The biological significance of the measurement of redox potential

Reducing potential can be defined as the tendency of a system to gain or donate electrons, and it is a derivation of the Gibbs free energy change equation applied to redox reactions. Redox reactions are those in which chemical species change their oxidation numbers and, generally, electrons are transferred between them. The Gibbs free energy change equation is derived from the First and Second Laws of classical thermodynamics and allows calculating whether or not a certain process is thermodynamically feasible (i.e. will occur spontaneously). Living organisms ob- tain their energy for maintenance, growth, reproduction, motility and other functions from redox reactions; therefore, it is of much interest to understand and measure reducing potential.

Redox reactions can be abstractly divided into two half reactions, one for the electron donor and another one for the electron acceptor. The reducing potential associated to a redox reaction depends on the chemical species involved and their chemical activity (approximate to concentration in dilute solutions). Each chemical species has a tendency to donate or accept electrons that defines its reducing potential. Re- lative reducing potential is measured by comparing the tendency of a solution to gain or accept electrons with respect a reference electrode. The standard reference electrode is the hydrogen electrode. The reaction:

H2 → 2 H+ + 2 e-

is defined by convention as having a reducing potential equal to 0 v. However, the hydrogen electrode is not generally used in measurements because of its fragility. Instead, other electrodes like silver chloride or mercury chloride (calomel) can be used, and measured values corrected by correcting for the difference between the electrode used and the standard hydrogen electrode, to obtain values that can be reported universally.

In aerobic metabolism, oxygen is the ultimate electron acceptor and water the ulti- mate electron sink. A large amount of Gibbs free energy is released in the aerobic oxidation of carbohydrates, lipids and proteins, for example glucose:

C6H12O6 + 6 O2 → 6 CO2 + 6 H2O ΔG°’ = -2823 kJ mol-1

Part of the Gibbs free energy released can be conserved in high-energy phosphate bonds and used to fuel anabolic reactions that otherwise would be thermodynami- cally unfeasible. However, in many environments devoid of oxygen, other com- pounds with lower reducing potential are used as electron acceptors; in fact, life on Earth is thought to have originated in the absence of oxygen. Electron acceptors in anaerobic oxidation can be mineral, such as sulphate, nitrate or ferric ion. In fer- mentation, electron acceptors are carbon compounds produced during the metabolic process itself; fermentation is an intra molecular oxidation. Fermentation is important biologically (e.g. anaerobic muscle metabolism, swamps and marshes, animal guts), in agriculture (domestic ruminants, silage, biogas) and industry (cheese, yogurt, al- coholic beverages).

In the rumen, the main electron donors are carbohydrates, and some important elec- tron acceptors are carbon dioxide, formate, oxaloacetate, fumarate, pyruvate, acry- lyl-CoA, β-hydroxybutyryl-CoA, and crotonyl-CoA. The main electron sinks are methane, propionate and microbial biomass, and the main carbon sinks are acetate, propionate, butyrate, carbon dioxide, methane and microbial biomass. Some im- portant cofactor pairs involved in intracellular redox reactions are NAD+/NADH, FAD/FADH2 and oxFed/redFed. Ruminal redox dynamics also involve interspecies electron-transfer, which is principally mediated by dihydrogen, formate, succinate and lactate.

Thus, ruminal fermentation pathways compete for electrons and carbon. It is of in- terest to understand how that competition is controlled i.e. what defines VFA pro- portions and the stoichiometrically associated methane production. Stoichiometries of VFA and methane production would theoretically allow for an infinite range of ra- tios between products that in reality is never observed, as ratios between VFA keep within a narrow range. Interconversion between VFA has been proven experimen- tally by infusing or dosing labelled VFA. Appearance of labelled carbon in other VFA pools allows calculating flows of VFA interconversion. The ratio between opposite flows of VFA interconversion was within one order of magnitude of predicted equili- brium in 16 out of 19 instances for acetate and propionate and 17 out of 18 instances for acetate and butyrate. This suggests that the narrow range observed in VFA ratios may be the result of thermodynamic equilibrium. Within that range shifts in VFA pro- file caused by dietary changes may be explained by the influence of factors like pas- sage rate and pH on methanogens growth rate, which in turn affects dihydrogen concentration and determines which pathways are thermodynamically favoured de- pending on their release or incorporation of reducing equivalents.

In summary, reducing potential defines which energy-generating reactions are ther- modynamically feasible in a biological system, where energy is generated through the transfer of electrons between chemical species. This in turn dictates which or- ganisms, depending on their metabolism, can generate enough energy so as to maintain and reproduce in the system under consideration. In turn, organisms them- selves affect the system’s reducing potential through their consumption and release of metabolites, resulting in a mutual interplay of the environment and community of living organisms that shapes their simultaneous evolution.