JPH01160491A - Continuous production of oxide - Google Patents

Abstract

PURPOSE:To obtain an oxide in high efficiency while regenerating microorganism in a short time, by adding benzoic acids to a reaction tank and a regeneration tank, contacting a methane-assimilating strain with an alkane, etc., in the presence of an electron donor, regenerating the microorganism having lowered activity in a regeneration tank and returning the activated microorganism to the reaction tank. CONSTITUTION:An oxide is produced by contacting an alkane, alkene or a cyclic compound with a methane-assimilation strain in a reaction tank in the presence of an electron donor, removing the produced oxide from the system, regenerating the methane-assimilation strain having lowered methane-oxidizing capability in a regeneration tank to recover the methane-oxidizing capability and returning the recovered strain to the reaction tank. In the above process, benzoic acid or its metal salt is added to the reaction tank and/or the regeneration tank to enable the continuous production of the oxide. The amount of the benzoic acid or its metal salt is preferably 0.1-8mmol/l.

Description

[Detailed Description of the Invention] [Industrial Application Field] The present invention relates to a method for continuously producing oxides, and more specifically, in continuously producing oxides by utilizing the co-oxidation ability of methane-assimilating bacteria, This invention relates to a method for efficiently producing oxides by adding benzoic acid or its metal salt to a reaction system to promote regeneration of bacterial cells with reduced methane oxidizing ability.

[Prior art and problems to be solved by the invention] Methane oxidase (methane monooxygenase) possessed by methane-assimilating bacteria converts alkanes and alkenes.

It is known that it has the ability to oxidize cyclic compounds and convert them into corresponding oxides (Japanese Patent Publication No. 58-31198
issue).

However, when producing oxides using methane-assimilating bacteria, the oxides produced inhibit the reaction or deactivate microorganisms and enzymes derived from microorganisms, so the oxides must be immediately removed from the system. need to be removed. As devices used in such cases, reactors using various membranes and reactors combined with extraction devices have been developed. However, even with the use of these devices, deactivation of microbial cells and enzymes is often unavoidable.

Moreover, when the reaction is carried out by adding oxygen as in the present invention, long-term operation is inherently difficult.

[Means for solving problems]

Therefore, when producing oxides using methane-assimilating bacteria, the present inventors removed the generated oxides from the system and regenerated the bacterial cells or enzymes with reduced activity, thereby producing oxides over a long period of time. We conducted repeated studies to develop a method for stably producing . During this process, the time it takes for the deactivated bacterial cells to start regenerating is greatly influenced by the conditions under which the bacterial cells were deactivated. It was found that it took some time for the bacterial cells to start regenerating. Therefore, we conducted research on a method for rapidly regenerating deactivated bacterial cells and found that adding benzoic acid to the reaction system was effective. By the way, as mentioned above, although methane assimilating bacteria have the ability to oxidize cyclic compounds, it has been found that benzoic acid is not oxidized and does not interfere with the co-oxidation reaction. The present invention was completed based on these findings.

That is, the present invention produces an oxide by bringing a methane-assimilating bacterium into contact with an alkane, alkene, or a cyclic compound in the presence of an electron donor in a reaction tank, and also removes the produced phosphorus oxide from the system. In a continuous method for producing oxides, the methane assimilating bacteria whose methane oxidation ability has decreased are regenerated in a regeneration tank to recover the methane oxidation ability, and then returned to the reaction tank for use in the production of oxides. This is a continuous method for producing oxides characterized by adding benzoic acid or its metal salt to a tank and/or a regeneration tank.

Alkanes used as raw materials in the present invention include methane, ethane, propane, butane, hexane, and octane, and alkenes include ethylene, propylene, butenes, and the like. Examples of the cyclic compound include alicyclic compounds such as cyclohexane; aromatic compounds such as benzene and toluene.

Oxides derived from these raw materials include alcohols from alkanes; epoxides from alkenes; and cyclic alcohols from cyclic compounds.

