Financial support for this study came from the National Natural Science Foundation of China (No. 41775148). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

1. PM Number Monitoring
<ol>
	<li>Use an airborne laser particle counter (see Table of Materials) to determine the total PM number. Collect the PM by the air sampling port on the top of the airborne laser particle counter. 
	NOTE: This particle counter is an automation equipment and it can work independently when the program including sampling time, interval, collection times, etc. has been set on its touch screen.
	<ol>
		<li>Equip the instrument with a sensor to monitor temperature and relative humidity. Measure and record temperature and relative humidity data simultaneously every 5 min.</li>
		<li>In total, measure and record 6 different particle size classes (0.3-0.5 &#956;m, 0.5-0.7 &#956;m, 0.7-2.5 &#956;m, 2.5-5 &#956;m, 5-10 &#956;m and &#62; 10 &#956;m) simultaneously every 5 min. Measure 4 other particle size classes (0.3-0.5 &#956;m, 0.5-1 &#956;m, 1-3 &#956;m and &#8805; 3 &#956;m).</li>
		<li>Collect the air samples though the sampling hole on the top of the sampler and use the test modules inside the sampler to measure the particle size of each PM. Then the data is stored automatically. All the processes above can be performed automatically after the relative parameters, including sampling time and counting range, are set though the mini touch displayer of the airborne laser particle counter. 
		NOTE: This particle counter is an automation equipment and it has test modules that count the number of PM of different particle size classes.</li>
	</ol>
	</li>
	<li>To assess experimental standard error, ensure that different particle size classes are counted with at least 15 replications. Ensure that readings are stored in the internal memory of the instrument and subsequently analyzed.
	<ol>
		<li>Ensure that analysis of the primary data includes PM number concentration, the ANOVA test and the percentage of PM in each size class. For example, as shown in Figure 1A, the PM number concentrations in December (237.6 particles/cm3) were significantly higher than those in October (average: 110.2 particles/cm3) (ANOVA; P-value &#60; 0.05). Figure 1B shows that the number of particles smaller than 3 &#956;m accounted for more than 99% of the total number.</li>
		<li>Use the laser particle counter to monitor the PM number concentrations and store the data automatically. Use a USB flash disk to export data in the computer and perform the ANOVA test.</li>
	</ol>
	</li>
</ol>
2. PM Collection by Cyclonic Aerosol Samplers
<ol>
	<li>Perform the PM sampling in an open area without any nearby major pollution sources at &#8764;2 m above the ground.
	<ol>
		<li>Record the position of the sampling site. For example, the campus of Beijing Institute of Technology is the following: 39&#176;57&#8217;51.0&#8217;&#8217; N; 116&#176;19&#8217;38.5&#8217;&#8217; E.</li>
	</ol>
	</li>
	<li>Use a cyclonic aerosol sampler to collect air samples. Use a sampler flow of 323 L/min, and a collection time of 6 h. 
	NOTE: The sampler flow rate is fixed and cannot be changed. The collection time is controlled by manual time-keeping as there is only a Start and Stop button on this sampler.
	<ol>
		<li>Use sterile water to wash the inside of the cyclonic aerosol sampler 3 times before collection, and use the automatic cleaning function 3 times by continuously pressing the collect button 3 times.</li>
		<li>Press the collect button to start sampling and press the pump button to stop sampling. Put the sampler on a shelf or floor with no one holding it in place.</li>
		<li>Preserve all samples in the dark at -20 &#176;C until the subsequent analyses.</li>
	</ol>
	</li>
</ol>
3. PM Collection by Filters
<ol>
	<li>Perform the PM sampling in an open area without any nearby major pollution sources at &#8764;2 m above the ground.
	<ol>
		<li>Record the position of the sampling site. For example, the campus of Beijing Institute of Technology is as follows: 39&#176;57&#8217;51.0&#8217;&#8217; N; 116&#176;19&#8217;38.5&#8217;&#8217; E.</li>
	</ol>
	</li>
	<li>Collect the PM samples on 20.32 &#215; 25.4 cm2 filters (see Table of Materials) by using a high-volume air sampler (see Table of Materials) at a flow rate of 1,000 L/min. Set the flow rate and collection time by relative auto-programming.
	<ol>
		<li>Preserve all sampled filters in the dark at -20 &#176;C until the subsequent analyses.</li>
	</ol>
	</li>
</ol>
4. Biological Composition Analysis
<ol>
	<li>For biological composition analysis, use each liquid sample from the cyclonic aerosol sampler or filter sample for DNA extraction by a multisource DNA extraction Kit (see Table of Materials) according to the manufacturer&#39;s protocols.
	<ol>
		<li>For samples from the high-volume air sampler with filters, use 1/8 of each filter sample for DNA extraction. Put the filter into a 50 mL tube with the sample facing inward and the back facing towards the tube wall. Add 10 beads in the central sites towards the side of the filter containing the sample.</li>
		<li>Vortex the tube for 15 min at room temperature. Pipette the liquid in the tube into a clean 50 mL centrifuge tube to continue the DNA extraction process.</li>
	</ol>
	</li>
	<li>Extract the DNA from the sample by the processes as follows.
	<ol>
		<li>Centrifuge the bacteria sample at 2,000 x g for 5 min, remove the supernatant, and suspend bacteria in 2 mL of sterile PBS buffer.</li>
		<li>Collect the suspension of bacteria in a 2 mL centrifuge tube and then centrifuge at 2,000 x g for 5 min to discard the supernatant solution.</li>
		<li>Add 350 &#956;L of sterile PBS buffer containing the suspended bacteria to the solution.</li>
		<li>Add 0.8 mL of RNase A.</li>
		<li>Add 150 &#956;L of Buffer CL and 8 &#956;L of proteinase K, and mix immediately by vortex shaking for 1 min. After brief centrifugation at 1,000 x g for 30 s (no residues on the wall), put the centrifugal tube in 56 &#176;C water for 10 min.</li>
		<li>Add 350 &#956;L of Buffer PD, and mix for 30 s and then centrifuge for 10 min at 12,000 x g.</li>
