Vol. 51, No. 6APPLIED AND ENVIRONMENTAL MICROBIOLOGY, June 1986, p. 1224-12290099-2240/86/061224-06$02.00/0Copyright C 1986, American Society for Microbiology

Isolation of Halotolerant, Thermotolerant, FacultativePolymer-Producing Bacteria and Characterization of the Exopolymer

S. M. PFIFFNER,t MICHAEL J. McINERNEY,l* GARY E. JENNEMAN,' AND ROY M. KNAPP2Department of Botany and Microbiologyl and School of Petroleum and Geological Engineering,2 University of Oklahoma,

Norman, Oklahoma 73019

Received 13 January 1986/Accepted 4 March 1986

Over 200 bacterial strains were selected for anaerobic growth at 50°C and extracellular polysaccharideproduction in a sucrose-mineral salts medium with NaNO3 and up to 10% NaCI. The predominant cell type wasan encapsulated gram-positive, motile, facultative sporeforming rod similar to BaciUlus species. Strain SP018grew and produced the polysaccharide on a variety of substrates at salinities up to 12% NaCl. Good polymerproduction only occurred anaerobically and was optimal between 4 and 10% NaCI. The ethanol-precipitatedSP018 polymer was a charged heteropolysaccharide that contained glucose, mannose, arabinose, ribose, andlow levels of allose and glucosamine. The SP018 polymer showed pseudoplastic behavior, was resistant toshearing, and had a higher viscosity at dilute concentrations and at elevated temperatures than xanthan gum.High-ionic-strength solutions reversibly decreased the viscosity of SP018 polymer solutions. The bacterium andthe associated polymer have many properties that make them potentially useful for in situ microbially enhancedoil recovery processes.

The success of a waterflood or an enhanced oil recoveryprocess ultimately depends on the sweep efficiency of thatprocess, that is, the fraction of the reservoir volume con-tacted by the recovery fluid. One of the most importantfactors affecting sweep efficiency is permeability variation(8). In an oil reservoir containing regions with differingpermeabilities, the injected fluid will preferentially movethrough the more permeable regions, and the oil entrapped inthe less permeable regions will be bypassed and unre-covered. Our work has focused on the in situ use ofmicroorganisms as selective plugging agents. Microorga-nisms, either indigenous to the reservoir or injected fromabove ground, are fed a carbohydrate-based medium such ascattle feed molasses to stimulate in situ microbial growth andextracellular polymer production to reduce the permeabilityof high permeability regions, thereby improving sweep effi-ciency. Laboratory studies using Berea sandstone coreshave shown that the in situ growth of microorganisms isselective for high permeability regions and does result inimproved sweep efficiency (17, 18).The in situ application of the microbial selective plugging

process requires that microorganisms grow and produceextracellular polymers under the environmental conditionsthat exist in the reservoir. Many oil reservoirs are anaerobicand have high salinities and temperatures (5). Little is knownabout bacteria that can grow and produce polymers underthese conditions. In this paper, we report the isolation andcharacterization of bacteria that grow rapidly and produceextracellular polymer in anaerobic medium at 50°C and up to10% (wt/vol) NaCl. These bacteria would be able to growunder conditions found in many oil reservoirs in Oklahoma(5, 23). The biopolymer produced by one of these bacteriawas isolated, and its rheological properties were compared

* Corresponding author.t Present address: Department of Biological Science, Florida

State University, Tallahassee, FL 32306.

with commercially available polymers used in enhanced oilrecovery.(A brief report of this work appeared previously [S. M.

Pfiffner, M. J. McInerney, R. M. Knapp, and D. E. Menzie,Abstr. Annu. Meet. Am. Soc. Microbiol. 1985, 150, p. 154].)

MATERIALS AND METHODS

Media and conditions of cultivation. Medium E (17) wassupplemented with 0.1% (wt/vol) NaNO3 and 2% (wt/vol)agar (Difco Laboratories, Detroit, Mich.) for the isolation ofpolymer-producing bacteria. Medium E is a sucrose-mineralsalts medium with 5% (wt/vol) NaCl. After initial isolation,the strains were grown in liquid cultures containing mediumE with 0.1% (wt/vol) NaNO3 and 0.05% yeast extract (Difco).All media were prepared anaerobically by the Hungatetechnique with an anaerobic chamber (1, 4). Liquid cultureswere grown in rubber-stoppered tubes or flasks, and agarplates were incubated anaerobically in Torbal jars (TorsionBalance Co., Clifton, N.J.). Cultures were routinely grown at50°C with shaking (100 rpm).

