Effects of sodium and phosphate on growth of cyanobacteria

War. Res. Vol. 21, No. 6, pp. 625-631, 1987 Printed in Great Britain

0043-1354/87 $3.00 +0.00 Pergamon Journals Ltd

EFFECTS OF SODIUM A N D PHOSPHATE ON GROWTH OF CYANOBACTERIA D. B. SEALE, M. E. BORAASand G. J. WARREN* Department of Biological Sciences and Center for Great Lakes Studies, University of Wisconsin-Milwaukee, Box 413, Milwaukee, WI 53201, U.S.A.

(Received August 1984) Abstract--We conducted laboratory experiments to evaluate the effects of NaC1 and phosphorus enrichments on natural phytoplankton assemblages from Lake Michigan in continuous-flow systems, at a dilution rate of 0.25 d -t. The experiment was repeated four times, 1981-1982, using freshly-collected natural lakewater inocula and temperature regimes typical of near-surface waters at initiation (6, 12, 16 and 20°C), at two levels of PO4-P (1-2 vs 91-92 #g 1-I) and ofNa ÷ (3-4 vs 9-10mg1-1) each time. As a single factor, sodium chloride enrichments had no significant effect on growth rates or densities of cyanobacteria in cultures containing natural phytoplankton assemblages from Lake Michigan. However, filamentous cyanobacteria proliferated in the presence of elevated phosphorus concentrations, both with and without concurrent NaC1 additions, particularly in warmer waters. Our laboratory results were consistent with the hypothesis that cyanobacteria are favored in phytoplankton of large lakes with low N:P ratios.

Key words---chemostat,phytoplankton, cyanobacteria, Lake Michigan, sodium, phosphorus, enrichment, competition

INTRODUCTION Sodium chloride from anthropogenic sources, such as road runoff and industrial wastes, may alter freshwater phytoplankton communities. Increased salinity should favor growth of cyanobacteria (cyanophytes or "bluegreen algae") because these are the only phototrophic plankton taxa requiring Na ÷ for growth (Allen and Arnon, 1955; Bostwick et al., 1968). When Na ÷ is present, cyanobacteria have higher nutrient uptake efficiencies and lower loss rates of fixed organic matter (Brownell, 1979). In the laboratory, Anabaena growth rates (Allen and Arnon, 1955; Ward and Wetzel, 1975) and biomass yields (Browneil and Nicholas, 1967) were maximized at 5-40 ppm NaC1. Phosphorus uptake by cyanobacteria was enhanced most by Na ÷ additions in the laboratory when PO4-P levels exceeded 50/.tgl -t (Mohleji and Verhoff, 1980). These laboratory studies imply that cyanobacteria may be stimulated when Na ÷ concentrations exceed 5 mg l -l, particularly if phosphorus also is increased, but they cannot predict when cyanobacteria should be competitively dominant over other phototrophs in a lake ecosystem. Increased salinity appears to be correlated with filamentous cyanobacteria in Lake Michigan (Stoermer and Ladewski, 1976; Baybutt and Makarewicz, 1981). Although diatoms are considered dominant in the net phytoplankton, many genera of cyanobacteria have been documented in the lake, including Oscil*Present address: Division of Biological Sciences, University of Michigan, Ann Arbor, MI 48109, U.S.A. 625

latoria, Anabaena, Aphanocapsa, Chroococcus, Coelosphaerium, Gleocapsa, Gomphasphaeria, Lyngbya, Microcystis, Pelogleoa, Spirulina, Aphanocapsa, Aphanothece and Doctylococcopsis (Brahce, 1980; Ristic, 1977; Stoermer and Ladewski, 1976; Stoermer

et al., 1978). Total PO4-P levels may be approaching 50/~g 1-~ in some nearshore waters of southern Lake Michigan (Makarewicz and Baybutt, 1981). Sodium and chloride concentrations have been increasing linearly in Lake Michigan for decades (Beeton, 1969). If these trends continue, the present concentrations of 4-5 mg 1-' Na + and 8-9 mg 1 l CI will double in 50 years. These trends have suggested a need to determine if filamentous cyanobacteria could dominate the phytoplankton in < 50 years if sodium chloride levels continue to increase in Lake Michigan at current rates (Brooks et al., 1984). We examined growth responses of natural phytoplankton assemblages to low ( 3 - 4 m g l - l ) vs projected (9-10 mg 1- l ) levels of Na+, in the presence of low (current offshore: 2/~gl -~) and of high (92/~gl - l ) PO4-P levels. The high PO4-P concentration was sufficient to produce a phosphorussaturated chemical environment (Sommer, 1983). We used continuous-culture methods to meter growth media to cultures containing Lake Michigan phytoplankton collected four times over an annual cycle. Cultures were held at the temperature typical of surface waters during the season of collection. All experiments were conducted at a dilution rate of 0.25d - l , approximating lower ranges of phytoplankton turnover rates in freshwaters (Sommer, 1986).

