Team:Lambert GA/NutrientMix
BACKGROUND
Testing nitrate and nitrite levels in an aquaponics system is vital to maintain a balance between the fish’s excretion of ammonia, which is converted to nitrite then eventually nitrate, and the plant’s uptake of those nutrients. The goal was to find a range of nitrite and nitrate concentrations that is not toxic to Lambert iGEM's Nar biosensor E.coli cells while being suitable for the plants and fish in the system. In order to be consistent with the Nar workflow, the team decided to test the effects of specific nitrate and nitrite concentrations at 0ppm to 300ppm because this range included the values measured by paper strips in the team's hydroponics and fish tank. The fish produced an average nitrate level of 250 ppm while 0ppm of nitrite. For the hydroponics system, the nitrite levels were approximately 10 ppm, and nitrate was above 250 ppm. Since the relative maximum was 250 ppm and the relative minimum was 0 pp for all nutrients, the team decided to extend the testing range to 300, allowing for more data regarding the effects of nitrate and nitrite on cells.
EXPERIMENTAL DESIGN
Lambert iGEM's experimental design initially started by creating a 6M concentrated stock solution for nitrite. The stock was then diluted to 4 molar concentrations in liquid culture tubes: 0M, 3M, 2M, 1M, and a control. After growing the liquid cultures for 12 hours, the 2M and 3M cultures showed no visible sign of cell growth. Following the same dilution method, the testing concentrations were 1M, 0.8M, 0.6M, 0.4M, 0.2M, and 0M. Despite showing some OD values, the results were overall inconclusive due to the immense variation in data. The same process of diluting and checking OD was repeated for 100um to 0um as the large intervals did not allow for a pattern to be observed. The team decided to change its test intervals from 20 to 40 due to the insignificant difference in OD600 measurements of cells, allowing for a wider range of data. In addition to this interval change, the team adjusted its original protocol to include technical triplets, testing for both nitrite and nitrate. This experiment gave an ideal value of 80µm before the graph dipped. The experiment was then repeated to measure nutrient concentrations in ppm rather than µm. In this experiment, the concentration of cells was measured before nitrite and nitrate were added. At both twelve and sixteen hours after the addition of nutrients, the concentration of cells was measured to test how nitrite and nitrate affected cell growth. As seen by the graph, the addition of nutrients allowed growth in all measured concentrations.
RESULTS
The nitrite results showed a significant increase in cell growth over the 16 hour period, with the greatest amount of growth occurring between 12 hours and 16 hours. Based on Figure 1 , 300 ppm resulted in the greatest increase in cell growth, which is followed by 120 ppm. 0 ppm was not included in our nitrate testing due to contamination during the dilution process. The results still showed that as time passed and nitrate concentrations increased, cell growth also increased. Based on Figure 2, 120 ppm shows the greatest increase of cells at the end of the 16 hour period. Figure 2. additionally shows that the overall nitrate concentration was higher than the overall nitrite concentration.
Figure 1. The graph above shows the different concentrations of nitrite levels over 16 hours and the effects on OD concentrations.
Figure 2. The graph above shows the different concentrations of nitrate levels over 16 hours and the effects on OD concentrations.