Uptake kinetics of nitrate,ammonia and phosphate by the dinoflagellate Heterocapsa circularisquama isolated from Hiroshima Bay,Japan

Heterocapsa circularisquama is a harmful dinoflagellate whose first bloom in Hiroshima Bay, Japan, appeared in 1992. As suggested by the authors’ group, in the Seto Inland Sea including Hiroshima Bay, oligotrophication particularly the reduction of phosphate starting 1980 is severe. The bloom caused serious damage to the bay's extensive oyster culture. In the present study, the uptake kinetics of nitrate, ammonia, and phosphate by this species were experimentally investigated. The maximum uptake rate (ρ_max) and the half‐saturation constant (Ks) were 0.41 pmol cell^?1 h^?1 and 4.45 μM, respectively, for nitrate, 2.02 pmol cell^?1 h^?1 and 11.1 μM for ammonium, and 0.079 pmol cell^?1 h^?1 and 1.79 μM for phosphate. The maximum specific uptake rates (V_max) for nitrate, ammonia, and phosphate were estimated to be 8.95, 44.1, and 21.3 day^?1, respectively. A comparison of V_max/Ks, which is also an index of affinity to nutrients, between this species and others suggested that H. circularisquama can utilize nitrate and ammonia efficiently, but not phosphate. Considering both reports describing that H. circularisquama has the ability to utilize dissolved organic phosphorus (DOP) and the DOP concentration is higher than phosphate in Hiroshima Bay, it was concluded that H. circularisquama became dominant due to the phosphate reduction measure.     

STUDY ON THE INHIBITION OF BRAIN GLUTAMATE DECARBOXYLASE BY PYRIDOXAL PHOSPHATE OXIME-O-ACETIC ACID

The kinetics of the inhibition of mouse brain glutamate decarboxylase by pyri-doxaI-5′-phosphate oxime-O-acetic acid (PLPOAA) was studied. The inhibition was noncompetitive with regard to glutamic acid; it could be partially reversed by pyridoxal phosphate, but only when the concentration of the latter in the incubation medium was higher than that of pyridoxal-5′-phosphate oxime-O-acetic acid. The inhibition produced by aminooxyacetic acid, which is remarkably greater than that produced by PLPOAA, was also partially reversed only when an excess of pyridoxal phosphate was added. Both in the presence and in the absence of a saturating concentration of pyridoxal phosphate, the activity of the enzyme was decreased by PLPOAA at a 10^?4m concentration to a value of about 50 per cent of the control value obtained without added coenzyme. This activity could not be further reduced even when PLPOAA concentration was increased to 5 × 10^?3m . This same minimal activity of glutamate decarboxylase was obtained after dialysis of the enzymic preparation, or after incubation with glutamic acid in the cold followed by filtration through Sephadex G-25. The addition of pyridoxal phosphate to the dialysed or glutamic acid-treated enzyme restored the activity to almost the control values. PLPOAA did not affect the activity of glutamate decarboxylase from E. coli or that of DOPA decarboxylase and GABA transaminase from mouse brain. To account for the results obtained it is postulated that brain glutamate decarboxylase has two types of active site, one with firmly bound, non-dialysable pyridoxal phosphate and the other with loosely bound, dialysable coenzyme; PLPOAA behaves as a weak inhibitor probably because it can combine mainly with the loosely bound coenzyme site, while aminooxyacetic acid is a potent inhibitor probably because it can block both the ‘loosely bound coenzyme’ and the ‘firmly bound coenzyme’ sites.     

