GB2153834A - Microbial enhancement of polymer viscosity - Google Patents

Abstract

A method for increasing the viscosity of a solution of a preformed polymer which comprises combining, under conditions favourable to microbial growth, (a) an aqueous solution of the preformed polymer; and (b) a microorganism incapable of de novo synthesis of the polymer, but capable of increasing the viscosity of the polymer solution is disclosed. The present invention may be applied, for example, to oil recovery, water clarification and paper production. Polymers which may be treated by this method include guar gum, hydroxypropyl guar gum, sodium carboxymethyl cellulose, hydroxyethyl cellulose, carboxymethyl hydexyethyl cellulose, xanthan gum, scleroglucan, locust bean gum, polyacrylamide, hydrolyzed polyacrylamide, poly (acrylic acid) and its salts, poly (2-acrylamido-2-methyl propane sulphuric acid (2 salt, ANPS), poly (acrylic acid-co-acrylate ester), poly (vinyl pyrrolidane), cellulose sulphate esters, poly (ethylene oxide), poly (vinyl alcohol), polyamine, poly (vinyl acetate-co-maleic anhydride) and poly (styrene sulphonic acid) and salts thereof.

Description

SPECIFICATION Microbial enhancement of polymer viscosity This invention relatesto microbial enhancement of polymer viscosity; more particularly, it relates to the action of microorganisms on polymers contained in solution and to a processforincreasing the viscosity of a polymer solution by the use of microorganisms.

For the present purposes, all polymers may be divided into two classes: biopolymers and synthetic polymers. "Biopolymers" are polymers produced at least in part by the action of biological processes, while "synthetic polymers" are produced by chemical processes.

Biopolymers may be obtained from a variety of natural plant sources, including free exudates, seed extracts, seaweed, starches and cellulose derivatives.

Some biopolymers, such as certain polysaccharides, may be produced by microorganisms.

A well-known example of a microbial biopolymer is xanthan gum, which is an anionic heteropolysaccharide made exo-cellularlyfrom carbohydratesubst- ances by organisms of the genus Xanthomonas, such as X. campestrisand X. begoniae. Xanthan and several other microbial biopolymers, such as the fungal productscleroglucan sold bythe Pillsbury Company underthe trade name "polytran" and a polysaccharide obtained from the soil bacteria Erwina marketed underthe trade name "Zanflo", are available in commercial quantities for applications including inks coatings, cosmetics, ceramics, paintthickeners, drilling muds, pharmaceuticals and foods. Some microbial biopolymershavetheabilitytofunctionas surfactants, such as surfactin produced by Bacillus subtilis.These surfactants are useful as flocculating agents, emulsifiers, demulsifiers, detergents, adhesives and enhanced oil recoveryfluids.

A microbial biopolymer is generally obtained by eitherfermentative or enzymatic syntheses. In fermentation, a microorganism that is genetically capable of building a specific polymer is placed in a medium with the necessary nutrients and substrate(s).The polysaccharide is recovered from this medium. In enzymatic synthesis, microbial cells are withdrawn from a cell culture, leaving a culture fluid containing the extra-cellurar enzyme. The enzyme is then contacted with the substrate(s) to produce the polysaccharide. Further information on polysaccha rides and the production thereof may be found in "Microbial Polysaccharides", Kirk-Othmer Encyclopedia of Chemical Technology, 3rd Ed., Vol. 15, at pages 439-58.

Synthetic polymers are prepared by the combination ofoneormoretypesofmonomericmaterials underconditions suitable to polymerization. They are generally less complex than biopolymers, usually consisting of an orderly progression of simple monomer units.

Both biopolymers and synthetic polymers may be added towater or othersolvents in ordertoform solutions having beneficial characteristics, such as increased solution viscosity. The viscosities ofthese solutions may vary greatly depending on the amount ofthe polymer in solution, the molecularweight and conformation ofthe polymer, temperature and the presence of salts (especially multivalent salts, such as magnesium and calcium), for example.

The continued stability of polymer solutions under conditions of severe temperature, salinities, press ures, shear forces and the presence of oxygen and chemical is a major concern for some applications.

Some microorganisms, whether naturally present in the solution environment or introduced by contamination, are also capable of degrading polymeric substances and thereby decreasing the solution viscosity.

For example, many microorganisms secrete enzymes (e.g. cellulase and amylase) that may hydrolyze polysaccharides into monosaccharides that may be used in the organism's metabolism. Synthetic polymers may also loose viscosity under microbial attack.

For the reasons given above, one of the objects of current research is to produce stable polymersolutions. The solution must be stable to the chemical, thermal and biological conditions encountered in various industrial processes. Of particular interest is the search for a polymer solution for use in enhanced oil recoverythatwill maintain its stability during pre-treatment steps, injection and throughout the reservoir.

For economic reasons, another object is to obtain the maximum viscosity for the least cost, i.e. the highest viscosity from the least amount of polymer in the solution.

The present invention relates to a method for enhancing the viscosity of a solution of preformed polymers by combining that solution with a microor ganism incapableofdenovosynthesisofthesid polymers but capable of increasing the viscosity of the said polymer solution, and maintaining that solution underconditionsfavourableformicrobial growth. The viscosity ofthis solution is increased or stabilized relative to a solution which does not contain the microorganism.

The microorganisms used to increase the viscosity of a chosen preformed polymer may be selected by using a microbial screening process characterized by: (a) preparing a growing culture ofthe microorganism; (b) preparing an aqueous solution ofthe preformed polymer and adding nutrients and minerals required for microbial growth; (c) separating that solution into two portions; a test and a control; (d) inoculating the test solution with the culture of the microorganism and incubating the mixture; and (e) measuring the viscosities of both test and control solutions after incubation to determine if the test solution is relatively more viscous than the control.

