Science of the Total Environment

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1 Science of the Total Environment 407 (2009) Contents lists available at ScienceDirect Science of the Total Environment journal homepage: Thermal effects on bacterial bioaerosols in continuous air flow Jae Hee Jung a,c, Jung Eun Lee b, Sang Soo Kim a, a Aerosol and Particle Technology Laboratory, Department of Mechanical Engineering, Korea Advanced Institute of Science and Technology (KAIST), Guseong-dong, Yuseong-gu, Daejeon , Republic of Korea b Public Health Microbiology Laboratory, Department of Environmental Health, Graduate School of Public Health, Seoul National University, Yeongeon-dong, Jongro-gu, Seoul , Republic of Korea c Center for Environmental Technology Research, Korea Institute of Science and Technology (KIST), Hawolgok-dong, Seongbuk-gu, Seoul , Republic of Korea article info abstract Article history: Received 16 September 2008 Received in revised form 28 April 2009 Accepted 6 May 2009 Available online 30 May 2009 Keywords: Bacterial bioaerosols Thermal electric heating system Air sterilization Airborne bacterial endotoxin Relative recovery Exposure to bacterial bioaerosols can have adverse effects on health, such as infectious diseases, acute toxic effects, and allergies. The search for ways of preventing and curing the harmful effects of bacterial bioaerosols has created a strong demand for the study and development of an efficient method of controlling bioaerosols. We investigated the thermal effects on bacterial bioaerosols of Escherichia coli and Bacillus subtilis by using a thermal electric heating system in continuous air flow. The bacterial bioaerosols were exposed to a surrounding temperature that ranged from 20 C to 700 C for about 0.3 s. Both E. coli and B. subtilis vegetative cells were rendered more than 99.9% inactive at 160 C and 350 C of wall temperature of the quartz tube, respectively. Although the data on bacterial injury showed that the bacteria tended to sustain greater damage as the surrounding temperature increased, Gram-negative E. coli was highly sensitive to structural injury but Gram-positive B. subtilis was slightly more sensitive to metabolic injury. In addition, the inactivation of E. coli endotoxins was found to range from 9.2% (at 200 C) to 82.0% (at 700 C). However, the particle size distribution and morphology of both bacterial bioaerosols were maintained, despite exposure to a surrounding temperature of 700 C. Our results show that thermal heating in a continuous air flow can be used with short exposure time to control bacterial bioaerosols by rendering the bacteria and endotoxins to a large extent inactive. This result could also be useful for developing more effective thermal treatment strategies for use in air purification or sterilization systems to control bioaerosols Elsevier B.V. All rights reserved. 1. Introduction Bacterial bioaerosols are found everywhere in both indoor and outdoor environments (Burge, 1990; Lee et al., 2006). Several studies have reported that the concentration of bacterial bioaerosols is relevant to the occurrence of human diseases and public health problems associated with tuberculosis (Riley et al., 1962), measles (Riley et al., 1978), and legionellosis (Fraser, 1980). Exposure to indoor aerosol pollutants has become a growing public and occupational health concern (Gammage and Berven, 1996; Samet, 1993). The outbreaks of emerging diseases and the threat of bioterrorism have generated special needs with respect to indoor air quality (IAQ) against respirable particles, especially those of biological origin (Jernigan et al., 2001; Lane et al., 2001; Nicas and Hubbard, 2003). Strategies that have been developed for protecting indoor environments from deliberately used aerosol agents require efficient air filtration and air sterilization systems (NIOSH, 2003). Several Corresponding author. Tel.: ; fax: address: sskim@kaist.ac.kr (S.S. Kim). methods have been suggested for controlling the viability of airborne microorganisms. The most common methods use ultraviolet germicidal irradiation (UVGI) (Lin and Li, 2002). Many researchers have studied how UVGI affects the viability of bioaerosols and how the experimental variables, such as level of irradiation, duration of exposure, pattern of air movement, and surrounding moisture