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

Journal of Cleaner Production 443 (2024) 141025 Available online 8 February 2024 0959-6526/© 2024 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). Biotechnological model for ubiquitous mixed petroleumand bio-based plastics degradation and upcycling into bacterial nanocellulose Jeovan A. Araujo a , 1 , * , George Taxeidis b , 1 , Everton Henrique Da Silva Pereira a , Muhammad Azeem a , Brana Pantelic c , Sanja Jeremic c , Marijana Ponjavic c , Yuanyuan Chen a , Marija Mojicevic a , ** , Jasmina Nikodinovic-Runic c , Evangelos Topakas b , Margaret Brennan Fournet a a Centre for Polymer Sustainability, PRISM Research Institute, Technological University of the Shannon: Midlands Midwest, N37 HD68, Athlone, Ireland b Industrial Biotechnology & Biocatalysis Group, Biotechnology Laboratory, School of Chemical Engineering, National Technical University of Athens, Iroon Polytechniou 9, 15772, Athens, Greece c Institute of Molecular Genetics and Genetic Engineering, University of Belgrade, Vojvode Stepe 444a, 11000, Belgrade, Serbia ARTICLE INFO Handling Editor: Jian Zuo Keywords: Mixed plastic waste Biodegradation Enzymatic synergism Plastic waste valorization Bio-upcycling Eco-polymers production ABSTRACT 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 (LCC ICCG ) 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 LCC ICCG and AMY, the LCC ICCG /AMY combined treatment synergistically produced a 2.6and 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 postenzymatic 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. 1. Introduction Managing plastic pollution is undeniably one of the greatest societal challenges of the century. While the use of plastic spans simple daily needs to modern high-tech applications, nations are struggling to meet ambitious environmental targets set to manage an ever-increasing amount of plastic waste being generated every year (Borrelle et al., 2020). The traditional, highly linear end-of-life (EoL) treatment of plastics leads to incontrollable accumulation in the environment and failure to provide sustainable waste management for the safety of future generations and the planet. The current figures recorded for the distribution of post-use and waste plastics are alarming with accumulation in closed landfills or natural environments such as soils and oceans (79 %), incineration (12 %), or recycling (9 %) (Geyer et al., 2017; Wang et al., 2023). Polyethylene terephthalate (PET), one of the most produced recalcitrant thermoplastics worldwide and the highest produced polyester, is often considered the most circular of all plastics. Consequently, the extensive use of PET products, lack of appropriate technology and * Corresponding author. ** Corresponding author. E-mail addresses: [email protected], [email protected] (J.A. Araujo), [email protected] (M. Mojicevic). 1 These authors contributed equally to this work. Contents lists available at ScienceDirect Journal of Cleaner Production journal homepage: www.elsevier.com/locate/jclepro https://doi.org/10.1016/j.jclepro.2024.141025 Received 25 October 2023; Received in revised form 24 January 2024; Accepted 28 January 2024 Journal of Cleaner Production 443 (2024) 141025 2 waste management have already caused serious environmental issues and, with increasing production, this problem is expected to be exacerbated in the foreseeable future (Darko et al., 2023; Rochman et al., 2013). Due to their biodegradability, renewability and versatility, the plastic industry has seen a surge in market demand for bioplastics (Filho et al., 2021; Friedrich, 2021; Muhammad Shamsuddin, 2017), with global production capacity projected to increase by a 3.1-fold from 2021 to 2026 (European Bioplastics Conference, 2021). Consequently, traditional plastic waste streams are increasingly permeated by an array of bioplastics as the scale of their production increases (Ansink et al., 2022), forming mixed (petro-bio)plastic waste. In 2021, around 30 and 26 % of the global production capacity for biodegradable plastics were dedicated to produce polylactic acid (PLA) and starch blends, respectively (European Bioplastics Conference, 2021). Thermoplastic starch (TPS) has received increased attention because starch is an abundant, renewable biopolymer (Shafqat et al., 2020), and most importantly, TPS is fully biodegradable regardless of the environment as opposed to PLA being biodegradable only under industrially controlled conditions (Narancic et al., 2018). Nevertheless, the absence of established recycling streams for re-using bioplastics and recovering bioplastics from mixed (petro-bio)plastic streams is one of the current key challenges to sustainably transitioning from fossilto bio-based plastics (Rosenboom et al., 2022). Post-consumer fossiland bio-based plastics that contain a range of physically and chemically incompatible polymers from different sources, with a variety of limited to high potential end-of-use options, and an array of chemical structures, are disposed of jointly, thus contributing to the deterioration of the original properties of the conventional petroleum-based plastic waste recycling streams. Concerning the EoL management of recalcitrant plastics, mixed plastic waste contains numerous challenging components, such as various synthetic plastics, non-plastic impurities, and bioplastics. As a first option for PET waste, mechanical recycling has emerged as an environmentally sustainable choice to enable the circularity of PET. However, the quality of the post-consumer PET recyclate must comply with specific standards. Expectedly, the success of mechanical recycling is severely limited by the low-quality of recyclate, resulting in degraded mechanical properties, and uneconomic costs (Schyns and Shaver, 2021). Alternatively, post-consumer PET can be chemically recycled via polymer breakdown into monomers, but the use of harmful chemicals and high temperatures makes it an environmentally hazardous and energy-intensive process (Thiounn and Smith, 2020). Furthermore, chemical recycling of mixed plastic waste result in complex mixtures of degradation products that require complicated additional separation steps to recover monomers