Experiment: Influence of CO2 and nitrogen limitation on the coccolith volume of Emiliania huxleyi (Haptophyta)

Coccolithophores, a key phytoplankton group, are one of the most studied organisms regarding their physiological response to ocean acidification/carbonation. The biogenic production of calcareous coccoliths has made coccolithophores a promising group for paleoceanographic research aiming to reconstruct past environmental conditions. Recently, geochemical and morphological analyses of fossil coccoliths have gained increased interest in regard to changes in seawater carbonate chemistry. The cosmopolitan coccolithophore Emiliania huxleyi (Lohm.) Hay and Mohler was cultured over a range of pCO2 levels in controlled laboratory experiments under nutrient replete and nitrogen limited conditions. Measurements of photosynthesis and calcification revealed, as previously published, an increase in particulate organic carbon production and a moderate decrease in calcification from ambient to elevated pCO2. The enhancement in particulate organic carbon production was accompanied by an increase in cell diameter. Changes in coccolith volume were best correlated with the coccosphere/cell diameter and no significant correlation was found between the coccolith volume and the particulate inorganic carbon production. The conducted experiments revealed that the coccolith volume of E. huxleyi is variable with aquatic CO2 concentration but its sensitivity is rather small in comparison with its sensitivity to nitrogen limitation. Comparing coccolith morphological and geometrical parameters like volume, mass and size to physiological parameters under controlled laboratory conditions is an important step to understand variations in fossil coccolith geometry.

Seawater carbonate chemistry and calcification during a study of barrier reef flat in Moorea, French Polynesia, 1998

The relative contribution of soft bottoms to the community metabolism (primary production, respiration and net calcification) of a barrier reef flat has been investigated at Moorea (French Polynesia). Community metabolism of the sedimentary area was estimated using in situ incubations in perspex chambers, and compared with estimates of community metabolism of the whole reef flat obtained using a Lagrangian technique (Gattuso et al., 1996. Carbon flux in coral reefs. 1. Lagrangian measurement of community metabolism and resulting air-sea CO2 disequilibrium. Mar. Ecol. Prog. Ser. 145, 109-121). Net organic carbon production (E), respiration (R) and net calcification (G) of sediments were measured by seven incubations performed in triplicate at different irradiance. Respiration and environmental parameters were also measured at four randomly selected additional stations. A model of Photosynthesis-irradiance allowed to calculate oxygen (O2), organic carbon (CO2) and calcium carbonate (CaCO3) evolution from surface irradiance during a diel cycle. As chlorophyll a content of the sediment was not significantly different between stations, primary production of the sediment was considered as homogeneous for the whole lagoon. Thus, carbon production at the test station can be modelled from surface light irradiance. The modelled respiration was two times higher at the test station than the mean respiration of the barrier reef, and thus underestimated sediment contribution to excess production. Sediments cover 40-60% of the surface and accounted for 2.8-4.1% of organic carbon excess production estimated with the modelled R and 21-32% when mean R value was considered. The sedimentary CaCO3 budget was a very minor component of the whole reef budget.

Seawater carbonate chemistry and biological processes during experiments with temperate coral Cladocora caespitosa, 2010

Atmospheric CO2 partial pressure (pCO2) is expected to increase to 700 µatm or more by the end of the present century. Anthropogenic CO2 is absorbed by the oceans, leading to decreases in pH and the CaCO3 saturation state of the seawater. Elevated pCO2 was shown to drastically decrease calcification rates in tropical zooxanthellate corals. Here we show, using the Mediterranean zooxanthellate coral Cladocora caespitosa, that an increase in pCO2, in the range predicted for 2100, does not reduce its calcification rate. Therefore, the conventional belief that calcification rates will be affected by ocean acidification may not be widespread in temperate corals. Seasonal change in temperature is the predominant factor controlling photosynthesis, respiration, calcification and symbiont density. An increase in pCO2, alone or in combination with elevated temperature, had no significant effect on photosynthesis, photosynthetic efficiency and calcification. The lack of sensitivity C. caespitosa to elevated pCO2 might be due to its slow growth rates, which seem to be more dependent on temperature than on the saturation state of calcium carbonate in the range projected for the end of the century.

