Nitrate and Phosphate Affect Cultivability of Cyanobacteria from Environments with Low Nutrient Levels

Nitrate and phosphate concentrations higher than those foundin the natural environment slowed down growth of two strainsof non-bloom-forming, phycoerythrin-rich Synechococcus spp.isolated from mesotrophic subalpine lakes. The results makeclear why isolation of these picocyanobacteria in standard cultivationmedia was difficult. At low concentrations, closely relatedstrains exhibited distinct growth characteristics with respectto these two nutrients, possibly explaining differences in theirseasonal appearance in the natural environment.

INTRODUCTION

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Introduction

At low levels of nutrient loading, small red-pigmented phycoerythrin(PE)-rich cyanobacteria of the Synechococcus type dominate theautotrophic picoplankton in freshwater ecosystems (10, 12, 16,21). These non-bloom-forming Synechococcus spp. (19) belongto the same phylogenetic clade as marine Synechococcus spp.and Prochlorococcus spp. (7, 8, 20). Despite their ubiquity,they were discovered in the late 1970s only, when epifluorescencemicroscopy and flow cytometry were introduced as counting techniques(see references in reference 18) and the first isolates frommarine and freshwater ecosystems became available (6, 23). Thedifficulty in cultivating red-pigmented freshwater Synechococcusspp. contrasts with the easy accessibility of blue-green, phycocyanin-richspecies, particularly those of the closely related genus Cyanobium(8, 22; for strain histories, see reference 14).

Recently, Becker et al. (3) reported that PE-rich picocyanobacteriafrom a biofilm of tiles deposited for 6 weeks in Lake Constanceformed small colonies on agar plates amended with a mineralmedium lacking nitrate, BG11₀, although none of them was capableof nitrogen fixation. A similar inoculum spread on plates withnitrate (BG11) produced blue-green colonies only. The mineralmedium BG11 was introduced by Stanier et al. (17) for the cultivationof coccoid cyanobacteria. In its original description, it isparticularly rich in nitrate (1.5 g/liter NaNO₃, equivalentto 247 mg/liter NO₃-N) and exhibits an N:P molar ratio of 100:1,which is far above the molar ratio of these elements in biomass(Redfield ratio, 16:1). Nevertheless, phosphate also is plentifulin BG11 compared to the environmental concentrations often limitingthe growth of photoautotrophs in meso- and oligotrophic lakes.

Cyanobacteria have developed highly efficient uptake and retentionmechanisms for three nutrient anions: bicarbonate, nitrate,and phosphate (1, 11, 15). The apparent competitive disadvantageof PE-rich Synechococcus spp. on a cultivation medium rich innitrate prompted us to examine the effect of high nutrient concentrationson the growth of two strains, Synechococcus rubescens strainSAG 3.81 and Synechococcus sp. strain BO 8807, isolated fromLake Zürich and Lake Constance, respectively.

Growth inhibition by phosphate and nitrate.

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Growth inhibition by phosphate...

The two red-pigmented Synechococcus strains BO 8807 and SAG3.81 of subalpine cluster I (8) were raised in 96-well microtiterplates. Before growth experiments were conducted, aliquots ofbatch cultures were diluted into fresh growth medium (BG11 amendedwith sterile solutions of sodium bicarbonate [0.168 g/liter]and vitamin B12 [0.02 mg/liter]) and maintained at least 20h under continuous light (10 ± 1 µmol quanta m^–2s^–1) at 19 ± 1°C, the conditions used duringgrowth experiments. After this preadaptation, cells were collectedby centrifugation (10 min; 10,000 x g), washed, and resuspendedin this medium lacking nitrate and phosphate. Sterile dilutionsof NaNO₃ and K₂HPO₄ were added, in a volume of 10 µl each,to the 96 wells of flat-bottom microtiter plates (Greiner).Finally, the resuspended cells were added in a volume of 130µl. The microtiter plates were taped at the long sidesand then placed on a microtiter plate vortexer (Applied QualityServices, United Kingdom) that was set to 2 min of vigorousshaking per h to counteract sedimentation of cells.

The 96-well format allowed not only the application of a widerange of nutrient loadings and N:P ratios but also monitoringof growth with a microplate reader (ELx808; Bio-Tek, Germany),a convenient and nonintrusive detection method. Growth was followedby measuring the optical density at 750 nm (OD₇₅₀). At thiswavelength, the pigments of cyanobacteria exhibit negligibleabsorbance and, hence, turbidity can be used as a measure forbiomass.

