Metabolic engineering of Halomonas cupida for co-mineralization of phenol and p-nitrophenol in high-saline wastewater

Phenol and p-nitrophenol (PNP) are often co-detected in high-saline phenolic wastewater and combined toxicity caused by phenolic compounds poses potential risks to higher organisms, which have aroused serious concern for phenolic wastewater treatment. In this study, a halophilic bacterium Halomonas cupida J9 was engineered by genomic integration of phenol and PNP biodegradation pathways and the gfp gene (coding for green fluorescent protein), resulting in a fluorescently-labeled and halotolerant degrader (named J9U-PP). The transcription of all nine exogenous genes was achieved in strain J9U-PP. Strain J9U-PP was able to degrade 25 mg/L phenol in high-salt media containing 6% NaCl, and it also had the ability to degrade 25 to 100 mg/L PNP in high-salt media containing 6 to 10% NaCl. Moreover, strain J9U-PP was capable of mineralizing phenol and PNP to CO₂ in high-salt media. More importantly, co-mineralization of phenol and PNP by strain J9U-PP was verified in laboratory-amended wastewater samples. We demonstrate for the first time co-mineralization of phenol and PNP by an engineered halotolerant chassis. Strain J9U-PP can be easily tracked by green fluorescence in natural environments. Strain J9U-PP displayed superior growth kinetics and excellent genetic stability during passage culture. These merits make strain J9U-PP an ideal candidate for the remediation of hypersaline environments co-contaminated with phenol and PNP. This study suggests that constructing composite degradation circuits in an extremophile chassis may serve as a promising strategy for the remediation of extreme environments co-contaminated with multiple pollutants.