JPH0712996B2 - Organic fertilizer manufacturing method - Google Patents

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for producing an organic fertilizer which uses livestock manure, human excrement, vegetable fiber substances and hard proteins as nitrogen sources.

[0002]

2. Description of the Related Art At present, animal organic materials such as livestock excrement and human excrement are often treated in septic tanks and are not often used as fertilizers. Since ancient times, it has been known that these manures are decomposed and ripened by using microorganisms in the natural world to be used as fertilizers.
And a strong odor is generated.

[0003]

SUMMARY OF THE INVENTION The problem to be solved by the present invention is to solve these problems in the past and to rapidly ripen animal organic materials such as livestock excrement, human excrement, hoof horn and leather scraps. An object of the present invention is to provide a method for producing an organic fertilizer that can be decomposed and produce a high-quality organic fertilizer with less bad odor.

[0004]

Means for Solving the Problems The gist of the present invention which has solved the above problems is as follows: 1) Clostriduim thermocem SK522 (Clostriduim thermocem)
llum biovar.SK522, Micro Engineering Research Center No. 3459)
And Thermus Aquatics SK542 described in 2 below
(Thermus aquaticus biovar SK542, Mikokenjoyori No. 3382) and a mixed culture obtained by symbiotic mixed culture in a thermophilic environment of 60 ° C. or higher, or a fermentative decomposing agent containing the mixed culture as an active ingredient. Manufacture of organic fertilizer characterized by adjusting the water content to the required level while mixing it with organic materials and animal organic materials such as human waste and excrement, and fermenting the organic materials by ripening and fermenting them with a mixed culture or fermentation decomposition agent. Method 1 has a lignin solubilizing ability, a suitable growth temperature is 65 to 72 ° C.,
A thermophilic fibrin-degrading bacterium that grows in the temperature range of 40 to 80 ° C. and ferments actively, Clostriduim thermocellum biovar.S
K522, Mikori Kenjo No. 3459) Note 2 Absolute aerobic, optimum growth temperature is 72 to 76 ° C, 40 to 82 ° C.
It grows in a medium of normal concentration in the temperature range of
Thermos aquaticus SK542 (Thermus aquaticus biovar S) which produces a proteolytic enzyme and a carotenoid-type yellow pigment with a thermophilic broad-range action hydrogen ion concentration of 85 ° C and a action hydrogen ion concentration pH = 4.0 to 11.3
K542, Micro Engineering Research Center No. 3382).

The thermophilic fibrinolytic bacterium used in the present invention, Clostridium thermocellum SK522 (Clostr
iduim thermocellum biovar. SK522, Micro Engineering Kenjoyori No. 3459) and Thermus Aquatics SK542
(Thermus aquaticus biovar SK542, Microtechnical Research Institute No. 3382) are all related to the discovery of the present inventors. In the following, they are simply referred to as SK522 strain and SK542, respectively.
It is called a strain.

[0006]

The present invention provides useful new strains of SK522 and SK5.
Effectively use a mixed culture of 42 strains. The synergistic effect of symbiotic mixed culture of SK522 strain and SK542 strain is
It enhances the fibrin degrading power and protein degrading function together with the remarkable solubilization of lignin. The present invention is a symbiotic mixed culture or a fermentative degrading agent containing the same as an effective main ingredient for animal organic materials such as manure, hoof horn, and leather scraps and sawdust.
It is mixed with a vegetable organic material such as rice bran and mixed while adjusting the water content to a required state of about 60%, and then mixed and fermented and ripened. Aerobic conditions are desirable for the environment, and switching is performed a required number of times. The new strain SK522 strain in the symbiotic mixed culture or the fermentation degrading agent solubilizes lignin, which is a plant organic material, and strongly decomposes fiber. In addition, the new strain SK542 strain produces a proteolytic enzyme to decompose proteins / hard proteins, and decomposes and ferments and ripens animal organic materials. And new strains SK522 and SK54
2 enhances their functions with each other, and powerfully and stably decomposes organic components such as lignin, fibrin and protein into a fertilizer by rapidly and strongly decomposing them.

The function of each new strain will be described below.
The SK522 strain has a strong fibrin-decomposing ability and a weak or weak lignin-solubilizing ability. The SK542 strain is an absolutely aerobic and moderately thermophilic bacterium, and produces a strong proteolytic enzyme having a thermophilic broad-acting hydrogen ion concentration activity and a carotenoid yellow pigment. Physicochemical properties of proteolytic enzyme produced by SK542 strain

[Table 1] It is a complex of several unstable enzymes in addition to protease A and protease B, which are considered to be novel enzymes having the above physicochemical properties. Physicochemical Properties of Pigment Produced by SK542 Strain The yellow pigment produced by this bacterium is a carotenoid yellow pigment, which is insoluble in water and soluble in the following solvents. Methanol, ethanol, petroleum ether, benzine,
A mixture of acetone, chloroform, acene + methanol (7: 3) and acetone + ethanol (1: 1).
Also, the infrared absorption maximum value is 450 nm. Other 430,
There is a small peak at 435,470 nm.

