# MIXed plastics biodegradation and UPcycling using microbial communities

Hendrik Ballerstedt, Till Tiso, Nick Wierckx, Ren Wei, Luc Avérous, Uwe Bornscheuer, Kevin O'Connor, Tilman Floehr, Andreas Jupke, Jürgen Klankermayer, et al.

► To cite this version:

Hendrik Ballerstedt, Till Tiso, Nick Wierckx, Ren Wei, Luc Avérous, et al., MIXed plastics biodegradation and UPcycling using microbial communities. European Journal of Futures Research, 2021, 33 (1), 10.1186/s12302-021-00536-5. doi: 10.34784/eh

HAL Id: hal-03478442

https://hal.science/hal-03478442

Submitted on 13 Dec 2021

HAL is a multi-disciplinary open access
archive for the deposit and dissemination of sci-
entific research documents, whether they are pub-
lished or not. The documents may come from
teaching and research institutions in France or
abroad, or from public or private research centers.

L'archive ouverte pluridisciplinaire HAL, est
destinée au dépôt et à la diffusion de documents
scientifiques de niveau recherche, publiés ou non,
émanant des établissements d'enseignement et de
recherche français ou étrangers, des laboratoires
publics ou privés.

<figure><img src="image_1.png" /></figure>

Distributed under a Creative Commons Attribution 4.0 International License
<figure><img src="image_2.png" /></figure>

DISCUSSION

Open Access

<figure><img src="image_3.png" /></figure>

Check for
updates

# MIXed plastics biodegradation and UPcycling using microbial communities: EU Horizon 2020 project MIX-UP started January 2020

Hendrik Ballerstedt<fnref n="1" />* , Till Tiso<fnref n="1" />, Nick Wierckx⁵, Ren Wei⁶, Luc Averous⁹, Uwe Bornscheuer⁶, Kevin O'Connor⁷, Tilman Floehr¹³, Andreas Jupke⁴, Jürgen Klankermayer³, Luo Liu¹⁷, Victor de Lorenzo⁸, Tanja Narancic⁷, Juan Nogales⁸, Rémi Perrin¹⁰, Eric Pollet⁹, Auxiliadora Prieto⁸, William Casey¹¹, Thomas Haarmann¹², Alexandru Sarbu¹⁰, Ulrich Schwaneberg², Fengxue Xin¹⁴, Weiliang Dong¹⁴, Jiamin Xing¹⁶, Guo-Qiang Chen¹⁵, Tianwei Tan¹⁷, Min Jiang¹⁴ and Lars M. Blank<fnref n="1" />*

## Abstract

This article introduces the EU Horizon 2020 research project MIX-UP, "Mixed plastics biodegradation and upcycling using microbial communities". The project focuses on changing the traditional linear value chain of plastics to a sustainable, biodegradable based one. Plastic mixtures contain five of the top six fossil-based recalcitrant plastics [polyethylene (PE), polyurethane (PUR), polypropylene (PP), polyethylene terephthalate (PET), polystyrene (PS)], along with upcoming bioplastics polyhydroxyalkanoate (PHA) and polylactate (PLA) will be used as feedstock for microbial transformations. Consecutive controlled enzymatic and microbial degradation of mechanically pre-treated plastics wastes combined with subsequent microbial conversion to polymers and value-added chemicals by mixed cultures. Known plastic-degrading enzymes will be optimised by integrated protein engineering to achieve high specific binding capacities, stability, and catalytic efficacy towards a broad spectrum of plastic polymers under high salt and temperature conditions. Another focus lies in the search and isolation of novel enzymes active on recalcitrant polymers. MIX-UP will formulate enzyme cocktails tailored to specific waste streams and strives to enhance enzyme production significantly. In vivo and in vitro application of these cocktails enable stable, self-sustaining microbiomes to convert the released plastic monomers selectively into value-added products, key building blocks, and biomass. Any remaining material recalcitrant to the enzymatic activities will be recirculated into the process by physicochemical treatment. The Chinese-European MIX-UP consortium is multidisciplinary and industry-participating to address the market need for novel sustainable routes to valorise plastic waste streams. The project's new workflow realises a circular (bio)plastic economy and adds value to present poorly recycled plastic wastes where mechanical and chemical plastic recycling show limits.

<figure><img src="image_4.png" /></figure>

<footnote>*Correspondence: Hendrik.Ballerstedt@rwth-aachen.de;
Lars.Blank@rwth-aachen.de</footnote>

<footnote n="1">¹ Institute of Applied Microbiology (iAMB), Aachen Biology and Biotechnology (ABBt), RWTH Aachen University, Worringerweg 1, 52074 Aachen, Germany
Full list of author information is available at the end of the article</footnote>
**Keywords:** PHA, Polyhydroxyalkanoate, Synthetic biology, Plastic recycling, Plastic crisis, Metabolic engineering, Microbial consortia, Mixed plastics valorisation, Biobased plastic, Depolymerisation

## Background

### General global plastic waste situation

Due to their benefits as a functional material, their extreme durability, longevity, low weight and low price, synthetic plastics, including polyvinyl chloride (PVC), PE, PS, PP, PUR, and PET, have become ubiquitous not only in work and social environments, but also in natural systems as contaminants. Plastic pollution has become a global threat, affecting all ecosystems, even remote ones like the pole regions, uninhabited atolls or deep ocean basins [1–6]. The global scale of plastics production increased by 21% in the last six years, reaching 368 million metric tons (Mt) in 2019 [7]. China and the European Union (EU) account for 31% and 16%, ranking first and third globally of all the world’s plastic production, respectively. Nonetheless, European plastic production revealed a decline since 2018 which has been significantly intensified by COVID-19 pandemic (estimated rate 2020: – 8.5%) [7]. Highest in plastic waste generation in 2016 were the United States, with 42 Mt followed by the EU (30 Mt), India (26 Mt) and China (22 Mt) [8]. All nations worldwide are struggling to manage the current volumes of plastic waste, making a highly efficient waste management system

increasingly important. A significant unintended drawback of the existing plastic economy is its linearity. Of all plastics produced globally, 83% has not been reused due to a lack of proper recycling technologies, and of the recycled 10%, only 15% has been reused more than once [9]. Seven main plastic polymers account for 92% of all primary produced plastic ever made (1950–2015: 8,300 Mt). The largest groups are the polyolefins, with PE (36%), and PP (21%), PVC (12%), followed by PET, PUR, or PS (less than 10% each) [7, 9]. Biobased plastics (bioplastics) with increasing volumes emerged as non-fossil alternatives on the last decennium’s plastic markets (Fig. 1A). Persistent biomass-derived plastic materials, non-biodegradable bioplastics derived from renewable resources represent 57% of all the bioplastics (2 Mt), including biobased PET, polyamides (PA), and PE [10]. Global bioplastics production capacities increase has been forecasted from around 2.1 Mt (biodegradable 1.3 Mt) in 2020 to approx. 2.9 Mt (1.8 Mt) in 2025, amounting to a 0.4% (2020) share of biodegradable plastics in general plastic waste streams [11].

