NO149079B - FINAL FITTINGS FOR BEARS OR HOSE - Google Patents

Abstract

End bracket for pipe or hose.

Description

Procedure for the production of L-glutamic acid by fermentation.

This invention relates to a method for the production of L-glutamic acid by cultivating a new strain, namely Microbacterium ammoniaphilum, ATCC 1535^ under aerobic conditions and using carbohydrate as substrate.

With the method according to the invention, L-glutamic acid can be accumulated to a high concentration after a shorter stay in the culture medium and thereby facilitate the production of L-glutamic acid on a technical scale.

The new strain ATCC 1535^, Microbacterium ammoniaphilum, was first isolated by the inventors from sewage water. It turned out that the Microbacterium ammoniaphilum that the applicants isolated is completely different from all the known microorganisms that can produce L-glutamic acid, e.g. from Micrococcus glutamicus, Brevibacterium divaricatum, Brevibacterium aminogenes, Brevibacterium glutamicus, Brevibacterium kawasaki, Brevibacterium lactofermentus nov. sp., Microbacterium fJavum var. glutamicum, Microbacterium salicinovorum nov. sp., Bacillus megatherium was Nr. 1066, Bacillus gigantus n. sp., Bacillus circulans.

The reason why Microbacterium ammoniaphilum ATCC 1535^ belongs to a new species will clearly appear from the following description of the strain's bacteriological properties, which were examined according to

to the manner indicated in the "Manuel of Microbiological Methods" published by the Society of American Bacteriologists (1957).

I. Microscopic observations:

1) Morphological characteristics:

Vegetative cells: rods, usually 0.6 - 0.8^u x 1.0 - 2.5/U pronounced pleomorphy, in a microscope, keel-shaped, slightly bent and swollen cells are observed, angular, palisade-like cells are observed and cells shaped like a Chinese character due to division by cleavage, no branching is observed, no life cycle or budding is observed at any stage of fermentation. 2) Gram test: positive, no change in colourability at any stage of the fermentation.

3) Spore formation: none.

H) Plagella: none.

5) Small grains (Refrel's or Albert's method): clearly observed at both cell poles, and in some cases only at one pole.

6) Mobility: none.

7) Acid resistance: negative.

II. Cultivation characteristics:

I) Agar colonies:

Growth: rather slow, after 24 hours colonies of 0.5-1 mm in diameter are formed, which gradually grow. Colonies: small circle, margin: entire, slightly raised, surface: slightly dry and smooth, opaque and lemon yellow, darkening with age.

2) Experiment with agar streak:

Ve.kst: Excellent;

Growth form: Wire-branched;

Odor: None;

Media colour: Unchanged.

3) Agar stick culture and thioglycollate stick culture:

Growth only on the surface; aerobic.

4), Nutritional supp e:

Moderate with formation of membrane-like sediment after growth, odorless.

5) Blood agar:

Hemolytic reaction: none.

6) Lifter medium:

Abundant growth.

7) Blood-tellurite medium:

Black colonies.

8) Potato medium:

Lemon yellow colonies from the original culture.

III. Physiological properties:

1) Temperature conditions:

Optimal temperature: 25 - 35°C.

Thermal resistance: survives for \ hour at 70°C and for T/M hour at 7-2°C in a culture of skimmed milk.

2) Catalase reaction: positive

3) Nitrite formation from nitrate-: none

4).) Hydrogen sulphide formation : none

5) IndoldanneTse-. negative

6) M. R. reaction-: positive

7) V.P. reaction: negative

8) Growth in Koser's medium: none

9) Growth in Hucker's medium: none

10) Urease: positive

11) Gelatin stick culture: The organism only grows on the surface and does not melt gelatin

12) Starch hydrolysis: none

13) Litmus milk culture: unchanged

14) Ammonia formation: Nothing from peptone

15) Utilization of cellulose: none

16) Methylene blue reduction: positive

17) Formed pigment: lemon yellow, water-soluble, the color of the medium unchanged. 18) Fermentation of different Sugars: Acid formation with rapid cleavage from glucose, fructose, sucrose, maltose and mannose; acid formation with late cleavage from trehalose and in-ositol; "no acid formation from lactose, xylose, arabinose, rhamnose, raffinose, galactose, glycerol, adonitol, mannitol, dulcitol, sorbitol, salicin, dextrin, starch, inulin and glycogen ; no gas formation from any sugar. 19) Actiological properties: Not pathogenic to guinea pigs and mice. 20) Nutrient requirements: Cystine and biotin are necessary for growth. 21) Lactic acid was observed in a standard culture where the medium contained glucose.

