Adaptability Of Anaerobic Bacteria To Different Salinities Biology Essay

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Microbial Enhanced oil Recovery (MEOR) is particularly suited for application in carbonate reservoir, after secondary oil recovery, there are still large amount of oil left in the reservoir. Some bacteria are able to increase the oil production when injected into the oil reservoir. To stimulate such anaerobic microbial increased oil recovery, nutrients is injected together with the injection water.

Oil recovery requires two to three stages which are briefly described below

Stage1: Primary Recovery - 12 - 15 % of the oil in the well is recovered without the need to introduce other substances into the well.

Stage 2: Secondary Recovery - The oil well is flooded with water or other substances to obtain an additional 15-20% more oil from the well.

Stage 3: Tertiary Recovery - This stage may be accomplished through several methods which includes MEOR to additionally recover up to 11% more oil from the well.

Figure 1.1: Flow diagram for different enhanced oil recovery processes

Layout for different recovery techniques are shown in figure 1. Primary and secondary recovery techniques are usually called conventional recovery. Primary recovery is done by natural flow which is usually enhanced by reservoir natural pressure, and artificial lift such as pumps and gas lift, etc. Secondary recovery is done by water folding and pressure maintenance by gas reinjection. Tertiary recovery techniques cover broad area which includes thermal recovery such as in-situ combustion and steam flooding, solvent recovery is done by methods such as polymer flooding and surfactant enhanced water flood. Chemical enhanced recovery methods include gas injection or hydrocarbon miscible injection and nitrogen and flue gas flooding. Microbial enhanced oil recovery which is the main focus of this project will be explained better in the next chapter; however, it is basically injection of microbes such as bacteria into oil reservoir to help recover oil.

1.1 AIM AND OBJECTIVES

The aim of this project is to study the adaptability of anaerobic bacteria (Clostridium Thyrobutyricum 633) to different salinities and check the effect of the microbial strain on permeability of the Danish Nord Sea Chalk.

To achieve this aim, the following objective has been set:

Check adaptability of microbial strain to high salinities

Microbial gas production and dynamics of metabolism

Carry out plate count experiment

Observation of fermentation process and microbial analysis

To determine and measure the volume of carbon dioxide gas produced by these microbes when exposed to different salinities

To determine the amount of acid produced during fermentation process

Statistical analysis of results to derive model

Improvement of experimental procedure

Chapter 2

Literature Review.

CHAPTER TWO

2.0 LITERATURE REVIEW

The project work is based on studying of the microbial enhanced oil recovery method and the possibilities of using this in the Danish sector of the Nord Sea. The project task applies experimental procedure and the specific to investigate if these microbes can survive under reservoir conditions and produce products important in oil recovery. However, it is worthwhile to discuss the various oil recovery techniques.

2.1 Oil recovery technique

All methods of oil recovery will be explained briefly.

2.1.1 Primary recovery

If the underground pressure in the oil reservoir is sufficient, then this pressure will force the oil out to the surface of the earth. Gaseous fuel, natural gas or water is usually present, which also supply needed underground pressure. In this situation, it is sufficient to place a complex arrangement of valves (Christmas tree) on the well head to connect the well to a pipeline network for storage and processing. Normally oil is recovered by natural means and artificial lift like pumps and gas lift.

2.1.2 Secondary recovery

Over a lifetime of an oil well, the pressure will fall and at some point there will be insufficient underground pressure to force the oil to the surface of the earth. If economical, as often is, more oil in the well is extracted using secondary recovery methods. Secondary oil recovery uses various techniques to aid in recovering oil from depleted or low pressure reservoir. Sometimes, pumps such as beam pumps and electric submersible pumps (ESPs) are used to pump the oil to the surface of the earth. Other secondary recovery techniques increases the reservoir's pressure by water injection, natural gas reinjection and gas lift, which inject air, carbon dioxide or some other gases into the reservoir. Together, primary and secondary recovery generally allows 25-35 % of the reservoir oil to be recovered.

2.1.2.1 Water injection

The productivity of existing oil wells can be significantly increased by the use of water injection. Statistics has shown that a reservoir produces just 37% oil in the first recovery. By using water injection, a reservoir can produce more than 50% of its oil. One of the most important issues during oil production is to keep the matrix/formation as clean as possible to maintain maximum oil production. Water is injected for two reasons: first is for pressure support of the reservoir. Second is to sweep or displace the oil from the reservoir, and push it outward.

Figure 2.1: illustration of Gas injection using carbon dioxide

Figure 1.2 shows carbon dioxide injection in reservoir to recover oil, carbon dioxide becomes miscible with oil to reduce viscosity and increase mobility of oil return with produced oil and separated from it.

2.1.3.2 Nitrogen and gas flooding

Nitrogen and flue gas about 87 % nitrogen and 12 % carbon dioxide is used in place of hydrocarbon gases because of economical reasons. Nitrogen competes with carbon dioxide because it is economical and its compressibility is much lower. For a given quantity at standard condition nitrogen will occupy much more space at reservoir pressure than carbon dioxide and methane at the same condition. Nitrogen has a poor solubility and lower viscosity in oil and requires much higher pressure to create miscibility.

