Protocol for Determining Ultraviolet Light Emitting Diode (UV-LED

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Environmental Measurements Methods

Protocol for Determining Ultraviolet Light Emitting Diode (UV-LED) Fluence for Microbial Inactivation Studies Ataollah Kheyrandish, Madjid Mohseni, and Fariborz Taghipour Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.7b05797 • Publication Date (Web): 01 Jun 2018 Downloaded from http://pubs.acs.org on June 1, 2018

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Environmental Science & Technology

Protocol for Determining Ultraviolet Light Emitting Diode (UV-LED) Fluence for Microbial Inactivation Studies

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Ataollah Kheyrandish, Madjid Mohseni, Fariborz Taghipour*

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Department of Chemical and Biological Engineering, The University of British Columbia,

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2360 East Mall, Vancouver, BC V6T 1Z3, Canada *[email protected]

Abstract

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Determining fluence is essential to derive the inactivation kinetics of microorganisms and to

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design ultraviolet (UV) reactors for water disinfection. UV light emitting diodes (UV-LEDs) are

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emerging UV sources with various advantages compared to conventional UV lamps. Unlike

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conventional mercury lamps, no standard method is available to determine the average fluence of

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the UV-LEDs, and conventional methods used to determine the fluence for UV mercury lamps

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are not applicable to UV-LEDs due to the relatively low power output, polychromatic

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wavelength, and specific radiation profile of UV-LEDs. In this study, a method was developed to

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determine the average fluence inside a water suspension in a UV-LED experimental setup. In

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this method, the average fluence was estimated by measuring the irradiance at a few points for a

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collimated and uniform radiation on a petri dish surface. New correction parameters were

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defined and proposed, and several of the existing parameters for determining the fluence of the

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UV mercury lamp apparatus were revised to measure and quantify the collimation and

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uniformity of the radiation. To study the effect of polychromatic output and radiation profile of

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the UV-LEDs, two UV-LEDs with peak wavelengths of 262 nm and 275 nm and different

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radiation profiles were selected as the representatives of typical UV-LEDs applied to microbial

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inactivation. The proper setup configuration for microorganism inactivation studies was also

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determined based on the defined correction factors.

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Keywords: UV disinfection; UV-LED; Fluence rate determination; Protocol; Water treatment;

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Degradation kinetics.

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1 Introduction

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Ultraviolet (UV) irradiation has emerged in the past years as one of the best water treatment

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alternatives, with many major cities, particularly in North America and Europe, adopting UV as

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their primary disinfection process. Recently, a newly employed UV radiation source is available

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for water treatment applications—ultraviolet light emitting diodes (UV-LEDs). UV-LEDs have

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many different features in terms of operation and radiation output compared to conventional UV

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lamps, including a smaller footprint, better robustness, potentially longer lifetime, instant on/off

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ability, tunable output wavelength, various radiation profiles, and mercury free structure

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Despite these advantages, the specific characterization, such as radiation profile, spectral power

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distribution, and operational requirements of UV-LEDs produce significant challenges to

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accurately measuring their efficiency and performance for water treatment applications.

2


1–4

.

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The inactivation kinetics study of the waterborne microorganisms usually takes place in a

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petri dish in a bench-scale setup wherein the petri dish is irradiated with UV radiation for

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different time periods. To determine the UV response of different microorganisms, different

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approaches have been taken in the literature. In some studies, the UV response is reported as a

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function of nominal or measured radiant power of the radiation source (e.g., 5). Since the radiant

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power of UV lamps is a function of the operational condition6 and the delivered radiation to the

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microorganism is dependent on the setup configuration, the results of these studies must be

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considered with caution. In some other studies, the UV response is presented as a function of

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exposure time (e.g.,

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inactivation kinetics of a microorganism is by determining the average fluence introduced to the

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target microorganism.

7–10

). However, the most accurate method to measure and report the UV

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Here we define two terms associated with the receipt of radiation, fluence rate and irradiance

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(the terms and symbols used in this article are described in Ultraviolet Applications

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Handbook)11. Fluence Rate (mW cm-2) is the radiant power passing from all directions through

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an infinitesimally small sphere of cross-sectional area, dA, divided by dA. Irradiance (mW cm-2)

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is the total radiant power incident on an infinitesimal element of surface of area dS containing

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the point under consideration divided by dS. For a parallel and perpendicularly incident beam

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(collimated beams), irradiance and fluence rate become identical. The appropriate term for UV

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inactivation of microorganisms is UV fluence rate, because a microorganism can receive UV

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rays from any direction. Consequently, if the fluence rate is constant over time, fluence (mJ cm-2)

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can be calculated as the fluence rate multiplied by the exposure time (s) of the sample.

