· Web viewMiss. Priya Bande, Department of Biotechnology & Environment Science MITCON, Pune,...

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

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

INDEX

Chapter

No.

Title Page

No.Abstract 2

List of Tables 4

List of figure 5

Acknowledgement 6

Abbreviation 8

1. Introduction:

Definition

Origin, constitution and use

Component of crude oil

Behavior of petroleum in Marine environment

9

9

9

16

19

2. Aims & Objectives 22

3. Material & method:

Collection of sample

Culture media

Biochemical reagents

Methods

23

23

23

25

26

4. Results & Discussion:

Physio-chemical characteristics of isolates.

Biodegradation efficiency.

Growth potential of isolates.

Identification of petroleum degrading isolated strains

30

30

33

36

38

5. Conclusion 39

6. Appendixes:

Appendix-1- Culture Medium

Appendix-2- Stains & Reagents

40

40

44

7. References 45

LIST OF TABLES

Table

No.

Title 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

incubation.

33

10. Petroleum degradation Efficiency. 36

LIST OF FIGURES

Figure

No.

Title 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

hydrocarbon (Biodegradation potential) at fifth days incubation

37

8. Bacterial growth on minimal medium containing different

hydrocarbon (Biodegradation potential) (A2 Culture)

37

9. Biodegradation potential of organisms(A3) on Different

Hydrocarbon source in minimal media ( After 21st days)

38

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.

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.

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

http://en.wikipedia.org/wiki/Wurster's_blue

Introduction

Chapter-1

INTRODUCTIONDefinitionBiodegradation 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)

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

RKJ4

Phenanthrene Samanta et al., 1999

Acidovorax delafieldii

P41

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 Phenanthrene Samanta et al., 1999Burkholderia sp.S3702,

RP007,2A12TNFYE5,

BS3770

Phenanthrene Kang et al., 2003,Balashova et al., 1999,

Laurie et al., 1999

Burkholderia sp. C3 Phenanthrene Seo et al., 2006Burkholderia cepacia

BU3Phenanthrene, Pyrene,

NaphthaleneKim et al., 2003

Burkholderia xenovoransLB400

Benzoate, Biphenyl Denef et al., 2005

Chryseobacterium sp. NCY

Carbazole Guo et al., 2008

Cycloclasticus sp. P1 Pyrene Wang et al., 2008Geobacillus sp. Napthalene, Phenanthrene,

Fluorene Bubians et al., 2007Geobacillus

stearothermophilus“AAP7919”

Anthracene Kumar et al., 2011

Janibacter sp. YY1 Phenanthrene, Fluorene,Anthracene, Dibenzofuran,

Yamazoe et al., 2004

Introduction

Dibenzopdioxin,Dibenzothiophene

Marinobacter NCE312 Naphthalene Hedlund et al., 2001Mycobacterium sp.PYR, Benzo[a]pyrene Cheung et al., 2001,

Grosser et al., 1991Mycobacterium sp. JS14 Fluoranthene Lee et al., 2007

Mycobacterium sp. 6PY1, KR2,AP1

Pyrene Rehmann et al., 1998,Vila et al., 2001,

Krivobok et al., 2003Mycobacterium sp.

RJGII135Benzo[a]pyrene,

Benz[a]anthracenePyrene

Schneider et al., 1996

Mycobacterium sp.PYR1,LB501T

Pyrene, Phenanthrene,Fluoranthene, Anthracene

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

Pyrene, Phenanthrene, Fluorene

Boldrin et al., 1993,Rehmann et al., 2001

Mycobacterium flavescens

Pyrene, Fluoranthene DeanRosset al., 2002,DeanRosset al., 1996

Mycobacterium vanbaalenii

PYR1

PhenanthrenePyrene,

Dimethylbenz[a]anthracene

Kim et al., 2005,Moody et al., 2003

Mycobacterium sp. KMS Pyrene Miller et al., 2004Nocardioides

aromaticivoransIC177

Carbazole Inoue et al., 2006

Pasteurella sp. IFA Fluoranthene Sepic 1999Polaromonas

naphthalenivorans CJ2Naphthalene Pumphrey et al., 2007

Pseudomonas sp. C18, PP2,

DLCP11

Phenanthrene, Naphthalene Denome et al., 1993,Prabhu et al., 2003

Pseudomonas sp. BT1d 3hydroxy2formylbenzothiophene

Bressler et al., 2001

Pseudomonas sp. HH69 Dibenzofuran Fortnagel et al., 1990Pseudomonas sp. CA10 Chlorinated dibenzopdioxin,

