JPH0522506B2 - - Google Patents

Abstract

PURPOSE:To carry out culture of anaerobe without necessitating particular inert gas, by supplying a culture medium of anaerobe with deoxygenated air obtained from a culture medium of aerobe. CONSTITUTION:Deoxygenated air is produced by repeatedly contacting air with a liquid containing an organic waste and aerobes (single species or mixture of plural species). A anaerobe is cultured in a liquid medium supplied with the deoxygenated air. The organic waste is preferably a filtrate of cultured product or microbial cell produced by the culture of anaerobe or a material obtained by removing produced substance from the above cultured product.

Description

[Detailed Description of the Invention] [Industrial Application Field] The present invention relates to a method and apparatus for culturing anaerobic bacteria;
In particular, the present invention relates to a culture method and apparatus suitable for liquid culture of anaerobic bacteria.

[Prior art]

Conventionally, aerobic bacteria have been used for fermentation production of useful substances. On the other hand, anaerobic bacteria, which have been little used until now, have different physiological activities, especially metabolism, from aerobic bacteria, so there is a strong possibility that they will be used in the future to produce useful substances that aerobic bacteria do not have. However, anaerobic bacteria are sensitive to molecular oxygen and cannot be cultured in air, so it is necessary to remove the dissolved oxygen in the liquid medium and the air in the gas phase of the culture tank before culturing.
Cultivation is carried out in batches, but conventionally an alkali salt of thioglycolic acid is added to the medium before culturing.
Furthermore, the air in the gas phase was replaced with a high-purity inert gas such as argon before culturing.

[Problem that the invention seeks to solve]

However, in the above-mentioned conventional anaerobic bacterial culture method, thioglycolic acid added to the medium is not only expensive, but also reduces the fermentation rate, although adding it shortens the time required to start fermentation after inoculation. It decreases and the fermentation completion time is extended. Furthermore, when organic wastes such as culture filtrate and bacterial cells are treated by methane fermentation treatment, a large amount of hydrogen sulfide gas is generated, which inhibits fermentation. In addition, gas replacement in the gas phase of a culture tank usually requires high-purity inert gas that is 10 times or more the volume of the culture tank.

The purpose of the present invention is to improve the drawbacks of the conventional anaerobic bacteria culture methods described above, and to provide a method for efficiently culturing anaerobic bacteria without using oxygen scavengers or inert gases, and a method thereof. The objective is to provide equipment used for implementation.

[Means for solving problems]

The inventors conducted intensive research with the aim of developing a new culture process for anaerobic bacteria that incorporates a new deoxidizing process. When I tried to culture anaerobic bacteria in batches in liquid, I mistakenly continued the culture without removing oxygen. As a result, the target anaerobic bacteria do not proliferate,
Instead, the aerobic bacteria that had been contaminated grew. When the gas composition of the gas phase was analyzed using a gas chromatograph, it was discovered that the oxygen concentration was significantly reduced. Furthermore, in a similar culture experiment, aerobic bacteria were cultured using the culture filtrate of anaerobic bacteria as a substrate while circulating the gas in the gas phase into the liquid using a tube pump, and the oxygen concentration decreased to 5 ppm. .
The discovery of this phenomenon led us to consider its application to the cultivation of anaerobic bacteria, and as a result, we arrived at the present invention.

A feature of the present invention is that aerobic bacteria are cultured using the remaining organic waste after separating the produced substances from the culture solution of anaerobic bacteria as a liquid culture medium, and the deoxygenated air generated at this time is used to cultivate the anaerobic bacteria. It is used as an inert gas for purging when starting the process.

A culture solution obtained by culturing anaerobic bacteria is separated into a culture filtrate and bacterial cells, and is used as a raw material for separating produced substances. At this time, the entire culture filtrate or bacterial cell slurry after separation is usually treated as waste by a method suitable for conventional concentrated organic waste treatment such as methane fermentation. In the present invention, a portion of these wastes is reused to generate purge gas during the cultivation of anaerobic bacteria. First, an inoculum of aerobic bacteria is added to a liquid containing waste, and the mixture is cultured through air. After removing the mist from the air that has passed through the liquid, it is forced into a compressor and stored under pressure. If the oxygen concentration of the stored gas is still higher than the target oxygen concentration, the flow of fresh air into the culture tank is stopped and the stored gas is circulated and aerated. The oxygen concentration that can be used as a purge gas before culturing anaerobic bacteria is 10 ppm or less, preferably 5 ppm.
The following is desirable. In liquid culture of anaerobic bacteria, the deoxygenated air described above is aerated into the liquid medium in a culture tank to remove dissolved oxygen in the liquid and expel air in the gas phase to the outside of the tank. The amount of gas used for aeration varies depending on the shape of the fermentation tank, the filling rate of the culture medium, the structure of the aeration system, etc., but it requires at least the same volume as the tank volume. After deoxidizing the tank, an inoculum solution of anaerobic bacteria is inoculated, and culture conditions such as temperature, pH, medium composition, and stirring speed are appropriately selected depending on the type of bacteria used.

