# Effect of Low pH on Carbohydrate Production by a Marine Planktonic Diatom (Chaetoceros muelleri)

Authors: Daniel C. O. Thornton
Journal: Research Letters in Ecology
 (2009)

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## Abstract

Rising carbon dioxide (CO2) concentrations in the atmosphere due to human activity are causing the surface ocean to become more acidic. Diatoms play a pivotal role in biogeochemical cycling and ecosystem function in the ocean. pH affected the quantum efficiency of photosystem II and carbohydrate metabolism in a planktonic diatom (Chaetoceros muelleri), representative of a widely distributed genus. In batch cultures grown at low pH, the proportion of total carbohydrate stored within the cells decreased and more dissolved carbohydrates were exuded from the cells into the surrounding medium. Changes in productivity and the way in which diatoms allocate carbon into carbohydrates may affect ecosystem function and the efficiency of the biological carbon pump in a low pH ocean.


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## Body

## 1. Introduction

Rising carbon dioxide (CO2)
concentrations in the atmosphere due to human activity are causing the ocean to become more acidic [1]. The pH of the upper ocean has decreased  from a preindustrial
value of 8.2 to approximately 8.1 today [1]. It is estimated that the pH of ocean surface waters will be 7.9 by the end of this century and 7.4 by the end of the millennium [2]. Despite the global biogeochemical significance of primary production by
diatoms, little is known about how they will respond to the decreasing pH of
the ocean. The carbohydrate glucan is the primary storage compound in diatoms for
carbon fixed during photosynthesis. Moreover,
diatoms exude a proportion of their photosynthate into the surrounding medium as extracellular
carbohydrates. The objective of this research was to determine how seawater pH
affects growth, photosynthesis, and carbohydrate metabolism in a common diatom.


## 2. Materials and Methods

Chaetoceros muelleri (Lemmermann) (CCMP 1316) was grown at pH 6.8, 7.4, 7.9, and 8.2. Cultures grown at pH 6.8 represented an
extreme beyond the current predictions for future ocean pH [2]. Batch cultures
were grown in artificial seawater salt base [3] to which nutrients, trace
metals, and vitamins were added as an f/2 enrichment [4]. Cultures were grown
at 20°C under a photosynthetic photon flux density of 50 μmol m−2s−1,
with a 14-hour daily light period. pH was maintained within the cultures using
a biological buffer (25 mM HEPES (Sigma-Aldrich, St. Louis, Mo, USA)) and by daily titration with a small
amount (<100 μL) of 1 M HCl.Growth each day was
followed in the cultures using in vivo chlorophyll fluorescence measured in a
small sample (200μL) using a spectrofluorometer plate reader (Molecular Devices, Sunnyvale, Calif, USA).The six replicate cultures grown at
each pH were extensively sampled during exponential growth phase on day 5. Samples were taken for cell counts [4] and
chlorophyll a concentrations [5]. The maximum quantum efficiency of photosystem
II (Fv/Fm) in Chaetoceros muelleri was measured in dark adapted (2 hours) cultures using a pulse
amplitude-modulated (PAM) fluorometer (Heinz Walz, Effeltrich, Germany) [6]. Carbohydrates were measured using the
phenol-sulfuric acid assay calibrated with D-glucose
[7]. Fractionation and extraction of the different carbohydrate pools were carried out using
established methods [8, 9]. Transparent exopolymer particles (TEP) were
quantified according to [10].