CarbazoleHabe et al., 2001

Pseudomonas sp. NCIB 98164

Fluorene, Dibenzofuran,Dibenzothiophene

Resnick et al., 1996

Pseudomonas sp. F274 Fluorene Grifoll et al., 1994

Introduction

Pseudomonas paucimobilis

Phenanthrene Weissenfels et al., 1990

Pseudomonas vesicularisOUS82

Fluorene Weissenfels et al., 1990

Pseudomonas putida P16,BS3701, BS3750,

BS590P,BS202P1

Phenanthrene, Naphthalene Kiyohara et al., 1994,Balashova et al., 1999

Pseudomonas fluorescensBS3760

Phenanthrene, Benz[a]anthracene,

Chrysene

Balashova et al., 1999

Pseudomonas stutzeri P15

Pyrene Kazunga et al., 2000

Pseudomonas saccharophilia


Pseudomonas aeruginosa Phenanthrene Romero et al., 1998Ralstonia sp. SBUG 290,

U2Naphthalene, Dibenzofuran Becher et al., 2000,

Zhou et al., 2002Rhodanobacter sp. BPC1 Benzo[a]pyrene Kanaly et al., 2002

Rhodococcus sp. Pyrene, Fluoranthene DeanRosset al., 2002,

Walter et al., 1991Rhodococcus sp.

WUK2RBenzothiophene,

NaphthothiopheneKirimura et al., 2002

Rhodococcus erythropolis I19

Alkylated dibenzothiophene Folsom et al., 1999

Rhodococcus erythropolis D1

Dibenzothiophene Matsubara et al., 2001

Staphylococcus sp. PN/Y Phenanthrene Mallick et al., 2007Stenotrophomonas

maltophiliaVUN 10,010

Benzo[a]pyrenePyrene, Fluoranthene

Boonchan et al., 1998

Stenotrophomonas maltophiliaVUN 10,003

Pyrene, Fluoranthene,Benz[a]anthracene

Juhasz et al., 2000

Sphingomonas yanoikuyae R1


Sphingomonas yanoikuyae

JAR02

Benzo[a]pyrene Rentz et al., 2008

Sphingomonas sp.P2, LB126

Phenanthrene, Fluoranthene,Fluorene, Anthracene

Pinyakong et al., 2003,Van et al., 2003,

Pinyakong et al., 2000Sphingomonas sp. Dibenzofuran, Carbazole,

DibenzothiopheneGai et al., 2007

Sphingomonas Phenanthrene, Fluoranthene, Story et al., 2001,

Introduction

paucimobilisEPA505

Anthracene, Naphthalene Mueller et al., 1990

Sphingomonas wittichii RW1

Chlorinated dibenzopdioxin Nam et al., 2006

Sphingomonas sp. KS14 Phenanthrene, Naphthalene Cho et al., 2001Terrabacter sp.DBF63 Fluorene, Dibenzofuran,

Chlorinated dibenzopdioxin,Chlorinated dibenzothophene

Habe et al., 2004, Habeet al., 2001, Habe et al.,

2002

Xanthamonas sp. Benzo[a]pyrenePyrene, Carbazole

Grosser et al., 1991

Table 2: Fungal genera capable of degrading PAHs (20):-

Name of Fungus PAH Reference

Phanerochaete

chrysporium

Anthracene Field et al.,1996

Bjerkandera sp. strainBOS55

Anthracene Field et al.,1996

Trametes versicolor Anthracene Collins et al., 1986

Cunninghamellaelegansoxidizes

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

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

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 (Hallier-

Soulier et al., 1999).

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).

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 andgreases, as viscosity stabilizers

3. Wax Used in the packaging of frozen foods4. Petroleum coke

(asphalt)Used in carbon products or as solid fuel, Paraffin waxes. Aromatic petrochemicals as precursors in other chemical synthesis.

5. Paraffin wax & aromaticpetrochemicals

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).

Introduction

1. Saturates are hydrocarbons containing no double bonds. They are further classified

according to their chemical structures into Alkanes (paraffin) and Cycloalkanes

(naphthenes).

