine oil as a carbon and energy source
.
Incubate flask at 37 c on shaker at 100rpm for 48hrs
After 48hrs streak loopfull of inoculum by from flask by four flame
method onto mineral salt agar plate containing Engine oil
.
Incubate plate at 37 c for 7 days in incubator
Obsevre for the growth of bacteria
[Note: Replace yeast extract with Engine oil as carbon source during
preparation of LB medium]
3.2.5 Procedure for Gram’s staining
After getting growth on LB and Mineral Salt agar medium, colony characteristics is
observed and Gram’staining of isolated colony is done.
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Prepare suspension of bacteria using single colony from plate in 2ml
sterile distilled water
Prepare a heat fixed smear from suspension
Cover smear with crystal violet stain for 1 min
Drain crystal violet and cover smear with Gram’s iodine for 1 min
Rinse slide in running water
Rinse slide with 95% ethanol for approximately 10-15 seconds
Rinse smear with water
Add counterstain safranin for 1 min
Rinse slide with water, air dry and observe under oil-immersion objective
3.2.6 Procedure for Biochemical Tests
Biochemical tests for bacteria is performed for identification of bacteria.
Prepare all biochemical media
Prepare suspension of bacteria using single colony from plate in 2ml
sterile distilled water
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Inoculate two loopfull of suspension into all biochemical media under
aseptic condition
Streak a loopfull of suspension on slants and plates under aseptic condition
Incubate all inoculated media at 37.c in incubator for 24hrs
After 24hrs, observe results
3.2.7 Procedure for biodegradation potential
After getting growth of bacteria on plates, their ability to degrade diesel, benzene,
toluene, petrol as carbon source is checked.
Take 10 ml Nutrient broth and LB broth in separate testtubes (5 tubes for
each broth)
Add 1 ml petrol, engine oil, diesel, toluene and benzene in each tube of
both the broths
Inoculate both broths with previously isolated bacteria
Incubate all tubes at 30.c for 72hrs in incubator
Take O.D. at 640nm
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[Note: Replace yeast extract with Engine oil, diesel, petrol, benzene,
and toluene in each separate tube of both broth as carbon source in
above procedure]
 Above all procedures are done for both the soil samples and
results are noted down.
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4. RESULTS AND DISCUSSION
4.1 Results of isolation procedure
 For sample 1
 On LB Agar plate greenish blue colonies of bacteria are observed. Clear
zone is observed around growth of bacteria that shows bacteria can degrade
engine oil. From LB plate one colony is streaked on Pseudomonas Agar
plate and well isolated colonies are observed on it.
No growth is observed on Mineral Salt Agar plate.
 For sample 2
 On Mineral Salt Agar plate small white colonies are observed.
(Sample 1)
(Sample 2)
[Fig. 4.1 Growth of hydrocarbon degrading bacteria on LB
agar plate (Sample 1) and on Mineral Salt agar plate
(Sample 2)]
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4.2 Colony characteristics
Characters
Size
Shape
Margin
Elevation
Texture
Opacity
Consistency
Pigmentation
Sample 1
Small
Round
Entire
Low convex
Smooth
Translucent
Moist
Greenish blue
Sample 2
Small
Round
Entire
Low convex
Smooth
Opaque
Moist
White
4.3 Results of Gram’s staining
Characters
Sample 1
Sample 2
Size
Shape
Arrangement
Gram’s reaction
Small
Short rods
Singly, chains, or clusters
Gram negative
Small
Oval, Round
Singly or clusters
Gram positive
4.4 Results of Biochemical Tests
No.
Test
Sample 1
Sample 2
1.
Carbohydrate
hydrolysis
Glucose
+
+ (G)
Sucrose
-
+ (G)
Maltose
-
+ (G)
Mannitol
-
+ (G)
Lactose
-
+ (G)
Xylose
-
+ (G)
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2.
Urea utilization
test
-
-
3.
H2S Production
test
-
-
4.
Gelatin
hydrolysis test
-
-
5
Citrate utilization
test
+
+
6.
Nitrate reduction
test
+
+
7.
Oxidase test
+
+
8.
Catalase test
+
+
9.
M-R test
-
-
10.
V-P test
-
+
11.
13.
15.
