2

DECLARATION

We hereby declare that the project entitled “BIODEGRADATION OF OIL

CONTAMINATED SITE” submitted in partial fulfillment for the degree of Bachelor of

Engineering in Biotechnology to Gujarat Technological University, Ahmadabad, is a bonafide

record of the project work carried out at V.V.P. ENGINEERING COLLEGE,RAJKOT under

the supervision of DR.KRISHNA JOSHI and that no part of the UDP has been presented earlier

for any degree, diploma, associate ship, fellowship or other similar title of any other university or

institution.

JARIWALA JENIL

090470104003

JOSHI RIDDHI

090470104011

3

V.V.P. ENGINEERING COLLEGE

DEPARTMENT OF BIOTECHNOLOGY

MAY, 2013

CERTIFICATE

Date: 20

th April, 2013

This is to certify that the UDP entitled “BIODEGRADATION OF OIL

CONTAMINATED SITE” has been carried out by JARIWALA JENIL AND

JOSHI RIDDHI under my guidance in fulfillment of the degree of Bachelor of

Engineering in BIOTECHNOLOGY (8th

Semester) of Gujarat Technological

University, Ahmadabad during the academic year 2012-13.

Guide: Dr. Krishna Joshi

Head of the Department: Prof. D. H. Sur

4

ACKNOWLEDGEMENT

It gives us to immense pleasure in expressing our sincere regards and gratitude to our

guide Dr. KRISHNA JOSHI for her valuable guidance, suggestions that encouraged us

throughout the course to improve our self and in completion of work.

We also thank to our principal Dr. SACHIN PARIKH and Head of Department Prof.

D. H. Sur for giving us suitable resources to work.

We are sincerely thankful to Dr. SUMITKUMAR TRIVEDI and Dr. RUSHI MEHTA

for his guidance.

We greatly thankful to GUJARAT TECHNOLOGICAL UNIVERSITY for

introducing UDP in our curriculum; our knowledge is greatly increased in the field of

Biotechnology.

So we glad to present this report in front of you.

Jariwala Jenil

(090470104003)

Joshi Riddhi

(090470104011)

5

ABSTRACT

Extensive hydrocarbon exploration activities often result in the pollution of

the environment, which could lead to disastrous consequences for the biotic and

abiotic components of the ecosystem if not restored. Remediation of Oil-

contaminated system could be achieved by either Physicochemical or biological

methods. Various mechanical and chemical methods are used for remove the

hydrocarbons from the contaminated site, but it is not so effective and expensive

too. Bioremediation methods are so applied to these contaminated sites because

this method of removal of hydrocarbons is cost-effective and give the complete

degradation of the Oil contaminant and site is mineralized. Bioremediation

functions basically on biodegradation, which may refer to complete mineralization

of organic contaminants into carbon dioxide, water, inorganic compounds, and

cell protein or transformation of complex organic contaminants to other simpler

organic compounds by biological agents like microorganisms. Many indigenous

microorganisms in water and soil are capable of degrading hydrocarbon

contaminants.

