Aerobic Sludge Granules: Macromolecular Associations in Extracellular Polymeric Substances

Thomas Seviour (2010). Aerobic Sludge Granules: Macromolecular Associations in Extracellular Polymeric Substances PhD Thesis, School of Chemical Engineering, The University of Queensland.

       
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Author Thomas Seviour
Thesis Title Aerobic Sludge Granules: Macromolecular Associations in Extracellular Polymeric Substances
School, Centre or Institute School of Chemical Engineering
Institution The University of Queensland
Publication date 2010-10
Thesis type PhD Thesis
Supervisor Prof Zhiguo Yuan
Dr Maite Pijuan
Prof Jurg Keller
Total pages 212
Total colour pages 10
Total black and white pages 202
Subjects 09 Engineering
Abstract/Summary The application of aerobic sludge granules in secondary activated sludge wastewater treatment processes has the potential to cope with substantially higher hydraulic and organic loading rates compared to the floccular aggregates used in conventional systems. However, very few aerobic full-scale systems use sludge granules globally. This probably reflects our current understanding of why, how and under which conditions granules form. This thesis aims to contribute to this understanding by investigating the extracellular polymeric substance (EPS) structure of aerobic granules. To achieve this it was important to understand what aerobic sludge granules are. The rheological profile of aerobic sludge granules performing biological removal of organic carbon, nitrogen and phosphorus from abattoir wastewater was indicative of a viscoelastic material exhibiting shear thinning behaviour. Granule strength depended on several environmental factors including temperature, pH and ionic strength, and gel-sol transitions were observed at pH 9-12 and 47 °C. The granules could be disrupted by exposure to proteolytic and amylolytic enzymes suggesting they are protein-polysaccharide composite physical hydrogels. A reversible, pH dependent sol-gel transition was observed subsequently with the extracellular polymeric substances (EPS) extracted from these granules, a behaviour analogous to that observed with the granules. This characteristic transition was not observed with any EPS samples extracted from floccular sludges. The granules are therefore distinguished from flocs by their synthesis of a gel-forming EPS. Granular EPS was found by analytical gel permeation chromatography (GPC) to comprise three components: high-molecular-weight (MW) exopolysaccharide, medium-MW proteins and glycosides, and low-MW proteins and glycosides. After this high-MW exopolysaccharide fraction was enriched by fractional precipitation, it possessed a critical overlap concentration (c* = 0.35 % w/w) consistent with the gel-forming constituent being polysaccharide not proteinaceous in nature. Following further purification by preparatory-scale GPC, Atomic Force Microscopy and rheological methods revealed it to have gel-like associative behaviour analogous to that of the granules. The low and medium MW material removed by GPC did not. Such behaviour indicates a structural role, and so it seems likely that the property of granular EPS to form gels appears to be attributable solely to the expression of this high-MW exopolysaccharide. 1D and 2D NMR spectral analyses could show that this gel-forming exopolysaccharide consisted of eight sugar residues. Carbon and proton shifts were assigned for each of these. Heteronuclear Multiple Bond Correlation (HMBC) and Nuclear Overhauser Enhancement Spectroscopy (NOESY) identified the linkage types between individual residues. Together these techniques showed this exopolysaccharide to be a complex single heteropolysaccharide with a repeat sequence of α-galactose, β-mannose, β-glucosamine, N-acetyl-β-galactosamine and 2-acetoamido-2-deoxy-α-galactopyranuronic acid. It possesses a disaccharide branch of β-galactose and β-glucuronic acid attached to 2-acetoamido-2-deoxy-α-galactopyranuronic acid and an α-rhamnose branch attached to α-galactose. To establish whether this exopolysaccharide was expressed in all granule types or restricted to particular groups of bacteria, granules were cultivated under different operating conditions. Candidatus "Competibacter phosphatis‟ and Candidatus "Accumulibacter phosphatis‟ had been identified by FISH as codominant populations in the original granules performing nutrient removal from abattoir wastewater. Granules generated under conditions unfavourable to Accumulibacter and Competibacter (i.e. synthetic feed with methanol and nitrate as sole carbon source and electron acceptor respectively) did not contain this gel-forming exopolysaccharide. It was also absent from Accumulibacter enriched granules. However, those granules enriched in Competibacter from a seed sludge consisting of the original granules (i.e. treating abattoir wastewater) continued to synthesise this exopolysaccharide. Similarly, granules formed from a floccular seed sludge induced the synthesis of the gel-forming exopolysaccharide under conditions of Competibacter enrichment, coincident with an increase in particle size and the onset of granulation. Although these results suggest this gel-forming exopolysaccharide is not synthesised by all granule communities it is not restricted to a single granule type and so may serve an important structural role in other granules forming under alternating anaerobic and aerobic conditions. The approach used in this study could be extended profitably to understanding the structure and role of individual macromolecules in other microbial aggregates.
Keyword activated sudge
aerobic granules
wastewater
gels
rheology
NMR
structure
hydrogels
biofilm
exopolysaccharides
Additional Notes 18, 63, 70, 142, 143, 144, 147, 148, 209, 210

 
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Created: Mon, 09 May 2011, 10:49:42 EST by Mr Thomas Seviour on behalf of Library - Information Access Service