The Role of Nitrogen Fixation in Cyanobacterial Bloom Toxicity in a Temperate,Eutrophic Lake

Toxic cyanobacterial blooms threaten freshwaters worldwide but have proven difficult to predict because the mechanisms of bloom formation and toxin production are unknown, especially on weekly time scales. Water quality management continues to focus on aggregated metrics, such as chlorophyll and total nutrients, which may not be sufficient to explain complex community changes and functions such as toxin production. For example, nitrogen (N) speciation and cycling play an important role, on daily time scales, in shaping cyanobacterial communities because declining N has been shown to select for N fixers. In addition, subsequent N pulses from N₂ fixation may stimulate and sustain toxic cyanobacterial growth. Herein, we describe how rapid early summer declines in N followed by bursts of N fixation have shaped cyanobacterial communities in a eutrophic lake (Lake Mendota, Wisconsin, USA), possibly driving toxic Microcystis blooms throughout the growing season. On weekly time scales in 2010 and 2011, we monitored the cyanobacterial community in a eutrophic lake using the phycocyanin intergenic spacer (PC-IGS) region to determine population dynamics. In parallel, we measured microcystin concentrations, N₂ fixation rates, and potential environmental drivers that contribute to structuring the community. In both years, cyanobacterial community change was strongly correlated with dissolved inorganic nitrogen (DIN) concentrations, and Aphanizomenon and Microcystis alternated dominance throughout the pre-toxic, toxic, and post-toxic phases of the lake. Microcystin concentrations increased a few days after the first significant N₂ fixation rates were observed. Then, following large early summer N₂ fixation events, Microcystis increased and became most abundant. Maximum microcystin concentrations coincided with Microcystis dominance. In both years, DIN concentrations dropped again in late summer, and N₂ fixation rates and Aphanizomenon abundance increased before the lake mixed in the fall. Estimated N inputs from N₂ fixation were large enough to supplement, or even support, the toxic Microcystis blooms.     

Seasonal Variation and Indirect Monitoring of Microcystin Concentrations in Daechung Reservoir, Korea

Physicochemical and biological water quality, including the microcystin concentration, was investigated from spring to autumn 1999 in the Daechung Reservoir, Korea. The dominant genus in the cyanobacterial blooming season was Microcystis. The microcystin concentration in particulate form increased dramatically from August up to a level of 200 ng liter⁻¹ in early October and thereafter tended to decrease. The microcystin concentration in dissolved form was about 28% of that of the particulate form. The microcystins detected using a protein phosphatase (PP) inhibition assay were highly correlated with those microcystins detected by a high-performance liquid chromatograph (r = 0.973; P < 0.01). Therefore, the effectiveness of a PP inhibition assay for microcystin detection in a high number of water samples was confirmed as easy, quick, and convenient. The microcystin concentration was highly correlated with the phytoplankton number (r = 0.650; P < 0.01) and chlorophyll-a concentration (r = 0.591; P < 0.01). When the microcystin concentration exceeded about 100 ng liter⁻¹, the ratio of particulate to dissolved total nitrogen (TN) or total phosphorus (TP) converged at a value of 0.6. Furthermore, the microcystin concentration was lower than 50 ng liter⁻¹ at a particulate N/P ratio below 8, whereas the microcystin concentration varied quite substantially from 50 to 240 ng liter⁻¹ at a particulate N/P ratio of >8. Therefore, it seems that the microcystin concentration in water can be estimated and indirectly monitored by analyzing the following: the phytoplankton number and chlorophyll-a concentration, the ratio of the particulate and the dissolved forms of N and P, and the particulate N/P ratio when the dominant genus is toxigenic Microcystis.     

Microcystin mcyA and mcyE Gene Abundances Are Not Appropriate Indicators of Microcystin Concentrations in Lakes

Cyanobacterial harmful algal blooms (cyanoHABs) are a primary source of water quality degradation in eutrophic lakes. The occurrence of cyanoHABs is ubiquitous and expected to increase with current climate and land use change scenarios. However, it is currently unknown what environmental parameters are important for indicating the presence of cyanoHAB toxins making them difficult to predict or even monitor on time-scales relevant to protecting public health. Using qPCR, we aimed to quantify genes within the microcystin operon (mcy) to determine which cyanobacterial taxa, and what percentage of the total cyanobacterial community, were responsible for microcystin production in four eutrophic lakes. We targeted Microcystis-16S, mcyA, and Microcystis, Planktothrix, and Anabaena-specific mcyE genes. We also measured microcystins and several biological, chemical, and physical parameters—such as temperature, lake stability, nutrients, pigments and cyanobacterial community composition (CCC)—to search for possible correlations to gene copy abundance and MC production. All four lakes contained Microcystis-mcyE genes and high percentages of toxic Microcystis, suggesting Microcystis was the dominant microcystin producer. However, all genes were highly variable temporally, and in few cases, correlated with increased temperature and nutrients as the summer progressed. Interestingly, toxin gene abundances (and biomass indicators) were anti-correlated with microcystin in all lakes except the largest lake, Lake Mendota. Similarly, gene abundance and microcystins differentially correlated to CCC in all lakes. Thus, we conclude that the presence of microcystin genes are not a useful tool for eliciting an ecological role for toxins in the environment, nor are microcystin genes (e.g. DNA) a good indicator of toxins in the environment.     

