Progressing Plastics Circularity: A Review of Mechano-Biocatalytic Approaches for Waste Plastic (Re)valorization

Inspirational concepts, and the transfer of analogs from natural biology to science and engineering, has produced many excellent technologies to date, spanning vaccines to modern architectural feats. This review highlights that answers to the pressing global petroleum-based plastic waste challenges, can be found within the mechanics and mechanisms natural ecosystems. Here, a suite of technological and engineering approaches, which can be implemented to operate in tandem with nature's prescription for regenerative material circularity, is presented as a route to plastics sustainability. A number of mechanical/green chemical (pre)treatment methodologies, which simulate natural weathering and arthropodal dismantling activities are reviewed, including: mechanical milling, reactive extrusion, ultrasonic-, UV- and degradation using supercritical CO2. Akin to natural mechanical degradation, the purpose of the pretreatments is to render the plastic materials more amenable to microbial and biocatalytic activities, to yield effective depolymerization and (re)valorization. While biotechnological based degradation and depolymerization of both recalcitrant and bioplastics are at a relatively early stage of development, the potential for acceleration and expedition of valuable output monomers and oligomers yields is considerable. To date a limited number of independent mechano-green chemical approaches and a considerable and growing number of standalone enzymatic and microbial degradation studies have been reported. A convergent strategy, one which forges mechano-green chemical treatments together with the enzymatic and microbial actions, is largely lacking at this time. An overview of the reported microbial and enzymatic degradations of petroleum-based synthetic polymer plastics, specifically: low-density polyethylene (LDPE), high-density polyethylene (HDPE), polystyrene (PS), polyethylene terephthalate (PET), polyurethanes (PU) and polycaprolactone (PCL) and selected prevalent bio-based or bio-polymers [polylactic acid (PLA), polyhydroxyalkanoates (PHAs) and polybutylene succinate (PBS)], is detailed. The harvesting of depolymerization products to produce new materials and higher-value products is also a key endeavor in effectively completing the circle for plastics. Our challenge is now to effectively combine and conjugate the requisite cross disciplinary approaches and progress the essential science and engineering technologies to categorically complete the life-cycle for plastics

Exploring the substrate spectrum of phylogenetically distinct bacterial polyesterases

The rapid escalation of plastic waste accumulation presents a significant threat of the modern world, demanding an immediate solution. Over the last years, utilization of the enzymatic machinery of various microorganisms has emerged as an environmentally friendly asset in tackling this pressing global challenge. Thus, various hydrolases have been demonstrated to effectively degrade polyesters. Plastic waste streams often consist of a variety of different polyesters, as impurities, mainly due to wrong disposal practices, rendering recycling process challenging. The elucidation of the selective degradation of polyesters by hydrolases could offer a proper solution to this problem, enhancing the recyclability performance. Towards this, our study focused on the investigation of four bacterial polyesterases, including DaPUase, IsPETase, PfPHOase, and Se1JFR, a novel PETase-like lipase. The enzymes, which were biochemically characterized and structurally analyzed, demonstrated degradation ability of synthetic plastics. While a consistent pattern of polyesters’ degradation was observed across all enzymes, Se1JFR stood out in the degradation of PBS, PLA, and polyether PU. Additionally, it exhibited comparable results to IsPETase, a benchmark mesophilic PETase, in the degradation of PCL and semi-crystalline PET. Our results point out the wide substrate spectrum of bacterial hydrolases and underscore the significant potential of PETase-like enzymes in polyesters degradation

Biotechnological model for ubiquitous mixed petroleum- and bio-based plastics degradation and upcycling into bacterial nanocellulose

Ubiquitous post-consumer plastic waste is often physically mixed combining recalcitrant petroleum-based plastics with bioplastics, forming (petro-bio)plastic streams. Finding appropriate end-of-life (EoL) strategies for mixed (petro-bio)plastic waste is highly pertinent in achieving environmental protection, sustainability for plastic value chain industries including recyclers and government policy makers worldwide. The presence of bioplastic mixed in with polyethylene terephthalate (PET) or other petroleum-based plastic streams poses a substantial drawback to mechanical recycling and strongly impedes the development of sustainable EoL routes. Here, we present a model system for the sustainable management of mixed (petro-bio)plastic waste, demonstrating a biotechnological route through synergy-promoted enzymatic degradation of PET–representing petrochemical polyester plastic–mixed with thermoplastic starch (TPS)–as a model bioplastic. Leaf-branch compost cutinase (LCCICCG) and commercial amylase (AMY) deliver effective depolymerization of this mixed (petro-bio)plastic material, with subsequent bio-upcycling of the mixed waste stream into bacterial nanocellulose (BNC) by Komagataeibacter medellinensis. Compared to LCCICCG and AMY, the LCCICCG/AMY combined treatment synergistically produced a 2.6- and 4.4-fold increase in enzymatic decomposition at 70 °C in four days, respectively, yielding sugars and terephthalic acid (TPA) as the main depolymerization building blocks. Bio-upcycling of post-enzymatic degradation hydrolysates resulted in a high BNC yield of 3 g L−1 after 10 days. This work paves the way for sustainable management routes for challenging mixed recalcitrant plastic and bioplastic waste and prepares opportunities for its participation in the circular production of sustainable eco-polymers

