TWI280977B - Novel microorganisms - Google Patents

Abstract

This invention provides novel ultra-thermophilic microorganisms. Ultra-thermophilic microorganisms (Caldothrix satsumae), originally from the compost fermented from organic waste above 85 DEG C, are belonged to new species and are able to growth above 80 DEG C. The microorganisms of this invention do not grow below 50 DEG C, actively grow at 70 to 85 DEG C, without spore formation and being Gram-negative absolute aerobic microorganisms. The microorganism of this invention can be used as the fermenting microorganisms in the production of organic waste compost by high temperature fermentation. Also, the microorganisms of this invention can be used in the production of enzymes with thermal stability.

Description

1280977 IX. Description of the invention: [Technical field to which the invention pertains] The enrollment of the present invention is derived from compost and is available. Growing above. The present invention relates to novel hyperthermophilic bacteria, particularly novel hyperthermophilic bacteria having a low salt concentration and their use. [Prior Art] Aerobic fermentation has been carried out by using hot microorganisms in organic waste such as livestock manure, urine, sludge, and garbage, to produce a odorless dry compost. Therefore, the thermophilic microorganism can be exemplified by a thermophilic radiobacteria belonging to the genus Thermoactinomyces or Thermomonospora (Patent Document 1); Bacillus, Bacillus licheniformis Genophyte spore-forming bacteria mixture of genus (Geobacillus), lactic acid bacteria, etc. (Patent Document 2); aerobic Bacillus subtilis (Patent Document 3), thermophilic decomposition of the lyophilized lignin ability, Thermusacticus (Patent Literature) 4), aerobic fiber decomposing bacteria 5) and the like.

Clostridium thermocellum thermusacticus) (Patent literature However, despite the use of these microorganisms, the acid fermentation heat generated by fermentation can increase the fermentation temperature to above 70 °C, but the highest is only up again. In particular, the useful bacteria in the compost of the bud-forming bacteria are only about 100 million per gram per gram (fertilizer), and can not be fully utilized as a fertilizer. Similar problems in sludge treatment, and the sludge _ sludge is subjected to acid fermentation at a temperature higher than 85 ° C, preferably above 95 ° C; the bacteria, grass seeds, etc. are eliminated, not only the sludge Purification, and can be obtained by people / 315988 5 1280977 Forced dioxin: study of mash. The results showed that in the sludge, Tim, and ^ two were raised from the soil of the volcanic area of Kirishima, Japan. The bacterial culture, aeration and fermentation to make the fermentation temperature of 85 〇C to the only person:: 3 of the mixed cockroach, the seeds are eliminated, the sludge is purified, and the sludge of most useful bacteria is obtained. Method and obtain a patent (specialized = 6). Sludge acid yeast is used in the manufacture of compost, and many kinds of aerobic spores, thermophilic aerobic spores, thermophilic bacteria of the genus BaciUus, Ge〇baciiius are found. In other words, the sludge fermentation product is about gram grams, and as shown in the table, the average sludge acid yield per liter contains about 1 billion bacteria mainly composed of aerobic bacteria, high temperature bacteria, and heat-resistant spores. Table 1 Aerobic bacteria Southern temperature bacteria Heat-resistant spores Intestinal bacteria Gram-negative Gram-positive lactic acid bacteria Anaerobic bacteria Medium temperature release High-temperature actinomy filamentous fungus Yeast bacteria 9·9χ 1 〇Γ 8·4χ 107 2.8 χ ΙΟ 7 100 or less 100 or less 2.8 χ ΙΟ 5 100 or less 100 or less l.lx ΙΟ 3 6. Οχ ΙΟ 2 100 or less 100 or less, and in this culture, colonies which are superior in breeding jiii are selected as separation The bacteria's observation of the morphology of the isolates, etc., clearly identified the presence of microorganisms such as r in the microbial search for the fermentation of 315988 6 1280977. Table 2 Approximate number of grams - 7x 102 3x 108 8x 107 Bacteria 107 lx 103 6x 1 02 Separation of high-temperature I-type thermophilic actinomycetes from high-temperature bacteria can clearly distinguish between pleomorphism, spore-free Gram-positive bacilli, and aerobic spores (medium temperature and high temperature). On the other hand, on the other hand, the thermophilic bacteria were measured by referring to the "Microbial Separation Method and the J-Knife Separation" edited by R.A. The dominant bacteria of thermophiles are aerobic spores (high temperature). In addition, in the above-mentioned microbial search, there will be an advantage to isolate 4 mesenchymal aerobic spores (isobacteria a), high temperature aerobic buds b), and thermophiles (isolated bacteria c Into the shape of the rod can be said ##园 (knife heart, physiology test and determination of the DNA of the bacteria in the human body L 3 1, the results are shown. 315988 7 1280977 Table 3 test project test results Isolated bacteria a isolated bacteria b isolated bacteria c. bacillus gram Gram stain + + + spore + + + shape circular to elliptical elliptical elliptical position central near end end to end sporangia not protruding prominently to slightly Prominent mobility - - + Demand for oxygen Aerobic aerobic aerobic catalase + + + Propagation under anaerobic - - - VP reaction - - - VP liquid medium 6.5 8.0*2 5.6 pH Acid production of glucose _*2 arabinose NT _*2 NT xylose NT _*2 NT mannitol NT _*2 NT by the gas of glucose - _*2 - the production of casein decomposition + pen NT*3 gelatin Gasification + - + Decomposition of starch 瞧 - - Utilization of citrate - _氺2 - utilization of propionate - _*2 - decomposition of tyrosine - NT phenylalanine deamination - NT NT reaction yolk reaction surface nitrate reduction + - - ρΗ6·8 Reproduction + - + 8 315988 1280977 (nutrition liquid medium) Propagation in ρΗ5·7 in 5% NaCl + + Subsequent reproduction in 7% NaCl presence + + subsurface reproduction at 10 ° C reproduction _ . NT 30 °c reproduction + slow - reproduction at 40 ° C + + + breeding surface at 50 ° C + NT propagation at 55 ° C NT + + reproduction at 65 ° C NT - + reproduction at 70 ° C NT NT + propagation at 71 ° C NT NT + reproduction at 72 ° C NT NT - 521i 521i 401 GC content of DNA in vivo (mole %) 9 315988 1 1 according to HPLC method. 12 use adjustment to ρ Η 8 · Medium of 0. 13 NT : No test. The isolate a is inconsistent with the characteristics of any strain, so the strain cannot be determined so far. The isolate b is shown to be weakly alkaline (pH 8.0 to pH 8.5). Good reproduction in the medium, but not at pH 7.0, but by other trait test results, it is considered to be with the brown pepper spore ⑽ do βίΖ / w) or Bacillus brevis (5. AreWs) similar. However, both are strains with atypical atypical traits that cannot be fully confirmed. Further, the isolated strain c was identified as the same species as the Bacillus thermophilus Bacillus stearothermophilus, but the GC content was large, and thus it was identified as a closely related species. 1280977 These isolates are deposited at the Institute of Biotechnology of the Industrial Technology Institute of Japan (now the Patent Microbiology Depository Center of the Institute of Industrial Technology, Independent Legal Corporation). The registration numbers are: Separation bacteria a is YM-01 registration number FERM P-15085, isolate B is YM-02 accession number FERM P-15086, and isolate c is YM-03 registration number FERM P-15087. DISCLOSURE OF THE INVENTION The present inventors further explored microorganisms that have been propagated in such high-temperature compost, and surprisingly found that they can grow actively at a temperature higher than 75, and can grow even at 85 ° C. Hyperthermophilic strain YM 081 (FERMP-18598) (FERM-18-8) belonging to the genus Hymena which cannot grow below 50 °C. Then, the hyperthermophilic bacteria in the compost were investigated, and the homologous bacteria (Co/doi/zr/x YM 081 had a homology of more than 98% in the base sequence of i6S rDNA, In addition, there are several types of microbial strains, such as a nutrient requirement or an optimum salt concentration, which are different from the YM 081. Patent Document 1 JP-A-55-121992 Patent Document 2 JP-A-51-129759 (Patent Document 3) Japanese Patent Application Publication No. Hei. No. Hei. No. Hei. No. Hei. No. Hei. No. Hei. No. Hei. No. Hei. A new strain belonging to the genus Hyperthermophilus obtained from the compost of 85 ° C or higher. Further, the object of the present invention is to provide growth at 10 315 988 1280977 8 (above TC, the optimum salt concentration is 1% or less) It belongs to the new strain of hyperthermophilic bacteria (Ca/t/oi/zrz.x sahi/wfle). The purpose of the present invention is to use such hyperthermophilic bacteria, especially the super-thermophilic with low optimum salt concentration. bacteria

