JP2000253868A - Microorganism inhibiting production of nitric acid state nitrogen or accumulation of the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide new microorganisms belonging to the genus Penicillium, genus Aspergillus, genus Pecilomyces, genus Streptomyces and genus Xanthomonas, having an inhibiting function of the production of a nitric acid state nitrogen or inhibiting its accumulation and reducing the environmental load such as pollution of underground water and rivers. SOLUTION: The microorganisms are Penicillium sp no. 3 (FERM P-17208), Aspergillus sp no.8 (FERM P-17205), Pecilomyces sp no.35 (FERM P-17207), Streptomyces sp-AC 5400 (FERM P-17202), Streptomyces sp-AC 5005 (FERM P-17203), Streptomyces sp-AC 5431 (FERM P-17204) or Xanthomonas maltophilia N (FERM P-17206). These microorganisms are isolated from the soils of a tea garden at Iwata city, Shizuoka prefecture, a tea garden at Ogasa town in Shizuoka prefecture, a tea garden and a vegetable field of Iruma city in Saitama prefecture, a wheat field of Atsugi city in Kanagawa prefecture and a strawberry field of Nirayama town in Shizuoka prefecture.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to microorganisms that reduce environmental load such as groundwater and river pollution caused by nitrate nitrogen flowing out of field soil, orchard soil, tea garden soil, and pasture soil. The present invention relates to a microbial material and a method for using the material to reduce nitrate nitrogen outflow to the environment.

[0002]

2. Description of the Related Art Nitrogen-containing fertilizers and nitrogen-containing organic substances are converted into ammonia nitrogen in soil by mineralization, and the ammonia nitrogen is converted into nitrite nitrogen by ammonia-oxidizing bacteria, and the nitrite nitrogen is converted into nitrite. It is known that oxidizing bacteria rapidly convert it to nitrate nitrogen, and this is called nitrification. The generated nitrate nitrogen is hard to be adsorbed on the soil, is easily leached out of the soil by precipitation, flows out into groundwater and rivers, causes groundwater and river pollution, and uses these contaminated water as drinking water for livestock. It is known that ingestion by humans causes nitrate poisoning, which is an important issue that must be solved as an environmental problem.

Conventionally, as a wastewater treatment technique containing a nitrogen component, a water treatment facility including an aerobic nitrification tank and an anaerobic denitrification tank has been put to practical use. That is, ammonia nitrogen,
Water containing nitrogen components such as nitrite nitrogen and nitrate nitrogen is converted to nitrate nitrogen by ammonium oxidizing bacteria and nitrite oxidizing bacteria in a nitrification tank, and then nitrate nitrogen is removed by denitrifying bacteria in a denitrification tank. It is reduced to nitrogen gas and released to the atmosphere. However, the method of denitrification of wastewater containing nitrogen component is suitable when the wastewater can be centrally controlled like a factory.However, in a field, orchard, tea plantation, etc. In the treatment of nitrate nitrogen derived from nitrogen fertilizers, pumping large amounts of groundwater, collecting surface water,
Alternatively, it requires large-scale civil engineering work and processing equipment, and cannot be said to be an easy method in terms of cost.

[0004] Also, a method is known in which the nitrification function of ammonia-oxidizing bacteria and nitrite-oxidizing bacteria in soil is suppressed by chemical substances. However, application of such chemical substances is not suitable for growing crops. Unresolved issues remain, including impacts on crop quality. In particular, the environmental burden due to the application of chemical substances to the natural world has recently become a major social problem, and there is a need for an environmentally friendly nitrification suppression method and a nitrate nitrogen accumulation prevention method.

[0005]

SUMMARY OF THE INVENTION The present inventors have paid attention to these points, and have made it possible to eliminate the need for large-scale wastewater treatment facilities and to use microorganisms without the use of chemical substances in soil or water. A method to reduce the environmental load caused by nitrate nitrogen was studied. That is, an object of the present invention is to provide a microorganism having a property of suppressing the production or accumulation of nitrate nitrogen. Another object of the present invention is to provide a microorganism material containing such a microorganism, whereby the production or accumulation of nitrate nitrogen can be suppressed. It is a further object of the present invention to provide a method for suppressing the generation or accumulation of nitrate nitrogen by applying such a material to soil or water, thereby suppressing the flow of nitrate nitrogen to the environment. .

