Aerobiology and Environmental Zonation in Gypsum Caves: A Comparative Study of Culturing and NGS Approaches

Classical aerobiological studies commonly use high-volume air samplers to quantify and identify cultivable airborne bacteria and fungi. However, this approach introduces a significant bias, as it overlooks the non-cultivable fraction, which likely constitutes a major component of the airborne microbiome. The advent of next-generation sequencing (NGS) has addressed this limitation, enabling a more comprehensive characterization of the cave aerobiome. This study analyzes both cultivable and non-cultivable airborne bacteria from Covadura and C3 caves, located in the Gypsum Karst of Sorbas (SE Spain). A total of 24 bacterial genera were identified using culture-based methods, whereas NGS revealed 749 genera. Culture-based methods using the surface air system (SAS) predominantly recovered Gram-positive spore-forming bacteria from the phyla Bacillota and Actinomycetota, which were largely absent or present in low relative abundances in the NGS datasets. In contrast, NGS revealed a broader diversity, including numerous Gram-negative and rare airborne bacteria not detected by culture. The NGS results from airborne samples showed greater similarity to the microbial communities found in cave biofilms and sediments, suggesting that a portion of airborne bacteria originates from within the cave and is influenced by microclimatic conditions such as ventilation and air stagnation. Although the short-read sequencing approach used in this study has limitations, such as reduced taxonomic resolution compared to the culture-based approach, it remains the most effective tool for capturing the diversity and ecological patterns of airborne microorganisms. The integration of gas tracers and other environmental data allowed the identification of zones within the cave with different ventilation patterns and degrees of isolation, which corresponded to different spatial distributions of airborne bacteria. Our findings underscore that reliable aerobiological studies in caves require the combination of non-culture dependent-based sequencing approaches and environmental monitoring to fully understand the origin, diversity, and ecological dynamics of airborne microbial communities.