Optimisation of the biocatalytic component in a ferricyanide mediated approach to rapid biochemical oxygen demand analysis
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A novel rapid method for the determination of biochemical oxygen demand (BOD) has been developed. By replacing oxygen, the terminal electron acceptor in the microbial oxidation of organic substrate, with the ferricyanide ion, a significant increase in the rate of the biochemical reaction could be achieved. This arises from the high solubility of the ferricyanide ion (compared to oxygen); therefore allowing for elevated microbial populations without rapid depletion of the electron acceptor. Therefore, the BOD of a sample can be determined within 1-3 hours compared to 5-days with the standard BOD5 assay. A range of microorganisms were shown to be able to use the ferricyanide ion as an alternative electron acceptor for the biodegradation of a range of organic compounds in the ferricyanide mediated BOD (FM-BOD) assay. The most suitable biocatalyst in the FM-BOD method, however, was shown to be a mixture of microorganisms that was capable of degrading large amounts and types of compounds. These mixed consortia of microorganisms included a synthetic mixture prepared in our laboratory and two commercially available consortia, BODseedTM and Bi-ChemTM. When these seed materials were employed in the FM-BOD assay, the method was shown to closely estimate the BOD5 values of real wastewater samples. The linear dynamic working range of the FM-BOD method was also greatly extended compared to the standard BOD5 assay (nearly 50 times greater) and other oxygen based BOD biosensors. The immobilisation of the microbial consortia by both gel entrapment and freeze-drying methods was shown to greatly reduce the preparation and handling time of the mixed consortia for use in the FM-BOD method. Immobilisation of the mixed microbial consortium in LentiKats®, a PVA hydrogel, resulted in a marked increase in the stability of the biocatalyst. Diffusion limitations resulting from the gel matrix, however, reduced the rate and extent of the bioreaction as well as the linear dynamic working range of the method. Freeze-drying techniques were shown to circumvent some of the limitations identified with gel entrapment for the immobilisation of the mixed consortia. The freeze-dried consortia could be used off-the-shelf and demonstrated reduced diffusional restrictions. A marked decrease in the viability of the microorganisms was observed directly following the freeze-drying process and in subsequent storage. Carrageenan, however, was shown to afford a significant degree a protection to the cells during the freeze-drying process.
Thesis (PhD Doctorate)
Doctor of Philosophy (PhD)
School of Environmental and Applied Science
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Biochemical oxygen demand