Evaluating use of ferricyanide-mediated respiration bioassays to quantify stimulatory and inhibitory effects on Escherichia coli populations

A number of recent studies have utilised ferricyanide as a respiratory mediator for microbial-based assays for determining water quality parameters such as biochemical oxygen demand (BOD) and toxicity. The majority of assays published to date obtain a result by determining the difference in ferrocyanide accumulation between a test sample and one or more control samples. However, a validation of the relationship between ferrocyanide accumulation and standard measures of cell density or viability has not yet been performed. To this end, a rapid microbially catalysed ferricyanide-mediated respiration (FM-RES) assay was compared with standard plate count (SPC) and spectrophotometer (OD600) measurements on a growing batch culture of Escherichia coli. Good agreement was observed between all techniques, with predictable deviations noted in different phases of the growth curves. Standardised FM-RES assays showed excellent correlations with the SPC method under controlled conditions, indicating that short-term changes in microbial activity are due to a change in per-cell respiration, rather than changes in cell numbers. The FM-RES assay was then used to observe the changes in the respiration of E. coli induced by the addition of a glucose–glutamic acid (GGA) mixture, 3,5-dichlorophenol (3,5-DCP) and Ag+ in various combinations and concentrations. Stimulation of respiration was pronounced in the presence of GGA while both 3,5-DCP and, in particular, Ag+ demonstrated inhibitory respiratory effects. The results highlight the validity and suitability of ferricyanide-mediated respiration bioassays, with appropriate modification, to monitor either stimulatory effects on microbial populations, such as occurs with BOD, or inhibitory effects, such as occurs with toxicity assays.