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 UV-

Introduction

absorption spectrometry, NMR and elemental analysis in combination with

appropriate separation techniques such as various chromatographic methods and/or

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. RadialDepiction for

PAH

PAH Name Molecularformula

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

Introduction

17. Tpl Triphenylene C18H12

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 low-

molecular-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

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 low-

molecular-weight carbonyl compounds (Ehrhardt and Weber, 1991).

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).

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.

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

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 medium-Component 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)

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)

Materials & Method

Table-6 Biochemical Reagents

Test Medium Reagent Observation1.Carbohydrate fermentation test

Glucose, maltose, Sucrose, Lactose, Mannitol, Xylose

Phenol red Red yellow color ( Gas production)

2. Urea utilization test

Urea broth, Phenol red Pinkish red color

3. H2S Production test

2% Peptone Lead acetate paper strip

Blackish of paper

4. Gelatin hydrolysis test

Nutrient gelatin broth – Liquefaction at 4°C

5. Citrate utilization test

Simmons Citrate agar Slant

Bromothymole blue

Green-Blue

6. Nitrate reduction test

Peptone nitrate broth Sulfanylic acid+

a-Naphylamine

Red color

7. Oxidase test Nutrient Agar Slant Oxidase strip Violet color8. Catalase test Nutrient Agar Slant 3% H2O2 Formation of

bubbles9. M-R test Glucose Phosphate

brothMethyl red Red color

10. V-P test Glucose Phosphate broth

40% KOH+

a- Naphthol

Pink color

11. Iodole production test

1% Peptone Kovac`s reagent Red ring production

12.TSI slant Triple Sugar iron agar Slant

– –

13. Macconkey`s Agar plate

Macconkey’s agar plate

– –

14. Gram`s stainining

– Grams iodine, Crystal violet, Ethanol, D/W,

Safranin

Microscopic observation.

Methods:

1. By using Oil Contaminated Soil Sample:

1gm sail in 100 ml Nutrient brothIncubation Temp. 30 CRotation 160 rpmTime – 3 Days

1 ml culture in 9 ml D/W

1. Physiological Characters2. Bio-chemical Characters

10-1 to 10-5

Oil Contaminated Soil

Enrichment

Dilution

Applied on Minimal Agar Plate containing hydrocarbon source (Crude oil)

Incubation Temp. – 30°CTime – 5-7 Days

Select Colony grown on plate

Culture it on Nutrient agar plate

Temp. – 30°CTime – 24 Hrs

Incubation

Study the Characteristics of colonies

BIODEGRADATION POTTENTIAL


Single colony 10 ml Nutrient brothInoculation

Materials & Method

Materials & Method

2. By using Petroleum Contaminated Sea-water Sample:

5ml Water in 100 ml Nutrient brothIncubation - Temp. 30 C Rotation–160 rpm Time – 3 Days

1 ml culture in 9 ml D/W

10-1 to 10-5

Oil Contaminated Sea Water

Enrichment

Dilution

Incubation Temp. – 30°CTime – 5-7 Days

Select Colony grown on plate

Temp. – 30°CTime – 24 Hrs Incubation


Applied on Nutrient Agar Plate

1. Physiological Characters2. Bio-chemical Characters

Study the Characteristics of colonies

Applied on Nutrient agar containing 3-5% NaClApplied on SWA (Sea Water Agar) plate containing Hydrocarbon source.

Incubation Temp. – 30°CTime –5-7 days

Observe the growth


Single colony 10 ml Nutrient brothInoculation

Materials & Method

Materials & Method

Results & Discussion

Chapter-4

RESULTS & DISCUSSIONPhysio- 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 IsolatesA1 A2 A3

Size Small Medium MediumShape Circular Circular CircularColor Yellow Yellow Colorless

Margin Entire Entire EntireElevation Convex Convex ConvexOpacity Opaque Opaque Opaque

Consistency Dry Moist Moist

Table-8 Biochemical Characteristics of organisms:

Test A1 A2 A3

1.Carbohydrate hydrolysis

Glucose + + +

Sucrose + – +

Maltose + + +

Mannitol + – +

Lactose – – +

Xylose + – +

2. Urea utilization test

– – -


3. H2S Production test

– – -

4. Gelatin hydrolysis test

– – -

5 Citrate utilization test

+ + + (Blue color)