The substrate for the anaerobic bacteria used in the present invention is not particularly limited as long as it is an organic substance discharged in the process after culturing the anaerobic bacteria, and may include culture filtrate, bacterial cells or crushed bacterial cells,
Alternatively, it may be the remaining culture filtrate or bacterial cells from which produced substances have been separated. As the aerobic bacteria, a single type of bacteria may be used, but a mixture of two or more types of bacteria may be used. The aerobic bacteria may be any type of bacteria, but for example, bacteria such as Bacillus subtilis and Escherichia coli, yeasts such as Satucharomyces cerevisiae, and mixed bacteria such as activated sludge are used. The culture temperature and pH are selected to be conditions suitable for the growth of the aerobic bacteria used. The anaerobic bacteria are not particularly limited, and obligate anaerobic bacteria such as Closturidium and Bifidobacterium can also be sufficiently applied.

After the cultivation is completed, the culture solution is transferred to a process for separating produced substances. Usually, it is separated into a filtrate and a bacterial cell slurry using a centrifuge, and the produced substances are extracted and separated from either of them. When extracting and separating produced substances,
A portion of the by-product organic waste is returned and reused as a medium for aerobic bacterial culture.

A system diagram of the anaerobic bacteria culturing apparatus of the present invention is shown in FIG.

The aerobic culture tank 4 has an aeration tube at the bottom of the tank, and by aerating oxygen-containing gas into the tank through the aeration tube, aerobic bacteria can be cultured. This culture tank is equipped with a temperature control device and a PH control device, making it possible to culture at a constant temperature and pH. As the aerobic bacteria to be cultured in the culture tank, bacteria such as those of the genus Bacillus, which grow either aerobically or anaerobically, are used. The anaerobic microbial cell slurry 30 and culture filtrate 28, which will be described later, are used as nutrients for the culture, and are supplied to the aerobic culture tank via the anaerobic microbial cell transfer piping 32 or the culture filtrate transfer piping 33. Aerobic culture is performed using a batch culture method. At the initial stage of culture, air is taken in through the air intake pipe 1, pressurized by the blower 2, and then pressurized into the aerobic culture tank 4 via the pipe 3 for aeration. After the culture of aerobic bacteria enters the logarithmic growth phase, the exhaust gas discharged from the aerobic culture tank is guided to a cyclone 6 through a pipe 5 to remove mist. Next, the exhaust gas is led to a filter 9 through a transfer pipe 8, and after removing solids such as bacterial bodies and mist from the exhaust gas,
The deoxygenated air is pressurized by a compressor 11 and stored in a pressurized storage tank 13 . Aerobic culture is continued until the amount of gas stored in the deoxygenated air pressurized storage tank 13 reaches the set amount. Once the set amount has been stored,
The air intake pipe 1 is closed, and the deoxygenated air stored in the deoxygenated air pressurized storage tank 13 is supplied to the aerobic bacteria culture tank 4 from the deoxygenated air transfer pipe 14.
The oxygen concentration in the gas in the deoxygenated air pressurized storage tank 13 is measured, and the deoxygenated air is circulated between the aerobic bacteria culture tank 4 and the deoxygenated air pressurized tank 13 until the oxygen concentration falls below the set value. to obtain deoxygenated air with a predetermined oxygen concentration.

