JP7710664B2 - Novel Kinneretia genus microorganism, composition containing said microorganism, and device using said microorganism - Google Patents

Description

NITE NITE P-03538

The present invention relates to a novel microorganism of the genus Kinneretia (accession number NITE P-03538) that has high organic matter reduction activity and high microbial coagulation activity, a composition containing the microorganism, and a device that utilizes the microorganism.

Currently, most organic wastewater discharged from food factories and sewage treatment facilities is treated with microorganisms. In particular, many food factories are increasing production efficiency by concentrating cooking in one location, and there is a strong trend toward larger manufacturing facilities and 24-hour operation. This has dramatically increased the burden on wastewater treatment facilities, and there is also a trend toward revising effluent control values to stricter values by laws and ordinances, making it necessary to make wastewater treatment facilities larger and more functional. However, expanding and building additional wastewater treatment facilities is very costly, so an alternative method was desired. One alternative is to use bacteria with highly functional wastewater treatment capabilities.

Attempts have been made to find useful microorganisms for efficient microbial treatment of organic matter. For example, Patent Document 1 discloses a microorganism belonging to the genus Candida for decomposing oil-containing substances, such as wastewater containing oils such as fats and oils, mineral oils, etc. However, in many cases, it is necessary to flocculate suspended solids contained in the wastewater with a flocculant and treat them separately as sludge before decomposition by the microorganisms, which leaves an issue in terms of cost. To date, practical microorganisms capable of treating wastewater containing organic matter and treatment methods using them have yet to reach a satisfactory level.

JP 2020-191802 A

In view of the above, the object of the present invention is to develop a microbial wastewater treatment technology that seeks out microorganisms with highly functional treatment capabilities and realizes high functionality of treatment facilities by utilizing these microorganisms without changing the existing facilities.

In order to solve the above problems, the inventors of the present invention have conducted extensive research, isolating microorganisms from Japanese rivers and succeeding in isolating a new microorganism that has the same high treatment capacity for meat processing wastewater as existing wastewater treatment materials. Furthermore, the microorganism in question has a coagulation property that is not found in the microorganisms contained in existing wastewater treatment materials. Furthermore, analysis of the base sequence of the 16S rDNA of the microorganism revealed that the microorganism in question is a new microorganism of the genus Kinneretia. Furthermore, when a wastewater treatment test was conducted using the microorganism in an existing facility using meat processing wastewater, it was possible to treat more wastewater than before while meeting the discharge standard value. As a result, the amazing high efficiency of the microorganism made it possible to reduce the running costs of the wastewater treatment facility to about one-quarter of the conventional cost.

Based on the above findings, the present invention has been completed.
That is, the present invention is as follows:
[1] An isolated microorganism having the following characteristics (1) or (2):
(1) The nucleotide sequence of 16S rDNA contains the nucleotide sequence shown in SEQ ID NO: 1.
(2) The base sequence of 16S rDNA has a homology of 90% or more with the base sequence shown in SEQ ID NO:1, and the microorganism belongs to the genus Kinneretia and has organic matter reduction activity and microbial coagulation activity.
[2] The microorganism described in [1], which exhibits the following bacteriological properties:
(1) Colony morphology Diameter: less than 1 mm Color: cream Shape: circular Protuberance: central depression Edge: entire edge Surface shape: smooth Transparency: opaque Viscosity: butter-like (2) Growth temperature: 20-45°C
(3) Cell morphology: Bacillus (4) Gram staining: -
(5) Presence or absence of spore formation: -
(6) Motility: +
(7) Catalase reaction: +
(8) Oxidase reaction: +
(9) Acid/gas production from glucose: -/-
(10) Glucose oxidation/fermentation test: -/-
(In the above, "+" indicates positive and "-" indicates negative.)
[3] An isolated Kinneretia genus microorganism having accession number NITE P-03538.
[4] An organic matter reducing agent comprising the microorganism according to any one of [1] to [3].
[5] A microbial flocculant comprising the microorganism according to any one of [1] to [3].
[6] A method for reducing organic matter, comprising contacting the microorganism according to any one of [1] to [3] with a sample containing organic matter.
[7] A method for agglutinating microorganisms, comprising contacting the microorganism according to any one of [1] to [3] with a sample containing the microorganisms.
[8] A wastewater purification apparatus, comprising:
(1) A microbial treatment tank for treating wastewater with a microorganism according to any one of (1) to (3) above, and (2) a microorganism according to (1) above, thereby reducing organic matter in the wastewater and/or flocculating microorganisms.
[9] The apparatus according to [8], further comprising at least one selected from the following:
(A) A pump tank for storing wastewater and pumping it to other tanks;
(B) a culture tank in which the microorganism according to any one of [1] to [3] is pre-grown;
(C) a solid-liquid separation tank for separating the flocculated microorganisms from the biologically treated wastewater;
(D) a discharge tank for storing and discharging the biologically treated wastewater from which the flocculated microorganisms have been separated;
(E) a sludge storage tank for storing the separated flocculated microorganisms;
(F) a reaction tank for flocculating suspended solids and n-Hex in the wastewater by treating the wastewater with a flocculant, and (G) a pressurized flotation device for separating the flocculated suspended solids and flocculated n-Hex from the flocculant-treated wastewater.
[10] The apparatus of [8], further comprising:
(A) A pump tank for storing wastewater and pumping it to other tanks;
(B) a culture tank in which the microorganism according to any one of [1] to [3] is pre-grown;
(C) a solid-liquid separation tank for separating the flocculated microorganisms from the biologically treated wastewater;
(D) a discharge tank for storing and discharging the biologically treated wastewater from which the flocculated microorganisms have been separated; and (E) a sludge storage tank for storing the separated flocculated microorganisms.

The isolated Kinneretia microorganism, with accession number NITE P-03538, has the same wastewater treatment capacity as existing wastewater treatment materials. Moreover, surprisingly, it can aggregate other types of microorganisms and solid components, so there is no need to treat wastewater with a flocculant before microbial treatment. As a result, by using this microorganism, it is possible to omit pretreatment and significantly reduce the cost of wastewater treatment without modifying existing drainage facilities. This microorganism contributes to the Sustainable Development Goals (SDGs), and in particular, it can contribute to the sixth SDG global indicator, "Ensure availability and sustainable management of water and sanitation for all," and the twelfth SDG indicator, "Ensure sustainable consumption and production patterns."

FIG. 1 shows an example of an embodiment of a wastewater purification apparatus according to the present invention. FIG. 2 shows an example of an embodiment of the wastewater purification apparatus of the present invention. FIG. 3 shows an example of an embodiment of the wastewater purification apparatus of the present invention. Figure 4 shows the relative TOC values of meat processing wastewater treated with each commercially available wastewater treatment material. Control: Untreated group. QqBiO, Ehime AI, Oil B: Commercially available wastewater treatment materials. 5 shows the relative TOC values of meat processing wastewater treated with the wastewater treatment material QqBiO or the microorganism E2 strain. Control: untreated group. QqBiO: commercial wastewater treatment material. E2: microorganism of the present invention. FIG. 6 shows the microbial agglutination properties of E2. FIG. 7 shows an electron microscope image of aggregated E2. FIG. 8 shows a molecular phylogenetic tree based on the 16S rDNA base sequence of E2. FIG. 9 shows a structural diagram of the meat processing wastewater treatment facility that was tested on-site. Figure 10 shows the change in biochemical oxygen demand (BOD) due to E2. The change in BOD is defined as outflow BOD/inflow BOD. In the "use of pressure flotation" case, no microorganisms were added, and the flocs generated by the coagulant were removed using a pressure flotation device. Figure 11 shows the change in n-hexane (n-Hex) due to E2. The change in n-Hex is defined as outflow n-Hex/inflow n-Hex. In the "pressure flotation use" case, no microorganisms were added, and the flocs generated by the coagulant were removed using a pressure flotation device. FIG. 12 shows the microbial aggregation properties of E2 for meat processing wastewater. FIG. 13 shows the cell count of E2 in a carrier fluidized bed in a meat processing wastewater treatment test.