Carbon uptake rate calculated for melt pond water samples during POLARSTERN cruise ARK-XXVII/3 (IceArc) in 2012

Net Primary Production was measured using the 14**C uptake method with minor modifications. Melt pond samples were spiked with 0.1µCi ml**-1 of 14**C labelled sodium bicarbonate (Moravek Biochemicals, Brea, USA) and distributed in 10 clear bottles (20 ml each). Subsequently they were incubated for 12 h at -1.3°C under different scalar irradiances (0-420 µmol photons m**-2 s**-1) measured with a spherical sensor (Spherical Micro Quantum Sensor US-SQS/L, Heinz Walz, Effeltrich, Germany). At the end of the incubation, samples were filtered onto 0.2 µm nitrocellulose filters and the particulate radioactive carbon uptake was determined by liquid scintillation counting using Filter count scintillation cocktail (Perkin Elmer, Waltham, USA). The carbon uptake values in the dark were subtracted from the carbon uptake values measured in the light incubations. Dissolved inorganic carbon (DIC) was measured for each sample using the flow injection system (Hall and Aller, 1992). The DIC concentration was taken into account to calculate the amount of labeled bicarbonate incorporated into the cell. Carbon fixation rates were normalized volumetrically and by chlorophyll a. Photosynthesis-irradiance curves (PI curves) were fitted using MATLAB® according to the equation proposed by Platt et al. (1980) including a photoinhibition parameter (beta) and providing the main photosynthetic parameters: maximum Chla normalized carbon fixation rate if there were no photoinhibition (Pb) and the initial slope of the saturation curve (alpha). The derived parameters: light intensity at which photosynthesis is maximal (Im), the carbon fixation rate at that maximal irradiance (Pbm) and the adaptation parameter or photoacclimation index (Ik) were calculated according to Platt et al. (1982).

Seawater carbonate chemistry, stable carbon isotope fractionation and growth rate during experiments with marine phytoplankton community, 1999

Stable carbon isotope fractionation (%) of 7 marine phytoplankton species grown in different irradiance cycles was measured under nutrient-replete conditions at a high light intensity in batch cultures. Compared to experiments under continuous light, all species exhibited a significantly higher instantaneous growth rate (pi), defined as the rate of carbon fixation during the photo period, when cultivated at 12:12 h. 16:8 h, or 186 h light:dark (L/D) cycles. Isotopic fractionation by the diatoms Skeletonema costatum, Asterionella glacialis, Thalassiosira punctigera, and Coscinodiscus wailesii (Group I) was 4 to 6% lower in a 16:8 h L/D cycle than under continuous light, which we attribute to differences in pi. In contrast, E, in Phaeodactylum tn'cornutum, Thalassiosira weissflogii, and in the dinoflagellate Scrippsiella trochoidea (Group 11) was largely insensitive to day length-related differences in instantaneous growth rate. Since other studies have reported growth-rate dependent fractionation under N-limited conditions in P. tricornutum, pi-related effects on fractionation apparently depend on the factor controlling growth rate. We suggest that a general relationship between E, and pi/[C02,,,] may not exist. For 1 species of each group we tested the effect of variable CO2 concentration, [COz,,,], on isotopic fractionation. A decrease in [CO2,,,] from ca 26 to 3 pm01 kg-' caused a decrease in E, by less than 3%0 This indicates that variation in h in response to changes in day length has a similar or even greater effect on isotopic fractionation than [COz,,,] m some of the species tested. In both groups E, tended to be higher in smaller species at comparable growth rates. In 24 and 48 h time series the algal cells became progressively enriched in 13C during the day and the first hours of the dark period, followed by l3C depletion in the 2 h before beginning of the following Light period. The daily amplitude of the algal isotopic composition (613C), however, was <1.5%0, which demonstrates that diurnal variation in Fl3C is relatively small.

Combined effects of different CO2 levels and N sources on the diazotrophic cyanobacterium Trichodesmium

To predict effects of climate change and possible feedbacks, it is crucial to understand the mechanisms behind CO2 responses of biogeochemically relevant phytoplankton species. Previous experiments on the abundant N2 fixers Trichodesmium demonstrated strong CO2 responses, which were attributed to an energy reallocation between its carbon (C) and nitrogen (N) acquisition. Pursuing this hypothesis, we manipulated the cellular energy budget by growing Trichodesmium erythraeum IMS101 under different CO2 partial pressure (pCO2) levels (180, 380, 980 and 1400?µatm) and N sources (N2 and NO3-). Subsequently, biomass production and the main energy-generating processes (photosynthesis and respiration) and energy-consuming processes (N2 fixation and C acquisition) were measured. While oxygen fluxes and chlorophyll fluorescence indicated that energy generation and its diurnal cycle was neither affected by pCO2 nor N source, cells differed in production rates and composition. Elevated pCO2 increased N2 fixation and organic C and N contents. The degree of stimulation was higher for nitrogenase activity than for cell contents, indicating a pCO2 effect on the transfer efficiency from N2 to biomass. pCO2-dependent changes in the diurnal cycle of N2 fixation correlated well with C affinities, confirming the interactions between N and C acquisition. Regarding effects of the N source, production rates were enhanced in NO3-grown cells, which we attribute to the higher N retention and lower ATP demand compared with N2 fixation. pCO2 effects on C affinity were less pronounced in NO3- users than N2 fixers. Our study illustrates the necessity to understand energy budgets and fluxes under different environmental conditions for explaining indirect effects of rising pCO2.

