Utilization of glutamate and glucose for heterotrophic growth by the marine pennate diatom Nitzschia laevis

Nitzschia laevis Hustedt grew in the dark in the presence of either glutamate or glucose as substrate. Complex mixtures of yeast extract or tryptone plus lactate also supported good heterotrophic growth, while tryptone alone only supported very slow growth in the dark. The observed growth rates of N. laevis in the dark at different concentrations of glutamate or glucose could be accounted for by the measured uptake rates of these compounds. The affinity of the uptake systems for glutamate and glucose (K _s =0.03 mM for each) was quite high, and similar for dark- and light-grown cells. The lack of a lag-phase when cells were transferred from photoautotrophic to heterotrophic growth conditions can be explained by the presence of uptake systems for glutamate and glucose in ligh--grown cells, as well as in dark-grown cells. However, the uptake capacity was generally higher in the latter than the former. N. laevis also took up alanine and lactate according to Michaelis-Menten kinetics, with a K _s for alanine of 0.02 mM and for lactate of 0.4 mM. Malate and glycerol were not taken up to a significant extent by the cells. Cells grown in continous light had a doubling time of 18 h. The shortest doubling time observed in the dark on glutamate was 48 h and on glucose 24 h. Glutamate was used for heterotrophic growth with an efficiency of 43% and glucose with an efficiency of 48%.Contribution No., 945 from the Department of Oceanography, University of Washington, Seattle, Washington 98195, USA.     

Implications of salinity fluctuation for growth and nitrogen metabolism of the marine diatom Ditylum brightwellii in comparison with Skeletonema costatum

In 1987 effects of salinity fluctuations on growth of Ditylum brightwellii (West) Grunow, isolated from the Eastern Scheldt estuary (SW Netherlands) in 1981, were studied. D. brightwellii was grown in a 12 h light: dark cycle at constant salinity in brackish media. Ammonium-limited cultures were subjected to a salinity fluctuation. By decreasing the salinity to 4.8 photosynthesis and cell division were inhibited; cells were deformed. Protein and carbohydrate contents increased slightly, dark respiration was stimulated and cellular levels of glucose decreased at low salinity; this indicated a possible role of sugars in osmoregulation. Ammonium was accumulated in cultures, amino acids may have been stored; the role of the vacuole as a storage compartment was discussed. Both the ammonium uptake capacity and the affinity for ammonium decreased. Nitrogen limitation was relieved in the transient state. [With the activity of the nitrogen assimilation enzymes glutamine synthetase (GS) and glutamate synthase (GOGAT) being uninhibited by lower salinity.] Recovery from hypo-osmotic stress during a salinity increase was initiated by stimulated photosynthesis; chlorophyll a increased, but persistant contractions of cytoplasm (with chloroplasts) may have delayed cell growth. The glutamate dehydrogenase (GDH) activity decreased further whereas the cellular level of alanine increased in the presence of large ammonium pools; this may indicate a temporary activity of ADH (alanine dehydrogenase). Skeletonema costatum (Greville) Cleve, recovered faster from hypoosmotic stress than did D. brightwellii. Due to an osmotic shock from 13.6 to 7.1 S both species excreted amino acids and glucose; S. costatum accumulated more glucose, D. brightwellii accumulated more amino acids. S. costatum may with the competition for nitrogen in waters with an unstable salinity; it will replace D. brightwellii.Contribution no. 427 Delta Institute for Hydrobiological Research, Yerseke, The Netherlands     

Kinetics of glucose and amino acid uptake by attached and free-living marine bacteria in oligotrophic waters

Kinetics of glucose and amino acid uptake by attached and free-living bacteria were compared in the upper 70 m of the oligotrophic north-western Mediterranean Sea. Potential uptake rates of amino acids were higher than those of glucose in all the samples analysed. Cell-specific potential uptake rates of attached bacteria were up to two orders of magnitude higher than those of total bacteria, both for amino acids and glucose (0.72–153 amol amino acids cell⁻¹ h⁻¹ and 0.05–58.42 amol glucose cell⁻¹ h⁻¹ for attached bacteria and 0.34–1.37 amol amino acids cell⁻¹ h⁻¹ and 0.07–0.22 amol glucose cell⁻¹ h⁻¹ for total bacteria). The apparent K _m values were also higher in attached bacteria than in total bacteria, both for amino acids and glucose. These results would reflect the presence of different uptake systems in attached and free-living bacteria, which is in accordance with the different nutrient characteristics of their microenvironments, ambient water and particles. Attached bacteria show transport systems with low affinity, which characterise a bacterial community adapted to high concentration of substrates. Received: 13 June 2000 / Accepted: 6 December 2000     

