Up-Cycling of PET (Polyethylene
Terephthalate) to the Biodegradable
Plastic PHA (Polyhydroxyalkanoate)
SHANE T. KENNY,
JASMINA NIKODINOVIC RUNIC,
RAMESH P. BABU,
CHRIS M. KEELY,
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, Institute for Technical
and Macromolecular Chemistry, University of Hamburg,
Bundesstrasse 45, 20146 Hamburg, Germany, and Materials
Ireland Polymer Research Center, School of Physics, Trinity
College, University of Dublin, Dublin-2, Ireland
Received April 11, 2008. Revised manuscript received
August 1, 2008. Accepted August 6, 2008.
The conversion of the petrochemical polymer polyethylene
terephthalate (PET) to a biodegradable plastic polyhydroxyal-
kanoate (PHA) is described here. PET was pyrolised at 450 °C
resulting in the production of a solid, liquid, and gaseous
fraction. The liquid and gaseous fractions were burnt for energy
recovery, whereas the solid fraction terephthalic acid (TA)
was used as the feedstock for bacterial production of PHA.
Strains previously reported to grow on TA were unable to
accumulate PHA. We therefore isolated bacteria from soil
exposed to PET granules at a PET bottle processing plant. From
the 32 strains isolated, three strains capable of accumulation
of medium chain length PHA (mclPHA) from TA as a sole source
of carbon and energy were selected for further study. These
isolates were identiﬁed using 16S rDNA techniques as P. putida
(GO16), P. putida (GO19), and P. frederiksbergensis (GO23).
P. putida GO16 and GO19 accumulate PHA composed
predominantly of a 3-hydroxydecanoic acid monomer while P.
frederiksbergensis GO23 accumulates 3-hydroxydecanoic
acid as the predominant monomer with increased amounts of
3-hydroxydodecanoic acid and 3-hydroxydodecenoic acid
compared to the other two strains. PHA was detected in all
three strains when nitrogen depleted below detectable levels
in the growth medium. Strains GO16 and GO19 accumulate
PHA at a maximal rate of approximately 8.4 mg PHA/l/h for 12 h
before the rate of PHA accumulation decreased dramatically.
Strain GO23 accumulates PHA at a lower maximal rate of
4.4 mg PHA/l/h but there was no slow down in the rate of PHA
accumulation over time. Each of the PHA polymers is a
thermoplastic with the onset of thermal degradation occurring
around 308 °C with the complete degradation occurring by
370 °C. The molecular weight ranged from 74 to 123 kDa.
X-ray diffraction indicated crystallinity of the order of 18-31%.
Thermal analysis shows a low glass transition (-53 °C) with
a broad melting endotherm between 0 and 45 °C.
PET is one of many petrochemical based plastics that
contribute greatly to the convenience of everyday life. Best
known for its use in plastic bottles it is produced on a
multimillion tonne scale worldwide. Like other petrochemical
plastics, the success of PET as a convenience bulk commodity
polymer has led to post consumer PET products becoming
a major waste problem. Greater than 5400 million lbs (2400
million kg) of PET bottles were on shelves in the U.S. in 2006
with only 23.5% of these bottles being recycled, and thus the
vast majority of PET bottles end up in the landﬁll (1). This
occurs despite a variety of recycling technologies being
available such as mechanical grinding for use in the ﬁber
industry and reprocessing for food contact usage. The current
technologies recycle PET to a low value product and thus
factors such as the high relative cost of sorting and the low
value of the downstream product contribute to the poor
recycling rates for PET. The conversion of PET to a high value
product (upcycling) should lead to higher levels of PET
recycling. The thermal treatment of PET in the absence of
air (pyrolysis) generates terephthalic acid (TA) as the major
product (Table 1). The TA generated is potentially a feedstock
for the microbial synthesis of the value added biodegradable
polymer polyhydroxyalkanoate (PHA). Consequently we are
investigating the conversion of PET to this desirable high
We have previously reported the conversion of polystyrene
to the biodegradable plastic PHA using a two step chemo-
biotechnological process which involves pyrolysis of poly-
styrene to styrene oil and subsequent feeding of that oil to
bacteria that can utilize it as a carbon source to make a
biodegradable carbon-based plastic (2). The conversion of
PET to a biodegradable plastic has never been demonstrated
and very few bacteria are known to degrade TA. Of those that
degrade TA, none make PHA and thus we have searched a
PET granule exposed soil to isolate new terephthalic acid
degrading bacteria capable of accumulating PHA.
PHA is the general term for a range of diverse polymers
that consist of polyesters of (R)-3-hydroxyalkanoic acids.
