Category Archives: Eberhard, Ean

Multiple Physiological Factors Underly Phytoplankton’s Response to Stress

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— Written by Ean Eberhard

When looking at the cell abundance across three light intensities, Low (60µmol(photon)/m2/s), Medium (400µmol(photon)/m2/s), and High (800µmol(photon)/m2/s) and four different temperatures, I found that the light intensity does have an effect on the cell abundance. At the lowest light intensity, the highest temperature of 30°C showed to have very little growth while all other temperatures steadily grew. However, when the light intensity increased at the highest temperature the cell abundance began to increase. This alone suggest that there is a complex response of phytoplankton to multiple stressors. If one was to do an experiment where all temperatures were only tested across the same low light intensity, then he would conclude that when temperatures are high there is very little growth in cell abundance and thus high temperatures are detrimental to phytoplankton. This statement is not true however because as stated previously, as the intensity of light increased, the high temperature cultures were growing well. The common trend across all three light intensities was that the 25°C temperature had the highest cell abundance. Now what about the growth rate?

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When looking at the growth rates across the four different temperatures I found a common trend that the lowest light intensity had the slowest growth rate. This low growth rate can be compared to the cell abundance and show a very steady increase in number. The other common trend is that the growth rate of both medium and high light intensities seems to be pretty similar across all four temperatures. Both these higher light intensities grow fast quick then decline in growth rate not soon after especially at 25°C. At 20°C the higher light intensity cultures do not decline in growth rate nearly as fast but also have smaller cell abundance. Again, this proves that there is an interaction between both temperature and light intensity in relation to both cell abundance and cell growth. So, we know the relationship between cell abundance and growth rate but what about the cell size? When the abundance increases are the cells increasing in size or decreasing in size?

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Taking a look at the cell size compared to the cell abundance across the three light intensities and four temperatures I found a general trend of a lower cell size when there is a higher abundance. However, there is more to look at. In the acclimation phase at the lowest light intensity there is reason to note that at the highest temperature of 30°C the cells abundance increases the least, as stated before. Although the cell abundance is not increasing the cell size is significantly increasing. The exact reason for this is unknown and further analysis is needed however, I can assume that the cells are allocating its resources towards cell growth and not cell division. This increase in the individual cell size may mean a greater number of chlorophyll, increasing the cells chance to obtain more light (as we know they want at the low light intensity and high temperature treatment) and grow in abundance.  The cell abundance in medium light shows that 30°C and 25°C have similar abundances at experimental day two but the 25°C has a much larger cell size. This means there is higher biomass at 25c. This higher biomass means that at a higher temperature there is less biomass. However, at the high light, the highest temperature of 30°C has the highest biomass as they have the largest cells while similar to the 25°C abundance. At medium light intensity, the 25°C has a higher biomass until we increase the light intensity and find that the 30°C has higher biomass. This again shows a more complex interaction between stressors; the higher light intensity counteracts the original assumption that the higher the temperature the lower the biomass. It seems that with a high light intensity and high temperature the cells seem to be doing fairly well in regard to biomass. The question is, is this biomass good biomass or not (quality food or bad food, this could affect the trophic cascade). In some cases, such as in medium light, the high temperature means less carbon will go into the system which means less carbon for trophic levels and less absorbed from atmosphere. In high light we can see that the 15 and 20 have larger cells than the 25 who has the highest abundance. Yet the highest temperature at highest light intensity is doing best, this means that with different combinations of stressors we have different complex responses. Again, we can ask are those larger cells better and more efficient than a bunch of smaller cells?

Does that mean that its producing more of lower quality cells? A further chemical analysis of the cells material composition is needed to understand the quality in regard to trophic cascade. Finally, I can take a look at the QY Max values of each culture to get an idea of the cells efficiency within its environment.

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A look at the QY Max values across the four temperatures at the three light intensities shows a general trend of higher light intensities across all temperatures had the lowest efficiency while the low and medium intensities would trade places for most efficient dependent on the temperature. When I compare the 25°C high light intensity treatments efficiency and cell size I find that it has a very small cell size with a low efficiency however when I compare this to the cell abundance I find that this treatment has a very high cell abundance. If one was to simply use cell abundance as proxy for the health of the phytoplankton than he would not have the full picture and say that these cells are doing very well, however, they are not. Another finding is that in the high light intensity at 20°C I found that as the cell size increases the cell efficiency also increases. This means that an overall finding is that the lower the cell size the lower the efficiency and vice versa.

