Note: Descriptions are shown in the official language in which they were submitted.
~.0~3 1~9
There are extensive published reports relating to the
production of hydrophilic colloids by the aerobic propagation of
bacteria of the genus Xanthomonas in aq~eous nutrient media. The
earliest work in this field was done at the Northern Rçgional
Research Labo~atory of the United States Department of Agriculture
at Peoria, Illinois and is described in United States Patent
3,000,790, Modified fermentation processes are described in
United States Patents 3,020,206; 3,391,060; 3,427,226; 3,433,708;
3,271,267; 3,251,749; 3,281,329; 3,455,766: 3,565,763; 3,594,280;
and 3,391,061.
~he hydrophilic colloid (xanthan gum) produced by
Xanthomonas campestris is a poly~accharide which contains mannose,
-
glucose, glucuronic acid, O-acetyl radi~als and acetal-linked
pyruvic acid in molar ratio o~ 2:2:1:1:0,5, This gum and its
lS derivatives have found wide food and industrial applications. Of
special interest is the increasing fo~us on tha use of xanthan gum
in displacement of oil from partially depleted reservoirs,
Typically, oil is recovered from underground reservoirs
vla a series of sequential operations. A new well will generally
produce a limited amount of oil as a result of release of internal
pressure in the well. As this pressure becomes depleted, it is
necessary to pump further quantities of oil by mechanical means.
These measures recover only about 25% of the total oil stored in
the reservoir. A great doal of oil is still trapped within the
pores of the formation. Further enhancement of recovery can then
C -2~
109 L~ 479
be effected by secondary recovery. In one method of recovery a
waterflood is carried out by pumping water into a well or series
of wells, displacing part of the trapped oil from the porous
rock and collecting the displaced oil from surrounding wells.
However, waterflooding still leaves about 55-60% of the availab~
oil trapped in the formation. The explanation for this phenomenon
is that the water has a very low viscoisty compared to the crude
oil and tends to follow the path of least resistance, fingering
through the oil and leaving large pockets untouched. In addition,
surface forces in the formation tend to bind the oil and prevent
its displacement.
A number of processes have been developed in recent
years to recover further quantities of oil from these reservoirs
by the use of mobility control solutions which enhance oil dis-
placement by increasing the viscosity or permeability of thedisplacing fluid. Of interest are those enhanced recovery pro-
cesses employing polymer flooding with a polysaccharide or poly-
acrylamide to increase the viscosity of the displacing fluid.
Variations of this process include the use of surfactants and
co-surfactants to release the oil from the reck formation. Poly-
acrylamides have been found to suffer such deficiencies as
~iscosity loss in brines and severe shear sensitivity. Since,
as was well documented in the prior art, xanthan gum is in-
sensitive to salts (does not precipitate or lose viscosity under
normal conditions), is shear stable, thermostable and viscosity
stable over a wide pH range, xanthan gum is a good displacing
agent. Moreover, the gum is poorly adsorbed on the elements of
the porous rock formations and it gives viscosities useful in en-
hanced oil recovery (5 to 90 Centipoise units at 1 32 sec. 1
30 shear rate) at low concentrations (100 to 3000 ppm).
-3-
. . ~ , .
..
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.
~09~1i47!~
The use of solutions of xanthan gum or derivatives of
xanthan g~m for oil recovery is described in United States Patents
3,243,000; 3,198,268; 3,532,166; 3,305,016; 3,251,417; 3,319,606;
3,319,715; 3,373,810, 3,434,542 and 3,729,460, It is suggested
S in United States Patent 3,305,016 that aqueous solution contain-
ing the heteropolysaccharide in sufficient ~uantity to increase
the viscosity be employed as the thickening agent in preparing
viscous waterflooding solutions. The polysaccharide may be pre-
pared, separated, purified and then added. Alternatively, accord-
ing to this reference, the entire culture, after adding a bacter-
icide (e.g., formaldehyde) to kill the bacteria, may be added to
the flood water.
