Hyperthermophile strain caldothrix satsumae capable of fermentation of organic waste at high temperatures

FIELD: biotechnology, agriculture.

SUBSTANCE: strain Caldothrix satsumae YM081 is isolated from compost by fermentation of organic waste at 85°C or higher. Strain is deposited under number FERM BP-8233.

EFFECT: hyperthermophile strain capable of fermentation of organic waste at high temperatures to produce compost.

6 dwg, 8 tbl, 3 ex

Technical area

The present invention relates to a new hyperthermophile obtained from compost, which can multiply at 80°C or more.

Previous research in this area

Previously thermophilic microorganisms designed to impact on organic waste, such as excrements of domestic animals, excrement and urine, sludge and municipal waste, for the aerobic fermentation of waste and to make them odorless and dry, and thus, compost. In addition, as of such thermophilic microorganisms known thermophilic actinomyces belonging to genera or Thermoactinomyces Thermomonospora (JP 55-121992 A), a mixture of thermophilic, aerobic and spore-forming bacteria, for example, bacteria belonging to the genera Bacillus or Geobacillus, or bacteria that produce lactic acid (JP 51-129759 A), aerobicBacillus subtilis(JP 6-5197 A), bacteria belonging to the genusThermus aquaticushaving the ability to dissolve the lignin (JP 6-105679 A), decomposing cellulose aerobic bacteriaClostridium,,Thermus aquaticus(JP 6-191977) and so on.

However, despite the use of these microorganisms, although the temperature of the fermentation in the allocation of the heat of fermentation during the fermentation rises to 70°C or more, the maximum temperature raises the I-80° C and thus cannot be destroyed saprophytes, in particular spore-forming saprophytes. In addition, the number of suitable bacterial cells in the resulting fertilizer is the largest approximately 100000000 per gram (dry manure), so that when cells are used as a fertilizer effect as fertilizer cannot be manifested sufficiently.

To resolve these problems concerning the disposal of sludge, the authors of the present invention conducted intensive studies to obtain a product of fermentation, cleaning sludge processing sludge fermenting at high temperatures, comprising 85°C or more, more preferably 95°C or more, for the destruction of saprophytes, weed seeds and the like, and which contains a large number of suitable bacterial cells. The authors found a way to obtain the product of fermentation of sludge containing a large number of extremely useful bacterial cells, including:

adding bacterial cultures growing at temperatures not less than 85°C, obtained from the soil of the volcanic area of Kirishima in the Prefecture of Kagoshima, Japan, in the raw sludge and mixing them and

processing the mixture obtained in aerobic enzymes for elimination of saprophytes and seeds contained in the sludge, p and the temperature of the fermenting 85° C or more for purification sludge, and received a patent for this method (JP 3064221 B). And fermented sludge was used as compost, in which large quantities were mesophilic aerobic spore-forming bacteria, thermophilic aerobic spore-forming bacteria, thermophily, etc. belonging to the genera Bacillus or Geobacillus.

That is, per gram of fermented sludge accounts for about 1000000000 bacterial cells where bacteria mainly include aerobic bacteria, thermophilic bacteria, and is heat-resistant spores, as shown in table 1.

On the other hand, in the culture of the selected colonies that grew on the culture of the tablet, to obtain individual bacteria, and the bacteria were subjected to morphological examination, etc. in order to find microorganisms that may be relevant to the enzymes.

As the description is but higher it is shown that mainly involved in the process polymorphic, do not form spores of gram-positive bacilli, aerobic spore-forming bacteria (mesophilic and thermophilic).

On the other hand, was carried out measurement of thermoflow, with reference to the description in "Methods for Isolating Microbes", YAMAZATO, Kazuhide and others, ed., published by R&D Planning. The main thermofill was an aerobic spore-forming bacteria (thermophilic).

In addition, mesophilic aerobic spore-forming bacterium (selected bacteria (a), thermophilic aerobic spore-forming bacterium (selected bacteria (b) and thermofill (selected bacterium c), which was mainly allocated during the above search microorganisms were subjected to morphological examination, testing, physiological properties and measurement of the fraction of GC in the DNA of bacterial cells. The results are presented in table 3.

The selected bacterium (a) did not meet any kind with respect to the properties, so that its appearance has not been determined. The selected bacterium (b) of prodemonstriruu is La good growth at slightly alkaline conditions (pH from 8.0 to 8.5), but it grew on medium at pH 7.0, and the results of tests of other properties suggested that it was a view that is close toBacillus badiusorB. brevis. However, bacteria (b) has properties that are not typical for any of them, so that identification of the species was not performed. In addition, because the selected bacterium (c) demonstrated bacterial properties, is identical to the bacteriological properties ofGeobacillus stearothermophilusit can be identified as the same species. However, a big difference in their share GC suggests that they are closely related species.

