Interactive Effects of Ocean Acidification and Nitrogen-Limitation on the Diatom Phaeodactylum tricornutum

Climate change is expected to bring about alterations in the marine physical and chemical environment that will induce changes in the concentration of dissolved CO₂ and in nutrient availability. These in turn are expected to affect the physiological performance of phytoplankton. In order to learn how phytoplankton respond to the predicted scenario of increased CO₂ and decreased nitrogen in the surface mixed layer, we investigated the diatom Phaeodactylum tricornutum as a model organism. The cells were cultured in both low CO₂ (390 μatm) and high CO₂ (1000 μatm) conditions at limiting (10 μmol L⁻¹) or enriched (110 μmol L⁻¹) nitrate concentrations. Our study shows that nitrogen limitation resulted in significant decreases in cell size, pigmentation, growth rate and effective quantum yield of Phaeodactylum tricornutum, but these parameters were not affected by enhanced dissolved CO₂ and lowered pH. However, increased CO₂ concentration induced higher rETR_max and higher dark respiration rates and decreased the CO₂ or dissolved inorganic carbon (DIC) affinity for electron transfer (shown by higher values for K_{1/2 DIC} or K_{1/2 CO2}). Furthermore, the elemental stoichiometry (carbon to nitrogen ratio) was raised under high CO₂ conditions in both nitrogen limited and nitrogen replete conditions, with the ratio in the high CO₂ and low nitrate grown cells being higher by 45% compared to that in the low CO₂ and nitrate replete grown ones. Our results suggest that while nitrogen limitation had a greater effect than ocean acidification, the combined effects of both factors could act synergistically to affect marine diatoms and related biogeochemical cycles in future oceans.     

Light-Modulated Responses of Growth and Photosynthetic Performance to Ocean Acidification in the Model Diatom Phaeodactylum tricornutum

Ocean acidification (OA) due to atmospheric CO₂ rise is expected to influence marine primary productivity. In order to investigate the interactive effects of OA and light changes on diatoms, we grew Phaeodactylum tricornutum, under ambient (390 ppmv; LC) and elevated CO₂ (1000 ppmv; HC) conditions for 80 generations, and measured its physiological performance under different light levels (60 µmol m⁻² s⁻¹, LL; 200 µmol m⁻² s⁻¹, ML; 460 µmol m⁻² s⁻¹, HL) for another 25 generations. The specific growth rate of the HC-grown cells was higher (about 12–18%) than that of the LC-grown ones, with the highest under the ML level. With increasing light levels, the effective photochemical yield of PSII (F_v′/F_m′) decreased, but was enhanced by the elevated CO₂, especially under the HL level. The cells acclimated to the HC condition showed a higher recovery rate of their photochemical yield of PSII compared to the LC-grown cells. For the HC-grown cells, dissolved inorganic carbon or CO₂ levels for half saturation of photosynthesis (K_1/2 DIC or K_1/2 CO₂) increased by 11, 55 and 32%, under the LL, ML and HL levels, reflecting a light dependent down-regulation of carbon concentrating mechanisms (CCMs). The linkage between higher level of the CCMs down-regulation and higher growth rate at ML under OA supports the theory that the saved energy from CCMs down-regulation adds on to enhance the growth of the diatom.     

The physiological response of marine diatoms to ocean acidification: differential roles of seawater pCO2 and pH