United States 3,000,790 describes the culturing of a
Xanthomonas bacterium in a well aerated medium containing commer-
cial glucoqe, an organic source of nitrogen, dipotassium phosphate
and appropriate trace elements. The source of organic nitrogen
usually employed is distillers' solubles. The use of this organic
nitrogen source contributes a substantial quantity of insolubles
to the fermentation broth.
The processes described in United States Patents
3,000,790, 3,391,060 and the other fermentation processes previous-
ly listed yield final fermentation broths that contain su~stantial
amounts of insoluble matter, even when diluted with water, for
injection into oil-bearing subterranean formations to impart the
necessary and desired mobility control for oil displacement. The
particulate matter and in certain c~ses the Xanthomonas cells in
such whole broth would soon plug the oil-bearing formation at the
site of injection and thus foul the well and prevent any further
oil recovery. Furthermore, the same problem would be encountered
with reconstituted xanthan gum precipitated and separated from
~, ~ 3
~- 4
, , .
,
:
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` 1094479
these fermentation broths. The plugging tendencies of these
fermentation broths can be obviated by filtration through diatom-
aceous earth leaf filters to remove the Xanthomonas cells and
particulate and colloidal matter. However~ suah additional
filtering steps are expensive and add significantly to the
overall cost factors for enhanced oil recovery.
United States 3,853,771 approach~s the plugging pro~lem
of whole fermentation broths by claiming a process for dissolving
or dispersing cellular microorganisms which comprises contacting
said materials with an aqueous solution containing at least one
surfactant effective for dispersing outer wall layers of micro-
organism cells, at least on~ chelating agent for dispersing the
inner wall layers of microorganism cells, and at least one alkali
metal hydroxide effective for enhanoing said dispersing actionsO
United States 4,010,071 describes a process for clarify-
ing fermentation broths and other aqueous suspensions containing
a dissolved xanthan gum and suspended solids resulting from the
fermentation by treatment with a minor amount of a pro~ease
enzyme. The injectivity of aqueous solutions containing xanthan
gum so clarified is improved in oil well flooding operations over
solutions not so treated, However, this treatment does not over-
come plugging problems due to the presence of insoluble inorganic
or non-proteinaceous organic materialæ present in the fermentation
medium or producçd during the course of the fermentation.
The object of the process of United States Patent
3,391,060 is to recover a polysaccharide product of substantial
purity without the use of extensive separation procedures. A
high quality, high viscosity, iight colored, high purity xanthan
gum is recovered from a Xanthomonas fermentation broth. According
to this patent, recovery i9 simplified and elaborate purification
i `` ` ~5~
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1' . '
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109~479
procedures are obviated by the replacement of the organic source
of nitrogen in the broth with an inorganic source of nitrogen
(i~e., ammonium nitrate). However, the fermentation process de-
scribed therein is not economical because of lengthy reaction
times required and low yields of biopolymer obtained.
The method of improving the permeability of mobility
control solutions by the addition of certain hydroxy substituted
carboxylic acids such as malic, tartaric, citric, gluconic, lactic
and salicylic is described in United States Patent 2,867,279.
This invention is concerned with an economical process
for preparing a Xanthomonas colloid-containing fermentation broth
suitable for preparation of mobility control solutions used in
oil recovery which comprises aerobically ~ermenting a Xant~Dmonas
organism in an aqueous nutrient medium whose ingredients comprise
a carbohydrate, a nitrogen source, a Krebs cycle acid, chelated
calcium, manganese ions and iron ions. The whole broth produced
provides mobility control solutions of about 100 to 3000 ppm
Xanthomonas colloid which are substantially free of insoluble
~; matter having a particle size greater than about 3 microns.
