Environ. Sci. Technol. 2006, 40, 2433-2437
A Two Step Chemo-biotechnological Conversion of Polystyrene to a Biodegradable Thermoplastic PATRICK G. WARD,† MIRIAM GOFF,† MATTHIAS DONNER,‡ WALTER KAMINSKY,‡ AND K E V I N E . O ’ C O N N O R * ,† 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 * Corresponding author phone: +353 1 716 1307; fax: +353 1 716 1183. e-mail:
[email protected]. † National University of Ireland. ‡ University of Hamburg. 10.1021/es0517668 CCC: $33.50 Published on Web 02/15/2006
2006 American Chemical Society
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 thermoplastic, 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 characterized 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 experiments, 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 VOL. 40, NO. 7, 2006 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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FIGURE 1. The conversion of polystyrene to polyhydroxyalkanoate. (a) Polystyrene beads. (b) Styrene oil derived from polystyrene by pyrolysis. (c) Film of PHA produced from polystyrene. provided by the preculturing of P. putida CA-3 for 16 h in 50 mL of E2 supplemented with 10 mM phenylacetic acid in a 250 mL Erlenmeyer flask. Fermentation Conditions Precultured P. putida CA-3 (as previously described in the shake flask conditions section) was used as an inoculum for the fermentor. All experiments were performed in a 7.5 liter continuously stirred tank reactor supplied with 5 liters of E2 mineral medium containing 67 mg nitrogen/L. The temperature was maintained at 30 °C, and the impellor speed was set to 500 rpm in all experiments. The air flow into the fermentor was kept constant at 5 l/min. Antifoam (polypropylene glycol (P2000)) was supplied at a concentration of 0.4 g/l. Styrene oil was supplied through the gaseous phase by using an additional airflow, controlled by a mass flow controller 5850S (Brooks Instrument), which was passed through styrene oil into the fermentor vessel. This airflow contained styrene at a concentration of approximately 9.5 mg/l. The flow rate was 0.15 l/min for the first 3 h of growth. This was increased to 0.25 l/min for the subsequent 3 h and, finally, to 0.65 l/min for the remainder of the fermentation. PHA Polymer Isolation and Monomer Determination. PHA polymer isolation and monomer determination was performed as previously described (18). Methanolysed PHA monomer samples were analyzed on a Fisons GC-8000 series gas chromatograph (GC) equipped with a 30 m by 0.25 mm HP-1-0.25 µm column (Hewlett-Packard) operating in split mode (split ratio 8:1) with temperature programming (50 °C for 1 min, increments of 10 °C/min up to 140 °C, 1 min at 140 °C). For peak identification, PHA standards from P. oleovorans were used. PHA monomer composition was confirmed by GC-MS as described previously (19). Nitrogen Determination Assay. The nitrogen concentration (as ammonium ion) in the medium was measured by the method of Scheiner (20). Determination of the Metabolic Activity of Whole Cells of P. putida CA-3. The metabolic activity of P. putida CA-3 can be measured by monitoring the rate of oxygen consumption by a washed cell suspension of P. putida CA-3 supplied with styrene. Cells were harvested at various time points from the fermentor, washed, and assayed in a biological oxygen monitor (Rank brothers Ltd., Cambridge, England) as reported previously (21). The cell dry weight in oxygen consumption assays varied between 0.15 g/l and 0.3 g/l.
