United States Patent 1191 [11] Patent Number: 5,071,976 Stirling [45] Date of Patent: Dec. 10, 1991

[ 54 1 No VEL H ETEROPOLYSACCHARIDE FOREIGN PATENT DOCUMENTS Inventor: David I. Stirling, Fanwood, 1589865 5/1981 United Kingdom _ [73] Assignee: Celgene Corporation, Warren, NJ. OTHER PUBLICATIONS [211 App}_ NOJ 270,404 Huq et al., Aust. J. BioL, 1978, vol. 31, pp. 311-316. Bergey’s Manual, 8th ed., 1979, Williams & Wilkins (p. [22] Filed: Nov. 7, 1988 268). Colby et al., Ann. Rev. Microbiol, 1979, vol. 33, pp. Related US. A lication Data 481-517 _ _ pp Byrom, D. in Microbial Growth on C1 Compounds, H. [63] Contmuauon o_f Ser. No. 820,535, Feb. 6, 1986, aban- Dalton ed.’ 1931’ pp. 278489. doned, wh1ch 1s a continuation of Ser. No. 700,564, Hackh’?’ 8 chemical Dictionary 4th Edition Feb. 11, 1985, abandoned. McGraw_Hin’ 1972. [51] Int. Cl.5 ...... C08B 37/00; CllP 19/04; Prima'y Examiner__D_ w_ Robinson c1 1N 1/20 Assistant Examiner-Irene Marx [52] U5. Cl...... 536/123; 435/ 101; Attorney, Agent, or Fl‘rm_-Mathews’ WOOdbI-idge’ & 435/2511 Collins [58] Field of Search ...... 435/101, 252.1; 536/123 [57] ABSTRACT _ This invention provides a fermentation process which [56] References C'ted involves aerobically cultivating a strain of Methyle U.S. PATENT DOCUMENTS philus viscogenes under growth conditions to produce an 3 346 463 10/1967 Goren ...... 435/101 accumula‘ed quantity Ofa novel type of exopolysaccha' 3,932,218 1/1976 Finn et al. , 435/101 ride’ Such as hetempdysacchafide Poly 54 4,514,563 4/1985 Fujiyama ...... 435/101 4,638,059 1/1987 Sutherland ...... 536/121 3 Claims, 2 Drawing Sheets US. Patent Dec. 10, 1991 Sheet 1 of 2 5,071,976

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80.9 nd US. Patent Dec. 10, 1991 Sheet 2 of 2 5,071,976

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00 ON adntmmmEhmribulose monophosphate pathway; gate methylotroph which uses the ribulose monophos (0) capable of growth on fructose; and phate pathway(RMP) cycle of formaldehyde ?xation. 25 (d) optimal growth rate at a cultivation medium temper Methylophilus methylotrophus kStrain AS~1 is a gram ature of 30°43“ C. negative, nonpigmented rod with a single polar ?agel Generally the novel bacterial strains of the present lum. invention exhibit glucose dehydrogenase activity. In SCP production, methanol feedstock constitutes In another embodiment this invention provides a the highest percentage of the operating costs, so that 30 bacterial culture having the identifying characteristics any increase in the microbial growth rate yield has a of strain ATCC 39893, said culture being capable of direct in?uence on the operating costs of the SCP pro aerobic bioconversion of methanol to an extracellular duction. For this reason I.C.I. has applied recombinant accumulation of heteropolysaccharide. DNA technology to increase the cell yield by altering Subcultures of accession Number ATCC 39893 strain the genome of the Methylophilus methylotrophus micro can be obtained upon request from the permanent mi organism. The genes for the more ef?cient glutamate croorganism collection of the American Type Culture dehydrogenase nitrogen assimilation system-from Esch Collection, 12301 Parklawn Drive, Rockville, Md. erichia coli were cloned and inserted into a Methyle 20852. The microorganism deposition is in accordance philus methylotrophus strain. This strain previously had with the requirements of the Budapest Treaty for the its less efficient glutamate synthase nitrogen assimilation purpose of patent procedure. system blocked by means of DNA mutation, as de The bacterial strains having the identifying character scribed by J. Windass et in Nature, 287 396(1980). istics of strain ATCC 39893 are not members of any of Although methanol generally is viewed as a substrate the known methylotrophic species such as Methylo for the production of single cell protein, the factors that phi/us methylotrophus. For purposes of taxonomic iden qualify it for SCP manufacture also recommend metha ti?cation herein, the name Methylophilus viscogenes is assigned to the.. new facultative methylotroph species nol as a potential feedstock for the production of accu which includes bacterial strains having the identifying mulated extracellular metabolites such as exopolysac characteristics of strain ATCC 39893. charides. A number of microbial