HU194941B - Process for preparing polysaccharides of xanthane type - Google Patents

Abstract

A PROCEDURE FOR THE PRODUCTION OF POLYACARIDES BY FERMENTATION OF AN AQUEOUS NUTRITIVE MEANS BY ACTION OF BACTERIA OF THE GENRE XAMTHOMONAS CAMPESTRIS. THE CULTIVATION MEANS COMPRISES AN EMULSION OF OIL-IN-WATER OF A COMPONENT CHOSEN BETWEEN VEGETABLE OILS AND LIQUID HYDROCARBONS WITH EBULLITION POINT GREATER THAN 140 ° C IN PRESENCE OF RENT FENOLES OR ETHODILATED FATTY ALCOHOLS COMCOMMONENT STABILIZER OF SUCH EMULSION AND EVENTUALLY CONCENTRATE THE ENVIRONMENT OF CULTIVATION TO TAKE THE CONTENT OF POLYACARIDOS PRODUCED BETWEEN 8 AND 60 ° IN WEIGHT.

Description

The present invention relates to a process for the production of xanthan-type polysaccharides by the fermentation of microorganisms on carbohydrates. More particularly, the invention relates to fermentation in an emulsion.

Heteropolysaccharides or biopolymers produced by fermentation of carbohydrates by bacteria of the genus Xanthomonas or Arthrobacter or by fungi of the genus Sclerotium or other similar microorganisms have industrial significance due to their thickening and viscosity-reducing properties.

The preparation of xanthan polymer by aerobic fermentation in an aqueous medium is described, for example, in U.S. Pat. No. 3,000,790, 3,020,206;

391,060, 3,433,708, 4,119,546, 4,154,654,

No. 296,203 and U.S. Pat. No. 4,377,637. United States and U.S. Patent No. 2,414,555. French patent specification.

At the end of the production process, the fermentation broth may contain about 15-50 g / l polysaccharide. At industrial scale it is very difficult to achieve levels above 30-35 g / l without the use of special methods. In fact, as a result of the formation of the polysaccharide, the viscosity of the reaction medium increases, slowing down oxygen delivery and inhibiting fermentation. Even if the reactors are equipped with strong stirrers, which is a costly solution in terms of energy consumption, it is difficult to achieve proper aeration and agitation of the reaction medium to such an extent that the desired concentration of polymer is raised.

Nos. 0 058 364, 0 074 775 and 0 098 473 EP patent applications propose a solution to increase the concentration of the polymer by fermentation in an oil-in-oil emulsion or dispersion of the aqueous medium, thereby reducing the viscosity of the fermentation broth and improving the oxygen transport.

However, it seems desirable to perform the first stage of growth of the microorganism in an aqueous medium before the oil is added and a water-in-oil emulsion is formed. Once a water-in-oil emulsion is formed, it is difficult to adjust the pH of the dispersed aqueous phase and there is a risk of formation of an acidic by-product which inhibits the growth of microorganisms. It is possible to work in a buffered medium, but the presence of a buffer can be detrimental to the resulting solution. In addition, a stable water-in-oil emulsion is difficult to dilute, which is disadvantageous if the emulsion is to be used directly, for example in oil wells.

It is an object of the present invention to provide a fermentation process which allows the growth of microorganisms in an emulsified medium.

The present invention thus relates to a process for the preparation of xanthan-type polysaccharides by fermentation, whereby a stable polysaccharide emulsion containing up to about θθ%, preferably 3-60% polysaccharides is obtained. According to the present invention, a polysaccharide-producing microorganism is fermented in an aqueous medium in which the oil is dispersed to form an oil-in-water emulsion in which the fermentation is carried out.

The aqueous phase may be any aqueous fermentation medium known in the art. For a detailed description, see, for example, U.S. Patent Nos. 3,391,060 and 3,433,708. United States and U.S. Patent No. 2,414,555. French patents. Conventional media include a source of carbohydrates, which may be sugars or other sources of carbohydrates, an organic and / or inorganic nitrogen source, and trace elements and, if desired, growth factors. Sugar, e.g. glucose or sucrose may be present in a concentration of 10 to 100 g / l, but may also be in the range of 150 to 200 g / l. A microorganism is a bacterium which is mentioned as a carbohydrate degrading agent in BERGEY'S MANUAL OF DETERMINATIVE BACTERIOLOGY (8th ed. (1974), Williams N., Wilkins Co., Baltimore), such as Xanthomonas begoniae, Xanthomonas campestris, Xanthom, Xanthom, Xanthomonas incanae, Xanthomonas malvacearum, Xanthomonas papavericola, Xanthomonas phaseoli, Xanthomonas pisi, Xanthomonas vasculorum, Xanthomonas vericatoria, Xanthomonas vitians, Xanthomonas pelargonii; bacteria of the genus Arthrobacter, in particular Arthrobacter stable, Arthrobacter viscosus; members of the Erwinia genus; members of the genus Azotobacter, in particular the strain Azotobacter indicus; members of the genus Agrobacterium, in particular Agrobacterium radiobacter, Agrobacterium rhizogenes Agrobacterium tumefaciens. Fungi of the genus Sclerotium may also be used.

