BIOAUGMENTATION OF Pseudomonas aeruginosa

AND Penicillium funiculoscum IN OIL

CONTAMINATED SOIL

Brion, Keano Miguel S.

Cabrillos, Nickcy Dulia Rose S.
Domiguez, Joaquin Marcelo C.

CHAPTER 1
INTRODUCTION
Background of the Study
The use of oils and fuels has been a necessity in our lives. These substances are used to
power industrial plants, automobiles, and other machines that we use to produce the things that
we need. However, despite its usefulness it strikes a major environmental impact especially when
these substances are mishandled or involved in accidents. Over the course of history, oil has
been one of the most common contaminant in the environment such as in the ground water,
marine environment and soil. Sources of oil contamination include leakages from oil tanks and
pipelines as well as from automobile accidents and pipe blowouts (Helmy et al., 2015).
Soil is prone to oil contamination because it has a direct contact with industrial sites, roads,
and gas stations. Oil contamination have adverse effects on the soil which includes increase in
toxicity levels and negative effects on the physicochemical properties of the soil. Apart from these,
oil contamination may also decrease the rate of germination of plants as well as decrease or
hinder the nutrients in the soil (Shayler, McBride, Harrison, 2009).
The oil contaminant in soil is usually referred to or measured as Total Petroleum
Hydrocarbon (TPH). TPH includes both aliphatic and aromatic hydrocarbons. The aliphatic
hydrocarbons includes both short and long chain hydrocarbons while aromatic hydrocarbons
include singles ring hydrocarbons and Polycyclic Aromatic Hydrocarbons (PAHs). Among these
types of hydrocarbons, long chain hydrocarbons containing 24 to 40 carbons are one of the most
difficult hydrocarbons to degrade or remediate. Branched alkanes, cycloalkanes as well as
aromatics are also difficult to remediate (Agency for Toxic Substances and Disease Registry
Atlanta, n.d.).
Several measures and technologies have already been explored or researched on to be
able to treat or mitigate oil contamination. One of the methods used to mitigate the spreading of
oil contaminants is by containment. This technology includes several methods such as
solidification, capping, micro containment by cold-mix asphalt, or land disposal or land filling.
Even though it can prevent the spreading of oil within the soil its major disadvantage is that it
doesn t treat nor degrade the contaminants (Friend, 1996).

Aside from the containment technology, several treatment technologies are also used in
remediating oil contaminated soil. These includes physical, chemical, thermal, and bioremediation
treatments. Physical treatment involves soil venting, flushing, and washing which helps prevent
contamination of adjacent sites. However, one of the disadvantage of physical treatment is that it
requires a lot of equipment which entails higher operating costs. On other hand, chemical
treatments involve the use of redox reactions within the oil as well as extracting the oil using
solvents. These methods can treat a wide range of petroleum hydrocarbons but the use of more
chemicals within the soil may increase the toxicity level. Thermal treatments include radio
frequency heating, vitrification, and thermal desorption. One of the major advantages of these
methods is that it can treat a wide range of TPH. However, these methods have high capital cost
and they are also energy extensive which entails a higher operating cost (Friend, 1996).
One of the promising techniques to remediate contaminated soil is bioremediation.
Bioremediation utilizes bioprocesses as well as organisms in order to degrade the oil
contaminants. It also includes bioventing, biopiling, as well as land farming. Moreover,
bioremediation doesn t use harmful substances which ensures that the soil properties will be
maintained. Bioremediation is also economical since the microorganisms needed for the
degradation of oil can be easily acquired and is also considered effective since specific
microorganisms can strive and survive in an environment where the only source of food is carbon
compounds or organic compounds (Friend, 1996).
Bioremediation may be categorized into two types namely, Ex

Situ and In

Situ. In

Situ refers to onsite treatment where the soil to be treated need not be excavated from the site to
be treated. This method also requires the necessary equipment for the bioremediation to be in
site. On the other hand, ex-situ refers to offsite treatment where the contaminated soil is removed
from its original location and is treated on site and off site. Usually these kinds of method involve
the use of bioreactors, where the soil with oil is mixed with microorganisms in a reactor where
necessary nutrients are fed to the reactor as well as proper aeration is provided. Apart from this,
the use of bioreactors would make varying the parameters easier thus, it would be easier to
optimize the parameters of the reactor or the parameters of the bioremediation process.
(EUGRIS: portal for soil and water management in Europe, n.d.)
Bioremediation may also involve two major techniques namely, biostimulation and
bioaugmentation. Biostimulation involves the introduction of substances which enhances the

