i

MICROBIAL ENHANCED OIL RECOVERY

ABSTRACT

The processes involving the use of microbes for improving oil recovery efficiency is

assessed from the perspective of reservoir engineering. Microbial enhanced oil recovery

(MEOR) is a method which utilizes a mixed microbial population (preselected

microorganisms or indigenous reservoir microorganisms) and their metabolic products

such as biomass, biosurfatants, biopolymers, gases and aids to increase the displacement

and/or volumetric efficiency of reservoirs. Chemical EOR methods such as polymer

flooding, surfactant flooding, alkaline flooding, etc. are the same as those of MEOR

processes. They are thus subject to the same technical difficulties, an example of which

is the retention and dissipation of chemicals within the reservoir. The major difference

between MEOR and chemical EOR however is the method by which the recovery-

enhancing chemicals are introduced into the reservoir.

An examination of literature reveals a large number of successful MEOR laboratory trials

but with very few field applications. This is as a result of a lack of understanding of the

mechanisms involved in MEOR. This dissertation thus covers a critical review of possible

microbial enhanced oil recovery methods and mechanisms in order to identify the most

feasible utilization of microbial technique to enhance oil recovery. Laboratory

experiments were conducted with the aim to investigate the rate of biodegradation of

dodecane using glass bioreactors over an incubation period of 31days. The results

obtained indicate that an increase in the rate of biodegradation can be achieved, thus

resulting in an increase in the oil recovery efficiency.

In conclusion, MEOR is a ‘’high-risk, high reward” process, depending on whether the

ii

microorganisms can produce oil recovery-enhancing chemicals by utilizing the residual

oil within the reservoir as a carbon source. The high risk in this context refers to the severe

constraints that the microbial system must satisfy in order to utilize an in situ carbon

source. The rewards however is that the logistical cost and difficulty in implementing the

process is similar to those of implementing a waterflood.

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TABLE OF CONTENT

ABSTRACT .................................................................................................................................. i

TABLE OF CONTENT ............................................................................................................. iv

LIST OF TABLES ..................................................................................................................... vi

TABLE OF FIGURES .............................................................................................................. vii

CHAPTER ONE .......................................................................................................................... 1

1.1 INTRODUCTION ......................................................................................................... 1

1.2 AIM OF THE PROJECT .............................................................................................. 2

1.3 JUSTIFICATION .......................................................................................................... 2

1.4 SCOPE OF WORK ....................................................................................................... 2

CHAPTER TWO ........................................................................................................................ 3

2 LITERATURE REVIEW ...................................................................................................... 3

2.1 OVERVIEW OF CRUDE OIL PRODUCTION ........................................................... 3

2.1.1 PRIMARY OIL RECOVERY............................................................................... 3

2.1.2 SECONDARY OIL RECOVERY ........................................................................ 4

2.1.3 TERTIARY/ ENHANCED OIL RECOVERY ..................................................... 5

2.2 EOR BY LITHOLOGY ................................................................................................ 5

2.3 TERTIARY/ ENHANCED OIL RECOVERY METHODS. ........................................ 6

2.3.1 MISCIBLE DISPLACEMENT ............................................................................. 6

2.3.2 THERMAL ........................................................................................................... 7

2.3.3 CHEMICAL .......................................................................................................... 9

2.3.4 MICROBIAL ...................................................................................................... 10

2.4 REVIEW OF MICORBIAL ENHANCED OIL RECOVERY ................................... 11

2.4.1 RELATED WORK DONE ON MEOR .............................................................. 12

2.5 CLASSIFICATION, MECHANISMS AND LIMITATIONS OF MEOR ................. 14

2.5.1 MEOR CLASSIFICATION ................................................................................ 14

2.5.2 MEOR MECHANISMS ...................................................................................... 17

2.5.3 MEOR LIMITATIONS ....................................................................................... 21

2.6 FIELD APPLICATIONS OF MEOR.......................................................................... 25

2.7 ADVANTAGES AND CHALLENGES OF MEOR .................................................. 26

CHAPTER THREE .................................................................................................................. 28

3 MATERIALS AND METHOD .......................................................................................... 28

3.1 CULTURES USED ..................................................................................................... 28

3.2 FLUIDS ....................................................................................................................... 28

3.2.1 HYDROCARBON SOURCE ............................................................................. 28

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3.2.2 DEIONISED WATER ........................................................................................ 29

3.3 GLASS BIOREACTORS ........................................................................................... 29

3.4 EXPERIMENTAL PROCEDURE ............................................................................. 29

3.4.1 MINERAL SOLUTION PREPARATION ......................................................... 29

3.4.2 PREPARATION OF UNCONSOLIDATED SAND .......................................... 30

3.4.3 MICROBIAL GROWTH EXPERIMENTS ....................................................... 30

3.4.4 SAMPLE ANALYSIS ........................................................................................ 30

CHAPTER 4 .............................................................................................................................. 36

4 RESULTS AND DISCUSSION ......................................................................................... 36

4.1 DODECANE DEGRADATION ................................................................................. 36

4.2 TOTAL SUSPENDED SOLIDS PRODUCTION ...................................................... 37

4.3 DISSOLVED OXYGEN ............................................................................................. 38

4.4 BIOFILM THICKNESS ............................................................................................. 39

4.5 MICROBIAL KINETICS. .......................................................................................... 40

4.6 BIOMASS YIELD COEFFICIENT YX/S .................................................................... 41

4.7 PROSPECTS FOR INDIVIDUAL MEOR MECHANISMS ..................................... 42

CHAPTER 5 .............................................................................................................................. 43

5 CONCLUSION AND RECOMMENDATIONS ................................................................ 43

REFERENCES ............................................................................................................................ 44

APPENDICES ........................................................................................................................... 53

v

LIST OF TABLES

Table 2.2.1: Microbial groups and their bioproducts formed ...................................................... 17

Table 2.2.2 : Permeability variations with lithology [64] ............................................................ 23

Table 3.1: Properties of the used fluids ....................................................................................... 28

Table 3.2: The compositions of the mineral solution .................................................................. 29

Table A.1: Day 0 Calibration ...................................................................................................... 54

Table A.2: Day 0 GC sample analysis ........................................................................................ 54

Table A.3: Day 8 Calibration Values .......................................................................................... 55

Table A.4: Day 8 GC Sample Analysis ....................................................................................... 55

Table A.5: Day 15 Calibration Values ........................................................................................ 56

Table A.6: Day 15 GC Sample Analysis ..................................................................................... 56

Table A.7: Day 23 Calibration Values ........................................................................................ 57

Table A.8: Day 23 GC Sample Analysis ..................................................................................... 57

Table A.9: Day 31 Calibration Values ........................................................................................ 58

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TABLE OF FIGURES

Figure 2.1: Waterflooding process ................................................................................................ 5

Figure 2.2: EOR field projects by lithology .................................................................................. 6

Figure 2.3: Viscous fingering ........................................................................................................ 7

Figure 2.4: Steam injection process .............................................................................................. 8

Figure 2.5: Steam assisted gravity drainage. ................................................................................. 8

Figure 2.6: In situ Combustion process ......................................................................................... 9

Figure 2.7: Surfactant flooding ................................................................................................... 10

Figure 2.8: Cyclic microbial oil recovery ................................................................................... 15

Figure 2.9: Microbial flooding recovery ..................................................................................... 16

Figure 2.10: Illustration of Selective plugging ............................................................................ 16

Figure 2.11: Residual oil saturation as a function of capillary number ....................................... 19

Figure 2.12: Range of pressures in various biological systems .................................................. 22

Figure 3.1: Mineral Solution on magnetic stirrer ........................................................................ 30

Figure 3.2:pH measurement ........................................................................................................ 31

Figure 3.3:Dissolved oxygen measurement ................................................................................ 31

Figure 3.4:Vacuum Filtration Process ......................................................................................... 32

Figure 3.5:Total Suspended solids on the filter paper ................................................................. 32

Figure 3.6: Liquid-Liquid extraction using Hexane .................................................................... 33

Figure 3.7:Examples of chromatograms. .................................................................................... 33

Figure 3.8: Example of Calibration curve. .................................................................................. 35

Figure 4.1: Dodecane concentration vs Time .............................................................................. 37

Figure 4.2: TSS concentration vs. Time ...................................................................................... 37

Figure 4.3: Dodecane concentration and TSS vs. Time .............................................................. 38

Figure 4.4: Growth on the hydrocarbon surface. ......................................................................... 39

Figure 4.5: Dissolved oxygen concentration vs. Time ................................................................ 39

Figure 4.6: Comparison between the heights of biofilm formed. ................................................ 40

Figure 4.7: Biofilm height vs. Time ............................................................................................ 40

Figure 4.8: Growth rate curve ..................................................................................................... 41

1

1.1 INTRODUCTION

CHAPTER ONE

Crude oil exists worldwide in an intricate network of oil reservoirs. This oil is brought to

surface facilities through production wells, using existent oil recovery technologies. As

defined by the Society of Petroleum Engineers (SPE), primary and secondary recovery

methods are used to target oil which can be produced due to viscous and capillary forces

in the in the reservoir [1, 2]. Primary recovery occurs due to the overburden pressure of

the earth on the oil bearing formation [3]. Overtime, the primary production rate

decreases, and some of the production wells are transformed to injection wells. Secondary

recovery however involves implementing either water flooding or gas flooding

techniques to boost the pressure in the reservoir. These injected fluids (water or gas) help

fracture the oil-bearing formation, and enhance the flow rate of oil and gas towards the

wellhead. Whereas primary recovery produces between 5-10% of the original oil in place,

secondary recovery produces between 10-40% of the total reserves [4]. Two-thirds of the

initial oil in place however remains in the reservoir after these conventional recovery

techniques have been applied.

The energy demand across the globe is increasing as a result of the increase in the

population. In order to meet these rising energy demands throughout the world, it is

therefore necessary that more attention be focused on techniques for recovering more

fraction of the initial oil in place from hydrocarbon reservoirs after secondary recovery.

