CA1309366C - Bacteria and its use in a microbial profile modification process - Google Patents

Abstract

ABSTRACT OF THE DISCLOSURE
The present invention is a bacteria and its use in a Microbial Enhanced Oil Recovery (MEOR) process. Any one of two newly isolated strains of bacteria are injected, along with a nutrient solution, downhole in a petroleum reservoir to modify its profile. This bacteria has the capability to plug the zones of higher permeability within the reservoir so that a subsequent waterflood may selec-tively enter the oil bearing less permeable zones. The injected water is used to drive this oil to an area where it may then be recovered.

Description

BACTERIA AND ITS USE IN A
MICROBIAL PROFILE MODIFICATION PROCESS
SBACKGROUND OF THE INVENTION
. _ This invention generally relates to the use of microorganisms to enhance oil recovery from petrolsum reservoirs. Here, a specific strain of bacteria is used along with a nutrient source to selectively plug high permeability zones and increase waterflood efficiency by redirecting the flood to lower permeability, oil bearing zones.
Petroleum that is in underground reservoirs is brought to the surface in a variety of ways. One of the more notable publicly held ideas of oil recovery is the "gusher," however, due to the changing nature of oil reserves, and economic and environmental policies, the gusher is the thing of the past. Surface pumps, which are a common highway site, oftentimes provide the lift force ~ necessary to bring oil to the surface in those reservoirs where the overburden pressure is insufficient. Addi-tionally, subsurface pumps can be coupled with the surface pumps to assist in the lifting duty. However, there comes a time in the life of many reservoir formations in which the overburden pressure and the pumping devices are not enough to overcome the oil viscosity and the capillary forces of the formation. At this point, enhanced oil recovery (EOR) techniques are useful to drive out that stubborn quantity that refuses to come to the surface by 3 the means described above.
The term "EOR" spans a panoply of techniques and devices that are used to recover the last bit of oil reserves. There are devices and methods for: steam injection, water injection, gas driving, emulsifying, injecting plugging agents, etc. One "device" that may perform many of these feats is a microorganism, most notably, a bacteria.
The idea of using bacteria to increase or enhance oil recovery is not new. Many laboratory i309366 investigations and a number of field tests have been per-formed both in the U.S. and elsewhere (see generally 05 J. Davis, Petroleum Microbiology [1967] and works collected in J. E. Zajic et al., Microbes and Oil Recovery, Bioresource Publications, El Paso [1985]).
Several technical meetings devoted exclusively to micro-bial enhanced oil recovery (MEOR) have been held. Some of the previous literature consists of anecdotal accounts or inadequately controlled studies, resulting in a skeptical appraisal of the technology. (See also D. Hitzman, Petroleum Microbiology and the History of its Role in Enhanced Oil Recovery, Proc. Int'l. Conf. on Micro. Enh.
15 Oil Rec., p 162, [May 16-21, 1982], and E. Donaldson et al., There are Bugs in My Oil ~ell, Chemtech, p 602 [Oct. 1985].) The principle behind MEOR is based on the fact that microbes can produce most of the agents now employed in chemical EOR; i.e., water-soluble polymers, surfac-tants, co-surfactants and solvents such as ethanol and acetone, and acids. (See M. Singer, Microbial Biosurfactants, in Zajic, Microbes and Oil Recovery; U.S.
Patent No. 4,522,261 to McInerney et al.; U.S. Patent 25 No. 2,807,570 to Updegraff and U.S. Patent No. 2,660,550 to Updegraff et al.) Some microbial-produced products, e.g., xanthan biopolymer, are now commercially used for EOR. Such use is dependent on the cost-effectiveness of the microbial product compared to competing non-microbial products, e.g., xanthan compared to polyacrylamide. In this application, the definition of MEOR applies to pro-cesses involving the in-situ application of microbial processes and usually excludes EOR processes which merely involve the use of chemical products which are produced in a fermentation plant.
The specific application of microorganisms for EOR in this invention is their use for the selective plugging of zones of high permeability (i.e., thief zones) in petroleum reservoirs. To back up a bit, when water injection is used to recover oil, it is injected downhole 1;~09366 01 ~3~
in an injection well to move any oil out of the formation to be recovered at a producing well. The water pushes the 05 oil out of the small interstices and pores of the rocks, but it pushes the oil out of wider spaces and larger pores (i.e., zones of higher permeability) first, leaving the smaller areas still filled with oil. Since petroleum is formed in stratified sedimentary deposits, several dis-tinct layers of oil-bearing sands are usually present over the vertical profile of an oil well. Different layers can vary widely in permeability and porosity, as well as other properties. Since a waterflood will naturally seek the zone of least resistance (or highest permeability), low permeability zones may be bypassed. After a time, recover-able oil is "watered out" of the high permeability zones, but the low permeability streaks still contain considerable recoverable oil. The way that the residual oil may be taken out of these lower permeability zones is by "profile modi-fication". Current technology involves the injection of water-soluble polymers, which selectively enter the high permeability zones. Cationic cross-linking agents, i.e., Cr+3, Ti+4, or Al+3, held in solution by a complexing agent (i.e., citrate) or by oxidation state, are co-injected with the polymer or are swept after the poly-mer. (See U.S. Patent No. 4,552,217 to Wu et al.). As the polymer gradually cross-links and gels into a water-insoluble 3-D matrix in the high permeability zones, the waterflood is channeled into zones of low permeability, thus increasing oil production. There are problems asso-ciated with the techniques of profile modification with cross-linking polymers. Such polymers are relatively expensive; they may shear-degrade upon injection at the wellhead and may not penetrate sufficiently before gelling. For this reason, the use of microorganisms may prove promising in profile modification because it may eliminate some of these problems.
As with other techniques, using microbes to plug high permeability zones is not exactly new. Some early researchers are listed in J. Davis, "Petroleum 01 ~4~
Microbiology" (1967) and more recently there is U.S.
Patent No. 4,558,739 to McInerney et al; and D. Revus, A
05 Study of Reservoir Selective Plugging Utilizing In Situ Growth of Bacteria to Improve Volumetric Sweep Efficiency, Masters Thesis, Univ. of Oklahoma (1982); P. Kalish et al., The Effect of Bacteria on Sandstone Permeability, 16 Jour.
Pet. Tech. 805 (July 1964); and C. Brierley et al., Investigation of Microbially Induced Permeability Loss During In-Situ Leaching, Bureau of Mines (NTIS Publication) (April 1982). They use microbes in a variety of ways to enhance oil recovery. Some researchers have used the bac-teria that naturally exists in the formation and have sim-ply injected nutrients downhole to get them to grow and plug the formation (see U.S. Patent No. 4,475,590 to Brown; and L. Allison, Effect of Microorganisms on Permeability of Soil Under Prolonged Submergence, 63 Soil Science 439 [1947]). Others have injected bacteria down-~0 hole and then followed by a nutrient solution. On anotherscore, some researchers depend on the biomass of the bac-teria for plugging purposes, while others show that exo-polymers produced by the bacteria are effective in closing off areas of high permeability.
Another factor in this plugging technique is the size of the organism that is being injected. For example, if a bacteria has a small enough size, it may penetrate the formation a bit easier to plug off the thief zones.
To that end, the spores of different bacteria may be used for injection to penetrate even deeper. Spores may pene-trate a reservoir formation and become lodged in these permeable zones, so that when they are stimulated to grow by a nutrient solution, they will plug more pores more effectively. To better achieve penetration, vegetative cells arising by germination of the spores should be motile so that they may propel themselves deeper into the pores.
Some problems exist with the environment in which the bacteria are injected. For example, downhole in a petroleum reservoir, there are conditions that put con-straints on microorganisms. More specifically, connate 13Q9;~66 01 _5_ water, in many formations, has both high concentrations ofsalt, (NaCl), alkaline earth ions (Ca+2, Mg+2, Ba+2), 05 transition metal, rare earth, and heavy metal ions. Such ions can form insoluble precipitates with many of the standard components of bacterial nutrient media. This plugs the wellbore and prevents the injection of cells or nutrients. Furthermore, some of these ions are inhibitory 0 or toxic to microbial cells and we have found that some ions (e.g., Ca+2) are inhibitory to biopolymer production by our microorganisms. Bacteria that are injected down-hole must be tolerant to these if they are to survive.
The downhole environment is usually anoxic, unlike the highly oxygenated condition above. To be able to survive and live in both environments, a bacteria must either be shielded from oxygen (which may be difficult and expensive) or must be tolerant to it (e.g., a facultative anaerobe). [Bacteria can be broadly divided into 3 cate-~0 gories based on their ability to utilize and tolerateoxygen: (1) obligately aerobic bacteria, which require molecular oxygen for growth; (2) obligately anaerobic bacteria, to which molecular oxygen is toxic; and (3) facultative anaerobes, which can grow either in the presence or absence of atmospheric oxygen. Of the three, facultative anaerobes appear to be the most suitable MEOR
