Team:Bristol/Description

Background: NOx

In recent years the environmental impact of nitrogen oxide (NOx) gases has become a pressing concern due to increases in anthropogenic sources, particularly diesel car emissions, and a lack of natural processes to remove it. NOx is a health risk, producing toxic ozone in the troposphere and particulate matter. This is a significant cause of morbidity and mortality; it irritates the lungs, exacerbating conditions such as asthma, and in Bristol alone kills 300 people per year (8.5% of deaths) - see Background for more information about NOx as an issue in Bristol. NOx also contributes to climate change, being an ozone depleting substance (ODS) in the stratosphere. NOx is now the third most detrimental greenhouse gas, behind only CO₂ and methane.

Biology

To tackle the problem of atmospheric NOx we aimed to engineer a strain of E. coli (DH10beta), derived from the widely used K12 strain, to upregulate components of the denitrification pathway. Our recombinant bacteria would be able to metabolise NOx compounds into ammonia (NH₃) at a much faster rate than wild type (WT) E. coli, once atmospheric NOx has been captured and dissolved in solution as nitrite and nitrate.

This aim would be achieved by transforming E. coli with synthetic constructs of two WT operons which code for the cytochrome c nitrite reductase (NrfA) and nitrate reductase (Nap), as well as their respective cytochrome c maturation (Ccm) complex. Using this combination of operons will maximise the amount of NOx consumed by our bacteria and maximise the efficiency of our system. In this system, Nap will be responsible for the reduction of nitrate (NO₃^-) to nitrite (NO₂^-); Nrf will then complete the reduction process taking nitrite to ammonia - see our Parts page for more detail. Two high copy number plasmids will be incorporated into E. coli. The first contains the NrfA and Nap operons under the control of a LacI inducible promoter, allowing us to regulate expression using IPTG. The operons are separated by a double terminator, comprised of two single terminators (BBa_B0021) separated by TACTAGAG; this ensures individual transcription of the operons. The second plasmid will contain the cytochrome c maturation complex essential for the covalent attachment of the c-type cytochromes into NrfA and Nap.

The enzymes NrfA and Nap are both membrane bound with active sites located in the periplasm. Once hemes from the cytochrome c maturation have been incorporated, NrfA is present in the periplasm as a hetero-tetramer comprising of 2 x NrfA and 2 x NrfB subunits. Although this enzyme works primarily on nitrite it exhibits some catalytic promiscuity, meaning there will be collateral reduction of both N₂O and NO.

Microbial Fuel Cell (MFC)

Once ammonia has been produced we intend to use it as a value added product within an MFC to produce electricity. To construct this we have taken inspiration from units made in the Bristol Robotics Lab and previous direct ammonia fuel cells in the literature. We have decided to make an anion exchange membrane MFC in which ammonia acts as a fuel, which reacts with a hydroxide group (OH^-) and undergoes an oxidation reaction at the anode to form water (H₂O) and nitrogen (N₂). The cathodic chamber will use an air exposed cathode made of manganese dioxide (MnO₂) coated carbon. Air will enter the system, undergo a reaction to form OH^-, H₂O and oxygen. The OH^- group will then travel across the anion exchange membrane to the anodic chamber whilst the other products exit the system. The anode itself will be made from platinum or could be constructed using chromium decorated nickel as a cost effective alternative. Read more about this on our Design page.

Implementation of our system in Bristol

We intend to incorporate our system into ‘pods’, which could be strategically placed in the city to reduce NOx air pollution in the worst polluted areas. Although there are multiple possible applications for which the system could be used, targeting NOx pollution in urbanised areas, such as Bristol city centre, will have most health benefits as these are more densely populated areas and tend to have higher levels of pollution. We intend to use an atmospheric pollution model to identify areas which would benefit most from our pods. Pods could then be either publicly or privately run with any electricity generated either powering electronic devices such as mobile phones or streetlights, or could be fed into the national grid.