CA1122134A - Amine salts of acidic microbial polysaccharides and their use in recovery of such polysaccharides - Google Patents

Abstract

Abstract of the Disclosure:
Acidic polysaccharides of the type obtainable by microbial fermentation of an organic material such as a carbohydrate (e.g., xanthan gwn) form novel amine salts with aliphatic or alicyclic polyamines having at least three amino nitrogen atoms and a molecular weight of at least 150.
The amine salts may be used for isolation of the microbial polysaccharide from its fermentation broth by a process comprising the steps of acidifying, forming the amine salt by adding the amine or a salt thereof, and reducing the inorganic salt concentration as necessary (e.g., by dilu-tion).

Description

Ll--l675--Ca AMINE SALTS OF ACIDIC MICROBIAL POLYSACCHARIDES
AND THEIR USE IN RECOVERY OF SUCH POLYSACCHARIDES

This invent:ion relates to new compositions oE matter and a method for their use. In particular, the invention comprises methanol-insoluble amine salts of acidic polysaccharides produced by microbial fermentation wherein the amine is at least one aliphatic or aliicyclic polyamine containing at least three amino nitrogen atoms.

The microbial production of polysaccharides by ermen-tation of organic compounds (especially carbohydrates suchas sugar and starch) in the pxesence of a suitable micro-organism is a well known procedure. The products (herein- :
after sometimes called "microbial polysaccharides"), typically acidic water-soluble gums, are useful in many applications including the preparation of foodstuffs and cosmetics and in secondary and tertiary oil recovery.

Several methods have been known for the isolation of the microbial polysaccharide from the Eermentat.ion broth.
': One requires dilution of the broth wi-th a large amount of a water-miscible solvent such as 2-propanol, whereupon the polysaccharide precipitates and may be removed ky filtration or an equivalent procedure. Others are the precipita,ion of an insolu~le calcium salt of the polysaccharide, followed by acidification, and preclpitation by the use of a long chain amine or quaternary ammonium salt. All of these methods are either cumbersome (e.g., because of the large amount of diluting solvent re~uired), eY.pensive (e.g., because of the relatively high cost of lime, quaternary ammonium salts, 10 etc.~, or both.
A principal object of the present invention, therefore, is to provide new compositions of matter com-prising amine salts of acidic microbial polysaccharides.
A further object is to provide a novel method for 15 recovering acidic microbial polysaccharides from the fer-mentation broth in which they are produced.
A further object is to provide a recovery method which requires the use of a minimum of processing steps and expensive chemicals.
A still further object is to provide a method for recovering acidic microbial polysaccharides in usable form by a relatively convenient, easily employed procedure.
Other objects will in part be obvious and will in part appear hereinater.
Microbial polysaccharides and the methods fox their production are well known, having been described in a larye number cf publications and patents. In general, they involve the cultivation of a suitable microorganism in an aqueous fermentation medium containing a carbohydrate, 30 normally under neutral or near-neutral conditions. The microoryanisms, many of which are disclosed in U.S. Patent 3,406,114, from the following list which produce acidic products are illustrative of those suitable for this pur-pose, with those maxked with an asterisk being illustrative of those especially suitableO
Algae:
Chlorella vulgaris Chlorella pyrenoidosa Chlorococcum 5p.
Bacteria:
Alcaligenes faecalis ATCC 212 Arthrobacter viscosus NRRL B-1973*, B-1797 Arthrobacter globiforme* NRCC
Arthrobacter stabilis NRRL B-3225 Azotobacter indicum* (Beijerinckia indicum) _ Azotobacter vinelandii Bacillus ethanicus Bacillus polymyxa Bacillus subtilis NRCC 2035 Bacterium ali~haticum liquefaciens Bacterium hedium Bacterium oligocarbophilus Beg~eotoa alba Chromobacterium violaceum subsp. mucilaginosus ATCC

Corynebacterium fascians Corynebacterium fiaccumfaciens*
Cor nebacterium insidiosum 110 Starr _Y
Corynebacterium michiganense Cory~ a terlum rathayii Corynebacterium sepedonicum Corynebacterium tritici -Klebsiella aero~enes Methanomonas methanica Pseudomonas methanlca*
Rh.izoblum le~uminosarum Sphaerotilus natans __.

