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
 xxxx 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
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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
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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.
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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
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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.
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