[0006]

Means for Solving the Problems The present inventors have searched for a large number of microorganisms to solve the above-mentioned problems, and as a result, discovered the existence of microorganisms that significantly reduce the amount of nitrate nitrogen in soil and water. Was reached. That is, the present invention
(1) a microorganism belonging to the genus Penicillium, Aspergillus, Pesiromyces, Streptomyces or Xanthomonas having an ability to inhibit the production or accumulation of nitrate nitrogen, (2) the microorganism of (1) and / or culture thereof A microbial material having an ability to inhibit the production or accumulation of nitrate nitrogen containing a substance as an active ingredient; (3) the microbial material according to (2) above, wherein the microorganism and / or a culture thereof is a mixture of two or more types; ) The microorganism of (1) above, (2) or
The present invention relates to a method for suppressing the production or accumulation of nitrate nitrogen by applying the microorganism material of (3) to soil or water.

[0007] It is generally known that the soil microflora changes depending on the temperature, moisture and nutrient of the soil, and in consideration of these soil conditions, the microorganism of the present invention comprises a fungus,
Microorganisms that reduce nitrate nitrogen were selected from each group of actinomycetes and bacteria. The microorganism according to the present invention was selected by the following method. In other words, soil can be grown at a pH of 5 or less according to the soil microbial experiment method (Soil Microorganisms Research Society, Yokido 1992) New fungi, actinomycetes and bacteria were isolated. The soil from which these microorganisms were isolated was Penicillium and Pesilomies, tea garden soil in Iwata City, Shizuoka Prefecture, and Aspergillus was tea garden soil in Ogasa Town, Shizuoka Prefecture. Also, Streptomyces A
C5400 is a tea garden soil in Iruma City, Saitama Prefecture.
Is vegetable field soil. Streptomyces AC5431
Is wheat field soil in Atsugi City, Kanagawa Prefecture. Furthermore, Xanthomonas is a strawberry field soil in Nirayama town, Shizuoka prefecture.

[0008] These microorganisms were further subjected to shaking culture using a yeast extract / malt extract medium (Soil microbial experiment method, page 384). After the cultivation, add a certain amount of the culture solution to the soil collected from the tea plantation in Makinohara, Shizuoka Prefecture,
The method of Hayatsu (Soil Sci. Plant Nutr.,
39, 209, 1993), the nitrification effect of the test soil was measured by ion chromatography. Microorganisms were selected. Microscopic observation of the properties of the selected seven types of microorganisms, and examination of their culture characteristics and biochemical properties, were identified as microorganisms belonging to the genera Penicillium, Aspergillus, Pesiromyces, Streptomyces and Xanthomonas, It is a novel microorganism in that it has the property of reducing or not producing and accumulating nitrogen.

In culturing the selected microorganism,
Normal carbon sources, nitrogen sources, inorganic ions, and, if necessary, may be cultured in a liquid or solid medium to which micronutrients have been added. As a carbon source, starch, sugars such as glucose, coffee waste, soybean meal, bran, vegetable waste such as bran, alcohol such as ethanol, organic acids such as acetic acid,
Oils such as lard can be used. As the nitrogen source, various ammonium salts, nitrates, amino acids, and animal waste such as fish meal, bone meal and meat extract can be used. As the trace nutrient, trace amounts of various vitamins and yeast extract may be added.

In the case of liquid culture, it is possible to perform submerged culture in a usual culture tank, and at 25 ° C. to 30 ° C. under aerobic conditions.
H4.0 to pH9.0, preferably pH4.0 to pH8.0, 4 days to 10
The culture is completed within days.

In the present invention, the culture solution of the liquid culture may be applied as it is to a soil surface as a microbial material, mixed with the soil, irrigated into the soil, or applied to the water. A carrier may be added for use.

When the cells are collected, ordinary centrifugation or filtration can be applied. Alternatively, a culture medium may be mixed with a filter aid such as celite or perlite for separation. These can be dried by freeze vacuum drying, spray drying or the like, or can be further adjusted by adding a new carrier.

In the solid culture, the present invention is applied to a mixture of a nutrient such as a carbon source, a nitrogen source and inorganic ions which can be used in a liquid culture, and a light and porous inorganic carrier such as zeolite or vermiculite. A method of inoculating a microorganism as an inoculum, a method of inoculating the microorganism of the present invention as an inoculum on a light-weight, porous inorganic carrier impregnated with the above-mentioned nutrient source, and further, plant fibers such as straw, etc. A method of impregnating a knitted material with a nutrient source and culturing is exemplified. In addition, in a container having the same hermeticity as that of an incubator in mushroom bottle culture or bag culture, culture similar to mushroom bed cultivation is possible.
The microorganism of the present invention grows for 15 days from the day. After the cultivation, crushing is preferably performed, and, similarly to the above, usually, a carrier of an inorganic substance such as zeolite or vermiculite or an organic substance such as bran or bran is mixed so that the water content is 30% or less. May be.

