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ISOLATION AND CHARCTERIZATION OF HYDROCARBON DEGRADING BACTERIA ISOLATED FROM SOIL CONTAMINATED

WITH ENGINE OIL.

SUBMITTED TO

VEER NARMAD SOUTH GUJARAT UNIVERSITY

GUIDED BY :

Miss. Priya Bande

Miss. Neha Vora

MITCON BIOPHARMA CENTER,

PUNE-411005,

MAHARASTRA,

INDIA.

SUBMITTED BY:

Hinal Desai

M.Sc. Biotechnology,

Department of Biotechnology,

Veer Narmad South Gujarat University,

Surat-395007.

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1. INTRODUCTION

Petroleum-based products are the major source of energy for industry and daily

life. Leaks and accidental spills occur regularly during the exploration, production,

refining, transport, and storage of petroleum and petroleum products. Release of

hydrocarbons into the environment whether accidentally or due to human activities is a

main cause of water and soil pollution. Soil contamination with hydrocarbons causes

extensive damage of local system since accumulation of pollutants in animals and plant

tissue may cause death or mutations. The technology commonly used for the soil

remediation includes mechanical, burying, evaporation, dispersion, and washing.

However, these technologies are expensive and can lead to incomplete decomposition of

contaminants. The process of bioremediation, defined as the use of microorganisms to

detoxify or remove pollutants due to their diverse metabolic capabilities is an evolving

method for removal and degradation of many environmental pollutants including the

products of petroleum industry. In addition, bioremediation technology is believed to be

noninvasive and relatively cost-effective. Biodegradation by natural populations of

microorganisms represents one of the primary mechanisms by which petroleum and other

hydrocarbon pollutants can be removed from the environment and is cheaper than other

remediation technologies.

The success of oil spill bioremediation depends on one’s ability to establish

and maintain conditions that favor enhanced oil biodegradation rates in the contaminated

environment. One important requirement is the presence of microorganisms with the

appropriate metabolic capabilities. If these microorganisms are present, then optimal rates

of growth and hydrocarbon biodegradation can be sustained by ensuring that adequate

concentrations of nutrients and oxygen are present and that the pH is between 6 and 9.

The physical and chemical characteristics of the oil and oil surface area are also important

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determinants of bioremediation success.

[Fig.1.1 A bird covered in the oil spill]

There are the two main approaches to oil spill bioremediation:

(a) bioaugmentation, in which known oil-degrading bacteria are added supplement the

existing microbial population, and

(b) biostimulation, in which the growth of indigenous oil degraders is stimulated by the

addition of nutrients or other growth-limiting cosubstrates.

Most existing studies have concentrated on evaluating the factors affecting oil

bioremediation or testing favored products and methods through laboratory studies.

Only limited numbers of pilot scale and field trials have provided the most convincing

demonstrations of this technology. The scope of current understanding of oil

bioremediation is also limited because the emphasis of most of these field studies has

been given on the evaluation of bioremediation technology for dealing with large-scale oil

spills on marine shorelines.

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http://microbewiki.kenyon.edu/index.php/File:Bird_in_oil_spill.jpg

1.1 WHAT ARE HYDROCARBONS????

A hydrocarbon is an organic compound consisting entirely of hydrogen and

carbon. They can be straight-chain, branched chain or cyclic molecules. Hydrocarbon

derivatives are formed when there is a substitution of a fuctional group at one or more

positions. An almost unlimited number of carbon compounds can be formed by the

addition of a functional group to hydrocarbon.

[Fig 1.2 Derivatives of hydrocarbon]

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1.2 TYPES OF HYDROCARBON

There are four types of hydrocarbon:

1. Saturated hydrocarbons2. Unsaturated hydrocarbons3. Cycloalkanes and4. Aromatic hydrocarbons

1. Saturated hydrocarbons (alkanes) are the simplest of the hydrocarbon

species and are composed entirely of single bonds and are saturated with

hydrogen. The general formula for saturated hydrocarbons is

CnH2n+2. Saturated hydrocarbons are the basis of petroleum fuels and are

found as either linear or branched species. Branched hydrocarbons can

be chiral. Chiral saturated hydrocarbons constitute the side chains of

biomolecules such as chlorophyll and tocopherol. Hydrocarbons with

the same molecular formula but different structural formulae are called

structural isomers.

