JP7010552B2 - Bacteria and water treatment system using them - Google Patents

Description

NPMD NITE P-02355 NPMD NITE P-02356

The present invention relates to a bacterium having N-acylated homoserine lactone (AHL) degrading activity and a water treatment system using the same.

Biofilms are formed by bacteria adhering to and multiplying on the surface of a solid phase of a substance to produce high molecular weight organic substances such as polysaccharides. A biofilm is a membranous structure, also called a biofilm or slime.

Biofilms are known to cause a decrease in heat transfer efficiency and clogging of pipes in a circulating water system such as a cooling water system. In addition, biofilm is known to cause obstacles (slime obstacles) such as productivity deterioration and quality deterioration in the papermaking process, and the flux of RO membranes in the water treatment process using reverse osmosis membranes (RO membranes). It is known to be a factor in the decline of.

Various treatment methods have been devised in order to prevent the above-mentioned problems caused by biofilms in the above-mentioned circulating water system, papermaking process, water treatment process and the like (hereinafter collectively referred to as "water treatment system").

Of these various treatment methods, the most common is a method of preventing the growth of bacteria by using a fungicide or a growth inhibitor. Methods using these bactericides and growth inhibitors have traditionally included chlorine, bromine and its derivatives, ClMIT (5-chloro-2-methyl-4-isothiazolin-3-one and 2-methyl-4-isothiazolin-3-one). Organic antibacterial agents such as on mixture) and DBNPA (2,2-dibromo-3-nitrilopropionamide) and oxidizing bactericides such as ozone and hydrogen peroxide are used.
In addition, dispersion treatment and stripping treatment using a surfactant are also performed, and in these methods, alkylbenzene sulfonic acid, polyoxyethylene alkyl ethers, polyethyleneimine and the like are used.

In recent years, it has become clear that cell-cell communication substances play an important role as a signal for biofilm formation, and technological developments are underway to decompose cell-cell communication substances and suppress biofilm formation. ..

A technology focusing on N-acylated homoserine lactone (AHL), which is a cell-cell communication substance widely used in Gram-negative bacteria, as a technology for decomposing cell-cell communication substances and suppressing the formation of biofilms. Is disclosed (Patent Document 1 etc.).

At the laboratory level, there is a technology that suppresses the formation of biofilm by introducing bacteria having AHL degrading activity (hereinafter, also referred to as "AHL degrading bacteria") into a water treatment system and causing AHL degrading bacteria to decompose AHL. It has been demonstrated (Non-Patent Document 1).

However, the actual water treatment system is an open system, and various microorganisms such as bacteria in the natural world exist in the system. Further, the conventional AHL-degrading bacteria have a problem that they cannot be stably established in an open water treatment system in which these various natural bacteria and the like exist.

Japanese Patent Publication No. 2013-540443

Environmental Science & Technology, Vol. 47, pp. 836-842, 2013

The present invention has been made in view of such circumstances, and the present invention solves the above-mentioned problems of the prior art and is stable even in an open treatment system in which various microorganisms such as bacteria in the natural world are present. It is an object of the present invention to provide an AHL-degrading bacterium that can be colonized and a water treatment system using the AHL-degrading bacterium.

As a result of diligent studies to solve the above problems, the present inventors have found that a novel strain of a bacterium belonging to the genus Sphingopixis isolated by the present inventors can achieve the above object, and completed the present invention. I let you.
The present invention provides the following [1] to [7].
[1] A bacterium belonging to the genus Sphingopicsis, which has chlorine resistance and N-acylated homoserine lactone (AHL) degrading activity.
[2] The bacterium of [1], wherein the 16SrDNA sequence has 97% or more sequence homology with the 16SrDNA sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2.
[3] Bacteria according to [1] or [2], which is a sphingopicsis FD7 strain (accession number: NITE P-02356) or a sphingopixis EG6 strain (accession number: NITE P-02355).
[4] A water treatment system containing any of the bacteria [1] to [3] in a water treatment system.
[5] The water treatment system according to [4], comprising a water treatment material in which the bacterium is immobilized on a carrier.
[6] The water treatment system of [4], comprising a bacterium addition means for adding the bacterium into the water treatment system.
[7] The water treatment method according to any one of [1] to [3], comprising a fungicide addition means for adding a chlorine-based agent as a fungicide into the water treatment system.

