Aerobic sludge granulation: A tale of two polysaccharides?

Abstract

Aerobic sludge granules are suspended biofilms with the potential to reduce the cost and footprint of secondary wastewater treatment. Attempts to answer how and why they form leads to a consideration of the role of their extracellular polymeric substances (EPS) in determining their physical and microbiological properties. The exopolysaccharide components of this matrix, in particular, have received attention as putative structural, gel-forming agents. Two quite different exopolysaccharides have been proposed as the gel-forming constituents, with their gel properties clearly different from those of activated sludge EPS. This review aims to address the question of whether more than one gel-forming exopolysaccharide exist in granules. Based on the available structural data, it seems likely that they are different gel-forming polymers and their differences are not artifacts of the analytical methods used. Nonetheless, both proposed structural gel polymers are extracted and purified based on procedures selecting for anionic polar polysaccharides soluble at high pH, and both contain hexuronic acids. Granulation does not result from EPS synthesis by any single microbial population, nor from production of a single exopolysaccharide. Future studies using solvents suitable for recalcitrant polysaccharides are likely to reveal important structural roles for other polysaccharides. It is hoped that this article will serve as a guide for subsequent studies into understanding the roles of exopolysaccharides in aerobic granular sludge.

Introduction

The application of aerobic sludge granules in wastewater treatment processes promises high rates of nutrient and organic carbon removal and a rapid separation of biomass and effluent liquid phases (de Kreuk et al., 2005; Liu and Tay, 2004). Granules seem to be an attractive, low-cost and low-footprint alternative to the now almost a century old flocculated sludge processes (Adav et al., 2008). Aerobic granular sludge has attracted considerable interest in recent years from research groups and design engineers alike (Bruin et al., 2004), and it is expected that the number of installations exploiting it will increase rapidly. Reluctance to accept this technology is largely from perceptions of low granule stability and long start-up times (Lee et al., 2010; Pijuan et al., 2011), notwithstanding that several large-scale reactors have now been operated in Europe and Africa where stable granule population has been maintained and fully compliant effluent levels achieved (www.dhv.com/nereda).

Flocculent sludge is believed to be the precursor of aerobic sludge granules, and fears of low granule stability reflect their reported propensity to revert to a floccular structure (Barr et al., 2010; Lee et al., 2010). It is important therefore to understand how and why these two microbial aggregates differ. Effort has been directed at comparing the physical and chemical attributes of their matrices, both of which consist of largely chemically undefined extracellular polymeric substances (EPS), to find the answer. Differences in the concentrations and distribution of proteins and polysaccharides, and levels of hydrophobicity have been cited as important (McSwain et al., 2005). A popular explanation has been that granular EPS is more adhesive than floccular EPS. Seviour et al. (2009a) provided a theoretical basis for this by demonstrating, with rheological methods, that granules could be distinguished from flocs by their gel-forming EPS, an important feature that explains the different macrostructures of granules and flocs. Hence, in granules the EPS acts as a real structural gel while with flocs the EPS is more like a paste. Seeking the chemical basis for such an important physical distinction is of considerable importance.

There seems to be a general agreement that polysaccharides are the major gel-forming constituents of the granular EPS (Lin et al., 2010a; Seviour et al., 2009a). Thus, the gel-forming characteristic of their EPS that distinguishes granules from flocs was attributed to exopolysaccharides. Subsequent characterization of gel-forming exopolysaccharides has led to an apparent and seemingly substantial discrepancy of the gel-forming exopolysaccharide components identified by two independent research groups. Lin et al. (2010a) claimed that in aerobic sludge granules it is alginate-like, while Seviour et al. (2010b) described it as a complex and highly novel heteropolysaccharide previously unreported in the literature. They named it Granulan (Seviour et al., 2011).

Several questions arise. Why have two very different polymers been proposed for the gel-forming matrix constituent? Has one or both been incorrectly identified? Are these the only EPS of importance in the matrix of aerobic sludge granules? This article attempts to answer these questions, by reviewing and analyzing published data related to the structural elucidation of these two exopolysaccharides in the context of what is known about other well-characterized bacterial exopolysaccharides. Specific reference will be made to their isolation, and their functional and structural characterization. It is hoped that this article will serve as a guide for subsequent studies into understanding the roles of exopolysaccharides in aerobic granular sludge and how this information can be exploited to improve granule formation and stability.