The anaerobic bacteria culture tank 18 has an aeration pipe at the bottom of the tank, and the aeration pipe is connected to the aseptic deoxygenated air transfer pipe 1
7, connected to a deoxygenated air pressurized storage tank 13 via a filter 16 and a deoxygenated air transfer pipe 15. In addition, this culture tank is equipped with a stirring device, temperature control device, automatic pH control device, and in-tank sterilization equipment, allowing pure culture under constant temperature and pH conditions. To start anaerobic culture, a liquid medium having a composition suitable for the bacteria to be cultured is placed in the anaerobic bacteria culture tank 18, and the inside of the tank is sterilized. The sterilization method is not particularly limited, and it is sufficient to use a commonly used high-pressure steam sterilization method. If there are substances unstable at high temperatures in the medium composition, sterilization by filtration may be used. After sterilizing the medium, set the temperature of the medium to the culture temperature. Next, deoxygenated air stored in the deoxygenated air pressurized storage tank 13 and sterilized by the filter 16 is vented into the anaerobic bacteria culture tank 18 to remove oxygen in the culture tank.
The amount of ventilation of deoxygenated air varies depending on the filling rate of the culture medium, but it is preferably at least the same volume as the culture tank volume. After removing the oxygen in the culture tank, inoculate the anaerobic bacteria inoculum and maintain the temperature at the specified temperature.
Start the anaerobic culture at pH, stirring speed. When fermentation gas is generated as the culture begins and anaerobic bacteria proliferate, the waste gas 21 is transferred to the waste gas transfer pipe 2.
0, it is discharged outside the tank. In addition, to prevent air from flowing back into the tank from the waste gas transfer piping,
It is desirable to provide a backflow prevention means in the middle of the piping 20. At the time when the anaerobic bacteria culture is completed, the anaerobic bacteria culture liquid is guided to the centrifuge 24 via the culture liquid transfer pipes 22 and 23 by the anaerobic bacteria culture liquid transfer pump 34, and is separated from the culture filtrate 28 and anaerobic bacteria. The bacterial cells are separated into a slurry 30. Culture filtrate 28
is temporarily stored in the culture filtrate storage tank 27 via the transfer piping 26, and the anaerobic bacterial cell slurry 30 is temporarily stored in the anaerobic bacterial cell slurry storage tank 31 via the transfer piping 29. If the produced substance is contained in the culture filtrate, the culture filtrate is transferred, and if it is contained in the bacterial slurry, the bacterial slurry is transferred to a produced substance extraction and separation process (not shown), and the desired product is extracted. obtain. The unnecessary culture filtrate or bacterial cell slurry is transferred to the transfer pipe 32 or 3.
3 is supplied to the aerobic bacteria culture tank 4 and used as a nutrient source for aerobic bacteria. Furthermore, organic waste produced as a by-product from the useful substance extraction and separation process can also be used as a nutrient source for aerobic bacterial culture.

[Effect]

By culturing aerobic bacteria such as Bacillus subtilis and Satucharomyces cerevisiae using the culture waste of anaerobic bacteria as a nutrient source, residual oxygen concentration of 1
Deoxygenated air with a concentration of less than ×10 ^-4 % is obtained, and anaerobic bacteria such as Crosssilidium can be efficiently cultured using this deoxygenated air. Then, substances produced by the anaerobic bacteria are recovered from the culture. Furthermore, the culture fluid of the anaerobic bacteria or the remaining culture waste from which the substances produced therefrom are recovered, are used cyclically as a nutrient source for the anaerobic bacteria, as described above. The growth rate of anaerobic bacteria using this method and device is significantly higher than that using impure cylinder nitrogen or oxygen scavengers, as shown in the following example, and is comparable to that when using expensive high-purity nitrogen. It was something to do.

[Examples and comparative examples]

Example 1 Soluble starch 1.5%, monopotassium phosphate 0.7
%, Sodium Phosphate 0.35%, Magnesium Sulfate
Using a synthetic medium containing 0.001% polypeptone, 0.5% polypeptone, and 0.5% yeast extract, under anaerobic conditions, 60°C, PH6.0.
Clostridium bacterium RS-0001 (Microtechnology Research Deposit No. 7918) was cultured at , and a culture solution was prepared.
This culture solution was centrifuged to prepare a culture filtrate and a bacterial cell slurry. Next, add 1.5 of the above culture filtrate to an internal volume of 30
Bacillus was placed in a stainless steel culture tank.
Inoculate the culture solution 750 2′ of S. subtilis as a seed,
Aerobic culture was performed at 30°C and pH 6.5 with an aeration rate of 0.5/min.
In addition, the exhaust gas from the culture tank is filtered by a cyclone with an internal volume of 10 mm and a 0.45 μm filter, after which mists and solids are removed, and then a compressor is used to reduce the exhaust gas to 10 mm.
It was compressed to kg/cm ² and stored in a pressurized tank with an internal volume of 1 m ³ . After the internal pressure of the storage tank reached 10 Kg/cm ² , the stored gas was circulated through the culture tank to consume oxygen in the gas. The oxygen concentration of the compressed deoxygenated air 24 hours after the start of the culture was measured and found to be 2 ppm.