2. Unsaturated hydrocarbons have one or more double or triple bonds

Between carbon atoms. Those with double bonds are called alkenes with

formula CnH2n. Those containing triple bonds are called alkynes, with

general formula CnH2n-2.

3. Cycloalkanes are hydrocarbons containing one or more carbon rings to

which hydrogen atoms are attached.

4. Aromatic hydrocarbons, also known as arenes, are hydrocarbons that

have at least one aromatic ring.

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[Fig 1.3 Types of Hydrocarbon]

Hydrocarbon can be gases (e.g. methane and propane), liquids (e.g. hexane and benzene),

waxes or low melting solids (e.g. paraffin wax and naphthalene) or polymer (e.g.

polyethylene, polypropylene and polystyrene).

[ Fig 1.4 Hydrocarbon Methane]

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1.3 GENERAL PROPERTIES OF HYDROCARBONS

Because of differences in molecular structure, the empirical formula remains different

between hydrocarbons, the amount of bonded hydrogen lessens in alkenes and alkynes

due to the "self-bonding" or catenation of carbon preventing entire saturation of the

hydrocarbon by the formation of double or triple bonds. This inherent ability of

hydrocarbons to bond to themselves is referred to as catenation and allows hydrocarbon

to form more complex molecules, such as cyclohexane,and in rarer cases, arenes such

as benzene. This ability comes from the fact that bond character between carbon atoms is

entirely non-polar, in that the distribution of electrons between the two elements is

somewhat even due to the same electronegativity values of the elements, and does

not result in the formation of an electrophile.

Hydrocarbons are hydrophobic and are lipids.

Some hydrocarbons also are abundant in the solar system. Lakes of liquid methane and

ethane have been found on Titan, Saturn's largest moon. Hydrocarbons are also abundant

in nebulae forming polycyclic aromatic hydrocarbons - PAH compounds.

1.4 USES OF HYDROCARBONS

Hydrocarbons are one of the Earth's most important energy resources. The predominant

use of hydrocarbons is as a combustible fuel source. In their solid form, hydrocarbons

take the form of asphalt. Mixtures of volatile hydrocarbons are now used in preference to

the chlorofluorocarbons as a propellant for aerosol sprays, due to chlorofluorocarbon's

impact on the ozone layer. Methane [1C] and ethane [2C] are gaseous at ambient

temperatures and cannot be readily liquefied by pressure alone. Propane [3C] is however

easily liquefied, and exists in 'propane bottles' mostly as a liquid.Butane [4C] is so easily

liquefied that it provides a safe, volatile fuel for small pocket lighters. Pentane [5C] is a

clear liquid at room temperature, commonly used in chemistry and industry as a powerful

nearly odorless solvent of waxes and high molecular weight organic compounds,

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including greases. Hexane [6C] is also a widely used non-polar, non-aromatic solvent, as

well as a significant fraction of common gasoline. The [6C] through [10C] alkanes,

alkenes and isomeric cycloalkanes are the top components of gasoline, naphtha,

jetfuel and specialized industrial solvent mixtures. With the progressive addition of

carbon units, the simple non-ring structured hydrocarbons have higher viscosities,

lubricating indices, boiling points, solidification temperatures, and deeper color. At the

opposite extreme from [1C] methane lie the heavy tars that remain as the lowest

fraction in a crude oil refining retort. They are collected and widely utilized as roofing

compounds, pavement composition, wood preservatives and as extremely high viscosity

sheer-resisting liquids.

1.5 MICROBIAL DEGRADATION OF PETROLEUM HYDROCARBON

Biodegradation of petroleum hydrocarbons is a complex process that depends on the

nature and on the amount of the hydrocarbons present. Petroleum hydrocarbons can be

divided into four classes: the saturates, the aromatics, the asphaltenes (phenols, fatty

acids, ketones, esters, and porphyrins), and the resins (pyridines, quinolines, carbazoles,

sulfoxides, and amides). Different factors influence hydrocarbon degradation. One of the

important factors that limit biodegradation of oil pollutants in the environment is their

limited availability to microorganisms. Petroleum hydrocarbon compounds bind to soil

components, and they are difficult to be removed or degraded. Hydrocarbons differ in

their susceptibility to microbial attack. The susceptibility of hydrocarbons to microbial

degradation can be generally ranked as follows: linear alkanes > branched alkanes > small

