Team:Victoria Wellington/Description
Team
Victoria
Description
WHAT IS AN EFC?
An Enzymatic Fuel Cell (or EFC) is a battery-like device that exploits enzymes' fantastic ability to catalyse organic reactions for power, instead of using traditional metal-based systems, such as lead and lithium. We are especially motivated by the idea of using enzymes to build greener energy sources and storage, in addition to, making bio-batteries competitive with 'traditional' batteries. Our contribution to this goal will be to improve an existing glycerol fuel cell, where one of the enzymes used in the fuel cell naturally has a slow rate of reaction, a small pH range, and (like most enzymes) is not very stable at room temperature.
There is a lot to consider when constructing a powerful, eco-friendly, EFC. What is the sustainable fuel powering it? What enzymes can work on this fuel? What are the waste products? While our project was in the planning phase, the team set about addressing these problems one at a time, starting with the most fundamental question: what would be our fuel?
The Fuel:
To make an effective EFC the fuel of choice should be; energy dense, sustainable to source and cheap to produce. Initially, we believed glucose to be the perfect candidate, as it is the human bodies choice of energy. However, glucose has a few critical issues which do not make it suitable for use in an EFC. Firstly, while glucose is energy dense, it is also a solid at room temperature, which means that it needs to be solvated. This dramatically reduces the energy density of glucose, as well as, adding extra weight, cost and volume. Secondly, obtaining a cheap, sustainable supply of glucose was problematic, as it is not a by-product of any industries.
However, glycerol overcame these issues. Unlike glucose, glycerol is liquid at room temperature, and because of this is far more energy dense. A comparison is given below:
Glycerol
Glucose
Enthalpy of Combustion (KJmol-1)
-1654
-2800
Mols in one litre of fuel
13.70 mol (pure solution)
5.05 mol (in 1L of water)
Energy per litre of fuel (KJ)
22,652
14,140
Weight per litre of fuel (Kg)
1.26
1.90
Thermodynamic data from the NIST Workbook
Glycerol is also a by-product of the biodiesel industry and so can be sourced sustainably and cheaply (see our human practices section for more details).
With the fuel decided, the team set about deciding how to oxidize it for energy generation.
The Pathway:
In our research we came across many possible pathways for many different fuels. These ranged in complexity and approach, from Zhul et al's monster thirteen-enzyme glucose degrading pathway, which used only enzymes, to the Minteer group's molecular catalyst-enzyme combination approach, which used a single enzyme, as well as small molecule oxidants. We decided that to achieve our project goal of making bio-batteries competitive with 'traditional' batteries, we needed to build the simplest EFC possible. After all, the less moving parts we had to incorporate, the less manufacturing and maintenance cost we would incur on the final product. Thus, we decided to research further into a small molecule-EFC combination approach.
There are many catalysts you can combine with enzymes to make hybrid EFCs. Palladium and gold catalysts make effective oxidizers of simple molecules. However, if we wish to achieve a sustainable and eco-friendly EFC, it would be wise to exclude all dependants on precious metals.
The better alternative we discovered was to use organic mild-radical oxidant such as TEMPO and TEMPO-NH2. As well as not being precious metal based, they are only very mildly toxic (category four), and so can be easily disposed of. Furthermore, these systems can work with a single enzyme which makes them more practical in a commercial environment. This is because TEMPO-NH2 and TEMPO oxidize alcohols to ketones/aldehydes, and these to carboxylic acids. As glycerol is a triol, it turns out that this almost completely oxidizes the entire molecule, with the enzymes only required for the breaking of the carbon-carbon bonds. Thus, with the correct choice, only a single enzyme is required.
However, the issue with this TEMPO hybrid EFC is that enzymes are limited to specific chemical parameters, for example pH and temperature, which do not overlap well with the parameters TEMPO (and its derivatives) require. From Hickey et al (2014), we know that TEMPO and TEMPO-NH2 both work faster the more basic the solution, with TEMPO starting to show activity at pH 6 and TEMPO-NH2 at pH 4. However, the pH optimum of oxalate oxidase, the enzyme Hickey et al used to break the carbon-carbon bonds of glycerol, has an optimum pH of 4.5. This is sub-optimal, as only half of the entire system can be working efficiently at one time.
Therefore, for our project we decided to focus on hybrid TEMPO-NH2-enzyme pathways and attempt to optimize enzymes that can break the carbon-carbon bonds of oxidized intermediates of glycerol, in as basic conditions as possible. The enzymes used in our hybrid TEMPO-NH2 EFC are Oxalate Decarboxylase, Formate Dehydrogenase and Diaphorase. Each one is discussed in more detail below.
Cell Mechanics:
Now that we had decided that we would build a hybrid TEMPO-EFC, it was time to decide the design of the cell itself, and again, there were a lot of factors to consider. To start us off, we interviewed Ashwath Sundaresan, our universities Senior Commercialisation Manager. He gave us many insights, including that the exact design of our EFC should come after iGEM, when we have data which shows us what market our EFC has a competitive advantage in. After all, different markets require different attributes, and the attributes of our EFC were not yet known. This simplified our discussion, leaving us with two questions. Firstly, if we are oxidizing glycerol, what are we reducing? Secondly, what electrode system do we employ?
