Apparatus for culturing microorganisms

Abstract: A method for producing L-sorbose by microbiological oxidation of D-sorbitol which comprises adding D-sorbitol to a culture liquid in such a concentration that it does not go up to over about 5% during and after the growth phase of a microorganism used, while circulating a culture exhaust gas enriched with oxygen gas, and releasing a part of said exhaust gas out of the system. An apparatus for culturing microorganisms suitable for the method is also disclosed.

Description: Field of the Invention The present invention relates to an apparatus for culturing microorganisms suitable for such a method. Background of the Invention L-Sorbose is one of naturally occurring ketohexoses, which is contained in juices of rowan tree, etc., and it is an important substance as a raw material in vitamin C synthesis. L-Sorbose is produced by fermentation wherein D-sorbitol is oxidized by microorganisms, for example, bacteria of the genus Gluconobacter . According to a conventional fermentation production of L-sorbose, a yield of conversion of the raw material, D-sorbitol, into L-sorbose is at highest about 93%, and there are formed considerable amounts of by-products such as 5-ketofructose, D-fructose, 2-ketogluconic acid, etc. In order to reduce the raw material cost of vitamin C, it is requested to increase the yield of conversion of D-sorbitol into L-sorbose to more than that in a conventional process and to inhibit formation of the above by-products to as little as possible. In our European Patent Application No. 233050 (A2), there is disclosed that L-sorbose can be produced in a higher yield from D-sorbitol by microbiological oxidation using a microorganism which belongs to the genus Gluconobacter and which is decreased in the ability to grow with D-sorbitol as the single carbon source compared with that of its parent strains. By the way, when the concentration of D-sorbitol in a culture medium goes up to over about 5% during and after the growth phase of the microorganisms, they are inhibited due to both high concentrations of the substrate (D-sorbitol) and the product (L-sorbose), which results in delay of the rate of oxidation. Accordingly, it is necessary to control the concentration of D-sorbitol below about 5%. However, when the concentration of the substrate is taken as a rate-determining step, there is a problem that the formation of by-products (fructose, 2-ketogluconic acid, etc.) is increased. In addition, in this type of a production method, sometimes, a culture exhaust gas containing oxgen in a high concentration is recovered by a compressor, and it is circulated and reused in a culture liquid to save resources. In this case, carbon dioxide gas generated by respiration of the microorganisms is accumulated in the exhaust gas and, when the concentration of carbon dioxide gas goes up to over 10%, the growth rate of the microorganisms and the rate of oxidation are strongly inhibited. Accordingly, in a conventional method, there is employed carbon dioxide gas removing means using a adsorbent such as sodium hydroxide, activated charcoal, etc. However, when such means are employed, there are many problems such as an increase in the production cost because of the necessity of an adsorption column, an adsorbent and the like and further necessity of maintenance thereof. Japanese Patent Kokoku No. 61-5709 discloses a method and an apparatus for culturing microorganisms such as fungi. In this publication, a gas enriched with oxygen is prepared from air by adsorbing nitrogen therein, and is used to control the partial pressure of carbon dioxide gas in a culture tank as well as to control the concentration of dissolved oxygen in a culture liquid. Objects of the Invention An object of the present invention is to reduce the production cost of L-sorbose by removing carbon dioxide gas in a culture exhaust gas to be reused without using any adsorbent to control the partial pressure of carbon dioxide gas properly. The object and advantage of the present invention will become apparent to those skilled in the art from the following description with reference to the accompanying drawings. Brief Description of Drawings Fig. 1 is a graph illustrating the relationship between an initial charge concentration of D-sorbitol and a specific growth rate; Fig. 2 is a graph illustrating the relationship between a concentration of D-sorbitol and a production rate of L-sorbose; Fig. 3 is a graph illustrating the relationship between a partial pressure of carbon dioxide gas and a specific growth rate; Fig. 4 is a graph illustrating the relationship between a partial