A DISSERTATION ON

Biochemical and Molecular

characterization of some diesel

degrading bacterial isolates from oil

contaminated soil sampleSUBMITTED TO THE

DEPARTMENT OF BIOTECHNOLOGY

INTEGRALUNIVERSITY, LUCKNOW

IN PARTIAL FULFILMENT

FOR THE

DEGREE OF MASTER OF SCIENCE

IN MICROBIOLOGY

BY

Hashim Ali

M. Sc. Microbiology (IV semester)

Department of Biosciences

Integral University, Lucknow

UNDER THE SUPERVISION

OF

Dr. Irafn Ahmed

Ansari AssistantProfessor

Department of

Biosciences

Integral

University

LUCKNOW

N INTEGRAL UNIVERSITYEstablished Under U.P. Act No 09 of 2004 by

State Legislation Approved by University Grants

Commission Phone No.: +91 (0552) 2890812,

2890730, 3296117, 6451039, Fax No.: 0522-

2890809

Kursi Road, Lucknow-226026, Uttar Pradesh (INDIA)

TO WHOM IT MAY CONCERN

This is to certify that Mr. Hashim Ali, a student of M.

Sc. Microbiology (IV semester), Integral University has

completed his/her three months dissertation work entitled

“Biochemical and molecular characterization of some

diesel degrading Bacterial isolates from oil contaminated

soil sample ” successfully. He has completed this work

from1st Jan to 31st March Integral University Lucknow under

the guidance of Dr.Irfan Ahmad Ansari. The dissertation

was a compulsory part of his M. Sc. degree.

I wish him good luck and bright future.

Prof. Dr. A.K. Srivastava

Head

Department of Biosciences

E-mail: info@integraluniversity.ac.in Web: www.integraluniversity.ac.in

E-mail: info@integraluniversity.ac.in Web: www.integraluniversity.ac.in

CERTIFICATE OF ORIGINAL WORK

This is to certify that the study conducted by Hashim Ali

during the months Jan – March, 2013 reported in the

E-mail: info@integraluniversity.ac.in Web:

present thesis was under my guidance and supervision. The

results reported by his are genuine and script of the

thesis has been written by the candidate his self. The

thesis entitled “Biochemical and molecular characterizationof some diesel degrading Bacterial isolates from oil

contaminated soil sample)” is therefore, being forwarded

for the acceptance in partial fulfillment of the

requirements for the award of the degree of Master of

Science in Microbiology, Department of Biosciences,

Integral University, Lucknow, (U.P).

Dr. Irfan Ahmad Ansari

Assistant Professor

(Department Of Biosciences)

E-mail: info@integraluniversity.ac.in Web:

www.integraluniversity.ac.in

List of abbreviations:

NA -

Nutrient agar.

NB -

Nutrient brroth

MSM -

Minral salt media

IMViC -

Indole, methyl red, Voges Proskauer, citrate.

MR-VP -

Methyl red, Voges Proskauer.

NaCl -

Sodium chloride

NaoH -

Sodium hydroxide.

K2HPO4 -

Dipotassium hydrogen phosphate.

SDS -

Sodium dodecyl Sulphate.

EDTA -

Ethylene diamine tetraacetic acid.

E-mail: info@integraluniversity.ac.in Web:

H2S -

Hydrogen sulfide.

H2O2 -

Hydrogen Peroxide.

CFU -

Colony forming units.

EtBr -

Ethidium Bromide.

µg -

Microgram.

µl -

Microliter.

CONTENTS

S.NO. Particulars

Page No.

1 List of Figures i-

ii

2 List of Tables

iii-vi

3 Introduction 1

4 Aims and Objective

5

5 Review of Literature 10

6 Methodology

18

7 Result 44

8 Discussions

50

9 Summary and Conclusion 57

10 Bibliography 59

Introduction

AIM AND OBJECTIVES

Review ofliterature

Material and

Methods

Result

Discussions

Summary and

Conclusion

References or

Bibliography

ACKNOWLEDGMENT

First of all I bow in reverence to the Almighty for blessing me with strong will

power, patience and confidence, which helped me in completing the present

work.

I would like to express my special thanks to Prof (Dr.) A. K.

Srivastava (Head, Department of Biosciences) for given me an opportunity

to join the department laboratory and providing all the necessary facilities

ever since I started my work.

I would like to express my deep sense of gratitude to Dr. Irfan Ahmad

Ansari (Department of Biosciences) for their invaluable guidance

throughout the course of my dissertation work and academic session. It

would have been impossible to complete this work in so short a time without

his constant guidance. I wish every trainee and research student were

fortunate enough to have such an affectionate guide.

I am elated with delight to avail this wonderful opportunity to express my

sincere thanks to Mr. Mohd Ali and Mr. Anil Pandey (lab instructors), for his

co-operation, affection, encourage me during my academic pursuit. I

gratefully acknowledge to Mr. Firoz Akhtar and Mr. Salman Khan (Ph D

Scholars) who inspired and encouraged me during various steps of my work.

Thanks are due to my friends who have helped me in my work. Other to whom

I am grateful and also like to put on the record Mr. Shadif (Computer- lab

assistant) for providing computer and internet facility during my whole

academic session and project work.

My acknowledgement will be incomplete if I do not mention my parents with

whose blessing I was able to achieve my goal successfully. There are no words

to express my feelings toward them. I silently acknowledge my debt to them.

Date

Name.

IntroductionPetroleum based products are the major source of energy

for industry and daily life. Leaks and accidental spills

occur regularly during the exploration, production,

refining, transport, and storage of petroleum products.

The amount of natural crude oil seepage was estimated to

be 600,000 metric tons per year with a range of

uncertainty of 200,000 metric tons per year (Kvenyolden &

Cooper, 2003). Release of hydrocarbons into the

environment whether accidentally or due to human

activities is a main cause of water and soil pollution

(Holliger et al 1997). Soil contamination with

hydrocarbons causes extensive damage of local system

since accumulation of pollutants in animals and plant

tissue may cause death or mutations (Alverase & Vogel,

1992). The technology commonly used for the soil

remediation includes mechanical, burying, evaporation,

dispersion, and washing. However, these technologies are

expensive and can lead to incomplete decomposition of

contaminants. The process of bioremediation, defined as

the use of microorganisms to detoxify or remove

pollutants owing to their diverse metabolic capabilities

is an evolving method for the removal and degradation of

many environmental pollutants including the products of

petroleum industry (Median et al., 2005). In addition,

bioremediation technology is believed to be noninvasive

and relatively cost-effective (April et al., 2000).

Biodegradation by natural populations of microorganisms

represents one of the primary mechanisms by which

petroleum and other hydrocarbon pollutants can be removed

from the environment (Ulrici et al., 2000) and is cheaper

than other remediation technologies (Leahy & Colwell,

1990).The success of oil spill bioremediation depends on

one’s ability to establish and maintain conditions that

favor enhanced oil biodegradation rates in the

contaminated environment. There are the two main

approaches to oil spill bioremediation: (a)

bioaugmentation, in which known oil degrading bacteria

are added to supplement the existing microbial

population, and (b) biostimulation, in which the growth

of indigenous oil degraders is stimulated by the addition

of nutrients or other growth-limiting co substrates.

Biodegradation of petroleum hydrocarbons is a complex

process that depends on the nature and on the amount of

the hydrocarbons present. Petroleum hydrocarbons can be

divided into four classes: the saturates, the aromatics

the asphaltenes (phenols, fatty acids, ketones, esters,

and porphyrins), and the resins (pyridines, quinolines,

carbazoles, sulfoxides, and amides (Venosa et al 2007).

Different factors influencing hydrocarbon degradation

have been reported by Cooney et al (Cooney et al., 1985).

One of the important factors that limit biodegradation of

oil pollutants in the environment is their limited

availability to micro organism diesel hydrocarbon

compounds bind to soil components, and they are difficult

to be removed or degraded (Barathi & Vasudevan, 2001).

Hydrocarbons differ in their susceptibility to microbial

attack. The susceptibility of hydrocarbons to microbial

degradation can be generally ranked as follows: linear

alkenes> branched alkenes>small aromatics> cyclic alkenes

[6, 22]. Some compounds, such as the high molecular

weight polycyclic aromatic hydrocarbons (PAHs), may not

be degraded at all (Atlas & Bragg, 2009).

