JP2006166875A - Denitrifying bacterial strain and method for removing nitric acid by using the same - Google Patents
Denitrifying bacterial strain and method for removing nitric acid by using the same Download PDFInfo
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Description
本発明は、脱窒菌株およびこれを用いた硝酸の除去方法に関するものである。 The present invention relates to a denitrifying strain and a method for removing nitric acid using the same.
「脱窒」は一般に、微生物が嫌気的条件下で硝酸態窒素あるいは亜硝酸態窒素を、N2あるいはN20を生成する過程のことをいう。この脱窒は、水中や土壌中においてN03等の窒素酸化物が、N2等のガス態の窒素に還元されるので、生物圏から大気圏へ放出される窒素の主要な経路として地球上の窒素循環に大きな役割を果たしている。この脱窒を行う微生物（細菌）のことを脱窒菌という。
The "denitrification" generally, microorganisms and nitrate nitrogen or nitrite nitrogen under anaerobic conditions refers to a process of generating a N 2 or
そして、現在、このような微生物（脱窒菌）の脱窒の原理を利用した上水用の硝酸の除去方法が開発されつつある。この方法では、亜硝酸還元酵素、硝酸還元酵素、一酸化窒素還元酵素、亜酸化窒素還元酵素等の一連の脱窒系酵素を産生する各種の微生物が用いられ、これらの酵素の働きにより処理水中の硝酸態・亜硝酸態窒素は全て分子状窒素として大気中に還元される。 Currently, a method for removing nitric acid for drinking water utilizing the principle of denitrification of such microorganisms (denitrifying bacteria) is being developed. In this method, various microorganisms that produce a series of denitrification enzymes such as nitrite reductase, nitrate reductase, nitric oxide reductase, and nitrous oxide reductase are used. All nitrate and nitrite nitrogen is reduced to the atmosphere as molecular nitrogen.
近年では、上記のような脱窒能を有する微生物（脱窒菌）およびこれを用いた硝酸の除去方法を改良した方法が開発されている。たとえば、亜硝酸の硝酸への酸化と、亜硝酸から亜酸化窒素への還元を短時間で同時に行うことのできる脱窒能を有する微生物（脱窒菌）およびこれを用いた硝酸の除去方法が提案されている（特許文献１）。
しかしながら、多くの微生物の至適増殖環境は中温中性条件（一般に、中温は30℃〜40℃程度、中性はpH 7前後である）であることを考慮すると、上記特許文献１のような脱窒能を有する微生物（脱窒菌）およびこれを用いた硝酸の除去方法は、その至適温度が30℃および至適pHが7.8（つまり、中温中性）の環境条件下で行われるため、自然環境下では他の雑菌も増殖してしまい、脱窒菌の脱窒能を十分に発揮できず、持続性が低いという問題があった。また、硝酸性窒素が多く含まれる低栄養の廃水を微生物で処理する場合においても、その微生物は中温中性菌であるため、上記と同様の問題があった。 However, considering that the optimal growth environment of many microorganisms is a neutral temperature neutral condition (generally, the intermediate temperature is about 30 ° C. to 40 ° C. and the neutral temperature is around pH 7), the above-mentioned Patent Document 1 Since the microorganism having denitrification ability (denitrification bacteria) and the method of removing nitric acid using the same are performed under environmental conditions of an optimum temperature of 30 ° C. and an optimum pH of 7.8 (that is, intermediate temperature neutrality) In the natural environment, other germs grew and the denitrifying ability of the denitrifying bacteria could not be fully exhibited, resulting in a problem that the sustainability was low. In addition, even when low-nutrition wastewater containing a large amount of nitrate nitrogen is treated with microorganisms, the microorganisms are mesophilic neutral bacteria, and thus have the same problems as described above.
さらに、たとえば、農畜産廃棄物の堆肥化過程における環境条件等のように、高温・高アルカリ（高pH）の環境条件下において、脱窒能を十分に発揮し得る脱窒菌およびこれを用いた効率のよい硝酸の除去方法は知られていない。 Furthermore, for example, a denitrifying bacterium capable of sufficiently exerting a denitrifying ability under high temperature and high alkali (high pH) environmental conditions, such as environmental conditions in the composting process of agricultural and livestock waste, and the like were used. An efficient nitric acid removal method is not known.
そこで、本発明は、以上の背景から、従来の問題を解消し、中温中性の環境条件でなくとも、特に高温・高アルカリの環境条件下であっても、脱窒能を十分に発揮でき、効率よく硝酸を除去できる、新しい脱窒菌株およびこれを用いた硝酸の除去方法を提供する。 Therefore, the present invention solves the conventional problems from the above background, and can sufficiently exhibit the denitrification ability even under medium temperature neutral environmental conditions, particularly under high temperature and high alkali environmental conditions. A new denitrifying strain capable of efficiently removing nitric acid and a method for removing nitric acid using the same are provided.
