Note: Descriptions are shown in the official language in which they were submitted.
~15~
PRODUCTION OF XANTHAN GUM FROM A
CHEMICALLY DEFINED MEDIUM
INTRODUCTION
The process of the present invention relates to
a method for the continuous production of high molecular
weight heteropolysaccharide polymers by the action of
15 bacteria of the genus Xanthomonas campestris on chemically
defined media containing certain organic supplements such
as vitamins with or without amino acids containing assim-
ilable nitrogen.
BACKGROUND OF THE INVENTION
Earlier work has indicated that heteropolysac-
charides produced by the action of Xanthomonas bacteria on
carbohydrate media have potential application as film
forming agents, as thickeners in edible products, cosmetic
preparations, pharmaceutical vehicles, oil field drilling
25 fluids, fracturing liquids, similar compositions, and as
emulsifying, stablizing, and sizing agents. Heteropoly-
saccharides, particularly, xanthan gum, have significant
potential as mobility control agents in micellar polymer
flooding. Xanthan gum has excellent viscosifying proper-
30 ties at low concentration, is resistant to shear degrada-
tion and exhibits only minimal losses in viscosity as a
function of temperature, pH, and ionic strength. For
these reasons, xanthan gum is an attractive alternative to
synthetic polyacrylamides for enhanced oil recovery opera-
35 tions.
Fermentation of the inoculated medium with Xan-
thomonas organisms for 36-72 hours under aerobic condi-
tions results in the formation of xanthan gum which is
~153
--2--
separated from other components of the medium by
precipitation with acetone or methanol in a known manner.
Because of time required to ferment each batch, the low
biopolymer content of the fermented medium and the proc-
5 essing required for the recovery and purification of theproduct, xanthan is relatively expensive.
Other investigators have produced xanthan heter-
opolysaccharide by means of single stage or multistage
"continuous" fermentation. In most instances, Xanthomonas
10 campestris was grown in a medium containing dried distil-
lers solubles (DDS) or other complex nutrient as a source
of nitrogen and growth factors. There has been no
instance of which we are aware, however, that xanthan was
produced by continuous fermentation with Xanthomonas cam-
15 pestris or any organism of the Xanthomonas genus using achemically defined medium containing glucose, ammonium
chloride, mineral salts J and an organic supplement con-
sisting of vitamins and/or amino acids.
It is well-known that continuous production of
20 xanthan can be hampered by a tendency of the culture (Xan-
thomonas campestris) to change or degenerate after a spe-
cific number of turnovers; i.e., the time required to com-
pletely replace one volume of broth in the fermentation
vessel, or the reciprocal of the dilution rate. Normally,
25 6 to 9 turnovers are the maximum that can be obtained
before degeneration of the culture occurs. Coincident
with degeneration, there is a decrease in xanthan vis-
cosity, a loss in volumetric productivity of xanthan,
i.e., grams of xanthan produced per liter of broth per
30 hour, and the appearance of variant strains that no longer
produce xanthan or else produce a poor quality of xanthan.
It has been demonstrated that this phenomenon occurs when
DDS is used as the complex nitrogen source whether in -the
whole form or as the water soluble extract.
The most pertinent publications of which we are
aware are as follows:
1. P. Rogovin, et al, 1970, "Continuous Fer-
mentation to Produce Xanthan Biopolymers: Laboratory
Investigation", Biotechnol. Bioeng., XII, pp. 75-83.
,
1~5
--3--
2. K. W. Silman, et al., 1972, "Continuous
Fermentation to Produce Xanthan Biopolymer: Effect of
Dilution Rate", Biotechnol. Bioeng., XIV, pp. 23-31.
3. P. Rogovin, U.S. 3,485,719, "Continuous
5 Production of Xanthan".
4. G. P. Lindblom, et al., U.S. 3,32~,262,
"Heteropolysaccharide Fermentation Process".
5. Netherlands Patent Application
No. 7,612,448, "Method for the Production of Bacterial
10 Polysaccharides".
6. "Production of Polysaccharides by Xantho-
monas campestris in Continuous Culture", FEMS Microbiology
Letters, 347-349 (1978) by I. W. Davidson.
7. Process for the Production of Xanthan Gum,
15 British Patent Application No. 2,008,138A (Tate and Lyle,
LTD).
SUMMARY OF THE INVENTION
In producing xanthan gum in accordance with our
invention, any species or strain of Xanthomonas organism
20 may be employed in continuous culture in a nutrient
medium, preferably under conditions of nutrient limited
growth, when the medium is chemically defined and contains
a defined organic supplement such as vitamins with or
without certain amino acids (which may also constitute a
25 defined nitrogen source) and in addition has present
essential inorganic salts, glucose and NH4Cl. Also,
citric acid is employed as a chelating agent to maintain
solubility of iron and other trace elements present in the
system.
