KR20150117594A - Algicidal bacteria for the prevention of algal bloom - Google Patents

Abstract

The present invention relates to algicidal bacteria among aerobic bacteria strains and random bacteria strains which can control algae exiting under water such as rivers. Particularly, the present invention relates to nature-friendly algicidal bacteria, capable of reducing and controlling generation of green algae, which can effectively control green algae, generated under water, before and after generation of the green algae. In order to innovatively control Microsystis sp. (particularity, Microsystis aeruginosa), which is a dominant species of blue-green algae, which generate green algal bloom, four strains Rhizobium sp, Aeromonas medis, Aeromonas caviae, and Stenotrophmonas maltophilia and six strains of Enterobacter sp., Arthrobacter sp., Comamonas sp., Enterobacter sp., Stenotrophomonas maltophilia, and Microbacterium sp. for effectively controlling Anabana Flos-aquae are selected to form a microorganism group. The strains are independently used or mixed appropriately for the purpose, are attached to the inside of a microspore (a piece of wood for example) which can provide a habitat or are sprayed to control green algal bloom. The strains can inhibit the growth of bacteria causing green algal bloom or can destroy the bacteria causing green algal bloom.

Description

{Algicidal bacteria for the prevention of algal bloom}

More particularly, the present invention relates to an algicidal bacterium or algicidal bacteria which controls algae present in water such as aerobic strains and arbitrary strains of rivers. It is possible to exclude environmental and secondary adverse effects by using microorganisms found in a natural environment rather than an artificial material, and it is possible to reduce and control the occurrence of green algae that can effectively control the green algae generated in the water as well as afterwards It is about nature-friendly killing bacteria.

When polluted wastewater exceeding the natural purification capacity flows into a river or a lake, nutrients accumulate in the water and the floor, and the water system becomes eutrophic, thereby causing an algal bloom or a water bloom. Thus, the sunlight blocking of the surface of the water, the restriction of oxygen transfer, etc., can reduce the dissolved oxygen in the water and destroy the aquatic ecosystem. In particular, in the artificial lake in Korea, the cause of algal bloom was Microsystis aeruginosa , Anabana Flos - aquae occur frequently, and poisonous substances are generated in this process, causing public health hygiene problems. For example, some algae in birds produce toxic substances that can damage the liver or nervous system. For example, in Pennsylvania, USA in 1979, about 60 people have experienced headache, abdominal pain, vomiting, diarrhea, Symptoms such as eye irritation, rash, and headache are known. In addition, these cyanobacteria directly affected all animals and livestock, including phytoplankton, zooplankton, protozoa and fish.

In addition, when the water treatment is performed, the function of the process may be deteriorated, and water use disorder may occur. For example, it causes problems such as coagulation sedimentation and filtration process, which are key processes in purification, and increases the cost of water purification. In addition, Geosmin (20 ppt), which is produced in the metabolic process of cyanobacteria, causes malodorous water in the tap water and causes an economic loss which lowers the water value of the river and lake (Y.-H. Kang, J.-D. Kim, B.-H. Kim, D.-S.Kong, M.-S.Han (2005), Isolation and characterization of a bio-agent antagonistic todiatom, Stephanodiscushantzschii , Journal of Applied Microbiology, 98 , 10301038, 2005). Above all, the water system stagnated at high temperatures is easy to generate green algae due to the eutrophication, and consequently, the harmonized water, which is covered with algae, will have a negative effect on the aesthetics of the people and will be a combination of culture, arts, tourism, The activation as a space can be made impossible. Also, from the aspect of ecology, there is a problem that the outbreak of algae plays a role of losing the self control ability to improve the water quality of the river itself and to restore the health of the aquatic ecosystem.

Currently, various physical, chemical and biological methods have been developed to control algae (especially cyanobacteria), which are the cause of algae, but they are not effective, and practical techniques that can control green algae efficiently and effectively There is little known, and in particular, there is no known technique to control the green algae generated in the water by using algae bacteria or algicidal bacteria.

BACKGROUND OF THE INVENTION [0002] There is a patent application No. 0356184 entitled " Biocidal composition comprising a mixture of halopropynil compound and sulfur-containing triazine ".

In the background mentioned above, a fungicidal or fungicidal composition comprising a mixture of a halopropynyl compound and sulfur-containing s-triazine is proposed.