pressure of carbon dioxide gas and a production rate of L-sorbose; and Fig. 5 is a general view of a preferred example of a culturing apparatus of the present invention. Summary of the Invention The present invention provides an apparatus for culturing microorganisms, particularly, those having L-sorbose producing ability, which comprises, a culture tank for culturing the microorganism equipped with a stirrer, a gas inlet provided at the lower part of the tank and an exhaust vent provided at the upper part of the tank; a substrate feeding device for adding a culture substrate to said culture tank; an oxygen supply device comprising a liquid oxygen storage tank and an evaporator for supplying said culture tank with a culture exhaust gas which has been enriched with oxygen gas; a dissolved oxygen concentration detector for detecting dissolved oxygen in a culture liquid in said culture tank to output a signal of the concentration of dissolved oxygen; a dissolved oxygen controller for controlling supply of dissolved oxygen by detecting said signal of the concentration of dissolved oxygen; a carbon dioxide gas measuring device for measuring the partial pressure of carbon dioxide gas in a culture exhaust gas, which is provided to an exhaust gas pipe from said culture tank; an exhaust gas release device for releasing a part of the culture exhaust gas out of the system by detecting said partial pressure of carbon dioxide gas; a conduit for circulating the remainder of said culture exhaust gas into the gas inlet of the culture tank; and an air supply device for supplying said culture tank with air. The yield of L-sorbose can be improved in comparison with a conventional method by using the above apparatus. That is, a yield of L-sorbose based on D-sorbitol is not more than about 93% in a conventional method, whereas, in the present invention, the yield can be increased to 2 to 3% more than that of the conventional method. Further, the production of by-products such as D-fructose, 2-ketogluconic acid, 5-ketofructose and the like can be minimized. Further, according to the present invention, a high concentration of D-sorbitol can be oxidized to L-sorbose within a short period of time and the yield per batch can be improved because the concentration of D-sorbitol in a culture medium during and after the growth phase is controlled so that it does not go up to over 5%. Furthermore, in the culture apparatus of the present invention, the exhaust gas is reused to save resources to enhance its economic advantages. Further, the apparatus of the present invention can be simplified and is advantageous in view of maintenance thereof because carbon dioxide gas in the exhaust gas is diluted and a part of the exhaust gas is released out of the system instead of using an adsorbent as in a conventional apparatus. By the above various advantages, L-sorbose the cost of which occupies a very high proportion in the raw material cost of vitamin C production can be supplied economically in comparison with a conventional method and, therefore, the production cost of vitamin C which has wide utility in medicines, food and the like can be reduced. Detailed Description of the Invention The present invention provides an apparatus for culturing microorganisms which is suitable for carrying out the method for producing L-sorbose as described above. That is, the present invention provides an apparatus for culturing a microorganism which comprises, a culture tank for culturing the microorganism equipped with a stirrer, a gas inlet provided at the lower part of the tank and an exhaust vent provided at the upper part of the tank; a substrate feeding device for adding a culture substrate (D-sorbitol) to said culture tank; an oxygen supply device comprising a liquid oxygen storage tank and an evaporator for supplying said culture tank with a culture exhaust gas which has been enriched with oxygen gas; a dissolved oxygen concentration detector for detecting dissolved oxygen of a culture liquid in said culture tank to output a signal of the concentration of dissolved oxygen; a dissolved oxygen controller for controlling supply of dissolved oxygen by detecting said signal of the concentration of dissolved oxygen; a carbon dioxide gas measuring device for measuring a partial pressure of carbon dioxide gas in a culture exhaust gas, which is provided to an exhaust gas pipe from said culture tank; an exhaust gas release device for releasing a part of the culture exhaust gas out of the system by detecting said partial pressure of carbon dioxide gas; a conduit for circulating the remainder of said culture exhaust gas to the gas inlet