UV-Tolerant Culturable Bacteria in an Asian Dust Plume Transported Over the East China Sea

UV-Tolerant Culturable Bacteria in an Asian Dust Plume Transported over the East China Sea

Kazutaka Hara1*†, Daizhou Zhang1, Hiromi Matsusaki1, Yasuhiro Sadanaga2, Keisuke Ikeda3, Sayuri Hanaoka3, Shiro Hatakeyama3

1 Faculty of Environmental and Symbiotic Sciences, Prefectural University of Kumamoto, Kumamoto 862-8502, Japan 2 Graduate School of Engineering, Osaka Prefecture University, Sakai, Osaka 599-8531, Japan 3 Graduate School of Agriculture, Tokyo University of Agriculture and Technology, Fuchu, Tokyo 183-8509, Japan

ABSTRACT

Airborne bacteria are dispersed along with Asian dust and have various influences on atmospheric characteristics, downwind ecosystems, and public health. In this study, aircraft observations were conducted during Asian dust passage and non-dust conditions over the East China Sea in December 2010, and the composition of culturable bacteria within the dust was investigated using culture-based gene analysis. The 16S rRNA gene analysis revealed that the 34 cultured strains isolated from the transported dust could be grouped into Bacillales and Actinomycetales. Highly tolerant endospore-forming bacteria (25 isolates) were predominant in the composition. On the other hand, all non-endospore-forming bacteria (9 isolates) were gram-positive bacteria with high guanine and cytosine contents, which have substantial ultraviolet (UV) resistance. Thus, the isolated culturable bacteria were strains having some tolerance for exposure to UV radiation. Although the atmosphere is an extreme environment for bacterial survival, Asian dust can facilitate atmospheric dispersion of culturable bacteria that are resistant to the harsh ambient air environment.

Keywords: Bioaerosols; Culturable bacteria; Asian dust; Long-range transport.

INTRODUCTION Yamada et al., 2010). Given the long-range diffusion of Asian dust in the Northern Hemisphere (Uno et al., 2009), Atmospheric dust derived from desert areas is frequently microorganisms internally and/or externally mixed with transported great distances (e.g., Uno et al., 2009). Dust dust are expected to be transported in the same manner. particles influence the global radiative balance (Satheesh Some field experiments coupled with 16S and 18S rRNA and Moorthy, 2005) and hydrologic cycle by acting as ice gene analysis revealed that Asian dust can possibly bridge nuclei and cloud condensation nuclei (Andreae and Rosenfeld, distant microbial ecosystems (Yukimura et al., 2009; Jeon 2008). Mineral dust also has a beneficial effect on fertilization et al., 2011; Yamaguchi et al., 2012; Jeon et al., 2013). It of downwind ecosystems (Bishop et al., 2002). However, has been also reported that biological materials in dust, some studies have shown that dust particles cause adverse such as bacteria and organic substances, might enhance the respiratory effects in humans (Kwon et al., 2002; Ichinose efficiency of ice-cloud formation and precipitation (Pratt et et al., 2005). al., 2009; Creamean et al., 2013). Biological substances in Recent studies have suggested that arid and semiarid dust can have positive and negative impacts on humans, regions of the Asian continent are potential sources of airborne and these substances increase adverse respiratory effects in microbes associated with desert dust (Kellogg and Griffin, comparison with pure minerals (Ichinose et al., 2008; Liu 2006; Kobayashi et al., 2007; Kakikawa et al., 2008; Maki et al., 2014). et al., 2008; Kenzaka et al., 2010; Nishimura et al., 2010; To estimate the influences of airborne microbes transported in Asian dust on downwind ecosystems, we need to consider what kind of microorganism can be transported with maintenance of microbial survivability through long-range † Now at Arid Land Research Center, Tottori University, atmospheric dispersion (Kellogg and Griffin, 2006; Iwasaka et Tottori 680-0001, Japan al., 2010). Studies have indicated that culturable halobacteria were sometimes dispersed with Asian dust (Echigo et al., * Corresponding author. 2005; Hua et al., 2007; Maki et al., 2010, 2011). On the Tel.: +81-(0)857-23-3411; Fax: +81-(0)857-29-6199 other hand, culture-independent methods revealed that various E-mail address: [email protected] types of bacteria were present in desert soils and were

