R ESEARCH ARTICLE

doi: 10.2306/scienceasia1513-1874.2019.45.458ScienceAsia 45 (2019): 458–464

Evaluation of the sterilization effect of a newmercury-free UVC light sourceHan Gaoa,b, Tongkuo Yuanb,c, Jian Zhaod, Hongye Liue, Huancai Yina,b,∗

a University of Science and Technology of China, Hefei, Anhui 230026 Chinab CAS Key Lab of Bio-Medical Diagnostics, Suzhou Institute of Biomedical Engineering and Technology,

Chinese Academy of Sciences, Suzhou, Jiangsu 215163 Chinac School of Life Sciences, Shanghai University, Shanghai 200433 Chinad NovelUv Co., Ltd, Shanghai 201108 Chinae Clinical Laboratory, Changchun Center Hospital, Jilin, Changchun 130051 China

∗Corresponding author, e-mail: yinhc@sibet.ac.cnReceived 13 May 2019Accepted 29 Oct 2019

ABSTRACT: So far, short-wave UV produced by low-pressure mercury lamps has been widely applied in sterilization,wastewater treatment, and bioassays, which could be due to its low cost, high conversion efficiency, and small size.However, metal pollution is caused by the evitable releasing of mercury. In this study, a new mercury-free lamp wasdeveloped, which generated UV light by the electron beam excitation (EBE) of YPO4:Bi3+ under vacuum conditions.Such lamps emit light at 241 nm and possessed a photoelectric effect. Excellent sterilization effect was obtained inCandida albicans, Staphylococcus aureus, Pseudomonas aeruginosa, and Escherichia coli, in the absence of ozone. Theseeffects positively correlated with the treatment distances and time. Furthermore, oxidative stress was found to playa major role in the sterilization process of our light sources, since no production of ozone was detected during eachtreatment. After all, EBE lamps could be a promising tool for sterilization, and our results provided the theoretical basisfor its microorganism-killing effects.

KEYWORDS: UV, mercury-free lamp, electron beam excitation (EBE) UV lamp, ozone-free, oxidative stress

INTRODUCTION

Nowadays, UV light sources have been widely ap-plied in industrial, agriculture, and medical fields,due to their fluorescent, biological, photochemicaland photoelectric effects1. In particular, short-waveUV with wavelengths of 200–280 nm has been founduseful in sterilization, wastewater treatment andbiological detection2. Among them, low-pressuremercury lamps producing 254 nm UV light weremostly accepted, which could be due to their longlifetime, high photoelectric conversion efficiency,mature manufacturing process, and low cost3. Un-fortunately, these lamps are being criticized for theirreleasing of mercury, which cause irreversible fataldamage to living organisms4. According to Mina-mata Treaty, mercury-containing UV light sourceswill be gradually replaced in 2020. Thus more andmore attentions are being paid on the developmentof mercury-free lamps.

The first developed light sources are mainlylight-emitting diodes (LEDs), which have advan-tages of long life, small size, short start-up time,

and no pollution5, 6. Despite the long-term devel-opment, UVC-producing LEDs are limited by theircomplicated manufacturing process, high cost, lowphotoelectric conversion efficiency, and severe heatradiation7. In fact, the thermal emission cathodeproduced by LG Company, which are of the most ma-tured manufactory techniques, only exhibits a lowphotoelectric conversion efficiency at 2%. Hencean efficient, non-polluting, and long-lifetime UVsources are still in demand.

Based on the recent publications, UV light couldbe generated by electron beam excitation of phos-phate, rare earth-doping material and ZnO8. Withsimilar mechanisms, Oto et al developed new UVlamps by electron beam excitation of AlGaN quan-tum wells, which exhibited a high power of 100 mWat 240 nm9. Max Shatalov et al used AlGaN todevelop a UV light source with a wavelength of278 nm10. Compared with UV LED, EBE-UV lightsources possessed simple production processes, nothermal effect, and high photoelectric conversionefficiency, thus exhibiting a promising application

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HV

UVC

VACUUM

ELECTRONBEAM

LUMINESCENT

MATERIAL

ELECTRON

SOURCE

(a)

