The overall goal of this protocol is to observe the contribution of proton transport to the rate of bacterial extracellular electron transport by the detection of the deuterium kinetic isotope effect. Our method reveals the impact of proton transport on the kinetics of extracellular electron transport or EET from bacteria to an electrode via outer membrane c-type cytochromes. Our assay reflects the native proton management in EET because we used living bacteria rather than purified enzyme.
However, our challenge was to remove the noises from other cellular processes. The deuterium kinetic isotope effect termed as KIE is one of the best technique to examine the contribution of proton transport on the associated electron transport process. To grow Shewanella MR-1 cells, transfer one colony of the cells grown on an agar plate into 15 milliliters of LB medium.
Grow the cells at 30 degrees Celsius for 24 hours in aerobic conditions with shaking at 160 rpm. Centrifuge the cell suspension at 6, 000 times gravity for 10 minutes. Then, resuspend the resultant cell pellet in 15 milliliters of Defined Medium, supplemented with 10 millimolar lactate as the source of carbon and 0.5 grams per liter yeast extract as trace elements for the cells.
After further cultivating the cell suspension aerobically at 30 degrees Celsius for 12 hours with shaking at 160 rpm, centrifuge again at 6, 000 times gravity for 10 minutes. Wash the resultant cell pellet twice with DM medium by centrifugation for 10 minutes at 6, 000 times gravity before the electrochemical experiment. To construct a three-electrode electrochemical reactor, put an ITO substrate as the working electrode at the bottom of the reactor.
Subsequently, insert a glass cylinder and a polytetrafluoroethylene cover and insert a platinum wire as the counter-electrode. Then, insert a silver chloride electrode into the reactor as the reference electrode. Next, add four milliliters of DM supplemented with 10 millimolar lactate and 0.5 grams per liter yeast extract into the electrochemical reactor.
After confirming that there is no leakage from the electrochemical reactor, flow the nitrogen gas into the electrochemical reactor over 20 minutes to maintain anerobic conditions inside the electrochemical reactor. Connect the electrochemical reactor to a potentiostat and apply a positive 0.4 volts to the ITO electrode, keeping the temperature of the electrochemical reactor at 30 degrees Celsius using an external water circulation system. To perform electrochemical cultivation, first adjust the cell density of the suspension to an optical density of 1.43 at 600 nanometers with DM supplemented by 10 millimolar lactate and 0.5 grams per liter yeast extract.
Add 0.3 milliliters of the cell suspension into the electrochemical reactor through the injection port using a syringe. The OD 600 in the reactor changes to 0.1. Continue the potential application at positive 0.4 volts to the ITO electrode for 25 hours.
Stop the potential application and disconnect the electrochemical reactor from the potentiostat and the water circulation system. Flow the nitrogen gas into a bottle containing DM with 10 millimolar lactate over 20 minutes to remove oxygen from the medium. Now, extremely slowly and gently, remove all the supernatant from inside the electrochemical reactor using a syringe under flowing nitrogen gas.
Then, add four milliliters of fresh DM containing 10 millimolar lactate using a syringe. Slant the electrochemical reactor to remove all of the supernatant on the wall of the reactor. Repeat these steps three times in total.
Stop the gas flow and connect the electrochemical reactor to the potentiostat again, applying a positive 0.4 volts to the ITO electrode at 303 Kelvin. 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 current stabilizes with a 5%increase over 10 minutes.
Extremely slowly and gently, add 40 microliters of anoxic 50 volume percent D2O into the electrochemical reactor using a syringe such that the concentration is 0.5 volume percent D2O in the reactor. To prevent damage to the biofilm by D20 addition, inject the D2O dropwise. Wait for the current to stabilize and subsequently add the D2O up to 4.4 volume percent to obtain the KIE value, which is the ratio of current production and presence of D2O and H2O.
Check the effect of the same volume of H2O addition on the current production. Here, the solid line is the representative result for the microbial current change induced by D2O addition. Addition of D2O sharply decreased the microbial current within 10 seconds, while H20 had almost no current decrease as shown with the dotted line.
To confirm that the observed current decrease by D2O is attributable to electron transport through outer membrane cytochromes, a diffusing electron mediator was used. Here addition of 100 micromolar alpha-AQS enhanced the current production by more than five times and the current production remained unaffected by D2O addition. These results indicate that the kinetics of the metabolic reactions are fast enough so that the extracellular electron transfer process is rate-limiting.
An alternative method for assessing this is the addition of the flavin molecule into the electrochemical reactor. An instant anodic current increase by flavin addition indicates rate-limitation by the electron transport process via outer membrane cytochromes. After watching this video, you should have a good understanding of how to examine the kinetic isotope effect on the extracellular electron transport process.
The most critical point to successfully obtain the kinetic isotope effect is to clarify and confirm the condition where the electron transport through the outer membrane cytochromes limits the rate of current production. In this video, we introduced two methods to confirm this condition, observation of current change by addition of flavin and by addition of diffusing extra mediator, such as alpha-AQS. Once the rate-limitation is confirmed, this system can be used not only for the deuterium experiment but also for the biochemical characterization of outer membrane cytochromes.
Furthermore, this protocol could be applicable to examining other electroactive microbes, as long the extracellular electron transport process is rate-limiting. Our methodology provides a basic and general technique to investigate the extracellular electron transport process via outer membrane cytochromes in vivo.