It has now been discovered that certain microorganisms are capable of increasing the viscosity of a polymer solution even when that microorganism is itself genetically incapable of de novo synthesis of the polymer. The term "de novo" is used herein to excludethose known techniques where a microorganism constructs polymeric material (such as polysac This print embodies corrections made under Section 117(1) of the Patents Act 1977.

charides)from monomers, and thereby increases the viscosity of a solution. As the present invention is defined,the microorganism cannot build the chosen polymer from monomers. This method is therefore most appropriate for preformed polymers (i.e. polymers which have previously been produced by either chemical or microbial syntheses). For example, a particular microorganism may be geneticallyincapable of producing a polysaccharide, but is capable of altering a solution ofthat polysaccharideto effectuate an increase in its viscosity.

While not being bound by a particular theory, it is believedthatthe microorganism acts to increase the average molecularweightofthe polymers in solution, possibly bythe action of one or more enzymes.

The polymers useful in accordance with the present invention include biopolymers and synthetic polymers that are compatible with water. Preferred polymers are not only compatible with water, but are readily soluble in water because they will thus be easily accessible to the microorganisms and accompanying extra-cellularenzymes in an aqueous media. However, even partially-soluble orwater-insoluble polymers are contemplated to be within the scope of the present invention, provided that an interface exists between the polymer and the microorganism or enzymes. For the present purposes, a "solution" of polymer means, in addition to dissolved polymer, water-wettable polymer surfaces, such as polymer suspensions, colloidal dispersions, polymeric gels or emulsions, and wettable polymeric solids.

In the preferred practice of the present invention, the polymer may be chosen from those known to be effective for a particular end use. For example, a polyacrylamide may be chosen if the desired use is water treatment, enhanced oil recovery, mineral processing or pulp and paper processing.

Extensive information has been published on ap plications for polymersolutions. Long-chain, watersoluble polymeric materials are extensively used as flocculation aids for solid-liquid separations. The use of aminated starches, polyamines and polyacryla midis in industrialwatertreatmentissummarizedin "Water (Industrial)", Kirk-Oth mer Encyclopedia of Chemical Technology, 2d Ed., Vol. 22. Other polymer flocculants are used in various processes, such as water clarification, sewage treatment, metal finishing, paper production, sugar refining, mineral extraction and food processing as discussed in "Flocculating Agents", Kirk-Othmer Encylopedia of Chemical Technology, 3d Ed., Vol. 10.

Enhanced oil recovery' (EOR) usually requires the injection of an aqueous fluid, such as water or brine, to push the oil ahead of it th rough the formation to a production well. A polymer is often added to at least a portion ofthe injected fluid in order to form a slug having increased viscosity. The polymer-thickened fluid moves in an even front and minimizes the by-passing and fingering that mightotherwise occur during conventional water-flooding.

Polymers considered to be useful for enhanced oil recovery(EOP) include guargum, hydroxypropyl guar, sodium carboxymethyl cellulose, hydroxyethyl cellulose, carboxymethyl hydroxyethyl cellulose, xanthan gum, glucan, locust bean gum, polyacrylamide, hydrolyzed polyacrylamide, poly(acrylic acid) and salts thereof, poly(2-acrylamido-2-methyl propane sulphuric acid) (2 salt) (AMPS). poly(acrylic acid-co-acrylate ester), poly (vinyl pyrrolidone), cellulose sulphate esters, poly (ethylene oxide), poly (vinly alcohol), polyamine, poly(vinly acetate-co-maleic anhydride), and poly(styrene sulphonic acid) and salts thereof. Preferred among these are hydroxyethyl cellulose, carboxymethyl hydroxyethyl cellulose, xanthan gum and hydrolyzed polyacrylamide.

The concentration of the polymer solution used in EOR is generally determined by the permeability of the rock and the viscosity of the oil in the reservoir. For economic reaso ns, the co ncentration iskepttothe minimal effective levels, which aretypicallyfrom 20to 3000 ppm, more typically from 50 to 1000 ppm and most typically from 200 to 600 ppm. High temperatures, shear stresses, high salinity, extreme pH values are other reservoir conditions which must be considered in choosing an effective polymer.

Polymers are otherwise used in oil fields in, for example, drilling, cementing, fracturing, acidizing, controlling water production, preventing sand production, clay stabilization and lost circulation. Guar, guar derivatives, cellulosics, xanthans, locust bean gums, starches and synthetic polymers, such as polyacrylamide, are commonly used, as more fully described in Chatterji, J. and Borchardt, J.K., "Applications ofwater-Soluble Polymers in the Oil Field", J.

Pet. Tech. 2042-2056, (Nov. 1981).

Preferred polymers for EOR mobility control agents will have all or most ofthefollowing characteristics: (1.) Ready solubility in water with a high positive affect on solution viscosity; (2.) Tolerance of dissolved salts (brine) in the aqueous solvent; !3. J High molecularweightfor maximum viscosity effects; (4.) Devoid of excessive branching or gels that might cause injection problems; (5.) Stability underthermal, biological and shear stresses in the reservoir environment and injection process; (6.) Potentially inexpensive in large volume production or having a high volume natural source.

The viscosity of a water-soluble polymer in solution is a function of manyfactors, chiefly molecularweight and electrolytic character. The greaterthe molecular weight of the polymer, the greater is the viscosity.

Charged polymers also tend to lose viscosity in highly saline water due to neutralization of the charges by the ions in solution.

