DE1909997A1 - Process for the production of microorganism cells - Google Patents

Description

- · ■ - ■ "

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Seh / Gl K 884

Kyowa Hakko Kogyo Co., Ltd., Tokyo / Japan

Process for the production of microorganism ropes (

The present invention relates to a method for producing co-organisms by carrying out the cultivation under aerobic conditions using gaseous ones. Hydrocarbons as the main source of carbon.

More recently, microorganisms, they are hydrocarbons Consume in fermentation media, for sine industrial brijg been used, ss wsrd © now found that in

gaseous hydrocarbons originating in iron from the Hatur fifo Microorganisms have been grown on the genera Nocardia, Corynebacterium and Brevibacteriuia gen <teen.

The aim of the invention is to provide an improved process for the production of new species of microorganisms; those belonging to the genera Nocardia, Oorynebaefcerium and Brovibacterium belong. The invention provides a method for producing these species in the presence of gaseous hydrocarbons as the main carbon source. This procedure can be carried out in an efficient and simple manner. This process can be made finer on an industrial scale by » are performed, with high product yields are obtained.

The invention is based on the knowledge that new Spaaios of microorganisms by growing a microorganism which is able to assimilate gaseous hydrocarbons and au the Genera Nocardia, Corynebacterium or Brevibacterium g © ~ - hears, in a watery environment during aerobic conditions köxinen are obtained in the presence of at least one gaseous hydrocarbon as the main carbon source,

Microorganisms used according to the invention are bacteria which are capable of assimilating gaseous hydrocarbons in a remarkable manner, these bacteria growing as the main carbon source with consumption of gaseous hydrocarbons. Your Wachstumsgeschwindigls ^ iteii are compared au those of _eight ma Mikroorganisaenaellen been bekaimt © 2i ₉ gaseous © hydrocarbons assimilating bacteria, "for example, = methane assimilierendea bacteria significantly pronounced" That arfindungsgemässe Züchtiragsveztfahren provides © represents the eshr favorable method οά®τ fias at », ven Milroor / ianisseasellen imtex? ¥ em-j © acLnag y

Λ j ¹ *. * \ t? ^ ! Λ Λ Λ S ~ k

ü rf ~ ä 4 5/0 6 8 y

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share out mild hydrocarbons. That way Microorganism cells obtained are suitable as a protein source, as amino acid sources, as nucleic acid sources, as Sources of vitamins or the like.

According to the invention, any microorganism can be used which belongs to the Genera Nocardia, Corynebaoterium or Brevi "bacteriun and is able to assimilate gaseous hydrocarbons Nooardia considered, it should be noted that both Nocardla paraffinica, AXCO 21198 and AXCO 21199 as well Nocardla butanioa, AXCO 21197 can be isolated from the ground. Their microbiological properties are like follows:

I. Nocardla paraffinica, AXCC 21198 and AXCC 21199 A) Morphological properties

1. Form of the microorganisms: usual short rods; young cells show the mycellular state; branched and comparatively long cells are found. The formation of spores depends on whether the mycelium breaks open,

2nd size: 0.5 - 0.75 x 2.5 - 16.0 u

3. ability to move; Non-movable

4. Spores: Formed

5. Flagellum: Not formed

6. Gram stain: positive

7. Säxirebes constant discoloration! positive

B) growing properties

1. The growth takes place very slowly in a meat broth agar plate culture (circular, wrinkled, vel " umbelfönigf yellowish-red, dull and un-

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2. In a Pleincabrulienagarsehragla * ltur that runs Slow growth (thread-like »glansjlos, yellowish ^ red ,, odorless, brittle). The culture medium is not changed.

3. The growth of the meat takes place in a meatbread culture Surface skin-like, with hardly any cloudiness and notices no settling.

4. In a gelatin puncture culture, the growth in the upper foil proceeds better than in the lower part, with no liquefaction taking place.

C) Physiological properties

1. Optimal temperature: 25 - 37 ° C (slight growth at 42 ° C).

2. Optimal pH: 6.0-9.0

3. Oxygen demand: aerobic

4. laokmusmiloh: Not altered or alkaline.

