CN115520931A - UVA-LED-based microorganism inactivation method - Google Patents
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- C—CHEMISTRY; METALLURGY
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- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F1/00—Treatment of water, waste water, or sewage
- C02F1/30—Treatment of water, waste water, or sewage by irradiation
- C02F1/32—Treatment of water, waste water, or sewage by irradiation with ultraviolet light
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- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F2103/00—Nature of the water, waste water, sewage or sludge to be treated
- C02F2103/007—Contaminated open waterways, rivers, lakes or ponds
Abstract
The invention discloses a microorganism inactivation method based on UVA-LED, belonging to the field of microorganism inactivation. The sterilization method of the present invention comprises the steps of: the microorganisms are irradiated by a UVA-LED light source in a single or multiple irradiation mode. The method provided by the invention is based on UVA-LED, is more green and safe, and has no reactivation phenomenon of microorganisms; through fractionated irradiation, the microbial inactivation efficiency is remarkably improved.
Description
Technical Field
The invention relates to the field of microbial inactivation, in particular to a microbial inactivation method based on UVA-LED.
Background
Water pollution seriously threatens human health, and water disinfection is a necessary process for water treatment to ensure safety. Recently, researchers have developed a range of water disinfection methods. Unlike chemical disinfectants, methods of disinfection by Ultraviolet (UV) light treatment are convenient to use, do not require the addition of chemicals, produce few by-products, and are efficient. Commonly used ultraviolet disinfection systems are Low Pressure (LP) mercury lamps with monochromatic ultraviolet radiation (254 nm) and Medium Pressure (MP) mercury lamps covering a broad spectrum. However, they are costly, have a short lifetime, and contain toxic mercury.
In recent years, ultraviolet light emitting diodes (UV-LEDs) have attracted much attention as a new type of ultraviolet light source due to their unique characteristics. Ultraviolet LEDs have lower energy requirements and longer lifetimes, and are more environmentally friendly and safer than low and medium pressure mercury lamps. In addition, UV-LEDs can emit light at any peak wavelength and have a narrow distribution range. Short wave ultraviolet (UVC, <280 nm) has been widely used for water disinfection, such as hospital waste water and purified water. However, UVC-LEDs are expensive and harmful to the human body. Furthermore, some microorganisms may be activated by photorepair/dark repair after UVC treatment.
Disclosure of Invention
The invention provides a UVA-LED-based microorganism inactivation method, which can realize inactivation of microorganisms in water, and has high inactivation rate and no microorganism revival phenomenon.
The invention firstly provides a method for sterilizing microorganisms, which comprises the following steps: and irradiating the microorganisms by using a UVA-LED light source in a single or multiple irradiation mode.
In the sterilization method, the maximum absorption wavelength of the UVA-LED light source is 365nm;
the irradiation power of the UVA-LED light source is more than or equal to 180mW/cm 2 。
In the above sterilization method, the number of times of the fractionated irradiation is 2; the irradiation dose of the first time is larger than that of the second time, and the time interval of the sub-irradiation is 5-30 min.
Preferably, the interval time of the fractionated irradiation is 15min.
In the above sterilization method, the microorganism is at least one of staphylococcus aureus, escherichia coli, and listeria monocytogenes.
The microorganisms are microorganisms in water.
The pH value of the water is 4-10, and specifically 5-8.
Compared with the prior art, the invention has the following advantages:
(1) UVC-LED light source is expensive and harmful to human body, and microorganisms can be reactivated through light repair/dark repair; the method provided by the invention is based on UVA-LED, is more green and safe, and has no reactivation phenomenon of microorganisms.
(2) The method provided by the invention is based on UVA-LED, and remarkably improves the microorganism inactivation efficiency through fractionated irradiation.
