Last updated: June 14, 2019
Topic: BusinessEnergy
Sample donated:

Microbial Enhanced oil Recovery ( MEOR ) is peculiarly suited for application in carbonate reservoir, after secondary oil recovery, there are still big sum of oil left in the reservoir. Some bacteriums are able to increase the oil production when injected into the oil reservoir. To excite such anaerobiotic microbial increased oil recovery, foods is injected together with the injection H2O.

Oil recovery requires two to three phases which are briefly described belowStage1: Primary Recovery – 12 – 15 % of the oil in the well is recovered without the demand to present other substances into the well.Phase 2: Secondary Recovery – The oil well is flooded with H2O or other substances to obtain an extra 15-20 % more oil from the well.Phase 3: Third Recovery – This phase may be accomplished through several methods which includes MEOR to to boot retrieve up to 11 % more oil from the well.

We Will Write a Custom Essay Specifically
For You For Only $13.90/page!

order now

Figure 1.

1: Flow diagram for different enhanced oil recovery procedures

Layout for different recovery techniques are shown in figure 1. Primary and secondary recovery techniques are normally called conventional recovery. Primary recovery is done by natural flow which is normally enhanced by reservoir natural force per unit area, and unreal lift such as pumps and gas lift, etc. Secondary recovery is done by H2O folding and force per unit area care by gas reinjection. Third recovery techniques cover wide country which includes thermic recovery such as unmoved burning and steam implosion therapy, solvent recovery is done by methods such as polymer implosion therapy and wetting agent enhanced H2O inundation. Chemical enhanced recovery methods include gas injection or hydrocarbon mixable injection and N and fluke gas implosion therapy. Microbial enhanced oil recovery which is the chief focal point of this undertaking will be explained better in the following chapter ; nevertheless, it is fundamentally injection of bugs such as bacteriums into oil reservoir to assist retrieve oil.



The purpose of this undertaking is to analyze the adaptability of anaerobiotic bacteriums ( Clostridium Thyrobutyricum 633 ) to different salts and look into the consequence of the microbic strain on permeableness of the Danish Nord Sea Chalk.To accomplish this purpose, the following aim has been set:Check adaptability of microbic strain to high saltsMicrobial gas production and kineticss of metamorphosisCarry out home base count experimentObservation of agitation procedure and microbic analysisTo find and mensurate the volume of C dioxide gas produced by these bugs when exposed to different saltsTo find the sum of acid produced during agitation procedureStatistical analysis of consequences to derive theoretical accountImprovement of experimental process

Chapter 2

Literature Review.

Chapter TWO


The undertaking work is based on perusal of the microbial enhanced oil recovery method and the possibilities of utilizing this in the Danish sector of the Nord Sea. The undertaking undertaking applies experimental process and the particular to look into if these bugs can last under reservoir conditions and produce merchandises of import in oil recovery.

However, it is worthwhile to discourse the assorted oil recovery techniques.

2.1 Oil recovery technique

All methods of oil recovery will be explained briefly.

2.1.1 Primary recovery

If the belowground force per unit area in the oil reservoir is sufficient, so this force per unit area will coerce the oil out to the surface of the Earth. Gaseous fuel, natural gas or H2O is normally present, which besides supply needed belowground force per unit area.

In this state of affairs, it is sufficient to put a complex agreement of valves ( Christmas tree ) on the well caput to link the well to a grapevine web for storage and processing. Normally oil is recovered by natural agencies and unreal lift like pumps and gas lift.

2.1.2 Secondary recovery

Over a life-time of an oil well, the force per unit area will fall and at some point there will be deficient belowground force per unit area to coerce the oil to the surface of the Earth.

If economical, as frequently is, more oil in the well is extracted utilizing secondary recovery methods. Secondary oil recovery uses assorted techniques to help in retrieving oil from depleted or low force per unit area reservoir. Sometimes, pumps such as beam pumps and electric submergible pumps ( ESPs ) are used to pump the oil to the surface of the Earth. Other secondary recovery techniques increases the reservoir ‘s force per unit area by H2O injection, natural gas reinjection and gas lift, which inject air, C dioxide or some other gases into the reservoir.

Together, primary and secondary recovery by and large allows 25-35 % of the reservoir oil to be recovered.


2.1 Water injection

The productiveness of bing oil Wellss can be significantly increased by the usage of H2O injection. Statistics has shown that a reservoir produces merely 37 % oil in the first recovery. By utilizing H2O injection, a reservoir can bring forth more than 50 % of its oil. One of the most of import issues during oil production is to maintain the matrix/formation every bit clean as possible to keep maximal oil production. Water is injected for two grounds: foremost is for force per unit area support of the reservoir. Second is to brush or displace the oil from the reservoir, and force it outward.

Figure 2.

