JPH04341392A - Control method of microbe in water - Google Patents

Abstract

PURPOSE:To prevent pollution of the surface of a structure in water due to living things by a clean and moderate electrochemical means. CONSTITUTION:The process to adsorb microbe 12 in water on the surface of a conductive substrate 3 and sterilize them by applying positive voltage to the conductive substrate and the process to detach the sterilized microbe which is adsorbed on the surface of the conductive substrate from the substrate are carried out. Consequently, without using chemical substances which cause residual toxicity, the microbe in water can be sterilized by a clean electrochemical method and thus the biological pollution is effectively prevented under the moderate conditions not like a severe electrode sterilizing method using high voltage.

Description

[Detailed description of the invention]

0001

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for electrochemically controlling microorganisms in water.

0002

BACKGROUND OF THE INVENTION In an oligotrophic environment such as distilled water, seawater or tap water, microorganisms gather and proliferate at the interface between a liquid layer and a solid layer. This is because the dilute organic matter present in water is adsorbed and concentrated at these interfaces, and microorganisms use this organic matter as a nutrient source. Microorganisms that have grown on the solid surface either diffuse back into the water and cause water pollution, or they secrete a slime-like substance while adsorbed on the solid surface and stain the surface. In particular, a large number of microorganisms exist in seawater, causing problems such as being pathogenic and adhering to underwater structures. Furthermore, since underwater structures are constantly in contact with vast amounts of seawater, the proliferation of various marine organisms, including the attachment of microorganisms, on their surfaces has become a problem. The mechanism by which these problems occur is as follows. First, adherent Gram-negative bacteria adsorb to the surface and secrete a large amount of slime-like material. Subsequently, other microorganisms gather in this slime layer and multiply, forming a microbial coating. Furthermore, large organisms begin to breed one after another on this microbial layer, and eventually the surface is covered with large organisms.

Conventionally, measures have been taken to prevent the adhesion and growth of microorganisms on the surfaces of underwater structures, such as injecting disinfectants such as chlorine gas or hypochlorous acid, or coating the surfaces with organic tin compounds, etc. For this reason, treatment methods using chemicals have been used. However, these methods have the problem of residual toxicity because the chemicals used diffuse and remain in the water.

0004

[Problems to be Solved by the Invention] The present inventor has conducted extensive research in order to develop a means that can effectively prevent the above-mentioned biological contamination without using chemical substances that cause residual toxicity. They discovered that it is possible to control the adhesion and growth of microorganisms by electrochemically sterilizing microorganisms that adsorb to the surface of underwater structures one after another in the initial stage and then detaching them from the surface. The present invention is based on this knowledge.

[0005]

[Means for Solving the Problems] Therefore, the present invention provides the steps of: (1) applying a positive potential to a conductive substrate in water to adsorb microorganisms in the water to the surface of the conductive substrate and sterilize them; , (2) a step of removing sterilized microorganisms adsorbed to the surface of the conductive substrate by applying a negative potential to the conductive substrate. Regarding.

The conductive substrate used in the method of the present invention may be entirely made of a conductive material, but it is only necessary that at least its surface (or a portion of the surface immersed in water) is conductive. Although the shape of the conductive substrate is not particularly limited, it is preferable that the conductive substrate has a large surface area, can efficiently adsorb microorganisms in water, can directly contact them, and can apply a potential. Preferred embodiments of the conductive substrate used in the method of the present invention include, for example, a sheet-like substrate immersed in a microorganism-containing liquid to be treated, or a substrate in which the underwater surface of an underwater structure or ship is made of the above-mentioned conductive substrate. be able to.

The material of the conductive substrate is not particularly limited, but it should have good plasticity and moldability so that a conductive surface layer can be formed on a wide surface such as a sheet-like substrate, an underwater structure, a ship, etc. For example, a conductive substrate formed by mixing a conductive material (e.g., graphite, carbon, platinum, or a conductive substance) with a binder polymer and forming it directly, or an underwater structure using a coating composition containing a conductive material. A conductive film formed on the surface of an object is preferred. Examples of the binder polymer include silicone resins, vinyl acetate resins, mixtures of vinyl chloride resins and polyurethane resins, styrene-butadiene rubber, urethane resins, chloroprene rubber, and epoxy resins, among which silicone resins are preferred. A conductive polymer (for example, polyaniline) may be added together with the binder polymer to lower the resistance value.

[0008] When a positive potential is applied to a conductive substrate in water containing microorganisms, the microorganisms in the water can be adsorbed onto the surface of the substrate. Furthermore, the positive potential applied to the substrate has the effect of electrochemically sterilizing microorganisms that adsorb to and come into contact with the substrate surface. That is, microorganisms are adsorbed to the substrate surface by the positive potential and are sterilized on the surface. The positive potential to be applied is +0 to +1.5 vs. SCE, preferably +0.5 to +1.5 vs. It is SCE. Applied potential is +
0 vs. If it is less than SCE, microorganisms cannot be adsorbed to the substrate and sterilized, and +1.5 vs. Exceeding the SCE is undesirable because water and dissolved salts will be electrolyzed to generate toxic gases or metals will be deposited on the surface of the conductive substrate.

