KR20170038453A - Compound fertilizer composition - Google Patents

Abstract

The present invention relates to a composite fertilizer composition comprising a culture liquid of Saccharomy cescerevisiae and mixed humus at a weight ratio of 5:2-5:7. The culture liquid of Saccharomy cescerevisiae comprises 4-6 wt% of nitrogen, 1-3 wt% of phosphoric acid and 3-5 wt% of potassium based on the total weight of the composition. The composition of the present invention can be used in all crops, has excellent quality, promotes growth of crops, and makes high quality crops harvested. Accordingly, the income of farms is increased; quality of crops can be improved; the good status of a soil ecosystem is maintained; and agricultural competitiveness can be established.

Description

[0001] Compound fertilizer composition [0002]

The present invention relates to a compound fertilizer composition, and more particularly, to a compound fertilizer composition containing a cultured yeast ( Saccharomyces cerevisiae ) as a microorganism and a corrosion acid and fulvic acid as an organic component.

As consumers' interest in safe food has increased in recent years, the use of organic synthetic pesticides and chemical fertilizers has been minimized, while complying with safety standards for pesticide use and standard application rates for each crop has resulted in comprehensive pest management that can preserve the environment and produce safe agricultural products (IPM), and integrated nutrient management (INM). As a result, agriculture in Korea uses organic fertilizer as a main component and uses fertilizer which is mainly used as a nutrient, a fourth type compound fertilizer as a nutrient source of plants.

These type 4 compound fertilizers are generally manufactured by dissolving raw materials for producing nutrient solutions in water. In order to reduce production costs as competition intensifies, there are many cases in which liquid fertilizers, which merely dissolve only solid fertilizers, are sold at high prices as functional fertilizers.

In this situation, it is urgent to develop a nutrient that can promote the growth of crops, harvest high quality crops, maintain the integrity of the soil ecosystem, and establish our agricultural competitiveness.

Corrosive acid is a raw material that improves the soil and is effective in the growth of crops. It is called humus or humic substances, and changes soil organic matter to produce humus. Organic matter is a compound containing a carbon atom originally and means all organic compounds that are naturally produced or artificially synthesized by an organism, but only organic compounds naturally derived from animals, plants and microorganisms in agriculture. Soil organic matter is simply an organic matter in the soil, so it is not different from organic matter in essence.

Soil organic matter is decomposed by various kinds of microorganisms and polymerized by the soil environment and is constantly changing. In this way, the soil organic matter changed in the soil environment is decomposed, condensed, polymerized and oxidized for a long time and put into the soil to form a brown-black organic chemically stable polymer compound (soil organic matter) (Humic acid), fulvic acid (FA), and ulmic acid (UA), depending on the type of fermentation.

Humic acid (HA) is almost insoluble in water but it is soluble in alkali such as potassium hydroxide (KOH) or sodium hydroxide (NaOH). It is brown-black brown and has many functional groups. Trace element is easily combined. Corrosive acids are aromatic aromatic macromolecules in which amino acids, amino sugars, peptides, and aliphatic compounds are complexly bound between aromatic rings and rings. They are nitrogen, oxygen, hydroxyl groups (OH groups), carboxy groups (COOH groups) (Stevenson, 1994).

Also. Corrosive acid is a kind of organic acid that is oxidized naturally and has a negative charge. Various cation and trace elements are bonded to this functional group, and various microorganisms are inhabited. Corrosive matter does not have a certain structure and molecular weight depending on the kind, so it is not easy to identify effectively. It should be evaluated comprehensively such as color, degree of polymerization, molecular weight, number of functional groups such as COOH group and OH group, number of carbon and oxygen and bending strength. Corrosive acids have been reported to improve soil chemical and soil buffering capacity and improve seed germination, seedling growth, balanced growth of plants and nutrient uptake by improving the water holding capacity and bending strength of soil (Chen, Y. and Aviad, T, 1990, Sparks, 1995, Varanini, Z. and Pinton, R., 1995). However, since the acid is weak acid, it can not dissolve the mineral particles, which are trace elements required for plant growth in the soil.

Fulvic acid, on the other hand, has a stronger acidity than a caustic acid and is highly hydrophilic, so it can dissolve mineral particles that are not soluble in the acid. Fulvic acid is a more beneficial substance for plants as compared with the acidic acid because it can function as a mineral dissolution and nutrient mediator. However, due to its high hydrophilicity, fulvic acid does not stay in the soil because it is highly soluble in water, and there is a problem that it penetrates deep into ground through rain or ground water before plants absorb it. Therefore, it is economically undesirable from the point of view of cost-effectiveness to spray an excessively high content of fulvic acid, and a more effective method of fertilizing is required.

Microorganisms that have the effect of promoting crop growth are microorganisms that live in the rhizosphere of plants. They are called Plant Growth Promoting Rhizobacteria (PGPR), which promote the supply of nutrients, induce metabolism by plant hormones, Maintains and regulates plant defense mechanisms such as Systemic Aquired Resistance (SAR) and Induced Systemic Resistance (ISR), and inhibits plant pathogens.

PGPR can be divided into two groups: bio control-PGPR, which indirectly helps plants by inhibiting plant pathogens, and direct influence on plant metabolism by inducing plant growth promotion, seed germination promotion and crop productivity increase There is PGPR. If the former acts as a biological control through ISR, SAR, and the release of antimicrobial substances, the latter may be a combination of indole-3-acetic acid (IAA), cytokinin, gibberellin octadecanoid ), Jasmonate analogue, Salicylate, or produce ethylene (Ethylene) to help improve plant growth, yield and resistance.

Among these, IAA is the most commonly found plant hormone, which is produced in the growing stem, root tip or young leaf, and is transferred to the stem or root elongation part and promotes cell growth of the part. Auxin is known to play a role in promoting growth, reproductive function, regulation of growth direction (luminous power), dominance of dominance, induction of root in the plant (Kwak and Yoon, 2004).

Although plants can synthesize auxin in the body, depending on the cultivation conditions, physiological response to exogenously supplied auxin can be greatly increased. Thianmann and Link in the 1930s first reported that auxin could be induced by Rhizobium sp., And many plant growth regulators and their derivatives have been reported to be produced by microorganisms afterwards Lumsden, 1981; Meyer, 1966). Denvender and David (1984) azo RY rilryum (Azospirillum sp.) Was confirmed this IAA is inducing the host bungeun of producing, Atzorn, etc. (1988) Rizzo Away (Rhizobium sp.) Produce IAA and leguminous plants Gardan et al. (1992) have shown that Pseudomonas savastanoi , a phytopathogenic fungus , produces high levels of IAA when plant tissues are infected, promoting the differentiation of the follicles Respectively.

The role of auxin in promoting growth, the growth of stem and apical shoot, the growth of root and root, and the rooting of root, depends on the concentration of auxin. In general, auxin growth promoting effect occurs at a very low concentration. In the case of stem, 10 ^-6 ~ 10 ^-5 M is the optimal concentration, and if it exceeds this concentration, kidney growth is inhibited. In the case of roots, when the concentration of oxine is higher than 10 ^-8 M, the growth of the main muscle tends to be inhibited by the inhibition of the growth of the main root, but the auricle is promoted rather than the high concentration of auxin (Kwon, 2003). Using these properties, synthetic phenoxy compounds such as 2,4-D (2,4-Dinitrophenol), PCP (Pentachlorophenol) and MCPA (2-Methyl-4-chlorophenoxyacetic Acid) On the other hand, it is also used as a plant-causing agent by using the effect of inhibiting growth (Kwak, 2004). Although these synthetic auxins are actually used as herbicides and plant growth agents, and many microorganisms that produce auxin have been isolated and studied, the relationship between these microorganisms and plants has not been studied yet (So et al., 2009 ).

1. Korean Patent Registration No. 10-0462953 2. Korean Patent Registration No. 10-0537986 3. Korean Patent Registration No. 10-1065539 4. Korean Patent Registration No. 10-1051666

It is an object of the present invention to provide a high-quality functional compound fertilizer composition, and more particularly, to a microorganism having a growth promoting effect of crops using a yeast ( Saccharomyces cerevisiae ) culture medium, a mixture of a combination of a corrosion acid and a fulvic acid It is an object of the present invention to provide a composite fertilizer composition which can accelerate the growth of crops by using the corrosion, maximize high quality crops, and maintain the integrity of the soil ecology.

According to an aspect of the present invention,

A composite fertilizer composition comprising a microbial component and an organic component,

The microbial component may be selected from the group consisting of yeast ( Saccharomyces cerevisiae culture medium,

Wherein the organic material component comprises mixed corrosion comprising a corrosive acid and fulvic acid,

The yeast culture broth and mixed corrosion thereof at a weight ratio of 5: 2 to 5: 7,

The yeast culture solution contains 4 to 6% by weight of nitrogen, 1 to 3% by weight of phosphoric acid and 3 to 5% by weight of potassium based on the total weight of the composition.

It is preferable that the composite fertilizer composition includes the yeast culture broth and mixed corrosion at a weight ratio of 5: 4 to 5: 5.

In the composite fertilizer composition, the mixed corrosion preferably comprises 20 to 90% by weight of caustic acid and 10 to 80% by weight of fulvic acid, and comprises 40 to 80% by weight of a caustic acid and 20 to 60% by weight of fulvic acid. Is more preferable.

In the composite fertilizer composition, the yeast culture solution and the mixed corrosion are preferably contained in an amount of 60 to 98 wt%, more preferably 80 to 95 wt%, based on the total weight of the composition.