These polymers are accumulated by bacteria as intracellular
carbon storage materials. It has been shown PHA accumula-
tion occurs in response to a range of environmental stress
factors such as inorganic nutrient limitation (3-5). The
substrates supplied to bacteria to accumulate PHA can be
divided into two groups: (1) PHA related substrates (fatty
acids) that resemble the monomers that make up PHA (i.e.,
alkanoic acids, fatty acids) and (2) PHA unrelated substrates
(i.e glucose, TA, etc.). There are two classiﬁcations of PHA
based on the length of the monomer chains, short chain
length PHA (sclPHA) which have monomers of 3-5 carbons
long and medium chain length PHA (mclPHA) which have
monomers of 6-14 carbons long (5). This variance in
monomer chain length gives rise to varying properties in the
polymer with sclPHA being more rigid and brittle than the
elastomeric mclPHA (6). These biopolymers are of interest
due to a broad range of applications and the fact that they
are completely biodegradable (3, 7, 8).
The cost of PHA production through fermentation is
inextricably linked to the cost of the starting substrate. We
investigate here the use of an easily sourced and inexpensive
* Corresponding author phone: +353 1 716 1307; fax +353 1 716
1183; e-mail: email@example.com.
University College Dublin.
University of Hamburg.
Environ. Sci. Technol. 2008, 42, 7696–7701
7696 9ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 42, NO. 20, 2008 10.1021/es801010e CCC: $40.75 2008 American Chemical Society
Published on Web 09/12/2008
post consumer petrochemical plastic waste as a feedstock
for the synthesis of PHA by bacteria.
Hydrolytic Pyrolysis of PET. Virgin PET Krupp-Formoplast
was supplied to a laboratory scale pyrolysis plant as previously
described (9-11) at a feed rate of approximately 1 kg/h. The
electrically heated ﬂuidized bed had a diameter of 130 mm.
Nine kg of quartz sand with diameters between 0.3 and 0.5
mm gave a height of 480 mm in the ﬂuidized bed, which was
maintained at a temperature of 450 °C. PET entered the
ﬂuidized bed reactor via a screw conveyor. The hot pyrolysis
products passed a cyclone to be cleaned by small amounts
of ﬁllers and then were cooled down by mixing up with cold
water to room temperature in a precipitator (desublimator).
In the precipitator the terephthalic acid and some other solids
(Table 1) were desublimated to generate a white powder.
The sand bed was ﬂuidized by steam with a ﬂow rate of 2.5
kg/h. The solids were analyzed by HPLC-MS-system (HP 1100,
column Multospher 100) using a diode array detector at 220
nm. The gas and oil fractions were characterized by gas
chromatography (GC-FID, HP 5890 Machery & Nagel SE 52)
and GC-MS (Fisons Instruments VG 70 SE, Machery & Nagel
Bacterial Growth Medium. The minimal mineral salts
media E2 was prepared as previously described (12) and used
as the media supplemented with sodium terephthalate as
the sole source of carbon and energy for all culture techniques
discussed in this work. The sodium terephthalate was
prepared by taking the solid fraction of PET pyrolysis and
dissolving in sodium hydroxide (Sigma). In addition, benzoic
acid (2.4 g/L) was also used as a carbon source.
Isolation of Bacteria from Soil. One kg of soil sample
was collected from PET exposed soil at an industrial site
used to mold PET granules to PET products. The granules
were present in the soil adjacent to the factory setting. We
reasoned that leaching of TA from PET may occur and that
this soil would be a good source of TA degrading bacteria.
The soil was sieved under aseptic conditions to a particle
size of 5 mm, and then 10 g of soil were added to 90 mL of
sterile Ringers solution (Sigma), this was vortexed for 5 min
to homogenize the sample. Serial dilutions were performed
to obtain a 10-5dilution of the original soil sample. The serial
dilutions were spread plated on solid E2 media containing
1.1 g/L of sodium terephthalate as the sole source of carbon
and energy. Plates were incubated at 30 °C for 48 h. Various
isolates were selected by visual differentiation of contrasting
colony morphology. These isolates were then assessed for
the ability to accumulate PHA.
Growth Conditions for PHA Accumulation. In addition
to microorganisms isolated from soil some bacterial strains,
known to utilize TA as a sole source of carbon and energy,
were tested for PHA accumulation. Both soil isolates and
known TA degraders were grown in shake ﬂask experiments,
where each strain was grown in a 250 mL Erlenmeyer ﬂask
containing 50 mL E2 medium (4.2 g/L of TA) at 30 °C with
shaking at 200 rpm. To screen for organisms capable of PHA
accumulation the inorganic nitrogen source sodium am-
monium phosphate (NaNH4HPO4.4H2O) was limited to 1 g/L
(67 mg nitrogen/L).
