MICROBIAL ENHANCEMENT OF OIL RECOVERY JOANNE ALI
UNIVERSJTY O FSURREYM ICROBIAL ENHANCEMENT OF OILRECOVERYJOANNE ALIA dissertation submitted to in partial fulfilment of the requirements for the Degree of Masterof PhilosophyFaculty of Engineering and Physical ScienceUniversity of SurreySupervised by:Prof. Adel SharifDr Aidong YangDr. Muhammed JavedAugust 2012J o an n e All 2 0 12
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Declaration of OriginalityABSTRACT:IIVVAIM AND OBJECTIVES:CHAPTER 1GENERAL INTRODUCTION1.1 Microbial Enhanced Oil Recovery (MEOR)VII881.1.1Primary Recovery81.1.2Secondary Recovery81.1.3Tertiary Recovery91.1.4History o f MEOR101.1.5Bio-surfactants111.1.6Alcohols, acids, solvents and surfactants131.1.7Acids and Gases131.1.8Polymers131.1.9In situ and in above ground facilities14Reservoir conditions1.1.10141.2 Crude Oil161.3 Oil Field181.4 The Reservoir181.4.1Reservoir Pressure191.4.2Reservoir Temperature191.4.3Reservoir Porosity201.4.4Reservoir Permeability201.5 Salinity211.6 Extraction of Oil211.7 History of oil well and drilling process of oil well221.8 Microorganisms used in the MEOR process231.9 The use of aerobic and anaerobic organisms251.9.1Microorganisms in Petroleum Wells and unsuccessful method271.10 Emulsification of crude oil281.11 Economics of MEOR291.12 Sour and Sweet Crude Oil30
CHAPTER 2MATERIALS AND METHODS312.1 The MEOR Process at the Universityof Surrey (UniS)322.2 Media, Buffers, and Solutions332.2.1 Nutrient Broth Medium332.2.2 Nutrient Agar332.2.3 Trace element stock solution (TE)342.2.4 Aerobic defined medium or Minimal Medium (MM)352.2.5 Blood agar352.2.6 Methylene Blue Agar352.2.7 Preservation o f the culture362.2.8LG solution362.3 Bacterial Strains362.4 Characterisation of the isolates372.4.1Procedure fo r Gram Staining2.5 Procedures for the isolation of microorganisms37372.5.1General procedures to isolate MEOR microorganisms382.5.2Tests fo r bio-surfactant activities382.6 Growth media for halpohile organisms392.6.1Isolation o f halophilic organisms392.6.2Luria- Bertani (LB) Medium392.6.3M-168 medium402.7 Preservation of Bacteria412.9 Bacterial growth measurement42CHAPTER 3RESULTS AND DISCUSSION433.1 Isolation of Microorganisms433.2 Characterisation of the Isolates463.3 Growth Characterisation of the Isolates473.4 Bacterial Identification673.4.1Morphology o f the isolates673.4.216 SrDNA analysis o f the isolates673.5 Discussion71II
CHAPTER 4CONCLUSIONS AND FUTURE WORK734.1 Conclusions734.2 Future work on MEOR764.2.1Measurement o f Viscosity764.2.2Use o f Mixed Culture764.2.3Consolidated MEOR Process774.2.4Identification and Characterisation o f the new isolate114.2.5Identification o f Bio-surfactants114.2.6Genetic Engineering and Recombinant DNA Technology784.2.7Pilot Scale Studies78CHAPTER 5REFERENCES79CHAPTER 6APPENDIX896.1 Samples at 37 C896.2 Samples at 55 C896.3 16SrDNA Sequences of the isolates916.3.1 4916.3.2926.3.3936.3.4JA3594III
Declaration of Originality“I hereby declare that this thesis entitled Microbial Enhancement of Oil Recovery for partialfulfilment of the degree of MPhil in Chemical and Process Engineering was composed by andoriginated entirely from me. Information derived from the published work of others has beenacknowledged in the text and references are given including those of the Internet. Allquotations have been distinguished and all sources of information have been acknowledgedby means of references”.Date: 31st August 2012Name: Joanne AllIV
ABSTRACT;MICROBIAL ENHANCEMENT OF OIL RECOVERYJoanne AliSupervised:Prof. Adel SharifDr Aidong YangDr. Muhammed JavedThe microbial enhanced oil recovery (MEOR) uses specialised microorganisms along withthe injection of certain nutrients such as sugars, phosphates, and nitrates into the oil well toproduce metabolic activities thus, forming products such as bio-surfactants, polymers, acidsand gases which, lead to an increase in oil recovery. Economically, MEOR is more attractivein comparison with other oil recovery processes because it requires less capital and operatingcosts.The present project is based on selecting particular microorganisms, which can produce bio surfactants and other useful products to enhance the oil recovery, by inevitably increasing thereservoir pressure, reducing interfacial tension of the oil, increasing the oil flow to theproduction well and plugging the high permeable zones.In this study, 42 strains were isolated from the mixture of the contaminated soil obtained froma car garage (Woking, Surrey) and from an oil reservoir obtained from the Iranian Oil field(based in Ahvaz). They were isolated using complex and minimal media with added crude oilor hexadecane. These strains were then studied for their biomass concentrations andmetabolic activities. The initial screen was based on their bio-surfactant production, asrevealed by the formation of a zone of clearance on Blood agar plates and results werecompared with those of Pseudomonas putida which was obtained from a culture collectioncompany based in UK. Strains which were unable to produce bio-surfactant were removedfrom the next round of screening and therefore, the results were narrowed to selected numberof strains. The production of other chemicals, such as acids, polymers, and gases were alsostudied to various levels. On the basis of this investigation, 5 strains were chosen for furtherV
