Bacteria for decomposing sludge, microbial decomposing bacteria, microbial preparation, sludge decomposing method and sludge decomposing device

Abstract

The present invention provides: a sludge decomposing bacterium having a 16S rRNA gene comprising a base sequence having 97% or more identity with the base sequence recorded in sequence number 1, a bacterium having a 16S rRNA gene comprising a base sequence having a base variation number of 2 bases or less relative to the base sequence recorded in sequence number 1 and having target microorganism decomposing ability, and a microbial preparation for decomposing a target microorganism comprising the bacterium (a1), wherein the bacterium (a1) is a bacterium having a 16S rRNA gene comprising a base sequence having 90% or more identity with the base sequence recorded in sequence number 1.

Description

Technical Field

[0001] The present invention relates to bacteria for decomposing sludge, microbial decomposing bacteria, microbial preparation, sludge decomposing method and sludge decomposing device. Background Art

[0002] When sewage is purified by the activated sludge process, sludge called excess sludge is produced, which contains microorganisms that proliferate by decomposing organic matter. Since excess sludge discharged from wastewater treatment equipment accounts for more than 40% of industrial waste, it is believed that reducing the volume of industrial waste is effective in reducing the volume of excess sludge. Generally, the excess sludge is incinerated after being dehydrated and dried (Non-Patent Document 1: "2015 Business and Industrial Waste Discharge and Treatment Status Survey Report, 2013 Performance (Summary Edition)" [Online], March 2016, Waste and Recycling Countermeasures Division, Minister's Office of the Ministry of the Environment, [retrieved on August 31, 2018], Internet <URL:https: / / www.e-stat.go.jp / stat-search / file-download?statInfId=000031403476&fileKind=2>; Non-Patent Document 2: Yamamoto Masayuki, "Regarding Sewage Combustion Technology", Journal of the Japan Society of Fuel, Japan Society of Combustion, 2011, Vol. 53, No. 164, p91-96).

[0003] However, if the excess sludge is incinerated, greenhouse gases will be produced, so it may not be a treatment method with less impact on the environment.

[0004] Prior art literature

[0005] Non-patent literature

[0006] Non-patent document 1: "2015 Business and Industrial Waste Discharge and Treatment Status Survey Report, 2013 Performance (Summary Version)" [Online], March 2016, Waste and Recycling Countermeasures Division, Minister's Secretariat, Ministry of the Environment, [retrieved on August 31, 2018], Internet <URL: https: / / www.e-stat.go.jp / stat-search / file-download?statInfId=000031403476&fileKind=2>

[0007] Non-patent document 2: Masayuki Yamamoto, "About Wastewater Combustion Technology", Journal of the Japan Society of Fuel Science, Japan Society of Combustion Science, 2011, Vol. 53, No. 164, p91-96 Summary of the invention

[0008] Problem that the invention aims to solve

[0009] In recent years, the entire society has become increasingly aware of the global environment, and has sought a method of reducing the volume of excess sludge with less impact on the environment, that is, a method of decomposing the microorganisms constituting the excess sludge.

[0010] The object of the present invention is to provide sludge decomposing bacteria, microbial decomposing bacteria, microbial preparation, sludge decomposing method and sludge decomposing device.

[0011] Means used to solve problems

[0012] That is, the present invention relates to [1] to

[18] exemplified below.

[0013] [1] A sludge-decomposing bacterium (hereinafter sometimes referred to as "the sludge-decomposing bacterium according to the present invention") having a 16S rRNA gene comprising a base sequence having 97% or more identity with the base sequence described in SEQ ID NO: 1.

[0014] [2] A bacterium (hereinafter sometimes referred to as "the bacterium of the present invention") having a 16S rRNA gene in which the number of base variations relative to the base sequence recorded in SEQ ID NO: 1 is 2 bases or less, and having the ability to decompose a target microorganism.

[0015] [3] The bacterium according to [2], wherein the target microorganism is a target bacterium.

[0016] [4] The bacterium according to [2] or [3], wherein the target microorganism is a target dead bacterium.

[0017] [5] The bacterium according to any one of [1] to [4], which has a 16S rRNA gene comprising the base sequence described in SEQ ID NO: 1.

[0018] [6] The bacteria deposited under the accession number NITE BP-03020.

[0019] [7] A microbial preparation for sludge decomposition (hereinafter sometimes referred to as "the microbial preparation of the present invention") comprising bacteria (a1),

[0020] Bacteria (a1): A bacterium having a 16S rRNA gene comprising a base sequence having 90% or more identity with the base sequence described in SEQ ID NO:1.

[0021] [8] The microbial preparation for sludge decomposition according to [7], wherein the bacterium (a1) has a 16S rRNA gene comprising a base sequence having 95% or more identity with the base sequence described in SEQ ID NO: 1.

[0022] [9] The microbial preparation for sludge decomposition according to [7], wherein the bacterium (a1) has a 16S rRNA gene comprising a base sequence having 97% or more identity with the base sequence described in SEQ ID NO: 1.

[0023]

[10] The microbial preparation for sludge decomposition according to [7], wherein the bacterium (a1) has a 16S rRNA gene containing the base sequence described in SEQ ID NO: 1.

[0024]

[11] The sludge decomposing microbial agent according to any one of [7] to

[10] , wherein the bacterium (a1) has a target microbial decomposition ability.

[0025]

[12] The sludge-decomposing microbial agent according to any one of [7] to

[11] , which decomposes sludge at a temperature of 20°C.

[0026]

[13] The microbial preparation for sludge decomposition according to [7], wherein the bacterium (a1) is a bacterium deposited under the accession number NITE BP-03020.

[0027]

[14] The microbial preparation for sludge decomposition according to any one of [7] to

[13] , further comprising a microorganism (a2), wherein the number of bacteria (a1) is 0.1% or more and less than 100% relative to the total number of bacteria (a1) and microorganisms (a2),

[0028] Microorganism (a2): A microorganism other than bacteria (a1).

[0029]

[15] A microbial preparation for sludge decomposition according to

[14] , wherein the microorganism (a2) comprises at least one selected from the group consisting of a bacterium having a 16S rRNA gene containing a base sequence having 90% or more identity with the base sequence recorded in SEQ ID NO: 7 and having the ability to decompose a target microorganism, a Bacillus bacterium having the ability to decompose a target microorganism, and a Paenibacillus bacterium having the ability to decompose a target microorganism.

[0030]

[16] A microbial preparation for sludge decomposition, comprising a culture of bacteria (a1),

[0031] Bacteria (a1): A bacterium having a 16S rRNA gene containing a base sequence having 90% or more identity with the base sequence described in SEQ ID NO: 1 and having target microbial decomposition ability.

[0032]

[17] A method for decomposing sludge (hereinafter sometimes referred to as "the decomposition method according to the present invention"), comprising the steps of allowing the bacteria according to any one of [1] to [6] or the sludge decomposing microbial preparation according to any one of [7] to

[16] to act on sludge.

[0033]

[18] A sludge decomposition device (hereinafter sometimes referred to as "the decomposition device according to the present invention"), which uses the bacteria according to any one of [1] to [6] or the sludge decomposition microbial preparation according to any one of [7] to

[16] .

[0034] Effects of the Invention

[0035] According to the present invention, it is possible to provide sludge decomposing bacteria, microbial decomposing bacteria, a microbial agent, a microbial decomposition method and a decomposition device. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 These are photographs showing the morphology of Brevibacillus parabrevis (hereinafter, sometimes referred to as “Bb. parabrevis”) NITE BP-03020 in Experiment 3.

[0037] Figure 2 These are photographs showing the results of Gram staining of Bacillus parabrevis NITE BP-03020 in Experiment 3.

[0038] Figure 3 This is a graph showing the target microbial decomposition ability of Bacillus parabrevis NITE BP-03020 in Experiment 4.

[0039] Figure 4 This is a graph showing the target microbial decomposition ability of Paenibacillus glycanilyticus in Experiment 4.

[0040] Figure 5 This is a graph showing the target microbial decomposition ability of the microbial preparation containing cultures of a plurality of Brevibacillus parasporum strains in Experiment 5.

[0041] Figure 6 This is a graph showing the degradation ability of the target microorganism (Micrococcus lysodeikticus) of the microbial preparation containing the culture of Bacillus parabrevis NITE BP-03020 in Experiment 6.

[0042] Figure 7 This is a graph showing the degradation ability of the microbial preparation containing the culture of Bacillus parabrevis NITE BP-03020 in Experiment 6 by the target microorganism (Saccharomyces cerevisiae).

[0043] Figure 8This is a graph showing the target microorganism (Escherichia coli) degradation ability of the microorganism preparation containing the culture of Bacillus parabrevis NITE BP-03020 in Experiment 6.

[0044] Fig. 9 This is a graph showing the target microorganism (Brevibacillus parabrevis) degrading ability of the microbial preparation containing the culture of Brevibacillus parabrevis NITE BP-03020 in Experiment 6.

[0045] Fig.10 This is a graph showing the degradation ability of the target microorganism (Gluconobacter oxydans) of the microbial preparation containing the culture of Bacillus parabrevicus NITE BP-03020 in Experiment 6.

[0046] Fig.11 This is a graph showing the sludge decomposition ability of the microbial preparation containing the culture of Bacillus parabrevis NITE BP-03020 in Experiment 7.

[0047] Fig.12 This is a graph showing the target microbial decomposition ability of the microbial preparation containing the culture of Brevibacillus parabrevis NITE BP-03020 under various temperature conditions in Experiment 8.

[0048] Fig.13 This is a graph showing the sludge decomposition ability of the microbial preparation containing a mixture of a culture of Bacillus parabrevicus NITE BP-03020 and a culture of other bacteria in Experiment 9.

[0049] Fig.14 This is a graph showing the dry weight of excess sludge after adding Tumebacillus sp. (NITE BP-02779) in Reference Experiment 7. DETAILED DESCRIPTION

[0050] Hereinafter, specific embodiments of the present invention will be described in detail. It should be noted that the present invention is not limited to the following embodiments.

[0051] (Explanation of general terms)

[0052] In this specification, "microorganisms" are those microscopic organisms whose structures cannot be discerned by the naked eye, and are organisms other than large multicellular organisms. "Target microorganisms" are microorganisms that are decomposed by the bacteria or microbial preparations described in the present invention. Examples of target microorganisms include bacteria and fungi, among which bacteria are preferred, and bacteria constituting excess sludge are more preferred. The target microorganisms may be live or dead.

[0053] "Target bacteria" refers to bacteria that become target microorganisms. Target bacteria may be live bacteria or dead bacteria, and may include live bacteria and dead bacteria. Live bacteria refer to living bacteria, such as bacteria that are metabolizing. Dead bacteria refer to dead bacteria, such as bacteria that are not metabolizing. Live bacteria and dead bacteria can be distinguished using pigments such as propidium iodide (PI). "Target dead bacteria" refers to dead bacteria among target bacteria. From the viewpoint of decomposition efficiency, target bacteria are preferably target dead bacteria. About 50% of the microorganisms in the residual sludge are dead bacteria. Target dead bacteria can also be obtained by, for example, heating the target bacteria, high-pressure steam sterilization, ultraviolet irradiation, formalin treatment, acid treatment, etc. In addition, the target dead bacteria can also be crushed.

[0054] Examples of target microorganisms include Gram-positive bacteria such as Micrococcus, Bacillus, Staphylococcus, Paenibacillus, and Lactobacillus; Gram-negative bacteria such as Escherichia, Acetobacter, and Gluconobacter; and fungi such as Saccharomyces cerevisiae.

[0055] In this specification, "target microorganism decomposition ability" refers to the ability to metabolize the target microorganism and convert part or all of the biomolecules constituting the target microorganism into different molecules. As biomolecules, for example, sugars, proteins, nucleic acids, lipids, etc. can be listed.

[0056] (Bacteria for decomposing sludge according to the present invention)

[0057] The sludge decomposing bacterium of the present invention has a 16S rRNA gene containing a base sequence having 97% or more identity with the base sequence described in SEQ ID NO: 1. Examples of the bacterium having a 16S rRNA gene containing a base sequence having 97% or more identity with the base sequence described in SEQ ID NO: 1 include bacteria belonging to the genus Brevibacillus. Examples of the Brevibacillus bacteria include Brevibacillus parabrevis, Brevibacillus choshinensis, Brevibacillus formosus, Brevibacillus agri, Brevibacillus nitrificans, Brevibacillus brevis, Brevibacillus reuszeri, Brevibacillus limnophilus, Brevibacillus panacihumi, and Brevibacillus gelatini.

[0058] The sludge decomposing bacteria of the present invention may have a 16S rRNA gene comprising a base sequence having an identity or homology of 97.5% or more, 98% or more, 98.5% or more, 98.8% or more, 99.0% or more, 99.3% or more, 99.5% or more, 99.8% or more, or 99.9% or more compared to the base sequence recorded in sequence number 1. The sludge decomposing bacteria may also be bacteria having a 16S rRNA gene comprising the base sequence recorded in sequence number 1, and representative strains thereof include Bacillus parabrevis (accession number NITE BP-03020) described later. The sludge decomposing bacteria of the present invention may include the bacteria of the present invention.

[0059] The sludge decomposing bacteria of the present invention may have a 16S rRNA gene comprising a base sequence in which one or several bases are substituted, deleted or added compared to the base sequence recorded in sequence number 1. One base or several bases may be, for example, 1 to 25 bases, 1 to 10 bases, or 1 to 5 bases. The above mutation is a mutation in which the expression and function of 16S rRNA are not lost.

[0060] The sludge decomposing bacteria of the present invention may also have a 16S rRNA gene, which contains a base sequence that hybridizes under stringent conditions to a continuous sequence of about 15 bases or more, preferably about 18 to about 500 bases, more preferably about 18 to about 200 bases, and even more preferably about 18 to about 50 bases contained in the base sequence recorded in SEQ ID NO: 1, or a complementary sequence thereof. Stringent conditions refer to conditions under which non-specific hybridization is not formed, and examples thereof include conditions of washing once or more in 60° C., 1×SSC, 0.1% SDS, preferably 68° C., 0.1×SSC, 0.1% SDS, and the like.

[0061] The sludge decomposing bacteria of the present invention have sludge decomposing ability. The sludge is preferably excess sludge. The sludge decomposing ability may vary depending on the reaction conditions (the type and concentration of the sludge and the target microorganisms contained in the sludge, the composition of the solution containing the sludge decomposing bacteria, the temperature, the pH, the number of bacteria, etc.). As a method for investigating whether there is sludge decomposing ability, for example, the following method can be cited: after reacting the sludge and the bacteria whose sludge decomposing ability is to be investigated in an appropriate culture medium or buffer for a certain period of time, the decomposition of the sludge in the culture medium or buffer is investigated. There are no particular limitations on the method for investigating decomposition, and examples thereof include the following methods: a method for measuring the turbidity of sludge, a method for detecting target microorganisms contained in sludge using an SLP reagent, a method for detecting DNA of target microorganisms contained in sludge using PCR, a method for measuring the dry weight of sludge, a method for measuring the dry weight of target microorganisms contained in sludge, and a method for detecting decomposition products from target microorganisms contained in sludge using high performance liquid chromatography (HPLC); mass spectrometry (MS); thin layer chromatography (TLC); nuclear magnetic resonance (NMR); gas chromatography (GC), etc.