5. Hydrogen Sulphide: Generated.

6. Indole: sight created.

7. Starch: decomposed spruce.

8. Hitrat is reduced.

9. Eatalaset positive.

10. Generation of Aanonium: Negative

11. Voges-Proskauer-Ieet: Negative

12. Consumption of sugars: from glucose, fructose »mannose, Acid is produced by drilling sugars, mannitol and sorbitol.

13. Hydrocarbon - Assimilating Properties: Assimilate xthane, propane, η-butane, n-dodocane, n-tridecane, n-tetradecane, n-pentadecane, n-hexadecane and n «hepta °> decan.

Hocardia paraffinica AICC 21198 and ASCC 21199 are, as far as studies have shown, only in consistency of the agar slant different. ATCC 21198 is bulky, while-

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rend ASCö 21139 is butter-like.

II. Nocardia butanica, ASCC 21197

A) Morphological properties

1. Form of the microorganisms: usually short rods? young Zollen segregate mycelium condition; branched and comparatively long cells are found. she Formation of spores depends on whether the mycelium aui ° breaks.

2nd large ©: 0.5 - 0.75 x 2.5 - 16.0 and 3 ο Movement variable arcs: Non-movable

4. Spores: Formed

5. Elagellum:! Spruce formed

6, Gram stain: positive

7, Acid-proof discoloration: Positive

B) Breeding properties

1. In a broth agar plate culture, this is Growth a little slow (circular * wrinkled, wel ~ ien-shaped, hub-shaped, matt red, “dull and un => transparent).

2. This takes place in a Pleisehbrtthenagarsenräk culture Grow slowly (thread-like, dull, matt red "goruchless, brittle). The culture medium is not changed.

3. Growth occurs in a meat broth culture d @ r Oberfläch® haue-shaped, with the broth table is klsir and no deposit found

k &: an.

4. In eiKiQ "/? Gelatinös äichkultür runs äas

t.n

than in that

0 0 9 0 A _{5/0 6 8 3 BAD 0RiG1MAL}

G) Physiological properties

1. Optimal temperature 25 - 35 ° E (slight growth at 42 ° 0).

2. Optimal pH: 6.0-9.0

3. Oxygen demand: aerobic

4. Laekrausmilch: Not changed or alkaline

5. Hydrogen Sulphide: Generated

6. Indole: Not generated

7. Starch: Not decomposed

8. Nitrate is reduced

9. Catalase: Positive

10. Ammonium generation: negative

11th Voges-Proskauer-Sest: negative

12. Consumption of sugars: Acid is made from glucose, fructose, Arablnose, mannose, cane sugar, galactose, xylose, mannitol and sorbitol.

13. Hydrocarbon assimilating properties: Assimilate ethane, propane, n «= butane, n ~ octane, n ~ undecsn _p n ° dodecane, n-tridecane, n-ietradecane, n-pentadecane, n ^ hexadecane and n-heptadecane.

The taxonomic positions of both microorganisms are determined » Determined according to Bergey's Manual of Determinative Bacteriology, 7th Edition.

Both bacteria belong to the order Actinomycetales! " ilij? e cells are stiff and in some growth phases a branched mycelium ^ -like structure is formed. Yes this? They belong to the 3rd order they belong to the 3rd amilie Actinojaycstaoeae, since an eighth mycelium is produced and, in addition, spores were formed on the breecheii of the KysaXium. In addition gehünan 3io in dieaer PamAlie au Ganus the liocartlia, since they ObIi-

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- * 7 approx

gatoriecher Voice are aerobic and acid resistant. In this genus they come Eooardia asteroidaa, Nocardia polyehrohogeneο, No car dia caprac and Iocardia minima closest. As from the The following table shows that they differ from Hocardia asteroides in their color and in their growth in one GluooBomedium as well as regarding their optimal growth temperature. They differ from Nocardia polychromogenes in terms of color, optimal vacuum temperature and Effect on milk. The difference su Nocardia oaprae consists in the wachetum in a Qluoosemedium as well as in a meat broth medium, in the production of a Lufticyzeliuma, in the action against Hiloh as well as with regard to an acid-resistant discoloration. Opposite Nocardia minima there is a difference in color, optimal growth temperature and acid-resistant discoloration. Furthermore, these new bacteria have an Aesimilations ability for gaseous hydrocarbons. They can be thought of as a new species.

They differ from each other in their SiOH Färb "Grosse su beginning of ZUohtens and in their sugar consumption _o Therefore, they were called Nocardia paraffinioa or Nocardia butanioa.

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Table I.