In the composite fertilizer composition, the corrosive acid and fulvic acid are prepared by adding 10 to 40% of nitric acid aqueous solution to lignite so that the ratio of the solid to the aqueous solution (S / L) is 0.5 to 3, and the mixture is heated at 10 to 90 ° C for 2 to 48 hours And then reacting and oxidizing the reaction mixture.

In the composite fertilizer composition, the yeast culture is preferably obtained by culturing yeast in a 10-15% carbon source medium at 28-32 ° C under anaerobic conditions.

In the composite fertilizer composition, the carbon source is preferably glucose or sucrose.

The compound fertilizer composition may further contain a stabilizer if necessary. Preferably, the stabilizer is sodium benzoate, potassium benzoate or resorcinol.

The composite fertilizer composition may further include a cryoprotectant if necessary, and the cryoprotectant is preferably at least one selected from propylene glycol and ethylene glycol.

The compound fertilizer composition is preferably applied at a fertilization rate of 0.1 to 1 L / 10 a in a foliar application, and preferably 1 to 3 kg / 10 a in a field application.

The compound fertilizer composition is preferably applied one to two times a week.

The fertilizer composition of the present invention can be used for all kinds of crops and is excellent in quality, thereby promoting the growth of crops and harvesting a lot of high quality crops, thereby increasing the income of farmers, improving the quality of crops, Agricultural competitiveness can be maintained.

FIG. 1 shows the results of investigating the growth of lettuce according to the kinds of microorganisms treated at various concentrations.
Fig. 2 shows the result of investigation of the growth of lettuce according to the treatment of microbial culture filtrate.
Fig. 3 shows the result of investigation of lettuce growth according to the treatment of the microbial mixed culture solution.
Figure 4 is an analytical chromatogram of IAA and IBA of the MGR strain in HPLC.
Fig. 5 shows the results of investigation of the growth of lettuce treated by the cultivation test for the final formulation.
FIG. 6 shows the results of investigation of the growth of the seedling seedlings in the crop cultivation test for the final formulation.
FIG. 7 shows the results of investigation of the growth of blueberry seedlings according to the application amount of the leaves of the final formulation.
FIG. 8 shows the results of investigation of the growth of Cheonggyeongchae of the final formulation and the treatments of the foliar application of other products.
FIG. 9 shows the results of investigation of the growth of cheonggyeongchae of the final formulation in terms of varietal treatment.
FIG. 10 shows the results of investigation of the growth of Cheonggyeongchae, which is a distinction between the final formulation and the cross fertilization treatment of other products.

The fertilizer composition of the present invention comprises a yeast culture liquid as a main component; Corrosion involving corrosion acids and fulvic acid.

1. Yeast culture

Preferably, the yeast culture is obtained by culturing yeast in a 10-15% carbon source medium at 28-32 ° C under anaerobic conditions. As the carbon source, it is preferable to use glucose or sucrose. In a preferred embodiment of the present invention, the cells were cultured at 30 ° C under anaerobic conditions using 12.4% glucose or 12.9% sucrose medium. When cultured under the above conditions, the highest concentration of IAA in the culture was 108.72 ppm (content in product: 29.35 ppm), which was 1.68 × 10 ^-4 M in terms of molar concentration (M) And 3.36 × 10 ^-7 M when used, which was consistent with the IAA concentration of the previous studies reported to promote crop growth. In addition, contamination could be prevented by anaerobic fermentation using the initial high carbon source concentration, and it was also possible to obtain the effect of emitting a fragrant aromatic alcohol odor during the production of the final product.

When the Bacillus subtilis culture and the yeast culture solution were mixed, the growth and yield of the crops were high. However, when the two kinds of microorganisms were mixed with each other, the mutual antagonistic action would result in deterioration of the efficacy. Therefore, Is preferably used.

The yeast culture liquid contains nitrogen, phosphoric acid and potassium as the assurance components. It is preferable that the nitrogen content is 4 to 6 wt%, the phosphoric acid is 1 to 3 wt%, and the potassium is 3 to 5 wt% based on the total weight of the mixed fertilizer composition, and 5 wt% 2% by weight and potassium 4% by weight.

2. Corrosion with corrosion acids and fulvic acid

The corrosion of the present invention containing a corrosive acid and fulvic acid can be carried out by mixing a conventionally used corrosion acid with fulvic acid, and preferably, a method of simultaneously obtaining a corrosion acid and a fulvic acid from the lignite disclosed in Japanese Patent Application Laid-Open No. 10-2015-0105509 It is more preferable to use mixed corrosion of the produced acid with fulvic acid. In the present invention, the term "mixed corrosion" means corrosion involving both a corrosive acid and a fulvic acid.

Hereinafter, the manufacturing method disclosed in the above-mentioned Patent Publication No. 10-2015-0105509 will be briefly described.

First, lignite is used as a raw material for extracting the caustic acid and fulvic acid.

Nitric acid is used as an oxidizing agent to increase the yield of the corrosion acid and fulvic acid by oxidizing the lignite and to control the ratio of the acid to the fulvic acid. It is preferable to use a 10 to 40% nitric acid aqueous solution. In a high concentration nitric acid solution, nitric acid is separated into two oxidizers, nitrogen dioxide and oxygen, and since nitrogen dioxide is a strong oxidizer, the carbon of the organic matter is oxidized.

10 to 40% nitric acid aqueous solution is mixed with the raw lignite so that the ratio of the solid to the aqueous solution (S / L) is 0.5 to 3, and the mixture is reacted at a temperature of 10 to 90 ° C for 2 to 48 hours.

By the above process, it is possible to efficiently produce the good acid and the fulvic acid by changing the carbon of the acid of the raw material and the carbon of the fulvic acid efficiently, and it is possible to obtain the acid and the fulvic acid with high yield.

The raw materials oxidized with nitric acid are titrated to pH 7.5 to 8 by adding an alkali solution to obtain a caustic acid and fulvic acid. As the alkali solution, it is preferable to use a sodium hydroxide solution or a potassium hydroxide solution, but it is not limited thereto.

If necessary, the ash that is not soluble in the alkali solution can be separated through ballast precipitation or a centrifuge.

The solution from which the residue is removed is titrated to pH 1-2 with inorganic acid without drying. As the inorganic acid, nitric acid, hydrochloric acid, sulfuric acid or the like is preferably used, but not limited thereto. When titrated with inorganic acids, the corrosive acid sinks in the form of precipitates and fulvic acid remains in the supernatant. The supernatant containing fulvic acid and the precipitate containing the corrosive acid can be separated by methods such as centrifugation.

When the pH is adjusted to 1 to 2 using sulfuric acid as the inorganic acid, the separated supernatant is treated with an alkali solution to separate the sulfate ion from the fulvic acid. As the alkali solution, it is preferable to use a barium hydroxide solution or the like, but it is not limited thereto.

The alkali solution is added to the separated supernatant to separate the acid ion from the fulvic acid. As the alkali solution, it is preferable to use a barium hydroxide solution or the like, but it is not limited thereto.

When the above-mentioned alkali solution-treated supernatant is dried at 90 to 100 ° C, more preferably at 95 ° C for 8 to 16 hours, more preferably 12 hours, a solid fulvic acid can be obtained. Flavic acid can be detected by absorbance at a wavelength of 465 nm. For example, when 1.5 g / l of fulvic acid is dissolved in water, the optical density is 0.256.

In order to simply separate the fugic acid from the nitrate-oxidized lignite, the lignite is washed with hot water. Fulvic acid dissolves in water, so it dissolves in hot water only in fulvic acid.

Further, the precipitated substance is dried at 90 to 100 캜, more preferably at 95 캜, for 8 to 16 hours, more preferably for 12 hours. After drying, the solid particles are washed with water, leaving only the corrosive acid.

The mixed corrosion of the present invention preferably comprises 20 to 90% by weight of a caustic acid and 10 to 80% by weight of fulvic acid, more preferably 40 to 80% by weight of a caustic acid and 20 to 60% by weight of fulvic acid .

3. Composite fertilizer composition

The fermentation broth with the yeast culture solution is mixed at a weight ratio of 5: 2 to 5: 7, more preferably 5: 4 to 5: 5.

Also, it is preferable that the fermentation broth and the mixed erosion are contained in an amount of 60 to 98% by weight, more preferably 80 to 95% by weight, based on the total weight of the mixed fertilizer composition.

The compound fertilizer composition of the present invention may further comprise an adjuvant. In particular, the compound fertilizer composition of the present invention is microbially used and is in a liquid form, so that it is more vulnerable to high temperatures in summer and low temperatures in winter than solid powder or granular formulations.

Examples of the auxiliary agent include a stabilizer for preventing decomposition of the main component, a surfactant for increasing the dissolution and dispersibility in water, an expanding agent for increasing convenience in use, an electrodeposition agent for increasing the adhesion to the target object, An ultraviolet screening agent for suppressing decomposition by ultraviolet rays when formulating or spraying, a cryoprotecting agent for preventing denaturation due to low temperature freezing in winter, and a sedimentation preventing agent for preventing layer separation by precipitation in a formulation.

As the stabilizer, it is preferable to use sodium benzoate, potassium benzoate or resorcinol having excellent gas generation inhibiting effect, and it is more preferable to use resorcinol considering the pH of the final formulation should be adjusted to 7 . The stabilizer is preferably contained in an amount of 0.5 to 1.5% by weight, more preferably 1% by weight, based on the total weight of the composition.

As the anti-freezing agent, propylene glycol or ethylene glycol is preferably used, and ethylene glycol is more preferably used. The cryoprotectant is preferably contained in an amount of 4 wt% or more based on the total weight of the composition.

