INOCULATION PROCEDURE FOR XANTHAN GUM PRODUCTION

Abstract: Inoculation of the nutrient medium for production of xanthan gum by aerobic fermentation is effected at a dilution of 0.5-0.01% using an inoculum comprising an organism of the genus Xanthomonas, wherein the total levels of iron, manganese, and zinc in said nutrient me­ dium are limited such that there is no more than 10 ppm iron, no more than 3 ppm zinc and the ppm ratio of iron : manganese is from 40 : 1 to 2 : 1.

Description: "INOCULATION PROCEDURE FOR XANTHAN GUM PRODUCTION" The present invention relates to the production of xanthan gum. Xanthan gum is a polysaccharide elaborated by many species of the genus Xanthamonas. The gum has interesting rheological properties, and is used particularly as a thickener in oil drilling and foodstuffs. Industrial production of xanthan gum is generally by a batch process, in which a culture of a polysaccharide-producing strain of Xanthamonas is Inoculated into a nutrient medium in a fermentation vessel and allowed to ferment for up to several days. Thereafter the gum is usually isolated from the fermented broth by addition of an organic solvent to precipitate the gum. An alternative to batch production production is continuous production, in which after inoculation and initial growth, fresh medium is continuously added to the fermentation vessel and a corresponding amount of fermented broth is continuously withdrawn. The advantages of continuous xanthan gum production, especially regular production, ease of control and smaller fermentation vessel, have been recognized for many years. However, for various reasons, particularly practical difficulties in maintaining extended, productive fermentation, batch production still predominates. An essential part of a process for production of xanthan gum is the inoculation step, in which a culture of the productive strain is added to the nutrient medium as a prelude to the onset of productive fermentation. Typically the nutrient medium used for production has a volume of 10 to 20 times the volume of the inoculum, corresponding to a dilution of 10 to 5%. Greater dilutions are avoided because of the strong possibility that the inoculating strain will not commence active growth in the production medium. Unexpectedly, as part of the discovery of the process described in our copending application of even date claiming priority from UK 81115856 and entitled "Process for Xanthan Gum Production", we have found that under certain conditions it is feasible to employ dilutions of less than 1%. According to the present invention we provide a method of inoculating a production medium with a polysaccharide-producing strain of Xanthomonas for subsequent culturing to produce a polysaccharide, xanthan gum, wherein an inoculum is mixed at dilution of 0.5% or less with a production medium containing iron, manganese,zinc, copper, cobalt and boron in controlled low levels. We have found that inocula ean be successfully employed to inoculate production media on a basis of 1 part by volume of inoculum to at least 200 parts by volume of production medium, provided that the production medium contains low levels of the trace elements. Without being bound by any theory, it appears that the customary higher levels of the specified trace elements have an inhibitory effect at high inoculation dilutions. More particularly, in accordance with the present invention the inoculum is used with a production medium which contains total levels of no more than 10 ppm of iron, no more than 3 ppm of manganese and no more than 3 ppm of zinc ('ppm' in this specification being parts by weight of the. element per million parts by volume of the medium). The volume ratio of inoculum: production medium is preferably 1:500 to 1:10,000, more preferably 1:1000 to 1:5000. By the use of such volume ratios there is the possibility of a substantial saving in manufacturing plant and a concomitant reduction in processing of the inoculum. Consider, for example, a plant wherein the main fermenter vessel has a liquid volume of 1Om . Starting with a culture of volume 1 litre, and assuming a conventional dilution at each stage of about 10%, it Is necessary to go through the sequence: EMI4.1 In contrast, with