Xanthomonas campestris ATCC 31601 and process for use

A new degenerative-resistant strain of Xanthomonas campestris is described which is suitable for long-term continuous fermentation to produce xanthan gum from a nutrient medium, typically containing inorganic salts, glucose, NH.sub.4 Cl, and citric acid, with or without yeast extract or yeast autolysate.

INTRODUCTION 
The present invention relates to the production of heteropolysaccharides by 
the action of certain novel degenerative resistant strains of Xanthomonas 
campestris on aqueous nutrient media. More particularly, it is concerned 
with the production of xanthan gum by the use of these novel bacteria. 
BACKGROUND 
Batch fermentation of an inoculated medium with Xanthomonas campestris NRRL 
B-1459 for 36-72 hours under aerobic conditions results in the formation 
of xanthan gum, which is separated from the 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 processing required for the recovery and 
purification of the product, xanthan gum produced by batch fermentation, 
hereinafter also referred to as xanthan, is relatively expensive. 
Because continuous operation of a fermentation process offers a number of 
potential advantages over conventional batch methods that could be 
reflected in lower costs, considerable effort has been put forth in the 
past to perfect conditions that would support a reliable continuous 
process. But even with a continuous process a cheap medium from which 
xanthan can be produced is required. In addition to the necessity of an 
inexpensive medium in the manufacture of a low cost xanthan product, the 
ratio of xanthan to cells (bacteria) should be as high as possible in 
order to reduce subsequent filtration costs for cell removal. The specific 
productivity of the culture employed also should be as high as possible in 
order to maintain the aforesaid high ratio as well as to reduce vessel 
volume and capital costs. The expression "specific productivity" as used 
in the present description is intended to mean the number of grams of 
xanthan produced/grams of cells/hour. The culture should be stable under 
continuous culture conditions on a long term basis to avoid frequent 
restarts and lost productivity. 
Although xanthan has been produced by continuous fermentation in the past, 
such methods have not met with unqualified success. In some cases, 
vitamins and/or amino acids had to be employed in the medium in 
substantial quantities in order to avoid culture degeneration or to 
improve specific productivity. Use of these additives, as well as soybean 
protein, cotton seed protein, etc., all tend to make the xanthan thus 
produced more costly. 
It is well known that the continuous production of xanthan by the use of 
Xanthomonas campestris B-1459 has been hampered by a tendency of the 
culture to change or degenerate after a fairly small and specific number 
of turnovers, the time required during the fermentation to completely 
replace one volume of broth in the fermentation vessel. Normally, 6-9 
turnovers are the maximum that can be obtained before degeneration of the 
culture occurs. At the same time, there is a decrease in viscosity, a loss 
in volumetric productivity of xanthan gum, i.e., grams of xanthan/liter of 
broth/hour, and appearance of a variety of culture variants or strains 
that no longer produce xanthan or else produce a xanthan of low quality. 
It has been demonstrated that culture degeneration occurs when dried 
distillers solubles (DDS) is used in the nutrient medium as the complex 
nitrogen source, whether in the whole form or as a water soluble extract. 
In other cases, certain strains of Xanthomonas have been grown 
successfully without culture degeneration in simple minimal media, but the 
xanthan:cell ratio and specific productivity have been low, on the order 
of 0.1-0.12 gm xanthan/gm of cells/hr. 
Earlier work has indicated that heteropolysaccharides produced by the 
action of Xanthomonas bacteria on assimilable carbohydrate containing 
media have potential applications as film forming agents, as thickeners 
for body building agents in edible products, cosmetic preparations, 
pharmaceutical vehicles, oil field drilling fluids, fracturing liquids and 
similar compositions and as emulsifying, stabilizing and sizing agents. 
Heteropolysaccharides, particularly xanthan gum, have significant 
potential as a mobility control agent in micellar polymer flooding. This 
gum has excellent viscosifying properties at low concentration, is 
resistant to shear degradation 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 operations. 