processes for the con The term “methylotroph” as empolyed herein refers version of methanol to value-added products have been to a microorgansim which is capable of growing non described but generally these are low-volume/high autotrophically on carbon compounds having one or priced compounds such as aminoacids. Thus, J. Bolbot more carbon atoms but no carbon-carbon bonds. “Au and C. Anthony in Proc. Soc. Gen. Microbial, 5, totrophic" refers to growth on a carbon dioxide sub 43(1978) found that a pyruvate dehydrogenase lacking strate. mutant of Pseudomonas AMI could accumulate the The term “facultative methylotroph" as employed aminoacids alanine and valine during growth on metha herein refers to a methylotroph which is capable of nol Y. Tani et al in Agric. Biol Chem, 42, 2275( 1978) growth on one or more heterotrophic substrates, e.g., have described the production of up to 5.2 grams per fructose. liter of L-serine from methanol employing an Arthrobac The term “Cl-compounds” as employed herein refers ter globlformi's strain. Up to the present time there has 60 to organic compounds which do not contain any car not been any report of methanol bioconversion to a bon-carbon bonds, such as methanol, formaldehyde, commodity type of bulk chemical. formate, forrnamide, carbon monoxide, dimethyl ether, Accordingly, it is an object of this invention to pro methylamine, dimethylamine, trimethylamine and tri vide a fermentation process for the bioconversion of a methylamine N-oxide. Cl-compound to an accumulated quantity of extracellu 65 The term “exopolysaccharide” as employed herein lar metabolite. refers to a polysaccharide which accumulates as an It is another object of this invention to provide a extracellular metabolite in a fermentation medium, as rapid growth culture medium for bio-oxidation of a exempli?ed by xanthan gum. 5,071,976 3 4 The term “heteropolysaccharide“ as employed facultative methylotroph which utilizes the ribulose herein refers to a polysaccharide which is composed of monophosphate pathway of C1 assimilation, and which at least two different kinds of monosaccharidic units, typically has a growth rate doubling time of 1-3 hours such as mannose and galactose. at 35°~40° C. The term “ribulose monophosphate pathway“ Strain ATCC 39893 grows on methanol, fructose and (RMP) as employed herein refers to the biochemical glucose. In addition, it will grow on a wider variety of cycle in which three molecules of formaldehyde are heterotropic substrates (e.g., succinate and pyruvate) condensed to produce either one molecule of pyruvate when an exogenous energy supply in the form of for or one molecule of dihydroxyacetone phosphate. mate or methanol is present. This is a property not Biochemical literature relating to elucidation of the previously described for any known microorganism. ribulose monophosphate pathway and its variations For purposes of identi?cation, bacteria manifesting this include Biochem. J., 144, 465(1974) by J. Strom et al; phenomenon are herein termed “latent facultative me Sci. Prog., 62, 167 (1975) by C. Anthony; and Biochem. thylotrophs”. J., 148, 513(1975) by Colby et al. Bacteria having the characteristics comprising those The ribulose monophosphate pathway involves en of strain ATCC 39893 are further identi?ed by non zymes which include G-phosphogluconate dehydrase/— slimy growth of pale orange colonies on solid media. phospho-Z-keto-3-digluconate aldolase; fructose di Another identifying characteristic of a strain ATCC phosphate aldolase; glucose-é-phosphate dehydro 39893 type of methylotrophic bacterium is a hexulose genase; 3-hexulose phosphate synthase; phosphofructo phosphate synthase/hexulose phosphate isomerase ac kinase; phosphoglucoisomerase; phospho-3-hexulose tivity (J. P. Van Dijken et al; FEMS. Microbiol. Lett., isomerase; phosphoriboisomerase; ribulose-S-phosphate 4, 97, 1978) of at least about 400 nanomoles of NADH 3-epimerase; transaldolasei transketolase; sedoheptulose formed per minute per milligram of protein. diphosphate aldolase; and sedoheptulose-1,4-diphospha Another signi?cant property of a strain ATCC 39893 tase. type of facultative methylotroph is the ability to biocon The RMP cycle effectively condenses 3 molecules of 25 formaldehyde with 3 molecules of ribulose 5-phosphate vert a methanol substrate into an accumulated quantity to form 