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In conclusion, it is clear that one cannot simply look at one factor of a phytoplankton’s cell response to stress when determining its health. There are multiple physiological changes to consider and that must be compared to one another to effectively interoperate what is happening to the cells under stress. It is also clear that there is a complex response induced by interactions between multiple stressors that are not simply addictive.  We know this because the combination of High light and high temperature was actually positive for cell abundance and because if one was to do this experiment at one light intensity they would find that the high temp is detrimental however that is not always the case, high temperature at high light intensities means high growth.

There is still plenty to be considered and analyzed in this experiment. A few things include POC (particulate organic carbon), where we can ask about C/N ratios and how much carbon is being taken up by the cells or is the biomass of the same quality? Secondly, we should consider biogenic silica and ask about the difference in cell structure. One other consideration is looking at the light curves where we can ask about photosynthesis rates and whether CO2 is being processed well by the cells. There are plenty of other questions to be answered and are underway. This was only the beginning.

Method to the Madness

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— Written by Ean Eberhard

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The experiment takes place in a cold room where the two multicultivators are shown side by side. Both are connected to the CO2 tank to the right which supplies a constant amount of CO2 to the 16 tubes.

The experimental design begins with the creation of artificial sea water. To this I add three different nutrient solutions; main stock, trace metal stock, and vitamin stock. This combination of nutrients and artificial sea water makes up F/2 media used for growing the culture of Phytoplankton that is specifically studied in this project. The name of this species is Thalassiosira pseudonana. Once the culture is healthy and acclimated the experiment can proceed. The experiment is split into two weeks, each week being split into two phases. The first phase is a three day long acclimating phase for the cultures to be monitored in their tubes. Monitoring the culture at this time assures that each tube is growing healthy before entering the second phase of 4 days, the experimental phase. The first week consist of two multicultivators (seen below), one set to maintain 15°C and the other 25°C. Each multicultivator contains eight tubes that are each inoculated with the same culture of Thalassiosira pseudonana. In each multicultivator there is a set of three tubes at low light intensities of 100µmol(photon)/m2/s, 200µmol(photon)/m2/s, and 300µmol(photon)/m2/s, a set of two tubes at medium light intensities of 400µmol(photon)/m2/s and 500µmol(photon)/m2/s, and a final a set of

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When taking a closer look at an individual multicultivator, one can see the aerators placed into each of the eight tubes. Behind the tubes are a set of LED lights which will be set to different intensities when running.

three tubes with high light intensities of 600µmol(photon)/m2/s, 700µmol(photon)/m2/s, and 800µmol(photon)/m2/s. Each tube is supplied with a continues flow of CO2 at 1000ppm with a pressure of 2psi. The multicultivators are set to begin a light cycle at 5am and go into a dark cycle at 5pm. These two multicultivators are designed to give us a total of 16 different environments for the phytoplankton to grow in, varying initially by temperature and light.

 

With the experiment started, the sampling begins. Each day, from each tube, I sample for, Non-photochemical quenching, light curve, chlorophyll, particulate organic carbon, nutrients, and pH. For the beginning, middle, and end of each experiment I sample for dissolved inorganic carbon, biogenic silica, and flow cytometry.

Sampling for Non-photochemical quenching and Light Curve

When sampling for NPQ and LC3, I remove the tubes and invert three times then pipette 3ml of the cultures from each tube into a cuvette, giving me a total of 16 cuvettes for NPQ measurements and 16 cuvettes for LC3 measurements. The cuvettes are placed in complete darkness for 30 minutes for a dark adaptation. After the 30 minutes the cuvettes are individually placed into an AquaPen, which will give a value for NPQ and LC3.

