Algal 14C and total carbon metabolisms. 1. Models to account for the physiological processes of respiration and recycling

A consistent set of equations has been written to describe thenet rate of algal ¹⁴CO₂ uptake (and where appropriate respirationand photosynthesis) which take into account separately complicationsdue to respiration of the labelled photosynthetic products andthe recycling of respiratory CO₂. Written specifically intothe equations is the concept of ‘new’ and ‘old’carbon, the coefficient q is used in the respiration model toallow for the differential respiration of organic material fromthe ‘new’ and ‘old’ carbon pools. Analyticalintegrals have been found for respiration and recycling models,and the behaviour of the models studied over periods of 12 h(i.e. up to 70% of the intrinsic generation time). The rateconstant for respiration has a greater effect on the behaviourof the recycling than the respiration model. Over short timecourses (up to 30% of the intrinsic generation time), the effectsof respiration and recycling on net ¹⁴CO₂ uptake are quite distinct,especially at high P/R ratios, and not complicated by assumptionsover the value of q. Although the value of q will have a time-dependentsecondary effect on the modelled total carbon-specific respirationrate, this was found not to give rise to major problems of interpretation.Beyond 50% of the intrinsic generation time, the separate treatmentof respiration and recycling in the models becomes less satisfactory.It was concluded that the present equations, which are not constrainedby mass balance considerations, would not be appropriate fora model that combines the two processes. The pattern of recyclingat low P/R values is identified as one of the major uncertaintiesin producing models of ¹⁴C uptake. The effect of the releaseof dissolved organic material can be anticipated in a generalway. The models have been used to define an experimental strategyto establish the separate effects of respiration and recyclingon the time course of net ¹⁴C uptake. The initial rates givethe clearest resolution of the two processes and it would appearthat with photosynthetic rates in the region of 1 day^–1,incubation periods up to 3–6 h would be suitable to determinethe importance of recycling in controlling net ¹⁴C uptake. Withthe present models, only in the absence of recycling could theeffect of respiration be studied and the value of q established.     

Isolation and characterization of glutamine synthetase from the marine diatom Skeletonema costatum.

Two peaks of glutamine synthetase (GS) activity were resolved by anion-exchange chromatography from the marine diatom Skeletonema costatum Grev. The second peak of activity accounted for greater than 93% of total enzyme activity, and this isoform was purified over 200-fold. Results from denaturing gel electrophoresis and gel-filtration chromatography suggest that six 70-kD subunits constitute the 400-kD native enzyme. The structure of the diatom GS, therefore, appears more similar to that of a type found in bacteria than to the type common among other eukaryotes. Apparent Michaelis constant values were 0.7 mM for NH4(+), 5.7 mM for glutamic acid, and 0.5 mM for ATP. Enzyme activity was inhibited by serine, alanine, glycine, phosphinothricin, and methionine sulfoximine. Polyclonal antiserum raised against the purified enzyme localized a single polypeptide on western blots of S. costatum cell lysates and recognized the denatured, native enzyme. Western analysis of the two peak fractions derived from anion-exchange chromatography demonstrated that the 70-kD protein was present only in the later eluting peak of enzyme activity. This form of GS does not appear to be unique to S. costatum, since the antiserum recognized a similar-sized protein in cell lysates of other chromophytic algae.     

Cytometric quantification of nitrate reductase by immunolabeling in the marine diatom Skeletonema costatum

BACKGROUND: The uptake of nitrate by phytoplankton is a central issue in biological oceanography due to its importance to primary production and vertical flux of biogenic carbon. Nitrate reductase catalyzes the first step of nitrate assimilation, the reduction of NO(3) to NO(2). A cytometric protocol to detect and quantify relative changes in nitrate reductase (NR) protein content of the marine centric diatom Skeletonema costatum is presented. METHODS: Immunolabeling of NR protein was achieved with polyclonal antibodies raised against S.costatum NR. Antisera specific to a NR protein subunit and to a NR polypeptide sequence were compared, and cytometric results of NR protein abundance were related to Western analyses. Changes in cellular NR abundance and activity were followed during an upwelling simulation experiment in which S. costatum was exposed to a shift from ammonia to nitrate as major nitrogen source. RESULTS: NR protein could be detected in NO(3)-grown cells and at extremely low levels hardly discernible by Western Blot densiometry in NH(4)-grown cells. The protocol allowed observation of early stages of NR induction during an upwelling simulation. NR abundance increased after the nutrient shift to reach a new physiological "steady-state" 96 hrs later. NR activity exhibited diel variation with maxima at mid-day. NR abundance as estimated by both flow cytometry and Western analysis exhibited a hyperbolic relationship to NR activity. This pattern suggests post-translational activation of NR protein. CONCLUSIONS: The presented protocol allows the differentiation of NH(4)- versus NO(3)-grown algae as well as the monitoring of early stages in the induction of nitrate assimilatory capacities.     

