Seawater carbonate chemistry and processes during experiments with Emiliania huxleyi, 2008

Ocean acidification in response to rising atmospheric CO2 partial pressures is widely expected to reduce calcification by marine organisms. From the mid-Mesozoic, coccolithophores have been major calcium carbonate producers in the world's oceans, today accounting for about a third of the total marine CaCO3 production. Here, we present laboratory evidence that calcification and net primary production in the coccolithophore species Emiliania huxleyi are significantly increased by high CO2 partial pressures. Field evidence from the deep ocean is consistent with these laboratory conclusions, indicating that over the past 220 years there has been a 40% increase in average coccolith mass. Our findings show that coccolithophores are already responding and will probably continue to respond to rising atmospheric CO2 partial pressures, which has important implications for biogeochemical modeling of future oceans and climate.

Global distributions of diazotrophs abundance, biomass and nitrogen fixation rates - Collection of source datasets - Contribution to the MAREDAT World Ocean Atlas of Plankton Functional Types

The MAREDAT atlas covers 11 types of plankton, ranging in size from bacteria to jellyfish. Together, these plankton groups determine the health and productivity of the global ocean and play a vital role in the global carbon cycle. Working within a uniform and consistent spatial and depth grid (map) of the global ocean, the researchers compiled thousands and tens of thousands of data points to identify regions of plankton abundance and scarcity as well as areas of data abundance and scarcity. At many of the grid points, the MAREDAT team accomplished the difficult conversion from abundance (numbers of organisms) to biomass (carbon mass of organisms). The MAREDAT atlas provides an unprecedented global data set for ecological and biochemical analysis and modeling as well as a clear mandate for compiling additional existing data and for focusing future data gathering efforts on key groups in key areas of the ocean. The present collection presents the original data sets used to compile Global distributions of diazotrophs abundance, biomass and nitrogen fixation rates

Global distributions of diazotrophs abundance and biomass - Depth integrated values computed from a collection of source datasets - Contribution to the MAREDAT World Ocean Atlas of Plankton Functional Types

The MAREDAT atlas covers 11 types of plankton, ranging in size from bacteria to jellyfish. Together, these plankton groups determine the health and productivity of the global ocean and play a vital role in the global carbon cycle. Working within a uniform and consistent spatial and depth grid (map) of the global ocean, the researchers compiled thousands and tens of thousands of data points to identify regions of plankton abundance and scarcity as well as areas of data abundance and scarcity. At many of the grid points, the MAREDAT team accomplished the difficult conversion from abundance (numbers of organisms) to biomass (carbon mass of organisms). The MAREDAT atlas provides an unprecedented global data set for ecological and biochemical analysis and modeling as well as a clear mandate for compiling additional existing data and for focusing future data gathering efforts on key groups in key areas of the ocean. The present data set presents depth integrated values of diazotrophs abundance and biomass, computed from a collection of source data sets.

Ocean acidification affects the phyto-zoo plankton trophic transfer efficiency

The critical role played by copepods in ocean ecology and biogeochemistry warrants an understanding of how these animals may respond to ocean acidification (OA). Whilst an appreciation of the potential direct effects of OA, due to elevated pCO2, on copepods is improving, little is known about the indirect impacts acting via bottom-up(food quality) effects. We assessed, for the first time, the chronic effects of direct and/or indirect exposures to elevated pCO2 on the behaviour, vital rates, chemical and biochemical stoichiometry of the calanoid copepod Acartia tonsa. Bottom-up effects of elevated pCO2 caused species-specific biochemical changes to the phytoplanktonic feed, which adversely affected copepod population structure and decreased recruitment by 30 %. The direct impact of elevated pCO2 caused gender-specific respiratory responses in A.tonsa adults, stimulating an enhanced respiration rate in males (> 2-fold), and a suppressed respiratory response in females when coupled with indirect elevated pCO2 exposures. Under the combined indirect+direct exposure, carbon trophic transfer efficiency from phytoplankton-to-zooplankton declined to < 50 % of control populations, with a commensurate decrease in recruitment. For the first time an explicit role was demonstrated for biochemical stoichiometry in shaping copepod trophic dynamics. The altered biochemical composition of the CO2-exposed prey affected the biochemical stoichiometry of the copepods, which could have ramifications for production of higher tropic levels, notably fisheries. Our work indicates that the control of phytoplankton and the support of higher trophic levels involving copepods have clear potential to be adversely affected under future OA scenarios.

