Structural Determination of a Key Exopolysaccharide in Mixed Culture Aerobic Sludge Granules Using NMR Spectroscopy

Abstract

Nuclear magnetic resonance (NMR) techniques were used to elucidate the structure of an exopolysaccharide material previously revealed to be important in formation of aerobic granules. The 1D NMR spectral data acquired showed that this gel-forming polysaccharide was a major component of granular EPS, while 1D and 2D NMR spectra showed it consisted of eight sugar residues. These were assigned as α-galactose, α-rhamnose, 2-acetoamido-2-deoxy-α-galactopyranuronic acid, β-mannose, β-galactose, β-glucuronate, β-glucosamine, and N-acetyl β-galactosamine. With the exception of 2-acetoamido-2-deoxy-α-galactopyranuronic acid, a highly unusual sugar, their presence was confirmed with high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD). Carbon and proton shifts were assigned for each sugar. Heteronuclear multiple bond correlation (HMBC) and nuclear Overhauser enhancement spectroscopy (NOESY) were used to identify linkage sites between individual sugar residues. This gel-forming exopolysaccharide appeared to be a highly 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 has 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.