JP4352146B1 - Highly functional nitrifying bacteria and method for purifying urea or ammonia using the bacteria - Google Patents

Abstract

[Problem] To provide a novel bacterium having nitrification ability, a growth rate faster than that of a chemotrophic autotrophic bacterium, and excellent storage and transport properties.
A novel bacterium belonging to Brevibacillus sp. And having spore-forming ability and nitrification ability.
[Selection figure] None

Description

  The present invention relates to a novel bacterium having a nitrifying action, a composition for nitrification containing the novel bacterium, and a method for purifying urea or ammonia using these.

  Introducing seeding agent containing microorganisms with excellent resolving power such as organic matter as one of the solutions when the microflora in the septic tank is brought up early or when the microbiota in the septic tank collapses due to some factors Can be considered (Patent Documents 1 and 2 and Non-Patent Document 1). The experience of using seeding agents so far has suggested that sludge volume reduction and nitrification / denitrification ability are promoted. However, details of the functions of individual microorganisms that provide the basis for these effects are not known. Not. Although the effectiveness of the seeding agent has been empirically proven, the use of the seeding agent imposes additional costs on wastewater treatment. The amendment of the Septic Tank Law, which was promulgated in May 2005, included an item that shortened the period of water quality inspection that is required for a certain period after the septic tank was installed. This is due to improved septic tank capacity and the start of effective seeding agents. On the other hand, the septic tank is required to exhibit a necessary capability in a shorter period of time, and as a result, there is a demand for supply of a seeding agent that can reliably improve the effect at low cost.

  In general, the nitrification reaction is carried out under the aerobic condition, ammonium ions are converted into nitrite ions by ammonia-oxidizing bacteria represented by bacteria of the genus Nitrosomonas. It is thought that it is converted into nitrate ions by nitrite-oxidizing bacteria represented by (Nitrobacter) genus bacteria (Patent Documents 3 and 4). Bacteria involved in these nitrification reactions are called chemotrophic autotrophic bacteria, which carry out biosynthetic reactions such as energy production and carbon dioxide fixation using only electrons received from ammonium ions and nitrite ions. Compared with such typical heterotrophic bacteria, it is characterized by extremely slow growth.

  As described above, since bacteria originally considered to perform nitrification reaction grow very slowly (Non-Patent Documents 2 and 3), they grow in an aerobic tank of a wastewater treatment facility until stable nitrification reaction is performed. It is thought that it takes time. It is possible to cultivate separately and put it into the start-up of the aerobic tank of the wastewater treatment facility, but it takes time to cultivate and it is desirable to operate in liquid, so time is required for adjustment. In addition, there are factors that are undesirable in terms of cost, such as the storage period being limited to some extent, and water being transported together.

JP 2002-316199 A Patent Publication 2003-334598 JP 2001-246397 A JP 2006-346589 A Improving the environment with microorganisms Is microbial preparation useful? Kazunori Nakamura (2002) p126 Japanese Society of Microbial Ecology Microbiology Ecology 19 The Role of Microorganisms in the Material Circulation Society Press Center (1995) p21 Biological wastewater treatment engineering by Takao Kitao Corona (2003) p159

  Therefore, in view of the above situation, the present invention is a novel bacterium capable of rapidly nitrifying ammonia and urea contained in wastewater, etc., in particular, a heterotrophic bacterium having a high growth rate compared to a chemotrophic autotrophic bacterium. It is an object of the present invention to provide a novel bacterium belonging to the above and having excellent storage and transport properties, and to provide a method for purifying urea or ammonia using them.

  In order to achieve the above object, the present inventors isolate and identify bacteria having the above characteristics using a seeding agent prepared by mixing livestock dung and sewage digested sludge, etc., and carrying out sedimentary primary fermentation. As a result of intensive studies, it was possible to isolate and identify a novel bacterium that was not classified as a known bacterium. The present invention is based on the finding that such novel bacteria belong to the heterotrophic bacterium Brevibacillus sp. And have spore-forming ability and nitrification ability.

  The novel bacterium according to the present invention belongs to Brevibacillus sp. And has spore-forming ability and nitrification ability. Among the novel bacteria isolated and identified by the present inventors as nitrifying bacteria having such characteristics, the Brevibacillus sp. DA2 strain has the mycological properties described in Table 1 below. .