The microorganism material of the present invention contains, as an active ingredient, a microorganism selected by the above-mentioned method and having an ability to inhibit the production or accumulation of nitrate nitrogen and / or a culture thereof. It is preferable to use a mixture of two or more different microorganisms and / or cultures in view of the difference in the properties of the various soils and drainage to be treated.

The microbial material of the present invention may be used in any suitable form, such as a liquid fluid, a powder, a granule, a pellet, or the like, depending on the application location. In order to simplify the location or operability where materials such as slopes may run away, in some cases, it is also preferable to adsorb or culture the microorganism of the present invention on a fibrous or net-like carrier. .
These carriers are preferably biodegradable fibers or nonwoven fabrics, for example, "Bennet" (Asahi Kasei Kogyo)
(Trade name, manufactured by KK Corporation) is a preferred example.

[0016] The microorganisms to reduce the soil or nitrate nitrogen in the water of the present invention, as microorganisms amount per land 1m ^2, 1.
0 × 10 ⁸ CFU (number of colonies formed; “New edition of soil microbial experiment method, edited by Soil Microorganisms Research Society, Yokendo” p. 62) When using the above-mentioned biodegradable fiber or nonwoven fabric, the same amount of microorganisms may be adsorbed. Examples of the carrier include inorganic substances such as zeolite and vermiculite, and bran and organic substances such as bran as described above.
In particular, it is extremely effective to mix a carrier which also serves as a nutrient that can be a nutrient source of the microorganism of the present invention.

The microorganism content of the microorganism material of the present invention is as follows:
There is no particular limitation as long as the effective amount of the microorganism can be sprayed, but if it is too thin, it is necessary to spray a large amount, and there are problems such as a large transport amount and a long spraying time. is necessary. This is not necessarily a problem when the culture solution is used as it is, but especially for solid microorganism materials, 1.0 ×
It is preferable to contain a microorganism of 10 ⁷ CFU or more. In addition, when the water content is 30% or less, particularly 15% or less, the stability of microorganisms is good and an effective amount of microorganisms can be maintained even when stored outdoors, which is particularly preferable.

The microorganisms of the present invention can be used alone or as a mixture of the microorganisms. The microorganisms can be used by spraying them on the surface of the soil, or by spraying when cultivating, mixing with the soil, or suspending them in water, Can be irrigated. As an application tool, a usual pesticide sprayer, a pesticide irrigation machine, a fertilizer sprayer and the like can be used. The time of spraying is not particularly limited, but it is effective to apply it during the time when the activities of nitrifying bacteria and nitrifying bacteria are active or immediately before the season. The reason why the microorganism of the present invention reduces the accumulation of nitrate nitrogen in soil or water is not fully understood in theory, but the utilization of nitrate nitrogen produced by nitrifying bacteria in soil or water, or nitrification Antagonism with bacteria can be considered.

[0019]

DESCRIPTION OF THE PREFERRED EMBODIMENTS Hereinafter, the present invention will be described specifically with reference to examples, but the present invention is not limited to these examples.

Example 1 A microorganism according to the present invention was selected by the following method. In other words, according to the soil microbial counting, separation, and identification methods described in the “Soil Microorganisms Experimental Method” (edited by the Society for the Study of Soil Microorganisms, Yokendo 1992), using soil in each region as the source of microorganisms,
Filamentous fungi, actinomycetes and bacteria capable of growing below 5 were isolated. These microorganisms were further subjected to shaking culture using a yeast extract / malt extract medium (Soil microbial experiment method, page 384). After completion of the culture, a certain amount of the culture solution was added to the soil collected from the tea plantation in Makinohara district, Shizuoka prefecture, and the method of Hayatsu was used at 25 ° C for 2 weeks (Soil Sci. Plant Nutr., 39, 209, 1993).
A nitrification experiment was carried out in accordance with the standards, and the amount of nitrate nitrogen in the test soil was measured by ion chromatography. Seven types of microorganisms that significantly reduced or did not produce or accumulate nitrate nitrogen were selected. Was done. Table 1 shows the results of analysis of the nitrate nitrogen produced by the filamentous fungi, Tables 2 and 3 for actinomycetes, and Table 4 for bacteria.

[0020]

[Table 1]

[0021]

[Table 2]

[0022]

[Table 3]

[0023]

[Table 4]

The properties of the selected seven types of microorganisms were observed under a microscope, and the culture properties and biochemical properties were examined. The properties were as follows.

