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Up-Cycling of PET (Polyethylene Terephthalate) to the Biodegradable Plastic PHA (Polyhydroxyalkanoate)


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The conversion of the petrochemical polymer polyethylene terephthalate (PET) to a biodegradable plastic polyhydroxyalkanoate (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.
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Up-Cycling of PET (Polyethylene
Terephthalate) to the Biodegradable
Plastic PHA (Polyhydroxyalkanoate)
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
value material.
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:
University College Dublin.
University of Hamburg.
Trinity College.
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.
Experimental Section
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
SE 52).
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
change, respectively.
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
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
both sources.
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 (%)
solids 77
terephthalic acid 51.0
oligomers 20.0
benzoic acid 1.0
others 5.0
oil 6.3
ethyleneglycol 0.75
acetic aldehyde 5.10
others 0.45
gases 18
co 3.5
hydrogen 0.18
ethene 1.0
others 0.34
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
GO23 AJ249382
frederiksbergensis 99 99
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
bacterial strain
P. putida GO16 27 1 21 48 14 16
P. putida GO19 23 1 23 45 14 17
P. frederiksbergensis
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.
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
reports (34).
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.
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... strain RHA1 degraded TPA via the PCA pathway and catechol pathway. Kenny et al. (2008) isolated three strains of the Pseudomonas genus that could utilize TPA to accumulate polyhydroxyalkanoate (PHA). Zhang et al. (2013) found that Arthrobacter sp. ...
... Through metabolically engineered microbes, TPA could be biotransformed into other added-value products, by various enzymes or pathways (Furtwengler and Avérous 2018;Salvador et al. 2019). Kenny et al. (2008) isolated three strains of the Pseudomonas genus, which could utilize TPA for growth and meanwhile accumulate large amount of PHA, a class of renewable and biodegradable polymer. After subsequent treatment, these Pseudomonas species were found to convert the TPA fraction to PHA with a maximal production rate at approximately 8.4 mg·L − 1 ·h − 1 . ...
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The polyethylene terephthalate (PET) is one of the major plastics with a huge annual production. Alongside with its mass production and wide applications, PET pollution is threatening and damaging the environment and human health. Although mechanical or chemical methods can deal with PET, the process suffers from high cost and the hydrolyzed monomers will cause secondary pollution. Discovery of plastic-degrading microbes and the corresponding enzymes emerges new hope to cope with this issue. Combined with synthetic biology and metabolic engineering, microbial cell factories not only provide a promising approach to degrade PET, but also enable the conversion of its monomers, ethylene glycol (EG) and terephthalic acid (TPA), into value-added compounds. In this way, PET wastes can be handled in environment-friendly and more potentially cost-effective processes. While PET hydrolases have been extensively reviewed, this review focuses on the microbes and metabolic pathways for the degradation of PET monomers. In addition, recent advances in the biotransformation of TPA and EG into value-added compounds are discussed in detail.
... The PET monomers TA and EG can be used as metabolic feedstocks for biochemical production pathways in engineered microbes. Recent examples include the engineering of bacterial strains capable of transforming TA and/or EG into value-added products such as β-ketoadipic acid, glycolic acid, and the bioplastics polyhydroxyalkanoate (PHA) and poly(amide urethane) [21][22][23][24][25]. ...
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Background Biocatalysis offers a promising path for plastic waste management and valorization, especially for hydrolysable plastics such as polyethylene terephthalate (PET). Microbial whole-cell biocatalysts for simultaneous PET degradation and growth on PET monomers would offer a one-step solution toward PET recycling or upcycling. We set out to engineer the industry-proven bacterium Pseudomonas putida for (i) metabolism of PET monomers as sole carbon sources, and (ii) efficient extracellular expression of PET hydrolases. We pursued this approach for both PET and the related polyester polybutylene adipate co -terephthalate (PBAT), aiming to learn about the determinants and potential applications of bacterial polyester-degrading biocatalysts. Results P. putida was engineered to metabolize the PET and PBAT monomer terephthalic acid (TA) through genomic integration of four tphII operon genes from Comamonas sp. E6. Efficient cellular TA uptake was enabled by a point mutation in the native P. putida membrane transporter MhpT. Metabolism of the PET and PBAT monomers ethylene glycol and 1,4-butanediol was achieved through adaptive laboratory evolution. We then used fast design-build-test-learn cycles to engineer extracellular PET hydrolase expression, including tests of (i) the three PET hydrolases LCC, HiC, and IsPETase; (ii) genomic versus plasmid-based expression, using expression plasmids with high, medium, and low cellular copy number; (iii) three different promoter systems; (iv) three membrane anchor proteins for PET hydrolase cell surface display; and (v) a 30-mer signal peptide library for PET hydrolase secretion. PET hydrolase surface display and secretion was successfully engineered but often resulted in host cell fitness costs, which could be mitigated by promoter choice and altering construct copy number. Plastic biodegradation assays with the best PET hydrolase expression constructs genomically integrated into our monomer-metabolizing P. putida strains resulted in various degrees of plastic depolymerization, although self-sustaining bacterial growth remained elusive. Conclusion Our results show that balancing extracellular PET hydrolase expression with cellular fitness under nutrient-limiting conditions is a challenge. The precise knowledge of such bottlenecks, together with the vast array of PET hydrolase expression tools generated and tested here, may serve as a baseline for future efforts to engineer P. putida or other bacterial hosts towards becoming efficient whole-cell polyester-degrading biocatalysts.
