JP2002325567A - Method for culturing microorganism by using sea water - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for culturing microorganisms capable of planning to stabilize proliferation with enhancing culture efficiency by culturing the microorganisms by using a medium prepared by dissolving ingredients of the medium in seawater. SOLUTION: This method for culturing microorganisms by using seawater is to culture the microorganisms by using a medium prepared by dissolving ingredients of the medium in ocean surface water or ocean deep water taken from the deep sea below 2000 m from the sea surface and maintain activities of the cultured microorganisms in a high level as basic means. Concretely the aforesaid medium is sterilized by high pressure steam in an autoclave and put in vessels which are previously sterilized with high pressure steam, subsequently seeds of the microorganisms are inoculated and stationarily cultured by standing in a water bath.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for culturing microorganisms using seawater, and more particularly to various fertilizers and soil improvement using marine surface water or deep-sea water, treatment of livestock feed and human waste, and improvement of water quality. The present invention relates to a culture method in which the microorganism used is cultured to increase the survival rate of the microorganism and maintain the culture efficiency and the activity of the cultured microorganism satisfactorily.

[0002]

2. Description of the Related Art Conventionally, chemical fertilizers mainly comprising nitrogen, phosphoric acid, and potassium, which are three main components of fertilization, have been used to enhance the growth of various crops and plants. With the growing need for fertilizers, there has been a shift from chemical fertilizers to organic fertilizers and a shift to crop cultivation using no pesticides. In response to the above, the thermophilic inoculum Miroku (Miroku fungus) (ATTC International Deposit No. PTA-1773) isolated from the body surface of shrimp, crab, small fish, etc. is cultured to be used as an organic fertilizer. Livestock excrement processing,
Means for use in soil improvement, water quality improvement, and the like have been attempted. The thermophilic inoculant Miroku is a thermophilic C-1 which is a closely related species of Bacillus brevis.
A thermophilic C-3 bacterium, a closely related species of the bacterium Bacillus brevis, Bacillus stearothermophilus CH-4
-4), and MH-1 (Thermophilic a
ctinomycetes MH-1) and a thermophilic bacterium,
It is a marine thermophilic microorganism that has the ability to decompose shrimp and / or crab residues under aerobic conditions and the ability to produce thermostable enzymes and chaperonins.

A thermophilic bacterium is a kind of microorganism close to a progenitor cell that has inhabited since the creation of the earth, and can inhabit even in an extreme environment of a high temperature of 60 ° C. to 90 ° C. Currently it inhabits hot springs and hydrothermal deposits. In recent years, it has been scientifically proved that it contains a useful functional component, such as a thermostable enzyme and chaperonin, as compared with normal-temperature bacteria. Examples of thermophilic bacteria include, in addition to the thermophilic inoculum Miroku described above, Streptococcus thermophilus (Streptococcus thermophus).
ilus), Bacillus acidocardrius (Bacillus ac)
idocaldarius) and Thermoanaerobacter italicus.

With respect to the above, Japanese Patent Application Laid-Open No. 2000-34224
No. 8 proposes a thermophilic inoculum Miroku as a thermophilic inoculum capable of producing a safe crop, an organic fertilizer using the thermophilic inoculum Miroku, a liquid organic fertilizer, and a method for producing the same. I have.

That is, the thermophilic inoculum is added to shrimp and / or crab residues, and is added under aerobic conditions at 50 ° C. to 9 ° C.
A method for producing an organic fertilizer by fermenting at 0 ° C., a method for producing a liquid organic fertilizer which is added to water and cultured at 30 ° C. to 70 ° C. under aerobic conditions, and Describes a method for producing a liquid organic fertilizer, which is added to a distillate collected at the time of the production of, and cultured at 30 ° C to 70 ° C under aerobic conditions. Table 1 shows the identification results of the thermophilic inoculum.

[0006]

[Table 1]

[0007] As described above, the thermophilic inoculant Miroku can be used as an organic fertilizer, besides excreting livestock from livestock, improving soil,
This thermophilic inoculant, Miroku, grows best at 50 ° C to 90 ° C and decomposes chitin, a body component of shrimp and crab. It has the characteristic of producing thermostable enzymes. Means for culturing the thermophilic inoculum miloc using a medium prepared by dissolving a medium component in well water or tap water in order to propagate the thermophilic inoculum miloc are known.

