Sweet Secrets: Exploring Novel Glycans and Glycoconjugates in the Extracellular Polymeric Substances of " Candidatus Accumulibacter"

Abstract

Biological wastewater treatment relies on microorganisms that grow as flocs, biofilms, or granules for efficient separation of biomass from cleaned water. This biofilm structure emerges from the interactions between microbes that produce, and are embedded in, extracellular polymeric substances (EPS). The true composition and structure of the EPS responsible for dense biofilm formation are still obscure. We conducted a bottom-up approach utilizing advanced glycomic techniques to explore the glycan diversity in the EPS from a highly enriched "Candidatus Accumulibacter" granular sludge. Rare novel sugar monomers such as N-Acetylquinovosamine (QuiNAc) and 2-O-Methylrhamnose (2-OMe-Rha) were identified to be present in the EPS of both enrichments. Further, a high diversity in the glycoprotein structures of said EPS was identified by means of lectin based microarrays. We explored the genetic potential of "Ca. Accumulibacter" high quality metagenome assembled genomes (MAGs) to showcase the shortcoming of top-down bioinformatics based approaches at predicting EPS composition and structure, especially when dealing with glycans and glycoconjugates. This work suggests that more bottom-up research is necessary to understand the composition and complex structure of EPS in biofilms since genome based inference cannot directly predict glycan structures and glycoconjugate diversity.

Figure 1

Reactor characteristics for the enrichments with the impeller rotating at 400 (top) and 800 (bottom) rpm at steady state. Each panel presents the activity test of a cycle by showing the concentrations of phosphate and acetate (mmol/gTSS) (left) and the microbial community abundance based on 16S rRNA amplicon sequencing (right). Both activity tests indicate that acetate was taken up during the anaerobic phase with the concurrent release of phosphate, typical for PAOs enrichments. For 16S rRNA results, the resolution at genus level indicates ≥1% abundance, otherwise genus with <1% abundance were clustered into the category “Others”.

Figure 2

Glycosyl composition of the extracted EPS as relative mole abundance from the total amount of carbohydrate monomers determined by GC-MS. Carbohydrate monomers detected: glucose (Glc), Rhamnose (Rha), Mannose (Man), Galactose (Gal), Ribose (Rib,) N-Acetylglucosamine (GlcNAc), N-Acetylquinovosamine (QuiNAc), and 2-O-Methylrhamnose (2-OMe-Rha).

Figure 3

Genetic potential for the biosynthesis of QuiNAc in “Ca. Accumulibacter”. (A) Biosynthetic pathway of the glycan QuiNAc in bacteria indicating the genes encoding each reaction step enzyme. (B) Presence (filled with blue) or absence (empty) of the genes involved in this biosynthetic pathway in multiple metagenome assembled genomes (MAGs) of “Ca. Accumulibacter” species. Genes with BLAST hit >40% identity but not annotated as such are filled with lighter blue.

Figure 4

Lectin microarray profile indicating the fluorescence intensity for binding of glycoproteins in the EPS to each individual lectin. The broad specificity of each lectin is shown, and more specific structural specificity is indicated in Supporting Information.

Figure 5

(A) Protein glycosylation mechanism present in C. jejuni (figure adapted from). (B) Presence (filled) or absence (empty) of the genes involved in this biosynthetic pathway in multiple metagenome assembled genomes (MAGs) of “Ca. Accumulibacter” species.