Microbial degradation is the major and ultimate natural

mechanism by which one can clean up the diesel

hydrocarbon pollutants from the environment (Atlas et

al., 1992). The recognition of biodegraded diesel-derived

aromatic hydro carbons in marine sediments was reported

by Jones et al. (Jones et al., 1993). They studied the

extensive biodegradation of alkyl aromatics in marine

sediments which occurred priodetectable biodegradation

of n-alkane profile of the crude oil and the

microorganisms, namely, Arthrobacter, Burkholderia, Mycobacterium,

Pseudomonas, Sphingomonas, and Rhodococcus were found to be

involved for alkyl aromatic degradation. Microbial

degradation of diesel hydrocarbons in a polluted tropical

stream in Lagos, Nigeria was reported by Adebusoye et al.

(Adebusoye et al., 2007). Nine bacterial strains, namely,

Pseudomonas fluorescens, P. aeruginosa, Bacillus subtilis, Bacillus sp.,

Alcaligenes sp., Acinetobacter lwoffi, Flavobacterium sp, Micrococcus

roseus, and Corynebacterium sp. were isolated from the

polluted stream which could degrade crude oil.

Hydrocarbons in the environment are biodegraded primarily

by bacteria, yeast, and fungi. The reported efficiency of

biodegradation ranged from 6% (Jones et al 1970) to 82%

[for soil fungi, 0.13% [Jones et al 1970] to 50% [Rinholt

et al 1979] for soil bacteria, and 0.003% [Hollaway et al

1980] to 100% [Mulkins Phillips& Stewart 1974] for marine

bacteria. Many scientists reported that mixed populations

with over all broad enzymatic capacities are required to

degrade complex mixtures of hydrocarbons such as crude

oil in soil [Bartha & Bossert 1984], freshwater [Coorey

et al 1984], and marine environments [Atlas & Floodagate

1984]. Bacteria are the most active agents in petroleum

degradation, and they work as primary degraders of

spilled oil in environment [Rahman et al 2003].

Several bacteria are even known to feed exclusively on

hydrocarbons [Broojmans et al 2009]. Floodgate [Flodgate,

1984] listed 25 genera of hydrocarbon degrading bacteria

and 25 genera of hydrocarbon degrading fungi which were

isolated from marine environment. In earlier days, the

extent to which bacteria, yeast, and filamentous fungi

participate in the biodegradation of diesel hydrocarbons

was the subject of limited study, but appeared to be a

function of the ecosystem and local environmental

conditions. Crude petroleum oil from diesel contaminated

soil from North East India was reported by Das and

Mukherjee . Acinetobacter sp .was found to be capable of

utilizing n-alkanes of chain length C10–C40 as a sole

source of carbon [Das Mukherji, 2007]. Bacterial genera,

namely, Gordonia, Brevibacterium, Aeromicrobium, Dietzia, Burkholderia,

and Mycobacterium isolated from diesel contaminated soil

proved to be the potential organisms for hydrocarbon

degradation [42]. The degradation of polyaromatic

hydrocarbons by Sphingomonas as was reported by Daugulis

and McCracken [43]. Fungal genera, namely, Amorphoteca,

Neosartorya, Talaromyces, and Graphium and yeast genera,

namely, Candida, Yarrowia, and Pichia were isolated from diesel

contaminated soil and proved to be the potential

organisms for hydrocarbon degradation [Chailan et al

2004]. Singh also reported a group of terrestrial fungi,

namely, Aspergillus, Cephalosporium and Pencillium which were also

found to be the potential degrader of crude oil

hydrocarbons. The yeast species, namely, Candida lipolytica,

Rhodoorula mucilaginosa, Geotrichumsp, and Trichosporon mucoides

isolated from contaminated water were noted to degrade

petroleum compounds. Though algae and protozoa are the

important members of the microbial community in both

aquatic and terrestrial ecosystems, reports are scanty

regarding their involvement in hydrocarbon

biodegradation. Walker et al isolated an alga, which was

capable of utilizing crude oil and a mixed hydrocarbon

substrate and exhibited extensive degradation of n-

alkanes and iso-alkane as well as aromatic hydrocarbons.

Review of Literature

Hydrocarbon degrading bacteria can be defined as bacteria

with the capability to degrade (leading to the formation

of less complex intermediate compounds) and/or to

mineralize (leading to the formation of water and carbon

dioxide) hydrocarbons. Heterotrophic bacteria depend on

carbon and electron sources from environment to be used

as their energy resource. Microbial degradation of

organic contaminants therefore occurs as result of

microorganisms using the contaminants as carbon, energy

or nutrient source for their own growth and reproduction.

Besides environmental factors such as oxygen,

temperature, soil physical‐chemical conditions,

bioavailability of the contaminant and available

nutrients (Romantschuk, et al., 2000), the capability to

degrade hydrocarbons in soil is also influenced by other

factors such as a) bacterial species‐dependent

capability; that makes every species differ in their

capability to metabolize hydrocarbons (Siciliano, et al.,

1998), b) the bacterial ability to quickly distribute

genetic information within a population and thereby to

adapt to environmental changes (van Elsas, et al., 2003),

c) the presence of the contaminant as selective pressure

to maintain their degrading capability (van der Lelie, et

al., 2005) and d) catabolic genes encoding degradation

enzymes (Romantschuk, et al., 2000).

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 then as a food source

(Harder, 2004). Microorganisms degrade these compounds by

using enzymes in the metabolism and can be useful in

cleaning up contaminated sites (Alexander, 1999).

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. These microorganisms can degrade a wide

range of target constituents present in oily sludge

(Barathi and Vasudevan, 2001; Mishra et al., 2001;

Eriksson et al., 1999). A large number of Pseudomonas

strains capable of degrading polyaromatic hydrocarbons

(PAHs) have been isolated from soil and aquifers (Johnson

et al., 1996; Kiyohara et al., 1992; Fall et al., 1979).

Other petroleum hydrocarbon degraders include Yokenella

spp.,Alcaligenes spp., Roseomonas spp., Stenotrophomonas spp.,

Acinetobacter spp., Flavobacter spp., Corynebacterium spp.,

Streptococcus spp., Providencia spp., Sphingobacterium spp.,

Capnocytophaga spp., Moraxella spp., and Bacillus spp.

(Rusanskyet al., 1987; Antai, 1990; Bhattacharya et al.,

2002).

The most rapid and complete degradation of the majority

of organic pollutants is brought about under aerobic

conditions. The initial intracellular attack of organic

pollutants is an oxidative process and the activation as

well as incorporation of oxygen is the enzymatic key

reaction catalyzed by oxygenases and peroxidases (Fig.

1). Peripheral degradation pathways convert organic

pollutants step by step in to intermediates of the

central intermediary metabolism, for example, the

tricarboxylic acid cycle. Biosynthesis of cell biomass

occurs from the central precursor metabolites, for

example, acetyl-CoA, succinate, pyruvate. Sugars required

for various biosynthesis and growth are synthesized by

gluconeogenesis. The degradation of diesel hydrocarbons

can be mediated by specific enzyme system. Fig. 2 shows

the initial attack on xenobiotics by oxygenases (Fritsche

et al., 2000).The mechanisms involved are (1) attachment

of microbial cells to the substrates and (2) production

of biosurfactants. The uptake mechanism linked to the

attachment of cell to oil droplet is still unknown but

production of biosurfactants has been well studied.

Fig 1: Main principle of aerobic degradation of

hydrocarbons by microorganisms.

Enzymes Participating in Degradation of Hydrocarbons

Cytochrome P450 alkenes hydroxylases constitute a super

family of ubiquitous Heme- thiolate Monooxygenases which

play an important role in the microbial degradation o

foil, chlorinated hydrocarbons, fuel additives, and many

other compounds. Depending on the chain length, enzyme

systems are required to introduce oxygen in the substrate

to initiate biodegradation. Higher eukaryotes generally

contain several different P450 families that consist of

large number of individual P450 forms that may contribute

as an ensemble of is forms to the metabolic conversion of

given substrate. In microorganisms such P450 multiplicity

can only be found in few species Zimmer et al., 1998.

Cytochrome P450 enzyme systems was found to be involved

in biodegradation of petroleum hydrocarbons (Table 1).

The capability of several yeast species to use n-alkanes

and other aliphatic hydrocarbons as a sole source of

carbon and energy is mediated by the existence of

multiple microsomal Cytochrome P450 forms. These

cytochrome P450 enzymes had been isolated from yeast

species such as Candida maltosa, Candida tropicalis, and Candida

apicola . The diversity of alkane oxygenases systems in

prokaryotes and eukaryotes hat are actively participating

in the degradation of alkanes under aerobic conditions

like Cytochrome P450 enzymes, integral membrane di-iron

alkane hydroxylases (e.g., alkB), soluble di-iron

methane Mono oxygenases, and membrane bound copper

containing methane Mono oxygenases have been discussed by

Van Beilen and Funhoff (2005).

