DE2857681A1 - PREPARATION OF MICROBIAL CELLS - Google Patents

Description

Τιεητιτπ - Rflui ιμλ - Kimme "patent attorneys and

IEDTKE DUHLING _IMNNE::: ^

Representative at the EPO

GD ::: ··.;: ■. öipl .- ^! ng. H.Tiedtke RUPE-Pellmann “j. _D S .'- chem. α

Dipl.-Ing. R. Kinne

9RR7RR1 Dipl.-Ing. R group

Z ö O / OO I Dipl.-Ing. B. Pellmann

Bavariaring 4, Postfach 202403 8000 Munich 2

Tel .: 089-5396 53

Telex: 5-24845 tipat

cable: Germaniapatent Munich

August 7, 1980 DE 0476 / case F8068-K70SK-PC3

description

Method for producing microbial cells 15th

Technological area

The invention relates to a method for producing microbial cells. It relates in particular to a method for economically producing microbial cells in large quantities using new microorganisms that aerobically assimilate methanol.

State of the art

In recent years , much work has been devoted to developing methods for effectively utilizing microbial cells as feed, food, medicine, industrial materials, etc. It is expected that methanol will be used as a major carbon source for industrial benefit production of microbial cells because of the good prospect of methanol being used in the chemical industry

H / 13 1306U / 0009

Danish Bank (Müncheni KIo. 51/61070 Diosdnor Bank (Munich) Kto. 3939 8 <M Postscheck (Munich) KIo. 670-43-804

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will be made available in large quantities and at low cost (see for example the Japanese Patent Laid-Open Nos. 143S9 / 1972 and 15490/1978.)

Disclosure of the invention

In view of these circumstances, the inventors have widely sought in nature for microorganisms which aerobically utilize methanol as a main carbon source and can be cultured with good efficiency, and as a result, the inventors found new microorganisms, namely Fiavobacterium tosaensis , Pseudomonas wakayamaensis , Fiavobacterium methanolicola , Pseudomonas kyotoensis , Pseudomonas aichiensis and Corynebacterium yamanasiensis . This led to the establishment of a method for economically producing microbial cells in large quantities by utilizing these microorganisms.

The invention accordingly provides a method for generating microbial cells available, which consists in that a bacterium belonging to strains that are derived from the by Fiavobacterium tosaensis , Pseudomonas wakayamaensis , Flavoba pterium methanolicol a, Pseudomonas kyotoensis , Pseudomonas aichiensis and Corynebacterium yamanasiensis are selected, cultivated in a nutrient medium containing methanol as a main carbon source, and the microbial cells obtained from the nutrient fluid.

As has been found, the microorganisms used according to the invention belong due to their bacteriological properties of the genus Pseudo-

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monas , the genus Flavobacterium or the genus Corynebacterium . They have been identified as new strains because they differ in various respects from known bacteria belonging to the genus Flavobacterium , from known bacteria belonging to the genus Pseudomonas and from known bacteria belonging to the genus Corynebacterium , and also because the microorganisms of the genus Flavobacterium used according to the invention

] Q can utilize methanol, while known bacteria of the genus Flavobacterium have not been found to assimilate methanol. These microorganisms have therefore been named Flavobacterium tosaensis , Pseudomonas wakayamaensis , Flavobacterium methanolicola , Pseudomonas kyotoensis , Pseudomonas aichiensis and Corynebacterium yamanasiensis . Typical bacterial strains belonging to the new microorganisms used according to the invention are Flavobacterium tosaensis DS-I (FERM-P No. 4058), Flavobacterium methanolicola DS-16 (FERM-P No. 4098), Pseudomonas wakayamaensis DS-25 (FERM- P No. 4100), Pseudomonas kyotoensis DS-22 (FERM-P No. 4099), Pseudomonas aichiensis DS-26 (FERM-P No. 4107) and Corynebacterium yamanasiensis DS-31 (FERM-P No. 4106), all of them in the Fermentation Research Institute, Agency of Industrial Science and Technology, Japan, 5-8-1, Higashi, Inage, Chiba-ken, Japan.

The bacteriological properties of these Bacterial strains are shown below.

(I) Flavobacterium tosaensis DS-I (FERM-P No. 4058):

(a) morphology

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At 37 ° C for 3 days in a nutrient medium and grown on a nutrient agar medium.

(1) Shape and size of cells: rods; 0.5 to 0.75 μm 1.6 to 2.2 pm

(2) Colonies of cells: singly or in pairs IQ (3) motility: none

(4) Spores: none

(5) Gram dye: negative

(6) Acid resistance: negative

(b) State of growth in various nutrient media

(1) Nutrient agar plate culture: Abundant growth at 37 ⁰ C for 3 TageC The colonies are circular with a diameter of 2 to 3 mm and a convex or buckelig having a uniform structure, a smooth surface and an entire edge. Yellowish white or milky white, shiny, translucent and slimy.

(2) Agar plate culture containing methanol: Das Growth is abundant at 37 ° C for 3 days. The colonies are circular with a diameter of up to 2.5 mm and convex or humped with a smooth surface and a whole edge. White or opalescent rend, shiny, translucent and slimy.

(3) Nutrient agar slant: thread-like growth

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• j at 37 ° C for 3 days. The colonies have a smooth surface and are moderately prominent with a yellowish white or milky white color and a glossy one Shine. Translucent and slimy.

(4) Methanol agar slant: thread-like growth at 37 ° C for 3 days. The colonies have a smooth Surface and are moderately prominent with a white or opalescent color and a glossy sheen.

IQ Translucent and slimy.

(5) Nutrient Broth: Abundant growth at 37 ° C for 3 days. The culture becomes cloudy. A sediment is formed. A white, ring-shaped membrane is formed on the surface.

(6) Peptone-water nutrient liquid: Abundant growth at 37 ^° C. over 3 days. The culture becomes cloudy. A sediment is formed. A ring-shaped membrane is formed on the surface.

(7) Nutrient fluid containing methanol: Abundant growth at 37 ° C for 3 days. The culture becomes cloudy. A Sediment is formed. A ring-shaped membrane is formed on the surface.

(8) Nutrient broth (stab culture): At 37 ° C for 3 days the growth occurs on the surface or it progresses to a depth of 2 to 3 mm.

(9) gelatin broth (stab culture): Surface growth at 30 ⁰ C for 5 days with sediment. Gelatin is liquefied.

(10) Litmus milk: Acid is formed at 37 ° C. The culture turns red and is coagulated.

(c) Biochemical properties

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(1) Reduction of nitrate: positive

(2) Denitrification reaction: positive

(3) MR test: negative

(4) VP test: positive (5) generation of indole: negative

(6) Generation of sulfur (!! hydrogen: negative

(7) hydrolysis of starch: negative

(8) Utilization of citra ':

(Koser's medium): negative

(9) Utilization of inorganic nitrogen

Ammonium salt: positive

Nitrate: positive

(10) Generation of pigment

(Kings Medium A and B): negative

(11) Urea reaction: positive

(12) Oxidase reaction: positive

(13) Catalase reaction: positive

(14) pH and temperature ranges for growth in methanol content: .ger nutrient liquid pH range: 5-9: good growth

6-8: optimum 4 and 10: no growth

Temperature range:

5-44 ° C: good growth

25-42 ° C: Optimum · 45 oikein growth

(15) Behavior towards oxygen: aerobic

(16) O-F test (using the Hugh-Leifson method):

Glucose is oxidatively metabolized (weak

Acid formation)

(17.) Generation of acid and gas from the following pickups (using peptone water at a sugar concentration of 1 % at 37 ° C for 10 days):

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Generation of
acid

Production of gas

* (1) L-arabinofic

(2) EUXyIose

(3) D-glucose
(L _1. ) D- ^lv 1annose

(5) D-fructose

(6) D-galactose

(7) maltose
] 5 (δ) sucrose

(9) lactose

(10) trehalose

(11) D-sorbitol
(12) D-Mann it

(13) inositol

(14) glycerin

(15) strength

+ (weak)

+ (weak) ■ f "* (weak)

+ (weak)

+ (weak)

+ (weak)

+ (weak)

4 (weak)

ι (weak)

+ (weak)

(weak) + (weak)

(18) Assimilation of sugars (using sugar in place of methanol in the methanoihaltigen NährflUssigkeit in a concentration of 1% at 37 ⁰ C over days):

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sugar

Assimilation

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(I) L-Arabinor.e
(2) D-xylose

(3) D-glucose
(A) D-Hannose

(5) D-fructose

(6) D-galactose

(7) maltose

(8) sucrose

(9) lactose

(10) trehalose

(II) D-sorbitol

(12) D-1'ianiiit.

(13) inositol

(14) glycerin

(15) strength

+ (weak)

+. (weak)

+ (weak)

ι- (weak)

+ (weak)

+ (weak)

+ (weak)

+ (weak)

+ (weak)

+ (weak)

-j- (weak)

+ (weak)

-f (weak)

(d) Source of insulation: soil

The methariol-containing agar medium and the methanol-containing Nutrient liquid used in the above culture tests had the following Composition:

(1) Methariol-containing agar medium

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1.5 g

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Ma ₂ HPC 3 0 2 g 0, 24 mg ° / G o _y 24 rag MgSO ₄ * O ₁ 5 g ^{1 line} 2 mg CaCi ₂ - 1 g 20th G FeSO ₄ - lated 01 g 1 1 ZnSO ₄ - ° / 4 mg CuSO ₄ ' 25 mg '4 , SO ₄ 7H ₂ O 2H ₂ O ■ 7H ₂ 0 water ■ 7H ₂ 0 "5H ₂ O _Well? Mo0 ₄ '2H ₂ CoCi ₂ MnSO ₄ Agar Destil '6H ₂ O ■ 5H ₂ 0

A nutrient medium consisting of the above-described ingredients was heated to 120 ° C. for 15 minutes sterilized, and then 20 g of methanol was added under sterile conditions.