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The collimated beam apparatus was established to facilitate the determination of the average

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fluence for the UV mercury lamps, and it has been adopted by the international ultraviolet

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association (IUVA) and the United States Environmental Protection Agency (US-EPA) as a

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standard protocol

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suspension, the sample needs to be positioned at least 20 cm away from the UV lamp, and petri

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factor is used to evaluate the uniformity of the incident radiation. At this distance, the incident

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radiation to the surface of the suspension was found to be collimated, which means that the

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fluence rate can be estimated by measuring irradiance 14. However, this method is not applicable

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to UV-LED systems. UV-LEDs have various radiation profiles

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power. At 20 cm, the incident radiation to the sample suspension is not effective to obtain

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meaningful inactivation in a reasonable time (few minutes), particularly for UV-resistant

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microorganisms. Thus, closer distances have been tried in the literature. At closer distances, the

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incident radiation might not be collimated due to the radiation profile of UV-LEDs (which

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usually have wide viewing angles of over 100°), and the reflected radiation from the suspension

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surface might not be negligible. While there have been several efforts studied in the literature for

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determining the fluence of UV-LEDs

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specific characteristics of UV-LEDs such as radiation profiles, spectral power distribution

78

(SPD), and operational conditions.

12,13

. In this method, to obtain uniform radiation distribution inside the sample

2,8,16–18

15

and a relatively low radiant

, there is no systematic approach considering the

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In this study, we developed a method to determine and measure the average fluence for

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microbiological experiments for various UV-LEDs by obtaining uniform radiation distribution

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inside a petri dish for microbial inactivation studies. Two UV-LEDs with radiation profiles that

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are representative of common UV-LEDs were selected, and the average fluence inside a petri

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dish at different setup configurations was determined. The definitions of the parameters proposed 4


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in determining the fluence of the mercury lamps are revised, and new parameters are proposed to

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measure and quantify the collimation and uniformity of the radiation. Development of these

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factors results in fluence determination by using the measured irradiance for studying the

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inactivation kinetics of the waterborne microorganisms.

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2 Methodology

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2.1 Setup configuration

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To measure the average fluence inside a petri dish and determine the UV response of a

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microorganism, a setup containing one UV-LED equipped with proper thermal management

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components was built and placed inside a box with UV absorbing wall surfaces (Figure 1). The

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UV-LED was secured to the ceiling of the box to minimize the reflection from the holder of UV-

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LEDs. In addition, the petri dish was placed on a lab jack under the UV-LED, which was used to

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regulate the distance between the UV-LED and petri dish/detector. This setup was designed to be

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modular to accommodate radiometry and actinometry experiments by interchanging the detector

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module (radiometry) and the petri dish module (actinometry)19.

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Figure 1. The schematic view for the setup of the radiometry and chemical actinometry experiments (in chemical actinometry experiments, the spectrometer was replaced with a petri dish containing the chemical actinometry suspension) – 1) UV-LED and its thermal management components, 2) Spectrometer, 3) Lab jack to change the UV-LED elevation, 4, 5) stepper motors and rails to move the detector, and 6) setup frame which was covered with a UV absorptive cloth.

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2.2 UV radiation source

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UV-LEDs’ radiation profile is a function of their structure-chip orientation and lens.

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Typically, UV-LEDs are categorized into two types based on their radiation profiles. UV-LEDs

106

with flip-chip have a heart-shaped radiation profile, and the maximum radiation intensity of these

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UV-LEDs is not normal to the UV-LEDs surface (LED1 in Figure 2). In contrast, UV-LEDs

108

with the lateral chip have a balloon-shaped radiation profile. The maximum radiation intensity of

109

these UV-LEDs is located normal to their surface (LED2 in Figure 2). A UV-LED from each

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type was selected to investigate the effect of the radiation profile on the uniformity and

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collimation of radiation (Table 1).

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112 113 114 115

Figure 2. Heart-shaped and balloon-shaped relative radiation profiles of LED1 and LED2. Most of the available UV-LEDs have a similar radiation profile to one of these UV-LEDs. Table 1. Studied UV-LED specification extracted from their manufacturers’ provided data sheet.

