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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.

Glycosaminoglycans (GAGs) are linear heteropolysaccharides containing a derivative of an amino sugar. The possibility of the presence of GAGs in aerobic granular sludge was studied by combining SDS-PAGE with Alcian Blue staining (at pH 2.5 and 1), FTIR, mammalian Hyaluronic acid and sulfated GAG analysis kits, enzymatic digestion and specific in situ visualization by Heparin Red and lectin staining. GAGs, including Hyaluronic acid-like and sulfated GAGs-like polymers were found in aerobic granular sludge. The sulfated GAGs-like polymers contained Chondroitin sulfate and Heparan sulfate/Heparin based on their sensitivity to the digestion by Chondroitinase ABC and Heparinase I & III. Heparin Red and lectin staining demonstrated that, the sulfated GAGs-like polymers were not only present in the extracellular matrix, but also filled in the space between the cells inside the microcolonies. The GAGs-like polymers in aerobic granules were different from those produced by pathogenic bacteria but resemble those produced by vertebrates. Findings reported here and in previous studies on granular sludge described in literature indicate that GAGs-like polymers might be widespread in granular sludge/biofilm and contribute to the stability of these systems. The extracellular polymeric substances (EPS) in granular sludge/biofilm are far more complicated than they are currently appreciated. Integrated and multidisciplinary analyses are significantly required to study the EPS., (Copyright © 2019 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Sialic acids have been discovered in the extracellular polymeric substances (EPS) of seawater-adapted aerobic granular sludge (AGS). Sialic acids are a group of monosaccharides with a nine-carbon backbone, commonly found in mammalian cells and pathogenic bacteria, and frequently described to protect EPS molecules and cells from attack by proteases or glycosidases. In order to further understand the role of these compounds in AGS, lectin staining, genome analysis of the dominant bacterial species, and shielding tests were done. Fluorescence lectin bar-coding (FLBC) analysis showed an overlap with protein staining, indicating presence of sialoglycoproteins in the EPS matrix. Genome analysis gives a positive indication for putative production of sialic acids by the dominant bacteria Candidatus Accumulibacter. FT-IR analysis shows upon selective removal of sialic acids a decrease in carbohydrates, extension of the protein side chain, and exposure of penultimate sugars. Enzymatic removal of sialic acids results in the removal of galactose residues from the EPS upon subsequent treatment with β-galactosidase, indicating a linkage between galactose and sialic acid at the terminus of glycan chains. This work indicates the importance of sialic acids in the protection of penultimate sugar residues of glycoproteins in EPS, and provides basis for future research in the composition of EPS from biofilms and granular sludge., (Copyright © 2019 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

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.

A new acid soluble extracellular polymeric substance (acid soluble EPS) was extracted from an acetate fed aerobic granular sludge reactor operated at 35 °C. Acid soluble EPS dominated granules exhibited a remarkable and distinctive tangled tubular morphology. These granules are dominated by Defluviicoccus Cluster II organisms. Acetic acid instead of the usually required alkaline extraction medium was needed to dissolve the granules and solubilise the polymeric matrix. The extracted acid soluble EPS was analysed and identified using various instrumental analysis including 1 H and 13 C Nuclear Magnetic Resonance, Fourier Transform Infrared Spectroscopy and Raman spectroscopy. In addition, the glycoconjugates were characterized by fluorescence lectin-binding analysis. The acid soluble EPS is α-(1 → 4) linked polysaccharide, containing both glucose and galactose as monomers. There are OCH 3 groups connected to the glucose monomer. Transmission and scanning electron microscopy (TEM, SEM) as well as confocal laser scanning microscopy (CLSM) showed that the acid soluble EPS was present as a tightly bound capsular EPS around bacterial cells ordered into a sarcinae-like growth pattern. The special granule morphology is decided by the acid soluble EPS produced by Defluviicoccus Cluster II organisms. This work shows that no single one method can be used to extract all possible extracellular polymeric substances. Results obtained here can support the elucidation of biofilm formation and structure in future research., (Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Magnetic resonance microscopy (MRM) was successfully applied for non-invasive online monitoring of biofilm development, sloughing, and forced detachment. Biofilm cultivation was performed in a tube reactor directly placed in the MRM scanner. Based on the differences in relaxation time of free and bound protons, the distributed water signal was allocated to the bulk and the biofilm phase. The velocity of the flowing water in the tube reactor was measured in all three directions (x, y, and z) at spatial resolutions of 78 microm. From the velocity data, maps of flow gradients (shear rates) were derived. The experiments showed that a more compact biofilm structure is sloughed off in total with nearly no biomass left on the substratum. Continued biofilm cultivation resulted in filamentous biofilm structures, which did not show any sloughing. Experiments at higher Reynolds numbers were performed in order to force biofilm detachment. Continuous measuring of proton velocity and biomass was used to characterize the different stages of biofilm development. The measurements revealed that biofilms are able to resist extremely high local shear stress being raised up to factor of 20 compared to the mean local shear stress acting on the complete biofilm surface. The maximum local shear stress of single biofilm structures exposed to flow was found to be on average seven times higher compared to the mean local shear stress of the entire biofilm surface. MRM was able to visualize and quantify the development of biofilms and interaction of biofilms with the surrounding fluid at the meso-scale. It is suggested that detachment and sloughing depends on both internal and external structural parameters., (2010 Wiley Periodicals, Inc.)

In this study an enrichment culture developed from activated sludge was used to investigate the architecture of fully hydrated multispecies biofilms. The assessment of biofilm structure and volume was carried out using confocal laser scanning microscopy (CLSM). Bacterial cell distribution was determined with the nucleic acid-specific stain SYTO 60, whereas glycoconjugates of extracellular polymeric substances (EPS) were stained with the Alexa-488-labeled lectin of Aleuria aurantia. Digital image analysis was employed for visualization and quantification of three-dimensional CLSM data sets. The specific volumes of the polymeric and cellular biofilm constituents were quantified. In addition, gravimetric measurements were done to determine dry mass and thickness of the biofilms. The data recorded by the CLSM technique and the gravimetric data were then compared. It was shown that the biofilm thicknesses determined with both methods agree well for slow-growing heterotrophic and chemoautotrophic biofilms. In addition, for slow-growing biofilms, the volumes and masses calculated from CLSM and the biomass calculated from gravimetric measurements were also comparable. For fast-growing heterotrophic biofilms cultivated with high glucose concentrations the data sets fit to a lesser degree, but still showed the same common trend. Compared with traditional gravimetric measurements, CLSM allowed differential recording of multiple biofilm parameters with subsequent three-dimensional visualization and quantification. The quantitative three-dimensional results recorded by CLSM are an important basis for understanding, controlling, exploiting, and modeling of biofilms.