On the other hand, 1 m ³ of the synthetic medium described above was placed in a culture tank with an effective volume of 1.5 m ³ , and steam was blown into the culture tank to raise the temperature to 121°C.
Autoclave sterilization was performed for 20 minutes. After cooling to 60℃,
9 m ³ of deoxygenated air passed through a sterilization filter was vented from the aeration tank at the bottom of the culture tank. The oxygen concentration in the gas phase after ventilation was 4 ppm. The dissolved oxygen concentration in the medium is
It was below 0.01ppm. This was inoculated with 10 inoculum of the Clostridia bacterium RS-0001, and 60
The cells were cultured anaerobically at ℃ and pH 6.0. After the start of the culture, bacterial cell proliferation started immediately and ended after about 16 hours. 19
After incubation for an hour, the culture solution was centrifuged, and the culture filtrate was
A bacterial slurry of 90% was obtained.

Comparative example 1 Soluble starch 1.5 in a culture tank with an effective volume of 1.5 ^m3
%, potassium phosphate 0.7%, sodium phosphate 0.35%, magnesium sulfate 0.001%, polypeptone 0.5%, yeast extract 0.5% 1 m ³ of synthetic medium
The container was then sterilized using high-pressure steam at 121°C for 20 minutes by blowing steam into the container. After cooling to 60° C., 9 m ³ of nitrogen (oxygen concentration 300 ppm) passed through a sterilization filter was aerated through the aeration tube at the bottom of the culture tank. The oxygen concentration in the gas phase after ventilation was 327 ppm. Furthermore, the dissolved oxygen concentration in the medium was 0.15 ppm. Add 10 inoculum of Clostridium bacteria RS-0001 to this.
It was inoculated and cultured anaerobically at 60°C and pH 6.0. Figure 2 shows the time course of bacterial cell proliferation at that time. After the start of culture, proliferation begins after a proliferation induction period of about 10 hours.
After 22 hours, the bacteria had grown to a concentration of 3.0 g/min.
After 24 hours of culture, centrifuge the culture solution and collect the culture filtrate.
950 and bacterial cell slurry 50 were obtained.

Comparative Example 2 Soluble starch 1.5% in a culture tank with an effective volume of 1.5 ^m3 .
Potassium phosphate 0.7%, sodium phosphate dibasic
1 m ³ of a synthetic medium containing 0.35% magnesium sulfate, 0.001% magnesium sulfate, 0.5% polypeptone, and 0.5% yeast extract was poured into the tube, and steam was blown into the tube to perform high-pressure steam sterilization at 121° C. for 20 minutes. After cooling to 60°C, 30 m ³ of nitrogen (oxygen concentration 300 ppm) passed through a sterilization filter was aerated through the aeration pipe at the bottom of the culture tank. The oxygen concentration in the gas phase after ventilation was 310 ppm. The dissolved oxygen concentration in the medium was 0.10 ppm. This was inoculated with 10 inoculum solutions of Clostridium bacteria RS-0001 and cultured anaerobically at 60°C and pH 6.0. Figure 2 shows the time course of bacterial cell proliferation at that time. After starting culture,
It took 9 hours for the bacteria to start growing, and after 21 hours, the bacteria reached a concentration of 3.0g/24.
After incubation for an hour. Centrifuge the culture solution and collect the culture filtrate 930
and a bacterial cell slurry of 70% was obtained.