Extraction of protease from Aspergillus tamarii URM 4634 in aqueous two-phase system under continuous and discontinuous process

Abstract

Aqueous two-phase systems have been studied for almost a century to separate biomolecules in harmless conditions. Proteases produced by Aspergillus tamarii URM 4634 were extracted in polyethylene glycol (PEG)/phosphate aqueous two-phase system under discontinuous and continuous (perforated rotative discs column) process. On the discontinuous process, it was evaluated the effect of operational conditions (PEG molar mass and its concentration, phosphate concentration and pH) over the partition coefficient, activity yield and purification factor. Protease partitioned to PEG-phase with partition coefficients up to 55.73. The best process parameters were 17.5% of PEG, with molar mass 8000?g·mol^?1, 15% of phosphate salt at pH 6, with 113.15% of activity yield and purification factor of 2.62. Under continuous extraction, hold up data showed that 57.1% of the discontinuous phase was available for protein extraction. Further, separation achieved 90.0% of efficiency. The yields surpassed 100% in almost all runs, and the best purification factor was 1.84, with both flows of 2?mL·min^?1. Thus, the best operational conditions reached an activity yield of 95.3% and 90.0% of separation efficiency. Hence, aqueous two-phase system PEG/phosphate extraction is an efficient process for separation of proteases produced by Aspergillus tamarii URM 4634, under continuous extraction likewise under discontinuous process.     

The rate of growth of asterionella formosa Hass. in relation to its ecology

Summary In Windermere, and some other lakes in the English Lake District, the vernal increase of Asterionella formosa ceases when the concentration of silica in the water falls to approximately 0.5 mg/l. When this planktonic diatom is cultivated in water from one of these lakes, Windermere, the addition of small amounts of phosphate phosphorus permits the growth of so large a crop that all the silica present is incorporated into the cells. The intensity and duration of the illumination and the temperature of the water effect this relationship between silica, phosphorus and the growth Asterionella. Therefore, the regular, observed relationship between the concentration of silica and the vernal maximum of Asterionella in nature may be related to the amount of this substance in the water, to the amount of phosphate phosphorus present and to the rate of growth imposed upon this diatom by the light and temperature conditions in the lake during the period concerned. The possible importance of the relatively low concentration of phosphate phosphorus in certain very unproductive lakes is discussed.Written in honour of Prof. Dr. E. G. Pringsheim.     

EFFECT OF THE SINKING RATE OF TWO DIATOMS (THALASSIOSIRA SPP.) ON UPTAKE FROM LOW CONCENTRATIONS OF PHOSPHATE1

The effect of the sinking rate, or rate of medium flow (φ) on the rate of phosphate incorporation (V) by the planktonic diatoms Thalassiosira fluviatilis Hust. and T. pseudonana Hasle & Heimdal in batch and chemostat cultures was determined by passing medium at defined flow rates (0.5–25.0 mm·min^?1) over algae on membrane filters. At concentrations from 1 to 100 μg phosphorus·l^?1 V, increases with increasing velocity of flow, approaching a maximum value (V_m) as described by the empirical relationship: where K_φ is the sinking rate value when V = 1/2 V_m+ V_o and V_o is the uptake at 0 rate of flow. By comparing uptake at controlled flow with uptake in a vigorously stirred medium, the phosphate concentration in the cell boundary layer can be determined. The sinking rate that reduces the phosphate concentration in the boundary layer to half of nominal concentration in the medium is much lower for the larger T. fluviatilis than for T. pseudonana. For both diatoms, it is inversely related to the nominal concentration.     

Diketopiperazine-mediated peptide formation in aqueous solution II. Catalytic effect of phosphate

The previous paper (I) reported that DKP (glycine anhydride) spontaneously reacts with glycine (Gly) or oligoglycines (Gly _n ) to produce longer oligoglycines (Gly _n+2). This paper presents that phosphate catalyzes the condensation reaction quite effectively.Formation of Gly₄ from DKP (0.1 M) and Gly₂ (0.1 M) in phosphate solution of various concentrations was investigated at a neutral pH at 41 °C. The yields of Gly₄ increased almost linearly with the concentration of phosphate from 0.06 M to 0.24 M. The yield in 0.24 M phosphate solution was approximately one hundred times as high as that in the absence of the phosphate, whereas in the case of Gly₃ formation from DKP and Gly the effect of the phosphate was of ten times lower than in the former case. Orthophosphate was the most effective catalyst among the various kind of chemicals tried in the present investigation including polyphosphates.     

