JPS5813386A - Continuous fermentation using immobilized microorganism - Google Patents

Abstract

PURPOSE:To suppress the contamination of the titled cntinuous fermentation system with various germs, and to enable the continuous operation stably for a long period, by accumulating and discharging the produced and precipitated floc from the reaction tank through the part placed in the tank. CONSTITUTION:A precipitate accumulation part 3 composed of inclined plates is attached to the bottom of the reactor (the sign 1 is the feeding port of the raw material and 5 is exhaust port of the produced gas) containing a number of vertical membranes 2 of immobilized microorganisms. The precipitating suspensoid (floc) produced in the tank is accumulated in the part 3, and the fermentation is continued while discharging the accumulated floc through the discharging line 4.

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 contamination with various bacteria. 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 carbon source consumption rate of the bacterial cells themselves is higher than in aerobic fermentation, and the reaction time is extremely shortened by the method using immobilized microorganisms. It has been known. However, although bacterial contamination can be somewhat reduced compared to conventional continuous fermentation even in such a large reaction tank, it is still a problem that cannot be avoided. 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 discharge outside 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 cypress 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, sedimentary suspended matter is collected at the bottom of the inclined plate, and the reaction tank effluent is extracted from there. (Fig. 1) (2) Method of using a conical sedimentation tank alone or in combination with an H inclined plate. (Figure 3) These fermenters will be explained below.

FIG. 1 shows a fermenter having a floc sedimentation and accumulation structure and using an immobilized membrane. The flocs are built up in the settling section 3 and discharged from the discharge log.

Figure 3 shows an example of using granular immobilized bacterial cells, and the flocs are collected through a wire mesh /≠ into a sedimentation accumulation part /l and an outlet /.
2 is discharged from the system.

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 substances is less likely to be disturbed, and the functionality of the sedimentation S tank can be expected to improve.

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 medium is usually determined based on the type of fermentation, the type of bacteria used, and the likelihood of bacterial contamination. For example, in continuous alcoholic fermentation using immobilized microorganisms, the method of sterilizing the raw material is ,
The sterilization process takes about minutes. However, in the case of 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 illegal use can be said to be of great significance as a concrete mitigation measure.

Aspects of the present invention will be explained below using examples.

Example t Each device shown in Figures 81-3 was sterilized.

A separately sterilized fc/0% sodium alginate aqueous solution/M volume of koji juice culture of Association No. 2 grape wine yeast was added and mixed. The mixed solution was sent to the fermenter shown in FIG. 1, and after the membrane was immersed in the liquid, a dibasic calcium chloride aqueous solution was sent to form a gel in the form of a membrane. After that, molasses containing sugar, which had been heated and maintained at 0° C. for 1 minute, was pumped in and continuous operation was performed (Reply 9/). Also, Figure 2 (Experiment, 2),
Fill each fermenter shown in Figure 3 (Experiment 3) with a sterilized calcium chloride aqueous solution, drop the above yeast-containing alginic acid mixture dropwise, and add the bead-like carriers to a bulk volume ( /, z t ) was prepared. Next, separately i,
Continuous operation was performed by supplying a sugar solution heated and sterilized for 2oC/J".The feed amount was gradually increased so that the sugar linearity at each fermenter outlet in Figures 1 to 3 was approximately 01/11. After 1 day, mash with alcohol concentration of tl, 2~A', 4 (V/V%) was obtained with 1roo waste 1/hr each. The effect of conversion was observed. Until the 00th hour, the conversion yield (ratio based on the theoretical value of 100%, hereinafter abbreviated as "yield") relative to consumed sugar remained constant, and no bacterial growth was observed. Therefore, from the j00th hour onwards, the sugar solution sterilization temperature in Experiment-13 was set at 20°C! minutes, the same as in the experiment.At the total 100th hour, the yield of experiment λ decreased to 10-1, and the number of bacteria in the column increased. Flocs were observed. On the other hand, in the <z> experiment/, three samples, the yield was 5% and no bacterial flocs were observed. Furthermore, the mucus sterilization temperature was lowered to 70°C! minutes, and the total / After a period of λ0θ, the yield was Li/T in run 1, J/excellent in run 2, and 13% in run 3.

