JP5334527B2 - Novel microorganism, wastewater treatment method and wastewater treatment apparatus using the novel microorganism - Google Patents

Description

  The present invention relates to a novel microorganism that can accumulate, for example, a phosphorus component contained in wastewater, and further relates to a wastewater treatment method and a wastewater treatment apparatus using the novel microorganism.

  Phosphorus is an indispensable element for the growth of animals and plants, but when the concentration in water increases, it will lead to eutrophication of the water area. Particularly, examples of the phosphorus component contained in the wastewater include orthophosphoric acid (orthophosphoric acid), polyphosphoric acid, phosphate, phosphate ester, phosphoprotein, glycerophosphoric acid, phospholipid, and the like. If the phosphorus component in the wastewater is excessive, phytoplankton may grow significantly due to water eutrophication.

  Conventionally, as a method for removing phosphorus components in wastewater (dephosphorization method), a method of adding a flocculant, a crystallization dephosphorization method, and an aerobic-anaerobic activated sludge method are known (Patent Document 1). .

  The flocculant addition method uses the fact that metal oxide cations such as aluminum ions and iron (III) ions react with normal phosphoric acid to form poorly soluble phosphate, and drains flocculants such as aluminum sulfate. In this method, flocs formed from poorly soluble phosphate (including biological flocs) are separated by precipitation. In this method, an increase in excess sludge of about 5 to 20% is recognized. Therefore, a large amount of excess sludge containing a large amount of phosphorus component is discarded, which is not a preferable method from the viewpoint of environmental conservation.

  The crystallization dephosphorization method is based on the reaction between orthophosphoric acid and calcium ions, and is preferable in that it does not involve an increase in excess sludge, but the conditions necessary for apatite crystallization (for example, carbonation by pretreatment). The removal of crystallization interfering substances such as ions, pH adjustment, temperature adjustment, etc.) must be strictly controlled, and its application is limited. In addition, since the processing cost is high, it is not preferable for large-scale processing.

  The aerobic-anaerobic activated sludge method utilizes the fact that microorganisms that have released polyphosphoric acid as normal phosphoric acid to gain energy in anaerobic conditions accumulate polyphosphoric acid after overdose and metabolism in the aerobic state. It is the method. In this method, waste water is subjected to repeated treatment in an anaerobic tank, an aerobic tank, and a sedimentation basin, and a phosphorus component is included in excess sludge to remove the phosphorus component in the treated waste water.

  That is, although the technology itself that removes the phosphorus component contained in the wastewater using a microorganism having phosphorus accumulation ability is known, it is sufficient in terms of the phosphorus accumulation ability, in other words, the phosphorus component removal ability. I could not say.

  On the other hand, in general, including biological reactions, the reaction rate increases at higher temperatures. Therefore, when removing the phosphorus component in wastewater using microorganisms, an increase in reaction rate can be expected if it can be performed under high temperature conditions. However, conventionally known microorganisms having phosphorus storage ability are only known to accumulate phosphorus components in wastewater under normal temperature conditions (for example, about 37 ° C.).

Japanese Patent Laid-Open No. 9-267099

  Therefore, in view of the actual situation as described above, the present invention has an object to provide a novel microorganism capable of accumulating phosphorus components contained in wastewater or the like under relatively high temperature conditions, and further, wastewater treatment using the microorganism. An object is to provide an apparatus and a wastewater treatment method.

  In order to achieve the above-described object, the present inventors have selected microorganisms having the desired characteristics using activated sludge collected from a wastewater treatment plant facility in Higashihiroshima City, Hiroshima Prefecture and soil samples collected from the Hiroshima University campus as separation sources. As a result of intensive investigations for isolation and identification, it was possible to isolate and identify a novel microorganism that was not classified as a conventionally known microorganism. The present invention has been made based on the phosphorus accumulating ability of these novel microorganisms. New microorganisms isolated and identified are not classified as conventionally known microorganisms.

