Ocean Iron Fertilization Project

[donnahardy2]

Hi Oriculum,

You have good questions.

1. I assume you have received an order for phytoplankton that has been actively growing before it was shipped to you. The algae will have to equilibrate for a day or two, and then start growing again in your ocean water samples. Phytoplankton will grow logarithmically within a short time if nutrients are available, but I'm not sure what the doubling time will be. A 1 ml sample in 1 liter would give you a 1:1000 dilution and a 10 ml sample diluted into 1 liter will give you a 1:100 dilution. If you have at least a week or two to conduct your experiment, I assume that this would give you a good starting number and there should be a difference in the growth curves with different iron concentrations. If you add a higher volume of sample, there will be fewer generations of phytoplankton to measure during your experiment. I have been looking for a reference that gives the generation time for phytoplankton, but I have not found one yet. This would be something good to look for.

2. Yes, why don't you try a centrifuge test with your sample and see if you can measure the volume with the equipment you have available? If you can measure phytoplankton volume, then you won't have to do the more time-consuming microscope counting project. Did the company that you ordered the sample from give any information about the numbers of phytoplankton in the sample? If not, call or e-mail the company and ask.

3. Yes, there should be a correlation between the mass of phytoplankton and the amount of CO2 consumed. This is another reference that you need to find (I haven't tried looking for this yet). But go ahead and proceed with your experiment, and I'm sure we'll find a conversion number to use before you have to turn in your project. This will be valid if you use a reference from the scientific literature, since you won't be able to measure CO2 directly.

4. The easiest way to check pH of water samples is with pH paper. You could check the pH on day one, and periodically during the experiment. It's difficult to measure the pH of water with a pH electrode, unless you have a special one that is designed for use with samples with low buffering capacity. pH paper would be good enough for your experiment. Do you have pH paper available?

Donna Hardy

[oriculumn]

Thank you for all the information!

Unfortunately, the company that sold me the phytoplankton did not give any information about the numbers of phytoplankton in the sample, and after emailing the company, I still haven't received a reply.

I'm not sure if I have pH paper available, but I will ask my teachers ASAP.

Thank you so much again!

[donnahardy2]

Hi Oriculum,

If you make a 1:100 dilution of the phytoplankton, your sample will be able to multiply for about 6.5 generations; if you make a 1:1000 dilution, the sample will be able to multiply for 10 generations, if the nutrients in your original sample are similar to the phytoplankton culture. I recommend trying the 1:1000 dilution.

Donna Hardy

[oriculumn]

Oh, my Biology teacher told me that phytoplankton need oxygen to survive and suggested that I use an oxygen pump (like for fishtanks) for each sample.

She also said to use empty plastic bottles filled with 1L seawater, phytoplankton, and then add iron to it. Punch two holes into each cap of each plastic bottle, and then stick some tubing into one of the holes that'll connect the oxygen pump to each bottle.

Is this a good plan to follow?

Wouldn't this affect the dissolved oxygen data?

[donnahardy2]

Hi,

Your biology teacher's suggestion to add oxygen to each container is excellent. The phytoplankton will grow much better, and the mixing that occurs will be similar to the natural mixing that occurs with the tides in coastal waters. The extra oxygen will affect the dissolved oxygen levels in the water samples, but since you will be setting up all of the bottles the same way, this will be a controlled parameter. The only factor that you want to vary in your samples if the iron concentration. If you will be measuring oxygen levels, then oxygen level is one of your dependent variables, along with the phytoplankton mass. You will just measure and record your results.

You topic is very timely. Here is an article on phytoplankton and global warming:

http://earthobservatory.nasa.gov/IOTD/view.php?id=7187

Donna Hardy

[oriculumn]

Oh, thank you so much!

I've run into another problem. I read through the instructions from the phosphate and dissolved oxygen kits used to measure the amount of phosphates and dissolved oxygen. Basically, I'll be needing about 40mL of water from each bottle for every time I want to test for phosphate and dissolved oxygen, and once the chemicals from the kits are used, I can't put the water back into the bottle; it needs to be disposed. I'm not sure what I can do about it now.

Would this affect my data? According to my calculations, if I were to continue with the daily tests for 2 weeks, I'll use up 560mL of water from each bottle.

What can I do to keep a consistent project? Should I just continue with daily tests despite the reducing amount of water in each bottle? or is there an alternative to testing for phosphates and dissolved oxygen?

[donnahardy2]

Hi,

I don't think it would be a good idea to use up half of your sample in testing. How much water are you going to remove for the phytoplankton measurement? I think you need to do daily testing on the phytoplankton levels, but perhaps for the oxygen and phosphate analysis, you could take a measurement at time zero, 7 days, and 14 days. That would give you an overview of the results without using up a significant volume of your sample.

