JP4614756B2 - Denitrification strain and method for removing nitric acid using the same - Google Patents

Description

  The present invention relates to a denitrifying strain and a method for removing nitric acid using the same.

The "denitrification" generally, microorganisms and nitrate nitrogen or nitrite nitrogen under anaerobic conditions refers to a process of generating a N ₂ or N ₂ 0. The denitrification, N0 nitrogen oxides such as ₃ in water and soil is, because it is reduced to nitrogen gas state, such as N _2, on earth as a major route of nitrogen released into the atmosphere from the biosphere It plays a major role in the nitrogen cycle. The microorganism (bacteria) that performs this denitrification is called denitrifying bacteria.

  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.

In recent years, a method has been developed in which a microorganism (denitrifying bacterium) having the above-described denitrifying ability and a method for removing nitric acid using the same are improved. For example, a microorganism capable of denitrification (denitrifying bacteria) that can simultaneously oxidize nitrous acid to nitric acid and reduce nitrous acid to nitrous oxide in a short time, and a method for removing nitric acid using this microorganism (Patent Document 1).
JP 2002-78479 A

  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.

  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.

  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.

In order to solve the above problems, the present invention includes, firstly, Anoxybacillus pushchinoensis AT-1 strain (NITE P-33), and secondly, Anoxybacillus pashkinoensis. (Anoxybacillus pushchinoensis) AT-2 strain (NITE P-34) is provided.

  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.

According to the first and second inventions, 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. . Moreover, it has excellent denitrification ability and can efficiently remove nitric acid.

  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.

  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.

  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.

  The present invention has the features as described above, and the embodiments thereof will be described in detail below.

Denitrificans strain of the present invention belongs to Ano carboxymethyl Bacillus (Anoxybacillus genus), reducing nitrate to nitrite, and Ri microorganisms der which is characterized by having the ability to reduce the nitrogen molecule, specifically Are Anoxybacillus pushchinoensis AT-1 (NITE P-33) and Anoxybacillus pushchinoensis AT-2 (NITE P-34). This strain can be isolated from the natural world such as soil (for example, field soil, hot spring soil, soil near a sewage treatment facility, ranch soil, etc.). In the denitrifying bacterium of the present invention, the atmospheric conditions in the process of reducing to nitrogen molecules are preferably aerobic conditions (for example, a dissolved oxygen concentration of 6 ppm or more) in order to efficiently reduce nitrogen molecules. Of course, the denitrifying strain of the present invention can sufficiently exhibit its function even under anaerobic conditions as in the prior art.

  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.

The isolation of AT-1 and AT-2 strains (single cell isolation) is specifically:
<1> Collect 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.

  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).

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 6 −
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 6 −
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 the grade which can fully exhibit denitrification activity, and culture | cultivating overnight normally. 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.

  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.

  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.

  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.

  Hereinafter, the present invention will be described more specifically with reference to examples, but the present invention is not limited to these examples.

  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.

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.

  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.

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 alignment of base sequences of the genus Anoxybacillus and representative bacterial species obtained from DDBJ, the position of the molecular phylogenetic tree of this strain was consistent with Anoxybacillus pushchinoensis.
(2) Results of classification and identification Based on the taxonomic properties of the AT-1 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. 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.

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 properties 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.

  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.

  Of course, the present invention is not limited to the above examples, and it goes without saying that various aspects are possible in detail.

It is the figure which showed the removal ability of nitrate nitrogen (nitric acid) in AT-1 stock | strain which is a denitrifying bacterium of this invention. It is the figure which showed the removal capability of nitrate nitrogen (nitric acid) in AT-2 stock | strain which is a denitrifying bacterium of this invention.

Claims (4)

Anoxybacillus pushchinoensis AT-1 strain (NITE P-33). Anoxybacillus pushchinoensis AT-2 strain (NITE P-34). A claim 1 or 2 method of removing nitric acid using bacterial strains described, the strain was added to the nitrate contamination, at a pH range of conditions temperature ranges and pH from 6.5 to 10.5 temperature 35 ° C. to 65 ° C. A method for removing nitric acid, comprising denitrifying nitric acid by reducing nitric acid to nitrous acid and reducing it to nitrogen molecules.   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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