It is, from the above-mentioned characteristics of Microbacterium ammoniaphilum, determined according to "Bergey's Manual of Determinative Bacteriology" (1957), 7th edition, published by the Society of American Bacteriologists, that Microbacterium ammoniaphilum belongs to the order Eubacteriales, as it is a bacterium that has the following characteristics: positive gram reaction; no sporulation; negative acid resistance; great pleomorphism, angular and palisade-like, is clearly observed.,

Based on the fact that among the families of L-glutamic acid-producing microorganisms, the family Micrococcaceae is globular or egg-shaped and has no thermal resistance, while the family Brevibacteriaceae has no pleomorphism and no thermal resistance, and the family Bacillusaceae provides spore formation, it can be concluded that the strain to which the present invention relates does not belong to any of the above-mentioned families, but that the strain belongs to the family Corynebacteriaceae. Furthermore, from the following facts 1) - 7) by careful comparison with genera of the family Corynebacteriaceae mentioned in Bergey's Manual, it is clear that the strain to which the present invention relates must belong to the genus Microbacterium. 1) The strain used according to the invention cannot belong to the genus Listeria, as it has no motility. 2) Nor can it belong to the species Erysipelothrix because its catalase reaction is positive and it grows under aerobic conditions.

3) Furthermore, it cannot belong to the genus Cellulomonas,

as it cannot utilize cellulose.

4) Furthermore, it cannot belong to the genus Arthrobacter, as morphological changes in the life cycle and changes in the gram reaction in each fermentation step are not observed. 5) Etiological nature and high requirements for nutrients - such as Corynebacterium - are not observed, but its requirements for nutrients are greater than those of Cellulomonas and Arthrobacter (both of these are soil microorganisms), i.e. the strain requires cystine and biotin. 6) It produces lactic acid in a medium in a stationary phase.

7) It withstands heating at 70°C for 30 minutes and i

15 minutes at 72°C.

The organism used according to the present invention differs both from Microbacterium lacticum and from Microbacterium flavum, which in Bergey's Manual are described as species belonging to the genus Microbacterium, due to the difference with regard to the cleavage of starch and maltose, and is considered as a key identification feature for the aforementioned two species. Furthermore, neither Microbacterium lacticum ATCC Nr. 8l80 or Microbacterium flavum ATCC No. 10340, which has been deposited as a standard strain belonging to the genus Microbacterium in the American Type Culture Collection, and has received its numbers there, is capable of producing L-glutamic acid. In contrast, the strain to which the present invention relates has a remarkable ability to produce and accumulate L-glutamic acid when the strain is grown under aerobic conditions in a medium containing carbohydrates, a nitrogen source and other nutrients. Furthermore, it is clear that the strain used according to the invention clearly differs from two previously known L-glutamic acid-producing species, namely Microbacterium flavum var. glutamicum (see English patent no. 885,611 and French patent no. 1,245,779) resp. Microbacterium salicinovorum nov. sp. _ IDoi, Kaneko; Journal of the Agricultural Chemical Society of Japan 34, (10) pp. 836 - 866/ belonging to the genus Microbacterium. This is evident from the properties indicated in the following table.

The strain used according to the present invention must therefore be considered a new species, which has never been described before. The applicants have given it the name Microbacterium ammoniaphilum, because it does not always require urea, but only aqueous ammonia or ammonia gas enables sufficient growth and thus the remarkable production of L-glutamic acid.

It is also clear from what has been stated above that Microbacterium ammoniaphilum differs clearly from the previously known L-glutamic acid-producing species which are gram-positive and have no spore formation.

The invention therefore relates to a method for the production of L-glutamic acid by cultivating a microorganism under aerobic conditions in a culture medium containing carbohydrate, nitrogen dioxide, a nutrient source and mineral salts, the method being characterized by the use of Microbacterium ammoniaphilum, ATCC 15354 as the microorganism.