Action Spectrum of Photosynthesis for Skeletonema costatum Obtained with Carbon-14

The quantized action spectrum of photosynthesis for Skeletonema costatum was obtained from values of photosynthetic ¹⁴CO₂ uptake at various wavelengths of light isolated “with a diffraction grating monochromator. The quantized action spectrum of photosynthesis exhibited maxima at wavelengths similar to maxima in the absorption spectrum, in vivo, of a suspension of S. costatum cells. While the ¹⁴CO₂ technique will provide an accurate action spectrum of photosynthesis for diatoms, a large number of samples is required in order to minimize sampling error.     

A photobioreactor with pH control: demonstration by growth of the marine diatom Skeletonema costatum

A photobioreactor with pH control for cultivation of algae isdescribed. The magnetically stirred culture flask is connectedto separate reservoirs for medium and for acid and base (dilutedHCl and NaOH, respectively). A pH electrode is inserted intothe culture flask and coupled to a pH controller, which activatesacid and base titration at set points of pH 8.1 and 7.8, respectively.Illumination is provided by light tubes with a diel light :dark cycle. The use of the photobioreactor in batch mode isillustrated by showing pH curves in different growth phasesof the marine diatom Skeletonema costatum. The photobioreactorcan also be run as a semicontinuous or continuous reactor withslight modifications.     

Lithium ions lengthen the circadian period of growing cultures of the diatom Skeletonema costatum

Turbidity measurements revealed that the circadian rhythm in the growth rate of the marine diatom Skeletonema costatum (Greville) Cleve was insensitive to temperature between 5 and 22°C. Growth of the alga was inhibited by lithium ions at concentrations higher than 2 m M . Lengthening of the circadian period was observed in the presence of 0.5–1.5 m M Li⁺. The results indicate that the lithium effect generally observed on circadian rhythms should not necessarily be ascribed to changes in an intercellular coupling of cellular oscillators.     

The effect of nutrient availability and temperature on chain length of the diatom, Skeletonema costatum

We determined the effects of temperature and nutrients on thechain length of a diatom, Skeletonema costatum, in batch cultureand enclosure experiments with estuarine water from San FranciscoBay, USA, using the recently developed CytoBuoy flow cytometer.Determination of the number of cells per diatom chain by CytoBuoyflow cytometer and associated software correlated well withbut was much more precise and time efficient than microscopicquantification. Increasing temperatures (from 6, 8 to 17°C)and nutrient concentrations induced high growth rates and dominanceby longer chains in a cultured S. costatum strain that was originallyacclimatized to a temperature range of 11–30°C. Similarly,a positive correlation between growth rate and chain lengthwas observed in S. costatum in batch culture and natural communitiesin enclosure experiments. Maximal chain lengths of S. costatumwere greater in natural populations than in the batch culture.Longer chains affect sinking rates and thus likely help thediatom remain suspended in the upper part of the water columnwhere physical and chemical parameters are more favorable forgrowth.     

High CO2 increases lipid and polyunsaturated fatty acid productivity of the marine diatom Skeletonema costatum in a two-stage model

Journal of Applied Phycology - Lipid and polyunsaturated fatty acids (PUFA) from microalgae can be used as biodiesel and health care products. How to enhance their productivity is crucial for...     

The role of complex lipids in the synthesis of bioactive aldehydes of the marine diatom Skeletonema costatum

Diatoms are unicellular plants broadly present in freshwater and marine ecosystems, where they play a primary role in sustaining the marine food chain. In the last 10 years, there has been accumulating evidence that diatoms may have deleterious effects on the hatching success of zooplankton crustaceans such as copepods, thus affecting dynamics of planktonic populations and limiting secondary production. At the molecular level, failure to hatch is ascribed to the presence of a family of inhibitory oxylipins, which we propose to collectively name polyunsaturated short-chain aldehydes (abbreviated here as PUSCAs). Here we describe the origin of PUSCAs produced by the marine diatom Skeletonema costatum via a lipoxygenase-mediated pathways involving non-esterified polyunsaturated fatty acids (PUFA). Experiments with complex lipids proved the pivotal role of chloroplast-derived glycolipids, especially monogalactosyldiacylglycerol (MGDG), in providing hexadecatrienoic acid (C16:3 omega-4), hexadecatetraenoic acid (C16:4 omega-1) and eicosapentaenoic acid (C20:5 omega-3) to the downstream process leading to 2E,4Z-octadienal (C8:2 omega-4), 2E,4Z,7-octatrienal (C8:3 omega-1) and 2E,4Z-heptadienal (C7:2 omega-3), respectively. Under physiological conditions, the hydrolytic process is associated to galactolipid hydrolyzing enzyme capable of removing fatty acids from both sn positions of glycerol.     

