— Written by Ean Eberhard
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
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.
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.