According to the present invention there is provided
a process for preparing a Xanthomonas colloid-containing ferment-
ation broth suitable for the preparation of mobility control
solutions used in oil recovery characterized in that a Xanthomonas
organism is fermented aerobically in an aqueous nutrient medium
whose ingredients comprise a carbohydrate, a nitrogen source, about
0.1 to 10 grams per liter of an assimilable Krebs cycle acid,
about 1 to 200 ppm chelated calcium, about 0.25 to 20 ppm iron
and trace elements until at least about 100 ppm colloid is
present in the broth, whereby the whole broth produced provides
mobility control solutions of about 100 to 3000 ppm Xanthomonas
C !, -6-
.. ,-- . , ~ , . ,
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109~79
colloid content which are substantially free of insoluble
matter having a particle size greater than about 3 microns,
The present invention now provides for the first time
a practical process for prepari~g a Xanthomonas fermentation
broth which without treatmant or clarification provides mo~ility
control solutions for use in oil recovery ha~ing a use concentra-
tion of about 100 to 3000 ppm Xanthomonas colloid which are sub-
stantially free of insoluble matter having a particle size greater
than about 3 microns. According to this invention, the whole
fermentation broth may be used directly as a mobility control
solution suoh as where the amount of colloid produaed during
fermentation is within the desired range of use concentration.
Alternatively, the broth may be diluted with water or a water
6a-
. . ' '' '
",
.
~0~4~79
solution such as ~rine to reduce the level of colloid to the
desired range, Additives to modify the properties of the mobility
control solutions may be employed in addition to the whole or un-
clarified broths of the present invention.
S It has been found that oil in cores from oil fields
generally will be effectively recovered by xanthan gum if the poly-
saccharide solution at use concentration can be made to pass a
Millipore filterability test as described later. Typical pore
sizes o~ Millipore filters used for this test are 0.45 to 3.0
m~crons. "Millipore" is a trademark.
In addition to Millipore filterability, core tests are
- performed as described by W.B. Gogarty in Mobility Control with
Polymer Solutions, Paper ~SPE 1566B) presented at the Society of
Engineers 41st Annual Fall Meeting held in Dallas, Texas,
lS October 2-5, 1966.
A novel feature of the fermentation broths of the present
invention, importantly distinguished from previously reported
Xanthomonas fermentation broths and preparations, is the obviated
need for expensive and time-consuming filtration or clarification.
Important factors that help make this possible are the following:
1. All nutrient ingredients are essentially water soluble
or become essentially water soluble during the course of the fer-
~ntation or prior to injection.
2. Absolute asepsis is maintained during the course of the
2S ~ermentation and a bactericide is preferably added at the end of
the fermentation.
3. Hard water with high concentrations of calcium ions is
; preferably not used for the make-up of fermentation media. Low
levels of calcium ions ~ess than about 100 mg./liter, preferably
less than about 60 mg./liter) can be sequestered by the addition
-7-
B
.i
. 10~7g
of a chelating agent such as ethylenediaminetetraacetic acid or
preferably citric acid.
4. Amounts of manganese ions may be limited to avoid seed
crystal formation and precipitation of insoluble calcium and
phosphate salts (typically about 0.75 to 60 ppm, preferably 1.5
to 2.4 ppm).
5. Amounts of trace iron are controlled to avoid seed
crystal formation and precipitation of insoluble calcium phosphate
salts.
Important to the process of the present invention is
the incorporation of the following ingredients to promote rapid
cell growth and increase xanthan vield while achieving the de-
sired mobility control solutions having substantially no insoluble
matter with a particle size above about 3.0 microns.
lS 1. Chelated calcium - about 1 to 200 ppm, preferably about
40 to 60 ppm.
2. Trace iron - typically about 0.25 to 20 ppm, preferably
0.5 to 8 ppm.
3. Krebs cycle acid - about 0.1 to 10 grams/li~er, prefer-
ably about 1 gram/liter.
Trace amounts of manganese and ferrous ions are added in the
form of such salts as MnSO4-H2O, MnC12, FeSO4-7H2O and FeC12.