On the other hand, recently, the cleanliness of the deep sea water and the abundant mineral components have attracted the attention of consumers and called a boom, and the water obtained by desalinating the deep sea water has entered the field of drinking water. It is in the present situation. The above-mentioned deep ocean water is currently being used practically in several places around the world, such as "off the coast of Norway", "off the coast of Hawaii", and "off the coast of Cape Muroto in Kochi Prefecture". Seawater in an environment where convection and mixing do not occur due to changes, and are not affected by marine pollution caused by various oils and chemicals used on the ground, and the dissolved organic matter in the seawater is extremely low. And is extremely clean.

Table 2 is a table showing the results of an analysis of various items of the ocean surface water and the deep sea water. In the general items, the average water temperature is 21 ° C. for the ocean surface water and 13 cm for the deep ocean water. The pH is as low as 0.1 ° C, the pH is 7.87 for 8.19, the DO (dissolved oxygen) is 7.28 mg / L for 8.33 mg / L, and the TOC is 1.60 mg / L. 0.98 mg / L, both of them are lower in deep sea water, but the viable cell count is 10 ² in deep sea water compared to 10 ³ to 10 ⁴ of sea surface water, which is one order of magnitude lower. Moreover, since it contains almost no pathogenic organisms, there is no concern about diseases caused by fish pathogens derived from seawater.

[0010]

[Table 2]

[0011] In the item of nutrients as minerals, NO ₃ -N in the deep sea water against 1.49μg-at / L ocean surface water _{25.9μg-at / L, PO 4} -
P is 1.65 μg-a against 0.34 μg-at / L.
t / L and SiO ₂ -Si are 13.6 μg-at / L and 64.2 μg-at / L, which is much larger in deep ocean water. Analysis results have also been obtained that the content of deep ocean water is higher than that of ocean surface water for other trace element items.

Analysis of the desalted water in deep sea water by atomic absorption spectrometry revealed that calcium contained 0.4 mg / L and magnesium contained 1.0 mg / L.
Furthermore, the concentrated water of the deep ocean water was similarly analyzed by atomic absorption spectrometry, and as a result, it was found that calcium contained 560 mg / L and magnesium contained 1700 mg / L.

[0013]

As described above, as a conventional method for cultivating a thermophilic inoculum Miroku, a thermophilic inoculum Miroku is cultured using a medium prepared by dissolving a medium component in well water or tap water. Although measures have been taken, the efficiency of cultivation cannot be sufficiently increased, and especially during long-term cultivation for one week or more, the number of surviving inoculum decreases rapidly, and the growth of the thermophilic inoculum Miroku is also increased. There is a problem of lack of stability.

Therefore, the present invention solves the problems of such a conventional method for culturing a thermophilic inoculant Miroku, and uses a medium prepared by dissolving a medium component in seawater to produce a thermophilic inoculant Miroku. It is an object of the present invention to provide a method for culturing a microorganism which can increase the culture efficiency and stabilize the growth by culturing another microorganism.

[0015]

SUMMARY OF THE INVENTION In order to achieve the above-mentioned object, the present invention provides a culture medium comprising a surface water of the ocean or a surface water below sea level.
The present invention provides, as basic means, a method of culturing microorganisms using a medium prepared by dissolving in deep ocean water taken from deep sea at a depth of 0 m or less, and maintaining the activity of the cultured microorganisms at a high level. Specifically, the medium was subjected to high-pressure steam sterilization by an autoclave, placed in a container previously subjected to high-pressure steam sterilization in a clean bench, inoculated with a seed of the microorganism, and then left standing in a hot water bath. Perform culture.

Means for using an effective and safe one of thermophilic microorganisms or room-temperature microorganisms or a coexisting complex microorganism system in which a plurality of species are combined, and a microorganism of Bacillus. Thermophilic C, a closely related species of Bacillus brevis
Bacillus stearothermophilus CH-4 (Bacillus stearothermophilus), a thermophilic C-3 bacterium which is a closely related species of Bacillus brevis.
CH-4) and thermophilic actinomycetes MH-1 (Thermophil
ic actinomycetes MH-1) and one or more microorganisms selected from thermophilic or thermotolerant microorganisms that cannot be separated and identified at this stage. / Thermophili
(C. inoculum MIROKU) or a deposited microorganism group of the thermophilic inoculum Miroku.

The medium component is a mixture of glucose, polypeptone, yeast extract, potassium dihydrogen phosphate, and magnesium sulfate heptahydrate. Glycerin, sucrose, and starch are used as the carbon source of the medium component. Further, corn steep liquor and cottonseed flour are used as the nitrogen source of the medium components, and metal salts of manganese and iron are used as the metal salts.

