Iron-Stimulated N(2) Fixation and Growth in Natural and Cultured Populations of the Planktonic Marine Cyanobacteria Trichodesmium spp

In light of recent proposals that iron (Fe) availability may play an important role in controlling oceanic primary production and nutrient flux, its regulatory impact on N(2) fixation and production dynamics was investigated in the widespread and biogeochemically important diazotrophic, planktonic cyanobacteria Trichodesmium spp. Fe additions, as FeCl(3) and EDTA-chelated FeCl(3), enhanced N(2) fixation (nitrogenase activity), photosynthesis (CO(2) fixation), and growth (chlorophyll a production) in both naturally occurring and cultured (on unenriched oligotrophic seawater) Trichodesmium populations. Maximum enhancement of these processes occurred under FeEDTA-amended conditions. On occasions, EDTA alone led to enhancement. No evidence for previously proposed molybdenum or phosphorus limitation was found. Our findings geographically extend support for Fe limitation of N(2) fixation and primary production to tropical and subtropical oligotrophic ocean waters often characterized by Trichodesmium blooms.     

Release of Dissolved Organic Nitrogen by Marine Diazotrophic Cyanobacteria, Trichodesmium spp

Trichodesmium sp. is a filamentous, colonial cyanobacterium which contributes substantially to the input of nitrogen in tropical and subtropical oceanic waters through nitrogen fixation (N(2) fixation). We applied a N tracer technique to assess the rate of release of dissolved organic nitrogen (DON) from this cyanobacterium and compared those rates with rates of N(2) fixation determined for the same assemblages at the same times of day. Rates of release of DON showed considerable variation within replicate experiments and were variable depending on time of day and duration of time course experiments. On average, rates of DON release were ca. 50% the rates of N(2) fixation. We also fractionated the DON released by using ultrafiltration and found that 60 to 80% of the total organic release was of the size class <10,000 Da. The release of these organic compounds by Trichodesmium spp. is likely a significant source of new nitrogen for the associated bacteria or the non-nitrogen-fixing filaments of the Trichodesmium colonies.     

Vertical migration of the toxic dinoflagellate Gymnodinium catenatum under different concentrations of nutrients and humic substances in culture

Vertical migration behaviour by the chainforming dinoflagellate Gymnodinium catenatum Graham was investigated using vertically-stratified laboratory columns. Under surface nutrient-deplete conditions, with nutrients added only at depth, 100% of cells underwent vertical migration (VM), starting downwards migration 3 h before the end of the light period and beginning upwards migration 3 h before the start of the light period. Cells in nutrient-replete columns showed no VM, but they were more dispersed in the upper layer during the dark compared to the light period. When surface layers (S) were nitrate-deplete (−N) and enriched with humic substances (H) contained in Huon River water and bottom waters (B) were nutrient replete (R) (SH_−NB_R), the pattern of VM was altered—50% of cells underwent migration and 50% remained at the pycnocline. In columns with nitrate-replete and humic-enriched surface layers (SH_RB_R), most cells underwent VM, while 30% remained at the surface. Cells in SH_−NB_R columns showed increased N quotas and intra-cellular nitrate concentrations after 4 days, indicating nitrate uptake by G. catenatum in bottom layers. The concomitant increase in particulate organic nitrogen (PON) with the decrease in external nitrate concentrations in bottom layers provide convincing evidence that VM by G. catenatum facilitates nutrient retrieval at depth. However, addition of humic substances (a potential source of organic nitrogen) to surface layers did not ameliorate G. catenatum N depletion sufficiently to preclude the need for NO₃⁻ uptake at depth. Furthermore, there was no detectable pattern of increasing carbon (C) quota during the day (photosynthate accumulation) or increasing N quota during the night (nitrate assimilation). Toxic dinoflagellate G. catenatum blooms are commonly associated with nitrate depletion in surface waters in south-east Tasmanian waters (Australia). Therefore, vertical migration, facilitating N uptake at depth, could play an important role in this organism's ecological strategy, enabling it to exploit environments where light and nutrients are vertically separated.     

The nitrogen physiology of the marine N2-fixing cyanobacteria Trichodesmium spp

Trichodesmium spp. have proved to be enigmatic organisms, and their ecology and physiology are unusual among diazotrophs. Recent research shows that they can simultaneously fix N2 and take up combined nitrogen. The co-occurrence of these two processes is thought to be incompatible, but they could be obligatory in Trichodesmium spp. if only a small fraction of cells within a colony or along a filament are capable of N2 fixation. Combined nitrogen is released from cells during periods of active growth and N2 fixation, and concomitantly taken up by Trichodesmium spp. or cells living in association with colonies. Although the nitrogenase of Trichodesmium spp. is affected by high concentrations of combined nitrogen, it might be relatively less sensitive to low concentrations of combined nitrogen typical of the oligotrophic ocean and culture conditions. Nitrogenase activity and synthesis exhibits an endogenous rhythm in Trichodesmium spp. cultures, which is affected by the addition of nitrogen.     

