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

Begin with electrochemical chambers containing a conductive electrode surface covered with a monolayer biofilm of electroactive microbes.

These microbes utilize outer membrane cytochromes to transfer intracellular electrons to the electrode via extracellular electron transport, or EET, generating current.

This electron flow is coupled with proton transfer to maintain charge balance across the membrane.

In the test chamber, introduce a small volume of deuterium oxide, or D₂O.

Deuterium replaces protons and moves slowly, reducing proton-coupled electron transfer and decreasing the current. Microbial adaptation leads to partial current recovery.

Sequentially add increasing concentrations of D₂O and monitor current changes.

In the control chamber, add an equal volume of H₂O to supply fast-moving protons that support electron flow and stabilize current.

Calculate the kinetic isotope effect, the ratio of current production in the presence of D₂O and H₂O, to confirm that proton transfer influences EET in electroactive biofilms.

Confirm that the current production from a monolayer biofilm of Shewanella MR-1 is stable and does not increase rapidly. If the current increases steeply, wait until the c