JPS6258986A - Cultivation of aerobic microorganism - Google Patents

Abstract

PURPOSE:To reduce the volume of oxygen to be supplied without reducing the amount of a useful substance produced, by providing an anode in a culture fluid, applying a voltage capable of oxidizing the components of the respiratory system of the microorganism to carry out cultivation. CONSTITUTION:An anode (working electrode) is provided in a culture fluid of an aerobic microorganism, e.g. aerobic bacterium, actinomyces, yeast, mold or basidiomycete, and a voltage is applied through a cathode (counter electrode) for passing through a current or a standard electrode to be a potential standard to the anode. The potential to be applied to the anode is a potential sufficient to oxidize an electron carrier of the respiratory system held by the microorganism to be cultivated, i.e. cytochrome, and is preferably such a potential as not cause the decomposition of water.

Description

[Detailed Description of the Invention] [Industrial Application Field] The present invention relates to a method for culturing aerobic microorganisms, and more specifically, a method for culturing aerobic microorganisms under a normal aeration rate or less to produce useful substances. Regarding the method.

[Conventional technology]

The method of culturing aerobic microorganisms to produce useful substances is
It is widely used industrially. In the cultivation of such aerobic microorganisms, aeration is generally essential, and if the amount of aeration is insufficient, the yield of fermentation products will not be sufficiently high. The cost required for this aeration accounts for a considerable proportion of the cost of producing useful substances by culturing microorganisms. Therefore, it would be extremely advantageous if the amount of aeration, ie, the amount of oxygen supplied, could be reduced without reducing the production of useful substances.

On the other hand, there has been an attempt to improve the production amount of useful substances by immersing an electrode in a culture solution of microorganisms and performing the culture while applying electricity. For example, Japanese Patent Publication No. 53-28992 describes a method for culturing glutamic acid-producing bacteria to produce glutamic acid, in which a cathode is installed in the culture solution,
An anode is installed in an electrolyte solution that is separated from this culture solution by an ion exchange membrane, and a soluble redox substance is added to the culture solution, which gives electrons to the glutamic acid production enzyme and reduces it, producing glutamic acid. A method to improve the ability is described. However, this method is not intended to reduce the amount of oxygen supplied, and it is not possible to reduce the amount of oxygen supplied without reducing the production amount of useful substances.

[Problem that the invention seeks to solve]

Therefore, an object of the present invention is to provide a method for culturing aerobic microorganisms in which the amount of oxygen supplied can be reduced without reducing the production amount of useful substances. As used herein, the term "orally useful substances" includes not only substances produced by microorganisms but also the microbial cells themselves.

[Means for solving problems]

As a result of various studies, the present inventor found that the above object can be achieved by installing an anode in the culture solution and culturing by applying a potential that can oxidize the respiratory system components of microorganisms to the anode, and the present invention. I was able to complete it.

The present invention is a method for culturing aerobic microorganisms, which comprises culturing by applying a potential capable of oxidizing respiratory system components of microorganisms to an anode placed in a culture solution.

Examples of aerobic microorganisms that can be cultured by the method of the present invention include aerobic bacteria, actinomycetes, yeast, filamentous fungi, and basidiomycetes. One or more of these types are inoculated into a medium containing carbon sources, nitrogen sources, etc. that are used for normal culture, and a potential is applied to the anode placed in the culture solution to supply oxygen or air. , or without feeding.

To carry out the present invention, three types of electrodes are available, each comprising an anode or working electrode, a cathode or counter electrode for passing a current, and a reference electrode serving as a potential reference, or two types of electrodes include only a working electrode and a counter electrode. Electrodes and the like are used.

The working electrode is an electrode made of a metal oxide semiconductor such as indium oxide, nickel oxide, tin oxide, or titanium oxide; or a carbonaceous conductive support such as graphite or carbon, or a metal support such as gold, platinum, silver, or nickel. Advantageously, electrodes are used in which the body surface is coated with a mediator or promoter.

The mediator is a redox substance such as methyl viologen or potassium ferricyanide, and is a substance that can mediate the transfer of electrons between a respiratory system component of a microorganism, that is, a cytochrome, and an electrode. Although the mediator may be present in the culture solution, it is advantageous to coat the surface of the conductive support as described above. The surface of the support can be coated by dissolving mesoiecous in a suitable solvent such as water and immersing the support in the solution to adsorb it onto the surface, or in the case of a mediator such as methyl viologen. is coated by electrolytic polymerization on the surface of the support, or in the case of a mediator such as potassium ferricyanide, a suitable polymer such as methanol of polyvinylpyridine is coated on the surface of the support in advance. After immersing the support in the solution and coating the surface with the polymer,
This can be carried out by various methods, such as immersing this polymer-coated support in a mediator solution to allow the mediator to be adsorbed onto the surface of the coated polymer.