		<li>Place the DNA preparation tube in a 2 mL centrifuge tube, and transfer the mixture made in step 4.2.6 to the preparation tube. Then centrifuge for 1 min at 12,000 x g.</li>
		<li>Discard the filtrate, put back the contents of the preparation tube into the original 2 mL centrifuge tube, and add 50 &#956;L of Buffer W1. Centrifuge the mixture for 1 min at 12,000 x g.</li>
		<li>Discard the filtrate, put back the contents of the preparation tube into the original 2 mL centrifuge tube, and add 700 &#956;L of Buffer W2. Centrifuge the mixture for 1 min at 12,000 x g. Repeat this step to wash again with 700 &#956;L of Buffer W2.</li>
		<li>Discard the waste liquid, and put back the contents of the preparation tube into the original 2 mL centrifuge tube. Centrifuge the mixture for 1 min at 12,000 x g.</li>
		<li>Put the contents of the DNA preparation tube into another clean 1.5 mL centrifuge tube, and add 100 &#956;L of eluent or deionized water to the middle of the membrane in the preparation tube (deionized water or eluent was heated to 65 &#176;C). Allow the mixture to stand at room temperature for 1 min, and Centrifuge the mixture for 1 min at 12,000 x g.</li>
	</ol>
	</li>
	<li>Perform quantitative real-time polymerase chain reaction (Q-RT-PCR) to quantify the relative abundances of bacteria and fungi on the sampling filters.
	<ol>
		<li>Use primers as follows: for bacterial 16S rDNA, 515F (5&#8217;- GTG CCA GCM GCC GCG GTA A-3&#8217;) and 806R (5&#8217;- GGA CTA CHV GGG TWT CTA AT-3&#8217;); and for the fungal ITS, ITS1 (5&#8217;-TCC GTA GGT GAA CCT GCG G-3&#8217;) and ITS1 (5&#8217;-GCT GCG TTC TTC ATC GAT GC-3&#8217;).</li>
		<li>Run the Q-RT-PCR assays on a Real-Time PCR System (see Table of Materials). RT-PCR condition: predegeneration, 95 &#176;C, 10 min; degeneration, 95 &#176;C, 15 s; anneal and extension, 60 &#176;C, 1 min; 40 cycles from degeneration to anneal and extension.</li>
	</ol>
	</li>
	<li>For bacterial and fungal community structure analysis, amplify the V1&#8211;V3 region of the bacterial 16S rDNA and the ITS region of the fungal rRNA operon by PCR.
	<ol>
		<li>Use the primers as follows: for bacteria, V1-9F (5&#8242;-CCT ATC CCC TGT GTG CCT TGG CAG TCT CAG ACG AGT TTG ATC MTG GCT CAG-3&#8242;) and V3-541R (5&#8242;-CCA TCT CAT CCC TGC GTG TCT CCG ACT CAG-barcode-ACW TTA CCG CGG CTG CTG G-3&#8242;); and for fungi, ITS-3F (5&#8217;-CCT ATC CCC TGT GTG CCT TGG CAG TCT CAG CAC ATC GAT GAA GAA CGC AGC-3&#8217;) and ITS-4R (5&#8217;-CCA TCT CAT CCC TGC GTG TCT CCG ACT CAG-barcode-GCT CCT CCG CTT ATT GAT ATG C-3&#8217;).</li>
		<li>Ensure that the 20 &#956;L mixture of PCRs included 2 &#956;L of 2.5 mM dNTPs, 4 &#956;L of 5&#215; FastPfu Buffer, 0.8 &#956;L of each primer (5 &#956;M), 0.4 &#956;L of FastPfu Polymerase, and 10 ng of template DNA (see Table of Materials).</li>
		<li>Use the PCR program as follows: 94 &#176;C for 5 min; followed by 10 cycles of 94 &#176;C for 30 s, 55-60 &#176;C for 45 s, and 72 &#176;C for 90 s; 20 cycles of 94 &#176;C for 30 s, 55 &#176;C for 45 s and 72 &#176;C for 90 s; and a final extension at 72 &#176;C for 5 min.</li>
	</ol>
	</li>
	<li>Perform DNA purification, DNA quantification and pyrosequencing as described in a previous study 21.</li>
</ol>
5. Bioaerosol Sampling and Cultivation
<ol>
	<li>Use an international standard Andersen six-stage sampler (see Table of Materials) to sample culturable airborne bacteria and fungi at a flow rate of 28.3 L/min. Use a sampling time of 35 min. Put the culture plate in each stage of the sampler. Sufficiently disinfect the sampler with 75% ethyl alcohol after each sampling. 
	NOTE: The sampler flow rate is fixed and the collection time is controlled by manual time-keeping.
	<ol>
		<li>Sample the six stages with the Andersen six-stage sampler, which is defined by the aerodynamic diameters of the airborne particles, including stage VI (0.65&#8211;1.1 &#956;m), stage V (1.1&#8211;2.1 &#956;m), stage IV (2.1&#8211;3.3 &#956;m), stage III (3.3&#8211;4.7 &#956;m), stage II (4.7&#8211;7.0 &#956;m) and stage I (&#8805;7.0 &#956;m).</li>
		<li>Collect the bacterial particles and deposit onto the culture plate in each stage containing Soybean-Casein Digest Agar (see Table of Materials). There is a container with many air intakes on the top in each stage of the sampler.</li>
		<li>Culture the airborne bacteria collection plates at 37 &#176;C for 24-48 h; then, count the colony numbers of bacteria on the sample dish for each stage.</li>
	</ol>
	</li>
	<li>To prevent the particles collected by the Andersen six-stage sampler from overlapping, correct the number of colonies at each sampler level by the following formula: 
	<img alt="Equation" src="/files/ftp_upload/58795/58795eq1.jpg" /> 
	where Pr: corrected number of colonies at each level, 
	N: number of sampling holes of the sampler at all levels (400), and 
	r: the actual count of colonies.</li>
	<li>Calculate the unit of colonies per m3 of air as follows: 
	<img alt="Equation" src="/files/ftp_upload/58795/58795eq2.jpg" /> 
	where C: concentration (CFU/m3), 
	N1-N6: the corrected number of colonies at each level, 
	T: sampling time (min), and 
	F: sampling flow rate (28.3 L/min).</li>
	<li>Use methods from Steps 5.1 - 5.3 to study the bioaerosol in livestock farm, including four types of piggeries. 
	NOTE: The livestock farm is located in Changchun, China. The central position 2 m above the ground in each piggery was selected as sampling location.) After culture of bacteria, treat all colonies in the plates as described in Steps 6.1 - 6.2.</li>
</ol>
6. Identification of Culturable Bacteria
<ol>
	<li>After 48 h or 72 h of culturing, place the bacteria into a 2 mL centrifuge tube. Extract the DNA of these bacteria or fungi by using a multisource DNA extraction Kit (see Table of Materials). The DNA extraction process is described in section 4.2.</li>
	<li>Use 200 &#956;L DNA extraction sample for 16S rDNA sequencing. Use the same processes as described in Steps 4.2, 4.3 and 4.4 on biological composition analysis.</li>
</ol>