The process of our invention can be either a
single-stage or two-stage continuous fermentation proce-
dure. Any conventional continuous stirred tank baffled
reactor can be employed with provision for aseptic opera-
tion, agitation, aeration, tempera-ture control, foam con-
35 trol, pH control, and measurement of dissolved oxygen ten-
sion. In carrying out our process, the medium is seeded
with an inoculum of culture grown in the same medium as
that used for the fermentation at an inoculum level of 5
1~53970
to 10% of the medium volume. The culture is grown in
batch mode for 24 to 48 hours until the desired cell
concentration is reached - usually 1.5 to 2.5 grams of
cells per liter. At that point, continuous flow of the
5 medium is started into the fermenter such that the dilu-
tion rate is about 75% or less of the specific growth rate
at which the organism is growing at that point. The
medium is diluted continuously in the fermentor with a
fermentable carbohydrate such as glucose. Continuous
10 recovery of a volume of culture broth equal to the volume
of medium introduced is also provided. Usually after two
culture turnovers, the dilution rate is adjusted as
desired. The process and microorganism may be regenerated
after the rate of Xanthan production has been reduced sub-
15 stantially by the addition of citric acid. The concentra-
tion of citric acid in the medium after the addition
should range from about 0.05 g/l to about 0.25 g/l. Pre-
ferably, the concentrate should be about 0.11 g/l.
20 Xanthan gum which is produced as a product of fermentation
can be used without further purification, or filtered to
remove cells, or can be precipitated with an alcohol such
as ethyl or isopropyl alcohols with or without initial
cell removal.
The following operating conditions may be
employed:
Dilution Rate: 0.01-0.14 hr.
Preferred Range 0.04-0.10 hr.
Temperature: 20-35C
Preferred Range 25-30C
pH: 5.5-8.0
Preferred Range 6.4-7.4
Air Rate: 0.2-2 vol/vol-min
Preferred Range 0.5-1 vol/vol-min
Agitation Rate: 200-1200 rpm
Preferred Range 500-800 rpm
11~3~0
The expression "chemically defined medium" as
used in the present description and claims is intended to
mean one that contains in known composition and proportion
essential mineral salts, trace elements, glucose or equi-
5 valent carbohydrate and defined supplemental organicgrowth factors; i.e., vitamins with or without appropriate
amino acids such as L-histidine, L-methionine, and L-tryp-
tophan. Other amino acids that may be included are glu-
tamic acid, tyrosine, threonine, aspartic acid, aspara-
10 gine, and arginine.
Representative species of the Xanthomonas genuswhich may be employed in carrying out our invention
include Xanthomonas carotae, Xanthomonas phaseoli, Xantho-
monas papavericola, Xanthomonas begoniae, Xanthomonas hed-
15 e _ , Xanthomonas translucens, Xanthomonas vasculorum,
Xanthomonas vesicatoria, Xanthomonas incanae, and Xantho-
monas malvacearum. Cultures of these organisms as well as
others of this genus may be obtained from the American
Type Culture Collection, Rockville, Maryland.
SPECIFIC EMBODIMENTS OF THE INVENTION
The process of our invention may be further
illustrated by reference to the following specific exam-
ples, the first of which demonstrates that when dried dis-
tiller solubles (a complex undefined nitrogen source) is
25 used in continuous culture, Xanthomonas campestris under-
goes culture degeneration after seven to nine culture
turnovers with subsequent loss of viscosity and xanthan
productivity.
EXAMPLE I
Xanthomonas campestris strain B-1459 originally
procured from the Northern Regional Research Center in
Peoria, Illinois, was maintained on YM agar slants at 4C
and transferred to fresh agar slants at bi-weekly inter-
vals.
For inoculum preparation, a loopful of culture
from a fresh slant culture (<3 days old) was incubated
into a 16 x 125 mm tube containing 7 ml of YM broth. The
culture was incubated at 28C, on a rotary shaker and
.
1~53~70
:150 rmp, at a 20 inclination for 18 hours. At this
point, the contents of the tube were transferred to 50 ml
of YM broth (Difco) in a 500 ml Erlenmeyer flask, which
was inoculated at 28C on a rotary shaker at 250 rpm for
5 18-24 hours. Next, the contents of the flask were trans-
ferred to 200 ml of YM broth contained in a 2000 ml Erlen-
meyer flask. This was incubated under the same conditions
as the 50 ml flask, for 18-24 hours.