The present invention provides an isolated microorganism (the microorganism of the present invention) having the following characteristics (1) or (2):
(1) The base sequence of 16S rDNA contains the base sequence shown in SEQ ID NO:1.
(2) The base sequence of 16S rDNA has a homology of 90% or more with the base sequence shown in SEQ ID NO:1, and the microorganism belongs to the genus Kinneretia and has organic matter reduction activity and microbial coagulation activity.

The microorganism of the present invention is a microorganism (microorganism I of the present invention) whose 16S rDNA base sequence includes the base sequence shown in SEQ ID NO:1.

The 16S rDNA fraction can be prepared using known techniques such as ethanol precipitation, but highly pure total DNA can also be prepared quickly and easily from a small amount of sample using a commercially available DNA extraction kit (e.g., achromopeptidase; manufactured by FUJIFILM Wako Pure Chemical, etc.). The base sequence of the obtained DNA can be determined by known means.

As described above, a BLAST homology search was performed using the nucleotide sequence shown in SEQ ID NO: 1 obtained from the 16S rDNA of the microorganism I of the present invention as a query sequence and the international nucleotide sequence database (DDBJ/ENA(EMBL)/GenBank). As a result, it was found that the nucleotide sequence of the 16S rDNA of the microorganism I of the present invention shows a homology rate of 99.6% to the nucleotide sequence of the 16S rDNA of the type strain KIN192 ^T (accession number AY136099) of Kinneretia asaccharophila. Furthermore, in a molecular phylogenetic tree analyzed based on the nucleotide sequence obtained by a homology search against DB-BA 15.0 (TechnoSuruga Laboratory) using a microorganism identification system (analysis software: ENKI), it was found that the microorganism I of the present invention is included in a cluster consisting of the family Comamonadaceae. Examples of microorganisms included in the family Comamonadaceae include microorganisms belonging to the genera Kinneretia (one genus, one species), Mitsuaria, Pelomonas, and Roseteles. From the above results, the microorganism I of the present invention is a novel microorganism belonging to the genus Kinneretia closely related to K. asaccharophila.

The present inventors deposited Microorganism I of the present invention on September 24, 2021 at the Patent Microorganism Depositary of the National Institute of Technology and Evaluation (2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture) under the accession number NITE P-03538.

The microorganism of the present invention may also be a microorganism (microorganism II of the present invention) whose 16S rDNA base sequence has 90% or more homology with the base sequence shown in SEQ ID NO:1, which belongs to the genus Kinneretia, and which has organic matter reduction activity and microbial coagulation activity.

The microorganism II of the present invention is a microorganism belonging to the genus Kinneretia, whose 16S rDNA base sequence has a homology of generally about 90% or more, preferably about 95% or more, and more preferably about 98% or more with the base sequence shown in SEQ ID NO:1.

The homology of the base sequences in this specification can be calculated using the homology calculation algorithm NCBI BLAST (National Center for Biotechnology Information Basic Local Alignment Search Tool) under the following conditions (expectation value = 10; gaps allowed; filtering = ON; match score = 1; mismatch score = -3).

Other algorithms for determining the homology of nucleotide sequences include, for example, the algorithm described in Karlin et al., Proc. Natl. Acad. Sci. USA, 90: 5873-5877 (1993) [this algorithm is incorporated into the NBLAST and XBLAST programs (version 2.0) (Altschul et al., Nucleic Acids Res., 25: 3389-3402 (1997))], the algorithm described in Needleman et al., J. Mol. Biol., 48: 444-453 (1970) [this algorithm is incorporated into the GAP program in the GCG software package], the algorithm described in Myers and Miller, CABIOS, 4: 11-17 (1988) [this algorithm is incorporated into the ALIGN program (version 2.0), which is part of the CGC sequence alignment software package], and the algorithm described in Pearson et al., Proc. Natl. Acad. Sci. USA, 85: 2444-2448 (1988) [this algorithm is incorporated into the FASTA program in the GCG software package], but is not limited thereto.

The microorganism of the present invention has organic matter reduction activity. Examples of organic matter that can be reduced by the microorganism of the present invention include lipids, proteins, organic acids, carbohydrates, and sugar alcohols. Examples of carbohydrates include D-glucose. Examples of organic acids include potassium gluconate.

The microorganism of the present invention also has microbial flocculation activity. The microorganism of the present invention secretes a biopolymer outside the microorganism in the growth environment, and the microorganisms adsorb each other via the biopolymer to form aggregates, which can adsorb other surrounding microorganisms and solid matter and precipitate. The other microorganisms are not particularly limited, but include microorganisms that are desirable to remove for sanitary reasons. Examples of such microorganisms include
Bacillus genus (e.g., Bacillus cereus, etc.), Candida genus (e.g., Candida, etc.), Campylobacter genus (e.g., Campylobacter jejuni, etc.), Clostridioides microorganisms (e.g., Clostridium difficile, etc.), Clostridium microorganisms (e.g., Clostridium perfringens, etc.), Cutibacterium microorganisms (e.g., Propionibacterium acnes, etc.), Enterococcus microorganisms (e.g., Enterococcus, etc.), Escherichia microorganisms (e.g., Escherichia coli, etc.), Helicobacter microorganisms (e.g., Helicobacter pylori, etc.), Pseudomonas microorganisms (e.g., Pseudomonas aeruginosa, etc.), Salmonella microorganisms (e.g., Salmonella enteritidis, etc.), Staphylococcus microorganisms (e.g., Staphylococcus, etc.), Streptococcus microorganisms (e.g., Streptococcus, etc.), Trichophyton microorganisms (e.g., Trichophyton, etc.), Vibrio microorganisms (e.g., Vibrio parahaemolyticus, etc.), etc., but are not limited thereto.

The microorganism of the present invention also exhibits the following mycological properties.
(1) Colony morphology When the microorganism of the present invention is cultured on a nutrient agar medium at 30° C. for 48 hours, the colonies formed exhibit the following characteristics when observed with a stereomicroscope:
Diameter: less than 1 mm Color: cream Shape: circular Protuberance: central depression Edge: entire edge Surface shape: smooth Transparency: opaque Viscosity: butter-like (2) Growth temperature The microorganism of the present invention can grow at 15 to 50°C.
(3) Cell Morphology The microorganism of the present invention has a cell morphology of a bacillus under morphological observation with an optical microscope.
(4) Gram Stainability The microorganism of the present invention is a gram-negative microorganism.
(5) Sporulation The microorganism of the present invention does not form spores.
(6) Motility
The microorganism of the present invention is motile.
(7) Catalase Reaction The microorganism of the present invention exhibits a catalase reaction.
(8) Oxidase Reaction The microorganism of the present invention exhibits an oxidase reaction.
(9) Acid/Gas Production from Glucose The microorganism of the present invention does not produce acid or gas from glucose.
(10) Glucose oxidation/fermentation test The microorganism of the present invention does not oxidize or ferment glucose.

The microorganism of the present invention further exhibits the following biochemical properties:
(1) Nitrate Reduction Reaction The microorganism of the present invention exhibits nitrate reduction reaction.
(2) Indole Production Reaction The microorganism of the present invention does not exhibit indole production reaction.
(3) D-glucose acidification reaction The microorganism of the present invention does not exhibit D-glucose acidification reaction.
(4) Arginine Dihydrolase The microorganism of the present invention does not exhibit arginine dihydrolase activity.
(5) Urease The microorganism of the present invention does not exhibit urease activity.
(6) Esculin Hydrolysis Reaction The microorganism of the present invention does not exhibit esculin hydrolysis reaction.
(7) Gelatin Hydrolysis Reaction The microorganism of the present invention does not exhibit gelatin hydrolysis reaction.
(8) Cytochrome oxidase The microorganism of the present invention exhibits cytochrome oxidase activity.
(9) Alkaline phosphatase The microorganism of the present invention exhibits alkaline phosphatase activity.
(10) Esterase (C4)
The microorganism of the present invention exhibits esterase (C4) activity.
(11) Esterase (C8)
The microorganism of the present invention does not exhibit esterase (C8) activity.
(12) Lipase (C14)
The microorganism of the present invention does not exhibit lipase (C14) activity.
(13) Leucine arylamidase The microorganism of the present invention exhibits leucine arylamidase activity.
(14) Valine aryl amidase The microorganism of the present invention exhibits valine aryl amidase activity.
(15) Cystine arylamidase The microorganism of the present invention does not exhibit cystine arylamidase activity.
(16) Trypsin The microorganism of the present invention exhibits trypsin activity.
(17) α-Chymotrypsin The microorganism of the present invention does not exhibit α-chymotrypsin activity.
(18) Acid Phosphatase The microorganism of the present invention exhibits acid phosphatase activity.
(19) Naphthol-AS-BI-phosphohydrolase The microorganism of the present invention exhibits naphthol-AS-BI-phosphohydrolase activity.
(20) α-galactosidase The microorganism of the present invention does not exhibit α-galactosidase activity.
(21) β-galactosidase The microorganism of the present invention does not exhibit β-galactosidase activity.
(22) β-glucuronidase The microorganism of the present invention does not exhibit β-glucuronidase activity.
(23) α-Glucosidase The microorganism of the present invention does not exhibit α-glucosidase activity.
(24) β-glucosidase The microorganism of the present invention does not exhibit β-glucosidase activity.
(25) N-acetyl-β-glucosaminidase The microorganism of the present invention does not exhibit N-acetyl-β-glucosaminidase activity.
(26) α-Mannosidase The microorganism of the present invention does not exhibit α-mannosidase activity.
(27) α-Fucosidase The microorganism of the present invention does not exhibit α-fucosidase activity.