(2) Nutrient fluid containing methanoi

This medium is similar to the methanol-containing agar medium described above, but the 20 g of agar not used while the amount of methanol used was changed to 5 g.

(II) Pseudomonas wakayamaensis DS-25 (FERM-P

No. 4100): (a) Morphology

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Grown in a nutrient broth and a nutrient agar medium at 37 ° C for 3 days.

(1) Shape and size of cells: rods; 0.3 to 0.4 pm χ 2.0 to 2.2 pm.

(2) CoJonies of cells: singly or in pairs

(3) Motility: freely moving with polar flagella.

(4) Spores: none

(5) Gram dye: negative

(6) Acid resistance: negative

(b) Growth catalyst in various nutrient media

(1) Nutrient agar plate culture: Abundant growth at 37 ° C for 3 days. The colonies are circular and 3 to 4 mm in diameter with a rough surface. Lobed edge, milky yellowish white color. Chalky, translucent and slimy.

(2) Agar plate culture containing methanol: Plentiful Growth at 37 ° C for 5 days. The colonies are circular, 3 to 4 mm in diameter and protrude like a navel (concave in the middle). Rough surface, whole edge, opalescent color and shimmering shimmer. Translucent and slimy.

(3) Nutrient agar slant: thread-like growth at 37 ° C for 3 days. The colonies have a smooth surface and are moderately prominent. More wavy or lobed edge, milky-yellowish white color and dull sheen. Translucent and slimy.

(4) Agar slant culture containing methanol: threadlike Growth at 37 ° C for 5 days. The colonies are

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moderately protruding and have a smooth surface. Whole edge, opalescent color and shiny shimmer. Translucent and slimy.

(5) Nutrient broth: Moderate growth at 37 ° C for 3 days. No sedimentation. On the surface a cuticle is formed.

(6) Nutrient bouillori (shaken): Abundant growth at 37 ° C for 3 days. The culture becomes cloudy. A

Sediment is formed. Translucent.

(7) PepLon water nutrient liquid (shaken): Abundant growth at 37 ° C for 3 days. The culture

becomes cloudy. A sediment is formed.

(8) Nutrient liquid containing methanol: Plentiful Growth at 37 ° C for 3 days. The culture becomes cloudy. A sediment is formed. On the surface will a thin, ring-shaped membrane is formed. Translucent.

(9) Nutrient liquid containing methanol (shaken): Abundant growth at 37 ° C for 3 days. The culture becomes cloudy. A sediment is formed. Translucent.

(10) Nutrient broth (stab culture): At 37 ° C for 3 days, growth takes place on the surface, or it progresses to a depth of 1 to 3 mm. Milky-yellowish white surface.

(11) Gelatin broth (stab culture): Surface growth at 30 ⁰ C for 5 days with sediment. Gelatin is liquefied.

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(12) Litmus milk: The culture is coagulated at 37 ° C.

(c) Biochemical properties

(1) Reduction of nitrate: positive

(2) Denitrification reaction: negative

(3) MR test: negative

(4) VP test positive

(5) Generation of indole: negative

(6) Generation of hydrogen sulfide: negative

(7) hydrolysis of starch: positive

(8) Utilization of citrate positive (Koser's medium):

(9) Utilization of inorganic nitrogen ammonium salt: positive

Nitrate: positive

(10) Generation of pigment

(Kings Medium A and B): negative

(11) Urea reaction: positive (12) Oxidase reaction: positive

(13) Catalase reaction: positive

(14) Ranges for growth: the temperature and pH are determined in the following nutrient liquid containing methanol

varied (the pH was adjusted by adding a

aqueous solution of NaOH or HCl). Good growth at a pH in the range of 6 to 9. No growth at a pH of 5 and 10. Good growth at a temperature of 5 to 40 ^° C. The optimal temperature be

carries 25 to 40 ° C. No growth at 41 ° C.

(15) Behavior towards oxygen: aerobic

(16) O-F test (according to the Hugh-Leifson method) :

Glucose is metabolized oxidatively.

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(17) Generation of acid and gas from the sugars given below (using of peptone water at a sugar concentration of 1% by weight at 37 ° C for 10 days):

Generation of generation of acid gas

(1) L-arabinose

(2) D-xylose

(3) D-glucose +

(4) D-mannose + 15th

(5) D-fructose +

(6) D-galactose

(7) maltose + (8) sucrose +

(9) lactose

(10) Trehalose +

(11) D-sorbitol

(12) D-mannitol +

(13) inositol

(14) Glycerin + (15.) - Strong +

(18) Assimilation of sugars (using sugar instead of methanol in the methanol-containing Nutrient fluid in an amount of 0.5% by weight at 37 ° C for 10 days):

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Assimi DE 0476 2857681 lation ₊ (l) L-arabinose + (2) D-xylose + (weak) (5) D-glucose + te (4) D-mannose + (weak) (5) D-fructose + (weak) (6) D-galactose + (7) maltose + (weak) (8) sucrose . - (9) lactose ■ + (10) trehalose + (11) D-sorbitol + (12) D-mannitol + (13) inositol + (14) glycerin (15) strength

(d) Source of insulation: soil

The ethanol-containing agar medium and the methanol-containing Nutrient fluids used in the breeding tests described above the following composition:

(1) Methariol-containing agar medium

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_4t- ■ ■ - ■■ ·· ■ · ■ - ■

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KH ₂ PO ₄ 1.5 g

Na ₂ HPO ₄ 3.2 g

(tJH ₄ ) ₂ S0 ₄ 3 g

MgSO ₄ '7H ₂ O 0.5 g

CaCi ₂ -2H ₂ OO _; lg

FeSO ₄ -7H ₂ O 0.01 β mg ZnSO ₄ '7H ₂ O 1.4 mg mg CuSO ₄ -5H ₂ O 0.25 mg Na ₂ HoO ₄ -2H ₂ O 0.24 1/2 mg CoCi ₂ -SH ₂ O 0.24 20 g MnSO ₄ * 5H ₂ O Agar 1 1 Distilled water

One of the ingredients given above Existing nutrient medium was sterilized for 15 minutes at 120 C, and then 20 g of methanol were added added sterile conditions.

(2) Nutrient liquid containing methanol

This medium was similar to the above-described, methanol-containing agar medium, but the 20 g Agar not used, and the amount of methanol used was changed to 5 g.

(III) Flavobacterium methariolicola DS-16 (FERM-P

No. 4098): 35

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(a) morphology

Grown in a nutrient broth and a nutrient agar medium at 37 ° C for 3 days.

(1) Shape and size of cells: rods; 0.5

up to 0.7 pm χ 1.5 to 1.8 / um.

(2) Colonies of cells: singly or in pairs.

(3) Motility: none
(4) Spores: none

(5) Gram dye: negative

(6) Acid resistance: negative

(b) State of growth in various nutrient media

(1) Nutrient agar plate culture: Abundant growth

at 37 ° C for 3 days. The colonies are circular with a diameter of 4 to 5 mm. The protruding one Part is convex or bumpy. Even structure door, smooth surface and entire edge. Yellowish brown, shiny, translucent and slimy.

(2) Agar plate culture containing methanol: Plentiful Growth at 37 C for 5 days. The colonies are circular with a diameter of 2 to 3 mm.

The protruding part is convex. Smooth surface, whole edge, milky-yellowish white color and glossier Shine. Translucent and slimy.

(3) Nutrient agar slant: filiform growth at 37 ° C for 3 days. The colonies are moderately prominent with one rough surface and one whole edge. Yellowish brown, shiny, translucent and slimy.

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] (4) Agar inclined culture door containing methanol: thread-like Growth at 37 ° C for 5 days. The colonies are moderately prominent with a smooth surface and a whole margin. Milky-yellowish white, shiny, translucent and slimy.

(5) Nutrient broth: Moderate growth at 37 ⁰ C for 3 days. A sediment is formed. A membrane is formed on the surface.

10

(6) Nutrient Broth (shaken): Abundant growth at 37 ° C for 3 days. The culture becomes cloudy. A Sediment is formed. Translucent.

(7) Peptone water nutrient liquid (shaken): Abundant growth at 37 ° C for 3 days. The culture becomes cloudy. A sediment is formed.

(8) Nutrient fluid containing methanol: Abundant growth at 37 C for 3 days. The culture becomes cloudy. A sediment is formed. A thin, ring-shaped membrane is formed on the surface. Translucent.

(9) Nutrient liquid containing mothanol (shaken): Abundant growth at 37 ° C for 3 days. The culture becomes cloudy. A sediment is formed. Translucent,

(10) Nutrient broth (stab culture): At 37 ° C for 3 days, growth takes place on the surface, or it progresses to a depth of 3 to 5 mm. Surface yellowish brown.