592 Hara et al., Aerosol and Air Quality Research, 15: 591–599, 2015 transported in Asian dust (Yamaguchi et al., 2012; Maki et al., then placed on sterile 0.22-µm pore size membrane filters 2014). Therefore, both tolerant and non-tolerant bacteria exist (Millipore) in the filter holders. After that, sampling units in deserts and dusty air, either viable or dead. Atmospheric were assembled and the units were stored in sterile plastic stressors such as ultraviolet (UV) radiation, low temperature bags at 4°C until sample collection. The above procedures and desiccation are harmful to airborne microbes, which can were performed in a laminar flow hood sterilized with 70% be critical to their viability in atmospheric transport (Lighthart, ethanol and UV (254-nm wavelength) radiation. 1997; Tong and Lighthart, 1997; Smith et al., 2011). However, Before sample collection, R2A agar media (BD Difco, there is limited information on the survival of airborne Detroit, MI) and tryptic soy agar media (TSA; BD Difco) bacteria transported with Asian dust. were prepared with 100 µg/mL cycloheximide added to Here we present qualitative information of culturable inhibit fungal growth. To rule out artificial contamination, bacteria associated with an Asian dust plume transported samples of all prepared agar media were incubated at room over the East China Sea. Aircraft observations were conducted temperature for more than 10 days before culture experiments over the sea offshore of western Japan during Asian dust were initiated. passage and non-dust conditions in December 2010, and isolated bacteria were analyzed using culture-based gene Airborne Observation and Sample Collection analysis. The purpose of this study is to indicate the Airborne observations were made over the East China composition of culturable bacteria transported in an Asian Sea (Fig. 1) near Fukue Island, on 11, 12 and 14 December dust plume over the East China Sea, and to estimate from 2010. The base was at Fukue Airport, Nagasaki, western the results bacterial tolerance associated with their survival Japan (128.83°E, 32.67°N; black asterisk in Fig. 1). A during atmospheric transport. Beechcraft King Air 200T was chartered from Diamond Air Service Inc. (Aichi, Japan) for observation flights. An inlet MATERIAL AND METHODS was installed on the upper portion of the airframe. Outside air from the inlet was introduced into a glass manifold and Preparation then supplied to air samplers and analyzers. During each Air samples were collected using sterile gelatin filters observation flight, airspeed was maintained at approximately (Sartorius AG, Germany). Sampling with these filters has 80 m/s. We assumed a particle density of 1.0 g/cm3 for bacteria high collection efficiency across a wide range of flow rates, and 2.3 g/cm3 for mineral dust particles. Given these two for example, > 90% for Bacillus endospores (Parks et al., airborne particle densities, estimated 50% cutoff aerodynamic 1996; Burton et al., 2005). All sampling apparatus such as diameters of the inlet at the aforementioned flight speed membrane filters, filter holders, tubes and tube clumps were were approximately 3.6 µm and 2.4 µm, respectively. first autoclaved at 121°C for 15 min. The sterile gelatin During the observation flights, sampling units and air filters were cut to fit on 47-mm filter holders (Millipore, samplers (Kobayashi et al., 2007) were connected to the Billerica, MA) using sterile instruments. Gelatin filters were manifold using sterile silicon tubes with a closed tube

Fig. 1. Flight and dust information. Flights on 11 and 12 December 2010 shown as red and blue lines, respectively. Flight on 14 December was similar to that on 12 December. Areas affected by Asian dust on 11 December marked by black dots (13 of 61 JMA monitoring sites; http://www.jma.go.jp/jma/index.html). Black asterisk indicates Fukue airport.