characterisation. These strains were initially identified by using staining techniques and otherbiochemical techniques and finally they were identified using 16SrDNA sequence analysis.These isolated strains were found to be closely related to Bacillus licheniformis, and Bacilluscereus. These strains grew on a variety of substrates and, at temperatures of 37 C and 55 C.However, one of the strains was not identified in this study and this strain was found growingat a temperature of 65 C.ACKNOWLEDGEMENTWould like to take this opportunity to thank Prof. Adel Sharif who has offered me assistanceall throughout these years and this study would have not been possible or successful withouthim. Also would like to express my deepest gratitude to my supervisor Dr. Muhammed Javedwho has been abundantly helpful and supportive.Lastly but not least, would like to express a sense of appreciation and love to my husband andmy two sons (Elias and Feras) for their constant support, love, and for their patience.VI
AIM AND OBJECTIVES:The aim of this research at the current stage is to isolate and characterise the bacteria whichcan be applied to the process of microbial enhancement of oil recovery (MEOR). The ultimateaim of this project at the university of Surrey is MEOR which is a tertiary oil recoveryprocedure and is usually performed by the injection of the bacteria and the required nutrientsto an oil well to enable bacteria to grow and carry their metabolic activities to enhance therecovery of the residual crude oil that is trapped in between the pores of the reservoir rocks.Trapped oil makes up about 70% of the total oil reserves. This value indicates that theprimary and secondary recoveries are to some extent inefficient. The aim is to selectmicroorganism(s) that will not cause any harm to the environment and not alter the physicaland chemical structure of the hydrocarbon chain of the crude oil. The selectedmicroorganisms must perform fermentation of inexpensive raw materials to produce bio surfactants. Most importantly the microbial cultures must survive under the extremeconditions of the oil well and reservoir.The first stage of this research was to carry out an experiment to isolate bacteria that havebeen cultured from a mixture of oil and soil. Properties such as growth behaviour, resistanceto the temperature, pressure, gas production, surfactant activity, and viscosity reduction (byvisual observation) were to be monitored.In the second stage (this procedure will be carried out for the PHD), these properties will beinvestigated thoroughly in a bio- reactor under specific conditions, pressures, andtemperatures similar to those of a reservoir. Eventually cultures will be applied to an injectionwell, and then the well will be shut off for several months to allow the bacteria to grow and toproduce its surfactants and to promote the mobility of oil to the production well by reducingthe oil viscosity. The current study is only focused on the first stage due to time limitationsand facilities availability.VII
CHAPTER 1GENERAL INTRODUCTION1.1 Microbial Enhanced Oil Recovery (MEOR)In the MEOR process, oil recovery is enhanced by the use of microbial activities. Initiallywhen oil pools in the ground (known as oil reservoir) are found, a well is drilled to thereservoir to bring the oil to the ground (known as oil recovery). Therefore, the utilisation ofthe MEOR process will help to recover this trapped oil and will extend the life of oil wells.Therefore, there is an enormous opportunity to improve this process in order to recover thereminder trapped oil in the ground which is unrecovered by other conventional methods and itis estimated to be about 70% (Khire and Khan, 1994).In the oil fields there are three stages of development:1.1.1Primary RecoveryIn the primary stage, the oil is recovered without the addition of water or gas into the reservoirsince there is enough pressure to force the oil upward to the ground. Primary recoveryinvolves the oil and gas production using the natural pressures of the reservoir. These naturalresources act as the driving forces and push the material to the surface of the wells. Oftenwells are injected with fluids, to disrupt hydrocarbon bonds to improve the flow rate of the oiland gas from the reservoir to the wellhead (Planckaert 2005).1.1.2Secondary RecoveryIn the secondary stage, water, steam and/or gas is injected into the well to encourage themovement of the oil to the ground. However, during primary and secondary stages only onethird to half of the oil can be recovered while the remaining oil is trapped in the reservoir.Secondary Recovery uses other mechanisms, such as re-injecting gas (methane) and floodingthe well with water, to increase the flow rate of the remaining oil and gas from the primaryprocess.