aromatics > cyclic alkanes. Some compounds, such as the high molecular weight

polycyclic aromatic hydrocarbons (PAHs), may not be degraded at all. Microbial

degradation is the major and ultimate natural mechanism by which one can cleanup

the petroleum hydrocarbon pollutants from the environment. Hydrocarbons in the

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environment are biodegraded primarily by bacteria, yeast, and fungi. The reported

efficiency of biodegradation ranged from 6% to 82% for soil fungi, 0.13% to 50% for

soil bacteria, and 0.003% to 100% for marine bacteria. Many scientists reported that

mixed populations with overall broad enzymatic capacities are required to degrade

complex mixtures of hydrocarbons such as crude oil in soil, fresh water, and marine

environments. Bacteria are the most active agents in petroleum degradation, and they

work as primary degraders of spilled oil in environment . Several bacteria are even known

to feed exclusively on hydrocarbons. In earlier days, the extent to which bacteria, yeast,

and filamentous fungi participate in the biodegradation of petroleum hydrocarbons was

the subject of limited study, but appeared to be a function of the ecosystem and local

environmental conditions. Though algae and protozoa are the important members of the

microbial community in both aquatic and terrestrial ecosystems, reports are scanty

regarding their involvement in hydrocarbon biodegradation. brown alga, and two diatoms

could oxidize naphthalene.

1.6 LIST OF MICROORGANISMS INVOLVED IN HYDROCARBON DEGRADATION

BACTERIA

Arthrobacter Burkholderia Mycobacterium Sphingomonas Pseudomonas fluorescens Pseudomonas aeruginosa Pseudomonas alcaligens Staphylococcus sp. Bacillus subtilis Bacillus sp. Alcaligenes sp. Flavobacterrium sp. Acinetobacterium sp.

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Micrococcus roseus Corynebacterium sp. Xanthomonas sp.

Fungi

Amorphoteca sp. Aspergillus Cephalosporium Penicillium Neosartorya sp. Talaromyces sp. Graphium sp.

Yeast

Candida sp. Yorrowia sp. Pichiya sp. Geotrichum sp.

1.7 MECHANISM OF PETROLEUM HYDROCARBON DEGRADATION

The most rapid and complete degradation of the majority of organic pollutants is brought

about under aerobic conditions. Figure 1.5 shows the main principle of aerobic

degradation of hydrocarbons. The initial intracellular attack of organic pollutants is an

oxidative process and the activation as well as incorporation of oxygen is the enzymatic

key reaction catalyzed by oxygenases and peroxidases. Peripheral degradation pathways

convert organic pollutants step by step into intermediates of the central intermediary

metabolism, for example, the tricarboxylic acid cycle. Biosynthesis of cell biomass occurs

from the central precursor metabolites, for example, acetyl-CoA, succinate, pyruvate.

Sugars required for various biosyntheses and growth are synthesized by gluconeogenesis.

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[Fig. 1.5 Main principle of aerobic degradation of hydrocarbon by microorganisms]

1.8 ENZYMES PARTICIPATING IN HYDROCARBON

DEGRADATION

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Cytochrome P450 alkane hydroxylases constitute a super family of ubiquitous Heme-

thiolate Monooxygenases which play an important role in the microbial degradation of

oil, chlorinated hydrocarbons, fuel additives, and many other compounds. Depending on

the chain length, enzyme systems are required to introduce oxygen in the substrate to

initiate biodegradation [Table 1]. Higher eukaryotes generally contain several different

P450 families that consist of large number of individual P450 forms that may contribute

as an ensemble of isoforms to the metabolic conversion of given substrate. In

microorganisms such P450 multiplicity can only be found in few species. Cytochrome

P450 enzyme systems was found to be involved in biodegradation of petroleum

hydrocarbons. The capability of several yeast species to use n-alkanes and other aliphatic

hydrocarbons as a sole source of carbon and energy is mediated by the existence of

multiple microsomal Cytochrome P450 forms. These cytochrome P450 enzymes had

been isolated from yeast species such as Candida maltose, Candida tropicalis,

and Candida apicola. The diversity of alkaneoxygenase systems in prokaryotes and

eukaryotes that are actively participating in the degradation of alkanes under aerobic

conditions like Cytochrome P450 enzymes, integral membrane di-iron alkane

hydroxylases (e.g., alkB), soluble di-iron methane monooxygenases, and membrane-

bound copper containing methane monooxygenases have also been studied.