Although increasing the pCO₂ for diatoms will presumably down‐regulate the CO₂‐concentrating mechanism (CCM) to save energy for growth, different species have been reported to respond differently to ocean acidification (OA). To better understand their growth responses to OA, we acclimated the diatoms Thalassiosira pseudonana, Phaeodactylum tricornutum, and Chaetoceros muelleri to ambient (pCO₂ 400 μatm, pH 8.1), carbonated (pCO₂ 800 μatm, pH 8.1), acidified (pCO₂ 400 μatm, pH 7.8), and OA (pCO₂ 800 μatm, pH 7.8) conditions and investigated how seawater pCO₂ and pH affect their CCMs, photosynthesis, and respiration both individually and jointly. In all three diatoms, carbonation down‐regulated the CCMs, while acidification increased both the photosynthetic carbon fixation rate and the fraction of CO₂ as the inorganic carbon source. The positive OA effect on photosynthetic carbon fixation was more pronounced in C. muelleri, which had a relatively lower photosynthetic affinity for CO₂, than in either T. pseudonana or P. tricornutum. In response to OA, T. pseudonana increased respiration for active disposal of H⁺ to maintain its intracellular pH, whereas P. tricornutum and C. muelleri retained their respiration rate but lowered the intracellular pH to maintain the cross‐membrane electrochemical gradient for H⁺ efflux. As the net result of changes in photosynthesis and respiration, growth enhancement to OA of the three diatoms followed the order of C. muelleri > P. tricornutum > T. pseudonana. This study demonstrates that elucidating the separate and joint impacts of increased pCO₂ and decreased pH aids the mechanistic understanding of OA effects on diatoms in the future, acidified oceans.     

Decreased photosynthesis and growth with reduced respiration in the model diatom Phaeodactylum tricornutum grown under elevated CO2 over 1800 generations

Studies on the long‐term responses of marine phytoplankton to ongoing ocean acidification (OA) are appearing rapidly in the literature. However, only a few of these have investigated diatoms, which is disproportionate to their contribution to global primary production. Here we show that a population of the model diatom Phaeodactylum tricornutum, after growing under elevated CO₂ (1000 μatm, HCL, pH_T: 7.70) for 1860 generations, showed significant differences in photosynthesis and growth from a population maintained in ambient CO₂ and then transferred to elevated CO₂ for 20 generations (HC). The HCL population had lower mitochondrial respiration, than did the control population maintained in ambient CO₂ (400 μatm, LCL, pH_T: 8.02) for 1860 generations. Although the cells had higher respiratory carbon loss within 20 generations under the elevated CO₂, being consistent to previous findings, they downregulated their respiration to sustain their growth in longer duration under the OA condition. Responses of phytoplankton to OA may depend on the timescale for which they are exposed due to fluctuations in physiological traits over time. This study provides the first evidence that populations of the model species, P. tricornutum, differ phenotypically from each other after having been grown for differing spans of time under OA conditions, suggesting that long‐term changes should be measured to understand responses of primary producers to OA, especially in waters with diatom‐dominated phytoplankton assemblages.     

Effects of ocean acidification and phosphate limitation on physiology and toxicity of the dinoflagellate Karenia mikimotoi

This work demonstrated a 10-day batch culture experiment to test the physiology and toxicity of harmful dinoflagellate Karenia mikimotoi in response to ocean acidification (OA) under two different phosphate concentrations. Cells were previously acclimated in OA (pH = 7.8 and CO₂ = 1100 μatm) condition for about three months before testing the responses of K. mikimotoi cells to a two-factorial combinations experimentation. This work measured the variation in physiological parameters (growth, rETR) and toxicity (hemolytic activity and its toxicity to zebrafish embryos) in four treatments, representing two factorial combinations of CO₂ (450 and 1100 μatm) and phosphate concentration (37.75 and 4.67 umol l⁻¹). Results: OA stimulated the faster growth, and the highest rETR_max in high phosphate (HP) treatment, low phosphate (LP) and a combination of high CO₂ and low phosphate (HC*LP) inhibited the growth and E_k in comparison to low CO₂*high phosphate (LCHP) treatment. The embryotoxicity of K. mikimotoi cells enhanced in all high CO₂ (HC) conditions irrespective of phosphate concentration, but the EC₅₀ of hemolytic activity increased in all high CO₂ (HC) and low phosphate (LP) treatments in comparison of LCHP. Ocean acidification (high CO₂ and lower pH) was probably the main factor that affected the rETR_max, hemolytic activity and embryotoxicity, but low phosphate was the main factor that affected the growth, α, and E_k. There were significant interactive effects of OA and low phosphate (LP) on growth, rETR_max, and hemolytic activity, but there were no significant effects on α, E_k, and embryotoxicity. If these results are extrapolated to the aquatic environment, it can be hypothesized that the K. mikimotoi cells were impacted significantly by future changing ocean (e.g., ocean acidification and nutrient stoichiometry).     