performance of the microorganisms. This method may include addition of nutrients to supplement
the needs of the microorganism or addition of surfactants which enhances the availability of oil in
the oil for degradation. The other technique is bioaugmentation where microorganisms such as
bacteria and fungi are added to the system to facilitate faster degradation by helping the natural
bacteria or microorganism in the soil to digest the food or oil (Maletic, Dalmacija, Roncevic, 2013).
Objectives
The general objective of this study is to determine the possibility of improving
degradation of petroleum hydrocarbons by bioaugmentation of Pseudomonas aeruginosa and

Penicillium funiculoscum.
The specific objectives are:
1. To monitor and determine the Total Petroleum Hydrocarbon (TPH) content of the soil for
one month by analyzing the soil in a Gas Chromatograph equipped with Flame Ionization
Detector.
2. To determine the effect of each organism in the system on the rate of degradation of TPH
by plotting the concentration of TPH with respect to time.
To determine if the mixture effects of both Pseudomonas aeruginosa and Penicillium

funiculoscum is significant in the degradation of oil in soil by using statistical methods specifically
full factorial analysis.
Significance of Study
While studies regarding mixing bacteria and fungal species are limited, the general results
of the present studies dealing with individual bacter and fungu show the effectivity of the
bioaugmentation of both species. Pseudomonas aeruginosa, for example, is known to release
biosurfactants, which enhances the bioavailability of the contaminants. The more available
contaminants can then be degraded by Penicillium funiculoscum which releases enzymes which
may be able to breakdown heavy and complex hidrocarbons. Despite the complementary nature
of both species, however, there are still several limitations for this bioaugmentation. The
petroleum hydrocarbon contaminant, for example, is not generally available for biodegradation,
unless acted upon by the bacteria.

Scope and Limitations

The experiment will only be limited to the use of Pseudomonas aeruginosa, Penicillium

funiculoscum, and the natural consortium present in the soil. Moreover, the experiment will be

limited to a simulated oil contaminated soil, such that petroleum will be added to the soil to be
able to simulate the oil contamination. Furthermore, the results for the TPH concentration will be
acquired from the Gas Chromatograph data and these results will be measured and monitored
throughout the 1 month experiment period.

Chapter 2
Review of Related Literature
Soil Contamination
Soil contamination is a major environmental issue that needs tackling. Contamination occurs
when any toxic or hazardous substance mixes with naturally occurring soil, either by spills or
leakage and direct burial of the contaminant in soil. Over the course of history, oil has been one
of the most common contaminants of soil. Sources of oil contamination include leakages from oil
tanks and pipelines as well as from automobile accidents and pipe blowouts (Helmy et al., 2015).
Oil is composed of several types of hydrocarbons referred to as Total Petroleum Hydrocarbons
(TPH). These TPHs can be classified as Gasoline Range Organics (GRO), which includes
alkanes with small chain, or Diesel Range Organics (DRO), which is composed of alkanes with
longer chains (Ezeonu et al., 2012). These oils may contaminate the soil physically or chemically.
Usually, these contaminants make contact with soil by spillage or leakage underground pipelines
or storage tanks. Once they make contact with soil, the contaminants will start to spread.
However, the degree of contamination or rate the rate of spreading of these contaminants
depends on several factors such as the soil type. Oil tends to spread faster on lighter soil and
travel slower in heavier soil (Helmy et al., 2015). Other factors which also affect the degree of
contamination include soil texture, acidity of soil, moisture levels, and temperature (Shayler et al.,
2009). Due to their low solubility, they tend to exist for a long time in affected areas (Capello,
2012). These contaminants can be transported and distributed to other areas by different physical,
chemical and biological processes and these may enhance the effects of oil contamination
(Ezeonu et al., 2012). Also, the degree of contamination may vary depending on the length of
the hydrocarbon chains present in the oil. The study of Malik and Ahmed (2012) showed that the
time required for the petroleum hydrocarbons to degrade depends on their structure. Alkanes with
single aromatic rings or with lengths less than C11 require 4 days to degrade. Those with lengths
between C12 and C18 require 8 to 16 days while those chains greater than C21 and those
containing multiple aromatic rings require at least 16 days to degrade.
The presence of oil in the soil may cause several environmental impacts. Since the oil may leach
through the soil, it may end up in the aquifers and contaminate the underlying groundwater. This
presents a threat to the health of populations living within the area of the contaminated site, as
petroleum hydrocarbons are typically carcinogenic, mutagenic and genotoxic (Kim, Lee, 2007).