  The novel microorganism according to the present invention belongs to Ureibacillus thermophilus and has the ability to accumulate polyphosphoric acid in the cells under conditions of 50 to 60 ° C. The novel microorganisms isolated and identified by the present inventors have the mycological properties described in any of Tables 1 to 3 below.

  The nucleotide sequence of a gene encoding 16S rRNA (hereinafter referred to as 16S rDNA) was determined for a novel microorganism isolated and identified by the present inventors. The base sequence is shown in SEQ ID NO: 1. When the homology search was performed using the database (GenBank / DDBJ / EMBL) and homology search software (BLAST) based on the base sequence shown in SEQ ID NO: 1, the base sequence shown in SEQ ID NO: 1 was “Ureibacillus thermophilus HC148 (Accession No. DQ348072) ”showed the highest homology (99.8%) with the nucleotide sequence registered.

  From the above bacteriological properties and knowledge based on the base sequence of 16S rDNA, the novel microorganism according to the present invention was classified as a novel strain belonging to Ureibacillus thermophilus. The new microorganism according to the present invention was named HTP-01, and it was established in July 2008 in the National Institute of Advanced Industrial Science and Technology, Patent Biological Deposit Center (Chuo 6-1, 1-1-1 Higashi 1-1-1, Tsukuba, Ibaraki Prefecture). Deposited as FERM P-21602 on the 10th.

  On the other hand, the wastewater treatment method and the wastewater treatment apparatus according to the present invention are for removing the present portion contained in the wastewater under high temperature conditions using the microorganisms according to the present invention described above. The wastewater treatment method and the wastewater treatment apparatus according to the present invention can remove phosphorus components under high temperature conditions, which could not be removed by the conventional method using microorganisms having phosphorus accumulation ability, by using the above-described microorganisms. Can do. Moreover, the wastewater treatment method and wastewater treatment apparatus according to the present invention can treat wastewater under high temperature conditions with the above-described microorganisms.

  ADVANTAGE OF THE INVENTION According to this invention, the novel microorganisms which can remove the phosphorus component contained in waste water etc. can be provided. In particular, the novel microorganism according to the present invention can remove phosphorus components even under high temperature conditions. By utilizing the novel microorganism according to the present invention, it is possible to provide a wastewater treatment method and a wastewater treatment apparatus that can remove phosphorus components contained in wastewater and the like. In these wastewater treatment methods and wastewater treatment apparatuses, the temperature of the wastewater to be treated can be set to a high temperature condition, so that it is possible to achieve excellent phosphorus removal efficiency and downsizing and cost reduction of the apparatus.

Hereinafter, the present invention will be described in detail.
The novel microorganism according to the present invention belongs to Ureibacillus thermophilus and has the ability to accumulate a phosphorus component in the microbial cells under high temperature conditions (for example, 50 to 60 ° C.). The novel microorganism according to the present invention does not grow under 5% NaCl conditions, but hydrolyzes gelatin, so that Ureibacillus thermophilus HC148 (Accession No. DQ348072) known as a microorganism belonging to Ureibacillus thermophilus Is different.

  In other words, the novel microorganism according to the present invention has the ability to accumulate a phosphorus component in the microbial cells under high temperature conditions (for example, 50 to 60 ° C.), does not grow under 5% NaCl conditions, and can hydrolyze gelatin. It can be said that this is Ureibacillus thermophilus. The novel microorganism according to the present invention having such characteristic properties can be isolated from the environment such as activated sludge, field, river water, and bottom soil of an anaerobic layer of a sewage treatment plant. As a specific method, first, samples collected from the above various environments are cultured under high temperature conditions, and selection is performed based on the presence or absence of phosphorus accumulating ability. Here, the sample is preferably stored under anaerobic conditions. In addition, when examining the phosphorus accumulation ability of the target microorganism, since the phosphorus component is accumulated as polyphosphoric acid in the bacterial body, 4,6-diamino-2-phenylindole (DAPI) It can be evaluated by staining polyphosphoric acid in the microbial cells using and measuring strong yellow fluorescence.

