JP4631082B2 - Nitrification and denitrification method that simultaneously removes NH4 + and NO3- using microorganisms - Google Patents

Description

The present invention is an ammonium ion of the object in utilizing microorganisms (NH ₄ ⁺⁾ and nitrate ion (NO ₃ ^-) at the same time nitrification-denitrification method for removing, nitrification and denitrification treatment agent used in the method , And a novel microbial strain that can be used in the method.

  In recent years, with industrial development, environmental pollution has expanded to the global scale. There are many types of pollutants, and the amount of pollutants is large and the range of pollution is widened. It is hard to say that it is advantageous from the viewpoint of energy and cost to carry out environmental purification using only physical and chemical methods. On the other hand, since organisms, especially microorganisms, can grow by decomposing and using pollutants as carbon sources, nitrogen sources, and energy sources, it is considered that the method of purifying the environment using microorganisms is more efficient. . Since the resolution of microorganisms is based on biological reactions, it is possible to treat contaminants under mild reaction conditions compared to physical and chemical methods. In addition, environmental purification using microorganisms is extremely advantageous compared to physical and chemical methods because it does not require complicated equipment and can reduce energy by-products.

  Currently, one of the environmental problems we face is eutrophication of rivers, lakes, and inland seas due to accumulation of nitrogen, phosphorus, and organic matter flowing from industrial wastewater, household wastewater, and farmland. In recent years, lakes such as Kasumigaura and Lake Biwa, inner harbors such as Tokyo Port and Ise Bay, and the Seto Inland Sea have suffered frequent damage due to eutrophication such as the occurrence of red tides and sea lions. Eutrophication increases the concentration of microbial growth-limiting substances such as nitrogen and phosphorus, so that algae grow, organic substances are accumulated by photosynthesis, and the color of water changes from green to brown. Moreover, since the microbial activity is also vigorous, the decomposition rate of organic matter is fast, and the growth of seafood is hindered by the lack of dissolved oxygen. In order to prevent eutrophication, it is necessary to treat industrial and domestic wastewater before flowing into lakes and the like to remove nitrogen, phosphorus, organic matter, and the like. Especially for nitrogen, an effective removal method has not been established so far, and establishment of a new nitrogen removal method has become an urgent issue.

  Currently, nitrogen removal methods currently used in wastewater treatment facilities include physicochemical treatment methods such as ion exchange, filtration using reverse osmosis membranes, and ammonia stripping, as well as nitrification and desorption using microorganisms. There is a biological treatment method that is a nitrogen process (see Non-Patent Documents 1 and 2 below). In the physicochemical treatment method, problems such as cost, influence on the environment, and unestablished regeneration method of the used resin have been pointed out. For this reason, biological treatment methods using microorganisms are widely used.

Nitrification and denitrification using microorganisms nitrifying bacteria (ammonium oxidizing bacteria, nitrite oxidizing bacteria) by the action of the denitrifying bacteria, NH in the waste water ₄ ⁺ and NO ₂ ^-, NO ₃ ^- in the method for removing a N ₂ is there. In this method, NH ₄ ⁺ by nitrifying bacteria NO ₂ ^- through the NO ₃ ^- is oxidized, the resulting NO ₃ ^- is reduced to the atmosphere as N ₂ by denitrificans. The nitrification process is generally performed by chemically synthesized autotrophic bacteria that grow under aerobic conditions, and the denitrification process is performed by denitrifying bacteria that grow under anaerobic conditions. Therefore, the current nitrification / denitrification method requires at least three types of microorganisms (nitrifying bacteria 2, denitrifying bacteria 1) and two treatment tanks (aerobic tank, anaerobic tank). In addition, since nitrifying bacteria have a very slow growth rate, there are disadvantages such as a long time required for treatment, and development of a new technique for improving these problems is demanded.

Recently, the NH ₄ ⁺ NO ₂ ^- and NO ₃ ^- presence of heterotrophic bacteria aerobic oxidation was reported (see below Non Patent Literature 3). Moreover, the microorganisms which show a denitrification action on aerobic conditions were also discovered (refer nonpatent literature 4 of the postscript). If these microorganisms can be applied to wastewater treatment, nitrification and denitrification can be performed in one treatment tank. However, it has been reported that the nitrification ability and denitrification ability of these microorganisms are inferior to those of conventional microorganisms, and that they do not grow under conditions of high concentrations of nitrogen compounds (non-patent documents 5 to 7 described later). reference).

  In addition, it has been attempted to produce a tube-shaped gel to immobilize nitrifying bacteria and denitrifying bacteria, create an aerobic and anaerobic environment in the tube, and perform the nitrification / denitrification process in a single treatment tank. (See Non-Patent Documents 8 and 9 below.) However, since a slow-growing nitrifying bacterium is used, no fundamental solution has been reached.

Nakamura Kazunori, Understanding of environmental preservation of microorganisms. In “Environment and Microorganisms” Sangyo Tosho, Tokyo, pp. 61-69 (1998). Satoshi Wakamatsu, Countermeasures against Contaminants-Treatment and Disposal. In "Environmental Lecture, Thinking about the Environment", eds University Waste Management Facility Council Environmental Education Subcommittee, Kagaku Shimbun, Tokyo, pp.145-147 (1999) Kuenen, J. G. and Robertson, L. A., Combined nitrification-denitrification processes. FEMS Microbiol. Rev., 15, 109-117 (1994). Robertson, L. A. and Kuenen, J. G., Aerobic denitrification: a controversy revived. Arch. Microbiol., 139, 351-354 (1984). Robertson, L. A. and Kuenen, J. G., Combined heterotrophic nitrification and aerobic denitrification in Thiosphaera pantotropha and other bacteria. Antonie van Leeuwenhoek., 57, 139-152 (1990). Yamada, T., Somiya, I., Tsuno, H., Screening of aerobic denitrifying bacteria and their physiological characteristics.Gesuido Kyoukaishi., 30, 118-130 (1992). Till, B. A., Weathers, L. J., and Alvarez, P. J. J., Fe (0) -Sopported autotrophic denitrification.Environ. Sci. Technol., 32, 634-639 (1998). Uemoto, H. and Saiki, H., Nitrogen removal reactor using packed gel envelopes containing Nitrosomonas europaea and Paracoccus denitrificans. Biotechnol. Bioeng., 67, 80-86 (2000). Uemoto, H. and Saiki, H., Nitrogen removal by tubular gel containing Nitrosomonas europaea and Paracoccus denitrificans.Appl.Environ.Microbiol., 62, 4224-4228 (1996). Kim, Y., Yoshizawa, M., Takenaka, S., Murakami, S., and Aoki, K., Isolation and culture conditions of a Klebsiella pneumoniae strain that can utilize ammonium and nitrate ions simultaneously with controlled iron and molybdate ion concentrations Biosci. Biotechnol. Biochem., 66, 996-1001 (2002)

  The present invention has been made in view of the above-described conventional problems, and the problems to be solved by the present invention are as follows.