To answer our first question, we first decided to again take our inspiration from the human body. In the electron transport chain, oxygen is used as the final electron acceptor, in turn being reduced to H2O. We thought that if we could somehow flood the batteries cathode with O2, we would have a limitless supply of strong reducing agent. However, while O2 is thermodynamically favoured to reduce, it is kinetically quite stable. Thus, our initial experiments were met with failure as TEMPO-NH2 could not provide enough energy to overcome this kinetic barrier.
It was decided that in the future either enzymatic or inorganic catalysts could be used to successfully oxidize oxygen. However, testing such a catalyst was leaving the scope of our iGEM project, so a place-holder copper cathode was decided upon, based upon the Daniel cell. This is a non-toxic, easy to work with cathode, that works without a catalyst, allowing us to focus on the anode, the main goal of this project.
Next, we can answer our second question, what electrode system to employ? There are two types; mediated and direct. A direct system is one where the catalyst is fused onto the electrode. While in a mediated electrode system the catalyst is in solution. Direct systems are preferable to mediated ones as they centre the reaction around the electrode, which greatly shortens diffusion times. However, the effective immobilisation of enzymes onto a surface could take an entire iGEM project of its own, and so it was decided that a mediated system will be used with the enzymes, but a direct system for TEMPO-NH2. However, after trying to bind TEMPO-NH2 into a carbon paste electrode unsuccessfully, we decided to let this fall by the wayside, as this kind of chemistry is outside of the scope of the iGEM project. Instead, we decided to make a fully mediated system, with TEMPO-NH2 and all our enzymes in solution. This allowed us to direct more time and resources to our enzymes.
HOW OXDC WORKS? (Our oxidation path)
Oxalate decarboxylase (or OxDc) is an enzyme found in a variety of species. Its primary substrate is oxalic acid, which it reacts to carbon dioxide and formic acid, using oxygen as a cofactor. In the case of mesoxalic acid, OxDc can react it to glyoxylic acid which in turn can be oxidised to oxalic acid by TEMPO, and then reacted on again by OxDc.
OxDc is thus useful because mesoxalic acid (see right) is the first roadblock TEMPO-NH2 meets on its oxidation of glycerol to carbon dioxide. This molecule consists of two carboxylic acid groups at both termini, as well as a ketone in the middle, making it immune to further oxidation by TEMPO-NH2. Also, a little further down the pathway, a similar scenario appears with oxalic acid. Oxalate decarboxylase can break the carbon-carbon bonds of these two oxidised intermediates allowing the pathway to proceed.
However, notice that OxDc is not a REDOX enzyme, and in fact harvest no energy from its reactions. Its sole purpose is to allow the continuation of the pathway, which does harvest more energy.
HOW DOES FDH WORK?
The primary substrate of FDH, or formate dehydrogenase, is formic acid, which it oxidises to CO2, reducing NAD+ to NADH in the process. Thus, FDH's purpose in our EFC is to complete the oxidation of glycerol to CO2, harvesting energy for the EFC in the process (two electrons per reaction). However, without our third enzyme diaphorase (DH), FDH would simply reduce all its NAD+ cofactor and become inert. We discovered that cbFDH has been shown to have weak specificity for mesoxalic acid. This makes cbFDH another candidate for enzyme for our oxidation pathway.
HOW DOES DIAPHORASE WORK?
Diaphorase is the final enzyme required in our EFC. Its task is not related directly to the oxidation of glycerol, but to support FDH in doing so. Diaphorase’s primary substrate is NADH, in which it abstracts a H- from, and donates to a H- donor. The donor in this case is the positively charged anode, which accepts the electrons and generates a current. In doing so diaphorase also regenerates NAD+, which FDH needs for a cofactor.
WHAT IS TEMPO'S ROLE?
TEMPO, and its derivative TEMPO-NH2, is the non-enzyme component of our hybrid-EFC. It is a mild radical oxidant that oxidises alcohols, aldehydes and ketones but not carbon-carbon bonds. Four methyl groups also defend the radical oxygen from side reactions, creating a small sterically hindered "barrel" in which only small hydroxy and carbonyl groups can fit down. TEMPO-NH2 provides several advantages over other oxidants. Firstly, due to its methyl groups, it cannot effectively oxidize our enzymes, increasing their stability in solution. Secondly, the reversible reduction of TEMPO is also quite facile. This is important because we need to eventually harvest the electrons, and so it is important that the reduced oxidant is not too stable. TEMPO works better at higher pHs, therefore to build a better EFC, we needed to find enzymes that can work in similar pH conditions so their activity was optimal.
References
Hickey, D., McCammant, M., Giroud, F., Sigman, M. and Minteer, S. (2014). Hybrid Enzymatic and Organic Electrocatalytic Cascade for the Complete Oxidation of Glycerol. Journal of the American Chemical Society, 136(45), pp.15917-15920.
Abdellaoui, S., Hickey, D., Stephens, A. and Minteer, S. (2015). Recombinant oxalate decarboxylase: enhancement of a hybrid catalytic cascade for the complete electro-oxidation of glycerol. Chemical Communications, 51(76), pp.14330-14333.
Zhu, Z., Kin Tam, T., Sun, F., You, C. and Percival Zhang, Y. (2014). A high-energy-density sugar biobattery based on a synthetic enzymatic pathway. Nature Communications, 5(1).
Shinde, P., Musameh, M., Gao, Y., Robinson, A. and Kyratzis, I. (2018). Immobilization and stabilization of alcohol dehydrogenase on polyvinyl alcohol fibre. Biotechnology Reports, 19, p.e00260.