BIOMEMES

Biofuel Cell

Background

Biofuel cells are energy devices for the production of electricity, inspired by nature in their use of naturally occurring food sources as fuel. Some examples of these fuels are the energy rich molecules available in food, such as carbohydrates and sugars. The inner working of a biofuel cell is typically based on the fundamental principles behind conversion of food into usable energy by the body. Food molecules are broken down into digestible molecules that are transported to the energy powerhouse of the cell Mitochondria, where they enter the well-known Krebs’s cycle. The digestible molecules are then further broken down through their stepwise oxidation into CO2 with the aid of specialized biocatalysts called redox enzymes. Redox enzymes utilize oxygen to “burn” the food into calories and yield water and CO2 as the by-products of this process called respiration. The “burning” of the food is actually the catalytic breakdown of food by redox enzymes into higher oxidation states, eventually CO2. Simultaneously, electrons are transferred down the electrochemical potential slope and stored into energy rich molecules called Adenosine triphosphate (ATP). These ATP molecules are like the energy currency of the body utilized by individual cells in the body to carry out their functions.

Figure 1. A schematic for a simple Glucose Oxidase/Bilirubin Oxidase based Enzymatic Biofuel Cell

Although the working principles of Biofuel cells are the similar to nature’s, the method for energy entrapment is different. Instead of the producing ATP during the electron-transfer cascade ,as in nature, a Biofuel cell couples the electron to an external electrode either directly or through mediator molecules. Thus extracting the energy inherent in the sugars into electrical energy through the use of an electrochemical cell. Such electrochemical coupling with the redox enzymes can be accomplished through immobilization of microorganisms containing these enzymes (Microbial Biofuel Cells or MBCs). However, because only a few enzymes on the outer edge of the cell membrane can be coupled at one time, cell efficiency is low. A more direct and efficient scheme is immobilization of redox enzymes directly on the electrodes and subsequent transfer of electrons through Direct Electron Transfer (DET) or Mediated Electron Transfer (MET) mechanisms. Such a scheme is known as an Enzymatic Biofuel Cell (EBC) and an example is shown in Figure 1. The basic reaction for a functioning EBC is a complete circuit comprised of the cathodic and anodic enzyme reactions that release and trap electrons respectively.
Glucose oxidase has been very commonly used as an anodic enzyme due to its high stability at physiological pH of 7.2 and high turnover rates. Bilirubin oxidase has emerged as the champion cathodic enzyme primarily because of its high stability at physiological pH. Many researches have chosen to work with a contending enzyme, Laccase. However, Laccase only performs with such outstanding efficiency at acidic pH of 5.2 and encounters a 10-fold drop in its performance at physiological pH of 7.2.

The redox reaction of these enzymes is shown below:

Glucose Oxidase Reaction (Anode)



Bilirubin Oxidase Reaction (Cathode)


The electrons exchanged at each electrode need to be “transported” to the electrode to make the cell active. This electron-coupling step is one of the most complicated and challenging, which has led to the development of several different strategies. We now review some of these schemes.

Electron Coupling Techniques

Figure 2. Typical electron coupling techniques used in enzymatic biofuel cells. (a) Direct electron transfer (b) Mediated electron transfer , Courtesy: (Calabrese Barton, Gallaway et al. 2004)

The bioelectrocatalytic efficiency of an enzyme electrode is largely governed by the electrical conductivity between the redox center of the enzyme and the electrode. The redox center of the majority of the redox enzymes is buried inside the protein matrix making free electrical communication with the electrode material nearly impossible. This can be overcome by Direct Electron Transfer (DET) or Mediated Electron Transfer (MET), both of which couple the electron transfer between the enzyme and electrode.

DET requires the redox center of the enzyme to be very close to the electrode so as to allow for electron tunneling, which is typically on the order of a few angstroms. Therefore, the enzyme must be very strongly adsorbed onto the electrode surface through physical or covalent bonding.  This technique has proven difficult to achieve and also has a detrimental effect of the enzyme’s activity. The DET rate between enzymes and electrode is typically sluggish as compared to MET, meaning only lower current densities can be extracted from the DET scheme.

An alternative approach to DET proposed by some other groups is the immobilization of the redox species of the enzyme through a conducting linker onto the electrode and then reconstitution of the apo-enzyme (enzyme with the redox species stripped). This provides a direct path for the electron to flow from the redox center of the enzyme to the electrode via the conducting linker (Willner and Willner 2001; Willner, Katz et al. 2006). However, this method is very expensive and non-viable for long-term use due to the reduced stability of the apo-enzyme.

For another method of DET, carbon nanotube matrix are used as the electrode and enzymes are immobilized onto the surface. Of the many rigid carbon nanotubes, statistics assumes on of them will “poke” into the redox center of the enzyme, thus establishing direct contact for the electrons to flow into the electrode (Ivnitski, Branch et al. 2006). However, this method is not very reproducible and depends on the statistical odds of a carbon nanotube reaching the redox center. Because of these disadvantages, the method cannot be systematically applied to the design and fabrication biofuel cells at large.

