KR20090047203A - Method and agents of the algae growth inhibition using nano-silver - Google Patents

Abstract

The present invention relates to a method for inhibiting algae causing algae and an algae inhibitor, and more particularly, to a method for inhibiting algae caused by cyanobacteria in fresh water using silver nano and an algae inhibitor. In the present invention, the algae control effect of silver nano is confirmed through various algae control experiments using silver nano, and based on this, a method of suppressing algae causing algae in fresh water is provided. The algal inhibition method of the present invention can selectively inhibit cyanobacteria (blue-green algae), which is a major cause of green algae.

Silver, nano, colloid, silver nano aqueous solution, algae suppression, green alga, cyanobacteria, fresh water

Description

Method of inhibiting algae using silver nano and algal inhibitors {Method and agents of the algae growth inhibition using nano-silver}

The present invention relates to a method for inhibiting algae causing algae and an algae inhibitor, and more particularly, to a method for inhibiting algae caused by cyanobacteria in fresh water using silver nano and an algae inhibitor.

Green algae phenomenon refers to a phenomenon in which green algae are colored green and blue as microalgae inhabiting fresh water such as rivers and lakes grow in large quantities. This green algae not only causes difficulty in using water resources, but also increases the cost of water purification and deteriorates the water quality, thereby making it difficult to provide high quality clean water in appearance.

The eutrophication of the appeal inevitably leads to algal blooms and hydration. The main source of algae in freshwater is called cyanobacteria (blue-green algae). Cyanobacteria are unicellular or multicellular, with many cells clustering together in the field. The breeding of blue algae has the problem of water flower phenomenon and water purification treatment, especially toxin production, which cause odor and coloration of water surface. Representative species of blue-green algae that cause algal blooms micro during seutiseu (Microcystis), ahnaba INC (Anabaena) Etc. When these cyanobacteria occur largely, they lose huge property damage and economic value, such as the collective death of fish and damage to farms.

In Korea, researches and applied methods to prevent eutrophication and algae control in reservoirs include the use of underwater aeration (e.g. Daecheong Dam), loess spray (e.g. Daecheongho, Paldangho), dredging, flocculant and microbubble. Pressure injuries (eg Daecheongho, Paldangho) to induce and remove sedimentation and flotation, and the use of bird fences (eg Seonakdonggang). In the case of foreign countries (US, Germany, Sweden, etc.), the basic policy of wastewater treatment in the watershed is generally set to the tertiary treatment which almost completely removes nutrients such as nitrogen and phosphorus to prevent eutrophication and algae control in the appeal. Doing. Physical and chemical methods to prevent eutrophication and algae control in the appeal are applied to techniques such as underwater aeration, dredging, stabilization of phosphorus, inactivation of phosphorus, dilution, etc. In addition, removal of deep appeal water, administration of algae, and artificial circulation There are also cases where such techniques have been implemented. However, most of the tidal current control methods listed above are at the level of utilization of individual technology rather than application of complex system technology. As a result, the applied methods lack the effects of eutrophication and algae control in the appeal, and cause secondary pollution, and thus do not have a clear expectation effect in improving water quality or supplying high quality purified water.

In addition, conventionally known algae control method, the use of antibiotics (Korean Patent Publication No. 10-1995-0702094), ultrasonic treatment (Republic of Korea Patent No. 443266, 504452), flocculation (Republic of Korea Patent No. 571519) ) And other complex treatments (Korean Patent No. 756282), but most of them are not satisfactory in terms of effects, and their impact on aquatic ecosystems and water quality is also problematic. Currently, the most commonly used method of removing hydration in Korea is the use of flocculant, which is a method of removing hydration from the surface of the water by flocculant, and cannot be a fundamental method of removing the causative species. It causes secondary pollution problem.

Silver nano technology called 'nano silver' is a particle that is harmless to human body and has strong sterilization function. It is known to have an excellent effect of preventing habitat. In particular, the bactericidal power of silver nanoparticles and silver ions against microorganisms (bacteria) has been studied a lot about pathogenic bacteria, and research on sterilization mechanisms against bacteria has been made steadily. However, the sterilization effect and sterilization mechanism of silver nano on algae have not been studied properly.

In the present invention, to determine the algae control effect of silver nano through a variety of algae control experiments using silver nano, and to provide an effective method of inhibiting algae using silver nano.

In particular, the present invention is to provide a method that can selectively inhibit cyanobacteria, which is the main cause of green algae in fresh water using silver nano.

Other objects and advantages of the present invention will be described below and will be better understood by practice of the present invention.

In the present invention, a method of suppressing algae for suppressing algae causing algae in fresh water using silver nano is provided. The silver nano may be used in the range of preferably 0.001 to 30 ppm, more preferably 0.001 to 10 ppm, particularly preferably 0.005 to 0.1 ppm.

In addition, the present invention provides a method for selectively inhibiting cyanobacteria causing green algae in fresh water by using silver nanoparticles at 0.005 to 0.05 ppm.

The silver nano used in the present invention is preferably added to water in the form of a silver nano colloidal aqueous solution.

In addition, the present invention provides an algae inhibitor containing silver nano as an active ingredient to inhibit algae causing algae in fresh water.

According to the present invention, it is possible to effectively suppress algae that cause green algae in fresh water using silver nano, and in particular, selectively inhibit cyanobacteria, which are the main cause of green algae.

Silver nano

In the present invention, silver nano is preferably added to water in the form of a silver nano colloidal aqueous solution. Colloidal silver nanoparticles are dispersed in an aqueous solution in which silver atoms are clustered and have a diameter of about several nm to about 120 nm. This silver colloidal solution is known to have a strong bactericidal power against various harmful bacteria and fungi. In the present invention, silver nano and silver nano colloidal aqueous solution (hereinafter, "silver nano aqueous solution" and "silver nano colloidal aqueous solution" are used in the same sense). The silver nano aqueous solution may be prepared by an electrolysis method. That is, when power is applied to the silver electrode in the reaction tank into which a certain amount of water is input, electrolysis is performed and a method of allowing silver ions to elute from the electrode is known in the art. In addition, the present inventors have developed another invention, "aqueous silver nano-aqueous solution manufacturing apparatus," which can produce a large amount of silver nano-aqueous solution required to remove algae generated from a reservoir or a lake. The silver nano-aqueous solution used in the present invention can be prepared using a conventional manufacturing method or an apparatus for producing a water-based silver nano-aqueous solution which is another invention of the present inventors.

