JPH0423979A - Methane-producing bacterium and production of vitamin b12 using same bacterium - Google Patents

Abstract

PURPOSE:To industrially produce vitamin B12 and enable stable supply of vitamin B12 by culturing methane-producing bacterial H117-1-2 and KN-15 having high vitamin B12 producing ability and high proliferation rate and collecting vitamin B12 from the cultured mixture. CONSTITUTION:H117-1-2 (FERM P-11356) and KN-15 (FERM P-11420) being gas-assimilating methane-producing bacterium belonging to the genus Methanobacterium and capable of producing methane from carbon dioxide and hydrogen and having high vitamin B12 producing ability and high proliferation rate are sufficiently cultured using No.1-modified Balch culture medium. Then the bacterial cell is directly extracted with a solvent or crushed by a well-known method such as ultrasonic wave or enzyme treatment and then extracted and the cultured liquid is directly extracted with a solvent. Then the extracted liquid is further properly treated by well-known separation and purification means such as precipitation and recrystallization to collect the vitamin B12. Thereby a large number of vitamin B12 can industrially be obtained from the cultured mixture.

Description

Detailed Description of the Invention (Industrial Application Field) The present invention relates to Mesanobacterium genus bacteria newly isolated from the soil near the hot spring spout and vitamin B12 using the same.
This invention relates to a new industrial manufacturing method.

(Prior art and problems to be solved by the present invention)
Generally, when microorganisms are used industrially, the properties of the microorganisms used, particularly their ability to proliferate and produce useful substances, are important factors. Among these, the inventors found that when microorganisms are used industrially, the ability to produce useful substances, that is, the ability to proliferate is high to some extent.

They also focused on the fact that the higher the production capacity, the lower the cost of raw materials, the shorter the culture time, and the smaller the required culture equipment.

Furthermore, when culturing microorganisms, it is desirable that the concentration of salts such as NaCl be as low as possible from the viewpoint of preventing corrosion of equipment and reducing the cost of auxiliary raw materials, and microorganisms that require NaCl are not industrially advantageous.

On the other hand, a large amount of gas is emitted as industrial waste and as a by-product, and only a portion is recovered as an energy source, so its utilization cannot necessarily be said to be high. The gas contains carbon dioxide, which can serve as a carbon source for microorganisms, and hydrogen or carbon monoxide, which can serve as an energy source.

Therefore, its effective use is a major issue from the perspective of reducing the burden on the global environment. The inventors aimed to effectively utilize gas using microorganisms.

We focused on methane-producing bacteria. Among this group of microorganisms, there are some that can produce methane from carbon dioxide, hydrogen, and carbon oxide, so in order to obtain useful microorganisms, we need to develop gas assimilation ability that grows on carbon dioxide and hydrogen as substrates. We searched for methane-producing bacteria.

Methane-producing bacteria produce vitamin D2, a useful substance.
．． It is known that the content of microorganisms is higher than that of other microorganisms (Current Microbiol,
.

3, 243 (191110)).

Traditionally, carbon dioxide and hydrogen are utilized to produce methane.

So-called gas-assimilating methanogenic bacteria include Methanobacterium genus,
Methanobrevibacter (Methanobrevibacter)
cter), Methanothe
rmus), Methanococcus
cus), Methanomicrobium (Methanomi
genus Crobium, Mesanospirillum (Aethan)
os irilum), Methanogenium, Methanoc.
orpusculum), Int.
, J. 5yst, Bacteriol, 38
．． 212-219 (1988)).

However, even if these have a high growth rate, the optimum NaCl concentration is high or the NaCl concentration is low.
On the other hand, if the growth cannot be achieved with
64.2.115-137 (1986). Furthermore, there were problems such as those with high vitamin 812 production ability but extremely low growth rate, and high proliferation rate but low vitamin B12 production (Current Micro
biol, 3.243 (1980)).

Vitamin B1□ is an important compound used as a therapeutic drug for pernicious anemia, etc., and as a growth promoting substance for animals, such as in feed additives. However, due to its complex structure, it is currently produced by fermentation using glucose as a raw material. The microorganisms used are propionic acid bacteria, that is, members of the genus Propionibacterium and genus Bacillus.