PHA Screening and Composition Analysis. Thirty-two
soil isolates and three commercially obtained strains were
grown in shaken ﬂasks for 48 h and tested for PHA
accumulation as previously described (13). The samples were
analyzed on an Agilent 6890N series GC ﬁtted with a 30 m
×0.25 mm ×0.25 µm HP-1 column (Hewlett-Packard) using
a split mode (split ratio 10:1). The oven method employed
was 60 °C for 2 min, increasing by 5 °C/min to 200 °C and
holding for 1 min. For peak identiﬁcation, PHA standards
from P. putida CA-3 (14) and (R)-3-hydroxydodecanoic acid
(3HDDA) (Sigma) were used. PHA monomer determination
was conﬁrmed using an Agilent 6890N GC ﬁtted with a 5973
series inert mass spectrophotometer, a HP-1 column (12 m
×0.2 mm ×0.33 µm) (Hewlett-Packard) was used with an
oven method of 50 °C for 3 min, increasing by 10 °C/min to
250 °C and holding for 1 min.
Nitrogen Determination Assay. The concentration of
nitrogen in the growth media was monitored over time using
the previously described method of Scheiner (15).
Determination of Terephthalic Acid Utilization during
Growth. The concentration of TA in the media was monitored
by taking 1 mL samples from the culture ﬂask at various time
points and centrifuging the samples at 14 000gfor 2 min.
The supernatant was retained, ﬁltered, and analyzed by
HPLC. In order to analyze the sample and maintain a linear
relationship between peak area on the HPLC chromatograph
and TA concentration, samples had to be diluted so that the
concentration of TA in the ﬁnal preparation did not exceed
0.63 g/L. An Agilent 1100series HPLC using a C18 ODS
Hypersil column (125 ×3 mm, particle size 5 µm) (Thermo)
was used, and samples were isocratically eluted using 0.2%
formic acid and acetonitrile (ratio 80:20, respectively) at a
ﬂow rate of 0.5 mL/min and read on a UV-vis detector at
230nm. The TA retention time under the above conditions
was 3 min.
Nuclear Magnetic Resonance (NMR). Solution NMR were
recorded on a Bruker DPX400 with 1H at 400.13 MHz and 13C
at 100.62 MHz. The solvent chloroform-d and tetramethyl-
silane (TMS) were used as internal references for chemical
shifts in 13C and 1H NMR, respectively. 13C NMR spectra were
recorded with proton-decoupling. Typically 2200 transients
were accumulated. Spectrometer peak areas were obtained
directly by standard signal integration.
Thermal Analysis. Differential scanning calorimetry (DSC)
was performed with Perkin-Elmer Pyris Diamond calorimeter
calibrated to Indium standards. The samples weighing 7-8
mg were encapsulated in hermetically sealed aluminum pans
and heated from -70 to 100 °C at a rate of 10 °C/min. To
determine the glass transition temperature (Tg) the samples
were held at 100 °C for 1 min and rapidly quenched to -70
°C. The samples were then reheated from -70 to 100 °Cat
10 °C/min to determine the melting temperature (Tm) and
Tg. The Tm was taken at the peak of the melting endotherm,
while the Tg was taken as the mid point of heat capacity
Thermogravimetric Analysis (TGA). To determine the
thermal stability and decomposition proﬁle of the samples,
TGA was carried out on a Perkin-Elmer Pyris 1 thermogravi-
metric analyzer calibrated using Nickel and Iron standards.
Each sample was weighed to ca. Seven mg and placed in a
platinum pan and heated from 30 to 700 °C at the heating
rate of 10 °C/min under an air atmosphere.