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[Table 1 Enzymes involved in petroleum hydrocarbon degradation]

1.9 FACTORS INFLUENCING PETROLEUM

HYDROCARBON DEGRADATION

A number of limiting factors have been recognized to affect the biodegradation of

petroleum hydrocarbons. The composition and inherent biodegradability of the petroleum

hydrocarbon pollutant is the first and foremost important consideration when the

suitability of a remediation approach is to be assessed. Among physical factors,

temperature plays an important role in biodegradation of hydrocarbons by directly

affecting the chemistry of the pollutants as well as affecting the physiology and diversity

of the microbial flora. At low temperatures, the viscosity of the oil increased, while the

volatility of the toxic low molecular weight hydrocarbons were reduced, this cause delay

of biodegradation. Temperature also affects the solubility of hydrocarbons . Although

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hydrocarbon biodegradation can occur over a wide range of temperatures, the rate of

biodegradation generally decreases with the decreasing temperature. Figure 1.6 shows

that highest degradation rates that generally occur in the range 30–40∘C in soil

environments, 20–30∘C in some freshwater environments and 15–20∘C in marine

environments. Ambient temperature of the environment affect both, the properties of

spilled oil and the activity of the microorganisms. Significant biodegradation of

hydrocarbons have been reported in psychrophilic environments in temperate regions.

[Fig. 1.6 Hydrocarbon degradation rate in soil, freshwater and marine environment]

Nutrients are very important ingredients for successful biodegradation of hydrocarbon

pollutants especially nitrogen, phosphorus, and in some cases iron. Some of these

nutrients could become limiting factor thus affecting the biodegradation processes. When

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a major oil spill occurred in marine and freshwater environments, the supply of carbon

significantly increases and the availability of nitrogen and phosphorus generally become

the limiting factor for oil degradation. In marine environments, it was found to be more

pronounced due to low levels of nitrogen and phosphorous in seawater. Freshwater

wetlands are typically considered to be nutrient deficient due to heavy demands of

nutrients by the plants. Therefore, additions of nutrients were necessary to enhance the

biodegradation of oil pollutant. On the other hand, excessive nutrient concentrations can

also inhibit the biodegradation activity. Use of poultry manure as organic fertilizer in

contaminated soil was also reported, and biodegradation was found to be enhanced in the

presence of poultry manure alone. Photo-oxidation also increases the biodegradability of

petroleum hydrocarbon by increasing its bioavailability and thus enhancing microbial

activities.

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2. OBJECTIVE

Isolation of hydrocarbon degrading bacteria from the soil of automobile

workshop.

Identification of bacteria by their morphological and colony characteristics and

biochemical tests.

To check ability of bacteria to utilize different hydrocarbon sources like

Benzene, Petrol, Engine oil, Diesel, Toluene as sole carbon source.

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3. MATERIALS AND METHODS

3.1 MATERIALS

Soil contaminated with diesel Luria Bertani (LB) Broth Luria Bertani Agar Plate Nutrient Agar Plate Nutrient Broth Psuedomonas Agar Plate Mineral salt medium 0.1 M phosphate buffer Media for Biochemical Tests Reagents for Biochemical Tests Reagents for gram’s staining

- Crystal violet stain- Gram’s iodine- 95% ethanol- Safranin stain

Different carbon sources (Petrol, Diesel, Engine oil, Toluene, Benzene)

All the instruments which were used are as following:

Weighing Balance pH meter Autoclave Oven Laminar Air Flow Incubator Shaker Water Bath Orbital shaker Microscope Centrifuge Spectrophotometer

Media and reagents for biochemical tests

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Test Medium Reagent Positive Results

Carbohydrate fermentation test

Glucose, maltose, Sucrose, Lactose, Mannitol, Xylose

Phenol red Red to Yellow (gas production in Durham’s tube)

Urea utilization test

Urea broth, Phenol red Pinkish red color

H2S Production test

2% Peptone Lead acetate paper strip

Blackening of paper

Gelatin hydrolysis test

Nutrient gelatin broth

– Liquefaction at 4°C

Citrate utilization test

Simmons Citrate agar Slant

Bromothymole blue

Green to Blue

Nitrate reduction test

Peptone nitrate broth

Sulphanilic acid+

a-Naphthalamine

Red color

Oxidase test Nutrient Agar Slant Oxidase strip Violet color

Catalase test Nutrient Agar Slant 3% H2O2 Formation of bubbles

M-R test Glucose Phosphate broth

Methyl red Red color

V-P test Glucose Phosphate broth

40% KOH+

a- Naphthol

Pink color

Iodole production test

1% Tryptone Kovac`s reagent Red ring production

TSI slant Triple Sugar iron agar Slant

– –

Macconkey`s Agar plate

Macconkeys agar plate

– –

3.2 METHOD

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3.2.1 Collection of soil :

Oil contaminated soil sample was collected from automobile work shop

from Pune (Sample 1) and Surat (Sample 2). Soil samples were used to isolate the

Bacteria. Samples were collected at a depth within 5cm from the surface of the

soil. They were collected in sterile polythene bags and tightly packed.