The classic definition of viscosity for Newtonian fluids will be employed for the present purposes, where viscosity equals shear stress over the rate of shear. Shear stress is further defined as the force per unit area required to produce the shearing action. The rate of shear is a measured value.

A suitable way to measure viscosity is to employ a viscometer, such as one manufactured by Brookfield Engineering Laboratories, Inc., of Stoughton, Massachusetts, U.S.A. The Brookfield Viscometer mea sures viscosity by measuring the force required to rotate a spindle in a fluid. Because practically all fluids become thinner astemperature increases and thicker as they cool, the temperature ofthe fluids being compared should be recorded and kept the same if possible.

The Brookfield LVTViscometerwith the UL (Ultra Low viscosity) adapter is especially suitable for this application. The UL adapter provides amplifying effects which makes possible measurements with a reproducibility of 0.2 centipoises (cps) or mPas in the ultra-low viscosity range of up to 10 cps or Mpas.

Calibration ofthe instrument showed an average error of + 0.2 cps or mPas at both ends ofthe shear rate range (10 sex~' and 70 sex~'). The shear rate on the instrument may be varied from 73.42 to 0.36 sex~, which covers the range of shear encountered underground in the flooding process (typically 10 sex~ 1).

Other measuring devices, such as capillary viscometers, plate and cone viscometers and concentric cylinders, may be used as appropriate.

Some polymer solutions will exhibitthixotropic properties, characterized by the ability of a solution to become fluid when subjected to shaking, stirring, or other stress and then returned to a gel when allowed to stand. Common examples ofthis are mayonnaise and paint. Thixotropy is also desirable for polymer solutions used in enhanced oil recover, in which the solution is morefluidtofacilitate pumping, but becomes more viscous once it enters the oil-bearing formation.

Non-Newtonian fluids are those in which a change in the rate of shear is not proportional to a change in the shear stress. For such fluids, the relationship between shear rate and shear stress is sometimes determined at several rates of shear. Although the polymeric solutions in accordance with the present invention may be non-Newtonian, shear rates which are obtained using a single set conditions for shear stress will usually be sufficient to use as a basis of comparison to determine any change in viscosity.

Additional measurements of shear rate with different shearforces may be made if desired. Thus, forthese purposes, all comparisons are made as if the fluids being measured behaved as Newtonian fluids. Proce dures for determining viscosity are easily within the skill of the analytical chemist and more detailed guidelines may be found in textbooks, journals and viscometer manufacturers' instructions.

For purposes of the present invention, a change in measured viscosity of about 10 percent is considered to be very significant when comparing a test polymer solution to a control solution. Since commonlyavailable equipment is incapable of accurately measuring the weight of polymers having a molecular weightapproaching one mil lion, the change in the polymer was measured indirectly by determining intrinsic viscosity.

The most common method of estimating the molecularweight of a polymer in solution is the use of "intrinsic viscosity". The intrinsic viscosity " [ N ] " may be related to the molecularweightofthe polymer by means of the Mark, Houwinkand Sakurada equation: [N] = KMa wherein K and a are constants being a function ofthe polymertype, temperature and solvent. (This equation is discussed in more detail in Billmeyer, Jr., F.W., TestbookofPolymerScience,3rd. Ed., NewYork, Interscience Publishers, 1965, pp 79-85.

The constants are initially determined by measuring polymers of known molecularweight. The determination of [N] is made by capillary viscometry of dilute polymer solutions at several concentrations. Viscosity data is extrapolated to zero concentration in order to produce a value related to molecular weight at a constant polymertype, solvent and temperature.

Even if constants are not known and the absolute values of molecularweight are unknown, the proce dure is still useful because the value of [ N ] is directly proportional to a low power ofthe molecularweight.

For a solvent-temperature-polymer system that does not have the constants available, the values of [ N ] may be used to determine increases or decreases of molecular weight, but not the actual values. Since only relative changes in molecularweight need to be determined, this procedure is satisfactory.

The microorganisms useful in accordance with the present invention will have the capability of increasing or at least stabilizing the viscosity of preformed polymers in solution.

In the broadest aspect of the present invention, a suitable microorganism may be chosen by selecting one or more candidates and performirig the screening test described below for determining the effect ofthe microorganism on the polymer solution. Candidate microorganisms may befound almost anywhere. Soil samples and plants are logical locations, as well as subterranean and surface water. Oxygen availability should not be a limiting factor in the selection, since both aerobes and anerobes are useful. Existing cultures of microorganisms may, of course, be tested for utility in accordance with the present invention.

Classes of microorganisms contemplated as useful for the present purposes include phototrophic bacteria, gliding bacteria, sheathed bacteria, budding or appendaged bacteria, spiral and curved bacteria, gram-negative aerobic rods and cocci, gram-negative facultatively anaerobic rods, gram-negative anaerobic bacteria, gram-negative cocci and coccobaccilli, gram-negative anaerobic cocci, grah-negative chemolithotrophic bacteria, methane-producing bacteria, gram-positive cocci, endospore-forming rods and cocci, gram-positive, asporogenous rod-shaped bacteria and actinomycetes and related organisms.Those classes unlikely to be useful in accordance with the present invention include rickettsias and mycoplasmas. Further information on the characteristics of each ofthese classes may be found in Bergey's Manual of Determinative Bacteriology (8th Ed.) R.E.

Buchanan and N.E. Gibbons, Co-editors (1974).

Once the sample has been obtained, the microorganism should be isolated and propagated on one or more types of laboratory media using conventional techniques.