The adsorption sterilization process of microorganisms, which involves applying a positive potential to the conductive substrate, is carried out for up to 6 hours, preferably for 5 to 30 minutes, although it varies depending on the concentration and type of microorganisms present in the water, the flow rate, and the temperature. It is preferable to do so. If the potential application time is longer than 6 hours, other microorganisms will be adsorbed onto the microorganisms sterilized on the substrate. Since the microorganisms that are subsequently adsorbed are not in direct contact with the conductive substrate, they are not subjected to the electrochemical sterilization effect caused by the positive potential. Therefore, living microorganisms are adsorbed onto the substrate surface one after another, and even if a negative potential is applied to remove the microorganisms, the microorganisms on the surface cannot be removed and the substrate is contaminated. I end up.

[0010] Subsequently, by applying a negative potential to the conductive substrate, the microorganisms adsorbed to the substrate surface can be removed. The negative potential to be applied is -0 to -0.4 vs. SCE
, preferably -0.1 to -0.2 vs. It is SCE. When the applied potential is -0 vs. If it is above SCE, microorganisms cannot be detached from the substrate, and -0.4 vs. If it is lower than SCE, the pH will increase, which is not preferable. In addition, the microbial desorption process, which involves applying a negative potential, varies depending on the amount and type of microorganisms attached to the surface, but
It is preferable to carry out the heating for 1 minute to 5 minutes, preferably for 1 minute to 5 minutes. If it is shorter than 30 seconds, the desorption of the sterilized microorganisms will not be sufficient, and when a next microorganism adsorption sterilization step in which a positive potential is applied is performed, other microorganisms will be adsorbed onto the sterilized microorganisms. Furthermore, if the time is longer than 30 minutes, the liquid to be treated cannot be effectively sterilized.

In the method of the present invention, it is preferable to repeat the microbial adsorption sterilization step and the desorption step. When the microorganism-containing water in the closed container is subjected to a process that repeats the cycle consisting of the adsorption sterilization step and the desorption step, the microorganisms contained therein are successively adsorbed to the substrate surface and sterilized. is desorbed, so that virtually all microorganisms can be sterilized in the end. Also,
When the method of the present invention is used in an open system such as a structure in seawater, the surface of the structure can be permanently protected from contamination by repeating the above cycle.

In the method of the present invention, a conductive substrate is used as a working electrode, and a suitable counter electrode, reference electrode, and potentiostat are used to control the potential applied to the conductive substrate. It is necessary. The counter electrode, reference electrode, and potentiostat that can be used are not particularly limited as long as they can apply a predetermined potential to the surface of the conductive substrate.

[0013] The water that can be treated by the method of the present invention is not particularly limited as long as it contains microorganisms, but includes, for example, seawater, river water, lake water, tap water, or drinking water. be. Furthermore, the target microorganisms are not particularly limited as long as they exist in water, and include bacteria, filamentous fungi, yeast, amphiphiles, algae, protozoa, and the like.

[0014]

[Examples] The present invention will now be explained in detail with reference to Examples, but these are not intended to limit the scope of the present invention.

In each of the following examples, the apparatus shown in FIG. 1 was used as a sterilization treatment apparatus. In this apparatus, a conductive resin plate electrode 3 molded on a glass substrate 2 is arranged in a sterilization treatment tank 1, and the plate electrode 3 is in communication with a potentiostat 4. The potentiostat 4 is in communication with a reference electrode 6 and a counter electrode 7 arranged in the control tank 5, respectively. The sterilization tank 1 and the control tank 5 are connected by a salt bridge 8, and a stirring device 9 and a stirring rod 10 are arranged at the bottom of the sterilization tank 1. The conductive resin plate electrode 3 is made of a material obtained by adding 50% by weight of graphite (Fluka Co., Ltd.) to silicone resin, and is placed on the glass substrate 2 in a plate shape (26 mm×
26 mm x 1.5 mm), and the counter electrode 7
is platinum, and reference electrode 6 is a saturated sweetened electrode (SCE).
) was used. Moreover, with this device, the water to be treated 11 in the sterilization treatment tank 1 is
sterilizes microorganisms 12 contained in the.

