HK1154397B - Method for production of seed material for microorganisms optimized as catalyst for parallel complex mineralization reaction - Google Patents

Description

Method for producing inoculum of microbial population optimized as catalyst for parallel multiple mineralization

[ technical field ] A method for producing a semiconductor device

The present invention relates to a method for producing an inoculum of a microorganism group optimized as a catalyst for a parallel multiple mineralization.

The present invention also relates to a method for producing a fertilizer containing nitrate nitrogen as an inorganic fertilizer component using the above inoculum.

[ background of the invention ]

In recent years, from the viewpoint of building a circulating society, the activity of controlling the use of chemical fertilizers and promoting the use of organic fertilizers has been actively carried out worldwide.

The production species of vegetables such as tomatoes, flowers, and the like are widespread, and improvement is expected for the purpose of utilizing organic fertilizers also in 'hydroponics' that does not use soil.

However, organic fertilizers have not been used in hydroponics. This is because, when organic substances are directly added to the nutrient solution, harmful intermediate decomposition products and plant root damage occur. Therefore, conventionally, only chemical fertilizers have been used in hydroponics.

Many researchers believe that in utilizing organic fertilizers in hydroponics, it is necessary to mineralize organic substances in advance to become a substance that is easily absorbed by plants.

As a technique for making organic substances inorganic, there is a wastewater treatment technique using a microbial group (see, for example, patent document 1).

However, these methods are not suitable for recovering nitrate nitrogen due to denitrification (a reaction in which nitrate nitrogen is reduced and released as nitrogen gas), and do not meet the purpose of being used in fertilizers containing nitrate nitrogen as an inorganic fertilizer component.

Accordingly, as a technique capable of recovering nitrate nitrogen (as nitrate ions) from an organic material and utilizing it as an inorganic fertilizer component, the "parallel multiple mineralization method" described in patent document 2 and non-patent document 2 has been invented.

This technique is a highly reproducible method of decomposing organic nitrogen while suppressing denitrification reaction and recovering nitrate ions of nitrate nitrogen as an inorganic fertilizer component. The technique is a technique in which microorganisms that decompose organic substances (ammonization) and generate nitrate ions (nitrification) are sequentially proliferated in the same reaction system, and ammonization and nitrification are performed in parallel in the same reaction liquid. This reaction can suppress the denitrification unlike the above-mentioned wastewater treatment techniques.

By using this parallel multiple mineralization technique, it becomes possible to directly add an organic fertilizer to the nutrient solution culture and use it, and by using this technique, it becomes possible to mineralize the organic matter into nitrate nitrogen to prepare an inorganic fertilizer component (see, for example, non-patent document 1 and non-patent document 2).

The invention described in patent document 2 is attracting attention as a technique for realizing nutrient solution cultivation using an organic fertilizer and for realizing the production of an inorganic fertilizer component such as nitrate nitrogen using an organic resource as a raw material. Therefore, the method has been concerned by farmers, plant factories, and enterprises planning recycling of organic resources, and has been expected to be a new nutrient solution cultivation technique.

However, in the method described in patent document 2, only "natural-derived microbial sources" such as soil and lake water are used as microbial sources.

These microbial sources are not considered to be optimized for the parallel multiple mineralization, and the following problems to be improved in terms of use remain in the conventional parallel multiple mineralization method.

That is, the first problem 1 is that it takes a long time to complete the reaction. This is because "a natural microorganism source (soil, lake water, etc.)" is not optimized for the parallel multiple mineralization reaction, and therefore, it is necessary to wait for microorganisms which have undergone the respective reactions to successively proliferate every time the amination reaction is carried out and then the reaction proceeds to the nitrification reaction.

Specifically, since the time required for completion of the reaction is about 2 weeks or more, problems such as unsatisfactory planting pot life of the seedlings cultivated with the nutrient solution occur.

The following problem 2 is that a large amount of organic substances cannot be added at once. As described above, the "naturally-derived microorganism source" is not optimized for the parallel multiple mineralization, and nitrifying bacteria (microorganisms that perform nitrification) contained therein are in a state of being weaker than the exposure to the organic component, and if a large amount of exposure to the organic component is received, they die. Therefore, when a large amount of organic substances are added at a time in the ` culturing process `, the nitrification reaction cannot be carried out, and nitrate nitrogen cannot be recovered.

Specifically, about 2g of organic matter per 1 addition was an upper limit for 1L of water (solution) in the reaction system. Therefore, when recovering nitrate nitrogen at a high concentration, the operation of adding the organic substance has to be divided into several times (preferably daily) and the operation becomes complicated.

Then, the 3 rd problem is that the amount of the microorganism source to be added is increased.

Since the natural microbial source is not optimized for the parallel multiple mineralization (microbial ecosystem) and the exposure to the nitrifying bacteria organic component is extremely weak, the large damage due to the exposure to the nitrifying bacteria organic component cannot be avoided, and the amount of the additive to be added in consideration of the loss due to the damage has to be large.

Specifically, about 5g or more of soil needs to be added to 1L of water (solution) in the reaction system. If the amount of the nitrate is less than the above amount, the nitrifying bacteria may die due to exposure to organic components, and nitrate nitrogen may not be recovered.

In addition, having to add large amounts of microbial sources is problematic at the site of nutriculture. In the field of nutrient solution cultivation, since a ton of nutrient solution is used, it is expected that thousands of soils equivalent to thousands of tons of nutrient solution have to be put into the nutrient solution. However, if such an amount of soil is added, problems such as turbidity of the soil particle flow path are likely to occur.

Further, if the amount of the soil to be charged is increased, the soil is likely to be anaerobic, denitrifying bacteria (microorganisms that perform denitrification) in an environment in which anaerobic conditions are preferred are propagated, and nitrate nitrogen gas is lost. In addition, anaerobic microorganisms are taken up by plants to secrete undesirable components (phytoxics) and deteriorate the growth of the plants.

Therefore, it is required to develop a technique for greatly reducing the amount of the microorganism source to be added.

Accordingly, there has been a demand for development of a method which can be effectively carried out at a level suitable for practical use, in order to carry out a multiple parallel mineralization to generate nitrate nitrogen as an inorganic fertilizer component from an organic material, thereby solving the above 3 problems.

Patent document 1: japanese laid-open patent publication No. 2001-300583

Patent document 2: japanese laid-open patent publication No. 2007-119260

Non-patent document 1: study ジヤ - ナル 2008, pages 31(1)44-46

Non-patent document 2: "the cultivation under the solution of the additional fertilizer module " and the harness yun, 81 th coil p.753-764 (2006)

[ summary of the invention ]

[ technical problem to be solved by the invention ]

In order to solve the above problems, the present invention provides the following method: in a parallel multiple mineralization of an organic material to produce nitrate nitrogen as an inorganic fertilizer component, the time until completion of the reaction for mineralizing the organic material into nitrate nitrogen can be significantly shortened, and a large amount of the organic material can be added at a time.

[ technical means for solving problems ]

The present inventors have found that, in a parallel multiple mineralization in which nitrate nitrogen is produced from an organic material as an inorganic fertilizer component, by using 'an inoculum of a microorganism group optimized as a catalyst for the parallel multiple mineralization' as a microorganism source, the time taken until the reaction for mineralizing the organic material into nitrate nitrogen is completed can be significantly shortened, and a large amount of the organic material can be added at a time.

Further, as a microorganism source including a microorganism group which performs an ammoniation action and a microorganism group which performs a nitrification action, an inoculum for feeding tropical fish (for filtering feed water) or activated sludge in a wastewater treatment plant is known.

However, since the users of tropical fish are forced to remove nitrogen for the purpose of preparing the growth environment of the fish, they often include microorganisms (denitrifying bacteria) for denitrification. In this case, no method has been provided for producing nitrate nitrogen at a high concentration because of the loss of nitrate nitrogen.

In addition, since the activated sludge in the wastewater treatment plant is also an important object to promote denitrification, it contains many microorganisms (denitrifying bacteria) that perform denitrification.

Therefore, these are not considered to be 'microbial populations optimized as catalysts for parallel multiple mineralization'.

The present invention has been completed based on these findings.

That is, the invention according to embodiment 1 is a method for producing an inoculum, characterized by placing water in a container capable of storing water, inoculating a microorganism group which performs a multiple parallel mineralization, and culturing the microorganism group which performs the multiple parallel mineralization by maintaining an environment in which the multiple parallel mineralization proceeds in the water; and forming a biofilm on the solid surface in contact with the water, and then recovering the biofilm; and using the collected biofilm as an inoculum of a microorganism group optimized as a catalyst for a parallel multiple mineralization.

An invention according to embodiment 2 is the method for producing an inoculum according to embodiment 1, wherein the inoculation of the microorganism group which performs the multiple parallel mineralization is performed by inoculating 1 or more species selected from the group consisting of: soil, composted bark, and naturally occurring water.

An invention according to embodiment 3 is the method for producing an inoculum according to embodiment 2, wherein the multiple parallel-type inorganic reaction is carried out in the water in an environment in which 0.05 to 1g of organic substances are added to 1L of the water every 1 to 7 days in terms of dry weight.

The invention according to embodiment 4 is a method for producing an inoculum, comprising placing water in a container capable of storing water, inoculating an inoculum obtained by the method for producing an inoculum according to any one of embodiments 1 to 3, and culturing a microorganism group that performs a parallel multiple mineralization in an environment in which the parallel multiple mineralization is performed in the water; and forming a biofilm on the solid surface in contact with the water, and then recovering the biofilm; and using the collected biofilm as an inoculum of a microorganism group optimized as a catalyst for a parallel multiple mineralization.