Fig. 1 (a) Schematic diagram of YPO4:Bi3+ EBE-UV lamp,HV = high voltage; (b) UV lamp body diagram.

prospect11, 12.In this study, a small sterilization device was

produced with an EBE-UV light source. After char-acterization of photoelectric conversion efficiencyand wavelength, the sterilization properties of thislamp were tested with four microorganisms, includ-ing Candida albicans, Staphylococcus aureus, Pseu-domonas aeruginosa, and Escherichia coli. Afterthen, the main sterilization mechanism of this lampwas detected, to provide the basis for its applicationin sterilization.

MATERIALS AND METHODS

EBE-UV characterization

The EBE-UV light was produced by electron beamexcitation on YPO4:Bi3+, and the cold cathode isprotected by vacuum environment4. In brief, theelectrons hit the matrix of YO4:Bi3+, causes energytransfer, and excites the fluorescence. With specificoptical filter, the wavelength of UV would be limitedaround 241 nm. By DC Ammeter and Voltmeter, theinput power of the UV lamp is 1.3 W, and outputpower is determined to be 52.13 mW. The photo-electric conversion rate η is calculated accordingto η =�

Plight/Pe

�

× 100%, where Plight and Pe are

Fig. 2 (a) Schematic diagram of sterilization device;(b) physical map of the equipment.

the electrical and optical powers, respectively. Aftercalculation, η should be 4.01%.

The schematic and digital illustrations of thelamp are shown in Fig. 1. Its emission spectra wereevaluated by a UV spectrometer (Gentec, Florida,USA) in a wavelength range of 200–360 nm. Ir-radiance power of EBE-UV was measured using aPH100-SiUV Probe accumulated UV meter (UshioInc., Japan), after the switch was turned on, theUV meter was set as zero first. Then the irradiancepower was randomly detected at 5 different posi-tions of the same height to calculate the averagevalue.

Sterilization device

To test the efficiency of UV light source, a smallsterilizer with adjustable distance and treatmenttime was designed. The structure is shown in Fig. 2.

The sterilizer consists of two parts: tank andshell. The tank was made of corrosion-resistantstainless-steel material (ISO11), with a hole in theupper field. The light sources located inside theshell, right on the upper of the tank holes. The light-ing time, interval time, and automatic light-off couldbe controlled automatically. A transparent viewingwindow is present on the outer shell, to observe

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the entire illumination process. Tin foil was usedto cover the shading treatment on the observationwindow, to avoid the light resurrection during UVtreatment. Finally, adjust the distances between thelight sources and the plane of the lowest-end plates,to determine the sterilization characteristics.

Culture of microorganisms

C. albicans (ATCC10231), S. aureus (ATCC25923),P. aeruginosa (ATCC9027), and E. coli (ATCC25922)obtained from American Type Culture Collection,were cultured at 37 °C in tryptic soy broth (pH 7.2,BD Diagnosis Systems, Sparks, MD)13–15. All thebacteria and yeast kept on a rotary shaker at220 rpm were cultivated for 8 h. The concentrationof cells was 109 CFU/ml as assessed by 600 nmabsorption.

Evaluation of sterilization effect

Microorganisms, including yeast (C. albicans),Gram-positive bacteria (S. aureus), and Gram-negative bacteria (E. coli and P. aeruginosa), wereused to evaluate the sterilization effect of EBE-UV. After reaching the set concentrations of 109

CFU/ml, the four strains of microorganisms werediluted by 1:10 00016. 100 µl of dilution was cul-tured on the solid nutrient agar medium, and thenimmediately placed in the sterilization device. Allagar media were treated with UV at 15 cm from theUV light source at 37 °C. According to the toleranceof each strain, different time gradients were usedfor C. albicans (0.0, 1.0, 2.0, 3.0, and 4.0 min),P. aeruginosa (0.0, 1.0, 1.5, 2.0, and 2.5 min),S. aureus (0.0, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, and3.5 min), and E. coli (0.0, 5.0, 10.0, 15.0, 20.0, 25.0,30.0, 35.0, and 40.0 s). Then the survived colonieswere counted and compared with untreated control.The differences were divided by the total colonies,and the results were defined as the disappearancerate. The obtained values were plotted against theirradiation dose (K , (mW.s/cm2)) of UV, which wascalculated as K = I × T/103, where I and T are theirradiation intensity (µW/cm2) and irradiation time(s), respectively. To prevent cells from reactivation,the processes of UV treatment and cultivating wereshaded away from light.