Data of the selected bacteria deposited in the Agency of Industrial Science and Technology, National Institute of Bioscience and Human-Technology (currently National Institute of Advanced Industrial Science and Technology, Patent Microorganism Depository, where they were assigned the corresponding inventory number: YM-01 inventory number FERM P-15085 for the selected bacteria (a), YM-02 inventory number FERM P-15086 for the selected bacteria (b) and YM-03 inventory number FERM P-15087 for the selected bacteria (c).

The authors present invention additionally undertook studies to determine the presence of such microorganisms growing at high temperatures in the compost, and suddenly discovered hyperthermophile belonging to new species, which is intensively propagated at high temperatures up to 75°C or more, still time is naujausia at 85° C, but does not propagate at 50°C or less.

Description of the invention

The aim of the present invention to provide a new thermofill, especially hyperthermophile, compost, obtained by fermenting sludge at 85°C or more.

To solve the above problems, the authors present invention undertook a search of thermophilic microorganisms present in the compost, obtained by fermenting sludge at 85°C or more (brand Satsuma Soil; made in the Bureau of Waterworks Department, Kagoshima City), and as a result, the authors found obligatorily aerobic bacterium, not razmnozhatsya at a temperature of cultivation for ordinary bacteria (from 30 to 40°C), but rapidly growing, and propagating at a temperature of from 70 to 85°C, in particular at 80°C or more. The authors then conducted a phylogenetic analysis of the systematics of bacteria, based on the nucleotide sequence of the 16S-rDNA. The authors of the present invention found that although this obligatorily aerobic bacterium is a gram-negative and does not possess the ability to form spores, it is closely related gram-positive soil bacteria belonging to the genera Bacillus or Geobacillus, but it is a bacterium, not dependent on the data bacteria, at least at the level of genus. The authors of the present invented what I called the bacterium Caldothrix satsumaeYM081 and deposited it in the International Patent Organism Depositary, the National Institute of Advanced Industrial Science and Technology, an Independent Administrative Institution under the Ministry of Economy, Trade and Industry, where she was assigned inventory number FERM P-18598. Subsequently, the bacteria were transferred to the international Depository, where she was assigned inventory number FERM BP-8233.

Thus, the present invention relates to a new hyperthermophile belonging to the genus Caldothrix, which breeds at a temperature of 80°C or more.

The present invention relates to a new hyperthermophile related toCaldothrix satsumae.

In particular, the present invention relates to a strain ofCaldothrix satsumaeYM081 (FERM BP-8233), representing a new hyperthermophile.

In addition, the complete nucleotide sequence of the 16S-rDNA of the bacteria has a nucleotide sequence shown in SEQ ID. No. 1 in the list of sequences.

Brief description of drawings

Fig. 1 is a phylogenetic tree of the genus Caldothrix of the present invention, based on 16S-rDNA. It should be noted that in Fig. 1 YM081 denotes the strain ofCaldothrix satsumaeYM081 representing hyperthermophile of the present invention.

Fig. 2 is an optical micrograph of strainCaldothrix satsumaeYM081 of the present invention.

Fig. 3 is an electronic micro is fotografiy strain Caldothrix satsumaeYM081 of the present invention.

Fig. 4 is a transmission electron micrograph of strainCaldothrix satsumaeYM081 of the present invention.

Fig. 5 is an electron micrograph (magnification of 5000 times) ultra-thin slice cells of strainCaldothrix satsumaeYM081 of the present invention.

Fig. 6 is a graph showing the relation between the time of generation of the strain ofCaldothrix satsumaeYM081 of the present invention on temperature.

The best way of carrying out the invention

Hyperthermophile of the present invention was isolated from compost (brand Satsuma Soil)obtained by fermentation of organic waste, such as feces and urine, Kagoshima city, Kagoshima Prefecture, Japan, at high temperatures in accordance with the method described in JP 3064221 B. as the allocation method used the following method.

To 5 ml of medium with the composition described in table 4 below, was added approximately 0.1 g of the above compost. Subcultivation repeated while maintaining the temperature at 80°C for the enrichment of bacteria, and then repeated the isolation and purification of the tablet containing the same medium as described above, to which was added Gellan gum.

Microbiological properties and taxonomic position thus obtained bacteria were as follows.

1) Morphologically it is a long, Bacillus, having a width of 0.5 μm and a length of 3 μm. The results of gram staining was negative. The observations of the ultra-thin slice of microbial cells at the electron microscope also indicates that the surface structure of the cell is a gram-negative, that is, except the cell membrane (plasma membrane) and the cell wall was observed by the presence of the outer membrane. She lacked the ability to form spores.

