JPS6358558B2 - - Google Patents

Description

[Detailed description of the invention]

Continuous fermentation using immobilized microorganisms has the same problem as conventional continuous fermentation in that the yield of the target product decreases due to bacterial contamination. The practical countermeasure that has been taken so far is to strengthen the sterilization of the routes of microbial invasion. Due to its purpose, continuous fermentation technology requires stable operation over a long period of time, and protection against bacterial contamination is necessary. This problem is also the same in continuous fermentation using immobilized microorganisms. In anaerobic fermentation such as alcohol fermentation, acetone/butanol fermentation, and lactic acid fermentation, the rate of carbon source consumption by the bacterial cells themselves is higher than in aerobic fermentation, and the residence time in the reaction tank is extremely shortened by the method using immobilized microorganisms. It is known that However, even in such a reaction tank, bacterial contamination is an unavoidable problem, although it is somewhat reduced compared to conventional continuous fermentation. One of the causes of bacterial contamination is that minute flocs are generated by the aggregation of production bacteria or bacteria used for immobilization, accumulate in the reaction tank, and are difficult to be discharged from the tank. As a result of research into a method for removing flocs, it was found that the influence of bacterial contamination could be greatly reduced by providing a sedimentary accumulation area for flocs in the reaction tank and discharging the flocs outside the tank for fermentation. According to the present invention, fermentation can be carried out stably for a long period of time by using a fermenter having a structure that allows the produced flocs to settle and accumulate, and by accumulating the flocs and appropriately discharging them outside the tank. Examples of tanks used in the present invention are shown below. (1) A reaction tank in which an inclined plate is installed at the bottom of the reaction tank to collect sedimentary suspended matter at the bottom of the inclined plate, and from which the reaction tank effluent is extracted. (Fig. 1) (2) Method using a conical sedimentation tank alone or in combination with the inclined plate of (1). (Figure 3) These fermenters will be explained below. FIG. 1 shows a fermenter having a floc sedimentation accumulation structure and using an immobilized membrane. The flocs are collected in the settling section 3 and discharged from the discharge port 4. FIG. 3 shows an example in which granular immobilized microbial cells are used, and the flocs are collected in the sedimentation accumulation section 11 through a wire mesh 14 and discharged from the system through the discharge port 12. For comparison, FIG. 2 shows an example using conventionally used granular immobilized bacterial cells. The reaction vessel used in the present invention can be applied not only to a fluidized bed reaction vessel using a carrier in the form of a film or a sphere, but also to a fixed membrane reaction vessel in which the carrier is fixed in the form of a membrane. The sedimentation of objects is less likely to be disturbed, and it is expected that the functionality of the sedimentation tank will be improved. According to the method of the present invention, there is almost no bacterial contamination even if the sterilization temperature of the raw material is lower than usual. In other words, the method of sterilizing the raw material culture medium is usually determined based on the type of fermentation, the type of bacteria used, and the degree of influence of bacterial contamination.For example, in continuous alcoholic fermentation using immobilized microorganisms, the method of sterilizing the raw material culture medium is carried out at 90 to 100°C for 5 minutes. It is carried out with a certain degree of sterilization. However, when using this method, no adverse effects of bacterial contamination are observed even when the temperature drops to about 70°C.
The energy required to sterilize raw materials to prevent bacterial contamination is a heavy burden, and this method can be said to be of great significance as a concrete mitigation measure. Aspects of the present invention will be explained below with reference to Examples. Example 1 Each device shown in FIGS. 1 to 3 was sterilized.
To 1 volume of a 10% sodium alginate aqueous solution that had been sterilized separately, 2 volumes of a koji broth culture of Japan Association No. 2 grape wine yeast were added and mixed. The mixed solution was sent to the fermentation tank shown in FIG. 1, and after the membrane was immersed in the liquid, a 2% aqueous calcium chloride solution was sent to form a gel in the form of a membrane. After that, 15% sugar-containing molasses heated and maintained at 90°C for 5 minutes was fed and continuous operation was performed (Experiment 1).
In addition, a sterilized calcium chloride aqueous solution was poured into each of the fermenters shown in Fig. 2 (Experiment 2) and Fig. 3 (Experiment 3), and the above alginic acid mixture containing yeast was added dropwise to the bead-shaped carrier. A bulk volume (1.5) corresponding to 1/2 of the volume was prepared. Next, a sugar solution that had been separately heated and sterilized at 120°C for 15 minutes was supplied, and continuous operation was performed. The sugar concentration at each fermenter outlet in Figures 1 to 3 is 20
Gradually increase the amount of feed so that it is about 8.2 to 800 ml/hr after 4 days
A mash with an alcohol concentration of 8.4 V/V% was obtained, and the operation was continued to observe the effects of bacterial contamination. Up to the 500th hour, the conversion yield (ratio based on the theoretical value of 100%, hereinafter abbreviated as "yield") relative to consumed sugar was maintained at 95%, and no bacterial growth was observed. Therefore, from the 500th hour, experiments 2 and 3
The sugar solution sterilization temperature was also the same as in Experiment 1, 90°C for 5 minutes. At the 800th hour in total, the yield in Experiment 2 decreased to 80% and flocs of bacteria were observed in the column.
On the other hand, in Experiments 1 and 3, the yield was 95% and no bacterial flocs were observed. Furthermore, the sugar solution sterilization temperature was lowered to 70℃ for 5 minutes, and after a total of 1200 hours,
In experiment 1, the yield was 91%, in experiment 2 61%, and in experiment 3 85%. Furthermore, in Experiment 1, a yield of 90% was maintained even after 1800 hours. At this time, in Experiment 2, the yield was 60%, and in Experiment 3, the yield was 82%. Example 2 A fermenter having an empty capacity of 3 as shown in FIG. 4 was filled with 2.5 Raschig rings made of wire mesh (manufactured by Tokyo Tokushu Kinami Co., Ltd., Dixon type, diameter 6 mm). First, the entire fermenter was sterilized. Seed culture solution of inosine producing bacterium Brevibacterium ammoniagenes ATCC15187 (medium composition: glucose 20g/, peptone 10g/, yeast extract 10g/, sodium chloride 3g/, 30
℃, 24 hour culture) 100 ml and commercially available sodium alginate (manufactured by Fuji Chemical Co., Ltd., Snow Algin L) 3.3%
A mixed solution of 900 ml of aqueous solution was evenly dropped into the fermenter from the top to wet the rough shilling with the mixed solution. Extract the excess mixed liquid from the bottom of the fermenter,
Next, a 2% aqueous solution of calcium chloride was introduced to effect gelation. Immediately after gelation, the following production medium was sent into the fermenter, and the flowing liquid was circulated at about 1/hr to start fermentation. Air is supplied to the air blowing pipe 25 and air blowing pipe 16 at 14/min and 1/min, respectively.
I sent min. Air is exhausted from the exhaust pipe 17.

