BATANGAS STATE UNIVERSITY

COLLEGE OF ENGINEERING, ARCHITECTURE, and

FINE ARTS

Mechanical and Petroleum Engineering Department

Microbial Degradation of Petroleum Hydrocarbon Contaminants

A Research Project

Presented to Dr.

Sicily Tiu

In Partial Fulfillment of the Requirement

for Research Project in Modern Biology

BUENVIAJE, BRIAN LOUIS L.

DATINGUINOO, NYIELS D.

LOGDAT, IVY MITCHELLE L.

PLATA, SHIELA MAE C.

PULON, RHOSE ANNE O.

TAYMEN, LIPY L.

March 2018
 Chapter I

The problem and its background

This chapter includes introduction, statement of the problem, scope and

delimitation, and significance of the study.

Introduction

Hydrocarbon contamination is one of the major environmental problems today

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

particular concern in the environment. Furthermore, release of hydrocarbons into the

environment whether accidentally or due to human activities is a main cause of water and

soil pollution .Soil contamination with hydrocarbons causes extensive damage of local

system since accumulation of pollutants in animals and plant tissue may cause death or

mutations. The technology commonly used for the soil remediation includes mechanical,

burying, evaporation, dispersion, and washing. However, these technologies are

expensive and can lead to incomplete decomposition of contaminants.

The process of bioremediation, defined as the use of microorganisms to detoxify or

remove pollutants 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. 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. In addition, bioremediation technology is believed to be noninvasive

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

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

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

technologies.

There are the two main approaches to oil spill bioremediation: (a) bio-

augmentation, in which known oil-degrading bacteria are added to supplement the

existing microbial population, and (b) bio-stimulation, in which the growth of indigenous

oil degraders is stimulated by the addition of nutrients or other growth-limiting co-

substrates.

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.

The accomplishment of bioremediation endeavors in the cleanup of the oil tanker Exxon

Valdez oil slick of 1989 in Prince William Sound and the Gulf of Alaska made colossal

enthusiasm for the capability of biodegradation and bioremediation innovation. Most

existing studies have concentrated on evaluating the factors affecting oil bioremediation

or testing favored products and methods through laboratory studies. Only limited

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

of this technology which 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 given on the evaluation of bioremediation

technology for dealing with large-scale oil spills on marine shorelines.

This research is done to provide updated information on microbial degradation of

petroleum hydrocarbon contaminants towards the better understanding in bioremediation

challenges and to gain knowledge about factors influencing Petroleum Hydrocarbon

degradation and its effects.

Statement of the problem

The purpose of this phenomenological study is to examine the naturally occurring

ability of microbial biodegradation activity to degrade, transform or accumulate

environmental pollutants more specifically, petroleum hydrocarbons. Thus, it aims to

answer the following questions:

1. How is petroleum hydrocarbon considered as a pollutant of the environment?

2. What are the factors influencing Petroleum Hydrocarbon degradation?

3. What are the effects of Microbial Degradation?

3.1 Positive Impacts

3.2 Negative Impacts

4. What is the advantage of using this microbial degradation method instead of the

others?

5. How does Microbial Degradation aid in conserving biodiversity?

Null Hypothesis

Microbial degradation of Petroleum Contaminants does not affect environmental

factors.

Significance of the Study

The importance of this study is to decrease the environmental problems brought

by hydrocarbon that produced from the petrochemical industry by the used of microbial

degradation.

This study is significant to the following:

Petroleum engineering students. In the future, these students could use this

study as their reference and to whatever necessary purpose to innovate or develop further

a solution to address the problems of Petroleum Industry. This will help the students to

have an idea on how to decrease environmental pollution brought by petrochemical

industries.

Petrochemical company. The direct recipients of the output of this research are

these companies since they are responsible for any pollution related activities brought by

petroleum exploration. Through this research they could gather ideas and develop

instruments that will help lessen the problem of environmental pollution due to oil and

petroleum chemicals.
 Community. They are the main reason why this study is being conducted as they

are the one being affected by the environmental pollution brought by hydrocarbon

chemical. The community is responsible for its environment and it’s important that they

know about this phenomenon and through this research they could be knowledgeable of

the possible solution to the environmental pollution.