Noomrdia aeteroidee

Hooardia

poly-

ohromogenes

Koeardia caprae

Nooardia

we) 4 τη «

Nooardia paraffinioa ATOO 21198 ATOO 21199

Nocardla butsnica ATCO 21197

colour

(O a & ** ■ · Waohstra in <* »a Gluoo- ** - eeaeditm ο en» ο approx

ltioht yellow «a growth, becomes deep-Mlb to yellowish-red

Form thin yellowish

Skins Burst colorless. In the

last yellowish-red Waking period fleieohrot or coral red

matt red

Grows in a ritual Sheaediua

Optimal Waohetms temperature

Minor deposit of fine 3? Lok ~ ken. Formation of Irtif ticyz a.

Lots of plok deposits. Formation of aerial mycelia.

Form yellow- Form matt-ro-ce lioh-red skin. No skin.KeI deposits that A deposit, the broth is ready, the broth is clear on the table. practically clear.

22-25 ° C 22-25 ° E

ibid

25-37 * 0

ibid

25-35 ° 0

(O O CD CO

Table I (continued)

Nocardia effect Nocardia Nocardia Nocardia You mei Nocardia not swine - Nocardia + 1 asteroides opposite to poly- oaprae minims most are paraffin! approx persist butanica 4th VO ο milk chromogenic AXOO 21198 dig ATCO 21197 + I. ο ATCO 21199 W. <*> Acid resistance Grow Solidify Do not change Not ver °° discoloration changes changes " ¹ ^ exercise cn O Easy Acid resistance Acid resistance «» Consumption of dig dig ^ω sugars - Arabinose Galactose Xylose

CO CO CO

- ίο -

Corynebacterium alkanium, AXCC 21194, Brevibacterium paraffinolyticum, AXCO 21195 and Brevibacterium butanioum, ASCC 21196 are each isolated from the ground. Their microbiological properties are given below:

I. Corynebaoterium alkanium, ATCC 21194 A) Morphological properties

1. form of microorganisms) Usually short rods; Incomplete cleavage cells, branched cells and ^n- snapping "-extige cleavage cells are often observed.

2nd size: 0.5 χ 2.5 - 5.0 a

3. Mobility Hioht-mobile

4. Spores: Not formed

5. ntgellum: Not formed

6. OrajB discoloration: positive

7. Acid-resistant «Discoloration: negative

B) Züohtungselgensohaften

1 · In a veal loaf agar plate cold bite growth (circular, smooth, complete, convex, yellowish «· gray, sparkling and opaque).

2. Massive growth (thread-like, sparkling, yellowish-gray, odorless, buttery). The culture medium is not changed.

3. Growth takes place in a meat brew culture the surface slowly, the broth becoming a little cloudy.

4. There is a minor one in a gelatin engraving culture Growth in the upper part with no gelatin liquefaction observed.

C) Physiological properties

1. Optimal temperature: 25 - 30 ° C, the waxing takes place

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extremely easy "at 35 ° 0.

2. Optimal pH: 5.0 - 9.0 (no growth at a pH of 4.0).

3. Oxygen demand: aerobic

4. Litmus miloh: Not altered or alkaline

5. Hydrogen Sulphide: Generated

6. Indole: Not generated

7. Strength: Not aersed Θ. Nitrate is reduced 9. Catalase: Positive

10. Generation τοη ammonium: negative

11th Voges-Proskauer-Sest: negative

12. Consumption of sugars: Acid is produced from glucose, fructose, mannose, cane sugar, lactose, mannitol and sorbitol.

13. Hydrocarbon -assimilating properties: Assimilate propane, η-butane, N-dodecane, n-trideoane, n-tetradeoane, n-pentadeoane, n-hexadecane and n-heptadeoane.

II. BrevibaoterluB butanieua, AIOC 21196 A) Morphologisoht properties

1. Form of microorganisms: Usually korae rods; Incomplete cleavage cells and "snapping" -like cleavage cells are often formed, but no areas of silence are found.

2. Capital 0.5 ζ 5.0 - 6.0 η

3. Movement arrows: light-movable

4. Sporent Mioht formed

5 «Flagellua: Not educated

6. Qraa stain: positive

7. Acid-resistant Ytr stain negative

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B) Breeding properties

1. In an ileisoe thorn agar plate culture, excessive Growth (circular, smooth, completely "humped, light brown" matt and opaque).

2. Too much in a sloping agar culture Growth (prickly, dull, light brown, odorless, buttery). The culture medium is not changed.