The fertilizer composition of the present invention can be appropriately selected and adjusted depending on the type and season of the crop. The amount of the compound fertilizer composition of the present invention is generally in the range of 0.1 to 4 L / 10 A, although it is not limited.

Hereinafter, the present invention will be described in more detail with reference to test examples. The following Test Examples illustrate the present invention, and the scope of the present invention is not limited thereto.

< Test Example  1>

Selection of functional microorganisms

In this study, 10 strains were firstly selected by assaying the auxotrophic growth promoting activity of about 800 strains which are part of many crop growth promoting microorganisms separated from the soil for many years. In consideration of crop growth test and convenience of formulation, two Bacillus subtilis strains, NM1-2 and YM1-1, were selected second. In addition, its Star ^™ Lactobacillus microorganisms (Bacillus subtilis, Accession No. KCTC 13706) and the bioactive substance producing ability is excellent yeast (Saccharomyces S. cerevisiae ) were selected.

< Test Example  2>

Growth of crops according to microbial treatment concentration

The crop cultivation test was carried out in the laboratory for 3 months from December 2013 to February 2014. The fertilizer used in this study was organic fertilizer (Yukbang Gold, Hyosung O & Nbi) and microorganism culture (13706, NM1-2, YM1-1, MGR) And the kinds of microorganisms are shown in Table 1 below.

division nitrogen Phosphoric acid potassium microbe (%) Yuzu Gold (AG) 4 2 One - 13706 - - - Lactostar strain NM1-2 - - - PGPR starter YM1-1 - - - PGPR starter MGR - - - yeast fungus

For the test, seeds of lettuce and bluegrass were planted on a tray on which the horticultural seeds (best soil: Hungong seed) were planted, and the seedlings were maintained for about 3 to 4 weeks. After the passage of time, seeds of same growth condition were settled in each port. After cultivation of the microorganism was completed, the culture of the microorganism was diluted 100 times and the culture was applied once a week for 4 times.

The treatments were classified into three groups according to the type of fertilizer: control (AG), three kinds of Bacillus microorganism treatment (13706, NM1-2, YM1-1) and one kind of yeast treatment (MGR) (10 ⁷ to 10 ¹⁰ cfu / ml) and yeast (MGR) were treated at a concentration of 10 ⁵ to 10 ⁸ cfu / ml, respectively. Five repeated cultivation experiments were carried out using a 4 - inch seedling pot using a fully randomized method. Irrigation during cultivation was carried out once or twice a day. Table 2 shows the treatment contents of each treatment.

Treatment ^* Grace Treatment concentration Kinds Concentration (cfu / ml) (kg / 10a) (cfu / ml) Control (AG) - 600 - 13706 10 ⁷ 600 10 ⁵ 10 ⁸ 600 10 ⁶ 10 ⁹ 600 10 ⁷ 10 ¹⁰ 600 10 ⁸ NM1-2 10 ⁷ 600 10 ⁵ 10 ⁸ 600 10 ⁶ 10 ⁹ 600 10 ⁷ 10 ¹⁰ 600 10 ⁸ YM1-1 10 ⁷ 600 10 ⁵ 10 ⁸ 600 10 ⁶ 10 ⁹ 600 10 ⁷ 10 ¹⁰ 600 10 ⁸ MGR


10 ⁷ 600 10 ³ 10 ⁸ 600 10 ⁴ 10 ⁹ 600 10 ⁵ 10 ¹⁰ 600 10 ⁶ * 13706 is a lactostar strain, NM1-1 and YM1-1 are PGPR selective strains, and MGR is a yeast strain.

1. Soil analysis before testing

The chemical properties of soil before lettuce cultivation were pH, electrical conductivity (EC), organic matter (OM), inorganic nitrogen (Inorg-N), available phosphate (Av-P ₂ O ₅ ), exchangeable potassium (K) and dehydrogenase were analyzed by soil chemical analysis method (National Institute of Agricultural Science and Technology, 1998). The results are shown in Table 3 below.

pH EC O.M. Inorg-N Av-P ₂ O ₅ Ex-K Dehydrogenase (1: 5) (dS / m) (5) (mg / kg) (cmol _c / kg) (Abs _{485 nm} ) 6.23 1.19 6.4 35 135 0.62 69

Soil before lettuce cultivation was pH 6.23, electrical conductivity 1.2 dS / m, organic matter 6.4%, and effective phosphoric acid 135 ㎎ / kg.

2. Soil analysis after lettuce cultivation test

The chemical properties of soil after lettuce cultivation were pH, electrical conductivity (EC), organic matter (OM), inorganic nitrogen (Inorg-N), available phosphoric acid (Av-P ₂ O ₅ ) The dehydrogenase was analyzed in accordance with a soil chemical analysis method (National Institute of Agricultural Science and Technology, 1998). The results are shown in Table 4 below.

Treatment * pH EC O.M. Inorg-N Av-P ₂ O ₅ Ex-K Dehydrogenase Kinds density (1: 5) (dS / m) (%) (Mg / kg) (cmol _c / kg) (Abs _{485 nm} ) Control 5.96 2.41 6.5 119 224 0.74 69 13706 10 ⁷ 6.37 0.71 6.4 28 164 0.76 75 10 ⁸ 6.44 0.73 6.6 28 138 0.82 78 10 ⁹ 6.35 0.66 5.1 28 147 0.82 72 10 ¹⁰ 6.21 1.02 5.2 42 138 0.84 75 NM1-2 10 ⁷ 6.27 0.70 5.6 35 155 0.83 92 10 ⁸ 6.23 0.63 6.0 28 138 0.87 74 10 ⁹ 6.23 0.67 6.8 42 155 0.82 78 10 ¹⁰ 6.18 0.67 6.7 28 181 0.74 75 YM1-1 10 ⁷ 6.27 0.82 7.5 28 138 0.69 95 10 ⁸ 6.27 0.92 5.5 28 173 0.79 105 10 ⁹ 6.30 1.09 7.2 35 147 0.74 79 10 ¹⁰ 6.20 1.28 6.0 35 164 0.78 90 MGR


10 ⁵ 6.42 0.65 6.3 42 147 0.77 80 10 ⁶ 6.42 0.54 6.4 21 147 0.82 84 10 ⁷ 6.39 0.93 6.0 28 155 0.64 92 10 ⁸ 6.35 0.87 7.0 21 173 0.68 88 * 13706 is Lactostar, NM1-1 and YM1-1 are PGPR, and MGR is yeast.

After the cultivation of lettuce, the electrical conductivity, inorganic nitrogen and available phosphorus of the treatments were lower than those of the control, and dehydrogenase was higher than that of the control.

The treatment of organic fertilizer by the application of organic fertilizer 15 days before the lettuce was improved by soil microbial fertilization, the soil microbial activity and the dehydrogenase activity increased, Nitrogen, phosphoric acid and potassium produced in the degradation process were used for microbial growth and crop growth, and the yield was increased.

3. Crop Growth Investigation

The crop growth survey was conducted to investigate the chlorophyll index, leaf number, leaf length, leaf width, biomass, and dry weight of crops harvested at each port after the end of the test. The mean of treatments was tested by Duncan's syndrome test using SPSS 12.0.1, and the results are shown in Table 5 below.

Treatment * chlorophyll ground game Leaf Leaf width In biological In building Kinds density (Mg / 100 cm ² ) (ea / plant) (cm) (g / plant) Control 29.1abc 10.8abc 21.3ab 13.1abc 48.8abc 3.00b 13706 10 ⁷ 27.7bc 9.8bc 18.8c 12.0c 43.5c 3.47ab 10 ⁸ 30.7ab 9.8bc 19.5bc 12.8bc 46.0 bc 3.26ab 10 ⁹ 28.7abc 9.3c 19.9abc 12.8bc 43.7c 3.02b 10 ¹⁰ 31.3a 11.0ab 20.8ab 13.3ab 52.2ab 3.54ab NM1-2 10 ⁷ 30.3ab 11.4a 20.6ab 12.8bc 51.6abc 3.24ab 10 ⁸ 28.8abc 9.8bc 20.7ab 13.1abc 47.1abc 3.02b 10 ⁹ 29.1abc 10.0abc 20.4abc 12.7bc 48.6abc 3.21ab 10 29.0abc 10.6abc 21.0ab 13.0 bc 47.0abc 2.98b YM1-1 10 ⁷ 26.7c 10.5abc 20.6abc 13.3a 53.5ab 3.52ab 10 ⁸ 29.1abc 10.2abc 21.1ab 13.0 bc 48.4b 3.06b 10 ⁹ 29.6abc 10.0abc 21.4ab 13.6ab 53.4ab 3.33ab 10 ¹⁰ 28.1 bc 10.5abc 21.8a 14.2a 54.3a 3.57ab MGR


10 ⁵ 29.4abc 10.5abc 21.7a 13.7ab 50.5abc 3.23ab 10 ⁶ 29.9ab 9.8bc 21.0ab 13.0 bc 48.4abc 3.54ab 10 ⁷ 29.0abc 10.5abc 21.0ab 13.1abc 53.1ab 3.61ab 10 ⁸ 30.7ab 10.8abc 20.9ab 12.9bc 50.6abc 3.79a * 13706 is Lactostar, NM1-1 and YM1-1 are PGPR, and MGR is yeast.