  Regarding Brevibacillus sp. DA2 strain, DNA is extracted, 16S rRNA gene is amplified by PCR, the base sequence of the obtained gene is analyzed with an auto sequencer, and similar base sequences are examined by blast search. It was. Based on the results, multiple alignments with similar base sequences were taken, and based on the obtained results, a phylogenetic tree was created by the neighbor-joining method. Based on the above mycological properties and the phylogenetic tree obtained by phylogenetic analysis based on the 16S rRNA gene sequence (FIG. 1), the Brevibacillus sp. DA2 strain is classified as a genus Brevibacillus. Identified as a new species.

  The Brevibacillus sp. DA2 strain was issued on September 25, 2008 at the National Institute of Advanced Industrial Science and Technology, Patent Biological Depositary Center (1st, 1st East, 1-chome, Tsukuba, Ibaraki, Japan). ) Under the deposit number FERM P-21693.

  The bacterium according to the present invention further includes a mutant strain of the above-mentioned Brevibacillus sp. DA2 strain, and this mutant strain has the same characteristics as the parent strain, that is, spore-forming ability and nitrification ability.

  The present invention also relates to a composition for nitrification containing the novel bacteria according to the present invention. The bacterium contained in the nitrification composition according to the present invention may be in a dry form or a form immobilized on a solid phase carrier.

  The present invention further relates to a method for purifying urea or ammonia, which comprises bringing the novel bacterium or nitrification composition according to the present invention into contact with a purification object containing urea or ammonia.

  ADVANTAGE OF THE INVENTION According to this invention, the novel bacterium which belongs to Brevibacillus sp. (Brevibacillus sp.) And has spore formation ability and nitrification ability, and the composition for nitrification containing this novel bacteria can be provided.

  The novel bacterium according to the present invention is a heterotrophic bacterium having a different growth mechanism from that of the conventionally known autotrophic nitrifying bacterium, so that its growth is quick and stable, and a bacterial preparation is produced. It is possible to significantly shorten the time for Moreover, since the novel bacterium according to the present invention has a spore-forming ability, the cultured microbial cells can be stored and transported as a dry powder or tablet, and the cost for management and transportation is compared with that of a liquid bacterial preparation. Can be significantly reduced.

  The novel bacterium according to the present invention having such characteristics and the nitrification composition containing the same can be widely used for treatment of waste water containing ammonia and urea discharged from various industries.

Hereinafter, the present invention will be described in detail.
The novel bacterium according to the present invention belongs to Brevibacillus sp. And has spore-forming ability and nitrification ability. As used herein, “nitrification ability” refers specifically to the ability to oxidize ammonium ions to nitrite ions.

  The novel bacteria according to the present invention can be isolated and identified from a seeding agent prepared by mixing livestock dung, dehydrated sewage digested sludge, etc., and performing sedimentary primary fermentation.

  As a specific isolation and identification method, a bacterial strain is isolated from the sheeting agent, cultured in a medium using ammonia as a substrate, and a bacterial strain having a high nitrite production activity using nitrite production activity as an indicator. And then, depending on the case, determination of the taxonomic position of the isolated cells, evaluation of spore-forming ability, etc. may be mentioned.

  Nitrite production activity is determined, for example, by detecting nitrite ions in the culture solution. For detection of nitrite, Griess-romijnnitrite reagent which is Griess-romijn (GR) reagent can be used. Specifically, the culture solution is centrifuged, the supernatant is collected in a test tube, a small amount of GR nitrite reagent is added and mixed, and then left to stand to observe the color change. Ions can be detected. Depending on the amount of nitrite ions, the solution turns from clear to light peach and dark red.

  In addition, nitrous acid can be detected by, for example, the naphthylethylenediamine method described in the following examples. This method is a diazo compound produced by adding aromatic amines (such as naphthylethylenediamine) to an azo compound produced by reacting nitrogen nitrite with an aromatic primary amine (such as sulfanilic acid) in an acidic solution. In this method, the concentration of nitrogen nitrite is detected by measuring the red absorbance of the nitrous acid.