本発明は、上記課題を解決するものとして、第１にはアノキシバチルス属（Anoxybacillus属）に属し、硝酸を亜硝酸に還元し、かつ、窒素分子に還元する能力を有することを特徴とし、第２には、菌株が、アノキシバチルス パシュキノエンシス（Anoxybacillus pushchinoensis）AT-1（NITE P-33）または、アノキシバチルス パシュキノエンシス（Anoxybacillus pushchinoensis）AT-2（NITE P-34）であることを特徴とする。 In order to solve the above problems, the present invention firstly belongs to the genus Anoxybacillus and is characterized by having the ability to reduce nitric acid to nitrous acid and to reduce to nitrogen molecules, Second, the strain is Anoxybacillus pushchinoensis AT-1 (NITE P-33) or Anoxybacillus pushchinoensis AT-2 (NITE P-34) It is characterized by that.
第３には、上記第１の発明または第２の発明の脱窒菌株を用いた硝酸の除去方法であって、脱窒菌株を硝酸汚染物に添加し、温度35℃〜65℃の温度条件範囲およびpH 6.5〜10.5のpH条件範囲で、硝酸を亜硝酸に還元させ、かつ、窒素分子に還元させて硝酸の脱窒処理を行うことを特徴とし、そして、第４には、窒素分子の還元過程は、好気条件下で行うことを特徴とする。 Third, there is a method for removing nitric acid using the denitrification strain of the first invention or the second invention, wherein the denitrification strain is added to a nitric acid contaminant and a temperature condition of 35 ° C. to 65 ° C. In the range of pH 6.5 to 10.5, nitric acid is reduced to nitrous acid and reduced to nitrogen molecules for denitrification of nitric acid. The reduction process is performed under aerobic conditions.
第１の発明によれば、中温中性の環境条件でなくとも、特に高温・高アルカリの環境条件下であっても、脱窒能を十分に発揮でき、効率よく硝酸を除去できる。 According to the first invention, denitrification ability can be sufficiently exerted and nitric acid can be efficiently removed even under medium temperature neutral environmental conditions, particularly under high temperature and high alkali environmental conditions.
第２の発明によれば、上記第１の発明の効果に加えて、脱窒能がさらに向上するとともに、さらに効率よく硝酸を除去できる。 According to the second invention, in addition to the effects of the first invention, the denitrification ability is further improved, and nitric acid can be removed more efficiently.
第３の発明によれば、中温中性の環境条件でなくとも、特に高温・高アルカリの環境条件下であっても、脱窒能を十分に発揮でき、効率よく硝酸を除去できる。 According to the third aspect of the invention, denitrification ability can be sufficiently exerted and nitric acid can be efficiently removed even under medium temperature and neutral environmental conditions, especially under high temperature and high alkali conditions.
第４の発明によれば、上記第３の発明の効果に加えて、脱窒能がさらに向上するとともに、さらに効率よく硝酸を除去できる。 According to the fourth invention, in addition to the effect of the third invention, the denitrification ability is further improved, and nitric acid can be removed more efficiently.
本発明は上記のとおりの特徴をもつものであるが、以下にその実施の形態について詳しく説明する。 The present invention has the features as described above, and the embodiments thereof will be described in detail below.
本発明の脱窒菌株は、アノキシバチルス属（Anoxybacillus属）に属し、硝酸を亜硝酸に還元し、かつ、窒素分子に還元する能力を有することを特徴とした微生物である。このとき、本発明の脱窒菌においては、窒素分子に還元する過程における雰囲気の条件は、好気的条件（たとえば、溶存酸素濃度6ppm以上）であることが、効率よく窒素分子を還元するために好ましい。もちろん、本発明の脱窒菌株は、従来のように嫌気条件下でもその機能を十分に発揮することができる。 The denitrifying strain of the present invention belongs to the genus Anoxybacillus and is a microorganism characterized by having the ability to reduce nitric acid to nitrous acid and to reduce nitrogen molecules. At this time, in the denitrifying bacterium of the present invention, the atmospheric conditions in the process of reducing to nitrogen molecules are aerobic conditions (for example, dissolved oxygen concentration of 6 ppm or more) in order to efficiently reduce nitrogen molecules. preferable. Of course, the denitrifying strain of the present invention can sufficiently exhibit its function even under anaerobic conditions as in the prior art.
