Trapping of redox-mediators at the surface of Chlorella vulgaris leads to error in measurements of cell reducing power

Abstract

The reduction of the redox mediator ferricyanide, [Fe(CN)6](3-), by a range of algal and bacterial species, is frequently measured to probe plasma membrane ferrireductase activity or to quantify the reducing power of algal/bacterial biofilms and suspensions. In this study we have used rotating disk electrochemistry (RDE) to investigate the reduction of ferricyanide by the model organism Chlorella vulgaris. Importantly, we have seen that the diffusion limited current due to the oxidation of ferrocyanide, [Fe(CN)6](4-), at the electrode decreased linearly as C. vulgaris was added to the solution, even though in a pure ferrocyanide solution the algae are not able to reduce the mediator further and are simply spectator 'particles'. We attribute this effect to trapping of ferrocyanide at the cell surface, with up to 14% of the ferrocyanide missing from the solution at the highest cell concentration. The result has important implications for all techniques that use electrochemistry and other concentration dependent assays (e.g. fluorescence and colourimetry) to monitor ferrocyanide concentrations in the presence of both biofilms and cell suspensions. Analyte trapping could lead to a substantial underestimation of the concentration of reduced product.

Iron reduction by the cyanobacterium Synechocystis sp. PCC 6803

“…Other Koutecky-Levich plots at higher cell and ferricyanide concentrations, shown in Figures 2b and 2c, had intercepts of zero -this means that for these conditions, the rate of ferricyanide reduction at the cell membranes was instead mass transport controlled. These latter results are similar to that found with C. vulgaris, where mass transport was also determined to be the rate controlling step for ferricyanide reduction [29].…”

“…Increasing the concentration of ferricyanide increased interactions between cyanobacteria and ferricyanide, thereby rates of reduction and hence bulk ferrocyanide concentration also increased. These trends were also obtained previously with C. vulgaris [29].…”

“…Each experiment lasted between 3-5 minutes and three repeats were performed using the same culture. Ferrocyanide diffusion coefficients were calculated previously by the authors [29] as 6.8x10 -6 cm 2 s -1 in cell media, decreasing to 5.48x10 -6 cm 2 s -1 with 7.5x10 7 cells ml -1 present, and were adjusted for each specific cell concentration in calculations to take into account any missing fraction trapped at the cell surface.…”

“…Electrochemical measurements are carried out by placing an electrode in cell suspensions and measuring the current due to oxidation of any reduced mediator in solution [27,28]. This was the approach taken in a previous paper by the authors to measure the reduction of ferricyanide by the algal species C. vulgaris [29]. In general, electrochemical methods measure the diffusion limited current, i.e.…”

“…A linear response is expected, where the slope contains information on the mass transport limited current and the intercept gives information on any electron transfer limited current. However, in all electrochemical measurements of this type, there will be some error due to trapping of iron at the cell surface [29].…”