  Specifically, in the present invention, the novel microorganisms according to the present invention are obtained from activated sludge collected from a sewage treatment plant in Higashihiroshima City, Hiroshima Prefecture and a soil sample collected from Kagamiyama Park in Higashihiroshima City, Hiroshima University Campus. It was isolated and named HTP-01, and the National Institute of Advanced Industrial Science and Technology, Patent Biological Depositary Center (Central 6th, 1-1-1 Higashi 1-1-1, Tsukuba City, Ibaraki Prefecture, 305-8566) was commissioned on July 10, 2008. Deposited as FERM P-21602. The mycological properties of this deposited strain are shown in Tables 4-6 below.

The base sequence of 16S rDNA of the deposited strain is shown in SEQ ID NO: 1.
The novel microorganism according to the present invention is not limited to the deposited strain specified by the deposit number FERM P-21602, and includes other microorganisms classified into the same strain as the deposited strain. For example, the novel microorganism according to the present invention has the bacteriological properties shown in Tables 1 to 3 above, and has the ability to accumulate phosphorus components in the microbial cells under high temperature conditions (for example, 50 to 60 ° C.). Contains filth. Further, the novel microorganism according to the present invention has a homology exceeding 99.8% with respect to the base sequence shown in SEQ ID NO: 1, and has an ability to accumulate a phosphorus component in the microbial cells under high temperature conditions (eg, 50 to 60 ° C.). Contains ureibacillus thermophilus.

  The novel microorganism according to the present invention described above has both a feature that it can accumulate a phosphorus component in a microbial cell under a high temperature condition and a feature that it is very excellent in the ability to accumulate a phosphorus component. Here, the phosphorus accumulating ability can be evaluated by the above-described method using DAPI. In particular, the novel microorganism according to the present invention can specifically accumulate 100 nmol / mg protein phosphate in the stationary phase. This accumulated amount can be said to be a very high value in bacteria that have not been genetically manipulated. Furthermore, the novel microorganism according to the present invention can accumulate polyphosphoric acid along with its growth. For example, E. coli can be said to have different characteristics compared to the ability to accumulate polyphosphate during amino acid starvation. From this, it can be said that the novel microorganism according to the present invention is advantageous because it does not need to be in an amino acid starvation condition when removing the phosphorus component contained in the wastewater.

  By using the novel microorganism according to the present invention, a novel wastewater treatment method and wastewater treatment apparatus can be constructed. That is, as long as the phosphorus component contained in the wastewater to be treated is removed using the microorganisms, they are all included in the wastewater treatment method and wastewater treatment apparatus according to the present invention regardless of differences in treatment steps and apparatus configuration. Examples of the phosphorus component contained in the wastewater include orthophosphoric acid (orthophosphoric acid), polyphosphoric acid, phosphate, phosphate ester, phosphoprotein, glycerophosphoric acid, and phospholipid.

  As a wastewater treatment apparatus, for example, as shown in FIG. 1, a dephosphorization tank 1 that performs dephosphorization by a novel microorganism according to the present invention, and a solid-liquid separation tank 2 that separates solids and liquids contained in wastewater. With. Further, the wastewater treatment apparatus includes a cogeneration apparatus 3 that supplies heat to the dephosphorization tank 1. The cogeneration apparatus 3 is a system that extracts a plurality of energies from one energy, and examples thereof include an engine, a turbine, and / or a fuel cell (FC). These engine, turbine, and / or fuel cell (FC) cogeneration apparatus 3 generates thermal energy by exhaust heat recovery in addition to electrical energy. The wastewater treatment apparatus shown in FIG. 1 is configured to supply thermal energy generated in the cogeneration apparatus 3 to the waste liquid in the dephosphorization tank 1. Note that a boiler may be used as a heat source for heating, and when the amount of heat from the cogeneration is less than the amount of heat for heating the dephosphorization tank 1, a deficient heating is performed with an attached boiler or the like. It is also possible to perform.

