JPS589673B2 - Biseibutsukintainoseizohou - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for producing bacterial cells using a novel microorganism.

More specifically, bacteria belonging to Pseudomonas denteformis or Alcaligenes autonitrificans that can grow autotrophically using hydrogen gas as an energy source are cultivated using hydrogen gas as the main energy source, and bacterial bodies are separated from the culture solution. The present invention relates to a method for producing bacterial cells characterized by the following.

Conventionally, molasses, sulfite pulp waste liquid, n-paraffin, etc. have been used as raw materials for producing bacterial cells.

However, the inventors of the present invention believe that there will be problems in terms of supply amount, price, etc. in the future when these substances are used as waste fermentation raw materials, and we have selected hydrogen gas as a raw material that can be stably supplied in the future. We conducted intensive research on the method for producing microbial cells.

As a result, it was discovered that a species of bacteria belonging to the genus Pseudomonas and Alcaligenes, which was newly isolated from nature, was suitable for this purpose, and based on this knowledge, the present invention was completed.

As shown below, according to its mycological properties, the strain used in the present invention is considered to belong to the genus Pseudomonas and the genus Alcaligenes; When compared as described above, there are differences in various points.

Therefore, it is clear that it is a new bacterial species, and it has been classified as Pseudomonas denteiformis (Pseudomonas denteiformis).
nas dentiformis nov-sp-)
and Alcaligenes autonitrificans (A
lcaligenes autonitrifican
It was named s nov- sp- ).

That is, the present invention involves culturing a bacterial strain belonging to Pseudomonas denteiformis or Alcaligenes autonitrificans that can grow using hydrogen gas as an energy source, and multiplying the bacterial cells using hydrogen gas as the main energy source. This is a bacterial cell production method characterized by collecting bacterial cells.

The following is a representative strain of this bacterial species, Pseudomonas dentiformis AJ3941
The mycological properties of No. 9) are shown below.

Pseudomonas dentiformis (Pseud)
omo-nas dentiformis nov・s
p・) AJ 3 9 4 1 Cell morphology: (cultured autotrophically in inorganic synthetic medium for 48 hours) 0.4-0.6 x 1.
Bacilli, singly or in pairs, 5-20 microns.

Motile (polar flagella). Durham negative.

It does not form spores, is non-acid-fast, and shows no cellular pleomorphism.

Colony morphology = (cultured autotrophically on an inorganic synthetic medium plate for 8 days) Irregular morphology, smooth, serrated edges, plate-like, translucent, grayish white, 5-6 mm in diameter, flesh juice without carotenoid pigments Agar culture: Good growth, smooth, serrated edges, grayish white, translucent broth Liquid culture: Good growth, liquid cloudy, no pigment formation;
Grow on top of gelatin puncture culture with sediment, aerobically, and liquefy.

Litmus milk: No change.

Starch degradability: positive Catalase activity: positive Oxidase activity: positive Denitrification reaction: negative MR test: negative vp test: negative Indole generation: none Inorganic nitrogen source: use Pigment generation: none 0-F test: No change Urease activity: positive Generation of hydrogen sulfide, weakly positive Formation of nitrite from nitrate double positive Utilization of citric acid: It is usable, but not very strong.

Optimal temperature: 30-37℃, does not grow at 40℃. optimal p
H: 6.0-7.5 Oxygen properties: Need for aerobic growth factors: No organic compounds are required as growth factors.

Autotrophic growth: Grows autotrophically in gases containing hydrogen gas, carbon dioxide gas, and oxygen gas.

Whether acid or gas is generated from various sugars: No acid or gas is generated from mannose, and glycose, fructose,
Does not produce arabinose, xylose, sucrose, trehalose, maltose or lactate.

Assimilation of carbon sources: Glucose, mannose, maltose, citric acid, succinic acid, acetic acid, hydroxybenzoic acid, phenol, glycerol, and ethanol are assimilated.

lactose, arabinose, xylose, trehalose, malonic acid, oxalic acid, formic acid, meth Gnolls are not assimilated.

