JP4475666B2 - Method of mass culture of holmium simple algae, liquid fertilizer containing this holmidium, and fertilization method thereof - Google Patents

Description

  The present invention relates to a method for easily and inexpensively culturing hormidium, a soil cyanobacteria, using an extract of a soil conditioner, a liquid fertilizer containing holmium obtained by this method, and a method for fertilizing the same.

Various proposals have been made regarding crop growth promoters, and many of them have been methods of diluting and spraying chemically synthesized drugs.
Recently, however, organic farming methods using compost and natural organic fertilizers have become popular, and attempts have been made to develop products that do not affect human health or the environment.

It has been studied in Japan that harvesting cyanobacteria on paddy fields has improved the harvesting rate, but research was discontinued with the spread of chemical fertilizers, and development research has been inherited and practiced exclusively in India. . However, there are few reports in recent years.
The purpose of adding cyanobacteria in paddy fields was thought to be supplementation of nitrogen using the nitrogen fixing ability of cyanobacteria, but now research on a wide range of material exchange between crops and soil microorganisms has been promoted, and the effect of promoting growth of crops by cyanobacteria Are also considered to be not only nitrogen (see Non-Patent Document 1 and Non-Patent Document 2).

  In addition, a pyrrole farming method has been proposed in which the growth of cyanobacteria and microorganisms growing in soil is promoted, thereby obtaining a high-calcium crop (see Non-Patent Document 3). The material used for the pyrrole farming method is an alkalinized mixture of organic matter such as manure with quick lime and trace elements by a unique method. For this reason, the pyrrole farming method has a lot of nuisance and is difficult to handle for the producers, such as the inflammation of the hands of the producers. In addition, the cyanobacteria in which growth is promoted by this method is not specified.

  On the other hand, a soil improvement method has been proposed in which soil algae is cultured and integrated with a superabsorbent resin and sprayed onto degraded soil and desertified soil, thereby converting these soils into arable land where plants can grow. (Refer to << Patent Document 1 >>). In this method, since the water-absorbent resin absorbs 500 to 1000 times its own weight, it is possible to promote the growth of algae even in soil having a high salt concentration by drying. However, this method has been tested for Anabaena and Nostoc, but does not mention holmium.

  Living holmium has the property that it adheres to the substrate or adheres to each other to form a lump (hereinafter this lump is also referred to as a “mat”) due to its secretory substances. For this reason, in liquid fertilizers in a state where live holmidium is suspended, for example, the liquid fertilizers clump together and adhere to the bottom of the liquid fertilizer storage container or spreader. There is an urgent need for improvement in handling (fertilization, storage, transportation, sales, etc.) and urgent development is required.

  However, holmidium forms filaments (trichomes) in which cells are connected in series, and adheres to the substrate by the secreted mucus substance and grows. Therefore, liquid suspension culture method (using liquid medium) used for microalgae such as chlorella A culture method with stirring or shaking is not possible. At the same time, since holmidium is a prokaryotic organism, antibiotics and the like are not used, pure culture is difficult, and a method for mass culture has not been established. For example, Non-Patent Document 4 proposes a method of growing colonies that grow in a spherical shape in the above-mentioned Nostock by a liquid aeration culture method (a method of culturing oxygen or a mixed gas in a liquid medium while forcibly aeration). However, this method is not effective for holmium that forms an adherent filamentous group as shown in FIG. 1, and does not lead to mass culture.

"Blue-green algae for rice production. A manual for its promotion." Venkataraman GS (1981) FAO Bulletin No. 46 FAO Rome "Soil and Rice Field. In The Ecology of Cyanobacteria. P.233-255" B.A.Whitton (2000) Kluwer Academic Publishers “Pyrrole farming to make high-calcium crops” by Sakai Ya (1996) Nobunbunkyo “Creation of soil sphere and cyanobacteria” Masayuki Omori, Kazuyuki Inubushi (2004) Chemistry and Biology Japanese Patent Laid-Open No. 6-80490

  An object of the present invention is to provide a method for easily and inexpensively culturing a large amount of soil cyanobacteria holmium using an extract of a soil conditioner. Another object of the present invention is to provide a fertilizer application method that is a liquid fertilizer containing holmium obtained by this culturing method and that can be easily and comfortably handled by farmers in agriculture and forestry.