1. As mentioned above, ammonia contained in the waste water (NH ₄ ⁺⁾ and nitrate (NO ₃ ^-) nitrogen compounds, such as the leading cause eutrophication in lakes and enclosed seas. In Japan, the regulation of nitrogen compounds in wastewater has been regulated for specific lakes since 1985 and for closed seas since 1993, but the problem of eutrophication has not been solved. For this reason, the development of an efficient removal technique for nitrogen compounds, particularly NH ₄ ⁺ and NO ₃ ⁻ in wastewater flowing into closed seas, has become an urgent issue.

2. Ammonium nitrate (ammonium, NH ₄ NO ₃ ) is produced as a by-product during the rare earth oxide production process. Industrial wastewater containing NH ₄ NO ₃ generally contains almost no substances other than NH ₄ NO ₃ , but the development of an efficient nitrogen removal method in this NH ₄ NO ₃ waste water utilizing microorganisms is also eutrophication. This is one of the important issues as a countermeasure.

3. As described above, the conventional biological nitrogen removal method requires at least three kinds of microorganisms (nitrifying bacteria 2, denitrifying bacteria 1) and two treatment tanks (aerobic tank and anaerobic tank). If microorganisms capable of removing NH ₄ ⁺ and NO ₃ ⁻ at the same time can be separated, nitrification and denitrification can be performed in one treatment tank, which is advantageous in terms of both time and cost.

4). In view of these problems, the present inventor previously isolated a microorganism (Klebsiella pneumoniae F-5-2 strain) that simultaneously removes NH ₄ ⁺ and NO ₃ ⁻ from soil and reported on its characteristics and the like (not above). (See Patent Document 10). The present invention aims to further advance this research and to find other microorganisms that have the ability to remove NH ₄ ⁺ and NO ₃ ⁻ simultaneously and can be used industrially, such as wastewater treatment. Further, since the summer becomes considerably high temperature in Japan, fully grown at high temperature conditions NH ₄ ⁺ and NO ₃ with ^- can be removed, and, so-called heat-resistant bacteria show similar biological activity at room temperature If obtained, its practicality will be high. Thus, the present invention also aims to search and isolate such heat-resistant bacteria.

In view of the above problems, the present inventor collected soil from various locations and searched for microorganisms having the ability to simultaneously remove NH ₄ ⁺ and NO ₃ ^−, and as a result, NH ₄ ⁺ and even at a high temperature of 50 ° C. NO ₃ ^- together with separating and identifying novel microorganism strain (T-7-2 strain described later) which simultaneously has a property as a heat-resistant bacteria can be removed, Bacillus, Pseudomonas, are classified to the genus Enterobacter or the like that NH ₄ ⁺ and NO ₃ from among stocks ^- found a strain capable of removing at the same time and completed the present invention.
That is, the present invention includes the following inventions A) to M) as industrially useful inventions.

A) A nitrification / denitrification method for simultaneously removing NH ₄ ⁺ and NO ₃ ^{− in} an object using at least one kind of microorganism selected from the following (a) to (d).
(A) a strain belonging to the genus Bacillus (b) a strain belonging to the genus Pseudomonas (c) a strain belonging to the genus Enterobacter (d) a strain Klebsiella pneumoniae IFO 3318

B) The nitrification / denitrification method according to A), wherein at least one microorganism selected from the following (a1) to (a3) is used as the strain of (a).
(A1) Bacillus licheniformis T-7-2 strain (FERM P-19418)
(A2) Bacillus subtilis IFO 13719 strain (a3) Bacillus subtilis IFO 3022 strain

C) The nitrification / denitrification method according to A), wherein at least one microorganism selected from the following (b1) to (b6) is used as the strain (b).
(B1) Pseudomonas aeruginosa IFO 12689 (b2) Pseudomonas aeruginosa IFO 13738 (b3) Pseudomonas aeruginosa IFO 3080 (b4) Pseudomonas aeruginosa IFO 3080 (b4) 13130 strain (b5) Pseudomonas fluorescens IFO 15833 strain (b6) Pseudomonas denitrificans IFO 13301 strain

D) The nitrification / denitrification method according to A), wherein at least one microorganism selected from the following (c1) to (c2) is used as the strain of (c).
(C1) Enterobacter aerogenes IFO 13534 strain (c2) Enterobacter cloacae IFO 13535 strain

E) The nitrification / denitrification method according to A) above, wherein a heat-resistant strain is used.

F) Any one of A) to E) above, which is used for removing nitrogen in water in wastewater treatment, etc., removing wastewater from factory wastewater and other inorganic substances, or removing nitrogen in manure and other organic substances. Nitrification and denitrification method.