At low initial nutrient concentrations (0.3 mg/liter K₂HPO₄and 15 mg/liter NaNO₃), biomass did not increase significantly.Both concentrations were higher than those measured in LakeConstance, which exhibited a total P concentration of 40 µg/literand 4.3 mg/liter NO₃ in 1988, when a maximum of 7.7 x 10⁵ cells/mlof autotrophic picoplankton was reported (9). The initial celldensity of (2 to 4) x 10⁷ cells/ml in our experiments may havealready exceeded the carrying capacity. Also, at an initialNaNO₃ concentration of 9.9 g/liter, the highest nitrate concentrationused, biomass of both strains did not increase during the durationof the experiments (10 to 14 days in different series). At intermediateconcentrations, growth strongly depended on the initial phosphateand nitrate concentrations (Fig. 1). The total biomass formationwas limited by nitrate if the molar N:P ratio was $≤$ 33 and waslimited by phosphate at higher N:P ratios, a factor two higherthan that expected from the Redfield ratio. For both strains,high initial nitrate and phosphate concentrations delayed growth.The initial phosphate concentration used in BG11 (0.03 g/literK₂HPO₄) inhibited growth of both strains even at low nitrateconcentrations (Fig. 1c and f). In combination with the highnitrate concentration of BG11 (1.5 g/liter; Fig. 1c and f),both strains did not reach stationary phase within 2 weeks.Similar growth characteristics were reported earlier for twoother strains of the subalpine cluster I (13).

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FIG. 1. Growth of Synechococcus sp. strain BO 8807 (a to c) and Synechococcus rubescens strain SAG (d to f) in microtiter plates. Initial concentrations of K₂HPO₄ (mg/liter) were 3.0 in a and d; 9.0 in b and e; and 30 in c and f. The initial NaNO₃ concentrations (g/liter) are indicated in panel c. Growth was recorded as absorbance at 750 nm. The initial concentrations of chlorophyll a (µg/ml in a methanol extract) varied between 0.23 and 0.31 in different experimental series. Continuous lines represent logistic fits of growth. Note that the scale of the y axis differs for the different initial phosphate concentrations.

In batch cultures, growth rates are not constant because theconcentration of the limiting nutrient decreases during growth.Growth limited by a vanishing resource is described by logisticgrowth models. For the logistic fit of the growth measured atOD₇₅₀, we applied the equation

(1)
Thesigmoid shape of the growth curve is characterized by four parameters:the upper asymptote, a, represented by the absorbance reachedin the stationary phase, a rate parameter, k, describing therate at which growth initially accelerates, and a time constant,c, that describes the time elapsing between the beginning ofthe growth experiment and the point of maximum increase in biomass(turning point). To fit the data of our experiments, an offsetin the absorbance (a₀), which remained constant throughout theexperiment, was added. In Fig. 1, the fits of individual growthcurves are depicted as continuous lines. The model fitted growthof the two PE-rich Synechococcus strains well except at highnutrient concentrations at which growth was inhibited and culturesdid not reach stationary phase before termination of the experiments.

From the parameters we calculated the biomass-specific initialgrowth rate at t = 0 (see Appendix). These calculations showedthat k is an excellent approximation of the initial, biomass-specificgrowth rate. Hence, we used k as an approximation for the maximumgrowth rate at the indicated nutrient concentration.

For both Synechococcus strains, we conducted two series of growthexperiments, both with an internal replicate for every concentrationused. To calculate the growth parameters a, a₀, c, and k formultiple experiments, the OD₇₅₀ values were imported into theprogram Origin (version 7.5; OriginLab Corp.). A script fordata management and analysis was written in LabTalk, a C-basedscripting language provided by this program. The script wasstructured in four subroutines: first, the extinction values(triplicates) were averaged and the intrinsic absorption ofeach well filled with 150 µl H₂O was subtracted. Next,time series were constructed with the net extinction values.Third, the time series were fitted assuming a logistic growthfunction (equation 1). Finally, the growth parameters of experimentswith identical initial nutrient concentrations were averaged,and standard deviations were calculated (the peripheral wellsof the 96-well plates were not considered). The results (Fig.2) show that carrying capacity (a) was similar for both strains.Growth rate constants k and time constants c (not shown) werealmost identical in multiple experiments conducted with thesame starting material (internal replicates) but differed betweenexperimental series. This variation is reflected in the errorbars of the growth rate constant (Fig. 2). However, this variationdid not obscure the effects of nutrient concentrations on growthoutlined above. The generation time of both strains was aboutsix times longer for BG11 than that in media with lower, optimalnutrient concentrations.

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FIG. 2. Carrying capacity a and growth rate constant k of Synechococcus strains BO 8807 (a) and SAG 3.81 (b) in media containing different initial concentrations of nitrate and phosphate. The figure is divided into six sections, each representing a series of experiments for which the initial K₂HPO₄ concentration is as indicated below the section. For each K₂HPO₄ concentration, six initial NaNO₃ concentrations were used as indicated in panel a. The growth parameters a (hatched bars) and k ( ${blacktriangledown}$ ) were derived from logistic fits of individual growth curves (see Fig. 1) and represented as an average with standard deviation for two to eight growth experiments. Note that in some experiments, the increase in biomass was too small to determine growth rate constants.