このような特徴を有する脱窒菌によって、中温中性の環境条件でなくとも、特に高温・高アルカリの環境条件下であっても、脱窒能を十分に発揮でき、効率よく硝酸を除去することができる。 With the denitrifying bacteria having these characteristics, even if it is not an intermediate temperature neutral environment condition, it can fully exhibit the denitrification ability even under high temperature and high alkali environment conditions, and efficiently remove nitric acid. Can do.
また、このような本発明の脱窒菌は、土壌等の自然界（たとえば、田畑の土、温泉の土、下水処理施設近辺の土、牧場の土等）から単離することができる。特に、田畑の土から得られた、アノキシバチルス パシュキノエンシス（Anoxybacillus pushchinoensis）AT-1（NITE P-33）およびアノキシバチルス パシュキノエンシス（Anoxybacillus pushchinoensis）AT-2（NITE P-34）は、本発明の脱窒菌株として好ましい。 Further, such denitrifying bacteria of the present invention can be isolated from the natural world such as soil (for example, field soil, hot spring soil, soil near sewage treatment facilities, ranch soil, etc.). In particular, Anoxybacillus pushchinoensis AT-1 (NITE P-33) and Anoxybacillus pushchinoensis AT-2 (NITE P-34) obtained from field soil The denitrifying strain of the present invention is preferable.
このAT-1株およびAT-2株の単離（単菌分離）は、具体的には：
＜１＞試料として各土壌を採取し、ペプトン硝酸培地（1Ｌ当たりの組成：0.3％ペプトンS（日本製薬）、0.02％酵母エキス（ディフコ）、0.1％硝酸カリウムを、水に溶解してオートクレーブ滅菌し、10％炭酸ナトリウム溶液でpHを10.0に調整した）に投入し、3日間60℃で培養した。
＜２＞培養3日後、ダ-ラム管で気泡が認められた試料を、脱窒反応陽性試料として、培養上清1％を新しいペプトン硝酸培地に植菌した。そして、上記と同様の培養を繰り返し、3回の培養で陽性が認められた1試料を、ペプトン硝酸寒天培地（1Ｌ当たりの組成：0.3％ペプトンS（日本製薬）、0.02％酵母エキス（ディフコ）、0.1％硝酸カリウム、2.0％寒天末を、水に溶解してオートクレーブ滅菌し、10％炭酸ナトリウム溶液でpHを10.0に調整した）に植菌し、単菌分離した。
＜３＞pH 10.0、50℃で単菌分離した結果、AT-1株およびAT-2株を取得できた。
＜４＞このAT-1株およびAT-2株をペプトン硝酸培地で培養し、生成されたガス成分をガスクロマトグラフィーで分析したところ、99％が窒素であることから、AT-1株およびAT-2株は脱窒菌であると確認できた。なお、生成ガスの分析条件の条件は、以下のとおりである。すなわち：
＜１＞ガスクロマトグラフィー装置：島津製作所、GC-14AT
＜２＞カラム：モレキュラーシーブ-5A、60〜80メッシュ、2m
＜３＞カラム温度：50℃、試料気化室温度：100℃、検出温度：100℃
＜４＞検出器：TCD、キャリーガス：He
＜５＞試料注入量：0.25ml
である。
The isolation of AT-1 and AT-2 strains (single cell isolation) is specifically:
<1> Take each soil as a sample, peptone nitrate medium (composition per liter: 0.3% peptone S (Nippon Pharmaceutical), 0.02% yeast extract (Difco), 0.1% potassium nitrate dissolved in water and autoclaved) The pH was adjusted to 10.0 with a 10% sodium carbonate solution) and cultured at 60 ° C. for 3 days.
<2> After 3 days of culture, 1% of the culture supernatant was inoculated into a new peptone nitrate medium using a sample in which bubbles were observed in a Durham tube as a positive sample for denitrification reaction. Then, the same culture as above was repeated, and one sample that was found to be positive in three cultures was added to a peptone nitrate agar medium (composition per liter: 0.3% peptone S (Nippon Pharmaceutical), 0.02% yeast extract (Difco)). 0.1% potassium nitrate and 2.0% agar powder were dissolved in water and sterilized by autoclaving, and the pH was adjusted to 10.0 with a 10% sodium carbonate solution.
<3> As a result of single bacterial isolation at pH 10.0 and 50 ° C., AT-1 and AT-2 strains were obtained.
<4> The AT-1 and AT-2 strains were cultured in a peptone nitric acid medium, and the generated gas components were analyzed by gas chromatography. As a result, 99% was nitrogen. -2 strain was confirmed to be denitrifying bacteria. In addition, the conditions of the analysis conditions of product gas are as follows. Ie:
<1> Gas chromatography equipment: Shimadzu Corporation, GC-14AT
<2> Column: Molecular sieve-5A, 60-80 mesh, 2m
<3> Column temperature: 50 ° C, sample vaporization chamber temperature: 100 ° C, detection temperature: 100 ° C
<4> Detector: TCD, Carry gas: He
<5> Sample injection volume: 0.25ml
It is.
