DISSERTATION ON
PETROLEUM BIODEGRADATION IN NATURAL
ENVIRONMENT
AS A PARTIAL REQUIREMENT
FOR FULFILMENT OF THE DEGREE OF
MASTER OF SCIENCE IN BIOTECHNOLOGY
(M. Sc. BIOTECHNOLOGY)
YEAR: 2011-2012
CARRIED OUT AT
MITCON BIOPHARMA INSTITUTE, PUNE,
MAHARASHTRA
GUIDED BY:
SUBMITTED BY:
Miss. PRIYA BANDE
PATEL JAYESHKUMAR C.
SUBMITTED TO
BHAGWAN MAHAVIR COLLEGE OF M. SC.
BIOTECHNOLOGY, SURAT
Page 1
Abstract
ABSTRACT
Petroleum-based products are the major source of energy for industry and daily life.
Petroleum is also the raw material for many chemical products such as plastics, paints,
and cosmetics. Due to widespread use of petroleum products, the number of petroleum
contaminated site has abounded. Natural attenuation, which relies on in situ
biodegradation of pollutants, has received a large amount of attention, especially for
petroleum contamination. Therefore in this work two different sources, soil and
marine water were chosen and oil degrading microorganisms were isolated using
different hydrocarbon containing minimal media. Two strains from soil and one strain
from marine water sample were selected according to their simultaneous good growth
on minimal medium with oil, sea-water agar and nutrient agar. Several physiological
and biochemical characteristics of isolated oil degrading strains were determined. Two
of them were Gram negative, oxidase positive, catalase positive & one was Gram
positive, Oxidase & catalase positive. By checking the petroleum degradation
potential of our selected oil degrading strains on individual hydrocarbon derivatives
for a period of 21 days, we showed that our strain decomposed diesel easily and very
fast. The strain also utilized petrol, engine oil, toluene, benzene, and Xylene.
Key words- Petroleum, in situ biodegradation, marine water, oxidase, Catalase,
Degradation, Toluene, Benzene, Xylene
Page 2
INDEX
Chapter
Title
No.
Page
No.
Abstract
2
List of Tables
4
List of figure
5
Acknowledgement
6
Abbreviation
8
Introduction:
9
Definition
9
Origin, constitution and use
9
Component of crude oil
16
Behavior of petroleum in Marine environment
19
2.
Aims & Objectives
22
3.
Material & method:
23
Collection of sample
23
Culture media
23
Biochemical reagents
25
Methods
26
Results & Discussion:
30
Physio-chemical characteristics of isolates.
30
Biodegradation efficiency.
33
Growth potential of isolates.
36
Identification of petroleum degrading isolated strains
38
5.
Conclusion
39
6.
Appendixes:
40
1.
4.
7.
Appendix-1- Culture Medium
40
Appendix-2- Stains & Reagents
44
References
45
Page 3
LIST OF TABLES
Table
Title
No.
Page
No.
1.
Bacterial genera involved in PAHs degradation.
10
2.
Fungal genera capable of degrading PAHs.
13
3.
Different distillations of Petroleum (Fuels) and their use.
16
4.
Parent Poly-aromatic hydrocarbons present in crude oil.
18
5.
Composition of Minimal agar medium.
24
6.
Biochemical Reagents.
15
7.
Colony Characteristics of isolates.
30
8.
Biochemical Characteristics of organisms.
30
9.
Liquid culture characteristics of Bacteria during 21 days
33
incubation.
10.
Petroleum degradation Efficiency.
36
Page 4
LIST OF FIGURES
Figure
Title
No.
Page
No.
1.
Gram Staining of A3 Organism: Gram Negative, Rod shape
32
2.
Growth of organisms(A3) on Sea-water agar media
32
3.
Oxidase positive test of organism
32
4.
Biodegradation of Engine oil by isolates
32
5.
Bacterial growth on Nutrient agar Plate
32
6.
Growth of A1 Culture on Nutrient agar media
32
7.
Bacterial growth on minimal medium containing different
37
hydrocarbon (Biodegradation potential) at fifth days incubation
8.
Bacterial growth on minimal medium containing different
37
hydrocarbon (Biodegradation potential) (A2 Culture)
9.
Biodegradation potential of organisms(A3) on Different
38
Hydrocarbon source in minimal media ( After 21st days)
Page 5
Acknowledgment
ACKNOWLEDGMENT
I humbly owe the completion of this dissertation work to the almighty whose
love and blessing was and will be with me in every moment of my life.
I am very much thankful to all my professors and my co-guidance Mr.
Naresh butani in our institute who made us work hard, taught us how to manage
everything skillfully and made us into confident individuals.
I gratefully acknowledge my deep sense of gratitude to my project guide
Miss. Priya Bande , Department of Biotechnology & Environment
Science MITCON, Pune, Maharashtra, for involving in our confidence and
essence of excitement about our work through her spontaneous encouragement and
inspiring guidance for which we shall always be grateful.
My special thanks to Dr.Chandrashekhar Kulkarni, HOD of department of
Biotechnology & Environment Science MITCON, Pune (Maharashtra) for providing
infrastructure and facilities required for this research work.
I sincerely extend thanks to Miss Neha Vora., Department of Biotechnology
& Environment Science MITCON, Pune, (Maharashtra) for his timely help during the
course of study and providing the necessary requirements & guidance.
I also express thanks to Mr. Sandeep & Mr. Amitbhai, store keeper who
helped me for providing the required chemicals and reagents needed for the project
work.
Page 6
Acknowledgment
I am especially thankful to my brother Mr. Satish Patel, M.Sc. Chemistry, and
Mr. Alkesh Nai for providing guidance in different chemical & reagent preparation.
I am very much thankful to my friends - Kamlesh Vasava, Snehal Patel and
P.D.Patel for helping in typing work & Collection the sample.
I am thankful to Falgun, Hemant, Sanjay, Hardik, Kuldeep, Nirav and all
other friends for their support and help during the course of studies.
I express my appreciated thanks to Lord Maa Narmada for showering his
infinite boundaries and grace upon me and for being my constant companion, the
strongest source of motivation and inspiration.
My acknowledgement won’t be complete without expressing deeply indebted
to My Parents and Family who stood as backbone and for their blessings, continuous
support and their unconditional everlasting love in my entire life.
Patel Jayesh C.
Page 7
Abbreviations
ABBREVIATIONS
BHM – Bushnell-Haas Media
CaCl2 – calcium chloride
D/W – Distilled water
FeSO4 – Iron sulfate
Gms – Grams
H2O2 – Hydrogen peroxide
H2S – Hydrogen sulfide
HCl – Hydrochloric acid
Inc. – Incubation
K2HPO4 – Di-potassium hydrogen phosphate
KH2PO4 – Mono potassium hydrogen phosphate
KOH – Potassium hydroxide
MgSO4 – Magnesium sulfate
MnSO4 – Manganese sulfate
M-R – Methyl red test
Na2HPO4 – Disodium hydrogen phosphate
NB/NA – Nutrient broth/Agar
NaCl – Sodium chloride
NaOH – Sodium hydroxide
NH4Cl – Ammonium Chloride
RPM – rotation per minutes
SWA – Sea water agar media
Temp. – Temperature
TMPD – N, N, N′, N′-tetra methyl-p-phenylenediamine
V-P – Voges-Proskauer
Page 8
Introduction
Chapter-1
INTRODUCTION
Definition
Biodegradation or biotic degradation or biotic decomposition is the chemical
dissolution of materials by bacteria or other biological means.
Petroleum is a viscous liquid mixture that contains thousands of compounds mainly
consisting of carbon and hydrogen.
Origin, constitution and use
Crude oil is the product of heating of ancient organic materials over geological period.
It is formed from pyrolysis of hydrocarbon, in a variety of reactions, mostly
endothermic at high temperature and/or pressure. Crude oil reserves were formed from
the preserved remains of prehistoric zooplankton and algae, which had settled to a sea
or lake bottom in large quantities under anoxic conditions. On the other hand, the
remains of prehistoric terrestrial plants led to form coal. During the formation of crude
oil, digenesis followed catagenesis. The studies documented that over a period, the
organic matter mixed with the mud and got buried under heavy layers of sediments
resulting in generation of high levels of heat and pressure (digenesis). This process
transformed the organic matter into a waxy material known as kerogen, followed by
its further conversion to liquid and gaseous hydrocarbons (catagenesis). The change
from kerogen to natural gas through oil is a temperature dependent event. Sometimes
the oil formed at extreme depths migrates and is entrapped at shallower depths. eg.
Athabasca oil sands. (20)
The crude oil is a heterogeneous entity, composed of hydrocarbon chains of varied
lengths. It contains hundreds of different hydrocarbon compounds such as paraffin,
naphthenes, aromatics as well as organic sulfur compounds, organic nitrogen
compounds and oxygen containing hydrocarbons (phenols).(20)
Page 9
Introduction
The most common distillations of petroleum are fuels. Fuels generally include, ethane
and other short chain alkanes, diesel fuel (petro diesel), fuel oils, gasoline (petrol), jet
fuel, kerosene, liquefied petroleum gas (LPG).
Table-1 Bacterial genera involved in PAHs degradation (20):Organisms
PAHs
References
Achromobacter sp. NCW