(11) Gelatin nutrient broth (stitch culture): At

30 ° C for 5 days there is no liquefaction of the gelatin

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(12) Litmus milk: The culture is coagulated at 37 ° C

(C) Biochemical properties

(1) Reduction of nitrate: positive

(2) Deritrification reaction: positive

(3) MR test: negative (4) VP test: positive

(5) Generation of indole: negative

(6) Generation of hydrogen sulfide: negative

(7) hydrolysis of starch: positive

(8) Utilization of citrate

(Koser's medium): positive

(9) Utilization of inorganic nitrogen

Ammonium salt: positive

Nitrate: positive

(10) Generation of pigment

(Kings Medium A and B): negative

(11) Urea reaction: positive

(12) Oxidase reaction: positive

(13) Catalase reaction: positive

(14) Ranges for growth: The temperature and pH are determined in the nutrient liquid containing methanol described below
varies. (The pH is adjusted by adding an aqueous solution of NaOH or HCl.) Good growth at a pH of 5 to 9. A pH range of 6 to 8 is preferred. No

Grows at a pH of 4 and 10. Good growth at a temperature of 15 to 40 ° C. Temperatures of 25 to 40 ° C are preferred, no growth at 42 ° C.

(15) Behavior towards oxygen: aerobic

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(16) 0-F test (according to the Hugh-Leifson method) :

Glucose is oxidatively metabolized.

(17) Generation of acid and gas from the following sugars (using peptone water with a sugar concentration ^of 1% by weight at 37 ° C for 10 days):

(I) L-arabinose (?,) D-xyIose

(3) D-glucose

(4) D-mannose

(5) D-fructose

(6) D-galactose

(7) maltose

(8) sucrose

(9) lactose

(10) trehalose

(II) D-sorbitol

(12) D-Manrii t

(13) inositol

(14) glycerin

(15) strength

Generation of generation of

Acid gas

+ (weak)

+ (weak) + (weak) + (weak)

+ (weak)

+ (weak)

(18) Assimilation of sugars (using sugar instead of methanol in the methanol-containing nutrient fluid in a concentration of 0.5% by weight at 37 ° C for 10 days):

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(1) L-arabinose

(2) D-xyIose
(3). D-glucose

(4) D-mannose

(5) D-fructose

(6) D-galactose

(7) Waltose

(8) sucrose

(9) lactose

(10) trehalose

(11) D-sorbitol

(12) D-l ^ annit

(13) inositol
(IA) glycerin
(15) strength

Assimilation

+ (weak) + ^: (weak)

+ (weak)

+ (weak)

+ (weak)

+ (weak)

+ (weak)

"■■ + (weak)

+ (weak) + (weak) + (weak) + (weak)

(d) Source of insulation: soil

That. methanol-containing agar medium and the methanol-containing Nutrient fluid used in those described above Breeding tests were used, had the following composition:

(1) Agar medium containing methanol

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KH ₂ PO ₄ 1.5 g Na ₂ HPO ₄ 3.2 g (NH ₄ ) ₂ S0 ₄ 3 B MgS0 ₄ -7I: I ₂ 0 0.5 g CaCi ₂ -2IT ₂ O 0.1 g FeSO ₄ '71I ₂ O 0.01 4 ZnSC ₄ -7I ^T ₂ 0 1/4 mg CuSO ₄ -5'I ₂ O 0.25 mg Na ₂ MoO ₄ -2H ₂ O 0.24 mg CoCip · 6HpO 0.24 mg IWSO ₄ -SH ₂ O 1.2 mg Agar 20 g Distilled water 1 1

A nutrient medium consisting of the components described above was heated to 120 ° C. for 15 minutes sterilized, and then 20 g of methanol was added under sterile conditions.

(2) Nutrient fluid containing methanol

; This medium was similar to the above-described, methanol-containing agar medium, but the 20 g Agar not used, and the amount of methanol used was changed to 5 g.

(IV? Pseudomonas kyotoensis DS-22 (FERM-P No. 4099):

(a) Morphology 35

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Grown in a nutrient broth and nutrient agar medium at 37 ° C for 3 days.

(1) Shape and size of cells: rods; 0.5 ρ χ 1.7 to 1.8 pm.

(2) Colonies of cells: singly or in pairs.

(3) Motility: freely moving with polar flagella.

(4) spores: small

(5) Gram dye: negative

IQ (6) Acid Resistance: negative

(b) State of growth in various nutrient media

(1) Nutrient agar plate culture: Abundant growth at 37 C for 3 days. The colonies are circular with a diameter of 3 to 4 mm and one smooth Surface as well as a whole or wavy edge. Milky-yellowish white, chalky, translucent and slimy.

(2) Agar plate culture containing methanol: Plentiful Growth at 37 ° C for 5 days. The colonies are circular with a diameter of 3 to 4.5 mm and protruding hub-shaped with a rough surface and a whole rim. Opalescent, chalky, translucent and slimy. .

(3) Nutrient agar slant: filiform growth at 37 ° C for 3 days. The colonies are moderately prominent with one smooth surface and one whole or wavy edge. Milky-yellowish white, chalky, translucent and slimy.

(4) Agar slant culture containing methanol: Filamentous Growth at 37 ° C for 5 days. The colonies

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are moderately protruding with a rough surface and a wavy or lobed edge, and they are opalescent. It becomes a pale pink pigment educated. Very dull shine. Translucent and slimy.

(5) Nutrient broth: Moderate growth at 37 ° C for 3 days. No sediment is formed. On the surface a cuticle is formed.

(6) Nutrient Broth (shaken): Abundant growth at 37 ° C for 3 days. The culture becomes cloudy. A sediment is formed. Translucent.

(7) Peptone water nutrient liquid (shaken): Abundant growth at 37 ° C for 3 days. The culture becomes cloudy. A sediment is formed.

(8) Nutrient liquid containing methanol: Plentiful Growth at 37 ° C for 3 days. The culture becomes cloudy. A sediment is formed. On the surface will a thin membrane is formed. Translucent.

(9) methanol mineral salt nutrient liquid (

shakes): Abundant growth at 37 ° C for 3 days. The culture becomes cloudy. A sediment is formed. Translucent.

(10) Nutrient bouillori (sting culture): Growth takes place at 37 ° C for 3 days only on the surface. Milky yellow surface.

(11) gelatin broth (stab): Oberle growth at 30 ⁰ C!
The gelatin is liquefied.

surface wax turn at 30 ⁰ C over 5 days with sediment.

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(12) Litmus milk: The culture is coagulated at 37 ° C.

(c) Biochemical properties i

(1) Reduction of nitrate: positive

(2) Denitrification reaction: negative

(3) MR test: negative

(4) VP test: positive (5) generation of indole: negative

(6) Generation of hydrogen sulfide: negative

(7) hydrolysis of starch: positive

(8) Utilization of citrate

(Koser's medium): positive

(9) Utilization of inorganic nitrogen

Ammonium salt: positive nitrate: positive

(10) Generation of pigment (Kings Medium A and B): negative (11) Urea reaction: positive

(12) Oxidase reaction: positive

(13) Catalase reaction: positive

(14) Ranges for growth: the temperature and the pH were varied in the nutrient liquid containing methanol described below (the pH was adjusted by adding an aqueous solution of NaOH or HCl). Grows well at a pH in the range of 5 to 9. A pH of 6 to 8 is preferred. No growth at pH 4 and 10. Good growth at temperature 5 to 45 ° C. Temperatures of 25 to 42 ° C. are preferred. No growth at 48 ° C. ■

(15) relate to oxygen: aerobic

(16) O-F test (using the Hugh-Leifson method): Glucose is oxidatively metabolized.

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(17) Generation of acid and gas from the sugars listed below (using peptone water at a sugar concentration of 1% by weight at 37 ° C for 10 days):

Generation of acid

Production of gas

(χ) L-arabinose (P.) D-xyIose (3) D-glucose

(/.<·) D-mannose

(5) D-fructose

(6) D-galactose

(7) maltose (8) sucrose

(9) lactose

(10) trehalose

(11) D-sorbitol (12) D-mannitol

(13) inositol

(14) glycerin

(15) Strength ^{V J}

(18) ABsitnilation of sugars (using of sugar instead of methanol in the methanol-containing nutrient fluid in a concentration of 0.5 35% by weight at 37 ° C for 10 days):

4 (weak)

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(1) L-arabinose

(2) D-xyIose

(3) D-glucose

(4) D-Hannose

(5) D-fructose

(6) D-galactose

(7) maltose

(8) sucrose

(9) lactose

(10) trehalose

(11) D-sorbitol

(12) D-mannitol

(13) inositol

(14) glycerin

(15) strength

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Assimilation

+ (weak)

25 (d) Source of insulation: soil

The methariol-containing agar medium and the methanol-containing Nutrient fluid used in those described above Breeding tests were used, 30 had the following composition:

(1) Methariol-containing agar medium

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2857681 KH ₂ PO ₄ 1.5 g Ma ₂ HPO ₄ 3.2 g (NH ₄ ) ₂ S0 ₄ 3 g MgSO ₄ -7Ii ₂ O 0.5 g Π Q Π P OXl C \ 0.1 g FeSO ₄ -7H ₂ O 0.01 g ZnSO ₄ -TH ₂ O 1.4 mg CuSO ₄ -5H ₉ O Oj 25 mg ! Ta ₂ -ToO ₄ -2H ₂ O 0 _y 24 mg CoC ^ ₉ -6H ₉ O 0.24 mg NInSO, -5H _o 0
H C. 1.2 mg 20 g Distilled water 1 1

A nutrient medium consisting of the components described above was heated to 120 ° C. for 15 minutes sterilized, and then 20 g of methanol was added under sterile conditions.