The inventors of the present invention have developed a soil conditioner containing igneous rock powder and clay as main components and mixed with ripe compost, sedimentary soil, uplifted coral powder, and the like. Focusing on promoting growth,
1) A method for mass cultivation using the extract of this soil conditioner and the characteristics of holmidium (the property of agglutinating and coagulating with secreted mucus substances while eliminating soil and other microorganisms) further,
2) Since cyanobacteria have strong resistance to the environment and can survive in a dry state, the present inventors have also found that a dry form of holmidium is prepared and used as a liquid fertilizer attached to the above extract, thereby completing the present invention. It came to do.

That is, the present invention
(1) An extract of a soil conditioner containing igneous rock powder and clay as the main components, matured compost matured to the extent that it can be applied as fertilizer, sedimentary soil containing colloidal micelles of humic acid, and raised coral powder. As a first culture solution, after transplanting holmium collected from the surface of the soil into the first culture solution, primary culture by stationary culture is performed, and holmidium grown in the primary culture is isolated from soil and other microorganisms. A method for mass-cultivating holmidium monoalgae, comprising performing secondary culture by static culture after transplantation to a second culture solution comprising the same components as the first culture solution.

(2) 1-50 parts by weight of fully matured compost matured to the extent that the soil conditioner can be applied as fertilizer to 100 parts by weight of igneous rock powder and clay, and sedimentary soil and uplift containing colloidal micelles of humic acid The method for mass-cultivating holmium simple algae according to (1), wherein 1 to 50 parts by weight of a mixture of coral powder is blended and mixed.

(3) The first and second culture liquids are supernatants obtained by mixing and agitating the soil conditioner with water and then allowed to stand; minerals eluted from igneous rock powder, clay and uplifted coral powder; And extracts from soil microorganisms and their metabolites contained in sedimentary soil, at least,
Calcium (Ca) 10-100mg / L (liter)
Nitrogen (N) 10-100 mg / L (liter)
Sodium (Na) 1-50 mg / L (liter)
Potassium (K) 1-50mg / L (liter)
Magnesium (Mg) 1-50mg / L (liter)
The method for culturing holmium simple algae as described in (1) or (2), comprising:

(4) A liquid fertilizer comprising the extract A as an agent A and the dried form of holmium obtained by the method according to any one of (1) to (3) as an agent B.

(5) A method for fertilizing a liquid fertilizer containing holmidium according to (4), wherein 0.2 to 0.5 g of dry form of holmium is added to 1 L (liter) of the extract and immediately sprayed. A fertilization method characterized by:
Can be provided.

  The method for culturing holmidium according to the present invention makes it possible to cultivate soil cyanobacteria holmidium simply and inexpensively.

  Moreover, the fertilizer application method of the liquid fertilizer containing the holmidium obtained by the mass culture method of this invention can be implemented simply and comfortably by an agricultural or forestry producer. By applying this fertilization method, holmium recovered from the dormant state grows while forming a mat on the surface of the hair follicle of the plant, and a physiologically active substance (such as a plant hormone) absorbed by the follicle promotes the growth of the plant. It is speculated that it is clear from experimental results that the quality of crops and the recovery of exhausted forests can be achieved by the growth of the cyanobacterium holmidium in the rhizosphere.

The inventors of the present invention are a soil conditioner comprising a mixture of ripened compost which is composed mainly of igneous rock powder and clay and can be applied as fertilizer, sedimentary soil containing humic acid colloid micelles, and raised coral powder. Found to promote plant growth.
As this igneous rock powder and clay, for example, those collected in Maniwa City, Okayama Prefecture can be suitably used. As for the fully matured compost, those containing plant raw materials such as cocoons, cocoons and cocoons are preferable.
As a mixing ratio in such a soil conditioner, 1 to 50 parts by weight of fully matured compost matured to the extent that it can be applied as fertilizer to 100 parts by weight of igneous rock powder and clay, and deposits containing colloidal micelles of humic acid A mixture of 1-50 parts by weight of a mixture of soil and raised coral powder is preferred.
The form of the soil conditioner may be powder (solid) or liquid (liquid).

  The method for obtaining the extract from the soil conditioner is not particularly limited. For example, after 10 g of the soil conditioner is soaked in 1 L of pure water, the supernatant that has been allowed to stand for a certain period of time with proper stirring is used as the extract. do it. If the extraction temperature is too high, there is a concern that the active ingredient of the extract is decomposed, and if it is too low, a long extraction time is required. Therefore, 10 to 100 ° C. is preferable, and room temperature ( About 25 ° C.). The longer the extraction time, the higher the concentration of components useful for the growth of cyanobacteria and crops. However, about 20 to 24 hours is sufficient for practical use.

