MIX-UP: MIXed plastics biodegradation and UPcycling using microbial communities

Poly(vinyl alcohol) (PVA) is a water‐soluble synthetic vinyl polymer with remarkable physical properties including thermostability and viscosity. Its biodegradability, however, is low even though a large amount of PVA is released into the environment. Established physical‐chemical degradation methods for PVA have several disadvantages such as high price, low efficiency, and secondary pollution. Biodegradation of PVA by microorganisms is slow and frequently involves pyrroloquinoline quinone (PQQ)‐dependent enzymes, making it expensive due to the costly cofactor and hence unattractive for industrial applications. In this study, we present a modified PVA film with improved properties as well as a PQQ‐independent novel enzymatic cascade for the degradation of modified and unmodified PVA. The cascade consists of four steps catalyzed by three enzymes with in situ cofactor recycling technology making this cascade suitable for industrial applications.

Poly(vinyl alcohol) (PVA) is a water‐soluble synthetic vinyl polymer with remarkable physical properties including thermostability and viscosity. Its biodegradability, however, is low even though a large amount of PVA is released into the environment. Established physical‐chemical degradation methods for PVA have several disadvantages such as high price, low efficiency, and secondary pollution. Biodegradation of PVA by microorganisms is slow and frequently involves pyrroloquinoline quinone (PQQ)‐dependent enzymes, making it expensive due to the costly cofactor and hence unattractive for industrial applications. In this study, we present a modified PVA film with improved properties as well as a PQQ‐independent novel enzymatic cascade for the degradation of modified and unmodified PVA. The cascade consists of four steps catalyzed by three enzymes with in situ cofactor recycling technology making this cascade suitable for industrial applications.

Microorganisms capable of decomposing polyurethane (PU) and other plastics have the potential to be used in bio-recycling processes. In this study, 20 PU-degrading strains were isolated, including 11 bacteria and 9 fungi, using a synthesized poly(1,4-butylene adipate)-based PU (PBA-PU) as the screening substrate. Three PU substrates with increasing structure complexities were used for a thorough evaluation of microbial degradation capacity: Impranil® DLN-SD, PBA-PU film and PU foam waste. After 4 days, the best fungal PBA-PU degrader, Cladosporium sp. P7, could degrade 94.5% of Impranil® DLN-SD. After 28 days of cultivation, 32.42% and 43.91% of solid PBA-PU film was converted into soluble small molecules when used as the sole carbon source or in a medium with other co-carbon sources, respectively. Accordingly, the weight loss of PU foam waste after 15 days was 15.3% for the sole carbon condition and 83.83% for the co-carbon conditions. Furthermore, PBA-PU was used for metabolic pathway analysis because of its known composition and chemical structure. Six metabolites were identified during the degradation process of PBA-PU, including adipic acid (AA), 1,4-butanediol (BDO), and 4,4'-methylenedianiline (MDA), which can also be used as the sole carbon source to grow the fungal strain P7, resulting in the discovery of two MDA metabolites during the cultivation processes. Based on the presence of these eight metabolites, we hypothesized that PBA-PU is first depolymerized by the fungal strain P7 via ester and urethane bond hydrolysis, followed by intracellular metabolism and mineralization of the three monomers to CO2 and H2O.

Enzymatic hydrolysis holds great promise for plastic waste recycling and upcycling. The interfacial catalysis mode, and the variability of polymer specimen properties under different degradation conditions, add to the complexity and difficulty of understanding polymer cleavage and engineering better biocatalysts. We present a systemic approach to studying the enzyme-catalyzed surface erosion of poly(ethylene terephthalate) (PET) while monitoring/controlling operating conditions in real time with simultaneous detection of mass loss and changes in viscoelastic behavior. PET nanofilms placed on water showed a porous morphology and a thickness-dependent glass transition temperature (Tg) between 40°C and 44°C, which is >20°C lower than the Tg of bulk amorphous PET. Hydrolysis by a dual-enzyme system containing thermostabilized variants of Ideonella sakaiensis PETase and MHETase resulted in a maximum depolymerization of 70% in 1 h at 50°C. We demonstrate that increased accessible surface area, amorphization, and Tg reduction speed up PET degradation while simultaneously lowering the threshold for degradation-induced crystallization.