The antimicrobial activity of silver nano aqueous solution is known to be due to the sterilization action by the deoxidation of SH-radical by silver nano ions and the action of active oxygen produced by the catalytic action of silver nano particles. Among these, the sterilization action of SH-radicals by silver nanoparticles is supplied with power to the silver nano-aqueous solution manufacturing apparatus, and electrolysis is performed in the electrolytic cell in which silver electrode is installed, so that a small amount of silver ions are released from the silver electrode. Silver nano-water solution with strong sterilization and antimicrobial is produced, and if silver ions or silver particles are adsorbed to proteins such as cell membranes of treated cells by treating them with harmful bacteria, bacteria and myriad cells, SH-radicals in protein of cell membranes It can be described as killing bacteria or sustaining high antimicrobial activity by breaking down or disrupting -S- crosslinking in proteins, making it difficult or interrupted by physiological activities such as metabolism and energy metabolism. In addition, sterilization by active oxygen generated by the catalytic action of silver nanoparticles causes silver nanoparticles to react with oxygen in the air to cause a catalytic action. As a result of this catalysis, oxygen on the surface of silver particles is partially active oxygen (active oxygen). ), And the generated free radicals are explained to have strong sterilization and antibacterial effects such as ozone and hydrogen peroxide.

Silver nano  Algal Growth Inhibitory Effect

In the present invention, the algae control effect of the silver nano is confirmed through the following experiment.

1. Selection and Cultivation of Bird Species

In order to control the algae causing hydration in fresh water using silver nano aqueous solution, an experiment was conducted to determine the concentration and treatment time of silver nano aqueous solution. In addition, an experiment was conducted to investigate the effect of silver nano solution on green algae and diatoms. The cyanobacteria were selected from Microcystis, Anabaena and Oscillatoria , green algae from Chlorella and Scenedesmus , and diatoms from Navicula and Thalassiosira .

Cultivation of microalgae was carried out by Allen medium for green algae and green algae, and Csi for Navicula which is freshwater species for diatoms. As a medium, Thalassiosira , a seawater diatom, used f / 2 medium. In addition, in order to examine the dynamics in the field, the water of Daecheongho was used for the experiment using a filtrate from which bacteria were removed using a 0.22 ㎛ membrane filter. The cyanobacteria and green algae selected for the experiment were used for 7 days spawn incubation using Allen medium, and diatoms were used for 21 days spawn in Csi medium and f / 2 medium. In this case, the degree of incubation is in vivo The fluorescence was measured and diluted to the same concentration before the experiment.

2. Survey on the amount of algae and physicochemical water quality

The present results and the physicochemical water quality of blue algae and other microalgae were investigated between August and October 2006 when green algae occurred. In August 2006, the temperature of Chusori site was 28 ~ 34 ℃ for one month. In the case of Chusori, the water temperature remained relatively high due to the shallow water depth. pH was 8-9. In September, the water temperature was lowered to 28-22 ° C, and the pH was kept relatively low at 7.8-8.2.

In order to find out the proper concentration of silver nano solution for the removal of blue algae, the killing rate was calculated by treating silver nano water solution for each concentration. In addition, the experiment was conducted in the same way to determine the resistance of silver nano solution of green algae and diatoms.

3. Effect analysis of concentration change of silver nano solution

In order to find out the resistance concentration of silver nano solution to the selected species, experiments were performed by treating the silver nano solution at high concentration (3.0 to 10.0 ppm) and low concentration (0.01 to 1.0 ppm). Experiments with high concentration silver nano solution showed a change in short time (5 minutes) after treatment in vivo Measured through fluorescence. In vivo As a result of fluorescence experiments, the concentration of silver nano solution in all species was increased in Microcystis and algae Chlorella and Scenedesmus . vivo The decrease in fluorescence tended to increase, but the effect on the silver nano-aqueous solution was difficult to observe clearly. When essential chlorophyll is observed with a fluorescence microscope for the photosynthesis and then in view of the ability to observe the fluorescence of red color processing the silver solution in a high concentration remaining one minute, green algae and diatoms all but one result Oscillatoria observed after 5 minutes Discoloration in the species could be observed. In the case of Oscillatoria , the length of filament was markedly shortened by fluorescence microscopy after treatment with silver nano aqueous solution. This is believed to have shortened the length of some cells of the filament due to the attack of silver ions. Low concentration silver nano solution treatment experiments compared survival rates of selected species over a long period of time (5 to 10 days). In the case of microcystis, the results of chl- a test showed that 0.01 ppm of silver nano solution in culture medium and natural water killed within 2 days. Therefore, it was judged that 0.01 ppm of silver nano-aqueous solution suitable for the control of the culture species Microcystis was the most suitable, and an appropriate concentration was calculated based on the experimental results. Chlorella, a green alga, was resistant to 0.01 ppm of silver nano-aqueous solution, and Scenedesmus was resistant to 0.05 ppm. Eukaryotic green algae are more resistant to silver nano solution than prokaryotic algae.

