Description: Highlights • Calcification rates are stimulated by CO2 and HCO3− and inhibited by H+. • This novel substrate–inhibitor concept is tested with experimental data. • The concept enables us to reconcile conflicting results among laboratory studies. • We illustrate how this physiological concept can be included in ecological theory. • We apply the concept to discuss coccolithophore dispersal in the oceans. Abstract Coccolithophores are a group of unicellular phytoplankton species whose ability to calcify has a profound influence on biogeochemical element cycling. Calcification rates are controlled by a large variety of biotic and abiotic factors. Among these factors, carbonate chemistry has gained considerable attention during the last years as coccolithophores have been identified to be particularly sensitive to ocean acidification. Despite intense research in this area, a general concept harmonizing the numerous and sometimes (seemingly) contradictory responses of coccolithophores to changing carbonate chemistry is still lacking to date. Here, we present the “substrate–inhibitor concept” which describes the dependence of calcification rates on carbonate chemistry speciation. It is based on observations that calcification rate scales positively with bicarbonate (HCO3−), the primary substrate for calcification, and carbon dioxide (CO2), which can limit cell growth, whereas it is inhibited by protons (H+). This concept was implemented in a model equation, tested against experimental data, and then applied to understand and reconcile the diverging responses of coccolithophorid calcification rates to ocean acidification obtained in culture experiments. Furthermore, we (i) discuss how other important calcification-influencing factors (e.g. temperature and light) could be implemented in our concept and (ii) embed it in Hutchinson’s niche theory, thereby providing a framework for how carbonate chemistry-induced changes in calcification rates could be linked with changing coccolithophore abundance in the oceans. Our results suggest that the projected increase of H+ in the near future (next couple of thousand years), paralleled by only a minor increase of inorganic carbon substrate, could impede calcification rates if coccolithophores are unable to fully adapt. However, if calcium carbonate (CaCO3) sediment dissolution and terrestrial weathering begin to increase the oceans’ HCO3− and decrease its H+ concentrations in the far future (10–100 kyears), coccolithophores could find themselves in carbonate chemistry conditions which may be more favorable for calcification than they were before the Anthropocene.

Description: A large-scale multidisciplinary mesocosm experiment in an Arctic fjord (Kongsfjorden, Svalbard; 78°56.2′N) was used to study Arctic marine food webs and biogeochemical elements cycling at natural and elevated future carbon dioxide (CO2) levels. At the start of the experiment, marine-derived chromophoric dissolved organic matter (CDOM) dominated the CDOM pool. Thus, this experiment constituted a convenient case to study production of autochthonous CDOM, which is typically masked by high levels of CDOM of terrestrial origin in the Arctic Ocean proper. CDOM accumulated during the experiment in line with an increase in bacterial abundance; however, no response was observed to increased pCO2 levels. Changes in CDOM absorption spectral slopes indicate that bacteria were most likely responsible for the observed CDOM dynamics. Distinct absorption peaks (at ~ 330 and ~ 360 nm) were likely associated with mycosporine-like amino acids (MAAs). Due to the experimental setup, MAAs were produced in absence of ultraviolet exposure providing evidence for MAAs to be considered as multipurpose metabolites rather than simple photoprotective compounds. We showed that a small increase in CDOM during the experiment made it a major contributor to total absorption in a range of photosynthetically active radiation (PAR, 400–700 nm) and, therefore, is important for spectral light availability and may be important for photosynthesis and phytoplankton groups composition in a rapidly changing Arctic marine ecosystem.

Description: The sea-surface microlayer (SML) is the ocean's uppermost boundary to the atmosphere and in control of climate relevant processes like gas exchange and emission of marine primary organic aerosols (POA). The SML represents a complex surface film including organic components like polysaccharides, proteins, and marine gel particles, and harbors diverse microbial communities. Despite the potential relevance of the SML in ocean-atmosphere interactions, still little is known about its structural characteristics and sensitivity to a changing environment such as increased oceanic uptake of anthropogenic CO2. Here we report results of a large-scale mesocosm study, indicating that ocean acidification can affect the abundance and activity of microorganisms during phytoplankton blooms, resulting in changes in composition and dynamics of organic matter in the SML. Our results reveal a potential coupling between anthropogenic CO2 emissions and the biogenic properties of the SML, pointing to a hitherto disregarded feedback process between ocean and atmosphere under climate change.

