GB2120274A - Process for producing microbial cells - Google Patents

Abstract

This invention relates to a process for producing microbial cells, and is characterized by the fact that the bacterium Corynebacterium yamanasiensis, which is a novel strain, is cultivated in a culture medium containing methanol as a major carbon source, and the resulting microbial cells are recovered from the culture broth. According to the process of this invention, bacterial cells can be produced in large quantities and in good yields and since the bacterial cells contain protein, they can be utilized as feeds, foods, materials for medicines, and industrial materials.

Description

SPECIFICATION Process for producing microbial cells This invention relates to a process for producing microbial cells economically in large quantities using novel microorganisms which aerobicallv assimilate methanol.

In recent years, much work has been done to develop methods for effectively utilizing microbial cells as, for example, feeds, foodstuffs, medicines and industrial materials. To produce microbial cells with industrial advantages, methanol is expected to be used as a major carbon source because there is a good prospect that methanol will be supplied in large quantities and at low costs by the chemical industry. [See, for example, Japanese Laid-Open Patent Publications Nos. 14389/1 972 and 1 5490/1978.] In view of these circumstances, the present inventors widely searched in nature for microorganisms which would aerobically utilize methanol as a major carbon source and be able to be cultivated with good efficiency, and consequently discovered a novel microorganism Corynebacterium yamanasiensis.This led to the establishment of a process for producing microbial cells economically in large quantities by utilizing this microorganism.

Thus, the present invention provides a process for producing microbial cells which comprises aerobically cultivating Corynebacterium yamanasiensis DS-31 (FERM-P No.4106) in a culture medium containing methanol as a major carbon source, a nitrogen source, a phosphorus source, a sulfur source and a metallic salt at a temperature of from 5 to 41 OC and a pH of 6 to 9; and recovering the microbial cells from the culture broth.

The microorganism used in this invention has been determined to belong to the genus Corynebacterium from its bacteriological characteristics. It has been identified as a new strain because it differs in various respects from known bacteria belonging to the genus Corynebacterium.

The new strain was deposited with the Fermentation Research Institute, Agency of Industrial Science and Technology, Japan at 5-8-1, Higashi, Inage, Chiba-ken, Japan on 28 June 1977 under FERM-P No.4106.

The bacteriological properties of this bacterial strain are shown below: CORYNEBACTERIUM YAMANASIENSIS DS-31 (FERM-P No.4106): (a) Morphology Cultivated in a nutrient broth and a nutrient agar medium at 370C for 3 days.

(1) Shape and size of cells: Short rods (0.5.6) by (0.6-0.9) micron.

(2) Colonies of the cells: Single or pair, and pleomorphic or curved and V-shaped divided cells.

(3) Motility: None (4) Spores: None (5) Gram strain: Positive (6) Acid-fastness: Negative (b) State of Growth on Various Culture Media (1) Nutrient agar plate culture: Growth abundant at 37 OC for 3 days. The colonies are circular having a diameter of 2-2.5 mm, and convex or umbonate with a smooth surface and an entire edge.

Yellow, glistening, translucent and butyrous.

(2) Methanol-containing agar plate culture: Growth abundant at 370C for 5 days. The colonies are circular having a diameter of 2-3 mm with a smooth surface and an entire edge. Opalescent, glistening, translucent and butyrous.

(3) Nutrient agar slant culture: Filiform growth at 370C for 3 days. The colonies are moderately protruded with a smooth surface and an entire edge. Yellow, glistening, translucent and butyrous.

(4) Methanol-containing agar slant culture: Hliform growth at 370C for 5 days. The colonies are moderately protruded with a smooth surface and an entire edge. Opalescent, glistening, translucent and butyrous.

(5) Nutrient broth: Growth rate moderate at 370C for 3 days. Sediment is formed. Slightly translucent. No ringed pellicle nor pellicle is formed on the surface.

(6) Nutrient broth (shaken): Growth abundant at 370C for 3 days. The culture becomes turbid.

Sediment is formed. Translucent.

(7) Peptone water broth: Growth abundant at 370C for 3 days. The culture becomes turbid.

Sediment is formed.

(8) Methanol-containing broth: Growth abundant at 37OC for 3 days. The culture becomes turbid.

Sediment is formed. A thin ringed pellicle is formed on the surface. Translucent.

(9) Methanol-containing broth (shaken): Growth abundant at 370C for 3 days. The culture becomes turbid. Sediment is formed. Translucent.

(10) Nutrient broth (stabbed): Growth is on the surface or proceeds to a 23 mm depth at 370C for 3 days. Opalescent surface.

(11) Nutrient gelatin broth (stabbed): surface growth at 300C for 5 days with sediment. No gelatin liquefaction.

(12) Litmus milk: At 37 OC, the culture is coagulated.