for recycling (Sullivan et al., 2022). For these reasons, recyclers rely on costly and complex sorting techniques to separate plastics of interest from the mixed solid waste. Failure to deliver a proper separation of the mixed plastic waste will result in techno-economic difficulties for the recovery of materials, or most certainly in landfilling or incineration (Kulas et al., 2023). Existing advanced sorting techniques are unable to economically separate recycling into distinct streams in the case of bioplastic presence. In this context, a recent report has concluded that if the EU is to meet its carbon emission reduction targets, an EU-wide roll-out of mixed plastic waste sorting would need to be in place (Grant et al., 2023). Nonetheless, the report falls short to recommend how the exorbitant monetary cost of such imposition would be met by EU’s member states. Thus, strategic development and implementation of sustainable pathways for mixed plastic waste is a pressing problem for current waste management practices. To overcome these shortfalls, we propose a biotechnological route for the biodegradation of a mixed (petro-bio)plastic system, and further upcycling into new bio-based polymers (Fig. 1). Over the past years, microorganisms and enzymes have been considered as an emerging alternative for bioremediation of plastics to prevent improper waste disposal to the environment (Lee et al., 2023; Yang et al., 2023). On the one hand, composting is a decomposition process that results in useful compost for soil fertilization (Gioia et al., 2021), where the addition of selected microorganisms can significantly speed up the partial or Fig. 1. End-of-life (EoL) management options for mixed (petro-bio)plastic waste: a) conventional destinations for mixed plastic waste; b) mechanical recycling of mixed plastic waste relies on expensive sorting techniques to provide partial recovery; and c) biotechnological approach proposed in this work to avoid expensive sorting and inappropriate disposal of mixed waste through tandem enzymatic degradation and bioconversion into eco-polymers. J.A. Araujo et al. Journal of Cleaner Production 443 (2024) 141025 3 complete disintegration process of biodegradable polymers (Quecholac-Pi˜ na et al., 2017). On the other hand, enzymatic degradation of PET still offers the eco-friendliest alternative for delivering PET circularity (Thomsen et al., 2022), as it requires mild reaction conditions, low energy and reagent consumption (Zhu et al., 2022). The PET backbone contains ester groups which are generally more biodegradable than C–C bonds. However, the substantial presence of aromatic terephthalate units in PET also decreases chain mobility which markedly restricts access to the hydrolysable ester bonds in the main chains. A number of studies was previously conducted to tune thermophysical properties of recalcitrant PET to make it amenable to biodegradation (Patel et al., 2023). The desired thermophysical characteristics of polyester substrates, such as high mobility of amorphous fraction, low glass transition temperature (T g ) and low crystallinity, directly influence the efficiency of enzymatic hydrolysis. For instance, higher amorphous fraction or lower crystallinity can accelerate enzyme penetration into crystalline lamellae, thereby increasing enzymatic depolymerization. Similarly, a low T g provides enzymes with greater accessibility to molecular chains due to enhanced chain mobility of polymers which becomes more mobile above T g (Patel et al., 2022). Wei et al. investigated biocatalytic efficiency of TfCut2 from Thermobifida fusca on postconsumer packaging and amorphous PET. It was concluded that amorphous regions of PET consist of both a rigid amorphous fraction and mobile amorphous fraction. Rigid amorphous fractions generally exhibit lower mobility of chains while mobile amorphous fraction provides chain mobility property to the total amorphous material. Results indicated that, compared to whole amorphous microstructures, the flexible mobile amorphous fraction is accessible to the polyester hydrolases (Wei et al., 2019a). A myriad of PET-hydrolyzing enzymes has been discovered and chemically characterized to date, including carboxylesterases, cutinases, and lipases (Tournier et al., 2023) that can cleave PET ester bonds resulting in bis(2-hydroxyethyl) terephthalic acid (BHET), mono-(2-- hydroxyethyl) terephthalic acid (MHET), terephthalic acid (TPA), and ethylene glycol (EG) as degradation products. Thermostable cutinases such as LCC ICCG and their homologues are the most efficient candidates due to their ability to operate at higher temperatures, closer to the T g of PET, at which the mobility of the polymer chain is increased, leading to improved accessibility to the ester linkages between monomeric units (Soong et al., 2022). Interestingly, it has recently been reported that LCC ICCG can also depolymerize biodegradable polyesters such as polycaprolactone (PCL), polybutylene succinate (PBS), polyhydroxybutyrate (PHB), and PLA (Makryniotis et al., 2023), making it a highly attractive enzyme for the treatment of mixed (petro-bio)plastic waste. Bio-upcycling is an important option to complement the EoL treatment of mixed plastic waste by revalorizing plastic debris. As opposed to harsh chemical treatments, enzymatic degradation is an advantageous approach that safely enables microorganisms to convert hydrolysates into added-value products (Kim et al., 2022). Among these products, bacterial nanocellulose (BNC) stands out as a material with immense potential for a wide range of applications due to its exceptional mechanical properties, making it a promising substitute for petroleum-based polymers (Navya et al., 2022). In spite of that, widespread use of BNC is restricted to excessive production costs (ca. 30 %) attributed to the culture medium (Jozala et al., 2016). Therefore, the use of post-degradation plastic debris as alternative cost-effective starting material for the production of BNC can be of great interest. Several bacterial genera including Komagataeibacter, Agrobacterium, Aerobacter, Achromobacter, Rhizobium, and Pseudomonas strains have been identified as BNC producers. Among these, Komagataeibacter is the most extensively studied and commonly used genus for BNC production. Besides BNC, biomaterials such as polyhydroxyalkanoates (PHAs) can potentially be obtained in similar process. Microbial production of these polymers was successfully implemented using TPS and PET monomers, separately (Gere and Czigany, 2018; Kourmentza et al., 2017). In this work, we implemented a biotechnological model using different commercially available enzymes and the benchmark PET degrading LCC ICCG to hydrolyze mixed (petro-bio)plastic materials (i.e. PET-TPS). Overall, this study presents a more sustainable EoL option for low-quality mixed (petro-bio)plastic (PET-TPS) waste streams using synergy-promoted enzymatic degradation, followed by a holistic biotransformation of the resulting by-products into BNC–a highperformance, multifunctional eco-polymer produced here adopting principles of a circular, bio-based economy for plastics. 