Semi-Continuous Method for Production of Xanthan Gum Using Xanthomanas Campestris ATCC 31600 and Xanthomanas Campestris ATCC 31602

The present invention relates to the manufacture of xanthan gum, hereinafter referred to as "xanthan". More particularly, it is concerned with an improved method for the production of xanthan by means of a semi-continuous fermentation process.

Xanthan gum and similar heteropolysaccharides are currently manufactured by means of fermenting a suitable nutrient medium with a Xanthomonas organism, typically Xanthomonas campestris B-1459. The process employed involves the use of a batch method in which the inoculum medium is allowed to ferment for a period of 36-72 hours under aerobic conditions. The xanthan gum thus produced is isolated from the other components of the medium by precipitation with acetone or a low molecular weight alcohol such as methanol, in a known manner. However, because of the time required to ferment each batch, the low xanthan content of the fermented medium, and the processing required for the recovery and purification of the product, xanthan produced by batch fermentation is relatively expensive.

Insofar as I am aware, all of the installed plant capacity presently in use for the manufacture of xanthan by fermentation methods is restricted to use of the batch fermentation process. In using this particular technique, one of the chief disadvantages is the lag time required to prepare adequate quantities of inoculum (seed) for each batch run. For the quantities of nutrient medium involved in industrial operations, as much as four days time is needed to provide enough inoculum. After the fermentation has been completed and the product xanthan separated, the spent mash in the fermenter must be withdrawn and the fermenter cleaned out and sterilized before a new charge of sterile medium can be introduced.

Xanthan gum has potential application as a film forming agent, as thickeners for bodybuilding agents in edible products, cosmetic preparations, pharmaceuticals, vehicles, oilfield drilling fluids, fracturing liquids, and similar compositions and as emulsifying, stabilizing and sizing agents. Xanthan also has a potential large volume use as a mobility control agent in micellar-polymer flooding. This gum possesses excellent viscosifying properties at low concentration and a resistance to shear degradation and exhibits only minimal losses in viscosity as a function of temperature, pH, and ionic strength. For these reasons, xanthan is an attractive alternative to synthetic polyacrylamides for enhanced oil recovery operations.

By the present invention, the efficiency of conventional batch-type plants can be substantially improved by employing semi-continuous fermentation. This process can be effectively conducted in existing batch-type plants with a minimum of modification. In semi-continuous fermentation, the fermenter is filled to the desired volume with a suitable sterile growth medium. A culture of the desired microorganism is then introduced into the growth medium and growth and/or product formation are allowed to occur under known conditions. When the fermentation is complete, a volume of culture broth, amounting to from 25 to 90 percent of the total volume, is withdrawn and the desired culture or product is recovered. Thereafter, a volume of sterilized fresh medium is introduced into the fermenter generally equivalent to the volume withdrawn. The microorganisims remaining in the fermenter resume growth and/or product formation when placed in contact with the sterile medium. After fermentation is complete, the cycle is repeated. This process is usually referred to as "serial culture" or "serial transfer". The precedure is considered to be semi-continuous in that no steady-state condition is reached as in continuous fermentation but the need for fermenter cleanup and preparation of fresh culture inoculum is avoided as is required in the batch process.

However, in the production of xanthan by semi-continuous fermentation using the common strains of Xanthomonas campestris, such as Xanthomonas campestris NRRL B-1459 which is generally used, are subjected to degeneration after just a few serial transfers. The result of this degeneration is a loss of xanthan-producing ability and the appearance of atypical bacterial variants. This phenomenon is documented by Norton, Faulk, & Luetzelschwab (SPE Paper No. 8420, 54th AIME Meeting, 1979).

I have discovered, however, that successful production of xanthan in good yield can be obtained by the use of a novel degenerative resistant Xanthomonas campestris organisms identified as Xanthomonas campestris XCP-1 ATCC 31600 and Xanthomonas campestris P-107 ATCC 31602, and fully described in copending applications Ser. No. 168,885, filed July 1980, and Ser. No. 168,882, filed July 14, 1980, respectively.

Subcultures of these living organisms can be obtained upon request from the permanent collection of the American Type Culture Collection, 12301 Parklawn Drive, Rockville, MD 20852. The accession numbers in this reponsitory for Xanthomonas campestris XCP-1 and P-107 are given above.

The medium employed may be an inexpensive minimal medium consisting primarily of essential 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. In general, it may be said that any nutrient medium containing essential salts and assimilable sources of carbon and nitrogen may be employed. 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 example, together with modifications apparent to those skilled in the art.

The operating conditions employed in this semicontinuous fermentation process may vary widely. In general, it may be said that such conditions include the following:

The ability of the XCP-1 and P-107 strains to produce xanthan under prolonged semi-continuous fermentation conditions is illustrated in the following example which also demonstrates the inability of the well-known Xanthomonas organism, Xanthomonas campestris B-1459, to produce xanthan under semicontinuous fermentation conditions after five serial transfers without degeneration of the culture. On the other hand, the XCP-1 and P-107 strains, when subjected to the same semi-continuous fermentation conditions, can undergo 10 or more serial transfers, thus forming the basis for a long-term economical fermentation process.

Inocula of Xanthomonas campestris B-1459 and Xanthomonas campestris XCP-1 ATCC 31600, and Xanthomonas campestris P-107 ATCC 31602, were prepared by transferring a loop full of each organism from a stock culture agar slant to 7 ml of EMSY-1 broth, the composition of which is shown in Table I, followed by incubation at 28.degree. C. and 150 rpm for 18 hours.

Next, the 7 ml cultures were transferred separately to 50 ml of EMS-2 medium, the composition of which is shown in Table II, followed by incubation at 28.degree. C. and 250 rpm for 18-20 hours.

To start the test, 10 ml of each culture were inoculated into 90 ml volumes of EMS-2 medium contained in 1000 ml Erlenmeyer flasks. These were incubated at 28.degree. C., and 250 rpm for 24 hours. At this point, 10 ml volumes of the cultures were inoculated into 1000 ml flasks containing 90 ml of fresh sterile medium and incubated as before. This serial transfer process was repeated a number of times. Xanthan yield was determined on each culture broth after the 24-hour incubation period.

The results given in the Table III show that xanthan productivity by strain B-1459 starts to decline after five serial transfers, whereas strains XCP-1 and P-107 continued to produce xanthan is good yields even after as many as 10 or more serial transfers.

The foregoing description and Example amply demonstrate the ability of strains XCP-1 and P-107 to produce xanthan by fermentation over an extended number of serial transfers, while its closely related strain, Xanthomonas campestris B-1459, is incapable of producing xanthan under semi-continuous fermentation conditions without degeneration.