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Journal of Occupational and Environmental Hygiene Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/uoeh20
Effects of Temperature, Humidity and Air Flow on Fungal Growth Rate on Loaded Ventilation Filters a
a
W. Tang , T. H. Kuehn & Matt F. Simcik
b
a
Department of Mechanical Engineering, University of Minnesota, 111 Church St SE, Minneapolis, MN 55455 b
Division of Environmental Health Sciences, School of Public Health, University of Minnesota, 420 Delaware St. SE, MMC 807, Minneapolis, MN 55455 Accepted author version posted online: 07 Apr 2015.
Click for updates To cite this article: W. Tang, T. H. Kuehn & Matt F. Simcik (2015): Effects of Temperature, Humidity and Air Flow on Fungal Growth Rate on Loaded Ventilation Filters, Journal of Occupational and Environmental Hygiene, DOI: 10.1080/15459624.2015.1019076 To link to this article: http://dx.doi.org/10.1080/15459624.2015.1019076
Disclaimer: This is a version of an unedited manuscript that has been accepted for publication. As a service to authors and researchers we are providing this version of the accepted manuscript (AM). Copyediting, typesetting, and review of the resulting proof will be undertaken on this manuscript before final publication of the Version of Record (VoR). During production and pre-press, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal relate to this version also.
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http:// www.tandfonline.com/page/terms-and-conditions
ACCEPTED MANUSCRIPT Effects of Temperature, Humidity and Air Flow on Fungal Growth Rate on Loaded Ventilation Filters
W. Tang1, T. H. Kuehn1, Matt F. Simcik2
1
Department of Mechanical Engineering
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University of Minnesota 111 Church St SE, Minneapolis, MN 55455
2
Division of Environmental Health Sciences
School of Public Health University of Minnesota 420 Delaware St. SE, MMC 807 Minneapolis, MN 55455
ABSTRACT This study compares the fungal growth ratio on loaded ventilation filters under various temperature, relative humidity (RH) and air flow conditions in a controlled laboratory setting. A new full size commercial building ventilation filter was loaded with malt extract nutrients and conidia of Cladosporium sphaerospermum in an ASHRAE Standard 52.2 filter test facility. Small sections cut from this filter were incubated under the following conditions: constant room temperature and a high RH of 97%; sinusoidal temperature (with an amplitude of 10 °C, an
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ACCEPTED MANUSCRIPT average of 23°C, and a period of 24 hours) and a mean RH of 97%; room temperature and step changes between 97% and 75% RH, 97% and 43% RH, and 97% and 11% RH every 12 hours. The biomass on the filter sections was measured using both an elution–culture method and by ergosterol assay immediately after loading and every 2 days up to 10 days after loading. Fungal growth was detected earlier using ergosterol content than with the elution–culture method. A student's t-test indicated that Cladosporium sphaerospermum grew better at the constant room
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temperature condition than at the sinusoidal temperature condition. By part-time exposure to dry environments, the fungal growth was reduced (75% and 43% RH) or even inhibited (11% RH). Additional loaded filters were installed in the wind tunnel at room temperature and an RH greater than 95% under one of two air flow test conditions: continuous air flow or air flow only 9 hours/day with a flow rate of 0.7m3/s (filter media velocity 0.15m/s). Swab tests and a tease mount method were used to detect fungal growth on the filters at day 0, 5, and 10. Fungal growth was detected for both test conditions, which indicates that when temperature and relative humidity are optimum, controlling the air flow alone can not prevent fungal growth. In real applications where nutrients are less sufficient than in this laboratory study, fungal growth rate may be reduced under the same operating conditions.
KEYWORDS
Fungal Growth · Ventilation Filters · Temperature · Humidity · Air Flow 5080 words
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INTRODUCTION Ventilation filters are used in heating, ventilating, and air conditioning (HVAC) systems to supply clean air to occupied spaces and maintain clean downstream HVAC components. Because microorganisms and particulate matter are ubiquitous in indoor and outdoor
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environments, ventilation filters in situ serve as sites that concentrate microorganisms and nutrient dust. Microorganisms on filters can proliferate when nutrients are available and environmental conditions are appropriate. Nutrients can come from the filter material itself such as cellulosic fibers or from captured dust. Several field studies conducted on ventilation filters have found evidence of microbial growth. A study by Simmons et al.(1) indicated that 8 out of 11 bag or panel filters removed from 7 hospitals in the eastern United States were colonized with fungi at the time of, or prior to, their manufacturers’ recommended replacement time. Price et al.(2) found 25% ~ 33% of untreated filters and 7~20% of chemically treated filters were colonized with mold. Ahearn et al.(3) observed Cladosporium and Penicillium growth on both sides of ventilation filters taken from office buildings. Microbial growth on ventilation filters may result in contaminating the air supplied to the occupied spaces with cells and or fragments of microorganisms, endotoxin, mycotoxin, and microbial volatile organic compounds that can lead to adverse health effects. The American Conference of Industrial Hygienists (ACGIH) emphasizes that “active fungal growth in indoor environments is inappropriate and may lead to exposure and adverse health effects".(4)
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ACCEPTED MANUSCRIPT Three environmental conditions that may affect microbial growth on filters are water available to microorganisms, air temperature, and air flow. Among these, water is the most critical parameter. Microbial growth on ventilation filters is usually observed under conditions of high relative humidity (RH): greater than 98%,(5) 90%,(6) 84%,(7) 80%,(8) and 70%.(9) A guideline published in Filtration and Separation (10) recommends keeping the three day averaged RH below 80% in all parts of an HVAC system whereas EPA suggests maintaining the RH below 60%
