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Requirements and solutions

Our pods must be able to:

Solutions:

Fragment design

We were told by IDT that neither the Nap nor Nrf operon could be synthesised in one piece, as at around 8 kb each they were too long. Each operon was therefore split into 4 fragments of < 2kb. These fragments were designed with overlapping sequences of approximately 40 bp, to allow subsequent Gibson assembly. This is shown below (the same was done for both Nrf and Nap).

We successfully used PCR to amplify each fragment for both operons, however Gibson assembly proved difficult (see Experiments for more detail).

Primer design

We planned to submit Nrf and Nap as one composite part, as well as individually (see Parts). In order to do this, we designed two different forward primers for the first of the four Nap fragments (fragment "Nap1"). The first primer annealed to the very start of the Nap1 fragment and contained an overlap allowing homology with the end of fragment Nrf4. We refer to this new fragment created after PCR as "Nap1H" (Nap1 homology). Subsequent Gibson assembly using this new fragment would result in the composite part, shown below.

The second primer annealed further along the Nap1 fragment, removing the double terminator which would not be required if Nap was used without Nrf preceding it. This primer had an overlap which contained the BioBrick prefix, allowing Nap to be used as a part on its own. We refer to this fragment as "Nap1-dt" (Nap1 minus the double terminator). Use of this fragment in Gibson assembly with Nap2, Nap3 and Nap4 would result in the following part:

Fuel cell design

To think about the future value and sustainability of our project we felt it was important to consider possible uses for the ammonia produced. Although ammonia itself is a useful by product and is used in fertilizers, cleaning products and industrial processes, harvesting the ammonia for these purposes would increase costs and most would lead to ammonia being fed back into the nitrogen cycle in the form of a nitrogen compound thus replenishing the nitrogen oxides (NOx) we are aiming to reduce. Therefore we took inspiration from the literature, and gained valuable insight from the Bristol Robotics Lab, to design a system in which the ammonia produced could be used to generate electricity.

This involves feeding ammonia into a fuel cell; there are multiple examples of ammonia being used within a fuel cell. However, most of these are within solid oxide fuel cells (SOFCs) which run at temperatures too high for our bacteria to survive. Bacteria would therefore need to be kept in separate chamber; this would increase both the cost of production, and the complexity of the design. SOFCs also present other problems such as start-up times and brittleness of components which are often made of ceramics. Traditional proton exchange membrane fuel cells (PEMFCs) are incompatible with using ammonia directly as a fuel due to the acidity of the membrane. Two options reported for using ammonia in PEMFCs are to convert the ammonia to hydrogen and nitrogen using either high temperatures or electrolysis, then using the hydrogen produced as fuel. Clearly this would be impractical as either of these would consume energy making the system less efficient as well as increasing cost.

We therefore chose to use a system based on an alkaline anion exchange membrane (Fig. 1) in which the bacteria could hopefully sit within the anodic chamber producing ammonia which could be directly oxidised with the aid of an OH- group at the anode, made of chromium decorated nickel (97.7:2.3). The cathode, made of manganese dioxide (MnO2) (20&percnt; wt MnO2) would be exposed to air; in the presence of water this would result in the reduction of oxygen, producing the OH- group which would subsequently be transported across the membrane to react with ammonia at the anode. The resulting products would be water and nitrogen from the anodic chamber, water and air produced in the cathodic chamber, and current resulting from the oxidation of ammonia.

Fig. 1 - Diagram illustrating functional flow of an ammonia fuelled alkaline anion exchange membrane fuel cell. Rong Lan, and Shanwen Tao Electrochem. Solid-State Lett. 2010;13:B83-B86

We chose the materials for our anode and cathode based on a paper written by Lan Rong and colleagues, who used these as substitutes for the traditional materials such as platinum to reduce cost.