Experimental evolution gone wild.

Because of their large population sizes and rapid cell division rates, marine microbes have, or can generate, ample variation to fuel evolution over a few weeks or months, and subsequently have the potential to evolve in response to global change. Here we measure evolution in the marine diatom Skeletonema marinoi evolved in a natural plankton community in CO2-enriched mesocosms deployed in situ. Mesocosm enclosures are typically used to study how the species composition and biogeochemistry of marine communities respond to environmental shifts, but have not been used for experimental evolution to date. Using this approach, we detect a large evolutionary response to CO2 enrichment in a focal marine diatom, where population growth rate increased by 1.3-fold in high CO2-evolved lineages. This study opens an exciting new possibility of carrying out in situ evolution experiments to understand how marine microbial communities evolve in response to environmental change.

Diurnal changes in seawater carbonate chemistry speciation at increasing atmospheric carbon dioxide.

Natural variability in seawater pH and associated carbonate chemistry parameters is in part driven by biological activities such as photosynthesis and respiration. The amplitude of these variations is expected to increase with increasing seawater carbon dioxide (CO2) concentrations in the future, because of simultaneously decreasing buffer capacity. Here, we address this experimentally during a diurnal cycle in a mesocosm CO2 perturbation study. We show that for about the same amount of dissolved inorganic carbon (DIC) utilized in net community production diel variability in proton (H+) and CO2 concentrations was almost three times higher at CO2 levels of about 675 ± 65 in comparison with levels of 310 ± 30 µatm. With a simple model, adequately simulating our measurements, we visualize carbonate chemistry variability expected for different oceanic regions with relatively low or high net community production. Since enhanced diurnal variability in CO2 and proton concentration may require stronger cellular regulation in phytoplankton to maintain respective gradients, the ability to adjust may differ between communities adapted to low in comparison with high natural variability.

Cellular pH measurements in Emiliania huxleyi reveal pronounced membrane proton permeability.

• 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.

Enhanced biological carbon consumption in a high CO2 ocean.

The oceans have absorbed nearly half of the fossil-fuel carbon dioxide (CO2) emitted into the atmosphere since pre-industrial times, causing a measurable reduction in seawater pH and carbonate saturation. If CO2 emissions continue to rise at current rates, upper-ocean pH will decrease to levels lower than have existed for tens of millions of years and, critically, at a rate of change 100 times greater than at any time over this period. Recent studies have shown effects of ocean acidification on a variety of marine life forms, in particular calcifying organisms. Consequences at the community to ecosystem level, in contrast, are largely unknown. Here we show that dissolved inorganic carbon consumption of a natural plankton community maintained in mesocosm enclosures at initial CO2 partial pressures of 350, 700 and 1,050 microatm increases with rising CO2. The community consumed up to 39% more dissolved inorganic carbon at increased CO2 partial pressures compared to present levels, whereas nutrient uptake remained the same. The stoichiometry of carbon to nitrogen drawdown increased from 6.0 at low CO2 to 8.0 at high CO2, thus exceeding the Redfield carbon:nitrogen ratio of 6.6 in today's ocean. This excess carbon consumption was associated with higher loss of organic carbon from the upper layer of the stratified mesocosms. If applicable to the natural environment, the observed responses have implications for a variety of marine biological and biogeochemical processes, and underscore the importance of biologically driven feedbacks in the ocean to global change.

Effect of trace metal availability on coccolithophorid calcification.