Drawings
FIG. 1 is a schematic illustration of the steps of single irradiation and fractionated irradiation according to the invention;
FIG. 2 is a wavelength spectrum of a 365nm UVA-LED used in the present invention;
FIG. 3 is a bar graph comparing the numbers of inactivated colonies by single irradiation and fractionated irradiation using Staphylococcus aureus as an indicator;
FIG. 4 is a graph showing the effect of pH change in an aqueous solution on the number of colonies inactivated by fractionated irradiation;
FIG. 5 is a graph of the effect of fractionated irradiation on different strains and the effect of different spacing patterns on inactivation of the strains; wherein (A) in FIG. 5 is the effect of fractionated irradiation on Escherichia coli inactivation; (B) Influence of fractionated irradiation on the inactivation of Listeria monocytogenes; (C) effects on inactivation of the strain at different time intervals; (D) effects of different spacing patterns on inactivation of the strain;
FIG. 6 is a graph of the effect of light and dark repair on fractional irradiation inactivation of a strain;
FIG. 7 is a graph showing the effect of fractionated irradiation on strain damage; wherein (a) in fig. 7 is the effect of a single irradiation on strain spill protein; (B) influence of fractionated irradiation on strain overflow protein; (C) The influence of single irradiation and multiple irradiation on the MDA content of the strain is avoided; (D) - (F) observing the influence of the fractionated irradiation on the cell morphology of the strain by using a scanning electron microscope; (G) - (I) is the influence of transmission electron microscope observation fractionated irradiation on the microscopic morphology of the strain;
FIG. 8 is the inactivation mechanism data of the fractionated irradiation in example 4; wherein (a) in fig. 8 is an interval irradiation sterilizer chart; (B) Pre-incubation for 30min for active oxygen scavenger, effect on the number of intermittently irradiated inactivated colonies; (C) Effect of direct irradiation on the number of intermittently irradiated inactivated colonies without incubation for addition of an active oxygen scavenger; (D) The relative content change of active oxygen at different time after the first irradiation; (E) The intracellular Glutathione (GSH) content of the strain is changed at different time after the first irradiation.
Detailed Description
The present invention is described in further detail below with reference to specific embodiments, which are given for the purpose of illustration only and are not intended to limit the scope of the invention.
The experimental procedures in the following examples are conventional unless otherwise specified.
In the quantitative tests in the following examples, three replicates were set up and the results averaged.
Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
The LB broth medium in the following examples was formulated as follows: 10g of tryptone (OXOID, USA), 5g of yeast extract (OXOID, USA), 10g of sodium chloride (Beijing Soilebao Tech., ltd., china), and 15-20 g of agar powder (Beijing Soilebao Tech., ltd., china) added into a solid culture medium; double distilled water was added to 1000mL.
The following examples used Staphylococcus aureus ATCC6538, escherichia coli O157H 7ATCC35150 and Listeria monocytogenes ATCC10403s, all purchased.
The experimental materials and experimental methods used in the following examples are as follows:
(1) The strain culture and counting method is as follows: staphylococcus aureus ATCC6538, escherichia coli O157: H7ATCC35150 and Listeria monocytogenes ATCC10403 were used by resuscitating twice in LB broth at 37 ℃ and 220 rpm. The number of colonies in water was calculated using the agar plate method. Briefly, serial dilutions were made in 0.9% normal saline and plated on LB agar medium. The plates were incubated at 37 ℃ until the colonies were clear enough to count (about 24 hours).
(2) Preparation of the microbial suspension: inoculating the twice recovered strain in (1) above into 0.9% normal saline, and diluting continuously to make initial concentration of Staphylococcus aureus, escherichia coli or Listeria monocytogenes about 10 8 CFU/mL, obtaining the microorganism suspension.
(3) FIG. 1 is a schematic diagram of the steps of single irradiation and fractionated irradiation according to the present invention, the single irradiation being continuous light treatment of a water sample; the split irradiation is the same as the single irradiation with the irradiation energy except that the light source is turned off for a period of time in the middle and then the second irradiation is performed.
(4) UVA-LED light source properties: the UVA-LED light source used in the examples is purchased from China, inc., of electronics and technology, of Zhongshan City.
The wavelength scan is shown in FIG. 2, the light intensity is strongest at 365nm, and the detailed properties of the light source are shown in Table 1.
TABLE 1UVA light source specific Properties
Example 1 Disinfection and Sterilization
The illumination intensity of the UVA-LED light source is 183mW/cm 2 . The microorganism suspension was prepared by diluting with 0.9% physiological saline. 2mL of the microorganism suspension was added at an initial concentration of 10 8 CFU/mL was added to a 6-well plate and then exposed to a UVA-LED light source. The dose of UVA irradiation varies depending on the type of bacteria. The time interval of the water sample fractional irradiation is 5 min-30 min, and the logarithmic value of the number of the inactivated colonies represents the inactivation efficiency.