25 (2) Nutrient liquid containing methanol

This medium was similar to the methanol-containing agar medium described above, but the 20 g Agar not used, and the amount of methanol used was changed to 5 g.

(V) Pseudomonas aichlensis DS-26 (FERM-P No. 4107):

35 (a) morphology

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Grown in a nutrient broth and nutrient agar medium at 37 ° C for 3 days.

(1) Shape and size of cells: rods; 0.3 to 0.4 pm χ 0.75 to 1.0 yum.

(2) Colonies of cells: singly or in pairs.

(3) Mütility: freely movable with polar flagella.

(4) Spores: none

(5) Gram dye: negative

(6) Acid resistance: negative

(b) State of growth in various nutrient media

(1) Nutrient agar plate culture: Abundant growth at 37 ° C for 3 days. The colonies are circular with a diameter of 4 to 6 mm and a hub-shaped shape, one smooth surface and one whole or lobed edge. Milky-yellowish white, shiny, translucent and slimy.

(2) Agar plate culture containing methanol: Plentiful Growth at 37 ° C for 5 days. The colonies are circular with a diameter of 2 to 3 mm and one smooth surface and a whole edge. Opalescent, shiny, translucent and slimy.

(3) Nutrient agar slant: filiform growth at 37 ° C for 3 days. The colonies are moderately prominent with one smooth surface and one whole or wavy edge. Milky-yellowish white, shiny, translucent and buttery.

(4) Agar slant culture containing methanol: Filamentous Growth at 37 ° C for 5 days. The colonies are moderately prominent with a smooth surface and

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.Jf.

a whole margin. Opalescent, shiny, translucent and buttery.

(5) Nutrient Broth: Abundant growth at 37 ° C for 3 days. A sediment is formed. Translucent. A membrane is formed on the surface.

(6) Nutrient Broth (shaken): Abundant growth at 37 ° C for 3 days. The culture becomes cloudy. A

Sediment is formed. Translucent.

(7) Peptone water nutrient liquid (shaken): Abundant growth at 37 ° C for 3 days. The culture becomes cloudy. A sediment is formed.

(8) Nutrient liquid containing methanol: Plentiful

Growth at 37 ° C for 3 days. The culture becomes cloudy. A sediment is formed. On the surface will a thin, ring-shaped membrane is formed. Translucent.

(9) Nutrient liquid containing methanol (shaken): Abundant growth at 37 ° C for 3 days. The culture becomes cloudy. A sediment is formed. Translucent.

(10) Nutrient broth (stab culture): At 37 ° C for 3 days, growth takes place on the surface, or it progresses to a depth of 2 to 3 mm. Milky white surface.

(11) Gelatin nutrient broth (stitch culture): top growth at 30 ° C above sea level ^:

Gelatin is liquefied.

surface growth at 30 ° C over 5 days with sediment.

(12) Litmus milk: The culture is coagulated at 37 ° C.

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de 047 ^ 857681

(c) Biochemical properties

(1) Reduction of nitrate: negative

(2) Denitrification: negative (3) MR test: negative

(4) VP test: positive

(5) Generation of indole: negative

(6) Generation of hydrogen sulfide: negative

(7) hydrolysis of starch: negative (8) utilization of citrate

(Koser's medium): negative (9) Utilization of inorganic nitrogen

Ammonium salt: positive

Nitrate: negative (10) generation of pigment

(Kings Medium A and B): negative

(11) Urea reaction: positive

(12) Oxidase reaction: positive

(13) Catalase reaction: positive

(14) Ranges for growth: the temperature and pH are varied (the pH is varied by Addition of an aqueous solution of NaOH or HCl adjusted). Good growth at pH im Range 5 to 9 but no growth at pH 4 and 10. Good growth at pH Temperature from 5 to 45 ° C, with temperatures from 25 to 43 ° C being preferred. No growth at 50 ° C.

(15) Behavior towards oxygen: aerobic

(16) O-F test (using the Hugh-Leifson method): Glucose is not metabolized aerobically or anaerobically (no acid or gas formation).

(17) Generation of acid and gas from the below specified sugars (using

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of peptone water at a sugar concentration of 1% by weight at 37 ° C over 10 days):

(1) L-arabinose

(2) D-xylose

(3) D-glucose

(4) D-mannose

(5) D-fructose

(6) D-galactose

(7) maltose

(8) sucrose

(9) lactose

(10) trehalose

(11) D-sorbitol

(12) D-mannitol

(13) inositol

(14) glycerin (15) starch

Generation of generation of acid gas

+ (weak) + (weak)

+ (weak) + (weak)

+ (weak)

+ (weak)

(18) Assimilation of sugars (using sugar instead of methanol in the methanol-containing nutrient fluid medium in a concentration of 0.5% by weight at 37 ^° C. for 10 days):

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(1) L-arabinose

(2) D-xylose

(3) D-clucose

(4) D-mannose

(5) D-fructose

(6) D-calactose

(7) maltose

(8) sucrose

(9) lactose

(10) trehalose

(11) D-sorbitol

(12) D-Marinite

(13) inositol

(14) glycerin

(15) strength

Assimilation
+ (very weak) + (very weak). + (very weak) + (very weak) + (very weak) + (very weak) + (very weak) + (very weak)

+ (very weak)

+ (very weak) + (weak)

+ (very weak)

(d) Source of insulation: soil

The methanoi-containing agar medium and the methanoi-containing nutrient liquid used in those described above Breeding tests were used, had the following composition:

(1) Agar medium containing methanol

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KH ₂ PO ₄ 1.5 g Na ₂ HPO ₄ 3.2 g (NH ₄ ) ₂ S0 ₄ 3 g MgSO ₄ -7H ₂ O 0.5 g CaCZ ₂ -2H ₂ O 0.1 g FeSO ₄ -7H ₂ O 0.01 g ZnSO ₄ -7H ₂ ⁰ 1.4 mg CuSO ₄ -5H ₂ O 0.25 rag Ha ₂ MoO ₄ ^ H ₂ O 0.24 mg CoCi ₂ '6K ₂ O 0.24 mg MnSO; * 5Η _ο 0 1/2 mg Agar 20 g Distilled water 1 1

A nutrient medium consisting of the above-mentioned components was heated to 120 ° C. for 15 minutes sterilized, and then 20 g of methanol was added under sterile conditions.

(2) Nutrient liquid containing methanol

This medium is similar to the methanol-containing agar medium described above, but the 2G g agar not used, and the amount of Methanois used was changed to 5 g.

fl / pCorynebacterium yamanasiensis DS-31 (FERM-P

No. 4106): (a) Morphology

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-> 4- - DE 0476

Grown in a nutrient broth and nutrient agar medium at 37 ° C for 3 days.

(1) Shape and size of cells: short rods; 0.5 to 0.6 µm χ 0.6 to 0.9 µm \.

(2) Colonies of cells: singly or in pairs and pleomorphic or curved and V-shaped

divided cells.

(3) Motility: none
(4) Spores: none

(5) Gram dye: positive

(6) Acid resistance: negative

(b) State of growth on different nutrient media 15

(1) Nutrient agar plate culture: Abundant growth at 37 ° C for 3 days. The colonies are circular, 2 to 2.5 mm in diameter and convex or humped with one smooth surface and one

whole edge. Yellow, shiny, translucent, and buttery.

(2) Methanol-containing agar plate culture: Abundant Growth at 37 ° C for 5 days. The colonies are circular with a diameter of 2 to 3 mm and a smooth surface and a whole edge. Opalescent, shiny, translucent and buttery.

Nutrient agar slant culture: thread-like growth at 37 ° C for 3 days. The colonies are moderately prominent with one smooth surface and one whole edge. Yellow, shiny, translucent, and buttery.

(4) Agar slant culture containing methanol: thread

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shaped growth at 37 ° C for 5 days. The colonies are moderately protruding with a smooth surface and a whole margin. Opalescent, shiny, translucent and buttery.

(5) Nutrient Broth: Moderate growth at 37 ° C above

3 days. A sediment is formed. Slightly translucent. There won't be any on the surface annular membrane and no membrane formed.

(6) Nutrient Broth (shaken): Abundant growth at 37 ° C for 3 days. The culture becomes cloudy. A Sediment is formed. Translucent.

(7) Peptone water nutrient liquid: Abundant growth at 37 ° C for 3 days
A sediment is formed.

Growth at 37 ° C for 3 days. The culture becomes cloudy.

(8) Nutrient fluid containing methanol: Plentiful Growth at 37 ° C for 3 days. The culture becomes cloudy. A sediment is formed. On the surface will a thin, ring-shaped membrane is formed. Translucent.

(9) Nutrient fluid containing methanol (shaken): Abundant growth at 37 ° C for 3 days. The culture becomes cloudy. A sediment is formed. Translucent,

(10) Nutrient broth (stab culture): At 37 ° C for 3 days, growth takes place on the surface, or it progresses to a depth of 2 to 3 mm. Opalescent surface.