Evidence for facultative heterotrophy in cultured zooxanthellae

The locus of symbiotic dinoflagellates within host cells provides a habitat which could potentially be exploited by the alga through heterotrophic uptake of host-derived organic substrates. Using zooxanthellae (Symbiodinium sp.) isolated from the tropical sea anemone Aiptasia pulchella collected from Kaneohe Bay, Hawaii, the effect of various potential organic substrates on growth in vitro was assessed in Erdschreiber seawater medium (ES) supplemented with organic compounds. Zooxanthellae maintained at 5 to 7 E m^-2 s^-1 (below compensation irradiance) grew heterotrophically when supplied with 100 M glycerol, glycolate, acetate, malate, or propionate, and grew in darkness on 100 M propionate. Zooxanthellae exposed to irradiance below compensation were able to utilize carbon sources in the unsupplemented ES medium for slow growth, but generally the growth rate of cultured zooxanthellae was a function of incubation irradiance. Zooxanthellae incubated for 10 wk in unsupplemented ES at 5 to 7 E m^-2 s^-1 were capable of growth at this low irradiance, but were also capable of net photosynthetic oxygen production at higher irradiances. This suggests that zooxanthellae can be photoautotrophic or facultatively heterotrophic. An estimate for the duration of mitosis (t _d ) is made on the basis of growth rate of cultured zooxanthellae in log-phase; this estimate of t _d =4.88 h is less than half the estimated t _d for zooxanthellae in situ.     

Sodium-dependent amino acid transport in the chlorophyte Platymonas subcordiformis

Sodium-dependent transport of alanine and serine by Platymonas subcordiformis was demonstrated by evaluating the kinetics of entry of these substrates over a range of concentrations of ambient sodium. The reciprocal of the concentration at which entry rate was half maximal (K _t ) was linearly related to ambient sodium concentration; maximum entry (V _max) was not affected. Entry of labeled amino acids as measured by determining radioactivity in the medium was close to but slightly less than rates of net entry as determined by high-performance liquid chromatography. This difference presumably reflects extrusion of labeled carbon by the cells in a form not detectable by the chromatographic techniques employed. The coupling coefficient (sodium ions per amino acid molecule) for entry of alanine and serine was evaluated and determined to be 2. Methylaminoisobutyric acid was not taken up by P. subcordiformis and had no inhibitory effect on uptake of alanine or serine. B-2-aminobicycloheptane-2-carboxylic acid competitively inhibited uptake of both substrates. The internal sodium concentration of P. subcordiformis was measured, and the maximum gradient energetically favorable for amino acid transport was calculated from data in the present work and drawn from the literature. Assuming that uptake of amino acids is strictly sodium-dependent, an amino acid concentration gradient of the order of 10⁶ (cell:medium) can be achieved. This concentration differential permits net uptake of amino acids by P. subcordiformis from ambient amino acid levels in the nanomolar range.     

Metabolic specialization of mitochondria from scallop phasic muscles

In fast, glycolytic muscles, oxidative phosphorylation presumably facilitates recuperation from exhaustive exercise and supports growth and maintenance metabolism. Given the shifts in pH with extensive glycolytic activity, the pH optima of mitochondrial processes should indicate whether mitochondria are adapted for recuperation from exercise or for growth and maintenance. We examined this question using mitochondria from the phasic adductor muscle of the scallop, Euvola (Pecten) ziczac, collected from the Golfo de Cariaco, Venezuela in 1992 and 1993. Scallop muscle mitochondria showed well coupled oxidation of glutamate and pyruvate at pH 7.0 and 6.4. The preferred substrates (glutamate, pyruvate and succinate) were oxidized at approximately 40 nmol O₂ min^-1 mg^-1 mitochondrial protein at 25°C, while malate and glutamine were oxidized at 75% and proline at 30% of these rates. Neither palmitoyl carnitine nor aspartate were oxidized. Succinate oxidation was not coupled to ADP utilization at pH 7.0 but was somewhat coupled at pH 6.4. Generally, State 3 rates of oxygen uptake were similar at pH 7.0 and 6.4. Maximal rates of oxidation of glutamate and pyruvate showed broad pH optima. For both glutamate and pyruvate, the highest respiratory control ratio (RCR) values were found at pH 6.5. The saturation curves of scallop muscle mitochondria for pyruvate, glutamate and ADP were well described by the Michaelis-Menten equation. The affinity for pyruvate was greater at pH 6.4 (apparent K _{m, app}=0.013 mM) than at pH 7.0 (K _{m, app}=0.026 mM) while the affinity for ADP (K _{m, app}=0.015 mM) and that for glutamate (K _{m, app}=0.55 mM) changed little with pH. The ADP affinity was the same whether pyruvate or glutamate was the carbon substrate. The combination of maintenance of sensitivity to ADP with an enhanced affinity for pyruvate at acidic pH values should facilitate recuperation from bouts of glycolytic activity. Scallops harvested in September and those harvested in January differed in the maximal rates of glutamate and pyruvate oxidation.     