influence the effectiveness of UVGI (Nicas, 1996; Nicas and Miller, 1999; Riley and Kaufman, 1972; Summer, 1962). However, although UVGI can be installed easily and consumes little energy, the 254 nm wavelength UV light, which offers a high inactivation effect on microorganisms, produces ozone and radicals, which can harm humans (Duthie et al., 1999; Longstreth et al., 1995). Electric ion emission has also been studied as a means of controlling bioaerosols. Air ion emissions can remove indoor aerosol particles (Grinshpun et al., 2005). In addition, when electric ion emission is used in various filtration systems, the efficiency of respiratory protection devices or air purifier systems against bioaerosols can be enhanced. Although air electric ions can decrease the viability of airborne bacteria (Huang et al., 2008), this method has a number of shortcomings; for instance, the generation of the ions produces ozone of high concentration and causes electric charges to accumulate on surrounding surfaces (Boelter and Davidson, 1997; Phillips et al., 1999). Antibacterial filters /$ see front matter 2009 Elsevier B.V. All rights reserved. doi: /j.scitotenv

2 4724 J.H. Jung et al. / Science of the Total Environment 407 (2009) Fig. 1. Schematic of experimental set up. are commonly associated with bactericidal methods. They have demonstrated good performance in initial trials. However, the antibacterial performance of these filters cannot be maintained for extended periods, as contact in air is a prerequisite to reduce the viability of bacteria (Jung et al., 2006; Ji et al., 2007). Recently, the microbial inactivation method using heat treatment has been used in continuous flow systems as a safe, effective, and environment-friendly method that does not use ozone, ions, or filter media (Fine and Gervais, 2005; Loss and Hotchkiss, 2004; Jung et al., 2009a). Thermal inactivation has long been considered to be a suitable and reliable method for controlling microorganisms. Two types of heat are generally used: moist and dry. Moist heat utilizes steam under pressure, whereas dry heat involves high temperature exposure without additional moisture. Several types of heat treatment are currently used for killing microorganisms, including incineration, tyndallization, pasteurization, and autoclaving (Madigan et al., 2003). However, most of these technologies were originally limited to controlling microorganisms in food or water. In addition, they may not be adequate for controlling bioaerosols because the environment of airborne microorganisms with high fluidity is vastly different from the conditions of food or water. Therefore, it is necessary to find adequate and practical conditions for controlling bioaerosols. Thus far, several investigations into using heat treatment against bioaerosols have been reported. Some of these studies have targeted airborne bacteria spores that have been widely used as surrogates for biological warfare agents (Decker et al., 1954; Gremillion et al., 1958; Mullican et al., 1971) and others have focused on a number of vegetative cells (Ehrlich et al., 1970a,b; Lee and Kaletunc, 2002; Peleg et al., 2005). Recently, we have studied the thermal effect on fungal bioaerosols by a high temperature, short-time (HTST) process in a continuous flow system (Jung et al., 2009a). We developed the fungal bioaerosol generator with stable and uniform fungal bioaerosol generation characteristics and examined the variation of size distributions, culturability, and concentration of (1 3)-β-D-glucan with thermal environment conditions (Jung et al., 2009b,c). The geometric mean diameter of the tested fungal bioaerosols decreased when they were exposed to increases in the surrounding temperature. More than 99.9% of the Aspergillus versicolor and Cladosporium cladosporioides bioaerosols lost their culturability in about 0.2 s when the surrounding temperature exceeded 350 C and 400 C, respectively. And, the HTST process produced a significant decline in the (1 3)-β-D-glucan of fungal bioaerosols. However, the bacterial bioaerosols are more thermo-sensitive than fungal bioaerosols; we could control the characteristics of bacterial bioaerosols such as bacterial viability or concentration of toxigenic substance with lower temperature range in comparison with fungal bioaerosols. In the study reported herein, the thermal effects of airborne bacteria were investigated when using continuous thermal inactivation. When the bacterial bioaerosol was passed through a thermal electric heating system, the bacteria were exposed to various temperatures for short periods. Then, we examined the bioaerosol and aerosol characteristics, including the relative recovery, relative injury, inactivation of endotoxin, aerosol size distribution, and aerosol morphology using a novel technique for sampling and measuring aerosols. 