JPS60172281A - Clostridium sp. no.68-2 - Google Patents

Abstract

NEW MATERIAL: Clostridium sp. No.68-2 (FERM-P No.7367), which is a Gram- negative sporangial bacillus belonging to the genus Clostridium, and growing on carbon dioxide and hydrogen without flagella. USE:Bacteria used for producing acetic acid and isobutyric acid from carbon dioxide and hydrogen. PREPARATION:Riverbed mud of the YAMATO river in OSAKA Prefecture is cultivated in a liquid culture medium in an atmosphere of a germfree gas containing conditions for example at 30 deg.C for 2 weeks by the stationary culture method.

Description

DETAILED DESCRIPTION OF THE INVENTION (Industrial Application Field) The present invention relates to a novel bacterium belonging to the genus Clostridium. This Ill bacterium can be used in a method for producing acetic acid and isobutyric acid from carbon dioxide and hydrogen.

(Conventional technology) Assimilates carbon dioxide and hydrogen under room temperature conditions.

Microorganisms that accumulate acetic acid in the growth medium include Clostridium 7ceticum, Acetobacterium uthudii,
Niebacterium Φlimosum, Butylibacterium methylotrophicum, Clostridium strain Cv
−^^1 etc. were known.

Also under high temperature conditions, Acedognium kiwi.

Clostridium thermoautotrophic cam.

I have Clostridium thermoaceticum. All four species of bacteria belonging to the Clostridium genus are flagellum-bearing bacteria.

Bacteria belonging to the genus Clostridium that have the ability to assimilate sugars and produce isobutyric acid include Clostridium stakelandii, Clostridium propionicum, and Clostridium goni 1.

(Objective of the Invention) Although the above-mentioned bacteria are known, there are still many problems to be solved in order to industrially produce acetic acid from carbon dioxide and hydrogen. It is important to obtain bacteria that have a high accumulation concentration and production rate of acetic acid. To this end, in addition to breeding and improvement based on known bacterial species, the creation of new bacterial species that can produce acetic acid from carbon dioxide and hydrogen is an extremely important means.

Furthermore, no bacteria capable of producing isobutyric acid from carbon dioxide and hydrogen has yet been reported.

The purpose of the present invention is to obtain a new microorganism that assimilates carbon dioxide and hydrogen and accumulates acetic acid and isobutyric acid in a culture medium.

(Structure of the Invention) The microorganism of the present invention is considered to be a bacterium belonging to the genus Clostridium in that it is an anaerobic bacterium that produces spores.
It is thought to be a new bacterial species, as it is different from known congener bacteria in various properties that will be described in detail later, such as growing on carbon dioxide and hydrogen and lacking flagella. Since the official species name has not yet been assigned, it is designated as Clostridium sp. No. in the present invention.

It is displayed as 68-2.

Next, we will show the creation method and mycological properties of Clostridium sp. No. 68-2 (hereinafter abbreviated as "home country"). separated. That is, after dispensing 5 ml of the liquid medium shown in Table 1 into test tubes and sterilizing them, approximately 0.3
0 soil was added, and after sealing with a butyl rubber stopper, the gas phase was replaced with a sterilizing gas containing hydrogen (67%) and carbon dioxide (33%), and the culture was left standing at 30°C, and every 3 weeks. I carried out succession planting. After sub-planting twice in a liquid medium, the roll tube method (
Methods in Microbiology, Volume 3, 13.1
17 (1969) Academic Press), a single bacterium was isolated and the present bacterium was obtained.

(Mycological Properties) For examination of the mycological properties that indicate the mycological properties of the strain of the present invention, please refer to [An Aerob Laboratory Manual (
Anaerobe Laboratory Manua
l ) 4th edition J (The V, 1.P, ^naero
be LaboratoryVirginia Po
1ytechnic In5titude and 5
tate University, Blacksburg
a (1972)) and ``Birshi's Manual.
of Determinative Bacteriology (Bcr)
gey's Manual of Determina
tive BacterioloOV) 8th Edition "Classification and Identification of Microorganisms" (written by Takeharu Hase, Gakkai Publishing Center)
Using the method and medium composition now described.

(Microscopic findings) 1. Cell shape and size: Single or double bacilli, width 1.3-1.6 μm, length 4.5-4.8 μm 2. Flagella: None 3, Spores: Present , Terminal 4, Durham staining: negative (medium composition) Examples are shown in Table 1.

(Growth status) Growth on an agar medium containing the composition shown in Table 1 plus 3% agar is as follows.

Bicircular shape: Smooth ridge: Slightly raised surface: Smooth Color tone: White (physiological properties) Attitude towards oxygen: Range of obligate anaerobic growth (pl-1) Optimum 1) H near, 7 growth pH
: 5.5-8.0 (Temperature) to 3a Temperature: 30℃ Growth temperature: 25-40℃ ■, Indole production: + ■, Gelatin liquefaction knee ■, Catalase production knee ■, Starch hydrolysis knee ■ , Hydrolysis of Aesculin ■ Formation of 0 Pigment (Negativity of carbon source) Add 5 ml of a liquid medium containing the following carbon source (1%) to the basic medium shown in Table 1 to a test tube with a straight rod of 18 mm.