1: illustration of Gas injection utilizing C dioxide

Figure 1.2 shows C dioxide injection in reservoir to retrieve oil, C dioxide becomes mixable with oil to cut down viscousness and increase mobility of oil return with produced oil and separated from it. Nitrogen and gas implosion therapy

Nitrogen and fluke gas about 87 % N and 12 % C dioxide is used in topographic point of hydrocarbon gases because of economical grounds. Nitrogen competes with C dioxide because it is economical and its squeezability is much lower.

For a given measure at standard status N will busy much more infinite at reservoir force per unit area than C dioxide and methane at the same status. Nitrogen has a hapless solubility and lower viscousness in oil and requires much higher force per unit area to make miscibility.


4 Solvent recovery Polymer implosion therapy

Both man-made polymer such as polyacrylamides and natural polymers are used for betterment of sweep efficiency. Extra polymer makes the H2O more syrupy so that oil is produced quicker.

Obviously, this is non a good thought n a low permeableness reservoir or one with high clay content that absorb the polymer. However, polymer-augmented H2O inundations can be profitable


3.4.2 Surfactant-Enhanced Water inundation

Three types of chemical inundations exist. The first is an alkaline-augmented polymer inundation. Another is an alkaline-surfactant polymer inundation.

The 3rd is a micellar or low interface tenseness inundation ( Donaldson, 1989 ) .Amongst the available third oil recovery techniques, MEOR is arguably the best for many grounds. One cardinal factor in the choice of microbic enhanced oil recovery is the economical potency, by which desirable chemicals and gases are produced to heighten oil recovery. MEOR procedures are besides energy efficient and environmental friendly as compared to other recovery techniques.

1 History of bugs used in MEOR

MEOR is a engineering that has a history based on over 60 old ages of research and field surveies. The earlier plants by ZoBell CE and Updegraff D ( USA ) , Kuznetsov SI and Shturn DL ( USSR ) , shows the international range of the work. This work was expanded in the 1950s chiefly by research workers Coty VF, Yarborough H and Hitzman DO in the major oil companies in the United States.

In MEOR, the procedure that facilitates oil production is complex and may affect multiple biochemical procedures. Microbial biomass or biopolymers may stop up high permeableness zones and lead to a redirection of H2O inundation, produce wetting agents which lead to increased mobilisation of residuary oil, addition gas force per unit area by the production of C dioxide or cut down the oil viscousness due to digestion of big molecules. Application of MEOR engineerings

MEOR engineerings have the common footing of presenting or exciting feasible micro beings in an oil well reservoir for the intent of heightening oil recovery. However, this wide generic definition of MEOR is non a individual methodological analysis but is a broader engineering which can be designed for different and selective applications.

It is convenient to split the MEOR engineering into the undermentioned application groups:Single good stimulationMEOR H2O inundationsParaffin ‘s remotionViscosity alterationWater recreationHeavy oil alterationThe categorization of MEOR engineering by the proposed oil let go ofing mechanism shows the scope of microbic effects which can be identified or expected to happen to which the MEOR system can be directed. MEOR Oil Releasing Mechanism

Gas coevals: The production of gases will help the supplanting of oil in the pore infinites.

Acerb production: Organic and inorganic acid production by bugs will fade out carbonate sedimentations, Fe sulfide and disintegration and sulphate stuffs.Surfactant production: Biosurfactants produced by the beings consequence in the decrease of interfacial surface tenseness of the oil/water bond.Other MEOR oil let go ofing mechanisms includes:Physical oil supplantingBiopolymer productionHydrocarbon alterationViscosity alterationSelective plugging of high permeableness zones within a reservoir is necessary to accomplish oil recovery. This is best achieved in MEOR procedure where cells stimulated to turn profoundly in a formation where production of biomass and merchandises will hold the greatest impact.

If growing occurs chiefly at the well bore, so face stop uping will ensue, and no extra oil will be recovered, go forthing the reservoir unproductive. The Science of MEOR

Figure 2.2: Conventional position of MEOR layout



5 Types of bugs and their choice

MEOR has gained much attending in recent times, but it is deserving observing that non all bugs can last in such conditions as found in an oil well, therefore the bug which are able to defy these conditions are discussed below:

6 Microbes used in MEOR

There are many types of bacteriums used in MEOR, they include aerophilic and anaerobiotic bacteriums and are divided on the footing of their demand for O. In this undertaking work, the bacteriums used were anaerobiotic from CHP-biogas works at Ribe in Denmark. Choice of Bacteria

The choice of specific bacteriums is considered in this method. There are a batch of bacteriums available, but the normal conditions for bulk of bacteriums is 5 % Sodium chloride, optimal temperature of 37 degree Celsius, pH less than seven. Factors impacting growing of bacteriums

There are many factors which affects the growing of bacteriums. Some of which are explained in the below:Salt: The term salt refers to the sum of dissolved salt that are present in H2O. Sodium chloride is the prevailing ions in sea H2O, the concentration of Mg, Ca and sulfate ions are besides significant. High salt and toxic substances are responsible for restricting the growing of bugs.