Abundances of iron-binding photosynthetic and nitrogen-fixing proteins of Trichodesmium both in culture and in situ from the North Atlantic

Marine cyanobacteria of the genus Trichodesmium occur throughout the oligotrophic tropical and subtropical oceans, where they can dominate the diazotrophic community in regions with high inputs of the trace metal iron (Fe). Iron is necessary for the functionality of enzymes involved in the processes of both photosynthesis and nitrogen fixation. We combined laboratory and field-based quantifications of the absolute concentrations of key enzymes involved in both photosynthesis and nitrogen fixation to determine how Trichodesmium allocates resources to these processes. We determined that protein level responses of Trichodesmium to iron-starvation involve down-regulation of the nitrogen fixation apparatus. In contrast, the photosynthetic apparatus is largely maintained, although re-arrangements do occur, including accumulation of the iron-stress-induced chlorophyll-binding protein IsiA. Data from natural populations of Trichodesmium spp. collected in the North Atlantic demonstrated a protein profile similar to iron-starved Trichodesmium in culture, suggestive of acclimation towards a minimal iron requirement even within an oceanic region receiving a high iron-flux. Estimates of cellular metabolic iron requirements are consistent with the availability of this trace metal playing a major role in restricting the biomass and activity of Trichodesmium throughout much of the subtropical ocean.     

Molecular analysis of the phosphorus starvation response in Trichodesmium spp.

The marine diazotroph Trichodesmium is a major contributor to primary production and nitrogen fixation in the tropical and subtropical oceans. These regions are often characterized by low phosphorus (P) concentrations, and P starvation of Trichodesmium could limit growth, and potentially constrain nitrogen fixation. To better understand how this genus responds to P starvation we examined four genes involved in P acquisition: two copies of a high-affinity phosphate binding protein ( pstS and sphX ) and two putative alkaline phosphatases ( phoA and phoX ). Sequence analysis of these genes among cultured species of Trichodesmium ( T. tenue, T. erythraeum, T. thiebautii and T. spiralis ) showed that they all are present and conserved within the genus. In T. erythraeum IMS101, the expression of sphX , phoA and phoX were sensitive to P supply whereas pstS was not. The induction of alkaline phosphatase activity corresponded with phoA and phoX expression, but enzyme activity persisted after the expression of these genes returned to basal levels. Additionally, nifH (nitrogenase reductase; involved in nitrogen fixation) expression was downregulated under P starvation conditions. These data highlight molecular level responses to low P and lay a foundation for better understanding the dynamics of Trichodesmium P physiology in low-P environments.     

High single-cell diversity in carbon and nitrogen assimilations by a chain-forming diatom across a century

Almost a century ago Redfield discovered a relatively constant ratio between carbon, nitrogen and phosphorus in particulate organic matter and nitrogen and phosphorus of dissolved nutrients in seawater. Since then, the riverine export of nitrogen to the ocean has increased 20 fold. High abundance of resting stages in sediment layers dated more than a century back indicate that the common planktonic diatom Skeletonema marinoi has endured this eutrophication. We germinated unique genotypes from resting stages originating from isotope-dated sediment layers (15 and 80 years old) in a eutrophied fjord. Using secondary ion mass spectrometry (SIMS) combined with stable isotopic tracers, we show that the cell-specific carbon and nitrogen assimilation rates vary by an order of magnitude on a single-cell level but are significantly correlated during the exponential growth phase, resulting in constant assimilation quota in cells with identical genotypes. The assimilation quota varies largely between different clones independent of age. We hypothesize that the success of S. marinoi in coastal waters may be explained by its high diversity of nutrient demand not only at a clone-specific level but also at the single-cell level, whereby the population can sustain and adapt to dynamic nutrient conditions in the environment.     

