Enzymatic Biofuel Cells by Plamen Atanassov, Chris Apblett, Scott Banta, Susan Brozik, Scott Calabrese Barton, Michael Cooney, Bor Yann Liaw, Sanjeev Mukerjee, and Shelley D. Minteer hat would it be like if you are also incredibly abundant and also green, and can be grown in could recharge your cell cheap to produce, something that the quantity whenever they are needed, Wphone battery instantly by wine and detergent industries have as opposed to the metal catalysts, pouring your soft drink into it? known for decades. But applying which need to be mined and Such applications may be a long way enzymes to power electronics is very purified using expensive and less off, but the U.S. Air Force Office of different from getting enzymes to environmentally friendly processes. Scientific Research is investing in clean our clothes. For one, enzymes They are also “selective,” a word such a future now. Under a Multi do not like to give up electrons as that scientists use to describe an University Research Initiative, easily as metal catalysts do, which enzyme’s ability to work with a very university professors from around the means that generating an electric specific fuel, and only that fuel, so country are now focused on a five- current from enzymes is much that the byproduct of one oxidation year research program to look at the tougher. Enzymes can be made step could be the fuel for another technical challenges surrounding a to give up their electrons with enzyme. By doing this step, enzymes fuel cell that will run on such simple mediators, but using mediators can could conceivably reproduce what sugars as those found in our everyday cause other problems in the fuel cell. animals already know how to do: foodstuffs. Enzymes also are not used to staying convert the sugars into just water The challenges are great. Most put. In animals, enzymes are floating and carbon dioxide. After all, there fuel cells in the world today run on freely in the cells of the body, but is as much energy in one jelly donut hydrogen. However, as the fuel gets to work in a fuel cell, they have to as you can find in 77 cell phone more complex, this oxidation process be put in a specific place and stay batteries. If you can get that energy becomes vastly more complicated. there, a process that scientists call out, it could have pretty, ahem, sweet Once carbon atoms are in the fuel, immobilizing the enzymes. Finally, consequences. The basic enzymatic biofuel cell contains many of the same components as a hydrogen/oxygen fuel cell: an anode, a cathode, and a separator. However, rather than employing metallic electrocatalysts at the anode and the cathode, the electrocatalyst used are oxidoreductase enzymes. This is a class of enzymes that can catalyze oxidation–reduction reactions. Since these enzymes are selective electrocatalysts, the separator could be an electrolyte solution, gel, or polymer. Figure 1 shows a schematic of a generic biofuel cell oxidizing glucose as fuel at the bioanode and reducing oxygen to water at the biocathode. Biofuel cells were first introduced FIG. 1. Generalized schematics of an enzyme biofuel cell consisting of an anode, catalyzed by in 1911 when Potter cultured yeast oxidases suitable for conversion of fuels of choice or a complex of such enzymes for a complete and E. Coli cells on platinum oxidation of biofuels. The cathode usually features an oxidoreductase that uses molecular oxygen as electrodes,1 but it was not until 1962 the ultimate electron acceptor and catalyzes reduction to water in neutral or slightly acidic media. that the enzymatic biofuel cell was invented employing the enzyme carbon monoxide poisoning of naturally occurring enzymes do not glucose oxidase to oxidize glucose at typical fuel cell catalysts becomes last that long. A typical enzyme in the anode.2 Over the last 45 years, problematic. Researchers are turning the human body lasts only a couple many improvements have been to the natural world in an effort of days, but to be effective in a laptop made in enzymatic biofuel cells and to see how sugars are oxidized by or an automobile, an enzyme is going those can be found in several review animals to produce power. to have to last for months or years articles.3-6 However, there are still Using enzymes (nature’s catalysts) before needing replacement. several main issues to consider with seems to be the answer, since they do The benefits of the technology biofuel cells. These include short not suffer from the contamination are as big as the risks, however. active lifetimes, low power densities, problems that more traditional Enzymes, as we mentioned before, and low efficiency due to normally metallic catalysts suffer from. They are cheap and plentiful. They are only incorporating a single enzyme 28 The Electrochemical Society Interface • Summer 2007 to do a partial oxidation of complex Electron Transport nanotube-modified porous matrix biofuels. There are a number of shows that we can make an