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continuously. Temperature affects the survival and growth of microorganisms. Temperature can affect the rate of enzyme-catalyzed reactions, and high temperatures can denature proteins. Each microbial species has minimum, optimum and maximum growth temperatures. Studies on the effects of air flow on microbial growth are mainly limited to ventilation filters. Maus et al. did not find a difference in viability of Bacillus subtilis when filter media were exposed to either air flow with RH greater than 85% or static air with RH greater than 98%.(5) However, they found growth of Aspergillus niger on some of the used media filters under static air but not on filters exposed to air flow. No clear inferences can be drawn because RH was not held constant across the different air flow conditions, and filters were obtained from different HVAC systems which may have collected dust with different nutrient compositions. In general, prior experimental studies fall into two categories: studies conducted on filters exposed to outdoor air without control of temperature, RH or captured dust composition(3, 7-9, 11-13) and studies conducted under controlled environmental conditions: 20~23°C and RH>98%(5), 22~24°C with a range of RH from 55% to 90%,(9), 70±3°F and 90%±2% RH.(14) For the studies conducted under controlled environmental conditions, the focus was on constant room temperature (around 20 to 24 °C) and constant RH. None investigated the effects of variable
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ACCEPTED MANUSCRIPT temperature or humidity on microbial growth. However, outdoor air temperature usually changes sinusoidally with time within one day, and this leads to a more damped variation in the temperature at the filters in an HVAC system. For example, the temperature measured at filter locations in the HVAC systems of a public building in Minneapolis, MN from early September to early October showed an approximate sinusoidal change with time on a daily basis. The outdoor air temperature varied between 33 and 87 °F and the mixed air (of outdoor and return air)
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temperature between 51 and 79 °F. Data from the same building indicated hourly variation of RH ranging from 23% to 93% within a single day. Thus, it is important to investigate the response of microorganisms to these variable environmental conditions. For some fungal species, growth in food at constant temperature and water activity has been well studied.(15) Microbial growth under variable environmental conditions has been limited to building materials such as gypsum-based materials(16), pine and spruce sapwood. (17) Li et al. concluded in their wind tunnel study that supply air velocity was not a direct factor that influenced microbial growth on duct surfaces, and that relative humidity was the main factor for fungal growth for supply air velocity of 3.0 m/s.(18) To our knowledge, no experimental studies on fungal growth on ventilation filters undergoing controlled but variable environmental conditions have been published. Starting with fungal growth under a reference condition (room temperature and high RH), this paper compares growth under sinusoidal temperature variation, step changes in RH and different air flow conditions, respectively. The goal of the research is to identify temperature, RH and airflow conditions that minimize fungal growth on HVAC filters.
METHODS
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ACCEPTED MANUSCRIPT The Challenge Microorganism and Nutrient Among the fungal species that were frequently found in buildings with mold problems, Cladosporium and Penicillium were particularly associated with HVAC systems.(19,
20)
Cladosporium sphaerospermum was selected in this study because it is more often found in indoor environments than outdoors(21) and it has greater tolerance of dry conditions than C. cladosporioides,(22) another Cladosporium species common in indoor environments. The wild C.
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sphaerospermum used in this study was isolated from indoor air sampling in a campus building at the University of Minnesota and was then incubated on malt extract agar (MEA) plates as the stock growth plate. The conidia of Cladosporium sphaerospermum suspended in 0.02% Tween 80 solution (by volume) are roughly spherical and range from 3 to 5 µm in diameter when observed under a microscope. An artificial nutrient, malt extract (BactoTM, Becton, Dickinson and Company, Sparks, MD), was selected to be loaded onto the test filters. Because malt extract is an optimum culture medium for C. sphaerospermum, it was reasoned that if a combination of environmental conditions were to reduce fungal growth on filters loaded with malt extract, the same reduction would be observed on filters loaded with ambient dust.
Experimental Facilities The experimental facilities consist of three major components: a wind tunnel, a static chamber, and incubation flasks. The wind tunnel was used to load test filters with the malt extract and C. sphaerospermum, and was used to vary air flow through test filters. The wind tunnel was built based on the requirements of ASHRAE Standard 52.2 1999 (see Figure 1a).(23) A Devilbiss 45
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ACCEPTED MANUSCRIPT nebulizer (Sunrise Medical, Somerset, Pennsylvania) was modified for the aerosolization system (see Figure 1b) and operated with compressed air of 1.93×105 Pa (28 psig) and dilution air of 0.0015 m3/s. The air temperature inside the wind tunnel was measured with a type T thermocouple and the RH was measured with an Omega HX 94V RH sensor. Facility qualification test results indicated that the aerosol concentration upstream of the test filter was uniform within the cross section of the tunnel with a coefficient of variance less than 12% for
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particle sizes ranging from 0.32 µm to 7.5 µm. This result indicates that the particulate concentration loaded on the test filters is uniform. A detailed description of the wind tunnel can be found in Tang et al. and Tang.(24, 25) A static chamber was used to provide a controlled sinusoidal temperature condition for filter media samples (see Figure 2a). The internal temperature was controlled by circulating water from a water bath through copper tubes wrapped around the stainless steel chamber. The incubation flask set up (see Figure 2b) included a 2000mL flask (Gledhill Shake Flask Assembly CO2, Ace Glass Inc., Vineland, NJ) and a customized stainless steel probe. The probe was built to seal one opening of the flask and to accommodate a Q-trak IAQ monitor (TSI, Inc., Shoreview, MN) for air temperature, RH, and carbon dioxide concentration monitoring inside the flask. The RH inside the flasks was maintained through saturated salt solutions.
Measurement Methods for Fungal Growth Two methods were used to quantify fungal mass present on filter samples: the elution-culture method and ergosterol essay. Microscopic observation was also used to observe overall fungal growth structure.