According to the present invention, it is possible to provide a bacterium having AHL degrading activity and chlorine resistance.
By introducing the bacterium of the present invention into a water treatment system as an AHL-degrading bacterium and coexisting a chlorine-based agent such as sodium hypochlorite as a bactericidal agent in the water treatment system at a low concentration, the bacterium of the present invention. In an open water treatment system, even in the presence of various indigenous bacteria, it is possible to overcome the competition with these bacteria and establish them, and to maintain the population predominantly. In this way, AHL-degrading bacteria that can continuously maintain a sufficient number of individuals in an open water treatment system are allowed to coexist in the water treatment system to perform water treatment, thereby decomposing AHL and biofilm. The effect of suppressing the formation can be continuously and stably obtained.

It is a figure which shows the result of the phylogenetic analysis of the sphingopicsis FD7 strain. It is a figure which shows the result of the phylogenetic analysis of the sphingopicsis EG6 strain.

The bacterium belonging to the genus Sphingopyxis of the present invention (hereinafter, also referred to as “the bacterium of the present invention”) has AHL degrading activity and chlorine resistance.

The AHL degradation activity can be quantified by a known method using a gene recombinant (AHL synthetic gene disruption strain) of Chromobacterium violaceus called a reporter bacterium.
Specifically, when a paper disk containing AHL is placed on an LB agar medium mixed with reporter bacteria, the reporter bacteria produce a purple pigment (violacein) in response to AHL. Therefore, the intensity of purple is used as an index for AHL. The amount can be semi-quantified.
Such reporter bacterial strains include CV026 strains (Microbiology, Vol. 143, pp. 3703-3711, 1997) that respond to short-chain AHL (C4 to C8: indicating the chain length of the acyl group) and long-chain AHL (C8 to C8 to). A VIR07 strain (FEMS Microbiol. Lett., Vol. 279, pp. 124-130, 2007) that responds to C18) is known and available.
The degradability of AHL can be evaluated by mixing and reacting the culture solution of the AHL-degrading bacterium and the AHL sample, and then performing the above-mentioned reporter assay using the reaction product. Specifically, the degradability can be semi-quantified by comparing and evaluating how much the color tone is lowered by the treatment with the culture solution with respect to the intensity of the purple pigment indicated by the unreacted AHL.

As used herein, having chlorine resistance means having resistance to sodium hypochlorite.
Specifically, bacteria having a survival rate of more than 50% after being contacted with an aqueous solution having a free residual chlorine concentration of 0.3 mg / L · as Cl ₂ at 30 ° C. for 15 minutes are referred to as “chlorine resistant” bacteria. Define. Free residual chlorine means what exists as hypochlorous acid or hypochlorite ion in water.

In addition to the above-mentioned properties, the bacterium of the present invention has 97% or more, preferably 98.7% or more, more preferably 99% or more sequence homology with the 16SrDNA sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2. be.

Furthermore, the bacterium of the present invention has the following biochemical properties.
Isolation source: Cooling water collected from a cooling tower Morphology: Aerobic gram-negative non-spore-forming spore-forming colonies: Yellow colonies formed on R2A agar medium

The bacterium of the present invention having the above-mentioned properties can be obtained, for example, by the following method.

Cooling water is collected from the cooling tower of a factory or building and used as a separation source sample. A cooling water system in which chlorine-based chemicals are continuously injected is desirable. Agar medium obtained by diluting the separation source sample and 10-fold diluted PY medium (1% polypeptone, 1% yeast extract, 0.5% NaCl, pH 7.0) with water, or R2A agar medium (Becton, Dickinson and Company). After smearing, incubate at 30 ° C. for 5 to 10 days. Then, the bacterium of the present invention can be isolated by observing the grown colonies visually or with a stereomicroscope, catching colonies having different morphologies, and repeating aerobic purification culture on the above-mentioned agar medium.

Specifically, examples of the bacterium having the above-mentioned properties include a sphingopicsis FD7 strain and a sphingopicsis EG6 strain isolated by the present inventors by the above-mentioned method. These were deposited on September 23, 2016 at the Patent Organism Depositary Center (1-1-1 Central 6 Higashi, Tsukuba City, Ibaraki Prefecture), National Institute of Advanced Industrial Science and Technology. The acceptance number is as follows.
Sphingolipidis FD7 strain: NITE P-02356
Sphingolipidis EG6 strain: NITE P-02355

Chromosome DNA was prepared from the isolates (FD7 strain of Sphingopixis and EG6 strain of Sphingopixis), and the entire length of the 16S rRNA gene was amplified by PCR using this as a template, and its nucleotide sequence was determined.
SEQ ID NO: 1 in the sequence listing shows the base sequence of 1411 bases of the Sphingopixis FD7 strain, and SEQ ID NO: 2 of the sequence listing shows the base sequence of 1411 bases of the Sphingopixis EG6 strain.