Molecular Response of the Bloom-Forming Cyanobacterium, Microcystis aeruginosa, to Phosphorus Limitation

Cyanobacteria blooms caused by species such as Microcystis have become commonplace in many freshwater ecosystems. Although phosphorus (P) typically limits the growth of freshwater phytoplankton populations, little is known regarding the molecular response of Microcystis to variation in P concentrations and sources. For this study, we examined genes involved in P acquisition in Microcystis including two high-affinity phosphate-binding proteins (pstS and sphX) and a putative alkaline phosphatase (phoX). Sequence analyses among ten clones of Microcystis aeruginosa and one clone of Microcystis wesenbergii indicates that these genes are present and conserved within the species, but perhaps not the genus, as phoX was not identified in M. wesenbergii. Experiments with clones of M. aeruginosa indicated that expression of these three genes was strongly upregulated (50- to 400-fold) under low inorganic P conditions and that the expression of phoX was correlated with alkaline phosphatase activity (p < 0.005). In contrast, cultures grown exclusively on high levels of organic phosphorus sources (adenosine 5′-monophosphate, β-glycerol phosphate, and d-glucose-6-phosphate) or under nitrogen-limited conditions displayed neither high levels of gene expression nor alkaline phosphatase activity. Since Microcystis dominates phytoplankton assemblages in summer when levels of inorganic P (P_i) are often low and/or dominate lakes with low P_i and high organic P, our findings suggest this cyanobacterium may rely on pstS, sphX, and phoX to efficiently transport P_i and exploit organic sources of P to form blooms.     

Nutrient limitation of Microcystis aeruginosa in northern California Klamath River reservoirs

Nutrient limitations were investigated in Copco and Iron Gate Reservoirs, on the Klamath River in California, where blooms of the toxin-producing cyanobacterium Microcystis aeruginosa were first reported in 2005. Nutrient enrichment experiments conducted in situ in June and August, 2007 and 2008, determined responses in phytoplankton biomass, Microcystis abundance and microcystin concentration to additions of phosphorus and different forms of nitrogen (NH₄⁺, NO₃, and urea). Microcystis abundance was determined using quantitative PCR targeting the phycocyanin intergenic spacer cpcBA.Total phytoplankton biomass increased with additions of N both before and during Microcystis blooms, with no primary effects from P, suggesting overall N limitation for phytoplankton growth during the summer season. NH₄⁺ generally produced the greatest response in phytoplankton growth, while Microcystis abundance increased in response to all forms of N. Microcystis doubling time in the in situ experiments was 1.24–1.39 days when N was not limiting growth. The results from this study suggest availability of N during the summer is a key growth-limiting factor for the initiation and maintenance of toxic Microcystis blooms in Copco and Iron Gate Reservoirs in the Klamath River.     

Application of Real-Time PCR for Quantification of Microcystin Genotypes in a Population of the Toxic Cyanobacterium Microcystis sp.

The cyanobacterium Microcystis sp. frequently develops water blooms consisting of organisms with different genotypes that either produce or lack the hepatotoxin microcystin. In order to monitor the development of microcystin (mcy) genotypes during the seasonal cycle of the total population, mcy genotypes were quantified by means of real-time PCR in Lake Wannsee (Berlin, Germany) from June 1999 to October 2000. Standard curves were established by relating cell concentrations to the threshold cycle (the PCR cycle number at which the fluorescence passes a set threshold level) determined by the Taq nuclease assay (TNA) for two gene regions, the intergenic spacer region within the phycocyanin (PC) operon to quantify the total population and the mcyB gene, which is indicative of microcystin synthesis. In laboratory batch cultures, the cell numbers inferred from the standard curve by TNA correlated significantly with the microscopically determined cell numbers on a logarithmic scale. The TNA analysis of 10 strains revealed identical amplification efficiencies for both genes. In the field, the proportion of mcy genotypes made up the smaller part of the PC genotypes, ranging from 1 to 38%. The number of mcyB genotypes was one-to-one related to the number of PC genotypes, and parallel relationships between cell numbers estimated via the inverted microscope technique and TNA were found for both genes. It is concluded that the mean proportion of microcystin genotypes is stable from winter to summer and that Microcystis cell numbers could be used to infer the mean proportion of mcy genotypes in Lake Wannsee.     

