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Journal of Aerosol Science 41 (2010) 319325

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Journal of Aerosol Science

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Technical note

Generation characteristics of fungal spore and fragment bioaerosols by airow control over fungal cultures
Jun Hyun Lee a, Gi Byung Hwang a, Jae Hee Jung b, Dae Hee Lee a, Byung Uk Lee a,
a b

Aerosol and Bioengineering Laboratory, College of Engineering, Konkuk University, 1 Hwayang-dong, Gwangjin-Gu, Seoul 143-701, Republic of Korea Center for Environmental Technology Research, Korea Institute of Science and Technology, 39-1 Hawolgok-dong, Seongbuk-gu, Seoul 136-791, Republic of Korea

a r t i c l e in fo
Article history: Received 7 July 2009 Received in revised form 5 November 2009 Accepted 5 November 2009 Keywords: Bioaerosol Aerosolization Fungal particle Fragment Spore Flow control

abstract
We investigated the generation characteristics of airborne fungal fragments and spores by means of a newly developed fungal bioaerosol generation system that directs airow to the fungal cultures horizontally. By controlling the airow over the fungal cultures inside the generation system, we can produce three aerosolization modes of fungal bioaerosols: (i) fungal spores only, (ii) fungal fragments only, and (iii) a mixture of fragments and spores. As concerns over fungal fragments and spores have grown due to the adverse health effects of fungal bioaerosols, this generation study can be applied to develop techniques for controlling airborne fungal particles through means such as antifungal material tests, fungal bioaerosol ltering or sampling tests, and fungal bioaerosol inactivation tests. In addition, the generation system presented here simulates the environmental dispersal of fungal bioaerosols. Therefore, the results of this study can be interpreted as equivalent to the natural dispersal characteristics of fungal bioaerosols. & 2009 Elsevier Ltd. All rights reserved.

1. Introduction Most fungal or mold species grow with multi-cellular laments called hyphae forming a mycelium (Bauman, MachunisMasuoka, & Tizard, 2007). During the process of fungal growth, fungal spores are formed at the hyphae and released to the surrounding environment by airow, water contact, animal activities, and movement of their hosts (Moore-Landecker, 1972). When the fungal particles are released by airow, fungal spores as well as fungal fragments are dispersed into the air (Cho, Seo, Schmechel, Grinshpun, & Reponen, 2005; Green et al., 2006; Seo et al., 2007) and these airborne spore and fragment particles are termed fungal bioaerosols. Several recent studies have shown that fungal fragment bioaerosols, which are o 1 mm size debris of fungal hyphae and rny et al., may contain fungal allergens and toxins, can be more common than spore bioaerosols in air environments (Go 2002; Green et al., 2006). The number concentration of fungal fragment bioaerosols dispersed by airow was found to be higher than that of fungal spores under several air velocity conditions such as an indoor air condition (1.4 m/s), an outdoor rny et al., 2002; Seo et al., air condition (1.4 and 5.8 m/s), and a ventilation duct condition (29.1 m/s) (Cho et al., 2005; Go 2007). Therefore, the study of fungal fragment bioaerosols, such as their generation characteristics and appropriate sampling methods, becomes important in the process of addressing public health issues caused by fungal bioaerosols. Thus far, the scientic research on fungal fragment bioaerosols has been restricted due to an incomplete understanding of their generation characteristics and technical problems with sampling the fragment bioaerosols. As a start, a few
Corresponding author. Tel.: + 82 2 450 4091; fax: +82 2 447 5886.

E-mail address: leebu@konkuk.ac.kr (B.U. Lee). 0021-8502/$ - see front matter & 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.jaerosci.2009.11.002

