Using Flow Cytometry and Light-Induced Fluorescence To
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Atmos. Chem. Phys., 20, 1817–1838, 2020 https://doi.org/10.5194/acp-20-1817-2020 © Author(s) 2020. This work is distributed under the Creative Commons Attribution 4.0 License. Using flow cytometry and light-induced fluorescence to characterize the variability and characteristics of bioaerosols in springtime in Metro Atlanta, Georgia Arnaldo Negron1,2, Natasha DeLeon-Rodriguez3,a, Samantha M. Waters1,b, Luke D. Ziemba4, Bruce Anderson4, Michael Bergin5, Konstantinos T. Konstantinidis6,3, and Athanasios Nenes1,7,8 1School of Earth and Atmospheric Sciences, Georgia Institute of Technology, Atlanta, GA 30332, USA 2School of Chemical and Biomolecular Engineering, Georgia Institute of Technology, Atlanta, GA 30332, USA 3School of Biology, Georgia Institute of Technology, Atlanta, GA 30332, USA 4School of Biological Sciences, Chemistry and Dynamics Branch/Science Directorate, National Aeronautics and Space Administration Langley Research Center, Hampton, VA 23681, USA 5Department of Civil and Environmental Engineering, Duke University, Durham, NC 2770, USA 6School of Civil and Environmental Engineering, Georgia Institute of Technology, Atlanta, GA 30332, USA 7Institute for Chemical Engineering Science, Foundation for Research and Technology Hellas, Patra, 26504, Greece 8Laboratory of Atmospheric Processes and their Impacts (LAPI), School of Architecture, Civil & Environmental Engineering, Ecole Polytechnique Fédérale de Lausanne, Lausanne, 1015, Switzerland acurrently at: Puerto Rico Science, Technology and Research Trust, Rio Piedras, 00927, Puerto Rico bcurrently at: Department of Marine Sciences, University of Georgia, Athens, GA 30602-3636, USA Correspondence: Konstantinos T. Konstantinidis ([email protected]) and Athanasios Nenes (athanasios.nenes@epfl.ch) Received: 9 October 2018 – Discussion started: 30 October 2018 Revised: 12 September 2019 – Accepted: 22 September 2019 – Published: 14 February 2020 Abstract. The abundance and speciation of primary biolog- HNA dominated the PBAP during humid days and follow- ical aerosol particles (PBAP) is important for understanding ing rain events, where HNA comprised up to 92 % of the their impacts on human health, cloud formation, and ecosys- PBAP number. Concurrent measurements with a Wideband tems. Towards this, we have developed a protocol for quan- Integrated Bioaerosol Sensor (WIBS-4A) showed that fluo- tifying PBAP collected from large volumes of air with a rescent biological aerosol particles (FBAP) and total FCM portable wet-walled cyclone bioaerosol sampler. A flow cy- counts are similar; HNA (from FCM) moderately correlated tometry (FCM) protocol was then developed to quantify and with ABC-type FBAP concentrations throughout the sam- characterize the PBAP populations from the sampler, which pling period (and for the same particle size range, 1–5 µm were confirmed against epifluorescence microscopy. The diameter). However, the FCM LNA-AT population, possibly sampling system and FCM analysis were used to study PBAP containing bacterial cells, did not correlate with any FBAP in Atlanta, GA, over a 2-month period and showed clearly type. The lack of correlation of any WIBS FBAP type with defined populations of nucleic-acid-containing particles: low the LNA-AT suggests that airborne bacterial cells may be nucleic acid-content particles above threshold (LNA-AT) and more difficult to unambiguously detect with autofluorescence high nucleic acid-content particles (HNA) likely containing than currently thought. Identification of bacterial cells even wet-ejected fungal spores and pollen. We find that the daily- in the FCM (LNA-AT population) is challenging, given that average springtime PBAP concentration (1 to 5 µm diameter) the fluorescence level of stained cells at times may be com- ranged between 1:4 × 104 and 1:1 × 105 m−3. The LNA-AT parable to that seen from abiotic particles. HNA and ABC population dominated PBAP during dry days (72 ± 18 %); displayed the highest concentration on a humid and warm Published by Copernicus Publications on behalf of the European Geosciences Union. 