The chlorophyll content of 13706 10 ⁸ cfu / ㎖, 10 ¹⁰ cfu / ㎖, NM1-210 ⁷ cfu / ㎖, MGR of 10 ⁶ cfu / ㎖ and 10 ⁸ cfu / And the number of leaves was increased at 13706 10 ¹⁰ cfu / ㎖ treatment and NM1-2 10 ⁷ cfu / ㎖ treatment. Leaf growth was increased in YM1-1 10 ¹⁰ cfu / ㎖ treatment and MGR 10 ⁵ cfu / ㎖ treatment And the leaf width was increased at 13706 10 ¹⁰ cfu / ㎖ treatment, NM1-2 10 ⁷ cfu / ㎖ treatment and MGR 10 ⁵ cfu / ㎖ treatment. The biomass was increased at 13706 10 ¹⁰ cfu / ㎖ treatment, YM1-1 10 ⁷ cfu / ㎖ treatment, 10 ⁹ cfu / ㎖ treatment and 10 ¹⁰ cfu / ㎖ treatment and MGR 10 ⁷ cfu / ㎖ treatment. In the building, 13706 10 ⁷ cfu / ㎖ treatment, 10 ⁸ cfu / ㎖ treatment and 10 ¹⁰ cfu / ㎖ treatments, NM1-2 10 ⁷ cfu / ㎖ treatments and 10 ⁹ cfu / ㎖ treatments, YM1-110 ⁷ cfu / ml treatment, was increased from 10 ⁹ cfu / ㎖ treatment groups and ¹⁰ 10 cfu / ㎖ treatment, MGR ⁵ 10 cfu / ㎖ treatment, 10 ⁶ cfu / ㎖ treatment, 10 ⁷ cfu / ㎖ treatment and 10 ⁸ cfu / ㎖ treatment.

The results of evaluation of the growth and production of lettuce on the basis of the structure of lettuce are shown in FIG.

MGR, a yeast strain, showed an increase in yields at all treatments when the yield was evaluated based on the dry weight of the building. As the concentration of microbial treatment increased, the yield tended to increase and the fertilizing effect was uniform according to the crop. Respectively. However, NM1-2 and YM1-1 showed no significant fertilization effect depending on the crops and were not suitable as a raw material for functional fertilizer.

< Test Example  3>

microbe In the culture filtrate  Growth of crops

In order to test the growth effect of the crops according to the microbial culture filtrate, the following experiment was conducted.

For the microbial culture filtrate, 13706 and MGR strains were cultured, respectively, and the cells were recovered by centrifugation, and the culture filtrate remaining after the recovery of the cells was used for the test. The cultured broth of 13706 strains and MGR strains were treated for 3 months from December 2013 to February 2014 and the same crops and the same fertilizer as the 'test for crop growth according to microbial treatment concentration' of Test Example 2 were used to cultivate the crop Test. The same seedlings were filled in each pot, and after the planting was completed, the microbial culture filtrate was diluted 200 times and 500 times with distilled water, and fed once a week for a total of 4 times. The samples used in the tests are shown in Table 6 below.

division nitrogen Phosphoric acid potassium microbe (%) Yuzu Gold (AG) 4 2 One - 13706 - - - Lactostar strain MGR - - - yeast fungus

As shown in Table 7, the treatments were as follows: untreated, control (oak gold treatment; AG), 13706 culture filtrate treatment and MGR culture filtrate treatment, 13706 culture filtrate and organic fertilizer mixture treatment, and MGR culture filtrate and organic fertilizer mixture And 4 - inch seedling pots were used for 5 repeated cultivation experiments.

Treatment * Dilution factor
(ship) Grace Culture filtrate (kg / 10a) Non-treatment - - - 13706 Culture filtrate 500 - 2 200 - 5 MGR culture filtrate 500 - 2 200 - 5 Control (AG) - 600 - 13706 Culture filtrate + AG 500 600 2 200 600 5 MGR culture filtrate + AG 500 600 2 200 600 5 * 13706 is lactosteum, and MGR is yeast culture filtrate.

After the end of the test, the chlorophyll index, leaf number, leaf length, leaf width, biomass and building weight were examined. The results are shown in Table 8 below.

Treatment * Dilution factor
(ship) chlorophyll ground game Leaf Leaf width In biological In building (Mg / 100 cm ² ) (ea / plant) (cm) (g / plant) Non-treatment - 16.7c * 0.0c 10.5c 4.9c 5.5c 0.50c 13706
Culture filtrate 500 19.8b 0.0c 11.2c 5.0c 6.4c 0.56c 200 18.3bc 0.0c 11.9c 5.7c 7.3c 0.59c MGR
Culture filtrate 500 18.3bc 0.0c 10.8c 5.0c 5.9c 0.54c 200 17.5 bc 0.0c 10.4c 4.9c 5.9c 0.52c Control (AG) - 25.1a 7.5b 19.4b 11.4b 40.0b 2.60b 13706
Culture filtrate + AG 500 24.6a 9.8a 21.8a 12.4a 48.9a 2.97a 200 25.3a 10.0a 21.8a 12.4a 48.5a 2.89ab MGR
Culture filtrate + AG
500 24.9a 10.0a 21.9ab 12.0ab 49.6a 2.88ab 200 23.5a 9.3a 21.9ab 12.2ab 44.1ab 2.70ab * 13706 was Lactostarum, MGR was yeast, and Duncan Syndaschia ( P <0.01 ).

Growth and yield of lettuce were lower than those of the control, because 13706 culture filtrate and MGR culture filtrate treated with lettuce alone had no effect on the growth of organic matter. However, when the culture filtrate was treated with the organic fertilizer, the yield increased. In particular, the treatment of the 13706 culture filtrate 500 times with the organic fertilizer was 5.9 times higher than that of the control and 14% higher than that of the control. The treatment of the MGR culture 500 times with the organic fertilizer was 5.8 times higher than the control, And 11%, respectively. These results are shown in Fig.

From the above results, it can be seen that the microbial culture filtrate is difficult to replace the organic fertilizer but the crop growth is improved when treated with the organic fertilizer.

< Test Example  4>

Growth of Crops by Microbial Mixing Treatment

In order to investigate the effect of mixing of yeast and Bacillus bacteria on the growth of crops, lettuce cultivation test was carried out in the same manner using the same microorganisms and fertilizers as the above 'Test for Growth of Crops according to Microbial Treatment Concentration'.

The treatments of the treatments are shown in Table 9 below, and the control group was treated with Yubac gold (AG). The microbial content was set at 80% in consideration of the inclusion of adjuvants (surfactants, stabilizers, dispersants, etc.) in the future formulation. The mixed microbial culture was diluted with distilled water and treated once a week for 4 times in total.

Treatment (microbial mixing ratio,%) * Grace Dilution factor (times)
Bacillus genus yeast fungus (kg / 10a) Control 600 - 13706 80 MGR 0 600 100 60 20 600 100 40 40 600 100 20 60 600 100 0 80 600 100 NM1-2 80 MGR 0 600 100 60 20 600 100 40 40 600 100 20 60 600 100 0 80 600 100 YM1-1 80 MGR 0 600 100 60 20 600 100 40 40 600 100 20 60 600 100 0 80 600 100 * 13706 is Lactostar, NM1-2 and YM1-1 are PGPR and MGR is yeast.

1. Growth of lettuce by treatments

After the cultivation of lettuce, the chlorophyll index, leaf number, leaf length, leaf width, biomass and building weight were examined. The results are shown in Table 10 below.

Treatment (Mixing ratio,%) * chlorophyll ground game Leaf Leaf width In biological In building Control 27.5a 9.0c 19.1b 12.7bc 40.0c 2.88 cd Bacillus genus yeast fungus (Mg / 100 cm 2) (ea / plant) (cm) (g / plant) 13706 80 MGR 0 25.6ab ** 10.5abc 22.2a 13.5a 64.3a 4.14a 60 20 25.7ab 10.3abc 21.4a 13.2abc 56.8ab 3.07bcd 40 40 26.1ab 10.5abc 22.2a 13.9a 61.4ab 3.57abc 20 60 25.8ab 10.5abc 21.1ab 13.5a 57.1ab 3.10bcd 0 80 26.3ab 11.1ab 21.7a 13.7a 57.6ab 3.30bcd NM1-2
80 MGR 0 26.6ab 10.3abc 22.2a 13.9a 61.2ab 3.56abc 60 20 25.7ab 10.5abc 22.2a 13.9a 61.5ab 3.59abc 40 40 26.0ab 11.3a 22.7a 14.0a 63.4ab 3.67ab 20 60 25.0ab 10.5abc 22.3a 13.2abc 59.0ab 3.12 acd 0 80 26.3ab 11.1ab 21.7a 13.7a 57.6ab 3.30bcd YM1-1

80 MGR 0 23.5b 9.3bc 21.5a 13.1abc 47.8 bc 2.70d 60 20 24.1b 10.5abc 22.1a 12.7bc 49.1abc 2.90 cd 40 40 25.3ab 11.3a 21.8a 13.8a 64.1a 3.66ab 20 60 24.5ab 10.5abc 21.2ab 12.2c 51.2abc 2.98bcd 0 80 26.3ab 11.1ab 21.7a 13.7a 57.6ab 3.30bcd * 13706 was Lactostar, NM1-1 and YM1-1 were PGPR, MGR was yeast, and Duncan syndrome was positive ( P <0.05 ).

(YM1-1 60% + MGR 20%) and (YM1-1 20% + MGR), which were some of the YM1-1 + MGR treatments, compared with the control, 60%), respectively.

The yields of 13706 and 13706 cultivars were higher than those of 13706 cultivars in the ratio of 1: 1 in 40% of the total cultivars. The lowest.

2. Lettuce Growth by Microbial Mixture Type

When 13706 medium was mixed with MGR medium, the growth and production of lettuce increased with 13706 alone treatment compared to MGR and MGR treatment. When 13706 medium and MGR medium were mixed, the effect was increased by 1: 1 ratio of 40% each, but the growth was not better than that of 13706 medium alone.