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sampling studies have recently been conducted for fragment bioaerosols ( o 1 mm). Seo et al. (2007) designed a collection system consisting of two types of Sharp-Cut cyclone samplers (PM2.5 and PM1.0) and an after-lter for fungal particle rny et al. (2002) used a seven-stage collection with three size fractions. For the sampling of fractional fungal particles, Go Andersen cascade impactor and Cho et al. (2005) used an electrical low-pressure impactor. However, generation studies for fungal fragments have thus far been insufcient. One of the most common aerosolization mechanisms of fungal particles is dispersion by environmental airow. The effects of air velocity and relative humidity on the release of fungal particles from agar (Pasanen, Pasanen, Jantunen, & Kalliokoski, 1991; Zoberi, 1961) have been studied using cultivation as the method of analysis of enumerating released fungal particles. In other rny, Reponen, Grinshpun, and Willeke (2001) and Kanaani, Hargreaves, Ristovski, and Morawska (2009) research, Go rny studied the relationship between air velocity and the particle number concentration of generated aerosols. However, Go et al. (2001) did not consider methods specically applicable to fungal fragments and Kanaani et al. (2009) considered three common fungal spore generation apparatus under the low airow rates which were o 10.2 m/s. Therefore, in this study, we developed a specic fungal bioaerosol generator to generate fragment bioaerosols and analyzed dispersal conditions for the fungal fragment particles under high airow velocity conditions. The common dispersal mechanism for fungal bioaerosols is the airow impaction on fungal cultures in real environmental conditions. To simulate the environmental aerosolization of fungal particles into the air, we designed a new generator that uses airow as a major dispersal principle. Especially, we suspected that fungal cultures are occasionally exposed to strong winds from sources such as typhoons or hurricanes (which feature air velocities of 4050 m/s, (Heckert, Simiu, & Whalen, 1998; Ou, Liau, Hsu, & Tzang, 2002) and the moving vehicles (over 30 m/s), therefore we designed and operated an experimental apparatus to include such conditions. In previous studies of fungal bioaerosol generators, airow in the generators descended vertically onto the culture plates from holes in the top of the generator, impacting the culture perpendicularly as it passed over the plate (Jung, Lee C.H. et al., 2009; Kanaani et al., 2009; Lee et al., 2008). However, in this study, the new generator blows airow to the fungal cultures horizontally and there is no vertical airow impact. We think that vertical and horizontal airows have distinctive effects on the aerosolization characteristics of fungal particles and that this generator design is closer to the true-environmental dispersal mechanism of fungal bioaerosols in a real environment due to the airow. The two fungal species Aspergillus versicolor and Cladosporium cladosporioides were used in this study because they are commonly found in indoor and outdoor environments (Chanda, 1996; Flannigan, 1997; Yeo & Kim, 2002). The two species were dispersed using the new fungal bioaerosol generator with varying air velocities measured at the nozzles of the generator as from 7 to 70 m/s. An aerodynamic particle spectrometer was used to measure the generated particle size distributions under several experimental conditions. A Nuclepore track-etched polycarbonate membrane lter and a scanning electron micrograph (SEM, JSM-6380, JEOL, Japan) were used to observe the generated fungal bioaerosols. It was discovered that it is possible to generate three modes of fungal bioaerosols using the new generator: (1) fungal fragments, (2) fungal spores, and (3) a mixed mode bioaerosol consisting of both fragments and spores. 2. Material and methods Fig. 1 shows a schematic diagram of the experimental setup. The experimental procedure is as follows. The airow, which passes through a HEPA lter and a diffusion dryer, enters the fungal bioaerosol generator and blows the fungal

Compressed Air

Diffusion Dryer

HEPA Filter

Mass Flow Controller

Membrane Filter Fungal Bioaerosol Generator

Particle Size Distribution Analyzer

Ventilation

Fig. 1. Schematic diagram of the experimental setup.

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particles. The ow-rate of the airow is adjusted using a Mass Flow Controller (MFC, Mykrolis, FC-280S, USA). The air velocity at the horizontal nozzles of the fungal bioaerosol generator ranged from 7 to 35.4 m/s for experiments with A. versicolor. For C. cladosporioides, the air velocity ranged from 17.7 to 70 m/s. We chose these ranges of velocities because we could obtain three modes of particle size distributions for each species with these ranges and we wanted to know the generation characteristics of fungal bioaerosols in rather high-speed environments. The airow emitted from the horizontal nozzles blows the fungal cultures and generates fungal bioaerosols, which include fungal fragments and spores. We measured the particle size distributions and the number concentrations of the generated fungal bioaerosols by means of a Particle Size Distribution analyzer (PSD 3603, TSI Inc., MN, USA). The PSD 3603 has 256 channels of particle size and can measure aerosol particles with aerodynamic diameters ranging from 0.1 to 1000 mm. We used particle size ranges smaller than this total size range in graphs of results depending on detected particle concentration at size channels. The minimum ow rate of the experimental system was 2 l/min (7 m/s case) and the sampling ow rate of the PSD 3603 was 1 l/min. Therefore, excess owing air through the system emitted through a ventilation tube with a lter. The relative humidity of the airow was 1020%, and this level was maintained by ensuring the airow passed through a diffusion dryer. The temperature was approximately 25 1C. Using a Nuclepore track-etched polycarbonate membrane lter (Whatman, Brentford, UK) with a pore size of 0.05 mm, we also sampled the fungal bioaerosols under each experimental condition; we then used a scanning electron microscope (SEM, JSM-6380, JEOL, Japan) to observe the shape of the sampled bioaerosols. The measurement of particle size distribution and the sampling was conducted for 3 min during the process of fungal bioaerosol generation. For all the experiments, we conducted at least three replications with three fungal plates under one experimental condition. In previous studies, the fungal generators produced bioaerosols, but with no control over whether they were fragments or spores (Jung, Lee C.H. et al., 2009; Lee et al., 2008). In this study, we designed a new fungal bioaerosol generator with the capability of producing: (i) fragment bioaerosols only, (ii) spore bioaerosols only, and (iii) mixed bioaerosols. Figs. 2(a) and (b) show three-dimensional drawings of the fungal bioaerosol generator. The inner size of the generator is tted to a commonly used Petri plate, which has a diameter of 90 mm and a height of 15 mm. A metal tube is located in the middle of the generator and the airow enters the generator through this metal tube. There are six holes, each with a diameter of 1 mm, at the end of this metal tube. The airow passes through these six holes and blows horizontally toward the fungal cultures. The horizontal ow carries detached fungal particles to the outlet of the fungal bioaerosol generator. This design