By Krebs cycle acid is meant an assimilable acid select-
ed from the group consisting of citric acid, oxaloacetic acid,
l-malic acid, fumaric acid, succinic acid and oxalosuccinic acid.
Cis-aconitic acid is a Krebs cycle acid that is not useful for the
process~of this invention. The preferred acid is citric acid be-
cause of its added efficiency as a sequestering agent for calcium
ions. Citric acid can be incorporated in the fermentation medium
at a concentration of about 0.5 to 2 grams/liter, preferably about
-8-
~. . . .
~ ......... ' ' ''~. . ` ' . `. `
10~479
l gram/liter.
In the practic~ of this invention, a suitable
fermentation medium is inoculated with an organism of the genus
Xanthomonas. The inoculum medium may be YM Broth tDifco) or a
medium containing crude glucose tcerelose), sodium and potassium
phosphates, magnesium sulfate and any of a variety of organic
sources of nitrogen such as an en2ymatic di~est of soybean ~Soy
Peptone Type T, Humko-Sheffield Chemical Co.) or an enzymatic
digest of casein ~NZ-Amine YT, Humko-Sheffield Chemical Co.).
A~ter aerobic propagation for about 30 hours at 28C., an aliquot
is transferred to a fermentor for~the second stage inoculum.
A suitable carbohydrate is present in the nutrient
medium at a concentration from about 1 to about 5% by weight.
Suitable carbohydrates include, for example, glucose, sucro6e,
maltose, fructose, lactose, processed inverted beet molasses,
invert sugar, high quality filtered thinned starch~or mixtures of
these carbohydrates. The preferred carbohydrates are glucose,
maltose, fructose, filtered starchhydrolyzates ~r mixtures thereof.
Inorganic nitrogen is present in the nutrient medlum
at a concentration of about 0.02 to about 0.35% by weight, prefer-
ably 0.07 to 0.25% by weight. Inorganic nitrate is the preferred
nitrogen source; ammonium nitrate at about l gram/liter, sodium
nitrate at about 2 gramæ/liter or potassium nitrate at about 2.4
grams/liter may be used. The preferred source of nitrogen in
this as well as the production medium is inorganic. However,
organic nitrogen sources can also be used although they enhance
large Xantho nas cell formation, provided the overall require-
ment of substantial freedom from insoluble materials with a
particle size above about 3 microns is maintained.
Magnesium in the form of MgSO4.7H2O or epsom salts, 0.1
g_
:`
10~!~479
to 1.0 grams/liter, is added along with trace manganese and
iron ions. A chelating agent such as ethylenediaminetetraacetic
acid or preferably citric acid which functions as a growth pro-
moting Krebs cycle acid and sequestering agent for any calcium
present is added.
Sufficient mono- and dipotassium phosphates are added ''
to buffer the medium at about pH 5.9 to 8.5, préfera~ly 6.0 to
7.5, After aerobic propagation for about 20-40 hours at 24 to
34C,, preferably 28-30C " an aliquot is transferred to a
fermentor containing the production medium.
The production medium i8 similar in composition to
that of ~he second stage inoculum medium with the exception
that sodium phosphates are preferably used'in place of potassium
phosphates because of their lower costs and a small amount of
calcium in the form of a salt such as calcium chloride or
calcium nitrate or oxide such as lime is added to increase
xanthan yield. The amount of calcium added is dependent on the
amount of calcium present in the water used for medium make-up,
the nitrogen source used and the species and strain of Xanthomonas
organism employed. When sodium nitrate or potassium nitrate
i8 used in place of ammonium nitrate, less calcium is required
(approximately 27 ppm). Deionized water, distilled water or
water containing less than about 20 ppm of calcium and other
phosphate precipitable cations may also be used for'medium
25 make-up. Calcium ions may be added to a desired concentration. `~
The role of calcium ions in the enhancement of xanthan produc-
tion is an important one but critical to the process of the
present invention is the prevention of the precipitation of
excess calcium cations and other eations as insoluble phosphate
salts, This is accomplished, when desired, by the addition of
1 0 -
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.