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The authors have nothing to disclose.

In this study, we provided some representative results that were obtained during hazy days and in livestock farms. The results from bioaerosol samples taken during Beijing hazy days facilitated a better understanding of the biological compositions of the biological composition of PM without PM present during Beijing hazy days. The results from samples taken from livestock farms will also provide basic data for environmental air quality control in piggeries and a theoretical foundation and technical support for healthy breeding and safe production in livestock farms. Many environmental factors, such as temperature, humidity and wind speed, might contribute to the distribution of aerosol particles in the cow house (Figure 1). Previous studies have revealed that the PM in livestock farms mainly comes from the fodder, feces, fur and feather, which may be related to the animal activities. The environmental factors can affect not only animal activities, but also the aggregation and diffusion of PM in a relatively closed cow house. Therefore, we detect different concentrations and size distributions of PM in four different months. Besides, the sanitary condition, feeding method and animal activity were different between the four types of piggeries, which might also affect their community structure of culturable bacteria in the air (Figure 4).
However, in this study, we mainly focused on the available methods that we could use to study the composition and distribution of bioaerosols under different environmental conditions. Compared with other microbial samples, those of airborne microorganisms have very low concentrations and are mixed with a large number of impurities, such as inorganic dust particles, which introduce certain difficulties during the collection and detection of such microorganisms21. Therefore, appropriate methods of collection and detection should be selected for microbial aerosols. The collection of microbial aerosol samples is generally performed by using the precipitation method or specialized equipment to collect the microorganisms in liquid, semi-solid or solid sampling medium22,23,24. Then, some corresponding technical treatment and specific testing and analysis are subsequently carried out25. The sampling medium should keep microorganisms intact to reduce the error associated with detection and analysis26. However, different aerosol microorganism samplers have different effects on the integrity of samples due to their different sampling principles and media. People have designed many kinds of bioaerosol samplers that use different sampling principles, such as inertial impaction, filtration resistance, and electrostatic precipitation27.
Impacting samplers can push airborne PM into the sampling medium at high speed by using extraction equipment. There are two types of impacting samplers: solid and liquid.Solid impacting samplers can be used to sample bioaerosols at a low concentration and can be almost impervious to air flow28. Aerosol particles of different sizes can be preliminarily screened, and microbes can be sampled directly into the culture medium, which increases the survival rate of culturable microorganisms10. Due to inertial impact, microbial aerosol particles easily collide at the same site, and colonies may overlap easily after culture. At present, the most common solid impacting sampler is the Andersen-6 microbial aerosol sampler. In this study, we used an Andersen six-stage sampler to study the culturable bacteria distributed in airborne PM of different sizes.
Cyclonic aerosol samplers use cyclones to spiral air at high speed into a cylinder or cone. Bioaerosol particles can be separated from the airflow by centrifugal force, which means that microbes can bump into the inner wall of the sampler and then be collected by the sampling buffer. This method is convenient and can be used for long sampling times in large flow and sampling operations. However, this method cannot be implemented at low temperatures because the operation is dependent on liquid. The cyclonic aerosol sampler used in this study is a cyclonic wetted-wall aerosol sampler that can extract and transfer airborne pathogens and particles from sampled air into a small volume of water for analysis29.
Filter samplers can work under low temperature conditions and can sample particles above a certain size. However, they have a great impact on microbial activity and are prone to damage, which will greatly influence subsequent studies on sampling activity. In this study, bioaerosol samples from Beijing hazy days were collected by using both a high-volume air sampler with filters and a cyclonic aerosol sampler. The aim of this experiment was to analyses the biological composition of PM without the separation and culturing of airborne microbes. Therefore, these two sampling methods were suitable for this study. For the cyclonic aerosol sampler method, airborne microbes at a low concentration can be easily extracted into the running buffer and then can be analyzed conveniently without the additional treatment that is commonly used for filter samples. In this study, these two sampling methods, cyclonic aerosol sampler and filter sampler, showed different sampling efficiency. The additional treatment that is commonly used for filter samples, such as recovering the sample from the filter, is one of the main differences between these two methods. Besides, the air samples were collected directly into the running buffer by cyclonic aerosol sampler while the other method collected samples on filters. The characteristics of different type of sampling method might contribute to this different sampling efficiency. We can assume that the cyclonic aerosol sampler is the better choice for microorganism collection, and this assumption needs further confirmation.
In this study, bacterial 16S rDNA and fungal ITS region sequencing were used to perform the biological analysis of bioaerosols. 16SrDNA sequencing is the determination of 16S rDNA segments in the microbial genome30. 16S rDNA widely exists in prokaryotes with high conservation and specificity, which makes it useful for the identification of microbial species31. Whole-genome sequencing requires only the extraction of genomic DNA and subsequent sequencing. In addition to producing a large amount of data, this process also allows for a more comprehensive analysis of microbial community structure. Besides, metagenomics can also be used in this field of study by providing more information in the future. Handelsman et al. first proposed the concept of the metagenome in a 1998 paper on microbes in soil32. In subsequent studies, the concept of the metagenome was gradually accepted, and much research was carried out on the microbes included in the human gut, ocean and soil33,34,35. With the support of high-throughput sequencing technology, metagenomics has developed rapidly, and it plays an increasingly important role in the study of pathogen detection. Traditional microbial research methods mainly rely on culture for separation and purification. However, many studies cannot be carried out because more than 99% of microbes cannot be cultured. In contrast to traditional methods, metagenomics can take the genetic information of all the microbes in the environment as a whole without the need to separate individual organisms36. A comprehensive analysis of all the resulting microorganisms can be conducted directly.
In summary, this study showed several detection, sampling and analysis methods that could be used for studies on the biological composition of environmental PM, including PM monitoring; PM sampling by an Andersen six-stage sampler, a high-volume air sampler with filters or cyclonic aerosol sampler; and subsequent biological analysis based on DNA sequencing. In practice, these methods can be used under different environmental conditions, such as many types of livestock farms. Our protocols and results may help other researchers all over the world further explore the health impacts of fungal and bacterial bioaerosols in the environment.