Isolation and characterization of strains. Environmentalsamples were obtained locally from aquifer material under-lying the Norman Landfill, coproduced water from an oilwell, brine injection water, anaerobic sewage sludge,eutrophic pond sediments, and soils in the vicinity of oilwells. Isolates were also obtained from effluents of Bereasandstone cores (Cleveland Quarries, Amherst, Ohio) inwhich medium E with 0.1% (wt/vol) NaNO3 was injected(17). The samples were serially diluted in medium E withoutsucrose and spread onto agar plates. Liquid enrichmentswere also obtained by inoculating serum bottles containingmedium E and NO3- with the environmental sample. Largemucoid colonies and viscous liquid enrichments wererestreaked onto plates of medium E with NO3 until a pureculture was obtained. The isolated strains were screened forrapid growth, viscosity increase, and extracellular polysac-charide production in medium E with NO3 and yeast

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extract. Strain SP018 consistently had good growth andpolymer production and was chosen for further study.

Characterization of SP018. Strain SP018 was tested forgrowth with different substrates in tubes with 10 ml ofmedium E without sucrose and with 0.1% NaNO3-0.05%yeast extract and substrate added at 2% for monosac-charides and 1% for the other substrates. Each tube wasinoculated with 0.1 ml of a late-log-phase culture of SP018and incubated at 50°C for 24 h. Tests for growth at differenttemperatures, pH values, or NaCl concentrations were donein a similar manner with sucrose as the energy source.Concentrated HCl or 10 N NaOH were used to adjust the pHof the medium. In some experiments the specific growth ratewas calculated from the increase in the absorbance of theculture with time. Samples of the culture broth were frozenat -20°C until viscosity was determined. Routine biochem-ical tests were performed at 30, 40, and 50°C according topublished procedures (26).Growth and polymer production of aerobic and anaerobic

cultures of SP018 were compared by inoculating 200 ml ofmedium E with 0.1% NaNO3-0.05% yeast extract-2% glu-cose instead of sucrose with 20 ml of an anaerobically grownculture. Samples (10 ml) were taken every 2 to 3 h. A sterilesyringe flushed with 02-free gas was used to sample theanaerobic culture.

Purification and characterization of the SP018 polymer. A7-liter batch culture of SP018 was grown anaerobically in aMicroferm fermentor (New Brunswick Scientific Co., Inc.,Edison, N.J.) until late exponential phase. Cells were re-moved by tangential flow filtration, using a 0.22-,um-porecassette filter (Millipore Corp., Bedford, Mass.). The filtratewas passed through a 100-kilodalton cassette filter (Milli-pore) to concentrate the polymer. The polymer solution wasdialyzed with 25 mM phosphate buffer (pH 7.0) with thefiltration apparatus and was brought to a final volume ofapproximately 200 ml in this buffer. Further purification ofthe polymer was achieved by ethanol precipitation anddrying in a vacuum desiccator (3).The purified polymer was subjected to slab gel electropho-

resis with gels with 10% (wt/vol) total acrylamide and 2%(wt/vol) N,N'methylenebisacrylamide and a constant cur-rent of 90 mA as described previously (9). The purifiedpolymer (10 to 50 ,ug [dry wt]) was applied to each lane. Gelswere stained with 0.4% (wt/vol) amido black 10B (Hartman-Leddon Co., Philadelphia, Pa.) in 7% (vol/vol) glacial aceticacid or with periodic acid-Schiff reagents as described byZacharius et al. (30). The gels were scanned at 500 or 550 nmwith a DU-8B spectrophotometer (Beckman Instruments,Inc., Palo Alto, Calif.).The amount of carbohydrate in the purified polymer was

determined with the anthrone method (13) with glucose asthe standard. Protein was determined by the method ofLowry et al. (22) with bovine serum albumin (Sigma Chem-ical Co., St. Louis, Mo.) as the standard. The UV absorptionspectrum of a 1.0% (dry wt/vol) solution of the purifiedpolymer was recorded with a Beckman DU-8B spectropho-tometer. Diffuse reflectance on the purified polymer wasperformed with Fourier transform infrared spectroscopy asdescribed previously (24). The purified polymer dissolved indistilled water was repeatedly applied to a metal disk andthen dried until the polymer film on the disk was thickenough to produce an infrared spectrum. Elemental analysisof the purified polymer for C, N, and H was done by MicroAnalytical Laboratory, Stanford University, Palo Alto,Calif.The carbohydrate composition of the purified SP018 poly-

mer was determined with glass capillary gas-liquid chroma-tography (10) by the derivatization and hydrolysis proce-dures of Fazio et al. (10). The amount of hexosamines in thepurified polymer was determined colorimetrically (6, 12)after acid hydrolysis of the polymer and separation of thehexosamines from neutral sugars by ion-exchange chroma-tography (13).