L~

Streptomyces sp.
Thixotrix nivea Xanthomonas campestris* NRRL B-1459 Xanthomonas carotae NRCC 105a7 Xanthomonas hederae Xanthomonas ~ hil NRCC 12612 Xanthomonas maculo~olii~ardeniae NRCC 10201 Xanthomonas malvaccarum NRCC 12131 Xanthomonas oryzae Xanthomonas papavericola Xanthomonas phaseoli NRCC 11766 Xan-thomonas pruni Xan~homonas stewartil Xanthomonas translucens NRCC 10772 Xanthomonas vesicatoria Xan~homonas vi~icola NRCC 11648 Zoo loca ramigera Fungi:
Aspergillus fischerii Asp-rgillus niger -~spergillus parasitious QM 884 Aspergillus sulphureous Aspergillus sydowi Candida heveanensis NRRL Y-1510 Claver~a cinerea Coccidioides_immi~is Coprinus atramentericus Cryptococcus albidus NRRL Y-1516, Y-1400 C~ptococcus diffluens* NRRL Y-1505, Y-1517 Cxyptococcus flavescens NRRL Y-laOl Cry~tococcus laurentii var. fla~escens Cryptoco-ccus luteolus NRRL Y-986 Cylindrocorpon radiciola Dacrymyces palmatus Dictyostelium discoldeum Fumago vagans Fusarium aquiductum tlo~ pH) Fusar.ium lini Fusarium moniliforme (Gibberel a ~ujikuroi) Fusarium solani*
Hansenula capsulata NRRL Y-1842 Hansenula holstii NRRL ~-2~48, Y-2154 Leptomitus lacteus L'pomyces lipofera NRRL Y-1351 Mucor racemosus*
-Penicillium brevi-compactum Penicillium capreolinum NRRL Y-1510 Penicillium charlesii Penicillium digitatum Penicillium expansum*
Penicillium javanicum Penicillium luteum Penicillium nigricans Penicillium rugulosum Penicillium schlerotiorium Penicillium varians .
Penicillium vinaceurn Pestalotia ramulosa _ymototrichum omnivorum Rhodotorula mucilaginosa*
_ Schizophyllum commune*
Selenaspora sp.
Stysanus stenionites Tremella braziliensis*
Tremella encephala Tremella ~oliaceae Tremella mesenterica*
_ Tremella subanomala Tricholoma personatum Trichoderma viric1e*
Tephrina sp. NRRL YB- 3638 Torulopsis rotundata NRRL Y-1510 Unidentified black yeast NRRL Y-6272 Protozoa: Carchesium sp.
For the purpose or this invention, the most suit-able polysaccharides are those produced by carkohydrate 10 fermentation in the presence o the Xanthomonas campestris bacteria, especially Xanthomonas campestris NRRL B-1459.
.. . ... ~
The acidic polysaccharide product produced by the last-named bacterium is commonly known as "xanthan gum."
As previously noted, the amines from which the 15 amine salts o~ this invention are prepared are aliphatic or alicyclic polyamines having a molecular weiyht of at least 150 and containing at least three amino (i.e., basic) ni-trogen atoms. Many amines of this type are known, including the following:
Alkylene polyamines, including the ekhylene, propylene, butylene and pentylene po'lyamines. Specific examples include di(heptamethylene)triamine, tripropylene tetramine, tetraethylene pentamine and pentaethylene hexa-mine, as well as similar compounds in which the various 25 alkylene groups are of differing chain lengths.
Aminoalkyl~substituted imidazolines and piper-azines related to the above-descrihed polyamines, such as 2-heptyl-1-(2-aminopropyl)imidazoline and 2-methyl-1-~2-aminobutyl) pipexazine.
Polyamines prepared hy cyanoethylation of such materials as ammonia, alkylene polyamines, alk.anolamines, aliphatic or alicyclic ketones, polyhydric alcohols, or heterocyclic amines (e.g., morpholine, piperidine, piper-azine) followed by reduction ~e.y., hydrogenation) of the 35 cyano groups.