Hara et al., Aerosol and Air Quality Research, 15: 591–599, 2015 593 clamp. Three trip blanks (gelatin filters in sterile 50 mL Taq Hot Start version (Takara Bio Inc.), 2.5 µL each primer, centrifugal tubes) were prepared for every flight. When the and 50 ng DNA extract. The PCR protocol consisted of 1 aircraft reached each sampling altitude, interiors of the cycle for 10 min at 94°C, 30 cycles of denaturation at 94°C inlet, pipe and manifold were primed with outside air for 5 for 40 seconds, annealing at 56°C for 1.5 min, extension at minutes. A tube clamp was then opened and outside airborne 72°C for 1.5 min, and 1 cycle for 10 min at 72°C. Each particles were collected on the filters through the manifold, amplified 16S rRNA gene was purified using the QIAquick at a rate of 16 L/min for 30 minutes. The tube clamp was then PCR Purification Kit (Qiagen Inc.) according to the kit closed until the next sample collection. After observations, protocol. Subsequently, each purified 16S rRNA gene was tube ends of the sampling units were wrapped with Parafilm, sequenced by Macrogen Inc., Japan (http://www.macrogen. and the units were placed into sterile plastic bags. The above com), and a phylogenetic tree including all sequences aligned procedures were done using sterile disposable gloves. using Crustal W was constructed according to the neighbor- Before observations began, an air sample from inside the joining method. Reported nucleotide sequence data are aircraft was collected on 10 December following the same available in the GenBank, EMBL and DDBJ databases under procedures, to evaluate the potential for contamination accession numbers AB748938 through AB748975. from inside the aircraft. All collected samples and trip blanks were stored at 4°C until further treatment. RESULTS Particle mass and number concentrations were also monitored during each flight. Particle mass concentrations On 11 December 2010, Asian dust was observed in were measured in diameter ranges 0.5–1.0 µm, 0.5–2.5 µm, western Japan. The airborne observation on that day was 0.5–7.0 µm and 0.5–10 µm, using a particle mass monitor conducted during Asian dust passage over the East China (GT-321; Shibata, Japan). Particle number concentrations Sea, at altitudes 500, 1000, 2000 and 3000 m. Table 1 shows with equivalent optical diameter Dp were measured in Dp > particle concentrations and flight observation data for each 2.5 µm, using an optical particle counter (GT-521; Shibata). level flight on 11, 12 and 14 December. Measurements by Ambient temperature and other flight observation data the particle mass monitor for each flight indicated that (e.g., atmospheric pressure and flight speed) were monitored concentrations in diameter range 0.5–10 µm were almost onboard the aircraft. The flight system is also described in equal to those of 0.5–7.0 µm (data not shown). In this study, other works (Hatakeyama et al., 2011; Fujiwara et al., 2014; measured concentrations within 0.5–7.0 µm were used as Hatakeyama et al., 2014). particle mass concentrations and shown in Table 1. Particle mass concentrations in level flights ranged between 1.3 and Cultivation 62 µg/m3, and number concentrations of coarse particles 3 5 The gelatin filters were removed from the filter holders in (Dp > 2.5 µm) were between 5.1 × 10 and 2.2 × 10 a sterilized laminar flow hood at the Prefectural University particles/m3. Particle mass and number concentrations in the of Kumamoto. Each gelatin filter was dissolved in sterile dust (11 December) were approximately 7–30 times higher Milli-Q water at 37°C, and 0.2 mL sample aliquots were than those in non-dusty air (12 and 14 December). streaked onto TSA and R2A agar media and incubated at Two samples collected at 500 and 1000 m altitudes on 11 25°C for approximately 1 month (e.g., van der Linde et al., December, a sample at 500 m on 12 December, a sample 1999). Single isolated bacterial colonies were identified and inside the aircraft (Control in Table 1), and trip blanks were transferred to fresh media three times to obtain pure cultures. used for cultivation. In total, 38 culturable bacterial strains were isolated from the four samples (Fig. 2). From Sample Analysis A, 15 strains were isolated on R2A agar and 10 strains DNA extraction and polymerase chain reaction (PCR) were isolated on TSA. From Sample B, five bacterial strains were carried out for bacterial isolates. Each isolated strain were isolated on R2A agar and four strains were isolated was incubated with R2A broth (Nihon Pharmaceutical Co., on TSA. No bacteria were isolated from Sample C. Four Ltd., Japan) or tryptic soy broth (BD Difco). A total 1 mL of strains were isolated on TSA from the control, but there broth culture was centrifuged at 6000 rpm for 5 min. was no bacterial growth in trip blanks. Supernatant was removed and bacterial cells were mixed with The result of gene and phylogenetic analysis is shown in 180 µL of lysis buffer, as recommended for DNA extraction Figs. 3(a) and 3(b). Based on 16S rRNA gene sequence from gram-positive bacteria in the DNeasy Blood and similarity, 38 bacterial isolates were classified into species. Tissue Kit (Qiagen Inc., Valencia, CA). Subsequent extraction For each isolate, the obtained sequences showed more than procedures were followed according to the kit protocol, and 99% sequence identity. All strains isolated from Samples A 200 µL of DNA elute was obtained. Extracts were concentrated and B were gram-positive and grouped into two orders, to 100 µL in TE buffer (pH.8.0) by ethanol precipitation, Bacillales and Actinomycetales. The 25 bacterial isolates and stored at −20°C until subsequent PCR amplification. obtained from Sample A were assigned to the genera Bacillus We used universal bacterial primers 27F (5’- (13 isolates), Streptomyces (6 isolates), Paenibacillus (2 AGAGTTTGATCCTGGCTCAG-3’) and 1492R (5’- isolates), Terribacillus (2 isolates), Gordonia (1 isolate), and TACGGYTACCTTGTTACGACTT-3’) for PCR Shimazuella (1 isolate). The nine strains obtained from amplification (Weisburg et al., 1991). The 50-µL reaction Sample B were in the genera Bacillus (5 isolates), mix included 5 µL 10X Ex Taq Buffer (Takara Bio Inc., Terribacillus (2 isolates), Saccharopolyspora (1 isolate), Japan), 4 µL dNTP mixture (Takara Bio Inc.), 0.25 µL Ex and Streptomyces (1 isolate).

594 Hara et al., Aerosol and Air Quality Research, 15: 591–599, 2015 Sample ) 3 particles/m Number 3 Japan StandardTime, Temp; (×10 ) 3 > 2.5 µm), NA; Not Available. Available. Not NA; > 2.5 µm), p D

Fig. 2. Photographs of bacterial colonies isolated from Asian dust samples. (a): Bacillus sp. (AECSB05) and (b) Streptomyces sp. (AECSS06) in Figs. 3(a) and 3(b).

Four bacterial strains obtained from the control were gram-positive and belonged to Actinomycetales (Fig. 3(a)). Isolated strains were assigned to the genera Streptomyces (3 isolates) and Micrococcus (1 isolate). Although Streptomyces species were also isolated from Samples A and B, these were not identical at the 16S rRNA gene level to Streptomyces species isolated from the control (Fig. 3(a)).