6

LIST OF TABLES

Table No Table Description Page No

4.1 Colonical Characteristics 16

4.3.1 Peanut oil degradation by growth 23

4.3.2 Engine oil degradation by growth 24

4.3.3 Break oil degradation by growth 25

4.4.1 Percentage degradation of Peanut oil 26

4.4.2 Percentage degradation of engine oil 26

4.4.3 Percentage degradation of break oil 26

7

LIST OF FIGURES

Figure

No

Figure Description Page

No

3.1 The main principle of aerobic degradation of hydrocarbon by

microorganisms

9

4.1(A) Isolation result 14

4.1(B) Isolation result 15

4.2(A) Peanut oil set 17

4.2(B) Growth at periphery 18

4.2(C) Engine oil set 19

4.2(D) Engine oil degraded 20

4.2(E) Engine oil degraded set 21

4.2(F) Break oil degradation set 22

4.3.1 Graph of peanut oil degradation 23

4.3.2 Graph of engine oil degradation 24

4.3.3 Graph of break oil degradation 25

8

LIST OF SYMBOLS, ABBREVIATIONS AND NOMENCLATURE

Sr. No. Keywords

1 Bioremediation

2 Aromatic hydrocarbon

3 Crude oil

4 Growth rate

5 Pollution

6 Bacteria

7 Culture Growth

8 Optical density

9 Solvent extraction

10 Percentage oil degradation

9

TABLE OF CONTENTS

Acknowledgement i

Abstract ii

List of Figures iii

List of Tables iv

List of Abbreviations v

Table of Contents vi

Chapter: 1 Introduction 1

Chapter: 2 History of work 4

Chapter: 3 Implementation of project 6

3.1 Literature survey 6

3.2 Implementation of work 11

3.2.1 Work flow 11

Chapter: 4 Result Analysis 14

4.1 Isolation results 14

4.2 Preparation of culture 17

4.3 Analysis of culture by measuring the growth 23

4.4 Calculation of percentage oil degradation 26

Chapter: 5 Conclusion 27

References

10

CHAPTER: 1 INTRODUCTION

The oil industries that are present only in the limited area of the world are responsible for the

high generation of contamination of soil, rivers and seas. They produce highly potent organic

residues that cause the severe damage to the environment at large aspect (Judith Liliana

Solórzano Lemos et al.). 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 (Nilanjana Das et al.). The biodegradation of oil pollutants is not a new

concept as it has been intensively studied in controlled conditions and in open field experiments,

but it has acquired a new significance as an increasingly effective and potentially inexpensive

cleanup technology. Its potential contribution as a countermeasure biotechnology for

decontamination of oil polluted systems could be enormous (Anthony I Okoh). In this project,

the fate of Oil in an environment is reviewed, with special emphasis placed on its biodegradation

(Shigeaki Harayama et al.).

Bioremediation methods are so applied to these contaminated sites because this method of

removal of hydrocarbons is cost-effective and give the complete degradation of the Oil

contaminant and site is mineralized. Bioremediation functions basically on biodegradation,

which may refer to complete mineralization of organic contaminants into carbon dioxide, water,

inorganic compounds, and cell protein or transformation of complex organic contaminants to

other simpler organic compounds by biological agents like microorganisms. Bioremediation,

which employs the biodegradative potentials of organisms or their attributes, is an effective

technology that can be used to accomplish both effective detoxification and volume reduction. It

is useful in the recovery of sites contaminated with oil and hazardous wastes. Besides,

bioremediation technology is believed to be non-invasive and relatively cost effective. In some

cases it may not require more than the addition of some degradation enhancers to the polluted

system. It could end up being the most reliable and probably least expensive option for

exploitation in solving some chemical pollution problems. No single microbial species has the

enzymatic ability to metabolize more than two or three classes of compounds typically found in

crude oil. A consortium composed of many different bacterial species is thus required to degrade

crude oil significantly. The use of a bacterial consortium provides certain advantages over

biostimulation in cases where pollutant toxicity or a lack of appropriate microorganisms (both

11

quantity and quality) is important. Determination of the potential success of application of

bacterial consortium requires an understanding of the survival and activity of the added

microorganism(s) or their genetic materials, and the general environmental conditions that

control the degradation rates such as the peculiarity of the contaminated site, for example, water

or soil systems. These factors may very well vary from place to place and from organism to

organism. It is a common stance that many farmers in the oil exploration areas in developing

countries are experiencing tremendous difficulties in restoring the fertility of pollution

devastated farmlands due to lack of knowledge on appropriate remediation procedures. This

problem could be attended to if adequate attention is given to the need for baseline data for the

evaluation of the application of bioremediation technology in the peculiar localities, using

indigenous isolates of microorganisms. The non-chalant attitude to the problem of oil pollution is

particularly of serious concern for food safety in such neglected areas as the Niger delta regions

of Nigeria as persistence of the pollution could result in the release of toxic pollutants into the

food chain and water products (Anthony I Okoh).