Regarding the production method of vitamin B1□ using methanogenic bacteria, Methanosarcina barkeri (Methanosarcina barkeri) is produced using methanol as a raw material.
It has been reported that
94. JP-A-63-91015, BIOINDUSTR
Y, 5.3.190 (1988), Vitamin, Kurashi,
12.657 (1988)), although this microorganism can utilize gas, its growth rate is extremely low and it is not suitable for industrial use. The production of vitamin B1□ by gas-assimilating methanogenic bacteria is also caused by Methanobacterium thermoautotrophicum (Methanobacterium thermoautotrophicum).
um therm++oautotrophicum)
It is reported in ΔH (DSM 1053) (Showa 63
Collected abstracts of the Annual Conference of the Japan Society for Fermentation Engineering, p137 (19
88)), but due to its low ability to produce vitamin 812,
Its productivity is low.

In this way, in the conventional technology, vitamin D2 is not available industrially.
There was no established method for manufacturing 2.

(Means for Solving the Problems) The present invention has been made in view of the current state of the technology described above, with the aim of stably supplying vitamin B1□, the demand for which is increasing.

A method for industrially producing vitamin B1□ that achieves the above objective has been investigated from various angles such as organic synthesis and extraction from natural products, but no success has been achieved.

We decided to focus again on fermentation methods that use microorganisms.

Therefore, regarding methane-producing bacteria, which is also known as vitamin B1□-producing bacteria, we will first discuss NaC1
As a result of extensive searches for gas-assimilating methanogens with low demands and high growth rates, we were able to isolate a new methane-producing bacterium with unprecedented properties from the soil near hot spring vents. Successful.

When we investigated these methane-producing bacteria in more detail, we unexpectedly discovered that they have an unprecedentedly high ability to produce vitamin B1□. Moreover, by culturing these methane-producing bacteria, vitamin D2 can be extracted from the culture.
They also confirmed that 2 can be industrially harvested, leading to the completion of the present invention.

Hereinafter, one aspect of the present invention will be explained in detail.

(1) Isolation of gas-utilizing methane-producing bacteria For isolation of gas-utilizing methane-producing bacteria, Balch et al.
crobiol, Rev., 43.260-296
(1979)) was used (Table 1). Samples collected from various parts of the country are inoculated into a separation medium anaerobically,
By performing shaking culture and inoculating those that have been observed to grow into a new medium aseptically and anaerobically, we aim to accumulate the target gas-assimilating methane-producing bacteria, and then transfer them to a roll tube. method (the medium used is the one in Table 1 with agar added,
Colonies were formed using agar (agar concentration 2.0%), and the target strain was finally isolated from a single colony. The culture temperature is
Among the gas-utilizing methanogens mentioned above, the optimal temperature (50 to 70°C
), the temperature was set at 60°C. In addition, obligate anaerobes such as methanogenic bacteria were handled according to conventional methods.

Balch et al.'s modified medium Table 1 21 (PO4 to H2PO4 CNH4)2 so4 H4Cl aCI MgSO,・7H20 CaCL　”　2H20 MnSO4・2H20 FeS04・7H20 oC1z ZnSO.

CuSO4・5H20 AIK (So,).

HlBO.

Na2MoO4・2H2O NiC1, 6H2O Na2Sea.

Na210.・2) 12O Nitrilotriacetic acid NaHC
O□ Yeast extract (Difco) Trypt
icase (BBL) L-cystein-HCI-H2O Na2S・9H20 0,3g/Q C03 0,3 1,0 0,61 0°16 0.009 o,oos 0.003 0.001 0.001 0.0001 0.0001 0.0001 0.0001 0.001 0.0017 0.001 0.015 5.0 0.05 0.05 0.5 0.5 ■) Composition of trace vitamins Folic acid 2 Pyridoxine/HCI 10 Thiamin・H
CI 5 Riboflavin
5 Nicotinic acid 5 Calcium pantothenate 5 Vitamin B120.1 Aminobenzoic acid 5 (2) Method for measuring specific growth rate and vitamin Bi2 The medium used for measuring specific growth rate contained the medium components listed in Table 1. Yeast extract (yeast extra)
ct (Difco)) and trypticase (Tryp
ticase (BBL)) at a concentration of 2 g/Il. A culture solution of methanogenic bacteria that had been cultured in advance (absorbance at 660 nm (A, o) of 0.5 to 0.8 was placed in a 125-volume bottle containing 20 ml of medium.
) was aseptically and anaerobically inoculated and cultured with shaking (160 rpm) at a predetermined temperature. To track the increase in bacterial cell concentration, the culture solution was extracted over time and AGGO was measured.
The specific growth rate μ was determined by plotting on a graph. In addition, in order to supplement the gas, a search table for methane-producing bacteria (Fermentation Engineering, 64゜2.115-137 (19
86)) and literature (Int, J, 5yst.

