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Effects of Nutrients on Growth and Nodularin Production of. Nodularia Strain GR8b. S. Repka, J. Mehtonen, J. Vaitomaa, L. Saari, K. Sivonen. Department of ...
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ECOLOGY Microb Ecol (2001) 42:606–613 DOI: 10.1007/s00248-001-0026-8 © 2001 Springer-Verlag New York Inc.

Effects of Nutrients on Growth and Nodularin Production of Nodularia Strain GR8b S. Repka, J. Mehtonen, J. Vaitomaa, L. Saari, K. Sivonen Department of Applied Chemistry and Microbiology, Biocenter Viikki, P.O. Box 56, 00014 University of Helsinki, Finland Received: 5 February 2001; Accepted: 6 June 2001; Online Publication: 22 October 2001

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To determine the effects of nutrients on growth and toxin production of Nodularia strain GR8b, several nutrient concentrations were tested in batch and chemostat cultures. In batch cultures, phosphate (55–5,500 µg L−1) and nitrate (100–30,000 µg L−1) concentrations were applied, whereas in chemostat cultures, phosphate concentrations (5–315 µg L−1) were tested. Intra- and extracellular toxin concentrations, together with biomass parameters, were measured. In the batch cultures with low phosphate concentrations, chlorophyll a and protein contents were reduced, but dry weights and cell numbers were not significantly affected. The highest nitrate concentrations resulted in reduced dry weight concentrations. Nodularin concentration per dry weight, nodularin to protein ratio, and dissolved nodularin were highest at the end of the experiment, but were not influenced by the nutrient concentrations. Nodularin concentration per cell was also rather constant under the varying nutrient concentrations. In the chemostat cultures, the biomass increased with high phosphate concentrations. However, the phosphate concentrations did not have statistically significant effects on nodularin production rates.

Introduction Cyanobacterial blooms are an annual phenomenon in the Baltic Sea. Their magnitudes vary according to the environmental conditions [1, 10, 16, 17, 20]. Toxic, N2-fixing cyanobacteria, Nodularia spp., are common in the open sea [18, 35, 36]. Nodularia spp. produce a hepatotoxin, nodularin [31, 35], that poses risks to the ecosystem and humans. Nodularin is most probably produced non-ribosomally by

Correspondence to: K. Sivonen; Fax: +358-9-19159322; E-mail: [email protected]

peptide synthethases [14, 27]. The toxic effects of nodularin and the chemically closely related microcystins are due to their protein phosphatase inhibition activity [12]. Quantification of Nodularia growth rates and nodularin production rates under variable growth conditions, especially in relation to nutrients, is needed when modeling the dynamics of cyanobacterial mass occurrences in the Baltic Sea [7]. The responses of nodularin production to nutrients and other environmental growth stimuli can also help us to understand the reasons behind its production. So far, the results on the influence of nutrients on nodularin production of Nodularia spp. have been contradictory. In some

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studies, more nodularin is produced in conditions promoting growth [21, 22]. Contrary to this, phosphate limitation has also been observed to increase toxin production in Nodularia [8]. The results may depend on the nutrient levels and other environmental conditions during the experiments, as well as on strain-specific differences [22]. In addition, the conclusions may depend on whether toxin concentrations are reported in terms of gravimetric concentrations, nodularin cell quota, or nodularin-to-protein ratios. Most studies have been performed in batch cultures, and the growth phases of the cells may have also affected the results. In batch cultures, the use of unnaturally high starting concentrations of nutrients is usually necessary. So far, there have not been studies on nodularin production in chemostat cultures. In this study, we cultured Nodularia strain GR8b in batch and chemostat cultures to ascertain the effects of phosphate and nitrate on growth and toxin production.

Methods Organism Nodularia strain GR8b was isolated from the Gulf of Finland [21, 22]. Because the taxonomy has not been settled [23] we do not use a species name. The morphology of Nodularia strain GR8b fits the species description of N. spumigena and N. littorae [15, 23]. Molecular evidence on the taxonomy of Nodularia, however, indicates that in Baltic Sea only one toxic Nodularia species exists [23, 24]. Our strain belongs to this species that probably could be called N. spumigena. An axenic strain was obtained by the soft agaroseplating method [33]. The strain has been maintained in the laboratory for c. 10 years with modified (without nitrogen and salt added) Z8 culture media [36].