  The taxonomic position of the isolated microbial cell may be determined according to a standard method known to those skilled in the art, and it is not necessary to use a special method. For example, it can be performed by phylogenetic analysis based on the sequence information of the 16S RNA gene of the isolated bacterial cell.

  Evaluation of the spore-forming ability of the isolated cells may be performed based on the determined taxonomic position, or the presence or absence of spore formation under a stress environment may be determined by a spore staining method (Wirtz method such as Schaeffer Fulton's modification of Wirtz method SCHAEFFER, A, B., and FULTON, M. 1933. A SIMPLIFIED MRTHOD OF STAINING ENDOSPORS. SCIENCE 77: 194). In addition, examples of the stress environment that can be used in this case include high temperature, exposure to toxic compounds, no-nutrition environment, and low humidity environment. In particular, it is preferable to use a low humidity environment.

  In addition, the novel bacterium according to the present invention may be isolated and identified by evaluating the isolated bacterial cells for various mycological properties described in Table 1 above. Among the new bacteria isolated and identified by the inventors of the present application, the bacteriological properties shown in Table 1 are those named as Brevibacillus sp. DA2 strain and deposited as FERM P-21693 belongs to.

  Therefore, the nitrifying bacteria of the present invention include, for example, the Brevibacillus sp. DA2 strain and mutants thereof having spore-forming ability and nitrifying ability. The mutant strain of the bacterium according to the present invention is derived from the DA2 strain by a mutation treatment known to those skilled in the art.For example, the mutation treatment is not limited thereto, but the parent strain , Irradiation with radiation (eg gamma rays), contact with mutagenic chemicals such as methylnitrosourea, N-methyl-N'-nitro-N-nitrosoguanidine (NTG) treatment, ethyl methanesulfonic acid (EMS ) Treatment. However, the mutant strain of the present invention preferably has the above-mentioned characteristics and has an activity equivalent to or higher than that of the wild type with respect to nitrite production activity.

  The bacterium according to the present invention can be prepared as an effective nitrifying composition in a short period of time because it has a high growth rate, unlike a conventionally known nitrifying bacterium which has been considered to have a low growth rate.

The composition of a liquid medium suitable for culturing bacteria according to the present invention is exemplified below:
Normal bouillon (liquid medium composition)
Casein peptone 10 g
Meat extract 5 g
Sodium chloride 5 g
Distilled water 1 L
(PH 7.0)

  In addition, culture conditions for the bacteria according to the present invention can be appropriately set by those skilled in the art based on the above-mentioned mycological properties. For example, the temperature during culture can be in the range of 16 to 44 ° C., and the pH can be in the range of 5.0 to 8.0.

The composition for nitrification according to the present invention (hereinafter also referred to as the composition of the present invention) is obtained by culturing the bacterium of the present invention, centrifuging the culture, collecting the cells, and adding physiological saline to It can be prepared by suspending so as to obtain a proper concentration. Moreover, the composition of this invention may also contain the additive well-known to those skilled in the art as needed. Examples of such additives include, but are not limited to, cytoprotective agents and pH adjusters. The concentration of the bacterium contained in the nitrification composition of the present invention can be appropriately selected depending on the culture method, but preferably contains about 1 × 10 ⁵ cells per 100 g of the composition. it can.

  The composition of the present invention may be formulated in any form of solid form, semi-solid form or liquid form. When preparing the composition of this invention as a liquid formulation, it can prepare by the following methods, for example. That is, it can be prepared by culturing the bacterium of the present invention, centrifuging the culture, collecting the cells, and suspending them in a liquid such as physiological saline or a medium to obtain an appropriate concentration. .

  The composition for nitrification of the present invention is preferably formulated in a solid form such as a powder preparation, a tablet, or a semi-solid form such as a cream-jelly preparation. Such formulations can significantly reduce the costs associated with management and transport compared to liquid formulations, and because the bacterium of the present invention has the ability to form spores, it does not lose activity even in dry form. is there.

  Powder formulations can be prepared by techniques known to those skilled in the art. For example, the bacterium according to the present invention is cultured, and the culture can be dried by granulation drying, freeze drying, or the like to obtain a powder preparation. For example, the cells may be collected from the culture solution by centrifugation, suspended in physiological saline or a fresh medium, and then dried by granulation drying or freeze drying. Additives such as pH adjusters and protective agents against drying can be added before drying.