Responses of the Emiliania huxleyi Proteome to Ocean Acidification

Ocean acidification due to rising atmospheric CO2 is expected to affect the physiology of important calcifying marine organisms, but the nature and magnitude of change is yet to be established. In coccolithophores, different species and strains display varying calcification responses to ocean acidification, but the underlying biochemical properties remain unknown. We employed an approach combining tandem mass-spectrometry with isobaric tagging (iTRAQ) and multiple database searching to identify proteins that were differentially expressed in cells of the marine coccolithophore species Emiliania huxleyi (strain NZEH) between two CO2 conditions: 395 (~current day) and ~1340 p.p.m.v. CO2. Cells exposed to the higher CO2 condition contained more cellular particulate inorganic carbon (CaCO3) and particulate organic nitrogen and carbon than those maintained in present-day conditions. These results are linked with the observation that cells grew slower under elevated CO2, indicating cell cycle disruption. Under high CO2 conditions, coccospheres were larger and cells possessed bigger coccoliths that did not show any signs of malformation compared to those from cells grown under present-day CO2 levels. No differences in calcification rate, particulate organic carbon production or cellular organic carbon: nitrogen ratios were observed. Results were not related to nutrient limitation or acclimation status of cells. At least 46 homologous protein groups from a variety of functional processes were quantified in these experiments, of which four (histones H2A, H3, H4 and a chloroplastic 30S ribosomal protein S7) showed down-regulation in all replicates exposed to high CO2, perhaps reflecting the decrease in growth rate. We present evidence of cellular stress responses but proteins associated with many key metabolic processes remained unaltered. Our results therefore suggest that this E. huxleyi strain possesses some acclimation mechanisms to tolerate future CO2 scenarios, although the observed decline in growth rate may be an overriding factor affecting the success of this ecotype in future oceans.

Seawater carbonate chemistry and Emiliania huxleyi biological processes during experiments, 2010

The calcifying phytoplankton species, coccolithophores, have their calcified coccoliths around the cells, however, their physiological roles are still unknown. Here, we hypothesized that the coccoliths may play a certain role in reducing solar UV radiation (UVR, 280-400 nm) and protect the cells from being harmed. Cells of Emiliania huxleyi with different thicknesses of the coccoliths were obtained by culturing them at different levels of dissolved inorganic carbon and their photophysiological responses to UVR were investigated. Although increased dissolved inorganic carbon decreased the specific growth rate, the increased coccolith thickness significantly ameliorated the photoinhibition of PSII photochemical efficiency caused by UVR. Increase by 91% in the coccolith thickness led to 35% increase of the PSII yield and 22% decrease of the photoinhibition of the effective quantum yield by UVR. The coccolith cover reduced more UVA (320-400 nm) than UVB (280-315 nm), leading to less inhibition per energy at the UV-A band.

Seawater carbonate chemistry, nutrients, and Calcidiscus leptoporus (strain RCC1135) growth rate and calcification rate during experiments, 2012

The coccolithophore Calcidiscus leptoporus was grown in batch culture under nitrogen (N) as well as phosphorus (P) limitation. Growth rate, particulate inorganic carbon (PIC), particulate organic carbon (POC), particulate organic nitrogen (PON), and particulate organic phosphorus (POP) production were determined and coccolith morphology was analysed. While PON production decreased by 70% under N-limitation and POP production decreased by 65% under P-limitation, growth rate decreased by 33% under N- as well as P-limitation. POC as well as PIC production (calcification rate) increased by 27% relative to the control under P-limitation, and did not change under N-limitation. Coccolith morphology did not change in response to either P or N limitation. While these findings, supported by a literature survey, suggest that coccolith morphogenesis is not hampered by either P or N limitation, calcification rate might be. The latter conclusion is in apparent contradiction to our data. We discuss the reasons for this inference.