Experiments The batch culture experiment was performed at 22° C and continuous light of 25 µE m−2 s−1 in 250-mL Erlenmeyer flasks with 100 mL of liquid medium. As the mineral culture medium, Z8 without N and P with salt concentration 2.5 ‰ was used. Ten mL of inoculum phosphorus-starved for 6 days was added to the Erlenmeyer flasks. Before starting the experiments, the absence of accompanying bacteria in the inoculum was microscopically verified after DAPI staining. During the experiment, all the cultures were tested for axenicity with TGY (tryptone–glucose–yeast extract) plates, which were incubated at room temperature for 5 days. The nutrient concentrations of the 10 treatments (Fig. 1) were determined by the Central Composite Design [25]. Growth was monitored by turbidity and dry weight on days 1, 2, 3, 4, 5, 8, 10, and 16. On sampling days 6, 13, 20, and 25 the following parameters were determined for the respective treatments (Fig. 1): dry weight, turbidity, Chl a, protein, dissolved protein, dissolved ni-

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Fig. 1. Experimental design of the batch culture experiments. Nine combinations of phosphate and nitrate concentrations are marked by •. There were 4 replicates (treatments 10–13) in the middle point. The phosphate and nitrate concentrations were determined by Central Composite Design (see Methods). Samples of treatments 1–4 were taken on days 6 and 20. On day 13, treatments 5–13 were sampled. On day 25, treatment 5 was sampled.

trate, dissolved phosphate, phosphatase enzyme activity (APA), cell numbers, and nodularin concentrations. Nodularia strain GR8b was also cultured in chemostats with a working volume of 2 L at a constant dilution rate of 0.15 d−1 at 21° C and a continuous illumination of 20 µE m−2 s−1. The mineral culture medium was the same as in the batch cultures. Because the results from the batch culture experiment indicated that nitrate had only marginal effects on growth and nodularin production, we decided to do the chemostat experiment without inorganic nitrogen source. Phosphate levels of 5, 10, 60, 100, 200, and 315 µg L−1 were used. A phosphate level of 100 µg P L−1 was replicated five times. When there was less than a 10% change in turbidity between three successive days, the cyanobacteria were considered to be in a steady state. From steady-state cultures, aliquots were used to measure biomass (optical density, dry weight, chlorophyll a, and protein) phosphatase enzyme activity (APA), and nodularin concentrations.

Analyses For dry weight measurements, 50–100 ml of culture was filtered on tared G52 (Scheichler & Schuell) filters. Subsequently, the filters were dried in an oven (105° C) for more than 2 hr and weighed again. Turbidity was measured at 750 nm with a spectrophotometer (Perkin Elmer). Chlorophyll a was measured according to [37]. Total protein content and dissolved proteins were determined at 500 nm by the method of Lowry using bovine serum albumin as a standard [11]. Nitrate was analyzed with a Lachat autosampler after

608 hydrazine reduction [13]. Soluble reactive phosphate was analyzed using the molybdate–ascorbic acid method [26]. Phosphatase exoenzyme activities were measured using 2 mmol L−1 fluorescent substrate and 4-methylumbelliferyl phosphate (MUF) according to [40]. Cell densities were enumerated from samples preserved with Lugol’s iodine solution. The cells were counted with a hemocytometer (Bu¨ rker, Marienfeld, Germany) and a light microscope at a magnification of 400× (Olympus BH-2). A minimum of 800 cells or 30 replicates were counted. Total nodularin was extracted from 50 mL of liquid culture by sonicating for 15 min (Braun Labsonic-U). The remaining cell material was removed by filtering with G52 filters (Schleicher & Schuell). Dissolved nodularin was determined from 400-mL samples after removing the cells by filtering with G52 filters. From these samples, nodularin was concentrated on C18 cartridges according to the manufacturer’s instructions (Oasis, Waters). The samples were subsequently dried in air stream and dissolved in 0.5 ml of 20% methanol. Nodularin was analyzed with a HewlettPackard HP1090 liquid chromatograph equipped with a HewlettPackard UV/VIS diode array detector and Hewlett-Packard ODS Hypersil column (100 mm × 4.6 mm). The mobile phase was a 77:23 (vol:vol) mixture of 10 mM ammonium acetate and acetonitrile. The flow rate was 1 ml min−1, injection volume 25 µl, and detection at 238 nm. Nodularin was identified by its retention time and UV spectrum. Purified nodularin was used as a standard.

Statistical Analyses For batch culture experiments, multivariate regression analyses, after linear transformation of the design matrices, were performed with MATLAB statistical software for Windows (MathWorks, Inc.; Natick, MA). Composite design matrices were created with prior transformation of the original variables into coded ones. The matrices were expanded to include the coeffects and quadratic (second power) effects of experimental factors. Multivariate regression models describing the variation of the measured parameters were obtained by omitting insignificant (p > 0.05) factors. The data from chemostat cultures were analyzed with regression analysis after logarithmic transformation.