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Elution-Culture Method The C. sphaerospermum cells were removed from the test filter sections and cultivated on nutrient agar plates. Filter sections each with a size of 38 mm × 51 mm were eluted with twenty mL of phosphate buffered saline (PBS) with 0.02% Tween 80 solution and shaken for 60 minutes using a wrist action shaker (model 75, Burrel Scientific, Pittsburgh, PA). A dilution
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series was prepared if needed and aliquots from the suspensions/dilutions were plated on MEA plates. After four days of incubation at room temperature, colony forming units (CFU) were counted and the number of viable C. sphaerospermum was calculated using equation (1). CFUs on filter sections = Averaged CFUs on growth plates × dilution factor × volume of dilution solution Eq. (1)
Ergosterol Essay The elution-culture method can underestimate the biomass because it only measures those viable cells removed from the filter samples that also survive the culturing procedure. Ergosterol assay was included in this project to obtain better information on the total amount of fungal biomass present. As a membrane constituent of most fungi except the subdivision Mastigomycotina and rust fungi,(26) ergosterol is often used as an indicator of fungal biomass(27-33) and living cells.(34) Ergosterol is negligible in bacteria(35) and either absent or in minor amounts in higher plants.(27, 34) Ergosterol was found to be very stable in heat killed fungi(36) but degraded when exposed to bright sunlight or short wave UV radiation.(36-38)
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ACCEPTED MANUSCRIPT The ergosterol essay method was based on the procedures of Axelsson et al.(28) and Dong et al.(39) with necessary modifications. The sample preparation and extraction was conducted under yellow light to minimize photodegradation. Hexane solutions containing 283.5 ng of 7Dehydrocholesterol (as internal standard for GC-MS analysis) were added to each of the two 50mL sterile polypropylene centrifuge tubes with polyethylene plugs and dried completely with a stream of nitrogen. Filter sections were cut into small pieces of about 13 mm × 10 mm and
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placed into two centrifuge tubes. Eighteen mL of 10% methanolic potassium hydroxide was added per tube and the tubes were heated at 80 °C for 2 hours. After cooling to room temperature, the mixture of the filter pieces and the methanolic potassium hydroxide suspension was filtered through a 250-mL Buchner funnel with a 25-50 µm disc filter (Ace Glass Inc., Vineland, NJ). The tube was rinsed with methanol twice and the rinsed methanol solution was also filtered through the 250-mL Buchner funnel. HPLC grade water with a volume of one third of the volume of collected solution was added. The aqueous methanolic solution was extracted with hexane three times and the hexane phases were pooled, transferred to two 12-mL glass vials, and evaporated with streams of nitrogen until about 0.5 mL of solution was left in each vial. The solutions in the glass vials were then transferred into new 3-mL glass vials. The 12-mL vials were rinsed with hexane twice and the rinsed solutions were also transferred to the 3-mL vial. The hexane was dried completely with a stream of nitrogen and the samples were derivatized by 0.3 mL of TMS (trimethylsilyl) reagent (TMSI/TMCS) (100/1, v/v) at room temperature for 15 minutes. 0.8 mL of hexane and 1 mL HPLC water were added. The liquid was vortexed until the top hexane phase became clear. The top phase was transferred into an amber vial with a 250 µL insert ready for GC-MS analysis.
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ACCEPTED MANUSCRIPT To quantify the mass of ergosterol using GC-MS, a series of ergosterol standards was made by adding hexane solution containing 0.567 µg of 7-dehydrocholesterol to a series of hexane solutions containing 0.1068, 0.267, 0.534, 1.068, 2.67, and 5.34 µg ergosterol. The ergosterol standard series were then dried with nitrogen and derivatized by TMS reagent. The ergosterol standard and ergosterol extracted from the filter samples were injected into a Hewlett-Packard Model 5890 gas chromatograph and analyzed by a Hewlett-Packard Model 5972 mass
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spectrometer. Injections were made in the splitless mode using an HP 6890 series autosampler. Helium was used as the carrier gas with a flow rate of 1 ml/min. A Restek Rtx-XLB column (0.25 µm in thickness, 30 m × 0.25 mm I.D.) was used and the oven temperature was programmed as 170 to 300°C at 20°C/min and kept at 300°C for 9 minutes. The injection temperature was 290°C and the interface temperature was 300°C. The electron ionization energy was 70 eV. An initial analysis of a mixture of dehydrocholesterol and ergosterol derivatives with the MS operated in the scan mode (m/z between 41 and 450) indicated that the dominant ions m/z were 325 and 351 for 7-dehydrocholesterol derivative and 337 and 363 for ergosterol derivative. The ergosterol standard and ergosterol extracted from the filter samples were analyzed in selected-ion monitoring (SIM) mode of mass spectrometer using these four ions. A calibration curve was obtained by correlating the peak-area ratio of ion 363 over 351 to the mass ratio of ergosterol/dehydrocholesterol from the series of ergosterol standards. The mass of ergosterol extracted from filter samples was calculated from the calibration curve and the peakarea ratio of ion 363 over 351 in the samples. The ergosterol content was found to be 0.19±0.02 pg/CFU or 0.13±0.01pg/cell. This conversion number is very close to the average value of 0.16 pg/cell reported by Lau et al. (2006).(40)
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Microscopy Observation Three microscopy methods were used to observe overall fungal growth structure: scanning electron microscope (SEM), swab test
(6)
and tease mount. For SEM, 10 mm × 10 mm filter
samples were cut and air dried inside a biosafety hood for 1 hour and then coated with gold. In the swab test, we used a sterile Q-tip soaked with sterile PBS with 0.02% tween 80 solution to
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swab the filter surface and then transferred the material to a 150 mm malt extract plate. The area of the swabbed filter surface was initially determined to be a circle of 13 mm diameter and later changed to be the inner pleat edge of the filter (96 mm in length) for better results. For the tease mount method, a drop of acid fuchsin-lactic acid staining solution (lactic acid: Fisher Scientific; acid fuchsin: Sigma Chemical Co. St. Louis, MD) was applied to a microscope slide, and then a clear adhesive tape was applied to the filter sampling location to pull away filter fibers and fungal structure if present and pressed onto the microscope slide. When observed under a light microscope (Olympus BX 50), the stained fungal structure was purple in color. Photos were taken from the eyepiece with a digital camera. The SEM method provides the best quality of images of mycelial structures but it is also a destructive method that requires filter sections to be cut from the test filter; thus it can only be used at the end of the experimental period. The swab method is a semi-quantitative method and its efficiency may be limited by the transfer of fungal spore and or mycelia from the filter to the Q-tip and from the Q-tip to the malt extract plates. The tease mount method qualitatively shows the mycelial structure of fungal growth during the experimental period.