Furthermore, since oil is made up of some volatile organic compounds some of the contaminants
may volatilize which allows them to escape from the soil into the air. Air-borne petroleum
hydrocarbons are odorous due to the presence of sulphur, which can cause illness at high
concentrations (Sider, 1994; Taylor et al., 1997). Lastly, the oil may bind tightly to the soil by
adsorption which may affect both physical and chemical properties of the soil (Shayler et al.,
2009). One adverse effect of the oil contaminant is the promotion of an anaerobic environment
within the soil, which is caused by blocked air diffusion in the soil pores, thus affecting soil fertility
(Wang et al., 2013).
Bioremediation
Microorganisms have a unique role in the detoxification of polluted soil environments. Organic
matter, from compounds naturally occurring in soils as well as those formed as by-product by
plants harnessing the energy of sunlight, are constantly recycled by various microorganisms such
as fungi and bacteria (Bollag et al., 1994). Bioremediation is the use of these biological systems
to detoxify or remediate the contaminants present in the medium. For soils, this is named soil
bioremediation (Bollag et al., 1994). Soil bioremediation has been proven to be an effective and
economic method to remediate contaminated soils. Several studies have resulted in the increase
in feasibility of reducing contaminant concentration and ecotoxicity in contaminated soils (Adetutu
et al, 2013; Gogoi et al., 2003; Sarkar et al., 2005).
There are three categories for bioremediation activities: intrinsic in situ, engineered in situ and
engineered ex situ. In situ refers to instances in which contaminants remain in place, while ex situ
refers to instances in which contaminants or parts of it are removed. Engineered in situ is a special
case of in situ instances, where the degree of human intervention is high (Kosaric, Sukan,
2015).Engineered bioremediation techniques can be processes that involve either biostimulation,
bioaugmentation or both.
Intrinsic in situ bioremediation refers to instances where in situ remediation is applied to systems
with no human intervention (Kosaric, Sukan, 2015). Though the process occurs naturally, it differs
with natural attenuation, since intrinsic in situ bioremediation focuses more on the microbial
transformation and biological reaction, while natural attenuation is the combination of the
biological, physical and chemical reactions in the overall remediation process. (Rittman &
McCarty, 2001)

Engineered in situ combines bioremediation techniques and engineering concepts to design

systems for large-scale biological processes. The combination of the two allows for the
maintenance of the overall degradation of the target contaminant (Cookson, 1995). An important
stage in engineered in situ bioremediation involves the technique selection for contaminant
isolation and control; thus, these techniques can be classified as to how stimulatory materials are
added to the system. The three frequently used methods for engineered in situ bioremediation
are bioventing, biosparging and liquid circulating systems (Kosaric, Sukan, 2015).
Engineered ex situ bioremediation involves the excavation of soil and treating it above ground
through aeration processes to increase the degradation of contaminants. Under aerobic
conditions, various organic contaminants can be treated and degraded to carbon dioxide and
water.

Engineered ex situ bioremediation techniques are processes that

stimulate

microorganisms to grow and to use the contaminants as a source of energy. Generally, there are
two types of techniques: solid phase and slurry phase (Kosaric, Sukan, 2015).

1.

Engineered In Situ Bioremediation Techniques

Bioventing systems inject atmospheric air in unsaturated soil, which provides a continuous source
of air streaming into the soil, which enhances the growth of microorganisms in the soil. Aside from
air, other gases influencing the growth of microorganisms can also be injected, such as nitrogen
and phosphorus. This technique is most applicable for soil systems where the water depth
exceeds 3 meters and superficial soil layers do not require treatment (Kosaric, Sukan, 2015).
Biosparging, also known as air sparging, is a useful mean for aerobic biodegradation. Unlike
bioventing, biosparging injects pressurized gas into a saturated soil zone for transfer of volatile
components to the unsaturated zone where biodegradation can easily occur. Similar to bioventing
though, biosparging also increases the concentration of oxygen in the soil, which enhances the
rate of biodegradation of contaminants by microorganisms. Biosparging is favorable for
remediation of systems contaminated with less volatile substances such as waste oils (Miiler,
1996). The injection of gases, however, needs an additional gas-recovery system to prevent offsite transport of the gaseous contaminants (Rittmann & McCarty, 2001).
Liquid systems circulating systems are for contaminants in saturated soil zones wherein a well
and underground air delivery system is designed to circulate air and other gases through the

contaminated zone to maximize degradation. Groundwater is also extracted and treated above
the ground, if necessary. Overall, the circulation system is designed to isolate the contaminated
zone hydraulically and to minimize contaminant transport out of the system (Kosaric, Sukan,
2015).
2.