The effect of phosphorylation on the conformation of oligo-peptides with Ser–Pro motif: a molecular dynamics simulation

Model tetrapeptide system was designed to investigate the cis/trans isomerization of peptidyl-prolyl imide bond of Ser–Pro motif. To establish the side-chain O-phosphorylation effect in regulating the peptides conformations, molecular dynamics (MD) simulations where carried out on the designed tetrapeptides and their corresponding phosphorylated forms by MD Insight II Discovery3 approach. The most stable configurations and the statistic cis/trans concentration distribution demonstrated that the phosphorylation evidently influences the peptidyl-prolyl imide bond isomerization and works as a key effect in regulating the peptide conformations. The charge state and the site provided for the charge of the phosphate moiety might be an important key. The results also demonstrated that phosphorylation changes the cis conformation ratio of the peptide and the maximum cis value is obtained when the phosphate group has no negative charge.     

Biological response to lake remediation by phosphate stripping: control of Cladophora

• 1 The North and South Basins of Windermere, Cumbria, have experienced a large increase in concentrations of nutrients, particularly phosphate, since 1945 when detailed measurements began. Over‐winter concentrations have increased from 1 to 3 mg PO₄‐P m^‐3 in the 1940s, up to 30 mg PO₄‐P m^‐3 in the South Basin of Windermere in the early 1990s where nutrient enrichment has been most marked. A visible manifestation of this ‘eutrophication’ in recent years has been the production of a large biomass by the green filamentous macroalga, Cladophora.
• 2 Since April 1992, tertiary chemical stripping of phosphate at the two sewage treatment plants on Windermere has reduced direct sources of phosphate to both basins. In the South Basin, over‐winter concentrations of phosphate have fallen to values similar to those in the early 1970s.
• 3 The biomass of Cladophora has declined markedly in response to the reduced phosphate availability. Significant relationships were found between the annual maximum biomass of Cladophora and two measures of phosphate availability: the over‐winter concentration and, more strongly, the day of year when the concentration fell below 1 mg m^‐3.
• 4 The annual biomass maxima of Cladophora since 1945, estimated from the regressions, showed a gradual increased potential for biomass production after 1965 as phosphate concentrations increased, followed by a striking and rapid biological response to lake remediation by phosphate stripping.

     

Growth optimization of the invasive cyanobacterium Cylindrospermopsis raciborskii in response to phosphate fluctuations

The role of ecophysiological traits in the success and expansion of the toxic cyanobacterium Cylindrospermopsis raciborskii is still under debate. One key factor appears to be the high physiological flexibility of this organism when obtaining limiting resources. Recent studies have found that filamentous bloom-forming cyanobacteria are able to optimize their growth by adjusting phosphate uptake during fluctuating nutrient conditions. We investigated the growth response of two phosphate-deficient C. raciborskii isolates (MVCC19 from Uruguay and CCMP1973 from USA) to short-term fluctuations in phosphate supply. These isolates were exposed to five phosphate concentrations which were provided in two supply modes: a single pulse (SingleP) versus the same amount divided in 10 pulses (TenP), with one pulse applied every 6 min. Morphological traits and changes in chlorophyll a and phycocyanin fluorescence were also evaluated. Growth rates of CCMP1973 and MVCC19 almost doubled and tripled, respectively, when exposed to multiple rather than single phosphate additions. Different growth rates were observed with the same total added resource, thus contradicting the classical model of dependence of growth rate on absolute external concentration. Phosphate-deficient C. raciborskii showed a remarkable physiological flexibility in adapting to phosphate availability on a timescale from minutes to hours. The TenP mode provided an extension of phosphate exposure time that allowed the energetic optimization of uptake and growth. The morphological plasticity of the species in response to phosphate supply mode was also shown by differences in trichome length and individual size. Our results are the first evidence of growth optimization of phosphate-deficient C. raciborskii to short-term nutrient fluctuations, revealing its physiological flexibility. This adaptive behaviour may help to explain the invasive success of this diazotrophic cyanobacterium in a wide range of aquatic ecosystems where phosphorus is frequently the limiting resource.     