of the culture tank; and an air supply device for supplying said culture tank with air. In the culturing apparatus of the present invention, a dissolved oxygen sensor composed of Galvani's oxygen electrode, etc. is used as the dissolved oxygen concentration detector in a culture liquid of a culture tank, and the divergence of an air flow valve or an oxygen gas control valve is controlled according to the signal of the concentration of dissolved oxygen through the dissolved oxygen controller which receives the detection signal from the dissolved oxygen sensor. Thereby, the concentration of dissolved oxygen in a culture liquid is controlled to proper conditions, that is, to 0.7 ppm or higher, preferably, 1 to 4 ppm. Further, feed of the culture substrate (D-sorbitol) to the culture tank is carried out by adding it continuously or intermittently according to a supply rate of oxygen to the culture tank during and after the growth phase of the microorganism with preventing that the concentration of D-sorbitol in the culture liquid goes up to over 5% so that a high concentration of D-sorbitol is oxidized to L-sorbose within a short period of time. Further, the apparatus of the present invention economizes resources by recovering the exhaust gas having increased partial pressure of oxygen with a compressor to circulate and reuse. In order to optimize the partial pressure of carbon dioxide gas in the circulating exhaust gas, an apparatus for measuring the partial pressure of carbon dioxide gas such as an infrared absorption analyzer or the like is provided at the pathway of the exhaust gas. According to the partial pressure of carbon dioxide gas measured by the measuring apparatus, the proper amount of exhaust gas is released by controlling the divergence of an air release valve provided at the pathway and thereby the partial pressure of carbon dioxide gas in the exhaust gas returned to the culture tank is controlled to a proper value (10% or less). Thus, in the present invention, the apparatus and maintenance are simplified by employing release of a part of the exhaust gas into the atmosphere instead of an adsorbent used in a conventional apparatus for removal of carbon dioxide gas. Furthermore, in the apparatus of the present invention, the partial pressure of oxygen gas in the exhaust gas is measured by means of a measuring apparatus of the partial pressure of oxygen gas composed of Zirconia's analyzer, etc., while the partial pressure of oxygen at the inlet to the culture tank is calculated from both circulating flow rate and fresh air aeration rate, to determine oxygen consumption. Then, the amount of the substrate to be oxidized is estimated from the oxygen consumption. Now, a preferred embodiment of the apparatus of the present invention is illustrated by using the accompanying Fig. 5. Fig. 5 shows a general view of a preferred example of a culturing apparatus of the present invention. The apparatus of Fig. 5 comprises a tank 1 for culturing a microorganism, a substrate feeding device 2 for adding a culture substrate (D-sorbitol) to the culture tank 1, an oxygen supply device 3 for supplying oxygen mixed with a culture exhaust gas, an air supply device 4 for supplying the culture tank 1 with air, a gas release device 5 for releasing a part of the exhaust gas from the culture tank 1 into the system, and a compressor 6 for circulating the exhaust gas to the culture tank 1. Inside the above culture tank 1, there is a stirrer 7 which is driven with a motor. The inlet 8a of a gas supply pipe 8 is connected to the lower part of the tank and the exhaust vent 9a of a gas exhaust pipe 9 is connected to the upper part. A substrate feeding device 2 is also connected to the upper part of the tank via a conduit 10. The substrate feeding device 2 comprises a substrate tank 11, a substrate feeding valve 12 and a substrate feeding pump 13 so that a culture substrate (D-sorbitol) is fed from the substrate tank 11 to the culture tank 1 through the substrate feed valve 12, the substrate feed pump 13 and the conduit 10. Feed of D-sorbitol is carried out by adding it continuously or intermittently with preventing that the concentration of D-sorbitol in the culture liquid does not go up to over about 5% during and after the growth period of the microorganism in the culture tank 1. The above gas inlet 8 is connected to the oxygen supply device 3 comprising a liquid oxygen storage tank 14, an emergency shut-off valve 15 and an evaporator 16 through a conduit 17, and oxygen gas formed by vaporizing liquid oxygen with the evaporator 16 is supplied to the culture tank 1 with controlling the supply through