JPS6225034B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a method for producing a methane-fermenting starter that has high methane production efficiency and is resistant to oxygen contamination. Methane is one of the promising energy alternatives to petroleum, and there has been much research into producing methane gas through fermentation methods. As a recent excellent high-speed methane fermentation method (1982, Society of Agricultural Chemistry, Proceedings, p. 82), a liquefaction fermentation tank of 0.5 m ³ and a 1.5 m ³
Continuous fermentation was carried out using a gasification fermenter with a total residence time of 8 days and an organic matter load of 15 kg/m ³ days. Fermentation gas with a methane concentration of 70% was
The amount of methane gas generated by this device is 15Kg/ ^m3・day×(0.5+1.5) ^m3 ×300/Kg×70
%=6.3m ³ (CH ₄ ). In this example, 8 days x 1.5 m ³ / (0.5 + 1.5) m ³ =
6 days, 2 days for liquefaction and 6 days for gasification. The methane gas production reaction is the rate-limiting step in the entire fermentation process. That is, the methane-producing ability of methane-producing bacteria is the most important factor governing the efficiency of the fermentation process. Furthermore, since this example is a continuous fermentation method, the concentration of methane-producing bacteria in the gasification tank is kept constant during steady-state operation. However, since the same amount of fermentation waste liquid flows out from the gasification fermenter as the new substrate flows in, the methane-producing bacteria proliferate by the amount that flows out. The division time of the methane-producing bacteria is 100
It is thought to be about an hour. Therefore, the growth of methane-producing bacteria during the fermentation process is negligible, and the methane-producing ability during the stationary growth phase is important. Now, as described in Bergier's manual, methane-producing bacteria are extremely strict anaerobic bacteria, and even if a very small amount of oxygen is mixed in, all the bacteria will die within a short period of time. This extremely sensitive and weak characteristic to oxygen causes significant difficulties when preserving methane-producing bacteria as seed bacteria for methane fermentation, culturing them in large quantities, and handling them such as transporting them. It is desired that the bacteria have considerable resistance to oxygen. Based on the above reasoning, the present inventors aimed to obtain methane-producing bacteria that have high methane-producing ability during the stationary growth phase and are resistant to mixed oxygen.
We isolated and searched for methane-producing bacteria from pond silt, anaerobic human waste digester sludge, etc., and obtained one strain of mesophilic (25-40°C) methane-producing bacteria that met the above characteristics. This is called Methanobacterium SP/ST-23. This bacterium was isolated as a dominant species in silt, with 10 ⁸ to 10 ⁹ living in 1 ml of silt. If this bacterium is cultured in liquid at 35°C using the medium listed above, it will reach the stationary growth phase in about 7 days. The methane production capacity of the culture on the 15th day of growth, which has completely entered the stationary growth phase, is 8.7× using hydrogen and carbon dioxide as substrates.
It showed a methane production capacity of more than 10 ^-8 ml/piece/day. The number of bacteria was measured by the plate culture method. Methane gas was analyzed by gas chromatography. This methane generating ability is the lower limit of the survival amount of this bacterium in silt.
Using 10 ⁸ /ml as the calculation basis and converting it to the tank capacity of 1.5 m ³ in the previous literature example, it corresponds to 13 m ³ /day, and the practical methane generation capacity is extremely large. Next, regarding the resistance of this bacteria to oxygen, 110ml
Transfer 50ml of medium to a vial (gas phase: 60ml)
Add 1 ml of hydrogen sulfide and cysteine as a reducing agent as equivalent to oxygen, add oxygen (0 to 12 ml) to the gas phase, and fill with a mixed gas of 80% H ₂ and 20% CO ₂ at 1.5 atm at 35°C. When cultured, a trace amount (0.05
ml/3 days) of methane production was confirmed. Furthermore, after 14 days, it was confirmed by the plate culture method that there was no contamination with other bacteria, and that there was no decrease in the number of bacteria even when up to 12 ml of oxygen was mixed in. The amount of oxygen of 12 ml in 60 ml of gas phase is equivalent to the oxygen concentration in the atmosphere, and this bacterium can withstand the same oxygen partial pressure as the atmosphere and survive. In this regard, Methanobacterium
Mobile (Journal of Bacteriology,
95, p. 1943, 1968) is said to die in the presence of less than the equivalent amount of oxygen as the reducing agent. The characteristics of this bacterium differ greatly from those of these bacterial species. This strain has the following mycological properties. For culture, a gas phase containing 80% H ₂ and 20% CO ₂ was used as a medium. (1) Light microscopic and electron microscopic findings a Straight to slightly curved rod with a rounded end, 0.3 μm in diameter and 1.3 to 3.3 μm in length. bIt has markedly unequal divisions, sometimes close to spheres. c No spores are formed, d no motility, e no hairs, f gram negative. (2) Agar plate culture findings a. Growth is poor, b. Small colonies appear 4 days after inoculation, c. Colonies with a maximum diameter of 0.7 to 1 mm in 14 days. d Young colonies are circular, with entire edges, and white to pale yellow-green in color; e As they get older, shallow, fine, radial folds appear on the surface. 3 Physiological properties a. A mesophilic anaerobic bacterium that grows near neutral pH. b. Grows by assimilating hydrogen and carbon dioxide, or formic acid or acetic acid. c For oxygen
It can withstand partial pressures up to 1/5 atmosphere and survive, and can withstand partial pressures up to 1/30 atmosphere and produce significant amounts of methane. d No growth promotion was observed by peptone and amino acids, e no growth promotion was observed by coenzyme M (CoM), and f slight growth promotion was observed by yeast extract. Although no growth-promoting effect is observed in the silt extract, growth is significantly promoted by mixed culture with certain types of bacteria. (4) Others The soluble fraction of crushed bacteria emits blue-green fluorescence when irradiated with near ultraviolet light. The taxonomic status of this strain was determined by Verges Annual of Determinative.
According to the 8th edition of Bacteriology, it is appropriate that it belongs to the genus Methanobacterium because it is a rod that produces methane. This strain produces methane from acetic acid.
Similar to Methanobacterium soehngenii,
The life style of Methanobacterium soehngenii is clearly different from this strain in that it cannot be isolated in its pure form. Furthermore, although it is thought to be close to Methanobacterium formicicum and Methanobacterium mobilee, both species have various differences as mentioned above, including the fact that they do not have the ability to assimilate acetic acid. It is considered appropriate to classify it as a new species of the genus Bacterium. Therefore, the new species name, Methanobacterium kademensis, was given after the name of the place where the silt from which this bacterium was isolated was produced. This bacterium was submitted to the Institute of Microbial Technology, Agency of Industrial Science and Technology with the microbial accession number No. 44 (FERM).
The storage of microorganisms was outsourced under the BP-44). Cultivation of this bacterium can be carried out, for example, in a medium having the composition shown in the table below. Increasing the amount of soda sulfide will make it even less susceptible to the effects of oxygen, but since a large amount of sodium sulfide inhibits the growth of this bacterium, the appropriate amount is about three times the amount shown in the table. As a culture substrate, any of hydrogen and carbon dioxide, formic acid, and acetic acid can be used, and it is appropriate to use hydrogen and carbon dioxide or formic acid. As is clear from thermodynamics, acetic acid is only used very slowly. A combination of formic acid and hydrogen is also possible, but in this case high partial pressure of hydrogen acts as an inhibitor to growth. When hydrogen and carbon dioxide are used as substrates, the substrate composition is 80% hydrogen and carbon dioxide according to the theoretical formula for methane production.
20% is appropriate. However, in the early stages of culture, growth is better if a mixed gas of hydrogen and carbon dioxide is diluted with nitrogen gas or the like. The appropriate culture temperature is a medium temperature range, 35° to 40°C. The appropriate medium pH is around neutrality, but it changes depending on the carbon dioxide concentration in the gas phase, and if formic acid is used, the medium pH will increase as the use of formic acid decreases, so a large buffering capacity should be added to the medium composition. Either the cells must be allowed to remain in the culture, or they must be constantly neutralized during the culture period.
Also, vitamin sources such as yeast extract may be used in place of the vitamins listed in the table. It takes about 7 days to grow. The microbial cells cultured in this way can be collected using known methods such as centrifugation or coprecipitation, or they can be mixed with other microorganisms constituting a methane fermentation system as a methane fermentation starter. Can be used.