D.B. SEALE et al.

626

We asked the questions: (1) Will e n r i c h m e n t o f NaC1 or P favor growth o f filamentous c y a n o b a c t e r i a in l a b o r a t o r y cultures o f n a t u r a l p h y t o p l a n k t o n assemblages from Lake Michigan? (2) Will g r o w t h o f cyanobacteria be e n h a n c e d m o r e if P a n d N a were a d d e d together t h a n if each were a d d e d alone? O u r experiments showed t h a t filamentous cyanobacterial growth was p r o m o t e d by elevating p h o s p h o r u s concentrations, b o t h with a n d w i t h o u t c o n c u r r e n t NaCI additions, particularly in w a r m e r waters. However, NaCI level, as a single factor, h a d no significant effect on relative growth rates o f Lake M i c h i g a n cyanobacteria. MATERIALS AND METHODS

Experimental system

perature regimes typical of near-surface waters at initiation. We examined the effects of sodium and phosphorus for each of the following four temperature and seasonal regimes: winter to early spring at 6°C; late spring to early summer at 12°C; late summer to autumn at 16°C; and summer at 20°C. The temperatures were maintained to ___I°C, on a 18 h light:6 h dark light regime within an environmental chamber. Inocula were collected from near-surface waters just before initiating each experiment, usually within 2 h, and placed in chemically-clean, sterile collection containers (polypropylene bottles or polyethylene bags). Raw water for inocula of experiments at 6 and 12°C were obtained in winter from the intake of the Linnwood Water Filtration Plant, Milwaukee, Wis., on December 1981, and 4 February 1982, respectively. The plant draws its water from an intake located north of Milwaukee, 1.6 km offshore, 17 m below the surface. Inocula for the 20 and 16°C experiments were collected with a Niskin bag sampler, about 5 km northeast of the Milwaukee harbor, from 5 m, on 28 June 1982, and 25 October 1982, respectively. The experimental flasks were filled with the inoculum within a laminar flow hood. lnocula were allowed to come to the chosen experimental temperature, at about 0.5°Cd -t, before media flow was initiated. From visual examinations, zooplankton were absent from inocula and subsequent samples.

