Microbial biosensors for the selective measurement of targeted environmental contaminants

Abstract

The inclusion of bacteriological components, with well characterised capabilities to recognise specific compounds or groups of compounds, underpins the development of many successful compound detecting biosensors. Certain bacteria have acquired the ability to catabolise otherwise potentially toxic aromatic compounds such as the BTEX compounds (benzene, toluene, ethyl benzene and the three isomers of xylene), poly aromatic hydrocarbons (PAH's) such as naphthalene and compounds based on the biphenyl structure. The catabolism of these compounds is generally initiated by a number of biochemically and genetically well characterised initial oxygenase enzymes and these oxygenase enzymes make ideal biocomponents in aromatic detecting whole cell biosensors due to their specificity, high stability and high activity levels in whole cells.

The biosensing approach for specific contaminant detection tested in this thesis was based on the production of two isogenic but phenotypically different bacterial strains of which only one strain expressed the enzymes required for catabolism). This strain pair was subsequently exposed to the target analyte of interest and differences in catabolic enzyme activity [due to the expression (or not) of the catabolic pathway] between these two strains were used to generate the analytical signal. The use of a differential signal allowed the discrimination between pathway and non-pathway responses to the target analyte.

To allow multiple simultaneous sample measurements, catabolic enzyme activity was measured using a redox active dye, resazurin, and a commercial multi well plate containing an oxygen sensitive fluorophore, BD Oxygen Biosensor ™ Plates. Unlike other growth and cytotoxicity assays, high cell densities were used to increase the rate of reaction and also to avoid cell adaptations that effectively displace available energy and carbon away from catabolism. Maximum rates of resazurin reduction and oxygen uptake were derived for four highly characterised toluene degrading strains (Pseudomonas putida mt-2, Burkholderia cepacia G4, Pseudomonas putida F1 and Pseudomonas mendocina KRl) in the presence or absence of toluene. These rates were used to determine the minimum concentration of the target analyte that could be detected relative to a no carbon control. Detection limits achieved with these two approaches were similar to those achieved with traditional oxygen electrode approaches and recombinant gene constructs.

The specificity of the assay was checked using a number of alternative aromatic compounds, a complex aromatic sample and a cyclic but nonaromatic compound. Alternative catabolic strains including Pseudomonas species strain CB406 and Burkholderia species RP007 were tested along with a range of non-catabolic capable strains (Pseudomonas putida PaW340, Escherichia coli K12 and Pseudomonas putida S12) for their response to a range of aromatic compounds. The contribution of the initial and ring cleavage oxygenase enzymes to the oxygen uptake signal was estimated compared to the contribution of the terminal electron accepting use.

The biosensing approach tested, showed potential to be used in a number of research settings. One area identified was its potential in bio-prospecting applications where high throughput screening of environmental samples is used to isolate oxygenase producing strains. The biosensor developed could also find application in monitoring the total remaining BTEX fraction in environmental samples after a BTEX contamination event.