Workers. This study will help them be aware and informant about the risk that

could cause them some health issues while they are working in their certain professions

specially those who are inclined to the petroleum industry. Through this research they

could avoid and be precautious of the environmental hazard brought by oil spill.

Future Researchers, The future researchers will benefit due to the source of

preliminary knowledge and studies for their own research. This research about the effects

of multivitamin pills may assist and guide them with their own research.

Scope and Limitation

This study is focused as to understand the Microbial Degradation of Petroleum

Hydrocarbons by which one can clean up the petroleum hydrocarbon pollutants from the

environment. This research involves a number of limiting factors that have been

recognized to affect the biodegradation of petroleum hydrocarbons. This study also

examines the positive and negative effects of microbial degradation and the advantage of

using its method. The study also covers a better understanding of the mechanism of

biodegradation that has a high ecological significance that can be considered as a key

component in the cleanup strategy for petroleum hydrocarbon remediation.

The study limits its coverage on the use of microorganisms to detoxify or remove

pollutants owing to their diverse metabolic capabilities in an evolving method for the

removal and degradation of many environmental pollutants particularly on the products

of petroleum industry.

Conceptual Framework

This section presents the paradigm which was illustrated by frames to show the

relationship of the input, process and output and has the concepts involved in the study.
The input refers to the technique to be use in biodegrading hydrocarbons such as,

use of microbial organisms that metabolize petroleum and degrading the hydrocarbon

content, use of bacteria as primary degraders of spilled oil in environment, attachment of

microbial cells to the substrates and production of biosurfactants. The input components

are important in the assembly and result of the output.

The second frame contains the process used to achieve the particular end product;

the researchers used library research, data gathering, analysis, and laboratory research in

order to process the input into results that are the main answer to this study.

The output is the end product of the research which is transform or mineralize the

organic contaminants, change waste into harmless or less toxic compounds, cleans up the

petroleum hydrocarbon pollutants from the environment, and elimination of spilled oil
 from the environment after critical removal of large amounts of the oil by various

physical and chemical methods

Figure 1. Paradigm of the Conceptual Framework

INPUT PROCESS OUTPUT

Transform or mineralize the

Use of microbial  Library Research organic contaminants
organisms that metabolize  Data Gathering Change waste into
petroleum and degrading  Analysis harmless or less toxic
the hydrocarbon content  Laboratory Experiment compounds
Use of bacteria as primary Cleans up the petroleum
degraders of spilled oil in hydrocarbon pollutants from
environment •Library Research the environment
Attachment of microbial •Data Gathering Elimination of spilled oil
cells to the substrates •Analysis and Observation from the environment after
Production of critical removal of large
biosurfactants amounts of the oil by
various physical and
chemical methods
Definition of Terms

Microbial biodegradation - is the use of bioremediation and biotransformation methods

to harness the naturally occurring ability of microbial xenobiotic metabolism to degrade,

transform or accumulate environmental pollutants, including hydrocarbons (e.g. oil),

polychlorinated biphenyls (PCBs), polyaromatic hydrocarbons (PAHs), heterocyclic

compounds (such as pyridine or quinoline), pharmaceutical substances, radionuclides and

metals.
Hydrocarbons - a compound of hydrogen and carbon, such as any of those which are the

chief components of petroleum and natural gas.

Bioremediation - the use of either naturally occurring or deliberately introduced

microorganisms or other forms of life to consume and break down environmental

pollutants, in order to clean up a polluted site.

Bio augmentation – A technique of bioremediation in which strains of natural or

genetically engineered bacteria with unique metabolic profiles are used to treat sewage or

contaminated water or soil.

Biostimulation – It involves the modification of the environment to stimulate existing

bacteria capable of bioremediation. This can be done by addition of various forms of rate

limiting nutrients and electron acceptors, such as phosphorus, nitrogen, oxygen, or

carbon.