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ACCEPTED MANUSCRIPT Experimental Procedure Filter Loading: Malt extract particles were loaded on each new test filter (clean 610 mm × 610 mm × 51 mm electrostatically charged synthetic media pleated filters with media area of 4.83m2, initial efficiency rating MERV-14). Two percent (by weight) autoclaved malt extract solution was aerosolized and injected into the wind tunnel. The malt extract aerosol concentration upstream
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and downstream of the test filter was monitored with an ultraviolet aerodynamic particle sizer (UVAPS 3314, TSI, Inc., Shoreview, MN). The mass concentration was calculated with the assumption that the particle density of malt extract is 1.0 g/cm3. The total mass of malt extract loaded on the test filter was calculated to be approximately 30 grams. After loading, the test filter was sterilized with ethylene oxide (Anprolene, H. W. Anderson Products Inc., Chapel Hill, NC). The sterilized test filter was again placed in the wind tunnel and loaded with C. sphaerospermum conidia at RH 85%. C. sphaerospermum conidia were harvested from MEA growth plates (3~4 weeks at 25 °C) by washing the plate with 0.02% Tween 80 solution and gently scratching the C. sphaerospermum colonies with a sterile cotton swab. The suspension was centrifuged at 2000 rpm for 10 minutes, the supernatant was discarded, and the pellet was re-suspended with sterile deionized water. This washing procedure was repeated twice to remove residual nutrients and the pellet was finally re-suspended with 0.02% Tween 80. The suspension was filtered through two layers of autoclaved Miracloth (EMD Biosciences Inc., La Jolla, CA) to remove the hyphae. An autoclaved magnetic stir bar with stir plate was used to mix the suspension during the aerosolization of C. sphaerospermum conidia. The conidia concentration in air flowing through the test filter was monitored with the UVAPS plus a six stage Andersen impactor for culturable
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ACCEPTED MANUSCRIPT conidia concentrations. The geometric mean aerodynamic diameter of the conidia was estimated to be 2.4 to 2.6 µm with a geometric standard deviation of 1.3 to 1.4. On average, 2.4 ×103 CFU/cm2 of conidia was loaded onto each test filter.
Procedure for Experiments on Variable Temperature Effect Small sections (38 mm × 51mm) were cut from the loaded filter and placed into five
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incubation flasks by hanging them from stainless steel probes with 8 sections per flask. The RH inside each flask was adjusted to be approximately 97% using a saturated potassium sulfate solution. Two temperature conditions with approximately constant RH were tested: room temperature with an average of 23 °C (mean RH=97%) and sinusoidal temperature according to equation (2) (sine temp) in the range 13 to 33 °C (mean RH=97%). For the room temperature condition, the incubation flasks were placed in a biosafety hood in a temperature controlled lab. For the sinusoidal temperature condition, the flasks were placed in the static chamber with controlled temperature. T = 23+10sin πt/12 where
Eq. (2)
T = temperature in °C
t = hour of the day, 0, 1, 2… 24 For each experimental replication, every two days until day 10, the eight filter sections in one flask were taken out for biomass analysis, six for ergosterol assay and two for the elution-culture method. The number of sections used for different biomass measurement methods was selected based on consideration of the lower detection limit of each method.
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ACCEPTED MANUSCRIPT Variable Humidity Tests For variable RH experimental conditions, filter sections were incubated in environments with a step change in RH every 12 hours. Three series each switching between 97% RH and a lower RH were investigated: 97-75% RH, 97-43% RH, and 97-11% RH. For each replication, 5 sets of filter sections were used. For every set of filter sections (8-pieces), two control incubation flasks were used with one maintained at 97% RH and the other maintained at the lower RH. The filter
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sections were switched between 97% RH and the lower RH flasks every 12 hours by moving the probe together with the filter sections with 97% RH as a start. The RH inside the incubation flasks was maintained at 97% RH, 75% RH, 43% RH, and 11% RH using a saturated potassium sulfate, sodium chloride, potassium carbonate, and lithium chloride solutions, respectively. The incubation flasks were placed inside a biosafety hood in a lab with temperature control. Every two days until day 10, one set of filter sections was removed for biomass assessment, six for ergosterol assay and two for the elution-culture method. For the RH=97-11% condition, the incubation period for the last replication was extended to 28 days with additional biomass measurement at day 14, 21, and 28 based on observation from the first two replications. Ergosterol analysis results from two trials of the RH=97-11% condition were not available because of instrument malfunction.
Viable Air Flow Tests Two experimental conditions with different fan operating patterns were tested at room temperature and RH greater than 95% for 10 days: fan continuously on and fan on 9 hours per day with a flow rate of 42 m3/min (1500 ft3/min), which corresponded to 9 m/min of air flow
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ACCEPTED MANUSCRIPT speed through filter media. For each replication, the biomass on a full size test filter loaded with malt extract particles and C. sphaerospermum conidia was detected with swab test and tease mount method immediately after loading and was then reinstalled in the wind tunnel at room temperature of 23 °C and RH of greater than 95% with the fan either continuous on or on 9 hours per day. After 5 days and 10 days, respectively, the biomass on the same test filter was measured with the swab test and tease mount methods on five randomly selected locations which were not
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re-used for subsequent tests. At the end of each replication test, filter sections were removed to measure biomass using both the elution–culture method and ergosterol assay.
RESULTS AND DISCUSSION Variable Temperature Effects For the three replicate tests under room temperature conditions, the temperature inside the incubation flasks was 24±1 °C, 23.3±0.3 °C, and 23.5±0.4 °C. For the sine temperature condition, the temperature inside the incubation flasks was well controlled to meet the target sinusoidal temperature condition given by equation (2). The carbon dioxide concentration was in the range of 250 to 600 ppm. This level of carbon dioxide concentration does not significantly impact growth of C. sphaerospermum (results not shown in this paper), and is much lower than the 200,000 ppm concentration that inhibits germination of fungal spores at ordinary temperatures.(41) The biomass (based alternatively on CFU counts and ergosterol content) at days 0, 2, 4, 6, 8, 10 of incubation divided by the biomass after initial loading was defined as the growth ratio. This ratio versus incubation time is plotted in Figure 3. The growth ratio based on ergosterol content was higher than that based on CFU counts during the first two days for the room
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ACCEPTED MANUSCRIPT temperature condition and during the first four days for the sinusoidal temperature condition. This is probably because hyphae extension is dominant compared to sporulation of new conidia during the initial growth period. Based on Student’s t-test (p
Journal of Occupational and Environmental Hygiene Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/uoeh20
Effects of Temperature, Humidity and Air Flow on Fungal Growth Rate on Loaded Ventilation Filters a
a
W. Tang , T. H. Kuehn & Matt F. Simcik
b
a
Department of Mechanical Engineering, University of Minnesota, 111 Church St SE, Minneapolis, MN 55455 b
Division of Environmental Health Sciences, School of Public Health, University of Minnesota, 420 Delaware St. SE, MMC 807, Minneapolis, MN 55455 Accepted author version posted online: 07 Apr 2015.