Uptake of Hydrocarbons by Biosurfactants

Biosurfactants are heterogeneous group of surface active

chemical compounds produced by a wide variety of

microorganisms. Surfactants enhance solubilization and

removal of contaminants. Biodegradation is also enhanced

by surfactants due to increased bioavailability of

pollutants. Bioremediation of oil sludge using

biosurfactants has been reported by Cameotra and Singh

[87]. Pseudomonas are the best known bacteria capable of

utilizing hydrocarbons as carbon and energy sources

producing biosurfactants.

Fig 2: Enzymatic reactions involved in the processes of

hydrocarbons degradation.

Table 1 : Enzymes involved in biodegradation of petroleum

hydrocarbons.

Among Pseudomonads, P. aeruginosa is widely studied for the

production of glycol lipid type biosurfactants. However,

glycol lipid type biosurfactant are also reported from

some other species like P. putida and P. chlororaphis.

Biosurfactants increase the oil surface area and that

amount of oil is actually available for bacteria to

utilize it. Biosurfactant can act as emulsifying agents

by decreasing the surface tension and forming micelles.

The micro droplets encapsulated in the hydrophobic

microbial cell surface are taken inside and degraded.

Although capability in degrading hydrocarbons spread

across wide range of bacterial species, but in general,

bacterial genera Pseudomonas, Arthrobacter, Alcaligenes,

Corynebacterium, Flavobacterium, Achromobacter,Micrococcus,

Mycobacterium, and Nocardia have reported as the most active

bacteria in the degradation of hydrocarbons in soil

(Frick et al., 1999), whereas Cellulomonas, Clavibacter,

Curtobacterium, Pseudomonas and Microbacteriumhave been

suggested as the most promising endophytic bacteria

(Ryan, et al., 2008). Endophytic bacteria are defined as

those bacteria that colonize the internal tissue of the

plant showing no negative effects on their host (Ryan,

2008).

Bacterial pathways for the degradation of hydrocarbons

contaminants have been the subject of intense study and

suggest several important physiological events as key

factors that lead to the efficient catabolism of these

compounds, which are: bioavailability, or the amount of a

substance that is physiochemically accessible to

microorganisms; chemotaxis, or the directed movement of

motile organisms towards or away from chemicals in the

environment; and transport mechanisms for the

intracellular accumulation of aromatic molecules

(Parales, et al., 2008). Degradation of hydrocarbon by

bacteria takes place through complex sequence of

reduction‐oxidation reactions, which are catalyzed by

enzymes. Aliphatic hydrocarbons are oxidized by several

alkane hydroxylase enzyme systems including cytochrome

P450 enzymes, an integral membrane mono or di‐iron alkane

hydroxylase (i.e. alkane monooxygenase), a soluble di‐

iron methane mono oxygenase (sMMO) and a membrane‐bound

copper‐containing (possibly iron‐containing) methane

monooxygenase (pMMO) (van Beilen and Funhoff, 2005). For

the reaction to take place,the compound must pass through

the bacteria’s cell membrane so the organism’s

electrontransport system can be used for energy storage.

Frequently, the bacteria are attached to alkanedroplets,

which make it more available to bacterial attack,

however, the enhanced bioavailability can cause a

toxicity problem for the bacteria. In addition, soil

moisture lower than 50% and pH above 8.5 appear to

inhibit hydrocarbon degradation (Cookson, 1995).The

degradation of hydrocarbons can be divided into aerobic

and anaerobic metabolic modes.

Aerobic metabolic mode:-

Alkane degradation pathway is performed by oxidation or

incorporating molecular oxygen in the hydrocarbon by a

membrane‐bound alkane mono oxygenase and two soluble

enzymes, rubredoxin and rubredoxireductase, which act as

electrons carriers between NADH and the hydroxylase for

conversion of alkane to alcohol. Commonly, the oxidation

takes place onone or both terminal methyl group or at a

sub‐terminal location .The alcohol can be further

oxidized to an aldehyde and acid prior to proceeding into

the β‐oxidation and tricarboxylic acid cycle (TCA cycle)

to produce energy (van Beilen, et al., 2003).

Figure 3: Metabolic pathway for degradation of alkanes by

terminal and sub‐terminal oxidation.

This pathway is best describing the alkane degradation by

Pseudomonas putidaGPO1, in which the genes coding for alkane

monooxygenase are located on a plasmid (Marín, et al.,

2001). Short chain alkanes except me thane are more

difficult to degrade and may require co‐metabolism‐

defined as the degradation of a compound only in the

presence of other organic material that serves as the

primary energy source. Branched alkanes and cyclic

alkanes are much less susceptible to degradation.

Anaerobic metabolic mode:

Degradation of hydrocarbons is restricted to anaerobic

photo heterotrophic bacteria (i.e. Blastochlorissulfoviridis),

Fe(III)‐reducing bacteria (i.e. Geobacter), denitrifying

bacteria (i.e. Azoarcus, Dechloromonas, Pseudomonas and Thauera),

and sulfate‐reducing bacteria (i.e. Desulfobacterium,

Desulfobacula)or to proton‐reducing and methano genic

bacteria living in syntrophic consortia (phenomenon that

one species lives off the products of another species).

It has been reported that hydrocarbons such as toluene,

alkyl benzenes, benzene, naphthalene, phenanthrene, >C6

n‐alkanes, branched alkanes and hydrocarbon mixtures can

be degraded under anaerobic conditions.

Diversity of Alkane Hydroxylase Systems

As described above, the first step for alkane degradation

under aerobic conditions is oxidation by an alkane

hydroxylase enzyme. Hydroxylases catalyze the addition of

hydroxyl groups (‐OH) by at taching oxygen atoms

(oxidation) during hydroxylation reactions. The terms of

hydroxylase andoxygenase is commonly used

interchangeably. However, oxygenases belong to the class

of oxidoreductases that catalyze the incorporation of

oxygen to the substrate.In prokaryotes and eukaryotes

several enzyme systems to mediate hydroxylation or

oxygenation of aliphatic hydrocarbons. Depending on the

chain‐length of the alkane substrate, different enzyme

systems are required to oxidate the irsubstrate and

thereby initiate the biodegradation process. In summary,

the responsible alkane hydroxylase enzymes, their

encoding genes, substrate range and examples of host

organisms areas follows:

1. Bacterial soluble di‐iron methane monooxygenase (sMMO)

encoded by the mmogenecluster (McDonald, et al., 1997);

substrate range C1‐C10 (Methylisinus trichosporium, Methylococcus

capsulatus), C2‐C8 for butane monooxygenase (Pseudomonas

butanovora) (van Beilen and Funhoff, 2005).

2. Bacterial integral membrane copper or iron‐containing

particulate methane mono oxygenase (pMMO) encoded by

pmoA, pmoB and pmoC genes (Hoffmann, et al.,2002); substrate

range for short alkanes C1–C5 (found in all known

methanothrops) (vanBeilen and Funhoff, 2005).

3. Cytochrome P450 alkane hydroxylase enzyme family; in

bacteria these enzymes are encoded by CYP153 genes

(encoding class I P450s), in eukaryotic yeast and fungi

are encoded by CYP52 genes, in mammals are CYP2E and

CYP4B (encoding class II P450s); substrate range for

bacteria C4 – C16 (e.g. Sphingomonas sp., Mycobacterium sp.,

Acinetobacter sp.), substrate range for eukaryote C10 – C16

(e.g. Candida maltose, Yarrowia lipolytica), substrate range for

mammals C6 – C10 (humans and rabbits) (van Beilen

andFunhoff, 2005).

4. Bacterial integral membrane non‐ heme iron AlkB‐

related alkane hydroxylase or alkane Monooxygenases

(alkB) encoded by a alk gene cluster; substrate range

between medium and long chain alkanes C5 – C16 (e.g.

Acinetobacter, Burkholderia, Mycobacterium, Pseudomonas) and for

RhodococcusC6 – C36 (van Beilen, et al., 2001, 2003, van

Beilen and Funhoff, 2005).

