Variation in the fluorescence intensity of thermally-exposed bacterial bioaerosols

Journal of Aerosol Science 65 (2013) 101–110

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Variation in the fluorescence intensity of thermally-exposed bacterial bioaerosols Jae Hee Jung a,b,n, Jung Eun Lee c,nn a Center for Environment, Health, and Welfare Research, Korea Institute of Science and Technology (KIST), Hawolgok-dong, Seongbuk-gu, Seoul 136-791, Republic of Korea b Department of Electrical Engineering, California Institute of Technology, 1200 E. California Blvd., Pasadena, CA, 91125, USA c Han-River Environment Research Center, National Institute of Environmental Research (NIER), Yangseo-myeon, Yangpyeong-gun, Gyeonggi-do 476-823, Republic of Korea

a r t i c l e i n f o

abstract

Article history: Received 21 May 2013 Received in revised form 17 July 2013 Accepted 17 July 2013 Available online 2 August 2013

This study describes the real-time fluorescence characteristics of bacterial bioaerosols (Escherichia coli and Bacillus subtilis) thermally inactivated to produce various degrees of cellular culturability. Bacterial bioaerosols were exposed to various temperatures for very short times in a thermal electric tube furnace and then passed into an aerosol fluorescence measurement system that measured the ultraviolet (UV) light-induced fluorescence intensity of airborne particles in real time. The fluorescence of particles in the optical sensing zone was continuously measured with two photomultiplier tubes (PMTs) equipped with optical filters to detect radiation in the UV and visible (Vis) bands. The results showed that both UV- and Vis-fluorescence intensities decreased with increasing deactivation temperature. Also, the ratio of UV- to Vis-fluorescence decreased with increasing temperature for each bacterial bioaerosol. Under the same experimental conditions, we found that the airborne aromatic amino acids (L-tryptophan and Ltyrosine) and ovalbumin particles showed similar reduction trends in their fluorescence characteristics, compared with the test bacterial bioaerosols. These results provide basic information on the feasibility of intrinsic fluorescence measurements for real-time characterization of biological particles. & 2013 Elsevier Ltd. All rights reserved.

Keywords: Real-time detection Fluorescence Bioaerosol Aerosol fluorescence sensor

1. Introduction Biological aerosols or biologically-derived airborne contaminants (termed bioaerosols) continue to threaten the public health and the environment (Eduard et al., 2012). Exposure to bioaerosols is relevant to human disease and various health problems associated with acute toxic reactions, allergies, and asthma (Cox & Wathes, 1995; Douwes et al., 2003; Pieckova & Jesenska, 1999). The importance of research on harmful bioaerosols has increased rapidly after the 2001 anthrax bioterrorism incidents in the United States (Jernigan et al., 2001; Yung et al., 2007). Recently, the pandemic outbreak of H1N1 influenza A in 2009 has served as a reminder of the importance of bioaerosol monitoring and transfer prediction research (Brownstein et al., 2009; Fraser et al., 2009; Yang et al., 2009). However, the detection and identification of bioaerosols (especially pathogenic biological aerosols) is still challenging. Conventional methods for monitoring bioaerosols

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Corresponding author. Tel.: +82 2 958 5718; fax: +82 2 958 5805. Corresponding author. Tel.: +82 31 770 7238; fax: +82 31 773 2268. E-mail addresses: [email protected] (J. Hee Jung), [email protected] (J. Eun Lee).

