Throughput and compartmentalisation shape bacterial recovery in droplet- and plate-based cultivation of hydrogen-oxidising communities

Abstract

Isolation methods are essential for recovering microbial diversity for physiological characterization. Droplet-based cultivation frequently shows greater taxonomic recovery than conventional plating, but the basis of this advantage remains unclear. Here, we used hydrogen-oxidising enrichment communities as a tractable model system to compare droplet- and plate-based cultivation and explicitly separate throughput from cultivation-format effects. Both methods combined achieved high recovery (~90%), with droplets recovering more OTUs, particularly under heterotrophic conditions (77.8 % versus 51.7%). However, much of this advantage was explained by the substantially higher number of cells screened in droplets. Comparison with previous droplet-cultivation studies showed that differences in founder-cell numbers are also common in the literature and are often not separated from cultivation-format effects, potentially inflating the apparent biological advantage of droplets. After throughput normalization, a significant residual droplet advantage remained under heterotrophic conditions (100% versus 90.0 %), whereas no clear difference remained under autotrophic conditions. Droplet growth showed compartmentalisation of nutrients, OTU growth tended to be more even in droplets, and the residual recovery advantage was concentrated among lower-abundance taxa, patterns consistent with reduced competitive dominance but not sufficient to establish competition as the causal mechanism. Overall, our results pick apart increased recovery in droplet cultivation as a combination of higher throughput and cultivation-format-specific effects.