Create a sterile medium, inoculate a water bottle, replace the gas phase with sterilizing gas containing nitrogen (67%) and carbon dioxide (33%), and incubate at 30°C.
The cells were statically cultured for 140 minutes. Growth was measured by measuring turbidity at 600 nm using a spectrometer (Spectronic 20°, manufactured by Shimadzu Corporation). Those whose turbidity at 600 nm differs from the control containing no carbon source by less than 0.1 are classified as "not assimilated", and those with a turbidity of 0.1 or more and less than 0.2 are classified as "slightly assimilated" (0.2 or more). 9 1Lt8G (Q nigercose, fructose, xylose, ribose, arabinose, rhamnose, galactose, maltose, sucrose).

Lactose, melibiose, trehalose, cellobiose, raffinose, melezitose, mannitol, and when hydrogen is used instead of nitrogen in the above test, carbon dioxide is also assimilated.

n (Is L/ lx 6% Gμ = sorbose, sorbitol.

Methanol, ethanol (generation of acids from sugars, etc.) Add 1% of the sugars that were confirmed to be assimilated in the above test to the basic medium shown in Table 1, and add nitrogen (67μ) and carbon dioxide (33μ) to the gas phase. %), and inoculate the water bottle with bacteria.
The cells were cultured statically at 30°C. Acetic acid and isorlIIM were produced as organic acids in the medium for all carbon sources.

Also, when peptone/yeast extract medium or peptone/yeast extract/glucose medium was used, acetic acid and isobutyric acid were produced in the medium as an organic acid.

(Comparison with similar conventional scales, etc.) Based on the above national characteristics, No. 68-2 is an obligate anaerobic Gram-negative spore bacillus whose main fermentation metabolites are acetic acid and isoacetic acid. This strain is characterized by Due to this property, Bersey's Manual of Determinative Bacteriology 8th edition and Aerob.
A search based on the 4th edition of the Laboratory Manual suggests that it is a strain belonging to Clostridium. Therefore, when identifying Clostridium sphenoides (C.
, spl+enoidcs), and in the 8th edition of the Bird's Manual of Determinative Bacteriology, there was no description of a bacterial species whose properties matched those of No. 68-2. When the properties of Clostridium sphenoides in No. 68-2 were compared, they both agreed that they were gram-negative right-spore bacilli, but the properties of the two samples were different in the points shown in Table 2.
The strain of the present invention grows on carbon dioxide and hydrogen to produce acetic acid and isoacetic acid.

There are four known types of bacteria belonging to the genus Clostridium that grow on carbon dioxide and hydrogen, but these can be distinguished from the present invention in that they all have flagella and all but one type are positive for Durham staining. Met.

From Table 2 and above, the nine domestic strains are considered to be new bacterial species belonging to the genus Clostridium.
It was named SP No. 68-2.

Furthermore, although this strain is similar to the one used in the Microbial Industrial Technology Research Institute of the Agency of Industrial Science and Technology, it is necessary to prevent oxygen from entering, and in the laboratory, it is recommended to leave it still or shake it in an incubator tightly closed with a rubber stopper. is used. On a slightly larger scale, a commonly used Wa fermenter can be used as is, and an anaerobic atmosphere can be created by replacing the oxygen in the device with an inert gas such as nitrogen or a raw material gas. The type of fermentation cypress is not particularly important, but in addition to the commonly used stirring mixing tank, it can also be a single-stage or multi-stage bubble column type.

A draft tube type SIYM can also be used.

The carbon source used for culture is usually supplied as carbon dioxide gas, but it can also be added to the medium as dissolved carbon dioxide, carbonate, or hydrogen carbonate. As the nitrogen source, various nitrogen compounds that can be used in conventional fermentation can be used, such as ammonium salts such as ammonium chloride and nitrates such as sodium nitrate.

Others as necessary: potassium dihydrogen phosphate, magnesium sulfate, manganese sulfate, sodium chloride, iron sulfate, cobalt chloride, calcium chloride, zinc sulfate.

The present invention will be explained below using specific examples.

Example 1 Stridium sp. No. 68-2 strain was cultured as follows. Transfer the culture medium shown in Table 1 to the test tube 5
m After dispensing and sterilizing, - Culture solution 10 cultured in the same medium
0 μm was added in an anaerobic glove box (1-Ma Co., Ltd., Anaero Box), and after sealing with a butyl rubber stopper, the gas phase was replaced with a sterilizing gas containing hydrogen (67%) and carbon dioxide (33%). Culture was carried out at 30°C.

Centrifuge a portion of the culture solution! Isolate the bacterial cells using a Li machine.

Furthermore, Fi'j was made acidic with phosphoric acid, and the product was quantified by gas chromatography.

As a result, 0.28 (J/1) of acetic acid and 0.480/I of isobutyric acid were produced in 10 days of static culture. (The products were confirmed by gas chroma 1 to graph-mass spectrometer. .

) Example 2 Using an L-shaped test tube, prepared in the same manner as in Example 1, Clostridium sp. N0.68-2 strain was incubated with shaking 3B. The measurement method was the same as in Example 1, and the product was analyzed. As a result, 0.42 g/l of acetic acid and 0.5 g/l of acetic acid were found in 10 days.
2o/l of isobutyric acid was produced r+v.

Patent applicant: Director of the Agency of Industrial Science and Technology

Claims (1)

[Claims] It belongs to the genus Clostridium and is grown in carbon dioxide and hydrogen.
Flagellated bacillus, Clostridium sp. No.
68-2