Dynamic Mechanical Analysis (DMA). DMA was carried
out on a Perkin-Elmer mechanical analyzer. Dynamic
measurements were made in extension mode on clamped
ﬁlm samples with dimensions of 5 ×2.8 ×0.5 mm. The
experiments were performed under nitrogen atmosphere at
a temperature range of -100 to 50 °C at a heating rate of 2
°C/min and frequency of 0.1, 1, and 10 Hz. The Tg was
identiﬁed by the sharp drop in storage modulus and the
corresponding the peak in the loss modulus (16). DMA glass
transition temperature is frequency dependent and detect-
able at higher temperature compared to the quasistatic DSC
data. The temperature at the maximum point of the loss
modulus (E′′) was taken as the measure of the glass transition
Gel Permeation Chromatography. Molecular weight
distribution were obtained by gel permeation chromatog-
raphy (GPC) using PL gel 5 mm mixed-C +PL gel column
(Perkin-Elmer) with PELV 290 UV-vis detector set at 254
VOL. 42, NO. 20, 2008 / ENVIRONMENTAL SCIENCE & TECHNOLOGY 97697
nm. Spectroscopic grade chloroform was used as the eluent
at ﬂow rate of 1.0 mL/min. Sample concentration of 1% (w/
v) and injection volumes of 500 µL were used. A molecular
weight calibration curve was generated with polystyrene
standards with low polydispersity using the Turbochrom 4.0
X-ray Diffraction (XRD) Analysis. XRD was performed at
room temperature and diffraction patterns were collected
on a Siemens D500 diffractometer ﬁtted with a Cu-KR
radiation source. The X-ray beam was Cµ-KR(λ)0.1514
nm) radiation operated at 40 KV and 30 mA. Data was
obtained from 2-60 °C(2θ) at a scanning speed of 0.1 °C/
16S rDNA Identiﬁcation. Three strains capable of ac-
cumulating PHA with TA as the sole carbon and energy source
were selected and identiﬁed by sequence analysis of 16S rRNA
genes. The genomic DNA of each bacterium was extracted
as previously described (17). The 16S rRNA genes were
ampliﬁed by PCR using primers 27F (agagtttgatcmtggctcag)
and 1392R (acgggcggtgtgtgtrc) (18) and the sequences were
determined by GATC-Biotech, Germany. The resulting
sequences were compared to known sequences in the NCBI
GenBank database by BLAST program (19).
Results and Discussion
Polyethyleneterephthalate Pyrolysis. The pyrolysis of PET
resulted in the generation of a solid, liquid and gaseous
fraction (Table 1). 72% w/v of the terephthalic acid present
in PET is recovered as monomeric TA (solid fraction).
Oligomers of terephthalic acid make up almost 26% of the
solid fraction. The addition of the solid fraction of PET
pyrolysis to a solution of sodium hydroxide resulted in the
hydrolysis of the oligomers and increased the proportion of
TA making up the solid fraction to 97% w/w. The pyrolysis
liquid fraction made up 6.3% w/v of the total weight of the
pyrolysis products and contained predominantly acetic
aldehyde and minor amounts of ethylene glycol. The gaseous
fraction made up 18% of the pyrolysis products and contained
predominantly CO2. The liquid and gaseous fraction were
burned to provide energy for the pyrolysis of PET while the
solid is desublimated, collected, and used as a feedstock for
biodegradable plastic synthesis by bacteria.
Isolation and Identiﬁcation of PHA Accumulating TA
Degraders from Soil. Three bacteria, known to utilize TA as
the sole source of carbon and energy, i.e. Comamonas
testosteroni YZW-D, C. testosteroni T-2, and C. testosteroni
PSB-4 (20-24) were tested for their ability to accumulate
PHA. However, these strains were incapable of PHA ac-
cumulation from TA derived from PET or commercially
available sodium terephthalic acid (control).
Since these strains were unable to synthesize PHA,
screening for TA degrading strains from soil was performed.
TA degrading strains were isolated from soil that had been
exposed to PET granules at a PET processing factory in
Ireland. From the PET exposed soil, 32 colonies with different
morphologies growing on E2 media with TA as the sole source
of carbon and energy were selected for further study. All of
these isolates were screened for PHA accumulation after
growth in shake ﬂasks with limited nitrogen (to stimulate
PHA production) and with TA as the sole source of carbon
and energy. Of the 32 isolates screened, only three ac-
cumulated detectable levels of PHA. These three organisms
were identiﬁed using 16S rDNA techniques. All three strains
shared 99% homology with known Pseudomonas species
(Table 2). Two of the three Pseudomonas strains are from the
species putida, which are known to degrade a wide variety
of aromatic compounds and more recently accumulate
aliphatic mcl-PHA from aromatic compounds such as styrene
and phenylacetic acid (14, 25). The other strain Pseudomonas
frederiksbergensis GO23 is from a species reported to degrade
the aromatic hydrocarbon phenanthrene (26). However this
is the ﬁrst report of any strain from this species accumulating
PHA and any microorganism accumulating PHA from TA.