It is known that the main microorganisms consuming petroleum hydrocarbons are bacteria and

fungi. However, the filamentous fungi possess some attributes that enable them as good potential

agents of degradation, once those microorganisms ramifies quickly on the substratum, digesting

it through the secretion of extracellular enzymes. Besides, the fungi are capable to grow under

environmental conditions of stress, for example: environment with low pH values or poor in

nutrients and with low water activity. Several authors have made lists containing bacteria and

fungi genera that are able to degrade a wide spectrum of pollutants, proceeding from marine

atmosphere as well as the soil. In accordance with several scientific publications, can be pointed

out that, amongst the filamentous fungi Trichoderma and Mortierella spp are the most common

ones isolated from the soil. Aspergillus and Penicillium spp have frequently been isolated from

marine and terrestrial environments. In this way, microbiology of hydrocarbons degradation

constitutes a field of research under development, once microbiological procedures may be used

in the decontamination processes (Judith Liliana Solórzano Lemos et al.).

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

industries. In addition bioremediation technology is believed to be non-invasive and relatively

12

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 (Nilanjana Das et al.).

Therefore, the objective of the present work was to identify microorganisms capable to degrade

petroleum hydrocarbons with views to a future employment in the bioremediation of polluted

soils (Judith Liliana Solórzano Lemos et al.).

13

CHAPTER: 2 HISTORY OF WORK

One of the major environmental problems today is hydrocarbon contamination resulting from the

activities related to the petrochemical industry. Accidental releases of petroleum products are of

particular concern in the environment. Hydrocarbon components have been known to belong to

the family of carcinogens and neurotoxic organic pollutants. Currently accepted disposal

methods of incineration or burial insecure landfills can become prohibitively expensive when

amounts of contaminants are large (Nilanjana Das et al.).

Petrochemical industries and petroleum refineries generate large amounts of priority pollutants.

The major pollutants found in these industries are petroleum hydrocarbons, specifically aliphatic

hydrocarbons, arising from storage of crude oil, spills, wash downs and vessel clean-outs from

processing operation. These processes are typically associated with numerous operational

problems, which include: poor settleability of the sludge due to low F/M (food to

microorganism) ratio; production of extra-cellular polymers consisting of lipids, proteins and

carbohydrates that adversely affect sludge settling; biological inhibition due to toxic compounds,

which necessitates very long sludge retention time; long period of acclimation or start-up and

production of large amount of biological sludge (Anal Chavan et al.).

The potentiality of the microorganisms, as agents of degradation of several compounds, indicates

biological treatments as the most promising alternative to reduce the environmental impact

caused by oil spills (Judith Liliana Solórzano Lemos et al.).

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. 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. 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 (Nilanjana

Das et al.).

14

The chemically and biologically induced changes in the composition of polluting petroleum

hydrocarbon mixture are known collectively as weathering. Microbial degradation plays a major

role in the weathering process. Biodegradation of petroleum in natural ecosystems is complex.

The evolution of the hydrocarbon mixture depends on the nature of the oil, on the nature of the

microbial community, and on a variety of environmental factors which influence microbial

activities (Ronald M Atlas).

The biodegradation denotes complete microbial mineralization of complex materials into simple

inorganic constituents such as carbon dioxide, water and materials as well as cell biomass. In

aquatic and terrestrial environments, the biodegradation of crude oil and other petroleum

complexes predominantly revolves around the action of bacterial and fungal populations.

Bioremediation refers to site restoration through the removal of organic contaminants by

microorganisms. It is a process that exploits the natural metabolic versatility of microorganisms

to degrade environmental contaminants. At present, bioremediation revolves around either

stimulating indigenous microbial population by environmental modification or introducing

exogenous microbial population that are known degraders to a contaminated site, a process also

known as seeding. Bioremediation potentially offers a number of advantages such as destruction

of contaminants, lower treatment costs, and greater safety and less environmental disturbance.

Bioremediation is not the universal remedy for organic contamination. Growth and survival of

microorganisms is affected by environmental factors like temperature, compostion of the

contaminant, soil type and nutrient and water availability. These factors affect the application of

bioremediation as a process of clean up. Similarly, petroleum hydrocarbons greatly vary in their

susceptibility to metabolic breakdown by bacteria. This can limit the scope and effectiveness of

bioremediation (Abu Bakar Salleh et al.).

15

CHAPTER: 3 IMPLEMENTATION OF PROJECT

3.1 LITERATURE SURVEY

Human activities constitute one of the major means of introduction of heavy metals into the

environment. One of the major development challenges facing this decade is how to achieve a

cost effective and environmentally sound strategies to deal with the global waste crisis facing

both the developed and developing countries (Soetan et al.).

