Many environmentally and industrially relevant microbes remain uncultured, limiting our ability to understand and use them. This is especially true in thermophilic anaerobic microbiomes, which are promising systems for producing sustainable chemicals from organic waste streams. In this study, we explored how different cultivation strategies influence which microbes can be isolated from a thermophilic chain-elongating reactor. By comparing traditional and novel methods, including droplet microfluidics, we showed that each method recovers a unique set of microbes. While droplet-based methods enable high sampling depth with minimal effort and excel at isolating rare microbes, we found that they also introduce clear biases, as certain organisms recovered by other methods did not grow in droplets. Our work highlights the importance of the cultivation method in isolation success and helps shine a light on the selective forces at play in droplet-based microbial isolation.

Abstract. Recent advances in single-cell technologies have profoundly impacted our understanding of microbial communities—shedding light on cell-to-cell va