Results Batch Cultures Specific growth rates of Nodularia strain GR8b varied from 0.6 to 1.3 d−1 (Fig. 2). Dry weights and cell numbers increased with culture age, but decreased with nitrate concentrations (Table 1, Fig. 3A for dry weight). The interaction of nitrate and phosphate was also negative (Table 1). Optical density, chlorophyll a, and protein contents of Nodularia strain GR8b were highest at the end of the experiment under high phosphate concentrations whereas the highest nitrate concentrations reduced them (Table 1, Fig. 2 for OD, Fig. 3B

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Fig. 2. Growth of Nodularia strain GR8b at varying phosphate and nitrate concentrations in batch cultures. The nutrient concentrations were determined by central composite design (Fig. 1). In the legend, P denotes mg L−1 phosphate and N mg L−1 nitrate.

for Chl a). Nodularia strain GR8b took up large amounts of phosphate and by day 13, phosphate from treatments 5 and 6 had been depleted (Table 2). By day 20, phosphate levels were close to the detection limit (Table 2). Nodularia strain GR8b also took up large amounts of nitrate, and nitrate was also depleted in treatments 2 and 8 (Table 2). The APA of Nodularia ranged from 0.03 to 0.27 nmol h−1 /µg Chl a. The APA was increased under low phosphate concentrations, the quadratic effect of phosphate (P2) was positive, and phosphate and culture age had also a negative coeffect on APA (Table 1, Fig. 3C). The Nodularia strain GR8b produced nodularin under all growth conditions. Total nodularin concentration (nodtot) was highest at the end of the experiment under low nitrogen levels, but phosphate had no effect on total nodularin content (Table 1, Fig. 3D). Nutrient concentrations had no effect on nodularin cell quota (nodularin concentration per cell) (Table 1). Nodularin cell quota varied from 1.0 × 10−4 to 4.4 × 10−4 ng cell−1. Gravimetric nodularin concentration (nodularin concentration per dry weight) as well as nodularin to protein ratio were highest at the end of the experiment, but the nutrient concentrations had no effects (Table 1). The amount of dissolved nodularin was also highest at the end of the experiment and not affected by the nutrient concentrations (Table 1). However, a higher percentage of the total nodularin pool was dissolved at the beginning of the experiment; at day 6, 50% of nodularin was dissolved and at day 25, only 25%. Nodularin production rates calculated until day 13 varied from 0.6 to 0.8 d−1. Nodularin production rates were correlated with the cell division rates (r = 0.76, p = 0.02).

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Table 1. Results of multiple regression analysis of Nodularia strain GR8b grown in batch cultures.

Optical density Dry weight (mg L−1) Cells (number L−1) Chl a (µg L−1) Protein (µg L−1) APA (nmol h−1/µg Chl a) Total nodularin (µg L−1) Nodularin cell quota (ng cell−1) Gravimetric nodularin (µg mg−1 DW) Nodularin to protein ratio (µg µg−1 prot) Dissolved nodularin (µg L−1)

t

P

N

t2

tP

tN

P2

PN

N2

30.1*** 16.1*** 6.3** 30.1*** 30.1*** 1.3 24.7*** −0.09 3.5* 2.8* 7.7**

10.2** 0.2 −0.08 10.2*** 3.6* −24.1*** 0.8 −0.5 −0.8 −1.4 2.2

−6.2** −3.1* −1.8 −6.2** −8.8** 1.5 −4.3* 0.8 −0.4 0.4 −0.8

3.4* 8.8** −0.6 3.4* 11.5** 4.8** 3.0* −3.9* −4.9** −4.5* 2.6*

7.8** −1.4 −0.08 7.8** 1.4 −14.3*** 0.3 −0.5 −0.006 −0.6 0.7

−4.2* −1.9 0.3 −4.2* −4.2* 1.7 −2.0 −0.7 0.2 0.4 −0.7

−4.8** 2.2 0.4 −4.8** 0.6 17.9*** −0.4 −1.1 −1.5 −0.7 −0.3

−4.7** −2.2* −2.2 −4.7** −7.4* 1.1 −4.1* 1.2 −0.8 0.08 −0.2

−4.1* −2.5* −1.6 −4.1* −2.6 −0.4 −2.7* 0.4 −0.7 −1.1 −2.3

* p ⱕ 0.05; **p ⱕ 0.01; ***p ⱕ 0.0001. The independent variables were culture age (t), phosphate concentration (P) and nitrate concentration (N). In addition, their coeffects: tP, tN, PN and quadratic effects: t2, P2 and N2 were included in the model. T-values for multiple regression coefficients and their significances are given.