LED1 LED2 116 117

Peak Wavelength (nm)

Nominal Radiant Power (mW)

FWHM* (nm)

Forward Voltage (V)

Forward Current (mA)

Viewing Angle

Efficiency (%)

275 262

10 12.5

12 11

8.5 11.6

100 300

124 127

1.2 0.4

* FWHM: Full width at half maximum

2.3 Radiometry

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Incident irradiance distribution at different distances from the UV-LED was measured with a

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spectrometer (Ocean Optics USB2000+). The detector (model number: ILX-511B) measurement

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range was 200–850 nm with an optical bench entrance aperture of 50-micron width. Since the

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radiation profile of a UV-LED is directional, the detector was equipped with a cosine corrector

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(with Spectralon diffusing material) to assure irradiance measurements

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reads the normal vector of the incident photon to its surface which is equal to the irradiance at

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that point. The setup illustrated in Figure 1 was used to measure the incident irradiance

125

distribution. The detector was then mounted on a motorized linear stage with two axes that could 7


20

. The cosine corrector


126

move the detector on a planar surface with high resolution (1 mm) and measure the spectral

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incident irradiance on the surface of the petri dish to calculate the average incident irradiance on

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the surface of the petri dish. In addition, the average irradiance in different depths of the petri

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dish was measured when it was needed.

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2.4 Chemical actinometry

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The average fluence rate on the surface of the solution was measured with iodide-iodate

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chemical actinometry in which the degradation of triiodide represents the fluence rate inside the

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well-mixed actinometry solution. The absorption of the actinometry solution at the germicidal

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range is high enough 21,22 to absorb almost all of the photons in a thin layer on the top of the petri

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dish. On the other hand, since the actinometry solution can absorb radiation from all direction, it

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indicates the fluence rate. Thus, it was assumed that the fluence rate measured with the chemical

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actinometry is equal to the fluence rate on the surface of the solution on the solution side.

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The quantum yield of iodide-iodate actinometry is a function of the wavelength. Since UV-

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LEDs are a polychromatic radiation sources, the quantum yield for each UV-LED was corrected

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based on their SPD utilizing the reported quantum yield in the literature22–26.

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2.5 UV fluence calculation

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To determine the UV response of a microorganism, uniform fluence rate must be introduced

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to each microorganism in the suspension. Consequently, mixing the suspension and obtaining

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uniform radiation distribution inside the petri dish is essential. Obtaining collimated and uniform

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radiation beams on the surface of a petri dish and utilizing the low water depth in the petri dish

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are among the methods to increase the uniformity of radiation distribution inside the petri dish. 8


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However, achieving a completely uniform irradiance distribution is always a challenge. To

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evaluate the uniformity of the radiation distribution inside the petri dish, collimation and

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homogeneity of the radiation on the surface of the petri dish were measured at different distances

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from the UV-LED for LED1 and LED2, which have different radiation profiles. To quantify the

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uniformity and collimation of the incident radiation and to determine the effect of radiation

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absorption of the suspension and the radiation reflection of the suspension surface, some

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correction factors were defined, and some of these correction factors presented in the report have

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been defined for UV mercury lamps 12. In our study, new correction factors are introduced, and

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some of the existing factors for the UV lamp setup were redefined to fit the special

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characteristics of UV-LEDs radiation pattern and/or to increase the accuracy of measuring the

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fluence rate. These correction factors are as follows:

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Petri factor (PF)–due to the non-uniform incident radiation distribution on the petri dish

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surface, a non-uniform fluence distribution is introduced to the microorganisms’ suspension.

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Petri factor is defined as the ratio of average irradiance on the petri dish surface to the irradiance

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at the center point of the petri dish to quantify the uniformity of incident radiation on the surface

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of the suspension. A simplified method to estimate PF is presented by Bolton et al. 27 that utilizes

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irradiance measurements every 5 mm on two perpendicularly crossed lines intersecting at the

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center of the petri dish surface. To test the applicability of PF for the systems of UV-LEDs, in

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this study, the PF was calculated at different distances (0.1–30 cm - It was found later that the PF

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become independent of distance after 20 cm) from the LED1 and LED2 on a 5.1-cm diameter

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petri dish using the PF definition and the simplified method. The comparison between these two

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values represents the accuracy of the simplified method to measure the PF for UV-LED systems.

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Water factor (WF)–due to the UV absorption of the microorganism suspension, irradiance

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decreases as photons pass through the suspension based on the Beer-Lambert law. WF is defined

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as the ratio of average fluence inside the petri dish to the average fluence at the surface of the

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petri dish. Since UV-LEDs are polychromatic radiation sources, the WF was weighted based on

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the SPD of the UV-LED and spectral absorption of the suspension, and WF at the wavelength of

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𝜆𝜆 was calculated using the following equation:

175

176 177

𝑊𝑊𝑊𝑊𝜆𝜆 =

𝐼𝐼𝜆𝜆 ×(1−10−𝑎𝑎𝜆𝜆 ×𝑙𝑙 )

Equation 1

𝐼𝐼×𝑎𝑎𝜆𝜆 ×𝑙𝑙×ln(10)

Where 𝐼𝐼 (mW.cm-2) and 𝐼𝐼𝜆𝜆 (mW.cm-2.nm-1) are the total radiant power of the UV-LED and the radiant power at 𝜆𝜆 of the UV-LED, respectively, 𝛼𝛼𝜆𝜆 (cm-1) is the absorption coefficient of the

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suspension at 𝜆𝜆, and 𝑙𝑙 (cm) is the depth of the microorganism suspension. To derive the WF

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that all the radiation incident to the bottom of the petri dish is absorbed by the wall of the petri

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dish. The impact of the monochromatic assumption of the UV-LED on WF was also evaluated

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for LED1 and LED2.

