Unveiling the hidden diversity and functional role of Chloroflexota in full-scale wastewater treatment plants through genome-centric analyses

Abstract

The phylum Chloroflexota has been found to exhibit high abundance in the microbial communities from wastewater treatment plants (WWTPs) in both aerobic and anaerobic systems. However, its metabolic role has not been fully explored due to the lack of cultured isolates. To address this gap, we use publicly available metagenome datasets from both activated sludge (AS) and methanogenic (MET) full-scale wastewater treatment reactors to assembled genomes. Using this strategy, 264 dereplicated, medium- and high-quality metagenome-assembled genomes (MAGs) classified within Chloroflexota were obtained. Taxonomic classification revealed that AS and MET reactors harbored distinct Chloroflexota families. Nonetheless, the majority of the annotated MAGs (166 MAGs with >85% completeness and < 5% contamination) shared most of the metabolic potential features, including the ability to degrade simple sugars and complex polysaccharides, fatty acids and amino acids, as well as perform fermentation of different products. While Chloroflexota MAGs from MET reactors showed the potential for strict fermentation, MAGs from AS harbored the potential for facultatively aerobic metabolism. Metabolic reconstruction of Chloroflexota members from AS unveiled their versatile metabolism and suggested a primary role in hydrolysis, carbon removal and involvement in nitrogen cycling, thus establishing them as fundamental components of the ecosystem. Microbial reference genomes are essential resources for understanding the potential functional role of uncultured organisms in WWTPs. Our study provides a comprehensive genome catalog of Chloroflexota for future analyses aimed at elucidating their role in these ecosystems.

Keywords: Chloroflexota; activated sludge; meta-analysis; metagenome assembled genomes; methanogenic reactors.

Figure 1

A) Geographic distribution of the 87 reactors. The numbers inside the circles indicate the number of reactors in each country. B) Violin plots showing the estimated completeness (%), estimated genome size (Mb) and GC (%) content for the representative species of Chloroflexota (MAGs_dRep = 264) obtained from all WWTPs. C) Violin plots showing the comparison of the estimated genome size (Mb) between MAGs_dRep from AS and MET reactors (Shapiro–Wilk normality test <0.05, non-parametric test Kruskal–Wallis P-value <0.05).

Figure 2

Heatmap showing the relative abundance of the Chloroflexota MAGs_dRep in activated sludge (AS) and methanogenic reactors (MET) classification at A) class level, B) order level, C) family level and D) genus level.

Figure 3

Phylogenomic tree of Chloroflexota MAGs_dRep (n = 264) and reference genomes retrieved from NCBI (n = 96). Letters from A to O indicate clusters containing high-quality (HQ) draft MAGs with no representative cultures. Genomes from the phylum Thermotogota were used as outgroup to root the tree.

Figure 4

Analysis of the CAZy enzymes in Chloroflexota MAGs from AS and MET reactors. CAZy families found in more than 50% of the MAGs_annot (annotated using the dbCAN2 carbohydrate-active enzyme (CAZy) domain HMM database). Glycoside hydrolase (GH), Glycosyl transferase (GT), carbohydrate esterase (CE), auxiliary activities (AA). Percentages of MAGs containing a Cazy family were calculated relative to the total number of MAGs in each reactor type (AS reactor vs MET reactors).

Figure 5

Metabolic model of the Chloroflexota MAGs_annot in activated sludge and methanogenic reactors. The percentage of MAGs_annot from AS and MET reactors appears in the boxes when the gene or metabolic pathway is present.

Figure 6

Hierarchical clustering (Euclidean distance) of the presence/absence of metabolic pathways in the MAGs_annot. In the right part of the figure taxonomic assignment is shown at the order level.