Fig. 3. Contour plots describing total nodularin concentration (µg L−1), dry weights (mg L−1), chl a concentrations (µg L−1), and phosphatase enzyme activities (nmol h−1/µg Chl a) of Nodularia strain GR8b in relation to nutrient concentrations (nitrate or phosphate) and culture age. In the formula, t denotes culture age, P phosphate, and N nitrate.

Chemostat Cultures In the chemostat cultures, growth measured as dry weight significantly increased with increasing phosphate concentration (regression coefficient t = 0.22, p = 0.003). This trend is obvious between phosphate concentrations 5 to 200 µg P L−1, but at the highest phosphate concentration (315 µg P L−1) the cells may have been light-limited (Fig. 4). However, the residual phosphate concentration was 2 µg

P L−1, close to detection level, indicating that almost all the phosphate was consumed at 315 µg P L−1. APA declined with increasing phosphate concentration (t = −2.5, p = 0.03), also indicating increasing phosphate limitation with decreasing phosphate concentrations. APA in chemostat cultures varied from 0.03 to 0.49 nmol h−1/µg Chl a and was thus higher than in batch cultures (Fig. 2). The Chl a concentrations (t = 0.39, p = 0.003), cell numbers (t = 0.37, p = 0.002, Fig. 4), and protein concentrations

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Table 2. Phosphate and nitrate concentrations (µg L−1) in batch-culture experiments at the beginning of the experiment (0) and on sampling days 6, 13, 20, and 25a Phosphate Treatment Day 0 6 13 20 25

1 110 40

2 550 401

3 110 7

4 550 349

2

2

1

9

Day 0 6 13 20 25

1 100 9

2 100 14

3 10000 2712

4 10000 2688

58

0

2894

2703

5 275

6 55

7 5500

8 275

9 275

0

0

3691

23

18

5 1000

6 1000

7 1000

8 50

9 30000

85

40

128

0

20934

6 Nitrate Treatment

2

a

The days of sampling and original phosphate and nitrate concentrations of the nine treatments were determined by using the central composite design (Fig. 1).

Fig. 4. Growth (dry weight and cell numbers) and nodularin concentration (gravimetric and nodularin cell quota) of Nodularia strain GR8b in chemostat cultures at 6 phosphate concentrations. There were 5 replicates at 100 µg P L−1.

(t = 0.34, p = 0.001) were higher at higher phosphate concentrations. Total nodularin production rates were significantly higher under higher phosphate concentrations (t = 0.4, p = 0.0009, Fig. 4). The gravimetric nodularin production rates were also higher at higher phosphate concentrations, but the differences were only marginally significant (t = 0.18, p = 0.1, Fig. 4). Nodularin to protein ratio (t = 0.06, p = 0.4) and nodularin cell quota (t = 0.02, p = 0.7, Fig. 4) were not influenced by the phosphate concentrations. Nodularin cell quotas between batch cultures (mean ± 95% conf. interval, 5.0 × 10−4 ± 7 × 10−5 ng cell−1) and chemostat cultures (4.3 × 10−4 ± 6 × 10−5 ng cell−1) did not differ (i.e., the confi-

dence intervals are overlapping). The percentages of dissolved nodularin in chemostat cultures varied from 28% to 63%, but they were not influenced by phosphate concentrations (t=0.001, p=0.97).

Discussion The specific growth rates of Nodularia strain GR8b from the Baltic Sea (0.6 to 1.3 d−1) correspond well to the growth rates of 1.2 d−1 that have been calculated in the field for Baltic Sea Nodularia sp. [19]. In batch cultures, only the lowest phosphate concentration limited growth measured as

