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

S1 Supplementary data for: Biotechnological Model for Ubiquitous Mixed Petroleumand Bio-Based Plastics Degradation and Upcycling into Bacterial Nanocellulose Jeovan A. Araujo,*‡a George Taxeidis,‡b Everton Henrique da Silva Pereira,a Muhammad Azeem,a Brana Pantelic,c Sanja Jeremic,c Marijana Ponjavic,c Yuanyuan Chen,a Marija Mojicevic,*a Jasmina Nikodinovic-Runic,c Evangelos Topakas,b and Margaret Brennan Fourneta a Centre for Polymer Sustainability, PRISM Research Institute, Technological University of the Shannon: Midlands Midwest, N37 HD68 Athlone, Ireland b Biotechnology Laboratory, Department of Synthesis and Development of Industrial Processes, School of Chemical Engineering, National Technical University of Athens, Athens, Greece c Institute of Molecular Genetics and Genetic Engineering, University of Belgrade, Vojvode Stepe 444a, 11000 Belgrade, Serbia ‡ Jeovan A. Araujo and George Taxeidis contributed equally to this work. * Email: [email protected], [email protected] (J.A.A.); [email protected] (M.M.) Note: S2 Number of pages: 17 Number of texts: 14 Number of figures: 9 Number of tables: 2 S3 Table of Contents Methods ................................................................................................................................... S4 Text S1: Method for model home composting bioaugmented with Bacillus sp. BPM12 ... S4 Text S2: Quantification of BHET, MHET, and TPA via HPLC analysis ........................... S5 Text S3: Quantification of reducing sugars via DNS colorimetric method ......................... S6 Characterization .................................................................................................................... S7 Text S4: Infrared spectrum of the starting TPS (model bioplastic) ..................................... S7 Text S5: Thermal properties of the mixed (petro-bio)plastic films ..................................... S8 Text S6: Mechanical properties of the mixed (petro-bio)plastic films .............................. S10 Biodegradation of the mixed PET-TPS materials ............................................................. S11 Text S7: Home composting bioaugmented with Bacillus sp. BPM12 ............................... S11 Text S8: Infrared spectra of post-composting neat PET vs. PET-TPS40 film .................. S11 Text S9: Microstructure analysis of post-composting neat PET vs. PET-TPS40 film ...... S12 Text S10: Enzymatic degradation study ............................................................................ S14 Text S11: Enzymatic degradation at 30 °C ........................................................................ S14 Text S12: Enzymatic degradation at 50 °C ........................................................................ S15 Bio-upcycling of mixed PET-TPS hydrolysates into bacterial nanocellulose (BNC) .... S18 Text S13: Infrared spectrum and visual appearance of the produced BNC ....................... S18 Text S14: Estimation of the mixed PET-TPS plastic conversion to BNC ......................... S20 References ............................................................................................................................. S21 Deleted: 3 Deleted: 3 Deleted: 4 Deleted: 5 Deleted: 6 Deleted: 6 Deleted: 7 Deleted: 8 Deleted: 9 Deleted: 9 Deleted: 9 Deleted: 10 Deleted: 11 Deleted: 11 Deleted: 12 Deleted: 15 Deleted: 15 Deleted: 16 Deleted: 17 S4 Methods Text S1: Method for model home composting bioaugmented with Bacillus sp. BPM12 Biodegradability of PET-TPS films was assessed in laboratory model compost during 10 weeks at 37 °C, according to a previously described protocol (Pantelic et al., 2021). The model composting system consisted of a commercial mixture of raw materials used for the cultivation of white button mushroom (80%, w/w) and universal soil for gardening and potting (20%, w/w), both obtained from ACS Garden (Belgrade, Serbia). Film samples weighing between 60–120 mg were rinsed with 70% EtOH and air dried, buried in compost (150 g) at a depth of 1 cm, in duplicates, and humidity was set up at approximately 50% with distilled water. Two types of compost were used: pristine non-treated compost and compost bioaugmented with Bacillus sp. BPM12 strain (log104 cells/gram of compost). Bacillus sp. BPM12 grown overnight in LB medium (Luria Bertani, 10 g·L−1 tryptone, 5 g·L−1 yeast extract, 10 g·L−1 NaCl) kept