The microorganism of the present invention further exhibits the following assimilation capabilities.
(1) D-Glucose The microorganism of the present invention exhibits D-glucose assimilation.
(2) L-Arabinose The microorganism of the present invention does not exhibit L-arabinose assimilation.
(3) D-Mannose The microorganism of the present invention does not exhibit D-mannose assimilation.
(4) D-Mannitol The microorganism of the present invention does not exhibit D-mannitol assimilation.
(5) N-acetyl-D-glucosamine The microorganism of the present invention does not exhibit N-acetyl-D-glucosamine assimilation.
(6) Maltose The microorganism of the present invention does not exhibit maltose assimilation.
(7) Potassium Gluconate The microorganism of the present invention exhibits potassium gluconate assimilation.
(8) n-Capric Acid The microorganism of the present invention does not exhibit the ability to assimilate n-capric acid.
(9) Adipic Acid The microorganism of the present invention does not exhibit adipic acid assimilation.
(10) dl-Malic Acid The microorganism of the present invention does not exhibit dl-malic acid assimilation.
(11) Sodium Citrate The microorganism of the present invention does not exhibit sodium citrate assimilation.
(12) Phenyl acetate The microorganism of the present invention does not exhibit phenyl acetate assimilation.

The microorganism of the present invention may be a mutant of the microorganism of the present invention (mutant of the present invention). The mutant is not particularly limited as long as it is a strain derived from the microorganism of the present invention, but may be a strain that exhibits one or more properties similar to those of the microorganism of the present invention in terms of, for example, morphological characteristics, staining, growth, nutritional requirements, genetic characteristics, physiological characteristics, and biochemical characteristics, and that retains the desired properties of the microorganism of the present invention. The desired properties include organic matter reduction activity, microbial flocculation activity, catalase reactivity, oxidase reactivity, nitrate reduction reactivity, cytochrome oxidase activity, alkaline phosphatase activity, esterase (C4) activity, leucine aryl amidase activity, valine aryl amidase activity, proteolytic activity, acid phosphatase activity, naphthol-AS-BI-phosphohydrolase activity, carbohydrate assimilation, organic acid assimilation, lipid assimilation, etc.

The mutant of the present invention can be obtained by subjecting the microorganism of the present invention to a mutagen treatment known per se, and selecting a strain having the desired effect equal to or greater than that of the parent strain. The organic matter reduction activity can be tested, for example, using total organic carbon, biochemical oxygen demand, or chemical oxygen demand as an indicator, but is not limited thereto, and any method known per se may be used.

The mutant of the present invention can be produced, for example, by modifying the microorganism of the present invention. Examples of such modification include culturing in the presence of a mutagenic substance, disrupting a gene possessed by the microorganism of the present invention, and a combination of these manipulations.

Examples of mutagens include, but are not limited to, alkylating agents (N-methyl-N'-nitro-N-nitrosoguanidine (NTG), ethyl methanesulfonate (EMS), etc.), nucleotide base analogs (bromouracil, etc.), nitroso compounds, DNA intercalators, DNA cross-linking agents, radiation, ultraviolet light, etc. Preferably, the mutant is a strain that has an increased desired effect compared to the parent strain, or has the same effect, but has other advantageous effects, such as the ability to grow over a wide pH range, a fast growth rate, or the ability to grow at low temperatures.

The microorganism or variant of the present invention can be cultured by a method known per se for culturing microorganisms belonging to the genus Kinneretia. Those skilled in the art can appropriately select a medium to be used for the culture, and examples of the medium include M9 medium (minimal medium), KL medium, BHI medium, 2YT medium, YPD medium, AM3 medium, LB medium, etc. The culture time and culture scale can be appropriately set depending on the use of the culture. The culture temperature is usually about 15 to about 50°C, preferably about 20 to about 45°C, more preferably about 25 to about 40°C, and even more preferably about 25 to about 35°C. The medium is usually adjusted to a pH of 3 to 10.

The microorganism of the present invention or its mutant can be cultured, for example, by placing a certain amount of medium in a culture vessel such as a test tube or culture flask, inoculating the microorganism of the present invention or its mutant, and culturing by shaking under aerobic conditions at about 15 to about 50°C using a test tube shaker, Symprocal shaker, rotary shaker, or the like. Larger scale culturing can be carried out by aeration and agitation culturing using a large scale culturing tank such as a jar fermenter of several liters or an industrial tank of several hundred liters to several hundred tons. There is no particular limit to the culturing time.

As described above, the microorganism of the present invention has organic matter reduction activity and microbial coagulation activity, and therefore can be used for the purpose of reducing organic matter and/or coagulating microorganisms from a sample containing organic matter and/or microorganisms. Therefore, the present invention also provides an organic matter reducer (organic matter reducer of the present invention) or a microbial coagulant (microbial coagulant of the present invention) that contains the microorganism of the present invention.

The organic matter reducing agent or microbial flocculant of the present invention can be obtained by preparing the culture solution obtained as described above into any suitable formulation as the organic matter reducing agent or microbial flocculant of the present invention, either as is or after suitable treatment. As a method for treating the culture solution, for example, the culture solution can be diluted with water or a suitable diluent (e.g., isotonic buffer solution, culture medium, etc.) to an appropriate microbial concentration. Alternatively, the culture solution can be filtered or centrifuged to recover the microorganisms and resuspended in an appropriate dispersion medium (e.g., isotonic buffer solution, fresh culture medium, etc.). Furthermore, the recovered microorganisms can be freeze-dried by a conventional method. Alternatively, the culture solution can be frozen and stored at -80°C with 10 to 20% glycerol added, and then thawed and resuspended in a culture medium or the like when used.

The organic matter reducing agent or microbial flocculant of the present invention may be in a variety of dosage forms, such as liquids, powders, granules, hydrated powders, granular hydrated powders (dry flowable powders), flowable powders (suspensions), emulsifiable concentrates, emulsions, and microcapsules, as long as the desired effect is not impaired.

The organic matter reducing agent or microbial flocculant of the present invention may contain a carrier in addition to the microorganism or mutant strain of the present invention, so long as the desired effect is not impaired. Any solid or liquid carrier that can be used is usable as long as it is commonly used when treating samples containing organic matter or microorganisms, and there is no particular limit to the carrier.

Examples of solid carriers include mineral carriers (clay, talc, calcium carbonate, diatomaceous earth, zeolite, bentonite, acid clay, activated clay, attapulgous clay, vermiculite, perlite, pumice, silica sand, silica, white carbon, titanium dioxide, etc.), plant carriers (inert powders or granules such as wood flour, corn stalks (cobs), walnut shells (nut husks), fruit kernels, rice husks, sawdust, bran, soy flour, powdered cellulose, starch, dextrin, sugars (glucose, maltose, lactose, sucrose, etc.), water-soluble polymer gels such as agar, and various polymer powders such as chlorinated polyethylene, chlorinated polypropylene, polyvinyl acetate, polyvinyl chloride, ethylene-vinyl acetate copolymer, and urea-aldehyde resins).

