DE3413687A1 - METHOD FOR THE MICROBIAL PRODUCTION OF POLYSACCHARIDES - Google Patents

Description

The invention relates to a method for the microbial production of polysaccharides by means of the bacterium Zoogloea ramigera strain 115.

5 It is known from the literature that polysaccharides im

Establish laboratory scale using the bacterium Zoogloea ramigera strain 115. However, this requires a very long cultivation time. Besides that
it was previously not possible to achieve a high polymer concentration. Likewise set the viscosity
poses a problem in the culture medium.

A disadvantage of the long cultivation time is that an infection can develop in the cultivation vessel. The low productivity is also a disadvantage.
5

The low polymer concentration that was achieved in the known processes has a corresponding effect high cost of extraction.

Due to the high viscosity in the cultivation medium, there were particular difficulties, the bacteria to be supplied with sufficient amounts of oxygen. That is particularly noteworthy because the metabolize special bacteria aerobically.

The disadvantages of the known methods have therefore not yet made it possible to use polysaccharides in large-scale production with the help of the bacterium Zoogloea ramigera strain 115.

According to the invention it has now surprisingly been found that you can avoid the disadvantages of the prior art and that you have a method for microbial production of polysaccharides with

25 tels of the bacterium Zoogloea ramigera strain 115 technical can perform. In the method according to the invention

(A) Zoogloea ramigera in a sterile aqueous medium containing a carbon source such as Glucose, starch or potatoes, a source of nitrogen,

such as Airunoniumchlorid, ammonium nitrate or potassium nitrate, nutrient salts such as phosphates and sulfates, and iron salts such as FeCl-, or Fe ^ (SO.) ^, in a concentration of 0.15 to 45 mg / 1 and preferably 0.5 to
5 mg / 1 increased, while obtaining a dense suspension of the bacterium and

(B) This suspension is then placed in a fermenter containing a sterile aqueous medium

above composition, transferred and left in the fermenter, the bacterium for a time of about 20 to 60 hours and preferably 30 to
50 hours until the main amount of the supplied carbon source is consumed, the initial concentration of the carbon source being about 10 to 50 g / l, to obtain a low-viscosity product containing 10 to 35 g / l of polymer, the polymer
is in the form of capsules.

The known strain 115 of the bacterium Zoogloea ramigera, which is used according to the present invention, was obtained from the American Type Culture Collection, USA, under the ATCC no. 25935 deposited.

The polysaccharide produced according to the invention builds generally consists of glucose, galactose and pyruvic acid, among other substances.

The addition of an iron salt to the culture medium increases the growth rate of the bacterium achieved. Preferably a sterile one is used

filtered iron solution. The amount of the iron salt, calculated as Fe, should be at least 0.15 mg / l and at most 4 5 mg / 1, based on the culture medium. It is preferably at least 0.5 mg / l and a maximum of 5 mg / 1.

The pH of the culture medium is generally at least 6.0 and not more than 8.0. It is preferably 6.5 to 7.5.

During the first stage of cultivation, the growth of the bacterium is stimulated. The growth stops when the initially added amount of nitrogen is consumed, since nitrogen is a growth-limiting one

15 element represents.

The next stage of cultivation involves the production of a polysaccharide, which is in the form of voluminous capsules surrounding the bacteria.

During the production of the polysaccharide, it is necessary that a carbon source is present in the cultivation medium, in part around the organism to be provided with the starting material from which the building blocks contained in the polysaccharides are built up, and in part to meet the energy needs of the bacterium.

According to an embodiment of the invention, can one the polymer concentration of the low viscosity

Increase the product from stage (B) by then checking in a subsequent one Step (F) adds another carbon source.

5 According to a further embodiment of the invention the polymer concentration of the low-viscosity product from stage (B) can be increased by that one controls one more carbon source and one more nitrogen source in succession 10 is added to a subsequent stage (F).

After the carbon source originally added in step (B) has been consumed, the addition from another carbon source in stage (F) or

(G) is added in a suitable manner from a separate vessel by means of a pump which is controlled by a signal from a COj analyzer. The analyzer continuously monitors the content of CO ₂ in the exhaust gas from the fermenter.