(11) Gelatin nutrient broth (stab culture): surface growth at 30 over 5 days with sediment.

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No liquefaction of gelatin.

(12) Litmus milk: The culture is coagulated at 37 ° C.

(c) Biochemical properties

(1) Reduction of nitrate: positive

(2) Denitrification reaction: positive

(3) MR test: positive

(4) VP test: positive

(5) Generation of indole: negative.

(6) Generation of hydrogen sulfide: negative

(7) hydrolysis of starch: positive

(8) Utilization of citrate

(Koser's medium): negative

(9) Utilization of inorganic nitrogen

Ammonium salt: positive

Nitrate: positive

(10) Generation of pigment

(Kings Medium A and B): negative

(11) Urea reaction: negative

(12) Oxidase reaction: negative

(13) Catalase reaction: positive

(14) Ranges for growth: The temperature and pH are varied in the nutrient liquid containing methanol described below. (The pH is adjusted by adding an aqueous solution of HCl or NaOH). Good growth at a pH in the range of 6 to 9, but no growth at pH 5 and 10, good growth at a temperature of 5 to 41 ⁰ C, said temperatures are preferably from 25 to 40 ° C * No growth at 42 ° C.

(15) Behavior towards oxygen: aerobic

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(16) 0-F test (according to the Hugh-Leifson method): glucose is neither metabolized aerobically nor anaerobically (no formation of acid and gas)

(17) Generation of acid and gas from the below indicated sugars (using peptone water with a sugar concentration of 1% by weight at 37 ° C for 10 days:

Generation of generation of acid gas

(I) L-arabinose (2) D-xyIose

(-) D-Clucose (U) D-Mannose (5) D-Fructose (5) D-Calactose (7) Maltose (β) Sucrose

(9) lactose

(10) trehalose

(II) D-sorbitol (12) D - * 'annitol

(13) inositol

(14) glycerin

(15) strength

(weak)

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(18) Assimilation of sugars (using sugar instead of methanol in the methanol-containing nutrient fluid medium in a concentration of 0.5% by weight at 37 ^° C. for 10 days):

(1) L-arabinose

(2) D-xylose

(3) D-glucose (A) D-mannose

(5) D-fructose

(6) D-C / alactose

(7) maltose

(8) sucrose

(9) lactose

(10) trehalose

(11) D-sorbitol

(12) D-mannitol

(13) inositol

(14) Glycerin

(15) strength

Assimilation

+ (weak)

+ (weak)

+ (weak)

+ (weak)

+ (weak)

+ (weak)

+ (weak)

+ (weak)

+ (weak)

+ (weak)

(d) Source of insulation: soil

The methanol-containing agar medium and the methanol-containing Nutrient fluid used in the growth tests described above the following composition:

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DE 0476 (ί) Agar medium containing methanol

KH ₂ PO ₄ 1.5 g Ma ₂ HPO ₄ 3.2 g (NH ₄ ) ₂ S0 ₄ 3 g MgSO ₄ * 7H ₂ O 0.5 g CaCi ₂ -2H ₂ O 0.1 g FeSO ₄ '7H ₂ O 0.01 g ZnSO ₄ '7H ₂ O 1 _; 4 mg CuSO ₄ * 5H ₂ O 0.25 mg Ma ₂ MoO ₄ -ZH ₂ O 0.24 mg CoCi ₂ '6H ₂ O 0.24 ng MnSO / 5Ηο0
* f 2 Iy 2 mg Agar 20 g Distilled water 1 1

A nutrient medium consisting of the components described above was heated at 120 ° C. for 15 minutes sterilized, and then 20 g of methanol was added under sterile conditions.

(2) Nutrient liquid containing methanol

This medium was similar to the above-described, methanol-containing agar medium, but the 20 g Agar was not used and the amount of methanol used was changed to 5 g.

The above experiments were carried out in agreement

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] Mood with "Bergey's Manual of Determinative Bacteriology, "8th Edition (1974)," Manual of Practical Bacteriology "(edited by Alumni Association of the Institute of Medical Science, revised editions, published by Maruzen Co., Ltd., 1975) and "Taxonomy and Determination of Microorganisms" (edited and written by Takeji Hasegawa, published by Tokyo University Press, 1975).

IQ From the facts given below became in connection with the bacteriological properties described above concluded that it The 6 bacterial strains mentioned above which are used according to the invention are new strains acts.

(A) An examination of the bacteriological properties of Flavobacterium tosaensis DS-I in accordance with the taxonomic rules described in Bergey's Manual of Determinative Bacteriology, 8th Edition (1974) shows that this strain is very similar to the genus Flavobacterium because it is rod-shaped, Gram-negative, aerobic and does not move freely, grows on a nutrient broth, does not form spores, produces acid from glucose and does not ferment lactose. The difference to the genus Flavobacterium is that the colonies of the present strain are not yellow, but milky yellow or opalescent. Assuming that this strain belongs to a genus other than Flavobacterium , its properties do not match the properties of any genus described in Sections 1-101, 102 and 103 of the Key to Genera of Manual in the Bergey's Manual mentioned above will. It was therefore found that this strain

13061 A / 00 09

DE 0476 is a bacterium of the genus Flavobacterium .

When the present strain is compared with the 12 main species of the genus Flavobacterium described in Bergey's Manual for the properties tabulated in this book, it turns out that Flavobacterium ferrugineuiT) and Flavobacterium rigenae differ from the present strain am at least differ.

In a further comparison of the present strain with these two species also with regard to the other properties described in the text of Bergey's Manual, it is seen that the present strain of Flavobacterium ferrugineum with regard to hydrolysis of starch and growth on a bouillon agar medium and from Flavobacterium rlgense with respect to motility, growth in litmus milk and behavior towards oxygen. In addition, no other known literature discloses a bacterium which has the same properties as the present strain. Therefore, it was found that the present strain is a new strain belonging to the genus Flavobacterium .

(B) An examination of Pseudomonas wakayamaensis DS-25 in accordance with the taxonomic rules described in Bergey's Manual shows that this strain belongs to the genus Pseudomonas .

The present strain was compared to the 29 major species of the genus Pseudomonas described in this manual for the properties tabulated in this book.

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Based on the comparison with respect to properties other than the utilization of carbon sources, it is believed that the present stemm is most similar to the species Pseudomonas facilis because it has flagella, does not grow at 41 ° C, and liquefies gelatin. However, when the present strain is compared with Pseudomonas facilis in all properties including the utilization of carbon sources, they differ not only in the utilization of methanol, which is a characteristic of the present strain, but also in the hydrolysis of Starch, the utilization of lactose and the utilization of sucrose.

. :, Relatively analogous stems that. in other known

References disclosed are Pseudomonas methanoloxidans described in Japanese Patent Application Laid-Open No. 6192/74, Pseudomonas aeruginosa described in Japanese Patent Application Laid-Open No. 154480/75, and Pseudomonas methanolitica described in Japanese Patent Application Laid-Open No. 41490/76.

However, the present strain differs from Pseudomonas methanoloxidans with respect to the reduction of nitrate, the positive urea reaction, the temperature and pH ranges for tias growth, the generation of acids from many kinds of sugars and the utilization of lactose, from Pseudomonas a <er »uginosa in relation to the negative result of the MR test, the positive result of the VP test, the temperature and pH ranges for growth, the extent of growth in a stationary nutrient broth culture, the absence of sediment and the non-utilization of lactose and of Pseudomonas methanolitica in relation to the

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Lack of hydrogen sulfide production, the hydrolysis of Starch, the utilization of citric acid, the temperature and pH ranges for the growth, the production of acids from many types of sugars and the non-utilization of lactose. ι

Therefore, it was confirmed that the present strain is highly different from all known strains of the genus Pseudomonas. Accordingly, it was found that the present strain is a new strain belonging to the genus Pseudomonas .

(C) An examination of the bacteriological properties of Flavobacterium methariolicola DS-16 in accordance with the taxonomic rules described in Bergey's Manual shows that this strain is most similar to the genus F1avobacterium because it is rod-shaped, Gram-negative, aerobic and not freely motile , grows on a nutrient broth medium, does not form spores, produces acid from glucose and does not ferment lactose. This strain differs from the genus Flavobacterium in that the colonies of the present strain are not yellow, but yellowish-brown. If it is assumed that this strain belongs to a genus other than Flavobacterium in terms of colony difference, it will be found that its properties do not coincide with the properties of any of the genera listed in Sections 1-101, 102 and 103 of the Key to Genera of Manual of Bergey's Manual. Therefore, it was found that the present strain is a bacterium belonging to the genus Flavobacterium.

If the present strain is associated with the 12 main species of the genus Fl avobacterium , those in Bergey's Manual

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are compared with respect to the properties tabulated in this book, it can be seen that Flavobacterium breve and Flavobacterium rlgense differ least from the present strain. A further detailed comparison of the present strain with these strains for all properties, including the properties described in the text of Bergey's Manual, shows that the present strain of Flavobacterium breve with respect to the generation of acid from maltose , the utilization of citrate and the growth on a bouillori agar nutrient medium and of Flavobacterium rigense with respect to the motility, the liquefaction of gelatin, the growth in litmus milk and the behavior towards oxygen. In addition, no other known literature discloses a strain which has the same properties as the present strain. Accordingly, it was found that this strain is a new strain belonging to the genus Flavobacterium .