When NM1-2 and MGR cultures were mixed, NM1-2 and MGR cultures were treated with NM1-2 and MGR cultures at a ratio of 1: 1, respectively, compared to NM1-2 and MGR cultures alone. But the other mixed treatments showed similar or lower growth and yield than NM1-2 alone. NM1-2 alone showed higher growth and production than MGR alone but the growth and production were lower than 13706 alone.

When the YM1-1 and MGR cultures were mixed, the growth and yield of YM1-1 and MGR were increased by 1: 1 mixture of YM1-1 and MGR by 40%, respectively. Growth and yield of lettuce decreased in YM1-1 treated group compared to MGR medium treated group, 13706 treated group and MGR treated group. These results are shown in Fig.

When two kinds of microorganisms are mixed in the production of functional fertilizer, the efficacy and effect are likely to be lowered due to the antagonistic action between different microorganisms. Considering the efficiency of the production process, it is effective to utilize the single microorganisms rather than the mixed microorganisms do.

< Test Example  5>

Plant growth hormone (IAA) production capacity of microorganisms

In order to confirm the plant growth hormone production ability of the selected four microorganisms, the production ability of indole-3-acetic acid (IAA), which is a kind of plant growth promoting hormone auxin, was evaluated by using Salkowski reagent .

To induce IAA production, microorganisms were inoculated on a King's B medium supplemented with 0.1% of L-tryptophan, which was a precursor, and cultured at 30 DEG C for 24 hours with shaking at 180 rpm. The culture broth was centrifuged at 6,000 rpm for 15 minutes at 4 ° C, and the supernatant was mixed with 1: 2 of Salskovski reagent (50% of 35% HClO ₄ and 1 ml of 0.5 M FeCl ₃ ) and reacted in a dark room for 30 minutes Absorbance was measured at 540 nm. The medium without microbial control was used as a control and IAA concentration was determined by comparing with IAA (Sigma-Aldrich) standard (Gorden, 1951; Son et al., 2012). The results are shown in Table 11 below.

microbe Class name IAA concentration (占 퐂 / ml) 13706 Bacillus subtilis 11.33 NM1-2 Bacillus subtilis 8.15 YM1-1 Bacillus subtilis 8.15 MGR Saccharomyces cerevisiae 19.16

The IAA production of the four microorganisms was highest at 19.16 μg / ㎖ of MGR, and the IAA production of 13706, NM1-2 and YM1-1 of Bacillus genus were 11.33 ㎍ / ㎖, 8.15 ㎍ / ㎖ , 5.22 / / ml.

< Test Example  6>

Tryptophan ( Tryptophan ) IAA Production capacity  Research

To investigate the effect of tryptophan, a precursor of IAA, on the production of IAA, L-tryptophan was added to the King's B medium at 0%, 0.1%, 0.2% and 0.5%, and MGR strain and 13706 strain were inoculated respectively After culturing at 30 DEG C for 72 hours with shaking at 180 rpm, the concentration of IAA was measured in the same manner as in Test Example 5 above. The results are shown in Table 12 below.

microbe Tryptophan concentration (%) IAA concentration (占 퐂 / ml) 13706 0 3.01 0.1 6.77 0.2 9.29 0.5 12.68 MGR 0 2.02 0.1 18.03 0.2 4.76 0.5 5.47

13706 strains of Bacillus spp. Were found to increase the production of IAA as the concentration of tryptophan increased. On the other hand, yeast ( Saccharomyces cerevisiae) strain is the MGR the production of IAA in 0.1% of the amount of tryptophan added was the highest in the higher concentration showed a tendency to inhibit the production of IAA.

< Test Example  7>

The concentration of peptone, IAA Production capacity  Research

Based on the results of Karadeniz (2006) study that the addition of peptone instead of expensive L-tryptophan can increase the productivity of IAA when cultivated with MGR, the productivity of IAA by peptone concentration in sucrose medium selected for industrial culture Respectively.

The MGR strain was inoculated with 6.45% of peptone, 0.5%, 1.0%, 1.5% and 2.0% of peptone, incubated at 30 ° C for 72 hours with shaking at 180 rpm, and the concentration of IAA was measured by the method of Test Example 5 . The results are shown in Table 13 below.

microbe Medium composition IAA concentration (占 퐂 / ml) MGR Sucrose 6.45% + peptone 0.5% 3.22 Sucrose 6.45% + peptone 1.0% 5.69 Sucrose 6.45% + peptone 1.5% 7.32 Sucrose 6.45% + peptone 2.0% 7.37

The effect of peptone on the auxin production of the MGR strain cultured in sucrose medium was investigated. As shown in Table 13, the higher the content of peptone, the higher the production of IAA. However, there was no effect of increasing the IAA production capacity in direct proportion to the peptone content, and it was confirmed that it was preferable to add 1% or less.

< Test Example  8>

HPLC  Using IAA and IBA Production capacity  Research

To investigate the production of IAA and IBA from MGR culture medium using HPLC (High Performance Liquid Chromatography), the following experiment was conducted.

As the culture medium, BHB medium (brain heart broth; 27.5 g of peptone, 2.0 g of D (+) glucose, 5.0 g of NaCl, and 2.5 g of Na ₂ HPO ₄ ) was used. To 100 ml of BHB medium was inoculated 0.4 ml of a culture solution of the isolated strain pre-cultured in NB medium (OD ₆₀₀ was adjusted to be 1), and cultured at 30 ° C for 3 days with shaking at 150 rpm, followed by analysis using HPLC. 100 ml of the shake cultured sample was centrifuged (5,000 × g, 4 ° C., 30 minutes), and the supernatant was recovered to adjust the pH to 2.5. 100 ml of ethyl acetate was added, And extracted three times to recover the ethyl acetate layer. The recovered 300 ml of ethyl acetate was evaporated, dissolved in 10 ml of methanol, and filtered through a filtration membrane having a pore size of 0.2 mu m to use as an analytical sample. The analysis was carried out on a Luna 5μ C18 250 × 4.6 mm column (Phenomenex, USA) with HPLC (FUTECS Co., Ltd. NS2100S). The flow rate was 1 ml / min and 35% methanol in 1% acetic acid was used as the mobile phase and analyzed at a wavelength of 280 nm (Lee and Song, 2007). The results are shown in Table 14, and the analytical chromatogram is shown in FIG.

microbe IAA concentration (占 퐂 / ml) IBA concentration (占 퐂 / ml) MGR 29.21 1.02

As can be seen from FIG. 4, in order to quantitatively determine the IAA and IBA production ability of the MGR strain, the ethyl acetate layer extracted from the culture supernatant was analyzed by HPLC, and the IAA peak was detected between 17 and 18 minutes, and the IBA peak The concentrations of IAA and IBA were 29.21 ㎍ / ㎖ and 1.02 ㎍ / ㎖, respectively.

IAA and IBA were analyzed by HPLC. However, IBA concentration was so low that precise quantitative analysis was difficult, IAA and IBA of the culture medium were lost during the pretreatment process of various stages, and analysis time Fell. Therefore, it was confirmed that it is faster and more accurate to use the color reaction using Salcovsky reagent to analyze IAA.

< Test Example  9>

Carbon source  Concentrations and culture conditions of IAA Production capacity  Research

The effect of sucrose concentration on the production of IAA was investigated in sucrose medium selected as an industrial medium for MGR strain culture. In addition, IAA production ability was compared between the stationary cultures and the shaking cultures in view of the fact that the production amount of aromatic substances including various alcohols increased when anaerobic culture conditions of yeast cells were changed by stationary culture.

0.5% of peptone was added to sucrose 6.45%, 12.9% and 25.8%, and the strain MGR was inoculated. The strain was incubated at 30 ° C with shaking at 180 rpm, and the IAA concentration was measured by the method of Test Example 5 on the first day and the third day. The results are shown in Table 15 below.

Sucrose concentration (%) Culture method IAA concentration (占 퐂 / ml) Day 1 Day 3 6.45 Anaerobic culture 9.35 98.36 Aerobic culture 35.25 70.77 12.9 Anaerobic culture 1071 108.72 Aerobic culture 55.19 74.04 25.8 Anaerobic culture 10.65 102.70 Aerobic culture 63.06 73.77

As a result of measuring the IAA production ability according to the carbon source concentration and culturing conditions of the MGR strain, the IAA concentration was high in the shaking culture on the first day of culture, but it was detected in the high culture condition on the third day. In addition, the carbon source concentration was 108.72 占 퐂 / ml at the third day in the medium supplemented with 12.9%.

 In the conventional MGR culture, a medium having a carbon source concentration of 6.4% is used. However, when the carbon source concentration is doubled, the IAA production is the highest, and the effect is increased by about 10.5%. However, in the medium with the carbon source concentration of 4 times (25.8%), the production amount was slightly increased, and the increase of the medium cost and the stabilization of the fermentation were insufficient, resulting in generation of gas even after 3 days.

< Test Example  10>

Selection of stabilizers

Lactivation using microorganisms or microbial metabolites causes odor and gas due to metabolism by microorganisms or denaturation and decomposition of internal materials over time. In order to prevent such denaturation and degradation, a method of inhibiting the metabolism by microorganisms by using an antiseptic or a protein chelating agent during the production of the liquor by inhibiting the action of the enzyme is used.