Effective plastic recycling poses a significant challenge for sustainability, as a plastic polymer currently degrades each time it is recycled [12]. Technological solutions as

<figure><img src="image_5.png" /></figure>
part of a circular economy can form only part of more
radical changes required in human behaviours like
throw-away mentality symbolised by single-use con-
sumer plastics or unnecessary packaging. Multilevel mit-
igation strategies to reduce the waste of natural resources
started with policymakers banning and placing levies
on single-use plastic consumer products to stimulate
sustainable alternatives and changed consumer behav-
iour. Upcycling plastic waste from fossil sources in an
open-loop process to biodegradable plastic and chemi-
cals to valorise post-consumer plastic should be part of a
rethinking towards a circular economy [13–15]. Essential
for a circular economy is the intense utilisation of every
side stream to minimise waste production or redundant
CO₂ release. Available recycling concepts are often not
cost-competitive and produce polymers of lower qual-
ity. The biotechnological recycling supplemented with
physicochemical techniques to tackle the more recalcii-
trant plastic polymers may promote new waste manage-
ment strategies. Promising new value-chains for plastic
waste and the increasing demand for recycled plastics
by the multinational brand owners, driven mainly by
the rising consumer awareness concerning sustainability
issues, shall urge the private sector to invest in a circu-
lar economy. The partners of the MIX-UP consortium
envision a better plastic future built on the '6 R' princi-
ples (rethink, refuse, reduce, reuse, recycle, replace) [16].
In the 2019 position paper "A circular economy for plas-
tics", the European Commission explained its vision for a
circular plastics system. The plastics should be produced
using renewable energy and feedstocks. The plastic prod-
ucts should be designed to be used, reused, repaired and
recycled (mechanically, chemically, biologically) so that
the material streams in society are fully circular, keeping
high value without posing risks for human health nor the
environment [17].

Chemical and mechanical plastic waste recycling

Crude oil and other fossil resources are the cost-effective
lifeblood of the chemical industry and have been exten-
sively used as energy and carbon feedstock for almost
90% of its products. One of the last boosts has been the
shale gas-driven multi-billion investments into the U.S.
chemical industry, leading to an acceleration of virgin
plastics manufacturing [18]. For decades, the traditional
economy of high-income countries followed the "take-
make-dispose" strategy creating economic value by man-
ufacturing and selling as many products as possible. The
envisioned global transition to a circular economy initi-
ated the founding of initiatives like, e.g. *The Global Plas-
tics Alliance* or *Alliance to End Plastic Waste* [19, 20]. It
activated private investors' investment or development

banks into recycling technology to recover and create
value from plastic waste.

The only widely applied large-scale technology to
treat solid plastic waste is mechanical recycling. Limi-
tations for mechanical recycling are temperature-sen-
sitive plastics, composites, and thermosets that cannot
be liquidised at high temperatures [21]. Differences in
mechanical behaviour and thermal properties of all the
different plastics require thorough sorting, washing to
remove organic residues and shredding of the collected
wastes. The melted and remoulded polymers are often
blended with virgin plastics to correct for lost proper-
ties. Two of the most prominent commodity plastics,
PET and PE, with annual EU market shares of about 8%
and 30%, respectively, mainly used in packaging, are the
only ones recovered by mechanical recycling [7, 21].

Chemical recycling has emerged as an alternative,
promising technology to valorise plastic waste. Plastic
wastes can be gasified into synthesis gas. Solvolytic pro-
cesses may convert polymers into monomers and oligom-
ers, subsequently re-polymerised after purification by,
e.g. precipitation combined with filtration [22]. Pyrolysis
(thermolysis) and hydrogenolysis using advanced cata-
lysts can selectively produce gases, fuels, or waxes. The
latter requires selective and efficient catalysts, preserving
critical functional groups [21]. High-energy costs but low
costs for competing virgin monomers from fossil-based
feedstocks often make chemical recycling commercially
unattractive [23]. Effective recycling processes within cir-
cular approaches should not only produce monomers for
later polymerisation ("bottle to bottle") but rather focus
on value-added products or intermediates for alternative
supply chains. The upcycling of PE into long-chain alkyl
aromatics ready to be sulfonated to make surfactants was
reported [24]. Others described the synthesis of inter-
mediary cyclic acetals, which are useful as solvents, fuel
additives or monomers for polymers [25]. The greatest
challenge is the chemical recycling of commingled plastic
waste, as even small amounts of the various polymer con-
taminants may change the properties of the end-product.
Therefore, chemical recycling requires often the use of
pure waste feedstocks obtained only after resource-
intensive sorting. The use of suitable compatibilisers for
upcycling recovered polymer mixtures can overcome this
problem [26–28].

Another feasible approach for PET/polylactate (PLA)
polyester mixtures is using a molecular ruthenium cata-
lyst for selective hydrogenolysis to separate the differing
sorts of monomeric diols, and methanol, respectively, at
varying reaction conditions (temperature, solvent) [29].
Alternatively, using pyrolysis oil in a naphtha cracker
might close the carbon loop, but much of the beneficial
molecular structure and plastic properties are lost in this
option.

