Increasing Lipid Yields in Chlorella vulgaris through Natural Nitrogen Depletion

Objectives/Goals

The objective was to increase the lipid per cell of Chlorella vulgaris through natural nitrogen depletion by 130% compared to the control.

 

Methods/Materials

A sample of Chlorella vulgaris was grown in a homemade photo-bioreactor over two 10 day trials. Cultures in both trials were grown in a vitamin enriched BBM media with varying amounts of nitrogen. The cultures were sampled and those samples were tested for cell counts, using a hemocytometer and microscope, and lipid content, using Nile red dye fluorescence assay. Graphs were made for each trial showing cells per mL, lipid indicated by AFUs, AFU per million cells, and the increase in lipid per cell over the 100% nitrogen control.

 

Results

The lipid per cell increased immensely in the cultures grown in 20%, 10% and 0% nitrogen levels. The highest value was 500% of the control, on day 10 in trial #2 by the 0% culture. This overshot the hypothesis of 130% by almost 4 fold. The 20% culture had the highest overall lipid in trial #2. This culture balanced cell numbers and the amount of lipid per cell.

 

Conclusions/Discussion

The data supported the hypothesis very strongly. The cultures depleted the nitrogen naturally in their media and significant lipid per cell increases were achieved. Cultures started with high nitrogen had the highest cell counts. Cultures started with limited nitrogen yielded the most lipid over a 10 day trial, because they balanced cell count and lipid per cell.

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My name is Gregory Martin and I go to Marshall Middle School in San Diego, CA. I am an avid fencer, and I also love doing science. I became interested in science because it allowed me to not only know the world, but understand it. Science gives an explanation to everything, which I have always sought after. I also love how what we know evolves over time, new hypotheses are made and old theories are disproved getting us closer to the answer. In my future I would love to be a microbiologist or organic chemist. These sciences explain some of the most complicated things in life. I would like to be able to start a company that researches algae based biofuels. If I were to win a prize it would mean a lot to me because it would mean that I was the best project in California.

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The purpose of this experiment is to explore the possibility of naturally depleting the nitrogen of algae’s media to boost its lipid yields. This is an improvement of the normal methods that involve harvesting the algae and physically transferring it into a nitrogen depleted environment. With my technique it should be possible to bypass the transferring step and grow the culture in a single media. This reduces the labor and material costs of growing algae for its lipid.

My hypothesis is that by using natural nitrogen depletion an increase in the algae’s lipid per cell content of 30% is expected. Natural nitrogen depletion is a process where nitrogen is depleted naturally through the algae’s consumption. This method allows the algae to stay in one media while naturally consuming the nitrogen, eventually depleting it. This process is an improvement over normal methods because it retains the higher lipid content but also gives higher cell counts in the later stages. By keeping up these two attributes it is possible that natural nitrogen depletion could yield higher overall lipid compared to that of normal methods. These higher lipid yields are expected because nitrogen depletion has already been proven to increase the lipid in algae, this is just an improved way of doing it.

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Algae is a promising source of lipid for biofuel. It is very efficient at producing oil at up to 5,000- 15,000 gallons per acre per year. Algae’s lipid content can also be easily increased through stressing. Nitrogen depletion is one of the most common forms of stressing and involves depleting the amount of nitrogen in the media. With this technique maximum lipid yields of Nannochlorpsis sp. were 1,400 g/l or 0.7 ml/g. using current techniques for nitrogen depletion, the algae must be transferred from a nitrogen rich media to a nitrogen depleted media. This is cumbersome and costly. 

Rukminasari’s research indicated that nitrogen depletion has a large impact on the lipid yields of the algae cells, up to 700%, but it also has a negative impact on the cell count. This is because when in a nitrogen depleted environment the algae cannot grow and will eventually die off. This loss of cells is not high enough to make nitrogen depletion unfeasible, but it could be improved. If the cell dying could be avoided the overall lipid content would be higher. To do this the cell must remain growing for a portion of the growth time to increase in population but then midway enter a nitrogen depleted environment. This would be conventionally done by harvesting the algae from the media and transferring it into a nitrogen depleted environment. An alternative way would be to let the algae deplete the nitrogen naturally through consumption, this is called natural nitrogen depletion. Natural nitrogen depletion involves adding a specific amount of nitrogen to the media. Because of the algae’s consumption, the nitrogen ideally runs out before harvesting, allowing time for nitrogen depletion. With this method, nitrogen depletion can be used without having to harvest the algae to do so. It also still allows for growth in the early stages, increasing the cell counts in the later stages. This could be one of the needed growing techniques to reduce the cost of algal biofuels.