Examples of methane-assimilating bacteria that can be used in the present invention include Methylococcus capsulatus (Meth 1ococcus).
Methylococcus bacteria such as NCI B 11132, Methylomonas ajire (Met
Bacteria of the genus Methylomonas, such as Methylomonas trichosporium (NCIB 11124), Trichos oriGm) NC
Methylocinus bacteria such as IB 11131, Methylocystis parvum (1 button 91n monthly ROM) NCIB
Methylocystis bacteria such as 11129, Methylobacter capsulata (around 1 anal opening) NCIB1
Examples include Methylobacter bacteria such as 1128.

The medium used for culturing the methane-assimilating bacteria may be any medium that allows the bacteria to sufficiently proliferate, and methane, methanol, etc. are usually used as the carbon source. Also,
As the nitrogen source, commonly used nitrogen sources such as ammonium chloride, potassium nitrate, and ammonium nitrate may be used. In addition, phosphoric acid, calcium salts, magnesium salts, trace amounts of inorganic salts (cupric salts, ferrous salts, cobalt salts, etc.), etc. are added as appropriate. A suitable medium is a medium such as Whittenberry (J, Gen, Microbiol, 61.2).
05-208, 1970). The space in the culture container containing the culture medium is replaced with a mixed gas of methane and oxygen-containing gas (such as air), and the culture medium in contact with the gas is inoculated with methane-assimilating bacteria.

The methane-assimilating bacteria used in the present invention are aerobic bacteria, and may be cultured by batch culture or continuous culture at 20 to 50°C under aerobic conditions.

Although the culture can be used as it is in the oxidation reaction of the raw material, it is also possible to use microbial cells obtained by solid-liquid separation by operations such as centrifugation.

Furthermore, microbial cells washed with a suitable solution such as a phosphate buffer and suspended in the solution may be used. In addition, microbial cells immobilized by conventional methods can also be used.

When bringing the methane-assimilating bacteria into contact with the raw material in the reaction tank, it is necessary to have an electron donor present. Here, as the electron donor, lower alcohols such as methyl alcohol and ethyl alcohol; formaldehyde,
Acetaldehyde.

Lower aldehydes such as propionaldehyde; Formic acid or formate salts such as sodium formate; Methane; Hydrogen; N
There are ADHz and NADPHz.

These may be used alone or in combination.

The reaction conditions vary depending on the type of microorganism 1 reaction tank used, etc.;
~50°C. In addition, in the present invention, in order to rapidly regenerate inactivated bacterial cells, benzoic acid or its metal salt (
For example, alkali metal salts.

alkaline earth metal salts). The addition timing is arbitrary, for example, when adding raw materials to the reaction tank.

It may be at any time such as the start of the reaction, an appropriate time during the reaction, or the time of regeneration. In addition, the amount of benzoic acid or its metal salt added is 0.1 to 8 rnmol/f, and the well-produced oxide must be quickly removed from the reaction tank. A portion of the reaction mixture containing bacterial cells is introduced into a substance recovery device to remove oxides. Part or all of the reaction mixture from which oxides have been removed is returned to the reaction tank.

On the other hand, in order to regenerate methane assimilating bacteria whose methane oxidation ability has decreased, a part of the reaction mixture containing the bacteria is extracted from the reaction tank and sent to the regeneration tank, where the methane oxidation ability of the bacteria is restored. let The regeneration tank contains substances such as methane, methanol, and formaldehyde as carbon sources, and gaseous nitrogen, nitric acid, L-ammonium nitrate, ammonium salts, peptone, casamino acids, yeast extract, and L-glutamine as nitrogen sources.

