In situ visualisation of the abundant Chloroflexi populations in full-scale anaerobic digesters and the fate of immigrating species

Anaerobic digestion is a key process for the conversion of waste organics to biogas for energy and is reliant on the synergistic activities of complex microbial communities. Members of the phylum Chloroflexi are often found to be abundant in these systems, yet little is known of their role, with most members yet to be cultured or identified. The aim of this study was to characterize the Chloroflexi communities present in full-scale anaerobic digesters receiving excess sludge from wastewater treatment plants. The core genus-level-phylotypes were identified from extensive 16S rRNA gene amplicon sequencing surveys of 19 full-scale systems over a 6 year period. The T78 and Leptolinea, and the RB349 and SJA-170, were found to be the most abundant genera of mesophilic and thermophilic digesters, respectively. With the exception of Leptolinea, these phylotypes are known only by their 16S rRNA gene sequence, and their morphology and metabolic potentials are not known. Fluorescence in situ hybridisation (FISH) probes were designed for these phylotypes, with their application revealing a similar thin filamentous morphology, indicating a possible role for these organisms in maintaining floc structure. The new FISH probes provide a useful tool for future efforts to characterize these organisms in situ. FISH also suggests that immigrating Chloroflexi species die off in the anaerobic digester environment and their high abundance in anaerobic digesters, observed with DNA based sequencing surveys, was quite possibly due to the persistence of their DNA after their death. This observation is important for the interpretation of popular DNA-based sequencing methods applied for the characterisation of communities with substantial immigration rates, such as anaerobic digesters.