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equation, it was assumed that the incident radiation on the petri dish surface is collimated and

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Divergence factor (DF)–due to the directional radiation of UV-LEDs, the incident radiation

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on a circular planar at different distances from the UV-LED is a function of distance, which

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means that a radiation gradient over the depth of the suspension exists. DF was defined as the

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ratio of the average fluence inside the petri dish in the absence of the suspension to the average

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fluence on the surface of the petri dish. DF was calculated with two methods. In the first method,

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the average fluence at different depths of the suspension was measured and integrated through

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the depth of the petri dish. In the second method, the point source assumption for UV-LEDs was

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made and the DF was calculated using the following equation:

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𝐷𝐷𝐷𝐷 =

𝐷𝐷

Equation 2

𝐷𝐷+𝑙𝑙

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where D (cm) is the distance between suspension surface and the UV-LED and l (cm) are the

193

suspension depth. These correction factors were measured at different distances from the UV-

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LED for different petri dish sizes and for LED1 and LED2.

195

Reflection factor (RF)–due to the differences in refractive indices of the suspension and air, a

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part of the incident radiation to the surface of the petri dish reflects back into the air. The

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reflected radiation is a function of the refractive indices of the two media and the incident angle

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of the photons. The refractive index is a function of temperature and wavelength, and the

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incident angle is a function of the distance between the UV-LED and the petri dish surface. In

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addition, the reflection factor at different distances was calculated by considering the incident

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angle distribution on the surface of the petri dish. In most microbial inactivation studies, UV-

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LEDs were considered a monochromatic radiation source, while the full width at half maximum

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(FWHM) of the spectrum of UV-LEDs was around 10 nm

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assumption on RF for LED1 and LED2 with different peak wavelengths was investigated in this

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study. In addition, the temperature impact on the refractive indices and consequently on the RF

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was investigated by using the refractive indices of water reported in the literature at different

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temperatures 28.

15

. The impact of monochromatic

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Collimation factor (CF) – due to the geometry of the radiation source, the incident radiation to

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the surface of the petri dish might not be collimated, resulting in a gradient of fluence inside the 11



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petri dish. With a collimated incident radiation, fluence rate can be estimated by measuring

211

irradiance. This concept has been used in conventional mercury lamp protocol to determine the

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average fluence rate on the petri dish surface by measuring the irradiance on the surface of the

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petri dish using a detector. In UV lamp protocol, collimation is achieved 20 cm away from the

214

UV lamp 14. Collimating the radiation for UV-LEDs has been tried recently by utilizing a column

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with a 3.3-cm diameter

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radiation collimation. In this study, we defined CF as the ratio of irradiance to fluence rate at the

217

surface of the petri dish to quantify the extent of collimation. CF of 1 means that the measured

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irradiance and fluence rate are equal and that a complete collimation occurred. Average

219

irradiance and average fluence rate were then measured by radiometry and chemical actinometry,

220

respectively.

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Consequently, the average fluence inside the microorganism suspension in a petri dish was

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calculated by using these correction factors, the measured irradiance, and the measured fluence

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rate on the surface of the suspension.

224

3 Results and discussion

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. However, no method was utilized to measure and quantify the

225

The correction factors were determined at different distances from the UV-LEDs for different

226

sizes of petri dishes to investigate whether an acceptable collimation and uniform radiation can

227

be obtained for the two UV-LEDs. The applicability of these factors was investigated for the

228

setup of UV-LEDs and, finally, a method was presented to determine the average fluence inside

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the petri dish for microbiological studies.

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3.1 Petri factor

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Comparing the calculated PF from the definition (using the physically validated model 19) and

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the simplified method, the results show a significant (up to 90%) difference between the

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calculated PFs from these two methods (Figure 3), while the acceptable uncertainty for PF is 5%

234

13

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irradiated area on the average irradiance estimation in the simplified method. In the simplified

236

method, the influence of a point irradiance measurement on the petri dish surface is considered to

237

be independent of its position, while the area irradiated with that irradiance is proportional to the

238

squared distance from the center of the petri dish. Another reason might be related to the detector

239

size and the measurement steps (5 mm intervals) in the simplified method. As explained in

240

previous research

241

Moreover, due to the non-uniform radiation distribution on the petri dish surface for the systems

242

of UV-LEDs, by decreasing the measurement step sizes, different PF values might be calculated.