2.1.3.4 Solvent recovery

2.1.3.4.1 Polymer flooding

Both synthetic polymer such as polyacrylamides and natural polymers are used for improvement of sweep efficiency. Additional polymer makes the water more viscous so that oil is produced faster. Obviously, this is not a good idea n a low permeability reservoir or one with high clay content that absorb the polymer. However, polymer-augmented water floods can be profitable

2.1.3.4.2 Surfactant-Enhanced Water flood

Three types of chemical floods exist. The first is an alkaline-augmented polymer flood. Another is an alkaline-surfactant polymer flood. The third is a micellar or low interface tension flood (Donaldson, 1989).

Amongst the available tertiary oil recovery techniques, MEOR is arguably the best for many reasons. One key factor in the selection of microbial enhanced oil recovery is the economical potential, by which desirable chemicals and gases are produced to enhance oil recovery. MEOR processes are also energy efficient and environmental friendly as compared to other recovery techniques.

2.1.3.5.1 History of microbes used in MEOR

MEOR is a technology that has a history based on over 60 years of research and field studies. The earlier works by ZoBell CE and Updegraff D (USA), Kuznetsov SI and Shturn DL (USSR), shows the international scope of the work. This work was expanded in the 1950s mainly by investigators Coty VF, Yarborough H and Hitzman DO in the major oil companies in the United States. In MEOR, the process that facilitates oil production is complex and may involve multiple biochemical processes. Microbial biomass or biopolymers may plug high permeability zones and lead to a redirection of water flood, produce surfactants which lead to increased mobilization of residual oil, increase gas pressure by the production of carbon dioxide or reduce the oil viscosity due to digestion of large molecules.

2.1.3.5.2 Application of MEOR technologies

MEOR technologies have the common basis of introducing or stimulating viable micro organisms in an oil well reservoir for the purpose of enhancing oil recovery. However, this broad generic definition of MEOR is not a single methodology but is a broader technology which can be designed for different and selective applications. It is convenient to divide the MEOR technology into the following application groups:

Single well stimulation

MEOR water floods

Paraffin's removal

Viscosity modification

Water diversion

Heavy oil modification

The classification of MEOR technology by the proposed oil releasing mechanism shows the range of microbial effects which can be identified or expected to occur to which the MEOR system can be directed.

2.1.3.5.3 MEOR Oil Releasing Mechanism

Gas generation: The production of gases will aid the displacement of oil in the pore spaces.

Acid production: Organic and inorganic acid production by microbes will dissolve carbonate deposits, iron sulphide and dissolution and sulphate materials.

Surfactant production: Biosurfactants produced by the organisms result in the reduction of interfacial surface tension of the oil/water bond.

Other MEOR oil releasing mechanisms includes:

Physical oil displacement

Biopolymer production

Hydrocarbon modification

Viscosity modification

Selective plugging of high permeability zones within a reservoir is necessary to achieve oil recovery. This is best achieved in MEOR process where cells stimulated to grow deeply in a formation where production of biomass and products will have the greatest impact. If growth occurs primarily at the well bore, then face plugging will result, and no additional oil will be recovered, leaving the reservoir unproductive.

2.1.3.5.4 The Science of MEOR

Figure 2.2: Schematic view of MEOR layout

2.1.3.5.5 Types of microbes and their selection

MEOR has gained much attention in recent times, but it is worth noting that not all microbes can survive in such conditions as found in an oil well, therefore the microbes which are able to withstand these conditions are discussed below:

2.1.3.5.6 Microbes used in MEOR

There are many types of bacteria used in MEOR, they include aerobic and anaerobic bacteria and are divided on the basis of their need for oxygen. In this project work, the bacteria used were anaerobic from CHP-biogas plant at Ribe in Denmark.

2.1.3.5.7 Selection of Bacteria

The selection of specific bacteria is considered in this method. There are a lot of bacteria available, but the normal conditions for majority of bacteria is 5 % Sodium chloride, optimum temperature of 37 degree Celsius, pH less than seven.

2.1.3.5.8 Factors affecting growth of bacteria

There are many factors which affects the growth of bacteria. Some of which are explained in the below:

Salinity: The term salinity refers to the amount of dissolved salt that are present in water. Sodium chloride is the predominant ions in sea water, the concentration of magnesium, calcium and sulphate ions are also substantial. High salinity and toxic substances are responsible for limiting the growth of microbes. Halophiles are salt loving microbes which use sodium chloride and also have complex nutrient requirements. Moderate halophiles can grow anaerobically at temperature greater than 50o C. The salinity in the northern part of Danish oil field is about 40g/l or more. Since salinity too high, formation water is diluted with sea water during injection in the well. In order to perform experimental and laboratory analysis, a sample of produce water is taken so as to know how much salinity can be controlled; therefore microbial gas production has been tested up to 140g/l.