2. Materials and methods The experimental system, shown schematically in Fig. 1, included four major components: (i) a system for generating bacterial bioaerosols, (ii) an aerosol measurement system that consisted of a condensation particle counter and a particle size distribution analyzer, (iii) a sampling system for various analyses of bacterial bioaerosols before and after exposure to thermal energy, and (iv) a thermal electric heating system Generation of bacteria bioaerosols Escherichia coli and Bacillus subtilis were used as bacteria particles. The Gram-negative E. coli are representatives of sensitive bacteria (Hamilton and Sale, 1967; Huang and Juneja, 2001; Palaniappan et al., 1992; Yao and Mainelis, 2007) and Gram-positive B. subtilis are known to be very resistant to many adverse conditions (Burton et al., 2005; Dhayal et al., 2006; Sneath et al., 1986). Stock cultures of E. coli (ATCC No.8739) and B. subtilis (KACC No.10111) were obtained from the Korean Agricultural Culture Collection (KACC, Suwon, South Korea). Both bacteria cultures were grown in Tryptic Soy Broth (TSB) (Becton Dickinson, NJ, USA) and Nutrient Broth (NB) (Becton Dickinson, NJ, USA) at 37 C for 18 h. The bacteria were harvested through centrifugation at 5000 g for 10 min (RC-5B, Sorval Co., CT, USA). Then, the pellets were washed twice with sterilized deionized distilled water, which was also used to dilute the cells to obtain a bacterial density that had an optical density of 0.89 to 0.91 at 600 nm. Then, a

3 J.H. Jung et al. / Science of the Total Environment 407 (2009) ml aliquot was taken and placed in a 6-Jet Collison Nebulizer (BGI Inc., Waltham, MA, USA). The cell concentration was approximately 10 8 CFU (colony-forming units) per ml. While bacterial particles were aerosolized from a liquid suspension through a Collison nebulizer stem at a flow rate of 5 L/min of dry, filtered, compressed air, the aerosolized bacterial particles were passed through a diffusion dryer to remove moisture and were diluted by an additional air flow of 30 L/ min. The air flow rates of the nebulizer and dilution were kept constant using a mass flow controller (Mykrolis, FC-280S, USA) Thermal electric heating system The designed thermal system consisted of a quartz tube (inner diameter, 29 mm; length, 700 mm; thickness, 1 mm) and an electric heating controller (Jung et al., 2009a). The system used electric heating coils as the source of thermal energy because they can be installed easily in existing air conditioning systems. The heating coils were located outside the quartz tube and were covered with insulating materials so that passing bioaerosols could be exposed to high temperatures without the bioaerosol flow stream being obstructed. For experimental temperature conditions from 17 C (room temperature) to 700 C, the residence time of the air flow between the inlet and outlet of the quartz tube in the thermal system was estimated to be from about 0.29 s (room temperature) to 0.17 s (700 C), allowing for expansion of the air volume due to the increase of temperature. The relative humidity in the inlet sampler was about 30 40% (SK-110TRH II Type1, SK SATO, Japan) Aerosol measurement system The aerodynamic particle size distributions of the bacterial bioaerosols were measured using a particle size distribution analyzer (PSD 3603, TSI Inc., MN, USA). The aerodynamic diameter, d a, of a particle is equivalent to that of standard-density spherical particles that have the same gravitational settling velocity (Hinds, 1999). The PSD 3603 has 128 size channels ranging from 0.3 to 700 µm. While measuring the particle size distributions, the total particle number concentration of the bacterial bioaerosols was also measured using a condensation particle counter (CPC 4330,HCTInc.,SouthKorea).TheCPC4330candetectamaximum concentration of 10,000 