Photosynthetic Algae, Thalassiosira weissflogii, Response to CO2 Change

Investigation #1 involved growing Thalassiosira weissflogii diatom cultures under different CO₂ concentrations. The hypothesis was that there would be greater diatom growth under higher C0₂ concentrations. This was measured by cell count and chlorophyll fluorescence.

Investigation #2 qualitatively determined the presence of carbonic anhydrase using a protein gel. The three different cultures of T. weissflogii were sampled to determine the influence of the different culture conditions on the activity of CA.

Investigation #3 quantitatively measured carbonic anhydrase activity by monitoring the rate of pH increase (from 6.2 to 6.7) as the enzyme converted bicarbonate HCO₃^- into CO₂ and H₂0.

Investigation #4 used thin layer chromatography to visualize the chlorophyll and other pigments in the diatoms and a spectrophotometer to measure the absorption spectrum of the diatom’s chlorophyll.

Activity #1 Thalassiosira weissflogii cell culture.

1. Twelve polycarbonate bottles were filled with 800 ml of sterile seawater.
2. Nutrients, vitamins, and trace metals were added to each bottle.
3. Each bottle was inoculated with 6 ml of a 1:100 stock culture of Thalassiosira weissflogii. These cultures were grown in CO₂ environments of: 100, 370, and 750 ppm by bubbling CO₂ in from tanks and room air. Four bottles were grown at each CO₂ concentration.
4. A 20 ml sample was taken from each bottle and the number of diatoms cells were counted using the Coulter Multisizer Counter. This same sample was also tested for chlorophyll fluorescence using a fluorometer.
5. A 300 ml sample from each bottle was filtered onto a 25mm diameter polycarbonate filter. The filters were placed in centrifuge tubes and stored in a freezer at –20° C for later pigment measurements.

1. 20 ml samples from each of the twelve bottles were taken and cell counts and chlorophyll fluorescence tests conducted.
2. 150 ml samples from each bottle were filtered onto a polycarbonate filter and stored for pigment measurement.
3. Another 150 mls from each bottle were filtered and the four samples grown at each of the three CO₂ levels were combined onto one filter. These filters were washed with 1ml of seawater.
4. These 1 ml diatom solutions were then sonicated to break open the silica shells and cell membranes to free the cytoplasm. Each sample was checked under microscope to insure that the cells were broken.
5. Gel electrophoresis using a polyacrylamide gel was used to separate the carbonic anhydrase from other cell proteins. The wells in the gel were loaded with different amounts of the sonicated diatom solution using a micropipette. These amounts were calculated to contain the same number of cells in each sample.
6. The gel was run at 200 volts for approximately 40 minutes with water cooling.
7. The gel was removed and then stained in a tray on a shaker table using bromthymol blue, an indicator solution. The gel was rinsed, and incubated with CO₂ gas for a few minutes using a simple plastic bag.
8. The gel was then placed on a light box to observe the CA band. The indicator is blue in a basic solution, and yellow in an acidic one (at pH 6). The CA band should have appeared yellow because as the CA converts the CO₂ to HCO₃^- and H⁺ ions, the solution becomes acidic. (This procedure did not work the first day, but was redone on day 6 using a freshly made gel and the yellow bands were seen.)
9. To measure carbonic anhydrase activity, 1.0 ml of bicarbonate was added to a phosphate buffer blank. The solution was continuously stirred with a stir bar and kept on ice. The time needed for the bicarbonate and CO₂ to come to equilibrium as shown by a change in pH from 6.2 to 6.7 was measured. This represented an uncatalyzed reaction and took about 95 seconds.
10. The same test was conducted using the three different cell lysates (broken cell solutions) containing CA, adding 1.0 ml bicarbonate to each sample. Compared to the blank, the reaction times for the diatom solutions were lower because CA was catalyzing the reaction.
11. Enzyme Units for the CA were calculated using the following equation:

U= [ (tuncatalyzed/tcatalyzed) -1] * 10

1. 20 ml samples from the original cell cultures were taken and cells counts and chlorophyll florescence were measured.
2. 50 ml of each original culture was filtered onto a polycarbonate filter for pigment analysis.
3. Ten ml of 90% acetone (in Q-water) was added to all of the 9 filters collected for pigment analysis over the three days. These were incubated in the dark for at least 15 minutes.
4. The supernatant was poured into 1 cm quartz cuvettes to be analyzed in a spectrophotometer.
5. The samples were scanned from 350 to 750 nm and the absorbance measured at 750, 664, 647, 510, and 480 nm. Thirty-six samples were measured altogether, four for each of the three CO₂ concentrations taken on the three different days. Chlorophyll a, b, c, and carotinoid concentrations in m g/ml were then calculated on a computer using standard calculations.
6. The 750 ppm C0₂ chlorophyll solutions were also used for thin layer chromatography. A small dot of the solution was applied to a silica gel plate using a thin glass pipette. For comparison, pigment solutions made from grass, and yellow and orange day lillies using 1 ml of a 2 parts acetone-1 part pentane solvent were also run.
7. The plates were placed in beakers containing a solvent (3 parts pentane-1 part acetone-1 part chloroform), covered with a watch glass, and placed under a hood for one hour.

The pigment bands were marked and the Retention factor (Rf) was calculated for each using the following equation: Rf = distance traveled by pigment/distance traveled by solvent front