Temperature: Extreme high temperature affects the growth of bacteria, although they need average temperature for growth. Thermopiles are bacteria which are heat loving; these bacteria have an optimum growth temperature of 45 o C - 80 o C. Their membranes are unusually stable at this extremely high temperature. Thus many important biotechnological processes utilise thermophilic enzymes because of their ability to withstand intense heat. So before injecting these bacteria into the reservoir, the temperature of the reservoir should be considered, therefore, selection of the right thermophilic bacteria for high temperature is very important.

Effect of pH: pH is the measure of acidity or alkalinity of a solution. Simply pH is the measure of concentration of hydrogen ions in a solution. It is a measure of the activity of dissolved hydrogen ion. In pure water at 25 o C, the concentration of hydrogen ion equals the concentration of hydroxide ions; this is known as "neutral" and corresponds to a pH level of 7.0. Solutions in which the concentration of hydrogen ions exceeds that of hydroxyl ion has a pH level lower than 7.0 and are known ad bases. The pH reading of a solution is usually obtained by comparing unknown solution to those of known pH, and there are several ways to do so. More favourable pH condition for micro organisms is about 7 and very few of them can grow below2 and above 10. Micro organisms capable of living at very low pH are called acidphilies and those which live at high pH are called alkaliphiles.

Pressure: Extreme pressure affects the growth and metabolism of micro organisms. A pressure lower than 100-200 atm has no effect on microbial metabolism, however, pressure of the range of 500-600 atm have limiting effect on growth of bacteria. The ocean floor possesses high pressure. For most MEOR processes barophilic organisms will not be necessary, instead, barotolerant microbes can grow at high pressures, but do not require these high pressures for optimal growth. The ability to grow pressure depends on the energy sources available, inorganic salts present, pH and temperature. Adaptation of microbial cultures to higher pressure therefore is possible.

Toxic elements: Chemicals which have toxic effects on micro organisms are found in some reservoirs. These chemicals include co-surfactant, surfactant, biocides, ethylenediaminetetraacetate, and toluene, many of which are used in various chemical EOR. Sodium and Potassium may be exchanged without impairing the growth of micro organisms. Magnesium has higher toxicity than sodium and potassium, but the most toxic formation water are those with high Calcium Chloride (CaCl2), so adaptability should be considered before injecting micro organisms in such toxic environment.

Nutrients: In MEOR recovery process, to achieve maximum level or required bacterial count, an optimum concentration and the right type of nutrient is desired. In this method, nutrient can be at the largest expense, so it is important to have the right combination and quantity available. Some of the most common nutrients are as follows:

Molasses

In-situ hydrocarbon (crude oil)

Molasses, nitrogen and phosphorus salt

In this experiment, only molasses have been used as nutrient. Molasses is easily available as slurry and in the real field; it can be pumped down easily into the well. The transport of the nutrient into the reservoir as well as and out of bacterial cell requires water. Water functions as a matrix through which the cellular chemistry takes place. The degree of water available for chemical activities and growth is called water activity. Osmotic pressure affects the water activity; the production of CO2 gas during the fermentation process can further increase this pressure around the system, therefore, water activity is inversely proportional to osmotic pressure.

2.2 Physical and environmental constraints

Gregory (1984) begins his exposition of the fundamentals of MEOR by identifying physical (temperature, pressure, and pore size/geometry), chemical (pH, h E, electrolyte composition) and biological factors that constrain microbial activity in hydrocarbon reservoirs. In the following subsections, these constraints, and their interactions, are discussed. More generally, it is to be observed that the same factors control the existence and behavior of bacteria in other subterranean environments, which are of relevance in other practical contexts - most notably the management and remediation of groundwater resources. The observation that numerous species of bacteria found in such environments can withstand, or even thrive, under physical conditions that are inimical to most life forms has given fresh impetus to study of this topic, in relation to research on the origins of life on earth and the possible existence of life on other planets.

2.2.1 Pore Size

The existence of bacteria in deep subsurface rocks has been disputed in the past, but since the advent of modern tracer techniques and improved sampling protocols (Frederickson and Phelps, 1996), is now generally accepted. Perhaps the most obvious constraint that applies to deep subsurface microbes is the size of the pores. In some studies, the lower limit of mean pore sizes has been shown to be smaller than the size of known bacteria. For example, through phospholipids fatty acid assays and measurements of 14 C acetate mineralization, Frederickson et al. (1997) assessed shale and sandstone cores from a site in northwestern New Mexico for microbial activity. They found no metabolic activity was detected in core samples with pore throats narrower than 0.2μ m, although in some cases it was after extended incubation. The observation of much higher levels of metabolic activity in more permeable samples led these authors to conclude that sustained bacterial activity require interconnected pores of diameter at least 0.2μ m.

2.2.2 Acidity

The acidity or (alkalinity) of the surrounding aqueous medium, measured by the pH, is significant in several respects

2.2.2.1 Surface Charge

On the cellular scale, pH controls the extent of ionization of the protein molecules that are embedded in the cell walls. As a result, cellular surfaces are generally charged and surrounded by diffuse double layers, the thickness of which is controlled by the overall electrolyte concentration. Interaction of these ionic space-charge regions with those that also surround small particles of mineral phases can strongly affect the motion of the cells through a natural porous medium. The effect of pH on the surface charge of a protein depends on the relative numbers of acidic and basic groups in the side chains. Protein molecules are often characterized by a pH called the isoelectric point, at which the positive and negative charges resulting from ionization of side chains are balanced.