1. Filamentous fungus No.3 (1) Growth state in each medium Characteristics on potato glucose agar medium (PGA) When cultured at 25 ° C for 7 days, the colony becomes 37-41 mm in diameter and the colony surface becomes felt-like. Colony color is white (1A1) to pale yellow paleyellow (1A1)
It is. When cultured for 14 days, colonies are 58-63 mm in diameter
And the color of the colony is yellowish yellowish white (4A2) ~
It is cream cream (4A3). In the colony, a faint ring pattern and a slightly raised striation extending from the center can be confirmed. When cultured at 37 ° C. for 7 days, the diameter becomes 3-3.5 mm, and deep wrinkles are formed on the colony surface. The color of the colony ranges from white white (1A1) to pale pink pinkish white (12A2). When cultured for 14 days, the colonies are 9-12 mm in diameter. Colony color is white (1A1) to light pinkish
white (12A2).

Characteristics on Malt / Yeast Extract Agar Medium (MYA) When cultured at 25 ° C. for 7 days, the colony becomes 45-50 mm in diameter, the surface is felt-like, and the color is white white (1A1) to pale yellow pale. It is yellow (1A1). When cultured for 14 days, the colony becomes 75-77 mm in diameter, and the color of the colony is yellowish white (4A2) to cream cream (4A2).
3). In the colony, a faint ring pattern and a slightly raised striation extending from the center can be confirmed. When cultured at 37 ° C. for 7 days, the diameter becomes 4-6 mm, and deep wrinkles are formed on the colony surface. The color of the colony is white (1A1). When cultured for 14 days, the colonies are 12-14 mm in diameter,
The color of the colony is white (1A1), but partially pale gray (1B1).

(2) Physiological properties When cultured in a potato glucose liquid medium: The range of pH at which growth is possible is 3-9, and the range of optimal growth pH is 6-8. When cultivated on potato glucose agar medium: The range of temperature that can grow is 6.9 to 37 ° C, and the optimum range of growth temperature is 24.
~ 30 ° C.

(3) Morphological characteristics and identification under a microscope The fungus forms ascocarp-like sclerotium, but ascospores cannot be confirmed. Therefore, since only the asexual generation (anamorph) exists, it belongs to the incomplete fungi. The fungus has a conidiophore erecting from the mycelium and branches off at the upper part of the conidia. The entire conidiophore is transparent, but shows a characteristic green color in the population. Conidiophores are rough and covered with fine grains. This bacterium has a phyarotype conidium formation mode, and produces 3 to 5 12 × 3 μm phialides. The surface structure of the conidium is smooth and 3 ×
3 μm spores are linked. These features are Penicillium
Good agreement with the genus features. The size of ascocarp-like sclerotium is 1
15 × 125 μm. The color is cream cream (4A3). This sclerotium is very likely to be an ascomycete but cannot be treated as Eupenicillium because it does not form ascospores. Therefore, Domsch, KH, Gams, W.an
d Anderson, Traute-Heidi 1980. ^ Compendium of Soil
Fungi "IHW-Verlag. Based on Fungi" IHW-Verlag., This bacterium was identified as Penicillium genus. This bacterium was designated as Penicillium sp. No. 3. The color of the growth status in each medium is indicated by Korne
rup A. and Wanscher JH1978 ^ Methuen Handbook of C
olour 3rd ed. "Eyre Methuen, London

2. Filamentous fungus No.8 (1) Growth state in each medium Characteristics on potato glucose agar medium (PGA) When cultured at 25 ° C for 7 days, the colony becomes 15-17 mm in diameter, and the colony surface has a fine-felt shape. Become.
The color is white (1A1). When cultured for 14 days, the colony is 28-30 mm in diameter and the color is white (1Al). No growth at 37 ° C.

Characteristics on Malt / Yeast Extract Agar Medium (MYA) When the colony is cultured at 25 ° C. for 7 days, the colony has a diameter of 15 to 16 mm, and the surface of the colony has a fine-grained felt shape.
The color is white (1A1). When cultured for 14 days, the colony is 28-30 mm in diameter and the color is white (1Al). No growth at 37 ° C.

(2) Physiological properties In the case of culturing in a potato glucose liquid medium: The range of pH at which growth is possible is 3 to 9, and the range of optimal growth pH is 5 to 8. When cultivated on a potato glucose agar medium: The growth temperature range is 14-35 ° C, and the optimal growth temperature range is 30-35 ° C.
34 ° C.