Example 1: Effect of applied potential on seawater The effect of applied potential on seawater was investigated using the apparatus shown in FIG. 50ml of seawater (pH 8.0) collected from Miura coast
was placed in the sterilization treatment tank 1, and while stirring at 350 rpm, a positive potential was applied to the plate electrode 3 at room temperature for 30 minutes, and changes in current density, residual chlorine concentration, and pH were measured. As is clear from FIG. 2 showing the measurement results, 1.6V vs. S.C.
Residual chlorine (▲ in FIG. 2) was not measured at a potential below E. Furthermore, −0.4V to +1.5V vs. No pH change (■ in Figure 2) was observed within the SCE range. Therefore, -0.4V to +1.5V vs. Sterilization by chlorine or pH changes does not occur within the SCE range.

Example 2: Preparation of plate electrodes The marine adhering bacterium Vibrio alginolyticus
inolyticus: ATCC 17749),
Marine broth 2216
(DIFCO Laboratory) at 25°C.
The cells were cultured aerobically for 10 hours. After culturing, the bacterial cells were collected by centrifugation (10 minutes; 2000 g), and then thoroughly washed with sterilized seawater. Then, using a hemacytometer,
Sample water of 0.05 cells/ml was prepared. The plate electrode 3 was immersed in 150 ml of this sample water, and left to stand for 90 minutes with gentle stirring at 350 rpm without applying an electric potential, so that the bacterial cells were sufficiently adsorbed onto the surface of the plate electrode 3. This plate electrode 3 was washed with sterilized seawater to remove bacterial cells not adsorbed to the surface, thereby preparing a bacterial cell-attached plate electrode 3.

Example 3: The above-mentioned microbial cell adhesion plate electrode 3 (amount of bacterial adhesion: 8.0
．． 0x104 CFU/cm2) and 350r
-0.4V vs. pm with gentle stirring. SCE~
+0.4V vs. A potential of SCE was applied. After applying a potential for 10 minutes, the plate electrode 3 was taken out from the sterilization tank 1. Bacteria present in the residual seawater in the sterilization tank 1 (bacterium detached from the plate electrode 3 during potential application) were collected by pipetting, and the number of viable bacteria was measured by a colony counting method. The measurement results are shown in FIG. 3 as the number of viable bacteria detached per unit area of the electrode. +0.4~0V
vs. In the range of SCE, approximately 5.0 x 104 CFU/cm2 of bacterial cells were recovered from the residual seawater in sterilization treatment tank 1, but -0.4 to 0V vs. In the SCE range, as the potential decreases, the number of bacterial cells recovered increases, and -0.2
V vs. 6.3×104 CFU/cm2 in SCE
of bacterial cells, and -0.4V vs. 7.2×1 in SCE
04 CFU/cm2 of bacterial cells were recovered.

Example 4: Sterilization by applying a positive potential The above-mentioned bacterial cell adhesion plate electrode 3 (amount of bacterial adhesion: 5
．． 6×103 CFU/cm2) and 350r
0V vs. with gentle stirring at pm. SCE~+1.
5V vs. The SCE potential was applied for 10 minutes. After applying positive potential, -0.4Vvs. The SCE potential is applied for 2 minutes to detach the bacterial cells from the plate electrode 3, and the plate electrode 3 is removed from the sterilization tank 1. Plate electrode 3
The bacterial cells detached from the cells were collected by pipetting, and the number of viable bacteria was determined by colony counting. Figure 4 shows the measurement results as the number of viable bacteria detached per unit area of the electrode.
Shown below. As the applied potential was increased, the number of viable cells recovered decreased. That is, 0V vs. Approximately 1.
Although 8×103 CFU/cm2 of bacterial cells were recovered,
+1.0V vs. Approximately 1.3 x 103 CFU in SCE
/cm2 of bacterial cells, and +1.5V vs. Approximately 0.2 x 103 CFU/cm2 of bacterial cells were recovered by SCE.

Example 5: Effect of positive potential application time In a sterilization tank 1 containing 150 ml of sterilized seawater (pH 8.0), a bacterial cell adhesion plate electrode 3 (amount of bacterial adhesion: 5.6×
103 CFU/cm2) and +1.5V vs. with gentle stirring at 350 rpm. After applying the SCE potential for 1 minute, 5 minutes, 10 minutes and 30 minutes, -0.4V vs. The SCE potential is applied for 2 minutes to detach the bacterial cells from the plate electrode 3, and the plate electrode 3 is removed from the sterilization tank 1. The bacterial cells detached from the plate electrode 3) were collected by pipetting, and the number of viable bacteria was measured by colony counting. The measurement results are shown in FIG. 5 as the number of viable bacteria detached per unit area of the electrode. Approximately 2.3×103 CFU/cm2, 5 after 1 minute
After a minute, it decreased to about 1.0×10 3 CFU/cm 2 .