Our continuous-culture system was based on a hydraulic pressure system (Tilman et al., 1982). A peristaltic pump (Harvard model 1203) continuously delivered modified lakewater growth media from large reservoirs (20-1. polycarbonate carboys) to culture vessels (two replicate 500-ml transparent polycarbonate Erlenmeyer flasks for each treatSampling, identification and analysis ment). The flasks, secured to a shaker table controlled by a The cultures were sampled 2-3 times a week. A subsample mechanical timer, were agitated for 20 s of each min at for phytoplankton counts was preserved in Lugol's solution. 120 rpm, promoting continuous, gentle mixing (S. Kilham, pers. comm.). Outflow from each flask was forced out by Phytoplankton from 25 ml of preserved sample was allowed to settle for at least 24 h before counting with a Zeiss hydraulic pressure at a rate equal to the input rate, and inverted microscope at 200 or 500 x . Randomly selected collected in a 500-ml, foil-covered Erlenmeyer flask. Teflon 8 "spaghetti" tubing was used, except for a short section of fields were counted until at least I00 of the most abundant silicon rubber tubing within the pump. Cool-white group were identified (Lund et al., 1958). Some organisms fluorescent lamps supplied light at about 100/~Einsteins were identified to genus, but most only classified to major m -2 s -I. Less than 5% of the incoming light was shaded by taxonomic group: (1) multicellular and filamentous cythe securing devices. The dilution rate (D = system flow anobacteria, predominantly Anabaena spp, Oscillatoria spp, rate/culture vessel volume) was held constant at 0.25 d -~ and colonial species; (2) green algae, primarily Anby delivering media to the culture vessels at 100mld -~. kistrodesmus spp; (3) diatoms, mostly Fragilaria, Tabellaria, Sampling continued 30-50d, until wall growth became various pennate diatoms, and a few centric forms; and (4) "other" algae, mostly Dinobryon spp and unidentified apparent. The water for media was pumped from the hypolimnion flagellated forms. Identification of cyanobacteria was aided (4~65 m below the surface), at least 30 m below the thermo- by epifluorescence microscopy. Very small unicellular cycline on each collection date (see below). Total nutrients in anobacteria were present in all treatments, particularly at the higher temperatures, but could not be counted with the hypolimnion and epilimnion are comparable, but hypolimnetic water provides more dissolved nutrients that are accuracy with these methods. Univariate analyses of variance (ANOVAs) and covarimmediately available for phytoplankton growth. Within a few hours of collection, the water was cleaned of particles iance (ANOCOVAs) as well as multivariate ones (MANby filtration through acid-washed and well-rinsed 0.22/~m OVAs and MANOCOVAs), were performed using SPSS cellulose acetate filters and then stored in the dark at 4°C. (Statistical Package for the Social Sciences) on a Sperry 1100 The basic medium contained 2 parts hypolimnetic Lake mainframe computer. To satisfy assumptions of these analMichigan water and 1 part distilled water. This dilution yses, data first were transformed by taking arcsines of reduced the [Na ÷ ] in our low-Na media, from a mean of proportions and square roots of counts (Sokal and Rohlf, 1969). In all ANOCOVAs and MANOCOVAs, main effects 4.7 ppm (our samples) to 3-4 ppm, which is lower than those stimulating growth of cyanobacteria in previous batch and interactions were evaluated after variations due to studies (Introduction). This basic medium was enriched with sample date (the covariate) were partialled out. 0.I ml 1-1 of Hutner's trace solution (Starr, 1978) to eliminate possible trace-element deficiencies. Four treatments RESULTS were used in all experiments: (l) basic medium (BM), (2) B M + 6 . 9 mg N a l -I (final [ N a + ] = 9 - 1 0 m g l - ~ ) , (3) O u r results showed n o tendency for filamentous B M + 6 / ~ m o l P1-1 (to 91-92/~gl-t), and (4) B M + cyanobacteria to d o m i n a t e in high-Na, low-P cultures 6 . 9 m g N a l -~ and 6 / t m o l P l -~. After preparation, the media were sterile-filtered into reservoirs through 0.22 ,am (Figs I-4). In the figures, m e a n entity c o u n t s for the filters, using either acid-washed cellulose acetate or poly- two replicate cultures are shown, with zero indicated carbonate filters. We used nonmetallic, autoclavable non- on the long scale as 1 cell m1-1. Relative densities toxic plastic ware rather than glassware for both sampling varied m o r e with P (1-2 vs 9 1 - 9 2 / ~ g l -~) at all four and culture devices, to minimize confounding effects of t e m p e r a t u r e s (6, 12, 16 a n d 20°C), t h a n with NaCI exogenous silicate and absorption of essential nutrients. levels. Time courses for cultures at the 2 N a ÷ levels, Experimental procedure within each t e m p e r a t u r e a n d p h o s p h o r u s levels, were The experiment was repeated four times, 1981-1982, virtual replicates o f each o t h e r (Figs 1-4). Densities using freshly-collected natural lakewater inocula and tem- o f c y a n o b a c t e r i a were consistently lower in h i g h - N a ,

Effects of sodium and phosphate on cyanobacteria growth

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l o w - P cultures than in a n y other treatment c o m b i n a tion (Figs 1-4). Elevated phosphorus levels promoted

We performed ANOVAs and ANOCOVAs to determine the statistical significance of the main effects of temperature (confounded with inoculum: see "Methods"), phosphorus level, and sodium level. A series of ANOVAs and of ANOCOVAs, with sample date as the covariate, showed the level of sodium was not associated with an2~change in densities (Table 1) of filamentous cyanobacteria or diatoms throughout the duration of the experiment. Phosphate level and temperature were the only

cyanobacterial proliferation in some cultures, both with and without high NaC1, particularly at higher temperatures (Figs 1-4). Cyanobacteria increased directly with temperature in high-P cultures. At 16°C, high cyanobacteria densities were observed at t e r m i n a t i o n in all cultures; h o w e v e r , h i g h - N a C 1 w a s a s s o c i a t e d with slightly lower relative densities o f

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628

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significant main effects on these two groups. We also computed proportions of each taxon in the final count (not shown). The tests of significance for main effects (p < 0.05) on these proportions were comparable to results in Table 1, except for a significant

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by determining the slope o f the line (In R vs time). These densities were monitored while the culture was under flow. Each panel shows a " w a s h o u t line," indicating the 0 . 2 5 d -] removal rate i m p o s e d by

effect on green algae (F = 6.61, p = 0.01). Further-

more, sodium level was not associated with variations in the vector of densities of cyanobacteria, diatoms, green algae, and "other" algae during the study (MANOCOVA: Wilk's ratio = 0.995, p = 0.475, n = 928).