Isotopic discrimination and kinetic parameters of RubisCO from the marine bloom‐forming diatom,Skeletonema costatum

The cosmopolitan, bloom‐forming diatom, Skeletonema costatum, is a prominent primary producer in coastal oceans, fixing CO₂ with ribulose 1,5‐bisphosphate carboxylase/oxygenase (RubisCO) that is phylogenetically distinct from terrestrial plant RubisCO. RubisCOs are subdivided into groups based on sequence similarity of their large subunits (IA–ID, II, and III). ID is present in several major oceanic primary producers, including diatoms such as S. costatum, coccolithophores, and some dinoflagellates, and differs substantially in amino acid sequence from the well‐studied IB enzymes present in most cyanobacteria and in green algae and plants. Despite this sequence divergence, and differences in isotopic discrimination apparent in other RubisCO enzymes, stable carbon isotope compositions of diatoms and other marine phytoplankton are generally interpreted assuming enzymatic isotopic discrimination similar to spinach RubisCO (IB). To interpret phytoplankton δ¹³C values, S. costatum RubisCO was characterized via sequence analysis, and measurement of its K_CO2 and V_max, and degree of isotopic discrimination. The sequence of this enzyme placed it among other diatom ID RubisCOs. Michaelis‐Menten parameters were similar to other ID enzymes (K_CO2 = 48.9 ± 2.8 μm ; V_max = 165.1 ± 6.3 nmol min^?1 mg^?1). However, isotopic discrimination (ε = [¹²k/¹³k ? 1] × 1000) was low (18.5‰; 17.0–19.9, 95% CI) when compared to IA and IB RubisCOs (22–29‰), though not as low as ID from coccolithophore, Emiliania huxleyi (11.1‰). Variability in ε‐values among RubisCOs from primary producers is likely reflected in δ¹³C values of oceanic biomass. Currently, δ¹³C variability is ascribed to physical or chemical factors (e.g. illumination, nutrient availability) and physiological responses to these factors (e.g. carbon‐concentrating mechanisms). Estimating the importance of these factors from δ¹³C measurements requires an accurate ε‐value, and a mass‐balance model using the ε‐value for S. costatum RubisCO is presented. Clearly, appropriate ε‐values must be included in interpreting δ¹³C values of environmental samples.     

The influence of salinity fluctuation on the ammonium metabolism of the marine diatom Skeletonema costatum grown in continuous culture

Ammonium-limited cultures of Skeletonema costatum were grownat dilution rates from 0.019 to 0.038 h^–1 at an averagesalinity of 22.4 For a few days cultures were exposed to a freshwaterpulse. When salinity was decreased to 8.6 (average minimum)photosynthesis and cell division were inhibited. Both in vivoand DCMU-enhanced fluorescence per cell were statistically constant:photosystems I and II were not inhibited by a gradual salinitydecrease. Ammonium assimilation was affected via an inhibitionof carbon fixation. Ammonium concentrations increased in thecontinuous cultures, whereas the overcapacity of ammonium uptakedeclined: the nitrogen limitation was relieved. When salinitywas increased again, photosynthesis and cell division were stimulated.Salinity fluctuations were accompanied by a fluctuation in thepools of aspartic acid (0.5–1.0 mM), glutamine acid (0.9–4.1mM) and glutamine (0.5–2.0 mM). The pool of glutamic acidfollowed the salinity pattern (r=0.67, P<0.05). The correlationbetween the amino acid pool and the osmotic value of the mediumwas significant (r=0.72, P<0.05). Cellular glutamic acidand glutamine levels increased until the nitrogen limitationwas restored.     