Liquid carriers include water, alcohols, polyhydric alcohols, polyhydric alcohol derivatives, ketones, esters, nitrogen-containing carriers, and oils and fats, and specific examples include ethanol, isopropanol, cyclohexanol, ethylene glycol, diethylene glycol, propylene glycol, hexylene glycol, polyethylene glycol, polypropylene glycol, propylene glycol ether, cyclohexanone, γ-butyrolactone, fatty acid methyl esters (coconut oil fatty acid methyl esters), dibasic acid methyl esters (dimethyl succinate, dimethyl glutamate, dimethyl adipate), N-alkylpyrrolidone, coconut oil, soybean oil, and rapeseed oil.

The organic matter reducing agent or microbial flocculant of the present invention may contain other organic matter reducing agents or microbial flocculants in addition to the microorganism or mutant strain of the present invention.

The sample containing organic matter and/or microorganisms may be a solid sample or a liquid sample. Examples of solid samples containing organic matter and/or microorganisms include food waste, compost, and feces. Examples of liquid samples containing organic matter and/or microorganisms include industrial wastewater, agricultural wastewater, domestic wastewater, and water from the ocean, rivers, lakes, and marshes.

The organic matter reducing agent or microbial flocculant of the present invention obtained as described above can reduce the organic matter in the sample and flocculate the microorganisms by contacting the sample with organic matter and/or microorganisms. Thus, the present invention also provides a method for reducing organic matter (organic matter reducing method of the present invention) which comprises contacting the microorganism of the present invention with a sample containing organic matter. Furthermore, the present invention provides a method for flocculating microorganisms (microbial flocculation method of the present invention) which comprises contacting the microorganism of the present invention with a sample containing microorganisms.

There are no particular limitations on the method of contacting the microorganism of the present invention with a sample containing organic matter and/or microorganisms, so long as the desired effect is obtained. For example, when the target sample is a solid sample containing organic matter and/or microorganisms, the organic matter reducer of the present invention or the microbial flocculant of the present invention may be directly sprayed onto the solid sample using a sprayer or the like, or the solid sample may be suspended in water or the like and mixed with the organic matter reducer of the present invention or the microbial flocculant of the present invention. When the target sample is a liquid sample containing organic matter and/or microorganisms, the organic matter reducer of the present invention or the microbial flocculant of the present invention may be directly added to the liquid sample and mixed.

When treating, for example, a liquid sample with the organic matter reducing agent of the present invention or the microbial flocculant of the present invention, the microbial concentration contained in the amount of the organic matter reducing agent of the present invention or the microbial flocculant of the present invention is typically 1.0 x 10 ⁶ cells/mL to 1.0 x 10 ⁸ cells/mL.

The timing of treating a sample with the organic matter reducing agent or microbial flocculant of the present invention is not particularly limited as long as it has the desired effect, and it can be used at any time.

There are no particular limitations on the frequency of use of the organic matter reducing agent or microbial flocculant of the present invention.

The present invention also provides a wastewater purification device (the wastewater purification device of the present invention) that utilizes the microorganism of the present invention. Examples of wastewater containing organic matter and/or microorganisms that can be treated by the wastewater purification device of the present invention include industrial wastewater, agricultural wastewater, domestic wastewater, and water from the ocean, rivers, lakes, and marshes. Purification refers to the complete or partial decomposition or removal of the amount of organic matter and/or microorganisms contained in the wastewater.

The wastewater purification apparatus of the present invention includes:
(1) The microorganism of the present invention.
(2) A microbial treatment tank in which wastewater is treated with the microorganism of the present invention to reduce organic matter in the wastewater and/or coagulate the microorganisms.

The wastewater purification apparatus of the present invention includes a microbial treatment tank (the microbial treatment tank of the present invention) that reduces organic matter in the wastewater and/or aggregates microorganisms by treating the wastewater with the microorganisms of the present invention (microbial treatment). The microorganisms of the present invention reduce organic matter contained in the wastewater by assimilating it, and can aggregate other microorganisms contained in the wastewater as aggregated microorganisms by the aggregation of the microorganisms of the present invention themselves, so that the microorganisms of the present invention can purify the wastewater. The wastewater purified by the microbial treatment tank of the present invention can be sent to a solid-liquid separation tank described below, or discharged into the sewer as long as it meets environmental standards. Examples of environmental standards include biochemical oxygen demand (BOD), suspended solids (SS), normal hexane extractables content (n-Hex), and coliform bacteria count.

The microbial treatment tank of the present invention may include an aeration device connected to an air agitation blower. When air is blown from the air agitation blower and ejected from the aeration device, the microorganisms of the present invention and the wastewater are agitated, thereby increasing the efficiency of wastewater treatment by the microorganisms of the present invention.

The microbial treatment tank of the present invention is classified into a carrier fluidized bed tank, an aeration tank, and a contact oxidation tank depending on its form. When the microbial treatment tank of the present invention is a carrier fluidized bed tank, it may contain a floating solid carrier. The solid carrier that can be used is not particularly limited as long as it is a floatable solid carrier that can support the microorganism of the present invention, and examples thereof include mineral-based carriers (clay, talc, calcium carbonate, diatomaceous earth, zeolite, bentonite, acid clay, activated clay, attapulgus clay, vermiculite, perlite, pumice, silica sand, silica, white carbon, titanium dioxide, etc.), plant-based carriers (inert powders or granular materials such as wood flour, corn stalks (cobs), walnut shells (nut husks), fruit kernels, rice husks, sawdust, bran, soybean flour, powdered cellulose, starch, dextrin, sugars (glucose, maltose, lactose, sucrose, etc.), water-soluble polymer gels such as agar, and various polymer powders such as chlorinated polyethylene, chlorinated polypropylene, polyvinyl acetate, polyvinyl chloride, ethylene-vinyl acetate copolymers, and urea-aldehyde resins). When the microbial treatment tank of the present invention is an aeration tank, instead of the above-mentioned carrier, a part of the aggregated microorganisms precipitated or captured in the solid-liquid separation tank described below can be used as the carrier carrying the microorganism of the present invention. When the microbial treatment tank of the present invention is a contact oxidation tank, it may contain a fixed bed. The fixed bed that can be used is not particularly limited as long as it is a fixed carrier capable of supporting the microorganism of the present invention, but examples thereof include the same as the floating carrier.

The wastewater purification apparatus of the present invention further includes at least one selected from the following:
(A) A pump tank that stores wastewater and pumps it to other tanks.
(B) A culture tank in which the microorganism of the present invention is pre-grown.
(C) A solid-liquid separation tank in which the flocculated microorganisms are separated from the biologically treated wastewater.
(D) A discharge tank for storing and discharging the biologically treated wastewater from which flocculated microorganisms have been separated.
(E) A sludge storage tank for storing the separated flocculated microorganisms.
(F) A reaction tank in which the wastewater is treated with a flocculant to flocculate suspended solids and n-Hex in the wastewater.
(G) A pressurized flotation unit which separates the flocculated suspended solids and flocculated n-Hex from the flocculant-treated wastewater.
In addition, the wastewater purification apparatus of the present invention preferably includes the following among the above:
(A) A pump tank that stores wastewater and pumps it to other tanks.
(B) A culture tank in which the microorganism of the present invention is pre-grown.
(C) A solid-liquid separation tank in which the flocculated microorganisms are separated from the biologically treated wastewater.
(D) A discharge tank for storing and discharging the biologically treated wastewater from which flocculated microorganisms have been separated.
(E) A sludge storage tank for storing the separated flocculated microorganisms.

The wastewater purification device of the present invention may include a pump tank (the pump tank of the present invention) that stores wastewater and sends it to another tank. The pump tank of the present invention can store a certain amount of wastewater to be treated by the microorganism of the present invention, homogenize it, and then send it to the other tank. The pump tank of the present invention may include an agitator to homogenize the wastewater. It may also include a pump for sending the homogenized wastewater. The pump may include the same number of pumps as the number of other tanks to which water is to be sent. Examples of other tanks to which water is sent from the pump tank of the present invention include the tanks or devices (A) to (G) above, and among these, preferred examples include a culture tank, a microbial treatment tank, and a reaction tank. Furthermore, the pump tank of the present invention may be equipped with a wastewater metering function (the flow rate adjustment tank of the present invention).