particles/cm 3 and can detect a minimum particle size of 15 nm with 95% accuracy. Before it was tested with bacterial bioaerosols, the sampling chambers and the quartz tube were flushed with HEPA-filtered clean air until no particles were detected by the PSD 3603 or CPC The total bacterial bioaerosol particle number concentrations in the inlet and outlet sampling chambers were used to determine the recovery and the endotoxin concentration of the bacterial bioaerosols Bioaerosol sampling A BioSampler (SKC Inc., PA, USA) was used to sample the bacterial bioaerosols before and after the thermal heating process. The BioSamplers were operated by pull vacuum pumps with flow meters (Gast IAQ Pump, EMS Inc., SC, USA). The bacterial bioaerosols were collected into 20 ml of phosphatebufferedsaline(pbs,ph7.4)eachatanominalflow rate of 12.5 L/min. The BioSampler was run for 15 min for each test Determination of recovery and injury Trypticase soy agar (TSA) (Becton Dickinson, NJ, USA) and nutrient agar (NA) (Becton Dickinson, NJ, USA) were used as complete, nonselective mediums of E. coli and B. subtilis, respectively. Metabolically and structurally injured bacteria were identified by performing additional colony counts on the two types of selective medium (Mainelis et al., 2002; Stewart et al., 1995). Minimal salts glucose agar (MA) (7.0 g of K 2 HPO 4,3.0gofKH 2 PO 4,0.1gofsodiumcitrate,0.1gofMgSO 4 7H 2 O, 1 g of (NH 4 ) 2 SO 4, 2.0 g of glucose, and 1.5% nutrient agar) was used to select against metabolically injured bacteria (Moss and Speck, 1963; Ray and Speck, 1973). For selection against structurally injured bacteria, MacConkey agar (Becton Dickinson, NJ, USA) and NA containing 2% NaCl were chosen for the collection of E. coli and B. subtilis, respectively (Stewart et al., 1995). The aliquots of ml of BioSampler suspensions before and after thermal treatment were serially diluted with PBS buffer. For E. coli bioaerosols, aliquots were plated on TSA, MA, and MacConkey agar. They were then incubated at 37 C for 12, 18, and 48 h, respectively. For B. subtilis bioaerosols, aliquots were plated on NA, MA, and NA plus 2% NaCl, and then incubated at 37 C for 24, 30, and 48 h, respectively. After incubation, the colonies that formed on the plate were enumerated. The bacterial recovery rate was assessed by the number of colonies on the complete and selective agar and is presented relative to the number of bacteria entering the sampler in the following formula: relative recovery = CFU Outlet CFU Inlet NInlet N Outlet where CFU Inlet and CFU Outlet is the number of bacteria colonies that were cultured from suspensions of inlet and outlet BioSampler, respectively, and where N Inlet and N Outlet are the total number of bacteria that entered the BioSampler. The level of injury was determined by the difference between the count on the complete medium (relative recovery Complete )andthaton minimal medium or selective medium (relative recovery Minimal or relative recovery Selective, respectively) (McFeters, 1990). For each series of three samples grown on complete, minimal, and selective agar, the percentage of metabolic injury and percentage of structural injury were calculated as follows, on the basis of differences in relative recovery on the minimal and selective medium: metabolic injury = relative recovery Complete relative recovery Minimal relative recovery Complete ð2þ structural injury = relative recovery Complete relative recovery Selective relative recovery Complete : 2.6. Assay of endotoxins In the case of E. coli, a part of the bacterial particles was analyzed for endotoxin by using the kinetic chromogenic Limulus Amebocyte Lysate (LAL) method (Pyrochrome, Associates of Cape Cod, East Falmouth, MA, USA). A liquid suspension of BioSampler was sonicated for 1 h at room temperature and shaken for 15 s every 15 min using a touch mixer (Fisher Scientific, USA). 