FR2465001A1 - HETEROPOLYSACCHARIDE S-88 - Google Patents

Abstract

THE NEW HETEROPOLYSACCHARIDE S-88, PREPARED BY FERMENTATION OF AN UNPATHED PSEUDOMON SPECIES, ATCC31554, HAS INTERESTING PROPERTIES AS A THICKENING, SUSPENDING AND STABILIZING AGENT IN AQUEOUS SYSTEMS. IT IS ESPECIALLY USEFUL IN THE PREPARATION OF FLUIDS AND DRILLING MUDS FOR OIL WELLS. ITS CHEMICAL COMPOSITION IS FULFILLED BETWEEN THE FOLLOWING LIMITS, IN WEIGHT, AS REGARDS SUGAR: 10-20 GLUCURONIC ACID; 10-30 OF MANNOSE; 30-40 GLUCOSE; 35-45 OF RHAMNOSE; THE POLYMER ALSO CONTAINS 3-7 ACETYL GROUPS.

Description

-1-

  The present invention relates to a hetero-

  polysaccharide, referred to as S-88 heteropolysaccharide.

  The product S-88 can be prepared by

  appropriate nutrient medium with an organism

  has not been described so far, according to advanced taxonomic studies, which is an unnamed species of Pseudomonas. An unrestricted permanent depot of this organism used in the production of the present heteropolysaccharide was made with the American Type Culture Collection on August 21, 1979

  under registration number ATCC 31554.

  Various classification criteria for the

  Pseudomonas genus and crop descriptions

  of Pseudomonas species are found in the 7th edi-

  Bergey's Manual (Breed et al. (1957)) and in the 8th edition of Bergey's Manual (Doudouroff and others, (1974)), as well as by other schools in

  various publications; Hugh and Gilardi, 1974, Pseudo

  Monas, Manual of Clinical Microbiology, 2nd Edition, Lennette et al., eds., pages 250-269; Iizuka

  et al., 1963, Attempt at Grouping the Pseudo Genus

  monas, J. Gen. Appl. Microbiolopy 9: 73-825 and Hendric et al., 1966, Identification of Certain Pseudomonas Species, Identification Methods for Microbiologists, Part A, Gibbs et al., Editors, pages 1-7, Academic

Press, New York.

  These criteria and descriptions were searched for

  for a species of Pseudomonas with characteristics

  morphological and cultural traits similar to those of ATCC 51554. The following considerations make

  definition of a new species of Pseudomonas justified

trusted and necessary.

-2-

Description of the strain

  A. Colon morphology characteristics

  Two types of colonies (typical and atypical

  c) appear on both nutrient agar and YM agar. On nutrient agar, small translucent yellow colonies develop in two days at room temperature; the diameter reaches about 1 mm in 5 days. The colonies are round, smooth-edged, and convex. Typical colonies are more shiny and the surface texture is extremely hard; whole colonies are removed if they are pushed by a loop; however, the colonies

  atypical ones do not have such characteristics.

  On YM agar, both typical and typical colony diameters are about 7-9 mm in days. Those of both types are round, smooth, convex, opaque and pigmented yellow. Typical colonies become more prominently shaped

  and the atypical colonies are flatter. The

  concentric keys are more important in the case of atypical colonies. The surface of typical colonies is more sticky and an elastic membrane is formed

with incubation-prolonged.

  B. Characteristics of Cell Morphology The S-88 strain is a gram-negative rod-shaped bacterium. There is no difference in the shape of cells between typical and atypical strains, except for the disposition of

  cells. Cells of a typical strain are ob-

  served in aggregation, as in a mass resembling Zoogloea. On nutrient agar, most cells have sizes of 0.5-0.6 on 2, G-2.51 iT;

  thinned at one end. Few cells are motile.

  Granules of poly-3-hydroxybutyrate can be

  observed in cells in the event of prolonged incubation

  -3- Sometimes, very long abnormal cells have been observed. Pleomorphism is common. Most cells are destroyed within 5 days at

Room temperature.