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Effects of sodium and phosphate on cyanobacteria growth

629

Table I. ANOCOVAs of the indicated sources of variation (main effects and covariate) in numbers of the four main taxonomic groups in each sample (N ~ 928) Degrees of freedom

Mean square

F

1 5 3 1 1

5089.228 1488.523 1129.831 4013.297 39.823

28.508 8.338 6.329 22.481 0.223

0.0001 0.0001 0.0001 0.0001 0.637

Covariate: days from to Main effects Temperature Phosphorus Sodium chloride

1 5 3 1 1

0.233 1370.962 825.305 3466.614 912.280

0.001 4.748 2.858 12.005 3.159

0.977 0.0001 0.036 0.001 0.076

C. Numbers of green algae Covariate: days from to Main effects Temperature Phosphorus Sodium chloride

1 5 3 1 1

0.035 933.124 266.163 3828.583 38.550

0.000 11.610 3.132 47.636 0.480

0.983 0.0001 0.020 0.0001 0.489

D. Numbers of diatoms Covariate: days from to Main effects Temperature Phosphorus Sodium chloride

1 5 3 1 1

4324.985 1040.019 399.436 3995.796 5.991

13.208 3A76 1220 12.203 0.018

0.0001 0.008 0.301 0.001 0.892

Source of variation

Significance of F

A. Numbers of cyanobacteria Covariate: Days from to Main effects Temperature Phosphorus Sodium chloride

**** **** **** ****

B. Numbers of "other algae" **** * *** *

**** ** ****

**** *** ***

*0.10 < p <0.05; **0.05 < p <<0.01; ***0.01 < p < 0.001; ****0.001 < p < 0.0001.

with /z's between 0 and 0.25 have slopes < 0 but greater than that shown by the "washout line." Therefore, the maximum growth rate, /M~, can be computed as the sum of D and the slope of the exponential line [(ln N 0 - 1 n N t ) / t ] + 0 . 2 5 or the difference between D and the slope of the line during washout (Pirt, 1975). We computed growth rates (not shown) from the time-course data (Figs 1-4). Excluding #'s for the interval to-t 1 (a period of acclimation), and other probably artifactual data, the highest average #'s (~max for each group) seen were: (1) Pennate diatoms--l.07d -~ at 6°C (d 25: high-Na, high-P) and 0.49d -t at 12°C (d 24: high-Na, high-P); (2) Greens--0.75 d -~ at 6°C (d 14: iow-Na, high-P); (3) "Other algae"--q).90 d -~ at 20°C (d 12: low-P, highNa) and 1.20 d -~ at 20°C at termination (low-P, low-Na); (4) Cyanobacteria--a pulse in # of I. 15 d at 20°C (d 40, low-Na, high-P). The large blooms of cyanobacteria in the final samples for the high-P cultures at 16°C (~u = 3 . 2 d -l on d 45 at low-Na, high-P) resulted from wall growth near termination. Results of ANOCOVAs (not shown) showed no significant Na + main effects for the t~'s of any taxonomic group (p > 0.05). DISCUSSION

Continuous-culture methods, used to examine relative growth rates of algae in two-species systems (Tilman et al., 1982) and natural phytoplankton assemblages (Jones et al., 1979; Sommer, 1983, 1986), are useful tools for examining the potential effects of nutrients and toxins. Continuous cultures minimize interference from factors confounding batch-culture