  The nitrification composition of the present invention can be produced by immobilizing the bacteria on a solid phase carrier. The solid phase carrier for immobilizing bacteria is not particularly limited as long as it can be used for immobilizing bacteria. For example, alginic acid, activated carbon, diatomaceous earth ceramic porous body, pumice and the like can be used. As the shape of such a solid phase carrier, a spherical shape or a cylindrical shape is suitable, and in the case of a spherical shape, the particle size is preferably about 0.3 to 2.8 mm, more preferably about 0.3 to 1.2 mm. Bacteria immobilization can be performed by a method known to those skilled in the art, such as a carrier binding method (for example, physical adsorption method, inclusion method, etc.).

  Since the bacterium isolated and identified as described above has excellent nitrification ability, a novel wastewater purification method for ammonia or urea can be constructed by using the bacterium or a nitrification composition containing the bacterium. it can. That is, as long as the purification object containing ammonia or urea is purified using the bacteria or nitrification composition, the ammonia or urea purification method according to the present invention (hereinafter referred to as the present invention) is used regardless of the difference in the treatment mode. Also referred to as purification method of the invention.

  For example, the purification target of the purification method of the present invention is not particularly limited as long as it contains urea or ammonia, and examples thereof include waste water containing urea or ammonia, soil, sludge, garbage, livestock manure, and the like. it can.

  For example, the contact between the bacterium or the nitrification composition according to the present invention and the object to be purified is performed by adding the bacterial culture solution itself or wet cells in the culture solution to the object to be purified, or on the immobilized carrier. This can be done by supplying the purification object to the nitrification composition containing the immobilized bacteria. In that case, based on the property of the bacteria of this invention, it is preferable that the temperature of a purification target object is the range of about 16-44 degreeC, and it is preferable that pH is the range of 5.0-8.0. Moreover, there is no restriction | limiting in particular in the amount of bacteria used for this invention, Those skilled in the art can set suitably according to the ammonia amount and urea amount which are contained in a process target object.

  Hereinafter, the present invention will be described more specifically with reference to examples. However, the technical scope of the present invention is not limited to these examples.

[Example 1] Separation of bacterial strain A seeding agent prepared by mixing beef dung, dewatered sewage digested sludge, wood chips, seed compost at a predetermined ratio, drying and sieving after sedimentary primary fermentation, and reducing to 1 mm or less. After three weeks of enrichment culture in three types of sewage, isolate a large number of bacterial strains, culture the isolates in a medium containing ammonia, then use the nitrite production activity as an indicator to identify bacterial strains with high nitrite production activity. separated.

  Specifically, first, a seeding agent was inoculated to the collected sewage sample, and accumulation culture was performed. During this period, nitrous acid was detected. Since nitrous acid was detected after 3 weeks of culture, about 80 bacterial strains were isolated from the main culture after 3 weeks of culture. Nitrous acid was detected by the naphthylethylenediamine method. Equal volume of 1st liquid (8 g of sulfanilic acid dissolved in 1000 ml of 5N acetic acid) and 2nd liquid (5 g of naphthylethylenediamine dissolved in 1000 ml of 5N acetic acid) were added, and 1 ml was added to 5 ml of culture medium. . Microorganisms were separated by GYP medium containing ammonia (1% glucose, 0.3% ammonium chloride, 0.05% potassium dihydrogen phosphate, 0.05% sodium hydrogen phosphate, 1.5% agar) and YP medium (0.3% ammonium chloride, 0.05% phosphorus). Potassium dihydrogen acid, 0.05% sodium hydrogen phosphate, 1.5% agar). The isolates were cultured in GYP medium and YP medium in liquid media not containing agar, and the production of nitrous acid was detected over time, and the DA2 strain was selected as a strain excellent in nitrification ability.

[Example 2] Classification of isolate strains The bacterial strain (DA2 strain) isolated in Example 1 had the mycological properties shown in Table 1 above when analyzed according to a conventional method.