The thermophilic symbiotic mixed culture of these two new strains is not an additive action of the functions of the respective strains, but a synergistic effect produced by the interaction, and enhances the functions of each other. It strongly and stably decomposes organic components such as lignin, fibrin, and proteins quickly and strongly. By using the mixed culture of the present invention as an effective main component, a fermentative decomposition agent for organic materials can be obtained. This fermentative decomposing agent for organic materials further enhances its effect when a useful microorganism is mixed in addition to the SK522 strain, the SK542 strain depending on the use, purpose, climate, climate and soil, and a new useful effect can be obtained. it can.

[0009]

EXAMPLES The present invention and examples of use thereof will be described in detail below. The microorganism used in the present invention is a new SK.
522 strain and SK542 strain. Properties of both new strains are as follows. In addition, all the tests and classification and identification of mycological properties described below are carried out by the Burgers Manual of Determinative Bacteriology 7th Edition (19
57), 8th Edition (1974) and Burgers Manual of Systematic Bacteriology No. 1
Volume (1984), Volume 2 (1986) (Bergey's Manu
al of Determinative Bacteriology 7th. Ed. (1957), 8t
h.Ed. (1974) .Bergey's Manualof Systematic Bacterio
logy Vol.1 (1984), Vol.2 (1986).).

[I] SK522 strain (Clostridium thermocellum biovar SK522, Mikori Kenjojo No. 3459, FERM BP-3459) Mycological properties It cannot grow alone without a concomitant bacterium. However, since the associated bacteria are easily isolated and pure-cultured, it is the result of testing various properties of the isolated associated bacteria while co-culturing them with the isolated associated bacteria as a basis. Shape There are also straight rod shape and slightly curved shape. Alone, sometimes two. 0.3-0.5 × 2.2-4.0 μ. When the culture becomes old, it extends into filaments, long-chain, and lengths of 5.7 to 12.8.
μ. Motility is not observed in pericardium and room temperature hanging drop specimens. Oval or circular spores are formed at the ends to expand the cells and form a club. Gram negative.

Culture characteristics (1) Plate culture It does not grow on plate culture using broth agar or other commonly used medium. However, while the medium presented by Viljoen et al., Tetrault circular filter paper agar plate forms colonies with associated bacteria and yellow spots, it cannot be produced by this bacterium alone. (2) Slope culture and stab culture Addition of Schweizer reagent-treated filter paper to Viljoe et al. Medium Agar slope culture and stab culture do not grow. (3) It does not grow on potato culture, gelatin culture surface, or puncture. (4) Liquid culture Viljoen et al. Medium, fibrin gravy,
Growth is observed only in a medium containing fibrin such as fibrin peptone water (poor growth). If a substance other than fibrin such as glucose or xylose is used as a carbon source, the degrading power of fibrin is lost.

Physiological and biochemical properties 1. Enzymatic action, etc. (1) Fibrinolysis The fibrinolytic enzyme possessed by this bacterium is essentially composed of some β
It is a complex of -1,4-glucanase. Secreted extracellularly, action temperature 80 ℃ or more, same hydrogen ion concentration pH = 1
It was confirmed that several kinds of enzymes having heat resistance and alkali resistance of 0.0 or more were contained. It cleaves from the ends of macromolecules of fibrin that form a micellar structure, producing glucose, cellobiose, cellooligosaccharides, and the like. When the substrates that act on these enzymes are mainly shown, it decomposes filter paper FP-ase: decomposes positive avicel Avicelase: positive decomposes cellobiose into two molecules of glucose cellobiase: positive (2) lignin-decomposition Inawala lignin solubilization : Positive (weak, weak) (3) Proteolytic enzymes: Negative peptidase: Positive (4) Starch hydrolysis test: Slightly positive (5) Hemicellulose, xylan, pectin hydrolysis test : Negative (6) Invertase, Maltase: Positive (7) Lipolysis test: Negative (8) Oxidation reaction Hydroquinone reaction: Positive Tyrosine reaction: Negative (9) Reduction action: Positive 2. Product test (1) Fibrin fermentation 78-91% fermentation rate, fermenting fibrin actively and ethanol, methanol, acetaldehyde, acetic acid, lactic acid, formic acid, lactic acid, succinic acid, fumaric acid, tartaric acid, gluconic acid, It produces glucose, cellobiose, cellooligosaccharides, cellodextrin, large amounts of carbon dioxide, hydrogen, and hydrogen sulfide. (2) Produced acid from other sugars and alcohol Produced acid from glucose, sucrose, maltose, and cellobiose. (3) Gas generation test Although gas is generated more violently than fibrin, no gas generation is observed from glucose, sucrose, maltose, and cellobiose. (4) Peptone water test Ammonia: Slight reaction Indole: Positive skatole: Positive Hydrogen sulfide: Positive (5) Pigment production Usually, carotenoid yellow pigment production 3. Growth conditions (1) Growth temperature Optimum temperature is 65-72 ° C, temperature range is 40-80 ° C, 4
It does not grow below 0 ° C. (2) Hydrogen ion concentration Optimum hydrogen ion concentration pH = 6.7-8.0, the range is 5.6-9.6. (3) Nitrogen source Peptone is the best, and urea, uric acid, asparagine, sodium glutamate, etc. are also good. Ammonium salts are also good sources of inorganic nitrogen. (4) Carbon source When the subculture is continued with carbohydrates other than fibrin, its growth and fermenting power are lost. (5) Relationship with oxygen Eh = 200 to −250 mV, which is an anaerobic bacterium. (6) Requirement of micronutrients Micronutrients such as biotin, pyridoxamine, vitamin B ₁₂ and _p -aminobenzoic acid are required. 4. Content of G + C of DNA mol% of G + C = 38-40 (Tm) is estimated.

