Edinburgh Napier University

  Bioluminescent microbial biosensors have become increasingly used in toxicity testing due to their apparent ease of use and potential to rapidly report on toxic events, as evidenced by the widespread implementation of such systems as the Microtox® field testing assay. However, disadvantages exist in using traditional marine-based organisms which tend to comprise the majority of commercially available sensors, including their high salt dependence and limited sensing (tolerance) range for toxicants. In order to address these problems a number of environmental pseudomonads, isolated from a highly toxic phenolic-remediating wastewater treatment system (WWTS), were genetically engineered to express a chromosomally located luxCDABE operon from Photorhabdus luminescens. Expression of this operon in Pseudomonas species has resulted in the development of a rapid, user-friendly, reproducible and reliable assay for toxicity measurement. In addition, the chromosomal location of the bioluminescence operon provides genetically stable constructs potentially minimising gene transfer through accidental release during environmental use. The developed panel of sensors encompasses a wide sensing range for phenolic pollutants (EC50 values for phenol in the range of 536-786 mg L-1) and may be used as bioindicators over a wide range of pH values without the need for buffering. During field trial analyses, the sensor panel were found to exhibit a linear response to changing phenolic concentration throughout the WWTS and displayed sufficient residual luminescence to allow them to be used in highly toxic compartments of the system . This is in direct contrast with the response of the marine bacterium Photobacterium fischeri which exhibited a non-linear response to wastewater samples and a very limited sensing range, with negligible luminescence in samples whose phenolic concentration was higher than 150-200 mg L-1. Furthermore, the luminescence responses of members of the developed sensor panel were found to accurately map changes in toxicity brought about by both a cascading pollution event which resulted in bioreactor failure, and a minor pollution event which resulted in system shutdown but not failure. These results indicate that the sensors are able to accurately predict toxicity of the major toxicants responsible for modulating microbial predict community activity and reactor process efficacy within the WWTS under investigation.