Characterization of the Atmospheric Microbiome at a high-altitude station in the eastern Mediterranean using Flow Cytometry during the fall season

Airborne biological materials (bioaerosols) disperse across ecosystems as they are transported through the atmosphere. This dispersal makes their detailed taxonomical composition characterization essential for understanding their ecological roles and potential impacts on public health. Although metagenomic approaches improved their characterization, complementary tools are needed to better understand their properties. One such tool is flow cytometry (FCM), an established method for analyzing microorganisms, but rarely applied to atmospheric bioaerosol studies (Negron et al., 2020, Liang et al., 2022, Abboud et al., 2026). This study aims to characterize and quantify bioaerosols using FCM to understand population variation at the Helmos Hellenic Atmospheric Aerosol & Climate Change Station (HAC²; Peloponnese, Greece, 2’314m asl, 42°N 05′, 34°E 14′). The sampling area is a typical free-tropospheric background site, with minimal influence from surface-polluted layers. It lies at the intersection of different air masses e.g., continental, Saharan and long-range biomass burning.

Sample collection (n = 55) was performed using a Coriolis µ high-volume wet cyclone over a period of 7 weeks in autumn (6 October to 28 November 2024) as part of the CleanCloud Helmos OrograPhic sIte experimeNt campaign (CHOPIN; http://go.epfl.ch/chopin-campaign). Combining nucleic acids for FCM staining with a self-organising map-based clustering algorithm (FlowSOM, Bioconductor - FlowSOM) after acquisition allowed us to identify populations characterized by low  and high nucleic acid content (LNA and HNA, respectively) (Abboud et al., 2026). The analysis included meteorological parameters and atmospheric pollutants, providing a comprehensive overview of these populations. Meanwhile, Oxford Nanopore Technologies (ONT) sequencing was employed to achieve in-depth taxonomic resolution.

The results show that the average number of bioaerosols collected in the planetary boundary layer (PBL, n = 39) was 1.5 ± 3.3 × 10⁵ m⁻³, compared to 8.7 ± 7.9 × 10³ m⁻³ in the free tropospheric layer (FTL, n = 13)., a decrease of two orders of magnitude between the layers. The LNA population dominated the bioaerosol fraction in both layers, accounting for 79% and 85% of the detected bioaerosols in the PBL and FTL, respectively, while intact cells represented 92% and 100%, respectively. In both layers, LNA population was smaller than the HNA, with mean diameters of 2.4 ± 0.9 µm and 4.4 ± 3.4 µm in the PBL, and 2.5 ± 1.3 µm and 4.3 ± 2.6 µm in the FTL, respectively. The different populations were taxonomically identified using ONT sequencing.

 

This work was supported by the Swiss National Science Foundation project LIPIC-Air (project number 215416) and the CleanCloud project, funded by the European Commission's Horizon Europe call for proposals, "Improved knowledge of cloud-aerosol interactions" (HORIZON-CL5-2023-D1-01-04).

References

Abboud E., Rossi P., Crouzy B., Nenes A.,Violaki K., (2026). Characterization of the Atmospheric Microbiome in a Semi-Rural Area of Central Europe Using Flow Cytometry. Under review in ISME Communication.

Liang L et al. (2022). The characterization and quantification of viable and dead airborne biological particles using flow cytometry and double fluorescent staining. J Aerosol Sci, 165.

Negron, A., Deleon-Rodriguez, N., Waters, S. M., Ziemba, L. D., Anderson, B., Bergin, M., Konstantinidis, K. T., & Nenes, A. (2020) Atmospheric Chemistry and Physics, 20(3), 1817–1838