A lux-based bioassay of heavy metal contamination of organic wastes
The luxCDABE genes (i.e. full cassette) were inserted into the bacterial strain used in this study, E. coli HB101, using a multi copy plasmid, (pUCD607). A number of experiments were carried out in this thesis to study the potential of using the biosensor, E. coli HB101 (pUCD607), for ecotoxicity testing. Growth and bioluminescence of E. coli HB101 (pUCD607) were characterised and optimised, as well as the stability of this biosensor to a range of environmental parameters. The biosensors were found to be sensitive to a range of pollutants and provided a highly consistent bioluminescence response under conditions likely to be encountered in environmental toxicity testing. Assessment of the potential of the lux-based bioassay for revealing the combination of toxicities of metals was carried out. This is necessary because samples in ecotoxicity testing may contain a number of types of pollutants. The biosensors showed high sensitivity of response to mixed metals and identified the combined toxicity of the mixture. Biosensor bioluminescence could also be used to diagnose the distinction between toxicity after 15 and 30 minutes exposure. Results suggested that exposure time is an important factor affecting on the toxicity of metals in mixtures. The results from challenging lux-based biosensors with heavy metals with various concentration of Cl- demonstrated the effect of Cl- ion complex formation for metals on the bioluminescence of E. coli HB101 (pUCD607). The bioluminescence response of the biosensor also related to the results from computer modelling (GEOCHEM) and was used to diagnose the effect of Cl- on metal toxicity. The results showed that E. coli HB101 (pUCD607) is sensitive to complexes of metals such as are formed by Cl- ion. Immobilised cells of E. coli HB101 (pUCD607) were used to investigate the toxicity of metals, both singly and in mixture. This work aimed to assess the potential of immobilised cells, as an alternative form of biosensor, for use in ecotoxicity testing. Bioluminescence response of immobilised cells varied with the test solution pH and showed that it has great potential for use in low pH environments. Immobilised cell biosensors demonstrated sufficient sensitivity to identify the toxicity of individual and mixed metals. High levels of bioluminescence of immobilised cells lasted up to 5 hours after bead production, suggesting that biosensor immobilisation increases the flexibility of the toxicity assay.