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Enzymatic 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 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 ’s ability to work with a very university professors from around the means that generating an electric specific , 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 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 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 . 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 , a , and a separator. However, rather than employing metallic at the anode and the cathode, the 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 , gel, or . 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 of choice or a complex of such enzymes for a complete and E. Coli cells on oxidation of . The cathode usually features an oxidoreductase that uses molecular oxygen as ,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 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 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 “burning” the fuel at a potential very of the enzymes, engineering of of 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 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 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 . large enzyme . 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, 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. In addition, peptides enzyme alone, there are things that the voltage of the cell will suffer (see that can bind redox mediators we can do with the electrode itself. Fig. 2). A group at Michigan State are also being engineered. This Nanostructured materials, specifically University is studying these effects by approach carbon nanotubes, could be of use. screening a library of redox mediators should dramatically simplify the At least they are comparable in size (based on osmium complexes) that fabrication, characterization, with the enzyme and their defects can assist both cathode and anode and reproducibility of the are probably a good part to interact processes. The redox potential of bioelectrocatalytic interface with the active site directly. Direct those mediators is tunable by slight employed in future devices. oxidation of glucose by glucose modifications in the chemical oxidase immobilized in a carbon (continued on next page)

The Electrochemical Society Interface • Summer 2007 29 Atanassov et al. between enzymes, and handling cell configurations that can be shared (continued from previous page) the fact that the enzymatic among laboratories to conduct activity of each enzyme is not comparative research work to facilitate structure of the organic ligand. They equivalent, so there are rate limiting the sharing and transfer of knowledge are all deployed as side chains of a enzymes within the system. The among collaborators. By using a redox polymer that forms a hydrogel researchers at Saint Louis University common geometry and dimensions, the on the electrode surface. Transport are employing click chemistry electrical field and reactor geometry is processes in those hydrogels and in to form enzyme complexes to maintained among experiments. The the porous corrugated media of the decrease transport limitations and results then can be compared with an enzyme biofuel cell electrode (think developing enzyme immobilization assumption of common geometry and of it as a sponge) is a topic of its membranes for ensuring the field to eliminate ambiguities around own. The deep understanding of the appropriate microenvironment, but these issues, so kinetic measurements transport effects on kinetics will give the electrochemists are turning to can be assessed. Figure 3 is a picture the team the means to address those metabolic engineers to learn how of the standardized cell configuration “simple” engineering problems and to to determine rate limiting enzymes being employed. produce as much current as possible within the system. from the bioenzymatic electrodes. Nature has evolved complex Conclusions The numbers are now moving from metabolic networks to extract µA to mA/cm2. from ambient fuel Although enzymatic biofuel cells sources. The enzymes involved in still have lifetime, , Complete Oxidation of Biofuels these networks have evolved to exhibit and efficiency issues that are a distributed control over the flux of currently being addressed, they have One of the main problems materials through these pathways so several attractive points including: plaguing enzymatic biofuel cells that no single node in the network the ability to operate optimally has been low energy densities due represents a dominant bottleneck. at temperatures between room to incomplete oxidation of biofuels Recently, there have been exciting temperature and body temperature; at the anode of the biofuel cell. The advances in the bioelectrochemical the flexibility of fuels that can be arena, as multi-enzyme systems are employed, including renewable being created for applications such fuels (e.g., , glucose, sucrose, as biofuel cells and biosensors. But, glycerol) and traditional fuels (e.g., these systems are often created using hydrogen, , etc.); and the enzymes from different organisms that use of non-platinum renewable did not evolve to function together catalysts. The two main application and are not optimized to function areas that are being considered for under in vitro conditions. This can enzymatic biofuel cells are in vivo, result in the creation of metabolic implantable power supplies for pathways where the kinetic control of sensors and pacemakers and ex vivo the system is not well-distributed, and power supplies for small portable a single node can dominate the overall power devices (wireless sensor performance. At Columbia University, networks, portable electronics, etc.). researchers are applying metabolic The implantable devices would most control analysis to better understand likely employ glucose as a fuel and and improve the kinetic performance recent advances by the Heller group of biofuel cells. These insights can are showing that biofuel cells can be be used to ensure optimal operating implanted and continue to function conditions as well as to drive the in a living organism (a grape).14 choice of enzymes that should be used Fig. 3. Standardized stack cell design for the For ex vivo applications, many biofuel cell. in these artificial metabolic networks. fuels are being considered, from alcohols to sugars. Figure 4 shows Standardization of an 8-cell ethanol/oxygen biofuel only example of complete oxidation Characterization Techniques cell stack prototype operating an in an enzymatic biofuel cell is for iPod. The optimal fuel choice will the oxidation of methanol using Due to the intricacy and sensitivity be a function of application for , aldehyde of enzymes toward the electrode these systems. All in all, biofuel dehydrogenase, and formate fabrication and modification, cells are a early stage technology 13 dehydrogenase. With complex characterizations of biofuels like glucose and sucrose, enzyme performance the enzymatic metabolic pathways are particularly are far more complex and contain difficult for cross both oxidoreductase enzymes platform comparison. and other enzymes (kinases, A possible solution is etc.) responsible for chemical to use standardized transformations. The cellular cell configurations pathways responsible for enzymatic and characterization breakdown of sugars are the techniques to allow glycolysis pathway and the Kreb’s such comparison to cycle. Mimicking these cellular the best faith. A team pathways at an electrode requires at the University both the ability to immobilize them of Hawaii has been in appropriate microenvironments engaged in such for enzyme activity, minimizing an endeavor to Fig. 4. Ethanol/air biofuel cell stack. This prototype, developed transport limitations associated design and develop by Akermin, Inc. in 2006, powers an iPod. Photograph courtesy of with oxidized products diffusing suitable modular Akermin, Inc.