  In the wastewater treatment apparatus shown in FIG. 1, first, wastewater to be treated is supplied to the dephosphorization tank 1, and then the solid and liquid contained in the wastewater are separated in the solid-liquid separation tank 2. In the dephosphorization tank 1, the reaction for removing the phosphorus component contained in the wastewater proceeds by the above-described new microorganism. Therefore, the solid content separated through the solid-liquid separation tank 2 contains the formula microorganism according to the present invention in which the phosphorus component contained in the wastewater is accumulated, and the solid content separated through the solid-liquid separation tank 2 This includes waste water from which the phosphorus component has been removed.

  In the wastewater treatment apparatus shown in FIG. 1, the thermal energy from the cogeneration apparatus 3 using city gas or the like as fuel is supplied to the dephosphorization tank 1, and the waste liquid in the dephosphorization tank 1 is set to the above-described high temperature condition. In the wastewater treatment apparatus according to the present invention, since the dephosphorization reaction can be performed under a high temperature condition as compared with the conventional case, excellent reaction efficiency can be achieved. Therefore, the wastewater treatment apparatus according to the present invention can reduce the size of the dephosphorization tank 1 and can reduce the cost. Furthermore, since the growth of other microorganisms is suppressed under high temperature conditions, inconveniences such as contamination in wastewater can be avoided.

  Note that the wastewater treatment apparatus according to the present invention is not limited to the configuration shown in FIG. 1. For example, as shown in FIG. 2, the phosphorus recovery apparatus that recovers the phosphorus component contained in the solid content separated in the solid-liquid separation tank 2. The structure which has this may be sufficient. According to the wastewater treatment apparatus shown in FIG. 2, the wastewater is purified by accumulating the phosphorus component contained in the wastewater in the cells of the novel microorganism according to the present invention, and the phosphorus component is recovered by collecting the accumulated phosphorus component. Effective use can be achieved. In addition, since the phosphorus component in wastewater accumulates in a microbial cell in the form of polyphosphoric acid, polyphosphoric acid can be isolate | separated as solid content by crushing a microbial cell.

  In the wastewater treatment apparatus and the wastewater treatment method according to the present invention, in particular, the treatment is performed under a high temperature condition such that the temperature in the dephosphorization tank 1 is 50 to 60 ° C. For this reason, in the wastewater treatment apparatus and the wastewater treatment method according to the present invention, most of the microorganisms mixed from the surrounding environment cannot grow, and even in an open system, it is possible to realize a process close to pure culture. is there.

EXAMPLES Hereinafter, although this invention is demonstrated in detail using an Example, the technical scope of this invention is not limited to a following example.
[Example 1] Isolation and identification of new microorganisms (1) Isolation of microorganisms from environmental samples The activated sludge sample was activated sludge from a wastewater treatment facility in Higashihiroshima City, Hiroshima Prefecture. Decomposing the agglomerates by performing a few seconds sonication was applied to 2 × YT agar medium to make a dilution series of approximately 10 to 10 ⁷ times. The composition of the 2 × YT agar medium was tryptone: 16 g, yeast extract: 10 g, NaCl: 5 g, gellan gum: 15 g, and distilled water: 1 L.

Soil samples were collected from the Hiroshima University campus. Samples were suspended in sterile water, to decompose the agglomerates by performing a few seconds sonication, making a dilution series of approximately 10 to 10 ⁷ times, was applied to 2 × YT agar medium. Each medium was cultured at 60 ° C. for 24 hours to isolate thermophilic bacteria.

(2) Screening for polyphosphate accumulating bacteria using DAPI staining as an indicator Thermophilic bacteria obtained from environmental samples were cultured at 60 ° C. for 12 hours. 1 ml of the culture solution was centrifuged (15,000 rpm × 3 min) to precipitate cells, washed with 1 ml of 0.8% NaCl, and resuspended in 1 ml of 0.8% NaCl.

  This solution was diluted 10-fold with 0.8% NaCl. When the bacterial cell concentration was high, it was diluted appropriately. 4 ', 6-diamino-2-phenylindole (DAPI) was mixed with the sample so that the final concentrations were 10, 20, and 30 μg / ml. Depending on the sample, there were bacteria that were easily stained and bacteria that were difficult to stain, so the DAPI concentration was divided into three levels. Staining was performed for 2 hours with gentle stirring in a cool dark room.