In order to study the new bacteria of the hyperthermophilic bacteria, the compost (c〇mPost) (trade name\samovar) obtained by fermenting the above sludge at 80 ° C. or higher, especially above 85 ° C, attempts to search for the existence. Among them, thermophilic microorganisms are found in a culture temperature (30 to 40 ° C) that cannot grow, at 70 to 85 ° C, ^ FERM P-18598 (FERM BP-8233) 〇 国 食品 food 910212 〇 special It is an aerobic bacteria that actively breeds above 80 °C. Therefore, the bacterium was classified and analyzed based on the sequence of the 16 S rDNA. As a result, it was found that the absolute aerobic bacteria were Gram-negative and spore-forming, as shown in Fig. 1, and the gram-positive soil of the gram-positive soil Bacillus genus, Bacillus licheniformis (Geobacillus) Genus is a close relative, and at least the genus is an independent bacterium. The inventor named it sabwwfle YM0 81, and deposited it in the Patent Biological Depository Center of the Institute of Industrial Technology and Technology, Independent Legal Person, with a registration number of 10. Also, the strain is deposited with China

Shown in the sequence listing number

''16S rDNA of the bacteria

The compost (trade name Samo) is separated. There is a machine waste method, which is obtained by high temperature fermentation of the household water. The separation method can use the following 315988 11 1280977 method. Add 5 ml of the above-mentioned compost to the medium of the composition described in Table 4, and add the above-mentioned compost to about 1 gram, and heat it to 8 〇r. After successively concentrating, add the gallan gum to the same medium to prepare the planar medium. On the addition, the separation and purification are repeated. Table 4 Medium composition soluble starch 〇·1 g casein 〇·3 g NaCl 3 g yeast extract 〇·2 g water 100 ml ρΗ7·2 The microbiological properties and taxonomic position of the obtained cells are as follows (1) The shape of sorrow is 0.5/zm wide and 3#m long. The results of Gram staining were negative. The ultrathin sections of the cells were observed under an electron microscope, and the cell surface structure was also Gram-negative. That is, it shows that there is an outer membrane other than the cell membrane and the cell wall. No spore formation ability. (2) It can be actively propagated from 70 to 85 C, and does not grow below 5 °C. At 75. (: The above high temperature can grow actively, and it can grow even if it is hungry. It is an aerobic bacteria. (3) The optimum pH for growth is neutral. The pH range of growth can be 6 to, and it shows weak salt. (4) It has the utilization of albumin, casein and various proteins and starches. (5) It has the productivity of urease. It has no reducing ability of nitric acid. It has no hydrogen sulfide, 吲315988 12 1280977 The productivity of mouth. (6) The G+C content of DNA was 70.0%. (7) Systematic classification analysis was performed based on the base sequence of 16SrDNA. The results are shown in Fig. 1 and Table 6. The complete base sequence of 16S rDNA is shown in the sequence table. The base sequence described in No. 1. Thus, although the bacterial cell of the present invention is Gram-negative, it has no sporulation ability, and is a spore-forming gram-positive soil bacterium Bacillus genus, ground spore The genus Geobacillus is similar, but at least it is a bacterium belonging to the genus. The microorganism of the present invention belongs to the eubacteria and is a hyperthermophilic bacterium. Furthermore, the sequence of the 16S rDNA is known. Although with the thermophilic bacillus The inventors formed a separate genus. Then, the inventors used the medium shown in Table 4 as a basic component to carry out research on new strains, and isolated several super-highly thermophilic bacteria. Named YMO 803, YMO 806, YMO 811, YMO 812, YMO 813. These strains were identified as the same strain as YMO 81. However, the optimum salt concentration of any of these strains is less than 1%, compared to super Thermophilic bacteria (Cfl/i/oi/zr/x sahwmfle) The optimum salt concentration of YMO 8 1 is 3%, which is characterized by a low optimum salt concentration. All of the strains are long bacilli, non-spore forming ability, Gram Negative highly thermophilic eubacteria and capable of growing at least 80 ° C. All strains use casein, soluble starch as a carbon source, nitrogen source, and have an optimum pH range of pH 7.5 to 8.5. However, for growth The nutritional requirements and the maximum salt concentration of 13 315988 1280977 are different. In addition, although the sequence of 16S rDNA is different from each strain, it has 98% or more sequence homology with YMO 81. Can be identified all the same species. [Brief Example] Next, the reference examples and the examples of the present invention