Set of Small Molecule Polyurethane (PU) Model Substrates: Ecotoxicity Evaluation and Identification of PU Degrading Biocatalysts

Polyurethanes (PUs) are an exceedingly heterogeneous group of plastic polymers, widely used in a variety of industries from construction to medical implants. In the past decades, we have witnessed the accumulation of PU waste and its detrimental environmental impacts. PUs have been identified as one of the most toxic polymers leaching hazardous compounds derived both from the polymer itself and the additives used in production. Further environmental impact assessment, identification and characterization of substances derived from PU materials and establishing efficient degradation strategies are crucial. Thus, a selection of eight synthetic model compounds which represent partial PU hydrolysis products were synthesized and characterized both in terms of toxicity and suitability to be used as substrates for the identification of novel biocatalysts for PU biodegradation. Overall, the compounds exhibited low in vitro cytotoxicity against a healthy human fibroblast cell line and virtually no toxic effect on the nematode Caenorhabditis elegans up to 500 µg mL−1, and two of the substrates showed moderate aquatic ecotoxicity with EC50 values 53 µg mL−1 and 45 µg mL−1, respectively, on Aliivibrio fischeri. The compounds were successfully applied to study the mechanism of ester and urethane bond cleaving preference of known plastic-degrading enzymes and were used to single out a novel PU-degrading biocatalyst, Amycolatopsis mediterranei ISP5501, among 220 microbial strains. A. mediterranei ISP5501 can also degrade commercially available polyether and polyester PU materials, reducing the average molecular number of the polymer up to 13.5%. This study uncovered a biocatalyst capable of degrading different types of PUs and identified potential enzymes responsible as a key step in developing biotechnological process for PU waste treatment options

New Labeled PET Analogues Enable the Functional Screening and Characterization of PET-Degrading Enzymes

The discovery and engineering of novel biocatalysts capable of depolymerizing polyethylene terephthalate (PET) have gained significant attention since the need for green technologies to combat plastic pollution has become increasingly urgent. This study focuses on the development of novel substrates that can indicate enzymes with PET hydrolytic activity, streamlining the process of enzyme evaluation and selection. Four novel substrates, mimicking the structure of PET, were chemically synthesized and labeled with fluorogenic or chromogenic moieties, enabling the direct analysis of candidate enzymes without complex preparatory or analysis steps. The fluorogenic substrates, mUPET1, mUPET2, and mUPET3, not only identify enzymes capable of PET breakdown but also differentiate those with exceptional performance on the polymer, such as the benchmark PETase, LCCICCG. Among the substrates, the chromogenic p-NPhPET3 stands out as a reliable tool for screening both pure and crude enzymes, offering advantages over fluorogenic substrates such as ease of assay using UV–vis spectroscopy and compatibility with crude enzyme samples. However, ferulic acid esterases and mono-(2-hydroxyethyl) terephthalate esterases (MHETases), which exhibit remarkably high affinity for PET oligomers, also show high catalytic activity on these substrates. The substrates introduced in this study hold significant value in the function-based screening and characterization of enzymes that degrade PET, as well as the the potential to be used in screening mutant libraries derived from directed evolution experiments. Following this approach, a rapid and dependable assay method can be carried out using basic laboratory infrastructure, eliminating the necessity for intricate preparatory procedures before analysis.The Supporting Information [https://pubs.acs.org/doi/10.1021/acssuschemeng.4c00143]This is the peer reviewed version of the paper: Taxeidis, G., Djapovic, M., Nikolaivits, E., Maslak, V., Nikodinovic-Runic, J., & Topakas, E. (2024). New Labeled PET Analogues Enable the Functional Screening and Characterization of PET-Degrading Enzymes. ACS Sustainable Chemistry & Engineering. [https://doi.org/10.1021/acssuschemeng.4c00143

A polyesterase from the Antarctic bacterium Moraxella sp. degrades highly crystalline synthetic polymers