Furthermore, homology search and phylogenetic analysis were performed based on the obtained sequence information. For homology search and phylogenetic analysis, use DDBJ (http://www.ddbj.nig.ac.jp/index-j.html), which is a database on the Internet, and a microorganism identification system manufactured by Technosuruga Lab Co., Ltd. , Apollon and other commercially available databases can be used. In this specification, the sequence homology is based on the value calculated by "Apollon, a microorganism identification system manufactured by Technosuruga Lab."

The results of homology search and phylogenetic analysis of the FD7 strain of the genus Sphingopicsis are shown in Table 1 and FIG. “D7” in FIG. 1 means the FD7 strain of the genus Sphingopicsis.

As shown in Table 1 and FIG. 1, the FD7 strain is most closely related to S. soli, and its homology is 98.8%. From this homology, the FD7 strain is judged to be either the same species as the sphingopicsis sled or a new species of bacterium.

Table 2 and FIG. 2 show the results of homology search and phylogenetic analysis of the sphingopicsis EG6 strain. “G6” in FIG. 2 means the EG6 strain of the genus Sphingopicsis.

As shown in Table 2 and FIG. 2, the EG6 strain is most closely related to S. chilensis and S. bauzanensis, with a homology of 98.4%. be. Since the homology is as low as 98.4%, the EG6 strain is judged to be a novel strain of the genus Sphingopicsis.

The bacterium of the present invention is suitable for use by containing the culture solution in a water treatment system.
According to the water treatment method in which the bacterium of the present invention is contained in the system of the water treatment system, AHL in the water to be treated can be decomposed in the water treatment system and the formation of a biofilm in the water treatment system can be suppressed. ..

The specific form for containing the bacterium of the present invention in the water treatment system is not particularly limited. For example, a water treatment system equipped with a bacterium addition means for adding the bacterium of the present invention into the water treatment system is constructed, and the bacterium of the present invention is intermittently or continuously introduced into the water treatment system by using the bacterium addition means. be able to. The bacterium of the present invention can be supplied in a suspended state in a solution such as a medium or a storage solution, or as a dry bacterium in the form of powder. It is also possible to provide culture equipment around the water treatment system to supply bacteria cultivated on-site. Alternatively, a water treatment system including a water treatment material in which bacteria are fixed to a carrier can be constructed, and the water to be treated in the water treatment system can be passed through the water treatment material.

It is preferable that the water treatment system is provided with a fungicide addition means for adding a fungicide in the water treatment system. As the disinfectant, sodium hypochlorite or a chloramine-based inorganic chlorine-based disinfectant can be used. The addition of the fungicide is preferable even intermittently, but continuous addition is desirable. It is desirable to control the concentration of the fungicide to a concentration at which the bacterium of the present invention can grow and other bacteria cannot grow.
In addition to the inorganic chlorine-based bactericide, an inorganic bromine-based bactericide, an oxidizing agent such as hydrogen peroxide, or an organic sterilizing agent such as DBNPA or Cl-MIT can also be used in combination. It can also be used in combination with water treatment agents such as anticorrosion agents and antiscale agents.
When the water treatment system is an open system, various microorganisms such as natural bacteria exist in the system, but these various indigenous bacteria do not have chlorine resistance. Therefore, if the bacteria of the present invention, various indigenous bacteria, and a chlorine-based bactericide such as sodium chlorite coexist in an open water treatment system, the bacteria of the present invention will compete with various indigenous bacteria. Overcome and settle.
According to such a water treatment system, even when the water treatment system is an open system and various microorganisms such as bacteria in the natural world are present in the system, the bacterium of the present invention is predominantly abundant in the system. In order to maintain the above, the effect of decomposing AHL and suppressing the formation of biofilm can be continuously and stably obtained.

The bacterium of the present invention can be used as it is, but a highly AHL active mutant strain or a highly chlorine-resistant mutant strain obtained by ultraviolet irradiation, treatment with a mutant agent, or a gene recombination operation can also be used.