4. Analysis of Treatment Effect of Silver Nano-Aqueous Solution on Field Sample (Daecheong Lake Microcystis )

Based on the experimental results of the above culture species, the silver nano aqueous solution was evaluated for the samples that had been hydrated in the field. The experiment was carried out to evaluate the silver nano-aqueous solution for the high concentration sample that forms the scum and the low concentration sample collected at 10 cm depth. As a result, high concentration samples (chl- a 1,500 ~ 1,900 ㎍ / ℓ) were measured for 24 hours at intervals of 1 hour and showed 40% mortality when using more than 3.0 ppm of silver nano solution. Therefore, it is considered unreasonable to treat the silver nano aqueous solution at the time of hydration. In the case of low concentration samples (chl- a 110-190 µg / l), 1.0 ppm of silver nano-aqueous solution was killed in 1 day. However, considering that the drinking water standard for silver is 0.1 ppm, applying it directly to the field seems problematic. Compared with the cultured species, the resistance of the silver nano-aqueous solution to the in situ sample is higher than that of the cultured species. Therefore, the field sample, which forms a large colony in the viscous layer, is advantageous to avoid the attack of silver ions, which is considered to be the result of the increased resistance to silver nano-aqueous solution. Nakdong River (waterfowl) is the area where diatoms grow in the winter. Nakdong River samples were collected and tested on November 24 for the evaluation of silver nano-aqueous solution for diatoms. Experimental results showed that diatoms, green algae, and cyanobacteria were diverse in the control group which was not treated with silver nano-aqueous solution, while diatoms and green algae became more diverse in the experimental group treated with 0.01 ppm of silver nano-aqueous solution, but did not increase in the case of southern algae. In case of treatment with silver nano-aqueous solution of 0.05 ppm or more, the species diversity of diatoms did not change significantly, but the species of green algae tended to be simple. Therefore, even in these experimental results, 0.01 ppm of silver nano solution is considered to be good for control of blue algae, and more than 0.05 ppm does not affect species diversity of diatoms, but significant species simplification occurs in green algae. It is deemed desirable to use at.

5. Analysis on the Killing Mechanism of Microalgae by Silver Nano-Aqueous Treatment

In order to observe the killing mechanism of Microcystis using silver nano-aqueous solution, it was observed by scanning electron microscope (SEM) and transmission electron microscope (TEM). As a result of SEM observation, after the silver nano solution was treated, a phenomenon in which a hole was formed on a smooth surface was observed. It is not clear whether the hole formed was due to the attachment of silver ions to the cell membrane or after cell necrosis by interfering with the function of ribosomes or proteins involved in ATP synthesis through the cell membrane. Therefore, the experiment should be performed for a time shorter than 10 minutes. TEM images were taken to determine the location of silver nanoparticles. Black spots, such as silver nanoparticles, were identified in the experimental zone treated with silver nano-aqueous solution. 10 minutes after the silver nano solution treatment, the destruction of the cell membrane was observed. As time went by, the destruction of the cell membranes increased, and after 1 hour, some of the cell membranes were depressed. Despite the hole of the cell membrane observed through SEM and the depression of the cell membrane observed through TEM, the organelles within the cells were relatively stable.

Silver nano  Inhibition of Algae

Based on the above experiments, the present invention provides a method for inhibiting algae using silver nanoparticles. In the present invention, "inhibition" means to kill algae or inhibit the growth and proliferation of algae to prevent the occurrence of green algae, including the increase, reduction, and extinction of the algae already generated. In the present invention, "control" is used in the same sense as the above suppression. In the algae suppression method using silver nano of the present invention, silver nano is preferably used at 0.001 to 30 ppm. If silver nano is used at less than 0.001 ppm, it is difficult to expect an algae inhibitory effect, and using at 30 ppm or more is not preferable in consideration of water effect and economical efficiency. In consideration of the influence on the water, economical efficiency, and the like, silver nano is more preferably used at 0.001 to 10 ppm. In consideration of the effective inhibitory concentration for algae identified through the experiments of the present invention and the silver concentration standard (0.1 ppm) of drinking water, silver nano is preferably used at 0.005 to 0.1 ppm.

In addition, as confirmed through experiments in the present invention, the algal growth inhibitory effect of the silver (Ag) aqueous solution depends on the type and density of microalgae (phytoplankton). In other words, silver nano was more sensitive to cyanobacteria such as algae, microcystis and Anabaena , which cause algal blooms. Therefore, by adjusting the concentration of the silver nano aqueous solution can be selectively controlled the algae, the algae cause algae, the present invention provides a method for selectively inhibiting the southern algae in algae using 0.005 to 0.05 ppm silver nano.

The silver nano used in the present invention is preferably added to water in the form of an aqueous silver nano colloidal solution.

Hereinafter, the present invention will be described in more detail with reference to specific examples. However, the scope of the present invention is not limited by the following examples, and those skilled in the art to which the present invention pertains should be within the equivalent scope of the technical concept of the present invention and the claims to be described below. Of course, various modifications and variations are possible.

For algae control Silver nano  Effect analysis of aqueous solution

1. Selection of microalgae and cultivation

end. Selection of microalgae

Examine the treatment effect due to the typical blue-green algae have a micro when stitcher's (Microcystis), a silver solution prepared by coming la thoria (Oscillatoria) and ahnaba electrolytic apparatus by selecting the (Anabaena) targeting species INC causing hydration in the domestic water system It was. Microcystis is a representative species that causes hydration annually near the inlet and water intake of the Daecheong Lake. Anabaena and Oscillatoria are species that cause algae phenomena near the deep dams in Daecheong Lake. The species dominate each year depending on the water quality and temperature of the Daecheong Lake.

On the other hand, Chlorella and Scenedesmus , the most common green algae in Korea's water system, were selected from freshwater Navicula and seawater Thalassiosira to investigate the effect on silver microalgae when controlling silver algae using silver nano aqueous solution. The resistance to the aqueous silver nano solution was evaluated. The list of southern algae and microalgae used in the experiment is shown in Table 1 below.

I. Microalgae Culture

(1) Culture medium

The cyanobacteria and green algae were cultured using Allen medium, and Navicula used Csi medium and Thalassiosira f / 2 medium. The components of each medium are shown in Tables 2-4. Tables 5 to 7 show the results of analyzing ^* Micro-element, ^* PIV metals of Table 3, and ^* f / 2 metals of Table 4, respectively, of the components of Table 2. In addition, the water of Daecheongho was used for the experiment using a filtrate from which bacteria were removed using a 0.22 μm membrane filter.

(2) Culture conditions

The cyanobacteria and green algae selected for the experiment were used for 7 days spawn incubation using Allen medium, and diatoms were used for 21 days spawn in Csi medium and f / 2 medium. At this time, the degree of incubation was used to measure in vivo fluorescence and diluted to the same concentration before the experiment.