また、このAT-1株およびAT-2株は、両者ともに、2004年11月9日付けで独立行政法人製品評価技術基盤機構特許微生物寄託センターに特許微生物として寄託されている（AT-1株の受託番号：NITE P-33；AT-2株の受託番号：NITE P-34）。 In addition, both AT-1 and AT-2 strains were deposited as patent microorganisms at the Patent Microorganism Depositary, National Institute of Technology and Evaluation on November 9, 2004 (AT-1 strain). No .: NITE P-33; AT-2 strain No .: NITE P-34).
このAT-1株の菌学的性質は、以下のとおりである。
１．表現形質
（１）細胞形態
桿菌（大きさ:0.7〜0.8× 2.0〜3.0 μm）
運動性 −
グラム染色 ＋
内生胞子 ＋
ＧＣ含量 42％
（２）生理的性質
ゼラチン液化 −
リトマスミルク −
インドールの生成 −
硝酸塩の還元 ＋
アセトインの産生 −
硫化水素の生成 −
デンプンの加水分解 ＋
クエン酸の利用 −
ウレアーゼ −
オキシダーゼ ＋
カタラーゼ ＋
酸素に対する態度 通性嫌気性
OFテスト（グルコース） 陰性
糖の分解 発酵
・Ｌ−アラビノース −
・Ｄ−キシロース −
・Ｄ−グルコース ＋
・Ｄ−マンノース −
・Ｄ−フラクトース ＋
・Ｄ−ガラクトース −
・マルトース ＋
・スクロース ＋
・ラクトース −
・トレハロース ＋
・Ｄ−ソルビトール −
・Ｄ−マンニトール ＋
・イノシトール −
・グリセリン −
・デンプン ＋
・ズルシトール −
・イヌリン ＋
２．生育温度とpH
（１）生育温度（ペプトン硝酸培地、pH 10）
30℃ −
37℃ ＋
60℃ ＋
70℃ −
（２）生育pH（ペプトン硝酸培地、60℃）
pH 6 −
pH 7 ＋
pH 10 ＋
pH 11 −
次に、AT-2株の菌学的性質は、以下のとおりである。
１．表現形質
（１）細胞形態
桿菌（大きさ:0.70〜0.8× 1.5〜2.0 μm）
運動性 ＋
グラム染色 ＋
内生胞子 ＋
ＧＣ含量 42％
（２）生理的性質
ゼラチン液化 −
リトマスミルク −
インドールの生成 −
硝酸塩の還元 ＋
アセトインの産生 −
硫化水素の生成 −
デンプンの加水分解 ＋
クエン酸の利用 −
ウレアーゼ −
オキシダーゼ −
カタラーゼ ＋
酸素に対する態度 通性嫌気性
生育温度 40℃〜60℃で良好な生育
生育pH pH 7〜10で良好な生育
OFテスト（グルコース） 陰性
糖の分解 発酵
・Ｌ−アラビノース −
・Ｄ−キシロース −
・Ｄ−グルコース ＋
・Ｄ−マンノース −
・Ｄ−フラクトース ＋
・Ｄ−ガラクトース −
・マルトース ＋
・スクロース ＋
・ラクトース −
・トレハロース ＋
・Ｄ−ソルビトール −
・Ｄ−マンニトール ＋
・イノシトール −
・グリセリン −
・デンプン ＋
・ズルシトール −
・イヌリン ＋
２．生育温度とpH
（１）生育温度（ペプトン硝酸培地、pH 10）
30℃ −
37℃ ＋
60℃ ＋
70℃ −
（２）生育pH（ペプトン硝酸培地、60℃）
pH 6 −
pH 7 ＋
pH 10 ＋
pH 11 −
上記のような菌学的性質を有する微生物（脱窒菌）は、通常の細菌用の培地で培養増殖させることができる。たとえば、ペプトン硝酸培地等であることが好ましい。そして、本発明の脱窒菌の培養に要する時間は、脱窒活性を十分に発揮できる程度まで培養し、通常は一晩培養する。そして、培養終了後、周知技術で培地から本発明の脱窒菌を採取し、硝酸汚染物（たとえば、堆肥化中の農畜産廃棄物や硝酸性窒素を含む低栄養性の廃水等）に添加して、硝酸の除去反応に用いることができる。
The bacteriological properties of this AT-1 strain are as follows.