Microbial and enzymatic plastics biotransformation

Facing the unabated growth of global plastic produc-
tion and considering the shortcomings of traditional
mechanical and chemical recycling technologies, bio-
logical depolymerisation and conversion technolo-
gies have been increasingly discussed, complementing
end-of-life plastic treatment options. With a view to
the economic circularity, selective removal of poly-
mer-building blocks using enzymatic treatments under
mild conditions and the ability to the selective recov-
ery of monomers from mixed plastic substrates would
be a real improvement [30–32]. Building blocks of
plastic polymers can, in general, be divided into dif-
ferent major groups as (i) monomers with vinyl groups
to produce PS, PE or PVC; (ii) bifunctional monomers
with terminal hydroxyl, amine, or carboxyl groups to
obtain polyesters or polyamides; (iii) diisocyanates for
PUR [33]. In recent years, considerable progress con-
cerning plastic polymers with hydrolysable groups in
their backbones, as PET, PA, or PUR were reported,
obtained mainly by polyaddition or polycondensation.
Several studies described the ability of microorganisms
and enzymes to degrade these plastics [34–47]. Typi-
cal enzymes are cutinases, lipases, and carboxylester-
ases [48]. The main challenge of enzymatic degradation
is the fraction of plastic polymers based on persistent
and robust chemical groups, which resist hydrolysis
with common biological enzymes that are highly recal-
citrant even under conditions favouring microbial pro-
cesses. These polymers (e.g. PE, PP, PS, PVC) obtained
by chain polymerisation comprise the major part of
the plastic waste market and are generally considered
non-biodegradable. The polymers possess extensive
inert C–C backbone structures, are completely devoid
of functional groups and might be only degraded by
high-energy redox reactions [48]. Only a few enzymes
have been reported to reduce the molar mass of PE and
PS. Alkane hydroxylase AlkB, a hydroquinone peroxi-
dase, laccases, and a laccase mediator system demon-
strated C–C-bond cleavage via autooxidation mediated
by putative radical mechanisms thought to occur ran-
domly, generating a large diversity of short-chain scis-
sion products [4, 48–55]. In addition to the description
of enzymatic activities towards PE and PS, several
reports described their mineralisation to CO₂ by insect
larvae and their enteric microbiome. The latter poten-
tially benefitting from the combined mechanical pre-
treatment and enzymatic hydrolysis [56–61]. Recently,
biodegradation of PVC in the gut of *Tenebrio molitor*
larvae has been described [62]. No biodegradation has

been demonstrated so far for the highly recalcitrant
polymer PP.

Mixed cultures in industrial applications

The application of microbial consortia in traditional
foods, such as bread, soy sauce, cheese and wine, have
been recorded for centuries. These bioprocesses were
realised with naturally occurring mixed cultures. Mixed
cultures were gradually replaced by pure cultures in fer-
mentation processes to avoid contaminations by food
spoilers or pathogenic microbes. Pure cultures have been
the workhorses for biotechnological processes to produce
bulk products like amino acids, antibiotics, enzymes or
organic acids. Fermentations based on pure cultures usu-
ally require strict aseptic conditions, purified substrates,
high operational energy costs, and gain in addition to
the targeted product high concentrations of by-products
in the form of biomass and potentially of organic acids
or alcohols. The traditional strategy of consolidated bio-
processing integrates all bioconversion reactions in one
step-bioprocesses using metabolically engineered whole-
cell biocatalysts hosting all required functional genes in
one consolidated strain.

Compared with the competing fossil-based chemical
production, industrial biotechnology lacks cheap, read-
ily available feedstocks to produce bulk biobased chemi-
cals using highly specialised whole-cell biocatalysts as
pure cultures. The main drawback for using lignocellu-
lose, molasses, sludge or organic wastes as feedstock in
pure-culture fermentations is the heterogeneity of the
feedstocks, non-aseptic conditions and the high costs
for substrate pre-treatments. Although mixed cultures as
industrial microbiomes are well established in the fields
of biofuels (biogas, bio-hydrogen, butanol-production),
biobased chemicals, and biopolymers, the emphasis in
industrial biotechnology still lies on pure cultures [63–
66]. The specific advantages of mixed-culture fermenta-
tion compared with pure culture are (i) the possibility of
utilising cheaper or mixed substrates (e.g. organic waste,
lignocellulose, raw glycerol); (ii) the synergies of different
enzymatic systems and combination of metabolic path-
ways of various microorganisms that can result in more
efficient utilisation of substrates and a narrow produc-
tion spectrum contributing to product purification; (iii)
shorter development times for mixed-culture design
compared with deep-genetic engineering to create uni-
versal "superbugs", and (iv) cost reduction, due to the
high microbial diversity with non-sterile requirements
[67]. An alternative for the latter is the use of robust
extremophilic strains able to produce the target com-
pounds (e.g. PHA) under simplified process conditions,
in open unsterile, continuous fermentation facilities
where most other organisms are unable to proliferate.
<figure><img src="image_6.png" /><figcaption>**Fig. 2** The generated MIX-UP workflow as combination of intensive protein engineering of different types of plastic depolymerising enzymes, metabolic engineering of defined mixed cultures and bioprocess-optimisation</figcaption></figure>

The extremophiles based process seems to be suitable for simple growth on mixed degradation products, including fatty acids, plastic monomers and food wastes [68–71]. In mixed cultures and consortia exist in addition to intraspecies interactions, e.g. quorum sensing, interspecies interactions between cells of the different species. Metabolite effects like mutualism, synergy, and competition for nutrients in an ecological niche might affect metabolisms and the yield of fermentation target products [72–75].

## EU H2020 Project "From Plastic Waste to Plastic value using Pseudomonas putida Synthetic Biology" (P4SB): achieved results embedded in MIX-UP

MIX-UP can, in part, build on the success of P4SB (grant no: 633962), an H2020 project in which several of the MIX-UP partners [RWTH-iAMB (coordinator, Aachen), University College Dublin, CIB-CSIC (Madrid), CNRS-University of Strasbourg; industrial partners: SOPREMA, Bioplastech] already worked together on plastic waste valorisation. The innovation radar has ranked P4SB as one of the top ten EU Biotechnology projects [76]. The main outcomes of P4SB regarding plastic hydrolysis are engineered PET degrading enzymes with significantly increased PET hydrolysing activity [77]. Furthermore, PUR hydrolases were identified [39]. In terms of monomer metabolism, *P. putida* strains for growth on all PET and PUR monomers tested could be isolated. However, efficient growth could not be achieved on all monomers. Subsequently, via genetic engineering, the P4SB partners could generate recombinant *P. putida* strains capable of efficient catabolism of ethylene glycol, terephthalic acid, and 1,4-butanediol [13, 14, 66, 78]. For the valorisation

