WO1992001780A1

WO1992001780A1 - Oil recovery using microorganisms

Info

Publication number: WO1992001780A1
Application number: PCT/AU1991/000317
Authority: WO
Inventors: Alan Sheehy
Original assignee: B.W.N. Live-Oil Pty. Ltd.
Priority date: 1990-07-16
Filing date: 1991-07-16
Publication date: 1992-02-06

Abstract

A method for recovering oil from an oil reservoir is described. The method involves the application of a biocide to a reservoir in an amount effective to cause limited microbial death and subsequent release of a utilisable microbial growth effective carbon source. One or more non-carbonaceous growth effective nutrients are added to the reservoir either before and/or after biocide addition so as to facilitate microbial growth. The reservoir is thereafter maintained for a time and under conditions sufficient for the substantial depletion of at least one of the added nutrients. Microbial growth following nutrient addition, followed by the depletion of at least one of the added nutrients results in microorganisms having reduced cell volume and/or increased surface active properties which facilitate oil recovery when the reservoir is subjected to oil recovery means.

Description

Oil Recovery Using Microorganisms

This invention relates to a method for the recovery of oil from oil containing reservoirs with assistance from microorganisms.

During primary oil production, the pressure within a reservoir decreases with a subsequent decline in oil production. To compensate for this production decrease water or gas is injected into the reservoir. This process is referred to as secondary oil production. During secondary production, the water to oil ratio increases until oil production is no longer economical. The residual oil, up to 65% of the original oil in place (OOIP), is distributed in a significantly different pattern to the OOIP. The failure of secondary oil production procedures to release trapped residual oil results from capillary forces in the oil/water/rock system and the failure of injected fluids to penetrate parts of the reservoir formation. Surfactants are often used to lower the interfacial tension between reservoir fluids and residual oil so that oil which cannot be removed by the injected fluids alone is displaced. Surfactants used in chemical EOR (enhanced oil recovery) show optimal activity over a narrow range of temperature, HLB (hydrophobic lipophilic balance) values, salinities and rock types. Thus, surfactant EOR processes are generally developed for individual reservoirs.

Surfactants derived from crude oil (e.g. petroleum sulphonates) have been shown in some field pilots to strip out residual oil but at a cost much higher than the market value of the oil recovered in this way. The surfactants are themselves expensive: they tend to adsorb to rock, and so large quantities are needed. Polymers, too, have had some successes, but again at a high cost. Both polyacrylamide, made from petroleum feedstocks, and the microbial product xanthan gum have been used: the former is less expensive but is not effective at the high temperatures and salinity levels common in many reservoirs. The latter is technically more satisfactory though there are problems of microgel formation causing blocking at the injection face, degradation may take place in the reservoir and, once again, the material is expensive.

It has been proposed to use microorganism derived surfactants of EOR. This technique is known as microbially enhanced oil recovery (MEOR). The production of surface active agents by microorganisms has been recognised for a number of years. These biosurfactant compounds almost universally contain a lipid component and are usually glycolipids. Other classes of biosurfactants are lipopeptides, phospholipids, fatty acids and neutral lipids.

There are several potential advantages in using MEOR processes. These include, the wide range of compounds with useful properties for EOR that can be produced by microbial biosynthesis, cost, and the ability to produce biometabolites within the reservoir and thus decrease the amount of chemical surfactants required.

Current MEOR techniques have involved the injection and establishment of an exogenous microbial population in an oil reservoir. This population is supplied with nutrients such as molasses or other fermentable sugars, a source of nitrogen and mineral salts as additives to the waterflood employed for secondary oil removal. Other hydrocarbon substrates have been researched, however, the economic advantage of fermentable sugars have made them the preferred substrate.

The development of methods utilizing the injection of microorganisms into oil reservoirs has been limited by the conditions which prevail in oil reservoirs. In particular, small and variable reservoir pore sizes together with extremely high temperatures, salinity/ionic strengths and pressures have severely limited the type, range and number of microorganisms that can be injected. Further, and of equal significance, is the highly reduced environment present in many reservoirs. The absence of oxygen severely limits the range of biometabolites that can be synthesized by organisms introduced into oil reservoirs.

A disadvantage of the microorganisms utilised in current MEOR technology is that they may tend to occlude the reservoir pores due to their large cell volume caused by the rich nutrient conditions provided in the waterflood. These large cells may also find it difficult to penetrate small pores in the rock.