Conversion of PET Derived Sodium Terephthalate to
PHA in Shake Flask Experiments. All three strains ac-
cumulated PHA to between 23 and 27% of the total cell dry
weight when supplied with TA either from a commercial
source or from TA derived from the pyrolysis of PET. We
used commercially available TA as a comparison to PET
derived TA. PHA levels and composition were identical from
PHA accumulation by each of the three strains was
monitored over time to determine when the onset of PHA
occurred as well as the time course for PHA production. All
three organisms were grown in shake ﬂasks under the
nitrogen limited conditions with 4.2 g/L of sodium tereph-
thalate (generated by PET pyrolysis). Nitrogen concentration
(as ammonium), TA concentration, cell dry weight and
quantity of PHA accumulated (Figure 1) were monitored. All
three bacteria had similar growth patterns, they showed a
long lag period in growth (of between 8 and 12 h) which
coincided with a lag in TA utilization, despite being grown
in precultures overnight on TA (4.2 g/L). During the
exponential phase of growth strains GO16, GO19, and GO23
consumed TA at 0.135 g/L/h, 0.157 g/L/h, and 0.121 g/L/h,
respectively, and had speciﬁc growth rates of 0.04 h-1, 0.043
h-1, and 0.049 h-1. All three strains consumed TA fully within
the same period of time (Figure 1). While GO23 had the lowest
rate of TA utilization during the exponential phase of growth
it achieved the highest ﬁnal cell dry weight and the highest
growth yield from TA. While benzoic acid is a minor
component of the solid fraction of the PET pyrolysis product,
all three strains were capable of utilizing it as a sole carbon
and energy source and thus it was also utilized during growth
of the bacteria when the pyrolysis product was used as the
substrate (data not shown). All three strains were also able
TABLE 1.Hydrolytic Pyrolysis of PET at 450°C
product composition weight percentage (%)
terephthalic acid 51.0
benzoic acid 1.0
acetic aldehyde 5.10
TABLE 2.Identification and Comparison of Bacterial Isolates,
From a Pet Exposed Soil, Capable of Growth and PHA
Accumulation with TA As the Sole Source of Carbon and
GO16 DQ133506 Pseudomonas putida 99 97
GO19 AY512611 Pseudomonas putida 99 99
frederiksbergensis 99 99
7698 9ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 42, NO. 20, 2008
to accumulate mclPHA when grown on benzoic acid (2.4
g/L) under nitrogen limited conditions to 20-27% of CDW.
Once the nitrogen concentration in the growth medium
was depleted below 15 mg/L at approximately 13 h, the onset
of PHA accumulation occurred in strains GO16 and GO19,
whereas PHA accumulation in GO23 began earlier. A
comparison of the rate of TA utilization (g/l/h) and PHA
accumulation (g/l/h) when PHA accumulation was occurring
maximally indicates that strain GO19 was the most efﬁcient
at converting TA to PHA during this time period at 8.4 mg
PHA/L/h. Strain GO23 appeared to accumulate PHA at a
lower rate of 4.4 mg PHA/l/h but maintained this rate of PHA
accumulation for a longer period of time compared to strains
GO16 and GO19 (Figure 1). Thus process development and
optimization of PHA production will be strain dependent.
PHA Composition. The 1H NMR spectra for each PHA
derived for strains GO16, GO19, and GO23 were established
(Figure 2a). Peak assignments were typical of medium chain
length PHA derivates (27-30). Terminal CH3protons were
detected by the strong resonance at 0.86 ppm. Another strong
signal from the methylene hydrogens associated with satu-
rated side chains occured at 1.25 ppm, whereas those
methylenes associated with double bonds were detected at
2.0 ppm. The resonance at 1.56 ppm was associated with the
methylene protons on C4. Methine protons were assigned
to the quadruplet resonance located at 5.2 ppm. Finally the
weak signal at 5.5 ppm was assigned to side-chains with
The 13C spectrum for PHA produced from GO19 is
illustrated (Figure 2b). Chemical shift assignments are
prominently associated with medium chain length monomer
structural units of 3-hydroxydodecenoic acid (3HDDE),
3-hydroxydecanoic acid (3HD), and 3-hydroxyoctanoic acid
(3HO). The percentage of unsaturated side chains was
estimated to be on the order of 5% by comparison of the
methine signal intensities (134-123 ppm) to those of the
methylene groups (10-40 ppm). The signal at 18.2 ppm is
less than 1% of the total methylene group intensities and
was previously assigned to 3-hydroxyhexanoic acid (3HH)
(30). GCMS analysis of the PHA samples conﬁrmed the
presence of 3HH, 3HO, 3HD, 3HDDA, and 3HDDE with 3HD
as the predominant monomer (Table 3). PHA from strain
GO23 contained a high proportion of 3HDDA compared to
PHA from strains GO16 and GO19. GCMS analysis indicated
a higher proportion of 3HDDE monomer than estimated by
13C NMR. PHA standards from P. putida CA-3, commercially
available 3HDDA, and a GCMS library (Agilent) were used
to determine peak identity.