DISCUSSION

Culturable bacterial strains isolated from the dust predominantly belonged to the order Bacillales (Fig. 3(b)). Bacteria such as Bacillus, Terribacillus, Paenibacillus and 870 870 31.5°N 32.7°N–126.5°E, 128.4°E, 11:06–11:48 4.1 0.55) (± 2.4 4.5) (± 7.7 550 550 32.6°E 31.9°E–128.4°N, 127.1°N, 17:45–18:06 3.8 4.3) (± 6.1 9.2) (± 15 1100 1100 32.5°N 31.3°N–128.3°E, 126.8°E, 10:07–10:52 4.5 8.8) (± 54 30) (± 220 B 2000 2800 128.2°E, 32.5°N–126.8°E, 31.2°N 31.7°N 31.1°N–127.3°E, 126.7°E, 10:55–11:38 11:43–12:37 3.0 –2.3 0.64) (± 2.4 1312) (± 5.9) (± 14 53 25) (± 1900 1900 2900 32.7°N 31.4°N–128.5°N, 126.6°N, 10:11–11:00 31.6°N 32.7°N–126.7°E, 128.6°E, 9:23–10:06 4.3 –1.7 0.93) (± 1.3 1.1) (± 2.1 3.7) (± 5.1 5.2) (± 8.0 2000 2000 3000 31.7°N 32.8°N–126.8°N, 128.7°E, 32.8°N 13:14–13:56 31.6°N–128.7°E, 126.8°E, 14:01–14:44 Shimazuella NA -0.17 1.0) (± 6.0 0.96) (± 2.2 species 2.4) (± 14 6.8) (± 12 can form highly tolerant endospores (Nicholson et al., 2000; Park et al., 2007). In the case of genus Bacillus, spore coats, specific proteins and DNA repair machinery in the endospores are important factors to prevent damage from solar UV radiation, temperature, desiccation, and chemical substances (Nicholson et al., 2000). It is obvious that the endospore state is advantageous to maintain bacterial survivability during atmospheric transport. In past

Air Air (m) Altitude End) to (Start Course Flight (JST) Time (°C) Temp (µg/m Mass studies, endospore-forming Bacillus species were frequently isolated from long-range transported dust using the culture-

Information on airborne observations. Altitude; Mean pressure altitude, Flight duration Time; or sampling time (Control), JST; dependent and independent methods (Hua et al., 2007; Dusty (Dec. 11) 11) (Dec. Dusty 490 31.4°N 32.6°N–127°E, 128.9°E, 9:21–10:03 8.7 34) (± 62 19) (± 170 A Non-dusty (Dec. 12) 12) (Dec. Non-dusty 440 32.6°N 31.5°N–128.2°E, 126.7°E, 11:51–12:33 8.1 NA 4.9) (± 5.7 C Non-dusty (Dec. 14) 14) (Dec. Non-dusty 460 31.7°E 32.7°E–126.9°N, 128.5°N, Maki 16:30–17:11 et 5.3 al., 2010; 0.82) (± 9.2 Jeon 3.4) (± 13 et al., 2011; Maki et al., 2011; Tanaka et al., 2011; Yamaguchi et al., 2012). The result of Inside of airplane (Dec. 10) airplane of Inside 15:40–16:10 Control Table 1. Mean temperature, Mass; Mean particle concentration,mass Number; Mean number concentration coarse of particles ( the present study is thus consistent with past investigations.

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We also isolated culturable non-endospore-forming bacteria cause the formation of reactive oxygen substances such as 1 – belonging to the order Actinomycetales (Streptomyces, •OH, H2O2, O2 and O2 in the cells, which trigger disruption Saccharopolyspora and Gordonia species) from the dust of cell membranes, inactivation of several enzymes, and (Fig. 3(a)). Characteristics of these species are high guanine DNA damage (Pattison and Davies, 2006). These damages and cytosine (GC) contents in their DNA (Singer and Ames, induced by both direct and indirect reactions with solar UV 1970; Arenskӧtter et al., 2004; Ventura et al., 2007; Qin et radiation lead to substantial cell death (Demidova and al., 2010). It has been reported that bacterial tolerance for Hamblin, 2004; Lukšienė, 2005). solar UV radiation increases with greater G + C content Culturable UV-tolerant bacterial strains were also isolated (Singer and Ames, 1970), i.e., high GC gram-positive bacteria in the previous studies carried on near grounds in the arrival have a certain level of solar UV resistance. Griffin et al. areas of Asian, African and Australian dust (Griffin et al., (2006) also mentioned that high GC contents might enhance 2006; Hua et al., 2007; Lim et al., 2011). However, these bacterial survival in atmospheric transport with African dust. studies could not omit the background or bacteria from Overall, all isolated culturable bacteria in the present study local sources, meaning that previous results could contain were strains with some tolerance for UV radiation exposure. not only the compositions of bacteria transported in dust but UV radiation can be separated into three wavelengths, UV-A also some few bacterial compositions aerosolized from near (400–320 nm), UV-B (320–280 nm) and UV-C (< 280 nm). sampling sites. Sample collections over the surfaces by Solar UV-A and UV-B radiation is able to penetrate the ozone using aircrafts and/or air balloons can decrease inputs of layer. UV-B radiation is absorbed by DNA in cells and microbes from various sources with respect to atmospheric inhibits DNA replication and RNA transcription (Mitchell samples obtained during dust passage, although those sample and Karentz, 1993). In addition, UV-A and UV-B radiation collections have other problems. One of the problems on

Fig. 3(a). Phylogenetic tree from bacterial isolates cultured on TSA and R2A agar media. The phylogenetic tree was constructed using the neighbor-joining method. Reference sequences (> 1400 bp) are shown in regular font with GenBank accession numbers. Sequences of isolated bacteria are shown in bold, together with sample and culture media information. Scale bar indicates 0.1 changes per nucleotide. Sequence assembly had > 99% sequence identity. Phylogenetic relationships were bootstrapped 1,000 times, and bootstrap values > 50% are shown.