U.S. Patent No. 4,475,590 (Brown) describes a method for increasing oil recovery from oil bearing formations by stimulating the growth of in-situ microbial populations by the injection into the formation of aqueous nitrogenous and aqueous phosphorus containing solutions in amounts to control the growth of the microbial population. A drive fluid is then injected into the formation to displace oil in a production well. The process of Brown is an aerobic one, as the in- situ microorganisms population degrades crude oil within the reservoir as a carbon source. The utilisation of crude oil as a carbon source has a strict requirement for oxidative metabolism, that is, metabolism in the presence of oxygen. The process of Brown also involves providing nutrients in the form of nitrogen and/or phosphorus in growth effective amounts throughout the process. This has the effect of producing bacteria with large cell volume which may occlude well pores and hamper oil recovery, such as in the situation where a drive fluid is injected into a well. The Brown process is also disadvantageous as the product desired to be obtained, that is, crude oil, is degraded.

It has previously been described in U.S. Patent No. 4,971,151 which is incorporated in its entirety by reference, that oil may be recovered from a reservoir utilising endogenous microorganisms to which is added a non-glucose-containing carbon source and at least one other non-glucose-containing nutrient, which nutrient is growth effective for endogenous microorganisms. The reservoir is then maintained for a time and under conditions sufficient for the substantial depletion of at least one of the added nutrients, wherein, the resultant microorganisms have reduced cell volume and increased surface active properties. The oil reservoir is subsequently subjected to oil recovery means. This invention is based, at least in part, on the observation that a proportion of oil wells contain significant numbers of microorganism, far in excess of levels previously thought to be present. The invention is further based on the finding that natural carbon sources within a well provided by the addition of biocides which cause limited microbial death, may be used, in combination with microbial growth limiting non- carbonaceous nutrients added to the well, to promote bacterial growth. Such microbial growth, in combination with a nutrient depletion step has been found to give rise to microorganisms having the advantageous properties of increased surface active properties, such as hydrophobicity, which is usually accompanied with a marked decrease in cellular volume.

Accordingly, the present invention contemplates in a first aspect a method for recovering oil from a reservoir having a population of endogenous microorganisms comprising adding to the reservoir one or more non- carbonaceous nutrients being growth effective for the endogenous microorganisms, and maintaining said reservoir for a time and under conditions sufficient for the substantial depletion of at least one of the added nutrients, wherein microbial growth following nutrient addition, followed by depletion of at least one of the added nutrients results in microorganisms having reduced cell volume and/or increased surface active properties, and thereafter subjecting said reservoir to oil recovery means; characterised in that a biocide in an amount effective to cause limited microbial death and subsequent release of a utilisable growth effective carbon source is added to said reservoir before and/or after the addition of said non-carbonaceous nutrients.

This invention may particularly be practised by removing a sample of liquid from the reservoir, in which microorganisms are resident, and analysing the liquid to determine what nutrients are likely to be growth limiting. An assessment of microbial growth is simply made by determining the numbers of organisms in culture following incubation with one or more nutrients.

"Non-carbonaceous nutrients" is used in its broadest sense and includes one or more inorganic and non-carbon- containing organic compounds required by a microorganism for growth or which facilitates microbial growth. Such nutrients include those containing at least one of the following elements: C, H, 0, P, N, S, Mg, Fe or Ca. By way of exemplification only, such inorganic compounds include P0 ^2", NH ⁺, N0 ~, NO3^" and SO4^2" and the like. A requirement for any of these nutrients (i.e., a deficiency of one or more such nutrients which inhibit microbial growth) may be readily determined according to standard microbial techniques as are well known in the art, and are hereafter described.

The levels of nutrients within a sample of fluid obtained from a reservoir are analysed by any number of well known analytical techniques, such as atomic absorption spectrophotometry (AAS), high pressure liquid chromatography (HPLC), magnetic measurements, infra-red spectometry, gravimetric analysis, electrochemical analysis, titration and other like methods, such as described, for example, in Analytical Chemistry, 3rd Edition, Saunders College Publishing, 1978, Skoog, D.A. and West, D.M.

At the time of sampling, the amount of assimilative organic carbon may also be determined.