an oxygen gas control valve 18 provided at the conduit 17. In the same manner, the gas supply pipe 8 is connected to the air supply device 4 through a conduit 20 and air is supplied to the culture tank 1 through an air flow control valve 21 provided at the conduit 20. The oxygen gas control valve 18 and the flow control valve 21, which control the supply of oxygen gas and air, are controlled by a dissolved oxygen controller 23 which receives a detection signal from a dissolved oxygen detector 22 installed in the culture tank 1. The dissolved oxygen detector is composed of, for example, Galvani's oxygen electrode. It detects the concentration of dissolved oxygen of the culture liquid in the culture tank to output a signal of the concentration of dissolved oxygen to the dissolved oxygen controller 23, and the divergences of the oxygen gas control valve 18 and the flow control valve 21 are controlled by the dissolved oxygen controller 23 which has detected the signal. Air is supplied from the air supply device 4 to the culture tank 1 so that the concentration of dissolved oxygen becomes a constant level until the growth phase of the microorganism. During and after the growth phase of the microorganism, the supply of air is stopped and oxygen gas is supplied from the oxygen supply device 3 to the culture tank 1 to control the concentration of dissolved oxygen to 0.7 ppm or higher, preferably, 1 to 4 ppm. The above exhaust vent 9 is connected to the gas release device via a mist separator 24 and a conduit 25, and is also connected to the compressor 6 via a conduit 26. A measuring apparatus for the partial pressure of oxygen 27 composed of Zirconia's analyzer, etc. and a measuring apparatus for the partial pressure of carbon dioxide gas such as an infrared absorption analyzer, etc. are provided to the conduit 25. The partial pressure of carbon dioxide gas in the exhaust gas is detected by the measuring apparatus for the partial pressure of carbon dioxide gas 28 and a signal of the detector is output to a carbon dioxide gas partial pressure controller 29. A divergence of a air release valve 30 of the gas release device 5 is controlled by the controller 29 to release a part of the culture exhaust gas out of the system. On the other hand, the compressor 6 is connected to the gas supply pipe 8 via a conduit for circulation 32 provided to an aeration circulating valve 31. The remainder of the exhaust gas which is not released from the gas release device 5 is circulated by the compressor 6 to return it to the culture tank 1 via the gas supply pipe 8 with controlling the flow rate by the aeration circulating valve 31. This exhaust gas is circulated and reused together with supply of pure oxygen gas during and after the growth phase of the microorganism. Thereby, the oxygen enriched gas which is a mixture of oxygen gas and the exhaust gas is circulated to the culture tank 1. The working of the above apparatus is illustrated in turn hereinafter. (a) Air is supplied from the air supply device 4 to the culture tank 1 with controlling the divergence of the flow control valve 21 by the dissolved oxygen controller 23 so that the concentration of dissolved oxygen of the culture liquid in the culture tank 1 becomes a prescribed constant level. At that time, for example, when the initial charge concentration of the substrate is 20%, air is supplied in the culture tank 1 so that the concentration of dissolved oxygen becomes 2 ppm. (b) Feed of the substrate from the substrate tank 11 to the culture tank 1 is started at the time when the oxygen consumption corresponding to 15% oxidation of the substrate concentration is calculated by the apparatus for measuring the partial pressure of oxygen 27. (c) When the flow rate of air from the air supply device 4 to the culture tank 1 reaches a maximum and the dissolved oxygen concentration can not be maintained at the prescribed level, the oxygen gas control valve 18 is opened by the dissolved oxygen controller 23 to supply the culture tank 1 with oxygen gas. (d) Immediately after the control of the concentration of dissolved oxygen by oxygen gas is started, the aeration circulating valve 31 is opened and the compressor 6 is started to circulate the exhaust gas to the culture tank 1, and the flow control valve 21 is closed. Thereafter, the concentration of dissolved oxygen is maintained only by oxygen gas. (e) When the circulation of the exhaust gas is started, carbon dioxide gas is accumulated in the aerated gas. Accordingly, the partial pressure of carbon dioxide gas in the exhaust gas is measured by the measuring apparatus 28 and then the air release valve 30 