Methods

Procedure for establishing T. weissflogii cultures:

Each culture was prepared in a 1-liter polycarbonate bottle.  Eight hundred milliliters of sterilized seawater was added to each bottle along with 800μl each of nutrients, trace metals, and vitamins as outlined in Price et al. (1988/1989).  Eight milliliters of T. weissflogii, obtained from a stock culture, was added to the seawater medium in each bottle.  These bottles were incubated in front of banks of fluorescent lights that were left on throughout the duration of the experiment.  One culture received 100ppm CO₂ (low CO₂ concentration), another culture received 370ppm CO₂ (current atmospheric CO₂ concentration), and the third culture received 750ppm CO₂ (high CO₂ concentration).  Atmospheric CO₂ was introduced to the culture via a simple air-pump, while gas canisters of the specified CO₂ concentration delivered gas to the other two cultures. For each CO₂ concentration, five duplicate cultures were established.  These conditions were maintained for 8 days.

Method for sampling population size:

At various intervals throughout the experiment, the size of each T. weissflogii population was measured using a Coulter Counter that was set to detect particles with a dimension similar to that of the diatom.  Each duplicate culture was sampled five times for a total of 25 samples per CO₂ condition.  

Method for determining pH of the culture medium:

pH measurements were conducted using a standard bench-top pH meter.

Method for determining the concentration of photopigments:

1.  Fluorescence

The fluorescence of chlorophyll was measured in fluorimeter. 

2.  Spectrophotometry

This method was used to determine the amount of pigments (chlorophylls and carotinoid) in each sample. Diatoms were separated from the medium by filtering three hundred milliliters of the culture through a polycarbonate filter with a pore diameter of 5μm.  The filtrate was stored at 4⁰C for later use in determining inorganic nutrient concentrations.  The filters, on the other hand were placed in various centrifugation flasks to which 10ml of 90% acetone was added.  After a 15 minutes incubation period, the supernatant from each flask was added to separate quartz cuvettes.  Following the calibration of the spectrophotometer to a known blank, the extinction of these samples was measured at λ = 750, 664, 647, 630, 510, and 480nm.  The readings were also adjusted to account for the small turbidity of each sample.  From the resulting data, the following calculations were performed to determine the concentrations of chlorophylls a, b, c, and carotinoids.

Chl. a = 11.85 E₆₆₄ – 1.54 E₆₄₇ – 0.08 E₆₃₀

Chl. b = 21.03 E₆₄₇ – 5.43 E₆₆₄ – 2.66 E₆₃₀

Chl. c = 24.52 E₆₃₀ – 1.54 E₆₆₄ – 7.60 E₆₄₇

Carotinoids = 7.6(E₄₈₀ – 1.49 E₅₁₀)

Pigment (µg/l) =

v = volume of acetone (ml)

V = volume of filtered seawater sample (l)

Method for determining carbonic anhydrase (CA) activity:

One hundred milliliters of culture from each duplicate sample was combined and filtered through a 5μl polycarbonate filter.  The filter cake was then washed with 1ml of seawater into an Eppendorf tube.  This sample was then centrifuged for 4 minutes after which time the supernatant was removed.  The cells were then resuspended in a Lysis buffer and sonicated on ice for 30-second intervals until microscopic inspection demonstrated that the cells had been sufficiently lysed. 

CA activity was evaluated by measuring the rate of H⁺ consumption as pH increases; an increase that is associated with the conversion of HCO₃^- to H₂CO₃/CO₂.  H⁺ consumption was measured by adding 3ml of phosphate buffer to 200μl of a sample (or 200μl phosphate buffer for the blank; uncatalyzed reaction).  Two milliliters of HCO₃^- saturated water was then added to the vial and the time it took the sample to go from pH 6.2 to pH 6.7 was recorded.  The amount of CA activity was then determined by the following equation:

Enzyme Units (U) =

Method for determining concentrations of inorganic compounds in culture medium:

Concentrations of nitrate, phosphate, and silicate in each culture were measured using protocol found in Parsons et al. (1984).  Culture samples for these measurements were obtained from the filtrate generated during the collection of diatoms for photopigment analysis.