Upcycling depolymerized PET waste into polyhydroxyalkanoates and triacylglycerols by a newly isolated Rhodococcus sp. strain.

Rebochoetal. Biotechnology for the Environment (2025) 2:5 https://doi.org/10.1186/s44314-025-00019-4 RESEARCH Open Access © The Author(s) 2025. Open Access This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which permits any non-commercial use, sharing, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if you modified the licensed material. You do not have permission under this licence to share adapted material derived from this article or parts of it. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by-nc-nd/4.0/. Biotechnology for the Environment Upcycling depolymerized PET waste intopolyhydroxyalkanoates andtriacylglycerols byanewly isolated Rhodococcus sp. strain Ana Teresa Rebocho1,2, Cristiana A. V. Torres1,2*, Helena Koninckx3, Lutgart Stragier3, Olivia A. Attallah4, Marija Mojicevic4, Cuneyt Erdinc Tas4, Margaret Brennan Fournet4, Maria A. Reis1,2 and Filomena Freitas1,2 Abstract The use of post-consumer polyethylene terephthalate (PET) wastes, which often contain various additives and contaminants such as metals and pigments that make mechanical recycling and reusability difficult, as feedstocks for microbial synthesis of value-added bio-based products is an emerging sustainable strategy for managing such wastes. This study evaluated the ability of a strain isolated from a plastic-contaminated site, Rhodococcus sp. isolate Ave7, to use terephthalic acid (TPA) obtained by chemically depolymerizing PET waste, as sole feedstock for cell growth and production of polyhydroxyalkanoates (PHAs) and triacylglycerols (TAGs) as intracellular storage compounds. The fed-batch bioreactor cultivation resulted in a cell dry weight production of 3.85 g/L, with PHA and TAG contents of 15.0 wt.% and 15.4 wt.%, respectively. Overall, the culture consumed 16.5 g/L TPA over a period of 73 h. The produced PHA was mainly composed of 3-hydroxyvalerate (3HV) monomers (> 90 wt.%). The accumulated TAGs presented a fatty acids profile rich in octadecenoic acid (C18:1; 52 wt.%), hexadecanoic acid (C16:0; 32 wt.%) and octadecanoic acid (C18:0; 12 wt.%). Overall, the strain Rhodococcus sp. Ave7 demonstrated a high capacity for TPA removal, converting it into cell biomass, PHA and TAGs, thus rendering this bioprocess a promising solution to reduce the plastic waste burden, in a circular and sustainable approach. Keywords Plastic upcycling, Biodegradation, Rhodococcus, Polyethylene terephthalate, Polyhydroxyalkanoates, Poly(3-hydroxybutyrate-co-3-hydroxyvalerate), Triacylglycerols Introduction Polyethylene terephthalate (PET) is a widely used thermoplastic, synthesized through the condensation of terephthalic acid (TPA) and ethylene glycol (EG) [1]. Given its simple synthesis, low-cost production, thermostability and durability, make it extensively used in packaging industries, namely plastic bottles of soft drinks, food jars, clothing, and plastic films [2, 3]. Despite accounting for 6.2% of the worldwide plastics’ production, only 25% of PET is recycled in Europe, with the majority discarded in the environment [4, 5]. The increasing demand and inadequate disposal of PET have led to severe environmental pollution, as large volumes escape proper waste management systems, contaminating oceans and ecosystems. Due to its resistance to microbial degradation, PET persist for centuries, intensifying plastic waste accumulation [6, 7]. Managing PET waste remains a critical challenge, as conventional *Correspondence: Cristiana A. V. Torres [email protected] 1 i4HB - Institute for Health and Bioeconomy, NOVA School of Science and Technology, NOVA University Lisbon, Caparica 2829-516, Portugal 2 UCIBIO – Applied Molecular Biosciences Unit, Department of Chemistry, NOVA School of Science and Technology, NOVA University Lisbon, Caparica 2829-516, Portugal 3 Avecom NV, Industrieweg 122P, Wondelgem 9032, Belgium 4 LIFE Research Institute, Technological University of the Shannon Midlands Midwest, Athlone N37 HD68, Ireland Page 2 of 21 Rebochoetal. Biotechnology for the Environment (2025) 2:5 disposal methods such as landfilling and incineration pose several disadvantages, including slow degradation rates in landfills, limited spaces, long-term risks of contamination of soils and groundwater with leachate containing toxic compounds (e.g. heavy metals, dioxins, furans and polychlorinated biphenyls), and the emission of greenhouse gases associated with incineration [2, 8]. Recycling is currently regarded as the most sustainable PET waste management strategy and is categorized into four types [9]. Primary recycling involves mechanical re-extrusion of clean and single-type PET into materials with similar properties [10], though it is limited by contamination and material degradation over multiple cycles [11]. Secondary recycling processes PET through cleaning, shredding and remelting into flakes or pellets, but it requires high water consumption to remove contaminants (e.g. polymers, dirt, and labels) and often results in downcycling due to thermal degradation [10, 12, 13]. Furthermore, tertiary recycling recovers monomers, oligomers or additives via chemical depolymerization methods such as hydrolysis, glycolysis, aminolysis and methanolysis [14, 15]. Although effective for heterogeneous PET waste streams, this process is costly, energy-intensive, susceptible to equipment corrosion, and generates waste solvents [16, 17]. Lastly, quaternary recovery harnesses PET’s