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OPEN Direct fermentative conversion

of poly(ethylene terephthalate)
into poly(hydroxyalkanoate)
by Ideonella sakaiensis
Ryoga Fujiwara 1, Rikako Sanuki 2, Hiroharu Ajiro 3, Toshiaki Fukui 4 & Shosuke Yoshida 1,5*
Poly(ethylene terephthalate) (PET) is a widely used plastic in bottles and fibers; its waste products
pollute the environment owing to its remarkable durability. Recently, Ideonella sakaiensis 201-
F6 was isolated as a unique bacterium that can degrade and assimilate PET, thus paving the
way for the bioremediation and bioconversion of PET waste. We found that this strain harbors a
poly(hydroxyalkanoate) (PHA) synthesis gene cluster, which is highly homologous with that of
Cupriavidus necator, an efficient PHA producer. Cells grown on PET accumulated intracellular PHA
at high levels. Collectively, our findings in this study demonstrate that I. sakaiensis can mediate
the direct conversion of non-biodegradable PET into environment-friendly plastic, providing a new
approach for PET recycling.

Petroleum-based plastics are used extensively worldwide. However, owing to their inertness, plastics entering
the environment are tough to degrade, and their accumulation has serious negative ­impacts1. Recent reports on
microbial degradation of plastics including p ­ olyethylene2, ­polystyrene3, and poly(ethylene terephthalate) (PET)4
highlight these problems, but the number of such reports is limited. Particularly, mechanisms underlying PET
biodegradation are best known by analyzing Ideonella sakaiensis strain 201-F64. This bacterium uses two unique
enzymes, PET hydrolase (PETase) and mono(2-hydroxyethyl) terephthalic acid (MHET) hydrolase (MHETase),
to hydrolyze PET into terephthalic acid (TPA) and ethylene glycol (EG) monomers, which are then assimilated.
Biodegradable plastics can be developed as more environmentally compatible and sustainable alternatives
to common plastics. Poly(hydroxyalkanoate) (PHA), a polyester synthesized by various microorganisms for
intracellular carbon and energy storage, is a promising biodegradable plastic, given its excellent b­ iodegradability5.
A representative PHA-producing strain, Cupriavidus necator H16 can produce and accumulate massive quantities
of the PHA poly(3-hydroxybutyrate) (PHB), of up to ≈80 w/w% of dry cell weight (DCW, ≈4 g/L culture),
using plant oils as the sole carbon source for ­growth6. A critical challenge in microbial fermentation-mediated
biodegradable plastic production is lowering the carbon source cost, as it influences the product p ­ rice5. PET is
cheap and abundant but deleterious to the environment; thus, the digestion of PET is expected to be a useful
carbon source for PHA production. Kenny et al.7 isolated Pseudomonas species, including the GO16 strain,
which can accumulate PHAs with monomers comprising more than six carbons (medium chain-length PHAs)
from TPA. These strains, when cultured using TPA derived from PET pyrolysis products, accumulated PHA at
≈25 w/w% of DCW (≈1 g/L culture), thereby achieving a PHA production of 0.25 g/L. Tiso et al.8 modified the
GO16 strain for EG and TPA assimilation. They cultured the strain with TPA and EG, which were obtained by
complete PET hydrolysis using a thermostable polyester hydrolase, to obtain PHAs at 7 w/w% of DCW (2.3 g/L
culture) with a PHA production of 0.15 g/L. Here, we investigated the use of I. sakaiensis for directly converting
PET into PHA, which does not require prior PET hydrolysis.

1
Graduate School of Biological Science, Nara Institute of Science and Technology, 8916‑5, Takayama‑cho, Ikoma,
Nara 630‑0192, Japan. 2Department of Applied Biology, Kyoto Institute of Technology, Saga Ippongi‑cho 1,
Ukyo‑ku, Kyoto 616‑8354, Japan. 3Division of Materials Science, Graduate School of Science and Technology, Nara
Institute of Science and Technology, 8916‑5, Takayama‑cho, Ikoma, Nara 630‑0192, Japan. 4School of Life Science
and Technology, Tokyo Institute of Technology, 4259 Nagatsuta, Midori‑ku, Yokohama 226‑8501, Japan. 5Division
for Research Strategy, Institute for Research Initiatives, Nara Institute of Science and Technology, 8916‑5,
Takayama‑cho, Ikoma, Nara 630‑0192, Japan. *email: [email protected]