TfCa, a promiscuous carboxylesterase from Thermobifida fusca, was found to hydrolyze polyethylene terephthalate (PET) degradation intermediates such as bis(2-hydroxyethyl) terephthalate (BHET) and mono-(2-hydroxyethyl)-terephthalate (MHET). In this study, we elucidated the structures of TfCa in its apo form, as well as in complex with a PET monomer analogue and with BHET. The structure–function relationship of TfCa was investigated by comparing its hydrolytic activity on various ortho- and para-phthalate esters of different lengths. Structure-guided rational engineering of amino acid residues in the substrate-binding pocket resulted in the TfCa variant I69W/V376A (WA), which showed 2.6-fold and 3.3-fold higher hydrolytic activity on MHET and BHET, respectively, than the wild-type enzyme. TfCa or its WA variant was mixed with a mesophilic PET depolymerizing enzyme variant [Ideonella sakaiensis PETase (IsPETase) PM] to degrade PET substrates of various crystallinity. The dual enzyme system with the wild-type TfCa or its WA variant produced up to 11-fold and 14-fold more terephthalate (TPA) than the single IsPETase PM, respectively. In comparison to the recently published chimeric fusion protein of IsPETase and MHETase, our system requires 10% IsPETase and one-fourth of the reaction time to yield the same amount of TPA under similar PET degradation conditions. Our simple dual enzyme system reveals further advantages in terms of cost-effectiveness and catalytic efficiency since it does not require time-consuming and expensive cross-linking and immobilization approaches.

Thermophilic polyester hydrolases (PES-H) have recently enabled biocatalytic recycling of the mass-produced synthetic polyester polyethylene terephthalate (PET), which has found widespread use in the packaging and textile industries. The growing demand for efficient PET hydrolases prompted us to solve high-resolution crystal structures of two metagenome-derived enzymes (PES-H1 and PES-H2) and notably also in complex with various PET substrate analogues. Structural analyses and computational modeling using molecular dynamics simulations provided an understanding of how product inhibition and multiple substrate binding modes influence key mechanistic steps of enzymatic PET hydrolysis. Key residues involved in substrate-binding and those identified previously as mutational hotspots in homologous enzymes were subjected to mutagenesis. At 72 °C, the L92F/Q94Y variant of PES-H1 exhibited 2.3-fold and 3.4-fold improved hydrolytic activity against amorphous PET films and pretreated real-world PET waste, respectively. The R204C/S250C variant of PES-H1 had a 6.4 °C higher melting temperature than the wild-type enzyme but retained similar hydrolytic activity. Under optimal reaction conditions, the L92F/Q94Y variant of PES-H1 hydrolyzed low-crystallinity PET materials 2.2-fold more efficiently than LCC ICCG, which was previously the most active PET hydrolase reported in the literature. This property makes the L92F/Q94Y variant of PES-H1 a good candidate for future applications in industrial plastic recycling processes.

Laccases are regarded as versatile green biocatalysts, and recent scientific research has focused on improving their redox potential for broader industrial and environmental applications. The density functional theory (DFT) quantum mechanics approach, sufficiently rigorous and efficient for the calculation of electronic structures, is conducted to better comprehend the connection between the redox potential and the atomic structural feature of laccases. According to the crystal structure of wild type laccase CueO and its variant, a truncated miniature cluster model method was established in this research. On the basic of thermodynamic cycle, the overall Gibbs free energy variations before and after the one-electron reduction were calculated. It turned out that the trends of redox potentials to increase after variant predicted by the theoretical calculations correlated well with those obtained by experiments, thereby validating the feasibility of this cluster model method for simulating the redox potentials of laccases.

Polybutylene adipate terephthalate (PBAT) is a biodegradable alternative to polyethylene and can be broadly used in various applications. These polymers can be degraded by hydrolases of terrestrial and aquatic origin. In a previous study, we identified tandem PETase-like hydrolases (Ples) from the marine microbial consortium I1 that were highly expressed when a PBAT blend was supplied as the only carbon source. In this study, the tandem Ples, Ple628 and Ple629, were recombinantly expressed and characterized. Both enzymes are mesophilic and active on a wide range of oligomers. The activities of the Ples differed greatly when model substrates, PBAT-modified polymers or PET nanoparticles were supplied. Ple629 was always more active than Ple628. Crystal structures of Ple628 and Ple629 revealed a structural similarity to other PETases and can be classified as member of the PETases IIa subclass, α/β hydrolase superfamily. Our results show that the predicted functions of Ple628 and Ple629 agree with the bioinformatic predictions, and these enzymes play a significant role in the plastic degradation by the consortium.

