JPS61199792A - Production of acetic acid - Google Patents

Abstract

PURPOSE:A specific microorganism is Clostridium, capable of assimilating carbon dioxide and hydrogen to produce acetic acid is cultured using carbon dioxide and hydrogen as substrates and the acetic acid accumulated is collected. CONSTITUTION:A microorganism in Clostridiuim, capable of assimilating carbon dioxide and hydrogen to produce acetic acid such as Clostridium sp. No.672 (FERM 8049) is cultured using carbon dioxide and hydrogen as substrates. Then, the acetic acid accumulated in the medium is collected to enable biochemical production of acetic acid.

Description

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

Acetic acid is used in fields such as food and industrial raw materials.

(Prior Art) Several microorganisms are known that assimilate carbon dioxide and hydrogen and accumulate acetic acid in the growth medium.

Among them, there are four types of bacteria belonging to the genus Clostridium that have been reported so far. And we newly created C1ostridium sp.
+a sp) N O, 68-2 Microtechnical Laboratory No. 7367
No., Clostridium φ sp.
um 5o) No. 307 Microtechnical Research Institute No. 7487, Clostridium sp.
sp) No. 484 and 7488, and there are 7 types in total including the 4 types mentioned above.

(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 each weighing industry, and aims to use it as a future energy source. This invention was achieved by studying a biochemical method for producing acetic acid by reacting it with hydrogen in the atmosphere. 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, especially when it comes to producing acetic acid from carbon dioxide and hydrogen. A bacterium that has an optimal growth p11 below p16 and has the ability to produce acetic acid from carbon dioxide and hydrogen has not yet been reported.

Under these circumstances, the present inventors aimed to provide a new method for producing acetic acid using carbon dioxide and hydrogen as substrates.

(Structure of the Invention) The present invention provides Clostridium m1. : Cultivates bacteria that have the ability to assimilate carbon dioxide and hydrogen to produce acetic acid.

This is a method for producing acetic acid, which is characterized by recovering the acetic acid that has been produced and accumulated.

The microorganism used in the present invention belongs to the genus Clostridium and is an acetic acid-producing bacterium that assimilates carbon dioxide and hydrogen, and the microorganism described in the Examples of the present invention is the first such microorganism. The microorganisms used in the examples are considered to belong to the genus Clostridium because they are anaerobic bacteria that produce spores.

It grows in an acidic region such as pH 4.0 to 7.0 with carbon dioxide and hydrogen, and has no flagella, and is different from known congener bacteria in various characteristics that will be described in detail later, and is considered to be a new bacterial species. Conceivable.

Since the official species name has not yet been assigned, the present invention uses Clostridium Sp.
) No. 672 Micro-Laboratory No. 8049.

Next, the production method and mycological properties of Clostridium S.B. No. 672 (hereinafter abbreviated as "canteen") will be shown.

(Creation method) The water bottle was isolated from Aso mud in Kumamoto Prefecture by the following method. That is, 5 ml of the liquid medium shown in Table 1 was dispensed into test tubes and sterilized. Approximately 0.3 g of soil was added in an anaerobic glove box, and after sealing with a butyl rubber stopper, the gas phase was diluted with hydrogen (67%
) and carbon dioxide (33%) with a sterilizing gas,
Static culture was carried out at 0°C, and subculture was performed at intervals of about 3 a.m. 2
After sub-planting in liquid medium, add 3% agar to the medium shown in Table 1.
Roll tube method using an agar medium supplemented with (Methods in Microbiology, Volume 3B).

117 (1969) Academic Press), a single bacteria was isolated and a water bottle 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 the “Aerob Laboratory Manual (
Anacrobe Laboratory Manua
l ) 4th edition J (The V, 1.P, Anacro
be LaboratoryVirginia Po
1ytecl+nic In5titudinal and
5tate university, Blacksbu
ro (1972)), rversies-'? Nuclear determinant bacteriology
(Bergey's Manual of Deter
minative Bacteri.

1oay) 8th Edition", [Birshi's Manual of Systematic Bacteriology (Volume 1)
) (Bergey's Manual of S
systematic Bactcriology (V
The method and culture medium composition described in "Classification and Identification of Microorganisms" (written by Takeharu Hase, Gakkai Publishing Center) were used.