6. Nitrate reduction test

- + +

7. 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/butt- Yellow

No gas prods.13. Macconkey`s

Agar plate No growth obtained

Yellowish color colony Grown

Pink colored colony grown

With pink centre

14. Gram`s stainining

Gram positive, Cocci

Gram negative, Rod shape

Gram negative, Short rod Shaped

15. Motility Non-motile Motile Non-motile

Keys- + -- Positive test – -- Negative test

Oxidase strip

Figure-3 Oxidase positive test of organism

Figure-4 Biodegradation of Engine oil by isolates ( A1 Culture)

Test Control

Engine oil


Figure 1 . Gram Staining of A3 Organism: Gram Negative, Rod shape

Figure 2. Growth of org. on Sea-water agar media. (A3 Culture)

Figure-5 Bacterial growth on Nutrient agar Plate (A3 Culture)

Figure-6 Growth of A1 Culture on Nutrient agar media


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)

Control (Petrol) Test ( Petrol) Control (Diesel)

Test (Diesel)

0 Clear particles of orange oil on top.

Clear particles of orange oil on top.

Clear particles of orange oil on

top.


top.1 Same as above Same as above Same as above Same as above5 Same as above Medium become

cloudySame as above Medium

become cloudy10 Same as above Same as above Same as above Same as above15 Same as above Same as above Same as above Increase growth21 Same as above more cloudy Same as above Become milky


Inc. period(Days)

Control (Engine oil)

Test (Engine oil) Control (Benzene)

Test (Benzene)



Clear particles on top.


1 Same as above Same as above Same as above Same as above5 Same as above Medium become


become cloudy10 Same as above Same as above Same as above Same as above15 Same as above Inc cloudiness Same as above Same as above21 Same as above Become milky Same as above Become milky



Incubation

period(Days)

Control (Toluene)

Test (Toluene) Control (Xylene)

Test (Xylene)

0 Clear particles on top.




1 Same as above Same as above Same as above Same as above5 Same as above Same as above Same as above Same as above10 Same as above Same as above Same as above Medium

become cloudy15 Same as above Same as above Same as above Same as above21 Same as above Medium become

slightly cloudySame as above Same as above


Inc. period(Days)


Test (Diesel)




top.




become cloudy10 Same as above Same as above Same as above Same as above15 Same as above Same as above Same as above More

cloudiness’21 Same as above more cloudy Same as above Become milky


Inc. period(Days)



Test (Benzene)







become cloudy10 Same as above Same as above Same as above Same as above

15 Same as above Increase cloudiness’

Same as above Same as above

21 Same as above Become milky Same as above more cloudiness’


Table-9.2.3 By using A2 Bacterial cultureInc.

(Days)Control

(Toluene)Test (Toluene) Control

(Xylene)Test (Xylene)





1 Same as above Same as above Same as above Same as above5 Same as above Same as above Same as above Same as above10 Same as above Same as above Same as above Same as above15 Same as above Medium become

slightly cloudy Same as above Medium

become cloudy 21 Same as above Same as above Same as above Same as above

Table-9.3.1 By using A1 Bacterial cultureInc.

period(Days)


Test (Diesel)




top.




become cloudy10 Same as above Same as above Same as above Same as above15 Same as above more cloudy Same as above More

cloudiness’21 Same as above Same as above Same as above Become milky


Inc. period(Days)



Test (Benzene)







become cloudy10 Same as above Same as above Same as above Same as above

15 Same as above Increase cloudiness’

Same as above Same as above

21 Same as above Become milky Same as above more cloudiness’



Inc. (Days)

Control (Toluene)

Test (Toluene) Control (Xylene)

Test (Xylene)





1 Same as above Same as above Same as above Same as above5 Same as above Same as above Same as above Same as above10 Same as above Same as above Same as above Same as above15 Same as above Same as above Same as above Medium

become cloudy 21 Same as above Medium become

slightly 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

Hydrocarbon sourcePetrol Diesel Engine oil Toluene Benzene Xylene

5th day15th day21st day

+++

+++

++++

++++

++++++++

––+

+++++

–++

Table-10.2 A2 Organism (From Soil Sample)

Incubation Period



+++

+++

+++

++++

+++

+++

––+

–++

+++

––+


Table-10.3 A3 Organism (From Marine Water Sample)

Incubation Period



+++

+++

+++

+++

+++

++++

–+

++

+++

+++

––+

Keys-: – -- No growth

+ -- Low growth ++ -- Medium growth

+++ -- High growth ++++ -- Very high growth

Figure-7 Bacterial growth on minimal medium containing different hydrocarbon (Biodegradation potential) at fifth days incubation (A2 Culture)

Figure-8 Bacterial growth on minimal medium containing different hydrocarbon (Biodegradation potential)(A2 Culture)

At 5th days incubation At 21st days incubation

Figure-9 Biodegradation potential of organisms(A3) on Different Hydrocarbon source in minimal media ( After 21st days)

Engine oilDieselPetrol

Test Control

Crude oil

TestTestTest TestControl Control

Benzene

ControlControl


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.