CHAPTER II
 This chapter includes review of related literature, conceptual literature and related

studies (foreign studies and local studies).

Related Literature

Environmental pollution with petroleum and petrochemical products has been

recognized as a significant and serious problem (Alexander, 1995, 2000). Most

components of oil are toxic to humans and wildlife in general, as it is easy to incorporate

into the food chain. This fact has increased scientific interest in examining the

distribution, fate and behavior of oil and its derivatives in the environment (Alexander,

1995, 2000; Semple et al., 2001, 2003; Stroud et al., 2007, 2009). Oil spills in the

environment cause long-term damage to aquatic and soil ecosystems, human health and

natural resources.

Bioremediation can be briefly defined as the use of biological agents, such as

bacteria, fungi, or green plants (phytoremediation), to remove or neutralize hazardous

substances in polluted soil or water. Bacteria and fungi generally work by breaking down

contaminants such as petroleum into less harmful substances. Plants can be used to aerate

polluted soil and stimulate microbial action. They can also absorb contaminants such as

salts and metals into their tissues, which are then harvested and disposed of.

Bioremediation is a complex process, with biological degradation taking place in the cells

of microorganisms which absorb pollutants, where if they have specific enzymes, the

degradation of pollutants and their corresponding metabolites will take place.

Hydrocarbons from oil are used as a source of nutrients and energy for microorganism

growth, and at the same time, microorganisms decompose them to naphthenic acids,
alcohols, phenols, hydro peroxides, carbonyl compounds, esters, and eventually to carbon

dioxide and water (Eglinnton, 1975; Marković et al., 1996).

Bioremediation is considered a non-destructive, cost- and treatment-effective and

sometimes logistically favorable cleanup technology, which attempts to accelerate the

naturally occurring biodegradation of contaminants through the optimization of limiting

conditions. Bioremediation is an option that offers the possibility to destroy or render

harmless various contaminants using natural biological activity. As such, it uses relatively

low-cost, low-technology techniques, which generally have a high public acceptance and

can often be carried out on site (Alexander, 1995). It will not always be suitable,

however, as the range of contaminants on which it is effective is limited, the time scales

involved are relatively long, and the residual contaminant levels achievable may not

always be appropriate (Maletić et al., 2009; Rončević et al., 2005).

Bioremediation can be briefly defined as the use of biological agents, such as

bacteria, fungi, or green plants (phytoremediation), to remove or neutralize hazardous

substances in polluted soil or water. Bacteria and fungi generally work by breaking down

contaminants such as petroleum into less harmful substances. Plants can be used to aerate

polluted soil and stimulate microbial action. They can also absorb contaminants such as

salts and metals into their tissues, which are then harvested and disposed of.

Bioremediation is a complex process, with biological degradation taking place in the cells

of microorganisms which absorb pollutants, where if they have specific enzymes, the

degradation of pollutants and their corresponding metabolites will take place.

Hydrocarbons from oil are used as a source of nutrients and energy for microorganism

growth, and at the same time, microorganisms decompose them to naphthenic acids,
alcohols, phenols, hydro peroxides, carbonyl compounds, esters, and eventually to carbon

dioxide and water (Eglinnton, 1975; Marković et al., 1996).

Bioremediation can be divided into two basic types: (1) natural attenuation, which

can be applied when the natural conditions are suitable for the performance of

bioremediation without human intervention, and (2) engineered bioremediation, which is

used when is necessary to add substances that stimulate microorganisms. The first one is

more attractive because of its low cost, minimum of maintenance and minimal

environmental impact. Still, this technology is applicable only in cases when the natural

level of biodegradation is higher than the degree of pollution migration. Nevertheless,

this technology is more often used as a supplement to the other technologies, or after

finished engineered bioremediation in order to prevent migration of pollution from the

treated area. Engineered bioremediation is faster than natural attenuation because it

includes microbial degradation stimulation, by controlling the concentrations of

pollution, oxygen, nutrients, moisture, pH, temperature, etc. (Rahman et al.,

2003; Yerushalmi et al., 2003). Engineered bioremediation is applied when it is essential

to carry out cleaning in a short time or when the pollution is very rapidly expanding. Its

application reduces the costs due to the shorter treatment of land and lower number of

sampling and analysis, and it is important for political and psychological needs when the

community is exposed to pollution.