Increased when the carbon source is added to the culture medium the CO content in the exhaust gas from the fermenter until the amount of substrate pumped into the fermenter is used up. Then the CO-

Content decreases rapidly and the addition of a carbon source is repeated. Through this special procedure one can monitor the addition of the carbon source so that the concentration of the carbon source within the desired range during

30 of the phase in which the polysaccharide is synthesized is present.

A suitable CO ^ level is at least 0.2 and at most 3.0% and is preferably 0.7 to 1.3%. ^y

In the embodiments according to steps (F) and (G), in which one another carbon source or another carbon source and nitrogen source admits, the total concentration of the carbon source in stages (B) plus (F) or (B) plus (G) 15 to 75 g carbon source per liter and preferably 20 to 50 g / l.

Lactose, glucose, starch, for example, can be used as the carbon source in stages (F) or (G) 15 or use potatoes.

The stages (B) plus (F) or (B) plus (G) are preferably completed within 30 to 48 hours.

Achieved by a combination of the addition of the carbon source and the nitrogen source in step (G) one has the further advantage that an even and high production of the polysaccharide during the Stage (G) is achieved.

According to a particularly advantageous embodiment if starch is used as a carbon source, ammonium chloride as a nitrogen source and FeCl., as an iron salt.

Generally one wins the polysaccharide and that Bacterium in stage (C) by using a

Alcohol, such as ethanol or isopropanol, performs a precipitation. In a suitable manner one obtains a Precipitate also by centrifugation (D), whereupon in a step (E) the finished product in general is dried.

The stages (C), (D) and (E) can, however, also in other, per se known manner, depending on the use of the end product.

The invention is explained in more detail with reference to the accompanying figure. In this is a procedural scheme shown for the preparation of a polysaccharide according to the invention. Refer to the examples below Examples 1 to 5 refer to comparative experiments, which are not inventive, and the examples 6 to 11 show methods according to the invention.

In the figure, three different embodiments according to the invention are shown. The different Letters in the figure have the following meanings:

(A) A propagation stage in which the bacterium is placed in an aqueous medium containing a

Carbon source, a nitrogen source, nutrient salts and iron salts, according to the invention.

(B) A fermenter cultivation stage in which the formation of the polysaccharide takes place.

(C) A recovery stage in which, for example, a precipitation stage is carried out, whereby the polysaccharide and bacteria are precipitated.

(D) A centrifugation step in which the precipitated product is collected.

(E) A drying stage. 10

(F) A fermenter cultivation stage in which a further subsequent addition of a carbon source takes place.

(G) A fermenter cultivation step in which a further subsequent addition of a carbon source and a nitrogen source takes place.

example 1

A colony of the bacterium Zoogloea ramigera strain 115 was removed from an agar plate in a 2 liter Erlenmeyer flask (A) containing 250 ml of a sterile medium of the following composition:

g / l aqueous solution glucose 25th K ₂ HPO ₄
KH ₂ PO ₄
NH ₄ Cl 2
1
1 MgSO ₄ -7H ₂ O 0.2

Yeast Extract (Difco) 0.01

The flask was placed for 18 hours to a shaking machine (100 to 300 ÜPM) and maintained at a temperature of 26 ⁰ C. The bacterial suspension obtained was used for inoculating (B) a sterile medium in a fermenter. The medium had the same composition as given above. 0.5 l of air per liter of medium and minute were blown through the fermenter and the mixture was stirred at 800 rpm. The temperature in the fermenter was kept at 26 ^° C. The pH of the medium was adjusted to 7.0 by automatically titrating with NaOH. 20th

After 95 hours of cultivation, the glucose was consumed and the concentration of the polysaccharide was reduced 11.5 g / l.

Example 2

The process according to Example 1 was repeated, except that a CaCl ₂ solution was added to the medium in step (B). The total amount of feed

Calcium was 3 mg / l culture medium. The glucose was consumed after 9 5 hours and the concentration of the polysaccharide was 10.1 g / l.