[0062] In the case of investigating the sludge decomposition ability by turbidity, having the sludge decomposition ability means that after adding sludge decomposing bacteria to the sludge, the turbidity after a given period of time is significantly reduced compared to the turbidity before addition. The turbidity after addition is, for example, less than 80% of the turbidity before addition, preferably less than 50%, and more preferably less than 30%. In the case of investigating the sludge decomposition ability by dry weight, having the sludge decomposition ability means, for example, that the dry sludge weight after addition is significantly reduced compared to the dry sludge weight before addition, for example, the dry sludge weight after addition is less than 95% of the weight before addition, preferably less than 90%. Generally speaking, bacteria have the sludge decomposition ability when they have the target microbial decomposition ability.

[0063] (Bacteria of the present invention)

[0064] The bacterium described in the present invention has a 16S rRNA gene with a base variation number of less than 2 bases relative to the base sequence recorded in sequence number 1, and has the target microorganism decomposition ability. In this specification, the 16S rRNA gene is preferably an endogenous 16S rRNA gene originally possessed by the bacterium. It can also be an artificially induced mutated 16S rRNA. As bacteria having a 16S rRNA gene with a base variation number of less than 2 bases relative to the base sequence recorded in sequence number 1, Brevibacillus bacteria can be listed, and Brevibacillus parabrevis can also be listed. The bacteria described in the present invention can include the sludge decomposition bacteria described in the present invention.

[0065] One embodiment of the bacterium according to the present invention includes a bacterium having a 16S rRNA gene including the base sequence described in SEQ ID NO: 1 and having the ability to decompose the target microorganism.

[0066] As a representative strain of bacteria having a 16S rRNA gene containing the base sequence recorded in sequence number 1, the following strain can be cited, which is Brevibacillus parabrevis (deposit number NITE BP-03020, original deposit date: September 17, 2019), deposited in the Patent Microorganism Depository of the Independent Administrative Institution Product Evaluation and Technology Foundation (NPMD, address: Room 2-5-8122, Kazusa Kamata, Kisarazu City, Chiba Prefecture, 292-0818) based on the Budapest Treaty. The mycological properties of this strain are described below.

[0067] The bacterium described in the present invention may have a 16S rRNA gene comprising a base sequence having an identity or homology of 98.5% or more, 98.8% or more, 99.0% or more, 99.3% or more, 99.5% or more, 99.8% or more, or 99.9% or more compared to the base sequence recorded in sequence number 1, or may have a 16S rRNA gene comprising the base sequence recorded in sequence number 1.

[0068] Bacteria with a base mutation number of 2 or less relative to the base sequence recorded in SEQ ID NO. 1 may have a 16S rRNA gene comprising a base sequence in which 1 or 2 bases are substituted, deleted, or added compared to the base sequence recorded in SEQ ID NO. 1. The above mutation is a mutation that does not cause loss of expression and function of 16S rRNA.

[0069] The bacterium of the present invention may also have a 16S rRNA gene, which comprises a base sequence that hybridizes under stringent conditions to a continuous sequence of about 15 bases or more, preferably about 18 to about 500 bases, more preferably about 18 to about 200 bases, and even more preferably about 18 to about 50 bases contained in the base sequence recorded in SEQ ID NO: 1, or a complementary sequence thereof. Stringent conditions refer to conditions under which non-specific hybridization is not formed, and examples thereof include conditions of washing once or more in 60° C., 1×SSC, 0.1% SDS, preferably 68° C., 0.1×SSC, 0.1% SDS, and the like.

[0070] The target microorganism decomposition ability of the bacteria of the present invention may vary depending on the reaction conditions (type and concentration of target microorganisms, composition of the solution containing bacteria, temperature, pH, number of bacteria, etc.) The target microorganism is, for example, a target bacterium, preferably a target dead bacterium.

[0071] For example, the representative strain of the bacteria described in the present invention, Bacillus parabrevis NITE BP-03020, and dead bacteria of the Micrococcus genus are prepared to have a turbidity (OD660) of 0.2, and are mixed at a volume ratio of 1:100. Then, the mixture is reacted at a temperature of 20 to 35°C and a pH of 6.5 to 8.0. This can reduce the turbidity by about 50% after one week and decompose the Micrococcus genus.

[0072] The bacteria of the present invention can be determined by analyzing the base sequence of the 16S rRNA gene of the target microorganism (the bacteria of the present invention or the microorganism to be investigated) and measuring the decomposition ability of the target microorganism. Specifically, if the target microorganism has a 16S rRNA gene with a base variation of 2 or less bases relative to the base sequence recorded in sequence number 1, and has the target microorganism decomposition ability, the microorganism can be determined to be the bacteria of the present invention.

[0073] The analysis of the base sequence of the 16S rRNA gene can be performed, for example, using the following method. First, using a known method, genomic DNA is extracted from the target microorganism, and the 16S rRNA gene is amplified. There is no particular limitation on the method for amplifying the 16S rRNA gene, and examples thereof include the PCR method using universal primers commonly used by those skilled in the art. The amplified product obtained by the PCR method can be purified as needed and used for determining the base sequence using a DNA sequencer, etc. The obtained base sequence is compared with the sequence described in SEQ ID NO. 1.

[0074] As a method for investigating whether the target microorganism has the ability to decompose, for example, the following method can be cited: after the object microorganism and the target microorganism react in an appropriate culture medium or buffer for a certain period of time, the decomposition of the target microorganism in the culture medium or buffer can be investigated. The method for investigating the decomposition is not particularly limited, and for example, the following methods can be cited: a method for measuring the turbidity of a solution containing the target microorganism, a method for detecting the target microorganism using an SLP reagent, a method for detecting the DNA of the target microorganism using PCR, a method for measuring the dry cell weight of the target microorganism, and a method for detecting the decomposition products from the target microorganism using high performance liquid chromatography (HPLC); mass spectrometry (MS); thin layer chromatography (TLC); nuclear magnetic resonance (NMR); gas chromatography (GC), etc.

[0075] In the case of investigating the decomposition of the target microorganism by turbidity, having the target microorganism decomposition ability means that after adding the bacteria described in the present invention to the solution containing the target microorganism, the turbidity after a given time is significantly reduced compared to the turbidity before addition. The turbidity after addition is, for example, less than 80% of the turbidity before addition, preferably less than 50%, and more preferably less than 30%. In the case of investigating the decomposition of the target microorganism by dry cell weight, having the target microorganism decomposition ability means, for example, that the dry cell weight after addition is significantly reduced compared to the dry cell weight before addition, for example, the dry cell weight after addition is less than 95% of the weight before addition, preferably less than 90%. Generally speaking, bacteria have the target microorganism decomposition ability when they have the ability to decompose sludge.

[0076] Based on the results of these base sequence analyses and the determination of the target microorganism's decomposition ability, it can be determined whether the target microorganism is the bacterium described in the present invention.

[0077] (Microbial preparation of the present invention)

[0078] One embodiment of the microbial preparation of the present invention comprises the following bacteria (a1).

[0079] <Bacteria (a1)>

[0080] The bacterium (a1) has a 16S rRNA gene comprising a base sequence having 90% or more identity with the base sequence described in SEQ ID NO:1.

[0081] The bacterium (a1) may have a 16S rRNA gene comprising a base sequence having an identity or homology of 93% or more, 95% or more, 97% or more, 98% or more, 99% or more, 99.5% or more, or 99.9% or more compared to the base sequence recorded in SEQ ID NO: 1, or may have a 16S rRNA gene comprising the base sequence recorded in SEQ ID NO: 1. Examples of the bacterium (a1) include bacteria of the genus Brevibacillus, and may also be Brevibacillus parabrevis. The bacterium (a1) may also be a bacterium deposited under the accession number NITE BP-03020.

[0082] In addition, the bacterium (a1) may have a 16S rRNA gene comprising a base sequence in which one or several bases are substituted, deleted or added compared to the base sequence recorded in sequence number 1. One base or several bases may be, for example, 1 to 135 bases, 1 to 100 bases, 1 to 50 bases, 1 to 25 bases, or 1 to 5 bases. The above mutation is a mutation in which the expression and function of 16S rRNA are not lost.

[0083] The bacterium (a1) may also have a 16S rRNA gene comprising a base sequence that hybridizes under stringent conditions to a continuous sequence of about 15 or more bases, preferably about 18 to about 500 bases, more preferably about 18 to about 200 bases, and even more preferably about 18 to about 50 bases contained in the base sequence recorded in SEQ ID NO: 1, or a complementary sequence thereof.

[0084] The base sequence of the 16S rRNA gene of the bacterium (a1) can be analyzed in the same manner as that of the bacterium of the present invention described above.

[0085] The bacteria (a1) preferably have target microbial decomposition ability. The target microbial decomposition ability of the bacteria (a1) can be evaluated in the same manner as the bacteria described in the present invention.

[0086] Since the target microorganism has good decomposition ability, the bacteria (a1) preferably include at least one of Bacillus parabrevis, the sludge decomposing bacteria of the present invention, the bacteria of the present invention, and bacteria selected from the following group A. The bacteria (a1) may be a single bacterium or a plurality of bacteria.

[0087] Group A: Brevibacillus invocatus, Brevibacillus centrosporus, Brevibacillus borstelensis, Brevibacillus levickii, Brevibacillus massiliensis, Brevibacillus ginsengisoli, Brevibacillus laterosporus, Brevibacillus fulvus, Brevibacillus fluminis, Brevibacillus sediminis, Brevibacillus thermoruber, and Brevibacillus saydinogluensis.

[0088] The microbial preparation described in the present invention has the ability to decompose sludge. The sludge decomposition ability can be evaluated using the method described in the column of sludge decomposing bacteria described in the present invention. The sludge decomposition ability of the microbial preparation is preferably derived from the bacteria (a1) or microorganisms (a2) contained in the microbial preparation or their cultures. The microbial preparation can decompose sludge at a temperature below 37°C, a temperature of 30°C, a temperature of 25°C or a temperature of 20°C, and can also decompose sludge in the temperature range of 20°C to 40°C. For example, after the microbial preparation described in the present invention is added to a solution containing a target microorganism, the turbidity of the solution on the 5th day at a temperature of 25°C is less than 2 times the turbidity of the solution at a temperature of 30°C, and at a temperature of 20°C is less than 3 times the turbidity of the solution at a temperature of 30°C, preferably less than 2.5 times.

[0089] In addition to the bacteria (a1), the microbial preparation may also contain a culture of the bacteria (a1), a microorganism (a2), an additive (b), a carrier (c), etc. Specific examples of the microbial preparation include a microbial preparation containing bacteria (a1), a microorganism (a2), an additive (b), and a carrier (c).

[0090] <Culture of bacteria (a1)>

[0091] One embodiment of the microbial preparation of the present invention comprises a culture of bacteria (a1). The culture of bacteria (a1) comprises, for example, secretions, metabolites, proteins, sugars, enzymes produced by bacteria (a1), and liquid culture medium in which they are suspended. Specifically, the culture comprises a culture of bacteria (a1) that proliferates in a given liquid culture medium or a liquid culture medium containing a carbon source and a nitrogen source under controlled conditions. The preparation for target microbial decomposition comprising a culture of bacteria (a1) may comprise live bacteria of bacteria (a1). The culture of bacteria (a1) may be a supernatant of cultured bacteria (a1). The culture supernatant may be obtained by, for example, removing bacteria (a1) from a liquid culture medium in which bacteria (a1) are cultured by centrifugation, filtration, etc. In addition, the culture may comprise fragments of bacteria (a1). Fragments of bacteria (a1) may also be obtained by, for example, ultrasonic pulverization, bead grinding, or chemical dissolution treatment of bacteria (a1). The culture of bacteria (a1) preferably has target microbial decomposition capability.

[0092] <Microorganisms (a2)>

[0093] Microorganism (a2) is a microorganism different from bacteria (a1). Microorganism (a2) is not particularly limited as long as it is a microorganism that does not cause the sludge decomposition ability of the microbial preparation to be lost. Microorganism (a2) may be a single microorganism or a plurality of microorganisms. Microorganism (a2) may be a bacterium that improves the proliferation ability or stability of bacteria (a1) or a bacterium that improves the stability of the culture of bacteria (a1).

[0094] The microorganism (a2) can be determined by analyzing the base sequence of the 16S rRNA gene of the microorganism, physiological and biochemical properties test, etc. The microorganism (a2) may or may not have the target microorganism decomposition ability. The analysis of the base sequence of the 16S rRNA gene of the target microorganism and the determination of the target microorganism decomposition ability can be carried out in the same manner as the bacteria described in the present invention.

[0095] The microorganism (a2) may be a gram-negative bacterium or a gram-positive bacterium, and examples thereof include bacteria of the genus Tumebacillus, bacteria of the genus Paenibacillus, bacteria of the genus Bacillus, bacteria of the genus Acetobacter, bacteria of the genus Gluconobacter, bacteria of the genus Lactobacillus, bacteria of the genus Gordonia, bacteria of the genus Microbacterium, bacteria of the genus Rhodococcus, bacteria of the genus Sphingomonas, bacteria of the genus Escherichia, and yeast of cerevisiae.

[0096] The microorganism (a2) preferably includes bacteria having the ability to decompose the target microorganism, for example, preferably includes at least one selected from the group consisting of bacteria having a 16S rRNA gene containing a base sequence having 90% or more identity with the base sequence recorded in SEQ ID NO: 7 and having the ability to decompose the target microorganism (hereinafter, sometimes described as "bacteria (a21)"), Bacillus bacteria having the ability to decompose the target microorganism, and Paenibacillus bacteria having the ability to decompose the target microorganism.

[0097] Examples of bacteria having a 16S rRNA gene containing a base sequence having 90% or more identity with the base sequence recorded in SEQ ID NO: 7 and having target microorganism decomposition ability include bacteria of the genus Tumebacillus. The bacterium (a21) may have a 16S rRNA gene containing a base sequence having an identity or homology of 93% or more, 95% or more, 98% or more, 99% or more, 99.5% or more, or 99.9% or more with the base sequence recorded in SEQ ID NO: 7, or may have a 16S rRNA gene containing the base sequence recorded in SEQ ID NO: 7. The bacterium (a21) may also be a strain deposited as a Tumebacillus sp. (deposit number NITE BP-02779, original deposit date: September 11, 2018) in the National Institute for Product Evaluation and Technology Patent Microbiological Depository (NPMD, address: Room 122, 2-5-8, Kazusa Kamata, Kisarazu City, Chiba Prefecture, 292-0818) based on the Budapest Treaty. The bacteriological properties of the strain are shown in Tables 6 to 9 described below.

