Evaluating Best Practices for Isolating Pyrophilous Bacteria and Fungi from Burned Soil

A live microbial culture is invaluable to assess traits and functions, yet culturing immediately from fresh soil is logistically challenging and media selection is not trivial. Deeper ecological understanding of pyrophilous microbes and their traits is hampered by a lack of culture-based work causing researchers to rely heavily on community sequence analysis. To improve our understanding of isolating pyrophilous bacteria and fungi after wildfires from burned soil, we tested which: 1) soil storage method and 2) media retained highest genera richness and highest culturable viability as measured by CFUs retained. We tested four soil storage methods (dried, refrigerated at 4{degrees}C, stored at -80{degrees}C with or without glycerol) using a well-homogenized soil sample, plated microbes on rich media, and compared to fresh soil obtained 6 months after a severe California shrubland wildfire. From the fresh soils, we tested 3 media types: rich (Lysogeny Broth (LB) for bacteria; Malt Yeast Agar (MYA) for fungi), oligotrophic (Reasoners 2 Agar (R2A)) and media made from pyrogenic organic matter (PyOM). For bacteria, storing soil frozen at -80{degrees}C without glycerol yielded the highest genera richness and with glycerol preserved the most species. For fungi, storing soil at -80{degrees}C without glycerol preserved the most species but preserved equivalent richness of genera as fresh and 4{degrees}C soil. R2A yielded the highest bacterial and fungal genus richness but some species of interest were only captured with PyOM. Using a combination of these storage and media methods along with a few additional techniques (mushroom cultivation, smoke cultivation, etc.) from 2018-2022, we cultured >500 isolates (286 bacteria; 258 fungi) after 7 California wildfires for testing pyrophilous microbial traits. ImportanceFires are increasing in frequency and severity across the globe, making the understanding of the traits of the pyrophilous microbes that survive and thrive post-fire and influence post-fire ecosystem regeneration critical and pressing. Yet, our ability to characterize the traits of pyrophilous microbes through genomics, transcriptomics, and phenotypic assays is hampered by lack of organisms in culture. Here, we debut a new medium created from pyrogenic organic matter that helps to cultivate fastidious pyrophilous microbes, and also describe a large-scale culture collection of pyrophilous bacteria and fungi (>500 isolates) that can be used for future collaborative research. We comprehensively test how soil storage and media type affect the cultivation of both bacteria and fungi, and for the first time, we test the best method to store soil to retain fungal diversity. Our work can be applied to improve culture collections of fastidious microbes from a variety of environments.