Aerotolerant growth inAzospirillum brasilense induced by dihydroxyphenyl iron-binding compound

High concentrations of phosphate drastically inhibited the aerobic growth ofAzospirillum brasilense in liquid medium having a limiting concentration of combined nitrogen. No growth inhibition occurred if a high concentration of combined nitrogen was present. Aerobic growth occurred only in the presence of a very low amount of phosphate and this could be enhanced by adding norepinephrine. Addition of norepinephrine enhanced the O₂ uptake by cell suspensions by almost a factor of 3. All of the intermediates of the tricarboxylic acid metabolic cycle were actively oxidized in cell-free extracts and their rate of oxidation increased in the presence of norepinephrine.     

Phosphate potential and phosphate capacity of soils

Summary The relationship between the phosphate potential (I) and the amount of phosphate (Q), added to the soil has been examined by equilibrating soil samples with 0.001M or 0.01M CaCl₂ solutions containing various amounts of phosphate. For one neutral and two alkaline soils the Q/I relationship depends on the CaCl₂ concentration and the pH in such a way that the apparent values of I decrease when the CaCl₂ concentration increases from 0.001 M to 0.01M. The difference between the two values increases when the pH increases. When correction is made for the formation of the soluble calcium phosphate complex, CaHPO₄, the Q/I relationship becomes independent of the CaCl₂ concentration. The initial phosphate potential (I₀) determined by interpolation, is also found to be independent of the CaCl₂ concentration. The necessary amount of phosphate to be added or removed per gram of soil in order to obtain a certain alteration of the phosphate potential is designated the differential phosphate potential buffering capacity, DPBC. For ten soils DPBC-values are determined on the basis of the Q/I relationships, (ΔQ/ΔI)_Io, and found to be independent of the CaCl₂ concentration. The content of colloids and of inorganic phosphate accounts for a significant part of the variation in the DPBC for different soils. The importance of the DPBC for characterization of the phosphate status of soils in respect to phosphate supply to plants is briefly discussed. The author is indebted to professor, Dr. H. C. Aslyng, head of the department for his suggestions and helpful criticism during the progress of this work.     

A study of N- and P-dependence of nikkomycin production in continuous culture with immobilized cells

Summary The influence of nitrogen and phosphate on the biosynthesis of nikkomycin was studied in chemically defined medium. Cells of Streptomyces tendae were immobilized on porous glass particles in a fluidized-bed reactor for continuous production of nikkomycin. Phosphate had no significant influence on the biosynthesis of nikkomycin. However, even a very low concentration of phosphate in the production medium (00.0125 mmol/l) resulted in microbial growth on the particles. The concentration of nitrogen was highly effective in the regulation of the biosynthesis of nikkomycin. A high level of antibiotic production (maximum 3.05 mg/g dry cell weight per hour) was maintained for a period of about 200 h in a medium that contained nitrogen at a concentration of 0.2 g NH₄NO₃/l. Offprint requests to: H. U. Trück     

Phosphate solubilization by <Emphasis Type="Italic">Rhizobium</Emphasis> strains

Forty-six Rhizobium isolates from legume root and stem nodules were examined for their phosphate-solubilizing ability on Pikovskaya’s agar medium. Rhizobium isolates from root nodules of Cassia absus, Vigna trilobata and three strains from Sesbania sesban showed zone of tricalcium phosphate (TCP) solubilization. The isolate from C. absus showed maximum solubilization (620 μg/ml) after 12 d of incubation, while the Rhizobium sp. strain 26 (from S. sesban) showed the least amount (150 μg/ml) of phosphate solubilization. Among the carbon sources tested for their ability to solubilize TCP, maximum solubilization (620 μg/ml) was observed in glucose by Rhizobium isolate from C. absus. Phosphate solubilization increased with increase in glucose concentration steeply up to 2% and slowly above this concentration in four isolates. Among the nitrogen sources tested, maximum solubilization (620 μg/ml) was observed in ammonium sulphate by Rhizobium isolate from C. absus.     