the present invention, the 1 litre can be used at 10% dilution to produce 10 litre of inoculum which at 0.1% dilution enables direct inoculation of 10 m3 of production medium in the main fermentation vessel. Thus, for the example under consideration, the vessels conventionally needed to hold the 100 litre and the 1 m3 stages as part of the inoculum preparation can now be dispensed with. Not only are there capital savings, but also there is less risk of contamination because there are fewer transfers and also there is generally less work involved. It will be appreciated that, with the tnoculation procedure of the invention, there is normally a longer growing-up period than before. Many hours are required before the bacteria present at a given concentration in 1 volume of inoculum will grow up to the same concentration in 200 or more volumes of production medium. The grow-up time is typically 10 to 100 hours. For this reason, the present invention is best suited to continuous fermentation processes. Measures can be taken to minimise the grow-up time. A particularly preferred measure is to use substantially the same medium for at least the final stage of the.inoculum preparation as is used for the production medium. Thus, for the example considered above where 10 m3 of medium is to be inoculated, the 1 litre of initial culture should be added to a sample of 9 litre of production medium, thereby giving the 10 litre inoculum grown in the same medium. This measure serves to pre-adapt the bacterium to growth in the production medium. Another preferred measure is for the inoculation to be effected when the bacterium is actively growing, rather than in any dormant phase. It is also preferred to minimise grow-up time by controlling the oxygen supply: the air flow and mixing conditions are preferably adjusted to keep the dissolved oxygen tension in the inoculated production medium at above the levels which cause oxygen limitation and below the levels which cause oxygen toxicity. Finally, it is also preferred to minimise grow-up times by use of a medium containing further trace elements and auxillary components at controlled levels. The production medium will contain assimilable sources of carbon and nitrogen. Glucose and ammonium phosphate resRectively, are suitable sources of these elements, but preferably these are supplemented with an auxilliary component which is a complex naturally-derived mixture containing organic nitrogen compounds. In particular, yeast extract should be added at O to 5 g/l, suitably 0 to 1 g/l. Yeast extract or similar auxilliary components appear to help minimise grow-up times, particularly under the 'stress' conditions encountered in the dilution which is employed in the present invention. Moreover, it is preferred that the production medium contains controlled, low total levels of the trace elements copper, cobalt and boron. The copper, cobalt and boron are each preferably at 2 ppm or less, better still 1 ppm or less. Thus, for preference, the production medium contains the following total levels of the trace elements specified in Table 1: Table 1 Trace Element Preferred Level Most Preferred (ppm) Level (ppm) Fe 0.1 to 10 0.5 to 5 Mn 0.01 to 3 0.1 to 1 Zn 0.01 to 3 0.1 to 0.5 Cu 0.001 to 2 0.01 to 0.1 Co 0.001 to 2 0.01 to 0.1 3 0 to 2 0.005 to 0.05 More generally, the present invention usually employs Iron at greater levels than manganese, with the iron: manganese ppm ratio preferably being from 40:1 to 2:1. In preparing a fermentation medium for use in the present process, the various trace elements are suitably added as watersoluble compounds, especially inorganic salts such as FeCl2.6H20 of FeS04.7H2O for iron; MnCl2.6H20 or MnS04.4H20 for manganese; ZnSO4.7H20 for zinc; CuCl2.2H20 or CuSO4.5H20 for copper; CoCl2.6H20 or CoC04.7H20 for cobalt; and H3803 for boron. In adding the trace elements, it is necessary to make due allowance for the amounts present in other components. It is greatly preferred to employ demineralized water prepared for example by passing tap water through an ion exchanger. Equally, it is greatly preferred to analyse the medium after it has been made up, in order to confirm that the controlled, low levels of trace elements have not been exceeded. Other