Up-Cycling of PET (Polyethylene
Terephthalate) to the Biodegradable
Plastic PHA (Polyhydroxyalkanoate)
S H A N E T . K E N N Y ,†
J A S M I N A N I K O D I N O V I C R U N I C ,†
W A L T E R K A M I N S K Y ,‡T R E V O R W O O D S ,§
R A M E S H P . B A B U ,§C H R I S M . K E E L Y ,§
W E R N E R B L A U ,§A N D
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, 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 carbon and energy were selected for further study. These
(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
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
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
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
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
investigating the conversion of PET to this desirable high
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
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.
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
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
* Corresponding author phone: +353 1 716 1307; fax +353 1 716
1183; e-mail: firstname.lastname@example.org.
†University College Dublin.
‡University of Hamburg.
Environ. Sci. Technol. 2008, 42, 7696–7701
76969ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 42, NO. 20, 200810.1021/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
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
maintained at a temperature of 450 °C. PET entered the
products passed a cyclone to be cleaned by small amounts
of fillers and then were cooled down by mixing up with cold
(Table 1) were desublimated to generate a white powder.
The sand bed was fluidized by steam with a flow rate of 2.5
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)
Bacterial Growth Medium. The minimal mineral salts
as the media supplemented with sodium terephthalate as
discussed in this work. The sodium terephthalate was
prepared by taking the solid fraction of PET pyrolysis and
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
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
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
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-
(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
analyzed on an Agilent 6890N series GC fitted with a 30 m
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
the previously described method of Scheiner (15).
Determination of Terephthalic Acid Utilization during
points and centrifuging the samples at 14 000g for 2 min.
The supernatant was retained, filtered, and analyzed by
HPLC. In order to analyze the sample and maintain a linear
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.
at 100.62 MHz. The solvent chloroform-d and tetramethyl-
silane (TMS) were used as internal references for chemical
recorded with proton-decoupling. Typically 2200 transients
were accumulated. Spectrometer peak areas were obtained
directly by standard signal integration.
calibrated to Indium standards. The samples weighing 7-8
and heated from -70 to 100 °C at a rate of 10 °C/min. To
were held at 100 °C for 1 min and rapidly quenched to -70
°C. The samples were then reheated from -70 to 100 °C at
10 °C/min to determine the melting temperature (Tm) and
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-
data. The temperature at the maximum point of the loss
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 9 7697
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
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-
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
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
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
species putida, which are known to degrade a wide variety
of aromatic compounds and more recently accumulate
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
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-
(as ammonium), TA concentration, cell dry weight and
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
it achieved the highest final cell dry weight and the highest
growth yield from TA. While benzoic acid is a minor
all three strains were capable of utilizing it as a sole carbon
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 compositionweight percentage (%)
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
GO19 AY512611 Pseudomonas putida
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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
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
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
GO16 and GO19 (Figure 1). Thus process development and
optimization of PHA production will be strain dependent.
PHA Composition. The1H NMR spectra for each PHA
derived for strains GO16, GO19, and GO23 were established
length PHA derivates (27-30). Terminal CH3protons were
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
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
13C spectrum for PHA produced from GO19 is
illustrated (Figure 2b). Chemical shift assignments are
structural units of 3-hydroxydodecenoic acid (3HDDE),
(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
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
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 MHz1H spectrum (b)13C
TABLE 3. Composition of PHA Accumulated from Terephthalic
(% CDW) 3HH 3HO 3HD 3HDDA 3HDDE
P. putida GO16
P. putida GO19
3HH ) 3-hydroxyhexanoic acid, 3HO ) 3-hydroxyoctan-
dodecanoic acid, 3HDDE ) 3-hydroxydodecenoic acid.
241 14 422221
VOL. 42, NO. 20, 2008 / ENVIRONMENTAL SCIENCE & TECHNOLOGY 9 7699
While the sequence homology of 16S rDNA indicated a
species have differing PHA accumulation abilities (25). The
PHA from P. putida GO23 contained a higher proportion of
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.
increased tackiness and malleability at room temperature
compared to plastic from GO16 and GO19.
in this study ranged in molecular weight (Mw) from 74 kDa
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
whereas PHA produced from longer-chain alkanoates ex-
changes in their Tm and ∆Hm values (Table 4).
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
All three PHA products had similar thermal degradation
with a high temperature shoulder most evident at ca. 350 °C
in the differential thermograph. Polymer degradation was
produced during thermal degradation being burnt at about
600 °C. Thus three thermoplastics were generated through
the chemo-biotecnological processing of PET.
that can offer a clean and cost-effective way of converting
PET to a high value polymer will generate a niche market in
as a carbon feedstock for the production of PHA, a biode-
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.
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