candidates, since they can survive exposure to air during storage and injection while retaining the ability to grow well anaerobically.] Furthermore, while most bacteria are "mesophilic", i.e., prefer to grow at temperatures in the range 15-37C, the environment in petroleum reservoirs, particularly at greater depths, frequently exceeds these temperatures and may easily be at 40-50C or even higher.
Therefore, it is advanatgeous to use for MEOR bacteria which are "thermotolerant" and can grow at 40-50C or even higher and can survive exposures to up to 100C.
The most important ingredient, i.e., the bac-teria, sometimes must be selected for these exact condi-tions that exist in a reservoir. Also, the nutrient's solution has to be tailored to both the bacteria and the reservoir in which it has to be injected. All theseconsiderations must be merged together to provide the 05 desired result in plugging the formation.
SUMMARY OF THE INVENTION
The ~resent microbial process improves sweep efficiency more effectively than is currently possible with injected polymer, and at a significantly lower cost.
We have isolated strains of bacteria from saline sediments which are halotolerant (able to live in a moderately salty environment), spore forming, thermotolerant (able to live within a wide temperature range), motile, biopolymer-pro-ducing facultative anaerobes; that is, they can grow and produce a viscous biopolymer within a petroleum reservoir.
When spores of these bacteria are pretreated and injected along with the specially designed nutrient solution of this disclosure, they selectively penetrate higher permeability zones. After germination and growth, their biomass, and the exopolymer (the polymer that is secreted outside of the cell) that is produced in-situ, gives the desired profile modi-fication. The effectiveness is superior to injected poly-mers since the non-viscous spore suspension penetrates further, and the chemicals cost one-tenth that of polymers. Since the polymer is formed in-situ, there is no degradation from injection shear or storage.
The nutrient medium of the present invention favors production of biopolymer by the microbe from cheap substrates such as sucrose and allows precise delivery of a gelation agent and control of gelation. This medium, among other things incorporates a unique polyphosphate compound, a constituent which serves several purposes: 1) it chelates cross-linking cations such as Cr+3, allowing their incorporation in the medium; 2) it serves as a phos-phorous source for growth and polymer production by themicrobes, and in this process it is consumed; 3) as con-sumed, it releases the chelated cation, stimulating gela-tion of the polymer; and 4) it also chelates other cations present in the rock and connate water, e.g., Ca+2, Mg+2, or Ba+2, which are inhibitory to polymer production by the 1;~09366 01 _7_ microbe. The medium of our invention, containing controlled amounts of polyphosphate, permits optimum poly-05 mer production in-situ, facilitates delivery of gelation agent, and the gelation reaction at the correct time and place.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is an example of a MEOR continuous testing unit.
FIG. 2 shows the pressure response of cumulative nutrient addition on growing cells in a core.
DETAILED DESCRIPTION OF THE INVENTION
MEOR requires the use of halotolerant, faculta-tive anaerobes since most oil field connate waters aresalty and oxygen tension is nil. The cells must be ther-mophilic (able to grow above 55C) or "thermotolerant"
(able to grow over a wide temperature range including 40-55C and survive exposure to above 55C) since petroleum reservoirs are most frequently within this tem-perature range. The cells (or spores from the cells) must be small-sized and mobile (or motile) so they can pene-trate far into the porous rock. They must have non-fastidious nutrient requirements since laboratory culture media would be prohibitively expensive for field applica-tion where huge volumes are injected. The cells must be able to grow and produce the desired product under in-situ conditions of pH, temperature, heavy metal ion concentra-tion, etc. Although some oil-bearing formations are too hot, impermeable or otherwise inhospitable to microbial presence, many are within a temperature range of 20-80C
and can support microbial presence and growth. Microbes used for MEOR must also be non-pathogenic and must not produce any animal or plant toxins, since they may be injected near water supply aquifiers.
Although many microbes can utilize hydrocarbons as the sole carbon and energy source, all known species that do this are aerobes which require molecular oxygen for the initial attack on hydrocarbons. If facultative anaerobes are used for MEOR, non-petroleum carbon sources should be supplied. Unless hydrocarbon-utilizing anaer-obes can be created through recombinant DNA techniques, 05 sufficient non-hydrocarbon metabolizable components must be present in the petroleum. Suitable carbon substrates are cheap carbohydrates such as molasses and whey and possibly inexpensive synthetic substrates such as methanol. Nitrogen, phosphorous and other nutrients must also be supplied if these are not present in the carbon substrate or in the rock. Nutrients must be supplied at the correct time that microbial activity is desired; loss or absorption of nutrients would be an economic debit.
Additionally, there is a problem when phosphates are added to the formation along with the bacteria. They may precipitate out of solution when injected downhole due to highly saline, acidic, or metal ion containing water.
A typical liter of nutrient medium may comprise: 200 g sodium chloride; 0.5 9 magnesium sulfate; 1.33 9 ammonium ~ nitrate; 2.0 g sodium nitrate; 1-5 9 yeast extract; 2.6 g citric acid; plus 5-20 9 of sodium tripolyphosphate as the phosphate source of the present invention.
Facultative anaerobic halophilic, or halotoler-ant, thermotolerant bacteria are preferred to achieve pro-file modification through the production of exopolymer and/or the growth of cells within highly permeable rock, thereby decreasing the permeability of this rock. The exopolymer forms an insoluble matrix within the rock pores which is resistant to bio-, shear, and thermal degrada-tion. Ionic or other cross-linking agents are preferably used to enhance polymer stability in-situ. However, since the polymer is produced in-situ and not injected from the surface (where water solubility is essential), such cross-linking agents are an optional feature of our invention.
The spores which are injected are small enough to pene-trate high permeability zones, but not oil-bearing low permeability zones.
The spores are prepared by growing cells on a sporulation medium to give a spore concentrate (109 spores/ml). This spore suspension is stable for long ~309366 01 _9_ periods of time and is pretreated (by aging, lysozyme or other enzyme treatment) and filtered to remove cellular 05 debris and improve injectivity. The spore suspension may be diluted 1:1000 with nutrient or brine prior to injec-tion, i.e., the injected spore concentration is approxi-mately 106/ml.
Natural Isolates from Enrichment Cultures To isolate bacteria which could survive high salt concentrations, environmental samples of oil reser-voir waters and sediments were obtained from saline lakes including the Great Salt Lake in Utah, the Salton Sea and Soda Lake in California. These samples were collected and lS shipped in a manner to preserve strictly anaerobic bacteria.
Enrichment cultures of each of the source sam-ples were set up in several different culture media con-taining 5, 10 and 15% salt. Nutrient broth glucose and the brain heart infusion enrichment culture media were ~0 used as culture media under sterile and anaerobic condi-tions (unless otherwise specified, all nutrient media are common formulations which are readily available from bac-terial nutrient supply companies, such as Difco or BBL).
For strict anaerobic bacteria, thioglycollate broth, which contains its own reducing agent, was used. All enrich-ments cultures were incubated at 40C.
After subculturing each enrichment medium at least once, the most recent subculture was streaked in duplicate to a variety of agar plates including nutrient agar, nutrient agar glucose, veal agar, and brain heart infusion agar containing salt in varying concentrations (i.e., 5, 10 and 15%). One set of plates was incubated aerobically at 40C; the other set was incubated in an anaerobic jar using a BBL ~asPac anaerobic system to create and maintain an anaerobic environment.
After incubation, the plates were examined and generally showed at least three morphologically distinct colony types. These colonies were picked from the agar surface and re-streaked in duplicate to plates of the same agar for both aerobic and anaerobic incubation. On the 1;~09366 o1 -1o-basis of these plates, strict aerobes and strict anaerobes were discarded.
05 The enrichment culture isolates were further screened for their ability to grow in a simple liquid medium. Bushnell-Haas medium, widely used for the culti-vation of hydrocarbon-degrading microbes (when supplemented with oil) is among the most simple media which provide the essential elements required by non-fastidious microorgan-isms. Its composition, per liter, is 0.2 g magnesium sulfate, 0.02 g calcium chloride, l.0 g monopotassium phosphate, 1.0 g dipotassium phosphate, l.0 g ammonium nitrate, and 0.05 g ferric chloride.
None of the enrichment culture isolates would grow anaerobically on the very simple Bushnell-Haas mineral salts medium containing 10% salt and molasses.
When this medium was supplemented with small amounts of yeast extract or tryptone, a very few cultures showed 20 moderate growth. These cultures included: SLS-l, which was isolated from sediment from the Great Salt Lake, Utah;
and Salton-l, which was isolated from the Salton Sea.
Continued culture in even this supplemented liquid medium resulted in less vigorous growth. For culture mainte-nance, Brain E~eart Infusion was added at l/20 its usualconcentration to facilitate continued good growth. This culture maintenance medium was called Inoculum Growth Medium (IGM) and had the following composition:

30Inoculum Growth Medium (IGM) Per liter distilled water 0.2 g Magnesium Sulfate 0.02 g Calcium Chloride l.0 g Monopotassium Phosphate Medium pH was adjusted l.0 g Dipotassium Phosphate to 7.0 (8.0 in later 35 l.0 g Ammonium Nitrate experiments) prior to 0.05 g Ferric Chloride autoclaving.
100 q Sodium Chloride 1.0 g Yeast Extract (Difco) 1.85 g Brain Heart Infusion ~BBL) 2.5 g Molasses (as solids) or Sucrose o1 -11-While IGM has been extensively used for culture maintenance, it contains a precipitate which makes it S unsuitable for injection into consolidated sandstone.
Molasses is low-cost and would be the preferred substrate for commercial field operation, but raw molasses contains insolubles, is variable in quality, and contains both sucrose and reducing sugars which complicates laboratory analyses for substrate consumption. For these reasons, most of the laboratory studies have been conducted usinq sucrose, or other simple sugars, as the growth substrates.
Characterization and Identification of MEOR Isolates Only two genera of bacillus-shaped, gram posi-tive, single endospore forming bacteria are recognized in Bergey's Manual for Determinative Bacteriology, the autho-ritative source for the classification of bacteria --there are: Clostridium and Bacillus. The two bacteria are differentiated by the ability of the Bacillus species ~ to survive and grow in the presence of molecular oxygen (they are facultative anaerobes); whereas Clostridia can-not (they are obligate anaerobes).
The two bacterial isolates showing the most promise for permeability modification were species of Bacillus. SLS-l INRRL No. B-18179) and Salton-l (NRRL
No. B-18178) show many similarities and a few differences.
Biochemical and morphological tests showed that the two isolates are indeed closely related and most closely resemble the species, Bacillus licheniformis, described in Bergey's Manual 1~09366 C CO
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136~9366 Bergey's Manual suggests that Bacillus licheniformis is a halotolerant (growth in 7~ NaCl) bac-05 terium which produces an extracellular glutamyl polypep-tide and a carbohydrate-based polymer of levan (repeating units of fructofuranose). The present isolates both pro-duce an extracellular polymer (which has been character-ized as polyglutamic acid for Salton-l), and both were isolated in medium containing 5-10% salt. There seems little doubt that these isolates are strains of Bacillus licheniformis though the cells of Salton-l are somewhat smaller than the dimensions given in Bergey's.
SPORULATION
lS Advantages of Spores over Vegetative Cells as Inoculum Vegetative cells of Salton-l have the following disadvantages as an inoculum for core or field experi-ments:
1. They tend to clump together in short chains or ~0 small masses, plugging injection line filters during cell injection and leading to inoculum losses when the cell suspension is pre-filtered. Adding commercially available surfactants such as magnesium lignosulfonate, Marasperse C-21 or N-22 (trademark of Marathon Co.), Reax 888 (trade-mark of West Virginia Pulp and Paper Co.), or Tween 80(trademark of Atlas Powder Co.) to either the cell suspen-sions prior to filtering or to the growth medium during cell growth does not effectively remedy the problem. When the surfactants were added to the growth medium, lower medium viscosities were recorded after cell growth.
2. Vegetative cells tend to rapidly lyse when they are stored. Polymer Medium-2 (PM-2) medium containing sucrose but no nitrogen proved to the best medium for maintaining viability of the cell suspension. (PM-2 medium contains [per liter of water]: 200 g NaCl; .5 g MgSO4; 1.33 g NH3NO4; 2.0 g NaNO4; 1 g yeast extract; and 2.6 9 citric acid, which is then adjusted to pH 8Ø) In PM-2 medium containing nitrogen but not sucrose, the viable cell count dropped from 108 to 104 per ml during the 72-hour storage. The availability of a carbon 130~366 substrate is apparen~ly very important in maintaining vegetative cell viability. Spores, on the other hand, are 05 stable and resistant to adverse environmental factors such as nutrient imbalances, desiccation, and high tempera-tures. Spores have been shown to survive for decades in soils, and our lab studies have shown no loss in germi-nation capacity when stored for over one month in brine at room temperature or at 4C.

3. Vegetative cells are considerably larger than the endospores that they produce, limiting their passage through narrow pore throats in rock formations. It also would seem logical that vegetative cells, coated with polymeric material, would tend to adsorb more readily to rock surfaces.
Inducing Sporulation in Salton-1 Species classification studies had shown that both Salton-l and SLS-l are spore-forming sacilli. Yet in PM-2, spores are formed in low numbers, generally less than 0.1% of the cells are present as spores, even in older cultures. (Spore percentages were determined by comparing plate counts on cell suspensions before and after heating the suspension to 80C for 20 minutes.) The sporulation literature indicates that ~acillus species vary widely in their propensity to sporu-late and that sporulation is influenced by dissolved oxygen, nutrition, and other factors. Examination of existing cultures showed that in IGM medium a greater proportion of cells produced spores than had been observed in PM-2 medium, which was interpreted as being due to the richer composition of the IGM medium. Experiments on sporulation included incubating PM-2 medium with and with-out supplementation with rich components aerobically and anaerobically, varying the salt concentration, and pre-paring and monitoring growth and sporulation in several media formulation taken from the literature.
In all of the above experiments, the only com-bination of medium formulation and incubation conditions that provided 100% sporulation of Salton-l cultures was aerobic incubation in chemically defined sporulation medium (CDSM~ of the composition similar to Hageman et al. (Hageman, J. H., Shankweiler, G. W., Wall, P. R., Franich, K., McCowan, G. W., Cauble, S. M., Grajeda, J., Quinones, C., 160, J. Bacteriol. 438 (1984), "Single chemically defined sporulation medium for Bacillus subtilis growth, sporulation and extracellular protease production". CDSM was prepared as follows:

10 x Macro Stock Solution 50 x Trace Element Solution 20.9 g MOPS buffer (40 mM) 10.17 g Magnesium chloride 1.36 g KH2P04 (40 mM) 1.94 g Calcium chloride 3.14 g Ammonium sulfate 0.25 g Manganese chloride (95 mM) 9.01 g glucose (200 mM) 0.0004 g Zinc chloride 1.13 g 1-Lactic acid 0.0203 g Ferric chloride (50 mM) 0.37 g 1-Glutamic acid 0.0169 g Thiamine HCl (10 mM) pH to 7.0 with KOH 0.5 ml of 1.0 N HCl Volume to 250 ml with Volume to 250 ml with distilled water distilled water Filter-sterilize Filter-sterilize CDSM Medium 50 ml Macro Stock Solutions 10 ml Trace Element Solution 0.050 g 1-Tryptophan 0.050 g 1-Isoleucine Volume to 500 ml with distilled water Filter-sterilize and dispense into sterile test tubes ~' Ol -17-Interestingly, growth of Salton-l is slow in this medium whether or not salt is added. But the final 05 cell density is higher than was observed with any other medium formulation, and after 3-4 weeks incubation, only small bright circular spores are observed under phase contrast microscopy; vegetative cell forms have essen-tially totally disappeared. Spore density in CDSM is around lO9 spores per ml.
Growth and spore production in aerobic CDSM
cultures is comparable whether or not salt is incorporated into the salt-free medium formulation. It is interesting to note, however, that spores transferred from salt-free CDSM medium to PM-2 medium produced significantly more polymer than those transferred from salt-containing CDSM, possibly indicating that a "cell shock" phenomenon is a factor in turning on polymer production.
Growth and sporulation are inhibited when CDSM
is incubated anaerobically or when 10% salt is added.
During sporulation the medium changes from a light yellow to a dark brown color. After spores are formed, the vege-tative cells lyse and slowly disintegrate.
Microscopic examination of freshly sporulated cells reveals that the optically refractile spores are surrounded with considerable cell debris and analysis shows that viscous cell proteins have been released into the medium. ~e have found that such fresh spore suspen-sions have poor injectibility and tend to plug at the face of cores when injected. Conversely, we have found that a short (e.g., 2 hrs.) treatment of fresh spores with the proteolytic enzyme lysozyme removes the adherent cell debris and solubilizes proteins suspended in the liquid, such~that the lysozyme-treated spores can be easily injected without face plugging. Further, it is known (e.g., ref. Hagemann et al) that sporulating cultures produce autogenous proteases, and we have observed that upon long-term aging (e.g., 1-2 mo.) of a fresh spore suspension, the adherent debris and suspended proteins are likewise solubilized as achieved by the brief lysozyme 130936Ç;

treatment. Assuming sufficient time is available, theaging procedure would be more economical, i.e., the spores 05 could be prepared several months prior to injection, since no added chemicals are required.
Germination of Salton-l Spores Some species of Bacillus produce spores which do not readily germinate even when environmental conditions and nutrients would favor the growth of vegetative cells.
These spores must be induced to break their dormant state by heat shocking or exposure to amino acids.
THE BIOPOLYMER
Polymer Recovery and Properties Salton-l produces a viscous, extra cellular, water-soluble polymer during the early stationary phase of its growth cycle. Many biopolymers, such as xanthan, can be precipitated from culture medium using alcohol, and it appears tllat the biopolymers produced by both bacteria (Salton-l and SLS-l) were no exception. Briefly, the method that was used to precipitate the biopolymer con-sisted of the following two steps: centrifugation of the culture medium containing the biopolymer at 7000 rpm for 20 minutes to remove the bacterial cells; and mixing the supernatant with an equal volume of 95% ethanol to pre-cipitate the polymer into slimy strands which can be quan-titatively recovered by filtration or low-speed centri-fugation (1500 rpm, 5 minutes).
Many microbes produce viscous polysaccharides and halophilic Bacilli are known to produce levans or polyfructoses. However, the identity of the polymer from both strands of bacteria was determined by spectroscopic tests to be primarily polyglutamic acid. Elemental ana-lysis of the polymer give the following results:

ElementWeight Percent Percent of Total Carbon 32.8 40.8 Hydrogen 5.1 6.1 Nitrogen 7.5 9.5 Phosphorus 0.7 Considering that the polymer was analyzed with contained moisture and impurities, and also that it had some phos-phorus content, the results agree with polyglutamic acid. The role of phosphorus is yet unclear; it may be merely ionically bound residual phosphate from nutrient medium or may play some biochemical or structural role.
Unlike polysaccharides, such as Xanthan Gum which are produced by some bacteria, polyglutamate is resistant to proteolytic enzymes and biological attack and is stable once formed [see Troy, F., 248, J. Biol.
Chem., 316 (1973) "Chemistry and biosynthesis of the poly(gamma-D-glutamyl) capsule in Bacillus licheniformis"].
Polymer Cross-linkinq Once the MEOR bacterial cultures have been injected downhole, induced to grow, and then induced to form a polymer, a way must be found to keep the high molecular weight, but water soluble, material from being washed from, or degraded within the areas in which they are injected. This usually means cross-linking the polymer with metal ions.
Cross-linking is dependent on pH, temperature, monomer, and metal concentration, as well as a number of other factors. Metal ions that are useful in cross-linking include: aluminium (+3), titanium (+3),titanium (+4), ferric iron, chromium (+3) chromium (+6), cobalt (+2), cupric copper (+2), La (+3) and (+4), Fe (+3) molybdate and tungstate. Organic cross-linking agents may also be used, such as Onomer-M, a polyamine produced by Gulf Oil Chemicals.
While it is encouraging that biopolymers react with a number of polyvalent metal ions to produce a more stable, cross-linked precipitate, the applicability of this knowledge to in situ application is questioned since the metal ion solutions are generally quite acid and they precipitate when neutralized. They may also 19 a exhibit a deleterious effect on the bacterial growth if they are freely present in too high a concentration.

1;3~)9366 Ol -2Q-Polyphosphate However, metal ions can be kept in solution at a 05 neutral pH when they are chelated with an inorganic poly-phosphate, such as sodium tripolyphosphate (TPP). When complexed with tripolyphosphate (TPP), the metals are not available to react with the polymers or the bacteria. So that when TPP is added to the nutrient medium the bacteria 0 may grow unencumbered and produce substantial quantities of biopolymer. At some point in time it will become necessary to release the metal ions to cross-link and gel the biopolymer (to plug the zones of high permeability).
This is where the TPP acts in a multifaceted role. It is added for three purposes: to chelate the metal ions for the purposes described above; to provide a metabolizable source of phosphate to fit the growth requirements of the bacteria; and to resist precipitation out of solution (due to the presence of other metal ions that we found in connate water, i.e., Ca+2 and Mg+2). Therefore, the TPP
is incorporated into the nutrient medium and may be injected downhole along with the bacteria and the metal ions that are used as biopolymer cross-linkers (it com-plexes these metal ions as well as connate water metal ions). As the bacteria grow they consume the TPP which releases the metal ions into the surrounding environment (as the TPP is depleted). As the metal ions are released they contact the biopolymer and cause it to gel and cross-link. An optimum concentration of TPP may be determined so that the chelation of the metal ions continues until such time when gellation of the polymer is desired and the adverse effects on the bacteria no longer matter.
One example of the preparation of a tripolyphos-phate may be as follows, but this is in no way the only preparation that can be made. If P205, phosphorus pent-oxide, is dissolved in H20, it reacts to form H3PO4, which is phosphoric or orthophosphoric acid. For example, 3H20 +
P205 --> 2H3PO4. If additional P205 is added, it can react to form condensed chain-type structures where tripolyphos-phate is the structure containing three phosphorus atoms:

~309366 O O O
--(O--P--O--P--O--P--O)--O O O
The tripolyphosphate anion forms salts such as sodium tripolyphosphate, Na5P3010 6H20. Trip yp phates (and other chain forms) are much better complexing agents for metal ions than an orthophosphate compound. They generally do not degrade very rapidly atalkaline pHs. Furthermore, the largest commercial application for polyphosphates is as a detergent builder where it serves to prevent ions that cause water hardness from precipitating a surfactant.
15For a more in-depth discussion on polyphosphate compounds, see J. Van Wazer, Vol. I, Phosphorus and Its Compounds, Interscience Publishers (1958). This reference discusses methods of preparation, crystallization, and identification of polyphosphates BENCH-SCALE CONTINUOUS CORE TESTS
The process was tested for profile modification as outlined below.
Automated Core Test Apparatus An automated bench-scale laboratory unit was operated continuously for performing core 2 and packed column testing to demonstrate the feasibility of this MEOR approach. A simplified flow schematic of the unit is shown in FIG. 1. Six core experiments can be run simultaneously and independently. Oil 4 and brine 6, bacteria and nutrients 8 were fed through separate feed pumping systems (oil was pumped through pump 10 and brine, bacteria, and nutrients through pump 12).
Sterile brine 6, bacterial cells (or spores) and nutrients 8 were pumped through 0.125" OD tubing using a Milton Roy or a peristaltic pump 12. To maintain anaerobic conditions, a small purge of nitrogen was bled into each vessel 6 and 8. Feed rates, depending on predetermined experimental 1;~09366 conditions normally ranged from 0.03-1.0 ml/min. This has a field correlation feed rate of 0.3-11 linear ft/day.
05 For temperature control, all cores 2 were placed in a constant temperature incubator 14. All experiments were conducted at 40C (104~). Differential pressure was recorded using transducers 16 with appropriate diaphragms.
In most experiments, pressure taps 18 were located at intervals along the length of the core 2. This was accom-plished by drilling a 0.125" OD hole into and through the resin and epoxy (on the surface of the core 2) and into the sandstone of the core 2 such that fluid transmitted through the core 2 flowed out the hole. The holes in the lS resin were tapped and threaded and fitted with gyrolock connectors. Nylon tubing was connected from these fittings to a pressure transducer. Pressure signals were processed and converted to a digital signal by a signal demodulator 20. Calibration of the transducers 16 were ~0 performed using a preset nitrogen calibration pressure setup. A computer 22 continuously monitored (and every 30 minutes printed and logged to hard copy or floppy disk) the cores' pressure and other readings. The printout gave a time-pressure log so as to follow the pressure differen-tial for the entire length of each core 2. This enabledan accurate compilation of pressure measurements for the entire duration of each core experiment independently.
Flow rates were measured both from time-pump feed rates and from effluent product collection rates; these rates were continuously taken. The effluent samples were collected continuously using a fraction collector 24.
CORE PREPARATION
Berea sandstone cores, obtained from Cleveland Quarries (Amherst, Ohio) were of 2" diameter and received as cylinders of specified lengths and permeabilities. A
specified core is coated with epoxy and cast in a resin mold. After cutting the core to a designated length and facing the ends, it was placed in a core holder to be attached to the continuous flow apparatus and then vacuum 130~366 saturated with brine for an accurate pore volume determi-nation. An oil-brine saturated core was prepared by 05 pumping several pore volumes of brine followed by adding several pore volumes of crude oil to a irreducible water saturation. Brine was then pumped through the core until no more oil was observed in the effluent. At this time, initial brine permeability was determined. For most experimental runs, the flow tubing, fittings, and valvings were disinfected and then completely flushed with sterile brine before each startup. Calculations for % porosity and % pore volume of oil saturation were also determined before starting each core experiment.
INJECTION OF CELLS AND NUTRIENT
Depending on the purpose of a designated experi-mental run, a core may or may not be oil saturated.
The sequence of additions for an experiment are as follows:
~0 1. srine in]ection to determine permeability.
2. Add 0.3-1.0 pore volumes of cell or spore suspension at a concentration of approximately 106 cells/ml.
3. Add 0.3-1.0 pore volumes of specifically formulated nutrient solution; this could be added with the cells (as has been demonstrated).

4. A cross-linker (Al+3 or Cr+3) could also be added either with a waterflush or in combination with cells and/or nutrient material. The concentration of cross-linker would be about 1000 ppm. Such cross-linker is preferably held in solubilized form by polyphosphate salt.

5. Incubation ("lock in") for 5-10 days.

6. Commence brine addition or add a second nutrient and/or cross-linker batch. At this time a check is also made for permeability reduction and effluent samples are analyzed for biopolymer concentration.

7. Incubation (repeated).

8. Steps 6 and 7 can be repeated.

)9366 9. Final brine addition to check for lasting permeability reduction.

Cell growth along with biopolymer production for the enhancement of lasting permeability reduction is the ultimate goal of our MEOR process. Monitoring the extent of this growth and production during an on-going core experiment can be accurately accomplished by recording gas pressure buildup as cells grow, calculating and evaluating pressure gradients and overall permeability calculations (while pumping) during sequential additions, and by per-forming analyses on effluent samples taken; i.e., cell plate count, biopolymer concentration, and residual sucrose (the most frequently used carbon source) concen-tration, etc. Several demonstration experiments were completed using this basic apparatus.
EXAMPLE 1 (Nutrient Utilization) ~0 FIG. 2 shows the pressure response of cumulative nutrient addition on growing cells in the core. Previ-ously added cells (to the core) were fed 4 separate batches of nutrient with approximately five days' incuba-tion between each addition. Separate curves are shown for the pressure response at: 1) the core's inlet, 2) 30~
length from the inlet, and, 3) 70% length from the inlet.
As shown, pressure response increases as a function of time as a result of step-wise nutrient addition to the core's previously added cells. The increase in static pressure during incubation is due to cell gas production (CO2) while the increase in dynamic (pumping) pressure with each injection is due to the accumulation of cell mass/biopolymer throughout the length of the core. It has been repeatedly demonstrated that when core effluent cell count and nutrient sucrose utilization increase, a desired reduction in core permeability results (as is shown here;
i.e., 367 millidarcy or md to 58 md).
EXAMPLE 2 (Long Distance Transport) To verify the presence of in situ produced bio-polymer at extended distances from the injection site, a 40 ft. packed Berea "slim tube" (crushed sandstone) wasconstructed with pressure transducers and sampling ports 05 at 10 ft. intervals. The initial brine permeability of the slim tube (76 cc pore volume) was 6.7 d (6700 md) and after feeding 2.3 pore volumes (abbreviated PV) of 6 x 107 cells/ml at a rate of 0.11 ml/min (80 ft/day) for 17 hours, the sample port at the end (40 ft. length) of the tube showed 104 cells/ml of cells identical to those fed and 113 ppm biopolymer that these cells produced. As time and subsequent additions proceeded, the overall per-meability was reduced to 0.4 d (400 md), and the biopoly-mer concentration at the end of the slim tube in the effluent was measured at 1222 ppm.
This experiment enabled measurement of polymer production-penetration and adsorption over a long dis-tance corresponding to a "thief zone" in the field. The results show that the cells and subsequently produced biopolymer completely penetrated to the end of the tube, demonstrating the effectiveness of the MEOR technique.
Total "plugging" of the slim tube is further evidenced by the following points:
1. Throughout the course of the experiment, the feed pump rate had to be reduced. The progressively slower rate was needed to stay below the pressure limita-tions on the equipment. The pressure buildup and slower feed rate demonstrates the effectiveness of biopolymer buildup in the total length of the slim tube.
2. Permeability reduction for the overall length of the slim tube is 94%.
3. Significant increases in the rate of change of pressure differentials occurred during the duration of the run.
In the "Slim Tube" experiment product samples were taken at specified times and locations along the packed core during the course of the experiment. The 40 ft. Berea-packed column had both transducers and sampling ports at the inlet and each subsequent 10 ft.
interval.

These results show that overall permeabilityreduction continues to increase due to the increase of in 05 situ produced biopolymer throughout the length of the 40 ft. slim tube. By continually monitoring pressure response and the concentration of biopolymer at the sample ports, a good representation of experimental progress (for permeability reduction) was continually available.
This experiment also demonstrated that the Salton bacteria cells will transport through high perme-ability Berea core at a reasonable concentration for pro-cess effectiveness.
CELL AND SPORE PENETRATION EXPERIMENTS
EXAMPLE 3 tCell Injection Phase) A cell penetration experiment using a moderately permeable Berea sandstone core (146 md, 9 cm in length) and a highly permeable core (1361 md, 9 cm long) was con-ducted to measure the flow properties of the cell suspen-sion. Three pore volumes of 4 x 105 cells/ml were addedto each core. High retention of the cells on the sand-stone with each core resulted; i.e., essentially 100% of the cells injected were retained on the respective cores.
A second cell migration experiment was run using a 9 cm long x 5 cm diameter Berea core to obtain more information on Salton cell penetration. Again, the major-ity of the cells were retained within the core. The following material balance was calculated:

Total Liquid recovery = 99.4%
Total vegetative cell count feed = 1.06 x 108 cells/ml Total recovered cell count = 3.06 x 105 cells/ml This showed that greater than 99% of the feed cells were retained on the core. From these two tests it became apparent that spores, not cells, should be the penetrating/
migrating species through the sandstone. The cells appear 13 [)9366 either too large and/or adhesive to the core material and cannot travel through the network of rock pores for any 05 appreciable distance.
EXAMPLE 4 (Spore Pretreatment) When cells of Bacillus bacteria are induceâ to sporulate, microscopic observation of fresh spores reveals small, optically refractile spores plus considerable adhering cell debris. When such "fresh" spores were diluted to approximately 106 spores/ml and injected into a Berea core of 1000 md permeability, poor injectability was obtained with almost immediate face plugging. Visual observation revealed a film of sticky proteinaceous matter on the face of the core. The concentrated spore suspen-sion was then treated by adding 1 mg/ml lysozyme at 40C
for 2 hours. The spores were then filtered through Whatman No. 1 paper. Microscopic observation revealed that the previous adherent material was no longer present.
Subsequent injection of these treated spores into a core gave good injectability with no buildup of any face-plugging layer.
It was later observed that aging of a fresh spore suspension for a period in excess of two months has a beneficial effect similar to that which can be obtained with lysozyme in a few hours.
EXAMPLE S (Spore Injection) Spore transport and spore adsorption/desorption characteristics were studied in a high and a low perme-ability core. A 107/ml concentration of pretreated sporeswas injected into each core for 23.33 hours. The low permeability core received 18.1 pore volumes (PV) of spores followed by the same amount of brine; the high permeability core received 8.5 PV spores followed by the same amount of brine.