Carbazole
Guo et al., 2008
Alcaligenes denitrificans
Fluoranthene
Weissenfels et al., 1990
Arthrobacter sp. F101
Fluorene
Casellas et al., 1997
Arthrobacter sp. P11
Phenanthrene, Carbazole,
Dibenzothiophene
Seo et al., 2006
Arthrobacter sulphureus
Phenanthrene
Samanta et al., 1999
Phenanthrene
Samanta et al., 1999
Bacillus cereus P21
Pyrene
Kazunga et al., 2000
Bacillus subtilis BMT4i
(MTCC9447)
Benzo[a]pyrene
Lily et al., 2009
Brevibacterium sp.HL4
Burkholderia sp.S3702,
RP007,
2A12TNFYE5,
BS3770
Burkholderia sp. C3
Burkholderia cepacia
BU3
Burkholderia xenovorans
LB400
Chryseobacterium sp.
NCY
Cycloclasticus sp. P1
Geobacillus sp.
Phenanthrene
Phenanthrene
Samanta et al., 1999
Kang et al., 2003,
Balashova et al., 1999,
Laurie et al., 1999
Phenanthrene
Phenanthrene, Pyrene,
Naphthalene
Benzoate, Biphenyl
Seo et al., 2006
Kim et al., 2003
Denef et al., 2005
Carbazole
Guo et al., 2008
Pyrene
Napthalene, Phenanthrene,
Fluorene
Anthracene
Wang et al., 2008
RKJ4
Acidovorax delafieldii
P41
Geobacillus
stearothermophilus
“AAP7919”
Janibacter sp. YY1
Phenanthrene, Fluorene,
Anthracene, Dibenzofuran,
Bubians et al., 2007
Kumar et al., 2011
Yamazoe et al., 2004
Page 10
Introduction
Marinobacter NCE312
Mycobacterium sp.PYR,
Mycobacterium sp. JS14
Mycobacterium sp. 6PY1,
KR2,
AP1
Mycobacterium sp.
RJGII135
Mycobacterium sp.PYR1,
LB501T
Dibenzopdioxin,
Dibenzothiophene
Naphthalene
Benzo[a]pyrene
Fluoranthene
Pyrene
Benzo[a]pyrene,
Benz[a]anthracene
Pyrene
Pyrene, Phenanthrene,
Fluoranthene, Anthracene
Hedlund et al., 2001
Cheung et al., 2001,
Grosser et al., 1991
Lee et al., 2007
Rehmann et al., 1998,
Vila et al., 2001,
Krivobok et al., 2003
Schneider et al., 1996
Mody et al., 2001,
Kelley et al., 1993,
Sepic et al., 1998,
Ramirez et al., 2001,
Van et al., 2003
Mycobacterium sp. CH1,
BG1,
BB1, KR20
Mycobacterium
flavescens
Pyrene, Phenanthrene,
Fluorene
Boldrin et al., 1993,
Rehmann et al., 2001
Pyrene, Fluoranthene
Mycobacterium
vanbaalenii
PYR1
Mycobacterium sp. KMS
Nocardioides
aromaticivorans
IC177
Pasteurella sp. IFA
Polaromonas
naphthalenivorans CJ2
Pseudomonas sp. C18,
PP2,
DLCP11
Pseudomonas sp. BT1d
Phenanthrene
Pyrene,
Dimethylbenz[a]anthracene
Pyrene
Carbazole
DeanRoss
et al., 2002,
DeanRoss
et al., 1996
Kim et al., 2005,
Moody et al., 2003
Pseudomonas sp. HH69
Pseudomonas sp. CA10
Pseudomonas sp. NCIB
98164
Pseudomonas sp. F274
Miller et al., 2004
Inoue et al., 2006
Fluoranthene
Naphthalene
Sepic 1999
Pumphrey et al., 2007
Phenanthrene, Naphthalene
Denome et al., 1993,
Prabhu et al., 2003
3hydroxy2formylbenzothioph
ene
Dibenzofuran
Chlorinated dibenzopdioxin,
Carbazole
Fluorene, Dibenzofuran,
Dibenzothiophene
Fluorene
Bressler et al., 2001
Fortnagel et al., 1990
Habe et al., 2001
Resnick et al., 1996
Grifoll et al., 1994
Page 11
Introduction
Pseudomonas
paucimobilis
Pseudomonas vesicularis
OUS82
Pseudomonas putida P16,
BS3701, BS3750,
BS590P,
BS202P1
Pseudomonas fluorescens
BS3760
Pseudomonas stutzeri
P15
Pseudomonas
saccharophilia
Pseudomonas aeruginosa
Ralstonia sp. SBUG 290,
U2
Rhodanobacter sp. BPC1
Rhodococcus sp.
Rhodococcus sp.
WUK2R
Rhodococcus erythropolis
I19
Rhodococcus erythropolis
D1
Staphylococcus sp. PN/Y
Stenotrophomonas
maltophilia
VUN 10,010
Stenotrophomonas
maltophilia
VUN 10,003
Sphingomonas
yanoikuyae R1
Sphingomonas
yanoikuyae
JAR02
Sphingomonas sp.P2,
LB126
Sphingomonas sp.
Sphingomonas
Phenanthrene
Weissenfels et al., 1990
Fluorene
Weissenfels et al., 1990
Phenanthrene, Naphthalene
Kiyohara et al., 1994,
Balashova et al., 1999
Phenanthrene,
Benz[a]anthracene,
Chrysene
Pyrene
Balashova et al., 1999
Pyrene
Kazunga et al., 2000
Phenanthrene
Naphthalene, Dibenzofuran
Romero et al., 1998
Becher et al., 2000,
Zhou et al., 2002
Kanaly et al., 2002
DeanRoss
et al., 2002,
Walter et al., 1991
Kirimura et al., 2002
Benzo[a]pyrene
Pyrene, Fluoranthene
Benzothiophene,
Naphthothiophene
Alkylated dibenzothiophene
Kazunga et al., 2000
Folsom et al., 1999
Dibenzothiophene
Matsubara et al., 2001
Phenanthrene
Benzo[a]pyrene
Pyrene, Fluoranthene
Mallick et al., 2007
Boonchan et al., 1998
Pyrene, Fluoranthene,
Benz[a]anthracene
Juhasz et al., 2000
Pyrene
Kazunga et al., 2000
Benzo[a]pyrene
Rentz et al., 2008
Phenanthrene, Fluoranthene,
Fluorene, Anthracene
Pinyakong et al., 2003,
Van et al., 2003,
Pinyakong et al., 2000
Gai et al., 2007
Dibenzofuran, Carbazole,
Dibenzothiophene
Phenanthrene, Fluoranthene,
Story et al., 2001,
Page 12
Introduction
paucimobilis
EPA505
Sphingomonas wittichii
RW1
Sphingomonas sp. KS14
Terrabacter sp.DBF63
Anthracene, Naphthalene
Mueller et al., 1990
Chlorinated dibenzopdioxin
Nam et al., 2006
Phenanthrene, Naphthalene
Fluorene, Dibenzofuran,
Chlorinated dibenzopdioxin,
Chlorinated dibenzothophene
Cho et al., 2001
Habe et al., 2004, Habe
et al., 2001, Habe et al.,
2002
Benzo[a]pyrene
Pyrene, Carbazole
Grosser et al., 1991
Xanthamonas sp.
Table 2: Fungal genera capable of degrading PAHs (20):Name of Fungus
PAH
Reference
Phanerochaete
Anthracene
Field et al.,1996
Bjerkandera sp. strain
BOS55
Anthracene
Field et al.,1996
Trametes versicolor
Anthracene
Collins et al., 1986
Cunninghamella
elegansoxidizes
Anthracene
Cernigilia, 1997
P. chrysosporium
Anthracene
Hammel et al., 1991
Aspergillus flavus
Benzo[a]pyrene
Romero et al., 2010
Paecilomyces farinosus
Benzo[a]pyrene
Romero et al., 2010
chrysporium
Oil fields are not uniformly distributed around the globe, but being in limited areas
such as the Persian Gulf region. The world production of crude oil is more than three
billion tons per year, and about the half of this is transported by sea. Consequently, the
international transport of petroleum by tankers is frequent. All tankers take on ballast
water which contaminates the marine environment when it is subsequently discharged.
The recent spill of more than 200,000 barrels of crude oil from the oil tanker Exxon
Valdez in Prince William Sound, Alaska, as well as smaller spills in Texas, Rhode
Page 13
Introduction
Island, and the Delaware Bay, has refocused attention on the problem of hydrocarbon
contamination in the environment.
Off-shore drilling is now common to explore new oil resources and this constitutes
another source of petroleum pollution. However, the largest source of marine
contamination by petroleum seems to be the runoff from land. Annually, more than
two million tons of petroleum is estimated to end up in the sea.
It is estimated that the annual global input of petroleum is between 1.7 and 8.8 million
metric tons, the majority of which is derived from anthropogenic sources.
Claude U. Sable had as far back as 1946, recognized that many microorganisms have
the ability to utilize hydrocarbons as the sole source of carbon and energy, and that
such microorganisms are widely distributed in nature. He further recognized that the
microbial utilization of hydrocarbons was highly dependent on the chemical nature of
the components within the petroleum mixture, and environmental determinants (Atlas
1981).
Biodegradation of hydrocarbons by natural populations of microorganisms represents
one of the primary mechanisms by which petroleum and other hydrocarbon pollutants
are eliminated from the environment.
Crude oil can be accidentally or deliberately released into the environment leading to
serious pollution problems (Thouand et al., 1999). Even small releases of petroleum
hydrocarbons into aquifers can lead to concentrations of dissolved hydrocarbons far in
excess of regulatory limits (Spence et al., 2005). These pollution problems often result
in huge disturbances of both the biotic and abiotic components of the ecosystems
(Mueller et al., 1992), more so that some hydrocarbon components have been known
to belong to a family of carcinogenic and neurotoxic organo-pollutants (HallierSoulier et al., 1999).
Page 14
Introduction
The currently accepted disposal methods of incineration or burial in secure landfills
(USEPA 2001; ITOPF 2006) can become prohibitively expensive when the amounts
of contaminants are large. This often results in cleanup delays while the contaminated
soil continues to pollute groundwater resources if on land, and death of aquatic life if
on waterways (Pye and Patrick 1983), thus necessitating speedy removal of the
contaminants.
Bioremediation, which employs the bio-degradative potentials of organisms or their
attributes, is an effective technology that can be used to accomplish both effective
detoxification and volume reduction. It is useful in the recovery of sites contaminated
with oil and hazardous wastes (Caplan 1993).
Biodegradation of hydrocarbons by natural populations of microorganisms is the main
process acting in the depuration of hydrocarbon-polluted environments.
There are many Bacteria (Table-1) & Fungi (Table-2) are identified which degraded
petroleum in natural environments
