Efficient recovery of low endotoxin medium-chain-length poly([R]-3-hydroxyalkanoate) from bacterial biomass Patrick Furrer a,b , Sven Panke b , Manfred Zinn a, ⁎ a Empa, Swiss Federal Laboratories for Materials Testing and Research, Laboratory for Biomaterials, CH-9014 St. Gallen, Switzerland b ETHZ, Swiss Federal Institute of Technology Zurich, Institute of Process Engineering, CH-8092 Zurich, Switzerland Received 23 July 2006; received in revised form 16 December 2006; accepted 5 January 2007 Available online 19 January 2007 Abstract Bacterial polyhydroxyalkanoate (PHA) is an attractive biopolyester for medical applications due to its biocompatibility. However, inappropriate extraction of PHA from bacterial biomass results in contamination by pyrogenic compounds (e.g. lipopolysaccharides) and thus influences medical testing. This problem was solved by a temperature-controlled method for the recovery of poly(3-hydroxyoctanoate-co-3- hydroxyhexanaote) (PHO) from Pseudomonas putida GPo1. In contrast to other methods, precipitation of PHO was triggered by cooling the hot solution to a particular temperature. N-hexane and 2-propanol were found to be optimal solvents for such procedure. Quantitative extraction with n-hexane took place at 50 °C and optimal precipitation occurred between 0 and 5 °C. The purity was > 97% (w/w) and the endotoxicity between 10 and 15 EU/g PHO. Additional re-dissolution in 2-propanol at 45 °C and precipitation at 10 °C resulted in a purity of close to 100% (w/w) and the minimal endotoxicity of 2 EU/g PHO. The polydispersity (M w /M n ) of PHO was decreased from 2.0 to 1.5 for this optimized procedure. © 2007 Elsevier B.V. All rights reserved. Keywords: Extraction; Low endotoxin; Medium-chain-length polyhydroxyalkanoate; Recovery; Temperature-controlled precipitation 1. Introduction New biomaterials, in particular degradable ones are needed for various medical applications (Hubbell, 1995; Ueda and Tabata, 2003). Today's biomaterials of the third generation (Hench and Polak, 2002) are designed to stimulate specific responses at the molecular level and to combine the basic concepts of bioactive and resorbable materials. Today's polymeric and biodegradable systems used in medicine are mainly based on poly(lactic acid) (PLA), on poly(glycolic acid) (PGA), and on their co-polymers (Gomes and Reis, 2004). Other biodegradable polymers have been proposed, but could not enter the market yet, due to lacking FDA approval (Gomes and Reis, 2004). One of the candidates is polyhydroxyalkanoate (PHA), a class of biodegradable and biocompatible polyesters with many potential applications in the medical field, such as heart valve scaffolds (Sodian et al., 2000a, 2002), pulmonary conduits (Stock et al., 2000), sutures, screws, bone plates, repair patches, stents, bone marrow scaffolds, and many others over the last years as recently reviewed by Chen and Wu (2005). In particular the scientific and industrial research on elastomeric PHA with medium-chain-length side-chains (mclPHA, containing 3- hydroxyalkanoate monomers ranging from C 6 –C 14 ) has been intensified in the last years (Chen et al., 2005; Sodian et al., 2002, 2000b; Stock et al., 2000; Williams et al., 1999; Zinn et al., 2001). MclPHA is synthesized by fluorescent pseudomonads belonging to the rRNA homology group I (Huisman et al., 1989) and serves as an intracellular carbon and energy storage compound. Its biosynthesis can be triggered by appropriate growth conditions such as phosphorus and nitrogen limitation (Witholt and Kessler, 1999). MclPHA is stored in distinct granules that are coated by amphiphilic phospholipids and proteins (Pötter and Steinbüchel, 2004). Along with cell growth, all of the Gram negative production strains produce lipopolysaccharides (LPS) as an integral part of the outer membrane (Petsch and Anspach, 2000). LPS are pyrogenic and their concentration in a medical product is strictly Journal of Microbiological Methods 69 (2007) 206 – 213 www.elsevier.com/locate/jmicmeth ⁎ Corresponding author. Tel.: +41 71 274 7698; fax: +41 71 274 7788. E-mail address: manfred.zinn@empa.ch (M. Zinn). 0167-7012/$ - see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.mimet.2007.01.002