(1) Comparison of Single and fractionated irradiation for Staphylococcus aureus inactivation
Treating a suspension of microorganisms containing Staphylococcus aureus with different UVA energies, with a single irradiation with an intensity of 183mW/cm 2 Irradiating for one time; the irradiation is divided into two parts with the illumination intensity of 183mW/cm 2 Irradiating for a certain time, then irradiating for 15min at intervals, and then irradiating for a certain time. The sum of the energy of the single irradiation and the energy of the fractionated irradiation is the same. At 37.8J/cm 2 In the irradiation group, the energy of the fractionated irradiation was 27.0J/cm 2 And 10.8J/cm 2 Two parts. At 43.2J/cm 2 The irradiation group has a fractional irradiation energy of 32.4J/cm 2 And 10.8J/cm 2 Two parts. At 54.0J/cm 2 The irradiation group has a fractional irradiation energy of 37.8J/cm 2 And 16.2J/cm 2 Two parts.
The results are shown in A-C in FIG. 3, and it can be seen from A-C in FIG. 3 that the number of inactivated colonies of Staphylococcus aureus gradually increases with increasing UVA irradiation dose. And the fractionated irradiation significantly increases the UVA inactivation efficiency as compared to a single irradiation, e.g., at 43.2J/cm 2 Under the condition of single irradiation, the number of inactivated colonies is 1.3 logs, and under the condition of fractionated irradiation, the number of inactivated colonies is 2.4 logs. The kinetic relationship between the irradiation dose and the number of inactivated colonies is shown as D in FIG. 3, and the irradiation dose and the number of inactivated colonies show a good linear relationship regardless of single irradiation or fractionated irradiation.
(2) The illumination intensity of a UVA-LED light source is 183mW/cm at different energies 2 The microorganism suspension was treated by fractionated irradiation with irradiation energy of 39.6J/cm, respectively 2 +19.8J/cm 2 ，32.4J/cm 2 +16.2J/cm 2 And 27.0J/cm 2 +10.8J/cm 2 The interval time is 15min; the pH of the staphylococcus aureus microbial suspension was adjusted to 5,6,7,8, and the effect of pH change in the aqueous solution on the inactivation of the strain was investigated. The results are shown in fig. 4, and in the range of pH 5.0-8.0, pH changes do not affect the inactivation of the strains by fractionated irradiation, which indicates that the fractionated irradiation mode can be applied to aqueous solutions with different pH.
(3) Escherichia coli (E.coli) and L.monocytogenes (L.monocytogenes) are taken as research objects, and the influence of fractionated irradiation on the inactivation of different strains is researched under the condition of different irradiation energies. The illumination intensity of a UVA-LED light source is 183mW/cm 2 The microbial suspension is irradiated for single and multiple times, and the interval time of the multiple times of irradiation is 15min. The total energy of irradiation of Escherichia coli was 21.6J/cm 2 And 14.4J/cm 2 At 21.6J/cm 2 The irradiation energy of the irradiation group is 14.4J/cm 2 And 7.2J/cm 2 Two parts. At 14.4J/cm 2 The irradiation energy of the irradiation group is 10.8J/cm 2 And 3.6J/cm 2 Two parts. The total irradiation energy of the Listeria monocytogenes is 54.0J/cm 2 And 37.8J/cm 2 . At 54.0J/cm 2 The irradiation energy is 37.8J/cm 2 And 16.2J/cm 2 Two parts. At 37.8J/cm 2 The irradiation energy of the irradiation group is 27.0J/cm 2 And 10.8J/cm 2 Two parts. The results are shown in A and B in figure 5, and the fractional irradiation of both Escherichia coli and Listeria monocytogenes significantly improves the inactivation efficiency of the UVA-LED light source on the strains.
(4) Optimizing the conditions of the fractionated irradiation:
the total energy of irradiation is 43.2J/cm by using Staphylococcus aureus as indicator 2 Under the condition of (2), the time intervals of the two times of irradiation are respectively 0min,5min,15min and 30min. The effect of time intervals on the inactivation of the strains was explored. Results C in FIG. 5 shows that the inactivation efficiency did not increase with time interval, but at 15min intervals, the number of inactivated colonies was the highest.