will be described. However, the present invention is not limited to the ones of the reference examples and the examples. Reference Example 1 37 to 40 C of the sulphur zone of the Kirishima volcanic belt in the Kagawa-gun, Kagawa-gun, Kagoshima, Kagoshima, and the soil of the nearby paddy field, and the soil mixture of 3 to 4 liters of dissolved sucrose water per cubic meter of soil mixture. (sucrose: water = 1: 500 to 1000), placed at 40 to 5 (rc placed in culture for up to % days). The sputum culture was mixed with several batches of raw sludge, which was blown to ferment under aerobic conditions, and the batch obtained at a fermentation temperature of 85 < t or higher was used as the corpus callosum culture. Adding slaked lime to the animal's manure, the sewer's dirt, and the long-term planting of the slaked lime, and adding 80 parts by weight of the mixture to the above-mentioned cell culture, 2 parts of the mixture, 'ventilation in the fermentation tank' The following is carried out: according to the fermentation, the temperature rises from room temperature to Μ3 at this temperature for 3 days. The fermentation is carried out on the 5th day after the start of the fermentation, and the temperature is lowered by about 16 , by the soil. About! Days> The degree of the dish rises to 85 to hunger. Maintain this temperature for 5 days for acid fermentation 315988 14 1280977 = After the fermentation and tumbling process is repeated several times, the temperature and fermentation temperature of the soil are gradually reduced. After repeating the above procedure four times, the temperature of the fermentation time was lowered to 35 ° C when the soil was turned to the final fermentation date. The obtained fermentation product was used as a carcass culture. The bacterial culture was dried to brown. Granular 'can be used directly as an organic fertilizer.

^The public sewage sludge of Kagoshima City, Kagoshima City, Japan was crushed = water, and the sludge with a water content of 68% was mixed with 20 parts by weight of the above-mentioned corpus callosum culture and placed in 酦. The yeast is placed in the field and fermented by the air below y ^. The temperature reached 7 after 7 days of fermentation. Continue to the island to ferment for 1 day, when the fermentation temperature begins to drop from 98t, stir it = its fermentation. After the machine was first reached, that is, the first day after Lin, the temperature dropped to 6〇 to 7 (rc.) At this time, the acid yeast of the yeast plant was set up to 'not expand', so that the temperature quickly fell to a long temperature. The brown sludge acid yeast powder is obtained, and the sludge powder can be used as a compost, or a carcass culture or a medium for the above acid fermentation.

The compost of the example was prepared into a sample about 〇.... inoculated in Table 4 ^ Maosheng' was kept at 80V, and after repeated submerged concentration, it was reversed on the plate medium prepared by adding the blue gum. The hyperthermophilic bacteria of the present invention are obtained by knife and chemical. ",, · ° 〇 · 3 /., ρ Η 7 · 2), cultured at 70 ° C, the 315988 15 1280977 long bacteria were separated on agar medium (pH 7.2, 70%), found at temperature 7 (above TC, in addition to the traditional Bacillus thermophilus (10)), there are many other novel thermophilic bacteria. 2. Microbiological properties of hyperthermophilic bacteria The super-therapeutic of the invention obtained in Example 1 The hot bacteria were inoculated on an agar medium consisting of casein 0.3%, yeast extract 0.2%, starch 0.1%, NaCl 3%, and ρΗ7 to 8, and cultured at 80 ° C for 24 hours to investigate the microbiological properties. It is shown in Table 5. The micrograph of this hyperthermophilic micrograph is shown in Fig. 2 to Fig. 5. Table 5 Test results Test results Stereobacteria (Blendon ·5 // m, length 3 // m) Gram Negative spore formation ability of S. sinensis has no motility, no need for oxygen. Absolute aerobic anaerobic reproduction does not grow. It is actively propagated at 70 to 85 °C, and pH is not grown below 50 °C. pH 6 to 9 can grow) The salt of growth is weak and salty (optimal salt concentration 3%) white Proteolysis + Decomposition of brewing protein decomposition sugar + L-arabinose + D-xylose - D-glucose + D-mannose soil D-fructose + 16 315988 1280977 D-galactose - maltose + Yan sugar · Lactose - trehalose (trehalose) + D-sorbitol-D-mannitol soil inositol - glycerol + temple powder + nitrate reduction - acetoin - the production of hydrogen sulfide - the formation of sputum - bacteria The DNA of the medium DNA contains 70.0 (mol%). The other base sequence is determined based on the base sequence of 16SrDNA, and the molecular phylogenetic tree is completed. The results are shown in Fig. 1 and Table 6. 17 315988 1280977