[Table] *Only for the first occurrence, excluded for continuous supply The production medium was sterilized in advance at 120°C for 30 minutes before use. The pH of the bottom liquid was adjusted to 7.4 using 22% aqueous ammonia. The medium was continuously supplied from 16 hours after the start of fermentation. The remaining amount of sugar in the fermented liquid from the discharge port 20 is 5 g/
The supply amount was gradually increased so that The fermentation liquid produced 35 g/hr of inosine at a feed rate of 60 ml/hr, and the inosine content decreased at a feed rate higher than this. Inosine was analyzed using high performance liquid chromatography. The above operations were carried out in parallel using two fermenters. One fermenter continued the above operation. The other fermenter has a discharge port, and after the start of feeding, 90
At the time point, the outlet was changed to the outlet 21, and the outlet 20 was closed. The inosine titer up to 240 hours after the start of supply was 35 g/in all cases, and no bacterial contamination was observed. After 240 hours in both fermenters, the medium sterilization conditions were changed to 100°C for 15 minutes. After easing sterilization conditions,
Bacterial contamination rapidly progresses in the fermenter extracted from the discharge port 21, and the inosine titer remains low after sterilization conditions are relaxed.
21g/ at 100th hour, 10g/ at 150th hour,
At the 200th hour, it became 0g/. On the other hand, in the device according to the present invention, bacterial contamination occurred in the same way, but the inosine titer was 32g/100 hours after relaxing the sterilization conditions.
The weight decreased to 30g/at the 150th hour and 28g/at the 200th hour, but deterioration due to bacteria was relatively slow. Example 3 150 ml of the following seed culture medium was placed in a 2-volume Erlenmeyer flask, and L-lysine producing bacteria Corynebacterium,
Glutamicum ATCC21513 was inoculated and cultured at a temperature of 28
Shaking culture was performed at ℃ for 24 hours. Separately, a fermenter shown in Figure 4 was prepared and sterilized.