This final volume was used to seed the fermenter
10 at a ratio of 5% of the total volume of the medium in the
fermenter. The culture was allowed to grow in a batch
mode for 24-48 hours. Initial environmental conditions
were as follows:
Agitation 300 rpm
Air Rate 0.5 vol/vol/min
pH 7.0
Temperature 28C
Dissolved Oxygen 90% saturation
After 15-18 hours, when viscosity and cell con-
20 centration increased to > 500 cp and > 1 gram/liter,respectively, the agitation was increased stepwise from
300 to 800 rpm and the air rate to 1 vol/vol/min.
When the cell concentration desired was reached
(usually 1.8-2.5 gram/liter), the fermenter was switched
25 to a continuous mode by pumping in fresh sterile medium at
a desired flow rate and removing product at the same rate
through an overflow level control device. The dilution
rate (flow rate divided by fermenter medium volwme) was
initially set to be about 75% of the maximum specific
30 growth rate of the culture. After two culture turnovers
(a culture turnover is the time required to completely
replace one volume of medium), the dilution rate was set
to that desired in the range of 0.04-0.14 hr.l.
11~39~0
The medium employed in this example had the fol-
lowing composition:
DDS Extract (10% sol'n)16 gm
K2HPO4 5 gm
MgS04 7H2o 0.2 gm
Glucose 20.0 gm
Water 1000 ml
The results of this run are shown in the fol-
lowing table:
1153~70
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EXAMPLE II
This example shows that degeneration of Xantho-
monas campestris NRRL B-1459 also occurs when it is grown
in continuous culture on a mineral salts, glucose, NH4Cl
5 medium. Procedural details employed in this example were
the same as for Example I, except for the following
changes:
During the inoculum preparation stage, the 7 ml
YM culture was inoculated into 70 ml of YM broth in a
10 500 ml Erlenmeyer flask and incubated at 28C, 250 rpm for
24 hours. This was then used to inoculate 700 ml of a
mineral salts-glucose-ammonium chloride medium in a
2000 ml Fernbach flask. The minimal medium used in this
last stage had the same composition as was used in the
15 fermentation step as shown below:
TABLE II
ComponentConcentration (ppm)
Glucose 22,000
NH4C1 300 as N
KOH 1000 + as K
H3PO4 150 as P
MgSO4 40 as Mg
CaC12 10 as Ca
NaCl 10 as Na
citric acid 500
FeCl3 3 as Fe
ZnSO4 1 as Zn
CuSO4 0.6 as Cu
MnSO4 0.3 as Mn
Na2MoO4 0.2 as Mo
H3BO3 0.1 as B
KI 0.1 as I
After the 700 ml culture was incubated at 28C,
250 rpm for 40 hours, the entire culture was used to ino-
culate 20 liters of mineral salts, glucose-NH4Cl medium
contained in a 23 liter New Brunswick fermenter (Model
CMF-128S). Initial environmental conditions were as fol-
lows:
.
~153~0
-10-
Temperature 28C
pH 6.0
Agitation 230 rpm
Air Rate 0.2-0.4 vol/vol/min
Dissolved 2 90lO saturation
After an initial lag of about 30 hours, cell
growth proceeded over the next 30 hours. When the cell
concentration reached 0.9 gram per liter, continuous oper-
ation was started at a dilution rate of 0.07 hr.l.
10 Within 48 hours, the cell concentration rose to 2.5
grams/liter. After about 10 culture turnovers, the vis-
cosity and specific productivity started to decline and
eventually was almost totally lost. The data for this run
are summarized in the Table below and clearly demonstrate
15 the inability of the B-1459 strain to produce xanthan in
continuous fermentation using a minimal medium.
11539~0
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-12-
EXAMPLE III
In this Example the inoculation procedure was
the same as outlined in Example I, as were the initial
environmental conditions. The medium employed was a chem-
5 ically defined medium having the following composition
similar to Yeast Carbon Base (Difco).