Polyurethane (PU) is one of the mass-produced recalcitrant plastics with a high environmental resistance but extremely low biodegradability. Therefore, improperly disposed PU waste adds significantly to plastic pollution, which must be addressed immediately. In recent years, there has been an increasing number of reports on plastic biodegradation in insect larvae, especially those that can feed on polyethylene and polystyrene. This study revealed that yellow mealworm (Tenebrio molitor) larvae can chew and ingest polyether-PU foams efficiently, resulting in a significant mass loss of nearly 67% after 35 days at a similar survival rate compared to when fed on bran. However, polyether-PU fragments were found in the frass of T. molitor, indicating that polyether-PU biodegradation and bioconversion in intestinal tracts were not complete. The scission of ether and urethane bonds in the polyether-PU can be evidenced by comparing polymer fragments recovered from frass with the pristine ones using Fourier transform infrared spectroscopy and X-ray photoelectron spectroscopy. Gel permeation chromatography suggested the release of low-molecular-weight oligomers as a result of the biodegradation, which also resulted in poor thermal stability of the polyether-PU foam as determined by thermogravimetric analysis. High-throughput sequencing of the gut microbiome revealed significant changes in the microbial community populations due to the polyether-PU diet, for example, an increase in the families Enterobacteriaceae and Streptococcaceae, suggesting that these microorganisms may contribute to the polyether-PU biodegradation.

Plastic waste imposes a serious problem to the environment and society. Hence, strategies for a circular plastic economy are demanded. One strategy is the engineering of polyester hydrolases towards higher activity for the biotechnological recycling of polyethylene terephthalate (PET). To provide tools for the rapid characterization of PET hydrolases and the detection of degradation products like terephthalic acid (TPA), we coupled a carboxylic acid reductase (CAR) and the luciferase LuxAB. CAR converted TPA into the corresponding aldehydes in Escherichia coli, which yielded bioluminescence that not only semi-quantitatively reflected amounts of TPA in hydrolysis samples but is suitable as a high-throughput screening assay to assess PET hydrolase activity. Furthermore, the CAR-catalyzed synthesis of terephthalaldehyde was combined with a reductive amination cascade in a one-pot set-up yielding the corresponding diamine, suggesting a new strategy for the transformation of TPA as a product obtained from PET biodegradation.

The environmental degradation and physical aging of microplastics (MP) caused by oxidative stress have not been thoroughly elucidated. In this study, we used different oxidative agents (Fe²⁺-activated peroxymonosulfate and Fenton reagents) that can form free radicals to study the degradation mechanisms of nylon 6 (PA6) and polystyrene (PS) MPs. After 4 cycles of treatment, mass losses of 25.6% and 22.1% were obtained with PA6 and PS MPs, respectively. Scanning electron microscopy (SEM), Fourier-transform infrared (FTIR) spectroscopy, Raman spectroscopy, and X-ray photoelectron spectroscopy (XPS) were employed to confirm the MP oxidation, and polymer chain scissions. FTIR data indicated the existence of oxygen-containing chemical groups resulting from MPs oxidation, and chain scissions, such as CO, C–O, and O–CO. Raman spectroscopy suggested the presence of exposed aromatic groups, and weakening of the relative intensity of C–H related to the oxidation, and chain scission of the MPs. SEM imaging revealed visible morphological changes on the surface of MPs as a result of degradation. XPS indicated that the O/C ratio could be used as an indicator for the degree of MPs oxidation. By analyzing the degradation products with surface-enhanced Raman scattering (SERS) and gas chromatography-mass spectrometry, low-molecular-weight alkanes, alcohols, aldehydes, carboxylic acids derived from the derivatization of alcohols, were detected. These findings confirmed the advantage of using multiple analytic methods in tandem to evaluate the degradation of environmental MPs.