Engineered Ex Situ Bioremediation Techniques

Solid phase techniques involve the placing of excavated contaminated soil into aboveground
enclosures. Treatment beds are constructed with a built-in aeration system as well as designed
such that parameters, like moisture and pH levels, are controlled and maintained by operators.
Solid phase techniques are useful for fuel hydrocarbon contaminants. There are three solid phase
techniques: land farming, biopile and composting (Kosaric, Sukan, 2015).
Land farming is a full-scale bioremediation technique where excavated contaminated soil are
placed in lined beds and periodically aerated. Nutrients, moisture and bulking agents are also
added to obtain optimal conditions for microbial growth. Lined beds are usually 50 centimeters
thick (Kosaric, Sukan, 2015).
Biopile is a composting process where composts are formed into piles on top of perforated
landings where blowers or vacuum pumps can aerate the piles. Biopiles are usually enclosed to
improve the control of moisture, pH levels, heat and oxygen to enhance the biodegradation
process. Biopiles are usually constructed to 6 meters in height, with 2 to 3 meters as the optimum
height (Kosaric, Sukan, 2015).
Composting is a biological process where organic contaminants are converted to stable byproducts by microorganisms. Excavated contaminated soils are mixed with bulking agents and
organic material to enhance the mixture porosity for composting. Aeration is also done to improve
degradation efficiency of the process (Kosaric, Sukan, 2015).
Slurry phase techniques involve the placing of excavated contaminated soil in a bioreactor for
treatment. The slurry is made by mixing the contaminated soil with water, nutrients and other
additives. The slurry is constantly mixed to maintain the contact of suspended solids and
microorganisms with the contaminants. Bioreactors are advantageous since operators have full

control on the parameters of the process; therefore, the process can be kept as close to the
optimal conditions as possible (Kosaric, Sukan, 2015).
Also, bioremediation can be done through biostimulation and bioaugmentation to enhance and
increase the rate of biodegradation (Nwadinigwe and Onyeidu, 2011).

Biostimulation

Biostimulation is a bioremediation technique which aims to enhance the activity of indigenous

microorganisms to improve treatment of contaminated sites. This technique offers faster
treatment and it is effective for the bioremediation of groundwater, wastewater and solid, oily
sludge (Suja et al., 2014). It is also the modification of environmental conditions to increase
bioactivity (Zawierucha and Malina, 2011). Biostimulation can be done by applying nutrients in
the affected area. In the study of Suja et al. (2014), inorganic nitrogen, phosphorus and potassium
were added at a CNPK ratio of 100:10:1:3. Also, biostimulation can be done by aeration. Aeration
ensures that enough amount of oxygen is present. It also allows more contact between the
hydrocarbons and the microorganisms thus, increasing bioavailability and biological activity.
Another way of increasing microbial activity is the addition of surfactants. Surfactants are used to
increase mobility, bioavailability and biodegradation by reducing surface and interfacial tensions
of the fluids and solids (Rahman et al., 2015). In the study of Rahman et al., rhamnolipid from
Pseudomonas aeruginosa was used as a biosurfactant for the biodegradation of oil.

Bioaugmentation

Bioaugmentation is a process of adding microorganisms into a contaminated site (Nwadinigwe

and Onyeidu, 2011). It can be done by introducing bacteria or fungi to increase the microbial
population and to improve the degradation of oil. Most of the time, local microbial strains are used
because it can easily adapt to the environment to facilitate biodegradation (Suja et al., 2014).
Bioaugmentation is done because indigenous species present in the contaminated area may
result to limited bioactivities since some of these microorganisms may not be capable of degrading
hydrocarbons and the population of the hydrocarbon-degrading bacteria may not be enough (Liu
et al, 2014). In the study of Okafor et al. (2009), fungal isolates of Aspergillus versicolor,
Aspergillus niger, Aspergillus flavus, Syncephlastrum spp., Trichoderma spp., Neurospora
sitophila, Rhizopus arrhizus and Mucor spp. showed biodegradation activities. Also, studies