An invention according to embodiment 5 is the method for producing an inoculum according to embodiment 4, wherein the environment in which the multiple parallel-type inorganic reaction is carried out in the water is an environment in which 0.01 to 10g of an organic substance in terms of dry weight is added to 1L of the water.

An invention according to embodiment 6 is the method for producing an inoculum according to any one of embodiments 1 to 5, wherein an environment in which the parallel multiple mineralization is performed in the water is an environment in which the water is maintained under aerobic conditions.

The invention according to embodiment 7 is the process for producing an inoculum according to embodiment 6, wherein the aerobic conditions are maintained by aeration or shaking.

An invention according to embodiment 8 is the method for producing an inoculum according to any one of embodiments 1 to 7, wherein the environment in which the multiple parallel mineralization is performed in the water is an environment in which the water is maintained at a water temperature of 15 to 37 ℃.

An invention according to embodiment 9 is the method for producing an inoculum according to any one of embodiments 1 to 8, wherein a solid surface in contact with the water is a wall surface and/or a bottom surface of the container.

An invention according to embodiment 10 is the method for producing an inoculum according to any one of embodiments 3, 5 to 9, wherein when the microorganisms capable of conducting the multiple parallel mineralization are cultured in the water, and when the concentration of nitrate ions generated in the water reaches 10 to 30mg/L, the microorganisms capable of conducting the multiple parallel mineralization are cultured while suppressing proliferation of the microorganisms capable of conducting the denitrification in the formed biofilm by stopping addition of the organic matter.

An invention according to embodiment 11 is the method for producing an inoculum according to any one of embodiments 1 to 10, wherein the collection of the biofilm is performed by discarding a supernatant of a culture solution obtained by culturing the microorganisms having undergone the multiple parallel mineralization, and then collecting the biofilm formed on the surface of the solid.

An invention according to embodiment 12 is the method for producing an inoculum according to any one of embodiments 1 to 11, wherein the recovery of the biofilm is performed as a mixed solution in which: the biofilm formed on the solid surface is a supernatant of a culture solution obtained by culturing the microorganism group which has undergone multiple parallel mineralization.

An invention according to embodiment 13 is the method for producing an inoculum according to any one of embodiments 11 or 12, wherein after the formed biofilm is collected, additional water is removed by centrifugation or filtration.

An invention according to embodiment 14 is the method for producing an inoculum according to embodiment 11, wherein a supernatant of the culture solution is discarded and then dried.

An invention according to embodiment 15 is the method for producing an inoculum according to embodiment 13, wherein the excess water is removed and then a drying treatment is performed.

An invention according to embodiment 16 is the method according to any one of embodiments 1 to 15, wherein the inoculum is an inoculum that does not lose a function as an inoculum optimized as a catalyst for a parallel multiple mineralization when heated at 50 to 80 ℃.

An invention according to embodiment 17 is the method according to any one of embodiments 1 to 16, wherein the inoculum includes a microorganism group that performs an ammoniation action, and the microorganism group that performs a nitrification action includes 1 ten thousand to 1 hundred million cells per 1g of the inoculum.

The invention according to embodiment 18 is an inoculum of a microorganism group optimized as a catalyst for a parallel multiple mineralization, and is obtained by the method for producing an inoculum according to any one of embodiments 1 to 17.

The invention according to embodiment 19 is a method for producing a fertilizer containing nitrate nitrogen as an inorganic fertilizer component, characterized by placing water in a container capable of storing water, and adding the above-mentioned inoculum described in embodiment 18 thereto; and allowing the multiple parallel mineralization to proceed in the water by maintaining an environment in which the multiple parallel mineralization proceeds in the water, the environment being: adding organic matter, maintaining the temperature at 15-37 ℃ and maintaining the condition of becoming aerobic; obtaining a reaction solution containing nitrate ions of more than 100 mg/L; the obtained reaction solution is used as a fertilizer, and the fertilizer comprises nitrate nitrogen as an inorganic fertilizer component.

An invention according to embodiment 20 is the method for producing a fertilizer according to embodiment 19, wherein the organic substance is added in an amount of not more than 10g in terms of dry weight to 1L of the water at a time.

An invention according to embodiment 21 is the method for producing a fertilizer according to any one of embodiments 19 and 20, wherein the reaction solution that produces nitrate ions at a concentration of 100mg/L or more can be obtained in a number of days not exceeding 8 days.

An invention according to embodiment 22 is the method for producing a fertilizer according to any one of embodiments 19 to 21, wherein the inoculum is added in an amount of not less than 0.01g per 1L of the water.

An invention according to embodiment 23 is the method for producing a fertilizer according to any one of embodiments 19 to 22, wherein the parallel multiple mineralization reaction is performed without a denitrification reaction.

The invention according to embodiment 24 is a fertilizer including nitrate nitrogen as an inorganic fertilizer component, and is obtained by the method for producing a fertilizer according to any one of embodiments 19 to 23.

The invention according to embodiment 25 is a method for cultivating a plant using the fertilizer according to embodiment 24, which contains nitrate nitrogen as an inorganic fertilizer component.

The invention according to embodiment 26 is a method of cultivating a plant, in which a fertilizer including an organic material is directly added to the reaction solution according to any one of embodiments 20 to 25, and the plant is cultivated in a nutrient solution.

An invention according to embodiment 27 is the method for cultivating a plant according to embodiment 25 or 26, wherein the plant is: leaf vegetables, fruit vegetables whose fruits are harvested, fruit trees, or flowers.

[ Effect of the invention ]

The present invention can provide an inoculum of a microorganism group optimized as a catalyst for a parallel multiple mineralization.

Thus, in the parallel multiple mineralization of nitrate nitrogen as an inorganic fertilizer component from an organic material, the present invention can significantly shorten the time until the completion of the reaction for mineralizing the organic material into nitrate nitrogen, and can add a large amount of organic material at a time.

In addition, the present invention can decompose organic matter resource containing much nitrogen and food waste fast to convert into inorganic fertilizer containing nitrate nitrogen.

[ embodiment ] A method for producing a semiconductor device

The present invention relates to a method for producing an inoculum of a microorganism group optimized as a catalyst for a parallel multiple mineralization in a parallel multiple mineralization for generating nitrate nitrogen as an inorganic fertilizer component from an organic material.

The present invention also relates to a method for producing a fertilizer containing nitrate nitrogen as an inorganic fertilizer component using the above inoculum.

FIGS. 1(a) to (c) are explanatory views showing an embodiment of the method for producing an inoculum of microorganisms optimized as a catalyst for a parallel multiple mineralization according to the present invention.

Specifically, FIG. 1(a) is an explanatory view of an embodiment using a natural source (soil, etc.)' as an inoculation source of the microorganism group. FIG. 1(b) is an explanatory view of an embodiment in which the ` inoculum of the present invention ` is used as an inoculum of a microorganism group. Fig. 1(c) is an explanatory view of an embodiment of using a 'biofilm remaining in a container (solid surface)' as an inoculation source of a microorganism group.

In the present invention, an inoculum of a microorganism group optimized as a catalyst for a parallel multiple mineralization is prepared as follows: placing water in a container capable of storing water, inoculating a microorganism group which performs a multiple parallel mineralization, and culturing the microorganism group which performs the multiple parallel mineralization by maintaining an environment in which the multiple parallel mineralization proceeds in the water; and forming a biofilm (microbial community) on the solid surface contacted with the water, and then, recovering the biofilm.

The first step in the preparation of the inoculum of the microorganism group optimized as the catalyst of the parallel multiple mineralization of the present invention is, first, a step of placing water in a container capable of storing water, inoculating a microorganism group for performing the parallel multiple mineralization for mineralizing an organic material to generate nitrate nitrogen, and culturing the microorganism group for performing the parallel multiple mineralization by maintaining an environment in which the parallel multiple mineralization proceeds in the water (culture step).

In the present culture step, the environment in which the multiple parallel mineralization is performed may be maintained after the inoculation of the microorganism group, but the environment may be maintained after the inoculation of the microorganism group into the water in a state in which the environment in which the multiple parallel mineralization is performed is maintained.

The "container capable of storing water" used in this step may be any container as long as it can store water.

Examples thereof include a container capable of storing a relatively large amount of water such as a water tank, a barrel, a tank, a water storage tank, a bath, a pool, and the like, a container capable of storing a relatively small amount of water such as a flask, a beaker, a test tube, and the like.

Specifically, cans, boxes, flasks may be used. In addition, in large-scale production or industrial application, a water storage tank or a pool can be used.

Further, it is preferable that the area of the solid surface of the container in contact with water is large for the volume, and the stagnant water portion is small for the difficulty of generating the anaerobic state.

In addition, the area of biofilm formation by the microorganisms can be increased by placing a solid carrier (by immersing the container in water) to which the microorganisms can easily adhere.

Specifically, a solid carrier such as bamboo charcoal, perlite, sea sand, vermiculite, ceramics, zeolite, glass, rock wool, urethane, nylon, melamine resin, or the like can be used.