3.2.2 Culture media

For Enrichment the culture LB broth and Mineral Salt Medium were used.

Isolation was carried out on LB agar plate and Mineral Salt agar medium

containing filtered engine oil.

Prepared media in D/W

Bring vol. 1 lit. & Autoclaving 15 psi, 121°C

Pour into sterile Petriplate

Allow to cool to room temp.

Invert Petri-plate

Spread 0.2 ml of hydrocarbon source on plate

3.2.3 Procedure for inoculum development :

1 gm soil sample (contaminated with diesel)

Vortex with 10ml distilled water in testtube

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Allow to settle

Use supernatent as inoculum

3.2.4 Procedure for Growth and Isolation of bacteria

100ml LB broth containing 1% Engine oil in flask

Add 10ml previously prepared supernatant into flask containing LB broth

Incubate flask at 37.c on shaker at 100rpm for 48hrs

Three successive subculture on same medium containing Engine oil

At every subculture, streak a loopfull of medium containing growth of

bacteria onto LB agar plate contaning Engine oil by four flame method

Incubate plate at 37.c in incubator for 48hrs

After 48hrs, observe plate for growth of bacteria

After three subculturing, centrifuge broth at 5000rpm for 10 min, collect cell pellets

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Wash cell pellets with 0.1 M phosphate buffer (pH 6.8) twice

Transfer pellets into 100ml Mineral salt medium in flask containing 1% Engine oil as a carbon and energy source

Incubate flask at 37.c on shaker at 100rpm for 48hrs

After 48hrs streak loopfull of inoculum by from flask by four flame

method onto mineral salt agar plate containing Engine oil

Incubate plate at 37.c for 7 days in incubator

Obsevre for the growth of bacteria

[Note: Replace yeast extract with Engine oil as carbon source during preparation of LB medium]

3.2.5 Procedure for Gram’s staining

After getting growth on LB and Mineral Salt agar medium, colony characteristics is

observed and Gram’staining of isolated colony is done.

Prepare suspension of bacteria using single colony from plate in 2ml

sterile distilled water

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Prepare a heat fixed smear from suspension

Cover smear with crystal violet stain for 1 min

Drain crystal violet and cover smear with Gram’s iodine for 1 min

Rinse slide in running water

Rinse slide with 95% ethanol for approximately 10-15 seconds

Rinse smear with water

Add counterstain safranin for 1 min

Rinse slide with water, air dry and observe under oil-immersion objective

3.2.6 Procedure for Biochemical Tests

Biochemical tests for bacteria is performed for identification of bacteria.

Prepare all biochemical media

Prepare suspension of bacteria using single colony from plate in 2ml sterile distilled water

Inoculate two loopfull of suspension into all biochemical media under aseptic condition

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Streak a loopfull of suspension on slants and plates under aseptic condition

Incubate all inoculated media at 37.c in incubator for 24hrs

After 24hrs, observe results

3.2.7 Procedure for biodegradation potential

After getting growth of bacteria on plates, their ability to degrade diesel, benzene,

toluene, petrol as carbon source is checked.

Take 10 ml Nutrient broth and LB broth in separate testtubes (5 tubes for each broth)

Add 1 ml petrol, engine oil, diesel, toluene and benzene in each tube of both the broths

Inoculate both broths with previously isolated bacteria

Incubate all tubes at 30.c for 72hrs in incubator

Take O.D. at 640nm

[Note: Replace yeast extract with Engine oil, diesel, petrol, benzene,

and toluene in each separate tube of both broth as carbon source in

above procedure]23 | P a g e

Above all procedures are done for both the soil samples and

results are noted down.

4. RESULTS AND DISCUSSION

4.1 Results of isolation procedure24 | P a g e

For sample 1

On LB Agar plate greenish blue colonies of bacteria are observed. Clear

zone is observed around growth of bacteria that shows bacteria can degrade

engine oil. From LB plate one colony is streaked on Pseudomonas Agar

plate and well isolated colonies are observed on it.

