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.

With the rapid growing availability of metagenome assembled genomes (MAGs) and associated metabolic models, the identification of metabolic potential in individual community members has become possible. However, the field still lacks an unbiassed systematic evaluation of the generated metagenomic information to uncover not only metabolic potential, but also feasibilities of these models under specific environmental conditions. In this study, we present a systematic analysis of the metabolic potential in species of "Candidatus Accumulibacter", a group of polyphosphate-accumulating organisms (PAOs). We constructed a metabolic model of the central carbon metabolism and compared the metabolic potential among available MAGs for “Ca. Accumulibacter” species. By combining Elementary Flux Modes Analysis (EFMA) with max-min driving force (MDF) optimization, we obtained all possible flux distributions of the metabolic network and calculated their individual thermodynamic feasibility. Our findings reveal significant variations in the metabolic potential among “Ca. Accumulibacter” MAGs, particularly in the presence of anaplerotic reactions. EFMA revealed 700 unique flux distributions in the complete metabolic model that enable the anaerobic uptake of acetate and its conversion into polyhydroxyalkanoates (PHAs), a well-known phenotype of “Ca. Accumulibacter”. However, thermodynamic constraints narrowed down this solution space to 146 models that were stoichiometrically and thermodynamically feasible (MDF > 0 kJ/mol), of which only 8 were strongly feasible (MDF > 7 kJ/mol). Notably, several novel flux distributions for the metabolic model were identified, suggesting putative, yet unreported, functions within the PAO communities. Overall, this work provides valuable insights into the metabolic variability among "Ca. Accumulibacter" species and redefines the anaerobic metabolic potential in the context of phosphate removal. More generally, the integrated workflow presented in this paper can be applied to any metabolic model obtained from a MAG generated from microbial communities to objectively narrow the expected phenotypes from community members.

The understanding of microbial communities and the biological regulation of its members is crucial for implementation of novel technologies using microbial ecology. One poorly understood metabolic principle of microbial communities is resource allocation and biosynthesis. Resource allocation theory in polyphosphate accumulating organisms (PAOs) is limited as a result of their slow imposed growth rate (typical sludge retention times of at least 4 days) and limitations to quantify changes in biomass components over a 6 hours cycle (less than 10% of their growth). As a result, there is no direct evidence supporting that biosynthesis is an exclusive aerobic process in PAOs that alternate continuously between anaerobic and aerobic phases. Here, we apply resource allocation metabolic flux analysis to study the optimal phenotype of PAOs over a temperature range of 4 °C to 20 °C. The model applied in this research allowed to identify optimal metabolic strategies in a core metabolic model with limited constraints based on biological principles. The addition of a constraint limiting biomass synthesis to be an exclusive aerobic process changed the metabolic behaviour and improved the predictability of the model over the studied temperature range by closing the gap between prediction and experimental findings. The results validate the assumption of limited anaerobic biosynthesis in PAOs, specifically “Candidatus Accumulibacter” related species. Interestingly, the predicted growth yield was lower, suggesting that there are mechanistic barriers for anaerobic growth not yet understood nor reflected in the current models of PAOs. Moreover, we identified strategies of resource allocation applied by PAOs at different temperatures as a result of the decreased catalytic efficiencies of their biochemical reactions. Understanding resource allocation is paramount in the study of PAOs and their currently unknown complex metabolic regulation, and metabolic modelling based on biological first principles provides a useful tool to develop a mechanistic understanding.