is opened by the controller for the partial pressure of carbon dioxide gas 29 to release a part of the exhaust gas into the atmosphere and to maintain the partial pressure of carbon dioxide gas in the aerated gas below a constant value (10%). (f) When the prescribed amount of substrate is fed into the culture tank 1, the feed is stopped. (g) When the fermentation proceeds in the culture tank 1 and reached toward the end, the required amount of oxygen is decreased. Accordingly, the inflow of oxygen into the culture tank 1 is stopped and the compressor is stopped. Further, the inflow of the exhaust gas into the culture tank 1 is stopped by closing the aeration circulating valve 31 and air supply from the air supply device 4 is reopened. (h) The concentration of dissolved oxygen in the culture tank 1 rises rapidly by the above aeration and the partial pressure of oxygen also rises. The fermentation is completed when the partial pressure of oxygen in the exhaust gas reaches a prescribed value (20%). The following Examples illustrate the process of the present invention in detail but are not to be construed to limit the scope thereof. In the following Examples, the apparatus as shown in Fig. 5 was used. Example 1 Gluconobacter suboxydans IFO 3254 was inoculated in 10 liters of a culture medium containing 20% of D-sorbitol, 0.2% of sodium glutamate, 0.018% of calcium carbonate, 0.003% of nicotinamide, 0.003% of calcium pantothenate, 0.0001% of vitamin B 2 , 0.0001% of p-aminobenzoic acid, 0.047% of potassium dihydrogen phosphate, 0.03% of yeast extract, 0.01% of magnesium sulfate, 0.00015% of ferrous sulfate, 0.00001% of manganese sulfate and 0.0005% of actocol and incubated at 30°C for 24 hours. As a seed, this was transferred to 49 liters of a culture medium containing 20% of D-sorbitol, 0.06% of ammonium acetate, 0.102% of sodium glutamate, 0.06% of calcium carbonate, 0.006% of nicotinamide, 0.0005% of calcium pantothenate, 0.0002% of vitamin B 2 , 0.00002% of p-aminobenzoic acid, 0.04% of potassium dihydrogen phosphate, 0.02% of magnesium sulfate, 0.0003% of ferrous sulfate and 0.00002% of manganese sulfate. This was incubated at 30°C and, after starting culturing, the amount of aeration from the air supply device 4 and the internal pressure of the culture tank were controlled in the range of 0.1 to 0.6 vvm and 0.1 to 1.8 kg/cm 2 G, respectively so that the concentration of dissolved oxygen was maintained within the range of 2.5 ± 0.5 ppm. After the maximum amount of aeration and the internal pressure reached 0.6 vvm and 1.8 kg/cm 2 G, respectively, oxygen gas formed by vaporizing liquid oxygen in the oxygen supply device 3 was introduced into the gas inlet 8 and then bubbled through the culture tank 1 so that the concentration of dissolved oxygen became 2.5 ± 0.5 ppm. At the same time, aeration was stopped and the compressor 6 was started to recover the exhaust gas. The exhaust gas was circulated to the culture tank 1. Thereafter, a part of circulating air (0.03 vvm) was released to the atmosphere by the exhaust gas release device 5 to prevent the accumulation of carbon dioxide gas. The concentration of dissolved oxygen was maintained by bubbling oxygen gas. The amount of air being insufficient to maintain the internal pressure was supplied by air from dwell. On the other hand, addition of D-sorbitol which was oxidized with progress of culturing was started through the substrate feeder 2 to the culture tank 1 at the time when the concentration thereof in the culture liquid became 5%, and the culturing was carried out with addition of 51 liters of a 60 w/w% aqueous D-sorbitol solution for 20 hours in all. According to the above culturing, it made possible to carry out the culturing with 50 w/v% of D-sorbitol charge concentration per batch in 20 hours, and L-sorbose was accumulated in the yield of 95% based on D-sorbitol. Example 2 Gluconobacter suboxydans IFO 3254 was inoculated into 10 liters of a culture medium containing 20% of D-sorbitol, 0.2% of sodium glutamate, 0.018% of calcium carbonate, 0.003% of nicotinamide, 0.003% of calcium pantothenate, 0.0001% of vitamin B 2 , 0.0001% of p-aminobenzoic acid, 0.047% of potassium dihydrogen phosphate, 0.03% of yeast extract, 0.01% of magnesium sulfate, 0.00015% of ferrous sulfate, 0.00001% of manganese sulfate and 0.0005% of actocol and incubated at 30°C for 24 hours. As a seed, this was transferred to 54 liters of a culture medium containing 25% of D-sorbitol, 0.08% ammonium acetate, 0.067% of alanine, 0.06% of calcium carbonate, 0.006% of nicotinamide, 0.0005% of calcium pantothenate, 0.0002% of vitamin B 2 , 0.00002% of p-aminobenzoic