No growth is observed on Mineral Salt Agar plate.

For sample 2

On Mineral Salt Agar plate small white colonies are observed.

(Sample 1) (Sample 2)

[Fig. 4.1 Growth of hydrocarbon degrading bacteria on LB agar plate (Sample 1) and on Mineral Salt agar plate (Sample 2)]

4.2 Colony characteristics

Characters Sample 1 Sample 2

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Size Small Small Shape Round Round

Margin Entire EntireElevation Low convex Low convexTexture Smooth Smooth Opacity Translucent Opaque

Consistency Moist MoistPigmentation Greenish blue White

4.3 Results of Gram’s staining

Characters Sample 1 Sample 2Size Small Small

Shape Short rods Oval, RoundArrangement Singly, chains, or clusters Singly or clusters

Gram’s reaction Gram negative Gram positive

4.4 Results of Biochemical Tests

No. Test Sample 1 Sample 2

1.Carbohydrate

hydrolysis

Glucose + + (G)

Sucrose - + (G)

Maltose - + (G)

Mannitol - + (G)

Lactose - + (G)

Xylose - + (G)

2. Urea utilizationtest

- -

3. H2S Production test

- -

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4. Gelatin hydrolysis test

- -

5 Citrate utilization test

+ +

6. Nitrate reduction test

+ +

7. Oxidase test + +8. Catalase test + +9. M-R test - -

10. V-P test - +

11. Iodole production test

- -

13. Macconkey`s Agar plate

Colourless colonies were observed

Pink colour colonies were

observed15. Motility Motile Non-motile

[Fig. 4.2 Results of biochemical tests for Sample 2] 4.4.1 Results of TSI slant

Sample 1 Sample 2

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Slant Pink (Alkaline) Yellow (Acidic)Butt Pink (Alkaline) Yellow (Acidic)

H2S production - -Gas production - (G)

Key : + Positive tests

- Negative test

(G) Gas production

(A) (B)

[Fig. 4.4 Results of TSI slant for Sample 1(B) and Sample 2(A)]

4.5 Identification of Hydrocarbon degrading isolated strain

The bacteria were distinguished on basis of their growth pigmentation

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and colony morphology on LB and Mineral Salt agar plate and selective media

(MacConkey’s Agar plate) at 37°c for 24hrs. Then the isolated bacteria were

identified by morphological and biochemical characteristics.

Bacteria isolated from sample 1 which was collected from the outer soil

of garage of pune were characterized as Pseudomonas sp. . The Pseudomonas

colonies were identified by the morphology, greenish blue, small colonies on

LB agar plate and greenish yellow colonies on Pseudomonas agar plate.

Organisms were Gram-negative, short rods arranged in clusters or in chain.

These bacteria were oxidase and catalase positive.

Bacteria isolated from sample 2 which was collected from the outer soil

of garage of surat were characterized as Stapylococcus sp.. The Stapylococcus

colonies were identified by the morphology, white small colonies on Mineral

salt Agar plate. Organisms were Gram-positive. Cocci shape arranged in

clusters. These bacteria were oxidase and catalase positive.

[Fig 4.5 Growth of Pseudomonas sp. isolated from Sample 1 on

Pseudomonas agar plate]

4.6 Results of biodegradation potential

Table 1 :

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Source Media O.D. at 640nm

Nutrient broth

Pseudomonas sp. Staphylococcus sp. Petrol 0.036 0.287

Benzene 0.303 0.193Toluene 0.023 0.030Diesel 0.265 0.067

Engine oil 0.236 0.128

Table 2 :

Source Media O.D. at 640nm

LB broth

Pseudomonas sp. Staphylococcus sp. Petrol 0.086 0.274

Benzene 0.338 0.129Toluene 0.045 0.010Diesel 0.251 0.140

Engine oil 0.203 0.109

From table 1 and 2, it was observed that Pseudomonas sp. are able to

degrade Benzene efficiently. These bacteria can degrade other sources

in following order :

Diesel > Engine oil > Petrol > Toluene

From table 1 and 2, it was observed that Staphylococcus sp. are able to

degrade Petrol efficintly. These bacteria can degrade other sources in

following order :

Benzene > Engine oil > Diesel > Toluene

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(A) (B) (C) (D)

Carbon sources

(A) Engine oil

(B) Petrol

(C) Diesel

(D) Benzene

[Fig. 4.6 Ability of Pseudomons sp. isolated from Sample 1 to use engine oil, petrol, diesel and benzene as sole carbon source]

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5. SUMMERY

Bacteria isolated from Sample 1 were identified as Psuedomonas sp. by their morphological and colony characteristics, gram’s reaction and biochemical tests.