1 T. thermo philus L _ 〇〇CD ο ii ( T. mariti j ma I g £2 / / / / a (D aw CO a: 茗si / / G. stearoth ermophilus QO ο 00 / / / / . E· coli OQ / / /, / B. subtilis ii / L turice nsis 〇0 i ! / / 4 C. satsu | mae | /: Ψ / / / / / / ..1 <D 目》 • fl W 0 ϋ 暴 1-t Vh 〇S § 杉〇铝 Racillus subtil is (Bacillus subtilis) Escherichra coli (E. coli) l §« 1 per IS • i—1 0 a CO cd g® § 酴 酴 Dm'w/ •^-4 •i-4 Θ 03® 〇馋名妒§ rH ·— ao IS 18 315988 1280977 The results show that the hyperthermophilic bacteria belong to eubacteria and are Gram-negative and have no stalk formation ability. It is closely related to the gram-negative soil of the gram-negative soil, Bacillus and Geobacillus, but belongs to the new genus. Furthermore, the GC content of the DNA is shown in Table 6. , not reaching 90% of the homologous bacteria, but also deviated from the genus Bacillus and the genus Bacillus, respectively (85% each), and judged It is a new genus (refer to Table 6 and Figure 1). Therefore, the genus is named Caldothrix. Since the optimum growth temperature is 80 ° C, it can be confirmed that the bacterium is a hyperthermophilic bacterium. Thermophilic sflhwmae) The comparison of the biochemical properties of YMO 81 with Bacillus subtilis is shown in Table 7.

Table 7 YMO 81 B. substilus oxidase activity - - hydrogen sulfide production capacity - - galactosidase activity + + leucine acid fermentation capacity - - ethoxylated sterol production capacity - + 吲哚 production capacity citric acid production Ability + + ability to produce lysine - - ability to produce ornithine - arginine production capacity - - urea decomposition ability + - malonate utilization capacity - - nitrate reduction ability + 19 315988 1280977 Example 2 Reference example The obtained compost is prepared into a sample of about 〇g, and is inoculated into the table: ΓΓ, 1 , and kept at 80 ° C, and after successively concentrating, ^ 2:: mixed with the glutinous rice to form a plane The medium was reversed: "^ purified to obtain the super-heat bacterium of the present invention. The isolated strains were named as YM〇8〇3, ΥΜ(10)6, γΜ〇chuan, ΥΜ0812, ΥΜ0813. The whole site is more than Daban & 士^.. Wang Chongxi is a long-growing bacterium, no spores form a sputum, a sputum-negative highly thermophilic eubacteria and at least a sail can be used. The soluble temple as a carbon source, nitrogen 仏 'has the optimum range of ΡΗ 7.5 ^ 8.5. However, as described below, the nutritional requirements and the optimum salt concentration are different. In particular, it has an optimum salt concentration of 1% or less, particularly G 3 to G W . The feature point. Among them, 'YM〇8G6|M especially shows that the optimum salt concentration is low and growth activity.

Furthermore, the base sequence of '16SrDNA is different from each other, but the j YM (10) strain contains 98% or more of sequence homology with each other, and ^ 315988 20 1280977 is the same species. These strains have different properties from the YM081 strain and are shown in Table 9. It can be seen from the table that some of the bacteriality is necessary for the addition of trace amounts of metal elements. The composition of the trace metal element is shown in Table 10. Some of the strains require composting extracts for growth. The preparation method of the compost extract is shown in Table 11. Table 9 Differences in growth properties of each strain Strain name optimum salt concentration Growth time Nutritional requirements YMO 803 0.4% 73〇C 38 points Minor metal element YMO 806 0.2% 80°C 35 points Minor metal element YMO 811 0.3% — Requires compost extract requires trace metal element YMO 812 0.5% 70°C 29 points Composting extract YMO 813 0.3% - Composting extract required Table 10 Trace metal composition Table concentration (mg/L) NaMo03· 2H20 1.2 V0S04· χΗ20 0.2 MnCl2 · 4H20 0.5 ZnS04 · 7H20 0.06 CuS04· 5H2〇0.015 CoC12 · 6H20 0.08 NiCl2 · 6H20 0.02 FeS04 · 7H20 10 MgCl2 · 6H20 125 CaCl2 · 2H20 25 21 315988 1280977 Table 11 Preparation of composting solution 1 Composting 5克 suspended in H20 50 ml, centrifuged to extract the supernatant through a sterilizing filter, and then stored in a refrigerated operation. 2 per 10 ml of the medium, added the above-mentioned preservation solution to the medium 0.4 ml of the inventors, and deposited the strains in an independent administrative entity industry. The Patent Biological Depository Center of the Institute of General Research has obtained the following registration number. Strain name Registration number YMO 803 PERM P-19412 YMO 806 FERM P-19413 YMO 811 FERM P-19414 YMO 813 FERM P-19415 The sequence of the 16S rDNA of these strains is shown in Sequence Listing Nos. 2 to 6. As shown in Table 12, although the nucleotide sequence of 16S rDNA differs from each strain, it has 98% or more sequence homology with YMO 81, and all of them can be identified as the same species. Thus, YMO806 was changed to an international deposit under the Budapest Treaty, and the registration number FERH ABP-10037 was obtained. The hyperthermophilic bacteria (Cfl/i/oMr/x(10)YMO806 strain has a particularly low optimum salt concentration (0.2%), has less nutrient requirements, and has the characteristic point of super-high heat bacteria which are easy to culture. Therefore, the strain is easy to grow. The heat-resistant enzyme can be obtained therefrom, and the strain can be added to organic waste (such as urine, livestock waste, municipal waste, sludge, etc.), and the organic waste can be at a high temperature of 80 ° C or higher, preferably 85 ° C. Above, it is better to decompose and ferment by ultra-high temperature fermentation of l〇〇°C or more, and to make compost. 22 315988 1280977 Table 12 Comparison of 16S rDNA base sequence homology (%) YMO 81 YMO 803 YMO 806 YMO 811 YMO 812 YMO 813 YMO 81 m~ 99 99 99 99 99 YMO 803 99"~' 100 99 99 99 99 YMO 806 99 99 100 99 99 99 YMO 811 99^~~ 99 99 100 99 99 YMO 812 99 99 99 99 100 99 YMO 813 99^~ ~~~ 99 99 99 99 100 Hjj in the industry uses organic waste such as urinary urinary as raw material, inoculates the fermented culture, and carries out vinegar's genus because it belongs to the genus Bacillus. , Geobacillus (Geobacillus), moderate thermophilic bacteria, etc. The temperature rises. After that, due to the increase in acid fermentation temperature, several super-thermophilic bacteria Caldothrix satsumae of the present month participate in the decomposition and fermentation of organic waste in the southeast compost. Therefore, the present invention is super Thermophilic bacteria decompose organic waste (purine, livestock, municipal waste, sludge, etc.) at high temperature to ferment it, and it is used as a strain, medium, etc. for composting. The produced protease and amylase are active at high temperatures, and it is desirable to use this property to produce a heat-resistant enzyme. [Simplified description of the drawing] Fig. 1 is a reference to 16S rDNA of the Caldothrix superthermophilic fungus like hwmae) of the present invention. Sub-system tree. Fig. 2 is an optical electron micrograph of the hyperthermophilic strain YMO 81 of the present invention. Fig. 3 is a scanning electron microscope photograph of the super-thermophilic strain 23 315988 1280977 YMO 81 of the present invention. Fig. 4 is a transmission electron micrograph of the hyperthermophilic bacteria (C^/(ic)//2Wx sahww(10)) YMO 81 strain of the present invention. Fig. 5 is an electron micrograph of the ultrathin section of the hyperthermophilic bacteria (Cfl/doMrz'x saiwm heart) YMO 81 of the present invention (magnification: 5000 times). 24 315988 1280977 Sequence Listing <110> Shanyou Co., Ltd. <120> Novel Microorganisms <130> SANYU3 <150> Japan's Special Purpose 2003 - 181282 <151> June 25, 2003 <160〉 6 <210〉 1 <211> 1541