The wastewater purification apparatus of the present invention may include a culture tank (culture tank of the present invention) in which the microorganism of the present invention is pre-grown. In the culture tank of the present invention, the microorganism of the present invention can grow until it reaches the logarithmic growth phase while decomposing the organic matter in the wastewater, since the microorganism of the present invention can assimilate the organic matter contained in the wastewater. For this reason, the culture tank of the present invention can grow the microorganism of the present invention in the culture liquid fed from another tank. In addition, like the microbial treatment tank of the present invention, the culture tank of the present invention may include an air diffuser connected to an air agitation blower. The air blown from the air agitation blower is blown out from the air diffuser, so that the microorganism of the present invention and the wastewater are agitated, and the microorganism of the present invention can be efficiently grown. Furthermore, for the purpose of increasing the contact efficiency between the microorganism of the present invention and the wastewater, the culture tank of the present invention may include a floating solid carrier capable of supporting the microorganism of the present invention. The solid carrier may be the same as that usable in the microbial treatment tank of the present invention. Alternatively, the microorganism of the present invention to be cultured in the culture tank of the present invention may be the organic matter reducer of the present invention or the microbial flocculant of the present invention that already contains a solid carrier. The initial concentration of the microorganism of the present invention cultured in the culture tank of the present invention is usually 1.0 x 10 ⁶ cells/mL to 1.0 x 10 ⁸ cells/mL. The timing of administration of the microorganism of the present invention to the culture tank of the present invention is not particularly limited as long as the desired effect is obtained, and it can be administered at any timing. There is no particular limit to the frequency of administration of the microorganism of the present invention, but since the cell number of the microorganism of the present invention is stably maintained as long as wastewater containing organic matter is continuously supplied, it is not necessary to administer it multiple times. The microorganism of the present invention grown in the culture tank of the present invention as described above is fed to another tank. Therefore, the culture tank of the present invention may include a pump for feeding the cultured microorganism of the present invention. An example of the other tank to which water is fed from the culture tank of the present invention is the microbial treatment tank of the present invention.

The wastewater purification apparatus of the present invention may include a solid-liquid separation tank (the solid-liquid separation tank of the present invention) that separates the flocculated microorganisms from the wastewater that has been treated with the microorganisms of the present invention in the microbial treatment tank of the present invention. The solid-liquid separation tank of the present invention can separate the flocculated microorganisms of the present invention and other microorganisms from the wastewater that has been treated with the microorganisms of the present invention in the microbial treatment tank of the present invention.

The solid-liquid separation tank of the present invention is classified into a settling tank and a membrane separation tank depending on its form. When the solid-liquid separation tank of the present invention is a settling tank (the settling tank of the present invention), the wastewater treated by the microorganism of the present invention is left standing for a certain period of time in the settling tank of the present invention, so that the flocculated microorganisms suspended in the wastewater are precipitated to the bottom of the settling tank of the present invention. In order to exert such a function, for example, the settling tank of the present invention has a structure that is a hopper having an angle of 60 degrees or more and has a water surface load of 8 m ³ /m ² ·day or less. The time for which the wastewater is left standing is not particularly limited as long as it is a time during which the flocculated microorganisms can be precipitated, and may be, for example, about 1.5 hours to about 4 hours. When the solid-liquid separation tank of the present invention is a membrane separation tank (the membrane separation tank of the present invention), the wastewater treated by the microorganism of the present invention is passed through a separation membrane having holes provided in the membrane separation tank, so that only the flocculated microorganisms can be captured in the separation membrane. Examples of the material of the separation membrane that can be used include, but are not limited to, polytetrafluoroethylene (PTFE), polyvinylidene fluoride (PVDF), polyethersulfone (PES), and the like. The pore size of the separation membrane is not limited as long as it is a size that does not allow the passage of aggregated microorganisms, and may be, for example, about 0.01 μm to about 2 μm.

The wastewater from which the coagulated microorganisms have been separated in the solid-liquid separation tank of the present invention is temporarily stored in the discharge tank and then discharged. Therefore, when the wastewater purification apparatus of the present invention includes the solid-liquid separation tank of the present invention, the wastewater purification apparatus of the present invention may further include a discharge tank (discharge tank of the present invention) for holding and discharging the microbially treated wastewater from which the coagulated microorganisms have been separated. The wastewater from which the coagulated microorganisms have been separated and stored in the discharge tank of the present invention can be discharged into the sewer as long as it satisfies environmental standards. Examples of environmental standards include biochemical oxygen demand (BOD), suspended solids (SS), normal hexane extractables content (n-Hex), coliform bacteria count, etc. The discharge tank of the present invention may include a pump for discharging the wastewater temporarily stored in the discharge tank of the present invention into the sewer (discharge pump tank of the present invention).

The flocculated microorganisms precipitated or captured in the solid-liquid separation tank of the present invention are temporarily stored in a sludge storage tank and then disposed of. Therefore, when the wastewater purification apparatus of the present invention includes the solid-liquid separation tank of the present invention, the wastewater purification apparatus of the present invention may further include a sludge storage tank (the sludge storage tank of the present invention) that stores the flocculated microorganisms separated from the microbially treated wastewater.

The wastewater purification apparatus of the present invention may include a reaction tank (the reaction tank of the present invention) that treats the wastewater with a coagulant (coagulant treatment) to coagulate suspended solids and n-Hex in the wastewater. The wastewater before microbial treatment contains suspended solids and n-Hex in addition to organic matter and microorganisms, and the suspended solids and n-Hex can be removed from the wastewater by coagulating them before microbial treatment. The coagulant used in the reaction tank of the present invention is not particularly limited as long as it can coagulate suspended solids and n-Hex, but examples of the coagulant include polyaluminum chloride, aluminum sulfate, ferric chloride, polyferric sulfate, and polysilica iron. The reaction tank of the present invention may include an agitator. The agitator agitates the coagulant and wastewater, promoting the coagulation of suspended solids and n-Hex.

When the wastewater purification apparatus of the present invention includes the reaction tank of the present invention, the wastewater purification apparatus of the present invention may further include a pressurized flotation device (the pressurized flotation device of the present invention) that separates the flocculated suspended solids and flocculated n-Hex from the flocculant-treated wastewater. In the pressurized flotation device of the present invention, the flocculated suspended solids and flocculated n-Hex contained in the wastewater are removed by the adhesion and floating of fine bubbles generated by pressurized air.

An example of an embodiment of the wastewater purification apparatus of the present invention is shown in Figure 1. The example of the wastewater purification apparatus of the present invention includes the following.
(1) Microorganism 1 of the present invention,
(2) A carrier fluidized bed tank 2 for treating wastewater with the microorganism 1 of the present invention to reduce organic matter in the wastewater and/or to coagulate the microorganisms.

Wastewater 3 containing organic matter and/or microorganisms is fed into the carrier fluidization tank 2, and the microorganisms 1 of the present invention are added. In the carrier fluidization tank 2, air is blown from the air mixing blower 2a and blown out from the air diffuser 2b, causing the microorganisms 1 of the present invention attached to the solid carriers 2c to grow, decompose the organic matter, and aggregate all the microorganisms, thereby treating the wastewater 3. The wastewater 4 that has been treated with microorganisms is discharged into the sewer.

Another embodiment of the wastewater purification apparatus of the present invention is shown in Fig. 2. The wastewater purification apparatus of the present invention includes the following components.
(1) The microorganism 5 of the present invention,
(2) a carrier fluidized bed 6 for treating wastewater with the microorganism 5 of the present invention to reduce organic matter in the wastewater and/or aggregate the microorganisms;
(A) A pump tank 7 for storing wastewater and sending it to a reaction tank 8;
(B) a culture tank 11 in which the microorganism 5 of the present invention is pre-grown;
(C) a settling tank 12 for separating the flocculated microorganisms from the biologically treated wastewater;
(D) a discharge pump tank 13 for storing and discharging the microbially treated wastewater from which the flocculated microorganisms have been separated;
(E) a sludge storage tank 14 for storing the separated flocculated microorganisms;
(F) a reaction tank 8, which treats the wastewater with a flocculant 9 to flocculate suspended solids and n-Hex in the wastewater, and (G) a flotation device 10, which separates the flocculated suspended solids and flocculated n-Hex from the flocculant-treated wastewater.

