Sampling spores and microorganisms in the stratosphere

Spores are the survival and dissemination units of fungi. Many are designed for optimal airborne dispersal while maintaining long-term survival. Depending on the chemical and structural nature of their walls, they are highly resistant to extreme temperatures and UV radiation. For example, Botrytis cinerea conidia stored dry at -80°C are still able to germinate after more than 20 years in storage. Given their anemochorous nature and resistance to abiotic factors, it would therefore be possible for spores of pathogenic fungi to be aeroported through the stratosphere. However, little is known about the spread of pathogenic fungi in high-altitude airspace.

 

In order to investigate the presence of fungal spores in the stratosphere and explore the diversity of viable and non-cultivable fungi, we designed a low-cost sampling device capable of sampling particles in the stratosphere. It consists in a sealed polystyrene box with two ports on the top and bottom sides, allowing air circulation. A rotating arm sampler spins in the resulting airflow, with four sticks coated with petroleum jelly. The opening of the ports is controlled by mobile covers driven by servomotors, managed by an Arduino Uno microcontroller connected to a high-pressure pressure sensor. Moreover, an on-board radiosonde continuously transmits GPS position, relative humidity, and temperature data. An internal camera captures the opening, closing, and sampling processes during the desired altitude segment. Additionally, a control box, that never opens during flight,  monitors potential contamination below the stratosphere.

 

Both the measurement and control boxes are sterilized under UV-C, sealed and attached to a meteorologic radiosonding balloon. Upon reaching an altitude of 12,000 meters, the covers open, and airborne particles are collected. Once the balloon bursts (at around 35,000 m; -63°C), a parachute deploys during the descent, and the cover closes at 12,000 meters.  The prompt recovery of the sample at landing is assisted by a specifically dedicated mobile app, that extrapolates the descent trajectory and guides the crew to the expected landing location.

 

Five test flights between October 2023 and June 2024 up to 35,000 meters altitude, allowed us to optimize and validate the device, the sampling conditions, and the sample recovery procedures and analysis. The collected samples were both cultured on fungal medium and prepared for deep DNA sequencing. The control box remained sterile, confirming the absence of contamination. Furthermore, several species of cultivable fungi were identified in the sample, demonstrating the viability of spores despite low pressure and temperature, while the DNA sequencing revealed the presence of many species, including exotic ones.

 

This setup opens the way to routine monitoring of stratospheric airborne fungi spores and other biological aerosols, in view of a better understanding of their dispersal and survival.