Technologies for the upcycling of recycled plastics materials (Public Summary)

This project has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement N° 101036838. D5.9 D86 – Technologies for the upcycling of recycled plastics materials (Public Summary) August 2025 Authors: Paola Branduardi (UNIMIB); Fuensanta Monzó (CETEC),Alejandro Arribas (CETEC), Pedro López (CETEC), María Nicolás (CETBIO, Raquel Barbosa (WETSUS) Ref. Ares(2025)6385012 - 05/08/2025 A2C – Deliverable D5.7v2.0 Page 2 І38 This project has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement N° 101036838 Technical references Project Acronym Agro2Circular Project Title TERRITORIAL CIRCULAR SYSTEMIC SOLUTION FOR THE UPCYCLING OF RESIDUES FROM THE AGRIFOOD SECTOR Project Coordinator Fuensanta Monzó CETEC [email protected] Project Duration October 2021 – March 2025 (42 months) Deliverable No. D5.9 D86 Dissemination level* PU Work Package WP 5 - A2C technologies for the upcycling of the recycled plastic materials Task All task in WP5 Lead beneficiary 8 (UNIMIB) Contributing beneficiary/ies Due date of deliverable 31 March 2025 Actual submission date 31 March 2025 PU = Public PP = Restricted to other programme participants (including the Commission Services) RE = Restricted to a group specified by the consortium (including the Commission Services) CO = Confidential, only for members of the consortium (including the Commission Services) A2C – Deliverable D5.7v2.0 Page 3 І38 This project has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement N° 101036838 Document history V Date Comments v0.1 28/03/2025 First draft of document v1.0 31/03/2025 First final version, approved by the WP leader and the project coordinator, (will be) submitted to EC. v2.0 04/08/2025 Second final version, approved by the WP leader and the project coordinator, (will be) submitted to EC. Verification and approval Name Date Verification Final Draft by WP leader Paola Branduardi 04/08/2025 Approval Final Deliverable by coordinator Fuensanta Monzo 04/08/2025 A2C – Deliverable D5.7v2.0 Page 4 І38 This project has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement N° 101036838 Disclaimer and acknowledgement This project has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement No 101036838 Disclaimer This document reflects only the views of the author(s) the European Research Executive Agency (REA) is not responsible for any use that may be made of the information it contains. Whilst efforts have been made to ensure the accuracy and completeness of this document, the A2C consortium shall not be liable for any errors or omissions, however caused. A2C – Deliverable D5.7v2.0 Page 5 І38 This project has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement N° 101036838 Table of contents 2 Executive summary ............................................................................................................. 9 3 Introduction ...................................................................................................................... 11 4 Key advancements in A2C technology for the valorisation of plastic and agrifood wastes by biomanufacturing ................................................................................................................... 14 4.1 Microbial fermentation for upcycling plastic and agrifood wastes ......................... 14 4.1.1 TPA, EG and F&V residues bioconversion to building blocks for cosmetic formulations ............................................................................................................ 14 4.1.1.1 Upcycling of ethylene glycol (EG) into glycolic acid (GA, commercial prize about 25 times the price of EG). ................................................................... 14 4.1.1.2 Upcycling of terephthalic acid (TPA) into protocatechuic acid (PCA, commercial prize about 500 times the price of TPA) .................................... 16 4.1.1.3 Upcycling citrus peel residues into microbial oil ....................... 17 4.1.2 PHBV range .............................................................................................. 18 4.1.3 PHBV green extraction, purification ........................................................ 21 4.2 High barrier recycled and recyclable compounds for food packaging and agricultural films 23 4.2.1 Key Advancements .................................................................................. 24 4.2.2 Main results achieved ............................................................................. 24 4.2.3 Experimental data ................................................................................... 27 4.3 Biodegradable compounds for food packaging and agricultural films ................... 29 4.3.1 Work Done and Challenges Encountered ................................................ 29 4.3.2 Results obtained ...................................................................................... 32 5 Conclusions ....................................................................................................................... 34 A2C – Deliverable D5.7v2.0 Page 6 І38 This project has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement N° 101036838 List of Tables Table 1 summary of DEMOs that saw the contribution of the work done in WP5 ..................... 13 Table 2 target properties for food packaging and agricultural film compounds ......................... 27 List of Figures Figure 1 diagram of Agro2Circular workflow .............................................................................. 11 Figure 2 WP5 workflow, with inputs (dark green) and outputs (orange ..................................... 12 Figure 3 Flowchart of the bioconversion of EG (derived from PET enzymatic hydrolysis) into GA ..................................................................................................................................................... 15 Figure 4 Flowchart of the conversion of LE into microbial oil ..................................................... 18 Figure 5. Scale-up process for PHBV production carried out by CETBIO. .................................... 20 Figure 6. Kneader elements to be assembled on the screws. ..................................................... 25 Figure 7. Screw area assembled only with Kneader elements .................................................... 25 Figure 8. Appearance of the mixture of all ground components, mixed and ready feed into the extruder ....................................................................................................................................... 26 Figure 9. Calendered film exiting through the nozzle, passing between the rolls. ..................... 26 Figure 10. Homogeneous appearance of the shading film with aluminium ............................... 