The pump tank 7 stores a certain amount of wastewater 15 containing organic matter, microorganisms, suspended solids, and n-Hex, homogenizes it using the agitator 7a, and then pumps it to the reaction tank 8 using the pump 7b.

The reaction tank 8 adds a flocculant 9 to the wastewater delivered from the pump tank 7, and agitates the flocculant 9 and the wastewater with an agitator 8a, flocculating the suspended solids and n-Hex. The reaction tank 8 then delivers the flocculant-treated wastewater to the pressurized flotation device 10.

The pressurized flotation device 10 causes the flocculated suspended solids and flocculated n-Hex contained in the flocculant-treated wastewater to float by attaching fine air bubbles to them, and removes them as excess sludge 16. The pressurized flotation device 10 then sends the wastewater, from which the suspended solids and n-Hex have been removed and which contains organic matter and microorganisms, to the culture tank 11.

In the culture tank 11, the microorganisms 5 of the present invention that have been introduced are mixed with the wastewater sent from the pressurized flotation device 10 by air blown from the air mixing blower 11a and sprayed out from the air diffuser 11b, and the microorganisms 5 of the present invention that have been attached to the solid carriers 11c are efficiently grown. After that, the culture tank 11 sends the microorganisms 5 of the present invention together with the wastewater to the carrier fluidization tank 6.

Wastewater containing organic matter and microorganisms together with the microorganisms 5 of the present invention is fed to the carrier fluidization tank 6, and air blown from the air agitation blower 11a is blown out from the air diffuser 6a, causing the microorganisms 5 of the present invention attached to the solid carriers 6b to grow, decomposing the organic matter, and flocculating all of the microorganisms including the microorganisms 5 of the present invention. The carrier fluidization tank 6 then sends the microbially treated wastewater to the sedimentation tank 12.

The wastewater that has been treated with microorganisms is left to stand in the sedimentation tank 12, causing the flocculated microorganisms suspended in the wastewater to settle to the bottom of the sedimentation tank 12. The sedimentation tank 12 then sends the wastewater from which the flocculated microorganisms have been separated to the discharge pump tank 13, and stores the settled flocculated microorganisms in the sludge storage tank 14.

The discharge pump tank 13 temporarily stores the wastewater from which the flocculated microorganisms have been separated, and then the wastewater 17 is discharged using pump 13a.

The sludge storage tank 14 temporarily stores the aggregated microorganisms.

Further, still another example of an embodiment of the wastewater purification apparatus of the present invention is shown in Fig. 3. The wastewater purification apparatus of the present invention includes the following.
(1) the microorganism 18 of the present invention,
(2) a carrier fluidized bed 19 for treating wastewater with the microorganisms 18 of the present invention to reduce organic matter in the wastewater and/or flocculate the microorganisms;
(A) A pump tank 20 for storing wastewater and supplying it to the carrier flow tank 19 and the culture tank 21;
(B) a culture tank 21 in which the microorganism 18 of the present invention is pre-grown;
(C) a settling tank 22 for separating the flocculated microorganisms from the biologically treated wastewater;
(D) a discharge pump tank 23 for storing and discharging the microbially treated wastewater from which the flocculated microorganisms have been separated;
(E) a sludge storage tank 24 for storing the separated flocculated microorganisms;

The pump tank 20 stores a certain amount of wastewater 25 containing organic matter and microorganisms, homogenizes it using the agitator 20a, and then pumps it to the carrier flow tank 19 using pump 20b and to the culture tank 21 using pump 20c.

In the culture tank 21, the wastewater sent from the pump tank 20 is mixed with the microorganisms 18 of the present invention by air blown from the air mixing blower 19a and sprayed from the air diffuser 21a, and the microorganisms 18 of the present invention attached to the solid carriers 21b are efficiently grown. After that, the culture tank 21 sends the microorganisms 18 together with the wastewater to the carrier flow tank 19 by the pump 21c.

In the carrier fluidization tank 19, wastewater containing organic matter and microorganisms is fed together with the microorganisms 18 of the present invention, and air blown from the air mixing blower 19a is blown out from the air diffuser 19b, causing the microorganisms 18 of the present invention attached to the solid carriers 19c to grow, decomposing the organic matter, and flocculating all of the microorganisms including the microorganisms 18 of the present invention. The carrier fluidization tank 19 then feeds the microbially treated wastewater to the sedimentation tank 22.

The wastewater that has been treated with microorganisms is left to stand in the sedimentation tank 22, causing the flocculated microorganisms suspended in the wastewater to settle to the bottom of the sedimentation tank 22. The sedimentation tank 22 then sends the wastewater from which the flocculated microorganisms have been separated to the discharge pump tank 23, and stores the settled flocculated microorganisms in the sludge storage tank 24.

The discharge pump tank 23 temporarily stores the wastewater from which the flocculated microorganisms have been separated, and then the wastewater 26 is discharged by pump 23a.

The sludge storage tank 24 temporarily stores the aggregated microorganisms.

The present invention will be described in more detail with reference to the following examples, but the examples are merely illustrative of the present invention and do not limit the scope of the present invention in any way.

Example 1 Comparison of wastewater treatment performance In order to objectively evaluate the performance of newly isolated microorganisms for the purpose of wastewater treatment, the inventors compared the performance of commercially available wastewater treatment materials that are mainly composed of microorganisms or enzymes for wastewater treatment. In the comparison test of wastewater treatment capacity, wastewater from a meat processing plant in Fukui Prefecture was used. 2 mL of various wastewater treatment materials were added to 100 mL of this meat processing wastewater (inoculation amount 2%), and wastewater treatment was performed using a baffled Erlenmeyer flask at 28°C for 24 hours at 120 rpm. The wastewater treatment capacity of the microorganisms was evaluated using the total organic carbon (TOC) in the wastewater after treatment as an indicator (a total organic carbon meter, SHIMADZU TOC-L, was used). TOC is the total amount of organic matter present in water expressed as the amount of carbon contained in the organic matter, and is used as one of the indicators of "water pollution." Compared to the traditional BOD (biochemical oxygen demand) and COD (chemical oxygen demand), TOC measurements have the advantage of being resistant to interference from coexisting substances in the sample and being able to accurately measure the amount of organic matter in real time. As a result of the comparative test, "QqBiO" (Quad Corporation) had the highest treatment capacity for meat processing wastewater (Figure 4). This is thought to be because QqBiO is a mixed microbial treatment material consisting of many different types of microorganisms (about 200 types).

Example 2 Search for wastewater treatment microorganisms As shown in Example 1, QqBiO is a mixed material containing many kinds of microorganisms, so it has a high wastewater treatment capacity, but it is difficult to mix different microorganisms evenly during the manufacturing process, so there is a problem that the quality of the product is not stable. Therefore, the inventors attempted to isolate a microorganism that has excellent wastewater treatment capacity even when used alone. Microorganisms with high wastewater treatment capacity were mainly searched for in water systems in Japan. As a result of an intensive search, they succeeded in isolating E2 strain, which has a high treatment capacity equivalent to QqBiO in meat processing wastewater (Figure 5). Furthermore, E2 strain has high coagulation property that QqBiO does not have. This is an extremely important characteristic in wastewater treatment, and it makes it easy to dispose of sludge and omits the treatment process using coagulants (Figure 6). When the coagulated and precipitated E2 strain was observed with an electron microscope, it was found that a large amount of biopolymers were secreted outside the bacterial cell, and these were adsorbed to each other to form aggregates (Figure 7). This flocculation property is an extremely advantageous characteristic for wastewater treatment microorganisms, as it not only causes the E2 strain to precipitate itself, but also other types of microorganisms and solid matter present in wastewater.

