Journal of Chromatography A, 1070 (2005) 131–136 Optimisation of poly--hydroxyalkanoate analysis using gas chromatography for enhanced biological phosphorus removal systems Adrian Oehmen, Beatrice Keller-Lehmann, Raymond J. Zeng, Zhiguo Yuan ∗ ,J¨ urg Keller Advanced Wastewater Management Centre (AWMC), The University of Queensland, St. Lucia, Brisbane 4072, Australia Received 21 October 2004; received in revised form 31 January 2005; accepted 9 February 2005 Available online 8 March 2005 Abstract Poly--hydroxyalkanoate (PHA) is a polymer commonly used in carbon and energy storage for many different bacterial cells. Polyphosphate accumulating organisms (PAOs) and glycogen accumulating organisms (GAOs), store PHA anaerobically through metabolism of carbon sub- strates such as acetate and propionate. Although poly--hydroxybutyrate (PHB) and poly--hydroxyvalerate (PHV) are commonly quantified using a previously developed gas chromatography (GC) method, poly--hydroxy-2-methylvalerate (PH2MV) is seldom quantified despite the fact that it has been shown to be a key PHA fraction produced when PAOs or GAOs metabolise propionate. This paper presents two GC-based methods modified for extraction and quantification of PHB, PHV and PH2MV from enhanced biological phosphorus removal (EBPR) systems. For the extraction of PHB and PHV from acetate fed PAO and GAO cultures, a 3% sulfuric acid concentration and a 2–20 h digestion time is recommended, while a 10% sulfuric acid solution digested for 20 h is recommended for PHV and PH2MV analysis from propionate fed EBPR systems. © 2005 Elsevier B.V. All rights reserved. Keywords: Poly--hydroxyalkanoate; Poly--hydroxy-2-methylvalerate; Enhanced biological phosphorus removal; Polyphosphate accumulating organisms; Glycogen accumulating organisms; Propionate 1. Introduction Enhanced biological phosphorus removal (EBPR) is a very commonly used and sustainable method for phosphorus removal from wastewater. A group of bacteria known as the polyphosphate accumulating organisms (PAOs) are pri- marily responsible for successful EBPR in activated sludge systems. Another group of bacteria known as the glycogen accumulating organisms (GAOs) have been shown to be able to compete with PAOs for the limiting carbon substrates in these systems. PAOs and GAOs are both able to anaerobically take up volatile fatty acids (VFAs) and convert them into intracellular poly--hydroxyalkanoates (PHAs). Although the VFA composition in wastewater systems can be diverse, acetate and propionate have been shown to be the primary fractions of VFA present in the influent to EBPR plants [1,2], ∗ Corresponding author. Tel.: +61 7 3365 7518; fax: +61 7 3365 4726. E-mail address: zhiguo@awmc.uq.edu.au (Z. Yuan). consequently most research has focused on the utilisation of these two carbon sources for PAO and GAO enrichment. PAOs tend to chiefly produce poly--hydroxybutyrate (PHB) from acetate [3], and mainly poly--hydroxyvalerate (PHV) and poly--hydroxy-2-methylvalerate (PH2MV) from propionate [4,5]. GAOs primarily convert acetate to PHB and PHV [6,7], while PHV and PH2MV are the major PHA fractions produced through propionate uptake [8,9]. Although most prior work in this field has focused on the utilisation of acetate as the sole carbon source, recent find- ings have suggested that a propionate feed source can provide PAOs an advantage over GAOs [8,10,11], resulting in more reliable EBPR operation. Despite this recent interest in pro- pionate as a carbon source, many researchers do not currently quantify PH2MV production [10,12–15], perhaps due to the lack of a proven method for analysing this particular PHA fraction. PH2MV has been shown to make up approximately half of the total PHA content when propionate is the sole car- bon source [4,5], therefore, the total PHA yield by PAOs and 0021-9673/$ – see front matter © 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.chroma.2005.02.020