Halophiles are salt loving bugs which use Na chloride and besides have complex food demands. Moderate halophiles can turn anaerobically at temperature greater than 50o C. The salt in the northern portion of Danish oil field is approximately 40g/l or more. Since salt excessively high, formation H2O is diluted with sea H2O during injection in the well. In order to execute experimental and laboratory analysis, a sample of green goods H2O is taken so as to cognize how much salt can be controlled ; hence microbic gas production has been tested up to 140g/l.

Temperature: Extreme high temperature affects the growing of bacteriums, although they need mean temperature for growing. Thermopiles are bacteriums which are heat loving ; these bacteriums have an optimal growing temperature of 45 Os C – 80 o C. Their membranes are remarkably stable at this highly high temperature. Therefore many of import biotechnological procedures utilise thermophilic enzymes because of their ability to defy intense heat.

So before shooting these bacteriums into the reservoir, the temperature of the reservoir should be considered, hence, choice of the right thermophilic bacteriums for high temperature is really of import.Consequence of pH: pH is the step of sourness or alkalinity of a solution. Simply pH is the step of concentration of H ions in a solution. It is a step of the activity of dissolved H ion.

In pure H2O at 25 o C, the concentration of H ion equals the concentration of hydroxide ions ; this is known as “ impersonal ” and corresponds to a pH degree of 7.0. Solutions in which the concentration of H ions exceeds that of hydroxyl ion has a pH degree lower than 7.0 and are known ad bases. The pH reading of a solution is normally obtained by comparing unknown solution to those of known pH, and there are several ways to make so. More favorable pH status for micro beings is about 7 and really few of them can turn below2 and above 10. Micro organisms capable of life at really low pH are called acidphilies and those which live at high pH are called alkaliphiles.Pressure: Extreme force per unit area affects the growing and metamorphosis of micro beings.

A force per unit area lower than 100-200 standard pressure has no consequence on microbic metamorphosis, nevertheless, force per unit area of the scope of 500-600 standard pressures have restricting consequence on growing of bacteriums. The ocean floor possesses high force per unit area. For most MEOR processes barophilic beings will non be necessary, alternatively, barotolerant bugs can turn at high force per unit areas, but do non necessitate these high force per unit areas for optimum growing. The ability to turn force per unit area depends on the energy beginnings available, inorganic salts present, pH and temperature.

Adaptation of microbic civilizations to higher force per unit area therefore is possible.Toxic elements: Chemicals which have toxic effects on micro beings are found in some reservoirs. These chemicals include co-surfactant, surfactant, biocides, ethylenediaminetetraacetate, and methylbenzene, many of which are used in assorted chemical EOR. Sodium and Potassium may be exchanged without impairing the growing of micro beings. Magnesium has higher toxicity than Na and K, but the most toxic formation H2O are those with high Calcium Chloride ( CaCl2 ) , so adaptability should be considered before shooting micro beings in such toxic environment.Foods: In MEOR recovery procedure, to accomplish maximal degree or required bacterial count, an optimal concentration and the right type of food is desired. In this method, food can be at the largest disbursal, so it is of import to hold the right combination and measure available. Some of the most common foods are as follows:MolassessIn-situ hydrocarbon ( rough oil )Molassess, N and P saltIn this experiment, merely molasses have been used as food.

Molassess is easy available as slurry and in the existent field ; it can be pumped down easy into the well. The conveyance of the food into the reservoir every bit good as and out of bacterial cell requires H2O. Water maps as a matrix through which the cellular chemical science takes topographic point. The grade of H2O available for chemical activities and growing is called H2O activity. Osmotic force per unit area affects the H2O activity ; the production of CO2 gas during the agitation procedure can farther increase this force per unit area around the system, hence, H2O activity is reciprocally relative to osmotic force per unit area.

2.2 Physical and environmental restraints

Gregory ( 1984 ) begins his expounding of the basicss of MEOR by placing physical ( temperature, force per unit area, and pore size/geometry ) , chemical ( pH, H E, electrolyte composing ) and biological factors that constrain microbic activity in hydrocarbon reservoirs.

In the undermentioned subdivisions, these restraints, and their interactions, are discussed. More by and large, it is to be observed that the same factors control the being and behaviour of bacteriums in other subterraneous environments, which are of relevancy in other practical contexts – most notably the direction and redress of groundwater resources. The observation that legion species of bacteriums found in such environments can defy, or even thrive, under physical conditions that are unfriendly to most life signifiers has given fresh drift to analyze of this subject, in relation to research on the beginnings of life on Earth and the possible being of life on other planets.