conducted by Al-Jawhari (2014) showed that the Aspergillus niger can thrive in contaminated
areas because it has a high resistance to pollution caused by crude oil. The ability of these
microorganisms to degrade oil is also affected by other organisms present in the site. In the study
of Suja et al. (2014), the consortium containing a combination of Acitenobacter sp. and
Pseudomonas sp. gave the highest degradation as compared to the consortium containing
another species of Pseudomonas and Acitenobacter, and another consortium containing two
species of Rodococcus.
Table 1 shows several studies on bioremediation of soils contaminated with petroleum
hydrocarbons. Table 1 summarizes the microorganisms used in the studies, the durations of the
experiments, the parameters changed during the experiments and the results of the remediation
of contaminated soil.

Physicochemical Environmental Characteristics

Bioremediation processes are successful only if preparations are made. Site characterization, or
the definition of the biological system to be studied, should be done to get the necessary data
needed to design the equipment needed for the bioremediation process. Several parameters and
characteristics are needed to be assessed in order to determine the potential success and
feasibility of the process.

1.

pH

pH is one of the major environmental factors which influences the bioremediation of oil
contaminants. Several factors affected by pH, such as bioavailability of contaminants, the
availability of other nutrients, and microbial activity, constitutes the dependence of the
effectiveness of a bioremediation process with pH. Based on studies, most of the hydrocarbondegrading microorganism thrives in neutral pH where they are most optimal. These
microorganisms are referred to as neutrophiles and usually optimal at pH range of 6.5 to 7.5.
However some microorganisms are well adapted on either acidic or alkaline environment and
these microorganisms are referred as acidophiles and alkaliphiles respectively. Acidophiles is
sufficient for optimal growth at pH below 5.5 while alkaliphiles are optimal at pH above 8.5 (Ajoku
& Oduola, 2013).

2.

Temperature

The temperature of the soil is another factor influencing the bioremediation of oil contaminants.
Naturally, microorganisms does not survive in extreme temperatures also, at these temperatures
the effect of enzymes are reduced. Moreover, enzymatic activity generally decreases with
decreasing temperature which results to a lowered rate of degradation of organic contaminants
(Ezeonu et al., 2012). However, some of the microorganisms may still thrive on either low or high
temperatures and be able to degrade hydrocarbons (Rike et al., 2008). In soils, most of the
microorganisms present are mesophilic bacteria which thrive and optimally degrade hydrocarbons
at moderate temperature environment which ranges from 30 to 40 C (Hesnawi & Mogadami,
2013).
Aside from the rate of degradation of contaminants, temperature may also affect both viscosity
and volatility of oil contaminants. Decreasing the temperature makes the oil more viscous which
is much harder for microorganisms to degrade (Ezeonu et al., 2012; Chen et al., 2010). Moreover,
decreasing the temperature also decreases the volatility of the contaminant thus the reduction of
hydrocarbons by evaporation or volatilization will also decreases (Enzeonu et al., 2012). These
finding shows that bioremediation is more optimal at warmer environment.
3.

Enzyme and Surfactants

The presence of enzymes in the contaminated area aids in the detoxification of the soil by
increasing the rate of degradation of the contaminants. However, environmental conditions such
as heat, pH, salt, strong oxidizing or reducing agents, organic chemicals and surfactants may
affect the activity of these enzymes (Ezeonu et. al, 2012). Moreover, surfactants increase the
degradation of oil by separating the oil from the soil such that it can be readily available to
microorganisms for degradation. On the other hand, surfactants to be used should not be toxic to
the native microorganisms present (Ezeonu et. al, 2012).
4.

Soil Type

All types of soils may undergo bioremediation however some considerations should be observed
in some specific type of soil. Such considerations include the use of additives or special
equipment. Clay soil is usually introduced with bulking agents to enhance the oxygen transport

within the soil. Sandy soil have generally low soil water holding capacity , low organic content and
low surface area available for microbial growth thus bioremediation in this type of soils is
comparably slower as compared to the other types. In order to improve the bioremediation in
sandy soils, organic matter may be added (Ezeonu et al., 2012).

5.

Moisture

Studies show that the rate of degradation increases as the amount of moisture in the soil
increases. However, this is only true for a certain range of moisture content (Shelton and Parkin,,
1991). Scholz et al. also reported that the degradation of contaminants decreases when the
moisture content of the soil exceeds 50%. Too much moisture in the soil may limit the amount of
oxygen available for the degradation of the toxins.
6.