  Observation was performed using a fluorescence microscope (BX-40, Olympus). The presence of yellow polyphosphoric acid granules can be confirmed in the cells that accumulate polyphosphoric acid. Using yellow fluorescence as an index, those having strong fluorescence intensity were selected as polyphosphate-accumulating bacteria.

(3) Determination of polyphosphoric acid
Extraction of polyphosphoric acid from bacterial cells 1 ml of the bacterial cell culture solution was centrifuged (15,000 rpm × 5 min) to collect the bacterial cells. 350 μl of GITC solution (4M guanidine isothiocyanate, 50 mM Tris-HCl, pH 7.4) was added to the precipitated cell pellet, and the mixture was incubated at 90 ° C. for 2 minutes. Furthermore, ultrasonic treatment was performed for 3 minutes to dissolve the cells. The mixture was further incubated at 90 ° C. for 2 minutes, 30 μl of 10% SDS (sodium dodecyl sulfate) and 300 μl of 99.5% ethanol were added, mixed well, and then incubated at 90 ° C. for 2 minutes.

  3 μl of glass milk (Gene Clean III KIT) was added to the sample solution, mixed well, and allowed to reach room temperature for several minutes. Glass milk was precipitated by centrifugation (15,000 rpm × 10 sec) and washed twice with 300 μl of NEW WASH solution (Gene Clean III KIT).

50 μl of nuclease solution (50 mM Tris pH 7.4, 10 mM MgCl ₂ , 20 μg / ml DNase / RNase) was added and mixed, and incubated at 37 ° C. for 15 minutes to completely decompose the nucleic acid.

  The sample solution was mixed with 150 μl of GITC solution and 150 μl of 99.5% ethanol, centrifuged to precipitate glass milk, and then washed twice with 200 μl of NEW WASH solution.

  100 μl of distilled water was added to the precipitate and mixed, and the mixture was kept at 90 ° C. for 2 minutes, then centrifuged (15,000 rpm × 4 min), and the supernatant was collected as a polyphosphoric acid solution and used for subsequent measurements.

Measurement of the amount of polyphosphate Polyphosphate was quantified by converting to ATP using polyphosphate kinase (PPK) and measuring the amount of ATP. 4 μl of polyphosphate solution prepared from bacterial cells, 2 μl of 0.5 mM ADP solution, 1 μl of PPK (15 μg / ml) and 3.3 × PPK buffer (50 mM HEPES-KOH, 40 mM (NH ₄ ) ₂ SO ₄ , 4 mM MgCl ₂ , 3 μl of pH 7.2) was added, and the mixture was incubated at 37 ° C. for 1 hour to convert polyphosphoric acid to ATP.

  40 μl of luciferase solution (ATP bioluminescence kit CLS II, manufactured by Roche, Cat # .1699695) was added to 5 μl of the sample, and the fluorescence value was immediately measured using a microplate reader.

(4) Identification of polyphosphate-accumulating bacteria Strains were identified for strains that accumulated a large amount of polyphosphate. For identification of the strain, a method was used in which a base sequence encoding 16S rRNA well conserved among species was compared on a database. In addition, morphological observation and physiological / biochemical tests were performed to determine the belonging taxon of the strain.

