JPH08291103A - Increase in yield of acetic acid by cellulase - Google Patents

Abstract

PURPOSE: To obtain clear high-quality acetic acid in high yield by making a cellulase exist during fermentation of acetic acid and culturing crystalline cellulose produced by lactic acid bacteria while hydrolyzing. CONSTITUTION: A cellulase (e.g. a cellulase having crystalline cellulose hydrolyzing activity, an enzyme of a cellulase producing fungus such as Trichoderma) is made to exist in fermentation of acetic acid and acetic acid bacteria are cultured while hydrolyzing crystalline cellulose produced by acetic acid bacteria belonging to the genus Acetobacter or Gluconobacter to give acetic acid. When cellulose is adsorbed on an anion exchanger and culture is carried out with shaking and stirring so as to always bring a culture solution of lactic acid bacteria into contact with an immobilized cellulase reacted with a polyfunctional reagent, the immobilized cellulase and immobilized lactic acid bacteria are obtained. The reaction can be continuously carried out while bringing the culture solution into contact with the immobilized cellulase and the immobilized lactic acid bacteria.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for increasing acetic acid yield by cellulase for culturing acetic acid bacteria to produce acetic acid.

[0002]

2. Description of the Related Art Generally, the method for producing acetic acid using acetic acid bacteria is roughly classified into a surface fermentation method and a full-scale fermentation method.

Of these, in the surface fermentation method, since the stirring and aeration of the fermentation solution are carried out gently so as not to destroy the pellicle on the surface of the solution, there is an advantage that acetic acid of clear quality can be obtained. However, the brewing period is from one month to several months, and there is a risk of spoilage during that period.

On the other hand, in the full-scale fermentation method, although it is possible to raise the concentration to a high concentration of, for example, about 5% in a short period of about 10 days, the acetic acid obtained tends to contain a large amount of impurities consisting of cellulose and microbial cells. It becomes turbid and of poor quality, which requires the addition of an adsorbent followed by filtration to obtain clear acetic acid. However, the filtration efficiency is not good because cellulose as an impurity contained in acetic acid obtained by the full-scale fermentation method forms a gel film and clogs the filter medium. This is in contrast to the fact that the acetic acid obtained by the surface fermentation method is so clear as to require no filtration in some cases, and the produced cellulose membrane contains a large amount of fermentation liquid, and this fermentation liquid is not recovered. It can be said that economic loss is also large.

[0005]

In recent years, large-scale factories have adopted a reactor-type production method, which is an improvement of the full-scale fermentation method, for mass production. In the case of this method, the production capacity of cellulose is increased. In order to cultivate non-living bacteria, strict temperature control and an atmosphere that is isolated from the surrounding environment are realized to minimize the generation of bacteria with cellulose production capacity. Management must be very strict and difficult to employ in small factories.

As another method for producing acetic acid bacteria, a method of culturing by adding cellulase to acetic acid bacteria has been reported, as disclosed in, for example, JP-A-63-74490.

This method shows a method for obtaining acetic acid bacteria at a high concentration in order to obtain crystalline cellulose. It is not an acetic acid fermentation in which ethanol is added to a culture solution, but a high acetic acid concentration state. The method of continuously using cellulase and acetic acid bacteria has not been sufficiently examined.

[0008] In addition, Rune standard
Et al. (Published in 1994, magazine “J.Bacterio”)
1 ”, 176, p. 665), the non-crystalline CMC produced by the acetic acid bacterium itself is used for the production of cellulose by the acetic acid bacterium.
There is also a report that cellulase (CMCase) that decomposes (carboxymethyl cellulose) is involved.

However, these reports are reported by Shigeru Yamanaka et al. (199
4th year, magazine "Chemistry and Biology", Vol. 32, p. 367), sheet-like crystalline cellulose acetate has excellent mechanical strength and is highly used as a speaker diaphragm material. So, while decomposing the non-crystalline cellulose,
It can be said that this is a part for obtaining crystalline cellulose with high purity and high yield.

[0010]

DISCLOSURE OF THE INVENTION It is an object of the present invention to find a method for obtaining clear, high-quality acetic acid in high yield while almost completely decomposing crystalline cellulose produced by acetic acid bacteria. The method for increasing the amount of acetic acid by cellulase according to the first invention is characterized in that cellulase is present during the fermentation of acetic acid and the crystalline cellulose produced by the acetic acid bacterium is decomposed and cultured.