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Fig. 3(b). Phylogenetic tree (continued). aircraft observations was inevitable particle loss in inlets and the present study. Recent works show that mineral particles pipes (e.g., Pui et al., 1987). Considering the cutoff diameter transported in long-range transported dust were dominated in of the inlet and measurements of particle mass concentrations, the range of particle diameter between 2 to 5 µm in remote the inevitable particle loss in our aircraft observations was areas (Zhang et al., 2003; Perry et al., 2004). Other studies occurred in the ranges of airborne particles larger than 7 µm. reported that almost bacteria in soils, water, atmosphere and As a result, most airborne particles > 7 µm and some airborne long-range transported dust were present in the size range particles < 7 µm in the air were not collected for analysis in smaller than 1 µm (Bae et al., 1972; Watson et al., 1977;

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Hara and Zhang, 2012). Therefore, our aircraft observations We acknowledge Prof. Fumihisa Kobayashi for his support were adequate to obtain airborne bacteria associated with or with sample collections. We also acknowledge Prof. Koji without mineral dust, even though the data included some Arizono and his laboratory staff for their support with sample underestimations. Another potential problem with aircraft analysis. In addition, we thank Prof. Yasunobu Iwasaka, observations could be contamination from inside the airplane. Prof. Atsushi Matsuki, Prof. Teruya Maki, Dr. Maromu In the present study, culturable bacteria isolated from the dust Yamada, and Dr. Yutaka Tobo for scientific discussions. were not identical at 16S rRNA gene level with culturable This study was supported by the Sumitomo Environmental bacteria isolated from air inside the aircraft. Additionally, no Foundation and the Grant-in-Aid for Scientific Research on bacterial growth was found in the trip blanks, indicating that Innovative Areas No. 4003 "Impacts of Aerosols in East Asia artificial contamination during sample preparation, collection on Plants and Human Health (ASEPH)" from the Ministry of and cultivation processes was minimal for at least the Education, Culture, Sports, Science and Technology of Japan. culturable bacterial composition. Thus probably up to the 34 types of cultured UV-tolerant bacteria traveled in the REFERENCES Asian dust plume over the East China Sea. Numerous case studies have indicated that solar UV Andreae, M.O. and Rosenfeld, D. (2008). Aerosol-Cloud- radiation can negatively influence microbial survival in Precipitation Interactions. Part 1. The Nature and various environments (e.g., Llabrés and Agusti, 2006). The Sources of Cloud-Active Aerosols. Earth Sci. Rev. 89: potential for bacterial cell membrane damage by the 13–41. atmospheric stressors through long-range atmospheric Arenskӧtter, M., Brӧker, D. and Steinbüchel, A. (2004). transport has been also reported (Hara and Zhang, 2012). Biology of the Metabolically Diverse Genus Gordonia. Even though the present study based on only one dust Appl. Environ. Microbiol. 70: 3195–3204. event over the East China Sea, the result indicates typical Bae, H.C., Cota-robles, E.H. and Casida, L.E. (1972). bacterial strains survived in long-range atmospheric transport Microflora of Soil as Viewed by Transmission Electron without viable bacterial strains of local sources and artificial Microscopy. Appl. Microbiol. 23: 637–648. contamination. However, a lot of case studies in various Bishop, J.K.B., Davis, R.E. and Sherman, J.T. (2002). dust events are needed to elucidate survival mechanisms of Robotic Observations of Dust Storm Enhancement of airborne bacteria transported in Asian dust. Furthermore, Carbon Niomass in the North Pacific. Science 298: 817– some laboratory experiments are also necessary to assess 821. bacterial UV-tolerance in the air. Nevertheless, bacterial Burton, N.C., Adhikari, A., Grinshpun, S.A., Hornung, R. UV-tolerance might be related to bacterial survival in and Reponen, T. (2005). The Effect of Filter Material on atmospheric transport from arid and semiarid regions of the Bioaerosol Collection of Bacillus subtilis Spores Used as Asian continent. a Bacillus anthracis Simulant. J. Environ. Monit. 7: 475– 480. CONCLUSIONS Creamean, J.M., Suski, K.J., Rosenfeld, D., Cazorla, A., DeMott, P.J., Sullivan, R.C., White, A.B., Ralph, F.M., We conducted aircraft observations in western Japan Minnis, P., Comstock, J.M., Tomlinson, J.M. and Prather, during Asian-dust passage and non-dust conditions over the K.A. (2013). Dust and Biological Aerosols from the East China Sea. Atmospheric samples were cultured for Sahara and Asia Influence Precipitation in the Western approximately 1 month, and 38 cultured bacterial isolates U.S. Science 339: 1572–1578. were studied through 16S rRNA gene analysis. The results Demidova, T.N. and Hamblin, M.R. (2004). Photodynamic show that culturable bacterial strains isolated from Asian Therapy Targeted to Pathogens. Int. J. Immunopathol. dust were different than strains isolated from air inside the Pharmacol. 17: 245–254. aircraft, and were dominated by highly tolerant endospore- Echigo, A., Hino, M., Fukushima, T., Mizuki, T., forming bacteria. Non-endospore-forming bacteria isolated Kamekura, M. and Usami, R. (2005). Endospores of from the dust were high G + C gram-positive bacteria, Halophilic Bacteria of the family Bacillaceae Isolated which have UV resistance. As a result, all cultured bacteria from Non-Saline Japanese Soil May be transported by were strains with substaintial tolerance for exposure to UV Kosa Event (Asian Dust Storm). Saline Systems 1: 8, radiation. Solar UV radiation likely affects the viability of doi: 10.1186/1746-1448-1-8. airborne bacteria, and bacterial UV-tolerance can be a key Fujiwara, H., Sadanaga, Y., Urata, J., Masui, Y., Bandow, factor for their survival in long-range atmospheric transport H., Ikeda, K., Hanaoka, S., Watanabe, I., Arakaki, T., with Asian dust. Kato, S., Kajii, Y., Zhang, D., Hara, K., Fujimoto, T., Seto, T., Okuyama, K., Ogi, T., Takami, A., Shimizu, A. ACKNOWLEDGMENTS and Hatakeyama, S. (2014). Aerial Observation of Nitorogen Compouds over the East China Sea in 2009 We thank participants (from Tokyo University of and 2010. Atmos. Environ. 97: 462–470. Agricultural and Technology, Kanazawa University, Griffin, D.W., Westphal, D.L. and Gray, M.A. (2006). Hiroshima University, Muroran Institute of Technology Airborne Microorganisms in the African Desert Dust and Osaka Prefecture University) and staff (Diamond Air Corridor over the Mid-Atlantic Ridge, Ocean Drilling Service Inc., Japan) involved in the airborne observations. Program, Leg 209. Aerobiologia 22: 211–226