A regenerating liquid containing substances such as L-asparagine and sulfur sources such as sulfuric acid, sulfates, hydrogen sulfide, sodium sulfide, hydrosulfide, and sodium hydrogen sulfide is charged, and this liquid is filled with oxygen (usually The regeneration operation is performed for a predetermined period of time while supplying air). In addition, the amount of carbon source supplied to the regeneration tank is 1O~600nlIlol/ll1
in・■ bacterial cells, preferably 30 to 400 n mol/
m1n-■ bacterial cells are appropriate, and the nitrogen source supply amount is 1 n
mol/min・■ bacterial cells or more, preferably 2 to 500
n mol/min・mg bacterial cells, but the amount of sulfur source supplied is 0.
0.2 n mol/min・■ bacterial cells or more, preferably 0.02 n mol/min.
1 to 150 nmol/min.2 bacterial cells are appropriate.

If the amount of the carbon source, nitrogen source, or sulfur source added is small, regeneration will be delayed, and if they are added in excess, there will be no further effect, and sometimes regeneration may be inhibited. As for the feeding method, as mentioned above, it is preferable to add at a certain rate per certain period of time,
It is also possible to temporarily add several hours at a time. - When adding at times, it is sufficient to add an amount commensurate with the amount added in the case of continuous feeding. carbon, nitrogen, and sulfur sources, as well as phosphoric acid.

By adding sufficient amounts of magnesium and trace metals, it is possible to simultaneously reproduce and multiply bacterial cells. Furthermore, if the amount of oxygen is insufficient, regeneration will be delayed. Preferably, the regeneration is carried out at a temperature of 15 to 60°C and a pH of 5.5 to 9.0.

The methane-assimilating bacteria thus regenerated are returned to the reaction tank and used for producing oxides.

According to the invention, alcohol corresponding to the raw materials used;
Epoxide and cyclic alcohol are obtained. These products are separated and collected using the recovery device described above.

〔Example〕

Next, the present invention will be explained in detail with reference to examples.

In addition, the methane-assimilating bacteria used in Examples etc. were cultured by the following method.

The medium shown in Table 1 and the medium shown in Table 2 were mixed at a ratio of 100:1.
The mixed medium 81 was charged into a 1O2 jar fermentor through a sterile filter.

Calcium chloride 1.0g Potassium nitrate 1.0g Calcium chloride 50■NaMo0a
1mgFeSO4'7Hz0
500μg Male 11 blades m ZnSO4'7HzO400, czg H3B0. 15H
gCoCfg・6HzO-50Hg MnCj! z・41(zo 20
HgNiCft・6H,010Hg CuSO4-5Hz0 200μ, g
EDTA 250H
g Distilled water 11NazH
PO,・12Hz0 43gKH,Po,
15.6gF e-EDTA
240mg distilled water
11 (pH 6, 8) Next, prepare 8 Meyer flasks containing 50 d of the medium shown in Table 1 and incubate at 120°C for 20 ml.
After sterilizing for minutes, the culture medium shown in Table 2 was heated at 120°C for 20 minutes.
0.5 d of sterilized material was added, and one platinum loop of methane-assimilating bacteria was inoculated thereto. After adding methane Sod, the mixture was sealed with a rubber stopper and cultured with shaking at 45°C for 3 days. After culturing, 8 flask culture fluids were used as seed bacteria, which were aseptically charged into the jar fermenter, and methane-air mixed gas (methane:air = 1:4) was added at a rate of 4 minutes per minute.
! and cultured for 3 days. Bacteria concentration is 0.5■
/ after reaching 10% of the culture medium shown in Tables 1 and 2.
Further, Cu5O,
- A culture medium to which 5H,0 was added at a rate of lx/l was supplied at a rate of 1.6ffi/hour while being sterilized with a sterile filter, and cultured continuously.

Examples 1 to 6 and Comparative Examples 1 to 3 200 d of culture solution of Methylococcus capsulatus NCIB 11132, which was continuously cultured according to the method for culturing methane-assimilating bacteria, was centrifuged to collect bacterial cells, and the reaction regenerated solution shown in Table 3 was obtained. The cells were suspended at a bacterial concentration of 1 mg/In1.