According to the method for culturing microorganisms, the turbidity of a culture solution when culturing the thermophilic inoculum Miroku and other microorganisms using a medium prepared by dissolving a medium component in surface ocean water or deep ocean water. Is not much different from the turbidity of the medium prepared by dissolving the medium components in deionized water or NaCl solution. The medium using the surface water of the ocean or the deep sea water shows higher values in each case, and is effective in maintaining a high survival rate of the thermophilic inoculum Miroku. In addition, although the CFU of the medium using the surface ocean water is slightly lower than that using the deep ocean water, it can be used without any problem in practical use.

[0019]

DESCRIPTION OF THE PREFERRED EMBODIMENTS Hereinafter, a specific embodiment of the method for culturing microorganisms using seawater according to the present invention will be described. Microorganisms to which the present invention can be applied include, among thermophilic microorganisms or room-temperature microorganisms, effective and safe microbial systems composed of one kind, or coexisting complex microbial systems in which a plurality of kinds are combined. Inoculum Miroku (International Deposit No. PTA-1773 / Thermophilic inoculum MIROK
U) or one or more microorganisms selected from the deposited microorganisms of the thermophilic inoculant Miroku. This thermophilic inoculum, Miroku, is Bacillus brevis
C-3, a closely related species of Bacillus brevis, and a thermophilic C-1 bacterium, a closely related species of Bacillus brevis
Bacteria, Bacillus stearothermophilus CH-4 (Baci
llus stearothermophilus, CH-4), and the thermophilic actinomycetes MH-1 (Thermophilic actinomycetes MH-).
1) and a marine thermophilic microorganism having the ability to decompose shrimp and / or crab residues under aerobic conditions and the ability to produce thermostable enzymes and chaperonins. In the present embodiment, the thermophilic inoculum Miroku (Miroku fungus) (ATTC International Deposit No. PTA-17) is used as a microorganism.
73), but the present invention can be similarly applied to culture of other microorganisms, particularly thermophilic microorganisms.

In the present invention, microorganisms are cultured using a medium prepared by dissolving the medium components in the surface water of the ocean or in the deep ocean water withdrawn from a depth of 200 meters or less below the sea surface, and the activity of the cultured microorganisms is maintained at a high level. Is one of the features.

An example of a method for preparing a seed of the thermophilic inoculant Miroku is described below. First, thermophilic bacteria are shrimp, crab,
The thermophilic inoculum, Miroku, which is added to the residue of the small fish and present on the body surface, is cultured under aerobic conditions, sprinkled on coffee grounds, and packed in an aluminum can to obtain a seed. Take out 1 gram of this inoculum, put it in a test tube, add 5 ml of deionized water which has been subjected to high-pressure steam sterilization treatment at 121 ° C. for 15 minutes, mix well, and allow to stand for 15 minutes. Used as seed mother.

Next, in order to culture the thermophilic inoculum Miroku,
The following four types of culture media were prepared, one liter at a time. A solution in which YPD medium components are dissolved in deionized water A solution in which YPD medium components are dissolved in 3.5% NaCl water A solution in which YPD medium components are dissolved in ocean surface water A solution in which YPD medium components are dissolved in deep ocean water

The components of the YPD medium in the 1 liter solution are as follows. Glucose: 10.0 g Polypeptone: 8.0 g Yeast extract: 7.0 g Potassium dihydrogen phosphate: 1.0 g Magnesium sulfate heptahydrate: 0.5 g

The YPD medium component is not limited to glucose as a carbon source, but may be other substances such as glycerin, sucrose,
Starch or the like can be used. The nitrogen source is not limited to polypeptone or yeast extract, and for example, corn steep liquor, cottonseed flour and the like can be used. Further, the metal salt is not limited to phosphorus, potassium and magnesium, and metal salts such as manganese and iron can be used.

The above four types of media were subjected to high-pressure steam sterilization at 121 ° C. for 15 minutes by an autoclave. A test tube or container previously covered with an aluminum cap is subjected to a high-pressure steam sterilization treatment for 15 minutes, and 10 ml of each of the four types of culture media is placed in a clean bench with three test tubes or containers each for one type of culture medium. Was dispensed.

The seed of the thermophilic inoculant Miroku 0.1 m
1 was inoculated into each test tube or container into which four types of media were dispensed, and static culture was started in a 50 ° C. water bath.
The turbidity of the culture at a wavelength of 660 nm was measured over time from day 0 to day 7. Table 3 shows the results.