28 A2C – Deliverable D5.7v2.0 Page 7 І38 This project has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement N° 101036838 1 List of abbreviations TPA terephthalic acid EG ethylene glycol GA glycolic acid PET polyethylene terephthalate PE polyethylene PCA protocatechuic acid F&V fruit & vegetables MPF multilayer plastic films SCO single cell oils C/N ratio Carbon:Nitrogen ratio LCA Life Cycle Assessment DoE Design of Experiment YTD/YT Yeast Extract/Tryptone/ Glucose medium / Yeast Extract/ Tryptone medium PPG Polypropylene glycol RID Refractive Index Detector VWD Variable Wavelength Detector LE Lemon extract Nlim Nitrogen limitation phase EVOH Ethylene vinyl alcohol PA Polyamides A2C – Deliverable D5.7v2.0 Page 8 І38 This project has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement N° 101036838 PET/PE Polyethylene terephthalate/Polyethylene PHBV Poli (3-hidroxibutirato-co-3-hidroxivalerato) PHAs: Polyhydroxyalkanoates A2C – Deliverable D5.7v2.0 Page 9 І38 This project has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement N° 101036838 2 Executive summary The general objective of WP5 was to obtain high added-value products & advanced packaging compounds from organic waste fractions deriving from fruit and vegetables production (F&V) and from plastic materials used along the same value chain, after they are collected, sorted and pretreated. The specific objectives were: - To upcycle PE and PET enzymatic degradation products into building blocks for cosmetic industry by microbial-based biotech processes; - To use by-products of organic waste fractions for the production of PHBV and carotenoids; - To obtain PHBV green extraction and purification; - To develop recycled and recyclable high barrier compounds for food packaging and agriculture films; - To develop biodegradable high barrier compounds for food packaging and agriculture films; The final aim of the WP5 was to describe all processes and develop protocols considering a possible industrial environment, and considering how to maximize quantitative evaluations and descriptions, in order to demonstrate the technology in relevant environment (pilot scale-up), which occurred with the DEMO units run into the frame of WP6. Overall, in WP5 we assessed the possibility to uncover the hidden values of waste and residues, closing loops and connecting different value chains. Indeed, the final products are recycled plastic blends, novel bioplastic blends, but also products/ingredients for the cosmetic and nutraceutical industry. Moreover, the target of the novel bioprocesses was to develop processes that can be ascribable to the logic of Safe and Sustainable by Design. In our case, this corresponds to processes that whenever possible are considering how to minimize the generation of waste and/or side stream, or to describe how new products and possible waste are recycled, recyclable and biodegradable. We succeeded in producing all the different products (new bioplastic blending, ingredients for cosmetics and nutraceuticals) all produced from waste and minimizing the generation of new waste. For upcycling waste into novel chemicals and polymers we rely on the power of microorganisms, which are notorious for their ability to metabolize and therefore transform a wide variety of substrates into a vast array of products. However, their natural abilities have to be further developed to match industrial requirements. For this reason, when microbial fermentations were used for biomanufacturing the products, microorganisms were selected based on their natural abilities and fermentation performances, including their minimal needs for specific A2C – Deliverable D5.7v2.0 Page 16 І38 This project has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement N° 101036838 Under the optimized condition all the EG (150 g) was consumed (100% consumption), and the yield of glycolic acid (% mol/mol) was 95%. The concentration of GA in the supernatant was 18 g/L for a total of 144 g of GA. 4.1.1.2 Upcycling of terephthalic acid (TPA) into protocatechuic acid (PCA, commercial prize about 500 times the price of TPA) Protocatechuic acid is a natural polyphenol antioxidant compound found in medicinal plants. It is known to have various pharmacological properties such as antibacterial, antiviral, anticancer, anti-inflammatory, anti-aging, and anti-atherosclerosis effects. These effects make it a promising compound if used as a pharmaceutical or functional food ingredient. In addition, more recently PCA was suggested as a staple ingredient in cosmetics thanks to its properties as a humectant that draws moisture to the other layers of the surface. This last use was investigated in this project, and for that reason activities were dedicated to producing it from TPA, similarly as GA from EG. Main results achieved As for EG conversion, the upcycling of TPA into PCA was first investigated in the baker’s yeast Saccharomyces cerevisiae, as yeasts were never described to be able to catabolize TPA and use it as carbon and energy source. Therefore, the strategy was to introduce heterologous genes encoding the bacterial activities known for the transformation of TPA into PCA in a host that does not contain other pathways for this transformation. The key enzymes involved in TPA metabolism are: TPA dioxygenase (TPA-DO), which catalyzes the oxidation of TPA to 1,2-dihydroxy-3,5cyclohexadiene-1,4-dicarboxylic acid (DCD), and DCD dehydrogenase (DCD-DH, or TphB), which catalyzes the decarboxylation of DCD to protocatechuic acid (PCA). We expressed these genes from two different bacterial sources, both described in literature as capable of performing the desired biotransformation. As in both strain series we did not see positive results, we proceeded in parallel by i) incrementing the gene copy number, thanks to the innovative development of a multy-cistronic element adapted to eukaryotic cells ii) investigating the TPA uptake. For the first task, we technically succeeded, as verified by the expression and translation of reporter genes, but still, we could not see any desired transformation. Going deeply in the literature, and also performing some in vitro analyses on cell extracts, we discovered that the heterologous enzymes are dependent on a sulfur-iron cluster that in yeasts is usually compartmentalized into the mitochondria. We tested the possibility of supporting these elements in the media, but with no success. In parallel, we discovered that the uptake of TPA is almost null at the operative pH, and only in mutants in specific membrane transporters we could indirectly infer a poor transport, but again no positive (or reproducible) data were collected. Also, the use of different permeabilizers did not change the findings. To