Oxygen Content

Most of the microorganisms degrading soil contaminants are aerobic thus they must obtain
oxygen from the soil as well as the water present in the soil; however, in most cases, oxygen is
supplied into the system through pumps (Cho et al., 2000). Therefore, bioremediation is generally
effective in soil environment with sufficient amount of oxygen content. The oxygen content in the
soil during bioremediation may be maintained through aeration. However, the contaminants may
also be degraded under anaerobic conditions but at a slower rate (Cho et al., 2000).
7.

Nutrients

Addition of nutrients in oil contaminated soil affects its availability to bacteria, which affects its
ability to consume contaminants. Nitrogen and phosphorus, for example, increases the
proliferation of bacteria which also increases biodegradation rates of contaminants. The major
sources of nutrients are fertilizers. Addition of fertilizers is usually gradual to avoid unnecessary
increases in pH levels as well as high nitrogen or phosphorus contents that can kill
microorganisms. (Prince & McMillen, 2002). Soil structure is also improved, thereby stimulating
microbial growth (Sublette, 2001).

Chapter 3
Methodology
A. Preparation of Inoculum
A.1 Agar and Broth Prepaation
Potato Dextrose Agar (PDA) will be prepared to culture fungi, Pseudomonas Isolation Agar
will be used to culture Pseudomonas aeruginosa and nutrient broth will be used to prepare
the inoculum which will be introduced to the reactor. The PDA, Pseudomonas Isolation Agar
and nutrient broth were prepared in three separate 250mL Erlenmeyer flasks by dissolving
the required amount of agar powder following the ratio specified by the manufacturer. The
solutions will be heated and stirred until the solutions boil. The solutions will be sterilized at
121C for 15 minutes and the agar will be transferred into petri dishes while the nutrient broth
will be placed in test tubes.

A.2 Preparing the Culture

Samples of Penicillium funiculoscum and Pseudomonas aeruginosa will be cultured in
PDA and Pseudomonas Isolation Agar, respectively. Cultures will be stored in an incubator
for 48 hours. Colonies of the bacteria and fungi and will be inoculated in separate test tubes
containing nutrient broth and these solutions will be homogenized. In another test tube, 10mL
of the fungi in broth and bacteria in broth will be mixed and homogenized using a rotary shaker
(120 rpm).
B. Reactor Design
4800 g of soil will be prepared. 2400 g will be sterilized for 20 minutes using an autoclave
at a temperature of 220C and a pressure of 15 psi. The sterilized soil will be divided into 12
containers and the unsterilized soil will be divided into the remaining 12 containers. 2g of
diesel, 250mg/kg soil of (NH4)2SO4 and 100 mg/kg soil of KH2PO4 will be added into 21 setups and the remaining 3 set-ups will serve as the control. 10 mL pure inoculation of bacteria
and fungi and 10 mL of the consortium containing bacteria and fungi will be added into the
set-ups specified in table 1.

Table 1. Set-up specifications.

Trial

The bottles will be covered with rubber stoppers and tubes will be attached on each bottle
to allow air to flow from the aerator to the bottles. Figure 1 shows the set-up for the reactors.

Figure 1. Experimental set-up.

C. Total Petroleum Hydrocarbon (TPH) Determination

The TPH determination will be done every 5 days for 30 days. One to two grams of soil
will be obtained from the set-up and it will be analysed using the methods described by Kaczorek
et al. (2014). TPH from the soil will be extracted by adding n-pentane into the soil sample at 1:4
ratio of soil to n-pentane. The soil will be filtered and the extract will be analysed for TPH. The

TPH analysis will be done using a gas chromatograph with flame ionization detector (GC-FID).
The capillary column which will be used will have 50% cyanopropylmethyl, 50% phenylmethyl
polisiloxane DB-225 (Agilent Technologies) 30m x 0.25mm I.D. with a film thickness of 0.25m.
The gas carrier will be Helium at 1.5mL/min and 90 kPa. The injector and detector will have a
temperature of 300C. The injection volume will be 5L. The temperature of the column will be
maintained at 60C for one minute and it will be increased at 10C/min until it reaches 220C.
The temperature will be maintained at 220C for 10.5 minutes.

