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Studying the bricks of microbial cities: characterization and structural properties of exopolysaccharides and their interaction with proteins and cations in anammox granular sludge

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Stability boosts wastewater treatment strategies

European policy objectives emphasise the need to increase the energy efficiency in industrial processes and to reuse waste materials. This requires a better understanding of the mechanisms in these biotechnology approaches.

Climate Change and Environment

Strategies for biological wastewater treatment employ microorganisms, which use pollutants as substrates and gain energy for growth from their transformation into harmless products. In particular, anaerobic ammonium oxidation (anammox) bacteria possess the unique ability to combine ammonium and nitrite to form nitrogen gas. This property has been exploited for the removal of nitrogen from wastewater with high ammonium concentrations. Due to the slow growth rate of anammox bacteria, efficient biomass retention is essential for reactor operation. The formation of large granular microbial aggregates characterised by excellent settling properties enhance reactor performance and contribute to stability of anammox technologies. Scientists with the EU-funded POLIS project wished to investigate the process of granulation and identify parameters that affect the formation of anammox granular sludge. “Our goal was to decipher the mechanism required for the mechanical stability of anammox bacteria, a prerequisite for the treatment of municipal wastewaters”, explains project coordinator Prof. Malpei. Anammox bacterial processes Granular stability is closely related to the properties of extracellular polymeric substances (EPS) secreted by anammox bacteria. POLIS researchers employed various methods including mass spectrometry and liquid chromatography to identify EPS structures and unravel the mechanisms central for granule stability. To investigate the interaction of EPS main components, proteins and polysaccharides, and cations, modern rheological techniques were combined with light scattering and microscopy. Following extraction, EPS analysis revealed the presence of proteins, polysaccharides and lipids as well as nucleic acids. Overall, 251 proteins were identified most of which were an integral part of the bacterial cell membrane. From a structural viewpoint, “the identification of EPS as amyloid-like fibrils will help improve anammox granular sludge formation and stability, key factors in current applications,″ continues Dr Tommaso Lotti, the principal investigator who was granted the prestigious Marie S.-Curie Actions Individual Fellowship. Interestingly, the EPS extracted from anammox granular sludge was used to form a viscoelastic biomaterial that could be applied in other industrial sectors. Physico-chemical characterisation of the recovered biomaterial determined its rheological properties as a function of the EPS concentration. Overall, the insight into EPS composition and function in anammox granules will contribute towards the innovative implementation of anammox-based processes in municipal treatment plants and the reuse of waste granular sludge generated. Promoting a circular economy In anammox applications, the biomass produced comprises a mixture of bacteria and EPS secreted by the bacteria themselves. Currently, this sludge is considered as a waste product and the related costs of disposal represents up to 50 % of the wastewater treatment operative costs. The POLIS project findings suggest that the recovery of EPS-based biomaterial can be applied in other industrial sectors, substantially increasing the sustainability and economics of wastewater treatment. As Dr Tommaso Lotti states, “this approach would promote the development of a circular economy contributing to the transformation of the conventional wastewater treatment plant into an efficient biorefinery.″ POLIS results on anammox EPS extend to bacterial biofilm formation and increase their scientific significance as they could be beneficial to other fields such as biofouling and biocorrosion. Prof. Malpei envisions that POLIS findings “will be utilised by existing companies and water administrations to direct more efficiently the research and development of future biofilm-based and anammox-based processes.″


POLIS, extracellular polymeric substances (EPS), wastewater, bacteria, anaerobic ammonium oxidation (anammox), biofilm, granular sludge

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