of plastic monomers, besides PHA synthesis, hydroxy alkanoyl oxy-alkanoic acids (HAA) synthesis has been successfully established. PHA synthesis could be shown on all PET and PUR plastic monomers [14]. Consolidated strains of *P. putida* engineered within P4SB for PET and PUR monomer metabolism will be used to benchmark the performance of defined mixed cultures in MIX-UP. Therefore, the strains will be combined with, e.g. engineered pseudomonads capable of metabolising oligo- and monomers from PS, PP, and PE degradation, and producing alternative novel biopolymers. In contrast to P4SB, MIX-UP focuses on consolidated bioprocesses in a combination of highly efficient enzymatic (pre-)treatment, defined mixed-culture biodegradation of released plastic monomers, additives and toxic constituents to biomass, value-added products, and building blocks using various engineered microbes. The increased interdisciplinary of the MIX-UP consortium was achieved by broadening the expertise's in the following fields: intensive protein engineering (University Greifswald, RWTH-BIOTEC); plastic biodegradation (Nanjing Tech University, Beijing University of Chemical Technology); enzyme production upscaling (AB Enzymes, IPE); bacterial stress-response and directed evolution (Research Center Jülich); environmental education and innovative clean-up technology (everwave); extremophilic PHA-bioproduction (Tsinghua University); product separation and chemical catalysis (RWTH-AVT, ITMC).

## Project aim, concept, and approach

The core aim of MIX-UP project is to establish mixed plastic waste as standard second-generation feedstock for industrial biotechnology—plastic waste as a valuable
resource. The bioconversion of unsorted, mixed plastic waste into value-added, sustainable biomaterials using heavily engineered enzyme mixtures for depolymerisation and mixed microbial cultures as whole-cell biocatalysts for biosynthesis is the way to achieve this goal as a contribution to the transition towards a low-fossil carbon circular bio-economy (Fig. 1B).

The main idea of MIX-UP is to showcase a novel approach to the circularity of the plastic life cycle. The overall concept is depicted in Fig. 2. MIX-UP will develop and use engineered polymer hydrolyzing and oxidising enzymes to depolymerise the mechanically sheared mixed plastic waste (e.g. marine litter, household) into their monomeric components (biotic plastics depolymerisation). These enzymes will be expressed in mixed microbial cultures, synthesised in an optimised production reactor (enzyme production) or as envisioned in a subsequently consolidated bioprocess with simultaneously implemented whole-cell biocatalysts biodegradation. The released metabolites, additives (e.g. stabilisers, plasticisers, and colourants), plastic monomers, and oligomers from the various plastics types will be transferred to the bioreactor (mixed culture). Here the plastic derived feedstock is fed to dedicated microbial communities converting the substrate into central metabolites, which provide afterwards the building blocks for the synthesis of novel polymers (e.g. HAA, PHAs), products (biosurfactants) or building blocks for chemo-catalysis (Fig. 2). The approach follows the bow-tie structure of metabolism [79]. Finally, MIX-UP will tackle downstream processing and recovery of the product by, for example, conditional release of the intracellular products and separation. The recalcitrant process residues will be separated and subjected to chemical transformation, also cracking persistent ester bonds, synthesising valuable chemicals, and closing the cycle by subsequent re-entering of the bioprocesses. The entire bioprocess will be optimised, performing metabolic engineering in an integrated manner by considering the upstream (strain/microbiome development, protein engineering), midstream (fermentation), and downstream (recovery and purification) processes altogether.

MIX-UP targets the engineering of a new-to-nature biological route to convert mixed plastic waste to value-added bio-products, which will enable the recycling industry a qualitatively new dimension. Furthermore, when successful, mixed plastic wastes can be established as novel second-generation carbon sources for bio-products, aiding to solve the conflict of food vs. fuel that is pervasive in contemporary Industrial Biotechnology. Thus, through a combination of metabolic engineering of mixed cultures, intensive protein engineering and bioprocess-optimisation, MIX-UP will enable new

value-chains within the framework of a sustainable knowledge-based bio-economy across sectors, including materials, chemicals, and environmental technologies. That will ultimately benefit the economy, environment, and society at large. The project has already produced a large number of publications that are available at the MIX-UP website www.mix-up.eu.

**Abbreviations**

EU: European Union; MIX-UP: Mixed plastics biodegradation and upcycling using microbial communities; PP: Polypropylene; PE: Polyethylene; PUR: Polyurethane; PET: Polyethylene terephthalate; PS: Polystyrene; PA: Polyamide; PLA: Polylactate; PHA: Polyhydroxyalkanoate; PBS: Polybutylene succinate; TPS: Thermoplastic starch; PVC: Polyvinyl chloride; HAA: Hydroxylalkanoyloxyalkanoic acids; Mt: Million metric tons; P4SB: From plastic waste to plastic value using *Pseudomonas putida* synthetic biology; H2020: Horizon 2020; *P. putida*: *Pseudomonas putida*.

**Acknowledgements**

We thank Burkhard Nobbe (Club of Communication GmbH, Wuppertal) for assistance with graphic design and Christine Kempchen (RWTH Aachen University) for comments that greatly improved the manuscript.

**Authors' contributions**

HB, TT, NW, RW, KC, LL, AP, AS, G-QC, MJ and LMB conceived the project and were in charge of overall direction and planning. HB and LMB compiled the manuscript and wrote the manuscript with contributions, critical feedback and input from all authors. All authors read and approved the final manuscript.

**Funding**

Open Access funding enabled and organized by Projekt DEAL. This project has received funding from the European Union's Horizon 2020 research and innovation programme under Grant Agreement No. 870294. In addition, this project is funded by the National Natural Science Foundation of China (Grant Numbers 31961133017, 31961133018, 31961133019).

**Availability of data and materials**

Not applicable.

**Declarations**

**Ethics approval and consent to participate**

Not applicable.

**Consent for publication**

Not applicable.

**Competing interests**

The authors declare that they have no competing interests.