conclude, despite all the strains we created and despite all the mitigation actions we tested, we still did not succeed in reaching the desired bioconversion. As a last mitigation action, we decided to reconsider engineered bacteria as cell factories. A fed-batch fermentation using an A2C – Deliverable D5.7v2.0 Page 17 І38 This project has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement N° 101036838 engineered strain of Pseudomonas putida was developed and optimized. After a growth phase, the feeding was initiated, and optimized in a way that cells were always capable to metabolise all the provided carbon source and transform all the provided TPA. Concurrently, PCA accumulates, reaching a final concentration of 10 g/L, with complete (100%) conversion of TPA. PCA, dissolved in the medium, was extracted using octanol (with the use of a rotavapor, solvent was 95% recovered). The extraction efficiency was enhanced by increasing the fraction of PCA in its acidic form, which approaches 100% at pH 2. Subsequently, acid precipitation (with 37% HCl) and lyophilisation were performed, resulting in a powder containing PCA and NH₄Cl, with a cumulative recovery of 62% PCA. Novelty and innovation: The work done was novel in respect to current literature, and it was also innovative as it allowed to develop microbial processes at lab scale, which were further expanded at higher scale with the DEMO, allowing to deliver products to the industrial partner in charge of producing novel cosmetic formulations. The main advancements can be summarised as follows, and have been published: - elucidate better than previously the catabolism of EG in S. cerevisiae (“Challenges in elucidating ethylene glycol metabolism in Saccharomyces cerevisiae” VG Senatore et al., FEMS Yeast Research, 2025, https://doi.org/10.1093/femsyr/foaf006) - describe the conversion of EG into GA in non-Saccharomyces yeasts (1) “Exploring yeast biodiversity and process conditions for optimizing ethylene glycol conversion into glycolic acid”. VG Senatore et al., FEMS Yeast Res. 2024 doi: 10.1093/femsyr/foae024. 2) “Ethylene glycol metabolism in the oleaginous yeast Rhodotorula toruloides.” Senatore VG, Reķēna A, et al., Appl Microbiol Biotechnol. 2025 doi: 10.1007/s00253025-13504-3.) 4.1.1.3 Upcycling citrus peel residues into microbial oil Oleaginous microorganisms are microbial cells in which oil content exceeds 20% and up to 70% of the dry cell mass. Microbial oils, also called single cell oils (SCOs), have many advantages, such as short production life cycle, cheap cultivation methods, less labor required, less affections by venue, season and climate, and easier to scale up (Ma, 2006; Yi and Zeng, 2006). Therefore, these SCOs are valid substitutes of the less sustainable vegetable oil as a cosmetic ingredient, playing a comparable oiling, softening, smoothing, antioxidant role, among others. Here we exploited citrus peel residues provided by CTNC to prepare cultivation medium for producing yeast biomass rich of lipids, using the oleaginous yeast Cutaneotrichosporon oleaginosum (previously known as Trichosporon oleaginosus). Moreover, we optimized oil extraction by comparing traditional protocols with new ones investigating green solvents. Main results achieved A2C – Deliverable D5.7v2.0 Page 18 І38 This project has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement N° 101036838 Lemon extract (LE) was successfully used for media preparation for fermenting an oleaginous yeast, Cutraneotrichosporon oleaginosus, with no additional needs of pretreatment or hydrolysis, as it already derives from extractive procedures. In fact, lemon extract served as a complete medium for microbial oil production, presenting a C/N ratio of 80, ideal for triggering oil accumulation. Overall, our process allowed us to reach 50% of the biomass in lipids by weight, in a relatively short process time (48 h) and without requiring the addition of any nutrients to the provided LE. After downstream 11.29 g of oil could be extracted with an extraction yield of 35 % (g of oil/g of biomass). Figure 4 Flowchart of the conversion of LE into microbial oil Novelty and innovation: The work done was novel in respect to current literature especially considering the possibility to valorise a waste that was already subjected to a first valorisation, within the same framework. The main advancements can be summarised as follows, and have been used for a joint effort among A2C partners, resulting in the preparation of an original research manuscript: - calculate impacts and CO2 footprint of microbial oil production from lemon extract (manuscript submitted). 4.1.2 PHBV range CETBIO focused on the development of an innovative bioprocess for the production of PHBV (poly(3-hydroxybutyrate-co-3-hydroxyvalerate)), a biodegradable and biocompatible bioplastic A2C – Deliverable D5.7v2.0 Page 19 І38 This project has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement N° 101036838 with promising industrial applications. PHBV belongs to the family of polyhydroxyalkanoates (PHAs), natural polyesters produced by microorganisms as energy reserves. Due to their renewable origin and environmental biodegradability, PHAs are seen as key materials for a circular bioeconomy. Within this project, we explored cost-effective and sustainable production pathways, making use of agro-industrial waste streams as feedstock and deploying the halophilic microorganism Haloferax mediterranei as a robust PHBV cell factory. The development process involved a series of laboratory and pilot-scale experiments at 30 L aimed at optimising key parameters for microbial growth and PHBV production. This included the assessment of different nutrient compositions and environmental conditions to enhance biopolymer yields. In parallel, the valorisation of by-products generated in Task 5.1.1 and the organic fractions from the agri-food processing industry (as outlined in Tasks 2.2 and 3.1) were explored as alternative feedstocks to improve the sustainability and cost-efficiency of the process. These efforts culminated in the successful demonstration of PHBV production at pilot scale, validating the technical feasibility of the approach and leading to the identification of scalable process conditions using agri-food waste. These developments have provided the technical basis for Demonstrator 5, where the process will be further scaled and validated under near-industrial conditions. Main results achieved The bioprocess developed for PHBV production using Haloferax mediterranei under high-salinity, non-sterile conditions has proven to be a robust and suitable platform. Systematic optimisation identified key fermentation parameters, such as temperature and salinity, that enhanced PHBV accumulation and biomass