Team:Bristol/Description

In recent years, the levels of nitrogen oxide (NOx) gases in our cities has become a pressing concern due to increases in anthropogenic sources, particularly diesel car emissions. NOx is a significant cause of morbidity and mortality; it irritates the lungs, exacerbating conditions such as asthma, and in Bristol alone NOx kills 300 people per year, equivalent to 8.5% of deaths. NOx also contributes to climate change by depleting ozone in the stratosphere. It is now the third most detrimental greenhouse gas, behind only CO2 and methane. In our project we aim to genetically engineer E. coli to metabolise NOx, using the cytochrome c nitrite reductase (NrfA), which reduces nitrite (NO2-) to ammonia, and the Nap periplasmic nitrate reductase for the reduction of nitrate (NO3-) to nitrite. As NOx dissolves in water to form both nitrite and nitrate, the use of Nap will improve the efficiency of our system. The ammonia produced could then be harvested and used to produce fertiliser, or incorporated into a microbial fuel cell (MFC) to produce electricity. An open-source agent-based bacterial model will be expanded upon to predict system efficiency and behaviour on a larger scale. We plan to use existing models and data of NOx concentrations in Bristol to strategically place our recombinant bacteria in problem areas within pod structures, and aim to assemble them into larger synthetic trees, which are either self-sustaining or require low maintenance.

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 can be constructed using chromium decorated nickel as a cost effective alternative.

The outer casing will be made from acrylic panels, which can be autoclaved. These are each separated by a gasket and held together using plastic bolts. The anodic and cathodic chambers will be separated by the anion exchange membrane and have an internal volume of 1400mm³ (10x70x70mm). The external construct was provided by the Bristol Robotics Lab (BRL) and from this we designed our own model of the MFC so we could create more if required. The two major benefits of using the BRL's design are:

Quick and easy to manufacture/build to allow for fast construction.

Made from cheap, easy to source materials due to a tight budget.

To capture the NOx and dissolve it in solution we aim to use a dry deposition and washing system which will feed into the MFC. One problem we have encountered whilst devising a solution to the capture and isolation of NOx is the separation of nitrogen and sulfur compounds as both elements are likely to stick onto our capture surface. Sulfur is undesirable as this may result in unwanted reactions within the fuel cell and have detrimental effects on the survival and metabolism of E. coli.

Upon completing this MFC design with sufficient power output using our recombinant E. coli the next step would be to combine individual units in an array connected in series-parallel to increase power output. This array would be contained within a ‘pod’, the design and placement of which will be determined alongside significant input from the general public and public officials.

Implementation of our system in Bristol

After successfully incorporating our system into ‘pods’ as described above, there are multiple possible applications for which the system could be used. A main target we have identified, although harder to achieve due to the complexity of NOx capture at the interface with the air, is reducing the levels of NOx pollution in urbanised areas such as Bristol City centre; targeting these areas will have most most health benefits as these are more densely populated areas and tend to have higher levels of pollution. Pods could then be either publicly or privately run with any electricity generated either powering electronic devices such as mobile phones or street lights, or could be fed into the national grid.