The biodegradation of oil pollutants is not a new concept as it has been intensively studied in

controlled conditions and in open field experiments, but it has acquired a new significance as an

increasingly effective and potentially inexpensive cleanup technology. Its potential contribution

as a countermeasure biotechnology for decontamination of oil polluted systems could be

enormous (Anthony I Okoh).

Bioremediation, which employs the biodegradative potentials of organisms or their attributes, is

an effective technology that can be used to accomplish both effective detoxification and volume

reduction. It is useful in the recovery of sites contaminated with oil and hazardous wastes.

Besides, bioremediation technology is believed to be non-invasive and relatively cost effective.

In some cases it may not require more than the addition of some degradation enhancers to the

polluted system. It could end up being the most reliable and probably least expensive option for

exploitation in solving some chemical pollution problems. Petroleum hydrocarbon especially in

the form of crude oil has been a veritable source of economic growth to society from the point of

view of its energy and industrial importance. These realizations, which have become more

pronounced in the last decade, have resulted in extensive exploration for more oil reserves. The

resultant effects of these exploratory activities have been the extensive pollution of the

environment. Bioremediation, which exploits the biodegradative abilities of live organisms and

their attributes have proven to be the preferred alternative in the long-term restoration of

petroleum hydrocarbon polluted systems, with the added advantage of cost efficiency and

environmental friendliness. Although extensive investigations have been carried out regarding

hydrocarbon biodegradation, these studies have been exhaustive, not exhausted. Nevertheless,

the effectiveness of this technology has only rarely been convincingly demonstrated, and in the

case of commercial bioremediation products, the literature is virtually completely lacking in

supportive evidence of success. Most existing studies have concentrated on evaluating the factors

16

affecting oil bioremediation or testing favored products and methods through laboratory studies.

Only limited numbers of pilot-scale and field trials, which may provide the most convincing

demonstrations of this technology, have been reported in the peer-reviewed literature. The scope

of current understanding of oil bioremediation is also limited because the emphasis of most of

these field studies and reviews has been on the evaluation of bioremediation technology for

dealing with large-scale oil spills on marine shorelines. Some shortcomings are evident in

petroleum hydrocarbons degradation studies. The identification of active strains is not always

ascertained to a sufficient degree, and misidentifications or incomplete identifications are

sometimes reported. Molecular techniques for the identification of hydrocarbon-degrading

bacteria have been only rarely used in environmental studies, and the biodegradation activities

are not always confirmed by chemical analyses of the degraded Hydrocarbon. Much need still

exist for the optimization of the process conditions for more efficient application of biological

degradation of oil pollutants under different climatic conditions and other diverse environmental

milieu (Anthony I Okoh).

It is usually difficult to get isolates with degradative abilities for all the components of

petroleum. Total degradation of oil component often results from the activities of consortium

consisting of mixture of organisms with degradative potentials for the diverse fractions of which

the oil is composed. Individual organisms are able to metabolize a limited range of hydrocarbon

substrates. Most of the bacteria frequently isolated from hydrocarbon-polluted sites belong to the

genera Pseudomonas, Sphingomonas, Acinetobacter, Alcaligenes, Micrococcus, Bacillus,

Flavobacterium, Arthrobacter, Alcanivorax Mycobacterium, Rhodococcus and Actinobacter[9]

.

The low solubility and high hydrophobicity of many hydrocarbon compounds make them highly

unavailable to microorganisms. Release of biosurfactants is one of the strategies used by

microorganisms to influence the uptake of PAHs and hydrophobic compounds in general. Many

hydrocarbon utilizing bacteria and fungi possess emulsifying activities, due to whole cell or to

extracellular surface active compounds. Microorganisms synthesise a wide variety of high and

low molecular mass bio-emulsifiers (Oluwafemi S et al.).

Hydrocarbons in the 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. Bacteria are the most active agents in

petroleum degradation, and they work as primary degraders of spilled oil in environment.

17

Several bacteria are even known to feed exclusively on hydrocarbons. Acinetobacter sp. Was

found to be capable of utilizing n-alkanes of chain length C10–C40 as a sole source of carbon.