FIGURE 1. PHA accumulation by (A) P. putida GO16, (B) P.
putida GO19, and (C) P. frederiksbergensis GO23 in shake ﬂask
containing growth medium consisting of 4.202 g/L of sodium
terephthalate and 67 mg/L of nitrogen at 30 °C. CDW g/L (]),
PHA accumulation g/l (∆), TA concentration (∆) and nitrogen
concentration g/L (•) supplied as sodium ammonium phosphate
were all monitored over a 48 h period. All data shown is the
average of at least three independent determinations.
FIGURE 2. NMR spectra of the mclPHA isolated from P. putida
GO19 recorded at 20°C in CDCl3(a) 400 MHz 1H spectrum (b) 13C
TABLE 3.Composition of PHA Accumulated from Terephthalic
(% CDW) 3HH 3HO 3HD 3HDDA 3HDDE
P. putida GO16 27 1 21 48 14 16
P. putida GO19 23 1 23 45 14 17
GO23 24 1 14 42 22 21
3HH )3-hydroxyhexanoic acid, 3HO )3-hydroxyoctan-
oic acid, 3HD )3-hydroxydecanoic acid, 3HDDA )3-hydroxy-
dodecanoic acid, 3HDDE )3-hydroxydodecenoic acid.
VOL. 42, NO. 20, 2008 / ENVIRONMENTAL SCIENCE & TECHNOLOGY 97699
While the sequence homology of 16S rDNA indicated a
strong similarity between these bacteria often closely related
species have differing PHA accumulation abilities (25). The
PHA from P. putida GO23 contained a higher proportion of
3HDDA (22%) and 3HDDE (21%) compared to PHA from the
other two strains (Table 3). It has been documented that the
monomer composition of the PHA dictates the polymer
properties (31) and while the difference between the GO23
polymer and the other two polymers presented in this study
may appear small, the PHA polymer isolated from P.
frederiksbergensis GO23 was physically different, showing an
increased tackiness and malleability at room temperature
compared to plastic from GO16 and GO19.
PHA Properties. GPC analysis showed the polymers tested
in this study ranged in molecular weight (Mw) from 74 kDa
to 123 kDa (Table 4). The molecular weight distribution (Mw/
Mn) of the PHAs ranged from 1.9 to 2.4 (Table 4) and these
values are typical for mclPHAs (32).
The DSC analysis shows that PHA polymers produced in
this study are partially crystalline, as evidenced by the
presence of a melting peak. Previous reports have shown
that short chain PHA produced by P. oleovorans from
hexanoate and heptanoate did not have a clear melting peak,
whereas PHA produced from longer-chain alkanoates ex-
hibited melting endotherms (28). All three polymers produced
with different bacterial strains showed similar Tg, with slight
changes in their Tm and ∆Hm values (Table 4).
X-ray diffraction (XRD) of cast ﬁlms was used to calculate
the crystallinity of the polymers (33). The strong diffraction
peaks were located at the 2θ)19.58, 21.38, and 19.38 for
PHA samples produced form GO16, GO19, and GO23,
respectively. The calculated crystallinity values are between
26.8 and 31.1% for GO16, GO19, and GO23. The presence of
higher amounts of 3HDDA and 3HDDE monomers leading
to a higher percentage crystallinity is in keeping with previous
All three PHA products had similar thermal degradation
patterns. Peak degradation maximum occurred at ca. 308 °C
with a high temperature shoulder most evident at ca. 350 °C
in the differential thermograph. Polymer degradation was
completed by 370 °C with all residual carbonaceous materials
produced during thermal degradation being burnt at about
600 °C. Thus three thermoplastics were generated through
the chemo-biotecnological processing of PET.
While certain avenues for PET recycling exist, a technology
that can offer a clean and cost-effective way of converting
PET to a high value polymer will generate a niche market in
PET recycling. Pyrolysis offers a means of streaming this waste
as a carbon feedstock for the production of PHA, a biode-
gradable polymer with broad uses encompassing biomedical
as well as packing applications (35-37).
This project has been funded under a grant from the
Environmental Protection Agency of Ireland (ERTDI 2005-
ET-LS-9-M3), we thank Dr Zysltra for the provision of strain
C. testosteroni YZW-D.
(1) NAPCOR 2006 Report on Post Consumer PET Container Recycling
Activity; NAPCOR: Sonoma, CA, 2006.
(2) Ward, P.; Goff, M.; Donner, M.; Kaminsky, W.; O’Connor, K. A
two step chemo-biotechnological conversion of polystyrene to
a biodegradable thermoplastic. Environ. Sci. Technol. 2006,40,
(3) Anderson, A.; Dawes, E. Occurrence, metabolism, metabolic
role, and industrial uses of bacterial polyhydroxyalkanoates.
