| Abstract | A novel whole-cell amperometric biosensor was developed and capable of detecting ammonium chloride, 2,4-dinitrophenol, phenol, formaldehyde, CMPP-K, chlorotoluron and simazine at the mgl-! level. The biosensor was optimised for performance in terms of the biocatalyst, the redox mediators and the biosensor construction and operation.
Ninety-six micro-organisms were isolated from a random soil sample and screened for sensitivity to CMPP-K, chlorotoluron and simazine using radial diffusion plates containing the herbicide, replica plating on herbicide agar, a novel rapid inhibition growth method and changes in respiratory activity using an oxygen electrode.
Nine micro-organisms were found to have properties that could be used. They were identified by standard microbiological tests (optical and scanning electron microscopy, Gram stain, catalase test, oxidase test, oxidative and fermentative activity, motility and spore stain) and by modern biochemical tests (API 20 NE strip and APi 50 CHB strip). The micro-organisms were identified as Pseudomonas fluorescens, Xanthomonas maltophilia, Acinetobacter baumannii, Bacillus cereus E, Bacillus cereus F, Bacillus £121 and Bacillus H910. Two of the Gram positive bacteria could not be identified by the API 50 CHB strip and these were named Gpnsr B12 and Gpnsr G34. All bacteria were at most members of the hazard group 2 ACDP categorisation and thus, safe to work with.
A three electrode bioelectrochemical cell was constructed in the reactor configuration. Three different carbon working electrodes and four agitation devices were tested to decrease the background noise. Cyclic voltammetry was performed on nine potential redox mediators (potassium ferricyanide, resorufin, p-benzoquinone, benzy| violiogen, 2,6-dichlorophenol-indophenol, brilliant cresyl blue, phenazine ethosulphate, methylene blue and thionine) and tested with each of the nine bacteria. Three mediators (potassium ferricyanide, p-benzoquinone and 2,6-dichlorophenol-indophenol) gave responses suitable for use in the biosensor. The greatest rate of mediator reduction was achieved by optimising the mediator concentration and time of biocatalyst sampling for each microorganism. Benzoquinone exerted a toxic effect on all the bacteria except the Bacillus cereus Strains.
The response of each combination of mediator and micro-organism when insulted by the pollutants was monitored using the biosensor. Measurement was based on a differential process where the rate of mediator reduction was compared before and after the addition of pollutant. Where possible, the limit of detection and concentration that caused either a 50% or 25% change in mediator reduction was calculated.
Permeability of the bacteria was artificially increased using either a lysozyme-EDTA preparation or | % (v/v) Tween 20. This treatment had the effect of increasing mediator reduction by some bacteria and decreasing mediator reduction by others. Several bacteria had increased sensitivity to the herbicides following permeation.
The responses of the bacteria to herbicides when monitored by respiratory activity measured on the oxygen electrode presented the possibility of distinguishing one herbicide from the other three herbicides. This was also possible using the mediated amperometric biosensor but much more complex. |
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