Zooxanthellae providing assimilatory power for the incorporation of exogenous acetate in Heteroxenia fuscescens (Cnidaria: Alcyonaria)

The soft coral Heteroxenia fuscescens (Ehrb.) and its isolated zooxanthellae (endosymbiotic dinoflagellates) were investigated with particular regard to uptake and utilization of exogenously supplied ¹⁴C-acetate in the light and in the dark. The incorporation of ¹⁴C from ¹⁴C-acetate into the host tissue and into the zooxanthellae was consistently much higher in the light than in the dark. The incorporated ¹⁴C-acetate was rapidly metabolized by the host and algae and was recovered from different assimilate fractions. The major proportion of radiocarbon from metabolized ¹⁴C-acetate was located in host tissue. The CHCl₃-soluble fraction composed of diverse lipids showed the strongest ¹⁴C-labelling. Zooxanthellae isolated prior to incubation accounted for about 80% of the acetate incorporation recorded for zooxanthellae in situ (in vivo). It is concluded from a comparison of acetate incorporation and conversion under light and dark conditions that most of the lipid reserve of the host tissue originates from fatty acids, which are synthesized within the algal symbionts and are then translocated to the heterotrophic partner via extrusion. The acetate units needed for lipid synthesis are obtained by absorption of free acetate from dissolved organic matter (DOM) in the seawater as well as by photosynthetic assimilation of inorganic carbon. Thus, in H. fuscescens, lipogenesis is operated as a light-driven process to which the zooxanthellae considerably contribute assimilatory power by performing fatty acid synthesis and translocation of lipid compounds to their intracellular environment (host cell). A metabolic scheme is proposed to account for the different pathways of carbon conversion observed in H. fuscescens. The incubations took place in August 1980 and the analytical part from October 1980 to January 1984.     

Uptake and accumulation of naphthalene by the oyster Ostrea edulis,in a flow-through system

A flow-through system was used to follow naphthalene and naphthalene metabolite accumulation in the seawater and in the tissue of the oyster Ostrea edulis. After 72 h, 82.5% of the naphthalene carbon was recovered from the system. Glucose was added to seawater to stimulate the pathways of glucose metabolism in the oysters. Streptomycin (100 ppm) reduced microbial oxidation of naphthalene and glucose, and reduced bacterial growth. However, even in the presence of streptomycin, microbial oxidation of naphthalene was considerable. The main oxidation product recovered from seawater was ¹⁴CO₂. Radioactivity was also associated with compounds which separated by TLC with 2- and 1- naphthol. The pattern of naphthalene uptake and accumulation in oyster tissues was relatively constant after only a few hours of exposure to naphthalene. The potential of tissues to accumulate naphthalene was shown to be a function of multiple variables such as nutritional state, lipid concentration, length of exposure to naphthalene, and the external naphthalene concentration. Carbon-14-labeled metabolites derived from ¹⁴C-naphthalene were consistently recovered from digests of the oyster tissues. Non-CO₂ alkaline-soluble substances were the primary metabolites. Hexane-extractable substances, which separated by TLC with known standards of 2- and 1- naphthol, were consistently recovered from seawater and tissue digests. It was not possible to conclude that these metabolites were a result of naphthalene metabolism by oyster enzyme systems.     