Among these microorganisms, it is preferred to use Xanthomonas campestris.

As for the oily phase, any mineral or vegetable oil that is immiscible with water, such as isoparaffins, deodorized kerosene, high, e.g. Hydrocarbons, rapeseed, soybean, maize, sunflower and peanut oils, boiling in the range of 150 ° C to 200 ° C, etc. It is preferred that the ratio of the oily phase to the total medium is less than 30% by volume, preferably 1 to 18% by volume, preferably 3 to 16% by volume.

Dispersion and stabilization of the oily phase in the aqueous phase may be facilitated by the use of a surfactant. Such a surfactant may be, for example, preferably a nonionic surfactant having an HLB value preferably below 11, more preferably between 6 and 11, this value being achieved using a single surfactant or a mixture of 2194941 surfactants. The selection and quantification of the surfactant can be readily accomplished by one skilled in the art according to the type of oil used and its concentration.

Nonionic surfactants include compounds which are prepared by condensation of alkylene oxide with aliphatic or alkyl aromatic organic compounds. Suitable surfactants that do not inhibit the growth of microorganisms include, for example, polyoxyethylene alkylated phenols, polyoxyethylene alcohols, polyoxyethylene fatty acids, polyoxyethylene triglycerides, and polyoxyethylene esters of sorbitan and fatty acids.

Said surfactants may be used singly or in combination. Depending on their affinity, the surfactant or all or a portion of the substances may be added to the oily or aqueous phase.

It is desirable to sterilize the aqueous and oily phases separately. After sterilization and cooling, the oily phase is admixed to the aqueous phase in the fermenter, but it is also possible to continuously introduce the aqueous and oily phases into the fermentor with stirring. A stable emulsion is formed immediately. Inoculation and fermentation are then carried out in the usual manner, with aeration and stirring. During the fermentation, the pH is maintained at optimum value by addition of alkali or ammonium hydroxide. It is also possible to periodically or continuously replenish the growth medium.

At the end of the fermentation, depending on the use, the emulsion itself can be used or it can be broken down in a known manner and the aqueous phase is separated from the oily phase and the fermentation broth can be used crude or precipitated therefrom. the polysaccharide, preferably a lower alcohol, e.g. isopropanol and optionally inorganic salts. The precipitated polysaccharide is separated off, washed with oil, dried and optionally ground. This provides a ready-to-use port.

According to a preferred embodiment of the invention, the emulsion obtained after completion of the fermentation is concentrated to a polysaccharide content of 8 to 60% by weight, preferably 15 to 60% by weight. In this case, the amount of oil used in the fermentation medium is calculated on the basis of the dry matter content at the end of the fermentation. Preferably, the amount of oil in the final emulsion should not exceed 45%.

The water can be removed, for example, by evaporation or distillation, optionally under reduced pressure. A portion of the oily phase may also be removed in the form of an azeotropic or non-azeotropic mixture with water. Alternatively, ultrafiltration may be carried out using conventional techniques and equipment, provided, of course, that a hydrophilic membrane with pores small enough to retain the biopolymer is selected.

The use of ultrafiltration ensures that the emulsion is maintained in the continuous aqueous phase. A further advantage of concentrating by ultrafiltration is that the inorganic salt content in the aqueous phase remains close to that of the original ferric liquor, regardless of the polymer content.

Alternatively, the emulsion may be concentrated first by ultrafiltration and then by some other means, such as evaporation or distillation.

It is also within the scope of the present invention to add bactericides, enzymes, or other additives to the resulting emulsion, provided that such additives do not adversely affect the stabilization of the emulsion.

The emulsion obtained by the process according to the invention has the advantage over the reverse emulsion that the microorganisms in the emulsified medium grow at a rate close to normal. The growth of microorganisms is not disturbed by the emulsion. Energy can be saved during the growth phase by reducing the energy required to mix the medium. It is possible to control the pH of the aqueous medium. It is possible to concentrate the emulsion without inverting the emulsion and precipitating the polysaccharide.