(3) Morphological characteristics and identification under a microscope The fungus does not form migratory spores and does not form sporangia. Only the asexual generation (anamorph) is formed, and no sexual generation (telemorph) is found. It has a conidiophore erecting from the mycelium, and the upper part of the conidia swells. The mode of conidial formation is of the phialo type, and phialides are formed on the swollen part. A clear foot cell is seen above the conidiophore. Conidia are transparent, but appear white in the population. Conidia are single cells and are formed on a chain from spheres and phialides. From these features, the ^ Comp
endium of Soil Fungi "
It was identified as a filamentous fungus belonging to the genus. Aspergillus
SP No.8. The color of the growth status in each medium is indicated by Kornerup A. and Wanscher JH1978 ^ Met
huen Handbook of Color 3rded. "Eyre Methuen Londo
n was followed.

3. Filamentous fungus No.35 (1) Growth state in each medium Characteristics on potato glucose agar medium (PGA) When cultured at 25 ° C for 7 days, the colony becomes 35-38 mm in diameter, and the colony surface becomes felt-like. The color of the colonies ranges from purple gray purplish grey (13B2) to grayish magenta (13B3). When cultured for 14 days, the colony is 68-71 mm in diameter, and the color of the colony ranges from purple gray purplish gray (13B2) to grayish magenta greyish Majenta (13B3). A ring pattern can be slightly observed in the colony. At 37 ° C., almost no growth is observed, but when cultured for 7 days, the diameter becomes 3-3.5 mm. When cultured for 14 days, the colonies are 3-5 mm in diameter. The color of the colony surface is white white (1Al).

Characteristics on Malt / Yeast Extract Doryen Medium (MYA) When cultivated at 25 ° C. for 7 days, the colony becomes 32-33 mm in diameter, and the colony surface becomes a felt-like. The color of the colonies ranges from purple gray purplish grey (13B2) to grayish magenta (13B3). When cultured for 14 days, the colonies are 61-65 mm in diameter, and the color of the colonies is purple gray purplish gray (13B2) to grayish magenta.
greyish Majenta (13B3). In the colony, a ring pattern can be slightly observed, and a thin radial streak can be recognized from the center of the colony in some cases. At 37 ° C, almost no growth is observed.
m. When cultured for 14 days, the colonies are 3-4 mm in diameter. The color of the colony surface is white white (1Al).

(2) Physiological properties In the case of culturing in a potato glucose liquid medium: The range of pH at which growth is possible is 3 to 9, and the range of optimal growth pH is 4 to 8. When cultivated on a potato glucose agar medium: The growth temperature range is 11 to 37 ° C, and the optimal growth temperature range is 23 to 37 ° C.
27 ° C.

(3) Morphological characteristics and identification under a microscope The bacterium belongs to imperfect fungi since it forms only asexual generation (anamorph) and no sexual generation (telemorph). It has a conidium stem erecting from the mycelium and branches off at the upper part of the conidiophore. Conidia are transparent, but the population shows a characteristic purple gray. The mode of conidial formation is of the firearo type, the size of the fluoride is 2 × 7 μm, and 3 to 5 strands are formed on metre. Conidia are single cells and are formed in a chain from lemon-shaped and fluoride.
4 μm. Morphologically similar to the genus Penicillium, but does not show the green color characteristic of the bacterium. From the above features ^
Compendium of Soil Fungi "
It was identified as a filamentous fungus belonging to the genus myces. This bacterium is designated as Pesilomies sp. The color of the growth state in each medium is indicated by Kornerup A. and Wanscher JH1.
978 "Methuen Handbookof Color3rded." EyreMethuen, L
Follow the ondon sign.

4. Actinomycetes No.15 (1) Morphological properties The cultures were cultured at 28 ° C. for 20 days on a starch / inorganic salt agar medium and a glycerol / asparagine agar medium, and the observations by an optical microscope and an electron microscope were as follows. The underlying mycelium extends in a curved or linear manner with branching and has a diameter of 0.3 to 0.4 μm. Usually, no hyphal disconnection is observed. The aerial hyphae formed from the basic hyphae extend linearly or gently into a curved line, form side yarns alternately or counterbalanced on the way, and have a diameter of 0.4 to 0.6 μm. Usually 50 to 100 or more linked spores are formed from the tip of the aerial hypha. Spores are short cylindrical, 0.6-0.8 × in size
The surface is smooth at 0.7-1.3 μm. No sporangia, motile spores, spinal silk and sclerotium are found.

(2) Chemical Classification (Diaminopimelin Shun) Method of StaneCk et al. (Appl. Microbiol. 28; 226-231 (1974))
As a result, LL-type diaminopimelic acid was detected and no meso-type was detected.

