Measuring the Kinetic Isotope Effect on Microbial Electron Transport Using Deuterium Oxide

Abstract

Source: Tokunou, Y., et al, Electrochemical Detection of Deuterium Kinetic Isotope Effect on Extracellular Electron Transport in Shewanella oneidensis MR-1. J. Vis. Exp. (2018)

This video demonstrates a whole-cell electrochemical method to assess how proton transfer affects extracellular electron transport (EET) in electroactive bacteria. By replacing protons with deuterons using deuterium oxide, the method measures changes in current to reveal a kinetic isotope effect, highlighting the role of proton movement in regulating electron flow.

Protocol

1. Addition of Deuterium Water to Measure the KIE on the EET Process (Figure 1)
   1. Confirm that the current production from a monolayer biofilm of Shewanella oneidensis MR-1 is stable and does not increase rapidly. If the current increases steeply, wait until the current stabilizes within a 5% increase over 10 min.adding shuttling electron mediators such as 100 µM anthraquinone-1-sulfonate (α-AQS).
      NOTE: The formation of a monolayer biofilm on an indium tin-doped oxide (ITO) electrode without contamination of other microbial strains was confirmed by rDNA sequences and a scanning electron microscopic image of the biofilm on an ITO electrode. For confirmation of the rate limitation by the EET process, monitor the effect of adding shuttling electron mediators such as 100 µM anthraquinone-1-sulfonate (α-AQS).
   2. Add 40 µL of anoxic 50% (v/v) D₂O into the electrochemical reactor using a syringe such that the concentration is 0.5% (v/v) D₂O in the reactor.
      NOTE: To prevent damage to the biofilm by D₂O addition, inject the D₂O drop-wise.
   3. Wait for the current to stabilize and subsequently add the D₂O up to 4.0% (v/v).
      NOTE: To obtain the KIE value (the ratio of current production in the presence of D₂O and H₂O), check the effect of the same volume of H₂O addition on the current production.

Results

Figure 1: Effect of D₂O addition on the current production from a monolayer biofi...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Indium tin-doped oxide (ITO) electrode	GEOMATEC	No.0001	Used as a working electrode, 5Ω/sq
Ag/AgCl KCl saturated electrode	HOKUTO DENKO	HX-R5	Used as a reference electrode, Φ0.30mm
Platinum wire	The Nilaco Cooporation	PT-351325	Used as a counter electrode
Anthraquinone-1-sulfonate (α-AQS)	TCI	A1428
Thermal water circulator	AS ONE	TR-1A	Used for maintanance of temperature of electrochemcial reactor
Deuterium oxide (D, 99.9%)	Cambridge Isotope Laboratories, Inc.	DLM-4-PK	Additive for kinetic isotope effect experiments