Secondly, the interval mode has great influence on the inactivation of the strain, staphylococcus aureus is taken as an indicator bacterium, and the irradiation energy is 43.2J/cm 2 Under the condition of 15min interval, the single irradiation is 43.2J/cm 2 The dose is divided into two parts of different irradiation doses, 10.8+32.4 represents that the first irradiation dose is 10.8J/cm 2 The second irradiation dose is 32.4J/cm 2 .21.6+21.6 indicates that the irradiation dose of the first time and the second time are both 21.6J/cm 2 .32.4+10.8 represents that the irradiation dose at the first time is 32.4J/cm 2 . The second irradiation dose is 10.8J/cm 2 。
The effect of different partitioning patterns on strain inactivation is shown in figure 5, D. The first irradiation dose is larger than the second irradiation dose, and the inactivation effect on the strain is the best.
Example 2 Effect of fractionated irradiation on Strain repair
A suspension of microorganisms containing Staphylococcus aureus was treated with different UVA energies, and 2mL of the suspension of microorganisms was added at an initial concentration of 10 8 CFU/mL was added to a 6-well plate and exposed to UVA-LED light source at 183mw/cm 2 . Treating the sample with single irradiation and multiple irradiation at interval of 15min and 54J/cm respectively 2 And 72J/cm 2 The microbial suspension is treated. At 54.0J/cm 2 Fraction of the irradiation group, irradiationEnergy is 37.8J/cm 2 And 16.2J/cm 2 Two parts. At 72.0J/cm 2 The irradiation energy is 43.2J/cm 2 And 28.8J/cm 2 Two parts. After the irradiation was stopped, 0.1mL of the irradiated microorganism suspension was counted (Nt), and 1.5mL of the irradiated suspension was transferred to a sterile transparent tube for a photoreactive test of Staphylococcus aureus. For the dark repair experiment of Staphylococcus aureus, 1.5mL of the irradiated microorganism suspension was transferred to a sterile aluminum foil wrapped clear tube. Since the bacteria after irradiation treatment are affected by temperature, the aluminum foil-wrapped and unwrapped transparent test tubes containing 1.5mL of irradiated bacteria liquid are placed in a constant temperature shaking table (25 ℃,60rpm, shanghai Zhichu desktop type full temperature shaking incubator shaking table ZQTY-90V) for revival experiment. 2 fluorescent tubes are arranged in the constant temperature shaking table, and after incubation for 0.5h,1h,2h,3h and 5h, sampling and plate coating are respectively carried out for counting (N1). The light rejuvenation efficiency of the bacterial liquid is evaluated according to the rejuvenation percentage, and the calculation method is as follows, wherein N0 represents the number of colonies (CFU/mL) in the stock solution before inactivation, nt represents the number of colonies in the sample after inactivation, and N1 represents the number of colonies after the sample is treated for a certain time under the condition of light or dark after inactivation.
Percent reactivation (%) = (Log) 10 N 1 -Log 10 N t )/(Log 10 N 0 -Log 10 N t )
The light repair/dark repair is one of the main factors influencing the UVC application, so that the influence of the fractionated irradiation on the light repair/dark repair of the strain is explored next, and the result is shown in figure 6, the fractionated irradiation has no influence on the light repair and dark repair of the strain, and the continuous inactivation effect on the strain is shown at the later stage; wherein (A) in FIG. 6 is 54J/cm 2 Performing light repairing; (B) Is 72J/cm 2 Performing light repairing; (C) Is 54J/cm 2 Dark repairing; (D) Is 72J/cm 2 And (5) dark repairing.
Example 3 Effect of fractionated irradiation on Strain Damage
2mL of the microorganism suspension was added at an initial concentration of 10 8 CFU/mL was added to a 6-well plate and exposed to UVA-LED light source at 183mw/cm 2 . At 43.2J/cm 2 And 72.0J/cm 2 Respectively processing microorganism suspension containing Staphylococcus aureus by single irradiation and multiple irradiation, wherein interval time of the multiple irradiation is 15min. At 43.2J/cm 2 The irradiation energy of the divided irradiation groups is 32.4J/cm and 10.8J/cm 2 Two parts. At 72.0J/cm 2 The irradiation energy of the irradiation group is 43.2J/cm 2 And 28.8J/cm 2 Two parts. After irradiation, the supernatant was centrifuged and the protein content was determined. The damage effect of the fractionated irradiation on the strain was explored from the microstructure. Proteins are an important component of bacteria, and when bacteria are destroyed, intracellular proteins are released. As shown in a and B in fig. 7, the intracellular protein release amount after fractionated irradiation was larger than that of single irradiation. Therefore, the damage effect of the fractionated irradiation on the strain is larger, and the inactivation of the strain is more facilitated.