The procedure is explained in more detail in the following description.

The carbohydrate and nitrogen sources that have previously been used for the production of L-glutamic acid by fermentation are also suitable in the present method. As a carbohydrate, it can e.g. glucose, fructose, mannose, sucrose, maltose, starch hydrolyzate liquid where the main component contains the above-mentioned carbohydrates, cane sugar, cane sugar molasses or sugar beet molasses are used. Furthermore, ammonia or ammonium salts can be used as a nitrogen source, e.g. ammonium chloride or sulfate; urea can also be used. Furthermore, nitrogen-containing organic materials containing different types of amino acids and vitamins can be used, e.g. peptone, meat extract, "NZ"-amine, hydrolyzate of wheat protein, corn-mold liquid. As mineral salts, e.g. magnesium salts, potassium salts, phosphates and other inorganic salts, together with the above sources of carbohydrates and nitrogen.

More specifically, a method according to the invention consists in cultivating the new strain of Microbacterium ammonia-philium at 27-33°C and pH 6.0-8.5 while adding air and stirring, stopping the cultivation after 28-60 hours, filters the bacterial cells from the broth, and precipitates the L-glutamic acid at the isoelectric point by neutralizing the filtrate to pH 3)2 or precipitates the hydrochloric acid salt of the L-glutamic acid by adding hydrogen chloride gas, or by treating the filtrate with an ion-exchange resin or a ion-exchange membrane, so that the L-glutamic acid is isolated.

The method according to the invention is unexpectedly superior to any corresponding method in certain respects due to the use of the new strain Microbacterium ammoniaphilum, ATCC 1535^. According to Norwegian patent no. 111,795, a method is known for the production of an amino acid, especially L-glutamic acid, by cultivating several microorganisms in a liquid culture medium, the microorganisms comprising various bacteria, yeast and fungi. According to the examples given in the patent, the best result is achieved using Torulopsis utilis ATCC 15239 under certain controlled bone gels which represent the production of L-glutamic acid with only 0.67 g per 100 ml of the liquid medium with a low glucose consumption of the order of 60% based on the given quantity and with a low yield of acid of 22.3$ based on glucose consumed. In contrast, the method according to the invention makes it possible to achieve a markedly higher production of L-glutamic acid, e.g. around 2 to 4 g or higher per 100 ml, with a higher yield, e.g. around 40 to 60%, or higher based on glucose consumed, as shown in the following examples.

In general, the method according to the invention differs from any corresponding known process in that the yield of L-glutamic acid based on consumed glucose is so high, which is unexpected from what is previously known. The other clear effect of the present invention in relation to the state of the art is that the new strain used according to the invention has an unusually higher efficiency in ammonia utilization than is known from other microorganisms. Furthermore, in light of the fact that ammonia is the cheapest, easily handled material as a nitrogen source in fermentations for the production of L-glutamic acid, the present invention entails a great advantage for the industrial production of L-glutamic acid by fermentation.

Example 1.

100 ml of one was placed in a 500-m-l rystko3.be

mixture, which contained 5% by volume of a technical amino acid solution - which had been produced by hydrolysis of wheat protein - from which L-glutamic acid had been removed, and 5% by volume of corn casting liquid, as well as 0.1 g of KH2PO^, - 0.1 g MgSOjj. 7H20. This mixture was adjusted to pH 6.4 -and sterilized- and then into it were inoculated two small portions (each of which could be taken in a loop of platinum wire) of Microbacterium ammoniaphilum ATCC 1535^ which had been incubated on slants- agar, and the mixture was then shaken and grown at 28°C for 24 hours. This culture was used as a pre-culture (seed culture). A culture medium containing 5% by weight glucose, 1.2% urea, 0.1$ ammonium sulfate, 0.7% KH^PO^, 0.1% MgSO^.7H2O, 0.2% of the above amino acid solution, and 0.4% corn liquor was placed in a 500 mL shake flask, adjusted to pH 6.8 and sterilized.The above pre-culture was inoculated in an amount of 2.5 ml in each 100 ml of the culture medium, and

the mixture was grown under shaking. After 36 hours, you received per 100 ml culture liquid 1.83 g L-glutamic acid, after 72 hours 2.11 g, and after 96 hours 2.12 g, i.e. approximately the same as after 72 hours. At this point the liquid contained 1.50 g/100 ml of residual sugar. The yield, calculated on the sugar used, was approx. 42% and calculated on sugar consumption approx. 62%.

