Creating a Halotolerant Degrader for Efficient Mineralization of p ‐Nitrophenol‐Substituted Organophosphorus Pesticides in High‐Saline Wastewater

The bioaugmentation performance is severely reduced in the treatment of high‐saline pesticide wastewater because the growth and degradation activity of pesticide degraders are significantly inhibited by high salt concentrations. In this study, a heterologous biodegradation pathway comprising the seven genes mpd/pnpABCDEF responsible for the bioconversion of p ‐nitrophenol (PNP)‐substituted organophosphorus pesticides (OPs) into β‐oxoadipate and the genes encoding Vitreoscilla hemoglobin (VHb) and green fluorescent protein (GFP) were integrated into the genome of a salt‐tolerant chassis Halomonas cupida J9, to generate a genetically engineered halotolerant degrader J9U‐MP. RT‐PCR assays demonstrated that the nine exogenous genes are successfully transcribed to mRNA in J9U‐MP. Gas chromatography analysis of methyl parathion (MP) and its intermediates demonstrated that the expressed MP hydrolase and PNP‐degrading enzymes PnpABCD show obvious degradation activity toward the specific substrates in J9U‐MP. Stable isotope analysis showed that J9U‐MP is able to efficiently convert 13 C 6 ‐PNP into 13 CO 2 , demonstrating the complete mineralization of MP in high‐salt media. J9U‐MP is genetically stable during passage culture, and genomic integration of exogenous genes does not negatively influence the growth of J9U‐MP. Under oxygen‐limited conditions, VHb‐expressing J9U‐MP does not show obvious growth inhibition and a significant reduction in the MP degradation rate. A real‐time monitoring system with enhanced GFP is used to track the motion and activity of J9U‐MP during bioremediation. Moreover, 50 mg/L MP and its intermediates (i.e., PNP and HQ) were completely degraded by J9U‐MP within 12 h in wastewater supplemented with 60 g/L NaCl. After 3 days of incubation, 25 mg/L 13 C 6 ‐PNP was converted into 13 CO 2 by J9U‐MP in wastewater supplemented with 60 g/L NaCl. Our results highlight the power of synthetic biology for creating new halotolerant pollutant‐mineralizing strains. The strong competitive advantages of J9U‐MP in high‐salinity and low‐oxygen environments make this degrader suitable for in situ bioaugmentation of OP wastewater.