**Author details**

<footnote n="1">¹Institute of Applied Microbiology (iAMB), Aachen Biology and Biotechnology (ABBt), RWTH Aachen University, Worringerweg 1, 52074 Aachen, Germany.</footnote>

<footnote n="2">²Institute of Biotechnology (BIOTEC), Aachen Biology and Biotechnology (ABBt), RWTH Aachen University, Worringerweg 3, 52074 Aachen, Germany.</footnote>

<footnote n="3">³Institute of Technical and Macromolecular Chemistry (ITMC), RWTH Aachen University, Worringerweg 2, 52074 Aachen, Germany. ⁴Fluid Process Engineering, Aachen Process Technology (AVT), RWTH Aachen University, Forckenbeckstraße 51, 52074 Aachen, Germany. ⁵Institute of Bio- and Geosciences IBG-1: Biotechnology, Research Center Jülich, Wilhelm Johnen Straße, 52428 Jülich, Germany. ⁶Institute of Biochemistry, University of Greifswald, Felix-Hausdorff-Str. 4, 17487 Greifswald, Germany. ⁷BiOrbic Bioeconomy SFI Research Centre, UCD Earth Institute and School of Biomolecular and Biomedical Science, University College Dublin, Belfield, Dublin 4, Ireland. ⁸Interdisciplinary Platform for Sustainable Plastics Towards a Circular Economy-Spanish National Research Council (SusPlast-CSIC), Biological Research Center (CIB-CSIC),</footnote>
28040 Madrid, Spain. ⁹BioTeam/ICPEES-ECPM, UMR CNRS 7515, Université de Strasbourg, 25 rue Becquerel, 67087 Strasbourg Cedex 2, France. ¹⁰SOPREMA, Direction R&D, 14 Rue Saint Nazaire, 67100 Strasbourg, France. ¹¹Bioplastech Ltd., Nova UCD, Belfield Innovation Park, University College Dublin, Belfield, Dublin 4, Ireland. ¹²AB Enzymes GmbH, Feldbergstraße 78, 64293 Darmstadt, Germany. ¹⁴College of Biotechnology and Pharmaceutical Engineering, Nanjing Tech University, No. 30 Puzhu Road, Nanjing 211816, PR China. ¹⁵School of Life Sciences (SLS), Tsinghua University, Beijing 100084, PR China. ¹⁶State Key Laboratory of Biochemical Engineering, Institute of Process Engineering (IPE), Chinese Academy of Sciences, 1 North 2nd Street, Zhongguancun, Beijing 100190, PR China. ¹⁷College of Life Science and Technology (CLST), Beijing University of Chemical Technology, Beishanhuan EastRoad 15, Chaoyang District, Beijing 100029, PR China.

Received: 9 May 2021 Accepted: 31 July 2021

Published online: 21 August 2021

## References

1. Ryan PG, Moore CJ, van Franeker JA, Moloney CL (2009) Monitoring the abundance of plastic debris in the marine environment. Phil Trans R Soc B 364:1999–2012. https://doi.org/10.1098/rstb.2008.0207

2. Dris R, Imhof H, Sanchez W, Gasperi J, Galgani F, Tassin B, Laforsch C (2015) Beyond the ocean: contamination of freshwater ecosystems with (micro-) plastic particles. Environ Chem 12:539–550. https://doi.org/10.1071/EN14172

3. Abel SM, Primpke S, Int-Veen I, Brandt A, Gerdts G (2021) Systematic identification of microplastics in abyssal and hadal sediments of the Kuril Kamchatka trench. Environ Poll 269:116095. https://doi.org/10.1016/j.envpol.2020.116095

4. Wright RJ, Erni-Cassola G, Zadjelovic V, Latva M, Christie-Oleza JA (2020) Marine plastic debris: a new surface for microbial colonization. Environ Sci Technol 54:11657–11672. https://doi.org/10.1021/acs.est.0c02305

5. Kane IA, Clare MA, Miramontes E, Wogelius R, Rothwell JJ, Garreau P, Pohl F (2020) Seafloor microplastic hotspots controlled by deep-sea circulation. Science. https://doi.org/10.1126/science.aba5899

6. Brahney J, Hallerud M, Heim E, Hahnenberger M, Sukumaran S (2020) Plastic rain in protected areas of the United States. Science 368:1257–1260. https://doi.org/10.1126/science.aaz5819

7. PlasticsEurope. (2020) Plastics—the facts 2020. Messe Düsseldorf and PlasticsEurope Deutschland e.V, Düsseldorf

8. Law KL, Starr N, Siegler TR, Jambeck JR, Mallos NJ, Leonard GH (2020) The United States' contribution of plastic waste to land and ocean. Sci Adv. https://doi.org/10.1126/sciadv.abd0288

9. Geyer R, Jambeck JR, Law KL (2017) Production, use, and fate of all plastics ever made. Sci Adv. https://doi.org/10.1126/sciadv.1700782

10. Andreessen C, Steinbüchel A (2018) Recent developments in non-biodegradable biopolymers: precursors, production processes, and future perspectives. Appl Microbiol Biotechnol. https://doi.org/10.1007/s00253-018-9483-6

11. European-Bioplastics (2020) https://www.european-bioplastics.org/wp-content/uploads/2020/11/Global_Production_Capacity_Total_2019-2025.jpg. Accessed 29 July 2021.

12. Stafford R, Jones PJS (2019) Viewpoint—ocean plastic pollution: a convenient but distracting truth? Mar Pol 103:187–191. https://doi.org/10.1016/j.marpol.2019.02.003

13. Blank LM, Narancic T, Mampel J, Tiso T, O'Connor K (2020) Biotechnological upcycling of plastic waste and other non-conventional feedstocks in a circular economy. Curr Opin Biotechnol 62:212–219. https://doi.org/10.1016/j.copbio.2019.11.011

14. Tiso T, Narancic T, Wei R, Pollet E, Beagan N, Schröder K, Honak A, Jiang M, Kenny ST, Wierckx N, Perrin R, Avérous L, Zimmermann W, O'Connor K, Blank LM (2021) Towards bio-upcycling of polyethylene terephthalate. Metab Eng 66:167–178. https://doi.org/10.1016/j.ymben.2021.03.011

15. Welsing G, Wolter B, Hintzen HMT, Tiso T, Blank LM (2021) Chapter Eighteen—Upcycling of hydrolyzed PET by microbial conversion to a fatty acid derivative. In: Weber G, Bornscheuer UT, Wei R (eds) Meth Enzymol, vol 648. Academic Press, Cambridge (MA), pp 391–421. https://doi.org/10.1016/bs.mie.2020.12.025