Other methods. Viscosity was measured at 50°C unlessindicated with a Wells-Brookfield microviscometer (Brook-field Engineering Laboratories, Stoughton, Mass.) at shearrates from 3.75 to 750/s. The viscometer was calibrated withstandards obtained from Brookfield Engineering Laborato-ries. The sample was allowed to equilibrate for 2 min beforethe viscosity was determined. The presence of cells orfreezing at -20°C did not affect the viscosity of the sample.Growth was measured spectrophotometrically at 660 nm.

Samples of cultures were centrifuged at 10,000 rpm for 10min at 4°C to remove cells. Glucose concentration wasdetermined colorimetrically with the glucose oxidasemethod (Technical Bulletin No. 635, Sigma Chemical Co.,St. Louis, Mo.). The amount of extracellular polymer pro-duced by the different isolates was determined by thephenol-sulfuric acid method (13) with glucose as the stan-dard. Cell-free samples were dialyzed for 24 h against 2 litersof distilled water before the amount of polymer was deter-mined.Commercial polysaccharides. Xanthan gum (grade II) and

guar gum were obtained from Sigma. Xanflood, a xanthangum product, was kindly provided by Halliburton Services,Duncan, Okla.

RESULTS

Isolation of strains. Over 200 strains of bacteria wereobtained that grew anaerobically in medium E with N02- at50°C. Aquifer material seemed to be the best source for theseisolates since agar plates inoculated with diluted aquifermaterial always had many large mucoid colonies. The pre-dominant morphological type was a gram-positive, faculta-tive, sporeforming, motile rod. Several strains grew rapidlywith specific growth rates of 0.9 to 1.6/h and reachedmaximum optical densities of 0.5 to 0.6 in 24 h. Theviscosities of these cultures ranged from 30 to 6 cP at 50°Cwith a shear rate of 19 or 150/s. The cultures exhibitedpseudoplastic behavior (decrease in viscosity with increas-ing shear rate). The amount of extracellular polysaccharideranged from 50 to 160 ,ug/ml. The viscosities and extracellu-lar polysaccharide concentrations were highest between 12to 24 h of growth and declined with longer incubationperiods. One of these strains, SP018, originally isolated froma Berea sandstone core, was chosen for further study.

Characterization of SP018. SP018 is a gram-positive, mo-

tile, rod-shaped bacterium. Spores were observed afterinitial isolation, but after repeated subculturing SP018 didnot produce spores under the cultural conditions. Colonieswere round and mucoid with undulate edges; the coloniesturned opaque and flattened out with longer incubationtimes. SP018 used a variety of sugars for growth, including(ranked by order of preference) fructose, glucose, mannose,cellobiose, maltose, sucrose, arabinose, mannitol, starch,galactose, and xylose, but not lactose or melibiose. Thespecific growth rate of SP018 with sucrose was 0.99/h. SP018used citrate, hydrolyzed urea and esculin, reduced nitrate,produced acetylmethylcarbinol, decarboxylated ornithine,and grew in the presence of bile salts. SP018 did nothydrolyze casein or produce indole or hydrogen sulfide.

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These properties are consistent with SP018 being a memberof the genus Bacillus (11).SP018 grew in medium with 0 to 12% (wt/vol) NaCl at

temperatures of 25 to 50°C and at pH values of 5.7 to 7.0, butnot 4.8. Higher pH values were not tested. The specificgrowth rates of SP018 in medium E with NO3 and yeastextract when incubated at 40, 45, and 50°C were 0.9, 1.3, and1.7/h, respectively.Polymer production. Aerobic cultures grew much more

rapidly on medium E than anaerobic cultures, but theviscosity of the medium did not increase (Fig. 1). However,mucoid colonies indicative of polymer production wereobserved on plates of nutrient agar incubated aerobically at40°C. The viscosity of anaerobically grown cultures in-creased during late exponential phase. In stationary phase,the viscosity of the anaerobic cultures decreased and nomaterial was recovered by ethanol precipitation at this time.About 150 mg (dry weight) of the polymer per liter wasobtained from cultures harvested in the late exponentialphase of growth.

