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Using a Cessna 404 aircraft (Nakanihon Air Service Co., Ltd.), bioaerosol collection was carried out as shown in Fig. 1(a). There is a 20 mm diameter hole in the roof of the craft (Fig. 1(b)). Before sampling, the hole was sealed. Upon beginning the sampling, the hole was opened, and the sterilized inlet was inserted as shown in Fig. 1(b). The inlet connected to a sterilized tube. The inlet and tube used for sampling were a conductive polytetrafluoroethylene (PTFE) tube (ASONE Co.) and a conductive silicon tube (SIBATA Sciencific Technology Ltd.), respectively. Since both products are highly conductive and can be sterilized with steam, they are suitable for collecting a sampling of bioaerosols. In calculating streamlines of air near the aircraft body, it was found that there was little influence when the inlet length was less than 10 mm. The sampling examined using 15 mm length of the inlet for no influence of air near the aircraft body. The tube was connected to a bioaerosol sampler.

Fig. 1. 
Cessna 404 aircraft used for the direct sampling of atmospheric bioaerosol (a) and the outside of the inlet (b). The inlet was sterilized just before sampling.

This bioaerosol sampler was the same as that used on a tethered balloon in previous studies (Chen et al., 2010; Yamada et al., 2010; Iwasaka et al., 2010, 2009; Kakikawa et al., 2010, 2008; Maki et al., 2010, 2008; Kobayashi et al., 2010, 2007). Atmospheric bioaerosols were collected on a 0.45 μm pore-size membrane filter. The filter was set into a filter holder (In-Line Filter Holder, 47 mm; Millipore Co., Ltd.) in the sampler under sterile conditions after autoclaving. The sampling volume of the air was estimated at 1.0m³ over a 60-minute sampling and flow rate of 17.0 L/min by an air pump. To avoid contamination during nonsampling, the inlet and outlet of the filter holder were closed by shutter valves.

The sampling of atmospheric bioaerosols was carried out using an aircraft over Noto Peninsula. Though Noto Peninsula juts out into the center of the Sea of Japan (Fig. 2), the collection of atmospheric bioaerosol samples while crossing the peninsula is not affected by microorganisms originating in the soil of the Japanese island. The sampling was carried out in the afternoon (15:00-16:00) on November 21, 2009. The weather on the sampling date was cloudy.

Fig. 2. 
Flight route (a) and altitude chart (b) for the direct sampling of atmospheric bioaerosol via aircraft.

Fig. 2 shows the flight route (a) and altitude chart (b). The number in Fig. 2(a) and horizontal axis in Fig. 2(b) indicate landmarks during our flight. The landmarks indicate Global Positioning System (GPS) locations at intervals of 2 minutes. The triangles indicate the beginning and conclusion of the sampling. The aircraft took off from Komaki Airport, and sampling was carried out for 1 hour in a circular flight over Noto Peninsula. The altitude for sampling was approximately 3,500 m.

In order to examine pre-existing microorganism contamination on the surface of the aircraft body, bioaerosol sampling was carried out just before takeoff using the same method as atmospheric sampling. To address the issue of microorganisms attaching to the surface of the aircraft, the aircraft surface was disinfected with ethanol before and after sampling (76.9-81.4 vol%, Wako Pure Chemical Industries, Ltd.).

For the estimation of origin of atmospheric bioaerosols, backward trajectories of air masses were calculated using the U.S. National Oceanic and Atmospheric Administration (NOAA) Air Resources Laboratory Hybrid Single Particle Lagrangian Integrated Trajectory (HYSPLIT) model (http://ready.arl.noaa.gov/HYSPLIT.php, Draxler and Hess, 1998). In this study, the sampling was carried out by wide field circling of the aircraft over Noto Peninsula. Thus, the backward trajectories were calculated from the trajectory ensemble as a trend.