16. Wei R, Tiso T, Bertling J, O'Connor K, Blank LM, Bornscheuer UT (2020) Possibilities and limitations of biotechnological plastic degradation and recycling. Nat Catal 3:867–871. https://doi.org/10.1038/s41929-020-00521-w

17. Crippa M, De Wilde B, Koopmans R, Leyssens J, Muncke J, Ritschkoff A-C, van Doorsselaer K, Velis C, Wagner M (2019) A circular economy for plastics—insights from research and innovation to inform policy and funding decisions. European Union. https://doi.org/10.2777/269031

18. Borrelle SB, Ringma J, Law KL, Monnahan CC, Lebreton L, McGivern A, Murphy E, Jambeck J, Leonard GH, Hilleary MA, Eriksen M, Possingham HP, De Frond H, Gerber LR, Polidoro B, Tahir A, Bernard M, Mallos N, Barnes M, Rochman CM (2020) Predicted growth in plastic waste exceeds efforts to mitigate plastic pollution. Science 369(6510):1515–1518. https://doi.org/10.1126/science.aba3656

19. Alliance to End Plastic Waste (2021) https://www.endplasticwaste.org. Accessed 28 Apr 2021.

20. Global Plastics Alliance, Marine Litter Solutions (2021) https://www.marinelittersolutions.com. Accessed 28 Apr 2021.

21. Garcia JM, Robertson ML (2017) The future of plastics recycling. Science 358:870–872. https://doi.org/10.1126/science.aaq0324

22. Walker TW, Frelka N, Shen Z, Chew AK, Banick J, Grey S, Kim MS, Dumesic JA, Van Lehn RC, Huber GW (2020) Recycling of multilayer plastic packaging materials by solvent-targeted recovery and precipitation. Sci Adv. https://doi.org/10.1126/sciadv.aba7599

23. Weckhuysen BM (2020) Creating value from plastic waste. Science 370:400–401. https://doi.org/10.1126/science.abe3873

24. Zhang F, Zeng M, Yappert RD, Sun J, Lee Y-H, LaPointe AM, Peters B, Abu-Omar MM, Scott SS (2020) Polyethylene upcycling to long-chain alkylaromatics by tandem hydrogenolysis / aromatization. Science 370:437–441. https://doi.org/10.1126/science.abc5441

25. Beydoun K, Klankermayer J (2020) Efficient plastic waste recycling to value-added products by integrated biomass processing. Chemuschem 13(3):488–492. https://doi.org/10.1002/cssc.201902880

26. Hamad K, Kaseem M, Deri F (2013) Recycling of waste from polymer materials: an overview of the recent works. Pol Deg Stab 98(12):2801–2812. https://doi.org/10.1016/j.polymdegradstab.2013.09.025

27. Eagan JM, Xu J, Di Girolamo R, Thurber CM, Macosko CW, LaPointe AM, Bates F, Coates GW (2017) Combining polyethylene and polypropylene: enhanced performance with PE/IPP multiblock polymers. Science 355:814–816. https://doi.org/10.1126/science.aah5744

28. Creton C (2017) Molecular stitches for enhanced recycling of packaging. Science 355:797–798. https://doi.org/10.1126/science.aam5803

29. Westhues S, Idel J, Klankermayer J (2018) Molecular catalyst systems as key enablers for tailored polyesters and polycarbonate recycling concepts. Sci Adv. https://doi.org/10.1126/sciadv.aat9669

30. Wei R, Zimmermann W (2017) Microbial enzymes for the recycling of recalcitrant petroleum-based plastics: how far are we? Microb Biotechnol. https://doi.org/10.1111/1751-7915.12710

31. Wierckx N, Narancic T, Eberlein C, Wei R, Drzyzga O, Magnin A, Ballerstedt H, Kenny ST, Pollet E, Avérous L, O'Connor KE, Zimmermann W, Heipieper HJ, Prieto A, Jiménez J, Blank LM (2018) Plastic biodegradation: Challenges and opportunities. In: Steffan R (ed) Consequences of microbial interactions with hydrocarbons, oils, and lipids: Biodegradation and bioremediation. Springer, Cham. https://doi.org/10.1007/978-3-319-44535-9_23-1

32. Wierckx N, Prieto AM, Pomposiello P, de Lorenzo V, O'Connor K, Blank LM (2015) Plastic waste as a novel substrate for industrial biotechnology. Microb Biotechnol 8(6):900–903. https://doi.org/10.1111/1751-7915.12312

33. Schaffer S, Haas T (2014) Biocatalytic and fermentative production of α,ω-bifunctional polymer precursors. Org Proc Res Dev 18(6):752–766. https://doi.org/10.1021/op5000418

34. Cregut M, Bedas M, Durand MJ, Thouand G (2013) New insights into polyurethane biodegradation and realistic prospects for the development of a sustainable waste recycling process. Biotechnol Adv 31(8):1634–1647. https://doi.org/10.1016/j.biotechadv.2013.08.011

35. Schmidt J, Wei R, Oeser T, Dedavid e Silva L, Breite D, Schulze A, Zimmermann W (2017) Degradation of polyester polyurethane by bacterial polyester hydrolases. Polymers 9(2):65. https://doi.org/10.3390/polym9020065
36. Wei R, Zimmermann W (2017) Biocatalysis as a green route for recycling the recalcitrant plastic polyethylene terephthalate. Microb Biotechnol. https://doi.org/10.1111/1751-7915.12714

37. Knott BC, Erickson E, Allen MD, Gado JE, Graham R, Kearns FL, Pardo I, Topuzlu E, Anderson JJ, Austin HP, Dominick G, Johnson CW, Rorrer NA, Szostkiewicz CJ, Copie V, Payne CM, Woodcock HL, Donohoe BS, Beckham GT, McGeehan JE (2020) Characterization and engineering of a two-enzyme system for plastics depolymerization. PNAS. https://doi.org/10.1073/pnas.2006753117

38. Tournier V, Topham CM, Gilles A, David B, Folgoas C, Moya-Leclair E, Kamionka E, Desrousseaux ML, Texier H, Gavalda S, Cot M, Guémard E, Dalibey M, Nomme J, Cioci G, Barbe S, Chateau M, André I, Duquesne S, Marty A (2020) An engineered PET depolymerase to break down and recycle plastic bottles. Nature 580:216–219. https://doi.org/10.1038/s41586-020-2149-4