Further, an assessment may be made as to the numbers of microorganisms present within a well sample. If a large number of microorganisms are present, it is possible then to simply add the missing non-carbonaceous nutrients directly to the reservoir to stimulate microbial growth for a selected period of time in conjunction with treating the well with a biocide so as to cause limited microbial death and subsequent release of a utilisable microbial growth effective carbon source. On the other hand, where there are only a small number of microorganisms present, the microorganisms may be grown such as in the laboratory or, where appropriate, on site, in an appropriate medium in which the missing nutrients are provided, in order to increase the numbers thereof. As mentioned above, however, we have surprisingly found that a proportion of oil reservoirs contain significant populations of microorganisms, far in excess of what was thought to be present. Accordingly, treatment of the well with a biocide in an amount effective to cause limited microbial death provides a ready source of utilisable carbon for the significant portion of non- lysed microorganisms within the well. In the inventors* experience, it has often been found that oil wells are deficient in nutrients such as nitrogen and phosphorus, as well as other trace metals. Non-carbonaceous nutrients required for endogenous microbial growth are added to the reservoir in growth effective amounts, that is in amounts capable of effecting microbial growth. Nutrients may be added to a reservoir in a solid or liquid form and may, for example, be pumped into a reservoir dissolved in a fluid such as production water, saline and the like.

Nutrients are generally provided in soluble form, that is, soluble in the fluids within an oil reservoir, to avoid precipitation of nutrients which would limit the availability of such nutrients. Nutrients are therefore added to an oil reservoir in any form which provides assimilable nutrient for use by microorganisms. For example, nutrients may be provided in the form of NaN03, Na2HPθ4, CaCl2, MgCaCθ3, ammonium nitrate, ferric chloride, manganese chloride, zinc chloride, copper acetate, etc.

The quantities of growth limiting non-carbonaceous nutrients added to a reservoir will depend upon a number of factors such as the levels of nutrients originally present, and amounts required for microbial growth over a determined time period. Quantities of nutrients required may be readily determined in any particular case, for example, by way of routine experimentation in which microorganisms from a reservoir are incubated in production or other fluid in the presence of varying amounts of nutrients, over various levels. In a situation where a reservoir has been shown by chemical analysis to be deficient in phosphorous, for example, microbes from the reservoir may be incubated in a medium containing various levels of phosphorous. Phosphorous levels in which maximum bacterial growth occurs may be selected for addition to a reservoir. Alternatively, less than optimum levels of phosphorous may be added to a reservoir to facilitate microbial growth for a shorter period of time. A similar approach may be used for any other nutrient.

As mentioned previously, we have surprisingly found that endogenous microorganisms resident within an oil reservoir, may themselves act as a carbon source for the growth of other microorganisms within the reservoir. Particularly, the use of biocide reagents in a localised portion of an oil reservoir, such as at a well head, leads to localised microbial lysis and/or fragmentation with the release/degradation of carbonaceous containing compounds which constitute the microbial cell wall, organells, cytoplasm, etc.

Biocides which may be used to cause localised microbial death and subsequent lysis include materials such as acids (for example hydrochloric acid, nitric acid, hydrofluoric acid), alkalis (for example sodium hydroxide and potassium hydroxide), detergents, osmotic lysis reagents, formaldehyde and like agents which cause microbial cell lysis. Examples of acids, alkalis, and detergents which may be used in this invention are described, for example, in the Kirk Othmer Encyclopedia of Chemical Technology, John Wiley & Sons, 1981.

One or more biocides may be added to a well to increase oil recovery following nutrient addition and subsequent nutrient depletion. Biocides are either added to the reservoir in soluble form, or in a form generally soluble within the reservoir. Biocides are added prefreably to the well head, although biocides may be added at any other access point to the well.

Biocides are added to the well in an amount effective to cause limited microbial death and subsequent nutrient release. The amount of biocide added to a well may vary with well volume, well topography and geography, potency of the biocide, solubility of the biocide, temperature of the well, salinity and the like. The approximate volume of the well-bore may be calculated according to well known procedures in the art, and an amount of biocide selected to constitute, for example, from 1 to 50% of such volume. It is to be stressed that the amount of biocide added to a well is selected so as to cause limited microbial death, generally around the well-head or other site of introduction of biocide to the oil well.

By way of example only, where formaldehyde is used as a biocide, from about 15 kg to about 150 kg of formaldehyde may be used. Such amounts of biocide are in no way limiting on this invention and are merely used in an exemplary sense. Clearly, the amount of biocide used in any particular oil well will be a routine matter based on an analysis of previously mentioned factors. When biocides are utilised, microorganisms resident within a limited area around the injection site of such material, such as a well head, would be lysed and/or degraded and their carbonaceous contents liberated for utilisation by other microorganisms remote from the area of biocide action. It is believed that the carbonaceous materials released on microbial cell lysis/degradation (that is microbial extracts) diffuse through the reservoir and are thus available for utilisation by other microorganisms. In some circumstances it may be necessary to add a non-glucose carbon source to the well to provide microbial growth effective conditions. For example, in the situation where a well contains relatively f" microorganisms, the population of organisms resident within the well may be expanded by supplying a non- glucose-containing carbon source and one or more growth limiting non-carbonaceous nutrients.