598 Hara et al., Aerosol and Air Quality Research, 15: 591–599, 2015

Hara, K. and Zhang, D. (2012). Bacterial Abundance and Diffusion of Bioaerosols in a Kosa Source Region. Viability in Long-Range Transported Dust. Atmos. Environ. Earozoru Kenkyu (J. Aerosol Res. Jpn.) 22: 218–227. 47: 20–25. Kwon, H.J., Cho, S.H., Chun, Y., Lagarde, F. and Pershagen, Hatakeyama, S., Hanaoka, S., Ikeda, K., Watanabe, I., G. (2002). Effects of the Asian Dust Events on Daily Arakaki, T., Sadanaga, Y., Bandow, H., Kato, S., Kajii, Mortality in Seoul, Korea. Environ. Res. 90: 1–5. Y., Sato, K., Shimizu, A. and Takami, A. (2011). Aerial Lighthart, B. (1997). The Ecology of Bacteria in the Alfresco Observation of Aerosols Transported from East Asia- Atmosphere. FEMS Microbiol. Ecol. 23: 263–274. Chemical Composition of Aerosols and Layered Structure Lim, N., Munday, C.I., Allison, G.E., O’Loingsigh, T., of an Air Mass over the East China Sea. Aerosol Air Deckker, P.D. and Tapper, N.J. (2011). Microbiological Qual. Res. 11: 497–507. and Meteorological Analysis of Two Australian Dust Hatakeyama, S., Ikeda, K., Hanaoka, S., Watanabe, I., Storms in April 2009. Sci. Total Environ. 412–413: 223– Arakaki, T., Bandow, H., Sadanaga, Y., Kato, S., Kajii, 231. Y., Zhang, D., Okuyama, K., Ogi, T., Fujimoto, T., Seto, Liu, B., Ichinose, T., He, M., Kobayashi, F., Maki, T., T., Shimizu, A., Sugimoto, N. and Takami, A. (2014). Yoshida, S., Yoshida, Y., Arashidani, K., Takano, H., Aerial Observations of Air Masses Transported from Nishikawa, M., Sun, G. and Shibamoto, T. (2014) Lung East Asia to the Western Pacific: Vertical Structure of Inflammation by Fungus, Bjerkandera abusta Isolated Polluted Air Masses. Atmos. Environ. 97: 456–461. from Asian Sand Dust (ASD) Aerosol and Enhancement Hua, N.P., Kobayashi, F., Iwasaka, Y., Shi, G.Y. and of Ovalbumin-Induced Lung Eosinophila by ASD and the Naganuma, T. (2007). Detailed Identification of Desert- Fungus in Mice. Allergy Asthma Clin. Immunol. 10: 10. Originated Bacteria Carried by Asian Dust Storms to Llabrés, M. and Agusti, S. (2006). Picophytoplankton Cell Japan. Aerobiologia 23: 291–298. Death induced by UV Radiation: Evidence for Oceanic Ichinose, T., Nishikawa, M., Takano, H., Sera, N., Sadakane, Atlantic Communities. Limnol. Oceanogr. 51: 21–29. K., Mori, I., Yanagisawa, R., Oda, T., Tamura, H., Lukšienė, Ž. (2005). New Approach to Inactivation of Hiyoshi, K., Quan, H., Tomura, S. and Shibamoto, T. Harmful and Pathogenic Microorganisms by (2005). Pulmonary Toxicity induced by Intratracheal Photosensitization. Food Technol. Biotechnol. 43: 411– Instillation of Asian Yellow Dust (Kosa) in Mice. Environ. 418. Toxicol. Pharmacol. 