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: 20th
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(540nm)
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.(540nm)
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.(540nm)
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
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