However, the above background art is mainly a specific chemistry using a chemical sterilizing function. In short, these chemicals have an antimicrobial function so that a plurality of microorganisms are temporarily killed. However, these chemicals are artificially developed to have antimicrobial function and are usually not present in the natural environment. Therefore, when the antimicrobial function disappears when it is artificially discharged into the natural environment, it becomes a new environmental pollutant, There is a problem in that it adversely affects.

Patent Registration No. 0356184 "Biocidal composition comprising a mixture of a halopropynyl compound and a sulfur-containing triazine"

Disclosure of Invention Technical Problem [8] Accordingly, the present invention has been made to solve the above problems, and it is an object of the present invention to provide a method and apparatus for discovery and separation of an optimal microorganism for decomposing a green tide in a natural environment, The object of the present invention is to provide a natural-friendly killing bacteria which can be effectively controlled even after death by reducing and controlling the occurrence of a greenhouse.

The present invention relates to a method for controlling microsystis sp., Which comprises mixing one or more of Rhizobium sp, Aeromonas medis strain, Aeromonas caviae strain, Stenotrophonas maltophilia strain for controlling Microsystis sp. We want to provide bacteria.

Also, it is preferable to use a mixture of one or more of Enterobacter sp., Arthrobacter sp., Comamonas sp., Enterobacter sp., Stenotrophomonas maltophilia strain and Microbacterium sp. For controlling Anabana Flos-aquae. And to provide a natural-friendly killer bacterium that can be reduced and controlled.

The natural-friendly killing bacteria capable of reducing and controlling the occurrence of the green tide of the present invention has an effect of effectively controlling the green tide generated in the water not only in advance of the control of the green tide in a natural environment, but also in post- Microsystis sp., The dominant species of cyanobacteria known as. (Especially, Microsystis aeruginosa) and Anabana Flos-aquae, so that there is a very useful effect that can very effectively inhibit or kill the growth of the green alga.

In addition, in order to effectively control the green algae, the present invention utilizes microorganisms found in a natural environment, rather than an artificial material, thereby exerting an environmentally-friendly and secondary adverse effect.

BRIEF DESCRIPTION OF THE DRAWINGS The accompanying drawings, which are incorporated in and constitute a part of the specification, illustrate exemplary embodiments of the invention and, together with the description, serve to explain the principles of the invention, Shall not be construed as limiting.
BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a view showing an example of a microbial separation experiment for decomposing cyanobacteria; FIG.
FIG. 2 is a comparison curve of growth at 30 DEG C for strain M1, M2, M3 and M4.
FIG. 3 is a comparison curve of growth at 35 DEG C for strains M1, M2, M3 and M4.
FIG. 4 is a graph showing growth curves of M3 strain by temperature.
FIG. 5 is a graph showing growth curves of M1, M2, M3, and M4 by mixed strain temperature.
FIG. 6 is a graph showing the growth curves of pH-dependent growth times of M3 strain.
FIG. 7 shows a microcystis sp. Chlorophyll-a and the number of individuals in biodegradation over time.

BRIEF DESCRIPTION OF THE DRAWINGS The present invention will be described in detail below with reference to embodiments thereof, but the present invention is not limited thereto.

More particularly, the present invention relates to an algicidal bacterium or algicidal bacteria that controls algae present in water such as aerobic strains and arbitrary strains among rivers. The present invention relates to a natural-friendly killing microorganism capable of reducing and controlling the occurrence of green algae, which can effectively control not only pre-control of green algae generated in water, but also post-event.

In particular, in the present invention, microsystis sp. , Aeromonas caviae strain, Stenotrophonas maltophilia strain and Rhizobium sp., Aeromonas medis strain, Stenotrophonas maltophilia strain, and Enterobacter sp., Arthrobacter sp., Comamonas sp., For effective control of Anabana Flos-aquae, ., Enterobacter sp., Stenotrophomonas maltophilia strain, Microbacterium sp. These six selected microorganism groups may be individually or appropriately mixed according to the purpose and fixed in a micropore (for example, a piece of wood) capable of providing a space that can be inhabited to control or spray the green tide, It has its own unique features in inhibiting or killing the growth of.

In the present invention, a suitable microorganism strain is firstly searched for and a microorganism capable of degrading the microorganism causing the algae is firstly searched for, and a suitable microorganism strain is secured from the algae producing area and sewage sludge.

Identification of the microbial strains was accomplished by sequencing the 16S rRNA gene. 16S rRNA was amplified using a pair of 20F (5M) (5'-AGAGTTTGATCCTGGCTC) and 1510R (5M) (5'-ACGGCTACCTT GTTAGA) primers following genomic extraction and corresponding PCR.

