JPH06105679A - Bacterium thermus aquaticus - Google Patents

Abstract

PURPOSE:To develop a method for remarkably solubilizing lignin with a bacterium and vigorously fermenting cellulose and proteins, rapidly decompose a hardly decomposable organic substance as its application and effectively utilize the decomposed substance thereof. CONSTITUTION:The strong decomposition and solubilization of a natural fibrous substance or hard proteinaceous material consisting essentially of hardly decomposable lignin or cellulose are facilitated by using a carotenoid-based yellow coloring matter and a strain Thermus aquaticus biovar. SK542 capable of producing a proteolytic enzyme having the high-temperature wide area acting hydrogen ion concentration activity and further utilizing a symbiotic mixed culture of the strain with a new bacterial strain of thermophilic cellulolytic bacterium Clostridium thermocellum biovar. SK522.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to solubilization of natural lignin, decomposition and fermentation of wood and other hardly decomposable fibrous substances, hard proteins and excess sludge, production of compost / manure, decomposition of human waste, humification and aging of biomass. The present invention relates to a new bacterium useful for promoting soil fertility and improving soil structure, and a technique for effective use thereof.

The term "hardly decomposable organic material" or "hardly decomposable fiber material" as used in the specification refers to sawdust, chip dust, planer waste, bark (bark) and other waste materials in the wood industry, straw rods such as rice husks, rice straws, wheat straws, etc. , Soybean, azuki bean, fallen seeds and soybean seed coats and pods, coffee grounds, deciduous leaves, bark, mountain grasses such as reeds and kaya, as well as abolished mushrooms such as shiitake mushrooms and other mushrooms 100
The above-mentioned various persistent plant bodies. The organic components that make up these plant remains are extremely complex and diverse, but generally, the main component is fibrin, which is about 30-75.
%, And then 15 to 40% of lignin and 45 to 90% or more when both are combined, and occupy most of them. Next is hemicellulose in the order of 7 to 25%, and in a small amount, nitrogen-containing compounds such as proteins, various sugars, organic acids, alcohols, and fats, waxes, essential oils and the like are contained.

[0003]

2. Description of the Related Art Conventionally, it has been very difficult to solubilize lignin or rapidly decompose fibrin with a degrading agent using microorganisms or enzymes, and hard proteins such as keratin and collagen are also subject to enzymatic action. hard. Therefore, effective solubilization and decomposition of natural refractory organic materials using microorganisms have not yet been put into practical use.

[0004]

The problem to be solved by the present invention is to provide a new strain having a high protein decomposing ability and capable of enhancing lignin solubilization and fibrinolysis, and enhancing the functions thereof. It is intended to provide a fermentative decomposing agent effective for decomposing fermented fermented materials and hard-to-decompose fiber materials, hard protein materials, soil improvement, and deodorizing decomposition of human waste.

[0005]

Means for Solving the Problems The gist of the present invention which has solved the above problems is as follows: 1) Thermus aquaticus biovar SK542, which is characterized by producing a carotenoid yellow pigment belonging to the genus Thermus Kokenjoyori No. 3382) 2) Thermus aquaticus bacteria (Thermus described in 1)
aquaticus biovar SK542, Micro Engineering Lab. No. 3382
No.) and lignin solubilizing ability, and the optimum growth temperature is 65-
The thermophilic fibrin-degrading bacterium Clostriduim thermocellum SK522 (Clostriduimthermocellum) that grows at 72 ° C. in the temperature range of 40 to 80 ° C. and actively ferments fibrin
biovar.SK522, Microtech Research Institute No. 3459) and 6
A mixed culture which is symbiotically mixed and cultivated in a thermophilic environment of 0 ° or more. 3) A fermentative decomposition agent for an organic material containing the mixed culture according to 2 as an effective main component. Hereinafter, Thermus aquaticus bacteria (Thermus of the present invention
aquaticus biovar SK542, Micro Engineering Lab. No. 3382
No.) and the thermophilic fibrinolytic bacterium Clostriduim thermocellum SK522 (Clostriduim thermocellum biova
r.SK522, Microtechnical Research Institute No. 3459) is related to the discovery of the present inventors.
It is referred to as the K542 strain and the SK522 strain. S of the present invention
The mycological characteristic of the K542 strain is that it is absolutely aerobic, has an optimum growth temperature of 72 to 76 ° C., grows in a medium having a normal concentration in the temperature range of 40 to 82 ° C., an effective temperature of 75 to 85 ° C., and action hydrogen ion. It produces a proteolytic enzyme and a carotenoid-based yellow pigment having a high temperature broad-range action hydrogen ion concentration activity with a concentration of pH = 4.0 to 11.3.

