The Effects of Varying CO₂ Concentrations on the Marine Diatom Thalassiosira  weissflogii

Kurt D. MacDonald, Bukky Okusanya,

Abstract

The effects of 100, 370, and 750ppm CO₂ on the biological, chemical, and enzyme activity of the marine diatom Thalassiosira weissflogii were investigated for seven days in sterilized seawater culture supplemented with nutrients, vitamins, and trace metals under laboratory conditions. 

There was a steady increase in the growth of these diatoms with time, with populations cultured in 370ppm CO₂ experiencing significantly more growth than the other cultures.  The pH of the culture medium was more basic with increased CO₂ concentration in response to the amount of carbonic acid formed in the culture medium.  However, this pH difference did not directly affect the growth of the diatoms maintained under different CO₂ concentrations.  While the fluorescence of chlorophyll also increased with elevated CO₂ concentrations, the total pigment content (Chlorophylls a, c, and carotinoid) generally decreased with increased CO₂ concentration.  This result indicates that fluorescence may not be a reliable indicator of photopigment content. 

The carbonic anhydrase (CA) activity was highest at the lowest CO₂ concentration and at more basic culture pH.  These results indicate that more CA was produced to utilize the bicarbonate ions that are more plentiful in media with low CO₂.  Over time, there was a decrease in the concentration of inorganic nutrients (nitrate, phosphate, and silicate) with increased CO₂, and this was correlated with increased diatom growth.  The decrease in phosphate and silicate was rather dramatic and their concentrations did not recover after being depleted, unlike nitrate.  The sigmoid shape of the growth curves suggest that one or more of these nutrients may be limiting population growth.

These results indicate that populations of marine diatom T. weissflogii did not respond positively to increased CO₂ concentration in terms of growth as a likely result of inorganic nutrient limitations.   There was, however, increased chlorophyll fluorescence, decreased photopigments and CA activity as CO₂ concentrations increased.  The possible roles of diatoms and other photosynthetic marine algae in the flow of carbon between the atmosphere and the ocean, and their involvement in global warming were discussed.

Introduction

The purpose of this study is to investigate how the marine diatom Thalassiosira weissflogii responds to varying concentrations of carbon dioxide (CO₂).  In particular, this study sought to determine how population growth rate, intracellular photopigment concentrations, and carbonic anhydrase enzyme activity were affected when populations were cultured in 100, 370, and 750 parts-per-million (ppm) CO₂.  Additionally, the culture medium was sampled for available nitrate, phosphate, silicate, and pH so that comparisons could be made between the observed biological parameters and the conditions of the sample media. 

This research seeks to investigate timely questions about the impact of increasing atmospheric CO₂ concentrations on the biosphere.  It has been widely documented that the concentration of atmospheric CO₂ has been steadily increasing since the turn of the 20^th century (Carbon Dioxide Information Analysis Center).  At the present time, atmospheric CO₂ is about 370 ppm, but different models suggest that atmospheric CO₂ concentrations could be 30-150% higher in 2100 (Environmental Protection Agency).  While there is little scientific disagreement over these facts, there is significant controversy concerning the effects that this increase will have on aquatic and terrestrial biomes.

One view focuses on the role of CO₂ as a greenhouse gas (GreenPeace).  Given the effectiveness of CO₂ at trapping heat, proponents of this view warn that increasing concentrations of CO₂ in the troposphere will result in significant increases in global average temperatures.  This, in turn, will likely generate profound changes in global climate that would threaten established agricultural practices in addition to the Earth’s natural flora and fauna.  The other view, however, suggests that increasing levels of atmospheric CO₂ will be beneficial to the environment (Greening Earth Society).  In particular, it is suggested that available CO₂ limits the rate at which photosynthetic organisms can grow, and, with increasing CO_2, the growth of primary producers will increase.  Because of the interdependence of all organisms on primary producers, increasing the biological energy store of primary producers would have large-scale, beneficial impacts on subsequent trophic levels.

In evaluating the merits of these two positions, it is vital that the marine ecosystem be studied.  Oceans account for approximately 66% of the earth’s surface, and it has been estimated that marine algae including diatoms, through CO₂ fixation, are responsible for approximately 40% of the earth’s primary production.  Given the significant rate of photosynthesis in the oceans, this biome is a large and important carbon sink.  Therefore, studying the effects of increased atmospheric CO₂ on marine photosynthetic diatoms seems prudent to understanding the effects of CO₂ on primary production.