20: 48–56. Maki, T., Susuki, S., Kobayashi, F., Kakikawa, M., Ichinose, T., Yoshida, S., Hiyoshi, K., Sadakane, K., Yamada, M., Higashi, T., Chen, B., Shi, G.Y., Hong, C., Takano, H., Nishikawa, M., Mori, I., Yanagisawa, R., Tobo, Y., Hasegawa, H., Ueda, K. and Iwasaka, Y. Kawazato, H., Yasuda, A. and Shibamoto, T. (2008). The (2008). Phylogenetic Diversity and Vertical Distribution Effects of Microbial Materials Adhered to Asian Sand of a Halobacterial Community in the Atmosphere of an Dust on Allergic Lung Inflammation. Arch. Environ. Asian Dust (Kosa) Source Region, Dunhuang City. Air Contam. Toxicol. 55: 348–357. Qual. Atmos. Health 1: 81–89. Iwasaka, Y., Kobayashi, F. and Minami, Y. (2010). Studies Maki, T., Susuki, S, Kobayashi, F., Kakikawa, M., Tobo, of KOSA-Bioaerosol: Micro-Biota Floating in the Y., Yamada, M., Higashi, T., Matsuki, A., Hong, C., Atmosphere. Earozoru Kenkyu (J. Aerosol Res. Jpn.) 25: Hasegawa, H. and Iwasaka, Y. (2010). Phylogenetic 4–21. Analysis of Atmospheric Halotolerant Bacterial Jeon, E.M., Kim, H.J., Jung, K., Kim, J.H., Kim, M.Y., Communities at High Altitude in an Asian Dust (Kosa) Kim, Y. P. and Ka, J.O. (2011). Impact of Asian Dust Arrival Region, Suzu City. Sci. Total Environ. 408: Events on Airborne Bacterial Community Assessed by 4556–4562. Molecular Analyses. Atmos. Environ. 45: 4313–4321. Maki, T., Aoki, K., Kobayashi, F., Kakikawa, M., Tobo, Y., Jeon, E.M., Kim, Y.P., Jeong, K., Kim, I.S., Eom, S.W., Matsuki, A., Hasegawa, H. and Iwasaka, Y. (2011). Choi, Y.Z. and Ka, J.O. (2013). Impacts of Asian Dust Characterization of Halotolerant and Oligotrophic Bacterial Events on Atmospheric Fungal Communities. Atmos. Communities in Asian Desert Dust (Kosa) Bioaerosol Environ. 81: 39–50. Accumulated in Layers of Snow on Mount Tateyama, Kakikawa, M., Kobayashi, F., Maki, T., Yamada, M., Central Japan. Aerobiologia 27: 277–290. Higashi, T., Chen, B., Shi, G.Y., Hong, C., Tobo, Y. and Maki, T., Puspitasari, F., Hara, K., Yamada, M., Kobayashi, Iwasaka, Y. (2008). Dustborne Microorganisms in the F., Hasegawa, H. and Iwasaka, Y. (2014). Variations in Atmosphere over an Asian Dust Source Region, Dunhuang. the Structure of Airborne Bacterial Communities in a Air Qual. Atmos. Health 1: 195–202. Downwind Area during Asian Dust (Kosa) Event. Sci. Kellogg, C.A. and Griffin D.W. (2006). Aerobiology and Total Environ. 488–489: 75–84. the Global Transport of Desert Dust. Trends Ecol. Evol. Mitchell, D.L. and Karentz, D. (1993) In Environmental 21: 638–644. UV Photobiology, Young, A.R., Bjorn, L.O., Moan, J. Kenzaka, T., Sueyoshi, A., Baba, T., Li, P., Tani, K., and Nultsch, W. (Eds.), Plenum Press, New York, p. Yamaguchi, N. and Nasu, M. (2010). Soil Microbial 345–377. Community Structure in an Asian Dust Source Region Nicholson, W.L., Munakata, N., Horneck, G., Melosh, H.J. (Loess Plateau). Microbes Environ. 25: 53–57. and Setlow, P. (2000). Resistence of Bacillus Endospores Kobayashi, F., Kakikawa, M., Yamada, M., Chen, B., Shi, to Extreme Terrestrial and Extraterrestrial Environments. G.Y. and Iwasaka, Y. (2007). Study on Atmospheric Microbiol. Mol. Biol. Rev. 64: 548–572.