(D) An examination of the bacteriological properties of Pseudomonas kyotoensis DS-22 in accordance with the taxonomic rules described in Bergey's Manual shows that this strain belongs to the genus Pseudomonas .

The present strain was compared with the 29 major species of the genus Pseudomonas classified in Bergey's Manual for the properties tabulated in this book. It is believed that the present strain is most similar to the species Pseudomonas alcaligenes with respect to properties other than the utilization of carbon sources because it has flagella, grows at 41 ° C,

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shows a negative result in the denitrification reaction and gelatin liquefies. However, when the present strain is compared with Pseudomonas alcaligenes for all properties including the utilization of carbon sources, it is seen that it differs not only for the utilization of methanol, which is a characteristic of the present strain, but also differ with regard to the hydrolysis of starch and the utilization of glucose, trehaiose and inositol.

Relatively analogous strains disclosed in other known literatures are Pseudomonas methacarbohylis disclosed in Japanese Patent Laid-Open No. 71886/75, Pseudomonas methanoloxidans disclosed in Japanese Patent Laid-Open No. 6192/74, and Pseudomonas methariolitica disclosed in Japanese Patent Application Laid-Open No. 41490/76.

However, the present strain differs from Pseudomonas methacarbohylis because it does not produce hydrogen sulfide, because it uses citrate, produces acids from many kinds of sugars and uses L-arabinose, inositol and glycerin, and because it does not use lactose, from Pseudomonas methanoloxidans because of its Reduction of nitrate is positive and its urea reaction is positive because its growth temperature is different and because it produces acids from many kinds of sugars and uses lactose, and of Pseudomonas methanolitica because it does not produce hydrogen sulfide because it hydrolyzes starch, uses citrate, acids out many types of sugars are produced and utilized and lactose

because its growth temperature is different. 35

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Accordingly, it was found that the present strain is largely different from all known strains of the genus Pseudomonas. Therefore . it was found that the present strain is a new strain belonging to the genus Pseudomonas .

(E) An examination of the bacteriological properties of Pseudomonas aichiensis DS-26 in accordance with the taxonomic rules described in Bergey's Manual has shown that this strain belongs to the genus Pseudomonas .

The present strain was compared with the 29 major species of the genus Pseudomonas classified in the aforementioned book for the properties tabulated in that book. As a result, it is believed that the present strain is most similar to the species Pseudomonas alcaligenes in that it has flagella, grows at 41 ° C, liquefies gelatin, and does not hydrolyze starch. However, when the present strain is compared with Pseudomonas alcaligenes for all properties including the ability to assimilate, it is seen that it differs not only for the utilization of methanol, which is a characteristic of the present strain, but also differ with regard to the growth temperature and the utilization of glucose, trehalose and inositol.

Relatively analogous strains disclosed in other known literature are Pseudomonas methanoloxidans (in Japanese Patent Laid-Open No. 6192/72) and Pseudomonas methanolitica (in Japanese Patent Laid-Open No. 41490/76). Of the

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] However, the present strain differs from Pseudomonas metharioloxidans with regard to the urea reaction, the growth temperature, the generation of acids from many types of sugars and the utilization of lactose, D-sorbitol and starch and from Pseudomonas methanolitica with regard to the reduction of nitrate, the generation of hydrogen sulfide, the growth temperature, the generation of acids from many types of sugars, and the utilization of lactose, D-sorbitol and starch.

Accordingly, it was found that the present strain is clearly different from all known strains of the genus Pseudomonas. Therefore, it was found that the present strain is a new strain belonging to the genus Pseudomonas .

(F) An examination of the bacteriological properties of Corynebacterium yamanasiensis DS-31 in accordance with the taxonomic rules described in Bergey's Manual has shown that this strain belongs to the genus Corynebacterium because it is in the form of short rods with respect to the Gram dye is positive, immobile, and aerobic because it has no flagella and has been found to have irregularly shaped or curved cells and divided V-shaped cells.

The present strain was compared with the species of the genus Corynebacterium described in Sections I and II of Bergey's Manual for properties other than the utilization of carbon sources and the generation of acids and gases from sugars. With the species described in Section III of Bergey's Manual, this strain can

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cannot be compared because it does not describe any properties.

Based on a comparison for the presence of flagella, the Gram dye, the catalase reaction, the indole reaction, the urea reaction, the hydrolysis of starch, the reduction of nitrate, the liquefaction of gelatin, the growth in litmus milk, the oxidase reaction and the temperature range for the growth, the present strain is believed to be similar to the species Corynebacterium pseudo tuberculosis , Coryriebacterium xerosis, Corynebacterium bovis , Corynebacterium enzymicum , Corynebacterium murisepticum , Corynebacterium nephridi and Corynebacterium diphteriae .

The present strain can not be fully distinguished from Corynebacterium enzymicum and Corynebacterium nephridi because very few properties are described, but an obvious difference is that these strains do not utilize methanol. The present strain differs from Coryriebacterium pseudo tuberculosis in terms of the utilization of methanol and the liquefaction of gelatin, from Corynebacterium bovis in terms of the utilization of methanol and the oxidase reaction , although only a few properties are described, from Coryriebacterium murisepticum in terms of to the recovery of methanol, the production of hydrogen sulfide, the growth in litmus milk and the production of acid from D-mannitol and from Coryriebacterium diphteriae with respect to the recovery of methanol and the production of acid from sucrose.

A relatively analogous strain to that in another

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known literature is Corynebacterium methanophilum described in Japanese Patent Laid-Open No. 41490/76. However, it was confirmed that the present strain of Corynebacterium methanophilum with respect to the temperature and pH ranges for growth, production of hydrogen sulfide, hydrolysis of starch, production of indole, reduction of nitrate, urea reaction , the VP test, the MR test, the generation of acids from many types of sugars, and the utilization of many carbon sources are clearly distinguished.

Accordingly, it was found that this strain is a new strain belonging to the genus Corynebacterium .

In growing the microorganisms in accordance with the invention is an aerobic liquid growing process suitable. The growth temperature is 5 to 45 ° C, and preferably 25 to 43 ° C. The pH of the culture system is 5 to 9, and preferably 6 to 8. The culture can be either batch or be carried out continuously.

In the nutrient medium, methanol is a major one Carbon source used. The preferred one The amount of methanol is not more than 50 g / liter.

Inorganic substances are used as nitrogen sources such as ammonium salts and nitrates and organic substances such as urea, casein, corn steep liquor, peptone, yeast extract and meat extract are used. Phosphorus sources are phosphate salts and suitable sulfur sources are sulfate salts. As a source of one Metals such as magnesium, potassium, calcium, sodium,

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Iron, manganese, copper, zinc, molybdenum, cobalt and boron will be an appropriate amount of a salt of the appropriate Metal added. If necessary, they become absolutely necessary for the growth of microbial cells Substances such as vitamins and amino acids or a material that promotes growth added.

The nutrient medium can be a natural nutrient medium if it is methanol, nitrogen sources, inorganic Substances and substances that are essential for growth, such as vitamins and amino acids or Contains appropriate amounts of growth promoting substances. The pH of the nutrient medium can be appropriately adjusted by a phosphate and ammonia.

The extraction of microbial cells from the .nutrient liquid can in the usual way, for example by filtration, by separation using a centrifuge etc. can be carried out. Washing and drying can also be used.

According to the invention methanol, which is provided by the chemical industry in copious amounts for disposal, be utilized as a primary carbon source, and microbial cells of strains tösaensis to Flavobacterium, Pseudomonas wakayamaensis, methanolicola Flavobacterium, Pseudomonas kyotoensis, Pseudomonas aichiensis and Corynebacteri.um yamanasiensis can be produced in large quantities and in good yields. Since the obtained microbial cells contain a lot of proteins, they can be used not only as feed and food, but also as materials for medicines and

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Best embodiment of the invention

The invention is further illustrated by the following examples.

example 1 G mg KH ₂ PO ₄ G Na ₂ HPO ₄ ^3/2 1 (W ₄ ) ₂ Sn ₄ 3 g G MgSO ₄ -7H ₂ O 0.5 G CaCi ₂ -2H ₂ O 0/1 ? G FeSO / -7HoO o, o; rag ZnSO ₄ -7H ₂ O ; ^2/8 mg CuSO, * 5W _o 0 O _x ^ S rag Wa ₂ MoO ₄ · 2Η _? 0 0jb8 mg LOL * o'DH ₀ U
C. C MnSO ₄ -SH ^ O Distilled 1 water

A nutrient medium (500 ml) consisting of the above ingredients was placed in a small 1 liter glass flask and sterilized at 120 ° C. for 15 minutes. Then 5 g of methanol were added under sterile conditions. 2% by volume of a preculture containing the cells of Fiavobacterium tosaensis DS-I (FERM-P No. 4058) were added to the nutrient medium obtained

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] inoculated by pre-cultivation for 24 hours in a nutrient medium with the same composition as above described had been obtained at 37 ° C. The cultivation was carried out at 37 ° C for 20 hours with aeration and stirring carried out while the pH of the nutrient medium was maintained at 7.0 by 13% strength by weight ammonia solution wupde. The nutrient liquid was centrifuged to separate the microbial cells. The cells were with Washed with water and dried for 24 hours at 100 ° C., dried cells in an amount of 4.5 g per liter of the nutrient liquid were obtained. The generation time of this culture was in the period of logarithmic growth 1.4 h. The cells were found to contain 80% by weight of crude proteins.

