WO1996041890A1

WO1996041890A1 - Method and medium for producing gellan in the presence of manganese

Info

Publication number: WO1996041890A1
Application number: PCT/FR1996/000755
Authority: WO
Inventors: Frédéric Monot; Francis X. Quinn
Original assignee: Institut Français Du Petrole
Priority date: 1995-06-13
Filing date: 1996-05-21
Publication date: 1996-12-27
Also published as: FR2735497A1; FR2735497B1

Abstract

A method for producing gellan, wherein an effective amount of manganese, i.e. at least 0.05 g/L, preferably at least 0.2 g/L, is added to a culture medium, is disclosed. A culture medium for producing gellan, comprising an effective amount of manganese, i.e. at least 0.05 g/L, preferably at least 0.2 g/L, is also disclosed.

Description

PROCESS AND MEDIUM FOR PRODUCING GELLANE IN THE PRESENCE OF MANGANESE

The present invention describes a process for obtaining gellan by growing a bacterium belonging to the species Pseudomonas elodea (also classified as Sphingomonas mobilis and in certain cases Auromonas elodea) on a culture medium containing significant concentrations of manganese. This process allows in particular to increase the concentration of gellan in the final fermentation must compared to that obtained after culture on fermentation media containing traces or free of manganese.

The production of gellan, also called heteropolysaccharide S 60 by Pseudomonas elodea (or Sphingomonas mobilis or Auromonas elodea) ATCC 31461 is described mainly in US Pat. article entitled "Agar-like polysaccharide produced by a Pseudomonas species: production and basic properties" published in "Applied and Environmental Microbiology", 1982, vol. 43 n ° 5, pages 1086-1091. Gellan is produced by aerobic fermentation on common culture media containing carbon sources, organic and mineral nitrogen sources and mineral salts.

The mineral salts usually incorporated in culture media for bacteria are sources of phosphorus (in the form of phosphate), of sulfur (in the form of sulfate) which are important mineral constituents of bacteria. Sodium, potassium, magnesium and chlorine salts, necessary for the physico-chemical balance of the bacterial cell, are also often introduced into the culture medium. Other elements called trace elements must also be present in the culture medium, because they play a role of cofactors or enzyme activators. Among the trace elements, mainly finds iron, calcium, cobalt, copper, molybdenum, manganese. A very low concentration of trace elements in the culture medium is sufficient for the development of microorganisms. Depending on the culture medium used, the trace elements are either introduced in a minimal quantity, or omitted considering that their presence in trace amounts in the other chemicals incorporated in the medium is sufficient to provide a sufficient quantity for the bacteria. Typically, the mineral salts contained in the culture medium recommended for the production of gellan by Pseudomonas elodea ATCC 31461 are ammonium nitrate (as a source of mineral nitrogen), potassium or sodium phosphate, sodium sulfate magnesium and traces of various salts such as ferrous sulphate, chlorides of copper, zinc, cobalt, manganese, and salts of boron and molybdenum. For example, the culture medium as described in the article entitled "Agar-like polysaccharide produced by a Pseudomonas species: production and basic properties" published in "Applied and Environmental Microbiology", 1982, vol. 43 No. 5, pages 1086-1091, initially contains 30 g / L of glucose, 0.5 g / L of promosoy (soy peptone, source of organic nitrogen), 0.9 g / L of NH4NO3, 0, 5 g / L of K2HPO4, 0.1 g / L of MgSθ4-7H2θ and 1 ml L of a salt solution itself containing 1.8 g / L of MnCl2-4H2θ, 2.487 g / L of FeSθ4-7H2θ, 0.285 g / L of H3BO3, 27 mg / L of CuCl2, 21 mg / L of ZnCl2, 74 mg / L of CoCl2-6H2θ, 23 mg / L of MgMoθ4 and 2.1 g / L of Na2C4H4θ6-2H2θ (tartrate of sodium dihydrate). The salt solutions described in the patents relating to the production of gellan have similar compositions. In such media, the initial concentration of MnCl2-4H2θ is 1.8 mg / L, or 0.50 mg / L of Mn. As mentioned above, the incorporation of trace amounts of manganese into culture media for microorganisms is a common technique.

The present invention relates to a process for the production of gellan by culture of the microorganism Pseudomonas elodea ATCC 31461 in a fermentation medium containing significant concentrations of manganese, that is to say concentrations greater than 50 mg / L of Mn. Surprisingly, the addition of such manganese concentrations makes it possible to obtain higher gellan concentrations. In the process, manganese can come from different compounds. These compounds can be, for example, manganese acetate tetrahydrate, manganese chloride • tetrahydrate or anhydrous, manganese nitrate, manganese dioxide, manganese sulfate tetrahydrate or anhydrous, these examples being given by way of illustration. The source of manganese is generally introduced all at once into the fermentation medium, before sowing.