Physicochemical properties of proteolytic enzyme produced by SK542 strain

[Table 8] Protease A and Protease B, which are considered to be novel enzymes having the above physicochemical properties. Physicochemical Properties of Yellow Pigment Produced by SK542 Strain The yellow pigment produced by this strain 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.

[0007]

The present invention provides a useful new strain SK542. It also provides a mixed culture of the same SK542 strain and SK522 strain and its useful use. SK542 strain and SK
The synergistic effect of the symbiotic mixed culture of 522 strain enhances the fibrinolytic activity and other functions together with the remarkable solubilization of lignin. 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. Further, the SK522 strain has a strong fibrin decomposing ability and a weak or weak lignin solubilizing ability.

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 SK542 strain, the SK522 strain depending on the use, purpose, climate, climate, 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.
542 strain and SK522 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] 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.

Cultural properties 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 The optimum temperature is 72 to 76 ° C, and the growth temperature range is 40 to 82 ° C.
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 the characteristic properties that cannot be found in the known bacterial species as described below, this strain is used as a new strain (strain) of Thermus aquaticus SK542 (Thermus aquaticus biovar SK54).
We conclude that it is appropriate to name it 2).

(1) The lignin solubilizing ability is remarkably enhanced by the 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).

Microbial Accession Number The microorganism accession number of this strain is Microtechnology Research Institute 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 the 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. This allows a substantially pure separation. 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 action 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.

[II] SK522 strain (Clostridium thermocellum biovar SK522, Mikoken Kenjoyori 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 properties (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: Positive Avicel is decomposed Avicelase: Positive Cellobiose is decomposed into two molecules of glucose Cellobiase: Positive (2) Lignin-decomposed 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 B12 and p-aminobenzoic acid are required. 4. Content of G + C of DNA mol% of G + C = 38-40 (Tm) is estimated.

Taxonomy position: SK522 which actively ferments fibrin at a growth optimum 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.

The results of comparative examination of various mycological properties as described above are summarized as follows: characteristic characteristics of this strain and known thermophilic fibrinolytic bacteria including Clostridium thermocellum. 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. This allows a substantially pure separation.
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 originally a vigorous fibrinolytic bacterium. 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.

[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 applied to the excipient (presented by the present invention)
(Refer to page) to make powder and granules, and make the product with water content of 5 to 7% or less, it can be stored for more than 2 years. 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 produced into a powder or granules using a excipient (see pages 3 and 4), The quality performance of the final product does not change at all. 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 genus Thermu (genus Thermu) including 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 this 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 of the SK522 strain and the SK542 strain referred to in claim 5 are as follows.

(1) Culture of Hemicellulose Degrading Bacteria Hemicellulose forms a plant (cell wall) together with fibrin, and is called xylan, araban, dextran, mannan, galactan, etc. depending on the sugars constituting the plant. 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 nature 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 (Pseudomonas), serratia (Serratia), phototrophic bacteria (Phototrophic bacteria) A culture of one or more strains belonging to, etc. is obtained.

[IV] Application Example of Symbiotic Mixed Culture of SK522 Strain and SK542 Strain Application Example 1. Application example of lignin solubilization method 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 analysis values are shown in Table 1.

[Table 1] 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 2] As is clear from Table 2, 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.

Application Example 2. Fermentation of sawdust by fermentation decomposer of organic materials 1. Fermentative decomposition agent: yeast extract 10% (wt / vo) in a symbiotic mixed culture of SK522 strain and SK542 strain.
l) and peptone 10% (wt / vol) are added to make a fermentation decomposer. 2. Sample: sawdust. Cedar tree, 17 to 20 years old, the wood part is pulled vertically, and the particle size is 3.0 to 1.0 mm.

[Table 3] 3. Display of test method and results 10 g of air-dried sample, 1000 ml Erlenmeyer flask, E-medium 7
Add 00 ml (filter paper, see page 19 of this specification), sterilize under pressure according to the usual method, and inoculate with an appropriate amount of fermentation decomposition agent, 65 ° C, 20
Day culture. Evaporation depletion is compensated and used for analysis. According to the result, the application example 1. The lignin solubilization rate, the fibrin fermentation rate, and the protein decomposition rate are calculated in the same manner as in (2nd in this specification).
(See page 4). 4. Test results The test results are shown in Table 4,

[Table 4] Fermentative decomposing agent for organic materials whose main component is symbiotic mixed culture of two new strains, SK522 strain and SK542 strain, uses natural refractory fiber material such as sawdust for about 10 to 12 days and 50% of lignin. The above was solubilized, and the fibrin and protein were fermented and decomposed similarly to the case of the SK522 strain alone, or more strongly and stably.