f~

Coupling products prepared by reaction o-E form-aldehyde or a formaldehyde-producing substance (e.g~, para-formaldehyde, trioxane) with any of the foregoing.
Homologs of the foregoing in which o~e or more hydrogen atoms bound to amino nitrogen are replaced by methyl groups.
An especially useful class of polyamines, par-tially by reason of thei.r ready availability, are those having the formula:
(R2)2N-(Rl-N)n-RIN(R2)2 in which n is an integer which is at least 1 and usually no higher than about 10, each Rl is independently a divalent aliphatic or alicyclic radical (usually an alkylene radical) ha~ing from ~ to about 18 and preferably from about 2 to 15 about 6 carbon atoms, and each R2 is independently hydrogen or lower alkyl (i.e., alkyl having at most 7 carbon atoms) and is usually hydrogen or methyl. Alkylene polyamines in which all Rl radicals are identical and each R2 is hydrogen are particularly preferred; and the ethylene polyamines, 20 examples of which are mentioned above, are especially de-sirable for reasons of cost and effectiveness. Ethylene polyamines are described in detail under the heading "Di-amines and Higher Amines" in Kirk-Othmer, Encyclopedia of Chemical Technolo~y, Second Edition, Vol. 7, pp. 22-39.
25 They are prepared most conveniently by the reaction oE
ethylene chloride or an ethylene imine with ammonia. ~hese reactions result in the production of complex mixtures of alkylene polyamines, including cyclic condensation products such as piperazines; and the mixtures are particularly 30 useful for the preparation of the amine salts of this in-vent.ion.
The minimum amine molecular weight of 150 is im~
portant, since amines with lower molecular weights are inoperative or, at ~est, marginally operative in the method for precipitating the microbial polysaccharide described hereinafter. ~owever, it is within the scope of the in-vention to use commercially available amine mi~tures con-sisting predominantly of amines (e.g., ethylene polyamines)with molecular weights higher than 150 but also containing lower molecular weight amines. The latter are not equi-valent to the former and are not included within the scope of the word "amine" or other terms used herein to refer to 10 specific amine subgenera and species with reference to the invention, but their presence is not detrimental.
~ he amine salts of this invention are insoluble in methanol, and this property is often crucial with respect to the utility of the arnine salts in the recovery of microbial 15 polysaccharides as described hereinafter. In this respect the amine salts of the present invention differ from the materials ohtained by reacting microbial polysaccharides such as xanthan gum with long chain amines or quaternary ammonium salts, since the latter are soluble in methanol.
Another aspect of the present invention is a novel method for recovering acidic microbial polysaccharides from an aqueous mixture comprising a fermentation broth which comprises the steps, performed in any order, of:
(A) Adjusting the mixture to a ~inal p~I within 25 the range of about 2.5-5.5;
(B) Adding to the mixture at least one amine as described hereinabove, or a salt of said amine, whereby an amine salt according to this invention is ultimately formed;
and (C) Reducing the inorganic salt concentration of the mixture a~ necessary to effect precipitation of said amine salt according to this invention therefrom~
This aspect is based on the aiscovery that acidic microbial polysaccharides, especially xanthan gum, may be precipitated as their amine salts by carrying o~lt a series of steps more fully de~cribed herelnafter. The material subject to these steps is a fermentation broth containing xanthan gum or a similar acidic microbial polysaccharide, the microbe which has produced the polysaccharide, and other materials including various inorganic ions such as phos-phate, nitrate, potassium and magnesium. The fermentation reaction is typically initiated by means of a seed culture which may contain an enzymatic protein material such as soy 10 peptone. The pH of the fermentation broth, as previously noted, is usually neutral or nearly so, typically 6.S-7.2.
Step A of the method of this invention is the re-duction of the pH of the mixture comprising the fermentation broth to a final value within the range of about 2.5-5.5.
15 (By "final value" is meant the value during or just prior to precipitation of the microbial polysaccharide; that is, after steps A, ~ and C as described herein have been com-pleted.) The preferred range is abou~ 2 8~4.2, and the optimum range is 3.7-3.8. The pH adjustment is normally 20 done by adding an acidic material to the mixture comprising the fermentation broth. The acid used may be an inorganic or an organic acid but is preferably inorganic; it may be monobasic or polybasic but is preferably monobasic. Within these classes, the identity of the acid is not critical 25 since the crucial ~act is reduction of the pHr not the identity of the acid used to effect the reduction. Typical suitable inorganic monobasic acids are hydrochloric and hydrobromic acids; suitable polybasic inorganic acids include sulfuric and phosphoric acids, with the latter being 30 preferred. Pmong organic acids, the carboxylic acids are preferred, especially monocarboxylic acids and particularly those with 1-10 carbon atoms such as formic, acetic~ pro-pionic, butyric, valeric and lactic acids, with lactic acid being especially suitable.