SUMMARY OF THE INVENTION 
We have now discovered a degenerative-resistant strain of Xanthomonas 
campestris and have developed a process for using this strain to 
effectively overcome the problems of continuous xanthan production recited 
above. This strain of Xanthomonas campestris which we have designated 
Xanthomonas campestris XCP-19 ATCC 31601 is capable of continuously 
producing xanthan at high specific productivities, i.e., 0.24 to 0.32 gm 
xanthan/gm cells/hr, for several hundred hours without culture 
degeneration from inexpensive aqueous nutrient media such as, for example, 
a minimal medium consisting primarily of inorganic salts, glucose and 
NH.sub.4 Cl. The medium may or may not also contain a yeast extract or 
yeast autolysate as a supplemental nitrogen source. Generally, it may be 
said that any medium having assimilable sources of carbon, nitrogen and 
inorganic substances will serve satisfactorily for use with this new 
organism. 
The process of our invention in which this new strain is utilized can be 
either a single stage or two-stage continuous fermentation process. In the 
single stage embodiment the organism is grown, preferably under conditions 
such that the quantity of one of the growth nutrients present is limited. 
The quantity of biomass obtained will be determined by the concentration 
of the limiting nutrient. A portion of the residual glucose or equivalent 
sugar present is converted to xanthan gum and the latter ultimately 
recovered from the fermentation effluent. In the two-stage process, the 
aforesaid fermenter effluent is taken to a second fermentation stage where 
additional glucose or equivalent sugar is introduced and converted to 
xanthan. In operation of the second stage, a balance of the flow of the 
first stage effluent and glucose solution must approximate the flow rate 
of the second stage effluent. The growth limiting nutrients normally 
employed are nitrogen, phosphorous or sulfur. 
SPECIFIC EMBODIMENTS OF THE INVENTION 
Subcultures of this living organism can be obtained upon request from the 
permanent collection of the American Type Culture Collection, 12301 
Parklawn Drive, Rockville, MD 20852. The accession number in this 
repository for Xanthomonas campestris XCP-19 is given above. The novel 
Xanthomonas campestris strain referred to was isolated from a pilot plant 
run in which a 28 liter fermenter was operated using a culture of 
Xanthomonas campestris NRRL B1459, growing in a suitable nutrient medium 
having a composition as shown in Table I. 
TABLE I 
______________________________________ 
Component Concentration (ppm) 
______________________________________ 
Glucose 22,000 
NH.sub.4 Cl 300 as N 
KOH 1000 + as K 
H.sub.3 PO.sub.4 300 as P 
MgSO.sub.4 100 as Mg 
CaCl.sub.2 10 as Ca 
NaCl 10 as Na 
FeSO.sub.4 2 as Fe 
ZnSO.sub.4 0.35 as Zn 
MnSO.sub.4 0.35 as Mn 
Na.sub.2 MoO.sub.4 
0.21 as Mo 
H.sub.3 BO.sub.3 0.07 as B 
KI 0.14 as I 
CuSO.sub.4 0.020 as Cu 
Citric Acid 250 
______________________________________ 
Continuous fermentation of the above medium was initially conducted at 
28.degree. C., a pH of 6.8, an aeration rate of 0.2-0.4 vol./vol./min., 
agitation rate of 230 rpm, dissolved oxygen of 90% saturation, and a 
dilution rate of 0.08 hr.sup.-1. After about 6 turnovers, the culture 
began to degenerate with lowered xanthan productivity and viscosity. At 
this point, several changes were made to the medium. The pH was lowered to 
6.4 from 6.8; iron, copper and zinc concentrations in the broth were 
raised to 3, 0.6 and 1 ppm, respectively, by adding these trace elements 
to the glucose feed tank (they were previously added with the rest of the 
inorganic salts), the citric acid level was doubled to 500 ppm, and 
H.sub.3 PO.sub.4 and MgSO.sub.4 levels were reduced by half. For 2 to 3 
culture turnovers after these changes the culture continued to degenerate 
with viscosity dropping to 70 cp and specific productivity decreasing to 
0.07 gm/gm cells/hr. 
During an overnight period, the exit line, from the fermenter plugged and 
the culture went into a semibatch/low dilution rate condition for about 10 
hours. The following morning, viscosity was up to 300 cp and when level 
and flow rates were corrected, it continued to increase. For the next 10 
turnovers, specific productivity gradually increased to over 0.35 gm 
xanthan/gm cells/hr, and culture morphology became normal again. The 
culture was maintained at this high productivity for some 30 turnovers 
after recovery without any evidence of culture degeneration before being 
terminated by a pH upset. This highly productive culture was saved in a 
viable state and used to isolate the novel Xanthomonas campestris strain 
referred to above. 