39. Magnin A, Pollet E, Perrin R, Ullmann C, Persillon C, Philip V, Avérous L (2019) Enzymatic recycling of thermoplastic polyurethanes: Synergistic effect of an esterase and an amidase and recovery of building blocks. Waste Manag 85:141–150. https://doi.org/10.1016/j.wasman.2018.12.024

40. Deguchi T, Kitaoka Y, Kakezawa M, Nishida T (1998) Purification and characterization of a nylon-degrading enzyme. Appl Environ Microbiol 64(4):1366–1371

41. Yoshida S, Hiraga K, Takehana T, Taniguchi I, Yamaji H, Maeda Y, Toyohara K, Miyamoto K, Kimura Y, Oda K (2016) A bacterium that degrades and assimilates poly(ethylene terephthalate). Science 351(6278):1196–1199. https://doi.org/10.1126/science.aad6359

42. Oda M, Numoto N, Bekker G-J, Kamiya N, Kawai F (2021) Chapter Eight—Cutinases from thermophilic bacteria (actinomycetes): From identification to functional and structural characterization. In: Weber G, Bornscheuer UT, Wei R (eds) Meth Enzymol, vol 648. Academic Press, Cambridge (MA), pp 159–85. https://doi.org/10.1016/bs.mie.2020.12.031

43. Magnin A, Pollet E, Avérous L (2021) Chapter Fifteen—Characterization of the enzymatic degradation of polyurethanes. In: Weber G, Bornscheuer UT, Wei R (eds) Meth Enzymol, vol 648. Academic Press, Cambridge (MA), pp 317–336. https://doi.org/10.1016/bs.mie.2020.12.011

44. Yan F, Wei R, Cui Q, Bornscheuer UT, Liu Y-J (2020) Thermophilic whole-cell degradation of polyethylene terephthalate using engineered *Clostridium thermocellum*. Microb Biotechnol. https://doi.org/10.1111/1751-7915.13580

45. Meyer-Cifuentes IE, Werner J, Jehmlich N, Will SE, Neumann-Schaal M, Öztürk B (2020) Synergistic biodegradation of aromatic-aliphatic copolyester plastic by a marine microbial consortium. Nat Commun 11(1):5790. https://doi.org/10.1038/s41467-020-19583-2

46. Magnin A, Pollet E, Philip V, Avérous L (2020) Evaluation of biological degradation of polyurethanes. Biotechnol Adv 39:107457. https://doi.org/10.1016/j.biotechadv.2019.107457

47. Li Z, Wei R, Gao M, Ren Y, Yu B, Nie K, Xu H, Liu L (2020) Biodegradation of low-density polyethylene by *Microbulbifer hydrolyticus* IRE-31. J Environ Manag 263:110402. https://doi.org/10.1016/j.jenvman.2020.110402

48. Inderthal H, Tai SL, Harrison STL (2020) Non-hydrolyzable plastics—an interdisciplinary look at plastic bio-oxidation. Trends Biotechnol. https://doi.org/10.1016/j.tibtech.2020.05.004

49. Krueger MC, Harms H, Schlosser D (2015) Prospects for microbiological solutions to environmental pollution with plastics. Appl Microbiol Biotechnol 99(21):8857–8874. https://doi.org/10.1007/s00253-015-6879-4

50. Krueger MC, Seiwert B, Prager A, Zhang S, Abel B, Harms H, Schlosser D (2017) Degradation of polystyrene and selected analogues by biological Fenton chemistry approaches: Opportunities and limitations. Chemosphere 173:520–528. https://doi.org/10.1016/j.chemosphere.2017.01.089

51. Yoon MG, Jeon HJ, Kim NM (2012) Biodegradation of polyethylene by a soil bacterium and AlkB cloned recombinant cell. J Bioremed Biodegrad. https://doi.org/10.4172/2155-6199.1000145

52. Nakamiya K, Sakasita G, Ooi T, Kinoshita S (1997) Enzymatic degradation of polystyrene by hydroquinone peroxidase of *Azotobacter beijerinckii* HM121. J Ferm Bioeng 84(5):480–482. https://doi.org/10.1016/S0922-338X(97)82013-2

53. Santo M, Weitsman R, Sivan A (2013) The role of the copper-binding enzyme—laccase—in the biodegradation of polyethylene by the actinomycete *Rhodococcus ruber*. Int Biodeter Biodegr 84:204–210. https://doi.org/10.1016/j.ibiod.2012.03.001

54. Kowalczyk A, Chyc M, Ryszka P, Latowski D (2016) *Achromobacter xylosoxidans* as a new microorganism strain colonizing high-density polyethylene as a key step to its biodegradation. Environ Sci Poll Res 23(11):11349–11356. https://doi.org/10.1007/s11356-016-6563-y

55. Erni-Cassola G, Wright RJ, Gibson MI, Christie-Oleza JA (2020) Early Colonization of weathered polyethylene by distinct bacteria in marine coastal seawater. Microb Ecol 79(3):517–526. https://doi.org/10.1007/s00248-019-01424-5

56. Yang Y, Yang J, Wu WM, Zhao J, Song Y, Gao L, Yang R, Jiang L (2015) Biodegradation and mineralization of polystyrene by plastic-eating mealworms: Part 1. Chemical and physical characterization and isotopic tests. Environ Sci Technol 49(20):12080–12086. https://doi.org/10.1021/acs.est.5b02661

57. Yang Y, Yang J, Wu WM, Zhao J, Song Y, Gao L, Yang R, Jiang L (2015) Biodegradation and mineralization of polystyrene by plastic-eating mealworms: Part 2. Role of gut microorganisms. Environ Sci Technol 49(20):12087–12093. https://doi.org/10.1021/acs.est.5b02663

58. Yang S-S, Brandon AM, Andrew Flanagan JC, Yang J, Ning D, Cai S-Y, Fan H-Q, Wang Z-Y, Ren J, Benbow E, Ren N-Q, Waymouth RM, Zhou J, Criddle CS, Wu W-M (2018) Biodegradation of polystyrene wastes in yellow mealworms (larvae of *Tenebrio molitor* Linnaeus): factors affecting biodegradation rates and the ability of polystyrene-fed larvae to complete their life cycle. Chemosphere 191:979–989. https://doi.org/10.1016/j.chemosphere.2017.10.117