MET, on the other hand, employs a mediator molecule which “carries” the electron from the redox center of the enzyme to the electrode. This is only possible if the Standard redox potential of the mediator molecule is less than the active redox species and greater than the electrode potential. MET has an obvious thermodynamic disadvantage of lower voltage output since a portion of the voltage is consumed in mediated transfer.

Figure 3. Schematic for the CMEMS fabrication process

Electrode Fabrication

The electrodes are manufactured using Carbon Micromachining or Far Field Electrospinning with PAN as the precursor polymer.

Carbon MEMS

      Carbon Micromachining is a carbon microfabrication technique based on the patterning of a UV sensitive epoxy resin or photoresist like SU8 (Figure 3). SU8-50 is patterned using Photolithography to produce posts with a height of 150µ and diameter 30µ. The resulting microstructures are then pyrolysed at a temperature of 900 °C or higher. During pyrolyization, the ramp rate is maintained such that at any given time the temperature of the furnace is lower than the glass transition temperature of the crosslinked epoxy [3, 4]. The heat leads to the vaporization of the non-carbonaceous components of the crosslinked epoxy, with only carbon in a mixed sp2/sp3 hybridized state remaining.

Carbon Nanofiber Electrodes

Preparation of porous carbon nanofibers is accomplished by electrospinning a blend of PAN-PMMA in different ratios. The PMMA phase is used as a sacrificial polymer for generation of pores. We create mat like sheets of electrospun polymeric nanofibers that are folded over onto themselves several times to create thick mats. The mats are then heat treated to decompose the PMMA phase. This results in porous “islands” in the PAN bulk phase of the nanofiber. The heat treated mats are pyrolysed in a reducing environment at 900 °C to obtain highly porous carbon nanofiber mats as a standalone electrode. Figure 4 shows the PAN nanofibers and the carbon nanofibers after the pyrolysis of the electrospun nanofibers. These nanofibers mats have been tested for their electrochemical activity and were found to be highly active.

Figure 4. SEM images of PAN: PMMA nanofibers in the ratio 9:1 (left) and Carbon Nanofibers obtained from pyrolysis of PAN: PMMA nanofibers (right)

These two micromachine techniques can be combined to create unqiue patterns.   PAN nanofibers are electrospun onto the Carbon Microposts arrays into multiscale fractal-like tree geometry as seen in Figure 5 below.

Figure 5. Multiscale CMEMS electrode with PAN nanofibers

Electrode Functionalization

The carbon electrodes are activated with oxygen plasma to generate carboxylic functional groups on the surface of the carbon electrodes.  These are used to conjugate the electrodes with amine terminated groups through carbodiimide chemistry. The plasma treated electrodes are then treated with a solution of Ethylene Diamine linker in EDC (which is used as the coupling reagent) for up to 3-4 hours. The EDOA functionalized carbon surface is then treated with a solution of 2,4 Dihydroxy-Benzaldehyde (Hydroquinone)/ Sodium Borohydride to initiate coupling of the hydroquinone mediator molecule to the EDOA linker (see Figure 6 for the reaction).

Figure 6. Reaction scheme for the attachment of hydroquinone mediator to the carbon electrode through EDOA linker

Figure 7. Proposed Mediated Electron Transfer pathway for the functionalized carbon electrode

The 2% (w/v) solution of enzyme Glucose Oxidase in PBS buffer is then incubated on the mediator functionalized electrode surface for several hours in order to couple it to the remaining carboxylic groups via the amide linkage. The enzyme attachment to the electrode is further improved by a cross-linking reaction with incubation in a 1% glutaraldehyde solution for up to 10 minutes.

Enzyme functionalization Results

The MET is tested by Cylic Voltammogram of the enzyme-mediator functionalized electrode in PBS buffer with and without Glucose after deaerating the solution by bubbling N2 for 30 minutes. Any traces of oxygen could lead to a competing reaction that produces H2O2 as a by-product. The Cyclic Voltammograms of the Mediator-Enzyme functionalized electrode reveal the phenomenon of MET through the appearance of redox peaks in the presence of Glucose which is illustrated in Figure 8. The CV revealed oxidation peaks when tested in 1M glucose in PBS solution. These peaks can be attributed to the MET since the absence of oxygen prohibits any H2O2 production peaks and no other interfering reactions are known to occur. The redox peaks for the MET between the electrode and the enzyme are weak due to the low quantity of enzyme that was attached to the electrode surface.  We believe the low functionality is caused by enzyme consumption during the attachment of the mediator in the course of Plasma treatment.

Figure 8. Cyclic Voltammogram of Enzyme functionalized carbon electrodes in the absence of glucose (flat curves) and presence of glucose (curved pointed with arrows) in 100mM PBS. The different CV curves are taken at different scan rates (100mV/s, 200mV/s, and 300mV/s)

 

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