In doing this project it is necessary to measure the lipid content of the algal cells, to do this a Nile red florescence assay was used. The research done by Wei Chen in, “A High Throughput Nile Red Method for Quantitative Measurement of Neutral Lipids in Microalgae”, evaluates the use of DMSO, an industrial solvent, to improve Nile red’s accuracy for measuring the lipid content per cell. DMSO is used to help the Nile red dye penetrate the cell wall. With the modified Nile red procedure more accurate lipid amounts were achieved compared to gravimetric method. Chen’s research gives detailed instructions on how the Nile red assay is performed. The paper includes dilution ratios for Nile red to DMSO to achieve the strongest fluorescence readings. The paper also documents lipid tests done on the Chlorella vulgaris, the same algae being used in this experiment. This project will follow the Nile red procedures described in Chens’ paper using the same algae, Chlorella vulgaris.

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Variables and Controls

Independent Variable

  • The percentage of nitrogen positive media (N+) that was in the growth media: 100%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 0%.

 

Dependent Variables

  • The florescence of the Nile red dye associating with lipid as measured over a 10 day period.
  • Cell counts per mL as tested over a 10 day period.

Procedures

Testing/growing

  1. Measured amounts of BBM (Bolds Basic Media) with, and without nitrogen were added to each growing chamber to make a total of 90ml at levels of 100%, 80%,70%, 60%, 50%, 40%, 30%, 20%, 10%, 0% nitrogen.
  2. Each tube received 30mL of dense algae culture.
  3. The tubes were placed in a homemade bioreactor under timed fluorescent light with an air stone bubbling air into them.
  4. A lead of air hose ending in an air stone was inserted into each test sample with the aquarium pump providing a steady supply of air. Foam plugs prevented contamination.
  5. Twice daily, the air stone was hit against the wall of the tube to dislodge any algae.
  6. At the end of the test run, the cultures were disposed of, and the tubes sterilized using a mild bleach solution of 1tbs of liquid bleach to 1 gallon of distilled water.

 

Sampling

  1. Air stones were bumped against the walls of the tubes 20 minutes before sampling.
  2. 5 mL round bottom tubes were labeled 1-10.
  3. A 2ml sample was taken from each sample and put into its respective tube.
  4. These samples were used for both the cell counts and the lipid analysis as described below.

Lipid analysis

  1. The fluorometer was turned on 15 minutes in advance to allow the lamp to heat up.
  2. 3µL of Nile red working stock solution (at 50µg/ mL of DMSO) was added to the top four rows of a black Costar 96-well plate.
  3. 5µL of the sample from the culture 1 was pipetted into each well.
  4. A solution of 25% DMSO was prepared in a 50mL conical tube by adding 10mL of DMSO to 30mL of distilled water and inverted to mix, then pipetted using an 8 channel multichannel pipetteman into each well.
  5. The plate was then shaken in a plate shaker at 300rpm set at 40°C for 10 minutes.
  6. The plate was placed in the fluorometer and read at emission set at 530nm and excitation, twice at 590nm at 80 and 50 gain.
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In this experiment Chlorella vulgaris was grown with varying amounts of nitrate or NaNO3. The goal was to increase the lipid per cell through natural nitrogen depletion, a variant of nitrogen depletion, while maintaining good cell counts. If successful, higher overall amounts of lipid could be achieved. The algae cultures were grown in a vitamin enriched BBM media.  A total of 20 cultures were grown over two 10 day trials. In each trial, 10 cultures were grown at nitrogen levels of 100%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 0%, with the 100% culture being the control.

In both trial 1 and trial 2, the 0% nitrogen culture had the highest peak lipid per million cells with 138 AFU/million cells and 321 AFU/million cells on day 5 and 3 respectively. In trial 1 the 70% culture had the highest overall florescence with 1369 AFU on day 7. The 70% culture also had the highest cell count and an average lipid per million cells. The 70% culture did not do well on day 10, where it recorded only 800 AFU. In trial 2 the 20% culture had the highest fluorescence reading 1851 AFU. Surprisingly, in trial 2, the 100% culture had the second highest overall florescence at 1731 AFU. The second trial started and ended with higher lipid values than the first trial.

At the beginning of each trial all the cultures were the same shade of green. By day 3 the last 3 cultures (20%, 10%, and 0%) were visibly less dense than the other cultures, and more of a pale, sickly green. Every other culture was a vibrant green. By day 6 there was an established color gradient. The 100%, 80%, and 70% cultures were a dark mossy green. The 60%, 50% and 40% cultures had a lighter color but still looked healthy. The last four cultures were lighter than the rest with a slight yellow brownish tinge. On day 10 most of the cultures looked the same, except the 10% and 0% cultures which looked almost clear with a green-yellow tinge.

A good number of the algae cultures exceeded the 130% that was anticipated in the hypothesis. The highest value achieved was observed in the 0% culture on the 10th day of trial 2, with a lipid per cell value of 500% of the control value. This was much higher than expected, and well over threefold the amount projected in the hypothesis. However, looking at the overall lipid content, the 0% culture was not as high as the 20% culture or the 100% culture, which had the highest overall lipid. Cultures that had a mid to high amount of nitrogen had lower amounts of lipid and cell counts than those at either extreme. This is thought to be due to them not having good cell counts or lipid per cell. The data also shows that cultures grown with extreme amounts of nitrogen did better than those in the mid ground.