... By distinct dioxygenases, protocatechuate may be degraded via ortho-, meta-, and para-cleavage pathways, rendering metabolites that will eventually be converted into acetyl-CoA and succinyl-CoA, which channel into the tricarboxylic acid (TCA) cycle for forming succinic acid (Hosaka et al., 2013;Salvador et al., 2019;Ru et al., 2020). P. putida GO16, P. putida GO19, and Pseudomonas frederiksbergensis GO23 are able to both metabolize and accumulate TPA, polymerizing medium chains of polyhydroxyalkanoate (PHA; Kenny et al., 2008). EG may be metabolized by acetogens pathway, where it is degraded to ethanol and acetaldehyde, then transformed to acetate via acetyl-CoA (Trifunović et al., 2016). ...
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Anthropogenic activities have extensively transformed the biosphere by extracting and disposing of resources, crossing boundaries of planetary threat while causing a global crisis of waste overload. Despite fundamental differences regarding structure and recalcitrance, lignocellulose and plastic polymers share physical-chemical properties to some extent, that include carbon skeletons with similar chemical bonds, hydrophobic properties, amorphous and crystalline regions. Microbial strategies for metabolizing recalcitrant polymers have been selected and optimized through evolution, thus understanding natural processes for lignocellulose modification could aid the challenge of dealing with the recalcitrant human-made polymers spread worldwide. We propose to look for inspiration in the charismatic fungal-growing insects to understand multipartite degradation of plant polymers. Independently evolved in diverse insect lineages, fungiculture embraces passive or active fungal cultivation for food, protection, and structural purposes. We consider there is much to learn from these symbioses, in special from the community-level degradation of recalcitrant biomass and defensive metabolites. Microbial plant-degrading systems at the core of insect fungicultures could be promising candidates for degrading synthetic plastics. Here, we first compare the degradation of lignocellulose and plastic polymers, with emphasis in the overlapping microbial players and enzymatic activities between these processes. Second, we review the literature on diverse insect fungiculture systems, focusing on features that, while supporting insects' ecology and evolution, could also be applied in biotechnological processes. Third, taking lessons from these microbial communities, we suggest multidisciplinary strategies to identify microbial degraders, degrading enzymes and pathways, as well as microbial interactions and interdependencies. Spanning from multiomics to spectroscopy, microscopy, stable isotopes probing, enrichment microcosmos, and synthetic communities, these strategies would allow for a systemic understanding of the fungiculture ecology, driving to application possibilities. Detailing how the metabolic landscape is entangled to achieve ecological success could inspire sustainable efforts for mitigating the current environmental crisis.
... 276 However, TPA from PET pyrolysis can be used as a feedstock for bacterial production of the biodegradable plastic polyhydroxyalkanoate (PHA). 293 When incorporated in feedstock mixtures, PET increases the CO and CO2 concentrations in product gases. 207,294 While some investigators have concluded that PET is not suitable for plastic pyrolysis, 272,295 others advocate for it based on the valorization of product char to activated carbon and product oil to fuel. ...
Less than 10% of the plastics generated globally are recycled, while the rest are incinerated, accumulated in landfills, or leach into the environment. New technologies are emerging to chemically recycle waste plastics that are receiving tremendous interest from academia and industry. Chemists and chemical engineers need to understand the fundamentals of these technologies to design improved systems for chemical recycling and upcycling of waste plastics. In this paper, we review the entire life cycle of plastics and options for the management of plastic waste to address barriers to industrial chemical recycling and further provide perceptions on possible opportunities with such materials. Knowledge and insights to enhance plastic recycling beyond its current scale are provided. Outstanding research problems and where researchers in the field should focus their efforts in the future are also discussed.