G) A nitrification / denitrification treatment agent that contains at least one kind of microorganism selected from the following (a) to (d), and simultaneously removes NH ₄ ⁺ and NO ₃ ⁻ in the object.
(A) a strain belonging to the genus Bacillus (b) a strain belonging to the genus Pseudomonas (c) a strain belonging to the genus Enterobacter (d) a strain Klebsiella pneumoniae IFO 3318

H) The nitrification / denitrification agent as described in G) above, which contains at least one kind of microorganism selected from the following (a1) to (a3) as the strain of (a).
(A1) Bacillus licheniformis T-7-2 strain (FERM P-19418)
(A2) Bacillus subtilis IFO 13719 strain (a3) Bacillus subtilis IFO 3022 strain

I) The nitrification / denitrification agent as described in G) above, which contains at least one kind of microorganism selected from the following (b1) to (b6) as the strain of (b).
(B1) Pseudomonas aeruginosa IFO 12689 (b2) Pseudomonas aeruginosa IFO 13738 (b3) Pseudomonas aeruginosa IFO 3080 (b4) Pseudomonas aeruginosa IFO 3080 (b4) 13130 strain (b5) Pseudomonas fluorescens IFO 15833 strain (b6) Pseudomonas denitrificans IFO 13301 strain

J) The nitrification / denitrification agent as described in G) above, which contains at least one kind of microorganism selected from the following (c1) to (c2) as the strain of (c).
(C1) Enterobacter aerogenes IFO 13534 strain (c2) Enterobacter cloacae IFO 13535 strain

K) The nitrification / denitrification agent as described in G) above, which contains a heat-resistant strain.

L) In any one of the above G) to K), which is used for removing nitrogen in water in wastewater treatment, etc., removing wastewater in factories and other inorganic substances, or removing nitrogen in manure and other organic substances. Nitrification / denitrification treatment agent.

M) Bacillus licheniformis T-7-2 strain (FERM P-19418).

According to the present invention, since a microorganism capable of simultaneously removing NH ₄ ⁺ and NO ₃ ⁻ is used, a nitrogen compound can be treated in one treatment tank. Therefore, compared with the conventional method which required two processing tanks, both the capital investment cost and the operating cost can be reduced, the processing time can be shortened, and the scale of the processing facility can also be reduced. In addition, when heat-resistant bacteria are used, treatment under high temperature conditions is possible, so that the conditions for treatment are widened, and for example, nitrogen removal treatment outdoors where the temperature changes drastically becomes possible.

Hereinafter, an embodiment of the present invention will be described.
The present inventor newly isolated a new strain that simultaneously removes NH ₄ ⁺ and NO ₃ ⁻ from soil collected from various places, and performed detailed examinations on its bacteriological properties. As a result, it was judged that it was appropriate to classify this new strain as Bacillus licheniformis, and “Bacillus licheniformis strain T-7-2: hereinafter simply referred to as T-7-2 strain”. ) ". The T-7-2 strain has been deposited as FERM P-19418 at the National Institute of Advanced Industrial Science and Technology (AIST) and the Patent Biological Deposit Center.

The T-7-2 strain, even at high temperatures such as 50 ° C., the growth NH in the environment ₄ ⁺ and NO ₃ ^- at the same time have the ability to remove and NH also in its colder example 30 ° C. _It has the ability to remove ₄ ⁺ and NO ₃ ^- at the same time (see FIGS. 2 and 3. The details will be described in the examples described later). In other words, the T-7-2 strain is considered to be a “heat-resistant bacterium” that grows only under high temperature and low temperature conditions, unlike so-called thermophilic bacteria that grow only under high temperature and exhibit its biological activity. The T-7-2 strain grows at approximately 25 to 55 ° C (preferably approximately 30 to 50 ° C) and can remove NH ₄ ⁺ and NO ₃ ^- at the same time. Can be used under both high temperature and low temperature conditions, and under severe temperature changes.

  The separation method of the above T-7-2 strain and its bacteriological properties will be described in the following examples, but here, the separation method will be briefly described with reference to FIG.

First, the collected soil (Soil) is suspended in, for example, 0.8% sodium chloride solution, allowed to stand, and an appropriate amount of supernatant (eg, 0.5 ml) is added to a test tube containing 0.1% ammonium nitrate (NH ₄ NO ₃ ) liquid medium. . For example, a medium having the composition shown in Table 1 below is used. However, NH ₄ NO ₃ is 1.0 g per 1,000 ml of water, and FeSO ₄ .7H ₂ O is 0.5 mM in this case, but it may be a lower concentration (for example, 0.1 mM). Further, for example, may be added Na ₂ MoO ₄ · 2H ₂ O to a concentration of 8 [mu] M.

  Thereafter, the test tube is sealed with a butyl rubber stopper or the like and then incubated at 50 ° C. After culturing for several days, a sample in which bubbles are actively observed is smeared on a 0.1% ammonium nitrate solid medium. A medium having the same composition as the above liquid medium may be used except that agar is added for solidification. In this solid medium, each sample was cultured at 30 ° C. under aerobic conditions, and the grown strain was then transferred to a gradient culture containing 0.1% ammonium nitrate, and the test tube was sealed with a screw cap and cultured again at 50 ° C., A strain showing good growth is selected. The present inventor obtained the T-7-2 strain showing good growth in a 0.1% ammonium nitrate medium at 30 ° C. and 50 ° C. by such a method.

Of course, it is possible to isolate the T-7-2 strain or a strain with similar properties from soil etc. by a method different from the above, and the isolation conditions such as the temperature conditions and the concentration conditions of each additive can be selected. By making various changes, it is possible to obtain strains that simultaneously remove NH ₄ ⁺ and NO ₃ ⁻ under different environments. For example, the above method includes a step of culturing at 50 ° C. and a step of culturing at 30 ° C. in order to obtain a thermostable bacterium capable of simultaneously removing NH ₄ ⁺ and NO ₃ ⁻ even at a high temperature of 50 ° C. Unless it is the purpose of acquiring thermostable bacteria, a high temperature culture process is not necessary. Of course, the temperature is 50 ° C. during the culture may be set to a temperature other than 30 ° C., the other conditions (e.g., iron ions in the medium (Fe ²⁺⁾ and molybdate ions (MoO ₄ ^2-) The concentration, the type and concentration of the carbon source, etc.) may be arbitrarily set according to the purpose.