Examples of such non-glucose containing carbon sources include lysed microbial cells (such as microbial cells lysed with a biocide), a protein hydrolysate or protein digest (such as produced by reacting animal or prokaryotic/eukaryotic proteins with a protease or treating proteins under acid or alkali conditions). It should also be recognised that reservoirs may contain non-glucose-containing carbon sources in the form of lactate, acetate, propionate, palmitate, benzoate, formate, hexadecane, hexadecene and various microbial metabolic products.

It is a principle feature of this invention that following the addition of the biocide and growth effective non-carbonaceous nutrients to a reservoir, the reservoir is subject to nutrient depletion conditions. Nutrient depletion conditions are generally effected by allowing microorganisms resident within a reservoir to exhaust one or more nutrients necessary for growth by virtue of their metabolic process. This is usually effected by sealing a well such that no further nutrients are added whereafter nutrient depletion takes place as a result of microbial metabolism. Alternatively, in oil reservoirs which are operated as a water flood, (where there are one or more wells through which a drive fluid, such as production water, is pumped into the well, and one or more wells through which fluid is recovered from the well) nutrient is released into the well for a determined period of time and then the nutrient source is cut off, such that nutrients within the reservoir are metabolised by resident microorganisms, and are flushed from the well by drive fluid of the water flood.

In the situation where a well is shut in (closed off) and resident bacteria allowed to metabolise nutrients within the reservoir, it is not uncommon for nutrient depletion to require from 14 to 300 or more days. This time period will depend, for example, upon the amount of growth effective nutrient added to the well, the physical and chemical conditions of the well, such as acidity, temperature, topography, etc. In an alternative embodiment, nutrient sequestering agents may be added to a well to withdraw nutrients from microbial availability thus creating a state of nutrient depletion.

Nutrient depletion may be readily assessed by a determination of the growth-limiting nutrients within the reservoir. This is carried out according to analytical techniques previously described.

Nutrient depletion may also be measured by determining the numbers of bacteria per unit volume of well fluid. When a steady state or relatively constant number of bacteria per unit volume is observed, this is indicative that nutrient limitation has occurred.

Microorganisms resident within an oil well which are subject to conditions of microbial growth as a result of the addition to the reservoir of growth-limiting nutrients, followed by conditions of nutrient depletion, undergo a surprising transformation to a form having increased surface active properties. These surface active properties are associated with the microbial cell rather than with a secreted surfactant. By "surface active property" is meant the property of a microorganisms which reduces surface tension, that is, increased hydrophobicity.

Microorganisms resident within an oil well generally occupy the boundary between the oil and water phases within the reservoir. As nutrient deprivation sets in, we have found that microorganisms become increasingly hydrophobic. This effect is not fully understood. Without wishing to limit this invention in any way, increased microbial hydrophobicity or surface active properties, may be associated with an increase in hydrophobic lipids in the microbial cell membrane, a change in configuration of membrane lipids to increased saturation, or other 1..ke mechanisms.

It is important to recognise that the features of increased hydrophobicity are themselves associated with the microbial cell. This is not to say, however, that microorganisms subject to growth in nutrient rich conditions, fr!lowed by nutrient depletion, will not themselves secrete surfactant like molecules.

Hydrophobicity of microorganisms may be measured by methods well known in the art, such as the hexane drop formation method or other like methods. Microorganisms subject to the method of this invention may, for example, be from 3 to 10 times more hydrophobic than non-treated microorganisms.

Microorganisms within a reservoir may be subject to several cycles of nutrient addition and nutrient depletion. Biocides may be added to the well before and/or after non-carbonaceous nutrient addition. It may only be necessary to add biocides during only one cycle. We have found that such cycling maximises surfactant properties, which as previously described can be readily ascertained, for example, by measuring a reduction in interfacial tension caused by the microorganisms or other well known methods in the art. It is believed that microorganisms having increased surface active properties permeate rock pores to act as surfactants themselves to enable trapped oil in the rock material to be readily flushed by outgoing water or other drive fluid from a well.