Field samples were collected twice on August 25 and September 3, 2006 when the sign language took place in Chusori (Daecheongho Water), Okcheon-gun, Chungbuk. In order to obtain a clean sample, the sample was taken in a boat where it was severely hydrated. In order to collect scum which formed a thick layer by hydration, only a surface layer was collected using a beaker, and a van don collector was used to collect a 10 cm deep sample from the water layer.

The final concentration of the silver nano-aqueous solution used in the experiment was about 30 ppm. Therefore, high concentration experiments in this experiment was used culture tube (13 × 100 ㎜, Corning). The culture tube sample was cultured in the culture tube at an appropriate concentration and inoculated with 2 ml, and the experiment was performed by adding a predetermined amount so that the concentration of the silver nano-aqueous solution became 3.0-10.0 ppm. Blue-green algae and to investigate the viability of the micro-algae After processing the silver solution for 5 minutes in 1-minute intervals In vivo fluorescence was measured. In the low concentration test, 50 ml of the seed cultured using a 125 ml Erlenmeyer flask was added, and the same amount was added by diluting appropriately so that the concentration of the silver nano solution was 0.01-1.0 ppm.

All. Analysis method

(1) growth curve

One) In vivo fluorescence

Since blue algae and microalgae contain chl- a , they fluoresce when exposed to UV light. A fluorometer (Turner Design) is used to read and display the fluorescence level. 2 ml of the culture solution was taken and placed in the cell for fluorescence measurement, the gain was fixed to 10, and the readings were read. The resulting value was calculated by the formula chl- a (µg / l) = FD × R × V / V. FD is the coefficient of door, R is the value measured with gain of 10, v is the amount of chloroform used (ml), and V is the amount of sample filtered on GF / C filter (ℓ).

2) Chl- a measurement

Measurement method of Chl- a : A solvent made of methanol and chloroform in a 1: 2 ratio is used. The sample is filtered using a GF / C filter and 1 ml is collected to collect cyanobacteria on the GF / C filter. Place the GF / C filter in a 15 ml corning tube. 6 ml of solvent is added to the corning tube containing the GF / C filter and the vortexer is used to completely immerse the GF / C filter. Chl- a is decomposed by sunlight, so wrap the Corning tube in foil and leave for 5 hours in a cold room at 4 ℃ to allow the chl- a to dissolve in the solvent. After 5 hours, 5.4 ml of water was added and mixed using a vortexer to stop the reaction and allowed to stand for 16 hours in a low temperature room. The liquid in the corning tube is divided into layers containing chloroform, water and methanol. Take chloroform in the lower layer carefully using pasteur pipette and measure fluorescence using fluorometer. At this time, the gain value was fixed at 10, and the experiment was performed. The sample with high value was measured after diluting with chloroform.

Silver nano  Control effect of microalgae in aqueous solution (cultured species)

One. Silver nano  Control effect by concentration of aqueous solution

In order to find out the proper concentration of silver nano solution for the removal of blue algae, the killing rate was calculated by treating silver nano water solution for each concentration. In addition, the experiment was conducted in the same way to determine the resistance of silver nano solution of green algae and diatoms.

end. High concentration (3.0-10.0 ppm) short term treatment

Since the silver nano aqueous solution was made while passing through the electrolysis device, the silver nano aqueous solution was used at a high concentration for the purpose of removing hydration generated in the field.

(1) mortality measurement

For the case of blue-green algae Microcystis to exist as single cells in Uniform in vivo fluorescence measurements were obtained, but because the filamentous Anabaena and Oscillatoria were grown in agglomerates, an error occurred according to the experiment, and only the change over time was observed through microscopic observation. For the green alga Chlorella and Scenedesmus with time in The increase in the value of in vivo fluorescence was observed, which is thought to increase as the chloroplasts in the cytoplasm were released out of the cell membrane.

The treatment time was determined to have a significant effect on the cells when the treatment at a high concentration was limited to 5 minutes and the measurement time was also performed at 1 minute intervals. Experimental method is 2 ml of the culture solution in a Pyrex tube and the silver nano aqueous solution was treated at concentrations of 3.0, 5.0, 10.0 ppm and in In vivo fluorescence was measured.

Treatment of high concentration silver nano solution (3.0, 5.0, 10.0 ppm) resulted in early in case of Microcystis (A). The in vivo fluorescence values started at 45-49. As a result of treatment with 3.0 ppm of silver nano aqueous solution, it increased to 2 minutes and became 51, after which the value was maintained (FIG. 1). However, when treated at 10 ppm, it decreased after 1 minute in In vivo fluorescence showed a 20% decrease.

On the other hand, the experiments on chlorella (B) showed a constant decrease with concentration and time (Fig. 2). Especially at high concentrations (10.0 ppm), the decreasing trend was initially noticeable compared to other concentrations.

For Scenedesmus (C), the initial 1 minute in all concentrations The value of in vivo fluorescence increased and then decreased (FIG. 3). Like chlorella, the decrease was slightly larger than that of other concentrations at 10.0 ppm. The experimental results were not obtained a clear result of the concentration with time, as the concentration of the silver solution increased in The magnitude of the decrease in vivo fluorescence value could be observed.

(2) microscopic observation

After 3 g / l of silver nano-aqueous solution, blue algae and diatoms were changed for 1 min after optical and epi-fluorescence conditions. Chlorophyll appears red under UV.

Blue-green algae (cyanobacteria): Figure 4 is silver solution (3 ppm, 1 min) after treatment Microcystis The change of sp was observed. A and B are optical micrographs and C and D fluorescence micrographs. In the case of microcystis, some bacteria were killed one minute after 3 ppm of silver nano-aqueous solution, and only the transparent form was observed under epi-fluorescence conditions. In case of cyanobacterial Microcystis , the result of 1 minute treatment at 3 ppm was not observed in the control group which was not treated with silver nano-aqueous solution. After treatment with silver nano-silver solution, some of the microcystis lost their chlorophyll and observed under epi-fluorescence microscope. Although it is indistinguishable by an optical microscope, chlorophyll appears red under UV by using a fluorescence microscope. In the case of cyanobacteria attacked by silver ions of silver nano solution, the cell membrane was destroyed and the cytoplasm leaked out of the cell and appeared white under the fluorescence microscope.