In addition, the promoter has an affinity for both the conductive support and the respiratory system components of microorganisms, ie, cytochromes, and has the function of forming a certain space between the two and facilitating the transfer of electrons, that is, oxidation. It has the following. Such promoters include bivirimidin and the Journal of Electroanalytical Chemistry (J, [Electroanal, Chem.
, ) 178. (1984), pages 69-86, such as mercaptopyridine, disulfide, etc., and especially those listed as suitable promoters in Table 1, pages 73-74, can be used. To coat these promoters on the surface of a conductive support,
The support is placed in an aqueous solution of bromocou for a predetermined period of time, e.g.
After soaking for ~3 minutes to adsorb the promoter to the surface,
Just wash with water.

Although it is preferable to use the electrode made of a metal oxide semiconductor as it is, it may be used after being coated with the above-mentioned mediator.

As the counter electrode, any of the above semiconductor electrodes, conductive support electrodes, and electrodes in which these electrodes are coated with a mediator or promoter may be used, and there are no particular limitations.

In the present invention, both the working electrode and the counter electrode may be placed in the culture medium, and in order to prevent a reverse reaction at the counter electrode,
The working electrode may be placed in a culture tank filled with a culture solution, and the counter electrode may be placed in a chamber separated from the culture solution by an ion exchange membrane or the like. The reference electrode is used by connecting it to the culture solution with a salt bridge.

The potential applied to the anode is preferably a potential sufficient to oxidize the respiratory electron carrier, ie, cytochrome, of the microorganism to be cultured, and a potential that does not cause water decomposition.

That is, it is desirable that the potential be more negative than the oxidation-reduction potential of cytochrome, and more base than the oxygen overvoltage in water electrolysis.

〔Effect of the invention〕

According to the method of the present invention, the amount of oxygen supplied can be reduced without reducing the production amount of useful substances, and the production cost of useful substances can be reduced.

〔Example〕

The method of the present invention will be explained in more detail with reference to Examples below.

Example 1 Production of glutamic acid by Brevibacterium lactofermentum culture Glucose 100 g/A, KH2PO, l g
/ 1, JS[], l g / 1, 300 mf of medium containing soy acid hydrolyzate (as total nitrogen) 480 mg/j2 and biotin 300 r7B! Brevibacterium lactofermentum (Breviba
cterium lactofermentum) (A
A 5% seed culture of TCC13869) was inoculated and cultured in an aerated dish at a temperature of 31.5°C for 30 hours. Culture 8
0.2% Tween 60 was added at the beginning of the test. The ventilation volume is Allvvm (voluma ver.
sus m1nute), B: 0.2vvm.

C: 0.2vνm, and the pH was adjusted to 7.5 using ammonia gas. Stirring was performed at 100 Qrρm.

No electrodes were placed in cultures tfA and B. Indium oxide was deposited on the inner wall of the culture tank C in the form of a horizontal strip (width: approximately 12 C:m), a lead wire was drawn out from a part of the strip, and a platinum piece was used as a counter electrode. The platinum piece was placed in the culture solution. After the start of the culture, a potential of +300 mV was applied to the indium oxide electrode with respect to the hydrogen standard electrode, which was connected to the culture solution via a salt bridge. After the culture was completed, the culture products were analyzed.
The results shown in Table 1 were obtained.

In B, where the aeration rate was 175 times that of A, the production of total organic acids increased due to the insufficient supply of oxygen, and the production of the target glutamic acid decreased to about 73%.

On the other hand, C
In this case, even though the aeration rate is set to A of 115, the production of total organic acids does not increase, that is, there is no shortage of oxygen supply, and the desired production of glutamic acid is:
It can be seen that it is almost the same as A.

Example 2 Bacterial cell production of glucose by Romyces cerevisiae (baker's yeast) 50 g/12, KH2PO 45 g
/It, medium containing MgSOsIg/A' and Cornsteepley force-10 g/β (pt(6,0)3
00- was placed in glass culture tanks A, B, and C similar to those used in Example 1, and Saccharomyces cerevisiae x (Saccharomyces cerevisiae) was added to this medium.
cerevisiae) (F E RM P-2
897) (AJ 14457) was inoculated at 10%, and cultured with aeration and stirring at a temperature of 30°C for 24 hours. The ventilation amount is A: 2. Ovvm, B:Q, 5vv
m, C: 0.5vvm, pH is lN-
Adjusted to 6.0 using NaOH. Stirring is 600 rpm
I went with m. As in Example 1, a potential of +300 mV with respect to the water standard electrode was applied to the indium oxide electrode of C. The analysis results of the culture products are shown in Table 2.

In case B, where the aeration rate is set to A, the amount of ethanol produced increases due to the lack of oxygen supply, and the desired amount of bacterial cell production is approximately 50.
%. On the other hand, in case C, in which an anode was installed in the culture medium and a potential was applied, no ethanol production was observed, even though the aeration rate was set to 2 in A, resulting in a lack of oxygen supply. It can be seen that the production amount of the target bacterial cells is almost the same as Δ.

Example 3 Production of protease by filamentous fungus culture using mediator-coated electrodes Pyrolitic graphite manufactured by Union Carbide (diameter 5 mm, length 12 cm) was used as a conductive support, and poly-4-vinylpyridine was applied to it. A methanol solution was applied and dried. This was immersed in a potassium ferricyanide solution (pH 1.5) with a concentration of 10-2 mol/l for about 10 minutes at room temperature, and then thoroughly washed with water to prepare a q% coated mediator. Small 11-volume glass culture tanks A, B, and C were prepared, and the 20 electrodes described above were arranged vertically so as to be in contact with the inner wall surface of culture tank C, and each electrode was connected.