It is to be appreciated that the microbial flora within an oil reservoir would be quite diverse, and may consist of representatives of a number of bacterial classes. For example, representative microorganism which may be present either singly or in combination in a reservoir may include the genera Arthrobacter, Vibiro, Bacillus, Pseudomonas and the like. Such microorganisms, as a result of the method of this invention, shift towards a highly hydrophobic state as previously described. The hydrophobic properties of microorganisms within a reservoir may be represented as a normal distribution, with few microorganisms possessing significant surface active properties. The method of this process shifts the microbial population to a position where a significant proportion of microorganisms within the reservoir possess increased surface active properties.

Microorganisms subject to one or more cycles of growth in growth-effective nutrient conditions, followed by nutrient depletion, have a considerably smaller cell volume than those microorganism which are subject only to conditions of nutrient addition. A cell volume reduction of 70% is not uncommon. Microorganisms have a requisite small cell volume are able to penetrate rock pores, which when coupled with surface-active properties of the microorganisms, facilitates oil recovery. It is likely that within an oil reservoir, bacteria are present in a number of shapes and sizes. The method of this invention predisposes bacteria towards a reduced cell volume state. The methods of this invention may be carried out under anaerobic conditions, as endogenous non-oil carbon sources and non-glucose containing carbon sources within a reservoir may be anaerobically utilised by microorganisms. As previously mentioned, desired nutrients may be dissolved in production water prior to introduction into a reservoir. By "production water" is mean the aqueous^' phase of an oil-aqueous mixture emitted from a reservoir. Production water may also be referred to as co-produced water. The production water may be buffered to be compatible to the ecology of the reservoir and frequently, carbonate or bicarbonate is used to prepare the buffering conditions. The choice of buffering compound is dependent on the ecological pH of the reservoir which can, range from pH 2 to 10. The desired nutrient(s) may be added to production water and injected into the reservoir under conditions and for a time sufficient in accordance with this invention. The emitted aqueous-oil mixture is collected and the phases separated. The aqueous phase may be collected and analysed to determine the concentration of nutrient(s) originally contained therein. If necessary, the concentrations) of additives are adjusted accordingly, and the buffering capacity may also be adjusted if necessary before being injected back into the reservoir. The term "reservoir" as used herein refers to any locus of oil-deposit. Additionally, "oil recovery means" refers to standard oil recovery practices, such as, but not limited to, the use of water or gas to generate pressure to eject oil containing liquid from a reservoir. According to another aspect of this invention there is provided a method for recovering oil from a reservoir comprising steps of:

(a) isolating endogenous microorganisms from said reservoir;

(b) ascertaining the limiting nutrient(s) for growth of said microorganisms;

(c) growing said microorganisms under growth effective conditions and thereafter subjecting said microorganisms to nutrient limiting conditions sufficient to produce a reduction in the mean cell volume to a level compatible with injection into said reservoir;

(d) supplying an amount of said nutrient(s) together with said microorganism to said reservoir for a time and under conditions sufficient to effect an increase in population of. endogenous microorganisms in said reservoir, said nutrients comprising a non-glucose containing carbon source and at least one of a non- glucose containing nutrient, said nutrients being growth effective for the endogenous microorganisms;

(e) maintaining said reservoir for a time and under conditions sufficient for the substantial depletion of at least one of the nutrients; wherein the addition of the non-glucose-containing carbon source, at least one other non-glucose containing nutrient, and the depletion of at least one of the nutrients results in microorganisms having reduced cell volume and increased surface active properties;

(f) optionally subjecting said reservoir to oil recovery means;

(g) adding to said reservoir one or more non- carbonaceous nutrients being growth effective for the microorganisms within the reservoir; (h) maintaining said reservoir for a time and under conditions sufficient for the substantial depletion of at least one of the added nutrients; wherein microbial growth following nutrient depletion, followed by depletion of at least one of the added nutrients results in microorganisms having a reduced cell volume and/or increased surface active properties; characterised in that a biocide in an amount effective to cause limited microbial death and subsequent release of a utilisable growth effective carbon source is added to said reservoir before and/or after the addition of said non-carbonaceous nutrients; and thereafter subjecting said reservoir to oil recovery means.

This invention will now be described by way of non- limiting example:

EXAMPLE

A number of oil reservoirs have been tested for oil recovery involving the addition of growth-effective nutrients to an oil well, followed by nutrient depletion. In the wells tested, including the Rankin Oil field in Texas (Rankin), increased oil recovery in the order of 5 to 15% was observed when a biocide was added to the oil well prior to nutrient addition and subsequent nutrient depletion.