(Microscopic findings) 1. Cell shape and large serrations: single or double straight bacilli, width 1.5 μm, serrations 6. O~8.0 μm 2, flagella: none 3, spores: present, terminal 4, Durham staining: phlegmatic (medium composition) Table 1 shows examples.

Table 1 Composition of basal medium (in deionized water 11) 0.1% indigo carmine solution 2 ml 110% N
8401 solution 10m 11MK) 12P
O4 (pH 7,0) solution 5ml 120% Mg5o -
7H○ solution Q, 5ml vitamin solution
20ml mineral solution
40ml cysteine hydrochloride (1H20)
0.5q Sodium sulfide 0
．． 250 Sodium hydrogen carbonate 1q6
001110 / I Sodium bromoethanesulfonate 1ml Fumo extract 0.2q
1) 115.3 Vitamin solution composition (mg/l) Biotin 2 Folic acid 2 Pyridoxine hydrochloride 10 Thiamine hydrochloride
5 Riboflavin 5 Nicotinic acid 5 Pan 1 Calcium acetate 5 Vitamin 812'
0.01p-aminobenzoic acid
5 Thioctic acid 1 Mineral solution composition (q/l) Nitrilotriacetic acid 0.25MQS○
-71-1200,1 Mn5O-4120 40,28 NaCI C95 "eSo
・7HO0,05 CoCl -6H00゜09 CaC1・2HO0907 ZnSO-7HOO,09 CuSO40,03 AIK(804)2-12H200-009H3[30
4,0,00,5 NaMoO-211200,006 (Growth condition) The growth n on an agar medium prepared by adding 3% agar to the composition shown in Table 1 is as follows.

Shape: 2 circular periphery: 2 smooth ridges: slightly raised surface 2: smooth Color tone: white (physiological properties) ■, Attitude towards oxygen = lliii anaerobic ■, Growth range (p. 11) Optimum Dll: 5.8 Growth pl-1
: 4.0 to 7.0 (Temperature) Optimum temperature = 30℃ Growth temperature: 25 to 40℃ ■, Indole production knee ■, Dilatin liquefaction knee ■, Kakrabi production knee ■, Starch hydrolysis knee ■, Hydrolysis of I screen ■ Formation of 0 pigment (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 mrn.

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 14 days. For growth, measure the turbidity at 600 nm using a spectrometer (Spectronic 20° Island) 1! (manufactured by 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.
1 or more and less than 0.2 I "slightly utilize" 0.
Items of 2 or more are considered to be ``capitalized''.

Assimilates 20-glucose, D-fructose, sorbose, xylose, and D-ribose.

Rhamnose, galactose, lactose, 170 bioses, and when hydrogen is used instead of nitrogen in the above test, carbon dioxide is assimilated.

Things that cannot be assimilated: sorbose, arabinose, maltose, sucrose, melibiose, trehalose, raffinose, melezitose, mannitol, methanol,
Ethanol (generation of acid from sugar, etc.) Add 1% of the sugar that was confirmed to be assimilated in the above test to the basic medium shown in Table 1, and add nitrogen (67%) and rA element dioxide (33%) to the gas phase. %), and inoculated with 1 wood fungus, which was then statically cultured at 30°C. Acetic acid and n-acetic acid were produced as organic acids in the medium using all carbon sources.

Also, peptone/yeast P4 extract medium or pair 1~N/M
R) Even when an extract/glucose medium is used, acetic acid and ()-butyric acid are produced as g1 acids in the medium.

(Comparison with similar native species, etc.) From the above mycological properties, No. 672 is a negative anaerobic Durham-negative right-spore bacillus, and its main fermentation metabolites are acetic acid, carbon dioxide and hydrogen. This strain is characterized by the fact that the other carbon sources it assimilates are acetic acid and PMIv. Because of this property, Bersey's Manual of Determining Bacteriology, 8th Edition, Bersey's
Manual of Systematic Bacariology (Volume 1) and Aerob Laboratory.
According to the 4th edition of the manual, it is considered to be a strain belonging to Clostridium (Clostridium+). Therefore, by following the genus identification key in the 4th edition of the Aerob Laboratory Manual, we identified Clostridium phalatucus (C, falta).
67.
There was no description of the bacterial species matching 2. No, (I compared the properties of Clostridium phalax with 372 and found that they were both obligately anaerobic, Gram-resistant right-spore bacilli, but as shown in Table 2, the properties of the two were different in some respects. was.