In recent reviews by van Beilen et al. (2003, 2007) it has

been suggested that in most bacterial strains, alkane

hydroxylase genes seem to be distributed over the genome,

they can be located on chromosomes, plasmids, or

transposons. Moreover, very frequent multiple alkane

hydroxylase genes are present in one alkane degrading

bacterium rendering the capactity to utilize a wide range

of alkanes.

Soluble and particulate methane monooxygenase (sMMO and

pMMO) are two types of enzyme systems that are known to

oxidize methane, propane and butane. Besides the

oxidation of methane, sMMO is able to oxidize saturated

and unsaturated alkanes as well as halogenated, aromatic

and heterocyclic compounds, whilst pMMO has a much

narrower substrate range, which appears to be restricted

to alkanes and alkenes up to C5 (van Beilen and Funh off,

2005).

The cytochrome P450 alkane hydroxylase enzyme system

comprises up to date more than 4000 different enzymes, of

which 10‐15% are found in prokaryotes. So far only few

P450s enzymes have been identified and characterized.

Class I P450 enzymes are soluble enzymes located in the

cytoplasm, and consists of 3‐component systems comprising

Cytochrome P450, ferredoxin and ferredoxin reductase

subunits. These enzymes need heme (conjugated protein) as

well as iron‐sulfur as cofactor during catalysis. This

enzyme system is found among bacteria that oxidize C5‐C10

alkanes, salicylic compounds and limonene, encoded by

CYP153 gene family (van Beilen and Funhoff, 2005) and is

commonly found in alkane degrading bacteria that lack the

integral membrane alkane hydroxylase (van Beilenet al.,

2006). Class II P450s enzymes are contained in the

microsome, consist of two‐component systems comprising a

membrane‐bound cytochrome P450 and a reductase, and need

heme as cofactor. The enzymes are encoded by genes

belonging to the CYP52 family and are usually found in

multiple copies in various yeast strains. In mammals, the

CYP2E1 gene is involved in the metabolism of endogenous

compounds and xenobiotics and is the key enzyme in the

microsomal pathway for ethanol oxidation.

AlkB‐related integral membrane alkane hydroxylase or

alkane mono oxygenase (alkB) is the most common enzyme

found in alkane degrading bacteria, first discovered in a

hexane‐degrading Fluorescent Pseudomonas strain now known

as P. putida strain GPo1.This enzyme system is a cytoplasmic

integral membrane protein comprising alkane monooxygenase

(AlkB), one or two rubredoxins and electron providing

rubredoxin reducates, and needs iron as cofactor.

Materials and Methodology:

Sample Collection:-

The study includes three types of samples to isolate the

hydrocarbon degrading bacteria. Soil sample extending

from the ground surface to a depth of 10–20 cm were

collected from diesel-contaminated areas near petrol

station, refining area. Samples were then transported to

laboratory under sterile conditions.

Serial dilution:

The technique involved the removal of a small amount of

an original solution to another container that is then

brought up to a predetermined volume using the working

solution (i.e. ddH2O). To make a 1:100 dilution (10-2),

remove 10μl and place this volume in a tube containing

990μl of ddH2O. This is often represented as 1:100 or 10-

2.

To dilute this by a factor of 1:1000, remove 1μl of the

1:100 dilutions and place it in a tube containing 999μl

of ddH2O or media. The secondary concentration (1:100) has

been diluted by a factor of 1,000 and the original

solution has been diluted by a factor of 100,000 (the

dilution factor). Same process was repeated to obtain 10-

2, 10-3, 10-4, 10-5, 10-6 and 10-7 dilutions.

Media:

Although Nutrient agar was the main medium used for the

isolation of bacteria, some other important selective

media were also involved in the bacterial isolation

process such as-

Media

Function

MSM (Minral salt media)

support oil degrading bacteria

Nutrient Agar

Nutrient Agar was used for the cultivation of less

fastidious microorganisms. Nutrient media are basic

culture media used for maintaining microorganism. This

relatively simple formula is still widely used in the

microbiological examination of variety of materials and

is also recommended by standard methods. It is one of the

several non-selective media useful in routine cultivation

of microorganisms. It can be used for the cultivation and

enumeration of bacteria which are not particularly

fastidious.

Composition of nutrient agar media (NAM) for one liter

(gm. /L)

Peptone :

5gm.

Beef extract :

3gm.

NaCl :

5gm.

Agar :

20gm.

Yeast extract :

10gm.

Isolation of Hydrocarbon Degrading Bacteria :

The bacteria were isolated by inoculating the soil

samples on enrichment medium that contains the autoclaved

mineral salt medium (MSM) supplemented with single

hydrocarbon compound as sole carbon source (1% liquid

diesel). The medium contains K2HPO4 (1.8 g/L); NH4Cl (4

g/L); MgSO4.7H2O (0.2 g/L); NaCl (0.1 g/L); Na2SO4.7H2O

(0.01 g/L); agar (20 g/L); carbon source (1% diesel);

and distilled water (1L) with pH 7.2. The medium without

hydrocarbons was sterilized by autoclaving at 121°C for

15 min. The medium was supplemented with 1% (v/v) filter

sterilized hydrocarbons ( diesel) to serve as the only

source of carbon and energy .the medium was incubated at

370C for 5 to 10 days .after the incubation period the

bacterial colonies that were grown on the medium were

identified by Gram`s staining and biochemical

characterization according to Bergy`s manual.

Isolation of bacterial strain by selective media-

The soil sample was collected from Petrol Pump. A

quantity of 1gm of soil 100 ml of minimal sample was

suspended in salt medium containing Na2HPO4 (6 g), KH2PO4

(3 g), NaCl(0.5 g), NH4Cl (1 g), CaCl2.2H2O (1 M)

andMgSO47.H2O (1 M) in 1000 ml double distilled water.1%

diesel was used as sole source ofCarbon and then

incubated in plate at 370C in incubator for a week.After

growth a single colony was picked and streak on a NA

plate and incubated at 37 0c for 24 hours. After the

incubation pure colony was isolated.

Determination of Bacterial Biodegradative Activity by

Turbidometry: Turbido metry is to determine the bacterial growth by

utilizing the hydrocarbons (1% diesel given as carbon

source in MSM broth. This shows whether the bacterium

possess the degrading activity of hydrocarbons like

phenol, petrol and diesel. The degrading activities of

each isolates were obtained by using Mineral salt broth

(MSB) in which 1% of each hydrocarbon (petrol and diesel)

was added and incubated at room temperature for 15 days.

The growth of the bacterium was measured by taking the

O.D readings at 595nm from 0hrs- 15 days at regular

intervals of 2 days against mineral salt medium as

bMaintenance of culture:

Experimental and stock cultures were grown and maintained

in nutrient agar slants. They were kept in the

refrigerator (40C).

GRAM.STAINING-

Gram staining is a technique which is used for

differentiation of Gram negative and Gram positive

Bacteria

Representation

Pink color – Gram negative

Purple color Gram positive

Procedure-

1: culture was taken on a sterilized slide with help of

inoculation loop.

2: Heat fixed takes the slide.

3: Add one or two drop of Cristal violet for 30 sec -1

min and then wash

water with Distilled.

4: Add one or two drop of iodine on slide for 30 sec – 1

min and then wash with 70% Alcohol.

5: Add one or two drop safranin for 30 sec – 1 min and

then wash with

distilled water.

6: Observation take place under the electron microscope.

BIOCHEMICAL CHARACTERIZATION-Oxidase test, catalase test, TSI test, Indole test,

methyl red test, Voges-Proskauer test, citrate test,

urease test, nitrate test and sugar fermentation tests

(glucose, lactose and mannitol) were carried out

according to standard procedure for the biochemical

characterization of isolated bacteria.

Oxidase Test

This test is used to identify microorganisms containing

the enzyme cytochrome oxidase (important in the electron

transport chain). It is commonly used to distinguish

between oxidase negative Enterobacteriaceae and oxidase

positive Pseudomadaceae.

Cytochrome oxidase transfers electrons from the electron

transport chain to oxygen (the final electron acceptor)

and reduces it to water. In the oxidase test, artificial

electron donors and acceptors are provided. When the

electron donor is oxidized by cytochrome oxidase it turns

a dark purple. This is considered a positive result. In

the picture below the organism on the right (Pseudomonas

aeruginosa) is oxidase positive.

Procedure

1.Placed a few amount of oxidase reagent on to watman

filter paper.

2. Add small amount of culture onto filter paper from

loop.

3. Positive result showed BLUE colour within a

minutes.