Click for updates To cite this article: W. Tang, T. H. Kuehn & Matt F. Simcik (2015): Effects of Temperature, Humidity and Air Flow on Fungal Growth Rate on Loaded Ventilation Filters, Journal of Occupational and Environmental Hygiene, DOI: 10.1080/15459624.2015.1019076 To link to this article: http://dx.doi.org/10.1080/15459624.2015.1019076
Disclaimer: This is a version of an unedited manuscript that has been accepted for publication. As a service to authors and researchers we are providing this version of the accepted manuscript (AM). Copyediting, typesetting, and review of the resulting proof will be undertaken on this manuscript before final publication of the Version of Record (VoR). During production and pre-press, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal relate to this version also.
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http:// www.tandfonline.com/page/terms-and-conditions
ACCEPTED MANUSCRIPT Effects of Temperature, Humidity and Air Flow on Fungal Growth Rate on Loaded Ventilation Filters
W. Tang1, T. H. Kuehn1, Matt F. Simcik2
1
Department of Mechanical Engineering
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University of Minnesota 111 Church St SE, Minneapolis, MN 55455
2
Division of Environmental Health Sciences
School of Public Health University of Minnesota 420 Delaware St. SE, MMC 807 Minneapolis, MN 55455
ABSTRACT This study compares the fungal growth ratio on loaded ventilation filters under various temperature, relative humidity (RH) and air flow conditions in a controlled laboratory setting. A new full size commercial building ventilation filter was loaded with malt extract nutrients and conidia of Cladosporium sphaerospermum in an ASHRAE Standard 52.2 filter test facility. Small sections cut from this filter were incubated under the following conditions: constant room temperature and a high RH of 97%; sinusoidal temperature (with an amplitude of 10 °C, an
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ACCEPTED MANUSCRIPT average of 23°C, and a period of 24 hours) and a mean RH of 97%; room temperature and step changes between 97% and 75% RH, 97% and 43% RH, and 97% and 11% RH every 12 hours. The biomass on the filter sections was measured using both an elution–culture method and by ergosterol assay immediately after loading and every 2 days up to 10 days after loading. Fungal growth was detected earlier using ergosterol content than with the elution–culture method. A student's t-test indicated that Cladosporium sphaerospermum grew better at the constant room
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temperature condition than at the sinusoidal temperature condition. By part-time exposure to dry environments, the fungal growth was reduced (75% and 43% RH) or even inhibited (11% RH). Additional loaded filters were installed in the wind tunnel at room temperature and an RH greater than 95% under one of two air flow test conditions: continuous air flow or air flow only 9 hours/day with a flow rate of 0.7m3/s (filter media velocity 0.15m/s). Swab tests and a tease mount method were used to detect fungal growth on the filters at day 0, 5, and 10. Fungal growth was detected for both test conditions, which indicates that when temperature and relative humidity are optimum, controlling the air flow alone can not prevent fungal growth. In real applications where nutrients are less sufficient than in this laboratory study, fungal growth rate may be reduced under the same operating conditions.
KEYWORDS
Fungal Growth · Ventilation Filters · Temperature · Humidity · Air Flow 5080 words
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INTRODUCTION Ventilation filters are used in heating, ventilating, and air conditioning (HVAC) systems to supply clean air to occupied spaces and maintain clean downstream HVAC components. Because microorganisms and particulate matter are ubiquitous in indoor and outdoor
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environments, ventilation filters in situ serve as sites that concentrate microorganisms and nutrient dust. Microorganisms on filters can proliferate when nutrients are available and environmental conditions are appropriate. Nutrients can come from the filter material itself such as cellulosic fibers or from captured dust. Several field studies conducted on ventilation filters have found evidence of microbial growth. A study by Simmons et al.(1) indicated that 8 out of 11 bag or panel filters removed from 7 hospitals in the eastern United States were colonized with fungi at the time of, or prior to, their manufacturers’ recommended replacement time. Price et al.(2) found 25% ~ 33% of untreated filters and 7~20% of chemically treated filters were colonized with mold. Ahearn et al.(3) observed Cladosporium and Penicillium growth on both sides of ventilation filters taken from office buildings. Microbial growth on ventilation filters may result in contaminating the air supplied to the occupied spaces with cells and or fragments of microorganisms, endotoxin, mycotoxin, and microbial volatile organic compounds that can lead to adverse health effects. The American Conference of Industrial Hygienists (ACGIH) emphasizes that “active fungal growth in indoor environments is inappropriate and may lead to exposure and adverse health effects".(4)
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ACCEPTED MANUSCRIPT Three environmental conditions that may affect microbial growth on filters are water available to microorganisms, air temperature, and air flow. Among these, water is the most critical parameter. Microbial growth on ventilation filters is usually observed under conditions of high relative humidity (RH): greater than 98%,(5) 90%,(6) 84%,(7) 80%,(8) and 70%.(9) A guideline published in Filtration and Separation (10) recommends keeping the three day averaged RH below 80% in all parts of an HVAC system whereas EPA suggests maintaining the RH below 60%