[Table] 900 ml of 6% wax methoxy pectin (manufactured by UniPectin) aqueous solution was added to the above culture solution, mixed well, and dripped onto the Raschig rings packed in the fermenter from the feed port 15 of the fermenter shown in FIG. Next, after sufficiently removing the excess mixed liquid, add 2% calcium chloride.
An aqueous solution was fed to perform gelation. After gelation, 500 ml of the following production medium (for up to 248 hours from the initial
sterilized at 120°C for 15 minutes) was sent and dropped onto the top of the immobilized microorganism gel layer. Ventilation was carried out at 18/min and 2/min from air inlets 25 and 16, respectively. Production medium composition Blackstrap molasses (as glucose) 150g / MgSO ₄ 7H ₂ O 0.3g / KH ₂ PO ₄ 0.7g / Soybean meal acid hydrolyzate 20g / Leucine 200mg / The flowing liquid of layer 18 is PH electrode 24 and 22% The pH was adjusted to 8.2 using the ammonia water supply port and the mixer 22, and the ammonia was returned to the top of the layer 18 through the recycle pipe 19. After 48 hours of culture, production medium was continuously supplied at a flow rate of 90 ml/hr. As in Example 2, the same operations as above were performed in parallel using two sets of devices. In each case, 120 hours after the start of continuous supply, 50 g of lysine hydrochloride was confirmed to be produced in the liquid extracted from the discharge port 20. Similarly, from the 150th hour onward, the discharge from one side was stopped from the discharge port 20, the discharge port 21 was opened, and the fermented liquid was discharged. On the other hand, the discharge port 21 was closed from the start of supply, and the fermented liquid was extracted from the discharge port 20. From the 200th hour after the start of continuous supply, the medium sterilization conditions were changed to 110℃15
Changed to minutes. Fifty hours after the sterilization conditions were relaxed, bacterial contamination was observed in the device that was extracted from the discharge port 21. In other words, the lysine content in the fermentation liquid was 48g/50 hours after relaxing the sterilization conditions, and 48g/100 hours after easing the sterilization conditions.
The amount was 35 g/, 29 g/ at the 150th hour, 18 g/ at the 200th hour, and 0 g/ at the 250th hour.
On the other hand, when using the method of extracting from the discharge port 20, 40g/200 hours after relaxing the sterilization conditions,
41g/at 300th hour, 39g/at 500th hour
No rapid deterioration was observed.

[Brief explanation of drawings]

FIG. 1 shows a fermenter with a settling section according to the invention. 1: Supply port, 2: Immobilized microorganism membrane, 3: Sedimentation accumulation section, 4: Discharge port, 5: Gas discharge port. FIG. 2 is a sectional view of a fermenter using a conventional immobilized spherical carrier. 6: supply port, 7: immobilized spherical carrier, 8: discharge port. FIG. 3 shows a fermenter with an inclined plate settling section according to the invention. 9: supply port, 10: immobilized spherical carrier, 12: discharge port, 13: gas discharge port, 14: wire mesh. FIG. 4 shows a fermenter according to the present invention and a conventional fermenter. 15: Supply port, 16: Air inlet, 17: Air discharge port, 18: Raschig ring, 19: Recycle pipe, 20, 21: Discharge port, 22: Ammonia water addition port and mixer for PH control, 23: Fermentation liquid liquid level, 24: PH electrode, 25: air inlet,
26: Sedimentation accumulation part. A conventional fermenter is shown when the discharge port 21 is used, and a fermenter used in the method of the present invention is shown when the discharge port 20 is used.

Claims (1)

[Claims] 1. A continuous fermentation method characterized in that fermentation is carried out while discharging flocs using a fermenter having a structure in which flocs produced in a fermenter using immobilized microorganisms are sedimented and accumulated.