Petri Dish

Fungal culture

Fig. 2. A three dimensional inside view of the fungal bioaerosol generator. The horizontal air jets come from six nozzles at the end of the inlet tube.

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is for simulation of the real environmental dispersal of fungal particles from one location due to airow in tunnels, pipes, and outdoor environments. A. versicolor (Korean Collection for Type Cultures (KCTC) 6987, Biological Resource Center, Korea) and C. cladosporioides (KCTC 16680) were selected as test fungal species. The Aspergillus spp. is common in air environments. Some Aspergillus species have harmful effects on human health, such as toxicity (aatoxin, carcinogenic sterigmatocystin, and so on) and allergic reactions. Also, the Aspergillus spp. is common when moisture problems occur in buildings. Cladosporium spp. is usually not pathogenic for humans, except for immuno-compromised patients. Cladosporium spp. has the ability to trigger allergic reactions in sensitive individuals (Samson & van Reenen-Hoekstra, 1988; Samson, van Reenen-Hoekstra, Frisvald, & Filtenborg, 1995). The fungal species were incubated and stored at 25 1C for 6 months in numerous Petri plates (test plates), each of which contained 20 ml of the malt extract agar. 3. Results and discussion Fig. 3 shows the particle size distributions of the generated A. versicolor fungal bioaerosols. As shown Fig. 3(a), the mode diameter of the size distribution is 0.32 mm and most of the generated particles are in the size range of 0.11 mm under the air velocity condition of 7 m/s at the nozzles. When we measured the particle size distributions under air velocity conditions of o 7 m/s, similar shapes of particle size distributions were obtained. These small particles were airborne fungal fragments with a size o 1 mm, and were also observed using a SEM as shown in Fig. 4(a). As the air velocity was increased to 14 m/s, different patterns of particle size distributions appeared. There are two mode diameters shown in Fig. 3(b), one around 0.3 mm (fragment size) and the other around 2.1 mm (spore size). This is in harmony with previous ndings that the diameter of A. versicolor spores ranges from 2 to 3.5 mm (Samson & van Reenen-Hoekstra, 1988). Under

350 500 300 dN/dlogDp (#/m3) dN/dlogDp (#/m3) 1 Aerodynamic diameter (m) 10 250 200 150 100 50 0 0.1 400 300 200 100 0 0.1 1 Aerodynamic diamter (m) 10

350 300 dN/dlogDp (#/m3) dN/dlogDp (#/m3) 0.1 1 Aerodynamic diameter (m) 10 250 200 150 100 50 0

1600 1400 1200 1000 800 600 400 200 0 0.1 1 Aerodynamic diameter (m) 10

Fig. 3. Particle size distributions of generated A. versicolor fungal bioaerosols under air velocity conditions of: (a) 7 m/s, (b) 14 m/s, (c) 21 m/s, and (d) 35.4 m/s.

