Optimisation of glycogen quantiﬁcation in mixed microbial cultures Ana B. Lanham a,1,2 , Ana R. Ricardo a,1,2 , Marta Coma b , Joana Fradinho a,2 , Mónica Carvalheira a,2 , Adrian Oehmen a,2 , Gilda Carvalho a,c,2 , Maria A.M. Reis a,⇑ a REQUIMTE/CQFB, Departamento de Química, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, 2829-516 Caparica, Portugal b LEQUIA, Universitat de Girona, Parc Cientíﬁc i Tecnològic, Ediﬁci Jaume Casademont, Porta B, Pic de Peguera, 15 La Creueta, 17003 Girona, Spain c Instituto de Biologia Experimental e Tecnológica (IBET), Av. da República (EAN), 2784-505 Oeiras, Portugal highlights " The method for glycogen analysis is optimised using design of experiments. " Different methods should be used for ﬂoccular and granular cultures. " The biomass concentration should be as low as possible, i.e. 1 mg/mL. " The acid concentration should be increased to 0.9 M HCl. " A global method can be used when the granulation is unknown or unstable. article info Article history: Received 13 January 2012 Received in revised form 16 April 2012 Accepted 18 May 2012 Available online 26 May 2012 Keywords: Glycogen quantiﬁcation Carbohydrate analysis Floccular and granular sludge Design of experiments Response surface modelling abstract This study addressed the key factors affecting the extraction and quantiﬁcation of glycogen from ﬂoccular and granular mixed microbial cultures collected from activated sludge, nutrient removal systems and photosynthetic consortiums: acid concentration, hydrolysis time and concentration of biomass in the hydrolysis. Response surface modelling indicated that 0.9 M HCl and a biomass concentration of 1 mg mL 1 were optimal conditions for performing acid hydrolysis. Floccular samples only needed a 2-h hydrolysis time whereas granular samples required as much as 5 h. An intermediate 3 h yielded an error of 10% compared to the results obtained with the hydrolysis times speciﬁcally tailored to the type of biomass and can thus be recommended as a practical compromise. Ó 2012 Elsevier Ltd. All rights reserved. 1. Introduction Glycogen, a polysaccharide formed of glucose units, is used as a storage compound by different organisms (Preiss, 1984). The reversible interconversion of glycogen to glucose and then to pyruvate through the glycolysis and the gluconeogenesis path- ways, provide cells with useful source of carbon, energy (ATP) and reducing equivalents (NADH). Therefore, the utilisation of stor- age compounds such as glycogen, but also polyphosphate and polyhydroxyalkanoate (PHA), is often used as a survival strategy for bacteria present in dynamic systems subjected to variable environmental conditions (Van Loosdrecht et al., 1997). The analysis and quantiﬁcation of bacterial glycogen has been essential to the understanding of the complex microbial communi- ties in various environments and glycogen has been included as a major component in metabolic models for activated sludge (Smolders et al., 1994; Kuba et al., 1996; Oehmen et al., 2006, 2010). Consequently, there is often a necessity to accurately quantify the glycogen content of cells and to estimate the kinetics and stoichiometry of the process. Nevertheless, metabolic modelling predictions for glycogen have often exhibited error rates as high as 20–25% exceeding those for PHA, phosphorus or acetate (Brdja- novic et al., 2000; Lopez-Vazquez et al., 2009). Therefore, an opti- misation of the analytical method for glycogen is needed. Glycogen analysis can be carried out in several ways, depending on the type of cells that are being investigated and also on whether the purpose of the analysis lies only on its quantiﬁcation or on its 0960-8524/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.biortech.2012.05.087 ⇑ Corresponding author. Tel.: +351 212 948 541; fax: +351 212 948 550. E-mail addresses: analanham@campus.fct.unl.pt (A.B. Lanham), arr@dq.fct.unl.pt (A.R. Ricardo), marta@lequia.udg.cat (M. Coma), j.fradinho@campus.fct.unl.pt (J. Fradinho), mic16141@campus.fct.unl.pt (M. Carvalheira), a.oehmen@fct.unl.pt (A. Oehmen), gs.carvalho@fct.unl.pt (G. Carvalho), amr@fct.unl.pt (M.A.M. Reis). 1 These authors contributed equally to this work. 2 Tel.: +351 212 948 541; fax: +351 212 948 550. Bioresource Technology 118 (2012) 518–525 Contents lists available at SciVerse ScienceDirect Bioresource Technology journal homepage: www.elsevier.com/locate/biortech