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The aim of this study was to investigate the pyrolysis products of polyhydroxyalkanoates (PHAs), polyethylene terephthalate (PET), carbon fiber reinforced composite (CFRC), and block co-polymers (PS-b-P2VP and PS-b-P4VP). The studied PHA samples were produced at temperatures of 15 and 50 oC (PHA15 and PHA50), and commercially obtained from GlasPort Bio (PHAc). Initially, PHA samples were analyzed by nuclear magnetic resonance (NMR) spectroscopy and size exclusion chromatography (SEC) to determine the molecular weight, and structure of the polymers. Thermal techniques such as thermogravimetry (TG) and differential scanning calorimetry (DSC) analyses were performed for PHA, CFRC, and block co-polymers to investigate the degradation temperature range and thermal stability of samples. Fast pyrolysis (500 oC, ∼10² °C s⁻¹) experiments were conducted for all samples in a wire mesh reactor to investigate tar products and char yields. The tar compositions were investigated by gas chromatography–mass spectrometry (GC–MS), and statistical modeling was performed. The char yields of block co-polymers and PHA samples (< 2 wt. %) were unequivocally less than that of the PET sample (~10.7 wt. %). All PHA compounds contained a large fraction of ethyl cyclopropane carboxylate (~ 38-58%), whereas PAH15 and PHA50 additionally showed a large quantity of 2-butenoic acid (~8-12%). The PHAc sample indicated the presence of considerably high amount of methyl ester (~15%), butyl citrate (~12.9%), and tributyl ester (~17%). The compositional analysis of the liquid fraction of the PET and block co-polymers have shown carcinogenic and toxic properties. Pyrolysis removed matrices in the CRFC composites which is an indication of potential recovery of the original fibers.
The grand challenge in chemical recycling of colored poly(ethylene terephthalate) (PET) wastes is to achieve cost-effective decolorization and purification of products. In the present work, an effective method combining hydrolysis, reactive processing and decoloration was developed to convert colored PET fabrics into high purity terephthalic acid (TPAca). Identification of intermediates and quantification of products were carried out by a range of characterizations, including SEM, EDS, TGA, FTIR, XRD, NMR and GPC. The color removal efficiency and selectivity and yield of the synthesized TPAca were calculated with the assistance of UV–vis, K/S values and chromaticity diagram to evaluate the viability of the method. It is found that disperse dyes with different structures (azo and anthraquinone) can be decolorized simultaneously with PET degradation under the joint effects of sublimation, pyrolysis and hydrolysis. In the process, calcium terephthalate (CaTP) acts as an essential medium to store and protect the synthesized TPAca. Overall, the purity of the obtained TPAca is comparable to that of commercial TPA. The monomer yield (88.51%) and decolorization rate (94.22–97.65%) are higher than TPAna produced from traditional hydrolysis. Along with future development towards sustainability, we anticipate that this method could be utilized as a basis for recycling and upcycling of complex colored PET wastes at an industrial level.
The chemical recycling of plastic wastes into value-added products is an attractive strategy to reduce the consumption of fossil fuels and reduce the plastic pollution. We report here the facile upcycling of poly(3-hydroxybutyrate) (P3HB) into value-added polymerizable monomers and subsequent polymerization toward degradable and recyclable polymers. The bicyclic monomer 4-methyloctahydro-2H-benzo[b][1,4]dioxepin-2-one (4-MOHB) was first synthesized from P3HB through efficient steps. The obtained monomers underwent bulk ring-opening polymerization (ROP) catalyzed by stannous octoate (Sn(Oct)2) to give the amorphous materials. With benzyl alcohol (BnOH) as an initiator, Sn(Oct)2 can effectively catalyze the ROP of bicyclic ether-ester monomers in a controllable fashion. Moreover, poly(4-methyloctahydro-2H-benzo[b][1,4]dioxepin-2-one) (P(4-MOHB)) showed a closed-loop recovery property due to the fused bicyclic monomer structure. The selective depolymerization of the P(4-MOHB) homopolymer back to 4-MOHB monomer can be easily realized using p-toluenesulfonic acid (TsOH) or Sn(Oct)2 as a catalyst in solution or in bulk. This strategy to recycle bioplastics into value-added materials has a promising application prospect and is beneficial to extend the life cycle of environment-friendly materials.