The atmospheric bioaerosols were cultivated immediately after collection in a clean booth. The filter sample was placed on a plate containing Nutrient Agar (Diffco BD Co., Ltd.) for general bacterium and Sabouraud dextrose agar (Wako Pure Chemical Industries, Ltd.) for the eumycetes, i.e., fungi. DNA was extracted from isolates on the plate using a cell-wall lytic enzyme, lysozyme, and proteinase K (Sigma-Aldrich). 16S rDNA for prokaryotes was amplified by polymerase chain reaction (PCR) as described by Weisburg et al. (1991). 18S rDNA for eukaryotes was amplified by PCR using primer F1 (5′-TGGTTGATCCTGCCAGAGG-3′) and R1 (5′-GGCTACCTTGTTACG ACTT-3′). The PCR reaction mixture (vol. 20 μL) included the following: 4 μL of 5×Buffer, 1.6 μL of 10×dNTP (2.5 mM each, dATP, dCTP, GTP, dTTP), 0.2 μL of each primer (20 mM), 12.8 μL of sterile deionized H₂O, 1 U of PrimeSTAR DNA polymerase (TAKARA BIO INC. Co., Ltd.), and 1 μL of DNA (~30 ng). A thermal cycler (Dice, TAKARA BIO INC. Co., Ltd.) was used under the following conditions for amplification: initial 2 min denaturation at 98°C; 35 cycle-10 s denaturation at 98°C; 10 s annealing at 54°C; 1.5 min extension at 72°C; and a final 3 min extension at 72°C.

The DNA sequencing of cloned rDNA was determined using a genetic analyzer (Applied Biosystems Co., Ltd.), and the related species of isolates were searched by Basic Local Alignment Search Tool (BLAST) analysis (http://www.ncbi.nlm.nih.gov/BLAST) against DNA databases (GenBank/EMBL/DDBJ).

In order to discuss the history of the sampled air mass, HYSPLIT backward-trajectories were tracked for a duration of 48 hours (Fig. 3). Since the sampling was conducted by circular flight, the center point of the flight was selected as the starting point of the trajectory analysis, and the trajectories were calculated with an ensemble option. This resulted in 27 members for all possible offsets in X, Y, and Z. As a trend, the trajectories suggested that the sampled air mass came to Noto Peninsula from Siberia through eastern Mongolia, northeastern China, the northern section of the Korean Peninsula, and the Sea of Japan. Altitudes of the trajectories decreased from about 5,000 and 7,000 m. Though KOSA seems to be transported through the Gobi desert region, as calculated by the trajectory analysis, the Japan Metrological Agency reported that the KOSA phenomenon was not present on the sampling date. Additionally, concentrated dust clouds were not observed from Total Ozone Mapping Spectrometer (TOMS, http://toms.gsfc.nasa.gov/index.html) data. Therefore, it was suggested that the sample in this study would not be related to KOSA bioaerosol.

Fig. 3. 
HYSPLIT back-trajectories of air masses arriving at the Noto Peninsula. Plots show 24-hour air mass back-trajectories on November 21, 2009.

In order to examine pre-existing microorganism contamination on the surface of the aircraft body, bioaerosol sampling was carried out just before takeoff using the same method as atmospheric sampling. First, the same sampling method as used in atmospheric conditions was carried out for 1 hour before takeoff. From the separated culture, two isolates grew from a Nutrient Agar medium and Sabouraud dextrose agar medium. They were named in order from A01 strain to A04 strain. Second, after the above sampling, the aircraft was cleaned and sterilized using an ethanol solution, particularly around the inlet. Using the same method, sampling was carried out just before takeoff. After separate culturing from a Nutrient Agar medium and Sabouraud dextrose agar medium, one and two isolates grew, respectively. They were named in order from B01 strain to B03 strain.

Fig. 4 shows a photograph of colonies of A01 strain (a) to A04 strain (d) before ethanol cleaning and B01 strain (e) to B03 strain (g) after ethanol cleaning. The A01 and B01 strain colonies, as shown in Fig. 4(a) and (e), were almost the same form and color. The colonies of these strains were pale yellow with a wrinkled surface. They seem to be a kind of actinomycetes. The colony of A02 strain, as shown in Fig. 4(b), was yellow, smooth, and convex with a regular edge. A03, A04, B02, and B03 strains, as shown in Fig. 4(c), (d), (f), and (g), respectively, were moderately expanding, velvety to powdery, and pale brown. They were observed to be of a kind of mold.

Fig. 4. 
Photographs of colonies of A01 (a), A02 (b), A03 (c), A04 (d), B01 (e), B02 (f), and B03 (g) strains on plates isolated from samplings just before takeoff. The A01, A02, A03, and A04 strains were isolated from sampling before ethanol cleaning. B01, B02, and B03 strains were isolated from sampling after ethanol cleaning.