2.2.1 Pore Size

The being of bacteriums in deep subsurface stones has been disputed in the past, but since the coming of modern tracer techniques and improved sampling protocols ( Frederickson and Phelps, 1996 ) , is now by and large accepted.

Possibly the most obvious restraint that applies to deep subsurface bug is the size of the pores. In some surveies, the lower bound of average pore sizes has been shown to be smaller than the size of known bacteriums. For illustration, through phospholipids fatty acerb checks and measurings of 14 C ethanoate mineralization, Frederickson et Al. ( 1997 ) assessed shale and sandstone nucleuss from a site in northwesterly New Mexico for microbic activity.

They found no metabolic activity was detected in nucleus samples with pore pharynxs narrower than 0.2I? m, although in some instances it was after extended incubation. The observation of much higher degrees of metabolic activity in more permeable samples led these writers to reason that sustained bacterial activity require interconnected pores of diameter at least 0.2I? m.

2.2.2 Sourness

The sourness or ( alkalinity ) of the environing aqueous medium, measured by the pH, is important in several respects


2.1 Surface Charge

On the cellular graduated table, pH controls the extent of ionisation of the protein molecules that are embedded in the cell walls. As a consequence, cellular surfaces are by and large charged and surrounded by diffuse dual beds, the thickness of which is controlled by the overall electrolyte concentration. Interaction of these ionic space-charge parts with those that besides surround little atoms of mineral stages can strongly impact the gesture of the cells through a natural porous medium. The consequence of pH on the surface charge of a protein depends on the comparative Numberss of acidic and basic groups in the side ironss.

Protein molecules are frequently characterized by a pH called the isoelectric point, at which the positive and negative charges ensuing from ionisation of side ironss are balanced.


2 Enzyme Function

Some of the embedded cell wall proteins play a important function in the consumption of foods, riddance of waste merchandises, and care of right electrolyte concentrations ; on a molecular graduated table, their ability to execute these maps besides depends on their extent of ionisation. The rates of the enzymic procedures that occur in respiration are strongly dependent on the pH. There by and large exists an optimum pH, lying between 2 and 9.5, for the rates of such procedures. The mineral stages in a porous medium ( peculiarly carbonates ) , and the proteins themselves can exercise a buffering consequence, which can extenuate the lowering of the pH by the acids generated by primary metamorphosis.

2.2.3 Oxidation Potential

Cellular respiration consists of enzymically mediated negatron transportations from an negatron giver ( cut downing agent, in chemical idiom ) to a terminal negatron acceptor ( oxidising agent ) .

Apart from a few rare instances where merely one mole of negatrons is transferred from each mole of reducing agent, this negatron transportation about ever involves a figure of intermediate negatron transportation stairss, which can be rather legion if the original negatron beginnings are complex molecules such as sugars. The thermodynamic drive force for these negatrons transfer processes is expressed quantitatively in footings of the oxidization potency, H E ( measured in V ) , which is the Gibbs energy alteration divided by the figure of moles of negatrons transferred. Harmonizing to the Nernst equation ( treatments of which can be found in most text editions of physical chemical science ) , this measure depends logarithmically on the concentrations ( purely talking, the activities ) of non merely the oxidized and reduced signifiers of the negatron acceptor, but besides of H ions and other species that might be involved. Therefore, for aerophilic respiration, the terminal negatron acceptor is O, which is reduced to H2O harmonizing to the overall equation

2.2.4 Oligotrophy and Heterotrophy

To explicate the being of active microbic communities in environments such as deep granitic and basaltic aquifers, where food degrees are expected to be highly low, it was suggested by Stevens and McKinley ( 1995 ) that such beings can be sustained by H generated by decrease of minerals by groundwater. Although many species of hydrogen-consuming lithotrophic bacteriums have been described, and it is good known that appreciable H fugacities can be ‘buffered ‘ by some naturally-occurring mineral gatherings, the suggestion that microbic communities could be sustained geochemically in this manner has, nevertheless, been disputed by Anderson et Al. ( 1998 ) .

These writers argued that basalt does non bring forth H under somewhat alkalic conditions, and that the production of H under somewhat acidic conditions can non be sustained over geological clip graduated tables. But a more recent treatment presented by Nealson et Al. ( 2005 ) points out that the most of import and hard issue to be established is the long-run independency of such communities from the merchandises of photosynthesis ; at nowadays this is best regarded as an unfastened inquiry.Considerable attending has been devoted to the survey of heterotrophic bugs in sandstones and shales, and the possibility that these beings are sustained by organic stuff carbon monoxide deposited with the deposits.

In a recent reappraisal, Krumholz ( 2000 ) considers formations incorporating jumping beds of sandstone and shale, and discusses experimental grounds that organic affair and agitation merchandises present in the shales can spread across sandstone shale boundaries and back up microbic communities in the sandstone, next 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 sedimentations.


