JPH053277B2 - - Google Patents

Description

[Detailed description of the invention]

[Industrial Application Field] The present invention relates to a method for producing oxides, and more specifically, in producing oxides by utilizing the co-oxidation ability of methane-assimilating bacteria, carbon dioxide and/or inorganic carbonates are added to the reaction system. The present invention relates to a method for producing an oxide, characterized in that the co-oxidation ability is improved by adding. [Prior art and problems to be solved by the invention] Methane oxidase (methane monooxygenase) possessed by methane-assimilating bacteria can produce alkanes, alkenes,
It is known that it has the ability to oxidize cyclic compounds and convert them into corresponding oxides (Special Publication No. 1983-
31198). However, when producing oxides using the co-oxidation ability of the methane-assimilating bacteria, the oxidation ability tends to decrease, making it impossible to stably obtain the desired oxide over a long period of time. [Means for Solving the Problems] Therefore, the present inventors have repeatedly investigated ways to increase the co-oxidation ability of methane-assimilating bacteria, and have found that by controlling the carbonate ion concentration in the reaction solution. It was discovered that the co-oxidation ability of the methane-assimilating bacteria could be increased, and the present invention was completed based on this knowledge. That is, the present invention provides an alkane, an alkene, or a cyclic compound in which at least one species selected from the genus Methylococcus, Methylomonas, Methylocinus, Methylocystis, and Methylobacterium has methane-assimilating properties. Provided is a method for producing an oxide, which comprises adding carbon dioxide gas and/or an inorganic carbonate to the reaction system when bringing bacteria into contact with each other to produce alcohol, epoxide, or cyclic alcohol that is an oxide corresponding to a raw material. It is something to do. 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. Also,
Examples of the cyclic compound include alicyclic compounds such as cyclohexane, and aromatic compounds such as benzene and toluene. As mentioned above, the oxides derived from these raw materials are alcohols from alkanes; epoxides from alkenes; and cyclic alcohols from cyclic compounds. Methane-assimilating bacteria that can be used in the present invention include:
For example, Methylococcus bacteria such as Methylococcus capsulatus NCIB 11132, Methylomonas bacteria such as Methylomonas albus NCIB 11123, Methylocinus bacteria such as Methylosinus sporium NCIB 11126, Methylocystis parbus ( Methylocystis bacteria such as NCIB 11129 (Methylocystis parvus), Methylobacterium organophyllum (Methylobacterium
Examples include Methylobacterium bacteria such as (organophilum) ATCC27886. The medium used for culturing the above-mentioned methane-assimilating bacteria may be any medium as long as the bacteria can sufficiently proliferate, and methane, methanol, etc. are usually used as the charcoal source. In addition, 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. As a suitable medium, whittenberry medium (J.Gen.Microbiol., 61 , pp. 205-208,
1970). The space in the culture container containing the medium is replaced with a mixed gas of methane and oxygen-containing gas (air, etc.), and the 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 preferable to use microbial cells obtained by solid-liquid separation by an operation such as centrifugation. Furthermore, microbial cells washed with a suitable solution such as a phosphate buffer and suspended in the solution are more suitable. In addition, it is also possible to use materials containing methane oxidase, such as crushed microbial cells and extracts thereof, or those obtained by immobilizing microbial cells by a conventional method. Here, the crushing treatment may be carried out by a conventional method, such as a method in which microbial cells are crushed by ultrasonic waves, a French press, or the like. Also,
For the extraction treatment, a method such as the above-mentioned crushing treatment followed by centrifugation to obtain a soluble extract can be adopted. When bringing the methane-assimilating bacteria into contact with the raw material, it is necessary to have an electron donor present.
Here, electron donors include lower alcohols such as methyl alcohol and ethyl alcohol; lower aldehydes such as formaldehyde, acetaldehyde, and propionaldehyde; formic acid or formate salts such as sodium formate; methane; hydrogen;
NADH ₂ ; NADPH ₂ , etc. These may be used alone or in combination. In order to obtain an oxide by bringing the above-mentioned raw material into contact with methane-assimilating bacteria, it is necessary to add carbon dioxide gas and/or inorganic carbonate to the reaction system. When using carbon dioxide gas, air: carbon dioxide gas = 1:0.1 ~
It is preferable to use it as a mixed gas of 0.75 (volume ratio). Furthermore, when carbon dioxide is added, the pH of the reaction solution increases.
The pH decreases, so neutralize it with basic substances such as potassium hydroxide, sodium hydroxide, ammonia, etc.
Should be used in the range 5.5 to 9.0. Examples of inorganic carbonates include potassium carbonate, sodium carbonate, sodium bicarbonate, and ammonium carbonate, which are added at a rate of 1 to 140 mmol/, preferably 1 to 130 mmol/. The optimal amount to add varies somewhat depending on the strain. The reaction between the above raw materials and methane-assimilating bacteria is as follows:
Although it varies depending on the type of microorganism used, generally the pH is 5.5 to 9.0, the temperature is 15 to 60℃, preferably
The temperature may be set so that the desired oxide can be obtained efficiently under conditions of 20 to 50°C. Through this reaction, as described above, alcohols, epoxides, and cyclic alcohols corresponding to the raw materials are obtained. These products can be phase separated, extracted, distilled,
It can be separated and collected by applying known techniques such as adsorption. [Example] Next, the present invention will be explained in detail with reference to Examples. Example 1 The following medium (1) 8 was placed in a 10 volume jar fermenter, sterilized at 120°C for 20 minutes, and then cooled. On the other hand, 85 ml of the following medium (2), which had been separately sterilized at 120°C for 20 minutes, was added to the medium (1). Similarly, 50 ml of the medium (1) was placed in a 500 ml Mayer flask, sterilized at 120°C for 20 minutes, and 0.5 ml of the separately sterilized medium (2) was added, followed by inoculation of one platinum loop of Methylococcus capsulatus NCIB 11132. After that, seal it tightly with a rubber stopper, add 50ml of methane, and raise the temperature to 45°C.
The cells were cultured with shaking for 3 days. After aseptically charging these 8 flask culture fluids into the jar fermenter as seed bacteria, a mixed gas of methane:air = 1:4 (volume ratio) was added at 4/min.
and cultured for 3 days. Bacteria concentration
After reaching 1.5 mg/ml, mix medium (1) and medium (2) to 100:
Add CuSO ₄ 5H ₂ O to the mixed medium at a ratio of 1.5
A medium prepared by adding 1 mg/h of the culture medium was supplied at a rate of 1.6/h while sterilizing the cells using a sterile filter, and cultured continuously. Medium (1) Magnesium sulfate/heptahydrate 1.0g Potassium nitrate 1.0g Calcium chloride 50 mg NaMoO ₄ 1 〃 FeSO ₄・7H ₂ O 500 μg ZnSO ₄・7H ₂ O 400 μg H ₃ BO ₄ 15 〃 CoCl ₂・6H ₂ O 50 〃 MnCl ₂・4H ₂ O 20 〃 NiCl ₂・6H ₂ O 10 〃 CuSO ₄・5H ₂ O 200 〃 EDTA 250 〃 Distilled water 1 Medium (2) Na ₂ HPO ₄・12H ₂ O 43g KH ₂ PO ₄ 15.6g Fe-EDTA 240mg Distilled water 1 PH 6.8 Culture solution of Methylococcus capsulatus NCIB 11132 strain continuously cultured according to the above method
20ml was centrifuged and suspended in 5mM pipe buffer (PH6.8, containing 10μM of CuSO ₄ .5H ₂ O) to a bacterial concentration of 0.5mg/ml. 1 ml of this suspension was placed in a 7 ml Mayer flask, sodium bicarbonate was added to it at various concentrations, the rubber stopper was sealed, and then 2 ml of propylene was added.
ml and stirred at 45°C for 30 seconds at 300 rpm. Immediately added methanol to 1mM and stirred at 45°C for 3 minutes. Propylene oxide was quantified by gas chromatography. . The results are shown in Table 1. Table 1 Sodium bicarbonate Amount of propylene oxide added Amount produced (m mol/) (n mol/ml) 0 410 5 542 12 603 24 645 50 698 120 668 140 590 Example 2 The culture method shown in Example 1 was used. Methylococcus capsulatus continuously cultured
The cells obtained by centrifuging 400 ml of NCIB11132 culture solution were added to a new medium (the above medium (1) and medium (2) were mixed at 100:1.5).
Add 1 mg/CuSO ₄ 5H ₂ O to the mixture at the ratio of
(prepared by adding at a ratio of
It was suspended at mg/ml. 400 ml of this suspension was placed in a 1 volume jar fermenter, and air was supplied at a rate of 80 ml per minute. The temperature was maintained at 45°C, carbon dioxide gas was supplied at various flow rates for 30 minutes, and the pH was adjusted to 6.8 with 4M potassium hydroxide. After 30 minutes of carbon dioxide gas supply, propylene 320ml/min,
Supply methanol at a rate of 120 μmol/min for 15 minutes,
Propylene oxide was produced. The amount of propylene oxide produced in the gas and reaction solution was determined by gas chromatography. The results are shown in Table 2. Table 2 Propylene oxide carbon dioxide supply amount Production amount (ml/min) (m mol/l) 0 1.10 8 1.59 30 1.98 60 1.67 Examples 3 and 4 The above medium (1) and medium (2) were mixed in a ratio of 100:1.5. A medium prepared by adding CuSO ₄ .5H ₂ O at a ratio of 0.3 mg/2.5 to a mixed medium at a ratio of 2.5
Pass it through a sterile filter into a Meyer flask.
I prepared 250ml. Add 50 ml of the same medium to a 500 ml Meyer flask.
Preparation, Methylomonas, albus NCIB 11123
and Metylocinus sporium NCIB 11126
One platinum loop of each was inoculated, 50 ml of methane was added, and the tubes were sealed with butyl rubber stoppers. Then, at 30℃
Sterilize the cultured product with shaking for 1 day, and sterilize it.
I put it in a Mayer flask. Furthermore, after adding 500ml of methane and sealing with a silicone rubber stopper, the temperature was raised to 30°C.
The cells were cultured with shaking for 24 hours. Thereafter, the bacterial cells were collected by centrifugation and treated with _CuSO4 .
It was washed twice with 4mM phosphate buffer (PH6.8) containing 0.5mg/ _5H2O . In addition, the bacterial concentration is 0.5
It was suspended in the same buffer at mg/ml. Pour 1 ml of this suspension into a 7 ml Mayer flask, add sodium bicarbonate at various concentrations, seal with a rubber stopper, and immediately add 2 ml of propylene.
Added ml. After stirring this suspension at 45℃ and 300 rpm for 30 seconds, methanol was immediately added to 1mM.
Add so that
The produced propylene oxide was quantified by gas chromatography. The results are shown in Table 4.

