Petra M. Visser

CHANGES IN TURBULENT MIXING SHIFT COMPETITION FOR LIGHT BETWEEN PHYTOPLANKTON SPECIES

The intriguing impact of physical mixing processes on species interactions has always fascinated ecologists. Here, we exploit recent advances in plankton models to develop competition theory that predicts how changes in turbulent mixing affect competition for light between buoyant and sinking phytoplankton species. We compared the model predictions with a lake experiment, in which the turbulence structure of the entire lake was manipulated using artificial mixing. Vertical eddy diffusivities were calculated from the measured temperature microstructure in the lake. Changes in turbulent mixing of the lake caused a dramatic shift in phytoplankton species composition, consistent with the predictions of the competition model. The buoyant and potentially toxic cyanobacterium Microcystis dominated at low turbulent diffusivity, whereas sinking diatoms and green algae dominated at high turbulent diffusivity. These findings warn that changes in the turbulence structure of natural waters, for instance driven by climate change, may induce major shifts in the species composition of phytoplankton communities.

Effects of Light on the Microcystin Content of Microcystis Strain PCC 7806

ABSTRACT Many cyanobacteria produce microcystins, hepatotoxic cyclic heptapeptides that can affect animals and humans. The effects of photosynthetically active radiation (PAR) on microcystin production by Microcystis strain PCC 7806 were studied in continuous cultures. Microcystis strain PCC 7806 was grown under PAR intensities between 10 and 403 μmol of photons m−2 s−1 on a light-dark rhythm of 12 h -12 h. The microcystin concentration per cell, per unit biovolume and protein, was estimated under steady-state and transient-state conditions and on a diurnal timescale. The cellular microcystin content varied between 34.5 and 81.4 fg cell−1 and was significantly positively correlated with growth rate under PAR-limited growth but not under PAR-saturated growth. Microcystin production and PAR showed a significant positive correlation under PAR-limited growth and a significant negative correlation under PAR-saturated growth. The microcystin concentration, as a ratio with respect to biovolume and protein, correlated neither with growth rate nor with PAR. Adaptation of microcystin production to a higher irradiance during transient states lasted for 5 days. During the period of illumination at a PAR of 10 and 40 μmol of photons m−2 s−1, the intracellular microcystin content increased to values 10 to 20% higher than those at the end of the dark period. Extracellular (dissolved) microcystin concentrations were 20 times higher at 40 μmol of photons m−2 s−1 than at 10 μmol of photons m−2 s−1 and did not change significantly during the light-dark cycles at both irradiances. In summary, our results showed a positive effect of PAR on microcystin production and content of Microcystis strain PCC 7806 up to the point where the maximum growth rate is reached, while at higher irradiances the microcystin production is inhibited.

Competition for light between toxic and nontoxic strains of the harmful cyanobacterium Microcystis

ABSTRACT The cyanobacterium Microcystis can produce microcystins, a family of toxins that are of major concern in water management. In several lakes, the average microcystin content per cell gradually declines from high levels at the onset of Microcystis blooms to low levels at the height of the bloom. Such seasonal dynamics might result from a succession of toxic to nontoxic strains. To investigate this hypothesis, we ran competition experiments with two toxic and two nontoxic Microcystis strains using light-limited chemostats. The population dynamics of these closely related strains were monitored by means of characteristic changes in light absorbance spectra and by PCR amplification of the rRNA internal transcribed spacer region in combination with denaturing gradient gel electrophoresis, which allowed identification and semiquantification of the competing strains. In all experiments, the toxic strains lost competition for light from nontoxic strains. As a consequence, the total microcystin concentrations in the competition experiments gradually declined. We did not find evidence for allelopathic interactions, as nontoxic strains became dominant even when toxic strains were given a major initial advantage. These findings show that, in our experiments, nontoxic strains of Microcystis were better competitors for light than toxic strains. The generality of this finding deserves further investigation with other Microcystis strains. The competitive replacement of toxic by nontoxic strains offers a plausible explanation for the gradual decrease in average toxicity per cell during the development of dense Microcystis blooms.

The Microcystin Composition of the Cyanobacterium Planktothrix agardhii Changes toward a More Toxic Variant with Increasing Light Intensity

ABSTRACT The cyanobacterium Planktothrix agardhii, which is dominant in many shallow eutrophic lakes, can produce hepatotoxic microcystins. Currently, more than 70 different microcystin variants have been described, which differ in toxicity. In this study, the effect of photon irradiance on the production of different microcystin variants by P. agardhii was investigated using light-limited turbidostats. Both the amount of the mRNA transcript of the mcyA gene and the total microcystin production rate increased with photon irradiance up to 60 μmol m−2 s−1, but they started to decrease with irradiance greater than 100 μmol m−2 s−1. The cellular content of total microcystin remained constant, independent of the irradiance. However, of the two main microcystin variants detected in P. agardhii, the microcystin-DeRR content decreased twofold with increased photon irradiance, whereas the microcystin-DeLR content increased threefold. Since microcystin-DeLR is considerably more toxic than microcystin-DeRR, this implies that P. agardhii becomes more toxic at high light intensities.