Reagents used for genomic DNA extraction

Bacteria targeted for the extraction of genomic DNA were cultured at 60 degrees for 12 hours and pelleted. 567 μl TE was added and well suspended by pipetting. 30 μl of 10% SDS and 3 μl of 20 mg / ml proteinase K were added, lightly vortexed and incubated at 37 degrees for 1 hour. 100 ml of 5M NaCl was added and mixed well by vortexing. 80 μl of CTAB / NaCl solution was added and mixed well, and incubated at 65 degrees for 10 minutes. Approximately equal volume (about 700 μl) of CI was added, vortexed for 20 seconds and mixed well to obtain a uniform emulsion, and centrifuged at 15,000 rpm for 5 minutes at room temperature. A white interface appeared after centrifugation. The aqueous layer was transferred to a new tube so as not to take the interface part. An approximately equal volume (700 μl) of PCI was added, vortexed for 20 seconds, mixed well to make a uniform emulsion, and centrifuged again at 15,000 rpm for 5 minutes at room temperature. The upper layer was transferred to a new tube. 0.6 volume (about 450 μl) of isopropanol was added, and the tube was turned over until the white string-like DNA was clearly visible. The mixture was allowed to stand at room temperature for 2-10 minutes, and centrifuged at 15,000 rpm for 10-15 minutes at room temperature to precipitate DNA. After dissolving in 10 μl of TE buffer, 1 μl of 10 mg / ml RNase solution was added and incubated at 37 degrees for about 1 hour. An equal volume (100 μl) of PCI was added, vortexed for 20 seconds to make a uniform emulsion, and centrifuged at 15,000 rpm for 15 seconds at room temperature. The upper layer was transferred to a new tube, 100 μl of TE was added again to the lower layer, and the mixture was centrifuged at vortex at 15,000 rpm for 15 seconds, and the upper layer was added to the previous one. 20 μl of 3M sodium acetate solution and 500 μl of 100% ethanol were added to 200 μl of this upper layer, mixed well, and then allowed to stand at room temperature for 2-10 minutes. After centrifugation at 15,000 rpm for 10-15 minutes, the DNA was pelleted, the supernatant was removed, 1 ml of 70% ethanol was added, and lightly mixed to wash the DNA. The mixture was centrifuged at 15,000 rpm for 5 minutes to precipitate again, the supernatant was removed, and the pellet was dried with a vacuum desiccator. It was dissolved in 100 μl of TE, the absorbance was measured at 260 nm, the concentration was determined, and the molecular weight of the DNA obtained by electrophoresis was checked.

Amplification of 16s rDNA region by PCR and determination of DNA sequence The target bacteria were cultured for 24 hours at 45 ° C in Nutrient agar medium (Oxoid). DNA was extracted from the cultured cells, and the 16s rDNA region was Amplification by PCR was followed by sequencing. Specifically, InstaGene Matrix (Bio Rad) was used for DNA extraction according to the attached protocol. For PCR, PrimeSTAR HS DNA Polymerase (manufactured by Takara Bio Inc.) was used according to the attached protocol. For cycle sequencing, BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems) was used according to the attached protocol. The primers used were 9F, 339F, disclosed in “Gene analysis method 16s rRNA gene base sequencing method, edited by Japanese Society for Actinomycetes, classification and identification of actinomycetes 88-117pp, Japan Society for Business Administration (2001)”, 785F, 1099F, 536R, 802R, 1242R and 1510R were used. The sequence used ABI PRISM 3100 Genetic Analyzer System (Applied Biosystems) according to the attached protocol. For sequencing, ChromasPro 1.4 (Technelysium Pty) was used according to the attached protocol.

  Based on the base sequence of the obtained 16s rDNA region, homology search and simple molecular phylogenetic analysis were performed. Apollon 2.0 (manufactured by Techno Suruga Laboratories) was used as software, and Apollon DB-BA3.0 and an international nucleotide sequence database (GenBank / DDBJ / EMBL) were used as databases.

Morphological observation and physiological / biochemical test Morphological observation by optical microscope and BARROW et al., "Cowan and Steel's Manual for the Identification of Medical Bacteria" 3rd edition. 1993. Catalase reaction based on the method described in Canibridge University Press, Tests were conducted for oxidase reaction, acid / gas production from glucose, and glucose oxidation / fermentation (O / F).
API50CHB (manufactured by BioMerieux) was used for physiological and biochemical tests.

