Wastewater Treatment
Our system would be most suited to be placed towards the end of the wastewater treatment process, during the tertiary treatment or even after, since the bacteria are sensitive to the concentration of phosphorous in the water. There are also less suspended solids at the end of the process which will reduce the damage to the bacteria and simplify our inputs and outputs.
A continuous stirred tank reactor, although commonly used in wastewater treatment processes, may not be suitable for our system since we aim to recover the bacteria which would disrupt the continuous nature of the process. Therefore, a batch process may be more appropriate, allowing us to manually place the bacteria into the reactor and remove when required. This could involve placing the bacteria into a suitable medium inside a membrane (imagine a dialysis bag!) and placing the membrane bag into a tank where the water can flow past, and phosphate can be taken up. The membrane containing the bacteria can then be physically removed from the tank to recover the organisms and allowing for cleaning to take place during the downtime.
The bacteria are encapsulated in a chitosan-sodium alginate matrix as bead form and directly placed into a bioreactor where the water flows in and phosphate can be taken up. The encapsulated bacteria could then be recovered using membrane technology or a settling tank. This could enable a continuous process where encapsulated bacteria are continuously fed in and removed without disruption to the treatment process. This would also reduce the amount of human involvement to become a more automated process.
Portable Phosphate Detection
We are also looking to apply the phosphate sensing ability of PhoBac to a portable phosphate detection device that could be used by farmers to detect areas of soil with excessive or insufficiently high P index without the need to pay for a soil company. Additionally, as sewage releases into British rivers and seas are an increasingly salient issue, we propose this detection device could be used by citizen scientists to monitor nutrient levels, faster, cheaper and more regularly than possible with lab testing. See Hardware. Together with our service for wastewater treatment, we believe this is a strong business model with multiple revenue streams.
Sustainable Fertilizer
While we received differing expert opinions on how to implement PhoBac as a biofertilizer, we believe implementing it as a foliar feed, a liquid spray applied to crops recommended by Joshua Gay from Newton Farms, see Human Practices, is the option that requires the least pre-processing and would have potentially the lowest cost. However, this raises concerns over runoff in the event of rainfall following application, and it may be less easy to transport than a solid fertilizer. As such, further work needs to be done to characterise the ecological activity of PhoBac, and of solid phase isolation of the bacteria.
Stable Expression: Cambridge Collaboration
A problem identified in discussions with Team Cambridge, see Collaboration, was the assumption of highly controlled warm and nutrient rich conditions embedded into our experimental design. We therefore collaborated with Team Cambridge to see how their antithetic integral controller circuit could be used as a homeostatic buffer to reduce the impact of extrinsic fluctuations. In particular putting the secreting enzymes of our phosphate release under control of our circuit should allow consistent and robust release of phosphate.
In brief, the antithetic integrated controller circuit pictured above improves upon an ordinary negative feedback loop that simply corrects for the direction or magnitude of the deviation. The circuit proposed by Cambridge employs a sigma factor (z1), upregulated by an input, which in our proposed combined implementation, is malate binding to the malate-sensitive promoter PmaeN. The sigma factor (z1) upregulates not only the target output genes, in our case phosphodiesterases glpQ and phoD, but also a feedback species (X) that indirectly downregulates output by upregulating anti-sigma factor (z2) which inactivates sigma factor (z1). Rather than simply engineering end-product inhibition directly, by working through the sigma/anti-sigma factor pair, the sizes of their pools in the cell give an indication of the history of deviation in output, allowing for much more rapid return to the baseline level without much overshoot, depicted below.
However, we found very few candidates in our literature search for safe orthologous, i.e., non-native, sigma/anti-sigma factor pairs for Bacillus. Cambridge purposely used a Bacillus sigma/anti-sigma factor pair to avoid interference with native gene regulation. This absence is partly due to the “narrow acceptance range” of Bacillus for heterologous sigma factors [1]. Therefore, in future to aid the widespread implementation of controller circuits in other gram-negative bacteria, more detailed characterisation of heterologous sigma factor function should be conducted.
Triple Implementation
In our partnership with BioBrussels, we found out about Maastrich who also had a project on water treatment. We decided to join forces in a collaboration to make a theoretical system that could implement all our designs together to reach our shared goal of clean and treated water.
BioBrussels
“So basically, what we've seen that our protein is able to do is remove supersaturated calcium carbonate from liquids, and the hotter a liquid is the lower the solubility of calcium carbonate becomes so the more calcium carbonate will be present in supersaturation aka. The more we can get out with our protein. That's why we suggest for now that our protein can be used on a heated liquid in either a membrane reactor (local high concentration of protein, sandwiched between two membranes) or a plug flow reactor (would need more protein and a way to immobilise it on beads but less complicated working overall) after the liquid passed through the reaction it should be softened and we wish to centrifuge it to then remove the calcium carbonate and the protein and re-use the calcium carbonate. In the future we'd like to step away from the heating part of our proposal by introducing things like carbonic anhydrases or local pH increased environments since these things also work on increasing supersaturation of calcium carbonate.”
iGEM Maastricht
“Our product will consist of alginate beads that have cyanobacteria embedded in them. These beads will be in a cube that has a fine mesh sheathing. This cuboid box will be placed into a batch of salt water. The water will then flow through the mesh and the polymer matrix until it reaches the bacteria. Once you shine green light on the bacteria, they will start to desalinate water. When the bacteria reach their maximum capacity of salt uptake, the cube will be taken out of the water. Next, the beads will be removed from the cube and a new set of beads will be added. Hence, desalinating the salt water will follow a typical batch process system.
The pore size of the alginate polymer beads will be precisely engineered to facilitate ion diffusion whilst restricting the diffusion of bacteria or other cell components out of the beads. Therefore, we built a three step protection system consisting of the polymer network, the cube and the kill switch to make sure that the bacteria do not end up in the furter processing parts. The bacteria should not be able to escape the beads and the beads should not be able to escape the cube system. If either of the events should take place causing the bacteria to escape the system, the kill switch will prevent them from proliferating.”
You can see and overview of how we see our projects working together over here!
References
- Pinto, D., Dürr, F., Froriep, F., Araújo, D., Liu, Q. and Mascher, T., 2019. Extracytoplasmic Function σ Factors Can Be Implemented as Robust Heterologous Genetic Switches in Bacillus subtilis. iScience [Online], 13, pp.380–390. Available from: https://doi.org/10.1016/j.isci.2019.03.001