3. The growth of the takes place in a flelsoh broth culture Skin-like surface. The broth is practically clear where " if no deposit is found.

4. In a Gelatinestiohkultur the growth proceeds on best in the upper part, no gelatin liquefaction being observed.

C) Physiological properties

1. Optimal temperatures: 25 - 30 ° C, easy growth at 37 ° C

2. Optimal pH: 6.0-9.0

3. Oxygen demand: aerobic

4. Laokausmiloh: Not altered or alkaline

5. Hydrogen Sulphide: Generated

6. Xndol: Not generated

7. Starch: Not .decomposed

8. Nitrate is reduced 9 «Catalase: Positive

10. Ammonium generation: negative

11. Toges-Proskauer test: negative

12. Consumption of sugars: Acid is extracted from glucose, fructose, arabinose, mannose, cane sugar, xylose, mannitol and sorbitol.

13.Hydrogen-assimilating properties: Assimilate propane, η-butane, n-decane, n-undecane * n ^ doßecane ,, n »tridecane, n-tetradecane, n-pentadecane, n-Hsza & ecan and n-heptadecane.

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III. Brevlbaeterium paraffinolyticum, AXCC 21193

A) Morphological properties

1. Form of the microorganisms: sticks, often "snapping" · * like cleavage cells, but none of them refused Cells are found.

2nd size: 0.5 x 2.5 - 5.0 u

3. Movement Ability: Hioht-agile

4. Spores: Not formed

5. Flagellum: Not formed

6. (iram discoloration: positive

7. Acid-resistant discoloration: negative

B) Breeding properties

1. Excessive in broth agar plate culture ¥ aohstum (circular, smooth, complete, convex to head-shaped, light pink, sparkling and opaque).

2. In a meat broth slant agar culture overoässlges Growth (thread-like, sparkling, yellowish-gray, noisy, buttery). The culture medium is not changed.

3. The growth takes place in a meat broth culture . the surface slowly, whereby the broth becomes cloudy.

4. It grows in a gelatin test culture upper part slightly. Gelatin liquefaction is not detected.

C) Physiological properties

1. Optimal temperature: 25-30 ⁰ C. The growth takes place very easily at 35 * 0.

2. Optimal pH: 5.0-9.0 (no growth at pH of 4 | O).

3. Oxygen demand: aerobic

4 "Iiackmusrailch: Not changed or alkaline

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-H-

5. Hydrogen Sulphide: Generated

6. Indole: Not generated

7. Starch: Not decomposed

8. Hitrat is reduced 9 Catalase positive

10. Generation of. inaoniuv: HegatiT

11. Yogee-Proslcauer tests Hegatiν

12. Consumption of. Sugaring: Acid is made from glucose> Produces fructose, cane sugar, lactose, mannitol and sorbitol.

13. Charcoal Vase Mineral Assinilizing Properties: Aeimilieren tropane »η-butane» n-undeoane, n-dodecane, n-trideoane, n-tetradeoane, n-pentadecane, n-hexadecane and n-heptadecane.

Si · taxonomic position of Oorynebaoterium alkanum, AXCO 21194 is according to Bergey's Manual of Determinative Bacteriology, 7th edition, and swar depending on the properties mentioned above. Ss belongs to the family Corynebaoteriaoeae, with regard to the rod cells which Acid instability, the lack of trichome formation, the gram positivity, ■ the lack of fermentation of sugars under aerobic conditions, the lack of formation of boundospores and formation of cell bifurcation. In this family it belongs to the genus Oorynebaoterium "since there is one curved and snapping-like cleavage shows »whereby the kata» lasetest is positive. In this genus, Corynebaoterium comes closest to Oorynebaoterium agropyri and Oorynebaoterium rathayi, However, there is a difference from Corynebacterium agropyri in terms of size, Gram discoloration and growth in elnor FlelschbrtOies slant agar culture. There is also a There is a difference compared to Corynebacterium rathayi with regard to size, growth and gelatin liquefaction. About that there is also a difference from the awsd mentioned above

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Species, in terms of the consistency of the Inclined agaro and the assimilation properties for gaseous and liquid hydrocarbons. Therefore one can from speak of a new species. This species was named Corynebacterium Alkanum, ATCC 21194.