Microbiol. Rev. 1990,54, 450–472.
(4) Doi, Y.; Kawaguchi, Y.; Koyama, N.; Nakamura, S.; Hiramitsu,
M.; Yoshida, Y.; Kimura, H. Synthesis and degradation of
polyhydroxyalkanoates in Alcaligenes eutrophus.FEMS Micro-
biol. Lett. 1992,103, 103–108.
(5) Reddy, C.; Ghai, R.; Rashmi; Kalia, V. Polyhydroxyalkanoates:
An overview. Biores. Technol. 2003,87, 137–146.
(6) Van der Walle, G.; de Koning, G.; Weusthuis, R.; Eggink, G.
Properties, modiﬁcations and applications of biopolyesters. Adv.
Biochem. Eng/biotechnol. 2001,71, 264–291.
(7) Hocking, P.; Marchessault, R., Biopolyesters in Chemistry and
Technology of Biodegradable Polymers; Chapman and Hall:
London, 1994; p 48-96.
(8) Lutke-Eversloh, T.; Fischer, A.; Remminghorst, U.; Kawadaj, J.;
Marchessault, R.; Bojershausen, A.; M, K.; Echert, H.; Reichelt,
R.; Liu, S. J.; Steinbuchel, A. Biosynthesis of novel thermoplastic
polythioesters by engineered Escherichia coli.Nat. Mater. 2002,
(9) Grause, G.; Kaminsky, W.; Fahrbach, G. Hydrolysis of poly-
(ethylene terephthalate in a ﬂuidized bed reactor. Polymer
Degrad. Stab. 2004,85, 571–575.
(10) Kaminsky,W.; Kim, S. Pyrolysis of mixed plastics into aromatics.
J. Anal. Appl. Pyrolysis 1999,51, 127–134.
(11) Yoshioka, T.; Grause, G.; Eger, C.; Kaminsky, W.; Okuwaki, A.
Pyrolysis of polyethylene terephthalate in a ﬂuidized bed plant.
Polymer Degrad. Stab. 2004,86, 499–504.
(12) Vogel, H.; Bonner, D. Acetylornithinase of E. coli: partial
puriﬁcation and some properties. J. Biol. Chem. 1956,218, 97–
(13) Braunegg, G.; Sonnleitner, B. Rapid gas chromatographic
method for the determination of poly-b-hydroxybutiric acid
in microbial biomass. Eur. J. Appl. Microbiol. 1978,6, 29–37.
(14) Ward, P.; de Roo, G.; O’Connor, K. Accumulation of polyhy-
droxyalkanoate from styrene and phenylacetic acid by Pseudomo-
nas putida CA-3. Appl. Environ. Microbiol. 2005,71 (4), 2046–
(15) Scheiner, D. Determination of Ammonia and Kjeldahl nitrogen
by indophenol method. Water Res. 1976,10, 31–36.
(16) Galego,N.; Rozsa, C.; Sanchez, R.; Fung, F.; Vazquez, A.; Tomas,
J. Characterization and application of poly(b-hydroxyalkanoates)
family as composite biomaterials. Polymer Testing 2000,19,
(17) Nikodinovic, J.; Barrow, K.; Chuck, J. High yield preparation of
genomic DNA from Streptomyces.Biotechniques 2003,35 (5),
(18) Lane, D., 16S/23S rRNA Sequencing. John Wiley & Sons:
Chichester, UK, 1991; p 115-175.
(19) Altschul,S.; Madden, T.; Schaffer, A.; Zhang, J.; Zhang, Z.; Miller,
W.; Lipman, D. Gapped BLAST and PSI-BLAST: a new generation
of protein database search programs. Nucleic Acids Res. 1997,
(20) Junker, F.; Saller, E.; Schlaﬂi Oppenberg, H.; Kroneck, P.;
Leisinger, T.; Cook, A. Degradative pathways for p-toluenecar-
boxylate and p-toluenesulfonate and their multicomponent
oxygenases in Comamonas testosteroni strains PSB-4 and T-2.
Microbiology 1996,142, 2419–2427.
(21) Patrauchan, M.; Florizone, C.; Dosanjh, M.; Mohn, W.; Davies,
J.; Eltis, L. Catabolism of benzoate and phthalate in Rhodococcus
sp. strain RHA1: redundancies and convergence. J. Bacteriol.
2005,187 (12), 4050–4063.