[Table] Example 5 In Example 1, the concentration of magnesium sulfate in the medium was set to 3.0 g/
Methylocystis parvus NCIB 11129 was continuously cultured in the same manner as in Example 1, except that the temperature was changed to .degree. Next, the bacterial cells were collected by centrifugation, and Example 3
The effect of adding sodium bicarbonate was investigated in the same manner as in 4. The results are shown in Table 5. Table 5 Sodium bicarbonate Amount of propylene oxide added Amount produced (m mol/l) (n mol/ml) 0 515 1 540 2 731 3 720 5 703 7 684 12 695 24 626 120 251 [Effects of the invention] Methane utilization When producing the corresponding oxide from raw materials such as alkanes using the co-oxidation ability of sex bacteria, by adding an inexpensive carbonate source to the reaction system,
It is possible to increase the activity of the microbial cells and improve the production amount of the target oxide per unit of microbial cells. In addition to being used to produce useful substances, this method is also applied to fields such as wastewater treatment.

Claims (1)

[Claims] 1. In the presence of an electron donor, at least one methane-assimilating bacterium selected from the genus Methylococcus, Methylomonas, Methylocinus, Methylocystis, and Methylobacterium is brought into contact with an alkane, alkene, or a cyclic compound. A method for producing an oxide, which comprises adding carbon dioxide gas and/or an inorganic carbonate to a reaction system when producing alcohol, epoxide, or cyclic alcohol, which is an oxide corresponding to a raw material.