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continuously. Temperature affects the survival and growth of microorganisms. Temperature can affect the rate of enzyme-catalyzed reactions, and high temperatures can denature proteins. Each microbial species has minimum, optimum and maximum growth temperatures. Studies on the effects of air flow on microbial growth are mainly limited to ventilation filters. Maus et al. did not find a difference in viability of Bacillus subtilis when filter media were exposed to either air flow with RH greater than 85% or static air with RH greater than 98%.(5) However, they found growth of Aspergillus niger on some of the used media filters under static air but not on filters exposed to air flow. No clear inferences can be drawn because RH was not held constant across the different air flow conditions, and filters were obtained from different HVAC systems which may have collected dust with different nutrient compositions. In general, prior experimental studies fall into two categories: studies conducted on filters exposed to outdoor air without control of temperature, RH or captured dust composition(3, 7-9, 11-13) and studies conducted under controlled environmental conditions: 20~23°C and RH>98%(5), 22~24°C with a range of RH from 55% to 90%,(9), 70±3°F and 90%±2% RH.(14) For the studies conducted under controlled environmental conditions, the focus was on constant room temperature (around 20 to 24 °C) and constant RH. None investigated the effects of variable
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ACCEPTED MANUSCRIPT temperature or humidity on microbial growth. However, outdoor air temperature usually changes sinusoidally with time within one day, and this leads to a more damped variation in the temperature at the filters in an HVAC system. For example, the temperature measured at filter locations in the HVAC systems of a public building in Minneapolis, MN from early September to early October showed an approximate sinusoidal change with time on a daily basis. The outdoor air temperature varied between 33 and 87 °F and the mixed air (of outdoor and return air)
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temperature between 51 and 79 °F. Data from the same building indicated hourly variation of RH ranging from 23% to 93% within a single day. Thus, it is important to investigate the response of microorganisms to these variable environmental conditions. For some fungal species, growth in food at constant temperature and water activity has been well studied.(15) Microbial growth under variable environmental conditions has been limited to building materials such as gypsum-based materials(16), pine and spruce sapwood. (17) Li et al. concluded in their wind tunnel study that supply air velocity was not a direct factor that influenced microbial growth on duct surfaces, and that relative humidity was the main factor for fungal growth for supply air velocity of 3.0 m/s.(18) To our knowledge, no experimental studies on fungal growth on ventilation filters undergoing controlled but variable environmental conditions have been published. Starting with fungal growth under a reference condition (room temperature and high RH), this paper compares growth under sinusoidal temperature variation, step changes in RH and different air flow conditions, respectively. The goal of the research is to identify temperature, RH and airflow conditions that minimize fungal growth on HVAC filters.
METHODS
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ACCEPTED MANUSCRIPT The Challenge Microorganism and Nutrient Among the fungal species that were frequently found in buildings with mold problems, Cladosporium and Penicillium were particularly associated with HVAC systems.(19,
20)
Cladosporium sphaerospermum was selected in this study because it is more often found in indoor environments than outdoors(21) and it has greater tolerance of dry conditions than C. cladosporioides,(22) another Cladosporium species common in indoor environments. The wild C.
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sphaerospermum used in this study was isolated from indoor air sampling in a campus building at the University of Minnesota and was then incubated on malt extract agar (MEA) plates as the stock growth plate. The conidia of Cladosporium sphaerospermum suspended in 0.02% Tween 80 solution (by volume) are roughly spherical and range from 3 to 5 µm in diameter when observed under a microscope. An artificial nutrient, malt extract (BactoTM, Becton, Dickinson and Company, Sparks, MD), was selected to be loaded onto the test filters. Because malt extract is an optimum culture medium for C. sphaerospermum, it was reasoned that if a combination of environmental conditions were to reduce fungal growth on filters loaded with malt extract, the same reduction would be observed on filters loaded with ambient dust.
Experimental Facilities The experimental facilities consist of three major components: a wind tunnel, a static chamber, and incubation flasks. The wind tunnel was used to load test filters with the malt extract and C. sphaerospermum, and was used to vary air flow through test filters. The wind tunnel was built based on the requirements of ASHRAE Standard 52.2 1999 (see Figure 1a).(23) A Devilbiss 45
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ACCEPTED MANUSCRIPT nebulizer (Sunrise Medical, Somerset, Pennsylvania) was modified for the aerosolization system (see Figure 1b) and operated with compressed air of 1.93×105 Pa (28 psig) and dilution air of 0.0015 m3/s. The air temperature inside the wind tunnel was measured with a type T thermocouple and the RH was measured with an Omega HX 94V RH sensor. Facility qualification test results indicated that the aerosol concentration upstream of the test filter was uniform within the cross section of the tunnel with a coefficient of variance less than 12% for
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particle sizes ranging from 0.32 µm to 7.5 µm. This result indicates that the particulate concentration loaded on the test filters is uniform. A detailed description of the wind tunnel can be found in Tang et al. and Tang.(24, 25) A static chamber was used to provide a controlled sinusoidal temperature condition for filter media samples (see Figure 2a). The internal temperature was controlled by circulating water from a water bath through copper tubes wrapped around the stainless steel chamber. The incubation flask set up (see Figure 2b) included a 2000mL flask (Gledhill Shake Flask Assembly CO2, Ace Glass Inc., Vineland, NJ) and a customized stainless steel probe. The probe was built to seal one opening of the flask and to accommodate a Q-trak IAQ monitor (TSI, Inc., Shoreview, MN) for air temperature, RH, and carbon dioxide concentration monitoring inside the flask. The RH inside the flasks was maintained through saturated salt solutions.
Measurement Methods for Fungal Growth Two methods were used to quantify fungal mass present on filter samples: the elution-culture method and ergosterol essay. Microscopic observation was also used to observe overall fungal growth structure.