[0098] The bacteria (a21) may also be, for example, Tumebacillus algifaecis, Tumebacillus avium, Tumebacillus flagellates, Tumebacillus ginsengisoli, Tumebacillus lipolyticus, Tumebacillus luteolus, Tumebacillus permanentifrigoris, or Tumebacillus soli.

[0099] Examples of Bacillus bacteria having target microbial decomposition ability include Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus sphaericus, and Bacillus licheniformis.

[0100] Examples of Paenibacillus bacteria having target microorganism decomposition capability include Paenibacillus polymyxa.

[0101] From the perspective of the target microorganism's decomposition ability, the number of bacteria (a1) relative to the total number of bacteria (a1) and microorganisms (a2) may be greater than 0.1%, greater than 1%, greater than 10%, greater than 50%, greater than 75%, greater than 90%, greater than 95%, less than 100%, or less than 100%.

[0102] The number of bacteria (a1) relative to the total number of bacteria (a1) and microorganisms (a2) is preferably the ratio of bacteria (a1) to all bacteria contained in the microbial preparation. Methods for calculating the number of bacteria (a1) relative to the total number of bacteria (a1) and microorganisms (a2) include methods such as calculation by clone library method, next generation sequencing analysis, and quantitative PCR.

[0103] As a method for calculating the existence ratio of bacteria by clone library method and next generation sequencing analysis, specifically, genomic DNA is extracted from bacteria (a1) and microorganisms (a2) contained in a microbial preparation to obtain a plurality of base sequences of 16S rRNA genes (hereinafter, the number of the plurality of base sequences obtained is referred to as the number of reads), and it is determined whether the obtained base sequence is a base sequence derived from bacteria (a1) or microorganisms (a2). Then, the number of base sequences derived from bacteria (a1) relative to the number of reads can be calculated as the number of bacteria (a1) relative to the total number of bacteria (a1) and microorganisms (a2).

[0104] The next generation sequencer used in the next generation sequencing analysis is not particularly limited as long as it can detect the fluorescence intensity of each base during resynthesis using a DNA fragment as a template to determine the base sequence. Examples of the next generation sequencer include MiSeq, HiSeq 2500 (manufactured by Illumina), 5500xl SOLiDTM, Ion ProtonTM, IonPGMTM (manufactured by Thermo Fisher Scientific), GS FLX+ (manufactured by Roche Diagnostics Co., Ltd.), and the like.

[0105] As a method for calculating the existence ratio of bacteria by quantitative PCR, specifically, the 16S rRNA gene copy number of bacteria (a1) contained in the microbial preparation, and the 16S rRNA gene copy number of bacteria (a1) and microorganism (a2) are calculated respectively. Then, the 16S rRNA gene copy number of bacteria (a1) relative to the 16S rRNA gene copy number of bacteria (a1) and microorganism (a2) can be calculated as the number of bacteria (a1) relative to the total number of bacteria (a1) and microorganism (a2).

[0106] As the real-time PCR device used in quantitative PCR, there is no particular limitation as long as it has a thermal cycler that can amplify DNA by PCR and a spectrofluorometer for detecting the amplified product. As the real-time PCR device, for example, StepOnePlus (Applied Biosystems), Thermal Cycler Dice Real Time System (Takarabio Co., Ltd.), LightCycler 96 System (Roche Diagnostics Co., Ltd.) etc. can be cited.

[0107] Examples of the reaction mixture (mastermix) used for quantitative PCR include Fast SYBR Green MasterMix, Power SYBR Green Master Mix, SYBR Select Master Mix, and PowerUp SYBR Green Master Mix (manufactured by Thermo Fisher Scientific).

[0108] <Additives (b)>

[0109] Examples of the additive (b) include surfactants, dispersants, adjuvants, protective agents, etc. The type and concentration of the additive (b) can be appropriately determined so as not to kill the bacteria (a1) or to prevent the bacteria from losing their target microbial decomposition ability.

[0110] <Carrier (c)>

[0111] As the carrier (c), for example, an inorganic microparticle carrier can be cited. The inorganic microparticle carrier can be a metal and its inorganic salts or oxides, or a substance that is chemically classified as an inorganic substance even if it contains carbon. In addition, it can also be a pure substance or a mixture in which the content of organic carbon is less than about 1%.

[0112] The center particle diameter of the inorganic microparticle carrier is preferably 1 μm to 100 μm, more preferably 4 μm to 75 μm, and further preferably 13 μm to 25 μm. When the center particle diameter is within this range, there is a tendency for bacteria (a1) to be easily loaded on the inorganic microparticle carrier. Here, the center particle diameter refers to the median particle diameter (D50) in the volume-based particle size distribution based on laser diffraction and light scattering methods. The specific gravity of the inorganic microparticle carrier is not particularly limited, and is preferably 1.2 to 3.5.

[0113] In order to improve the yield rate in the initial stage of culture, various aggregating agents may be used to aggregate the inorganic microparticle carriers as needed. Examples of the aggregating agent include nonionic, cationic, and anionic polymer aggregating agents.

[0114] Examples of a method for producing a microbial preparation include a method in which bacteria (a1) and an inorganic fine particle carrier are mixed, bacteria (a1) are supported on the inorganic fine particle carrier, and the mixture is cultured, and the obtained microbial preparation is recovered.

[0115] As a culture method, any of batch, semi-batch, fed-batch, and continuous methods can be used. As a culture method, for example, from the viewpoint of effectively preparing microorganisms that proliferate slowly and have low bacterial yield, a continuous culture method can be used in which the concentration of the target compound supplied to the container (hereinafter referred to as the reaction tank) for culturing the microorganism increases logarithmically over the culture time, as described in Japanese Patent Laid-Open No. 9-187272 Gazette.

[0116] In the present invention, as long as the target microbial decomposition ability of the bacterium (a1) is not lost, the formulation method of the microorganism is not particularly limited, and a known formulation method can be used. As the form of the microbial preparation, it can be a liquid or solid (including encapsulated bodies, agar-like, powdered, etc.), or it can be their frozen bodies, freeze-dried bodies, etc. When the microbial preparation is a liquid, it can be set to a bacterial suspension suspended in a culture medium, a buffer solution, a physiological saline solution, etc. The liquid can also be acidic or neutral. When the microbial preparation is a solid or a freeze-dried body, for example, after the cultured bacteria are concentrated, appropriate drying or freeze-drying can be carried out to make a solid or a freeze-dried body. At this time, an excipient, etc. can also be added.

[0117] (Sludge decomposition method)

[0118] The decomposition method of the present invention includes a step of allowing the sludge decomposing bacteria of the present invention, the bacteria of the present invention, or the microbial preparation of the present invention to act on sludge. The target microbial decomposition ability of the sludge decomposing bacteria of the present invention, the bacteria of the present invention, and the microbial preparation of the present invention is useful for treating excess sludge containing target microorganisms, and the volume of excess sludge can be reduced by the decomposition method of the present invention.

[0119] Allowing the sludge-decomposing bacteria, the bacteria or the microbial preparation of the present invention to act on sludge means: for example, the sludge-decomposing bacteria, the bacteria or the microbial preparation of the present invention or a solution in which they are suspended is brought into contact with the sludge. The sludge is decomposed by the sludge-decomposing bacteria, the bacteria or the microbial preparation of the present invention decomposing the target microorganisms contained in the sludge.

[0120] The process of allowing the sludge decomposing bacteria of the present invention, the bacteria of the present invention or the microbial preparation of the present invention to act on the sludge is not particularly limited as long as the sludge decomposing bacteria of the present invention, the bacteria of the present invention or the bacterium (a1) does not die or the target microbial decomposition ability of the bacterium is not lost. The process may be carried out at a temperature of 20 to 40° C. or 25 to 30° C. In addition, the process may be carried out at a pH of 6.5 to 8.0 or 7.0 to 7.5.

[0121] The amount of the sludge-decomposing bacteria of the present invention, the bacteria of the present invention, or the microbial agent of the present invention added to the sludge can be appropriately set in consideration of the type and concentration of the target microorganisms, the volume of the reaction system, and the like.

[0122] In the decomposition method of the present invention, the method for confirming that sludge decomposition has occurred is not particularly limited and can be performed by methods commonly used by those skilled in the art. Examples of such confirmation methods include the above-mentioned evaluation method for sludge decomposition ability or the evaluation method for target microorganism decomposition ability.

[0123] (Sludge decomposition device)

[0124] The sludge decomposition device is not particularly limited as long as it is a device that can reduce excess sludge using the sludge decomposition bacteria of the present invention, the bacteria of the present invention, or the microbial preparation of the present invention, and examples thereof include sludge treatment devices and wastewater treatment devices that produce excess sludge. In addition, the sludge decomposition bacteria of the present invention, the bacteria of the present invention, or the microbial preparation of the present invention may be added to an existing sludge treatment device, wastewater treatment device, etc. that has excess sludge, to thereby form a sludge decomposition device.

[0125] Conventionally, in order to decompose excess sludge, chemical treatments such as ozone, alkali, hypochlorous acid, physical treatments such as bead mills and ultrasonic pulverization, and biological treatments using microorganisms that function under high temperature conditions or alkaline conditions have been carried out. In order to carry out these treatments, the excess sludge must be transferred to other tanks, which consumes a lot of equipment costs and operating costs. The sludge decomposing bacteria, the bacteria and the microbial preparation of the present invention can be directly added to the excess sludge, so the cost of preparing new equipment can be reduced, and there is no need to change the sludge decomposition method that has been carried out in the past.

[0126] Example

[0127] [Experiment 1. Exploration of microorganisms with target microbial decomposition capabilities]

[0128] (method)

[0129] Several strains were isolated from microbial flora present in the environment (water).

[0130] (result)

[0131] The decomposition ability of micrococcal bacteria was investigated for each of the isolated strains, and the decomposition ability of micrococcal bacteria was confirmed in one strain. Hereinafter, the strain for which the decomposition ability of micrococcal bacteria was confirmed is referred to as "strain A".

[0132] [Experiment 2. Analysis of the base sequence of the 16S rRNA gene of strains having the ability to decompose Micrococcus bacteria]

[0133] (Material)

[0134] 802 medium: 10 g polypeptone, 2 g yeast extract, and 1 g magnesium sulfate heptahydrate were dissolved in ultrapure water, the pH was adjusted to 7.0, and the resulting volume was 1000 mL. The medium was then sterilized by high pressure steam.

[0135] Forward primer for cloning (27f: SEQ ID NO: 2)

[0136] Reverse primer for cloning (1492r: SEQ ID NO: 3)

[0137] Primers for sequence analysis (339F: SEQ ID NO: 4, 536R: SEQ ID NO: 5, 907F: SEQ ID NO: 6)

[0138] (method)

[0139] DNA was extracted from strain A using EZ-EXTRACT for DNA (manufactured by AMR Corporation) and used as template DNA for amplifying the 16S rRNA gene by PCR.

[0140] PCR was performed under the following conditions. 25.0 μL of 2×PCR buffer for KOD FX (manufactured by Toyobo Co., Ltd.), 10.0 μL of dNTP mix (2 mM), 1.5 μL of cloning forward primer and cloning reverse primer (10 pmol / μL each), 0.58 μL of template DNA, 10.4 μL of sterile water, and 1.0 μL of DNA polymerase (KOD FX, 1 U / μL, manufactured by Toyobo Co., Ltd.) were added to a microtube and mixed. The microtube was supplied to a PCR apparatus to perform a template DNA amplification reaction. The reaction was performed at (1) 94°C for 2 minutes, (2) 98°C for 10 seconds, (3) 50°C for 30 seconds, and (4) 68°C for 1.5 minutes, and the steps (2) to (4) were repeated for 35 cycles. The amplified product after PCR was purified.

[0141] The following reaction solution was prepared, which was mixed with 150 ng of purified PCR amplification product, 0.32 μL of primers for sequence analysis (10 μM), and 8.0 μL of BigDye Terminator v3.1 (Applied Biosystems), and sterile ultrapure water was added to adjust the liquid volume to 20.0 μL. The reaction solution was supplied to a PCR device for amplification reaction. The reaction was carried out at (1) 96°C for 1 minute, (2) 96°C for 10 seconds, (3) 50°C for 5 seconds, and (4) 60°C for 4 minutes, and the steps (2) to (4) were repeated for 25 cycles. The obtained reaction solution was purified, and the purified solution was supplied to DNA sequence analysis (3730xl DNA analyzer) to determine the base sequence of the 16S rRNA gene of the template DNA extracted from strain A.

[0142] (result)

[0143] The obtained base sequence was subjected to homology analysis with respect to the international base sequence database (DDBJ / ENA (EMBL) / GenBank). In the reference strain, it showed 99.8% identity with the base sequence of the 16S rRNA gene of Bacillus parabrevis IFO12334. However, there is no microorganism having a 16S rRNA gene that is completely identical to the obtained base sequence.

[0144] The base sequence of the 16S rRNA gene in this case is represented by Sequence No. 1. The above shows that the bacteria having the 16S rRNA gene having the base sequence described in Sequence No. 1 have the target microorganism decomposition ability.

[0145] [Experiment 3. Morphological observation and physiological and biochemical property tests of a strain of Brevibacillus parabrevis having a 16S rRNA gene containing the base sequence described in SEQ ID NO: 1]

[0146] (method)

[0147] Morphological observation and physiological and biochemical properties of strain A were performed. These tests were performed by morphological observation using an optical microscope, the method of Barrow et al. (Cowan and Steel's Manual for the Identification of Medical Bacteria 3rd Edition 1993, Cambridge University Press.) and API50CHB (manufactured by bioMerieux, Lyon, France). The test results are shown in Tables 1 to 3.

[0148] (result)

[0149] like Figure 1 As shown, the colonies of strain A have a round and creamy morphology. Figure 2 As shown in Table 4, strain A is a bacillus and is negative for Gram staining. In the aspects shown in Table 4, strain A has characteristics different from the parabrevis strain IFO12334 with the highest homology of 16S rRNA gene. Therefore, it is shown that strain A is a new strain different from the previous parabrevis strain. Strain A was deposited as parabrevis NITE BP-03020.

[0150] [Table 1]

[0151]

[0152]

[0153] +: positive, -: negative

[0154] [Table 2]

[0155]

[0156]

[0157] *Fermentation test, **Biochemical test +: positive, -: negative

[0158] [Table 3]

[0159]

[0160]

[0161] [Table 4]

[0162]

[0163] [Experiment 4. Evaluation of the target microbial decomposition ability of Bacillus parabrevis NITE BP-03020]

[0164] (Material)

[0165] 802 medium: 10 g polypeptone, 2 g yeast extract, and 1 g magnesium sulfate heptahydrate were dissolved in ultrapure water, the pH was adjusted to 7.0, and the resulting volume was 1000 mL. The medium was then sterilized by high pressure steam.