~3Q9366 .
05 Low Permeability High Permeability Core Core 9.2 cm -Length- 16.3 cm 5.1 cm -Diameter- 5.0 cm 35.0 ml -Pore Volume- 74.4 ml PERMEABILITY
(feed rate - 5.2 ft/d (field)) 123 md -Brine- 800 md 105 md -50~ Spores Added- 800 md 82 md -100% Spores Added- 686 md The pressure drop data for the high permeability core indicate that the spores penetrated farther than in the low permeability core. After completing the material balance for the high permeability core, the following 2U conclusions have been made:
1. Breakthrough of injected spores in the core effluent occurred at 0.5 PV which is a measure of tne "inaccessible pore volume" for spore transport.
2. Over the course of the experiment, spore concentration in the effluent increased from 4 x 102 to 1.8 x 106 spores/ml.
3. 95% of the injected spores were retained by the high permeability core even after extensive brine flushing. A negligible concentration of spores was detected in the effluent during the injection and subse-quent brine flushing from the low permeability core.
Significant overall permeability reduction was observed in both cores but the pressure drop data suggest some spore accumulation at the front of each core.
EXAMPLE 6 (Spore Injection) In proposed field application, large volumes of fluids will pass through the well bore surface where any face plugging mechanism is a concern. A Salton spore sus-pension (conc. = 1.3 x 106 spores/ml) was injected into a small cross section, low permeability (141 md) Berea core Ol -29-at a rate of 1 PV/hr in two operational modes; i.e., con-tinuous recycle and straight-through single pass.

PERMEABILITIES (recycle) 1. Brine 141 md 2. After 138 pv 140 md IO Spores Added 3. After 337 pv 132 md Spores Added PERMEA~ILITIES (single pass) 1. After 91 pv 124 md Spores Added (428 pv cum.) 2.After 151 pv 115 md Spores Added (488 pv cum.) 3.After 184 pv 110 md ~O Spores Added (521 pv cum.) Core Parameters: L = 8.5 cm D = 1.4 cm PV = 6.5 ml The results of Table 2 show the permeability calculations for the single pass and recycle experiments.
Further it was observed that no feed spores were detected in the effluent sent back to recycle. It can thus be safely assumed that for the 365 hours of recycling 337 PV
of spores, essentially 100% of the spores remained on the core. Also, by carefully reviewing the pressure drop data along the length of the core, the predominant weight frac-tion of the feed was in the very front (face) of the core.
This almost certainly accounts for the 6% permeability reduction after 337 PV were recycled.
The same core was then used for straight-through, single pass operation and was continuously injected with 184 PV of a 1.35 x 106/ml spore feed.

~309366 Again, no spores were detected in the effluent. After 184 PV were added in the single-pass mode and 337 PV in 05 the recycle mode (521 total PV added) a 22~ permeability reduction was measured. After feeding over 500 PV of the spore suspension, no indication of any face plugging was evident; i.e., only a +0.3 psig pressure change at the face of the core.
EXAMPLE 7 (Spore Retention) Spores were injected in a single pass to a Berea core of relatively high starting brine permeability (approximately 2100 md). The essential core parameters are listed below:

Core length = 10.85 cm Diameter = 5.04 cm Pore volume = 49.7 ml Only two pressure transducers, inlet and outlet, were ;!0 attached to the core; 145 PV of spores were injected (average conc. = 1.9 x 10/6ml) into the core over 339 hours at an average feed rate of 0.36 ml/min. The results show that spore breakthrough (2.5 x 102 spores/ml) occurred after 20.7 ml (0.42 PV) and the concentration of spores continued to increase during pumping to an effluent concentration of 5.6 x 105 spores/ml at the conclusion of the experiment. An accurate material balance was com-pleted for this run as follows:

Total spores fed (145 PV) = 1.43 x 101 Total spores recovered (effluent) = 5.73 x 108 Spores recovered = 4%
Spores remaining on core = 96%

This result indicates that the majority of the spores are 35 retained by the core after feeding 145 PV. Permeability has continued to decrease throughout the duration of the run from approximately 2100 md at the beginning to 60 md at the end of the experiment. No plugging was observed at the core face; injection rate decreased only 13%; and the 40 injection pressure increased less than 1 psig.

1~09366 EXMAMpLE 8 (Growth of Bacterial Isolates in Polyphsophate) A solution was prepared and named PM-2 which 05 comprised: 200 g sodium chloride; 0.5 9 magnesium sulfate; 1.33 g ammonium nitrate; 2.0 g sodium nitrate;
1 g yeast extract; 2.6 g citric acid and was adjusted to pH 8Ø Phosphate (as KH2PO4 or sodium tripolyphosphate) was added to PM-2 to give 5 or 10 g/l as indicated in Table I. The total volume of each tube was 35 ml. A
0.1 ml inoculum of either SLS (NRRL No. B-18179) or Salton-l (NRRL No. B-18178) was added to each tube. The results are in Table 6.

Growth SLS Salton Medium having 3.5 ml PO4 17 hr 2 day visc. 17 hr 2 day visc.
~0 PM-2 Control (with +++ 4.2 +++ 3.02 10 g/l KH2PO4) PM-2 with 5 g/l sodium tripolyphos-phate and 5 g/l KH2 25 pO4 +++ 4.06 +++ 4.24 PM-2 with 5 g/l sodium tripolyphos-phate +++ 1.9 +++ 2.8 PM-2 with 10 g/l sodium tripolyphos-phate and 5 g/l KH2 PO4 +++ 5.66 +++
PM-2 with 10 g/l sodium tripolyphos-phate +++ 4.02 +++ 3.40 EXAMPLE 9 (Chelation of Ions in Connate ~Jater) A solution of sodium tripolyphosphate was prepared in distilled water and the addition of a small amount of connate field water (with contained calcium and magnesium ions) caused a 13~g366 dense white precipitate to form. The addition of additonal sodium tripolyphosphate caused the precipitate to dissolve.
05 To determine the amount of polyphosphate required to prevent precipitation of this field water, 10 ml of PM-2 nutrient medium was combined with varying amounts of polyphos-phate and field water. For the results see Table 7.

10 ml PM-2 (without Amount of PO4) plus: Soluble Field Water Result _ 0.05 g polyphsophate Yes 0.5 ml ppt redissolved*
1.0 ml ppt remained 0.15 g polyphosphate yes 1.0 ml ppt redissolved*
1.5 ml ppt remained 0.20 g polyphosphate partially 1.0 ml ppt redissolved*
1.5 ml ppt remained ~0 *A precipitate initially formed, but was redissolved upon stirring.

EXAMPLE 10 (Growth of Bacterial Isolates in Crushed Berea) Salton and SLS bacteria were added to PM-2 medium containing 5% sucrose. The phosphate source was either 10 g/l KH2PO4 or sodium tripolyphosphate (as indicated below). Each culture tube was grown in the presence of 5 g crushed Berea sandstone.