Ocean acidification stimulates particulate organic carbon accumulation in two Antarctic diatom species under moderate and high natural solar radiation

Impacts of rising atmospheric CO₂ concentrations and increased daily irradiances from enhanced surface water stratification on phytoplankton physiology in the coastal Southern Ocean remain still unclear. Therefore, in the two Antarctic diatoms Fragilariopsis curta and Odontella weissflogii, the effects of moderate and high natural solar radiation combined with either ambient or future pCO₂ on cellular particulate organic carbon (POC) contents and photophysiology were investigated. Results showed that increasing CO₂ concentrations had greater impacts on diatom physiology than exposure to increasing solar radiation. Irrespective of the applied solar radiation regime, cellular POC quotas increased with future pCO₂ in both diatoms. Lowered maximum quantum yields of photochemistry in PSII (F_v/F_m) indicated a higher photosensitivity under these conditions, being counteracted by increased cellular concentrations of functional photosynthetic reaction centers. Overall, our results suggest that both bloom‐forming Antarctic coastal diatoms might increase carbon contents under future pCO₂ conditions despite reduced physiological fitness. This indicates a higher potential for primary productivity by the two diatom species with important implications for the CO₂ sequestration potential of diatom communities in the future coastal Southern Ocean.     

Carbon acquisition by diatoms

Diatoms are responsible for up to 40% of primary productivity in the ocean, and complete genome sequences are available for two species. However, there are very significant gaps in our understanding of how diatoms take up and assimilate inorganic C. Diatom plastids originate from secondary endosymbiosis with a red alga and their Form ID Rubisco (ribulose-1,5-bisphosphate carboxylase-oxygenase) from horizontal gene transfer, which means that embryophyte paradigms can only give general guidance as to their C acquisition mechanisms. Although diatom Rubiscos have relatively high CO₂ affinity and CO₂/O₂ selectivity, the low diffusion coefficient for CO₂ in water has the potential to restrict the rate of photosynthesis. Diatoms growing in their natural aquatic habitats operate inorganic C concentrating mechanisms (CCMs), which provide a steady-state CO₂ concentration around Rubisco higher than that in the medium. How these CCMs work is still a matter of debate. However, it is known that both CO₂ and HCO₃⁻ are taken up, and an obvious but as yet unproven possibility is that active transport of these species across the plasmalemma and/or the four-membrane plastid envelope is the basis of the CCM. In one marine diatom there is evidence of C₄-like biochemistry which could act as, or be part of, a CCM. Alternative mechanisms which have not been eliminated include the production of CO₂ from HCO₃⁻ at low pH maintained by a H⁺ pump, in a compartment close to that containing Rubisco.     

The effect of pCO2 on carbon acquisition and intracellular assimilation in four marine diatoms

The effect of pCO₂ on carbon acquisition and intracellular assimilation was investigated in the three bloom-forming diatom species, Eucampia zodiacus (Ehrenberg), Skeletonema costatum (Greville) Cleve, Thalassionema nitzschioides (Grunow) Mereschkowsky and the non-bloom-forming Thalassiosira pseudonana (Hust.) Hasle and Heimdal. In vivo activities of carbonic anhydrase (CA), photosynthetic O₂ evolution, CO₂ and HCO₃⁻ uptake rates were measured by membrane-inlet mass spectrometry (MIMS) in cells acclimated to pCO₂ levels of 370 and 800 μatm. To investigate whether the cells operate a C₄-like pathway, activities of ribulose-1,5-bisphosphate carboxylase (RubisCO) and phosphoenolpyruvate carboxylase (PEPC) were measured at the mentioned pCO₂ levels and a lower pCO₂ level of 50 μatm. In the bloom-forming species, extracellular CA activities strongly increased with decreasing CO₂ supply while constantly low activities were obtained for T. pseudonana. Half-saturation concentrations (K_1/2) for photosynthetic O₂ evolution decreased with decreasing CO₂ supply in the two bloom-forming species S. costatum and T. nitzschioides, but not in T. pseudonana and E. zodiacus. With the exception of S. costatum, maximum rates (V_max) of photosynthesis remained constant in all investigated diatom species. Independent of the pCO₂ level, PEPC activities were significantly lower than those for RubisCO, averaging generally less than 3%. All examined diatom species operate highly efficient CCMs under ambient and high pCO₂, but differ strongly in the degree of regulation of individual components of the CCM such as C_i uptake kinetics and extracellular CA activities. The present data do not suggest C₄ metabolism in the investigated species.     

