Rational Combination of Promiscuous Enzymes Yields a Versatile Enzymatic Fuel Cell with Improved Coulombic Efficiency

Abstract

Enzymatic fuel cells (EFCs) utilize enzymatic catalysts to convert chemical energy to electrical energy, typically by performing a 2e − oxidation of saccharides. In the case of sugars, a single 2e − oxidation does not fully exploit this energy-dense fuel that is capable of producing 24e − from its complete oxidation to CO 2 . Here, we propose an efficient approach to design a versatile EFC that can produce electrical energy from 12 (oligo)saccharides by combining two enzymes that possess diverse substrate specificities: pyranose dehydrogenase (PDH) and a broad glucose oxidase (bGOx). Additionally, PDH is able to perform single or two sequential oxidations of glucose (at C2 and/or C3) yielding up to 4e − , whereas bGOx only performs a single 2e − oxidation at the anomeric (C1) position. By combining PDH and bGOx, we demonstrate the ability to achieve deep oxidation of glucose and xylose, whereby each is able to undergo sequential oxidations by PDH and bGOx. Additionally, we demonstrate that this deep oxidation can yield improved performances of EFCs. For example, an EFC comprised of a bi-enzymatic PDH/bGOx bioanode using xylose as a fuel yields a maximum current density of 586 ± 3 μAcm −2 whereas mono-enzymatic PDH or bGOx EFC bioanodes result in current densities of 440 ± 4 μAcm −2 and 120 ± 1 μAcm −2 , respectively. Owing to their high selectivity, enzymatic fuel cells (EFCs) are devices that offer multiple advantages over traditional fuel cell systems (i.e. H 2 /O 2 ) such that they are able to operate under physiological temperature and pH and are able to produce electrical energy from common energy-dense fuels, such as glucose. [1][2][3][4][5] In contrast to traditional fuel cells that operate on precious metal catalysts, EFCs employ enzymes as biocatalysts at the anode and cathode. In addition to mild operational requirements, inherent substrate specificity of enzymes can allow for the operation of EFCs in the absence of a membrane separator between the anodic and cathodic compartments. 6 Theoretically, the complete oxidation of a single glucose molecule to CO 2 yields 24e − that could be harnessed within an EFC. 7 While many examples of high power EFCs can be found, [8][9][10][11][12] the vast majority of glucose EFCs report only a single 2e − oxidation, thereby operating at <10 % efficiency. In the recent past, alternative enzymes to the commonly used glucose oxidase (GOx) for glucose oxidation have been explored, namely flavin adenine dinucleotide-dependent glucose dehydrogenase (FAD-GDH), 8,[13][14][15] nicotinamide adenine dinucleotidedependent glucose dehydrogenase (NAD-GDH), [16][17][18] pyrroloquinoline quinone-dependent glucose dehydrogenase (PQQ-GDH), 19,20 cellobiose dehydrogenase (CDH), 21,22 pyranose oxidase (POx) 23 and pyranose dehydrogenase (PDH).24-27 FAD-GDH and CDH, which both oxidize their substrates at the anomeric (C1) position, are promising alternatives to GOx, because they do not utilize molecular oxygen as their electron acceptor, thus bioanode efficiency is retained in the presenc...

Recent advances in the electrooxidation of biomass-based organic molecules for energy, chemicals and hydrogen production

“…33,[281][282][283][284][285][286] Furthermore, since most oxidase and dehydrogenase enzymes only catalyze twoelectron oxidation of substrates, enzyme cascades appear to be a solution for a deep oxidation and even complete oxidation of fuels, which require advancements in scaffolding for substrate channeling. 274,282,[287][288][289] The majority of bioelectrocatalysts (GOx, GDH, CDH, FDH) is limited to specific substrates. Minteer's group reported a commercial genetically modified GOx (Amano Enzyme Inc., Japan) with enhanced promiscuity that is capable of oxidizing multiple mono-, di-, and poly-saccharides.…”

Direct enzymatic bioelectrocatalysis: differentiating between myth and reality

“…We recently demonstrated an MET-type bioelectrode that simultaneously employed two oxidoreductases, where heatdenatured proteins were prepared to confirm the bioelectrochemical activity of each counterpart [105]. An alternative to heat denaturation would be to digest the oxidoreductase with a promiscuous protease (such as trypsin).…”

Efficient Biomass Fuel Cell Powered by Sugar with Photo‐ and Thermal‐Catalysis by Solar Irradiation

“…2) In enzymatic glucose fuel cells (EFCs), the power density only reaches 0.003–0.16 mW cm −2 ; the glucose oxidation reaction generally involves a two‐electron transfer process to form gluconolactone when glucose oxidase is used as the enzymatic catalyst, which means a low coulombic efficiency of 8.3 %; the coulombic efficiency can be improved to 90 % by mixing multi oxidase enzymes, but the power density is still as low as 0.16 mW cm −2 . 3) For microbial glucose fuel cells (MFCs) in which living microorganism cells are used, the glucose can be completely oxidized into CO 2 , but the power density is very low (usually only 0.003 to 0.6 mW cm −2 ) . 4) For alkaline dye‐mediated glucose fuel cells, the glucose is partially oxidized and the coulombic efficiency was measured as 37 % when methyl viologen (MV) was used as electron shuttle in the fuel cell .…”

“…The performances of four types of glucose fuel cells recently reported are summarized in Figure C. 1) For direct glucose alkaline fuel cells (DGAFCs) where noble metal catalysts are used, the power density is in the range from 0.9 to 38 mW cm −2 but the electron utilization efficiency (coulombic efficiency) of glucose is low as mentioned above . 2) In enzymatic glucose fuel cells (EFCs), the power density only reaches 0.003–0.16 mW cm −2 ; the glucose oxidation reaction generally involves a two‐electron transfer process to form gluconolactone when glucose oxidase is used as the enzymatic catalyst, which means a low coulombic efficiency of 8.3 %; the coulombic efficiency can be improved to 90 % by mixing multi oxidase enzymes, but the power density is still as low as 0.16 mW cm −2 . 3) For microbial glucose fuel cells (MFCs) in which living microorganism cells are used, the glucose can be completely oxidized into CO 2 , but the power density is very low (usually only 0.003 to 0.6 mW cm −2 ) .…”