Example 3 Microorganism Identification Test
Because strain E2 showed extremely high treatment capacity for meat processing wastewater, we performed 16S rDNA (16S rRNA gene, approximately 1,500 bp) sequence analysis, morphological observation, and physiological and biochemical property tests to infer its taxonomic group.
(Partial 16S rDNA sequence analysis)
・DNA extraction: Achromopeptidase (FUJIFILM Wako Pure Chemical, Japan)
・PCR amplification: Tks Gflex DNA Polymerase (Takara Bio, Japan)
・ Cycle sequencing: BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, USA)
Primers used: PCR amplification: 9F, 1510R
Sequence: 9F, 515F, 1099F, 536R, 926R, 1242R 1510R
・ Sequencing: ABI PRISM 3130 xl Genetic Analyzer System (Applied Biosystems)
・Sequencing: ChromasPro 2.1 (Technelysium, AUS)
- BLAST homology search: The analysis software used was ENKI (TechnoSuruga Laboratory, Japan), and the databases used were the international nucleotide sequence database (DDBJ/ENA(EMBL)/GenBank) and DB-BA15.0 (TechnoSuruga Laboratory).
- Molecular phylogenetic analysis: The neighbor-joining method was used to estimate the phylogenetic tree, the Kimura-2-parameter base substitution model was used, and the bootstrap method (1,000 iterations) was used to evaluate the reliability of the tree shape.
(16S rDNA base sequence of E2 strain) (SEQ ID NO: 1)

A BLAST homology search using the microbial identification system (analysis software: ENKI) against DB-BA 15.0 (TechnoSuruga Laboratory) and the international nucleotide sequence database (DDBJ/ENA(EMBL)/GenBank) showed that the 16S rDNA sequence of E2 strain was 99.6% identical to the type strain KIN192 ^T (accession number AY136099) of Kinneretia asaccharophila (Tables 1 and 2). In addition, in the molecular phylogenetic tree (Fig. 8) analyzed based on the nucleotide sequence obtained by the BLAST homology search against DB-BA 15.0, E2 strain was included in the cluster consisting of the Comamonadaceae family, which includes the genera Kinneretia (one genus and one species), Mitsuaria, Pelomonas, and Roseteles, and formed a cluster with K. asaccharophila KIN192 ^T (AY136099), indicating that it is closely related to K. asaccharophila. Based on the results of 16S rDNA sequence analysis, it was estimated that strain E2 is a new species of bacteria, Kinneretia sp., closely related to K. asaccharophila.

Example 4 Physiological and Biochemical Property Tests Colonies were observed using a stereomicroscope, morphological observation was performed using an optical microscope, and tests were performed on catalase reaction, oxidase reaction, acid/gas production from glucose, and oxidation/fermentation (O/F) of glucose based on the method of Barrow & Feltham (Cowan and Steel's Manual for the Identification of Medical Bacteria, 3rd ed. Cambridge: Cambridge University Press; 1993).
・ Gram staining: Faber G "Nissui" (Nissui Pharmaceutical, Japan)
・Microscope: Optical microscope BX50F4 (Olympus, Japan)
・ Stereo microscope: SMZ800N (Nikon, Japan)
The following kits were used for physiological and biochemical property tests:
API 20 NE (bioMerieux, FRA) [SIID30828-01]
API ZYM (bioMerieux, FRA) [SIID30828-01]
API 20 E (bioMerieux, FRA) [SIID30828-02, 03]
The physiological and biochemical properties of strain E2 were determined to be a motile Gram-negative rod-shaped bacterium that did not form spores, and showed positive catalase and oxidase reactions (Table 3). These properties were consistent with those of the genus Kinneretia, which was suggested to be a possible member of the species by 16S rDNA base sequence analysis, except for the catalase reaction. In addition, strain E2 reduced nitrate, did not produce indole, did not ferment glucose, showed negative urease and β-galactosidase activities, did not hydrolyze esculin or gelatin, and utilized glucose and potassium gluconate, but did not utilize L-arabinose, maltose, or DL-malic acid (Table 4). Furthermore, strain E2 showed alkaline phosphatase, trypsin, and acid phosphatase activities, but did not exhibit esterase (C8) activities, and did not grow at 10°C (Tables 3 and 5). Although these characteristics were consistent with those of K. asaccharophila, which was shown to be closely related based on 16S rDNA sequence analysis, there were also many differences.

As described above, strain E2 is included in the Kinneretia genus, and is most closely related to K. asaccharophila among known species. However, because the results of 16S rDNA sequence analysis and physiological and biochemical property tests indicate that it is different from K. asaccharophila, it was not possible to estimate its taxonomic group at the species level. Therefore, based on the results of the above identification tests, strain E2 was identified as a new species of bacteria, Kinneretia sp. To date, only one genus and one species of Kinneretia has been found, K. asaccharophila, discovered in Lake Kinneret in Israel, and strain E2 is the second species of Kinneretia found in Japan.

Example 5 Wastewater treatment performance test The microflora of the wastewater treatment facility is composed of a wide variety of microorganisms, and we investigated how much wastewater treatment effect can be achieved by adding a single microorganism to it. The field test was carried out using a wastewater treatment facility attached to a meat processing plant in Fukui Prefecture. The average amount of wastewater treated in this facility is about 120 ^m3 /day. Wastewater (raw water) that flows into the wastewater treatment facility from the meat processing plant is temporarily stored in a raw water tank, and then pumped by a raw water pump to a "flow rate adjustment tank," where the wastewater condition is leveled, and a certain amount of water is transferred to a reaction tank using a pump and a metering device (Figure 9). A flocculant is added to the reaction tank to flocculate suspended solids (SS) and n-Hex in the wastewater, and caustic soda (sodium hydroxide) is used to adjust the pH to promote the flocculation reaction. Next, a part of the pressurized flotation treated water is treated with pressurized air to obtain fine bubble water, which is mixed with wastewater pumped from the reaction tank. Fine bubbles are generated in the wastewater in the pressurized flotation device, and the bubbles are attached to the organic matter (flocs) formed by the coagulant, causing the flocs to float and separate and remove oil and fat solids. In this way, the biochemical oxygen demand (BOD), normal hexane extract (n-Hex), and suspended solids (SS) of the pressurized flotation treated water that has passed through the pressurized flotation device are reduced to a certain extent. The pressurized flotation treated water is then transferred to the "first chamber of the wastewater treatment tank (carrier fluidized bed)", and while passing through multiple wastewater treatment tanks, the BOD and n-Hex are treated by microorganisms until they reach or fall below the discharge standard value. Meanwhile, the flocs separated and removed as sludge (which is uniformly spread in the water and can be measured as a concentration) by the pressurized flotation device are temporarily stored in the sludge storage tank and then transferred to the dehydrator by the sludge transfer pump. In the dehydrator, dehydration is carried out using cationic and anionic polymer flocculants, and the dehydrated sludge is disposed of off-site as industrial waste. Thus, wastewater treatment facilities have had problems such as "large amounts of chemicals used" in the pressurized flotation device, "industrial disposal of the sludge generated," and "bad odors dispersing outside the facility." Furthermore, a large amount of energy is required to "dehydrate the sludge" and "pressurize large amounts of water."
To solve these problems at once, the E2 strain culture solution was directly added to the first chamber of the wastewater treatment tank at a rate of 100 L/ ^m3 in the field test without using a flotation device. As a result, it was possible to clear the discharge standards for biochemical oxygen demand (BOD) and n-hexane extract (n-Hex) without using a flotation device and even with a wastewater treatment load about 1.6 times higher than that during normal operation (Figures 10 and 11). In addition, flocculation was maintained (Figure 12). These effects of the E2 strain lasted for about one month with a single addition. Due to the amazing efficiency of the E2 strain, the running costs of the wastewater treatment facility could be reduced to about one-quarter of the conventional level.