After standard plate counts were made on the culture, using YM agar 
(Difco), 9 isolated colonies of varying sizes were picked and subcultured 
on YM agar for purification. Three isolates were discarded as obvious 
duplicates, and the remaining six isolates P-101, P-102, P-104, P-105, 
P-107 and P-108 were maintained for three bi-weekly transfers on YM agar 
slants. The isolates were then subcultured to EMSY-1 broth and then 
maintained on EMSY-1 agar slants as well as on the YM agar slants. In 
addition, cultures grown in EMSY-1 broth were frozen in liquid nitrogen. 
The composition of this (EMSY-1) broth is given in Table II. 
TABLE II 
______________________________________ 
Component Concentration (ppm) 
______________________________________ 
Glucose 10,000 
NH.sub.4 Cl 112 as N 
KH.sub.2 PO.sub.4 386 as P 
Na.sub.2 HPO.sub.4 
390 as P 
MgSO.sub.4.7H.sub.2 O 
40 as Mg 
CaCl.sub.2.2H.sub.2 O 
10 as Ca 
NaCl 10 as Na 
FeCl.6H.sub.2 O 1 as Fe 
ZnSO.sub.4.7H.sub.2 O 
0.33 as Zn 
MnSO.sub.4.H.sub.2 O 
0.1 as Mn 
Na.sub.2 MoO.sub.4.2H.sub.2 O 
0.067 as Mo 
H.sub.3 BO.sub.3 0.033 as B 
KI 0.033 as I 
CuSO.sub.4.5H.sub.2 O 
0.2 as Cu 
Citric Acid 500 
Yeast Extract 400 
______________________________________ 
The Xanthomonas campestris XCP-1 strain was obtained by pooling five of the 
six remaining isolates, i.e., P-101, P-102, P-104, P-105, and P-108, into 
one culture. The XCP-19 strain was obtained from the XCP-1 culture, after 
several transfers, as a single large (4 mm) light yellow colony and this 
isolate was propagated separately. 
STRAIN DESCRIPTIONS 
The above-mentioned strain of Xanthomonas campestris was characterized with 
respect to to Xanthomonas campestris strain NRRL B-1459 from which strain 
XCP-19 was originally derived. The following descriptions characterize the 
strain XCP-19 ATCC 31601. 
I. Cell Morphology 
A. After growth in EMSY-1 broth for 18 hours at 28.degree. C., cells appear 
singly and in pairs with infrequent chains of 3 or 4 cells also occurring. 
Cell dimensions are 0.3-0.6 microns in width by 0.5-1.5 microns in length. 
Often cells in pairs appear almost coccoid. 
B. After growth in YM broth (Difco) for 18 hours at 28.degree. C., cells 
appear singly or in pairs, with chains of 3-4 cells. Chaining is more 
abundant than in EMSY-1 broth. Cell dimensions are larger than in EMSY-1 
broth, being 0.5-0.7 microns in width and 0.75-3 microns in length. 
C. Strain XCP-19 does not exhibit motility, has a negative Gram stain 
reaction, and does not form endospores. These bacteria may be reproduced 
only asexually by means of binary fission. 
II. Colony Morphology 
A. After 72 hours growth at 28.degree. C. on EMSY-1 agar plates, isolated 
colonies are pale cream in color, circular, entire, mucoid, and raised. 
Colony diameter is 2-3 mm. 
B. After 72 hours growth at 28.degree. C. on YM agar (Difco) isolated 
colonies are whitish to creamy in color, circular, entire, mucoid, and 
raised. Typical colony diameter is 3-4 mm. 
C. After 72 hours growth at 28.degree. C. on Nutrient Agar (Difco) 
containing 1% dextrose, isolated colonies are pale yellow in color, 
circular, entire, mucoid, and raised. Colony diameter is about 0.5-2 mm. 
III. Biochemical Characteristics 
In order to determine whether strain XCP-19 is physiologically different 
from Xanthomonas campestris NRRL B-1459, the following tests were 
conducted. 
A. Growth at 35.degree. C. Inoculated slants of YM agar (Difco) and EMSY-1 
agar (see Table II for composition) were incubated at 35.degree. C. for 
five days and the results are shown in Table III. 
