Evaluation of immobilized redox indicators as reversible, in situ redox sensors for determining Fe(III)-reducing conditions in environmental samples

An in situ methodology based on immobilized redox indicators has been developed to determine when Fe(III)-reducing conditions exist in environmental systems. The redox indicators thionine (Thi, formal potential at pH 7 (E70′) equals 66 mV), toluidine blue O (TB, E70′=31 mV), and cresyl violet (CV, E70′=−75 mV) have been immobilized to 40–60 μm agarose beads via an amine–aldehyde coupling reaction. These beads were packed into a flow cell to allow spectrophotometric monitoring of the redox state of simple solutions and wastewater slurries pumped from in a bioreactor. Fe(II), a product of microbial activity, at levels observed in real systems reduces both the free (non-immobilized) and immobilized redox indicator to different degrees for samples with pH 6.5 or higher. At pH 7, immobilized Thi and TB are significantly reduced at Fe(II) concentrations greater than 0.1 and 0.3 mM, respectively. CV, with the lowest formal potential, requires Fe(II) levels in excess of 10 mM. The degree of reduction of the indicators (i.e. the fraction of indicator oxidized) observed during titrations can be qualitatively modeled with a simple equilibrium model based on ferrihydrite or lepidocrocite as the Fe(III)-solid phase. The reversibility of Fe(II)-indicator reactions was also demonstrated by showing that the reduced indicator becomes re-oxidized when Fe(II) levels decrease.