≪ 212 > DNA < 213> Caldothrix satgumae YM081 < 400> 1 agagtttgat gfgfcttttc cgtaacacgt ccggatagga ggatgggccc gtagccggcc g £ gaggcagc cctggctcag gcgtgaagcc gggcaaccta cggcggaccg gcggcccatt tgagagggtg agtagggaat gacgaacgct ttcgggcgga ccccgaggac i .. catggtccgc agcttgttgg accggccaca cttccgcaat ggcggcgcgc tcgcggggag cgggataact cgtggaaagg tggggtaacg ctgggactga gggcgaaagc ctaatacatg agcctagcgg ccgggaaacc cggcgcaagc gcccaccaag gacacggccc ctgacggagc caagtcgagc 60 cgaacgggtg 120 ggggctaata 180 tgccacctcg 240 gcgacgatgg 300 agactcctac 360 gacgccgcgt 420 1 315988 1280977 gagggaggaa aagagggccg ccgcggtaaa gcggcctctt ggg ^ ggcttg gatcgggagg S gaaagcgtgg gctaggtgtg ggggagtacg v gagcatgtgg ggccttcggg tcgtaaacct ctgttgtcag ggacgaaccc gtgcggttcg 480 cgcgctgacg gtacctgacg aggaagcccc ggctaactac gtgccagcag 540 Acgtaggggg cgagcgttgt ccggaattac tgggcgtaaa gcgcgcgtag 600 aagtccggtg tgaaagcccg cggctcaacc gcgggaggcc actggaaact 660 agggcaggag aggggagtgg aattcccggt gtagcggtga aatgcgtaga 720 aacaccagtg gyg aaggcgg ctccctggcc tgtacctgac gctgaggcgc 780 ggagcaaaca ggattagata ccctggtagt ccacgccgta aacgatgggt 840 aggggcgttt ggcccttcgt gccgaagcta acgcgataag caccccgcct 900 gccgcaaggc tgaaactcaa aggaattgac gggggcccgc acaagcggtg 960 tttaattcga agcaacgcga agaaccttac cagggcttga catcccgctg 1020

accgccccag agatggggtt tccctccttt cggagggcag cggtgacagg tggtgcatgg 1080 ttgtcgtcag ctcgtgtcgt gagatgttgg gttaagtccc gcaacgagcg caacccctgc 1140 caagccggag 1200 cacgtgctac 1260 aagccggtct 1320 gtaaatccgc 1380 cgtcacacca 1440 agccgccgaa 1500 1541 ccctagttgc gaaggtgggg aatggccggt cagttcggat gggatcagca cgagagtctg ggtggggcag cagcgggtga atgacgtcaa acaaagggtt tgcaggcttg tgccgcggtg caacacccga atgattgggg ggccgggcac atcatcatgc gcgaacccgc caactcgcct aatacgttcc agtcggtgcg tgaagtcgta tctaggggga cccttatgcc Gagggggaggc gcatgaaggc cgggccttgt ccaacccctt acaaggtaac ctgccggcga ctgggctaca caatcccaaa ggaatcgcta acacaccgcc acggggaggc c <210> 2 <211> 1523

&lt;212> DNA &lt;213&gt; Caldothrix satgumae YM0803 <400> agagtttgat cctggctcag gacgaacgct ggcggcgcgc ctaatacatg caagtcgagc 60 ggggcttttc gcgtgaagcc ttcgggcgga tcgcggggag cctagcggcg aacgggtgcg 120 taacacgtgg gcaacctacc ccgaggaccg ggataactcc gggaaaccgg ggctaatacc 180 2