elements will usually be present in the production medium, especially potassium, phosphorus and sulphur. These elements can be added at conventional levels since it is not apparent that any advantage stems from altering the levels. The medium preferably has a pH of 6 to 8, and is preferably held at 29 to 320C for the growing up. The present invention is suited for use with any polysaccharide-producing strain of Xanthomonas, but for preference a strain of X.campestris is used. Once the growing up of the bacterium has been completed to the desired level in the intended volume of medium, the production fermentation can be commenced. With a continuous process, extra medium is continuously added and fermented broth is continuously drawn off. For preference the dilution rate is 0.01 to 0.12 h-l. The fermented broth can be further purified if desired so as to give a solid xanthan gum product. In a preferred process, the broth is heat-treated at 100 to 1400C for 1 to 10 minutes, more preferably 125 to 1300C for 1 to 5 minutes, and then mixed with IPA causing precipitation of the gum which can be filtered off or otherwise separated. The present invention is illustrated by the following examples. Examples 1 to 4 Small scale fermentations Working stocks of X.campestris (derived from ATCC 13951) were maintained on TGY agar slopes for a maximum of 14 days. The TGY medium had the composition shown in Table 2: Table 2 Tryptone 5 gf Glucose 2 gel 1 Yeast Extract 3 gi Agar 20 gl-1 Fermenter inocula for examples 1 and 3 were prepared by suspending the growth on a TGY slope in 10 ml Ringers solutions (1/4 strength) and using 2 ml to inoculate 100ml MYGP in 500ml shake flasks. The MYGP medium had the composition shown in Table 3: Table 3 Malt Extract 3 gl-1 Yeast Extract 3 gl-1 Peptone 5 gl-1 Glucose 10 gl-1 After 24 h incubation at 300C on a gyrotatory shaker the MYGP broth was used for inoculation. For Examples 2 and 4 a sample from a continuous culture was used as an inoculum. The continuous culture had been set in operation in the way described for Example 1 except that 10% inoculum level was employed. For each of Examples 1 to 4, 2 litre scale Xanthomonas fermentations were started using 0.1 or 0.01% inoculum levels to inoculate media of the composition shown in Table 4 when read in conjunction with Table 5. Table 4 Medium composition for fermenter cultures Examples 1 to 4 Example 5 Dexyme 081 Glucose Syrup 60 gl 53 gl-1 Citric Acid 1 gl-1 1.0 gl -1 Acetic Acid 0.45 ml Ca(OH)2 0.05 gl-1 0.05 gl-1 Mg(OH)2 0.0775 gl-1 0.07 gl-1 MgSO4#7H2O 0.058 gl-1 0.086 gl-1 (NH4)2HPO4 3.0 gl-1 2.38 gl-1 K2HPO4 1.0 gl-1 1.0 gl-1 MnSO4#H2O 0.83 mgl-1 0.83 mgl-1 ZnSO4#7H2O 1.42 mgl-1 0.71 mgl-1 CuSO4#5H2O 0.25 mgl-1 0.25 mgl-1 CoSO4#7H2O 0.28 mgl-1 0.28 mgl-1 H3BO4 0.06 mgl-1 0.06 mgl-1 FeSO4#7H2O 35.6 mgl-1 35.6 mgl-1 Yeast Extract O or 0.2 l-1 0.2 2 gl-1 Deionized Water to 1000 ml to 1000 ml The glucose syrup was selected because of its purity, the Dexyme 081 being low in trace elements. In ppm, the syrup contained the following: Zn, less than 0.01; Cu, less than 0.2; Mn, less than 0.04; Co, less than 0.08; Fe, less than 0.18. The yeast extract was a special low salts yeast extract, and contained in ppm: Zn, 60; Cu, 10; Mn, 15; Co, less than 5; Fe, 100. The other reagents were of analytical grade. Table 5 EMI11.1 <tb> Example <SEP> No. <SEP> Inoculum <SEP> preparation <SEP> Inoculum <SEP> Yeast <SEP> Extract <tb> <SEP> level <SEP> (% <SEP> v/v) <tb> <SEP> 1 <SEP> Shake <SEP> flask; <SEP> MYGP <SEP> 0.1 <SEP> No <tb> <SEP> 2 <SEP> Continuous <SEP> culture; <tb> <SEP> Production <SEP> medium <SEP> 0.1 <SEP> Yes <tb> <SEP> 3 <SEP> Shake <SEP> flask; <SEP> MYGP <SEP> 0.1 <SEP> No <tb> <SEP> 4 <SEP> Continuous <SEP> culture; <tb> <SEP> Production <SEP> medium <SEP> 0.01 <SEP> No <tb> Time <SEP> to <SEP> reach <SEP> E <SEP> 540 <SEP> of <SEP> 3.0 <SEP> (h) <tb> <SEP> 86 <tb> <SEP> 50 <tb> <SEP> 90 <tb> <SEP> 106 <tb> Each culture was monitored during batch phase by measuring regularly the cell mass using turbidity measurements (adsorption at 540 nm(E540)) and dissolved oxygen tension (DOT). To avoid oxygen limitation or oxygen toxicity, the air flow and stirring speed were adjusted to keep the DOT within the range 