The deposition of atmospheric dust into the ocean has varied considerably over geological time. Because some of the trace metals contained in dust are essential plant nutrients which can limit phytoplankton growth in parts of the ocean, it has been suggested that variations in dust supply to the surface ocean might influence primary production. Whereas the role of trace metal availability in photosynthetic carbon fixation has received considerable attention, its effect on biogenic calcification is virtually unknown. The production of both particulate organic carbon and calcium carbonate (CaCO3) drives the ocean's biological carbon pump. The ratio of particulate organic carbon to CaCO3 export, the so-called rain ratio, is one of the factors determining CO2 sequestration in the deep ocean. Here we investigate the influence of the essential trace metals iron and zinc on the prominent CaCO3-producing microalga Emiliania huxleyi. We show that whereas at low iron concentrations growth and calcification are equally reduced, low zinc concentrations result in a de-coupling of the two processes. Despite the reduced growth rate of zinc-limited cells, CaCO3 production rates per cell remain unaffected, thus leading to highly calcified cells. These results suggest that changes in dust deposition can affect biogenic calcification in oceanic regions characterized by trace metal limitation, with possible consequences for CO2 partitioning between the atmosphere and the ocean.

Determination of the complex stability of zinc with carbonic anhydrase in sea-water.

Carbonic anhydrase (CA) is inactive unless associated with zinc, with possible substitution by cobalt. In this work, the complexation of zinc by CA was determined in sea-water using cathodic stripping voltammetry (CSV) with ligand competition. The zinc was found to be released from the CA over a period of 3 h when equilibrated with a competing complexing ligand and the complex was re-formed with the CA when zinc was added. A value of 8.90+/-0.27 was found for logK'ZnCA where K'ZnCA is the conditional stability constant for the complex of Zn2+ with CA in pH 8 sea-water. A value for the molecular weight of CA was calculated from its equivalent ligand concentration (in nM) obtained by titrations with zinc at various CA concentrations (1-4 mg l(-1)). The value found (34740 g mol(-1)) for the molecular weight is consistent with values found previously by other methods (29000-31000 g mol(-1)) confirming that the stoichiometry of the complex between zinc and CA is 1:1. This work confirms that the zinc-CA complex is reversible and that the interaction between zinc and CA can be determined using CSV with ligand competition.

Reduced calcification of marine plankton in response to increased atmospheric CO2.

The formation of calcareous skeletons by marine planktonic organisms and their subsequent sinking to depth generates a continuous rain of calcium carbonate to the deep ocean and underlying sediments. This is important in regulating marine carbon cycling and ocean-atmosphere CO2 exchange. The present rise in atmospheric CO2 levels causes significant changes in surface ocean pH and carbonate chemistry. Such changes have been shown to slow down calcification in corals and coralline macroalgae, but the majority of marine calcification occurs in planktonic organisms. Here we report reduced calcite production at increased CO2 concentrations in monospecific cultures of two dominant marine calcifying phytoplankton species, the coccolithophorids Emiliania huxleyi and Gephyrocapsa oceanica. This was accompanied by an increased proportion of malformed coccoliths and incomplete coccospheres. Diminished calcification led to a reduction in the ratio of calcite precipitation to organic matter production. Similar results were obtained in incubations of natural plankton assemblages from the north Pacific ocean when exposed to experimentally elevated CO2 levels. We suggest that the progressive increase in atmospheric CO2 concentrations may therefore slow down the production of calcium carbonate in the surface ocean. As the process of calcification releases CO2 to the atmosphere, the response observed here could potentially act as a negative feedback on atmospheric CO2 levels.

Temporal trends in deep ocean Redfield ratios.

The Redfield ratio [carbon:nitrogen:phosphorus (C:N:P)] of particle flux to the deep ocean is a key factor in marine biogeochemical cycling. Changes in oceanic carbon sequestration have been linked to variations in the Redfield ratio on geological time scales, but this ratio generally is assumed to be constant with time in the modern ocean. However, deep-water Redfield ratios in the northern hemisphere show evidence for temporal trends over the past five decades. The North Atlantic Ocean exhibits a rising N:P ratio, which may be related to increased deposition of atmospheric nitrous oxides from anthropogenic N emissions. In the North Pacific Ocean, increasing C:N and C:P ratios are accompanied by rising remineralization rates, which suggests intensified export production. Stronger export of carbon in this region may be due to enhanced bioavailability of aeolian iron. These findings imply that the biological part of the marine carbon cycle currently is not in steady state.