Effect of growth media on the diversity of Neocallimastigomycetes isolates from non-rumen habitats. undefined

Anaerobic fungi (AF) in the phylum Neocallimastigomycota are pivotal components of the microbiota in the digestive tract of various herbivore animals. These fungi play a major role in the breakdown of crude ingested forage due to physical penetration in conjunction with a unique extracellular hemicellulolytic machinery. Studies aiming to understand the role of AF require robust and reliable isolation and culturing techniques, which are unchanged since many years. However, evidence is emerging that even slight variations in isolation methodology and culture media composition affect in vitro AF composition, especially in AF from non-rumen habitats. Using amplicon sequencing we show that the addition of rumen fluid to the culture medium compromises successful culturing of AF isolated from non-rumen habitats. We compared composition and growth of AF isolated from the feces of Przewalski’s horses and cultured in a medium with rumen fluid, one with depleted and one without rumen fluid. Unweighted UniFrac distance analysis showed that all AF growing on the three media are phylogenetically related to each other. Shown in a Jaccard emperor plot, AF from all three media formed one cluster, whereby AF from the medium without rumen fluid had the highest sequencing depth, followed by those with rumen fluid. These results clearly show that medium composition affects AF composition. Therefore, optimizing culture media is important to draw informed conclusions about the microbiota present in mammalian guts.