Comparative Example 3 Soluble starch 1.5% in a culture tank with an effective volume of 1.5 ^m3 .
Potassium phosphate 0.7%, sodium phosphate dibasic
0.35% magnesium sulfate, 0.001% magnesium sulfate, 0.5% polypeptone, 0.5% yeast extract, and ^0.3 % sodium thioglycolate. After cooling to 60℃, add nitrogen (oxygen concentration 1 ppm) through a sterilization filter from the aeration pipe at the bottom of the culture tank9
m ³ was vented. The oxygen concentration in the gas phase after ventilation is
It was 280ppm. The dissolved oxygen concentration in the medium is
It was 0.1 ppm. This was inoculated with 10 inoculum of the Clostridia bacterium RS-0001, and incubated at 60°C.
Anaerobic culture was performed at pH 6.0. Figure 2 shows the time course of bacterial cell proliferation at that time. After the start of culture, it takes 11 hours for the bacteria to begin to grow, and after 28 hours the bacterial concentration
The growth was completed to 2.3g/. After 30 hours of culture,
The culture solution was centrifuged to obtain 960 ml of culture filtrate and 40 ml of bacterial cell slurry.

Comparative Example 4 Soluble starch 1.5% in a culture tank with an effective volume of 1.5 ^m3 .
Potassium phosphate 0.7%, sodium phosphate dibasic
1 m ³ of a synthetic medium containing 0.35% magnesium sulfate, 0.001% magnesium sulfate, 0.5% polypeptone, and 0.5% yeast extract was poured into the tube, and steam was blown into the tube to perform high-pressure steam sterilization at 121° C. for 20 minutes. After cooling to 60°C, 9 m ³ of nitrogen (oxygen concentration 1 ppm) passed through a sterilization filter was aerated through the aeration tube at the bottom of the culture tank. The oxygen concentration in the gas phase after ventilation was 3 ppm. The dissolved oxygen concentration in the medium was below 0.01 ppm. This was inoculated with 10 inoculum solutions of Clostridium bacteria RS-0001 and cultured anaerobically at 60°C and pH 6.0. Figure 2 shows the time course of bacterial cell proliferation at that time. After starting culture,
Bacterial growth started immediately, and after 15 hours the bacterial concentration was 3.5g/
It grew until then and then stopped. After culturing for an hour, the culture solution is centrifuged, and the culture filtrate 905 and bacterial cell slurry 95
I got it.

As described above, in Example 1 according to the present invention, oxygen replacement was performed with impure cylinder nitrogen as shown in Comparative Example 1, replacement with a large amount of impure cylinder nitrogen as in Comparative Example 2, and replacement with a large amount of impure cylinder nitrogen as in Comparative Example 3. The growth rate is higher than in any case where a reducing agent is added.
In addition, the effects of shortening culture time and increasing bacterial cell concentration were observed. In order to obtain the same culture efficiency as in this example, as shown in Comparative Example 4, a large amount of expensive high-purity nitrogen must be used.

Example 2 Soluble starch 1.5%, potassium monophosphate 0.7
%, Sodium Phosphate 0.35%, Magnesium Sulfate
Using a synthetic medium containing 0.001% polypeptone, 0.5% polypeptone, and 0.5% yeast extract, under anaerobic conditions, 60°C, PH6.0.
Clostridium bacterium RS-0001 (Feiberal Institute deposited No. 7918) was cultured and a culture solution was prepared. This culture solution was centrifuged to prepare a culture filtrate and a bacterial cell slurry. Next, put the above culture filtrate 10 into a stainless steel culture tank with an internal volume of 20, and add the E. coli culture solution to it.
500 2′ was inoculated as a seed, aeration rate was 0.5/min,
The cells were cultured aerobically at 37°C and pH 6.5. In addition, the exhaust gas from the culture tank is a cyclone with an internal volume of 5 and a 0.45μ
After removing mist and solid matter with a filter, it is compressed to 10Kg/ ^cm2 using a compressor.
It was stored in a pressurized tank with an internal volume of 1 m ³ . After the internal pressure of the storage tank reached 10 Kg/cm ² , the stored gas was circulated through the culture tank to consume oxygen in the gas. The oxygen concentration of the compressed deoxygenated air was measured 15 hours after the start of the culture and found to be 3 ppm.

