The concentration of heterotrophic growth substrates in culture medium is a crucial determinant of the culturability of subsurface soil microbes

Soil microbial communities have crucial roles in soil fertility and biogeochemistry. While surface soils have been relatively well characterized, we know far less about the microbiology of oligotrophic shallow subsurface soils that often contain microbial taxa that are distinct from surface soils. We hypothesized that the concentration of heterotrophic growth substrates in microbiological growth medium is a critical determinant of the ability to culture subsurface soil microbes. To test this, we conducted dilution-to-extinction experiments with microbial cells separated from subsurface soils collected in a mid-elevation conifer forest in Arizona, USA. We diluted cells into two defined growth media with identical concentrations of inorganic nutrients and vitamins but contained a 100-fold difference in the availability of an amino acid and organic carbon mixture. Across both media formulations, we isolated a total of 133 pure cultures. The concentration of heterotrophic growth substrate in the medium facilitated the isolation of taxonomically distinct microbes. We isolated representative cultures of the most abundant member in the soil community (Bradyrhizobium spp.) and representatives of five of the top 10 most abundant Actinobacteria phylotypes, including Nocardioides spp., Mycobacterium spp., and several other phylogenetically divergent lineages. Flow cytometric analysis of the cultures showed that SYBR green-stained cells isolated on medium with reduced heterotrophic substrate concentrations had significantly lower nucleic-acid fluorescence than those isolated on medium with higher substrate concentrations. These results show that medium substrate concentration can facilitate the isolation of certain microbial lineages and that dilution-to-extinction is an effective method to isolate abundant soil microbes.