[0166] Solution A: Dissolve 4.35 g of dipotassium hydrogen phosphate, 1.70 g of potassium dihydrogen phosphate, 8.92 g of disodium hydrogen phosphate 12 hydrate, and 0.34 g of ammonium chloride in ultrapure water to prepare a 200 mL solution, which was sterilized by high pressure steam.

[0167] Solution B: Dissolve 4.50 g of magnesium sulfate heptahydrate in ultrapure water to prepare 200 mL of solution, and sterilize with high pressure steam.

[0168] Solution C: Dissolve 5.50 g of anhydrous calcium chloride in ultrapure water to prepare 200 mL of solution, and sterilize with high pressure steam.

[0169] Solution D: Dissolve 0.05 g of ferric chloride hexahydrate in ultrapure water to prepare 200 mL of the solution, which was sterilized by filtration using a 0.2 μm syringe filter.

[0170] (method)

[0171] Micrococcus (target microorganism) was cultured in 802 medium at a temperature of 25°C, and after bacteria collection and washing, the mixture was mixed in a manner that the turbidity (OD660) became 0.2 with respect to 986 mL of ultrapure water, and sterilized by high pressure steam to obtain a matrix solution. 3.0 mL of A solution, 3.0 mL of B solution, 3.0 mL of C solution, 3.0 mL of D solution, and 1.8 mL of 1% phosphoric acid solution were mixed in 986 mL of the matrix solution to obtain a target microorganism solution using an inorganic medium as a solvent. The target microorganism contained in the target microorganism solution is a target dead bacterium.

[0172] The parabrevicaulis NITE BP-03020 was inoculated into 802 medium and cultured at 30°C for 24 to 48 hours. After the culture, 50 μL of the culture solution of parabrevicaulis NITE BP-03020 was added to 5.0 mL of the target microorganism solution, and the mixture was shaken at 25°C and 200 rpm. The turbidity (OD660) of the test tube was measured over time using a simple turbidity meter (simple OD minitor, miniphoto 518R, manufactured by TAITEC). The results are shown in Figure 3 In addition, 50 μL of the culture solution of Paenibacillus glycanilyticus cultured in the same manner was added to 50 mL of the target microorganism solution, and the turbidity was measured. The results are shown in Figure 4 The negative control was the turbidity (OD660) of the target microorganism-containing solution to which the culture solution of Bacillus parabrevicus NITE BP-03020 was not added.

[0173] like Figure 3 As shown in FIG. 1 , the turbidity of the target microorganism-containing solution of the negative control remained almost unchanged, whereas the turbidity of the target microorganism-containing solution to which the culture solution of Bacillus parabrevis NITE BP-03020 was added was reduced to less than 50% of that before addition. Figure 4 As shown, no decrease in turbidity was observed in the target microorganism-containing solution to which the culture solution of Paenibacillus sp. was added. From the above results, it was found that Bb. parabrevis has the target microorganism decomposition ability.

[0174] [Experiment 5. Evaluation of the target microbial decomposition ability of a microbial preparation containing a culture of Bacillus parabrevicaulis NITE BP-03020]

[0175] (method)

[0176] Bacillus parabrevis NITE BP-03020, Bacillus parabrevis NBRC3331, Bacillus parabrevis NBRC12333, Bacillus parabrevis NBRC12334, and Bacillus parabrevis NBRC12374 were inoculated into 802 medium, respectively, and cultured at 30°C for 24 to 48 hours. After the culture, the culture solution was filtered with a 0.2μm syringe filter to obtain the culture supernatant (culture). 50μL of the culture supernatant was added to 5.0mL of the target microorganism solution, and the mixture was shaken at 25°C and 200rpm. The turbidity (OD660) of the test tube was measured over time using a simple turbidity meter (simple OD minitor, miniphoto 518R, manufactured by TAITEC). The results are shown in Figure 5 The negative control was the turbidity (OD660) of the target microorganism solution without the addition of the culture supernatant of Bacillus parabrevis. The target microorganism solution used was the same solution as in Experiment 4. Bacillus parabrevis NBRC3331, Bacillus parabrevis NBRC12333, Bacillus parabrevis NBRC12334, and Bacillus parabrevis NBRC12374 were obtained from the Institute of Technology and Product Evaluation.

[0177] (result)

[0178] For each Brevibacillus parasporin strain other than Brevibacillus parasporin NITE BP-03020, the turbidity of the target microorganism-containing solution was also reduced by adding the culture supernatant. Therefore, it was found that Brevibacillus parasporin strains other than Brevibacillus parasporin NITE BP-03020 also have the ability to decompose the target microorganism. In addition, it was found that the culture of Brevibacillus parasporin strains also has the ability to decompose the target microorganism.

[0179] Furthermore, the target microorganism solution to which the culture supernatant of Bacillus parabrevis NITE BP-03020 was added had lower turbidity than the target microorganism solution to which the culture supernatant of other Bacillus parabrevis strains was added. It is known that Bacillus parabrevis NITE BP-03020 has a superior target microorganism decomposition ability than other Bacillus parabrevis strains.

[0180] [Experiment 6. Evaluation of the decomposition ability of a microbial preparation containing a culture of Bacillus parabrevicaulis NITE BP-03020 against various target microorganisms]

[0181] (Material)

[0182] LB liquid medium: LB broth and 1.1 g of PERTABLET (manufactured by SIGMA) were dissolved in a ratio of 10 to 500 mL of ultrapure water and sterilized by high pressure steam.

[0183] 802 medium: same as experiment 4

[0184] Bacterial culture medium: 20 g of glucose, 5 g of polypeptone, 3 g of yeast extract, 3 g of meat juice, 2 g of ammonium sulfate, 1 g of potassium dihydrogen phosphate, and 0.5 g of magnesium sulfate heptahydrate were dissolved in ultrapure water, the pH was adjusted to 7.0, and the medium was prepared to 1000 mL and sterilized by high pressure steam.

[0185] YM medium: 20g glucose, 5g peptone, 3g yeast extract, and 3g malt extract dissolved in ultrapure water and sterilized by high pressure steam

[0186] The target microorganisms described in Table 5 were cultured in the culture medium and culture temperature described in the table, and after the bacteria were collected and washed, they were mixed in a manner such that the turbidity (OD660) became 0.2 to 0.3 with respect to 986 mL of ultrapure water, and sterilized by high pressure steam to obtain a matrix solution. The same A liquid to D liquid and phosphoric acid solution as in Experiment 4 were mixed in the matrix solution to obtain a target microorganism-containing solution with various types of bacteria (dead bacteria) as target microorganisms.

[0187] [Table 5]

[0188]

[0189] Except for the type of target microorganism solution, the culture supernatant of Bacillus parabrevis NITE BP-03020 was added to each target microorganism solution in the same manner as in Experiment 5, and the turbidity was measured over time. The results are shown in Figure 6 to Figure 10 .

[0190] (result)

[0191] The turbidity of the target microorganism-containing solution to which the culture supernatant of Bacillus parabrevis NITE BP-03020 was added was reduced compared with the negative control, indicating that Bacillus parabrevis and its culture can decompose various target microorganisms.

[0192] [Experiment 7. Evaluation of sludge decomposition ability of a microbial preparation containing a culture of Bacillus parabrevicaulis NITE BP-03020]

[0193] (Material)

[0194] After washing the excess sludge, the excess sludge was mixed with 986 mL of ultrapure water so that the turbidity (OD660) was 0.2, and sterilized by high pressure steam to obtain an excess sludge matrix solution. The target microorganisms in the excess sludge matrix solution were dead bacteria. A solution containing excess sludge was prepared, which was prepared by mixing 3.0 mL of solution A, 3.0 mL of solution B, 3.0 mL of solution C, 3.0 mL of solution D, and 1.8 mL of 1% phosphoric acid in 986 mL of excess sludge matrix solution.

[0195] (method)

[0196] The culture supernatant of Bacillus parabrevicus NITE BP-03020 was added to the excess sludge-containing solution in the same manner as in Experiment 5, except that the excess sludge-containing solution was used as the solution containing the target microorganism, and the turbidity of the excess sludge-containing solution was measured over time.

[0197] (result)

[0198] like Fig.11 As shown, it was confirmed that the turbidity of the excess sludge-containing solution decreased by adding the culture supernatant of Bacillus parabrevis NITE BP-03020. Therefore, it was found that the culture of Bacillus parabrevis NITE BP-03020 has the ability to decompose sludge.

[0199] [Experiment 8. Evaluation of Temperature Sensitivity of Target Microbial Decomposition Ability of Microbial Preparations Containing Cultures of Bacillus parabrevicaulis NITE BP-03020]

[0200] (method)

[0201] The turbidity of the target microorganism-containing solution was measured over time in the same manner as in Experiment 5 except that the shaking temperature after adding the culture supernatant of Bacillus parabrevicus NITE BP-03020 to the target microorganism-containing solution was changed. The shaking temperature was 20°C, 25°C, 30°C, or 37°C.

[0202] (result)

[0203] like Fig.12 As shown, the turbidity of the target microorganism-containing solution to which the culture supernatant of Bacillus parabrevis NITE BP-03020 was added decreased at a shaking temperature of 20° C. to 37° C. It was found that the microorganism preparation containing the culture of Bacillus parabrevis NITE BP-03020 also had the target microorganism decomposition ability at a temperature of 20° C.

[0204] [Experiment 9. Evaluation of sludge decomposition ability of composite microbial preparations containing culture supernatants of Bacillus parabrevis NITE BP-03020, Bacillus subtilis subsp. inaquosorum DSM 22148, and Bacillus expansus NITE BP-02779]

[0205] (Material)

[0206] After washing the excess sludge, the excess sludge was mixed with 986 mL of ultrapure water so that the turbidity (OD660) was 0.15, and sterilized by high pressure steam to obtain an excess sludge matrix solution. The target microorganisms in the excess sludge matrix solution were dead bacteria. A solution containing excess sludge was prepared, which was prepared by mixing 3.0 mL of A solution, 3.0 mL of B solution, 3.0 mL of C solution, 3.0 mL of D solution, and 1.8 mL of 1% phosphoric acid in 986 mL of excess sludge matrix solution.

[0207] Bacillus subtilis desert subsp. DSM 22148 was isolated from the environment. The base sequence of the 16S rRNA gene of Bacillus subtilis desert subsp. DSM 22148 is shown as Sequence No. 8. The identity of the 16S rRNA gene sequences of Bacillus parabrevis NITE BP-03020 and Bacillus expansus NITE BP-02779 was 87%, and the identity of the 16S rRNA gene sequences of Bacillus parabrevis NITE BP-03020 and Bacillus subtilis desert subsp. DSM 22148 was 89%.

[0208] (method)

[0209] Bacillus parabrevis NITE BP-03020 was inoculated into 802 medium and cultured at 30°C for 24 to 48 hours. Bacillus subtilis subsp. deserticola DSM 22148 was inoculated into 802 medium and cultured at 37°C for 24 to 48 hours. Bacillus expansum species NITE BP-02779 was inoculated into R2A medium and cultured at 25°C for 24 to 48 hours. After the culture was completed, the culture solution was filtered with a 0.2μm syringe filter to obtain a culture supernatant (culture). 50μL of each culture supernatant was added to 5.0mL of the excess sludge solution, and the solution was shaken at 25°C and 200rpm. The turbidity (OD660) of the test tube was measured over time using a simple turbidity meter (simple ODminitor, miniphoto 518R, manufactured by TAITEC). 16.7μL of each culture supernatant was added to the three mixed supernatant addition zones. The results are shown in Fig.13 The negative control was the turbidity (OD660) of the target microorganism-containing solution without the addition of culture supernatant.

[0210] (result)

[0211] like Fig.13 As shown in the figure, compared with the culture supernatant of Bacillus parabrevis NITE BP-03020, Bacillus subtilis subsp. deserticola DSM 22148, and Bacillus expansum NITE BP-02779 added alone, the turbidity of the excess sludge-containing solution was significantly reduced by mixing and adding each of them. Therefore, it is known that the composite microbial preparation containing the culture of Bacillus parabrevis NITE BP-03020 and the other two bacteria also has the ability to decompose sludge.

[0212] [Reference Experiment 1. Exploration of microorganisms with target microbial decomposition capabilities]

[0213] (method)

[0214] Microorganisms that decompose Micrococcus genus bacteria are enriched and cultured by culturing microorganisms existing in the environment (water) using a culture medium containing Micrococcus genus bacteria as a carbon source. Then, several strains that proliferate well are isolated from the enriched and cultured microorganisms.

[0215] (result)

[0216] The decomposition ability of micrococcus bacteria was investigated for each of the isolated strains, and the decomposition ability of micrococcus bacteria was confirmed in one strain. Hereinafter, the strain for which the decomposition ability of micrococcus bacteria was confirmed is sometimes referred to as "strain B".

[0217] [Reference Experiment 2. Analysis of the base sequence of the 16S rRNA gene of strains having the ability to decompose micrococcal bacteria]

[0218] (Material)

[0219] R2A medium: 3.2 g of R2A broth (DAIGO (manufactured by Nippon Pharmaceutical Co., Ltd.)) was dissolved in 1000 mL of ultrapure water and sterilized by high pressure steam.

[0220] Forward primer for cloning (27f: SEQ ID NO: 2)

[0221] Reverse primer for cloning (1492r: SEQ ID NO: 3)

[0222] Primers for sequence analysis (339F: SEQ ID NO: 4, 536R: SEQ ID NO: 5, 907F: SEQ ID NO: 6)

[0223] (method)

[0224] DNA was extracted from strain B using EZ-EXTRACT for DNA (AMR Corporation) and used as template DNA for amplifying the 16S rRNA gene by PCR.

[0225] PCR was performed under the following conditions. 25.0 μL of 2×PCR buffer for KOD FX (manufactured by Toyobo Co., Ltd.), 10.0 μL of dNTP mix (2 mM), 1.5 μL of cloning forward primer and cloning reverse primer (10 pmol / μL each), 0.58 μL of template DNA, 10.4 μL of sterile water, and 1.0 μL of DNA polymerase (KOD FX, 1 U / μL, manufactured by Toyobo Co., Ltd.) were added to a microtube and mixed. The microtube was supplied to a PCR apparatus to perform a template DNA amplification reaction. The reaction was performed at (1) 94°C for 2 minutes, (2) 98°C for 10 seconds, (3) 50°C for 30 seconds, and (4) 68°C for 1.5 minutes, and the steps (2) to (4) were repeated for 35 cycles. The amplified product after PCR was purified.

[0226] The following reaction solution was prepared, which was mixed with 150 ng of purified PCR amplification product, 0.32 μL of primers for sequence analysis (10 μM), and 8.0 μL of BigDye Terminator v3.1 (Applied Biosystems), and sterile ultrapure water was added to adjust the liquid volume to 20.0 μL. The reaction solution was supplied to a PCR device for amplification reaction. The reaction was carried out at (1) 96°C for 1 minute, (2) 96°C for 10 seconds, (3) 50°C for 5 seconds, and (4) 60°C for 4 minutes, and the steps (2) to (4) were repeated for 25 cycles. The obtained reaction solution was purified, and the purified solution was supplied to DNA sequence analysis (3730xl DNA analyzer) to determine the base sequence of the 16S rRNA gene of the template DNA extracted from strain B.