In natural environments, various kinds of microorganisms exist in bioaerosols, including fungi, bacteria, viruses and other microorganisms1. Airborne microorganisms, which can be emitted from some human activities such as animal feeding operations in livestock farms, are the important contents of the atmospheric environment2. These microorganisms may not only play important roles in the atmospheric environment but also have significant impacts on human health and diseases spread.
As an important way of spreading diseases, microbial aerosols have attracted wide attention throughout the world. In recent studies, many human diseases were found to be associated with the complex composition of environmental particulate matter (PM) in different locations, such as chemical factories, livestock farms and smoggy cities3,4. The biological composition of PM may contribute to some respiratory and cardiovascular diseases in PM-exposed humans5. Different body regions, such as the mucosa, skin, digestive tract and respiratory tract, can be potential targets of microbes attached to PM6,7. An increased risk of lung cancer might be caused by prolonged exposure to PM2.58.
Airborne bacteria have been surveyed at various locations in several countries around the world, including in subway stations, veterinary hospitals, slaughterhouses, composting facilities, tanneries, milk-processing facilities, coal mines, dental clinics and indoor environments9,10,11,12,13,14,15,16,17, generating a large number of reports about biological aerosols. Crowded places associated with campuses, livestock farms and large cities during hazy days are three particularly important public conditions for which we need to explore the connections between human health and the potential effects of PM exposure. Furthermore, during winter days in the northern cities of China, high PM2.5 values may affect human health. Although PM2.5 can generate toxic effects by targeting respiratory surfaces and dissolving into blood18, it is still not clear whether and how the microbes attached to PM2.5 could potentially affect human health19,20. Livestock farms are one of the main sources of PM and microbial aerosols in the air. The large numbers of pathogens, such as influenza virus and Brucella melitensis, that are carried by aerosols in the fields around livestock farms are important factors causing respiratory diseases in both livestock and poultry workers.
In this study, we explored multiple types of analyses of bioaerosols, including PM number monitoring, bioaerosol collection and biological composition analysis. Air samples were collected by a cyclonic aerosol sampler, a high-volume air sampler with filters and an Andersen six-stage sampler. Then, the samples collected by these three samplers were analyzed by biological analysis including bacterial 16S rDNA and fungal ITS sequencing to determine their biological compositions. Herein, we show representative results from the bioaerosol samples collected during Beijing hazy days and from livestock farms indicating that bioaerosols might have great impacts on human and animal health. The comparison between liquid and filter sampling methods were also explored in this study mainly based on the data from 16S DNA and fungal ITS sequencing.

<table><tbody><tr><td>airborne laser particle counter</td><td>TSI Inc, MN, USA</td><td>model 9306</td><td></td></tr><tr><td>Andersen six-stage sampler</td><td>Tisch Inc, USA</td><td>TE-20-600</td><td></td></tr><tr><td>AxyPrep multisource DNA Miniprep Kit</td><td>Axygen, NY, USA</td><td>AP-MN-MIS-GDNA-50G</td><td></td></tr><tr><td>FastPfu Polymerase</td><td>TransGen Inc., Beijing, China</td><td>AP221-01</td><td></td></tr><tr><td>High-volume air sampler</td><td>Beijing HuaRui HeAn Technology Co., Ltd., China</td><td>HH02-LS120</td><td></td></tr><tr><td>Real-Time PCR System</td><td>Thermo Fisher Scientific, USA</td><td>Applied Biosystems&amp;reg; 7500</td><td></td></tr><tr><td>Soybean-Casein Digest Agar</td><td>Becton, Dickinson and company, MD, USA</td><td>211043</td><td></td></tr><tr><td>Tissuquartz filters</td><td>Pall, NY, USA</td><td>7204</td><td></td></tr><tr><td>Wetted-Wall Air Sampler</td><td>Research International, Inc. 17161 Beaton Road SE&lt;br/&gt; Monroe, Washington 98272-1034 USA</td><td>SASS 2300</td><td></td></tr></tbody></table>

composition and distribution analysis of bioaerosols under different environmental conditions