Figure 5 Anabaena after treatment with silver nano aqueous solution (3 ppm, 1 min) The changes of sp. were observed. A and B are optical micrographs and C and D were fluorescence micrographs. In the case of Anabaena , microscopic observation after 1 minute showed little effect on filament and chlorophyll loss. These results indicate that filamentous bodies are not separated due to the short treatment time, but it is considered that chlorophyll in the cells was not observed because cytoplasm was leaked out of the cells due to cell membrane degradation due to the action of silver ions.

Figure 6 shows the change in Oscillatoria after treatment with silver nano-aqueous solution (3 ppm, 1 min), A, B is an optical microscope picture, C, D is a fluorescence microscope picture. Oscillatoria showed a tendency of shortening of filamentous body compared with the untreated control group after 1 min of silver nano solution treatment with 3 ppm. However, no trends such as chlorophyll loss observed in Microcystis and Anabaena were observed.

Green algae ( Chlorophyceae ) : Figure 7 Scenedesmus after treatment with silver nano aqueous solution (3 ppm, 5 min) The changes of sp. were observed. A and B are optical micrographs and C and D were fluorescence micrographs. In the case of Scenedesmus , the concentration of silver nano solution was treated with 3 ppm and observed after 5 minutes. As a result, some cells could be discolored. The green algae, Scenedesmus, was more resistant than the southern algae, so after 1 minute, it had the same chlorophyll as the initial appearance, and at 5 minutes, some cells were discolored. Scenedesmus are characterized by four cells that live together to form an individual. Treatment with silver nano-aqueous solution showed that four cells were not affected and some cells first bleached. After treatment with the silver nano aqueous solution, the shape was clearly present in the optical micrograph, but there were also individuals that appeared white due to fluorescence microscopic observation (Fig. 7 B, D). Untreated silver nano-aqueous solution was clearly out of focus on the optical microscope, and the fluorescence microscope showed a reddish color. Therefore, when treated with silver nano-aqueous solution, the chloroplasts in the cells were destroyed and chlorophyll was lost.

Diatoms ( Bacillariophyceae ) : Figure 8 shows the change in Navicula sp. After treatment with silver nano-aqueous solution (3 ppm, 5 min), A, B is an optical microscope picture, C, D is a fluorescence microscope picture. In the case of Navicula , the discoloration phenomenon was observed in some cells as a result of 3 ppm of silver nano-aqueous solution. Similarly, diatoms had some resistance to the silver nano-aqueous solution, and after 1 minute, no discoloration was observed, but after 5 minutes, the discoloration was observed through a fluorescence microscope. Unlike the cyanobacteria and green algae, it was difficult to observe the shape through the optical microscope and the color was severely discolored through the fluorescence microscope, but the shape remained (FIG. 8B and D). The central part of the figure was not seen at all under the optical microscope, but it was discolored under the fluorescence microscope and the shape remained.

9 is a change of Thalassiosira sp. After treatment with silver nano-aqueous solution (3 ppm, 5 min). A and B are optical micrographs and C and D are fluorescence micrographs. Seawater diatoms Thalassiosira also showed similar results with Navicula . After treatment with 3.0 ppm of silver nano aqueous solution, 5 minutes after fluorescence microscopy, the phenomenon of discoloration was observed.

In conclusion, green algae and diatoms were more resistant to silver nano solution than silver algae, so chlorophyll discoloration could be observed after a certain time after treatment. Observed.

I. Low concentration (0.01 to 1.0 ppm) long term treatment

The drinking water standard for silver (Ag) concentration in water is 0.1 ppm. Therefore, in order to investigate the effect on silver algae and microalgae between 0.01 ppm and 10 ppm 1.0 times lower than drinking water standard, the concentration of silver nano aqueous solution was diluted to 0.01, 0.05, 0.1, 0.5 ppm. Experiment. In the low concentration experiment, the experiment was conducted to find out the survival pattern over a long period of time (5 to 10 days), unlike the high concentration experiment.

The experiment using low concentration of silver nano aqueous solution was conducted by inoculating a predetermined concentration of microalgae into the culture solution and natural water prepared so that the silver ion concentration was 0.01, 0.05, 0.1, and 0.5 ppm (the intensity was 100 μmol E / m ² / Survival was measured by incubating for 5 and 10 days in a fluorescent lamp irradiated for 24 hours to maintain s. The experiment was performed in a 250-mL Erlenmeyer flask with a 50 mL experimental volume, in 5 ml samples were taken for the measurement of in vivo fluorescence and chl- a .

In the low concentration experiment of silver nano solution for the blue alga, Microcystis , culturing for 5 days showed changes over time. In the case of Allen medium, the control group which did not add the silver nano-aqueous solution was shown to grow with time, but the killing rate of the silver nano-aqueous solution was changed according to the concentration. Microcystis experiments using cultured species were carried out at 30 to 40 ㎍ / ℓ of the initial chl- a .

In the case of Allen medium, the control group grew chl- a to 720 μg / L after one week. However, when treated with an aqueous solution of silver nano it was shown to die within 2 days after inoculation at all concentrations (Fig. 10). In vivo fluorescence measurement showed a value of 0 in 1 day treated with silver nano-aqueous solution of 0.05 ppm or more, and became 0 ㎍ / ℓ even in the case of 0.01 ppm. However, experiments measuring chl- a showed a value of 0 μg / l at one day at all concentrations (FIG. 11). On the other hand, in the experiments treated with natural water, the control group did not show a growing pattern and survived in the same amount (FIGS. 12 and 13). This may be because the collected natural water lacks the nutrients necessary for the growth of Microcystis . Experimental results of a natural number in the in vivo fluorescence measurements showed a 50% death rate in one day at 0.01 ppm, 3 days, showed a tendency to kill 100%. At 0.05 ppm or higher, the mortality rate was about 70% per day and 100% mortality on 2 days. The reason why the resistance to silver nano solution is stronger in natural water than that of Allen medium is because it is combined with anions in natural water and converted to salts, which weakens the sterilizing power.