The taxonomic position of Brevib ac terium butanicum, ATCC 21196 is determined in the manner described above. It belongs to the Brevibacteriaceae family, namely from for the following reasons: presence of rod cells, acid instability, no formation of triohomes, Grampoeitiv, none Formation of endospores and no formation of branched cells. In this family it belongs to the genus Brevibacterium, because of the non-branched rods, which have none Form threads. In this genus it comes Brevibaoterium marle, Brevibaoterium fusourn and Brevibaoterium ammonlagenes (compare the following table) are closest. Like iodooh This table shows that there is a difference to Brevibacterlum maris in terms of size, color and height of the colonies, the production of sulfur hydrogen and the effect on cane sugar. There is also a difference to Brevibac terium fusourn in terms of size, color and height of the Colonies as well as growth of a sting culture and a broth culture. The difference to Bre " vibacterium ammoniagenes is large and high as well as in the color of the colonies and also in the consistency of the colonies and in relation to the assinilation properties for gaseous and liquid hydrocarbons. Therefore one can speak of a new species which has been designated as Brevi "bacterium butanloum, ATCC 21196.

The taxononic position of Brevibacterium paraffinoly » ticum, ATCO 21195 is made in the manner described above

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"Definitely. It belongs to the family Brevibaeterisceae" for the following reasons: rod cells, Säwreunbes ability, no formation of (erichomes, Grara positive, no formation of endospores and no formation of branched cells. In this family it belongs to the genus Brevibacteriui ^ Because of the non-branched rods that do not form threads. In this genus it comes closest to Brevibacterium marie, Brevibacterium iuscum and Brevibacterium ammoniagenes. As can be seen from the following table, it differs from Brevibacterium maris in terms of size, color, Growth in a dilatation broth, effect on sugar bites and production of hydrogen sulfide. Compared to Brevibacterium fueoum there is a difference in size and growth in a gelatinous culture. In addition, there is a difference compared to Brevibacterium ammoniagenes in terms of size, color and consistency of the colonies and the eggallation properties for gaseous ones and liquid hydrocarbons. Therefore one can speak of a new species »This was named Brevibacterium paraffinolyticuH, ATCC 21195. Brevibacterium butan! Cum, ATCC 21196 and Brevibacterium paraffinolyticum, ASCC 21195 are different species from one another, since they differ in terms of Color _t distinguish between gloss and zuoker consumption.

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! Table II

Color is. a PlaischbrüiiS inclined agar culture Gram discoloration sticker consumption G-lucoss Eear sugar lactose

EeraetsimgsvermÖgön from Strong gelatin culture

I work and growth in a beef stock agar plate culture

Corynebacterium agropyric

Corynebacterium rathayj

Corynebacterium
alkantane. ASCC 21

0.4 - 0 _β 6 χ 0.6 - 1.1 ju 0.6 - 0.75 x 0.75 0.5 χ 2.5 - 5.0

yellow pissing off

Acid is generated Acid is generated

weak not liquefied

yellow

slow

very tough growth yellow
positive

yellowish-gray
positive

Acid is generated Acid is generated
Acid is generated Acid is generated
Acid is generated Acid is generated

gradually liquefied after 7 weeks

slow growth

none
not liquefied

yellowish-gray
massive Vachsinxai
buttery

CD co cc

cn

O cr> σ>

CD CD. OO

«Η

ο σ> σ>

Size

Meat broth * agar plate

Culture

Gelatin culture

IPleiGrowth culture

table III

Brevibaoterium Brevibacterium marls fuscum Brevlbacterliim Brevibacterium Brevibacterium ammoniagenea paraffinolybutanictun

ticnm AXCC 21196 ATCC 21195

0.6 χ 1.5) i

0.7 - 0.8 χ 1 _S O - 1.2 Ji

orange-yellow, brownish-yellow, convex slightly convex 1.4 -

gray or

pale yellow,

0.5 x 2.5 - 5.0 a

not damned gradually Bigt liquefied

forms a forms a skin · orange colored, sediment, tes membrane no cloudiness and a sediment, no cloudiness 0.5 x 2.5 -5.Ou

light pink safaris light brown, humped ben, convex to head-shaped

not liquefied not liquefied not liquefied liquefied

-massive distribution the growth of the ω

in the height of the surface in front of _:

Surface runs slowly,

moderate cloudiness

none

Lactose none

Soliwefelvjasser- no acid is generated acid is generated

Acid is generated

Acid is generated generated

To carry out the fermentation can either one. synthetic Culture medium or a natural nutrient medium can be used "provided that it is suitable for the growth of the microorganisms used" contains essential nutrients. Ser-like nutrients are known.These include, for example, substances such as a carbon source, a material source, inorganic compounds or the like, these substances being of the microorganisms used are consumed in the appropriate quantities