Variable microorganisms in particulate matter (PM) under different environmental conditions may have significant impacts on human health. In this study, we described a protocol for multiple analyses of the biological compositions in environmental PM. Five experiments are presented: (1) PM number monitoring by using a laser particle counter; (2) PM collection by using a cyclonic aerosol sampler; (3) PM collection by using a high-volume air sampler with filters; (4) culturable microbes collection by the Andersen six-stage sampler; and (5) detection of biological composition of environmental PM by bacterial 16SrDNA and fungal ITS region sequencing. We selected hazy days and a livestock farm as two typical examples of the application in this protocol. In this study, these two sampling methods, cyclonic aerosol sampler and filter sampler, showed different sampling efficiency. The cyclonic aerosol sampler performed much better in terms of collecting bacteria, while these two methods showed the same efficiency in collecting fungi. Filter samplers can work under low temperature conditions while cyclonic aerosol samplers have a sampling limitation for temperature. A solid impacting sampler, such as an Andersen six-stage sampler, can be used to sample bioaerosols directly into the culture medium, which increases the survival rate of culturable microorganisms. However, this method mainly relies on culture while more than 99% of microbes cannot be cultured. DNA extracted from the culturable bacteria collected by the Andersen six-stage sampler and samples collected by cyclonic aerosol sampler and filter sampler were detected by bacterial 16S rDNA and fungal ITS region sequencing.All the methods above may have wide application in many fields of study, such as environmental monitoring and airborne pathogen detection. From these results, we can conclude that these methods can be used under different conditions and may help other researchers further explore the health impacts of environmental bioaerosols.

In this study, we performed an assessment of the overall PM distribution and a comprehensive analysis of the bioaerosols in a dairy farm from September to December. Many environmental factors contribute to the distribution of aerosol particles. We studied the concentration and size distributions of PM in a cow house by using a TSI laser particle counter. As shown in Figure 1A, the concentration of aerosol particles was highest in December and lowest in October, which might be caused by changes in temperature and humidity (Table 1). The concentration of inhalable aerosol particles (0.3-3.0 &#181;m) accounted for more than 99% of the total particle concentration (Figure 1B), and the particles in this range could reach the deep respiratory tract, causing serious hazards for humans and animals.
Biological composition analysis of samples can be performed by DNA extraction, bacterial 16SrDNA and fungal ITS region sequencing instead of microorganism culture. From the biological analysis of bioaerosol samples collected by using a cyclonic aerosol sampler or a high-volume air sampler with filters, we can preliminarily compare the efficiencies of these two methods in collecting bacteria and fungi. Figure 2 showed the analysis results of the bioaerosol samples collected during Beijing hazy days in the campus of Beijing Institute of Technology in December 20, 2016. For bacteria collection, the results indicated that the cyclonic aerosol sampler collected many more genera than the high-volume air sampler with filters (Figure 2A). For fungi collection, these samplers showed equal collection efficiencies and almost the same genus abundances (Figure 2B). From the results presented in Figure 2, we were able to measure the different collection efficiencies of these two methods for bacteria and fungi. For bacteria collection, the cyclonic aerosol sampler performed much better than the high-volume air sampler with filters because the samples from the former showed a higher genus abundance (Figure 2A). However, the fungal sequencing analysis of the two samples from different sampling methods showed nearly identical community structures (Figure 2B).
We studied airborne culturable bacteria by using an Andersen six-stage sampler. As shown in Figure 3, the colony numbers of culturable bacteria for stage I-VI particles was reduced. Stage I particles (particle size &#62; 8.2 &#181;m) had the highest numbers of culturable bacteria colonies. The percentage of stage I colonies in four different types of piggeries including farrowing house, pregnant sow house, fattening house and weaning house was 33%, 30%, 26% and 34%, respectively. The percentage of stage II colonies in four different types of piggeries was 20%, 22%, 19% and 20% respectively. The percentage of stage III colonies in four different types of piggeries was 18%, 18%, 18% and 19% respectively. The percentage of stage IV colonies in four different types of piggeries was 17%, 16%, 16% and 16% respectively. The percentage of stage V colonies in four different types of piggeries was 10%, 10%, 14% and 6% respectively. Stage VI particles (particle size &#60; 1.0 &#181;m) had the lowest numbers of culturable bacteria colonies. The percentage of stage VI colonies in the four different types of piggeries was 3%, 5%, 6% and 5%, respectively.
The air samples were collected in four different types of piggeries by using an Andersen six-stage sampler and then cultured under suitable conditions. The whole-genome DNA of the culturable bacteria collected from each particle stage was extracted and detected by bacterial 16S rDNA and fungal ITS region sequencing. A total of 91 genera and 158 species of bacteria were identified in the culturable bacteria in piggeries. The culturable bacteria community structures in four different types of piggeries, including farrowing house, pregnant sow house, fattening house and weaning house are shown in Figure 4 with data from stage I to stage VI. The content of different predominant bacterial genera is not the same among different piggeries.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58795/58795fig1v4.jpg" /> 
Figure 1: The concentration and size distribution of PM in four different months. (A) Boxplot of PM number during the study period. Each boxplot includes the Maximum, minimum, median, two quartiles and abnormal value of the database. (B) Average size distribution maps of PM. There were eighty cows in the house from September to December.&#160;The percentage of PM (&#8805; 3 &#181;m) in four months (September, October, November and December) was 0.005, 0.005, 0.002 and 0.002 respectively. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/58795/58795fig1v4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/58795/58795fig2v4.jpg" /> 
Figure 2: Biological analysis of bioaerosol samples collected by two samplers. (A) and (B) show the abundances of bacterial or fungal genera in bioaerosol samples obtained with different collection methods. Wetted-Wall Air Sampler represents the cyclonic aerosol sampler. Quartz Filters represents the high-volume air sampler with filters. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/58795/58795fig2v4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/58795/58795fig3v4.jpg" /> 
Figure 3: Average hierarchical distribution maps of culturable bacteria in four types of piggeries. The four types of piggeries are&#160;farrowing house, pregnant sow house, fattening house and weaning house. S1 to S6 represent the six particle stages (I to VI) collected by the Andersen six-stage sampler. The stages were defined by the aerodynamic diameters of the airborne particles, including stage VI (0.65-1.1 &#181;m), stage V (1.1-2.1 &#181;m), stage IV (2.1-3.3 &#181;m), stage III (3.3-4.7 &#181;m), stage II (4.7-7.0 &#181;m) and stage I (7.0 &#181;m). <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/58795/58795fig3v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/58795/58795fig4v3.jpg" /> 
Figure 4: Bacterial community structures with different abundances in the air samples. The air samples were collected in four different types of piggeries by using an Andersen six-stage sampler and then cultured under suitable conditions. The whole-genome DNA of the culturable bacteria in each particle stage collected by the Andersen six-stage sampler was extracted and detected by bacterial 16S rDNA sequencing. The numbers 1 to 6 in each type of piggery represent particle stages I to VI measured by the Andersen six-stage sampler. The text on the right side includes the genus name for each bacterium. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/58795/58795fig4v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Composition and Distribution Analysis of Bioaerosols Under Different Environmental Conditions at JoVE.com

