Single‐Cell Identification of Bacterivorous Mixotrophs in a Freshwater Habitat

The nutritional strategy of phago‐mixotrophy among pigmented eukaryotes has been widely recognized and characterized across various taxonomic lineages. While their ecological role as bacterivores in different aquatic environments is acknowledged, establishing a clear connection between phago‐mixotrophic activities and taxonomy within natural communities remains challenging. In this study, we utilized a combination of microscopy and fluorescence‐activated single‐cell sorting techniques to investigate the diversity and temporal variations of bacteria‐feeding mixotrophs in a freshwater habitat. We identified four groups of mixotrophs using both methods: Pedinellales (Dictyochophyceae), Cryptomonas (Cryptophyceae), Dinobryon (Chrysophyceae) and a single‐chloroplast bearing group within Chlorophyta. Together, these groups accounted for 58%–96% and 54%–95% of total mixotrophs detected by microscopy and cell sorting, respectively. Samples collected in different seasons demonstrated that Pedinellales and Chlorophyta were more abundant in spring and summer/autumn, while Dinobryon was only present in winter when temperatures were lowest and light intensity was moderate. Our findings, with 6 classes and 12 genera identified as bacterivorous mixotrophs, highlight the promising potential of single‐cell techniques in aquatic microbiology. This approach reveals the taxonomic identity and feeding activities of phago‐mixotrophs in situ, thereby enhancing our understanding of their functional ecological roles within microbial food webs.