Bioremediation technology utilizes microorganisms to degrade toxic pollutants to

harmless products such as CO2, H2O, and other inorganic compounds and these

processes are environmentally safe and cost efficient (Philip et al., 2005). It has been

reported that roughly 25% of all petroleum-contaminated land is being bio remediated
using natural attenuation processes thus underscoring the importance of microorganisms

in remediation strategies (Holden et al., 2002). However, application of microbial

technologies for treating contaminated high salinity or fluctuating salinity environment is

limited due to the detrimental effects of salt on microbial life including disruption cell

membrane, denaturation of enzymes, low solubility of oxygen, low solubility of

hydrocarbons, and desiccation (Pernetti and Di Palma, 2005). Therefore, bioremediation

of saline environments without costly dilution of salt-laden soil and water requires

halophilic or halotolerant organisms that tolerate high salt concentrations. Halophiles are

classified into three groups according to their optimal salt concentration for growth

Related Studies

Sunita J. Varjani study is an approach of Microbial degradation of petroleum

hydrocarbons. Petroleum hydrocarbon pollutants are recalcitrant compounds and are

classified as priority pollutants. Cleaning up of these pollutants from environment is a

real world problem. Bioremediation has become a major method employed in restoration

of petroleum hydrocarbon polluted environments that makes use of natural microbial

biodegradation activity. Petroleum hydrocarbons utilizing microorganisms are

ubiquitously distributed in environment. They naturally biodegrade pollutants and

thereby remove them from the environment. Removal of petroleum hydrocarbon

pollutants from environment by applying oleophilic microorganisms (individual

isolate/consortium of microorganisms) is ecofriendly and economic. Microbial

biodegradation of petroleum hydrocarbon pollutants employs the enzyme catalytic

activities of microorganisms to enhance the rate of pollutants degradation.

Furthermore, the enhancement of natural biological degradation processes, in

what is termed bioremediation, can be a preferred cost-effective method of removing

contaminants from soil-contaminated and other contaminated environments. The role of

microorganisms has been shown to be essential in the remediation of, at least, organic

pollution, and the activity of microbial types naturally present can be enhanced by

bioremediation techniques which include increased aeration of the polluted material and

nutrient additions (Christofi et al. 1998; Ivshina et al. 1998). Supplementing the source of

microorganisms capable of degrading particular pollutants (bioaugmentation) and the

enhancement of the desorption of pollutants from particulates using surfactants can

increase hydrocarbon degradation

Biodegradation, is a mineralization of organic chemicals, which ultimately

leading to the formation of CO2, H2O and biomass (Hamdi et al.,2007). Biodegradation

being an economical and eco-friendly approach, has emerged as the most advantageous

soil and water clean-up technique for contaminated sites containing oil spills, is applied

with different strategies, but well accomplished with a process called bio-stimulation, that

is, the addition of several nutrients and fertilizers to a contaminated matrix (Tyagi et al.,

2011).

Microbial treatment of industrial effluents is also possible. Fundamental work has

revealed that a wide variety of microorganisms are capable of degrading an equally wide

range of organic pollutants. Pure and mixed cultures of microorganisms have been
studied and degradation is observed under both aerobic and anaerobic conditions.

Breakdown products have been found during work on the degradative pathways involved

and toxicological assessments using bacteria and higher organisms (fish, plants) have

been used to determine the toxicity of these intermediates. Many of the degradative genes

responsible for xenobiotic metabolism are present on plasmids, transposons or are

grouped in clusters on chromosomes. This provides clues to the evolution of degradative

pathways and makes the task of genetic manipulation easier such that new microbial

strains capable of efficiently degrading pollutants can be developed (Ian Singleton et al.,

2004 ).
 CHAPTER III

Research Design and Process

This chapter presents the research methodologies used in the study. This research

includes the research design and the statistical treatment.