Glucose, sucrose, arabinose, and maltose supported thebest polymer production with viscosities of 3 to 5 cP at 25°Cwith a shear rate of 150/s. Increasing sucrose or nitrateconcentrations did not affect polymer production. Increasing

5-

C.)

0

C')

4-

3-

2-

I-

0-.

Ec

0

(D

0)

0

.0

0(0

.0

Time (h)

5-

4-

_>' 3-cnCo0

u)

> 2-

O-

EC

0

(D(0

a)0

csa.00

.0

0 10 20 30 45

Time (h)

FIG. 1. Growth and viscosity increases by aerobic (A) andanaerobic (B) cultures of SP018. Each culture of strain SP018 was

grown at 50°C in glucose-mineral salts medium supplemented withnitrate and yeast extract. Aerobic cultures were shaken at 200 rpm.Symbols: 0, absorbance; A, viscosity at 150/s shear rate; Ol,glucose concentration.

' 0.4-0

4) 0.2-0C:0

-0

.0

-01o.,

-40c0

200C'

8 2 6 24 8 12 16 20D

NaCI Concentration (g/100 ml)FIG. 2. Effect of salt concentration on the growth and viscosities

of SP018 cultures. The A660 (0) and viscosity (0) at 150/s shear rateat 25°C were measured after 24 h of incubation of 50°C. Values areaverages of three cultures.

the C to N ratio of the medium by decreasing the amount ofammonium and nitrate to as low as 0.02% (wt/vol) of bothcomponents did not affect polymer production (data notshown).Optimal polymer production occurred in medium with 4 to

10% NaCl (Fig. 2). In other experiments, viscosities of 4 to5 cPs were observed in medium containing 10% NaCl.

Characteristics of the SP018 polymer. About 185 ml of aconcentrated polymer solution was obtained from 7 liters ofculture after tangential ultrafiltration. This solution con-tained 12 mg of carbohydrate per ml as determined by theanthrone reaction and had a viscosity of 140 cP at 25°C witha shear rate of 150/s. About 1.5 g (dry weight) of the purifiedpolymer was obtained after ethanol precipitation. This rep-resented 75% of the total carbohydrate present in the solu-tion. The polymer was retained by a 100-kilodalton filter,indicating a molecular weight in excess of 100,000.

Polyacrylamide electrophoresis of the purified polymershowed one slowly migrating diffuse band that stained onlywith the periodic acid-Schiff reagent specific for carbohy-drates; this band accounted for 95% of the area when the gelwas scanned at 500 or 550 nm (data not shown). Twofast-moving bands were observed at the bottom of the gel.Both of these bands stained with amido black and periodicacid-Schiff reagents indicating the presence of both proteinand carbohydrate. A UV absorption spectrum of a 1-mg/mlsolution of the purified polymer had no absorbance atwavelengths specific for proteins or nucleic acids (data notshown). This solution did absorb strongly at 200 to 210 nm,which is characteristic of carbohydrates. When the colori-metric test of Lowry et al. (22) was used to detect protein,about S ,ug of protein per mg (dry weight) of the purifiedpolymer was found. The purified polymer did not react asreadily as xanthan gum with the anthrone reagent (13). Thepolymer contained 44 p.g of carbohydrate per mg (dryweight) compared with 1.2 mg of carbohydrate per mg (dryweight) for xanthan gum.The purified SP018 polymer contained the following sug-

ars (picomole per milligram [dry weight]): mannose, 94.1 ±26.2; glucose, 86.9 ± 15.2; arabinose, 11.5 ± 11.6; ribose,7.6 + 3.0; allose, 3.3 ± 0.8; and glucosamine, 1.0. Eachvalue is a mean (± standard deviation) of three determina-tions. These monosaccharides accounted for approximately80% of the dry weight of the polymer. The SP018 polymercontained an unusually large amount of mannose. The SP018polymer contained arabinose, ribose, allose, and galactose.Elemental analysis of the purified SP018 polymer showedthat it contained 8.93% C, 3.16% H, and 1.26% N.