Isolation source: soil As mentioned above, this bacterial strain is considered to belong to the genus Pseudomonas because it is a Gram-negative, aerobic bacillus with polar flagella.

The major characteristics of this strain are that it grows autotrophically and hydrolyzes gelatin and starch.

Among the known Pseudomonas species that can grow autotrophically, there is a report by Davis et al. According to Pseudomonas satsukalophylla
S saccharophila ) hydrolyzes starch but not gelatin, and Pseudomonas facilis ( Pseudomonas f.
Acilis) is said to hydrolyze gelatin but not starch, as described in Bergey's Manual of Determinative Bacteriology.
Even in Erminative Bact-6riology, 8th edition, there is no known bacterial species that grows autotrophically and has both starch and gelatin hydrolyzing activities.

The colony morphology is also specific, and this strain has been determined to be a new bacterial species.Since it forms serrated colonies, it is considered to be Pseudomonas denteifornemis.
dentiformisnov-sp-).

Next, the mycological properties of Alcaligenes autonitrificans, another type of bacterial strain, are shown below.
enes autonitrificans nov-
sp-atrain M15 (Alcaligenes autonitrificans isolation number M15)
No. 180 (AJ - Morphological characteristics Durham negative, motile bacilli, 0.6-0.8 x 2
．． It has a circumflagellum that is 5-4.0 microns long.

No cell pleomorphism, no sporulation, non-acid fast.

When grown on an agar medium containing 0.4% yeast extract, 1.0% malt extract, and 0.4% glucose, young cells are characterized by the appearance of similar grains that are stained with Sudan Black B. It is.

Meat juice agar culture: No growth Meat juice liquid culture: No growth Meat juice gelatin puncture culture: No growth Colony morphology Grow autotrophically on an inorganic synthetic medium for 14 days using hydrogen as an energy source and carbon dioxide as a carbon source. The morphology of the colony is circular, about 3 mm in diameter, the surface is smooth, semi-lenticular, the periphery is complete, yellowish gray and opaque.

When grown on broth agar for 10 days, yellow, opaque colonies with a diameter of about 1 rILm were formed in the shape of flowers, wrinkles, cones, serrations, and granules.

Physiological properties It uses the energy of oxidizing hydrogen to fix carbon dioxide and grows autotrophically.

Catalase activity: positive Oxidase activity: positive Urease: negative 0-F test: No change Starch hydrolysis activity: negative Gelatin hydrolysis activity: negative Litmus milk: unchanged Hydrogen sulfide generation: negative Production of nitrite from nitrate: positive Denitrification reaction: negative MR test: negative VP test: negative Indole production: negative Use of inorganic nitrogen sources: Use Pigment formation: None.

When growing autotrophically using H2 as an energy source and carbon dioxide as a carbon source, nitrogen gas can be used as the only nitrogen source.

No acid or gas production was observed from various sugars.

It has the ability to assimilate succinic acid, acetic acid, methanol, ethanol and glycerol.

Without using citric acid, gluconic acid, malonic acid, oxalic acid,
Assimilation of formic acid, para- and meta-hydroxybenzoic acid, and phenol was not observed.

aerobic Optimum pH range for growth is 5.5-7.5 Suitable growth temperature: 30-35°C, grows even at 37°C.

GC content of DNA 64.4% This bacterium is considered to be a strain belonging to the genus Alcaligenes based on its morphological and physiological characteristics.As a hydrogen bacterium belonging to the genus Alcaligenes, the Verge Eighth Manual 8th edition describes it as Alcaligenes euthrohus.
es eutrophus) and alcaligenes
Alcaligenes parad
oxus), but this bacterium differs from Alcaligenes varadoxas in that it does not have dylotinoid pigments, does not have the ability to assimilate sugars, and has the ability to fix atmospheric nitrogen.Methanol It is different from Alcaligenes eutrophus in that it has no ability to assimilate para- and meta-hydroxybenzoic acid, phenol, etc.