  On medium IY, the cells are larger and longer (0.6-0.8 on 2.5-35.07m), in sticks of regular shapes. Most cells are in a palisade setting. Granules of

  poly-p-hydroxybutyrate accumulate. The cells

  come very long (more than 5 / am) during a

prolonged bation.

  The coloring of the flagella is extremely difficult because relatively few cells are flagellated and the formation of gum gene the observation of the flogging mode. According to the methods of Iniss and Mayfield, the majority of flagellated cells are monotriched; insertion is polar

and / or sub-polar.

  C. Physiological and biochemical characteristics

  Low or negative result with cyto-

  chromium oxidase; positive with catalase. The organism is capable of development at 37C, but not at 40C or 410C. No tolerance at 3% NaCl and pH

  of 4 and 12. No survival at 60 C for 50 minutes.

  aerobics; acid is produced from various carbohydrates, but not from gases. Nitrate can be reduced. The sunflower milk was reduced, but not peptonized. There was no H2S product (lead acetate method). Phosphatase and lipase were produced. There was hydrolysis of esculin

  and gelatin (very weakly), but not

  midon or casein. The results with ADH, LDC and ODC were negative. Tolerance at 0.1% o of triphenyltetrazolium chloride. No development on SS agar media, Pseudosel or Aconkey. Acid -4-

  was produced in slant culture; no development

in the TSI environment.

  D. Antibiotic sensitivity test

  The S-88 strain is susceptible to antibiotic

  following: Carbenicillin (100 μg) Gentamicin (10 μg) Chlorotetracycline (51 μg) Penicillin (10 units) Polymyxin B (300 units) Erythromycin (15 μg) Novobiocin (30 μg) Kanamycin (30 μg) Tetracycline ( 3 μg) Neomycin (30 μg and is not susceptible to: Streptomycin (10g) Colistin (10μg) E. Nutrition Characteristics Vitamins are not needed for development Ammonium salts serve as the sole source of At least 26 out of 123 organic compounds

  are used as the sole source of carbon and energy.

  gy; most are carbohydrates.

  F. G + C content of DNA The molar G + C content of strain S-88

is 69.8 5.

  G. Identification by API tube systems and

OXI / FERM

  The S-88 strain was not identified by API or OXI / FERMI tube methods. This suggests that the organism could not be isolated from a

clinical source.

H. Identification

  Since strain S-88 is flagellated with

  -5- monotric polar or subpolar, the organism belongs to one of the members of the genus Pseudomonas,

  to judge from the phenotypic characteristics.

  The G + C content of the DNA (69.8 mol%) is in the range of G + C molar levels of the genus Pseudomonas. TABLE I: Biochemical and other various assays used for strain S-88 Oxidase: Kovac Pathotech Catalase Medium OF: fermentative oxidant Gases from glucose Produce H2S: TSI from cystine Ammonium from peptone β-galactosidase ( ONPG) Arginine dihydrolase Lysine decarboxylase Ornithine decarboxylase Tryptophan deaminase Phenylalanine deaminase Urease Indole MR test VP test Nitrate reduction Nitrite reduction Denitrification N2 fixation: Development in Burk medium Nitrogenase activity Malonate (oxidation) Phosphatase + (weak) + (weak + + + + Hydrolysis of Gelatin Casein Starch Tween 80 Pectin Alginate Cellulose Chitin DNA Esculin + (weak) + NT NT + Development on various media EMB agar +

MacConkey Agar -

SS agar -

Mannitol salt agar -

TCBS agar -

Agar with blood and

Tellurite of Tinsdale -

Pseudosel agar -

  Pigment production: Middle King A Middle King B + NT + Dye reaction: Red Congo Blue Nil NT ru J "0% Ln H3 + I Il Il Il Fd Fd ti:

P) O (D)

  ca or as P. Pa -J. m Fb Ft (D cc CD ,, I O I TABLE I (Continued)