The oil-in-water emulsion produced by the process of the present invention can be used in any area where high viscosity aqueous liquids are required, e.g. construction works, paints, cosmetics, plant protection, oil industry, etc. These emulsions have the advantage that they can be used directly in dilute aqueous solutions without the need for an intermediate phase change. They are well dispersible in water and have a dissolution rate at least as good as the original fermentation broth. I can pump well and remain stable over a wide concentration range. Their viscosity is substantially lower than that of an aqueous solution containing the same concentration of biopolymer, so that we have an emulsion that can be filled and pumped and contains high concentrations of the polysaccharide in the aqueous phase.

In view of the above-mentioned advantages, the emulsions produced by the process of the invention are particularly suitable for use in oil extraction for the preparation of aqueous liquids for enhanced oil extraction.

The following examples illustrate the process of the invention.

The following general methods are used in the methods described in the examples:

-319494 1 - Fill the aqueous medium with the fermenter and sterilize.

The oil, optionally containing the surfactant (s), is sterilized and poured into the aqueous medium in the fermenter with stirring at room temperature.

- The medium is inoculated with a pre-culture of sucrose in MY medium at 10 g / l Xanthomonas camiq pestris.

Example 1

A 14 L sterile oil / water emulsion containing 6.25% oily phase and 93.75% aqueous phase in a 20 liter fermenter was inoculated with 210 g pre-culture.

Composition of the aqueous phase:

Glucose 61 g / l

Yeast extract 7 g / l

Na ₂ HPO ₄ 2.35 g / l (NH ₄ ) ₃ PO ₄ 2.2 g

MgSO ₄ 7H ₂ O 0.35 g / L

Composition of the oily phase:

Deodorized aliphatic hydrocarbon 70% by weight.

(Exsol D, Esso Chimie)

30% by weight of ethoxylated nonylphenols (67:33 Cemulsol NP4 and Cemulsol NP17 (SFOS))

HLB value: 11 Fermentation conditions:

Temperature 28 ° C pH adjusted to 7 with addition of NaOH Mixing 400 rpm

aeration

0.24 vol / vol / min for 0-17 hours

0.48 vol / vol / min for 17-23 hours

0.98 vol / vol / min for 23-90 hours

Fermentation while the sugar is completely depleted,

Time is 90 hours

The characteristics of culture development and media specific resistance over time are summarized in Table 1.

The yield was 425 g (30.3 g / kg), yield 56% based on sugar.

After the fermentation, the emulsion has a viscosity of ignition of ⁵⁰ 8.1 Pa · s (Brookfield viscosimeter No. 4-20 ° - 30 rpm).

Example 2

The procedure of Example 1 was followed using a starting medium containing 29% by volume of Exsol D 100 hydrocarbon and 71% by volume of an aqueous phase. The composition of the aqueous phase is as follows:

Glucose 64 g / l

Yeast extract 9.24 g / l

Na ₂ HPO ₄ 3.08 g / l (NH ₄ ) ₃ PO ₄ 3.08 g / l

MgSO ₄ -7H ₂ O 0.35 g / L

No surfactant is added.

The fermentation conditions are the same as in Example 1, except that a constant aeration of 0.98 v / v / min is maintained. Fermentation takes 85 hours until the sugar is consumed.

The changes in culture and specific resistance over time are summarized in Table 1.

333 g of product are obtained, which corresponds to a yield of 51.3% of the sugar. The final emulsion has a viscosity of 10.2 Pa (conditions identical to those of Example 1).

Example 3

The initial oil / water emulsion consists of 14,125 t of an aqueous phase and 0.875 l of an oil phase.

Composition of the aqueous phase:

Sucrose 85 g / l

Soluble maize extract 25.5 g / l

MgSO ₄ 7H ₂ O 0.28 g / l

The oily phase contains 70% rapeseed oil and 30% ethoxylated nonylphenols (Cemulsol NP4) with an HLB of 9.

The medium was inoculated as in Example 1.

Conditions for fermentation:

Temperature 28 ° C pH adjusted to 7 with NaOH Stirring 0-17 h 390 rpm

17-24 hours at 500 rpm

600 rpm for 24-84 hours

aeration

For 0-17 hours, 0.92 v / v / min

1.4 vol / vol / min for 17-84 hours

Average Energy Requirement At 6.09 kW / m ³ , 51 g of xanthan polymer per kg of medium are obtained. Yield / Sugar Consumption: 64%.