Effect of nutrients and light intensity on growth of Mallomonascaudata (Synurophyceae)

We used batch cultures of three strains of the unicellular synurophyte Mallomonascaudata to investigate the effects of nitrate, phosphate, silicate and light intensity on population growth and growth rate. The three strains were isolated from three different reservoirs in Kyungpook Province, Korea. For all three strains, we observed high population growth under all nutrient concentrations studied, except at nitrate concentration below 0.8 μM. The maximum growth rate (μ_max) occurred at 8.2 μM or 16.5 μM nitrate, depending on the strain, and at 11.5 μM phosphate. Silicate concentration had no effect on growth rate. With respect to light intensity, the maximum population growth and maximum growth rates (μ_max) occured between 42 and 104 μmol m^?2 s^?1 depending on strain and culture temperature. Population growth of these three strains under batch culture occurred over a wide range of nutrient and light intensities, but there seemed to be strain‐specific differences that may represent adaptations to local environments.     

Solute distribution between vacuole and cytosol of sugarcane suspension cells: Sucrose is not accumulated in the vacuole

The compartmentation of solutes in suspension cells of Saccharum sp. during different growth phases in batch culture was determined using CuCl₂ to permeabilize the plasma membrane of the cells. The efflux of cytosolic and vacuolar pools of sugars, cations and phosphate was monitored, and the efflux data for phosphate were compared and corrected using data from compartmentation analysis of phosphate as determined by ³¹P-nuclear magnetic resonance spectroscopy. The results show that sucrose is not accumulated in the vacuoles at any phase of the growth cycle. On the other hand, glucose and fructose are usually accumulated in the vacuole, except at the end of the cell-culture cycle when equal distribution of glucose and fructose between the cytosol and the vacuole is found. Both Na⁺ and Mg²⁺ are preferentially located in the vacuoles, but follow the same tendency as glucose and fructose with almost complete location in the vacuole in the early culture phases and increasing cytosolic concentration with increasing age of the cell culture. Potassium ions are always clearly accumulated in the cytosol at a concentration of about 80 mM; only about 20% of the cellular K⁺ is located inside the vacuole. Cytosolic phosphate is little changed during the cell cycle, whereas the vacuolar phosphate pool changes according to total cellular phosphate. In general there are two different modes of solute compartmentation in sugarcane cells. Some solutes, fructose, glucose, Mg²⁺ and Na⁺, show high vacuolar compartmentation when the total cellular content of the respective solute is low, whereas in the case of ample supply the cytosolic pools increase. For other solutes, phosphate and K⁺, the cytosolic concentration tends to be kept constant, and only excess solute is stored in the vacuole and remobilized under starvation conditions. The behaviour of sucrose is somewhat intermediate and it appears to equilibrate easily between cytosol and vacuole.Abbreviation NMR nuclear magnetic resonance The very cooperative help by Dr. J. Reiner with the ³¹P-NMR measurements and the technical assistance by D. Keis are gratefully acknowledged. This research was supported by the Deutsche Forschungsgemeinschaft and by Fonds der Chemischen Industrie.     

Regulation of phosphate uptake kinetics inOscillatoria agardhii

In order to study phosphate uptake kinetics the cyanobacteriumOscillatoria agardhii was grown in continuous culture under a phosphorus limitation. The affinity of the uptake system reflected in the initial slope of the uptake rate versus external substrate concentration curve (dV/ds) was found to be unaffected by the growth wate.The maximum phosphate uptake rate (V _m ) decreased as the growth rate was increased. Attempts were made to relate the decrease ofV _m to the increase in phosphorus content of the cells that occurred a higher growth rates. Accumulation of phosphate during pulse experiments indeed resulted in a decrease ofV _m . However feedback regulation ofV _m by accumulated phosphorus was found to occur only to a small extent in steady state growing cells. The main part of the regulation of the activity of the phosphate uptake system seemingly is determined by a long term process that is, at least longer than 2 h. The presence of short term feedback inhibition by accumulated phosphorus on the activity of the uptake system provides an explanation of the phenomenon thatOscillatoria agardhii is not able to grow at near _max growth rates under a phosphorus limitation.     