The method by which this is achieved is termed tertiary recovery, also known as

Enhanced Oil Recovery (EOR). This implies that the target for EOR methods is

significant (two-thirds of the total reserves). EOR however has a close relationship with

the prevailing oil price and the general economics associated with the technique.

Classification of EOR methods are based on the oil displacement mechanism [5, 6]. These

methods alter the viscous and capillary forces between the oil, rock surface and injected

fluid. The classification of EOR include thermal methods (heat injection), miscible gas

injection (solvent injection), chemical methods (injection of surfactant/polymer) and

microbial (injection of microorganism). Microbial enhanced oil recovery (MEOR) is in

the research and development stage, and that is the purpose of this project.

2

MEOR refers to the use of microorganisms and their metabolic by-products to extract the

residual oil from reservoirs. Metabolic by-products include a range of compounds

produced through microbial metabolism, an example of which is biosurfactant. The

produced biosurfactant reduces the interfacial tension at the oil-rock interface, thereby

increasing the oil recovery efficiency [7]

1.2 AIM OF THE PROJECT

There are various mechanisms by which MEOR operates. This research work presents a

broad overview of EOR technologies, with focus on Microbial Enhanced Oil Recovery

(MEOR), its mechanisms and the rate of biodegradation of hydrocarbon, field

applications and its’ challenges.

1.3 JUSTIFICATION

MEOR has obtained very few applications in the oil industry. Considering that MEOR is

a novel technology still in the R & D phase. This research work was therefore carried out

in order to add to the already existing body of knowledge on the subject matter, so as to

increase the application of MEOR in depleting oilfields. This investigation has been

carried out by potential investigation of the rate of biodegradation of hydrocarbons, with

the aim of gaining more insight to the mechanisms of MEOR. In this research work,

dodecane represents the hydrocarbon used, and the microbial activity is subjected to

aerobic conditions. Mineral water is used to stimulate the microbial growth.

1.4 SCOPE OF WORK

This research work is limited to laboratory experiments to investigate the rate of

biodegradation of dodecane under aerobic conditions. The research work terminates with

additional study to investigate which MEOR mechanism plays a major role in

biodegradation of hydrocarbon

3

CHAPTER TWO

2 LITERATURE REVIEW

2.1 OVERVIEW OF CRUDE OIL PRODUCTION

In the late nineteenth century, the demand for crude oil began to increase as a result of

the invention of the internal combustion engine, and has come to be one of the most

significant commodities traded worldwide. Modern civilization is thus heavily dependent

on crude oil and its byproducts. Conventional and unconventional hydrocarbons will most

likely remain the major component in the energy mix. One of the major aims of the

petroleum industry is to sustain the world’s energy demand, by a guaranteed flow of

hydrocarbon fluids, and at the same time, make profits.

Crude oil is produced by creating pressure gradients within the reservoir which results in

the migration of oil through the interconnected pore spaces towards one or more

production wells. This production is characterised by three main phases. They include

production buildup, constant peak production, and production decline. An in-depth

understanding of the various recovery mechanisms is essential in order to sustain the

required production levels over the period of the lifecycle of the oilfield. Primary recovery

which utilizes the natural reservoir pressure has a short lifecycle, and a maximum

recovery factor of 20%. Secondary recovery depends on the injection of artificial water

or gas, and this increases the primary recovery factor by 15 to 20% [8]. Tertiary or

enhanced oil recovery processes are implemented in order to maximize hydrocarbon

recovery beyond secondary recovery method. It has a recovery factor of approximately

60%, thus has the potential of significantly contributing to the much-desired energy

supply.

2.1.1 PRIMARY OIL RECOVERY

Oil is present in the interconnected pore spaces within the reservoir rocks which are

beneath the earth’s surface. In this recovery process, production is dependent on the

natural pressure of the reservoir. This natural pressure in the reservoir causes oil to flow

up production wells, and its origin is form the following forces:

Expanding force of high-pressure natural gas

Buoyant force of underground flowing water (aquifer drive)

Gravitational force

4

A displacement force as a result of compaction of unconsolidated reservoir rocks.

Depending on the properties and composition of the reservoir, the above mentioned forces

can act either concurrently, or progressively. It is believed that the expanding forces of

high-pressure natural gas has a greater contribution to oil production. However in inclined

reservoirs, gravitational force is more effective, as it enhances oil drainage [9]. As

hydrocarbon fluids flow up the production wells to the surface, the initial pressure of the

reservoir continually decreases, until a point where pumping or artificial lift is required

to sustain the production rate. As the pressure in the reservoir falls below the bubble point

pressure, some gas bubbles are released form the solution. These bubbles are initially

trapped within the pores of the reservoir, and their expansion results in displacement of

oil (dissolved gas drive) [10]. This is not always the case, as in some reservoirs, the gas

bubbles coalesce, forming large pockets of continuous gas phase which flows to the upper

part of the reservoir. As the reservoir pressure continues to decrease, the gas cap expands,

thereby displacing more oil (gas cap drive).

2.1.2 SECONDARY OIL RECOVERY

As the reservoir pressure reduces during primary recovery, it gets to a critical point where

the initial pressure of the reservoir is no longer sufficient to act as to act as a natural force

resulting in the movement of hydrocarbons towards the production wells. It becomes

necessary at this point to provide the reservoir with external energy, by injecting water

(waterflooding) so as to maintain or increase the reservoir pressure

2.1.2.1 Waterflooding

Waterflooding is the most commonly explored secondary oil recovery technology for

conventional and/or heavy oil reservoirs. This is as a result of availability of water, its

simplicity, and cost-effectiveness. In the case of heavy oil reservoirs, thermal energy is

combined with the injection water, although this is regarded as a tertiary oil recovery

method. As illustrated in Figure 2.1, the waterflooding process involves injecting water

is introduced into the reservoir through injection wells. This water increases the pressure

within the formation, and drives oil through the permeable reservoir rocks towards the

production wells, thereby increasing the oil production rate. Nonetheless, waterflooding

has some disadvantages, some of which include

Corrosion of topside and subsurface facilities

5

The injected water may react with the formation water, thereby resulting in the

damage of the formation.

Figure 2.1: Waterflooding process. Adapted from [11]

2.1.3 TERTIARY/ ENHANCED OIL RECOVERY

Approximately two thirds of the original oil in place is economically unrecoverable after

the implementation of primary and secondary recovery techniques [12]. It is the desire of

operators of depleting oilfields to maximize the cost-effective recovery of unrecoverable

oil after the natural pressure of the reservoir is depleted, and secondary recovery

techniques have been implemented. Tertiary oil recovery, often termed enhanced oil

recovery in the ideal sense means reducing the unrecoverable oil and/or increasing the

sweep (volumetric) efficiency (residence time). It involves injecting a fluid into the

reservoir in order to increase the amount of oil produced by the traditional primary and

secondary production methods. Thus, the target for EOR processes is this substantial

volume of residual oil in the reservoir and it involves employing physical, chemical or

thermal mechanisms so as to improve the microscopic oil displacement [13].

2.2 EOR BY LITHOLOGY

Reservoir lithology is a very important variable in determining the applicability of

specific EOR techniques. From Figure 2.2 below, most EOR applications have been in

sandstone reservoirs in comparison with other lithologies. Sandstone reservoirs generally

6

have the highest potential for EOR projects due to the fact that the technologies have been

tested on a commercial scale in this lithology.

Figure 2.2: EOR field projects by lithology. Adapted from [14].

2.3 TERTIARY/ ENHANCED OIL RECOVERY METHODS.

For conventional oil reservoirs, the methods involve flow diversion through polymer gels,

polymer flooding, miscible gas injection, and the use of surfactants. However, for heavy

oil reservoirs characterised by more viscous oil, the EOR methods employed involve

steam injection and air injection, thereby resulting in the in situ combustion of the

hydrocarbons. Most EOR methods are more expensive to implement, as compared with

primary and secondary recovery methods, and are only economically viable for large

oilfields when the oil price is high. EOR processes are categorised into three main

methods. They include miscible displacement, thermal method, and chemical methods.

These are discussed in details below.

2.3.1 MISCIBLE DISPLACEMENT

Miscible displacement is an EOR technique that enhances the microscopic sweep

efficiency by reducing or eliminating the interfacial tension between the oil and the

displacing fluid (gas). When implemented after waterflooding, it has the ability to re-

establish a channel through which the remaining oil flows, thus leading to very low

residual oil saturation (approximately 2% has been recorded [15] ). The gas injected is

dependent on availability and the reservoir conditions, and may be hydrocarbon gas,

7

nitrogen or CO2. At a low temperature and pressure, CO2 is miscible with oil, and requires

a regular supply source. Previous applications have been on fields with close proximity

with the natural source of CO2 [16, 17] . The use of CO2 can lead to corrosion of the steel

pipes and this problem can be overcome by careful design of wells, flowlines and facilities

[18]. Nitrogen requires reservoirs with high pressures, and additional equipment for its

separation from air. As a result, it is not widely used [16]. Hydrocarbon gas is usually

obtainable from the oilfield itself or neighbouring fields, and is employed in fields where

there is no available market for the hydrocarbon gas [15, 16, 19, 20].

A disadvantage of miscible displacement is that the gas is less dense and less viscous than

the oil. Thus, the macroscopic sweep efficiency is low, as it is affected by heterogeneity

[21, 22], gravity [8, 23] and viscous fingering (Fig 2.3) [8, 24, 25].

Figure 2.3: Viscous fingering observed when low viscosity gas displaces higher

viscosity oil. Flow is from left to right. Adapted from [26].

2.3.2 THERMAL

The basic principle of thermal methods used to improve the recovery of heavy oils is to

supply the reservoir with heat. This increases the temperature within the reservoir, thereby

reducing the oil viscosity, and causes an increase in the mobility of the oil. The two

methods used to conduct thermal recovery are steam flooding and in situ combustion.