Major cellular and physiological impacts of ocean acidification on a reef building coral

As atmospheric levels of CO2 increase, reef-building corals are under greater stress from both increased sea surface temperatures and declining sea water pH. To date, most studies have focused on either coral bleaching due to warming oceans or declining calcification due to decreasing oceanic carbonate ion concentrations. Here, through the use of physiology measurements and cDNA microarrays, we show that changes in pH and ocean chemistry consistent with two scenarios put forward by the Intergovernmental Panel on Climate Change (IPCC) drive major changes in gene expression, respiration, photosynthesis and symbiosis of the coral, Acropora millepora, before affects on biomineralisation are apparent at the phenotype level. Under high CO2 conditions corals at the phenotype level lost over half their Symbiodinium populations, and had a decrease in both photosynthesis and respiration. Changes in gene expression were consistent with metabolic suppression, an increase in oxidative stress, apoptosis and symbiont loss. Other expression patterns demonstrate upregulation of membrane transporters, as well as the regulation of genes involved in membrane cytoskeletal interactions and cytoskeletal remodeling. These widespread changes in gene expression emphasize the need to expand future studies of ocean acidification to include a wider spectrum of cellular processes, many of which may occur before impacts on calcification.

Seawater carbonate chemistry and carbon allocation, growth and morphology of the coccolithophore Emiliania huxleyi (calcifying strain CCMP 371) during experiments, 2011

Coccolithophores are unicellular phytoplankton that produce calcium carbonate coccoliths as an exoskeleton. Emiliania huxleyi, the most abundant coccolithophore in the world's ocean, plays a major role in the global carbon cycle by regulating the exchange of CO2 across the ocean-atmosphere interface through photosynthesis and calcium carbonate precipitation. As CO2 concentration is rising in the atmosphere, the ocean is acidifying and ammonium (NH4) concentration of future ocean water is expected to rise. The latter is attributed to increasing anthropogenic nitrogen (N) deposition, increasing rates of cyanobacterial N2 fixation due to warmer and more stratified oceans, and decreased rates of nitrification due to ocean acidification. Thus future global climate change will cause oceanic phytoplankton to experience changes in multiple environmental parameters including CO2, pH, temperature and nitrogen source. This study reports on the combined effect of elevated pCO2 and increased NH4 to nitrate (NO3) ratio (NH4/NO3) on E. huxleyi, maintained in continuous cultures for more than 200 generations under two pCO2 levels and two different N sources. Here we show that NH4 assimilation under N-replete conditions depresses calcification at both low and high pCO2, alters coccolith morphology, and increases primary production. We observed that N source and pCO2 synergistically drive growth rates, cell size and the ratio of inorganic to organic carbon. These responses to N source suggest that, compared to increasing CO2 alone, a greater disruption of the organic carbon pump could be expected in response to the combined effect of increased NH4/NO3 ratio and CO2 level in the future acidified ocean. Additional experiments conducted under lower nutrient conditions are needed prior to extrapolating our findings to the global oceans. Nonetheless, our results emphasize the need to assess combined effects of multiple environmental parameters on phytoplankton biology in order to develop accurate predictions of phytoplankton responses to ocean acidification.

