KR100693865B1 - 0210-09 [-11368] Development of the isolate Pseudomonas fluorescens HYK0210-SK09 against the harmful diatom in fresh water growth control method and bio-agent using the isolate - Google Patents

Abstract

A novel Pseudomonas fluorescens HYK0210-SK09 is provided to show excellent diatom growth inhibition effect capable of environment-friendly controlling the growth of the diatom against harmful diatom in fresh water, thereby effectively solving the green tide caused by the diatom in fresh water. The Pseudomonas fluorescens HYK0210-SK09 against harmful diatom such as Stephanodiscus hanzschii UTCC 267 in fresh water is deposited as a deposition number of KFCC-11368P. To control the diatom, the Pseudomonas fluorescens HYK0210-SK09 is applied to water system where the diatom occurs.

Description

Pseudomonas fluorescens HYK0210-SK09 against the harmful diatom in fresh strain Pseudomonas fluorescens HB0210- ス Ｋ09 [ＫFCC-11368P], a method for controlling diatoms using the same, and a biologic for controlling diatoms using the same. water, growth control method and bio-agent using the isolate}

Figure 1 shows the DNA homology of Pseudomonas fluorescens HYK0210-SK09 (KFCC-11368P) and Pseudomonas fluorescens MM-B16.

FIG. 2 is a graph comparing diatom blood cell counts of a treatment group inoculated with Pseudomonas fluorescens HYK0210-SK09 and treated with only diatoms.

FIG. 3 is a photograph confirming that Pseudomonas fluorescens HYK0210-SK09 is attached to the surface of Steppanoscus Hantzsig UTCC 267 through pili after 4 hours of inoculation through SEM (Scanning Electronic Microscope).

Figure 4 is a photograph confirmed by SEM (Scanning Electronic Microscope) that Steppanoscus Hantsuji UTCC 267 was destroyed 12 hours after Pseudomonas fluorescens HYK0210-SK09 inoculation.

5 is a photograph taken with the control (A) incubated with steppanodiscus Hantzji UTCC 267 million three days after Pseudomonas fluorescens HYK0210-SK09 inoculation (B).

FIG. 6 is a photograph of a field in which mesocosm tests have been performed. FIG.

Figure 7 shows a mesocosme installation diagram in the field.

FIG. 8 is a graph comparing phytoplankton populations of treatment group 1 inoculated with Pseudomonas fluorescens HYK0210-SK09 in mesocosm and control group not inoculated with mycobacteria.

FIG. 9 is a graph comparing phytoplankton populations of treatment group 2 treated with inoculation of biologics adsorbed with Pseudomonas fluorescens HYK0210-SK09 on a mesocosm and a control group not inoculated with the new strain.

The present invention relates to a new strain Pseudomonas fluorescens HYK0210-SK09 [KFCC-11368P], which has excellent killing ability of freshwater harmful diatoms, and more particularly to diatoms, especially steppano, which is a cause of freshwater algae phenomenon. Pseudomonas fluorescens HYK0210-SK09 [KFCC-11368P], a new strain Pseudomonas fluorescens HYK0210-SK09 to effectively remove the Discus Hantzuji, and the present invention relates to a diatom control method and biologics for diatom control.

In the eutrophic aquatic ecosystem of temperate regions like Korea, the generation of algae has exploded every year, creating an environment favorable for the growth of primary producers such as phytoplankton based on abundant nutrients. In particular, algal blooms occurring in rivers and lakes in Korea are mainly due to the occurrence of cyanobacteria in summer and diatoms in spring and winter.

The large outbreaks of diatoms in the eutrophication system in winter and spring are mainly small diatoms, the representative species of which are produced in major rivers such as the Han and Nakdong rivers by Stephanodiscus hantzschii . As a result, freshwater ecosystems can cause:

(1) Diatoms are used by other aquatic organisms in the upper part of the food chain, and various organic matters and inorganic salts such as nitrogen and phosphorus are eluted in the process of organic matter generation and decomposition by microorganisms. Deepen.

(2) Off-flavor occurs, causing severe odor.

(3) Prevents settling sand in water purification plant and causes economic loss.