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Results and discussion

We found a gene cluster on the Ideonella sakaiensis 201-F6 genome essential for converting acetyl coenzyme
A (acetyl-CoA) into PHAs (Fig. 1). The cluster comprises phaC, phaA, and phaB encoding PHA synthase,
β-ketothiolase, and acetoacetyl-CoA reductase, respectively. The amino acid sequences of these proteins are
highly similar to those of the proteins produced by C. necator, indicating that this bacterium is a potential PHB
producer. I. sakaiensis was cultured on PET granules in a minimal medium (yeast extract-sodium carbonate-
vitamins-oyster shell [YSVO] medium) for 3, 6, and 9 days, and stained with Nile red, a lipophilic fluorescent
dye. Microscopy revealed that fluorescence intensity in the intracellular regions increased over time with the
highest abundance and intensity on day 6 (Fig. 2a,c). This was likely to have affected the bacterial cell area,
which increased 1.3-fold between days 3 and 6 (Fig. 2a,b). Transmission electron microscopy (TEM) of the
6-day-cultured cells showed multiple large granules in the cytoplasm (Fig. 2d), which were consistent with
those observed using fluorescence microscopy (Fig. 2a). The chloroform-soluble fraction was extracted from the
lyophilized I. sakaiensis cells cultured on PET and analyzed via proton nuclear magnetic resonance (1H NMR)
spectroscopy and size exclusion chromatography (SEC) to determine the chemical structure and molecular
weight, respectively. The 1H NMR spectrum showed typical signals of poly(3-hydroxybutyrate) (PHB), as noted
­previously9 (Fig. 3a). SEC revealed a number-average-molecular weight (Mn) of 3.2 × ­105 and a weight-average-
molecular weight (Mw) of 8.0 × ­105 (Fig. 3b), which are similar to that of PHB produced by C. necator (Mn,
4.0 × ­105; Mw, 1.0 × ­106)6. To quantify the PHA content, I. sakaiensis was cultured in PET granules-added YSVO
medium while monitoring the DCW. We have previously shown that I. sakaiensis degrades PET films, exhibited
by a significant reduction in their ­weight4 and that it causes an increase in the proportion of the functional
(hydroxyl and carboxyl) groups through surface h ­ ydrolysis10. However, the amount of PET hydrolysates in
the culture supernatant was small (0.003% of MHET units of the degraded PET), indicating their rapid uptake
into the cell. Therefore, we used the weight loss of PET as another measure of the degradation. I. sakaiensis
exhibited minimal growth on the culture medium without PET, with the highest DCW of 0.040 g/L, but much
higher growth on the medium with PET with DCW of up to 1.6 ± 0.1 g/L, concurrent with a PET weight loss
of 7.6 ± 0.6 g/L (Fig. 4a), indicating that PET was a major carbon source for its growth; oyster shell, consisting
mostly of calcium carbonate and various trace amounts of m ­ inerals11, has little to no factors that contribute
to cell growth. The PHB content was quantified by performing gas chromatography (GC) of the lyophilized
cells after methanolysis. Methyl 3-hydroxybutyrate (3HB) (~ 100 mol%) along with trace amounts of methyl
3-hydroxyvalerate (3HV) were detected among the potential methylated-PHA monomers (Table 1), indicating
that the synthesized PHA was nearly a PHB homopolymer. The highest amount of accumulated PHA was
observed on day 6, accounting for 48 ± 5 w/w% of the DCW (Fig. 4b, Table 1). The highest PHA production
rate was 0.75 ± 0.09 g/L on day 6. Subsequently, the carbon yield of PHA from PET on day 6 was calculated
as 9.0 ± 1.5 % (Table 1), indicating that a majority of the carbon in PET was converted into other compounds
including carbon dioxide through respiration. We further examined cultivation of I. sakaiensis in YSVO medium
supplemented with the PET monomer(s); 0.5 w/v% disodium terephthalate (TPA-2Na), 0.5 w/v% EG, or a
mixture of both. The DCWs were significantly higher when compared to the culture without carbon source:
0.21 ± 0.03 g/L on TPA-2Na, 0.15 ± 0.02 g/L on EG, and 0.33 ± 0.02 g/L on TPA-2Na/EG for 2 days, and small
amounts of PHA (< 5.0 wt%) were detected (Fig. 5 and Table 1). These facts indicated that I. sakaiensis was able
to incorporate extracellular TPA and EG and then metabolized them to acetyl-CoA and other intermediates.
However, considering much high cell growth and PHA accumulation on PET, the cellular ability to assimilate
the monomer(s) appeared to be poor. One of explanations for the observations would be better preference of the
uptake system of I. sakaiensis to PET hydrolytic oligomer products rather than TPA and EG.
In conclusion, we demonstrated that I. sakaiensis could not only metabolize PET, but also produce PHA,
and that these two pathways were functionally linked. This information can contribute to the development
of new PHA production pathways, which can help reduce plastic pollution and increase the production of
inexpensive biodegradable plastics and PET recycling. Compared with previous studies on PHA production
from the digestion of PET by Pseudomonas ­GO167 and the modified s­ train8, I. sakaiensis produced 3.0- and
5.0-fold more PHA, respectively, from PET. However, scaling up the production levels to an industrial scale
remains challenging. The metabolic and fermentation engineering strategies previously applied for C. necator,
which enable the production of PHA copolymer (> 100 g/L) by high-density fed-batch cultivation under nitrogen
­limitation12, offers a potentially useful approach.