Example 2

A culture medium (1.2 1) with the same composition as in Example 1 was filled therein in a 2.5-1 fermenter serving as a glass flask and 20 min sterilized at 120 ⁰ C. Then 12 g of methanol were added under sterile conditions. The nutrient medium obtained was inoculated with 5% by volume of a preculture containing the cells of Flavobacterium tosaensis DS-I (FERM-P No. 4058), which was cultivated for 20 hours at 37 ° C. in a nutrient medium with the same composition as in Example 1 had been received. Cultivation was carried out at 37 ° C. with aeration and stirring while the pH of the nutrient medium was maintained at 7.0 using 13% by weight ammonia solution. At the end of 20 hours from the start of cultivation, the concentration of the cells was 4.5 g / l. The following aqueous metal salt solutions were used per liter of the nutrient liquid

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added.

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18 ml of a 10% strength by weight aqueous solution of KH ₂ PO ₄ ,
77 ml of a 5% strength by weight aqueous solution of

Na ₂ HPO ₄ ,

18 ml of a 10% strength by weight aqueous solution of

MgS0 ₄ -7H ₂ 0,

3.5 ml of a 10% strength by weight aqueous solution of CaCl ₂ - 2H ₂ O,

5 ml of a 1% strength by weight aqueous solution of

FeSO ₄ -7H ₂ O,

4 ml of a 1% strength by weight aqueous solution of ZnSO ₄ '7H ₂ O,
0.7 ml of a 1% strength by weight aqueous solution of

CuSO ₄ -5H ₂ O,

1.1 ml of a 1% strength by weight aqueous solution of

MnSO ₄ -5H ₂ O,

2 ml of a 0.1% strength by weight aqueous solution of ^H 3 ^B0 3 ^and

0.5 ml of a 0.1% strength by weight aqueous solution of

CoCi ₂ -6H ₂ O.

20 hours after the start of the cultivation, methanol was automatically added so that the concentration of the methanol in the nutrient liquid then to 1 until 5 g / l was maintained. The amount of added at the end of h after the start of the cultivation Methanol reached 96 g per liter of nutrient fluid, At the end of 29 hours after the cultivation, the nutrient liquid was centrifuged to separate the cells of the microbes. The cells were washed with water and dried for h at 100 ° C., with 32 g dried Cells per liter of the nutrient fluid were obtained. The cells were found to contain 77% by weight of crude proteins.

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held.

Example 3 KH ₂ PO ₄ 1.5 g 3.2 g \ ™ / J O "- / 3 g MgSO ₄ -7H ₂ O 0.5 g CaCi ₂ * 2HpO 0/1 g FeSO ₄ -7HpO 0.02 g ZnSO ₄ -7H ₂ O 2.0 mg CuSO ₄ -5HpO 0.5 mg Na ₂ HoO ₄ • 2H ₂ O 0.48 rag CoCi ₂ -OH ₂ O 0.48 mg MnSO ₄ '5HpO . 2 ₇ 4 mg Distilled water 1 1

A nutrient medium (100 ml) composed of the above ingredients was charged into a 0.5 liter Sakaguchi flask and sterilize for 15 minutes at 120 ° C! .. Then 0.2 g of methanol were added added sterile conditions. In the nutrient medium obtained, 0.5% by volume of one of the cells of

"" Pseudomonas wakayamaensis DS-25 (FERM-P No. 4100) was inoculated, which had been obtained by preculturing for 24 hours at 35 ° C in a nutrient medium which had the same composition as described above, but in which the amount

"C of the methanol had been changed to 0.5% by weight.

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] The cultivation was carried out with shaking at 35 ° C for 24 hours. The nutrient liquid was used for separation the microbial cells centrifuged. The cells were washed with water and dried at 100 ° C. for 24 hours, whereby 0.9 g of dried cells per liter of the nutrient fluid were formed. The generation time of this culture was 1.5 hours in the period of logarithmic growth. It was found that cells 78 Weight-9% crude proteins.

Example 4

A nutrient medium (500 ml) with the same composition as in Example 3 was poured into a small 1-1 glass flask and sterilized at 120 ° C. for 15 minutes. Then 5 g of methanol were added under sterile conditions. In the nutrient medium obtained, 2% by volume of a preculture containing the cells of Pseudomonas wakayamaensis DS-25 (FERM-P No. 4100) was inoculated, which was obtained by preculturing for 24 hours at 37 ° C. in a nutrient medium with the same composition as described above had been received. Cultivation was carried out at 37 ° C for 20 hours while maintaining the pH of the nutrient medium at 7.0 using 13% by weight ammonia solution.

The nutrient liquid was used to separate the Centrifuged microbes. The cells were with Washed with water and dried at 100 ° C. for 24 hours, with 4.2 g of dried cells per liter of the nutrient fluid were obtained. The generation time of this culture was in the logarithmic period Growth 1.6 h. It was found that the cells

1306U / 0009

DE contained 78 wt% crude proteins.

Example 5 KH ₂ PO ₄ 1.5 g Na ₂ HPO ₄ 3, 2 g MgSO ₄ -7 ^ ₂ O 0.5 g CaC £ _? '2H _? 0 0.1 g FeSO ₄ -7H ₂ O 0.02 g ZnSO ₄ * 7H _? 0 2.0 mg CuSO ₄ -5H ₂ O 0.5 mg Na ₂ MoO ₄ * 2H ₂ O 0.48 mg CoC ^ p * 6ttp0 0 _; 48 mg MnSO ₄ * 5H ₂ O 2 ^ 4 mg Distilled water 1 1

One of the above ingredients -25 Existing nutrient medium (100 ml) was placed in a 0.5 liter Sakaguchi flask filled in and sterilized at 120 ° C for 15 min.

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Then 0.2 g of methanol was added under sterile conditions. In the nutrient medium obtained, 0.5 % by volume of a preculture containing the cells of Flavobacterium methanolicola DS-16 (FERM-P No. 4098), which had been obtained by preculture for 24 hours at 35 ° C. in a nutrient medium which had the same composition as described above, but in which the amount of methanol added had been changed to 0.5% by volume. The cultivation was carried out with shaking at 35 ° C for 24 hours.

The nutrient fluid was used to separate the microbial cells centrifuged. The cells were washed with water and dried at 100 ° C. for 24 hours, whereby 0.85 g of dried cells per liter of the nutrient fluid

were obtained.
20th

The generation time of this culture was 1.5 hours in the period of logarithmic growth. It was found that the cells contained 75% by weight of crude proteins.

Example 6

A nutrient medium (500 ml) with the same composition as in Example 5 was in a small 1-1-

Filled with glass flask and ^{sterilized at 120 °} C. for 15 min. Then 5 g of methanol were added under sterile conditions. 2% by volume of a preculture containing the cells of Flavobacterium methano I lcola DS-16 (FERM-P No. 4098) was inoculated into the nutrient medium obtained.

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setting was obtained as described above. The cultivation was carried out for 20 hours with aeration and stirring at 37 ^° C. while the pH of the nutrient medium was kept at 7.0 using a 13% strength by weight ammonia solution.

• The nutrient fluid was centrifuged to separate the microbial cells. The cells were washed with water and dried for 24 hours at 100 ⁰ C h to give 4.2 g of dried cells was obtained per liter of the NährflUssigkeit. The generation time of this culture in the period of logarithmic growth was 1.6 hours. The cells were found to contain 78% by weight of crude protein.

Example 7

[UT ₂ JO ₄ 1/5 g Ma ₂ HPO ₄ 3.2 g O ¹ V ₂ SO ₄ 3 g PgS0 ₄ -7n ₂ 0 0.5 g CaC .C ₂ '2H ₂ O 0.1 g FeS0 ₄ -7 ^T * ₂ 0 0.02 g Zn ^ y ₇ H ₂ O 2.8 mg CuSO ₄ -5H ₂ O 0.5 mg Ha ₇ MoO ₄ * 2H ₂ O 0.48 mg CoCi ₂ -OH ₂ O 0.48 mg ^ nSO ₄ '5H ₂ O 2.4 mg Distilled Z 1 V / ater

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A nutrient medium (500 ml) consisting of the above components was filled into a small 1-1 glass flask and ^{sterilized at 120 °} C. for 15 minutes. Then 5 g of methanol were added under sterile conditions. In the resulting culture medium VoI .-% preculture containing were subjected to the cells of Pseudomonas kyotoensis DS-22 (4099 FERM-P Nr.) Inoculated, the hour 24 by preculture at 37 ⁰ C in a growth medium having the same composition as described above had been received. Cultivation was carried out at C for 20 hours with aeration and stirring while the pH of the nutrient medium was maintained at 7.0 using a 13% by weight ammonia solution. The nutrient fluid was centrifuged to separate the microbial cells. The cells were washed with water and ^{dried for 24 hours at 100 °} C., 4.3 g of dried cells per liter of the nutrient fluid being obtained. The generation time of this culture was 1.5 hours in the period of logarithmic growth. It was found that the cells 79 wt -.% Crude protein contained.