Description: Coccolithophores are important calcifying phytoplankton predicted to be impacted by changes in ocean carbonate chemistry caused by the absorption of anthropogenic CO2. However, it is difficult to disentangle the effects of the simultaneously changing carbonate system parameters (CO2, bicarbonate, carbonate and protons) on the physiological responses to elevated CO2. Here, we adopted a multifactorial approach at constant pH or CO2 whilst varying dissolved inorganic carbon (DIC) to determine physiological and transcriptional responses to individual carbonate system parameters. We show that Emiliania huxleyi is sensitive to low CO2 (growth and photosynthesis) and low bicarbonate (calcification) as well as low pH beyond a limited tolerance range, but is much less sensitive to elevated CO2 and bicarbonate. Multiple up-regulated genes at low DIC bear the hallmarks of a carbon-concentrating mechanism (CCM) that is responsive to CO2 and bicarbonate but not to pH. Emiliania huxleyi appears to have evolved mechanisms to respond to limiting rather than elevated CO2. Calcification does not function as a CCM, but is inhibited at low DIC to allow the redistribution of DIC from calcification to photosynthesis. The presented data provides a significant step in understanding how E. huxleyi will respond to changing carbonate chemistry at a cellular level.

Description: About 50 Gt of carbon is fixed photosynthetically by surface ocean phytoplankton communities every year. Part of this organic matter is reprocessed within the plankton community to form aggregates which eventually sink and export carbon into the deep ocean. The fraction of organic matter leaving the surface ocean is partly dependent on aggregate sinking velocity which accelerates with increasing aggregate size and density, where the latter is controlled by ballast load and aggregate porosity. In May 2011, we moored nine 25 m deep mesocosms in a Norwegian fjord to assess on a daily basis how plankton community structure affects material properties and sinking velocities of aggregates (Ø 80–400 µm) collected in the mesocosms' sediment traps. We noted that sinking velocity was not necessarily accelerated by opal ballast during diatom blooms, which could be due to relatively high porosity of these rather fresh aggregates. Furthermore, estimated aggregate porosity (Pestimated) decreased as the picoautotroph (0.2–2 µm) fraction of the phytoplankton biomass increased. Thus, picoautotroph-dominated communities may be indicative for food webs promoting a high degree of aggregate repackaging with potential for accelerated sinking. Blooms of the coccolithophore Emiliania huxleyi revealed that cell concentrations of ~1500 cells/mL accelerate sinking by about 35–40%, which we estimate (by one-dimensional modeling) to elevate organic matter transfer efficiency through the mesopelagic from 14 to 24%. Our results indicate that sinking velocities are influenced by the complex interplay between the availability of ballast minerals and aggregate packaging; both of which are controlled by plankton community structure.

Description: Coccolithophore calcite refers to the plates of calcium carbonate (CaCO3) produced by the calcifying phytoplankton, coccolithophores. The empirical study of the elemental composition has a great potential in the development of paleoproxies. However, the difficulties to separate coccolithophore carbonates from organic phases hamper the investigation of coccoliths magnesium to calcium ratios (Mg/Ca) in biogeochemical studies. Magnesium (Mg) is found in organic molecules in the cells at concentrations up to 400 times higher than in inorganically precipitated calcite in present-day seawater. The aim of this study was to optimize a reliable procedure for organic Mg removal from coccolithophore samples to ensure reproducibility in measurements of inorganic Mg in calcite. Two baseline methods comprising organic matter oxidations with (1) bleach and (2) hydrogen peroxide (H2O2) were tested on synthetic pellets, prepared by mixing reagent grade CaCO3 with organic matter from the non-calcifying marine algae Chlorella autotrophica and measured with an ICP-AES (inductively coupled plasma-atomic emission spectrometer). Our results show that treatments with a reductive solution [using hydroxylamine-hydrochloride (NH2OH·HCl + NH4OH)] followed by three consecutive oxidations (using H2O2) yielded the best cleaning efficiencies, removing 〉99% of organic Mg in 24 h. P/Ca and Fe/Ca were used as indicators for organic contamination in the treated material. The optimized protocol was tested in dried coccolithophore pellets from batch cultures of Emiliania huxleyi, Calcidiscus leptoporus and Gephyrocapsa oceanica. Mg/Ca of treated coccolithophores were 0.151 ± 0.018, 0.220 ± 0.040, and 0.064 ± 0.023 mmol/mol, respectively. Comparison with Mg/Ca literature coccolith values, suggests a tight dependence on modern seawater Mg/Ca, which changes as a consequence of different seawater origins (〈10%). The reliable determination of Mg/Ca and Sr/Ca, and the low levels of organic contamination (Fe/Ca and P/Ca) make this protocol applicable to field and laboratory studies of trace elemental composition in coccolithophore calcite

