Bio-upcycling of polyethylene terephthalate

TLDR: A novel value-chain for PET upcycling is presented, adding technological flexibility to the global challenge of end-of-life management of plastics.

Abstract: Over 359 million tons of plastics were produced worldwide in 2018, with significant growth expected in the near future, resulting in the global challenge of end-of-life management. The recent identification of enzymes that degrade plastics previously considered non-biodegradable opens up opportunities to steer the plastic recycling industry into the realm of biotechnology. Here, we present the sequential conversion of polyethylene terephthalate (PET) into two types of bioplastics: a medium chain-length polyhydroxyalkanoate (PHA) and a novel bio-based poly(amide urethane) (bio-PU). PET films were hydrolyzed by a thermostable polyester hydrolase yielding 100% terephthalate and ethylene glycol. A terephthalate-degrading Pseudomonas was evolved to also metabolize ethylene glycol and subsequently produced PHA. The strain was further modified to secrete hydroxyalkanoyloxy-alkanoates (HAAs), which were used as monomers for the chemo-catalytic synthesis of bio-PU. In short, we present a novel value-chain for PET upcycling, adding technological flexibility to the global challenge of end-of-life management of plastics. Graphical abstract

Figures

Figure 5: Novel bio-PU synthesis. Polymerization reaction between the hydroxy fatty acid ester HAA, and MDI diisocyanate. The resulting prepolymer is submitted to chain extension with BDO to form a partly bio-based poly(amide urethane) (bio-PU).

Figure 2: PET film hydrolysis. Time courses of PET hydrolysis into (soluble) ethylene glycol (EG), terephthalic acid (TA), and mono-(2-hydroxyethyl)TA (MHET) (primary y-axis) catalyzed by purified LCC in a stirred tank

Figure 4: a) HAA synthesis from hydrolyzed PET. Pseudomonas sp. GO16 KS3 pSB01 was cultivated in shake flasks with 50 mL of mineral salts medium (MSM) at 30 °C. The PET hydrolysate was added to a concentration of 15-18 mM TA and EG. Growth (cell dry weight, CDW), HAA, substrates terephthalic acid (TA) and ethylene glycol (EG) depletion. The error bars represent the deviation from the mean of two biological replicates. b) Molecular diversity of the HAA congeners synthesized from PET monomers. The error bars represent the standard deviation from the mean of two independent fermentations (as indicated in a) at four time points, i.e. eight biological replicates.

Figure 1: Two alternative bio-upcycling routes for PET waste to novel biopolymers. PET hydrolysis is catalyzed by a thermostable polyester hydrolase. The resulting monomers EG and TA (blue and red, respectively) are fed to an engineered P. putida, which synthesizes either extracellular HAA (green), or the intracellular biopolymer PHA (cyan). While PHA is already a polymer, HAA is co-polymerized with a diisocyanate and butanediol (orange) to yield a novel bio-based poly(amide urethane) (bio-PU).

Figure 3: Growth, PHA accumulation, and substrate depletion by Pseudomonas sp. GO16 KS3 when enzymatically hydrolyzed PET was used under nitrogen limiting conditions. Pseudomonas sp. GO16 KS3 was cultivated in a 5 L bioreactor with 3 L of mineral salts medium (MSM) at 30 °C. The hydrolyzed PET was added to a concentration of approximately 30 mM TA and EG. Growth (cell dry weight, CDW), polyhydroxyalkanoate (PHA, %CDW), substrates terephthalic acid (TA) and ethylene glycol (EG). The error bars represent the standard deviation from the mean of three independent biological replicates.

Figure 6: Confirmation of bio-PU synthesis. 1H NMR spectrum of the final polymer showing the presence of NH signals of both amide (circled in red) and urethane (circled in blue) bonds.