, . . .
; :
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a chelating agent such as ethylenediaminetetraacetic acid or
other suitable compound known to those skilled in the art a~
a concentration of about 1 to 20 millimolar, preferably 2 to 8
millimolar.
The pH of the fermentation medium is quite important
to suitable growth of the Xanthomonas bacteria. The preferred
range is about 6.0 to 7.5. Control of the pH within this range
can be obtained by the use of a buffer compound such a~ disodium
acid phosphate. Ethylenediaminetetraacetic acid or other suit-
able chelating agent is also added in the buffer solution used
for pH control to prevent the precipitation of calcium ions
introduced in the water used for medium make-up as insoluble
calci~m salts. The p~ is preferably controlled during the
fermçntation cycle by the addition of sodium or potassium
hydroxide solution which has the added advantages of decreasing
broth viscosity without affec~ing xanthan yield and the elimin-
ation of the need for chelation of the buffer solution.
In order to obtain a rapid fermentation, it is
essential to have the correct amount of oxygen a~ailable for
the growing bacterial culture. The fermentation medium is
aerated to provide sufficient oxygen to produce a sulfite oxida-
tion value within the range of about 1,5 to about 3.5 millimole~
of oxygen per liter per minute. A description of sulfite oxida-
tion value is set forth in Industrial Engineering Chemistry 36,
504 tl936). The sulfite oxidation value is a measure of the
rate of oxygen uptake in the fermentor under the agitation
and aeration conditions employed.
The fermentation is allowed to proceed at a tempera-
ture of about 30C. until the broth has a xanthan concentration
of at least about 100 ppm, preferably at least about 1.0% and
C : :
~ .
~ 0~4479
more preferably at least about 1.4% (30-96 hours). Viscosities
of the broth are typically at least about 4,000 Centipoise
units and preferably at about 7,000 Centipoise units.
It is desirable to kill the microbial cells by the
addition of a bactericide such as formaldehyde, glutaraldehyde,
phenol or substituted phenol such as a cresol or hydroxybenzene
or a polyhalogenated phenol such as Corexit ~Exxon Corporation~,
or any other preservative well known in the art. The preferred
preservative is formaldehyde at a concentration of from about
200 to 10,000 ppm, preferably about lOOO to 3000 ppm, which
can be added to the final fermentation broth before or during
storage.
The process of this invention works well ~or many of
the various species of Xanthomonas bacteria. Illustrative
species include Xanthomonas ~haseoli, Xanthomonas malvacear~m,
Xanthomonas carotae, Xanthomonas begQniae, Xanthomonas incanae
and Xanthomonas vesicatoria. The preferred species is Xanthomonas
campestris.
The Millipore filterability test i5 an experimental pro-
cedure that measures flow rate through a Millipore filter (0O45
to 3.0 ~ pore size) as a function of volume under a constant ~ "'
pressure of 40 psig. The filter ratio is the ratio of the time
to collect the fourth 250 ml. of mobility control solution to
the time to collect the first 250 ml. of mobility control solu-
tion. A filter ratio of 1.0 indicates that the solution has no
plugging tendencies. An acceptable mobility control solution
has a filter ratio of 1 to 3 (0.45 to 3 ~ Millipore fiter), pre-
ferably <1.7.
The desired filter ratio and Millipore filter size for
testing of a particular mobility control solution are dependent
~! -12-
;;
'
.
10~3~479
on the permeability of the subterranean stratum of the oil
field for which oil displacement is planned.
