Reservoirs around the world are facing serious pollution: eutrophication. Once the water body accumulates excessive amounts of nutrients, algae grow rapidly and cover the whole area. This coverage will block aquatic plants from light and they cannot photosynthesize oxygen. Moreover, when a large amount of algae decomposes, oxygen in the water is consumed. The large and abrupt consumption of oxygen will lead to serious environmental problems such as fish kill and dead zones.

Our team developed an engineered E. coli, TripleP, that can hydrolyze paraoxon (PXN), organic phosphate, into inorganic phosphate, and fixate inorganic phosphate in the form of polyphosphate. To implement our synbiotic solution in the real world, we developed a filtering device hardware containing TripleP cells.

To ensure the filters stay around at the water surface, we chose an artificial ecological island to support our filters. The island can mitigate eutrophication through ecological methods. The aquatic plants on the island, such as common Rush, water arum, water chestnut, and hygrophila, absorb phosphates and nitrogen in the eutrophic water. The total area of the island is 25 square meters, and we further designed 4 solar panels on the island to provide the energy required for the additional functions of the device.

The filtering tubes are connected to the upper part of the device, the ecological island with threads. (We chose nylon threads due to its prestigious flexibility and stretch.) The engineered bacteria TripleP are contained in the filter tubes, which are attached with filter papers on both sides. The filter tube would continue to float in the water with the support of the outer tube. The function of the engineered bacteria in the filter tube would be monitored by the light source, light sensor, and the Arduino chip designed in the water-proof container.

The filter tube is composed of two filter papers and a transparent acrylic tube. The filter papers cover the top and bottom of the tube. Eutrophic water will flow through the tube, while the engineered bacteria (TripleP cells) absorb the phosphate in the water. The filter papers are composed of cellulose acetate, whose hydrophilic property will allow water to pass easily, and they have a pore size of 0.45μm. As E. coli has a diameter of about 1.0-2.0 micrometers, the holes allow water, and phosphate particles to pass but not bacteria. So, when the phosphate concentration in the tube decreases, the phosphate outside the tube will diffuse into the tube, and TripleP will continue to hydrolyze and uptake phosphate. The TripleP cells in the filter tubes will work until they overaccumulate polyphosphate, which will be monitored by the Arduino chip.

In our original hardware design, we contained the TripleP cells in the tube without other materials. However, bacteria are inclined to adhere to nearby surfaces. Therefore, if we let bacteria disperse freely in the tube, they would attach to the filter paper and block the diffusion, leading to the malfunction of our device.

As a result, we took advice from the HNU_China team during our collaboration and decided to use sodium chloride to fix bacteria into spheres. We mix sodium alginate solution with a culture medium and add the solution to a calcium oxide solution. The spheres can keep their shape for 12 hours, and both water and phosphate can pass through the material of the spheres. We'll then fix the spheres containing the engineered bacteria in the center of the filtering tube by restricting them in an additional permeable sack, refraining them from directly contacting and causing damage to the filter paper on both sides of the tube. The design would allow water flow to be free from bacteria’s biofilm blockade while making it easier for users to organize and replace the engineered bacteria in the tube.

The caps that cover both ends of the tube are designed to tighten the filter papers. They will push the paper against the tube wall such that the water will not escape through the space between the filter paper and the tube. This design can ensure that the engineered bacteria would not escape from the filter and leak into the environment. Furthermore, to protect filter papers from being broken by stones and branches in the waterbody, we added an additional web to protect the filter paper. We design another cap that can cover the filter paper with filter screen made of nylon. The filter screen is tightened and fixed between the space of to cap such that it will not fall off even under strong impact.

Parts of the hardware have been 3D printed as our prototype. For more information on the design and 3D printing of our device, please visit our Contribution Page for more.

Arduino Uno is a microcontroller board used in the device. It contains 14 input/output pins which are ample for the modules used in the project. The chip can be programmed and connected to different modules and sensors to perform various tasks. In this project, the Arduino chip will monitor the filtering status and return information back to the users. The specific steps will be explained below: In our device, the Arduino controller is used with a laser, light sensors, and a wifi module. The laser will emit light into the filter tube continually with an interval of two hours and excite mCherry, a fluorescent protein whose production is, based on the biosensor plasmid design, would be inhibited by polyphosphate. If the engineered bacteria TripleP absorbs inorganic phosphate and fixates it into polyphosphate, the production of mCherry would decrease. The light sensor connected to the Arduino chip will detect mCherry fluorescence, whose information will then be processed by the Arduino chip and sent to users via the wifi module. The effectiveness of the bacteria’s filter can hence be determined by the continuous monitoring of mCherry. If the software’s calculation of the mCherry level has lowered to a minimum, an alert would be sent to remind the user of the necessity to replace the filter, thus providing a convenient way for the users to ensure the efficiency of the device and the safety of the environment. Since the filtering tubes are designed under the ecological island, the island itself creates a dark environment suitable for the sensor chip to detect the fluorescent protein generated by TripleP.

For more information about the polyphosphate biosensor design and the experimental validation, please visit our Engineering Success Page .

the composition of an island:

the composition of a filter tube