JPS60192595A - Production of acetic acid - Google Patents

Abstract

PURPOSE:To enable the biochemical production of acetic acid utilizing CO2 available as an industrial waste, by culturing a novel Clostridium sp. No.307 strain using CO2 and H2 as the substrate. CONSTITUTION:Novel Clostridium sp. No.307 strain (FERM-P No.7487) is cultured in a fermentation tank, etc. in an anaerobic atmosphere using CO2 and H2 as the substrate, and the objective acetic acid produced and accumulated in the medium is recovered. The above novel strain can be obtained by culturing the soil collected from the Hiraito Field, Hokkaido Prefecture, Japan, in an anaerobic glove box in an aseptic gas atmosphere containing H2 and CO2 at 30 deg.C.

Description

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

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

Microorganisms that accumulate acetic acid in the growth medium include Crystridium aceticum, Acetobacterium uthudii,
Niebac millirum limosum, Butylibacterium methylotrophicum, Clostridium strain CV
-AA1 etc. were known.

Also under high temperature conditions, Acetogenium kiwi.

Clostridium thermoautotrophicum.

I have Clostridium thermoaceticum.

(Purpose of the Invention) The present inventor focused on carbon dioxide, which is a renewable resource that exists in abundance in nature, and is also the final waste product of various industrial processes, and hopes that it will be supplied in large quantities as a future energy source. This invention was achieved by studying a method for biochemically producing acetic acid by reacting it with hydrogen.

Until now, the above-mentioned bacteria have been known as microorganisms that produce acetic acid from carbon dioxide and hydrogen. What is the assignment?

Among these, it is important to obtain bacteria that have a high accumulation concentration and production rate of acetic acid. To do this, we need a new bacterial species #J that can produce acetic acid from carbon dioxide and hydrogen! ! With this intention in mind, the present invention utilizes carbon dioxide and hydrogen to obtain a new microorganism that accumulates acetic acid in the ground, and uses this to produce acetic acid. The purpose is to provide a new manufacturing method.

(Structure of the Invention) The present invention is a method for producing acetic acid, which is characterized by culturing Clostridium sp. No. 307 using carbon dioxide and hydrogen as substrates, and recovering the produced and accumulated acetic acid.

The microorganism used in the present invention belongs to the genus Clostridium, grows on carbon dioxide and hydrogen, has no ability to assimilate methanol or liquefy gelatin, and exhibits enteric properties in an indole production test.
Clostridium sp. No. 307 (hereinafter abbreviated as "home country"), a bacterium with flagella and a slight curve, is thought to belong to the genus Clostridium, as it is an anaerobic bacterium that produces spores. It grows on carbon dioxide and hydrogen, and is different from known congener bacteria in various properties that will be described in detail later, and is considered to be a new bacterial species.

Since the official species name has not yet been assigned, it is designated as Clostridium sp. No. 307 in the present invention.

Next, we will show the method of creation in water and the mycological properties.

(Creation method) Mizuuchi was isolated from the soil of Hiraito Plain in Hokkaido by the following method. That is, 5 ml of the liquid medium shown in Table 1 was dispensed into test tubes, sterilized, and heated to about 0.3C in a Hibatsu glove box.
After adding the soil in I and sealing with a butyl rubber stopper, the gas phase was replaced with a sterilizing gas containing hydrogen (67%) and carbon dioxide (33%), and cultured at 30°C. I carried out succession planting. After sub-planting twice in a liquid medium, the roll tube method (
Methods in Microbiology, Volume 3B, 11
7 (1969) Academic Press), single bacteria were isolated and water was obtained.

(Mycological properties) In order to check the mycological properties of the strain of the present invention, [Aerob Laboratory-? Knee L
Full (Anaerobe Laboratory Ma
nual ) 4th edition J (The V, 1.P, Ana
erobe Laboratory V”irgini
a Polytechnic In5titut a
nd 5tate University, Black
Bergey's Manual of Determination (1972) and Bergey's Manual of Determination.
The method and culture medium composition described in "Classification and Identification of Microorganisms" (written by Takeharu Hase, published by Gakkai Publishing Center) were used.

(Microscopic findings) 1. Shape and size of 11A vacuole: Slightly curved rod, singly or in pairs, 0.6-0.8 μm wide.

Length 2.0-3.4μm 2. Flagellum: periflagella 3. Spores: Yes, terminal 4. Durham staining: negative (Medium composition) Examples are shown in Table 1.