1.1.3Tertiary RecoveryThe MEOR is a tertiary stage process of the oil recovery. In the MEOR process, suitablemicroorganisms, nutrients, oxygen (in some cases), heat, steam and/ or hot water are injectedinto the reservoir through the oil well to stimulate the flow of oil and gas to the surface of thewell. The microorganisms multiply and produce chemicals to move the trapped oil into thewater phase. The microorganisms grow in the interface between the oil and water, and releasethe metabolic products due to their metabolic activities. Such products are helpful for the oilrecovery. The microbes oxidise the oil to fatty acids which, act as detergents (surfactant) andtherefore, reduces the interfacial tension and oil viscosity. They can also produce smallamounts of carbon dioxide (CO2 ) gas, due to beta-oxidation of the fatty acids and the gas canpressurise the oil while their biomass between oil and rock physically displaces the oil. Thesemicrobial activities thus enhance the oil recovery. In EOR process fluids are moved from aninjection to a production well and physical and chemical interaction may occur as these fluidsencounter different rocks and or other fuels fluid.There are two methods by which the MEOR process is applied. The first method is the mostcommon and popular approach, where the oil well is injected by a small volume of microbialcultures with the nutrients then the well is shut for a period of time to ensure the grov h of themicrobes prior to extraction of oil recovery. This technique is relatively inexpensive and givesa quick response (Sheely, 1990). In the second method the microbial culture and nutrients areinjected with water flooding to improve both sweep efficiency and displacement efficiency(this process involves the displacing of oil with water and gas that occurs microscopically andmacroscopically in the reservoir). Sweep efficiency (SE), is a measurement of theeffectiveness of an enhanced oil recovery that depends on the volume of the reservoircontacted by the injected fluid. The volumetric sweep efficiency is an overall result thatdepends on the injection pattern selected by considering the fractures in the reservoir, positionof oil/gas and oil/water contacts, reservoir thickness, permeability and a real verticalheterogeneity (which is the variation in the rock properties and variable properties of thereservoir from one place to another), mobility ratio, density difference between the displacingand the displaced fluid, and the flow rate.However, the displacement efficiency (DE) can be improved by the reduction of the residualoil saturation. DE refers to the fraction of the oil in place that is swept from a unit volume of
the reservoir, and is a function of fluid viscosities and the relative permeability characteristicsof the reservoir rock. (Archer and Wall, 1986).1.1.4History o f MEORThe idea of MEOR was first introduced in 1926 by Beckman, but was not studied further until1950’s when ZoBell and his colleagues (Desouky, et. al., 1996) proposed that the microbialaction degraded the organic matrix surrounding the hydrocarbons produced by plants andanimals and as a result, oil was released, migrated, and accumulated to form a petroleumreservoir. Hitzman (1962) and co-workers patented a process for the injection of bacterialspores along with nutrient into a reservoir. The spores would germinate and enhance theremoval of oil from the reservoir. In 1983 Rountree applied the first MEOR process to 3 oilfields and obtained an increase in the production level of oil from 26 to 60 bbl (blue barrel)/day (Hitzman, 1962). Bryant and associates demonstrated that the spores within the reservoirwill germinate and enhance the recovery of oil from the reservoir rocks. In another report, theapplication of different bacterial strains to heavy oil of an API (American Petroleum Institute)of 14 to 17, up to 70% of the crude oil was recovered when the selected strains were appliedwith water flooding (Bryant, et. ah, 1998). 78% of the MEOR projects are reported successfulin reducing the water production rate and showing an increase in oil production.The most likely implementation of MEOR will entail inoculation of injection wells withmicrobes, a suitable shut- in period for incubation and then resumption of water floodingcontaining appropriate nutrients (Lin, et. al, 1994; Banat, 1995). The continual presence ofmicroorganisms ensures further reduction of viscosity of the residual oil that is left in thereservoirs and therefore, secondary recovery methods may no longer be required. However,MEOR has certain constraints which mean that all other conventional methods as areplacement cannot be abandoned. Nevertheless, this process is unique because, bacterial cellsare injected to the reservoir and they multiply in situ.The production of acids, solvents, gases, and surfactants in or near the well bore preventsscale and paraffin deposition. They also, change the wettability of the rock, and change thefluid saturations. These characteristics enable the well to improve its oil drainage (Adkins, et.al., 1993; Bryant and Douglas, 1998; Mclnemey et. al., 1990).Wettability of a reservoir is the actual process when a liquid spreads on (wet) a solid substrateor surface. Wettability can be determined by measuring the displacement effectiveness of10