Taxonomic position: SK522 which actively ferments fibrin at an optimum growth temperature of 65 to 72 ° C. and a temperature range of 40 to 80 ° C.
The following thermophilic fibrinolytic bacteria are similar to the strains. Clostridium ther
mocellum Viljoen, Fred and Peterson, 1926 .: Jour. A
gr. Sci. (London), 16,7 (1926).) C. dissolvens
Bergey et al. 1925, aka Bacillus cellulose dessolve
ns Khouvine, 1923 .: Ann. Inst. Past.37,711 (1923); Be
rgey's Manual, 2nd.Ed. (1925) p.344.) C. thermocellulas
eum Enebo, 1951.: Bergey's Manual, 7th.Ed. (1957) p.68
9.) Bacillus thermocellolitex
ellulolyticus Coolhaas, 1928.: Cent Bakt.II, 75,101
(1928), 76,38 (1929).) Bacillus thermofibrinco
lus Itano and Arakawa, 1929: Farming, 5,816,921 (1929); 6,
248,257 (1930).) Among these, Clostridium disorbens is a bacterium that is very similar to this strain in morphological, culture, and physiological biochemical tests except for the following four differences. (1) It has a lignin-solubilizing ability although it is weak or weak. (2) Suitable growth temperature of 65 to 72 ° C, growth temperature range of 40 to 80
Does not grow at temperatures below 40 ° C. (3) Actively ferment fibrin, but not hemicellulose, xylan, pectin, etc. (4) Lignin solubilization ability is the genus Thermus
It is prominently symbiotically emphasized by certain types of bacteria, and at the same time has a positive effect on the fibrinolytic activity and other action functions of this bacterium. However, at present, Berges' Manual (Bergsy's Manual
of Systematic Bacteriology Vol.2 (1986) p.1104, p.11
In 41.), only Clostridium thermocellum is described as a thermophilic degrading bacterium. All of them are treated as subspecies or variants of Clostridium dissolvens, or incompletely studied. Therefore, for the classification and identification of this strain, it was decided to compare and examine Clostridium thermocellum and various mycological properties thereof, and at the same time, other thermophilic fibrinolytic bacteria were also compared for reference.

As described above, the results of comparative examination of various mycological properties are summarized as follows. The characteristic properties of this strain and known thermophilic fibrinolytic bacteria including Clostridium thermocellum are listed. The differences are the above-mentioned four items, and they can be enumerated in various ways such as the action on various other carbohydrates, the requirement of micronutrients, etc., but what I would like to emphasize especially is the problem with lignin. This strain has a lignin solubilizing ability, and the solubilizing ability is thermus aquaticus SK542 (Thermus aquaticus bi
It is significantly enhanced by the symbiotic mixed culture with ovar SK542). In contrast, there is no description about lignin of other thermophilic fibrinolytic bacteria including Clostridium thermocellum. If so, it is about its inhibitory effect on fibrin degradation. For this reason, a new strain (Strain) was established based on the physiological and biochemical properties of the lignin-solubilizing ability of this strain as one basis, and Clostridium thermocellum SK522 (Clostridium thermocellum biova
r SK522).

Microorganism Accession Number The microorganism accession number of this strain is Microtechnology Research Institute No. 3459 (FERMBP-3459). This strain is the standard strain, and Clostridium thermocellum (Clostrid
ium thermocellum Viljoen, Fred and Peterson, 192
It has the ability to solubilize lignin among bacterial species belonging to 6.), and significantly enhances the ability to solubilize lignin by symbiotic mixed culture with certain bacteria belonging to the genus Thermus Brock and Freeze1969. It is a novel strain with physiological and biochemical properties that characterizes the SK522 strain and natural and artificial mutants.

Screening of SK522 strain Soil, beach sludge, manure / manure, human / livestock faeces are used as separation sources, and concentrated culture is repeated several times at 55 to 65 ° C., then presented by Viljoen et al. Medium Tetrault circular filter paper Agar plates are used for isolation according to standard methods. The bacterium was able to be isolated substantially purely. The salt composition suggested by Viljoen et al. Is suitable as the medium, but if necessary, a trace amount of inorganic metal salts, vitamins, growth promoting factors such as yeast extract, etc. may be added. Viljoen, Fred and Peterson 1926 Peptone 5.0 g Calcium carbonate Excess ammonium phosphate sodium 2.0 g Potassium acid phosphate 1.0 g Magnesium sulfate 0.3 g Calcium chloride 0.1 g Ferric chloride Trace fiber Element (filter paper) 15.0 g Imizu 1000 ml

Utility of SK522 Strain The present SK522 strain is a fibrin-degrading bacterium that is originally active. The present inventors have found that this strain has a lignin solubilizing ability, although this strain is weak or weak, and the solubilizing ability of the strain is symbiotic mixing of this strain with a certain bacterium of the genus Thermus (genus Thermus). It was remarkably enhanced by culturing, and also had a favorable effect on the fibrinolytic activity and other action functions of this strain. In addition, the success of this mixed culturing enables the decomposition and utilization of many types of persistent organic materials, improves their productivity, and ensures the nodoko composting method. The manufacturing method was established. And SK
The formation of a healthy active soil microbial flora mainly consisting of the 522 strain and the SK542 strain stabilized the growth and action function of effective bacteria against changes in environmental factors, and eliminated or overcame soil diseases and continuous cropping disorders. In addition, it is expected that new technology of microbial utilization such as new technology of direct spraying on soil or leaf surface of plant and deodorization of human waste and decomposition of hard protein will be developed.