On the other hand, fill a culture tank with an effective volume of 1.0 m ³ with 0.7 m ³ of the synthetic medium described above, blow steam into it, and
High-pressure steam sterilization was performed at ℃ for 20 minutes. After cooling to 60°C, 8 m ³ of deoxygenated air passed through a sterilization filter was aerated through the aeration tube at the bottom of the culture tank. The oxygen concentration in the gas phase after ventilation was 5 ppm. The dissolved oxygen concentration in the medium was below 0.01 ppm. Seven inoculum solutions of Clostridium bacteria RS-0001 were inoculated into this,
Anaerobic culture was performed at 60°C and pH 6.0. Figure 2 shows the time course of bacterial cell proliferation at that time. Bacterial growth begins immediately after culture begins, and the maximum bacterial concentration reaches 3.4 after 14 hours.
Proliferation was terminated upon reaching g/g/. After 18 hours of incubation,
The culture solution was centrifuged to obtain a culture filtrate 630 and a bacterial cell slurry 70.

Example 3 Soluble starch 1.5%, potassium phosphate 0.7
%, Sodium Phosphate 0.35%, Magnesium Sulfate
Using a synthetic medium containing 0.001% polypeptone, 0.5% polypeptone, and 0.5% yeast extract, under anaerobic conditions, 60°C, PH6.0.
Clostridium bacterium RS-0001 (Feiberal Institute deposited No. 7918) was cultured and a culture solution was prepared. This culture solution is centrifuged to prepare a culture filtrate and bacterial cell slurry.The above culture filtrate 15 is placed in a stainless steel culture tank with an internal volume of 30, inoculated with culture solution 750 2' of Satucharomyces siervisiae as a seed, and aerated. amount
Aerobic culture was performed at 0.5/min, 30°C, and PH5.5. In addition,
After removing mist and solid matter from the exhaust gas from the culture tank using a cyclone with an internal volume of 10 and a 0.45 μm filter, it is compressed to 10 kg/cm ² by a compressor and then transferred to a pressurized tank with an internal volume of 1 m ³ . I stored it.
After the internal pressure of the storage tank reached 10 Kg/cm ² , the stored gas was circulated through the culture tank to consume oxygen in the gas. The oxygen concentration of the compressed deoxygenated air 24 hours after the start of the culture was measured and found to be 4 ppm.

On the other hand, 1 m ³ of the synthetic medium described above was placed in a culture tank with an effective volume of 1.5 m ³ , and steam was blown into the culture tank to raise the temperature to 121°C.
Autoclave sterilization was performed for 20 minutes. After cooling to 60℃,
9 m ³ of deoxygenated air passed through a sterilization filter was vented through the aeration tube at the bottom of the culture tank. The oxygen concentration in the gas phase after ventilation was 5 ppm. The concentration of dissolved acid in the medium is
It was below 0.01ppm. This was inoculated with 10 inoculum of the Clostridia bacterium RS-0001, and 60
The cells were cultured anaerobically at ℃ and pH 6.0. Figure 2 shows the time course of bacterial cell proliferation at that time. After the start of culture, bacterial cells begin to proliferate immediately, reaching a maximum bacterial concentration of 3.5 after 15 hours.
The cells grew to 1.9 g/g/g/g/g/g/g/g/g/g/g/g/g/g/g/g/g/g/g/g/g/g/g/g/g/g/g/g/g/g/g/g/g/g/g/g/g/g/g/g/g/g/g/g/g/g/g/g/g/g/sg/g/g/g/g/g/g/g/g/g/g/g/g/g/g/g/g/g/g/g/g/g/g/g/g/g/g/g/g/g/g/g/g/g/g/g/g/g/g/g/g/g/g/g/g/g/g/g/g/g/g/g/g/g/g/g/g/g/g/g/g/g/g/g/g/g/g/g/g/g/g/g/g/g/. After culturing for 18 hours, the culture solution was centrifuged, and the culture filtrate 908 and bacterial cell slurry were separated.
Got 92.

Example 4 Soluble starch 1.5%, monopotassium phosphate 0.7
%, Sodium Phosphate 0.35%, Magnesium Sulfate
Using a synthetic medium containing 0.001% polypeptone, 0.5% polypeptone, and 0.5% yeast extract, under anaerobic conditions, 60°C, PH6.0.
Clostridium bacterium RS-0001 (Feiberal Institute deposited No. 7918) was cultured and a culture solution was prepared. This culture solution was centrifuged to prepare a culture filtrate and a bacterial cell slurry. Next, the above culture filtrate 10 was placed in a stainless steel culture tank with an internal volume of 30, and activated sludge 10 was inoculated as a seed culture at an air flow rate of 1.0/min, 25°C, and pH 6.5.
Cultured aerobically. In addition, the exhaust gas from the culture tank is
After removing mist and solid matter using a cyclone with an internal volume of 10 and a 0.45 μm filter, it was compressed to 10 Kg/cm ² using a compressor and stored in a pressurized tank with an internal volume of 1 m ³ . The internal pressure of the storage tank is 10Kg/cm ²
After reaching this point, the stored gas was circulated through the culture tank to consume the oxygen in the gas. As a result of measuring the oxygen concentration of compressed deoxygenated air 24 hours after the start of culture,
It was 6ppm.