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with fundamental scientific and engineering hurdles to overcome, but About the Authors they are a promising technology for certain applications. n The authors are all members of a U.S. Air Bor Yann Liaw is a specialist at the Force Office of Scientific Research MURI University of Hawaii. His research Acknowledgments project on biofuel cells. interests are in electrochemical power systems, including advanced batteries, Plamen Atanassov is an associate micro-fuel cells, and ultracapacitors, The authors would like to thank the professor of at especially in modeling, characterization, U.S. Air Force Office of Scientific the University of New Mexico. His and microsystem integration. He may be Research for funding. research interests are in materials design reached at [email protected]. of electrocatalysts for fuel cells, bio- References electrocatalysis and its applications Sanjeev Mukerjee is a professor in the in biosensors and biofuel cells, and department of chemistry and chemical 1. M. C. Potter. Proc. Royal Soc., B84, in electrochemical microfabrication biology at Northeastern University. His 260 (1911). technologies that allow integration of research interests are electrocatalysis, 2. Y. B. Davis and H. F. Yarborough, sensors and power sources in microsystems transport phenomenon in solid Science, 137, J. Kim, H. Jia, and P. devices. He may be reached at plamen@ state , in situ synchrotron Wang, Biotech. Advances, 24, 296 unm.edu. spectroscopy, MEMS devices for micro-fuel (2006). cells, and sensors. He may be reached at 5. R. A. Bullen, T. C. Arnot, J. B. Scott Banta is an assistant professor [email protected]. Lakeman, and F. C. Walsh, Biosensors of chemical engineering at Columbia and Bioelectronics, 21, 2015 (2006). University (New York). His research group Chris Apblett is a principal member 6. S. D. Minteer, B. Y. Liaw, and M. J. uses protein and metabolic engineering of technical staff at Sandia National Cooney, Current Opinion in Biotech, in tools and techniques to address a Laboratories. His research interests are press, 2007. variety of important bioengineering in microsystem and microfabrication 7. C. M. Moore, N. L. Akers, A. D. Hill, problems including drug delivery, technologies for various energy harvesting Z. C. Johnson, and S. D. Minteer, bionanotechnology, biosensing, and and sensing devices and systems. He may Biomacromolecules, 5, 1241 (2004). bioelectrochemistry. He may be reached at be reached at [email protected]. 8. N. L. Akers, C. M. Moore, and S. D. [email protected]. Minteer, Electrochim. Acta, 50, 2521 Susan Brozik is a senior member (2005). Scott Calabrese Barton is an assistant of technical staff at Sandia National 9. S. Topcagic, B. L. Treu, and S. D. professor of chemical engineering at Laboratories. Her programs include Minteer, in Electrode Processes VII, Michigan State University. His research a broad range of bioengineering and V. Birss, et al., Editors, PV 2004-18, concerns materials and transport processes biotechnology projects targeting p. 230, The Electrochemical Society in bioelectrodes and porous electrodes. He integration of biotechnology into Proceedings Series, Pennington, NJ may be reached at [email protected]. microsystems. She may be reached at (2004). [email protected]. 10. T. L. Klotzbach, M. Watt, and S. D. Michael Cooney is an associate researcher Minteer, J. Membrane Sci., 282, 276 at the Hawaii Natural Energy Institute, Shelley D. Minteer is an associate (2006). University of Hawaii. His research interests professor of chemistry at Saint Louis 11. D. Ivnitski, B. Branch, P. Atanassov, address biological energy systems, University. Her research focus has been and C. Apblett, Electrochem. Comm., including enzyme fuel cells, and fuels on enzyme immobilization membranes 8, 1204 (2006). production from microbial and microalgal for bioanodes and biocathodes, along 12. G. Gupta, V. Rajendran, and P. systems. He may be reached at mcooney@ with multi-enzyme systems for complete Atanassov, Electroanalysis, 16, 1182 hawaii.edu. oxidation of simple biofuels. She may be (2004). reached at [email protected]. 13. G. T. R. Palmore, H. Bertschy, S. H. Bergens, and G. H. Whitesides, J. Electroanal. Chem., 443, 155 (1998). 14. N. Mano, F. Mao, and A. Heller, J. Am. Chem. Soc., 125, 6588 (2003). Future Technical Meetings

Oct. 7-12, 2007 Oct. 12-17, 2008 Oct. 4-9, 2009 Washington, DC PRiME 2008 Vienna, Honolulu, Hawaii May 18-23, 2008 April 25-30, 2010 Phoenix, Arizona May 24-29, 2009 Vancouver, BC San Francisco, California Oct. 10-15, 2010 Las Vegas, NV For more information on these future meetings, contact ECS Tel: 609.737.1902 Fax: 609.737.2743 www.electrochem.org

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