One-third of food produced for human consumption is lost or wasted globally, which amounts to about 1.3 billion tons per year. Food loss and waste (FLW) causes serious social, economic, and environmental issues undermining our planet’s sustainability. Therefore actions aimed at FLW prevention and reduction must be urgently taken. The unsustainability of the current economic model, based on a linear system of production and consumption, has emerged above all in the food sector, where little is being done to upcycle residues generated along the supply chain. In this context the shift to a circular economy (CE) model applied to this sector has become urgent, to promote the transition to a more sustainable agro-food system, moving toward a zero-waste industry. Recently, scientific research is examining the adoption of CE models, thanks to the “Sustainable Development Goals” publication. In this chapter, a review of the literature focused on the CE approach along the whole food production chain was performed, highlighting its benefits, with the perspective of developing a closed-loop agri-food system. Finally, we look at the key role played by nonconventional membrane process technology that constitutes a promising tool for waste valorization through the recovery of high-added-value compounds.
The vast majority of commodity plastics do not degrade and therefore they permanently pollute the environment. At present, less than 20% of post-consumer plastic waste in developed countries is recycled, predominately for energy recovery or repurposing as lower-value materials by mechanical recycling. Chemical recycling offers an opportunity to revert plastics back to monomers for repolymerization to virgin materials without altering the properties of the material or the economic value of the polymer. For plastic waste that is either cost prohibitive or infeasible to mechanically or chemically recycle, the nascent field of chemical upcycling promises to use chemical or engineering approaches to place plastic waste at the beginning of a new value chain. Here state-of-the-art methods are highlighted for upcycling plastic waste into value-added performance materials, fine chemicals and specialty polymers. By identifying common conceptual approaches, we critically discuss how the advantages and challenges of each approach contribute to the goal of realizing a sustainable plastics economy. Methods for the transformation of plastics into materials with value, known as plastic waste upcycling, are outlined, and their advantages and challenges in terms of a sustainable plastics economy are discussed.
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The biosynthesis of poly(β-hydroxyalkanoates) (PHA's) by P. oleovorans was carried out by using the sodium salt of n-alkanoic acids as carbon sources. The PHA's produced contained at least two major (>5 mol%) monomer units. That is, depending on the carbon source used, the PHA's can have n-alkyl pendant groups with chain lengths from methyl to nonyl. The maximum cellular yield and polymer content (in percent of the cellular dry weight) obtained were 1.5 g/L and 49%, respectively, using nonanoate as the carbon source. The M̄w values of these poly-β-esters measured by GPC ranged from 160,000 to 360,000.
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Medium-chain-length polyhydroxyalkanoates are produced by Pseudomonas putida strain IPT046 growing on carbohydrates. Analysis by gas chromatography and nuclear magnetic resonance of the elastomeric material revealed that PHAmcl is composed from essentially hydroxydecanoate (60–70%) and hydroxyoctanoate (20–25%) sequence units with a non-terminal double bond in about 6% of the side chains. The average molecular weight of PHAmcl is 223 kDalton and the X-ray diffractogram showed that 24% of the solid phase is crystalline. This biodegradable polyester presents a relative low glass transition temperature (−39.7 °C) and melting point (56 °C), is thermically stable (234 °C) and displays appropriate thermomechanical properties for potential use as packaging film.
Polyhydroxyalkanoates (PHAs), of which polyhydroxybutyrate (PHB) is the most abundant, are bacterial carbon and energy reserve materials of widespread occurrence. They are composed of 3-hydroxyacid monomer units and exist as a small number of cytoplasmic granules per cell. The properties of the C4 homopolymer PHB as a biodegradable thermoplastic first attracted industrial attention more than 20 years ago. Copolymers of C4 (3-hydroxybutyrate [3HB]) and C5 (3-hydroxyvalerate [3HV]) monomer units have modified physical properties; e.g., the plastic is less brittle than PHB, whereas PHAs containing C8 to C12 monomers behave as elastomers. This family of materials is the centre of considerable commercial interest, and 3HB-co-3HV copolymers have been marketed by ICI plc as Biopol. The known polymers exist as 2(1) helices with the fiber repeat decreasing from 0.596 nm for PHB to about 0.45 nm for C8 to C10 polymers. Novel copolymers with a backbone of 3HB and 4HB have been obtained. The native granules contain noncrystalline polymer, and water may possibly act as a plasticizer. Although the biosynthesis and regulation of PHB are generally well understood, the corresponding information for the synthesis of long-side-chain PHAs from alkanes, alcohols, and organic acids is still incomplete. The precise mechanisms of action of the polymerizing and depolymerizing enzymes also remain to be established. The structural genes for the three key enzymes of PHB synthesis from acetyl coenzyme A in Alcaligenes eutrophus have been cloned, sequenced, and expressed in Escherichia coli. Polymer molecular weights appear to be species specific. The factors influencing the commercial choice of organism, substrate, and isolation process are discussed. The physiological functions of PHB as a reserve material and in symbiotic nitrogen fixation and its presence in bacterial plasma membranes and putative role in transformability and calcium signaling are also considered.