Steam Flooding

This process is illustrated in Figures 2.4. High quality steam is injected into the reservoir,

and this can be done either in cycles or continuously. Whereas cyclic injection involves

using one well as both injection well and production well, continuous injection uses both

injection and production wells.

8

Figure 2.4: Steam injection process. Adapted from [27]

As this steam condenses, the thermal energy is transferred to the reservoir rocks and

fluids. This causes the expansion of the oil, thereby reducing its viscosity, and release of

dissolved gases. The sweep efficiency of the reservoir is limited by gravity segregation,

and can be overcome by steam assisted gravity drainage.

Figure 2.5: Steam assisted gravity drainage. Adapted from [27]

In situ Combustion

This method is often referred to as fire flood. In in-situ combustion, heat is generated in

the reservoir by burning some of the reservoir oil, thereby resulting in the displacement

9

of the residual oil towards the production wells. This method is not widely applied, as

controlling the process is a challenge [13].

Figure 2.6: In situ Combustion process. Adapted from [27]

2.3.3 CHEMICAL

Chemical flooding involves the addition of chemicals such as polymers and surfactants

to the displacing water. Depending on the method used, these may increase the viscosity

of the water such that it matches that of the oil (polymers), and/or reduce the interfacial

tension between the water and the oil (surfactant). Chemical flooding has been an

alternative for EOR since mid-1960s.

Surfactant flooding

As shown in Figure 2.7, this method involves injecting a surfactant solution (water,

surfactant, and electrolyte) into the reservoir. This reduces the interfacial tension between

the oil and the rock, and also the interfacial tension between the oil and the injected water

[28], thereby resulting in the displacement and recovery of residual oil.

10

Figure 2.7: Surfactant flooding. Adapted from [11]

Polymer flooding

This method involves adding water soluble polymers to the injection water. This method

improves the volumetric sweep efficiency, and as a result, the oil is pushed towards the

production wells. The application of this method is limited, as higher polymer

concentrations are required by heavy oils with high viscosities.

2.3.4 MICROBIAL

One of the available EOR methods is microbial enhanced oil recovery commonly referred

to as MEOR, which utilizes microorganisms and their metabolites to extract the residual

oil from reservoirs. MEOR and chemical flooding both have the same function, the

difference being that in the case if MEOR, the chemicals are produced in the reservoir as

a result of the microbial activity.

The use of microorganisms in enhanced oil recovery is based on two justifications. The

first is oil migration through the porous media by the alteration of the interfacial

properties between the oil-water interface. In this type of system, the bacterial activity

modifies the displacement efficiency (increase in permeability, decrease in interfacial

tension); fluidity (miscible flooding, viscosity reduction); sweep efficiency (selective

11

plugging, mobility control); and the natural force (reservoir pressure). The second

rationale is referred to as upgrading. In this scenario, heavy oils are degraded into light

oils as a result of the microbial activity in the reservoir. It can also result in the removal

of heavy metals, as well as sulphur from heavy oils.

In order to sustain the growth and metabolism of these microbes, nutrients of different

types are injected into the reservoir. Whereas some processes utilize fermentable

carbohydrate such as molasses as the nutrient, some others require water containing a

source of phosphates, vitamins and nitrates, so as to enable the growth of anaerobic

bacteria. The microorganisms employed in MEOR are generally anaerobic extremophiles

[29, 30]. These bacteria are usually non-pathogenic, hydrocarbon-utilizing, and occur

naturally in petroleum reservoirs. Bacillus strains cultured with glucose mineral salt are

the most common bacteria used in MEOR technologies, especially when reduction in the

oil viscosity is not the main aim of the operation [31].

2.4 REVIEW OF MICORBIAL ENHANCED OIL RECOVERY

Microbial enhanced oil recovery (MEOR) is a process which utilizes microorganisms to

improve the recovery of oil from the reservoir. These microorganisms may be indigenous

to the reservoir, or are present in the drilling mud or injection water which are introduced

through the wellbore into the reservoir. They feed on the hydrocarbons, undergo

metabolism, and produce by-products such as gases, organic acids, polymers,

Biosurfactants and biomass. These by-products help reduce the viscosity of the oil,

thereby enhancing its flow. Whereas primary and secondary recovery methods recover

up to 35 – 45% of the original oil in place, MEOR helps increase production by 5- 15%.

The idea of MEOR was put forward first by Beckman in 1926, when he suggested that

microorganisms can help improve the oil production rate form porous media ref.

However, it was not until a series of laboratory investigations by Zobell in the 1940s that

any serious considerations was given to the process [32]. Based on the method of

implementation, MEOR can be classified as either cyclic, microbial flooding or selective

plugging recovery [33]. Microbial enhanced oil recovery has many unique advantages.

It has low toxicity, is less dependent on crude oil prices and consumes low amounts of

energy [34] . Also microbial growth occurs at an exponential rate, hence additional

volumes of oil can be produced at cheap rates. In spite of existing disagreements by some

groups[19], continuous research in addition to a number of successful field applications

12

confirm that MEOR is thus a viable alternative in improving the hydrocarbon recovery

efficiencies from reservoirs [35, 36].

2.4.1 RELATED WORK DONE ON MEOR

In 1926, after Beckham postulated that microorganisms could be used to increase the

amount of oil recovered from reservoirs, very little consideration was given to the process,

as the ability of microorganisms to use hydrocarbons was viewed as a biological curiosity.

In the late 1940s, Zobell and his research team from Russia began conducting research in

university laboratories. The results obtained from these laboratory experiments showed

that certain microorganisms, together with adequate nutrients and reservoir conditions

such as temperature, pressure, salinity and dissolved gases and produce biopolymers,

biosurfactants, acids and gases as their metabolic byproducts. Zobell further discussed

and patented the mechanisms behind which oil is released form porous sand columns by

utilizing the byproducts of the microorganisms (refer to Table 2.1 below).

Experimental results obtained by Zobell and his team laid the foundation upon which

other researchers worldwide began to their own experiments. In the USSR, Kuznetsov

concluded that bacteria which is capable of degrading oil to form gases (H2, CH4, N2, and

CO2) are present in oil deposits. His work led to the authentication of the technology of

activation of reservoir microorganisms, which was later developed by Ivanov and his

research team [37].

Between the 1960s and 1970s, a significant number of research activities were carried out

in certain European countries such as Hungary, Poland and former Czechoslovakia [38,

39]. Based on the results obtained, some field trials were carried out in these countries,

and these were done by injecting certain preselected bacteria (Pseudomonas,

Micrococcus, Peprococcus, Mycobacterium, Bacillus, etc.) based on their ability to

produce large amounts of acids, solvents, polymers, gases, surfactants and biomass. The

oil recovery efficiency is however increased by either one or a combination of

mechanisms such as selective plugging, interfacial tension reduction, gas production or

biomass formation [40]. The results obtained from some of these field trials produced

different results from what was obtained from laboratory experiments. This deviation in

results was attributed to the lack of proper understanding of the physical and chemical

processes which occur within the reservoir where in situ metabolism occurs [41].

According to Hitzman [37], a number of reasons have been identified for these observed

13

variations. An important factor he reported was the changing environment that normally

occurs within the reservoir which is difficult to simulate in the laboratory using core

samples and reactors. These changes in the reservoir occur as a result of the interaction

of the exponentially growing microorganisms in the reservoir. Another reason he pointed

out for the failure of field trials is the inadequate consideration of the prevailing

conditions of the reservoir [42]. These conditions include pH, salinity, temperature,

pressure, nutrients and dissolved oxygen. Another reason he identified is insufficient

knowledge on the growth of microorganisms in hydrocarbons under anaerobic conditions.

However, recent research has proven that bacteria have the ability to metabolize the

hydrocarbons in an anaerobic environment [43]. It has been suggested that careful

planning can help overcome some of the problems associated with MEOR field

applications [44].

Studies carried out between 1970 and 2000 have verified the basic nature of existence of

indigenous microorganisms in hydrocarbon reservoirs, and the reservoir characteristics

necessary for successful MEOR applications [37]. Research about MEOR is still on

going, and this is because of the increasing oil prices and increase in the number of

matured oilfields. Some recent studies include the works of McInerney et al, 2002; Bryant

and Lockhart, 2002; Brown et al. 2002; Kowalski et al, 2006; Lazar et al. 2007; Kaster et

al. 2009 and Rudyk and Sogaard, 2011. These studies were attempts to eliminate the

variations in laboratory experiments and field applications of MEOR.

Research into area of MEOR modelling is increasing rapidly. It has been observed that a

mathematical model can be used to predict the most important parameters and their

relationships for the successful application of MEOR [45]. Development of mathematical

models for MEOR is however a challenging task as a result of dynamic physical and

chemical variables that govern the activities of the microbes in reservoirs. Microbial

activity has however been successfully modelled mathematically in the work of Monod

[46]. Following his publication of his research work, several other mathematical models

have been developed to simulate MEOR processes. Examples include models that

incorporate the effect of salinity on interfacial tension reduction and adsorption of

microorganisms [37], models for changes in relative permeability [47, 48], and basic

equations for the adsorption and diffusion of microorganisms [49, 50].

14

In conclusion, the ability of microbes to enhance oil recovery is not questionable, rather

the challenge is how to apply this ability in an economic and practical manner obtain

results which can be transferred from laboratory scale to field applications. Research

projects to support MEOR have been carried out worldwide in countries such as US,

China, Russia, Canada, Hungary, Norway, Great Britain, Poland, Czech Republic,

Germany, Bulgaria and Australia. It is pertinent to recognise the role of the US

Department of Energy (DOE), which funded a number of MEOR projects and organised

various international symposia. MEOR technologies today are fit for application as the

demand for oil rises and the production remains constant or decreases. However despite

the extensive history of MEOR activities, MEOR technologies have gained very little

acceptance by the oil industry. This may be attributed to the fact that most available

literature on MEOR is based on laboratory experiments or field applications with

insufficient duration, and little collaboration between reservoir engineers,

microbiologists, geologists, and owner operators. Many researchers have made

recommendations necessary to validate MEOR as a feasible method to improve oil

recovery efficiencies, and until these tests are carried out, MEOR may not attain a highly

desirable recognition in the oil industry.