2.2.2.2 Enzyme Function

Some of the embedded cell wall proteins play a crucial role in the uptake of nutrients, elimination of waste products, and maintenance of correct electrolyte concentrations; on a molecular scale, their ability to perform these functions also depends on their extent of ionization. The rates of the enzymic processes that occur in respiration are strongly dependent on the pH. There generally exists an optimal pH, lying between 2 and 9.5, for the rates of such processes. The mineral phases in a porous medium (particularly carbonates), and the proteins themselves can exert a buffering effect, which can mitigate the lowering of the pH by the acids generated by primary metabolism.

2.2.3 Oxidation Potential

Cellular respiration consists of enzymically mediated electron transfers from an electron donor (reducing agent, in chemical parlance) to a terminal electron acceptor (oxidizing agent). Apart from a few rare cases where only one mole of electrons is transferred from each mole of reductant, this electron transfer almost always involves a number of intermediate electron transfer steps, which can be quite numerous if the original electron sources are complex molecules such as sugars. The thermodynamic driving force for these electron transfer processes is expressed quantitatively in terms of the oxidation potential, h E (measured in volt), which is the Gibbs energy change divided by the number of moles of electrons transferred. According to the Nernst equation (discussions of which can be found in most textbooks of physical chemistry), this quantity depends logarithmically on the concentrations (strictly speaking, the activities) of not only the oxidized and reduced forms of the electron acceptor, but also of hydrogen ions and other species that might be involved. Thus, for aerobic respiration, the terminal electron acceptor is oxygen, which is reduced to water according to the overall equation

2.2.4 Oligotrophy and Heterotrophy

To explain the existence of active microbial communities in environments such as deep granitic and basaltic aquifers, where nutrient levels are expected to be extremely low, it was suggested by Stevens and McKinley (1995) that such organisms can be sustained by hydrogen generated by reduction of minerals by groundwater. Although many species of hydrogen-consuming lithotrophic bacteria have been described, and it is well known that appreciable hydrogen fugacities can be 'buffered' by some naturally-occurring mineral assemblages, the suggestion that microbial communities could be sustained geochemically in this way has, however, been disputed by Anderson et al. (1998). These authors argued that basalt does not produce hydrogen under slightly alkaline conditions, and that the production of hydrogen under slightly acidic conditions cannot be sustained over geological time scales. But a more recent discussion presented by Nealson et al. (2005) points out that the most important and difficult issue to be established is the long-term independence of such communities from the products of photosynthesis; at present this is best regarded as an open question.

Considerable attention has been devoted to the study of heterotrophic microbes in sandstones and shales, and the possibility that these organisms are sustained by organic material co deposited with the sediments. In a recent review, Krumholz (2000) considers formations containing alternating layers of sandstone and shale, and discusses experimental evidence that organic matter and fermentation products present in the shales can diffuse across sandstone shale boundaries and support microbial communities in the sandstone, adjacent to the sandstone-shale interfaces. Similar phenomena have been identified by McMahon and Chapelle (1991) and McMahon et al. (1992) in clay-sand sequences and by Ulrich et al. (1998) in lignite/clay deposits.

2.2.5 Water and Electrolytes

The concentrations of electrolytes and other dissolved species required for proper cellular function is maintained by enzymically mediated exchange of solutes or solvent with the surrounding medium. Dissolution of electrolytes reduces the thermodynamic activity of water, w a, Dissolution of electrolytes reduces the thermodynamic activity of water, aw. This effect is measured by the ratio of the fugacity of water above the solution to that of pure water. At temperatures far below the critical point of water, the fugacity of water is approximately equal to the vapor pressure For example, w a in sea water is about 0.98, while in inland salt lakes it can be as low as 0.75. Since the water activity corresponding to appreciable electrolyte concentrations differs only slightly from 1, an alternative measure is provided by the osmotic pressure of the solution, which is defined as the hydrostatic pressure that must applied to a solution to raise its vapor pressure to that of pure water. Thus, for sea water (of approximately 3.3% salinity), the osmotic pressure estimated from van't Hoff's equation is about 2.8 MPa. Differences in ionic strength across membranes provide a powerful driving force for diffusion of water into cells (when the surrounding medium is a more dilute electrolyte) or out of cells (when it is more concentrated.) While most bacteria are incapable of surviving in media with w a below about 0.95, minimum water activities for Pseudomonas species (which are of interest as candidates for MEOR) are considerably lower (0.91). Extreme halophiles, such as Halococcus, can survive when w a =0.75 (Todar, 2008). Aerobic degradation of benzene, toluene and xylene by halotolerant Marinobacter species in soil contaminated with oilfield brines was demonstrated by Nicholson and Fathepure (2004), suggesting potential usefulness in environmental remediation. Anaerobic bacteria from hypersaline environments are of particular interest to MEOR, considering the high salinity of connate water often found in oil-bearing formations. A review of such organisms by Ollivier et al. (1994) devoted considerable attention to sulfate-reducing organisms that feed on polymeric substrates such as starch, cellulose, and chitin, and the work of McMeekin et al. (1993) on anaerobic microorganisms isolated from concentrated salt lakes in Antarctica suggests applications to hydrocarbon degradation. In addition to MEOR and environmental remediation, the study of halotolerant bacteria is also relevant to food preservation (Vilhelmsson et al., 1997).