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Biotechnol. Lett., Volume 20, Pages 749-751
27. Shayler H., McBride M., Harrison E. (2009).
Cornell Waste Management Institute, Department of Crop & Soil Science, Pages
1-6

28. Shelton D., Parkin T. (1991).

carbofuran in soil. J. Agric. Food Che., Volume 39, Pages 2063-2068

Fundamentals of Bioremediation of Hydrocarbon Contaminated

29.

Soils
Texas
30. Suja, F., Rahim, F., Taha, M.R., Hambali, N., Razali, M.R., Khalid, A., Hamzah, A.
(2012)

bioaugmentation and biostimulation on the

bioremediation of total petroleum hydrocarbons (TPH) in crude oil contaminated soil

International Biodeterioration &
Biodegradation
31. Wang Y., Feng J., Lin Q., Lyu X., Wang X., Wang G. (2013).
Chinese Geographical Science, Volume 23, Pages 708

715

Microorganisms

Trichoderma sp.

Gordonia
alkanivorans CC-JG39,
Rhodococcus erythropolisCCBC11,Acinetobacter junii CCFH2,
Serratia marcescens KH1, and
ExiguobacteriumaurantiacumCCLSH-4, Pseudomonas
aeruginosa CC-RS1; fungal sp.
(Candida guilliermondii
and
Candida parapsilosis)

Pseudomonas Putida F1

Duration

270 days

280days

Specific
Component
of oil?

Results/Conclusion

PAHs

Biostimulation caused a decrease of PAHs throughout

the first
two months of treatment whilst the natural attenuation
did not.
Soil inoculation with Trichoderma sp. Evx1 also
triggered a more
efficient degradation of the C12-40 fraction in
comparison to the
results obtained in

soils with
different soil
organic
matter
content

bacterial community shift significantly associated with

the TPH degradation stages; (2) when SOM levels
were close to each other, their degradation
performance and bacterial communities were similar
to each
other, and (3) dynamics of bacterial communities
could influence the TPH degradability

BenzeneToluenePhenol
mixture

Toluene significantly inhibite biodegradation rate of

other substrates
Benzene slowed down consumption of phenol, but not
toluene
Phenol had little effect in biodegradation of Benzene
and toluene

Parameters

15-30%
moisture
content

Sum kinetics with interaction paramaters model was

the best model

Acinetobacter sp. and

Pseudomonas sp.

up to 90% degredation after 45 days using

biostimulation witn N and P

45 days

Comparison between bacteria in river water, sterilized

culture media and in field
20 mg/L NB:
14 hours - river water
20 hours - culture media
- no difference between bacterial growth in river and
culture: nutrients in river water was not the growth
limiting factor for cell growth
- direct inoculation in river water for large-scale
remediation of NB-contaminated river water

Pseudomonas putida

Bioaugmentation:
10 days in sediment and 8 days in water

native microorganisms

8 weeks

diesel oil

biosolids-amended soils and the high-rate

fertilizer-treated soils degraded more than 96% of the
original TPH contamination whereas the control soils
(MNA) (monitored natural attenuation) degraded 94%
of the original TPH contamination.

30 C
Temperature
Fertilizer as
nutrient
source

Pseudomonas aeruginosa AS03,

P. aeruginosa N108
P. aeruginosa N002
Achromobacter xyloxidans N78,

native microorganisms were

identified by extracting nucleic
acids; growth of the isolates was
also measured at the
temperatures 5, 20,
30, 40, 50 and 60 C.

28 Weeks

80% degredation using combined treatment

of bacterial consortia supplemented with nutrients

88 days

-gram negative and rod in shape strains were isolated.

most from Gammaproteobacteria (Pseudomonas,
Halomonas, Marinobacter, Hahella and
Alcanivorax);bioaugmentation of contaminated
sediments with a mixture of these strains can
accelerate the
biodegradation processes, however biostimulation
through the
addition of inorganic nutrients still seems to be a more
efficient
approach;CO2 method also fails to measure the
carbon fraction
that could be assimilated in biomass. It has been
demonstrated that
isolates of bacteria and yeasts assimilate as much as
50% of the
supplied carbon

silt loam
texture

pH = 8.9

30 C
Temperature

Recycler 102 and RemActiv

(culture concentrates)

addition of surfactant showed highest degradation of

TPH while Daramend (commercial organic
amendment for biostimmulation) showed highest
degradation of soil with TPH and PAH. however, PAH
degradation is insignificant compared to the control;
reccommendations: Inoculation of soil with indigenous
microbes directly isolated and cultured from the same
soil has generally delivered better results [49] and
should be the focus of future bioaugmentation
attempts, since the strains are already adapted to their
environment

195 days

15 bacteria isolated by
enrichment technique from the
sample
collected from an oil
contaminated site.