Chemical recycling of poly(ethylene terephthalate) (PET) materials was carried out by pyrolysis with a fluidised bed reactor between 510 and 730°C. Several PET materials, with and without fillers, were successfully decomposed. Almost all products were gases and solids. The yield of gases was between 38 and 49wt%. Pyrolysis gas consisted chiefly of CO2 and CO. Other gases like methane, ethane and hydrogen occurred in small yields. The other major fraction was formed by solid products. Above all, the organic residues in the reactor increased strongly with temperature. Just 5wt% of organic residues were found in the reactor at a temperature of 510°C, but 40wt% at 730°C. The amount of oxygen containing compounds decreased with increasing temperature due to the decomposition of terephthalic acid and benzoic acid. The amount of liquid products was small. The aliphatic hydrocarbons in oil fraction were slight, but aromatic hydrocarbons were found between 2 and 5wt% of all organic products due to formation of benzene, benzene derivatives and naphthalene derivatives, respectively.
Pseudomonas sp. 61-3 is able to produce a blend of poly(3-hydroxybutyrate) [P(3HB)] homopolymer and a random copolymer [P(3HB-co-3HA)] consisting of 3-hydroxyalkanoate units of 4–12 carbon atoms. In a cell accumulating polyhydroxyalkanoates upon glucose or alkanoic acids, both needle-type and mushroom-type structures were observed as PHA granules by freeze-fracture electron microscopy, indicating that Pseudomonas sp. 61-3 synthesized and stored both P(3HB) and P(3HB-co-3HA) granules simultaneously as separate granules in the same cell. 13C-NMR analysis of polyhydroxyalkanoates synthesized from 13C-labeled octanoate revealed that 3-hydroxybutyrate units in the resultant polyhydroxyalkanoates were not only supplied via fatty acid β-oxidation but also via dimerization of two acetyl-CoA molecules in Pseudomonas sp. 61-3. Approximately 26% of 3-hydroxybutyrate units was found to be generated via dimerization of acetyl-CoA when octanoate was fed as a carbon source.
Pseudomonas sp. 61-3 is able to produce a blend of poly(3-hydroxybutyrate) [P(3HB)] homopolymer and a random copolymer [P(3HB-co-3HA)] consisting of 3-hydroxyalkanoate units of 4–12 carbon atoms. In a cell accumulating polyhydroxyalkanoates upon glucose or alkanoic acids, both needle-type and mushroom-type structures were observed as PHA granules by freeze-fracture electron microscopy, indicating that Pseudomonas sp. 61-3 synthesized and stored both P(3HB) and P(3HB-co-3HA) granules simultaneously as separate granules in the same cell. 13C-NMR analysis of polyhydroxyalkanoates synthesized from 13C-labeled octanoate revealed that 3-hydroxybutyrate units in the resultant polyhydroxyalkanoates were not only supplied via fatty acid β-oxidation but also via dimerization of two acetyl-CoA molecules in Pseudomonas sp. 61-3. Approximately 26% of 3-hydroxybutyrate units was found to be generated via dimerization of acetyl-CoA when octanoate was fed as a carbon source.
The hydrolysis of poly(ethylene terephthalate, PET) is the reverse of the direct esterification of terephthalic acid (TA) and ethylene glycol (EG). By this means the recovery of monomers is possible. These experiments have shown that 60%–72% of TA were recovered from virgin PET in a temperature range between 400 and 500 °C. Another 22%–27% TA remained in oligomers. The highest yield of TA (72%) was found at 450 °C. When real materials were used, still 60%–69% of TA were found at 450 °C. There was also a catalytic effect of transition metal oxides which reduced the content of TA in oligomers to 8%. The yield of EG was less than 10% in all experiments. Most EG reacted with water to form carbon oxides and hydrogen. Other decomposition products were acetaldehyde and ethene.