Mobility control solutions pr~pared from the whole
fermentation broths of the present invention have filter ratios
suitable for use in most oil fields. Where subterranean strata
are highly impervious, mobility control solutions with low
filter ratios must be employed. Under such circumstances,
the whole or unclarified broths acco,rding to the present inven-
tion preferably have substantially no insoluble matter with a
particle size in excess of about 0.65 u. This can be accomplish-
ed simply by storage of the final fermentation broth in the
presence of about 200 at 10,000 ppm formaldehyde for a period
of about 3 to 4 days. This aging process shrinks the Xanthomonas
cells so that their minor dimension does not exceed 0,65 ~ in
size. Xanthomonas cells are rod-shaped and normally the longer
dimension of the cells is greater than 0.65 ~ whereas the shorter
dimension of the cells is about 0.4 to 1.0 u. ~he storage
temperature is n~tcritical and may range from 0 to 135C., pre-
ferably 20 to 45C~ A practical expedient is storage at room
temperature for a sufficient period of time so that microscopic
examination reveals that the minor dimensions of the Xanthomonas
cells are not greater than 0,65 ~ in size ~3-4 days).
The following data show the lnfluence of medium in-
gredients on the filter ratios of mobility control solutions pre-
pared from aged fermentation broths:
-13-
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Alternatively to the aging process, an alkali metal
hydroxide such as sodium or potassium hydroxide is added to the
final fermenta~ion broth at a concentration of 0.1 to 2.0% w/v,
pr~ferably ~.5% w/v, along with an alkali metal salt such as
sodium or potassium chloride or sulfate at a concentration of O
to 5% w/v, preferably 1.0% w/v. The preferred salt is sodium
chloride. The broth is heated under an oxygen free atmosphere
~e.g., nitrogen~ at a temperature of 55-121C., preferably 85-95C.
The heating time is not crucial and may be extended for several
hours. A practical time is 1 to 30 minutes, preferably about 10
minutes. The broth is cooled to room temperature and adjusted to
pH 3 to 12, preferably 7, with an acid such as hydrochloric acid.
This alkaline heat treatment hydrolyzes the bacterial cells with-
out affecting the xanthan content. Deacetylation of the xanthan
occurs during this process and the deacetylated product is quite
suitable for use in oil recovery.
The suitably aged or alkaline heat-treated fermentation
broth is generally diluted with water or brine to a xanthan con-
centration of about 400 to 1000 ppm for use in enhanced oil re-
covery. Optionally, a surfactant may be added to enhance the re-
covery of oil. Representative surfactants include various
petroleum sulfonates well known to those versed in the art of oil
recovery.
It i6 understood that there may be conditions and
factors that make impractical or expensive the transportation of
large volumes of fermentation broth for injection into oil-con-
taining reservoirs. For æuch purposes a special composition is
provided. To the alkaline heat-treated broth previously described
i8 added a water miscible solvent such as methanol, ethanol,
acetone, t-butyl alcohol or isopropanol sufficient to precipitate
-16-
.
.
-` 10~4~79
the xanthan gum which is separated by centrifugation or filtra-
tion and dried. The preferred water miscible solvent is isopropanol
at a concentration of 20-75% w/w, preferably about 38% w/w. Re-
constitution with water or brine to a xanthan concentration of 100
to 3000 ppm provides a composition for enhanced oil recovery that
is comparable in performance with that o diluted whole fermenta-
tion broth. During the process of re-dissolution, it is important
to provide sufficient shear to cause adequate dispersion of the
polysaccharide and prevention of clump formation.
TEST PROCEDU~ES
Millipore Filterability Test
Prepare 1000 ml. of 750 ppm xanthan solution in 500 ppm
salt solution ~10:1 - NaCl:CaC12) as follows:
In a Waring type blender equipped with a rheostat,
measure sufficient broth ~ased on xanthan content) to make 0.75 g.
xanthan solids. Dilute 1 to 6 with salt solution. Shear this mix-
ture as follows:
40% power/2 minutes
60~ power/2 minutes
80% power/2 minutes
Dilute in the blender to 750 ppm of xanthan and shear
at 40% power for 2 minutes. ~Solution also used for viscosity
determination.)
