Growth characteristics and growth modeling of <Emphasis Type="Italic">Microcystis aeruginosa</Emphasis> and <Emphasis Type="Italic">Planktothrix agardhii</Emphasis> under iron limitation

Although iron is a key nutrient for algal growth just as are nitrogen and phosphorus in aquatic systems, the effects of iron on algal growth are not well understood. The growth characteristics of two species of cyanobacteria, Microcystis aeruginosa and Planktothrix agardhii, in iron-limited continuous cultures were investigated. The relationships between dissolved iron concentration, cell quota of iron, and population growth rate were determined applying two equations, Monod’s and Droop’s equations. Both species produced hydroxamate-type siderophores, but neither species produced catechol-type siderophores. The cell quota of nitrogen for both M. aeruginosa and P. agardhii decreased with decreasing cell quota of iron. The cell quota of phosphorus for M. aeruginosa decreased with decreasing cell quota of iron, whereas those for P. agardhii did not decrease. Iron uptake rate was measured in ironlimited batch cultures under different degrees of iron starvation. The results of the iron uptake experiments suggest that iron uptake rates are independent of the cell quota of iron for M. aeruginosa and highly dependent on the cell quota for P. agardhii. A kinetic model under iron limitation was developed based on the growth characteristics determined in our study, and this model predicted accurately the algal population growth and iron consumption. The model simulation suggested that M. aeruginosa is a superior competitor under iron limitation. The differences in growth characteristics between the species would be important determinants of the dominance of these algal species.     

Temperature effects on steady-state growth,phosphorus uptake,and the chemical composition of a marine phytoplankter

The marine chrysophyteMonochrysis lutheri was grown in phosphorus-limited continuous cultures at temperatures of 15°, 18.8° and 23°C. The effect of temperature on the maximum growth rate was well-defined by the Arrhenius equation, but the Q₁₀ for this alga (1.7) was somewhat lower than has been determined previously for many other phytoplankton species (2.0–2.2). The minimum phosphorus cell quota was relatively unaffected by temperature at 18.8°C and 23°C, but doubled in magnitude at 15°C. As a result, the internal nutrient equation of Droop described the relationship between specific growth rate and phosphorus cell quota well at 18.8° and 23°C, but was less successful at 15°C. The major limitation in using the Droop equation is that the ratio between the minimum and maximum cell quotas must be known, thus necessitating the need to establish the true maximum growth rate by the cell washout technique. In addition, the phosphorus uptake rate on a cell basis at a given steady state growth rate (=specific uptake rate) increased dramatically at 15°C, whereas the turnover rate of total available phosphorus was unaffected by temperature. Both the nitrogen and carbon cell quotas were relatively unaffected by growth rate at a given temperature, but the average values increased slightly with decreasing temperature. The overall conclusion is that phytoplankton growth and limiting-nutrient uptake rates are only synchronous at or near the optimum temperature. Because these types of responses are species specific, much additional data on temperature effects will be required before the importance of including such effects in phytoplankton-nutrient models can be determined.     

COMPARATIVE KINETIC STUDIES OF NITRATE-LIMITED GROWTH AND NITRATE UPTAKE IN PHYTOPLANKTON IN CONTINUOUS CULTURE1

A comparative study of nitrate-limited growth and nitrate uptake was carried out in chemostat cultures of Ankistrodesmus falcatus (Corda) Ralfs., Asterionella formosa Hass., and Fragilaria crotonensis Kit. In each species growth rate (μ) was related to total cell nitrogen or cell quota (q) by the empirical Droop growth function. Nitrate uptake was a function of both external N concentration and q. The apparent maximum uptake rate (V_m') at a given μ was inversely related to q – q₀, where q₀ is the minimum quota. The apparent half-saturation constant for uptake, (K_m') appears to show a slight inverse trend with μ, although statistical analysis shows that this trend is inconclusive. When q approaches q₀, V_m' is several orders of magnitude greater than μq, the calculated steady-state uptake rate. As q increases, however, the difference between these two variables decreases sharply until q approaches q_m, the cell quota for nitrogen-rich cells. At this point the difference between μq and V_m' disappears. This behavior is explained by the feedback regulation of N uptake. The inverse relationship between V_m' and q – q₀ can be described by an empirical three-parameter equation.     