3 molecules of fructose 6-phosphate, which in of an exopolysaccharide which imparts pseudoplastic turn regenerates 3 molecules of ribulose S-phosphate via and thioxtropic properties to aqueous solutions. A strain the cycle with the overall production of one molecule ATCC 39893 bacterium is particularly unique in its of dihydroxyacetone phosphate or pyruvic acid. These ability to produce a large quantity of accumulated ex metabolites function as substrates for cellular biosynthe opolysaccharide in the cultivation medium, in the pres $15. ence or absence of cell growth and under a variety of The two key enzymes of the cycle are envisioned to culture conditions. A strain ATCC 39893 bacterium has be hexulose phosphate synthase and hexulose phosphate the inherent ability to divert a large percentage of avail isomerase, which are the enzymes responsible for con able carbon source to biosynthesis of an exopolysaccha densing formaldehyde and ribulose S-phosphate and ride. converting the product to fructose 6-phosphate. These A further chacteristic ofa strain ATCC 39893 type of are two recognized variants of the RMP cycle, i.e., the bacteria is a non-logarithmic pattern of growth, as op fructose bisphosphate variant which gives rise to dihy posed to an exponential growth phase. A strain ATCC droxyacetone phosphate, and the Entner-Doudoroff 39893 culture exhibits a natural metabolic dysfunction variant which produces pyruvic acid. which prevents unrestricted logarithmic growth. When the fermentation medium is supplemented with a vari Species Methylophilus viscogenes ety of growth factors such as vitamins, aminoacids, or A series of isolations were conducted on samples of yeast extract, the non-logarithmic growth is not af soil and water which originated in the operating vicin 45 fected. ity of a methanol manufacturing plant in Bishop, Texas. A Methylophilus viscogenes strain can be cultured aer The samples were screened for the presence of me obically in a nutrient medium comprising a carbon thanol-utilizing microorganisms. source, nitrogen source, salts, and various growth pro A portion of each sample was inoculated into 250 ml moters. Erlenmeyer ?asks containing methanol (0.5% v/v) and Suitable nitrogen sources include ammonium sulfate, an appropriate nutrient medium, e.g., the mineral ammonium chloride, ammonia, diammonium phos salts(MS) medium illustrated in Table I, plus one gram phate, ammonium nitrate, sodium nitrate, urea, corn per liter of ammonium chloride. The flasks were incu steep liquor, casein, peptone yeast extract, meat extract, bated at various temperatures ranging from 30° C. to and the like. 55° C. After detectable turbidity indicative of growth Suitable mineral salts include calcium salts, magne occurred, 2 ml samples were removed and employed to sium salts, potassium salts, phosphate salts, iron salts, inoculate similar sterile ?asks of medium. A number of manganese salts, zinc salts. copper salts, and the like. subsequent subcultures were taken for each original Bacterial growth promoters include soy bean protein sample, then the cultures were streaked out on medium hydrolysate yeast extract, vitamins, and aminoacids. agar plates to obtain single microorganism-derived col~ 60 Cultivation of the microorganism in the nutrient me onies from which pure cultures of methanol-assimilat dium typically is conducted aerobically at a tempera~ ing microorganisms were obtained. Strain AICC 39893 ture of 35°40" C. and a pH between about 6.7-7.1 by was isolated and obtained as a biologically pure culture means of shaken or submerged cultivation. in this manner. Strains of Methylophilus viscogenes exhibit a novel Exopolysaccharide Production combination of properties that distinguish them from Employing the cultivation conditions previously de other methylotrophic bacteria. A Methylophilus visco scribed, a present invention strain of Methylophilus visco genes bacterium such as strain ATCC 39893 is a type 1 genes produces and accumulates an exopolysaccharide 5,071,976 5 6 in a high concentration and with a high rate of carbon xanthan gum and tamarind gum, and polygalactoman source utilization. nan gums such as guar gum and locust bean gum. Strain ATCC 39893 is capable of producing the ex A synergistic enhancement of viscosity thickening opolysaccharide in an amount up to about 10 grams per