As described above, the present inventor has first found a microorganism strain (ie, T-7-2 strain) that simultaneously removes NH ₄ ⁺ and NO ₃ ⁻ from the genus Bacillus. Therefore, it was investigated whether among the stocks classified into the genus Bacillus, there was a strain that simultaneously removes NH ₄ ⁺ and NO ₃ ⁻ (the investigation method will be described in Examples described later). One of NH to strain ₄ ⁺ and NO ₃ ^- were found property of simultaneously removing.
1. Bacillus subtilis IFO 13719 strain2. Bacillus subtilis IFO 3022

In addition, the number attached | subjected to these strains is a preservation | save stock number of a foundation for fermentation (IFO). Of the above two strains, the IFO 3022 strain showed the property of a thermostable bacterium capable of simultaneously removing NH ₄ ⁺ and NO ₃ ⁻ under both conditions of 30 ° C. and 45 ° C. Therefore, like the T-7-2 strain, the IFO 3022 strain can be used at high temperatures.

Moreover, it is possible to select a strain having the property of simultaneously removing NH ₄ ⁺ and NO ₃ ⁻ from existing Bacillus species by the same investigation method. The present invention may utilize a Bacillus genus strain obtained by such a strain selection method.

Further, as a result of investigating the conserved strains other than the genus Bacillus by the same investigation method as described above, a total of 6 Pseudomonas genera, 2 Enterobacter genera and 1 Klebsiella genera described below were obtained. Nine strains were found to simultaneously remove NH ₄ ⁺ and NO ₃ ⁻ .
1. 1. Pseudomonas aeruginosa IFO 12689 strain 2. Pseudomonas aeruginosa IFO 13738 strain 3. Pseudomonas aeruginosa IFO 3080 strain 4. Pseudomonas aeruginosa IFO 13130 strain 5. Pseudomonas fluorescens IFO 15833 strain 6. Pseudomonas denitrificans IFO 13301 strain Enterobacter aerogenes IFO 13534 strain8. Enterobacter cloacae IFO 13535 strain9. Klebsiella pneumoniae IFO 3318 strain

  The present invention may use these nine strains, or use a strain of Pseudomonas, Enterobacter, or Klebsiella obtained by a similar strain selection method. Also good.

Nitrification and denitrification process of the present invention, using at least one microorganism strain wherein the other above T-7-2 strain, NH ₄ ⁺ and NO ₃ in the object ^- and are simultaneously process for removing, A plurality of types of strains may be used in combination. Further, “removing NH ₄ ⁺ and NO ₃ ⁻ at the same time” means performing nitrification / denitrification using a microorganism that can remove both NH ₄ ⁺ and NO ₃ ⁻ .

  The nitrification / denitrification treatment agent of the present invention may basically contain any of the T-7-2 strain and at least one microbial strain described above, in addition to a protective agent, a PH adjuster, a medium. It may contain components and the like.

The present invention is useful for wastewater treatment (meaning widely including sewage treatment, wastewater treatment, sewage treatment, etc.) and uses microorganisms that can remove NH ₄ ⁺ and NO ₃ ^- at the same time. It can be processed. Therefore, compared with the conventional method which required two processing tanks, cost reduction and processing time can be shortened, and the scale of a processing facility can also be reduced. In addition, when heat-resistant bacteria are used, processing under high-temperature conditions is possible, so the conditions that can be processed are widened, for example, processing outdoors where the temperature changes drastically, and processing without air conditioning during the summer season. Become.

  In addition, the present invention can be used for wastewater treatment in factories, removal of nitrogen in inorganic substances, excreta treatment of livestock, sludge treatment, and removal of nitrogen in other organic substances, and also for aquaculture such as aquariums, squid and octopus. It can also be used to remove nitrogen (especially ammonia) from water in the field. Thus, the present invention can be widely used in industry.

  The use conditions (use temperature, medium composition, culture conditions (for example, aerobic or anaerobic), etc.) when using microorganisms in the present invention are not particularly limited, and the nature of the microorganism used What is necessary is just to select the optimal use conditions for nitrogen removal according to the characteristics.

  EXAMPLES Hereinafter, although an Example demonstrates this invention more concretely, this invention is not limited at all by these Examples.

[Example A: Isolation of thermotolerant bacteria that simultaneously remove NH ₄ ⁺ and NO ₃ ⁻ , their bacteriological properties, and simultaneous removal test of NH ₄ ⁺ and NO ₃ ⁻ using the same bacteria]
(A-1) Isolation of heat-resistant bacteria 1 g of soil collected from various places was suspended in 9 ml of 0.8% (w / v) sodium chloride solution, allowed to stand, and then 1 ml of supernatant was added to 7 ml of ammonium nitrate liquid medium (medium composition) Were added to the test tube containing Table 1 above). After sealing with a butyl rubber stopper, static culture was performed at 50 ° C. After culturing for several days, a sample in which generation of bubbles was actively observed was smeared on a solid ammonium nitrate medium (see Table 2 below for the composition of the medium). Each of these samples was cultured at 30 ° C under aerobic conditions, and the grown strain was transferred to a slant (gradient culture) containing ammonium nitrate solid medium. After sealing the test tube with a screw cap, it was cultured again at 50 ° C. A strain showing growth was selected. As a result, T-7-2 strain showing good growth in the same medium was obtained even at 30 ° C. and 50 ° C. (see FIG. 1).

(A-2) Mycological properties of T-7-2 strain and its identification The results of investigation on the mycological properties of T-7-2 strain (hereinafter referred to as the present fungus) isolated by the above method are as follows. Show.

(1) Morphological properties The size of the vegetative cell of this bacterium is 0.64 × 1.6 to 3.2 μm and is a gonococcus. There is no polymorphism.
The fungus exhibits motility. This bacterium has periflagellum.
When this bacterium is cultured on a broth agar medium for 2 days, a spore is formed in the central part of the vegetative cell. Spores of this bacterium are stained red when stained using the Dorner method. The spore shape is almost elliptical, and its size is 0.6 × 0.8 μm.

(2) Properties under various culture conditions The bacterium grows well in a broth agar medium, a synthetic medium using glucose as a carbon source and energy source, and ammonium nitrate as a nitrogen source.
The fungus grows at ˜55 ° C. in a broth liquid medium.
In this culture, the shape of the colony is irregular, the surface elevation of the colony is trapezoidal, the periphery of the colony is wavy, and the surface of the colony is rough. The surface of the colony has a dull luster and is buttery. The colony color is white-gray several days after inoculation, but turns brown after one week.
The fungus exhibits a cotton-like or wooly shape in the broth liquid culture.
The bacterium grows in the form of a sac in the broth gelatin puncture culture and liquefies gelatin.
This bacterium shows acidity in culture using litmus milk, and the culture solution coagulates.