The fermentation according to the invention is carried out in an aqueous nutrient medium which is a gaseous hydrocarbon or a mixture of gaseous hydrocarbons as Contains main source of carbon. The gaseous hydrocarbons used include ethane, propane and butane. Sources of carbon that can be used in small amounts in the fermentation medium are, for example, liquid ones Hydrocarbons such as 8.B. n-dodeoane, n-trideoane, n-xetradeoane, n-pentadeoane, n-hexadeoane, n-heptadeoane or the like, carbohydrates such as, for example. Glucose, pruotoee, Maltose, cane sugar, starch hydrolyzate, molasses or the like, or other suitable carbon sources, such as glycerine, mannitol, sorbitol, organic acids or like that.

Graining as a nitrogen source, various kinds of inorganic or organic salts or compounds into carrier, for example, urea, or Aanoniak Amoniumsalse such as ammonium chloride, ^ τρ ^ μ ^ Ι ¹ WXII * "· * =, ammonium nitrate, Ammoniunphosphat or the like. In addition, one or more amino acids can be used, and natural substances that contain nitrogen can also be used, for example corn liquid, yeast extract, meat extract, fish meal, peptone,

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Meat broth, chasuble hydrolysates, soluble fish ingredients, Rice bran extract or the like. These substances can also either alone or in combination two or more substances can be used.

Inorganic compounds that can be added to the culture medium in appropriate amounts are, for example, phosphates, such as sodium phosphate, disodium monohydrogen phosphate, Potassium dihydrogen phosphate, potassium monohydrogen phosphate, metal · » compounds such as magnesium sulphate, manganese sulphate fat, zinc sulfate, copper sulfate, iron sulfate or other iron salts, manganese chloride, cobalt chloride, nickel chloride, calcium = Chloride, oaloium carbonate, boric acid, sodium aolybdate, or the like, may also be necessary to add certain essential nutrients to the culture medium, depending on the microorganism used, for example amino acids such as aspartic acid, threonine, methionine or the like, and / or vitamins such as biotin, xhiamine, Cobalamin or the like may be mentioned.

Dae Zttohten is under aerobic conditions at a ZUchtungstemperatur of about 20 - 50 ⁰ C and carried out at a pH of about 4.0 * 9.0. The gaseous hydrocarbon used is supplied in a gaseous state. Sr is used mixed with air or oxygen. For example, ethane, propane and butane may be mentioned as the gaseous hydrocarbons used. The gas concentration is not subject to any particular one

Different fermentation methods that are more common in performing Fermentation process using saccharin materials or liquid hydrocarbons as the main carbon swell

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can be used. For example, the fermentation rate can be significantly accelerated by adding small amounts of unused liquid hydrocarbons. "Further, substances _β a similar effect have, or surfactants Kittel to increase the dissolution rate of the gaseous hydrocarbons can be added in the culture medium. After completion of the fermentation, the microbial cells may be separated from the culture broth by conventional methods.

The following examples illustrate the invention without limiting it restrict. Unless otherwise stated, the percentages relate to weight.

example 1

2.7 1 of a culture medium consisting of 0.05 $> KH ₂ PO., 0.05 £ Na ₂ HPO ₄ .12H ₂ O, 0.01 36 MgSO ₄ .7H ₂ O, 0.001 56 MnSO ₄ .4HgO, 0.001 * PeS0 ₄ .7H ₂ 0, 0.001 # ZnSO ₄₁ THgO, 0.001 £ OaCIg.2HgO, 10 / Vl H ₅ BO ₅ , 10 $ 7l Na ₂ MoO ₄ .2H ₂ O, 50 Γ / 1 CuSO ₄ .5H ₂ O.10 J7l CoCl ₂ .2H ₂ 0 _f 50 371 NiCl ₂ .6H ₂ O, 0.05 # corn liquor and 0.2 $> NH ₄ NO ₃ at a pH of 7.2 are put into a 5 1- Fermentation vessel given and sterilized.