To determine the type and concentration of the stabilizer, the medium containing 257 g / L of glucose, 5 g / L of peptone and 5 g / L of soybean meal was autoclaved, and then the MGR strain was inoculated and cultured at 30 ° C. for 24 hours under the maximum condition of gas evolution . Sodium benzoate, potassium sorbate, etc., which are widely used as microbial stabilizers after placing 60 ml of the MGR culture under these conditions in a 100 ml HDPE (High Density Poly Ethylene) , 0.1%, 0.5%, 1%, 3% and 5%, respectively, of sodium propionate, sodium-EDTA, methyl paraben and resorcinol, %, And incubated at 30 ° C for 24 hours, and odor and gas evolution were observed over time at intervals of 1 day. The results of confirming the gas evolution of the MGR culture liquid according to the kind and content of the stabilizer are shown in Table 16 below.

content
(%) Stabilizer type Sodium
Benzoate Potassium
Sorbate Sodium
Propionate The sodium-
EDTA methyl
Paraben Resorcinol Control 0.1 ++ ++ ++ ++ ++ ++ ++ 0.5 ++ ++ ++ ++ ++ ++ 1.0 + ++ ++ ++ ++ + 3.0 - - ++ ++ ++ - 5.0 - - - + ++ - * Degree of expansion: ++ severe, + weak, - no change.

As a result of measuring the gas generation pattern of MGR culture liquid according to the type and content of stabilizer, it was confirmed that no gas was generated when 3% or more of sodium benzoate, potassium sorbate and resorcinol were added. It was expected that sodium-EDTA, which was previously used in our laboratory as a stabilizer, and methylparaben, which was widely used as a preservative, would be effective at low concentrations. However, even if 5% was added to the culture medium in which the gas evolution of the MGR culture reached its maximum value It was confirmed that there was no effect. .

< Test Example  11>

Stabilizer effect by pH

Considering that the metabolic activity is rapidly lowered at a low pH, the microorganism is the same as the experimental method of Test Example 10 to select an optimal stabilizer capable of suppressing gas generation while reducing the amount of the expensive stabilizer, The content of sodium benzoate, potassium sorbate, and resorcinol was varied, and the pH of the culture was adjusted to 4, 5, 6, and 7, followed by incubation at room temperature for 24 hours. The odor and gas evolution were observed at intervals of one day, and the results are shown in Table 17 below.

stabilizator content(%) Degree of expansion * pH 4 pH 5 pH 6 pH 7 No treatment - ++ ++ ++ ++ Potassium sorbate 0.3 ++ ++ ++ ++ 0.5 - ++ ++ ++ One - - + ++ Sodium benzoate 0.3 + ++ ++ ++ 0.5 - ++ ++ ++ One - ++ ++ ++ Resorcinol 0.3 ++ ++ ++ ++ 0.5 ++ ++ ++ ++ One + + + + * Degree of expansion: ++ severe, + weak, - no change.

Sodium benzoate, potassium sorbate and resorcinol were added to the culture at concentrations of 0.3%, 0.5% and 1%, and the pH was adjusted to 4, 5, 6, and 7, respectively. As a result, it was confirmed that the gas generation was suppressed even with a low concentration of potassium sorbate and sodium benzoate as the pH was low.

Considering that the pH of the liquid fertilizer added with the final mixed corrosion should be adjusted to 7, it was found that the use of resorcinol with less gas evolution at pH 4 ~ 7 was most preferable when 1% was added as the stabilizer.

< Test Example  12>

Freezing agent selection

When freezing the microbial and microbial metabolic product liquids at low temperatures in winter, it is necessary to add a cryoprotectant in order to prevent the decrease of the microbial count and the precipitation due to the denaturation of the components.

The MGR medium, which has a high content of about 26.5% in the medium, is not easily frozen at low temperatures. Therefore, the culture medium of 13706 strain, which is easily frozen due to low content of the medium in the medium, is used as a culture solution for selection of the cryoprotectant. (Ethylene glycol) and propylene glycol (propylene glycol), which have been widely used as antifreeze agents since they are not toxic to microorganisms.

Ethylene glycol and propylene glycol were added to 10 ml of 13706 culture medium in an amount of 1 to 10% and placed in a 15 ml tube. Freezing was confirmed at 5 ° C, -10 ° C and -13 ° C, and the results are shown in Table 18 .

Kinds Temperature
(° C) Cryoprotectant content (%) * One 2 3 4 5 6 7 8 9 10 Ethylene glycol -5 × × × × × × × × × × -10 ○ ○ ○ × × × × × × × -13 ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ Propylene glycol -5 × × × × × × × × × × -10 ○ ○ ○ ○ ○ × × × × × -13 ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ * ○: Freezing, ×: Non-freezing.

As a result, it was found that neither ethylene glycol nor propylene glycol was frozen at 5 ° C, and ethylene glycol was not frozen at 4% or more and propylene glycol was frozen at 6% or more at -10 ° C. At -13 ℃, all treatments containing 10% or less of freezing agent were frozen. Therefore, ethylene glycol was found to be more effective in preventing freezing than propylene glycol.

< Test Example  13>

Toxicity to microbial strains

Three sodium benzoate, potassium sorbate and resorcinol stabilizers and ethylene glycol, which is a cryoprotectant, were added to the formulation to test for toxicity to microorganisms.

13706 strains belonging to the fungi and MGR strains belonging to the fungus were cultured in YM medium for 24 hours, 40% of the culture medium, 5% of ethylene glycol, 54% of water, and 1% of sodium benzoate, potassium sorbate or resorcinol The mixture was allowed to stand at room temperature, and then diluted on NA medium and YM medium for 5 days and 7 days, and the viable cell count was measured. In addition, a control was added to the culture solution in which the sterilized water only was added to the control solution and the pH of the control solution was adjusted to 4 to inhibit microbial growth. The results are shown in Table 19 below.

Incubation period Processing contents Viable cell count (cfu / ml) 13706 7248 0 days Microbial culture stock solution 1.27 x 10 ⁷ 3.43 × 10 ⁹ 5 days The control (pH 4) 9.60 × 10 ⁶ 8.77 × 10 ⁸ Non-treatment 7.75 × 10 ⁶ 1.88 × 10 ⁹ Sodium benzoate 8.30 × 10 ⁶ 4.35 × 10 ⁷ Resorcinol 1.38 × 10 ⁷ 2.71 × 10 ⁸ Potassium sorbate 1.32 x 10 ⁷ 9.38 × 10 ⁸ 7 days The control (pH 4) 7.65 × 10 ⁶ 4.15 x 10 ⁸ Non-treatment 5.60 × 10 ⁶ 1.38 × 10 ⁹ Sodium benzoate 1.10 x 10 ⁷ 3.30 × 10 ⁷ Resorcinol 1.13 x 10 ⁷ 1.43 x 10 ⁹ Potassium sorbate 9.45 × 10 ⁶ 8.71 × 10 ⁸

Microbial toxicity test showed that the viable cell count was high even after 7 days of treatment with resorcinol, and that potassium sorbate was less toxic to 13706 and MGR. In the case of sodium benzoate, there was no toxicity to 13706 belonging to the fungus, and the MGR strain belonging to the fungus was weakly toxic, indicating that the bacterial concentration dropped to 1/100 level on the third day. However, after 7 days, the bacterial concentration was not decreased any more, so that the toxicity to the immature cells appeared at the beginning of the experiment, and the toxicity to the mature cells was not serious.

< Test Example  14>

Crop Cultivation Test

(1) Preparation of formulations

The following formulation was prepared by mixing the microbial culture with mixed corrosion (70% by weight of caustic acid + 30% by weight of fulvic acid)

Preparation Example 1: 50% by weight of the microorganism culture + mixed corrosion 43% by weight (MH)

Production Example 2: 45% by weight of microorganism culture + mixed corrosion 43% by weight + 5% by weight amino acid (MHA)

Production Example 3: Including Assurance Component 50% by weight of microbial culture + mixed corrosion 43% by weight (MH524)

The amino acid of Preparation Example 2 was a blood amino acid, and the culture broth of Preparation Example 3 was adjusted to include 5% by weight of nitrogen, 2% by weight of phosphoric acid and 4% by weight of potassium.

(2) Test method

The crop cultivation test was carried out in the green house for Hyosung O & N eco-friendly lab for three months from February 2014 to April 2014. The disclosed crops, the disclosed fertilizers and the test methods were the same as those of the 'Test for Growth of Crops According to Microbial Treatment Concentration' in Test Example 2, and each liquid was diluted with distilled water and treated once a week for 4 times in total.

As shown in Table 20 below, the microorganism control (M8, K company), the 4th type compound fertilizer control (NA, D company), Production Example 1 (microorganism + mixed corrosion), Production Example 2 Corrosion + amino acid) and Production Example 3 (microorganism + mixed corrosion, 5-2-4 correction + guaranteed trace element) were used.

division nitrogen Phosphoric acid potassium microbe (%) Microbial + mixed corrosion MH - - 0.6 yeast fungus Microbial + mixed corrosion + amino acid MHA - - 0.7 yeast fungus Microbial + mixed corrosion (5-2-4 calibration) MH524 5 2 4 yeast fungus Compound fertilizer control NA (D company) NA 6 3 4 - Microbial control M8 (K) M8 - - - Bacillus genus

Production Example 1 (MH), Production Example 2 (MHA) and Production Example 3 (MH524) were classified into a quantitative treatment (2 kg / 10 a) and a dose treatment (4 kg / 10 a) according to the application amount. The treatment contents of each treatment are shown in Table 21 below.

Treatment Grace Functional fertilizer (fertilizer) * Remarks (kg / 10a) Control (AG) 600 - AG AG + M1 600 2 MA-8 AG + Na-D 600 1.7 Na-D AG + MH 250 times 600 4 MH 500 times 600 2 2MH AG + MHA 250 times 600 4 MHA 500 times 600 2 2MHA AG + MH524 250 times 600 4 MH524 500 times 600 2 2MH524

The 4-inch seedling pot was used to formulate the crops. Each treatment was carried out in a completely randomized manner (6 replicates). In the irrigation and growth survey during the cultivation period, the test method was as follows: Black '.