Composition and Distribution Analysis of Bioaerosols Under Different Environmental Conditions

Understanding the biological composition of environmental particulate matter is important for the study of its significant impacts on human health and disease spread. Here, we used three types of bioaerosol sampling methods and a biological analysis of airborne microbes to better explore airborne microbial communities under different environmental conditions.

Variable microorganisms in particulate matter (PM) under different environmental conditions may have significant impacts on human health. In this study, ...

composition-distribution-analysis-bioaerosols-under-different

Universidad San Sebastian

Research

JoVE Journal

Environment

10.5K Views.  Academy of Military Medical Sciences. Understanding the biological composition of environmental particulate matter is important for the study of its significant impacts on human health and disease spread. Here, we used three types of bioaerosol sampling methods and a biological analysis of airborne microbes to better explore airborne microbial communities under different environmental conditions.

Video: Composition and Distribution Analysis of Bioaerosols Under Different Environmental Conditions

Air-sampled Filter Analysis for Endotoxins and DNA Content

Outdoor aerosol research commonly uses particulate matter sampled on filters. This procedure enables various characterizations of the collected particles to be performed in parallel. The purpose of the method presented here is to obtain a highly accurate and reliable analysis of the endotoxin and DNA content of bio-aerosols extracted from filters. The extraction of high molecular weight organic molecules, such as lipopolysaccharides, from sampled filters involves shaking the sample in a pyrogen-free water-based medium. The subsequent analysis is based on an enzymatic reaction that can be detected using a turbidimetric measurement. As a result of the high organic content on the sampled filters, the extraction of DNA from the samples is performed using a commercial DNA extraction kit that was originally designed for soils and modified to improve the DNA yield. The detection and quantification of specific microbial species using quantitative polymerase chain reaction (q-PCR) analysis are described and compared with other available methods.

Two complementary analyses of atmospheric biological particles from air sampled filters are described herein: the extraction and detection of endotoxin, and of DNA.

Outdoor aerosol research commonly uses particulate matter sampled on filters. This procedure enables various characterizations of the collected particles ...

Production and Measurement of Organic Particulate Matter in a Flow Tube Reactor

Organic particulate matter (PM) is increasingly recognized as important to the Earth's climate system as well as public health in urban regions, and the production of synthetic PM for laboratory studies have become a widespread necessity. Herein, experimental protocols demonstrate approaches to produce aerosolized organic PM by &#945;-pinene ozonolysis in a flow tube reactor. Methods are described for measuring the size distributions and morphology of the aerosol particles. The video demonstrates basic operations of the flow tube reactor and related instrumentation. The first part of the video shows the procedure for preparing gas-phase reactants, ozonolysis, and production of organic PM. The second part of the video shows the procedures for determining the properties of the produced particle population. The particle number-diameter distributions show different stages of particle growth, namely condensation, coagulation, or a combination of both, depending on reaction conditions. The particle morphology is characterized by an aerosol particle mass analyzer (APM) and a scanning electron microscope (SEM). The results confirm the existence of non-spherical particles that have grown from coagulation for specific reaction conditions. The experimental results also indicate that the flow tube reactor can be used to study the physical and chemical properties of organic PM for relatively high concentrations and short time frames.

This paper describes the operation procedure for the flow tube reactor and related data collection. It shows the protocols for setting the experiments, recording data and generating the number-diameter distribution as well as the particle mass information, which gives useful information about chemical and physical properties of the organic aerosols.

Organic particulate matter (PM) is increasingly recognized as important to the Earth's climate system as well as public health in urban regions, and the ...

Production and Measurement of Organic Particulate Matter in the Harvard Environmental Chamber

The production and the evolution of atmospheric organic particulate matter (PM) are insufficiently understood for accurate simulations of atmospheric chemistry and climate. The complex production mechanisms and reaction pathways make this a challenging research topic. To address these issues, an environmental chamber, providing enough residence time and close-to-ambient concentrations of precursors for secondary organic materials, is needed. The Harvard Environmental Chamber (HEC) was built to serve this need, simulating the production of gas and particle phase species from volatile organic compounds (VOCs). The HEC has a volume of 4.7 m3 and a mean residence time of 3.4 h under typical operating conditions. It is operated as a completely mixed flow reactor (CMFR), providing the possibility of indefinite steady-state operation across days for sample collection and data analysis. The operation procedures are described in detail in this article. Several types of instrumentation are used to characterize the produced gas and particles. A High-Resolution Time-of-Fight Aerosol Mass Spectrometer (HR-ToF-AMS) is used to characterize particles. A Proton-Transfer-Reaction Mass Spectrometer (PTR-MS) is used for gaseous analysis. Example results are presented to show the use of the environmental chamber in a wide variety of applications related to the physicochemical properties and reaction mechanisms of organic atmospheric particulate matter.