Some reviews focused on the examination of factors, are including nutrients, physical
state of the oil, oxygen, temperature, salinity and pressure influencing petroleum
biodegradation rates, with a view to developing environmental applications (Atlas,
1981; Jonathan et al., 2003).
Bioremediation makes use of indigenous oil–consuming microorganisms, called
petrophiles, by enhancing and fertilizing them in their natural habitats.
Petrophiles are very unique organisms that can naturally degrade large hydrocarbons
and utilize them as a food source (Harder, 2004). Microorganisms degrade these
compounds by using enzymes in their metabolism and can be useful in cleaning up
contaminated sites (Alexander, 1999).
Page 15
Introduction
Microbial remediation of a hydrocarbon–contaminated site is accomplished with the
help of a diverse group of microorganisms, particularly the indigenous bacteria present
in soil.
Other organisms such as fungi are also capable of degrading the hydrocarbons in
engine oil to a certain extent. However, they take longer periods of time to grow as
compared to their bacterial counterparts (Prenafeta- Boldu et al., 2001).
Table 3: Different distillations of Petroleum (Fuels) and their use.
S. No.
Fuel/ Derivatives
Uses
1.
Alkenes (Olefins)
Manufacture of plastics or other compounds
2.
Lubricants
Synthesis of light machine oils, motor oils
and
greases, as viscosity stabilizers
3.
Wax
Used in the packaging of frozen foods
4.
Petroleum coke
(asphalt)
5.
Paraffin wax & aromatic
petrochemicals
Used in carbon products or as solid fuel,
Paraffin waxes. Aromatic petrochemicals as
precursors in other chemical synthesis.
As precursor in chemical production
Components of petroleum:
All petroleum products are derived from crude oil whose major constituents are
hydrocarbons. Petroleum components can be separated into four fractions, the
Saturated,
Aromatic,
Resin
and
Asphaltene
fractions,
by
absorption
chromatography. Each of these fractions contains a large number of compounds
(Karlsen and Larter, 1991).
1. Saturates are hydrocarbons containing no double bonds. They are further classified
according to their chemical structures into Alkanes (paraffin) and Cycloalkanes
(naphthenes).
Page 16
Introduction
Alkanes have either a branched or unbranched (normal) carbon chain(s),
and have the general formula CnH2n+2.
Cycloalkanes have one or more rings of carbon atoms (mainly
cyclopentanes and cyclohexanes), and have the general formula CnH2n. The majority
of Cycloalkanes in crude oil have an alkyl substituent(s) (Figure 1).
2. Aromatics have one or more aromatic rings with or without an alkyl substituent(s).
Benzene is the simplest one (Figure 1), but alkyl-substituted aromatics generally
exceed the non-substituted types in crude oil (Mater and Hatch, 1994).
3. Asphaltene consists of high-molecular weight compounds which are not soluble in
a solvent such as n-heptanes, while resins are n-heptanes-soluble polar molecules.
4. Resins contain heterocyclic compounds, acids and sulfoxides.
In contrast to the saturated and aromatic fractions, both the resin and
asphaltene fractions contain non-hydrocarbon polar compounds. Their elements
contain, in addition to carbon and hydrogen, trace amounts of nitrogen, sulfur and/or
oxygen. These compounds often form complexes with heavy metals.
The components of petroleum in crude oil have been analyzed mainly by using gas
chromatography in combination with mass spectrometry (GC/MS). Consequently, the
chemical structures of the higher molecular- weight components (the heavy fractions)
that cannot be identified by GC are mostly unknown.
Furthermore, the compositions of many branched alkanes and alkyl cyclo-alkanes
have not been determined because their isomers are numerous and cannot be resolved
by GC (Killops and Al-Juboori, 1990; Gough and Rowland, 1990). Therefore, a
multitude of analytical techniques such as flame ionization detection, IR- and UVabsorption spectrometry, NMR and elemental analysis in combination with
appropriate separation techniques such as various chromatographic methods and/or
Page 17
Introduction
chemical conversion is necessary to characterize petroleum, and especially its heavy
fractions.
Various petroleum products are produced by refining crude oil. Refining is essentially
a fractional distillation process by which different fractions or cuts are produced.
Alkenes, a series of unsaturated hydrocarbons including ethylene, are not found in
crude oil, but are produced during the cracking of crude oil.
Table 4: Parent Poly-aromatic hydrocarbons present in crude oil.
S.N.
Radial
Depiction for
PAH
PAH Name
Molecular
formula
1.
Pen
Pentalene
C8H6
2.
Ind
Indene
C9H8
3.
Nap
Naphthalene
C10H8
4.
Azu
Azulene
C10H8
5.
Hep
Heptalene
C12H10
6.
Bip
Biphenylene
C12H8
7.
aIn
as-Indacene
C12H8
8.
sIn
s-Indacene
C12H8
9.
Can
Acenaphthylene
C12H8
10.
Flu
Fluorene
C13H10
11.
Phe
Phenalene
C13H10
12.
Phr
Phenanthrene
C14H10
13.
Ant
Anthracene
C14H10
14.
Flt
Fluoranthene
C16H10
15.
Acp
Acephenanthrylene
C16H10
16.
Aca
Aceanthrylene
C16H10
17.
Tpl
Triphenylene
C18H12
Page 18
Introduction
18.
Pyr
Pyrene
C16H10
19.
Chr
Chrysene
C18H12
20.
Npc
Naphthacene
C18H12
21.
Ple
Pleiadene
C18H12
22.
Per
Perylene
C20H12
23.
Pic
Picene
C22H14
24.
Pen
Pentaphene
C22H14
25.
Pec
Pentacene
C22H14
26.
Tpl
Tetraphenylene
C24H16
27.
Hep
Hexaphene
C26H16
28.
Hex
Hexacene
C26H16
29.
Rub
Rubicene
C26H14
30.
Cor
Coronene
C24H12
31.
Trp
Trinaphthylene
C30H18
32.
Hep
Heptaphene
C30H18
33.
Hec
Heptacene
C30H18
34.
Pya
Pyranthrene
C30H16
35.
Ova
Ovalene
C32H14
Behavior of Petroleum in Marine Environment:
When petroleum is spilled into the sea, it spreads over the surface of the water. It is
subjected to many modifications, and the composition of the petroleum changes with
time. This process is called weathering, and is mainly due to evaporation of the lowmolecular-weight fractions, dissolution of the water-soluble components, mixing of
the oil droplets with seawater, photochemical oxidation, and biodegradation.
Those petroleum components with a boiling point below 250 °C are subjected to
evaporation. Therefore, the content of n-alkanes, whose chain length is shorter than
C14, is reduced by weathering. The content of aromatic hydrocarbons within the same
Page 19
Introduction
boiling point range is also reduced as they are subjected to both evaporation and
dissolution.
The mixing of oil with seawater occurs in several forms. Dispersion of the oil droplets
into a water column is induced by the action of waves, while water-in oil
emulsification occurs when the petroleum contains polar components that act as
emulsifiers. A water-in-oil emulsion containing more than 70% of seawater becomes
quite viscous; it is called chocolate mousse from its appearance. After the light
fractions have evaporated, heavy residues of petroleum can aggregate to form tar balls
whose diameter ranges from microscopic size to several tenths of a centimeter.
After a large oil spill, the oil slick is sometimes treated with a dispersant. Dispersants
emulsify petroleum by reducing the interfacial tension between petroleum and water.
The small droplets that are formed are dispersed into a water column to a depth of
several meters, preventing wind-induced drift of the oil slick. It is claimed that
treatment by a dispersant enhances the biodegradation of petroleum. However, the
results of such tests are controversial (Tjessem et al., 1984). The original dispersants
used were highly toxic; however, less toxic dispersants have subsequently been
developed.
Under sunlight, petroleum discharged at sea is subjected to photochemical
modification. Some reports have suggested the light-induced polymerization of
petroleum components, while others have suggested their photo degradation. An
increase in the polar fraction and a decrease in the aromatic fraction have also been
observed. Aliphatic components do not significantly absorb solar light, and are by
themselves photonic chemically inert. However, they can be degraded by
photosensitized oxidation. The aromatic or polar components in petroleum and
anthraquinone that is present in seawater can provoke the degradation of n-alkanes
into terminal n-alkenes (a carbon carbon double bond at position 1) and lowmolecular-weight carbonyl compounds (Ehrhardt and Weber, 1991).
Page 20
Introduction
The water-soluble components of petroleum exert a toxic effect on marine organisms.
In general, aromatic compounds are more toxic than aliphatic compounds, and smaller
molecules are more toxic than larger ones in the same series. Solar irradiation affects
oil toxicity: Surface films become less toxic due to the loss of polycyclic aromatic
hydrocarbons, but the toxicity of the water-soluble fraction increases as its
concentration increases (Nicodem et al., 1997).
Page 21
Aims & Objective
Chapter-2
AIMS & OBJECTIVE