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0021-8502/$ - see front matter & 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.jaerosci.2013.07.008

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include two stages: sample collection and laboratory analysis. These methods are both labor-intensive and time-consuming, which often leads to a limited number of samples or low accuracy in the rate of detection (Jung et al., 2011). Ideally, a bioaerosol detection system should be capable of rapidly detecting suspicious airborne particles with a high true-positive rate and confirm the identity of those bioagents at relatively low concentrations, compared with background atmospheric materials (Sivaprakasam et al., 2009). Among the conventional methods, polymerase chain reaction (PCR)-based analyses and enzyme-linked immunosorbent assays (ELISAs) are quantitative and accurate (Ho, 2002). However, prior to identification, these methods require a variety of sample processing steps to remove inhibiting factors introduced while acquiring the ambient sample (Madigan et al., 2003; Murray et al., 2003; Sivaprakasam et al., 2009). In the search for methods by which an ambient environment can be continuously monitored for potentially harmful bioaerosols, particle fluorescence optical sensors, based on ultraviolet laser-induced fluorescence (UV-LIF), have been used in rapid front-end warning/alarm systems. Such systems have been deployed for many years, affording genuine, real-time bio-detection in several military defense systems (Hill et al., 1995; Ho, 2002; Pinnick et al., 1995), including for example, the fluorescence aerodynamic particle sizer (FLAPS) developed by the Canadian Defense Ministry (Agranovski et al., 2003b; Ho, 2002), the ultraviolet aerodynamic particle sizer (UVAPS) manufactured by TSI Inc. (Hairston et al., 1997), the biological agent warning sensor (BAWS) developed by MIT Lincoln Laboratory (Jeys et al., 2007; Primmerman, 2000), the wide issue bioaerosol sensor (WIBS) developed by the Defense Science and Technology Laboratory, UK (Kaye, 1999; Kaye et al., 2004; Kaye et al., 2005), and the single particle fluorescence analyzer (SPFA) developed at the Naval Research Laboratory (Eversole et al., 2001; Eversole et al., 1999). Although such UV-LIF systems do not have sufficient specificity to identify biological particles at the genus and species levels, the intrinsic particle fluorescence induced by UV radiation can be used to help differentiate biological particles from nonbiological particles. In this way, a UV-LIF instrument can act as a trigger for the aforementioned biochemical assays (Kaye et al., 2004). Such a design would reduce analysis costs, decrease detection times, and increase the detection accuracy of target bioagents (Sivaprakasam et al., 2009). However, many common environmental pollutants, as well as some natural atmospheric components, may exhibit similar fluorescence profiles and light-scattering properties, making them easily mistaken for target microorganisms (Pinnick et al., 1999). Also, changes to the physical and/ or metabolic state of bioaerosols resulting from various environmental and nutritional conditions can make their intrinsic fluorescence measurement more difficult to interpret. To resolve this critical problem, many researchers have recently begun focusing on improving particle differentiation via individual fluorescence spectral analysis and fluorescence-based optical sensors (Sivaprakasam et al., 2009). Pan and coworkers evaluated single atmospheric aerosol particles using a dualwavelength-excitation particle fluorescence spectrometer with a 32-anode photomultiplier array (Pan et al., 2011, 2010). They observed different