growth. Among the culture media evaluated, formulations with balanced carbon to nitrogen ratios were found to promote efficient PHBV synthesis and cell growth. This optimisation was successfully scaled up from laboratory shaker flasks to a 30 L pilot bioreactor, where further refinement of aeration and agitation parameters was conducted to maintain optimal dissolved oxygen levels. These efforts resulted in PHBV titres exceeding 2 g/L. In terms of feedstock valorisation, agro-industrial residues, specifically lemon waste and apple pomace were confirmed as effective and sustainable alternative carbon sources for H. mediterranei. Lemon waste showed the best results at moderate concentrations (11 % v/v), supporting high PHBV yields, whereas apple pomace (22 % v/v) also supported growth and production, though to a lesser extent. Conversely, TPA, a by-product from enzymatic PET recycling, was found unsuitable as a carbon source for this microorganism under the tested conditions. A2C – Deliverable D5.7v2.0 Page 20 І38 This project has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement N° 101036838 Figure 5. Scale-up process for PHBV production carried out by CETBIO. The successful demonstration at pilot scale has laid the groundwork for further upscaling to larger bioreactors (300 L), integrating lemon waste as the primary feedstock. This approach not only utilises waste streams effectively but also leverages the extremophilic nature of H. mediterranei to enable sustainable and cost-effective PHBV production in less stringent sterile conditions. The PHBV production process developed by CETBIO introduces multiple layers of innovation, both in its technological design and in its alignment with the principles of the circular bioeconomy. CETBIO demonstrated that certain agri-food residues, such as lemon waste, can serve as fully viable carbon sources for Haloferax mediterranei, achieving PHBV yields comparable to or exceeding those obtained with commercial media. By replacing these with unrefined, renewable feedstocks, the process not only reduces costs but also enhances the environmental sustainability and scalability of bioplastic manufacturing. From a product innovation standpoint, the process developed by CETBIO allows the production of PHBV with a significantly higher hydroxyvalerate (3-HV) content (>10%) than what is currently available on the market (typically around 1%). This elevated 3-HV fraction greatly improves the polymer’s mechanical flexibility, impact resistance, and thermoplastic behaviour, making it more comparable to fossil-derived plastics and better suited for flexible packaging applications. Challenges encountered • Identification of suitable renewable feedstocks: One of the initial challenges was selecting an alternative carbon source that was not only compatible with the growth of Haloferax mediterranei but also soluble in the saline culture medium and capable of sustaining or improving PHBV yields compared to commercial substrates. While lemon A2C – Deliverable D5.7v2.0 Page 21 І38 This project has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement N° 101036838 waste and apple pomace showed promise, variability in agro-industrial residues requires continuous monitoring and process adaptability. • Equipment limitations and material compatibility: The high salinity of the culture medium, though beneficial in reducing contamination, presents serious corrosion risks for conventional stainless-steel equipment. This limits the use of standard downstream units such as centrifuges and necessitates reliance on slower alternatives like tangential flow filtration. Furthermore, the project employed non-conventional plastic bioreactors, which are uncommon in industrial biotechnology and require tailored design solutions. • Industrial integration barriers: Introducing biotechnology-based production processes into sectors such as the plastics industry, where conventional methods are significantly more cost-effective, remains a major challenge. Bridging the gap between lab-scale feasibility and economically viable industrial application requires not only technical refinement but also demonstration of clear added value and sustainability. Lessons learned • Halophilic fermentation offers clear operational advantages: The use of H. mediterranei under high-salinity, non-sterile conditions confirmed that halophilic systems can reduce both sterilisation requirements and freshwater dependency. However, these benefits must be weighed against infrastructure limitations, particularly the need for corrosion-resistant equipment in both upstream and downstream operations. • Nutrient optimization is fundamental to performance: The balance of carbon, nitrogen, and phosphorus significantly influences microbial growth and PHBV synthesis. Specifically, a C:N ratio of 5 was shown to favour biomass accumulation, while the C:P ratio had less impact within the tested range. This highlights the importance of nutrient tuning for scalable and cost-effective production. • Feedstock characterisation and selection are essential. Feedstock selection plays a decisive role in process success. While some by-products like lemon waste and apple pomace proved highly suitable, others (e.g. TPA) were incompatible with microbial growth. Thorough feedstock characterisation is essential to avoid wasted resources and ensure process reliability. • Early integration of valorisation strategies across project tasks has maximised synergies and resource efficiency, setting a solid basis for scale-up and future exploitation. 4.1.3 PHBV green extraction, purification Solvent recovery from PHA containing biomass produced under the A2C project was challenged not only by the high salt concentrations required for halophilic organisms to thrive, but also by the nature of the biomass itself, which consisted of dispersed, free-living single cells. The elevated salt content resulted in dried solids containing substantial amounts of readily soluble inorganic material, which have a direct negative impact on downstream processing. Furthermore, the presence of cations is known to reduce the thermal stability of the polymer. Compared to flocculating biomass, the dispersed nature of halophilic biomass made dewatering A2C – Deliverable D5.7v2.0 Page 22 І38 This project has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement N° 101036838 and filtration significantly more difficult. To overcome these challenges, a substantial initial effort focused on creating broadly applicable methods for purifying halophilic, PHA-containing biomass. An additional aim of Task 5.1.3 was to explore and advance downstream processing strategies that would highlight the unique benefits of solvent-based recovery. Conventionally, plastic production