In Korea, diatoms in Stephanodiscus occur largely from the late 1980s for the Han River and from 1991 after the construction of the Nakdong Estuary Dam in the Nakdong River. Since then, major rivers in Korea have been showing up, causing water quality deterioration. Therefore, research on the regulation of eutrophication and the control of diatoms is an urgent task for the smooth use of social health and water resources.

In order to prevent eutrophication and hydration of the aqueous system, physicochemical methods such as those described in Table 1 below are used. However, these methods have limitations in terms of technical and economic aspects. Especially, lakes in Korea are constructed in the middle of rivers, so the storage period is short, there are many pollutant loads from the outside, the basin area is large, and the land is narrow. There are a lot of difficulties.

Green algae control technology Characteristic Algae through chemicals Algae algae using calcium salts, copper sulfate (CuSO ₄ ), and chlorine Precipitation by Algae Adsorbent Adsorption using adsorption principles such as siliceous porous body, loess and copper sulfate Phosphorus removal method by adding flocculant Aluminum sulfate, ferrous chloride, etc. are used to prevent eutrophication. Dissolved Air Pressurization Removal of volatile organic contaminants and trace organic contaminants through pressurization of air filtration Filtration with Granular Activated Carbon and Charcoal Shorten residence time Inhibits algal growth by reducing the residence time of the water system, causing dilution of pollutants Biological manipulation and biological restoration technology Multifunctional complex ecological clean technology using aggressive function micro organisms and natural enemies

Chemicals such as copper sulfate (CuSO ₄ ), chlorine dioxide (ClO ₂ ), ozone (O ₃ ) and simazine are used in part to control hydration in eutrophic waters. . Copper sulfate, which is widely used because it is the least expensive, is not specific to diatoms, so invertebrates such as rotifera and cladocera, green algae, diatoms and flakes By removing phytoplankton such as imitation, it is known to destroy the food chain of aquatic ecosystems and to be toxic to fish. In addition, it has to be used repeatedly because of its temporary effect, and it becomes unstable under the high basic conditions of the environment involved in the hydration of algae, so a large amount of treatment is inevitable, and economic and toxicity to other organisms in the water is inevitable. The problem becomes even worse.

Research on the control of algae growth through biological methods using microorganisms present in the ecosystem is inadequate at home and abroad, but the research on the new strains and the method of controlling algae using them are as follows.

Korean Patent Registration No. 10-0433384 of the present inventors has proposed a bacterium cynorizobium genus KFCC11297 which has excellent degrading activity against algae, particularly anavena silindrika and algae removal method using the same.

In addition, Korean Patent Registration No. 10-0468048 of the present inventors proposed a new strain Pseudomonas putida HY0203-SK02 having excellent killing ability against diatoms, particularly Steppanoscus Hantzuji, and a method for controlling diatom growth using the same.

In addition, the Republic of Korea Patent Registration No. 10-0461620 proposed a bacillus Bacillus subtilis C1 and the growth control method of cyanobacteria using the same to induce the growth and suppression of the growth of cyanobacteria genus cyanobacteria.

However, all of the above-described inventions have only confirmed algae killing ability in the laboratory by separating algae bacteria from algae in freshwater, and there is no example confirming the application of the algae through mesocosm testing.

An object of the present invention is excellent ability saljo of fresh water diatoms hazardous novel strain Pseudomonas Monastir fluorenyl sense HYK0210-SK09 ( Pseudomonas fluorescens HYK0210-SK09) [KFCC-11368P].

Another object of the present invention is to provide a method for controlling diatoms using the strain applicable to the field.

Still another object of the present invention is to provide a bio-agent for diatom control including the strain.

In order to achieve the above object, the present invention provides Pseudomonas fluorescens HYK0210-SK09 [KFCC-11368P] excellent in the killing ability of freshwater harmful diatoms.

In addition, the present invention provides a diatom control method characterized in that the strain is administered to the diatom generating system.

In addition, the present invention provides a diatom control method characterized in that for administering a diatom control bio-agent (bio-agent) comprising the strain to the diatom generating system.

In addition, the present invention provides a diatom control bio-agent (bio-agent) comprising the strain.