effect in aqueous solutions is observed when hetero liter, based on the culture medium, when methanol is polysaccharide Poly 54 is utilized in combination with used as the growth carbon source. The yield of ex guar gum, locust bean gum, tara gum, starch or carbox opolysaccharide typically will vary in the range be ymethylcellulose. tween about 30-60 percent, based on the quantity of Other physicochemical and structural features of methanol utilized. heteropolysaccharide Poly 54 are as follows: Accordingly, in another embodiment this invention 10 provides a process for producing a heteropolysaccha ride which comprises aerobically cultivating a strain of A. Constituent Sugars (molar ratio) Methylophilus viscogenes in a nutrient medium contain glucose 10 ing a growth carbon source (e.g., a cl-compound or galactose 7-10 mannose 1—3 fructose) to yield the heteropolysaccharide as an accu uronic acid 1-3 mulated extracellular product. In a further embodiment, this invention provides a process for producing a heteropolysaccharide which Poly 54 contains between about 1-3 weight percent comprises aerobically cultivating Methylophilus visco of nitrogen. Poly 54 has no pyruvate or protein content, genes strain ATCC 39893 or a mutant thereof in a nutri and it contains about one acetyl group per four mono ent medium containing methanol as a growth carbon saccharide units. The uronic acid constituent comprises source to yield heteropolysaccharid Poly 54 as an accu glucuronic acid and/or galacturonic acid and/or man mulated extracellular product, and optionally recover nuronic acid. ing the said product from the culture medium. . . 7 After the completlon of a fermentation run, the whole Bi Elemental Analysis (%)' cells are removed from the broth by conventional C 39.2 means such as centrifugation, ultra?ltration or heat H 6.1 sterilization. The heteropolysaccharide is recovered O 43.1 from the supernatant by any convenient procedure such N 2.17 as freeze-drying, or precipitation with a water-soluble ‘Average values: ash corrected. organic solvent, e.g., acetone, methanol, ethanol, and the like. Precipitation of the heteropolysaccharide can C. Melting Point also be effected by treatment of the solution with a calcium salt. " No clear melting point. and decomposes above a 35 The crude heteropolysaccharide can be redissolved temperature of about 150° C. in water, and the aqueous solution then subjected to dialysis and lyophilization to provide a purified prod D. Infrared Spectrum uct. Band at 1740 cm—l which may indicate aliphatic ester The novel heteropolysaccharide Poly 54 product has groups. an average molecular weight in the range between Bands at 1650 cm-1 and 1540 cm—I which may indi about 800,000 and 1,500,000. ' cate amide groups. Heteropolysaccharide Poly 54 exhibits a 0.5% viscos- “ ity of 2100 cps at 12 RPM, as measured with Brook?eld E. Ultraviolet Absorption Spectrum RVT viscometer Spindle No. 4 at 25° C. An aqueous 45 No detectable peaks in ultraviolet range of wave solution of heteropolysaccharide Poly 54 is colorless. lengths transparent and exhibits pseudoplastic and thixotropic viscosity properties. The aqueous solution viscosity F. Solubility Properties properties are stable at temperatures up to about 130° Soluble in water, but insoluble in all common organic C., and at pH values up to about 12. ~ 50 solvents. Under low shear rate conditions, heteropolysaccha ride Poly 54 exhibits an apparent viscosity which typi G. Speci?c Rotation cally is about 5-30 times that of commercial xanthan No measurable speci?c rotation. gum. The comparative viscosity properties of hetero The present invention heteropolysaccharides differ in polysaccharide Poly 54 and commerical xanthan gum 55 composition and properties from the polysaccharides (e.g., Kelzan) under shear conditions are illustrated in described in US Pat. No. 4,514,563. A polysaccharide FIG. 1. of US. Pat. No. 4,514,563 is composed mainly of (a) The pseudoplastic properties of an aqueous solution glucose, (b) galactose, (c) mannose, and (d) glucuronic of heteropolysaccharide Poly 54 are illustrated in FIG. acid. The molar ratio of (a):(b):(c):(d) is 10:3—6:0.5—2:0 2, which is a flow curve of 1%(W/V) Poly 54. The .5-2. difference between the up curve and the down curve in A typical polysaccharide of US. Pat. No. 4,514,563 is FIG. 2 demonstrates that