The in situ characterization of the Chloroflexi communities of full-scale anaerobic digesters and the influence of immigration.

Filamentous Chloroflexi are often abundant in activated sludge and anaerobic digesters, where they have an important role in floc formation and degradation of complex matter. They are also well-known to cause bulking and foaming problems. Some members of the phylum have been isolated and characterized in situ, however, more insight is needed to understand their function. The aim of this study was to characterize the Chloroflexi community in anaerobic digesters, identify novel taxa and design FISH probes for their in situ detection. Furthermore, as it was concluded that the microbial community in anaerobic digesters was strongly influenced by migration, the goal was to investigate the survival of Chloroflexi abundant in the activated sludge and potentially able to survive in anaerobic conditions, when fed into the digesters. 16S rRNA amplicon sequencing was used to identify the most abundant phylotypes in 32 Danish anaerobic digesters. T78 and Leptolinea were the dominant Chloroflexi in mesophilic digesters, and RB349 and SJA-170 were the most abundant in thermophilic digesters. Phylogenetic analysis showed their affiliation with the Anaerolineae class, which contains several cultured filamentous bacteria with anaerobic metabolism. All the novel phylotypes were short and thin filaments, often found on the surface of sludge flocs. This specific spatial arrangement may be linked to their functional role, as they likely contribute to the hydrolysis of complex matter. Filamentous Chloroflexi fed into the digesters with activated sludge were quantified with q-FISH and were shown to die off once they reach the new environment, suggesting detrimental effect of high temperature and/or salt concentration.