TABLE III* 
______________________________________ 
Strain No. YM Agar EMSY-1 Agar 
______________________________________ 
XCP-19 0 0 
B-1459 2+ 0 
______________________________________ 
*0 = no growth; 1+ = slight growth; 4+ = heavy growth 
B. Growth Characteristics in Minimal Medium. Inoculated tubes of liquid 
EMS-2 medium shown in Table IV were incubated at 28.degree. C. for 96 
hours. 
TABLE IV 
______________________________________ 
Component Concentration (ppm) 
______________________________________ 
Glucose 22,500 
NH.sub.4 Cl 224 as N 
KH.sub.2 PO.sub.4 150 as P 
MgSO.sub.4.7H.sub.2 O 
40 as Mg 
CaCl.sub.2.2H.sub.2 O 
10 as Ca 
Citric Acid 500 
FeCl.sub.3.6H.sub.2 O 
2 as Fe 
ZnSO.sub.4.7H.sub.2 O 
0.66 as Zn 
CuSO.sub.4.5H.sub.2 O 
0.4 as Cu 
MnSO.sub.4.H.sub.2 O 
0.2 as Mn 
Na.sub.2 MoO.sub.4.2H.sub.2 O 
0.13 as Mo 
H.sub.3 BO.sub.3 0.066 as B 
KI 0.066 as I 
NaCl 10 as Na 
______________________________________ 
Strain XCP-19 showed heavy growth throughout the tubes with a ragged 
surface pellicle and clumping in the broth. Strain B-1459 showed less 
growth overall and only a slight surface growth. 
C. Hydrolysis of Gelatin, Casein, and Starch. Solid agar media individually 
containing 0.4% gelatin, 0.4% casein, or 0.3% soluble starch were prepared 
and used according to the procedure in "Identification Methods for 
Microbiologists", 1966, B. M. Gibbs and F. A. Skinner, eds., Academic 
Press, p. 12. 
TABLE V 
______________________________________ 
Strain No. Gelatin Casein Starch 
______________________________________ 
XCP-19 4+ 2+ 4+ 
B-1459 4+ 4+ 4+ 
______________________________________ 
As shown in Table V, strain B-1459 showed complete hydrolysis of all three 
substrates, whereas XCP-19 showed lesser degrees of hydrolysis of casein. 
D. Action on Litmus Milk. Cultures inoculated into Litmus Milk medium 
(Difco) were incubated at 28.degree. C. for three weeks, according to the 
method of Ivanoff et. al. (1938, J. Bacteriol. 35 235). Strains XCP-19 and 
B-1459 were both active on litmus milk with peptonization, litmus 
reduction, and precipitate formation. 
E. Hydrogen Sulfide Production. The medium for H.sub.2 S production was 
prepared according to the method of Hayward and Hotchkiss (1961, J. Gen. 
Microbiol. 26, 133-140). H.sub.2 S production was determined by the use of 
lead acetate paper strips suspended over the medium in loosely capped 
tubes. The cultures were incubated for six days at 28.degree. C. and 
observed for blackening of the strips. Each of the strains produced 
hydrogen sulfide. 
F. Urease Production. Urea medium was prepared according to the method of 
Christensen (1946, J. Bacteriol. 52 461-466). The slants were inoculated 
and incubated at 28.degree. C. for 14 days. A red to violet color in the 
medium would be indicative of urea hydrolysis. Urease production was found 
to be negative for each of the strains tested. 
G. Growth in Presence of Salt. Basal media containing NaCl at 
concentrations of 1, 2, 3, 4, and 5% were prepared according to the method 
of Hayward and Hotchkiss (1961. J. Gen. Microbiol. 26 133-140). Cultures 
were inoculated and incubated at 28.degree. C. for 14 days. Both strains 
tested gave an identical growth pattern as shown in Table VI. 
TABLE VI 
______________________________________ 
Salt Conc'n 
Strain No. 