[0227] (result)

[0228] The obtained base sequence was subjected to homology analysis with respect to the international base sequence database (DDBJ / ENA (EMBL) / GenBank). In the reference strain, it showed 98.1% identity with the base sequence of the 16S rRNA gene of Tumebacillus permanentifrigoris Eurl_9.5. However, there is no microorganism with a 16S rRNA gene that is completely identical to the obtained base sequence.

[0229] The base sequence of the obtained 16S rRNA gene is represented by Sequence No. 7. This indicates that bacteria containing the 16S rRNA gene having the base sequence described in Sequence No. 7 have microbial decomposition ability.

[0230] [Reference Experiment 3. Morphological observation and physiological and biochemical property test of Tumebacillus bacteria containing the 16S rRNA gene having the base sequence described in SEQ ID NO: 7]

[0231] (method)

[0232] Morphological observation and physiological and biochemical properties of strain B were performed. These tests were performed by morphological observation using an optical microscope, the method of Barrow et al. (Cowan and Steel's Manual for the Identification of Medical Bacteria 3rd Edition 1993, Cambridge University Press.) and API50CHB (manufactured by bioMerieux, Lyon, France). The test results are shown in Tables 6 to 9.

[0233] (result)

[0234] Strain B does not grow at 10°C, but this feature is not found in the Tumebacillus permanentifrigoris Eurl_9.5, which has the highest homology of 16S rRNA gene. Therefore, it is shown that strain B is a new species different from the previous Tumebacillus. Strain B is deposited as Tumebacillus species NITE BP-02779.

[0235] [Table 6]

[0236]

[0237]

[0238] +: positive, -: negative, +w: weak reaction

[0239] [Table 7]

[0240]

[0241]

[0242] +: positive, -: negative

[0243] [Table 8]

[0244]

[0245]

[0246] +: positive, -: negative

[0247] [Table 9]

[0248] Test Project Test results Grow at 10°C - Growth at pH 9.0 + Starch hydrolysis +

[0249] +: positive, -: negative

[0250] [Reference Experiment 4. Evaluation of the Decomposition Ability of Bacillus expansus NITE BP-02779 on Target Bacteria (Dead Bacteria)]

[0251] (Material)

[0252] R2A medium: 3.2 g of R2A broth (DAIGO (manufactured by Nippon Pharmaceutical Co., Ltd.)) was dissolved in 1000 mL of ultrapure water and sterilized by high pressure steam.

[0253] LB liquid medium: LB broth and 1.1 g of PERTABLET (manufactured by SIGMA) were dissolved in a ratio of 10 to 500 mL of ultrapure water and sterilized by high pressure steam.

[0254] 802 medium: 10 g polypeptone, 2 g yeast extract, and 1 g magnesium sulfate heptahydrate were dissolved in ultrapure water, the pH was adjusted to 7.0, and the resulting volume was 1000 mL. The medium was then sterilized by high pressure steam.

[0255] Bacterial culture medium: 20 g of glucose, 5 g of polypeptone, 3 g of yeast extract, 3 g of meat juice, 2 g of ammonium sulfate, 1 g of potassium dihydrogen phosphate, and 0.5 g of magnesium sulfate heptahydrate were dissolved in ultrapure water, the pH was adjusted to 7.0, and the medium was prepared to 1000 mL and sterilized by high pressure steam.

[0256] Inorganic culture medium containing target bacteria (dead bacteria): a mixture of 986 mL of substrate solution, 3.0 mL of solution A, 3.0 mL of solution B, 3.0 mL of solution C, 3.0 mL of solution D, and 1.8 mL of 1% phosphoric acid

[0257] In addition, the following solutions were used as the above-mentioned base solution and solution A to solution D.

[0258] Matrix solution: The target bacteria listed in Table 10 were cultured under the conditions listed in the table, the bacteria were collected and washed, and then mixed with 986 mL of ultrapure water so that the turbidity (OD660) was 0.2, and then sterilized by high pressure steam.

[0259] Solution A: Dissolve 4.35 g of dipotassium hydrogen phosphate, 1.70 g of potassium dihydrogen phosphate, 8.92 g of disodium hydrogen phosphate dodecahydrate, and 0.34 g of ammonium chloride in ultrapure water to prepare a 200 mL solution that was sterilized by high pressure steam.

[0260] Solution B: Dissolve 4.50 g of magnesium sulfate heptahydrate in ultrapure water to prepare 200 mL of solution, and sterilize with high pressure steam.

[0261] Solution C: Dissolve 5.50 g of anhydrous calcium chloride in ultrapure water to make 200 mL of solution, and sterilize with high pressure steam.

[0262] Solution D: 0.05 g of ferric chloride hexahydrate was dissolved in ultrapure water to prepare 200 mL of the solution, which was sterilized by filtration using a 0.2 μm syringe filter.

[0263] [Table 10]

[0264]

[0265] (method)

[0266] Bacillus expansum sp. NITE BP-02779 was inoculated into R2A medium and cultured at 25° C. for 24 to 48 hours.

[0267] After the culture was completed, 50 μL of the culture solution of Bacillus expansus species NITE BP-02779 and 5.0 mL of the inorganic culture medium containing the target bacteria (dead bacteria) were added to the test tube, and the reaction was carried out at 25°C and 200 rpm. The turbidity (OD660) of the test tube was measured over time using a simple turbidity meter (simple ODminitor, miniphoto 518R, manufactured by TAITEC). The number of days after the turbidity (OD660) of the inorganic culture medium containing the target bacteria (dead bacteria) showed 50% of the turbidity (OD660) of the negative control was calculated, and the presence or absence of decomposition ability was determined based on the following criteria.

[0268] A: More than 0 days and less than 2.0 days

[0269] B: more than 2.0 days and less than 5.0 days

[0270] C: 5.0 days or more

[0271] In addition, the turbidity (OD660) of the inorganic culture medium containing the target bacteria (dead bacteria) to which Bacillus sp. NITE BP-02779 was not added was used as the negative control.

[0272] (result)

[0273] The results are shown in Table 11. It was confirmed that the turbidity of the inorganic culture medium containing the target bacteria (dead bacteria) decreased by adding Bacillus expansus NITE BP-02779. Therefore, Bacillus expansus NITE BP-02779 can decompose the target microorganisms (target dead bacteria).

[0274] [Table 11]

[0275] Target bacteria (dead bacteria) result Cell morphology Gram stain Micrococcus A(0.7 days) cocci Positive Bacillus A(0.8 days) Bacillus Positive staphylococcus A(1.9 days) cocci Positive Lactobacilli A(1.9 days) Bacillus Positive Paenibacillus A(1.1 days) Bacillus Positive Escherichia A(0.6 days) Bacillus Negative Acetobacter A(0.7 days) Bacillus Negative

[0276] [Reference Experiment 5. Evaluation of the Decomposition Ability of Bacillus expansus NITE BP-02779 on Target Bacteria (Live Bacteria)]

[0277] (Material)

[0278] Inorganic culture medium containing target bacteria (live bacteria): a mixture of 986 mL of sterile water, 3.0 mL of solution A, 3.0 mL of solution B, 3.0 mL of solution C, 3.0 mL of solution D, 1.8 mL of 1% phosphoric acid, and a solution containing target bacteria (live bacteria)

[0279] In addition, the same solutions as those in Reference Experiment 4 were used as solutions A to D. In addition, the target bacteria (live bacteria) were mixed so that the turbidity (OD660) of the inorganic culture medium containing the target bacteria (live bacteria) was 0.2.

[0280] (method)

[0281] The decomposition ability of Bacillus expansum sp. NITE BP-02779 on the target bacteria (live bacteria) was evaluated in the same manner as in Reference Experiment 4 except that an inorganic culture medium containing the target bacteria (live bacteria) was used.

[0282] (result)

[0283] The results are shown in Table 12. It was confirmed that the turbidity of the inorganic culture medium containing the target bacteria (live bacteria) decreased by adding Bacillus expansus NITE BP-02779. Therefore, Bacillus expansus NITE BP-02779 can decompose the target bacteria (live bacteria).

[0284] [Table 12]

[0285]

[0286] [Reference Experiment 6. Evaluation of the Decomposition Ability of Bacillus expansus NITE BP-02779 on Excess Sludge (Dead Bacteria) (1)]

[0287] Inorganic culture medium containing excess sludge (dead bacteria): a mixture of 986 mL of substrate solution, 3.0 mL of solution A, 3.0 mL of solution B, 3.0 mL of solution C, 3.0 mL of solution D, and 1.8 mL of 1% phosphoric acid

[0288] · Base solution: A solution prepared by washing excess sludge, mixing it with 986 mL of ultrapure water so that the turbidity (OD660) becomes 0.2, and sterilizing it with high pressure steam

[0289] The same solution as in Reference Experiment 4 was used except that the above solution was used as the base solution.

[0290] (method)

[0291] The decomposition ability of Bacillus expansum sp. NITE BP-02779 on excess sludge (dead bacteria) was evaluated in the same manner as in Reference Experiment 4 except that an inorganic culture medium containing excess sludge (dead bacteria) was used.

[0292] (result)

[0293] The results are shown in Table 13. It was confirmed that the turbidity of excess sludge (dead bacteria) was reduced by adding Bacillus expansus NITE BP-02779. Therefore, Bacillus expansus NITE BP-02779 can decompose excess sludge (dead bacteria).

[0294] [Table 13]

[0295] Target bacteria (dead bacteria) result Cell morphology Gram stain Excess sludge B (3.2 days) - -

[0296] [Reference Experiment 7. Evaluation of the Decomposition Ability of Bacillus expansus NITE BP-02779 on Excess Sludge (Dead Bacteria) (2)]

[0297] (Material)

[0298] The same materials as in Reference Experiment 6 were used.

[0299] (method)

[0300] The reaction was carried out in the same manner as in Reference Experiment 4. The reaction was carried out five times in succession, and all the excess sludge remaining in the test tube was recovered on the fourth day after the start of the reaction. After the recovered excess sludge was dried, the dry weight of the excess sludge was measured, and a significant difference test (t-test, one-sided) was performed between the negative control group and the group added with Bacillus sp. NITE BP-02779.

[0301] (result)

[0302] The results are shown in Fig.14Compared with the negative control group, the group to which Bacillus expansum NITE BP-02779 was added showed a significant decrease in the dry weight of excess sludge (p<0.01). Therefore, by adding Bacillus expansum NITE BP-02779, excess sludge can be reduced.

[0303] [Reference Experiment 8. Evaluation of the Decomposition Ability of Various Bacteria on Micrococcus]

[0304] (Material)

[0305] As the target bacteria-containing inorganic culture medium, a target bacteria-containing (dead bacteria) inorganic culture medium containing the Micrococcus bacteria used in Reference Experiment 4 as target bacteria was prepared. The bacteria shown in Table 14 were prepared as bacteria for evaluating the presence or absence of microbial decomposition ability.

[0306] [Table 14]

[0307]

[0308]

[0309]

[0310] (method)

[0311] The target bacteria-containing inorganic culture medium and the culture solution containing the bacteria to be evaluated were mixed in the same manner as in Reference Experiment 4, and the microbial decomposition ability of the bacteria to be evaluated was evaluated. The number of days until the turbidity (OD660) of the target bacteria-containing inorganic culture medium reached 50% of the turbidity (OD660) of the negative control was calculated, and the presence or absence of decomposition ability was determined based on the following reference microorganisms.

[0312] A: more than 0 days and less than 1.0 days

[0313] B: more than 1.0 days and less than 4.0 days

[0314] C: 4.0 days or more

[0315] In addition, the turbidity (OD660) of the inorganic culture medium containing the target bacteria to which the culture solution containing the bacteria to be evaluated was not added was used as a negative control.

[0316] (result)

[0317] The results are shown in Table 15. In addition, a homology search was performed between the base sequence of the 16S rRNA gene of the bacteria to be evaluated listed in Table 14 and the base sequence of the 16S rRNA gene of Bacillus expansus species NITE BP-02779 (sequence number 7), and the calculated identity ratio is shown in Table 15. The base sequences of the 16S rRNA gene of the bacteria listed in Table 15 are represented by sequence numbers 9 to 18. These are sequences included in the online catalog of the National Institute of Technology for Product Evaluation (NBRC) <https: / / www.nite.go.jp / nbrc / catalogue / NBRCDispSearchServlet?lang=jp> or the database catalog of the National Center for Biotechnology Information (NCBI) <https: / / www.ncbi.nlm.nih.gov / >.

[0318] Bacillus expansum species NITE BP-02779 and algae expansum Bacillus NBRC108765t were judged as A, while Bacillus bacteria and Paenibacillus bacteria were judged as B or C. Therefore, Bacillus expansum species NITE BP-02779 and algae expansum Bacillus NBRC108765t had a superior target microorganism (target dead bacteria) decomposition ability compared with Bacillus bacteria and Paenibacillus bacteria.

[0319] The identity of the base sequence of the 16S rRNA gene of Bacillus expansus species NITE BP-02779 and the base sequence of the 16S rRNA gene of Bacillus expansus NBRC108765t is 92.8%. On the other hand, the identity of the base sequence of the 16S rRNA gene of Bacillus expansus species NITE BP-02779 and the base sequence of the 16S rRNA gene of Bacillus bacteria or Paenibacillus bacteria is less than 90%. It is believed that the target microbial decomposition ability of bacteria with a base sequence of 16S rRNA gene of 90% or more identity with Bacillus expansus species NITE BP-02779 is excellent. Bacillus expansus NBRC108765t can be obtained from NBRC, and the base sequence of its 16S rRNA gene is included in the GenBank / EMBL / DDBJ database with accession number JX110710.

[0320] [Table 15]

[0321]

[0322]

[0323] [Reference Experiment 9. Evaluation of target microbial decomposition ability of a mixed solution containing Bacillus expansus NITE BP-02779 and Escherichia coli]

[0324] (Material)

[0325] As the target bacteria-containing inorganic culture medium, a target bacteria-containing (dead bacteria) inorganic culture medium containing the Micrococcus bacteria used in Reference Experiment 4 as the target bacteria was prepared. In addition, a solution containing Bacillus expansum sp. NITE BP-02779 and Escherichia coli DH5α was prepared.

[0326] (method)

[0327] First, Bacillus expansum sp. NITE BP-02779 was cultured at 25°C for 24 hours, and Escherichia coli DH5α was cultured at 37°C for 24 hours.