40 µl of the Pychrome reagent was added to 40 µl of the sample from the BioSampler in a microplate and incubated in a reader for 150 min at 37±1 C. At 405 nm, the absorbance measurements (TECAN, Infinite M200, TECAN Ltd., Männedorf, Switzerland) were collected with time after the addition of Chromo-LAL and were analyzed with software (Magellan V6, TECAN Ltd., Männedorf, Switzerland). The range of endotoxin concentration for the standard curve was EU/mL. The sensitivity of the assay is defined as the lowest concentration used in the standard curve. Then, the maximum sensitivity of this test was EU/mL. The endotoxin results were expressed as concentrations (Endotoxin concen Inlet or Outlet, EU/m 3 ), using the sampling flow rate and the sampling time as a basis. The relative endotoxin ratio was determined as the ratio of the endotoxin concentration of the sampled bacterial particles in the inlet and outlet BioSamplers. Finally, the endotoxin inactivation ratio by thermal heating process was obtained from the relative endotoxin ratio according to the following formula: Relative endotoxin ratio = Endotoxin concen: Outlet Endotoxin concen: Inlet ð1þ ð3þ ð4þ

4 4726 J.H. Jung et al. / Science of the Total Environment 407 (2009) Endotoxin inactivation ratio = ½1 Relative endotoxin ratioš 100: ð5þ 2.7. Analysis by scanning electron microscopy The morphology of bacterial bioaerosol was investigated using scanning electron microscopy (SEM) (XL30S FEG, Philips, Netherlands). For this purpose, bacterial bioaerosols that had been exposed at various temperatures were sampled onto a 13-mm mixed cellulose ester (MCE) filter with a pore size of 1.2 µm (Millipore Corporation, Bedford, MA) downstream from the outlet sampling chamber for 5 min. After sampling, the MCE filters were coated with Osmium coater using the chemical vapor deposition method (HPC-1SW, Vacuum Device Inc. Japan) and then analyzed using SEM Statistical analysis The data were analyzed statistically by analysis of variance (ANOVA), t-test, and linear regression using the software package SAS 9.1 for Microsoft Windows. 3. Results 3.1. Temperature characteristics of the thermal electric heating system Fig. 2 shows (a) the centre temperature distributions and (b) the maximum centre temperature of the inner thermal tube under the experimental set temperature (wall temperature) conditions. As shown in Fig. 2(b), the maximum temperature of each of the centre temperature distributions was smaller than the set temperature (wall temperature) due to thermal heat transfer from the quartz tube wall to the air flow with rapid velocity (thus, the residence time was short). Fig. 3. Variation in the relative recovery of (a) E. coli and (b) B. subtilis with various temperature conditions. The error bars indicate standard deviations (number of samples, n=5). In contrast to the confined thermal systems for food and liquid, a thermal system for a continuous stream of airborne particles cannot, in practice, maintain a single constant temperature uniformly for an entire system because of the fluid flow and the continuous heat transfer in the system. That being the case, we regarded the set temperature (wall temperature) as a possible parameter for describing the thermal heat treatment of the bioaerosols Effect of temperature on bacterial recovery and injury Fig. 2. Temperature distributions of thermal electric tube furnace: (a) centre temperature distribution, (b) maximum centre temperature via. set temperature (wall temperature). As shown in Fig. 3, an increase in the surrounding temperature produced a considerable decline in the relative recovery rates of both E. coli and B. subtilis. We performed the bacterial recovery experiment five times (Number of samples, n=5). The highest relative recovery rates of E. coli on TSA (approximately 95%) and of B. subtilis on NA (approximately 98%) were found at normal temperature (20 C). All microorganisms were rendered inactivate (N99.9%) above 160 C for E. coli and above 350 C for B. subtilis. As expected, the relative recovery rate was higher on the complete (TSA and NA) medium than on the minimal and selective medium for both microorganisms. In Fig. 3(a), which shows data for the E. coli bioaerosol, the relative recovery rate on MA was higher than that on selective medium (MacConkey agar). However, in Fig. 3(b), which shows data for the B. subtilis bioaerosol, the relative recovery rate on MA was lower than that on selective medium (NA plus 2% NaCl). In Fig. 4, which shows data for bacterial injury, a trend may be seen towards increased injury as the surrounding temperature increases. These results show that E. coli is highly sensitive to structural damage, with injury of more than approximately 80% at temperatures above 80 C (Fig. 4(a)), while structural injury for B. subtilis was found to be between 4.6±4.35% at 20 C and 51.4±23.31% at 300 C (Fig. 4(b)). Metabolic injury rates for E. coli and B. subtilis were found in the