1. Phenotypic traits (1) Neisseria gonorrhoeae (size: 0.7-0.8 × 2.0-3.0 μm)
Mobility −
Gram staining +
Endospores +
GC content 42%
(2) Physiological properties Gelatin liquefaction −
Litmus milk −
Indole generation −
Reduction of nitrate +
Production of acetoin −
Production of hydrogen sulfide −
Starch hydrolysis +
Use of citric acid −
Urease −
Oxidase +
Catalase +
Attitude to oxygen facultative anaerobic
OF Test (Glucose) Degradation of Negative Sugar Fermentation / L-arabinose-
-D-xylose-
・ D-glucose +
・ D-Mannose-
・ D-fructose +
・ D-galactose-
・ Maltose +
・ Sucrose +
・ Lactose −
・ Trehalose +
・ D-sorbitol-
・ D-mannitol +
・ Inositol −
・ Glycerin −
・ Starch +
・ Zulcitol −
・ Inulin +
2. Growth temperature and pH
(1) Growth temperature (peptone nitrate medium, pH 10)
30 ° C −
37 ℃ +
60 ℃ +
70 ° C −
(2) Growth pH (peptone nitrate medium, 60 ° C)
pH 7 +
pH 10 +
pH 11 −
Next, the mycological properties of the AT-2 strain are as follows.
1. Phenotypic traits (1) Neisseria gonorrhoeae (size: 0.70-0.8 × 1.5-2.0 μm)
Motility +
Gram staining +
Endospores +
GC content 42%
(2) Physiological properties Gelatin liquefaction −
Litmus milk −
Indole generation −
Reduction of nitrate +
Production of acetoin −
Production of hydrogen sulfide −
Starch hydrolysis +
Use of citric acid −
Urease −
Oxidase −
Catalase +
Attitude toward oxygen, facultative anaerobic growth temperature, good growth at 40 ° C to 60 ° C, good growth at pH 7-10
OF Test (Glucose) Degradation of Negative Sugar Fermentation / L-arabinose-
-D-xylose-
・ D-glucose +
・ D-Mannose-
・ D-fructose +
・ D-galactose-
・ Maltose +
・ Sucrose +
・ Lactose −
・ Trehalose +
・ D-sorbitol-
・ D-mannitol +
・ Inositol −
・ Glycerin −
・ Starch +
・ Zulcitol −
・ Inulin +
2. Growth temperature and pH
(1) Growth temperature (peptone nitrate medium, pH 10)
30 ° C −
37 ℃ +
60 ℃ +
70 ° C −
(2) Growth pH (peptone nitrate medium, 60 ° C)
pH 7 +
pH 10 +
pH 11 −
Microorganisms having the above mycological properties (denitrifying bacteria) can be cultured and grown in a normal bacterial medium. For example, a peptone nitrate medium is preferable. And the time required for culture | cultivation of the denitrifying bacteria of this invention is culture | cultivated to such an extent that denitrification activity can fully be exhibited, and it culture | cultivates normally overnight. Then, after completion of the culture, the denitrifying bacteria of the present invention are collected from the culture medium by well-known techniques and added to nitrate contaminants (for example, agricultural and livestock waste during composting or low nutrient wastewater containing nitrate nitrogen). Thus, it can be used for nitric acid removal reaction.
すなわち、本発明は、上記の脱窒菌株を用いた硝酸の除去方法も提供する。具体的には、上記のとおりの脱窒菌株を硝酸汚染物に添加し、硝酸を亜硝酸に還元させ、かつ、窒素分子に還元させて硝酸の脱窒処理を行う。その際の環境条件は、温度35℃〜65℃の温度条件範囲およびpH 6.5〜10.5のpH条件範囲であることが好ましい。特に、温度条件については、40℃〜60℃の範囲であることが好ましく、またpH条件については、pH 7〜10の範囲であることが好ましい。これによって、従来のように中温中性の環境条件でなくとも、特に高温・高アルカリの環境条件下であっても、脱窒能を十分に発揮させて、効率よく硝酸を除去することができ、硝酸性窒素による汚染物や汚染水の浄化に貢献することができる。 That is, this invention also provides the removal method of nitric acid using said denitrification strain. Specifically, the denitrification strain as described above is added to the nitric acid contaminant, nitric acid is reduced to nitrous acid, and reduced to nitrogen molecules to perform denitrification of nitric acid. The environmental conditions at that time are preferably a temperature condition range of 35 ° C. to 65 ° C. and a pH condition range of pH 6.5 to 10.5. In particular, the temperature condition is preferably in the range of 40 ° C to 60 ° C, and the pH condition is preferably in the range of pH 7 to 10. This makes it possible to remove nitric acid efficiently by fully exerting the denitrification ability even under the medium temperature neutral environment conditions as in the past, especially under high temperature and high alkali environment conditions. It can contribute to the purification of pollutants and contaminated water by nitrate nitrogen.
