Cold Active Enzyme Booster Technology (EnBooT) for Biodegradation of P-Xylene

p-xylene is used as a solvent in medical technology, the leather, paint, and rubber industries. The principal pathway of human contact to p-xylene is via soil and groundwater contamination. Bioremediation offers potential advantages such as being cost-effective and environmentally friendly with lesser undue damage to environments. The main aim of this project is to find an enzyme mixture for biodegradation of p-xylene contaminated sites. In this regard, screening of indigenous bacteria, identification of involved enzymes, and biodegradation tests were carried out. The results showed that xylene monooxygenase (XMO) and catechol 2,3-dioxygenase (C2,3D) have a matching end product, they acted in symphony to degrade p-xylene. The mixture of these enzymes confirmed the complete degradation of p-xylene within 48 h in groundwater (initial concentration of 200 mg/L), 7 days in soil tests (initial concentration of 10,000 -12,000 mg/kg of soil) at 15°C, which is revolutionary for the industrial sector. In soil column tests, different concentrations of the enzyme mixture were used (1x, 5x, and 10x dilution). In this test, 92-94% p-xylene removal was achieved in the treated soil with a 5x diluted enzyme mixture (contained 10 U/mL of XMO and 20 U/mL of C2,3D). Our results showed that biodegradation is a scale-dependent phenomenon and the maximum degradation rate decreased from ~90% to 68% from the soil column to tank tests. It is due to limited access of enzymes to trapped p-xylene in soil pores, low dissolved oxygen, soil heterogeneity, and free phase contaminant. In addition, one of the major challenges in the practical and commercial application of these enzymes is their inherent instability. Our results showed that immobilization improved the stability of enzymes. For example, micro/nano biochar-chitosan matrices increased the stability of enzymes with more than 50% residual activity after 30 days at 4±1 ºC, while the free enzymes had less than 10% of its activity. Overall, this cold-active enzyme mixture can be applied for the biodegradation of all BTEX compounds (benzene, toluene, ethylbenzene, and xylenes). This study could set the guideline for the enzymatic bioremediation of mono-aromatic pollutants in contaminated soil and groundwater under cold conditions.