The use of phosphate as a fertiliser is essential for maximizing crop plant yields in many agricultural settings. Phosphate runoff from fields can cause eutrophication. This leads to algal blooms and the deterioration of aquatic ecosystems. It is estimated that the damage costs of freshwater eutrophication in England and Wales is £75.0−114.3 million per year (Pretty et al., 2003). Moreover phosphate rock is a finite and increasingly scarce resource; with peak phosphate production estimated to be reached in 2030. In 50-100 years, phosphate reserves will be depleted if current rates of extractions continue. Together, these two issues suggest a closed-loop solution (Cordell and White, 2014).
Our solution aims to address this issue through a system which could sequester and store high levels of phosphate: Phosphostore. Phosphostore uses genetically-modified bacteria that can accumulate phosphate from wastewater in protein-based microcompartments for future recycling. In our project, we expressed Eut (Ethanolamine utilisation) bacterial microcompartments and a polyphosphate kinase (PPK) enzyme with a Pdu (1,2-propanediol utilisation) localization tag. This enables the encapsulation of the PPK enzyme within the microcompartment, allowing the formation of polyphosphate chains to be built and stored safely from degradation by endogenous exopolyphosphatase (PPX). We were able to demonstrate the proof of concept through fluorescent microscopy, which can be seen here.
Improving our System
We utilized a Design of Experiments approach to optimise the production of key components in our system. Firstly, we explored the expression of our key PPK enzyme. Through two rounds of DoE we were able to identify the optimal conditions for the expression of this enzyme, within the design space explored. DoE was also used to identify the optimum expression conditions for microcompartment expression. The five Eut proteins were split into 3 orthogonally controlled expression modules to allow us to tune the expression ratio of the microcompartment subunits.
An innovative ensemble modelling approach was also utilized to predict the operating characteristics of our phosphate starvation operon as a regulatory system for controlling microcompartment synthesis.
In interviews with a wide range of stakeholders, we explored the economic and regulatory constraints that would influence the implementation of our system in real-world water treatment plants, giving us detailed insights into the practices of innovation management in the water industry.
We further investigated three key areas: intellectual property, GMO legislation and industrial scale-up. This research informed us in the development of our business plan.
Investigation of intellectual property and GMO legislation in the UK inspired us to develop our international collaboration with other iGEM teams to generate a worldwide legislation map, with the aim of assessing potential international markets where our project could be implemented. As a result, we have managed to analyse multiple markets, which has directed us to set up our Phosphostore business model in Canada, where the legislative approach towards GMMs is both safe and open as our collaborative friends convinced us.
A continuous culture model was also incorporated into the development of Phosphostore. Taking into consideration industrial scale-up and media supplementation enabled us to predict the rate of bacterial culture and phosphate remediated (g/L), allowing us to estimate the yearly cost of treating wastewater using Phosphostore.
Cordell, D. and White, S. (2014). Life's Bottleneck: Sustaining the World's Phosphorus for a Food Secure Future. Annual Review of Environment and Resources, 39(1), pp.161-188.
Pretty, J., Mason, C., Nedwell, D., Hine, R., Leaf, S. and Dils, R. (2003). Environmental Costs of Freshwater Eutrophication in England and Wales. Environmental Science & Technology, 37(2), pp.201-208.