It was only after the sinking of the super tanker Torney Canyon in the English
Channel that the attention of the scientific community was drawn towards the
problems of oil pollution. Thereafter, several studies have examined the fate of
petroleum in various ecosystems (Boehm et al., 1995; Whittaker et al., 1999).
The development of petroleum industry into new frontiers, the apparent
inevitable spillages that occur during routine operations, and records of acute
accidents during transportation has called for more studies into oil pollution
problems (Timmis et al., 1998), which has been recognized as the most
significant contamination problem on the continent (Snape et al., 2001). Also,
the extensive use of petroleum products leads to the contamination of almost
all compartments of the environment, and biodegradation of the hydrocarbons
by natural populations of microorganisms has been reported to be the main
process acting in the depuration of hydrocarbon-polluted environments
(Challain et al., 2004), the mechanism of which has been extensively studied
and reviewed (van Hamme et al., 2003).
Mechanical method to reduce hydrocarbon pollution is expensive and time
consuming. Hydrocarbons including PAHs have been long recognized as
substrates supporting microbial growth (Bushnell and Haas, 1941; Speight,
1991; Ehrlich, 1995).
The objective of this work is:

To isolates the petroleum degrading microbes from petroleum contaminated
samples (Soil & Sea water).

To identify the isolates by physiological & biochemical characteristics.