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Elution-Culture Method The C. sphaerospermum cells were removed from the test filter sections and cultivated on nutrient agar plates. Filter sections each with a size of 38 mm × 51 mm were eluted with twenty mL of phosphate buffered saline (PBS) with 0.02% Tween 80 solution and shaken for 60 minutes using a wrist action shaker (model 75, Burrel Scientific, Pittsburgh, PA). A dilution
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series was prepared if needed and aliquots from the suspensions/dilutions were plated on MEA plates. After four days of incubation at room temperature, colony forming units (CFU) were counted and the number of viable C. sphaerospermum was calculated using equation (1). CFUs on filter sections = Averaged CFUs on growth plates × dilution factor × volume of dilution solution Eq. (1)
Ergosterol Essay The elution-culture method can underestimate the biomass because it only measures those viable cells removed from the filter samples that also survive the culturing procedure. Ergosterol assay was included in this project to obtain better information on the total amount of fungal biomass present. As a membrane constituent of most fungi except the subdivision Mastigomycotina and rust fungi,(26) ergosterol is often used as an indicator of fungal biomass(27-33) and living cells.(34) Ergosterol is negligible in bacteria(35) and either absent or in minor amounts in higher plants.(27, 34) Ergosterol was found to be very stable in heat killed fungi(36) but degraded when exposed to bright sunlight or short wave UV radiation.(36-38)
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ACCEPTED MANUSCRIPT The ergosterol essay method was based on the procedures of Axelsson et al.(28) and Dong et al.(39) with necessary modifications. The sample preparation and extraction was conducted under yellow light to minimize photodegradation. Hexane solutions containing 283.5 ng of 7Dehydrocholesterol (as internal standard for GC-MS analysis) were added to each of the two 50mL sterile polypropylene centrifuge tubes with polyethylene plugs and dried completely with a stream of nitrogen. Filter sections were cut into small pieces of about 13 mm × 10 mm and
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placed into two centrifuge tubes. Eighteen mL of 10% methanolic potassium hydroxide was added per tube and the tubes were heated at 80 °C for 2 hours. After cooling to room temperature, the mixture of the filter pieces and the methanolic potassium hydroxide suspension was filtered through a 250-mL Buchner funnel with a 25-50 µm disc filter (Ace Glass Inc., Vineland, NJ). The tube was rinsed with methanol twice and the rinsed methanol solution was also filtered through the 250-mL Buchner funnel. HPLC grade water with a volume of one third of the volume of collected solution was added. The aqueous methanolic solution was extracted with hexane three times and the hexane phases were pooled, transferred to two 12-mL glass vials, and evaporated with streams of nitrogen until about 0.5 mL of solution was left in each vial. The solutions in the glass vials were then transferred into new 3-mL glass vials. The 12-mL vials were rinsed with hexane twice and the rinsed solutions were also transferred to the 3-mL vial. The hexane was dried completely with a stream of nitrogen and the samples were derivatized by 0.3 mL of TMS (trimethylsilyl) reagent (TMSI/TMCS) (100/1, v/v) at room temperature for 15 minutes. 0.8 mL of hexane and 1 mL HPLC water were added. The liquid was vortexed until the top hexane phase became clear. The top phase was transferred into an amber vial with a 250 µL insert ready for GC-MS analysis.
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ACCEPTED MANUSCRIPT To quantify the mass of ergosterol using GC-MS, a series of ergosterol standards was made by adding hexane solution containing 0.567 µg of 7-dehydrocholesterol to a series of hexane solutions containing 0.1068, 0.267, 0.534, 1.068, 2.67, and 5.34 µg ergosterol. The ergosterol standard series were then dried with nitrogen and derivatized by TMS reagent. The ergosterol standard and ergosterol extracted from the filter samples were injected into a Hewlett-Packard Model 5890 gas chromatograph and analyzed by a Hewlett-Packard Model 5972 mass
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spectrometer. Injections were made in the splitless mode using an HP 6890 series autosampler. Helium was used as the carrier gas with a flow rate of 1 ml/min. A Restek Rtx-XLB column (0.25 µm in thickness, 30 m × 0.25 mm I.D.) was used and the oven temperature was programmed as 170 to 300°C at 20°C/min and kept at 300°C for 9 minutes. The injection temperature was 290°C and the interface temperature was 300°C. The electron ionization energy was 70 eV. An initial analysis of a mixture of dehydrocholesterol and ergosterol derivatives with the MS operated in the scan mode (m/z between 41 and 450) indicated that the dominant ions m/z were 325 and 351 for 7-dehydrocholesterol derivative and 337 and 363 for ergosterol derivative. The ergosterol standard and ergosterol extracted from the filter samples were analyzed in selected-ion monitoring (SIM) mode of mass spectrometer using these four ions. A calibration curve was obtained by correlating the peak-area ratio of ion 363 over 351 to the mass ratio of ergosterol/dehydrocholesterol from the series of ergosterol standards. The mass of ergosterol extracted from filter samples was calculated from the calibration curve and the peakarea ratio of ion 363 over 351 in the samples. The ergosterol content was found to be 0.19±0.02 pg/CFU or 0.13±0.01pg/cell. This conversion number is very close to the average value of 0.16 pg/cell reported by Lau et al. (2006).(40)
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Microscopy Observation Three microscopy methods were used to observe overall fungal growth structure: scanning electron microscope (SEM), swab test
(6)
and tease mount. For SEM, 10 mm × 10 mm filter
samples were cut and air dried inside a biosafety hood for 1 hour and then coated with gold. In the swab test, we used a sterile Q-tip soaked with sterile PBS with 0.02% tween 80 solution to
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swab the filter surface and then transferred the material to a 150 mm malt extract plate. The area of the swabbed filter surface was initially determined to be a circle of 13 mm diameter and later changed to be the inner pleat edge of the filter (96 mm in length) for better results. For the tease mount method, a drop of acid fuchsin-lactic acid staining solution (lactic acid: Fisher Scientific; acid fuchsin: Sigma Chemical Co. St. Louis, MD) was applied to a microscope slide, and then a clear adhesive tape was applied to the filter sampling location to pull away filter fibers and fungal structure if present and pressed onto the microscope slide. When observed under a light microscope (Olympus BX 50), the stained fungal structure was purple in color. Photos were taken from the eyepiece with a digital camera. The SEM method provides the best quality of images of mycelial structures but it is also a destructive method that requires filter sections to be cut from the test filter; thus it can only be used at the end of the experimental period. The swab method is a semi-quantitative method and its efficiency may be limited by the transfer of fungal spore and or mycelia from the filter to the Q-tip and from the Q-tip to the malt extract plates. The tease mount method qualitatively shows the mycelial structure of fungal growth during the experimental period.