Nocardia paraffinica, AICC 21198 is previously cultivated with shaking in a culture medium containing 0.25 i> yeast extract, meat extract 0.5 56 0.5 $ peptone, sodium chloride, and 0.25 # $ 2.0> sorbitol. The pH of this medium is 7.2 and the cultivation is carried out for 24 hours. These bacteria are seeded in the first culture medium in an amount of 10 $> and? 0 ° C under Bühren bred (about 600 ÜPM). Dabsi aa? Is followed by aeration at 1 1/1 per minute of a mixed gas (the concentration of η-butane in air is

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1.3? O during a period of 72 hours · Dar pH wirci during breeding by adding ammonia was-ae :? avf held a value of 7.2 .

After breeding is over, the increased amount is « of the bacterium 10 rag in the form of dried ropes per ml of the medium. After a centrifuge, 30 g of the cells are yaw, washing with water and drying obtained.

Example 2

Hocardia butanica, ASGC 21197 is grown in the same way as described in Example 1, with a mixed gas (the concentration of propane in the air is 1.5 SO for a period of yqto. 72 hours, with the exception that propane is used as the carbon source.After the end of the breeding process, the amount of bacterium that has grown in the form of dried balls is 10 mg / ml of the medium.

Example 3

Nocordia paraffin! Ca, AXCC 21199 is prepared according to the method in Example 2 working method described, with the exception that a high concentration of η-butane is used as the carbon source. Using a mixed gas is made ventilated (the concentration of n ~ butane in air is 20 To. 72 * hours of cultivation will yield $ 2 mg of the dried len / ml of the medium obtained,

Example 4

Brevibaoterlum paraffinolyticum, ASJCC 21195 will sash mean? The procedure described in Example 3 is cultured, ice culture medium is used which consists of 0.05 #

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0.05 $ Na ₂ HPO ₄ .12H ₂ O, 0.01 £ HgSO ₄ .7HgO, 0.001 # MnSO ₄ . 0.001? S EeSO _4. ? H ₂ 0, 0.001 % ZnS0 ₄ .7H ₂ 0 _f 0.001 jS OaOIg.g 10 TA H ₃ BO ₅ , 10371 Ha ₂ MoO ₄ .2H ₂ O, 50 J7l CuSO ₄ .5H ₂ O , 10? 7l GoOl ₂ .2H ₂ O, 50? 7l HiOl ₂ .6H ₂ O, 0.05 # corn liquor and 0.2 i (NlI ^) ₂ SO ₄ , with the exception that a mixed gas with a 50% butane concentration in air is used for ventilation. After 96 hours of cultivation, 4 mg of the dried cells / ml of the medium are obtained.

Example 5

Breribaoterium butanioum, ASGC 21196 is grown according to the procedure described in Example 4. After 96 hours of cultivation, 5 mg of the dried cells / ml of des Medium received.

Example 6

Corynebacterium alkanum, ATCC 21194 is dried according to the procedure described in Example 4. After a 96 hour culture, 3 mg of the dried cells / ml the medium received.

OQSBL 5/0663

BATH ORIGINAL

Claims (9)

- 24 patent claims 1. Process but production of microorganism cells which are able to assimilate gaseous hydrocarbons and BU belong to the genera Hooardia, Coxynebäcterium or Brevibacterium, daduroh characterized that the microorganisms in an aqueous nutrient medium under aerobic conditions be cleared in the presence of at least one gaseous hydrocarbon as the main carbon source, whereupon the these finely produced microorganism cells are isolated and obtained. 2. The method according to claim 1, characterized in that Kooardia paraffinica, ATOO 21198 is used as the microorganism. 3. The method according to claim 1, characterized in that as a microorganism Hocardi «, paraffinioa, AXCO 21199 is used. 4. The method according to claim 1, characterized in that Nocardia butane! ca, ASOO 21197 is used as the microorganism will. 5. The method according to claim 1, characterized in that as a microorganism Brevibacterium paraffinolytioum »AXOC 21195 is used. 6. The method according to claim 1, characterized in that as a microorganism Brevibaoterium butanioum, ASOO 21196 is used. 009845/0663 BATH ORIGINAL 7. Procedure naoh Anepruoh 1, dadaroh marked that ale microorganism Qorynebaoterium alkanum, AXCC 21194 is used. 8. The method naoh claim 1 «daduroh characterized daas the used gaseous · hydrocarbons from xthane «propane or η-butane consists. 9. The method naoh claim 1, characterized gekennseioh & et that the Microorganism under aerobic conditions at a sea temperature of about 20 - 50 ° G and at a pH of about 4-9.