This Small Business Innovation Research Phase I project will develop high-power and high-energy-density enzymatic fuel cells (EFCs) that can completely oxidize low-cost maltodextrin (i.e., a partially hydrolyzed starch fragment). EFCs have received increasing interest as a next-generation, environmentally friendly (micro-)power source. Compared to microbial fuel cells, EFCs have much higher power densities suitable for more applications. However, current EFCs are limited by the partial oxidization of hexose molecules by one or two redox enzymes (i.e., 2-4 mol of electrons produced per mol of glucose) and a short enzyme lifetime. The goal of this project is to demonstrate the technical feasibility of the complete oxidation of maltodextrin in EFCs through a patent-pending synthetic enzymatic pathway. The technological innovation of this project is the construction of an ATP-free and CoA-free pathway by an assembly of thermostable enzymes to generate 24 electrons per glucose unit and increase power density. As a result, EFCs are expected to feature high energy density due to the complete oxidization of the fuel, high-power density due to substrate channeling among cascade enzymes and the mitigation of product inhibition of the enzymes, and a long lifetime due to the use of thermostable enzymes.
The broader impact/commercial potential of this project is developing bio-inspired sugar biobatteries featuring four appealing advantages: (i) biodegradability, (ii) safety, (iii) high energy storage density (e.g., 400 Wh electricity/kg for a 20% (w/v) maltodextrin solution, nearly three times that of lithium ion batteries), and (iv) fast refilling by adding a sugar solution. EFCs would have broad potential applications, such as rechargeable battery chargers (e.g., cellular phone chargers for outdoor uses or portable military devices), educational toy kits, and disposable (primary) batteries. In the future, miniaturized sugar-powered EFCs could potentially replace some secondary (rechargeable) batteries. Sugar-powered EFCs would be nearly 100% biodegradable, with the exception of the electrodes and wires, and are based on non-toxic and earth-abundant elements. The maltodextrin solution is neither toxic nor flammable. The innovation of EFCs equipped with this in vitro synthetic pathway would greatly promote the concept of in vitro synthetic biology and demonstrate another advantage a faster reaction rate than that of microbes due primarily to the absence of a cellular membrane. In addition, the generation of electricity from renewable and low-cost sugars, namely maltodextrin or future cellulosic materials, would decrease greenhouse gas emissions, increase national energy security, and promote rural economies.
Enzymatic fuel cells (one of biofuel cells) are a type of devices converting chemical energy into electrical energy with the utilization of enzymes as catalysts under mild conditions (near neutral pHs and 20-60oC). Enzymatic fuel cells are an excellent alternative to conventional fuel cells or batteries because they feature several advantages such as high-energy storage density, high-specificity, biodegradability and safety. As a result, enzymatic fuel cells are expected become a promising micro-power device for use in military and civilian applications. However, many biofuel cells studied/developed in the academic or industrial areas are still susceptible to the problem of the incomplete oxidation of fuels and short lifetimes because only one or two mesophilic oxidative enzymes are used to extract electrons from fuels. CFB9 has recently developed enzymatic fuel cells which can produce a high power output by completely oxidizing the low-cost fuel, maltodextrin (a short chain of starch). To efficiently utilize the fuel, we employed a non-natural synthetic enzymatic pathway containing 12 thermoenzymes (enzymes can work at elevated temperature) to demonstrate the feasibility of the complete oxidation of glucose units from maltodextrin for producing high power outputs. The synthetic enzymatic pathway contains four sub-modules: 1) conversion of maltodextrin into glucose-6-phosphate (g6p) catalyzed by phosphoglucomutase (PGM) and a-glucan phosphorylase (a-GP), 2) production of NADH catalyzed by two dehydrogenases, 3) regeneration of g6p from ribulose-5-phosphate (ru5p) catalyzed by the eight enzymes of the pathway, and 4) the oxidation of NADH with diaphorase and vitamin K (VK3) to produce electrons. Our data also shows that our enzymatic pathway can extract more than 86.0% of electrons from a glucose unit. In addition, our biofuel cell utilizing thermoenzymes as catalysts has demonstrated prolonged operation times for weeks, which is cardinal to make commercialization of biofuel cells viable. The first prototype of biofuel cells developed by CFB9 is based on a "cuvette" set-up. The VK3/CNTs-adsorbed carbon paper electrodes and the two cathode electrodes (each with the active area of 1.08 cm2) affixed on two separated open windows (0.6 × 1.8 cm each) of a cuvette are combined to construct a one-compartment biobattery (Fig. 1A and B). We have demonstrated that our prototype biofuel cell can light LED lights and power digital clocks. (Fig. 1C). Further applications of EFCs are being explored. In the SBIR II proposal, we expect to generate enough power for running an iPad (~2 mW). The size of a "cuvette" cell is similar to an ordinary AA battery, and a single cell can be filled with up to 4 mL of the reaction solution containing the enzymes and sugar. The power output of the biofuel cell in the presence of only dehydrogenases (#3-4) was tested. The data (Fig. 2A and B) shows that a single biobattery can achieve the open-circuit voltage of 0.8 V, the short-circuit current of 0.82 mA cm-1 and the maximum power density of 0.23 mW cm-1 at 0.42 V at room temperature.