In addition, in the above-mentioned container, by immersing an immersing material such as a plate (plate-like material) or a columnar structure (by immersing when water is placed therein), the area of biofilm formation by the microorganism group can be increased. The impregnated material is preferably one which is easily detached and easily pulled out from water. Specifically, the impregnated material may be a material which does not spoil or corrode in water, such as glass, acrylic resin, plastic, ceramic sheet, or ceramic.

As the water used in this step, tap water, distilled pure water, well water, river water, lake water, sea water, etc. may be used.

The amount of water is not particularly limited as long as it is 50 times or more the dry weight of the organic material to be added, and in order to obtain a sufficient amount of inoculum, specifically, 0.001 to 10000L, preferably 0.01 to 1000L, of water is left.

In the present invention, the "parallel multiple mineralization" refers to a reaction in which an organic substance is mineralized to generate nitrate nitrogen, and the decomposition (ammoniation) of the organic substance into ammonia nitrogen and the nitrification (nitrification) of the ammonia nitrogen into nitrate nitrogen are continuously performed in the same reaction system.

Specifically, the term "decomposition reaction" refers to a reaction in which, in the decomposition of an organic substance, organic nitrogen contained in the organic substance is decomposed into ammonia nitrogen, and the ammonia nitrogen is nitrified (oxidized) by nitrification to form nitrate nitrogen.

In the present invention, nitrate nitrogen generated by mineralizing an organic substance means a nitrate ion or a nitrate, and specifically, a nitrate ion is assumed.

The "microorganism group which performs multiple parallel mineralization" inoculated in the present culture step may be any microorganism group which can perform multiple parallel mineralization when cultured in a predetermined environment, including a microorganism group which performs ammoniation and a microorganism group which performs nitrification.

Further, as the types of microorganisms constituting the microorganisms, there can be mentioned: microorganisms capable of ammonifying such as protozoa, bacteria, and ammonifying bacteria such as funiculus; examples of the microorganisms (nitrifying bacteria) that perform nitrification include nitrifying bacteria such as nitrifying bacteria, nitrifying bacteria belonging to the genus nitrosocholinesterase of ammonia-oxidizing bacteria (or nitrite-producing bacteria), nitrifying bacteria belonging to the genus nitrosococcus, nitrifying bacteria belonging to the genus nitrosospirillum (including the genus nitrosophyllum and the genus nitrosovibrio), and nitrifying bacteria belonging to the genus nitrifying spirillum.

The inoculation source of the microorganism group which performs the multiple parallel mineralization in this step may be specifically a "natural source" such as compost such as soil and bark compost, activated sludge, and naturally occurring water (specifically, lake and marsh water, spring water, well water, river water, sea water, etc.).

However, these naturally derived sources of seeding are not necessarily optimized as catalysts for parallel multiple mineralization reactions. Therefore, in the case of culturing the microorganism group which performs a multiple parallel inorganic reaction using these as an inoculum, specifically, in the whole step up to the completion of the step of preparing the inoculum of the present invention, the amount of the organic substance added is at most 10 days in the case of 1g or less at a reaction temperature of 25 ℃ with respect to 1L of water, and usually 15 to 20 days are necessary.

Therefore, as the inoculum of the microorganism group in this step, it is preferable to appropriately inoculate the "microorganism group optimized as a catalyst for the parallel multiple mineralization" obtained in the present invention.

The "inoculum" is, as described in detail below, one obtained by recovering a biofilm (microbial colony structure) formed on the solid surface after the culture step, or one obtained by recovering the biofilm so as to contain the biofilm.

The culture step can be terminated quickly by adding the inoculum as an inoculation source for the microorganism group in this step. Specifically, the organic matter is added in the amount of not more than 8 days at a reaction temperature of 25 ℃ or less of 1g per 1L of water, and the total step of preparing the inoculum of the present invention can be completed in 4 to 8 days in general.

That is, the time required for the whole steps of the preparation of the inoculum of the present invention can be reduced to about half, and the preparation efficiency can be greatly improved by increasing the number of operations.

After the biofilm is collected, the biofilm remaining in the container (solid surface) can be used as an inoculation source in the present culture step as a substance having the same function as the "inoculum".

In the actual production, in a stage where the "inoculum" of the present invention is not obtained, the "naturally-derived material" (soil, bark compost, naturally-occurring water, etc.) is used as an inoculum of the microorganism group in the present step, and after the "inoculum" of the present invention is obtained, the "inoculum" is preferably used from the viewpoint of efficiency as described above.

In the inoculation of the microorganism group in the present culture step, the amount of the inoculum added is not particularly limited, but when "natural sources" (soil, bark compost, natural water, etc.) are added, it is necessary to add a large amount of water to the container.

Specifically, in the case of soil or bark compost, 1 to 10g of water is added to 1L of water placed in the container, and in the case of naturally occurring water, the water may be added in the container in a manner of replacing 100 to 1000ml of water (in a manner of 10 to 100% of the total amount of naturally occurring water) with 1L of water placed in the container.

On the other hand, when the "inoculum" of the present invention is added as the inoculation source, the amount of the inoculum can be greatly reduced, and 0.005 to 1g in terms of dry weight may be added to 1L of water placed in the container.

Specifically, 0.005 to 1g of water is added to 1L of water placed in the container, and 0.05 to 10g of water is added to dry and wet bacteria. In addition, in the case of adding the mixed solution of the biofilm and the supernatant of the culture solution, 1 to 500ml of water (1 to 50% of the mixed solution based on the total amount) may be replaced with 1L of water placed in the container.

When the biofilm remaining in the container (solid surface) after the recovery of the biofilm is used as the inoculation source, water for inoculating the microorganism group can be prepared by adding 1L of water to the container in an amount of 0.005 to 1g in terms of dry weight of the biofilm.

In the present culture step, the "environment in which the multiple parallel mineralization is carried out in the water" means, specifically, an environment in which the water is maintained under aerobic conditions, the water is supplemented with an organic substance, and the water is maintained at a temperature of 15 to 37 ℃.

The microbial population that performs the parallel multiple mineralization can be cultured by maintaining such an environment.

In the present culture step, the conditions that are "maintained in an aerobic condition" in the water are set to conditions suitable for the activity of the microorganism group that performs the multiple parallel mineralization by increasing the dissolved oxygen concentration in the water.

In addition, since the microorganisms (denitrifying bacteria) that perform denitrification reaction are easily activated under anaerobic conditions, they are also suitable for suppressing the proliferation of microorganisms that perform denitrification reaction.

As a method for maintaining the water in an aerobic condition, aeration, shaking, dissolution of high-concentration oxygen, utilization of high-concentration oxygen-containing water, and the like can be carried out. Preferably, the aeration and the shaking are performed.

In the present culture step, the "water temperature" suitable for the multiple parallel mineralization is a water temperature suitable for the growth of the microorganisms capable of performing the multiple parallel mineralization. Specifically, the temperature is preferably 15 to 42 ℃, more preferably 15 to 37 ℃, even more preferably 20 to 37 ℃, and most preferably maintained at about 25 ℃.

Further, when the temperature is lower than 15 ℃, the growth of the microorganism is delayed and the time required for the culture is not preferable. When the temperature is higher than 42 ℃, part of the microorganisms necessary for the multiple parallel mineralization is killed, which is not preferable.

In this culture step, as the "organic material" to be added to the water, for example, an organic fertilizer, a food residue, a plant residue, a livestock waste, an organic resource of excrement, or the like can be used, but it is preferable to use a high nitrogen-containing organic material having a carbon/nitrogen content ratio of 11 or less, preferably 10 or less, in terms of improving the recovery efficiency of nitrate nitrogen.

The organic material preferably includes proteins, protein decomposition products, amino acids, and the like, and specifically includes food residues such as fish sauce, corn steep liquor, oil meal, fish meal, cow milk, soybean meal, yeast meal, distiller's grains, distilled grains, and raw garbage. These are waste products obtained in the process of producing food products, and are preferably free of toxic components.

In addition, fish gravy, corn steep liquor and oil meal are preferably used. Specifically, the fish cooking juice may be bonito cooking juice. Further, examples of the corn steep liquor include corn steep liquor (CSL: corn steep liquor which is a by-product in the production of corn starch). Further, rapeseed oil meal is exemplified as the oil meal.

Further, as the organic substance, an organic substance which may be in a liquid state or a powder state is used, and particularly, bonito boiled juice and corn steep liquor are preferable because they are easily uniformly diffused in the water which is a liquid.

In this culture step, the method of "adding an organic substance" to the water varies depending on the kind of the inoculum of the microorganism group.

That is, when "a naturally-derived microorganism source" (soil, bark compost, natural water, etc.) is added as the inoculation source, in order to prevent nitrifying bacteria contained therein from being exposed to organic substances and dying, the organic substances are added to 1L of water at a rate of 1 day and 2g or less in a little (slowly added).

Specifically, each of 0.01 to 2g (in terms of dry weight), preferably 0.05 to 1g (in terms of dry weight) of the water is preferably added to 1L of the water every 1 to 14 days, preferably every 1 to 7 days, and more preferably every day. For example, 0.01-2 g of rapeseed oil meal can be added.

When the organic substance is in a liquid state, the value in terms of dry weight may be in the above range. For example, when using bonito cooking juice, 0.01 to 2g (0.007 to 1.4g in terms of dry weight) of liquid can be added, and when using corn steep liquor, 0.01 to 2g (0.005 to 1g in terms of dry weight) of liquid can be added.