Have you done a trial run on phytoplankton measurement? I recommend doing this before you set up your experiment. I think you were going to take a sample of water and centrifuge it and measure the volume of the phytoplankton pellet. Is this correct?

Donna Hardy

[oriculumn]

Hi,

I was thinking of taking 5mL of each sample for phytoplankton measurement. Would that be enough?

I am about to do the trial run on phytoplankton measurement. Yes, I plan to take 5mL of each water sample and centrifuge it, and then measure the volume of phytoplankton pellet.

Thank you so much!

[donnahardy2]

Hi,

Why don't you do a trial run on 5 ml and see if that is enough? You might not get enough to measure at first, but it would probably be plenty for later samples. If it takes a larger volume for the phytoplanton measurements, you might have to make adjustments in the frequency of your sampling, or the initial water volume.

Donna Hardy

[oriculumn]

Hello! I'm now on the calculation portion of my experiment. I can't exactly find the correlation between CO2 absorption and phytoplankton growth. Could I please get some help with this?

Also, could anyone suggest any extra calculations I can do to improve my project? I've only measured pH, phosphate, and dissolved oxygen content. So far, it's basically been the same. The pH became slightly acidic. There was no difference in phosphate, and some samples lowered the dissolved oxygen content.

[donnahardy2]

Hi Oriculum,

I'm sorry I suggested that there was a correlation between CO2 consumption and phytoplankton growth. There is, of course, because phytoplankton use CO2 as a carbon source to grow, but I can't find a paper where this is quantitated. Why don't you send an e-mail message to the author of the original paper and ask this specific question? Here's another website with author contact information. I think you need a real subject matter expert for this part of the question.

http://www.pubmedcentral.nih.gov/articl ... id=1913777

Don't worry about your project write up if you don't get an answer. If your phytoplankton increased in volume or numbers, you can still conclude that that more CO2 was consumed, because it is a known scientific fact that plants use CO2 for growth.

However, it sounds like you have some excellent results. You can plot the pH, phosphate, and dissolved oxygen. It's OK that there's no change. Data is data, and you can plot those results. You should have seen an increase in the phytoplankton volume. What did you observe with the phytoplankton results?

Donna Hardy

[oriculumn]

Well, what had actually resulted was an increase in the amount of phytoplankton over the course of one week. Afterward, the amount of phytoplankton began to decrease. This probably has something to do with how when phytoplankton decompose aerobically, they use oxygen and release CO2, which lowers the pH of the water. CO2 is probably being released rather than sinking to the bottom since the vessel used (plastic bottle) is not deep enough or because of the decrease in water volume after each centrifuge test.

The amount of phytoplankton unfortunately was too small to actually record the volume. If were to have used larger water samples to centrifuge, I would have run out of water for each sample. Is there another way to calculate the volume of phytoplankton? or should I just observe any general growth or decline in phytoplankton amount?

I looked over my results again, and I realized that I had made a mistake with the phosphates. The amount of phosphate actually increased over the course of two weeks. I'm not sure why though. I couldn't really find anything related to an increase in phosphates in my research. Could you explain or send me a link about this?

I'm assuming the fluctuations in dissolved oxygen was due to the differences in amount of oxygen pumped into each bottle. Sometimes, when I would check on the experiment, no oxygen would be pumping into the bottle. This could be one reason for the fluctuations, but are there any other reasons?

Also, I'm not sure how I should display all of my phytoplankton data. Should I display one graph for each day, displaying an increase or decrease in phytoplankton amount depending on concentration? or Should I display one graph for each concentration of iron, displaying an increase and/or decrease in phytoplankton amount per day? Which would be best?

Since I did 3 simultaneous trials, should I average out this data for the graphs? or should I separate them?
or if there's a better way, could you give me suggestions?

[donnahardy2]

Hi Oriculum,

If you couldn't measure the volume of the phytoplankton then do include as much information as you can, but be sure and mention that you would have made this measurement quantitative if you could have. Do you still have the samples available so you could at least measure the volume of each concentration of iron at the end of the experiment? You could use the whole sample if you needed to compare results if needed. If not, then just make the best qualitative description that you can.

How much did the phosphate increase? What kind of containers were you using? I would have guessed that the phytoplankton would have used phosphate and incorporated it into the cells. Did you filter out the phytoplankton before you measured phosphate in the water, or were the phytoplankton part of the phosphate samples? What method of phosphate analysis were you using? This is an exceptionally interesting result, and I'm sure there is a good explanation that will make your write up fascinating, but a little more information is needed to figure this one out.