The role of external carbonic anhydrase in photosynthesis during growth of the marine diatom Chaetoceros muelleri

Carbon dioxide concentrating mechanisms (CCMs) act to improve the supply of CO₂ at the active site of ribulose‐1,5‐bisphosphate carboxylase/oxygenase. There is substantial evidence that in some microalgal species CCMs involve an external carbonic anhydrase (CA_ext) and that CA_ext activity is induced by low CO₂ concentrations in the growth medium. However, much of this work has been conducted on cells adapted to air‐equilibrium concentrations of CO₂, rather than to changing CO₂ conditions caused by growing microalgal populations. We investigated the role of CA_ext in inorganic carbon (C_i) acquisition and photosynthesis at three sampling points during the growth cycle of the cosmopolitan marine diatom Chaetoceros muelleri. We observed that CA_ext activity increased with decreasing C_i, particularly CO₂, concentration, supporting the idea that CA_ext is modulated by external CO₂ concentration. Additionally, we found that the contribution of CA_ext activity to carbon acquisition for photosynthesis varies over time, increasing between the first and second sampling points before decreasing at the last sampling point, where external pH was high. Lastly, decreases in maximum quantum yield of photosystem II (F_v/F_m), chlorophyll, maximum relative electron transport rate, light harvesting efficiency (α) and maximum rates of C_i‐ saturated photosynthesis (V_max) were observed over time. Despite this decrease in photosynthetic capacity an up‐regulation of CCM activity, indicated by a decreasing half‐saturation constant for CO₂ (K_0.5CO₂), occurred over time. The flexibility of the CCM during the course of growth in C. muelleri may contribute to the reported dominance and persistence of this species in phytoplankton blooms.     

The dinoflagellate Akashiwo sanguinea will benefit from future climate change: The interactive effects of ocean acidification,warming and high irradiance on photophysiology and hemolytic activity

Due to global climate change, marine phytoplankton will likely experience low pH (ocean acidification), high temperatures and high irradiance in the future. Here, this work report the results of a batch culture experiment conducted to study the interactive effects of elevated CO₂, increased temperature and high irradiance on the harmful dinoflagellate Akashiwo sanguinea, isolated at Dongtou Island, Eastern China Sea. The A. sanguinea cells were acclimated in high CO₂ condition for about three months before testing the responses of cells to a full factorial matrix experimentation during a 7-day period. This study measured the variation in physiological parameters and hemolytic activity in 8 treatments, representing full factorial combinations of 2 levels each of exposure to CO₂ (400 and 1000 μatm), temperature (20 and 28 °C) and irradiance (50 and 200 μmol photons m⁻² s⁻¹). Sustained growth of A. sanguinea occurred in all treatments, but high CO₂ (HC) stimulated faster growth than low CO₂ (LC). The pigments (chlorophyll a and carotenoid) decreased in all HC treatments. The quantum yield (F_v/F_m) declined slightly in all high-temperature (HT) treatments. High irradiance (HL) induced the accumulation of ultraviolet-absorbing compounds (UV_abc) irrespective of temperature and CO₂. The hemolytic activity in the LC treatments, however, declined when exposed to HT and HL, but HC alleviated the adverse effects of HT and HL on hemolytic activity. All HC and HL conditions and the combinations of high temperature*high light (HTHL) and high CO₂*high temperature*high light (HCHTHL) positively affected the growth in comparison to the low CO₂*low temperature*low light (LCLTLL) treatment. High temperature (HT), high light (HL) and a combination of HT*HL, however, negatively impacted hemolytic activity. CO₂ was the main factor that affected the growth and hemolytic activity. There were no significant interactive effects of CO₂*temperature*irradiance on growth, pigment, F_v/F_m or hemolytic activity, but there were effects on P_m, α, and E_k. If these results are extrapolated to the natural environment, it can be hypothesized that A. sanguinea cells will benefit from the combination of ocean acidification, warming, and high irradiance that are likely to occur under future climate change. It is assumed that faster growth and higher hemolytic activity and UV_abc of this species will occur under future conditions compared with those the current CO₂ (400 μatm) and temperature (20 °C) conditions.     