Description: To understand the influence of changing surface ocean pH and carbonate chemistry on the coccolithophore Emiliania huxleyi, it is necessary to characterize mechanisms involved in pH homeostasis and ion transport. Here, we measured effects of changes in seawater carbonate chemistry on the fluorescence emission ratio of BCECF (2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein) as a measure of intracellular pH (pH(i)). Out of equilibrium solutions were used to differentiate between membrane permeation pathways for H+, CO(2) and HCO(3)-. Changes in fluorescence ratio were calibrated in single cells, resulting in a ratio change of 0.78 per pH(i) unit. pH(i) acutely followed the pH of seawater (pH(e)) in a linear fashion between pH(e) values of 6.5 and 9 with a slope of 0.44 per pH(e) unit. pH(i) was nearly insensitive to changes in seawater CO(2) at constant pH(e) and HCO(3)-. An increase in extracellular HCO(3)- resulted in a slight intracellular acidification. In the presence of DIDS (4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid), a broad-spectrum inhibitor of anion exchangers, E. huxleyi acidified irreversibly. DIDS slightly reduced the effect of pH(e) on pH(i). The data for the first time show the occurrence of a proton permeation pathway in E. huxleyi plasma membrane. pH(i) homeostasis involves a DIDS-sensitive mechanism.

Description: Experimental setups to study modes of inorganic carbon acquisition and fixation rates by marine phytoplankton commonly make use of so-called disequilibrium techniques. The chemical or isotopic disequilibrium, either caused by phytoplankton cells taking up inorganic carbon or by a small disturbance of the isotopic equilibrium in the carbonate system, requires to account for the relatively slow chemical interconversion of carbon dioxide (CO2) to bicarbonate (HCO3−) in seawater. Because in such experiments a constant pH is a prerequisite, pH buffers are generally used. However, a possible influence of such buffers on the kinetics of the carbonate system has hitherto not been investigated. Here, a model of the carbonate system in seawater is employed to show how pH buffers are operating. Furthermore, a new approach is presented to determine the rate constants, k+ and k−, for the conversion reaction of CO2 to HCO3− and vice versa, by means of membrane inlet mass spectrometry (MIMS). For the two pH buffers tested (HEPES and BICINE) it is shown that measured rate constants are in good agreement with calculated values for k+ and k− in a pH range of 7 to 8.5 and at temperatures from 10 to 25 °C.

Description: Throughout the last similar to 900 kyr, the Late Pleistocene, Earth has experienced periods of cold glacial climate, punctuated by seven abrupt transitions to warm interglacials, the so-called terminations. Although most of glacial ice is located in the Northern Hemisphere (NH), the Southern Hemisphere (SH) seems to play a crucial role in deglaciation. Variation in the seasonal distribution of solar insolation is one candidate for the cause of these climatic shifts. But so far, no simple mechanism has been identified. Here we present a mathematical analysis of variations in midsummer insolation in both hemispheres at 65 degrees latitude. Applying this analysis to the entire Pleistocene, the last 2 Myr, we find that prior to each termination the insolation in both hemispheres increases in concert, with a SH lead. Introducing time and energy thresholds to these overlaps, calculated times for the onsets of the seven terminations by this insolation canon (exceptional overlaps meeting the two threshold prerequisites) are similar to 23, 139, 253, 345, 419, 546 and 632 kyr BP, perfectly matching the geologic record. The timing originates from the interplay between the two orbital parameters obliquity and precession, explaining why terminations occur at integer multiple of the precessional cycle. There is no such constellation between I and 2 Myr BP, the Early Pleistocene, in agreement with Earth's climate at that time. This change in orbital forcing coincides with the Mid Pleistocene Revolution, separating the Late from the Early Pleistocene. Therefore, we hypothesize that the insolation canon is the trigger for glacial terminations. (c) 2006 Elsevier B.V. All rights reserved.

Description: Iron is limiting phytoplankton productivity in large parts of today's oceans, the so-called HNLC (high nutrient low chlorophyll) areas. It is a key component in photosynthesis during which inorganic carbon fixation in most phytoplankton species is sustained by so-called carbon concentrating mechanisms (CCMs). Here we investigate CCM regulation in the coccolithophore Emiliania huxleyi in response to varying degrees of iron limitation by means of membrane-inlet mass spectrometry. Compared to iron replete conditions rates of both active CO2 and HCO-3 uptake were markedly reduced under iron limitation leading to significantly diminished growth rates. Moreover, there was a concomitant decrease in CCM efficiency, reflected in an increased CO2 loss from the cell in relation to carbon fixation. Under such conditions higher values for carbon isotope fractionation (∈P) would be expected. However, direct measurements of ∈P showed that carbon isotope fractionation was insensitive to changes in growth rates and CCM activity. This can be explained by concomitant changes in internal DIC fluxes in and out of the chloroplast as demonstrated with a simple cell model comprising two compartments. Thus, carbon isotope fractionation reflects the ability of phytoplankton to actively control their inorganic carbon acquisition depending on environmental conditions. The insensitivity of carbon isotope fractionation to changes in the availability of iron could be of interest for paleoreconstructions in the HNLC areas of today's oceans.