Study of Nitrobacter and Spirulina Algae for Conversion of Nitrites and Nitrate Salts to Harmless Gases.

Abstract

The goal of my project is to find a way to remove the NOx emissions from air, particularly those emitted from automobile exhaust. NOx is one of the most dangerous pollutants in existence, not only being highly corrosive and toxic itself, but also catalyzing the formation of ground level ozone from oxygen. The system that I have designed converts the NO2 into nitrite and nitrate salts. To remove the salts, biological methods were used. Nitrifying bacteria convert nitrites to nitrates and denitrifying organisms convert nitrates to nitrogen gas. The nitrifying bacteria used was nitrobacter and the denitrifying organisms used was spirulina platensis, a type of algae. A photonic density measurement device was designed to measure the algae growth. A beaker with water containing nitrobacter and algae was initially set up. Then a salt solution with nitrite and nitrate which was obtained from step 2 of the experiment was added to this beaker. This solution had nitrite at 5 ppm (parts per million) and the nitrate was at 10 ppm. The volume of the solution was 325 mL. A nitrite and nitrate reading was taken every 12 hours and the growth of algae was measured every 6 hours using an arduino based photonic density device. Both the nitrite and the nitrate readings fell to zero after 5 days and the algae grew.

Background

Nitrobacter converts the nitrite salts to nitrate salt. It oxidizes the nitrite ion into the nitrate ion (Ferguson and Nicholls, 2013). To do this, it first reacts water with the ion. The oxygen is transferred to the nitrite ion, converting it to nitrate. This reaction leaves 2 hydrogen ions and two electrons. These ions can then be used by the cell's mitochondria to produce energy. The oxidation reaction is as follows,

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The hydrogen plus ions and the two electrons can be reacted with oxygen gas from the atmosphere to produce water and energy. The reaction is as follows,

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These reactions combined look like this,

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This reaction gives , 74kJ of energy per mole of nitrite.

As well as using nitrite as an energy source, nitrobacter is also a carbon sequestering bacteria, taking in carbon dioxide to fulfill its carbon needs. This makes it an optimal choice for nitrite removal.

Spirulina converts nitrate to nitrogen gas (Bothe et al., 2007). The equation can be written out as follows.

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This reaction has four parts to it. Each requires a separate enzyme. First, the nitrate is converted to nitrite. This causes the combination of electrons, hydrogen cations, and the liberated oxygen to produce water. This is performed by nitrate reductase, Nar.

This charge difference allows the cell’s mitochondria to create ATP. Then, the nitrite is converted to nitric oxide. This liberates still more oxygen, creating water and allowing ATP synthesis. This is performed by nitrite reductase, Nir.

After that, two molecules of NO are converted to one molecule of N2 O, nitrous oxide. This is performed by nitric oxide reductase, Nor.

This liberates yet another oxygen which the cell uses in the same fashion. The final step is taking the oxygen from the nitrous oxide, converting it to nitrogen gas and using the oxygen for energy. This is performed by nitrous oxide reductase, Nos.

This process can release small amounts of NO and N 2O, however, it is a small amount and both NO and N2 O can be reused by other algae to produce N 2 and energy.

Culture Creation

The algae was purchased as a small starter colony with a growth media. The nitrobacter was purchased as a fish tank supplement. It was contaminated with nitrosomonas, however, since there was no ammonia in the solution, the nitrosomonas should have quickly died off. The algae was started in a small colony that is shown in figure 1. A nitrobacter colony was also set up in a fish tank full of distilled water. It is shown in figure 2. Nitrobacter will die if exposed to sunlight or other bright light sources within four days of moving to a new habitat.

To keep the nitrobacter alive, KNO2 was added to the

tank. The KNO2 was made by heating KNO3 to make it

decompose into oxygen and KNO2 . Since algae need sun to thrive and the nitrobacter need to stay in the dark, the two were grown separately, then combined. The algae colony was left to grow from December 1st 2019 to December 15th 2019 in a sunny spot. The nitrobacter was given the same amount of time under an aluminum cover. Then, a new fish tank was filled halfway with distilled water, a quarter way with the nitrobacter tank’s water, and most of the algae was poured in. A small amount was left in the original bottle in case something went wrong.