315988 1280977 ggataggacg gcggaccgca tggtccgccg tggaaaggcg gcgcaagctg ccacctcggg 240 atgggcccgc ggcccattag cttgttggtg gggtaacggc ccaccaaggc gacgatgggt 300 agccggcctg agagggtgac cggccacact gggactgaga cacggcccag actcctacgg 360 gaggcagcag tagggaatct tccgcaatgg gcgaaagcct gacggagcga cgccgcgtga 420 flgaggaagg ccttcgggtc gtaaacctct gttgtcaggg acgaacccgt gcggttcgaa 480 gagggccgcg cgctgacggt acctgacgag gaagccccgg ctaactacgt gccagcagcc 540 gcggtaaaac gtagggggcg agcgttgtcc ggaattactg ggcgtaaagc gcgcgtaggc 600 ggcctcttaa gtccggtgtg aaagcccgcg gctcaaccgc gggaggccac tggaaactgg 660 gaggcttgag ggcaggagag gggagtggaa ttcccggtgt agcggtgaaa tgcgtagaga 720 tcgggaggaa caccagtggc gaaggcggct ccctggcctg tacctgacgc tgaggcgcga 780 aagcgtgggg agcaaacagg attagatacc ctggtagtcc acgccgtaaa cgatgggtgc 840 taggtgtgag gggcgtttgg cccttcgtgc cgaagctaac gcgataagca ccccgcctgg 900 ggagtacggc cgcaaggctg aaactcaaag gaattgacgg gggcccgcac aagcggtgga 960 gcatgtggtt taattcgaag caacgcgaag aaccttacca gggcttgaca tcccgctgac 1020 Cgcc ccagag atggggtttc cctcctttcg gagggcagcg gtgacaggtg gtgcatggtt 1080 gtcgtcagct cgtgtcgtga gatgttgggt taagtcccgc aacgagcgca acccctgccc 1140 ctagttgcca gcgggtgagg ccgggcactc tagggggact gccggcgaca agccggagga 1200 aggtggggat gacgtcaaat catcatgccc cttatgccct gggctacaca cgtgctacaa 1260 tggccggtac aaagggttgc gaacccgcga gggggagcca atcccaaaaa gccggtctca 1320 gttcggattg caggctgcaa ctcgcctgca tgaaggcgga atcgctagta atcgcggatc 1380 agcatgccgc ggtgaatacg ttcccgggcc ttgtacacac cgcccgtcac accacgagag 1440 tctgcaacac Ccgaagtcgg tgcgccaacc ccttacgggg aggcagccgc cgaaggtggg 1500 gcagatgatt ggggtgaagt cgt 1523 &lt;210> 3 <211> 1537 '

<212> DNA &lt; 213 &gt; Caldothrix satgumae YM0806 3 315988 1280977 &lt; 400> agagtttgat gcggggcttt cgtaacacgt ccggatagga ggatgggccc gtagccggcc gggaggcagc gagggaggaa aagagggccg ccgcggtaaa gcggcctctt gggaggcttg gatcgggagg gaaagcgtgg gctaggtgtg ggggagtacg gagcatgtgg accgccccag ttgtcgtcag ccctagttgc gaaggtgggg aatggccggt cagttcggat tcagcatgcc agtctgcaac gggcagatga cctggctcag tcgcgtgaag gggcaacctg cggcggaccg gcggcccatt tgagagggtg agtagggaat ggccttcggg cgcgctgacg acgtaggggg aagtccggtg agggcaggag aacaccagtg ggagcaaaca aggggcgttt gccgcaaggc tttaattcga agatggggtt ctcgtgtcgt cagcgggtga atgacgtcaa acaaagggtt tgcaggctgc gcggtgaata acccgaagtc ttggggtgaa gacgaacgct ccttcgggcg ccccgaggac catggtccgc agcttgttgg accggccaca cttccgcaat tcgtaaacct gtacctgacg cgagcgttgt tgaaagcccg aggggagtgg gcgaaggcgg ggattagata ggcccttcgt tgaaactcaa agcaacgcga tccctccttt gagatgttgg ggccgggcac atcatcatgc gcgaacccgc aactcgcctg cgttcccggg two ggtgcgccaa gtcgtaacaa ggcggcgcgc gatcgcgggg cgggataact Cgtggaaagg tggggtaacg ct gggactga gggcgaaagc ctgttgtcag aggaagcccc ccggaattac cggctcaacc aattcccggt ctccctggcc ccctggtagt gccgaagcta aggaattgac agaaccttac cggagggcag gttaagtccc tctaggggga cccttatgcc gagggggagc catgaaggcg ccttgtacac ccccttacgg ggtaacc ctaatacatg agcctagcgg ccgggaaacc cggcgcaagc gcccaccaag gacacggccc ctgacggagc ggacgaaccc ggctaactac tgggcgtaaa gcgggaggcc gtagcggtga tgtacctgac ccacgccgta acgcgataag gggggcccgc cagggcttga cggtgacagg gcaacgagcg ctgccggcga ctgggctaca caatcccaaa gaatcgctag accgcccgtc ggaggcagcc tctaagtcga 60 cgaacgggtg 120 ggggctaata 180 tgccacctcg 240 gcgacgatgg 300 agactcctac 360 gacgccgcgt 420 gtgcggttcg 480 gtgccagcag 540 gcgcgcgtag 600 actggaaact 660 aatgcgtaga 720 gctgaggcgc 780 aacgatgggt 840 caccccgcct 900 acaagcggtg 960 catcccgctg 1020 tggtgcatgg 1080 caacccctgc 1140 caagccggag 1200 cacgtgctac 1260 aagccggtct 1320 taatcgcgga 1380 acaccacgag 1440 gccgaaggtg 1500 1537 <210> 4 4 315988 1280977 &lt;211> 1511