## 3. Results and Discussion

Acid titration was
used to maintain the desired pH [11], which tended to drift upward due to the
removal of aqueous CO2 during photosynthesis. Future work should aim to decrease the pH by bubbling CO2-enriched
air through seawater [12], which both reduces pH and increases the dissolved
inorganic carbon (DIC) concentration. This simulates the effect of rising
atmospheric pCO2 and
provides control over DIC concentration.Photosynthesis was
affected by pH as determined byFv/Fm (see Figure 1(a)). Fv/Fm was inversely correlated with seawater pH (r=0.90, P<.001, n=24).
These data show that the photosynthetic efficiency of C. muelleri decreases as the environment surrounding the cells
becomes more acidic. Therefore, less energy was available to the cells to fix
carbon at relatively low pH. Growth rate was not affected by pH between 7.4 and
8.2; however, growth rate at pH 6.8 was significantly lower (analysis of
variance; F3,20=24.2, P<.001) (see Table 1).Table 1
Chaetoceros muelleri biomass
and growth. Actual pH was the pH
measured over the course of 6 days in culture. Growth rate derived from changes in in vivo chlorophyll fluorescence between days 2 and 5. Biomass is expressed as diatom concentration
(cells mL−1) and chlorophyll a (chl. a) concentration (μg L−1). These data were used to calculate chl. a content per cell (fg cell−1)
on day 5. Values are means ± standard
deviation (n=6 replicate cultures).
pHActual pHGrowth rate (μ′)Doubling time (days)Cells mL−1chl.a (μg L−1)chl.a
(fg cell−1)6.86.82±0.040.19±0.011.55±0.051.36×106±6.89×104518 ± 89378 ± 507.47.42±0.040.24±0.021.27±0.101.83×106±9.26×104687 ± 141375 ± 767.97.93±0.040.25±0.011.22±0.031.84×106±8.84×104644
± 80351 ± 458.28.23±0.050.25±0.011.20±0.071.65×106±8.08×104637 ± 68388
± 55Effect of medium pH on photosynthesis and carbohydrate metabolism inChaetoceros muelleri. Samples taken after 5 days growth in batch culture.
(a) Maximum quantum efficiency of photosystem II (Fv/Fm).
(b) Total, cell and dissolved extracellular carbohydrate concentrations.
(c) Cell-associated carbohydrates. (d) Proportion of carbohydrates in the
dissolved extracellular and cell storage pools. (e) Extracellular carbohydrate
pools. (f) Concentration of transparent exopolymer particles (TEP).
Bars show mean + standard deviation (n=6 replicate cultures).
(a)(b)(c)(d)(e)(f)Total carbohydrate
was the greatest in cultures grown at the preindustrial pH of 8.2 (see Figure1(b)).
There was a significant decrease in
total (F3,20=21.6, P<.001) and cell (F3,20=22.7, P<.001) carbohydrate as the diatoms
were grown at lower pH. In contrast, there was a significant increase
(Kruskal-Wallis ANOVA; H=18.6, 3 degrees of freedom, P<.001) in dissolved extracellular carbohydrates with decreasing pH. The ratio of
cell carbohydrate to dissolved extracellular carbohydrate was 2.60±0.26 (mean
± SD) at pH 8.2; decreasing to 1.99±0.41 at pH 7.9, and 1.24±0.41 at pH 7.4
(F3,20=30.3, P<.001). There was a significant difference (F3,19=6.55, P=.003) in non-storage cell carbohydrates at different growth pH (see Figure 1(c)).As pH decreased, a
decreasing proportion of the total carbohydrate was stored within the cells as
glucan, and a greater proportion of the total carbohydrate was exuded from the
cells (see Figure1(d)). There was a significant inverse correlation between pH
and the ratio of dissolved extracellular carbohydrate to glucan (r=0.91, n=23, P<.001). The
increase in dissolved extracellular carbohydrate concentrations with decreasing
pH was mainly in the form of low molecular weight carbohydrates rather than
exopolymers (see Figure 1(e)). This is supported by the measurements of
transparent exopolymer particle (TEP) concentrations within the cultures, which
were not significantly different at different pH (see Figure 1(f)).The shift in
carbon allocation to different pools by diatoms at low pH may have implications
for the ecosystem function, the efficiency of the biological carbon pump, and
the resulting sequestration of CO2 in the deep ocean [13]. Less
carbohydrate stored within the cells, and a greater production of low-molecular-weight
dissolved extracellular carbohydrates at low pH, will result in less particulate
organic carbon in the water column, potentially reducing the efficiency of the
biological carbon pump during diatom blooms. The reduction in Fv/Fm
and the
increase in the proportion of carbohydrates exuded from the cells suggest that low pH affects
membrane function in Chaetoceros muelleri.
Thylakoid membrane integrity is vital to maintain photosynthetic capability and
the plasmalemma controls exchange between the intra- and extracellular
environments. Further work, including larger-scale mesocosm experiments in situ, will be needed to test these
hypotheses.



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*Source: 105901-2009-03-05.xml*