Acclimatization of the crustose coralline alga Porolithon onkodes to variable pCO2

Ocean acidification (OA) has important implications for the persistence of coral reef ecosystems, due to potentially negative effects on biomineralization. Many coral reefs are dynamic with respect to carbonate chemistry, and experience fluctuations in pCO2 that exceed OA projections for the near future. To understand the influence of dynamic pCO2 on an important reef calcifier, we tested the response of the crustose coralline alga Porolithon onkodes to oscillating pCO2. Individuals were exposed to ambient (400 µatm), high (660 µatm), or variable pCO2 (oscillating between 400/660 µatm) treatments for 14 days. To explore the potential for coralline acclimatization, we collected individuals from low and high pCO2 variability sites (upstream and downstream respectively) on a back reef characterized by unidirectional water flow in Moorea, French Polynesia. We quantified the effects of treatment on algal calcification by measuring the change in buoyant weight, and on algal metabolism by conducting sealed incubations to measure rates of photosynthesis and respiration. Net photosynthesis was higher in the ambient treatment than the variable treatment, regardless of habitat origin, and there was no effect on respiration or gross photosynthesis. Exposure to high pCO2 decreased P. onkodes calcification by >70%, regardless of the original habitat. In the variable treatment, corallines from the high variability habitat calcified 42% more than corallines from the low variability habitat. The significance of the original habitat for the coralline calcification response to variable, high pCO2 indicates that individuals existing in dynamic pCO2 habitats may be acclimatized to OA within the scope of in situ variability. These results highlight the importance of accounting for natural pCO2 variability in OA manipulations, and provide insight into the potential for plasticity in habitat and species-specific responses to changing ocean chemistry.

Impact of temperature and species interaction on filamentous cyanobacteria may be more important than salinity and increased pCO2 levels

A future business-as-usual scenario (A1FI) was tested on two bloom-forming cyanobacteria of the Baltic Proper, Nodularia spumigena and Aphanizomenon sp., growing separately and together. The projected scenario was tested in two laboratory experiments where (a) interactive effects of increased temperature and decreased salinity and (b) interactive effects of increased temperature and elevated levels of pCO2 were tested. Increased temperature, from 12 to 16 °C, had a positive effect on the biovolume and photosynthetic activity (F v/F m) of both species. Compared when growing separately, the biovolume of each species was lower when grown together. Decreased salinity, from 7 to 4, and elevated levels of pCO2, from 380 to 960 ppm, had no effect on the biovolume, but on F v/F m of N. spumigena with higher F v/F m in salinity 7. Our results suggest that the projected A1FI scenario might be beneficial for the two species dominating the extensive summer blooms in the Baltic Proper. However, our results further stress the importance of studying interactions between species.

Seawater carbonate chemistry and physiological responses of three temperate coralline algae in a laboratory experiment

Coralline algae are major calcifiers of significant ecological importance in marine habitats but are among the most sensitive calcifying organisms to ocean acidification. The elevated pCO2 effects were examined in three coralline algal species living in contrasting habitats from intertidal to subtidal zones on the north-western coast of Brittany, France: (i) Corallina elongata, a branched alga found in tidal rock pools, (ii) Lithophyllum incrustans, a crustose coralline alga from the low intertidal zone, and (iii) Lithothamnion corallioides (maerl), a free-living form inhabiting the subtidal zone. Metabolic rates were assessed on specimens grown for one month at varying pCO2: 380 (current pCO2), 550, 750 and 1000 µatm (elevated pCO2). There was no pCO2 effect on gross production in C. elongata and L. incrustans but L. incrustans respiration strongly increased with elevated pCO2. L. corallioides gross production slightly increased at 1000 µatm, while respiration remained unaffected. Calcification rates decreased with pCO2 in L. incrustans (both in the light and dark) and L. corallioides (only in the light), while C. elongata calcification was unaffected. This was consistent with the lower skeletal mMg/Ca ratio of C. elongata (0.17) relative to the two other species (0.20). L. incrustans had a higher occurrence of bleaching that increased with increasing pCO2. pCO2 could indirectly impact this coralline species physiology making them more sensitive to other stresses such as diseases or pathogens. These results underlined that the physiological response of coralline algae to near-future ocean acidification is species-specific and that species experiencing naturally strong pH variations were not necessarily more resistant to elevated pCO2 than species from more stable environment.

Carbon uptake rate calculated for CTD water samples during POLARSTERN cruise ARK-XXVII/3 (IceArc) in 2012