2. Materials and methods 2.1. Materials PET granules from Alpek Polyester UK Ltd. (Lazenby, UK) and thermoplastic potato starch (TPS) commercialized as Solanyl® C2002 by Rodenburg Biopolymers (Oosterhout, The Netherlands) were used to prepare a model mixed (petro-bio)plastic waste material. Commercial thermostable alpha amylase (AMY) (Liquozyme® SC DS) and glucoamylase (Spirizyme® Fuel) were obtained from Novozymes (Bagsværd, Denmark) and used as received. The gene encoding the quadruple variant of leaf branch compost cutinase (LCC ICCG , variant F243I/D238C/ S283C/Y127G) was provided by GenScript Biotech B.V. (Rijswijk, Netherlands). Ethanol (EtOH), isopropyl β-D-1-thiogalactopyranoside (IPTG), HPLC grade acetonitrile and sulfuric acid, tris hydrochloride (Tris-HCl), sodium chloride (NaCl), and phosphate-buffered saline (PBS) buffers, potassium hydroxide (KOH), and other chemicals used for preparation of bacterial growth medium, buffers, and degradation experiments were purchased from Sigma-Aldrich (Burlington, MA). Hestrin–Schramm (HS) (2 % glucose, 0.5 % yeast extract, 0.27 % disodium phosphate (Na 2 HPO 4 ), and 0.15 % citric acid) was used as a control medium for the production of BNC by Komagataeibacter medellinensis ID13488 strain (Spanish Type Culture Collection, CECT 8140, Valencia, Spain). Ultrapure water was obtained from an Accu100 Ultrapure Water System (Fisher Scientific, Loughborough, UK). 2.2. Fabrication of the mixed PET-TPS films PET-TPS materials were prepared from commercial resins as a model mixed (petro-bio)plastic waste. PET and TPS pellets were physically mixed and processed by hot-melt extrusion at different TPS weight ratios (wt.%), namely 5, 10, 30 and 40 %. Films were prepared using a benchtop Prism™ twin-screw extruder by Thermo Electron GmbH (Karlsruhe, Germany), with 16 mm diameter screws at a temperature profile of 270, 250, 200, 180, and 70 ◦C, respectively from feeding to die zone, with a screw speed of 60 rpm. The extruder was coupled with a 3-roll calendar system to form films. The thickness of all films, measured by Vernier caliper, was below 1 mm. The fabricated films were shredded into small pieces, frozen in liquid nitrogen, then milled into powdered samples by using an Ultra Centrifugal Mill ZM 200 from Retsch GmbH (Haan, Germany), operating at a rotor speed of 17,000 rpm. Resulting films and powdered samples were stored at room temperature until further use. 2.3. Characterization 2.3.1. Fourier transform infrared (FTIR) spectroscopy The chemical changes of the PET-TPS samples were investigated by FTIR carried out on a PerkinElmer Spectrum One FTIR spectrometer (PerkinElmer Inc., Washington, USA) fitted with an attenuated total reflectance (ATR) sampling accessory. Spectra were recorded before and after mixed plastic degradation using a spectral resolution of 4 cm −1 , with 20 scans acquired for each spectrum (4000–650 cm −1 ), and air as background at room temperature. From the FTIR results, it was possible to determine the carbonyl index value, CI, according to equation (1) (Fotopoulou and Karapanagioti, 2017). CI =A1712 cm−1 A1408 cm−1 (1) J.A. Araujo et al. Journal of Cleaner Production 443 (2024) 141025 4 where A1712 cm−1 corresponds to the intensity of band typical of carbonyl group, and A1408 cm−1 is the intensity of deformation vibration of C–H band. 2.3.2. Differential scanning calorimetry (DSC) The thermal behavior of the mixed plastic films was studied by DSC thermograms recorded using a DSC 2920 Modulated DSC (TA Instruments, New Castle, DE, USA) with a nitrogen flow rate of 30 mL min −1 to prevent oxidation. Calibration of the instrument was performed using indium as standard. All the samples were dried at 60 ◦C for 8 h prior to testing. Test specimens weighed 6–9 ±0.01 mg and were measured on a Sartorius scale (MC 210 P; Goettingen, Germany). Measurements were carried out with samples crimped in non-perforated aluminum pans, against an empty pan as a reference, heated from 30 to 275 ◦C, at a 10 ◦C min −1 heating rate. The percentage degree of crystallinity (X c ) of each sample was calculated from the second heating cycle, according to equation (2): Xc(%) = ΔHm−ΔHc ΔH◦ m x100 (2) where, ΔH m is the apparent melt enthalpy of the test samples, ΔH c is the cold crystallization, ΔH ◦ m is the melt enthalpy of 100 % crystalline PET, which has been reported as 140 J g −1 (Wunderlich, 1973). 2.3.3. Scanning electron microscopy (SEM) The morphology of the mixed plastic films was analyzed by SEM images obtained using a Tescan Mira XMU (Tescan™, Brno, Czech Republic) in back scattered electron mode for surface analysis, with an accelerating voltage of 9 kV. Prior to analysis, tested samples were placed on individual aluminum stubs and sputtered with a thin layer of gold using a Baltec SCD 005 sputter for 110 s under vacuum at 0.1 mbar. 2.3.4. Tensile testing The mechanical properties of the mixed plastic films were evaluated using a Lloyd Lr10k tensiometer (Ametek Ltd., West Sussex, UK) with a 2.5 kN load cell on standard test specimens. Each sample was measured using a Vernier caliper, and tests were recorded at room temperature, with a strain rate of 10 mm min −1 , initial gauge length of 20 mm, and using a pull-to-limit setup (stop at a strain of 30 %). The tensile tests were carried out in accordance with ASTM D882, recorded using Nexygen™ software, and a minimum of five replicates per sample were analyzed and presented as mean values ±standard deviation. From the stress-strain curves recorded, the tensile strength and Young’s modulus of each sample were determined. 