2.5 CLASSIFICATION, MECHANISMS AND LIMITATIONS OF MEOR

2.5.1 MEOR CLASSIFICATION

The main aim of microbial enhanced oil recovery is to reduce the viscous and capillary

forces in heavy oil reservoirs, by the use of microorganisms, thereby increasing the

amount of oil produced form the reservoir. The methods in which these microorganisms

are introduced into the reservoir vary. MEOR is however classified based on the method

in which the microorganisms are introduced into the reservoir. The first method, the

microorganisms and their bio-products are cultured on surface facilities and are injected

into the target zones in the reservoir. This method is referred to as surface MEOR. The

second method however entails the injection of nutrients into the reservoir to stimulate

indigenous microorganisms in the reservoir. This method is referred to as underground

MEOR.

According to the implementation method, underground MEOR is classified as follows:

Cyclic Microbial Recovery (huff and puff)

Microbial Flooding Recovery

15

Selective Plugging Recovery

Cyclic microbial recovery is a single well simulation method in which microorganisms

together with some nutrients are injected into an oil reservoir through a single well. Upon

completion of the injection, the well is shut in for a period of 3-5 days. During this period,

the microorganisms feed on the nutrients, grow and produce metabolites. The metabolites

produced however vary, depending on the microorganisms used, and may be surfactants,

acids or gases such as hydrogen, methane and carbondioxide. After this period is the oil

production stage, and this may take several weeks or months. This method eliminates the

need for continual injection. However when production rate starts to decrease, the whole

process is repeated with a new supply of microorganisms and the nutrients. The area in

the reservoir which is covered by these microorganisms is however limited by the rate of

injection and the rate at which the microbial process progresses [51].

Figure 2.8: Cyclic microbial oil recovery Adapted from [52]

In microbial flooding recovery, nutrients are added to the injection water which is

introduced to the oil reservoir through injection wells. This helps stimulate the growth of

indigenous microorganisms within the reservoir. However, in an event where the

necessary microbial activity is not present within the formation, microorganisms can be

injected together with the nutrients. As these microorganisms feed on the oil, they produce

metabolic by-products, which act on the oil, reducing the viscosity, interfacial surface

tension, and increasing the pressure within the reservoir. These changes to within the

formation cause the heavy crude oil to flow towards the production wells. This method is

16

most probably less expensive, as microbial growth is stimulated in larger portions of the

reservoir where the residual oil (carbon source) is located.

Figure 2.9: Microbial flooding recovery. Adapted from [8]

In selective plugging, the water is diverted to low permeability zones through microbial

processes. Injected nutrients thus flow to the high permeability zones, resulting in the

stimulation of biopolymer production in these zones, resulting in the reduction of rock

permeability [53].

Figure 2.10: Illustration of Selective plugging Adapted from [11]

17

2.5.2 MEOR MECHANISMS

In order for MEOR to be generally accepted in the petroleum industry as a sustainable

technology, it is imperative that the mechanisms behind the process be understood.

Microbial activity within the reservoir results in chemical changes of the formation fluids.

Improvements in the oil recovery factor as a result of the microbial activities can be

achieved through several mechanisms such as selective plugging by microorganisms and

their metabolites, reduction in the interfacial tension at the oil-water interface by

surfactant production, acid production which enhances the absolute permeability of the

rock and reduction in the oil viscosity by gas production. It is believed however, that the

first two mechanisms have the largest effects on improving oil recovery [11, 48, 54-56].

Microorganism have the capacity to produce large quantities of the same type of

compounds as those used in conventional EOR methods, the only difference between both

oil recovery enhancement methods being the means by which these compounds are

introduced into the reservoir [51]. Different microbial groups, their metabolites and

applications in MEOR are summarised in Table 2.1[31]

Table 2.2.1: Microbial groups and their bioproducts formed

Microbial Product Microbes Application in MEOR

Surfactants Bacillus, Pseudomonas, Acinetobacter Reduction of Interfacial

tension

Biomass Xanrhomonas, Leuconostoc and

Biomass Bacillus

Wettability alteration and

selective plugging

Acids Mixed acidogens, Clostridium,

Enterobacter

Permeability increase.

Emulsification

Gases

Clostridium, Enterobacter

Methanobacterium

Increase in pressure, oil

swelling, and viscosity

reduction.

Solvents Zymomonas, Clostridium, Klebsiella Increased permeability

Viscosity reduction

Polymers Maacillus, Xanthomonas, Leuconostoc Viscosity reduction and

selective plugging.

18

2.5.2.1 Biosurfactant Application

Chemical surfactants are toxic and expensive compounds that are non-biodegradable ref.

An increasing concern about protecting the environment has led to the development of

cost- effective bioprocesses for the production of biosurfactant. Biosurfactants are

efficient alternatives to chemical surface-active agents, as they possess better

characteristics, such as high biodegradability, low toxicity, ease of application, and high

tolerance under extreme conditions of temperature, pH, and salinity. There are three main

ways for the application of biosurfactants in oil recovery.

I. Injection of microorganisms capable of producing biosurfactants into the reservoir

via the wellbore, with subsequent growth and multiplication of the

microorganisms within the reservoir.

II. Injection of nutrients into the reservoir, which is capable of stimulating the growth

of indigenous biosurfactant producing microorganisms.

III. Ex-situ production of biosurfactants, and subsequent injection into the reservoir.

Biosurfactants improve oil recovery by reducing the interfacial tension and altering the

wettability of the porous media. A reduction in the interfacial tension at the oil-rock

results in a reduction in the capillary forces that trap interface residual oil in the porous

rocks thereby causing additional oil to be displaced from the capillary network.

2.5.2.1.1 Requirements to mobilize oil by IFT reduction.

Residual oil exists as globules within the pore spaces in the reservoir. After primary and

secondary recovery from the reservoir, the pressure gradient required to transport these

globules through the pore throats is insufficient, and the viscous force promoting flow is

opposed by capillary forces acting on the globule. In this situation, a correlating parameter

is the capillary number Nca. This is defined as the ratio of viscous to capillary forces, and

is expressed mathematically as:

Nca

u

Equation 2.1

Where u is the velocity (m/s), µ is the viscosity (Pa.s), and is the interfacial tension

(N/m). The larger the capillary number, the lower the viscosity of the oil, thus the greater

the possibility of mobilization of residual oil. Surfactants reduce the interfacial tension,

thereby increasing the capillary number. As illustrated in Figure2.11, core floods for

19

miscible flooding show that in order to mobilize oil, the capillary number has to be

increased to the range between 10-5 - 10-4 [54]. Generally, the capillary number for most

reservoirs under waterflood is in the range of 10-7. This implies that the surfactants have

to reduce the interfacial tension by a minimum of two orders of magnitude in order to

mobilize residual oil. For example, the interfacial tension at the oil-water interface in

sandstone reservoirs is usually in the order of 30 to 40mN/m [55]. Thus the surfactants

used to have any effect on the residual oil saturation, it must be capable of reducing the

interfacial tension to below 0.4mN/m.

Figure 2.11: Residual oil saturation as a function of capillary number Adapted from [54]

For chemical surfactant flood to have any positive effect on the oil mobility, the

recommended interfacial tension must be reduced in the range between 0.01 to

0.001mN/m [57]. Biosurfactants which are capable of lowering the interfacial tension to

such reduced values have been reported [58].

2.5.2.2 Clogging Mechanism

The MEOR mechanism which has gotten the most attention is the plugging of high

permeability zones in the reservoir. Microorganisms alongside the nutrients are injected

into the reservoir in order to stimulate growth and metabolism. These injected nutrients

and microorganisms flow preferentially into high permeability zones within the reservoir.

Overtime, the microbial cell mass increases, and this selectively plugs these zones,

reducing the permeability below that of moderate or low permeability zones [59]. This

alters the fluid flow in the reservoir, by moving fluid flow form regions of high

permeability to regions of low permeability, thereby increasing the sweep efficiency.

Experiments using sandstone cores have shown that injecting microbes along with

nutrients led to clogging of 60%- 80% of the pore spaces in the reservoir [60].

20

A critical analysis of the feasibility of this mechanism reveals that the oil recovery is a

function of the volume of the high permeability zone in relation to the overall pore volume

of the reservoir. The yield becomes more attractive as the volume of the high permeability

zones decreases. Reservoirs which have less than 6% of the total pore volume in the high-

permeability zones are better prospects for bacterial plugging [54].

2.5.2.3 Application of gases and solvents

As a result of metabolism of microorganisms within the reservoir, gases such as

carbondioxide, hydrogen, and methane are produced. These gases exist in a free gas phase

at low pressures in the reservoir. These gases produced have the ability to increase the oil

recovery based on mechanisms such as reduction in residual oil saturation,

repressurization and reduction in viscosity. However, it is necessary to state that if the

microorganisms feed on some oil to produce these gases, the viscosity of the residual oil

will be greater than that of the initial oil. This is because the microorganisms will feed

on the lighter fractions of the oil, and the produced microbial gas will be insoluble in the

remaining heavier fraction. This will result in an immobile residue of heavy oil, with

only gas flowing. Using microorganisms to convert immobile oil to flowing gas is not

within the scope of this research.

During the stationary growth phase, microbial process yields some solvents such as

ethanol, acetone, and butanol in smaller amounts. These dissolve in the oil, reducing its

viscosity and increasing its mobility ratio. Gases and solvents both have the ability to

dissolve carbonate rock, thus resulting in an increase in its porosity and permeability [61].