[0328] After the culture, a mixed solution was prepared, which was mixed with a culture solution of Bacillus expansum species NITEBP-02779 (prepared to OD660 = 0.2) and a culture solution of Escherichia coli DH5α (prepared to OD660 = 0.2) at the ratio shown in Table 16. 50 μL of the prepared microbial preparation and 5.0 mL of the inorganic culture medium containing the target bacteria were added to a test tube, and the reaction was carried out at 25°C and 200 rpm. After 2 days, the turbidity (OD660) of the mixed solution was measured using a simple turbidity meter (simple OD minitor, miniphoto 518R, manufactured by TAITEC). The ratio of the turbidity (OD660) of the inorganic culture medium containing the target bacteria to which the microbial preparation was added to the turbidity (OD660) of the negative control was calculated as the residual rate of the target microorganism, and the target microorganism decomposition ability was evaluated according to the following criteria.

[0329] A: 0% or more and less than 50%

[0330] B: 50% or more and less than 70%

[0331] C: 70% or more

[0332] It should be noted that the turbidity (OD660) of the inorganic culture medium containing the target bacteria to which no mixed solution was added was used as a negative control.

[0333] (result)

[0334] The results are shown in Table 16. The solution containing Bacillus expansum NITE BP-02779 and Escherichia coli showed good target microorganism decomposition ability.

[0335] [Table 16]

[0336]

[0337] T: Bacillus expansus species NITE BP-02779 bacterial count

[0338] E: The number of Escherichia coli DH5α

[0339] Residual rate of target microorganism (%) = OD660 (with added microbial preparation) / OD660 (negative control) × 100

[0340] [Reference Experiment 10. Evaluation of target microbial decomposition ability of Bacillus expansus NITE BP-02779 culture]

[0341] (Material)

[0342] As the phosphate buffer containing the target bacteria (live bacteria), a solution prepared by mixing 10 mL of 66 mM potassium phosphate buffer (pH 6.24) and 10 mg of dried cells of Micrococcus bacteria (manufactured by SIGMA-ALDRICH) was used.

[0343] (method)

[0344] First, Bacillus expansum sp. NITE BP-02779 was cultured in R2A medium for a certain period of time.

[0345] After the culture, the culture solution was centrifuged and the culture supernatant from which the bacterial cells were removed was recovered. 100 μL of the recovered culture supernatant and 100 μL of the target bacteria phosphate buffer were added to a 96-well plate, and the ABS450 nm was measured over time using an ELISA reader (Molecular Device). The decomposition capacity of micrococcal bacteria (UNITS / mL) was calculated, and the target microbial decomposition capacity was evaluated according to the following criteria.

[0346] S: 200 (UNITS / mL) or more

[0347] A: 100 (UNITS / mL) or more and less than 200 (UNITS / mL)

[0348] B: 20 ​​(UNITS / mL) or more and less than 100 (UNITS / mL)

[0349] C: less than 20 (UNITS / mL)

[0350] In addition, the decomposition ability (UNITS / mL) of the Micrococcus bacteria was calculated using the following formula.

[0351] Micrococcus bacteria decomposition capacity (UNITS / mL)

[0352] ={ΔABS450 nm / min (phosphate buffer containing target bacteria with culture supernatant added) - ΔABS450 nm / min (phosphate buffer containing target bacteria without culture supernatant added)} / (0.001×0.1)

[0353] (result)

[0354] The results are shown in Table 17. It was found that the culture supernatant obtained after culturing Bacillus expansum species NITE BP-02779 for a certain period of time had target microorganism decomposition ability.

[0355] [Table 17]