そして、このとき、窒素分子の還元過程における雰囲気の条件は、脱窒能をさらに向上するとともに、さらに効率よく硝酸を除去することを考慮すると、好気条件（たとえば、溶存酸素濃度6ppm以上）の下で行うことが好ましい。 At this time, the conditions of the atmosphere in the reduction process of the nitrogen molecules further improve the denitrification ability, and considering the removal of nitric acid more efficiently, aerobic conditions (for example, dissolved oxygen concentration of 6 ppm or more) It is preferable to carry out below.
なお、本発明の脱窒菌株は、公知の凍結法（たとえば、-80℃のディープフリーザーや液体窒素にて凍結。保護剤として30％グリセリン液や市販の保護剤を使用）により保管することができる。凍結した脱窒菌株は、常温に戻して、培養または硝酸の除去反応に用いることができる。 The denitrifying strain of the present invention can be stored by a known freezing method (for example, freezing in a deep freezer or liquid nitrogen at −80 ° C., using a 30% glycerin solution or a commercially available protective agent as a protective agent). it can. The frozen denitrifying strain can be returned to room temperature and used for culture or nitric acid removal reaction.
以下に実施例を示して、本発明の脱窒菌についてさらに具体的に説明するが、本発明はこの例によって限定されるものではない。 Hereinafter, the present invention will be described more specifically with reference to examples, but the present invention is not limited to these examples.
実施例では、本発明の脱窒菌であるAT-１株およびAT-2株を用い、それぞれの作用効果を解析した。 In the examples, AT-1 and AT-2 strains, which are the denitrifying bacteria of the present invention, were used, and their respective effects were analyzed.
AT-1株およびAT-2株を培養するにあたって、共通事項として、まず培地は、ペプトン硝酸培地を用いた。その組成は（1Ｌ当たり）：
0.3％ ペプトンS、
0.02％ 酵母エキス、および
0.1％ 硝酸カリウム、
を蒸留水に溶解して混合し、オートクレーブ滅菌後、10％炭酸ナトリウム溶液を用いてpHを10.0に調製した。
In culturing AT-1 and AT-2 strains, as a common matter, first, a peptone nitrate medium was used as the medium. Its composition (per liter):
0.3% peptone S,
0.02% yeast extract, and
0.1% potassium nitrate,
Was dissolved in distilled water and mixed, and after autoclaving, the pH was adjusted to 10.0 using a 10% sodium carbonate solution.
次に、培養方法は、11ml容量の試験管に上記のペプトン硝酸培地8mlを入れ、同培地で一晩培養した全培養液を終濃度1％になるように添加し、60℃で静置培養した。 Next, the culture method is to put 8 ml of the above peptone nitrate medium into an 11 ml test tube, add the whole culture medium cultured overnight in the same medium to a final concentration of 1%, and culture at 60 ° C for stationary culture. did.
そして、測定方法については、本発明の脱窒菌株の生育度合いは、分光光度計を用いて波長A660の吸光度を測定した。硝酸性窒素の濃度は、塩化バナジウム-ジアゾ化カップリング法（たとえば、Doane T A., and Horwath W R., Analytical Letters, Vol. 36, No. 12, pp. 2713-2722, 2003）で測定した。
実施例１：AT-1株
（１）16S rDNAの塩基配列に基づく分子系統解析
本発明のAT-1株の16S rDNAの塩基配列を定法に従って決定し、DNAデータベース（DDBJ）にアクセスし、FASTAプログラムを用いて、16S rDNA の塩基配列の相同性検索を行った。その結果、公知の菌株Anoxybacillus pushchinoensis K-1と本発明のAT-1株の16S rDNAの相同性が99.1%であることが確認できた。また、DDBJより入手したAnoxybacillus 属および代表的な細菌種の塩基配列を多重整列後、NJ法により分子系統解析を行った結果、本菌株の分子系統樹の位置はAnoxybacillus pushchinoensisと一致した。
（２）分類および同定の結果
本発明のAT-1株の表現形質による分類学的性質に基づき、公知技術（たとえば、Bergey's Manual of Systematic Bacteriology、Bergey's Manual of Determinative Bacteriology （9th ed.）等）に従って分類および同定を行った。その結果、AT-1株はAnoxybacillus pushchinoensisと同定することができた。また、AT-1株の16S rDNAの塩基配列に基づく分子系統解析の結果においても、Anoxybacillus pushchinoensisであることが示された。
（３）硝酸性窒素の除去
AT-1株を60℃の温度条件およびpH 10のpH条件の下で、上記のとおりのペプトン硝酸培地で培養した（培養時間：0時間、1時間、2時間、4時間、6時間、8時間）。結果は、表１および図１に示したとおりであった。
And about the measuring method, the growth degree of the denitrification strain | stump | stock of this invention measured the light absorbency of wavelength A660 using the spectrophotometer. The concentration of nitrate nitrogen was measured by the vanadium chloride-diazotization coupling method (for example, Doane T A., and Horwath W R., Analytical Letters, Vol. 36, No. 12, pp. 2713-2722, 2003). did.