Furthermore, in the experiment/100/100th hour, the yield oqtr could be maintained. At this time, the yield was 10% in experiment-3, and the yield was r-1 in experiment 3.

Example 2 A wire mesh Raschig ring (manufactured by Tokyo Tokushu Kin #j Koji, DiXOn type diameter jm) was placed in a fermenter consisting of the empty capacity 31 shown in Figure V.
22 was filled. First, the entire fermenter was sterilized. Inosine-producing bacterium Brevibacterium ammoniagenes ATCC
/j/17 seed culture solution (medium composition Nigel course 20 (1
/l, peptone 10i/l, yeast extract 109/l! ,
Sodium chloride 311/l, 30°C, time culture) 1
00111 and commercially available sodium alginate (Fuji Chemical @#
Snow Algin L) 3.3% aqueous solution o.

The mixed solution (j) was evenly dropped into the fermenter from the top, and the mixed solution was added to the lashings. Excess liquid mixture is extracted from the lower part of the fermenter, and then calcium chloride 2
A water attack solution was sent in to form a gel. Immediately after gelation, the following production medium is sent to the fermenter, and the flowing liquid is approximately /lj
/br to start fermentation. Air blowing pipe-2
J and air blowing pipe /l respectively /4'//'m
in, /l/min. Air is a discharge pipe/
It is discharged from 7.

Production Medium Glucose l 011/1. ChiHPO□ j
l/IKB, Po, 31/l, Mt3SO
,・7H20311/1 meat extract 101//l,
Fe50. -7H2010*ti/1ZndO, -7H
, O/Q/l, Mn804t・! H,O/OR9
/1 Vitamin B, /41 Calcium zeta-nocytothenate/L cystine 20m9/1. Biotin 30γ/10aOz, ・jH, 2rJ (separately steamed #)
, tl/l, urine] o++i boiled) λ11/l *first onset only,
For continuous feeding, the excluded production medium was sterilized in advance at 120°C for 30 minutes before use. The p 11 of the bottom liquid was converted to 711 using 2.2% aqueous ammonia.
It was adjusted to After and after the start of fermentation, the medium was continuously supplied. The remaining amount of sugar in the fermented liquid from the discharge port-20 is 111/
The supply amount was gradually increased to 1. In the fermentation liquid, 311/l of inosine was produced at a feed rate of IQul/hr, and the inosine content decreased when the feed rate was higher than this.

High performance liquid chromatography was used for analysis of inosine. The above operations were carried out in parallel using a Koren fermenter. One fermenter continued the above operation. The discharge port of the other fermenter was changed to the discharge loco/at time O after the start of supply, and the discharge port 20 was closed. The inosinium value from the start of supply to the end of the hour was 3jg7t in all cases, and no bacterial contamination was observed.

In both fermenters, the medium sterilization conditions were changed to ioo°C/) minutes from the 24th tO hour. After relaxation of sterilization conditions, bacterial contamination rapidly progresses in the fermenter from which discharge 0.2>1.↓ is extracted, and the inosinka value is 2/,! //l, /jθ time 1011/1,2
It became 0971 at the 00th hour. On the other hand, with the device according to the present invention, bacterial contamination similarly occurred, but after relaxing the sterilization conditions,
Inosinka titer: t, zll/l at 00 hours, decreased to 3o11/l at /j0 hours, and 21 #/l at 2oo hours, but the deterioration due to contaminants was relatively slow.

Example 3 The following seed culture medium /60- was placed in a 21-volume Erlenmeyer flask, and the L-lysine producing bacterium Corynebacterium glutamicum ATOOλ/j/3 was inoculated and cultured at a culture temperature of 2j°C.
Shaking culture was performed for 4A hours. Separately, a fermenter shown in Figure V was prepared and sterilized.