243

Another reason might be related to the radiation distribution on the petri dish surface that is not

244

fully considered in the simplified method. In the simplified method, the irradiance is measured

245

on an 𝑥𝑥–𝑦𝑦 axis every 5 mm on the petri dish surface (Figure 4). For the other points on the petri

246

. One reason for this major difference might be related to not considering the effect of the

19

, the irradiance measurement is highly related to the detector surface size.

dish surface, e.g., the point with 𝑥𝑥𝑖𝑖 and 𝑦𝑦𝑗𝑗 coordinates (Figure 4), irradiance is estimated with the

248

geometric average between the irradiances of the (𝑥𝑥𝑖𝑖 , 0) and (0, 𝑦𝑦𝑗𝑗 ) points. However, the

249

the polar radiation symmetry. This approach of calculating the overall irradiance does not result

250

in an accurate average irradiance; while in the PF measuring method using the PF definition, the

247

irradiance at 𝑥𝑥𝑖𝑖 and 𝑦𝑦𝑗𝑗 coordinates are more similar to those of (𝑥𝑥́ 𝑖𝑖 , 0) and (0, 𝑦𝑦́𝑗𝑗 ) points due to

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average irradiance on the surface of the petri dish (using a radiation distribution model 19) is used

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instead of the average irradiance of limited points.

253

To improve the simplified method, polar coordinates with weighted irradiance on the area is

254

suggested. In this proposed method, irradiance is measured for a polar discretized mesh and

255

weighted for the mesh area with the following equation:

256

𝑃𝑃𝑃𝑃𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝 =

2𝜋𝜋 ∑𝑅𝑅 𝑟𝑟=0 ∑𝜃𝜃=0 𝐴𝐴(𝑟𝑟,𝜃𝜃)×𝐸𝐸(𝑟𝑟,𝜃𝜃)

Equation 3

𝐸𝐸(0,0)

258

where 𝐴𝐴(𝑟𝑟, 𝜃𝜃) (cm2) and 𝐸𝐸(𝑟𝑟, 𝜃𝜃) (mW.cm-2) are the area and the irradiance of the polar mesh at 𝑟𝑟

259

calculated based on the definition and the proposed method in this study using polar coordinates.

257

and 𝜃𝜃 (Figure 4), respectively. As shown in Figure 3, there is close agreement between the PFs

260 261 262 263

Figure 3. Calculated petri factor and coefficient of variation (CV) of a) LED1 and b) LED2 at various distances from the UV-LED for a petri dish with 5.1-cm diameter. The petri factor was calculated based on the PF definition, the simplified method, and the newly proposed method in the polar coordinate system.

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264 265 266

Figure 4. The big circle shows the petri dish surface. Filled/empty dots represent the detector position for irradiance measurement to calculate petri factor with the simplified method and the proposed method.

267

Although using the proposed method increased the accuracy of the PF determination, the PF

268

of LED1 with a heart-shaped radiation profile shows a PF equal or greater than 1 for some

269

distances from the UV-LED (as seen in Figure 3, this is because the irradiance is higher at

270

locations other than the center of the petri dish), while the non-uniformity of radiation

271

distribution at these distances was observed (the radiation distribution is provided in the

272

Supplementary Information). Thus, the PF fails to quantify the radiation uniformity for UV-LED

273

setup and needs to be revised. Based on the PF definition, PF is a unitless average irradiance on

274

the petri dish surface and is not a true representative of the uniformity. However, PF can show

275

the uniformity of radiation distribution in a collimated beam apparatus to some extent because of

276

the specific configuration of the collimated beam apparatus. We proposed the coefficient of

277

variation (CV) as a new parameter to represent the uniformity of radiation distribution for UV-

278

LEDs. This suggestion considers the various radiation profiles of UV-LEDs for any general

279

cases, including where there is no maximum irradiance at the center of the petri dish. 15



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280

The weighted CV is proposed to substitute the PF for UV-LED systems to measure the

281

uniformity of radiation on the petri dish surface. CV is the ratio of the standard deviation of the

282

sample to the mean value of the sample, and the standard deviation statistically shows the

283

dispersion/uniformity of a sample, but it is sensitive to the data magnitude. Dividing the standard

284

deviation of the samples by the mean value of the samples makes it possible to compare the

285

uniformity of different systems. CV is widely utilized in polymerization and microsphere size

286

distribution due to its advantages over other methods of dispersity evaluation 29,30.