Oligotrophic waters of the Northwest Atlantic support taxonomically diverse diatom communities that are distinct from coastal waters

Diatoms are important components of the marine food web and one of the most species-rich groups of phytoplankton. The diversity and composition of diatoms in eutrophic nearshore habitats have been well documented due to the outsized influence of diatoms on coastal ecosystem functioning. In contrast, patterns of both diatom diversity and community composition in offshore oligotrophic regions where diatom biomass is low have been poorly resolved. To compare the diatom diversity and community composition in oligotrophic and eutrophic waters, diatom communities were sampled along a 1,250 km transect from the oligotrophic Sargasso Sea to the coastal waters of the northeast US shelf. Diatom community composition was determined by amplifying and sequencing the 18S rDNA V4 region. Of the 301 amplicon sequence variants (ASVs) identified along the transect, the majority (70%) were sampled exclusively from oligotrophic waters of the Gulf Stream and Sargasso Sea and included the genera Bacteriastrum, Haslea, Hemiaulus, Pseudo-nitzschia, and Nitzschia. Diatom ASV richness did not vary along the transect, indicating that the oligotrophic Sargasso Sea and Gulf Stream are occupied by a diverse diatom community. Although ASV richness was similar between oligotrophic and coastal waters, diatom community composition in these regions differed significantly and was correlated with temperature and phosphate, two environmental variables known to influence diatom metabolism and geographic distribution. In sum, oligotrophic waters of the western North Atlantic harbor diverse diatom assemblages that are distinct from coastal regions, and these open ocean diatoms warrant additional study, as they may play critical roles in oligotrophic ecosystems.     

Size scaling of extracellular carbonic anhydrase activity in centric marine diatoms

Many microalgae have a surface‐associated extracellular carbonic anhydrase (eCA) that converts HCO₃^? to CO₂ for uptake and subsequent photosynthetic fixation. We investigated eCA activity and assessed its importance for photosynthetic CO₂ supply in six centric diatom species spanning nearly the full range of cell sizes for centric diatoms (equivalent spherical radius 3–67 μm). Since larger cells are more susceptible to diffusion limitation, we hypothesized that eCA activity would increase with cell size as would its importance for CO₂ supply. eCA activity did increase with cell size, increasing with cell radius by a size‐scaling exponent of 2.6 ± 0.3. The rapid increase in eCA activity with cell radius keeps the absolute CO₂ concentration difference between bulk seawater and the cell surface very low (<~0.2 μM) allowing high rates of CO₂ uptake even for large diatoms. Although inhibiting eCA did reduce photosynthesis in the diatoms, there was no overall relationship between the extent of inhibition of photosynthesis and cell size. The only indication that eCA may be more important for larger diatoms was that photosynthesis in the smallest diatoms (<4 μm radius) was only affected by eCA inhibition when CO₂ concentrations were very low, while photosynthesis in some larger diatoms was affected even at typical seawater CO₂ concentrations. eCA is ubiquitous in centric marine diatoms, in contrast to other taxa where its presence is irregularly distributed among different species, and plays an important role in supplying CO₂ for photosynthesis across the size spectrum.     