Intracellular ROS assay

After sterilization, the microorganisms on the platewere scraped off with 1.0 ml PBS. About 100 µlof the treated suspensions were taken out fortotal number counting, and the remaining sus-pensions were collected into 1.5 ml eppondorf

tubes (EP), to measure intracellular ROS with com-mercial Kits (Beyotime, China)17. 10 µm 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA)was added to the treated suspensions and incubatedfor 20 min at 37 °C. Then the cells were washed andsuspended in 1.0 ml of PBS. The pictures of accu-mulated 2′,7′-dichlorofluorescein (DCF) inside cellswere taken with a digital camera (Olympus Corpo-ration, Tokyo) and a Zeiss axiophot fluorescence mi-croscopy (Carl Zeiss, Oberkochen, Germany) witha green filter. After then, the fluorescence of sus-pension was detected with a Multi-Mode microplateReader (BioTek, US) using an excitation/emissionwavelength of 488/525 nm. Relative fluorescenceintensity (RFI) of the intracellular ROS level wasnormalized to the total number of microorganism18,and then expressed as the fold increase of controlgroup.

RFI=DCFEX/(1−Disappearance%)

DCFControl,

where DCFEX and DCFControl are the detected fluores-cence of the treated groups and the control group,respectively.

Ozone content detection

The light resource of the UV sterilizer was fixed at15 cm from the bottom of the plate. Since all themicroorganisms died after 20 min irradiation. Theozone production was detected after then. Ozoneinstrument (JSA8–O3, Shenzhen, China) was usedto detect the ozone content at 6 random points toobtain an average value. The enclosed space of thisdevice is 15 cm in height, 23 cm in length, 10 cmin width. The total volume is about 3.45 l. As apositive control, the ozone produced by an ordinary254 nm lamp (TUV, 8 W, Philips) was detected in thesame condition, and the results indicated a 2.7 ppmproduction of ozone.

DNA damage analysis

After UVC irradiation for indicated time, the fourmicroorganisms were collected and extracted fortheir DNA with a commercial kit. The degree of DNAdamage was determined by the alkaline versionof the Comet Assay (single-cell gel electrophoresisSCGE) as described by Lanier et al19. The irradia-tion time was selected for each microorganism withthe occurrence of significant death. The obtainedcomets were visualized with a fluorescent micro-scope (Carl Zeiss, Oberkochen, Germany) at 40×magnification (Achroplan and ECPlan-Neofluar ob-jectives) with the red filter (excitation 546 nm and

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Fig. 3 Emission spectra of YPO4:Bi3+ EBE-UV lamp.

Table 1 Radiation intensity at different distance (DIST)from the lighting tube.

DIST/cm 3 6 9 12 15Irradiance (µW/cm2) 651.1 280.6 139.1 84.2 55.5

emission 590 nm). The obtained images were anal-ysed using AxioVision software version 4.7.2 (CarlZeiss, Oberkochen, Germany).

Statistical analysis

All data represent here are mean±SD of three inde-pendent experiments with each in triplicate (n= 9).Statistical analysis was performed using SPSS Statis-tic 15.0 Software (SPSS Inc., Chicago, Illinois, USA).Comparisons were conducted between control andtreated groups with student’s t-test, *p < 0.05 wasconsidered significant.

RESULTS

Characterization of EBE-UV light sources

The diameter of the front-edge of UV light sourceis 50 mm, and the length of the whole light sourceis 155 mm. The emission spectrum is shown inFig. 3, which indicates a core wavelength of 241 nmwith a half-peak width of 15 nm. The photoelectricconversion efficiency was determined to be 4.01%.After 1000-h operation, the photoelectric conver-sion efficiency will be reduced by about 75%. The ir-radiance at different distances from the light sourceis shown in Table 1, which decreases gradually withthe increase in distance.