[0356] Cultivation time (hours) Micrococcus bacteria decomposition ability 0 C(13UNITS / mL) 21 A(121UNITS / mL) 28 S(249UNITS / mL) 46 S(250UNITS / mL) 53 S(252UNITS / mL) 68 A(153UNITS / mL) Sequence Listing <110> Sumitomo Chemical Co., Ltd. Kataoka Microbiology Research Institute Co., Ltd. <120> Bacteria for decomposing sludge, microbial decomposing bacteria, microbial preparation, sludge decomposing method and sludge decomposing device <130> S44179WO01 <150> JP2019-233401 <151> 2019-12-24 <160> 18 <170> PatentIn version 3.5 <210> 1 <211> 1486 <212> DNA <213> Brevibacillus parabrevis NITE BP-03020 <400> 1 cgaacgctgg cggcgtgcct aatacatgca agtcgagcga gggttttcgg accctagcgg 60 cggacgggtg agtaacacgt aggcaacctg cctctcagac cgggataaca tagggaaact 120 tatgctaata ccggataggt ttttggattg catgatccga aaagaaaaga tggcttcggc 180 tatcactggg agatgggcct gcggcgcatt agctagttgg tggggtaacg gcctaccaag 240 gcgacgatgc gtagccgacc tgagagggtg accggccaca ctgggactga gacacggccc 300 agactcctac gggaggcagc agtagggaat tttccacaat ggacgaaagt ctgatggagc 360 aacgccgcgt gaacgatgaa ggtcttcgga ttgtaaagtt ctgttgtcag ggacgaacac 420 gtrccgttcg aatagggcgg yaccttgacg gtacctgacg agaaagccac ggctaactac 480 gtgccagcag ccgcggtaat acgtaggtgg caagcgttgt ccggatttat tgggcgtaaa 540 gcgcgcgcag gcggctatgt aagtctggtg ttaaagcccg gagctcaact ccggttcgca 600 tcggaaactg tgtagettga gtgcagaaga ggaaagcggt attceacgtg tagcggtgaa 660 atgcgtagag atgtggagga acaccagtgg cgaaggcggc tttctggtct gtaactgacg 720 ctgaggcgcg aaagcgtggg gagcaaacag gattagatac cctggtagtc cacgccgtaa 780 acgatgagtg ctaggtgttg ggggtttcaa taccctcagt gccgcagcta acgcaataag 840 cactccgcct ggggagtacg ctcgcaagag tgaaactcaa aggaattgac gggggcccgc 900 acaagcggtg gagcatgtgg tttaattcga agcaacgcga agaaccttac caggtcttga 960 catcccgctg accgctctgg agacagagct tcccttcggg gcagcggtga caggtggtgc 1020 atggttgtcg tcagctcgtg tcgtgagatg ttgggttaag tcccgcaacg agcgcaaccc 1080 ttatctttag ttgccagcat tcagttgggc actctagaga gactgccgtc gacaagacgg 1140 aggaaggcgg ggatgacgtc aaatcatcat gccccttatg acctgggcta cacacgtgct 1200 acaatggttg gtacaacggg atgctacctc gcgagrggac gccaatctct gaaaaccaat 1260 ctcagttcgg attgtaggct gcaactcgcc tacatgaagt cggaatcgct agtaatcgcg 1320 gatcagcatg ccgcggtgaa tacgttcccg ggccttgtac acaccgcccg tcacaccacg 1380 ggagtttgca acacccgaag tcggtgaggt aaccgcaagg agccagccgc cgaaggtggg 1440 gtagatgact ggggtgaagt cgtaacaagg tatccgtacc ggaagg 1486 <210>2 <211>20 <212>DNA <213>Artificial Sequence <220> <223>Synthetic <400>2 agrgtttgat cmtggctcag 20 <210> 3 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Synthetic <400> 3 ggytaccttg ttacgactt 19 <210> 4 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Synthetic <400> 4 ctcctacggg aggcagcag 19 <210> 5 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Synthetic <400> 5 gtattaccgc ggctgctg 18 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic <400> 6 aaactyaaag gaattgacgg 20 <210> 7 <211> 1471 <212> DNA <213> Tumebacil lus sp. NITE BP-02779 <400> 7 acgaacgctg gcggcgtgcc taatacatgc aagtcgagcg gagtgtmgag agcttgctct 60 cwraggtcag cggcggacgg gtgagtaaca cgtgggtaac ctgcctggca gactgggata 120 acgcttggaa acgagtgcta ataccggata atctcttgga tcgcatggtc tgagagtaaa 180 aggagctttt gcttcactgc yagatggacc cgcggcgcat tagctagttg gtgaggtaat 240 ggctcaccaa ggcgacgatg cgtagccgac ctgagagggt gaccggccac actgggactg 300 agacacggcc cagactccta cgggaggcag cagtagggaa tcttccgcaa tgggcgcaag 360 cctgacggag caacgccgcg tgagtgatga aggccttcgg gttgtaaaac tctgtcttct 420 gtgaagaacc atcctgtgca gagaaagctc aggacctgac ggtaacagag gaggaagccc 480 cggctaacta cgtgccagca gccgcggtaa tacgtagggg gcaagcgttg tccggaatca 540 ctgggcgtaa agcgcgcgca ggcggtctct cacgtccggg gtgaaagccc agagctcaac 600 tctgggattg ccttggatac ggggagactt gaggatcgga gaggcaaggg gaattccacg 660 tgtagcggtg aaatgcgtag agatgtggag gaacacctgt ggcgaaggcg ccttgctggc 720 cgatttctga cgctgaggcg cgaaagcgtg gggagcaaac aggattagat accctggtag 780 tccacgccgt aaacgatgag tgctaggtgt tagggggccc accccttagt gccgaagcta 840 acgcattaag cactccgcct ggggagtacg gtcgcaagac tgaaactcaa aggaattgac 900 gggggcccgc acaagcagtg gagcatgtgg tttaattcga agcaacgcga agaaccttac 960 caagacttga catcccgctg accrctctag agatagagyt tcccttcggg gcagcggtga 1020 caggtggtgc atggttgtcg tcagctcgtg tcgtgagatg ttgggttaag tcccgcaacg 1080 agcgcaaccc ctatgttgtg ttgctaccat ttagttgagc acttacaaca gactgccggt 1140 gacaaaccgg aggaaggcgg ggatgacgtc aaatcatcat gccccttatg tcttgggcta 1200 cacacgtgct acaatggacg gtacaaaggg ttgcgaagcc gcgaggtgaa gccaatccca 1260 gaaagccgtt cgtagttcgg attgcaggct gcaactcgcc tgcatgaagc cggaattgct 1320 agtaatcgcg gatcagcatg ccgcggtgaa ttcgttcccg ggccttgtac acaccgcccg 1380 tcacaccatg ggagttggta acacccgaag tcggtgaggt aactgcttgc agagccagcc 1440 gcctaaggtg ggatcgataa ctggggtgaa g 1471 <210>8 <211>1538 <212>DNA <213>Bacillus subtilis subsp. inaquosorum DSM022148 <400>8 tttgatcctg gctcaggacg aacgctggcg gcgtgcctaa tacatgcaag tcgagcggac 60 agatgggagc ttgctccctg atgttagcgg cggacgggtg agtaacacgt gggtaacctg 120 cctgtaagac tgggataact ccgggaaacc ggggctaata ccggatgctt gtttgaaccg 180 catggttcaa acataaaagg tggcttcggc taccacttac agatggaccc gcggcgcatt 240 agctagttgg tgaggtaacg gctcaccaag gcaacgatgc gtagccgacc tgagagggtg 300 atcggccaca ctgggactga gacacggccc agactcctac gggaggcagc agtagggaat 360 cttccgcaat ggacgaaagt ctgacggagc aacgccgcgt gagtgatgaa ggttttcgga 420 tcgtaaagct ctgttgttag ggaagaacaa gtaccgttcg aatagggcgg taccttgacg 480 gtacctaacc agaaagccac ggctaactac gtgccagcag ccgcggtaat acgtaggtgg 540 caagcgttgt ccggaattat tgggcgtaaa gggctcgcag gcggtttctt aagtctgatg 600 tgaaagcccc cggctcaacc ggggagggtc attggaaact ggggaacttg agtgcagaag 660 aggagagtgg aattccacgt gtagcggtga aatgcgtaga gatgtggagg aacaccagtg 720 gcgaaggcga ctctctggtc tgtaactgac gctgaggagc gaaagcgtgg ggagcgaaca 780 ggattagata ccctggtagt ccacgccgta aacgatgagt gctaagtgtt agggggtttc 840 cgccccttag tgctgcagct aacgcattaa gcactccgcc tggggagtac ggtcgcaaga 900 ctgaaactca aaggaattga cgggggcccg cacaagcggt ggagcatgtg gtttaattcg 960 aagcaacgcg aagaacctta ccaggtcttg acatcctctg acaatcctag agataggacg 1020 tccccttcgg gggcagagtg acaggtggtg catggttgtc gtcagctcgt gtcgtgagat 1080 gttgggttaa gtcccgcaac gagcgcaacc cttgatctta gttgccagca ttcagttggg 1140 cactctaagg tgactgccgg tgacaaaccg gaggaaggtg gggatgacgt caaatcatca 1200 tgccccttat gacctgggct acacacgtgc tacaatggac agaacaaagg gcagcgaaac 1260 cgcgaggtta agccaatccc acaaatctgt tctcagttcg gatcgcagtc tgcaactcga 1320 ctgcgtgaag ctggaatcgc tagtaatcgc ggatcagcat gccgcggtga atacgttccc 1380 gggccttgta cacaccgccc gtcacaccac gagagtttgt aacacccgaa gtcggtgagg 1440 taacctttta ggagccagcc gccgaaggtg ggacagatga ttggggtgaa gtcgtaacaa 1500 ggtagccgta tcggaaggtg cggctggatc acctcctt 1538 <210> 9 <211> 1470 <212> DNA <213> Tumebacillus algifaecis NBRC108765t <220> <221> misc_feature <222> (45)..(45) <223> n is a, c, g, or t <220> <221> misc_feature <222> (159)..(159) <223> n is a, c, g, or t <220> <221> misc_feature <222> (161)..(161) <223> n is a, c, g, or t <220> <221> misc_feature <222> (172)..(173) <223> n is a, c, g, or t <220> <221> misc_feature <222> (443)..(443) <223>n is a, c, g or t <220> <221>misc_feature <222>(1427)..(1427) <223>n is a, c, g or t <400>9 gacgaacgct ggcggcgtgc ctaatacatg caagtcgagc ggacnggagg gagcttgctc 60 ccaaaggtta gcggcggacg ggtgagtaac acgtgggcaa tctgcccgac agactgggat 120 aacgcttgga aacgagtgct aataccggat aagcgattnc ntcgcatgag gnnatcgaga 180 aagaagcttt cgcttcactg tcggatgagc ccgcggcgca ttagctagtt ggtgaggtaa 240 cggctcacca aggcgacgat gcgtagccga cctgagaggg tgatcggcca cactgggact 300 gagacacggc ccagactcct acgggaggca gcagtaggga atcttccaca atgggcgcaa 360 gcctgatgga gcaacgccgc gtgaatgatg aaggccttcg ggttgtaaaa ttctgtcttc 420 tgtgaagaac aagtgtgaga agngaatgct cacaccctga cggtaacaga ggaggaagcc 480 ccggctaact acgtgccagc agccgcggta atacgtaggg ggcaagcgtt gtccggaatc 540 actgggcgta aagcgcgcgc aggcggccat ctgcgtccgg ggtgaaagcc caaggctcaa 600 ccttgggact gccttggata cgggatggct tgaggatcgg agaggcaagg ggaattccac 660 gtgtagcggt gaaatgcgta gagatgtgga ggaacacctg tggcgaaggc gccttgctgg 720 ccgatttctg acgctgaggc gcgaaagcgt ggggagcaaa caggattaga taccctggta 780 gtccacgccg taaacgatga gtgctaggtg ttggggggta ccaccctcag tgccgaagct 840 aacgcattaa gcactccgcc tggggagtac ggtcgcaaga ctgaaactca aaggaattga 900 cgggggcccg cacaagcagt ggagcatgtg gtttaattcg aagcaacgcg aagaacctta 960 ccaagacttg acatcccgct gaccggttta gagatagacc ttcccttcgg ggcagcggtg 1020 acaggtggtg catggttgtc gtcagctcgt gtcgtgagat gttgggttaa gtcccgcaac 1080 gagcgcaacc cctaaattgt gttgccatca ttcagttggg cactcacaat tgactgccgg 1140 tgacaaaccg gaggaaggcg gggatgacgt caaatcatca tgccccttat gtcttgggct 1200 acacacgtgc tacaatgggc ggtacaaagg gttgcgaggc cgcgaggcgg agccaatccc 1260 aaaaagccgc tcacagttcg gattgcaggc tgcaactcgc ctgcatgaag ctggaattgc 1320 tagtaatcgc ggatcagcat gccgcggtga attcgttccc gggccttgta cacaccgccc 1380 gtcacaccat gggagttggc aacacccgaa gccggtgagg taaccgnaag gagccagccg 1440 tctaaggtgg ggtcgatgac tggggtgaag 1470 <210>10 <211>1479 <212>DNA <213>Paenibacillus polymyxa JCM 2507t <220> <221>misc_feature <222>(1004)..(1004) <223>n is a, c, g or t <220> <221>misc_feature <222>(1248)..(1249) <223>n is a, c, g or t <220> <221>misc_feature <222>(1254)..(1255) <223>n is a, c, g or t <400>10 gacgaacgct ggcggcgtgc ctaatacatg caagtcgagc ggggttattt agaagcttgc 60 ttctaattaa cctagcggcg gacgggtgag taacacgtag gcaacctgcc cacaagacag 120 ggataactac cggaaacggt agctaatacc cgatacatcc ttttcctgca tgggagaagg 180 aggaaaggcg gagcaatctg tcacttgtgg atgggcctgc ggcgcattag ctagttggtg 240 gggtaaaggc ctaccaaggc gacgatgcgt agccgacctg agagggtgat cggccacact 300 gggactgaga cacggcccag actcctacgg gaggcagcag tagggaatct tccgcaatgg 360 gcgaaagcct gacggagcaa cgccgcgtga gtgatgaagg ttttcggatc gtaaagctct 420 gttgccaggg aagaacgtct tgtagagtaa ctgctacaag agtgacggta cctgagaaga 480 aagccccggc taactacgtg ccagcagccg cggtaatacg tagggggcaa gcgttgtccg 540 gaattattgg gcgtaaagcg cgcgcaggcg gctctttaag tctggtgttt aatcccgagg 600 ctcaacttcg ggtcgcactg gaaactgggg agcttgagtg cagaagagga gagtggaatt 660 ccacgtgtag cggtgaaatg cgtagagatg tggaggaaca ccagtggcga aggcgactct 720 ctgggctgta actgacgctg aggcgcgaaa gcgtggggag caaacaggat tagataccct 780 ggtagtccac gccgtaaacg atgaatgcta ggtgttaggg gtttcgatac ccttggtgcc 840 gaagttaaca cattaagcat tccgcctggg gagtacggtc gcaagactga aactcaaagg 900 aattgacggg gacccgcaca agcagtggag tatgtggttt aattcgaagc aacgcgaaga 960 accttaccag gtcttgacat ccctctgacc ggtctagaga tagncctttc cttcgggaca 1020 gaggagacag gtggtgcatg gttgtcgtca gctcgtgtcg tgagatgttg ggttaagtcc 1080 cgcaacgagc gcaaccctta tgcttagttg ccagcaggtc aagctgggca ctctaagcag 1140 actgccggtg acaaaccgga ggaaggtggg gatgacgtca aatcatcatg ccccttatga 1200 cctgggctac acacgtacta caatggccgg tacaacggga agcgaagnng cganntggag 1260 ccaatcctag aaaagccggt ctcagttcgg attgtaggct gcaactcgcc tacatgaagt 1320 cggaattgct agtaatcgcg gatcagcatg ccgcggtgaa tacgttcccg ggtcttgtac 1380 acaccgcccg tcacaccacg agagtttaca acacccgaag tcggtgaggt aaccgcaagg 1440 agccagccgc cgaaggtggg gtagatgatt ggggtgaag 1479 <210>11 <211>1475 <212>DNA <213>Bacillus amyloliquefaciens NBRC3037 <220> <221>misc_feature <222>(152)..(152) <223>n is a, c, g or t <400>11 gacgaacgct ggcggcgtgc ctaatacatg caagtcgagc ggacagatgg gagcttgctc 60 cctgatgtta gcggcggacg ggtgagtaac acgtgggtaa cctgcctgta agactgggat 120 aactccggga aaccggggct aataccggat gnttgtttga accgcatggt tcagacataa 180 aaggtggctt cggctaccac ttacagatgg acccgcggcg cattagctag ttggtgaggt 240 aacggctcac caaggcaacg atgcgtagcc gacctgagag ggtgatcggc cacactggga 300 ctgagacacg gcccagactc ctacgggagg cagcagtagg gaatcttccg caatggacga 360 aagtctgacg gagcaacgcc gcgtgagtga tgaaggtttt cggatcgtaa agctctgttg 420 ttagggaaga acaagtgccg ttcaaatagg gcggcacctt gacggtacct aaccagaaag 480 ccacggctaa ctacgtgcca gcagccgcgg taatacgtag gtggcaagcg ttgtccggaa 540 ttattgggcg taaagggctc gcaggcggtt tcttaagtct gatgtgaaag cccccggctc 600 aaccggggag ggtcattgga aactggggaa cttgagtgca gaagaggaga gtggaattcc 660 acgtgtagcg gtgaaatgcg tagagatgtg gaggaacacc agtggcgaag gcgactctct 720 ggtctgtaac tgacgctgag gagcgaaagc gtggggagcg aacaggatta gataccctgg 780 tagtccacgc cgtaaacgat gagtgctaag tgttaggggg tttccgcccc ttagtgctgc 840 agctaacgca ttaagcactc cgcctgggga gtacggtcgc aagactgaaa ctcaaaggaa 900 ttgacggggg cccgcacaag cggtggagca tgtggtttaa ttcgaagcaa cgcgaagaac 960 cttaccaggt cttgacatcc tctgacaatc ctagagatag gacgtcccct tcgggggcag 1020 agtgacaggt ggtgcatggt tgtcgtcagc tcgtgtcgtg agatgttggg ttaagtcccg 1080 caacgagcgc aacccttgat cttagttgcc agcattcagt tgggcactct aaggtgactg 1140 ccggtgacaa accggaggaa ggtggggatg acgtcaaatc atcatgcccc ttatgacctg 1200 ggctacacac gtgctacaat ggacagaaca aagggcagcg aaaccgcgag gttaagccaa 1260 tcccacaaat ctgttctcag ttcggatcgc agtctgcaac tcgactgcgt gaagctggaa 1320 tcgctagtaa tcgcggatca gcatgccgcg gtgaatacgt tcccgggcct tgtacacacc 1380 gcccgtcaca ccacgagagt ttgtaacacc cgaagtcggt gaggtaacct ttatggagcc 1440 agccgccgaa ggtgggacag atgattgggg tgaag 1475 <210>12 <211>1475 <212>DNA <213>Bacillus subtilis IFO 3134 <400>12 gacgaacgct ggcggcgtgc ctaatacatg caagtcgagc ggacagatgg gagcttgctc 60 cctgatgtta gcggcggacg ggtgagtaac acgtgggtaa cctgcctgta agactgggat 120 aactccggga aaccggggct aataccggat gcttgtttga accgcatggt tcaaacataa 180 aaggtggctt cggctaccac ttacagatgg acccgcggcg cattagctag ttggtgaggt 240 aatggctcac caaggcaacg atgcgtagcc gacctgagag ggtgatcggc cacactggga 300 ctgagacacg gcccagactc ctacgggagg cagcagtagg gaatcttccg caatggacga 360 aagtctgacg gagcaacgcc gcgtgagtga tgaaggtttt cggatcgtaa agctctgttg 420 ttagggaaga acaagtaccg ttcgaatagg gcggtacctt gacggtacct aaccagaaag 480 ccacggctaa ctacgtgcca gcagccgcgg taatacgtag gtggcaagcg ttgtccggaa 540 ttattgggcg taaagggctc gcaggcggtt ccttaagtct gatgtgaaag cccccggctc 600 aaccggggag ggtcattgga aactggggaa cttgagtgca gaagaggaga gtggaattcc 660 acgtgtagcg gtgaaatgcg tagagatgtg gaggaacacc agtggcgaag gcgactctct 720 ggtctgtaac tgacgctgag gagcgaaagc gtggggagcg aacaggatta gataccctgg 780 tagtccacgc cgtaaacgat gagtgctaag