Characterization of Dominant Heterotrophic Flagellates in Anaerobic Digesters Using Combined Culture-based and Metabarcoding Approaches

While bacteria and archaea are regarded as major components of anaerobic ecosystems, eukaryotes have received less attention, especially in engineered ecosystems such as waste reclamation plants. Previous work on these environments has tended either to focus on individual organism strains in culture or on metabarcoding sequence analysis of uncultured material; we employed both approaches. Interestingly, the culture-independent analysis of a full-scale anaerobic digester mostly detected aerobic eukaryotes that originated directly from feed material, such as cabbage, sweet potato, aerobic fungi, and mites. Remarkably, the anaerobic protist Tetratrichomonas was the dominant organism in all samples, consistently present but showing a more pronounced peak in April than in other months; its bloom is likely associated with increased levels of volatile fatty acids and biogas production. Also, we surveyed the protist communities in full-scale anaerobic digesters microscopically, from which we isolated novel heterotrophic flagellates; based on sequence identity, molecular phylogeny, and morphology, we identify as these Tetratrichomonas spp. Although neither culture-dependent nor culture-independent approaches give a complete picture of the communities within these ecosystems, both suggest that Tetratrichomonas may be a key genus in Korean mesophilic (36.6-38.9 ˚C) anaerobic digesters treating a mixture of livestock manure, food waste, and sewage sludge.