287

The CV for UV-LEDs was calculated using the weighted measured irradiance distribution on

288

the surface of the petri dish in a polar coordinate system, and the ratio of the standard deviation

289

of these weighted measurements (samples) to their mean value was presented as CV

290

the PF, the smaller CV presents a better uniformity. A PF of 0.9 (90%) was presented as the

291

minimum acceptable PF by IUVA, and this value was also adopted by the US-EPA 13,27 for low-

292

pressure mercury lamps. To find the maximum value for CV, the radiation distribution of LED2

293

on the petri dish at different distances from the UV-LED was considered. This approach was

294

taken because the radiation distribution of LED2 on the petri dish surface is similar to that of

295

low-pressure mercury lamps. The CV and PF of the setup at different distances between the UV-

296

LED and petri dish are presented in Figure 3, and the maximum acceptable CV was determined

297

when the PF is higher than 0.9. Furthermore, a CV of 6.7% was determined to be the maximum

298

CV value to obtain a uniform irradiance distribution.

299

3.2 Water factor

31

. Unlike

300

To consider the radiation gradient through the depth of suspension, WF was calculated for

301

LED1 and LED2 with/without the monochromatic radiation assumption. With the 16


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monochromatic assumption, the WF was calculated at the peak wavelength of the LED1 (275

303

nm) and LED2 (262 nm) using Equation 1. Then, the calculated WF was compared to the

304

weighted WF utilizing the SPD of each UV-LED. As can be seen in equation 1, WF is a function

305

of the sample absorption, SPD of the source, and the depth of the microorganism suspension.

306

The impact of the sample depth is the same as that of the conventional UV dose determination

307

method, and it can be easily shown that for smaller depth the WF is higher. For the UV-LED

308

systems, the absorption of the sample at different wavelengths and polychromatic radiation of the

309

UV source may affect the WF calculation.

310

The results showed (the data are provided in the Supplementary Information) that at lower

311

absorbance (e.g. for a sample of E.coli with absorption of 0.0211 at 285nm), the monochromatic

312

assumption for both UV-LEDs is valid (differences were around 0.1%). However, for higher

313

absorption of the suspension, e.g., a high concentration of microorganisms32, the monochromatic

314

assumption caused more than 8% error. Hence, the UV absorption of the microorganism

315

suspension should be measured at different wavelengths for each experiment prior to considering

316

the monochromatic assumption for the experiments of the UV-LEDs.

317

3.3 Divergence factor

318

Figure 5 indicates that there is a minimum distance from the UV-LEDs after which the point

319

source assumption becomes valid and that the DF can be calculated from Equation 2. This

320

distance was 5.5 cm and 8 cm for LED1 and LED2 (tolerance = 1%), respectively. In addition, at

321

closer distances, the irradiance gradient along the suspension depth was considerable, leading to

322

non-uniform irradiance distribution inside the petri dish. Thus, a proper distance that is

323

dependent on the radiation profile, petri dish size, and the suspension depth of UV-LEDs must be 17



324

determined for each UV-LED setup to obtain a uniform radiation distribution inside the petri

325

dish. A sample of DF calculation is provided in the Supplementary Information.

326 327 328 329

Figure 5. Divergence factor calculated with the point source assumption (PS) for a 1-cm depth petri dish in different distances from the UV-LED and calculated divergence factor for LED1 and LED2, integrating the irradiance through the depth of the suspension.

330

3.4 Reflection factor

331

The effect of temperature, radiation profile, and SPD of the UV-LEDs were investigated on

332

the RF determination. The effect of the temperature on RF determination was negligible (less

333

than 1%) since the refractive index of the suspension of the microorganism (phosphate buffered

334

saline, PBS, and microorganisms) was not sensitive to temperature in the range of 10–30°C.

335

To investigate the validity of monochromatic assumption for UV-LEDs, RF was calculated

336

with a monochromatic assumption, and the results were compared to the weighted RFs based on

337

the SPD of the UV-LEDs. Furthermore, a negligible difference (0.7%) between these results

338

were observed, indicating that at relatively long distances to LED, the monochromatic

339

assumption is valid for reflection factor calculation of UV-LEDs.

18


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340

The impact of the radiation profile of UV-LEDs on the reflection factor was investigated by

341

changing the distance between UV-LED and petri dish to obtain different radiation distributions

342

and consequently different incident angles on the petri dish surface. RF was measured at

343

different distances from the UV-LED for a 5.1-cm diameter petri dish, and the reflection factor

344

was considered to be 2.5% for low-pressure mercury lamps. However, as shown in Figure 6, for

345

closer distances to UV-LEDs, the reflection factor is higher than 2.5%, meaning more radiation

346

were reflected from the petri dish surface compared to the UV-lamp setup. Although reflection

347

factor does not limit the setup designs, the reflection of the petri dish must be considered when

348

the irradiance measured with a detector is used to determine the average fluence inside the

349

suspension.