Nutrient limitation of phytoplankton in a naturally acidic lake (Lake Caviahue, Argentina)

As a result of a low pH, the inorganic carbon of acidic lakes is present as CO₂ at air-equilibrium concentration and is substantially lower than the inorganic carbon concentration in higher-pH waters with bicarbonate. This situation is quite common in artificially acidified lakes and where inorganic carbon is considered the limiting factor in phytoplankton growth. Apart from low inorganic carbon content, Lake Caviahue in Argentina has low nitrogen and high phosphorus content. The aim of this work was to assess the importance of inorganic carbon, phosphorus, and nitrogen, relating data on lake nutrients to phytoplankton species requirements. Lake samples taken in the 2004–2006 period did not show any particular trend in the vertical distribution of the water column of ammonium, inorganic carbon, and phosphorus with reference to either seasonality or depth. A decrease of some 15% in the lake’s phosphorus concentration was observed over the same period. Although the total phytoplankton biomass in Lake Caviahue was similar throughout the period, a seasonal variation was observed. Lab bioassays were carried out with solutions of bicarbonates, ammonium, nitrates, and phosphate. We worked with three species separately, namely, two chlorophytes, Keratococcus rhaphidioides and Watanabea sp.; and one euglenophyte, Euglena mutabilis. Answers to specific nutrient requirements differed for each algal species: both chlorophytes prefer ammonium or nitrates added on their own, whereas the euglenophyte registered a higher growth rate with the joint addition of ammonium and phosphorus. Even when the limiting nutrient(s) for phytoplankton yield and rate varied between species, we observed a tendency for nitrogen limitation in Lake Caviahue.     

A molecular assessment of the iron stress response in the two phylogenetic clades of Trichodesmium

Trichodesmium spp. play key roles in global carbon and nitrogen budgets and thus defining what controls their productivity is important for understanding climate change. While iron availability has been shown to be an important chemical factor for controlling both growth and nitrogen fixation rates in Trichodesmium , all culture experiments to date have focused solely on representatives from one clade of Trichodesmium . Genomic sequence analysis determined that the Trichodesmium erythraeum (IMS101) genome contains many of the archetypical genes involved in the prokaryotic iron stress response. Focusing on three of these genes, isiB , idiA and feoB , we found that all three showed an iron stress response in axenic T. erythraeum (IMS101), and their sequences were well conserved across four species in our Trichodesmium culture collection [consisting of two T. erythraeum strains (IMS101 and GBRTRLI101), two Trichodesmium tenue strains (Z-1 and H9-4), Trichodesmium thiebautii and Trichodesmium spiralis ]. With clade-specific quantitative PCR (qPCR) primers for one of these genes, isiB , we found that high isiB expression at low Fe levels corresponded to specific reductions in N₂ fixation rates in both major phylogenetic clades of Trichodesmium (the T. erythraeum clade and T. tenue clade). With regard to the two clades, the most significant difference determined was temperature optima, while more subtle differences in growth, N₂ fixation rate and gene expression responses to Fe stress were also observed. However the apparent conservation of the Fe stress response in the Trichodesmium genus suggests that it is an important adaptation for their niche in the oligotrophic ocean.     

Modeling the dynamic regulation of nitrogen fixation in the cyanobacterium Trichodesmium sp

A physiological, unbalanced model is presented that explicitly describes growth of the marine cyanobacterium Trichodesmium sp. at the expense of N(2) (diazotrophy). The model involves the dynamics of intracellular reserves of carbon and nitrogen and allows the uncoupling of the metabolism of these elements. The results show the transient dynamics of N(2) fixation when combined nitrogen (NO(3)(-), NH(4)(+)) is available and the increased rate of N(2) fixation when combined nitrogen is insufficient to cover the demand. The daily N(2) fixation pattern that emerges from the model agrees with measurements of rates of nitrogenase activity in laboratory cultures of Trichodesmium sp. Model simulations explored the influence of irradiance levels and the length of the light period on fixation activity and cellular carbon and nitrogen stoichiometry. Changes in the cellular C/N ratio resulted from allocations of carbon to different cell compartments as demanded by the growth of the organism. The model shows that carbon availability is a simple and efficient mechanism to regulate the balance of carbon and nitrogen fixed (C/N ratio) in filaments of cells. The lowest C/N ratios were obtained when the light regime closely matched nitrogenase dynamics.     