Iodole production
test
Colourless colonies
Macconkey`s
were observed
Agar plate
Motility
Motile
Pink colour
colonies were
observed
Non-motile
[Fig. 4.2 Results of biochemical tests for Sample 2]
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4.4.1 Results of TSI slant
Slant
Butt
H2S production
Gas production
Sample 1
Sample 2
Pink (Alkaline)
Pink (Alkaline)
Yellow (Acidic)
Yellow (Acidic)
-
(G)
Key : + Positive tests
- Negative test
(G) Gas production
(A)
(B)
[Fig. 4.4 Results of TSI slant for Sample 1(B) and Sample 2(A)]
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4.5 Identification of Hydrocarbon degrading isolated strain
The bacteria were distinguished on basis of their growth pigmentation
and colony morphology on LB and Mineral Salt agar plate and selective media
(MacConkey’s Agar plate) at 37°c for 24hrs. Then the isolated bacteria were
identified by morphological and biochemical characteristics.
Bacteria isolated from sample 1 which was collected from the outer soil
of garage of pune were characterized as Pseudomonas sp. . The Pseudomonas
colonies were identified by the morphology, greenish blue, small colonies on
LB agar plate and greenish yellow colonies on
Pseudomonas agar plate.
Organisms were Gram-negative, short rods arranged in clusters or in chain.
These bacteria were oxidase and catalase positive.
Bacteria isolated from sample 2 which was collected from the outer soil
of garage of surat were characterized as Stapylococcus sp.. The Stapylococcus
colonies were identified by the morphology, white small colonies on Mineral
salt Agar plate. Organisms were Gram-positive. Cocci shape arranged in
clusters. These bacteria were oxidase and catalase positive.
[Fig 4.5 Growth of Pseudomonas sp. isolated from Sample 1 on
Pseudomonas agar plate]
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4.6 Results of biodegradation potential
Table 1 :
Source
Media
Petrol
Benzene
Toluene
Diesel
Engine oil
Nutrient broth
O.D. at 640nm
Pseudomonas sp.
Staphylococcus sp.
0.036
0.303
0.023
0.265
0.236
0.287
0.193
0.030
0.067
0.128
Table 2 :
Source
Media
Petrol
Benzene
Toluene
Diesel
Engine oil
LB broth
O.D. at 640nm
Pseudomonas sp.
Staphylococcus sp.
0.086
0.338
0.045
0.251
0.203
0.274
0.129
0.010
0.140
0.109
 From table 1 and 2, it was observed that Pseudomonas sp. are able to
degrade Benzene efficiently. These bacteria can degrade other sources
in following order :
Diesel > Engine oil > Petrol > Toluene
 From table 1 and 2, it was observed that Staphylococcus sp. are able to
degrade Petrol efficintly. These bacteria can degrade other sources in
following order :
Benzene > Engine oil > Diesel > Toluene
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(A)
(B)
(C) (D)
 Carbon sources
(A) Engine oil
(B) Petrol
(C) Diesel
(D) Benzene
[Fig. 4.6 Ability of Pseudomons sp. isolated from Sample 1 to
use engine oil, petrol, diesel and benzene as sole carbon source]
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5. SUMMERY

Bacteria isolated from Sample 1 were identified as Psuedomonas sp. by their
morphological and colony characteristics, gram’s reaction and biochemical tests.

Bacteria isolated from Sample 2 were identified as Staphylococcu sp. by their
morphological and colony characteristics, gram’s reaction and biochemical tests.
 Pseudomonas sp. and Staphylococcus sp. both are able to use petrol, engine oil,
diesel, benzene and toluene as carbon source.
 Pseudomonas showed maximun growth in presence of Benzene than other
Sources while Staphylococcus showed maximum growth in presence of Petrol
than other sources.
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6. CONCLUSION
The microbial degradation of oil pollutants is a complex process and the rates of
biodegradation of hydrocarbons from oil spills appear to be highly dependent on
localized environmental conditions. The fate of many components in petroleum, the
degradative pathways which are reactive in the environment, the importance of
cooxidationin natural ecosystems, and the role of microorganisms in forming
persistent environmental contaminants from hydrocarbons such as the compounds
found in tar balls are unknown and require future research. Although a number
of rate-limiting factors have been elucidated, the interactive nature of microorganisms,
oil, and environment still is not completely understood, and further examination of
case histories is necessary to improve predictive understanding of the fate of oil
pollutants in the environment and the role of microorganisms in biodegradative
environmental decontamination. With an understanding of the microbial hydrocarbon
degradation process in the environment, it should be possible to develop models for
predicting the fate of hydrocarbon pollutants and to develop strategies for utilizing
microbial hydrocarbon degrading activities for the removal of hydrocarbons from
contaminated ecosystems.