Figure 1.  Conservation of poly(hydroxyalkanoate) (PHA) synthesis gene clusters on the genomes of I.

sakaiensis 201-F6 (GenBank Accession no. NZ_BBYR01000000) and C. necator H16 (NZ_CP039287). Open
reading frame numbers without locus tag prefixes (ISF6 for I. sakaiensis; H16 for C. necator) are indicated in the
genes. Gene products and amino acid sequence identities are indicated between the genes.

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Figure 2.  Microscopy of I. sakaiensis grown on poly(ethylene terephthalate) (PET). (a) Bright field and
fluorescence microscopy of Nile red-stained I. sakaiensis cells after growth on PET for 0 (pre-cultured cells in
802 nutrient-rich medium for 1 day), 3, 6, and 9 days. Box-and-whisker plots show the distribution of cell areas
(b) and average fluorescence intensity of Nile red in cells (c); median values, 25th to 75th percentiles, and range
are shown. n, number of cells or fluorescent dot assemblies. ND, not detected. Different letters above the plots
indicate significant differences among groups (Tukey’s multiple comparisons test, P < 0.0001). (d) Transmission
electron microscopy image of cells grown on PET granules for 6 days. Scale bar, 600 nm.

Methods
Culture
I. sakaiensis (NITE Biological Resource Center, NBRC 110686) was cultured as described ­previously4,13 with
several modifications. Briefly, I. sakaiensis was pre-cultured in NBRC 802 medium (1.0 w/v% polypeptone, 0.2
w/v% yeast extract, and 0.1 w/v% M ­ gSO4) at 30 °C and harvested by centrifugation (5000×g, 5 min, 4 °C). The
cell pellet was resuspended in YSV medium (0.01 w/v% yeast extract, 0.02 w/v% sodium hydrogen carbonate,
0.1 w/v% ammonium sulfate, 0.01 w/v% calcium carbonate, 0.1 v/v% vitamin mixture [0.25 w/v% thiamine-
HCl, 0.005 w/v% biotin, and 0.05 w/v% vitamin B12], and 1 v/v% trace elements [0.1 w/v% ­FeSO4·7H2O, 0.1
w/v% ­MgSO4·7H2O, 0.01 w/v% C ­ uSO4·5H2O, 0.01 w/v% M ­ nSO4·5H2O, and 0.01 w/v% Z ­ nSO4·7H2O] in 10 mM
phosphate buffer; pH 7.4). The suspension was inoculated into a Petri dish (90 mm in diameter) containing

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Figure 3.  Analyses of chloroform-soluble fraction of I. sakaiensis cells grown on poly(ethylene terephthalate)

(PET) granules for 6 days. (a) Proton nuclear magnetic resonance (1H NMR) spectrum. Inlet shows the
chemical structure of poly(3-hydroxybutyrate) (PHB). (b) Size exclusion chromatography (SEC). Mn, number
average molecular weight; Mw, weight average molecular weight; PDI, polydispersity index. Molecular weight
values were calculated by comparison with poly(methylmethacrylate) (PMMA) calibration standards.

