JPS5988093A - Production of acetone and butanol - Google Patents

Abstract

PURPOSE:To shorten foaming time and to reduce fear of contamination by various germs, by immobilizing a bacterium capable of producing acetone and butanol to a carrier to give an immobilized mold, cultivating it by a batch method, feeding a medium to it continuously, fermenting it. CONSTITUTION:A bacterium producing acetone and butanol (e.g., Clostridium saccharoperbutyacetoniucum ATCC27022) is blended with an aqueous solution of sodium alginate, agar, etc., and the mixture is added dropwise to an aqueous solution of a gelatinizing agent (e.g., CaCl2), to give an immobilized mold. The immobilized mold is packed into a reaction column, cultivated by a batch method in a medium having 4-10w/v% (calculated as glucose) carbon source concentration at about 6.5pH at 28-38 deg.C, a medium is fed to it at 0.1-0.4l/hr speed based on 1l packed immobilized mold, and fermentation is carried out.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for continuous fermentation production of acetone and butanol using immobilized acetone/butanol producing bacteria.

There was a time when acetone-butanol fermentation was carried out in batches. However, the yield of acetone and butanol obtained by this method is only about 0.4 f/7 hr as a solvent, and the production of acetone and butanol has since been carried out by petrochemistry (1).

Recently, the production of various fermentation products using immobilized bacterial cells has been studied, and acetone-butanol fermentation has also been studied from this point of view [motθchn -010gy L
etters VOl, 2 (45), 241-24
6 (1980)).

The fermentation method adopted in the above literature is a method of repeated batch operation (repeated use in a bat).
chprocess).

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, When performing continuous fermentation, it takes a considerable amount of time, up to 9 days, before the grooved phenomenon (foaming due to fermentation gases such as hydrogen and carbon dioxide 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 required. Therefore, the present inventors further studied to shorten the oiling time and found that - (2) When culturing immobilized bacterial cells in a medium in batches prior to continuous fermentation, the grooved time can be significantly shortened. They discovered what could be done and completed the present invention.

There is an advantage in that the total fermentation time can be shortened by shortening the grooving time, but an even greater advantage is that the risk of bacterial contamination can be greatly reduced. 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. No.

Therefore, shortening the grooving time leads to reducing 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, Clostridium (C1ostri+iium)
) various fungi belonging to the genus Clostridium acetobutyllcum
l Clostridium saccharoper'butyacetonicum (3)
um ) l Clostridium saccharobutylacetonicum (ElaCCharObutylacetonicum) l Clostridium saccharobutylacetonicum (sacc
harobutylacetonicum) e Clostridium saccharoacetobutylicum
Acetone/butanol producing bacteria belonging to the genus etc. are used. Specifically, Clostridium acetobutylicum ATCC824, 4259110132, IFO385
41 Clostridium Saccharoperbutiacetonicum ATCIC27022 and the like.

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. Gel entrapment methods include calcium alginate gel, polyacrylamide gel, collagen, and fibrin.

Method using agar, carrageenan, cellulose, etc. (4) There is.

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.

Entrapment by polyacrylamide gel consists of acrylamide monomer, N + N'-methylenebisacrylamide as a cross-linking agent and live bacterial cells (may be as a culture medium).
is suspended in a buffer solution, and ammonium persulfate as a polymerization initiator and N, NI N as a polymerization accelerator are added to the suspension.
Add 'IN'-tetramethylethylenediamine to 1
Immobilized bacterial cells can be obtained by carrying out the polymerization reaction at 5 to 23°C for about 10 minutes.

According to the present invention, the immobilized bacterial cells (5) 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 carried out by filling a reaction tower with immobilized bacterial cells and continuously supplying a medium (fermentation medium), but batch culture can also be carried out 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.

Carbon sources include glucose and shakerose.

Various carbohydrates can be used, such as fructose, mannose, starch, starch hydrolysate, and blackstrap molasses.

Nitrogen sources include various inorganic and organic ammonium salts such as ammonia, ammonium chloride, ammonium sulfate, ammonium carbonate, ammonium acetate, or urea and other nitrogen-containing compounds, as well as peptone, meat extract, yeast extract, corn extract, etc. Various natural products such as steep liquor, casein hydrolyzate, fishmeal or its digested product, defatted soybean meal or its digested product, and soybean hydrolyzate can be used.