Nutrition and growth kinetics in nitrogen- and phosphorus-limited cultures of the novel red tide flagellate Chattonella ovata (Raphidophyceae)

In the summer of 2004, a harmful bloom caused by Chattonella ovata (Raphidophyceae) occurred over almost the entire area of the Seto Inland Sea and caused fishery damages. This incident was the first record of a bloom and damage to the fisheries caused by this species in Japanese waters. In order to elucidate the mechanism of the bloom outbreak, we examined the nutrition and the growth kinetics in nitrogen (N)- and phosphorus (P)-limited semi-continuous cultures of this species. Inorganic N compounds, such as nitrate, nitrite, and ammonium, were found to be good nitrogen sources for the growth of C. ovata, while organic nitrogen (urea and uric acid) was not utilized. This species was capable of using ATP, ADP and inorganic phosphorus compounds, but did not utilize phosphate monoesters as a sole P source. Under both N-limited and P-limited steady state conditions, the growth rate as a function of cell nitrogen and phosphorus quota, respectively, followed the Droop equation. Kinetic parameters μ_m (maximum growth rate) and k_q (minimum cell quota) obtained for N- and P-limited cultures were 0.79 day⁻¹ and 5.5 pmol N cell⁻¹ and 0.86 day⁻¹ and 0.48 pmol P cell⁻¹, respectively. The minimum cell quotas were 23–30% lower than those of C. antiqua. The nutrient availability and kinetic parameters of C. ovata are compared with other harmful algae and the ecological implications of these characteristics discussed.     

The ferrous iron oxidation kinetics of Thiobacillus ferrooxidans in batch cultures

The ferrous iron oxidation kinetics of Thiobacillus ferrooxidans in batch cultures was examined, using on-line off-gas analyses to measure the oxygen and carbon dioxide consumption rates continuously. A cell suspension from continuous cultures at steady state was used as the inoculum. It was observed that a dynamic phase occurred in the initial phase of the experiment. In this phase the bacterial ferrous iron oxidation and growth were uncoupled. After about 16 h the bacteria were adapted and achieved a pseudo-steady state, in which the specific growth rate and oxygen consumption rate were coupled and their relationship was described by the Pirt equation. In pseudo-steady state, the growth and oxidation kinetics were accurately described by the rate equation for competitive product inhibition. Bacterial substrate consumption is regarded as the primary process, which is described by the equation for competitive product inhibition. Subsequently the kinetic equation for the specific growth rate, μ, is derived by applying the Pirt equation for bacterial substrate consumption and growth. The maximum specific growth rate, μ _max, measured in the batch culture agrees with the dilution rate at which washout occurs in continuous cultures. The maximum oxygen consumption rate, q _O2,max, of the cell suspension in the batch culture was determined by respiration measurements in a biological oxygen monitor at excess ferrous iron, and showed changes of up to 20% during the course of the experiment. The kinetic constants determined in the batch culture slightly differ from those in continuous cultures, such that, at equal ferric to ferrous iron concentration ratios, biomass-specific rates are up to 1.3 times higher in continuous cultures. Received: 8 February 1999 / Accepted: 17 February 1999     

NUTRIENT-LIMITED GROWTH OF AUREOUMBRA LAGUNENSIS (PELAGOPHYCEAE), WITH IMPLICATIONS FOR ITS CAPABILITY TO OUTGROW OTHER PHYTOPLANKTON SPECIES IN PHOSPHATE-LIMITED ENVIRONMENTS

Growth and chemical compositional characteristics of the brown tide-forming alga, Aureoumbra lagunensis Stockwell, DeYoe, Hargraves et Johnson, were studied through a series of nitrogen-limited and phosphate-limited continuous cultures over a range of growth rates. The specific growth rate of A. lagunensis was hyperbolically related to the cell quota of the limiting nutrient in ammonium-limited cultures. In phosphate-limited cultures, the relationship was best described by a straight line. The N cell quota of A. lagunensis ranges from about 20 fmol at zero growth rate under N-limited conditions to a high of roughly 85 fmol under N-replete conditions. Similarly, the P cell quota of A. lagunensis ranges from about 0.15 fmol at zero growth rate under P-limited conditions to a high of 2 fmol under P-replete conditions. Aureoumbra lagunensis has a very high N:P critical ratio (>100). The high N:P critical ratio, as well as the organism's apparent ability to use forms of phosphorus other than phosphate under severe phosphate deficiency, may partially explain its success in P-limited environments, such as the Laguna Madre. In addition, a uniqe quadratic relationship between the productivity index (PI) and growth rate was discovered. Such a relationship supports an earlier argument that PI may not be a good indicator of nutritional status.     