Bacterial genera, namely, Gordonia, Brevibacterium, Aeromicrobium, Dietzia, Burkholderia,

and Mycobacterium isolated from petroleum contaminated soil proved to be the potential

organisms for hydrocarbon degradation. Fungal genera, namely, Amorphoteca, Neosartorya,

Talaromyces, and Graphium and yeast genera, namely, Candida, Yarrowia, and Pichia were

isolated from petroleum contaminated soil and proved to be the potential organisms for

hydrocarbon degradation (Nilanjana Das et al.).

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, delaying the onset of biodegradation. Temperature also affects the solubility of

hydrocarbons. Although hydrocarbon biodegradation can occur over a wide range of

temperatures, the rate of biodegradation generally decreases with the decreasing temperature.

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 (Nilanjana Das et al.).

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

under aerobic conditions. Figure 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.

18

Fig. 3.1 Indicates the main principle of aerobic degradation of hydrocarbon by microorganisms

(Nilanjana Das et al.).

Microbiological cultures, enzyme additives, or nutrient additives that significantly increase the

rate of biodegradation to mitigate the effects of the discharge were defied as bioremediation

agents by U.S.EPA. Bioremediation agents are classified as bioaugmentation agents and

biostimulation agents based on the two main approaches to oil spill bioremediation. Numerous

bioremediation products have been proposed and promoted by their vendors, especially during

early 1990s, when bioremediation was popularized as “the ultimate solution” to oil spills.

Compared to microbial products, very few nutrient additives have been developed and marketed

specifically as commercial bioremediation agents for oil spill cleanup. It is probably because

common fertilizers are inexpensive, readily available, and has been shown effective if used

properly. However, due to the limitations of common fertilizers several organic nutrient

products, such as oleophilic nutrient products, have recently been evaluated and marketed as

bioremediation agents (Nilanjana Das et al.).

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.

19

Numerous scientific review articles have covered various factors that influence the rate of oil

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

appropriate metabolic capabilities (Nilanjana Das et al.).

Cleaning up of petroleum hydrocarbons in the subsurface environment is a real world problem.

A better understanding of the mechanism of biodegradation has a high ecological significance

that depends on the indigenous microorganisms to transform or mineralize the organic

contaminants. Microbial degradation process aids the elimination of spilled oil from the

environment after critical removal of large amounts of the oil by various physical and chemical

methods. This is possible because microorganisms have enzyme systems to degrade and utilize

different hydrocarbons as a source of carbon and energy. The use of genetically modified (GM)

bacteria represents a research frontier with broad implications. The potential benefits of using

genetically modified bacteria are significant. But the need for GM bacteria may be questionable

for many cases, considering that indigenous species often perform adequately but we do not tap

the full potential of wild species due to our limited understanding of various phytoremediation

mechanisms, including the regulation of enzyme systems that degrade pollutants. Therefore,

based on the present review, it may be concluded that microbial degradation can be considered as

a key component in the cleanup strategy for petroleum hydrocarbon remediation (Nilanjana Das

et al.).

20

3.2 IMPLEMETATION OF WORK

3.2.1 WORK FLOW:

(1) Collection of the soil sample from food making industries in Saurashtra region, hotels

and restaurants.

(2) Isolation of microorganism by preparing Bushnell and Hass agar plate:

Composition of media in gm per lit.:

(1) MgSo4 - 0.2

(2) CaCl2 -0.02

(3) KH2Po4 -1.0

(4) K2HPO4 -1.0

(5) NH4NO3 – 1.0

(6) FeCl3 -0.05

(7) Agar-Agar-20.0

(8) PH-7.0 at 25°C

(9) Assume 20% degradation capacity so oil – 50 gm

NOTE: Here we have used peanut oil as a nutrient for the microbes for isolation

(3) Phase -1

(1) Dilution of the sample from 10-1

to 10-10

and isolation by spread plate method.

(2) Incubation period of 1 week.

(4) Phase -2

(1) Enrichment of the microbes, obtained in Phase – 1 by four flame strick plate

method.

NOTE: In this Phase we have not added peanut oil in the media preparation.

We have added peanut oil after 3 days incubation period. Hence this

phase is Control Phase.