Fourier transform-infrared spectroscopy of the purified

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.05-

0 078-

0

-oL4

0.03-4000 3200 2400 1600 800

Wavenumbers (per cm)FIG. 3. Fourier transform-infrared spectrum of the purified

SP018 polymer. The predominant carbinyl stretch typical of uronicacid-containing polymers is shown at 1,550/cm.

polymer showed a weak symmetrical stretching band atabout 1,406/cm and an asymmetrical stretching band at1,619/cm (Fig. 3), which is consistent with the presence ofcarboxylate ion. The carboxylate ion has two stretchingbands, a strong asymmetrical band between 1,550 and1,650/cm and a weaker symmetrical band near 1,400/cm(P. D. Nichols, personal communication). Other peaks ob-served with the SP018 polymer were the 0-H stretching at3,300/cm and the C-H stretch at 2,950/cm.

Rheological properties of SP018 polymer. The SP018 poly-mer had higher viscosities than commercially obtained poly-mers had at each concentration tested (Fig. 4). Guar gum andXanflood were also tested at these concentrations (data notshown). The Xanflood preparation had viscosities similar tothat of xanthan gum (Sigma). Guar gum had much lowerviscosities than did the SP018 polymer or either of thexanthan gums. Kennedy and Bradshaw (21) found that a0.5% (dry wt/vol) solution of xanthan gum has a viscosity of175 cP at a shear rate of 10/s which is lower than weobserved for xanthan gum (Fig. 4).The SP018 polymer exhibited pseudoplastic behavior (Fig.

5). The viscosity of the solution was restored when the shearrate was decreased (data not shown). The SP018 polymerhad lower viscosities at 50 (Fig. 5) or at 75 than at 25°C.

800-

X 600-

0

C.) 2000

0 0.1 0.2 0.3 0.4 0.5

Concentration (mg dry wt./ml)FIG. 4. Effect of concentration on the viscosities of xanthan gum

and SP018 polymer solutions. The viscosities of xanthan gum (l)and SP018 (0) solutions were determined at 25°C at a shear rate of7.5/s.

60-0

40

20-

0-0.5 1 1.5 2 2.5 3

Log Shear Rate (per sec)FIG. 5. Effect of shear rate and temperature on the viscosities of

xanthan gum and SP018 polymer. The concentration of each poly-mer was 0.1%. Xanthan gum viscosities at 25 (0) and 50°C (0);SP018 polymer viscosities at 25 (U) and 50°C (O).

However, the SP018 polymer had higher viscosities thanxanthan gum had at each shear rate and temperatpre tested.The rates of viscosity decrease at 75°C of xanthan gum and

SP018 polymer dissolved in distilled water were compared(Fig. 6). The viscosity of the xanthan gum solution decreasedmore rapidly than did the viscosity of the SP018 polymersolution. After 1 h at 75°C, about a 50% decrease in viscositywas observed for xanthan gum compared with about a 25%decrease in viscosity for the SP018 polymer. Thermal deg-radation of xanthan gum at 60°C in distilled water has beenpreviously observed (7, 19-21).Xanthan gum solutions have high viscosities even at

relatively high salt concentrations (7, 14, 19-21), and wefound that a 0.25% (dry wt/vol) xanthan gum solution hadhigh viscosities even at 15% (wtlvol) NaCl concentration(Fig. 7). However, increasing salt concentration greatlyreduced the viscosities of SP018 polymer solutions (Fig. 7).A decrease in viscosity was also observed when the SP018polymer was dissolved in 25 mM sodium phosphate buffer(pH 7.0) compared with the same concentration of thepolymer dissolved in distilled water. When the SP018 poly-mer with 10% NaCl was dialyzed against water, the initialviscosity of the polymer was restored. Thus, the SP018polymer lost viscosity as the ionic strength of the solution

> 60

50

0O 40-C

30

20-

C10

0 10 20 30 40 50 60 70

Time (min)FIG. 6. Effect of thermal stress on the viscosities of xanthan gum

and SP018 polymer solutions. The percent reduction in viscosity at75°C of a 2.5-mg/ml solution of xanthan gum (O) or SP018 polymer(0) was measured at 10-min intervals with a shear rate of 37.5/s.

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was increased, but the process was reversible, and the initialviscosity was restored after dialysis with water.