Hemolysis (sheep blood) -

  Sunflower milk: acid, reduction only

Production of 5-ketolactose -

  Survival at 600C pend.0 min. + TSI: Slant culture "ButteuD Gas Yolk reaction Acid No development -'C! Os c :, -8- Fermentation conditions Heteropolysaccharide S-88 is produced during the aerobic fermentation of appropriate aqueous nutrient media in conditions controlled by inoculation of the organism of the nonspecific species of Pseudomonas.The media are fr d usual media, containing a source of carbon, nitrogen and inorganic salts.In general, carbohydrates (by

  glucose, fructose, maltose,

  crose, xylose, mannitol, etc.) can be used

  singly or in combination as sources of

  assimilable carbon in the nutrient medium. The quantity

  The exact source of the carbohydrate source or sources used in the medium depends in part on the other ingredients in the medium, but in general the amount of carbohydrate usually varies.

  about 2% to 4% of the weight of the medium. These sources

  These carbon sources can be used individually or several of these carbon sources can be combined in the medium.

  protein materials can be used as sources of

  these nitrogen in the fermentation. Nitrogen sources

  can be used, for example, hydrolysates of

  primary yeast, soy flour, cottonseed meal, casein hydrolysates, maize

  soluble residues of distillation,

  matte, etc. Nitrogen sources, alone or in combination

  boring, are used at a rate of 0.5 to 0.2%

from the weight of the aqueous medium.

  Among the inorganic nutrient salts that can be incorporated in the culture media, are the usual salts capable of giving ions of sodium, potassium, ammonium, calcium and phosphate, sulfate, chloride, carbonate ions. ,

  etc. Also included are the metals present in

  trace elements such as cobalt, manganese,

iron and magnesium.

  It should be noted that the media described in the examples are only illustrative of the wide variety of media that can be used, and

  should not be considered as limiting.

  An important feature of media is that when strain S-88 is grown under conditions of low Ca ++ content, i.e. in deionized water or in an aqueous system having less than 200 ppm of ions Ga ++, the resulting gum

  is easily soluble in non-gel solutions

fication.

  Fermentation is carried out at temperatures

  between 25 and 355C; however,

  for the best results, it is best to

  induce fermentation at temperatures between 28 and 32 0 approximately. The pH of nutrient media for the development of Pseudomonas culture and for the production of S-88 polysaccharide can be

vary between 6 and 8 approximately.

  Although the S-88 polysaccharide is produced

  both surface and immersed, it is preferable

  to lead the fermentation to the submerged state.

  A fermentation is conveniently carried out

  small scale by inoculating an appropriate nutrient medium

  prayed with the culture and, after transfer to a medium

  of production, allowing the fermentation to

  at a constant temperature of about 300C

  on a shaker for several days.

  Fermentation is started in a sterilized bottle of medium by one or more stages of

  germs development. The nutrient medium for the

-10-

  stage of germ development can be a combination of

  any suitable source of carbon and

  zote. The sprouting flask is shaken in a constant temperature chamber of about 300 ° C for 1 to 2 days, or until the development is satisfactory, and a small amount of water is used.

  resulting vegetative development to inoculate one

  xth stage of sprout development or the production medium. The flasks of the intermediate stages of germ development, when used, are

  developed essentially in the same way; it is-

  that is, a portion of the contents of the last stage seed development flask is used to inoculate the production medium. Inoculated flasks

  are shaken at a constant temperature for several

  three days and at the end of the incubation period

  the contents of the flasks are collected by precipitation.

  by a suitable alcohol such as isopropanol.

  For a large-scale job, it is

  it is preferable to conduct the fermentation in

  Suitable vessels equipped with an agitator and means for aerating the fermentation medium. In this process, the nutrient medium is prepared in the reservoir and

  sterilized by heating at temperatures up to

  than about 1210C. After cooling, the medium

  sterilized is inoculated with previously obtained

  of the producing culture and is left

  carry out the fermentation for a period of, for example, 2 to 4 days while stirring and / or

  aerating the nutrient medium and maintaining the temperature

  at around 3OC. This production process of the

  S-88 is particularly suitable for the preparation

large quantities.

  The product is collected from the medium

  fermentation by precipitation with a suitable alcohol

-he-

required, such as isopropanol.