injected fluids and ultimate oil recovery. Kovscek et. al., (1993) observed that wettability is aprime factor in controlling multi phase flow and phase trapping since wetting fluids occupythe smallest and tiniest and most hydro-dynamically resistant pore channel.Furthermore Banat, et. al, (1995) patented four different species of common soil organismssuch as Pseudomonas fluorescens. Bacillus cereus, Bacillus thuringiensis, and Bacillussphaericus which are expected to have applications in MEOR because, of their bio-surfactantsproduction.Carbon dioxide and/ or methane production resulting from the bacterial fermentation and theneutralisation of acid products by the reservoir rock would re-pressurise the reservoir.Solvents, mainly alcohols (methanol, ethanol, propanol, isobutanol, and butanol) and to someextent formaldehyde and acetone could reduce oil viscosity.Hence, the candidate microorganisms in the MEOR process would be expected to producesone or more of the following products:1. Bio-surfactant2. Solvents, such as Alcohols, Ketones3. Acids and gases, such as carbon dioxide4. Polymers1.1.5Bio-surfactantsBio-surfactants are produced by microorganisms and they are lipids and glyco-lipids. Theyare substances that adsorb to and alter the conditions prevailing/-exist at interfaces.Furthermore, they have very versatile chemical structures and surface properties (Rosenberg,el a\., 1997). Bio-surfactants are amphiphilic compounds produced on living surfaces, mostlythey are microbial cell surfaces, or excreted extra-cellular materials and composed of ahydrophobic and hydrophilic layer moieties that reduce the surface tension and interfacialtension between individual molecules at the surface and interface respectively (Lin, et al,1994; Karanth et. al, 2005). The polar layer can be carbohydrate, an amino acid, a phosphategroup, or some other compound. However, the non-polar part of this layer is composed of along chain of fatty acid. The non-pathogenic microorganisms can ingest on the hydrocarbonswith the injected nutrients as a food source which, they metabolise and excrete bio-products(Brown, 1992, Portwood, 1995).11
Bio-surfactants help to disperse oil and this is the main reason for utilising bio-surfactants inthe MEOR work. They have very diverse chemical structures and surface properties(Rosenberg, et. a\., 2001).Bio-surfactants are used for environmental application because, they are easily biodegradable(Oberbremer et. al., 1990; Harvey et. a\., 1990; Van Dyke et. al., 1993). One of the organismsthat have shown to produce an effective bio-surfactant under aerobic and an-aerobicconditions is Bacillus licheniformis (Jenneman et. al., 1983). This fact has attracted manyresearches in the MEOR field. However, the organisms that are selected for the MEOR mustbe tolerant to crude oil and be able to tolerate the reservoir hostile conditions (Premuzic E. Tet. al., 1996).The bio-surfactants are divided into three categories and generally they comprise: Low molecular weight bio-surfactants:Low molecular weight bio-surfactants are generally lipo-peptides or glycol-lipids,which have mono or disaccharide unit linked to the fatty acid moiety. They function tolower the interfacial tensions efficiency. The amphipathic nature of surfactin maycontribute to some of its interesting biological properties, such as the formation of ionconducting pores in membranes (Rosenberg et. al., 2001). High molecular weight bio-surfactants:High molecular weight or polymeric bio-surfactants include polysaccharide,polysaccharide protein complex, lipo-polypeptides or protein with emulsifyingproperties. These polymers are tightly bound to the surface of the crude oil (Passeri et.al., 1991). The microbes which produce these surfactants, under suitable conditions,release the capsule polysac
2.6.1 Isolation of halophilic organisms 39 2.6.2 Luria- Bertani (LB) Medium 39 . microorganism(s) that will not cause any harm to the environment and not alter the physical . resources act as the driving forces and push the material to the surface of the wells. Often wells are injected with fl
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PART I The Scope of Microbiology 1 1 Evolution, Microbial Life, and the Biosphere 3 2 Historical Overview 31 3 Fundamental Chemistry of the Cell 55 4 Structure and Function of Bacteria and Archaea 75 PART II Microbial Physiology: Nutrition and Growth 123 5 Nutrition, Cultivation, and Isolation of Microorganisms 125 6 Microbial Growth 153 7 Control of Microbial Growth 179
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