In addition, when the "inoculum" of the present invention is added as the above-mentioned inoculum, the nitrifying bacteria contained therein can be added to water 1L 'at a time' in an amount of not more than 10g of organic matter due to the increased resistance to exposure to organic components.

Specifically, the ` initial day of culture ` is preferably added to 1L of water in an amount of 0.01 to 10g (in terms of dry weight), more preferably 0.05 to 5g (in terms of dry weight). For example, 0.01-10 g of rapeseed oil meal can be added.

In the case where the organic substance is in a liquid state, the value in terms of dry weight may be in the above range. For example, when using bonito cooking juice, 0.01 to 10g (0.007 to 7g in terms of dry weight) of the liquid can be added, and when using corn steep liquor, 0.01 to 10g (0.005 to 5g in terms of dry weight) of the liquid can be added.

In this culture step, the "culture time" of the microorganisms which perform the multiple parallel mineralization is preferably set until half of the nitrogen contained in the added organic material is produced as nitrate ions, and the concentration of the nitrate ions in the culture solution rises to a peak.

Further, as a specific number of days elapsed in the case of the culture under the above-mentioned environment (the case of culturing until the concentration of nitrate ions in the culture solution rises to the top), the shortest time to 10 days, and usually 15 to 20 days, were observed when the amount of the organic matter added was 1g or less with respect to 1L of water and the reaction temperature was 25 ℃ and "a natural source of microorganisms" (soil, bark compost, natural water, etc.) was added as the above-mentioned inoculation source.

On the other hand, the amount of organic substances added is 1g or less for 1L of water, the reaction temperature is 25 ℃, and the number of days of addition of the "inoculum" of the present invention is not more than 8 days at the longest, and is usually 4 to 8 days.

In the present culture step, when the microorganisms capable of conducting the multiple parallel mineralization are cultured, the microorganisms capable of conducting the multiple parallel mineralization are cultured while suppressing proliferation of the microorganisms capable of conducting the denitrification in the formed biofilm.

The "denitrification reaction" refers to a phenomenon in which nitrate nitrogen is lost by reduction of nitrate nitrogen to nitrogen gas, nitrous oxide gas, or the like by a microorganism group (denitrifying bacteria) that performs a denitrification reaction, and this reaction is easily induced when two conditions, namely, a condition for existence of an organic component that is an energy source of the denitrifying bacteria and a condition for formation of nitrate nitrogen that is an oxygen donor of the denitrifying bacteria, are satisfied at the same time.

Therefore, in the present invention, it is preferable that the microorganisms capable of conducting multiple parallel mineralization be cultured while suppressing the proliferation of microorganisms capable of conducting denitrification (denitrifying microorganisms) by stopping the addition of the organic substance before or immediately after the formation of nitrate nitrogen in the water is started.

Specifically, it is preferable that the addition of the organic substance is stopped when the nitrate nitrogen generated in the water reaches 10 to 50mg/L, preferably 10 to 30mg/L (before or immediately after the nitrate nitrogen is generated) in terms of nitrate ions.

Further, since the microorganisms (denitrifying bacteria) which perform denitrification are easily activated under anaerobic conditions, it is preferable to maintain the water under aerobic conditions.

By performing the culturing step, a biofilm of the microorganisms capable of conducting the multiple parallel mineralization is formed on the solid surface in contact with the water, and then a step of collecting the biofilm (collecting step) is performed.

The "solid surface" in contact with the water means, specifically, a surface of the solid carrier immersed in water differently from the wall surface, bottom surface, and container of the container, and means a surface of the plate immersed in water differently from the container.

Furthermore, from the viewpoint of the operability of biofilm recovery, it is preferable that the solid surface formed by the biofilm of the microorganisms has a structure in which water flow is difficult to accumulate in the wall surface and/or the bottom surface of the container, and it is preferable that the solid carrier or the plate immersed in water be easily detached and easily removed from the water.

In this step, the term "recovery of a biofilm" means that a biofilm formed on the surface of the solid is recovered or that the biofilm is recovered so as to contain the biofilm.

Specifically, there are (1) a case where a supernatant of a culture solution obtained by culturing the microorganisms having undergone the multiple parallel mineralization is discarded and then the biofilm formed on the solid surface is recovered, and (2) a case where the biofilm formed on the solid surface is recovered as a mixed solution of the biofilm and a supernatant of the culture solution obtained by culturing the microorganisms having undergone the multiple parallel mineralization. FIG. 2(c) is a schematic diagram showing an embodiment of the method for collecting a biofilm according to the present invention.

(1) The method (4) is a method of collecting a biofilm formed on a solid surface after discarding a supernatant of a culture solution.

In this case, the culture supernatant is preferably discarded by draining from the drain port, pouring (a method of discarding the supernatant by tilting the vessel), suction-discarding, evaporation-drying, or the like, and the drainage from the drain port, pouring, suction-discarding, or the like is preferably performed in a simple structure and easy handling.

After the supernatant of the culture solution is discarded, specifically, the biofilm can be collected and recovered by scraping the surface of the vessel, the surface of the impregnated solid carrier, the surface of the impregnated matter, or the like. The biofilm thus recovered was recovered as a 'wet bacterial mass'. In addition, the carrier to which the biofilm is attached may be collected as the wet bacterial cell of the present invention so as to be retained.

(2) The method (4) is a method in which the biofilm formed on the solid surface is mixed with a supernatant of a culture solution obtained by culturing the microorganism group which has undergone the multiple parallel mineralization, and the mixture is recovered as a "mixed solution".

In this case, the mixing of the formed biofilm with the supernatant of the culture solution means that, specifically, the formed biofilm can be mixed by physically scraping with a brush or a wiper to be sufficiently mixed with the supernatant of the culture solution, or by scraping the biofilm against a water stream to be mixed, or by scraping the biofilm by vibrating the entire container.

In the mixed solution, the amount of the supernatant of the culture solution for the biofilm is preferably about 0.5 to 100ml per 1g of the biofilm.

The "biofilm" formed in the above-mentioned culture step often includes a microorganism group which performs a multiple parallel mineralization, and often includes a microorganism group which performs an ammoniation and a microorganism group which performs a nitrification (nitrifying bacteria). In particular, it is suitable for a microorganism group (nitrifying bacteria) which is nitrified as a solid surface stationary recovery.

The "supernatant of the culture solution" obtained by culturing the microorganisms that have undergone the multiple parallel mineralization includes microorganisms that have undergone ammoniation but hardly any microorganisms that have undergone nitrification (nitrifying bacteria).

Therefore, the ` supernatant of the culture broth ` is hardly active in the nitrification reaction, and is not suitable for the inoculum of the microorganism group which performs the multiple parallel mineralization.

That is, in the present invention, as in the method (1) or (2) described above, a biofilm formed on a solid surface or a biofilm-containing biofilm can be recovered as an inoculum of a microorganism group optimized as a catalyst for a parallel multiple mineralization.

Furthermore, considering the problems of storage and transportation of the inoculum, it is preferable to use the biofilm method which is adopted in the method (1) in which the volume and weight are easily reduced.

In addition, in the case where the purpose of storage or transport is not particularly required, the method (2) of mixing the biofilm with the supernatant of the culture solution is preferable in view of the easiest operability.

The biofilm recovered by the method (1) or (2) can be used as the 'inoculum of the microorganism group optimized as a catalyst for the parallel multiple mineralization' of the present invention, and further, the recovered biofilm can be recovered as a 'wet bacterial mass' from which additional water is removed by centrifugation or filtration. In this treatment, a combination of both centrifugation and filtration may be performed.

In this treatment, the centrifugation can be performed at a centrifugation speed (2000 to 20000 Xg) at which the microorganism is not stressed. The filtration can be performed by filtering the mixture of the wet biofilm or the supernatant of the biofilm and the culture medium using filter paper, cloth, or the like.

In the present treatment, it is preferable to collect the wet bacterial material having a moisture content of 90% or less.

In this recovery step, the seed of the microorganism group optimized as a catalyst for the multiple parallel mineralization can be recovered in the form of a "dried microorganism" by performing a drying treatment.

When the present collection step is carried out by the method of the above (1), the supernatant of the culture solution may be discarded and then dried. Specifically, after the supernatant of the culture solution is discarded, the formed biofilm may be dried in a state of adhering to the solid surface. Alternatively, the wet biofilms formed may be collected and then dried.

In addition, after the present collection step is carried out by the method of the above (1) or (2), and when additional water is removed by centrifugation or filtration, the wet bacterial substance is collected and then dried, and the wet bacterial substance can be used as a dry bacterial substance.

The drying treatment may be air drying, dry heat treatment, drying under reduced pressure, or the like. Specifically, the air drying can be performed at room temperature (15 to 37 ℃) for about 1 hour to one night (about 6 to 14 hours), preferably for about one night (about 6 to 14 hours).

In the drying treatment, it is preferable that the dried microbial cell has a moisture content of 20% or less.

The "inoculum of microorganism group optimized as a catalyst for parallel multiple mineralization" of the present invention can be prepared by going through the above-described steps.

The shape of the seed bacteria can be prepared as a wet bacteria, a dried bacteria (bacteria after drying treatment), a liquid (mixture of a biofilm and a supernatant of a culture solution), a solid carrier to which a biofilm is attached, or the like.

Preferably, the preparation is in the form of a 'dried bacterial product' from the viewpoint of storage and distribution (for the purpose of transportation and storage in another place), from the viewpoint of heat resistance of a dried bacterial strain, or from the viewpoint of improving workability by reducing the amount of inoculation.

