Rapid Measurement of Microbial Extracellular Respiration Ability Using a High-Throughput Colorimetric Assay

Abstract

Microbial extracellular respiration (MER) involves the transfer of electrons to extracellular substrates and has significant environmental implications. Conventional methods for MER ability determination are reagent- and time-consuming, have a low throughput, or require noncommercial instruments. In this study, a plate-based colorimetric assay is proposed to measure MER ability. This method utilizes the peroxidase activity of the key components (multi-heme <i>c</i>-type cytochromes) of the extracellular electron-transfer network. The bacterial intrinsic peroxidase-catalyzed oxidation of chromogen (e.g., tetramethylbenzidine) resulted in a measurable color change correlated with the MER ability of the tested microorganisms. The results of the proposed colorimetric assay correspond well with those of traditional methods, such as the dissimilatory Fe­(III) reduction method (Spearman’s ρ of 0.946; <i>P</i> < 0.01) and the electricity generation method (Spearman’s ρ of 0.893; <i>P</i> < 0.01). The proposed method allows researchers to identify extracellular respiring bacteria within several minutes and to measure their MER ability quantitatively by a plate-based assay