Evaluation of sterilization effect

To evaluate the microorganism-killing effect ofdeep-UV light, four strains of microorganisms(yeast, Gram-positive and Gram-negative bacteria)were irradiated with 241 nm UV at different doses.

It could be seen that the sterilization efficiencyof this EBE-UV light was dose-dependent, and allthe microorganisms could be killed under the ir-radiation lower than 66.6 mW.s/cm2. As shownin Fig. 4a, the most tolerant microorganism wasC. albicans, while E. coli was the most easily killedbacteria (Fig. 4d). The tolerance of P. aeruginosaand S. aureus was found to be between C. albicansand E. coli (Fig. 4bc).

Sterilization mechanism

The changes of ROS curve in the four microorgan-isms after UV treatment were shown in Fig. 5a. Asthe irradiation time and dose increase, the levels ofROS increase to different degrees for each microor-ganism. To more intuitively show the effect of irra-diation on cells, the fluorescence intensity of DCF incells was photographed by fluorescence microscopyin the experiment, and the results were shown inthe following Fig. 5b, which became brighter withthe increase of treatment time. Surprisingly, noproduction of ozone was detected during the treat-ment of EBE-UV lights, as the data kept below zeroduring the detection. In addition, significant DNAdamages were noticed after UVC irradiation, as DNAmigration exhibited an obvious comet style (Fig. 6and Table 2).

DISCUSSION

With the development of science and technology,the demands for human health continue to increase.As an important environmental pollutant, mercuryis posing a huge threat to human health due totheir extensive applications in the industry20. On19 January 2013, the United Nations EnvironmentProgramme adopted Minamata Convention, to re-duce the releasing of mercury in the world21. Forthe UV light sources, the convention clearly statesthat after 2020, low-cost mercury lamps will beunavailable. Hence all countries are developinghigh-efficiency and mercury-free UV light sources,to occupy a huge market including lighting, steril-ization, and wastewater treatment.

In this study, a new sterilization device wasconstructed with new mercury-free UV sources.The lamp has a maximum emission wavelength of241 nm with a half-width of 15 nm. The pho-toelectric conversion efficiency was calculated tobe 4%. After improvement on the matrix, muchhigher photoelectric conversion efficiency of even12% could be achieved, which is much higher thanLG’s commercial products (data to be published).

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462 ScienceAsia 45 (2019)

Fig. 4 The sterilization efficiency at 241 nm was examined by disappearance of (a) C. albicans, (b) P. aeruginosa,(c) S. aureus, and (d) E. coli. Data are expressed as means±SD of 3 samples, ***p < 0.001 compared with untreatedcontrol.

S. aureus

E. coli

Control 1 min 1.5 min 2.5 min

Control 10 s 20 s 40 s30 s

Control

Control

1 min

1 min

2 min

1.5 min

3 min 4 min

2 min 2.5 min

C. albicans

P. aeruginosa

(b)

Fig. 5 (a) Intracellular ROS concentration after the treatment with 241 nm EBE-UV lamp in yeast, Gram-positive,and Gram-negative bacteria: ROS value of C. albicans (�), P. aeruginosa ( ), S. aureus (Î), and E. coli (È). (b) DCFfluorescence produced by bacteria with fluorescence microscope.

In the previous reports, the effective radiationpower of conventional sterilization lamp (254 nm)was 150 mJ/cm2 (1.5×103 mW · s/cm2), whichcould kill C. albicans up to 10−5 CFU/ml22. The doseused by Mohr H and Gravemann U was even higher,

300 mJ/cm2 (3.0×103 mW · s/cm2) to inactivate10 bacteria species in platelet concentrates23. Onthe other hand, Dotson et al used doses from 0.1–0.2 J/cm2 (1.0×103 to 2.0×103 mW · s/cm2) fordrinking water disinfection24. All these irradiation

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Table 2 Analytical results of comet assay.

