Sustainable production and co-immobilization of cold-active enzymes from Pseudomonas sp. for BTEX biodegradation

Toluene/o-Xylene Monooxygenase (ToMO) is equipped with a broad spectrum of aromatic substrate specificity (such as BTEX; benzene, toluene, ethylbenzene, and isomers of xylenes). TOMO has can hydroxylate more than a single position of aromatic rings in two consecutive monooxygenation reactions. Catechol 1,2-dioxygenase (C1,2D) is an iron-containing enzyme able to cleave the ring of catechol (the converted product from ToMO) for complete detoxification of BTEX. In this study, cold-active ToMO and C1,2D were produced using newly isolated psychrophilic Pseudomonas S2TR-14 in the minimal salt medium supplemented with crustacean waste and different concentrations of used motor oil (0.2-2% (v/v)). Crude ToMO and C1,2D were immobilized into micro/nano biochar-chitosan matrices and used for BTEX biodegradation. The results showed that the highest enzyme production (12 U/mg for ToMO and 22 U/mg for C1,2D) was achieved at the presence of 0.5% v/v used motor oil compared to the control group without motor oil (0.07 and 0.06 U/mg). High immobilization yield was achieved due to covalent bonding of ToMO (92.26% for micro matrix and 77.20% for nano matrix) and C1,2D (87.57% for micro matrix and 74.79% for nano matrix) with matrices. FTIR spectra confirmed the immobilization of enzymes on the surface of microbiochar and nanobiochar-chitosan matrices as proper support. The immobilization increased the storage stability of the enzymes with more than 50% residual activity after 30 days at 4 ± 1 °C, while the free form of enzymes had less than 10% of its activity. Immobilized enzymes degraded more than 80% of BTEX (~200 mg/L in groundwater and ~10,000 mg/kg in soil) at 10 ± 1 °C in groundwater and soil. Therefore, integrated use of microbiochar and nanobiochar with chitosan for co-immobilization of ToMO and C1,2D can be a potential way to remove petroleum hydrocarbons with higher efficiency from contaminated groundwater and soil.