from polymers begins with the dried material, which is combined with additives and processed at high temperatures to achieve a uniform melt. However, solid gels naturally formed during WETSUS extraction can also serve as the primary purified starting material after recovering PHA from biomass using PHA-poor solvents. In this context, the A2C project also investigated a novel approach: utilizing solid gels formed during the extraction as a primary purified input for melt processing techniques. Main results achieved • Dried solids quality assessment from CETBIO production batches Dried solid samples were analysed by TGA, then subjected to acid washing, rinsing, re-drying, and reassessment. PHA extraction was performed at laboratory-scale using standard dimethyl carbonate (DMC) methods at 140 °C for 20 minutes. The extracted polymer was then evaluated using TGA, DSC, and dilute solution rheology. While the dried solids consistently contained the same type of PHA, both molecular weight and PHA content varied, and thermal stability was generally suboptimal. Pretreatment significantly improved the quality of the PHA-rich biomass by removing unwanted fractions, resulting in higher PHA content and improved thermal stability. This pretreatment step is essential immediately after PHA accumulation and but requires greater removal of process water prior to drying. As part of the work conducted for D5.5, mild SDS rinsing of freshly produced PHA-rich halophilic biomass was shown to yield higher-quality dried solids than post-washing CETBIO material. Results from both D5.3 and D5.5 indicate that it is technically feasible to produce highly upgraded PHA-rich dried solids with improved PHA content, thermal stability, and preserved high molecular weight. • General principles for optimized CETBIO PHBV recovery DSC analysis proved to be a reliable method for identifying the optimal temperature range for extracting PHAs from biomass using a specific solvent. However, the rate of extraction was primarily influenced by the polymer’s intrinsic viscosity, which in turn depends on factors such as co-polymer composition, blend characteristics, average molecular weight, and degree of crystallinity. The PHBV produced in the A2C project exhibited higher intrinsic viscosity at equivalent molecular weights when compared to either more crystalline or more amorphous PHBVs. To enhance extraction efficiency (assuming all other variables are constant) extraction using PHA-poor solvents will require either elevated temperatures or reduced biomass loading to achieve effective recovery and purification of A2C PHBVs. A2C – Deliverable D5.7v2.0 Page 23 І38 This project has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement N° 101036838 • Pilot scale recovery towards facilitating a PHBV supply A successful pilot-scale campaign was carried out to process batches of PHA-rich biomass derived from two distinctly different sources, each producing a different type of PHA. This demonstrated the proof of concept for the proposed supply chain model, featuring a centralized processing facility supporting regionally distributed PHA production from organic waste streams. The recovered polymers were further upgraded through an innovative pelletisation step, adding value to the final product. As per the DoA, 500 grams of PHBV pellets were delivered to support further research and development in downstream formulation and processing. • Gel extrusion with melt processing and concurrent solvent recovery. Wet melt-processing techniques for solvent-rich PHA were successfully developed and validated. This approach enables simultaneous melt processing and solvent recovery, allowing the polymer to be directly upgraded and formulated into melt-processed products without the need for an intermediate drying step. The elimination of this step has the potential to simplify the process and therefore reduce energy use. These innovations not only enhance the overall efficiency of PHA recovery but also increase the value of the end product, strengthening the economic feasibility of PHA recovery within the supply chain model explored in this project. 4.2 High barrier recycled and recyclable compounds for food packaging and agricultural films Based on the chosen formulation in previous work packages consisting of LDPE, EVOH and PA, the worst possible case in terms of mechanical properties and oxygen transfer rate (OTR). This formulation is used as a simulator to study how the properties of recycled plastics from food or agriculture can be improved and what their possible application would be. The materials contained in the multilayer and multimaterial plastic packaging and agricultural plastic waste were separated and cleaned thanks to the sorting and delamination technology developed in WP3. Three main streams reached WP5 from WP3: 1. A non-metallised recycled plastic fraction, made of LDPE, PA and EVOH, being LDPE the main material and PA and EVOH present in smaller proportions. 2. Aluminium recovered from the metallised fraction after saperatec delamination process. 3. LDPE recycled plastic fraction separated in saperatec delamination process. Two different ways were followed to improve the mechanical and gas barrier properties of these recycled plastic fractions: 1. The use of compatibilisers to improve the miscibility of the different materials that make up the plastic fractions. A2C – Deliverable D5.7v2.0 Page 24 І38 This project has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement N° 101036838 2. The modification of the aluminium particles to enhance its compatibility with the plastic matrix and subsequent preparation of an aluminium/recycled plastic composite. The aluminium, modified to enhance its compatibility with plastic, was added as a filler to plastic blends, the latter optimized by adding compatible agents that improve miscibility between PE, PA and EVOH plastics. The project initially explored commercial compatibilizers for high barrier recycled and recyclable compounds. However, these proved unsatisfactory, as they did not meet the specified requirements for barrier packaging or agricultural uses. This led to the investigation of a composite material incorporating modified aluminium nanoparticles made by the University of Boku. 4.2.1 Key Advancements • Recycled Plastic Optimization: Three strategies were employed for developing recycled and recyclable compounds: ◦ Blends made with LDPE, PA, EVOH and aluminium nanoparticles modified by Boku. ◦ Blends made with LDPE, PA, EVOH, aluminium nanoparticles from BOKU, and “last generation” compatibilizers. ◦ Blends made with LDPE, PA, EVOH, compatibilizer and aluminium from saperatec • Processing Techniques: The mixtures were initially ground to achieve similar particle sizes, which was intended to favour the impregnation of the aluminium. The next step involved extruding these mixtures in a twin-screw extruder with a special arrangement of screw blocks to obtain an extensional flow. After extrusion and drying, the material was processed into film by calendering. 