Purdue Biomakers





Our system for phosphorus removal is designed to be modular in order to work efficiently and effectively in many different environments. In our operons, promoters can be switched and new genes can be added to our E. coli in order to alter the expression levels of our proteins or to solve additional problems related to specific application. We envision that our E. coli would be especially useful in several applications, such as methane digesters, tile drainage systems, wastewater treatment plants, home bioreactors, and even a floating phosphorus collector unit.

Methane Digesters


In anaerobic methane digesters, acidogenic bacteria convert organic material in cow manure to volatile fatty acids, which are later broken down into acetic acid and hydrogen gas. Methanogenic bacteria then consume acetic acid and hydrogen gas to produce methane gas and carbon dioxide. [2]


The methane gas is then collected for use as bioenergy. An effluent containing most of the nutrients in manure passes through the digester and is collected for use in fields. During this process bacteria do not consume phosphorus. While a small amount of phosphorus settles to the bottom of the digester, the rest of the phosphorus is in the effluent. Another major nutrient for crop, nitrogen, is also present in the effluent, although some of the organic nitrogen is converted to ammonium. Some of the ammonium is released as ammonia gas, and the rest of the ammonia will be incorporated into the soil, where microorganisms convert it to nitrite and then nitrate, the form in which most plants uptake nitrogen.[4]

Still, most of the nutrient content from the manure stays in the effluent. This is a problem because when cow manure is used as fertilizer, nitrogen is the limiting nutrient. Excess phosphorus then accumulates on the soil, and surface runoff can carry phosphorus to bodies of water. [5]

Therefore, our phosphorus-accumulating E. coli could be applied to remove some phosphorus from the effluent after the effluent leaves the digester. This way, the effluent contains both necessary nutrients, but there would be less excess phosphorus. We could achieve this partial removal of phosphorus by choosing the weaker constitutive σS promoter BBa_J45992. Also, the physical design of the system could allow for reduced contact between our E. coli and water in order to achieve this partial removal of phosphorus. Our system can also be adapted for nitrification, converting ammonia to water soluble nitrite, by adding the genes for ammonia monooxygenase (BBa_K1067003), hydroxylamine oxidoreductase(BBa_K1067004), and cytochromes A and X (BBa_K1067002). (These parts came from DTU Denmark’s 2013 iGEM project.[6]) Therefore, ammonia in the effluent will not be released as gas, and microorganisms in the soil can convert the nitrite to nitrate. This would increase the amount of nitrogen, the limiting nutrient in the effluent, that plants can use, and therefore, less effluent (and less excess phosphorus) would need to be applied.

Tile Drainage


Tile drainage systems are subsurface drainage systems used in agricultural fields that lack adequate natural drainage, and therefore remain wet several days after heavy rains. About 25% of farmland in the United States and Canada uses tile drainage because over-saturated soils provide insufficient aeration and thus inhibit crop growth. The subsurface pipes collect the excess water and carry it to an outlet pipe, where the water is emptied into a ditch or natural waterway.[8]

While tile drainage systems are beneficial due to their ability to increase crop yields and reduce surface runoff, they also are problematic due to their export of nitrogen, phosphorus, and pesticides, such as atrazine, into waterways.[9] An eight-year study on six different tile drains and a watershed outlet within the Upper Big Walnut Creek watershed in central Ohio concluded that tile drainage systems’ contribution to phosphorus in surface water is not negligible, as previously thought. More than 90% of all measured concentrations exceeded 0.03 mg/L, the recommended level for minimizing algae growth. The annual flow-weighted mean for dissolved reactive phosphorus in tile drainage systems was 0.12 mg/L, and the annual flow-weighted mean for total phosphorus in tile drainage systems was 0.15 mg/L. Results indicated that tile drainage accounted for 48% of the monthly dissolved phosphorus and 40% of the monthly total phosphorus exported from the watershed. While the amount of phosphorus that enters the tile drain system varies with soil type and season, the researchers concluded that, based on similar studies, the observed relationship between phosphorus in tile drainage systems and watersheds are likely relevant for many other watersheds in the midwestern United States.[10]

Therefore,our phosphorus-accumulating E. coli could be used to remove phosphorus from the water before the water leaves the outlet pipe. Our system would be designed to attach to the end of the outlet pipe. Also, we would add genes (based on Nanjing-China’s 2013 iGEM project) to our E. coli to absorb and degrade atrazine. These would include a transporter for atrazine (BBa_K1145001), trzN, an enzyme which degrades atrazine (BBa_K1145002), and a ribosome switch opened by atrazine (BBa_K1145003). By placing trzN downstream of the ribosome switch, we would only produce trzN when necessary. [11] Also, in the future, we could work on developing a genetic system for nitrogen remediation to incorporate into our E. coli.