Vse an experimental set-up that allows the flow rate
through a Millipore filter disc ~47 mm, 0.45-3.0 y pore size) as
a function of volume under a constant pressure of 40 psig. Use a
reservoir that will accommodate >1000 ml.
Charge the reservoir with a liter of xanthan solution
(750 ppml. Set pressure at 40 psig. Open valve and start record-
ing volume filtrate and time ~seconds).
-17-
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10~4479
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Filter Ratio-time to collect the 4th 250 ml, of solution
time to collect the 1st 250 ml, of solution
Viscosity Determination
Measure the viscosity with a Brookfield synchro-lectric
viscometer, model LVT, using a UL adapter. Measure at 25C. at
6 and 12 RPM. Viscosity is expressed in Centipoise units.
Xanthan Determination
Highly purified xanthan contains about 18.4% glucuronic
acid. Glucuronic acid in xanthan compositions i8 determined in
the absence of formaldehyde and without borate at 100C. by the
method of Knutson and Jeanes, Anal. Biochem., 24, 470 (1968);
ibid., 482.
% Glucuronic Acid x 100
% Xanthan = - -
18.4
EXAMPLE I
Cells of Xanthomonas campestris from a YM agar slant
are transferred to 300 ml. of YM broth contained in a 2.8 liter
Fernbach flask and shaken on a rotary shader for about 31 hours
at 28C. A 25 ml. aliquot is transferred to a 2.8 liter Fernbach
flask containing 500 ml. of a medium of the following composition:
20 Ingredient Grams/liter
Glucose-fructose (Isosweet 100,
Corn Products)autoclave 10,1
Crude glucose (cerelose) _ separately 25.7
NH4N3 1,0
MgS04-7H20 0,10
MnS04 H20
FeS4'7H2 0,01
Anhydrous citric acid 1.0
K2HP04 4.1
RH2P04 0.69
-18-
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10~4~9
The cerelose and Isosweet 100 are dissolved in
distilled water and autoclaved. The rest of the ingredients
are combined, adjusted to pH 6.4 an~ autoclaved. The separate-
ly autoclaved materials are then combined.
After shaking at 28C, for about 33 hours a 200 ml.
portion is transferred to a 4-liter, mechanically agitated
fermentor containing 2 liters of medium:
Ingredient Grams/litler
Cerelose~ 25.7
autoclave separately
10 Iæosweet 100 ~ 10.1
NH4NO3 1.0
MgS04 ~7H20 0 .10
MnSO4 H2O a .03
FeSO '7H O 0.~1
15 Anhydrous citric acid 1.0
CaC12 2 2 0.20
Na2HPO4 3-34
NaH2PO4 0~70
The sugars dissolved in 300 ml. of water are auto-
claved separately. The rest of the ingredients dissolved in
1700 ml. of water are autoclaved, and the two solutions then
combined. Aeration is at a rate to provi~e 1.5 to 3.5 milli~
moles of oxygen par liter per minute. The fermentation is conduct-
ed at 30C. for 48 hours during which time the pH of the medium
is maintained betwee~ 5.9 and 7.5 by the addition of sodium
phosphate buffer made up with tap water. Ethylenediaminetetra-
acetic acid is also added to ~he sodium phosphate buffer to
prevent the precipitation of calcium phosphate salts. At the
end of the fermentation, the viscosity of the broth is >7800
Centipoise units (at 6.27 sec. 1 shear rate) and the xanthan
19-
: ,
10~479
.
yield is >l.5%.
A mobility control solution has a filter ratio of ~1.7
(3.0 ~ Millipore filter). ,
A mobility control solution prepared from whole fermen-
tation broth aged for 4 days has a filter ratio of ~1.7 ~0.65
Millipore filter).