5 Water and Electrolytes

The concentrations of electrolytes and other dissolved species required for proper cellular map is maintained by enzymically mediated exchange of solutes or dissolver with the environing medium. Dissolution of electrolytes reduces the thermodynamic activity of H2O, w a, Dissolution of electrolytes reduces the thermodynamic activity of H2O, aw. This consequence is measured by the ratio of the fugacity of H2O above the solution to that of pure H2O. At temperatures far below the critical point of H2O, the fugacity of H2O is about equal to the vapour force per unit area For illustration, w a in sea H2O is about 0.98, while in inland salt lakes it can be every bit low as 0.75.

Since the H2O activity matching to appreciable electrolyte concentrations differs merely somewhat from 1, an alternate step is provided by the osmotic force per unit area of the solution, which is defined as the hydrostatic force per unit area that must use to a solution to raise its vapor force per unit area to that of pure H2O. Therefore, for sea H2O ( of about 3.3 % salt ) , the osmotic force per unit area estimated from Virginia n’t Hoff ‘s equation is about 2.8 MPa. Differences in ionic strength across membranes provide a powerful drive force for diffusion of H2O into cells ( when the environing medium is a more dilute electrolyte ) or out of cells ( when it is more concentrated. ) While most bacteriums are incapable of lasting in media with tungsten a below about 0.95, minimal H2O activities for Pseudomonas species ( which are of involvement as campaigners for MEOR ) are well lower ( 0.91 ) .

Extreme halophiles, such as Halococcus, can last when w a =0.75 ( Todar, 2008 ) . Aerobic debasement of benzine, methylbenzene and xylol by halotolerant Marinobacter species in dirt contaminated with oilfield seawaters was demonstrated by Nicholson and Fathepure ( 2004 ) , proposing possible utility in environmental redress. Anaerobic bacterium from hypersaline environments are of peculiar involvement to MEOR, sing the high salt of connate H2O frequently found in oil-bearing formations. A reappraisal of such beings by Ollivier et Al. ( 1994 ) devoted considerable attending to sulfate-reducing beings that feed on polymeric substrates such as amylum, cellulose, and chitin, and the work of McMeekin et Al. ( 1993 ) on anaerobiotic micro-organisms isolated from concentrated salt lakes in Antarctica suggests applications to hydrocarbon debasement. In add-on to MEOR and environmental redress, the survey of halotolerant bacterium is besides relevant to nutrient saving ( Vilhelmsson et al.

, 1997 ) .In add-on to the specific chemical and biochemical effects that are frequently associated with high electrolyte concentrations, non-specific effects can be expected. Solubility of the huge bulk of non electrolytes decreases with increasing ionic strength. This phenomenon, which is known as the ‘salting out ‘ consequence, is peculiarly pronounced for non polar solutes ( which tend to hold low solubilities in pure H2O ) . Important illustrations are O ( the concentration of which controls the thermodynamic drive force for aerophilic metamorphosis ) , and C dioxide, ionisation of which controls the pH of many natural Waterss. In this manner, high electrolyte concentration could impact both pH and H E.

2.2.6 Temperature

The addition in random molecular gesture ensuing from an addition in temperature by and large exerts negative effects on enzyme map, since the active-site constellations required for contact action are disrupted. At still higher temperatures, the hydrogen-bonded 3-dimensional agreement of the amino-acid ironss besides becomes disordered, ensuing in irreversible denaturation. This molecular image of the effects of temperature on enzyme map is by and large accepted, but it is besides to be observed that the temperatures at which these phenomena occur vary widely between beings. In general, bugs can be classified harmonizing to their optimal temperature scope as psychrophiles ( & lt ; 25oC ) , mesophiles ( 25-45oC ) , and thermophiles ( 45-60oC ) . The comparatively recent find of bugs that can last in H2O at temperatures above 100oC has well extended the scope of conditions under which life can be expected to be.

Microbes that thrive under such utmost conditions are by and large referred to as ‘extremophile ‘ . Since the mean geothermic gradient beneath the continents is of the order of 25oC per kilometer, and presuming more conservative upper bound of 110oC for bacterial activity, as suggested by the work of Blochl et Al. ( 1995 ) , the biosphere could widen up to 5 kilometers beneath the surface of the Earth ( compare Gold, 1992 ) .


2.7 Pressure

The effects of force per unit area on micro-organisms are closely associated with those of temperature, since elevated force per unit areas in natural environments are ever associated with temperature fluctuations. Specifically the force per unit area in the ocean additions by about 10 MPa for every kilometre of deepness, while the temperature of the ocean is about 3oC below approximately 100 m. On land, the force per unit area additions by about 3 MPa per kilometer deepness, but the temperature additions by about 25oC per kilometer. Therefore, in footings of the earlier nomenclature introduced to depict the temperature tolerance of bacteriums, a marine bacteria that thrives on the seafloor at a deepness of 3 kilometers would be a psychrophile, while its tellurian opposite number at the same deepness resistance would be a thermophile. An obvious exclusion to this generalisation would be the bacterium in the locality of hydrothermal blowholes on the seafloor ( the alleged ‘black tobacco users ‘ ) , some of which can defy temperatures every bit hot as 121oC ( Miroshnichenko and Osmolovskaya, 2006 ) .

