JPH0365153B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a method for continuously fermentatively producing acetone and butanol using immobilized acetone/butanol producing bacteria. There was a time when acetone-butanol fermentation was carried out batchwise. However, the yield of acetone and butanol obtained by this method is
The production rate of acetone and butanol was only about 0.4g/hr, and after that, the production of acetone and butanol was carried out by petrochemicals. Recently, the production of various fermentation products using immobilized bacterial cells has been studied, and acetone-butanol fermentation is also being considered from this point of view [Biotechnology Letters vol. 2 (No. 5), 241-246
(1980)]. The fermentation method adopted in the above literature is a repeated use in a batch operation.
process). The present inventors investigated a method for continuous fermentation production of acetone and butanol using immobilized acetone/butanol-producing bacteria, and found that, unlike the case of immobilized yeast for alcohol fermentation, continuous fermentation production was performed immediately after immobilization. When carrying out fermentation, it may take a considerable amount of time or days before the bubbling phenomenon (foaming caused by fermentation gases such as hydrogen and carbon gas produced with the production of acetone and butanol), which is an indicator of the start of normal fermentation, is observed. It turned out that it was necessary. Therefore, the present inventors conducted further studies to shorten the fermentation time, and found that the fermentation time could be significantly shortened if the immobilized bacterial cells were once cultured in a medium in a batch manner prior to continuous fermentation. The invention has been completed. By shortening the fermentation time, there is the advantage of shortening the total fermentation time, but the biggest advantage is that the risk of bacterial contamination can be completely eliminated. In other words, in continuous fermentation, once normal fermentation has started, the inside of the device is maintained at positive pressure by the fermentation gas, and there is little risk of contamination with bacteria, but until normal fermentation has started, sufficient care must be taken to avoid contamination with bacteria. Must be. Therefore, shortening the springing time will lead to eliminating the risk of bacterial contamination. The present invention will be explained in more detail below. Known acetone/butanol producing bacteria can be used in the present invention. For example, various fungi belonging to the genus Clostridium, namely Clostridium acetobutylicum, Clostridium saccharoperbutyacetonicum, Clostridium saccharoperbutyacetonicum, and Clostridium saccharoperbutyacetonicum. Acetone-butanol-producing bacteria belonging to C. cum (saccharobutylicum), Clostridium saccharobutylacetonicum, Clostridium saccharoacetobutylicum, etc. are used. Specifically, Clostridium acetoptylicum
ATCC824, 4259, 10132, IFO3854, Clostridium satucaloperptiacetonicum
Examples include ATCC27122. Immobilization of acetone-butanol-producing bacteria can be performed by a general method. For example, a carrier binding method, a crosslinking method, a gel entrapment method, etc. can be applied. Among these, the gel entrapment method is preferably used because it facilitates immobilization and provides stable activity. Calcium alginate gel, polyacrylamide gel, collagen, fipurin, agar,
There are methods using carrageenan, cellulose, etc. The operation of entrapping with these gels may be carried out by known operations, but specific examples are as follows. That is, by adding live microbial cells or a culture solution to be included in a sodium alginate aqueous solution to prepare a mixture, and dropping the mixture dropwise into an aqueous solution of a gelling agent such as calcium chloride, immobilized microbial cells can be obtained. When the third component is added, the third component may be further included in the mixture. Encapsulation by polyacrylamide gel involves suspending acrylamide monomer, N,N ¹ -methylenebisacrylamide as a crosslinking agent, and live bacterial cells (which may be used as a culture medium) in a buffer solution, and adding a polymerization initiator to the suspension. Immobilized bacterial cells are obtained by adding ammonium persulfate and N,N,N ¹ ,N ¹ -tetramethylethylenediamine as a polymerization accelerator and carrying out a polymerization reaction at 15 to 23°C for about 10 minutes. According to the present invention, the immobilized bacterial cells prepared as described above are first cultured in a medium in a batch manner, and then the medium is continuously supplied to carry out acetone-butanol fermentation. This continuous fermentation is performed by filling a reaction tower with immobilized bacterial cells and continuously supplying a medium (fermentation medium), but batch culture may be performed using a separate device outside the reaction tower. Alternatively, the reaction may be carried out in the reaction tower. Considering the operational effort, it is preferable to perform batch culture in the reaction tower and then switch to continuous fermentation. As the medium for batch culture and the medium for continuous fermentation, the same ones can be used. That is, either a synthetic medium or a natural medium can be used as long as it contains a suitable amount of a nitrogen source, inorganic substances, and other nutrients in addition to the main carbon source. As the carbon source, various carbohydrates such as glucose, sucrose, fructose, mannose, starch, starch hydrolyzate, and blackstrap molasses can be used. Nitrogen sources include ammonia, ammonium chloride,
various inorganic and organic ammonium salts such as ammonium sulfate, ammonium carbonate, ammonium acetate or urea and other nitrogen-containing compounds;
Also peptone, meat extract, yeast extract, corn steep liquor, casein hydrolyzate,
Various natural products can be used, such as fish meal or its digested product, defatted soybean meal or its digested product, and pupa hydrolyzate. Furthermore, as an inorganic substance,
Potassium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, calcium carbonate, etc. can be used. As the culture medium, a solution containing a considerable amount of suspended matter produced from Hiomas etc. can also be used. An example of such a liquid is found in JP-A-53-136585. The temperature, pH, carbon source concentration, etc. of batch culture and continuous culture are not particularly limited as long as the growth of bacteria and the production of acetone and butanol are carried out appropriately.
~38℃, pH around 6.5, carbon source concentration 4~10% (w/
v) (in terms of glucose) is preferred. The termination of batch culture or the switch to continuous fermentation is usually carried out at or slightly after the start of fermentation. Continuous fermentation can be carried out in upflow or downflow mode. There are no particular limitations on the medium supply rate, but a suitable rate is 0.1 to 0.4/hr per 1 packed and immobilized bacterial cell. Furthermore, in order to increase the utilization rate of the supply base, a plurality of reaction towers may be connected in series. Next, examples of the present invention will be shown. Example 1 (1) Preparation of immobilized bacterial cells A small amount of soil stock of Clostriderium satucaloperptiacetonicum ATCC 27022 was placed in 10 ml of an activation medium having the following composition in a test tube and heated in boiling water for 1 hour. minute heat treatment) so-called heat treatment
I was shocked. Next, it was cooled and cultured anaerobically at 30°C for 24 hours. Then, the obtained activated culture solution was
Seed culture medium with the following composition in a ml Erlenmeyer flask
The mixture was added to 150 ml and cultured anaerobically at 30°C for 24 hours. On the other hand, add 3% sodium alginate aqueous solution.
The mixture was sterilized at 110°C for 10 minutes, and after cooling, a certain amount of the above seed culture solution was added, and the mixture was dropped into a 1% calcium chloride aqueous solution to obtain immobilized bacterial cells. Activation medium: 150g potato, 3g glucose, 0.6g ammonium sulfate, 1.2g calcium carbonate and 420g water
Consists of ml. Seed culture medium: Molasses 6g/dl (as glucose), ammonium sulfate 0.5g/dl, calcium carbonate 0.3g/dl
dl, lime superphosphate 0.03 g/dl (2) Brewing period when continuous fermentation is performed immediately Addition ratio of seed culture solution to 3% sodium alginate aqueous solution at the time of immobilized bacterial cell preparation (v/
v) was set to 1%, 5%, 10%, and 20%. Fill 60ml of immobilized bacterial cells into a reaction tower as shown in Figure 1, and fill with the following fermentation medium.Immobilized bacterial cells 1
The water was continuously fed at a feed rate of 0.2/hr per hour, and the welling up state in the reaction tower was visually examined. The fermentation temperature was 32°C, and the medium was supplied in an ascending manner. The results are shown in Table 1.