  In the present invention, the extract thus obtained is at least calcium (Ca) 10-100 mg / L, nitrogen (N) 10-100 mg / L, sodium (Na) 1-50 mg / L, potassium (K). It is preferable to contain 1-50 mg / L and magnesium (Mg) 1-50 mg / L, and as other minerals, iron (Fe), phosphorus (P), etc. are preferably contained in a trace amount of 1 mg or less.

  In the holmidium mass culture method of the present invention, after transferring holmidium to the first culture solution, which is such an extract, primary culture is performed by stationary culture, and holmidium grown in the primary culture is used as the first culture. It is important to plant a large amount of the second culture solution composed of the same components as the solution and perform secondary culture by static culture.

  The purpose of primary culture with the first culture solution is to remove holmium collected from the soil and remove it purely from mixed soil and other microorganisms. It grows until it can be transformed. Since the secondary culture is a large-scale culture, a large amount of the secondary culture solution is required, and a large number of secondary culture vessels are used.

  In addition, holmidium is more prone to grow if it is not suspended in the liquid, and it is better to have a large liquid surface area in contact with air for photosynthesis, so both primary and secondary culture vessels have a shallow liquid depth (specifically In this case, a flat container is suitable.

In this way, by using stationary culture (method of culturing the liquid medium in a stationary state without stirring and shaking) instead of suspension culture or aeration culture, holmium is attached to the container and proliferated. Can do. In addition, by repeating stationary culture twice, soil and foreign matter are more reliably eliminated, and a large amount of pure culture is possible without using antibiotics or the like.

  About the holmidium used for culture | cultivation of this invention, after raining, the surface of the soil which turns green is peeled off, this is observed with a microscope, and after confirming that it is holmidium, it transfers to primary culture | cultivation. The holmium used in the present invention was collected in Maniwa City, Okayama Prefecture. Moreover, since what is hold | maintained by indoor culture | cultivation for a long term has weak activity, what was extract | collected from soil one by one and carried out primary culture | cultivation is used.

  The fertilizer application method of liquid fertilizer containing holmium of the present invention is prepared by blending 0.2 to 0.5 g (dry weight) of holmium obtained by the mass culture method with respect to 1 L of the aforementioned extract, It is preferable to spray immediately. If the dry state of holmium mixed in 1 L of the extract is too small, it takes a long time to form a mat on the hair root of the crop, and it does not give a sufficient effect for promoting the growth of the crop, and is too much. However, since the growth promotion effect of the crop is saturated, the above range is preferable in the present invention.

  Regarding the dry state of holmidium, holmium obtained by the mass culture method of the present invention may be naturally dried or forcedly dried by a dryer or the like. Since holmium is sufficiently resistant to drying, it becomes dormant. In consideration of ease of storage and transportation, the dry state is preferably powdered. As a condition for preserving this dry state, it is easy to store in a plastic bag or the like, sealed and shielded from moisture in the atmosphere, and from the viewpoint of ease of transportation and handling, preferable.

In the fertilization method of the present invention, the dry powder form of holmium is mixed in the extract immediately before use (spreading) at the above blending ratio and sprayed well after stirring. Thus, by mixing in the extract immediately before use, it is possible to reliably prevent the formation of lumps in the extract and aggregation on the bottom surface of the spreader.
In the present invention, the extract is A agent, the dried form of holmium is B agent, and A / B is within the range of 10000 parts by weight / 2 to 5 parts by weight. It is also possible to select as appropriate according to the condition and provide it as a liquid fertilizer.

  The method for spraying the liquid fertilizer of the present invention is not particularly limited, and a common watering device or sprayer may be used. The soil after spraying is supplemented with sufficient water in addition to the liquid fertilizer of the present invention to form holmium. It is also possible to promote the growth of.

  The fields and forests that can use the liquid fertilizer of the present invention do not require specific conditions, but are generally effective in existing low-nutrition soils, and may be effective even in extremely poorly nourished dune sand. Has been demonstrated.