Further, in order to carry out the multiple parallel mineralization again in the same place, it is preferable to prepare the inoculum in a form of a 'wet bacterial material' or a 'liquid state' because of the simplicity of the procedure for preparing the inoculum.

The inoculum of the present invention is represented by the following microbial composition: the microorganism group (nitrifying bacteria) for nitrification is included, and 1g of the obtained seed bacteria include 1 ten thousand to 1 hundred million cells.

When the inoculum of the present invention is not subjected to the multiple parallel mineralization and is not a synergy between the microorganisms capable of nitrification and the microorganisms capable of nitrification, the microorganisms capable of nitrification are easily killed by the exposure of a large amount of organic components, and the nitrification is lost, so that the multiple parallel mineralization cannot be performed, which is not preferable.

That is, in the above-mentioned inoculum of the present invention, the nitrification activity of the nitrifying microorganisms is maintained even in the presence of an organic component by the synergy or interaction between the ammoniating microorganisms and the nitrifying microorganisms.

The inoculum of the present invention does not lose the function of an inoculum of a microorganism group optimized as a catalyst for a parallel multiple mineralization even when heated at 50 to 80 ℃, preferably 50 to 60 ℃, and more preferably 50 ℃. The time for the heat resistance is about 0.1 to 12 hours, preferably about 30 minutes.

This heat resistance is effective in preventing the inactivation of the seed bacteria at high temperatures in the vehicle or warehouse during transportation and storage.

The inoculum of the present invention can be used as a 'microorganism source' of a "microorganism group optimized as a catalyst for a multiple parallel mineralization" when nitrate nitrogen as an inorganic fertilizer component is produced from an organic material using a microorganism group that performs a multiple parallel mineralization.

In the present invention, by using the ' microorganisms which perform multiple parallel mineralization ' to generate nitrate nitrogen as an inorganic fertilizer component from an organic material ', specifically, "a fertilizer including nitrate nitrogen as an inorganic fertilizer component" can be prepared.

In the present invention, the preparation of a fertilizer containing nitrate nitrogen as an inorganic fertilizer component (fertilizer preparation step) is carried out as follows: placing water in a container capable of storing water, and adding the 'above-mentioned inoculum' of the present invention thereto; and performing a multiple parallel mineralization in the water by maintaining an environment in which the multiple parallel mineralization proceeds in the water, the environment being an environment in which an organic substance is added, the temperature is maintained at 15 to 37 ℃, and the environment is maintained under aerobic conditions; obtaining a reaction solution containing nitrate ions of more than 100 mg/L; the obtained reaction solution was recovered.

In the present fertilizer production step, the environment in which the multiple parallel mineralization proceeds may be maintained 'after the addition of the above-mentioned inoculum,' but the environment may be maintained 'after the addition of the above-mentioned inoculum to water in a state in which the environment in which the multiple parallel mineralization proceeds is maintained'.

The "container capable of storing water" used in this step may be any container as long as it can store water and has a structure that allows dissolved oxygen to easily diffuse.

For example, there may be mentioned a container capable of storing a relatively large amount of water such as a water tank, a barrel, a tank, a water storage tank, a bath, a pool, etc., a container capable of storing a relatively small amount of water such as a flask, a beaker, a test tube, etc., and the like.

Specifically, cans, boxes, and flasks may be used. In addition, in large-scale production or industrial application, a water storage tank or a pool can be used.

As the water used in this step, tap water, distilled pure water, well water, river water, lake water, sea water, etc. may be used.

The amount of water is not particularly limited as long as it is 50 times or more the dry weight of the organic matter to be added, and in order to obtain a sufficient amount of fertilizer, specifically, 0.001 to 10000L, preferably 0.01 to 1000L, is allowed to stand.

In the present fertilizer preparation step, the amount of the inoculum of the present invention added as a 'microorganism source' is not less than 0.01g, preferably not less than 0.2g, per 1L of water placed in the above container. When the amount of the inoculum added is less than 0.2g, the time for completion of the multiple parallel mineralization is delayed, which is not preferable.

Further, in the conventional method, when bark compost or the like is added as a microorganism source, it is necessary to add about 5g or more to 1L of water.

That is, by using the inoculum of the present invention as a microorganism source, the amount of the microorganism source to be added can be reduced to about 1/50 times, preferably about 1/25 times, more than the conventional methods.

In order to maintain the environment in which the parallel multiple inorganic reactions are performed in the present fertilizer production step, it is necessary to 'maintain an environment in which an organic substance is added, the temperature is maintained at 15 to 37 ℃, and the condition of becoming aerobic is maintained'.

By maintaining such an environment, the microorganisms optimized as a catalyst for the multiple parallel mineralization in the water are proliferated, so that the multiple parallel mineralization can be rapidly performed, and nitrate nitrogen of the inorganic fertilizer component can be generated 'without accompanying denitrification reaction'.

In the present fertilizer production step, by "maintaining the conditions for becoming aerobic" in the water, the dissolved oxygen concentration in the water can be increased, and conditions suitable for the activity of the microorganism group which performs the multiple parallel mineralization can be obtained.

In addition, since the microorganisms (denitrifying bacteria) that perform denitrification reaction are easily activated under anaerobic conditions, they are also suitable for suppressing the proliferation of microorganisms that perform denitrification reaction.

As a method for maintaining the water in an aerobic condition, aeration, shaking, dissolution of high-concentration oxygen, utilization of high-concentration oxygen-containing water, and the like can be carried out. Preferably, the aeration and the shaking are carried out.

In the present fertilizer production step, the "water temperature" suitable for the parallel multiple mineralization is a water temperature suitable for the growth of the microorganisms involved in the parallel multiple mineralization. Specifically, the temperature is preferably 15 to 42 ℃, more preferably 15 to 37 ℃, even more preferably 20 to 37 ℃, and most preferably maintained at about 25 ℃.

Further, in the case where the temperature is lower than 15 ℃, it is not preferable because the growth of the microorganism is delayed and the reaction takes time. In addition, in the case where the temperature is higher than 42 ℃, it is not preferable because a part of microorganisms necessary for performing the parallel multiple mineralization is dead.

In the fertilizer preparation step, the addition of the "organic substance" to the water is optimized and completed by the inoculum serving as a catalyst for the parallel compound mineralization, and a large amount of the "organic substance" can be added 1 time.

Specifically, the amount of the surfactant may be added to 1L of the water in an amount of not more than 20g (in terms of dry weight), preferably not more than 10 g.

In this step, the organic substance may be added before the start of the reaction or may be added after the start of the reaction.

The organic substance may be added in a liquid state or a powder state.

Specifically, when the organic substance is in a liquid state, 0.1 to 10g (liquid weight (0.07 to 7g in terms of dry weight)) of bonito cooking liquor can be added, 0.1 to 10g (liquid weight (0.05 to 5g in terms of dry weight)) of corn steep liquor can be added, and 0.1 to 10g of rapeseed oil meal can be added.

Further, in the conventional method, when bark compost or the like is added as a microorganism source, the amount of water is added to 1L at a time only up to about 2 g.

When the above-mentioned other species are added as a 'microorganism source', if more than 2g of organic matter is added per 1L of water, the inherent nitrifying bacteria die due to exposure to a large amount of organic components and lose their nitrification activity, and thus the parallel multiple mineralization cannot be performed, not to mention a microorganism group suitable for the parallel multiple mineralization.

That is, by using the inoculum of the present invention as a microorganism source, the amount of organic substances that can be added at one time can be increased by about 10 times, preferably about 5 times, as compared with the conventional method.

In the fertilizer production step, a culture solution containing nitrate nitrogen in terms of nitrate ions at 100mg/L or more, preferably 200mg/L or more can be obtained on days not exceeding 8 days, preferably 4 to 8 days, from the start of the culture.

In addition, the time from the start of the reaction 'to the completion of the mineralization of the organic substance into nitrate nitrogen' is not more than 10 days, preferably not more than 8 days, and more preferably 4 to 8 days, when the target nitrate ion concentration is 400 mg/L.

Note that the term "until the mineralization of nitrate nitrogen from the organic material is completed" means "until the concentration of the produced nitrate nitrogen reaches the peak".

Further, in the conventional method, when bark compost or the like is added as a 'microorganism source', the time from the start of the reaction 'to the completion of the mineralization of organic matter into nitrate nitrogen' is at least 10 days, and usually 15 days or more are necessary.

That is, by using the inoculum of the present invention as a ' microorganism source ', the inoculum can be decomposed at a rate of about 2 times or more as compared with the conventional method, and ' the reaction time can be greatly shortened ' until the mineralization of the organic matter into nitrate nitrogen is completed '.

By this fertilizer production step, a reaction solution containing nitrate nitrogen in terms of nitrate ions at a 'high concentration' of 100mg/L or more, preferably 200mg/L or more, and more preferably 400mg/L or more in the reaction solution can be obtained 'effectively'. By recovering this reaction solution, it is possible to obtain a fertilizer containing nitrate nitrogen as an inorganic fertilizer component.

The prepared fertilizer may be a liquid fertilizer mixed with a liquid fertilizer or a chemical fertilizer diluted 2 to 10 times, although the reaction solution obtained in the above step may be used as it is as a raw solution. Alternatively, the dried product may be processed into a concentrated solution, a solid powder, or a solid tablet.