  Identification of the taxonomic group of this DA2 strain was performed as follows. First, the isolated strain (DA2 strain) was cultured on an ordinary agar slant medium at 27 ° C for 1-2 days, and observed with an optical microscope and an electron microscope. It has been found. In addition, the obtained physiological and biochemical property test results are shown in Shida et al. (1996) Proposal for two new genera, Brevibacillus gen. Nov. And Aneurinibacillus gen. Nov. Int. J. Syst. Bacteriol. 46, Consistent with the nature of Brevibacillus bacteria described in 939-946.

Subsequently, the isolated strain was subjected to molecular phylogenetic analysis based on the 16S rRNA gene sequence. DNA is extracted from the cultured DA2 strain by a conventional method, and a 16S rRNA gene fragment is amplified by PCR using a primer specific for the 16S rRNA gene as a template, and then its base sequence is analyzed using an automatic sequencer did. The base sequence of the obtained 16S rRNA gene fragment was subjected to homology search by BLAST search in a sequence database, and similar sequences of bacteria-derived 16S rRNA were examined. An alignment between the hit similar sequence and the 16S rRNA gene sequence of the DA2 strain was made, and a phylogenetic tree was created by the neighborhood joining method, which showed that the bacterium belongs to the genus Brevibacillus (FIG. 1). Among the bacterial strains of the same genus, they showed high homology of 99.1% and 98.9%, respectively, with Brevibacillus choshinensis and Brevibacillus formosus. Furthermore, DNA-DNA homology to the phylogenetic related strains Brevibacillus choshinensis NBRC 15518 ^T and Brevibacillus formosus NBRC 15716 ^T based on 16S rRNA gene sequences When the sex was examined, it was shown that the DA2 strain was not the same species as any of the strains, since the values considered to be the same species at 25% and 6%, respectively, were less than 70%.

  From the above results, the strain (DA2 strain) isolated in Example 1 was identified as belonging to a new strain of Brevibacillus. The present inventors named this isolated strain as the Brevibacillus sp. DA2 strain. Brevibacillus sp. DA2 was issued on September 25, 2008 at the National Institute of Advanced Industrial Science and Technology, Patent Biological Depositary Center (1st, 1st East, 1st Street, Tsukuba City, Ibaraki, Japan). -Deposited as 21693.

[Example 3] Nitrification experiment
Three types of culture media were prepared by adding 0.3% ammonium chloride, ammonium sulfate, and urea to YP medium (peptone, 0.15%; yeast extract, 0.2%, pH 7.5) in an Erlenmeyer flask with 100 ml baffle. Brevibacillus sp. DA2 strain was inoculated and cultured at a shaking speed of 120 rpm at 27 ° C., and the amount of nitrite produced was measured on the 1st, 3rd and 6th days of culture. For the determination of nitrous acid, add 1 ml of sulfanilamide solution to 10 ml of sample, mix and leave for 15 minutes at room temperature, then add 1 ml of naphthylethylenediamine solution, mix and leave for 20 minutes at room temperature, then spectrophotometer By measuring the absorbance value at 540 nm.

  The experimental results are shown in FIG. In FIG. 2, the horizontal axis represents the number of culture days, and the vertical axis represents the amount of nitrous acid purified. Triangles are the results of culturing in a medium supplemented with urea, squares with ammonium sulfate, and diamonds with ammonium chloride. From the first day of culture, the amount of nitrous acid produced by the decomposition of urea was more than twice that of ammonium chloride and ammonium sulfate as substrates. As a result of further culturing until 3 to 6 days later, the amount of nitrous acid produced increased in the order of urea, ammonium sulfate, and ammonium chloride.

It is a figure which shows the systematic position based on 16S rRNA gene of Brevibacillus sp. (Brevibacillus sp.) DA2 strain | stump | stock. It is a figure which shows the time-dependent change of the production amount of nitrous acid when a different ammonia related compound is used as a substrate.

Claims (5)

Brevibacillus sp. DA2 strain (FERM P-21693), or a mutant thereof having spore-forming ability and nitrification ability . A composition for nitrification containing the bacterium according to claim 1 . 3. The nitrifying composition according to claim 2, which is in a solid or semi-solid form. 3. The nitrification composition according to claim 2, wherein the bacterium is in a form immobilized on a solid support. Claim 1, wherein the bacteria or the nitrification composition according to any one of claims 2-4, wherein the contacting the cleaning object containing urea or ammonia purification process of urea or ammonia .

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