30 mL YSV medium supplemented with ≈ 300 mg of oyster shells as a pH adjusting agent (YSVO medium) in
the presence or absence of 10 g (≈ 560 pieces) of PET granules shaped like an elliptic cylinder [2 (minor axis) × 3
(major axis) × 3 (height) mm]; 5.8% crystallinity determined by differential scanning calorimetry (Bell Polyester
Products, Inc., Yamaguchi, Japan), 0.5 w/v% of disodium terephthalate (TPA-2Na), EG, or a mixture of 0.5
w/v% TPA-2Na and 0.5 w/v% EG as the carbon source to adjust the absorbance measured at 660 nm to 0.002.
The culture dish was placed in a reciprocal shaker set at 50 strokes/min at 30 °C. After filtering the culture fluid
through a 5 µm pore size poly-vinylidene difluoride filter (Merck Millipore, Billerica, MA) to remove the small
broken pieces of oyster shells, cells were harvested, lyophilized using an FZ-2.5 freeze-dry system (Labconco,
Kansas City, MO), and weighed. As a portion of the cells was trapped by the filter, the DCW was corrected
using the amounts of protein before and after filtration. Briefly, cells were mixed with an equal volume of 10
w/v% sodium dodecyl sulfate (SDS) and heated at 90 °C for 10 min to lyse the cells and solubilize the cellular
protein, and the protein concentration was determined with a DC protein assay kit (BioRad, Hercules, CA) with
comparison to a calibration curve using bovine serum albumin. The PET granules were washed with 1 w/v% SDS,
distilled water, and then ethanol. After drying in air and a desiccator, the granules were weighed to determine
the weight reduction.

Fluorescence microscopy
PHA that accumulated in the cells was stained with Nile red (Fujifilm Wako Pure Chemical, Osaka, Japan)14.
A glass slide was coated with 0.01 w/v% poly-l-lysine (Sigma-Aldrich, St. Lois, MO) for cell attachment. The
culture suspension (100 µL) was placed on the poly-l-lysine-coated glass slide for 10 min and then aspirated. The
cells attached to the glass slide were incubated with a drop of 10 µg/mL Nile red in D-PBS(−) (Nacalai Tesque,
Kyoto, Japan) for 30 min, washed with D-PBS(−), mounted with Prolong Diamond antifade medium (Thermo
Fisher Scientific, Waltham, MA) for immobilization, and covered with a coverslip. Cells were observed using
a fluorescence microscope (BZ-X800, Keyence, Osaka, Japan). The frame of each individual cell was manually
identified, and the cell area was calculated using the BZ-X800 Analyzer software (Keyence). PHA formation was

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Figure 4.  Time course of poly(hydroxyalkanoate) (PHA) production by I. sakaiensis grown on poly(ethylene

terephthalate) (PET). (a) Growth of I. sakaiensis on PET. The cells were cultured with and without PET granules
(10 g) in YSVO medium for 3, 6, and 9 days at 30 °C. Dry cell weight (DCW; blue closed squares, with PET; blue
open squares, without PET) and weight loss of PET granules (red closed triangles) were measured. (b) Time
course of PHA production. PHA production with and without PET shown as blue closed squares and open
squares, respectively. PHA content per DCW shown as red closed circles. Data are also shown in Table 1. Error
bars in represent the standard error among three independent biological replicates.

Composition
(mol%)
*Carbon yield of PHA from
Carbon source Cultivation time (days) DCW (g/L) PHA (g/L) PHA content (wt%) 3HB 3HV PET weight loss (g/L) PET (%)
3 1.3 ± 0.1 0.54 ± 0.05 42 ± 3 ~ 100 Trace 3.3 ± 0.1 15 ± 2
PET 6 1.6 ± 0.1 0.75 ± 0.09 48 ± 5 ~ 100 Trace 7.6 ± 0.6 9.0 ± 1.5
9 1.2 ± 0.0 0.32 ± 0.00 27 ± 1 ~ 100 Trace 7.9 ± 0.5 3.6 ± 0.2
TPA-2Na 2 0.21 ± 0.03 0.00059 ± 0.00006 0.28 ± 0.01 100 0
EG 2 0.15 ± 0.02 0.0073 ± 0.0006 4.9 ± 0.1 100 0
TPA-2Na/EG 2 0.33 ± 0.02 0.0033 ± 0.0002 1.0 ± 0.0 100 0
3 0.014 0.00039 2.8 100 0
None 6 0.039 0.0019 5.0 100 0
9 0.040 0.00022 0.56 100 0

Table 1.  PHA biosynthesis by Ideonella sakaiensis. Error bars represent the standard error among three
independent biological replicates. DCW dried cell weight, EG ethylene glycol, ND not detected, PET
poly(ethylene terephthalate), PHA poly(hydroxyalkanoate), TPA-2Na disodium terephthalate, 3HB
3-hydroxybutyrate, 3HV 3-hydroxyvalerate. *Calculated as [(carbon weight of PHA produced with PET—
carbon weight of PHA produced without PET)/carbon weight in degraded PET]×100%.