Bacteria isolated from Sample 2 were identified as Staphylococcu sp. by their

morphological and colony characteristics, gram’s reaction and biochemical tests.

Pseudomonas sp. and Staphylococcus sp. both are able to use petrol, engine oil,

diesel, benzene and toluene as carbon source.

Pseudomonas showed maximun growth in presence of Benzene than other

Sources while Staphylococcus showed maximum growth in presence of Petrol

than other sources.

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6. CONCLUSION

The microbial degradation of oil pollutants is a complex process and the rates of

biodegradation of hydrocarbons from oil spills appear to be highly dependent on

localized environmental conditions. The fate of many components in petroleum, the

degradative pathways which are reactive in the environment, the importance of

cooxidationin natural ecosystems, and the role of microorganisms in forming

persistent environmental contaminants from hydrocarbons such as the compounds

found in tar balls are unknown and require future research. Although a number

of rate-limiting factors have been elucidated, the interactive nature of microorganisms,

oil, and environment still is not completely understood, and further examination of

case histories is necessary to improve predictive understanding of the fate of oil

pollutants in the environment and the role of microorganisms in biodegradative

environmental decontamination. With an understanding of the microbial hydrocarbon

degradation process in the environment, it should be possible to develop models for

predicting the fate of hydrocarbon pollutants and to develop strategies for utilizing

microbial hydrocarbon degrading activities for the removal of hydrocarbons from

contaminated ecosystems.

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7. APPENDIX I CULTURE MEDIA

1. LB broth / Agar

Components gmCasein enzyme

hydrolysate10

Yeast extract 5Distilled water 1000 ml

Agar 20NaCl 10pH 7.5

Dissolve all ingredients (exept agar) by heating and adjust to pH 7.5. Add agar

powder and digest it by boiling in waterbath. Sterilize it by autoclaving (121.c for

15 min). LB broth has the same composition except that it does not contain the

solidifying agent agar.

[Note : Here replace yeast extract with engine oil/ petrol/ diesel/ benzene/ toluene

during preparation of LB broth / agar ]

2. Mineral Salt Medium

Components gmKNO3 1.0

MgSO4.7H2O 1.0CaCl2.6H2O 0.1

FeSO4 0.05Trace element

solution250 ml

Phosphate buffer(1 M, pH 6.8)

20 ml

Distilled water 730 mlpH 7.5

Dissolve all ingredients and adjust to pH 7.5. Sterilize it by autoclaving (121.c

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for 15 min). For preparation of Mineral Salt agar, add 20 gm agar with above

components.

Trace element solution

Components gmSnCl2 0.05

KI 0.05LiCl 0.05

MnSO4.4H2O 0.08HBO3 0.50

ZnSO4.7H2O 0.10CoCl2.6H2O 0.10NiSO4.6H2O 0.10

BaCl2 0.05Ammonium molybdate

0.05

Distilled water 1000 ml

Add all salts one by one

3. Pseudomonas agar

Components gmCasein enzyme

hydrolysate

10.0

K2HPO4 1.5

Proteose Peptone 10.0

MgSO4 1.5

Agar 15


pH 7.0Dissolve by heating, and adjust the pH. Sterilize by autoclaving at 15 lbs pressure

(121°C) for 15 min.

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Media for Biochemical Tests

1. Glucose Phosphate Broth

Components gmGlucose 5.0

K2HPO4 5.0

Peptone 5.0


pH 6.9-7.0Dissolve by heating, and adjust the pH. Sterilize by autoclaving at 15 lbs pressure

(121°C) for 15 min.

2. MacConkey’s Agar Media

Components gmPeptone 20.0

Lactose 10.0

NaCl 5.0

Bile salts 3.0-5.0

Neutral red 30.0 mg

Crystal violet 10.0 mg


Agar 30.0

pH 7.4Dissolve by heating, adjust pH to 7.4 and sterilize by autoclaving.

3. Nutrient sugar broth

Components ml

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10% aq. test sugar

solution (e.g. glucose)

10.0

1% peptone water 90.0

Phenol red 1.0

pH 7.4Mix components given in table. Sterilize by autoclaving at 10 psi for 10 minutes.