350 351 352

Figure 6. Reflectance (reflection factor = (100-reflectance)/100) calculated for LED1 and LED2 at different distances for a 5.1-cm petri dish.

19



353

3.5 Collimation factor

354

CF was used to find the extent of collimation at different distances (0.1–30 cm) from the UV-

355

LED. In addition, the impact of the radiation profile on the radiation collimation on the petri dish

356

surface was studied by calculating CF for LED1 and LED2 (Figure 7).

357

The fluence rate measurements for UV-LED systems have been made at 2–5 cm distances

358

between the UV-LED and the suspension with microorganism, as reported in the literature (e.g.,

359

8,10,33–35

360

indicating that the measured average irradiance or the measured average fluence rate must be

361

used with caution to estimate the average fluence inside the petri dish. Non-collimated radiation

362

on the petri dish surface causes two main issues for determining the average fluence rate. First,

363

the average fluence rate and average irradiance on the surface of the petri dish are not equal;

364

consequently, unlike the conventional method for mercury lamps, the average fluence rate cannot

365

be estimated by measuring the average irradiance with a detector. Second, non-collimated

366

radiation causes radiation gradients inside the petri dish, resulting in different radiation

367

pathlengths for photons and the reflection of photons from the petri dish wall. The radiation

368

distribution will not be uniform inside the petri dish, and this setup cannot be used for measuring

369

the inactivation kinetics of a microorganism. Determining the average fluence, in this case, needs

370

a complex model to consider the petri dish wall reflection and radiation pathlengths.

). However, as presented in Figure 7, poor collimation occurs at these distances,

371

The minimum distance to obtain CF of more than 0.99 was determined for LED1 and LED2

372

to be 12.5 cm and 12.3 cm, respectively. Even without a collimating column, quasi-collimated

373

radiation can be obtained from UV-LEDs, and this distance can be determined for different petri

374

dish sizes and UV-LED systems utilizing the physically validated model. Given the radiation 20


Page 20 of 39

Page 21 of 39


375

profile of the studied UV-LEDs covered the typical commercially available UV-LEDs, for a petri

376

dish with 5.1-cm diameter, a 13-cm distance provides a uniform fluence distribution inside the

377

petri dish for performing inactivation kinetics studies.

378 379 380

Figure 7. Collimation factor (irradiance–fluence rate ratio) for LED1 and LED2 at different distances from the UV-LED.

3.6 Fluence determination

381

Accurate determination of fluence is essential for establishing the kinetics of microbial

382

inactivation with UV. This, in turn, provides the necessary information for determining the log

383

inactivation of target microorganisms as a function of fluence and calculating the reduction

384

equivalent dose (RED) that is needed for the design and validation of UV reactors.

385

In this study, the average fluence in a petri dish for a UV-LED setup was determined by using

386

the average irradiance on the surface of the petri dish utilizing a spectrometer. Collimation and

387

uniformity of the incident radiation were evaluated by defining and utilizing the CF and the CV.

388

For CV lower than 6.7% and CF higher than 99%, the average fluence inside the petri dish was

389

calculated using the following equation:

21



390

𝐹𝐹�0 =

�� 𝐸𝐸 0 ×𝐷𝐷𝐷𝐷×𝑊𝑊𝑊𝑊×𝑅𝑅𝑅𝑅×𝑡𝑡

Page 22 of 39

Equation 4

𝐶𝐶𝐶𝐶

392

where, 𝐹𝐹�0 (mJ.cm-2) is the average fluence inside the petri dish, �𝐸𝐸��0� (mW.cm-2) is the average

393

parameters in Equation 4 were estimated based on the measured irradiance and fluence rate on

394

the surface of the petri dish.

391

incident irradiance on the surface of the petri dish, and t (s) is the exposure time. All the

395

For the studied UV-LEDs and a petri dish of 5.1 cm diameter, at distances greater than 12.5

396

cm from the UV-LED, collimation was achieved and all the DF, WF, and RF correction factors

397

were in a comparable range with those reported in the literature for the standard collimating

398

apparatus method for the mercury lamps. Thus, even without a collimating column, the

399

microbiological test can be performed at appropriate distances between the UV-LED and the

400

suspension surface. The average fluence rates at the distance of 13 cm from LED1 and LED2

401

were 0.01 mW.cm-2 and 0.02 mW.cm-2, respectively, and these values correspond to utilizing

402

about 3% of the radiant power of the UV-LED on the surface of the petri dish. Note that fluence

403

rate in the collimated beam apparatus for low-pressure mercury lamps is usually an order of

404

magnitude higher. Considering the low power output of current UV-LEDs, to achieve a

405

comparable fluence from a UV-LED system, longer exposure times, utilizing optical devices, or

406

using multiple UV-LEDs are recommended.