This paper describes operation procedures for the Harvard Environmental Chamber (HEC) and related instrumentation for measuring gaseous and particle species. The environmental chamber is used to produce and study secondary organic species produced from the organic precursors, especially related to atmospheric organic particulate matter.

The production and the evolution of atmospheric organic particulate matter (PM) are insufficiently understood for accurate simulations of atmospheric ...

Experimental Protocol to Investigate Particle Aerosolization of a Product Under Abrasion and Under Environmental Weathering

The present article presents an experimental protocol to investigate particle aerosolization of a product under abrasion and under environmental weathering, which is a fundamental element to the approach of nanosafety-by-design of nanostructured products for their durable development. This approach is basically a preemptive one in which the focus is put on minimizing the emission of engineered nanomaterials' aerosols during the usage phase of the product's life cycle. This can be attained by altering its material properties during its design phase without compromising with any of its added benefits. In this article, an experimental protocol is presented to investigate the nanosafety-by-design of three commercial nanostructured products with respect to their mechanical solicitation and environmental weathering. The means chosen for applying the mechanical solicitation is an abrasion process and for the environmental weathering, it is an accelerated UV exposure in the presence of humidity and heat. The eventual emission of engineered nanomaterials is studied in terms of their number concentration, size distribution, morphology and chemical composition. The purpose of the protocol is to study the emission for test samples and experimental conditions which are corresponding to real life situations. It was found that the application of the mechanical stresses alone emits the engineered nanomaterials' aerosols in which the engineered nanomaterial is always embedded inside the product matrix, thus, a representative product element. In such a case, the emitted aerosols comprise of both nanoparticles as well as microparticles. But if the mechanical stresses are coupled with the environmental weathering, the experimental protocol reveals then the eventual deterioration of the product, after a certain weathering duration, may lead to the emission of the free engineered nanomaterial aerosols too.

In this article, an experimental protocol to investigate particle aerosolization of a product under abrasion and under environmental weathering is presented. Results on the emission of engineered nanomaterials, in the form of aerosols are presented. The specific experimental set-up is described in detail.

The present article presents an experimental protocol to investigate particle aerosolization of a product under abrasion and under environmental ...

Engineering

Collection and Extraction of Occupational Air Samples for Analysis of Fungal DNA

Traditional methods of identifying fungal exposures in occupational environments, such as culture and microscopy-based approaches, have several limitations that have resulted in the exclusion of many species. Advances in the field over the last two decades have led occupational health researchers to turn to molecular-based approaches for identifying fungal hazards. These methods have resulted in the detection of many species within indoor and occupational environments that have not been detected using traditional methods. This protocol details an approach for determining fungal diversity within air samples through genomic DNA extraction, amplification, sequencing, and taxonomic identification of fungal internal transcribed spacer (ITS) regions. ITS sequencing results in the detection of many fungal species that are either not detected or difficult to identify to species level using culture or microscopy. While these methods do not provide quantitative measures of fungal burden, they offer a new approach to hazard identification and can be used to determine overall species richness and diversity within an occupational environment.

Determining the fungal diversity within an environment is a method utilized in occupational health studies to identify health hazards. This protocol describes DNA extraction from occupational air samples for amplification and sequencing of fungal ITS regions. This approach detects many fungal species that can be overlooked by traditional assessment methods.

Traditional methods of identifying fungal exposures in occupational environments, such as culture and microscopy-based approaches, have several ...

A System to Create Stable Nanoparticle Aerosols from Nanopowders

Nanoparticle aerosols released from nanopowders in workplaces are associated with human exposure and health risks. We developed a novel system, requiring minimal amounts of test materials (min. 200 mg), for studying powder aerosolization behavior and aerosol properties. The aerosolization procedure follows the concept of the fluidized-bed process, but occurs in the modified volume of a V-shaped aerosol generator. The airborne particle number concentration is adjustable by controlling the air flow rate. The system supplied stable aerosol generation rates and particle size distributions over long periods (0.5-2 hr and possibly longer), which are important, for example, to study aerosol behavior, but also for toxicological studies. Strict adherence to the operating procedures during the aerosolization experiments ensures the generation of reproducible test results. The critical steps in the standard protocol are the preparation of the material and setup, and the aerosolization operations themselves. The system can be used for experiments requiring stable aerosol concentrations and may also be an alternative method for testing dustiness. The controlled aerosolization made possible with this setup occurs using energy inputs (may be characterized by aerosolization air velocity) that are within the ranges commonly found in occupational environments where nanomaterial powders are handled. This setup and its operating protocol are thus helpful for human exposure and risk assessment.

We designed and developed an effective nanopowder aerosolization setup and operating protocol. The system generated nanoparticle aerosols with stable number concentrations and size distributions for long durations, requiring only small quantities of test material (min. 200 mg).

Nanoparticle aerosols released from nanopowders in workplaces are associated with human exposure and health risks. We developed a novel system, requiring ...