The ecological stoichiometry of toxins produced by harmful cyanobacteria: an experimental test of the carbon-nutrient balance hypothesis

The elemental composition of primary producers reflects the availability of light, carbon and nutrients in their environment. According to the carbon-nutrient balance hypothesis, this has implications for the production of secondary metabolites. To test this hypothesis, we investigated a family of toxins, known as microcystins, produced by harmful cyanobacteria. The strain Microcystis aeruginosa HUB 5-2-4, which produces several microcystin variants of different N:C stoichiometry, was cultured in chemostats supplied with various combinations of nitrate and CO(2). Excess supply of both nitrogen and carbon yielded high cellular N:C ratios accompanied by high cellular contents of total microcystin and the nitrogen-rich variant microcystin-RR. Comparable patterns were found in Microcystis-dominated lakes, where the relative microcystin-RR content increased with the seston N:C ratio. In total, our results are largely consistent with the carbon-nutrient balance hypothesis, and warn that a combination of rising CO(2) and nitrogen enrichment will affect the microcystin composition of harmful cyanobacteria.

Toxic and Nontoxic Microcystis Colonies in Natural Populations Can Be Differentiated on the Basis of rRNA Gene Internal Transcribed Spacer Diversity

ABSTRACT Assessing and predicting bloom dynamics and toxin production by Microcystis requires analysis of toxic and nontoxic Microcystis genotypes in natural communities. We show that genetic differentiation of Microcystis colonies based on rRNA internal transcribed spacer (ITS) sequences provides an adequate basis for recognition of microcystin producers. Consequently, ecological studies of toxic and nontoxic cyanobacteria are now possible through studies of rRNA ITS genotypic diversity in isolated cultures or colonies and in natural communities. A total of 107 Microcystis colonies were isolated from 15 lakes in Europe and Morocco, the presence of microcystins in each colony was examined by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS), and they were grouped by rRNA ITS denaturing gradient gel electrophoresis (DGGE) typing. Based on DGGE analysis of amplified ITSa and ITSc fragments, yielding supplementary resolution (I. Janse et al., Appl. Environ. Microbiol. 69:6634-6643, 2003), the colonies could be differentiated into 59 classes. Microcystin-producing and non-microcystin-producing colonies ended up in different classes. Sequences from the rRNA ITS of representative strains were congruent with the classification based on DGGE and confirmed the recognition of microcystin producers on the basis of rRNA ITS. The rRNA ITS sequences also confirmed inconsistencies reported for Microcystis identification based on morphology. There was no indication for geographical restriction of strains, since identical sequences originated from geographically distant lakes. About 28% of the analyzed colonies gave rise to multiple bands in DGGE profiles, indicating either aggregation of different colonies, or the occurrence of sequence differences between multiple operons. Cyanobacterial community profiles from two Dutch lakes from which colonies had been isolated showed different relative abundances of genotypes between bloom stages and between the water column and surface scum. Although not all bands in the community profiles could be matched with isolated colonies, the profiles suggest a dominance of nontoxic colonies, mainly later in the season and in scums.

Selective suppression of harmful cyanobacteria in an entire lake with hydrogen peroxide

Although harmful cyanobacteria form a major threat to water quality, few methods exist for the rapid suppression of cyanobacterial blooms. Since laboratory studies indicated that cyanobacteria are more sensitive to hydrogen peroxide (H(2)O(2)) than eukaryotic phytoplankton, we tested the application of H(2)O(2) in natural waters. First, we exposed water samples from a recreational lake dominated by the toxic cyanobacterium Planktothrix agardhii to dilute H(2)O(2). This reduced the photosynthetic vitality by more than 70% within a few hours. Next, we installed experimental enclosures in the lake, which revealed that H(2)O(2) selectively killed the cyanobacteria without major impacts on eukaryotic phytoplankton, zooplankton, or macrofauna. Based on these tests, we introduced 2 mg L(-1) (60 μM) of H(2)O(2) homogeneously into the entire water volume of the lake with a special dispersal device, called the water harrow. The cyanobacterial population as well as the microcystin concentration collapsed by 99% within a few days. Eukaryotic phytoplankton (including green algae, cryptophytes, chrysophytes and diatoms), zooplankton and macrofauna remained largely unaffected. Following the treatment, cyanobacterial abundances remained low for 7 weeks. Based on these results, we propose the use of dilute H(2)O(2) for the selective elimination of harmful cyanobacteria from recreational lakes and drinking water reservoirs, especially when immediate action is urgent and/or cyanobacterial control by reduction of eutrophication is currently not feasible. A key advantage of this method is that the added H(2)O(2) degrades to water and oxygen within a few days, and thus leaves no long-term chemical traces in the environment.