4. A negative result showed COLOURLESS colour within a

minutes.

Catalase test-

This test is used to identify organisms that produce the

enzyme, catalase. This enzyme detoxifies hydrogen

peroxide by breaking it down into water and oxygen gas.

2H2o2 --------catalase

--------------->2H2o

and o2

The bubbles resulting from production of oxygen gas

clearly indicate a catalase positive result. The sample

on the right below is catalase positive.

TheStaphylococcus spp. and the Micrococcus spp. are catalase

positive. The Streptococcus and Enterococcus spp. are catalase

negative.

Procedure:-

1. Placed a small amount of growth from culture onto a

clean microscope slide.

2. Added a few drops of H2O2 onto the smear. If needed,

mix with a toothpick. DO NOT use a metal loop or needle

with H2O2; it will give a false positive and degrade the

metal.

3. A positive result showed rapid evolution of O2 as

evidenced by bubbling.

4. A negative result showed no bubbles or only a few

scattered bubbles.

ISOLATION OF BACTERIAL GENOMIC DNA-

Introduction-

Genomic DNA……

The isolation of total genomic DNA from a culture of

bacterial cell comprises of following steps-

1.Growing and harvesting a bacterial culture

2. Purification of DNA from a cell extract

3. Concentration of DNA samples

1. Growing and harvesting a bacterial culture-

The bacteria can be grown in a liquid LB medium (broth

culture) at 370C with constant shaking at 150-250 rpm on a

rotatory platform. The growth of the culture can be

monitored by reading the optical density at 600 nm.

Preparation of cell extract: In order to prepare a cell

extract, the bacterial cells are harvested by spinning

the culture in a centrifuge. The bacterial cell is

enclosed in a cytoplasmic membrane and an outer rigid

cell wall. In some E. coli, the cell wall may be

enveloped by a second, outer membrane. These barriers are

disrupted by the use of lysis solution which contains

Nacl, Tris, EDTA, SDS & Protease k.

Tris – maintain the optimum pH of cell.

EDTA – chelates the divalent cations like Mg+2, which is

an activator of nucleases.

Nacl- maintains the osmotic pressure so that cells do not

burst open and genome does not get sheared.

SDS- help in lysis by removing the lipid molecules and

thereby causing the disruption of the cell membranes.

Lysozyme- degrade the membrane proteins.

For its activity, the cells are incubated for 4-6hrs at

500C.

2. Purification of DNA from a cell extract:

Besides genomic DNA, significance amount of protein and

RNA contamination is observed at the time of isolation.

The cell extract is deproteinized by adding a mixture of

phenol: chloroform: isoamylalcohol (25:24:1). These

organic solvents precipitate proteins but leave the

nucleic acids (DNA & RNA) in aqueous solution.

Centrifugation precipitates the contaminating proteins as

a white coagulated mass at the in the interface between

the aqueous and organic layers. The interphase being

produced as result of isoamylalcohol which acts as a

antifoaming agent. The aqueous layer of nucleic acid can

then be removed with a micropipette.

4 Concentration of DNA sample:

Mainly DNA samples are concentrated by ethanol

precipitation. In the presence of salt (mainly monovalent

cations) and at low temperature, absolute ethanol

precipitates nucleic acids. The acetate group replaces

the hydroxyl group of water and DNA is able to make

hydrogen bonds and becomes heavier. It can be easily

separated by centrifugation or by spooling.

Spooling is the sticking of DNA threads to the glass rod.

A glass rod can be directly put into the DNA containing

absolute ethanol solution and as a result DNA molecules

get adhered to the glass rod in form of fibers. DNA is

then dried in air and with appropriate amount of TE

buffer (pH =8.0) it is dissolved and can be at -200C or

lyophilized ( for long term storage) or can be kept at 40C

(for regular use).

REQUIREMENTS:

Equipment:

Refrigerated centrifuge, Incubator shaker, Laminar air

flow, micropipettes, Refrigerator, Deep freezer, Agarose

gel electrophoresis, UV-Transilluminator, Power pack,

Autoclave, pH meter, Electronic balance,

Magneticstirrer,Vortexer, Hot plate (Microwave oven),

Incubator, UV-spectrophotometer.

Stock Solution-

TE buffer (pH=8.0)

1M Tris –Cl (pH=8.0)

0.5 M EDTA(pH=8.0)

RNA ase A (Bovine pancreatic)

Phenol: Chloroform: Iso-amyl alcohol (25:24:1)

3 M Potassium acetate (pH=4.8)

5 M NaCl

Lysozyme (20mg/ml)

10 % SDS

Lysis Solution:

50 x TAE

0.5 M EDTA

Ethidium Bromide (10mg/ml)

6 x DNA loading dye

OR….Preparations: stock solutions……….

For 40ml SET Buffer:

50mM Sucrose: Dissolved 0.171gm of sucrose in 10ml

distilled water.

25mM EDTA: Dissolved 0.104gm of EDTA in 10ml distilled

water.

50mM Tris HCl: Dissolved 0.031gm of Tris HCl in 10ml

distilled water (pH 7.5).

75mM NaCl: Dissolved 0.0438gm of NaCl in 10ml distilled

water.

(Final pH should be 8.0)

Lysozyme: 1mg/ml.

10% SDS: 55µl.

5M NaCl: Dissolved 2.922gm of NaCl in 10ml distilled

water.

Chloroform: Isoamyl alcohol (24:1): used in 1:1

ratio

Isopropanol: 300µl/0.5ml.

Ethanol: 1ml

For 100µl TE Buffer:s

1M Tris HCl: Dissolved 7.88gm in 25ml distilled water (pH

7.5).

0.5M EDTA (pH 8.0).

Procedure:

1. Bacteria were cultured in Nutrient broth.

2. Cells were harvested from 1-5ml of culture at

12,000rpm for 5 minutes.

1. Pellet was suspended in 0.5ml (500µl) of SET buffer

with 500µl lysozyme.

2. Incubated for 1hr at 370C.

3. Added 1/10th volume of 10% SDS and incubated for 30

minutes at 370C followed by addition of 100µl of 5M

NaCl and then chloroform: Isoamyl alcohol (24:1) in

1:1 ratio.

4. Incubated at room temperature for 30 minutes.

5. Centrifuged at 12,000rpm for 5 minutes.

6. Aqueous phase was transferred to new tube.

7. DNA was precipitated by adding 0.6th volume of

isopropanol and incubated at -200C for overnight.

8. Recovered the precipitate by centrifugation at

15,000rpm for 5-10 minutes.

9. Discard aqueous solution and add 1ml ethanol.

10. Added 40µl of TE buffer.

ResultThe study was aimed to isolate and characterize of

different micro organism isolated from the oil

contaminated soil the result of current study have shown

some interesting data

First of all micro organism was isolated on NA agar

media, serially diluted samples where poured and spreaded

on NA plates after 24 hrs of incubation at 37 degree. We

observed different colonies of bacteria in different

texture, color shape and size. The number of the CFU

forming unit form to dependent upon dilution factor .in

which sample was diluted

The number of CFU was counted on each plate .all the

experiment was performed in triplicate.

The number of CFU where formed to be in different samples

CFU formula Formula Dilution factor (1: 10):

No. Of microorganisms / gram ═ No. of Colonies ×

Dilution Factor

Volume of sample added

Dilution of the sample was made by adding 1ml of the

sample to 9ml of Distilled water to make a sample solution

of 1:10.

The no. of CFU was found to be in oil contaminated soil

sample.

Cfu/ml = (no. of colonies x dilution factor) / volume

inoculated

OBSERVATION RESULTS:

SAMPLE No. of

colonies

Cfu

count 1 Diesel

Soil on NA

60 6 x 10 -7 CFU

/100ui2 Diesel Soil on

MSM

115 11.5x 10-8 CFU/100ui

1. Petrol soil

sample on

NA

54 5.4 x108 CFU/100ui

2 Petrol soil

sample on

MSM

5 O.5x10-7CFU/100ui

2 further more when samples poured and spreaded

on NA plate we observed for texture , color shape

and size

TEXTURE AND MORHOLOGICAL CHARACTERISTICS

SAMPLE MEDIUM ELEVATION EDGE SIZE COLONY COLOR Soil NA small yellow

soil NA large yellow

soil

NA large white

3. The different micro organism isolated from the

NA plate where further grown on some specific

media such as MSM, mineral salt media for their

further identification

Serially diluted sample was spread on NA medium

and we observed that

Diesel colony on NA media

Fig A

Three type colony growths take place on NA medium

1 White colony

2 Yellow small colony

3 Yellow large colony

Streaking- All type colony streaked on NA

plate and we observed that result

Fig1

Fig2

Fig3

Fig 1- Showing yellow large colony streaked on NA plate

Fig 2- Showing yellow small colony streaked on NA plate

Fig 2- Showing white colony streaked on NA plate

4.Then thease three different colony isolated from NAplate where streaked on MSM media and we observed that

Yellow small colony

Yellow small colony

Fig 1 Fig 2

Fig 3 Fig 4

Yellow small colony

white colony

Fig 1- showing yellow large colony streaked on

MSM plate

Fig 2&3- Showing yellow small colony streaked

dill 105 &106 on MSM plate

Fig 3- Showing white colony streaked on MSM plate

5.We further wanted to find out the presence of somedifferent bacterial species in our sample.