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.

Appendixes

Chapter-6

APPENDIXESAppendix-1 Culture media:

1. Bushnell-Haas Media:-Directions-

Suspend 3.270 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.

Take 990 ml BHM +10 ml

Hydrocarbon source (Oil, Petrol etc.)

.

2. Glucose Phosphate Broth:-Directions-

Suspend 15 grams in 1000 ml distilled water.


completely.

Sterilize by autoclaving at 15 lbs

pressure (121°C) for 15 minutes.

3. Macconkeys Agar 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

Final pH 7.0 ± 0.2


Glucose 5.0 gm

K2HPO4 5.0 gm

Peptone 5.0 gm

D/W 1000 ml

Final pH 6.9-7.0

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

Agar 20.0 gm

Final pH 7.1 ± 0.2

Appendixes

Suspend 56.53 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.

4. Nutrient Agar Media:-Directions-

Suspend 38 gms in 1000 ml distilled water.


completely.



5. Nutrient Gelatin broth:-Directions-

Suspend 163.0 Gms in 1000 ml distilled

water.


completely.



6. Nutrient sugar Broth:-Directions-

Mixed the components given in table.




Peptone 10 gm

NaCl 5 gm

Beef Extract 3 gm

Agar 20 gm

Final pH 7.4 ± 0.2


Meat extract 3.0 gm

Peptone 10.0 gm

Gelatin 150.0 gm

D/W 1000.0 ml

Final pH 7.2

Components Amt.

1% Peptone 90 ml

10% Sugar ( E.g.

Glucose- 10 Gms in

100 ml distilled

water)

10 ml

Phenol red 0.01 gm

Final pH 7.4 ± 0.2

Appendixes

7. Peptone Nitrate Broth:-Directions-

Suspended 9 gm component in 1000 ml D/W.



8. Sea-Water Agar Media:-Directions-

Dissolve the content in filtered sea water.

After streaking the different strains onto

quadrants of SWMA agar, a carbon source

was added to the center, and the plates were

incubated at 32 C for 1 week.

9. Simmons Citrate Agar:-Directions-

Suspended 29.28 gm component in 1000 ml

D/W.



Pour into sterilized petriplates & solidified

it.


Meat extract 3.0 gm

Peptone 5.0 gm

Potassium nitrate 1.0 gm

D/W 1000 ml

Final pH 7.5


K2HPO4.3H2O 0.01 gm

Urea 0.45 gm

Sea water 1000ml

Agar 20 gm

Final pH 7.5 ± 0.2


Sodium citrate 2.0 gm

MgSO4 0.2 gm

NaCl 5.0 gm

Ammonium Dihydrogen phosphate

1.0 gm

K2HPO4 1.0 gm

Bromothymole blue 0.08 gm

Agar 20.0 gm

Final pH 6.9

Appendixes

10.1% Tryptone broth:-Directions-

Suspended 15 gm component in 1000 ml D/W.



11.Urea Broth:-Directions-

First mix the component in 950ml distilled

water.

Then add 50 ml 40% Urea in it. and adjust the

pH 6.8.



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


Tryptone 10.0 gm

NaCl 5.0 gm

D/W 1000 ml

Final pH 7.5


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

Appendixes

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.

References

Chapter-7

REFERENCES

1. Abraham, W.R., Meyer, H., and Yakimov, M. 1998. Novel glycine containing

glucolipids from the alkanes using bacterium Alcanivorax borkumensis. Biochim.

Biophys. Acta 1393: 57-62.

2. Adeline, S. Y. Ting, Carol, H. C. Tan and Aw, C. S. Hydrocarbon-degradation by

isolate Pseudomonas lundensis UTAR FPE2. Malaysian Journal of Microbiology,

Vol 5(2) 2009, pp. 104-108.