the polymer solution has produced by an acetic acid bacteria such as Acetobacter thixotropic properties. l-leteropolysaccharide Poly 54 polysaccharogenes MT-ll-Z or MF~8. The polysaccha has excellent properties for application as a thickening ride does not contain acetyl or pyruvic acid constitu agent in aqueous solutions. Heteropolysaccharide Poly ents, and it does not contain any nitrogen. The viscosity 54 can be employed alone or in combination with one or of a 1% aqueous solution is 500—1200 centipoises as more other water-soluble polysaccharides such as hy determined by a Brook?eld type viscometer at 25° C. drophilic gums. Illustrative of hydrophilic gums are and a spindle rate of 30 revolutions per minute. 5,071,976 7 8 The following examples are further illustrative of the trap/111s viscogenes strain cultivation run. The broth is present invention. The components and speci?c ingredi removed from the fermentor, and whole cells are re ents are presented as being typical. and various modifi moved by centrifugation (13,000 xg for 10 minutes). cations can be derived in view of the foregoing disclo The exopolysaccharide usually is isolated by isopropa sure within the scope of the invention. nol precipitation from solution. For growth of methanol-assimilating bacteria a min Rheological measurements on aqueous solutions of eral salts medium(MS) (Table I) is employed. The me exopolysaccharides are made on a Wells/Brook?eld dium is either supplemented with 1 g/L potassium ni cone/plate digital viscometer or a Rheomat 30 viscome trate, giving nitrate mineral salts medium(NMS) or 1 ter ?tted with a coaxial cylinder sensor system. g/ L ammonium chloride, giving ammonium mineral The xanthan gum employed for comparative viscos salts medium (AMS). The carbon source, methanol, ity measurements is Kelzan, which is a commercial preferably is present at a concentration between product sold by Kelco. 0.2—O.5%(v/v). Analysis of the monosaccharide content of an ex For solid media, 17 g/ L of Difco bacto-agar is added opolysaccharide is accomplished by a hydrolysis-gas to the basic mineral salts medium (minus phosphates) liquid chromatography method. The method involves prior to sterilization. Sterile phosphate solution is added hydrolyzing the exopolysaccharide in aqueous tri?uo aseptically to the sterile mineral salts medium on cool roacetic acid solution, then reducing with sodium boro lng. hydride and acetylating with acetic anhydride. The Fermentation procedures are conducted in a New polyacetate derivatives of the monosaccharides are then Brunswick Microferm (14 liters). A 10 liter working analyzed by gas chromatography in comparison to stan volume is used, and methanol is added aseptically after _ dards. sterilization of the fermentor. A stirring rate of 200 rpm is routinely used with an air delivery rate of two liters EXAMPLE tn- 1. The fermentor is equipped with pH and dissolved This Example illustrates the culturing of a Methylo oxygen control. 25 phi/is viscogenes strain to form an accumulated quantity Hexulose phosphate synthase/hexulose phosphate of exopolysaccharide metabolite. isomerase are assayed simultaneously, since there is no A 500 ml inoculum of strain ATCC 39893 is grown assay for hexulose (“a-phosphate which is the product of employing MS medium, one gram per liter of ammo hexulose phosphate synthase activity. Hexulose phos nium chloride, and 0.5% (v/v) methanol as a growth phate isomerase converts hexulose é-phosphate to glu 30 carbon source. The ?ask is incubated at 37° C. for two cose 6-phosphate which can be estimated using glucose days. A 14 liter New Brunswick Microferm fermentor 6-phosphate dehydrogenase and following the concom containing 10 liters of MS medium, one gram per liter of itant NADP+ reduction at 340 nm. The enzyme assay ammonium chloride,.and 0.5% (v/v) methanol is inocu solution contains (?nal conc.): phosphate buffer 50 mM, lated with the above culture. pH 7.2; magnesium chloride 2.5 mM; glucose 6-phos 35 Initial fermentation conditions are as follows: 37° C., phate dehydrogenase 0.7 units; phosphoglucoisomerase agitation 200 rpm, air flow 2 liters/minute. and pH 7.0. 