2.3.5. Statistical analysis Paired t-test (two sample for means) significance method was used to test the means of mechanical properties of PET with and without the presence of bioplastic, being considered statistically significant when the probability value (p) was found to be p ≤0.05, and tests were performed using the data analysis tools add-in feature on MS Excel Software (Microsoft Corporation, Redmond, WA, USA). 2.4. Biodegradation of the mixed PET-TPS materials 2.4.1. Model home composting bioaugmented with Bacillus sp. BPM12 Biodegradation of the studied mixed PET-TPS materials was first assessed by implementing a laboratory scale model home composting, using bioaugmented compost treated with a novel Bacillus sp. BPM12 strain (Pantelic et al., 2023) for 10 weeks at 37 ◦C, according to a previously described protocol (Pantelic et al., 2021) (Text S1, supplementary data). 2.4.2. Preparation of recombinant LCC enzyme In order to examine the biodegradability of the mixed PET-TPS films, genes coding LCC ICCG were synthesized following a known method (Tournier et al., 2020), codon optimized for expression in E. coli, and cloned in the expression vector pET26b(+) (GenScript Biotech, Rijswijk, Netherlands). The enzyme expression was performed in E. coli BL21 induced by 0.2 mM IPTG at 16 ◦C for 20 h, according to a previously described protocol (Dimarogona et al., 2015). After enzyme expression, the cells were harvested by centrifugation at 4000g for 15 min at 4 ◦C (Eppendorf Centrifuge 5417 R, Hamburg, Germany), and resuspended in 50 mM Tris-HCl, 300 mM NaCl pH =8 buffer. Cell suspension was disrupted by sonication during four 1-min cycles (8 s pulses and 5 s pause) at 40 % amplitude using a 20 kHz high intensity (400 W) ultrasonic processor (VC 400, Sonic & Materials, Newtown, CT, USA). After cell lysis, the cell debris was removed by centrifugation at 20,000g for 30 min at 4 ◦C, and the liquid fraction was purified using an immobilized metal-ion (Co 2+ ) affinity chromatography (IMAC) (Nikolaivits et al., 2022). The purity of isolated enzymes was checked on SDS-PAGE electrophoresis (Bio-Rad, Hercules, CA, USA) (12.5 % poly-acrylamide) and protein concentration was determined by measuring the absorbance at 280 nm. The molar extinction coefficient used (LCC ICCG : 28,836 M −1 cm −1 ) was calculated by ProtParam tool from ExPASy (Gasteiger et al., 2005). 2.4.3. Enzymatic degradation study PET-TPS film samples were rinsed with 70 % EtOH, air dried, weighted and inserted in glass flasks. The following enzyme preparations were used for degradation experiments: (1) LCC ICCG ; (2) AMY; and (3) LCC ICCG /AMY. Degradation experiments with LCC ICCG and AMY were performed using film samples hydrolyzed in 0.1 M phosphate buffer, pH =7, under agitation (1200 rpm) in an Eppendorf Thermomixer Comfort (Hamburg, Germany). Typically, 10 mg of each sample was added to 1 mL of reaction medium, and enzymatic treatments were optimized by conducting reactions at three different temperatures, namely 30, 50 and 70 ◦C. All tests were carried out on a continuous base for a period of four days. Hydrolysis reaction was initiated by adding 0.12 μ g of LCC ICCG which had been dialyzed in Tris-HCl buffer, pH =7. Moreover, 30 μ g of AMY was also added to the reaction mixture to assess the combined effect of these enzymes on the degradation of mixed PETTPS materials. This addition was also made in the case of PET, primarily for comparative purposes with the other tested materials. In control reactions, an equal amount of Tris-HCl buffer or deactivated AMY (boiled for 15 min) was added to the reaction mixture. After 24, 48, and 72 h, half of the initial amount of each enzyme was added to the reaction mixture to sustain the enzyme activity, thus mitigating enzyme issues related to thermal inactivation, and adsorption onto the material surface (Makryniotis et al., 2023). At the end of the reactions, the residual material was isolated by centrifugation, washed with ultrapure water, freeze-dried and its weight was measured. The reaction supernatant was analyzed by high-performance liquid chromatography (HPLC) and the dinitrosalicylic acid (DNS) colorimetric method to detect degradation products from the mixed PET-TPS plastic films. 2.4.4. Quantification of BHET, MHET, and TPA via HPLC analysis Samples were prepared by adding 1 μ L of 6 M HCl (50 % aqueous solution of concentrated HCl) to 1 mL of reaction supernatant, vortexed and centrifuged for 15 min at 12,000 rpm. The supernatant was filtered through 0.2 μ m syringe filters. For the HPLC analysis, an UltiMate 3000 HPLC (Thermo Fisher Scientific, Waltham, USA) coupled with an Eurospher II 100-3 C18A 150 ×4.6 mm column (Knauer, Germany) was used. The mobile phase consisted of 20 % (v/v) acetonitrile and 80 % (v/ v) 2.5 mM sulfuric acid in ultrapure water at a flow rate of 0.8 mL min −1 under isocratic conditions. Detection of reaction products was carried out using an ultraviolet–visible (UV–vis) detector at 241 nm and the total run time was set at 25 min. Quantification of TPA, MHET, and BHET was performed following the same HPLC protocol described above (Fig. S1, supplementary data). J.A. Araujo et al. Journal of Cleaner Production 443 (2024) 141025 5 2.4.5. Quantification of reducing sugars via DNS colorimetric method The total amount of reducing sugars was quantified using the DNS colorimetric method. The DNS method was performed according to the traditional protocol (Miller, 1959), with modifications made to reduce the reaction volumes. Sugar levels were quantified at 540 nm using the SpectraMax® ABS Plus microplate reader (Molecular Devices, Sunnyvale, CA, USA), and data were processed using SoftMax® Pro 7 software (Molecular Devices, Sunnyvale, CA, USA). A calibration curve was constructed using glucose as a standard (Fig. S2, supplementary data). 