4. Urea broth

Components gmKH2PO4 9.1

Na2HPO4 9.5

Yeast extract 0.1

Phenol red 0.01


40% Urea 50 ml

pH 6.8Heat to dissolve and adjust pH to 6.8. Sterilize by autoclaving and allow to cool to

55.c. Add 50 ml sterile urea solution.

5. 2% Peptone broth

Components gmPeptone 20.0

NaCl 5.0


pH 7.5Heat to dissolve and adjust pH to 7.5. Sterilize by autoclaving.

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6. Nutrient Gelatin broth

Components gmMeat extract 3.0

Peptone 10.0

Gelatin 150.0



7. Simmon’s citrate agar slant

Components gmSodium citrate 2.0

MgSO4 0.2

NaCl 5.0

NH4H2PO4 1.0

K2HPO4 1.0

Bromothymol blue 0.08

Agar 20.0



8. Peptone Nitrate broth


Peptone 5.0

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Potassium nitrate 1.0



9. Nutrient agar slant


Peptone 10.0

NaCl 5.0


Agar 20.0

pH 7.4Heat to dissolve and adjust pH to 7.5. Sterilize by autoclaving. Pour medium into

sterile testtubes under aseptic condition and place tubes in slant position and allow to

solidify.

10. 1% Tryptone broth

Components gmTryptone 10.0

NaCl 5.0



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11. TSI [Triple Sugar Iron] Agar


Yeast extract 3.0

Peptone 15.0

Proteose peptone 5.0

Lactose 10.0

Glucose 1.0

Sucrose 10.0

Ferrous sulphate 0.2

Na2S2O3 0.3

NaCl 5.0

Agar 20.0

Phenol red 0.24


pH 7.4Heat to dissolve the ingredients and adjust the pH. Distribute the medium in

testtubes and sterilize by autoclaving. Allow the tubes to solidify in manner which will

give butt and slant.

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8. APPENDIX II STAINS AND REAGENTS

REAGENT FOR GRAM STAINING:

(A) Crystal Violet Staining Reagent:Crystal violet : 2.0 g

Ethanol (95%) : 20.0ml

Ammonium oxalate : 0.8 g

Distilled water : 80ml

Dissolve the dye in alcohol and ammonium oxalate in distilled water. Mix two

solutions and allow it to stand for 24 hrs. Filter and use.

(B) Iodine Solution:Iodine : 1.0 gm

Potassium iodide : 2.0 gm

Distilled water : 300 ml

Dissolve KI and iodine in little amount of water and adjust to 300 ml with water.

(C) Safranin Solution:Safranin : 0.25 gm

95% ethanol : 10.0 ml


Dissolve safranin in ethanol and make final volume to 100 ml with distilled water.

REAGENT FOR BIOCHEMICAL TEST:

(A) Methyle Red Indicator:

Methyl red : 0.1 gm

95% ethanol : 300 ml

Dissolve the dye in alcohol and use.

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(B) Phenol red indicator:

Phenol red : 0.2 gm

95% ethanol : 500 ml


Dissolve the phenol red in alcohol. Add distilled water and filter and use.

(C) 40% Potassium Hydroxide Solution:

KOH : 40.0 gm


Dissolve KOH in water to make the final volume to 100 ml.

(D) Kovac’s Reagent:ρ-dimethylaminobenzaldehyde :5.0 gm

95% ethanol : 75 ml

Conc. HCl : 25 ml

Dissolve the aldehyde in ethanol by gently warming in a waterbath (about 50-55.c).

Cool and add the acid. Protect the reagent from light and store in brown glass

bottle.

(E) Sulphanilic Acid:Sulphanilic acid : 1.0 g

5N Acetic acid :100 ml

Dissolve sulphanilic acid in distilled water. Filter and use.

(F) a-naphthalamine

N, N- Dimethyle-1-naphthalamine : 1 gm

5N acetic acid :1000 ml

Store at -2 to -8.c for upto 3 months in dark.

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Other solutions

1. 1 N NaOH 4 gm in 100 ml Distilled water.

2. 1 N HCl 8.8 ml Conc. HCl in 91.2 ml Distilled water.

3. 40% Urea 40 gm in 100 ml Distilled water.

4. 0.1 M phosphate buffer 49.7 ml 1 M K2HPO4 + 50.3 ml 1 M KH2PO4 Dilute the combined 1 M stock solution to 1000 ml with distilled water.

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