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Figure 5.  Growth of I. sakaiensis on poly(ethylene terephthalate) (PET) monomers. The cells were cultured
with or without 0.5 w/v% disodium terephthalate (TPA-2Na), 0.5 w/v% ethylene glycol (EG), and a mixture of
the two compounds (TPA-2Na/EG) in YSVO medium at 30 °C, and then their dry cell weight was measured.
Error bars in represent the standard error among three independent biological replicates.

visualized using a tetramethylrhodamine isothiocyanate filter set (λem ≈ 554 nm, λex ≈ 570 nm). The fluorescence
intensity of the Nile red-stained dot assembly was calculated on average using the BZ-X800 Analyzer software.

TEM
The I. sakaiensis cells grown on PET granules for 6 days were harvested by centrifugation, fixed with phosphate-
buffered 2% glutaraldehyde, and post-fixed with 2% osmium tetraoxide for 3 h on ice. Next, the samples were
dehydrated using an ethanol gradient and embedded in epoxy resin. Ultrathin sections of the specimen were
stained with uranyl acetate and lead, and subjected to TEM using the H-7600 transmission electron microscope
(Hitachi, Tokyo, Japan).

Polymer extraction and analyses

Samples were prepared as described ­previously15 with several modifications. The lyophilized cells of I.
sakaiensis were suspended in chloroform with vigorous stirring for 1 day, and the extract was filtered
through a polytetrafluoroethylene filter (pore size, 0.2 µm). The filtrate was mixed with twofold volume of
35% methanol:35% ethanol:30% water (v/v/v), and the precipitate was dried in vacuo and then dissolved in
chloroform. To determine the molecular weight of the polymer, the chloroform-soluble fraction was analyzed
using a size exclusion chromatography system equipped with a RI-2031Plus refractive detector (Jasco, Tokyo,
Japan) and two in-line TSKgel G
­ MHHR-M columns with a TSK-guard column ­HHR-H (Tosoh, Tokyo, Japan) at
40 °C. Chloroform was used as the mobile phase and passed through the column at a flow rate of 1 mL/min. The
molecular weight was estimated by comparison with poly(methylmethacrylate) (PMMA) standards (PMMA
calibration kit M-M-10, Agilent Technologies, Santa Clara, CA). To analyze the polymer chemical structure,
the chloroform-soluble fraction was further purified by precipitation in cold methanol. The precipitate was
dissolved in deuterated chloroform (­ CDCl3) and analyzed via proton nuclear magnetic resonance (1H NMR)
(JNM-ECX400P, Jeol, Tokyo, Japan).

PHA quantification and composition analysis

The cellular PHA content and composition were determined by GC after direct methanolysis of the dried cells in
the presence of 15% sulfuric acid at 100 °C for 140 min, as described ­previously16. The GC analysis was performed
using GC-2014 (Shimadzu, Kyoto, Japan) equipped with an InertCap 1 capillary column (30 m × 0.23 mm; GL
Sciences, Tokyo, Japan) and a flame ionization detector. PHA composition was further determined by mass
spectrometry.

Received: 23 June 2021; Accepted: 28 September 2021

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Acknowledgements
We thank Dr. Tsuyoshi Ando, Yumiko Kawakami, and Mari Nakagawa for research assistance.

Author contributions
S.Y. designed the study. R.F. conducted the growth experiments and analyzed the data. R.S. conducted
fluorescence microscopy and analyzed the data. H.A. and R.F. conducted the 1H NMR and SEC experiments
and analyzed the data. T.F. conducted the GC experiments and analyzed the data. S.Y., R.F, and T.F. wrote the
manuscript. All authors discussed the results and commented on the manuscript.

Competing interests
The authors declare no competing interests.

Additional information
Correspondence and requests for materials should be addressed to S.Y.
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