The fertilizer containing nitrate nitrogen as an inorganic fertilizer component obtained in the fertilizer preparation step can be used as a fertilizer for cultivation of vegetables, fruits, flowers, foliage plants, and all plants.

In particular, it is suitable for cultivation of leaf vegetables such as cabbage, komatsuna, lettuce, spinach, etc., vegetables used as harvested fruits such as tomato, fruit trees, flowers, etc. In particular, the method is suitable for cultivating cabbage and Chinese juniper.

Further, the fertilizer containing nitrate nitrogen as an inorganic fertilizer component can be used for cultivation of plants in general, such as cultivation in a nutrient solution, cultivation using ordinary soil, and the like.

In addition, in the present invention, by supplying the 'reaction solution' obtained in the above-described fertilizer preparation step to the nutrient solution for nutriculture, 'nutriculture by directly adding a fertilizer including an organic material,' which has been difficult in the past, is possible.

Specifically, by directly performing the fertilizer preparation of the above steps in the nutrient solution for nutriculture, direct addition of a fertilizer including organic matter to the nutrient solution becomes possible.

In addition, the container (reaction tank) in which the fertilizer preparation step is performed can be used as a device for performing nutriculture by directly adding a fertilizer including an organic material to the container.

In the nutrient solution cultivation method, when conventional bark compost or the like is used as a 'microorganism source' for constructing a microbial ecosystem in a nutrient solution, about 5g or more is required to be added per 1L of the addition amount, about 2g or more of organic matter is added per 1L, and a period of 15 to 20 days or more is usually required until the reaction is completed (time required for completing the microbial ecosystem in the nutrient solution).

On the other hand, by adding the inoculum of the present invention as a "microorganism source", the amount of the "microorganism source" added is about 1/50 times, preferably about 1/25 times as much as that of the conventional method, and the addition of the organic substance can be made up to 10g at a time, and completed by about half or less days (not more than 8 days, preferably 4 to 8 days) after the reaction is completed.

The nutrient solution cultivation method can be used for cultivating vegetables, fruits, flowers and trees, foliage plants and the like, and cultivating all plants.

In particular, it is suitably used for cultivation of leaf vegetables such as cabbage, komatsuna, lettuce, spinach, vegetables for harvesting fruits such as tomato, fruit trees, and flowers. In particular, the method is suitable for cultivating cabbage and Chinese juniper.

[ examples ] A method for producing a compound

The present invention will be described in more detail with reference to the following examples, but the present invention is not limited to these examples.

[ example 1] preparation of inoculum: biofilm formation and recovery

The step of ` culturing ` a microorganism group which performs a parallel multiple mineralization and ` recovering ` a biofilm by forming the same (culturing step and recovering step) are used as a step for preparing an inoculum of a microorganism group optimized as a catalyst for the parallel multiple mineralization.

10L of water was put into a Huaganna pot (made by Taurus) and bark was composted (trade name Golden bark, made by Shuihong Timber industries Co., Ltd.) to add 5g for 1L of water.

To this, about 1g of bonito boiled juice (a by-product of bonito construction) was added (added in a small amount) to 1L of water on a daily basis, and the microorganisms subjected to the parallel multiple mineralization were cultured at 25 ℃ for 2 weeks while maintaining aerobic conditions by aeration with an air pump. After the culture, biofilm formation was observed on the wall surface of the vessel. Then, the supernatant of the culture solution obtained after the culture was removed by pouring and discarded.

In the adhesion, the biofilm formed on the wall surface of the container was air-dried overnight and then recovered by scraping with a metal spatula, and a dried microbial substance (inventive example 1-1) was obtained.

FIG. 1(a) is a schematic diagram showing a method for forming and recovering a biofilm in this example. In addition, FIG. 2 is a photograph showing the formation and recovery process of the biofilm in this example.

In addition, after the supernatant of the culture solution was removed by pouring and discarded in the above-mentioned step, the biofilm formed on the wall surface of the container was scraped with a brush without air drying and mixed with the supernatant of the culture solution, and recovered as a mixed solution, and the excess water was removed by centrifugation, and a wet bacterial body as a settling vessel was obtained (inventive example 1-2).

[ example 2] addition amount and reaction time of inoculum

A check was made as to whether or not the biofilm formed by the process of conducting the multiple parallel mineralization was usable as a new microbial source for conducting the multiple parallel mineralization.

50mL of distilled pure water was put into a triangular flask (200mL volume), and 0.2g, 0.4g, or 1.0g of the dried microbial substance obtained in example 1 (inventive example 1-1) was added as a microorganism source in an amount of 1L per water.

To this, bonito boiled juice (a by-product of bonito construction) was added 1L per water, and the mixture was soaked at 120rpm and kept aerobic, and the reaction was carried out at 25 ℃ for 16 days.

Further, as a control experiment, an experiment in which bark compost (trade name Golden bark, manufactured by sanchi industries co-ordination system) was reacted at a rate of 5g per 1L of water as a microorganism source was also carried out. The results are shown in FIG. 3.

As a result, when the dry microbial cell obtained in example 1 (inventive example 1-1) was added as a microorganism source, the reaction time (time until the peak of the nitrate ion concentration reached) to the completion of the mineralization of the organic matter into nitrate nitrogen was completed in 6 to 8 days.

On the other hand, in the case where the bark compost was added as a microorganism source as a control experiment, it took 13 days.

Therefore, it was found that the addition of the dried microbial biomass obtained in example 1 as a microbial source can shorten the reaction time from the completion of mineralization of organic matter into nitrate nitrogen by approximately half the number of days as compared with the case where the bark compost is added as a microbial source.

Furthermore, in the case of using a microorganism source, such as soil or bark compost, which has not been optimized for parallel multiple mineralization, if a relatively large amount of organic matter is added, nitrifying bacteria contained in the microorganism source are less exposed than organic components and easily die, and it is necessary to add a large amount of the microorganism source (when the amount of the added microorganism source is small, nitrifying bacteria die and nitrification does not proceed). Specifically, when about 1g of organic matter is added to 1L of water, about 5g of microorganism source needs to be added to 1L of water, but when the amount of microorganism source is excessively added (specifically, when soil is added in an amount exceeding 10g to 1L of water), the microorganism source becomes self-block and anaerobic inside, and denitrifying bacteria easily grow.

However, as shown in the present example, when the biofilm formed in the process of the parallel multiple mineralization was added as the microorganism source, the parallel multiple mineralization could be performed without any problem even with an addition amount of 0.2g to 1L of water, and the reaction time to the end of the reaction could be shortened as compared with the case of adding the conventional microorganism source (from 14 days to 8 days when using the inventive example 1-1).

That is, it was found that the amount of the microorganism source added can be reduced to about 4% in the conventional case, and the reaction time can be shortened to about half.

From these results, it was found that the dried microbial cells of example 1-1 of the present invention can be used as a seed of a microbial population optimized as a catalyst for a multiple parallel mineralization.

Example 3 Mass addition of organic substances

A test was conducted to determine whether a biofilm formed by a process of parallel multiple mineralization was used as a microbial source and whether a large amount of organic substances could be added at once.

50mL of distilled water was put into a triangular flask (200mL), and 5g of the wet bacterial substance (inventive example 1-2) obtained in example 1 was added as a microorganism source in an amount of 1L per water.

To this, bonito decoction (a by-product of bonito workshop) was added at 1L 10 g/water, and the mixture was soaked at 120rpm to maintain aerobic conditions, and the reaction was carried out with water at 25 ℃ for 14 days.

Further, as a control experiment, an experiment was also performed in which bark compost (trade name Golden bark, manufactured by sanchi harbor wood industries co-ordination) was added to 1L of water as a microorganism source and reacted with 5g of the added water. The results are shown in FIG. 4.

As a result, when the moist microbial biomass (inventive examples 1 to 2) obtained in example 1 was added as a microbial source, when bonito boiled juice (a by-product of a bonito workshop) was added in an amount of '10 g with respect to 1L of water' (a large amount of an organic substance was added), microbial groups optimized as a catalyst for a parallel multiple mineralization were also proliferated without any problem, and nitrate nitrogen was generated as an inorganic fertilizer component from the organic substance.

On the other hand, when the bark compost of the control experiment was added as a microbial source, the generation of ammonia was confirmed, and the generation of nitrate ions (nitrate nitrogen) was not detected. Therefore, the control experiment showed that the reaction was stopped at the amination completion stage and the nitration reaction was not carried out (parallel multiple inorganic reactions were not carried out to the final product).

Furthermore, conventionally, when soil or bark compost or the like, which is not optimal for multiple parallel mineralization reactions, is used as a microorganism source, the allowable amount of organic substances to be added is limited to 'about 2g for 1L of water'.

However, as shown in the present example, it was found that when a biofilm formed in the process of performing multiple parallel mineralization is added as a microorganism source, even if a large amount of organic matter (about 5 times the amount of the conventional organic matter) is added, the multiple parallel mineralization can be performed without any problem, and nitrate nitrogen as an inorganic fertilizer component can be produced from the organic matter.

Example 4 Complex parallel mineralization of moist bacteria and reaction rate

The inventors examined at what speed the multiple parallel mineralization was performed by using the 'wet microbial biomass' of the biofilm formed in the process of the multiple parallel mineralization as a microbial source.