[0027]

[Table 3]

According to Table 3, the turbidity of the culture of the thermophilic inoculum Miroku in the four media reached a maximum of 0.30 after 1 day and changed little from the second day to the seventh day. It turned out there was no.

Next, the number of colonies of the thermophilic inoculum Miroku in the four types of media was measured using the stationary culture solutions on the second and seventh days. First, 1 liter of a YPD agar medium was prepared. The components of the YPD agar medium are as follows. Glucose: 10.0 g Polypeptone: 3.0 g Yeast extract: 7.0 g Potassium dihydrogen phosphate: 1.0 g Magnesium sulfate heptahydrate: 0.5 g Agar powder: 10.0 g Deionized water: 1 liter

The above agar medium was autoclaved for 12 hours.
A high-pressure steam sterilization treatment was performed at 1 ° C. for 15 minutes, and each 20 ml of the agar medium was dispensed into a petri dish in a clean bench. After the lid of this petri dish was opened and dried for one day, it was used for CFU assay.

1 ml of each of the three test tubes containing the stationary culture solution on the second and seventh days was placed in a sterile plastic tube, and a 100-fold dilution and a 10000-fold dilution were prepared from the four tubes. After that, 1 ml of each diluted solution was placed in each of three Petri dishes, and cultured at 30 ° C. for 24 hours. Then, the number of colonies on the Petri dishes was counted, and the average value of the three Petri dishes was calculated. Table 4 shows the results.

[0032]

[Table 4]

According to Table 4, the value of CFU when the culture solution on the second day was used was 1 in the four types of mediums.
No significant difference was observed between the 00-fold dilution and the 10000-fold dilution. However, the CFU value when using the culture solution on the 7th day, which is the latter stage of the culture, is 27, 42 for the 100-fold diluted solution when the medium is used.
In the case of a medium diluted with a surface water of the ocean and a deep ocean water, the concentration is 10 or less, respectively.
The ratio was 374,688 for the 0-fold diluted solution, and 6,10 for the 10,000-fold diluted solution, and a large difference was observed from the medium of either the 100-fold diluted solution or the 10,000-fold diluted solution.

In particular, the medium obtained by dissolving the YPD medium in deionized water and the medium obtained by dissolving the YPD medium in 3.5% NaCl water show a significant decrease in CFU on day 7, whereas the marine surface layer The CFU shows a high value in both the medium using water and the medium using deep sea water, and particularly, the medium using deep sea water maintains high survival rate of the thermophilic inoculum Miroku. That is, it was found to be effective in maintaining the activity of the cultured microorganism high. Although the CFU of the medium using the surface ocean water is slightly lower than that using the deep sea water, it can be used without any problem in practical use.

The deep ocean water employed in the present invention is seawater taken from a depth of 370 meters off Cape Muroto, and among the tri-state nitrogen contained in the deep-layer water, ammonia nitrogen and nitrite nitrogen are extremely small. The effect on living organisms was slight, and the amount of nitrate nitrogen was very small in the surface layer. However, as the water depth increased, the concentration increased,
More than 95% of inorganic dissolved nitrogen in water at a depth of 0 meters or less is nitrate nitrogen at 24 μM. In addition, 1.7 μM of phosphoric acid phosphorus and 41 μM of silicate silicon are dissolved, and each has a nutrient concentration of 5 to 10 times or more that of the surface layer.

The natural elements contained in deep sea water are Fe (iron), I (iodine), Cu (copper), Mn (manganese), Zn (zinc), Co (cobalt), and Mo (molybdenum). , Se (selenium), Cr (chromium), Sn (tin), V (vanadium), F (fluorine), Si (silicon), Ni (nickel), and As (arsenic). A major feature is that all of them are contained in deep ocean water in a well-balanced manner. Therefore, it can be said that deep sea water has great potential for culturing and growing microorganisms. Such potential
In recent years, it has been proved that it has been used in cultivation experiments on sea bream, kelp, deep-sea coral, etc. and has achieved great results. In particular, it has been reported that the deep ocean water off the coast of Norway is called fjord deep water and is suitable for salmon farming.

As shown in Table 2 above, the number of viable bacteria in the deep sea water is one or more orders of magnitude lower than that in the surface water, and contains almost no pathogenic organisms. There is a great feature that the safety when employed for culturing microorganisms is extremely high. According to the present invention, by adopting such a natural element contained in deep ocean water as a medium, it is possible to increase the culture efficiency and stabilize the growth of microorganisms, which cannot be achieved with a conventional medium.