In addition to the specific chemical and biochemical effects that are often associated with high electrolyte concentrations, non-specific effects can be expected. Solubility of the vast majority of non electrolytes decreases with increasing ionic strength. This phenomenon, which is known as the 'salting out' effect, is particularly pronounced for non polar solutes (which tend to have low solubilities in pure water). Important examples are oxygen (the concentration of which controls the thermodynamic driving force for aerobic metabolism), and carbon dioxide, ionization of which controls the pH of many natural waters. In this way, high electrolyte concentration could affect both pH and h E.

2.2.6 Temperature

The increase in random molecular motion resulting from an increase in temperature generally exerts negative effects on enzyme function, since the active-site configurations required for catalysis are disrupted. At still higher temperatures, the hydrogen-bonded three-dimensional arrangement of the amino-acid chains also becomes disordered, resulting in irreversible denaturation. This molecular picture of the effects of temperature on enzyme function is generally accepted, but it is also to be observed that the temperatures at which these phenomena occur vary widely between organisms. In general, microbes can be classified according to their optimum temperature range as psychrophiles (< 25oC), mesophiles (25-45oC), and thermophiles (45-60oC). The relatively recent discovery of microbes that can survive in water at temperatures above 100oC has considerably extended the range of conditions under which life can be expected to exist. Microbes that thrive under such extreme conditions are generally referred to as 'extremophile'. Since the mean geothermal gradient beneath the continents is of the order of 25oC per km, and assuming more conservative upper limit of 110oC for bacterial activity, as suggested by the work of Blöchl et al. (1995), the biosphere could extend up to 5 km beneath the surface of the earth (compare Gold, 1992).

2.2.7 Pressure

The effects of pressure on microorganisms are closely associated with those of temperature, since elevated pressures in natural environments are always associated with temperature variations. Specifically the pressure in the ocean increases by about 10 MPa for every kilometer of depth, while the temperature of the ocean is about 3oC below about 100 m. On land, the pressure increases by about 3 MPa per km depth, but the temperature increases by about 25oC per km. Thus, in terms of the earlier terminology introduced to describe the temperature tolerance of bacteria, a marine bacterium that thrives on the seafloor at a depth of 3 km would be a psychrophile, while its terrestrial counterpart at the same depth underground would be a thermophile. An obvious exception to this generalization would be the bacteria in the vicinity of hydrothermal vents on the seafloor (the so-called 'black smokers'), some of which can withstand temperatures as hot as 121oC (Miroshnichenko and Osmolovskaya, 2006). Indirect and direct effects of pressure on cellular function can be identified. Growth rates of normal bacteria decrease to zero as hydrostatic pressure approaches about 40 MPa. ZoBell and Johnson used the term 'barophilic' to describe bacteria whose growth rate is enhanced at elevated pressure. (The prefix 'baro-' is sometimes replaced by 'piezo-'.) It is also customary to refer to bacteria for which the diminution of growth rate commences at pressures above 40 MPa as 'piezotolerant'. A third class of bacteria, which cannot be grown under ambient conditions, are referred to as 'obligatory piezophiles'.

The microbiology of bacteria isolated from the deepest oceans has been reviewed by Jannasch and Taylor (1984) and Yayanos (1995). Kato and Bartlett (1997) describe the identification of Pressure-regulated genes from deep-sea bacteria of the genus Shewanella. Imposition of high pressure affects the fluidity and water-permeability of the cell walls, causing the phospholipids bilayers to pack more tightly and assume a more ordered configuration. Piezotolerant organisms apparently compensate for this by increasing the proportion of unsaturated fatty acids, which have a much lower tendency towards such packing (DeLong and Yayanos, 1985, 1986; Kamimura et al., 1993). A more recent review by Daniel et al. (2006) describes other impressive advances that have been made in the molecular-level understanding of pressure effects on aspects of bacterial physiology. Under high pressures, the DNA double helix becomes denser, which can interfere with gene expression and the associated protein synthesis. Another important factor is the sterol content; membrane lipids that have high cholesterol content are more pressure resistant that those that contain ergosterol instead.

DNA analysis reveals the extremophile to be among the most ancient life forms known. This fact has given rise to intriguing speculations that life on earth could have originated in these extreme environments. The idea that life originated in the depths of the oceans about 3.8 billion years ago is also explored in some detail by Daniel et al. (2006). In a more practical context, pressure-induced deactivation of bacteria has been investigated as a possible way of sterilizing food (Spilimbergo et al., 2002; Aoyama et al., 2004).