2) It is extensively grown at temperatures from 70 to 85°C; 50°C or less growth was observed. At high temperatures up to 75°C or more it intensively bred, and even at 85°C observed reproduction. It is an obligate aerobe.

3) Optimum pH for reproduction is neutral. The pH range in which it can multiply, represents from 6 to 9. So the e she showed weak halophilia.

4) It demonstrates the ability to learn various proteins, such as albumin and casein, and starch.

5) She has the ability to produce urease, but not the ability to restore nitrates. It does not have the ability to produce sulfides or indoles.

6) the Proportion of G+C DNA is 70,0%.

7) Conducted phylogenetic systematic analysis based on the nucleotide sequence of the 16S-rDNA. The results are shown in Fig. 1 and in table 6. Additionally in SEQ ID No. 1 in the list of sequences presents full nucleotide sequence of the 16S-rDNA.

As described above, the bacterium of the present invention is close to the genera Bacillus or Geobacillus gram-positive soil bacteria with the ability to form spores, although the bacterium is gram-negative and does not possess the ability to form spores. However, the bacterium is independent of the data bacteria, at least at the level of the genus.

Here it is shown that the microorganism of the present invention belongs to the Eubacteriales and represents hyperthermophile. Also, based on the nucleotide sequence of the 16S-rDNA, it is shown that the microorganism is close toGeobacillus stearothermophilusbut forms an independent genus.

Examples

The present invention will be described by way of examples and comparison examples is s, outlined below. However, the present invention should not be construed as limited to these examples and comparison examples.

Example comparison 1

1. Obtaining a cell culture

At a temperature of from 37 to 40°C mixed soil from sulfuric zone volcanic belt in Kirishima city Makizono, Aira district, Kagoshima Prefecture, Japan, and the soil from the rice field next to it, where there are green lichens. To the mixture was added an aqueous solution of sucrose in an amount of from 3 to 4 l/m³the sucrose solution was obtained by dissolving sucrose in water that exceeded the amount of sucrose in the amount of 500 to 1000 times. The resulting mixture was cultured, leaving for a period of from 30 to 50 days at a temperature of from 40 to 50°C. Aliquots of the culture were mixed with raw sludge at a few parties, which then allowed ferentiates in aerobic conditions with blowing air. Party with the temperature of the fermenting 85°C or more was chosen as a cell culture.

2. Processing of raw sewage sludge

To a mixture of animal manure, draught wastewater, residual starch and kitchen waste was added slaked lime to conduct a deodorizing treatment. Then 80 mass parts of their aliquot was mixed with 20 mass parts of a cell culture obtained in the above stud and 1, and in aerobic conditions was conducted by fermentation in the fermenter. After performing this step, the temperature of the fermented product was increased from room temperature to values from 85 to 95°C during the day. When the temperature of the fermentation mixture is maintained for about 3 days, and 5 days after the start of the fermentation product was ground (mixed). In the mixing temperature of the product of fermentation decreased to about 60°C, but in about 1 day it increased to values from 85 to 95°C. Fermentation was continued for 5 hours while maintaining this temperature. Repeating the operations of fermentation and stirring several times, the temperature of the fermentation product during mixing and the temperature of the fermentation was gradually reduced. Day, when the temperature of the fermentation product during mixing was reduced to approximately 35°C after repeating these operations four times, determined as of the last day of fermentation. The resulting fermentation product dried up prior to the formation of brown granules, which can be used as organic fertilizer.

3. Getting compost from raw materials

Mixed 80 mass parts, raw sewage sludge, obtained by processing sieges is and municipal wastewater of the city of Kagoshima, Prefecture of Kagoshima, Japan, compression drying to reduce the moisture content up to 68%, and 20 mass parts of a cell culture obtained in the above stage 2. The mixture was placed in a fermenter, where for fermenting bottom blowing in the air. On the seventh day from the beginning of the fermentation the temperature reached 98°C. After fermenting for 10 days, when the temperature of the fermentation was started to decline from 98°C, were stirring again to continue fermenting. After the temperature first reached 99°C, i.e. 10 days after mixing, the temperature was rapidly decreased to values between 60 and 70°C. At this point the fermentation product was distributed in the fermenter for rapid cooling to ambient temperature, to obtain a brown powder fermented sludge. Powder fermented sludge can be used as compost or cell culture, or environment for carrying out the above fermentation.

