Description: Nitrogen fixation by diazotrophic cyanobacteria is a critical source of new nitrogen to the oligotrophic surface ocean. Research to date indicates that some diazotroph groups may increase nitrogen fixation under elevated pCO2. To test this in natural plankton communities, four manipulation experiments were carried out during two voyages in the South Pacific (30–35oS). High CO2 treatments, produced using 750 ppmv CO2 to adjust pH to 0.2 below ambient, and ‘Greenhouse’ treatments (0.2 below ambient pH and ambient temperature +3 °C), were compared with Controls in trace metal clean deckboard incubations in triplicate. No significant change was observed in nitrogen fixation in either the High CO2 or Greenhouse treatments over 5 day incubations. qPCR measurements and optical microscopy determined that the diazotroph community was dominated by Group A unicellular cyanobacteria (UCYN-A), which may account for the difference in response of nitrogen fixation under elevated CO2 to that reported previously for Trichodesmium. This may reflect physiological differences, in that the greater cell surface area:volume of UCYN-A and its lack of metabolic pathways involved in carbon fixation may confer no benefit under elevated CO2. However, multiple environmental controls may also be a factor, with the low dissolved iron concentrations in oligotrophic surface waters limiting the response to elevated CO2. If nitrogen fixation by UCYN-A is not stimulated by elevated pCO2, then future increases in CO2 and warming may alter the regional distribution and dominance of different diazotroph groups, with implications for dissolved iron availability and new nitrogen supply in oligotrophic regions.

Description: The availability and composition of dissolved nitrogen in ocean waters are factors that influence species composition in natural phytoplankton communities. The same factors affect the ratio of organic to inorganic carbon incorporation in calcifying species, such as the coccolithophore Emiliania huxleyi (Lohman) W. W. Hay et H. Mohler. E. huxleyi has been shown to thrive on various nitrogen sources, including dissolved organic nitrogen. Nevertheless, assimilation of dissolved nitrogen under nitrogen-replete and -limited conditions is not well understood in this ecologically important species. In this study, the complete amino acid sequences for three functional genes involved in nitrogen metabolism in E. huxleyi were identified: a putative formamidase, a glutamine synthetase (GSII family), and assimilatory nitrate reductase. Expression patterns of the three enzymes in cells grown on inorganic as well as organic nitrogen sources indicated reduced expression levels of nitrate reductase when cells were grown on NH(4)+ and a reduced expression level of the putative formamidase when growth was on NO(3)-. The data reported here suggest the presence of a nitrogen preference hierarchy in E. huxleyi. In addition, the gene encoding for a phosphate repressible phosphate permease was more highly expressed in cells growing on formamide than in cells growing on inorganic nitrogen sources. This finding suggests a coupling between phosphate and nitrogen metabolism, which might give this species a competitive advantage in nutrient-depleted environments. The potential of using expression of genes investigated here as indicators of specific nitrogen-metabolism strategies of E. huxleyi in natural populations of phytoplankton is discussed.

Description: We examined the diel variation in nitrogen and carbon metabolism in Crocosphaera watsonii WH8501 at the physiological and gene expression level in order to determine the temporal constraints for N2 fixation and photosynthesis. N2 fixation and photosynthesis were restricted to the dark and light periods, respectively, during a 24 h light–dark cycle. All genes studied here except one (psbA2) showed diel variations in their expression levels. The highest variation was seen in nifH and nifX relative transcript abundance with a factor of 3–5 × 103 between light and dark periods. Photosynthesis genes showed less variation with a maximum factor of about 500 and always had high relative transcript abundances relative to other genes. At the protein level, the photosystems appeared more stable than the nitrogenase complex over a 24 h light–dark cycle, suggesting that C. watsonii retains the ability to photosynthesize during the dark period of the diel cycle. In contrast, nitrogenase is synthesized daily and exhibits peak abundance during the dark period. Our results have implications for field studies with respect to the interpretation of environmental gene expression data.