SIZE-DEPENDENT PHOSPHORUS UPTAKE KINETICS AND CELL QUOTA IN PHYTOPLANKTON1

The allometric equation, y = aX^b, described the interspecific variation of phosphate uptake kinetics and cell quota with phytoplankton cell size and showed that smaller cells are superior in uptake rate to large. Species-specific measurements, made by track autoradiography in phosphorus deficient cultures of communities from a phosphorus-limited lake, revealed that eight different species did not differ significantly in the Michaelis-Menten half-saturation constant, K_m. However, both saturated uptake rates (V_max) and the initial slope of the uptake curve (V_max:K_m) decreased per unit biomass with increasing cell size. Biomass-specific cell phosphorus quotas also decreased with increasing cell volume, but less rapidly than did V_max or V_max: K_m. Comparable data from the literature showed that marine species were superior in phosphorus uptake to freshwater species of similar size, but allometric variation of kinetics appeared to exist within both groups. Together with a variable internal stores model of phosphorus-limited growth, the allometric relationships of uptake kinetics and quotas predicted competition to favor smaller cells, with a differential in growth rate diminishing as competitive intensity increased.     

Nutrient Requirement and Growth of a Synechococcus Species Isolated from Lake Balaton

A pico sized Synechococcus species isolated from Lake Balaton was studied in batch and continuous cultures. This picocyanobacterium had a pH optimum at 8.5 and a temperature optimum at 28-30°C. The I_k value for growth was 52 μEinstein m⁻² S⁻¹, the maximum growth rate 2.27 d⁻¹, the half saturation Constant of growth 1.2 μg PO₄-P I⁻¹ and the minimal cell quota 1.74 nig P g dry weight⁻¹. The dry weight of cells showed a minimum, the chlorophyll-a/biomass ratio a maximum as a function of growth rate. Above the quota of 3.4 fg P Cell⁻¹ significant amounts of non-reactive dissolved Phosphorous were released.     

GROWTH RATE VARIATION IN THE N:P REQUIREMENT RATIO OF PHYTOPLANKTON1

Theoretical considerations predict that the cell N:P ratio at transition from nitrogen limitation to phosphorus limitation of phytoplankton growth (critical ratio, R_c) varies, as a function of population growth rate. This prediction is confirmed by experimental, data from the literature along with new experimental data for the marine, prymnesiophyte Pavlova lutheri (Droop) Green. R_c passes through a maximum at intermediate growth rates for the three phytoplankton species for which data, are available, but there is significant interspecific variability in its value. There is no theoretical or experimental evidence to support the idea that the ratio of subsistence N and P cell quotas is equal to R_c over the range of growth rates, or that the subsistence quota ratio equals the ratio of the N and P cell quotas minus a storage fraction. Examination of N:P composition ratios can be used to determine which nutrient is limiting, but cannot be used to determine relative growth rates or competitive advantage between species limited by the same nutrient. Growth rates are determined by environmental conditions and by the cell quota of the limiting nutrient, without reference to the cell quota of the non-limiting nutrient.     

Towards a more biologically realistic use of Droop's equations to model growth under multiple nutrient limitation

Droop's model was originally designed to describe the growth of unicellular phytoplankton species in chemostats but it is now commonly used for a variety of organisms in models of trophic interactions, ecosystem functioning, and evolution. Despite its ubiquitous use, Droop's model is still limited by several simplifying assumptions. For example, the assumption of equal theoretical maximum growth rates for all nutrients is commonly used to describe growth limited by multiple nutrients. This assumption, however, is both biologically unrealistic and potentially misleading. We propose the alternative hypothesis of equal realized maximum growth rates for all nutrients. We support our hypothesis with empirical and theoretical arguments and discuss how it may improve our understanding of the biology of growth, while avoiding some of the pitfalls of the previous assumption.     