* PCR (polymerase chain reaction) mixture included 2 μl dNTP, 2 μl buffer, 0.2 μl of 5, 35 M primer, 10 μl DNA template and 5 μl rTaq. PCR samples were first denatured at 94 for 5 min, annealed at 60 for 30 s, extended at 72 for 90 s, repeated 30 cycles and then extended at 72 for 5 min.

The plasmid vector containing 16S rRNA was used to transform the pGEM-T Easy vector (Promega, Madison, WI, USA) and E. coli cells for nucleotide sequence analysis of selected cells. 16S rRNA was sequenced using an automated sequencer (Model 4200; DNA Analyzer Gene READIR, Li-COR, Inc., USA). Using 16S rRNA sequence analysis, we compared the results from the database using the National Center for Biotechnology Information (NCBI, USA) Blast search program for comparison with the nearest fungi in the nucleotide sequence.

After identification of the microorganism strains as described above, the obtained microorganism strains were selected from aerobic bacteria and arbitrary strains decomposing the green algae. Among them, Microsystis sp . (Particularly, Microsystis aeruginosa ) and Anabana A strain capable of effectively removing Flos - aquae was selected.

The selected microorganism strains were cultured, the strains were selected for the purpose of use, and then experiments were carried out after inoculation on various carriers in order to provide an appropriate habitat environment of the selected microorganism strains.

For example, a microorganism strain appropriately selected from materials such as porous wood chips (a kind of wood pieces and a plurality of fine pores) capable of providing a plurality of microorganism habitats is inoculated, Was investigated. Inoculated microbial strains were investigated by quantitative observation of microbial strain inoculation and fixed environment using Field Emission Scanning Electron Microscopy (FE-SEM), S-4800 model, Hitachi.

In addition, the morphological characteristics of the selected microorganism strains were examined with an optical microscope (Olympus CX40, Japan) to observe the morphology and growth state of the strains.

The following is a description of the cultivation of microorganisms and the degradation of microorganisms, which are capable of reducing and controlling the generation of green algae of the present invention through the preferred experimental examples.

1. Cyanobacterium culture

In the present invention, Microsystis sp. (KMMCC-1520) and Anabana Flos-aquae (KCCM-11922), which are known as the public algae, were purchased from the Korea Marine Microalgae Bank (KMCC) .

Cyanobacteria were obtained by mixing 4.0 g of Jaworski`s Medium (Stocks per 200 ml of Ca (NO3) 2.4H2O, 2.48 g of KH2PO4, 10.0 g of MgSO4.7H2O, 3.18 g of NaHCO3, 0.45 g of EDTAFeNa, 0.45 g of EDTANa2, 0.496 g of H3BO3, 0.28 g of (NH4) 6Mo7O24. 4H2O 0.20 g, Cyanocobalamin 0.008 g, Thiamine HCl 0.008 g, Biotin 0.008 g, NaNO3 16.0 g, Na2HPO4 7.2 g / medium per liter to stock solution 1 ml each). Cultivation conditions of cyanobacteria were cultured by shaking at 25, 100 rpm.

2. Isolation of Cyanobacterial Degrading Microorganisms

The source of the strain was activated sludge of sewage treatment plant. Microsystis sp. And Anabana Flos-aquae were cultured in C-medium for 7 days and centrifuged at 8000 rpm for 10 minutes. The composition of C-midium is shown in Table 1 below. 3% cyanobacterial C-medium was inoculated with 0.3 ml (3%) of activated sludge and cultured for 5 days under conditions of 25 to 100 rpm and 12D: 12L. When compared with the control group in which the activated sludge was not inoculated by the naked eye, the bird-specific index was selected as green and transparent, and pure cultured in LB agar (Table 2).

C-Medium composition Component Amount (NH _{4) 2} SO ₄ 5.0g KH ₂ PO ₄ 1.0 g K ₂ HPO ₄ 2.0 g O MgSO ₄ 7H ₂ O 0.2 g NaCl 2.0 g CaCl ₂ 쨌 2H ₂ O 10 mg FeSO ₄ O 7H ₂ O 10 mg Trace element solution 2.0ml Distilled water 1000ml pH 7.0

LB medium composition Component Amount Trypton 10.0 g yeast extract 5.0g NaCl 10.0 g Agar 15.0 g Distilled water 1000ml pH 7.0

3. Identification of Cyanobacterial Microorganisms

Gram strains were isolated from Gram - negative bacteria and Gram - positive bacteria by optical microscope.

