<p> Supplementary Information Prepared for Applied Microbiology and Biotechnology</p><p>Selectively inducing the synthesis of a key</p><p> structural exopolysaccharide in aerobic granules</p><p> by enriching for Candidatus ‘Competibacter</p><p> phosphatis’</p><p>Thomas W. Seviour1, Lynette K. Lambert 2, Maite Pijuan3, Zhiguo Yuan1*</p><p>1 The University of Queensland, Advanced Water Management Centre (AWMC), St. Lucia, QLD 4072,</p><p>Australia. </p><p>2 The University of Queensland, Centre for Advanced Imaging, St. Lucia, QLD 4072, Australia.</p><p>3 Catalan Institute for Water Research (ICRA), Technology Park of the University of Girona, 17003, Spain</p><p>Applied Microbiology and Biotechnology</p><p>Preparation date: April 15, 2011</p><p>* Corresponding Author</p><p>Email: [email protected]</p><p>Tel: +61-7-33654730</p><p>FAX: +61-7-33654726</p><p>Number of pages; 7</p><p>Number of figures; 4</p><p>Number of tables; 0 Introduction</p><p>International Union Pure and Applied Chemistry (IUPAC) Name</p><p>(2S,3S,4S,5S,6R)-2-{[(2S,3S,4R,5R,6S)-2-{[(2S,3S,4S,5S,6S)-2-(carbonyl-$l^{1}- oxidanyl)-4-{[(2R,3R,4S,5S,6R)-4-{[(2R,3R,4R,5R,6R)-6-(carbonyl-$l^{1}-oxidanyl)-3,4,5- trihydroxyoxan-2-yl]oxy}-3,5-dihydroxy-6-(hydroxymethyl)oxan-2-yl]oxy}-6-</p><p>{[(2S,3R,4R,5S,6R)-6-{[(2R,3S,4S,5S,6S)-4,5-dihydroxy-2-(hydroxymethyl)-6- methoxyoxan-3-yl]oxy}-4-hydroxy-2-(hydroxymethyl)-5-{[(2R,3S,4R,5R,6S)-3,4,5- trihydroxy-6-methyloxan-2-yl]oxy}oxan-3-yl]oxy}-5-acetamidooxan-3-yl]oxy}-3- acetamido-5-hydroxy-6-(hydroxymethyl)oxan-4-yl]oxy}-4-hydroxy-6-(hydroxymethyl)-5- methoxyoxan-3-aminium</p><p>Materials and Methods</p><p>Reactor 1: Methanol driven denitrification</p><p>A synthetic wastewater feed was used consisting of 91 % w/w solution A and 9 % w/w solution B. Solution A (adapted from Baytshtok et al., 2009) contained 1.348 g/L NaNO3,</p><p>0.222 g/L MgSO4∙7H2O, 0.022 g/L CaCl2∙2H2O, 0.096 g/L K2HPO4 0.157 g/L NH4Cl and</p><p>2.2ml of nutrient solution. Solution B contained, per litre: 8.42 L methanol. The nutrient solution contained 1.5 g/L FeCl3∙6H2O, 0.15 g/L H3BO3, 0.03 g/L CuSO4∙5H2O, 0.18 g/L KI,</p><p>0.12 g/L MnCl2∙4H2O, 0.06 g/L Na2MoO4∙2H2O, 0.12 g/L ZnSO4∙7H2O, 0.15 g/L CoCl2∙6H2O and 10 g/L ethylene-diamine tetraacetic acid (EDTA) (based on Zeng et al., 2003). </p><p>Results</p><p>Reactor 1: Denitrification with methanol as the carbon source and nitrate as electron acceptor 1000</p><p>800 ) L / g 600 m (</p><p> n o i t</p><p> a 400 r t n e c</p><p> n 200 o c</p><p>0 Decant Feed End-feed End-anoxic End-oxic</p><p> nitrate COD</p><p>Fig. SI-1 Typical cycle transformation of granules achieving denitrification with methanol as the carbon source and nitrate as electron acceptor Reactor 3: CCP enrichment from CCP-/CAP-dominated granular sludge</p><p>Fig. SI-2 CLSM images of FISH micrographs of granules enriched in CCP from a CCP/CAP dominated granular sludge. Competibacter spp. cells are magenta (overlay of red GAOmix and blue EUBmix) and other bacteria are blue. Scale bar 20 μm Reactor 4: CCP enrichment from floccular sludge</p><p>Fig. SI-3 CLSM images of FISH micrographs of granules enriched in CCP from a floccular sludge at t = 15 days (a) and t = 83 days. Competibacter spp. cells are magenta (overlay of red GAOmix and blue EUBmix), Accumulibacter spp. cells are green (overlay of yellow PAOmix and blue EUBmix) and other Bacteria are blue. Scale bar 20 μm 1H NMR spectroscopy to detect the exopolysaccharide “Granulan” in crude EPS</p><p>A </p><p>Anomeric region </p><p>B β-Glucuronic acid 2-acetoamido-2-deoxy-α- β-Mannose (β-GlcA) α-Galactose (α-Gal) galactopyranuronic acid (β-Man) (α-GalANAc) α-Rhamnose (α-Rha) </p><p>N-acetyl-β- galactosamine (β-GalNAc) β-Galactose (β-Gal) </p><p>Figure SI-4 (A) 1H NMR spectrum of “Granulan” purified by fractional precipitation and gel permeation Fig. SI-4 (A) 1H NMR spectrum of “Granulan” purified by fractional precipitation and gel permeation chromatography (B) Expansion of the anomeric proton region of the spectrum of purified “Granulan”. chromatography (B) Expansion of the anomeric proton region of the spectrum of purified “Granulan”. References</p><p>Baytshtok, V., Lu, H., Park, H., Kim, S., Yu, R., Chandran, K., (2009) Impact of varying electron donors on the molecular microbial ecology and biokinetics of methylotrophic denitrifying bacteria. Biotechnology and Bioengineering 102 (6), 1527-1536.</p><p>Zeng, R. J., Lemaire, R., Yuan, Z., Keller, J., (2003) Simultaneous nitrification, denitrification, and phosphorus removal in a lab-scale sequencing batch reactor. Biotechnology and Bioengineering 84 (2), 170-178.</p>