(5) Experimental results
Screening for polyphosphate-accumulating thermophilic bacteria from environmental samples Plate the suspension of environmental samples on a 2xYT plate and incubate at 60 degrees, diluting 10 ² -10 ⁴ times for activated sludge samples, 10- 10 for soil samples 10 A single colony of thermophilic bacteria could be obtained at a ^3- fold dilution. The obtained thermophilic bacteria were inoculated into 2 × YT, cultured at 60 ° C., and DAPI-stained ones were observed with a fluorescence microscope, and polyphosphate-accumulating bacteria were isolated using yellow fluorescence as an indicator. Tests were performed on 204 colonies of thermophilic bacteria obtained from activated sludge and soil. As a result, of the 204 samples tested, yellow samples were observed in 22 samples. Of these, 2 strains showed very strong yellow fluorescence, 5 strains showed relatively strong yellow fluorescence, and 15 other strains showed weak yellow fluorescence. When a reproducibility test was conducted on these, there were many cases in which the fluorescence intensity changed from one test to another, and there were some in which reproducibility could not be obtained. This may indicate that polyphosphate accumulation changes depending on the culture conditions and cell growth stage, and was considered unsuitable for use in a stable phosphate recovery system. . Therefore, a strain in which yellow fluorescence was observed stably every time was selected as a candidate strain of a polyphosphate high accumulation strain.

Measurement of polyphosphate accumulation amount of candidate strains of polyphosphate-accumulating thermobacteria Measure the amount of polyphosphate accumulated by the strain that stably accumulates polyphosphate and emits yellow fluorescence. Examined. The test strain was cultured at 60 ° C., sampled over time, and the value of OD600 and the amount of polyphosphoric acid were measured. The result is shown in FIG. As can be seen from FIG. 3, the amount of polyphosphate accumulated in the test strain started to increase from the middle of the logarithmic growth phase and became constant in the stationary phase. Its value reached about 100 nmol / mg protein. This accumulation amount is very high in bacteria that have not been genetically manipulated. The test strain accumulated polyphosphoric acid with growth. This is interesting because E. coli does not accumulate polyphosphate during amino acid starvation. Further, from the viewpoint of phosphate recovery, it was considered that there was an advantage in that it was not necessary to use special culture conditions.

  On the other hand, when the optimum culture temperature of the test strain was examined, it grew at 50 ° C. and 60 ° C., but could not grow at 37 ° C. and 70 ° C. Further, when the amount of polyphosphoric acid accumulated at this time was measured, it was found that polyphosphoric acid accumulated more at 60 ° C. than at 50 ° C. (FIG. 4).

SEQ ID NO: 1 shows the result of determining the base sequence of the 16s rDNA region of the test strain 16s rDNA . The results of physiological and biochemical tests are shown in Tables 9-11.

  As a result of homology search for Apollon DB-BA3.0 using BLAST, the 16S rDNA nucleotide sequence of the test strain showed high homology to 16S rDNA derived from Ureibacillus and Bacillus etc., and U. suwonensis 6T19 strain (KIM et al., Ureibacillus suwonensis sp. nov., isolated from cotton waste composts. Int. J. Syst. Evol. Microbiol., 2006, 56, 663-666) with the highest homology of 98.7%. showed that. As a result of homology search against GenBank / DDBJ / EMBL, the 16S rDNA of the test strain showed high homology to 16S rDNA derived from Ureibacillus, and the reference strain was U. thermophilus HC148 strain (WEON et al., Ureibacillus composti). nov. and Ureibacillus thermophilus sp. nov., isolated from livestock-manure composts. Int. J. Syst. Evol. Microbiol., 2007, 57, 2908-2911), homology 99.8%, U. composti HC145 (WEON et al., Ureibacillus composti sp. nov. and Ureibacillus thermophilus sp. nov., isolated from livestock-manure composts. Int. J. Syst. Evol. Microbiol., 2007, 57, 2908-2911) A high homology with 98.3% homology to 16S rDNA was shown. As a result of a simple molecular phylogenetic analysis performed by adding U. thermophilus HC148 strain and U. composti HC145 strain 16S rDNA to 16S rDNA of the top 10 strains of 16S rDNA of the test strain and Apollon DB-BA3.0. Is contained in a cluster formed by all five known species of Ureibacillus, forms a cluster with 16S rDNA of U. thermophilus, and shows the same molecular systematic position as U. thermophilus (Fig. 5) .