(5) Phase – 3

(1) Growth of the 3rd

generation of the oil degrading microbes was obtained.

(6) Phase - 4, 5, 6

(1) Growth of 4th

, 5th and 6th

generations were obtained respectively.

21

In the NEXT LEVEL of the project we followed these methodologies:

• After obtaining the strain of bacteria on the plates using Bushnell and Hass agar

composition, we decided to degrade oil at different level of oil concentration by preparing

culture.

• So those at first we prepared an inoculum of bacterial strain and transferred 3 loop full

colonies into it.

• At regular interval we measured the optical density of the inoculum at 540nm to measure

the growth of the strain we inoculated.

• It was carried out for 4 days of incubation period at 37°C and 100 rpm.

• We prepared two flasks of inoculum and after 4 days of incubation we select the flask on

the basis of the growth rate of strain by measuring optical densities at 540nm for both the

flask.

In the next stage we prepared cultures using peanut oil at DIFFERENT VALUES OF

CONCENTRATIONS.

• The concentrations of oil were 10ml, 20ml and 40ml in 200 ml of media.

• We used Bushnell and Hass medium for culturing.

• We transferred the 20 ml of inoculum having optical density value 1.02 after 4 days of

incubation period to these flasks.

• We provide 7 days of incubation time at 37°C and 100 rpm and also measured optical

density at 540nm for each flask for 7 days.

• By measuring optical density the growth rate of microbes decided and oil degradation

was observed.

After completion of peanut oil degradation set we took the sample of hydrocarbon oil i.e. engine

oil.

• For this sample we took concentrations of 10ml, 20ml and 40ml in 100ml media.

22

• Here media composition used is Bushnell and Hass.

• To these cultures we transferred 20ml inoculum having an optical density value 1.48.

• 6 days of incubation time was provided at 37°C and 100 rpm and took the optical density

at 540nm for each sample for 6 days.

• We obtain the growth of strain in cultures and oil degradation was observed.

In third stage we took sample of another hydrocarbon oil generally use as break oil in

automobiles.

• In this stage of experiment we took two concentrations of oil, 10 ml and 20ml in 100ml

of BH media.

• To these we transferred 20ml of inoculum having optical density 1.87.

• At present we are measuring growth rate by measuring optical density at 540nm and this

sample is in incubation.

For CALCULATION OF PERCENTAGE OIL DEGRADATION

• We provided 1 month incubation time for the oil degradation.

• We used solvent extraction method. Toluene used as solvent.

• We took toluene as the equal amount of the culture in the separation funnel.

• We provided 24hr incubation time for vaporization of Toluene.

• After that we measured the quantity of oil remained in the extract.

23

CHAPTER: 4 RESULT ANALYSIS

4.1 ISOLATION RESULTS

Fig. 4.1 (A)

Colonies of the bacteria obtained in BH media by striking method. Here oil is used as a carbon

source.

24

Fig. 4.1(B)

Colonies of the bacteria obtained in BH media by striking method. Here oil is used as a carbon

source.

25

Table 4.1: Colonical Characteristics

Sr.No.

Colonical

Characteristic

Colony

Phase-1

Colony

Phase -2

Colony

Phase-3

Colony

Phase-4

Colony

Phase-5

Colony

Phase-6

1 Size Large Small

Small,

Medium Small Small Small

2 Shape

Round,

Oval

Round,

Oval Round

Small

Round

with

Spores

Small

Round

Small

Round

3 Elevation Raised Raised

Raised,

Flat Raised

Raised,

Flat

Raised,

Flat

4 Surface Texture Rough Rough Rough

Waxy,

Rough

Waxy,

Rough

Waxy,

Rough

5 Margin Broad Broad

Broad to

Medium Small Small Small

6 Growth Pattern Colony Colony Colony Colony Colony Colony

7 Opacity Opaque Opaque Opaque Opaque Opaque Opaque

8 Pigmentation

Yellow &

White

Yellow &

White

Yellow &

White

Creamish

White

Creamish

White

Creamish

White

26

4.2 PREPARATION OF CULTRURE

Fig. 4.2(A) Peanut Oil set

Here we prepared a culture medium using BH media and N-broth. We took different quantities

of oil i.e. 10, 20, 40ml in each flask. We incubate culture in the environment incubator at 37° C

and 100 rpm.