The manganese concentration in the culture medium, counted in manganese Mn, is at least equal to 0.05 g / L, preferably greater than or equal to 0.2 g / L.

The invention also relates to a culture medium for producing gellan. The culture medium comprises a source of manganese whose concentration counted in manganese Mn is at least equal to 0.05 g / L, preferably greater than or equal to 0.2 g / L.

Furthermore, as regards the other ingredients of the culture medium, the nutritive medium allowing the growth of the microorganism and the production of gellan by the strain can be chosen from the culture media described in the literature. The conventional medium contains a carbon substrate which can be a sugar such as glucose, fructose, maltose, sucrose, xylose or mannitol (this list not being limiting) or another carbohydrate, used alone or in mixture. The initial concentration of carbohydrate in the medium partly depends on the concentration of the other constituents of the medium, in particular on the nitrogen concentration. Thus, to promote the production of polysaccharide, it is preferable to have initial concentrations of carbon and nitrogenous substrates such that the molar ratio of the quantities of initial carbon and nitrogen (C / N) is much greater than 1. However, the initial concentration of carbohydrate must be chosen so as to minimize as much as possible the residual concentration (that is to say at the end of fermentation) of carbohydrate. The initial carbohydrate concentration generally varies from 10 to 60 g / l, preferably from 20 to 40 g / l. The source of nitrogen in the nutrient medium can be a source of organic and / or mineral nitrogen. The sources of organic nitrogen, compounds rich in proteins or protein hydrolysis residues, can be yeast extract, malt extract, meat extracts, fishmeal, flour soybeans, cotton flour, corn steep liquor, soluble distillation residues, casein hydrolysates, peptones, etc. A source of mineral nitrogen, in the form of ammonium salt or nitrate salt, can also be used alone or in addition to sources of organic nitrogen. Thus, culture media such as those described in patent FR 94/15 742 which comprise a nitrogen source consisting mainly of nitrate ions, can be used in the present invention. The nitrogen concentration, counted in nitrogen N can be between 0.01 and 5 g / 1 in the culture medium.

Other mineral salts can be introduced into the nutrient medium, such as magnesium, potassium, sodium, sulfur, iron, boron, zinc, cobalt, molybdenum ... A typical composition of the mixture of these salts is given in l 'example 1. This composition is given by way of illustration and is not limiting.

The fermentation according to the present invention is carried out at temperatures between 25 and 35 ° C, preferably between 28 and 32 ° C. The pH of the medium is initially adjusted to a value between 6 and 8. In the case of shake flask cultures, the medium is preferably buffered, for example with phosphate buffer, to avoid an excessive drop in pH which would inhibit growth of the strain. In the case of cultures in a stirred reactor, the pH can be regulated to a value of between 6 and 8 throughout the fermentation. The culture medium must be shaken and ventilated. When flask cultures are used, these are incubated on reciprocating or rotary shakers. This agitation also ensures ventilation of the medium. The exchange surface between the air and the liquid medium must be large enough to allow good exchanges. The so-called Fernbach flasks are well suited to this type of culture. In the case of fermentations carried out in a reactor, the production medium is agitated using agitation modules ensuring good transfer to the within the reactor. The stirring speed is generally between 50 and 1000 rpm. The medium is aerated at a rate of between 0.1 and 2 vvm (volume of air per volume of liquid and per minute).

Fermentation is generally carried out in batch. For this, the culture medium contains all the nutrients, then is sterilized and, once returned to the fermentation temperature, is then seeded with an active culture. An aqueous solution containing manganese and the nutrient medium can be sterilized separately. The manganese solution is then added sterile to the culture medium in an amount such that the final concentration in the culture medium is that desired. The introduction of manganese into the culture medium can be carried out initially or during fermentation. The addition of this solution can be carried out at once or in several successive operations distributed during the culture.

The seeding rate, that is to say the ratio of the volumes of seeding and medium after seeding, is between 0.1 and 10%. In the case of reactor cultures, this can be equipped with sterilizable probes making it possible to record and regulate parameters such as pH, temperature, dissolved oxygen level. It is also possible to know the oxygen and carbon dioxide levels in the outlet gases using analyzers placed on the outlet of the reactor gases.

After 1 to 4 days of culture, the culture can be stopped. The main criterion for stopping fermentation is low glucose consumption. The fermentation must contains raw gellan. This can be recovered by precipitation with an appropriate solvent, most often isopropanol. The recovered precipitate can be dried. The total dry matter contains both so-called native gellan and cells from the culture of Pseudomonas elodea. This method of precipitation with isopropanol followed by drying is the conventional method for determining the concentration of crude gellan in the fermentation must. The present invention will be better understood and its advantages will appear more clearly on reading the following examples. These are given by way of illustration and without limitation of the invention.