high calorific value through combustion, but it is often associated with the release of toxic fumes [18]. The complexity of PET waste, often mixed with municipal waste and containing multilayered plastics, rubber, aluminium and functional additives, that are designed for specific packaging functions, poses significant challenges to effective recycling [19]. To address these challenges, upcycling strategies combining depolymerization and bioconversion are being explored to enhance PET waste valorisation [20, 21]. PET depolymerization yields intermediates, such as TPA, which can serve as microbial substrates, suitable for bioconversion by TPA-metabolizing microorganisms via specific metabolic pathways [21]. This biological approach enables the transformation of PET waste into high-value products with diverse applications [22, 23]. Recent studies have confirmed the microbial metabolism of TPA, EG and other PET degradation products into compounds, such as polyhydroxyalkanoates (PHA) [24–28], hydroxyalkanoyloxy-alkanoates (HAAs) [27], bacterial cellulose [29], muconic acid [30], vanillic acid [31], β-ketoadipic acid [32, 33], catechol, gallic acid and pyrogallol [33, 34]. Among microbial candidates for PET bioconversion, Rhodococcus has emerged as a promising genus due to their remarkable metabolic versatility and environmental resilience. Commonly found in contaminated sites, Rhodococcus species are known for degrading various recalcitrant compounds [35]. Several strains have been identified for their ability to metabolize pollutants and convert complex substrates into valuable compounds via diverse catabolic pathways [36]. For example, Rhodococcus jostii RHA1 and Rhodococcus sp. SSM1 were reported to degrade TPA [37, 38], while Rhodococcus sp. DK17 and R. erythropolis PR4 can degrade aromatic and alicyclic rings, as well as alkanes, respectively [39]. Additionally, R. aetherivorans IAR1 was reported to convert toluene into PHA and triacylglycerols (TAGs) [40], while R. eryhthropolis MTCC3951 and R. pyridinivorans P23 exhibit both TPA degradation and PHA production capabilities [41, 42]. Furthermore, Rhodococcus sp. can synthesize other valuable compounds with environmental and industrial relevance, including biosurfactants and carotenoids [35], PHA [43], wax esters (WEs) and TAGs [44, 45]. Their metabolic versatility makes them strong candidates for bioconversion and bioremediation, with potential applications in breaking down persistent plastic pollutants like polyethylene [46] and producing sustainable bioproducts such as PHA and TAGs [47]. This aligns with circular economy principles, offering an eco-friendly alternative to traditional PET waste management [35]. In this study, Rhodococcus sp. isolate Ave7 was cultivated in bioreactor using chemically depolymerized postconsumer PET waste, with TPA as the sole carbon source. The strain’s ability to produce intracellular reserves of PHA and TAGs was evaluated under different cultivation modes, including batch and fed-batch processes. Following cultivation, the bioproducts were extracted and characterized, including an analysis of their composition, as well as the molecular mass distribution and thermal properties for PHA. Materials andmethods Feedstock processing andcharacterization Chemical depolymerization ofpost‑consumer PET waste Post-consumer PET waste (Fig.1(A)), containing approximately 2–5% polyethylene (PE), 1–2% pigments, metallic ingredients and carbon black additives, was supplied by Novelplast (Ireland). This material was used as the standard substrate without further processing. Depolymerization experiments via reactive extrusion (REX) were conducted using a bench-top PrismTM twin-screw extruder (Thermo Electron GmbH, Karlsruhe, Germany) following a modified procedure from Fournet etal. [48]. The PET waste feedstock was mixed with solid NaOH in a 2:1 (wt%/wt%) ratio. The well-mixed depolymerization reaction mixture was then dispensed through the main shaft into the barrel, which was maintained at a constant temperature of 250 °C, while the screw rotational speed was set at 20 rpm. The resulting REX product, named REX-PET, was subsequently processed to extract and Page 3 of 21 Rebochoetal. Biotechnology for the Environment (2025) 2:5 separate its terephthalic acid (TPA) content, named reactive extrusion-terephthalic acid (REX-TPA) (Fig.1(B)). Characterization ofdepolymerized PET waste The depolymerized PET waste was characterized in terms of particle size distribution, moisture and inorganic content, elemental analysis and FT-IR. The particle size distribution of REX-PET was determined by sieving a sample (~ 100 g) in a mechanical sieving shaker, equipped with sieves of pore sizes ranging from 106 to 2000 µm. The sample was agitated for 10 min, and the material collected on each sieve was weighed separately to determine its respective fraction in the sample. For the moisture content determination, REX-PET (~ 50 mg) was subjected to a temperature of 100 °C until a constant weight was achieved. Afterwards, the dried sample was placed at a temperature of 550 °C for 24 h, and the inorganic salts content was determined gravimetrically by weighing the resulting ashes. The elemental analysis of REX-PET was performed using an elemental analyzer (Thermo Finnigan-CE Instruments, Flash EA 1112 CHNS series, Italy). A REX-PET sample was characterized via Fourier-transform infrared spectroscopy (FT-IR) with a spectrum two spectrometer (PerkinElmer, Waltham, MA, USA) equipped with the attenuated total reflectance (ATR) accessory. The spectra were recovered based on eight scans between a resolution of 4000 and 400 cm−1, at room temperature. Depolymerized PET waste processing For the bacterial cultivation experiments, REX-PET was processed into an aqueous solution (named REX-TPA), which was obtained by preparing a 3.33% (w/v) mixture of REX-PET (5 g) (Fig. 1(B)) in deionized water (150 mL), followed by homogenization at 600 rpm during 1 h. The mixture was filtered (using paper filters with pore size 20 µm) followed by pH 7 normalization by HCl 5 M addition. The final solution contained approximately 20 mg/mL of REX-TPA, representing a recovery of 60% (Fig.1(C)). The solution of REX-TPA was assessed for its pH, moisture and inorganic content, ICP, total carbon and TPA quantification. Moisture and inorganic content were also determined for REX-TPA samples (~ 5 mL) as determined as described in the previous section. REX-TPA samples (~ 5 mL) were filtered (0.2 µm nylon, Whatman) and analysed by inductively coupled plasma-atomic emission spectrometry (ICP-AES) (Horiba Jobin–Yvon, France, Ultima, equipped with a 40.68 MHz RF generator, Czerny-Turner monochromator with 1.00 m (sequential) and autosampler AS500). The Total Carbon (TC) was analysed in a TOC-VCSH Analyser (Shimadzu) with a combustion catalytic oxidation at a temperature of 680 °C. High purity air served as carrier gas at a flow rate of 150 mL/min. TPA concentration was determined by high performance liquid chromatography (HPLC) using an Agilent Eclipse C18 250 × 4.6 mm, coupled to a UV detector. The analysis was performed at 50 °C, with samples eluted in isocratic mode using methanol (Fisher Chemical, HPLC grade) and 0.1% formic acid (Sigma-Aldrich, HPLC grade) solution (1:1, v/v). The flow rate was set to1 mL/min, and the injection volume 5 µL [49]. TPA detection was obtained at 240 nm. A TPA stock solution (1 g/L) (Merck Millipore, 98%) was prepared in a phosphate buffer (containing per liter: (NH4)2HPO4, 1.1 g (PanReac AppliChem, 99%); K2HPO4, 5.8 g (PanReac AppliChem, 99%); KH2PO4, 3.7 g (ChemLab, 99.5%)) and adjusted to pH 7. From this stock solution, TPA standards were prepared by serial dilution with a water–methanol mixture (10% methanol, Fisher Chemical, HPLC grade) to achieve TPA concentrations ranging from 4 to 400 mg/L. Similarly, the cell-free supernatant samples were diluted in the same 10% methanol aqueous solution to ensure consistency of Fig. 1 Post-consumer PET waste (A), REX-PET chemical depolymerized sample obtained from reactive extrusion of post-consumer PET waste (B) and REX-TPA solution used for bioreactor cultivations (C) Page 4 of 21 Rebochoetal. Biotechnology for the Environment (2025) 2:5 the standards and samples matrix for the HPLC analysis. All measurements were done in triplicate. Microorganism Rhodococcus sp. Ave 7 was isolated from landfill soil, by serially diluting and cultivating a sample on selective plates, containing commercial TPA (Merck Millipore, 98%) as carbon source. Single colonies were isolated and used for the amplification of the 16S rRNA gene via colony PCR using a T100 Thermal Cycler (Bio-Rad). The amplified 16S rRNA genes were then sent to Macrogen Europe (The Netherlands) for Sanger sequencing. Culture media Mineral salt medium (MSM) containing 8.86 g/L K2HPO4 (PanReac AppliChem, 99%), 2.80 g/L KH2PO4 (ChemLab, 99.5%), 0.50 g/L NaCl (PanReac AppliChem, 99.5%), 0.10 g/L MgSO4·7H2O (Biochem Chemopharma, 99.5%) and 0.10 g/L NH4Cl (Biochem Chemopharma, 99.5%), was used for all experiments. The micronutrients’ solution was added to the medium at a concentration of 10 mL/L. The micronutrients solution contained the following (per liter): FeSO4·7H2O, 1.83 g; MnSO4·1H2O, 0.56 g; ZnSO4·7H2O, 1.35 g; CaCl2·2H2O, 0.067 g; CoSO4 7H2O, 0.036 g; CuSO4 5H2O, 0.036 g; H3BO3, 0.65 g; EDTA dissodium·2H2O, 1.104 g. This medium was used for inocula preparation of all assays. The medium was supplemented with terephthalic acid (TPA) (Merck Millipore, 98%) as carbon source at a concentration of 0.5 g/L for inocula preparation. For bioreactor assays, MSM was supplemented with REX-TPA solution (prepared as described above), to achieve a final TPA concentration of 12 g/L. All media were sterilized by autoclaving at 121 °C and 1 bar, for 30 min. Bioreactor cultivations Three bioreactor experiments were performed under different modes of cultivation: batch (Assay A) and fedbatch with pulse feeding (Assay B) or continuous feeding (Assay C). The inocula for the bioreactor assays were prepared by inoculating 1 mL of the cryopreserved culture into 200 mL MSM, prepared as described above, in a 500 mL baffled shake flask. The flasks were incubated in a rotary shaker (200 rpm), at 30 °C, for 48 h. For the bioreactor assays, MSM supplemented with REX-TPA (12 g/L) and ammonia (0.3 g/L), yielding a C/N ratio of 29.7 (gC/ gN), was prepared. After sterilization in an autoclave at 121 °C, 1 bar, for 30 min, the medium was inoculated with 200 mL of the prepared bacterial culture to initiate the experiments. Throughout the assays, the pH was monitored but not controlled. The temperature was controlled at 30 ± 0.1 °C in all assays. An aeration rate of 2 SLPM (standard liters per minute) was kept during the experiments. The dissolved oxygen (DO) concentration was controlled at 20% of the air saturation, by automatically adjusting the stirring rate between 300 and 1000 rpm. Foam formation was automatically suppressed by addition of Antifoam 204 (Sigma-Aldrich). Assay A, under batch mode, was conducted