Heteropolysaccharide S-88

  The heteropolysaccharide produced by a

  unnamed species of Pseudomonas is mainly

  carbohydrate with 3-7% of groups

  acetyl as the 0-glycosidically bonded ester.

  The carbohydrate portion of the polysaccharide

  S-88 wrinkle contains 10-20% glucuronic acid, -30% mannose, 30-40% glucose and 35-45%

rhamnose.

  Table II shows results of

  specific lyses on six fermenta-

  including the triplicate analysis of two samples

  lons. TABLE II: Composition of S-88 Gum

PARTICULAR ORGANIZATIONS

  Echan- Glucuronic acid glucuronic acid Pyruvate no (%) p (%) Acetyl (%) s Neutral sugar constituents (%) Glu Man Fuc Rha Ara Protein (% o)

- 45

- 59

- 57

- 36

3 37

- 41

1 37

I 40

I 44

0 41 I

  11.8 11.9 11.5 11.7 12.7 12.7 12.7 14.0 14.0 14.0 a by decarboxylation y by colorimetric analysis 4.b 0% uo o> CO ABC 6A 6B 6C 11.8 18.7 18.0, 8, 1, 1, 1 13.7 13.7 13.7 0.1 0.1 0.1 0.1 0, I 0.1 0.1 0.1 0.1, 0.2, 2.7, 0.6, 6.1, 6.1, 4.1, 4.9, 4.9, 4.9. The acetyl group content of 5-10. % was determined by treating an aqueous solution & 0.2% S-88 gum with an alkaline reagent, hydroxylamine,

  this being followed by treatment with a chlorine reagent

  ferric acid acid [S. Hestrin (1949) J. Biol. Chem.

pages 249-261].

  The neutral sugars of the polysaccharide S-88 were determined by dissolving 10 mg of the product in

  2 cm3 of 2E solution of H2SO04 and heating the

  lance at 100 C for 4 hours. The resulting solution was cooled, neutralized with barium hydroxide

  and the pH was raised to 5-6 by means of carbon dioxide.

  solid. The resulting barium sulphide precipitate was removed by centrifugation and the supernatant liquid was concentrated as a syrup under reduced pressure. An attempt has been made to identify the sugars in the hydrolyzate by gas-liquid chromatography of

  their derivatives aldononitrile acetate on a chromato-

  Model 5750 from Hewlett-Packard using 3% by weight of 0V-225 on Gas Chrom Q particles

  from 0.149 to 0.177 mm at 210 C. It has been identified and determined

  quantitated sugars by comparison with authentic standards [J.K. Baird, M.J. Holroyde,

  and D.C. Ellwood (1973) Carbohydr. Res. 27 pages 464-

467j.

  The various neutral sugars of polysaccharides

  have also been characterized by chromatic

  descending tography on paper on paper for

  Uhatman # 1 chromatography using as sol-

  the top layer of a pyridine mixture:

  ethyl acetate: water (2: 5: 5). The colors were colored

  tograms using a silver nitrate dip and a reagent for acidic alanine phthalate spray. The constituent sugars were identified by co-chromatography with sugar standards and by the specific color reaction with

the alanine phthalate reagent.

  The glucuronic acid content of polysaccharide

  wrinkle was determined by two separate methods. In one method, the sample was decarboxylated with 19% hydrochloric acid and the liberated carbon dioxide was collected in normal sodium hydroxide and made by back titration determinations [B.L. Browning (1967) Methods of Wood

  Chemistry II, pages 632-633] and by the coloring method

  metric to carbazole [T. Bitter and H.M. Muir (1962)

Anal. Biochem. 4 pages 330-334J.

  Paper electrophoresis was used for separating and attempting to identify glucuronic acid present in the hydrolyzate

  neutralized acid described above. Food portions

  Quotas of the latter and known standards of glucuronic acid were applied on Camag electrophoresis paper NI 68-011 and electrophoresis was performed for 2.0 hours in a pH 2.7 buffer using an apparatus. Camag electrophoresis model HVE. Chromatograms were stained with Silver Nitrate Reagent

  in order to locate the separated glucuronic acids.