Application Example 3. Fermented decomposing agent in the form of powder and Noduki composting method Manufacture of Fermentation Decomposition Agent And, the manufacture of the powdery fermentation decomposition agent of the present invention is SK5
Along with the large-scale cultures of 22 strains and SK542 strains, some kinds of micronutrients and trace minerals were selected, added to the excipient such as limestone rock powder, and well mixed by stirring to obtain the shape established in the manufacturing standard. It is used as a fermentative decomposition agent. An example of the blending of raw materials is as follows, and it is a powdery product. If necessary, a binder or the like may be added to form a granule. Mixing ratio of raw materials (vehicle: based on 1000 kg of limestone rock powder) Large-scale culture of SK522 strain 2 kg Large-scale culture of SK542 strain 2 kg Biotin 1 g Vitamin B ₁₂ 1 g Cystine 5 g Methionine 10 g Manganese 10 g Cobalt 1 g Yeast extract 100 g Soil 50kg 2. Manufacture of compost Using bark made of cedar wood as the main raw material and a powdery main fermentation decomposing agent, compost was manufactured by the conventional Nozomi composting method, and its application effect was observed. (1) Composition of bark

[Table 5] (2) Blending of raw materials Burk (20 mm mesh size) 1000 kg Rice bran 250 kg Ammonium sulphate 2 kg Coal superphosphate (added during the second turning) 20 kg Main fermentation decomposer 60 kg Moisture approx. 60% (3) Compost loading Burk crushed Using 1000 kg of the product, water was supplied so that the water content was about 60% while thoroughly mixing the main fermentation decomposing agent and the like, the product was loaded into a wooden frame of 180 × 180 cm, and the upper part was covered with a vinyl sheet. (4) Manufacturing progress The loading operation will be carried out on May 10, and 20kg of superphosphate will be added at the time of turning back three times and the time of turning the second time. The product temperature rises quickly, reaching 61 ° C on the 5th day after loading, and continuing to be 60 ° C or higher after that. After the second turning, the maximum temperature reached 72 ° C, the product temperature gradually decreased, and it entered the post-ripening period. When the integrated temperature up to July 10 is calculated, it will be 4,383 ° C / day. Simply averaging, it will be 2.7 to 3.2 ° C per day compared to the conventional case.
Shows an increase in temperature. In addition, the change in color at the time of switching back is as shown in Table 6 when viewed in the Munsell color diagram.

[Table 6] (5) Results The results are shown in Table 7. Bark compost, which used to take 3 to 6 months or more to manufacture, can now be done in 60 days. In addition, the accumulated temperature for 60 days is 4,383 ° C / day, which means that the product temperature rises by 2.7 to 3.2 ° C per day compared to the conventional case, and the change in color due to aging is also favorable. The results are shown. As described above, it is considered that the fermentation effect of the present fermentation decomposing agent was sufficient for bark composting. This is finally well understood by the analysis results of the product.
All of the analysis values show excellent results exceeding the unified standard values of the Japan Burk Compost Association and the National Burk Compost Industry Association.

[Table 7]

The excipients used in the present specification include limestone rock powder, dolomite rock powder, shell fossil powder, crab shell, shellfish powder such as shellfish, calcium carbonate, slaked lime, perlite, vermiculite and zeolite. , Diatomaceous earth, basic rock rock powder, crushed peat moss, charcoal, charcoal powder, etc. are present, and a mixture of one or more of these is shown. Micronutrients and trace minerals in the present specification include vitamins such as biotin, nicotinic acid amide, thiamine, pyridoxamine, vitamin B12, balaaminobenzoic acid, arginine, cystine, glutamic acid, isoleucine, leucine, lysine, methionine, phenylalanine, There are amino acids such as threonine, tryptophan, tyrosine and valine, and trace minerals such as iron, manganese, cobalt and calcium. As described above, these excipients, micronutrients and trace minerals are appropriately selected according to effective bacteria, purpose of use, soil environment, climate and climate.