You need to find out why the phytoplankton numbers decreased. One possibility is that phytoplankton will undergo autolysis (breaking down) when they are under stress. Here’s a reference that includes a little information on this. If you are sure that the phytoplankton disappeared, then this would explain the phosphate increase also, because the cells would release all of the cell components, including the phosphate that is part of DNA and molecules used for energy transfer like ATP. If you use this explanation, you will need to explain why your phytoplankton were so stressed.

http://www.sciencedirect.com/science?_o ... 5cc78df9da

Here’s another reference that includes information on phytoplankton autolysis:

http://www.sciencedirect.com/science?_o ... 29601a1f44

I'm sure the oxygen levels were a reflection of the aeration of the sample, and you would expect it to decrease if the pumps stopped working. This would definitely affect your results if it happened frequently.

For your data, it would be best to put everything on one graph with the x axis containing:
1. estimate of phytoplankton numbers.
2. oxygen levels
3. phosphate levels


The y axis would be time.
Use a different color line for every concentration of iron and the control. To make it clear make all 3 replicates similar and label each line clearly. If the graph looks too complicated, then average the 3 replicate results so you have just one line for each concentration of iron. Do a rough draft and show it to someone who is not familiar with your project and see if understand what happened. The science fair judges will spend a minute or two looking at your results, and you want your presentation to be very clear and concise. This graph is the most important part of your project.

You have some excellent results and your presentation of the results and analysis and discussion of the data could make this an exceptional project. Do you need any additional information?

Donna Hardy

[oriculumn]

Thank you so much! And yes, I will be needing additional information.

What sort of qualitative description could I use for the amount of phytoplankton?

Well, on Day 0, the phosphate was a 0.3ppm. By Day 14, some had increased to about 1.5ppm. I was using plastic 1L bottles in this experiment. I did not filter out the phytoplankton when taking the phosphate measurement. I was using a LaMotte Phosphate test kit to test for phosphates. So, what could have caused this increase?

I'll research more on what could have caused the stress on the phytoplankton.

I got lost on the explanation of the graph. So, I should put everything (phytoplankton, pH, oxygen levels, phosphate levels) on the x-axis? How do I do that? Wouldn't the graph look quite cluttered with all the different lines?
So, in the end it'll be one graph per sample? I'm still very confused. Sorry.

[donnahardy2]

Hi Oriculum,

You are right; putting everything on one graph will be too cluttered. Here is an example of a line graph, which will work with your data:

http://www.ais.msstate.edu/AEE/Tutorial/pdfs/line.pdf

You should put each data set on one graph, so you will have one graph for pH, one for phosphate, and one for oxygen. Look at the example, and label the graph clearly. For example, for the pH chart, the title will be "pH change"
the y axis will be "pH," the x axis will be time. You will have one line for each concentration of iron. You should have a key identifying the concentration of iron (e.g. use green (or circles) for the control, red (or squares) for the lowest concentration of iron, etc.) You will have one line for the control, and one for each concentration of iron. This will unclutter the data and communicate your results clearly.

Now for the phytoplankton. Your results are qualitative, but you could make another line graph, and label the graph "Phytoplankton population estimation," label the y axis "approximate phytoplankton numbers, with an arrow pointing up". Then mark in the approximate population for every time point day, using the same scheme to identify you used for the other graphs. Anyone looking at your 4 graphs will be able to sort out the results for each sample. Does this help, or do you need more help on graphing?

Now a question about the disappearance of the phytoplankton. Did the phytoplankton disappear from the control sample and from each concentration of iron? If so, then they may have starved to death due the lack of a critical nutrient, possibly nitrogen or silica. Nitrogen is used to manufacture proteins and silica is used for the support structure of phytoplankton, kind of like human bones. Or, maybe something else was growing in the sample that may have been toxic. For example, here is a website that summarizes a PhD thesis done on the interaction and competition of various marine microorganisms. The term used to describe this is allelopathy, or inhibition of plant growth due to biomolecules released by other organisms.

http://www.fimr.fi/en/ajankohtaista/mtl ... sertation/

The phytoplankton had enough oxygen, and plenty of light, so these parameters were not limiting the growth.

Another possibility. I think you said that you collected the samples some time before you started the experiment, so something else may have been growing in the samples and using the nutrients before you added the phytoplankton. Was there any difference between the no-iron control sample and the samples with added iron? If there was some growth in the controls, then perhaps the concentration of iron was too high.

The increase in phosphate concentration supports the theory that the increase in phosphate was due to the autolysis (death) of the phytoplankton. Did the phytoplankton disappear shortly after you started the experiment, or did they die gradually over several days. What difference in the samples did you see that indicated the phytoplankton were disappearing?

Since adding iron to your samples did not increase the growth of phytoplankton, as you had proposed in your hypothesis, you should include an analysis that would explain what happened, and why your phytoplankton did not grow. You can add a short paragraph to state what you would do differently in the next experiment to prove your original hypothesis.

Does this help?

Donna Hardy