An experimental test of the keystone species concept in microbial food webs using a synthetic soil pore space system

The keystone species concept suggests that certain members of an ecological community, despite their low abundance, exert disproportionately large effects on species diversity and composition. In microbial ecology, experimental validation of this concept is limited because targeted removal of individual species remains technically challenging. Here, we developed a new experimental procedure to test the keystone species concept within a soil microbial food web by selectively suppressing a protist predator at the microscale via UV phototoxicity in a microfluidic soil chip system. We targeted a Hypotrichia ciliate, which was presumed to be primarily bacterivorous under our experimental conditions, and combined microscopy with DNA metabarcoding across multiple trophic levels to evaluate how suppression affected microbial community abundance, diversity, and composition. Over the 20-day incubation, the chip system supported complex communities of bacteria, fungi, and protists. Following Hypotrichia suppression, two distinct ecological responses were observed: first, an increase in the relative abundance of flagellates consistent with mesopredator release, accompanied by a significant rise in overall protist diversity; second, a convergence in protist community composition, indicative of biotic homogenization. Surprisingly, bacterial community abundance, richness, and composition remained unaffected, likely due to compensatory predation by a relative increase in other bacterivorous flagellates. In contrast, fungal diversity decreased, presumably because the altered protist community favored facultative fungal consumers. Collectively, these findings provide direct experimental evidence that low-abundance microbial predators can function as keystone species, modulating predator community composition and diversity and exerting cascading effects on lower trophic levels within a brown microbial food web.