Inorganic nitrogen metabolism in marine bacteria: The intracellular free amino acid pools of a marine pseudomonad

The marine pseudomonad bacterium PL₁ contains an intracellular pool of free amino acids which consist mainly of glutamate with small amounts of glutamine and aspartate when grown in a nutrient medium containing 0.2 M NaCl. When the NaCl concentration of the growth medium is increased to 0.8 M, proline becomes a major component of the intracellular pool together with glutamate—at this molarity and under suitable nutrient conditions these amino acids comprise 20% of total bacterial amino acid nitrogen. When grown in a nutrient growth medium containing a constant level of NaCl, the intracellular pool size can vary by a factor of 4 depending on the concentration of carbon and nitrogen in the medium. Experiments show that the amino acid pool can act as a nitrogen reserve but has little function as a carbon reserve. At high NaCl concentrations there is a marked dependence for growth on the presence of sufficient potassium in the medium. However, no correlation between K⁺ and glutamate concentration in either nitrogen or K⁺-limited cultures has been found. None of the enzymes associated with glutamate biosynthesis was influenced by NaCl levels between 0.2 and 0.5 M. Neither Na⁺ or K⁺ stimulated the activity of these enzymes when tested in vitro.     

An examination of photosynthetic production,excretion of photosynthetic products,and heterotrophic utilization of dissolved organic compounds with reference to results from a coastal subtropical sea

The rate of primary production, excretion of photosynthetic products and turnover of glucose and amino acids was measured at a station in a coastal region in the Bahamas. Over the depths 0 to 50 m, total photosynthetic rates varied from 1.7 to 12.7 gC fixed 1^-1day^-1, averaging 4.3. The extent of extracellular photosynthetic products ranged from undetectable to 23%, averaging 6.9%. Neither the field data nor studies with axenic cultures of Dunaliella tertiolecta, Skeletonema costatum, and Monochrysis lutheri showed any evidence of an increase in the percentage excretion at low population densities or low photosynthetic rates. Rates of amino acid turnover varied from 21 to 168% day^-1, and that of glucose from 8.3 to 41% day^-1. Light seems to have little effect on the uptake and respiration of these substrates by the planktonic population. There was a significant relationship between the fraction of the substrate used for respiration and that retained by the cell. On average, 42% of the glucose taken up was respired and 21% of the amino acid mixture. Tentative calculations suggest that the production of dissolved organic material as extracellular photosynthetic products would be insufficient to supply the heterotrophic population, and it was concluded that some other route(s) must be of major importance.     

Light-dependency of nitrate uptake by phytoplankton over the spring bloom in Auke Bay,Alaska

The effect of light intensity on nitrate uptake by natural populations of phytoplankton was examined by ¹⁵N traceruptake experiments during the spring (March–May 1987) in Auke Bay, Alaska. The data were fit to a rectangular hyperbolic model which included a term for dark uptake. Three types of curves described nitrate uptake as a function of light intensity. The first (Type I) had a low half-saturation light intensity (K _I), low chlorophyll-specific uptakes rates, no dark uptake and occasional photoinhibition. These were observed during a period of biomass decrease, accompanied by low daily light and strong wind, prior to the major bloom. The second type (Type II) had relatively high K _I, high chlorophyll-specific uptake rates, and no dark uptake. Type II curves were observed during most of the period prior to nitrate depletion in the surface waters. Types I and II both appeared prior to nitrate depletion in the water and reflected variations in the light history of the phytoplankton population. The third type (Type III) occurred in nitrate-deplete conditions, when nitrate uptake was less dependent on light intensity (i.e., high rates of dark uptake and lower K _I). Decreased light-dependency during this period was coupled with physiological nitrogen deficiency in the population. Comparing these parameters to those of photosynthetic carbon fixation, K _Ivalues of nitrate uptake were generally higher than those of photosynthesis prior to nitrate depletion, and lower during nutrient-deplete conditions.     