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7. APPENDIX I CULTURE MEDIA
1. LB broth / Agar
Components
gm
Casein enzyme
hydrolysate
Yeast extract
Distilled water
Agar
NaCl
pH
10
5
1000 ml
20
10
7.5
Dissolve all ingredients (exept agar) by heating and adjust to pH 7.5. Add agar
.
powder and digest it by boiling in waterbath. Sterilize it by autoclaving (121 c for
15 min). LB broth has the same composition except that it does not contain the
solidifying agent agar.
[Note : Here replace yeast extract with engine oil/ petrol/ diesel/ benzene/ toluene
during preparation of LB broth / agar ]
2. Mineral Salt Medium
Components
gm
KNO3
MgSO4.7H2O
CaCl2.6H2O
FeSO4
Trace element
solution
Phosphate buffer(1
M, pH 6.8)
Distilled water
pH
1.0
1.0
0.1
0.05
250 ml
20 ml
730 ml
7.5
.
Dissolve all ingredients and adjust to pH 7.5. Sterilize it by autoclaving (121 c
34 | P a g e
for 15 min). For preparation of Mineral Salt agar, add 20 gm agar with above
components.
 Trace element solution
Components
gm
SnCl2
KI
LiCl
MnSO4.4H2O
HBO3
ZnSO4.7H2O
CoCl2.6H2O
NiSO4.6H2O
BaCl2
Ammonium
molybdate
Distilled water
0.05
0.05
0.05
0.08
0.50
0.10
0.10
0.10
0.05
0.05
1000 ml
Add all salts one by one
3. Pseudomonas agar
Components
gm
Casein enzyme
10.0
hydrolysate
K2HPO4
1.5
Proteose Peptone
10.0
MgSO4
1.5
Agar
15
Distilled water
1000 ml
pH
7.0
Dissolve by heating, and adjust the pH. Sterilize by autoclaving at 15 lbs pressure
(121°C) for 15 min.
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 Media for Biochemical Tests
1. Glucose Phosphate Broth
Components
gm
Glucose
5.0
K2HPO4
5.0
Peptone
5.0
Distilled water
1000 ml
pH
6.9-7.0
Dissolve by heating, and adjust the pH. Sterilize by autoclaving at 15 lbs pressure
(121°C) for 15 min.
2. MacConkey’s Agar Media
Components
gm
Peptone
20.0
Lactose
10.0
NaCl
5.0
Bile salts
3.0-5.0
Neutral red
30.0 mg
Crystal violet
10.0 mg
Distilled water
1000 ml
Agar
30.0
pH
7.4
Dissolve by heating, adjust pH to 7.4 and sterilize by autoclaving.
3. Nutrient sugar broth
Components
ml
36 | P a g e
10% aq. test sugar
10.0
solution (e.g. glucose)
1% peptone water
90.0
Phenol red
1.0
pH
7.4
Mix components given in table. Sterilize by autoclaving at 10 psi for 10 minutes.
4. Urea broth
Components
gm
KH2PO4
9.1
Na2HPO4
9.5
Yeast extract
0.1
Phenol red
0.01
Distilled water
950 ml
40% Urea
50 ml
pH
6.8
Heat to dissolve and adjust pH to 6.8. Sterilize by autoclaving and allow to cool to
.
55 c. Add 50 ml sterile urea solution.
5. 2% Peptone broth
Components
gm
Peptone
20.0
NaCl
5.0
Distilled water
1000 ml
pH
7.5
Heat to dissolve and adjust pH to 7.5. Sterilize by autoclaving.
37 | P a g e
6. Nutrient Gelatin broth
Components
gm
Meat extract
3.0
Peptone
10.0
Gelatin
150.0
Distilled water
1000 ml
pH
7.2
Heat to dissolve and adjust pH to 7.2. Sterilize by autoclaving.
7. Simmon’s citrate agar slant
Components
gm
Sodium citrate
2.0
MgSO4
0.2
NaCl
5.0
NH4H2PO4
1.0
K2HPO4
1.0
Bromothymol blue
0.08
Agar
20.0
Distilled water
1000 ml
pH
6.9
Heat to dissolve and adjust pH to 6.9. Sterilize by autoclaving.