Further, the method for increasing the yield of acetic acid by cellulase of the second invention is characterized by culturing while contacting the culture solution with the immobilized cellulase and the immobilized acetic acid bacterium,
The method for increasing the amount of acetic acid by cellulase of the third invention is characterized by adsorbing cellulase on an anion exchanger and adsorbing acetic acid bacteria on immobilized cellulase reacted with a polyvalent reaction reagent.

[0012]

[Function] When acetic acid is fermented and cellulase is present and cultured while degrading crystalline cellulose produced by acetic acid bacteria, cellulose of acetic acid bacteria is not produced, and clear acetic acid is easily obtained. Since the sugar resource used is used for the production of acetic acid bacteria and acetic acid, high-concentration culture and high-concentration production are possible, and a culture solution is prepared using immobilized cellulase and immobilized acetic acid bacteria. If the culture is carried out while contacting with the immobilized carrier, the reaction can be continued.

As the acetic acid bacterium used in the present invention, any one can be used as long as it can acetic acid-ferment with a bacterium belonging to the genus Acetobacter or Gluconobacter, and the cellulase used in the present invention. As the cellulase having the activity of degrading crystalline cellulose, an enzyme secreted by a cellulase-producing bacterium such as Trichoderma (CELLULYSIN ^™ , manufactured by Wako Pure Chemical Industries, Ltd.), etc., As the cellulase preparation, a crude enzyme preparation containing CMCase, β-glucosidase and the like may be used.

The cellulase reaction method of the present invention is to allow cellulase to act at the same time as culturing the acetic acid bacterium so that the acetic acid bacterium can decompose crystalline cellulose at the same time. Since cellulase tends to act in the early stage of crystalline cellulose production, in order to achieve acetic acid production without the accumulation of crystalline cellulose, it is necessary to use it in the early stage of crystalline cellulose production so that even a very small amount of cellulase is used. It is possible to achieve the purpose of the period.

As for the existing amount of cellulase, the production of crystalline cellulose was hardly observed even when the amount of cellulase was such that it was reacted in a test tube to decompose 8.2 × 10 ^-4 % of Avicel (crystalline cellulose) in 18 hours. Inferring from the examples, the reaction in the test tube was optimally 1 × 10 in 18 hours.
^An amount of cellulase that decomposes Avicel by ^-4 % or more will be sufficient.

As a comparative experiment, a cellulase (0.1%) containing a high concentration of cellulase (avicerase) having a high activity of decomposing crystalline cellulose, using 10 mg / ml of cellulose of acetic acid bacteria as a substrate, was used in the test. Was disassembled. The decomposition rate was 0.05 to 0.08% even after decomposition at pH 5 at 50 ° C for 18 hours. It has been shown that the produced cellulose of acetic acid bacterium having a high mechanical strength is difficult to be decomposed even by using cellulase having a strong crystalline cellulose decomposing activity.

When soluble cellulase is used,
An easy method is to add the enzyme solution of about 1% of enzyme powder obtained by powder-drying the culture filtrate, which is sterilized by using a sterilizing filter, and simultaneously adding it to the culture. In addition, the same effect can be obtained by introducing a gene encoding avicelase onto the gene of acetic acid bacterium.

Here, when the immobilized cellulase and the immobilized acetic acid bacterium are used, a sterilized immobilized cellulase which easily adsorbs the acetic acid bacterium is added at the same time as the culture, and the immobilized cellulase is constantly contacted with the culture solution of the acetic acid bacterium. As described above, by culturing with shaking, stirring, or circulating, the immobilized cellulase and the immobilized acetic acid bacterium in which the immobilized cellulase adsorbs the acetic acid bacterium can be obtained. Sterilized cellulase is a method of gas sterilizing with formalin, ethylene oxide, or the like, or as shown in the examples, sterilized cellulase is adsorbed on an anion exchanger sterilized by pressure steam, and sterilized multivalent. Immobilized cellulase that has been dried under a germicidal lamp while aerating sterile reaction gas with sterilizing air may be used.