27

4.2 (B) Growth of organism at the periphery

We obtained the growth of the microbes at the periphery

28

4.2(C) Engine Oil set



and 100 rpm. The significant results were obtained in all the flasks. We were able to get visible

growth of the microbes.

29

4.2(D) Engine oil degraded

Here the colour of the engine oil was changed and it was degraded in clumps.

30

4.2(E) Engine oil degraded set




growth of the microbes. In the picture it is shown that the oil in the first two flaks has been

degraded significantly.

31

4.2(F) Break oil degradation set




growth of the microbes. In the picture it is shown that the oil in the flaks has been degraded

significantly.

32

4.3 ANALYSIS OF DEGRADATION BY MEASURING THE GROWTH

(1) Peanut oil

Table 4.3.1

O.D.(10ml) O.D.(20ml) O.D.(40ml) Time(Day)

0.54 0.43 0.63 1

0.65 0.48 0.63 2

0.68 0.53 0.66 3

0.68 0.77 0.68 4

0.73 0.77 0.7 5

0.44 0.69 0.72 6

0.56 0.69 0.7 7

Fig. 4.3.1

From the graph it is concluded that in the 7 days of incubation time, the growth of the microbes

increase with the incubation time and the maximum average growth is observed on 5th

day of

incubation.

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

0 2 4 6 8 10

O.D

(540n

m)

Time(Days)

O.D. V/S Time

O.D.(10ml)

O.D.(20ml)

O.D.(40ml)

33

(2) Engine oil

Table 4.3.2

O.D.(10ml) O.D.(20ml) O.D(40ml) Time (Day)

0.52 0.75 0.7 1

0.65 0.86 0.83 2

0.65 0.89 1.03 3

0.68 0.89 1.04 4

0.69 0.89 1.04 5

0.71 0.9 0.91 6

Fig. 4.3.2



day of

incubation. At the 5th

day we got the constant growth.

0

0.2

0.4

0.6

0.8

1

1.2

0 1 2 3 4 5 6 7

O.D

.(540n

m)

Time (days)

O.D V/S Time

O.D.(10ml)

O.D.(20ml)

O.D(40ml)

34

(3) Break oil

Table 4.3.3

O.D.(10ml) O.D.(20ml) Time(days)

0.81 0.63 1

0.86 0.65 2

0.93 0.69 3

0.97 0.69 4

1.13 0.71 5

Fig. 4.3.3



day of

incubation.

0

0.2

0.4

0.6

0.8

1

1.2

0 2 4 6

O.D

.(540n

m)

Time(Days)

O.D. V/S Time

O.D.(10ml)

O.D.(20ml)

Time(days)

35

4.4 CALCULATION OF PERCENTAGE OIL DEGRADATION

Table 4.4.1 Peanut oil

Sr. No. Oil added(ml) Oil remained after extraction(ml) Percentage(%) oil degradation

1 10 2 80

2 20 5 75

3 40 15 62.5

Table 4.4.2 Engine oil

Sr. No. Oil added(ml) Oil remained after extraction(ml) Percentage(%) oil degradation

1 10 0 100

2 20 5 75

3 40 7.28 81.8

Table 4.4.3 Break oil

Sr. No. Oil added(ml) Oil remained after extraction (ml) Percentage(%) oil degradation

1 10 6.5 65

2 20 13 65

36

CHAPTER: 5 CONCLUSION

Our aim of the study was to isolate the microbes having potential of oil degradation. We used

three types oil as a sample to check the potential of isolate obtained during study. Peanut oil is

the major food source of Saurashtra region. As, Rajkot city has well developed automobile

industrial zone, so we took engine oil as our second source of oil for degradation. Same way our

third oil sample was break oil from auto garages. We used concentration of above mentioned oil

samples in a proportion of 10%, 20% and 40% of 100 ml of total BH medium. Inoculum was

added 20% of total medium. We obtained significant result in degradation of engine oil, i.e.