4.2.2 Main results achieved Extrusion Process and Extensional Flow-Mixing The extrusion process involves two main types of mixing: dispersive and distributive. Dispersive blending focuses on breaking down components within the polymer matrix. Twin-screwextruders (TSEs) are considered highly effective for blending polymers, fillers and additives due to their modularity, allowing for modification of screw elements based on mixing requirements. Kneading blocks are specifically designed elements in modular twin-screw-extruders that, with suitable arrangement and high screw speeds, help achieve similar extensional flow conditions. The Leistritz ZSE 18 HP extruder, a co-rotating twin extruder, was used for all blends. A2C – Deliverable D5.7v2.0 Page 25 І38 This project has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement N° 101036838 Figure 6. Kneader elements to be assembled on the screws. Figure 7. Screw area assembled only with Kneader elements The mixing process for aluminium nanoparticles and other components involved grinding them using a Retsch SM300 cutting mill to achieve a homogeneous mixture with a particle size suitable for continuous feeding. A Brabender DDSR20 twin-screw feeder, designed for powdered substances, was employed for continuous feeding into the extruder. The extruder was configured with numerous kneading elements to maximize conditions similar to extensional flow. High temperatures (up to 230 °C) and a high extrusion speed (400 rpm) were used, particularly due to the presence of polyamide, which has a high melting point, to achieve the extensional flow in the areas near the kneading blocks. A2C – Deliverable D5.7v2.0 Page 32 І38 This project has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement N° 101036838 • Melt Flow Rate (MFR) and Density: Density was determined following ASTM D792. MFR, indicating polymer flowability and mechanical strength/molecular structure, was measured according to ISO 1133 at 160°C under a 2.16 kg load. MFR values were generally suitable for material processing. 4.3.2 Results obtained Film Properties and Performance: • Differential scanning calorimetry (DSC): Due to the immiscibility of the two polymers in the composition range studied, the thermal behaviour observed in the DSC curves corresponded to that of the individual polymers. Thus, the data extracted from the calorimetric studies were assigned to the individual components. Melting point values between 164 and 185 °C have been obtained for PHBV component and 90 and 140 °C for the PBAT component. • Rheological properties: a decrease in storage modulus and loss modulus is observed in all samples. • Tensile test: a range between 17 and 26 MPa has been observed for the tensile strength of the blends in the machine direction while a range between 21 and 24 MPa were observed in the transverse direction. • Barrier properties. Permeability to oxygen: Oxygen permeability values for LDPE-PAEVOH were 55400±300 cm3⋅μm⋅m−2⋅day−1⋅atm−1 and for LDPE-PA-EVOH-AL were 41800±390cm3⋅μm⋅m−2⋅day−1⋅atm−1. While the addition of aluminium reduced permeability, it was still considered high and not sufficient for a "barrier" designation, primarily because the aluminium did not form a continuous layer. PHBV-PBAT1 had an oxygen permeability of 35800±100m3⋅μm⋅m−2⋅day−1⋅atm−1 and PHBV-starchPBAT1 had 40200±250 cm3⋅μm⋅m−2⋅day−1⋅atm−1. Pure PHBV showed a lower permeability of 2770 cm3⋅μm⋅m−2⋅day−1⋅atm−1, but was still not considered a barrier material. The incorporation of unmodified lemon fibres (PHBV+5ULF) further reduced permeability to 1685 cm3⋅μm⋅m−2⋅day−1⋅atm−1. However, with modified lemon fibres (PHBV+5MLF), permeability could not be measured as it fell outside the measuring device's range, likely due to the fibres not forming a continuous layer, similar to the aluminium issue. Increasing the concentration of aluminium and fibres to enhance barrier properties negatively impacted mechanical properties for final applications. • Melt flow rate (MFR) and density: all blends range between 1,8 and 3 g/10 min for MFR. Regarding density, LDPE blends range between 0,971 and 0,990 g/cm3 while PHBV blends range between 1,16 and 1,26 g/cm3. A2C – Deliverable D5.7v2.0 Page 33 І38 This project has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement N° 101036838 Validation by End-Users (Food Packaging): • Citromil (Lemon Concentrate): Lemon concentrate was packaged in PHBV-StarchPBAT1, LDPE-PA-EVOH, and LDPE-PA-EVOH-AL bags. Stored at 2°C, the evolution of parameters was normal and stable over 3,5 months. Vitamin C decreased normally but remained sufficient at the end of shelf life. PHBV-Starch packaging seemed to maintain higher vitamin C levels. Microbiological content decreased due to high acidity and refrigeration. Organoleptic analysis by expert tasters (scoring >3 for acceptance) showed that quality decreased during storage, but similarly to commercial plastic bags. PHBV-Starch packaging showed less colour degradation and maintained good scores across all parameters, including commercial suitability. • Almond (Coconut Oil): Samples packaged in LDPE-PA-EVOH-AL and LDPE-PA-EVOH arrived "broken" due to sealing problems. The seals were weak and gave way easily under slight pressure, rendering the containers unsuitable for coconut oil. No migration was observed from the container. • Mocitos (Apple Cream/Pear Cream): Biodegradable PHBV-StarchPBAT1 packaging for apple cream had a very short shelf life. At 27ºC, bubbles and mould appeared within days. Recycled LDPE-PA-EVOH-AL containers also showed oxidation, albeit slower, but still unacceptable compared to commercial aseptic bags that preserve puree for 2-3 years. Validation by End-Users (Agricultural Film): • Proexport (Lettuce Crops with PHBV-PBAT Mulch Film): PHBV-PBAT biodegradable films were tested on lettuce crops in the Campo de Cartagena area to assess performance under real conditions. Lettuce was chosen for its fast growth, allowing rapid evaluation due to project time constraints. After one month, the lettuce grew normally, and the mulch film showed clear signs of biodegradation, confirming the task's objectives were met. Recyclability: The mechanical properties of both PHBV-PBAT1 and PHBV-StarchPBAT1 formulations showed no significant differences after five cycles of extrusion and injection. This indicates that the materials can undergo at least five recycling cycles without losing key mechanical properties. Similarly, rheology tests revealed no significant