2.2.8 Relation to MEOR

The purpose of the preceding discussion was to identify the factors that constrain the growth of bacteria in subsurface environments, thereby providing a set of criteria by which the suitability of organisms for use in EOR can be assessed and compared. For oil-bearing formations, it is to be observed that some of these constraints are somewhat less rigorous. For example, the salinities of connate brines are typically greater than that of seawater, but much less than those occurring in salt lakes, and pressures of up to 20 MPa and temperatures to 80 o are well within the limits observed for survival of bacteria. But the combination of these constraints can be expected to limit the number of suitable organisms. Among the best 'all-round performers' are the Bacillus bacteria. Yakimov et al. (1997) described a detailed study of several strains of Bacillus licheniformis, and concluded that this organism is potentially useful for enhanced oil recovery. It is capable of functioning at moderately elevated temperatures (55 o C) and salinities to 12% NaCl, produces significant quantities of biomass, and a surfactant similar to surfactin (produced by B. subtilis) which is known to possess antimicrobial properties. A more recent report by McInerney et al. (2004) describes a particularly thorough examination of over 200 strains of Bacillus subtilus, B. licheniformis, B. mojavensis, and B. sonorensis, which were compared with respect to surfactant production under anaerobic conditions at 5% salinity. In the course of their work, these authors developed new and improved procedures for isolating biosurfactants produced by the organisms, established quantitative relationships between the surfactant concentration and interfacial tensions, and performed numerous experiments involving mobilization of oil from Berea sandstone cores.

2.3 The choice of Clostridium Tyrobutiricum

Thousands of bacteria have been investigated for MEOR purpose, but the fermentation bacteria remain the most popular especially Clostridia specie because they produce large volume of gas which include CO2, H2 and CH4. These gases produced, decrease the oil viscosity and increase the pressure in the oil reservoir.

2.4 Fermentation

Waste products formed in this way include gases, ethyl alcohol, butyl alcohol, organic acids, acetone and others. Molasses fermentation generates energy rich metabolic product, which may react in the final decomposition line of sulphate reduction under anaerobic formation condition. With sulphate ion in the formation water, sulphur reduction predominates. Hydrogen sulphide produced is actually not desirable. The organic acids are formed through fermentation of the molasses by the bacteria in the reservoir do cause a rock dissolving process.

Figure 2.3: Fermentation bacteria

2.5 Dorben field (Germany), 1982, Dr. Wagner

Another reason for using fermentation bacteria is Dr. Wagner field trail. If we make comparison between Danish north oil field formation and Zechstein evaporates rocks which are similar to the Danish North Sea formation. Dolomite is also similar to Danish north field chalk. Formation temperature is quite similar and of course has a high salinity. Clostridia Tyrobutiricum was selected for Dr. Wagner's experiment.

The characteristics of Dr. Wagner's experiment field are as follows:

Dolomite of Zechstein formations

Depth of 1240m

Formation temperature 53 oC

High salinity formation water, even the fissures and fractures are partially filled with salt.

The result of Dr. Wagner's MEOR well experiments:

Water cut decreased from 80 to 60 %

Average annual oil production:

Before microbial treatment - 50 tons per month

3 months after injection - 150 tons per month

1 year after injection - 300 tons per month

Since all these conditions are similar to Danish North Sea formation and other factors are also same, so we can use fermentation bacteria for MEOR experiment.

2.6 Adaptation of bacteria to high salinities

Majority of the bacteria cannot withstand high salinity, from the time of ancient civilization; it is known that adding 50 g/l of salt in food preserves it from spoiling. This means that fermentation bacteria which normally populate organic substances have a challenge of adaptation in high salinity. The spore forming bacteria like clostridium form spores in extreme conditions. These conditions allow bacteria to survive but they will not be active and would not be productive. Under extremely high salinities, bacteria undergo osmotic stress which is expressed in osmotic pressure. Osmotic pressure affects the water activity and production of CO2 gas during the fermentation process.

2.6.1 Osmosis

Osmosis is the passage of water from region of high concentration through a semi-permeable membrane to a region of lower water concentration.

Semi permeable membrane are very thin layers of material (cell membrane are semi-permeable) which allow some substances to pass through them and prevent other substances from passing through. Cell membranes will allow small molecules like oxygen, water, CO2, ammonia, glucose, amino acid, etc. to pass through; meanwhile, cell membranes do not allow passage of larger molecules like sucrose, starch, protein, etc.

2.6.2 Osmotic pressure

2.6.3 Potential osmotic pressure

2.6.4 Osmotic properties of cells

The wall of bacteria and growing plant cells are not completely rigid, and the turgor pressure has been proposed to provide the mechanical force for the expansion of the cell walls during cell growth. The uptake or biosynthesis of osmotically active solutes causes an increase in the cells, thus providing the necessary tugor pressure for expansion of the cell walls. Although the suggestion that turgor pressure is the driving force for cell wall expansion would imply that the mechanisms that regulate the osmotic balance of organisms are central to the very process of cell growth.