Cooccurrence of ScDSP gene expression, cell death, and DNA fragmentation in a marine diatom, Skeletonema costatum

A novel death-specific gene, ScDSP, was obtained from a death stage subtraction cDNA library of the diatom Skeletonema costatum. The full length of ScDSP cDNA was 921 bp in length, containing a 699-bp open reading frame encoding 232 amino acids and two stretches of 66 and 156 bp in the 5' and 3' untranslated regions, respectively. Analysis of the peptide structure revealed that ScDSP contained a signal peptide domain, a transmembrane domain, and a pair of EF-hand motifs. When S. costatum grew exponentially at a rate of 1.3 day(-1), the ScDSP mRNA level was at 2 mumol . mole 18S rRNA(-1). In contrast, when the culture entered the death phase with a growth rate decreasing to 0.5 day(-1), ScDSP mRNA increased dramatically to 668 mumol . mole 18S rRNA(-1), and a high degree of DNA fragmentation was simultaneously observed. Under the influence of a light-dark cycle, ScDSP expression in both exponential and stationary phases clearly showed a diel rhythm, but the daily mean mRNA level was significantly higher in the stationary phase. Our results suggest that ScDSP may play a role in the molecular mechanism of self-destructive autolysis in phytoplankton under stress.     

Characterization of exudates released by the marine diatom Skeletonema costatum exposed to copper stress: a 3D-fluorescence spectroscopy approach

In a laboratory study, metal contamination experiments were conducted to investigate the effects of two free copper concentrations (10^?9 and 10^?8 M) on cell growth and on dissolved organic matter exudation by a marine diatom Skeletonema costatum. Throughout incubation, the growth kinetics and exudation of extracellular molecules (i.e. dissolved organic carbon (DOC) and the fluorescent organic matter) were determined. Results revealed an inhibition of S. costatum growth when the free copper level increased (from 10^?9 to 10^?8). Furthermore, DOC release was more significant in cultures contaminated by 10^?9 M Cu²⁺ than in control, suggesting a coping mechanism developed by this species. In this study, samples were daily analysed by 3D-fluorescence and PARAFAC algorithm, in order to compare the fluorescent material produced during growth under different contaminations. PARAFAC treatment revealed two main contributions: one related to the biological activity (C1), the other linked to the marine organic matter (C2). The third component C3 was typically protein-like. This fluorophore was considered as a tryptophan-like fluorophore, whereas the C1 and the C2 components were associated to marine production such as humic matter.     

The regulation of photosynthetic rate and activation of extracellular carbonic anhydrase under CO2-limiting conditions in the marine diatom Skeletonema costatum

In the marine diatom Skeletonema costatum , carbonic anhydrase activity exterior to the plasma membrane (CA_ext) was detected only when the available CO₂ concentration was less than 5·0 mmol m^–3, this activity being unaffected by the total dissolved inorganic carbon concentration. The inhibition of CA_ext by dextran bound sulphonamide (DBS) demonstrated the key role of this enzyme in maintaining photosynthetic rate under CO₂-limited conditions. Treatment with trypsin followed by affinity chromatography on p-aminomethylbenzene-sulphamide agarose and subsequent SDS-PAGE analysis revealed a polypeptide from carbon-replete cells of identical molecular mass to the CA_ext released by trypsin from CO₂-limited cells. Redox activity in the plasma membrane of intact cells was measured by following the light-dependent reduction of ferricyanide or NADP, the greatest activity being shown by CO₂-limited cells. Overall the results suggest that high rates of redox activity under conditions of CO₂-limitation were required for the activation of CA_ext.     

Nitrate Utilization by the Diatom Skeletonema costatum: I. Kinetics of Nitrate Uptake

Nitrate uptake has been studied in nitrogen-deficient cells of the marine diatom Skeletonema costatum. When these cells are incubated in the presence of nitrate, this ion is quickly taken up from the medium, and nitrite is excreted by the cells. Nitrite is excreted following classical saturation kinetics, its rate being independent of nitrate concentration in the incubation medium for nitrate concentration values higher than 3 micromolar. Nitrate uptake shows mixed-transfer kinetics, which can be attributed to the simultaneous contributions of mediated and diffusion transfer. Cycloheximide and p-hydroxymercuribenzoate inhibit the carrier-mediated contribution to nitrate uptake, without affecting the diffusion component. When cells are preincubated with nitrate, the net nitrogen uptake is increased.     

Nitrate Utilization by the Diatom Skeletonema costatum: II. Regulation of Nitrate Uptake

Nitrate utilization has been characterized in nitrogen-deficient cells of the marine diatom Skeletonema costatum. In order to separate nitrate uptake from nitrate reduction, nitrate reductase activity was suppressed with tungstate. Neither nitrite nor the presence of amino acids in the external medium or darkness affects nitrate uptake kinetics. Ammonium strongly inhibits carrier-mediated nitrate uptake, without affecting diffusion transfer. A model is proposed for the uptake and assimilation of nitrate in S. costatum and their regulation by ammonium ions.     