2.4.6. Degree of synergism Based on the detected concentration of degradation products from the mixed PET-TPS films, the degree of synergism (DS) between LCC ICCG and AMY to degrade the different films was determined. The DS was calculated using equation (3): DS =DPLCC/AMY DPLCC +DPAMY (3) where, DP LCC/AMY is the degradation products released by LCC ICCG / AMY, DP LCC is the degradation products released by LCC ICCG , and DP AMY is the degradation products released by AMY. 2.5. Bio-upcycling of PET-TPS degradation products 2.5.1. Scale-up of the enzymatic degradation After enzymatic treatment was proven to be successful on examined materials, experiment was set up in larger volumes using 1 g of each PET-TPS powdered sample. Samples were treated in 50 mL of 0.1 M phosphate buffer, pH =7, at 50 ◦C in a shaking incubator (200 rpm) for four days. More specifically, material hydrolysis was initiated after the addition of 1.2 μ g of LCC ICCG dialyzed in Tris-HCl buffer, as mentioned previously, while an equal amount of the enzyme was supplemented after 48 h. TPS liquefaction was accomplished by adding 300 μ g of AMY, whereas in control reactions, an equal amount of Tris-HCl or deactivated AMY was added. After four days, 500 mg of glucoamylase was added to each of the reactions, and the reaction mixture was kept under the same conditions for an additional hour to ensure the total conversion of released reducing sugars into glucose, facilitating their quantification and subsequent valorization. Finally, the residual material was isolated by centrifugation, washed twice with ultrapure water, and freeze-dried before its weight was measured. The reaction supernatant was analyzed by HPLC and DNS methods to detect the released products after the mixed PET-TPS degradation according to the protocols aforementioned. Hydrolysates were kept for composition analysis, and further bioupcycling into BNC. 2.5.2. Bacterial nanocellulose (BNC) production Liquid residues obtained from the enzymatically degraded mixed PET-TPS materials were used as growing medium for the production of BNC by Komagataeibacter medellinensis ID13488. Typically, 2 mL of each liquid residue was poured into glass tubes, and pH was adjusted to 4.5 by using 0.05 mol L −1 HCl and NaOH solutions, then 10 % bacterial inoculum was added. Prior to inoculation, bacterial culture was rinsed with PBS buffer followed by centrifugation, to secure the removal of any additional carbon and energy sources from the production media. All samples were statically incubated at 28 ◦C for 10 days. HS medium was used as a positive control. After 10 days, the BNC obtained was treated with 5 % KOH, rinsed with distilled water until neutral pH was reached, air dried at 60 ◦C, and weighted. All BNC growth trials were performed in triplicates. 3. Results and discussion In order to attain plastic circularity, this study proposes an alternative EoL management to prevent incineration or landfilling of lowerquality mixed plastics by demonstrating a biotechnological route to degrade mixed (petro-bio)plastic material (i.e. PET-TPS), and further bio-upcycle into high added value BNC. In our pursuit of a more sustainable option for such mixed (petro-bio)plastic waste, this work was divided into three main stages, as depicted in Fig. 2. 3.1. Fabrication and characterization of mixed (petro-bio)plastic films In the first part of this work, we used TPS as a model bioplastic to simulate the problem of fossil-based PET recycling streams that are unrestrained mixed with bio-based plastics (Siddiqui et al., 2021). Hot-melt extrusion was used to compound PET and TPS at different weight ratios prior to enzymatic depolymerization, as presented in Table 1. Recently, Patel et al. have studied melt processing of PET as a pretreatment for enzymatic depolymerization of single plastic waste, while Åkesson et al. have suggested that even at 1 % contamination the highly dense hydroxyl groups present in TPS are chemically incompatible with the ester linkages of PET backbone (Åkesson et al., 2021; Patel et al., 2022). To verify the above claim, the effect of varying the TPS content mixed with PET in the extruded materials was evaluated in terms of changes in the films’ chemical, thermal, and mechanical properties which were unveiled by FTIR, DSC, and tensile testing, respectively. Chemical changes in the functional groups and fingerprint regions of the extruded mixed PET-TPS films (Fig. 3, top) were inspected by FTIRATR (Fig. 3, bottom). Characteristic bands of PET that were observed in the range from 3055 to 2850 cm −1 were assigned to aromatic and aliphatic –C–H bond stretching, at 1712 cm −1 was attributed to the stretching of the ester carbonyl group –C – – O, and at 1244 and 1095 cm −1 were assigned to the C–O stretching and the methylene group vibrations, respectively (Chen et al., 2012). Also, the band at 871 cm −1 was indicative of aromatic ring C–H out-of-plane bending (Donelli et al., 2009), while the bands at 1370 and 1340 cm −1 were attributed to gauche and trans conformations of the CH 2 wagging vibrations. Moreover, change in the intensity of these two bands reveals the occurrence of an amorphous or semi-crystalline phase, so the ratio of these two bands indicates a change in the crystalline domains of PET (Sammon et al., 2000). As the amount of TPS present in PET increased from 0 to 40 %, a decrease of the 1340 cm −1 /1370 cm −1 absorbance ratio was clearly noted, which declined from 1.99 in the reference neat PET film to 1.11 in the mixed PET-TPS40 material. Therefore, it is suggested that the presence of TPS mixed in the PET stream has affected the close packing nature of the polyester crystalline phase and, consequently, promoted an amorphization process of the PET content. The spectrum of neat TPS film (Fig. S3, supplementary data) has been previously examined elsewhere in the literature (Araujo et al., 2021). In comparison to the spectrum of the neat PET (Fig. 3a, bottom), the spectra of the mixed PET-TPS films exhibited four distinct bands due to the reactive interactions between the two polymeric components. As it is clearly shown in the spectrum of the PET-TPS40 film (Fig. 3e, bottom), the presence of Fig. 2. Schematic detailing the main stages of the proposed biotechnological route for the end-of-life (EoL) management of mixed plastic waste: a) preparation