Full text

1
Bio-upcycling of polyethylene terephthalate
Till Tiso
1,§
, Tanja Narancic
2,3,§
, Ren Wei
4,#
, Eric Pollet
5
, Niall Beagan
2
, Katja Schröder
1
, Annett
Honak
4
, Mengying Jiang
5,8
, Shane T. Kenny
6
, Nick Wierckx
1,7
, Rémi Perrin
8
, Luc Avérous
5
,
Wolfgang Zimmermann
4
, Kevin O’Connor
2,3
*, and Lars M. Blank
1
*
1
iAMB - Institute of Applied Microbiology. ABBt - Aachen Biology and Biotechnology, RWTH
Aachen University, Worringerweg 1, D-52074 Aachen, Germany
2
BEACON - SFI Bioeconomy Research centre, University College Dublin, Belfield, Dublin 4,
Ireland
3
School of Biomolecular and Biomedical Science and UCD Earth Institute, University College
Dublin, Belfield, Dublin 4, Ireland
4
Department of Microbiology and Bioprocess Technology, Institute of Biochemistry, Leipzig
University, Johannisallee 23, D-04103 Leipzig, Germany
5
BioTeam/ICPEES-ECPM, UMR CNRS 7515, Strasbourg University, 25 rue Becquerel, F-
67087 Strasbourg Cedex 2, France
6
Bioplastech Ltd., NovaUCD, Belfield Innovation Park, University College Dublin, Belfield,
Dublin 4, Ireland
7
Institute of Bio- and Geosciences IBG-1: Biotechnology, Forschungszentrum Jülich, 52425
Jülich, Germany
8
SOPREMA, 14 rue de Saint-Nazaire, F-67025 Strasbourg Cedex, France
§
These authors contributed equally to the work
#
Current address for Ren Wei: Department of Biotechnology and Enzyme Catalysis, Institute
of Biochemistry, University of Greifswald, Felix-Hausdorff-Str. 4, 17487 Greifswald, Germany
.CC-BY-NC-ND 4.0 International licensemade available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is
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2
*Corresponding authors:
Lars M. Blank
iAMB - Institute of Applied Microbiology. ABBt - Aachen Biology and Biotechnology, RWTH
Aachen University, Worringerweg 1, D-52074 Aachen, Germany
Phone: +49 241 80 26600 (office), +49 241 80 622180 (fax); e-mail: lars.blank@rwth-
aachen.de
Kevin O’Connor
UCD Earth Institute and School of Biomolecular and Biomedical Science, BEACON -
Bioeconomy Research Centre, University College Dublin, Belfield, Dublin 4, Ireland
Phone: +353 1 716 4000, e-mail: kevin.oconnor@ucd.ie
Abbreviations: EG – ethylene glycol, TA – terephthalic acid terephthalate, PET - polyethylene
terephthalate, PHA – polyhydroxyalkanoate, HAA – hydroxyalkanoyloxy-alkanoate, MHET –
mono-(2-hydroxyethyl)TA
Keywords: polyethylene terephthalate (PET) degradation, metabolic engineering, biopolymers,
polyhydroxyalkanoate (PHA), bio-upcycling, Pseudomonas putida, bioplastic, synthetic biology
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(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is
The copyright holder for this preprintthis version posted March 18, 2020. ; https://doi.org/10.1101/2020.03.16.993592doi: bioRxiv preprint

3
Graphical abstract
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4
Abstract
Over 359 million tons of plastics were produced worldwide in 2018, with significant growth
expected in the near future, resulting in the global challenge of end-of-life management. The
recent identification of enzymes that degrade plastics previously considered non-biodegradable
opens up opportunities to steer the plastic recycling industry into the realm of biotechnology.
Here, we present the sequential conversion of polyethylene terephthalate (PET) into two types
of bioplastics: a medium chain-length polyhydroxyalkanoate (PHA) and a novel bio-based
poly(amide urethane) (bio-PU). PET films were hydrolyzed by a thermostable polyester
hydrolase yielding 100% terephthalate and ethylene glycol. A terephthalate-degrading
Pseudomonas was evolved to also metabolize ethylene glycol and subsequently produced PHA.
The strain was further modified to secrete hydroxyalkanoyloxy-alkanoates (HAAs), which were
used as monomers for the chemo-catalytic synthesis of bio-PU. In short, we present a novel
value-chain for PET upcycling, adding technological flexibility to the global challenge of end-
of-life management of plastics.
.CC-BY-NC-ND 4.0 International licensemade available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is
The copyright holder for this preprintthis version posted March 18, 2020. ; https://doi.org/10.1101/2020.03.16.993592doi: bioRxiv preprint

5
1 Introduction
One of the challenges humankind faces is the shift to a sustainable plastic industry. In 2018,
359 million tons of plastics have been produced worldwide and this number is growing at a rate
of approximately 3% per annum
1
. Of all the plastic ever produced, only 9% was recycled and
12% was incinerated. The remaining majority is either in use or was landfilled, with a chance
to be released into the environment
2
. Indeed, in 2010 an estimated 5-13 million tons of plastic
ultimately ended up in the ocean
3
. While plastic, due to its lightweight and sturdiness, has many
environmentally beneficial applications, the environmental damage caused by plastic must be
arrested by addressing the end-of-life challenge.
State-of-the art plastic recycling is either via mechanical or chemical methods, or a combination
thereof
4
. An ideal plastic for recycling is polyethylene terephthalate (PET). The main PET
product, beverage bottles, can be specifically collected, avoiding mixed material challenges. In
addition, with its thermoplastic properties such as high melting temperature and the possibility
to process it without the use of additives, PET fulfils many technical recycling criteria. While
in some European countries, PET is collected at quotas above 95%, only approximately 30%
of it is recycled, even under these ideal conditions
5
. Reasons are manifold including cost,
consumer acceptance, and safety regulations surrounding recycled material, to name a few. An
alternative way to increase plastic recycling is to add additional value to the plastic waste, not
aiming for the same material or consumer good (e.g., bottle-to-bottle recycling), but rather
upcycling to chemicals and materials of higher value. This concept has already been
demonstrated using chemical methods as glycolization
6
, alcoholysis
7
, glycolysis
8,9
, and
organocatalysis
10
.
This upcycling can potentially be achieved by using carbon-rich plastic waste streams as a
substrate for biotechnological processes
5
. Here, PET is degraded into its monomers terephthalic
.CC-BY-NC-ND 4.0 International licensemade available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is
The copyright holder for this preprintthis version posted March 18, 2020. ; https://doi.org/10.1101/2020.03.16.993592doi: bioRxiv preprint

Frequently asked questions

Q1. What are the contributions in "Bio-upcycling of polyethylene terephthalate" ?

