Complete+Oxidation



=The Complete Oxidation Problem=

Glucose powered enzymatic fuel cells have been plagued by low power densities due to their incomplete oxidation of glucose. Most use glucose dehydrogenase or glucose oxidase (GOX) to oxidize glucose to gluconolactone.

This generates 2 electrons out of a possible 24 electrons that can be liberated from glucose.[1] The chart to the right compares the energy density of a one step oxidation vs complete oxidation of common fuels used in enzymatic fuel cells. Glucose has the highest energy density close to 7000 Whr/L upon complete oxidation, but has the lowest energy density of all the examined fuels upon a single oxidation. This makes it clear that if glucose is to be a viable fuel for enzymatic fuel cells the issue of deep and complete oxidation must be dealt with.[1][2]

=Why not mimic the metabolic pathway?=

The natural pathway of oxidizing glucose to CO2 utilizes 19 enzymes of which only six are those that are oxidoreductase.[1] Oxidoreductase enzymes are those enzymes that catalyze electron transfers; It is these oxidoreductase enzymes that produce the useful electron flow. This translates into a very low oxidoreductase/ nonoxidoreducatse enzyme ratio, and the power density produced per unit area of electrode would be negatively affected. The figure to the right and the one below outline the main oxidation pathway of glucose in living organisms. Glucose is first broken down into pyruvate through a ten enzyme cascade known as glycolysis (schematic to the right). Out of the ten enzymes that transform glucose into pyruvate only one enzyme, glyceraldehyde 3-phosphate dehydrogenase, is an oxidoreductase enzyme. The remaining nine enzymes, although necessary for metabolism and inducing chemical transitions, do not produce electrical energy.[1] If the natural glycolysis pathway is utilized very poor power densities would be inevitable.

Upon Pyruvate formation the next step in the metabolic pathway is the Kreb`s or citric acid cycle. The cycle, shown in the diagram below, is a nine enzyme cascade in which all of the carbons and oxygens of pyruvate end up as carbon dioxide and water. Of the nine enzymes of the citric acid cycle five contribute to the release of electrons. Although this results in a much greater power density than the glycolysis stage the citric acid cycle is unnecessarily complex. Enzymes that catalyze the reaction pathway have widely varying enzymatic activities that can lead to bottlenecks at various stages in the cycle.[1] In addition, some of the enzymes require the oxidized byproducts of other biofuels to regenerate and extract electrons from the system. Taking into account the bottleneck problem as well as the need for the introduction of other biofuels or their oxidized byproducts the system quickly becomes very complex. This once more leads to poor power densities and leads to the conclusion that the metabolic pathway for the oxidation of glucose is not suitable for enzyamtic biofuel cell applications.



=A Non-natural Six Enzyme Cascade that Converts Glucose to CO2=

A recent publication by S. Xu et al. (University of Utah) demonstrated how the full potential of glucose could be unlocked through the use of a non-natural six enzyme cascade. The working bio-fuel cell they produced consisted of an air-breathing platinum cathode separated from the anolyte by a Nafion 212 PEM. The enzyme cascade was immobilized on a Toray electrode with tetrabutylammonium bromide(TBAB)-modified Nafion polymer. As fuel the anodic compartment of the cell contained 100 mM 13C-labeled glucose in pH 7.2 phosphate buffer.[3]



The six enzyme cascade can be divided into three steps:
 * Step1:** Use two-enzymes cascade extracted from //Gluconobacter sp//. (pyrroloquinoline quinone (PQQ)-dependent glucose dehydrogenase and PQQ-dependent 2-gluconate dehydrogenase) to oxidize glucose to gluconolactone and then glucuronic acid.


 * Step 2:** Cleave the ring structure of glucuronic acid with an aldolase from //Sulfolobus solfatricus// to form two smaller molecules, glyceraldehydes and hydroxypyruvate.[3][4]


 * Step 3:** Three enzyme cascade utilizes alcohol dehydrogenase, aldehyde dehydorgenase and oxalate oxidase to complete the oxidation to CO2. This cascade had been earlier developed by at the same university during their study of glycerol and its complete oxidation.[5]



Comparing the six-enzyme cascade to the two-enzyme cascade of the first step a 46.8-fold power density increase and a 33.9-fold current density increase is observed. The graph below shows power curves for the two-enzyme cascade (red) and the six-enzyme cascade (black).

It is clear that the complete oxidation of glucose is needed if glucose enzymatic fuel cells are to be utilized. Although the complete oxidation of other bio-fuels is being examined there is a lack of literature on the complete oxidation of glucose. Nevertheless, glucose, upon complete oxidation, still has one of the highest energy densities and remains a promising fuel for enzymatic bio-fuel cells application.

= = =References:=
 * 1) Sokic-Lazic, D.; Arechederra R.L, Treu B.L, Minteer S.D. 2009. Oxidation of biofuels: fuel diversity and effectiveness of fuel oxidation through multiple enzyme cascades. Electroanalysis 22: 757-764.
 * 2) Miyake T, Haneda K, Nagai N, Yatagawa Y, Onami H, Yashino S, Abe T, Nishizawa M. 2011. Enzymatic biofuel cells designed for direct power generation form biofluids in living organisms. Energy Environ. Sci. 4: 5008-50012.
 * 3) Xu S. Minteer S.D. 2012. Enzymatic biofuel cell for oxidation of glucose to CO2. ACS Catal. 2: 91-94.
 * 4) Buchanan C.L, Connaris H, Danson M.J, Reeve C.D. 1999. An extremely thermostable aldolase from sulfolobus solfataricus with specificity for non-phosphorlyated substrates. Biochem. J. 343: 563-570.
 * 5) Arechederra R.L. Minteer S.D. 2009. Complete oxidation of glycerol in an Enzymatic biofuel cell. Fuel Cells. 9: 63-69.