  Based on the above, it was considered that the test strain was included in Ureibacillus and likely to belong to U. thermophilus. A difference of 3 bases between the 16S rDNA sequences was confirmed, but 2 of these were primer regions and the remaining 1 base was a mixed base in the test strain. It was considered inappropriate to regard it as a clear difference between the two. Therefore, since both 16S rDNA sequences are substantially the same, the test strain was estimated to be a strain belonging to U. thermophilus from the results of 16S rDNA nucleotide sequence analysis.

  As a result of morphological observation and physiological and biochemical tests, the test strain showed good growth at 45 ° C and aerobic conditions. Gram-negative bacilli with motility showed circular spore formation. Body swelling was not observed. Catalase reaction and oxidase reaction were both positive. These properties were considered to be consistent with the general properties of Ureibacillus whose assignment was suggested in the results of 16S rDNA nucleotide sequence analysis. As a result of API tests as physiological and biochemical tests, the test strains oxidize glycerol, ribose, sorbose, etc., do not oxidize D-arabinose, glucose, fructose, etc., hydrolyze gelatin, exhibit urease activity There wasn't. As a result of the additional test, the test strain did not grow under anaerobic conditions, grew at 35 ° C. and 65 ° C., did not grow at 5% NaCl, and did not hydrolyze casein and starch. Although these properties are consistent with the properties of U. thermophilus, which was suggested to be related in the results of 16S rDNA nucleotide sequence analysis, it does not grow on 5% NaCl, and hydrolysis of gelatin is not possible with U. thermophilus. The properties were slightly different.

  From the above, it was determined that the test strain was included in Ureibacillus at the genus level and belonged to U. thermophilus. However, since it was different as a microorganism from known strains belonging to U. thermophilus, it was determined that the test strain was a new strain belonging to U. thermophilus.

  A new strain of U. thermophilus isolated and identified in this example and capable of accumulating phosphorus components was named HTP-01, and the National Institute of Advanced Industrial Science and Technology, Patent Biological Deposit Center (Tsukuba, Ibaraki, 305-8566) Deposited on July 10, 2008 as deposit number FERM P-21602 at City East 1-1-1 Central No. 6).

It is a block diagram which shows an example of the waste water treatment apparatus to which this invention is applied. It is a block diagram which shows an example of the waste water treatment apparatus to which this invention is applied. It is a characteristic view which shows the growth curve and phosphorus accumulation amount of a test strain. It is a characteristic figure which shows the phosphorus accumulation amount in 50 degreeC or 60 degreeC of a test strain. It is a phylogenetic tree including a test strain (HTP-01 strain) and related species.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 ... Dephosphorization tank, 2 ... Solid-liquid separation tank, 3 ... Cogeneration apparatus, 4 ... Phosphorus collection | recovery apparatus

Claims (8)

Belongs to the Ureibachirusu-thermophilus (Ureibacillus thermophilus), polyphosphoric acid under the conditions of 50~60 ℃ possess the ability to accumulated in the cells, microorganisms which are accession number FERM P-21602. A wastewater treatment method comprising a step of bringing the microorganism according to claim 1 into contact with wastewater containing a phosphorus component. The wastewater treatment method according to claim 2, wherein the wastewater is set to 50 to 60 ° C. The wastewater treatment method according to claim 2 , further comprising a step of separating the microorganism that has accumulated the phosphorus component contained in the waste liquid after contacting the microorganism and recovering the phosphorus component from the separated microorganism. A wastewater treatment apparatus comprising a dephosphorization tank for bringing the microorganism according to claim 1 into contact with wastewater containing nitrate ions. The wastewater treatment apparatus according to claim 5 , further comprising temperature control means for controlling the temperature of the wastewater to a desired temperature. The waste water treatment apparatus according to claim 6 , wherein the temperature control means controls the temperature of the waste water in the dephosphorization tank to 50 to 60 ° C. The wastewater treatment apparatus according to claim 5 , further comprising a device connected to the dephosphorization tank and configured to separate and collect microorganisms that accumulate phosphorus components contained in the waste liquid.

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