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

Abstract

A live microbial culture is invaluable to assess traits and functions via 'omics and biophysical assays. However, it is not always logistically feasible to culture immediately from freshly obtained soil, and selecting the proper media for culturing is not trivial. While building a culture collection of pyrophilous microbes obtained from burnt soil, we tested the best 1) method of storing soil to retain culturable viability and 2) media to garner the most microbial diversity. We tested four methods of soil storage (dried, stored at 4°C, stored at -80°C alone or in glycerol) and compared to fresh soil obtained 6 months after a severe California chapparal shrubland wildfire. For bacteria, soil frozen at -80°C with glycerol preserved the greatest diversity (25 species, 13 genera) compared to fresh soil (26 species, 13 genera). For fungi, soil stored at -80°C alone preserved the greatest diversity (10 species, 3 genera) compared to fresh soil (13 species, 7 genera). We also tested 3 media types: rich media (Lysogeny Broth (LB) for bacteria; Malt Yeast Agar (MYA) for fungi), oligotrophic media (Reasoner's 2 Agar (R2A) and media made from pyrogenic organic matter (PyOM). For bacteria, culturing on LB and R2A garnered the greatest diversity (LB = 26 species, 13 genera, R2A = 27 species, 15 genera). For fungi a combination of R2A and PyOM captured the greatest diversity (R2A = 15 species, 8 genera, PyOM = 12 species, 6 genera). For both bacteria and fungi, some species of interest were only captured using the PyOM media. Using a combination of these methods from 2018-2022, we cultured >500 isolates (286 bacteria; 258 fungi) from burned soils of 7 Southern California wildfires.