2.5.2.4 Biopolymers application

Certain bacteria have the ability to produce water soluble biopolymers which can increase

the oil recovery using the mechanism of selective plugging by cell growth [62]. These

biopolymers plug the high permeability zones in waterflooding operations, thus changing

the path of the water-flood to oil-rich regions in the reservoir and providing a better sweep

efficiency in the low permeability layers [28]. However, for this mechanism to be

successful in improving oil recovery, the biopolymers produced must not be degradable

by the indigenous microbes, else a more frequent injection of nutrients will be required

to sustain the required level of biopolymers within the reservoir [36].

21

2.5.3 MEOR LIMITATIONS

In order to achieve maximum results in any process, it is important that parameters

involved be outlined. Optimization of MEOR can be achieved once the detrimental

factors are identified and analysed. It is however important to be aware of the limiting,

rather than simply the beneficial factors of employing microbes in enhanced oil recovery.

One of the reasons why MEOR technology has not been widely accepted for field

applications is because of inadequate consideration of the prevailing conditions in the

reservoir, and the physiology of the microorganisms which are able to survive in these

conditions. The physical and chemical conditions of the reservoir determine the activities

of the microbes used in the MEOR process. These conditions include pH, temperature,

salinity, pore size, etc. These factors unarguably affect the growth of microorganisms,

their metabolism and survival within the reservoir, thereby reducing their ability to

produce metabolites in the desired amounts necessary for enhanced oil recovery. Some

of these factors which are considered as constraints to MEOR process are discussed

below.

Temperature

The in situ temperature to be considered in reservoir increases as it has become necessary

to explore deeper reservoirs. The average temperature gradient is 25⁰C for every 1000m

of burial [63]. The demand for hydrocarbon is on an increase, as a result of increase in

population. Oil companies are thus forced to drill to deeper into the earth’s surface. The

temperature gradients at these depths are considered inhospitable, and do not support

bacterial growth and their metabolism. MEOR however cannot be ruled out even at these

high temperatures, as cold water injection, which is a secondary oil recovery technique,

results in temperature drops within the well [64]. Depending on the temperature ranges

for survival, microbes are classified into three categories:

Psychrophiles (grow below 25⁰C)

Mesophiles ( grow between 25 - 40⁰C)

Thermophile (grow between 45 – 60⁰C)

Most oil reservoirs are situated at depths with temperatures higher than 37⁰C, and this is

considered as optimum temperature for bacterial growth and proliferation [65] .

22

Pressure

Although the limiting boundaries for most biological processes is set by temperature, the

importance of pressure cannot be neglected [66]. The average depth of the oceans is

3800m, and this corresponds to an average pressure of 38MPa , approximately 380 times

greater than atmospheric pressure (0.1MPa) [67]. Communities of subsurface

microorganisms have been discovered 3500m below the sea level [68]. Different

magnitude of pressures exists in different biological systems [69, 70], and these pressures

however exert different effects on microorganisms (Fig 2.11).

Figure 2.12: Range of pressures in various biological systems. Adapted from [71]

Adaptation to pressure changes is the characteristic of all life, with evidence of this

adaptation observed in deep sea microorganisms [72]. However, in the case of

microorganisms that are already adapted to atmospheric pressure, adaptation to high

hydrostatic pressures are assumed to be non- harmful, but this may have negative effects

on cell growth, as most mesphilic microbes (E. coli cells) become filamentous at high

pressures [63].

It is important to note that the effect pressure has on microorganisms does not only depend on the

magnitude, but also other factors such as the length of time over which the pressure is appplied,

the temperature at which these pressues are applied, pH, the composition of the bacterial culture

media, and the oxygen supplied [67] .

Salinity

The concentration of salt in reservoirs is another factor that determines the survival of

microorganisms in the subsurface. The total dissolved solids in reservoir brine are made

up of 90% sodium chloride, hence it important that microbes employed in MEOR have a

high salinity tolerance. The extent to which salinity modifies growth and metabolism of

bacteria depends on the osmotic balance necessary for such growth. Clostridia species

capable of growth were cultured at 45⁰C, but it was observed that their solvents and gas

producing ability were significantly reduced at high sodium chloride concentrations (5%

23

w/v) [73]. However, in order to adapt to the high concentrations of sodium chloride in

reservoirs, microorganisms have the ability to accumulate a variety of small organic

solutes in the cytoplasm. These organic solutes are referred to as osmolytes, and they have

the ability to maintain cell volume and turgor pressure – essential elements for cell

proliferation [74].

pH

pH is an important environmental factor that affects microbial activity. The optimum pH

for the growth of microbes is in the range of 5-8, and this is because nearly all microbial

metabolic reactions are enzymic [64]. However for microbial growth to be sustained, the

minimum and maximum pH possible is 2 and 9.5 respectively. The concentration of

dissolved carbondioxide, to a large extent, has an effect on the pH of a system. Reduction

in pH, caused by microbial growth, occurs either as a result of microbial respiration, or

the production of acids, which react in carbonate structures to form carbondioxide. A

reduction in the pH is reported to have occurred in all MEOR field trials [75].

Pore size

Based on rock lithology, reservoirs are classified into three groups namely sandstone,

carbonate and igneous/metamorphic rocks. These rocks must possess adequate

permeability in order for fluids to be injected or produced form the reservoir.

Igneous/metamorphic rocks however possess very low permeability, and thus are not

suitable hydrocarbon reservoirs. Permeability values for sandstone and carbonate

reservoirs are listed in Table 2.2 below:

Table 2.2.2 : Permeability variations with lithology [64]

Sandstone (mD) Carbonate (Limestone/

Dolomite) (mD)

Min 11 10

Med 564 127

Max 4000 1600

24

It has been proven experimentally that reservoirs with permeability values of less than

100mD are constricting for the free movement of the microbes. MEOR is thus applicable

mainly in sandstone reservoirs.

A permeability of 100mD corresponds to a porosity of approximately 5µm. Bacteria are

unicellular, with dimensions of length of around 0.2-10 µm and width in the range of 0.5-

2.0 µm [60] .The use of microorganisms in MEOR is however limited to bacteria, as yeast

and other members of the Protista kingdom have widths larger than 5µm, and hence are

larger than the average reservoir pore space [64]. The pore size must be twice the size of

the bacteria in order for effective transportation to occur within the reservoir [76] . It has

thus been concluded that the pore size or the pore throat diameter is an important factor

in determining the microbial activity within the reservoir [77].

Dissolved Gases.

The dissolved gases that are of importance in MEOR are oxygen, carbondioxide,

hydrogen sulphide, methane, hydrogen and nitrogen. Of the above mentioned gases,

oxygen is the most significant, then carbondioxide, and hydrogen sulphide [63]. In terms

of oxygen usage, microbes are classified into four groups. They include:

I. Aerobic, which require oxygen for growth

II. Anaerobic, which do not require oxygen for growth

III. Microaerophilic, growth and metabolism is best sustained in small amounts of

oxygen

IV. Facultative Anaerobic, can survive under either aerobic or anaerobic conditions.

Aerobic microbes cannot be sustained in a hydrocarbon reservoir in which surface water

is not injected. This is because the dissolved oxygen tension will be too low to sustain the

growth and metabolism of these microbes. Dissolved oxygen concentration in reservoirs

is controlled by the use of deaerator towers or chemicals, and in some cases, a

combination of both. 6-8 ppm of dissolved oxygen is however considered to be corrosive

[32].

Whereas oxygen is required mainly as a hydrogen acceptor in the microbial cytochrome

system, carbondioxide and methane act as the main carbon source for the microbes. The

concentration of hydrogen sulphide in the reservoir is important as well. Sulphate ions

25

are converted into hydrogen sulphide by the action of sulphate reducing bacteria, thereby

forming sour hydrocarbons. This results in corrosion problems within the reservoir.

Nutrients

The availability of essential nutrients is a major requirement in order to sustain the growth

and metabolism of microbes used in the MEOR process. Organic carbon (fatty acids and

sugar) and mineral ions (e.g. calcium and phosphorus) are basic requirements for bacterial

growth, and they are usually transported in an aqueous phase in the reservoir. The nature

of the bioproducts from different bacteria is dependent on the nature and concentration of

the nutrient provided. Updegraff and Wren were the first to propose the use of molasses

as a substrate [76]. Molasses, which is a by-product of sugar, has been used in various

field applications as the carbon source for the microbes. Some microbes on the other hand

also utilize the oil as the carbon source. This is essentially beneficial in heavy oil

reservoirs, as it reduces the viscosity of the oil, thereby increasing its quality [78].

2.6 FIELD APPLICATIONS OF MEOR.

With a lot of research and various field trials, MEOR has been established to have great

potentials in improving the amount of oil extracted from reservoirs. Field tests run in the

US by the National Institute for Petroleum and Energy research were completed with an

increase in the amount of oil produced from flood water that was treated with

microorganisms at a reduced cost [79]. Cyclic microbial recovery technique have also

been implemented commercially on hundreds of wells, with the aim of controlling

paraffin deposition [80, 81]. Activities towards controlling the deposition of paraffin and

sludge in the bottom of tanks were carried out between 1986 and 1990, by MICRO-BAC

International Inc. in Austin, Texas [37]. Microbial permeability modification has been

conducted by Phillips Petroleum Company in the North Burbank Uni. Osage County,

Oklahoma [60]. As patented by Phillips, the MEOR method employed was using injecting

nutrients into the reservoir in order to stimulate the indigenous bacteria. The first injection

turned out unsuccessful, however the second showed a 20% decrease in permeability [79].

A method has successfully been established in Russia, based on the principle of

introducing some salts and oxygen along with injection water into the reservoirs, so as to

stimulate the indigenous microorganisms [82, 83].

In the last 20-30 years, China has been very active in MEOR, and is still active in this

field till today, and may be recognised as one of the leaders in this field [84]. Reservoir

26

engineers in China have reported on microbial simulation carried out in the Fuyu field.