Example 3

The process according to Example 1 was repeated, but using a MnSO. Solution to the medium in the stage (B) was supplied. The total amount of the supplied manganese was 3 mg / 1 culture medium. The glucose was consumed after 85 hours and the concentration of the polysaccharide was 10.2 g / l. 15th

Example 4

The process of Example 1 was repeated, except that a CuCl. Solution was added in step (B) became. The total amount of the supplied copper was 3 mg / 1 culture medium. The glucose was at 88 hours consumed and the concentration of the polysaccharide

25 was 11.0 g / l.

Example 5 30

The process according to Example 1 was repeated, except that a ZnCl ^ solution was added in step (B)

became. The total amount of zinc supplied was 3 mg / 1 culture medium. The glucose was after 90 hours consumed and the concentration of the polysaccharide was 9.2 g / l.

Example 6

The process according to Example 1 was repeated, except that an FeCl ₃ solution was added to the culture medium in steps (A) and (B). The total amount of iron added was 3 mg / liter of the culture medium. The glucose was already consumed after 45 hours and the concentration of the polysaccharide was 13.2 g / l. The viscosity was 40 centipoise.

20 Example 7

In order to achieve the optimal C / N ratio in the culture medium for the production of an extracellular polysaccharide means To achieve Z. ramigera, the strain became 115 prepared in a separate batch culture medium without further feeding. In this attempt a glucose concentration of about 25 g / l was used. By changing the amount of Source of nitrogen (0.25 to 4 g NH.Cl/1) to the medium In the various cultivations it was found that the optimal yield of the polysaccharide from the

added glucose is generally achieved with an amount of 0.5 to 2 g NH ₄ ClA. The total cultivation time is 36 to 60 hours in most cases.

Example 8

The optimal C / N ratio of the culture medium at which a polysaccharide is produced by means of Z. ramigera strain 115 in a batch system, adding another carbon source by adding glucose is generally between 10 and

25. Applying higher nitrogen levels during the first batch procedure will result in a larger than normal cell mass. After the original glucose has been used up, the addition of glucose is started. The existing cell mass effectively converts the supplied glucose into the polymer. The dosage of the glucose is automatically monitored by means of a signal _{value from a CO analyzer, which measures the CO 2} content in the exhaust gas from the fermenter. The addition of glucose

25 starts when the signal drops below a previously set value. The cultivation time is generally about 36 to 54 hours.

Example 9

The optimal content of dissolved O ₂ in a culture medium with Z. ramigera strain 115 was determined in a batch cultivation. The stirring, by which the content of dissolved oxygen is also influenced, was kept constant at 800 rpm. Is the air supply to the fermenter
0.25 to 1.5 vvm (volume of air per volume of medium

and minute), then the best conditions for converting glucose into polymer are present. In these tests the dissolved O _{2 content was}
never less than 20% of the saturation value and
good conversion of glucose to

15 polymer.

Example 10

According to the invention it was found that iron is an essential trace element in order to achieve effective polymer production by means of Z. ramigera. the
Iron should be added during the bacterium's growth phase. This is conveniently done by adding an iron solution (e.g.
0.01 M) dosed to the fermentation medium at the beginning of the cultivation. The content of iron salt in the fermentation medium should be 0.15 to 45 mg / l, preferably

0.5 to 5 mg / l calculated as iron. the
Examples 7 to 9 above were carried out by

the iron to the medium in the form of a FeCl -.- solution was fed in a concentration of 2.5 mg / 1 fermentation medium. The addition of the iron resulted in that shortened the total process time from normally about 100 hours to about 36 to 60 hours could be.

10 Example 1-1

The cultivation with the addition of glucose with an optimal C / N ratio was carried out:

attempt

The initial glucose concentration was 25 g / l and the nitrogen content was 0.26 g / l.
20th

The medium also contained the following compounds

(g / i):

K ₂ HPO ₄ (Difco) 2 2 KH ₂ PO ₄ 1 01 MgSO ₄ -7H ₂ O 0, Yeast extract 0,

2.5 mg / l FeCl - 6H "0 were metered in. The first supplied glucose was consumed within 50 hours. By adding more glucose (5 g / L) to the

3413637

Fermentation medium, an increased polymer yield was obtained, however, polymer synthesis was slow.

final polymer concentration: 18 g / l final cell mass: 2.3 g / l
Viscosity: 250 centipoise.