The present invention will be described in detail as follows.

In order to find an environmentally friendly method for restoring the ecology of the contaminated water, the present inventors have separated the bacteria having excellent function of inhibiting diatom growth from fresh water and tested their killing ability, which is the most representative in the large-scale breeding of diatoms. Isolates mycobacteria showing strong killing ability against the steppenodiscus Hantzuji UTCC 267, and confirmed that the mycobacterial killing ability was excellent in mesocosm test. The present invention was completed by establishing Pseudomonas fluorescens HYK0210-SK09 useful for controlling, a method for controlling diatom growth using the same, and a biological material for diatom control.

Pseudomonas fluorescens of the present invention HYK0210 - SK09 [KFCC-11368P] The separation process is as follows.

First, for the separation of algae bacteria, 70 species of bacteria were isolated from surface waters in Paldang Lake, where diatom hydration occurred in early spring and winter, using the Stephanodiscus hantzschii lawn method. Tsujii was inoculated in the UTCC 267 culture to determine whether each of the killing effect, and found a strain SK09 showing a strong killing power.

16s rDNA sequencing was performed to confirm the identification of the bacterium showing excellent algal degradation activity. As a result, Pseudomonas fluorescens showed more than 99% concordance with Pseudomonas fluorescens MM-B16 species. HYK0210 - SK09 ( Pseudomonas fluorescens HYK0210 - SK09) was named, and the strain was deposited on March 24, 2006 to the Korea Microorganism Conservation Center was given a deposit number of KFCC-11368P.

In order to confirm the diatom killing ability of the new strain, the result of the inoculation of the new strain against incubation step of the diatom step panodiscus Hantsuji UTCC 267, it was confirmed that the diatoms are completely removed by the new strain according to the present invention.

By administering the new strain of the present invention and a bio-agent including the same to the water system in which diatom hydration has occurred, it is possible to control the growth of diatoms, particularly Steppanoscus Hantzuji UTCC 267, to control the green alga.

As the bio-agent of the present invention, preferably, the new strain is adsorbed onto a biologically acceptable carrier. Here, the carrier may be preferably selected from any one or a mixture of cellulose (Cellulose), CelCaSi, Zeolite, activated carbon, polyvinyl alcohol (Polyvinyl alcohol), the kind thereof It is not specifically limited.

As a result of confirming the diatom control activity in the 2 ton mesocosm of the new strain of the present invention and the biological product adsorbed on the carrier, the phytoplankton cell number decreased by more than 60% in 9 days after inoculation of the new strain, When applied, it showed high algae control ability.

Therefore, when the new strains and biologics of the present invention are directly applied to the water system in which diatom hydration has occurred, it is possible to control the growth of diatoms, particularly Steppanoscus Hantzuji UTCC 267, more effectively than the conventional method, thereby controlling green alga. It is expected to be.

Hereinafter, preferred embodiments of the present invention are described. However, the following examples are only one preferred embodiment of the present invention and the present invention is not limited to the following examples.

(Example)

(Example 1)

(Separation of microorganisms)

10 liters of surface water of the Paldang Lake Guangdong Bridge branch were collected, stored in an ice box and transported to the laboratory within 4 hours, and then inoculated with 0.2 ml of the sample collected on the prepared Stephanodiscus hantzschii lawn, Tsujii ( Stephanodiscus hantzschii ) It was visually observed for 15 days in 15 degreeC of culture conditions, Light: Dark = 12: 12 cycles, and the quantity of light of 50 micromol m <^-2> s- ¹ .

When a clear zone is formed on the lawn, the clear zone is scraped slightly with platinum and inoculated onto a new nutritious agar (15%), which is 29 ° C., dark for normal growth conditions of bacteria. Colonies formed by incubation for 2 days were separated. Stephanodiscus hantzschii isolated from these algae bacteria Blood cells under an optical microscope for 24 hours by inoculating 150 ml of UTCC 267 medium (culture condition: 15 ℃, Light: Dark = 12: 12 cycles, 50 μmol m ^-2 s ^-1 ) at a concentration of 10 ⁶ cells / ml The most active strains were isolated by counting using a haemocytometer. Finally, among the 70 kinds of bacteria isolated from the sample, the strain having the best killing ability was isolated.