Also, in the epiphytic state of flagella, Alcaligenes eutrophus has a small number of flagella, so-called degen.
erte peritrichoua flagell
In contrast, this bacterium has normal periflagellated peritri
It is different in that it is Chous flagella, and can be considered a new bacterial species belonging to the genus Alcaligenes.

In addition, Inuyama et al., which has the ability to fix atmospheric nitrogen,
Strains of Hydrogenomonas 083 and Gogotofura (Archief Fair Microbiology 97, 359-36
2 (1974)) in that it is motile and does not produce carotenoid pigments.This strain is a previously unknown hydrogen bacterium, and the present inventors believe that it is a hydrogen bacterium that has not been previously known. It was named Nitrificanth.

Next, a method of implementing the present invention will be described.

The microorganisms used in the present invention are those belonging to Pseudomonas denteiformis or Alcaligenes autonitrificans, and for example, the former is FERM.
P-3179, the latter as FERMP-3180, etc.

A medium containing at least a carbon source, a nitrogen source, and inorganic salts is used.

Carbon gas is usually used as a carbon source, but glucose,
Organic carbon sources such as acetic acid and methanol may also be used;
Carbon dioxide gas and an organic carbon source may be used together.

Carbon dioxide gas may be supplied by aeration like hydrogen gas or the like, or may be added to the medium in the form of baking soda or the like.

As the nitrogen source, inorganic nitrogen compounds such as ammonium salts and nitrates, or organic nitrogen compounds such as cornstarch liquor, peptone, and yeast extract are used.

Inorganic salts include phosphates, magnesium salts, calcium salts,
Use iron salt etc.

Furthermore, growth promoting substances such as vitamins may be added as necessary.

Culture temperature is 20-38°C, preferably 30-37°C
And the culture pH is 5-9, preferably 6-8.

The culture medium is placed in a sealable container, sterilized, and then inoculated with microorganisms.

These gases are introduced so that the gas phase within the container contains at least hydrogen gas and oxygen gas.

Air may be used instead of oxygen gas. In addition to these, the gas may also contain impurities that do not inhibit the growth of microorganisms.

As a culture method, after introducing gases into the container, the container may be sealed, allowed to stand still, and shaken, but it is preferable to culture by aeration and agitation.

The culture format may be either batch culture or continuous culture.

The grown bacterial cells are collected by an appropriate method such as centrifugation.

It is also possible to collect metabolic products from the culture supernatant.

The obtained bacterial cells have a crude protein content of 80% or more and contain extremely high quality proteins.

In this way, the present invention makes it possible to easily produce microbial cells even in the absence of other energy sources, as long as at least hydrogen gas is present.

Hereinafter, it will be explained in more detail using examples.

Example 1 As a medium, (NH4) 2 so42 g/l
KH2PO40.3 g/L Na2HPO4 1
2H, 20 1.8 g/IMgS04・7H2O0.
2g/LFeS04・7H2010mg/l, CaCl
2.2H2O 10mg/l, deionized water 1l pH 7.2
Pour 20 ml of the composition into a 500 ml volumetric flask, sterilize it, and add Pseudomonas denteiformis A.
J3941 was inoculated and the gas phase inside the flask was changed to 10% CO2.
Replaced with a mixed gas of 70% H2 and 220% H2, and after sealing 3
Culture was carried out with shaking at 5°C.

Growth rate (specificgro
wth rate) 0. Logarithmic growth was observed at 25 hr-1, and after 36 hours of culture, 10 ml of the culture solution in the flask was taken, the cells were collected by centrifugation, and dried to obtain 17.5 μm of dry cells.

The nitrogen content of this bacterial cell is 13.07%, and the crude protein content is 8.
It was 1.7%.