The wells located in this field contained 18-23% paraffin. As a result of the microbial

stimulation, the viscosity reduced by 8.2% (from 45 to 36.8cP) and 42 out if a total of 44

wells responded positively [79]. There was also an increase in the CO2 content in the

production wells by 21%. Some of the wells sustained an increase in production for up to

357 days [79] . In Australia, a concept involving the use of ultra microbacteria generated

through nutrient manipulation has been developed. The outer cells of ultra microbacteria

possess some surface-active properties, which help reduce the interfacial tension of the

oil in the reservoir. This developed concept was successfully verified in increasing the

amount of oil produced from the Alton oil field in Queensland, Australia [42, 85]. In

Romania, successful MEOR field trials have been reported using both microbial flooding

and single well stimulation recovery technologies on a number of Romanian oilfields by

the injection of molasses into the reservoir [86]. A successful increase in the amount of

oil produced from carbonate reservoirs by utilizing molasses and inoculum as the nutrient

support has been reported in Germany [87]. This method was also used in MEOR

applications in oilfields in Russia.

Conducting successful MEOR applications on water floods, and the minimal cost of

MEOR is the foundation on which MEOR field experiments have been based after 1990.

Also technologies involving the use of ultrabacteria have the potential for increasing the

field applications of MEOR [37]. It is believed that as the demand for oil continues to

increase with an increase in the number of stripper wells, MEOR will gain the much

desired recognition as a means of tertiary oil recovery, and will be applied to more oil

fields across the world.

2.7 ADVANTAGES AND CHALLENGES OF MEOR.

Some of the major advantages of MEOR technologies in comparison with other EOR

technologies are listed below [37] :

1. It is a sufficient alternative before abandoning stripper wells, as the cost is

potentially low.

2. The bacteria and nutrients which are injected into the reservoirs are inexpensive

and easy to handle in the field.

27

3. Since the fluids injected into the reservoir are not petrochemicals as is the case

with chemical EOR technologies, their costs do not depend on the prevailing oil

prices.

4. In the case of carbonate oil reservoirs, MEOR technologies are suitable where

some EOR processes cannot be implemented.

5. Only slight modifications to the existing facilities on the field are required where

MEOR process is to be implemented. When compared with other EOR

technologies, it is less costly

6. Based on available statistical evaluation in the US, 81% of MEOR projects

resulted in the increase in the amount of oil produced with no recorded decrease

in the oil production as a result of the application of MEOR processes

7. As a result of the exponential growth of the microorganisms, the effect of their

activity within the reservoir increases overtime, whereas in EOR technologies, the

effect the additives have on the reservoir diminishes with time.

8. Microbes which are indigenous to the reservoir can be utilized, thereby

eliminating the possibility of adsorption and losses as a result of degradation.

9. The products of MEOR are biodegradable, thus will not accumulate in the

environment, so it is environmentally benign.

Irrespective of the above mentioned advantages, there are some problems surrounding

MEOR. A major challenge is reservoir heterogeneity and varying oil complexities. A

method which appears optimal for one reservoir may not necessarily be optimal for

another reservoir. Other challenges with MEOR include souring of wells by sulphate

reducing bacteria (SRB) and undesirable plugging of pores caused by large microbial

cells. Well souring can be reduced by using nitrate-reducing bacteria to get rid of the

SRBs. Unwanted plugging can be avoided as well by injecting microorganisms with one

tenth of the pore entry diameter.

Some technical and economic issues have also been identified. In the case of

neighbouring production wells with different owners, if one well is treated with MEOR,

it may lead to an increase in the oil recovery form the neighbouring wells, and this can

result in economic and legal issues.[32].

28

CHAPTER THREE

3 MATERIALS AND METHOD

MEOR experiments were carried out to complement the studies reported in this

dissertation. The experiments served as the basis for testing and improving considered

methods for field applications and additional experiments. Soil sample collected from

Aberdeen city was used to culture microorganisms, which were incubated for a total

period of 31 days. The ability of these microorganisms to degrade dodecane was tested,

and the results are detailed in Chapter 4 of this report. A brief outline of the experiments

is thus provided below.

3.1 CULTURES USED

A total of 5 enrichment cultures of bacteria isolated from soils sample were used in this

research. The bacteria were cultured aerobically at 27⁰C. Enrichment cultures were used

rather than pure cultures because it is almost impossible to maintain pure cultures in field

applications of a microbial oil recovery method.

3.2 FLUIDS

The fluids used in this experiment include a source of hydrocarbon (dodecane) and

deionised water. The physical properties of these fluids are summarised in Table 3.1.

Table 3.1: Properties of the used fluids

Property Dodecane Water

Density @ 20⁰C (g/l) 749 1000

Viscosity (mPa.s) 29 1

Flash point (⁰C) 74 -

Pour point (⁰C) -9.6

3.2.1 HYDROCARBON SOURCE

Dodecane used was as a pure carbon source free from crude oil components such as salts,

sulphur, resins, asphaltene and ash contents, thus the effects these components have on

microbial activities have not been considered in this research.

29

3.2.2 DEIONISED WATER

Deionised water with a pH of 8 was used in the preparation of the mineral solution.

Deionised water was selected because it is free from contaminants such as lead, mercury

nitrates, phosphates, as well as pesticides.

3.3 GLASS BIOREACTORS

Ten glass bioreactors were used in this experiment. Five of them were used to culture

bacteria, and the other five were used for the control experiment.

3.4 EXPERIMENTAL PROCEDURE

The experimental procedure used in this research is as described below:

3.4.1 MINERAL SOLUTION PREPARATION

The mineral solution was prepared based on the reports that minerals solutions containing

nitrates and phosphates are effective for in situ MEOR process. The solution was prepared

for all the bioreactor samples by mixing the mineral salts and their corresponding

concentrations as listed in Table 3.2 with 1000ml of deionised water. This mixture was

then placed on a magnetic stirrer and allowed to thoroughly stir for 1 hour.

Table 3.2: The compositions of the mineral solution

Compositions Concentration (g/l)

Ammonium chloride (NH4Cl) 30

Potassium hydrogen phosphate (K2HPO4) 68

Sodium dihydrogen phosphate

(NaH2PO4)

60

Magnesium chloride (MgCl2.H2O) 0.05

Calcium chloride hexahydrate

(CaCl2.6H2O)

0.035

30

Figure 3.1: Mineral Solution on magnetic stirrer

3.4.2 PREPARATION OF UNCONSOLIDATED SAND

Clean dry sand which was collected from Aberdeen city was properly crushed so as to

reduce the grain size. The crushed sand was then sieved and dried in the oven to a

temperature of 500⁰C for 4 hours. The sand was then cooled and packed into a beaker.

3.4.3 MICROBIAL GROWTH EXPERIMENTS

Microorganisms were cultured under standard aerobic conditions at room temperature for

a period of 31 days. These enrichment cultures were grown in their respective bioreactors.

The method involved growing the cultures in in a mineral solution media containing

dodecane as the sole carbon source. Each bioreactor had 100ml of solution (95ml of

mineral solution and 5ml of dodecane). The experiment was performed in ten replicates,

five of which had 0.5g of soil added to it, and the other five serving as the control

experiment had no sand added to them. The control experiment was to serve as the basis

for comparison with the soil sample experiments so as to be able to draw a valid

conclusion on the rate of biodegradation experienced in the soil sample experiments. In

order to ensure maximum contact between the hydrocarbon, media and microorganisms,

all the cultures were incubated with continuous agitation in sealed bioreactors containing

a magnetic stirring bar. Samples were taken from the rotary shaker at pre-determined

intervals and analysed using a gas chromatograph.

3.4.4 SAMPLE ANALYSIS

3.4.4.1 pH and Dissolved oxygen measurement

After every incubation interval, the content of the bioreactors to be analysed were

transferred to a clean beaker, and the pH and dissolved oxygen in the samples were

measured using litmus paper and dissolved oxygen meter respectively.

31

Figure 3.2:pH measurement Figure 3.3:Dissolved oxygen

measurement

3.4.4.2 Visual volumetric observation

A visual observation is made using a measuring cylinder. This is necessary in order to

check for any abiotic effects such as evaporation of the dodecane or mineral solution.

This was also necessary procedure in order to monitor the bacterial activity at the oil-

water interface. The biofilm growth at this interface was monitored as a function of height

for every incubation interval over the 31 day period.

3.4.4.3 Filtration and Extraction.

Vacuum filtration technique was used to separate the suspended solids from the incubated

mineral solution mixture. The experimental setup is shown in Figure 3.4 below. The

samples were poured through a filter paper into a Buchner funnel. The suspended solids

were trapped by the filter paper, and the liquid was drawn through the funnel into the

conical flask below, by an electric vacuum pump. The total suspended solids (biomass)

on the filter paper was dried at a 104⁰C for 4 fours, and weighed using an electronic

weighing balance in order to obtain the weight of the additional biomass formed after

every incubation interval. The total suspended solids (biomass) yield was expressed as

g/l.

32

Figure 3.4:Vacuum Filtration

Process

Figure 3.5:Total Suspended

solids on the filter paper

In order to completely separate the dodecane present from the mineral solution mixture,

liquid-liquid extraction was carried out using a separating funnel. This process involved

using a volatile solvent (hexane) which is miscible with the mineral solution to extract

the dodecane. 20ml of hexane was added to the liquid which was drawn off from the

vacuum filtration process. In comparison with the mineral solution, dodecane has a higher

solubility in hexane. As shown in Figure 3.6, two clear separate layers were observed in

the separation funnel, with the ether mixture at the top, and the denser aqueous layer

(mineral solution) at the bottom of the separation funnel. This was let to run-out into an

appropriate sized conical flask aby opening the tap, while carefully watching as the

interface between the two layers moved towards the tap so as to stop the flow once all of

the lower layer had been removed. A second clean conical flask was used to collect the

upper layer (hexane-dodecane mixture), and this was measures into vials for analysis

using the gas chromatograph (GC).