acid, 0.04% of potassium dihydrogen phosphate, 0.02% of magnesium sulfate, 0.0003% of ferrous sulfate and 0.00002% of manganese sulfate. This was incubated at 30°C and after starting culturing, the amount of aeration from the air supply device 4 and the internal pressure of the culture tank were controlled in the range of 0.1 to 0.6 vvm and 0.1 to 1.8 kg/cm 2 G, respectively, so that the concentration of dissolved oxygen became 2.5 ± 0.5 ppm. After the maximum amount of aeration and the internal pressure reached 0.6 vvm and 1.8 kg/ch 2 G, respectively, oxygen gas formed by vaporizing liquid oxygen in the oxygen supply device 3 was introduced into the gas inlet 8 and then bubbled through the culture tank 1 so that the concentration of dissolved oxygen reached 2.5 ± 0.5 ppm. At the same time, aeration was stopped and the compressor 6 was started to recover the exhaust gas. The exhaust gas was circulated to the culture tank 1. Thereafter, a part of circulating air (0.03 vvm) was released to the atmosphere by the exhaust gas release device 5 to prevent the accumulation of carbon dioxide gas. The concentration of dissolved oxygen was maintained by bubbling oxygen gas. The amount of air being insufficient to maintain the internal pressure was supplied by air from dwell. On the other hand, addition of D-sorbitol oxidized with progress of culturing was started through the substrate feeder 2 to the culture tank 1 at the time when the concentration thereof in the culture liquid became 5%, and culturing was carried out with addition of 51 liters of a 60 w/w% aqueous D-sorbitol solution for 20 hours in all. According to the above culturing, it made possible to carry out the culturing with 50 w/v% of D-sorbitol concentration charged per batch in 20 hours, and L-sorbose was accumulated in the yield of 95% based on D-sorbitol. Example 3 Gluconobacter suboxydans BL-9 (IFO 14489, FERM BP-1241) was inoculated in 10 liters of a culture medium containing 20% of D-sorbitol, 0.3% of glycerin, 0.2% of sodium glutamate, 0.018% of calcium carbonate, 0.003% of nicotinamide, 0.003% of calcium pantothenate, 0.0001% of vitamin B 2 , 0.0001% of p-aminobenzoic acid, 0.047% of potassium dihydrogen phosphate, 0.01% of magnesium sulfate, 0.00015% of ferrous sulfate, 0.00001% of manganese sulfate and 0.0005% of actocol and incubated at 30°C for 24 hours. As a seed, this was transferred to 49 liters of a culture medium containing 20% of D-sorbitol, 0.05% of glutamic acid, 0.06% of ammonium acetate, 0.102% of sodium glutamate, 0.06% of calcium carbonate, 0.006% of nicotinamide, 0.0005% of calcium pantothenate, 0.0002% of vitamin B 2 , 0.00002% of p-aminobenzoic acid, 0.04% of potassium dihydrogen phosphate, 0.02% of magnesium sulfate, 0.0003% of ferrous sulfate and 0.00002% of manganese sulfate. This was incubated at 30°C and, after starting culturing, the amount of aeration from the air supply device 4 and the internal pressure of the culture tank were controlled in the range of 0.1 to 0.6 vvm and 0.1 to 1.8 kg/cm 2 G, respectively so that the concentration of dissolved oxygen was maintained within the range of 2.5 ± 0.5 ppm. After the maximum amount of aeration and the internal pressure reached 0.6 vvm and 1.8 kg/cm 2 G, respectively, oxygen gas formed by vaporizing liquid oxygen in the oxygen supply device 3 was introduced into the gas inlet 8 and then bubbled through the culture tank 1 so that the concentration of dissolved oxygen became 2.5 ± 0.5 ppm. At the same time, aeration was stopped and the compressor 6 was started to recover the exhaust gas. The exhaust gas was circulated to the culture tank 1. Thereafter, a part of circulating air (0.03 vvm) was released to the atmosphere by the exhaust gas release device 5 to prevent the accumulation of carbon dioxide gas. The concentration of dissolved oxygen was maintained by bubbling oxygen gas. The amount of air being insufficient to maintain the internal pressure was supplied by air from dwell. On the other hand, addition of D-sorbitol which was oxidized with progress of culturing was started through the substrate feeder 2 to the culture tank 1 at the time when the concentration thereof in the culture liquid became 5%, and the culturing was carried out with addition of 51 liters of a 60 w/w% aqueous D-sorbitol solution for 20 hours in all. According to the above culturing, it made possible to carry out the culturing with 50 w/v% of D-sorbitol charge concentration per batch in 20 hours, and L-sorbose was accumulated in the yield of 97.5% based on D-sorbitol. Example 4 Gluconobacter suboxydans BL-115 (IFO 14490, FERM BP-1240) was inoculated into 10 liters of