Using Electrochemical Techniques to Elucidate Electron-Transport Mechanisms in Microbial Anode Respiration
Authors: Yoho Rachel , Lusk Bradley , Popat Sudeep , Torres César I 
Published: February 2020

Anode-respiring bacteria (ARB) catalyze the complete oxidation of organic compounds (e.g. acetate, glucose) into electrical current and carbon dioxide.  ARB naturally produce a biofilm at the electrode surface of up to 100 micrometers, where even cells on the outer part of the biofilm are participating in current production.  The specific pathway by which ARB transport electrons intra- and extracellularly is still largely unknown.  This is largely due to the fact that many ARB have redundant respiratory mechanisms, leading to a large set of possible proteins involved in these mechanisms.  Our research utilizes a variety of electrochemical techniques in order to characterize electron transport responses from various ARB.  Our goal is to provide insights into the possible mechanisms of electron transfer from the electrochemical responses of ARB biofilms.

Through these experiments, we have observed a complex response to anode potential that allows ARB, such as G. sulfurreducens, to optimize their efficiency in electron transport.  In G. sulfurreducens, two pathways were identified for anode respiration with midpoint potentials of – 0.155 ±0.005 and –0.095 ± 0.003 V versus SHE.  G. sulfurreducens can shift between these two pathways depending on the anode potential in order to optimize its energy recovery from anode respiration.  Pathway shifts were observed within 5-20 minutes of a shift in anode potential, suggesting that this microorganism has a potential-sensing mechanism.  The fast shifts also confirm that the slow-scan cyclic voltammograms (~1 mV/s or slower) commonly performed in our field can be the result of transient responses by ARB, who can shift pathways within the experimental time.  Multiple redox pathways have also been observed in thermophile T. ferriacetica and alkaliphile Geoalkalibacter ferrihydriticus, suggesting that most ARB can have multiple anode-respiration pathways.

While the topic of electron transport is the focus of most ARB research, ionic transport is an important factor in determining rate-limiting and potential loss processes.  ARB require near-neutral pH in the medium to grow, differing from chemical fuel cells commonly employed, which run under acidic or alkaline conditions.  This pH requirement results in a major transport limitation, as H+ ions (now in mM range) should be transported from anode to cathode to achieve electron neutrality.  In an MXC anode, H+ ions accumulate in the ARB biofilm, creating an acidification that limits current generation.  This behavior has been observed with several ARB, including T. ferriacetica and Glk. ferrihydriticus.  Through these experiments, we determined that the rate-limiting protein that is responsible for the potential response of ARB is a proton-coupled reaction.  Experiments at different pH values result in shifts in the midpoint potential response of ARB, suggesting a proton coupled redox reaction.  The specific stoichiometry of the reaction is difficult to elucidate since the H+ accumulation in the biofilm causes a pH gradient.  Nonetheless, our studies provide insights into the characteristics of the rate-limiting proteins in anode respiration which are yet to be identified.