in a 3 L bioreactor (Jupiter 3, Solaris, Italy), with initial working volume of 2 L. Assay B was performed in a 5 L bioreactor (Jupiter 6.0, Solaris, Italy) with an initial working volume of 2 L. After initial TPA depletion, signalled by an abrupt increase of DO concentration, REX-TPA pulse (1 L) containing 20 g/L TPA was fed to the bioreactor. Assay C was performed in a 3 L bioreactor (Bionet F1, Spain), with an initial working volume of 1.5 L. A continuous feeding of a REX-TPA solution containing 20 g/L TPA and 0.01 g/L of ammonia, was fed to the bioreactor at a 0.1 L/h flow rate for 15 h. Samples (10–20 mL) were periodically taken from the bioreactor for quantification of the cell dry weight (CDW), TPA, ammonium, PHA and TAG. Analytical methods CDW quantification For determination of the CDW, culture broth samples (5 mL) were centrifuged (20 min at 18,516 g, 4 °C). The cell pellet was washed with deionized water (5 mL) twice and lyophilized for 48 h. The CDW was determined gravimetrically by weighing the dried cell pellets. All measurements were done in triplicate. Quantification ofTPA TPA concentration was determined using HPLC, as described above, with an Agilent Eclipse C18 column (250 × 4.6 mm) and UV detection at 240 nm. The analysis was conducted at 50 °C in isocratic mode with a mobile phase of methanol and 0.1% formic acid (1:1, v/v), at a flow rate of 1 mL/min and an injection volume of 5 µL. A TPA stock solution (1 g/L) was prepared in phosphate buffer (pH 7), and TPA standards were prepared through serial dilution to obtain concentrations ranging from 4 to 400 mg/L. Cell-free supernatant samples were diluted with a 10% methanol–water solution to match the matrix of the standards. All measurements were performed in triplicate. Ammonium quantification Ammonia concentration was determined by colorimetry using a flow segmented analyser (Skalar 5100, Skalar Analytical, Netherlands). NH4Cl (Biochem Chemopharma, 99.5%) samples at concentrations ranging from 2 to 20 mg/L were used as standards. All measurements were done in triplicate. Page 5 of 21 Rebochoetal. Biotechnology for the Environment (2025) 2:5 PHA andTAGs quantification Storage compounds content in the biomass, namely PHA and TAGs, and their composition were determined by gas chromatography (GC) after acidic methanolysis of freeze-dried cells’ samples. Freeze dried samples (3 to 5 mg) were mixed with 2 mL 20% (v/v) sulphuric acid (Honeywell Fluka, HPLC grade) in methanol (Fisher Chemical, HPLC grade) and 2 mL benzoic acid in chloroform (0.5 g/L) (Fisher Chemical, HPLC grade) and heated at 100 °C, for 4 h. Benzoic acid (Sigma-Aldrich, ≥ 99.5%) acted as internal standard. The calibration curve for PHA quantification was prepared using a standard solution of P(HB-co-HV) (Sigma-Aldrich) composed of 14 mol% 3-hydroxyvalerate (3HV). For TAGs quantification, a mixture of fatty acid methyl esters (FAME) composed of C14-C22 (Sigma-Aldrich) at concentrations ranging from 0.1 to 1.0 g/L was used. The methyl esters obtained from the methanolysis, derived simultaneously from both PHA and TAGs, were analysed in a single run using a Trace 1300 GC apparatus (Thermo Fisher Scientific, US) equipped with a flame ionization detector (FID) (Thermo Fisher Scientific, US) and a Restek column (Crossbond, Stabilwax). The system operated at constant pressure (96 kPa) using helium as carrier gas. The oven temperature program was the following: 20 °C/min until 100 °C; 3 °C/ min until 155 °C and, finally, 20 °C/min until 230 °C with a holding time of 30 min. All measurements were done in triplicate. Glycogen analysis Glycogen accumulation was analysed following the protocol described by [50]. The previously weighed freezedried biomass was treated with 2 mL of a dilute solution of HCl. The tubes were incubated at 100 °C for 3 h. The samples were filtered (filter with 0.2 µm pore size, Whatman) and analysed by high-performance liquid chromatography (HPLC) using a chromatograph equipped with an Aminex HPX-87H HPLC column (Bio-Rad, USA). A solution of 0.01 N of H2SO4 was used as a mobile phase with a flow rate of 0.5 mL/min and a 30 °C operating temperature. The detection wavelength was set at 210 nm. Glucose (Scharlau, Barcelona, Spain) was used as standard ranging from 0.06 to 1 g/L. Samples were analysed in triplicate. Polyphosphate staining method For staining polyphosphate inclusions, samples were fixed with gentle heat on glass microscopic slides and exposed to Loeffler’s methylene-blue staining in each case with light washing in distilled water [51]. Calculations The maximum specific cell growth rate (μmax, h−1) was calculated by determining the linear regression slope of the exponential phase of Ln Xt/X0 versus time curve, where Xt /X0 (g/L) is the active cell biomass concentration at time t (h) and at the beginning of the run (t0), respectively. The active cell biomass (X, g/L) (without PHA and TAG) used for yield calculations, at time t, was determined by Eq. (1). where CDWt (g/L), PHAt (g/L) and TAG t (g/L) represent the CDW and the concentrations of PHA and triacylglycerol (TAG) at time t(h), respectively. The overall volumetric productivity (rP, g/ L.