 

 

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In this experiment, two trials of 10 cultures each were grown in a vitamin enriched BBM media with starting levels of nitrogen at 100%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, and 0%, with the 100% being the control. The hypothesis was that by using natural nitrogen depletion it would be possible to get a 30% increase in the amount of lipid per cell over the control culture. In trial 1 the highest yield was 300% of the control value on day 7 by the 0% culture. In trial 2 the highest yield was from the 0% culture on day 10 which reached over 500% of the control’s value. These results were clearly much higher than projected.

Cell counts could have been more accurate. It was recommended to count 5 5x5 squares on the hemocytometer. Because of time constraints, this was impossible, so only 2 5x5 squares were counted. Some of the airstones did not bubble as well as others. In trial 2, the 50% culture almost died after not getting enough air. To ensure that the cultures are getting enough air in equal amounts, more consistent airstones and a stronger air pump might be needed. These factors might have led to non-consistent data but overall the data is strong enough to be not entirely due to errors.  

The lipid gains through natural nitrogen depletion were much higher than hypothesized. The hypothesis was that using natural nitrogen depletion it would be possible to increase the lipid by 30%, to 130% of the control culture. Peak values from the 60%, 50%, 20%, 10%, and 0% cultures were around or above 200% of the control with some as high as 350% and 500%. It is worth noting that when running the lipid assays, the results varied as much as 500 AFU in either direction. Also, the cell counts could be off as much as half a million cells per mL. However, even with these deviations, the peak lipid yields are high enough to suggest that natural nitrogen depletion works. In testing, cultures with a small amount of nitrogen were able to retain higher cell counts during the early to mid-stages. At the end of each trial, they would convert to a nitrogen depleted environment, boosting the lipid per cell. As a result of this, the 30% culture in trial 1 showed total florescence readings of 237 AFU above the 0% culture. In trial 2 the 20% culture got up to 686 AFU above the 0% culture.

Natural nitrogen depletion is an effective way to increase the lipid yields in algae. By using the techniques described in this experiment it should be possible to lower the cost of creating biofuel from algae by eliminating the need to transfer the algae to a separate nitrogen depleted environment. Natural nitrogen depletion could be used as an end step in the growing phase to maximize the yields while keeping costs down. It would reduce the amount of processing but also increase lipid yields.

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I would like to give thanks to my parents and my science teacher Mrs.Gillum. They were my editors, they stayed up late to keep me motivated and took time out of their life to help me. Without my mom there is no way I would have actually made science fair. She was able to lend lab equipment to me without which the project would have been impossible. My mom was able to loan me hemocytometers, a microscope, pipetteman, pipette-aid, kimwipes, gloves, and assortment of conical tubes.

 

Bibliography

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Chen, Wei, Cheng wu Zhang, Lirong Song, Milton Sommerfeld, and Qiang Hu. "A High Throughput Nile Red Method for Quantitative Measurement of Neutral Lipids in Microalgae." Journal of Microbiological Methods 77.1 (2009): 41-47. Print.

 

Croft, M. T., M. J. Warren, and A. G. Smith. "Algae Need Their Vitamins." Eukaryotic Cell 5.8 (2006): 1175-183. Print.

 

Frans, Annaliese K. "Phenotypic Screening with Oleaginous Microalgae Reveals Modulators of Lipid Productivity." ACS Chemical Biology 8 (2013): 1053-062. Print.

 

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Javanmardian, Minoo, and Bernhard O. Palsson. "High-Density Photoautotrophic Algal Cultures: Design, Construction, and Operation of a Novel Photobioreactor System." Biotechnology and Bioengineering 38 (1991): 1182-189. Deep Blue. Web. 3 Nov. 2013.

 

Kalpesh, Sharma K. "High Lipid Induction in Microalgae for Biodiesel Production." Energies 5 (2012): 1532-553. Print.

 

Kubler, Janet E., Andrew M. Johnston, and John A. Raven. "The Effects of Reduced and Elevated CO2 and O2 on the Seaweed Lomentaria Articulata." Plant, Cell and Environment 22.10 (1999): 1303-310. Print.

 

Maxwell, Denis P., Stefan Falk, Charles C. Trick, and Norman P. A. Huner. "Growth at Low Temperature Mimics High-Light Acclimation in Chlorella Vulgaris." Plant Physiology 105 (1994): 535-43. Print.

 

Rukminasari, Nita. "Effect of Nutrient Depletion and Temperature Stressed on Growth and Lipid Accumulation in Marine-green Algae Nannochloropsis Sp." .American Journal of Research Communication USA Journals.  2013. Web. <http://www.usa-journals.com/wp-content/uploads/2012/11/Rukminasari.pdf>. 9 Sept. 2013.

 

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