The major sources for release of hydrocarbons into the environment include the effluents generated from chemical processing industries and ports. The introduction of such hazardous compounds into natural water bodies creates considerable disturbances in aquatic life and causes a threat to humans. Thus, it is essential to detect and quantify pollutants at various stages of the wastewater generation and treatment before they reach natural aquatic environments and contaminate them. This study reports the development of "biosensing strains" by cloning hydrocarbon recognizing promoter-operator and a reporter gene in bacterial strains for sensing the presence of pollutants at their lowest possible concentration. So far, various biosensing strains have been constructed with a fused promoter-operator region of the hydrocarbon degrading operons, but most of them use luxAB as a reporter gene. A novel approach in the present study aimed at constructing strains harboring two different fluorescent protein (FP)-based reporter genes for the quantification of multiple pollutants at a time. Two vectors were designed with a fusion of tbuT-gfp and phnR-cfp for the quantification of mono- and poly-aromatic hydrocarbons, respectively. The designed vectors were transformed into E. coli DH5á, and these strains were designated as E. coli DH5á 2296-gfp (containing pPROBE-Tbut-RBS-gfp-npt) and E. coli DH5á 2301-cfp (containing pPROBE-phn-RBS-cfp-npt). Both the developed recombinant strains were capable of successfully detecting mono- and poly-aromatic hydrocarbons in the range of 1-100 ìM. The sensing capacity of recombinant strains was successfully validated with actual wastewater samples against available physico-chemical analytical techniques. The development of such recombinant microbial strains indicates the future for online contaminant detection, treatment quality monitoring and protection of aquatic flora and fauna.
Biosensing Techniques Escherichia coli