The isolates were identified by polymerase chain reaction (PCR) amplification through 16s rRNA sequncing analysis with SolGent. Based on the results of bi - directional sequencing of primers 27F and 1492R, the names of the strains were determined by comparative searching through the NCBI database of gene banks.

4. Growth characteristics of cyanobacterial microorganisms

1) Optimal initial pH survey

To determine the optimal initial pH, 100 ml of LB medium was placed in a 300 ml Erlenmeyer flask, and the culture was inoculated at 1% concentration in the LB medium. The pH range was adjusted with 10N NaOH and HCl at pH 6, pH 7, pH 8, pH 9 and pH 10, and cultured at 30 ° C and 120 rpm under shaking conditions. For the growth of other cells at different times, 1 ml of the culture was taken every 2 hours and optical density was measured at 600 nm using a spectrophotometer (UV / vis spectrophotometer, Shimadzu, Japan).

2) Optimal temperature survey

In order to investigate the optimum culture temperature of the strain, 100 ml of LB medium was placed in a 300 ml Erlenmeyer flask, and the strain pre-cultured in LB medium was inoculated at 1% concentration and cultured at each temperature condition. Temperature conditions were adjusted to 25 ° C, 30 ° C, 35 ° C, 40 ° C and 45 ° C. For the growth of different microorganisms during the incubation time, 1 ml of the culture solution was sampled every 2 hours and the optical density was measured at 600 nm using a spectrophotometer.

3) Optimum substrate concentration investigation

In order to investigate the optimum substrate concentration of the strain, 100 ml of C-medium, a low-nutrient salt, was added to a 300 ml Erlenmeyer flask, and the strain cultured in LB medium was inoculated with 1% Substrate conditions were adjusted to 0%, 1%, 5%, 10% and 15%. For the growth of different microorganisms during the incubation time, 1 ml of the culture solution was sampled every 2 hours and the optical density was measured at 600 nm using a spectrophotometer.

5. On the house  Cyanobacterial decomposition experiment by

3L of JM medium was added to each of four 5-liter beakers at a concentration of 15% each, and cultured for 7 days at 25 ° C in 12D: 12L. Then, a strain having a cyanobacterial degrading microorganism 1.8 OD (600 nm Optical density) And fixed to 1 kg of the porous ceramic microorganism carrier.

The chlorophyll-a of cyanobacteria, which is a typical indicator of green algae, was measured by using a hemocytometer at intervals of 1 day to measure the change in the number of cyanobacteria, and the amount of chlorophyll was measured by using an acetone solution The amount of chlorophyll-a was determined by measuring the absorbance of the extract at 663, 645, 630 and 750 nm.

1) Isolation and Culture of Cyanobacterial Degrading Microorganisms

Microsystis sp. And Anabaena Flos-aquae, which are cyanobacteria in C-medium, were separated using activated sludge at 25 ℃, 100 rpm, 12D: 12L with 3% Microsystis sp. Obtained by centrifugation of the isolated strain in C-medium was used as a substrate. The result of culturing in a test tube with the addition of substrate (3%) is shown in Fig.

In Fig. 1, C was added with only 3% Microsystis sp. As a control group without adding a strain. In FIG. 1, test tubes 1, 2 and 3 show the results obtained by adding 3% of substrate Microsystis sp., And then inoculating 1% of the effluent of activated sludge. In order to isolate microorganisms degrading cyanobacteria, 3% of Microsystis sp. Was added and 1% of activated sludge was inoculated. When the cyanobacteria were compared with C, which was not inoculated with activated sludge, Were selected. The isolated strains were named M1, M2, M3, and M4, and the 16S rRNA sequence of the strain was decoded by commissioning the digestion strain with Solgent. Table 3 shows the results of sequencing and NCBI BLAST search program. Table 3 shows the results of discrimination between Gram-negative bacteria and then positive bacteria by microscopic observation of this strain. Table 4 shows the morphological results of the microorganisms degrading Anabaena Flos-aquae through the same experiment.