Example 2 400 ml of the same medium as in Example 1 was placed in a 1 liter small glass jar fermenter, and after sterilization, Pseudomonas
Inoculated with Denteiformis AJ3941, CO210
ml/min, H270ml/min, oxygen 20m
1/min, and stirred at 1400 rpm.

The culture temperature was 35°C, and during the culture, the pH of the medium decreased as the bacteria grew, but the culture was carried out while adjusting the pH to 6.5 with ammonia gas, which also served as a nitrogen source.

After 48 hours of culture, 100 ml of the culture solution was taken, and the cells were collected by centrifugation and dried to obtain 950 mg of dry cells.

The amino acid composition of this bacterial cell was as shown in Table 1.

Example 3 Batch culture was carried out in a small jar fermenter in the same manner as in Example 2, and after 36 hours of culture, dilution was performed with the same medium as in Example 1, and continuous culture was started.

The dilution rate with the medium is 0. 1 br-1, a steady state was reached after 72 hours of culture, and culture was continued for two weeks thereafter.

During this period, the bacterial concentration in the culture solution can be maintained at 6.3 g/l of dry bacterial cells, and the hydrogen yield relative to consumed bacterial cells is 1.9 g.
Fungal body/. 9H2, the yield relative to consumed carbon dioxide is 0.58 g bacterial cells/gCO2, and the yield relative to consumed carbon dioxide is 0.45 g bacterial cells/
It was g02.

Furthermore, the bacterial cell production rate was 0.63 g/l/hr.

Example 4 Using the same medium as in Example 1, inoculating Alcaligenes autonitrificans AJ3942 after sterilization, and replacing the gas phase in the flask with CO2 in the same manner as in Example 1.
Replaced with a mixed gas of 10%, H270%, 0220%,
After sealing, the culture was carried out with shaking at 35°C.

Growth rate (specific gr
low ratc) 0. Logarithmic growth was observed at 2 3 hr=.

After 36 hours, 10 ml of the culture solution in the flask was taken, and the bacterial cells were collected by centrifugation and dried to obtain 16.8 ml of dried bacterial cells.

The nitrogen content of this bacterial cell is 11.7%, and the crude protein content is 73%.
．． It was 1%.

Example 5 400 ml of the same medium as in Example 1 was placed in a 1 liter glass jar fermenter, and after sterilization, Alcaligenes autonitrificans AJ 39 4 2 was inoculated, and C.
O2 10 ml/min, H2 70 m
Aerate l/min, 0 2 2 0 ml/min,
Culture was carried out with stirring at 1400 rpm.

The culture temperature was 35℃, and as the bacteria grew during the culture,
Although the pH of the culture medium decreased, the culture was carried out while adjusting the pH to 6.8 with ammonia gas, which also served as a nitrogen source.

After 48 hours of culture, 100 ml of the culture solution was taken, and the cells were collected by centrifugation and dried to obtain dried cells of 840rn9.

The amino acid composition of this bacterial cell was as shown in Table 2.

Example 6 Batch culture of Alcaligenes autonitrificans AJ3942 was carried out in a small jar fermenter in the same manner as in Example 5, and after 40 hours of culture, dilution was performed with a medium having the same composition as in Example 1, and continuous culture was started. Ta.

When the dilution rate with the medium was set to 0.1 hr-1, a steady state was reached after 80 hours of culture, and the culture was continued for two weeks thereafter.

During this period, the bacterial concentration in the culture solution can be maintained at 5.2 g/l of dry bacterial cells, and the bacterial cell production rate is 0.52 g/l.
l/hr.

Claims (1)

[Claims] 1 Pseudomonas pseudomonas is grown in a nutrient medium containing at least a carbon source, a nitrogen source, and inorganic salts using hydrogen gas as the main energy source.
1. A method for producing microbial cells, which comprises culturing Denteiformis or Alcaligenes autonitrificans to produce cells, and collecting the cells.