of a model mixed (petro-bio)plastic (PET-TPS) system; b) biodegradation of mixed (petro-bio)plastics resulting in low-cost starting materials; and c) bioupcycling of low-cost waste-derived materials into new high-value eco-polymers. J.A. Araujo et al. Journal of Cleaner Production 443 (2024) 141025 6 TPS is confirmed by the bands present at 1758 cm −1 that was assigned to the stretching of C – – O group, at 1454 cm −1 which corresponded to C–H bending vibrations, and at 1184 and at 1044 cm −1 that were assigned to changes in the C–O and C–O–H stretching vibrations of TPS molecules (Bergel et al., 2018; Diop et al., 2011). The thermal properties of the mixed (petro-bio)plastic films with varying bioplastic content were evaluated by DSC. The results obtained from the second heating step of the thermal analysis are shown in Table 1. The thermogram of the neat PET film showed its glass transition temperature (T g ) at about 84 ◦C, a characteristic endothermic melting peak (T m ) at almost 253 ◦C, and a resulting X c =37 %, which are in agreement with previous reports in the literature (Del Mar Castro L´ opez et al., 2014). When the amount of TPS bioplastic mixed in the PET material was increased, T g and X c were clearly reduced, confirming the decline in PET crystalline phase as previously indicated by the FTIR results. Particularly, T g reduced to around 50 ◦C and X c decreased to only 26 % in the PET material with the highest bioplastic content (i.e. PET-TPS40), respectively. In addition, a lower X c was observed following the thermomechanical processing used prior to enzymatic degradation (Fig. S4, Table S1, supplementary data). PET monomeric units are linked by ester linkages resulting from the polymerization reaction between TPA and EG. X c can be used to describe the level of mobility of the ester linkages that link PET monomeric units, which can be associated to a measure of the polymer segmental motion (Zekriardehani et al., 2017). Thus, the reduced X c indicated lesser rigidity in the linkages, lowering the restriction in polymer chain motion. Furthermore, T g is the temperature at which a transition from the glassy to the rubbery state occurs and, consequently, the mobility of the polymer chains is improved. Following the thermomechanical extrusion process, the decrease in T g and X c reported for the mixed PET-TPS films indicated that the ester linkages in PET became more accessible at lower temperatures. As a result, the increased mobility could enhance the activity of polyester hydrolases by allowing their access to the target ester linkages. Patel et al. have recently reported on the importance of thermally pretreating single plastic PET to enhance enzymatic depolymerization by promoting irreversible macromolecular changes caused by thermal and mechanical shear (Patel et al., 2022), therefore our findings indicate that a similar pretreatment concept could be used to advance the depolymerization of complex mixed PET streams using enzymes. In agreement with this decay of the thermal properties, Åkesson et al. recently investigated the effect of up to 5 wt% bioplastic content on the mechanical recycling of conventional plastics, and they reported suggestive decreases in the T g and X c values of PET (Åkesson et al., 2021). To gain more insights into the effect of bio-based polymers present in PET recycling streams, the mechanical properties of the fabricated films were assessed in terms of Young’s modulus (E) and tensile strength ( σ ) (Text S6, supplementary data). In general terms, it is clear that the mixed (petro-bio)plastic with increased TPS content triggered the deterioration of mechanical properties, which is consistent with the decay of thermal properties previously presented, particularly the T g and X c , suggesting that such plastic streams would be unsuitable for mechanical recycling as an EoL option for the mixed (petro-bio)plastic waste. 3.2. Biodegradation of the fabricated mixed PET-TPS films A recent study has isolated and identified a novel bacterium strain named Bacillus subtilis BPM12 that efficiently interacts with PET and can hydrolyze its intermediate compounds (Pantelic et al., 2023). Therefore, in the second stage of our study we first developed a model home composting system bioaugmented with Bacillus sp. BPM12 to evaluate the biodegradation of mixed PET-TPS materials, which were initially assessed in terms of biodegradation and further characterized by FTIR (Fig. S5, supplementary data) and SEM (Fig. S6, supplementary data) analysis, but decomposition was limited to only 3.4 and 4.1 % weight loss from nontreated and bioaugmented compost, respectively (Text S7, supplementary data), making home composting an unsuitable route for the biodegradation of the studied mixed (petro-bio)plastic system. A reasonable explanation is that home composting is a bioremediation process for plastic treatment that occurs under uncontrolled conditions–hence, degradation rates can be very unpredictable. A more interesting alternative to delve into treatment options for such mixed Table 1 Composition by weight and thermo-mechanical properties of the fabricated mixed (petro-bio)plastic films. Sample PET (wt.%) TPS (wt.%) T g (◦C) T m (◦C) ΔH m (J g −1 ) X c (%) E (MPa) σ (MPa) PET 100 83.6 252.9 52.4 37.5 2239 ±105 38 ±5 PET-TPS5 95 5 79.2 246.9 49.0 35.0 2093 ±215 21 ±1 PET-TPS10 90 10 76.7 247.2 48.5 34.6 2028 ±61 21 ±2 PET-TPS30 70 30 54.2 248.4 45.2 32.3 1985 ±135 19 ±3 PET-TPS40 60 40 50.7 245.4 36.6 26.1 1628 ±83 13 ±2 T g , glass transition temperature; T m , melting temperature; ΔH m , melting enthalpy; X c , degree of crystallinity; E, Young’s modulus; σ , tensile strength. Fig. 3. Digital images (top) and FTIR spectra (bottom) of the studied mixed (petro-bio)plastic films: (a) neat PET, (b) PET-TPS5, (c) PET-TPS10, (d) PETTPS30, and (e) PET-TPS40 films. J.A. Araujo et al. Journal of Cleaner Production 443 (2024) 141025 7 (petro-bio)plastic streams following a thermomechanical extrusion pre-treatment could be industrial composting, however such assessment is out of the scope of this work. Conversely, various enzymes have also shown the ability to degrade PET, including natural or engineered esterases such as PETase (Yoshida et al., 2016), carboxylesterase (von Haugwitz et al., 2022), and cutinases (Sintawee et al., 2012; Tournier et al., 2020). Therefore, we turned our efforts to investigate the biodegradation of the mixed (petro-bio)plastic waste by exploring the biocatalytic potential of three different enzymatic systems (i.e. AMY, LCC ICCG , and LCC ICCG /AMY). 