tgttaggggg tttccgcccc ttagtgctgc 840 agctaacgca ttaagcactc cgcctgggga gtacggtcgc aagactgaaa ctcaaaggaa 900 ttgacggggg cccgcacaag cggtggagca tgtggtttaa ttcgaagcaa cgcgaagaac 960 cttaccaggt cttgacatcc tctgacaatc ctagagatag gacgtcccct tcgggggcag 1020 agtgacaggt ggtgcatggt tgtcgtcagc tcgtgtcgtg agatgttggg ttaagtcccg 1080 caacgagcgc aacccttgat cttagttgcc agcattcagt tgggcactct aaggtgactg 1140 ccggtgacaa accggaggaa ggtggggatg acgtcaaatc atcatgcccc ttatgacctg 1200 ggctacacac gtgctacaat ggacagaaca aagggcagcg aaaccgcgag gttaagccaa 1260 tcccacaaat ctgttctcag ttcggatcgc agtctgcaac tcgactgcgt gaagctggaa 1320 tcgctagtaa tcgcggatca gcatgccgcg gtgaatacgt tcccgggcct tgtacacacc 1380 tcgctagtaa tcgaggatca gcatgccgcg gtgaatacgt tcccgggcct tgtacacacc 1380 gcccgtcaca ccacgagagt ttgtaacacc cgaagtcggt gaggtaacct tttaggagcc 1440 gcccgtcaca ccacgagagt ttgtaacacc cgaagtcggt gaggtaacct tttaggagcc 1440 agccgccgaa ggtgggacag atgattgggg tgaag 1475 agccgccgaa ggtgggacag atgattgggg tgaag 1475 <210>13<210>13 <211>1475<211>1475 <212>DNA<212>DNA <213>枯草芽孢杆菌(Bacillus subtilis)IFO 3026<213>Bacillus subtilis IFO 3026 <400>13<400>13 gacgaacgct ggcggcgtgc ctaatacatg caagtcgagc ggacagatgg gagcttgctc 60 gacgaacgct ggcggcgtgc ctaatacatg caagtcgagc ggacagatgg gagcttgctc 60 cctgatgtta gcggcggacg ggtgagtaac acgtgggtaa cctgcctgta agactgggat 120 cctgatgtta gcggcggacg ggtgagtaac acgtgggtaa cctgcctgta agactgggat 120 aactccggga aaccggggct aataccggat ggttgtttga accgcatggt tcaaacataa 180 aactccggga aaccggggct aataccggat ggttgtttga accgcatggt tcaaacataa 180 aaggtggctt cggctaccac ttacagatgg acccgcggcg cattagctag ttggtgaggt 240 aaggtggctt cggctaccac ttacagatgg acccgcggcg cattagctag ttggtgaggt 240 aacggctcac caaggcaacg atgcgtagcc gacctgagag ggtgatcggc cacactggga 300 aacggctcac caaggcaacg atgcgtagcc gacctgagag ggtgatcggc cacactggga 300 ctgagacacg gcccagactc ctacgggagg cagcagtagg gaatcttccg caatggacga 360 ctgagacacg gcccagactc ctacgggagg cagcagtagg gaatcttccg caatggacga 360 aagtctgacg gagcaacgcc gcgtgagtga tgaaggtttt cggatcgtaa agctctgttg 420 aagtctgacg gagcaacgcc gcgtgagtga tgaaggtttt cggatcgtaa agctctgttg 420 ttagggaaga acaagtaccg ttcgaatagg gcggtacctt gacggtacct aaccagaaag 480 ttagggaaga acaagtaccg ttcgaatagg gcggtacctt gacggtacct aaccagaaag 480 ccacggctaa ctacgtgcca gcagccgcgg taatacgtag gtggcaagcg ttgtccggaa 540 ttattgggcg taaagggctc gcaggcggtt tcttaagtct gatgtgaaag cccccggctc 600 aaccggggag ggtcattgga aactggggaa cttgagtgca gaagaggaga gtggaattcc 660 acgtgtagcg gtgaaatgcg tagagatgtg gaggaacacc agtggcgaag gcgactctct 720 ggtctgtaac tgacgctgag gagcgaaagc gtggggagcg aacaggatta gataccctgg 780 tagtccacgc cgtaaacgat gagtgctaag tgttaggggg tttccgcccc ttagtgctgc 840 agctaacgca ttaagcactc cgcctgggga gtacggtcgc aagactgaaa ctcaaaggaa 900 ttgacggggg cccgcacaag cggtggagca tgtggtttaa ttcgaagcaa cgcgaagaac 960 cttaccaggt cttgacatcc tctgacaatc ctagagatag gacgtcccct tcgggggcag 1020 agtgacaggt ggtgcatggt tgtcgtcagc tcgtgtcgtg agatgttggg ttaagtcccg 1080 caacgagcgc aacccttgat cttagttgcc agcattcagt tgggcactct aaggtgactg 1140 ccggtgacaa accggaggaa ggtggggatg acgtcaaatc atcatgcccc ttatgacctg 1200 ggctacacac gtgctacaat ggacagaaca aagggcagcg aaaccgcgag gttaagccaa 1260 tcccacaaat ctgttctcag ttcggatcgc agtctgcaac tcgactgcgt gaagctggaa 1320 tcgctagtaa tcgcggatca gcatgccgcg gtgaatacgt tcccgggcct tgtacacacc 1380 gcccgtcaca ccacgagagt ttgtaacacc cgaagtcggt gaggtaacct tttaggagcc 1440 agccgccgaa ggtgggacag atgattgggg tgaag 1475 <210>14 <211>1475 <212>DNA <213>Bacillus subtilis IFO 13169 <220> <221>misc_feature <222>(257)..(257) <223>n is a, c, g or t <400>14 gacgaacgct ggcggcgtgc ctaatacatg caagtcgagc ggacagatgg gagcttgctc 60 cctgatgtta gcggcggacg ggtgagtaac acgtgggtaa cctgcctgta agactgggat 120 aactccggga aaccggggct aataccggat ggttgtttga accgcatggt tcaaacataa 180 aaggtggctt cggctaccac ttacagatgg acccgcggcg cattagctag ttggtgaggt 240 aacggctcac caaggcnacg atgcgtagcc gacctgagag ggtgatcggc cacactggga 300 ctgagacacg gcccagactc ctacgggagg cagcagtagg gaatcttccg caatggacga 360 aagtctgacg gagcaacgcc gcgtgagtga tgaaggtttt cggatcgtaa agctctgttg 420 ttagggaaga acaagtaccg ttcgaatagg gcggtacctt gacggtacct aaccagaaag 480 ccacggctaa ctacgtgcca gcagccgcgg taatacgtag gtggcaagcg ttgtccggaa 540 ttattgggcg taaagggctc gcaggcggtt tcttaagtct gatgtgaaag cccccggctc 600 aaccggggag ggtcattgga aactggggaa cttgagtgca gaagaggaga gtggaattcc 660 acgtgtagcg gtgaaatgcg tagagatgtg gaggaacacc agtggcgaag gcgactctct 720 ggtctgtaac tgacgctgag gagcgaaagc gtggggagcg aacaggatta gataccctgg 780 tagtccacgc cgtaaacgat gagtgctaag tgttaggggg tttccgcccc ttagtgctgc 840 agctaacgca ttaagcactc cgcctgggga gtacggtcgc aagactgaaa ctcaaaggaa 900 ttgacggggg cccgcacaag cggtggagca tgtggtttaa ttcgaagcaa cgcgaagaac 960 cttaccaggt cttgacatcc tctgacaatc ctagagatag gacgtcccct tcgggggcag 1020 agtgacaggt ggtgcatggt tgtcgtcagc tcgtgtcgtg agatgttggg ttaagtcccg 1080 caacgagcgc aacccttgat cttagttgcc agcattcagt tgggcactct aaggtgactg 1140 ccggtgacaa accggaggaa ggtggggatg acgtcaaatc atcatgcccc ttatgacctg 1200 ggctacacac gtgctacaat ggacagaaca aagggcagcg aaaccgcgag gttaagccaa 1260 tcccacaaat ctgttctcag ttcggatcgc agtctgcaac tcgactgcgt gaagctggaa 1320 tcgctagtaa tcgcggatca gcatgccgcg gtgaatacgt tcccgggcct tgtacacacc 1380 gcccgtcaca ccacgagagt ttgtaacacc cgaagtcggt gaggtaacct tttaggagcc 1440 agccgccgaa ggtgggacag atgattgggg tgaag 1475 <210>15 <211>1478 <212>DNA <213>Bacillus sphaericus IFO 3341 <400>15 gacgaacgct ggcggcgtgc ctaatacatg caagtcgagc gaacagaaaa ggagcttgct 60 cctttgacgt tagcggcgga cgggtgagta acacgtgggc aacctaccct atagtttggg 120 ataactccgg gaaaccgggg ctaataccga ataatctctt ttacttcatg gtgaaagact 180 gaaagacggc atctcgctgt cgctatagga tgggcccgcg gcgcattagc tagttggtga 240 ggtaacggct caccaaggcg acgatgcgta gccgacctga gagggtgatc ggccacactg 300 ggactgagac acggcccaga ctcctacggg aggcagcagt agggaatctt ccacaatggg 360 cgaaagcctg atggagcaac gccgcgtgag tgaagaaggt tttcggatcg taaaactctg 420 ttgtaaggga agaacaagta cagtagtaac tggctgtacc ttgacggtac cttattagaa 480 agccacggct aactacgtgc cagcagccgc ggtaatacgt aggtggcaag cgttgtccgg 540 aattattggg cgtaaagcgc gcgcaggcgg tcctttaagt ctgatgtgaa agcccacggc 600 tcaaccgtgg agggtcattg gaaactgggg gacttgagtg cagaagagga aagtggaatt 660 ccaagtgtag cggtgaaatg cgtagagatt tggaggaaca ccagtggcga aggcgacttt 720 ctggtctgta actgacgctg aggcgcgaaa gcgtggggag caaacaggat tagataccct 780 ggtagtccac gccgtaaacg atgagtgcta agtgttaggg ggtttccgcc ccttagtgct 840 gcagctaacg cattaagcac tccgcctggg gagtacggtc gcaagactga aactcaaagg 900 aattgacggg ggcccgcaca agcggtggag catgtggttt aattcgaagc aacgcgaaga 960 accttaccag gtcttgacat cccgttgacc actgtagaga tatagtttcc ccttcggggg 1020 caacggtgac aggtggtgca tggttgtcgt cagctcgtgt cgtgagatgt tgggttaagt 1080 cccgcaacga gcgcaaccct tgatcttagt tgccatcatt tagttgggca ctctaaggtg 1140 actgccggtg acaaaccgga ggaaggtggg gatgacgtca aatcatcatg ccccttatga 1200 cctgggctac acacgtgcta caatggacga tacaaacggt tgccaactcg cgagagggag 1260 ctaatccgat aaagtcgttc tcagttcgga ttgtaggctg caactcgcct acatgaagcc 1320 ggaatcgcta gtaatcgcgg atcagcatgc cgcggtgaat acgttcccgg gccttgtaca 1380 caccgcccgt cacaccacga gagtttgtaa cacccgaagt cggtgaggta accttttgga 1440 gccagccgcc gaaggtggga tagatgattg gggtgaag 1478 <210>16 <211>1477 <212>DNA <213>Bacillus sphaericus IFO 3528 <400>16 gacgaacgct ggcggcgtgc ctaatacatg caagtcgagc gaacagagaa ggagcttgct 60 ccttcgacgt tagcggcgga cgggtgagta acacgtgggc aacctacctt atagtttggg 120 ataactccgg gaaaccgggg ctaataccga ataatctgtt tcacctcatg gtgaaacact 180 gaaagacggt ttcggctgtc gctataggat gggcccgcgg cgcattagct agttggtgag 240 gtaacggctc accaaggcga cgatgcgtag ccgacctgag agggtgatcg gccacactgg 300 gactgagaca cggcccagac tcctacggga ggcagcagta gggaatcttc cacaatgggc 360 gaaagcctga tggagcaacg ccgcgtgagt gaagaaggat ttcggttcgt aaaactctgt 420 tgtaagggaa gaacaagtac agtagtaact ggctgtacct tgacggtacc ttattagaaa 480 gccacggcta actacgtgcc agcagccgcg gtaatacgta ggtggcaagc gttgtccgga 540 attattgggc gtaaagcgcg cgcaggtggt ttcttaagtc tgatgtgaaa gcccacggct 600 caaccgtgga gggtcattgg aaactgggag acttgagtgc agaagaggat agtggaattc 660 caagtgtagc ggtgaaatgc gtagagattt ggaggaacac cagtggcgaa ggcgactatc 720 tggtctgtaa ctgacactga ggcgcgaaag cgtggggagc aaacaggatt agataccctg 780 gtagtccacg ccgtaaacga tgagtgctaa gtgttagggg gtttccgccc cttagtgctg 840 cagctaacgc attaagcact ccgcctgggg agtacggtcg caagactgaa actcaaagga 900 attgacgggg gcccgcacaa gcggtggagc atgtggttta attcgaagca acgcgaagaa 960 ccttaccagg tcttgacatc ccgttgacca ctgtagagat atagtttccc cttcgggggc 1020 aacggtgaca ggtggtgcat ggttgtcgtc agctcgtgtc gtgagatgtt gggttaagtc 1080 ccgcaacgag cgcaaccctt gatcttagtt gccatcattt agttgggcac tctaaggtga 1140 ctgccggtga caaaccggag gaaggtgggg atgacgtcaa atcatcatgc cccttatgac 1200 ctgggctaca cacgtgctac aatggacgat acaaacggtt gccaactcgc gagagggagc 1260 taatccgata aagtcgttct cagttcggat tgtaggctgc aactcgccta catgaagccg 1320 gaatcgctag taatcgcgga tcagcatgcc gcggtgaata cgttcccggg ccttgtacac 1380 accgcccgtc acaccacgag agtttgtaac acccgaagtc ggtgaggtaa ccttttggag 1440 ccagccgccg aaggtgggat agatgattgg ggtgaag 1477 <210>17 <211>1475 <212>DNA <213>Bacillus licheniformis IFO 12197 <400>17 gacgaacgct ggcggcgtgc ctaatacatg caagtcgagc ggacagatgg gagcttgctc 60 cctgatgtca gcggcggacg ggtgagtaac acgtgggtaa cctgcctgta agactgggat 120 aactccggga aaccggggct aataccggat gcttgattga accgcatggt tcaattataa 180 aaggtggctt ttagctacca cttacagatg gacccgcggc gcattagcta gttggtgagg 240 taacggctca ccaaggcaac gatgcgtagc cgacctgaga gggtgatcgg ccacactggg 300 actgagacac ggcccagact cctacgggag gcagcagtag ggaatcttcc gcaatggacg 360 aaagtctgac ggagcaacgc cgcgtgagtg atgaaggttt tcggatcgta aaactctgtt 420 gttagggaag aacaagtacc gttcgaatag ggcggtacct tgacggtacc taaccagaaa 480 gccacggcta actacgtgcc agcagccgcg gtaatacgta ggtggcaagc gttgtccgga 540 attattgggc gtaaagcgcg cgcaggcggt ttcttaagtc tgatgtgaaa gccccggct 600 caaccgggga gggtcattgg aaactgggga acttgagtgc agaagaggag agtggaattc 660 cacgtgtagc ggtgaaatgc gtagagatgt ggaggaacac cagtggcgaa ggcgactctc 720 tggtctgtaa ctgacgctga ggcgcgaaag cgtggggagc gaacaggatt agataccctg 780 gtagtccacg ccgtaaacga tgagtgctaa gtgttagagg gtttccgccc tttagtgctg 840 cagcaaacgc attaagcact ccgcctgggg agtacggtcg caagactgaa actcaaagga 900 attgacgggg gcccgcacaa gcggtggagc atgtggttta attcgaagca acgcgaagaa 960 ccttaccagg tcttgacatc ctctgacaac cttagagata gggcttcccc ttcgggggca 1020 gagtgacagg tggtgcatgg ttgtcgtcag ctcgtgtcgt gagatgttgg gttaagtccc 1080 gcaacgagcg caacccttga tcttagttgc cagcattcag ttgggcactc taaggtgact 1140 gccggtgaca aaccggagga aggtggggat gacgtcaaat catcatgccc cttatgacct 1200 gggctacaca cgtgctacaa tgggcagaac aaagggcagc gaagccgcga ggctaagcca 1260 atcccacaaa tctgttctca gttcggatcg cagtctgcaa ctcgactgcg tgaagctgga 1320 atcgctagta atcgcggatc agcatgccgc ggtgaatacg ttcccgggcc ttgtacacac 1380 cgcccgtcac accacgagag tttgtaacac ccgaagtcgg tgaggtaacc ttttggagcc 1440 agccgccgaa ggtgggacag atgattgggg tgaag 1475 <210>18 <211>1475 <212>DNA <213>Bacillus subtilis 168 NBRC11470 <220> <221>misc_feature <222>(437)..(437) <223>n is a, c, g or t <400>18 gacgaacgct ggcggcgtgc ctaatacatg caagtcgagc ggacagatgg gagcttgctc 60 cctgatgtta gcggcggacg ggtgagtaac acgtgggtaa cctgcctgta agactgggat 120 aactccggga aaccggggct aataccggat ggttgtttga accgcatggt tcaaacataa 180 aaggtggctt cggctaccac ttacagatgg acccgcggcg cattagctag ttggtgaggt 240 aacggctcac caaggcaacg atgcgtagcc gacctgagag ggtgatcggc cacactggga 300 ctgagacacg gcccagactc ctacgggagg cagcagtagg gaatcttccg caatggacga 360 aagtctgacg gagcaacgcc gcgtgagtga tgaaggtttt cggatcgtaa agctctgttg 420 ttagggaaga acaagtnccg ttcgaatagg gcggtacctt gacggtacct aaccagaaag 480 ccacggctaa ctacgtgcca gcagccgcgg taatacgtag gtggcaagcg ttgtccggaa 540 ttattgggcg taaagggctc gcaggcggtt tcttaagtct gatgtgaaag cccccggctc 600 aaccggggag ggtcattgga aactggggaa cttgagtgca gaagaggaga gtggaattcc 660 acgtgtagcg gtgaaatgcg tagagatgtg gaggaacacc agtggcgaag gcgactctct 720 ggtctgtaac tgacgctgag gagcgaaagc gtggggagcg aacaggatta gataccctgg 780 tagtccacgc cgtaaacgat gagtgctaag tgttaggggg tttccgcccc ttagtgctgc 840 agctaacgca ttaagcactc cgcctgggga gtacggtcgc aagactgaaa ctcaaaggaa 900 ttgacggggg cccgcacaag cggtggagca tgtggtttaa ttcgaagcaa cgcgaagaac 960 cttaccaggt cttgacatcc tctgacaatc ctagagatag gacgtcccct tcgggggcag 1020 agtgacaggt ggtgcatggt tgtcgtcagc tcgtgtcgtg agatgttggg ttaagtcccg 1080 caacgagcgc aacccttgat cttagttgcc agcattcagt tgggcactct aaggtgactg 1140 ccggtgacaa accggaggaa ggtggggatg acgtcaaatc atcatgcccc ttatgacctg 1200 ggctacacac gtgctacaat ggacagaaca aagggcagcg aaaccgcgag gttaagccaa 1260 tcccacaaat ctgttctcag ttcggatcgc agtctgcaac tcgactgcgt gaagctggaa 1320 tcgctagtaa tcgcggatca gcatgccgcg gtgaatacgt tcccgggcct tgtacacacc 1380 gcccgtcaca ccacgagagt ttgtaacacc cgaagtcggt gaggtaacct tttaggagcc 1440 agccgccgaa ggtgggacag atgattgggg tgaag 1475

Claims

1. Bacteria, which is Bacillus parabrevis deposited under the accession number NITE BP-03020 ( Brevibacillus parabrevis ).

2. A microbial preparation for decomposing excess sludge, comprising bacteria (a1), wherein the bacteria (a1) is Bacillus parabrevis deposited under the accession number NITE BP-03020.

3. A composite microbial preparation comprising Bacillus parabrevis NITE BP-03020, Bacillus subtilis deserticola ( Bacillus subtilis subsp.inaquosorum )DSM 22148, Bacillus expansus species ( Tumebacillus sp. ) Each culture supernatant of NITE BP-02779.

4. A microbial preparation for decomposing excess sludge, comprising a culture of bacteria (a1), wherein the bacteria (a1) is Bacillus parabrevis deposited under the accession number NITE BP-03020.

5. A method for decomposing excess sludge, comprising the step of allowing the bacteria according to claim 1 or the composite microbial preparation according to claim 3 or the microbial preparation for decomposing excess sludge according to claim 2 or 4 to act on excess sludge.

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