Research Questions and Hypotheses

This study attempts to address the following questions:

·        Is there a relationship between the concentration of atmospheric CO₂ and the growth of T. weissflogii?

It follows that if atmospheric CO₂ is a limiting factor, then increasing the concentration of CO₂ in a culture medium would increase growth rate.

·        Does varying levels of atmospheric CO₂ affect the pH of seawater, and if so, how does this change in water chemistry affect the availability of CO₂ for T. weissflogii?

Because CO₂ forms carbonic acid in the presence of water, increased CO₂ concentrations should lower the pH of the culture medium and affect the type and ratio of dissolved inorganic carbon in the medium (CO₂, HCO₃^-, and CO₃^2-).

·        Does varying CO₂ concentrations affect the amount of photopigments in the chloroplasts of T. weissflogii?

If the concentration of atmospheric CO₂ limits the rate of photosynthesis and hence the demand on photopigments, then increasing atmospheric CO₂ should cause an increase in the photopigment concentrations so that the diatoms can take full advantage of the available CO₂.

·        Does varying CO₂ concentrations affect the activity and abundance of the enzyme carbonic anhydrase (CA)?

Because carbonic anhydrase is primarily responsible for converting bicarbonate (HCO₃^-) to carbon dioxide (CO₂), when atmospheric CO₂ is abundant, CA activity should be reduced.

·        Is there a relationship between the population growth dynamics of T. weissflogii and the levels of various inorganic compounds present in the culture medium?

Given that diatoms require specific inorganic compounds to maintain sustainable populations, there should be a strong correlation between the presence of these compounds and the growth of the diatom population.

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.

Results

The following results were obtained:

Population Size:

Our results (Fig. 1) indicate that the populations cultured in 370 and 750ppm CO₂ experienced significantly greater population growth (P < .01) throughout the duration of the experiment than the culture incubated in 100ppm CO₂.   The most prolific growth at current concentrations of atmospheric CO₂ (370ppm), whereas those populations cultured in abnormally low CO₂ concentrations (100ppm) experienced the most limited growth.  On the last day of the experiment, the population of all three cultures was significantly different from each other (100 vs. 370ppm, P<.001; 100 vs. 750ppm, P<.001; 370 vs. 750ppm, P<.01) with the most prolific growth occurring at current concentrations of atmospheric CO₂ (370ppm).

pH of Culture Medium:

The results of this analysis (Fig. 2) reveal that the culture medium incubated in 100ppm CO₂ was more basic than the 370ppm CO₂ culture, which, in turn, was more basic than the culture maintained at 750ppm CO₂.

Photopigment Concentrations:

Fluorescence:

Analysis of the fluorescence of chlorophyll (Fig. 3) showed a steady increase throughout the experiment in all culture conditions.  The data suggests, however, that there is no difference in the fluorescence of the chlorophyll between the cultures incubated in 370ppm and 750ppm CO₂.  The populations in these two culture conditions do, however, appear to have more chlorophyll fluorescence than the diatom population maintained at 100ppm CO₂^.

Direct Photopigment Analysis:

Our data shows that chlorophyll a is the most abundant photopigment (58%), followed by carotinoid (35%), and chlorophyll c (7%).  Within each culture condition (100ppm, 370ppm, and 750ppm CO₂) chlorophylls a and c behaved similarly over the course of the experiment with both steadily increasing in concentration except for the 750ppm culture where no increase in chlorophyll a was recorded on the last day of the experiment (Figs. 4&5, respectively).  Additionally, the concentration of chlorophyll a on the last day of the experiment was significantly higher in the 100ppm culture than the 750ppm culture (P<.05).  There was, however, no statistically significant difference in the concentrations of chlorophyll c between the culture conditions.

Carotinoid concentrations increased over the course of the experiment in all culture conditions (Fig. 6).  On the last day, the concentration of carotinoids in the 750ppm culture was significantly higher than that found in the 100ppm culture (P<.05).

The total photopigment concentration was highest in cultures maintained at 100ppm CO₂ and lowest in the 750ppm culture.

Inorganic Nutrient Concentrations:

The concentrations of inorganic nutrients (nitrate, phosphate, and silicate) in the 100ppm CO₂ culture decreased at a gradual rate for the duration of the experiment (Figs. 7, 8, and 9).  The concentrations of nitrate, phosphate, and silicate in the 370ppm and 750ppm CO₂ cultures, however, decreased rapidly to zero during the first four days of the experiment.  After this point, nitrate recovered slightly to approximately 40ppm (Fig. 9).