--10~

Step ~ is the addition to the mixture comprisiny the fermentation broth of at least one amine as descriked hereinabove, or a salt of said amine (preferably a salt of a mineral acid such as hydrochloric acid). Since the pH es-tablished in step A is in the acidic range, the amine willbe present as its salt with the acid used in step ~ until acid exchanye with the acidic microbial polysaccharide takes place to form the amine salt of this invention.
~he amount of amine used in step B will depend to 10 some extent on the pH established by step A and the amount of dilution or equivalent operation employed in step C. In general, it has been found that a minimum of about 0.08 part by weight of amine should be used per part of acidic miro-bial polysaccharide in the aqueous mixture. On the other 15 hand, it is rarely necessary to use more than about 0.5 part of amine, and most often not more than 0.2 part, per part of microbial polysaccharide, although higher amounts are not detrimental.
In step C, the inorganic salt concentration of the 20 mixture comprising the fermentation broth is reduced. The reduction may be accomplished by such methods as ion ex-change, but i~c is usually convenient merely to dilute the mixture with water, ahout 0.5-5.0 a:nd most often about 1.0-5.0 parts by weight of water generally being employed per 25 par~ of fermentation broth.
The presence of step C in the method of this invention is based on the discovery that, in many instances, the presence of inorganic salts in concentrations comparable to those in the fermentation broth increases the solubility 30 of the amine salt of the microbial polysaccharide. It ls frequently possi.ble, however, to adjust the parameters and conditions of steps ~ and B in order to make dilution (step C) unnecessary. For example, the xanthan ~um amine salts of this invention will precipitate without diluti.on or its --ll--equivalent if a monobasic acid (e.g., hydrochloric or lactic acid) is used for pE~ adjustment in step A and the amount of amine used in step B is at least about 0.14 part by weight per part of xanthan gum. Depending upon the conditions of steps A and B, therefore, step C may be optional. Even if it is unnecessary, howevex, it is frequently preferred for reasons explained hereinafter.
As previously noted, the order in which the steps of the method of this invention are carried out is im-10 material for the purposes of this invention; regardless ofthe order, precipitation of the amine salt will ultimately occur after all the steps described herein have been com-pleted~ Several factors may govern the order of steps. For example, the amine may be non-toxic to the microorganism 15 employed for fermentation, and in that event the amine may be used to maintain the neutral environment needed durin~
fermentation. (Use thereof perforce decreases the inorganic salt content of the mixture, thus providing conditions particularly conducive to precipitat:ion.) Thus, the ethyl 20 ene polyamines are non-toxic to many microorganlsms in-cluding Xanthomonas campestris NRRL B-1459, and if they are used for pH control during the fermentation and are present in the requisite amount when fermentation is completed, later addition of amine will be unnecessary and steps B and 25 C may immediately ~e performed to effect precipitation of the amine salt. On the other hand, some amines are toxic to the microorganism and if they are used, they must be added after fermentation is complete and pH control during fer-mentation must be effected by adding some other alkaline 30 reagent such as sodium hydroxide or potassium hydroxide.
When this is the case, steps A and B may be combined as ky adding an acidic solution of the amine salt. This may also be done when the amine has been used for pH control during the fermentation but additional amine must subsequently be 35 added.