33

Figure 3.6: Liquid-Liquid extraction using Hexane

3.4.4.4 Gas Chromatography (GC) Analysis.

Gas Chromatography (GC) is a method for separating and analysing the components of a

mixture. It works on the principle of using a mobile gaseous phase to transport

components of a sample through a column containing the stationary phase – a microscopic

layer of liquid. The different components of the mixture travel through this stationary

phase at different speeds, thereby causing them to separate. Since they get through the

column at different times, they are identified at the outlet of the column by a detector.

The GC produces a plot of detector signal against of time. This plot is referred to as a

chromatogram, with different peaks analogous to different components. Examples of

chromatograms are shown in Figure 3.7 below.

Figure 3.7:Examples of chromatograms. (a) Single component (Hexane) sample and (b)

Two component (hexane and dodecane) mixture.

34

The GC profile of dodecane was monitored before and after incubation in the presence of

microbes. Microbial growth was predicted only when changes in the profile were

observed. The experimental protocol used in this analysis is as described below:

Calibration of GC.

Before analysing the composition of the samples, it was essential to carry out the

following:

1. Identify the retention time for each of the components to be analysed.

2. Generate a calibration curve, i.e. a curve which shows the relationship between

the peak areas on the chromatograms and the composition of the mixtures. The

calibration curve generated was then used to analyse the samples.

In order to complete the first task, GC analysis was performed for pure components

(hexane and dodecane). Each of these chromatograms contain only one large peak, with

the retention time described as the time at which these peaks were observed. Identifying

the retention times for hexane and dodecane was necessary in order to be successfully

identify these components in the mixture. Figure 3.8 shows a chromatogram of the toe-

component mixture. Knowing the retention times reveals that the first and second peaks

are hexane and dodecane respectively.

A calibration was generated by performing GC analysis of samples of known

concentrations. The concentrations used for the calibration are 20g/l, 50g/l, 100g/l, 200g/l

and 400g/l. An example of calibration curve plotted is shown in Figure 3.8. Two GC runs

were performed for each concentration. The peak areas obtained yielded high

reproducibility, hence the error bars in this experiment are reduced to a minimum.

35

Figure 3.8: Example of Calibration curve. The line indicates results of a least squares fit

to the experimental data.

6000

5000

4000

3000

2000

1000

0

y = 13.72x R² = 0.9933

0 50 100 150 200 250 300 350 400 450

Dodecane in Hexane (g/l)

Pea

k A

rea

36

CHAPTER 4

4 RESULTS AND DISCUSSION

Microbial activity was detected to have occurred in the bioreactors during the incubation

period. This was evident in parameters such as the concentrations of dodecane and

dissolved oxygen, which decreased throughout the experiment. The following items are

therefore highlighted in this section

Dodecane degradation

TSS production

Dissolved oxygen

Biofilm Height

4.1 DODECANE DEGRADATION.

The results obtained over the 31day incubation period is illustrated graphically in Figure

4.1 below. The concentration of dodecane used at the start of this experiment was 37.5g/l.

It is observed that the dodecane concentration in the soil sample on the zeroth day falls

below 37.5g/l. During the visual observation in the measuring cylinder, there was no

noticeable effect of evaporation in the dodecane phase. Hence, it is assumed that this

decrease in concentration may be due to the adsorption of the dodecane on the sand

particles. However, in order to account for these losses, optimistic evaluations were

carried out as the analysis in every case assumed a 1% loss of dodecane due to adsorption.

Thus this phenomena is unlikely to alter the conclusions of this research. Over the total

incubation period of 31 days, the dodecane concentration decreased at a rather slow rate,

the final concentration being 29.5g/l. Conversely, the dodecane concentration in the

control experiment remained fairly constant, with maximum variations of about 2%.

37

Figure 4.1: Dodecane concentration vs Time

4.2 TOTAL SUSPENDED SOLIDS PRODUCTION.

Figure 4.2 shows a graph of TSS formed against time. From the graph, it is observed that the TSS

produced increases as the incubation period increases. This can be attributed to the growth and

metabolism of the microorganisms over time within the bioreactors. It was also observed that the

TSS concentration increased at a slower rate after the fifteenth day. This can be attributed to a

decrease in metabolic activities of the microorganisms, which may be either as a result of a

decrease in the amount of nutrients present in the samples, or a depletion in oxygen concentration

in the bioreactors.

Figure 4.2: TSS concentration vs. Time

In order to establish a relationship between dodecane consumption and biomass formed,

a graph showing the relationship between the TSS and the dodecane concentration over

time was plotted. This is illustrated in Figure 4.3. It is observed that as the TSS

37

35

33

31

29

27

25

0 5 10 15 20 25 30 35

Time (Days)

Soil Sample Control

Do

dec

ane

in 1

00

ml o

f w

ater

(g/

l)

5

4

3

2

1

0

0 5 10 15 20 25 30 35

Time (days)

TSS

(g/l

)

38

concentration increases, the dodecane concentration decreases. This relationship is an

indication that the dodecane present in the soil samples has undergone biodegradation.

However, in large scale MEOR field applications, an increase in TSS may not necessarily

indicate that the hydrocarbon present has undergone biodegradation. This is because the

microorganisms present may feed on carbon sources other than the hydrocarbon within

the reservoir, undergo metabolism and still produce biomass.

Figure 4.3: Dodecane concentration and TSS vs. Time

4.3 DISSOLVED OXYGEN

Microorganisms are described by their ability to use oxygen. Obligate aerobes require

oxygen for growth, and cannot survive unless the concentration is high enough. In the

soil samples analysed, their presence was evident by only growth at the surface as shown

in Figure 4.4. The dissolved oxygen concentration in the soil sample and control

experiments were measured. The results obtained are illustrated graphically in Figure 4.5.

It is evident that the oxygen concentration in the soil sample decreases with time. This is

because the oxygen present is used by the microbes, as their growth is a function of

oxygen availability.

37

36

35

34

33

32

31

30

5

4

3

2

1

0

0 5 10

Time (days)

15 20 25

Dodecane Concentration TSS

Do

dec

ane

in 1

00

ml o

f w

ater

(g

/l)

TSS

(g/l

)

39

Figure 4.4: Growth on the hydrocarbon surface.

Figure 4.5: Dissolved oxygen concentration vs. Time

4.4 BIOFILM THICKNESS

The activity at the dodecane-water interface was monitored by the thickness of biofilm

growth measured from the bottom of the oil-water interface. Figure 4.6 shows a

comparison between the heights of the biofilms formed on different incubation days. A

graphical illustration of the heights obtained through the total incubation period is shown

in Figure 4.7. It shows that the highest thickness of 2.5ml was observed on the thirty-first

day, while no biofilm growth was observed on the zeroth and eighth day.

8.5

8

7.5

7

0 5 10 15 Soil sample

20 25 30 35 Control

40

Figure 4.6: Comparison between the heights of biofilm formed.

Figure 4.7: Biofilm height vs. Time

This biofilm formation can cause an increase in the adherence of the microbial cells to

the oil- water interface, and can act as a surfactant with the ability to extract small oil

droplets from the water phase. Growth within the biofilm is however not uniform as

channels are formed which allow for the combination of fresh nutrients and oxygen.

4.5 MICROBIAL KINETICS.

The concentration of biomass produced is defined in terms of cell dry weight

measurements, measured in g/l. The biomass growth curve is illustrated in Figure 4.8

below.

3

2.5

2

1.5

1

0.5

0

0 5 10 15 20 25 30 35

Time (days)

Bio

film

hei

ght

(ml)

41

Figure 4.8: Growth rate curve

The specific growth rate is defined as any point during the growth cycle. From Figure 25

above, the R2 value of 0.8884 indicates a fairly strong correlation between the TSS

produced and the incubation period. It is observed that on an average, the TSS increases

at a rate of 0.0066day-1, and this growth rate is constant during the exponential growth

phase. From the equation of the regression line, the y intercept value of 1.2866 is the

Monod’s prediction for the growth rate on day 0. It is observed that the regression line

does not pass through all of the points on the graph. This regression line however can be

used to make predictions about TSS increase after particular incubation periods into the

near future, but it is not a very good model to make a prediction very far into the future.

4.6 BIOMASS YIELD COEFFICIENT YX/S

A quantitative assessment of microbial growth and/or biomass formation is necessary in

order to establish a relationship between the biomass formation and the consumption of

substrate, which in this case is dodecane. In relation to microbial biomass production, the

amount of cell mass formed is proportional to the mass of substrate consumed. This is

usually referred to as the yield coefficient, and is expressed mathematically as

YX / S

X

S

……………………………………………………………………………………………………………………………

….Equation 4.1

where

1.6

1.4

1.2 y = 0.0066x + 1.2866

R² = 0.8884

1.0

0.8

0.6

0.4

0.2

0.0

0 5 10 15 20 25 30 35

Time (days)

LoG

TSS

42

X is the amount of biomass produced

S is the amount of dodecane consumed

Over the 31 days incubation period, the total amount of biomass formed is 0.81g/l and

the total amount of dodecane which was biodegraded is 6.8g/l. These are illustrated in

Figure 4.3. Applying equation 2 above, the yield coefficient gives a value of 0.1191. This

means that for every unit of dodecane consumed by the microorganisms, 1.191g/l of

biomass is formed. It can also mean that for every unit of biomass formed, 8.395g/l of

dodecane will be consumed.

In field applications however, it is expected that there will be variations in yield

coefficients. For instance, the yield coefficients vary with growth rate. These variations

may occur as a result of differences in the assimilation of the substrate into the cell mass,

and the different energy requirements of the microorganisms (energy for growth and

maintenance).

4.7 PROSPECTS FOR INDIVIDUAL MEOR MECHANISMS

The following analysis is based on the results obtained from the laboratory experiments

conducted. From the experiments, two main mechanisms are observed. They are

modification of interfacial properties by biosurfactant production and modification of

reservoir properties by selective plugging of high permeability zones. These two

mechanisms will be the basis for comparison.