To check the biodegradation efficiency of each isolates.

To check the biodegradation potential of each isolates in different hydrocarbon
sources.
Page 22
Materials & Method
Chapter-3
MATERIALS & METHOD
Collection of soil & water Sample:

Oil contaminated-Soil sample was collected from automobile work shop from
Surat. 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.

Petroleum Contaminated-Sea water Sample was collected from Reliance Ltd.
Dahej. Sample were collected in polythene bottle & tightly packed. They were
then carefully transferred to the laboratory for analysis and stored at 4°C
aseptically before processing.
Culture Media:
Enrichments & Isolation of Microorganisms from sample:
For Enrichment the culture Nutrient broth medium was used.

Isolation and enumeration of bacteria from soil sample were performed by soil
dilution plate technique using Minimal agar medium containing filtered crude
oil. The composition of minimal agar media was given following table-3.(17)
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 with tween-20 on plate
Page 23
Materials & Method

The isolation of bacteria from marine sample was performed by following
method: First Enrichment the culture in nutrient agar medium containing NaCl.
Then this culture was spreader on sea water agar media containing
hydrocarbon sources, as sole sources of carbon.
Table– 5 Composition of Minimal agar mediumComponent
Amt. per lit.
Agar
20 g
K2HPO4
4.4 g
NH4cl
2.1 g
KH2PO4
1.7 g
100X Salt medium
10.0 ml
100X Salt medium (per lit.)
MgSO4
19.5 g
FeSO4.7H2O
5.0 g
MnSO4.H2O
5.0 g
Ascorbic acid
1.0 g
CaCi2.2H2O
0.3 g
Basic tests for identification of isolates:
The isolates were identified by various morphological & biochemical test were
performed in this work including: Colony Morphology, Cell Micro
morphology, Grams reaction, motility tests, Fermentation of different sugar,
oxidase, Catalase test & other biochemical test. The Biochemical test was
described in Table-6.(25)
Growth potential of hydrocarbon degrading bacteria:
Growth potential was carried out by using Bushnell-Hass medium with fresh
culture of bacteria. The hydrocarbon substrates (10% v/v; diesel and petrol &
other hydrocarbon sources) were used as sole carbon source.(17)
Page 24
Materials & Method

They were incubated at 30°C at 160rpm for 21 days. A control devoid of the
bacterial isolate was prepared for each set of experiments.(17)
Table-6 Biochemical Reagents
Test
1.Carbohydrate
fermentation test
2. Urea utilization
test
3. H2S Production
test
4. Gelatin hydrolysis
test
5. Citrate utilization
test
6. Nitrate reduction
test
Medium
Reagent
Observation
Glucose, maltose,
Sucrose, Lactose,
Mannitol, Xylose
Urea broth,
Phenol red
Red
yellow
color ( Gas
production)
Pinkish red color
2% Peptone
Lead acetate
paper strip
–
Nutrient gelatin broth
Simmons Citrate agar
Slant
Peptone nitrate broth
7. Oxidase test
8. Catalase test
Nutrient Agar Slant
Nutrient Agar Slant
9. M-R test
Glucose Phosphate
broth
Glucose Phosphate
broth
10. V-P test
11. Iodole
production test
12.TSI slant
13. Macconkey`s
Agar plate
14. Gram`s
stainining
1% Peptone
Triple Sugar iron
agar Slant
Macconkey’s agar
plate
–
Phenol red
Bromothymole
blue
Sulfanylic acid
+
a-Naphylamine
Oxidase strip
3% H2O2
Methyl red
40% KOH
+
a- Naphthol
Kovac`s reagent
Blackish of
paper
Liquefaction at
4°C
Green-Blue
Red color
Violet color
Formation of
bubbles
Red color
Pink color
–
Red ring
production
–
–
–
Grams iodine,
Crystal violet,
Ethanol, D/W,
Safranin
Microscopic
observation.
Page 25
Materials & Method
Methods:
1. By using Oil Contaminated Soil Sample:
Oil Contaminated Soil


1gm sail in 100 ml Nutrient broth
Incubation
Temp. 30 C
Rotation 160 rpm
Time – 3 Days
Enrichment