Example 1 Preparation of Gellan According to the Prior Art, in the Presence of Traces of Manganese

(fermentation 1)

The Pseudomonas elodea ATCC 31461 strain is stored in tubes at -22 ° C on a complex agar medium (Nutrient Agar, Difco). For the re-culture of this strain, a tube is returned to ambient temperature for a few hours and a sample of approximately one ose is taken on the agar surface and spread on a Petri dish containing agar medium (Nutrient Agar , Difco). After 48 hours of incubation at 30 ° C., a development of yellow colonies is observed on the surface of the agar medium. An isolated colony is removed and suspended in 20 ml of liquid medium (Nutrient Broth, Difco) in Erlenmeyer flasks. These vials are stirred for about 30 hours at 30 ° C. The entire contents of a vial, i.e. 20 mL, are used to inoculate 180 mL of preculture medium placed in a Fernbach vial. The composition of this preculture medium is given in Table 1. All of these transfer operations are carried out under sterile conditions and the media and containers have previously been sterilized under conditions usual for those skilled in the art. The Fernbach flasks are stirred for about 40 hours at 30 ° C. After incubation, approximately 180 mL of the contents of a Fernbach flask are used to seed 1.82 liters of production medium contained in a fermenter. The composition of the production medium is indicated in Table 1 and corresponds to the optimal composition given in the prior art. The 20 ml of remaining medium are used to carry out routine analyzes and to verify the good progress of the preculture. The fermenter used is equipped with a multi-hole aerator, temperature regulation, a pH probe connected to a regulator controlling the injection of KOH IN or H3PO4 IN, a dissolved oxygen probe, d '' a stirring system consisting of an axis provided with two centripetal turbines and driven by a stirring motor. The temperature of the medium is regulated at 30 ° C., the pH at 6.5, the air flow rate is 2 vvm and the stirring speed is 900 revolutions / minute.

The main parameters determined during fermentation are the concentrations of glucose, of crude gellan and the optical density of the medium read at 620 nm.

Table 1: Composition of preculture and fermentation media

The salt solution comprises: 1.8 g / L MnCl2-4H2θ 2.5 g / L FeSθ4-7H2θ 0.28 g / L H3BO3

0.027 g / L CuCl2 0.021 g / L ZnCl2 0.074 g / LC ₀ Cl2-6H2θ 0.010 g / L (NH4) 6M ₀ 7θ24-4 (HO) 2.1 g / L sodium tartrate

It is important to note that the manganese concentration is very low. Indeed, the culture medium initially contains 1.8 mg / L of MnCl2-4H2θ, or 0.50 mg / L of manganese Mn.

The glucose concentration is determined using an automatic analyzer (Glucose Analyzer II, Beckman, U.S. A.) by an enzymatic method with glucose oxidase.

The concentration of crude gellan is determined on the fermentation must by precipitation of one volume of medium with two volumes of isopropanol. The precipitation is carried out hot with vigorous stirring. The precipitate is recovered by filtration on a glass microfiber filter, of porosity 1.2 μm, previously dried and tared. The precipitate is washed with isopropanol. The precipitate is then dried and weighed in an infrared desiccator mounted on a balance. This gives the concentration of crude gellan, that is to say the polysaccharide and cell debris.

The concentration of so-called native gellan, that is to say containing no cellular debris, is determined by subtracting from the concentration of crude gellan the concentration in cellular dry matter. This is evaluated according to a correlation established between the optical density of the medium measured at 620 nm against water and the dry weight of cells. To establish this correlation, samples of fermentation wort of known optical density were diluted 20 times in deionized water, stirred for a long time and then centrifuged. The pellets obtained were then put back into solution then filtered hot on filters previously dried and tared. The dry matter of the retentates makes it possible to determine the dry weight of the cells, the gellan having been removed during the centrifugation and filtration operations. The correlation obtained gives: cellular dry weight (in g / 1) = 0.26 x (optical density of the medium at 620 nm).