Selective plugging of high permeability zones is rated as a better MEOR mechanism than

interfacial tension reduction. Although literature has it that biosurfactant contributes

significantly to MEOR, the production of these biosurfactant alone is not enough to cause

an increase in oil mobilization. Moreover key problems such as the biodegradation of

surfactants by other species of microbes which are indigenous to the reservoir have not

been considered in literature. Also, it is expected that an increased stimulation of

biosurfactant production over a period long enough to cause an increase in the volumetric

sweep efficiency of a reservoir will inevitably stimulate the growth of biosurfactant

degraders. In contrast, selective plugging requires has the potential to cause significant

improvements in the areal sweep efficiency. The selection of suitable microorganisms to

produce biopolymers is however necessary for the success of this method.

43

CHAPTER 5

5 CONCLUSION AND RECOMMENDATIONS

MEOR is an eco-friendly, cost effective and highly attractive process which has diverse

advantages and great potential over other EOR chemical methods. However, there are

uncertainties in attaining the engineering design criteria for microbial processes

application, and this has resulted to its low acceptance in the petroleum industry.

The results obtained from the laboratory experiments indicate that dodecane is

biodegradable under aerobic conditions. Based on the observations, the prominent

recovery mechanism is selective plugging Thus in relation to oilfields, biodegradation has

a potential to be useful for MEOR if the crude oil present in the reservoir is used as the

sole carbon source. However, the growth rate value calculated from the results obtained

is not applicable in large scale oilfields. To overcome this limitation, it is recommended

that further laboratory research with specific reservoir conditions be carried out, after

which experiments must. Be carried out in the actual oil fields. Provisions should also be

made in the laboratory for the identification of the bioproducts formed. This will enable

allow for a better comparison of the oil recovery mechanisms.

44

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53

APPENDICES

Appendix 1

CALCULATIONS USED IN PREPARATION OF SAMPLES FOR

CALIBRATION.

Density = mass

volume

Density of Dodecane=750g/l

Initial volume of Dodecane = 5ml = 0.005l

The mass of Dodecane equivalent to 0.005l is calculated using

Mass= density × volume

Mass = 750 × 0.005 = 3.75g

The concentration of Dodecane in 100ml of water is given as

Concentration = mass

total volume of water =

3.75 = 37.5g/l

0.1

Extraction was carried out using 20ml of hexane.

Concentration of Dodecane in 20ml of hexane = 3.75

= 187.5g/l 0.02

Appendix 2

CALIBRATION CALCULATIONS

In order to prepare the samples for the GC calibration. It was essential to determine the

volume of Dodecane present in 1ml of hexane.

For the first sample point 20g/l;

Concentration = mass

volume

Mass = concentration × volume = 20 × 0.02 = 0.4g

This implies that 20g/l of dodecane-hexane mixture contains 0.4g of dodecane.

The above calculation is repeated for subsequent sample points (50g/l, 100g/l, 200g/l, and

400g/l respectively).

54

Appendix 3

CALIBRATION AND SAMPLE ANALYSIS RESULTS.

Day 0

Table A.1: Day 0 Calibration

Peak Area (1) Peak Area (2) Retention

Time (1)

Retention

Time (2)

Pure Hexane 11247.2957 11347.5844 1.198 1.197

Pure Dodecane 12478.7292 12478.5679 1.243 1.243

20g/l 299.9561 278.6321 1.260 1.255

50g/l 637.6055 599.4325 1.263 1.263

100g/l 1199.4624 987.2349 1.280 1.282

200g/l 3148.2884 3321.4633 1.273 1.277

400g/l 5211.2865 5813.7652 1.193 1.272

Table A.2: Day 0 GC sample analysis

Peak Area (1) Peak Area (2)

Soil Sample 2488.905 2401.628

Control 2509.14 2495.65

Dodecane concentration calculation.

SOIL SAMPLE.

From the calibration curve, Y= 13.49x

If Y=2445.267, x= 181.27g/l – Concentration of dodecane in 20ml of hexane

Concentration of dodecane in 100ml of solution (H2O) = (181.27×0.02)/0.1 = 36.3g/l

CONTROL

Y=2502.395

If Y =2502.395, x= 185.5 g/l – Concentration of dodecane in 20ml of hexane

55

Concentration of dodecane in 100ml of solution (H2O) = (185.5×0.02)/0.1 = 37.1g/l

DAY 8

Table A.3: Day 8 Calibration Values

Peak Area (1) Peak Area (2) Retention

Time (1)

Retention

Time (2)

Pure Hexane 11347.5844 11729.8940 1.197 1.198

Pure Dodecane 12920.8400 12706.7292 1.252 1.250

20g/l 260.1586 231.4328 1.255 1.255

50g/l 581.2318 549.3821 1.265 1.263

100g/l 958.1462 1216.9344 1.285 1.275

200g/l 3015.9115 3421.6911 1.278 1.277

400g/l 5408.4588 5843.7822 1.272 1.272

Table A.4: Day 8 GC Sample Analysis

Peak Area (1) Peak Area (2)

Soil Sample 2401.5976 2315.6489

Control 2463.539 2648.248

Dodecane concentration calculation

SOIL SAMPLE.

From the calibration curve, Y= 13.72x

If Y=2358.6233, x= 171.91g/l – Concentration of dodecane in 20ml of hexane

Concentration of dodecane in 100ml of solution (H2O) = (171.91×0.02)/0.1 = 34.38g/l

CONTROL

Y=2502.395

If y =2555.894, x= 186.3 g/l – Concentration of dodecane in 20ml of hexane

56

Concentration of dodecane in 100ml of solution (H2O) = (185.5×0.02)/0.1 = 37.0g/l

DAY 15

Table A.5: Day 15 Calibration Values

Peak Area (1) Peak Area (2) Retention

Time (1)

Retention

Time (2)

Pure Hexane 10217.3235 9893.5152 1.198 1.200

Pure Dodecane 11632.5458 11164.7423 1.258 1.258

20g/l 296.6242 325.3303 1.267 1.263

50g/l 671.8631 666.9514 1.275 1.275

100g/l 1272.7241 1156.7544 1.280 1.267

200g/l 3015.9115 2835.2771 1.273 1.277

400g/l 4858.956 4505.4242 1.273 1.275

Table A.6: Day 15 GC Sample Analysis

Peak Area (1) Peak Area (2)

Soil Sample 2077.2532 2153.2486

Control 2324.863 2341.575

Dodecane concentration calculation

SOIL SAMPLE.

From the calibration curve, Y= 12.578x

If Y=2115.251, x= 168.17g/l – Concentration of dodecane in 20ml of hexane

Concentration of dodecane in 100ml of solution (H2O) = (168.17×0.02)/0.1 = 33.63g/l

CONTROL

Y=2333.219

57

If y =2333.219, x= 185.5 g/l – Concentration of dodecane in 20ml of hexane

Concentration of dodecane in 100ml of solution (H2O) = (185.5×0.02)/0.1 = 37.2g/l

DAY 23

Table A.7: Day 23 Calibration Values

Peak Area (1) Peak Area (2) Retention

Time (1)

Retention

Time (2)

Pure Hexane 10217.3235 9893.5152 1.198 1.200

Pure Dodecane 11632.5458 11164.7423 1.258 1.258

20g/l 296.6242 325.3303 1.267 1.263

50g/l 671.8631 666.9514 1.275 1.275

100g/l 1272.7241 1156.7544 1.280 1.267

200g/l 3015.9115 2835.2771 1.273 1.277

400g/l 4858.956 4505.4242 1.273 1.275

Table A.8: Day 23 GC Sample Analysis

Peak Area (1) Peak Area (2)

Soil Sample 2094.593 2375.807

Control 1989.460 2501.266

Dodecane concentration calculation.

SOIL SAMPLE.

From the calibration curve, Y= 13.217x

If Y=2145.1191, x= 154.5g/l – Concentration of dodecane in 20ml of hexane

Concentration of dodecane in 100ml of solution (H2O) = (154.5×0.02)/0.1 = 30.9g/l

CONTROL

Y=2438.537

58

If y =2438.537, x= 184.5 g/l – Concentration of dodecane in 20ml of hexane

Concentration of dodecane in 100ml of solution (H2O) = (184.5×0.02)/0.1 = 36.9g/l

DAY 31

Table A.9: Day 31 Calibration Values

Peak Area (1) Peak Area (2) Retention

Time (1)

Retention

Time (2)

Pure Hexane 10181.1738 1.201.2547 1.198 1.197

Pure Dodecane 10987.2493 9501.2184 1.258 1.252

20g/l 226.0226 274.6572 1.265 1.260

50g/l 697.3319 637.2487 1.275 1.283

100g/l 1149.2987 947.1451 1.280 1.274

200g/l 2715.6303 2541.3849 1.277 1.273

400g/l 4266.5408 4795.8462 1.272 1.275

Table A.10: Day 31 GC Sample Analysis

Peak Area (1) Peak Area (2)

Soil Sample 1772.6951. 1661.6951

Control 2998.2208 1272.1049

Dodecane concentration calculation

SOIL SAMPLE.

From the calibration curve, Y= 11.655x

If Y=1716.8499, x= 147.31g/l – Concentration of dodecane in 20ml of hexane

Concentration of dodecane in 100ml of solution (H2O) = (147.31×0.02)/0.1 = 29.5g/l

CONTROL

Y=2135.1629

If Y =2135.1629, x= 183.197 g/l – Concentration of dodecane in 20ml of hexane

59

Concentration of dodecane in 100ml of solution (H2O) = (183.197×0.02)/0.1 = 36.6g/l

Table A.11: Dodecane concentration over the incubation period

Time (days) Soil sample (g/l) Control (g/l)

0 36.30 37.10

8 34.38 36.90

15 33.63 37.24

23 30.90 36.91

31 29.50 36.60