DISCUSSIONSeveral bacterial strains have been isolated that have

useful characteristics for an in situ microbial selective plug-ging process. These characteristics include rapid growth andthe ability to produce extracellular polymer in anaerobic,carbohydrate-based medium with up to 10% NaCl at 50°C.Zajic and Mesta-Howard (31) have isolated several Bacillusspecies which can grow under these conditions and whichhave culture viscosities of 14 to 45 cP. These bacteria wouldbe able to grow under conditions that exist in many oilreservoirs (5, 23). Another useful property of these bacteriais their ability to produce spores which would facilitate theinjection and penetration of the bacteria in the oil reservoir(15, 28). The fact that bacteria with the above properties arefound in many environments, particularly oil reservoirbrines and cores and aquifer material, suggests that thesekinds of bacteria may be indigenous to many oil reservoirs.Thus, the injection of bacteria or spores into the reservoirmay not be required. All that may be needed is the stimula-tion of the indigenous populations by nutrient injection. Wehave found that the addition of medium E with NO3 withoutthe injection of cells or spores to Berea sandstone cores doesresult in large permeability reductions, and large numbers ofbacteria are found in the effluent of the core (17, 18). Thesebacteria are similar to those described in this paper. Theability of SP018 to grow and produce the polymer on a widevariety of substrates indicates that many commercialsources of carbohydrate could be used as substrates in fieldtrials.The fact that low culture viscosities were observed does

not necessarily preclude the use of SP018 or similar orga-nisms in a microbial selective plugging process. Good mo-bility control is obtained when viscosity of the fluid isbetween 10 and 50 cP at a shear rate of 1/s (14). We routinelymeasured culture viscosities at much higher shear rates 19 to150/s or greater. Although the culture viscosities were notmeasured at lower shear rates, the purified SP018 polymerdid have much higher viscosities at lower shear rates, and wewould expect this to be the case if the culture viscositieswere measured at lower shear rates. We have shown thatsome of the strains in our study are good plugging agents,reducing the permeability of Berea sandstone cores by 70%or more (17, 18). Both the bacterium with its attachedexopolymer and the exopolymer itself could be involved inplugging the 10- to 20-,um pores of Berea sandstone (28).The biochemical characterization of SP018 suggests that it

is a member of the genus Bacillus (11). However, additionalwork is required to more accurately determine the taxo-nomic status of this strain. One interesting feature of SP018was the cultural conditions that affect polymer production.Polymer production at 50°C occurred only under anaerobicconditions. Polymer production was not enhanced by lowerconcentrations of nitrogen in the medium and was better inmedium with 4 to 10% NaCl. These data suggest that culturalconditions important for polymer production in SP018 aredifferent than those for other bacteria (19, 25, 27, 29). Zajicand Mesta-Howard (31) also found that polymer productionby the Bacillus species they studied was better at higher saltconcentrations.The biochemical characterization of the SP018 polymer

shows that it is a heteropolysaccharide composed mainly ofglucose and mannose in approximately equal amounts.Arabinose, ribose, allose, and glucosamine were also found

0-0

U,0C.)C

0 3 6 9 IZ 1D

NaCI Concentration (%)FIG. 7. Effect of salt on the viscosities of xanthan gum and

SP018 polymer. The viscosity of a 2.5 mg/ml solution of xanthangum (I) or SP018 polymer (0) was determined at 25°C at a shearrate of 18.75/s in the presence of the indicated salt concentrations.

in lower amounts. The carbohydrate composition of theSP018 polymer is very different than that found for otherBacillus species (19). Fourier transform-infrared spectros-copy of the SP018 polymer indicates the presence ofcarboxylate ion, suggesting that some of the monosac-charides may be uronic acids. The monosaccharides mayalso be acetylated or contain ketal-linked pyruvates oruronic acids. Further evidence for the presence of uronicacids is resistance to acid hydrolysis or decreased reactive-ness with anthrone reagent, both of which were observed forthe SP018 polymer. The SP018 polymer showed low valueswhen the anthrone procedure was used to determine carbo-hydrate content. Uronic acid-containing polymers are im-portant in and may contribute to adhesion of marine sedi-ments (A. R. M. Nowell, D. Thistle, J. D. Davis, P. D.Nichols, M. B. Trexler, and D. C. White, Estuarine CoastalShelf Sci., in press).The SP018 polymer does have many properties that make