Example 8

A nutrient medium (1.2 l) with the same composition as in Example 7 was filled into a 2 , bJ glass flask ^{serving as a fermenter and sterilized for 20 minutes at 120 °} C. Then 12 g of methanol were sterile added conditions. in the resulting culture medium was 5 vol .-% of an inoculated, the microbial cells of Pseudomonas kyotoensis DS-22 (FERM-P no. 4099) preculture containing the hour 24 by preculture at 37 C in a culture medium with the same

1306U / 0009

Chen composition was obtained as described above. The cultivation was carried out with aeration and Stirring is carried out at 37 ° C while adjusting the pH of the nutrient medium using a 13% strength by weight ammonia solution was held at 7.0.

. At the end of 20 hours after the start of the cultivation, the concentration of the cells was 4.3 g / l. Per liter the following aqueous metal salt solutions were added to the nutrient liquid.

18 ml of a 10% strength by weight aqueous solution of KH ₂ PO ₄ ,

77 ml of a 5% strength by weight aqueous solution of Na ₂ HPO ₄ ,

18 ml of a 10% strength by weight aqueous solution of MgSO ₄ .7H ₂ O,

3.5 ml of a 10% strength by weight solution of

5 ml of a 1% strength by weight aqueous solution of FeSO ₄ .7H ₂ O,

3 ml of a 1% strength by weight aqueous solution of ZnSO ₄ .7H ₂ O,

0.6 ml of a 1% strength by weight aqueous solution of CuSO ₄ -SH ₂ O,

0.7 ml of a 1% strength by weight aqueous solution of 35

MnSO ₄ .5H ₂ O and

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2 ml of a 0.1% strength by weight aqueous solution of H ₃ BO ₃ .

h after the initiation of the cultivation, methanol was automatically added so that its concentration was then kept at 1 to 5 g / l in the nutrient fluid. The total amount of the added Methanol at the end of 32 hours after the initiation of the cultivation reached 92 g / L of the nutrient liquid. At the end 32 hours after the initiation of the cultivation, the nutrient liquid was centrifuged to separate the microbial cells. The cells were washed with water and dried at 100 ° C. for h, with 31 g of dried Cells per liter of the nutrient fluid were obtained.

The cells were found to contain 78% by weight of crude protein.

Example 9

WIpPO ₄ 1.5 g

TiaJiPO / 3.2 g

2 η- ι

(i-iii ₄ ) ₂ so ₄ 3 g

MgSO ₄ · 7Η ₂ 0 0.5 g

CaCi ₂ -2H ₂ O 0.1 g

FeSO ₄ '7H ₂ O 0.02 g

ZnSO ₄ '7H ₂ O 2.8 mg

CuSO ₄ .5Η _? 0 0.5 mg

Ha "MoO / · 211 ~ 0 0.48 mg - 2. i ± έ ι

CoC ^ ₂ x 6Η ₂ 0 0.48 mg

NInSOι · 5Η ~ 0 2.4 mg

H- C- I.

Distilled water 1 Z

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A nutrient medium (100 ml) consisting of the above components was filled into a 0.5-1 Sakaguchi flask and ^{sterilized at 120 °} C. for 15 minutes. Then 0.2 g of methanol was added under sterile conditions. In the nutrient medium obtained, 0.5% by volume of a preculture containing the microbial cells of Pseudomonas aichiensis US-26 (FERM-P No. 4107) was inoculated, which was followed by 24-hour pre-cultivation
37 ⁰ C in a nutrient medium that the

had the same composition as described above except that the amount of methanol was changed to 0.5% by weight. The cultivation was for 24 hours
carried out with shaking at 37 C. The nutrient fluid was centrifuged to separate the microbe cells

fused. The cells were washed with water and

Dried for 24 hours at 100 ^° C., 0.88 g of dried cells per liter of the nutrient liquid being obtained. The generation time of this culture was in the
Period of logarithmic growth 1.5 h. It was found

that the cells contained 79% by weight of crude proteins.

Example 10

A nutrient medium (500 ml) with the same composition as in Example 9, but in which the amount

of the added methanol has been changed to 5 g
was filled into a small 1-1 glass flask and ^{sterilized at 120 °} C. for 15 minutes. then
methanol was added in the amount indicated above under sterile conditions. In the received
Nutrient medium were 2 .Vo 1 .-% of the cells of
Pseudomonas aichiensis DS-26 (FERM-P No. 4107) containing preculture, which had been obtained by preculturing at 37 ° C. for 24 hours in a nutrient medium having the same composition as in Example 9, was inoculated.
Cultivation was carried out for 20 hours with aeration and stirring

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carried out at 37 ° C, while the pH of the nutrient medium using a 13 wt .-% ammonia solution was held at 7.0.

The nutrient fluid was centrifuged to separate the microbe cells. The cells were washed with water and ^{dried for 24 hours at 100 °} C., 4.1 g of dried cells per liter of the nutrient liquid being obtained. The generation time of this culture in the period of logarithmic growth was 1.6 hours. The cells were found to contain 79% by weight of crude protein.

Example 11 1.5 g KH ₂ PO ₄ 3.2 g Na ₂ HPO ₄ 3 g (IVTH ₄ ) _? S0 ₄ 0.5 g MgSO, 7Η _ο 0
M ei OjI g CaCf ₂ -2H ₂ O 0.02 ß FeSO, 7Η _Ο ° 2 _r 8 mg ZnSO ₄ -7H ₂ O 0.5 mg CuSO ₄ • 5H ₂ O 0.48 rag Na ₂ HoO ₄ -2H ₂ O 0-48 rag CoCi ₂ -6H ₂ O 2.4 mg MnSO ₄ '5HpO £ 1 Distilled water

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A nutrient medium (100 ml) consisting of the above components was filled into a 0.5-1 Sakaguchi flask and ^{sterilized at 120 °} C. for 15 minutes. Then 0.2 g of methanol was added under sterile conditions. In the resulting culture medium containing 0.5 vol .-% pre-culture were added to a cell of Corynebacterium yamanasiensis DS-31 (4106 FERM-P Nr.) Inoculated which had been obtained by 24-stUndige preculture at 35 ⁰ C in a nutrient medium, which had the same composition as described above, but in which the amount of methanol added was changed to 0.5% by weight. Cultivation was carried out with shaking at 35 ° C. for 24 hours. The nutrient fluid was centrifuged to separate the microbial cells. The cells were washed with water and dried for 24 hours at 100 C, with 0.84 g of dried cells was obtained per liter of ~ ö ^v he ~ ^r iJahrflüssigkeit. The generation time of this culture was 1.5 hours in the period of logarithmic growth. The cells were found to contain 76% by weight of crude protein.

Example 12

A nutrient medium (500 ml) with the same composition as in Example 11, but with the amount of methanol being changed to 5 g, was filled into a small 1-1 glass flask and ^{sterilized at 120 °} C. for 15 minutes. Then methanol was in the

The amount indicated above was added under sterile conditions. 2% by volume of a preculture containing the cells of Corynebacterium yamanaslensis DS-31 (FERM-P No. 4106) was inoculated into the nutrient medium obtained.

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Settlement had been obtained as described above. Cultivation was carried out for 20 hours with aeration and stirring carried out at 37 ° C, while the pH of the nutrient medium using a 13% strength by weight ammonia solution was held at 7.0.

ι ■

• The nutrient fluid was used to separate the microbial cells centrifuged. The cells were washed with water and dried at 100 ° C. for 24 hours, whereby 4.0 g of dried cells per liter of the nutrient fluid were obtained. The generation time of this culture was in the period of logarithmic growth 1.6 h. The cells were found to contain 77% crude protein by weight contained.

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Claims (5)

Claims ■■ // '■ 1. Process for the production of microbes <slJ. «N, characterized in that a bacterium belonging to a species that is derived from the F1 avob ac ter ium '. tosaensis , Pseudomonas wakayamaensis , Flavobacterium methanolicola , Pseudomonas kyotoensis , Pseudomonas aichiensis and Corynebacterium yamanasiensis is grown in a nutrient medium containing methanol as a main carbon source, and the microbial cells are recovered from the nutrient fluid. 2. The method according to claim 1, characterized in that the bacterium belonging to Flavobacterium tosaensis is Flavobacterium tosaensis DS-I (FERM-P No. 4050). 3. The method according to claim 1, characterized in that the bacterium belonging to Pseudomonas wakayaaiaensis is Pseudomonas wakayamaensis DS-25 (FERM- *]? No. 4100). ■ ■ '* -> i · ■ 4. The method according to claim 1, characterized in net that that to Flavobacterium methanolicola . belonging bacterium Flavobacterium methanolicola DS-16 (FERM-P No. 4098). ' 5. The method according to claim 1, characterized gekeiwzeich- ■■ '*' net that the bacterium belonging to Psei iomonas kyotoensis is Pseudomonas kyotoensis DS-22 (FERM-P No. 4099). ■ 1306U / 0009 DE 04762857681 1 6, «Method according to claim 1, characterized in that the bacterium belonging to Pseudomonas aichiensis is Pseudomonas aichiensis DS-26 (FERM-P No. 4107). ■,. ' 7. The method according to claim 1, characterized / that the bacterium belonging to Corynebacterium yamanasiensis is Corynebacterium yamanasiensis DS-31 (FERM-P No. 4106). 10 130614/0009