However, the results of chl- a showed a value of 0 ㎍ / ℓ in 24 hours in the same manner as Allen media when treated with silver nanoparticle solution of 0.05 ppm or more, and decreased by 40% per day even when 0.01 ppm was treated. 100% decrease on day 2. Therefore, it has been shown that microcystis can be sufficiently controlled by treating 0.01 ppm, which is 10 times lower than 0.1 ppm of silver drinking water. Photographs of the growth of the microalgae were inhibited by the silver nano aqueous solution were taken on the 2nd and 5th samples (FIG. 14). In FIG. 14, A and C are Allen medium, and B and D are natural water, and control (B, D) was darker than 2 days according to the growth of Microcystis , but was treated with silver nano-aqueous solution ( A, C) also showed a transparent even in the sample of 2 days showed no growth at all.

In conclusion, the concentration of silver nano solution for the initial growth inhibition of cyanobacteria was 0.01 ppm, and was killed within 2 days after silver nano solution treatment.

The green algae Chlorella and Scenedesmus grew green in Allen medium without the addition of silver nanoaqueous solution. When the silver nano aqueous solution was added at a concentration of 0.05 ppm, the growth rate was slightly slowed, but the growth was overcome. However, at 0.1 ppm or more, no growth occurred. The natural water prepared by filtering the water of Daecheongho showed no visible growth even when the silver nano solution was not added, but the killing phenomenon was clearly observed according to the concentration of the silver nano solution.

Nokjoin In the case of Allen Chlorella culture medium in 4 days In vivo fluorescence showed an 10% growth compared to the control and, 8 days, 80%, 10 days 95% and little or no growth in the control group compared to the control In vivo fluorescence values were shown (FIG. 15). Chl- a also showed a slight lag time when treated with 0.05 ppm silver nano-aqueous solution, but showed similar growth as the control (FIG. 16). In the case of natural water, the growth of the control, like Microcystis , tended to be slow, and after 8 days, the growth was slowed (Figs. 17 and 18). In all the experiments the concentration of the silver solution was added to four days in demonstrate a tendency to decrease fluorescence vivo showed that the aspect death by the silver nano solution and, 0.05 ppm up to 60% compared to the control by the re-growth after 4 days in In vivo fluorescence value increased. After 8 days in control and 0.05 ppm treated in The decrease in in vivo fluorescence is believed to be due to the depletion of N and P required for growth of chlorella . In the experimental group treated with 0.05 ppm silver nano-aqueous solution, 40% of chl- a was present at 4 days compared to the control group, but more than 90% was present at 8 days, and the lag time was longer when treated with silver nano-water solution as in the case of Microcystis . Although it is resistant to silver nano aqueous solution, it showed growth again.

However, at 0.1 ppm and higher, chl- a continuously decreased to 0 ㎍ / ℓ at 8 days. In conclusion, Chlorella showed resistance up to 0.05 ppm of silver nano aqueous solution and was killed at more than 0.1 ppm.

In 4 and 10 days after inoculation of silver nano aqueous solution, it was observed that the green color increased with the growth of Chlorella in the experimental group treated with 0.05 ppm at 10 days in the case of Allen medium on day 10 (Figure 19). In the case of natural water, growth was not achieved even in the control group. In conclusion, green alga Chlorella is resistant to 0.05 ppm of silver nano aqueous solution and has little effect on growth.

The results of silver nano solution concentration on green alga Scenedesmus showed different results from chlorella. Scenedesmus was more resistant to silver nano-solution than Chlorella , so growth was similar to the control after 10 days in Allen medium even at 0.1 ppm (FIGS. 20, 21). Scenedesmus initially had some lag time for silver nano-aqueous solutions, but on day 8 at 0.05 and 0.1 ppm in In vivo fluorescence values were the same as the control (Fig. 20). In particular, 0.1 ppm showed little growth at 4 days and showed rapid growth at 4-8 days. In addition, even after treatment with 0.5 ppm showed a slight growth after 8 days, showing that the lag time by the aqueous solution of silver nano is significantly longer. Since then, growth is likely to continue. Also in the experiment of natural numbers in In vivo fluorescence showed a tendency to decrease until 10 days after growth similar to the control from 0.1 ppm to 8 days (Fig. 22, 23). It is believed that the resistance to the silver nano-aqueous solution decreases as N and P in the medium are depleted. In the case of natural numbers, the results of chl- a showed different results. In the control group, chl- a increased from 60 to 140 μg / l, but for 0.05 ppm, the initial inoculation concentration of 63 μg / l was maintained during the measurement time (FIG. 23). Also, 0.1 ppm decreased to 4 ㎍ / ℓ on the 4th day, but on the 8th day, 52 ㎍ / ℓ was obtained due to the regrowth of Scenedesmus . And the media in Allen As with the results of in vivo fluorescence, the results of chl- a increased slightly after 8 days and showed 2 ㎍ / ℓ.

The reason Scenedesmus with a high resistance to the aqueous solution as compared with silver Chlorella is determined because if the grown Scenedesmus four cells as compared with the united Chlorella present in a single cell with high resistance to the silver solution. In the micrographs of the high concentration experiments, not all four cells die at the same time, but some of them die first. Therefore, the resistance to the silver nano-aqueous solution is expected to increase. The rapid killing after 8 days in natural water experiments is attributed to the lack of N and P needed for growth. In conclusion, Scenedesmus was more resistant to silver nano-solution than Chlorella , the same green algae, so that the lag time was longer at 0.1 ppm.

By field sample Silver nano  Analysis of microalgae control effect in aqueous solution

Cultured in the laboratory In the case of microcystis , long-term passages result in morphological modifications, such as the presence of a single cell rather than colonization. Therefore, present in the field Microcystis was analyzed for the control effect of the concentration of silver nano solution. 24 is a micrograph of Microcystis that exist in a variety of forms that exist in the picture and then water hydration chusori occurred in August 2006. As shown in the photograph, the colony is surrounded by a mucous layer and forms a colony.