3. Akio Ueno1, Yukiya Ito2, Isao Yumoto3, Hidetoshi Okuyama. Isolation and

characterization of bacteria from soil contaminated with diesel oil, and the

possible use of these in autochthonous bioaugmentation

4. Amikam Horowitz, David Gutnick, & Eugene Rosenberg. Sequential Growth of

Bacteria on Crude Oil. Appuan Microbiology, July 1975, p. 10-19

5. Anthony I Okoh. Biodegradation alternative in the cleanup of petroleum

hydrocarbon pollutants. Biotechnology and Molecular Biology Review Vol. 1 (2),

pp. 38-50, June 2006

6. Anupama mittal & Padma Singh, Department of Microbiology, Jwalapur,

Haridwar, India. Isolation of hydrocarbon degrading bacteria from soil

caontaminated with crude oil spills. Sep.2005, Vol.47.

7. Chenli Liu and Zongze Shao. Alcanivorax dieselolei sp. nov., a novel alkane-

degrading bacterium isolated from sea water and deep-sea sediment. International

Journal of Systematic and Evolutionary Microbiology (2005), 55, 1181–1186

References

8. David R. Arahal, Itziar Lekunberri, Jose´ M. Gonza´ lez, Javier Pascual, Marı´a J.

Pujalte, Carlos Pedro´ s-Alio´ and Jarone Pinhassi. Neptuniibacter caesariensis

gen. nov., sp. nov., a novel marine genome-sequenced gammaproteobacterium.

International Journal of Systematic and Evolutionary Microbiology (2007), 57,

1000–1006

9. Esin Eraydin Erdoğan1*, Fikrettin Sahin2 and Ayten Karaca1. Determination of

petroleum degrading bacteria isolated from crude oil-contaminated soil in Turkey.

African Journal of Biotechnology Vol. x(xx), 2011

10. Ganesh R. Bartakke, Mangesh V. Suryavanshi, Avinash A. Raut and Mohanlal B.

Gandhi. Isolation & Characterization of Camphor degrading bacteria.

International Journal of Advanced Biotechnology and Research ISSN 0976-2612,

Vol 2, Issue 4, 2011, pp 468-472

11. Jackie Aislabie á Julia Foght á David Saul. Aromatic hydrocarbon-degrading

bacteria from soil near Scott Base, Antarctica. Polar Biol (2000) 23: 183±188

12. J. D. Walker' & R. R. Colwell, Enumeration of Petroleum-Degrading

Microorganisms. Applied & Environmental Microbiology, Feb. 1976, p. 198-207

13. J. D. Walker and R. R. Colwell. Appl. Microbial. 1974, 27(6):1053. Microbial

Petroleum Degradation: Use of Mixed Hydrocarbon Substrates

14. Jahir Alam Khan and Shrashe Singh. Evaluation of oil degradination potential of

Micrococcus varians. International journal of applied biology & Pharmaceuticalk

technology, Volume: 2: Issue-4: Oct - Dec -2011 ISSN 0976-4550

15. Joseph G. Leahy & Rita R. Colwell. Microbial Degradation of Hydrocarbons in

the Environment. Microbiological Reviews, Sept. 1990, p. 305-315

References

16. K. Jirasripongpun. The characterization of oil-degrading microorganisms from

lubricating oil contaminated (scale) soil. Applied Microbiology 2002, 35, 296–

300.

17. K. Santhini, J. Myla, S. Sajani and G.Usharani. Screening of Micrococcus Sp

from Oil Contaminated Soil with Reference to Bioremediation. Botany Research

International 2 (4): 248-252, 2009 ISSN 1995-8951.

18. Kastburi venkaieshwaran, Tokuro Iwabuchi, Yasuko Matsui, Haruhisa Toki,

Eisuke Hamada & Hiroki Tanaka. Distribution & Biodegradation potential of oil-

degrading bacteria in North Eastern Japanese coastal waters, 1991

19. Kenneth Lee†and Francois Xavier Merlin. Bioremediation of oil on shoreline

environments: development of techniques and guidelines. Pure Appl. Chem., Vol.

71, No. 1, pp. 161–171, 1999.

20. Kumar Arun 1 , Munjal Ashok 1 , Sawhney RajeshCrude oil PAH constitution,

degradation pathway and associated bioremediation microflora: an overview.