fluorescence spectra for different materials and demonstrated that effective differentiation of interfering compounds should be possible with broadband spectral data (Kaye et al., 2005; Pan et al., 1999). For field applications, the focus has been on developing simpler, more robust, and inexpensive real-time sensors with minimal maintenance and fewer false alarms for intelligent network deployments (Kaye et al., 2004). Jeys and coworkers investigated the use of dual-wavelength, single-element light-emitting diodes (LEDs) (Jeys et al., 2003), and Davitt and coworkers demonstrated a compact system operating with a linear array of UV-LEDs (Davitt et al., 2005). The aerosol fluorescence sensor (AFS; 105974; Biral, Bristol, UK) system was developed for unattended deployment in medium-to-large area networks as a low-cost prototype bioaerosol sensor based on UV-LIF. The AFS sensor uses a xenon flash-lamp source, instead of the more common solid-state UV laser, such as a frequency quadrupled Nd-YAG laser. An optically-filtered, 280nm excitation waveband targeted the intrinsic fluorescence of common amino acids, for example, tryptophan and tyrosine, and other components found in living matter (Jung et al., 2011, 2010a). The induced fluorescence was collected and measured on two wideband fluorescence detectors (dual bands). A previous study used the AFS system to demonstrate the different fluorescence characteristics of different bacterial bioaerosols, such as Escherichia coli and Bacillus subtilis, and nonbacterial aerosols such as polystyrene latex (PSL) spheres (Jung et al., 2012a). Although several studies have demonstrated the feasibility of detecting bioaerosols with the AFS, few investigations have been focused on the quantitative relationship between fluorescence and the viability of airborne microorganisms. The fluorescence of excited biomolecules within the microorganism depends strongly on environmental and biomolecular factors. Using a UV-APS, Agranovski and coworkers found that bacterial injury by heat stress and the bacterial growth phase can impact the intrinsic fluorescence of individual bioparticles (Agranovski et al., 2003a). Kanaai and coworkers discovered a significant relationship between variations in fluorescence and the age of fungal spores (Kanaani et al., 2007). Santarpia and coworkers observed that the UV-LIF spectra of Yersinia rohdei and bacterioparge MS2 bioaerosols were altered upon ozone exposure (Santarpia et al., 2012). The concentration of intracellular fluorophores within a cell varies, depending on changes in bacterial viability induced by various environmental and nutritional conditions. Therefore, to use the AFS quantitatively, it is important to know the contributions of individual AFS fluorescence signals that can be obtained from test microorganisms exposed to various environmental conditions. As part of the research on the effect of environmental conditions on bioaerosol fluorescence characteristics, the present study describes the variation in the fluorescence intensity of thermally-exposed bacterial bioaerosols in high-temperature short-time (HTST) processes. The HTST process utilizes dry heat for the short thermal exposure process and is a well-known heat-based bioaerosol control method (Mullican et al., 1971). Previous researchers have shown that the HTST technique can effectively inactivate fungal, bacterial and viral bioaerosols in a continuous-flow system (Grinshpun et al., 2010a, 2010b, 2010c Jung et al., 2009c, 2010b, 2009d; Lee & Lee, 2006). This HTST technique has important applications for the biodefense/ counter-terrorism field, in which biological warfare agents can be exceptionally resistant to various stresses, such as “dry”