Responses of Two Scleractinian Corals to Cobalt Pollution and Ocean Acidification

The effects of ocean acidification alone or in combination with warming on coral metabolism have been extensively investigated, whereas none of these studies consider that most coral reefs near shore are already impacted by other natural anthropogenic inputs such as metal pollution. It is likely that projected ocean acidification levels will aggravate coral reef health. We first investigated how ocean acidification interacts with one near shore locally abundant metal on the physiology of two major reef-building corals: Stylophora pistillata and Acropora muricata. Two pH levels (pH_T 8.02; pCO₂ 366 μatm and pH_T 7.75; pCO₂ 1140 μatm) and two cobalt concentrations (natural, 0.03 μg L^-1 and polluted, 0.2 μg L^-1) were tested during five weeks in aquaria. We found that, for both species, cobalt input decreased significantly their growth rates by 28% while it stimulated their photosystem II, with higher values of rETR_max (relative Electron Transport Rate). Elevated pCO₂ levels acted differently on the coral rETR_max values and did not affect their growth rates. No consistent interaction was found between pCO₂ levels and cobalt concentrations. We also measured in situ the effect of higher cobalt concentrations (1.06 ± 0.16 μg L^-1) on A. muricata using benthic chamber experiments. At this elevated concentration, cobalt decreased simultaneously coral growth and photosynthetic rates, indicating that the toxic threshold for this pollutant has been reached for both host cells and zooxanthellae. Our results from both aquaria and in situ experiments, suggest that these coral species are not particularly sensitive to high pCO₂ conditions but they are to ecologically relevant cobalt concentrations. Our study reveals that some reefs may be yet subjected to deleterious pollution levels, and even if no interaction between pCO₂ levels and cobalt concentration has been found, it is likely that coral metabolism will be weakened if they are subjected to additional threats such as temperature increase, other heavy metals, and eutrophication.     

CO2 concentrations in perennially ice-covered lakes of Taylor Valley, Antarctica

Lakes in Taylor Valley, southern Victoria Land,Antarctica, are unusual in that they areperennially covered by a 3–5 m thick ice layer.Previous work on gas concentrations in theselakes has shown that the surface waters aresupersaturated with respect to O₂,N₂O, as well as the noble gases. Our datashow that the dissolved CO₂ (CO_2(aq))concentrations, calculated from pH andCO₂, can be highly undersaturatedat shallow depths of the lakes. CO₂partial pressure values (pCO₂) are as lowas 10^–4.3 atm and 10^–4.2 atm in theeast and west lobes of Lake Bonney,respectively, and 10^–3.8 atm in LakeHoare. CO_2(aq) depletion occurred only inthe uppermost part of the water column, inassociation with elevated primary productivity(PPR). The upward diffusion of CO_2(aq)from the aphotic zone, and the annual input ofCO₂ via glacial meltwater can notreplenish the amount of CO_2(aq) annuallylost to primary productivity in the uppermostmeters of the water column. Calcification is alimited source of CO_2(aq), since the lakesare undersaturated with respect to calcitethrough portions of the austral summer.Preliminary respiration rates have been used toobtain an annual inorganic carbon balance.Further down in the water column, at the sitesof the deep-water maximum in primary production(PPR_max), which in Lakes Bonney andFryxell is associated with nutrient gradients,CO_2(aq) is not undersaturated. A largeupward flux from CO₂-supersaturatedaphotic waters provides a surplus ofCO_2(aq) at the PPR_max. Lake Fryxell,unlike the other lakes, is supersaturated withCO_2(aq) throughout the entire water column.     