[II] SK542 strain (Thermus aquaticus biovar SK542, Mikoken Kenjoyori No. 3382, FERM BP-3382) Mycological properties Long rod-shaped, 0.4-0.6 × 3.0-5 0.0μ. Under certain conditions, for example, when culture becomes old, filamentous, length 20 to 130
μ. No flagella and no motility in room temperature hanging drops.
No endospores. Gram negative.

Culture property Proliferation is active. Generation time is 20 to 50 minutes. (1) Plate culture 3% agar, 60 ° C culture: yellow, relatively dense, small round colonies. (2) Agar stab culture Only surface growth, yellow slightly expanded. (3) Liquid culture Grows in a film on the surface (static culture).

Physiological and biochemical properties 1. Enzyme action, etc. (1) Proteolytic proteolytic enzyme: Positive (strong) (proteolytic enzymes) Gelatin hydrolysis: Positive peptidase: Positive (2) Starch hydrolysis: Slightly positive (3) Pectin degradation: Positive (weak) ) (4) Degradation of fibrin: Negative (5) Degradation of lignin: Negative Degradation of both fibrin and lignin is negative, but lignin digestion of SK522 strain is possible by symbiotic mixed culture with thermophilic fibrinolytic SK522 strain. It significantly enhances the solubilizing ability, and at the same time has a favorable effect on the fibrin decomposing power and other action functions. (6) Lipolysis: Negative (7) Invertase, maltase: Positive (8) Catalase, oxidase reaction: Positive (9) Hydrogen sulfide production: Positive (weak) (10) Sulfate reduction reaction: Negative 2. Product test (1) Raw acid and gas generation from sugar and alcohol No gas generation. A raw acid from glucose, galactose, maltose, lactose, and glycerol. (2) Formation of indole and skatole: Negative (3) Pigment production Yellow pigment (carotenoid pigment, maximum absorbance 450n
m, others with small peaks at 430, 435, 470 nm). However, it is scarcely produced in a synthetic medium containing glucose and other sugars as a carbon source.

3. Growth conditions (1) Medium concentration Bacteria of the genus Thermus are generally sensitive to organic matter concentrations. It is said that the less nutrients and the lower the concentration, the better. However, this strain is different from these in that it grows well even at normal concentrations. It grows even with salt NaCl 5% or more. Optimal concentration 2.
0-3.0%. In general, Genus Thermus bacteria do not grow in the presence of more than 2% NaCl. It grows even with sodium glutamate of 3% or more. It grows on sucrose or maltose 5% or more. Its growth is not inhibited even in the presence of 2% peptone + 1% yeast extract. Generally the genus Thermu
For the bacteria of s), trypton and yeast extract concentrations of about 0.1% are suitable, and they do not grow when they exceed 1%. (2) Growth temperature Optimum temperature is 72-76 ℃, growth temperature range is 40-82 ℃,
It cannot grow below 40 ° C. (3) Hydrogen ion concentration Optimal hydrogen ion concentration pH = 6.0 to 10.0. Growth hydrogen ion concentration range pH = 4.0 to 11.0. (4) Nitrogen source Proteins such as gelatin, glutamate, urea, and ammonium salt are often used as an inorganic nitrogen source. (5) Carbon source Glucose, sucrose, maltose, galactose, cellobiose, raffinose, stachyose, starch, glycerol, acetic acid, lactic acid, malic acid, glutamate are used. (6) Relationship with oxygen Absolutely aerobic. (7) Demand for micronutrients This strain has high demands for micronutrients. In addition to vitamins such as biotin, nicotinamide, and thiamine, a relatively high concentration of metal ions such as iron, manganese, and calcium is required for good growth of this strain. It is also sensitive to the amount of these minerals. (8) Susceptibility to antibiotics Like general Thermus genus (genus Thermus) bacteria,
It shows high sensitivity to penicillin G, chloramphenicol, tetracycline, streptomycin, kanamycin and other antibiotics. 4. Content of G + C of DNA mol% of G + C = 68 (Tm) is estimated.