changes in melt viscosity after five extrusion and injection cycles, suggesting no significant molecular weight degradation. This confirmed that the materials can withstand at least five cycles without significant property loss based on this criterion. Compostability: Both PHBV+PBAT and PHBV+Starch PBAT mixtures were found to be compostable according to UNE-EN ISO 14855. Both materials exhibited a biodegradation degree with less than 20% difference compared to the reference material (cellulose). The cellulose A2C – Deliverable D5.7v2.0 Page 34 І38 This project has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement N° 101036838 reference material showed 87.3% biodegradation, while PHBV+PBAT achieved 78.9% and PHBV+StarchPBAT achieved 81.3% over 80 days. Biodegradability in Soil: The PHBV+PBAT and PHBV+Starch PBAT mixtures demonstrated biodegradability in soil according to UNE-EN ISO 17556. Their biodegradation degree was less than 20% different from the reference material (cellulose). Over 250 days, cellulose showed 87% biodegradation, PHBV+PBAT showed 72%, and PHBV+StarchPBAT showed 77%. The reference material's biodegradation degree exceeded 60% in the plateau phase, validating the test outcomes. Biodegradability in Seawater: The PHBV-StarchPBAT mixture demonstrated biodegradability in seawater, fulfilling the UNE-EN ISO 19679 standard. The PHBV-PBAT mixture also approached compliance with this standard. Over 250 days, cellulose showed 78.3% biodegradation, PHBV+PBAT showed 57%, and PHBV+StarchPBAT showed 69%. The test was considered valid as the reference material's biodegradation rate was greater than 60% after 180 days. 5 Conclusions Within the framework of Agro2Circular project, several technologies were developed as part of two value chains in the Region of Murcia, the upcycling of multilayer plastic waste and the upcycling of fruit and vegetable plastic waste, respectively. For the plastic waste value chain: • Novel processes using microorganisms able to convert the hydrolysate after PET enzymatic degradation into building blocks that can be natural ingredients for the cosmetic industry. • New plastic formulations were developed from the recycled plastic separated from the multilayer plastic waste, to be used again as packaging and film with barrier properties to IR radiation. From the fruit and vegetable waste value chain: • A highly biodegradable plastic, of the kind of polyhydroxyalkanoates, was produced from high sugary waste used as the sole carbon source in a fermentation process using halo-archaeas. Simultaneously, a most sustainable process was developed for the extraction of the polymer from the biomass, by using chlorine free solvents. • The same high sugary wastes were used as medium unbalanced in the carbon source and therefore suitable for the production of microbial oils in a yeast fermentation. All processes have been developed using secondary raw materials as the feedstock, following circular economy strategies. A2C – Deliverable D5.7v2.0 Page 35 І38 This project has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement N° 101036838 For biodegradable compounds for food packaging and agricultural films we have the following conclusions and future outlook: The project successfully scaled up two recycled plastic mixtures from agricultural waste (LDPEPA-EVOH-AL and LDPE-PA-EVOH) and two biodegradable blends from organic agricultural wastes (PHBV-starch-PBAT1 and PHBV-PBAT1). All chosen materials were thoroughly characterized and met milestone MS5 requirements. Key Findings: • Recycled Blends for Food Packaging: Recycled blends, particularly those made with conventional materials and suitable compatibilizers, demonstrated suitability for use as food packaging. • Limited Barrier Improvement with Aluminium: While the addition of aluminium reduced oxygen permeability in recycled containers, the reduction was insufficient to classify them as high-barrier materials. This limitation is attributed to the aluminium not forming a continuous or sufficiently tortuous layer to impede oxygen diffusion. • Biodegradable Materials for Food Packaging: Biodegradable PHBV materials were also found to be suitable for food packaging applications. • Challenges in Barrier Improvement with Lemon Fibers: An intended goal was to enhance the barrier properties of PHBV using lemon fibres derived from the project's own waste, aiming to demonstrate circularity in additive use. However, this objective was not fully achieved, as the lemon fibres did not sufficiently improve PHBV's barrier properties to qualify it as a high-barrier material. • Food Compatibility Limitations: The shelf-life studies revealed significant limitations. The developed blends were not resistant to fatty foods like coconut oil, and their high permeability prevented effective preservation of foods such as apple sauce. • Suitability for Lemon Juice: Conversely, the blends proved particularly favourable for storing lemon juice, with the PHBV-Starch blend maintaining higher vitamin C values and less color degradation compared to other materials. • Agricultural Film Success: The biodegradable PHBV-PBAT agricultural film successfully demonstrated biodegradation in horticultural soils under real-life conditions, with positive performance observed on lettuce crops. • Recyclability validated: Both PHBV-PBAT1 and PHBV-StarchPBAT1 formulations maintain their mechanical and rheological properties through at least five recycling cycles, making them viable for repeated use. This is a significant finding as it addresses the crucial aspect of maintaining material integrity through multiple reprocessing steps in a circular economy. A2C – Deliverable D5.7v2.0 Page 36 І38 This project has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement N° 101036838 • Broad biodegradability: The PHBV-based formulations are confirmed to be compostable and biodegradable in soil and, largely, in seawater. This multi-environment biodegradability is essential for materials intended to reduce environmental impact and prevent microplastic accumulation across various disposal scenarios. • PHBV-StarchPBAT performance: The PHBV-StarchPBAT mixture consistently performed well across all biodegradability tests, meeting or exceeding the required standards. This specific blend shows great promise for environmentally friendly applications. Unresolved Problems or New Challenges Encountered: • Sealing Issues: A critical unresolved issue is the weakness of the seals in the developed packaging, particularly for recycled plastic containers. This led to package failure with products like coconut oil and significantly shortened the shelf life of other packaged foods. This highlights a need for further research and development in optimizing sealing parameters and