Description: There has been a widespread increase in the reporting of harmful and ‘nuisance’ algal blooms in the coastal ocean over the past few decades. On the global scale this is suspected to be a consequence of coastal eutrophication, however, on a case-by-case basis there is usually insufficient evidence to discriminate between the effects of human and natural causal factors. Intense blooms of the ‘Brown Tide’ unicellular algae (Aureococcus anophagefferens) have occurred sporadically since 1985 in coastal waters of Eastern Long Island and have devastated the local commercial scallop fishery. Analysis of an 11-year time-series dataset from this region indicates that bloom intensity is correlated with higher salinities and inversely correlated with the discharge of groundwater. Laboratory and field studies suggest that whereas salinity is unlikely to represent a direct physiological control on Brown Tide blooms, the addition of inorganic nitrogen tends to inhibit Brown Tide blooms. Budget calculations indicate that the inorganic nitrogen supply from groundwater is 1–2 orders of magnitude higher than any other external source of nitrogen for this ecosystem. Biweekly time series data collected in 1995 demonstrate that Brown Tide blooms utilize dissolved organic nitrogen (DON) for growth, as evidenced by a large decrease in DON parallel with an increase in cell abundance. On an interannual basis, bloom intensity was also positively correlated with mean DON concentrations. We hypothesize that bloom initiation is regulated by the relative supply of inorganic and organic nitrogen, determined to a large extent by temporal variability in groundwater flow. The 1980s and 1990s were characterized by exceptionally high and interannually variable groundwater discharge, associated with a large-scale climate shift over the North Atlantic. This, coupled with the time-lagged discharge of groundwater with high nitrate concentrations resulting from increased fertilizer use and population increase during the 1960s and 1970s, may have been a key factor in the initiation of Brown Tide blooms in 1985

Description: Available data support a mechanism of buoyancy‐mediated vertical migration by large‐sized diatoms of Rhizosolenia spp. as a means to access “new” nitrogen from deep waters. To assess whether phytoplankton simultaneously satisfy their Fe requirements by this mechanism, field samples collected during summer 1996 at stations located along a transect through the central North Pacific gyre were assayed for the presence of flavodoxin and ferredoxin via Western blot analysis. All samples, regardless of their buoyancy status and the station from which they were collected, had accumulated flavodoxin but not ferredoxin. To understand better the significance of the field results, cultures of Rhizosolenia formosa H. Peragallo were grown in the laboratory with varying levels of total Fe (200 nM–10,000 nM). Fe had little effect on the physiological and photochemical parameters measured for each treatment. Growth rates did not exceed 0.17 d−1 and values of Fv/Fm ranged from 0.48 to 0.62. In addition, R. formosa accumulated only flavodoxin at each level of Fe addition. From these results, it appears that for some rhizosolenids, flavodoxin is constitutively expressed. The underlying basis for the constitutive nature of this flavodoxin is unclear at present, although it is likely that it is ultimately related to chronic Fe deficit incurred in natural waters.

Description: We examined the influence of forecasted changes in global temperatures and pCO2 on N2 fixation and assimilation in the ecologically important cyanobacterium Trichodesmium spp. Changes of mRNA transcripts (nifH, glnA, hetR, psbA, psaB), protein (nitrogenase, glutamine synthetase) pools and enzymatic activity (nitrogenase) were measured under varying pCO2 and temperatures. High pCO2 shifted transcript patterns of all genes, resulting in a more synchronized diel expression. Under the same conditions, we did not observe any significant changes in the protein pools or in total cellular allocations of carbon and nitrogen (i.e. C : N ratio remained stable). Independently of temperature, high pCO2 (900 µatm) elevated N2 fixation rates. Levels of the key enzymes, nitrogenase and glutamine synthetase that mediate nitrogen assimilation did not increase, implying that the high pCO2 allowed higher reaction turnover rates through these key enzymes. Moreover, increased temperatures and high pCO2 resulted in higher C : P ratios. The plasticity in phosphorous stoichiometry combined with higher enzymatic efficiencies lead to higher growth rates. In cyanobacteria photosynthesis, carbon uptake, respiration, N2 fixation and nitrogen assimilation share cellular components. We propose that shifted cellular resource and energy allocation among those components will enable Trichodesmium grown at elevated temperatures and pCO2 to extend its niche in the future ocean, through both tolerance of a broader temperature range and higher P plasticity.

Description: The nitrogen cycling of Lake Cadagno was investigated by using a combination of biogeochemical and molecular ecological techniques. In the upper oxic freshwater zone inorganic nitrogen concentrations were low (up to ∼3.4 μM nitrate at the base of the oxic zone), while in the lower anoxic zone there were high concentrations of ammonium (up to 40 μM). Between these zones, a narrow zone was characterized by no measurable inorganic nitrogen, but high microbial biomass (up to 4 × 107 cells ml−1). Incubation experiments with 15N-nitrite revealed nitrogen loss occurring in the chemocline through denitrification (∼3 nM N h−1). At the same depth, incubations experiments with 15N2- and 13CDIC-labelled bicarbonate, indicated substantial N2 fixation (31.7–42.1 pM h−1) and inorganic carbon assimilation (40–85 nM h−1). Catalysed reporter deposition fluorescence in situ hybridization (CARD-FISH) and sequencing of 16S rRNA genes showed that the microbial community at the chemocline was dominated by the phototrophic green sulfur bacterium Chlorobium clathratiforme. Phylogenetic analyses of the nifH genes expressed as mRNA revealed a high diversity of N2 fixers, with the highest expression levels right at the chemocline. The majority of N2 fixers were related to Chlorobium tepidum/C. phaeobacteroides. By using Halogen In Situ Hybridization-Secondary Ion Mass Spectroscopy (HISH-SIMS), we could for the first time directly link Chlorobium to N2 fixation in the environment. Moreover, our results show that N2 fixation could partly compensate for the N loss and that both processes occur at the same locale at the same time as suggested for the ancient Ocean.