1818 A. Negron et al.: Characterization of bioaerosols in Metro Atlanta day after a rain event (14 April 2015), suggesting that both molecules (e.g., DNA/RNA) which subsequently fluoresce populations correspond to wet-ejected fungal spores. Over- when excited by the FCM lasers. The resulting scattering and all, information from both instruments combined reveals a fluorescent light emissions are then detected by an array of highly dynamic airborne bioaerosol community over Atlanta, sensors to allow the differentiation of biological and abiotic with a considerable presence of fungal spores during humid (e.g., dust) particles according to the characteristic specific days and an LNA-AT population dominating the bioaerosol to the stain used. FCM has proven to be as reliable as EPM, community during dry days. but with the advantage of lower uncertainty, higher quantifi- cation efficiency, and requiring considerably less time and effort than EPM per sample (Lange et al., 1997). FCM is frequently used in biomedical research to quantify eukary- 1 Introduction otic cell populations and in microbiology to quantify a wide variety of yeast and bacterial cells (Nir et al., 1990; Van Primary biological aerosol particles (PBAP), also called Dilla et al., 1983). FCM is also used to study environmental bioaerosols, are comprised of airborne microbial cells (e.g., samples, e.g., to differentiate low nucleic acid (LNA) from bacteria, diatoms), reproductive entities (e.g., pollen, fun- high nucleic acid (HNA) phytoplankton in aquatic environ- gal spores), viruses, and biological fragments. Bioaerosols ments (Y. Wang et al., 2010; Müller and Nebe-von-Caron, are ubiquitous, with potentially important impacts on human 2010). Despite its advantages, FCM has seen little use in the health, cloud formation, precipitation, and biogeochemical bioaerosol field to date, owing in part to the challenges asso- cycles (Pöschl, 2005; Hoose et al., 2010; DeLeon-Rodriguez ciated with collecting sufficient PBAP mass for robust count- et al., 2013; Morris et al., 2014; Longo et al., 2014; Fröhlich- ing statistics to be obtained (Chen and Li, 2005; Liang et al., Nowoisky et al., 2016; Myriokefalitakis et al., 2016). Despite 2013). Chen and Li (2005) determined that, for counting pur- their low number concentration relative to abiotic particles, poses, the SYTO-13 nucleic acid stain is the most effective PBAP possess unique functional and compositional charac- (among five different nucleic acid stains studied) for deter- teristics that differentiate them from abiotic aerosol. For ex- mining reliable concentration of bioaerosols. ample, certain PBAP constitute the most efficient of atmo- Light-induced fluorescence (LIF) is an increasingly uti- spheric ice nucleators, affecting the microphysics of mixed lized technique for bioaerosol quantification, and it relies phase clouds and precipitation (Hoose and Möhler, 2012; on measuring the autofluorescence intensity of specific high- Sullivan et al., 2018). The mass and nutrient content of PBAP yield fluorophores (e.g., nicotinamide adenine dinucleotide may suffice to comprise an important supply of bioavailable – NADH co-enzyme – flavins, and amino acids like trypto- phosphorous to oligotrophic marine ecosystems (Longo et phan and tyrosine) present in PBAP. The major advantage of al., 2014; Myriokefalitakis et al., 2016). In addition, the con- the technique is that it is fully automated, does not require a currence of disease outbreaks during dust storms has been at- liquid suspension (i.e., it directly senses particles suspended tributed to pathogenic microbes attached to airborne dust that in air), and provides high-frequency measurements (∼ 1 Hz), are subsequently inhaled (Griffin et al., 2003; Ortiz-Martínez making it ideal for continuous monitoring and operation in et al., 2015; Goudie, 2014). highly variable environments (e.g., aircraft operation). Par- Quantification of the concentration and size of PBAP is ticles detected by LIF, called fluorescent biological aerosol critical for understanding their environmental impacts. Mea- particles (FBAP), although not equal to PBAP, may still con- suring PBAP however poses a challenge for established mi- stitute a large fraction of the biological particles (Healy et crobiology tools, owing to their low atmospheric concentra- al., 2014; Gosselin et al., 2016). Using LIF, FBAP diurnal tion (103–106 cells m−3 air; Fröhlich-Nowoisky et al., 2016) cycles showing maximum concentrations during evenings and wide diversity of airborne particle types and sizes. For in- and minima around middays, especially in heavily vege- stance, only a fraction of microorganisms (an estimated 5 %; tated environments, have been observed. This behavior has Chi and Li, 2007) can be cultured, and cultivation cannot been related to known temperature and relative humidity re- be used to quantify dead organisms, viruses, or fragments, lease mechanisms of certain fungal spore species (Wu et while most culture-independent methods are optimized for al., 2007; Gabey et al., 2010; Tropak and Schnaiter, 2013). more abundant microbial populations. Epifluorescence mi- Huffman et al. (2010) used a Ultraviolet-Aerodynamic Par- croscopy (EPM) is the standard for bioaerosol quantification, ticle Sizer (UV-APS) to show that the concentration and fre- but is not high throughput and requires considerable time for quency of occurrence of 3 µm FBAP particles at Mainz, Ger- quantification of a concentration per sample.