possibly developing new sealing technologies compatible with these novel materials. • Limited Shelf Life for Certain Foods: The short shelf life of biodegradable packaging and even the oxidation observed in recycled plastic containers for apple puree pose a significant challenge. Achieving shelf lives comparable to conventional aseptic packaging (2-3 years) remains a major hurdle, especially for highly perishable or sensitive food products. • Achieving High Barrier Properties: The project faced difficulties in achieving "high barrier" properties for both recycled and biodegradable films, even with additives like aluminium and lemon fibres. This suggests that simply incorporating these fillers might not be enough to create a continuous barrier layer. Future work needs to explore alternative strategies, such as multi-layer structures or advanced coating technologies, to create effective barrier layers without compromising mechanical properties. • Thermal Stability of PHBV with Fibers: Rheological tests indicated that both modified and unmodified lemon fibres did not provide thermal stability to PHBV at the processing temperature, suggesting a potential for material degradation during high-temperature processing. This necessitates careful optimization of processing conditions or the development of new additives that can enhance thermal stability. • While most biodegradability criteria were met, the PHBV-PBAT formulation narrowly missed complying with the UNE EN ISO 19679 standard for biodegradability in seawater. This was primarily due to limitations in the project's execution time, preventing an extended test period. The biodegradation trends observed suggest that with more time, this specific formulation would likely have met the standard. This highlights a challenge in aligning rigorous, long-duration biodegradation testing with project timelines, especially for materials that degrade at a slower, albeit still significant, rate. A2C – Deliverable D5.7v2.0 Page 37 І38 This project has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement N° 101036838 Lessons Learned: • Material Compatibility is Key: The project successfully demonstrated the importance of material compatibilisation in recycled blends to achieve suitable properties for food packaging. • Additive Functionality: While natural additives like lemon fibres offer circularity benefits, their effectiveness in achieving specific technical properties (e.g., high barrier) might require further in-depth research into their dispersion, interfacial adhesion, and interaction within the polymer matrix. Surface modification of fibres, while attempted, did not yield the desired barrier improvement, suggesting that the "grafting from" strategy may need refinement or other modification techniques should be explored. • Validation with End-Users is Crucial: The direct involvement of end-users (Mocitos, Almond, Citromil, Proexport) in the validation process provided invaluable real-world feedback, quickly identifying critical performance gaps such as sealing issues and shelflife limitations that laboratory tests alone might not fully capture. • Tailoring Materials for Specific Applications: The varying success rates with different food products (lemon juice vs. coconut oil or apple sauce) underscore that there is no "onesize-fits-all" solution for food packaging. Materials need to be precisely tailored to the specific preservation requirements of different food types. • Importance of Blending: The project underscores the effectiveness of blending PHBV with other commercial and biodegradable polymers like PBAT and starch-PBAT to optimize cost and performance, making PHBV more applicable for a wider range of uses. • Testing Protocol Rigor: The adherence to international standards (UNE EN ISO 14855, 17556, 19679) for testing provided robust and reliable data on the materials' end-of-life behaviour. This highlights the necessity of standardized testing for credible environmental claims. • Time Constraints in Biodegradation Studies: The slight shortfall of the PHBV-PBAT in the seawater biodegradation test due to time limitations emphasizes that long-term environmental degradation processes require adequate study durations, which can conflict with project deadlines. Future projects should ideally factor in longer timelines for such comprehensive assessments. Technologies Identified as Appropriate: • Blown Extrusion: Proven effective for producing both food packaging films and agricultural films at small scale, demonstrating its versatility and industrial relevance. • Cast Extrusion: Utilized for producing flat films, particularly for barrier-improved PHBV formulations with lemon fibres. A2C – Deliverable D5.7v2.0 Page 38 І38 This project has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement N° 101036838 • Compounding Extrusion (Twin-Screw): Essential for blending and incorporating additives like lemon fibres into polymer matrices, allowing for good mixing and control over the composite formulation. • Bag Heat Sealing Machines: Standard equipment for sealing flexible packaging, though further optimization of their use with novel materials is needed. • Advanced Characterization Techniques: DSC, rheology, tensile testing, and oxygen permeability measurements (ASTM D3418, D882, D3985; ISO 1133, D792) were critical for understanding the material properties and guiding development. The MOCON Oxtran gas permeability tester, for example, provided quantitative data on oxygen barrier performance. • Respirometry for biodegradation: The ECHO respirometer was instrumental in accurately measuring CO2 production and % biodegradation in compostability, soil, and seawater tests, providing continuous data under controlled conditions. • Standardised testing methods: The consistent application of UNE EN ISO standards for mechanical, rheological, and biodegradation testing ensured comparability and reliability of results, crucial for validating new material formulations. The Agro2Circular project has made significant strides in developing recycled and biodegradable plastic compounds from agrifood waste. While impressive progress has been made in demonstrating recyclability and general biodegradability, challenges remain in achieving highperformance barrier properties and ensuring adequate shelf life for a broad range of food products, as well as refining sealing processes. These areas present clear avenues for future research and development, building upon the foundational knowledge and the promising materials identified in this project. The ultimate vision of a truly circular plastics economy, where materials from agrifood waste are continuously reused and responsibly managed at their endof-life, moves closer with each step taken in projects like Agro2Circular.