[0042]

INDUSTRIAL APPLICABILITY As described above, according to the present invention, a useful new strain SK542 is provided. In addition, new bacteria SK542 and S
The success of the symbiotic mixed culture of both strains of K522 enabled the following applications of the action system of microbial flora in natural ecosystems, agricultural ecosystems, and factory production systems, and ensured its practical application. . (1) It enabled the decomposition and utilization of various kinds of natural persistent organic substances, and not only decomposed and digested it, but also produced some useful substances. (2) The decomposition was quick and stable, the productivity was improved, and the field composting method was secured. (3) Furthermore, a thermophilic rapid decomposition method was established in which a complicated decomposition process of persistent organic substances was carried out in one step in the shortest possible time. (4) The formation of a strong and active soil microbial flora mainly consisting of the SK522 strain and the SK542 strain stabilized the growth and action function of effective bacteria against changes in environmental factors. (5) At the same time, it was possible to enhance the resistance to contamination by various bacteria and harmful bacteria, and eliminate and overcome soil diseases and continuous crop damage. (6) The application of good quality compost produced as described in the preceding paragraph, composting, or the development of a sprinkling technique to be applied directly to the foliage of soil or plants improves the physical properties of the soil, especially in the case of green farmland. It was possible to activate plants and increase the fertility for maintaining productivity. (7) It is also effective for deodorizing human waste and decomposing hard proteins and excess sludge, and effectively decomposes hardly decomposable fiber substances by using these as nitrogen sources.

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[Procedure amendment]

[Submission date] November 12, 1992

[Procedure Amendment 1]

[Document name to be amended] Statement

[Name of item to be corrected] Claim 1

[Correction method] Change

[Correction content]

[Procedure Amendment 2]

[Document name to be amended] Statement

[Name of item to be corrected] 0005

[Correction method] Change

[Correction content]

[0005]

Means for Solving the Problems The gist of the present invention which has solved the above problems is as follows: 1) Absolute aerobic, optimum growth temperature of 72 to 76 ° C., 40 to 40 ° C.
It grows in a medium with a normal concentration in the temperature range of 82 ° C
75-85 ° C, working hydrogen ion concentration pH = 4.0-1
1.3 High temperature broad-range action hydrogen ion concentration active protein
Therma that produces cytolytic enzymes and carotenoid yellow pigments
Scan aquaticus bacteria (Thermus aquaticusbiovar SK
542, Micro Engineering Research Institute No. 3382) 2) Thermus aquaticus bacteria (Thermu) described above.
s aquaticus biovar SK542, Micro Engineering Kenjoyori 338
No. 2) and lignin solubilizing ability, and the optimum growth temperature is 65
The thermophilic fibrinolytic bacterium Clostriduum thermocellum SK522 (Clostriduim thermocell), which grows in the temperature range of 40 to 80 ° C. at a temperature of up to 72 ° C.
um biovar.SK522, Microtechnical Research Institute No. 3459) and a mixed culture obtained by symbiotic mixed culture in a thermophilic environment of 60 ° or more 3) An organic material containing the mixed culture according to 2 as an effective main component It is in the fermentation decomposition agent. Hereinafter, Thermus aquaticus bacteria (Thermus of the present invention
aquaticus biovar SK542, Micro Engineering Lab. No. 3382
No.) and the thermophilic fibrinolytic bacterium Clostriduim thermocellum SK522 (Clostriduim thermocellum biova
r.SK522, Microtechnical Research Institute No. 3459) is related to the discovery of the present inventors.
It is referred to as the K542 strain and the SK522 strain.

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Claims (3)

[Claims] 1. A Thermus aquaticus biovar SK542, which is characterized by producing a carotenoid-type yellow pigment belonging to the genus Thermus (Thermus aquaticus biovar SK542, Microtechnology Research Institute No. 3382). 2. The Thermus aquaticus biovar SK542, Mikori Kenjoyori No. 3382, which has the ability to solubilize lignin, and a suitable growth temperature at 65 to 72 ° C. The thermophilic fibrinolytic bacterium Clostridium thermocellum SK522 (Clostridui) that grows in the temperature range of -80 ° C and ferments fibrin actively
m thermocellum biovar.SK522, Micro Engineering Lab. No. 34
No. 59) and a mixed culture obtained by symbiotic mixed culture under a thermophilic environment of 60 ° or more. 3. A fermentative decomposition agent for an organic material, which comprises the mixed culture according to claim 2 as an effective main component.