50mL of distilled pure water was put into a triangular flask (200mL volume), and 5g of the wet bacterial substance obtained in example 1 (inventive example 1-2) was added to 1L of water as a microorganism source.

To this, 1L/water of bonito cooking juice (a by-product of bonito workshop) was added, and the mixture was soaked at 120rpm to maintain aerobic conditions, and the reaction was carried out at 25 deg.C for 16 days.

Further, as a control experiment, an experiment was also conducted in which bark compost (trade name Golden bark, manufactured by sanchi industries co-ordination system) was added at 1L/water 5g as a microorganism source and reacted. The results are shown in FIG. 5.

As a result, when the wet bacterial cells (inventive example 1-2) obtained in example 1 were added as the microbial source, the reaction time (time until the peak of the nitrate ion concentration reached) to the completion of the mineralization of the organic matter into nitrate nitrogen was completed in 4 days.

On the other hand, when the bark compost of the control experiment was added as a microorganism source, it took 11 days.

Therefore, from the results of the present example, when a biofilm that is a microorganism source optimized for the multiple parallel mineralization is added in the shape of a "moist bacterial mass", it was found that the number of days until completion of the mineralization of organic matter into nitrate nitrogen can be shortened to approximately 1/3, compared to when a microorganism source that is not optimized for the multiple parallel mineralization, such as bark compost, is used.

Comparative example 1 the supernatant of the culture solution was not suitable for inoculation

A check was made as to whether or not the multiple parallel mineralization can be performed by using the 'supernatant of the culture solution' obtained after culturing the microorganisms that have undergone the multiple parallel mineralization as a microorganism source.

First, as an organic material, microorganisms which were subjected to a parallel multiple mineralization were cultured in the same manner as in example 1, except that 1g of CSL (trade name "organic liquid fertilizer", manufactured by Sakata seeds) was added to 1L of water. Then, the supernatant of the culture solution obtained after the culture was aspirated and discharged by a pipette and recovered (comparative preparation 1).

Next, 6 flasks were prepared for autoclaving by adding 0.5g of any solid carriers of bamboo charcoal, perlite, sea sand, bark compost, horticultural culture (seedling-one-double) and without solid carriers, and by adding 50ml of distilled water.

Then, 0.5ml (10 ml for 1L of water) of the supernatant (comparative preparation 1) after the culture was added as a microorganism source to each flask.

To this was added 0.5g (1L/water, 10g) of CSL (trade name: organic liquid fertilizer, manufactured by Sakata seeds) and the mixture was shaken at 120rpm to maintain aerobic conditions, and the reaction was carried out at 25 ℃ for 17 days. The results are shown in FIG. 6.

As a result, although a solid carrier which is thought to be readily attached to the microorganisms (nitrifying bacteria) that have performed nitrification was added, only nitrification was performed in any of the bottles, and the formation of nitrate ions (nitrate nitrogen) was not observed.

Therefore, the microorganisms (nitrifying bacteria) that have performed nitrification activity hardly float in the supernatant of the culture solution obtained after the microorganisms that have performed multiple parallel mineralization are cultured, and even if they are present, they are considered to be extremely small and are considered to die due to exposure of the added organic component.

Therefore, it was found that the supernatant of the culture solution obtained by culturing the microorganisms which have undergone the multiple parallel mineralization is not suitable for use as an inoculum of the microorganisms which catalyze the multiple parallel mineralization.

Example 5 method of Using a mixture of supernatant of culture solution after culture and biofilm as inoculum, and type of organic substance in preparation of inoculum

The 'mixed solution' in which the supernatant of the culture solution obtained after culturing the microorganisms that have undergone the multiple parallel mineralization and the formed biofilm is mixed is used as a microorganism source to carry out the multiple parallel mineralization. In addition, it was also examined that when the parallel multiple mineralization reaction was performed by adding an organic substance other than bonito sauce, a microorganism group optimized as a catalyst for the parallel multiple mineralization reaction could be prepared in the same manner as when using bonito sauce.

First, as an organic material, a microorganism group which was subjected to a parallel multiple mineralization was cultured in the same manner as in example 1, except that rapeseed oil meal (manufactured by Rinoru oil and fat co., ltd.) was added to 1L of water in an amount of about 1 g. Then, the biofilm formed on the wall surface was physically scraped by a brush to be sufficiently mixed with the supernatant of the culture solution, and the mixed solution was recovered (inventive example 5).

Next, 9L of water was put into a Huaganna pot (manufactured by Taurus nobilis Co., Ltd.), and 1L of the mixed solution (inventive example 5) obtained in the above-described steps (culture step and recovery step) was added as a microorganism source.

To this, 10g (1 g for 1L of water) of rapeseed oil meal (manufactured by Rinoru oil & fat Co., Ltd.) was added, and the reaction was carried out at 25 ℃ for 10 days while maintaining aerobic conditions by aeration with an air pump. The results are shown in FIG. 7.

As a result, when the mixed solution (example 5 of the present invention) was added as a microorganism source, the reaction time from the completion of the mineralization of the organic matter into nitrate nitrogen (the time until the peak of the nitrate ion concentration was reached) was completed in 6 days. In addition, nitrate ions in excess of 350mg/L were produced in the reaction solution after the completion of the reaction.

Therefore, it was shown that the mixture of the supernatant of the culture solution obtained after the culture and the formed biofilm was usable as an inoculum optimized as a microorganism source for catalyzing a parallel multiple mineralization.

In addition, biofilms other than bonito sauce can be similarly used as inoculum.

Example 6 filtration of a liquid mixture of a supernatant of a culture solution after culture and a biofilm

A moist microbial material obtained by filtering a mixed solution of a biofilm formed by culturing a microbial population which has undergone multiple parallel mineralization and a supernatant of the obtained culture solution is used as a microbial source, thereby performing multiple parallel mineralization.

Then, the dried microbial cell mass (inventive example 7) was suspended in 200mL of distilled pure water, and 50mL of the suspension (including 37.5mg of the wet microbial cell mass per 1L of water) was placed in a triangular flask.

To this was added 0.05g (1 g for 1L of water) of bonito cooking juice (Nakazaki fishery Co., Ltd.), and the reaction was carried out at 25 ℃ for 6 days while maintaining aerobic conditions by shaking at 120 rpm. The results are shown in FIG. 8.

As a result, when the mixed solution of the supernatant of the culture solution and the biofilm obtained in the above-described step was filtered and the obtained dried microbial biomass (inventive example 7) was added as a microorganism source, the formation of nitrate ions (nitrate nitrogen) was observed 6 days after the start of the reaction.

Therefore, it was shown that the dried microbial cell obtained by filtering the mixed solution of the supernatant of the culture solution and the biofilm can be used as an inoculum of a microbial population optimized as a catalyst for a parallel multiple mineralization.

[ example 7] Heat resistance of inoculum

The inventors carried out an examination of how much the activity as an inoculum is affected by applying a 'heat treatment' to an inoculum of a microorganism group optimized as a catalyst for a parallel multiple mineralization.

Each 100mg of the dried microbial cells (inventive examples 1 to 1) obtained in example 1 was allowed to stand at room temperature (about 25 ℃ C.), 50 ℃ C., and 80 ℃ C. for 30 minutes.

Next, 30mL of distilled pure water was put into a triangular flask (200mL volume), and 30mg (1L 1g per water) of each of the dried microbial cells after the heat treatment was added as a microbial source.

To this was added 0.03g (1L/water) of bonito cooking juice (Naokazai fishery co-Ltd.) and the mixture was soaked at 120rpm to maintain aerobic conditions, and the reaction was carried out at 25 ℃ for 15 days.

Further, the reaction was carried out by adding 30mg (1L/water) of bark compost (trade name: Golden bark, manufactured by Shuichong Timber industries Co., Ltd.) as a microorganism source as a control experiment. The results are shown in FIG. 9.

As a result, when the dried microbial substance subjected to the heat treatment at 50 ℃ for 30 minutes was added as a microorganism source, the reaction time (time until the peak of the nitrate ion concentration was reached) from the completion of the mineralization of the organic substance into nitrate nitrogen ended in 5 days.

Therefore, the dried microbial cells subjected to the heat treatment at 50 ℃ for 30 minutes show that the function as a "microbial population optimized as a catalyst for the multiple parallel mineralization" is maintained without decrease as compared with the case of standing at room temperature (about 25 ℃).

On the other hand, when the dried biomass heated at 80 ℃ for 30 minutes was added as the microorganism source, the reaction time (time until the peak of the nitrate ion concentration was reached) required 9 days to complete the mineralization of the organic matter into nitrate nitrogen, and the reaction time was about the same as that required when the bark compost was used as the microorganism source.

From the above results, it was found that the heat resistance was achieved without lowering the function of the inoculum of the microorganism group optimized as a catalyst for the multiple parallel mineralization in the heat treatment at 50 ℃ for 30 minutes.

It was also found that the amount of nitrate ions (nitrate nitrogen) produced after the completion of the reaction was as high as that produced by leaving the reactor at room temperature (about 25 ℃ C.) when the reactor was heated at 80 ℃ for 30 minutes, and that the commercial value as inoculum was not lost when the reactor was exposed to only a high temperature of 80 ℃ for a while.

Further, in the case of the bark compost as a microorganism source, the amount of nitrate nitrogen produced at the end of the reaction was small, and the content of organic components was more than that of the dried bacterial product (inventive example 1-1) obtained in example 1, and it was estimated that the concentration of nitrate ions that could be recovered was decreased by consuming nitrate ions with the microorganisms used for the production.

