Membraneless Ethanol/O2 Biofuel Cell Using PQQ-Dependent Alcohol and Aldehyde Dehydrogenase Along with Au Nanoparticles
Authors: Aquino Neto Sidney , Milton Ross D , Hickey David P , De Andrade Adalgisa Rodrigues , Minteer Shelley D. 
Published: February 2020

Introduction
Among the various alternative technologies for efficient energy conversion, the use of enzymes immobilized onto the surface of electrodes as the main catalyst in biological fuel cells has been extensively reported [1]. The biofuel cell provide a means to obtain clean, renewable energy and have great potential for maybe in the future be used as alternative energy source for low power devices. Some key issues in the development of this device, such as lifetime, stability of enzymes, achievement of higher power densities, overcoming difficulties in the electron transfer between enzymes and electrodes, and the improvement of techniques for enzyme immobilization are still important objects of research. We have previously studied the bioelectrooxidation of ethanol using pyrroloquinoline quinone (PQQ)-dependent alcohol and aldehyde dehydrogenase (ADH and AldDH) enzymes with both direct electron transfer (DET) and mediated electron transfer (MET) mechanisms employing high surface area materials such as multi-walled carbon nanotubes (MWCNTs) and MWCNT-decorated gold nanoparticles along with different immobilization techniques [2]. Here, we investigated the use of these bioelectrodes in a complete membrane-less enzymatic biofuel cell employing bilirubin oxidase-based biocathodes that use anthracene-modified MWCNTs to allow for the electrocatalytic oxygen reduction.
Experimental
The PQQ-dependent enzymes ADH and AldDH were isolated and purified from Gluconobacter sp.33 (DSM 3504) as previously described [2]. MWCNTs-decorated gold nanoparticles were synthesized by the dendrimer-encapsulated Au nanoparticle method, in which the metallic species are firstly stabilized onto the NH2-functionalized PAMAM dendrimers, reduced with NaBH4, extracted with n-dodecanethiol, and finally supported onto the surface of MWCNTs for a final 2 wt% metal catalyst loading. DET bioanodes were prepared by immobilizing the purified enzymes onto Toray carbon paper along with the MWCNTs-decorated gold nanoparticles and Tbab-modified Nafion. MET bioanodes were obtained by a redox polymer based on ferrocene-modified linear polyethyleneimine (LPEI), which was prepared by the attachment of 3-(di-methyl ferrocenyl) propyl groups to ca.17% of its nitrogen atoms (FcMe2-C3-LPEI). Enzyme immobilization was achieved by pipetting 40 µL of a vortex-mixed solution containing 6.75 mg mL-1 of the redox polymer, 2.9 mg mL-1 purified PQQ-containing enzymes, and 0.15 vol% ethyleneglycoldiglycidylether (EGDGE) directly onto the carbon paper electrode containing MWCNTs-decorated gold nanoparticles. Bilirubin oxidase-based biocathodes were achieved by immobilizing the enzymes onto the same carbon paper support using Tbab-modified Nafion and anthracene-modified MWCNTs that orientate the enzymes for DET bioelectrocatalytic oxygen reduction. Biofuel cell tests were conducted in a single compartment cell containing 0.2M Tris–HCl buffer pH 7 and 1 mM CaCl2, controlled by a CH Instruments 611C potentiostat.
Discussion
Figure 1 show representative polarization and power curves for the membrane-less ethanol/O2 biofuel cell test in 0.2M Tris–HCl buffer pH 7, 1 mM CaCl2, and 20 mM ethanol, using the hybrid PQQ-dependent ADH/AldDH bioanode in MET architecture and the bilirubin oxidase-based biocathode. In this situation, an open circuit potential of 657 mV and maximum current and power density of 0,354 mA cm-2 and 85 µW cm-2, were obtained respectively. Complete membrane-less enzymatic biofuel cell tests employing DET bioanodes showed that this bioanode configuration still lacks of high current density, in fact, power density values as low as 12 µW cm-2were registered. Those data are mainly related to the poor electronic communication between the enzymes’ redox centers and the electrode surface, as well as the minor control of the enzyme arrangement for reaching efficient DET. In the case of the bioanodes operating via MET employing ferrocene-modified LPEI redox polymer, efficient energy conversion capability in ethanol/air biofuel cells is observed.
Acknowledgements
This work was supported by FAPESP, CNPq, and CAPES.
References
1) M. Rasmussen, S. Abdellaoui, S.D. Minteer, Biosens. Bioelectron. 76 (2015) 91-102.
2) S. Aquino Neto, R. D. Milton, D. P. Hickey, A. R. De Andrade, S.D. Minteer, Biosens. Bioelectron. 72 (2015) 247-254.
Figure 1. Polarization and power curves for the membrane-less ethanol/O2 biofuel cell test in 0.2M Tris–HCl buffer pH 7, 1 mM CaCl2, and 20 mM ethanol.

Figure 1