  S-88 polysaccharide gives viscosity to an aqueous medium when dissolved in water at low concentrations. For this reason and also for its shear sensitivity and overall rheology, it is useful as a thickener, suspending agent, emulsifier, stabilizer, lubricant,

  film-forming agent or binder, especially in systems

  aqueous systems. In particular, it has uses

  in the following applications or the following products

  adhesives, cement for joint walls, mortar

  liquid and water-retaining mortars, Z-F65O1 -15- coatings for sealing cracks, sealing of cans, boiler products, latex creaming, solder flux melts, solder pastes, ceramic varnishes and extrusions, products cleaning and brightening, toys, emulsions (latex, asphalt, silicone), silver recovery, seed coatings, spray regulation for pesticides

  or herbicides, pesticides and herbicides fluids and

  emulsifiable concentrates, binders for tobacco, inks for

  water base, solutions for lithographic dispensers

  phics, leather finishing products, hydro-mulching

  and hydro-seeding, printing and finishing of fabrics, addi-

  for the paper to be added in the wet part, ad-

  a retention and training machine for paper for

  in the wet area, anti-adhesive products, release agents, liquid resins, slurry and packaged explosives, oil well drilling muds and water wells, petroleum stimulating fluids cosmetics, suspensions and emulsions

pharmaceuticals.

  Moreover, this eraser is useful in

  food products such as jellies and other products

  of high sugar content, drinks consisting of

  citric acid beverages, dairy products including ice cream and yoghurt, salad dressings, dry mixes, icings, syrups, so-called puddings, flour foods, canned foods preserved and autoclaved and filling products

for bakery.

  A particularly useful utility is in the field of drilling muds for oil wells and water wells. Following are more detailed examples illustrating this use.

preferred.

  -16- Although the S-88 gum has a property

  the general viscosity

  particular thread of properties in solution is a

  distinctive feature that distinguishes it from other heteropolysaccharides.

  In the dry state, the gum has a content of

  solid 85-90%; in its most useful particle size, it passes 100% through a sieve with a mesh size of 0.42 mm and not more than 30%

  through a sieve of 0.044 mm. It has a 1% visco-

  from 1200 to 2500 cPo in a Brook-

  field at 60 rpm and gives the following results to a

  concentration of 0.4% in tap water containing

  2% KCl, Famm (F = 0.2): 600 rpm> 120; 300 rpm> 92; 200 rpm> 76; 100 rpm> 70; 6 rpm

> 32; and 3 rpm> 28.

  The gum also has excellent heat stability and no loss of viscosity by changing to

  autoclave at 1210 C and 1.05 kg / cm 2 for 15-20 minutes.

  utes. It produces a firm gel with 15% NaOH and

* incubation at 800 C for two hours. It is incompa-

  Suspended with saturated CaCl2, ammonium polyphosphate

and NH4NO3 60%.

  In addition, the rubber has the following profile of

  properties: 1. Viscosity and shear A. Brookfield 1. 1.0% 60 rpm I 520 cPo 6 rpm 11 600 cPo Rotor N j 2. 0.1% (UL adapter) at 295 cPo 3. 0.5% Wells Brookfield at 9.6 s- 474 cPo -17- B. Shearb 1. n at 1.92 s-1 8512 cPo 2. n at 9.6 s-1 2342 cPo 3. n at 76.8 s-1 320 cPo 4. n at 384 s-1 64 cPo 5. n at 3842 s-1 64cPo 6. n at 9.6 s-1 1558 cP C. Storage at 4.4oC

  1450 cPo, rotor N 3 at 60 rpm, no gelation

tion, jerky flow.