[0038]

As described in detail above, according to the present invention, the thermophilic inoculum inoculated by culturing the thermophilic inoculant Miroku using a medium prepared by dissolving the medium components in the surface ocean water or deep sea water. The number of colonies (CFU) of the inoculant Miroku shows a higher value than the conventional medium prepared by dissolving the medium components in well water or tap water, and it is possible to sufficiently increase the cultivation efficiency and for one week or more. To maintain high viability of the thermophilic inoculum Miroku even during long-term culture of
That is, the activity of the cultured microorganism can be maintained at a high level, and the growth can be stabilized.

Although the CFU of the medium using the surface ocean water is slightly lower than that using the deep ocean water, it can be used without any problem from a practical point of view, and is an effective means when applied to the culture of microorganisms. I will provide a.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) (C12N 1/00 C12R 1:04) (71) Applicant 000148221 Izumi Ironworks Co., Ltd. 18 Ukizu, Muroto-shi, Kochi Prefecture (72) Inventor Hisashi Miyamoto 706 Iwatani 27, Kitsuki-shi, Oita Pref. 36 (36) Inventor Hirokuni Miyamoto 9-11-11 Miyamoto, Funabashi-shi, Chiba 72) Inventor Hiroyasu Tobe 587, Kambara, Okatoyo-cho, Nankoku-shi, Kochi Pref.Medical University Dormitory A-504 (72) Inventor, Yasuhisa Izui 18, Ukizu, Muroto-shi, Kochi 18th Izui Iron Works Co., Ltd.F-term (reference) 4B065 AA06X AA15X AA18X BB40 CA60

Claims (10)

[Claims] 1. A medium component comprising a surface water of the sea or a subsea surface.
A method for culturing microorganisms using seawater, which comprises culturing microorganisms using a medium prepared by dissolving in deep sea water taken from deep sea at a depth of 00 meters or less. 2. A medium component comprising: surface water of the sea or below sea level.
Culture of microorganisms using a medium prepared by dissolving in deep-sea water taken from deep sea below 00 meters, thereby maintaining the activity of the cultured microorganisms at a high level. Method. 3. The method according to claim 1, wherein the medium components are surface water of the sea or below sea level.
A medium was prepared by dissolving the medium in deep sea water taken from the deep sea at a depth of 00 meters or less, and this medium was subjected to high-pressure steam sterilization by an autoclave, and placed in a container previously subjected to high-pressure steam sterilization in a clean bench. A method for culturing a microorganism using seawater, comprising inoculating a seed of the microorganism and performing stationary culture in a hot water bath. 4. The microorganism according to claim 1, wherein the microorganism is an effective and safe one of thermophilic microorganisms or room-temperature microorganisms, or a coexisting complex microorganism system in which a plurality of species are combined. 3. The method for culturing microorganisms using seawater according to 3. 5. A microorganism such as Bacillus brevis (Ba)
cillus brevis), thermophilic C-3 bacterium related to Bacillus brevis, Bacillus stearothermophilus CH-4 (Bacillus brevis). stearothermophilus, CH-
4) and MH-1 (Thermophilic actin) of thermophilic actinomycetes
The method for culturing microorganisms using seawater according to claim 1, 2, or 3, wherein one or a plurality of microorganisms selected from the group of thermophilic or heat-resistant microorganisms which cannot be separated and identified at this stage is used. . 6. The microorganism may be a thermophilic inoculant Miroku (International Deposit No. PTA-1773 / Thermophilic inoculum MI).
The method for culturing microorganisms using seawater according to claim 1, 2, or 3, wherein one or more microorganisms selected from the group of deposited microorganisms of ROKU) or the thermophilic inoculant Miroku are used. 7. The medium component is a mixture of glucose, polypeptone, yeast extract, potassium dihydrogen phosphate, and magnesium sulfate heptahydrate.
7. The method for culturing microorganisms using seawater according to 5 or 6. 8. The method for culturing microorganisms using seawater according to claim 7, wherein glycerin, sucrose, and starch are used as a carbon source of the medium component. 9. The method for culturing microorganisms using seawater according to claim 7, wherein corn steep liquor and cottonseed flour are used as a nitrogen source of the medium component. 10. Manganese, as a metal salt of a medium component,
The method for culturing microorganisms using seawater according to claim 7, wherein a metal salt of iron is used.