results, including nutrient depletion, constantlychanging levels of toxins, and self-inhibition (Fredrickson and Tsuchiya, 1977). Unlike batch cultures, only reproductive algae can maintain themselves in continuous culture; senescent or slow-growing forms are flushed from the system. Theoretically, in a continuous culture system without predators, only one species per limiting nutrient can exist at a particular dilution rate at steady state (Tilman et al., 1982; Sommer, 1983). We attempted to maximize the opportunity for the proliferation of cyanobacteria by selecting a low dilution rate (D) of 0.25 d -~. The dilution rate does affect the outcome of competition; Sommer (1986) showed a regular transition in diatom steady-state dominants along a dilution rate gradient, from 0.3 to 1.6d -~. The mechanism for this transition appears to be that nutrient uptake processes determine the competitive outcome at low values of D while the maximum specific growth rate, # ~ , is more important at high values of D. A high D imposes a high density-independent mortality rate that shifts the predicted competitive dominant (Tilman, 1982). Earlier work had shown that uptake of PO4-P by cyanobacteria was enhanced by the presence of sodium (Brownell, 1979), which might give cyanobacteria a competitive advantage at slow D's. Therefore, we chose a value of D that is at the lower end for actively-growing phytoplankton in the field (Sommer, 1986). Since cyanobacteria did dominate in some of our high-P cultures, D = 0 . 2 5 d -t obviously was small enough. Increasing D above 0.25 d -~ should select more strongly against filamentous cyanobacteria.

630

D.B. SEALEet al.

Field predictions from laboratory data must be made with caution. Our studies emphasized the effects of nutrient modifications on phytoplankton growth and competitive interactions. They ignored some factors that are known to alter cyanobacteria proportions, such as differential grazing (Scale, 1980). Flow-through systems favor actively-growing algae; however, senescent forms, induced to proliferate by added nutrients, may dominate some blooms in the field. Our semi-logarithmic plots tend to emphasize slight differences in numbers at lower cell number, exaggerating trends toward cyanobacteria dominance in some of these cultures, in particular the high-Na, high-P cultures at 16 and 20°C. Although not significant, these data could indicate suppression of cyanobacteria under certain low-P, high-Na conditions and enhancement with concurrent Na and P enrichment in warm nearshore waters. Replication at 20°C (Fig. 4) was imprecise for unknown reasons, perhaps obscuring some significant relationships. After completing the study, re-examination of some samples from the 20°C cultures at high magnification revealed many ( > 106 ml ~) small unicellular cyanobacteria, which were not counted in the study. Phagotropic microflagellates (included in "other algae" for this study) are common in Lake Michigan (Boraas et al., 1985); and they could have affected our results by consuming cells <3/~m. Also, interactions with fish and zooplankton can influence Lake Michigan's phytoplankton community structure (Brooks et al., 1984). The enhancement of cyanobacterial growth at elevated phosphate concentrations (Figs 1-4) is consistent with some theories of eutrophication in large lakes. Cyanobacteria are favored in epilimnetic phytoplankton of lakes with extremely low N:P ratios (Schindler, 1977), dominating only in lakes with Total N:Total P (TN:TP) ratios <29 by mass (Smith, 1983). Our low-P media had a TN:TP ratio >40, and cyanobacteria were not the dominant phytoplankters. In contrast, cyanobacteria dominated in the high-P cultures, with T N : T P ratios <4. In low-nitrogen waters, some cyanobacteria can fix nitrogen (Fogg et al., 1973), giving them a clear competitive edge. Furthermore, cyanobacteria apparently are inferior to diatoms in competing for phosphorus (Tilman et al., 1982). Diatoms grew well in both high and low sodium chloride concentrations (Figs 1-4). Their competitive superiority at low-P was not altered by NaC1 levels. In high-P spring and summer cultures (Figs 2-4), diatoms grew well initially, but cyanobacteria eventually dominated. Similar negative relationships between cyanobacteria and diatoms have been attributed to allelopathy, with cyanobacteria (e.g. Oscillatoria) supposedly releasing inhibitory metabolites (Keating, 1978). However, since the rate of decline in diatom numbers was less than the washout rate represented by the washout line, diatoms clearly could sustain positive p's, even in the presence of

cyanobacteria (Fig. 3). These dynamics are consistent with an hypothesis of exploitative or resource competition (Fredrickson and Tsuchiya, 1978; Kilham and Kilham, 1978; Tilman, 1982). Allelopathy need not be invoked to interpret our results. In conclusion, elevating NaCI levels alone probably will not induce a shift toward cyanobacterial domination in natural phytoplankton assemblages from open waters of Lake Michigan. The temperatures, sodium levels, and phosphorus levels in this study bracket minimum and maximum values likely to be seen in Lake Michigan within this century. However, phosphorus enrichments, both with and without increased salinity, could lead to proliferation of cyanobacteria in Lake Michigan, particularly in warm littoral or surface waters during summer and early autumn. Acknowledgements--This research was supported in part by

the U.S. Environmental Protection Agency Great Lakes National Program Office under Grant No. R005 655-01. W. Monagle counted the phytoplankton. Contribution No. 302, Center for Great Lakes Studies. REFERENCES

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