Database Mining to Unravel the Ecology of the Phylum Chloroflexi in Methanogenic Full Scale Bioreactors

Abstract

Although microbial communities of anaerobic bioreactors have been extensively studied using DNA-based tools, there are still several knowledge gaps regarding the microbiology of the process, in particular integration of all generated data is still limited. One understudied core phylum within anaerobic bioreactors is the phylum Chloroflexi, despite being one of the most abundant groups in anaerobic reactors. In order to address the abundance, diversity and phylogeny of this group in full-scale methanogenic reactors globally distributed, a compilation of 16S ribosomal RNA gene sequence data from 62 full-scale methanogenic reactors studied worldwide, fed either with wastewater treatment anaerobic reactors (WTARs) or solid-waste treatment anaerobic reactors (STARs), was performed. One of the barriers to overcome was comparing data generated using different primer sets and different sequencing platforms. The sequence analysis revealed that the average abundance of Chloroflexi in WTARs was higher than in STARs. Four genera belonging to the Anaerolineae class dominated both WTARs and STARs but the core populations were different. According to the phylogenetic analysis, most of the sequences formed clusters with no cultured representatives. The Anaerolineae class was more abundant in reactors with granular biomass than in reactors with disperse biomass supporting the hypothesis that Anaerolineae play an important role in granule formation and structure due to their filamentous morphology. Cross-study comparisons can be fruitfully used to understand the complexity of the anaerobic digestion process. However, more efforts are needed to standardize protocols and report metadata information.

Keywords: 16S rRNA amplicon sequence; Chloroflexi; filamentous bacteria; full-scale methanogenic reactors; meta-analysis; wastewater.

FIGURE 1

Boxplot of the total community for STARs and WTARs showing the phyla with a relative abundance higher than 2% in at least one reactor.

FIGURE 2

PCoA analysis and PERMANOVA test of the total community. The different colors indicate the different wastes or wastewaters used to fed the reactors. The PERMANOVA test results are shown at the top of the figure (see Table 3).

FIGURE 3

Boxplot of the Chloroflexi phylum for STARs and WTARs showing the relative abundance at: (A) class level (showing all the classes present in the systems); (B) genus level (showing the 25 most abundant genus which belong to the Anaerolinea class).

FIGURE 4

PCoA analysis and PERMANOVA test of the phylum Chloroflexi populations, the different colors indicate the different hypervariable regions. The PERMANOVA test results are shown at the top of the figure (see Table 3).

FIGURE 5

Boxplot of the Chloroflexi phylum for STARs and WTARs showing the relative abundance at species level (25 most abundant).

FIGURE 6

Phylogenetic tree of the phylum Chloroflexi inferred from the 16S rRNA gene sequences. The tree was reconstructed using the ML method and the GTR model. ML bootstrap values greater than or equal to 70% are shown at each node. Bar, 0.01 substitutions per site. The reference OTUs with a relative abundance greater than 5% in at least one reactor are represented by their GenBank accession numbers. Only OTUs from the core microbiome are indicated in color: from STARs are in blue color, from WTARs are in red color. Thermotoga spp. sequence served as an outgroup for rooting tree.