Effects of temperature and pH on growth and photosynthesis of the thermophilic cyanobacterium Synechococcus lividus as measured by pulse-amplitude modulated fluorometry

In this study, the effects of five different temperatures and pH conditions on growth and photosynthetic performance of Synechococcus lividus Copeland from Taiwan were monitored in the field and the laboratory by using an underwater pulse‐amplitude modulated (Diving‐PAM) fluorometer. In the field, the optimal growth temperature of S. lividus was found to be 57°C. Such a finding was congruent with the growth rate in the laboratory culture, in which the optimal growth temperatures ranged from 45 to 60°C. In photosynthetic performance, the light‐saturated maximum relative electron transport rate (ETR_max) and the light‐limited slope (α^ETR) exhibited highest values at 50°C. At five different pH conditions, higher ETR_max and α^ETR were observed from pH 7 to 9. In addition, regression analysis demonstrated a significant positive relationship between the growth rate and the ETR_max values (R² = 0.9527), indicating that the growth of S. lividus was largely restricted to its photosynthetic performance. In conclusion, the photosynthetic performance and growth of the thermophilic cyanobacterium S. lividus were sensitive to fluctuations in temperature but not in pH. The present investigation offers a better understanding of the photosynthetic physiology.     

GROWTH OF VITAMIN B12-REQUIRING MARINE DIATOMS IN MIXED LABORATORY CULTURES WITH VITAMIN B12-PRODUCING MARINE BACTERIA12

The vitamin B₁₂ requirement of several marine diatoms can be satisfied in B₁₂^?limited laboratory cultures by heterotrophic marine bacteria isolated from the same waters and from sediments. The bacteria can utilize diatom excretory products, or the remains of dead diatom cells, in the production of the vitamin. The growth of 12 B₁₂¹? requiring diatoms (7 genera) in mixed cultures with 14 different bacteria (without added B₁₂) was compared to the growth of those same diatoms in axenic cultures with excess added B₁₂. Diatom growth was generally rapid in the first few days, followed by sustained, slower growth. The diatom yields in mixed cultures ranged from 0.8 to 84% of the yields in axenic cultures with added B₁₂. In a detailed study of one mixed culture, increases in diatom densities were paralleled by increases in cell densities of the bacterium during the first few days of exponential diatom growth. During the period of slow diatom growth, when diatom densities oscillated but steadily increased, the decreases in diatom densities were associated with increased bacterial growth. This suggests that death of a fraction of the B₁₂-limited diatom population releases sufficient organic matter to stimulate growth of the bacteria and their subsequent excretion of B₁₂; this B₁₂ in turn stimulates further growth of the diatoms. Diatom-bacteria interactions leading to the production of B₁₂ may be important in maintaining viable populations of B₁₂-requiring diatoms in nutrient-poor waters during periods between blooms, when conditions are unfavorable for rapid growth.     

Effect of elevated CO<Subscript>2</Subscript> levels on leaf starch,nitrogen and photosynthesis of plants growing at three natural CO<Subscript>2</Subscript> springs in Japan

Plant communities around natural CO₂ springs have been exposed to elevated CO₂ levels over many generations and give us a unique opportunity to investigate the effects of long-term elevated CO₂ levels on wild plants. We searched for natural CO₂ springs in cool temperate climate regions in Japan and found three springs that were suitable for studying long-term responses of plants to elevated levels of CO₂: Ryuzin-numa, Yuno-kawa and Nyuu. At these CO₂ springs, the surrounding air was at high CO₂ concentration with no toxic gas emissions throughout the growth season, and there was natural vegetation around the springs. At each site, high-CO₂ (HC) and low-CO₂ (LC) plots were established, and three dominant species at the shrub layers were used for physiological analyses. Although the microenvironments were different among the springs, dicotyledonous species growing at the HC plots tended to have more starch and less nitrogen per unit dry mass in the leaves than those growing at the LC plots. In contrast, monocotyledonous species growing in the HC and LC plots had similar starch and nitrogen concentrations. Photosynthetic rates at the mean growth CO₂ concentration were higher in HC plants than LC plants, but photosynthetic rates at a common CO₂ concentration were lower in HC plants. Efficiency of water and nitrogen use of leaves at growth CO₂ concentration was greatly increased in HC plants. These results suggest that natural plants growing in elevated CO₂ levels under cool temperate climate conditions have down-regulated their photosynthetic capacity but that they increased photosynthetic rates and resource use efficiencies due to the direct effect of elevated CO₂ concentration.