(3) Physiological properties Gram staining: Positive nitrate reduction: Positive denitrification: Positive
MR test: negative
VP test: Production of positive indole: Production of negative hydrogen sulfide: Hydrolysis of positive starch: Use of positive citric acid: Use of positive inorganic nitrogen sources (nitrate and ammonium salt): Use both salts.
Pigment production: Insoluble brown pigment produced Urease: Negative oxidase: Negative catalase: Positive growth range: 25-55 ° C, pH 6-9
OF test (Hugh Leifson method): Whether acid and gas are produced from fermented sugars: D-glucose and L-arabinose produce acid, but no gas.

(4) Chemical taxonomic properties As a result of analysis of 1,544 bases of the 16S ribosomal RNA gene of this bacterium, the nucleotide sequence shows a high homology value with the following strains by searching the DNA database (B. is Bacillus) Abbreviation, the same applies hereinafter)
B. licheniformis strain B16 99.7%
B. licheniformis strain PR-1 99.7%
B. licheniformis strain KL-185 99.4%
B. licheniformis strain Mo1 99.1%
B. subtilis strain 09 98.1%

(5) Date of isolation of the source strain: August 2002 Source: Soil collection site: Kobe

(6) Identification of the bacterium Based on the above results, the bacterium was identified as belonging to the genus Bacillus, based on the description in the Burgy's Manual of Systematic Bacteriology (Volume 2). Furthermore, from the analysis of the 16S ribosomal RNA gene, this bacterium is considered to belong to B. licheniformis or B. subtilis, but this bacterium is a facultative anaerobe and can be grown at 55 ° C. Therefore, it is appropriate to identify this bacterium as B. licheniformis. Therefore, the present inventor named this bacterium "Bacillus licheniformis strain T-7-2".

(A-3) Removal test of NH ₄ ⁺ and NO ₃ ⁻ by this bacterium As shown in FIG. 2, in the medium of Table 1 containing 0.1% NH ₄ NO ₃ , 3% glucose and 0.5 mM Fe ²⁺ , In this bacterium, NH ₄ ⁺ (Ammonia in the figure) and NO ₃ ⁻ (Nitrate in the figure) were almost removed from the medium at 50 ° C. for 24 hours. Further, as shown in FIG. 3, also substantially eliminates the NH ₄ ⁺ in the same medium for 48 hours at 30 ° C., NO ₃ ^- was removed up to 40%. From this result, it was found that this bacterium removes both ions from the medium more rapidly at 50 ° C than at 30 ° C.

_(A-4) NH 4 ⁺ and NO ₃ ^- Effect of Fe ²⁺ and MoO ₄ ^{2-concentration} giving the simultaneous removal of the
In a medium containing 0.1 to 0.2% NH ₄ NO ₃ and 3 to 5% glucose, the influence of the Fe ²⁺ and MoO ₄ ²⁻ concentration on the simultaneous removal of NH ₄ ⁺ and NO ₃ ⁻ was examined. Fig. 4 shows the results of investigating the effect of divalent iron ions (Fe ²⁺ ), in the growth medium (OD ₆₆₀ ) of the bacterium (T-7-2 strain) on the third day of culture (after 72 hours). The remaining amount (%) of NH ₄ ⁺ , NO ₃ ⁻ , and NO ₂ ⁻ is shown. FIG. 4A shows the result of culturing at 50 ° C., and FIG. 5B shows the result of culturing at 30 ° C. As shown in the figure, the addition of Fe ²⁺ was very important in order to remove both ions. In particular, this tendency was remarkable at 50 ° C. That is, in a medium containing 0.1% NH ₄ NO ₃ , both ions are hardly removed unless Fe ²⁺ is present, but both ions are completely removed in 3 days in the presence of 0.2 mM or more Fe ^2+. I understood. In addition, in the medium in which the concentration of NH ₄ NO ₃ was increased to 0.2%, both NH ₄ ⁺ and NO ₃ ⁻ ions were removed at 0.5 mM Fe ²⁺ under 50 ° C. culture.

FIG. 5 shows the results of examining the influence of molybdate ions (MoO ₄ ²⁻ ). (A) shows the result of culturing the bacterium (T-7-2 strain) at 50 ° C., (b) shows the result of 30 ° C. It is the result of having been cultured with. As shown in the figure, the effect of MoO ₄ ^{2− on} the removal of NH ₄ ⁺ and NO ₃ ⁻ was not as significant as Fe ²⁺ . Moreover, when MoO ₄ ^{2− of} 10 nM or more was present, the removal of both ions by this bacterium was rather inhibited. It was found that effective removal of both ions by this bacterium can be achieved by adjusting the concentration of MoO ₄ ^2- appropriately.

(A-5) Change in total nitrogen amount in the culture solution of the present bacterium Next, changes in the total nitrogen amount in the culture solution of the present bacterium were examined. The total nitrogen was quantified by the following method.

  Organic nitrogen was converted to ammonia nitrogen by Kjeldahl decomposition, and the amount of ammonia nitrogen produced was determined by Conway's micro diffusion analysis method. The absorbent is 2 g boric acid dissolved in 20 ml of 99% (w / w) ethanol and 2 ml of pH indicator (0.2% (w / v) methyl red-0.2% (w / v) bromocresol green (1: After adding 5)), the pH was adjusted to 3.5 to 3.6 with 0.05 N HCl solution, and the total amount was adjusted to 100 ml with deionized water. 1 ml of sample was added to the outer chamber and 1 ml of absorbent was added to the inner chamber. 1 ml of 12% (w / v) MgO light suspension was added to the outer chamber, immediately capped, and allowed to stand at 30 ° C. overnight to absorb the generated ammonia in the inner chamber. After adding 1 ml of deionized water to the inner chamber, titration with 1/500 N HCl solution was performed to quantitate ammonia nitrogen.

