The atmosphere is the most underexplored microorganism-harbouring ecosystem due to the technological challenges inherent in the sampling of an ecosystem of global dimensions, that also contains ultra-low biomass. The Meta-’omics & Microbiome team’s advancements in biomass collection, DNA extraction, library preparation and sequencing techniques have enabled a metagenomics time course study of unprecedented temporal and taxonomic resolution, examining the time scales at which airborne microbial community composition fluctuates throughout a day, between days and across months.

The microbial composition of tropical air in Singapore was assessed by collecting samples 12 times per day, for two hours per time point, over five consecutive days. This sampling regime was repeated in three-month intervals, resulting in four independent time series over 13 months, covering the wet and dry seasons with opposing wind directions. The resulting 795 air metagenome samples, from 265 time points, generated a total of 2.27 tera base pairs – the equivalent to about 760 human genomes – which were analysed for community composition and dynamics.

This study demonstrated for the first time that the microorganisms in air follow a precise diel cycle, with larger variation in microbial diversity observed within a single day, than was observed on a day-to-day or month-to-month basis. These within-day observations showed significant and consistent day-night differences, with various microbial groups increasing up to 10-fold at midday or during rain events, while others dominated during the night. Surprisingly, the study revealed larger microbial diversity in air samples compared to those collected in aquatic, terrestrial or human host environments.

Temperature was one of the key driving factors for the observed diel dynamics and the pattern of stable diel fluctuations has also been demonstrated in temperate climates.

Taxonomical breakdown and temporal resolution of the air microbial communities. (a) Relative abundance of seven taxonomic groups of organisms in air. Rain events with recorded amounts of precipitation (blue bars) and temperature distribution (red buttons) are denoted for each time series. The timescale is indicated (bottom), as well as day and night samples denoted by sun and moon symbols. (b) Observed number of species by taxonomical groups in each of the four-day variation sampling (DVE). The graph depicts the change in species abundance per group, indicating the robustness of the Basidiomycota community versus strong daily variation of the community composition for the other six phyla.

The dynamics and variability of airborne microbial communities associated with different layers of the vertical air column were investigated using the methods established for sequencing low biomass environments. An innovative sampling methodology was devised that would not restrict air flow and could truly capture the air microbiome composition of different air layers in the vertical air column. This vertical testing array was established in Germany and comprised a 200 m meteorological tower (Karlsruhe Institute of Technology, Institute of Meteorology and Climate Research, Karlsruhe, Germany) and a research aircraft (Technische Universit.t Braunschweig, Institute of Flight Guidance, Braunschweig, Germany). Synchronised measurements of meteorological parameters and microbial biomass were taken from ground level up an altitude of 3,500 m.

The temporal and spatial resolution of this study demonstrated that the diel cycle of airborne organisms is a ground-based phenomenon and is absent at heights more than 1,000 m. This finding led to the discovery that temperature, not sunlight or humidity, is the single most important parameter that influences the composition of airborne microbial communities. Hence, rising global temperatures are predicted to alter the existing air microbiome and lead to a much wider dispersal of airborne microorganisms.

This is study also showed for the first time that the atmosphere acts as a sink in the sky for microorganisms. As a result, higher air layers aggregate a large microbial diversity, which eventually reaches a global range of dispersal. This explains the observation of highly similar microbial taxa in aquatic and terrestrial ecosystems around the globe, and provides a working model for airborne microbial communities as key drivers for the biological organisation of the planetary air ecosystem.

Association of airborne microorganisms with different altitudes in the lower troposphere. Images taken of field sampling using (a) meteorological tower (MT) and (b) research aircraft (RA). Taxa cloud graphs plotting the mean relative abundance of the species identified in the (c) MT sampling at ground level (x-axis) and 200-metre tower-top air layer (y-axis) and (d) RA sampling on the ground level (x-axis) and 1,000- to 3,500-metre air layer (y-axis). Adapted from PNAS (2022) 19: e2117293119.