On the other hand, 1 m ³ of the synthetic medium described above was placed in a culture tank with an effective volume of 1.5 m ³ , and steam was blown into the culture tank to raise the temperature to 121°C.
Autoclave sterilization was performed for 20 minutes. After cooling to 60℃,
9 m ³ of deoxygenated air passed through a sterilization filter was vented through the aeration tube at the bottom of the culture tank. The oxygen concentration in the gas phase after ventilation was 7 ppm. The oxygen concentration in the medium is
It was below 0.01ppm. This was inoculated with 10 inoculum of the Clostridia bacterium RS-0001, and 60
The cells were cultured anaerobically at ℃ and pH 6.0. Figure 2 shows the time course of bacterial cell proliferation at that time. After the start of culture, bacterial cells begin to proliferate immediately, reaching a maximum bacterial concentration of 3.5 after 15 hours.
The culture was completed when the cells proliferated to 1.5 g/g/g/g/g/g/g/g/g/g/g/g/g/g/g/g/g/g/g/g/g/g/g/g/g/g/g/g/g/g/g/g/g/g/g/g/g/g/g/g/g/g/g/g/g/g/g/g/g/g/g/g/g/g/g/g/g/g/g/g/g/g/g/g/g/g/g/g/g/g/g/g/g/g/g/g/; After culturing for 15 hours, the culture solution was centrifuged to obtain culture filtrate 920 and bacterial cell slurry 30.

Example 5 Soluble starch 1.5%, potassium phosphate 0.7
%, Sodium Phosphate 0.35%, Magnesium Sulfate
Using a synthetic medium containing 0.001% polypeptone, 0.5% polypeptone, and 0.5% yeast extract, under anaerobic conditions, 60°C, PH6.0.
Clostridium bacterium RS-0001 (Feiberal Institute deposited No. 7918) was cultured and a culture solution was prepared. This culture solution was centrifuged to prepare a culture filtrate and a bacterial cell slurry. Next, the above culture filtrate 10 and bacterial cell slurry 5
was placed in a stainless steel culture tank with an internal volume of 30 mm, inoculated with culture solution 750 2' of Bacillus subtilis as a seed, and aerobically cultured at 30° C. and pH 6.5 at an air flow rate of 0.5/min. The exhaust gas from the culture tank is used to remove mist and solid matter using a cyclone with an internal volume of 10 and a 0.45 μm filter, and then compressed to 10 Kg/cm ² by a compressor and then pressurized with an internal volume of 1 m ³ . stored in a tank. After the internal pressure of the storage tank reaches 10Kg/ ^cm2 , the stored gas is circulated through the culture tank.
The oxygen in the gas was consumed. As a result of measuring the oxygen concentration of compressed deoxygenated air 24 hours after the start of culture,
It was 1ppm.

On the other hand, 1 m ³ of the synthetic medium described above was placed in a culture tank with an effective volume of 1.5 m ³ , and steam was blown into the culture tank to raise the temperature to 121°C.
Autoclave sterilization was performed for 20 minutes. After cooling to 60℃,
9 m ³ of deoxygenated air passed through a sterilization filter was vented through the aeration tube at the bottom of the culture tank. The oxygen concentration in the gas phase after ventilation was 3 ppm. The dissolved oxygen concentration in the medium is
It was below 0.01ppm. This was inoculated with 10 inoculum of the Clostridia bacterium RS-0001, and 60
The cells were cultured anaerobically at ℃ and pH 6.0. Figure 2 shows the time course of bacterial cell proliferation at that time. Bacterial growth begins at the same time as culture begins, and the maximum bacterial concentration reaches 3.6 g/14 hours later.
The culture was completed when the cells proliferated until the end of the culture. After culturing for 17 hours, the culture solution was centrifuged and the culture filtrate 940 and bacterial cell slurry were separated.
Got 60.

