Insights into the phylogenetic and metabolic diversity of Planctomycetota in anaerobic digesters and the isolation of novel Thermoguttaceae species

Abstract

Studying bacteria in anaerobic digestion (AD) is crucial for optimizing microbial processes. While abundant taxa are often studied, less abundant groups may harbour novel metabolic potential. This study fills the gap by focusing on the Planctomycetota phylum, known to encode diverse carbohydrate-active enzymes (CAZymes). Despite their common presence in diverse aerobic and anaerobic environments, their role in AD is relatively unexplored. We utilized both culture-dependent and culture-independent techniques to investigate the phylogenetic and metabolic diversity of Planctomycetota within AD reactors. Our findings revealed that among the diverse planctomycetotal operational taxonomic units present, only a few are prevalent and abundant community members. Planctomycetota share functional traits with e.g. Verrucomicrobiota exhibiting distinct CAZyme gene repertoires that indicates specialization in degrading algal polysaccharides and glycoproteins. To explore the planctomycetotal metabolic capabilities, we monitored their presence in algal-fed digesters. Additionally, we isolated a strain from mucin-based medium, revealing its genetic potential for a mixotrophic lifestyle. Based on the genomic analysis, we propose to introduce the Candidatus Luxemburgiella decessa gen. nov. sp. nov., belonging to the Thermoguttaceae family within the Pirellulales order of the Planctomycetia class. This study enhances our understanding of Planctomycetota in AD by highlighting their phylogenetic diversity and metabolic capabilities.

Keywords: Planctomycetes; Planctomycetota; CAZymes; algal polysaccharides; anaerobic digestion; bacterial utilization of sulphated glycans; encoded metabolic potential; exoglycosidases; mucins.

Figure 1.

Planctomycetotal OTU diversity based on the 16S rRNA gene amplicon sequencing and SILVA taxonomy. (a) Total number of OTUs (in brackets) across all studied reactors found within each taxonomic order of the Planctomycetia, Phycisphaerae, and other minor classes. (b) Number of OTUs within each of the abundance categories: RT—rare taxa, CRT—conditionally rare taxa, and CRAT—conditionally rare and abundant taxa. (c) Fraction of OTUs within the set categories for each family level. (d) Total number of unique planctomycetotal OTUs detected across all samples, categorized by reactor type. (e) Number of OTUs in the individual sample per AD category. (f) Relative abundance of Planctomycetota (%) within the total bacterial community per reactor. The number of samples is indicated in brackets. (g) Visualization of PCoA based on the planctomycetotal OTUs Bray–Curtis distance matrix, coloured by the sampling location unit (‘U’).

Figure 2.

Phylogenetic relationship of AD Planctomycetota with previously isolated strains, coloured by family level as indicated in the legend. (a) Relative abundance (%) of the closest cultured relatives to the AD OTUs, grouped by family level and displayed for different reactor categories. (b) Fraction of OTUs likely falling within genera characterized by cultured representatives, shown for each family level. (c) OTU prevalence in AD reactors, e.g. fraction (%) of reactors containing assigned OTUs, displayed by genus level. (d) Relative abundance (%) of OTUs per sample within the planctomycetotal community. (e) Number of OTUs assigned to the listed genera across all the samples. In panels (b–d), only the most abundant genera are shown (>1% of planctomycetotal relative abundance). All records are detailed in Supplementary File 1.

Figure 3.

General encoded metabolic potential of Planctomycetota compared to other bacteria and archaea. (a) Venn diagram showing the number of shared and unique KOs. (b) PCoA plot comparing the presence–absence of KOs across the different bacterial genomes coloured by phylum taxonomic level. The plot includes ellipses representing 95% confidence around the group centroids and only the phyla with >90% ellipse intersections with Planctomycetota are coloured: Acidobacterota, Hydrogenedentota, Desulfobacterota, and Verrucomicrobiota. (c) The same PCoA plot highlighting the Planctomycetota MAGs only.

Figure 4.

Putative substrates specific for Planctomycetota based on the genomic capacity for targeting glycosidic linkages. Lysosomal and other minor activities including oligosaccharidases are omitted. RFO—raffinose family oligosaccharides, HMO—human milk oligosaccharides, GAG—glycosaminoglycans, EPS—extracellular polymeric substances, and BGA—blood group antigen.

Figure 5.

Metabolic reconstruction of AD Planctomycetota MAGs assembled in this study, including isolated/enriched planctomycetotal strains (M10, M16, and M17). The clade colour on the tree denotes taxonomic affiliation (Planctomycetia in blue, Phycisphaerae in yellow), with stars indicating MAGs reconstructed from wastewater ADs. The following colour strip designates MAGs taxonomy and the planctomycetotal orders: Pirellulales in blue, UBA1845 in orange, Sedimentisphaerales in green, and the other minor orders in grey. Square symbols represent metabolic traits based on the genomic annotation: fully coloured symbols indicate complete pathways (all required genes present, or only one is missing), empty symbols denote the partially encoded pathways (some required genes missing), and missing symbols indicate that no genes related to the pathway were found. Abbreviations: ASR—assimilatory sulphate reduction, DSR—dissimilatory sulphate reduction, ANR—assimilatory nitrogen reduction, and DNR—dissimilatory nitrogen reaction. Further details on the prediction of functional traits can be found in Supplementary File 2, Table S3.

Figure 6.

Enrichment and isolation of Planctomycetota; 1, 2, and 3 designate different inocula used. (a) Relative abundance of Planctomycetota in all the enrichments in our study. (b) Community analysis at the end of the incubation period, with each bar representing a single OTU; only the significantly enriched samples on mucin, fucoidan, and NAG substrates are shown. (c) Gram-stained microscopy image of the purified SKZ1R strain isolated from the SKZ1R enrichment. (d) Gram-stained microscopy image of the SKZ5-1 enrichment, Pirellula-like shape cells are captured.

Figure 7.

16S rRNA phylogeny of SKZ strains and type strains of Pirellulales.