We further diluted sample spreaded on MSM media and

incubated at 28 degree centigrade for

seven days and observed that

Diesel colonies on

MSM

Gram staining – After the gram staining we observed that some

intresting result

Diesel white

colony

Fig 1a- Gram negative Coco Fig 2b-

Gram negative Coco Bacilli

Bacilli.

Fig 3c- Gram negative

Coco Bacilli

Fig 4d-Gram

positive Coco

Bacilli suspected Staphylococcus aureus

Diesel yellow colony

Fig 5e- Gram negative Coco Bacilli. Fig 6f-Gramnegative Coco Bacilli

Diesel colony on MSM media

Fig 7g- Gram negative Coco Fig 8h- Gram

negative Coco Bacilli.

Bacilli.

Petrol colony

Gram negative Bacilli

Gram negative Bacilli

Gram negative Bacilli Gram

negative Bacilli

CATALASE TESTSample culture was taken on the sterilized slide

by inoculation loop and then add one or two drop

of H2O2 and we observed bubble formation take

place

Catalase positive

OXIDASE TEST - Oxidase reagent adds 2 or 4 drop on the filter paper and then

the samples are put on this

filter paper. and observe the color

Change.

Oxidase positive

Bacterial typing by 16s r DNA amplification-In order to identify bacterial isolate at species

level .we further aimed to analyse the 16 s Ribosomal DNA

amplification for the Bacterial typing species in this

scenario .we first of all isolated Bacterial Genomic DNA

from different isolates by following a generalized

protocol for the isolation of Genomic DNA

The isolated bacterial DNA was visualized on agarose gel

containing eithedium bromide as shown in following figure

Quantification –The amount of genomic DNA was quantified are to the

formula as earlier discussed in material and methods. and

the purity of DNA was also checked and was found to be

between the range

Isolates absorbence 260 to 280

References

[1] K. A. Kvenvolden and C. K. Cooper, “Natural seepage

ofcrude oil into the marine environment,” Geo-Marine

Letters,vol. 23, no. 3-4, pp. 140–146, 2003.

[2] C. Holliger, S. Gaspard, G. Glod et al.,

“Contaminatedenvironments in the subsurface and

bioremediation: organiccontaminants,” FEMS Microbiology

Reviews, vol. 20, no. 3-4,pp. 517–523, 1997.

[3] P. J. J. Alvarez andT.M. Vogel, “Substrate

interactions of benzene,toluene, and para-xylene during

microbial degradationby pure cultures and mixed culture

aquifer slurries,” Applied

and Environmental Microbiology, vol. 57, no. 10, pp. 2981–2985,

1991.

[4] J. I.Medina-Bellver, P.Mar´ın, A. Delgado et al.,

“Evidence forin situ crude oil biodegradation after the

Prestige oil spill,” Environmental Microbiology, vol. 7, no. 6,

pp. 773–779, 2005.

[5] T. M. April, J. M. Foght, and R. S. Hurrah,

“Hydrocarbondegradingfilamentous fungi isolated from

flare pit soilsin northern and western Canada,” Canadian

Journal of

Microbiology,vol. 46, no. 1, pp. 38–49, 2000.

[6] W. Ulrici, “Contaminant soil areas, different

countries andcontaminant monitoring of contaminants,” in

EnvironmentalProcess II. Soil Decontamination Biotechnology, H. J. Rehm

and G. Reed, Eds., vol. 11, pp. 5–42, 2000.

[7] J. G. Leahy and R. R. Colwell, “Microbial degradation

ofhydrocarbons in the environment,” Microbiological

Reviews,vol. 54, no. 3, pp. 305–315, 1990.

[12] J. M. Foght and D. W. S. Westlake, “Biodegradation

ofhydrocarbons in freshwater,” in Oil in Freshwater:

Chemistry,Biology, Countermeasure Technology, J. H. Vandermeulen

and

S. R. Hrudey, Eds., pp. 217–230, Pergamon Press, New

York,NY, USA, 1987.

[13] R. M. Atlas and R. Bartha, “Fundamentals and

applications,”in Microbial Ecology, pp. 523–530,

Benjamin/Cummings, SanFrancisco, Calif, USA, 4th edition,

1998.

[14] A. J. Mearns, “Cleaning oiled shores: putting

bioremediationto the test,” Spill Science and Technology Bulletin,

vol. 4, no. 4,pp. 209–217, 1997.

[15] R. C. Prince, “Petroleum spill bioremediation in

marineenvironments,” Critical Reviews in Microbiology, vol. 19,

no.4, pp. 217–242, 1993. [18] A. D. Venosa, D.W. King,

and G. A. Sorial, “The baffled flask test for dispersant

effectiveness: a round Robin evaluation of

Reproducibility and repeatability,”Spill Science and Technology

Bulletin, vol. 7, no. 5-6, pp. 299–308, 2002.

[19] R. R. Colwell, J. D. Walker, and J. J. Cooney,

“Ecologicalaspects of microbial degradation of petroleum

in the marineenvironment,” Critical Reviews in Microbiology, vol.

5, no. 4,pp. 423–445, 1977.

[20] J. J. Cooney, S. A. Silver, and E. A. Beck, “Factors

influencinghydrocarbon degradation in three freshwater

lakes,” MicrobialEcology, vol. 11, no. 2, pp. 127–137, 1985.

[21] S. Barathi and N. Vasudevan, “Utilization of

petroleumhydrocarbons by Pseudomonas fluorescensisolated from

apetroleum-contaminated soil,” Environment International,

vol. 26, no. 5-6, pp. 413–416, 2001.

[23] R. Atlas and J. Bragg, “Bioremediation of marine oil

spills:when and when not—the Exxon Valdez experience,”

MicrobialBiotechnology, vol. 2, no. 2, pp. 213–221, 2009.

[24] R. M. Atlas, “Petroleum microbiology,” in Encyclopedia

ofMicrobiology, pp. 363–369, Academic Press, Baltimore,

Md,USA, 1992.

[26] B. Lal and S. Khanna, “Degradation of crude oil

byAcinetobactercalcoaceticusand Alcaligenesodorans,” Journalof Applied

Bacteriology, vol. 81, no. 4, pp. 355–362, 1996.

[27] D. M. Jones, A. G. Douglas, R. J. Parkes, J. Taylor,

W.Giger, and C. Schaffner, “The recognition of

biodegradedpetroleum-derived aromatic hydrocarbons in

recent marine

sediments,” Marine Pollution Bulletin, vol. 14, no. 3, pp. 103–

108, 1983.

[28] S. A. Adebusoye, M. O. Ilori, O. O. Amund, O. D.

Teniola,and S. O. Olatope, “Microbial degradation of

petroleumhydrocarbons in a polluted tropical stream,”

World Journal ofMicrobiology and Biotechnology, vol. 23, no. 8, pp.

1149–1159,2007.

[29] J. Jones, M. Knight, and J. A. Byron, “Effect of

grosspopulation by kerosene hydrocarbons on the

microflora ofa moorland soil,” Nature, vol. 227, p. 1166,

1970.

[30] Y. Pinholt, S. Struwe, and A. Kjoller, “Microbial

changesduring oil decomposition in soil,” Holarctic Ecology,

vol. 2,pp. 195–200, 1979.

[31] S. L. Hollaway, G. M. Faw, and R. K. Sizemore,

“Thebacterial community composition of an active oil

field in theNorthwestern Gulf of Mexico,”Marine Pollution

Bulletin, vol.11, no. 6, pp. 153–156, 1980.