Example 6: Adsorption, sterilization and desorption treatment of bacterial cells and comparative example 1.0×103 cells using a hemacytometer
Plate electrode 3 with no bacterial cells attached was inserted into sterilization tank 1 containing 50 ml of seawater (pH 8.0) adjusted to pH 8.0.
1) +1.0V vs. Adsorption and sterilization step of applying SCE positive potential for 10 minutes, and (2) -0.2V vs. A treatment cycle consisting of a desorption step in which a negative potential of SCE was applied for 2 minutes was repeatedly performed, and the number of viable bacterial cells in 1 ml was counted by colony counting method. In FIG. 6 showing the results, the case where the above treatment cycle was repeated is plotted with ■, and the case where no potential was applied (control) is plotted with ●. In the control, 1.1 x 10 after 1 hour
3 cells/ml, 1.2 x 103 after 6 hours
cells/ml, 5.0×103c after 10 hours
cells/ml and 9.3 x 103 in 12 hours
The number of bacterial cells increased to cells/ml. On the other hand, when carrying out the above-mentioned treatment cycle in which positive potential and negative potential are applied alternately, viable bacteria are no longer observed after 6 hours;
It can be seen that electrochemical sterilization was performed efficiently.

[0022] Also, as a comparative example, +1.0V vs. S
Positive potential of CE or -0.2V vs. The same treatment was carried out while continuously applying the negative potential of SCE. In FIG. 7 showing the results, +1.0V vs. When the positive potential of SCE is continuously applied (1.3 × 103 cells after 1 hour)
/ml, 2.0×103 cells/m after 6 hours
l, 7.0×103 cells/ml after 10 hours
, and 12.4×103cells/m in 12 hours
▲, -0.2V vs. If the negative potential of SCE is continuously applied (1.4× after 1 hour)
103 cells/ml, 1.6 x 10 after 6 hours
3 cells/ml, 5.6 x 103 after 10 hours
cells/ml, and 11.8 x 1 in 12 hours
03 cells/ml) is plotted as ■, and the case where no potential was applied (control) is plotted as ●.

Reference example: Study of conductive plate electrode materials Silicone resin, ethylene vinyl acetate copolymer resin, mixture of vinyl chloride and polyurethane resin, styrene-ptadiene rubber, urethane resin, chloroprene rubber, epoxy resin, graphite ( Fluka Inc.), 49
% by weight, 64% by weight, 59% by weight, 63% by weight, 70% by weight, 60% by weight, and 46% by weight were mixed, formed into a plate shape on a glass substrate, and made into an electrode using carbon fiber as a lead wire. When the resistance value between two points between the electrode surface and the lead wire was measured, it showed a low resistance value of about 20 to 50 Ω. In addition, when cyclic voltammetry measurement of ferricyanide was performed, the redox peak was confirmed when silicone resin and chloroprene rubber were used.

Next, when the resistance value of the plate electrode using silicone resin as the binder polymer was measured with respect to the graphite content, it was found that the graphite content was 3.
As the resistance increases to 5%, 42%, and 50% by weight, the resistance increases to 160Ω, 42Ω, and 11%, respectively.
It was found that the value decreases as Ω. Also, the content rate is 50
It showed a nearly constant resistance value of about 11Ω at weight % or more. Furthermore, when polyaniline, a conductive polymer (prepared by stirring purified aniline in 1N HCl with ammonium persulfate as an oxidizing agent and stirring overnight at -5°C), the resistance value further decreased. Understood.

[0025]

[Effects of the Invention] According to the method of the present invention, microorganisms in water can be sterilized using a clean electrochemical method that does not cause water pollution, unlike conventional methods that use chemical substances that cause residual toxicity. Moreover, biological contamination can be effectively prevented under mild conditions, which is different from the extremely harsh conventional electrode sterilization method using high voltage.

[Brief explanation of the drawing]

FIG. 1 is an explanatory diagram schematically showing an apparatus for carrying out the method of the present invention.

FIG. 2 is a graph showing the effect of applying a potential to seawater.

FIG. 3 is a graph showing the effects of positive and negative potentials on microorganisms.

FIG. 4 is a graph showing the effect of positive potential on microorganisms.

FIG. 5 is a graph showing the effect of applying a positive potential for a long time.

FIG. 6 is a graph showing the effect of applying a positive potential and a negative potential alternately.

FIG. 7 is a graph showing the effect of performing a process of continuously applying a constant positive potential or negative potential.

[Explanation of symbols]

1 Sterilization treatment tank 2 Glass substrate 3 Conductive resin plate electrode 4 Potentiostat 5 Control tank 6. Opposite 7 Reference pole 8　Salt Bridge 9 Stirring device 10 Stirring rod 11　Water to be treated 12 Microorganisms

Claims (1)

[Claims] 1. A step of sterilizing microorganisms in the water by adsorbing them to the surface of the conductive substrate by applying a positive potential to the conductive substrate in water, and a step of sterilizing microorganisms in the water by applying a negative potential to the conductive substrate. A method for controlling microorganisms in water, comprising the steps of: desorbing sterilized microorganisms adsorbed on the surface of the conductive substrate.