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and “wet” heat (Setlow, 1995). It can also be used to assess indoor air quality and sterilization systems (Grinshpun et al., 2010b). In addition, thermal processes are believed to be effective, safe and environmentally-friendly for controlling viable biological particles in continuous-flow settings, such as heating, ventilation and air-conditioning systems. In this study, the UV- and Vis-band fluorescence intensities of test particles (E. coli and B. subtilis) thermally inactivated at various temperatures were measured using an AFS. The fluorescence characteristics of the particles, including the ratio of UV- to Vis-fluorescence, were evaluated. We also tested the airborne aromatic amino acids (L-tryptophan and L-tyrosine) and ovalbumin particles using the same experimental conditions. 2. Materials and Methods 2.1. Test bacteria, aromatic amino acids, and protein Test bacterial bioaerosols were generated using E. coli (ATCC No. 8739) and B. subtilis (KACC No. 10111). Gram-negative E. coli is an environmentally sensitive bacterium that has been evaluated in numerous microbiological and bioaerosol studies (Huang & Juneja, 2001; Jung et al., 2009c; Lee et al., 2010b; Palaniappan et al., 1992). Airborne E. coli has been found in indoor environments. Varma and coworkers reported that pathogenic E. coli O157:H7 can be spread in an airborne manner (Varma et al., 2003). Gram-positive B. subtilis is resistant to many adverse conditions and commonly found in a variety of environments (Agranovski et al., 2003a, 2003b; Jung et al., 2009c; Lee et al., 2010b; Yao & Mainelis, 2006). E. coli cultures were grown in tryptic soy broth (TSB; Becton Dickinson, Franklin Lakes, NJ, USA) at 37 1C for 18 h. B. subtilis cultures were grown in nutrient broth (NB; Becton Dickinson) at 30 1C for 24 h. To obtain bacterial pellets, stationary phase organisms with an optical density (OD) of 0.89–0.91 at 600 nm were harvested by centrifugation (5000 g, 10 min). For the test aromatic amino acids and protein, L-tryptophan (93659; Sigma Chemicals, St. Louis, MO, USA; 499.5%), L-tyrosine (93829; Sigma Chemicals; 499.0%), and ovalbumin (A5503; Sigma Chemicals; 4 98%) were used in this study. 2.2. Bioaerosol generation Fig. 1 shows a schematic diagram of the experimental setup. For bacterial bioaerosol generation, bacterial pellets were carefully washed three times with sterilized deionized water (SDW) using a centrifuge (5000 g, 10 min) to remove the residual medium. Dilutions to obtain the final bacterial suspensions were performed with SDW. A 30-mL aliquot was placed in a 6-jet Collison Nebulizer (BGI Inc., Waltham, MA, USA) and nebulized at an air flow rate of 5 L/min. For aerosol generation of aromatic amino acids and proteins, we used a 0.025% (w/v) L-tryptophan, 0.05% (w/v) L-tyrosine and 0.25% (w/v) ovalbumin suspension diluted with SDW. For each of the test material cases, the aerosolized particles were first passed through a diffusion dryer to remove all moisture and then circulated through an electrostatic charge neutralizer (two 210Po radioactive sources, each with an activity of 500 mCi) to minimize the electrostatic removal of particles by the inner surfaces of the test system. The air flow rate in the nebulizer and the dilution flow were held constant by mass flow controllers (FC-280S; Mykrolis, Billerica, MA, USA). 2.3. Thermal electric heating system Test bioaerosols introduced into the electric heating tube were exposed to an HTST environment (Grinshpun et al., 2010a, 2010b; Jung et al., 2009d). The thermal heating system consisted of a quartz tube (inner diameter: 28 mm; length: 700 mm; thickness: 1 mm) and an electric heat controller. The length of the heating zone was 400 mm. A 16-channel thermocouple receiver was used to measure the temperature distribution along the center axis at a constant interval (5 cm) under the HEPA filter