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dry weight or cell numbers. The P-starvation period of 6 days was probably too short. A starvation period of 7 days to deplete the cellular phosphate reserves of Nodularia sp. and Aphanizomenon sp had been previously successfully used [22]. In this study, however, the phosphate at the lowest concentration was consumed by day 13 to below the detection level, but Nodularia strain GR8b still continued to grow for 12 days, either on cellular phosphorus storage or recycling phosphorus from decaying cells by phosphatase enzyme. Phosphatase enzyme activities (APA) were clearly elevated in cells under the lowest phosphate concentrations indicating phosphate limitation. The Chl a and protein contents were also reduced under low phosphate concentrations, which suggests that phosphate was limiting the synthesis of these cellular components more than dry weight or cell numbers. Both Chl a and protein content of cyanobacteria are usually reduced under nutrient limitation [9]. In batch cultures, the total nodularin concentration increased toward the end of the experiment. The dissolved nodularin concentration also increased with time, due to lysing cells and possible transport of nodularin to the culture medium [32]. Earlier, it had been observed that the quantity of dissolved nodularin is highest in Nodularia sp. cultures in stationary phase [21, 22]. Because the cultures were axenic, the degradation of nodularin was probably of minor importance [39]. In batch cultures, the release of nodularin by the starved mother culture explains the highest observed percentage of dissolved nodularin at the beginning of the experiment. The specific nodularin concentrations were not influenced by nutrient concentrations. The highest gravimetric nodularin concentrations and nodularin-to-protein ratios were observed at the end of the experiment. The nodularin production rates of Nodularia strain GR8b were higher in cultures where also growth rates were higher. Similarly, a positive correlation has been found for growth rates and microcystin production rates of Microcystis [29]. This may indicate that peptide toxin production is coupled to cell division and before cell division more toxin is produced to supply both daughter cells with toxin. In chemostat cultures it is possible to apply stringent phosphate limitation on cells at close to natural phosphate concentrations. Phosphate limitation decreases and light limitation increases with the phosphate concentration in the medium. In this study, decreasing phosphate limitation was evident from increasing biomass production rates and decreasing phosphatase enzyme activities with increasing phosphate concentration. Thus, it is possible to compare the toxin production of cells in different physiological states.

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The general conclusions on toxin production of Nodularia strain GR8b were the same in chemostat cultures and batch cultures. Gravimetric nodularin concentrations, nodularinto-protein ratios, or nodularin cell quotas did not differ between phosphate concentrations. There are no previous chemostat culture experiments on Nodularia, but the concentration of the closely related toxin microcystin has been observed to increase under phosphate limitation in chemostats [28]. The concentrations of dissolved nodularin were high in chemostat cultures. It is generally believed that cyanobacterial hepatotoxins are not actively transported outside cells, but are released mainly after lysis of the cells [2]. However, it has recently been observed that microcystins may be actively transported outside the Microcystis cells [32], and this may also be true for Nodularia. In this study, biomass measured as dry weight or cell numbers responded differently to nutrient limitation than biomass measured as Chl a or protein. Under limiting nutrient concentration, cell biomass and carbohydrate and protein concentration may change [5, 6] and thus gravimetric toxin concentration or toxin to protein ratio may not be good measures of toxin production. In earlier studies, the gravimetric nodularin concentration was reduced at low phosphate levels [21, 22]. In this study, the gravimetric nodularin concentration was also reduced at low phosphate levels, but the decrease was not statistically significant. The average nodularin cell quotas from batch and chemostat cultured cells were not different. Because the growth conditions in batch cultures differ from those in chemostat cultures, it can be concluded that growth conditions had only marginal effects on nodularin cell quota. This study provides information on growth rates and toxin production rates of Nodularia. These are needed when modeling Nodularia bloom dynamics in the Baltic Sea. In addition, responses of nodularin production to nutrients can shed light on the significance of nodularin to the cell. Because nodularin cell quota remained stable even under phosphate-limited concentrations, nodularin and other bioactive peptides produced by Nodularia [4, 30] probably are important for the cell. For the biosynthesis of peptide toxins such as nodularin, nitrogen is needed, and establishing and maintaining heterocysts and nitrogen fixation is costly for the cyanobacterium [38]. However, the viability of nonmicrocystin-producing M. aeruginosa mutant [3] and good growth performance of non-anabaenopeptilide-producing Anabaena sp. mutant [34] demonstrate that the specific compound was not essential for the cell in the laboratory environment. Whether this is true for the chemically closely

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related nodularin needs to be studied by disrupting the nodularin synthesis gene from Nodularia. The peptides and other bioactive compounds are probably beneficial to the cyanobacteria in their natural environment (e.g., chemical communication) and studies with a more diverse plankton community are needed to verify this.

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Acknowledgments This research is funded by the EU research program “Preserving the Ecosystem” under BASIC project contract ENV4-CT97-0571, The Maj and Tor Nessling Foundation, The Applied Bioscience Graduate School and the Academy of Finland. The authors thank Jaana Lehtima¨ ki for her comments. This is contribution ELOISE No. 227.

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