Carbonic Anhydrase Activity:

The results of the experiment reveal that carbonic anhydrase (CA) activity varied inversely with the concentration of CO₂ in the culture medium, with the 100ppm culture having the most CA activity and the 750ppm culture having the least amount of CA activity (Fig. 10).

Discussion

The significantly larger populations cultured in 370ppm CO₂ than at 750ppm CO₂ demonstrates that T. weissflogii populations did not respond to concentrations of atmospheric CO₂ above those currently present in the atmosphere.  At the conclusion of the experiment, the nutrients appear to be limiting population growth (Figs.7, 8 & 9). Data collected over the last two days of the experiment seem to suggest that all populations, regardless of culture condition, appear to be leveling off.  That noted, it would be interesting to prolong the experiment to determine more precisely when and where the different cultures reach their carrying capacity.  This is particularly relevant because if all populations ultimately plateau at the present level, with the 370ppm culture producing more cells  than either the 750ppm and 100ppm cultures, then marine diatoms may not serve as a long-term sink for atmospheric CO_2.  These results did not support our hypothesis that there is a direct relationship between the concentration of atmospheric CO₂ and the growth of T. weissflogii.

The pH of the culture media was directly correlated with the amount of CO₂ introduced into the medium.  This result occurred because CO₂ forms carbonic acid in the presence of water.   As a result, the medium exposed to 750ppm CO₂ was the least basic (pH≈8.0) whereas the most basic medium was that which was incubated with 100ppm CO₂ (pH≈9.1).  Despite these differences in pH, they did not appear to affect the growth rates of the experimental populations.

The CO₂ concentrations of 370ppm and 750ppm produced diatom cultures whose chlorophyll yielded the greatest fluorescence (Fig. 3), but subsequent spectrophotometer-based determination of the chlorophyll concentration did not support this finding (Figs. 4, 5, and 6).  This disparity was also noted by Lee (1997) and could have resulted from the different sampling techniques.  Fluorescence indirectly measures chlorophyll concentration by recording the fluorescence generated when light-activated electrons fall back to their original orbital, but direct measurements of chlorophyll concentration rely on spectrophotometer readings that record the amount of light absorbed by the sample at specified wavelengths.  Fluorescence, then, may not be a reliable indicator of chlorophyll concentration.  As a result of this inconsistency, more research needs to be done to either justify or refute a link between atmospheric CO₂ concentrations and photopigment quantities. 

The highest rate of carbonic anhydrase (CA) activity in the diatom cultures maintained at 100ppm CO₂ agrees with principles of aquatic chemistry (Fig. 10).  In particular, it is known that in more basic media, there are a higher percentage of bicarbonate ions than carbon dioxide.  Diatoms use carbonic anhydrase to convert bicarbonate into useable carbon dioxide.  Therefore our results show a direct relationship between low CO₂ concentrations, more basic pH (Fig. 2), and increased CA activity (Fig. 10).  Similar research performed in 1999 yielded similar findings, but in 2000 different results were obtained.  The results of this study support our hypothesis that increased pH should yield more active CA enzymes because of the low CO₂ concentration present in this environment.

The drastic reduction in nitrate, phosphate, and silicate experienced in the first four days of the experiment in the 370ppm and 750ppm CO₂ cultures was largely caused by the exponential growth of these diatom populations.  Those cultures reared in 100ppm CO₂ experienced less population growth and, as a result, consumed less inorganic nutrients (Figs. 7, 8, and 9).  Additionally, it was discovered that phosphate and silicate, unlike nitrate, did not recover after being depleted.  This may be explained by the relative insolubility of phosphate and silicate; once living diatoms have absorbed these nutrients they are not readily released even following death of the diatom.  The continued growth of the experimental populations after inorganic nutrient levels approached zero may be explained by metabolic inertia of the diatoms (Latasa, 1995).  Overall, there seems to be a strong relationship between diatom growth rate and available nutrients.

In all, the data from this short duration experiment suggest that T. weissflogii may play a role in the global carbon cycle, but may not have the potential to increase their growth rates in conditions of elevated atmospheric CO₂ concentrations.  Consequently, marine diatoms may not help to lessen the impact of global warming by increasing carbon storage during periods of increased atmospheric CO₂.  Longer duration studies should be performed to quantify the long-term impact of diatoms on atmospheric CO₂ given the finite availability of inorganic nutrients especially in light of previous research, which has indicated that inorganic nutrients can limit T. weissflogii growth Lee (1997).

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