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ACCEPTED MANUSCRIPT Experimental Procedure Filter Loading: Malt extract particles were loaded on each new test filter (clean 610 mm × 610 mm × 51 mm electrostatically charged synthetic media pleated filters with media area of 4.83m2, initial efficiency rating MERV-14). Two percent (by weight) autoclaved malt extract solution was aerosolized and injected into the wind tunnel. The malt extract aerosol concentration upstream
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and downstream of the test filter was monitored with an ultraviolet aerodynamic particle sizer (UVAPS 3314, TSI, Inc., Shoreview, MN). The mass concentration was calculated with the assumption that the particle density of malt extract is 1.0 g/cm3. The total mass of malt extract loaded on the test filter was calculated to be approximately 30 grams. After loading, the test filter was sterilized with ethylene oxide (Anprolene, H. W. Anderson Products Inc., Chapel Hill, NC). The sterilized test filter was again placed in the wind tunnel and loaded with C. sphaerospermum conidia at RH 85%. C. sphaerospermum conidia were harvested from MEA growth plates (3~4 weeks at 25 °C) by washing the plate with 0.02% Tween 80 solution and gently scratching the C. sphaerospermum colonies with a sterile cotton swab. The suspension was centrifuged at 2000 rpm for 10 minutes, the supernatant was discarded, and the pellet was re-suspended with sterile deionized water. This washing procedure was repeated twice to remove residual nutrients and the pellet was finally re-suspended with 0.02% Tween 80. The suspension was filtered through two layers of autoclaved Miracloth (EMD Biosciences Inc., La Jolla, CA) to remove the hyphae. An autoclaved magnetic stir bar with stir plate was used to mix the suspension during the aerosolization of C. sphaerospermum conidia. The conidia concentration in air flowing through the test filter was monitored with the UVAPS plus a six stage Andersen impactor for culturable
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ACCEPTED MANUSCRIPT conidia concentrations. The geometric mean aerodynamic diameter of the conidia was estimated to be 2.4 to 2.6 µm with a geometric standard deviation of 1.3 to 1.4. On average, 2.4 ×103 CFU/cm2 of conidia was loaded onto each test filter.
Procedure for Experiments on Variable Temperature Effect Small sections (38 mm × 51mm) were cut from the loaded filter and placed into five
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incubation flasks by hanging them from stainless steel probes with 8 sections per flask. The RH inside each flask was adjusted to be approximately 97% using a saturated potassium sulfate solution. Two temperature conditions with approximately constant RH were tested: room temperature with an average of 23 °C (mean RH=97%) and sinusoidal temperature according to equation (2) (sine temp) in the range 13 to 33 °C (mean RH=97%). For the room temperature condition, the incubation flasks were placed in a biosafety hood in a temperature controlled lab. For the sinusoidal temperature condition, the flasks were placed in the static chamber with controlled temperature. T = 23+10sin πt/12 where
Eq. (2)
T = temperature in °C
t = hour of the day, 0, 1, 2… 24 For each experimental replication, every two days until day 10, the eight filter sections in one flask were taken out for biomass analysis, six for ergosterol assay and two for the elution-culture method. The number of sections used for different biomass measurement methods was selected based on consideration of the lower detection limit of each method.
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ACCEPTED MANUSCRIPT Variable Humidity Tests For variable RH experimental conditions, filter sections were incubated in environments with a step change in RH every 12 hours. Three series each switching between 97% RH and a lower RH were investigated: 97-75% RH, 97-43% RH, and 97-11% RH. For each replication, 5 sets of filter sections were used. For every set of filter sections (8-pieces), two control incubation flasks were used with one maintained at 97% RH and the other maintained at the lower RH. The filter
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sections were switched between 97% RH and the lower RH flasks every 12 hours by moving the probe together with the filter sections with 97% RH as a start. The RH inside the incubation flasks was maintained at 97% RH, 75% RH, 43% RH, and 11% RH using a saturated potassium sulfate, sodium chloride, potassium carbonate, and lithium chloride solutions, respectively. The incubation flasks were placed inside a biosafety hood in a lab with temperature control. Every two days until day 10, one set of filter sections was removed for biomass assessment, six for ergosterol assay and two for the elution-culture method. For the RH=97-11% condition, the incubation period for the last replication was extended to 28 days with additional biomass measurement at day 14, 21, and 28 based on observation from the first two replications. Ergosterol analysis results from two trials of the RH=97-11% condition were not available because of instrument malfunction.
Viable Air Flow Tests Two experimental conditions with different fan operating patterns were tested at room temperature and RH greater than 95% for 10 days: fan continuously on and fan on 9 hours per day with a flow rate of 42 m3/min (1500 ft3/min), which corresponded to 9 m/min of air flow
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ACCEPTED MANUSCRIPT speed through filter media. For each replication, the biomass on a full size test filter loaded with malt extract particles and C. sphaerospermum conidia was detected with swab test and tease mount method immediately after loading and was then reinstalled in the wind tunnel at room temperature of 23 °C and RH of greater than 95% with the fan either continuous on or on 9 hours per day. After 5 days and 10 days, respectively, the biomass on the same test filter was measured with the swab test and tease mount methods on five randomly selected locations which were not
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re-used for subsequent tests. At the end of each replication test, filter sections were removed to measure biomass using both the elution–culture method and ergosterol assay.
RESULTS AND DISCUSSION Variable Temperature Effects For the three replicate tests under room temperature conditions, the temperature inside the incubation flasks was 24±1 °C, 23.3±0.3 °C, and 23.5±0.4 °C. For the sine temperature condition, the temperature inside the incubation flasks was well controlled to meet the target sinusoidal temperature condition given by equation (2). The carbon dioxide concentration was in the range of 250 to 600 ppm. This level of carbon dioxide concentration does not significantly impact growth of C. sphaerospermum (results not shown in this paper), and is much lower than the 200,000 ppm concentration that inhibits germination of fungal spores at ordinary temperatures.(41) The biomass (based alternatively on CFU counts and ergosterol content) at days 0, 2, 4, 6, 8, 10 of incubation divided by the biomass after initial loading was defined as the growth ratio. This ratio versus incubation time is plotted in Figure 3. The growth ratio based on ergosterol content was higher than that based on CFU counts during the first two days for the room
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ACCEPTED MANUSCRIPT temperature condition and during the first four days for the sinusoidal temperature condition. This is probably because hyphae extension is dominant compared to sporulation of new conidia during the initial growth period. Based on Student’s t-test (p