Another factor affecting the order of steps is the desired physical state of the amine salt as precipitated.
If step C is the last step performed or if it is unnecessary because the conditions of steps A and B are adjusted in accordance with the description hereinabove, a fine pre-cipitate is formed which is relatively free from entrained cells of microorganism but which may be somewhat difficult to remove by filtration or the like. On the other hand, if step C precedes either or both of steps A and B the preci-10 pi~ate is fre~uently somewhat coarser and easier to filter,but may contain a substantial number of entrained micro-organisrn cells.
The nature of the acid used in step A may also affect the coarseness of the precipitate. ~onobasic acids 15 generally give a coarse, easily separated precipitate while polybasic acids yield a fine precipitate. For certain uses, such as secondary or tertiary oil recovery, the presence of microbial cells in the product is ~mdesirable and it will then be preferred to recover the microbial polysaccharide as 20 a fine precipitate. In other uses, the presence of micro-bial cells is not detrimental and t:he coarser, more easily isolated precipitate is satisfactory. In any event, it is usually found that precipitation is improved if the aqueous mixture is gently agitated, as by gentle stirring, during 25 the practice of the method of this invention.
Other factors which are frequently interrelated are the amount of amine used and the pH at which preci-pitation takes place. Thus, the use of relatively large amounts of amine in step B will result in precipitation at 30 somewhat higher pH values (as provided by step A~ than when lower amounts of amine are used. It has also been found that the use or a monocarboxylic acid such as lactic acid results in precipitation at somewhat higher pE values, frequenty as high as 5.4-5.5 when the amount of amine is relatively high. For lower amounts of amine, a pH value of 4.2 or less r..ay be necessary for precipitation.
The amine salts of this invention are easily clis-persed in neutral or slightly acidic aqueous solutions, yielding low viscosity disperslons which do not exhibit the agglomeration or "clumping" phenomenon usually encountered when xanthan gum and similar acidic rnicrobial polysaccha--rides are contacted with water. Upon addition to the aqueous dispersion of strong bases (e.g., sodium or potassium 10 hydroxide), salts (e.g., sodium chloride, potassium nitrate) or an aqueous formaldehyde solution, a thic}cened aqueous solution i9 obtained by the action of the freed xanthan gum or similar polysaccharide.
If recovery of the amine from the amine salt is 15 desired, the latter may be treated as a solid with a solu-tion of strong base in a non-solvent for the free acidic polysaccharide (e.g., a~ueous methanol), whereupon the amine is released in an interchange reaction.
The method of this invention is illustrated by the 20 following examples. All percentages are by weight.
Example 1 _ _ _ A seed culture is prepared from sterile solutions comprising 860 yrams of water, 19.4 grams of glucose, ~6 grams of an aqueous solution comprising 2~ dipotassium 25 hydrogen phosphate and 0.~5% ammonium nitrate, 86 grams of an aqueous solution comprising 0.1~ magnesium sulfate hepta-hydrate, and 2.6 grams of soy peptone. One hundred grams of the resulting solution is innoculated with a fresh culture of Xanthomonas campestris NRRL B-1459 and shaken in the dar]c 30 at 2~C. for 25 hours. A 70-gram portion of the resulting broth is comkined with the remainder of the aqueous solution and shaken at 29C. for 54-1/2 hours. The pH of the solu-tion ls periodically measured and adjusted to 6.8-7.2 by the ~2~