407

3.7 Proposed protocol flowchart for fluence determination in UV-LED

408

setups

409

The diagram presented in Figure 8 proposes a protocol for the estimation of the UV fluence

410

and calculation of related factors, as discussed in this study. Although radiation distribution on 22


Page 23 of 39


411

the surface of the suspension (e.g. petri dish) is affected by the radiation profile of the source, the

412

proposed protocol is independent of the UV source radiation profile, radiant power, and spectral

413

power distribution of the UV source. Thus, this protocol can be implemented for any UV

414

radiation source. The only consideration would be accurately measuring the irradiance

415

distribution and fluence rate on the surface of the suspension under study (e.g., microorganism

416

suspension in a petri dish). Consequently, all correction factors can be calculated independent of

417

the UV source radiation profile.

23



418 419

Figure 8. Proposed protocol for fluence (UV dose) determination for the system of the UV-LED.

420 24


Page 24 of 39

Page 25 of 39

421


4 Acknowledgments

422

This research was sponsored by the Natural Sciences and Engineering Research Council of

423

Canada.

424

5 Supporting information available

425

The information regarding the UV-LED characterization, the examples of actinometry and

426

radiometry experiments, and a glossary of terminology are included in the supplementary

427

information. A numerical example is also presented for calculating each correction factor.

428

6 References

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Jefferson, B. Evaluation of a UV-Light Emitting Diodes Unit for the Removal of

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Micropollutants in Water for Low Energy Advanced Oxidation Processes. Chemosphere

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Bulb Development on the Economic Viability of Ultraviolet Technology for Disinfection.

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Characterization and Operation of UV-LED for Water Treatment. Water Res. 2017, 122,

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Chevremont, A.-C.; Farnet, A.-M.; Coulomb, B.; Boudenne, J.-L. Effect of Coupled UV-

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A and UV-C LEDs on Both Microbiological and Chemical Pollution of Urban

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Li, J.; Hirota, K.; Yumoto, H.; Matsuo, T.; Miyake, Y.; Ichikawa, T. Enhanced Germicidal

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Effects of Pulsed UV-LED Irradiation on Biofilms. J. Appl. Microbiol. 2010, 109 (6),

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2183–2190.

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Bolton, J. R.; Linden, K. G. Standardization of Methods for Fluence (UV Dose)

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Determination in Bench-Scale UV Experiments. J. Environ. Eng. 2003, 129 (3), 209–215.

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Semiconductor Light-Emitting Diode. J. Environ. Eng. 2011, 137 (12), 1215–1218.

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Environmental Science & Technology Page 30 of 39 Coefficient of Variation (Petri Factor)

Water Absorption Spectrum

Weighted Water Factor

Inactivation

UV-LED Fluence Determination Protocol Radiation Uniformity

Fluence Radiation Collimation

Collimation Factor (New)

In Depth Radiation Gradient

Divergent Factor (New Method)

Surface Radiation Reflected

Reflection Factor (New Method)

Accurate fluence determination

Reliable and Reproducible Photo-Kinetic Results


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Page 33Environmental of 39 Science & Technology


Environmental Science & Technology Page 34 of 39


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Page 39 of 39

Start


Measure incident average fluence rate with KI/KIO3 actinometry Æ '4

Revise the setup configuration (increase the distance between radiation source and petri dish, use smaller petri dish, or use optical devices)

Report CV

Measure incident irradiance distribution on the petri dish surface using a cosine corrector equipped detector

Calculate the average incident irradiance Æ' 4

CV90% Yes

Calculate the reflection factor based on the incident irradiance distribution Æ RF

Reduce the sample depth

Calculate collimation factor L

Report RF

No

Report DF

¾$ ¾,

CF>99% Yes

Measure the spectral absorbance of the sample

Report CF

Calculate water factor w/o monochromatic assumption + :s F sr?Ô ß ; 9( L += H Ž•:sr; s F sr?Ôß 9( L = H Ž•:sr;

Report WF

Í 9( L 9(

No

(4 L Í

' 4 H &( H 9( H 4( H P %(

Yes

(4 L

' 4 H &( H 9( H 4( H P %(


Report the Fluence

Protocol for Determining Ultraviolet Light Emitting Diode (UV-LED

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