(3) Culturing properties The results of culturing on various media at 28 ° C. for 20 days and observations are as shown in Table 5. The color display is Color harmony
manual 4th edition 1958 Container Corporation of Am
Followed erica.

[0040]

[Table 5]

(4) Physiological properties Growth temperature range: 13 to 40 ° C. Production of melanin-like pigment: on tyrosine agar medium: false positive: on peptone / east extract / iron agar medium; availability of positive element: L -Arabinose, D-fructose, D-glucose, inositol, D-mannite-
L, raffinose, L-rhamnose, sucrose and D-sylose. Table 5 shows the culture characteristics of this strain.

As described above, the actinomycete strain No. 15 forms aerial hyphae that form a large number of linked spores from true basal hyphae, does not form flagellar spores or spores, and diaminopimelic acid. Has characteristics such as being LL-type, so Bergey's Manual of systematic Bacter
iology vol.4 (1989) by Streptomyces (Strep
(Tomyces) genus. Therefore, actinomycetes N
o.15 strains from Streptomyces sp. AC5431 (Stre
ptomyce s sp. AC5431 ) strain.

5. Actinomycetes No.49 (1) Morphological properties East extract / malt extract agar medium, oatmeal agar medium, starch / inorganic salt agar medium and glycerol / agar medium
The cells were cultured on asparagine agar medium at 28 ° C. for 20 days, and the observations by an optical microscope and an electron microscope are as follows. The underlying mycelium extends in a curved or linear manner with branching and has a diameter of 0.3 to 0.4 μm. Usually, no hyphal disconnection is observed. The aerial hyphae formed from the basic hyphae is formed of a long curved or linear main shaft and a short side yarn alternately or counterbalanced, and has a diameter of 0.4 to 0.6 μm.
Normally, the end of the side thread has a spiral shape wound tightly three to four turns, and forms about 10 to 20 linked spores. The spores are ovoid, 0.6-0.8 × 0.7-1.0 μm in size, and spiny on the surface. No sporangia, motile spores, spinal silk and sclerotium are found.

(2) Chemical classification (diaminopimelic acid) The method of Staneck et al. (Appl. Microbiol. 28; 226-231 (197
As a result of analysis according to 4)), LL-type diaminopimelic acid was detected, but no meso-type was detected.

(3) Culturing properties The results of culturing on various media at 28 ° C. for 20 days and observations are as shown in Table 6. The color display is Color harmony
manual 4th edition 1958 Container Corporation of Amer
Followed ica.

[0046]

[Table 6]

(4) Physiological properties Growth temperature range: 13-45 ° C. Production of melanin-like pigment: Tyrosine agar agar; Negative: on beptone-east extract / iron agar medium; Negative Use of carbon source: L- Arapinose, D-fructo-
, D-glucose, D-mannitol and D-xylose. Inositol is questionable. Rough inose, L-rhamnose and sucrose are not used. Table 6 shows the culture characteristics of this strain.

As described above, the actinomycete No. 49 strain produces aerial hyphae which is more branched than the true basal hyphae, the tip of which is spiralized to form linked spores, flagella spores and spore spores. Is not formed, and the amino acid is diaminopimelic acid is LL-type.
s It was identified as belonging to the genus Streptomyces according to the Manual of systematic Bacteriology. Therefore, the actinomycete No. 49 strain is referred to as Streptomyces sp. AC5400 strain.

6. Actinomycetes No.60 (1) Morphological properties East extract / malt extract agar medium, oatmeal agar medium, starch / inorganic salt agar medium, glycerol /
The cells were cultured on asparagine agar medium at 28 ° C. for 20 days, and the observations by an optical microscope and an electron microscope are as follows. The underlying mycelium extends in a curved or linear manner with branching and has a diameter of 0.3 to 0.4 μm. Usually, no hyphal disconnection is observed. The aerial hyphae formed from the basic hyphae is formed of a long curved or linear main shaft and a short side yarn alternately or counterbalanced, and has a diameter of 0.4 to 0.6 μm.
Normally, the end of the side thread has a spiral shape wound tightly three to four turns, and forms about 10 to 20 linked spores. The spores are ovoid, 0.6-0.8 × 0.7-1.0 μm in size, and spiny on the surface. No sporangia, motile spores, rota silk and sclerotium are found.

(2) Chemical separation (diaminopimelic acid) The method of StaneCk et al. (Appl. Microbiol. 28; 226-231 (19)
As a result of analysis according to 74)), LL-type diaminopimelic acid was detected, but no meso-type was detected.