Sampling for Chlorophyll, Particulate Organic Carbon, Biogenic Silica, and Nutrients

Using a filtering apparatus and filter papers, I collect samples for Chlorophyll, POC, and Bsi by filtering the NPQ and LC3 samples. Each tube gets its own filter for Chlorophyll, POC, and Bsi. The Chlorophyll and Bsi samples are stored in the freezer for a later analysis while the POC filters are incubated and left to dry. The filtrate from these tubes are pooled together by light intensity, the low light intensities (100, 200, 300µmol(photon)/m2/s) are combined for a nutrient sample while the medium light intensities are pooled together for another nutrient sample and finally a third nutrient sample is taken from the high light intensities of 600, 700, and 800µmol(photon)/m2/s. These nutrient samples are also stored in the freezer for later analysis.

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The filtering apparatus uses a pump to create suction. A filtering paper specific to the material being collected is placed on a surface above the vacuum. The samples of NPQ and LC3 are filtered through the papers. The filtrate is collected for nutrients below and the material collected rest on the filter paper.

Sampling for Flow Cytometry

For flow cytometry measurements I pipette 1ml of the culture from each of the 16 tubes. I then fix these samples with 50µl of CH2O to kill the cell while preserving its structure. These samples are then stored in the freezer until processed and counted with the flow cytometer.

Sampling for Dissolved Inorganic Carbon

To sample for dissolved inorganic carbon (DIC) I fill a serum bottle full of the culture from each tube and add 50µl of mercury chloride. This gives me a total of 16 samples. The samples are then crimped shut to exclude gas exchange and stored in freezer for later analysis.

Sampling for pH

Sampling for pH is done for every tube. I pipette 3ml of the growing cultures from each tube into their own glass cuvette. These cuvettes are then placed in a dry bath for 5 minutes or until they reach room temperature. An initial reading is then taken by placing the cuvettes into a spectrophotometer where each cuvette is given three values at three different wavelengths. The cuvettes are then removed and treated with 50µl of m-cresol and then left for 5 minutes longer. The cuvettes are placed back into the spectrophotometer for another set of readings. Both the initial and treated values for each tube are entered into an excel spreadsheet with a preexisting equation that gives the value of pH. I should end with a total of 16 pH readings.

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To the left is the spectrophotometer that gives the values that are then used to calculate the pH for each tube. Above are 5 cuvettes demonstrating the visual grade of variance amongst pH when treated with m-cresol.

 

What does a changing climate mean for Phytoplankton and what does this mean for our planet?

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— Written by Ean Eberhard

The microscopic marine algae known as Phytoplankton are incredibly important to our planet. Although minuet in size these organisms have an immense amount of responsibility within both aquatic and terrestrial ecosystems.

Phytoplankton are primary producers, the foundation of the marine food web, feeding everything from zooplankton to massive whales. As a primary producer these phytoplankton create the foundation of the aquatic food web and thus bare the weight of the rest of the aquatic food web. In other words, the fluctuation in phytoplankton abundance could mean a collapse in the web.  Phytoplankton are not only a source of food but are also a large contributor to the biological carbon pump, responsible for most of the carbon dioxide transfer from the atmosphere to the ocean. For this reason, changes in the growth of phytoplankton could have a great effect on atmospheric concentrations of carbon dioxide. Phytoplankton can also be detrimental. When nutrients levels are high, phytoplankton can grow out of control, creating algal blooms which produce extremely toxic compounds with many harmful effects.   There is no doubt that the balance in phytoplankton abundance is crucial to all life. Unfortunately, phytoplankton are experiencing more stress due to a changing climate.

For the next seven weeks I will be studying the photochemistry of phytoplankton in response to three different stressors. This experiment will be a multifactorial design that will give a more realistic look into how the stress of a changing climate is much more complex than rising temperatures. These three stressors include variances in light intensity, temperature, and CO2 levels. Each of these stressors correspond to variances in the phytoplankton’s environment due to the change in climate. I will dive deeper into the response of these phytoplankton by measuring cell abundance, cell efficiency, Dissolved Inorganic Carbon and pH. This experiment will give us an insight into just how exactly phytoplankton may respond to a changing world and the findings may provide an insight into how the effects on phytoplankton could have a ripple effect on all other life.