Decorating the Anammox House: Sialic Acids and Sulfated Glycosaminoglycans in the Extracellular Polymeric Substances of Anammox Granular Sludge.

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.

Extracellular protein isolation from the matrix of anammox biofilm using ionic liquid extraction.

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.

Identification of Glycoproteins Isolated from Extracellular Polymeric Substances of Full-Scale Anammox Granular Sludge.

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.

Purification of Biotinylated Proteins Using Single Walled Carbon Nanotube-Streptavidin Complexes.

In this study, Single walled carbon nanotube (SWNT)-streptavidin complexes were used to capture and purify biotinylated proteins, including bio-GFP and bio-DBS using a pull-down method. The purification conditions were systematically studied, including surface blocking of SWNT using chicken egg albumin (CEA), the ratio of SWNT-streptavidin complexes to the cell lysate, as well as the centrifugation speed. Optimization of the protein purification using SWNT-streptavidin complexes shows the possibility of carbon nanotubes as a promising candidate for protein purification applications. The SWNT-streptavidin could be used as a scaffold to analyze protein structure directly by cryo-transmission electron microscopy, which provides better understanding in protein­protein interactions and biological processes.

Solubilization and characterization of extracellular proteins from anammox granular sludge.

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 ß-sheet dominated secondary structures. These ß-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. ß-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.

DNRA and Denitrification Coexist over a Broad Range of Acetate/N-NO3- Ratios, in a Chemostat Enrichment Culture.

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.