Taxonomic position The bacterium does not grow in absolute aerobic condition, optimum growth temperature of 72 to 76 ° C, growth temperature range of 40 to 82 ° C, 40 ° C or lower. Since it is a non-spore-free, Gram-negative long bacillus, etc., in consideration of other mycological properties, the genus Thermus (genus Thermus Broc
k and Freege 1967). And, as a similar bacterium of the genus Thermus, Thermus aquaticus Broc
k and Freege 1969.:Bergey's Manual (1984) Vol.1, P.3
37.) Thermus thermophilus Osh
ima and Imahori 1974.:Int.J.Bacteriol.,24,102(197
4)) Thermus Flavus Saiki, Kimura an
d Arima 1972.:Agr.Biol.Chem.,36,2357(1972)), etc., but currently Bersey's Manual
s Manual of Systematic Bacteriology Vol.1 (1984) P.33
3) Genus Thermus Brock and Freeg 196
Bacterial species belonging to 7) are Thermus aquatics (The
rmus aquaticusBrock and Freege 1969),
Moreover, since it has a characteristic property that cannot be found in the known bacterial species as described below, this strain is used as a new strain of Thermus aquaticus (strain), and Thermus aquaticus biovar SK54 (Thermus aquaticus biovar SK54
We conclude that it is appropriate to name it 2).

(1) The lignin-solubilizing ability is remarkably enhanced by symbiotic mixed culture with the thermophilic fibrinolytic bacterium SK522 strain. At the same time, it has a positive effect on the ability to decompose fibrin and other functions. (2) It has a strong proteolytic activity with a wide range of working hydrogen ion concentration and working temperature. (3) The genus Thermus (genus
(Thermus) bacteria are very weak and grow well in medium of normal concentration. (4) Strong demand for micronutrients, demand for various vitamins and metal ions, and sensitivity to their mineral contents. (5) Yellow pigment (carotenoid pigment, maximum absorbance 45)
0 nm and others have small peaks at 430, 435 and 470 nm).

Microorganisms accession number The microorganisms accession number of this strain is Micro Engineering Research Article No. 3382 (FERMBP-3382). The SK542 strain is a standard strain of this strain, and by symbiotic mixed culture with a thermophilic fibrinolytic bacterium (SK522 strain) among bacterial species belonging to Thermus aquaticus Brock and Freege 1969, A new strain with a physiological and biochemical property that significantly enhances its lignin solubilizing ability and at the same time has a favorable effect on the fibrin degrading power, and has physiobiochemical properties that embraces the SK542 strain and natural and artificial mutants ( strain).

Screening of SK542 strain Isolate sources are hot spring sources in various places in Kyushu, soils near hot spring sources, humus and the like. The separated sample is precultured at 55 to 60 ° C. and then isolated by 3% agar plating according to a standard method. The bacterium was able to be isolated substantially purely. Separation medium composition (Castenholz medium: Castenholz, RW; Bacteriol.
Rev., 33, 467 (1969)) Nitrilotriacetic acid 100 mg CaSO ₄ .2H ₂ O 60 mg MgSO ₄ .7H ₂ O 100 mg NaCl 8 mg KNO ₃ 103 mg NaNO ₃ 689 mg Na ₂ HPO ₄ 111 mg FeCl ₃ 0.28 mg MnSO ₄ .H ₂ O 2.2 mg ZnSO ₄ .7H ₂ O 0.5 mg H ₃ BO ₃ 0.5 mg CuSO ₄ 0.016 mg Na ₂ M ₀ O ₄ .2H ₂ O 0.025 mg (yeast extract 5000 mg) (tryptone 5000 mg) (sucrose 10000 mg) ) Total volume (with pure water, pH = ~ 8) 1000 ml Note: The composition in () is modified by the inventors.

Utility of SK542 Strain The utility of this new strain is characterized by the following three functions. (1) By symbiotic mixed culture with a thermophilic fibrinolytic bacterium (SK522 strain), the lignin degrading ability of the bacterium is remarkably enhanced. At the same time, it also has a positive effect on its fibrin decomposing power and other action functions. (2) It has a strong proteolytic activity with a wide range of action hydrogen ion concentration and action temperature. (3) It produces an insoluble yellow carotenoid pigment. This yellow pigment in the fungus body is decomposed by other microorganisms and becomes a water-soluble low molecular weight substance, which is absorbed by the plant,
It helps improve the quality of fruits such as taste, color and storability. When the SK542 strain is applied, it promotes the growth of general heterotrophic microorganisms and soil-effective bacteria that propagate using this strain as a substrate. The above is the known genus Thermu (genus Thermu
In combination with the extremely weak sensitivity of organic matter concentration unlike bacteria of (s), in natural ecology and agricultural ecosystem, various kinds of persistent organic substances, hard proteins and surplus sludge, and various other organic materials are widely used. Is now available. Furthermore, this leads to the development of a new dimension of microbial utilization, such as productivity improvement, overcoming soil diseases and continuous crop damage, and deodorizing and decomposing human waste, and developing new technology for direct spraying on soil or plant foliage. Connect.

[III] Mixed culture of SK522 strain and SK542 strain 1. Large-scale culture of SK522 strain Using the medium of Viljoen et al., 1926 described above, under anaerobic or semi-anaerobic conditions, 65-70 ° C, 48-
After culturing for 60 hours, the culture solution is used as a culture containing cells. 2. Large-scale culture of SK542 strain Using the culture medium of Castenholz (1969), 70-75 under aerobic conditions such as aeration or shaking
Cultivate at 24 ° C for 24 hours, and use the culture solution as a culture.