(3) Culture properties Table 7 shows the findings observed on various media at 28 ° C for 20 days. (Color display is Color harmony man
ual 4th edition 1958 Container Corporation of America
Followed. )

[0052]

[Table 7]

(4) Physiological properties Growth temperature range: 15 to 43 ° C. Production of melanin-like pigment: on tyrosine agar medium; negative: on bepton / yeast extract / iron agar medium: negative Use of carbon source: L-arapino -S, D-Fract-
, D-glucose, inositol, D-mannitol and D-xylose. Rough inose, L-rhamnose and sucrose are not used. Table 7 shows the culture characteristics of this strain.

As described above, the actinomycete No. 60 strain produces aerial hyphae which is more branched than the true basal hyphae, the tip of which is spiralized to form linked spores, flagella spores and spore spores. Is not formed, and the amino acid is diaminopimelic acid is LL-type.
s It was identified as belonging to the genus Streptomyces according to the Manual of systematic Bacteriology. Therefore, the actinomycete No. 60 strain is referred to as Streptomyces sp. AC5005 (Streptomyces sp. AC5005) strain.

7. Xanthomonas maltophilia-N The mycological properties of this bacterium are as follows. (1) Characteristics of growth Ordinary agar slant medium Good growth in rod form. wet. Pale yellow to orange. No soluble dye. Ordinary agar plate medium Round and irregular. Flat and swelling in the center. Pale yellow to orange. No soluble dye. Translucent. Litmus milk alkaline. Peptone conversion.

(2) Morphological features Cell shape: straight or slightly curved bacilli with rounded edges, no polyplasia of cells. Size: 0.4-0.5 x 1.5-4.0 μm Motility: non-motile Spore: no spore formation Gram stain: negative Acid-fast stain: negative

[0057]

[0058]

The feature of this bacterium is that it is non-motile and has the ability to produce acid from rhamnose. The main properties of this strain are gram-negative bacilli, oxidase-negative (or weakly positive), catalase-positive, and oxidatively degrade glucose to produce acids. The properties of this strain are B
ergey's Manual of systematic Bacteriology Vol. 1 (19
84), Vol.2 (1986) and Int.J.Syst.Bacteriology 3
3, 409-413 (1983), etc., and is a mutant of Xanthomonas maltophilia and named Xanthomonas maltophilia N.

Tables 8 and 9 compare various properties of this strain with Pseudomonas vesicaris and Xanthomonas maltophilia.

[0061]

[Table 8]

[0062]

[Table 9]

From the results shown above, each of these microorganisms was Penicillium sp. No. 3 (FERM P-1720).
8), Aspergillus SP No. 8 (FERM P-17205),
Pesilomies sp.No.35 (FERM P-17207), Streptomyces espi AC5400 (FERM P-17202), Streptomyces espi AC5005 (FERM P-17203), Streptomyces espi-AC5431 (FERM) P-17204) and Xanthomonas maltophilia-N (FERM P-17206).

[0064]

[Example 2] Liquid culture was carried out on each of the seven selected microorganisms, and the relationship between the number of microorganisms and the production rate of nitrate nitrogen was tested by the Hayatsu method (Soil Sci. Plant Nutr., 39, 209, 1993). That is, each of the seven microorganisms was inoculated into a yeast extract / malt extract medium (Soil microbial experiment method, p. 384), and liquid culture was performed at 28 ° C. Alternately mixed into the test soil described above. After mixing, the test soil was stored in a thermostat at 25 ° C. After 3 weeks, the amount of generated nitrate nitrogen was measured by ion chromatography. The ratio was determined and defined as the nitrate nitrogen generation rate. Filamentous fungus Penicillium sp.No.3 (FERM P
-17208), Aspergillus SP No.8 (FERM P-1720)
5), the suppression of nitrate nitrogen accumulation by Pesiromyces sp. No. 35 (FERM P-17207), actinomycetes Streptomyces espi-AC5400 (FERM P-17202), Streptomyces espi-AC5005 (FERM P- 17203), Streptomyces espi-AC5431 (FERM P-17204) results of inhibition of nitrate nitrogen accumulation and bacteria Xanthomonas maltophilia-N (FERM P-17206) nitrate nitrogen accumulation suppression results in Table 10, respectively. The results are shown in Tables 11 and 12.

[0065]

[Table 10]

[0066]

[Table 11]

[0067]

[Table 12]

From the results of this experiment, it was found that the inoculation rate of 1.0 × 10 ⁸ CFU / m ² or more of the fungi, actinomycetes and bacteria showed that the production rate of nitrate nitrogen was 10% or less, and that the accumulation of nitrate nitrogen was It was confirmed that it decreased.

