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.

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Polyphosphate accumulating organisms (PAOs) are responsible for enhanced biological phosphate removal (EBPR) from wastewater, where they grow embedded in a matrix of extracellular polymeric substances (EPS). EPSs comprise a mixture of biopolymers like polysaccharides or (glyco)proteins. Despite previous studies, little is known about the dynamics of EPS in mixed cultures, and their production by PAOs and potential consumption by flanking microbes. EPSs are biodegradable and have been suggested to be a substrate for other organisms in the community. Studying EPS turnover can help elucidate their biosynthesis and biodegradation cycles. We analyzed the turnover of proteins and polysaccharides in the EPS of an enrichment culture of PAOs relative to the turnover of internal proteins. An anaerobic-aerobic sequencing batch reactor (SBR) simulating EBPR conditions was operated to enrich for PAOs. After achieving a stable culture, carbon source was switched to uniformly ¹³C-labeled acetate. Samples were collected at the end of each aerobic phase. EPSs were extracted by alkaline treatment. ¹³C enrichment in proteins and sugars (after hydrolysis of polysaccharides) in the extracted EPS were measured by mass spectrometry. The average turnover rate of sugars and proteins (0.167 and 0.192 d⁻¹ respectively) was higher than the expected value based on the solid removal rate (0.132 d⁻¹), and no significant difference was observed between intracellular and extracellular proteins. This indicates that EPS from the PAO enriched community is not selectively degraded by flanking populations under stable EBPR process conditions. Instead, we observed general decay of biomass, which corresponds to a value of 0.048 d⁻¹. Key Points: • Proteins showed a higher turnover rate than carbohydrates. • Turnover of EPS was similar to the turnover of intracellular proteins. • EPS is not preferentially consumed by flanking populations.

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The majority of bacteria grow in the form of microbial aggregates known as biofilms. In these biofilms, microorganisms are embedded in a mixture of extracellular polymeric substances (EPS) produced by the microorganisms themselves. EPS is a complex mixture of biopolymers of different nature, such as polysaccharides, proteins, nucleic acids or lipids, among others. In spite of the significant progress over the last decades, EPS is still a black box waiting to be opened, in terms of specific composition, function, structure and production. Biofilms have great importance in many environmental engineering processes, as for example, aerobic granular sludge (AGS). AGS is a novel biological wastewater treatment where microorganisms are stimulated to form compact granules. Among the complex microbial community in AGS, polyphosphate accumulating organisms (PAOs) are of great importance, due to their role in phosphate removal and granule stabilization. Because of their dominance in AGS and their rapid anaerobic carbon sequestration, they are assumed to be the main EPS producer in AGS. Therefore, PAOs (specifically the well-studied “Candidatus Accumulibacter phosphatis”) can be used as model microorganism for the study of EPS of AGS. The goal of this thesis is to study the EPS of “Ca. Accumulibacter” in terms of specific composition, application and synthesis/consumption. A better characterization of the EPS of “Ca. Accumulibacter” will lead to a comprehensive understanding of this microorganism and further optimization of the granular sludge processes, and their application... @en

Abstract: Pseudaminic and legionaminic acids are a subgroup of nonulosonic acids (NulOs) unique to bacterial species. There is a lack of advances in the study of these NulOs due to their complex synthesis and production. Recently, it was seen that “Candidatus Accumulibacter” can produce Pse or Leg analogues as part of its extracellular polymeric substances (EPS). In order to employ a “Ca. Accumulibacter” enrichment as production platform for bacterial sialic acids, it is necessary to determine which fractions of the EPS of “Ca. Accumulibacter” contain NulOs and how to enrich and/or isolate them. We extracted the EPS from granules enriched with “Ca. Accumulibcater” and used size-exclusion chromatography (SEC) to separate them into different molecular weight (MW) fractions. This separation resulted in two high molecular weight (> 5500 kDa) fractions dominated by polysaccharides, with a NulO content up to 4 times higher than the extracted EPS. This suggests that NulOs in “Ca. Accumulibacter” are likely located in high molecular weight polysaccharides. Additionally, it was seen that the extracted EPS and the NulO-rich fractions can bind and neutralize histones. This opens the possibility of EPS and NulO-rich fractions as potential source for sepsis treatment drugs. Key points: • NulOs in “Ca. Accumulibacter” are likely located in high MW polysaccharides • SEC allows to obtain high MW polysaccharide-rich fractions enriched with NulOs • EPS and the NulOs-rich fractions are a potential source for sepsis treatment drugs.

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Sialic acids are a family of nine-carbon negatively charged carbohydrates. In animals, they are abundant on mucosa surfaces as terminal carbohydrates of mucin glycoproteins. Some commensal and pathogenic bacteria are able to release, take up and catabolize sialic acids. Recently, sialic acids have been discovered to be widespread among most microorganisms. Although the catabolism of sialic acids has been intensively investigated in the field of host-microbe interactions, very limited information is available on microbial degradation of sialic acids produced by environmental microorganisms. In this study, the catabolic pathways of sialic acids within a microbial community dominated by 'Candidatus Accumulibacter' were evaluated. Protein alignment tools were used to detect the presence of the different proteins involved in the utilization of sialic acids in the flanking populations detected by 16S rRNA gene amplicon sequencing. The results showed the ability of Clostridium to release sialic acids from the glycan chains by the action of a sialidase. Clostridium and Chryseobacterium can take up free sialic acids and utilize them as nutrient. Interestingly, these results display similarities with the catabolism of sialic acids by the gut microbiota. This study points at the importance of sialic acids in environmental communities in the absence of eukaryotic hosts.

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Nonulosonic acids (NulOs) are a family of acidic carbohydrates with a nine-carbon backbone, which include different related structures, such as sialic acids. They have mainly been studied for their relevance in animal cells and pathogenic bacteria. Recently, sialic acids have been discovered as an important compound in the extracellular matrix of virtually all microbial life and in “Candidatus Accumulibacter phosphatis”, a well-studied polyphosphate-accumulating organism, in particular. Here, bioaggregates highly enriched with these bacteria (approx. 95% based on proteomic data) were used to study the production of NulOs in an enrichment of this microorganism. Fluorescence lectin-binding analysis, enzymatic quantification, and mass spectrometry were used to analyze the different NulOs present, showing a wide distribution and variety of these carbohydrates, such as sialic acids and bacterial NulOs, in the bioaggregates. Phylogenetic analysis confirmed the potential of “Ca. Accumulibacter” to produce different types of NulOs. Proteomic analysis showed the ability of “Ca. Accumulibacter” to reutilize and reincorporate these carbohydrates. This investigation points out the importance of diverse NulOs in non-pathogenic bacteria, which are normally overlooked. Sialic acids and other NulOs should be further investigated for their role in the ecology of “Ca. Accumulibacter” in particular, and biofilms in general. Key Points: •“Ca. Accumulibacter” has the potential to produce a range of nonulosonic acids. •Mass spectrometry and lectin binding can reveal the presence and location of nonulosonic acids. •The role of nonulosonic acid in non-pathogenic bacteria needs to be studied in detail.

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