at 180 rpm and 30 ℃ was used for inoculation, while 20% glycerol stocks were prepared and stored at -80 ℃ for continuous replenishment of model home bioaugmented composting. Samples were inspected weekly, when fresh aliquots of bacterial culture were added to bioaugmented compost, and distilled water was added to pristine compost, in order to maintain constant humidity. After 10 weeks of incubation, samples were removed, rinsed with 70% EtOH, air dried, and stored before further tests were carried out. S5 Text S2: Quantification of BHET, MHET, and TPA via HPLC analysis The quantification of TPA, MHET, and BHET present in the degradation products released after enzymatic treatment was performed by constructing the calibration curves below ( Fig. S1 ), which were based on the HPLC peak area (AUC) method. Fig. S1. Calibration curves used for HPLC quantification of (a) TPA, (b) MHET, and (c) BHET. Standard concentrations were used in the range of 0.01–0.8 mM. Inserts show the details related to the linear fit applied to each curve, including the goodness of fit (R2). Deleted: Fig. S1 S6 Text S3: Quantification of reducing sugars via DNS colorimetric method A calibration curve of the absorbance at 540 nm (a.u.) was plotted against the glucose concentration (mg·mL-1), which was used as a standard (Fig. S2). Fig. S2 . Calibration curve used to determine the amount of reducing sugars via DNS method. The insert shows the details of the linear fit applied to the curve, including the goodness of fit (R2). Formatted: Font: Not Italic, Check spelling and grammar Deleted: Fig. S2 S7 Characterization Text S4: Infrared spectrum of the starting TPS (model bioplastic) The spectrum of neat TPS film (Fig. S3) has been previously examined elsewhere in the literature (Araujo et al., 2021). Thermoplastic potato starch (TPS) was used as a model bioplastic in the mixed (petro-bio)plastic materials. The spectrum of TPS showed a strong band at 1758 cm−1 attributed to the stretching of C=O group that might have resulted from the plasticization process of starch. The typical band observed at 1454 cm−1 was attributed to C–H bending vibrations, while bands at 1184 and 1044 cm−1 were due to changes in the C–O and C–O–H stretching vibrations. Fig. S3. FTIR spectrum of TPS in the wavenumber range from 4000 to 650 cm−1. Formatted: Font: Not Italic, Check spelling and grammar Deleted: Fig. S3 S8 Text S5: Thermal properties of the mixed (petro-bio)plastic films The thermal properties of the mixed (petro-bio)plastic materials with varying bioplastic content were evaluated by DSC (Fig. S4). Fig. S4. First heating scan from DSC curves of (a) PET pellets, (b, b') neat PET, (c, c') PETTPS5, (d, d') PET-TPS10, (e, e') PET-TPS30, and (f, f') PET-TPS40 materials. Double line, starting PET pellets used as received; solid lines, extruded mixed (petro-bio)plastic films; dashed lines, powder samples obtained from the extruded films after milling process. Table S1. Thermal properties of the starting PET pellets, film and powder PET and PET-TPS samples calculated from the first heating DSC scan. Sample Processing Sample form Melting enthalpy (J.g-1) Crystallinity (%) PET none (used as received) pellet 72.8 52.0 PET hot-melt extrusion film 53.6 38.3 PET-TPS5 hot-melt extrusion film 52.2 37.3 PET-TPS10 hot-melt extrusion film 44.2 31.5 PET-TPS30 hot-melt extrusion film 38.5 27.5 PET-TPS40 hot-melt extrusion film 30.5 21.8 PET extrusion + milling powder 39.1 27.9 PET-TPS5 extrusion + milling powder 37.4 26.7 PET-TPS10 extrusion + milling powder 32.8 23.4 PET-TPS30 extrusion + milling powder 31.6 22.6 Formatted: Font: Not Italic, Check spelling and grammar Deleted: Fig. S4 S9 PET-TPS40 extrusion + milling powder 28.5 20.4 S16 Then, the degradation reaction of the mixed PET-TPS films was studied at 50 °C. As it can be clearly seen in Fig. S8 a, the overall performance of the enzymatic treatments was improved with the temperature increase, indicating that both AMY and LCCICCG enzymes are active at 50 °C. The improved degradation was attributed to the activation of LCCICCG, as demonstrated by a 15% depolymerization of neat PET film treated with only LCCICCG. Moreover, the combined LCCICCG/AMY treatment synergistically improved the enzymatic decomposition of the PET-TPS films, with depolymerization rates up to 31%. This was supported by an increase in the total concentration of TPA, MHET, and BHET released from PET-TPS films after