Attempt_2 £

The nitrogen concentration in the culture medium was 0.52 g / l, all other conditions were as in example 1. Cell production stopped after 14 hours. The original glucose was after 22 hours consumed. The addition of fresh glucose resulted in rapid conversion of the glucose to the polymer. In the finished cultivation (48 hours) the polymer concentration was 32 g / l. The target mass concentration was 4.2 g / l. The viscosity was 225 centipoise.

Attempt_3_ £

The nitrogen concentration in the culture medium was

1.04 g / l, all other conditions were as in Example 1. The formation of the cell mass stopped after 18
Hours on. The initially supplied glucose was used up after 20 hours. The addition of fresh GIu-

30 cose gave very rapid conversion of glucose to polymer, but large amounts of glucose

- 20 -

were lost as CO-. After finishing cultivation (4 5 hours) the polymer concentration was 25 g / l. The cell mass was 9.8 g / l. The viscosity was 230 centipoise.

The above embodiment shows that the absence of iron in the culture medium or the use

from other metal salts, such as calcium salts, manganese salts, copper salts, zinc salts, an extension the cultivation times results, in contrast to the invention Procedure where a special amount of iron salt is used.

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Claims (9)

HOFFMANN · ΕΓ · "! _ Ε <$ ■ PARTNER PATENT AND LAWYERS OH 1 OOO / PATENTANWÄLTE DIPL.-ING. W. EITLE. DR. RER. NAT. K. HOFFMANN DIPL-ING. W. LEHN DIPL.-ING. K. FOCHSLE. DR. RER. NAT. B. HANSEN. DR. RER. NAT. H -A. BRAUNS DIPL.-ING. K. GDRG DIPU-ING. K. KOHLMANN · LAWYER A. NETTE September 11, 1984 P 34 13 687.8 40 029 o / kü PERSTORP AB, S-284 00 PERSTORP / SWEDEN Process for the microbial production of poly saccharides PATENT CLAIMS . Process for the microbial production of polysaccharides using the bacterium Zoogloea ramigera strain 115 (ATCC 25935), characterized in that one 5 (A) Zoogloea ramigera in a sterile aqueous medium containing a carbon source such as glucose, starch or potatoes, a nitrogen source such as ammonium chloride, ammonium nitrate or potassium nitrate, nutrient salts such as phosphates and sulfates, and an iron salt such as FeCl ₃ or Fe "( S0.) _, At a concentration of 0.15 to 45 mg / 1, preferably 0.5 to 5 mg / 1, with a dense suspension of the bacterium being obtained lets and (B) the suspension in a fermenter containing a sterile aqueous medium, the
Above composition transferred, wherein in the fermenter the bacterium for 20 to hours and preferably 30 to 50 hours, the main amount of the supplied carbon source can consume that in an initial concentration of about 10 to 50 g carbon source per Liter of suspension is present, and a low-viscosity product containing 10 to 35 g / l of poly mer in the form of capsules wins. 2. The method according to claim 1, characterized in that one 20 (C) the product from step (B) wins, the recovery being a precipitation with an alcohol, such as ethanol or isopropanol, including, (D) a separation including a centrifuge separation to collect the obtained Precipitation carries out and (E) preferably drying the separated product into an end product. 3. The method according to claim 1 or 2, characterized characterized in that one can further increase the polymer concentration of the low-viscosity Product from stage (B) (F) makes a subsequent controlled addition of the carbon source. 4. The method according to claim 1 or 2, characterized marked that one 10 ter-increase in the polymer concentration of the low-viscosity product from stage (B) (G) then a controlled addition of a carbon source and a nitrogen source undertakes. 5. The method according to any one of claims 1 to 4, characterized in that one Starch as the carbon source, ammonium chloride as the nitrogen source and FeCl., Used as the iron salt . 6. The method according to claim 3, characterized in that g e k e η η indicates that steps (B) and (F) together are completed within 30 to 48 hours. 7. The method according to claim 4, characterized in that the steps (B) and (G) are finished together within 30 to 48 hours, 8. The method according to claim 3 or 4 thereby characterized in that lactose is used as a carbon source in stages (F) or (G) admits. 9. The method according to claim 3 or 4, characterized in that the following Adding a carbon source in stages (F) or (G) by means of a signal is monitored by a CO_ analyzer.