&Lt; 212 &gt; DNA &lt; 213 &gt; Caldothrix satgumae YM0811 &lt; 400 &gt; aacgctggcg ggcggatcgc ggaccgggat ccgccgtgga ttggtggggt cacactggga caatgggcga gcgcgcctaa ggggagccta aactccggga aaggcggcgc aacggcccac ctgagacacg aagcctgacg tacatgcaag gcggcgaacg aaccggggct aagctgccac caaggcgacg gcccagactc gagcgacgcc tcgagcgggg ggtgcgtaac aataccggat ctcgggatgg atgggtagcc ctacgggagg gcgtgaggga cttttcgcgt acgtgggcaa aggacggcgg gcccgcggcc ggcctgagag cagcagtagg ggaaggcctt gaagccttcg 60 cctaccccga 120 accgcatggt 180 cattagcttg 240 ggtgaccggc 300 gaatcttccg 360 cgggtcgtaa 420 acctctgttg gacgaggaag ttgtccggaa cccgcggctc gtggaattcc gcggctccct gataccctgg tcgtgccgaa tcaaaggaat gcgaagaacc tcagggacga ccccggctaa ttactgggcg aaccgcggga cggtgtagcg ggcctgtacc tagtccacgc gctaacgcga tgacgggggc ttaccagggc acccgtgcgg ctacgtgcca taaagcgcgc ggccactgga gtgaaatgcg tgacgctgag cgtaaacgat taagcacccc ccgcacaagc ttgacatccc ttcgaagagg gcagccgcgg gtaggcggcc aactgggagg tagagatcgg Gcgcgaaagc gggtgctagg gcctggggag ggtggagcat gctgaccgcc gccg cgcgct taaaacgtag tcttaagtcc cttgagggca gaggaacacc gtggggagca tgtgaggggc tacggccgca gtggtttaat ccagagatgg gacggtacct 480 ggggcgagcg 540 ggtgtgaaag 600 ggagagggga 660 agtggcgaag 720 aacaggatta 780 gtttggccct 840 aggctgaaac 900 tcgaagcaac 960 ggtttccctc 1020 ctttcggagg gcagcggtga caggtggtgc atggttgtcg tcagctcgtg tcgtgagatg 1080 ttgggttaag tcccgcaacg agcgcaaccc ctgcccctag ttgccagcgg gtgaggccgg 1140 gcactctagg gggactgccg gcgacaagcc ggaggaaggt ggggatgacg tcaaatcatc 1200 atgcccctta tgccctgggc tacacacgtg ctacaatggc cggtacaaag ggttgcgaac 1260 ccgcgagggg gagccaatcc caaaaagccg gtctcagttc ggattgcagg ctgcaactcg 1320 cctgcatgaa ggcggaatcg ctagtaatcg cggatcagca tgccgcggtg aatacgttcc 1380 cgggccttgt acacaccgcc cgtcacacca cgagagtctg caacacccga agtcggtgcg 1440 5 315988 1280977 ccaacccctt acggggaggc agccgccgaa ggtggggcag atgattgggg tgaagtcgta 1500 acaaggtaac c 1511 &lt; 210> 5 &lt; 211 &gt; 1535

&Lt; 212> DNA &lt; 213 &gt; Caldothrix satgumae YM0812 &lt; 400 &gt; agagtttgat cctggctcag gacgaacgct ggcggcgcgc ctaatacatg caagtcgagc 60 ggggcttttc gcgtgaagcc ttcgggcgga tcgcggggag cctagcggcg aacgggtgcg 120 taacacgtgg gcaacctacc ccgaggaccg ggataactcc gggaaaccgg ggctaatacc 180 ggataggacg gcggaccgca tggtccgccg tggaaaggcg gcgcaagctg ccacctcggg 240 atgggcccgc ggcccattag ctagttggtg gggtaacggc ccaccaaggc gacgatgggt 300 agccggcctg agagggtgac cggccacact gggactgaga cacggcccag actcctacgg 360 gaggcagcag tagggaatct tccgcaatgg gcgaaagcct gacggagcga cgccgcgtga 420 gggaggaagg ccttcgggtc gtaaacctct gttgtcaggg acgaacccgt gcggttcgaa 480 gagggccgcg cgctgacggt acctgacgag gaagccccgg ctaactacgt gccagcagcc 540 gcggtaaaac gtagggggcg agcgttgtcc ggaattactg ggcgtaaagc gcgcgtaggc 600 ggcctcttaa gtccggtgtg aaagcccgcg gctcaaccgc gggaggccac tggaaactgg 660 gaggcttgag ggcaggagag gggagtggaa ttcccggtgt agcggtgaaa tgcgtagaga 720 Tcgggaggaa caccagtggc gaaggcggct ccctggcctg tacctgacgc tgaggcgcga 780 aagcgtgggg agcaaacagg att agatacc ctggtagtcc acgccgtaaa cgatgggtgc 840 taggtgtgag gggcgtttgg cccttcgtgc cgaagctaac gcgataagca ccccgcctgg 900 ggagtacggc cgcaaggctg aaactcaaag gaattgacgg gggcccgcac aagcggtgga 960 gcatgtggtt taattcgaag caacgcgaag aaccttacca gggcttgaca tcccgctgac 1020 cgccccagag atggggtttc cctcctttcg gagggcagcg gtgacaggtg gtgcatggtt 1080 gtcgtcagct cgtgtcgtga gatgttgggt taagtcccgc aacgagcgca acccctgccc 1140 ctagttgcca gcgggtgagg ccgggcactc tagggggact gccggcgaca agccggagga 1200 aggtggggat gacgtcaaat catcatgccc cttatgccct gggctacaca cgtgctacaa 1260 6 315988 1280977 tggccggtac gttcggattg agcatgccgc tctgcaacac gcagatgatt aaagggttgc caggctgcaa ggtgaatacg ccgaagtcgg ggggtgaagt gaacccgcga ctcgcctgca ttcccgggcc tgcgccaacc cgtaacaagg gggggagcca atcccaaaaa gccggtctca 1320 tgaaggcgga atcgctagta atcgcggatc 1380 ttgtacacac cgcccgtcac accacgagag 1440 ccttacgggg aggcagccgc cgaaggtggg 1500 taacc 1535 &lt; 210> 6 &lt; 211> 1502