Growth characteristics of two bloom‐forming synurophytes (Mallomonas elongata and Synura petersenii) at different nitrate and phosphate concentrations

Two dominant planktonic bloom‐forming algal species in a small shallow eutrophic pond were identified as Mallomonas elongata and Synura petersenii by electron microscopy. Their growth requirements were investigated as uni‐algal cultures in a laboratory study. The maximum population growth and maximum growth rate of M. elongata occurred at concentrations of 24 μM nitrate (NO₃) and 5 μM phosphate (PO₄) at a temperature of 15°C and a pH of 6. Synura petersenii grew maximally and exhibited the highest growth rate at a NO₃ concentration of 24 μM and a PO₄ concentration of 2 μM. Mallomonas elongata and S. petersenii had similar nutrient requirements for optimum growth, suggesting that the biomass of these two species can be controlled by nutrient gradients.     

Further evidence for a phycobilisome model from selective dissociation,fluorescence emission,immunoprecipitation, and electron microscopy

Phycobilisomes, isolated in 500 mM Sorensen's phosphate buffer pH 6.8 from the red alga, Porphyridium cruentum, were analyzed by selective dissociation at various phosphate concentrations. The results are consistent with a structural model consisting of an allophycocyanin core, surrounded by a hemispherical layer of R-phycocyanin, with phycoerythrin being on the periphery. Such a structure also allows maximum energy transfer.Intact phycobilisomes transfer excitation energy ultimately to a pigment with a fluorescence emission maximum at 675 nm. This pigment is presumed to be allophycocyanin in an aggregated state. Uncoupling of energy transfer among the pigments, and physical release of the phycobiliproteins from the phycobilisome follow a parallel time-course; phycoerythrin is released first, followed by R-phycocyanin, and then allophycocyanin. In 55 mM phosphate buffer, the times at which 50% of each phycobiliprotein has dissociated are: phycoerythrin 40 min, R-phycocyanin 75 min, and allophycocyanin 140 min.The proposed arrangement of phycobiliproteins within phycobilisomes is also consistent with the results from precipitation reactions with monospecific antisera on intact and dissociated phycobilisomes. Anti-phycoerythrin reacts almost immediately with intact phycobilisomes, but reactivity with anti-R-phycocyanin and anti-allophycocyanin is considerably delayed, suggesting that the antigens are not accessible until a loosening of the phycobilisome structure occurs. Reaction with anti-allophycocyanin is very slow in P. cruentum phycobilisomes, but is much more rapid in phycobilisomes of Nostoc sp. which contains 6–8 times more allophycocyanin. It is proposed that allophycocyanin is partially exposed on the base of isolated intact phycobilisomes of both algae, but that in P. cruentum there are too few accessible sites to permit a rapid formation of a precipitate with anti-allophyocyanin.Phycobilisome dissociation is inversely proportional to phosphate concentration (500 mM to 2 mM), and is essentially unaffected by protein concentration in the range used (30–200 μg/ml). Phycobiliprotein release occurs in the same order (phycoerythrin > R-phycocyanin > allophycocyanin) in the pH range 5.4–8.0.     

NITRATE AND PHOSPHATE UPTAKE INTERACTIONS IN A MARINE PRYMNESIOPHYTE1

The short- and long-term uptake of nitrate and phosphate ions, and their interactions, were studied as functions of the preconditioning of Pavlova lutheri (Droop) Green. Populations were preconditioned in continuous culture at a variety of growth rates and N:P supply ratios. The maximum uptake rates cell^?1 for nitrate and phosphate were of similar magnitudes, in spite of the forty-fold smaller requirement for phosphorus. Short-term phosphate uptake was independent of the nitrate concentration, but the short-term nitrate uptake rate was reduced in the presence of phosphate. The severity of inhibition of nitrate uptake by phosphate was positively correlated with the preconditioning N:P supply ratio and the preconditioning growth rate. In response to large additions of nutrients, P. lutheri was able to increase its phosphorus content sixty-fold, but was only able to take up enough nitrate to double its nitrogen content. The high rate of phosphate uptake relative to its requirement, the inhibition of nitrate uptake by phosphate, and the large capacity for phosphorus storage relative to its requirement, all of which were observed even under N limitation, may imply that even where nitrogen is limiting there can be interspecific competition for available phosphate.