Description: The effects of nitrate, phosphate, and iron starvation and resupply on photosynthetic pigments, selected photosynthetic proteins, and photosystem II (PSII) photochemistry were examined in the diatom Phaeodactylum tricornutum Bohlin (CCMP 1327). Although cell chlorophyll a (chl a) content decreased in nutrient‐starved cells, the ratios of light‐harvesting accessory pigments (chl c and fucoxanthin) to chl a were unaffected by nutrient starvation. The chl a‐specific light absorpition coefficient (a*) and the functional absorption cross‐section of PSII (σ) increased during nutrient starvation, consistent with reduction of intracellular self‐shading (i.e. a reduction of the “package effect”) as cells became chlorotic. The light‐harvesting complex proteins remained a constant proportion of total cell protein during nutrient starvation, indicating that chlorosis mirrored a general reduction in cell protein content. The ratio of the xanthophylls cycle pigments diatoxanthin and diadinoxanthin to chl a increased during nutrient starvation. These pigments are thought to play a photo‐protective role by increasing dissipation of excitation energy in the pigment bed upstream from the reaction centers. Despite the increase in diatoxanthin and diadinoxanthin, the efficiency of PSII photochemistry, as measured by the ration of variable to maximum fluorescence (Fv/Fm) of dark‐adapted cells, declined markedly under nitrate and iron starvation and moderately under phosphate starvation. Parallel to changes in Fv/Fm were decreases in abundance of the reaction center protein D1 consistent with damage of PSII reaction centers in nutrient‐starved cells. The relative abundance of the carboxylating enzyme, ribulose bisphosphate carboxylase/oxygenase (RUBISCO), decreased in response to nitrate and iron starvation but not phosphate starvation. Most marked was the decline in the abundance of the small subunit of RUBISCO in nitrate‐starved cells. The changes in pigment content and fluorescence characteristics were typically reversed within 24 h of resupply of the limiting nutrient.

Description: What limits phytoplankton growth in nature? The answer is elusive because of methodological problems associated with bottle incubations and nutrient addition experiments. We are investigating the possibility that antibodies to proteins repressed by a specific nutrient can be used as probes to indicate which nutrient limits photosynthetic carbon fixation in the ocean. The diatom Phaeodactylum tricornutum Bohlin and the chlorophyte Dunaliella tertiolecta Butcher were grown in batch cultures in artificial seawater and f/2 nutrient lacking either phosphorus, iron, or nitrogen. Chlorosis was induced by nutrient limitation in both species with the exception of phosphorus‐limited D. tertiolecta. The synthesis and appearance of specific proteins were followed by labeling with 14C‐bicarbonate. Nutrient limitation in general leads to a decrease in the quantum efficiency of photosystem II, suggesting that deficiency of any nutrient affects the photosynthetic apparatus to some degree: however, the effect of nitrogen and iron limitation on quantum efficiency is more severe than that of phosphorus. A crude fractionation of the soluble and membrane proteins demonstrated that the large proteins induced under limitation by phosphorus and iron were associated with the membranes. However, small iron‐repressible proteins were located in the soluble fraction. Isolation with anion‐exchange chromatography and N‐terminal sequencing of iron‐repressible, 23‐kDa Proteins from D. tertiolecta, P. tricornutum, and Chaetoceros gracilis revealed that these small soluble proteins have strong homology with the N‐terminal sequence of flavodoxins from Azotobacter and Clostridium. The identity of the flavodoxin from D. tertiolecta was confirmed by immunodetection using antiflavodoxin raised against Chlorella. Flavodoxin was detected only under iron deprivation and was absent from nitrogen‐and phosphorus‐limited algae. Flavodoxin is a prime candidate for a molecular probe of iron limitation in the ocean. The requirements to confirm its utility in nature are discussed.