<Creation of extract>
Mainly composed of igneous rock powder and clay collected in Maniwa City, Okayama Prefecture, ripe compost ripe to the extent that it can be applied as fertilizer, sedimentary soil containing colloidal micelles of humic acid, and raised coral powder were dispersed and mixed As a soil conditioner, a commercially available “Ginga no Hoshi (Midori Symbiotic Co., Ltd.)” was used.
The above soil conditioner is immersed in pure water at a room temperature of 25 ° C. or more at a rate of 10 g per liter, allowed to stand for 24 hours with proper stirring, and the filtered supernatant is used as a “first culture solution” in a mass culture method of holmium. The “second culture solution” and the “liquid fertilizer” accompanying the dry state of holmidium are used.

<Collecting holmium from soil>
Soil (the soil where holmidium appears on the surface of the soil after rain and greening is observed) is enough to mix “Galaxy Stars” so that the soil / “Galaxy Stars” is about 1/100 (weight ratio). If pure water is given and left for 2 weeks in natural light avoiding direct sunlight, holmium will grow as a green film on the soil surface. FIG. 2 shows holmidium grown on the soil surface.

<Primary culture>
Since the film-form holmidium thus obtained is easily peeled off, it is peeled off from the soil surface and transferred to the first culture solution.
It is preferable to use a culture solution of about 20 to 30 mL (milliliter) with respect to a holmium medium mass of about 3 mm ³ .
As the culture container, a flat container having a shallow liquid depth, specifically, a petri dish or a vat with a transparent lid is preferable.
The culture condition is preferably natural light avoiding direct sunlight, the culture temperature is 20 to 30 ° C., preferably about 25 ° C. (room temperature), and the culture period is about 2 weeks. The holmidium mass spreads and grows in one layer on the bottom of the culture vessel.

<Secondary culture>
Only the thin layered and massive holmidium grown in the primary culture is peeled off to form a small lump and transplanted to the second culture solution. FIG. 3 shows holmium deposited in a petri dish (about 30 pieces) containing the second culture solution.
The amount of the culture solution for transplanted holmium, preferred culture vessels, and preferred culture conditions are the same as in the primary culture.
FIG. 4 shows holmium obtained as monoalgal culture 14 days after planting in the second culture solution. From about 0.1 g (wet weight) of holmidium nodules after primary culture, 5 to 10 g (wet weight) of pure holmium was harvested after 14 days.

  For large-scale cultivation of holmidium, thin petri dishes are placed in units of 100 on a flat surface, cultured at 22 ° C in natural light abundant in direct sunlight, freshly cultured every two weeks, and mass harvested by regular rotation. Get.

<Creation of dry form of holmidium>
After the obtained holmidium was dried at room temperature and dried for 2 days, 5 days, 15 days, and 20 days, the culture medium was returned to the same culture medium as the first and second culture mediums, One week later, it was revived. However, those dried for 20 days have been delayed in recovery, so it is preferable to use a dried product of up to about 2 weeks. The dried product was stored at room temperature away from direct sunlight.

<Fertilization of liquid fertilizer containing holmium>
Immediately before spraying, 5 g of dry holmium powder was mixed in 10 L of the above liquid fertilizer, stirred well, placed in a sprayer, and sprayed on the target soil. Sufficient water was supplied to the soil after spraying.

Examination example “Ginga no Hoshi” (manufactured by Midori Symbiotic Co., Ltd.), a soil conditioner composed mainly of igneous rock powder and clay collected in Maniwa City, Okayama Prefecture and mixed with fully-ripened compost, etc., was used.
The extraction temperature and time were investigated as the best conditions for obtaining an extract from this “Galaxy Star” (powder). Extraction was performed under the conditions shown in Table 1 at a rate of 10 g of “galactic star” per liter of water. Each extract was poured into sterilized dune sand, seeded with komatsuna, and the growth of seedlings was compared. The results are shown in Table 1. In Table 1, + indicates the degree of growth, and the more +, the better the growth.

  As shown in Table 1, it can be seen that the extraction has a strong influence not on the extraction temperature but on the immersion time, that is, the extraction time. I found out. Practically, the immersion treatment may be performed at room temperature for about 6 to 20 hours.

Example 1
100 g of “Galaxy Star” (powder) was soaked in 10 L of pure water, kept at 25 ° C. for 20 hours, and then filtered to obtain a supernatant (hereinafter referred to as “Gex”).
In 20 mL of this Gex, the dry state of holmium cultivated by the mass culture method of the present invention was mixed in the amount shown in Table 2 and immediately sprayed on 60 mL of sterilized dune sand. The case where holmium was not added was used as a comparative example.
Each of the komatsuna seeds was sown 10 times after soaking in water for 24 hours, kept for 2 weeks at room temperature under natural light while replenishing only water, and the growth of seedlings of komatsuna was compared for the presence or absence of holmium after 2 weeks. The results are shown in Table 2.