Ammonium regeneration and carbon utilization by marine bacteria grown on mixed substrates

We examined the impact of exposing natural populations of marine bacteria (from seawater collected near Woods Hole, Massachusetts, USA) to multiple nitrogen and carbon sources in a series of batch growth experiments conducted from 1989 through 1990. The substrate C:N ratio (C:N_s) was varied from 1.5:1 to 10:1 either with equal amounts of NH ₄ ⁺ and different amino acids or an amino acid mixture, all supplemented with glucose to maintain the C:N_s ratio equal to that of the respective amino acid, or with combinations of glucose and NH ₄ ⁺ alone. A common feature of the experiments involving amino acids was the concurrent uptake of NH ₄ ⁺ and amino acids that persisted as long as a readily assimilable carbon source (glucose in our case) was taken up. There was no net regeneration of NH ₄ ⁺ , even though catabolism of amino acids occurred. Regeneration of NH ₄ ⁺ was evident only after glucose was completely utilized, which usually occurred at the end of exponential growth. The contribution of¹⁵NH ₄ ⁺ to total nitrogen uptake by the end of exponential growth varied from ~60 to 80% when individual amino acids were present and down to ~24% when the amino acid mixture was added. These estimates are conservative because we did not account for possible isotope dilution effects resulting from amino acid catabolism. When NH ₄ ⁺ and glucose were the sole nitrogen and carbon sources, there was a stoichiometric balance between glucose and NH ₄ ⁺ uptake over a wide range of C:N_s ratios, leading to a constant bacterial biomass C:N ratio (C:N_B) of ~4.5:1. As a result NH ₄ ⁺ usage varied from 50% when the C:N_s ratio was 3.6:1, to 100% when the C:N_s ratio was 10:1. Gross growth efficiency varied from ~60% when NH ₄ ⁺ plus glucose were added alone or with the amino acid mixture, to 47% when the individual amino acids were used in place of the mixture. It is thus evident that actively growing bacteria will act as sinks for nitrogen when a carbon source that can be assimilated easily is available to balance NH ₄ ⁺ uptake, even when amino acids are available and are being co-metabolized.     

Comparison between the carbon-14 and oxygen consumption method for the determination of the activity of heterotrophic bacterial populations

The activity of the heterotrophic microbial population in the saline Lake Grevelingen (The Netherlands) and the Mediterranean Etang Salses Leucate (France) was determined by measuring the oxygen consumption rate, and the uptake of ¹⁴C-labelled glycollate, pyruvate and an amino acid mixture. The maximum uptake rate of the applied organic compounds in Lake Grevelingen was generally less than 10% of the carbon mineralization rate calculated from the oxygen consumption experiments. Only for pyruvate and glycollate higher values were found of about 30 to 40% with one exceptionally high value for pyruvate of 149%. However, these higher percentages were found in winter, when the activity of the heterotrophic microbial population was very low. In Etang Salses Leucate higher maximum uptake rates of the ¹⁴C-labelled compounds were found, relating this uptake to the oxygen consumption rate. Yet the maximum uptake rate is still always lower than 35% of the carbon mineralization calculated from the oxygen uptake rate. Taking into account that maximum uptake rates were considered, the results demonstrate that the uptake of ¹⁴C-labelled organic compounds represents a serious underestimation of the activity of the bacterial population in situ. The extent of the underestimation depends on the water type. It was concluded that the determination of the heterotrophic activity by measuring oxygen consumption rates offers a better insight into the carbon mineralization process in natural waters than the uptake experiments with ¹⁴C-labelled substrates.Communication no. 228 of the Delta Institute for Hydrobiological Research, Yerseke, The Netherlands     

Salinity effects on glucose uptake and catabolism in the obligately psychrophilic marine bacterium Vibrio marinus

Studies on the effects of various salinities on the uptake and catabolism of glucose in Vibrio marinus MP-1 revealed several significant shifts in total uptake and respiration as the cells were subjected to increasingly greater concentrations of NaCl. As the salinity increased from 0.30 to 1.0 M NaCl, there was a decrease in the C₆/C₁ (CO₂) ratio. The resulting patterns suggests that the relative participation of the hexose monophosphate pathway in glucose catabolism was altered. This pathway is apparently shut down in the region of the minimum-growth salinity, and may be related to growth limitation at rower salinities. The shift in C₆/C₁ ratio was not affected by changing the incubation temperature, nor was it dependent specifically on the presence of Na⁺ or Cl^-. As the salinity increased from 0.15 to 0.30 M NaCl, there was a shift in the total uptake patterns which suggests the formation and loss of metabolic by-products derived from the first, second, sixth, and presumably fifth carbons of glucose.This paper was taken in part from a dissertation by the senior author, submitted in partial fulfillment of the requirement for the Ph.D. degree, Oregon State University, Corvallis. Published as technical paper No. 3647, Oregon Agricultural Experiment Station.     