Example 1: AT-1 strain (1) Molecular phylogenetic analysis based on the base sequence of 16S rDNA The base sequence of 16S rDNA of the AT-1 strain of the present invention was determined according to a standard method, accessed to the DNA database (DDBJ), and FASTA Using the program, the homology search of the base sequence of 16S rDNA was performed. As a result, it was confirmed that the homology between the known strain Anoxybacillus pushchinoensis K-1 and the 16S rDNA of the AT-1 strain of the present invention was 99.1%. In addition, as a result of molecular phylogenetic analysis by NJ method after multiple alignments of Anoxybacillus genus and representative bacterial species obtained from DDBJ, the position of the molecular phylogenetic tree of this strain coincided with Anoxybacillus pushchinoensis.
(2) Results of classification and identification Based on the taxonomic characteristics of the AT-1 strain of the present invention based on the phenotypic traits, according to known techniques (for example, Bergey's Manual of Systematic Bacteriology, Bergey's Manual of Determinative Bacteriology (9th ed.), Etc.) Classification and identification were performed. As a result, the AT-1 strain could be identified as Anoxybacillus pushchinoensis. In addition, the results of molecular phylogenetic analysis based on the base sequence of 16S rDNA of AT-1 strain showed that it was Anoxybacillus pushchinoensis.
(3) Removal of nitrate nitrogen
The AT-1 strain was cultured in the peptone nitrate medium as described above under a temperature condition of 60 ° C. and a pH of 10 (culture time: 0 hour, 1 hour, 2 hours, 4 hours, 6 hours, 8 hours time). The results were as shown in Table 1 and FIG.
AT-1株を培養する時間が長いほど、AT-1株は、吸光度の増加のとおり当然に増殖し、これに比例して、硝酸性窒素(mg/ml)は減少することが確認できた。つまり、本発明のAT-1株によって、効率よく硝酸を除去することができた。
実施例２：AT-2株
（１）16S rDNAの塩基配列に基づく分子系統解析
実施例１のAT-1株と同様に、AT-2株の16S rDNAの塩基配列を決定し、DDBJにアクセスして、FASTAプログラムを用いて、16S rDNA の塩基配列の相同性検索を行った。その結果、AT-2株においても、Anoxybacillus pushchinoensis K-1との塩基配列の相同性は99.1%であった。また、DDBJより入手したAnoxybacillus 属および代表的な細菌種の塩基配列を多重整列後、NJ法により分子系統解析を行った結果、AT-2株の分子系統樹の位置についても、AT-1株と同様にAnoxybacillus pushchinoensisと一致した。
（２）分類および同定の結果
本発明のAT-2株の表現形質による分類学的性質に基づき、公知技術（たとえば、Bergey's Manual of Systematic Bacteriology、Bergey's Manual of Determinative Bacteriology （9th ed.）等）に従って分類および同定を、実施例１のAT-1と同様に行った。その結果、AT-2株は、AT-1と同様にAnoxybacillus pushchinoensisと同定することができた。また、AT-2株の16S rDNAの塩基配列に基づく分子系統解析の結果においても、Anoxybacillus pushchinoensisであることが示された。
（３）硝酸性窒素の除去
AT-2株を60℃の温度条件およびpH 10のpH条件の下で、上記のとおりのペプトン硝酸培地で培養した（培養時間：0時間、1時間、2時間、4時間、6時間、8時間）。結果は、表２および図２に示したとおりであった。
It was confirmed that the longer the AT-1 strain was cultured, the more naturally the AT-1 strain grew as the absorbance increased, and the nitrate nitrogen (mg / ml) decreased proportionally. . That is, the AT-1 strain of the present invention was able to remove nitric acid efficiently.
Example 2: AT-2 strain (1) Molecular phylogenetic analysis based on the 16S rDNA base sequence In the same manner as the AT-1 strain of Example 1, the base sequence of the AT-2 strain 16S rDNA was determined and DDBJ was accessed. Then, using the FASTA program, the homology search of the base sequence of 16S rDNA was performed. As a result, also in the AT-2 strain, the base sequence homology with Anoxybacillus pushchinoensis K-1 was 99.1%. In addition, as a result of molecular phylogenetic analysis by NJ method after multiple alignment of base sequences of Anoxybacillus genus and representative bacterial species obtained from DDBJ, the position of the molecular phylogenetic tree of AT-2 strain Similar to Anoxybacillus pushchinoensis.
