Multiple EPS interactions involved in the cohesion and structure of aerobic granules.

This study aims to clarify the biochemical nature and interactions of Extracellular Polymeric Substances (EPS) involved in the structure and cohesive properties of aerobic granules. Granules were incubated with selective hydrolytic enzymes or with chemicals and the resistance of digested granules to shear stress was evaluated. After α-amylase digestion, the hydrodynamic stress released macro-particles (>315 µm) while soluble molecules (<1.5 µm) and micro-particles (1.5-315 µm) where mainly recovered after savinase and EDTA treatments. These data show that α (1-4) glucans and proteins are key polymers for granule cohesion and that divalent cationic bridging is a major aggregative mechanism. On the basis of these experiments and microscopy observations, a model is proposed for the spatial organization of EPS in the granular structure, in which α glucans are arranged in a capsular layer surrounding bacterial clusters while anionic proteins constitute the intercellular cement that may reinforce cohesion inside the bacterial clusters.

Extracellular polymeric substances (EPS) from aerobic granular sludges: extraction, fractionation, and anionic properties.

A multi-method protocol previously proposed for the extraction of extracellular polymeric substances (EPS) from flocculated sludges was investigated on dense aerobic granules. The protocol combines mechanical disruption by sonication and chemical extraction using the Tween detergent and the cation chelator, EDTA. Polysaccharides were mainly recovered during the first sonication step while proteins were recovered all along the extractive procedure with a high prevalence in the EDTA step. These data confirmed the interest of the multi-method protocol for harvesting a diversified pool of EPS from dense granules and for fractionation of the polymers according to their physicochemical properties. In addition, the high extractability of proteins with EDTA confers a specific behavior of the aerobic granules towards the multi-method extraction protocol, supporting the idea that proteins are associated in the granule matrix through ionic interactions involving divalent cations. Analysis of the extracted EPS by anionic exchange chromatography confirmed the presence of highly anionic proteins that were specifically detected in the extracts obtained from granules. One important question is now to investigate whether these highly anionic proteins are involved in the aggregation and densification process and if their presence is related to the cohesive properties of these particles.