1% 2% 3% 4% 5% 
______________________________________ 
XCP-19 4+ 3+ 3+ 2+ 0 
B-1459 4+ 3+ 3+ 2+ 0 
______________________________________ 
H. Carbohydrate Assimilation Pattern. A basal carbohydrate assimilation 
medium was prepared according to the method of Hayward and Hotchkiss 
(1961. J. Gen. Microbiol. 26 133-140). Each strain was inoculated into 
replicate tubes containing the carbohydrates shown in Table VII and 
incubated for 14 days at 28.degree. C. 
TABLE VII 
______________________________________ 
Carbohydrate XCP-19 B-1459 
______________________________________ 
Glucose + + 
Galactose + + 
Arabinose + + 
Mannose + + 
Melibiose + + 
Cellobiose + + 
Sucrose weak weak 
Fructose weak weak 
Trehalose + + 
Xylose - - 
Mannitol + + 
Lactose - - 
Maltose + + 
______________________________________ 
As can be seen in Table VII, both strains gave an identical assimilation 
profile. 
I. Oxidase Production. Using isolated colonies from 72 hour old YM agar 
(Difco) plates, the strains were tested for presence of indophenol oxidase 
using the method of Gaby and Hadley (1957. J. Bacteriol. 74 356-358). Each 
strain was positive for oxidase. 
J. Catalase Production. Growth from a 48 hour YM agar (Difco) slant was 
tested for catalase activity by emulsifying a loopful of culture in a drop 
of 3% H.sub.2 O.sub.2 and observing for effervescence. Strains XCP-19 and 
B-1459 were weakly positive. 
K. Utilization of Organic Acids. EMS-2 basal medium without glucose was 
prepared. Replicate tubes containing 1% citric, malic, succinic, benzoic, 
and tartaric acids were inoculated and incubated at 28.degree. C. for 14 
days and observed for extent of growth. As shown by the results given in 
Table VIII, both strains were identical except that XCP-19 gave slightly 
less growth on citric acid. 
TABLE VIII 
______________________________________ 
Organic Acid XCP-19 B-1459 
______________________________________ 
Citrate 2+ 3+ 
Malate 4+ 4+ 
Succinate 4+ 4+ 
Benzoate 0 0 
Tartrate 1+ 1+ 
______________________________________ 
L. Indole Production. The strains were tested for indole production for the 
same peptone-water medium used to test for H.sub.2 S production, following 
the method of Hayward and Hotchkiss (1961. J. Gen Microbiol. 26 133-140). 
All strains were negative for indole production. 
M. Acetoin Production. The strains were tested for acetoin production using 
MRVP medium (Difco) after incubation of the inoculated cultures for six 
days at 28.degree. C., following the method given in the reference in (L) 
above. Neither strain tested positive for acetoin. 
Summary of Characterization Studies 
Strain XCP-19 is essentially indistinguishable from X. campestris strain 
NRRL B-1459 on the basis of cell morphology. However, definite differences 
in colonial morphology make this strain distinguishable from B-1459. 
XCP-19 produces larger, paler yellow colonies than B-1459 on EMSY-1 and 
Nutrient+Glucose Agar, and almost colorless colonies on YM agar. 
In terms of physiological characteristics, this strain is very similar to 
NRRL B-1459 except that B-1459 grows poorly on a minimal medium. In 
addition, XCP-19 is distinguishable in that it shows no growth on YM agar 
at 35.degree. C., exhibits less active casein hydrolysis and gives less 
growth on citrate. 
The foregoing is intended to point out that, while the major distinguishing 
characteristics of the XCP-19 strain lie in its high xanthan specific 
productivity and resistance to degeneration in continuous culture, other 
distinguishing characteristics nevertheless are present. 
In carrying out the process of the present invention, the fermenter medium 
is seeded with an inoculum of culture grown in the same medium as that to 
be used for fermentation at an inoculum level of 5-10% of the medium 
volume. The culture is grown in a batch mode for 24-48 hours, until a 
desired cell concentration is reached (usually 1.5-2.5 gm cells/l). 
Thereafter, continuous flow of medium is started into the fermenter such 
that the dilution rate is 75% or less of the specific growth rate at which 
the organism is growing at that point. Continuous harvesting of a volume 
of culture broth equal to the volume of medium introduced is also carried 
out. After approximately two culture turnovers, the dilution rate is 
adjusted as desired. Xanthan gum, which exists in the recovered broth, can 
be used without further purification, or filtered to remove cells, or can 
be precipitated with an alcohol, such as ethyl or isopropyl alcohol, with 
or without initial cell removal. The medium used in this process is a 
preferably minimal medium consisting primarily of inorganic salts, 
NH.sub.4 Cl, glucose, and citric acid, with or without additional yeast 
extract or yeast autolysate. 