Microorganisms that degrade Microcystis sp Carbon source Isolated
strain The closest relative based on 16S rDNA Max
ident Liquid culture Solid culture Microcystis sp M1 Rhizobium sp 99%

M2 Aeromonas medis strain 99%

M3 Aeromonas caviae strain 99%

M4 Stenotrophmonas maltophilia strain 99%

Microbes that break down Anabaena Flos-aquae Carbon source Isolate strain The closest relative based on 16S rDNA Max
ident Liquid culture Solid couture Anabaena
Flos - aquae A1 Enterobacter sp 87%

A2 Arthrobacter sp 99%

A3 Comamonas sp 99%

A4 Enterobacter sp 99%

A5 Stenotrophomonas maltophilia strain 99%

A6 Microbacterium sp 99%

2) Microcystis sp.

In order to investigate the optimal culture temperature of strain, 100% of LB medium was inoculated at 1% concentration, and optical density was measured at 600nm using UV (sizuma, Japan). The strains M1, M2, M3 and M4 were cultured under the conditions of 30 ℃ and 35 ℃ for the selection of the mycelial cells. The strains M3, M1, M2, M3 and M4, 30 ° C, 35 ° C, 40 ° C and 45 ° C (FIGS. 2 to 5).

3) Microcystis sp.

To determine the optimal initial pH, 100% of LB medium was inoculated at a concentration of 1% and the growth was observed at each initial pH. The pH range was adjusted with 10N NaOH and HCl at pH 6, pH 7, pH 8, pH 9 and pH 10, and cultured at 30 ° C and 120 rpm under shaking conditions. Optical density was measured at 600 nm using UV (sizuma, Japan) for different cell growth at the time of culture (Fig. 6).

4) Microscopy of Microcystis sp.

In order to observe the biodegradation of Microcystis sp, it was observed with an optical microscope (olimpus, japan) at magnification of 1000 times. Microcystis sp was degraded by each M1, M2, M3, and M4 microorganisms, confirming that the cells were detached (Table 5).

Microcystis sp Biodegradation Microscopy Observation Strain Optical microscope (x 1000) A Optical microscope (x000) B Microcystis sp

M1

M2

M3

M4

6. Killing bacteria  Microbial decomposition experiment to decompose cyanobacteria

A strain of cyanobacterial degrading microorganism 1.8 O.D. (600 nm Optical density) in the logarithmic growth phase was fixed to 1 kg of the carrier described above.

Microsystis sp was inoculated 15% (6.5x106 cells / mL) in JM medium 3L. In order to measure the variation of the number of microsystis sp. With time, the result of measurement using a hemocytometer for 15 days at 25 ° C 12D: 12L is shown in FIG. 7a. And increased to 47.70 × 06 cells / mL after 5 days from the initial 6.5 × 06 cells / mL. Chloropyr-a was measured by increasing the amount of microsystis sp. According to the increase of microsystis sp., And the microsystis sp. As a control group, only 15% was taken as in FIG. 7B. 85 to 90% decomposition. Fig. 7b shows the result of algae digestion after 15 days of immobilization of the carrier organism immobilized with the strain M3 microorganism having excellent algal degradation.

As described above, the natural-friendly killing bacteria capable of reducing and controlling the occurrence of the green algae of the present invention have an effect of effectively controlling the green algae generated in the water not only in advance of the control of the green algae in a natural environment, but also in post- Microsystis , the dominant species of cyanobacteria, sp . (Particularly, Microsystis aeruginosa , and Anabana Flos - aquae , which can effectively inhibit or kill the growth of green algae. In order to effectively control the algae, microorganisms found in natural environments, rather than artificial substances, are used to make environmentally friendly and secondary It also has the effect of eliminating adverse effects.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is evident that many alternatives, modifications and variations will be apparent to those skilled in the art in light of the above teachings. will be. The invention is not limited by these variations and modifications, but is limited only by the claims appended hereto.

The Center for Agricultural Genetic Resources (KACC) KACC91864P 21130909 The Center for Agricultural Genetic Resources (KACC) KACC91852P 21130909 The Center for Agricultural Genetic Resources (KACC) KACC91853P 21130909 The Center for Agricultural Genetic Resources (KACC) KACC91855P 21130909 The Center for Agricultural Genetic Resources (KACC) KACC91856P 21130909 The Center for Agricultural Genetic Resources (KACC) KACC91857P 21130909 The Center for Agricultural Genetic Resources (KACC) KACC91858P 21130909

Claims (2)

Wherein the microorganism is a mixture of one or more of Rhizobium sp., Aeromonas medis strain, and Aeromonas caviae strain for controlling Microsystis sp. Anabana Flos-aquae, Arthrobacter sp., Comamonas sp., Enterobacter sp., And Microbacterium sp. For controlling anabana Flos-aquae. Friendly killing bacteria.

Images