1. Lettuce  Cultivation test

(1) Growth of lettuce grown by treatments

The results of cultivation of lettuce by each treatment are shown in Table 22 and FIG.

Treatment * chlorophyll ground game Leaf Leaf width In biological In building (Mg / 100 cm 2) (ea / plant) (cm) (g / plant) Control (AG) 26.5ab 8.5bc 13.0abcd 9.6ab 41.8abc 4.16abc M8 27.0ab 7.8bc 12.6bcd 9.2abc 41.1 bc 4.11abc NA 24.3b 8.1bc 12.2d 8.5bc 36.9cd 3.98bc MH MH 500 times 25.7ab 7.6bc 12.3cd 8.3bc 37.2cd 4.07bc 2MH MH 250 times 24.3b 5.2c 12.1d 7.7c 29.0d 3.29c MHA MHA 500 times 25.8ab 9.8ab 13.0abcd 9.3abc 43.0abc 4.62ab 2MHA MHA 250 times 27.1ab 11.3ab 13.9a 10.6a 52.1ab 5.06ab MH524 MH524 500 times 26.7ab 11.4ab 13.7ab 9.8ab 51.2ab 4.83ab 2MH524 MH524 250 times 29.2a 12.3a 13.5abc 10.5a 53.8a 5.26a M: Microorganism (45%) + Mixing: AG: Control, M8: Control of microorganism (K) Corrosion (43%) + amino acid (5%), MH524: Microorganism containing assurance component (50%) + mixed corrosion (43%), Duncan syndrome test ( P <0.05 ).

As shown in Table 22, when the lettuce growth of the three kinds of prototypes was compared with that of the control, the chlorophyll index was 2MH524, the leaf number and the dry matter were MHA, 2MHA, MH524 and 2MH524, , MH524 and 2MH524, respectively.

The highest yields were obtained from MH524 (MH524 250), and the yields were higher than those of control (AG) in HA, 2MHA, MH524 and 2MH524 treatments. However, the MHA treatment showed the growth promoting effect on lettuce. However, the precipitation occurred due to the mixing corrosion and the reaction of amino acid during the manufacture of the product, and the gas was generated by the decomposition of the amino acid by the microorganism even though the stabilizer was added. There was no.

(2) Between microbial fertilizers  Comparison (M8, MH , MHA )

The chlorophyll index, leaf number, leaf width, leaf width, biomass, and dry matter in the lettuce after MH fertilization were similar or slightly lower than those of M8 in comparison with the fertilizing effect of the microorganism control fertilizer M8 (K) and the preparation example 1 (MH) . In addition, the growth and production of crops were decreased in 2MH (treatment) than in MH (control) treatment, which is considered to be a result of nutrient competition between crops and microorganisms due to high microbial concentration. When comparing the fertilizing effect of the microorganism control fertilizer M8 with that of Production Example 2 (MHA), the growth and production of lettuce leaf, leaf width, leaf weight, biomass, and dry weight of the lettuce were higher than those of M8 (MHA) and 2MHA Respectively.

(3) Four species Between compound fertilizers  Comparison (Na, MH524)

(MH524) was higher in chlorophyll index, leaf number, leaf width, leaf width, living organism, and building than the control NA (D5) In comparison, the number of leaves, leaves, leaf width, biomass, and dry matter were higher in the treated plants.

2. Cheonggyeongae  Cultivation test result

(1) Treatment distinction Serpentine  Growth survey result

Table 23 and FIG. 6 show the results of growing the seedlings in each treatment plot.

Treatment * chlorophyll ground game Leaf Leaf width In biological In building (Mg / 100 cm 2) (ea / plant) (cm) (g / plant) Control (AG) 41.1cd 7.6ab 14.8ab 6.8abc 50.0 bc 5.38bc M8 40.3d 7.0ab 14.2ab 6.7bc 47.4c 5.15bc NA 43.4abcd 8.8a 14.7ab 6.9ab 56.0abc 5.30bc MH MH 500 times 47.7a 9.0a 15.1a 7.2ab 68.6a 6.61a 2MH MH 250 times 48.4a 8.0a 14.8ab 7.1ab 58.5abc 5.81ab MHA MHA 500 times 41.7 bcd 5.8b 13.3b 6.3c 45.0c 4.44c 2MHA MHA 250 times 45.8abcd 7.8ab 14.9a 6.6bc 53.0 bc 5.38bc MH524 MH524 500 times 46.8ab 8.7a 14.8ab 7.3a 62.7ab 5.82ab 2MH524 MH524 250 times 46.4abc 8.2a 15.1a 7.0ab 61.9ab 5.83ab M: Microorganism (45%) + Mixing: AG: Control, M8: Control of microorganism (K) Corrosion (43%) + amino acid (5%), MH524: Microorganism containing assurance component (50%) + mixed corrosion (43%), Duncan syndrome test ( P <0.05 ).

2, 2MHA, 2MHA and 2MH524, leaf width was Na, MH, 2MH, MHA, 2MHA and 2MH524, leaf width was Na- In the case of D, MH, 2MH, MH524 and 2MH524, biomass was increased in the MH, 2MH, MH524 and 2MH524 in NA, MH, 2MH, 2MHA, MH524 and 2MH524.

MH, 2MH, MH524, and MH524 were the most effective treatments for MH, respectively, when the production was evaluated based on the weight of the building. 2MH524 treatment. MH treatment increased the growth of the seedlings, but decreased in the treatments, indicating that the growth rate of the crops according to the application amount was large, whereas the MH524 showed excellent growth without the variation of the crop growth according to the application amount.

(2) Between microbial fertilizers  Comparison (M8, MH , MHA )

The yields of chlorophyll index, leaf number, leaf width, leaf width, biomass, and biomass were higher than those of M8 in the case of MH fertilization after comparing with the fertilizing effect of the microorganism control fertilizer M8 (K) and the preparation example 1 (MH). In addition, the growth and yield of crops were decreased in 2MH (treatment) than in MH (control) treatment, which is considered to be the result of nutrient competition between crops and microorganisms due to high microbial concentration.

When comparing the fertilizing effect of the microorganism control fertilizer M8 with that of the production example 2 (MHA), the growth and production including the leaf number, leaf area, living organism, and building weight of the blueberry plant were increased in the MHA (quantitative) and 2MHA Respectively.

(MHA), but the yield was lower than that of MH or 2MH treatment. Therefore, the effect of the addition of amino acid on growth was not observed.

(3) Comparison among four kinds of compound fertilizers (Na, MH524)

The chlorophyll index, leaf number, leaf width, leaf width, biomass, and dry matter were increased in the treatments of Preparation Example 3 (MH524) compared to NA (Control D) When 2MH524 (Dose) was compared, the growth and yield of bluegrass were not significantly affected by fertilizer application.

3. Results

The fertilizing effect of lettuce and cheongchyeongchae of three kinds of preparations produced by mixing starch microorganism, mixed corrosion and amino acid with crop growth promoting effect is as follows.

Crop growth and yields were higher in MHA, 2MHA, MH524 and 2MH524 than in control (AG) in both lettuce and bluegrass. The growth and yield of crops were increased in MH, 2MH, MH524 and 2MH524 than control. The treatments in which the growth and production of the crops increased in the lettuce and the bluegrass were higher than those in the MH524 and 2MH524 treatments.

The growth and yield of crops were increased compared to that of control (M8) in both of the two crops, which was a mixed fertilizer mixed with microbial culture and mixed corrosion. However, the difference in growth and production was significant depending on the type of crop, It was not suitable as an applicable functional fertilizer.

In addition, in the case of Production Example 2 (MHA) in which microorganism culture, mixed corrosion and blood amino acid were mixed, the growth and yield of the crops were higher than that of the control M8, It was not suitable as a functional fertilizer because heavy gas and sediment were continuously generated and the safety of the product could not be secured.

On the other hand, Production Example 3 (MH524) containing 5%, 2% and 4% of nitrogen, phosphoric acid and carly, respectively, after mixed with microbial culture and mixed corrosion increased the crop growth and yield of the two crops compared to NA , The variability of the fertilizer according to the crops was small, so that both the lettuce and the bluegrass were able to produce high quality crops. In addition, no precipitation or gas was generated during product manufacture and storage, and it was stable even after long-term storage.

Of the three tested prototypes, the best fertilizer for growing crops was Type 4 compound fertilizer (for leaf surface) produced by mixing mixed microbial cultures with the assurance components in Mixture 3 in Production Example 3. The recommended dose of prototype 3 obtained from the test results was 2kg / 10a (300 pyeong), and the application period was appropriate once a week.

< Test Example  15>

Of the final formulation  Growth of crops according to fertilization and fertilization method

The growth of the crops according to the application amount and the fertilization method of the final formulation, Production Example 3 (hereinafter referred to as "Manchun"), was confirmed by comparison with other products.

The test was carried out in the green house for testing of environmentally friendly laboratories for 2 months from June 2014 to July 2014, and the public crops were selected from Cheonggyeongchae (Nongwoo Cheongcheongjeong, Nongwoo Bio) (C), IS (I), and KI (U), which are amino acid fertilizers, and M8 (K), a microbial fertilizer (NA) And the ingredient contents and characteristics of each fertilizer are shown in Table 24 below.

division nitrogen Phosphoric acid potassium Remarks (%) Marbled gold 4 2 One - Enchant 5 2 4 Yeast + mixed corrosion NA 6 3 4 Type 4 compound fertilizer M8 - - - Bacillus CO - - - amino acid IS 6 - - amino acid KI 10 3 4 amino acid

The crop cultivation test was carried out in the same manner as in the test of "Growth of crops according to microbial treatment concentration" in Test Example 2, and each of the liquids was diluted with distilled water, .