addition o a sterile 10~ aqueous solution of a commercial ethylene pol~amine mixture approximately correspondiny in molecular weight to pentaethylene hexamine (and referred to as such hereinafter); a total of 8 ml. of the pentaethylene hexamine solution is added. The resulting broth i5 used as a seed culture in later fermentations.

Exa~e 2 A sterile system comprising a resin flask, stir-ring means, liauid and gas addition means, temperature 10 measuring means and reflux condensing means is charged with sterile solutions comprising 270 grams of glucose, 12 grams of dipotassium hydrogen phosphate, 7O2 grams of ammonium nitrate, 1.2 grams of magnesium sulfate heptahydrate, 3.6 grams of soy peptone and 12 liters of water. To the mixture 15 is added 750 grams of the seed culture of Example 1, and the solution is purged with air and stirred at 28C. in the dark for about 49 1/2 hours. Periodic pH measurements are made and the pH is adjusted to 6.8-7.2 by the addition of a sterile 10~ aqueous solution o pentaethylene hexamine. The 20 glucose content of the solution is also checked periodically by means of Clinistix.
After 49-1/2 hours, the broth tests negative for glucose an~ 800 ml~ of a 2.6% aqueous phosphoric acid solu-tion is ac1ded to reduce the pH to 3.5. Water, 18 liters, is 25 added slowly followed by 50 grams of the pentaethylene hexamine solution. The total pentaethylene hexamine charged to the system by this time is 22.8 grams. The desired xanthan gum precipitates as a fine precipitate which is separated by centrifuying; the supernatant liquid is cloudy, 30 indicating the presence therein of substantial quantities of microhial cells. The xanthan gum amine salt is washed with a methanolic solution of sodium hydroxide and then with methanol, and is dried in a vacuum oven. The yield is 150 grams.

Example 3 A fermentation is carried out usiny the procedure of Example 2, except that 6 grams of soy peptone is used and the total weight of pentaethylene hexamine adcled during the fermentation is 15.9 grams. When the fermentation i9 com-plete, 11,200 ml. of water and A5 ml. o-~ 10~ aqueous penta-ethylene hexamine solution are added, followed hy a solution of 240 ml. of l M phosphoric acid in 800 ml. of water. The desired xanthan yum amine salt precipitates and the super-lO natant liquid is decanted. The product is washed withdilute phosphoric acid and slurried with methanol; to the slurry is added 22.8 grams of 50% aqueous sodium hydroxide solution. The xanthan gum is finally washeA with methanol again and dried in a vacuum oven. The yield is 157 grams.

15 Example 4 Following a procedure slmilar to that of Example

2, xanthan gum is prepared from 9.7 grams of glucose, d3 grams of an aqueous solution comprising 1% dipotassium hydrogen phosphate and 0.45% ammonium nitrate, 43 grams of a 20 0.1% aqueous solution of magnesium sulfate heptahydrate, 0.22 gram of sov peptone and 430 grams of water. pH adjust-ment during the fermentation is effected by the addition of 10% aqueous pentaethylene hexamine solution; a total of 12 ml. of such solution is added during the fermentation.
25 Following the fermentation, an additional 1.2 ml. of penta-ethylene hexamine solution is added and the pH is adjusted to 3.8 with 1 M hydrochloric acid. The desired xanthan gum amine salt precipitates and i5 separated; washed with water, dilute hydrochloric acid, a methanolic solution of sodium 30 hydroxide, and aqueous methanol; and vacuum dried. The yield is 5.1 grams.