TABLE IV
Ingredient Amt./Liter
Dextrose 22.5 gm
L-Histidine-HCl 1 mg
DL Methionine 2 mg
L Tryptophan 2 mg
Biotin 2 mcg
Calcium Pantothenate 400 mcg
Folic Acid 2 mcg
Inositol 2000 mcg
Niacin 400 mcg
p-Aminobenzoic Acid 200 mcg
Pyrid~xine HCl 400 mcg
Riboflavin 200 mcg
Thiamine HCl 400 mcg
Boric Acid 500 mcg
Copper Sulfate 40 mcg
Potassium Iodide 100 mcg
Ferric Chloride 3.98 mg
Manganese Sulfate 400 mcg
Sodium Molybdate 200 mcg
Zinc Sulfate 400 mcg
Potassium Phosphate Monobasic 1 gm
Magnesium Sulfate 0.5 gm
Potassium Chloride 0.57 gm
Sodium Chloride 0.1 gm
Calcium Chloride 0.1 gm
Ammonium Chloride 1.53 gm
~153~0
The culture of Xanthomonas campestris B-1459 was
grown under batch conditions for 40 hours at which time
the ce]l concentration was 2.4 gm/liter. Continuous oper-
ation was then initiated at a dilution rate of
5 0.056 hr.l. There was a slight precipitate present in
the medium upon makeup which was removed by sterile fil-
tration. After approximately six turnovers the culture
began to degenerate as indicated by a decrease in xanthan
concentration and productivity. At this poin-t the compo-
10 sition of the medium was changed by increasing the concen-
tration of all components by 1.33 except for glucose, KCl,
and NH4Cl. Thereafter a sixfold concentrate of the medium
was filtered through glass wool, which removed additional
precipitate. Then the medium was diluted to final volume
15 and subjected to sterile filtration. The result was a
drastic decrease in cell concentration and almost complete
loss of xanthan and viscosity. Thereafter citric acid was
added to give a clear medium. The ultimate concentration
of citric acid was 0.11 gm/l. Use of this revised medium
20 resulted in a completc regain of prior cell concentration
followed by a recovery of xanthan productivity. There-
after the culture was maintained for an additional 18
turnovers, which was approximately three times the number
obtained in previous runs with dried distillers solubles.
25 At the time the run was terminated, the culture was
entirely normal. The results are shown in more detail in
the following table:
1153~'~
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1153970
-15-
EXAMPLE IV
Inoculum preparation was conducted in the same
manner as in Example I as were the initial fermenter envi-
ronmental conditions. Batch growth lasted 29 hours at the
5 end of which time the cell concentration reached ~1.0
gram/liter. However, initial dilution rate was set too
high at 0.08 hr.l and the culture washed out over the
next 40 hours, even after lowering the dilution rate to
0.05 hr.l. The culture was allowed to regrow in batch
10 mode for 24 hours at which time the cell concentration
reached 3.2 grams/liter. Continuous operation was started
again at a dilution rate of 0.06 hr.l and this time the
culture lined out satisfactorily.
The culture was grown in batch mode and ini-
15 tially in continuous mode on Yeast Carbon Base medium
(Difco) which composition is given below:
TABLE VI
Component Concentration (per liter)
KH2PO4 1.0 gm
MgSO4 0.5 gm
NaCl 0.1 gm
CaC12 0.1 gm
H3BO3 500 mcg
CuSO4 40 mcg
KI 100 mcg
FeC13 3.98 mg
MnSO4 400 mcg
Na2MoO4 200 mcg
ZnSO4 400 mcg
NH4C1 1.53 gm
L-Histidine 1 mg
L-Methionine 2 mg
L-Tryptophan 2 mg
Glucose 22.5 gm
Biotin 2 mcg
Calcium Pantothenate 400 mcg
Folic Acid 2 mcg
Inositol 2000 mcg
Niacin 400 mcg
11~3g7
-16-
p-Aminobenzoic Acid 200 mcg
Pyridoxine HCl 400 mcg
Riboflavin 200 mcg
Thiamine HCl 400 mcg
Citric Acid 167 mg
Yeast Carbon Base medium is a chemically defined
medium containing organic supplements in -the form of vita-
mins with or without certain amino acids and does not con-
10 tain yeast or other undefined nitrogen source. The term"Yeast Carbon Base" indicates that this medium was used
originally to determine the carbon requirement of yeasts.
After 146 hours of continuous operation, the medium was
changed to Yeast Carbon Base at 1.5 times normal strength.
15 After 240 hours of operation, 0.21% L-glutamic acid was
added to the medium. Finally, after 453 hours of opera-
tions, the medium was replaced by one having the composi-
tion given in the table below.
TABLE VII
Component Concentration (per liter)
KH2P04 0.88 gm
K2SO4 0.54 gm
MgS04 7H2 0.41 gm
CaC12 2H2 0.056 gm
Citric acid 0.167 gm
NH4C1 1.53 gm
L-glutamic acid 2.1 gm
Glucose 22.5 gm
FeC13 10.1 mg
Trace Elements Same as in Table II
Pertinent operating data for this run are sum-
marized below.
11539~
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~1539
-18-
In this case the vitamins were removed and glu-
tamic acid was included to provide 10% of the total carbon
(90% from glucose) and 33% of the total nitrogen.
Examples III and IV show that when a Xanthomonas
5 organism is used to ferment a chemically defined medium,
the process lends itself well to continuous fermentation
conditions to produce xanthan gum of good quality. The
process described above, while a single-stage method, can
also be operated as a two-stage procedure using any medium
10 coming within the scope of that defined herein. In a
two-stage process, cell production and xanthan production
would both occur in the first stage, while xanthan produc-
tion primarily would occur in the second stage since the
cell growth limiting nutrient would be exhausted from the
15 medium.
