The application of lux-marked bacteria for terrestrial ecotoxicity testing
The introduction of lux genes, able to express bioluminescence, into terrestrial bacteria enabled the optimisation of a bioluminescence-based bioassay that was environmentally relevant. Individual assay parameters such as growth phase, cell washing, lyophilisation, pH tolerance and temporal response to a range of metal and xenobiotic pollutants were evaluated. The effects of a range of pollutants upon the metabolic response of the lux-marked organisms were assessed using declines in bioluminescence. The lux -based bioassay proved more sensitive to the sub-lethal effects of metal pollutants than tests relying on culturability. Uncontaminated soils were spiked with metal and xenobiotic solutions both as single pollutants and in combination with other contaminants. Relative toxicity of metal and xenobiotic pollutants in soil systems were investigated using ecotoxicity assays based upon lux-marked constructs of Rhizobium leguminosarum biovar trifolii (an important associative nitrogen fixer) and the respiration of the microbial community. The lux-marked bioassay proved to be more sensitive than the community microbial assay to the presence of multiple contaminants at sub-lethal concentrations. The relative toxicities of metal and organic xenobiotic compounds were shown to be time dependent and better represented using chronic assaying of lux-marked microorganisms. Following a field trial involving the application of paper mill sludge to land and subsequent crop failure a rapid diagnosis of soil pollutants was required. A suite of ecotoxicity assays including lux-based bioassays, respirometry and enzyme activity were used to assess the toxicity of paper mill sludge to the soil microbial biomass. The selected lux-marked soil bacteria showed potential for use as rapid, field-based screening techniques to provide early warning of the potential hazards of waste application.