(day)), where P isindicative of PHA or TAG, were determined by Eq. (2): where ΔP (g/L) is the product (PHA or TAG) produced in time interval Δt (h). The yields of active biomass (YX/S, gX/gTPA) and the products (P) on substrate basis (YP/TPA, gP/gTPA) were determined by Eqs. (3) and (4): where ΔX and ΔP are the active biomass and the PHA and/or TAG produced (g/L), respectively, and ΔS (g/L) is the concentration of TPA from REX-TPA residue consumed during the same time range of the cultivation run. Statistical analysis The statistical differences for the mean and standard deviation of the kinetic and stoichiometric parameters obtained from the three assays performed by Rhodococcus sp. Ave7 using REX-TPA were assessed using one-way ANOVA followed by Bonferroni’s multiple comparison tests in GraphPad Prism 5 with the criteria for statistical significance set at p < 0.05. Bioproducts extraction andcharacterization The cultivation broth was centrifuged (10,350 g, 20 min, 4 °C) and the obtained cell pellets were freeze-dried and milled. The bioproducts were extracted from the freezedried biomass by Soxhlet extraction with chloroform (Fisher Chemical, HPLC grade), at 80 °C, for 48 h. Later, (1) Xt = CDW t − (PHAt + TAGt) (2) r p= P t (3) Y X/S= �X �S (4) Y P/S= �P �S Page 6 of 21 Rebochoetal. Biotechnology for the Environment (2025) 2:5 the PHA was precipitated in ice-cold ethanol (1:10, v/v), under vigorous stirring, and dried in a fume hood at room temperature [52]. The ethanol used for PHA precipitation was collected and allowed to evaporate at room temperature in a fume hood, to recover the produced TAGs. For further purifying the PHA, the sample was mixed with 1-butanol (≥ 99.5%, PanReac AppliChem) at a concentration of 0.3% (w/v), and heated to 75 °C for 2 h, under constant stirring, to dissolve the TAGs fraction. The solvent was removed while still hot and the insoluble PHA was recovered and left to dry at room temperature in a fume hood. FT‑IR FT-IR analysis were conducted for the recovered TAGs and PHA samples in a Perkin-Elmer Spectrum two spectrometer coupled with ATR accessory. The polymer was directly analysed on the FTIR cells. The spectra were recorded between 400 and 4000 cm−1 resolution and 10 scans were conducted at room temperature. Thermal properties Differential scanning calorimetry (DSC) was carried out with a DSC Q2000 instrument (TA Instruments, New Castle, FL, USA). Hermetic aluminium pans were used to place the samples, and the analysis was performed with a heating and cooling rate of 10 °C/min over a temperature range of − 90 °C to 180 °C, through three heating cycles. The endotherm peak’s temperature and area of the first heating cycle were used to determine melting temperatures (Tm) and melting enthalpies (∆Hm), respectively, while the glass transition temperature (Tg, °C) was taken as the midpoint of the heat flux step. The crystallinity (XC, %) was estimated as the ratio between the obtained melting enthalpy and the melting enthalpy of 100% crystalline PHB, estimated as 146 J/g [53]. Thermogravimetric analysis (TGA) was performed using a thermogravimetric Analyzer Labsys EVO (Setaram, France), with weighing precision of + / − 0.01%. Samples were placed in aluminium crucibles and analysed in argon atmosphere with temperature range between 25 and 800 °C, at a rate of 10 °C/min. The maximum thermal degradation temperature (Tdeg, °C) corresponds to the temperature value obtained for the maximum decreasing peak of the sample mass. Molecular mass distribution Size-exclusion chromatography (SE-HPLC) was performed to determine the number average molecular weight (Mn), weight average molecular weight (Mw) and polydispersity index (Mw/Mn) of the PHA. Monodisperse polystyrene standards (370–2,520,000 Da) and the biopolymer were prepared at a concentration of 0.2% (w/v) in chloroform. Analysis was conducted using a KNAUER Smartline SE-HPLC system (Berlin, Germany) equipped with a Phenomenex Phenogel Linear Liquid Chromatographic Column (300 × 7.8 mm; Torrance, CA, USA), operated at 30 °C with a 1 mL/min chloroform flow rate as the mobile phase, using a Waters2414 refractive index detector (RID) (Milford, CT, USA). Results anddiscussion Feedstock characterization The residue obtained from the depolymerization of PET waste, named REX-PET, was a uniform dark powder (Fig.1(B)). The majority of the material comprised particles in the 1000–2000 µm size range (46 ± 2.0% w/w), followed by particles larger than 2000 µm (28 ± 5.7% w/w) and those at 500–1000 µm (16 ± 1.4% w/w) (Fig.2). Smaller particles (< 500 µm) collectively accounted for less than 12 ± 1.1% w/w. This particle size distribution indicates that the material is predominantly granular, with larger size fractions dominating the sample. The moisture and inorganic content of REX-PET is shown in Table1, and it presents a high inorganic salt content for REX-PET (46.46 ± 4.69 wt.%), since NaOH was used as a catalyst for the depolymerization of the PET waste, forming a sodium salt of TPA [54]. The elemental analysis (Table 1) revealed that REX-PET was mainly composed of carbon (44.21 ± 2.4%), with a minor hydrogen content (2.97 ± 0.39%) of trace of nitrogen (0.04 ± 0.02%), while no sulphur was detected. As shown in Fig.3, the spectral peaks of all REX-PET batches exhibited high similarity among the batch samples, although they differ from those reported for commercial TPA. The carboxylic group (-OH) stretching peak appears around 3000–2800 cm−1, but it is less intense in the REX-PET samples, suggesting a lower concentration of free carboxylic acid groups compared to pure TPA [55]. The intense zone of peaks between 1718 and 1270 cm−1 correspond to the C = O and C = C bonds of the benzene ring in TPA, with intense peaks at 1557 and 1391 cm−1 indicative of the acidic carbonyl group (-C = O) and aromatic ring vibrations [55–57]. Notably, this region shows