FIG. 6 shows the results of the above experiment conducted under the culture condition of 50 ° C. As shown in the figure, as NH ₄ ⁺ and NO ₃ ⁻ in the culture solution decrease, the total nitrogen amount in the medium decreases and the total nitrogen amount in the cells increases. NH in the medium ₄ ⁺ and NO ₃ ^- is the sum total amount of nitrogen during the bacterial cell culture at the time it was completely consumed, the amount was reduced to 70% of the total amount of nitrogen added to the medium at the beginning ing. The remaining 30% is considered denitrified. Also in 30 ° C. culture, along with the growth of the fungus NH ₄ ⁺ and NO ₃ in the medium ^- is consumption, the total nitrogen content at the time of culture 48 hours is reduced to 80% at the start of cultivation ( (See FIG. 7). Therefore, a part of the nitrogen compound consumed even at 30 ° C. is considered to be denitrified.

As described above, this bacterium was currently isolated and identified (i.e., Bacillus licheniformis strain T-7-2) is, (1) even at a high temperature such as 50 ° C., NH ₄ ⁺ and NO ₃ ^- to simultaneously remove And (2) NH ₄ ⁺ and NO ₃ ⁻ can be simultaneously removed not only at 50 ° C. but also at 30 ° C. This result indicates that the present bacterium is a “heat-resistant bacterium” which is different from so-called thermophilic bacterium which grows only at high temperature and does not grow so much at normal temperature and exhibits its biological activity. As described above, the bacterium grows at about 30 to 50 ° C. and can remove NH ₄ ⁺ and NO ₃ ⁻ at the same time. Therefore, it can be used even under conditions where the temperature changes drastically.

[Example B: Selection of strains that simultaneously remove NH ₄ ⁺ and NO ₃ ⁻ among conserved strains belonging to the genus Bacillus and simultaneous removal test of NH ₄ ⁺ and NO ₃ ⁻ by the same strain]
As described above, the present inventor found for the first time a Bacillus genus microorganism that simultaneously removes NH ₄ ⁺ and NO ₃ ⁻ (ie, T-7-2 strain). Therefore, next, of the stocks that are classified to the genus Bacillus, NH ₄ ⁺ and NO ₃ ^- was investigated by the following experiment whether strains of removing simultaneously present.

(B-1) Strain used In this experiment, the strains (1) to (10) specified by the following display and stored in a microorganism storage organization were used.
(1) B. subtilis IFO 13721 (2) B. subtilis IFO 3013 (3) B. subtilis IFO 13719
(4) B. subtilis IFO 3335 (5) B. subtilis IFO 3009 (6) B. subtilis IFO 3022
(7) B. megaterium (8) B. sphaericus IFO 3525 (9) Bacillus. Sp. AM-23
(10) Bacillus. Sp. FL-90 (Hereafter, IFO may be omitted.)

(B-2) Preparation of medium The composition of the medium used in this experiment is shown in Table 3 below. Prepare 7 ml of 0.1% (w / v) NH ₄ NO ₃ medium containing Na ₂ MoO ₄ · 2H ₂ O at a concentration of 0.8 pM, 0.8 nM, or 8 μM, and adjust the FeSO ₄ · 7H ₂ O was added.

(B-3) Cultivation After inoculation of each bacterium, shaking culture was performed at 30 ° C. under aerobic conditions, and turbidity of the culture solution and NH ₄ ⁺ , NO ₃ ⁻ , NO remaining in the medium every day ₂ ^- was quantified.

(B-4) NO ₂ ^- Determination of
0-0.6 mM of NO ₂ ^- To a solution of 0.15 ml containing, after adding _{H 2 O 0.35 ml, 0.1 M} potassium-sodium phosphate buffer (pH 7.1) and 0.5 ml, 1% of 0.5 ml (w / v) A sulfanilamide-9% HCl solution and a 0.02% N-naphthylethylenediamine · 2 HCl solution were added, and the mixture was allowed to stand at room temperature for 10 minutes, and the absorbance was measured at 540 nm.

(B-5) NO ₃ ^- Determination of
After adding 0.2 ml of 2% (w / v) salicylic acid-concentrated sulfuric acid to a solution containing 0.25-3 mM NO ₃ ⁻ , 3.7 ml of 10% (w / v) NaOH solution was added, and then 410 nm Absorbance was measured at.

(B-6) Determination of NH ₄ ⁺
To 1 ml of a solution containing NH ₄ ⁺ of 0.01-0.30 mM, was added to the 0.4 ml phenol-nitroprusside sodium solution (see Table 4), 0.6 ml of antiformin solution (Table 5 Reference: antiformin 4ml And 40 ml of 1N NaOH were mixed and made up to 100 ml with H ₂ O.) was added, stoppered, allowed to stand at room temperature for 45 minutes, and the absorbance was measured at 635 nm.

(B-7) Experimental Results The experimental results are summarized in Table 6 below.

As shown in Table 6, among the strains of the genus Bacillus (1) to (10) investigated this time, NH ₄ ⁺ , NO ₃ ⁻ in two strains (3) B. subtilis 13719 and (6) B. subtilis 3022 Decrease was observed. As for (3) B. subtilis 13719, NH ₄ ⁺ and NO ₃ ⁻ decreased most when the concentration of MoO ₄ ²⁻ was 0.8 pM (the results are shown in FIG. 8). On the other hand, regarding (6) B. subtilis 3022, NH ₄ ⁺ and NO ₃ ⁻ decreased most when the concentration of MoO ₄ ²⁻ was 0.8 nM (the results are shown in FIG. 9).

Next, 7 ml of 0.1% (w / v) NH ₄ NO ₃ medium containing Na ₂ MoO ₄ .2H ₂ O at a concentration of 0.8 pM, 0.8 nM, or 8 μM is prepared, and 0.1 mM FeSO ₄ is added to each. • 7H ₂ O was added. This (6) B. subtilis 3022 and after inoculation, cultured with shaking at 45 ° C., NH remaining in turbidity and medium culture each day ^{_{4 +, NO 3 -, NO}} 2 - Quantitative did. As a result, NH ₄ ⁺ and NO ₃ ⁻ were reduced most at 0.8 nM MoO ₄ ²⁻ (FIG. 10 shows the results at 0.8 nM. In FIGS. 4 and 8 to 10, NO 2, NO3 and NH4 represent NO ₂ ⁻ , NO ₃ ⁻ and NH ₄ ⁺ , respectively).