Growth Phase and Elemental Stoichiometry of Bacterial Prey Influences Ciliate Grazing Selectivity

ABSTRACT. Protozoa are known to selectively graze bacteria and can differentiate prey based on size and viability, but less is known about the effects of prey cellular composition on predator selectivity. We measured the effect of growth phase and elemental stoichiometry of Escherichia coli on grazing by two ciliates, Euplotes vannus and Cyclidium glaucoma . Bacterial cells of a single strain were transformed with green and red fluorescent protein and harvested from culture at differing growth stages. Cells in exponential growth phase had low carbon:phosphorus (39) and nitrogen:phosphorus (9) ratios, while cells from stationary phase had high carbon:phosphorus of 104 and nitrogen:phosphorus of 26. When offered an equal mixture of both types of bacteria, Cyclidium grazed stationary phase, high carbon:phosphorus, high nitrogen:phosphorus cells to 22% of initial abundance within 135 min, while Euplotes reduced these cells to 33%. Neither ciliate species decreased the abundance of the exponential phase cells, lower carbon:phosphorus and nitrogen:phosphorus, relative to control treatments. Because protozoa have higher nitrogen:phosphorus and carbon:phosphorus ratios than their prokaryotic prey, this study raises the possibility that it may be advantageous for protozoa to preferentially consume more slowly growing bacteria.     

Diel vertical migration thresholds of Karenia brevis (Dinophyceae)

Light and nutrient availability change throughout dinoflagellate diel vertical migration (DVM) and/or with sub-population location in the water column along the west Florida shelf. Typically, the vertical depth of the shelf is greater than the distance a sub-population can vertically migrate during a diel cycle, limiting the ability of a sub-population to photosynthetically fix carbon toward the surface and access nutrients sub-surface. This project investigated changes of Karenia brevis (C.C. Davis) G. Hansen et Moestrup intracellular carbon, nitrogen, internal nitrate (iNO₃), free amino acid (FAA), and total lipid concentrations in high-light, nitrate-replete (960 μmol quanta m⁻² s⁻¹, 80 μM NO₃), and high-light, nitrate-reduced (960 μmol quanta m⁻² s⁻¹, <5 μM NO₃) mesocosms. The nitrate-reduced mesocosm had a slowed cell division rate when compared to the nitrate-replete mesocosm. Minimum intracellular carbon, nitrogen, iNO₃, FAA, and total lipid concentrations during the largest surface sub-population aggregations led to the conclusion that daughter cells resulting from cell division received unequal shares of the parental resources and that this inequality influenced migration behavior. Nutrient reduced daughter cells were more strongly influenced by light and phototaxis for carbon production than their replete same cell division sister cells during vertical migration thus rapidly increasing the fulfillment of constituents through photosynthesis. Vertical migration was consistent with an optimization scheme based on threshold limits through utilization or formation of photosynthate. We propose a simplified conceptual model describing how K. brevis is transported along the benthos of the west Florida shelf from off-shore to on-shore. Dynamic carbon thresholds are also suggested for future DVM modeling efforts on K. brevis populations transported between nitrogen replete and nitrogen reduced environmental conditions.     

On the vertical distribution of microorganisms in lake Baikal during spring deep-water renewal

The vertical distribution of microorganisms during spring deep-water renewal in Lake Baikal was studied. The downward advection of trophogenic waters was found to create conditions for the extensive growth of microorganisms capable of decomposing and mineralizing organic carbon, nitrogen, and phosphorus in deep water layers. These processes occur annually at spring thermal bars near the underwater slope of Lake Baikal, whereas in its pelagic zone, the deep intrusions of waters rich in organic material are observed only in the years when enhanced deep-water renewal is accompanied by a high spring yield of phytoplankton.     

on the vertical distribution of microorganisms in lake baikal during spring deep-water renewal

The vertical distribution of microorganisms during spring deep-water renewal in Lake Baikal was studied. The downward advection of trophogenic waters was found to create conditions for the extensive growth of microorganisms capable of decomposing and mineralizing organic carbon, nitrogen, and phosphorus in deep water layers. These processes occur annually at spring thermal bars near the underwater slope of Lake Baikal, whereas in its pelagic zone, the deep intrusions of waters rich in organic material are observed only in the years when enhanced deep-water renewal is accompanied by a high spring yield of phytoplankton.     

Diversity and distribution of diazotrophic communities in the South China Sea deep basin with mesoscale cyclonic eddy perturbations