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

Shape: Bi-circular periphery: Smooth Protrusion: Slightly raised surface: Smooth Color tone: White to cream (Physiological properties) ■, Attitude towards oxygen resistance: Obligately anaerobic ■, Growth range (pH) Optimum 1) H Near, 7 growth pH
:6. o ~ 8.0 (Temperature) Optimal humidity: 30℃ Growth temperature = 25-40℃ ■, Indole production: + ■, Gelatin liquefaction knee ■, Catalase production knee ■, Starch hydrolysis knee ■, Aesculin hydration Decomposition: + ■ Formation of 9 pigments (assimilability 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 cultured statically for 14 days. Growth was measured by measuring turbidity at 600 nm using a spectrometer (Spectronic 20, manufactured by Shima 41 Seisakusho). Those whose turbidity at 600 nm differs from the control containing no carbon source by less than 0.1 are classified as "non-assimilated J", 0.
0.2 to “slightly utilize” those with a value of 1 or more and less than 0.2
The above-mentioned items were defined as ``capitalize''.

Assimilated substances: nigerose, fructose, xylose, ribose, arabinose, rhamnose, marl-s, melibiose, trehalose.

Cellobiose, mannitol, and when hydrogen is used instead of nitrogen in the above test, carbon dioxide is also assimilated.

Slightly utilizable: Not applicable Things that cannot be assimilated: sorbose, galactose.

Saccharose, lactose, raffinose, melezitose, sorbitol, methanol, starch (generation of acids from sugars, etc.) 1% of the sugars that were confirmed to be assimilated in the above test were added to the basic medium shown in Table 1, and the gas phase was replaced with sterilizing gas containing nitrogen (67%) and carbon dioxide (33%), and nine countries were inoculated.
'Statically cultured at 30°C. For all carbon sources, acetic acid was produced as an organic acid in the medium.

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

(Comparison with similar native species, etc.) Based on the above mycological properties, No. 307 is an obligate anaerobic, slightly curved Gram-negative sporobacillus that shows a positive indole production test. fil? B is a strain whose metabolite is acetic acid. Due to this property, Bersey's Manual of Determinative Bacteriology 8th Edition and Aerob Laboratory Manual 4th Edition
If you search based on the edition, you will find Clostridium (CIost).
ridiuIll).

Therefore, I identified Clostridium sphenoides (C, 5phcnoides) according to the genus identification key in the 4th edition of the Aerob Laboratory Manual.
I've come across a lot of different types of books, including the 8th edition of Barsey's Manual of Bacteriology! The condition is No, and there was no description of the bacterial species matching 307.N
A comparison of the properties of Clostridium sphenoides and Clostridium o, 307 revealed that both were in agreement that they were obligate anaerobic spore-bearing bacilli, but the properties of the two paintings differed in the points shown in Table 2.

The strain of the present invention grows on carbon dioxide and hydrogen to produce acetic acid. There are four known types of bacteria belonging to the genus Clostridium that grow on carbon dioxide and hydrogen;
The species is a straight bacillus, and the remaining species (Clostridium strain CV-AA1) has the ability to assimilate methanol and liquefy gelatin.

This was distinguishable from the present invention.

Table 2 It was named Trijiram sp. No. 307.

This strain of Dorani was sent to the Institute of Microbial Technology, Agency of Industrial Science and Technology.
FERN-P No. 7487 (FERN-P No. 74)
87) How was it deposited?

(Cultivation method) The cultivation method is basically the same as that for general microorganisms, but it is necessary to prevent oxygen from entering, and in the laboratory, culture is carried out in an incubator tightly closed with a rubber stopper. , leaving it still or shaking it. On a slightly larger scale, two commonly used fermenters 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. W
The type of J fermenter is not particularly important, but it is a commonly used stirring mixing tank [Yaka, one-stage or multi-stage bubble column type].

A draft fermenter 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 bicarbonate. As the nitrogen source, various nitrogen compounds that can be used in ordinary fermentation can be used, such as ammonium salts such as ammonium chloride and nitrate salts such as sodium nitrate.

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

Addition of inorganic compounds such as copper sulfate, alum, sodium molybdate, and boric acid, or vitamins such as biotin and Nambo extract is generally carried out.

The present invention will be explained below using specific examples.

Example 1 Clostridium sp. No. 307 strain was cultured as follows. After dispensing 5 ml of the medium shown in Table 1 into a test tube and sterilizing it, transfer the 100 μm culture solution cultured in the same medium to an anaerobic glovebox (Pharma, Anaerobox).
After adding it in the tank and sealing it with a butyl rubber stopper, the gas phase was replaced with hydrogen (
The cells were replaced with a sterilizing gas containing 67%) and carbon dioxide (33%), and cultured at 30'C.

A portion of the culture solution is centrifuged to separate the bacterial cells.

The supernatant was acidified with phosphoric acid and the product was analyzed by gas chromatography.

As a result, 1.70 g/l of acetic acid was produced during 10 days of static culture. (The product was confirmed using a gas chromatograph-mass spectrometer.) Example 2 Using a single-shaped test tube, preparation was made in the same manner as in Example 1, and the patent applicant: Director of the Agency of Industrial Science and Technology

Claims (1)

[Claims] A method for producing acetic acid, which comprises culturing Clostridium sp. No. 307 using carbon dioxide and hydrogen as substrates, and recovering the produced and accumulated acetic acid.