SMPS

Clean air tank

MFC

Temperature Controller

Diluting air

210Po Diffusion dryer neutralizer

6-Jet Collison nebulizer

AFS

APS

Pull air pump

Exhaust Thermal electric tube furnace

BioSampler

Fig. 1. Experimental configuration.

Pull air pump

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Centerline temperature (°C)

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experimental temperature conditions. For temperatures ranging from room temperature (17–20 1C) to 200 1C, the air flow residence time between the inlet and outlet of the heating zone of the quartz tube ranged from 3.48 s (17 1C) to 2.37 s (200 1C), allowing for air volume expansion due to increased temperature. Fig. 2 shows centerline temperature distributions in the inner thermal tube, under the experimental set temperature (wall temperature) conditions. 2.4. Real-time bioaerosol measurement system After heating, the aerosol stream passed through a sampling chamber. Real-time aerosol data and fluorescence measurements were obtained using an aerodynamic particle sizer (APS; model 3321; TSI Inc., Shoreview, MN), a scanning mobility particle sizer (SMPS) system, and the AFS, respectively. The size and number of bacterial bioaerosols were measured using the APS. The APS is capable of measuring the aerodynamic size and concentration of airborne particles within the target size range of 0.5–10 μm (Peters & Leith, 2003). The APS overlaps two beams from a laser diode (maximum power: 30 mW; wavelength: 655 nm), producing one double-crested beam profile. As each particle passes through the double crest, it produces two scattering events, which can be converted into time-of-flight information. This information is related to both particle velocity and its aerodynamic size. The size distributions of the aromatic amino acids and protein aerosols were measured using an SMPS system (Jung et al., 2009b, 2007, 2006; Wang & Flagan, 1990), consisting of a differential mobility analyzer (DMA 3081; TSI Inc.) (Chen et al., 1998) and an ultra-condensation particle counter (UCPC 3776; TSI Inc.) (Jung et al., 2009a); the system characterizes particle size distributions in the range of 14–673 nm, based on the particle electrical mobility. The details of the AFS have been described previously (Jung et al., 2012a). The volume of the sensing zone was approximately 1.3 cm3, and the pulse rate of excitation light from the xenon flash lamp was 10 Hz. The fluorescence produced is measured on two channels, each equipped with specific optical filters and a PMT module. The short-wavelength band range was 305–385 nm (UV channel), and the visible band range was 415–550 nm (Vis channel). Final fluorescence intensities are presented in arbitrary units (a.u.). 2.5. Bioaerosol sampling A BioSamplers (SKC Inc., Eighty Four, PA, USA) was used to sample the bacterial bioaerosols before and after the thermal heating process (Willeke et al., 1998). The BioSamplers were operated by pull vacuum pumps with flow meters (Gast IAQ Pump; EMS Inc., Charleston, SC, USA). The bacterial bioaerosols were collected into 20 mL of phosphate-buffered saline (PBS, pH 7.4) at a nominal flow rate of 12.5 L/min. The BioSampler was run for 10 min per test. The overall particle collection efficiency (η) of the BioSampler is defined as η ¼ 1C down =C up

ð1Þ (particles/cm3air)

of the bioaerosols measured downstream and upstream where Cdown and Cup are the particle concentrations of the BioSampler by APS, respectively. The physical collection efficiency of the BioSampler was 89.8% for E. coli particles and 90.5% for B. subtilis particles (Jung et al., 2012b). 2.6. Determination of cell culturability Bacterial culturability, defined as recovery from culture, was determined by culturing samples of bacterial suspensions. Aliquots (0.1 mL) of the BioSampler suspensions were serially diluted with PBS, plated on tryptic soy agar (nutrient agar for B. subtilis), and incubated at 37 1C for 12 h (at 30 1C for 24 h for B. subtilis). After incubation, the colonies that formed on the plates were enumerated. The relative bacterial cell culturability was determined by the number of colonies on the agar plate

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and is presented relative to the number of bacteria entering each sampler according to the following formula: Relative bacterial cell culturability ¼

CFUOutlet NInlet U CFUInlet NOutlet

ð2Þ

where CFUInlet and CFUOutlet are the number of bacterial colonies that were cultured from suspensions of the inlet and outlet BioSampler, respectively. NInlet and NOutlet are the total number of bacteria that entered the BioSampler at the inlet and outlet of thermal electric tube furnace.

3. Results and Discussion 3.1. Cell culturability measurement Fig. 3 shows the reduction in relative cell culturability (V) for each test bacterial bioaerosol, following treatment at various temperatures in the thermal electric tube furnace. As the set temperature of the tube furnace increased, the degree of inactivation increased linearly at temperatures below 100 1C and logarithmically at temperatures above 100 1C. Colony cultures after inactivation showed that nearly 99% of the bacteria were inactivated at 115 1C (E. coli) and 130 1C (B. subtilis). As expected, the cell culturability of the Gram-positive B. subtilis was higher than that of the Gram-negative E. coli under similar conditions. Gram-positive cells have a fairly rigid and protective membrane wall (Neidhardt et al., 1990) that may provide better protection against thermal inactivation (Jung et al., 2009c).

