JPS6274282A - Cultivation of obligatory anaerobic bacteria - Google Patents

Abstract

PURPOSE:To rapidly cultivate obligatory anaerobic bacteria in a high concentration, by cultivating the bacteria in a liquid culture medium mixed with a gelatinized natural polysaccharide. CONSTITUTION:A natural polysaccharide, e.g. agar, carrageenan or alginic acid, capable of forming gel is gelatinized and obligatory anaerobic bacteria, e.g. bacteria of the genus Clostridium or Eubacterium or sulfuric acid reducing bacteria, are cultivated in a liquid culture medium while bringing the gelatinized natural polysaccharide into contact with the liquid culture medium. The growth rate of the obligatory anaerobic bacteria reaches about 1.2-2.5 times of that obtained by ordinary liquid cultivation and the maximum yield of the bacterial cells reaches about 5-17 times that obtained by ordinary liquid cultivation.

Description

DETAILED DESCRIPTION OF THE INVENTION Field of Industrial Application The present invention relates to a method for culturing obligate anaerobic bacteria. Specifically, the present invention relates to a method for rapidly culturing obligate anaerobic bacteria at a high concentration by mixing a gelled natural polysaccharide and a liquid medium, and culturing microorganisms in the liquid medium.

[Conventional technology and problems]

Obligate anaerobic bacteria cannot grow in the presence of oxygen, so
All culture operations must be performed under anoxic environment (anaerobic conditions). In addition to the difficulty of culturing caused by this, obligate anaerobic bacteria generally have lower growth rates and lower maximum cell concentrations than other microorganisms such as aerobic bacteria, and obligate anaerobic bacteria at high concentrations Obtaining anaerobic bacterial cells is often difficult. For this reason, obligate anaerobic bacteria, aerobic bacteria, etc.
Compared to other microorganisms, research is currently lagging behind in both basic fields such as biochemistry and physiology, as well as applied fields such as fermentation engineering.

[Means for solving problems]

The present inventors have conducted a series of studies on high-concentration culture of microorganisms such as obligate anaerobic bacteria.

As a result, it was discovered that by bringing the natural polymorph gel into contact with a liquid medium and growing microorganisms in the liquid medium, it is effective in improving the growth rate and maximum bacterial cell yield.Based on this, the present invention was developed. I ended up completing it. That is, the present invention is a method for cultivating obligate anaerobic bacteria in a liquid medium by gelling a gel-forming natural polysaccharide such as agar, galrageenan, or alginic acid, and contacting the gel with a commonly used liquid medium. The present invention provides a novel method for culturing anaerobic bacteria.

The obligate anaerobic bacteria used in the present invention refers to bacteria that cannot grow in the presence of oxygen among so-called anaerobic bacteria that grow under anoxic conditions, such as Clostridium. Bacteria belonging to the genera Eubacterium (E(11)3Cterium), Acetobacterium, Selenomonus, Butyribacterium, etc., methanobacteria, sulfate reduction Examples include bacteria and most photosynthetic bacteria.

The natural Pis used in the present invention include agar,
Any natural polysaccharide having gel-forming properties such as galrageenan, alginic acid, xanthan gum, and locust bean gum can be used. Among these, agar, galrageenan, and alginic acid are preferred. These natural polysaccharides can be used singly or in combination of several types.

The method for forming the natural poly(+)In gel in the present invention may be performed using any known method depending on the type of natural poly(vMR) used. For example, in the case of agar, after heating an agar-containing solution of an appropriate concentration, Also, is alginic acid suitable? A gel can be obtained by contacting a solution containing agricultural alginic acid with a solution of a divalent or trivalent metal salt such as a calcium salt, iron salt, or aluminum salt. Furthermore, by adding medium components and other specific components to the gel, the effect on the growth rate and maximum bacterial cell yield can be further improved. The natural polysaccharide concentration of the gel can be arbitrarily selected as long as it is at least the minimum concentration that allows gel formation. An appropriate concentration may be selected depending on the type of i. Although the volume ratio of the gel to the liquid medium can be selected arbitrarily, it is usually preferable that the volume of the gel is 1 to 5 times that of the liquid medium. The shape of the gel can be arbitrary, and in addition to forming a gel layer at the bottom of the culture vessel and culturing by layering the liquid medium, it is also possible to use gel molded into any shape such as spherical or rectangular and floating it in the liquid medium. You can also.

The liquid medium used in the present invention may be any liquid medium in which obligate anaerobic bacteria can grow. For example, Acetobacterium
erium), Eubacterium
rium) genus, Butyri-b.
For bacteria belonging to the genus A.
ivesof Microbiology) 120L2
01-204 (1979) is used.