100% degradation was observed in that case within 12 days of incubation for 10 ml. similarly for

20 ml of engine oil 75% oil degradation observed within 12 days of incubation period. For 40 ml

81.8% degradation was obtained within the same incubation time period. The least degradation

was obtained in peanut oil in the range of 62.5% for 40 ml, break oil 65% for 20 ml and 10 ml

respectively for incubation time of 12 days. We would be able to isolate the potential strain of

an organism for their oil degradation capacity, which is highest for engine oil and moderate for

peanut oil and break oil. By considering the environmental issues bioremediation is the most

potential process for cleaning up the environment. As the isolated strain obtained during this

study showed significant potency for oil degradation further study required to be carried out in

terms of characterization, identification and further explored for cellular level activity.

37

REFERENCES

[1] Abu Bakar Salleh, Farinazleen Mohamad Ghazali, Raja Noor ZalihaAbd Rahman and

Mahiran Basri, Bioremediation of Petroleum Hydrocarbon pollution, Enzyme and

Microbial Technology Research, Faculty of Science and Environmental Studies,

Universiti Putra Malaysia

[2] Anal Chavan, Suparna Mukherji, Treatment of hydrocarbon-rich wastewater using oil

degrading bacteria and phototrophic microorganisms in rotating biological contactor:

Effect of N:P ratio, Centre for Environmental Science and Engineering (CESE), Indian

Institute of Technology (Bombay), Powai, Mumbai 400076, India

[3] Anthony I Okoh , “Biodegradation alternative in the cleanup of petroleum hydrocarbon

pollutants”.

[4] Debajit Borah and R.N.S. Yadav Centre for Studies in Biotechnology, Dibrugarh

University, Dibrugarh-786004, India, UV Treatment Increases Hydrocarbon Degrading

Potential of Bacillus spp. Isolated from Automobile Engines.

[5] Judith Liliana Solórzano Lemos, Andrea C. Rizzo, Valéria S. Millioli , Adriana Ururahy

Soriano, Maria Inez de Moura Sarquis

& Ronaldo Santos .

Centro de Tecnologia

Mineral - CETEM, Rio de Janeiro;

Centro de Pesquisas e Desenvolvimento Leopoldo

Américo M. de Mello - CENPES, Rio de Janeiro;

Instituto Oswaldo Cruz, Fundação

Oswaldo Cruz - FIOCRUZ, Rio de Janeiro , “Petrolium degradation by filamentous

fungi”.

[6] Nilanjana Das and Preethy Chandran, Microbial Degradation of Petroleum Hydrocarbon

Contaminants, Environmental Biotechnology Division, School of Biosciences and

Technology, VIT University, Vellore, Tamil Nadu 632014, India

[7] Nilanjana Das and Preethy Chandran , “Microbial degradation of petroleum hydrocarbon

contaminants: An Overview, SAGE-Hindawi Access to Research Biotechnology

Research International, Volume 2011, Artical ID 941810, 13 pages ,

doi:10.4061/2011/941810.

[8] Oluwafemi S., Obayori Æ Matthew O., Ilori Æ Sunday A., Adebusoye Æ Ganiyu O.,

Oyetibo Æn Ayodele E., Omotayo Æ Olukayode O. Amund, Degradation of

hydrocarbons and biosurfactant productionnby Pseudomonas sp. strain LP1.

[9] Ronald M Atlas, Microbial degradation of Petroleum Hydrocarbons: An Environmental

38

Perspective, Department of biology, Univesity Of Loisville, Loisville, Kentucky.

[10] Sayyed Hossein Mirdamadian, Giti Emtiazi, Mohammad H. Golabi and Hossein

Ghanavati, Biodegradation of Petroleum and Aromatic Hydrocarbons by Bacteria

Isolated from Petroleum-Contaminated Soil.

[11] Shigeaki Harayama, Hideo Kishira, Yuki Kasai and Kazuaki Shutsubo, “Petroleum

biodegradation in marine environments” Marine Biotechnology Institute 3-75-1 Heita,

Kamaishi, Iwate 026-0001, Japan. (JMMB symposium).

[12] Soetan, K.O., The role of biotechnology towards attainment of a sustainable and safe

global agriculture and environment – A review, Department of Veterinary Physiology,

Biochemistry and Pharmacology, University of Ibadan, Nigeria.

biodegradation of oil contaminated site

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