The fluid has a K resistance (Ostwald's) of 37 Pa and a pseudoplasticity index of 0.195.

for comparison, the K resistance of an aqueous solution containing 48.5 g / kg of polymer

Pa-s and n have an index of 0.24.

Fermentation was continued for 113 hours, yielding 66 g / kg polymer, 83% yield / sugar consumed.

Examples 4 and 5

Two fermentations were carried out in an oil / water emulsion containing 0.875 liters of oil and 14.125 liters of an aqueous phase.

The composition of the medium is as follows: saccharose 8% Soluble corn extract 4% MgSO ₄ 7H ₂ O 0.05% Oil 3.5% Surfactant L5% The oily phase consists of rapeseed oil and ethoxylated

consists of natural fatty alcohols (Cemulsol AS 5 - SFOS) in Example 4 and ethoxylated nonylphenols (50:50 mixture of Cemulsol NP4 and Cemulsol NP17) in Example 5.

-4194941

Fermentation Temperature

- after 5 pm after 5 pm the circumstances are as follows:

28 ° C

Aeration Mixing

0.92 v / v / min 390 rpm

1.39 rpm 500 rpm

1.39 rpm 500 rpm (example 4) 600 rpm (example 5)

Fermentation is continued until the sucrose is completely consumed. Results:

Example 4 Example 5

Time 68 hours 80 hours

Yield 47.4 g / kg 51 g / kg

Yield / sucrose 60% 64%

6-14. Example 1 Inoculate 500 ml of Xanthomonas campestris 10 g / l sucrose ΜΫ with a 500 ml

100 ml of oil / water emulsion medium in an Erlenmeyer flask.

The aqueous phase is the same as that used in Example 3.

The oily phase is variable. Each flask contains 95% aqueous phase and 5% by weight oily phase. Stirring was carried out with a rotary agitator of 220 rpm with a 20 g gel and 5 cm amplitude and incubated at 28 ° C for 89 hours.

The results are shown in Table 2.

Table 1

Age Xanthomonas cell count / ml Specific resistance (hours) in emulsion & cm

First example 2. ' example 1. example Example 2 0 1 ' 6 10 ⁸ 3.9 10 ⁷ 216 17 4 ' 0 10® 3.0 10 ® 216 23 8 ' 0 10® 5.4 10 ® 370 354 43 7 / 0 10® 2.0 10 ¹⁰ 216 354 66 4 / 0 10® 1.2 1O ¹⁰ 216 277 90 1 / 0 10® 185 262 Table 2 example Number 6 * 7 8 9 10 11 12 13 14 Rape 3.5 3.5 Soy 3.5 3.5 Oil ** Exsol D 80 3.5 3.5 (g) Isopar M 3.5 3.5 Cemulsol NPA 1.5 1 0.75 1 0.75 Surface- Cemulsol NP17 0.5 0.75 0.5 0.75 ^aktlv,. * Cemulsol R5 0.35 substances * _{Cemulsol T} 0.15 % Cemulsol NP5 1.5 Cemulsol AS5 1.5 PH 6.75 6.2 6.5 6.55 6.35 6.3 5.75 6 5.9 Viscosity *** (mPas) 9600 11700 7400 8700 11.00 7000 , 0500 2900 12900 Sugar lost (g / 1) 45.6 49.5 60.5 47.2 48.4 54.5 63.3 62.2 67.1 Polymer (g / kg) 32.4 41.3 33.9 35 36 35.9 41.2 41.2 42.8 Yield / out of sugar 71 83 89 74 74 66 65 66 64

Notes to Table Z: Without control oil

- ** Exsol D 80 - Aliphatic Hydrocarbon - Esso Chimie by Isopar M - Flavoring Paraffins - Esso Chimie

Cemulsol NP4, NP5, NP17 - Ethoxylated Nonyl Phenols - Made by SFOS Cemulsol R5, AS5, T - Ethoxylated Oleocetyl Alcohols - Made by SFOS *** Brookf Ield Viscometer No. 4, 20'C, 30 RPM

-5194941

Example 15

Fermentation with Arthrobacter viscosus (ATCC 19584) A. Preparation of inoculum

Inoculate with the following medium:

Glucose monohydrate 15 g

Yeast extract 4 g

K ₂ HPO ₄ 4 g

MgSO ₄ -7H ₂ O 0.8 g

MnSO ₄ -H ₂ O ppm (expressed in Mn) per liter

At C for 29 hours

Distilled water

The fermentation is carried out 28 '.