Further, as inorganic substances, potassium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, calcium carbonate, etc. can be used.

As the culture medium, a liquid containing suspended matter produced from biomass or the like can also be used. An example of such a liquid is disclosed in JP-A-5
3-136585.

Temperature, pH, carbon source concentration, etc. for batch culture and continuous culture are based on bacterial growth, acetone and butano-10%/V.
(in terms of glucose) is preferable.

(7) Termination of batch culture or switching to continuous fermentation is usually carried out at or slightly after the start of grooving.

Continuous fermentation can be carried out in upflow or downflow mode.

The medium supply rate is not particularly limited, but is suitably 0.1 to 0.4 //hr (11 hours F) of filled and immobilized bacterial cells.

Furthermore, in order to increase the utilization rate of the supplied medium, a plurality of reaction towers may be arranged in series.

Next, the implementation side numbers of the present invention are shown.

Example 1 (1) Preparation of immobilized bacterial cells Clostridium saccharoperbutiacetonium A
A small amount of TCC27022 soil stock was placed in an activation medium IQd having the following composition in a test tube and heat-treated in a boiling water bath for 1 minute (so-called heat 5hoclc).

Next, it was cooled and cultured anaerobically at 30°C for 24 hours. The obtained activated culture solution was then added to 150 d of a seed culture medium having the following composition in a 250 d Erlenmeyer flask, and cultured under air at 30° C. for 24 hours.

On the other hand, aqueous solution of sodium 3-thyalginate was heated to 110°C.
After sterilization for 0 minutes, a certain amount of the above seed culture solution was added after cooling, and the mixture was dropped into a 1% calcium chloride aqueous solution to obtain immobilized bacterial cells.

Activation medium: Potato 9150 f+ Dextrose 3fe Ammonium sulfate 0.6ft Calcium carbonate 1.2f and Water 420w
It's RL.

Seed culture medium: molasses 6 tldi (as glucose), amthonium sulfate 0, stldi, calcium carbonate 0°3
tldi.

Lime superphosphate 0.03 rAtl (2) Casting period when continuous fermentation is performed immediately The addition ratio (v/v) of the seed culture solution to the 3% sodium alginate aqueous solution during the preparation of immobilized bacterial cells is 1 es. %+1
o%, 20chi.

Immobilized bacterial cells 6011j were packed into a reaction tower as shown in Fig. 1, and the following fermentation medium was filled at a rate of 6.21 per 11 immobilized bacterial cells.
(9) The condition of oil in the reaction tower was visually inspected. Fermentation temperature: 32℃
The culture medium was supplied using an ascending method.

The results are shown in Table 1.

Table 1 Fermentation medium: Molasses 6 V'dl (as glucose), ammonium carbonate o, stldi, calcium carbonate o, 3
y/di.

Lime superphosphate 0.03 fi (3) When performing batch culture, the immobilized bacterial cells 6 (117) from (2) above were packed into a 100 WLt capacity reaction tower as shown in Figure 1, and the above (2) A fermentation medium with the same composition as above was supplied to make 100 d, and batch culture was performed at 32°C.

The grooving time was as shown in Table 2.

(lO) Table 2 A comparison of Tables 1 and 2 shows that the casting 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, immobilized bacterial cells 601Rt obtained with the above seed culture solution addition ratio of 10
was packed into a 100 d reaction tower, and the fermentation medium was supplied to bring the internal volume of the reaction tower to 100 d, and batch culture was carried out at 32°C. From 20 hours after the start of culture, the fermentation medium was added to the immobilized bacterial cells. Continuous fermentation was started by continuously feeding in an upward flow manner at a rate of 0.2 J/hr. The fermentation temperature was 32°C. Figure 2 shows the yield of solvent (acetone/butanol and low-t ethanol) and residual sugar (as glucose) when continuous fermentation was carried out for 21 days.

[Brief explanation of the drawing]

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. 1 l figure

Claims (1)

[Claims] 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 the culture medium is continuously supplied to carry out fermentation. ! A method for producing acetone and butanol as jgL.