As described above, (1) B. subtilis IFO 13719 and B. subtilis IFO 3022 simultaneously removed NH ₄ ⁺ and NO ₃ ⁻ at 30 ° C. among the Bacillus species examined this time. Further, (2) B.subtilis IFO 3022 NH even in 45 ° C. ₄ ⁺ and NO ₃ ^- to simultaneously removed. That is, this strain exhibited the properties of a so-called heat-resistant bacterium that can simultaneously remove NH ₄ ⁺ and NO ₃ ⁻ under both conditions of 30 ° C. and 45 ° C.

[Example C: Selection of strains that simultaneously remove NH ₄ ⁺ and NO ₃ ⁻ among conserved strains such as Enterobacter and Pseudomonas, and simultaneous removal test of NH ₄ ⁺ and NO ₃ ⁻ by the same strain]
The present inventor further determines whether or not there is a strain that simultaneously removes NH ₄ ⁺ and NO ₃ ⁻ among the conserved strains classified into the genus Enterobacter, Pseudomonas, Klebsiella, Serratia, Proteus, Alcaligenes, or Acetobacter. It was investigated by the experiment.

(C-1) Strain used In this experiment, a storage strain specified by the following display was used.
Enterobacter aerogenes IFO 13534
Enterobacter cloacae IFO 13535
Pseudomonas aeruginosa IFO 12689
Pseudomonas aeruginosa IFO 13738
Pseudomonas aeruginosa IFO 3080
Pseudomonas aeruginosa IFO 13130
Pseudomonas putida IFO 3738
Pseudomonas fluoresscens IFO 15833
Pseudomonas denitrificans IFO 13301
Klebsiella pneumoniae IFO 3318
Klebsiella planticola IFO 3317
Serratia marcescens IFO 3736
Proteus vulgaris IFO 3851
Alcaligenes faecalis IFO 13111
Alcaligenes xylosoxidans subsp.denitrificans IFO 15125

(C-2) Preparation of medium The composition of the medium used in this experiment is shown in Table 7 below. NH ₄ NO ₃ having a concentration of 0.1 to 0.4% (w / v) and Na ₂ MoO ₄ .2H ₂ O having a concentration of 0.8 pM, 0.8 nM, or 8 μM were used.

(C-3) Culture After inoculation of each bacterium, shaking culture is performed at 30 ° C. under aerobic conditions, and turbidity of the culture solution and NH ₄ ⁺ , NO ₃ ⁻ , NO remaining in the medium every day ₂ ^- was quantified.

(C-4) Determination of NO ₂ ⁻ , NO ₃ ⁻ , NH ₄ ⁺ and determination of total nitrogen
Each quantification of NO ₂ ⁻ , NO ₃ ⁻ , and NH ₄ ⁺ was carried out in the same manner as the method described in (B-4), (B-5), and (B-6). Moreover, the determination method of total nitrogen was performed similarly to the method as described in said (A-5).

(C-5) Experimental results Among the two Enterobacter genera, 7 Pseudomonas genera, 2 Klebsiella genera, 1 Serratia genera, 1 Proteus genera, and 2 Alcaligenes genera, which were investigated as a result of this experiment, 2 Enterobacter genera strains 6 strains of Pseudomonas except P. putida IFO 3738 and 9 strains of Klebsiella pneumoniae IFO 3318 were able to remove NH ₄ ⁺ and NO ₃ ⁻ simultaneously in 0.1% NH ₄ NO ₃ medium. NH by these strains ₄ ⁺ and NO ₃ ^- The results of simultaneous removal shown in Table 8.

The above results are the results on the second day of culture (48 hours), and NH ₄ ⁺ and NO ₃ ⁻ indicate the amount (mM) remaining in the medium.

  As a representative example, the results of E. cloacae IFO 13535 are shown in FIG. 11, and the results of P. denitrificans IFO 13301 are shown in FIG. E. is an abbreviation for Enterobacter, and P. is an abbreviation for Pseudomonas.

Among the strains that could simultaneously remove NH ₄ ⁺ and NO ₃ ⁻ , ₄ strains of P. aeruginosa IFO 12689, P. aeruginosa IFO 13738, P. aeruginosa IFO 3080, P. aeruginosa IFO 13130 were 0.4% NH ₄ In NO ₃ medium, NH ₄ ⁺ and NO ₃ ^- were almost completely removed within 2 days from the start of culture. At this time, NO ₂ ^- accumulation was observed. As a typical example, the result of P. aeruginosa IFO 3080 is shown in FIG.

Further, as shown in FIG. 13 (b), as NH ₄ ⁺ and NO ₃ ⁻ in the culture solution decrease, the total nitrogen amount in the medium decreases and the total nitrogen amount in the cells increases. To do. Medium NH ₄ ⁺ and NO ₃ in ^- is the sum of the total amount of nitrogen during the bacterial cell culture at the time it was completely consumed (2 days), the amount of the total amount of nitrogen added to the medium at the beginning It has decreased to 45%. The remaining 55% is considered denitrified.

The results of P. aeruginosa IFO 3080 are summarized as follows: (1) By adjusting the concentrations of Fe ²⁺ and MoO ₄ ^2- , NH ₄ NO ₃ as high as 0.4% could be removed from the medium. (2) At the time when NH ₄ ⁺ and NO ₃ ^- were removed, nitrogen compounds were hardly present in the medium (in the culture supernatant). 45% of the total nitrogen added is accumulated in the cells, and the remaining 55% is considered to be denitrified.

As described above, the present invention relates to a nitrification / denitrification method that simultaneously removes NH ₄ ⁺ and NO ₃ ⁻ using microorganisms. As described above, the present invention can be used for wastewater treatment, It can be used for excrement treatment, and also for removing ammonia in water in aquariums and farms, and can be used widely in industry.

