Atmospheric Bioaerosol , Bacillus sp . , at an Altitude of 3 , 500 m over the Noto Peninsula : Direct Sampling via Aircraft

This work focuses on the analysis of bioaerosols in the atmosphere at higher altitudes over Noto Peninsula, Japan. We carried out direct sampling via aircraft, separated cultures, and identified present isolates. Atmospheric bioaerosols at higher altitudes were collected using a Cessna 404 aircraft for an hour at an altitude of 3,500 m over the Noto Peninsula. The aircraft-based direct sampling system was devised to improve upon the system of balloon-based sampling. 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. Identification was carried out by a homology search for 16S or 18S rDNA isolate sequences in DNA databases (GenBank). Isolate sampling just before takeoff revealed Stretpomyces sp., Micrococcus sp., and Cladosporium sp. One additional strain, Bacillus sp., was isolated from the sample after bioaerosol collection at high altitude. As the microorganism contamination on the aircraft body before takeoff differed from that while in the air, the presence of additional, higher atmosphere-based microorganisms was confirmed. It was found that Bacillus sp. was floating at an altitude of 3,500 m over Noto Peninsula.


INTRODUCTION
Bioaerosols, a group of organic aerosols ranging from 10 nm to 100 μm, are airborne particles or large molecules that carry living organisms or are released from living organisms (e.g., bacteria and fungi) (Aria and Amyot, 2004).Many fields of study have focused on atmospheric bioaerosols.In geophysics and meteorology, there is a substantial body of work in progress on the importance of atmospheric bioaerosols as ice nuclei and cloud condensation nuclei (Bowers et al., 2009;Aria and Amyot, 2004).In medicine, Ichinose et al. (2008) reported that Asian sand dust containing microbiological materials causes adverse respiratory health effects (Ichinose et al., 2005).In biology, microorganisms (Atmosphere bioaerosols) can be transported by KOSA (yellow dust events) not only in North East Asia but also in Arctic areas (Yukimura et al., 2009;Hua et al., 2007).Based on this evidence, the longrange transportation of atmospheric bioaerosols seems to have a great influence on ecosystem of source and deposit regions.
In clarifying the behavior of atmospheric bioaerosols, especially their long-range transport, it is necessary to directly collect them at very high altitude.Griffin (2008Griffin ( , 2004) ) collected atmospheric bioaerosols at an altitude of 20,000 m using an aircraft flying through African dust.In East Asia, we collected atmospheric bioaerosols using a tethered balloon below an altitude of 1,000 m over Noto Peninsula, Japan, and the east side of the Tarim Basin (Taklamakan desert), China, and discovered many kinds of atmospheric bioaerosols (Chen et al., 2010;Yamada et al., 2010;Iwasaka et al., 2010Iwasaka et al., , 2009;;Kakikawa et al., 2010Kakikawa et al., , 2008;;Maki et al., 2010Maki et al., , 2008;;Kobayashi et al., 2010Kobayashi et al., , 2007)).However, in East Asia, very few papers have reported on the collection and analysis of atmospheric bioaerosols at high altitudes.
Using a Cessna 404 aircraft, we carried out the direct sampling of atmospheric bioaerosols at an altitude of 3,500 m.In order to examine microorganism contamination at other altitudes, bioaerosols were collected just before takeoff.The identification of viable bacteria and/or fungi in the form of bioaerosols in these samples was reported.

1 Use of an Aircraft-Based Sampling
Device 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.
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., 2010Iwasaka et al., , 2009;;Kakikawa et al., 2010Kakikawa et al., , 2008;;Maki et al., 2010Maki et al., , 2008;;Kobayashi et al., 2010Kobayashi et al., , 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 1.0 m 3 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.

2 Sampling
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 Atmospheric Bioaerosol, Bacillus sp., at an Altitude of 3,500 m 165 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 (76.9-81.4vol%, Wako Pure Chemical Industries, Ltd.).

3 Trajectory Analysis
For the estimation of origin of atmospheric bioaero-sols, 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 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).

1 Trajectory Analysis
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 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.

2 Bioaerosols Just Before Takeoff
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.
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.

3 Direct Sampling of Atmospheric
Bioaerosol via Aircraft 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.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.

CONCLUSIONS
A system of direct bioaerosol sampling via aircraft was developed and carried out at an altitude of 3,500 m over Noto Peninsula on November 21, 2009.From the trajectory analysis, the sampled air mass appeared to come from the Siberia through the Sea of Japan to Noto Peninsula.Just before takeoff, Streptomyces sp., Micrococcus sp., and Cladosporium sp. were isolated through sampling.Micrococcus sp.appeared deceased from ethanol cleaning.Bio-analyzing high-altitude samples for cultivable microorganisms revealed the formation of a colony identified as Bacillus sp.In this aircraft-based sampling system, there was no contamination by other microorganisms because high-altitude atmospheric bioaerosol sampling results did not agree with those just before takeoff.It was found that there was a living bacterium, Bacillus sp., present as an atmospheric bioaerosol at an altitude of 3,500 m over Noto Peninsula.

Fig. 1 .
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.

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

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

Table 2 .
Atmospheric Bioaerosol, Bacillus sp., at an Altitude of 3,500 m 169 Isolates of atomospheric bioaerosol at an altitude of 3,500 m over Noto peninsula.