Photosynthetic Algae, Thalassiosira weissflogii, Response to CO2 Change

Glossary

The results from this experiment were divided into the following six categories:

1. Cell Counts

2. Fluorescence

3. Protein Gel Electrophoresis

4. Enzyme Assay

5. Pigment/Chromatography

6. Chlorophyll Concentration/Spectrophotometry

Cell counts increased in all twelve bottles over the six-day treatment period. As expected, cell counts were highest at the 750 ppm concentration and lowest at the 100 ppm. A log graph of the cell growth showed a rapid increase in cells between day 0 and day 3, the growth phase, then a slowing of the growth rate, the lag phase of the population curve. By day 6, the cell counts were very close among the three CO₂ levels as the populations leveled off. This point on the graph represents the point at which nutrients became limiting to the diatoms. Although CO₂ was continually supplied, the diatoms no longer had enough of other necessary nutrients to continue to grow.

2. Fluorescence:

Fluorescence was non-detectable on day 0 and increased in all twelve bottles over the six days. At day 3, when the cells were in their most active growth phase, the 100 ppm had the lowest fluorescence, and 750 ppm had the most. This was true both for measured fluorescence and when corrected for cell numbers. This test was a simple method of estimating numbers of cells and amounts of chlorophyll.

The gel showed three distinct bands of carbonic anhydrase activity. The enzyme appeared as yellow bands on the gel because the indicator used is yellow in an acidic solution. When CO₂ was bubbled over the carbonic anhydrase bands, the enzyme catalyzed the conversion of CO₂ to HCO₃^- and H⁺, thereby causing the solution to become more acidic. This was a qualitative test for the presence of carbonic anhydrase in the diatoms.

The uncatalyzed reaction of HCO₃^- to CO₂ occurred in 95 seconds. The time needed for this reaction decreased with increasing CO₂ concentration under which the cells were grown. The greater the carbonic anhydrase activity, the less time needed for the reaction. Enzyme units were calculated for the three samples. The enzyme units likewise decreased with the increasing CO₂ concentrations under which the cells were grown. In other words, the diatoms grown at 100 ppm had less carbonic anhydrase activity than those grown at 750 ppm. This was directly opposite of had been hypothesized.

Cells grown under low CO₂ conditions should produce more carbonic anhydrase in order to be able to convert more bicarbonate to CO₂ when CO₂ is limited. However, in this experiment, the cells grew so fast that they used up all the CO₂ readily available to them. They then started producing carbonic anydrase, so they could use bicarbonate in addition to the CO₂ bubbled into the bottles. The exceedingly rapid growth of these cell cultures may be attributable to nutrients present in the natural seawater in addition to those added to the cultures.

The diatom solutions analyzed by chromatography showed no pigment bands. There may not have been enough chlorophyll in the solution for good separation. The orange day lily started as a purple dot and showed one band of yellow. The yellow day lily showed no separation. The most interesting sample was the grass which showed four distinct bands of yellow/green. The solvent traveled 4.7 cm and the Rf values for the grass pigments were calculated at .98, .94, .38, and .28 respectively.

A total of 36 pigment samples were collected during the six days. These were analyzed in the spectrophotometer and the absorbance of light measured for each sample at wavelengths of 750, 664, 647, 630, 510, and 480 nm. The absorption spectrum for chlorophyll shows peaks in the 470 and 670 range, which correspond to blue and red wavelengths respectively. Chlorophyll absorbs very little in the 500 to 650 range (green and yellow). Results were than averaged for the three CO₂ level groups. Using standard calculations, the concentrations of chlorophyll a, chlorophyll c, and carotenoid were calculated. Concentrations of all three pigments increased between day 0 and day 6 in each of the three treatment groups. When normalized for cell number, the 750 ppm group had the highest concentrations of all three pigments, and the 100 ppm group had the lowest for all three.

From Robert Bidigare’s Pigment and Production Group