Indirect and direct effects of force per unit area on cellular map can be identified. Growth rates of normal bacteriums lessening to zero as hydrostatic force per unit area approaches about 40 MPa. ZoBell and Johnson used the term ‘barophilic ‘ to depict bacteriums whose growing rate is enhanced at elevated force per unit area. ( The prefix ‘baro- ‘ is sometimes replaced by ‘piezo- ‘ . ) It is besides customary to mention to bacteriums for which the decline of growing rate commences at force per unit areas above 40 MPa as ‘piezotolerant ‘ . A 3rd category of bacteriums, which can non be grown under ambient conditions, are referred to as ‘obligatory piezophiles ‘ .The microbiology of bacteriums isolated from the deepest oceans has been reviewed by Jannasch and Taylor ( 1984 ) and Yayanos ( 1995 ) .

Kato and Bartlett ( 1997 ) depict the designation of Pressure-regulated cistrons from deep-sea bacteriums of the genus Shewanella. Imposition of high force per unit area affects the fluidness and water-permeability of the cell walls, doing the phospholipids bilayers to pack more tightly and presume a more ordered constellation. Piezotolerant organisms seemingly compensate for this by increasing the proportion of unsaturated fatty acids, which have a much lower inclination towards such wadding ( DeLong and Yayanos, 1985, 1986 ; Kamimura et al. , 1993 ) . A more recent reappraisal by Daniel et Al. ( 2006 ) describes other impressive progresss that have been made in the molecular-level apprehension of force per unit area effects on facets of bacterial physiology. Under high force per unit areas, the DNA dual spiral becomes denser, which can interfere with cistron look and the associated protein synthesis.

Another of import factor is the steroid alcohol content ; membrane lipoids that have high cholesterin content are more pressure resistant that those that contain ergosterol alternatively.Deoxyribonucleic acid analysis reveals the extremophile to be among the most ancient life signifiers known. This fact has given rise to challenging guesss that life on Earth could hold originated in these utmost environments. The thought that life originated in the deepnesss of the oceans about 3.8 billion old ages ago is besides explored in some item by Daniel et Al. ( 2006 ) .

In a more practical context, pressure-induced inactivation of bacterium has been investigated as a possible manner of sterilising nutrient ( Spilimbergo et al. , 2002 ; Aoyama et al. , 2004 ) .

2.2.8 Relation to MEOR

The intent of the predating treatment was to place the factors that constrain the growing of bacteriums in subsurface environments, thereby supplying a set of standards by which the suitableness of beings for usage in EOR can be assessed and compared. For oil-bearing formations, it is to be observed that some of these restraints are slightly less strict. For illustration, the salts of connate seawaters are typically greater than that of saltwater, but much less than those happening in salt lakes, and force per unit areas of up to 20 MPa and temperatures to 80 Os are good within the bounds observed for endurance of bacteriums.

But the combination of these restraints can be expected to restrict the figure of suited beings. Among the best ‘all-round performing artists ‘ are the Bacillus bacteriums. Yakimov et Al. ( 1997 ) described a elaborate survey of several strains of Bacillus licheniformis, and concluded that this being is potentially utile for enhanced oil recovery. It is capable of working at reasonably elevated temperatures ( 55 o C ) and salts to 12 % NaCl, produces important measures of biomass, and a surfactant similar to surfactin ( produced by B. subtilis ) which is known to possess antimicrobic belongingss. A more recent study by McInerney et Al. ( 2004 ) describes a peculiarly thorough scrutiny of over 200 strains of Bacillus subtilus, B.

licheniformis, B. mojavensis, and B. sonorensis, which were compared with regard to surfactant production under anaerobiotic conditions at 5 % salt. In the class of their work, these writers developed new and improved processs for insulating biosurfactants produced by the beings, established quantitative relationships between the surfactant concentration and interfacial tensenesss, and performed legion experiments affecting mobilisation of oil from Berea sandstone nucleuss.

2.3 The pick of Clostridium Tyrobutiricum

Thousands of bacteriums have been investigated for MEOR intent, but the agitation bacterium remain the most popular particularly Clostridia coinage because they produce big volume of gas which include CO2, H2 and CH4. These gases produced, diminish the oil viscousness and increase the force per unit area in the oil reservoir.