Research Design

Research is a process of searching logically and systematically the new and useful

information on a particular topic. Research is done with the aid of study, experiment,

observation, analysis, comparison and reasoning. (S. Rajasekar, 2013). Ary et al 2006

states that “Experimental research design is to enable researcher to estimate the effect of

an experimental treatment”. The researchers chose to conduct the study using

experimental methods in order to determine the validity of conclusions that can be drawn

from the study. This research employs a qualitative method. This research design is

efficient in this study because it’s more effective in acquiring the desired output.

Qualitative data source for the study includes library research, data gathering, and

analysis and laboratory experiment. Qualitative researchers aim to gather an in-depth

understanding of the microbial degradation of petroleum hydrocarbon contaminants.

An experimental usually involves two groups of subject, an experimental group

and a comparison group, although it is possible to conduct an experiment with one group.

This study uses pre-experimental design in the form of one- group Pretest- Posttest

design using quantitative approach. In Pre-test and Post-test group the observation do two

times, before giving treatment called Pre-test and after giving treatment called Post-test.
 This study is classified as experimental design because it is little or no control of

extraneous variable. In the one-group Pretest-Posttest design, a single group is measured

or observed not only after being exposed to a treatment.

Data Gathering Procedure

The researcher will conduct an experiment wherein they will compare two

samples. Both samples will contain equal amount of petroleum hydrocarbons and to

determine the reaction of microbial degradation, one container will contain

pseudomonads. This certain bacteria is the most effective bacteria capable in utilizing

hydrocarbons as carbon and energy sources and producing biosurfactants which will be

acquired in soil, water, and plants. Through observation the researcher will gather

information and compare the treated sample with the untreated sample.

Statistical Treatment

The data gathered was treated by using statistical tools dealing with the ways on

how the respondents evaluate the overall benefits and disadvantage of using microbial

degradation for petroleum hydrocarbon contaminants.

The one-way analysis of variance (ANOVA) is used to determine whether there

are any statistically significant differences between the means of two or more

independent (unrelated) groups (although you tend to only see it used when there are a

minimum of three, rather than two groups).

Bibliography

Fakhruddin, F. A. (2018, May 24). A Review on Environmental Contamination of.

Retrieved from juniperpublishers.com.

Vijay Kothari, M. P. (2018). Microbial Degradation of Hydrocarbons . Retrieved from

semanticscholar.org:
https://pdfs.semanticscholar.org/5515/3033100ef5dc816f6d8251ba83e44330be9b.pdf

Atlas R., Ronald M., (1991) “Microbial hydrocarbon degradation-bioremediation of oil

spills”. Journal of Chemical Technical Biotechnology.

Eweis J. B., Ergas S.J., Chang D.P.Y., Schroeder E.D. (2000). “Biodegradation of
selected compounds”. In: Bioremediation Principles: ch:. McGraw-Hill international
editions, Singapore.

Singh A., Ward O.P., (Eds.) (2004). “Biodegradation and Bioremediation”, Springer-
Verlag Berlin Heidelberg.

Van der Heul R. M., (2009) “Environmental degradation of petroleum hydrocarbons”.

Utrecht university.(http://studenttheses.library.uu.nl/search.php?
language=en&qry=Environ mental+Degradation+of+petroleum+hydrocarbons)

Beam H. W., and Perry J. J. (1999) “Microbial degradation of cycloparaffinic

hydrocarbons via cometabolism and commensalism”. Journal of General Microbiology,

Van Beilen J. B. and Funhoff E. G., (2005) “Expanding the alkane oxygenase toolbox:
new enzymes and applications”. Current Opinion in Biotechnology).

Zhang Y., and Miller R. M., (1994) “Effect of a Pseudomonas ramnolipid biosurfactant
on cell hydrophobicity and biodegradation of octadecane”. Applied and Environmental
Microbiology.