(c) Biochemical Properties (1) Reduction of nitrate: positive (2) Denitrofication reaction; positive (3) MR test: positive (4) VP test: positive (5) Production of indole: negative (6) Production 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) Production of pigment (King's A and B media): negative (11) Urease reaction: negative (12) Oxidase reaction: negative (13) Catalase reaction: positive (14) Ranges for growth: The temperature and pH are varied in the methanol-containing broth described hereinbelow (the pH is adjusted by adding an aqueous solution of HCI or NaOH). Good growth at a pH in the range of 6 to 9, but not growth at a pH of 5 and 10.Good growth at a temperature of 5 to 41 OC, and temperatures of 25 to 400C are preferred. No growth at 42 OC.

(15) Relation to oxygen: Aerobic (16) 0-F test (by the Hugh Leifson method): Glucose is neither aerobically not anaerobically metabolized. (No formation of acid gas) (17) production of acid and gas from the following sugars (using peptone water in a sugar concentration of 1 wt% at 370C for 10 days): Production Production of Acid of Gas (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 + (weak) (11) D-Sorbitol (12) D-Mannitol (13) Inositol (14) Glycerin + (15) Starch (18) Assimilation of sugars (using sugar in place of methanol in the methanol-containing broth medium in a concentration of 0.5 wt% at 370C for 10 days):: Assimilation (1) L-Arabinose + (2) D-Xylose + (weak) (3) D-Glucose + (weak) (4) D-Mannose + (weak) (5) D-Fructose + (weak) (6) D-galactose + (weak) (7) Maltose + (weak) (8) Sucrose + (weak) (9) Lactose + (weak) (10) Trehalose + (11) D-sorbitol (12) D-Mannitol + (weak) (13)Inositol + (14) Glycerin + (weak) (15) Starch (d) Source of Isolation: Soil The methanol-containing agar medium and the methanol-containing broth used in the cultivation tests above are as follows: (1) Methanol-containing Agar Medium KH2PO4 1.5 g Na2HPO4 3.2 g (NH4)2SO4 3 9 MgSO4 7H20 0.5 g CaCl2.2H20 O.1 2H2O 0.1 g FeSQ.7H2O 0.01g ZnSO4.7H2O 1.4 mg CuSO4.5H2O 0.25 mg Na2MoO4. 2H20 0.24 mg CoCI2 6H2O 0.24 mg MnSO4 .5H2O 1.2 mg Agar 20 g Distilled water 11 A culture medium consisting of the above ingredients was sterilized at 1 200C for 1 5 minutes, and then 20 g of methanol was aseptically added thereto.

(2) Methanol-containing Broth The same medium as the methanol-containing agar medium above except that 20 g of agar was not used and the amount of methanol used was changed to 5 g.

The above experiments were performed in accordance 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 edition published in 1975 by Maruzen Co., Ltd) and "Taxonomy and Determination of Microorganisms" (edited and written by Takeji Hasegawa, published in 1975 by Tokyo University Press).

That the bacterial strain used in this invention is novel was judged from the following facts taken in conjunction with the above bacteriological properties.

A study of the bacteriological properties of Carynebacterium yamanasiensis DS-31 in accordance with the taxonomical standards described in the Bergey's Manual has shown it to be of the genus Corynebacterium because it is short-rod-shaped, Gram stain positive, non-motile and aerobic, does not have flagella and it is seen to have irregularly-shaped or curved cells and V-shape divided cells.

The present strain has been compared with the species of the genus Corynebacterium which are described in Section I and II of the Bergey's Manual in regard to properties other than the utilization of carbon sources and the formation of acids and gases from sugars. It cannot be compared with the species described in Section Ill of the Bergey's Manual because it does not describe properties.

On comparison with regard to the presence of flagella, Gram-stain, cataiase reaction, indole reaction, urease reaction, the hydrolysis of starch, the reduction of nitrate, the liquefaction of gelatin, growth in litmus milk, oxidase reaction, and the growth temperature range, the present strain is considered to be close to Corynebacterium pseudo tuberculosis, Corynebacterium xerosis, Corynebacterium bovis, Corynebacterium enzymicum, Corynebacterium murisep tic urn, Corynebacterium nephridi, and Corynebacterium diphteriae.

The present strain cannot be fully differentiated from Corynebacterium enzymicum and Corynebacterium nephridi because there are a very few properties described, but an apparent difference is that these strains do not utilize methanol. The present strain differs from Corynebacterium pseudo tuberculosis in regard to the utilization of methanol and the liquefaction of gelatin; from Corynebacterium bovis in regard to the utilization of methanol and oxidase reaction although only a few properties are described; from Corynebacterium murisepticum in regard to the utilization of methanol, the formation of hydrogen sulphide, growth in litmus milk, and the formation of acid from D-mannitol; and from Corynebacterium diphteriae in regard to the utilization of methanol and the formation of acid from sucrose.