It is a figure which shows the outline of the isolation | separation method of the microorganisms (T-7-2 strain) based on this invention. 3 is a graph showing the nitrification / denitrification ability of the T-7-2 strain under 50 ° C. culture. It is a graph which shows the nitrification / denitrification ability in 30 degreeC culture | cultivation of the said T-7-2 strain | stump | stock. NH ₄ ⁺ and NO ₃ of the T-7-2 strain ^- a graph showing the results of examining the effect of Fe ²⁺ concentration giving the simultaneous removal of, (a) shows the result of incubation at 50 ℃, (b) Is the result of culturing at 30 ° C. NH ₄ ⁺ and NO ₃ of the T-7-2 strain ^- a graph showing the results of examining the effect of MoO ₄ ^{2-concentration} giving the simultaneous removal of, (a) shows the result of incubation at 50 ° C., (b ) Is the result of culturing at 30 ° C. It is a graph which shows the result of having investigated the change of the total nitrogen amount in the culture solution under 50 degreeC culture | cultivation of the said T-7-2 strain | stump | stock. It is a graph which shows the result of having investigated the change of the total nitrogen amount in the culture solution under 30 degreeC culture | cultivation of the said T-7-2 strain | stump | stock. It is a graph which shows the nitrification and denitrification ability of B. subtilis IFO 13719 strain. It is a graph which shows the nitrification / denitrification ability in 30 degreeC culture | cultivation of B.subtilis IFO 3022 strain | stump | stock. It is a graph which shows the nitrification / denitrification ability in 45 degreeC culture | cultivation of B.subtilis IFO 3022 strain | stump | stock. It is a graph which shows the nitrification and denitrification ability of E. cloacae IFO 13535 strain. 2 is a graph showing the nitrification / denitrification ability of P. denitrificans IFO 13301 strain. (A) is a graph showing the nitrification / denitrification ability of P. aeruginosa IFO 3080 strain, and (b) is a graph showing the results of examining the change in the total nitrogen amount in the culture medium in the process.

Claims (13)

Using at least one microorganism selected from the following (a) ~ (c), NH 4 + and NO ₃ in the object ^- and at the same time nitrification-denitrification method for removing.
(A) NH ₄ ⁺ and NO ₃ ^- at the same time strain belonging to the genus Bacillus can be removed (b) NH ₄ ⁺ and NO ₃ ^- strain belonging to the genus Pseudomonas capable of simultaneously removing (c) NH ₄ ⁺ and NO ₃ ^- at the same time Strains belonging to the genus Enterobacter that can be removed 2. The nitrification / denitrification method according to claim 1, wherein at least one microorganism selected from the following (a1) to (a3) is used as the strain (a).
(A1) Bacillus licheniformis T-7-2 strain (FERM P-19418)
(A2) Bacillus subtilis IFO 13719 strain (a3) Bacillus subtilis IFO 3022 strain The nitrification / denitrification method according to claim 1, wherein at least one microorganism selected from the following (b1) to (b6) is used as the strain (b).
(B1) Pseudomonas aeruginosa IFO 12689 (b2) Pseudomonas aeruginosa IFO 13738 (b3) Pseudomonas aeruginosa IFO 3080 (b4) Pseudomonas aeruginosa IFO 3080 (b4) 13130 strain (b5) Pseudomonas fluorescens IFO 15833 strain (b6) Pseudomonas denitrificans IFO 13301 strain The nitrification / denitrification method according to claim 1, wherein at least one microorganism selected from the following (c1) to (c2) is used as the strain (c).
(C1) Enterobacter aerogenes IFO 13534 strain (c2) Enterobacter cloacae IFO 13535 strain   2. The nitrification / denitrification method according to claim 1, wherein a heat-resistant strain is used.   It is used for nitrogen removal in water in wastewater treatment, etc., nitrogen removal in factory wastewater treatment and other inorganic substances, or nitrogen removal in manure and other organic substances, according to any one of claims 1 to 5. Nitrification / denitrification method. A nitrification / denitrification agent containing at least one microorganism selected from the following (a) to ( c ) and simultaneously removing NH ₄ ⁺ and NO ₃ ⁻ in an object.
(A) NH ₄ ⁺ and NO ₃ ^- at the same time strain belonging to the genus Bacillus can be removed (b) NH ₄ ⁺ and NO ₃ ^- strain belonging to the genus Pseudomonas capable of simultaneously removing (c) NH ₄ ⁺ and NO ₃ ^- at the same time Strains belonging to the genus Enterobacter that can be removed The nitrification / denitrification treatment agent according to claim 7, wherein the strain (a) contains at least one kind of microorganism selected from the following (a1) to (a3).
(A1) Bacillus licheniformis T-7-2 strain (FERM P-19418)
(A2) Bacillus subtilis IFO 13719 strain (a3) Bacillus subtilis IFO 3022 strain The nitrification / denitrification treatment agent according to claim 7, wherein the strain (b) contains at least one microorganism selected from the following (b1) to (b6).
(B1) Pseudomonas aeruginosa IFO 12689 (b2) Pseudomonas aeruginosa IFO 13738 (b3) Pseudomonas aeruginosa IFO 3080 (b4) Pseudomonas aeruginosa IFO 3080 (b4) 13130 strain (b5) Pseudomonas fluorescens IFO 15833 strain (b6) Pseudomonas denitrificans IFO 13301 strain The nitrification / denitrification treatment agent according to claim 7, wherein the strain (c) contains at least one microorganism selected from the following (c1) to (c2).
(C1) Enterobacter aerogenes IFO 13534 strain (c2) Enterobacter cloacae IFO 13535 strain   The nitrification / denitrification treatment agent according to claim 7, comprising a heat-resistant strain.   It is used for nitrogen removal in water in wastewater treatment, etc., wastewater treatment in factories and other inorganic substances, or removal of nitrogen in manure and other organic substances, according to any one of claims 7-11. Nitrification / denitrification treatment agent. Bacillus licheniformis T-7-2 strain (FERM P-19418).

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