(Example 2)

(Identification of microorganisms)

16S rDNA of the isolated strain was isolated through DNA prep to analyze the base sequence. Genomic DNA was extracted from the culture medium of the strain using a DNeasy Plant Mini Kit (Qiagen, Germany). Then, polymerase chain reaction (PCR) was performed using the following primers to obtain 16S rDNA.

The primer used in the analysis was used a universal primer (nucleotide primer), the base sequence and PCR performance conditions are as follows.

primer :

       5'-GAGTTGGATCCTGGCTAG-3 '

       5'-AAGGAGGGGATCCAGCC-3 '

PCR  Reactant:

1 μl of genomic DNA

100 pmol 2 μl

100 pmol 2 μl

2 μl 10 mM dNTP mix

5 μl of 10 × Han-OMNI PCR buffer

0.2µl of Taq DNA polymerase (Genenmed, Korea)

38.8 μl dH ₂ O

50 μl total

PCR  Reaction condition 25 cycles ):

Initial denaturation process 95 ℃, 3 minutes

Denaturation process 94 ℃, 40 seconds

Annealing process 67 ℃, 1 minute

Extension (polymerization) process 72 ℃, 1 minute

Final extension process 72 ℃, 4 minutes

The base sequence of the 16S rDNA obtained through the PCR reaction was analyzed and the results are shown in SEQ ID NO: 1. The base sequence is blast search (blast search) on Genebank of the National Center for Biotechnology Information (NCBI). As a result of identifying the strain through), it showed 99% homology with Pseudomonas fluorescens MM-B16 strain.

Therefore, the isolated strain was identified as mycorrhizal fungus with similar properties to Pseudomonas fluorescein , and this was named Pseudomonas fluorescens HYK0210-SK09 ( Pseudomonas fluorescens HYK0210-SK09). It was deposited on March 24 and was given accession number KFCC-11368P.

(Example 3)

(Checking diatom killing ability of mycobacteria)

Pseudomonas fluorescens in 100 ml of stephanodiscus hantzschii UTCC 267 strain of 10-day logarithmic growth stage

HYK0210-SK09 ( Pseudomonas fluorescens HYK0210-SK09) strains were inoculated to a final concentration of 5 × 10 ⁶ cells / ml and counted using a haemocytometer and an optical microscope compared to the control group not inoculated with SK-09 every 24 hours. 2 is shown.

As shown in FIG. 2, the cell number of the Steppanidiscus Hantsuji UTCC 267 strain rapidly decreased in one day after the inoculation, and the cell number of the cells was reduced by more than 80%.

FIG. 3 is a photograph confirming that Pseudomonas fluorescens HYK0210-SK09 is attached to the surface of Steppanoscus Hantzsig UTCC 267 through pili after 4 hours of inoculation through SEM (Scanning Electronic Microscope).

Figure 4 is a photograph confirming that the diatoms steppanodiscus Hantzuji UTCC 267 due to the killing ability of Pseudomonas fluorescens HYK0210-SK09 12 hours after inoculation through SEM (Scanning Electronic Microscope).

FIG. 5 is a photograph taken with a control group incubated with Steppanoscus Hantzsig UTCC 267 only 3 days after Pseudomonas fluorescens HYK0210-SK09 inoculation. As shown in Figure 3, "A" is a control, "B" shows the appearance of the steppanodiscus Hantzuji UTCC 267 strain treated with the new strain. The control group of the flask A was brown in color, showing that the StepCanodiscus Hantssey UTCC 267 was a large breed, and the flask B was transparent and the StepCanodiscus Hantzsey UTCC 267 was confirmed to be degraded.

In other words, the flask experiment showed that algae can be completely removed by Pseudomonas fluorescens HYK0210-SK09 [KFCC-11368P].

(Example 4)

(Checking algal control activity of new strains and biologics in mesocosms)

Mesocosm, which is a site-like ecosystem, was constructed to investigate the practicality of the new strains in the field of winter algae.