3. Mixed culture of SK522 strain and SK542 strain Using the following medium, appropriate amounts of large-scale cultures of the SK522 strain and the SK542 strain were inoculated, respectively, to obtain 65-70.
Cultivate at 24 ° C for 36 to 36 hours to obtain a mixed culture of both strains. Then, this is made into a powder or granular material by using a shaping agent, and a product having a water content of 5 to 7% or less can be stored for 2 years or more. In addition, just before application, a large amount of the cultures of both strains are mixed in equal amounts and mixed well, and immediately applied, or even if it is manufactured into a powder or granule using a excipient, the quality performance of the final product is completely Unchanged. Further, the following E-medium for mixed culture is simply referred to as E-medium. E-medium for co-cultivation mixed culture K ₂ HPO ₄ 5 kg (NH ₄ ) ₂ SO ₄ 2 kg Urea 2 kg Peptone 5 kg Yeast extract 5 kg Filter paper (fibrin) 15 kg CaCO ₃ excess water (~ pH 7.0) 1000l 4. Utility of symbiotic mixed culture of both strains Under the condition of high temperature environment, symbiotic mixed culture with SK542 strain of absolutely aerobic Genus Thermus is anaerobic thermophilic fibrinolytic bacterium SK522. It remarkably enhances the decomposition and fermentation of natural refractory organic materials, which is almost impossible with the strain alone. First, the growth of the SK542 strain is S
It complements the proteolytic activity of the K522 strain.
Moreover, the decomposition and fermentation of persistent organic materials such as waste wood, manure, waste wood, and municipal sewage sludge are all solid-state fermentations, and are in a well-ventilated environment. And
The growth of the SK542 strain in the early stages is transformed into a high temperature anaerobic environment, which allows the SK522 strain to grow in nature.

(1) Solubilization of lignin Here, it is particularly noted that the SK542 strain exerts a remarkable lignin-solubilizing ability of the SK522 strain. The persistent organic material actually exists in a state in which the fibrin is strongly bound to lignin and other organic components. Such natural lignocellulose can hardly be decomposed by the single application of the SK522 strain having a strong fibrinolytic activity. Although the chemical structure of lignin has not been completely clarified yet, phenylpropane, which has a side chain with three carbon atoms in the benzene ring, serves as the basic unit, and this unit is formed by a radical reaction catalyzed by peroxidase. It is a polymer compound that is randomly and three-dimensionally polymerized.
It is a consensus view of many researchers and engineers that resistance to microbial decomposition is extremely strong and extremely difficult to decompose. However, SK5 used in the present invention
Twenty-two strains solubilize lignin, though weakly or weakly. And, this lignin solubilizing ability, by symbiotic mixed culture with the SK542 strain, for example, that 50% or more of Inawara lignin is solubilized in a short time around 10 days, this time, in the present invention,
It was revealed for the first time. Its practical application is an important characteristic aspect of the present invention, and its action and effect are 50 to 80 ° C.
It is exerted at the high temperature stage mentioned above, and the humification progresses rapidly to the decomposition fermentation.

(2) Production of yellow pigments The genus Thermu including the SK542 strain
The yellow pigment produced by s) is a carotenoid insoluble pigment. This yellow pigment in the bacterial cells is decomposed by other microorganisms to become a water-soluble low molecular weight substance, which is absorbed by the plant body and transferred to a necessary site, which is just a convenient precursor. That is, such a yellow pigment not only promotes the growth and proliferation of rhizomes along with other lysed cell contents and secretions, and fermentation products, but also causes the formation of flower buds, fruit set, fruit enlargement, etc. It has been clarified by research conducted at the molecular biological level that it is deeply involved in the metabolic system of reproductive growth, and it has been developed into its application and has already been improved in quality such as fruit taste, color and storage quality. Is useful to.

(3) Mixed Complexation of Microbial Ecosystem Further, when the bacterial cells of the SK542 strain containing the yellow pigment are applied, the growth of general heterotrophic microorganisms, soil effective bacteria and the like that propagate using the same as substrates is promoted. More importantly,
In the field such as compost production in the natural world, thermophilic microorganisms such as the SK522 strain do not immediately proliferate and activate. Initially, general room temperature heterotrophic microorganisms grow,
Fermentation heat due to these actions is accumulated to help increase product temperature, and only a single strain of thermophilic fibrinolytic bacteria and a small number of microorganisms are involved in the humification of natural persistent organic materials. Do not mean. It is required to mix and diversify the microbial flora. It is desirable to have a complex content that there are many effective bacteria of various types and that "feed (substrate)" is also appropriate. Thus, the high-temperature decomposition of persistent organic matter is
The bacterial flora centering on the K522 strain is formed first, and the humification process not only involves simple changes in the constituent components, but also the extremely complex interactions and transitions of the microbial flora, which are most efficiently and closely related. Of. Other effective bacteria to be mixed with the SK522 strain and the SK542 strain include the following.

(1) Culture of Hemicellulose Degrading Bacteria Hemicellulose forms a plant (cell wall) with fibrin, and is called xylan, araban, dextran, mannan, galactan, etc. depending on the sugars that compose this. As a culture of hemicellulose-decomposing bacteria, Iwata's medium (1936) added to a concentration of rice xylan about 1% was used, and the cells were facultatively and anaerobically collected at 30 to 38 ° C. Usually Bacterium vulgatus, Bac. Prodigiosum, Bacteria mesentericu
s ruber), Microspyra Agariquefisense (M
A mixed culture of one or more strains such as icrospira ager-liquefaciens) is obtained. (2) Cultures of pectin substance-degrading bacteria Many of the bacteria that strongly decompose pectin substances belong to the Bacillus subtilis group bacteria and ethanol / acetone bacteria when they are aerobic, and to the lactic acid bacteria when they are anaerobic. In the present invention, using a Morish medium (Molisch 1939), soil, compost,
Separation sources such as horse manure, bagasse and chomp are 27
The inoculum is obtained by collecting culture several times in a thick layer and a thin layer at ˜35 ° C. for 3 to 5 days of culturing. In the present invention, cultures of one or more strains are used.