With these results in mind, we prepared agarose plates withBG11 (plus vitamin B12) and four versions of the bicarbonate-enrichedmedium, one with original concentrations of nutrients and threein which phosphate was reduced to 30% and nitrate to either10%, 3%, or 1%. These plates were inoculated with serial dilutionsof the two red-pigmented Synechococcus strains used in thisstudy and a blue-green isolate, strain BO 8806, which is closelyrelated to Cyanobium gracile PCC 6307 (8). The blue-green strainwas able to form colonies with high plating efficiency (>90%)on all variants of the medium (in Table 1, only colony numbersof a 10⁴-fold dilution of the preadapted cultures are shown).In contrast, dilutions of the red-pigmented strains failed toform colonies in full-strength BG11 with or without bicarbonate.However, both strains grew with only slightly lower platingefficiency on all plates with lowered nutrient concentrations(Table 1). Apparently, on the surfaces of agarose plates, theinhibitory effect of high nutrient concentrations on the growthof the red-pigmented strains was enhanced by evaporation, resultingin a very strong, negative isolation bias. The results are infull agreement with the observations of Becker et al. (3) reportinga selective suppression of colony formation of red-pigmentedpicoplankton by BG11.

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TABLE 1. Number of colonies of Synechococcus sp. strains BO 8806 and BO 8807 and Synechococcus rubescens strain SAG 3.81 on agarose plates amended with variations of the mineral medium BG11

Clonal effects of nitrate and phosphate.

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Clonal effects of nitrate...

Figures 1 and 2 indicate that nitrate and phosphate affectedgrowth of the two Synechococcus strains differently. Increasingnitrate concentrations delayed growth of strain BO 8807 (Fig.1a to c and 3a) even at low initial phosphate levels, whilegrowth of strain SAG 3.81 was affected by high nitrate only.On the other hand, growth of strain SAG. 3.81 was delayed andinhibited by increasing phosphate (Fig. 1d to f and 3b), whilestrain BO 8807 not only needed higher initial phosphate concentrationsto obtain highest growth rates but also tolerated high phosphateconcentrations for cultivation, provided nitrate was low. Thus,in strain BO 8807 the tolerance towards phosphate seemed tobe paired with a high sensitivity towards nitrate, while instrain SAG 3.81 tolerance of nitrate is paired with high sensitivitytowards phosphate. Contour plots summarize the combined effectof nitrate and phosphate on carrying capacity and growth rateconstants of the two strains (Fig. 3).

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FIG. 3. Contour plots depicting the growth parameters a and k of Synechococcus strains BO 8807 (a, b) and SAG (c, d) as a function of the initial concentrations of nitrate and phosphate. Contour plots were constructed from the growth parameters shown in Fig. 2, placed at the positions indicated by black dots. The lines indicate constant values of a or k, respectively, that were calculated using the weighted distance algorithm provided by the program Statistica with a stiffness of 0.2. Note that the logarithmic scales in a and c (carrying capacity a) deviate from those of b and d (growth rate constant k).

The observation that strain BO 8807 tolerates high concentrationsof phosphate but is highly sensitive towards nitrate, whilestrain SAG 3.81 can tolerate high nitrate if phosphate is low,indicates that uptake and assimilation of these two anions isendogenously limited by a common resource. Thus, the questionremains why non-bloom-forming Synechococcus spp. cannot copewith high nutrient levels or what is the limiting endogenousresource. Most studies of the regulation of N, P, and C assimilationwere conducted with blue-green freshwater cyanobacteria growingwell in presence of high concentrations of nitrate and phosphate.Presumably, all these strains are capable of efficiently limitinguptake and assimilation of N (via NtrA [11]) and P (via theP-regulon [4]) when high concentrations (at low light intensity)threaten to impair photosynthetic assimilation of CO₂. Sucha control may be missing not only in the oceanic Synechococcussp. strain WH 8103 (5) but also in non-bloom-forming freshwaterSynechococcus spp., with the reported effects with cultivationin the presence of high nutrient levels. However, at ambientnutrient levels, the particular growth characteristics of thetwo strains may be of relevance. In summer picoplankton, whenphosphate reaches a minimum in the euphotic zone of Lake Constance(9), genotypes closely related to that of strain SAG 3.81 areabundant, while the genotype of isolate BO 8807 reaches a relativeand absolute minimum (2; S. Becker, P. Richl, P. Boger, andA. Ernst, unpublished data). As growth of this genotype is moreconstrained by low levels of phosphate, this may reflect itsparticular growth characteristics.

APPENDIX

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Appendix

The growth rate of a logistic model can be calculated from theequation

From this equation, the initialbiomass-specific growth rate (IGR) at t = 0 and y = y₀ can becalculated as

For c >> k, the biomass-specificIGR approaches the value of the growth rate constant k, andthe biomass-specific growth rate at the turning point is halfthat of the initial growth rate. In all our experiments, k providedan excellent approximation of IGR.

ACKNOWLEDGMENTS

This work was partly financed by the Commission of the EuropeanCommunity through RTD project MIRACLE (contract: EVK3-CT-2002-00087).

The comments of L. Stal during preparation of the manuscriptwere greatly appreciated.

FOOTNOTES

* Corresponding author. Mailing address: NIOO-CEME, P.O. Box 140, NL-4400 AC Yerseke, The Netherlands. Phone: 31 (0) 113 577300. Fax: 31 (0) 113 573616. E-mail: a.ernst{at}nioo.knaw.nl.

This is publication 3506 NIOO-KNAW of the Netherlands Instituteof Ecology.

REFERENCES

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