As the immobilization carrier, diaion H
PA-25, WA-21 (Mitsubishi Kasei), Diolite A-4, A-7 (Sumitomo Chemical), Spherocyl QMA
(Made by Ron-Ne-Roulen), Biosupport CPG500
(Electronucleonic Co., Ltd.), Celite biocatalyst support R635 (Manville Co., Ltd.) and the like are used.

As the above carrier, a carrier of polystyrene or celite having a pore size of 100 angstroms or more and having a wide area for cellulase binding is used. Besides, since many anion exchange groups have been introduced,
Even under a low pH condition in which acetic acid is present, it is possible to lower the proton concentration in the microenvironment on the surface of the carrier by the electrostatic repulsion of cations such as amino groups. Due to the above-mentioned microenvironmental effect, the environment around the cellulase and acetic acid bacteria bound to the carrier does not become so low in pH even under a high concentration of acetic acid, and the carrier has an advantage that the activity can be maintained for a long time. is there.

Further, since the acetic acid bacterium is easily adsorbed to the above-mentioned immobilized carrier, the immobilized acetic acid bacterium can be easily obtained by adsorbing the acetic acid bacterium to the immobilized cellulase, and the continuous use of the immobilized cellulase and the acetic acid bacterium. Acetic acid fermentation is also possible.

For other culture conditions, medium composition, etc., known methods used for ordinary acetic acid fermentation (issued in 1957,
Asai and Takai, Magazine "Fermentation Engineering", Vol. 35, p. 229)
The latest technology for high-concentration fermentation (published in 1988, written by Akihiko Mori, magazine "Microorganisms", 4
Vol., P. 229).

[0023]

EXAMPLES The present invention will be described in detail below with reference to examples.

[Example 1] Cellulase is a cellulase having a strong crystalline cellulose degrading activity (avicellase activity) (CELLULYSIN ^™ , manufactured by Wako Pure Chemical Industries, Ltd.).
Was dissolved in 1/15 mol of McIlvain buffer of pH 4.5, and when it was added to the medium as 1% concentration, it was sterilized by filtration with a sterilizing filter (0.22 μm Millipore).

For culturing acetic acid bacteria, yeast extract was added to 0.
5%, glucose 3.0% and polypeptone 2.0%
After sterilizing the culture medium containing the above with autoclave, ethanol filtered through a sterilizing filter was sterilized to a concentration of 4.0% by weight, and ethanol was added continuously. Acetobacter is Acetobacterium xylinum (RIKEN preservation number JCM7644
ATCC 23767) was used. A slant medium was prepared by adding 2% of powdered agar to the above medium. When using,
As a preculture, one inoculated ear was taken in 5 ml of medium and shaken overnight at 30 ° C. to inoculate a culture solution at 1%.

To a 200 ml Erlenmeyer flask, 50 ml of the above-mentioned medium, 5 ml of sterilized cellulase solution, and 0.5 ml of precultured mother liquor were added, and static culture was carried out at 30 ° C.
Sampling on days 1, 2, 3, 4, 7, 10
The ethanol concentration was adjusted. The results of culture on the 10th day are shown in Table 1. The comparative example is the result of static culture in which cellulase was not added (5 ml of 1/15 McIlbein buffer solution of 5 ml was added). Acetic acid and ethanol are
It was measured by gas chromatography using methanol as an internal standard. The cell weight was obtained from the amount of reduction of the wet weight obtained by centrifugation when the alkali heat treatment was performed, and the measurement of crystalline cellulose was obtained from the residual wet weight after the alkali heat treatment.

[0027]

[Table 1]

As is clear from Table 1, by adding soluble cellulase and statically culturing, almost no crystalline cellulose was produced, the cell yield was increased to 123%, and the acetic acid yield was 138%. Increased. Incidentally, according to the observation with a transmission electron microscope, in the comparative example, it was found that a large amount of crystalline cellulose was emitted from the side surface of the bacterial cell, but in the system to which cellulase was added, an amorphous secretory substance was observed. However, no fibrous structure was observed.

Example 2 In this example, acetic acid bacteria were cultured in the same manner as in Example 1 except that the culture was carried out with shaking. The results of the 10th day of culture are shown in Table 2.