Study on phosphate uptake of the marine cyanophyte Synechococcus sp. NIBB 1071 in relation to oligotrophic environments in the open ocean

Inorganic phosphate (Pi) uptake by the marine cyanophyte Synechococcus sp. NIBB 1071 was studied using cells grown in an artificial seawater medium. The phosphate uptake was markedly enhanced in cells grown in the medium of low phosphate concentrations (phosphate-limited cells) than in cells grown in the phosphate-rich medium (phosphate-replete cells). The diagnosis of kinetics of instantaneous phosphate-uptake showed that V _max of the former was more than two orders of magnitude greater than that of the latter, and the k _m of the former was about 1/20 of that of the latter. The enhancement of the phosphate uptake was completed after a 40-h incubation of phosphate-replete cells in the phosphate-free medium. The activation was suppressed by chloramphenicol, an inhibitor of protein synthesis. The uptake developed in phosphate-limited cells was energy dependent and susceptive to osmotic shock, which suggests the involvement of a periplasmic phosphate-binding protein, analogous to that found in heterotrophic gram-negative eubacterial cells. The relationship between phosphate quota and growth rate, together with the kinetical data for phosphate uptake, predicted that ambient phosphate as low as 0.5 nM could support cell growth at a rate of one division per day. Results indicate that cells can grow rapidly even at phosphate concentrations as low as nanomolar levels. A possible regulatory mechanism of phosphate uptake in marine Synechococcus spp. is discussed in relation to a wide distribution of this picophytoplankton in the ocean environment. Received: 19 March 1997 / Accepted: 2 April 1997     

Impact of a temporal salinity decrease on growth and nitrogen metabolism of the marine diatom Skeletonema costatum in continuous cultures

In 1987 effects of salinity fluctuations on growth of the centric diatom Skeletonema costatum (Greville) Cleve, isolated from the brackish Krammer estuary (SW Netherlands) in 1981, were investigated. Continuous cultures (12 h light: dark cycle) of S. costatum were adapted to constant salinity in natural (16.1) and synthetic (13.5) media. For several days the ammonium-limited cultures were exposed to a salinity fluctuation (minimum 4.8). Decreasing salinity caused an inhibition of photosynthesis, dark respiration and cell growth. Cellular pools of glucose decreased. While the carbohydrate content remained constant, the protein content increased slightly. Net carbon fixation was more inhibited than nitrogen assimilation. Ammonium accumulated during a salinity decrease; a total decline of the overcapacity of ammonium uptake was noticed and nitrogen limitation was relieved. Amino acid pools decreased, probably as a result of excretion (osmoregulation). The enzymes invoilved in ammonium assimilation showed an increased activity. Cellular activities were resumed during a salinity increase. Chlorophyll a increased; photosynthesis, ammonium uptake and growth were stimulated. The ammonium uptake capacity recovered completely; glutamic acid accumulation and increased glutamate-dehydrogenase (GDH) activity indicated supplementary ammonium assimilation via GDH. The activities of glutamine synthetase/glutamate synthase (GS/GOGAT) and GDH stabilized, and the cells returned to steady state under ammonium limitation.Communication no. 426 Delta Institute for Hydrobiological Research, Yerseke, The Netherlands     

Carbon and nitrogen metabolism by Monochrysis lutheri: Measurement of growth-rate-dependent respiration rates

Dark respiration rates were measured and carbon-excretion rates calculated for a nitrate-limited population of the marine chrysophyte Monochrysis lutheri grown in continuous culture at 20°C on a 12 h light-12 h dark cycle of illumination and over a series of 4 growth rates. A significant (P<0.05) positive correlation was found between dark respiration rate and growth rate. From a simple linear fit to the data, the respiration rate at maximum growth rate was estimated to be roughly 10.5% of the maximum gross-carbon-production rate, and more than three times higher than the extrapolated respiration rate at zero net-growth rate. Carbon-excretion rates showed no significant correlation with growth rate, and averaged less than 5% of the maximum gross-carbon-production rate. Mean cell nitrogen to carbon ratios were correlated in a virtually linear manner (r=0.994) with growth rate, and at a given growth rate were consistently higher than nitrogen to carbon ratios for the same species grown on continuous light. A comparison of carbon and nitrogen quotas as a function of growth rate for M. lutheri and other species suggests that the increase of cellular nitrogen at high growth rates under nitrate-limited growth conditions may be associated with the storage of cellular protein or amino acids rather than the presence of an inorganic nitrogen reservoir. The maximum nitrate uptake rate per cell during the day changed very little over the range of growth rates studied, and was comparable to the maximum uptake rate found for cells grown on continuous light. However, the cell nitrogen quota increased steadily with growth rate, causing a reduction in the maximum specific-uptake rate of nitrate during the day at high growth rates. The dark nitrate-uptake capacity of the population was clearly exceeded by the supply rate at the two higher growth rates, leading to a buildup of nitrate during the night which amounted to as much as 21% of the particulate nitrogen in the growth chamber by morning.Hawaii Institute of Marine Biology Contribution No. 478.     