it suitable for enhanced oil recovery or other applications (7,14, 19, 20). These properties include high viscosities at diluteconcentrations and at elevated temperatures, pseudoplastic-ity, and resistance to shear and thermal degradation. How-ever, increasing the ionic strength reversibly reduces theviscosity of SP018 polymer solutions. This would make thepurified polymer unsuitable as a mobility control agent inchemical-enhanced oil recovery processes where concen-trated polymer solutions are injected into an oil reservoir,since many oil reservoirs have high salinities (5). Our resultsshow that salt-tolerant polymers may not always be obtainedby selecting for salt-tolerant organisms.The viscosities and extracellular polymer concentrations

of the cultures were low compared with those of otherpolymer-producing bacteria (2, 3, 7, 19-21, 25, 27). Manyaerobic bacteria produce large amounts of polymer (inexcess of 10 g/liter) and have very high viscosities (1 to 10 P).However, these aerobic bacteria would not be suitable for insitu microbially enhanced oil recovery, since oil reservoirsare generally anoxic environments with elevated tempera-tures and salinities (5, 23). Anaerobic or facultative polymerproducers include Leuconostoc and Streptococcus species(16). Although these bacteria grow anaerobically, they donot grow well at high salinities and temperatures. We testedthe ability of four Leuconostoc strains to grow in 2 Ind 5%NaCl at room temperature (data not shown). Although all thestrains produced extracellular polymer and had viscous

.o

IO-

.o ------O

'o-

10-

0 -00- I I I I I1- e% eN =

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cultures in medium without salt, the addition of 2 and 5%NaCl to the medium either inhibited growth or polymerproduction by these strains.

ACKNOWLEDGMENTS

We thank R. Lee Mosley for the polyacrylamide electrophoreticstudies of the SP018 polymer, David P. Nagle for his assistance in theuse ofthe DU-8B spectrophotometer, and Karen Kealy for assistancewith the routine biochemical tests performed on strain SP018.Furthermore, we thank D. C. White for the use of his laboratory atFlorida State University, and the assistance of John D. Davis andPeter D. Nichols with the carbohydrate composition and the Fouriertransform-infrared analysis of the SP018 polymer.The research was supported by Department of Energy contracts

DE-AS19-80B10300 and DE-AS05-83ER-13053 and the ResearchCouncil of the University of Oklahoma.

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cultivation of methanogenic bacteria: 2-mercaptoethanesulfonicacid (HS-CoM)-dependent growth of Methanobacteriumruminantium in a pressurized atmosphere. Appl. Environ. Mi-crobiol. 32:781-791.

2. Bodie, E. A., R. D. Schwartz, and A. Catena. 1985. Productionand characterization of a polymer from Arthrobacter sp. Appl.Environ. Microbiol. 50:629-633.

3. Boyle, C. D., and A. E. Reade. 1983. Characterization of twoextracellular polysaccharides from marine bacteria. Appl.Environ. Microbiol. 46:392-399.

4. Bryant, M. P. 1972. Commentary on the Hungate technique forculture of anaerobic bacteria. Am. J. Clin. Nutr. 25:1324-1328.

5. Clark, J. B., D. M. Munnecke, and G. E. Jenneman. 1981. In situmicrobial enhancement of oil production. Dev. Ind. Microbiol.22:695-701.

6. Clark, J. M., Jr., and R. L. Switzer. 1977. Experimentalbiochemistry. W. H. Freeman and Co., San Francisco.

7. Cottrell, I. W., and K. S. Kang. 1978. Xanthan gum, a uniquebacterial polysaccharide for food application. Dev. Ind. Micro-biol. 19:117-131.

8. Craig, F. F., Jr. 1974. Secondary and tertiary oil recoveryprocesses. Interstate Oil Compact Commission, OklahomaCity, Okla.

9. Davis, B. J. 1964. Disc electrophoresis. II. Method and applica-tion to human serum proteins. Ann. N.Y. Acad. Sci.121:404-427.

10. Fazio, S. A., D. J. Uhlinger, J. H. Parker, and D. C. White.1982. Estimations of uronic acids as quantitative measures ofextracellular and cell wall polysaccharide polymers from envi-ronmental samples. Appl. Environ. Microbiol. 43:1151-1159.

11. Gibson, T., and R. E. Gordon. 1974. Genus I. Bacillus Cohn1872, 174, p. 529-550. In R. E. Buchanan, and N. E. Gibbons(ed.), Bergey's manual of determinative bacteriology, 8th ed.The Williams & Wilkins Co., Baltimore.

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