1. Field sample chl - a Control effect of different concentrations (Daecheongho)

When hydration occurs, the microcystis in the water moves to the surface of the water, and when it grows in large quantities, it forms a green band, and moves in accordance with the wind to form a thick layer in contact with the surface to form a scum. In order to investigate the killing effect of the silver nano-aqueous solution in the thick layered samples due to hydration in the Chusori field, the experiment was conducted under the following conditions. When the hydration of Chusori occurred, the removal effect using silver nano-aqueous solution was measured at the concentration of 150 µg / l, the concentration of chl- a of Microcystis in water, and the concentration of 1,600 µg / l, the concentration of scum in the aqueous layer.

end. High concentration spot sample

(1) mortality measurement

Microcystis was present in the surface where hydration occurred, and the aqueous solution of silver nano solution was treated by concentration to see the survival rate of Microcystis . Experimental method is the same as the previous method using a conical flask. The field sample was prepared in an Erlenmeyer flask so that the initial inoculation amount was about 1,500-1,900 µg / L in chl- a . At this time, chl- a has a large error because it is not easy to inoculate homogeneously because it is layered. The silver nano-aqueous solution was added and initially measured at 1 hour intervals, and measured after 12 hours and 24 hours after 4 hours. It was expected that there would be a drastic change in the experimental section treated with high concentration, but the experiment was conducted at short intervals, but the change was not as severe as expected. Experimental results of silver nano aqueous solution treatment for high concentration of microcystis showed the same survival rate as the control group up to 0.05 ppm of silver nano aqueous solution, and low concentration of silver nano aqueous solution was judged to have insufficient control effect for high concentration of microcystis (FIG. 25).

In the case of 0.5 and 1.0 ppm of silver nano aqueous solution, the reduction was maintained up to 4 hours and after 24 hours, it was expected that regrowth would occur and the lag time was longer even at 1.0 ppm. However, at 3.0 ppm or more, it showed a sharp decrease, showing a survival rate of 60% compared to the control at 24 hours, and showing a decreasing trend. To remove scum formed by on-site hydration, a high concentration (more than 3.0 ppm) of silver nano solution I think you should use. When 5.0 ppm of silver nano aqueous solution was used, survival rate of 40% was shown in 24 hours, and it tended to decrease continuously. When the silver nano aqueous solution was treated with 3.0 ppm and 5.0 ppm, it showed a sharp decrease with time. Therefore, the concentration of the initial silver ion should be more than 3.0 ppm for the killing of algae after hydration. However, when hydration has already occurred, the treatment of the silver nano-aqueous solution may affect the environment because the treatment concentration of silver ions is too high.

(2) electron microscope observation

Cell surface and cell cross-sections were examined by scanning electron microscope (SEM) and transmission electron microscope (TEM) in order to observe the morphological changes of cells and the attachment position of silver nano solution after treatment with silver nano aqueous solution. Observed.

In the experiment, 50 μl of high concentration of Microcystis was added using 1.5-ml Eppendrof tube, and the concentration of silver nano solution was added to 10.0 ppm, and it was fixed in fixed solution every 10, 20, 30 and 60 minutes, and then refrigerated and sample preparation of SEM and TEM The procedure was carried out in an electron microscope room in the Korea Research Institute of Bioscience and Biotechnology. SEM was taken in an electron microscope room in KAIST, and the TEM was taken in an electron microscope room in the Korea Institute of Biotechnology. 26 and 27 are photographs of surface changes of cells with time after 10 mg / l of silver nano solution was treated using a scanning electron microscope. The shape of the cells was severely distorted over time. The degree of degradation of the cell surface gradually changed with the treatment time. Already within 10 minutes after treatment, most bacteria were observed to have holes. It is believed that the change in particle shape is caused by the silver nano-aqueous solution, and silver ions bind to the SH group of the protein and thus are a result of necrosis of bacteria by interfering with respiration, enzyme activity, and electron transfer.

Figure 28 looks at the effect of the silver nano solution on the field sample using a transmission electron microscope. After treatment with silver nano aqueous solution, the results were compared at 10, 20, 30, and 60 minute intervals. In the beginning, the microcystis cell membrane structure was clearly divided into three layers, but the shape was severely distorted with time. The cell shape of was initially observed a phenomenon that one side is severely depressed in a round shape (Fig. 28C). This mechanism is thought to be the result of the breakage of the cell membrane by disrupting the energy balance of the cell membrane by combining with the negative charge portion of the cell membrane since silver ions have a positive charge. However, there was no outflow of organelles inside the cell compared to the degree of loosening of the cell membrane, which may be due to the effect of standing with the silver nano solution. In addition, black granules are seen in the cells of cyanobacteria, which are believed to be silver nanoparticles penetrating into the cells. Penetration of silver nanoparticles is observed 10 minutes after treatment with silver nano-aqueous solution. The infiltrated silver nanoparticles are believed to interfere with the synthesis of ribosomes from which proteins are made, as previously identified. It has the property of binding to proteins with SH ^- groups. Of course, not all proteins with SH ^- groups bind, but only with specific proteins. In particular, it binds to the well-known NADH dehydrogenase. However, there are no reports of binding to DNA yet. Therefore, silver ions are assumed to work in conjunction with proteins.

I. Low concentration (0.01 to 1.0 ppm) long term treatment

(1) mortality measurement

The resultant treatment of the silver nano-aqueous solution for 7 days was performed with low concentrations of chl- a (110-190 ㎍ / L) in situ (Fig. 29). At a relatively high silver ion concentration of 1 ppm or more of silver nano-aqueous solution, it was observed to die within 1 day after treatment, but it was resistant to 0.1 ppm or less. When treated with 0.05 ppm silver nano-aqueous solution, it showed 80% growth compared to the control which was not treated with the silver nano-aqueous solution for 7 days, indicating a relatively low killing effect.

The treated group treated with 0.1 ppm silver nano-aqueous solution showed the same value as the control after 1 day, but it was judged that the amount of initially inoculated cyanobacteria was higher than that of the control, and it was higher than 0.05 ppm over time. The growth was further inhibited by 70% after 7 days. In the case of the field sample, the biggest cause of the inhibition effect of the silver nano aqueous solution compared with the cultured species is considered to be that the cultured species are not only separated by single cells but also do not have a viscous layer that is a polysaccharide to aggregate together.

