JPH053279B2 - - Google Patents

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, benzoic acid or its benzoic acid is added to the reaction system. The present invention relates to a method for efficiently producing oxides by adding metal salts to promote regeneration of bacterial cells with reduced methane oxidizing ability. [Prior art and problems to be solved by the invention] Methane oxidizing oxygen (methane monooxygenase) possessed by methane-assimilating bacteria is capable of oxidizing alkanes, alkenes,
It is known to have the ability to oxidize cyclic compounds and convert them into corresponding oxides (Special Publication No. 1983-
31198). 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 the problem] Therefore, when producing oxides using methane-assimilating bacteria, the present inventors removed the produced oxides from the system and removed bacterial cells or enzymes with reduced activity. We conducted repeated studies to develop a method for producing oxides stably over a long period of time by regenerating them. 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 the more the bacterial cells were treated, the longer it took for them 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, we have found that methane-assimilating bacteria have the ability to oxidize cyclic compounds, and 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 involves 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 to produce an alcohol, epoxide, or cyclic alcohol that is an oxide corresponding to the raw material, and The generated oxide is removed from the system, and 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 and used for oxide production. This continuous method for producing an oxide is characterized by adding benzoic acid or a metal salt thereof to a reaction 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. Also,
Examples of the cyclic compound include alicyclic compounds such as cyclohexane; 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 agile NCIB 11124, Methylosinus trichosporium NCIB 11131
Methylocinus bacteria such as Methylocystis parvus, Methylocystis bacteria such as NCIB11129, Methylobacter capsulata
Examples include Methylobacter bacteria such as NCIB11128. The medium used for culturing the methane-assimilating bacteria may be any medium as long as the bacteria can sufficiently proliferate, and methane, methanol, etc. are usually used as the carbon 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. A suitable medium is the medium of Whittenberry et al. (J. Gen. Microbiol., 61 , pp. 205-208,
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, wash with a suitable solution such as phosphate buffer,
Microbial cells suspended in the solution may also 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. Examples of electron donors include lower alcohols such as methyl alcohol and ethyl alcohol; formaldehyde, acetaldehyde,
Examples include lower aldehydes such as propionaldehyde; formic acid salts such as formic acid or sodium formate; methane; hydrogen; NADH ₂ ; NADPH ₂ . These may be used alone or in combination. Reaction conditions vary depending on the type of microorganism used, reaction tank, etc., but generally pH 5.5-9.0, preferably 6.0-8.5, temperature 15-60°C, preferably 20-
It is 50℃. In the present invention, benzoic acid or a metal salt thereof (for example, an alkali metal salt or an alkaline earth metal salt) is added to the reaction system in order to rapidly regenerate the inactivated bacterial cells. The timing of its addition is arbitrary, for example, when adding raw materials to the reaction tank, at the start of the reaction,
It may be carried out at any appropriate time during the reaction or during regeneration. Further, the amount of benzoic acid or its metal salt added is 0.1 to 8 mmol/, preferably 0.2 to 8 mmol/
5 mmol/ is appropriate. The generated oxides need to be removed quickly from the reaction tank, for example, a part of the reaction mixture containing bacterial cells is introduced into a product recovery device such as a scrubber, extraction tower, or membrane separation device to remove the 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, nitrates, ammonia, ammonium salts, peptone, casamino acids, yeast extract, L-glutamine, L-asparagine, etc. as nitrogen sources. The liquid is filled with a regenerating liquid containing substances such as sulfuric acid, sulfates, hydrogen sulfide, sodium sulfide, hydrosulfides, and sodium bisulfide as a sulfur source, and this liquid is filled with oxygen (usually air). The regeneration operation is performed for a predetermined period of time while supplying. The appropriate carbon source supply amount to the regeneration tank is 10 to 600 nmol/min・mg bacterial cells, preferably 30 to 400 nmol/min・mg bacterial cells, and the nitrogen source supply amount is 1 nmol/min・mg bacterial cells or more. , preferably 2 to 500 nmol/min・mg bacterial cells, and the sulfur source supply amount is 0.02 nmol/min・mg bacterial cells or more, preferably
0.1 to 150 nmol/min/mg 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 described above, the feeding method is preferably to add at a constant rate over a certain period of time, but it is also possible to add for several hours at a time. When added temporarily, it is sufficient to add an amount commensurate with the amount added when continuously supplied. In addition to carbon sources, nitrogen sources, and sulfur sources, if enough phosphoric acid, magnesium, and trace metals are added, it is possible to simultaneously reproduce and multiply bacterial cells. Furthermore, if the amount of oxygen is insufficient, regeneration will be delayed. The regeneration is preferably 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 present invention, alcohols, epoxides, and cyclic alcohols corresponding to the raw materials used can be 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. Note that 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
Medium 8 mixed at a ratio of 100:1 was charged into a 10 volume jar fermenter through a sterile filter. Table 1 Magnesium nitrate heptahydrate 1.0g Potassium sulfate 1.0g Calcium chloride 50mg NaMoO ₄ 1mg FeSO ₄・7H ₂ O 500μg Table 1 (continued) ZnSO ₄・7H ₂ O 400μg H ₃ BO ₄ 15μg CoCl ₂・6H ₂ O 50μg MnCl ₂・4H ₂ O 20μg NiCl ₂・6H ₂ O 10μg CuSO ₄・5H ₂ O 200μg EDTA 250μg Distilled water 1 Table 2 Na ₂ HPO ₄・12H ₂ O 43g KH ₂ PO ₄ 15.6g Fe-EDTA 240mg distilled water 1 (PH6.8) Next, prepare eight 500ml Meyer flasks containing 50ml of the culture medium shown in Table 1, and heat at 120°C.
After sterilizing the media shown in Table 2 for 20 minutes at 120℃
0.5 ml of sterilized for 20 minutes was added, and one platinum loop of methane-assimilating bacteria was inoculated thereto. methane 50 here
ml was added, the tube was sealed with a rubber stopper, and cultured with shaking at 45°C for 3 days. Eight flask culture fluids after completion of cultivation were used as seed bacteria, which were aseptically charged into the jar fermenter, and methane-air mixed gas (methane:air = 1:4) was supplied at a rate of 4/min. ,
It was cultured for 3 days. After the bacterial concentration reached 0.5mg/ml,
Add 1 part of CuSO ₄ 5H ₂ O to a medium prepared by mixing the media shown in Tables 1 and 2 at a ratio of 100:1.5.
The culture medium was added at a rate of 1.6 mg/hour while being sterilized using a sterile filter, and cultured continuously. Examples 1 to 6 and Comparative Examples 1 to 3 200 ml of the culture solution of Methylococcus capsulatus NCIB11132, which was continuously cultured according to the method for culturing methane-assimilating bacteria, was centrifuged to collect the bacterial cells, and the reaction regeneration shown in Table 3 was carried out. The bacteria were suspended in a solution at a concentration of 1 mg/ml. 400 ml of this suspension was charged into a 1-volume jar fermenter, and the temperature was raised to 45° C. while supplying air at a rate of 100 ml per minute. Next, add sodium benzoate in the amount shown in Table 4, stir for 10 minutes, and then add 320 ml/min of propylene and methanol.
Propylene oxide was produced by supplying 350 nmol/min·mg 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/min to remove propylene oxide remaining in the reaction solution from the water. After 15 minutes, reduce the air flow rate to 35ml/min, and after confirming that the temperature is 45℃, methane
It was supplied at a rate of 120 ml/min, 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. Table 3 (reaction regeneration solution) Magnesium sulfate heptahydrate 1.0g Potassium nitrate 1.0g Calcium chloride 100mg NaMoO ₄ 1mg FeSO ₄・7H ₂ O 500μg ZnSO ₄・7H ₂ O 400μg Table 3 (continued) H ₃ BO ₄ 15μg CoCl ₂・6H ₂ O 50μg MnCl ₂・4H ₂ O 20μg NiCl ₂・6H ₂ O 10μg CuSO ₄・5H ₂ O 500μg EDTA 250μg Na ₂ HPO ₄・12H ₂ O 645mg KH ₂ PO ₄ 234mg Fe−EDTA 3.6mg Distillation Water 1 (PH6.8)