3.2.1. Enzymatic hydrolysis of the mixed PET-TPS films To investigate the breakdown of the mixed PET-TPS samples, we selected LCC ICCG and AMY as these enzymes show great performance in PET degradation (Tournier et al., 2023) and starch hydrolysis (Miłek and Lamkiewicz, 2022). Given that there are no similar materials mentioned in the literature, the enzyme/substrate ratio utilized was almost 80 times lower to what is usually mentioned in low crystallinity PET degradation (Tournier et al., 2023), significantly contributing to the feasibility and viability of the process. The degradation of prepared mixed (petro-bio)plastic films was determined by analyzing the percentage enzymatic depolymerization of PET-TPS films in comparison to that of neat PET. Post-enzymatic degradation amounts of BHET, MHET, and TPA were determined via HPLC analysis, while the amount of total reducing sugars released in the reaction medium was monitored using the DNS method. Finally, the synergism between LCC ICCG and AMY was calculated to further examine the performance of the combined enzymatic treatment in the biodegradation of mixed PET-TPS materials. LCC ICCG has a known optimal temperature at around 70 ◦C (Tournier et al., 2020), while AMY shows a wide temperature range from 20 to 70 ◦C (Tomasik and Horton, 2012). Here, we sought to investigate the impact of the temperature on the synergistic effect of LCC ICCG /AMY to convert mixed PET-TPS plastics into their basic building blocks by performing degradation tests at three different temperatures, i.e. 30, 50, and 70 ◦C. The degradation studies at 30 and 50 ◦C are discussed in details in Texts S10–S12, Figs. S7–8, supplementary data. The enzymatic degradation of the mixed PET-TPS films treated with AMY, LCC ICCG , and LCC ICCG /AMY was assessed at 70 ◦C. It was clear that this temperature exhibited the best overall biodegradation results in our study (Fig. 4), which correlated to improved enzymatic activity for both enzymes at temperatures exceeding 60 ◦C (Ruiz et al., 2011; Tournier et al., 2020). As expected, LCC ICCG alone showed an improved performance of 53.5 ±0.1 % enzymatic decomposition for neat PET film at 70 ◦C. Similarly, this enzyme increased the degradation of the mixed PET-TPS films from around 19 % at 50 ◦C up to 32 % at 70 ◦C, as depicted in Fig. 4a. Most importantly, the synergistic action of LCC ICCG /AMY was evident at 70 ◦C, at which a significant improvement in depolymerization was noted for all mixed PET-TPS samples. The combined LCC ICCG /AMY action remarkably increased enzymatic Fig. 4. Enzymatic degradation study on the mixed PET-TPS films using AMY, LCC ICCG , and LCC ICCG /AMY at 70 ◦C: (a) Enzymatic decomposition (%) calculated from the total degradation products (BHET, MHET, TPA, and reducing sugars); (b) Total PET products released detected by HPLC analysis; (c) Reducing sugars detected via DNS method; and (d) Comparison of DS for the different mixed PET-TPS samples. The concentration of 100 % PET-TPS degradation products is equivalent to 10 mg mL −1 . Tests were carried out in triplicate; error bars represent the standard deviation of the mean values. J.A. Araujo et al. Journal of Cleaner Production 443 (2024) 141025 8 decomposition percentages to 56, 60, 70, and 77 % for the mixed PET-TPS5, PET-TPS10, PET-TPS30, and PET-TPS40 materials, respectively. Compared to the LCC ICCG single treatment, the LCC ICCG /AMY synergistic action exhibited a 2.0-, 1.9-, 2.2-, and 2.6-fold increase in enzymatic decomposition. Thus, this was a clear evidence of the synergism promoted by the dual-enzyme preparation on the depolymerization of the mixed (petro-bio)plastic samples. Moreover, this exceptional overall performance was attributed to a noteworthy boost observed for degradation products released during the hydrolysis reaction deriving from both PET and TPS counterparts in the mixed (petro-bio)plastic materials. The detection of total PET intermediate products (TPA, MHET, and BHET) is shown in Fig. 4b. It is worth mentioning that EG is another commonly expected degradation product when TPA is detected, however the HPLC protocol used in this work is not suitable for quantifying EG. AMY does not degrade PET, so no release of TPA, MHET or BHET was observed. It can be noted that the PET degradation products released from the neat PET film remained practically the same after both LCC ICCG and LCC ICCG /AMY treatments at around 5.2 mg mL −1 , which was equivalent to a 56 % PET conversion. However, it is evident that the presence of this enzyme in the LCC ICCG /AMY treatment had an important role in synergistically increasing the total mixed PET-TPS film conversion. The addition of AMY to the LCC ICCG enzyme led to an increase in the total amount of reducing sugars (Fig. 4c). As expected, the amount of total reducing sugars detected for the mixed PET-TPS40 film after enzymatic treatment with AMY at 70 ◦C increased to 1.74 ±0.1 mg mL −1 . Notably, when PET-TPS40 was treated with LCC ICCG /AMY, the amount of total reducing sugars surged to 3.24 ±0.3 mg mL −1 , a highest in the extent of saccharification among all enzymatic treatments. The identification of reducing sugars provides insights into the simultaneous degradation of the mixed PET-TPS materials, and proves that the synergistic action of these enzymes is of vital importance in order to achieve higher degradation yields. Regarding DS of LCC ICCG /AMY at 70 ◦C, it varied from 1.4 to 1.8, with the highest value observed in the case of PET-TPS30 (Fig. 4d). It is worth noting that the DS does not appear to be influenced by the temperature, with similar values observed at 30 (Fig. S7d, supplementary data) and 50 ◦C (Fig. S8d, supplementary data). However, the overall enzymatic depolymerization showed a strong correlation with the reaction temperature change, indicating optimal enzymatic activity of LCC ICCG /AMY at 70 ◦C. These