The growth effect of Komatsuna seedlings was based on the comparative example.

  In both cases, Komatsuna seedlings grew, but as shown in Table 2 and FIG. 5, there was a clear difference in growth with and without holmidium.

  The capillary roots of Komatsuna seedlings were observed with a fluorescence microscope by DAPI staining. This result shows that holmium, which looks like a red thread due to the autofluorescence of chlorophyll, is arranged on the surface of the capillary root by a mucus substance secreted by itself (a substance for lectin that is an agglutinin) to form a mat. When Gex was added, it was observed that holmium was growing more than when the extract Gex was not added.

The liquid fertilizer containing holmium of the present invention is a state in which both can be sprayed in combination to enhance the fertilizer effect of the soil improver and the soil improvement effect of cyanobacteria holmidium more rationally.
For example, it can be suitably used for purification and restoration of fields that have been devastated by typhoon damage, restoration of soil hardened by administration of chemical fertilizer for many years, and the like.
Moreover, by applying the application method, apart from being mixed in the soil before cultivation, it can be applied to the soil where the crops already exist as additional fertilizer to contribute to improvement and reinforcement of growth.
Furthermore, in recent years, devastation of forests has become a major issue, and many fallen trees due to weakened forest soils are also seen. Therefore, it is possible to use liquid fertilizers containing holmium of the present invention over a wide area to improve forest soils. it can.

  Cyanobacteria are not only the evolutionary creatures of this planet, but also the creatures that brought “soil” to this planet made of rocks. Even on today's earth, it is cyanobacteria that first appears in degraded soil where living things do not grow and makes soil where plants can grow. It is human desire to expand the fertile soil by increasing cyanobacteria, maintain a rich environment and a rich green earth, and fertilize the liquid fertilizer containing holmium of the present invention. Method development is part of that.

It is a microscope picture of holmidium which forms an adherent filamentous group. It is the photograph of the holmium which propagated in the film form on the soil surface layer. It is the photograph at the time of the start of the secondary culture after peeling off the thin layer of the holmidium which spread and spread on the bottom of the petri dish in the primary culture and planted it in the second culture solution. It is a photograph which shows the breeding situation of holmidium two weeks after the start of secondary culture. It is the photograph which investigated that the growth of seedlings of Komatsuna differed depending on the presence or absence of dry holmium.

Claims (5)

The first culture is an extract of a soil conditioner that is composed of igneous rock powder and clay as main components, matured to the extent that it can be applied directly as fertilizer, sedimentary soil containing colloidal micelles of humic acid, and raised coral powder. Liquid,
After transplanting holmium collected from the soil surface layer to the first culture solution, primary culture by stationary culture is performed,
The holmidium grown in the primary culture is isolated from soil and other microorganisms, transplanted to a second culture solution composed of the same components as the first culture solution, and then subjected to secondary culture by static culture. A method for mass culture of holmium simple algae.   1-50 parts by weight of fully matured compost matured to the extent that the soil conditioner can be applied as fertilizer to 100 parts by weight of igneous rock powder and clay, and sedimentary soil and raised coral powder containing humic acid colloid micelles. The method for mass-cultivating holmidium monoalgae according to claim 1, wherein 1 to 50 parts by weight of the mixture is mixed. The first and second culture liquids are the supernatants that have been allowed to stand after mixing and stirring the soil improver with water, minerals eluted from igneous rock powder, clay and uplifted coral powder, and fully matured compost and sedimentary soil. Including extracts from soil microorganisms and their metabolites contained therein, at least,
Calcium (Ca) 10-100mg / L
Nitrogen (N) 10-100 mg / L
Sodium (Na) 1-50mg / L
Potassium (K) 1-50mg / L
Magnesium (Mg) 1-50mg / L
The method for mass culture of holmium simple algae according to claim 1 or 2, wherein   The liquid fertilizer which makes the said extract liquid A agent and the dry state of the holmium obtained by the method as described in any one of Claims 1-3 as B agent. 5. The method of fertilizing a liquid fertilizer according to claim 4, wherein 0.2 to 0.5 g of dried holmium is added to 1 L of the extract and immediately sprayed.