Hereinafter, the present invention will be described in detail with reference to examples, but the present invention is not limited to these examples.
The following DSM strains were obtained from the German strain preservation organization DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH) as standard strains of the genus Sphingopicsis.
S. witflariensis DSM14551
S. chilensis DSM14889
S. taejonensis DSM15583
S. bauzanensis DSM22271

<Example 1: Evaluation of chlorine resistance>
The obtained DSM strain, Sphingopixis FD7 strain, and Sphingopixis EG6 strain were each grown on 1/10 concentration PY agar medium, and each bacterial cell was suspended in 150 mM phosphate buffer (pH 7.0). It became cloudy.
Sodium hypochlorite was added so that the final concentration was 0.3 mg / L · as Cl ₂ (free chlorine concentration), and the mixture was kept warm at 30 ° C. for 15 minutes.
After dilution, it was applied to 1/10 concentration of PY agar medium and cultured at 30 ° C. for 1 week. After culturing, the number of colonies generated was measured to obtain CFU (colony forming unit), and CFU was used as the viable cell count.
The survival rate was determined by comparison with the target experiment (without addition of hypochlorous acid). Survival rate was calculated as the ratio of chlorine-free control system to CFU.

As shown in Table 3, the FD7 strain and the EG6 strain showed survival rates of 69% and 74%, respectively, and were confirmed to have strong resistance. It was also confirmed that the DSM strain, which is a standard strain of the genus Sphingopicsis, has no resistance to chlorine.

<Example 2: Evaluation of AHL decomposition activity>
The obtained DSM strain, Sphingopixis FD7 strain, and Sphingopixis EG6 strain were precultured in TSB liquid medium (Becton, Dickinson and Company) at a concentration of 1/5, respectively, at 30 ° C. for 16 hours.
The preculture was transferred to a fresh 1/5 concentration TBS liquid medium containing a 20 μM C6-AHL aqueous solution (C6 indicates the chain length of the acyl group) and cultured at 30 ° C. for 3 hours.
The culture was centrifuged at 12000 xg for 5 minutes to obtain a supernatant. 0.03 ml of the supernatant after centrifugation was applied to a paper disk (diameter 8 mm: Advantech), placed on an LB agar medium mixed with Chromobacterium violaceum CV026 strain in advance, and cultured at 30 ° C. for 16 hours. ..
After culturing, the purple color tone intensity around the paper disc was observed.

Further, for each strain, C10-AHL was used instead of the C6-AHL aqueous solution, and VIR07 strain was used instead of the CV026 strain, and the same culture and observation as described above were carried out.

As shown in Table 4, it was observed that the control (control system) in which AHL was placed directly on the paper disc showed a strong purple color.
No purple pigment production was observed in the system in contact with the FD7 strain culture solution and the EG6 strain culture solution, confirming that AHL was completely degraded.
It was confirmed that the purple color tone intensity differs depending on the strain in the system contacted with the DSM strain culture medium, which is a standard strain of the genus Sphingopicsis. Among the DSM strains, S. bauzanensis DSM22271 was confirmed to have AHL degrading activity.

The present invention can be used, for example, to suppress the formation of a biofilm in a circulating water system such as a cooling water system, a paper making process, or a water treatment process using a reverse osmosis membrane (RO membrane).

Claims (6)

It belongs to the genus Sphingopixis, has chlorine resistance, has N-acylated homoserine lactone (AHL) degrading activity, and is in an aqueous solution having a free residual chlorine concentration of 0.3 mg / L · as Cl ₂ at 30 ° C. for 15 minutes. Bacteria with a survival rate of 69% or more after contact
A bacterium that is a sphingopicsis FD7 strain (accession number: NITE P-02356) or a sphingopixis EG6 strain (accession number: NITE P-02355) . The bacterium according to claim 1, which has 97% or more sequence homology with the 16SrDNA sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2. A water treatment system containing the bacterium according to claim 1 or 2 in a water treatment system. The water treatment system according to claim 3 , further comprising a water treatment material in which the bacteria are immobilized on a carrier. The water treatment system according to claim 3 , further comprising a bacterial addition means for adding the bacteria into the water treatment system. The water treatment system according to any one of claims 3 to 5 , further comprising a fungicide addition means for adding a chlorine-based agent which is a fungicide in the water treatment system.

Images