[32] G. J. Mulkins Phillips and J. E. Stewart,

“Distributionof hydrocarbon utilizing bacteria in

Northwestern Atlanticwaters and coastal sediments,”

Canadian Journal of Microbiology,

vol. 20, no. 7, pp. 955–962, 1974.

[33] R. Bartha and I. Bossert, “The treatment and

disposal ofpetroleum wastes,” in Petroleum Microbiology, R.

M. Atlas,Ed., pp. 553–578, Macmillan, New York, NY, USA,

1984.

[34] J. J. Cooney, “The fate of petroleum pollutants

infresh water ecosystems,” in Petroleum Microbiology, R.

M.Atlas, Ed., pp. 399–434, Macmillan, New York, NY,

USA,1984.

[35] R. M. Atlas, “Effects of hydrocarbons on micro-

organismsand biodegradation in Arctic ecosystems,” in

PetroleumEffects in the Arctic Environment, F. R. Engelhardt, Ed.,

pp.63–99, Elsevier, London, UK, 1985.

[36] G. Floodgate, “The fate of petroleum in marine

ecosystems,”in Petroleum Microbiology, R. M. Atlas, Ed., pp.

355–398,Macmillion, New York, NY, USA, 1984.

[37] K. S. M. Rahman, T. J. Rahman, Y. Kourkoutas, I.

Petsas,R. Marchant, and I. M. Banat, “Enhanced

bioremediationof n-alkane in petroleum sludge using

bacterial consortium

amended with rhamnolipid and micronutrients,”

BioresourceTechnology, vol. 90, no. 2, pp. 159–168, 2003.

[38] R. J. W. Brooijmans, M. I. Pastink, and R. J.

Siezen,Hydrocarbon-degrading bacteria: the oil-spill

clean-upcrew,” Microbial Biotechnology, vol. 2, no. 6, pp.

587–594,2009.

[39] M. M. Yakimov, K. N. Timmis, and P. N. Golyshin,

“Obligate

oil-degrading marine bacteria,” Current Opinion in

Biotechnology,vol. 18, no. 3, pp. 257–266, 2007.

[40] K. Das and A. K.Mukherjee, “Crude petroleum-oil

biodegradationefficiency of Bacillus subtilisand Pseudomonas

aeruginosastrains isolated from a petroleum-oil

contaminated soil

from North-East India,” Bioresource Technology, vol. 98, no.7,

pp. 1339–1345, 2007.

[41] M. Throne-Holst, A. Wentzel, T. E. Ellingsen, H.-

K.Kotlar, and S. B. Zotchev, Identification of novel

genes involved inlong-chain n-alkane degradation by

Acinetobactersp. StrainDSM 17874,” Applied and Environmental

Microbiology, vol.73, no. 10, pp. 3327–3332, 2007.

[42] F. Chaillan, A. Le Fl`eche, E. Bury et al.,

“Identification andbiodegradation potential of tropical

aerobic hydrocarbondegradingmicroorganisms,” Research in

Microbiology, vol.155, no. 7, pp. 587–595, 2004.

[44] H. Singh, Mycoremediation: Fungal Bioremediation, Wiley-

Interscience, New York, NY, USA, 2006.

[45] E. Bogusławska-Was and W. Da¸browski, “The

seasonalvariability of yeasts and yeast-like organisms in

water andbottom sediment of the Szczecin Lagoon,”

InternationalJournal of Hygiene and Environmental Health, vol. 203,

no.5-6, pp. 451–458, 2001.

[51] J. D. Walker, R. R. Colwell, Z. Vaituzis, and S. A.

Meyer,Petroleum degrading achlorophyllous alga

Protothecazopfii,”Nature, vol. 254, no. 5499, pp. 423–424,

1975.

[52] C. E. Cerniglia, D. T. Gibson, and C. Van Baalen,

“Oxidation of naphthalene by cyanobacteria and

microalgae,” Journal ofGeneral Microbiology, vol. 116, no. 2,

pp. 495–500,

1980.

[53] M. L. Brusseau, “The impact of physical, chemical

andbiological factors on biodegradation,” in Proceedings of

the International Conference on Biotechnology for Soil Remediation:Scientific

Bases and Practical Applications, R. Serra, Ed.,pp. 81–98,

C.I.P.A. S.R.L., Milan, Italy, 1998.

[54] R. M. Atlas, “Effects of temperature and crude oil

compositionon petroleum biodegradation,” Journal of

AppliedMicrobiology, vol. 30, no. 3, pp. 396–403, 1975.

[62] J. M. Foght, D. W. S. Westlake, W. M. Johnson, and

H.F. Ridgway, “Environmental gasoline-utilizing isolates

andclinical isolates of Pseudomonas aeruginosaare

taxonomically

indistinguishable by chemotaxonomic and molecular

techniques,”Microbiology, vol. 142, no. 9, pp. 2333–2340,

1996.

[63] A. D. Venosa and X. Zhu, “Biodegradation of crude

oilcontaminating marine shorelines and freshwater

wetlands,”Spill Science and Technology Bulletin, vol. 8, no. 2, pp.

163–

178, 2003.

[64] E. Pelletier, D. Delille, and B. Delille, “Crude oil

bioremediationin sub-Antarctic intertidal sediments:

chemistry andtoxicity of oiled residues,” Marine

Environmental Research,

vol. 57, no. 4, pp. 311–327, 2004.

[65] D. Delille, F. Coulon, and E. Pelletier, “Effects of

temperaturewarming during a bioremediation study of

naturaland nutrient-amended hydrocarbon-contaminated sub-

Antarctic soils,” Cold Regions Science and Technology, vol.

40,no. 1-2, pp. 61–70, 2004.

[66] W. J. Mitsch and J. G. Gosselink, Wetlands, John

Wiley &Sons, New York, NY, USA, 2nd edition, 1993.

[67] S.-C. Choi, K. K. Kwon, J.H. Sohn, and S.-J. Kim,

“Evaluationof fertilizer additions to stimulate oil

biodegradation insand seashore mesocosms,” Journal of

Microbiology and

Biotechnology, vol. 12, no. 3, pp. 431–436, 2002.

[68] S.-J. Kim, D. H. Choi, D. S. Sim, and Y.-S. Oh,

“Evaluationof bioremediation effectiveness on crude oil-

contaminatedsand,” Chemosphere, vol. 59, no. 6, pp. 845–

852, 2005.

[69] F. Chaillan, C. H. Chaˆıneau, V. Point, A. Saliot,

and J. Oudot,“Factors inhibiting bioremediation of soil

contaminated withweathered oils and drill cuttings,”

Environmental Pollution,vol. 144, no. 1, pp. 255–265, 2006.

[70] J. Oudot, F. X. Merlin, and P. Pinvidic, “Weathering

rates of oil components in a bioremediation experiment in

estuarinesediments,” Marine Environmental Research, vol. 45,

no. 2,pp. 113–125, 1998.

[71] C. H. Chaˆıneau, G. Rougeux, C. Y´epr´emian, and J.

Oudot,“Effects of nutrient concentration on the

biodegradation ofcrude oil and associated microbial

populations in the soil,”Soil Biology and Biochemistry, vol. 37,

no. 8, pp. 1490–1497,2005.

[72] L. M. Carmichael and F. K. Pfaender, “The effect of

inorganicand organic supplements on the microbial

degradation ofphenanthrene and pyrene in soils,”

Biodegradation, vol. 8, no.1, pp. 1–13, 1997.

[73] J. C. Okolo, E. N. Amadi, and C. T. I. Odu, “Effects

ofsoil treatments containing poultry manure on crude

oildegradation in a sandy loam soil,” Applied Ecology

andEnvironmental Research, vol. 3, no. 1, pp. 47–53, 2005.

[74] H. Maki, T. Sasaki, and S. Haramaya, “Photooxidation

ofbiodegradable crude oil and toxicity of the

photooxidizeproducts,” Chemosphere, vol. 44, pp. 1145–

1151, 2005.

[75] W. Fritsche and M. Hofrichter, “Aerobic

degradationby microorganisms,” in Environmental Processes-

SoilDecontamination, J. Klein, Ed., pp. 146–155, Wiley-VCH,

Weinheim, Germany, 2000.

[76] R. K. Hommel, “Formation and phylogenetic role of

biosurfactants,”Journal of Applied Microbiology, vol. 89, no.

1,pp. 158–119, 1990.

[77] J. B. Van Beilen and E. G. Funhoff, “Alkane

hydroxylasesinvolved in microbial alkane degradation,”

AppliedMicrobiology and Biotechnology, vol. 74, no. 1, pp. 13–

21,2007.