(0.1 ml) 3-day 3-day Additive Phosphate Inoculum Growth Viscosity Crushed aerea KH2PO4 SLS - 0.96 35 Crushed Berea KH2PO4 Salton - 1.2 Crushed Berea polyphosphate SLS ++++ 2.62 Crushed Berea polyphosphate Salton ++++ 4.0 13 [)9366 01 _33_ EXAMPLE 11 (Metal Ions as Cross-linking Agents for the Blopolymer) 05 Purified, dialyzed solutions of biopolymer from SLS and Salton bacteria were prepared and 10 ml was dis-pensed in each tube. The following metal ions were tested for cross-linking ability: Titanium in the +3 (as TiC13 or TioSo4) and +4 (as TiC14) valence state, Aluminum in the +3 (Aluminum citrate) valence state, Chromium in the +3 (Cr2 (SO4)3) and +6 (CrO3) valence state, Fe in the +3 (FeC13) valence state, Copper in the +1 (CuSO4) valence state, and Cobalt in the +2 (CoC12) valence state. For the results see Table 9.

Cross-linking Metal Ion ~ SLS Polymer Salton Polymer 300 ppm Ti+4 YesYes (TiC14 used) 300 ppm Al+3 as citrate, adjusted to pH 6.2 Yes/NoYes/No 116 ppm Cr+3as Cr2(SO4)3 Yes/No Yes/No 0.1 ml of 20% TiC13 Yes 10 ml 20~ TiC13 YesYes 1 ml 0.2% TiC13 YesYes 50 ml saturated FeC13 Yes Yes 0.3 ml Ti+3 as TioSo4 Yes Yes 0.1 ml of 1% CrO3 No Yes 0.0 ml lN Thiourea Al(SO4)3 YesYes 0.5 ml of 1% CoSO4 . 5H2O No data Yes 247 ppm Co+2 (500 ml of 2% CoC12) No data Yes The use of TPP in a MEOR nutrient medium means that metal ions or other compounds (that are used to gel 40 and stabilize a biopolymer) may be directly added to the ~31[1936~

Ol -34~
microbial growth medium itself. It also means that the bacteria that are injected into a petroleum reservoir can 05 be protected from the deleterious effects of the ambient metal ions while being able to use a form of phosphate that is not precipitated out of solution by these same ambient conditions.
It has been experimentally shown that under conditions of our invention, cells and spores can easily penetrate serea core material. These conditions are:
1) Use of selected bacteria such as our SLS and SALTON-l strains which form small, compact spores and motile cells.
2) Pretreatment of spores with a proteolytic enzyme such as lysozyme (or with autogenous proteases generated by long-term aging of spores) so as to remove adhering cell debris and sticky proteins.
3) Use of polyphosphate ion in the nutrient solution which chelates and prevents precipitation with ions present in the connate brine such as calcium and magne-slum.
In the Examples, cells and viscous biopolymer were evident the total length of the core. Since the experiment used a high permeability core, it is safe to say that field "thief zones" can be altered successfully by this microbial profile modification process. In no experiments where our preferred procedures have been used has there been any significant face plugging. Cells and spores have easily gone into the respective cores of low or high permeability. If continuous flow measurements are taken in low permeability cores using cells or spores, little or no penetration is observed by pressure drop data, but no face plugging is evident after many pore volumes of nutrient or recycle solution has been added.
In contrast to this, continuous injection experiments of spores into high permeability cores, develop a gradient of spore concentration (by observing pressure drop data) with some fraction (e.g., 5%) of feed spores collected at the effluent after a suitable period.

01 _35_ Our proposed MEOR process will give the germinated cells time to grow, multiply, and produce bio-05 polymer. This "incubation" time is, if all componentshave been properly selected, less than a week in duration.
We have observed substantial incubation gas production over the total length of core, cell growth followed by biopolymer production and lasting permeability reduction to continual brine flushing.
The magnitude of profile modification with our process can easily be as low as 65% permeability reduction and as high as 95%. The level of reduction depends on several factors; i.e., beginning permeability, amount of bacteria added, incubation time, and obviously, proper nutrient. The ease of reducing permeability seems to increase when using cores that are in excess of 600 md, which is desirable since the "thief zones" we wish to plug are high permeability. Also, a certain level ~concentra-tion) of cells may be necessary before substantial amounts of biopolymer are formed and permeability is reduced.
The first amount of injected cells may act as a "conditioner" for the sandstone, enabling further spore/
cell addition to perform their required tasks, i.e., germination, reproduction, and biopolymer production.
Incubation times of 5-10 days allows processing steps to be completed. Nutrient composition and the quantity added are "critical" process requirements. All nutrient for-mulations must be optimized to maximize biopolymer produc-tion. Also, when more than one addition of nutrient ismade, more than one cell/spore addition may be required to achieve maximum permeability reduction. Alternatively, profile modification can be accomplished by injecting the nutrient solution into an injection well and the spores, or cells, or mixtures thereof, into an adjacent production well, or vice versa. If all steps have been achieved to ensure significant profile modification, permeability reduction will be more resistant to erosion due to con-tinual water flooding and elapsed time.

Since many modifications and variations of the present invention are possible within the spirit of this 05 disclosure, it is intended that the embodiments that are disclosed are only illustrative and not restrictive.
Reference is made to the following claims rather than the specific description to indicate the scope of the inven-tion.

~(~

Claims (44)

1. A process for isolating bacteria selected from the group consisting of biologically pure strains of Bacillus licheniformis, NRRL No. B-18178, biologically pure strains of Bacillus licheniformis, NRRL No. B-18179, and spores of these two strains, wherein said bacteria is a halotolerant, thermotolerant, facultative anaerobe, the process comprises:
(a) sampling saline, anaerobic environments;
(b) growing bacteria from the samples of step (a) at or above a temperature of 40°C in enrichment media which contains sodium chloride;
(c) growing identical samples of the bacteria from step (b) anaerobically and aerobically; and (d) selecting bacteria from step (c) which grow both anaerobically and aerobically.

2. A process for isolating the bacteria as recited in Claim 1, in which the bacteria also sporulate and produces an exopolymer, the method further comprises:
(a) examining colonies of the bacteria for exopoly-mer production;
(b) microscopically examining bacteria for spore production; and (c) testing bacteria for motility.

3. A process as described in Claim 2 wherein the concentration of salt in the enrichment media is between 5% and 15%.

4. A process as described in Claim 1 wherein the enrichment media are selected from the group consisting of nutrient agar, nutrient agar glucose, veal agar, and BHI agar.

5. A process for producing a spore of bacteria selected from the group consisting of biologically pure strains of Bacillus licheniformis NRRL No. B-18178, and biologically pure strains of Bacillus licheniformis, NRRL No. B-18179, comprising:
(a) contacting the bacteria with CDSM medium comprising 50 ml Macro Stock Solutions, 10 ml Trace Element Solution, 0.050 g 1-Tryptophan, 0.050 g 1-Isoleucine, volume to 500 ml with distilled water, (b) filter-sterilizing said contacted bacteria, and (c) dispensing said bacteria into sterile test tubes.

6. An enhanced oil recovery process comprising selectively plugging relatively highly permeable zones within a petroleum reservoir using a biologically pure strain of Bacillus licheniformis, NRRL No. B-18179 or its spores or mixtures thereof.

7. An enhanced oil recovery process comprising selectively plugging relatively highly permeable zones within a petroleum reservoir using a biologically pure strain of Bacillus licheniformis, NRRL No. B-18178 or its spores or mixtures thereof.

8. An enhanced oil recovery process comprising selectively plugging relatively highly permeable zones within a petroleum reservoir using a biologically pure strain of Bacillus licheniformis, NRRL Nos. B-18178 and 18179 or their spores or mixtures thereof.

9. The process of Claim 6, 7 or 8 wherein the spores have been pretreated with proteolytic enzymes.

10. The process according to Claim 6, 7 or 8, wherein the spores have been aged in suspension for a period of at least one month.

11. The process according to Claim 6, 7 or 8, wherein the relatively highly permeable zones are selectively plugged by the growth of the bacteria and by the production of a bacterial exopolymer.

12. The process according to Claim 6, 7 or 8, further comprising injecting a nutrient solution into the relatively highly permeable zones.

13. A process according to Claim 6, 7 or 8, further comprising injecting a nutrient solution into the relatively highly permeable zones comprising polyphosphate and a polyvalent crosslinker.

14. A process as recited in Claim 6, 7 or 8, further comprising injecting a nutrient solution into the relatively highly permeable zones comprising polyphosphate and a polyvalent crosslinker and wherein the polyvalent crosslinker is an ion selected from the group consisting of aluminum, titanium, or chromium.