The South China Sea (SCS) is an oligotrophic subtropical marginal ocean with a deep basin and a permanently stratified central gyre. Upwelling and nitrogen fixation provide new nitrogen for primary production in the SCS. This study was aimed at an investigation of phylogenetic diversity and quantification of the diazotroph community in the SCS deep basin, which is characterized by frequent mesoscale eddies. The diazotroph community had a relatively low diversity but a distinct spatial heterogeneity of diversity in the SCS deep basin. The potential for nitrogen fixation consistently occurred during cyclonic eddies, although upwelling of nutrient-replete deep water might have alleviated nitrogen limitation in the SCS. However, diazotrophic proteobacteria were dominant, but neither Trichodesmium nor heterocystous cyanobacterial diatom symbionts. Quantitative PCR analysis using probe-primer sets developed in this study revealed that the nif?H gene of the two dominant alpha- and gammaproteobacterial groups was at the highest abundance (up to 10(4) to 10(5) copies?L(-1) ). Trichodesmium thiebautii was detected with an average density of 10(2) trichomes?L(-1) in the euphotic waters, while Richelia intracellularis was observed sporadically under the microscope. The unicellular cyanobacterial groups A and B were not detected in our libraries. Our results suggested that diazotrophic proteobacteria were significant components potentially contributing to nitrogen fixation in this oligotrophic marginal ocean ecosystem.     

Nitrate metabolism of the red tide dinoflagellate Gonyaulax polyedra Stein

Certain idiosyncracies in the metabolism of nitrogen and diurnal vertical migration may account for the development and persistence of Gonyaulax polyedra Stein red tides along the Southern California Coast.In culture, G. polyedra has the ability for both uptake and assimilation of nitrate in the dark and this together with its enhancement by previous N-starvation could enable these dinoflagellates to meet 50–100 % of their daily nitrogen requirements for growth from dark assimilation alone. Less pronounced light-dark variations in nitrate assimilation and a greater stability of the nitrate assimilatory enzymes, together with the ability to migrate into nitrate-rich subsurface waters at night, probably give G. polyedra and other red tide dinoflagellates a competitive advantage over coastal diatoms during the ‘upwelling season’, when most red tides occur.     

The Ratios of Carbon,Nitrogen, and Phosphorus in a Wetland Coastal Ecosystem of Southern India

The fertility of the coastal and estuarine waters is of great concern because of its influence on the productivity of these waters. Seasonal variations in the distribution of organic carbon, total nitrogen and total phosphorus in the sediments of Kuttanad Waters, a part of the tropical Cochin Estuary on the south west coast of India, are examined to identify the contribution of sediments to the fertility of the aquatic systems. The adjoining region has considerable agricultural activity. The fresh water zones had higher quantities of silt and clay whereas the estuarine zone was more sandy. Organic carbon, total phosphorus and total nitrogen were higher in the fresh water zones and lower in the estuarine zones. Total phosphorus and organic carbon showed the lowest values during monsoon periods. No significant trends were observed in the seasonal distributions of total nitrogen. Ratios of C/N, C/P and N/P, and the phosphorus and nitrogen content indicate significant modification in the character of the organic matter. Substantial amounts of the organic matter can contribute to reducing conditions and modify diagenetic processes.     

氮磷营养限制影响三角褐指藻光合无机碳利用和碳酸酐酶活性

以海洋硅藻三角褐指藻为实验材料, 研究了不同氮磷比培养对其光合无机碳利用和碳酸酐酶活性的影响, 结果显示三角褐指藻生长速率在N:P=16:1时最大, 高于或低于16:1时明显下降, 表明其最适生长受到氮磷的限制。氮限制(N:P=4:1或1:1)导致叶绿素a含量分别下降30.1% 和47.6%, 磷限制(N:P=64:1或256:1)下降39.1%和52.4%, 但氮或磷限制对叶绿素c含量并没有明显影响。不同营养水平培养对光饱和光合速率具有明显的影响, 与营养充足培养相比, 在严重氮磷限制(N:P=1:1或256:1)培养下光饱和光合速率分别下降39.7%和48.0%, 光合效率与暗呼吸速率也明显下降。在氮磷限制培养下藻细胞pH补偿点明显下降; K0.5CO2值在磷限制下降低30%, 表明磷限制有助于提高细胞对CO2的亲和力, 但氮限制并没有明显影响。在氮磷限制培养的细胞反应液中Fe (CN)63-浓度下降速率较慢, 表明在氮磷限制环境中生长的细胞质膜氧化还原能力明显低于营养充足条件下生长的细胞。氮磷限制也导致胞内、外碳酸酐酶活性明显下降, 其中在氮限制下胞外碳酸酐酶活性分别下降50%和37.5%, 在磷限制下下降22.3%和42.1%。严重的氮(N:P=1:1)或磷(N:P=256:1)限制导致胞内碳酸酐酶活性下降36.5%和42.9%。研究结果表明, 三角褐指藻细胞在氮磷营养限制的环境中, 可以通过调节叶绿素含量、无机碳的利用方式和碳酸酐酶的活性以维持适度的生长。