  2. Stability to acids, bases and heat A. Stability

1. Acetic acid plus heat -

  at. initial value of n: 1620 cPo b. final value of n: 2450 cPo c. % change: +51% (gelation)

2. 10% HC01 plus heat -

  at. initial value of n: t cPo b. final value of n: Pt cPo c. % change: Ppt%

3. 15% NaOH plus heat -

  at. initial value of n: 1,869 cPo b. final value of n: Gel cPo

4. Heat only -

  at. initial value of n: 2061 cPo b. final value of n: 2266 cPo c. % change: + 1_0% B. Effect of pH 1. 5% acetic acid pH 2.69 1741 cPoc 2. NH40H at 5% pH 10.99 1741 cPo

  3. The S-88 solution is stable in the

  pH range 1.25 to 12.3 -18- Compatibility with salts and A. Salts 1. CaC12 (saturated) 2. Ammonium polyphosphate

3. INH403 at 60%

  4. 1 / Al12 (SC4) 3.18H2O 5. I% CaCl2.2H2O 6. 1% KCl 7. 0.1% KCl 8. 2.5% KCl B. Dyes 1. Green "Milling" 2. Methylene Blue 4. Texture / flow properties Discontinuous, discontinuous, gelatinous, gummy to the touch 5. Synergy and Enzymesc dyes Pre-precipitated Precipitated Precipit Compatible Compatible Compatible 2214 cPoc 922 cPoc Compatible Precipitated high viscosity, A. Guar BHP Guar

C. CMC

D. HEC

E. LAS 1-1

A. Guar B. H. P. Guar C. CmC

D. HEC

Nope

1 0 O]

  D 1600 cP 170; 1715 cPo 174 '- 852 cPo 108e 602 cPo 122 <225 cPo Predictable viscosity 1900 cPo 1i975 cPo 1375 cPo 1175 cPo hour mix 2 cPo 1 cPo 38 cPo) cPo n after 2 hours of mixing 1741 cPo 1741 cPo 1665 cPo 998 cPo Synergy None Nil +21 Nil 6. Reactivity with milk A. Dispersion: poor - fine granular precipitate B. Separation of dolphin: 1st day C. Other observations:

A65001

  -19 - 7. Film formation The eraser extends irregularly, a film

  forms, irregular consistency, weakly

tick, brittle.

  aViscosity measured on a Brookfield viscometer

  LVF model at 6 rpm with rotor n 'I and an adaptation

UL.

  bAll measurements performed on a micro-viscometer

Wells-Brookfield Model RVT-C / p.

  CViscosity measured on a Wells viscometer

-1

  Brookfield model RVT-c / p at 9.6 s-1.

Example I

  Fermentation technique for producing hetero

  polysaccharide S-88 A. Care of culture The organism Pseudomonas, not known as ATCC

  31554, grown on NA agar at incubation temperature

  This organism produces a carotin pigment

  yellow tapeworm. Colonies on NA are small (only

  1-3 mm) after 48 hours are convex and have a gelatinous texture. The typical colony tends

  stick stubbornly to the surface of the gen-

  lose. Sometimes a morphological variant can be

  velopper, which is easy to spot on NA. The variant has a flat colony and does not adhere stubbornly

  on the surface of the agar plate. It has been found that this

  It has a reduced activity in the production of S-88 gum. B. Preparation of germs Ballooned germs are prepared in

YI broth incubated at 30 C.

  In this environment, culture will typically give

  development of the flocculant type followed by

  viscosity growths with a gravitational appearance

  nulaire. The seeds in Y. are then used at 24-30 hours to inoculate a seed production medium which is the same as in the final fermentor, except that the phosphate content is increased to 0.5%. Fermentors of 3.785 liters are used as containers for the production of germs for

  fermenters of 20 liters and 70 liters.

C. Medium in the final fermenter

  The following medium gives results

  ceptables in fermentors as much as 20 liters as

70 liters.

  Glucose ............................ 3.0%

K2HP04. .................... 0.05%

AM .......................... 0.05%

  NH4N03 ............................... 0, 09%

  MgSO 4.7H20 ......... 0.01% Fe ++ ............ i ........ 1 ...... ii 1 PPMi F e . .1 ppm Salts HoLe ...................... I ml / L A stirring speed set at 500 rpm

  in fermentors of both 20 liters and 70 liters

  very is advantageous. Fermentation times can range from 45 to 70 hours, with viscosity

  beer between 3000 cPo and 5000 cPo.