2.4 Agitation

Waste merchandises formed in this manner include gases, ethyl intoxicant, butyl intoxicant, organic acids, propanone and others. Molassess agitation generates energy rich metabolic merchandise, which may respond in the concluding decomposition line of sulphate decrease under anaerobiotic formation status. With sulphate ion in the formation H2O, sulfur decrease predominates. Hydrogen sulfide produced is really non desirable. The organic acids are formed through agitation of the molasses by the bacteriums in the reservoir do do a stone fade outing procedure.

Figure 2.

3: Agitation bacteriums

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

Another ground for utilizing agitation bacteriums is Dr. Wagner field trail. If we make comparing between Danish north oil field formation and Zechstein evaporates stones which are similar to the Danish North Sea formation. Dolomite is besides similar to Danish north field chalk. Formation temperature is rather similar and of class has a high salt. Clostridia Tyrobutiricum was selected for Dr.

Wagner ‘s experiment.The features of Dr. Wagner ‘s experiment field are as follows:Dolomite of Zechstein formationsDepth of 1240mFormation temperature 53 oCHigh salt formation H2O, even the crevices and breaks are partly filled with salt.

The consequence of Dr. Wagner ‘s MEOR good experiments:Water cut decreased from 80 to 60 %Average one-year oil production:Before microbic intervention – 50 dozenss per month3 months after injection – 150 dozenss per month1 twelvemonth after injection – 300 dozenss per monthSince all these conditions are similar to Danish North Sea formation and other factors are besides same, so we can utilize agitation bacteriums for MEOR experiment.


6 Adaptation of bacteriums to high salts

Majority of the bacteriums can non defy high salt, from the clip of ancient civilisation ; it is known that adding 50 g/l of salt in nutrient conserves it from botching. This means that agitation bacteriums which usually populate organic substances have a challenge of version in high salt. The spore organizing bacteriums like clostridia signifier spores in utmost conditions. These conditions allow bacteriums to last but they will non be active and would non be productive. Under highly high salts, bacteriums undergo osmotic emphasis which is expressed in osmotic force per unit area. Osmotic force per unit area affects the H2O activity and production of CO2 gas during the agitation procedure.


1 Osmosis

Osmosis is the transition of H2O from part of high concentration through a semi-permeable membrane to a part of lower H2O concentration.Semi permeable membrane are really thin beds of stuff ( cell membrane are semi-permeable ) which allow some substances to go through through them and forestall other substances from go throughing through. Cell membranes will let little molecules like O, H2O, CO2, ammonium hydroxide, glucose, amino acid, etc. to go through through ; meanwhile, cell membranes do non let transition of larger molecules like saccharose, amylum, protein, etc.

2.6.2 Osmotic force per unit area


6.3 Potential osmotic force per unit area

2.6.4 Osmotic belongingss of cells

The wall of bacteriums and turning works cells are non wholly stiff, and the turgor force per unit area has been proposed to supply the mechanical force for the enlargement of the cell walls during cell growing. The consumption or biogenesis of osmotically active solutes causes an addition in the cells, therefore supplying the necessary tugor force per unit area for enlargement of the cell walls. Although the suggestion that turgor force per unit area is the driving force for cell wall enlargement would connote that the mechanisms that regulate the osmotic balance of beings are cardinal to the really procedure of cell growing.Lipid membranes allow rapid diffusion of H2O molecules into or out of cells while showing an effectual barrier to most other biological molecules. Membranes that exhibit selective permeableness for different substances are called semi permeable, and the osmotic belongingss of cells derive from this belongings of the membranes.

2.7 Thermophyllic and Halophyllic bacteriums

There are bacteriums which need high salts and high temperatures for their growing. In order to look into and enter the conditions of bugs at high salts and high temperature it is better to cognize about the bacteriums which can defy on these conditions. Important information has been given about these type of bacteriums is discussed below.


In nature, if certain bacteriums are Halophyllic so they are non Thermophyllic, so in order to choose the right version for recovery procedure, bugs which can turn in the reservoir environment should be considered. Pressure, temperature and salt are the most of import factors to see so it is necessary to roll up a aggregation of micro being with desirable features. Growth restrictions would necessitate to be established for each civilization.

For such a aggregation, the best being for a peculiar application would be chosen. To find the conditions under which microbic processs would be preferred which require an accretion of information ensuing from field application of the procedure.

Chapter 3

Materials And Method.

Chapter THREE


In order to look into the adaptability of the considered bacteriums and see how effectual MEOR method could turn out for the Danish field, a research lab based experiment was carried out at Aalborg University Esbjerg, Denmark

3.1 Materials

The undermentioned stuffs were used at one point or the other during this undertaking work:Anaerobic bacteriums ( Clostridium Thyrobutyricum 633 )MolassessWater bath ( MGW LAUDA M20 )Sodium chloride ( NaCl )Autoclave machineTest tubingOne liter bottle for mensurating H2O supplantingVolumetric flaskpH metreConductivity metreGas chromatography ( GC ) Shimadzu GC14 with on-column injectorFlow metreEppendorf pipettes with polypropene tips

3.2 Operating parametric quantities for gas chromatography

Carrier gas H ( safety guidelines ) , force per unit area 0.4 saloonDetector temperature: 350 A°CColumn temperature: 40 A°C ( 5 min ) , 20A°/min to 340 A°C, 340 A°C ( 15 min )Entire running clip: 41 min + equilibration clip ( about 10 min )Injector temperature: column temperature + 5 A°CDetector: FID, Range 0, makeup gas N 20 ml/minInjection volume: 2I?l


3 Sample aggregation

The anaerobiotic bacteriums used for the intent of this experiment were collected from CHP-biogas works at Ribe in Denmark on August 10, 2010.