Bacteriol, 38.212-219 (19
As a result of the search based on 88)), Mesanobacterium (
It was thought that it belonged to the genus Methanobacterium or Methanotherinus.

Next, since the optimal growth temperatures of H117-1-2 and KN-15 are 60°C and 65°C, respectively, these strains are not of the genus Mesanothermus.

It was thought to be a microorganism belonging to the genus Mesanobacterium. - As shown in Table 2, a comparison was made with two strains that belong to the genus Mesanobacterium and have an optimal growth temperature of 50 to 70°C.

H117-1-2 KN-15 Yumai Shiken DSM 1105 3tiolfe iDS 2970 Morphology during growth MipFI Methane substrate H, /4C00H CH3coDH CI (30H α to μm, (h-1) GC content (%) Motility 6 .0~8.5 6.0~8.8 7.4~7.5 7.4~7.8 0.45 (60°C) 0.62 (65°C) 7.2~7.6 0. 43 rate 48-52 7.0-7.7 Fist) In the medium used for specific growth rate measurement, o, 4h-1
Met.

As a result of the comparison in Table 2, H117-1-2 is a thermoautotrophicum of the genus Methanobacterium.
hicum), and tyolfei
Methanobacterium thermoautotrophicum is similar to Methanobacterium thermoautotrophicum in terms of substrate assimilation, optimal temperature, and optimal pH, but its specific growth rate is considerably higher than that of Wolfei (%1olfei).
iu+s thermoautotrophicun+
) is considered to be close to. However, it is considered to be different from Mesanobacterium thermoautotrophicum DSM 1053 because of its cell morphology and optimum growth temperature of 60°C.

In addition, KN-15 showed a growth rate of 0.62h-'', which was higher than the growth rate of Mesanobacterium wolfei.
It has distinctly different properties, being much higher. In addition, when compared with Mesanobacterium thermoautotrophicum, the growth rate and cell morphology are clearly different.

Growth rate is particularly important for methanogenic bacteria as a phenotype of overall cellular metabolism. From the above, H117
-1-2 and N-15 were identified as new species belonging to the genus Mesanobacterium and close to Mesanobacterium thermoautotrophicum. The physiological properties of these bacteria are shown in Tables 3 and 4.

These bacteria were submitted to the Institute of Microbial Technology, Agency of Industrial Science and Technology [Microorganism accession number, Microbiological Research Institute No. 11356 (FERM
P-11356) and Microtechnical Research Institute No. 11420 (FE
The storage of microorganisms was entrusted to RM P-11420)J.

1 (117-1-2), no growth was observed in a medium with a high NaCl concentration (2% or more), and a high growth rate was obtained with a low NaCl concentration (0.1% or less).

The high growth rate of the KN-15 strain is due to the low N
It is obtained in aC1 concentration medium, but not in high NaC1 concentration medium, and the proliferation of KN-15 is inhibited.

Table 3 Physiological properties of 8117-1-21. Morphological properties: The medium composition is based on meat juice and meat juice agar, but for those that do not grow on these, an appropriate medium may be used. Ba1ch Na is shown separately because of the noise.
1 modified medium was used.

■Shape: Bacillus longus (logarithmic growth phase), no flagella Size: 0.3
~0.5X1-3 ■Cell pleomorphism: None ■Motility: None ■Spore formation: None ■Durham staining: Positive 2, cultural properties; Those that do not grow in the following media are suitable for other growth Describe the growth status in the appropriate medium.

Therefore, the modified Balch & 1 medium shown separately was used as the basic medium, and colonies were formed by the roll tube method for colony observation.

■ Broth medium; no growth ■ Modified Balch Na 1 agar medium (culture temperature 60°C
, culture time: 5 days); Good growth, smooth periphery, light greenish milky color, diameter 2-4 mm, no pigment formation 3, Physiological properties: ① Utilization of inorganic nitrogen source; Uses ammonium salts.

■Production of pigment: After culturing, the liquid medium becomes yellow-green, which is characteristic of methanogenic bacteria.

■Growth range temperature: 40-70℃, below 40℃, no growth above 75℃ Optimum growth temperature: 60℃ ■Growth range pH: 6.0-8.5 Optimum growth pH: 7.4-7.5 ■Oxygen Requirements: Absolutely anaerobic ■Generation of gas: (1) L-arabinose, (2) D-xylose, (3
) D-glucose, (4) D-mannose, (5) D
-fructose, (6) D-galactose, (7) maltose, (8) sucrose, (9) lactose, (1
0) )-levalose, (11) D-sorbitol, (1
2) Neither oxidation nor fermentation is performed from D-mannite, (13) inosit, (14) glycerin, and (15) starch.