Seasonal variation of cyanobacteria and microcystins in the Nui Coc Reservoir, Northern Vietnam

In order to understand the environmental variables which promote the proliferation of cyanobacteria and variation in microcystin concentrations in the Nui Coc reservoir, Vietnam, physicochemical parameters, the occurrence, and abundance of phytoplankton, cyanobacteria, and microcystin concentration were monitored monthly through the year 2009–2010. The relationships between these parameters were explored using principal component analysis (PCA) and Pearson correlation analysis. The phytoplankton community was mainly dominated by the cyanobacterium Microcystis with higher cyanobacteria abundance during summer and autumn season. PCA and Pearson correlation results showed that water temperature and phosphate concentration were the most important variables accounting for cyanobacteria, Microcystis, and microcystin occurrence. Analysis of the toxins by high-performance liquid chromatography demonstrated the presence of two microcystin variants: microcystin-LR (MC-RR) and microcystin-ddRR (MC-ddRR) with total concentrations of the toxins in filtered samples from surface water ranging from 0.11 to 1.52 μg MC-LR equiv L^?1. The high concentrations of microcystin in the Nui Coc reservoir highlighted the potential risk for human health in the basin. Our study underlined the need for regular monitoring of cyanobacteria and toxins in lakes and reservoirs, which are used for drinking water supplies, not only in Vietnam but also in tropical countries.     

A review of the global ecology,genomics, and biogeography of the toxic cyanobacterium,Microcystis spp.

This review summarizes the present state of knowledge regarding the toxic, bloom-forming cyanobacterium, Microcystis, with a specific focus on its geographic distribution, toxins, genomics, phylogeny, and ecology. A global analysis found documentation suggesting geographic expansion of Microcystis, with recorded blooms in at least 108 countries, 79 of which have also reported the hepatatoxin microcystin. The production of microcystins (originally “Fast-Death Factor”) by Microcystis and factors that control synthesis of this toxin are reviewed, as well as the putative ecophysiological roles of this metabolite. Molecular biological analyses have provided significant insight into the ecology and physiology of Microcystis, as well as revealed the highly dynamic, and potentially unstable, nature of its genome. A genetic sequence analysis of 27 Microcystis species, including 15 complete/draft genomes are presented. Using the strictest biological definition of what constitutes a bacterial species, these analyses indicate that all Microcystis species warrant placement into the same species complex since the average nucleotide identity values were above 95%, 16S rRNA nucleotide identity scores exceeded 99%, and DNA–DNA hybridization was consistently greater than 70%. The review further provides evidence from around the globe for the key role that both nitrogen and phosphorus play in controlling Microcystis bloom dynamics, and the effect of elevated temperature on bloom intensification. Finally, highlighted is the ability of Microcystis assemblages to minimize their mortality losses by resisting grazing by zooplankton and bivalves, as well as viral lysis, and discuss factors facilitating assemblage resilience.     

Competition for Light between Toxic and Nontoxic Strains of the Harmful Cyanobacterium Microcystis

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.     

Evidence of freshwater algal toxins in marine shellfish: Implications for human and aquatic health

The occurrence of freshwater harmful algal bloom toxins impacting the coastal ocean is an emerging threat, and the potential for invertebrate prey items to concentrate toxin and cause harm to human and wildlife consumers is not yet fully recognized. We examined toxin uptake and release in marine mussels for both particulate and dissolved phases of the hepatotoxin microcystin, produced by the freshwater cyanobacterial genus Microcystis. We also extended our experimental investigation of particulate toxin to include oysters (Crassostrea sp.) grown commercially for aquaculture. California mussels (Mytilus californianus) and oysters were exposed to Microcystis and microcystin toxin for 24 h at varying concentrations, and then were placed in constantly flowing seawater and sampled through time simulating riverine flushing events to the coastal ocean. Mussels exposed to particulate microcystin purged the toxin slowly, with toxin detectable for at least 8 weeks post-exposure and maximum toxin of 39.11 ng/g after exposure to 26.65 μg/L microcystins. Dissolved toxin was also taken up by California mussels, with maximum concentrations of 20.74 ng/g after exposure to 7.74 μg/L microcystin, but was purged more rapidly. Oysters also took up particulate toxin but purged it more quickly than mussels. Additionally, naturally occurring marine mussels collected from San Francisco Bay tested positive for high levels of microcystin toxin. These results suggest that ephemeral discharge of Microcystis or microcystin to estuaries and the coastal ocean accumulate in higher trophic levels for weeks to months following exposure.     

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

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.