Seawater carbonate chemistry and effects of CO2 and their modulation by light in the life-cycle stages of the coccolithophore Emiliania huxleyi strains RCC 1216 and 1217 during experiments, 2012

The effects of ocean acidification on the life-cycle stages of the coccolithophore Emiliania huxleyi and their by light were examined. Calcifying diploid and noncalcifying haploid cells (Roscoff culture collection 1216 and 1217) were acclimated to present-day and elevated CO2 partial pressures (PCO2; 38.5 vs. 101.3 Pa, ., 380 vs. 1000 matm) under low and high light (50 vs. 300 mmol photons m-2 s-1). Growth rates as well as quotas and production rates of C and N were measured. Sources of inorganic C for biomass buildup were using a 14C disequilibrium assay. Photosynthetic O2 evolution was measured as a function of dissolved inorganic C and light by means of membrane-inlet mass spectrometry. The diploid stage responded to elevated PCO2 by shunting resources from the production of particulate inorganic C toward organic C yet keeping the production of total particulate C constant. As the effect of ocean acidification was stronger under low light, the diploid stage might be less affected by increased acidity when energy availability is high. The haploid stage maintained elemental composition and production rates under elevated PCO2. Although both life-cycle stages involve different ways of dealing with elevated PCO2, the responses were generally modulated by energy availability, being typically most pronounced under low light. Additionally, PCO2 responses resembled those induced by high irradiances, indicating that ocean acidification affects the interplay between energy-generating processes (photosynthetic light reactions) and processes competing for energy (biomass buildup and calcification). A conceptual model is put forward explaining why the magnitude of single responses is determined by energy availability.

Seawater carbonate chemistry and biological processes of Emiliania huxleyi (strains RCC1256 and NZEH) during experiments, 2011

With respect to their sensitivity to ocean acidification, calcifiers such as the coccolithophore Emiliania huxleyi have received special attention, as the process of calcification seems to be particularly sensitive to changes in the marine carbonate system. For E. huxleyi, apparently conflicting results regarding its sensitivity to ocean acidification have been published (Iglesias-Rodriguez et al., 2008a; Riebesell et al., 2000). As possible causes for discrepancies, intra-specific variability and different effects of CO2 manipulation methods, i.e. the manipulation of total alkalinity (TA) or total dissolved inorganic carbon (DIC), have been discussed. While Langer et al. (2009) demonstrate a high degree of intra-specific variability between strains of E. huxleyi, the question whether different CO2 manipulation methods influence the cellular responses has not been resolved yet. In this study, closed TA as well as open and closed DIC manipulation methods were compared with respect to E. huxleyi's CO2-dependence in growth rate, POC- and PIC-production. The differences in the carbonate chemistry between TA and DIC manipulations were shown not to cause any differences in response patterns, while the latter differed between open and closed DIC manipulation. The two strains investigated showed different sensitivities to acidification of seawater, RCC1256 being more negatively affected in growth rates and PIC production than NZEH.