The treatments were as follows: control (brown gold treatment), microbial fertilizer control (M8), type 4 compound fertilizer control (NA), amino acid fertilizer treatment (CO) The fertilizer was fertilized on the basis of fertilizer. In order to test the growth of crops according to the amount of application, Manchurong was divided into two groups: semi - treated (1,000 times), quantitative (500 times), double (250) and quadrupled (125) In order to evaluate the effect according to the method, we compared the crop growth effect according to foliar fertilization and cultivation.

Table 25 shows the contents of the treatments for confirming the crop growth effect by the foliar fertilization, and Table 26 shows the contents of the treatments for confirming the crop growth effect by the cultivation of the rivers.

Treatment Grace Functional fertilizer * Remarks (kg / 10a) No treatment - - - Control 600 - - M8 600 0.4 Bacillus NA 600 0.4 Type 4 compound fertilizer CO 600 0.4 amino acid IS 600 0.2 amino acid KI 600 0.4 amino acid Only 600 0.2 1000 times Quantification 600 0.4 500 times Twice the full capacity 600 0.8 250 times Quadruple 600 1.6 125 times * Functional fertilizer fertilization of other company is applied with recommended fertilizer of product.

Treatment Grace Functional fertilizer * Remarks (kg / 10a) No treatment - - - Control 600 - AG M8 600 2 Bacillus NA 600 2 Type 4 compound fertilizer CO 600 2 amino acid IS 600 One amino acid KI 600 2 amino acid Only 600 One 1000 times Quantification 600 2 500 times Twice the full capacity 600 4 250 times Quadruple 600 8 125 times * Functional fertilizer fertilization of other company is applied with recommended fertilizer of product.

4-inch seedling pots were used to plant the crops. Each treatment was carried out in a completely randomized manner (5 repetitions). The irrigation and growth test during the cultivation period was carried out in the same manner as in Test Example 2, Black '.

One. On foliar fertilization  Effect of crop growth

(1) Results of crop growth survey

Table 27 shows the results of examining the growth of the cheonggyeongseong after fertilizing the third party product according to the application amount and fertilizing method.

Treatment chlorophyll ground game Leaf Leaf width In biological In building (mg / 100 cm ² ) (ea / plant) (cm) (g / plant) No treatment 35.2c ^* 0.0d 9.1d 4.1c 7.6e 0.81c Control 41.3ab 10.0c 15.2c 7.7b 51.7d 4.27b M8 41.9ab 11.8ab 16.4bc 8.0ab 63.2c 4.34b NA 39.5b 8.6c 16.5c 7.6b 63.5c 4.38b CO 45.7a 12.2a 17.7ab 8.3ab 70.6abc 4.51ab IS 44.3a 12.2a 16.3bc 8.6a 79.7a 4.60ab KI 45.1a 12.2a 17.0ab 8.4ab 67.0 bc 4.53ab Only 43.2ab 10.6ab 16.5 bc 8.4ab 63.8c 4.49ab Quantification 43.9ab 11.2ab 16.5 bc 8.6a 72.7abc 4.68a Lot 43.5ab 11.6ab 18.0a 8.6a 76.0ab 4.68a Quadruple 42.7ab 10.8ab 17.4ab 8.3ab 62.4c 4.59a * Duncan syndrome test ( P <0.05 )

 The chlorophyll, leaf number and leaf width of M8, CO, IS, KI, and M8, NA, CO, IS, KI, and total manganese (half, fixed, multiply, quadrupled).

 Leaf length and leaf width were similar to or slightly higher than those of amino acid treatments, and those of dry leaves were higher than those of amino acid treatments. Growth and yield of the crops were higher than that of the other functional fertilizers.

(2) Enchant Foliar fertilization  Determination of proper dosage

In order to determine the optimum fertilizing rate for the application of mungtong, it was investigated the production amount of bluegrass bran according to the application amount. The highest treatments in the building were the Manchurian quantitative treatments and the dose treatments. The recommended fertilizer application was evaluated as a fertilization treatment with a 500 - fold dilution and foliar application. At this time, the recommended foliar application rate of mungtong was 0.4ℓ / 10a, and the fertilization period was once a week.

(3) Only through  Third-party Foliar fertilization  Comparison of crop growth

Fertilizer application of foliar application of mungtong showed that the yield of crops was higher than that of control. Compared with the control group, the amount of other fertilizers increased by 1.6 ~ 7.1% compared to the control group, and the increase was 9.6% higher than that of the control group (Figure 8). Compared with other farm products and crop production (in buildings) based on the recommended fertilizer rate of 500 times, the NKW increased about 1.7 ~ 7.8%.

2. In the township fertilization  Effect of crop growth

(1) Results of crop growth survey

Table 23 shows the results of the growth survey on the seedlings with other products after fertilizing the seedlings according to the application amount and fertilizing method.

Treatment chlorophyll ground game Leaf Leaf width In biological In building (mg / 100 cm ² ) (ea / plant) (cm) (g / plant) No treatment 35.2d * 0.0c 9.1d 4.1b 7.6e 0.81e Control 41.3bc 10.0b 15.2c 7.7a 51.7d 4.27bcd M8 40.8c 10.8ab 16.5abc 7.7a 60.9d 4.22cd NA 44.4abc 11.6ab 15.8bc 8.1a 66.4bcd 4.17d CO 43.8abc 10.2ab 16.3abc 8.2a 65.5cd 4.05d IS 43.6abc 9.8b 16.9abc 8.6a 73.1abc 4.62abcd KI 45.9a 11.4ab 17.0abc 8.5a 77.6ab 4.80abc Only 41.5 bc 12.2ab 17.8a 8.6a 77.5ab 4.84ab Quantification 45.3ab 13.2a 17.9a 8.1a 80.0a 4.89a Lot 43.2abc 12.2ab 17.5ab 8.6a 77.4ab 4.89a Quadruple 46.9a 12.2ab 18.2a 8.3a 78.0a 4.83ab * Duncan syndrome test ( P <0.05 )

The chlorophyll and leaf widths were compared with those of the control, and the number of leaves was M8, NA, CO, KI, and total treatments (half, dose, (4 times the amount), the leaves and the biomass were treated with M8, NA, CO, IS and KI, and the treatments were IS, KI, , 4-fold).

 The growth and production of blueberry seedlings were improved in all treatments, such as leaf, leaf, biomass, and building, compared with other fertilizer treatments.

(2) Enchant Cross-fertilization  Determination of proper dosage

In order to determine the optimum fertilizer application rate when manchae were applied to the myeongchong, the amount of production of blueberry seedlings was increased by 13.1 ~ 14.5% than that of the control plot (Fig. 9). The highest amount of the treatments was Manchurian quantitative treatment and dose treatment, and the recommended fertilizer dose was evaluated as a quantitative treatment with dilution of 500 times. At this time, the recommended gypsum application rate was 2ℓ / 10a, and the fertilization cycle was once a week.

(3) Only through  Third-party Cross-fertilization  Comparison of growing crops

As a result of comparisons of the mushroom cultivars with the other crops and the cheonghyeongchae, the crop yields of IS, KI and Manchon were higher than those of the control. IS and KI increased by 8.2% and 12.4%, respectively, while those of Manchon were 14.5% higher than those of control (Figure 10). The yields of Manchon were increased by 5.8% and 1.8%, respectively, compared to IS and KI, and the yield of crops increased when manchuria were foliated.

Claims (10)

A composite fertilizer composition comprising a microbial component and an organic component,
The microbial component is composed of a yeast ( Saccharomyces cerevisiae ) culture,
Wherein the organic material component comprises mixed corrosion comprising a corrosive acid and fulvic acid,
The yeast culture broth and mixed corrosion thereof at a weight ratio of 5: 2 to 5: 7,
Wherein the yeast culture liquid comprises 4 to 6% by weight of nitrogen, 1 to 3% by weight of phosphoric acid and 3 to 5% by weight of potassium based on the total weight of the composition. The method according to claim 1,
Wherein the yeast culture solution and the mixed corrosion are contained at a weight ratio of 5: 4 to 5: 5. The method according to claim 1,
Wherein the mixed corrosion comprises 20 to 90 wt% of a caustic acid and 10 to 80 wt% of fulvic acid. 4. The method according to any one of claims 1 to 3,
Wherein said yeast culture broth and mixed erosion are comprised between 80 and 95 wt% based on the total weight of the composition. 4. The method according to any one of claims 1 to 3,
The step of oxidizing the fatty acid and the fumaric acid comprises adding 10 to 40% of nitric acid solution to the lignite so that the ratio of the solid to the aqueous solution (S / L) is 0.5 to 3, and the reaction is carried out at 10 to 90 ° C for 2 to 48 hours &Lt; / RTI &gt; wherein the composite fertilizer composition is prepared by a method comprising: 4. The method according to any one of claims 1 to 3,
Wherein the yeast culture broth is obtained by culturing yeast in a 10-15% carbon source medium at 28-32 ° C under anaerobic conditions. The method according to claim 6,
Wherein the carbon source is glucose or sucrose. 4. The method according to any one of claims 1 to 3,
Wherein the composition further comprises a stabilizer. 9. The method of claim 8,
Wherein the stabilizer is sodium benzoate, potassium benzoate or resorcinol. 4. The method according to any one of claims 1 to 3,
Wherein the composition further comprises at least one cryoprotectant selected from propylene glycol and ethylene glycol.

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