[Table] Fermentation medium: Molasses 6g/dl (as glucose), ammonium carbonate 0.5g/dl, calcium carbonate 0.3g/dl
dl, superphosphate lime 0.03 g/dl (3) When performing batch culture 60 ml of the immobilized bacterial cells from (2) above are filled into a 100 ml reaction tower as shown in Figure 1, and the above (2) is carried out. A fermentation medium with the same composition was supplied to make the total volume 100 ml, and batch culture was carried out at 32°C. The springing time was as shown in Table 2.

[Table] From a comparison of Tables 1 and 2, it can be seen that the fermentation time is faster when batch culture is carried out than when continuous fermentation is carried out immediately. (4) When continuous fermentation is performed after batch culture, the immobilized bacterial cells obtained by adding the seed culture solution at a rate of 10% as described above
60 ml was filled into a 100 ml reaction tower, and the fermentation medium was supplied to bring the internal volume of the reaction tower to 100 ml, and batch culture was performed at 32°C. From 20 hours after the start of culture, the fermentation medium was continuously supplied in an upward flow manner at a rate of 0.2/hr per immobilized bacterial cell to start continuous fermentation. The fermentation temperature was 32°C. Figure 2 shows the yield of solvent (acetone, putanol and a small amount of ethanol) and residual sugar (as glucose) when continuous fermentation was carried out for 21 days.

[Brief explanation of drawings]

FIG. 1 shows an example of a fermentation apparatus used in carrying out the present invention. Each number in the figure has the following meaning. 1: Supply sugar solution, 2: Metering pump, 3: Reaction tower,
4: Immobilized bacterial cells, 5: Receiver Figure 2 shows the relationship between the number of fermentation days, yield of solvent (acetone/butanol and a small amount of ethanol), and residual sugar when continuous fermentation is performed according to the present invention.

Claims (1)

[Claims] 1. In a method for producing acetone and butanol using acetone/butanol producing bacteria immobilized on a carrier, the immobilized bacteria are first cultured in a batch manner, and then fermentation is carried out by continuously supplying a medium. Characteristic methods for producing acetone and butanol.