(2) Results of classification and identification Based on the taxonomic characteristics of the AT-2 strain of the present invention based on the phenotypic characteristics, according to known techniques (for example, Bergey's Manual of Systematic Bacteriology, Bergey's Manual of Determinative Bacteriology (9th ed.), Etc.) Classification and identification were performed in the same manner as AT-1 in Example 1. As a result, the AT-2 strain could be identified as Anoxybacillus pushchinoensis in the same manner as AT-1. In addition, the results of molecular phylogenetic analysis based on the base sequence of 16S rDNA of AT-2 strain showed that it was Anoxybacillus pushchinoensis.
(3) Removal of nitrate nitrogen
The AT-2 strain was cultured in the peptone nitrate medium as described above under a temperature condition of 60 ° C. and a pH of 10 (culture time: 0 hour, 1 hour, 2 hours, 4 hours, 6 hours, 8 hours time). The results were as shown in Table 2 and FIG.
表２および図２に示したとおり、AT-2株を培養する時間が長いほど、AT-1株と同様にAT-2株は、吸光度の増加のとおり当然に増殖し、これに比例して、硝酸性窒素(mg/ml)は減少することが確認できた。つまり、本発明のAT-2株によっても、効率よく硝酸を除去することができた。 As shown in Table 2 and FIG. 2, the longer the AT-2 strain is cultured, the more naturally the AT-2 strain grows as the absorbance increases, and the proportion of this increases. Nitrate nitrogen (mg / ml) was confirmed to decrease. That is, the AT-2 strain of the present invention was able to remove nitric acid efficiently.
もちろん、本発明は以上の例によって限定されるものではなく、その細部については様々な態様が可能であることはいうまでもない。 Of course, the present invention is not limited to the above examples, and it goes without saying that various aspects are possible in detail.
Claims (4)
The method for removing nitric acid according to claim 3, wherein the reduction process of nitrogen molecules is performed under aerobic conditions.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| JP2006272321A (en) * | 2005-03-04 | 2006-10-12 | Hitachi Plant Technologies Ltd | Treatment method of ammonia-containing liquid and its treatment apparatus |
| JP2008023485A (en) * | 2006-07-24 | 2008-02-07 | Japan Organo Co Ltd | Biological denitrification method and apparatus therefor |
| JP2011010594A (en) * | 2009-07-01 | 2011-01-20 | National Agriculture & Food Research Organization | Microorganism reducing nitrate nitrogen and nitrite nitrogen in fermented feed |
| CN102337232A (en) * | 2011-04-13 | 2012-02-01 | 吉林农业大学 | Aerobic denitrification psychrotolerant bacterium and preparation method thereof |
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Non-Patent Citations (2)
| Title |
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| JPN6010037661, Ｉｎｔ Ｊ Ｓｙｓｔ Ｅｖｏｌ Ｍｉｃｒｏｂｉｏｌ． ２００４ Ｊｕｌ；５４（Ｐｔ ４）：１２３９−４２． * |
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| JP2006272321A (en) * | 2005-03-04 | 2006-10-12 | Hitachi Plant Technologies Ltd | Treatment method of ammonia-containing liquid and its treatment apparatus |
| JP2008023485A (en) * | 2006-07-24 | 2008-02-07 | Japan Organo Co Ltd | Biological denitrification method and apparatus therefor |
| JP2011010594A (en) * | 2009-07-01 | 2011-01-20 | National Agriculture & Food Research Organization | Microorganism reducing nitrate nitrogen and nitrite nitrogen in fermented feed |
| CN102337232A (en) * | 2011-04-13 | 2012-02-01 | 吉林农业大学 | Aerobic denitrification psychrotolerant bacterium and preparation method thereof |
| CN102337232B (en) * | 2011-04-13 | 2013-02-13 | 吉林农业大学 | Aerobic denitrification psychrotolerant bacterium and preparation method thereof |
| CN103881949A (en) * | 2014-03-21 | 2014-06-25 | 浙江工业大学 | Anoxybacillus contaminans HA and application thereof in degrading oxynitrides |
| CN103881949B (en) * | 2014-03-21 | 2016-06-15 | 浙江工业大学 | The application of anaerobic spore-bearing bacilli HA and degraded oxynitride thereof |
| CN116376756A (en) * | 2023-03-09 | 2023-07-04 | 清远海贝生物技术有限公司 | Aerobic denitrifying bacteria and application thereof in sewage treatment |
| CN116376756B (en) * | 2023-03-09 | 2023-10-03 | 清远海贝生物技术有限公司 | Aerobic denitrifying bacteria and application thereof in sewage treatment |
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