(22) Shigematsu, T.; Yumihara, K.; Ueda, Y.; Morimura, S.; Kida,
K. Puriﬁcation and gene cloning of the oxygenase component
of the terephthalate 1,2-dioxygenase system from Delftia
tsuruhatensis strain T7. FEMS Microbiol. Lett. 2003,220 (2),
(23) Sugimori, D.; Dake, T.; Nakamura, S. Microbial degradation of
disodium terephthalate by alkaliphilic Dietzia sp. strain GS-1.
Biosci. Biotechnol. Biochem. 2000,12, 2709–2711.
(24) Wang,Y.; Zhou, Y.; Zylstra, G. Molecular analysis of isophthalate
and terephthalate degradation by Comamonas testosteroni YZW-
D. Environ. Health Perspect. 1995,103 (5), 9–12.
(25) Tobin, K.; O’Connor, K. Polyhydroxyalkanoate accumulating
diversity of Pseudomonas species utilizing aromatic hydrocar-
bons. FEMS Microbiol. Lett. 2005,253 (11), 111–118.
(26) Andersen, S.; Johnsen, K.; Sorensen, J.; Nielsen, P.; Jacobsen, C.
Pseudomonas frederiksbergensis sp. nov., isolated from soil at
TABLE 4.Properties of PHA Polymer Extracted from P. putida
GO16, P. putida GO19, and P. frederiksbergensis GO23
(°C) MW MN PD
GO 16 12 35 -53 7.4 ×1043.7 ×1041.97 26.8
GO 19 10 34 -53 12.3 ×1045.2 ×1042.37 18.7
GO 23 11 35 -53 9.3 ×1044.4 ×1042.10 31.1
7700 9ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 42, NO. 20, 2008
a coal gasiﬁcation site. Intern. J. Syst. Evol. Microbiol. 2000,50
(27) Fukui, T.; Kato, M.; Matsusaki, H.; Iwata, T.; Doi, Y. Morphological
and 13C-nuclear magnetic resonance studies for polyhydroxy-
alkanoate biosynthesis in Pseudomonas sp. 61-3. FEMS Mi-
crobiol. Lett. 1998,164, 219–225.
(28) Gross, R.; DeMello, C.; Lenz, R.; Brandl, H.; Fuller, R. The
biosynthesis and characterization of poly (β-hydroxyalkanoates)
produced by Pseudomonas oleovorans.Macromolecules 1998,
(29) Hab, E.; Vidal-Mas, J.; Bassas, M.; Espuny, M.; Llorens, J.;
Manresa, A. Poly 3-(hydroxyalkanoates) produced from oily
substrates by Pseudomonas aeruginosa 47T2 (NCBIM 40044):
Effect of nutrients and incubation temperature on polymer
composition. Biochem. Eng. J. 2007,35, 99–106.
(30) Sanchez, R.; Schripsemaa, J.; da Silva, L.; Taciro, M.; Pradella,
J.; Gomez, J. Medium-chain-length polyhydroxyalkanoic acids
(PHAmcl) produced by Pseudomonas putida IPT 046 from
renewable sources. Eur. Polym. J. 2003,39, 1385–1394.
(31) Yoshie, N.; Inoue, Y., Structure, Composition and Soultion
Properties of PHA’s; Wiley-VCH: Weinheim, Germany, 2002; Vol
2, p 133-157.
(32) Jiang, X.; Ramsey, J.; Ramsay, B. Acetone extraction of mcl-PHA
from Pseudomonas putida KT2440. J. Microbiol. Methods 2006,
(33) Rabek,J., Experimental Methods in Polymer Chemistry: Physical
Principles and Applications. Wiley: New York, 1980; p 507.
(34) Ouyang, S.; Luo, R.; Chen, S.; Liu, Q.; Chung, A.; Wu, Q.; Chen,
G. Production of polyhydroxyalkanoates with high 3-Hydroxy-
dodecanoate monomer content by fadB and fadA knockout
mutant of Pseudomonas putida KT2440. Biomacromolecules
(35) Valappil, S.; Misra, S.; Boccaccini, A.; Roy, I. Biomedical
applications of polyhydroxyalkanoates, an overview of animal
testing an in vivo responses. Expert Rev. Med. Dev. 2006,3(6),
(36) Misra, S.; Valappil, S.; Roy, I.; Boccaccini, A. Polyhydroxyal-
kanoate (PHA)/inorganic phase composites for tissue engineer-
ing applications. Biomacromolecules 2006,7, 2249–2258.
(37) Chen, G.-Q.; Wu, Q. Microbial production and applications of
chiral hydroxyalkanoates. Appl. Microbiol. Biotechnol. 2005,67,
VOL. 42, NO. 20, 2008 / ENVIRONMENTAL SCIENCE & TECHNOLOGY 97701