(3) Soil actinomycete culture It is generally difficult to say about the action of actinomycetes (Actinomycetales Buchanan, 1917) in soil. But,
It decomposes various organic substances, especially incombustible fibrin and lignin, together with other microorganisms, and plays an important role in the formation of humus, which is the source of soil fertility. Certainly, it has important significance in terms of microflora control. The culture of actinomycetes is Waxman's medium (Waksman 1919), fertilized soil as a separation source,
In addition, strong bacteria are collected by using the manure and manure. (4) Cultures of soil filamentous fungi and yeasts The most abundant locations of soil filamentous fungi are soils like bacteria and actinomycetes, and filamentous fungi in soils are naturally abundant in cultivated soil with plant roots, especially rhizosphere. Then, the work is also active. It is involved in the decomposition of organic substances such as plant remains and is related to soil fertility. It is considered that filamentous fungi are mainly active in the initial stage of decomposition. Next, there are many unclear points about the function of soil yeast. However, there are quite a few yeasts in the soil,
In addition, it is certain that the abundant vitamins and growth factors contained therein will affect coexistence with other microorganisms and soil activity. Soil filamentous fungi and yeast cultures are separated and cultured from soil or manure / manure using a Tuapeck Dox medium (Czapek & Dox, 1910).

(5) Culture of thermophilic Bacillus genus Bacillus which is generally aerobic, motile, and has endospores, and is a group of bacteria widely distributed in the natural world such as soil, leaf surface, and hay.
It is also reported that most of the bacterial strains have strong heat resistance and can grow even at 55 ° C. or higher, and thus are major microorganisms during compost production. In addition, secreting antibacterial substances such as bacitracin and basilicin has attracted attention in the field of soil plant pathology in recent years. From these reasons, it is possible to try to utilize this bacterial group for crop production, and fertilized soil or a suspension of manure / manure is heat treated at 80 ° C. for 10 minutes to be used as a separation source. Waxman's medium (Waksman, 19
22), and aerobically collect the bacterial group at 50 to 60 ° C. In the present invention, single or mixed culture of these bacteria is used. (6) Culture of yellow pigment-producing bacteria Yellow pigment produced by bacteria is a carotenoid pigment. There are many types of ripe compost, and many types. It is also often found on the leaves of plants. Using these as a separation source, a normal meat extract medium or peptone-yeast extract medium,
Incubate and separate at 25-35 ° C under aerobic conditions. It is easily identified by colors such as yellow-green, yellow, yellow-brown, and red, and is generally flavobacterium (Flavobacterium), chromobacterium (Chromobacterium), Pseudomonas, serratia, phototrophic bacteria. A culture of one or more strains belonging to, etc. is obtained.

[IV] Symbiotic mixed culture of SK522 strain and SK542 strain 1. Inoculum: SK522 strain and SK542 strain symbiotic high-volume mixed culture Large-scale culture of SK522 strain Large-scale culture of SK542 strain Each culture is as described above in the present specification. 2. Specimen: The raw material used for the test is rice straw (rice cake),
The analytical values are shown in Table 2.

[Table 2] 3. Test method 10.0 g Erlenmeyer flask was injected with 10.0 g of air-dried sample, 700 ml of E-medium (without filter paper), sterilized under pressure as usual, and inoculated with an appropriate amount of inoculum, and cultured at 65 ° C for 20 days. Evaporation depletion is compensated and used for analysis. 4. Display results

[Equation 1]

[Equation 2]

[Equation 3] 5. Test results

[Table 3] As can be seen from Table 3, this 522 strain and 542 strain
Thermophilic symbiotic mixed culture of two new strains enhances each other's functions not only by the additive effects of their respective functions but also by the synergistic effect of the interaction, which makes them naturally persistent such as rice straw. In a short time of 10 to 12 days, fibrous material solubilizes 50% or more of lignin and 90% fibrin.
As described above, 70% or more of the protein is fermented and decomposed strongly and stably. There are remarkable differences from the individual cultures of the 522 strain and the 542 strain, respectively.