Wastewater Treatment Plants


Our phosphorus-accumulating E. coli would be useful during tertiary treatment of the wastewater treatment process. Tertiary treatment refers to any additional treatment after secondary treatment, and this often involves removal of impurities and nutrients, such as phosphorus.[13] The Environmental Protection Agency and state agencies are requiring reductions in the amount of phosphorus in wastewater discharge [14]. Phosphorus can be removed by either a chemical or biological process. The chemical removal of phosphorus involves the formation of a precipitate by the addition of metal salts. The precipitate, and therefore the phosphorus, can then be removed. The traditional biological method of phosphorus removal, enhanced biological phosphorus removal, phosphorus-accumulating organisms are present in sludge. The phosphorus-accumulating organisms (PAOs) then store 5 to 30% of their dry weight as phosphorus. The exact composition of PAOs is unknown, but likely includes Acinetobacter, Rhodocyclus and various coccus-shaped bacteria. [15]

Our phosphorus-accumulating E. coli system provides another option for biological phosphorus removal in tertiary treatment. Our system would be designed in a way for easy removal of the E. coli from the wastewater in order to harvest the phosphorus for future work. Also, due to the ease of engineering E. coli, in the future, we can add to our system in order to handle several other aspects often involved in tertiary treatment, such as nitrogen removal, chlorine removal, [16] and antibiotic and pharmaceutical removal [17].

Home Bioreactors

In a bioreactor, bacteria can remove nutrients such as phosphorus and nitrogen [18]. A home bioreactor can use our phosphorus-accumulating E. coli to remove phosphorus from a house’s wastewater before it reaches the treatment plant. In the future, a system for nitrogen removal can also be designed and incorporated into the home bioreactor.

Floating Phosphorus Collector Unit

Dundee 2013 developed a prototype of an electronic environmental sensor to collect and relay real-time data from water reservoirs. We see this design as another platform to apply our modified E. coli in an on-site location. The Dundee design is already modulated with sensors for light, dissolved oxygen, temperature and humidity, and pH. Adding to this design, we could create a compartment housing the bioreactor with modified E. coli for uptaking phosphorus at the contaminated source of water.


[1] US Department of Agriculture,. Anaerobic Digester On Pennwood Dairy Farms. 2012. Web. 3 June 2016.
[2] S. M. Mitchell, N. Kennedy, J. Ma, G. Yorgey, C. Kruger, J. L. Ullman, and C. Frear, “Anaerobic Digestion Effluents And Processes: The Basics,” Washington State University Extension, FS171E, pp. 1–16, 2015.
[3] Wikipedia,. Key Process Stages Of Anaerobic Digestion. 2016. Web. 3 June 2016. [4] “Biogas and Anaerobic Digestion,” PennState Extension. [Online]. Available: [Accessed: 02-Jun-1996].
[5] “Managing Phosphorus for Agriculture and the Environment,” PennState Extension. [Online]. Available: [Accessed: 02-Jun-2016].
[6] DTU Denmark's 2013 iGEM Team, “Requiem for a Stream,” 2013. [Online]. Available: [Accessed: 02-Jun-2016].
[7] Commonwealth Scientific and Industrial Research Organization,. Tile Drainage Pump Flow Into Drainage Channel. 2007. Web. 3 June 2016.
[8] “J. Wright and G. Sands, “Planning an agricultural subsurface drainage system,” University of Minnesota Extension, 2009.”
[9] Panuska, John. "An Introduction To Agricultural Tile Drainage". 2012. Presentation.
[10] K. King, M. Williams and N. Fausey, "Contributions of Systematic Tile Drainage to Watershed-Scale Phosphorus Transport", Journal of Environmental Quality, vol. 44, no. 2, p. 486, 2015.
[11] Nanjing-China's 2013 iGEM Team ,“Atrazine Elf,” 2013. [Online]. Available: [Accessed: 02-Jun-2016].
[12] Annabel. Overview Of Wastewater Treatment Plant Of Antwerpen-Zuid. 2009. Web. 3 June 2016.
[13] “Introduction to Wastewater Treatment Processes,” World Bank Group. [Online]. Available: [Accessed: 03-Jun-2016].
[14] Environmental Protection Agency, "Advanced Wastewater Treatment to Achieve Low Concentration of Phosphorus", Office of Water and Watersheds, Seattle, WA, 2007.
[15] Minnesota Pollution Control Agency, "Phosphorus Treatment and Removal Technology", Minnesota Pollution Control Agency, St. Paul, MN, 2006.
[16] "Stage 3 - Tertiary Treatment", Sydney Water, 2010. [Online]. Available: [Accessed: 04- Jun- 2016].
[17] "Lecture 38: Tertiary Wastewater Treatment", NPTEL IIT Kharagpur Web Courses.
[18] T. Yamashita and R. Yamamoto-Ikemoto, “Nitrogen and phosphorus removal from wastewater treatment plant effluent via bacterial sulfate reduction in an anoxic bioreactor packed with wood and iron,” International Journal of Environmental Research and Public Health, vol. 11, pp. 9835-9853, 2014.