Hara et al., Aerosol and Air Quality Research, 15: 591–599, 2015 599

Nishimura, Y., Kenzaka, T., Sueyoshi, A., Li, P., Fujimura, H., Layers on Mt. Tateyama, Japan. Bull. Glaciol. Res. 29: Baba, T., Yamaguchi, N. and Nasu, M. (2010). Similarity of 31–39. Bacterial Community Structure between Asian Dust and Its Tong, Y. and Lighthart, B. (1997). Solar Radiation has a Sources Determined by rRNA Gene-Targeted Approaches. Lethal Effect on Natural Populations of Culturable Outdoor Microbes Environ. 25: 22–27. Atmospheric Bacteria. Atmos. Environ. 31: 897–900. Pattison, D.I. and Davies, M.J. (2006). In Experientia Uno, I., Eguchi, K., Yumimoto, K., Takemura, T., Shimizu, Supplenmentum, Vol. 96, Leon, P.B. (Eds.), Birkhäuser A., Uematsu, M., Liu, Z., Wang, Z., Hara, Y. and Sugimoto, Verlag, Switzerland, p. 131–157. N. (2009). Asian Dust Transported One Full Circuit around Park, D.J., Dastager, S.G., Lee, J.C., Yeo, S.H., Yoon, J.H. the Globe. Nat. Geosci. 2: 557–560, doi: 10.1038/ngeo583. and Kim, C.J. (2007). Shimazuella Kribbensis gen. nov., Van der Linde, K., Lim, B.T., Rondeel, J.M.M., Antonissen, sp. nov., a Mesophilic Representative of the Family L.P.M.T. and de Jong, G.M.T. (1999). Improved Thermoactinomycetaceae. Int. J. Syst. Evol. Microbiol. Bacteriological Surveillance of Haemodialysis Fluids: a 57: 2660–2664. Comparison between Tryptic Soy Agar and Reasoner’s Parks, S.R., Bennett, A.M., Speight, S.E. and Benbough, 2A Media. Nephrol. Dial. Transplant. 14: 2433–2437. J.E. (1996). An Assessment of the Sartorius MD8 Ventura, M., Canchaya, C., Tauch, A., Chandra, G., Fitzgerald, Microbiological Air Sampler. J. Appl. Bacteriol. 80: 529– G.F., Chater, K.F. and van Sinderen, D. (2007). Genomics 534. of Actinobacteria: Tracing the Evolutionary History of an Perry, K.D., Cliff, S.S. and Jimenze-Cruz, M.P. (2004). Ancient Phylum. Microbiol. Mol. Biol. Rev. 71: 495–548. Evidence for Hygroscopic Mineral Dust Particles from the Watson, S.W., Novitsky, T.J., Quinby, H.L. and Valois, F.W. Intercontinental Transport and Chemical Transformation (1977). Determination of Bacterial Number and Biomass Experiment. J. Geophys. Res. 109, doi: 10.1029/2004JD in the Marine Environment. Appl. Environ. Microbiol. 004979. 33: 940–946. Pratt, K.A., DeMott, P.J., French, J.R., Wang, Z., Westphal, Weisburg, W.G., Barns, S.M., Pelletier, D.A. and Lane, D.J. D.L., Heymsfield, A.J., Twohy, C.H., Prenni, A.J. and (1991). 16S Ribosomal DNA Amplification for Prather, K.A. (2009). In situ Detection of Biological Phylogenetic Study. J. Bacteriol. 173: 697–703. Particles in Cloud Ice-Crystals. Nat. Geosci. 2: 398–401, Yamada, M., Iwasaka, Y., Kobayashi, F. and Zhang, D. doi: 10.1038/ngeo521. (2010). Challenge of Measuring Bioaerosols at KOSA Pui, D.Y.H., Romay-Novas, F., and Liu, B.Y.H. (1987). Source Areas: Tethered Balloon Observation Coupled Experimental Study of Particle Deposition in Bends of with Individual Particle Analysis. Earozoru Kenkyu (J. Circular cross Section. Aerosol Sci. Technol. 7: 301–315. Aerosol Res. Jpn.) 25: 13–22. Qin, S., Chen, H.H., Klenk, H.P., Kim, C.J., Xu, L.H. and Yamaguchi, N., Ichijo, T., Sakotani, A., Baba, T. and Nasu, Li, W.J. (2010). Saccharopolyspora Gloriosae sp. nov., M. (2012). Global Dispersion of Bacterial Cells on and Endophitic Actinomycete Isolated from the Stem of Asian Dust. Sci. Rep. 2: 525, doi: 10.1038/srep00525. Gloriosa superba L. Int. J. Syst. Evol. Microbiol. 60: Yukimura, K., Nakai, R., Kohshima, S., Uetake, J., Kanda, 1147–1151. H. and Naganuma, T. (2009). Spore-Forming Halophilic Satheesh, S.K. and Moorthy, K.K. (2005). Radiative Bacteria Isolated from Arctic Terrains: Implications for Effects of Natural Aerosols: A Review. Atmos. Environ. Long-Range Transportation of Microorganisms. Polar Sci. 39: 2089–2110. 3: 163–169. Singer, C.E. and Ames, B.N. (1970). Sunlight Ultraviolet Zhang, D., Iwasaka, Y., Shi, G.Y., Zang, J., Matsuki, A. and and Bacterial DNA Base Ratios. Science 170: 822–826. Trochkine, D. (2003). Mixture State and Size of Asian Smith, D.J., Griffin, D.W., McPeters, R.D., Ward, P.D. and Dust Particles Collected at Southwestern Japan in Spring Schuerger, A.C. (2011). Microbial Survival in the 2000. J. Geophys. Res. 108, doi: 10.1029/2003JD003869. Stratosphere and Implications for Global Dispersal. Aerobiologia 27: 319–332. Tanaka, D., Tokuyama, Y., Terada, Y., Kunimochi, K., Received for review, March 31, 2014 Mizumaki, C., Tamura, S., Wakabayashi, M., Aoki, K., Revised, July 27, 2014 Shimada, W., Tanaka, H. and Nakamura, S. (2011). Accepted, August 8, 2014 Bacterial Communities in Asian Dust-Containing Snow