Redox Coupling to Microbial Respiration: an Evaluation of Secondary Mediators as Binary Mixtures with Ferricyanide

Abstract

MICREDOX is a rapid microbial-based assay, which was originally developed at Lincoln Technology to monitor biochemical oxygen demand (BOD).[1] The assay is characterized by high levels of biocatalyst (microorganisms) and redox mediator; these facilitate a fast reaction in which the microbial oxidation of an organic substrate is coupled with the reduction of the mediator. Previous efforts toward optimizing this assay have principally been directed at the selection and performance of different bacterial strains, either singly[2] or as a consortium.[3] Here we report the effect of adding a second mediator to the assay by comparing the coulombic responses and the substrate stoichiometric conversion efficiencies. Escherichia coli and half-strength standard BOD substrate (75 mg glucose L−1, 75 mg glutamic acid L−1; GGA) were incubated for one hour at 37°C in the absence of oxygen with an excess of redox mediator, potassium hexacyanoferrate(iii) (HCF), either individually or as a binary mixture that included a secondary mediator. Secondary redox mediators investigated were p-benzoquinone (BQ), 2,6-dichlorophenolindophenol (DPIP), menadione (MD), neutral red (NR), N,N′-tetramethyl-1,4-phenylenediamine (N-TMPD), phenazine ethosulphate (PES), and 2,3,5,6-tetramethyl-1,4-phenylenediamine (TMPD). When HCF was supplemented with TMPD or PES the stoichiometric conversion efficiency for the oxidation of the GGA by E. coli increased, whereas when supplemented with N-TMPD, MD, DCIP, or BQ the conversion efficiency decreased. No effect was evident when NR was used as the supplement.