The culture temperature may be any temperature as long as the bacteria to be cultured can grow, but it is preferable to use the optimal growth temperature for each bacteria.Culture must be performed under anoxic conditions. , Other conditions are optional, and depending on the type of bacteria to be cultured, cultivation can be done by methods such as standing still, stirring, gas aeration, shaking, etc. under conditions suitable for each type of growth, and continuous cultivation can be performed. is also possible.

The present invention relates to a method for growing bacterial cells at a high concentration only in a liquid medium without the presence of bacterial cells in a gel, and the immobilization method of enclosing bacterial cells in a gel is the purpose·
The method is also different.

〔Effect of the invention〕

By using the present invention, the growth rate of obligate anaerobic bacteria in liquid culture is about 1.2 to 2.5 times that of normal liquid culture, and the maximum bacterial cell yield is about 1.2 to 2.5 times that of normal liquid culture. Approximately 5-17
It is possible to double the amount and obtain extremely high concentrations of obligate anaerobic bacterial cells.

〔Example〕

The present invention will be explained below with reference to Examples, but the present invention is not limited to these Examples.

Example 1 100 ml of a solution prepared by adding 4 g of agar to the "methanol D medium" shown in Table 1 was placed in a 500 ml flask, brought to an anaerobic state in an atmosphere of 67% nitrogen and 33% carbon dioxide, and sealed with a butyl rubber stopper. Thereafter, the mixture was autoclaved at 120° C. for 20 minutes, and allowed to cool and solidify at room temperature to form an agar gel layer containing medium components on the bottom of the flask. "Methanol D liquid medium" 100 which was subjected to anaerobic operation and sterilization operation separately
m1 was added in a glove box, and the liquid medium was layered on top of the agar gel layer. This was supplemented with Acetobacterum wo, which had been precultured for 3 days in methanol/D liquid medium °.
odiii) DSM1030. Acetobacterium woodii
05M2396+Butyribacterium methylotrophicum (Butyribacterium IIImeth)
ylotro-phicum) 8TCC33266, Eubacterium limousum
1imosum)05M20543. Clostridium formicoaceticum (C1ost-ridiu)
m formicoaceticu+w)05M205
Approximately 0.3 to 1 liter of obligate anaerobic bacterial strains of 43 4 genera, 4 species, 5 strains
The cells were inoculated under anaerobic conditions and cultured at 30° C. under static conditions. The culture medium for Clostridium formicoaceticum was prepared by adding 10 g of fructose instead of methanol. In addition, as a control experiment, culture using only a liquid medium without the addition of agar gel was simultaneously conducted under the same conditions.

The bacterial cell concentration during the culture was measured using a spectrophotometer.
The absorbance was measured using an ICI+ cell at a wavelength of .

Table 2 shows the growth rate and maximum cell yield for each strain. In both cases, the presence of agar gel resulted in clearly higher growth rates and maximum bacterial cell yields than in normal liquid culture, especially for Acetobacterium utdii DSM.
1030, the growth rate reached approximately 2.2 times and the maximum bacterial cell yield reached approximately 5.2 times.

Table 2 Culture results Example 2 Using Acetobacterium utsudi DSM2396,
The procedure was carried out in the same manner as in Example 1 except that k-garrageenan was used instead of agar as the gelled natural polysaccharide. As a result, the growth rate and maximum bacterial cell yield were 3.65 and 12 in the medium containing k-garrageenan gel. .4, about 1.9 times and about 6.7 times that of normal liquid culture, respectively.

Example 3 Using Acetobacterium utsudii DSM2396,
Example 1 except that agar gel without added medium components was used.
As a result of carrying out the same procedure as above, the growth rate and maximum bacterial cell yield were as follows.
In the medium containing agar gel, the values were 3.27 and 4.05, which were approximately 1.7 times and 2.2 times that of normal liquid culture, respectively.

Example 4 Using Acetobacterium utsudii DSM2396,
The procedure was carried out in the same manner as in Example 1, except that the volume ratio of the agar gel containing the medium components and the liquid medium was 5:1. As a result, the growth rate and maximum bacterial cell yield were 4.3 for the medium containing the agar gel.
and 30.9, respectively, which is about 2.3 of normal liquid culture.
This amount reached approximately 16.6 times.

Claims (1)

[Scope of Claims] 1. A method for culturing obligate anaerobic bacteria, which comprises bringing a gelled natural polysaccharide into contact with a liquid medium and culturing obligate anaerobic bacteria in the liquid medium. 2. The method for culturing obligate anaerobic bacteria according to claim 1, wherein the gel-forming natural polysaccharide is at least one selected from agar, galrageenan, and alginic acid.