2mL of the microorganism suspension was added at an initial concentration of 10 8 CFU/mL was added to a 6-well plate and exposed to UVA-LED light source at 183mw/cm 2 . At 72.0J/cm 2 Respectively treating the microorganism suspension containing staphylococcus aureus by single irradiation and multiple irradiation, wherein the interval time of the multiple irradiation is 15min. At 72.0J/cm 2 The irradiation energy of the irradiation group is 43.2J/cm 2 And 28.8J/cm 2 Two parts. After the irradiation is finished, centrifuging and taking the supernatant, and determining the content of malonaldehyde. Malondialdehyde (MDA) as a cytotoxic substance represents the degree of lipid peroxidation of cell membranes. The MDA content is determined by TBA (thiobarbituric acid) method. As shown in fig. 7C, the MDA content of the strain increased more after fractionated irradiation compared to the single irradiation, indicating that the strain suffered more severe oxidative damage.
2mL of the microorganism suspension at an initial concentration of 10 8 CFU/mL was added to a 6-well plate and then exposed to a UVA-LED light source at 183mw/cm 2 . At 43.2J/cm 2 Respectively processing microorganism suspension containing Staphylococcus aureus by single irradiation and multiple irradiation, wherein interval time of the multiple irradiation is 15min. At 43.2J/cm 2 The irradiation energy is 32.4J/cm 2 And 10.8J/cm 2 Two parts. After the irradiation is finished, the precipitate is centrifuged and usedThe control group samples were not irradiated (i.e. 10) as observed by scanning electron microscope and transmission electron microscope 8 CFU/mL microorganism suspension is directly fixed by an electron microscope fixing solution and then used for electron microscope observation treatment). The influence of the fractionated irradiation on the strain morphology was observed by a scanning electron microscope, and as shown in the scanning electron microscope image in fig. 7 (see D-F in fig. 7, where D is a control group, E is a sample after single irradiation, and F is a sample after fractionated irradiation), the staphylococcus aureus cells in the control group were intact and spherical, and the surface of the staphylococcus aureus cells began to wrinkle after UVA treatment. The cell surface of the fraction had more debris. The same results were observed by transmission electron microscopy, as shown in the transmission electron microscopy image of fig. 7 (see G-I in fig. 7, where G is the control, H is the sample after single irradiation, and I is the sample after fractionated irradiation), the staphylococcus aureus cells of the control were in good condition, and the intracellular environment, cell membrane and cell wall were also good. In contrast, the cytoplasm of staphylococcus aureus in the single irradiation group was lost. In addition, the S.aureus cells of the fractionated irradiation group were incomplete and had lysis.
Example 4 inactivation mechanism by fractionated irradiation
We hypothesized that some of the actives interact with s.aureus cells during the interval, increasing their sensitivity to UVA, eventually leading to strains that are more easily inactivated (a in figure 8).
2mL of the microorganism suspension was added at an initial concentration of 10 8 CFU/mL was added to a 6-well plate and exposed to UVA-LED light source at 183mw/cm 2 . At 43.2J/cm 2 Respectively processing microorganism suspension containing Staphylococcus aureus by single irradiation and multiple irradiation, wherein interval time of the multiple irradiation is 15min. At 43.2J/cm 2 The irradiation energy of the irradiation group is 32.4J/cm 2 And 10.8J/cm 2 Two, the mechanism of inactivation was explored under this condition.