Dilution
1 ml culture in 9 ml D/W
10-1 to 10-5
Applied on Minimal Agar Plate containing
hydrocarbon source (Crude oil)
Incubation
Temp. – 30°C
Time – 5-7 Days
Select Colony grown on plate
Culture it on Nutrient agar plate
Incubation
Temp. – 30°C
Time – 24 Hrs
Study the Characteristics of colonies
1. Physiological Characters
2. Bio-chemical Characters
BIODEGRADATION POTTENTIAL
Page 26
Materials & Method
BIODEGRADATION POTTENTIAL
Single colony
10 ml Nutrient broth
Inoculation
Temp. – 30°C
Time – 24 Hrs
Incubation
Test
1 ml Bacterial Culture
+
5.0 ml BH Medium
+
0.5 ml Hydrocarbon source
(Petrol, Diesel, Engine oil,
Toluene, Benzene, Xylene)
Control
(No Bacterial culture)
5.0 ml BH Medium
+
0.5 ml Hydrocarbon source
(Petrol, Diesel, Engine oil,
Toluene, Benzene, Xylene)
Incubation
Temp. – 30°C
Time – 21 Days
Rotation– 160 rpm
Observe the tubes at 5 Days
time interval
Page 27
Materials & Method
2. By using Petroleum Contaminated Sea-water Sample:
Oil Contaminated Sea Water


5ml Water in 100 ml Nutrient broth
Incubation Temp. 30 C
Rotation–160 rpm
Time – 3 Days
Enrichment

1 ml culture in 9 ml D/W
Dilution
10-1 to 10-5
Applied on Nutrient Agar Plate
Incubation
Temp. – 30°C
Time – 5-7 Days
Study the Characteristics of colonies
1. Physiological Characters
2. Bio-chemical Characters
Select Colony grown on plate
Applied on Nutrient agar
containing 3-5% NaCl
Incubation
Applied on SWA (Sea Water
Agar) plate containing
Hydrocarbon source.
Temp. – 30°C
Time – 24 Hrs
Incubatio
n
Temp. – 30°C
Time –5-7 days
Observe the growth
BIODEGRADATION POTTENTIAL
Page 28
Materials & Method
BIODEGRADATION POTTENTIAL
Single colony
10 ml Nutrient broth
Inoculation
Temp. – 30°C
Time – 24 Hrs
Incubation
Test
Control
1 ml Bacterial Culture
+
5.0 ml BH Medium
+
0.5 ml Hydrocarbon source
(Petrol, Diesel, Engine oil,
Toluene, Benzene, Xylene)
(No Bacterial culture
added)
5.0 ml BH Medium
+
0.5 ml Hydrocarbon source
(Petrol, Diesel, Engine oil,
Toluene, Benzene, Xylene)
Incubation
Temp. – 30°C
Time – 21 Days
Rotation– 160 rpm
Observe the tubes at 5 Days
time interval
Page 29
Results & Discussion
Chapter-4
RESULTS & DISCUSSION
Physio- chemical characteristics of isolates:
There were total three bacteria, two from soil sample(A1 & A2) & one from
sea water sample (A3), isolated.

They were identified by physiological morphology (Table-7) & Biochemical
characteristics (Table-8).
Table-7 Colony Characteristics of isolates:Characteristics
Size
Shape
Color
Margin
Elevation
Opacity
Consistency
A1
Small
Circular
Yellow
Entire
Convex
Opaque
Dry
Isolates
A2
Medium
Circular
Yellow
Entire
Convex
Opaque
Moist
A3
Medium
Circular
Colorless
Entire
Convex
Opaque
Moist
Table-8 Biochemical Characteristics of organisms:
A1
A2
A3
Glucose
+
+
+
Sucrose
+
–
+
Maltose
+
+
+
Mannitol
+
–
+
Lactose
–
–
+
Xylose
+
–
+
Urea utilization
test
–
–
-
Test
1.
2.
Carbohydrate
hydrolysis
Page 30
Results & Discussion
3.
H2S Production
test
–
–
-
4.
–
–
-
+
+
+ (Blue color)
-
+
+
7.
Gelatin
hydrolysis test
Citrate
utilization test
Nitrate
reduction test
Oxidase test
+
+
+
8.
Catalase test
+
+
+
9.
M-R test
–
–
+
10. V-P test
–
–
–
11. Iodole
production test
12. TSI slant
–
–
–
No color change
No color change
Slant/buttYellow
No gas prods.
Pink colored
colony grown
With pink
centre
Gram negative,
Short rod
Shaped
Non-motile
5
6.
13. Macconkey`s
Agar plate
14. Gram`s
stainining
15. Motility
Keys-
No growth
obtained
Yellowish color
colony Grown
Gram positive,
Cocci
Gram negative,
Rod shape
Non-motile
Motile
+ -- Positive test – -- Negative test
Page 31
Results & Discussion
Figure 1. Gram Staining of A3 Organism:
Figure 2. Growth of org. on Sea-water
Gram Negative, Rod shape
agar media. (A3 Culture)
Oxidase strip
Engine oil
Test
Contro
l
Figure-3 Oxidase positive test of
Figure-4 Biodegradation of
organism
Engine oil by isolates ( A1
Culture)
Figure-5 Bacterial growth on
Nutrient agar Plate (A3 Culture)
Figure-6 Growth of A1 Culture on
Nutrient agar media
Page 32
Results & Discussion
Biodegradation efficiency:
By means of liquid culture characteristics (Table 9) to degrade different
hydrocarbon sources in minimal medium was noted.

