Ribosomal RNA-based and bioluminescence-based techniques for the monitoring of heavy metal bioavailability in ecological systems
The aim of this research was the development of a novel microbial biosensor system based on the cellular rRNA contents of bacteria. This would be used for the assessment of cadmium toxicity and bioavailability to bacteria, and compared with the more widely used lux-based microbial biosensors. Experimentation was carried out to develop an efficient and reproducible protocol for the extraction and quantification of bacterial rRNA. This enabled the determination of cellular rRNA contents during growth of Pseudomonas fluorescens. The bacteria were then grown in cadmium supplemented medium to determine if any change in growth rate imposed by the presence of the metal was reflected by a change in cellular rRNA levels. It was found that in bacteria exposed to cadmium, an increase in rRNA content in comparison to control cells was observed. If this change in rRNA content and could be shown to be dose responsive, with respect to cadmium concentration, this could prove useful for the assessment of bacterial metabolic activity in response to heavy metals. In situ hybridization cellular rRNA and quantification using confocal microscopy were also carried out within these experimental systems. The toxicity of cadmium on bacterial metabolic activity was then assessed by the use of Escherichia coli and P. fluorescens lux-marked biosensors. EC50 values for cadmium were determined for each of the bacterial species. In order to determine how the bioavailability of a metal effects its toxicity, an experiment was carried out in which the toxicity of cadmium to lux-marked E. coli and P. fluorescens was assessed in the presence of environmentally relevant concentrations of sulphate. The speciation of cadmium within each experimental system was predicted using computer program, GEOCHEM. It was shown that increasing concentrations of sulphate effectively reduced the toxicity of cadmium to the bacteria and that this was due to a reduction in the concentration of free cadmium ions within the experimental systems. It was found that using the techniques employed within this project, cellular rRNA content was of little use for the further development of a bioassay system for the assessment of bacterial metabolic activity in response to cadmium. Lux-marked microbial biosensors did, however, appear to give a reliable assessment of bacterial metabolic activity in response to the heavy metal, and also allowed the toxicity of cadmium in the presence of other inorganic ions to be determined.