Note: Descriptions are shown in the official language in which they were submitted.
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
--2--
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
--3--
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
21521
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~
--4--
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)
NRRL B-1951
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*
_
--6--
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
R2
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
9--
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.
-12-
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~
-14-
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
,.
-16-
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.