Results and Discussion Polystyrene Pyrolysis. The pyrolysis of polystyrene resulted in a pyrolysis oil containing 82.8% w/w styrene as well as low levels of R-methylstyrene, toluene, styrene dimer, and traces of other aromatic compounds (Table 1). This method resulted in the complete conversion of polystyrene to styrene oil, with only traces of aliphatic waste emitted during the process. In previous reports the noncatalytic pyrolysis of polystyrene has resulted in lower yields of styrene monomer (55-65%) and the generation of pyrolysis oils containing lower levels 2434
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of styrene, as well as higher levels of styrene dimer, R-methylstyrene and toluene (22-24). Thus the method described here is the most efficient noncatalytic pyrolysis method for styrene retrieval from polystyrene reported to date (22-24). Conversion of Styrene Oil to PHA in Shake Flask Experiments. P. putida CA-3 is capable of growth and PHA accumulation with commercially pure styrene (17). The pathway from styrene to PHA has been partially elucidated and involves metabolism through phenylacetic acid and the de novo fatty acids synthesis pathway (21, 25). However, the ability of the organism to grow when supplied with a complex mixture of compounds (styrene oil) has previously been untested. Often mixtures of aromatic compounds can have a negative effect on the growth of a microorganism due to competitive inhibition (26), toxicity (27), or the formation of toxic intermediates generated by the nonspecific action of metabolic enzymes (28). Thus it was expected that P. putida CA-3 would not grow when supplied with styrene oil as the sole source of carbon and energy, and that the pyrolysis oil would need to be further purified by distillation. Surprisingly the untreated styrene oil supported the growth of P. putida CA-3 in shake flask experiments. Thus, in subsequent experiments, the styrene oil was used without further treatment as the growth substrate for P. putida CA-3. PHA accumulation in P. putida CA-3 was induced by limiting the concentration of nitrogen (67 mg nitrogen/l) in the growth medium (17). Cells grown in shake flask cultures, consumed 1 g of styrene oil to generate 62.5 mg of PHA and 250 mg of bacterial biomass (6.25% conversion rate). A low level of PHA accumulation from styrene oil was observed in the first 10 h of growth in shake flask cultures, followed by a dramatic rise in the level of PHA accumulation between 16 and 24 h, after which very little PHA was accumulated. This is a pattern similar to that observed when commercially pure styrene was supplied to P. putida CA-3 in shake flasks (17). However, growth on styrene oil yielded a lower level of PHA (0.14 g/l) after 48 h when compared to commercially pure styrene (0.18 g/l). The lower level of PHA from the styrene oil may be explained by the presence of nonvolatile compounds in the oil (Table 1), which lowers the vapor pressure of the liquid and thus results in a lower concentration of styrene being supplied to the bacteria in the growth medium. Consequently, the styrene oil was distilled, increasing the proportion of styrene to 90%, and removing all nonvolatile compounds from the oil. In shake flask experiments P. putida CA-3 accumulated the same level of PHA from the distilled styrene oil (0.18 g/l) as cells supplied with commercially pure styrene. Conversion of Styrene Oil to PHA in Stirred Tank Reactor. A 2.3-fold increase in the level of PHA accumulation from nondistilled styrene oil (0.32 g/l) was observed when P. putida CA-3 was grown in a 7.5 liter stirred tank reactor (fermentor) (Figure 3). In fermentation experiments there was no PHA accumulated by P. putida CA-3 in the first 10 h of growth (Figure 3). PHA accumulation initially occurred to a low level when the nitrogen concentration dropped below 5 mg/l (16 h) and increased rapidly once the nitrogen was no longer detectable in the growth medium (24 h). While similarities exist between the growth patterns and PHA accumulation in the shake flask and fermentation experiments, there is a significant lengthening of the period in which PHA accumulation occurs in the fermentor (Figure 3). The accumulation of biodegradable plastic in the fed batch fermentor from styrene oil occurred over a period of 32 h (16-48 h) (Figure 3), compared to PHA accumulation over 14 h in shake flasks. There was no further increase in the level of PHA accumulated from styrene oil in the fermentor after 48 h. The extended period of PHA accumulation could be explained by the continuous controlled supply of styrene oil to the bacterial cells in the fermentor compared to the
FIGURE 2. Schematic diagram of fluidized bed plant for the pyrolysis of polystyrene. M, motor; PIR, pressure indicator recording; PI, pressure indicator; TIR , temperature indicator recording; PSV, pressure safety valve; NV, needle valve; MV, Magnetic Valve; BV, ball valve; ROTA, rotameter. VOL. 40, NO. 7, 2006 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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TABLE 1. Composition of Styrene Oil Generated from Polystyrene by Pyrolysis oil component
% (w/w) composition
styrene benzene toluene ethylbenzene R-methylstyrene 1-ethyl-2-methyl-benzene biphenyl R-methyl-biphenyl styrene dimer R-methyl-stilbene 1-butene-1,3-diphenyl unidentified
82.8 < 0.1 1.7 0.8 5.8 < 0.1 0.3 0.3 1.3 1.6 1.4 3.8