Hereinafter, an example of the method for producing an organic fertilizer using this symbiotic mixed culture or a fermentative decomposing agent containing the same as an effective main component and its effects will be described. Application example. Example of production method of organic manure using livestock manure, persistent fiber material, and hard protein as nitrogen source Test purpose: In the production method of livestock manure, persistent fiber material, and organic fertilizer containing hard protein as nitrogen source Examination of application effects ((1) deodorizing effect, (2) ripening effect) of the fermentation decomposition agent of the present invention. Fermentation decomposer: Immediately mix the following raw materials to make a powdery product. Mixing ratio of fermentative decomposing agent (vehicle: per 1000 kg of limestone rock powder) Mixed culture of SK522 strain and SK542 strain 4 kg (mixed culture described in 0028) Soil actinomycete culture 2 kg (culture described in 0033) Material) Yellow pigment-producing bacterium culture 2 kg (culture described in 0034) Trace element complex 200 g (Co, B, Mo, Mn)

1. Manufacture of organic fertilizer (1) Manufacturing equipment: rigid co-flow fermenter, 200 l capacity. (2) Sample raw materials: Mixed powder of pig manure (urine), sawdust, hoof horns and leather scraps. An example of these analyzes is as follows: Component composition of test raw material (% based on dry matter) Moisture C N Ash C / N Pig manure (urine) 80.6 36.9 3.4 22.2 10.9 Sawdust 17 .3 50.4 0.1 0.8 504.0 (mixed with conifers and leaf trees) Hoof horn / leather scrap mixed powder 14.2 31.7 9.2 7.4 7.4 4.6 (3) Blend of raw materials 100 kg Rice bran 10 kg Pig manure (urine) 30 kg Hoof horns and leather scraps 5 kg Superphosphate (1 kg is added at the time of turning back) 1 kg + 1 kg Fermentation decomposer 2 kg Water content adjusted to 60% (4) Loading of organic fertilizer 100 kg of the present invention Approximately 60% moisture while mixing fermented decomposing agent, hoof horn / leather scrap, pig manure (urine), etc.
Water and load it in the fermenter. (5) Production progress Even in the production of this organic fertilizer, microorganisms are rapidly decomposed at high temperature under aerobic conditions as much as possible even by the high-speed composting method. The maximum temperature is 78 to 86 ° C., which is continued for 10 days, the cut-off is performed once, the remaining 1 kg of superphosphate is added, and the main fermentation is completed in 2 weeks. Almost completely matured at 30 ° C. or higher and after-ripening fermentation for 2 weeks.

2. Results and discussion (1) Deodorizing effect The results are shown in Table 4 below. As you can see. The result is remarkable. The indicator compound, dimethyl sulfide, was significantly reduced by the application of the fermentative decomposition agent of the present invention, and was hardly detected after the beginning of the post-ripening period. This is also organoleptically clear, and there is no fear of a bad odor during ripening and fermentation, and the organic fertilizer used as a product has a characteristic compost odor, rather an aroma. (2) Effect of ripening The effect is as shown in the analysis results of the product organic fertilizers in Tables 5 and 6 below. It exceeds the provisional standard value for wood compost and no abnormalities are observed in the seedling test.
Generally, animal material fertilizers are fast-acting, but hoof horns, leather scraps, etc. have a high nitrogen content and have a low carbon content, but are difficult to decompose and are extremely slow-acting. However, as can be seen from this application example, by selecting the effective microorganism of the present invention, decomposition fermentation proceeds rapidly, and its excellent ripening effect is shown in Tables 5, 6 and 7 below. Is shown in.

3. Main test data The main test data is the deodorizing effect of this fermentation decomposition agent. Tables 5 and 6 showing the chemical composition of the organic fertilizer used as a product, and Table 7 showing the dynamics of microbial groups that appear during decomposition and aging of this organic fertilizer. Is.

[Table 4]

[Table 5]

[Table 6]

[Table 7]

[0040]

As described above, according to the present invention, by using the symbiotic mixed culture of the two new strains SK522 and SK542, animal organic matter such as livestock excrement, human excrement, hoof horn, leather scraps, etc. can be obtained. It was possible to quickly decompose and ripen the material and the plant-based organic material containing a large amount of fibrous material with little odor and to obtain a high-quality organic fertilizer. In addition, the resources of animal organic materials and plant organic materials as raw materials can be effectively used and treated.

Claims (1)

[Claims] 1. A thermophilic fibrinolytic bacterium as described in 1 below, Clostridium thermocellum SK522 (Clostriduim).
thermocellum biovar.SK522, Micro Engineering Kenjiro 345
9) and Thermus Aquatics SK described in 2 below.
542 (Thermus aquaticus biovar SK542, Mikori Kenjoyori No. 3382) and a mixed culture obtained by co-cultivating them in a thermophilic environment at 60 ° C. or higher, or a fermentative decomposing agent containing the mixed culture as an active ingredient. Adjust to the required water content while mixing with the organic organic material and the animal organic material of human waste and excrement,
A method for producing an organic fertilizer, which comprises fermenting an organic material to ripen and ferment with a mixed culture or a fermentation decomposer. Note 1 having a lignin solubilizing ability, a suitable growth temperature is 65 to 72 ° C.,
A thermophilic fibrinolytic bacterium that grows in the temperature range of 40 to 80 ° C. and fermented fibrin actively, Clostriduim thermocellum biovar.S
K522, Mikori Kenjo No. 3459) Note 2 Absolute aerobic, optimum growth temperature is 72 to 76 ° C, 40 to 82 ° C.
It grows in a medium of normal concentration in the temperature range of
Thermos aquaticus SK542 (Thermus aquaticus biovar S) which produces a proteolytic enzyme and a carotenoid-type yellow pigment with a thermophilic broad range action hydrogen ion concentration activity of 85 ° C and a action hydrogen ion concentration pH = 4.0 to 11.3
(K542, Micro Engineering Research Center No. 3382)