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Fig. 4. Sampled A. versicolor fungal particles under air velocity conditions of (a) 7 m/s and (c) 35.4 m/s. Sampled C. cladosporioides fungal particles under air velocity conditions of (b) 17.7 m/s and (d) 70 m/s. (a and b) Have 0.5 mm scale bars, (c) has a 5 mm scale bar and (d) has a 1 mm scale bar.

the air velocity conditions between 7 and 14 m/s, we observed patterns similar to those in Fig. 3(b). Therefore, under such conditions, we can generate a mixture of fungal spores and fragments of A. versicolor. Fig. 3(c) shows the particle size distribution under an air velocity of 21 m/s. Under this condition, the amount of generated fragment particles is signicantly reduced, and most of the generated fungal bioaerosols are fungal spores. Fig. 3(d) shows the results at the air velocity of 35.4 m/s. A pattern of size distributions similar to those in Fig. 3(c) can be observed. The sampled fungal particles were observed via a SEM, as shown in Fig. 4(c), and fungal spores were observed. Overall, we can generate fragment bioaerosols, spore bioaerosols, and mixed particles of A. versicolor by varying the air velocity conditions in the generator. Fig. 5 shows the particle size distributions of generated C. cladosporioides fungal bioaerosols. Although the total aerosol concentration of C. cladosporioides is much higher than in the A. versicolor case, we can observe a pattern of generated particles similar to that of A. versicolor: fragments, spore bioaerosols, and a mixture of both. Fungal fragment bioaerosols, as shown in Fig. 5(a), were generated under air velocity conditions o 17.7 m/s at the nozzles of the inlet, which is a higher value than in the A. versicolor case. These small particles were airborne fungal fragments with a size o 1 mm, and were also observed using a SEM as shown in Fig. 4(b). A bioaerosol mix of fragments and spores was generated under an air velocity condition of 35.4 m/s. Under the air velocity conditions of 53.1 and 70 m/s, fungal spore bioaerosols were generated. The sampled fungal particles were observed via a SEM, as shown in Fig. 4(d), and fungal spores were observed. As the air velocity condition varied from 17.7 to 70 m/s at the nozzles of the inlet tube, the mode diameters of the particle size distribution changed from 0.3 to 3 mm. The results presented in Figs. 3 and 5 are not only verication that the fungal bioaerosol generator introduced here generates three modes of fungal bioaerosols, but also an important clue of the dispersal mechanism of fungal bioaerosols. When the air velocity over the culture is low, fragment particles are detached from the fungal hyphae and dispersed to air environments. Under low air velocity conditions, the spores do not respond to the airow. As the airow becomes stronger, the spore bioaerosols, which have sizes larger than 2 mm, are detached and dispersed into the air. The air velocity required to detach spores into the air depends on the species: A. versicolor spore bioaerosols start to be generated at an air velocity of 14 m/s, while C. cladosporioides began to generate spore bioaerosols at 35.4 m/s under these experiment conditions. Another observation is that when the spore concentration increases, the concentration of fragments is decreased. This result can be explained as follows: most of fragment bioaerosols may come from the spore regions of the fungal hyphae, and when spores are detached from the fungal hyphae, the fragments are just attached to the detaching spores and have less opportunities to be dispersed independently.

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3500 3000 dN/dlogDp (#/m3) dN/dlogDp (#/m3) 1 Aerodynamic diameter (m) 10 2500 2000 1500 1000 500 0 0.1

1.2e+7 1.0e+7 8.0e+6 6.0e+6 4.0e+6 2.0e+6 0.0 0.1 1 Aerodynamic diameter (m) 10

3e+7 3e+7 dN/dlogDp (#/m3) dN/dlogDp (#/m3) 2e+7 2e+7 1e+7 5e+6 0 0.1 1 Aerodynamic diameter (m) 10

6e+8 5e+8 4e+8 3e+8 2e+8 1e+8 0 0.1 1 Aerodynamic diameter (m) 10

Fig. 5. Particle size distributions of generated C. cladosporioides fungal bioaerosols under air velocity conditions of: (a) 17.7 m/s, (b) 35.4 m/s, (c) 53.1 m/s, and (d) 70 m/s.

The experimental results of this study also suggest that the air velocity conditions in indoor and outdoor environments will inuence the composition of fungal fragment and spore bioaerosols present in those air environments. 4. Conclusion In this study, we designed a fungal bioaerosol generator that can produce three modes of fungal particles: fragment bioaerosols, spore bioaerosols, and particles that are a mix of fragments and spores, by adjusting air ow velocities over the fungal cultures inside the generator. Fragment bioaerosols are generated at relatively low velocities (7 m/s for A. versicolor and 17.7 m/s for C. cladosporioides), and the generation of fungal spore bioaerosols requires higher velocities. The air velocity conditions over the fungal cultures will play an important role in determining the compositions of fungal bioaerosols in indoor and outdoor environments.

Acknowledgment This work was supported by Konkuk University in 2009. References

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