15-45X of air saturation. For the 0.1% inoculum level cultures this meant an initial air flow of 1.0 1 min and a stirring speed of 50 rpm. For the culture inoculated at 0.01% level no stirring was necessary until 85 h, the agitation from the air flow of 1.01 min -1 providing adequate mixing and oxygen transfer. The air flow was later turned up to 2.01 mien 1 and the stirring speed increased as the DOT dropped below 15%. Other fermentation conditions were temperature 30 + 0.050C, pH 7.0 + 0.1 and ungassed working volume 2 litre. For each of Examples 1 to 4, the inoculation was successful and continuous fermentation to produce xanthan gum was possible. The cultures differed in the time taken to reach the desired working biomass level before the switch from batch to continuous operation and feasible, as indicated in Table 5 for the time to reach E540 of 3.0, corresponding to a cell mass of approximately 2g dry weight per litre. Example 5 Pilot Scale Fermentation An inoculum was prepared in a 20 l vessel containing the medium indicated in Table 4. The medium was Inoculated with 100 ml of MYGP broth from a shake flask incubated at 30 C for 48 h following inoculation from a TGY slope. Initial fermentation conditions were: temperature, 30 + 0.05 C, pH, 7.0 + 0.1; air flow, 2.0 litre rnn1; impeller speed, 50 rpm. As the DOT fell the air was turned up to 4.0 litre mi and the stirring increased in steps to 350 rpm keeping the DOT between 10 and 50% of air saturation. The culture was ready for inoculating a 1000 litre culture after 58 h when E 540 was 2.3. The inoculum was added at 0.1% to 1000 litre of the same medium. Initial conditions were:temperature, 31 + 1 0C; pH, 6.8 + 0.1;' Impeller speed, 35 rpm; working volume, 980 1; overpressure, 27 kPa. After 21 h the air flow and impeller speed were stepped up to keep the DOT at about 30%. As the DOT fell the stirring was stepped up gradually to a maximum of 280 rpm and the air flow and overpressure were increased to a maximum of 1080 litre mien¯1 and 100 kPa, such that DOT was maintained at 40-60% of saturation under the initial operating conditions. The procedure produced a cell doubling time of 5 h. The product concentration reached 24 gel 1 at 46 h after inoculation, at which time the broth viscosity was about 26000 cps. Medium inflow for continuous operation was then begun, leading to production of xanthan gum broth on a continuous basis.

Claims: CLAIMS: 1. A process of producing xanthan gum by aerobic fermentation of a nutrient medium, in which inoculation of the nutrient medium is effected at a dilution of 0.5-0.01% using an inoculum comprising an organism of the genus Xanthomonas, wherein the total levels of iron, manganese, and zinc in said nutrient medium are limited such that there is no more than 10 ppm iron, no more than 3 ppm zinc and the ppm ratio of iron:manganese is from 40:1 to 2:1. 2. A process of Claim 1, wherein the organism has the identifying characteristics of X. campestris, preferably the strain ATCC 13951, and the dilution is 0.1-0.2%. 3. A process of Claim 1, wherein the composition of the inoculum is substantially the same as the composition of the nutrient medium. 4. A process of Claim 1, wherein the inoculation is effected while the organism is actively growing. 5. A process of Claim 1, wherein the nutrient medium further comprises a complex naturally derived mixture of organic nitrogen compounds. 6. A process of Claim 1, wherein the nutrient medium comprises no more than 2 ppm copper, cobalt or boron. 7. A process of Claim 1, wherein the nutrient medium comprises: ppm iron 0.1 - 10 manganese 0.01 - 3 zinc 0.01 - 3 copper 0.001 - 2 cobalt 0.001 - 2 boron 0-2 8. A process of Claim 6, wherein the nutrient medium comprises: ppm iron 0.5 - 5 manganese 0.1 - 1 zinc 0.1 - 0.5 copper 0.01 - 0.1 cobalt 0.01 - 0.1 boron 0.005 - 0.05 9. A process of Claim 1 which is a continuous process with a dilution rate of 0.01 to 0.12 h-1, 10. A process of Claim 1 comprising the further step cf treating the fermented medium at 100 - 140 C for 1 - 10 minutes prior to recovery of the xanthan gum.

Inventors: RODNEY, JARMAN TREVOR
Applicants: KELCO BIOSPECIALTIES LIMITED
Application year: 1982
Main classification: C12P19
IPC codes: C12P19, C12R1