1 BIODEGRADATION OF OIL CONTAMINATED SITE A PROJECT THESIS Submitted by JARIWALA JENIL (090470104003) JOSHI RIDDHI (090470104011) In fulfillment for the award of the degree of BACHELOR OF ENGINEERING in BIOTECHNOLOGY V.V.P. ENGINEERING COLLEGE, RAJKOT Gujarat Technological University Ahmadabad May, 2013
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BIODEGRADATION OF OIL CONTAMINATED SITE
A PROJECT THESIS
Submitted by
JARIWALA JENIL (090470104003)
JOSHI RIDDHI (090470104011)
In fulfillment for the award of the degree
of
BACHELOR OF ENGINEERING in
BIOTECHNOLOGY
V.V.P. ENGINEERING COLLEGE, RAJKOT
Gujarat Technological University
Ahmadabad
May, 2013
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
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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
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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)
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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.
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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
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LIST OF FIGURES
Figure
No
Figure Description Page
No
3.1 The main principle of aerobic degradation of hydrocarbon by
microorganisms
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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
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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
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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
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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
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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
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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.).
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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.).
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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.).
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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
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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.
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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.