Example 2.

A culture medium containing 10.02% by weight glucose, 0.1% ammonium sulfate, 0.2% KHgPOjj, 0.05% MgSO^.THgO, 0.2% of the above amino acid solution, and 0.4% corn liquor, in a 10 liter container, was adjusted to pH 7.0. In 5 liters of the resulting medium, which had been sterilized, the same pre-culture as in ex. 1 inoculated in an amount of 100 ml, after which the mixture was grown at 28°C with shaking at 400 rpm. minute, while air was blown in at a rate of 5 liters per minute. After 6 hours from the inoculation, ammonia gas was continuously introduced into the liquid, as an amino source, until pH = 8.0. After 24 hours from the start of the fermentation, 2.79 g of L-glutamic acid had been formed per 100 ml, and after 36 hours 4.13 g - After 72 hours the content of L-glutamic acid was 4.11 g per 100 ml, and this amount remained practically unchanged. The volume of the fermentation liquid at this time was 4.9 litres. The yield, calculated on sugar used, was therefore 40.2% and calculated on sugar consumed 43.1%. The fermentation liquid obtained was filtered and concentrated, after which the L-glutamic acid was precipitated by adjusting the pH to 3.2, filtered off and dried, whereby 185.3 g of L-glutamic acid of 96% purity was obtained.

Example 3-

500 ml of the same pre-culture as in ex. 1 was inoculated in 50 liters of the same culture liquid as in ex. 1, which had been produced in a fermentation vessel of volume 100 litres. The mixture was grown under shaking at 280 rpm. minute, at 28°C for 180 hours, while air was blown into the liquid in a quantity of 30 litres/minute. The liquid was used as pre-culture for the second step. A culture medium containing 9.97% glucose based on starch hydrolysis liquor, 0.2% by weight KH2P0^, 0.05% MgSOjj.7H2O, and 0.5% corn liquor was prepared in a 2 m'' fermentation tank, after which ammonia gas was introduced into the liquor to pH 7.2. The resulting liquid was inoculated with 50 liters of said second-stage culture and animal

ket at 28°C with shaking at 190 rpm. minute while 500 liters of air were blown in per minute. From 3 hours after the inoculation, ammonia gas began to be introduced, until pH = 8. In this way, after 24 hours from the start of the fermentation, 2.75 g of L-glutamic acid per 100 ml liquid and after 42 hours 4.26 g/100 ral. The remaining sugar after 42 hours was 0.28 g/100 ml, calculated as glucose, and the amount of fermentation liquid was 975 liters. The yield calculated on sugar used was 4l.6% and on sugar consumed 42.8%. The fermentation liquid was treated in the same way as in example. 2, whereby 37.3 kg of crude L-glutamic acid with a purity of 96% was recovered.

Example 4.

100 ml of a culture medium which contained 5% by weight beet molasses, calculated as glucose, and which had been treated so that it was suitable for the production of L-glutamic acid, 0.2% by weight KH^POjj, 0.05% by weight MgSO^.7H20, 1.2 weight percent urea and 0.1 weight percent (NH1|)2S01| was poured into a 500 ml shake flask, adjusted to pH 6.4 and sterilized. 2.5 ml of the same pre-culture as in ex. was inoculated. 1, and the mixture was grown with shaking at 28°C. After 72 hours of cultivation, the yield of L-glutamic acid was 2.64 g/l. The concentration of the remaining carbohydrates in the liquid was at this time 0.51% by weight. The yield calculated for used sugar was 53% and for consumed sugar 58%.

Claims (1)

Process for the production of L-glutamic acid by cultivating a microorganism under aerobic conditions in a culture medium containing carbohydrate, nitrogen source, nutrient source and mineral salts, characterized in that Microbakterium ammoniaphilum, ATCC 15354 is used as the microorganism.