All. Microscopic observation

After the field sample was treated with 3 ppm of silver nano-aqueous solution, the change pattern over time was observed through a fluorescence microscope at 30 second intervals (FIG. 30). 30 seconds after the silver nano solution was treated, the color began to fade from the microcystis at the edge of the viscous layer, and after 1 minute and 30 seconds, the color faded. As time passed, the disappearance of chlorophyll of Microcystis was confirmed to be the death of silver nano solution.

Pilot - scale  Microalgae Control Effect (Culture Sample)

For the continuous study of field samples, the change over time according to the concentration of silver nano solution was observed while culturing 120 ℓ using 200-ℓ photoincubator. Previously, 0.1 ppm of silver nano-aqueous solution, which was obtained based on the experiment of an Erlenmeyer flask, was applied to a 120 L optical incubator. As a result of the experiment, incubation in Allen medium without treatment of silver nanoin vivo fluorescence is 4 (chl-a, Inoculated at about 50 ㎍ / ℓ) and incubated for 10 daysin vivo fluorescence is 28 (chl-a, About 500 μg / L) (FIG. 31). After five days of incubation, the greens became quite thick. In the experiment where the initial concentration was inoculated the same as the control and the silver nano aqueous solution was treated with 0.1 ppm of drinking water,in vivo The fluorescence tends to decrease by about 1 day, after 5 daysin vivo The fluorescence level was nearly zero, which was observed to die without growth.

The algae control method using the silver nano of the present invention can be used to improve the water quality of rivers, lakes, dams, reservoirs, etc., in addition to improving the water quality of aquaculture, industrial and agricultural water, water quality of sewage treatment plant effluent. Can be utilized.

Figures 1 to 3 is in a high concentration when processing an aqueous solution of silver Microcystis (A), Chlorella (B ), Scenedesmus (C) respectively, A graph showing the change in vivo fluorescence.

Figure 4 Microcystis after treatment with silver nano aqueous solution (3 ppm, 1 min) The change of sp was observed. A and B are optical micrographs and C and D fluorescence micrographs.

Figure 5 Anabaena after treatment with silver nano aqueous solution (3 ppm, 1 min) The changes of sp. were observed. A and B are optical micrographs and C and D were fluorescence micrographs.

Figure 6 shows the change in Oscillatoria after treatment with silver nano-aqueous solution (3 ppm, 1 min), A, B is an optical microscope picture, C, D is a fluorescence microscope picture.

Figure 7 Scenedesmus after treatment with silver nano-aqueous solution (3 ppm, 5 min) The changes of sp. were observed. A and B are optical micrographs and C and D were fluorescence micrographs.

8 shows the change of Navicula sp. After treatment with silver nano-aqueous solution (3 ppm, 5 min). A and B are optical micrographs and C and D are fluorescence micrographs.

9 is a change of Thalassiosira sp. After treatment with silver nano-aqueous solution (3 ppm, 5 min). A and B are optical micrographs and C and D are fluorescence micrographs.

10 and 11 are graphs showing the growth change (Allen medium) according to the concentration of silver nano solution of Microcystis ,

12 and 13 are graphs showing the growth change (Natural water) according to the concentration of silver nano solution of Microcystis .

Figure 14 Microcystis 2 days and 5 days after the silver nano solution treatment Photo of the culture sample, A and C are Allen medium, B, D is Natural water.

15 and 16 are graphs showing the growth change (Allen medium) of the concentration of silver nano solution of Chlorella,

17 and 18 are graphs showing the growth change (Natural water) according to the concentration of silver nano solution of chlorella.

19 is Chlorella 4 days and 10 days after the silver nano solution treatment Photo of the culture sample, A, C is Allen medium, B, D is Natural water.

20 and 21 are graphs showing the growth change (Allen medium) of the silver nano solution concentration of Scenedesmus ,

22 and 23 are graphs showing the growth change according to the concentration of silver nano solution of Scenedesmus .

24 is a Chusori field sample picture.

25 is a high-concentration field samples (Microcystis) various silver solution concentration variation curve according to the growth on: a (initial inoculation density of Microcystis Chl- a 1,500~1,900 ㎍ / ℓ) .

FIG. 26 shows SEM photographs (× 10.0K) after 10 ppm of silver nano-aqueous solution of field samples ( Microcystis ).

FIG. 27 is a SEM photograph (× 20.0K) result of 10 ppm silver nano-aqueous solution of a field sample ( Microcystis ).

FIG. 28 is a TEM photograph of 10 ppm silver nano-aqueous solution of a field sample ( Microcystis ) (upper row: 10.0K, lower row: 20.0K) (A, D: 10min, B, E: 20min, C, F: 1hrs) to be.

FIG. 29 is a growth change curve (initial inoculum concentration of Microcystis : Chl- a : 110 to 190 μg / L) according to various concentrations of silver nano solution concentrations for low concentration field samples ( Microcystis ).

30 is a microscope showing the change over time (3 ppm) after treating the silver nano-aqueous solution in the field sample.

Figure 31 is a growth change curve in a mass culture sample of Chucyori field sample ( Microcystis ).

Claims (8)

A method of inhibiting algae, characterized in that to suppress algae causing algae in fresh water using silver nano. The method of claim 1, wherein the silver nano is used at 0.001 to 30 ppm. The method of claim 1, wherein the silver nano is used at 0.001 to 10 ppm. The algae control method according to claim 1, wherein the silver nano is used at 0.005 to 0.1 ppm. The method of suppressing algae according to claim 1, wherein the silver alga is selectively inhibited using 0.005 to 0.05 ppm of silver nano. The method of any one of claims 1 to 5, wherein the silver nano is introduced into water in the form of an aqueous silver nano colloidal solution. Algae inhibitor containing silver nano as an active ingredient to inhibit algae causing algae in fresh water. The algal inhibitor according to claim 7, wherein the silver nano is an aqueous silver nano colloidal solution.

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