【table】

[Table] The activity of the bacterial cells was measured by the method shown below. Adjust the bacterial cells to a concentration of 0.5 mg/ml.
7 ml of 1 ml suspended in mM pipes buffer
ml Meyer flask, add 2 ml of propylene, stopper with a rubber stopper, and heat at 45℃ for 30 seconds.
The mixture was shaken and stirred at 300 revolutions per minute. Next, methanol was added to a concentration of 1mM, and after further shaking culture for 3 minutes, the amount of propylene oxide produced was quantified using gas chromatography and expressed as the amount of propylene oxide produced per 1 mg of bacterial cells per minute. Ta. Examples 7 to 10 and Comparative Examples 4 and 5 Except for using 1-butene instead of propylene in Example 1 and supplying air at a rate of 4/min for 30 minutes to remove butylene oxide after stopping the reaction. The same operation as in Example 1 was performed. The results are shown in Table 5.

[Table] Examples 11 to 13 and Comparative Examples 6 and 7 Same as Example 1 except that benzoic acid was added instead of sodium benzoate and the pH was adjusted to 7.0 with 0.5M caustic potassium. I performed the following operations. The results are shown in Table 6.

[Table] [Effects of the Invention] According to the present invention, the regeneration of bacterial cells with reduced methane oxidizing ability can be started in a short time, so not only does the energy required for regeneration be small, but also a small amount of bacterial cells can be used. The desired oxide can be continuously produced by effectively utilizing the oxide. Therefore, the present invention is useful in fields such as chemical industry, fermentation industry, and wastewater treatment.

Claims (1)

[Claims] 1. Production of alcohol, epoxide, or cyclic alcohol, which is an oxide corresponding to the raw material, by bringing methane-assimilating bacteria into contact with an alkane, alkene, or a cyclic compound in the presence of an electron donor in a reaction tank. At the same time, the generated oxides are removed from the system, and the methane assimilating bacteria whose methane oxidation ability has decreased are regenerated in the regeneration tank to recover the methane oxidation ability, and then returned to the reaction tank to remove the oxides. A continuous method for producing an oxide, which comprises adding benzoic acid or a metal salt thereof to a reaction tank and/or a regeneration tank. 2 0.1 to 8 m of benzoic acid or its metal salt
2. The method according to claim 1, wherein the addition is carried out at a ratio of mol/mol. 3. The method according to claim 1, wherein the methane-assimilating bacterium belongs to any one of the genus Methylococcus, the genus Methylomonas, the genus Methylocinus, the genus Methylocystis, and the genus Methylobacter.