[Table] Next, the method of the present invention will be specifically explained with reference to Examples of the production of this seed culture, but this is not intended to limit the method of the present invention. Put the liquid medium with the composition shown in the table into a 700ml vial.
Take 500ml and sterilize in an autoclave at 120℃ for 10 minutes. However, vitamins, sodium carbonate, sodium sulfide, and cysteine hydrochloric acid should be sterilized separately and added to the sterilized liquid medium. Seal the tube with a butyl rubber stopper, cover with an aluminum cap, replace the gas phase with a mixed gas of 80% hydrogen and 20% carbon dioxide through the syringe needle, and fill with the mixed gas until the concentration reaches 1.5 atm. Inoculate a liquid culture of this bacterium at a rate of 1% using a syringe, and culture it statically at 35°C. During this time, add mixed gas once every two days.
After 10 days, the turbidity measured at 660 nm was 0.01, and the bacterial concentration counted by plate culture was 10 ⁶ /ml.
200 mg of wet bacterial cells were obtained by centrifugation at 8000×G for 10 minutes. In addition, sterilize the liquid medium by adding thorium formate, sodium acetate, sodium propionate, sodium butyrate, methanol or ethanol.
This bacterium was cultured under the same conditions as in the previous example, except for the addition of 0.1% gas and the replacement of the gas phase with N ₂ gas, and an increase in medium turbidity after 30 days of culture. The presence or absence of growth was determined qualitatively.
As a result, growth was observed in the plants added with sodium formate and those added with sodium acetate, but not in the others.
Therefore, this bacterium can effectively produce formic acid and acetic acid as substrates. As described above, according to the present invention, a strain belonging to the genus Methanobacterium and having high methane production ability during the stationary growth phase and exhibiting oxygen tolerance is used to produce hydrogen and carbon dioxide, or from the three groups of formic acid or acetic acid. Methane-producing bacterial cells are obtained by culturing anaerobically in a mesotemperature range using at least one of the selected substrates and a medium to which at least a nitrogen source, an inorganic salt, and a reducing agent are added. , it is possible to obtain a methane-fermenting starter that has high methane production efficiency and is resistant to oxygen contamination.

Claims (1)

[Claims] 1 Using a strain that belongs to the genus Methanobacterium and has a high methane production ability during the stationary growth phase and is resistant to oxygen, at least one of the following three groups is used: hydrogen and carbon dioxide, or formic acid or acetic acid. A method for producing a methane-fermenting inoculum, which comprises culturing anaerobically in a meso-temperature range to obtain methane-producing microorganisms using a medium containing a methane as a substrate and at least a chisel source, an inorganic salt, and a reducing agent.