As shown in Fig. 6, from February 14 to 27, in the Nakdong River basin of Bonpo, Gyeongsangnam-do, Changnyeong-gun, Gyeongsangnam-do, Korea, where the harmful green algae Steppanoscus Hantzuji occurs in the winter of more than 10 ⁴ cells / ml, mesocosm experiment of 2 ton scale Was performed.

2 days after the mesocosm was installed as shown in Figure 7, Pseudomonas fluorescens HYK0210-SK09 strain (treatment group 1) and the strain mixed with activated carbon and polyvinyl alcohol (size: 1.5 cm x 1.5 cm x 1.5 cm) Inoculate the biological product (treatment group 2) adsorbed to the final concentration of 5 × 10 ⁶ cells / ml, and compared with the control group that did not inoculate the new strain every 24 hours, compared with the counting chamber (Sedgwick-Rafter Counting Chamber) and Counting was performed using an optical microscope, which is shown in FIGS. 8 and 9.

As shown in FIG. 8, in the field control experiment of Steppanoscus Hantsujii, phytoplankton population rapidly decreased within 9 days of inoculation, and the cell number was reduced by more than 67%.

As a result of experimenting with the biological material adsorbing the new strain to the carrier (activated carbon + polyvinyl alcohol), as shown in Figure 9, it can be confirmed that the phytoplankton population decreases by more than 60% in 9 days after inoculation, high green algae in the field application It showed controllability.

 That is, the field application experiments showed that algae control is possible by Pseudomonas fluorescens HYK0210-SK09 [KFCC-11368P] and biomaterials including the same.

As described above, the new strain Pseudomonas fluorescens HYK0210-SK09 according to the present invention can effectively decompose diatoms, particularly Steppanoscus Hantsujii, and this strain is conventionally used in water systems where eutrophication is caused by diatoms. It is expected to control the growth of diatoms more economically and environmentally than the physical and chemical methods. In addition, since the green algae control ability was confirmed in the mesocosm test, it is expected that the restoration of the ecosystem can be more effective than the conventional method in the direct field application.

Attach sequence list electronic file

Claims (10)

Pseudomonas fluorescens HYK0210-SK09 [KFCC-11368P] with excellent killing ability of freshwater harmful diatoms. According to claim 1, The diatoms, Stephanodiscus Hantzujii UTCC 267 ( Stephanodiscus hanzschii UTCC 267) Pseudomonas fluorescens HYK0210-SK09 [KFCC-11368P]. A method for controlling diatoms, comprising administering Pseudomonas fluorescens HYK0210-SK09 [KFCC-11368P] with excellent killing ability of freshwater harmful diatoms. Diatoms characterized by administering a diatom control bio-agent containing Pseudomonas fluorescens HYK0210-SK09 [KFCC-11368P], which has excellent killing ability of freshwater harmful diatoms, to diatoms generating water system. Control method. The diatom according to claim 3 or 4, Stephanodiscus Hantzujii UTCC 267 ( Stephanodiscus hanzschii UTCC 267). The method of claim 4, wherein the diatoms control bio-agent (bio-agent), Pseudomonas fluorescein sense HYK0210-SK09 (Pseudomonas fluorescens HYK0210-SK09) [KFCC-11368P] was adsorbed on a biologically acceptable carrier. The method of claim 6, wherein the carrier, Diatoms control method, characterized in that selected from the group consisting of cellulose (Cellulose), CellCaSi, Zeolite, activated carbon, polyvinyl alcohol and mixtures thereof. Pseudomonas fluorescens ( Pseudomonas fluorescens) with excellent killing ability of freshwater harmful diatoms Bio-agent for diatom control including HYK0210-SK09) [KFCC-11368P]. In claim 8, Pseudomonas fluorescein sense HYK0210-SK09 (Pseudomonas fluorescens HYK0210-SK09) [KFCC-11368P] A bio-agent for diatom control, characterized by adsorbing a biologically acceptable carrier. The method of claim 9, wherein the carrier, Bio-agent for controlling diatoms, characterized in that selected from the group consisting of cellulose (Cellulose), CellCaSi, zeolite, activated carbon, polyvinyl alcohol and mixtures thereof.

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