results emphasize the significance of utilizing enzymatic combinations to enhance the recovery of depolymerization products from mixed (petro-bio)plastic waste, which could then be used as low-cost starting materials for the sustainable production of new synthetic or bio-based polymers. The yield of PET intermediates TPA, MHET, and BHET resulting from the enzymatic degradation of the neat PET and mixed PET-TPS films was examined at 50 and 70 ◦C (Fig. 5). The total amount of detected products at 30 ◦C was approximately 200and 500-times lower than at 50 and 70 ◦C, respectively, thus it was intentionally neglected in the interest of calculating the yield of hydrolysis products. TPA was the main PET degradation product for all mixed PET-TPS samples treated by LCC ICCG , from which between 69–100 % and 80–100 % TPA was detected at 50 and 70 ◦C, respectively (Fig. 5a, c). Interestingly, the treatment of mixed PET-TPS films with LCC ICCG /AMY resulted in slightly higher TPA yields Fig. 5. Yield of PET degradation products (%) detected by HPLC analysis of the neat PET and mixed PET-TPS films: (a) LCC ICCG and (b) LCC ICCG /AMY treatments at 50 ◦C; (c) LCC ICCG and (d) LCC ICCG /AMY treatments at 70 ◦C. J.A. Araujo et al. Journal of Cleaner Production 443 (2024) 141025 9 in comparison to LCC ICCG alone, from which minimum TPA yields of 94 % (PET-TPS30 at 50 ◦C, Fig. 5b) and 86 % (PET-TPS5 at 70 ◦C, Fig. 5d) were measured. Such findings were correlated to the synergistic action of the dual-enzymatic treatment that simultaneously promoted the degradation of the mixed (petro-bio)plastics, where the increased degradation of TPS by AMY enabled the polymer chains of PET to become more easily accessible for the LCC ICCG hydrolytic action, thus increasing the TPA yield. Further investigation including chemical, thermal, and microstructural analysis assessed the extent of the enzyme-catalyzed depolymerization of the mixed PET-TPS materials and provided a deeper understanding of the hydrolysis reaction. FTIR-ATR was used to enlighten the changes in functional groups of selected PET-TPS40 material compared to that of neat PET films after enzymatic degradation by AMY, LCC ICCG , and LCC ICCG /AMY treatments at 70 ◦C (Fig. 6). The hydrolytic depolymerization promoted by the enzymatic attack can cleave PET backbone ester bonds and lead to a reduction of carbonyl and subsequent formation of carboxylic and hydroxyl end groups (Brueckner et al., 2008), which contributed to the broad band centered at 1244 cm −1 . This agreed with observations reported by Du et al. for PET hydrolysis at 90 ◦C (Du et al., 2014). This was shown by the shift of the carbonyl ester band from 1712 to 1718 cm −1 in the neat PET films treated with LCC ICCG (Fig. 6a–iii) and LCC ICCG /AMY (Fig. 6a–iv), and complete disappearance in the mixed PET-TPS40 film treated with LCC ICCG (Fig. 6b–iii), which confirmed the enzyme ability to degrade PET from the mixed (petro-bio)plastic sample. Donelli et al. have also studied the hydrolysis of PET by cutinase and they observed a similar decrease in the intensity of the carbonyl stretching band associated with the enzymatic attack of PET polymer chains (Donelli et al., 2010). Interestingly, the strong band at 1758 cm −1 assigned to the C – – O group vibration of TPS could not be detected after the combined LCC ICCG /AMY treatment (Fig. 6b–iv), suggesting the complete decomposition of TPS from the mixed PET-TPS40 waste. Another important aspect to consider, as previously mentioned, is the presence of trans and gauche rotational conformers of EG in the hydrolyzed materials. As demonstrated in studies by Guo et al. and Wei et al., the conformation of PET oligomer chains simultaneously occurs to varying extents in amorphous (enhanced in gauche conformation) and semi-crystalline (enhanced in trans conformation) PET materials, and it could be a critical contributor to the enzymatic degradation (Guo et al., 2022; Wei et al., 2019b). The bands at 1471, 1340, 972, and 849 cm −1 are associated with the existence of trans conformers, whereas gauche conformation bands were observed at 1370 and 1044 cm −1 , as depicted in Fig. 6a and b. In particular, the spectra of mixed post-enzymatic degradation leftovers from PET-TPS40 treated with LCC ICCG displayed bands enriched in gauche conformation, while the LCC ICCG/ AMY treatment resulted in enriched trans conformation, which suggested the existence of phases predominantly amorphous and crystalline, respectively. In the case of PET, the amorphous regions are considered more susceptible for enzymatic hydrolysis via cleavage of backbone ester bonds (Wei et al., 2019a). As suggested by the FTIR and DSC results for the mixed PET-TPS films prior to biodegradation studies, the increase of TPS content mixed in PET has resulted in a decrease in the X c . The reduced crystallinity of the starting PET-TPS films before enzymatic degradation enabled the enhancement of biocatalytic activity (Fig. 4a), resulting in 77 % decomposition of the PET-TPS40 material, which showed the lowest crystallinity value (X c =26 %, Table 1), in contrast with a 56 % depolymerization observed for the neat PET film (X c =37 %, Table 1), showing a correlation between the mixed materials’ low crystallinity and their susceptibility to enzymatic degradation. Upon biocatalytic treatment with LCC ICCG /AMY, noticeable changes in the CI and X c values were observed (Table 2). A rise of up to 5 % in X c in the neat PET samples treated with AMY or LCC ICCG was noted. In contrast, no significant difference in crystallinity was observed between the non-treated control and that of PET treated with LCC ICCG /AMY. Interestingly, the PETFig. 6. FTIR spectra of (a) PET and (b) PET-TPS40 films subjected to enzymatic degradation treatments at 70 ◦C: (i) non-treated control, (ii) AMY, (iii) LCC ICCG , and (iv) LCC ICCG /AMY. Detailed wavenumber range shown: 2000–650 cm −1 . Table 2 Properties of the PET and PET-TPS40 materials after enzymatic treatment. Enzymatic treatment PET PET-TPS40 CI X c (%) CI X c (%) Non-treated 2.6 37.1 3.5 26.0 AMY 3.3 41.4 3.1 27.8 LCC ICCG 2.2 42.5 2.1 24.0 LCC ICCG /AMY 2.7 37.8 1.9 20.7 CI, carbonyl index; X c , degree of crystallinity. J.A. Araujo et al.