All three microbes used different hydrocarbon as sole sources of carbon and
degraded it so the medium became cloudy from cleared particles. and it was
noted by comparing controls with tests.
Table-9 Liquid culture characteristics of Bacteria during 21 days
incubation:
Table-9.1.1 By using A3 Bacterial culture
Inc.
period
(Days)
0
Control (Petrol)
Test ( Petrol)
Control
(Diesel)
Test (Diesel)
Clear particles of
orange oil on top.
Clear particles of
orange oil on top.
1
5
Same as above
Same as above
10
15
21
Same as above
Same as above
Same as above
Same as above
Medium become
cloudy
Same as above
Same as above
more cloudy
Clear particles
of orange oil on
top.
Same as above
Same as above
Clear particles
of orange oil on
top.
Same as above
Medium
become cloudy
Same as above
Increase growth
Become milky
Same as above
Same as above
Same as above
Table-9.1.2 By using A3 Bacterial culture
Inc.
period
(Days)
0
Control (Engine
oil)
Test (Engine oil)
Control
(Benzene)
Test (Benzene)
1
5
Clear particles of
orange oil on top.
Same as above
Same as above
Clear particles
on top.
Same as above
Same as above
10
15
21
Same as above
Same as above
Same as above
Clear particles of
orange oil on top.
Same as above
Medium become
cloudy
Same as above
Inc cloudiness
Become milky
Clear particles
on top.
Same as above
Medium
become cloudy
Same as above
Same as above
Become milky
Same as above
Same as above
Same as above
Page 33
Results & Discussion
Table-9.1.3 By using A3 Bacterial culture
Incub
ation
period
(Days)
0
Control
(Toluene)
Test (Toluene)
Control
(Xylene)
Test (Xylene)
1
5
10
Clear particles on
top.
Same as above
Same as above
Same as above
Clear particles on
top.
Same as above
Same as above
Same as above
Clear particles
on top.
Same as above
Same as above
Same as above
15
21
Same as above
Same as above
Same as above
Same as above
Same as above
Clear particles
on top.
Same as above
Same as above
Medium
become cloudy
Same as above
Same as above
Medium become
slightly cloudy
Table-9.2.1 By using A2 Bacterial culture
Inc.
period
(Days)
0
Control (Petrol)
Test ( Petrol)
Control
(Diesel)
Test (Diesel)
Clear particles of
orange oil on top.
Clear particles of
orange oil on top.
1
5
Same as above
Same as above
10
15
Same as above
Same as above
Same as above
Medium become
cloudy
Same as above
Same as above
Clear particles
of orange oil on
top.
Same as above
Same as above
Clear particles
of orange oil on
top.
Same as above
Medium
become cloudy
Same as above
More
cloudiness’
Become milky
21
Same as above
more cloudy
Table-9.2.2 By using A2 Bacterial culture
Inc.
period
(Days)
0
Same as above
Same as above
Same as above
Control (Engine
oil)
Test (Engine oil)
Control
(Benzene)
Test (Benzene)
1
5
Clear particles of
orange oil on top.
Same as above
Same as above
Clear particles
on top.
Same as above
Same as above
10
Same as above
Clear particles of
orange oil on top.
Same as above
Medium become
cloudy
Same as above
Same as above
Clear particles
on top.
Same as above
Medium
become cloudy
Same as above
15
Same as above
Same as above
Same as above
21
Same as above
Increase
cloudiness’
Become milky
Same as above
more
cloudiness’
Page 34
Results & Discussion
Table-9.2.3 By using A2 Bacterial culture
Inc.
Control
Test (Toluene)
(Days)
(Toluene)
0
Clear particles on Clear particles on
top.
top.
1
Same as above
Same as above
5
Same as above
Same as above
10
Same as above
Same as above
15
Same as above
Medium become
slightly cloudy
21
Same as above
Same as above
Table-9.3.1 By using A1 Bacterial culture
Inc.
Control (Petrol)
Test ( Petrol)
period
(Days)
0
Clear particles of Clear particles of
orange oil on top. orange oil on top.
1
5
Same as above
Same as above
10
15
Same as above
Same as above
Same as above
Medium become
cloudy
Same as above
more cloudy
21
Same as above
Same as above
Control
(Xylene)
Clear particles
on top.
Same as above
Same as above
Same as above
Same as above
Same as above
Test (Xylene)
Clear particles
on top.
Same as above
Same as above
Same as above
Medium
become cloudy
Same as above
Control
(Diesel)
Test (Diesel)
Clear particles
of orange oil on
top.
Same as above
Same as above
Clear particles
of orange oil on
top.
Same as above
Medium
become cloudy
Same as above
More
cloudiness’
Become milky
Same as above
Same as above
Same as above
Table-9.3.2 By using A1 Bacterial culture
Inc.
period
(Days)
0
Control (Engine
oil)
Test (Engine oil)
Control
(Benzene)
Test (Benzene)
1
5
Clear particles of
orange oil on top.
Same as above
Same as above
Clear particles
on top.
Same as above
Same as above
10
Same as above
Clear particles of
orange oil on top.
Same as above
Medium become
cloudy
Same as above
Same as above
Clear particles
on top.
Same as above
Medium
become cloudy
Same as above
15
Same as above
Same as above
Same as above
21
Same as above
Increase
cloudiness’
Become milky
Same as above
more
cloudiness’
Page 35
Results & Discussion
Table-9.3.3 By using A1 Bacterial culture
Inc.
(Days)
0
1
5
10
15
Control
(Toluene)
Clear particles on
top.
Same as above
Same as above
Same as above
Same as above
21
Same as above
Test (Toluene)
Clear particles on
top.
Same as above
Same as above
Same as above
Same as above
Medium become
slightly cloudy
Control
(Xylene)
Clear particles
on top.
Same as above
Same as above
Same as above
Same as above
Test (Xylene)
Clear particles
on top.
Same as above
Same as above
Same as above
Medium
become cloudy
Same as above
Same as above
Growth potential of isolates in different hydrocarbon sources:
The growth potential of hydrocarbon utilizing bacteria on different
hydrocarbon sources were tested and results were observed. (Table-10)
Table-10 Petroleum degradation potential:
Table-10.1 A1 organism (From soil Sample)
Incubation
Period
5th day
15th day
21st day
Petrol
+
++
+++
Diesel
+
+++
++++
Hydrocarbon source
Engine oil
Toluene
++
+++
+++
–
–
+
Benzene
+
++
++
Xylene
–
+
+
Table-10.2 A2 Organism (From Soil Sample)
Incubation
Period
5th day
15th day
21st day
Petrol
+
++
+++
Diesel
+
++
++++
Hydrocarbon source
Engine oil
Toluene
+
++
+++
–
–
+
Benzene
–
++
+++
Xylene
–
–
+
Page 36
Results & Discussion
Table-10.3 A3 Organism (From Marine Water Sample)
Incubation
Period
5th day
15th day
21st day
Petrol
Diesel
+
++
+++
+
++
+++
Hydrocarbon source
Engine oil
Toluene
+
++
++++
Benzene
–
+
++
Keys-:
Xylene
+
++
+++
–
–
–
+
-- No growth
+ -- Low growth
++ -- Medium growth
+++ -- High growth
++++ -- Very high growth
At 5th days incubation
At 21st days incubation
Figure-7 Bacterial growth on minimal medium
Figure-8 Bacterial growth on
containing different hydrocarbon
(Biodegradation potential) at fifth days
incubation (A2 Culture)
minimal medium containing
different hydrocarbon
(Biodegradation potential)
(A2 Culture)
Page 37
Results & Discussion
Petrol
Test
Diesel
Control Test
Engine oil
Control Test
Control
Crude oil
Test
Control
Benzene
Test Control
Figure-9 Biodegradation potential of organisms(A3) on Different Hydrocarbon
source in minimal media ( After 21st days)
Identification of Hydrocarbon degrading isolated strain:
The bacteria were different based on their growth pigmentation and colony
morphology on nutrient agar and selective media at 37°c for 24hrs.Then the
isolated bacteria were identified by morphological, biochemical characteristics.

An A1 bacterium isolated from oil contaminated soil sample was characterized
as Micrococcus sp., The Micrococcus colonies were identified by the
morphology, yellow color, smaller colonies on nutrient agar.
Cells were
Gram-positive Cocci arranged in tetrads. It was oxidase & catalase positive.

An A2 bacterium also isolated from contaminated soil sample was
characterized as pseudomonas sp. Pseudomonas sp. oxidized glucose, reduced
nitrate and was oxidase positive. These bacteria have been described as the
most common bacteria isolated in terrestrial as well aquatic areas of
hydrocarbon contamination.