The term "minimal medium" as used throughout the present description and 
claims should be interpreted to cover media of the type generally referred 
to herein and specifically in the Examples, together with modifications 
apparent to those skilled in this field. 
Operating conditions to be employed in the process of our invention include 
the following: 
Agitation: 100-2000 rpm 
Preferably: 500-1000 rpm 
Air Rate: 0.1-2 vol./vol./min. 
Preferably: 0.5-1 vol./vol./min. 
Temperature: 20.degree.-35.degree. C. 
Preferably: 25.degree.-30.degree. C. 
pH: 5-8 
Preferably: 6.4-7.4 
Dissolved Oxygen: 10-90% saturation 
Preferably: 20-60% saturation 
Dilution Rate: 0.01-0.15 hr.sup.-1 
Preferably: 0.04-0.1 hr.sup.-1 
Our invention will be illustrated by reference to the following specific 
examples:

EXAMPLE I 
This Example shows that when Xanthomonas campestris NRRL B-1459, is grown 
in a minimal medium in continuous culture, the organism exhibits only low 
specific productivity and degenerates in a short time. The culture was 
grown in a 28 liter fermenter in a minimal medium having a composition 
shown in Table IX. 
TABLE IX 
______________________________________ 
Component Concentration (ppm) 
______________________________________ 
Glucose 22,000 
NH.sub.4 Cl 300 as N 
KOH 1,000 as K 
H.sub.3 PO.sub.4 150 as P 
MgSO.sub.4 40 as Mg 
CaCl.sub.2 10 as Ca 
NaCl 10 as Na 
Citric Acid 500 
FeSO.sub.4 3 as Fe 
ZnSO.sub.4 1 as Zn 
MnSO.sub.4 0.3 as Mn 
Na.sub.2 MoO.sub.4 
0.2 as Mo 
H.sub.3 BO.sub.3 0.1 as B 
KI 0.1 as I 
CuSO.sub.4 0.6 as Cu 
______________________________________ 
Xanthomonas campestris NRRL B-1459 was maintained on YM agar (Difco) slants 
at 4.degree. C. and transferred to fresh agar slants at bi-weekly 
intervals. For inoculum preparation, a loopful of culture from a fresh (&lt;3 
day old) slant was inoculated into a 16.times.125 mm tube containing 7 ml 
of YM broth. The culture was incubated at 28.degree. C. on a rotary shaker 
at 150 rpm, at a 20.degree. inclination for 18 hours. At this point, the 
contents of the tube were transferred to 50 ml YM broth in a 500 ml 
Erlenmeyer flask, and incubated at 28.degree. C. on a rotary shaker at 250 
RPM for 18-24 hours. Next, the contents of the flask were transferred to a 
2000 ml Fernbach flask containing 700 ml of a mineral 
salt-glucose-NH.sub.4 Cl medium, of the composition given above in Table 
III. This was incubated under the same conditions as for the 50 ml flask, 
but for a total of 40 hours. Next, the entire culture was used to 
inoculate 20 liters of the same medium contained in a 28 liter New 
Brunswick fermenter (Model CMF-128S). The initial operating conditions 
employed were as follows: 
Temperature--29.degree. C. 
pH--6.0 
Agitation--230 rpm 
Air Rate--0.2-0.4 vol/vol/min 
Dissolved O.sub.2 --90% saturation 
After an initial growth lag of about 30 hours, cell growth proceeded over 
the next 30 hours. When the cell concentration reached 0.9 gm/liter, 
continuous operation was started at an initial dilution rate of 0.07 
hr.sup.-1. Within 48 hours, the cell concentration rose to 2.5 gm/liter. 
After about 10 culture turnovers, the viscosity and specific productivity 
started to decline and were eventually almost totally lost. Cell 
morphology became abnormal and gum quality deteriorated badly. All of 
these changes proved to be irreversible and the culture did not revert to 
normal. The results obtained in this run are shown in Table X. 
