Biodegradation of Poly (Ethylene Terephthalate) by Bacteria Isolated from Soil

Abstract

Polyethylene terephthalate (PET) is one of the most widely used synthetic polymers due to its desirable properties, including excellent mechanical strength, chemical resistance, and low production cost. However, the persistent accumulation of PET in the environment has raised concerns about its long-term impact on ecosystems. In recent years, there has been increasing interest in finding sustainable solutions for the management of PET waste through biodegradation. The aim of this study was to investigate the biodegradation of PET by bacteria and its enzyme from plastic contaminated soil. A polyethylene terephthalate (PET) degrading bacterium Stenotrophomonas maltophilia PRS8 was isolated from soil of a waste-dumping site located in Peshawar, Pakistan at N 34.0074° and E 71.6194°. The degradation of PET was evaluated using various qualitative assays such as weight loss, biofilm formation on PET pieces, Fourier-transform infrared (FTIR) spectroscopy and Scanning Electron Microscopy (SEM). S. maltophilia PRS8 strain demonstrated its ability to attach to PET pieces and form biofilm that could be helpful in its degradation. Approximately 1.2% reduction in weight of PET pieces was measured in comparison to control where no loss was found. A change in intensity of FTIR peaks of the treated PET was observed at wavenumber 722, 1098, 1242, and 1714 cm-1 compared to the untreated PET that indicates polymer chain scission by S. maltophilia PRS8. SEM images also showed cracks and roughness on surface of PET pieces that clearly indicates its deterioration after treatment with S. maltophilia PRS8. The degradation medium was checked for presence of enzymes at the end of experiment, strain PRS8 expressed cutinase activity during PET degradation. Different physico-chemical conditions were optimized for better enzyme yield using one time one factor and multiple factor one-time statistical models [Placket-Burman Design (PBD) and Central Composite Design (CCD) software]. The involvement of each ingredient in production of cutinase is depicted in a Pareto chart acquired from PBD. Cutin, NaNO3, and [(NH4)2SO4] were discovered to have a considerable influence on cutinase synthesis. S. maltophilia PRS8 cutinase was optimized via PBD and CCD, which led to a 5-fold increase in enzyme activity. Cutinase was purified to homogeneity by column chromatography using Sephadex G-100 resin and molecular weight was found to be approximately 58 kDa. The specific activity of purified cutinase was estimated as 450.58 xvii U/mg with 6.39-fold purification and 48.64% yield. The Km and Vmax values were determined as 0.703 mg.ml−1 and370.37μmol.mg−1min−1, respectively. The enzyme was stable at various temperatures (30-40°C) and pH (8.0-10.0). The cutinase activity was significantly enhanced by organic solvent (Formaldehyde), surfactants (Tween-40 and Triton X-100) and metals (NiCl2 and Na2SO4). The purified cutinase hydrolyzed PET piece at 40°C and approximately 18.3% reduction in weight of PET was measured. The degradation products; terephthalic acid (TPA), mono(2-hydroxyethyl)-TPA (MHET) and bis(2-hydroxyethyl)-TPA (BHET) were confirmed through liquid-chromatography mass spectrometry (LC-MS). Furthermore, the activity of PET hydrolyzing enzyme was enhanced by cloning and expression of gene for PET hydrolyzing enzyme into expression host. The PET hydrolyzing enzyme was successfully cloned into PET-21b and expressed into BL21 (DE3). HiPrep 16/60 Sephacryle S-300 High resolution gel filtration column chromatography resin was used for the purification of recombinant enzyme and fractions were collected for measuring enzyme activity. The molecular weight of recombinant cutinase was found to be approximately 58 kDa. The specific activity of purified cutinase was estimated as 595.93 U/mg with 6.19-fold purification and 23.89% yield. The maximum PET hydrolyzing enzyme activity was observed at temperature 30-40˚CandpH 8.0-9.0, while SDS, CTAB, ethanol and Hg+ strongly inhibited its activity. The PET depolymerization products were detected by HPLC and LC-MS, and were identified as TPA, MHET, and BHET. However, an outstanding performance was achieved on PET reaching 22.5 % depolymerization activity after treated with recombinant PET hydrolyzing enzyme for 72 hours, while 18.3% depolymerization activity was achieved after PET treated with native enzyme which is lower than that of recombinant enzyme. The crytallinity of treated and untreated PET pieces with native and recombinant enzyme were analyzed by Differential Scanning Calorimetry (DSC). About 4.1 % reduction in crystallinity of PET pieces was observed after treatment with recombinant cutinase which was slightly higher than that of the native enzyme (3.4%). The recombinant PET hydrolyzing enzyme showed a depolymerization yield of the PET comparable to that of Idonella skaiensis IsPETase and significantly higher than that of Humicola insolens thermostable HiCut (HiC) cutinase. This study suggests that S. maltophilia PRS8 possesses xviii the ability to degrade PET at mesophilic temperature and could be further explored for sustainable management of plastic waste.