For example, in this paper, the authors reported that the larvae of the meal moth Plodia interpunctella can degrade polyethylene, a trait also discovered later in related species. 

Q2. What are the future works mentioned in the paper "Bio-upcycling of polyethylene terephthalate" ?

Currently further metagenome and mechanistic studies of this important enzyme class are carried out by the scientific community, most likely discovering protein family members with superb activities or at least interesting amino acid variations. Accessing non-biodegradable plastics of petrochemical origin ( and in the future of biological origin ) as carbon source for fermentations enables biotechnology to valorize enormous waste streams for the sustainable production of many valuable products by exploiting the metabolic versatility of microorganisms. While the mesophilic PET hydrolase from I. sakaiensis15 suggests consolidated hydrolysis and utilization, the authors focused on sequential plastic depolymerization and monomer conversion on purpose. 

Q3. What is the potential of enzyme cocktails?

The use of enzyme cocktails will also enable feedstock flexibility, especially when combined with microbes engineered to accept other plastic-derived substrates. 

Q4. What is the synthesis of a poly(amide urethane)?

Since an isocyanate moiety can react with both an hydroxyl and a carboxylic acid group, and HAA is an hydroxy acid, its direct polymerization with 4,4’- methylene diphenyl diisocyanate (MDI) and butanediol (BDO) was performed and led to the formation of a poly(amide urethane). 

Q5. How did the thermal stability of the polymer change?

The polymer started to degrade and to lose volatile products at 160 °C and then showed a multi-step degradation profile with the main mass loss occurring between 250 and 350 °C. 

Q6. How many plastics are currently being used in the packaging industry?

Renewable plastics including PHA have already been proposed to effectuate a shift of the packaging industry, which consumes over 38% of the plastics produced53. 

Q7. What is the important advantage of lignocellulose-derived substrates?

Lignocellulose-derived substrates come with a large fraction of solids, which are not completely degraded impeding the application of, e.g., enzyme immobilization or in situ removal of formed monomers (by e.g., precipitation or extraction). 

Q8. What is the main reason for the lack of recycling of plastic?

While plastic, due to its lightweight and sturdiness, has many environmentally beneficial applications, the environmental damage caused by plastic must be arrested by addressing the end-of-life challenge. 

Q9. How many ml samples were taken for each time point?

Three 2 ml samples were taken at regular intervals for the analysis of TA, EG and nitrogen concentrations, biomass, and PHA accumulation for each time point. 

Q10. What is the way to increase plastic recycling?

An alternative way to increase plastic recycling is to add additional value to the plastic waste, not aiming for the same material or consumer good (e.g., bottle-to-bottle recycling), but rather upcycling to chemicals and materials of higher value. 

Q11. What is the way to degrade PET?

The use of enzyme engineering and enzyme cocktail formulation will enable an even more efficient PET degradation, for instance using specialized enzymes of the various types of PET, i.e. high molecular weight PET and PET oligomers mono-(2-hydroxyethyl)TA (MHET) and bis2(hydroxyethyl)TA (BHET); possibly combined with chemical hydrolysis methods such as glycolysis56. 

Q12. How much of the TA and EG concentrations increased during the hydrolysis?

The concentration of TA and EG showed a steep near-linear increase within the first 24 h of the hydrolytic reaction and weakened to a markedly lower rate until 120 h. 

Q13. Who supervised the experiments regarding monomer metabolism and synthesis?

TT supervised the experiments regarding monomer metabolism and HAA synthesis, drafted the manuscript, and coordinated the study, TN provided strain Pseudomonas sp. GO16, supervised the experiments regarding PHA synthesis and drafted parts of the manuscript, RW supervised the experiments regarding depolymerization and drafted parts of the manuscript, EP supervised the experiments regarding polymerization and drafted parts of the manuscript, KS carried out the experiments regarding monomer metabolism and HAA synthesis, NB carried out the experiments regarding PHA synthesis, AH carried out the experiments regarding depolymerization, MJ carried out the experiments regarding polymerization, SK was involved in PHA bioprocess design, NW was involved in designing and coordinating the study, drafted parts of the manuscript and critically read the manuscript, RP was involved in designing the study and critically read the manuscript, LA was involved in designing the study and critically read the manuscript, WZ was involved in designing the study and critically read the manuscript, KOC designed the study and critically read the manuscript, LMB designed and coordinated the study and critically read the manuscript. 

Q14. What is the main reason for the biodegradation of plastic waste?

For obvious reasons, the biodegradation of these recalcitrant plastics are exciting discoveries that give hope for the natural bioremediation of sites contaminated with plastic waste in the environment, although plastic degradation in the ocean seems to be slow at best and the anthropogenic dissemination of new plastic pollution likely far exceeds its decay18. 

Q15. How many tons of plastics were produced in 2018?

In 2018, 359 million tons of plastics have been produced worldwide and this number is growing at a rate of approximately 3% per annum1.