Example 6 Analysis of the microbial flora (community) in wastewater As mentioned above, wastewater (raw water) flowing into the wastewater treatment facility from a meat processing plant is temporarily stored in the raw water tank and then pumped to the "flow adjustment tank" by the raw water pump, but it is thought that a wide variety of microorganisms are already multiplying at this stage. Therefore, in order to understand how the E2 strain functions in such a complex microbial environment, we performed "microbiota (community structure)" in the wastewater and "temporal dynamics of the E2 strain." The community structure in the wastewater (raw water) was analyzed by amplicon sequencing, and the dynamics of the E2 strain introduced into the wastewater treatment tank was analyzed by real-time PCR.
(1) Amplicon sequence analysis
1. DNA extraction/extraction method: MORA-EXTRACT kit (Kyokuto Pharmaceutical, Japan), cell disruption device: FastPrep-24 5G (MP-Biomedicals, USA)
・Absorbance measurement supplement: NanoDrop ND8000 (Thermo Fisher Scientific, USA), DNA concentration (ng/μl), DNA purity (A260/A280)
2. PCR
Primers used: Pro341F - Pro805R (approximately 430 bp excluding the primer sequence for bacterial and archaeal 16S rDNA)
・ PCR conditions: Takahashi S, Tomita J, Nishioka K, Hisada T, Nishijima M. Development of a prokaryotic universal primer for simultaneous analysis of Bacteria and Archaea using next-generation sequencing. PLoS One 2014; 9: e105592.
3. Amplicon sequence analysis: Sequencer: MiSeq (Illumina, USA), sequence excluding primer sequence determined (approximately 380-430 bp)
・ Sequencing Kit: MiSeq Reagent Kit v3 (600 cycles) (Illumina)
4. Fastq paired-end ligation Software: fastq-join
5. Sequence preprocessing (quality filtering)
・ Software: FASTX-Toolkit
6. Chimera check: Remove chimeric sequences detected by software: QIIME1.8.0 and usarch6.1.544_i86
7. Homology search
1) Search using the Ribosomal Database Project (RDP) and the software Metagenome@KIN (World Fusion, Japan)
・ Database RDP MultiClassifier ver.2.11 (16S rDNA)
Cut Off: Phylum: 0.8 (Extract taxa with confidence of 0.8 or higher)
Taxonomic group: Kingdom - Genus
2) Search using microbial identification database Software: Metagenome@KIN (World Fusion)
・ Database: Microbial identification database DB-BA13.0 (TechnoSuruga Laboratory, Japan)
Extract the highest-ranking taxon with a homology rate of 97% or more as a closely related species. Taxon name: Kingdom - Species
8. Comparative analysis between samples Software: Metagenome@KIN (World Fusion)
・Analysis method: Principal component analysis and cluster analysis (clustering method: group average method, distance function: Pearson's correlation coefficient)
(2) Real-time PCR analysis
1. DNA extraction/extraction method: MORA-EXTRACT kit (Kyokuto Pharmaceutical, Japan), cell disruption device: FastPrep-24 5G (MP-Biomedicals, USA)
・Absorbance measurement: NanoDrop ND8000 (Thermo Fisher Scientific, USA), DNA concentration (ng/μL), DNA purity (A260/A280)
2. PCR Conditions and Primers
The combination of 341f - 534r comprehensively amplifies 16S rDNA from all eubacteria (Muyzer G, de Waal EC, Uitterlinden AG. Profiling of complex microbial populations by denaturing gradient gel electrophoresis analysis of polymerase chain reaction-amplified genes coding for 16S rRNA. Appl Environ Microbiol 1993;59:695-700.)
Kasaccharo_F (GTGGGGGATAACTGCTCGAAAGAGCAG (SEQ ID NO: 2)) and Kasaccharo_R (TACCCCGAGGTATTAACCCAGAGCG (SEQ ID NO: 3)) were designed and amplified to specifically amplify only the 16S rDNA of the E2 strain. Standard: Escherichia coli JCM 1649 ^T (for measuring the copy number of 16S rDNA in all eubacteria)
Plasmid DNA derived from E2 strain (for measuring K. asaccharophila 16S rDNA copy number)
3. Real-time PCR
Reagent: TB Green Premix Ex Taq II (Tli RNaseH Plus) (Takara Bio, Japan)
・ Equipment: Rotor-Gene Q (QIAGEN, DEU)

Analysis of the wastewater (raw water) microflora using a next-generation sequencer confirmed that more than 300 to 400 species (genera level) of bacteria and archaea were always present in the "flow control tank," indicating an extremely biologically diverse environment. Furthermore, real-time PCR analysis was used to count the number of microorganisms present in the wastewater being treated, and the number was 7.08 x 10 ⁸ /mL at the time of introduction, and bacteria of the order of 10 ⁸ /mL were constantly detected thereafter (Figure 13). In addition, the bacterial cell concentration of the E2 strain introduced into the wastewater treatment tank in the form of a culture solution was 2.12 x 10 ⁷ /mL immediately after introduction at a rate of 100 L/m ³ , which was approximately 3% of the total bacterial count, but this decreased to the order of 10 ⁵ /mL after 24 hours and to the order of 10 ⁴ /mL after 48 hours, and was then maintained stably at the order of 10 ⁴ /mL for one month thereafter, demonstrating the high wastewater treatment effect (Figure 13). In this way, it was confirmed that strain E2 functions stably as a wastewater treatment microorganism at a cell concentration of 10 ⁴ /mL in wastewater that constantly contains more than 300 species at the genus level and bacteria at a concentration of 10 ⁸ /mL.

The microorganisms of the present invention have the same wastewater purification ability as existing wastewater treatment materials. Moreover, surprisingly, they can aggregate other types of microorganisms and solid components together, so there is no need to treat the wastewater with a flocculant before treating it with the microorganisms of the present invention. As a result, by using these microorganisms, it is possible to drastically reduce the cost of wastewater treatment without having to renovate existing drainage facilities while omitting pretreatment.

1: Microorganism of the present invention 2: Carrier fluidized bed 2a: Air agitator 2b: Air diffuser 2c: Solid carrier 3: Wastewater containing organic matter and/or microorganisms 4: Microorganism-treated wastewater 5: Microorganism of the present invention 6: Carrier fluidized bed 6a: Air diffuser 6b: Solid carrier 7: Pump bed 7a: Stirrer 7b: Pump 8: Reaction tank 8a: Stirrer 9: Flocculant 10: Pressurized flotation device 11: Culture tank 11a: Air agitator 11b: Air diffuser 11c: Solid carrier 11d: Pump 12: Settling tank
13: Discharge pump tank 13a: Pump 14: Sludge storage tank 15: Wastewater containing organic matter, microorganisms, suspended solids and n-Hex 16: Excess sludge 17: Wastewater 18: Microorganism of the present invention 19: Carrier fluidized tank 19a: Air mixing blower 19b: Air diffuser 19c: Solid carrier 20: Pump tank 20a: Agitator 20b: Pump 20c: Pump 21: Culture tank 21a: Air diffuser 21b: Solid carrier 21c: Pump 22: Sedimentation tank
23: Discharge pump tank 23a: Pump 24: Sludge storage tank 25: Wastewater containing organic matter and microorganisms 26: Wastewater

Claims (8)

The isolated Kinneretia microorganism has accession number NITE P-03538. An organic matter reducing agent comprising the microorganism according to claim 1 . A microbial flocculant comprising the microorganism according to claim 1 . A method for reducing organic matter, comprising contacting the microorganism according to claim 1 with a sample containing organic matter. A method for agglutinating microorganisms, comprising contacting the microorganism according to claim 1 with a sample containing the microorganism. Wastewater purification equipment, including:
(1) A microbial treatment tank for treating wastewater with the microorganism according to claim 1 , and (2) a microorganism according to (1) to reduce organic matter in the wastewater and/or aggregate the microorganisms. The apparatus of claim 6 , further comprising at least one selected from the following:
(A) A pump tank for storing wastewater and pumping it to other tanks;
(B) a culture tank in which the microorganism according to claim 1 is pre-grown;
(C) a solid-liquid separation tank for separating the flocculated microorganisms from the biologically treated wastewater;
(D) a discharge tank for storing and discharging the biologically treated wastewater from which the flocculated microorganisms have been separated;
(E) a sludge storage tank for storing the separated flocculated microorganisms;
(F) a reaction tank for flocculating suspended solids and n-Hex in the wastewater by treating the wastewater with a flocculant, and (G) a pressurized flotation device for separating the flocculated suspended solids and flocculated n-Hex from the flocculant-treated wastewater. 7. The apparatus of claim 6 , further comprising:
(A) A pump tank for storing wastewater and pumping it to other tanks;
(B) a culture tank in which the microorganism according to claim 1 is pre-grown;
(C) a solid-liquid separation tank for separating the flocculated microorganisms from the biologically treated wastewater;
(D) a discharge tank for storing and discharging the biologically treated wastewater from which the flocculated microorganisms have been separated; and (E) a sludge storage tank for storing the separated flocculated microorganisms.