The present invention may be practiced using various production methods suitable for microbiological synthesis. Conventional methods are divided into two categories: batch and continuous. In a batch process, all ofthe starting materials including the microogranisms are placed in a vessel where they remain until the desired product is formed, whereupon the vessel is emptied and the product is separated.

In a continuous process, the raw materials are added and the product is withdrawn at a steady rate. A continuous method is preferred for making or treating large amounts of material, although batch processes are especially useful when close control overthe process conditions is desired.

The process is typically operated at conditions which are optimal for growth ofthe microorganism, since the maximum results are obtained at a high metabolic rate. These optimal conditions are usually similarto those found in the microorganism's natural environment. The environment is duplicated if possibleto provide the same temperature, pH, salinity, trace elements, and other materials.

If the environment cannot be readily duplicated, a conventional nutrient media should be used to provide water, sugars, amino acids, vitamins, minerals and trace elements. Such media are available at biological supply houses.

The optimal range of pH and temperature for rapid growth, as well as the range oftolerable conditions, may easily be determined by common tests known to those skilled in the art. A more detailed description of these tests is given in "Growth", Ralph N. Costilow, Ed. Manual of Methods for General Bacteriology, pp.

65-179, (1981).

The following procedure may be used to screen candidate polymers to determine if a given microorganism will enhance the viscosity of a solution of that polymer. It may also be used to screen candidate microorganisms to determine iftheywill enhance the viscosity of a given polymer. The procedure may also be used to screen entirely new combinations of polymers and microorganisms.

(A.) Preparation of Culture: A growing culture of the microorganism is prepared in a suitable growth medium. The specific pH and ingredients of the medium will vary depending upon the requirements ofthe particular microorganism. This culture is preferably incubated fortwenty-four hours at the organism's optimum growth temperature.

(B.) Inoculation ofthe Polymer Solution: The polymersolution is preferably concentrated enough to provide measurable viscosities for the viscosity test, but sufficiently dilute so thatthe amount of polymer is not toxic to the microorganism. The polymer solution may be sterilized by steam or other means to avoid the effects of competing growth of undesired microbial contaminants. A basal salts solution is optio ally added to the polymer to ensure essential nutrients and mineralsforthe microorganism.

The growing cell culture maythen be centrifuged, washed to removeanyresidual growth medium and resuspended in a minimal amount of distilled water.

For optimal results, a cell count is performed to determine the concentration of cells. A measured amount of inoculum, containing a given concentration of cells, is then added to the solution containing the preformed polymer. A control solution is also pre pared which has the same concentration of polymer solution and basal salts, but does not contain microor ganisms. An equivalent volume of sterile distilled water is added to the control to match the volume of inoculum added to the solutions containing the preformed polymer.

(C.) Incubation of the Solution: The control and the polymer solution with the microorganism are incubated atthe optimum growth temperatureforthe microorganism. A sample of the solution may be removed periodically to show trends in viscosity change, if desired. Definitive results are typically obtained after an incubation of from 2 to 7 days.

After incubation, the microorganisms are preferably removed by centrifugation (or other methods compatible with the polymer and microorganism), leaving a microorganism-free solution of the polymer.

(D.) Determination of Changes in Viscosity: The viscosity ofthe microorganism-free solution may be determined by the methods discussed above, such as a Brookfield viscometer equipped with an ultra-low viscosity cell which will produce viscosity data (centipoise or mPas) at a variety of shear rates.

The viscosity ofthe test solution is compared to the viscosity ofthe control solution. wif the viscosity ofthe test solution is greater than that of the control solution, the microorganism may be considered capable of increasing the viscosity of a solution of that polymer.

Several microorganisms were screened to determine the effectthereof on polymer solutions, using the following general procedure, exceptwhere specifically noted.

Preparation of a Medium: A basal salts medium was prepared at a double-strength concentration for later dilution with the appropriate polymer solution. The basal salts solution consisted ofthe following: Yeast extract 0.1 g K2HPO4 1.Og KH2PO4 1.Og (NH4)2SO4 0.59 CaCI2 .2H2O 0.1 g Deionized H20 500 ml The exact composition of the basal salts medium is usually not critical and it may be altered depending upon the requirements of the microorganism being tested. The same basal salts medium should be used throughout a similar series of viscosity tests, however, because the presence of salts may affect the viscosity characteristics of a polymer in solution.

The CaCI2 .2H20 was autoclaved separately to avoid precipitation with the other components upon heating. After both solutions had cooled to room tempera ture,theCaCl2 2H20wasaddedtotheotheringre- dients. The basal salts medium was chekced for purity by streaking an aliquot onto a nutrient agar plate and incubating the plate at 30"C for 24 hours.

Preparation of a Culture: Bacteria and fungi to be screened were grown on nutrient agar slants and Sabouraud dextrose agar slants, respectively. The microorganisms were incubated at 300C for 24 hours, aerobically and1or anaerobically depending on the physiological properties thereof. Culture purity was determinedviathegram stain and microscopic examination. 5 ml of the basal salts medium were added to each slant and the organisms were removed from the surface by vortexing. 0.05 ml ofthe cell suspension was used as the inoculum.

Preparation of the Polymer Solution: A quantity of the polymer was added to deionized waterto achieve a concentration of 2400 ppm (weightto volume). The polymer solution was adjusted to pH 7.0 and was steam sterilized at 15 psi (1.03 x 105 Pa) and 121 C for 60 minutes. The solution was then tested for purity as describedforthe basal salts solution.

Incubation: The basal salts solution and polymer solution were mixed in a 1:1 ratio, by volume, to provide a test solution of 5 ml. 0.05 ml of microbial suspension (inoculum) was added to each of three tubes. Thesetubes were incubated for 14 days under conditions described for the inoculum preparation.