the LCCICCG/AMY treatment at 50 °C, which ranged from 1.9 to 2.3 mg·mL−1 ( Fig. S8 b). Reducing sugars were detected in all treatment scenarios at 50 °C, however this was limited to only 1.3 mg·mL−1 was detected for PET-TPS40 treated with AMY alone ( Fig. S8 c). As suggested above, increased enzymatic depolymerization was correlated with the enzymatic degradation synergy for the mixed PET-TPS films, particularly for the PET-TPS10 sample that showed a DS = 1.5 ( Fig. S8 d). Deleted: Fig. S8 Deleted: Fig. S8 Deleted: Fig. S8 Deleted: Fig. S8 S17 Fig. S8. Enzymatic degradation study on the mixed PET-TPS films using AMY, LCCICCG, and LCCICCG/AMY at 50 ℃: (a) Enzymatic decomposition (%) calculated from the total degradation products (BHET, MHET, TPA, and reducing sugars); (b) Total PET products released detected by HPLC analysis; (c) Reducing sugars detected via the DNS method; and (d) Comparison of degree of synergism (DS) for the different mixed PET-TPS samples. DS was calculated from the total decomposition by LCCICCG/AMY in relation to each individual enzymatic treatment. Reactions were carried out for four days under constant agitation (1200 rpm) using 30 μg of AMY, 0.12 μg of LCCICCG, or a combination of both enzymes in 0.1 M phosphate buffer, pH = 7. The concentration of 100% PET-TPS degradation products is equivalent to 10 mg·mL-1. Tests were carried out in triplicate; error bars represent the standard deviation of the mean values. S18 Bio-upcycling of mixed PET-TPS hydrolysates into bacterial nanocellulose (BNC) Text S13: Infrared spectrum and visual appearance of the produced BNC The harvested BNC films were air-dried appropriately at 60 ℃, and cut into tapes before FTIR analysis (Fig. S9). For comparison purposes, two BNC samples were selected: (a) BNC obtained from the control HS medium, and (b) BNC grown from the mixed PET-TPS40 hydrolysates obtained after the synergetic degradation by the LCCICCG/AMY treatment. Both samples are visually similar with subtle color differences. FTIR spectra of both BNC samples from (a') control HS medium and (b') PET-TPS40 hydrolysates showed analogous characteristic bands, including a broad O−H stretching vibration centered at 3330 cm−1, C−H aliphatic stretching between 2985−2850 cm−1, O−H bending at 1626 cm−1, CH2 bending at 1427 cm−1, C−O stretching vibrations at 1270, 1240, and 1160 cm−1, and C−O−C pyranose ring skeletal stretching vibrations at 1052 and 1028 cm−1 (Molina-Ramírez et al., 2018; Rozenberga et al., 2016). Fig. S9. Digital images (top) and FTIR spectra (bottom) of the BNC produced from (a, a') HS medium used as glucose-supplemented control and (b, b') PET-TPS40 hydrolysates obtained from the LCCICCG/AMY enzymatic treatment at 50 ℃. Formatted: Font: Not Italic, Check spelling and grammar Deleted: Fig. S9 S19 S20 Text S14: Estimation of the mixed PET-TPS plastic conversion to BNC The conversion of mixed (petro-bio)plastic to BNC was estimated by taking the amount of BNC produced (g) after each enzymatic treatment in relation to the initial amount of mixed plastic substrate (g) from which the hydrolysates were obtained via enzymatic degradation. The conversion expressed in percentage (%) is measured as g of BNC per g of PET-TPS substrate used, and the results are presented below in Table S2 . Table S2. Conversion of mixed PET-TPS hydrolysates to BNC following AMY, LCC, and LCCICCG/AMY enzymatic treatments at 50 ℃. Sample Amount of plastic substrate (g) Conversion to BNC (%) AMY LCC LCC/AMY PET 1 0.00% 0.00% 0.00% PET-TPS5 1 0.00% 0.00% 0.41% PET-TPS10 1 0.38% 0.28% 0.58% PET-TPS30 1 0.38% 0.27% 0.53% PET-TPS40 1 0.41% 0.34% 0.58% S21 References Araujo, J.A., Cortese, Y.J., Mojicevic, M., Brennan Fournet, M., Chen, Y., 2021. Composite Films of Thermoplastic Starch and CaCl2 Extracted from Eggshells for Extending Food Shelf-Life. Polysaccharides 2, 677–690. https://doi.org/10.3390/polysaccharides2030041 Maurya, A., Bhattacharya, A., Khare, S.K., 2020. Enzymatic Remediation of Polyethylene Terephthalate (PET)–Based Polymers for Effective Management of Plastic Wastes: An Overview. Front Bioeng Biotechnol 8. https://doi.org/10.3389/fbioe.2020.602325 Molina-Ramírez, C., Enciso, C., Torres-Taborda, M., Zuluaga, R., Gañán, P., Rojas, O.J., Castro, C., 2018. Effects of alternative energy sources on bacterial cellulose characteristics produced by Komagataeibacter medellinensis. 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