Polyethylene terephthalate (PET) is the most widespread synthetic polyester, having been utilized in textile fibers and packaging materials for beverages and food, contributing considerably to the global solid waste stream and environmental plastic pollution. While enzymatic PET recycling and upcycling have recently emerged as viable disposal methods for a circular plastic economy, only a handful of benchmark enzymes have been thoroughly described and subjected to protein engineering for improved properties over the last 16 years. By analyzing the specific material properties of PET and the reaction mechanisms in the context of interfacial biocatalysis, this Perspective identifies several limitations in current enzymatic PET degradation approaches. Unbalanced enzyme−substrate interactions, limited thermostability, and low catalytic efficiency at elevated reaction temperatures, and inhibition caused by oligomeric degradation intermediates still hamper industrial applications that require high catalytic efficiency. To overcome these limitations, successful protein engineering studies using innovative experimental and computational approaches have been published extensively in recent years in this thriving research field and are summarized and discussed in detail here. The acquired knowledge and experience will be applied in the near future to address plastic waste contributed by other mass-produced polymer types (e.g., polyamides and polyurethanes) that should also be properly disposed by biotechnological approaches.

The plastic crisis requires drastic measures, especially for the plastics’ end-of-life. Mixed plastic fractions are currently difficult to recycle, but microbial metabolism might open new pathways. With new technologies for degradation of plastics to oligo- and monomers, these carbon sources can be used in biotechnology for the upcycling of plastic waste to valuable products, such as bioplastics and biosurfactants. We briefly summarize well-known monomer degradation pathways and computed their theoretical yields for industrially interesting products. With this information in hand, we calculated replacement scenarios of existing fossil-based synthesis routes for the same products. Thereby, we highlight fossil-based products for which plastic monomers might be attractive alternative carbon sources. Notably, not the highest yield of product on substrate of the biochemical route, but rather the (in-)efficiency of the petrochemical routes (i.e., carbon, energy use) determines the potential of biochemical plastic upcycling. Our results might serve as a guide for future metabolic engineering efforts towards a sustainable plastic economy.

Polyethylene terephthalate (PET) is a mass-produced petroleum-based synthetic polymer. Enzymatic PET degradation using, for example, Ideonella sakaiensis PETase (IsPETase) can be a more environmentally friendly and energy-saving alternative to the chemical recycling of PET. However, IsPETase is a mesophilic enzyme with an optimal reaction temperature lower than the glass transition temperature (Tg) of PET, where the amorphous polymers can be readily accessed for enzymatic breakdown. In this study, we used error-prone PCR to generate a mutant library based on a thermostable triple mutant (TM) of IsPETase. The library was screened against the commercially available polyester-polyurethane Impranil DLN W 50 for more thermostable IsPETase variants, yielding four variants with higher melting points. The most promising IsPETaseTMK95N/F201I variant had a 5.0°C higher melting point than IsPETaseTM. Although this variant showed a slightly lower activity on PET at lower incubation temperatures, its increased thermostability makes it a more active PET hydrolase at higher reaction temperatures up to 60°C. Several other variants were compared and combined with selected previously published IsPETase mutants in terms of thermostability and hydrolytic activity against PET nanoparticles and amorphous PET films. Our findings indicate that thermostability is one of the most important characteristics of an effective PET hydrolase.

Polyethylene terephthalate (PET) is a mass-produced petroleum-based non-biodegradable plastic that contributes to the global plastic pollution. Recently, biocatalytic degradation has emerged as a viable recycling approach for PET waste, especially with thermophilic polyester hydrolases such as a cutinase (LCC) isolated from a leaf-branch compost metagenome and its variants. To improve the enzymatic PET hydrolysis performance, we fused a chitin-binding domain (ChBD) from Chitinolyticbacter meiyuanensis SYBC-H1 to the C-terminus of the previously reported LCC ICCG variant, demonstrating higher adsorption to PET substrates and, as a result, improved degradation performance by up to 19.6% compared to with its precursor enzyme without the binding module. For compare hydrolysis with different binding module, the catalytic activity of LCC ICCG -ChBD, LCC ICCG -CBM, LCC ICCG -PBM and LCC ICCG -HFB4 were further investigated with PET substrates of various crystallinity and it showed measurable activity on high crystalline PET with 40% crystallinity. These results indicated that fusing a polymer-binding module to LCC ICCG is a promising method stimulating the enzymatic hydrolysis of PET.