8. Peptone Nitrate broth
Components
gm
Meat extract
3.0
Peptone
5.0
38 | P a g e
Potassium nitrate
1.0
Distilled water
1000 ml
pH
7.5
Heat to dissolve and adjust pH to 7.5. Sterilize by autoclaving.
9. Nutrient agar slant
Components
gm
Meat extract
3.0
Peptone
10.0
NaCl
5.0
Distilled water
1000 ml
Agar
20.0
pH
7.4
Heat to dissolve and adjust pH to 7.5. Sterilize by autoclaving. Pour medium into
sterile testtubes under aseptic condition and place tubes in slant position and allow to
solidify.
10. 1% Tryptone broth
Components
gm
Tryptone
10.0
NaCl
5.0
Distilled water
1000 ml
pH
7.5
Heat to dissolve and adjust pH to 7.5. Sterilize by autoclaving.
39 | P a g e
11. TSI [Triple Sugar Iron] Agar
Components
gm
Meat extract
3.0
Yeast extract
3.0
Peptone
15.0
Proteose peptone
5.0
Lactose
10.0
Glucose
1.0
Sucrose
10.0
Ferrous sulphate
0.2
Na2S2O3
0.3
NaCl
5.0
Agar
20.0
Phenol red
0.24
Distilled water
1000 ml
pH
7.4
Heat to dissolve the ingredients and adjust the pH. Distribute the medium in
testtubes and sterilize by autoclaving. Allow the tubes to solidify in manner which will
give butt and slant.
40 | P a g e
8. APPENDIX II STAINS AND REAGENTS
 REAGENT FOR GRAM STAINING:
(A) Crystal Violet Staining Reagent:
Crystal violet
: 2.0 g
Ethanol (95%)
: 20.0ml
Ammonium oxalate
: 0.8 g
Distilled water
: 80ml
Dissolve the dye in alcohol and ammonium oxalate in distilled water. Mix two
solutions and allow it to stand for 24 hrs. Filter and use.
(B) Iodine Solution:
Iodine
Potassium iodide
Distilled water
: 1.0 gm
: 2.0 gm
: 300 ml
Dissolve KI and iodine in little amount of water and adjust to 300 ml with water.
(C) Safranin Solution:
Safranin
: 0.25 gm
95% ethanol
: 10.0 ml
Distilled water
: 100 ml
Dissolve safranin in ethanol and make final volume to 100 ml with distilled water.
 REAGENT FOR BIOCHEMICAL TEST:
(A) Methyle Red Indicator:
Methyl red
: 0.1 gm
95% ethanol
: 300 ml
Dissolve the dye in alcohol and use.
41 | P a g e
(B) Phenol red indicator:
Phenol red
: 0.2 gm
95% ethanol
: 500 ml
Distilled water
: 500 ml
Dissolve the phenol red in alcohol. Add distilled water and filter and use.
(C) 40% Potassium Hydroxide Solution:
KOH
: 40.0 gm
Distilled water
: 100 ml
Dissolve KOH in water to make the final volume to 100 ml.
(D) Kovac’s Reagent:
ρ-dimethylaminobenzaldehyde
:5.0 gm
95% ethanol
: 75 ml
Conc. HCl
: 25 ml
Dissolve the aldehyde in ethanol by gently warming in a waterbath (about 50-55.c).
Cool and add the acid. Protect the reagent from light and store in brown glass
bottle.
(E) Sulphanilic Acid:
Sulphanilic acid
: 1.0 g
5N Acetic acid
:100 ml
Dissolve sulphanilic acid in distilled water. Filter and use.
(F) a-naphthalamine
N, N- Dimethyle-1-naphthalamine
: 1 gm
5N acetic acid
:1000 ml
Store at -2 to -8.c for upto 3 months in dark.
42 | P a g e

Other solutions
1. 1 N NaOH
 4 gm in 100 ml Distilled water.
2. 1 N HCl
 8.8 ml Conc. HCl in 91.2 ml Distilled water.
3. 40% Urea

40 gm in 100 ml Distilled water.
4. 0.1 M phosphate buffer


49.7 ml 1 M K2HPO4 + 50.3 ml 1 M KH2PO4
Dilute the combined 1 M stock solution to 1000 ml with distilled water.
43 | P a g e
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