a culture medium containing 20% of D-sorbitol, 0.3% of glycerin, 0.2% of sodium glutamate, 0.018% of calcium carbonate, 0.003% of nicotinamide, 0.003% of calcium pantothenate, 0.0001% of vitamin B 2 , 0.0001% of p-aminobenzoic acid, 0.047% of potassium dihydrogen phosphate, 0.01% of magnesium sulfate, 0.00015% of ferrous sulfate, 0.00001% of manganese sulfate and 0.0005% of actocol and incubated at 30°C for 24 hours. As a seed, this was transferred to 54 liters of a culture medium containing 25% of D-sorbitol, 0.05% of glycerin, 0.08% ammonium acetate, 0.067% of alanine, 0.06% of calcium carbonate, 0.006% of nicotinamide, 0.0005% of calcium pantothenate, 0.0002% of vitamin B 2 , 0.00002% of p-aminobenzoic acid, 0.04% of potassium dihydrogen phosphate, 0.02% of magnesium sulfate, 0.0003% of ferrous sulfate and 0.00002% of manganese sulfate. This was incubated at 30°C and after starting culturing, the amount of aeration from the air supply device 4 and the internal pressure of the culture tank were controlled in the range of 0.1 to 0.6 vvm and 0.1 to 1.8 kg/cm 2 G, respectively, so that the concentration of dissolved oxygen became 2.5 ± 0.5 ppm. After the maximum amount of aeration and the internal pressure reached 0.6 vvm and 1.8 kg/ch 2 G, respectively, oxygen gas formed by vaporizing liquid oxygen in the oxygen supply device 3 was introduced into the gas inlet 8 and then bubbled through the culture tank 1 so that the concentration of dissolved oxygen reached 2.5 ± 0.5 ppm. At the same time, aeration was stopped and the compressor 6 was started to recover the exhaust gas. The exhaust gas was circulated to the culture tank 1. Thereafter, a part of circulating air (0.03 vvm) was released to the atmosphere by the exhaust gas release device 5 to prevent the accumulation of carbon dioxide gas. The concentration of dissolved oxygen was maintained by bubbling oxygen gas. The amount of air being insufficient to maintain the internal pressure was supplied by air from dwell. On the other hand, addition of D-sorbitol oxidized with progress of culturing was started through the substrate feeder 2 to the culture tank 1 at the time when the concentration thereof in the culture liquid became 5%, and culturing was carried out with addition of 51 liters of a 60 w/w% aqueous D-sorbitol solution for 20 hours in all. According to the above culturing, it made possible to carry out the culturing with 50 w/v% of D-sorbitol concentration charged per batch in 20 hours, and L-sorbose was accumulated in the yield of 97.3% based on D-sorbitol.

Claims: An apparatus for culturing a microorganism which comprises, a culture tank for culturing the microorganism equipped with a stirrer, a gas inlet provided at the lower part of the tank and an exhaust vent provided at the upper part of the tank; a substrate feeding device for adding a culture substrate to said culture tank; an oxygen supply device comprising a liquid oxygen storage tank and an evaporator for supplying said culture tank with a culture exhaust gas which has been enriched with oxygen gas; a dissolved oxygen concentration detector for detecting dissolved oxygen in a culture liquid in said culture tank to output a signal of the concentration of dissolved oxygen; a dissolved oxygen controller for controlling supply of dissolved oxygen by detecting said signal of the concentration of dissolved oxygen; a carbon dioxide gas measuring device for measuring the partial pressure of carbon dioxide gas in a culture exhaust gas, which is provided to an exhaust gas pipe from said culture tank; an exhaust gas release device for releasing a part of the culture exhaust gas out of the system by detecting said partial pressure of carbon dioxide gas; a conduit for circulating the remainder of said culture exhaust gas into the gas inlet of the culture tank; and an air supply device for supplying said culture tank with air. An apparatus for culturing the microorganism according to claim 1, wherein an air supply pipe is provided between said air supply device and said culture tank through an air flow control valve and the air flow control valve is controlled by said dissolved oxygen controller so that a concentration of dissolved oxygen is controlled to a constant level by regulation of air flow by means of the dissolved oxygen controller until the microorganism has reached the growth phase.

Inventors: KINTAKA KAZUHIKO, KISHIMOTO KATSUMITSU, YOSHINAGA HIROYUKI, KINTAKA, KAZUHIKO, KISHIMOTO, KATSUMITSU, YOSHINAGA, HIROYUKI
Applicants: TAKEDA CHEMICAL INDUSTRIES LTD, TAKEDA CHEMICAL INDUSTRIES, LTD.
Application year: 1987
Main classification: C12M1
IPC codes: C12P19, C12M1