FIGURE 3. PHA accumulation by P. putida CA-3 when styrene oil was supplied to a fermentor containing 5 liters of growth medium supplied with 67 mg nitrogen/L, at 30 °C. Biomass (cell dry weight (CDW)) (g/L) ([), PHA accumulation (9), and nitrogen (supplied as sodium ammonium phosphate) concentration (mg/L) (b) were all monitored over a 48 h period. All data are the average of at least three independent determinations. less ideal shake flasks experiments where the styrene oil is supplied to a central glass tubing in the Erlenmeyer flask from which styrene partitions into the air and subsequently into the liquid growth medium (17). The metabolic activity of P. putida CA-3 cells in the fermentor may also contribute to the differences in PHA accumulation compared to cells cultured in shake flasks. Consequently P. putida CA-3 cells were harvested at various time points from the fermentor, and the metabolic activity of the cells analyzed by measuring the rate of oxygen consumption by whole cells when supplied with styrene or other compounds present in the styrene oil. Washed cell suspensions of P. putida CA-3, when supplied with styrene, consumed oxygen at 150, 250, and 130 nmoles/min/mg cell dry weight (figures shown represent averages of triplicate experiments) after 6, 10, and 16 h of growth, respectively. Thus P. putida CA-3 cells maintain a higher rate of oxygen consumption for a longer period in the fermentor compared to shake flasks cultures grown on commercially pure styrene (17). Similar to these cultures (17), the depletion of nitrogen and the onset of PHA accumulation coincided with a decrease in the biochemical activity of P. putida CA-3 cells toward styrene (35 nmoles of oxygen consumed/min./mg cell dry weight after 24 h of growth). The biochemical activity of cells continued to fall over time to 20 nmoles oxygen consumed/ min./mg cell dry weight at 30 h after which the rate of oxygen consumption remained low. Prolonging a high level of biochemical activity of the cells in the fermentor should increase the final bacterial cell mass and the level of biodegradable plastic accumulated by the bacteria and thus increase the yield of PHA from polystyrene. The slow feeding 2436
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of nitrogen into the bacterial growth medium has previously been shown to improve PHA accumulation by bacteria and thus such a strategy will be attempted in the future (29). The biochemical activity of P. putida CA-3 cells toward the volatile compounds R-methylstyrene, toluene and ethylbenzene was below detectable levels. Thus P. putida CA-3 appears to convert only the styrene component of the oil to PHA. Consequently, a pyrolytic process that generates higher levels of styrene from polystyrene or a bacterium capable of utilizing a broader range of substrates is needed to increase the efficiency of polystyrene conversion to PHA. The former approach can be achieved by using a known catalytic pyrolysis method, which yields styrene oil containing up to 98% styrene monomer (30). However this method will produce spent catalyst which may generate further waste problems. After 48 h of fermentation 1.6 g of PHA and 2.8 g of bacterial biomass was accumulated from 16 g styrene oil (10% conversion). This equates to a 1.6-fold increase in the PHA yield from nondistilled styrene oil compared to shake flask experiments. Four fermentation runs were completed to generate 6.4 g of PHA from 64 g of styrene oil. The PHA produced from styrene oil, as measured by GC-MS, is composed of (R)-3-hydroxyhexanoate, (R)-3-hydroxyoctanoate, and (R)-3-hydroxydecanoate monomers in a molar ratio of 0.046:0.436:1.126. This is referred to as medium chain length PHA. Medium chain length PHAs are thermoplastics and elastomers that have applications as plastic coatings and pressure sensitive adhesives, as well as medical applications in wound management, drug delivery, and tissue engineering (12). Furthermore, medium chain length PHA is composed of chiral hydroxy acids that have potential as synthons for anti-HIV drugs, anti-cancer drugs, antibiotics, and vitamins (31, 32). Thus polystyrene has been converted to a polymer with very different end uses. 14 million metric tons of polystyrene are produced annually worldwide, most of which ends up in landfill. Hence, the conversion of waste polystyrene (a dead end product) into a useful commodity is desirable. As a result of its widespread use and poor rate of recycling, polystyrene is viewed as a major post-consumer waste product (1). However, due to the biotechnological conversion of polystyrene to PHA, post consumer polystyrene is, potentially, a starting material for the synthesis of biodegradable plastic. Due to the general applicability of pyrolysis for plastic conversion to an oil (2) and the large number of microorganisms capable of PHA accumulation from a vast array of molecules, the principle of the process described here can be applied for the recycling of any petrochemical plastic waste into PHA (33). Indeed this work creates a substantive link between petrochemical and biological polymers and potentially opens up a new area of exploration for the petrochemical industry.
Acknowledgments This project is funded under the Higher Education Authority (HEA) Program for Research and Training at Third Level Institutions phase III (PRTLI III).
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Received for review September 6, 2005. Revised manuscript received January 10, 2006. Accepted January 11, 2006. ES0517668
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