■ Salt tolerance: Grows up to 2%.

■Usability of carbon sources: Methane is produced using CO□ (in the presence of H2), but formic acid, acetic acid, methanol, and methylamine are not used.

4. Separation source: Soil near the hot spring outlet Table 4 Physiological properties of KN-15 1. Morphological properties: The medium composition is based on meat juice and meat juice agar, but for those that do not grow on this, use an appropriate medium. may also be used. Because of the confusion, I have separately shown Ba
A 1ch Nα1 modified medium was used.

■Shape: Bacillus longus (logarithmic growth phase), no flagella Size: 0.3
~0.5X1-10 ■ Cell pleomorphism: None ■ Motility: None ■ Sporulation: None ■ Durham staining: Positive 2, culture properties: Those that do not grow in the following media are suitable for other growth Describe the growth status in the appropriate medium.

Therefore, the Ba1ch&1 modified medium shown separately was used as the basic medium, and colonies were formed by the roll tube method for colony observation.

■ Broth medium; no growth ■ Modified Balch Na 1 agar medium (culture temperature 60°C
, culture time: 5 days); Good growth, smooth periphery, pale milky color with a green tinge, diameter 2-5 mm, no pigment formation 3. Physiological properties: ■Use of inorganic nitrogen sources; use of ammonium salts.

■Production of pigment: After culturing, the liquid medium becomes yellow-green, which is characteristic of methanogenic bacteria.

■Growth range temperature: 40-70℃, below 40℃, no growth above 75℃ Optimum growth temperature: 65℃ ■Growth range pH: 6.0-8.8 Optimum growth pH: 7.4-7.8 ■Oxygen Requirements: Absolutely anaerobic ■Generation of gas: (1) L-arabinose, (2) D-xylose, (3
) D-glucose, (4) D-mannose, (5) D-
Fructose, (6) D-galactose, (7) Maltose, (8) Sucrose, (9) Lactose, (10
) )-levalose, (11) D-sorbitol, (12
) D-Mannit, (13) Inosit, (14) Glycerin, (tS) No oxidation or fermentation from starch.

■ Salt tolerance: Grows up to 2%.

■Usability of carbon sources: Co-(in the presence of uz) is used to produce methane, but formic acid, acetic acid, methanol, and methylamine are not used.

4. Separation source: soil near the hot spring spout After these old 17-1-2 and/or Ni-I5 are sufficiently cultured as described above, vitamin 8□2 is collected from the culture. Conventional methods are used as appropriate for this purpose; for example, in addition to extracting the bacterial cells directly with a solvent, they are crushed using known physical or chemical methods such as ultrasound or enzymes, and then extracted.
In addition, the culture solution is directly extracted with a solvent. However, it is also possible to prepare a concentrated solution by concentrating by vacuum concentration, ultrafiltration, etc. before and/or after the solvent extraction. The extract is
Further, it is appropriately processed by known separation and purification means such as chromatography, precipitation with a solvent, and recrystallization.

Vitamin 812 can be purified and used as described above, as well as
When used as a feed additive, etc., it can be used in unpurified form, such as bacterial cells, culture solution, a mixture thereof, or the above-mentioned concentrate.

Next, the bacteria according to the present invention have a superior ability to produce vitamin B1□ than known gas-assimilating bacteria, and
The manufacturing method of No. 2 will be explained in Examples.

(Example 1) First, for comparison, production of vitamin B12 from carbon dioxide and hydrogen by known gas-assimilating methanogenic bacteria was investigated. Select representative strains from among the gas-assimilating bacteria preserved at DSM, a German microbial preservation organization, and for those that grow on the medium in Table 1, use the medium in Table 1 and the storage medium specified by 05M. Those that do not grow on the medium in Table 1 are 05M
In the specified storage medium, culture at the specified temperature for 7 days or 3 days, and the bacterial cell concentration (A-S.) and vitamin B1□
The concentration was measured as described above. The results are shown in Tables 4-1 and 4-2. From the amount of vitamin B12 produced and the amount of bacterial cell growth, it is clear that Methanobacterium thermoautotrophicum (DSM 1053)
autotrophicum) is the strain with the highest vitamin-producing ability.