Example 1

The selection of hyperthermophile

Approximately 0.1 g of compost obtained in example comparison 1 was inoculable in 5 ml of the medium described in table 4, and repeatedly transferred and cultured at 80°C for the enrichment of bacteria. Then, to obtain hyperthermophile really and is finding carried out isolation and purification of bacteria on tablets, containing the medium with the same composition as described above, to which was added Gellan gum.

It should be noted that the sample of compost was added to the medium with peptone/yeast extract (0.5% peptone, 0.3% of yeast extract, pH 7,2) and the final mixture was maintained at 70°C, followed by separation multiplication of bacterial cells on the tablet with agar (pH of 7.2, 70°C). The result foundGeobacillus stearothermophiluswhich, as previously thought, was responsible for fermenting the compost at a temperature of 70°C or more, and a number of other new thermoflow.

Example 2

Microbiological properties of hyperthermophile

Hyperthermophile of the present invention obtained in example 1 was inoculable in agar medium with a pH of 7 to 8, containing 0.3% casein, 0.2% of yeast extract, 0.1% starch and 5% NaCl, and were cultured at 80°C for 24 hours followed by analysis of microbiological properties. The results are shown in table 5.

Images of hyperthermophile obtained by means of the microscope shown in Fig. 2-5.

The result found that although the bacterium of the present invention belongs to the Eubacteriales, is gram-negative and does not possess the ability to form spores, the isolate is closely related to the gram-positive soil bacteria that have the ability to form spores and belong to the genera Bacillus or Geobacillus, however the isolate is not monophyletic with them and thus, belongs to another genus. In addition, as regards the share of GC in DNA that did not exist bacteria that would demonstrate 90% or more sequence homology with the sequence of bases of the 16S-rDNAC.satsumaeYM081, as shown in table 1, and the bacterium of the present invention was equidistant (85% each) from these two genera, i.e Bacillus and Geobacillus, so it was identified as a new genus (see table 6 and Fig. 1). Thus, this genus was named Caldothrix. As the temperature optimum reproduction is 80°C, it is shown that this bacterium is hyperthermophile.

It should be noted that the comparison of the biochemical properties ofCaldothrix satsumaeYM081 of the present invention and Bacillus subtilus shown in table 7.

The above bacteria, belonging to a new genus of the present invention, was given the name of a strain ofCaldothrix satsumaeYM081 and deposited in the Patent Microorganism Depository, National Institute of Advanced Industrial Science and Technology, where she was assigned inventory number FERM P-18598. Subsequently, the bacteria were transferred to the international Depository, where she was assigned inventory number FERM BP-8233.

Example 3

The relationship between temperature and growth in strainCaldothrix satsumaeYM081

The strain ofCaldothrix satsumaeYM081 was inoculable in a medium containing starch/casein/yeast extract, and the culture was stirred at 120 rpm at respective predetermined temperatures and measured the doubling time (the time to doubling of the number of cells). The results are presented in Fig. 6.

As can be seen from Fig. 6, the optimum temperature for reproduction of bacteria was 78° C. the doubling Time when 78°C was approximately 26 minutes, whereas the doubling time at 82°C was approximately 55 minutes, and even at 82°C bacteria continues to multiply at a speed that made up half the rate under the optimal condition. Also, adding to the aqueous solution extracted from the compost, made possible the reproduction even at 85°C, as shown in table 8.

Applicability in industry

When culture for fermenting inoculant in organic waste, such as feces and urine, as raw material for fermentation, the temperature of fermentation is increased because of a number of mesophilous species belonging to the genera Bacillus or Geobacillus. After the temperature of fermentation is increased, hyperthermophileC. satsumaeaccording to the present invention begins to participate in the otlozhenii and fermenting organic waste. Therefore, hyperthermophile of the present invention is mainly used, for example, as seed bacteria or the environment for decomposition and fermentation of organic waste at high temperatures to obtain compost.

In addition, protease and amylase produced by hyperthermophiles have activities at high temperatures and, therefore, using this property, it is possible to obtain enzymes that are resistant to heat.

Comments on the deposited biological materials

A. the Name and address of the organization where the deposited biological materials:

Title: Patent Microorganism Depository, National Institute of Advanced Industrial Science and Technology.

Address: zip code: 305-8565, Chuo No. 6, 1, Higashi 1-Chome, Tsukubashi, Ibaragiken, Japan.

B. date of Deposit of the organization A:

November 7, 2002.

(Date of original Deposit: November 13, 2001).

C. the Number of Deposit issued by the organization A:

FERM BP-8233.

Hyperthermophilic strain Caldothrix satsumae YM081 (FERM BP-8233), is able to ferment organic waste at a temperature of 85°C or higher.