[Effects of the present invention]

According to the present invention, anaerobic bacteria can be cultured at a high concentration in a short time without adding an expensive reducing agent that inhibits growth or using a large amount of high-purity inert gas. .

[Brief explanation of the drawing]

FIG. 1 shows the system of the anaerobic bacteria culturing apparatus of the invention, and FIG. 2 shows the time course of cell growth in the anaerobic bacteria culture according to the invention. 1...Air intake piping, 2...Blower, 3...
Air and deoxygenated air transfer piping, 4...Aerobic bacteria culture tank, 5...Deoxygenated air transfer piping, 6...Cyclone,
7...Aerobic bacteria culture solution, 8...Deoxygenated air transfer piping,
9... Filter, 10... Deoxygenated air transfer piping, 11...
Compressor, 12...Deoxygenated air transfer piping, 13
...Deoxygenated air pressurized storage tank, 14...Deoxygenated air transfer piping, 15...Deoxidized air transfer piping, 16...Filter,
17... Aseptic processing deoxygenated air transfer piping, 18... Anaerobic bacteria culture tank, 19... Anaerobic bacteria culture solution, 20... Waste gas transfer piping, 21... Waste gas, 22... Anaerobic bacteria culture solution transfer piping, 23... Anaerobic bacteria culture solution transfer piping, 2
4...Centrifugal separator, 25...Recovered liquid transfer piping, 26...
Culture filtrate transfer piping, 27... Culture filtrate storage tank, 28...
Culture filtrate, 29...anaerobic bacterial cell slurry transfer piping,
30... Anaerobic bacterial cell slurry, 31... Anaerobic bacterial cell slurry storage tank, 32... Anaerobic bacterial cell slurry transfer piping, 33... Culture filtrate transfer piping, 34... Pump for anaerobic bacterial culture transfer, A... Implementation Culture curve described in Example 1, B... Culture curve described in Example 2, C... Culture curve described in Example 3, D... Culture curve described in Example 4, E... Culture curve described in Example 5, F... Comparison Culture curve according to Example 1, G: Culture curve according to Comparative Example 2, H: Culture curve according to Comparative Example 3, I: Culture curve according to Comparative Example 4.

Claims (1)

[Claims] 1. Air is repeatedly brought into contact with a liquid containing organic waste in the presence of aerobic bacteria, deoxygenated air obtained by the contact is aerated into a liquid medium, and the liquid medium is anaerobic. A method for culturing anaerobic bacteria characterized by culturing sexual bacteria. 2 Reduce the residual oxygen concentration of deoxygenated air to 1×10 ^-4 % (V/
V) The method for culturing anaerobic bacteria according to claim 1, which is characterized by the following. 3. The method for culturing anaerobic bacteria according to claim 1, characterized in that a single bacterium or a mixed bacterium is used as the aerobic bacterium. 4. Claim No. 4, characterized in that the organic waste is a culture filtrate or bacterial cells produced by culturing anaerobic bacteria, a culture filtrate from which produced substances have been removed, or a mixture of one or more types of bacterial cells. The method for culturing anaerobic bacteria according to item 1. 5 An aerobic bacteria culture tank in which a liquid containing organic waste generated by culturing anaerobic bacteria is brought into contact with air in the presence of anaerobic bacteria, and the air that has come into contact with the liquid in the culture tank is collected and repeatedly combined with the liquid. A deoxygenated air transfer pipe for contact, a storage tank for storing the deoxygenated air obtained in the aerobic bacteria culture tank under pressure, and a culture medium containing the stored deoxygenated air obtained in the storage tank. A deoxygenated air transfer pipe that ventilates the tank, an anaerobic bacteria culture tank that inoculates anaerobic bacteria into a liquid medium through which deoxygenated air is aerated, and cultivates it under anaerobic conditions; A centrifugal separator that separates and collects the culture filtrate and bacterial cell slurry containing the produced substances in bacterial culture, and the culture filtrate obtained by the centrifugal separator or the remaining culture filtrate from which the bacterial cells or produced substances are recovered. It is combined with an anaerobic bacterial culture filtrate or bacterial slurry transfer tube that returns organic waste consisting of one or more types of bacterial cells to the aerobic bacterial culture tank as a substrate for aerobic bacterial culture. Characteristic anaerobic bacteria culturing device.