Lipid membranes allow rapid diffusion of water molecules into or out of cells while presenting an effective barrier to most other biological molecules. Membranes that exhibit selective permeability for different substances are called semi permeable, and the osmotic properties of cells derive from this property of the membranes.

2.7 Thermophyllic and Halophyllic bacteria

There are bacteria which need high salinities and high temperatures for their growth. In order to investigate and record the conditions of microbes at high salinities and high temperature it is better to know about the bacteria which can withstand on these conditions. Important information has been given about these type of bacteria is discussed below.

.

In nature, if certain bacteria are Halophyllic then they are not Thermophyllic, so in order to select the right adaptation for recovery process, microbes which can grow in the reservoir environment should be considered. Pressure, temperature and salinity are the most important factors to consider so it is necessary to accumulate a collection of micro organism with desirable characteristics. Growth limitations would need to be established for each culture. For such a collection, the best organism for a particular application would be chosen. To determine the conditions under which microbial procedures would be preferred which require an accumulation of information resulting from field application of the process.

Chapter 3

Materials And Method.

CHAPTER THREE

3.0 MATERIALS AND METHOD

In order to check the adaptability of the considered bacteria and see how effective MEOR method could prove for the Danish field, a laboratory based experiment was carried out at Aalborg University Esbjerg, Denmark

3.1 Materials

The following materials were used at one point or the other during this project work:

Anaerobic bacteria (Clostridium Thyrobutyricum 633)

Molasses

Water bath (MGW LAUDA M20)

Sodium chloride (NaCl)

Autoclave machine

Test tubes

One litre bottle for measuring water displacement

Volumetric flask

pH meter

Conductivity meter

Gas chromatography (GC) Shimadzu GC14 with on-column injector

Flow meter

Eppendorf pipettes with polypropylene tips

3.2 Operating parameters for gas chromatography

Carrier gas hydrogen (safety guidelines), pressure 0.4 bar

Detector temperature: 350 °C

Column temperature: 40 °C (5 min), 20°/min to 340 °C, 340 °C (15 min)

Total running time: 41 min + equilibration time (about 10 min)

Injector temperature: column temperature + 5 °C

Detector: FID, Range 0, make-up gas nitrogen 20 ml/min

Injection volume: 2μl

3.3 Sample collection

The anaerobic bacteria used for the purpose of this experiment were collected from CHP-biogas plant at Ribe in Denmark on August 10, 2010.

3.4 Sample preparation

Sodium chloride (NaCl) was first added to each flask according to the salinity desired (i.e. 20 g/l, 40g/l etc.) except for the control (0 g/l).

700 ml of water was then added to all flasks.

50 ml of molasses was added to each flask.

Flasks were then placed in the water bath and heated until a temperature of 53oC is attained.

50 ml of the anaerobic bacteria was added to the flask.

All flasks were mixed properly to attain a homogenous content within the each flask and placed back in the water bath.

The initial pH and conductivity of all flasks were measured and recorded

From the above figure, it can be observed that four flask containing bacteria with different salinities are placed in a water bath. The bath is filled up with water up to three quarter of the tank level. The temperature of the bath is maintained at 53o C with the aid of a special heater placed inside the bath. Each of these flasks is tightly closed with a rubber cork to maintain anaerobic conditions. The cork has a special arrangement to mount the pH meter and conductivity meter. Daily monitoring of the system is carried out to ensure proper maintenance of anaerobic condition and working environment. The experiment was carried out in two batches because the water bath is not big enough to all flasks used for this experiment. Therefore the first batch was for salinity of 0g/l (control), 20g/l, 40 g/l, 60 g/l, the second batch was for salinity of 80 g/l, 100 g/l, 120 g/l, 140 g/l. For each of the salinity there was a replicate.

Each experiment was conducted for 120 hours (5 days) and during this period no nutrient (molasses) was added, this was to estimate the frequency of consumption of nutrient by these bacteria and to study the quantity of gases and acids they can produced with a specified amount of nutrient within the stipulated 120 hours. Every 24 hours, the pH and conductivity as well as volume of gas produced for each flask was measured and recorded.

Chapter 4

Results And Discussion.

CHAPTER FOUR

4.0 RESULTS AND DISCUSSION

Table 4.1: Quantity of NaCl added to each flask

SALINITY

QUANTITY OF NaCl

(g/l)

ADDED (g)

 

Batch 1

Batch2

0

0.00

0.00

20

14.05

14.04

40

28.03

28.02

60

42.03

42.02

80

56.04

56.03

100

70.03

70.03

120

84.03

84.02

140

98.01

98.03

Table 4.2: Initial pH and Conductivity reading

SALINITY

INITIAL pH

INITIAL

(g/l)

 

CONDUCTIVITY (ms/cm)

0

7.83

10.36

20

7.57

35.17

40

7.48

55.40

60

7.39

75.70

80

7.00

99.20

100

6.97

113.40

120

7.02

128.00

140

7.01

139.60

Table 4.3: Results for first batch

SALINITY

pH

CONDUCTIVITY

TEMPERATURE

GAS VOLUME

HOURS

DAYS

(g/l)