[0069]

Example 3 A filamentous fungus Aspergillus sp. No. 8 (FE
RM P-17205) and Pesilomies SP No. 35 (FERM P
-17207) was inoculated into a liquid culture medium of pH 6.8 containing 2% malt extract (DIFCO), 0.2% yeast extract (DIFCO) and 2% dextrose (Wako Pure Chemical). Culture was performed at 26 ° C. for 6 days under aerial conditions. At this time, the bacterial count of Aspergillus sp. No. 8 (FERM P-17205)
The concentration of 1.8 × 10 ⁶ CFU per 1 ml of the culture solution was 9.68 × 10 × 10 ⁵ CFU for Pesilomies sp. No. 35 (FERM P-17207). Solid culture was performed using this culture solution as a seed fungus. Rice bran and rice straw (volume ratio 1: 1), 1 l of culture medium adjusted to 60% water content, polypropylene bag for mushroom cultivation (Sanbuck;
(Trade name; manufactured by Mitomi Corporation) and sterilized at 121 ° C. for 2 hours. After completion of the sterilization, the solution was allowed to cool, and 20 ml of the liquid culture seed solution was inoculated into a solid culture medium and cultured at 25 ° C for 2 weeks.

By this solid culture, 6.8 × 10 ⁷ CFU of Aspergillus sp.
o.8 (FERM P-17205) containing 45% water in solid culture and 1.45 × 10 ⁸ CFU of Pesilomyces sp.
A 40% water solid culture containing ERM P-17207) was obtained. Dried vermiculite was added to each of the solid cultures in a ratio of 1: 2 (w
/ w) at a ratio of 2.3 ×
10 ⁷ CFU Aspergillus SP No.8 (FERM P-17
205) containing 15% moisture and microbial material A and 4.8 x 10 ⁷
A 14% moisture microbial material P containing CFU Pesiromyces sp. No. 35 (FERM P-17207) was produced.

Using the microbial materials A and P, a nitrate nitrogen production test was carried out by the Hayatsu method (Soil sci, Plant Nut, 39, 209, 1993). The results are shown in Table 13. Similar to liquid culture even in solid culture from this experimental result, inoculation bacteria count per 1 m ² the accumulation of nitrate in 1 × 10 ⁸ CFU or 9
It was shown that 0% or more was suppressed.

[0072]

[Table 13]

[0073]

According to the present invention, a microbial material is prepared by using a microorganism that suppresses the production or accumulation of nitrate nitrogen and is applied to soil or water. Generation and accumulation can be suppressed and the environment can be prevented from being contaminated with nitrate. Moreover, the application method is simple, and the pollution of the environment can be efficiently prevented by the simple method.

 ────────────────────────────────────────────────── ─── Continuing on the front page (72) Inventor Toshiaki Araki One of 632 Mifuku, Oni-cho, Tatagata-gun, Shizuoka Prefecture F-term in Asahi Kasei Kogyo Co., Ltd. 4B065 AA50X AA56X AA58X AA60X AA67X AC12 AC20 BA22 CA54 4D040 DD01 DD14

Claims (5)

[Claims] 1. A microorganism belonging to the genus Penicillium, Aspergillus, Pesiromyces, Streptomyces or Xanthomonas having the ability to inhibit the production or accumulation of nitrate nitrogen. 2. A microorganism belonging to the genus Penicillium, Aspergillus, Pesiromyces, Streptomyces or Xanthomonas having an ability to inhibit the production or accumulation of nitrate nitrogen is Penicillium sp. No. 3 (FERM).
P-17208), Aspergillus SP No.8 (FERM P-1
7205), Pesilomies SP No.35 (FERM P-1720
7), Streptomyces SP AC 5400 (FERM P-17
202), Streptomyces SP AC 5005 (FERM P-
17203), Streptomyces SP AC 5431 (FERM
P-17204), or Xanthomonas maltophilia -N
A microorganism that is (FERM P-17206). 3. The microorganism according to claim 1 or 2 and / or
Alternatively, a microbial material having an ability to inhibit the production or accumulation of nitrate nitrogen, comprising using a culture thereof as an active ingredient. 4. The microorganism material according to claim 3, wherein the microorganism and / or a culture thereof is a mixture of two or more kinds. 5. A method for suppressing the production or accumulation of nitrate nitrogen, which comprises applying the microorganism according to claim 1 or 2 or the microorganism material according to claim 3 to soil or water.