EXAMPLE II
The method of Example I is repeated employing the
following fermentation media:
Inoculum Medium (lst Stage~
Ingredient Grams/liter
Cerelose lO.0
(NH4)2HPo4 2.0
KH2P4 l.0
MgSO 7H2O 0 5
NZ-Amine Y~ pH - 7.0 ll.0
Inoculum Medium (2nd Sta~e)
Ingredient Grams/liter
D-Glucose ~ autoclave 27,0
~r separately
20 D-Fructose 3.0
NH4NO3 1.0
MgSO 7H2O O.lO
MnSO4 2
FeSO 7H O O.Ol
25 Anhydrous citric acid 1.O
K2HPO4 4.l
XH2PO4 pH - 6.4 0.69
,1 ~i -20-
~ ' '
,
J10~44~9
Production Medium
-
Ingredient Grams/liter
D-Glucose ~ 27.0
~ autoclave separately
5D-FructoseJ 3.0
Anhydrous citric acid 1.0
NH4NO3 1.0
MgSO4~7H2O 0.10
MnS4~H2 0.03
10 FeS04 ~ 7H20 0 . 01
CaC12~2H2O 0.20
Na2~PO4 3.34
NaH2PO4 0.70
pH - 6.4
The fermentation is conducted as in Example I, with com-
parable results, with the exception that pH adjustment is made with
sodium hydroxide solution without the concommitant addition of
ethylenediaminetetraacetic acid.
EXAMPLE III
A large scale fermentation is conducted in the following
manner:
Inoculum M~dium ~lst Stage)
Ingredient Grams~liter
Cerelose 10.0
25 ~NH4)2HPO4 2.0
KEI2P4 1. 0
MgS~4 ~H20
NZ-Amine YT 11~0
~ pH - 7O0
1 30 The medium is dispensed in 500 mlO portions into two 2.8liter Fernbach flasks. After autoclaving and cooling, the flasks
are inoculated with cells of Xanthomonas campestris. After shaking
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.~ .
" ~ :
: .' . ~, ~ ' ,' ; .
;' ? ~ '
~05`~79
for 30 hours at 28C., the two flasks are combined and used to
inoculate a 200 gallon fermentor containing the following ingredients
in 100 ga~lons of medium:
Inoculum Medium (2nd Stage~
Ingredient A
Cerelose 10~0
~I4)2HPo4 2.0
KH2P4 1 o O
Epsom salts 0.5
NZ-Amine YT 11.0
pH - 7.0
~ he fermentation medium is stirred with a mechanical
agitator and aerated to provide l.S to 3O5 millimoles of oxygen
per liter minute. Sodium hydroxide solution is added at intervals
to maintain the pH at 6O0 to 7.5~ Soybean oil is added to control
excessive foam. After about 48 hours fermentation, a volume
sufficient to provide a 5~ v/v inoculum is transferred to a 2000
gallon fermentor containing 800 gallons of medium of the following
composition: `
~
~ Amount
. ~
NH4NO3 (50~ solution) 1c33 gallons
R2HPO4 27O5 lbs.
KH2PO4 4~75 lbs,
Epsom salts 306 grams
MnSO4 H2O 94 grams
FeSO4~7H2O 30~5 grams
Cerelose ~ 175 lbso
~ autoclave separately
Isosweet 100~ 70 lbs.
Anhydrous citric acid 6O75 lbs~
pH - 7.0
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' '
1094~79
After fermentation under the previously described condi-
tions for about 31 hours, a sufficient volume to provide a 10% v/v
inoculum is transferred to a 2000 gallon fermentor containing 1200
gallons of the following medium:
Production Medium
.
I dient Amount
nqre - -
cerelose-______ 297 lbs,
~ autoclave separately
Isosweet lOL~ - 117 lbs.
NH4NO3 ~50% solution~ 2 gallons
Hydrated lime 0.73 lbs.
Anhydrous citric acid 10.0 lbs~
Na2HPo4 33~5 lbs~
NaH2P04 7~0 lbs~
Epsom salts ~MgS04~7H20) 454 grams
MnSO4 H20 141 grams
FeSO4 7H2O 45~ grams
pH - 7.0
.~ .
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.:
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