24 days

removal of aliphatic and aromatics was 94.64% and

93.75% respectively; the biotic removal of alkanes
was
maximum, 90.96% for tridecane (C13) followed by
pentadecane (C15) at 77.95%, octadecane (C18) at
74.1%,
while other alkanes showed 56 to 69% after 24 days
of incubation

Pseudomonas genera and

Enterobacteriaceae family
(natives)

21 days

95% to 97% (Lab scale)

In terms of 5 to 6 ring PAH: 14 - 39%

24 days

pH: 7.45 to 7.25

TOC: 3.32% to 3%
TN: 34.17ppm to 6.05 ppm
Sulfate: 21.52 to 18 ppm
Phosphate: 2.29 to 0.75 ppm
Moisture content: 38.88% to 58.73%

P. aeruginosa and S.
marcescens - gasoline,
kerosene, diesel oil, lubricating
oil

diesel

40-60%
moisture
content
Nitrogen and
Phosphorus
as nutrients
Neutral pH

37 C
Temperature

39-59%
moisture
content

Pseudomonas, Acinetobacter,
Rhodococcus--> 3 combinations
of these sp.

70 days

TPH lang

degradation of TPHs in the top soil was highest in

columns combining
bioaugmentation and nutrient addition, whereas in the
bottom soil, the degradation of TPHs was highest
in columns combining bioaugmentation with the
addition of both nutrients and Oxygen releasing
compounds; highest degradation for set-up with
species of Rhodococcus

Lactobacillus pentosus(releases
biosurfactants) and native

45 days

Octane

surfactants acclerates biorem

Waste
engine oil

This study has shown that the soils with previous

contact with hydrocarbons can have enhanced
hydrocarbon-degrading capacity because of the
presence
of hydrocarbonoclastic bacterial and fungal species

COM001 which is a
hydrocarbonoclastic fungus
(Australian provisional patent no.
2009900493)

3 months

25-80%
moisture
content

Highest total petroleum hydrocarbon (TPH)

degradation with the combined treatment
First 2 weeks: all treatments - bacterial count
increased then stabilized

Nocardia sp. H17-1 for BA and

CT in Luria-Bertani medium

First 4 weeks: addition of Noccardia sp. H17-1,

biosurfactant and nutrients caused rapid reduction in
hydrocarbon content

120 days

16 weeks (TPH reduction):

NA - 8378 - 1608 mg/kg
BS - 7452 - 1469 mg/kg
BE - 6978 - 2563 mg/kg
BA - 10111 - 1362 mg/kg
CT - 8948 - 2489 mg/kg
CT had highest TPH degradation rate

Rahnella sp. strain EK12

isolated form soil contaminated
with crude oil

21 daysbioremediation
alone; 6
months for
preparation of
bacterial strain

consortium was prepared from

six
potent hydrocarbon utilizing
bacterial (HUB) isolates obtained
after screening sixty nine (69)
isolates.

75 days

Diesel oil

50%
maximum
moisture
content
0.1% KNO3
and
K2HPO4,
and MEL
supplement
as nutrients
sandy loam
pH = 7.1

70% degradation by Rahnella in the presence of

saponins after 21 days; 50% degradation was
observed in the presence of rhamnolipids; change in
hydrophobicity is less than 10% in the presence of the
surfactants

83.70% degradation was recorded

37 C
Temperature

16-20%
moisture
content
Aeromonas hydrophila,
Alcaligenes xylosoxidans,
Gordonia sp., Pseudomonas
fluorescens, Pseudomonas
putida, Rhodococcus equi,
Stenotrophomonas maltophilia,
Xanthomonas
sp.

365 days
(field) lab
scale as not
indicated

bioaugmentation success
biosurfactant no significant contri

N (4.34
g/kg), P
(0.07 g/kg),
K (1.56 g/kg)
as nutrients
20 C
Temperature
pH = 7.48 7.64

Pseudomonas Putida with an

inducible toluene dioxygenase
enzyme
P. Putida UV 4 strain

Complete removal of TCE from assay mixtures at a

concentration of 10uM. Higher concentrations, slower
TCE removal rate
Bioconversion and cytotoxicity of TCE are mediated
by toluene dioxygenase
TCE removal kinetics linar for P. stutzeri IBB2