TABLE X 
__________________________________________________________________________ 
Xanthan 
Xanthan 
Time Cell Xanthan Volumetric 
Specific Total 
Period 
Conc'n 
Conc'n 
Viscosity 
Productivity 
Productivity 
Dilution 
Culture 
(Hrs) 
(gm/l) 
(%) (cp) (gm/l/hr) 
(gm/gm cells/hr) 
Rate (hr.sup.-1) 
Turnovers 
__________________________________________________________________________ 
0-34 
1.3-1.9 
.26-.30 
120-340 
.20 .11-.15 .07-.08 
0-2.5 
34-130 
2.6-2.56 
.32-.39 
640-850 
.23-.31 
.10-.13 .07-.085 
2.5-9.7 
130-178 
1.7-2.17 
.23-.265 
420-430 
.17-.22 
.09-.10 .07-.08 
9.7-13.7 
178-202 
1.0 .187 160 .13 .13 .07 13.5-15 
202-266 
.6-1.1 
.12-.14 
28-48 
.1-.11 
.09-.175 .077 15-20.1 
__________________________________________________________________________ 
EXAMPLE II 
In this example, Xanthomonas campestris XCP-19 ATCC 31601 was grown in 
continuous culture in a medium (EMS-2) supplemented with 0.04% yeast 
extract having the following composition: 
TABLE XI 
______________________________________ 
Composition of EMS-2 Medium 
Component Concentration (ppm) 
______________________________________ 
NH.sub.4 Cl 224 as N 
KH.sub.2 PO.sub.4 150 as P 
MgSO.sub.4.7H.sub.2 O 
40 as Mg 
CaCl.sub.2.2H.sub.2 O 
10 as Ca 
NaCl 10 as Na 
Citric Acid 500 
FeCl.sub.3.6H.sub.2 O 
2 as Fe 
ZnSO.sub.4 0.66 as Zn 
MnSO.sub.4 0.2 as Mn 
CuSO.sub.4 0.4 as Cu 
Na.sub.2 MoO.sub.4 
0.13 as Mo 
H.sub.3 BO.sub.3 0.066 as B 
KI 0.066 as I 
Glucose 22,500 
______________________________________ 
For inoculum preparation, a loopful of culture from a fresh (&lt;3 day old) 
slant was inoculated into a 16.times.125 mm tube containing 7 ml of EMSY-1 
broth. The culture was incubated at 28.degree. C. on a rotary shaker at 
150 rpm, at a 20.degree. C. inclination for 18 hours. At this point, the 
contents of the tube were transferred to 50 ml EMS-2 broth (containing 
0.04% YE (see Table XI) in a 500 ml Erlenmeyer flask, and incubated at 
28.degree. C. on a rotary shaker at 250 RPM for 18-24 hours. Next, 10 ml 
volumes of the culture were inoculated into each of two 1000 ml Erlenmeyer 
flasks containing 100 ml of EMS-2 medium, plus 0.04% yeast extract. These 
were incubated at 28.degree. C. on a rotary shaker at 250 rpm for 18 to 24 
hours. The culture contents of both flasks were combined and 150 ml of the 
culture was used to seed 3000 ml of EMS-2 medium, plus 0.04% yeast 
extract, contained in a 7.5 liter fermenter (New Brunswick Model MF-107). 
The culture of Xanthomonas campestris XCP-19 ATCC 31601 was grown for 56 
culture turnovers (720 hours) without degeneration under essentially the 
same conditions as employed in Example I. For the first 24 turnovers, the 
specific productivity averaged 0.253 gram xanthan/gram cells per hour. 
When yeast extract concentration was reduced to 0.02%, specific 
productivity was adversely affected and decreased to an average of 0.23 gm 
xanthan/gm cells/hr. When the yeast extract concentration was restored to 
0.04% again, the specific productivity came back to 0.25 gram xanthan/gram 
cells/hour. This shows that 0.04% yeast extract is optimum for this 
particular strain, and when deprived of yeast extract under conditions 
where it is present initially, the culture is adversely affected but 
improves upon restoration of the yeast extract to original levels. The 
data obtained in this run are given in Table XII below and demonstrate 
that Xanthomonas campestris XCP-19 ATCC 31601 is stable at high specific 
productivities under conditions where the parent would degenerate and 
exhibit lower specific productivity. 