NEUTRAL LIPID AND CARBOHYDRATE PRODUCTIVITIES AS A RESPONSE TO NITROGEN STATUS IN ISOCHRYSIS SP. (T‐ISO; HAPTOPHYCEAE): STARVATION VERSUS LIMITATION1

Partitioning of the carbon (C) fixed during photosynthesis between neutral lipids (NL) and carbohydrates was investigated in Isochrysis sp. (Haptophyceae) in relation to its nitrogen (N) status. Using batch and nitrate‐limited continuous cultures, we studied the response of these energy reserve pools to both conditions of N starvation and limitation. During N starvation, NL and carbohydrate quotas increased but their specific growth rates (specific rates of variation, μ_CAR and μ_NL) decreased. When cells were successively deprived and then resupplied with NO₃, both carbohydrates and neutral lipids were inversely related to the N quota (N:C). These negative relationships were not identical during N impoverishment and replenishment, indicating a hysteresis phenomenon between N and C reserve mobilizations. Cells acclimated to increasing degrees of N limitation in steady‐state chemostat cultures showed decreasing NL quota and increasing carbohydrate quota. N starvation led to a visible but only transient increase of NL productivity. In continuous cultures, the highest NL productivity was obtained for the highest experimented dilution rate (D = 1.0 d^?1; i.e., for non N‐limited growth conditions), whereas the highest carbohydrate productivity was obtained at D = 0.67 d^?1. We used these results to discuss the nitrogen conditions that optimize NL productivities in the context of biofuel production.     

RESPONSES OF FRESHWATER ALGAE TO INHIBITORY VANADIUM CONCENTRATIONS: THE ROLE OF PHOSPHORUS1

We found that species-specific differences exist among a variety of freshwater algae and cyanobacteria in the extent to which growth and photosynthesis are inhibited by vanadium. A major factor controlling the degree of inhibition by vanadium was the phosphorus state (P-sufficient vs. P-deficient) of the organisms. In P-sufficient cultures, vanadium was inhibitory when the vanadium concentration exceeded the phosphate concentration. In P-deficient cultures, the depression of photosynthesis by vanadium increased with increasing phosphorus deficiency. Our conclusion that vanadium competed with phosphate for uptake sites was supported by the following three observations: 1) the decreased influx of ³²P-PO ₄ into P-deficient cells in the presence of vanadium, 2) the amelioration of vanadium inhibition of photosynthesis by the addition of phosphate, and 3) the accumulation of vanadium by cells. At vanadium concentrations that severely inhibited growth, the cells of Scenedesmus obliquus (Turp.) Kruger were larger than normal and contained more vacuoles, lipid, and starch bodies than normal cells. Four-celled coenobia were replaced by unicells. Scenedesmus acutusf: alternans Hortobagyi cells from vanadium-inhibited cultures had 7.5 times more vanadium per cell than control cultures and contained numerous granules that did not stain for polyphosphate and may be composed of condensed vanadate molecules. The cellular P quota and turnover time of PO₄in the medium are important regulators of the extent of inhibition by vanadium.     

STEADY-STATE LUXURY CONSUMPTION AND THE CONCEPT OF OPTIMUM NUTRIENT RATIOS: A STUDY WITH PHOSPHATE AND NITRATE LIMITED SELENASTRUM MINUTUM (CHLOROPHYTA)1