&lt;212&gt; DNA &lt;213&gt; Caldothrix satgumae YM0813

<400> aacgctggcg gcgcgcctaa tacatgcaag tcgagcgggg cttttcgcgt gaagccttcg 60 ggcggatcgc ggggagccta gcggcgaacg ggtgcgtaac acgtgggcaa cctaccccga 120 ggaccgggat aactccggga aaccggagct aataccggat aggacggcgg accgcatggt 180 ccgccgtgga aaggcggcgc aagctgccac ctcgggatgg gcccgcggcc cattagctag 240 ttggtggggt aacggcccac caaggcgacg atgggtagcc ggcctgagag ggtgaccggc 300 cacactggga ctgagacacg gcccagactc ctacgggagg cagcagtagg gaatcttccg 360 caatgggcga aagcctgacg gagcgacgcc gcgtgaggga ggaaggcctt cgggtcgtaa 420 acctctgttg tcagggacga acccgtgcgg ttcgaagagg gccgcgcgct gacggtacct 480 gacgaggaag ccccggctaa ctacgtgcca gcagccgcgg taaaacgtag ggggcgagcg 540 ttgtccggaa ttactgggcg taaagcgcgc gtaggcggcc tcttaagtcc ggtgtgaaag 600 cccgcggctc aaccgcggga ggccactgga aactgggagg cttgagggca ggagagggga 660 gtggaattcc cggtgtagcg gtgaaatgcg tagagatcgg gaggaacacc agtggcgaag 720 gcggctccct ggcctgtacc tgacgctgag gcgcgaaagc gtggggagca aacaggatta 780 gataccctgg tagtccacgc cgtaaacgat gggtgctagg tgtgaggggc gtttggccct 840 tcgtgccgaa Gctaacgcga taagcacccc gcctggggag tacggccgca aggctgaaac 900 tcaaaggaat tgacgggggc ccgcacaagc ggtggagcat gtggtttaat tcgaagcaac 960 gcgaagaacc ttaccagggc ttgacatccc gctgaccgcc ccagagatgg ggtttccctc 1020 7

315988 1080 1280977 ctttcggagg gcagcggtga ttgggttaag tcccgcaacg gcactctagg gggactgccg atgcccctta tgccctgggc ccgcgagggg gagccaatcc cctgcatgaa ggcggaatcg cgggccttgt acacaccgcc ccaacccctt acggggaggc ac caggtggtgc atggttgtcg agcgcaaccc ctgcccctag gcgacaagcc ggaggaaggt tacacacgtg ctacaatggc caaaaagccg gtctcagttc ctagtaatcg cggatcagca cgtcacacca cgagagtctg agccgccgaa ggtggggcag tcagctcgtg tcgtgagatg ttgccagcgg gtgaggccgg ggggatgacg tcaaatcatc cggtacaaag ggttgcgaac ggattgcagg ctgcaactcg tgccgcggtg aatacgttcc caacacccga agtcggtgcg Atgattgggg tgaagtcgta 1140 1200 1260 1320 1380 1440 1500 1502 8 315988

Claims (1)

1280977 Patent Application No. 93118451 Patent Revision (March 2, 1996) 1. A hyperthermophilic bacterium (Cladothrix satsumae, accession number BCRC 910278) having the following sequence of 16S rDNA at 80° Proliferation above C, and the optimum salt concentration is 1% or less agagtttgat cctggctcag gacgaacgct ggcggcgcgc ctaatacatg tctaagtcga 60 gcggggcttt tcgcgtgaag ccttcgggcg gatcgcgggg agcctagcgg cgaacgggtg 120 cgtaacacgt gggceacctg ccccgaggac cgggataact ccgggaaacc ggggctaata 180 ccggatagga cggcggaccg catggtccgc cgtggsaagg cggcgceagc tgccacctcg 240 ggatgggccc gcggcccatt agcttgttgg tgggftaacg goccaccaag gcgacgatgg 300 rtagcccgcc tgaeagggtg accegccaca ctgggactga gacacggccc a ^ actcctac 360 SESaggcagc agtagggaat cttccgcaat gggcgaaagc ctgacggagc gacgccgcgt 420 gagggaggaa ggocttcggg tcgtaaacct ctettgtcag ggacgaaccc gtgcggttcg 480 aagagggccg cgcgctgacg gtacctgacg aggaagcccc £ gctaactac gtgccagcae 540 ccgcggtaaa acetaggggg cgagcgtt ^ t ccggaattac tgggcgtaaa gcgogcgtag 600 gcggcctott azgtocggtg tgaaagoccg cggctceecc gcgggaggcc actggeaact 660 gggaggcttg agggcaegag aggegaetgg a &amp; ttcccegt gtagcggtga aatgcgtega 720 eatcgggagg aacaccagtg ecgaaggcgg ctccctggcc tgtacctgac gctgaggcgc 780 gaaagcgtgg ggagcaaaca ggattagata ccctggtagt ccacgccgta aacgatgggt 840 gctagctgtg aggggcgttt ggcccttcgt gccgaagcta acgcgataag caccccgcct 900 ggggagtacg gccgcaaggc tgaaactcaa aggaattgac gggggcccgc acaagcggtg 960 gagcetgtgg tttaattcga agcaacgcga agaaccttac c &amp; gggcttga catcccgctg 1020 accgccccag agatggggtt tccctccttt cggagggcag cggtgacagg tggtgcatgg 1080 ttgtcgtcag ctcgtgtcgt gagatgttgg gtta &amp; gtccc gcaacgagcg caacccctgc 1140 ccctagttgc csgcfggtga ggccgggcac tctaggggge ctfccggcga caagccggag 1200 gaaggtgggg atgacgtcaa atcatcatgc cccttatgcc ctgggctaca cacgtgctac 1260 aatggocggt acaaagggtt gcgaacccgc gagggggagc caatcccaaa aagccggtct 1320 cagttcggat tgcaggctgc aactcgcctg catgaaggcg gaatcgctag taatcgcgga 1380 tcagcatgcc gcggtgaata cgttcccggg ccttgtacac accgcccgtc acaccacga ^ 1440 agtctgcaac acccgaagtc ggtgcgccaa ccccttacgg ggaggcagcc gccgaaggtg 1500 gggcagatga ttggggtg &amp; a gtcgtaacaa ggtaacc 1537 2 · - types of compost (compost) the method for producing It is added to organic waste (屎315988 revised this 1280977 urine, livestock manure, sludge, etc.) When a composting mixture is produced by a medium-temperature aerobic spore, a thermophilic aerobic spore, or a thermophilic substance, the temperature rise phase is the super-hobby of the first item of the patent application scope. Thermobacteria (Ca/t/oi/zr/x action, and ultra-high temperatures above 85 °C occur. 315988 amendment