considerable differences from commercial TPA, as the peaks correspond to the formation of TPA disodium salt, a product of the depolymerization process [58]. This is evidenced by the absence of -O–H bending bond in REX-PET, at 940 cm−1 attributed to the presence of disodium terephthalate [59], and the disappearance of the carboxylic acid groups (-COOH) at 1625 and 1423 cm−1 [60]. Moreover, peaks displayed at 1088 and 1023 cm−1 can be attributed to the = C-H bending vibrations of the aromatic ring [56]. The FT-IR spectra of the three REXPET samples in Fig.3 show strong similarity among the Page 7 of 21 Rebochoetal. Biotechnology for the Environment (2025) 2:5 batches, indicating consistent chemical structures with no significant variations in peaks or intensities, reflecting a stable and reproducible depolymerization process. The REX-TPA solution obtained from REX-PET presented a dark, translucid without visible suspended particles (after filtration) as can be observed in Fig.1(C). The pH of the REX-TPA solution was found to be 11.05 (Table2), which correlates with the dry REX depolymerization process that follows the hydrolysis method under alkaline conditions provided by adding NaOH [43]. This pH value is comparable to that obtained for a solution containing PET depolymerized upon relatable hydrolysis [61]. The REX-TPA solution had a total carbon (TC) content of 12.06 ± 0.24 g/L (as determined by the TOCVCSH Analysis) and a TPA concentration of 19.69 ± 0.09 g/L (as determined by the HPLC analysis). This TPA concentration accounts for a carbon content of 11.39 g/L which shows that the solution predominantly comprised TPA, with only a minor content of other carbonaceous compounds. The moisture and inorganic content (Table 2) for REX-TPA was of 96.20 ± 0.43 wt.% and 2.21 ± 0.13 wt.%, respectively. As expected, REX-TPA showed a high content in Na (461.12 ± 71.71 mg/L) (Table2), which can be attributed to the depolymerization procedure conducted under alkaline conditions. Thus, HCl was used to neutralize the aqueous solution [62]. The main advantage of using this type of depolymerization conditions is its suitability for complex PET waste streams, which often contain significant amounts of metals, pigments or other types of plastic [9, 63]. Other elements found in REXTPA were Fe (8.84 ± 2.45 mg/L), Ti (4.16 ± 1.48 mg/L), Sb (3.82 ± 1.23 mg/L), Si (2.12 ± 0.84 mg/L) and traces of Cr, Al, Zn, W and Mo (< 0.5 mg/L) (Table2). This wide range of components found in REX-TPA reveals the high heterogeneity of additives that can be found in mixed plastic litter samples [64]. Batch bioreactor cultivation Rhodococcus sp. Ave7 was cultivated under batch mode using an initial TPA concentration of 12 g/L as sole carbon source, under a controlled temperature of 30 °C and an initial pH of 7. The initial concentrations of TPA (12 g/L) and ammonium (0.3 g/L) in the bioreactor medium were determined based on prior findings from an RSM study (TableS1 and Fig. S1, Supplementary Material). This optimization study identified these parameters as most suitable for enhancing cell growth and the accumulation of intracellular storage compounds, namely PHA and TAGs. After a 10-h lag phase, Rhodococcus sp. Ave7 entered an exponential phase, reaching a maximum specific cell growth rate of 0.18 ± 0.05 h−1 and a CDW of 1.78 ± 0.08 g/L at 20 h of cultivation, when ammonia was exhausted Fig. 2 Particle size distribution for REX-PET Table 1 Characterization of REX-PET feedstock n.d. not detected Parameter REX-PET Moisture (wt.%) 1.07 ± 0.41 Organic content (wt.%) 52.47 ± 4.92 Inorganic salts (wt.%) 46.46 ± 4.69 Elemental analysis (%): C 44.21 ± 2.4 H 2.97 ± 0.39 N 0.04 ± 0.02 S n.d Page 8 of 21 Rebochoetal. Biotechnology for the Environment (2025) 2:5 (Fig.4(A1)). A final CDW of 2.67 ± 0.06 g/L was reached at the end of the assay. This value is slightly higher than the 2.3 g/L of CDW reported for Pseudomonas umsongensis G016 KS3 grew in a batch reactor, with similar duration, using as carbon source TPA and EG monomers obtained by enzymatic PET hydrolysis [27]. PHA accumulation started during the exponential cell growth phase, at 13 h of cultivation, and continued (Fig.4(A2)) until the end of the assay, reaching a maximum PHA content in the biomass of 4.22 ± 0.03 wt.%, corresponding to a PHA concentration of 0.11 ± 0.02 g/L (Table3). Slightly higher values were reported for P. umsongensis G016 KS3 (7 wt.%), corresponding to 0.15 g/L of PHA [27]. TAGs synthesis was initiated later, at around 21 h of cultivation (Fig.4(A2)), reaching an intracellular content of 13.45 ± 0.69 wt.% and a concentration of 0.15 g/L by 21 h of cultivation (Table3). This corresponds to an overall volumetric productivity of 0.305 g/(L day). During the first 21 h, the bacterial strain consumed 7.74 g of TPA, for both cellular growth and PHA accumulation, resulting in growth yield of 0.19 gX/gTPA. After ammonium depletion, the culture used the available TPA for PHA and TAG accumulation. The PHA yield was 0.011 ± 0.00 gPHA/gTPA, while a higher yield was reached for TAGs (0.031 ± 0.002 gTAG /gTPA). An overall consumption of 10.7 g/L of TPA was achieved over 28 h. Similar values Fig. 3 Fourier-transform infrared (FT-IR) spectra of three batches of REX-PET samples derived from PET waste depolymerization under similar conditions, and of commercial TPA (Merck Millipore, 98%) Table 2 Characterization of REX-TPA solution used in bioreactor media cultivation as feedstock Parameter REX-TPA pH 11.05 Moisture (wt.%) 96.20 ± 0.43 Organic content (wt.