3.4 Sample readying

Sodium chloride ( NaCl ) was foremost added to each flask harmonizing to the salt desired ( i.e. 20 g/l, 40g/l etc.

) except for the control ( 0 g/l ) .700 milliliter of H2O was so added to all flasks.50 milliliter of molasses was added to each flask.Flasks were so placed in the H2O bath and heated until a temperature of 53oC is attained.50 milliliter of the anaerobiotic bacterium was added to the flask.All flasks were assorted decently to achieve a homogeneous content within the each flask and placed back in the H2O bath.The initial pH and conduction of all flasks were measured and recordedFrom the above figure, it can be observed that four flask incorporating bacteriums with different salts are placed in a H2O bath.

The bath is filled up with H2O up to three one-fourth of the armored combat vehicle degree. The temperature of the bath is maintained at 53o C with the assistance of a particular warmer placed inside the bath. Each of these flasks is tightly closed with a gum elastic cork to keep anaerobiotic conditions. The cork has a particular agreement to mount the pH metre and conduction metre.

Daily monitoring of the system is carried out to guarantee proper care of anaerobiotic status and working environment. The experiment was carried out in two batches because the H2O bath is non large plenty to all flasks used for this experiment. Therefore the first batch was for salt of 0g/l ( control ) , 20g/l, 40 g/l, 60 g/l, the 2nd batch was for salt of 80 g/l, 100 g/l, 120 g/l, 140 g/l.

For each of the salt there was a replicate.Each experiment was conducted for 120 hours ( 5 yearss ) and during this period no food ( molasses ) was added, this was to gauge the frequence of ingestion of food by these bacteriums and to analyze the measure of gases and acids they can bring forth with a specified sum of food within the stipulated 120 hours. Every 24 hours, the pH and conduction every bit good as volume of gas produced for each flask was measured and recorded.

Chapter 4

Consequences And Discussion.

Chapter FOUR


Table 4.

1: Measure of NaCl added to each flask



( g/l )

ADDED ( g )


Batch 1





Table 4.2: Initial pH and Conductivity reading




( g/l )






























Table 4.

3: Consequences for first batch








( g/l )


( ms/cm )

( 0C )

( milliliter )





















































Table 4.4: Consequences for 2nd batch








( g/l )


( ms/cm )

( 0C )

( milliliter )













































4.1 Analysis of consequence

Figure 4.1: Accumulative Gas production for each salt

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

Figure 4.2: Accumulative Gas production for each salt 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 salt 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 tendency of salt measured during this experiment. The tendency shows a diminution in pH with clip across all salt. The highest pH was measured at 140 g /l. It can so be concluded that pH has a direct correlativity with salt. The higher the salt the higher the pH.

Figure 4.4: Electrical conduction vs. pH

The above graph of Electrical conduction against pH shows that electrical conduction additions as pH additions

Figure 4.5: Gas production per twenty-four hours

Figure 4.6: Gas production per twenty-four hours without 0 and 20 g/l

Figure 4.7: Gas production with pH

4.2 Gas composing analysis

The gas produced was ab initio analyzed and consequence shows that 70 % of the entire gas produced was C dioxide, but the staying 30 % was non known. Therefore farther analysis with more gas sensing technique was proposed. However, equipment for this analysis is non yet available as of day of the month but is expected within the first hebdomad of October 2010.

4.3 Discussion

Microbial enhanced oil recovery procedure with multiple mechanisms happening at the same time depends on many factors to be successful. In this probe, the experiment showed that it was possible to increase the adaptability bound of the considered bacteriums growing to salinities up to 140 g/l. this was higher than the salt of 45 g/l where the pure civilizations were able to turn. The add-on of molasses further increased the bound significantly. During the agitation yearss the sum of C dioxide gas produced is limited due to the fact that it was carried out in a limited sum of substrate solution. Therefore more probe has to be carried out on mass balance of this procedure to hold a better apprehension of the production of byproducts.During informations aggregation like the pH, a small sample has to be removed from each flask on a day-to-day footing. This often disrupts the anaerobiotic conditions inside the flask. Therefore more sophisticated agreement has to be made so as to guarantee proper anaerobiotic status is maintained.