[78] T. Zimmer, M. Ohkuma, A. Ohta, M. Takagi, and W.-H.

Schunck, “The CYP52 multigene family of Candidacytochromes

p450,” Biochemical and Biophysical ResearchCommunications, vol.

224, no. 3, pp. 784–789, 1996.

[79] U. Scheuer, T. Zimmer, D. Becher, F. Schauer, and

W.-H.Schunck, “Oxygenation cascade in conversion of n-

alkanes toα,ω-dioic acids catalyzed by cytochrome P450

52A3,” Journal

of Biological Chemistry, vol. 273, no. 49, pp. 32528–

32534,1998.

[80] J. B. Van Beilen and E. G. Funhoff, “Expanding the

alkaneoxygenase toolbox: new enzymes and applications,”

CurrentOpinion in Biotechnology, vol. 16, no. 3, pp. 308–314,

2005.12 Biotechnology Research International

[81] M. O. Ilori, S. A. Adebusoye, and A. C. Ojo,

“Isolation and characterization of hydrocarbon-degrading

andbiosurfactant-producing yeast strains obtained from a

polluted lagoon water,” World Journal of Microbiology

andBiotechnology, vol. 24, no. 11, pp. 2539–2545, 2008.

[82] G. S. Kiran, T. A. Hema, R. Gandhimathi et

al.,“Optimization and production of a biosurfactant from

thesponge-associated marine fungus

AspergillusustusMSF3,”Colloids and Surfaces B, vol. 73, no. 2, pp.

250–256, 2009.

[83] O. S. Obayori, M. O. Ilori, S. A. Adebusoye, G. O.

Oyetibo,A. E. Omotayo, and O. O. Amund, “Degradation

ofhydrocarbons and biosurfactant production by

Pseudomonassp. strain LP1,” World Journal of Microbiology

andBiotechnology, vol. 25, no. 9, pp. 1615–1623, 2009.

[84] M. L. Brusseau, R. M. Miller, Y. Zhang, X. Wang, and

G. Y.Bai, “Biosurfactant and cosolvent enhanced

remediation ofcontaminated media,” ACS Symposium Series, vol.

594, pp.82–94, 1995.

[85] G. Bai, M. L. Brusseau, and R. M. Miller,

“Biosurfactantenhancedremoval of residual hydrocarbon

from soil,”Journal of Contaminant Hydrology, vol. 25, no. 1-2,

pp.157–170, 1997.

[86] T. Barkay, S. Navon-Venezia, E. Z. Ron, and E.

Rosenberg,“Enhancement of solubilization and

biodegradation ofpolyaromatic hydrocarbons by the

bioemulsifieralasan,”Applied and Environmental Microbiology, vol.

65, no. 6, pp.2697–2702, 1999.

[87] S. S. Cameotra and P. Singh, “Bioremediation of oil

sludgeusing crude biosurfactants,” International

Biodeteriorationand Biodegradation, vol. 62, no. 3, pp. 274–280,

2008.

[88] R. Beal and W. B. Betts, “Role of

rhamnolipidbiosurfactantsin the uptake and mineralization

of hexadecane inPseudomonas aeruginosa,” Journal of Applied

Microbiology,vol. 89, no. 1, pp. 158–168, 2000.

[89] O. Pornsunthorntawee, P. Wongpanit, S. Chavadej,

M.Abe, and R. Rujiravanit, “Structural and

physicochemicalcharacterization of crude biosurfactant

produced byPseudomonas aeruginosaSP4 isolated from petroleum

—contaminated soil,” Bioresource Technology, vol. 99, no. 6,

pp.1589–1595, 2008.

[90] M.NikolopoulouandN. Kalogerakis, “Biostimulation

strategiesfor fresh and chronically polluted marine

environmentswith petroleum hydrocarbons,” Journal of Chemical

Technologyand Biotechnology, vol. 84, no. 6, pp. 802–807,

2009.

[91]:Butler CS, JR Mason (1997). Structure–functionanalysis of the bacterial aromatic ring–hydroxylatingdioxygenase. Advanced Microbial Physiology. 38: 47–84.

[92]:Hag well IS, LM Delfino, JJ Rao (1992). Partitioningof Polycyclic Aromatic Hydrocarbons from oil into water.Environ. Sci. Technol. 26: 2104–2110.

[93]:Mishra S, J Jyot, RC Kuhad, B Lal (2001). Evaluationof inoculum addition to stimulate in situ Bioremediationof oily–sludge–contaminated soil. Appl. Environ.Microbial. 67(4): 1675–1681.

[94]:Propst TL, RL Lochmiller, CW Qualis, Jr. K McBee(1999). In situ (mesocosm) assessment of immunotoxicityrisks to small mammals inhabiting petrochemical wastesites. Chemosphere. 38: 1049–1067.

[95]:Lloyd CA, TA Cackette (2001). Diesel Engines:Environmental Impact and Control. Air and WasteManagement Association. 51: 805–847.

[96]: Van Hamme JD, Singh A, Ward OP (2003). RecentAdvances in Petroleum Microbiology. Microbial. Mol. Biol.Rev. 67(4): 503–549.

[97]:Blodgett WC (2001). Water–soluble mutagen productionduring the bioremediation of oil–contaminated soil.Florida Scientist. 60(1):28–36.

98:Roling WFM, Milner MG, DM Jones, K Lee, F Daniel,Swannell RJP, Head IM (2002). Robust hydrocarbondegradation and dynamics of bacterial communities duringnutrient–enhanced oil spill bioremediation. Appl.Environ. Microbial. 68 (11): 5537-5548.

[99]:Bushnell LD, HF Haas (1941). The utilization ofhydrocarbons by microorganisms. J. Bacterial. 41: 653–673.

[100]:Speight JG (1991). The chemistry and technology ofpetroleum. Marcel Dekker, New York, N.Y.

[101]: Ehrlich HL (1995). Geomicrobiology. Marcel Dekker,Inc., New York, N.Y.

[102]: Harder E (2004). Bioremediation of engine oil.Little Flower Academy. Dallas, Texas.

[103]:Alexander M (1999). Biodegradation andBioremediation (2nd edition) Academic Press, San Diego

[104]:Barathi S, N Vasudevan (2001). Utilization ofpetroleum hydrocarbons by Pseudomonas fluoresces isolatedfrom a petroleum–contaminated soil. Environ. Int. 26: 413– 416.

[105]:Eriksson M, Dalhammer G, AK Borg–Karlson. (1999).Aerobic degradation of a hydrocarbon mixture in naturaluncontaminated potting soil by indigenous microorganismsat 20oC and 6oC. Appl.Microbial. Biotechnology. 51: 532–535

[106]:Johnson K, Anderson S, CS Jacobson (1996).Phenotypic and genotypic characterization ofphenanthrene–degrading fluorescent Pseudomonas biovars.Appl. Environ. microbial. 62: 3818–3825.

[107]:Kiyohara H, Takizawa N, Nagao K (1992). Naturaldistribution of bacteria metabolizing many kinds ofpolyaromatic hydrocarbons. J. Ferment. Bioeng. 74: 49–51.

[108]:Fall RR, JL Brown, TL Schaeffer (1979). Enzymerecruitment allows the biodegradation of recalcitrant–branched hydrocarbons by Pseudomonas citronellals. Appl.Environ. Microbial. 38: 715–722.

[109]:Rusansky S, Avigad R, Michaeli S, Gutnick DL(1987). Involvement of a plasmid in Growth on andDispersion of Crude Oil by Acinetobactersp calcoaceticusRA57. Appl. Environ. Microbial. 53: 1918-1923.

[110]: Prenafeta–Boldu XF, Kuhn A, DMAM L, Anke H, JW VanGroenestijn, JAM De Bont (200)1. Isolation andCharacterization of fungi growing on volatile aromatichydrocarbons as their sole carbon and energy source.Mycological Res. 4: 477–484.

[111]:McLaughlin B (2001). Soil Remediation. Enginr. Sci.Rev. 2:69–77. Mishra S, J Jyot, RC Kuhad, B Lal (2001).Evaluation of inoculum addition to stimulate in situBioremediation of oily–sludge–contaminated soil. Appl.Environ.Microbiol. 67(4): 1675–1681.

[112]:Swannell RPJ, K Lee K, McDonagh M (1996). Fieldevaluation of Marine Oil Spill Bioremediation. Microbial,Rev. 60: 342–365.