B) Production

A 20 liter fermenter containing 15 liters of oil-in-water emulsion was inoculated with 300 g of pre-culture. The emulsion consists of 0.875 l of an oil phase and 14.125 l of an aqueous phase, i.e. 5.8% by volume of an oil phase and 94.2% of an aqueous phase.

Composition of the aqueous phase:

Glucose monohydrate 40 g / l 10 g / l

0.8 g / l ppm (expressed in Mn) g / l

Corn Soaking Juice MgSO ₄ -7H ₂ O MnSO ₄ -7H ₂ OK ₉ HPO ₄

Drinking water per liter

Composition of the oily phase:

Rape oil 70% by weight

30% by weight of ethoxylated fatty alcohols (CEMULSOL AS 5, manufactured by SFOS)

Temperature: 28 ° C pH: 6.8-7.0, adjusted with NaOH Mixing and aeration:

For 0-15 hours, 0.25 v / v / min to 300 rpm

For 15-50 hours, 0.50 v / v 400 rpm

50 hours 0.85 vol / vol / min

450 rpm

Results:

Fermentation time: 68 hours Product obtained: 31.6 g / kg Yield for consumed glucose: 87.7% Viscosity of final emulsion: 6.8 Pa

Example 16

Fermentation with strain Sclerotium rolfsii (ATTCC 15205)

A) Inoculum preparation

Inoculate with the following medium:

saccharose 10 g / 1 yeast Extract 1 g / 1 MgSO ₄ -7H ₂ O 0.5 g / 1 KH ₂ PO ₄ 1 g / 1 NaNO ₃ 3 g / 1 KCI 0.5 g / 1 FeSO ₄ -7H ₂ O 0.01 g / l HCl 10% 0.5 ml / 1 Distilled water 1 liter

The fermentation is carried out at 26 ° C for 48 hours.

B) Production

A 20 liter fermenter containing 15 liters of oil-in-water emulsion was inoculated with 300 g of pre-culture. The emulsion contains 1.5 L (10%) oil phase and 13.5 L (90%) aqueous phase.

Composition of the aqueous phase:

saccharose 60 g / 1 yeast Extract 1.5 g / 1 MgSO ₄ -7H ₂ O 0.5 g / 1 KH ₂ PO ₄ 1 g / 1 NaNO ₃ 3 g / 1 KCI 0.5 g / L FeSO ₄ -7H ₂ O 0.01 g / 1 Drinking water 1 liter

Composition of the oily phase:

Aliphatic Hydrocarbon (EXSOL Dl00) 70%

Ethoxylated nonylphenols 30% (1: 1 mixture of Cemulsol NP4 and Cemulsol NP17)

Fermentation conditions:

Temperature: 26 ° C pH: not regulated (3.5 at start of fermentation)

Mixing and aeration:

0.25 vol / vol / min for 0 to 20 hours

350 rpm

0.50 vol / vol / min at 400 rpm for 20-60 hours

From 60 hours to 1 v / v / min

500 rpm

Fermentation is continued until all sucrose is consumed.

Results:

Fermentation time: 113 hours Product obtained: 40.5 g / kg Yield: 75% sucrose

The viscosity of the final emulsion is 12.1 Pa

Claims (8)

PATENT CLAIMS A process for the preparation of xanthan type polysaccharides by fermentation of a microorganism of the genus Xanthomonas, Arthrobacter or Sclerotium, characterized in that the fermentation is carried out in an aqueous medium in which an oil-in-water emulsion is formed by dispersing the oil and optionally worked. 2. A process according to claim 1, wherein less than 30% by weight of the emulsion is an oil phase. The process according to claim 2, wherein the oil phase comprises from 1% to 18% by weight of the emulsion. The process according to claim 3, wherein the emulsion is in the range of 3% to 16% by weight of the emulsion. 5. Process according to any one of claims 1 to 6, characterized in that the oil is a-6194941 a water-in-water emulsion is stabilized with at least one nonionic surfactant. 6. A process according to claim 5, wherein the HLB is less than II or a mixture of surfactants. 7. The method of claim 6, wherein the HLB is 6 to 11 surfactants. 8. Figures 1-7. A process according to any one of claims 1 to 3, characterized in that after the production of the polysaccharide, the emulsion is concentrated to a content of 8 to 60% by weight of the polysaccharide by removing water. Without drawing