The effect of nitrate and phosphate availability on Emiliania huxleyi(NZEH) physiology under different CO2 scenarios

Growth and calcification of the marine coccolithophorid Emiliania huxleyi is affected by ocean acidification and macronutrients limitation and its response varies between strains. Here we investigated the physiological performance of a highly calcified E. huxleyi strain, NZEH, in a multiparametric experiment. Cells were exposed to different CO2 levels (ranging from 250 to 1314 µatm) under three nutrient conditions [nutrient replete (R), nitrate limited (-N), and phosphate limited (-P)]. We focused on calcite and organic carbon quotas and on nitrate and phosphate utilization by analyzing the activity of nitrate reductase (NRase) and alkaline phosphatase (APase), respectively. Particulate inorganic (PIC) and organic (POC) carbon quotas increased with increasing CO2 under R conditions but a different pattern was observed under nutrient limitation. The PIC:POC ratio decreased with increasing CO2 in nutrient limited cultures. Coccolith length increased with CO2 under all nutrient conditions but the coccosphere volume varied depending on the nutrient treatment. Maximum APase activity was found at 561 ?atm of CO2 (pH 7.92) in -P cultures and in R conditions, NRase activity increased linearly with CO2. These results suggest that E. huxleyi's competitive ability for nutrient uptake might be altered in future high-CO2 oceans. The combined dataset will be useful in model parameterizations of the carbon cycle and ocean acidification.

Reduced resilience of a globally distributed coccolithophore to ocean acidification: Confirmed up to 2000 generations

Ocean acidification (OA), induced by rapid anthropogenic CO2 rise and its dissolution in seawater, is known to have consequences for marine organisms. However, knowledge on the evolutionary responses of phytoplankton to OA has been poorly studied. Here we examined the coccolithophore Gephyrocapsa oceanica, while growing it for 2000 generations under ambient and elevated CO2 levels. While OA stimulated growth in the earlier selection period (from generations 700 to 1550), it reduced it in the later selection period up to 2000 generations. Similarly, stimulated production of particulate organic carbon and nitrogen reduced with increasing selection period and decreased under OA up to 2000 generations. The specific adaptation of growth to OA disappeared in generations 1700 to 2000 when compared with that at 1000 generations. Both phenotypic plasticity and fitness decreased within selection time, suggesting that the species' resilience to OA decreased after 2000 generations under high CO2 selection.

Seawater carbonate chemistry and combined physiological effects of CO2 and light on the N2-fixing cyanobacterium Trichodesmium IMS101 during experiments, 2010

Recent studies on the diazotrophic cyanobacterium Trichodesmium erythraeum(IMS101) showed that increasing CO2 partial pressure (pCO2) enhances N2 fixation and growth. Significant uncertainties remain as to the degree of the sensitivity to pCO2, its modification by other environmental factors, and underlying processes causing these responses. To address these questions, we examined the responses ofTrichodesmium IMS101 grown under a matrix of low and high levels of pCO2 (150 and 900 µatm) and irradiance (50 and 200 µmol photons m-2 s-1). Growth rates as well as cellular carbon and nitrogen contents increased with increasing pCO2 and light levels in the cultures. The pCO2-dependent stimulation in organic carbon and nitrogen production was highest under low light. High pCO2 stimulated rates of N2fixation and prolonged the duration, while high light affected maximum rates only. Gross photosynthesis increased with light but did not change with pCO2. HCO3- was identified as the predominant carbon source taken up in all treatments. Inorganic carbon uptake increased with light, but only gross CO2 uptake was enhanced under high pCO2. A comparison between carbon fluxes in vivo and those derived from 13C fractionation indicates high internal carbon cycling, especially in the low-pCO2treatment under high light. Light-dependent oxygen uptake was only detected underlow pCO2 combined with high light or when low-light-acclimated cells were exposed to high light, indicating that the Mehler reaction functions also as a photoprotective mechanism in Trichodesmium. Our data confirm the pronounced pCO2 effect on N2fixation and growth in Trichodesmium and further show a strong modulation of these effects by light intensity. We attribute these responses to changes in the allocation of photosynthetic energy between carbon acquisition and the assimilation of carbon and nitrogen under elevated pCO2. These findings are supported by a complementarystudy looking at photosynthetic fluorescence parameters of photosystem II, photosynthetic unit stoichiometry (photosystem I:photosystem II), and pool sizes of key proteins in carbon and nitrogen acquisition.