The tank was left in a sunny spot to grow. Figure 3 shows the algae on Jan 15th 2020. There was a large amount of growth but the algae was not growing fast. This was because the light source, the sun, was not strong enough. So, instead, I set up a light with an automatic timer. This drastically increased the growth, in a few days, the tank was fully green. The bubbling comes from the increased

creation of N2 and O2 gas from denitrification and photosynthesis respectively. The tank is shown in figure 4. To further help the growth rate, a fish tank heater was added to keep the temperature at 78 ˚F. While the optimal temperature is around 90 ˚F, the beaker was kept closer to the optimal than it would have been without the heater.

Experiment

When all the other parts of the experiment were complete, a small colony of algae was scooped out of the larger tank with a spoon and put into a beaker filled with 200 mL of distilled water. The algae clump was then broken into smaller pieces with a glass stir rod. More nitrobacter containing water was added to the beaker and 250 mL of space were left. The salts produced earlier in the project were added to the beaker and the volume was topped off to 325 mL with distilled water. The salt that was made from the

NO2 had a nitrite level of 5 ppm and a nitrate level of 10 ppm. The beaker is shown in figure 5. To take the measurement, other devices were connected. The setup with all the parts is shown in figure 6. A diagram of the system is in figure 7.

The system was then left on for 5 days. Every day at 6am, midday, 6pm and midnight it took a reading of the optical density, the murkiness of the water. Every 12 hours, a nitrite/nitrate test strip was dipped in the solution. This happened at 8am and 8pm. The experiment ran for 4 days before the nitrite and the nitrate level reached zero.

Results

Graph 1 shows the nitrite and nitrate level. It took 4 days for the nitrite level to reach zero and 4 for the nitrate level to reach zero. Since the nitrite and nitrate chart shows the values in larger increments, I estimated the value based on the average of the two

values the strip was closest to. I also used the value of other strips for this process, for example, the value 2.5 was calculated from the fact that the strip was between 5 and 0. The average of these values is 2.5, so I estimated that the strip, whose color was in the middle of the two, was 2.5. The data from the optical density measurement device is shown below in graph 2. The blue line shows the difference between the optical density above and below the beaker.

Discussion

Graph 1 shows the data from the nitrite and nitrate test strips. The salt that was made from the NO2 had a nitrite level of 5 ppm and a nitrate level of 10ppm. The tank, however, already had leftover nitrate and nitrite from the KNO 2 that was added. This is why the initial nitrite and nitrate ppm are so high. The lines are not linear, because the nitrobacter converts the nitrite to nitrate, adding to the nitrate line. The algae destroy the nitrate. The nitrobacter are slower at metabolizing the nitrite, it took 24 hours for them to start consuming it. The algae, however, are much faster, destroying 58% of nitrate within 12 hours. After 24 hours, the nitrobacter started to convert the nitrite to nitrate, so the nitrite level drops during the next 12 hours but the algae compensate for the excess of nitrate. This happened overnight, when the algae are least active so the two effects cancelled out and the nitrate remained constant. When the light turned back on in the morning, the algae sped up their metabolism and the nitrate level went down. The nitrite level also decreased. The metabolism rate for the nitrobacter went down as the nitrite concentration went down, because nitrite conversion is the only energy source for the

nitrobacter. The same is true for algae, but not quite so drastically, because algae can photosynthesis for energy as well. While the nitrite and nitrate value was most likely above zero at 8pm day 5, it was so low that the value is practically undetectable. Graph 2 shows the difference in optical density across the algae. The algae grew mostly during 6am to 6pm, when the light was on. The algae feed on nitrate, and each night the nitrobacter would convert the nitrite to nitrate. This meant that each morning, when the algae were working, there was always nitrate for them. This went on until day 3 between 6 pm and 12 am. At that time, the nitrate value dropped close to zero and so did the nitrite. This caused the growth to slow considerably, causing the algae to grow mostly due to photosynthesis, not denitrification.

Conclusion

The nitrobacter and algae successfully destroyed the nitrite and the nitrate. They did this over the course of 96 hours, 4 and ½ days. In the future, I would like to test the nitrite and nitrate electronically, allowing me to take measurements every 6 hours, instead of every 12. The optical density measurement device was a success, it took the measurements every 6 hours. In all, since this is the final step in the entire project, the entire project was a success.

References

● Nicholls , D. G., & Ferguson, S. J. (2013). Respiratory Chains. In Bioenergetics (Fourth Edition) 2013 (4th ed., pp. 91–157). Academic press. doi: https://doi.org/10.1016/C2010-0-64902-9 ● Bothe, H., Ferguson, S., & Newton, W. E. (2006). Biology of the Nitrogen Cycle (1st ed.). Elsevier Science