At Rankin the addition of 5 gallons of 36% formaldehyde to the well head prior to growth-effective nutrient addition, when compared to the addition to the well of only growth-effective nutrients followed by nutrient depletion, demonstrated increased oil recovery.

Claims

THE CLAIMS DEFINING THE INVENTION ARE AS FOLLOWS:

1. A method for recovering oil from a reservoir having a population of endogenous microorganisms comprising adding to the reservoir one or more non- carbonaceous nutrients being growth effective for the endogenous microorganisms, and maintaining said reservoir for a time and under conditions sufficient for the substantial depletion of at least one of the added nutrients, wherein microbial growth following nutrient addition, followed by depletion of at least one of the added nutrients results in microorganisms having reduced cell volume and/or increased surface active properties, and thereafter subjecting said reservoir to oil recovery means; characterised in that a biocide in an amount effective to cause limited microbial death and subsequent release of a utilisable growth effective carbon source is added to said reservoir before and/or after the addition of said non-carbonaceous nutrients.

2. A method according to claim 1, wherein said biocide is added to the reservoir before the addition of said one or more non-carbonaceous nutrients.

3. A method according to claim 1, wherein said biocide is added to the reservoir after the addition of said non-carbonaceous nutrients.

4. A method according to claim 1, wherein said biocide is added to the reservoir both before and after the addition of said non-carbonaceous nutrients.

5. The method according to claim 1 wherein said one or more non-carbonaceous nutrients comprise one or more of the elements selected from H, 0, P, N, S, Mg, Fe or Ca.

6. The method according to claim 1, wherein said biocide is selected from one or more agents which cause microbial cell lysis.

7. A method according to claim 6, wherein said agents causing microbial cell lysis are selected from acids, alkalis, detergents, osmotic lysis reagents and formaldehyde.

8. A method according to claim 7, wherein said acids are selected from hydrochloric acid, nitric acid and hydrofluoric acid.

9. A method according to claim 7, wherein said alkalis are selected from sodium hydroxide and potassium hydroxide.

10. A method according to claim 1, wherein depletion of at least one of the added nutrients is effected by incubating the reservoir for a time sufficient to allow endogenous microorganisms to deplete one or more nutrients necessary for microbial growth.

11. A method according to claim 1, wherein said oil recovery means comprises injecting water or gas into or adjacent the reservoir.

12. A method according to claim 1, which comprises additionally adding to the reservoir one or more non- glucose containing carbon sources.

13. A method according to claim 12, wherein said non-glucose carbon source comprises lysed prokaryotic cells.

14. A method according to claim 12, wherein said carbon source comprises a protein hydrolysate or protein digest .

15. A method according to claim 1, wherein said method is repeated one or more times.

16. A method according to claim 1, wherein the non- carbonaceous nutrients for microbial growth are added in production water.

17. A method according to claim 1, wherein said biocide is added to the well in production water.

18. A method according to claim 1, wherein said method additionally comprises the steps of:

(a) isolating endogenous microorganisms from said reservoir;

(b) ascertaining the limiting non-carbonaceous nutrient(s) for growth of said microorganisms;

(c) and thereafter adding to said reservoir said limiting nutrient(s) in an amount effective for growth of endogenous microorganisms in the said reservoir.

19. A method for recovering oil from a reservoir comprising steps of:

(a) isolating endogenous microorganisms from said reservoir;

(b) ascertaining the limiting nutrient(s) for growth of said microorganisms;

(d) supplying an amount of said nutrient(s) together with said microorganism to said reservoir for a time and under conditions sufficient to effect an increase in population of endogenous microorganisms in said reservoir, said nutrients comprising a non-glucose containing carbon source and at least one of a non- glucose containing nutrient, said nutrients being growth effective for the endogenous microorganisms;

(f) optionally subjecting said reservoir to oil recovery means;

(g) adding to said reservoir one or more non- carbonaceousnutrients being growth effective for the microorganisms within the reservoir;

(h) maintaining said reservoir for a time and under conditions sufficient for the substantial depletion of at least one of the added nutrients; wherein microbial growth following nutrient depletion, followed by depletion of at least one of the added nutrients results in microorganisms having a reduced cell volume and/or increased surface active properties; characterised in that a biocide in an amount effective to cause limited microbial death and subsequent release of a utilisable growth effective carbon source is added to said reservoir before and/or after the addition of said non-carbonaceous nutrients; and thereafter subjecting said reservoir to oil recovery means.

20. A method according to claim 19, wherein step (g) and (h) are repeated one or more times.