 

(ms/cm)

(0C)

(ml)

 

 

 

5.70

12.54

43.0

1300

24

20-Aug

 

5.55

13.31

36.8

0

48

21-Aug

0

5.15

12.81

36.9

0

72

22-Aug

 

5.08

13.25

39.5

40

96

23-Aug

 

5.10

13.07

39.2

0

120

24-Aug

 

6.12

34.90

41.8

2000

24

20-Aug

 

5.66

34.70

39.5

1100

48

21-Aug

20

5.76

36.50

38.8

850

72

22-Aug

 

5.10

35.10

39.9

20

96

23-Aug

 

5.16

37.40

42.2

0

120

24-Aug

 

6.46

56.00

39.5

0

24

20-Aug

 

5.85

57.20

40.7

0

48

21-Aug

40

5.52

57.20

38.6

0

72

22-Aug

 

4.93

57.10

41.4

250

96

23-Aug

 

5.08

58.00

42.4

10

120

24-Aug

 

6.50

77.10

41.9

0

24

20-Aug

 

6.42

75.90

39.5

0

48

21-Aug

60

6.44

76.60

38.2

0

72

22-Aug

 

6.16

76.60

42.1

130

96

23-Aug

 

6.04

76.90

43.9

50

120

24-Aug

Table 4.4: Results for second batch

SALINITY

pH

CONDUCTIVITY

TEMPERATURE

GAS VOLUME

HOURS

DAYS

(g/l)

 

(ms/cm)

(0C)

(ml)

 

 

 

6.74

94.40

38.2

60

24

27-Aug

 

6.74

93.30

39.7

40

48

28-Aug

80

6.40

93.20

39.6

70

72

29-Aug

 

6.88

95.60

40.2

20

96

30-Aug

 

6.59

95.40

40.2

5

120

31-Aug

 

6.87

109.40

41.5

230

24

27-Aug

 

6.94

112.40

42.6

0

48

28-Aug

100

6.90

107.70

41.4

60

72

29-Aug

 

6.50

114.40

42.3

20

96

30-Aug

 

6.43

111.70

43.6

10

120

31-Aug

 

7.00

123.50

42.5

40

24

27-Aug

 

7.11

122.70

40.9

50

48

28-Aug

120

7.02

124.60

44.2

40

72

29-Aug

 

7.07

121.90

43.7

20

96

30-Aug

 

6.91

118.40

43.0

5

120

31-Aug

 

7.96

139.20

42.2

30

24

27-Aug

 

7.27

138.80

43.4

50

48

28-Aug

140

7.24

139.00

43.6

20

72

29-Aug

 

7.37

138.80

45.3

10

96

30-Aug

 

7.39

138.70

45.4

0

120

31-Aug

4.1 Analysis of result

Figure 4.1: Cumulative Gas production for each salinity

From the figure above, it can be seen that at 20 g/l salinity, has the highest gas production (3970 ml), followed by 0 g/l salinity with 1340 ml, the others produced less gas. For the sake of clarity, another cumulative graph was drawn without 0 g/l and 20 g/l salinity, and as such, the other salinity will be shown clearly.

Figure 4.2: Cumulative Gas production for each salinity without 0 and 20 g/l

The above figure 4.6 shows the cumulative gas production without 0 g/l and 20 g/l. it can be observed that at salinity of 140 g/l has the least gas production, this is due high salt content.

Figure 4.3: pH against Salinity

The above figure 4.7 shows the trend of salinity measured during this experiment. The trend shows a decline in pH with time across all salinity. The highest pH was measured at 140 g /l. It can then be concluded that pH has a direct correlation with salinity. The higher the salinity the higher the pH.

Figure 4.4: Electrical conductivity vs. pH

The above graph of Electrical conductivity against pH shows that electrical conductivity increases as pH increases

Figure 4.5: Gas production per day

Figure 4.6: Gas production per day without 0 and 20 g/l

Figure 4.7: Gas production with pH

4.2 Gas composition analysis

The gas produced was initially analyzed and result shows that 70 % of the total gas produced was carbon dioxide, but the remaining 30 % was not known. Therefore further analysis with more gas detection technique was proposed. However, equipment for this analysis is not yet available as of date but is expected within the first week of October 2010.

4.3 Discussion

Microbial enhanced oil recovery process with multiple mechanisms occurring simultaneously depends on many factors to be successful. In this investigation, the experiment showed that it was possible to increase the adaptability limit of the considered bacteria growth to salinities up to 140 g/l. this was higher than the salinity of 45 g/l where the pure cultures were able to grow. The addition of molasses further increased the limit significantly. During the fermentation days the amount of carbon dioxide gas produced is limited due to the fact that it was carried out in a limited amount of substrate solution. Therefore more investigation has to be carried out on mass balance of this process to have a better understanding of the production of by-products.

During data collection like the pH, a little sample has to be removed from each flask on a daily basis. This frequently disrupts the anaerobic conditions inside the flask. Therefore more sophisticated arrangement has to be made so as to ensure proper anaerobic condition is maintained.

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