11.400 d of this suspension. Yong's Jarfa Mentor
The temperature was raised to 45° C. while supplying air at a rate of 100 ml per minute. Next, sodium benzoate in the amount shown in Table 4 was added, and after stirring for 10 minutes, propylene 3
20 IR1/win and methanol 350n mo
Propylene oxide was produced by supplying l/min·■ bacterial cells for 30 minutes.

After 30 minutes, the supply of propylene and methanol was stopped, and air was supplied at a rate of 3.2 l/min to remove propylene oxide remaining in the reaction solution from the water. 15
After a few minutes, the air flow rate was reduced to 35 d/1IIin, and after checking that the temperature was 45°C, 120 m of methane was added.
/win, and the regeneration operation of the bacterial cells was started.

Thereafter, the activity of the bacterial cells was measured sequentially, and the time until the bacterial cells started to reproduce was measured. The results are shown in Table 4.

3,・') Magnesium sulfate heptahydrate 1.0g Potassium nitrate 1.0g Calcium chloride
100 ■ N a M OOa
1 mgFeSOn・7HzO50
0Hg ZnSOa'IHz0 400Hg range] Bye noon! Seimu H3B0. 15HgCo
C1z-6HzO50Hg MnC1z・4 HzO20u g NiCl,z・6H2010Hg CuSOa・5H20500Hg EDTA 250HgN
azHPO<・12 Hz 0 645mgK
H2P 04 234mgFe
-EDTA 3.6 mg Distilled water 11 (ptl 6.8
) The activity of the bacterial cells was measured by the method shown below.

Cells were suspended in 5mM Pipes buffer at a concentration of 0.5μ/d, then 1ml was placed in a 7-capacity Mayer flask, 2ml of propylene was added thereto, and the flask was sealed with a rubber stopper. 30 seconds per minute at 45°C.
Shake and stir at 0 rotations. Next, add methanol to 1 m
M, and after further shaking culture for 3 minutes,
The amount of propylene oxide produced was determined by gas chromatography and expressed as the amount of propylene oxide produced per minute per 1 mg of bacterial cells.

Examples 7 to 10 and Comparative Example 4.5 Example 1 except that 1-butene was used instead of propylene and that air was supplied at 42/min for 30 minutes to remove butylene oxide after the reaction was stopped. teeth,
The same operation as in Example 1 was performed. The results are shown in Table 5.

Examples 11-13 and Comparative Examples 6 and 7 Example 1 except that benzoic acid was added instead of sodium benzoate and the pH was adjusted to 7.0 with 0.5 M caustic potassium. A similar operation was performed. The results are shown in Table 6.

〔Effect of the invention〕

According to the present invention, the regeneration of bacterial cells with reduced methane oxidation ability can be started in a short time, so the energy required for regeneration is small and a small amount of bacterial cells can be used effectively to achieve the desired purpose. oxides can be produced continuously.

Therefore, the present invention is useful in fields such as chemical industry, fermentation industry, and wastewater treatment.

Claims (3)

[Claims] (1) In the presence of an electron donor in a reaction tank, an alkane,
Oxides are produced by bringing methane-assimilating bacteria into contact with alkenes or cyclic compounds, and the generated oxides are removed from the system, and the methane-assimilating bacteria whose methane oxidizing ability has decreased are regenerated in a regeneration tank. Adding benzoic acid or its metal salt to the reaction tank and/or regeneration tank in a continuous method for producing an oxide, which involves restoring the methane oxidizing ability by using the oxidation tank and then returning it to the reaction tank for use in the production of the oxide. A continuous production method for oxides characterized by: (2) 0.1 to 8 mmol of benzoic acid or its metal salt
2. The method according to claim 1, wherein the addition is carried out in a proportion of 1/1. (3) The method according to claim 1, wherein the methane-assimilating bacterium belongs to any one of the genus Methylococcus, genus Methylomonas, genus Methylosinus, genus Methylocystis, and genus Methylobacter.