[0030]

[Table 2]

As is clear from Table 2, when the soluble cellulase was added and shake-cultured, crystalline cellulose was hardly produced and the cell yield and acetic acid yield were increased. Comparing the increased amount with Table 1, the cell yield was 1
Shaking culture was higher than static culture at 40% and acetic acid yield of 132%.

[Example 3] In this example, the immobilized cellulase was prepared by using the same 1% cellulase solution as in Example 1 with an anion exchanger (Diaion HPA25 manufactured by Mitsubishi Kasei) sterilized under pressure steam as a carrier. Cellulase was adsorbed to the carrier by adding 1 ml per 1 g of the carrier and shaking for 18 hours.
Then, divalent reaction reagent glutaraldehyde was added to 1/1
1. With 5 mol of McIlvein buffer (pH 4.5)
0.8m per 1g of carrier after adjusting to 5% and sterilized
1 was added and shaken for 10 minutes to react. Next, sterilized 20% sodium bisulfite solution is added to 0.2 g per 1 g of the carrier.
ml was added and shaken for 10 minutes to decompose excess glutaraldehyde. Finally, washing with water, filtration and air-drying were performed under a germicidal lamp while performing suction filtration using sterile air.

50 ml of the culture solution was placed in a 200 ml Erlenmeyer flask, 5 g of the immobilized cellulase prepared by the above-mentioned method and 0.5 ml of the seed liquor of acetic acid bacteria were added, and the mixture was shaken and cultured as in Example 2. Since the acetic acid bacterium was easily adsorbed to the above-mentioned immobilized carrier, the acetic acid bacterium was adsorbed to the immobilized cellulase, and the immobilized acetic acid bacterium was easily obtained. The results of culturing with immobilized cellulase and immobilized acetic acid bacteria for 10 days are shown in Table 3.

[0034]

[Table 3]

As is clear from Table 3, crystalline cellulose was hardly produced even with the immobilized cellulase, and the cell amount and acetic acid production increased. Although the amount of increase is smaller than that in Table 2, the immobilized cellulase and the immobilized acetic acid bacterium can be repeatedly used, so that the amount can be increased or continuous fermentation can be easily performed.

[0036]

INDUSTRIAL APPLICABILITY As described above, the present invention is capable of almost completely decomposing crystalline cellulose accompanying acetic acid fermentation, contributing to improvement of filterability and clarification of acetic acid, and was applied to cellulose production. Since the energy can be applied to the production of acetic acid, the yield of acetic acid can be improved. Furthermore, since the fermentation membrane, which was conventionally difficult to collect because it was contained in the cellulose membrane, lost the cellulose membrane, the acetic acid recovery rate was improved. Can be planned.

Further, in the culture surface, also called konjac bacterium, since the whole fermentation method can be applied even under the growth conditions of a strong cellulose-producing bacterium such as Acetobacterium xylinum which was a harmful bacterium at the time of acetic acid fermentation, the konjac bacterium can be applied. The labor of strict production control to prevent the generation of lactic acid can be omitted, and high-concentration fermentation of acetic acid can be facilitated even in a small-scale factory.Furthermore, cellulase and acetic acid bacteria can be reduced in pH even in the presence of acetic acid. Since it is immobilized on a carrier with a small microenvironmental effect, it greatly contributes to the efficiency of high-concentration continuous fermentation.

The above-mentioned intended purpose can be sufficiently achieved.

 ─────────────────────────────────────────────────── ─── Continued Front Page (72) Inventor Katsuji Kasahara 2-7-13 School Town, Mitsuke City, Niigata Prefecture Niigata Prefectural Industrial Technology Center, Mitsuke Proving Ground

Claims (3)

[Claims] 1. A method for increasing the yield of acetic acid by cellulase, which comprises culturing while degrading crystalline cellulose produced by an acetic acid bacterium in the presence of cellulase during fermentation of acetic acid. 2. The method for increasing the amount of acetic acid by cellulase according to claim 1, wherein the cellulase and the immobilized acetic acid bacterium are cultured while contacting the culture solution. 3. The method for increasing the amount of acetic acid by cellulase according to claim 2, wherein the cellulase is adsorbed on an anion exchanger, and the acetic acid bacterium is adsorbed on the immobilized cellulase reacted with the polyvalent reaction reagent.