15. A process as recited in Claim 6, 7 or 8, further comprising injecting a nutrient solution into the relatively highly permeable zones comprising polyphosphate and a polyvalent crosslinker, wherein the polyvalent crosslinker is an ion selected from the group consisting of aluminum, titanium, or chromium and further comprising flowing water into the relatively highly permeable zones before the introduction of the bacterial spores or cells.

16. A process as recited in Claim 6, 7 or 8, further comprising injecting a nutrient solution into the relatively highly permeable zones comprising polyphosphate and a polyvalent crosslinker, wherein the polyvalent crosslinker is an ion selected from the group consisting of aluminum, titanium, or chromium, and further comprising flowing water into the relatively highly permeable zones after the introduction of the nutrient medium.

17. A process as recited in Claim 6, 7, or 8, further comprising injecting a nutrient solution into the relatively highly permeable zones comprising polyphosphate and a polyvalent crosslinker, wherein the polyvalent crosslinker is an ion selected from the group consisting of aluminum, titanium, or chromium, and further comprising flowing a flooding agent into the petroleum reservoir after the relatively highly permeable zones have been plugged.

18. A process as recited in Claim 6, 7 or 8, further comprising injecting a nutrient solution into the relatively highly permeable zones comprising polyphosphate and a polyvalent crosslinker, wherein the polyvalent crosslinker is an ion selected from the group consisting of aluminum, titanium, or chromium, and wherein the nutrient solution is flowed into the relatively highly permeable zones prior to the introduction of the bacterial cells or spores.

19. A process as recited in Claim 6, 7 or 8, further comprising injecting a nutrient solution into the relatively highly permeable zones comprising polyphosphate and a polyvalent crosslinker, wherein the polyvalent crosslinker is an ion selected from the group consisting of aluminum, titanium, or chromium, and wherein the nutrient solution is flowed into the relatively highly permeable zones simultaneously with the introduction of the bacterial cells or spores.

20. A process as recited in Claim 6, 7 or 8, further comprising injecting a nutrient solution into the relatively highly permeable zones comprising polyphosphate and a polyvalent crosslinker, wherein the polyvalent crosslinker is an ion selected from the group consisting of aluminum, titanium, or chromium, and wherein the nutrient solution is flowed into the relatively highly permeable zones after the introduction of the bacterial cells or spores.

21. A process as recited in Claim 6, 7, or 8, further comprising injecting a nutrient solution into the relatively highly permeable zones comprising polyphosphate and a polyvalent crosslinker, wherein the polyvalent crosslinker is an ion selected from the group consisting of aluminum, titanium, or chromium, wherein the nutrient solution is flowed into the relatively highly permeable zones after the introduction of the bacterial cells or spores, and further com-prising flowing the nutrient solution into a production well and the bacterial spores or cells or mixtures thereof are injected into an adjacent injection well.

22. A process as recited in Claim 6, 7 or 8, further comprising injecting a nutrient solution into the relatively highly permeable zones comprising polyphosphate and a polyvalent crosslinker, wherein the polyvalent crosslinker is an ion selected from the group consisting of aluminum, titanium, or chromium, wherein the nutrient solution is flowed into the relatively highly permeable zones after the introduction of the bacterial cells or spores, and further com-prising flowing the nutrient solution into a production well and the bacterial spores or cells or mixtures thereof are injected into an adjacent injection well, and wherein the nutrient solution is flowed into an injection well and the bacterial spores or cells or mixtures thereof are injected into an adjacent production well.

23. A process for modifying the profile of a petroleum reservoir to enhance oil recovery from relatively high permeability zones, comprising:
preparing a biologically pure form of Bacillus licheniformis, NRRL No. 8-18178 or 8-18179;
forming spores of B-18178 or B-18179;
separating the spores from cell debris and proteins; flowing the spores into a relatively high permeability zone in a petroleum reservoir; and contacting the spores with a nutrient solution to facilitate growth and exopolymer production.

24. A process for enhancing the oil recovery in an oil producing reservoir, where there is saline connate water, by improving the sweep efficiency of a waterflood, comprising the steps of:

isolating a bacterium that has the following characteristics:
it is motile;
it is a faculatitive anaerobe;
it is at least halotolerant;
it is at least thermotolerant;
it produces an exopolymer; and it sporulates;
preparing a suspension of said bacterial spores or cells or mixtures thereof;
removing cell debris and proteins from said suspen-sion;
injecting said suspension into an oil producing reservoir where said spores may become lodged in zones of high permeability; and flowing a polyphosphate-containing nutrient solution into said zones where said spores are lodged to cause said spores to grow into vegetative cells which may move further into said zone and may produce an exopolymer which, in combination with the vegetative cell itself, will plug said zones.

25. The method of Claim 24 further comprising:
flowing water downhole into an oil producing reser-voir prior to injecting said bacterial spores.

26. The method of Claim 24 further comprising:
flowing water into said zones of high permeability after said nutrient medium.

27. The method of Claim 24 further comprising:
flowing a flooding agent into said zones of high permeability after said spores have germinated and plugged said zones so that said flooding agent may push out any oil that is in said reservoir.

28. The method of Claim 24 wherein said nutrient solution is injected prior to the injection of said bac-terial spores or cells or mixtures thereof.

29. The method of Claim 24 wherein said nutrient solution is injected simultaneously with said bacterial spores or cells or mixtures thereof.

30. The method of Claim 24 wherein said nutrient solution is injected after said bacterial spores or cells or mixtures thereof.

31. The method of Claim 24 where said nutrient solu-tion is injected into a production well and said bacterial spores or cells or mixtures thereof are injected into an adjacent injection well.

32. The method of Claim 24 where said nutrient solu-tion is injected into an injection well and said bacterial spores or cells or mixtures thereof are injected into an adjacent production well.

33. A nutrient medium that is capable of flowing downhole into a petroleum reservoir and is capable of providing a metabolizable source of phosphate for microorganisms without precipitating on contact with connate water, said nutrient medium comprising a tripolyphosphate.

34. The nutrient medium of Claim 33 wherein the tripolyphosphate is sodium tripolyphosphate, potassium tripolyphosphate, or ammonium tripolyphosphate.

35. The nutrient medium of Claim 34 further comprising metabolizable phosphate in forms that are not polyphosphates.

36. A nutrient medium to provide essential nutrients to induce bacterial spores to grow, replicate, and produce an exopolymer, comprising:
a metabolizable source of tripolyphosphate in a form so that the phosphate will not precipitate out of solution when the nutrient medium is flowed into a highly saline environment that has ions selected from the group consisting of alkaline earth, rare earth, transition and heavy metal ions, said phosphate compound being able to chelate polymer cross-linking agents that are deleterious to the growth of the bacteria; and sources for remaining metabolic requirements.

37. The nutrient medium of Claim 36 wherein one liter of nutrient medium comprises an approximate amount of the following compounds:
200 g sodium chloride;
0.5 g magnesium sulfate;
1.33 g ammonium nitrate;
2.0 g sodium nitrate;
1-5 g yeast extract;
2.6 g citric acid; and 5-20 g/liter Sodium tripolyphosphate;
whereby the pH of said solution is adjusted to approximately 8Ø

38. The nutrient medium of Claim 37 wherein inorganic sources of phosphate are also added that are non-polyphosphates.

39. The nutrient medium of Claim 37 wherein the concentration of sodium tripolyphosphate is 10 to 20 g/liter.

40. The nutrient medium of Claim 36 wherein a polymer cross-linking agent is added and wherein said cross-linking agent is selected from the group consisting of: (a) polyamide, and (b) ions of aluminum, chromium, titantium, lanthanum, cesium, iron, cobalt and copper.

41. The nutrient medium of Claim 37 wherein a polymer cross-linking agent is added and wherein said cross-linking agent is selected from the group consisting of: (a) polyamide, and (b) ions of aluminum, chromium, titanium, lanthanum, cesium, iron, cobalt and copper.

42. The nutrient medium of Claim 33 wherein the tripolyphosphate concentration is from 5 to 190 grams per liter.

43. The nutrient medium of Claim 34 wherein the tripolyphosphate concentration is from 5 to 190 grams per liter.

44. The nutrient medium of Claim 36 wherein the tripolyphosphate concentration is 5 to 190 grams per liter.