Detection of Viruses from Bioaerosols Using Anion Exchange Resin

This protocol demonstrates a customized bioaerosol sampling method for viruses. In this system, anion exchange resin is coupled with liquid impingement-based air sampling devices for efficacious concentration of negatively-charged viruses from bioaerosols. Thus, the resin serves as an additional concentration step in the bioaerosol sampling workflow. Nucleic acid extraction of the viral particles is then performed directly from the anion exchange resin, with the resulting sample suitable for molecular analyses. Further, this protocol describes a custom-built bioaerosol chamber capable of generating virus-laden bioaerosols under a variety of environmental conditions and allowing for continuous monitoring of environmental variables such as temperature, humidity, wind speed, and aerosol mass concentration. The main advantage of using this protocol is increased sensitivity of viral detection, as assessed via direct comparison to an unmodified conventional liquid impinger. Other advantages include the potential to concentrate diverse negatively-charged viruses, the low cost of anion exchange resin (~$0.14 per sample), and ease of use. Disadvantages include the inability of this protocol to assess infectivity of resin-adsorbed viral particles, and potentially the need for the optimization of the liquid sampling buffer used within the impinger.

An anion exchange resin-based method, adapted to liquid impingement-based bioaerosol sampling of viruses is demonstrated. When coupled with downstream molecular detection, the method allows for facile and sensitive detection of viruses from bioaerosols.

This protocol demonstrates a customized bioaerosol sampling method for viruses. In this system, anion exchange resin is coupled with liquid ...

Methods for Characterizing the Co-development of Biofilm and Habitat Heterogeneity

Biofilms are surface-attached microbial communities that have complex structures and produce significant spatial heterogeneities. Biofilm development is strongly regulated by the surrounding flow and nutritional environment. Biofilm growth also increases the heterogeneity of the local microenvironment by generating complex flow fields and solute transport patterns. To investigate the development of heterogeneity in biofilms and interactions between biofilms and their local micro-habitat, we grew mono-species biofilms of Pseudomonas aeruginosa and dual-species biofilms of P. aeruginosa and Escherichia coli under nutritional gradients in a microfluidic flow cell. We provide detailed protocols for creating nutrient gradients within the flow cell and for growing and visualizing biofilm development under these conditions. We also present protocols for a series of optical methods to quantify spatial patterns in biofilm structure, flow distributions over biofilms, and mass transport around and within biofilm colonies. These methods support comprehensive investigations of the co-development of biofilm and habitat heterogeneity.

Biofilms have complex interactions with their surrounding environment. To comprehensively investigate biofilm-environment interactions, we present here a series of methods to create heterogeneous chemical environment for biofilm development, to quantify local flow velocity, and to analyze mass transport in and around biofilm colonies.

Biofilms are surface-attached microbial communities that have complex structures and produce significant spatial heterogeneities. Biofilm development is ...

Bioengineering

Breath Collection from Children for Disease Biomarker Discovery

Breath collection and analysis can be used to discover volatile biomarkers in a number of infectious and non-infectious diseases, such as malaria, tuberculosis, lung cancer, and liver disease. This protocol describes a reproducible method for sampling breath in children and then stabilizing breath samples for further analysis with gas chromatography-mass spectrometry (GC-MS). The goal of this method is to establish a standardized protocol for the acquisition of breath samples for further chemical analysis, from children aged 4-15 years. First, breath is sampled using a cardboard mouthpiece attached to a 2-way valve, which is connected to a 3 L bag. Breath analytes are then transferred to a thermal desorption tube and stored at 4-5 &#176;C until analysis. This technique has been previously used to capture breath of children with malaria for successful breath biomarker identification. Subsequently, we have successfully applied this technique to additional pediatric cohorts. The advantage of this method is that it requires minimal cooperation on part of the patient (of particular value in pediatric populations), has a short collection period, does not require trained staff, and can be performed with portable equipment in resource-limited field settings.

This protocol describes a simple method for the acquisition of breath samples from children. Briefly, samples of mixed air are pre-concentrated in sorbent tubes prior to gas chromatography-mass spectrometry analysis. Breath biomarkers of infectious and non-infectious diseases can be identified using this breath collection method.

Breath collection and analysis can be used to discover volatile biomarkers in a number of infectious and non-infectious diseases, such as malaria, ...

Chemistry

Use of Capillary Aerosol Generator in Continuous Production of Controlled Aerosol for Non-Clinical Studies

The capillary aerosol generator (CAG) is operated with the principal of thermal liquid evaporation through heating of e-liquid in the initial phase, followed by nucleation and condensation regulated through a mixture of airflow to generate aerosols, such as in an electronic cigarette (EC). The CAG is particularly useful in generating aerosols of large volumes in a continuous manner, for instances such as in vivo inhalation toxicology studies, where usage of ECs is not feasible. The thermal effects of generating aerosol from the CAG are similar in terms of temperature applied in an EC, thus allowing investigators to assess the vapors of e-liquids at scale and reproducibility. As the operation of the CAG allows users to control critical parameters such as the flow rate of e-liquid, heating temperatures and dilution air flows, it allows investigators to test various e-liquid formulations in a well-controlled device. Properties, such as aerosol particle size, are demonstrated to be regulated with the air flow rate with respect to the e-liquid flow and e-liquid composition. The CAG, however, is limited in assessing common EC-related issues, such as overheating of its elements. We seek to demonstrate that the CAG can generate aerosol that is reproducible and continuous, by assessing the chemical and physical aerosol characteristics with a chosen e-liquid formulation. The protocol describes the operating parameters of liquid flow rate, dilution air-flow rates and operating procedures needing to optimize the aerosol concentration and particle size required for an in vivo toxicology study. Presenting the representative results from the protocol and discussing the challenges and applications of working with a CAG, we demonstrate that CAG can be used in a reproducible fashion. The technology and protocol, that has been developed from prior work, serve as a foundation for future innovations for laboratory-controlled aerosol generation investigations.

The protocol describes the settings and use of a capillary aerosol generator for continuous production of controlled aerosol from a multispecies liquid solution, suitable for steady large-volume aerosol delivery (e.g., in vivo inhalation studies).

The capillary aerosol generator (CAG) is operated with the principal of thermal liquid evaporation through heating of e-liquid in the initial phase, ...