0.75 units; phosphoribose isomerase 1.75 units; ribose The pH is controlled at about 7.0 during the fermenta 5-phosphate 5 mM; NADP+0.25 mM; and formalde tion. Methanol is added intermittenly to provide a con hyde 5 mM. centration of 0.5% (v/v) whenever the fermentation Any presence of hydroxypyruvate reductase is indic 40 medium becomes carbon exhausted. The concentration ative of the serine pathway of formaldehyde assimila of methanol is determined by gas-liquid chromatogra tion in the microorganism. This is determined by an phy. After 24-36 hours of fermentation, the agitation enzyme assay solution which contains (?nal conc.): rate is increased to 400 rpm. potassium phosphate 1 M, pH 6.3; lithium hydroxy The fermentation is terminated when methanol is no pyruvate 0.01 M; and NADH 2 mM. NADH disappear 45 longer being utilized by the culture (usually 5-7 days) ance is measured at 340 nm. The highly viscous fermentation broth is then diluted The estimation of glycerol and dihydroxyacetone is with isopropanol (2:1 alcoholzbroth) to facilitate precip accomplished with glycerol dehydrogenase/dihydrox itation of the exopolysaccharide. The resulting precipi yacetone reductase (glycerolzNAD+2-oxidoreductase, tate is ?ltered and then washed with 100% isopropanol, EC 1.1.1.6). The enzyme is derivated from Enterobacler and then with 70% isopropanol. The precipitated ex aerogenes, and is available from Sigma Biochemicals. opolysaccharide is then dried at 55° C. in a forced air For the estimation of glycerol, the reaction mixture oven. The dried exopolysaccharide subsequently is (1.0 ml) contains 50 umole TRlS-HCl buffer (Sigma ground to a powder in a Wiley Mill. The physicochemi Biochemicals), pH 9.7; 0.2 nmole NAD+§ 1 unit en cal properties of the exopolysaccharide product corre zyme; and 15 umole glycerol (or test solution/fermenta spond to those described hereinabove for heteropoly tion broth, 20-50 11.1). Assays are initiated by addition of saccharide Poly 54. substrate and followed by monitoring increasing absor In an alternative process embodiment, heteropoly bance at 340 nm in a Beckman Model 25 UV spectro saccharide Poly 54 is recovered from a fermentation photometer. broth and purified in accordance with the following For the estimation of dihydroxyacetone, the reaction 60 procedure. mixture (1.0 ml) contains 50 umole phosphate buffer, At the termination of the fermentation. the fermenta pH 6.0; 0.5 umole NADH; 1 unit enzyme; 15 umole tion broth is diluted 1:1 with deionized water and centri dihydroxyacetone (or test solution/fermentation broth, fuged at 10,000 revolutions per minute (rpm) for 20 20—50 [.LI‘l'll). Assays are initiated by addition of substrate minutes to separate microbial cells and solid materials. and followed by monitoring decreasing absorbance at 65 The broth then is dialyzed with deionized water for 72 340 nm. hours with frequent changes of the water volume. The exopolysaccharide (e. g., heteropolysaccharide The addition of 2 volumes of isopropanol to the broth Poly 54) is recovered after the completion of a Methylo yields heteropolysaccharicle Poly 54 as a white fibrous 5,071,976 10 precipitate. The precipitate is collected, washed with What is claimed is: isopropanol, and dried in a vacuum oven. The white 1. A heteropolysaccharide free of protein and con precipitate is redissolved in deionized water and the taining (i) nitrogen in an amount of from about 1 to solution is centrifuged as described above. The product about 3 weight percent. (ii) as constituent monosaccha is precipitated with isopropanol and then ?ltered and rides, glucose, galactose, mannose, and at least one dried to produce puri?ed heteropolysaccharide Poly uronic acid selected from the group consisting of glucu 54. ronic acid, galaturonic acid, and mannuronic acid, the TABLE I molar ratio of said constituent monosaccharides to one another being: MS MEDIUM MgSO4.7HZO I g CaClz 0.2 g glucose 10 Na2HPO4 0.33 g galactose 7 to 10 KHZPO4 0.26 g mannose l to 3 FeEDTA 5.0 mg uronic acid 1 to 3. Na2MoO4.2I-l2O 2.0 mg CuCl2.2H1O 1.0 mg FeSO4.7H2O 500 pg and (iii) approximately one acetyl group for every four ZnSO4.7H2O 400 pg monosaccharides. MnCIZAHZO 20 pg 2. A thickening composition comprising a blend of a P131304 15 pg CoClg.6l-lzO 50 pg heteropolysaccharide according to claim 1 and at least 20 NiClz?HzO 10 pg one water soluble polysaccharide. EDTA 250 pg 3. An aqueous solution of a composition according to H20 1 liter pH 6.8 claim 2. i * $ * $

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