Seed culture medium composition - Glucose 011/l, 2HPO,/!
g/IKH, PO4Io, zl/l, urea 3
11/lMg5O, 7H:20 0. J′ll/l
, Peptone, 20II/I! Meat extract II/
1. Biotin jOf, add 6% wax methoxy pectin (manufactured by Unipectin) aqueous solution to the culture medium on Q00d
was added, mixed well, and dripped onto the Raschig rings packed in the fermenter from the feed port /j of the fermenter shown in Figure 1. Next, after sufficiently removing the excess liquid mixture, a J% calcium chloride aqueous solution was fed to perform gelation. After gelation, the following production medium z
oow (changed after sterilization at 0° C. lj minutes from initial onset to firming time) and dropped onto the top of the immobilized microorganism gel layer. From air blowing loco j and 16 respectively / r /
Aeration was performed at 2 l/min and 2 l/min.

Production medium composition Blackstrap molasses (as glucose) 1101171Mg
5Ogej7HJOO, 39/11#KH, 2POda
0.711/l soybean meal acid hydrolyzate, 2011/10 Ishin
+200 layers/l of flowing liquid is I) H electrode, 2
+ and 22% ammonia water supply port and mixer, 22
was used to adjust the pH to tλ and returned to the top of layer/r through the recycle pipe. After culturing for the at waiting period, the production medium was continuously supplied at a flow rate of 20+j/hr. As in Example λ, the same operations as above were carried out in parallel (/17) using Koren's apparatus. After the start of continuous supply, the production of 109/l of lysine hydrochloride was confirmed in the liquid extracted from the discharge port ◯ at every date and time. From the same </jO time, one side is discharge port 2
The extraction from 0 was stopped, the discharge port λ/ was opened, and the fermented liquid was extracted. On the other hand, the discharge port 2/ was closed from the start of supply, and the fermented liquid was extracted from the discharge 0.20. From 200 hours after the start of continuous supply, the medium sterilization conditions were changed to 110°C for 71 minutes. Ten hours after the sterilization conditions were relaxed, bacterial contamination was observed in the device for extracting from the discharge port 21. In other words, the lysine content in the fermentation liquid was a
rl/ノ, 311/l at ioo hour, richness at /jO hour, 9/11, /ri/l, 210 at λoo hour
At the hour, it was 01/l. On the other hand, when using the discharge loco O method, -200
Time, ≠oi7t for eye 1.

300 hours above /i/l, 3ri1 at 100th hour
1/l, and no rapid deterioration was observed.

[Brief explanation of the drawing]

FIG. 7 shows a fermenter with a settling section according to the invention. l: Supply port, 2 = Immobilized microorganism membrane, 3: Sedimentation accumulation part, 4
t=discharge port, j: gas discharge port. FIG. 2 is a sectional view of a fermenter using a conventional immobilized spherical carrier. t: supply port, 7: immobilized spherical carrier, r: discharge port. FIG. 3 shows a fermenter with an inclined plate settling section according to the invention. P: Supply port, 10: Immobilized spherical carrier, //: Inclined plate settling part, / Coni discharge port, 13: Gas discharge port, / Dust mite wire mesh. FIG. 7 shows a fermenter according to the present invention and a conventional fermenter. /j: Supply port, /F: Air inlet, 17: Air outlet,
/l: Raschig ring, /li: recycling pipe, 20. λ/
:1□'1□1・14. Discharge port, ammonia water addition port for λKoni pH control, and mixer. 3: Liquid level of fermentation liquor, λ4': pH electrode, 2: Air inlet, -6: Sedimentation accumulation section. A conventional fermenter is shown when a discharge of 0.2/ is used, and a fermenter used in the method of the present invention is shown when a discharge port of 20 is used. Patent applicant: New Fuel Oil Development Technology Research Association (/3)

Claims (1)

[Claims] A continuous fermentation method characterized by carrying out fermentation while discharging flocs using a fermenter having a structure in which flocs generated in a fermenter using immobilized microorganisms are sedimented and accumulated.