Net Primary Production was measured using the 14**C uptake method with minor modifications. Seawater samples were spiked with 0.1µCi ml**-1 of 14**C labelled sodium bicarbonate (Moravek Biochemicals, Brea, USA) and distributed in 10 clear bottles (20 ml each). Subsequently they were incubated for 12 h at -1.3°C under different scalar irradiances (0-420 µmol photons m**-2 s**-1) measured with a spherical sensor (Spherical Micro Quantum Sensor US-SQS/L, Heinz Walz, Effeltrich, Germany). At the end of the incubation, samples were filtered onto 0.2 µm nitrocellulose filters and the particulate radioactive carbon uptake was determined by liquid scintillation counting using Filter count scintillation cocktail (Perkin Elmer, Waltham, USA). The carbon uptake values in the dark were subtracted from the carbon uptake values measured in the light incubations. Dissolved inorganic carbon (DIC) was measured for each sample using the flow injection system (Hall and Aller, 1992). The DIC concentration was taken into account to calculate the amount of labeled bicarbonate incorporated into the cell. Carbon fixation rates were normalized volumetrically and by chlorophyll a. Photosynthesis-irradiance curves (PI curves) were fitted using MATLAB® according to the equation proposed by Platt et al. (1980) including a photoinhibition parameter (beta) and providing the main photosynthetic parameters: maximum Chla normalized carbon fixation rate if there were no photoinhibition (Pb) and the initial slope of the saturation curve (alpha). The derived parameters: light intensity at which photosynthesis is maximal (Im), the carbon fixation rate at that maximal irradiance (Pbm) and the adaptation parameter or photoacclimation index (Ik) were calculated according to Platt et al. (1982).

Iron limitation modulates ocean acidification effects on southern ocean phytoplankton communities

The potential interactive effects of iron (Fe) limitation and Ocean Acidification in the Southern Ocean (SO) are largely unknown. Here we present results of a long-term incubation experiment investigating the combined effects of CO2 and Fe availability on natural phytoplankton assemblages from the Weddell Sea, Antarctica. Active Chl a fluorescence measurements revealed that we successfully cultured phytoplankton under both Fe-depleted and Fe-enriched conditions. Fe treatments had significant effects on photosynthetic efficiency (Fv/Fm; 0.3 for Fe-depleted and 0.5 for Fe-enriched conditions), non-photochemical quenching (NPQ), and relative electron transport rates (rETR). pCO2 treatments significantly affected NPQ and rETR, but had no effect on Fv/Fm. Under Fe limitation, increased pCO2 had no influence on C fixation whereas under Fe enrichment, primary production increased with increasing pCO2 levels. These CO2-dependent changes in productivity under Fe-enriched conditions were accompanied by a pronounced taxonomic shift from weakly to heavily silicified diatoms (i.e. from Pseudo-nitzschia sp. to Fragilariopsis sp.). Under Fe-depleted conditions, this functional shift was absent and thinly silicified species dominated all pCO2 treatments (Pseudo-nitzschia sp. and Synedropsis sp. for low and high pCO2, respectively). Our results suggest that Ocean Acidification could increase primary productivity and the abundance of heavily silicified, fast sinking diatoms in Fe-enriched areas, both potentially leading to a stimulation of the biological pump. Over much of the SO, however, Fe limitation could restrict this possible CO2 fertilization effect.

Strong shift from HCO3- to CO2 uptake in Emiliania huxleyi with acidification: new approach unravels acclimation versus short-term pH effects

Effects of ocean acidification on Emiliania huxleyi strain RCC 1216 (calcifying, diploid life-cycle stage) and RCC 1217 (non-calcifying, haploid life-cycle stage) were investigated by measuring growth, elemental composition, and production rates under different pCO2 levels (380 and 950 µatm). In these differently acclimated cells, the photosynthetic carbon source was assessed by a (14)C disequilibrium assay, conducted over a range of ecologically relevant pH values (7.9-8.7). In agreement with previous studies, we observed decreased calcification and stimulated biomass production in diploid cells under high pCO2, but no CO2-dependent changes in biomass production for haploid cells. In both life-cycle stages, the relative contributions of CO2 and HCO3 (-) uptake depended strongly on the assay pH. At pH values =< 8.1, cells preferentially used CO2 (>= 90 % CO2), whereas at pH values >= 8.3, cells progressively increased the fraction of HCO3 (-) uptake (~45 % CO2 at pH 8.7 in diploid cells; ~55 % CO2 at pH 8.5 in haploid cells). In contrast to the short-term effect of the assay pH, the pCO2 acclimation history had no significant effect on the carbon uptake behavior. A numerical sensitivity study confirmed that the pH-modification in the (14)C disequilibrium method yields reliable results, provided that model parameters (e.g., pH, temperature) are kept within typical measurement uncertainties. Our results demonstrate a high plasticity of E. huxleyi to rapidly adjust carbon acquisition to the external carbon supply and/or pH, and provide an explanation for the paradoxical observation of high CO2 sensitivity despite the apparently high HCO3 (-) usage seen in previous studies.