Impact of irradiance on the C allocation in the coastal marine diatom Skeletonema marinoi Sarno and Zingone

Elemental stoichiometry and organic composition were investigated in an Adriatic strain of Skeletonema marinoi, cultured at 25 [low light (LL)] and 250 [high light (HL)]µmol photon m^?2 s^?1. Inorganic carbon acquisition, fixation and allocation, and silicic acid and orthophosphate uptake were also studied. The C : P ratio was below the Redfield ratio, especially at LL. In HL cells, N quota was halved, C quota was similar, silica quota was lower, growth rate and long‐term net primary productivity were almost doubled, relative to LL cells. The HL : LL cell quota ratios were 6 for lipid, 0.5 for protein and 0.4 for carbohydrate. Phosphoenolpyruvate carboxylase (PEPc) and glutamine synthetase (GS) activities were unaffected by the growth irradiance; phosphoenolpyruvate carboxykinase (PEPck) was 2.5‐fold more active in LL cells. This suggests that in S. marinoi, C₄ photosynthesis is unlikely, PEPc is anaplerotic and PEPck may be involved in the conversion of lipid C to carbohydrates, especially in LL cells. Because about 50% of the cost for the production of an HL cell is caused by lipid biosynthesis, we propose that the preferential allocation of C to lipid at HL takes advantage of the relatively high volume‐based energy content of lipids, in an organism that reduces its size at each vegetative cell division.     

Physiology of diatom Skeletonema costatum (Grev.) Cleve photosynthetic extracellular release: evidence for a novel coupling between marine bacteria and phytoplankton

The photosynthesis of cellular materials by phytoplankton isaccompanied by release of organic molecules from the algal cellsinto the water. The patterns of carbon fixation in particulateand dissolved pools were investigated in Skeletonema costatumcultured under 12 h light/12 h dark cycles. The short-term production(1–15 min) of particulate organic carbon (POC) and extracellularorganic carbon (EOC) compounds was studied by measuring theuptake of ¹⁴C-labelled sodium bicarbonate and its subsequentincorporation and release into organic compounds. Slightly modifiedtraditional ¹⁴C radiotracer protocols were used, including separationby electrophoresis and thin-layer chromatography and detectionby autoradiography. Results indicated that there was a distinctdifference between radiolabelled compounds in the POC and EOCpools. Several metabolites found in the EOC pool were not presentin the POC pool, indicating the active release of these productsfrom the cells into the ambient water during short-term incubations,and indicating that inorganic carbon fixation pathways in marineautotrophs might be partly extracellular.     

Photosynthetic parameters, pigment composition and respiration rates of the marine diatom Skeletonema costatum grown in continuous light and a 12:12 h light-dark cycle

Nutrient-sufficient cultures of a Trondheimsfjord (Norway) cloneof the marine centric diatom Skeleionema costatum (Grev.) Clevewere grown at 75 µmol m^–2 s^–1 and 15C at24 and 12 h daylength to study diurnal variations and the effectof daylength on pigment and chemical composition, photosyntheticparameters, dark respiration rates and scaled fluorescence excitationspectra (F), the latter used as estimates for the absorptionof energy available to Photosystem II. Specific growth rateswere 1.06 and 0.56 day^– in 24 and 12 h daylength, respectively,while dark respiration rates were generally 85% of the net growthrate. The Chla-normalized photosynthetic coefficients P^B_m anda^B were {small tilde}20–25% higher in continuous lightthan at 12 h daylength, while the Chla:C ratio was {small tilde}15%lower (0.051 versus 0.061 w:w). Thus, the carbon-normalizedcoefficients P^c_m and a^c were <11% lower at 24 h than at 12h daylength. The maximum quantum yield _max, the Chla:C ratioand F differed negligibly, as did the light saturation indexl_k, the N:C ratio and the ratios Chlc:Chla and Fucoxanthin:Chla. P^B_m and l_k did not exhibit diurnal variations at 24 hdaylength, and varied within 23% of the daily mean at 12 h daylength.Predictions of the daily gross photosynthetic rate based ondata for a given time of the day should thus not be >10%in error relative to an integrated value based on several datasets collected through 24 h. _max was 0.084–0.117 mol O₂(mol photons)^– for gross oxygen evolution. However, ifused in mathematical models for predicting the gross and netgrowth rates (i.e. the gross and net carbon turnover rates),‘practical’ values of 0.076 and 0.040 g-at C (molphotons)^–, respectively, should be employed. Correspondingly,values for a^B and P^B_m should be adjusted pro rata. ¹Present address: College of Marine Studies, Sjmannsveien 27,N-6008 lesund, Norway