Photosynthetic carbon dioxide fixation in zooxanthellae

The carbon-fixation patterns of freshly isolated zooxanthellae from the hermatypic coral Acropora formosa were examined during a 15 min exposure to sodium mosa were examined during a 15 min exposure to sodium [¹⁴C]bicarbonate. The labelling pattern during the first 60 s exposure showed that the C₃ carbon-fixation pathway is the major route for photosynthetic carbon fixation in Symbiodinium sp. 3-Phosphoglyceric acid, which constituted >50% of the label after 5 s, steadily decreased over the first 60 s. Hexose phosphates, aspartate, malate and glucose were the other main products during the first 60 s. Over longer periods, significant amounts of the organic acids succinate, aspartate and glutamate were found in the extract along with glucose; but no glycerol.     

Dark CO2 uptake by the diatom Chaetoceros simplex in response to nitrogen pulsing

In a continuing investigation of dark CO₂ uptake by nitrogen-limited cultures of the marine diatom Chaetoceros simplex (Bbsm), we expanded on several of our earlier conclusions regarding the potential application of this physiological response for measuring the degree and type of nitrogen limitation in phytoplankton populations. First, the duration over which the maximal enhancement of dark ¹⁴CO₂ uptake was sustained after NH ₄ ⁺ enrichment was a function both of the concentration of added NH ₄ ⁺ and the standing crop of phytoplankton nitrogen — in effect, the total N demand. Second, pulsing with NH ₄ ⁺ for a given degree of N-limitation always produced the same level of enhanced dark CO₂ uptake regardless of whether the cultures were preconditioned with oxidized or reduced nitrogen. In contrast, urea pulsing led to reduced dark CO₂ uptake, but the effect was most pronounced in cells grown on NO ₃ ^– . And third, the assay could be used to distinguish readily between no, moderate, and severe N limitation. The degree of severe N limitation was quantitatively correlated with the degree of enhanced dark CO₂ uptake, but this relationship was not so clear in the region of moderate N limitation. The main advantage of the assay is that it is a relatively simple and effective alternative to more complicated techniques for gauging the degree and form of N limitation in phytoplankton. Further evaluation will be required, both in the laboratory and field, before the assay can be calibrated for quantitative use.Contribution No. 5982 from the Woods Hole Oceanographic Institution     

Role of glutamine synthetase in ammonia assimilation by symbiotic marine dinoflagellates (zooxanthellae)

The dinoflagellate symbionts (zooxanthellae) present in many reef corals aid in the survival of the symbiotic unit in nitrogen deficient tropical waters by providing additional routes of nitrogen uptake and metabolism. The enzymatic pathway of ammonia assimilation from seawater and the re-assimilation of coral ammonium waste by zooxanthellae was studied by examining the affinity of glutamine synthetase for one of its substrates, ammonia. Glutamine synthetase activity was measured in dinoflagellates of the species Symbiodinium microadriaticum found in symbiotic association with various marine coelenterates. Michaelis-Menten kinetics for the substrate ammonia were determined for freshly isolated dinoflagellates from Condylactis gigantea (apparent NH₃ K_m=33 M) and for cultured dinoflagellates from Zoanthus sociatus (apparent NH₃ K_m=60 M). On the basis of the low apparent K_ms for NH₃, it appears that ammonia assimilation by these symbiotic dinoflagellates occurs via the glutamine synthetase/glutamate synthase pathway. Additionally, the uptake of exogenous ammonium by an intact coelenterate-dinoflagellate symbiosis was strongly inhibited by 0.5 mM methionine sulfoximine, and inhibitor of glutamine synthetase.