After incubation, each tube was examined forturbidity indicative of microbial growth.

Intrinsic Viscosity: For these experiments, results were used for comparative changes of molecular weights. Low molecularweight metabolic products were not removed and were assumed to make no contribution to viscosity. Because of the washing procedure to remove the microbial cells from the polymer solution, viscosity change may be attributed to an alteration ofthe polymer itself and not to the presence of biological residue. The temperature for the intrinsic viscosity determination was 25 C.

EXAMPLES 1 to 4 A gram-negative rod characterized as a strian from the genus Enterobacterwas isolated from oil field produced water and was assigned code number MO6882. In earlier laboratory experiments this strian had demonstrated the ability to use the partially hydrolyzed polyacrylamide, "Dow Pusher 500", as its solecarbonsourceforgrowth.

The media used in this experiment were prepared in two parts. Afirst polymer solution was made by dissolving "Dow Pusher 500", as obtained commercially in distilled water at a concentration of 2400 ppm (weight to volume) and pH 7.0. The second solution was made in the same way, but after purifying the polymer by alcohol extraction. Both solutions were stirred for 24 hours, weighed before sterilization and then autoclaved (sterlized) for 1 hour at 121 0C and 15 Psi (1.03 x 105 Pa). All water lost due to autoclaving was returned by adding sterile distilled water until the polymer solutions reached original weight.

A basal salts solution was prepared at a double concentration, as explained above. It contained 0.02% yeast extract manufactured by BBL (a division of Becton, Dickinson & ompanyofCockeysville, M D.

USA), 0.2% KH2PO4, 0.2% K2HPO4, 0.1% (N H4)2 S04 and 0.02% CaCal2 -2H20 in distilled water at pH 7.0. The CaCl2 was made as a sepa rate stock solution and added to the salts solution after both were sterilized and cooled to room temperature.

The two separate "Dow Pusher 500"/basal salts solutions forthis experiment were made by aseptically mixing 1 :1 solutions of basal salts medium (2X) and 2400 ppm (w.v) polymer solution.

The EnterobacterM06882 culture was grown aerobically on a nutrient agar (Difco) slant at 300C for 24 hours. The cells were harvested by adding 5 ml of sterile basal salts solution to thetesttube slant and vortexing the tube gently. This cell suspension was used as the inoculum forthis experiment.

All bottles were incubated at 30 C for 14 days.

Aerobic incubation included rotary shaking ofthe flasks at 200 rpm. Anaerobic incubation was carried out in an anaerobicchambersystem (manufactured by Forma Scientific). Control solutions were incubated both aerobically and anaerobically.

After incubation, all test solutions and controls were centrifuged(10,000 x g, 300C, 20 min) and the supernatant aseptically decanted into clean, sterile sample bottles. The viscosities of the solutions were measured.

The molecular weight of the "Dow Pusher 500" was assumed to be 3.4 x 106, based upon published data (F.D. Martin and J.S. Ward, Polymer Preprints, Vol.22, No.2, American Chemical Society Meeting, August, 1981, p.24). The control solution viscosity was also assumed to be constant. Detailed data showing measured viscosity, intrinsic viscosity and estimated molecularweight ofthe polymerforcontrol solution A and Examples 1 and 2 are given in Table 1 below.

Table 1 Enterobacter MO6882 with "Dow Pusher 500" Intrisec Measured viscosity Viscosity (cps or mPar) Estimare Example Incubation [N] 10sec-1 70sec-1 Mol.wt.

A. Aerobic 16.6 9.7 7.0 3.4 x 106 1. Aerobic 18.1 9.2 6.3 3.7 x 106 2. Aerobic 27.6 17.1 7.3 5.7 X 106 Compared tothe control, the organism causes an increase in viscosity or stabilizes the purified polymer from decomposition over the 14 days. Under anaerobic conditions, Enterobacter M 06882 increases the apparent molecularweight of "Dow Pusher 500" from 3.4 x 106to5.7 x 106, an increaseofabout68%.

Estimates of molecularweightwere madefrom changes in intrinsic viscosity.

EXAMPLES 5 to 8 The procedures above were repeated to determine changes in intrinsic viscosity as discussed above. All conditions were the same except where noted.

Table 2 below summarises the intrinsic viscosity results ofthese solutions incubated with Enterobac ter M06882 for 2 weeks. Relative viscosity increases were found in all samples tested. The greatest enhancement in solution viscosity was with the samplesthatwere incubated anaerobically. Because a control was not available to compare the effects of a purified polymer under anaerobic incubation, the effects of purification were deemed secondary.

Exa m ples 2 and 6 were therefore compared to Control C, the closest comparison.

The intrinsic viscosity data from Examples 1 to 4 and Controls A and B are also shown in Table 2 below.

Toble 2 Enterobacter MO6882 and Polyacrylamide" Incubrition Intrinsic Example Polymer Conditions Viscosity in Viscosity A. Pure Aerobic 16.6 CONTROL B. Comm. Aerobic 15.5 CONTROL Comn. Aerobic 16.9 CONTPOL 1. Pure Aerobic 18.1 9 2. Pure Anaerobic 27.6 63 3. Com . Aerobic 17.0 10 4. Conni. Aerobic 26.6 57 5. Por'. Aerobic 18. i 9 6. are Acoerobic 17.8 5 7. Co ro. Aerobic 18.5 16 Anaerobic 19.1 18 *,,Dow Pusher 500": "Comm." means as commer cially obtained; "Pure" means the polymer was purified by extraction with methanol.