The uncontrolled release of plastics in the environment has rendered them ubiquitous around the planet, threatening the wildlife and human health. Biodegradation and valorization of plastics has emerged as an ecofriendly alternative to conventional management techniques. Discovery of novel polymer-degrading enzymes with diversified properties is hence an important task in order to explore different operational conditions for plastic-waste upcycling. In the present study, a barely studied psychrophilic enzyme (MoPE) from the Antractic bacterium Moraxella sp. was heterologously expressed, characterized and its potential in polymer degradation was further investigated. Based on its amino acid composition and structure, MoPE resembled PET-degrading enzymes, sharing features from both mesophilic and thermophilic homologues. MoPE hydrolyzes nonbiodegradable plastics, such as polyethylene terephthalate and polyurethane, as well as biodegradablePeer-reviewed manuscript: [https://imagine.imgge.bg.ac.rs/handle/123456789/1630

Triggering and identifying the polyurethane and polyethylene-degrading machinery of filamentous fungi secretomes

The uncontrollable disposal of plastic waste has raised the concern of the scientific community, which tries to face this environmental burden by discovering and applying new techniques. Regarding the biotechnology field, several important microorganisms possessing the necessary enzymatic arsenal to utilize recalcitrant synthetic polymers as an energy source have been discovered. In the present study, we screened various fungi for their ability to degrade intact polymers, such as ether-based polyurethane (PU) and low-density polyethylene (LDPE). For this, ImpranIil® DLN-SD and a mixture of long-chain alkanes were used as sole carbon sources, indicating not only the most promising strains in agar plate screening but also inducing the secretion of depolymerizing enzymatic activities, useful for polymer degradation. The agar plate screening revealed three fungal strains belonging to Fusarium and Aspergillus genera, whose secretome was further studied for its ability to degrade the aforementioned non-treated polymers. Specifically for ether-based PU, the secretome of a Fusarium species reduced the sample mass and the average molecular weight of the polymer by 24.5 and 20.4%, respectively, while the secretome of an Aspergillus species caused changes in the molecular structure of LDPE, as evidenced by FTIR. The proteomics analysis revealed that the enzymatic activities induced in presence of Impranil® DLN-SD can be associated with urethane bond cleavage, a fact which was also supported by the observed degradation of the ether-based PU. Although, the mechanism of LDPE degradation was not completely elucidated, the presence of oxidative enzymes could be the main factor contributing to polymer modification.The authors would like to thank the VIB Proteomics Core for the contribution regarding the mass spectrometry-based proteomics experiments (EPIC-XS, project number 823839, funded by the Horizon 2020 programme of the European Union).This is the peer reviewed version of the paper: Taxeidis, G., Nikolaivits, E., Siaperas, R., Gkountela, C., Vouyiouka, S., Pantelić, B., Nikodinović-Runić, J., & Topakas, E. (2023). Triggering and identifying the polyurethane and polyethylene-degrading machinery of filamentous fungi secretomes. Environmental Pollution, 325, 121460.[ https://doi.org/10.1016/j.envpol.2023.121460]Related to published version: [https://imagine.imgge.bg.ac.rs/handle/123456789/1797]Related to other materials: [https://zenodo.org/record/7767083#.ZCP8WXZBxPa

The Impressive Healing Power of Autologous Fibroblasts Isolated from Early Cultures of Skin Biopsies for the Treatment of Diabetic Foot Ulcers: Preliminary Results Regarding 2 Cases

Aims: The purpose of this study was to investigate whether we can quickly, effectively, and with relatively low cost, heal long-standing (>8 months) diabetic foot ulcers using autologous skin fibroblasts. Place and Duration of Study: Immunology & National Histocompatibility Department and 2nd Department of Surgery, ‘G. Gennimatas’ General Hospital, ‘Demetrios Voyatzoglou’ Diabetic Foot Clinic, ‘A. Fleming’ General Hospital, Department of Biology, National and Kapodistrian University of Athens, Athens, Greece, between June 2011 and May 2012. Study Design and Methodology: Early, autologous skin fibroblasts arisen in large numbers from small split-thickness skin biopsies, cultured in high concentration of fetal bovine serum, and dispersed in patients΄ own serum, were injected subcutaneously into the surrounding healthy tissue of uninfected diabetic foot ulcers of two type 2 diabetic patients without peripheral angiopathy. Results: There was complete healing in 11 and 27 weeks in patients 1 & 2, respectively. The early cultured fibroblasts showed impressive healing power for diabetic foot ulcers. On the contrary, the power of the prolonged cultured fibroblast diminished steadily, while the fibroblasts undergone the freezing-thawing procedure were not effective. Conclusion: The healing was complete, quick, safe, permanent, without scars or hyperkeratosis, and relatively inexpensive