In biofilms, microorganisms are embedded in a hydrated matrix that provides a stable structure and protection against influences fromthe environment. Thismatrix is formed by extracellular polymeric substances (EPS) that are produced by the microorganisms of the biofilm. A major part of the microorganisms in nature lives in aggregated forms like biofilms. Yet, knowledge about biofilm formation, composition and structure is limited. A specific formof biofilm is granular sludge. A granule is a spherical biofilmthat is not attached to a surface or carrier. In wastewater treatment, granular sludge systems are used for efficient wastewater treatment. Due to the high settling velocity of granules, granular sludge-based plants can be built smaller, compared to conventional plants (with flocculent sludge). Anaerobic ammonium oxidizing (anammox) bacteria are applied in granular sludge systems in wastewater treatment. Anammox bacteria are important players in the nitrogen cycle in wastewater treatment, as well as in the natural environment. Although the formation of granular sludge is not completely understood, EPS are the key components in the formation of the matrix that provides a stable structure wherein the bacteria are embedded. The aim of this thesis was to characterize the EPS composition of anammox granular sludge. @en

Anaerobic ammonium oxidation (anammox)-performing bacteria self-assemble into compact biofilms by expressing extracellular polymeric substances (EPS). Anammox EPS are poorly characterized, largely due to their low solubility in typical aqueous solvents. Pronase digestion achieved 19.5 ± 0.9 and 41.4 ± 1.4% (w/w) more solubilization of laboratory enriched Candidatus Brocadia sinica anammox granules than DNase and amylase, respectively. Nuclear magnetic resonance profiling of the granules confirmed proteins as dominant biopolymer within the EPS. Ionic liquid (IL) 1-ethyl-3-methylimidazolium acetate and N,N-dimethylacetamide (EMIM-Ac/DMAc) mixture was applied to extract the major structural proteins. Further treatment by anion exchange chromatography isolated homologous serine (S)- and threonine (T)-rich proteins BROSI_A1236 and UZ01_01563, which were major components of the extracted proteins, and sequentially highly similar to putative anammox extracellular proteins KUSTD1514 and WP_070066018.1 of Ca. Kuenenia stuttgartiensis and Ca. Brocadia sapporoensis, respectively. Six monosaccharides (i.e., arabinose, xylose, rhamnose, fucose, galactose, and mannose) were enriched for BROSI_A1236 against all other major proteins. The sugars, however, contributed < 0.5% (w/w) of total granular biomass and were likely co-enriched as glycoprotein appendages. This study demonstrates that BROSI_A1236 is a major extracellular component of Ca. B. sinica anammox biofilms that is likely a common anammox extracellular polymer, and can be isolated from the matrix following ionic liquid extraction.

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Anammox (anaerobic ammonium oxidation) bacteria are important for the nitrogen cycle in both natural environments and wastewater treatment plants. These bacteria have a strong tendency to grow in aggregates like biofilms and granular sludge. To understand the formation of anammox aggregates, it is required to unravel the composition of the extracellular polymeric substances (EPS), which are produced by the bacteria to develop into aggregates and granules. Here, we investigated anionic polymers in anammox granular sludge, focussing on sialic acids and sulfated glycosaminoglycans. Quantification assays and fluorescent stains indicated that sialic acids and sulfated glycosaminoglycans were present in the anammox EPS (1.6% equivalents of sialic acids and 2.4% equivalents of sulfated glycosaminoglycans). Additionally, the potential genes for the biosynthesis of sialic acids and sulfated glycosaminoglycans were analyzed in the anammox draft genomes. The finding of these components in anammox granular sludge and previously in other nonpathogenic bacteria pointed out that sialic acids and sulfated glycosaminoglycans are worth investigating in the context of a broader function in microbial communities and biofilm systems in general.

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Elucidating the extracellular polymeric substances (EPS) of anammox granular sludge is important for stable nitrogen removal processes in wastewater treatment. However, due to a lack of standardized methods for extraction and characterization, the composition of anammox granule EPS remains mostly unknown. In this study, alkaline (NaOH) and ionic liquid (IL) extractions were compared in terms of the proteins they extracted from different “Candidatus Brocadia” cultures. We aimed to identify structural proteins and evaluated to which extend these extraction methods bias the outcome of EPS characterization. Extraction was focussed on solubilization of the EPS matrix, and the NaOH and IL extraction recovered on average 20% and 26% of the VSS, respectively. Using two extraction methods targeting different intermolecular interactions increased the possibility of identifying structural extracellular proteins. Of the extracted proteins, ∼40% were common between the extraction methods. The high number of common abundant proteins between the extraction methods, illustrated how extraction biases can be reduced when solubility of the granular sludge is enhanced. Physicochemical analyses of the granules indicated that extracellular structural matrix proteins likely have β-sheet dominated secondary structures. These β-sheet structures were measured in EPS extracted with both methods. The high number of uncharacterized proteins and possible moonlighting proteins confounded identifying structural (i.e. β-sheet dominant) proteins. Nonetheless, new candidates for structural matrix proteins are described. Further current bottlenecks in assigning specific proteins to key extracellular functions in anammox granular sludge are discussed.