The strain of the present invention grows on carbon dioxide and hydrogen and produces acetic acid. Seven species of bacteria belonging to the genus Crossi.Rhydium were known to grow in carbon dioxide and hydrogen, but all of these grow in a neutral pH region (= near I) and do not grow in acidic regions. It can be distinguished from the present invention in that it cannot.

Table 2 C.]7 Lux No. 670 (literature value)
*(Actual measurements) Morphology: Direct bacillus Direct bacillus alone or 2 to 3 cells Single or 2 to 3 cells without flagella
Size without flagella: 0.6~0.9 1.5X 6.0~
8.0 (μm) x 2.0-10.0 Durham staining: Positive Negative pigment production: None
None Carbon source assimilation Xylose assimilation: None Rhamnose assimilation that is well assimilated: None Maltose assimilation that is well assimilated: Well assimilated None Products: In addition to acetic acid, butyric acid, from carbon dioxide and hydrogen Production of lactic acid 4 produces acetic acid and butyric acid from other utilizable carbon sources.

*Birshi's Manual Determinative Bacteriology (8th Edition) Based on the above, it is believed that the 1-Hydrogen strain is a new bacterial species belonging to the genus Clostridium, so it was named Clostridium SB No. 672.

Furthermore, this strain was sent to the Institute of Microbial Technology, Agency of Industrial Science and Technology.
FERN-P No. 80
49)".

(Cultivation method) The cultivation method is basically the same as for general microorganisms, but it is necessary to prevent the contamination of M elements, and in the laboratory, culture is carried out in an incubator sealed with a rubber stopper etc. In this case, a method of standing still or shaking is used. On a slightly larger scale, a commonly used mH tank can be used as is, and an anaerobic atmosphere can be created by replacing the M element in the device with an inert gas such as nitrogen or a raw material gas. The type of fermentation tank is not particularly important, but in addition to the commonly used stirring mixing tank, it can be a single-stage or multi-stage bubble column type.

A draft tube type wJ 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 commonly used in fermentation can be used, such as ammonium salts such as ammonium chloride and nitrates such as sodium nitrate.

Potassium dihydrogen phosphate, magnesium sulfate, manganese sulfate, sodium chloride, iron sulfate, cobalt chloride, calcium chloride, zinc sulfate, as required.

The addition of sterile compounds such as copper sulfate, alum, sodium molybdate, boric acid, or vitamin sources such as yeast extract is conventional practice.

The present invention will be explained below using specific examples.

Example 1 Clostridium SB No. 672 strain was cultured as follows. After dispensing 5 ml of the medium shown in Table 1 into test tubes and sterilizing them, 100 μm of a culture solution cultured in the same medium was added in an anaerobic glow box.

After sealing with a butyl rubber stopper, the gas phase was replaced with a sterilizing gas containing hydrogen (67%) and carbon dioxide (33%).

It was statically cultured at 30°C.

Part of the culture solution was centrifuged to separate the bacterial cells.

The supernatant was acidified with phosphoric acid and the product was quantified by gas chromatography.
f1101r0.78Q/1 of acetic acid was produced.

Example 2 Using a single-shaped test tube, Clostridium SB No. 672 strain was cultured with shaking in the same manner as in Example 1. The measurement method was the same as in Example 1, and the product was analyzed. As a result, 0.98 o/I of acetic acid was produced in 10 days.

Claims (1)

[Claims] Clostridium SB No., a bacterium that belongs to the genus Clostridium and has the ability to assimilate carbon dioxide and hydrogen to produce acetic acid, using carbon dioxide and hydrogen as substrates. 67
A method for producing acetic acid characterized by culturing 2 and recovering the produced and accumulated acetic acid.