3.2. Size distribution measurement

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Relative bacterial cell culturability, V (%)

We evaluated variations in the normalized aerodynamic particle size distributions of E. coli and B. subtilis bioaerosols at 20 1C and 200 1C using APS. The geometric mean diameter (GMD) and the geometric standard deviation (GSD) of the two species of bacterial bioaerosols were maintained almost uniformly, regardless of the surrounding temperature. The GMD and GSD of E. coli bioaerosols were 0.92 70.002 μm and 1.24 70.001 at 20 1C, and 0.91 70.002 μm and 1.24 70.001 at 200 1C, respectively. In the case of B. subtilis, the GMD and GSD were 0.85 70.002 μm and 1.17 70.001 at 20 1C, and 0.8470.002 μm and 1.20 70.003 at 200 1C, respectively. Compared with room-temperature conditions (20 1C), the GMD reduction ratios [1–(GMD200 1C/GMD20 1C)] of E. coli and B. subtilis were about 1.1% and 1.2%, respectively. Although little variation was observed in the GSD of E. coli as a function of temperature, the GSD of B. subtilis was about 2.5% larger at 200 1C than at room temperature. However, the size distributions of test bacterial bioaerosols were nearly maintained at both temperatures. This indicates that the HTST thermal inactivation process did not affect the bacterial cell through fragmentation or shape deformation, which can affect the aerodynamic size of the particles (Jung et al., 2009c). Fig. 4 shows that the aromatic amino acids and protein aerosols have very broad size distributions. Under higher temperature conditions, the size distributions of L-tryptophan and ovalbumin shifted to a smaller size, while that of L-tyrosine increased to a larger size. However, the GSD of all three materials decreased with increasing temperature. Thermal exposure of HTST could lead to physical size variations of these materials by thermal oxidation and pyrolysis, or thermal decomposition processes. We observed that the physical variation of bacterial bioaerosol was relatively less than that observed for amino acids and protein, under HTST conditions. This result can be explained by the cell wall structure of the bacteria. Bacteria cells have a fairly rigid, protective membrane that provides better protection against thermal exposure (Jung et al., 2009c; Ray, 1984).

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Surrounding temperature (°C) Fig. 3. Reductions in relative bacterial cell culturability (V) of bacterial bioaerosol tests at various temperatures.

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Normalized particle number concentration, (dN/dLog(D e))/Max.

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GMD: 52.4 ± 0.60 nm GSD: 2.07 ± 0.013

GMD: 61.4 ± 1.44 nm GSD: 2.07 ± 0.010

GMD: 60.8 ± 1.79 nm GSD: 2.19 ± 0.006

200°C

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GMD: 26.6 ± 0.47 nm GSD: 1.54 ± 0.009

GMD: 69.4 ± 1.17 nm GSD: 1.98 ± 0.016

GMD: 52.7 ± 1.44 nm GSD: 2.15 ± 0.003

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Electrical mobility diameter, De(nm) Fig. 4. Variations in the normalized particle size distributions of (a) L-tryptophan, (b) L-tyrosine and (c) ovalbumin aerosols at 20 1C and 200 1C, measured with a scanning mobility particle sizer (SMPS).

Fluorescence intensity (x100, Arb. unit)

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Fig. 5. Variation in the ultraviolet (UV) and visible (Vis) fluorescence response of the bacterial bioaerosols: (a) the UV-and Vis-fluorescence intensities of various particle concentrations at room temperature, and (b) the normalized UV- and Vis-fluorescence intensities of test bacterial particles at various temperatures.

3.3. Real-time fluorescence measurement Fig. 5 shows the fluorescence response (UV and Vis) of test bacterial bioaerosols at various particle concentrations and temperatures. As the concentration of particles in the chamber approached zero, the UV and Vis fluorescence intensities approached  140 a.u. and 190 a.u., respectively (Jung et al., 2012a). Fig. 5(a) shows that the room-temperature (20 1C)