The effect of active oxygen in fractionated irradiation was measured by using free radical scavenger, mannitol to scavenge OH, mannitol concentration of 0.5M (Beijing Solebao science and technology Co., ltd., china, CAS: 69-65-8), and catalase to scavenge H 2 O 2 The catalase concentration was 1mg/mL (CAS: 9001-05-2, china, beijing Solape technologies, ltd.). In order to completely dissolve the scavenger in water and penetrate into the cells, the scavenger was incubated with the S.aureus cells in a dark, thermostatted shaker for 30min (25 ℃,80 rpm) prior to the light treatment. After the incubation is finished, single irradiation or multiple irradiation is carried out, and the irradiation energy is 43.2J/cm 2 The irradiation energy is 32.4J/cm in the fractional irradiation group 2 And 10.8J/cm 2 Two parts, with 15min interval. After the irradiation is finished, the number of coated plates is counted, and the influence of the free radicals on the fractionated irradiation is judged. As shown in B in fig. 8, the survival rate of staphylococcus aureus was significantly increased when mannitol cleared OH even higher than the single irradiation group compared to the control group, indicating an important role of OH in fractionated exposure. Furthermore, in the presence of catalase, no significant difference was observed in the inactivation of the strain, indicating that hydrogen peroxide hardly participates in the inactivation of the strain. Interestingly, when the strain was directly irradiated in a single or divided dose without pre-incubation with scavenger (irradiation energy of 43.2J/cm) 2 The irradiation energy is 32.4J/cm in the fractional irradiation group 2 And 10.8J/cm 2 Two parts, with 15min interval. ) There was no difference between the mannitol group and the control group (C in fig. 8). This result indicates that intracellular OH is critical for water disinfection, not water OH.
Staphylococcus aureus is used as indicator bacteria, and irradiation energy is 43.2J/cm 2 The irradiation energy is 32.4J/cm in the fractional irradiation group 2 And 10.8J/cm 2 Two parts, with 15min interval. Under fractionated irradiation conditions, the ROS and GSH changes over time after the first irradiation are determined. ROS is determined by a fluorescence probe DCFH-DA method, and GSH is determined by a colorimetric method (purchased from Nanjing institute of bioengineering). As shown in D and E in fig. 8, the fluorescence intensity decreased with increasing time interval, indicating a decrease in ROS in the water sample. In contrast, GSH content increases with increasing time. Glutathione is a free radical scavenger and cytoprotectant. Thus, an increase in glutathione levels may reduce cell damage. Long time interval is not favorable for multiple irradiation inactivationThe reason for the strain may be due to a gradual decrease in intracellular ROS and a gradual increase in GSH content.
Claims (7)
1. A method of sterilizing a microorganism comprising the steps of: and irradiating the microorganisms by using a UVA-LED light source in a single or multiple irradiation mode.
2. A sterilization method according to claim 1, characterized in that: the maximum absorption wavelength of the UVA-LED light source is 365nm;
the irradiation power of the UVA-LED light source is more than or equal to 180mW/cm 2 。
3. A sterilization method according to claim 1 or 2, characterized in that: the times of the fractionated irradiation are 2 times; the irradiation dose of the first time is larger than that of the second time, and the time interval of the sub-irradiation is 5-30 min.
4. A sterilization method according to claim 3, characterized in that: the interval time of the fractionated irradiation is 15min.
5. Sterilization method according to any one of claims 1 to 4, characterized in that: the microorganism is at least one of staphylococcus aureus, escherichia coli and listeria monocytogenes.
6. Sterilization method according to any one of claims 1 to 5, characterized in that: the microorganisms are microorganisms in water.
7. The sterilization method according to claim 6, wherein: the pH value of the water is 4-10.
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| US20110226966A1 (en) * | 2008-11-21 | 2011-09-22 | The University Of Tokushima | Outdoor water treatment apparatus to kill bacteria with ultraviolet light |
| JP2019129751A (en) * | 2018-01-31 | 2019-08-08 | マルハニチロ株式会社 | Fish storage method and fish storage apparatus |
| CN111511409A (en) * | 2017-10-11 | 2020-08-07 | 香港科技大学 | Asynchronous intermittent illumination for rapid surface disinfection |
| CN111787956A (en) * | 2018-01-26 | 2020-10-16 | 优格创新与发展研究 | Photo-biological conditioning device |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| US20110226966A1 (en) * | 2008-11-21 | 2011-09-22 | The University Of Tokushima | Outdoor water treatment apparatus to kill bacteria with ultraviolet light |
| CN111511409A (en) * | 2017-10-11 | 2020-08-07 | 香港科技大学 | Asynchronous intermittent illumination for rapid surface disinfection |
| CN111787956A (en) * | 2018-01-26 | 2020-10-16 | 优格创新与发展研究 | Photo-biological conditioning device |
| JP2019129751A (en) * | 2018-01-31 | 2019-08-08 | マルハニチロ株式会社 | Fish storage method and fish storage apparatus |
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