59. Kim HR, Lee HM, Yu HC, Jeon E, Lee S, Li J, Kim D-H (2020) Biodegradation of polystyrene by *Pseudomonas* sp. isolated from the gut of superworms (larvae of *Zophobas atratus*). Environ Sci Technol 54(11):6987–6996. https://doi.org/10.1021/acs.est.0c01495

60. Peng B-Y, Su Y, Chen Z, Chen J, Zhou X, Benbow ME, Criddle CS, Wu W-M, Zhang Y (2019) Biodegradation of polystyrene by dark (*Tenebrio obscurus*) and yellow (*Tenebrio molitor*) mealworms (*Coleoptera: Tenebrionidae*). Environ Sci Technol 39(9):5256–5265. https://doi.org/10.1021/acs.est.8b06963

61. Wu W-M, Criddle CS (2021) Chapter Five—Characterization of biodegradation of plastics in insect larvae. In: Weber G, Bornscheuer UT, Wei R (eds) Meth Enzymol, vol 648. Academic Press, Cambridge (MA), pp 95–120. https://doi.org/10.1016/bs.mie.2020.12.029

62. Peng B-Y, Chen Z, Chen J, Yu H, Zhou X, Criddle CS, Wu W-M, Zhang Y (2020) Biodegradation of polyvinyl chloride (PVC) in *Tenebrio molitor* (*Coleoptera: Tenebrionidae*) larvae. Environ Internat 145:106106. https://doi.org/10.1016/j.envint.2020.106106

63. Dias JM, Lemos PC, Serafim LS, Oliveira C, Eiroa M, Albuquerque MG, Ramos AM, Oliveira R, Reis MA (2006) Recent advances in polyhydroxyalkanoate production by mixed aerobic cultures: from the substrate to the final product. Macromol Biosci 6(11):885–906. https://doi.org/10.1002/mabi.200600112

64. Dietz D, Zeng AP (2014) Efficient production of 1,3-propanediol from fermentation of crude glycerol with mixed cultures in a simple medium. Bioproc Biosyst Eng 37(2):225–233. https://doi.org/10.1007/s00449-013-0989-0

65. Moita R, Freches A, Lemos PC (2014) Crude glycerol as feedstock for polyhydroxyalkanoates production by mixed microbial cultures. Water Res 58:9–20. https://doi.org/10.1016/j.watres.2014.03.066

66. Utomo RNC, Li W-J, Tiso T, Eberlein C, Doeker M, Heipieper HJ, Jupke A, Wierckx N, Blank LM (2020) Defined microbial mixed culture for utilization of polyurethane monomers. ACS Sust Chem Eng. https://doi.org/10.1021/acssuschemeng.0c06019

67. Jiang L-L, Zhou J-J, Quan C-S, Xiu Z-L (2017) Advances in industrial microbiome based on microbial consortium for biorefinery. Biores Bioproc. https://doi.org/10.1186/s40643-017-0141-0

68. Zheng Y, Chen JC, Ma YM, Chen GQ (2020) Engineering biosynthesis of polyhydroxyalkanoates (PHA) for diversity and cost reduction. Metab Eng 58:82–93. https://doi.org/10.1016/j.ymben.2019.07.004

69. Chen GQ, Jiang XR (2018) Next generation industrial biotechnology based on extremophilic bacteria. Curr Opin Biotechnol 50:94–100. https://doi.org/10.1016/j.copbio.2017.11.016

70. Tan D, Wang Y, Tong Y, Chen G-Q (2021) Grand challenges for industrializing polyhydroxyalkanoates (PHAs). Trends Biotechnol. https://doi.org/10.1016/j.tibtech.2020.11.010
71. Jiang XR, Yan X, Yu LP, Liu XY, Chen GQ (2021) Hyperproduction of 3-hydroxypropionate by *Halomonas bluephagenesis*. *Nat Commun* 12(1):1513. https://doi.org/10.1038/s41467-021-21632-3

72. Federle MJ, Bassler BL (2003) Interspecies communication in bacteria. *J Clin Invest* 112(9):1291–1299. https://doi.org/10.1172/jci20195

73. Nowak MA (2006) Five rules for the evolution of cooperation. *Science* 314(5805):1560–1563. https://doi.org/10.1126/science.1133755

74. Du R, Yan J, Li S, Zhang L, Zhang S, Li J, Zhao G, Qi P (2015) Cellulosic ethanol production by natural bacterial consortia is enhanced by *Pseudomonas taiwanensis*. *Biotechnol Biofuels* 8(1):10. https://doi.org/10.1186/s13068-014-0186-7

75. Stolyar S, Van Dien S, Hillesland KL, Pinel N, Lie TJ, Leigh JA, Stahl DA (2007) Metabolic modeling of a mutualistic microbial community. *Mol Syst Biol* 3:92. https://doi.org/10.1038/msb4100131

76. DECHEMA Gesellschaft für Chemische Technik und Biotechnologie e.V. (2019) KETBIO. Top Ten EU Biotech projects. https://ketbio.eu/KETBIO_Parade. Accessed 23 Nov 2020

77. Wei R, Breite D, Song C, Gräsing D, Ploss T, Hille P, Schwerdtfeger R, Matysik J, Schulze A, Zimmermann W (2019) Biocatalytic degradation

efficiency of postconsumer polyethylene terephthalate packaging determined by their polymer microstructures. *Adv Sci* 6(14):1900491. https://doi.org/10.1002/advs.201900491

78. Li W-J, Narancic T, Kenny ST, Niehoff P-J, O'Connor K, Blank LM, Wierckx N (2020) Unraveling 1,4-butanediol metabolism in *Pseudomonas putida* KT2440. *Front Microbiol* 11:382. https://doi.org/10.3389/fmicb.2020.00382

79. Sudarsan S, Dethlefsen S, Blank LM, Siemann-Herzberg M, Schmid A (2014) The functional structure of central carbon metabolism in *Pseudomonas putida* KT2440. *Appl Environ Microbiol* 80(17):5292–5303. https://doi.org/10.1128/aem.01643-14

**Publisher's Note**

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Submit your manuscript to a SpringerOpen journal and benefit from:

► Convenient online submission

► Rigorous peer review

► Open access: articles freely available online

► High visibility within the field

► Retaining the copyright to your article

Submit your next manuscript at ► springeropen.com