A Two Step Chemo-biotechnological Conversion of Polystyrene to a Biodegradable Thermoplastic PATRICK G. WARD, † MIRIAM GOFF, † MATTHIAS DONNER, ‡ WALTER KAMINSKY, ‡ AND KEVIN E. O’CONNOR* ,† School of Biomolecular and Biomedical Sciences, Centre for Synthesis and Chemical Biology, Conway Institute for Biomolecular and Biomedical Research, Ardmore House, National University of Ireland, University College Dublin, Belfield, Dublin 4, Republic of Ireland, and Institute for Technical and Macromolecular Chemistry, University of Hamburg, Bundesstrasse 45, 20146 Hamburg, Germany. A novel approach to the recycling of polystyrene is reported here; polystyrene is converted to a biodegradable plastic, namely polyhydroxyalkanoate (PHA). This unique combinatorial approach involves the pyrolysis of polystyrene to styrene oil, followed by the bacterial conversion of the styrene oil to PHA by Pseudomonas putida CA-3 (NCIMB 41162). The pyrolysis (520 °C) of polystyrene in a fluidized bed reactor (Quartz sand (0.3-0.5 mm)) resulted in the generation of an oil composed of styrene (82.8% w/w) and low levels of other aromatic compounds. This styrene oil, when supplied as the sole source of carbon and energy allowed for the growth of P. putida CA-3 and PHA accumulation in shake flask experiments. Styrene oil (1 g) was converted to 62.5 mg of PHA and 250 mg of bacterial biomass in shake flasks. A 1.6-fold improvement in the yield of PHA from styrene oil was achieved by growing P. putida CA-3 in a 7.5 liter stirred tank reactor. The medium chain length PHA accumulated was comprised of monomers 6, 8, and 10 carbons in length in a molar ratio of 0.046:0.436:1.126, respectively. A single pyrolysis run and four fermentation runs resulted in the conversion of 64 g of polystyrene to 6.4 g of PHA. Introduction Petrochemical based plastics, produced annually on the 100 million ton scale, pervade modern society as a result of their versatile and highly desirable properties. However, once disposed of, many of these plastics pose major waste management problems due to their recalcitrance. In the U.S. alone, over 3 million tons of polystyrene are produced annually, 2.3 million tons of which end up in a landfill (1). Furthermore only 1% of post-consumer polystyrene waste was recycled in the U.S. in 2000. The poor rate of polystyrene recycling is due to direct competition with virgin plastic on a cost and quality basis (2). Consequently, there is little or no market for recycled polystyrene (3). As an alternative to polymer recycling, polystyrene can be burned to generate heat and energy (4) or converted back to its monomer components for use as a liquid fuel (4-6). A number of techniques for converting plastic back to its monomer components have been developed, one of which, pyrolysis, involves thermal decomposition in the absence of air to produce pyrolysis oils or gases (4). In addition to their use as fuels, pyrolysis oils may also have a biotechnological use, i.e., as a starting material for the bacterial synthesis of value added products. Consequently, we report here on the conversion of polystyrene to PHA, a biodegradable thermo- plastic, through a combination of pyrolysis and bacterial catabolism (Figure 1a-c). PHAs are highly diverse and desirable polymers with a broad range of applications (7-9). They are polyesters of (R)-3-hydroxyalkanoic acids accumulated by bacteria as intracellular storage materials and are accumulated in response to a variety of stressful environmental conditions, such as inorganic nutrient limitation (e.g., nitrogen or oxygen) (8, 10, 11). PHAs are divided into two groups: short chain length PHAs, which contain monomers of 3-5 carbons in length, and medium chain length PHAs, which contain monomers of 6-14 carbons in length (11). The physical and mechanical properties of these polymers, such as stiffness, brittleness, and melting point are dramatically affected by the monomer composition of the polymer (12). While many studies have focused on the conversion of sugars and fatty acids to PHA, a limited number of studies have investigated the conversion of waste materials to PHA (13-15). However, to the best of our knowledge, this is the first study to investigate the conversion of a petrochemical plastic to a biodegradable plastic. Polystyrene was identified as a potentially attractive starting material for PHA production due to its widespread use and the waste management issues associated with it. Experimental Section Polystyrene Pyrolysis. Virgin polystyrene (Ultra polymers PSGP172L) was supplied to the pyrolysis plant (Figure 2), at a feed rate of 1.5 kg/hour. The electrically heated fluidized bed had a diameter of 130 mm. Quartz sand with diameters between 0.3 and 0.5 mm led to a height of 480 mm in the fluidized bed, which was maintained at a temperature of 520 °C. The polystyrene entered the reactor via a screw conveyor system. Further distillation of the pyrolysis oil to achieve a more purified liquid is carried out after the pyrolysis by merging all liquid phases and distilling it at a pressure of 2 hPa, up to 120 °C representing a boiling point of around 300 °C under atmospheric pressure. The oil fraction was char- acterized by gas chromatography-flame ionization detector (GC-FID) (HP 5890, Macherey & Nagel SE 52) and gas chromatography-mass spectrometry (GC-MS) (GC: HP 5890, MS: Fisons Instruments VG 70 SE, Macherey & Nagel SE 52). Media. E2 medium was prepared as previously described (16). Shake Flask Growth Conditions. In shake flask experi- ments, P. putida CA-3 cultures were grown in 250 mL Erlenmeyer flasks containing 50 mL of E2 medium at 30 °C, with shaking at 200 rpm. The inorganic nitrogen source sodium ammonium phosphate (NaNH4HPO4‚4H2O) was supplied at 1 g/l (67 mg nitrogen/l). Styrene oil was supplied to a central glass column (10 mm in diameter by 60 mm in length) fused to the central base of the growth flasks. Styrene and other volatile compounds that are present in the styrene oil partition into the air and, subsequently, into the liquid medium where the bacterial cells utilize the compounds as carbon and energy sources (17). Fermentation inoculum was * Corresponding author phone: +353 1 716 1307; fax: +353 1 716 1183. e-mail: kevin.oconnor@ucd.ie. † National University of Ireland. ‡ University of Hamburg. Environ. Sci. Technol. 2006, 40, 2433-2437 10.1021/es0517668 CCC: $33.50  2006 American Chemical Society VOL. 40, NO. 7, 2006 / ENVIRONMENTAL SCIENCE & TECHNOLOGY 9 2433 Published on Web 02/15/2006