A relatively analogous strain described in another known literature reference is Corynebacterium methanophilum described in Japanese Laid-Open Patent Publication No. 41490/76. It has been ascertained, however, that the present strain differs markedly from Corynebacterium methanophilum in regard to the growth temperature and pH ranges, the formation of hydrogen sulfide, the hydrolysis of starch, the formation of indole, the reduction of nitrate, urease reaction, VP test, MR test, the formation of acids from many kinds of sugars, and the utilization of many carbon sources.

Accordingly, this strain has been determined to be a novel strain of the genus Corynebacterium.

Cultivation of the microorganism in accordance with this invention can be performed either batchwise or continuously. Methanol is used as a major carbon source in the culture medium, preferably in amount not more than 50 g/liter.

The nitrogen sources used are inorganic substances such as ammonium salts and nitrates and organic substances such as urea, casein, corn steep liquor, peptone, yeast extract and meat extract.

Phosphorus sources are phosphate salts, and useful sulfur sources are sulfate salts. As a source of a metal such as magnesium, potassium, calcium, sodium, iron, manganese, copper, zinc, molybdenum, cobalt and boron, a suitable amount of a salt of the corresponding metal is added. If required, a substance required for the growth of microbial cells such as vitamins and amino acids, or a growth promoting material is added to the culture medium.

A natural culture medium may be used if it contains all of the essential ingredients. The pH of the' culture medium can be conveniently adjusted by a phosphate and ammonia.

Recovery of microbial cells from the culture broth can be effected in a customary manner, for example, by filtration or centrifugal separation. Washing and drying can also be applied.

According to this invention, methanol supplied abundantly by the chemical industry can be utiiized as a major carbon source, and microbial cells of Corynebacterium yamanasiensis can be produced in large quantities and in good yields. Since the microbial cells obtained contain much protein, they can be utilized not only as feeds and foods, but also as materials for medicines and industrial materials.

The following Examples further illustrate the present invention.

EXAMPLE 1 KH2PO4 .1.5 g Na2HPO4 3.2 g (NH4)2SO4 3 g MgSO4. 7H2O 0.5 g CaCI2 2H2O 0.1 g FeSO4 7H2O 0.02 g ZnSO4.7H20 2.8 7H2O 2.8mg CuSO4. 5H2O 0.5 mg Na2MoO4 2H2O 0.48 mg CoCi2 6H2O 0.48 mg MnSO4 5H20 2.4 mg Distilled water 11 A culture medium (100 ml) consisting of the above ingredients was placed into a 0.51 Sakaguchi flask, and sterilized at 1 200C for 15 minutes. Then, 0.2 g of methanol was aseptically added.Into the resulting culture medium was inoculated 0.5% by volume of a pre-culture containing the cells of Corynebacterium yamanasiensis DS-31 (FERM-P No. 4106) which has been obtained by pre-cultivation at 350c for 24 hours in a culture medium having the same ccmposition as above except that the amount of methanol added was changed to 0.5% by weight. Cultivation was performed with shaking at 350C for 24 hours. The culture broth was centrifuged to separate the microbial cells. The cells were washed with water, and dried at 1000C for 24 hours to afford 0.84 g of dried cells per liter of the culture broth. The generation time of this culture in the logarithmic growth period was 1.5 hours. The cells were found to contain 76% by weight of crude proteins.

EXAMPLE 2 A culture medium (500 ml) having the same composition as in Example 1 except that the amount of methanol was changed to 5 g was placed into a 1 I small jar, and sterilized at 1209C for 15 minutes.

Then, methanol in the above-indicated amount was added aseptically. Into the resulting culture medium was inoculated 2% by volume of a pre-culture containing the cells of Corynebacterium yamanasiensis DS-31 (FERM-P No. 4106) which had been obtained by pre-cultivation at 370C for 24 hours in a culture medium having the same composition as above. Cultivation was performed with aeration and stirring at 370C for 20 hours while maintaining the pH of the culture medium at 7.0 by using 13% by weight ammonia solution.

The culture broth was centrifuged to separate the microbial cells. The cells were washed with water, and dried at 1000C for 24 hours to afford 4.0 g of dried cells per liter of the culture broth. The generation time of this culture in the logarithmic growth period was 1.6 hours. The cells were found to contain 77% by weight of crude proteins.

Claims (3)

1. A process for producing microbial cells which comprises aerobically cultivating Corynebacterium yamanasiensis DS-31 (FERM-P No.4106) in a culture medium containing methanol as a major carbon source, a nitrogen source, a phosphorus source, a sulfur source and a metallic salt, at a temperature of from 5 to 41 C and a pH of 6 to 9; and recovering the microbial cells from the culture broth.

2. A process according to claim 1 wherein the culture medium contains a growth promoting material.

3. A process according to claim 1 substantially as described in Example 1 or 2.