%) 1.58 ± 0.55 Inorganic salts (wt.%) 2.21 ± 0.13 Total carbon (g/L) 12.06 ± 0.24 [TPA] (g/L) 19.69 ± 0.09 Element (mg/L) Na 461.12 ± 71.71 K 9.30 ± 3.91 Fe 8.84 ± 2.45 Ti 4.16 ± 1.48 Sb 3.82 ± 1.23 P 3.74 ± 0.98 Mg 3.11 ± 0.86 Ca 2.15 ± 0.36 Si 2.12 ± 0.84 Cr 0.51 ± 0.28 Cu 0.29 ± 0.03 Al 0.27 ± 0.09 Zn 0.25 ± 0.18 W 0.25 ± 0.06 Mo 0.13 ± 0.06 Page 9 of 21 Rebochoetal. Biotechnology for the Environment (2025) 2:5 were obtained for P. umsongensis GO16 KS3 cultivated in TPA and EG obtained by enzymatic PET hydrolysis, (0.21 gCDW/gsubstrate and 0.014 gPHA/gsubstrate, respectively) [27]. Fed-batch cultivation withpulse feeding In Assay B (Fig.4(B1)), Rhodococcus sp. Ave7 entered the exponential growth phase after 9 h of cultivation, presenting a maximum specific growth rate of 0.18 ± 0.02 h−1, which was not significantly (p > 0.05) different from Assay A (0.18 ± 0.05 h−1). Ammonia depletion was observed after 17 h of cultivation, resulting in a CDW of 2.18 ± 0.13 g/L. By 24 h, the culture achieved a maximum CDW of 3.17 ± 0.03 g/L (Fig.4(B1)), with PHA and TAGs contents of 2.77 ± 0.01 wt.% and 11.03 ± 0.36 wt.%, respectively. At this moment, dissolved oxygen concentration started to increase (data not shown), indicating depletion of the carbon source. Therefore, a 1 L REX-TPA pulse (containing 20 g/L TPA) was fed to the culture, rising the TPA concentration to 6.76 ± 0.87 g/L. During the first 24 h, Rhodococcus sp. Ave7 produced 0.09 ± 0.00 g/L of PHA (Fig.4(B2)). Despite the subsequent feeding of a TPA pulse, no further increase in Fig. 4 Cultivation profiles for batch (A), fed-batch with pulse feed (the dashed line denotes the time the REX-TPA pulse was given) (B) and fed-batch with continuous feed (the grey area denotes the time REX-TPA was fed to the bioreactor, at a rate of 0.1 L/h) (C) of Rhodococcus sp. Ave7 using REX-TPA as feedstock. Error bars correspond to triplicate measurements Page 16 of 21 Rebochoetal. Biotechnology for the Environment (2025) 2:5 content (58 to 98 mol%) [85]. Moreover, the Mw and PDI obtained was also comparable to the PHBV, 260 kDa and PDI of 1.9, produced by mixed microbial cultures with 82 mol% 3HV [84]. Nevertheless, the Mw attained for Rhodococcus sp. Ave7 PHBV was low compared to the produced by R. pyridinivorans P23 (Mw of 600 kDa) using TPA, but still within the same order of magnitude [42]. The PDI of the PHBV attained in assay C suggests a good polymer uniformity, which may facilitate its processing and biodegradation rate [95, 96]. Thermal properties The biopolymer presented a melting temperature (Tm) of 95.1 °C that is within the range of several PHBV with 3HV contents varying between 58 and 98 mol% (89.9– 109.4 °C) [85], and similar to the Tm (101 °C) reported for PHBV produced with TPA as feedstock [42]. The high Fig. 7 FT-IR spectra for (a) PHBV produced by Rhodococcus sp. Ave7 during fed-batch with continuous feed conditions and (b) PHBV with 11% 3HV content (adapted from [94]) Table 6 Physicalchemical and thermal properties of PHBV produced in Assay C from Rhodococcussp. Ave7 Mw molecular weight, PDI polydispersity index, Tm melting temperature, Tg glass transition temperature, Tdeg degradation temperature, ΔHm melting enthalpy, Xc crystallinity fraction Characterization Value Mw (kDa) 277 PDI 1.5 Tm (°C) 95.1 ∆Hm (J/g) 26.3 Xc (%) 18.0 Tg (°C) − 21.1 Tdeg (°C) 270.0 395.5 503.6 Page 17 of 21 Rebochoetal. Biotechnology for the Environment (2025) 2:5 3HV content in the copolymer lowers the Tm significantly, thus broadening its processability window, which facilitates the polymer processing in comparison to the homopolymer composed by 3HB, characterized by a Tm very close to the degradation temperature [97]. The PHBV also exhibited a glass transition temperature (Tg) of − 21.1 °C (Table6), which is significantly lower than the typical values reported for PHBV with 3HV contents of 82 mol% (− 13.2 °C) [84]. The low Tg observed for PHBV may result from TAGs still present in the polymer matrix after 1-butanol precipitation, potentially acting as plasticizers that increase the free volume between PHBV chains and enhance their mobility at lower temperatures, as reported for PHBV and plasticizers blends [89, 98]. The polymer presented a crystallinity of 18.0% (Table6), indicating it was more amorphous than other 3HV-rich PHBV (40–50 mol % of 3HV) that present crystallinities within 50% [85]. For copolymers where 3HV content was higher, a higher crystallinity was expected since it would mainly take the crystal structure of the P(3HV) homopolymer lattice [99]. This decrease in crystallinity may be related to the presence of remnants of TAGs that were not completely removed from the sample [100], as previously detected in the biopolymer FT-IR spectrum. Nevertheless, the biopolymer’s low crystallinity may provide more flexibility compared to other PHBV blends with lower 3HV content, making it suitable for applications that require softer, more flexible materials [84]. The biopolymer was thermally stable until 270 °C (Table6), in concomitant with degradation temperature (Tdeg) (279 °C) for other 3HV-rich PHBV, where it suffered a weight loss of 46% [101]. Above this temperature, a second weight loss of 15.5% was attained at 395.5 °C followed by a third observed at 503.6 °C for 33.8% of weight loss. These later stages of degradation are attributed to the degradation of fatty acids, confirming the decomposition of TAGs monounsaturated and saturated fatty acids, mainly composed by C18:1, C16:0 and C18:0 [102]. The presence