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.

Recalcitrant plastic waste has caused serious global ecological problems. There is an urgent need to develop environmentally friendly and efficient methods for degrading the highly stable carbon skeleton structure of plastics. To that end, we used a quantum mechanical calculation to thoroughly investigate the oxidative scission of the carbon-carbon (C–C) backbone in polyethylene (PE). Here, we studied the reaction path of C–C bond oxidation via hydroxyl radical in PE. The flexible force constants and fuzzy bond orders of the C–C bonds were calculated in the presence of one or more carbocations in the same PE carbon chain. By comparison, the strength of the C–C bond decreased when carbocation density increased. However, the higher the density of carbocations, the higher the total energy of the molecule and the more difficult it was to be generated. The results revealed that PE oxidized to alcohol and other products, such as carboxylic acid, aldehyde and ketone, etc. Moreover, the presence of carbocations was seen to promote the cleavage of C–C backbones in the absence of oxygen.

Microbial polyhydroxyalkanoates (PHA) containing short- and medium/long-chain-length monomers, abbreviated as SCL-co-MCL/LCL PHAs, generate suitable thermal and mechanical properties. However, SCL-co-MCL/LCL PHAs with carbon chain longer than nine are difficult to synthesize due to the low specificity of PHA synthase PhaC and the lack of either SCL- or MCL/LCL monomer precursor fluxes. This study succeeds in reprogramming a β-oxidation weakened Pseudomonas entomophila containing synthesis pathways of SCL 3-hydroxybutyryl-CoA (3HB) from glucose and MCL/LCL 3-hydroxyalkanoyl-CoA from fatty acids with carbon chain lengths from 9 to 18, respectively, that are polymerized under a low specificity PhaC61-3 to form P(3HB-co-MCL/LCL 3HA) copolymers. Through rational flux-tuning approaches, the optimized recombinant P. entomophila accumulates 55 wt% poly-3-hydroxybutyrate in 8.4 g L⁻¹ cell dry weight. Combined with weakened β-oxidation, a series of novel P(3HB-co-MCL/LCL 3HA) copolymers with over 60 wt% PHA in 9 g L⁻¹ cell dry weight have been synthesized for the first time. P. entomophila has become a high-performing platform to generate tailor-made new SCL-co-MCL/LCL PHAs.

This article introduces the EU Horizon 2020 research project MIX-UP, “Mixed plastics biodegradation and upcycling using microbial communities”. The project focuses on the ambitious vision to change the traditional linear value chain of plastics to a sustainable, biodegradable based one. In MIX-UP, plastic mixtures containing five of the top six fossil-based recalcitrant plastics (PE, PUR, PP, PET, and PS), along with upcoming biobased and biodegradable plastics (bioplastics) such as PHA and PLA, will be used as feedstock for microbial transformations. The generated new workflow increases recycling quotas and adds value to present poorly recycled plastic waste streams. Consecutive controlled enzymatic and microbial degradation of mechanically pre-treated plastics waste combined with subsequent microbial conversion to polymers and value-added chemicals by mixed cultures. Through optimization of known plastic-degrading enzymes by integrated protein engineering, high specific binding capacities, stability, and catalytic efficacy towards a broad spectrum of plastic polymers under high salt content and temperature conditions will be achieved. Another focus lies in the search and isolation of novel enzymes active on recalcitrant polymers. MIX-UP will also enhance the production of enzymes and formulate enzyme cocktails tailored to specific waste streams. In vivo and in vitro application of these cocktails enables stable, self-sustaining microbiomes to convert the released plastic monomers selectively into value-added products, key building blocks, and biomass. Any of the remaining material recalcitrant to the enzymatic activity will be recirculated into the process by physicochemical treatment. The Chinese-European MIX-UP is a multidisciplinary and industry-participating consortium to address the market need for novel sustainable routes to valorize plastic waste streams. MIX-UP realizes a circular (bio) plastic economy and will contribute where mechanical and chemical plastic recycling show limits.