An A3 Bacterium isolated from petroleum contaminated sea water was
characterized as Marinobacter sp. Oxidase- and catalase-positive & Urease
negative. Cells are rod-shaped and motile. They can also grow on standard
medium, without hydrocarbons.
Page 38
Conclusion
Chapter-5
CONCLUSION
The ability to isolate high numbers of certain oil degrading microorganisms from oil
polluted environment is commonly taken as evidence that these microorganisms are
the active degraders if the environment.
Isolation was carried out using the traditional microbiological technique with
petridishes containing selective agar with hydrocarbons, as the sole source of carbon.
The soil sample which showed higher contaminated age, yield more numbers of
colonies.
In the present study, 2 species of bacteria (Micrococcus, Pseudomonas) were isolated
from contaminated soil sample and one species of bacterium (Marinobacter sp). was
isolated from marine sample and all of them were cultivated on BHA media with
hydrocarbon as the sole source of carbon.
Here, the degradation efficiency of hydro-carbon degrading bacteria was analyzed
using liquid culture characteristics and emulsification activity.
Page 39
Appendixes
Chapter-6
APPENDIXES
Appendix-1 Culture media:
1. Bushnell-Haas Media:Directions-
Components
Amt. (Gms/Lit.)
MgSo4
0.200 gm
CaCl2
0.020 gm
K2HPO4
1.0 gm
KH2PO4
1.0 gm
Ammonium Nitrate
1.0 gm
Ferric Chloride
0.050 gm
15 lbs pressure (121°C) for 15
Final pH
7.0 ± 0.2
minutes.

Suspend 3.270 grams in
1000 ml distilled water.

Heat to boiling to dissolve
the medium completely.


Sterilize by autoclaving at
Take 990 ml BHM +10 ml
Hydrocarbon source (Oil, Petrol etc.)
.
2. Glucose Phosphate Broth:Components
Amt. (Gms/Lit.)
Glucose
5.0 gm
K2HPO4
5.0 gm
Peptone
5.0 gm
D/W
1000 ml
Final pH
6.9-7.0
Directions
Suspend 15 grams in 1000
ml distilled water.

Heat to boiling to dissolve
the medium completely.

Sterilize by autoclaving at
15 lbs pressure (121°C) for 15
minutes.
Page 40
Appendixes
3. Macconkeys Agar Media:Directions-
Components
Amt. (Gm/Lit.)
Peptone
17.0 gm
Protease peptone
3.0 gm
Lactose
10.0 gm
Bile salt
1.5 gm
NaCl
5.0 gm
Neutral red
0.03 gm
15 lbs pressure (121°C) for 15
Agar
20.0 gm
minutes.
Final pH
7.1 ± 0.2

Suspend 56.53 Gms in 1000
ml distilled water.

Heat to boiling to dissolve
the medium completely.

Sterilize by autoclaving at
4. Nutrient Agar Media:Components
Amt. (Gms/Lit.)
Peptone
10 gm
NaCl
5 gm
Beef Extract
3 gm
Agar
20 gm
Final pH
7.4 ± 0.2
Directions
Suspend 38 gms in 1000 ml
distilled water.

Heat to boiling to dissolve
the medium completely.

Sterilize by autoclaving at
15 lbs pressure (121°C) for 15
minutes.
5. Nutrient Gelatin broth:Components
Amt. (Gms/Lit.)
Meat extract
3.0 gm
Peptone
10.0 gm
Gelatin
150.0 gm
D/W
1000.0 ml
Final pH
7.2
Directions
Suspend 163.0 Gms in 1000
ml distilled water.

Heat to boiling to dissolve
the medium completely.

Sterilize by autoclaving at
15 lbs pressure (121°C) for 15
minutes.
Page 41
Appendixes
6. Nutrient sugar Broth:Components
Amt.
1% Peptone
90 ml
10% Sugar ( E.g.
10 ml
Directions-

100 ml distilled
the
components
given in table.

Glucose- 10 Gms in
Mixed
Sterilize by autoclaving at
10 lbs pressure (121°C) for 10
water)
minutes.
Phenol red
0.01 gm
Final pH
7.4 ± 0.2
7. Peptone Nitrate Broth:Components
Amt. (Gms/Lit.)
Meat extract
3.0 gm
Peptone
5.0 gm
Potassium nitrate
1.0 gm
D/W
1000 ml
Final pH
7.5
Directions
Suspended 9 gm component
in 1000 ml D/W.

Sterilize by autoclaving at
15 lbs pressure (121°C) for 15
minutes.
8. Sea-Water Agar Media:Components
Amt. (Gms/Lit.)
Directions
Dissolve
the
content
in
K2HPO4.3H2O
0.01 gm
Urea
0.45 gm
Sea water
1000ml
strains onto quadrants of SWMA
Agar
20 gm
agar, a carbon source was added to
Final pH
7.5 ± 0.2
the center, and the plates were
filtered sea water.

After streaking the different
incubated at 32 C for 1 week.
Page 42
Appendixes
9. Simmons Citrate Agar:Components
Amt. (Gms/Lit.)
Sodium citrate
2.0 gm
MgSO4
0.2 gm
NaCl
5.0 gm
Ammonium
Dihydrogen
phosphate
K2HPO4
1.0 gm
Bromothymole blue
0.08 gm
Agar
20.0 gm
Final pH
6.9
Directions
Suspended
29.28
gm
component in 1000 ml D/W.

Sterilize by autoclaving at
15 lbs pressure (121°C) for 15
minutes.

1.0 gm
Pour into sterilized
petriplates & solidified it.
10.1% Tryptone broth:Components
Amt. (Gms/Lit.)
Tryptone
10.0 gm
NaCl
5.0 gm
D/W
1000 ml
Final pH
7.5
Directions
Suspended
15
gm
component in 1000 ml D/W.

Sterilize by autoclaving at
15 lbs pressure (121°C) for 15
minutes.
11.Urea Broth:Components
Amt. (Gms/Lit.)
KH2PO4
9.1
Na2HPO4
9.5
Yeast extract
0.1
Phenol red
0.01
Distilled water
950.0ml
40% Urea
50.0ml
Final pH
6.8
Directions
First mix the component in
950ml distilled water.

Then add 50 ml 40% Urea
in it. and adjust the pH 6.8.

Sterilize by autoclaving at
10 lbs pressure (121°C) for 10
minutes.
Page 43
Appendixes
Appendix-2 Stains & Reagents:
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. Gram`s Iodine:
Dissolve Potassium Iodide (2.0 gm) & Crystal Iodine (1.0 gm) in
some amount of water & then make up 300 ml with D/W. Protect
from sunlight.
5. Sulfanilic acid:
Dissolve 8 g of Sulfanilic acid in 1 liter 5N acetic acid. Store Reagent
A at room temperature for up to 3 months, in dark. Reagents may be
stored in dark brown glass containers; bottles may be wrapped in
aluminum foil to ensure darkness.
6. a-Naphylamine:
Dissolve 6 g of N, N-Dimethyl-1-naphthylamine in 1 liter 5N acetic
acid. Store Reagent B at 2 to 8°C for up to 3 months, in dark.
Reagents may be stored in dark brown glass containers; bottles may
be wrapped in aluminum foil to ensure darkness.
Page 44
References
Chapter-7
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