TABLE XII 
__________________________________________________________________________ 
Xanthan 
Xanthan 
Time Cell Xanthan Volumetric 
Specific Total 
Period 
Conc'n 
Conc'n 
Viscosity 
Productivity 
Productivity 
Dilution 
Culture 
(Hrs) 
(gm/l) 
(%) (cp) (gm/l/hr) 
(gm/gm cells/hr) 
Rate (hr.sup.-1) 
Turnovers 
__________________________________________________________________________ 
0-325 
2.3-2.55 
.71-.87 
1970-2570 
.57-.64 
0.253 .07-.083 
0-23.9 
reduced YE to 0.04% 
325-509 
2.08-2.19 
.58-.79 
1345-2347 
.48-.64 
0.23 .078-.085 
27.9-39.9 
restored YE to 0.04% 
509-720 
2.09-2.29 
.63-.72 
1490-1735 
.50-.58 
0.25 .078-.082 
39.9-56.1 
__________________________________________________________________________ 
EXAMPLE III 
In this example, Xanthomonas campestris XCP-19 ATCC 31601 was grown in 
continuous culture in the same minimal medium as employed in Example II, 
EMS-2, but without yeast extract. 
Inoculation procedures were the same as in Example II except that the 50 ml 
and 100 ml cultures were grown in the EMS-2 medium mentioned immediately 
above. The fermentation was inoculated and the culture was grown in a 
batch mode under operating conditions identical to those in Example II. 
Batch growth proceeded only very slowly for the first 44 hours. At that 
point, the concentration of iron and trace elements (Zn, Cu, Mn, Mo, B, I) 
was doubled in the fermenter. Over the next six hours, growth was more 
rapid and the cell concentration reached about 1.5 gm/liter. Continuous 
operation was started at a dilution rate of 0.046 hr.sup.-1 again under 
operating conditions identical to those in Example II. This run lasted 
1,216 hours for a total of 92 culture turnovers. A very high specific 
productivity of 0.3 to 0.33 gm xanthan/gm cells/hr was maintained for 386 
hours during part of the run. At no time was the specific productivity 
less than 0.22. The results for this particular run are given in Table 
XIII. 
TABLE XIII 
__________________________________________________________________________ 
Xanthan 
Xanthan 
Time Cell 
Xanthan Volumetric 
Specific Total 
Period 
Conc'n 
Conc'n 
Viscosity 
Productivity 
Productivity 
Dilution 
Culture 
(Hrs) (gm/l) 
(%) (cp) (gm/l/hr) 
(gm/gm cells/hr) 
Rate (hr.sup.-1) 
Turnovers 
__________________________________________________________________________ 
(2X conc'n of TM, Fe)* 
0-152 
1.96 
0.82 2180 0.43 0.22 0.05 0-8.2 
(1X conc'n of TM, Fe) 
152-338 
1.98 
0.89 2535 0.49 0.25 0.055 8.2-18.8 
(2X conc'n of TM, Fe) 
338-505 
2.0 0.72 1680 0.59 0.30 0.08 16.8-32.6 
505-646 
1.92 
0.67 1630 0.59 0.31 0.087 32.6-43.9 
646-724 
1.97 
0.65 1400 0.64 0.33 0.10 43.9-51.3 
(3X conc'n of TM, Fe) 
724-817 
1.95 
0.53 980 0.51 0.26 0.10 51.3-61.3 
817-913 
2.07 
0.67 1430 0.55 0.27 0.082 61.3-69.2 
(2X conc'n of TM, Fe) 
913-1117 
1.93 
0.60 1170 0.48 0.25 0.08 69.2-84.5 
1117-1216 
1.87 
0.55 1000 0.45 0.24 0.08 84.5-92.6 
__________________________________________________________________________ 
*The concentrations of trace elements (TM = Zn, Cu, Mn, Mo, B, I) and Fe 
were altered periodically between singlestrength (IX) as shown above and 
2X and 3X. 
It will be apparent from the foregoing description that by the use of the 
above-mentioned novel strain it is now possible to design and operate a 
long-term continuous culture process for xanthan production in which the 
culture does not degrade, cheap simple media are used, and xanthan can be 
produced at a high specific productivity, thus lowering the overall 
economics of the process.