Seawater carbonate chemistry, growth rate and morphology of Calcidiscus leptoporus (RCC1135) during experiments, 2011

The coccolithophore Calcidiscus leptoporus (strain RCC1135) was grown in dilute batch culture at CO2 levels ranging from ~200 to ~1600 µatm. Increasing CO2 concentration led to an increased percentage of malformed coccoliths and eventually (at ~1500 µatm CO2) to aggregation of cells. Carbonate chemistry of natural seawater was manipulated in three ways: first, addition of acid; second, addition of a HCO3/CO3 solution; and third, addition of both acid and HCO3/CO3 solution. The data set allowed the disentangling of putative effects of the different parameters of the carbonate system. It is concluded that CO2 is the parameter of the carbonate system which causes both aberrant coccolithogenesis and aggregation of cells.

Seawater carbonate chemistry and biological processes of coccolithophore (Gephyrocapsa oceanica and Coccolithus pelagicus ssp. braarudii) during experiments, 2011

All species of coccolithophore appear to respond to perturbations of carbonate chemistry in a different way. Here, we show that the degree of malformation, growth rate and stable isotopic composition of organic matter and carbonate produced by two contrasting species of coccolithophore (Gephyrocapsa oceanica and Coccolithus pelagicus ssp. braarudii) are indicative of differences between their photosynthetic and calcification response to changing DIC levels (ranging from ~1100 to ~7800 µmol/kg) at constant pH (8.13 ± 0.02). Gephyrocapsa oceanica thrived under all conditions of DIC, showing evidence of increased growth rates at higher DIC, but C. braarudii was detrimentally affected at high DIC showing signs of malformation, and decreased growth rates. The carbon isotopic fractionation into organic matter and the coccoliths suggests that C. braarudii utilises a common internal pool of carbon for calcification and photosynthesis but G. oceanica relies on independent supplies for each process. All coccolithophores appear to utilize bicarbonate as their ultimate source of carbon for calcification resulting in the release of a proton. But, we suggest that this proton can be harnessed to enhance the supply of CO2(aq) for photosynthesis either from a large internal HCO3- pool which acts as a pH buffer (C. braarudii), or pumped externally to aid the diffusive supply of CO2 across the membrane from the abundant HCO3- (G. oceanica), likely mediated by an internal and external carbonic anhydrase respectively. Our simplified hypothetical spectrum of physiologies may provide a context to understand different species response to changing pH and DIC, the species-specific delta p and calcite "vital effects", as well as accounting for geological trends in coccolithophore cell size.

Seawater carbonate chemistry, growth rate and Emiliania huxleyi (strain AC481) biological processes during experiments, 2010

The impact of ocean acidification and increased water temperature on marine ecosystems, in particular those involving calcifying organisms, has been gradually recognised. We examined the individual and combined effects of increased pCO2 (180 ppmV CO2, 380 ppmV CO2 and 750 ppmV CO2 corresponding to past, present and future CO2 conditions, respectively) and temperature (13 °C and 18 °C) during the exponential growth phase of the coccolithophore E. huxleyi using batch culture experiments. We showed that cellular production rate of Particulate Organic Carbon (POC) increased from the present to the future CO2 treatments at 13 °C. A significant effect of pCO2 and of temperature on calcification was found, manifesting itself in a lower cellular production rate of Particulate Inorganic Carbon (PIC) as well as a lower PIC:POC ratio at future CO2 levels and at 18 °C. Coccosphere-sized particles showed a size reduction with both increasing temperature and CO2concentration. The influence of the different treatments on coccolith morphology was studied by categorizing SEM coccolith micrographs. The number of well-formed coccoliths decreased with increasing pCO2 while temperature did not have a significant impact on coccolith morphology. No interacting effects of pCO2 and temperature were observed on calcite production, coccolith morphology or on coccosphere size. Finally, our results suggest that ocean acidification might have a larger adverse impact on coccolithophorid calcification than surface water warming.