The results obtained after 47 hours of culture are as follows: Residual glucose: 7 g / L

Cell concentration (dry cell weight): 3.3 g / L Crude gellan: 12.5 g / L Native gellan: 9.2 g / L

Example 2 Preparation of Gellan in Culture Media Containing Significant Concentrations of Manganese

( ^" fermentations 2 to 5)

Four other fermentations (2, 3, 4 and 5) were carried out under conditions strictly identical to those described in Example 1 with the exception of the amount of manganese present in the preculture and production media. The manganese concentrations used are shown in Table 2. Table 2: Manganese concentration used in preculture and fermentation 2, 3, 4 and 5 production media

FERMENTATION 2 3 4 5

MnCl2-4H2θ in the medium of 0.16 g / L 1.6 g / L 2.4 g / L 3.2 g / L preculture

MnCl2-4H2θ in the medium of 0.16 g / L 1.6 g / L 2.4 g / L 3.2 g / L production

Mn equivalent in the medium of 0.044 g / L 0.44 g / L 0.67 g / L 0.88 g / L production

The fermentations are all stopped after approximately 48 hours of culture, except fermentation 3 for which the residual glucose content was zero after 31.5 hours of culture. The initial glucose concentrations were slightly higher than 30 g / L in the case of fermentations 4 and 5. The durations of the fermentations, the concentration of glucose consumed and the final concentrations of cells, crude gellan and native gellan determined according to the procedures described in Example 1 is shown in Table 3.

Table 3: Concentrations of glucose consumed, cells, raw and native gellan obtained in fermentations 2 to 5

FERMENTATION 2 3 4 5

Duration (h) 47.5 31.5 48 47.5

Glucose consumed (g / L) 24.2 30.7 32.8 34.3

Dry cell weight (g / L) 3.4 3.8 4.2 4.3

Raw gellan (g / L) 12.5 18.3 16.8 14.8

Native Gellan (g / L) 9.1 14.5 12.6 10.5 It can be seen that the concentrations of crude gellan and native gellan obtained during fermentations 3, 4 and 5 are much higher than those obtained during fermentation 1. In addition, the durations of the fermentations being equivalent, there is therefore also an increase in the gellan productivity (quantity of gellan produced per unit of reaction volume and per unit of time). This example clearly shows the beneficial influence of adding significant amounts of manganese to the culture medium.

Example 3:

Preparation of gellan in so-called synthetic culture media containing different concentrations of manganese

(fermentations 6 to 8)

Fermentations 6, 7 and 8 were carried out on culture media containing no organic nitrogen. This type of medium is called synthetic medium and its exact composition can be perfectly defined. In the present case, the nitrogen source is potassium nitrate, KNO3, at a concentration of 2.3 g / L.

Compared to the previous examples, only the composition of the fermentation medium is changed, all the other conditions also being identical to those described in Example 1, including the composition of the preculture medium.

The composition of the fermentation media used in fermentations 6 to 8 is described in Table 4. Table 4: Composition of the culture media used in fermentations 6 to 8.

The salt solution used is identical to that given in Example 1. The durations of fermentations 6 to 8 as well as the concentrations of glucose consumed and the final concentrations in cellular dry weight, in crude and native gellan are indicated in Table 5 .

Table 5: Concentrations of glucose consumed, cells, raw and native gellan obtained in fermentations 6 to 8

FERMENTATION 6 7 8

Duration (h) 50 23 40

Glucose consumed (g / L) 19.7 26.7 28.5

Dry cell weight (g / L) 4.3 3.7 4.3

Raw gellan (g / L) 12.4 15.5 16.4

Native Gellan (g / L) 8.1 11.8 12.1 It can be seen that the concentrations of crude gellan and native gellan obtained during fermentations 7 and 8, carried out in the presence of significant concentrations of MnCl ₂ , are clearly higher than those obtained during fermentation 6, carried out in the presence of traces of manganese. This example clearly shows the beneficial influence of adding significant quantities of manganese to a culture medium containing only mineral nitrogen, in this case in the form of nitrate, as a source of nitrogen.

Claims

1) Process for producing gellan by culture in a reaction medium, characterized in that a useful quantity of manganese greater than or equal to 0.05 g / L is added to said medium.

2) Method according to claim 1, characterized in that a useful quantity of manganese greater than or equal to 0.2 g / L is added to said medium.

3) Method according to one of the preceding claims, characterized in that the source of manganese is added all at once and before sowing.

4) Culture medium for producing gellan, characterized in that it comprises a Peudomonas elodea microorganism (ATCC 31461) and a useful quantity of manganese greater than or equal to 0.05 g / L.

5) Culture medium according to claim 4, characterized in that it comprises a useful quantity of manganese greater than or equal to 0.2 g / L.

6) Culture medium according to one of claims 4 or 5, characterized in that the source of manganese comes from at least one of the following compounds: manganese acetate tetrahydrate, manganese chloride tetrahydrate or anhydrous, manganese nitrate, manganese dioxide, manganese sulfate tetrahydrate or anhydrous.

7) Culture medium according to one of claims 4 to 6, characterized in that the nitrogen source of said medium comes mainly from nitrate ions.