anode strategies for solving these problems, The key issue in biofuel cells, as operational at about 400 mV (vs. Ag/ but our group is focused on strategies well as in any other type of low- AgCl).11 This means that we can start for immobilization and stabilization temperature fuel cells, is the catalysis “burning” the fuel at a potential very of the enzymes, engineering of of electrode processes. Oxidation of close to the one provided to us by the enzymes to function optimally at the fuel, let say glucose, catalyzed thermodynamics of the system (see electrode surfaces, electron transport by enzymes such as glucose oxidase Fig. 2). between the enzyme and the current (the biosensor’s favorite enzyme of all When we combine this with collector, optimization of multi- times) may be accomplished directly direct reduction of oxygen, which enzyme systems, and developing or may involve redox mediators. is catalyzed by copper-containing standardized characterization Direct enzymatic oxidation requires enzymes (laccases, bilirubin protocols for the biofuel cell research that the active site of the enzyme oxidases, or ascorbate oxidases) at community at large. communicates immediately with a potential just 50 mV below the the electrode surface. That seemed thermodynamic value,12 this gives us Enzyme Immobilization and Stabilization rather impossible for the best beloved the option of making a biofuel cell glucose oxidase because it flavin- with as high open circuit voltage as One strategy for enzyme type active site is “buried” too deep 1 V. For all this to happen, a lot of immobilization and stabilization has in the protein “shell” of the quite things need to be fine-tuned, the been the use of micellar polymers. large enzyme molecule. Sandia enzyme surface interactions most Enzymes in solution are typically National Laboratories addresses of all. Charge transfer processes and active for a few hours to a few this issue by genetically modifying enzyme orientation are of immense days. This lifetime can be extended to 7-20 days by entrapment in hydrogels and binding to electrode surfaces.4 However, researchers at Saint Louis University have extended active enzyme lifetimes at electrode surfaces to greater than one year by immobilization within hydrophobically modified micellar polymers.7-9 Micellar polymers, such as, Nafion and chitosan, can be hydrophobically modified to tailor the micellar pore or pocket structure to be the optimal size for enzyme immobilization, while also ensuring a hydrophobic and buffered pH microenvironment for optimal enzyme activity. This strategy has been shown both to increase the active lifetime of the enzyme, but also to increase the enzymatic activity of IG 10 F . 2. Principles of biofuel cell design indicting the maximum oxidation potentials for glucose the enzyme by up to 2.5-fold. and the corresponding thermodynamic potential for oxygen reduction at neutral pH. Redox The appropriate design of the potentials of several enzymes and their corresponding co-factors are shown along with the enzyme–electrode interface is a key potential “zone” containing the redox potentials of the usual mediators. Polarization curves depict consideration for the creation of typical current performances for direct and mediated electron transfer in biofuel cell electrodes. new bioelectrochemical systems like biofuel cells and biosensors. These systems generally involve complex the enzyme to make its active site importance in direct electron transfer. arrangements of immobilization more accessible for communication The rest is “simple” engineering of polymers, redox mediators, and with the electrode. A lot of mutant the materials, which should allow us enzymes that must interact with proteins can be made in a day. to draw as much current as possible. the electrode substrate. At Columbia Knowing which one will work is a big There is indeed another way: we University, researchers are using part of the problem. Fast screening can use mediators. These are redox protein engineering to design of genetically modified enzymes for couples that can easily communicate multifunctional proteins and their electrocatalytic properties is the with both the enzyme and the peptides that can both simplify and task of a collaborative effort between electrode. The use of mediators improve enzymatic electrodes. To Sandia National Laboratories and the should be well measured because if this end, they are creating proteins University of New Mexico. their potential is too close to that of that can self-assemble into bioactive Researchers at the University of New the enzyme, the driving force of the hydrogels with redox enzyme Mexico are also looking at the problem enzyme/mediator “cascade” will be activities. This eliminates the need of enzyme/electrode interactions too low. If their potential falls too far for the incorporation of polymers from a different angle: let us leave the from that of the enzyme active site, in the system.