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Ratio of UV-FL. and Vis-FL. intensities, R

fluorescence intensity of both channels increased with particle concentration. The increase in UV fluorescence intensity was greater than that of the Vis fluorescence. The slopes of the curves in Fig. 5(a) correspond to the mean fluorescence intensities per unit particle concentration. Fig. 5(b) shows the variation in normalized UV- and Vis-fluorescence intensities of test bacterial particles at various temperatures (a.u./particle). When the temperature increased from room temperature to 200 1C, the normalized UV fluorescence intensities of both bacteria decreased linearly, from about 0.52 to 0.34 for E. coli (y¼  1.37  10  3x+0.557) and from about 0.42 to 0.16 for B. subtilis (y ¼  1.92  10  3x+0.455). However, the normalized Vis fluorescence intensities of both bacteria were similar. Previous studies have shown that common amino acids, such as tryptophan and tyrosine, are responsible for producing the fluorescence signal in the UV. Pinnick and coworkers reported that washed bacteria typically have a fluorescence spectrum dominated by tryptophan, even though many other aromatic compounds exist in bacteria (Pinnick et al., 1999). Pan and coworkers also reported that the broad fluorescence near 340 nm mainly originates from tryptophan and that the fluorescence response between 400 and 500 nm may be attributable to reduced nicotinamide compounds (Pan et al., 2009). HTST thermal inactivation environments could alter the levels of common amino acids in bacteria, resulting in a reduction of UV fluorescence intensity per particle. AFS analysis detected the auto-fluorescence of test bioaerosols (e.g., bacterial cells, aromatic amino acids, and ovalbumin) through UV emission despite the exposure to 200 1C and inactivation of nearly 99% of bacterial at 115 1C (E. coli) and 130 1C (B. subtilis) (Fig. 3). Lee and co-workers showed similar results through the detection of fluorescence signals from airborne fungal spores despite rapid thermal inactivation at  400 1C using the ultraviolet aerodynamic particle sizer spectrometer (UVAPS, TSI Inc.) (Lee et al., 2010a). However, our results show that an elevation of the surrounding temperature can lead to a reduction of auto-fluorescence intensity resulting from the thermal denaturation and decomposition of various cellular fluorophores, including DNA, proteins and other enzymes. Grinshpun and co-workers described the dry heat inactivation of bacterial bioaerosols through the following mechanisms: (1) DNA damage, (2) damage to essential proteins and (3) breaking of the cell membrane coat layers (Grinshpun et al., 2010a). Heat exposure exceeding the specific threshold level results in irreversible changes to indispensible proteins, causing cellular inactivation (Setlow & Setlow, 1995; Setlow, 2006). Under our experiment conditions, the normalized fluorescence intensity of bacterial cells did not reach zero. Even after reaching the specific threshold level of thermal inactivation, a low-level fluorescence signal from the cell residue remains measurable by AFS. Fig. 6 shows the variation in the ratio (R) of UV- to Vis-fluorescence intensities as a function of temperature. The measurements of R by the AFS are independent of particle concentration, and the variation of R is related to the rearrangements or transformation of the chemical structure of specific components in the material under thermal exposure. Fig. 6(a) shows the distinct R values of the test bacteria at each temperature. As the temperature increased, R decreased linearly for both bacteria from about 9.91 (20 1C) to 5.32 (200 1C) for E. coli (y¼ 0.026x+10.551) and from about 10.69 (20 1C) to 4.26 (190 1C) for B. subtilis (y¼ 0.038x+11.816) (Fig. 6(a)). The decrease in R at higher temperatures was more apparent for B. subtilis than for E. coli. In other words, the fluorescence characteristics of B. subtilis bioaerosols were more affected by temperature than those of E. coli. Seaver and coworkers reported that the molar absorptivity and quantum yield per molecule were 5  higher for tryptophan than tyrosine, and that B. subtilis produces slightly more fluorescence emission per particle than E. coli (Seaver et al., 1998). Although fluorescence excitation at 260–280 nm has been the standard method for measuring protein concentrations in conventional quantitative fluorescence analytical biochemistry (Groves et al., 1968; Kreusch et al., 2003; Noble & Bailey, 2009), little is known about the changes in the overall fluorescence spectra of Grampositive/negative bacteria that occur as a function of cell culturability or viability. In Fig. 6(a), the amount of common amino acids was likely to decrease more significantly in B. subtilis than in E. coli. The physicochemical mechanisms that result in the observed differences in the fluorescence characteristics of thermally-inactivated E. coli and B. subtilis are beyond the scope of this study.