DNA was extracted from the isolates; the 16S rDNA for prokaryotes and 18S rDNA for eukaryotes were amplified by PCR, sequenced, and analyzed for homologies using a BLAST program. Table 1 shows the homology analysis of isolates collected before takeoff. The partial DNA sequence data of the A01 (Accession no. AB603644) and B01 strains (Accession no. AB6 03648), 299 and 334 base pairs, were closely related to Streptomyces sp. SOK4/28-05 (Accession no. EU098 024, 100%). Observation of colony forms, as shown in Fig. 4(a) and (b), confirmed the homology analysis results (Tresner and Backus, 1963). The A01 and B01 strains appeared to be Streptomyces sp. The DNA of the A02 strain (Accession no. AB603645), 312 base pairs in length, was closely related to Micrococcus sp. PA-E028 (Accession no. FJ233852, 100%). The observation results of colony forms of the A02 strain identified it as Micrococcus sp. (Fig. 4(b), Baird-Parker, 1974). The DNA of A03 (Accession no. AB603646), A04 (Accession no. AB603647), and B02 strains (Accession no. AB603649), 391, 405, and 391 base pairs in length, respectively, were related to Cladosporium sp. 6027 (Accession no. FJ235525, 98, 99, and 99%, respectively). The B03 strain (Accession no. AB603650), 369 base pairs in length, was closely related to Cladosprorium cladosporioides (Accession no. EU723484, 100%). From the observation of colonies of the B03 strain, as shown in Fig. 4(g), it was not clear that the B03 strain could be identified as Cladosporium “cladosporioides”. Thus, A03, A04, B02, and B03 strains suggested to be Cladosporium sp.

From the results of colony form observation and homology analysis in 16S or 18S rDNA before takeoff, Streptomyces sp., Micrococcus sp., and Cladosporium sp. were present before ethanol cleaning, and Streptomyces sp. and Cladosporium sp. were present after. It was found that Micrococcus sp., present on the body of the aircraft, was dead after the ethanol cleaning.

Direct sampling using an aircraft was carried out at an altitude of 3,500 m over Noto Peninsula (Fig. 2). After separate culturing overnight, one strain grew from the Nutrient Agar medium. Fig. 5 shows the colony of isolated atmospheric bioaerosol on a plate. This isolate was named BASZHN 0901. The colony of this isolate was observed as thick, opaque, and creamcolored. Table 2 shows the results of the homology analysis of the 16S rDNA sequence of the BASZHN 0901 strain. The partial DNA sequence data of the BASZHN 0901 strain (Accession no. AB603643), 372 base pairs in length, showed close relation to Bacillus subtilis (Accession no. FR729926, 99%). The DNA identity was a high value (99%), but it was difficult to be identified as Bacillus subtilis. The BASZHN 0901 strain was identified as Bacillus sp. The observation of a colony of BASZHN 0901 (Fig. 5) indicated this result (Gibson and Gordon, 1974). As there was no Bacillus sp. among the isolated microorganisms from just before takeoff samples, the contamination, i.e. in other altitude or attaching the aircraft body, was not occurred.

Fig. 5. 
Photograph of a colony of BASZHN 0901 strain as an atmospheric bioaerosol at an altitude of 3,500 m over Noto Peninsula.

Griffin (2004) reported that Bacillus is present at an altitude of 20,000 m in Earth’s atmosphere. He noted in the paper mentioned above that Bacillus is a sporeformer and that its spores are egg-like vesicles that can protect microorganisms from physical stress such as ultraviolet light-induced DNA damage, desiccation, and extreme temperatures (Saffary et al., 2002; Setlow, 2001). In East Asia, Bacillus sp. was found to be a spore-former. The possible origin of Bacillus sp. BASZHN 0901 at altitude of 3,500 m over Noto Peninsula is very interesting. From the trajectory analysis (Fig. 3), the air containing Bacillus sp. BASZHN 0901 came from Siberia, eastern Mongolia, northeast China, the northern section of the Korean peninsula, or the Sea of Japan. Analyses of soil and/or seawater have shown that Bacillus exists in Siberia (Rikhvanov et al., 1999), northeast China (Wang et al., 2010; Carrasco et al., 2007), and the Sea of Japan (Yoon et al., 2004). In order to clarify the origin of high-altitude atmospheric bioaerosols such as Bacillus sp. BASZHN 0901, it is necessary to obtain more detailed data on the DNA of various strains of Bacillus in East Asia. We hope that future study will focus on this effort.