International Journal of environmental sciences vol.1,Nov. 7, 2011

21. L. A. Nwaogu1*, G. O. C. Onyeze1 and R. N. Nwabueze. Degradation of diesel

oil in a polluted soil using Bacillus subtilis. African Journal of Biotechnology

Vol. 7 (12), pp. 1939-1943, 17 June, 2008

22. Lies Indah Sutiknowati. Hydrocarbon degrading bacteria: Isolation &

Identification. Makara, Sains, Vol. 11, No. 2, Nov. 2007: 98-103

23. Lyel G. Whyte, Luc Bourbonnie`re, & Charles W. Greer* NRC—Biotechnology

Research Institute, Montreal, Quebec, Canada H4P 2R2. Biodegradation of

Petroleum Hydrocarbons by Psychotropic Pseudomonas Strains Possessing Both

References

Alkanes (alk) and Naphthalene (nah) Catabolic Pathways. Applied &

Environmental Microbiology, 0099-2240/97/$04.0010, Sept. 1997, p. 3719–3723

24. M. C. Ma´rquez and A. Ventosa. Marinobacter hydrocarbonoclasticus Gauthier

et al. 1992 and Marinobacter aquaeolei Nguyen et al. 1999 are heterotypic

synonyms. International Journal of Systematic and Evolutionary Microbiology

(2005), 55, 1349–1351

25. M.Mashreghi & K.Marialigeti,Department of biology, Faculty of Science,

Ferdwosi of Mashhad, Iran,Characterization of Bacteria Degrading Petroleum

derivative isolated from contaminated soil & Water(2005)

26. Manoj Kumara, Vladimir Leona, Angela De Sisto Materanoa, Olaf A. Ilzinsa,

Ivan Galindo-Castroa, and Sergio L. Fuenmayora. Polycyclic Aromatic

Hydrocarbon Degradation by Biosurfactant-Producing Pseudomonas sp. IR1

27. P.O. Okerentugba & O.U. Ezeronye. Petroleum degrading potentials of single and

mixed microbial cultures isolated from rivers and refinery effluent in Nigeria. July

2003. African Journal of Biotechnology Vol. 2 (9), pp. 288-292, September 2003

28. R. Margesin · F. Schinner. Biodegradation and bioremediation of hydrocarbons

in extreme environments. Appl Microbiol Biotechnol (2001) 56:650–663.

29. Ronald M. Atlas. Microbial Degradation of Petroleum Hydrocarbons: an

Environmental Perspective. Microbiological Reviews, March 1981, p. 180-209

Vol. 45

30. Ruma Roy, Raja Ray, Ranjana Chowdhury, Pinaki Bhattacharya. Degradation of

polyaromatic hydrocarbons by mixed culture isolated from oil contaminated soil

—A bioprocess engineering study. Indian Journal of Biotechnology Vol 6,

January 2007, pp 107-113

References

31. Shigeaki Harayama*, Hideo Kishira, Yuki Kasai and Kazuaki Shutsubo,

Petroleum Biodegradation in Marine Environments.

32. Sayyed Hossein Mirdamadian1, Giti Emtiazi2, Mohammad H. Golabi3 and

Hossein Ghanavati. Biodegradation of Petroleum and Aromatic Hydrocarbons by

Bacteria Isolated from Petroleum-Contaminated Soil.

33. T. Mandri and J. Lin. Isolation and characterization of engine oil degrading

indigenous microorganisms in Kwazulu-Natal, South Africa. African Journal of

Biotechnology Vol. 6(1), pp. 023-027, 4 January 2007

34. WOLFGANG FRITSCHE MARTIN HOFRICHTER Jena, Germany, Aerobic

Degradation by Microorganisms.

35. Yin Shen,1 Lester G. Sthemeier,1,2 & Gerrit Voordoum. Identification of

Hydrocarbon-Degrading Bacteria in Soil by Reverse Sample Genome Probing.

November 1997

36. ZHANG Guo-liang, WU Yue-ting 1, QIAN Xin-ping, MENG Qin.

Biodegradation of crude oil by Pseudomonas aeruginosa in the presence of

rhamnolipids. Journal of Zhejiang University SCIENCE ISSN 1009-3095.

· Web viewMiss. Priya Bande, Department of Biotechnology & Environment Science MITCON, Pune,...

Documents

Transcript of · Web viewMiss. Priya Bande, Department of Biotechnology & Environment Science MITCON, Pune,...