Selenastrum minutum (Naeg.) Collins was grown over a wide range of growth rates under phosphate or nitrate limitation with non-limiting nutrients added to great excess. This resulted in saturated luxury consumption. The relationships between growth rate and cell quota for the limiting nutrients were well described by the Droop relationship. The observed variability in N cell quota under N limitation as reflected in k_Q·Q_max^?1*, was similar in magnitude to previously reported values but k_Q·Q_max^?1* for P under P limitation was greater than previously reported for other species. These results were evaluated in light of the optimum ratio hypothesis. Our findings support previous work suggesting that the use of a single optimum ratio (k_Qi·K_Qj^?1) is inappropriate for dealing with a species growing under steady-state nutrient limitation. Under these conditions the optimum ratio should be viewed as a growth rate dependent variable. Two approaches for testing the growth rate dependency of optimum ratios are proposed. The capacity for luxury consumption differed between nutrients and was growth rate dependent. At low growth rates, the coefficient of luxury consumption (R_sat) for P was ca. four times that for N. The set of all possible relationships between N and P cell quota under these conditions was reported and these values were then used to establish the cellular N:P niche boundaries for S. minutum. Cell quotas of non-limiting nutrients were not described by the Droop equation. Analysis showed that as the cellular N:P ratio deviates from the optimum ratio, the ability of the Droop equation to describe the relationship between growth rate and non-limiting cell quotas decreases. When non-limiting nutrient cell quotas are saturated, the Droop equation appears to be invalid. Previously reported patterns of non-limiting nutrient utilization are summarized in support of this conclusion. The physiological and ecological consequences of luxury consumption and growth rate dependent optimum ratios are considered.     

PHOSPHATE UPTAKE AND GROWTH KINETICS OF TRICHODESMIUM (CYANOBACTERIA) ISOLATES FROM THE NORTH ATLANTIC OCEAN AND THE GREAT BARRIER REEF,AUSTRALIA1

We compared inorganic phosphate (P_i) uptake and growth kinetics of two cultures of the diazotrophic cyanobacterium Trichodesmium isolated from the North Atlantic Ocean (IMS101) and from the Great Barrier Reef, Australia (GBRTRLI101). Phosphate‐limited cultures had up to six times higher maximum P_i uptake rates than P‐replete cultures in both strains. For strain GBRTRLI101, cell‐specific P_i uptake rates were nearly twice as high, due to larger cell size, but P‐specific maximum uptake rates were similar for both isolates. Half saturation constants were 0.4 and 0.6 μM for P_i uptake and 0.1 and 0.2 μM for growth in IMS101 and GBRTRLI101, respectively. Phosphate uptake in both strains was correlated to growth rates rather than to light or temperature. The cellular phosphorus quota for both strains increased with increasing P_i up to 1.0 μM. The C:P ratios were 340–390 and N:P ratios were 40–45 for both strains under severely P‐limited growth conditions, similar to reported values for natural populations from the tropical Atlantic and Pacific Oceans. The C:P and N:P ratios were near Redfield values in medium with >1.0 μM P_i. The North Atlantic strain IMS101 is better adapted to growing on P_i at low concentrations than is GBRTRLI101 from the more P_i‐enriched Great Barrier Reef. However, neither strain can achieve appreciable growth at the very low (nanomolar) P_i concentrations found in most oligotrophic regimes. Phosphate could be an important source of phosphorus for Trichodesmium on the Great Barrier Reef, but populations growing in the oligotrophic open ocean must rely primarily on dissolved organic phosphorus sources.     

Modeling continuous cultures of microalgae colimited by nitrogen and phosphorus

It is well documented that the combination of low nitrogen and phosphorus resources can lead to situations where colimitation of phytoplankton growth arises, yet the underlying mechanisms are not fully understood. Here, we propose a Droop-based model built on the idea that colimitation by nitrogen and phosphorus arises from the uptake of nitrogen. Indeed, since N-porters are active systems, they require energy that could be related to the phosphorus status of the cell. Therefore, we assumed that N uptake is enhanced by the P quota. Our model also accounts for the biological observations that uptake of a nutrient can be down-regulated by its own internal quota, and succeeds in describing the strong contrast for the non-limiting quotas under N-limited and P-limited conditions that was observed on continuous cultures with Selenastrum minutum and with Isochrysis affinis galbana. Our analysis suggests that, regarding the colimitation concept, N and P would be better considered as biochemically dependent rather than biochemically independent nutrients.     

