Improving DTA and BOD MICREDOX® for commercial application

MICREDOX® is a rapid microbial-based assay developed by Lincoln Ventures Ltd to monitor biochemical oxygen demand (BOD). Although originally designed for BOD monitoring, this assay was extended to perform direct toxicity assessment (DTA) of toxic chemicals on biological materials resident in the environment. Previous efforts toward optimising this assay have principally been directed at the selection and performance of different bacterial strains, either singly or as a consortium. The research presented in this thesis further investigated optimising MICREDOX® to report reproducible results that correlate with those reported by other recognised DTA and BOD methods. The miniaturised MICREDOX® DTA assay reported EC₅₀ (concentration where effect is 50%) values that were in close accord to those previously reported with other recognised methodology for a group of chlorophenols (4-chlorophenol, 2,4-dichlorophenol, 3,5- dichlorophenol and pentachlorophenol). Good correlation indices indicated that the miniaturised assay was capable of determining reproducible EC₅₀ values. The ability for this assay to report EC₅₀ values for other groups of toxicants, however, was shown to be limited due to the mediator potassium hexacyanoferrate III (HCF). Use of alternative redox mediators (hexamine ruthenium chloride II, methylene blue, neutral red, phenazine ethosulfate, resazurin, toluidine blue-O and TIRON® showed that those chosen could not report accurate bacterial respiration, and were therefore unable to be utilised as a reliable primary mediator in the MICREDOX® assay. MICREDOX® can report accurate BOD results when the glucose glutamic acid standard (GGA) is used as the substrate. apiZYM analysis indicated that preconditioning of the bacterial biocomponent in 10 mM glucose produced "favourable" exo-enzymes. These were altered upon washing. The MICREDOX® washing protocol was therefore modified to increase the retention of the "favourable" exo-enzymes and reduce the release of "putative" endo-enzymes. The re-addition of lost exo-enzymes into the MICREDOX® assay further increased the synthetic sewage standard substrate (OECD) metabolism by Arthrobacter globiformis and Bacillus subtilis. However, the presence of Escherichia coli exo-enzymes in a MICREDOX® assay was detrimental to the biocomponent, reducing its ability to metabolise OECD. In an effort to further increase the OECD metabolised by both B.subtilis and E.coli biocomponents, A.globiformis exo-enzymes were added. This procedure increased the OECD metabolism 75% of the time by E.coli, but not by B.subtilis. In order to increase protease production, the biocomponents were pre-conditioned in growth media containing complex peptides, but growth in 10 mM glucose still produced the most active protease(s). Enzyme hydrolysis of the OECD standard did not significantly increase the OECD/GGA ratio for any of the three biocomponents. To reduce the time lag associated with the current free cell biocomponent protocol, two different techniques using bacterial cell pellets and bacterial membrane fragments were investigated. A.globiformis cell pellets reported OECD/GGA ratios that were similar to those reported previously by free A.globiformis cells, whereas Klebsiella oxytoca membrane fragments reported a greater sensitivity to the standard 3,5-DCP. Overall, the manipulations to the MICREDOX® assay within this research project provided valuable information about the potential for the MICREDOX® assay to reproducibly report both EC₅₀ values and BOD readings, if the correct experimental conditions were employed