of fatty acids significantly affected the thermal properties of the sample. The fatty acids within the polymer matrix exhibited higher Tdeg than the individual oleic, stearic and palmitic methyl esters (210–229 °C for 5% weight loss) or their ethylene glycol esters (228–248 °C) tested as potential PHBV plasticizers [89]. This shows that the presence of TAGs-derived fatty acids in the polymer provide a higher window of thermal stability compared to previously PHBV composites with similar fatty acids. This demonstrates that PHBV materials with high 3HV content can be tailored by adding natural fatty acids as plasticizers, enhancing the biopolymer’s performance, particularly in the thermal stability for end-use applications [89]. Conclusions This work validated Rhodococcus sp. Ave7 as a promising microorganism for the bioremediation of PET waste, given its high capacity for TPA bioconversion. The culture efficiently upcycled depolymerized PET waste into biomass, PHA and TAGs. The produced co-polyester PHBV, with a 3HV content up to 90 wt.%, has potential for being used in PHBV copolymer blends. The biosynthesized TAGs, on the other hand, were enriched in octadecenoic and hexadecanoic acids, which might be of interest to pair with the existing production from vegetable oils sources. Overall, this study demonstrated the potential of Rhodococcus sp. Ave7 for effective biodegradation of chemically depolymerized PET waste into value-added bio-based products, thus contributing to reduce the impact of PET waste and valorising it into value-added products, within the circular economy concept. Through the bioconversion of PET waste into sustainable alternatives to petroleum-derived plastics and oils, this research supports efforts to reduce plastic waste and dependence on fossil-based resources, underlining its role in advancing sustainable waste management solutions and reinforcing its environmental and societal relevance. Abbreviations PET Polyethylene terephthalate TPA Terephthalic acid REX Reactive extrusion REX-TPA Reactive extrusion-terephthalic acid PHAs Polyhydroxyalkanoates TAGs Triacylglycerols PHBV Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) 3HV 3-Hydroxyvalerate C18:1 Octadecenoic acid C16:0 Hexadecanoic acid Supplementary Information The online version contains supplementary material available at https:// doi. org/ 10. 1186/ s4431402500019-4. Supplementary Material 1. Table S1 Matrix of the Response Surface Methodology (RSM), composed of eleven experiments: four factorial design points; four experiments of axial level; and a central point with three replicas. The two independent variable, X1 (REX-TPA concentration, g/L) and X2 (Ammonium concentration, g/L) and the response Y1 (CDW, g/L), Y2 (PHA wt.%) and Y3 (TAG, wt.%) for Rhodococcus sp. Ave7. Table S2 One-way ANOVA results for kinetic and stoichiometric parameters of the three assays performed by Rhodococcus sp. Ave7 using REX-TPA. Fig. S1 Three-dimensional response surface and contour plots show the interactive effects of different concentrations of ammonium and REX-TPA on CDW (g/L) (a), PHA (wt.%) (b) and TAG (wt.%) (c) for Rhodococcus sp. Ave7. Acknowledgements Not applicable. Authors’ contributions Conceptualization: ATR, CT, and FF; formal analysis: ATR, CT and FF; investigation: ATR; methodology: ATR; HK, LS, OA; writing—original draft: ATR; writing— review and editing: MF, CT, CET, MM, MR and FF. All authors have read and agreed to the published version of the manuscript. Page 18 of 21 Rebochoetal. Biotechnology for the Environment (2025) 2:5 Funding This work was financed by the European Union’s Horizon 2020 research and innovation program through Project Bio Innovation of a Circular Economy for Plastics (BioICEP), under grant agreement No. 870292, supported by the National Natural Science Foundation of China (grant numbers: Institute of Microbiology, Chinese Academy of Sciences: 31961133016; Beijing Institute of Technology: 31961133015; Shandong University: 31961133014). European Union’s Horizon Europe EIC Pathfinder programme, Eco conversion of lower grade PET and mixed recalcitrant PET plastic waste into high performing biopolymers (EcoPlastic), under grant agreement No 101046758. By national funds from FCT—Fundação para a Ciência e a Tecnologia, I.P., in the scope of the projects UIDP/04378/2020 and UIDB/04378/2020 of the Research Unit on Applied Molecular Biosciences—UCIBIO and the project LA/P/0140/2020 of the Associate Laboratory Institute for Health and Bioeconomy—i4HB. A.T. R. acknowledges FCT I.P. for the Ph.D. grant 2020.06470.BD. Data availability No datasets were generated or analysed during the current study. Declarations Ethics approval consent to participate Not applicable. Consent for publication Not applicable. Competing interests The authors declare no competing interests. Received: 8 July 2024 Accepted: 3 March 2025 References 1. Nisticò R. Polyethylene terephthalate (PET) in the packaging industry. Polym Test. 2020;90: 106707. https:// doi. org/ 10. 1016/j. polym ertes ting. 2020. 106707. 2. Maurya A, Bhattacharya A, Khare SK. Enzymatic remediation of polyethylene terephthalate (PET)–based polymers for effective management of plastic wastes: an overview. Front Bioeng Biotechnol. 2020;8: 602325. https:// doi. org/ 10. 3389/ fbioe. 2020. 602325. 3. Samak NA, Jia Y, Sharshar MM, Mu T, Yang M, Peh S, et al. Recent advances in biocatalysts engineering for polyethylene terephthalate plastic waste green recycling. 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