Light/Dark Cycle and Temperature-Their Impact on Phosphate-Limited Growth of Oscillatoria redekei VAN GOOR in Semicontinuous Culture

Phosphate-limited growth of Oscillatoria redekei in semicontinuous culture has been studied under conditions of continuous illumination at 20 °C as well as in a 12/12 hours light-dark cycle at temperatures between 5 °C and 20 °C. The subsistence quota (q₀) amounted to 0.052 μmol mm⁻³ under all conditions, when the phosphate quota was expressed on the basis of cell volume. The interaction between temperature and phosphate quota and its impact on growth rate are described by the following model: Parameter values are t_opt=24.5 °C, t_min=0.95 °C, μ_max =0.873 d⁻¹. The maximum phosphate quota was found to depend on temperature and to increase along with declining temperature.     

The substrate constant for the ammonium ion of growing

The relation between ammonium concentration and growth rate was studied in steady state continuous cultures of Saccharomyces cerevisiae in nitrogenlimited glucose ammonium medium. This relation could be described by the Monod equation. A maximum specific growth rate of 0.41 h^-1 and a substrate constant for ammonium of 5–11 M were calculated. Ammonium was determined by a modification of the phenol hypochlorite method. A discussion of the results in view of literature data on the substrate constants for other nutrients is given.     

The unsteady continuous culture of phosphate-limited Monochrysis lutheri droop: Experimental and theoretical analysis

A three-state variable model for phosphate-limited phytoplankton growth in a continuously lit continuous culture is proposed. In the model, the phosphate uptake rate per cell is a Michaelis-Mententype hyperbolic function of ambient nutrient concentration and the growth rate is a Droop-type hyperbolic function of cell quota. Steady-state and short-term uptake experiments with unialgal cultures of Monochrysis lutheri Droop, a marine chrysophyte, were used to calibrate the proposed model. For the long-term unsteady experiments, the model predicts well the culture's dynamic response in terms of cell density to steps down and up in influent concentration of limiting nutrient. For step changes in dilution rate, the model predicts well the culture's response to a step down but predicts poorly the culture's response to a step up. The long-term responses of the cultures to impulses in influent concentration show that the model fails to predict, even qualitatively, the behavior of the phytoplankton. Not unexpectedly, the model fails most dramatically in those experiments involving a rapid increase in cell quota, thereby demonstrating both the inherent flaws in the concept of the instantaneous growth rate as a function of instantaneous cell quota and the need for further dynamic characterization of phytoplankton behavior.     

Alexandrium ostenfeldii growth and spirolide production in batch culture and photobioreactor

Growth and spirolide production of the toxic dinoflagellate Alexandrium ostenfeldii (Danish strain CCMP1773) were studied in batch culture and a photobioreactor (continuous cultures). First, batch cultures were grown in 450 mL flasks without aeration and under varying conditions of temperature (16 and 22 °C) and culture medium (L1, f/2 and L1 with addition of soil extract). Second, cultures were grown at 16 °C in 8 L aerated flat-bottomed vessels using L1 with soil extract as culture medium. Finally, continuous cultures in a photobioreactor were conducted at 18 °C in L1 with soil extract; pH was maintained at 8.5 and continuous stirring was applied.This study showed that A. ostenfeldii growth was significantly affected by temperature. At the end of the exponential phase, maximum cell concentration and cell diameter were significantly higher at 16 °C than at 22 °C. In batch culture, maximum spirolide quota per cell (approx. 5 pg SPX 13-desMeC eq cell⁻¹) was detected during lag phase for all conditions used. Spirolide quota per cell was negatively and significantly correlated to cell concentration according to the following equation: y = 4013.9x^−0.858. Temperature and culture medium affected the spirolide profile which was characterized by the dominance of 13,19-didesMeC (29–46%), followed by SPX-D (21–28%), 13-desMeC (21–23%), and 13-desMeD (17–21%).Stable growth of A. ostenfeldii was maintained in a photobioreactor over two months, with maximum cell concentration of 7 × 10⁴ cells mL⁻¹. As in batch culture, maximum spirolide cell quota was found in lag phase and then decreased significantly throughout the exponential phase. Spirolide cell quota was negatively and significantly correlated to cell concentration according to the equation: y = 12,858x^−0.8986. In photobioreactor, spirolide profile was characterized by higher proportion of 13,19-didesMeC (60–87%) and lower proportions of SPX-D (3–12%) and 13-desMeD (1.6–10%) as compared to batch culture.