Further, as the counter electrode, a platinum piece was used as in Example 1, and it was placed in the culture solution.

Seed culture agar slant medium (glucose 1.0
%, peptone 0.2%, malt extract 0.1%, yeast extract 0.1%, agar 1.5%, pH 7.0) was inoculated with Aspergillus feinisis (ATCCI 4332) and incubated at 27°C for 10 days. Cultured. Collect spores and add 0.1M
The spores were suspended in phosphate buffer (p) 16.8) so that the number of spores was approximately 107/ml.

This suspension 30- was added to the main medium (adipron (soybean extract protein) 1.5%, lecithin for food additive 3) in each of the culture tanks.
．． 0%, KH2PO40.5%, pH6.6) 300
- and cultured with aeration at 27°C for 4 days without adjusting the pH. A potential of +300 mV was applied to C with respect to a hydrogen standard electrode. The ventilation amount is A:1. Ov
vm SB: 0.1vvm SC: 0.1vvm,
Stirring was performed at 700 rpm. The protease activity of the supernatant obtained by centrifuging the culture solution was measured according to a conventional method. The results are shown in Table 3.

Table 3 IU shows that in B, where the active aeration rate to produce 1 μg of tyrosine per minute is 1/10 that of A, the production amount of protease decreases to about 175, whereas the anode is placed in the culture medium, In C, where a potential is applied to this, the ventilation amount is 1710 that of A.
It can be seen that the production amount of protease is higher than that of A.

Example 4 Production of antibacterial substances by culturing actinomycetes using promoter-coated electrodes After polishing the surface of a gold electrode with a diameter of 4 mmφ and a length of 12 cm with alumina, bis(4-pyridine) disulfide at a concentration of 1 mmol/Il was applied. A promoter-coated electrode was prepared by immersing it in the solution for 2 minutes and then thoroughly rinsing with water. Example 3
Similarly, three small 11-volume glass culture tanks were prepared, and electrodes were placed in one of them in the same manner.

500ml! Culture medium (A) 3 with the following composition in Yosaka Lof flask
0rr11, inoculated with 1 platinum loop of Streptomyces glyceroincartanus 819C (FERM P4O10), and incubated at 30°C for 24 hours, 120 times/min, 7
Shaking culture (seed culture) was performed at an amplitude of cm. This culture solution is 30mj! 300mj of culture medium (B) with the following composition was added to each culture tank. and cultured with aeration at 30° C. for 50 hours without adjusting the pit. C
A potential of +300 mV was applied to the hydrogen standard electrode. The ventilation amount is A: 0.5vvm, B: 0.0
5vvm, C: 0.05vvm, stirring at 70
This was done at 0 rpm.

The culture solution was centrifuged, and the resulting supernatant was measured for antibacterial activity against Escherichia coli MP-2.

The results are shown in Table 4.

Table 4 B, which has an air flow rate of 1710, has an antibacterial activity of 20%.
However, even if the aeration rate is set to 1710, an anode is installed in the culture solution and a potential is applied to it.
It can be seen that this shows almost the same antibacterial activity as A.

Measuring method for antibacterial activity: A medium consisting of 1.75% Bacto Antibiotic Medium 3 (Difco product) and 1.0% agar (M3
After heat sterilizing the culture medium at 120° C. for 15 minutes, it is dispensed into 20-mL petri dishes and left to cool to prepare a plate culture medium.

On the other hand, peptone 0.5%, meat extract 0.5%, Naα 0
．． A medium consisting of 3% agar and 0.8% agar is heat sterilized at 120°C for 15 minutes. After that, it was kept in a constant temperature bath at 42°C, and when the temperature of the culture medium reached 42°C, 11 Tll of Escherichia coli MP-2, which had been previously cultured at 37°C for 17 hours, was added. 5 X
Add to the medium so that 10" cells are present. Take 2 ml with a pipette and add it to the surface of the M3 medium prepared in advance, and spread quickly and uniformly to solidify.

Dilute the test solution and make 0.06ml of the solution! Soak it into a Penoku disc (Toyo filter paper). This paper disk was placed on the preparation plate and heated at 37°C for 17 days.
Incubate for a time and measure the size of the inhibition circle created by the test solution. That is, P,<-・The distance from one end of the disk to the end of Escherichia coli MP-2 growth inhibition
The antibacterial activity of ml of test solution is expressed as U/-.

Claims (4)

[Claims] (1) A method for culturing aerobic microorganisms, which comprises culturing by applying a potential capable of oxidizing respiratory system components of microorganisms to an anode placed in a culture solution. (2) The method according to claim (1), wherein the anode is made of a metal oxide semiconductor. (3) The method according to claim (1), wherein the anode is an electrode coated with a mediator. (4) The method according to claim (1), wherein the anode is an electrode coated with a promoter.