5 J.H. Jung et al. / Science of the Total Environment 407 (2009) Fig. 4. Variation in the injury of (a) E. coli and (b) B. subtilis with various temperature conditions. The error bars indicate standard deviations (n=5). ranges from 2.4±4.09% (room temperature) to 90.0±1.31% (160 C) and from 7.3±3.91% (room temperature) to 60.4±14.64% (300 C), respectively. In addition, both metabolic and structural injuries were approximately 99.9% at temperatures above 160 C for E. coli and above 350 C for B. subtilis. At these temperatures, all microorganisms were rendered inactive. For E. coli, the structural injury appeared to be greater than the metabolic injury, whereas for B. subtilis, the metabolic injury appeared to be greater than the structural injury Endotoxin inactivation using thermal heating process Thermal heating is one of the most effective depyrogenation methods. We tested the efficiency of the thermal heating process on Fig. 6. Variation in the aerodynamic particle size distribution of (a) E. coli and (b) B. subtilis with various temperature conditions. The error bars indicate standard deviations (n=3). the endotoxin inactivation of E. coli bioaerosols. As seen in Fig. 5, an increase in the surrounding temperature produced a significant decline in the concentration of endotoxins in E. coli. The ranges of the concentration of endotoxins, measured at the inlet and outlet sampling chambers, were EU/m 3 and EU/m 3, respectively. Then, the endotoxin inactivation ratio was found in ranges from 9.2% (200 C) to 82.0% (700 C) Aerodynamic size distribution and morphology Fig. 6 shows the variations in the normalized aerodynamic particle size distribution of (a) E. coli and (b) B. subtilis bioaerosols according to the surrounding temperature. In Fig. 6, it may be seen that constant size Table 1 Variation in the GMD and GSD of E. coli and B. subtilis bioaerosols with various temperature conditions. Fig. 5. Variation in the E. coli endotoxin inactivation ratio with various temperature conditions. The error bars indicate standard deviations (n=3). Mean±standard deviation, n=3 Temperature E. coli B. subtilis ( C) GMD a GSD b GMD GSD ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±0.017 a b Geometric mean diameter (μm). Geometric standard deviation.

6 4728 J.H. Jung et al. / Science of the Total Environment 407 (2009) distributions of E. coli and B. subtilis bioaerosols were maintained at both room temperature and 700 C (p-value N0.05 about GMD, p-value N0.05 about GSD by paired t-test). Details are shown in Table 1. In the case of E. coli, thecoefficients of the variance (CV) of GMD and GSD with temperature were 2.79% and 1.11%, respectively. In the case of B. subtilis, the CV of GMD and GSD with temperature were 3.15% and 2.20%, respectively. SEM analysis was used to observe the change in morphology of bacterial bioaerosols in response to exposure to high temperature. Fig. 7 shows the microphotographs of E. coli and B. subtilis at 20, 400, and 700 C, respectively. Rod-shaped E. coli ranges from 0.6 to 0.8 µm in diameter and 0.8 to 1.2 µm in length. B. subtilis is also rod-shaped and approximately 0.7 to 0.8 µm in width and 1.3 to 1.6 µm in length. The particle size distribution from PSD 3603 and micrograph from SEM analysis confirmed that the thermal exposure had little effect on the size distribution and cell surface of bacterial bioaerosols. 4. Discussion The effects on bacterial bioaerosols of continuous exposure to high temperature were studied experimentally. Fig. 4 shows a considerable trend towards greater bacterial injury as the surrounding temperature increases. As expected, these results agree well with the relative recovery results in Fig. 3, which show lower recovery rates at high temperature conditions. As the surrounding temperature of bacterial bioaerosols increased, the recovery rate decreased as a result of the greater number of cells lethally injured by the stress that was induced by the high temperature. Lethal injury to bacterial cells has also been Fig. 7. SEM pictures (Mag. 30,000).; (a) 20 C, (b) 400 C and (c) 700 C of E. coli, respectively, and (d) 20 C, (e) 400 C and (f) 700 C of B. subtilis, respectively.