DSM: Deutsche Sammlung v
on MikroorganismenGesells
chaft fiir Biotechnology
che Forschungm, b, H, (International Depositary, Budapest Report No. 22).

Next, strain H117-1-2 (FERN P-11356
) and DSM 1053 strain were compared.

The medium used was yeast extract (Yeas) among the medium components in Table 1.
extract (Difco) and Trypticase (BBL) each having a concentration of 2 gIQ were used. A suspension of gas-assimilating methanogenic bacteria) 1117-1-2 (absorbance A at 660 nm that had been cultured in advance) was placed in a 125 m 12 vial containing a 20 mff culture medium (absorbance A at 660 nm was 0.5 to 0.8). ) to 0
．． 5a+Q inoculated, and shaken culture solution (160 rp) at 60°C.
m) did. In order to track the increase in bacterial cell concentration, the culture solution was withdrawn over time, As5o was measured, and substrate gas (CO2/H2 = 1/4) was added to the vial until the gas pressure inside the vial reached 1.8 kg/cxl. The supply was pressurized as follows.

After culturing for a fixed period of time, vitamin Lx in the culture solution was converted to cyano form, and then E. coli E and coli215 were cultured using a conventional method.
It was measured by a bioassay method using the strain. For comparison, Mesanobacterium thermoautotrophicum (M
ethanobacterium thermoauto
The production of vitamin Elzz in C. trophicum was investigated using the same experimental system. The first is the change in absorbance As5o over time.
The results for the vitamin B12 concentration are shown in the figure and in Table 5.

Table 5 Production of vitamin B12 by gas-assimilating methanogens H117-1-2M, thermoautotr
ophicum DSM 1053 A66oO30270,0307 hours after inoculation,
,, 0.695 0.571
B12 concentration (mg/12) 0.139
0.099B12 production ability (mg/Q/AGG
) 0.200 0.173 Specific growth rate (h-1) 0.42 0.
39 As is clear from the results in Table 5, H117-1-2
The vitamin B12 concentration and the amount of vitamin Lx produced per bacterial cell (vitamin B12 concentration/bacterial cell concentration (AG,
. )), and the proliferation rate was also shown to be high. That is, by using the H117-1-2 strain, low NaC
It has been found that vitamin B1□ can be produced in a short time, at a higher concentration, and at a higher yield using a single concentration medium.

(Example 2) Vitamin B1□ was produced in the same manner as in Example 1 except that strain N-15 (FERM P-11420) was used and the culture temperature was 65°C. Absorbance A, &. Figure 2 shows the changes over time, and Table 6 shows the results of the vitamin StZ concentration.

Table 6 Production of vitamin 812 by gas-assimilating methanogens KN-15M, thermoautotrophy
cumDSM 1053 A, G, 01028 at the time of inoculation
A1° after 0.0306 hours 0.591
0.263B12 concentration (mg/12)
0.100 0.046 specific growth rate (
h-1) 0.618 0.389
As is clear from Table 6, the KN-15 strain is better.

B vitamins, concentration and growth rate were also shown to be high. In other words, it has been revealed that the KN-15 strain can produce vitamin D22 more efficiently from carbon dioxide and hydrogen than conventional gas-assimilating bacteria. We were able to newly isolate a methanogenic bacterium that proliferated at a very high rate in the culture medium and had an excellent ability to produce vitamin La. By culturing these bacteria, they succeeded in industrially obtaining vitamin D22 from the culture.

That is, after culturing, the culture solution is extracted by a conventional method, and the bacterial cells are either directly extracted or crushed by known means such as ultra-sonic waves, and then extracted, and then extracted by a conventional method. A large amount of vitamin D22 can be obtained by separating and purifying it according to the method.

In other words, the present invention was the first to successfully develop an industrial method for producing vitamin B1□.

[Brief explanation of drawings]

Figures 1 and 2 are graphs illustrating changes in absorbance over time for microorganisms according to the present invention. Agent: Patent attorney 1) Parent: Male chart culture time (h)

Claims (5)

[Claims] (1) Methanogenic bacterium KN-15. (2) Methanogenic bacteria H117-1-2. (3) Vitamin B_1_2 producing bacteria characterized by using bacteria H117-1-2 and/or KN-15 as effective bacteria. (4) A method for producing vitamin B_1_2, which comprises culturing bacteria H117-1-2 and/or KN-15. (5) A method for producing vitamin B_1_2, which comprises culturing bacteria H117-1-2 and/or KN-15 and collecting vitamin B_1_2 from the culture.