[ INDUSTRIAL APPLICABILITY ]

The inoculum of the present invention is valuable as a technique for solving the problem of poor operability in nutrient solution cultivation of organic fertilizers (which has been recently noticed in practical use (about 2 weeks of reaction time is required, the amount of microorganism source is about 5g per 1L, and about 2g of organic substances added at a time is a limit). By using the inoculum provided by the invention, the reaction time is shortened to be less than half, the inoculation amount of a microorganism source is reduced to 4%, the addition amount of organic matters can be increased to 5 times, and the operability is greatly improved.

At present, nutrient solution cultivation using organic fertilizers is concerned, producers are rapidly increasing, 150ha and 4000ha in the domestic and the netherlands, and a considerable part of continuously expanding nutrient solution cultivation is expected to be replaced by using organic fertilizers. The seed bacteria provided by the present invention are expected to be widely used because they greatly contribute as a technique for assisting the production activities of these producers, and the market size thereof is expected to become very large. The seed bacteria of the present invention can be used not only in nutrient solution culture but also in water culture for exhibition such as indoor greening and indoor greening, and the marketability is not limited to the agricultural field.

Further, the present invention can be applied to a technique for producing an inorganic fertilizer using organic waste as a raw material. The waste recycling industry is expected to expand to a market scale of 2 million-5 billion circles in the future, and the present invention is a technology for rapidly and effectively recycling a large amount of organic resources into inorganic fertilizer components, and has a very high industrial applicability.

Further, the present invention can newly prepare an inoculum by using the inoculum of the present invention. Since the preparation can be carried out in half a day as compared with the conventional multiple parallel mineralization, the rapid mass production of the inoculum becomes possible. As described above, the need for the seed bacteria is expected to increase from the wide marketability of the seed bacteria itself, and the technology itself for rapidly mass-producing the seed bacteria is expected to have a large marketability.

[ description of the drawings ]

FIG. 1(a) to (c) are explanatory views showing various embodiments of the method for producing an inoculum of microorganisms optimized as a catalyst for a parallel multiple mineralization according to the present invention. Further, (a) is a schematic diagram showing a method for forming and recovering a biofilm in example 1.

FIG. 2 (a) is a schematic view showing an embodiment of the present invention in which a biofilm is formed on a solid surface. Further, (b) is a photograph showing the formation and recovery process of the biofilm in example 1. Further, (c) is a schematic diagram showing an embodiment of the method for collecting a biofilm of the present invention.

FIG. 3 is a graph showing the measurement results of the nitrate ion concentration in example 2.

FIG. 4 is a graph showing the measurement results of the nitrate ion concentration, nitrite ion concentration and ammonia concentration in example 3.

FIG. 5 is a graph showing the measurement results of the nitrate ion concentration in example 4.

FIG. 6 is a graph showing the results of measuring the nitrate ion concentration and the ammonia concentration in comparative example 1.

FIG. 7 is a graph showing the measurement results of the nitrate ion concentration in example 5.

FIG. 8 is a graph showing the measurement results of the nitrate ion concentration in example 6.

FIG. 9 is a graph showing the measurement results of the nitrate ion concentration in example 7.

Claims (9)

1. The preparation method of the inoculum comprises the following steps:

water is placed in a container in which water can be stored,

if necessary, impregnating the substrate with any one or more of the following (A1) to (A3),

adding 1 to 10g of a microorganism source described in the following (B) containing microorganisms capable of conducting a multiple parallel mineralization of an organic substance to form nitrate nitrogen, and

culturing the microorganisms capable of conducting multiple parallel mineralization by maintaining the environment in the water so as to satisfy all of the conditions (C1) to (C4) below;

forming a biofilm on a solid surface described in the following (E) which is in contact with the above water, and then,

discarding a supernatant of a culture solution obtained by culturing the microorganisms which have undergone the multiple parallel mineralization, and then collecting a biofilm formed on the surface of the solid; and

the collected biofilm is used as an inoculum of a microorganism group optimized as a catalyst for a parallel multiple mineralization that mineralizes an organic substance to produce nitrate nitrogen,

(A1) bamboo charcoal, perlite, sea sand, vermiculite, ceramic, zeolite, glass, rock wool, urethane, nylon, or melamine resin;

(A2) a plate made of glass, acrylic, plastic, ceramic plate, or ceramic;

(A3) a pillar of glass, acrylic, plastic, ceramic sheet, or ceramic;

(B) a microbial source selected from one or more of the following: soil, compost, activated sludge, or naturally occurring water;

(C1) the water temperature is 15-37 ℃;

(C2) maintaining aerobic conditions by aeration and/or agitation;

(C3) adding 0.01 to 2g of the organic substance (D) to 1L of the water in terms of dry weight per 1 to 14 days;

(C4) stopping adding the organic matter when the concentration of nitrate ions generated in the water reaches 10-50 mg/L;

(D) one or more organic substances selected from the group consisting of: fish cooking juice, corn steep liquor, oil meal, fish meal, cow milk, soybean meal, yeast meal, wine meal, distilled liquor meal, or household garbage;

(E) a solid surface selected from one or more of the following: the wall surface of the container, the bottom surface of the container, the surface of the solid carrier, the surface of the plate-like object, or the surface of the column-like object.

2. The preparation method of the inoculum comprises the following steps:

water is placed in a container in which water can be stored,

if necessary, impregnating the substrate with any one or more of the following (A1) to (A3),

adding 0.005 to 1g of the inoculum obtained by the method for producing the inoculum according to claim 1 as a microorganism source in terms of dry weight to 1L of the water, and

culturing the microorganisms that perform a multiple parallel mineralization of an organic substance to form nitrate nitrogen by maintaining the environment in the water so as to satisfy all of the conditions (C1), (C2), (C3-2), and (C4) below; and

forming a biofilm on a solid surface described in the following (E) which is in contact with the above water, and then,

discarding a supernatant of a culture solution obtained by culturing the microorganisms which have undergone the multiple parallel mineralization, and then collecting a biofilm formed on the surface of the solid; and

the collected biofilm is used as an inoculum of a microorganism group optimized as a catalyst for a parallel multiple mineralization for mineralizing an organic matter to produce nitrate nitrogen,

(A1) bamboo charcoal, perlite, sea sand, vermiculite, ceramic, zeolite, glass, rock wool, urethane, nylon, or melamine resin;

(A2) a plate made of glass, acrylic, plastic, ceramic plate, or ceramic;

(A3) a pillar of glass, acrylic, plastic, ceramic sheet, or ceramic;

(C1) the water temperature is 15-37 ℃;

(C2) maintaining aerobic conditions by aeration and/or agitation;

(C3-2) adding 0.01 to 10g of the organic substance described in the following (D) in terms of dry weight to 1L of the water every 1 to 14 days;

(C4) stopping adding the organic matter when the concentration of nitrate ions generated in the water reaches 10-50 mg/L;

(D) one or more organic substances selected from the group consisting of: fish cooking juice, corn steep liquor, oil meal, fish meal, cow milk, soybean meal, yeast meal, wine meal, distilled liquor meal, or household garbage;

(E) a solid surface selected from one or more of the following: the wall surface of the container, the bottom surface of the container, the surface of the solid carrier, the surface of the plate-like object, or the surface of the column-like object.

3. A method for producing an inoculum according to any one of claims 1 to 2, wherein the biofilm is recovered and then dried.

4. An inoculum having the characteristics described in the following (F) and (G), which is obtained by the method for producing an inoculum according to any one of claims 1 to 3,

(F) a microbial composition optimized as a catalyst for a parallel multiple mineralization in which an organic material is mineralized to produce nitrate nitrogen;

(G) the function of an inoculum, which is an inoculum of a microorganism group optimized as a catalyst for a parallel multiple mineralization that mineralizes an organic substance to produce nitrate nitrogen, is not lost even when the heating is performed at 50 to 80 ℃.

5. A method for preparing a fertilizer comprising nitrate nitrogen as an inorganic fertilizer ingredient, comprising:

water is placed in a container in which water can be stored,

adding the above-mentioned inoculum according to claim 4 thereto; and

maintaining the environment in the water so as to satisfy all of the conditions (C1), (C2), and (C3-3) below, thereby performing a parallel multiple mineralization of organic matter in the water to generate nitrate nitrogen;

obtaining a reaction solution containing nitrate ions of more than 100 mg/L;

using the obtained reaction solution as a fertilizer, wherein the fertilizer comprises nitrate nitrogen as an inorganic fertilizer component,

(C1) the water temperature is 15-37 ℃;

(C2) maintaining aerobic conditions by aeration and/or agitation;

(C3-3) adding an organic substance in an amount of not more than 20g in terms of dry weight to 1L of the water.

6. The method for producing a fertilizer according to claim 5, wherein the inoculum is added in an amount of not less than 0.01g to 1L of the water.

7. A fertilizer containing nitrate nitrogen as an inorganic fertilizer component, which is obtained by the method for producing a fertilizer according to claim 5 or 6.

8. A method for cultivating a plant, which comprises using the fertilizer according to claim 7, wherein the fertilizer comprises nitrate nitrogen as an inorganic fertilizer component.

9. A method for cultivating a plant by adding a fertilizer containing an organic substance directly to the reaction solution according to any one of claims 5 to 8, and performing nutrient solution cultivation.