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Fig. 6. Variation in the ratio (R) of UV- and Vis-fluorescence intensities at various temperatures: (a) E. coli and B. subtilis, (b) L-tryptophan, L-tyrosine and ovalbumin.

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Many bacteria exhibit spectra attributable primarily to protein and flavin fluorescence. Most proteins show intrinsic fluorescence originating from the aromatic amino acid residues of tryptophan, tyrosine, and phenylalanine. When one or more tryptophan residues are present in a protein, the fluorescence from phenylalanine is usually negligible, and the fluorescence from tyrosine rarely comprises more than 10% of the total emission (Dalterio et al., 1986). Fig. 6(b) shows that as the temperature increased, R decreased for L-tryptophan, L-tyrosine, and ovalbumin aerosols from about 10.18 (20 1C) to 6.03 (200 1C) for L-tryptophan (y¼  0.018x+10.544), from about 5.71 (20 1C) to 3.79 (200 1C) for L-tyrosine (y¼  0.013x +6.008), and from about 4.34 (20 1C) to 3.38 (200 1C) for ovalbumin (y¼ 0.005x+4.541). L-tryptophan exhibited the highest R at 20 1C and retained a greater sensitivity to the thermal exposure, compared with L-tyrosine and ovalbumin (Fig. 6(b)). It is well documented that the indole groups of tryptophan residues are the dominant source of UV absorbance and emission in cell proteins. Previous studies have shown that the emission of tryptophan is highly sensitive to its local environment; spectral shifts in its emission have been observed as a result of a range of phenomena, such as the binding of ligands and protein–protein association (Bhatta et al., 2006). Gally and Edelman reported that the emission spectra shape of tyrosine and tryptophan remained unaltered; however, the quantum yield of emission decreased as the temperature increased from 25 to 90 1C (Gally & Edelman, 1962). Permyakov and coworkers reported that thermal denaturation of a protein results in both a decrease of the tryptophan fluorescence quantum yield and a redshift in tryptophan fluorescence (Permyakov & Burstein, 1984; Permyakov et al., 1982). In our study, we measured the fluorescence characteristics of thermally-exposed bioaerosols in real time using AFS. In the airborne state, the particles experience a very high temperature of 4100 1C, compared with the liquid state. In this case, thermal exposure by HTST could lead to intensive variation in the biochemical components of proteins by thermal oxidation or decomposition processes. Future work using more quantitative analyses of fluorescence spectra will help to establish the fundamental nature of thermal denaturation of both airborne and waterborne microorganisms.

4. Conclusions Intracellular fluorophore content depends on bacterial culturability as well as various environmental and nutritional conditions. Therefore, the real-time and continuous measurement of the intrinsic fluorescence of bacterial bioaerosols can provide useful information about the physical and/or metabolic state of the cells. The present report describes the variation in the fluorescence intensity of thermally-exposed bacterial bioaerosols using an AFS with dual UV and Vis channels. The results demonstrate that the variation in UV- and Vis-fluorescence intensities is related to the surrounding thermal environment. Also, the ratio of UV- to Vis-fluorescence decreased with increasing temperature for each bacterial bioaerosol. Under the same experimental conditions, we found a similar reduction in the fluorescence characteristics of airborne aromatic amino acids (L-tryptophan and L-tyrosine) and ovalbumin particles compared to the test bacterial bioaerosols. These results provide information useful for improving the resolution of real-time bioaerosol sensor systems. This study may be broadened further by incorporating multi-wavelength excitation using several UV excitation sources (e.g., 263 nm and 351 nm) as a means of acquiring additional diagnostic information.

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