  Conversion efficiencies range from 31% to 52% with 3% c glucose. Small amounts of a commercially available antifoaming agent can be used. Gram stains performed on S-88 fermentation beer show gram-negative cells with sizes of 1.25 Px 2.5x

  with polar bodies taking a dark color.

  HoLe salts are an oligo-

  containing tartrate, magnesium molybdate

  sium, CoCl3, ZnCl2, CuCl2, boric acid,

  manganese chloride and ferrous sulphate. When we want a product of a low

-21-

  calcium, a fermentor medium of 50

  the following is the case: Medium in a 30-liter fermentor for a product with a low calcium content Deionized water Glucose ..............

  ............. 3.0 a..DTD: K2HP04 ......... 0.05%

ALIP .................. 0.05%

  MgS04.7H20 ......................... 0.02%

  1TE4N03 ............................ 0.09%

  Yeast Extract ......... 0.01% HoLe Salts .......................... 40 ml Vitamin Blend. ............... 25 ml Fe ++ ....................... 1 ppm Ca ++ ... ... .............. 2 ppm The vitamin mixture is a mixture of 1 / cm3 of each of the following compounds: thiamine,

  cyanocobalamin, patothenate, riboflavin, nicotine

  tin, choline and pyridoxamine; 0.05 / u / cm3 of folic acid and paminobenzoic acid; and 0.005 / u / cm3

of biotin.

  D. Recueil Fermentation beer is pasteurized at C for 10 to 15 minutes. Due to the excellent heat stability exhibited by this product, higher pasteurization temperatures with shorter times would be acceptable. Good fibers are typically produced under conditions

  of precipitation giving 58-60% of used IPA.

  E. Drying All the product thus collected was dried at 50-55 ° C for up to one hour in a drying kiln.

forced air trays.

  The properties of the S-88 gum produced under these fermentation conditions were indicated above.

Z465001

  When the low calcium gum S-88 is prepared, it has a similar chemical analysis and properties profile as the S-88 gum, but has an unusual response in NaCl solution. . Although the initial viscosity with 0% salt is slightly low, a viscosity

  stable is maintained at 6%, see Table III.

TABLE III

  Salt response: NaCl NaCl concentration,% by weight

0 2 4 6 8 10 15 20

  600 "13.4 14.2 14.8 15.8 11.4 10.2 9.5 9.0

pn

  300 20.1 21.4 122.0 20.6 20.2 19.7 19.4 19.0

_-4 - -i -.-

  26.0 26.1 27.1 25.4 24.3 524.3 1.8 23.6

  41.7 '42, 0 42.5 41.2 40.6 39.6 39.1 38.5

6 33552 339 342 526 289 276 254 252

3,578,577 586,563 510,482,441,410

  Viscosity, Co, Fann, 35 rpm Compositions Using S-88 As mentioned above, S-88 gum, both of the normal form and the calcium form, can be used in base-based drilling muds.

freshwater or saltwater.

  A typical formula for a fresh water sludge is: S88 1.0 kg Bentonite 10.0 kg Fresh water 350 liters Viscosity results Fann: Speed (rpm) 3 6 100 200 300 600 Reading on the dial 7 , 6 7.9 16, 8 21.5 25.6 35.3 pH = 8.5; Filtrate API = 12.0 cm3 -23-

  Another formula, for a mud with salt water

  lee, is: S-88 1.0 kg Seawater 350 liters Viscosity results Fann: Speed (rpm) 3 6 100 200 300 600 Reading on the dial 2, 8 3.2 9.0 12.8 15 , 6, 23.6

pH = 7.2.

-24-

-REVEEIDICATIONS -

  I - Heteropolysaccharide S-88, containing -20% glucuronic acid; 1030% mannose, -40% glucose; 35-45% rhamnose; and 3-7% acetyl groups, prepared by fermentation under controlled conditions of a culture of ATCC 31554,

a species of Pseudomonas.

  2 - Product according to claim 1, characterized

  teris6 in that it contains less than 200 ppm of Ca ++ ions