EXAMPLES 9 to 130 Eight strains of bacteria in addition to Enterobacter MO6882 were chosen for testing. All were capable of growing aerobically and/or anaerobically on various water-soluble polymers. The nine strains are identified bythefollowing reference numbers: fam-negative rods: MO6882 PO4062 PO3482 P04282 20582 cocci: MO6682 MO6782 MO6182 rod: I6782 The organisms PO4082, PO3482 and PO4282 were laboratory contaminants isolated from aqueous polymer solutions. All other bacteria were isolated from oilfield produced water and drilling muds.

All strains (exept MO6182) were grown aerobically in nutrient broth (Difco) at 30 for 24 houres. Organism MO61 82 was incubated anaerobically at 30 C for 24 hours using thioglycollate broth.

After incubation, the bacteria were washed three times by centrifugation (5,000 x g,5'C, 10 min) with sterile distilled water. The washed cells were resuspended in minimal sterile distilled water. Cell counts were done on each cell suspension using a Petroff Haussercounting chamber.

Media were preapred forthis experiment in two parts. Polymer solutions were made in distilled water at a concentration of 2400 ppm (w/v) and pH 7.0. The polymers in Table 3 below were used in commercial form and after purification by extraction.

T0o 1, 3 Polymer Source Polyacrylamide, hydrolyzed "Dow Pusher" Polyvinyl alcohol Polysciences, Inc., Cot.#2015 Poly[AMPS] poly(2-octylamido-2-methyl propone sulphonic acid) from Lubrixal Corporation Xanthan @@@ "Xanflood":Kelca Hydroxyethyl cellulose Nutrosol 250 HHR, from Hercules, Inc.

Carboxyacthyl cellulose CHC 9H4 from Hercules, Inc.

Glucon Actigum CS 1-L abtoined from Jerco Chemicals, Inc.

Polyacrylamido syntherized unhydralyzed polyacrylamide molecular weight of 3.5 x 10 Polyacrylic acid Scientific Polymer products, Inc., catalog #026 All solutions were stirred for 24 hours, weighed and then autoclaved (sterilized) for 1 hour at 1 210C and 15 psi (1.03x105Pa). All water lost due to autoclaving ws returned by adding sterile distilled water until the polymer solutions reached original weight.

A basal salts medium was prepared as described in Example 1. Eighteen different polymer media forthis experiment were made by aseptically mixing 1:1 (v/v) solutions of basal salts medium and 2400 ppm polymer solution.

50 ml aliquots of polymer media were aseptically transferred to clean, sterile 150 ml Erlenmeyerflasks.

Each flask (except controls) was inoculated to a concentration of 103 bacteria/ml with the appropriate cell suspension. Controls were inoculated with equivalent volumes ofsterile distilled water.

All flasks were incubated at 30 C for 2 weeks.

Aerobic incubation included rotary shaking ofthe flasks at 200 rpm and anaerobic incubation was conducted in an anaerobic chamber system. Controls were incubated aerobically.

After incubation, all test solutions and controls were centrifuged (10,000 x g,30 C, 20 min) and the supernatant aseptically decanted into a clean, sterile sample bottle. Apparent viscosity determination were made using a Brooksfield viscometer. Table 4 below shows the results for Examples 9to 130 and also incorporates data from Examples 1 to 8.