SampleL-head L-tail L-comet Head-DNA Tail-DNA Tail Olivetail(µm) (µm) (µm) (%) (%) moment moment

C. albicans 0 min 27.0±2.4 4.3±0.6 31.4±3.3 97.53±3.96 2.46±0.85 0.09±0.02 0.32±0.02C. albicans 1 min 31.6±3.8 37.7±4.0* 69.3±9.8* 73.33±5.95 26.66±3.05* 8.93±2.08* 9.20±2.92*

C. albicans 3 min 27.3±3.6 33.5±5.5* 60.8±4.2 73.54±8.57 26.45±3.43* 10.72±1.99* 4.03±0.12*

P. aeruginosa 0 min 25.7±3.4 3.0±1.1 28.7±3.7 99.95±8.78 0.04±0.02 0.02±0.003 0.01±0.09P. aeruginosa 1 min 23.9±3.6 21.7±4.8* 45.7±3.3 79.37±6.99 20.62±3.01* 4.33±2.02* 3.93±1.33*

P. aeruginosa 2 min 19.7±3.9 29.8±6.2* 49.5±4.8 38.10±4.22* 61.89±5.78* 17.95±2.04* 9.98±1.96*

S. aureu 0 min 27.1±2.4 3.7±1.6 30.7 ±7.6 99.93±12.55 0.06±0.45 0.009±0.0003 0.009±0.0003S. aureu 0.5 min 26.0±8.8 18.7±7.3* 44.7±6.9 84.28±7.98 15.71±4.02* 2.82±1.78* 2.73±1.71*

S. aureu 2 min 25.6±5.8 50.9±7.4* 76.5±9.7* 63.83±4.72 36.16±3.28* 18.08±9.14* 12.81±7.65*

E. coli 0 min 23.2±3.3 4.0±1.4 27.2±4.1 99.97±9.38 0.02±0.01 0.007±0.003 0.003±0.002E. coli 10 s 19.4±4.8 3.9±2.4 23.4±8.6 99.92±27.72 0.07±0.02 0.002±0.001 0.007±0.006E. coli 40 s 19.4±4.0 9.6±2.1* 28.9±5.1 74.16±8.23 25.83±7.77* 2.32±1.54* 2.56±1.35*

* p < 0.05 versus control.

Fig. 6 Representative comet assay images for the fourstrains: C. albicans, P. aeruginosa, S. aureus, and E. coliafter the treatment with EBE-UV light.

doses were much higher than that in this experi-ment, where the new UV sources killed all the fourmicroorganisms after irradiation of 20 min and thedose reached 66.6 mW · s/cm2. Such phenomenoncould be due to the narrow spectral peak at 241 nmcompared with broad-spectrum of traditional lamps.

According to literature, DNA damage, ROS ef-fects, and production of ozone could be the rea-sons of UV sterilization. In this experiment, ROSeffects and DNA damages have been found in thisexperiment (Fig. 5 and Fig. 6). However, no ozonewas detected after the irradiation of UV lights for20 min. Such phenomenon could be occurred onthe fact that the emission spectrum of EBE lightsdoes not contain 185 nm, which ionizes the air to

generate ozone25. Although ozone can be usefulfor sterilization, it has strong oxidizing properties,which can cause ageing of equipment and is not suit-able for human environment26, 27. In this respect,the application of new UV light sources could beadvantageous.

CONCLUSION

In this paper, a novel sterilization device was con-structed with EBE-UV light sources, which gen-erated UV light with an electron beam to exciteYPO4:Bi3+ material. This device had a narrow emis-sion peak (241±25 nm), high photoelectric con-version efficiency (4.01%), environmental friend-liness (without mercury) and low cost (around0.5 $/mW). This device exhibited an excellent sen-sitization effect at 15 cm from the light source, withoxidative stress as the major mechanism. Henceit has great application prospects for the surfacesterilization of objects.

Acknowledgements: This work was supported bygrants from National Science and Technology MajorProject of China (2017ZX10302301-003) and NationalKey R&D Programme of China (2017YFF0108600 &2017YFC0110400).

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