7 J.H. Jung et al. / Science of the Total Environment 407 (2009) investigated in other studies in which microorganisms have been subjected to various stressors, such as aerosolization (Jung et al., 2009d; Cox, 1966; LeChevallier and McFeters, 1985; Walter et al., 1990; Webb, 1959), collection (Cox, 1966; Stewart et al., 1995), freezing (Moss and Speck, 1963; Moss and Speck, 1966), and UV irradiation (Fujioka and Narikawa, 1982; Kapuscinski and Mitchell, 1981). Sublethally injured bacterial bioaerosols are capable of growing on selective medium, given suitable growth conditions (LeChevallier and McFeters, 1985). At the same time, the recovery rate is expected to be higher on complete medium than on selective medium. The relatively low recovery of E. coli on the selective MacConkey agar illustrates the fact that Gram-negative bacteria are more susceptible than Gram-positive bacteria to damage as a result of the thermal stress induced by the surrounding high temperature. This result can be explained in terms of the difference in cell wall structure between Gram-positive and Gram-negative bacteria. Gram-positive cells have a fairlyrigidandprotectiveshellmembranewall.thisshellwallmay provide better protection against structural injury than against metabolic injury during exposure for short periods at high temperatures (Ray, 1984). For instance, the cell envelope of Gram-positive bacteria is similar to a thick insulator that protects the cell against changes in the environment. However, the Gram-negative bacteria cell envelope acts like a thin insulator, which offers considerably less protection against adverse environmental conditions (Neidhardt et al., 1990). Therefore, the overall recovery rate of Gram-positive B. subtilis is greater than that for Gram-negative E. coli. It has been shown that the outermost lipid membrane (e.g. an endotoxin component) of Gram-negative bacteria becomes a primary target for temperature-induced damage with respect to rendering endotoxins inactive (Morrissey and Phillips, 1993; Perkins, 1969). In previous research, dry heat has been used to render endotoxins inactive (Robertson et al., 1978; Tsuji and Harrison, 1978; Tsuji and Lewis, 1978). The standard depyrogenation treatment in the pharmaceutical and medical industry for heat-stable materials was to expose the drug or surgical equipment to a temperature of above 250 C for more than 30 min. By contrast, in our study, an endotoxin inactivation ratio of over 80% was obtained by exposing the E. coli bioaerosols for about 0.3 s at 700 C. However, it is difficult to compare our results with those of previous studies directly, due to the difference in the temperature to which and the time for which the endotoxin materials were exposed. In addition, thermal inactivation tests for various bacterial bioaerosols are needed for comparison to previous studies. We think that this thermal heating system could be useful for rendering airborne endotoxins inactive in continuous air flow. The aerodynamic diameter of bioaerosols, which may differ from their physical size, determines their behavior and transport in the air. Therefore, the deposition of particles in the human respiratory system or the filtration efficiency of ventilation system may be affected by this aerodynamic diameter (Hinds, 1999). Prior to conducting our study, we expected that the size distributions of bacterial bioaerosols would tend to decrease (due to desiccation and the oxidation process related to the thermal effect) when they passed through a thermal electric heating system under a very high surrounding temperature. However, under this experimental condition, the GMD, GSD, and morphology of the two bacteria changed little, as shown in Table 1 and the SEM images. The lack of change may be explained in terms of the thermal characteristics that accompany the surroundings of bacterial bioaerosols. The desiccation and oxidation of microorganisms require a certain minimum residence time in a high temperature environment. However, in our experiment, the residence time was less than about 0.3 s. It is likely that the short residence time is a major cause of the lack of change in the GMD, GSD, and morphology of the bacteria. However, other explanations such as those that consider the structure, inner vapor pressure, and outer and inner components of the bacteria must also be considered. A thermal heating process has long been considered a suitable and reliable method for controlling microorganisms. However, most such technologies were originally limited to controlling microorganisms in liquid or on material surfaces such as food and medicine. In continuous air flow, there have been few studies on the use of thermal processes for controlling microbial bioaerosols. Although the high temperature short-time pasteurization technique, which is based on high temperature stresses for very short periods, has been used for continuous microbial decontamination in the pharmaceutical and beverage industry, the thermal process has not been investigated sufficiently in the context of sterilizing air (Fine and Gervais, 2005; Grant et al., 2002; Xu et al., 2008). The continuous surrounding environment of airborne bioaerosols is significantly different from the conditions in liquid and on solid surfaces. Hence, in order to apply thermal heating to the control of an airborne microorganisms in a continuous flow system, such as a heating, ventilation, and air conditioner system (HVAC), it is necessary to design a thermal heating process that consumes little energy while rendering the microorganisms inactive with a high degree of efficiency. In addition, it is also necessary to find adequate and practical conditions for controlling microbial bioaerosols and surrounding air quality. In this study, we tested thermal effects on the vegetative cells of E. coli and B. subtilis bacteria in continuous air flow. Our results could be useful for developing more effective thermal treatment strategies that could be used in air purification or sterilization systems to control bioaerosols. Further study is required to elucidate in detail the effects of thermal heating on the inactivation of bacterial spores and endotoxins. 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