TABLE 4 Toble 4 Continued Selected Microbe/Polymer Combinations Percent Incu- Change in Example Polymers Organism bation Viscosity Percent 48 Poly(AMPS), purified4 MO6682 A -1.9 Incu- Change in Example Polymer Organism bation Viscosity 49 " MO6682 An -0.5 1 Poylacrylamide , purified MO6882 A 9. 50 " MO6882 A -12.9 2 ,. " M06882 An 63. 51 .. M06882 An -10.0 3 Polyacylamide (comm.) MO6882 A 10. 52 " PO4082 A -29.7 4 " " MO6882 An 57. 53 " PO4082 An -29.2 5 Polyacrylamide , purified MO6882 A 9. 54 " PO3482 A -25.4 6 l ., M06882 A 5.0 55 ' P03482 An -23.4 7 Polyacryamide (comm.) MO6882 A 16. 56 " PO4282 A -27.3 8 ' MO6882 An 18.0 57 " P04282 An 19.6 9 P04882 A -8.0 58 Xapthon Gum P04082 An -3.0 10 " P04082 An +3.5 59 " P04282 A -3.0 11 1. P04282 A -4.4 60 " P04282 An -6.1 12 " P04282 An +4.4 61 " 20582 An -3.0 13 .. 16782 A 0 62 Xanitron Gum, purified P04082 An 0 14 " 16782 An -28.3 63 " PO4082 A +6.7 15 " 20582 A +6.2 64 .. 16782 An 0 16 " 20582 An +4.4 65 " 16782 A +6.7 17 (purified) PO4282 A -1.0 66 Hydroxyethyl cellulose M06882 A -10.7 18 " PO4282 An +35.6 67 " MO6882 An -10.7 19 ' P04082 A 4.8 68 " P04082 A 0 20 " Po4082 An 9.6 69 " P04082 An -7.1 21 Polyvinyl Alcohol M06682 An 7.7 70 .P04282 An 1.3 22 ' M06682 A 0 Toble 4 (Continued) Table 4 (Continued) Percent Percent Incu- Change in Incu- Change in Example Polymer Organism bation Viscosity Example Polymer Organism bation Viscosity 71 Hydroxyalkyl cellulose MO6882 A +10.0 23 Polyvinyl Alcohol MO6882 A 0 24 " MO6882 An 0 72 Hydroxylalkyl cellulose MO4082 An -13.3 25 " P04082 A 0 73 " P04082 A -16.7 26 " PO4082 An +7.7 74 " PO4082 An -3.3 27 ' 16782 A 0 75 ' P04282 An -6.7 28 ' 16782 An -7.7 76 Carboxymethyl cellulose M06882 A -8.3 29 - 20582 A -7.7 77 " P04082 A -11.1 30 " 20582 An -7.7 78 ' P04282 An -11.1 31 " PO4282 A +7.7 74 "(purified)4 MO6782 A -3.3 32 " P04282 An +7.7 80 ' M06782 An -10.0 33 Polyvinyl Alcohol (purified) M06682 A 0 81 .. M06882 A -3.3 34 ' M06882 A -7.7 82 " PO4082 A -3.3 35 " M06882 An 0 83 . P04082 An 0 36 " P04082 A 0 84 .. P04282 An -6.7 37 " PO4082 An -7.7 85 Glucon MO6882 A -10.5 38 . P03482 A 0 86 " M06882 An -6.7 39 ' P03482 An -7.7 87 " P04082 A -7.7 40 " P04282 A 0 88 ' P04082 An -3.4 41 " PO4282 An +7.7 89 " PO4282 A +15.9 42 Poly(AMPS) MO6882 An -3.8 70 " 16782 A +17.4 43 ' M06882 A 0.6 91 .. 20582 A +16.1 44 . P04082 A -8.3 22 . P04282 An -17.2 45 " PO4082 An +1.9 73 " 14782 An -18.1 46 .P04282 An O 47 " MO6682 A +7.0 Table 4 (Continued) P f Incu- Change in Example Polymer Organism bation Viscosity 94 Glucon 20582 An -19.8 95 " (purified)4 MO6882 A -18.2 96 " M06882 An +36.7 97 " PO4082 A -8.5 98 " PO4082 An +51.4 99 n P06282 A +19.4 100 " PO4282 A -13.8 101 " PO3482 A +33.4 102 " 16782 An -13.0 103 " 16782 A +46.6 104 " 20582 An -13.6 ies " 20582 A o16.0 106 Polyaxylamida5 M06882 An -5.3 107 " PO4802 A -5.8 108 PO4302 An 0 129 " PO4282 A -10.5 110 u PO4282 An -5.2 111 MO6182 An -10.5 132 Polyacrylamide (purified)4 MO6882 A -13.6 113 PO4082 A -9 1 114 a PO4082 An 4.5 115 " PO3422 A -18.2 116 " PO3482 An -4.5 117 u PO4282 A 4.5 118 = PO4282 An -9.1 Table 4 (Continued) 9 Incu- Change in Example Polymer Organism bation Viscosity 119 Polyacrylic Acid7 MO6832 A -3.3 120 MO6882 An +3.3 12' PO4082 A -3.3 122 PO4082 An -3.3 123 PO3482 An -3. 124 " PO4282 An 16.7 125 "(purified) MO682 A -3.3 126 " M06882 An -6.7 127 " PO4082 A -3.3 128 PO4082 A -6.7 129 PO4282 An +3.3 130 " P04282 A +16.7 "A" denotes Aerobic; "An" denotes eccecobic 2 Intrinsic viscosity; indicates relative change in molecular weight of the polymer compared to the control 3 "Dow Pucher 500", a partially-hydralyzed acrylamide.

4 Purified by extraction with nzethonol 5 Synthsized in "Sohio" laboratorier; unhydrolyzed polyacrylamide having a molecular weight of 3.5 x 105.

6. Purified by extraction with isoproponol.

7 Purified by extraction with ocetonitrile.

MicrobialEnhancemenf ofPolym erSolution Viscosity Several samples tested in this polymer/bacteria screen showed solution viscosity increases after 2 weeks of incubation. Some of the samples that showed microbial enhancementof polymer solution viscosity may be found in Table 5 below, grouped according to the code numberforthe bacterium.

TABLE 5 Percent Incu- Change in Example Polymer Organism bation Viscosity 1 Polyacrylamide , purified MO6882 A +9.

2 " " MO6882 An +63.

5 " " MO6882 A +9.

6 " " MO6882 A +5.0 3 Polyacrylamide (comm.) MO6882 A +10.

4 " " MO6882 An +57.

7 " " MO6882 A +16.

8 " " MO6882 An +18.0 43 Poly(AMPS) MO6882 A +0.6 96 Glucon (purified)4 MO6882 An +34.7 120 Polyacrylic Acid MO6882 An +2.2 65 Xanshan Gum, purified4 16782 A +6.7 50 Glucon 16782 A +17.4 103 Glucon (purified)4 16782 A +46.4 47 Poly'AMPS) MO6882 A +7.8 15 Polycrylamide (Dow) ZO582 A +4.2 16 " ZO582 An +4.4 91 Glucon ZO582 A +16.1 105 " ZO582 A +16.0 101 Glucon (purified)4 PO3482 A +33.4 10 Polyacrylamide PO4082 AN +3.5 19 Polyacrylamide (purified) PO4082 A +4.2 20 " " PO4082 An +2.6 26 Polyvinyl Alcohol PO4082 An +7.7 45 Poly(AMPS) PO4082 An +1.9 63 Kon+kon Gum, purified4 PO4082 A +6.7 98 Glucon (purified)4 PO4082 An +51.4 12 Polyacrylamide PO4282 An +4.4 18 Polyacrylamide (purified) PO4282 An +35.6 31 Polyvinyl Alcohol PO4282 A +7.7 32 " PO4282 An +7.7 41 " (purified)7 PO4282 An +7.7 89 Glucon PO4282 A +15.7 99 Glucon (purified)4 PO4282 A +19.4 124 Polycrylic Acid PO4282 An +16.7 120 " (purified) PO4282 A +16.7 Priorto 28th December 1983,the microorganisms In Table 6 below were deposited with the American Type Culture Collection (ATCCj in Rockville, Mary land, U.S.A.