Example 5 A xanthan gum amine salt is prepared by a method similar to that of Example 2, except that the pentaethylene ,.

hexamine is replaced by an approximately equivalent amount of tetraethylene pentamine.

Example 6 A xanthan ~um amine salt is prepared by a method similar to that of Example 2, except that the pentaethylene hexamine is replaced by an approxirnately equivalent amount of a commercial polyethylene polyamine mixture consisting principally of polyamines having a molecular weiyht between 150 and 275.

Claims (22)

LF-1675-Ca THE EMBODIMENTS OF THE INVENTION IN WHICH AN EXCLUSIVE
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:

1. A metahanol-insoluble amine salt of an acidic polysaccharide produced by microbial fermentation, wherein the amine is at least one aliphatic or alicyclic polyamine having a molecular weight of at least 150 and containing at least three amino nitrogen atoms.

2. An amine salt according to claim 1 wherein the amine is at least one alkylene polyamine of the formula wherein n is an integer which is at least 1, each R1 is independently a divalent hydrocarbon radical having about 2-18 carbon atoms, and each R2 is independently hydrogen or lower alkyl.

3, An amine salt according to claim 2 wherein the polysaccharide is xanthan gum.

4. An amine salt according to claim 3 wherein each Rl is the same and is an alkylene radical having 2-6 carbon atoms and each R2 is independently hydrogen or methyl.

5. An amine salt according to claim 4 wherein each R2 is hydrogen.

6. An amine salt according to claim 5 wherein the amine is at least one ethylene polyamine.

7. A method for recovering an acidic poly-saccharide produced by microbial fermentation from an aqueous mixture comprising a fermentation broth which comprises the steps, performed in any order, of:

(A) Adjusting the mixture to a final pH within the range of about 2.5 5.5;
(B) Adding to the mixture at least one aliphatic or alicyclic polyamine having a molecular weight of at least 150 and containing at least three amino nitrogen atoms, or a salt of said amine, whereby an amine salt according to claims l, 4, 5 or 6 is ultimately formed;
and (C) Reducing the inorganic salt concentration of the mixture as necessary to effect precipitation of the amine salt of said acidic polysaccharide therefrom.

8. A method according to claim 7 wherein step A comprises adding an acid to the mixture.

9. A method according to claim 8 wherein the amount of acid added is such as to attain a final pH of 2.8-4.2.

10. A method according to claim 9 wherein the acid is a monobasic inorganic acid.

11. A method according to claim 10 wherein the acid is hydrochloric acid.

12. A method according to claim 11 wherein the acid is a monocarboxylic acid.

13. A method according to claim 12 wherein the acid is lactic acid.

14. A method according to claim 9 wherein step C 15 effected by diluting the mixture with water.

15. A method according to claim 14 wherein the amount of water used for dilution is about 0.1-5.0 parts by weight per part of broth.

16. A method according to claim 15 wherein the acid used in step A is a monobasic inorganic acid.

17. A method according to claim 16 wherein the acid is hydrochloric acid.

18. A method according to claim 15 wherein the acid is a monocarboxylic acid.

19. A method according to claim 16 wherein the acid is lactic acid.

20. A method according to claim 15 wherein the acid used in step A is a polybasic inorganic acid.

21. A method according to claim 9 wherein the final PH after step A is 3.7-3.8.

22. A method for recovering xanthan gum from an aqueous mixture comprising a fermentation broth containing Xanthomonas campestris NRRL B-1459 which comprises the steps, performed in any order, of (A) adding hydrochloric acid to the mixture in an amount to attain a final pH of 3.7-3.8; (B) adding a commercially available amine mixture consisting predominantly of ethylene polyamines having a molecular weight of at least 150, or a salt of asid amine mixture, whereby an amine salt of said xanthan gum is ultimately formed; (C) diluting with about 0.5-5.0 parts by weight of water per part of broth, and recovering the precipitated product.