Modelling vertical migration of the cyanobacterium Microcystis

Computer models can be helpful tools to provide abetter understanding of the mechanisms responsible forthe complex movements of cyanobacteria resulting fromchanges in buoyancy and mixing of the water column ina lake. Kromkamp & Walsby (1990) developed a verticalmigration model for Oscillatoria, that wasbased on the experimentally determinedrelationship between the rates of density change andphoton irradiance in this cyanobacterium. To adaptthis model to Microcystis, we determinedrelated changes in carbohydrate content in cultures ofMicrocystis. Samples were incubated at variousconstant values of photon irradiance and then placedin the dark. The changes in carbohydrate content ofthe cells during these incubations were investigated.The relationship between the ratio of carbohydrate toprotein and cell density in Microcystis wasestablished to permit conversion of the rates ofcarbohydrate change to rates of density change. Byplotting the calculated rates of density changeagainst the values of photon irradiance experiencedduring the incubations, an irradiance-response curveof density change was established. The curve showed adistinct maximum at 278 µmol photons m-2s-1. At higher values of photon irradiance, therate of density change was strongly inhibited. Apositive linear correlation was found between celldensity and the rates of density decrease in the dark.The validity of the use of rate equations of densitychange, which are based on short-term incubations atconstant values of photon irradiance, to predictdensity changes in Microcystis in fluctuatinglight regimes was tested. This was accomplished bymeasuring the time course of change in carbohydratecontent of two continuous cultures of Microcystis, which were submitted to fluctuatinglight regimes, and comparing the results with thechanges in the carbohydrate contents of these culturespredicted by the rate equations of carbohydratechange. The results showed good agreement: the rateequations of density change were therefore introducedinto the model to simulate vertical migration of Microcystis. The model predicts that the maximummigration depth of Microcystis will increasewith colony size up to a maximum of 200 µm radius.The effect of colony size on the net increase in celldensity during the light period was also investigatedwith the model. It predicts that small colonies havea higher net increase in cell density than largecolonies, but are inhibited at high photon irradiancesat the surface.

Reversal in competitive dominance of a toxic versus non-toxic cyanobacterium in response to rising CO2

Climate change scenarios predict a doubling of the atmospheric CO2 concentration by the end of this century. Yet, how rising CO2 will affect the species composition of aquatic microbial communities is still largely an open question. In this study, we develop a resource competition model to investigate competition for dissolved inorganic carbon in dense algal blooms. The model predicts how dynamic changes in carbon chemistry, pH and light conditions during bloom development feed back on competing phytoplankton species. We test the model predictions in chemostat experiments with monocultures and mixtures of a toxic and non-toxic strain of the freshwater cyanobacterium Microcystis aeruginosa. The toxic strain was able to reduce dissolved CO2 to lower concentrations than the non-toxic strain, and became dominant in competition at low CO2 levels. Conversely, the non-toxic strain could grow at lower light levels, and became dominant in competition at high CO2 levels but low light availability. The model captured the observed reversal in competitive dominance, and was quantitatively in good agreement with the results of the competition experiments. To assess whether microcystins might have a role in this reversal of competitive dominance, we performed further competition experiments with the wild-type strain M. aeruginosa PCC 7806 and its mcyB mutant impaired in microcystin production. The microcystin-producing wild type had a strong selective advantage at low CO2 levels but not at high CO2 levels. Our results thus demonstrate both in theory and experiment that rising CO2 levels can alter the community composition and toxicity of harmful algal blooms.

Recruitment of benthic Microcystis (Cyanophyceae) to the water column: internal buoyancy changes or resuspension?

In some lakes, large amounts of the potentially toxic cyanobacterium Microcystis overwinter in the sediment. This overwintering population might inoculate the water column in spring and promote the development of dense surface blooms of Microcystis during summer. In the Dutch Lake Volkerak, we found photochemically active Microcystis colonies in the sediment throughout the year. The most vital colonies originated from shallow sediments within the euphotic zone. We investigated whether recruitment of Microcystis colonies from the sediment to the water column was an active process, through production of gas vesicles or respiration of carbohydrate ballast. We calculated net buoyancy, as an indication of relative density, using the amounts and densities of the major cell constituents (carbohydrates, proteins, and gas vesicles). Carbohydrate content of benthic Microcystis cells was very low throughout the year. Buoyancy changes of benthic Microcystis were mostly a result of changes in gas vesicle volume. Before the summer bloom, net buoyancy and the amount of buoyant colonies in the sediment did not change. Therefore, recruitment of Microcystis from the sediment does not seem to be an active process regulated by internal buoyancy changes. Instead, our observations indicate that attachment of sediment particles to colonies plays an important part in the buoyancy state of benthic colonies. Therefore, we suggest that recruitment of Microcystis is more likely a passive process resulting from resuspension by wind‐induced mixing or bioturbation. Consequently, shallow areas of the lake probably play a more important role in recruitment of benthic Microcystis than deep areas.