Table 6 Code Number vs. ATCC Number MC5882 39553 PO4082 39955 PO3482 39560 PO4282 39558 20582 39554 MO6682 39557 16782 39556 MO6182 39559 MO6782 39552

Claims (19)

1. A method for increasing the viscosity of a solution of a preformed polymerwhich comprises combining, under conditions favourable to microbial growth, (a) an aqueous solution ofthe preformed polymer; and (b) a microorganism incapable of de novo synthesis of the polymer, but capable of increasing the viscosity of the polymer solution.

2. A method as claimed in claim 1 wherein the microorganism has been selected by a screening process comprising: (a) preparing a growing culture of the microor ganism; (b) preparing an aqueous solution of preformed polymer and adding nutrient(s) and mineral(s) re quired for microbial growth; (c) separating that solution into two portions: a test and a control; (d) inoculating thetest solution with theculture of the microoganism and incubating the mixture; and (e) measuring the viscosities of both test and control solutionsafterincubationto determine if the test solution is relatively more viscous than the control.

3. A method as claimed in claim 1 or claim 2 wherein the microorganism is a bacterium.

4. A method as claimed in claim 3 wherein the bacterium is selected from gram-negative rods, gram-positive rods and gram-positive cocci.

5. A method as claimed in claim 3 or claim 4 wherein the bacterium is selected from MO6882, PO4082, PO3482, PO4282, 20582, MO6682, MO6782, 16782 and MO6182.

6. A method as claimed in any of claims 1 to 5 wherein the polymer is a biopolymer or a synthetic polymer.

7. A method as claimed in any of claims 1 to 6 wherein the polymer is selected from guar gum, hydroxypropyl guar, sodium carboxymethyl cellulose, hydroxyethyl cellulose, carboxymethyl hydroxyethyl cellulose, xanthan gum, scleroglucan, locust bean gum, polyacrylamide, hydrolyzed polyacrylamide, poly(acrylic acid) and salts thereof, poly(2 acrylamido-2-methyl propane sulphuric acid) (2 salt) (ANPS), poly(acrylic acid-co-acrylate ester), poly (vinyl pyrrolidone), cellulose sulphate esters, poly (ethylene oxide), poly (vinyl alcohol), polyamine, poly (vinyl acetate-co-malleic anhydride) and poly(styrene sulphonic acid) and salts thereof.

8. A method as claimed in claim 7 wherein the polymer is selected from polyacrylamide, hydrolyzed polyacrylamide, polyvinyl alcohol, xanthan gum, poly(2-acrylamido-2- methyl -propane sulphonic acid, hydroxyethyl cellulose, poly(acrylic acid), carboxymethyl cellulose and glucan.

9. A method as claimed in claim 8 wherein the polymer is selected from hydrolyzed polyacrylamide, polyvinyl alcohol, poly(2 - acrylamido - 2 - methylpropane sulphonic acid), xanthan gum, poly(acrylic acid) and glucan.

10. A method as claimed in claim 9 wherein the polymer is selected from hydrolyzed polyacrylamide, poly(acrylic acid) and glucan.

11. A method as claimed in any of claims 1 to 10 wherein: the polymer is hydrolyzed polyacrylamide and the microorganism is selected from PO4082, PO4282, 20582 and MO6882; or the polymer is polyvinyl alcohol and the microorganism is selected from PO4082 and PO4282; or the polymer is poly(AMPS) and the microorganism is selected from PO4082, MO6682, and MO6882; or the polymer is xanthan gum and the microorganism is selected from PO4082 and 16782; or the polymer is glucan and the microorganism is selected from PO4082, 16782, PO4282, 20582, MO6882 and PO3482; orthe polymer is poly(acrylic acid) and the microoganism is selected from PO4282 and MO6882.

12. A method as claimed in claim 1 substantially as herein described with particular reference to the Examples.

13. Apolymersolution having enhanced viscosity characteristics which is obtainable by combining an aqueous solution of a preformed polymerwith a microorganism incapable of do novo synthesis of the polymer, but capable of increasing the viscosity of the polymer under solution and maintaining the solution under conditions conducive to microbial growth.

14. A solution as claimed in claim 13 from which the microorganism is subsequently removed.

15. A solution as claimed in claim 13 substantially as herein described with particular reference to the Examples.

16. A method for the enhanced recovery of oil which comprises combining a preformed polymer with an aqueous fluid to form a polymer solution, introducing the polymer solution into an oil-bearing reservoir via at least one injection well and recovering oil from a production well,there being added to the polymer solution a microorganism incapable of de novosynthesis ofthe polymer, but capable of increasing the viscosity of the polymer solution.

17. A method forthe clarification of water by flocculation which comprises mixing a preformed polymer with an aqueous fluid and thereby forming a floc in which particulate matter is suspended, there being added to the polymer a microorganism incapable of de novo synthesis ofthe polymer, but capable of increasing the viscosity of the polymer.

18. A methodforthe manufacture of paper which comprises preparing a pulp slurry and adding an aqueous solution of preformed polymerto the slurry, there being added to the polymersolution a microorganism incapable of de novo synthesis ofthe polymer, but capable of increasing the viscosity of the polymer solution.

19. A method as claimed in any of claims 16 to 18 substantially as herein described with particular reference to the Examples.