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ANaerobic AMMonium OXidation (anammox) is an established process for efficient nitrogen removal from wastewater, relying on anammox bacteria to form stable biofilms or granules. To understand the formation, structure, and stability of anammox granules, it is important to determine the composition of the extracellular polymeric substances (EPS). The aim of this research was to elucidate the nature of the proteins, which are the major fraction of the EPS and were suspected to be glycosylated. EPS were extracted from full-scale anammox granular sludge, dominated by "Candidatus Brocadia", and subjected to denaturing polyacrylamide gel electrophoresis. By further analysis with mass spectrometry, a high abundant glycoprotein, carrying a heterogeneous O-glycan structure, was identified. The potential glycosylation sequence motif was identical to that proposed for the surface layer protein of "Candidatus Kuenenia stuttgartiensis". The heavily glycosylated protein forms a large fraction of the EPS and was also located by lectin staining. Therefore, we hypothesize an important role of glycoproteins in the structuring of anammox granules, comparable to the importance of glycans in the extracellular matrix of multicellular organisms. Furthermore, different glycoconjugates may have distinct roles in the matrix of granular sludge, which requires more in-depth characterization of different glycoconjugates in future EPS studies.

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Denitrification and dissimilatory nitrate reduction to ammonium (DNRA) compete for nitrate in natural and engineered environments. A known important factor in this microbial competition is the ratio of available electron donor and elector acceptor, here expressed as Ac/N ratio (acetate/nitrate-nitrogen). We studied the impact of the Ac/N ratio on the nitrate reduction pathways in chemostat enrichment cultures, grown on acetate mineral medium. Stepwise, conditions were changed from nitrate limitation to nitrate excess in the system by applying a variable Ac/N ratio in the feed. We observed a clear correlation between Ac/N ratio and DNRA activity and the DNRA population in our reactor. The DNRA bacteria dominated under nitrate limiting conditions in the reactor and were outcompeted by denitrifiers under limitation of acetate. Interestingly, in a broad range of Ac/N ratios a dual limitation of acetate and nitrate occurred with co-occurrence of DNRA bacteria and denitrifiers. To explain these observations, the system was described using a kinetic model. The model illustrates that the Ac/N effect and concomitant broad dual limitation range related to the difference in stoichiometry between both processes, as well as the differences in electron donor and acceptor affinities. Population analysis showed that the presumed DRNA-performing bacteria were the same under nitrate limitation and under dual limiting conditions, whereas the presumed denitrifying population changed under single and dual limitation conditions.@en

Anammox (anaerobic ammonium oxidation) bacteria belong to the phylum of Pianctomycetes. They are able to convert ammonium and nitrite to nitrogen gas. Anammox bacteria have a strong tendency to grow in aggregates. To have a better understanding of the formation of aggregates like flocs and granules, it is important to elucidate the composition of extracellular polymeric substances (EPS) However, due to the limitation in the present method ologies of extraction and characterization, the EPS of anammox granules are still a black box. The aim of this study was to explore the EPS of anammox granules and to get more insights into the structure of the EPS matrix An alkaline extraction was used to extract EPS from the granules. To get a high extraction yield while avoiding protein hydrolysis, different concentrations of NaoH were tested. The extracted EPS were characterized by elemental composition analysis and by quantification of the proteins and polysaccharides with the BCA and phenol sulphuric acid assay, respectively, The proteins were further analysed using SDS-PAGE. Besides Coomassie blue staining for the detection of proteins, periodic acid-Schiff (PAS) and Alcian blue staining were applied to detect respectively neutral and acidic glycoproteins. The monomer composition of polysaccharides was determined using high-perfor mance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD). The mechanical properties of the extracted EPs were investigated by rheological characterization. The results showed that extraction with 0,1 M NaOH for 5 hours gives a relatively high yield of 205,21 mg/g VSS of which the main part is proteins. Using SDS-PAGE a glycoprotein of 8o kDa was found to be a dominant protein. Different monomers of sugars were detected by HPAEC-PAD. The rheology measurements showed that the EPS form a gel at pH 8 and 25 degrees. The strength of the gel increased with multiple frequency sweeps. This research provides first insights into the investigation of glycoproteins in EPS of anammox granules, and the gel forming property of the EPS. The gel-forming property of EPS is important since it contributes to a gel matrix in granular sludge for anammox bacteria to be imbedded in. Furthermore, the finding of a dominant glycoprotein in the EPs is a very interesting starting point to study the EPS composition. Future research will focus on identifying if the glycoprotein of 8o kDa is a surface layer protein of anammox bacteria, and its function in anammox granule formation.@en