Shifts in phytoplankton and zooplankton communities in three cyanobacteria-dominated lakes after treatment with hydrogen peroxide.

Cyanobacteria can reach high densities in eutrophic lakes, which may cause problems due to their potential toxin production. Several methods are in use to prevent, control or mitigate harmful cyanobacterial blooms. Treatment of blooms with low concentrations of hydrogen peroxide (H2O2) is a promising emergency method. However, effects of H2O2 on cyanobacteria, eukaryotic phytoplankton and zooplankton have mainly been studied in controlled cultures and mesocosm experiments, while much less is known about the effectiveness and potential side effects of H2O2 treatments on entire lake ecosystems. In this study, we report on three different lakes in the Netherlands that were treated with average H2O2 concentrations ranging from 2 to 5 mg L-1 to suppress cyanobacterial blooms. Effects on phytoplankton and zooplankton communities, on cyanotoxin concentrations, and on nutrient availability in the lakes were assessed. After every H2O2 treatment, cyanobacteria drastically declined, sometimes by more than 99%, although blooms of Dolichospermum sp., Aphanizomenon sp., and Planktothrix rubescens were more strongly suppressed than a Planktothrix agardhii bloom. Eukaryotic phytoplankton were not significantly affected by the H2O2 additions and had an initial advantage over cyanobacteria after the treatment, when ample nutrients and light were available. In all three lakes, a new cyanobacterial bloom developed within several weeks after the first H2O2 treatment, and in two lakes a second H2O2 treatment was therefore applied to again suppress the cyanobacterial population. Rotifers strongly declined after most H2O2 treatments except when the H2O2 concentration was ≤ 2 mg L-1, whereas cladocerans were only mildly affected and copepods were least impacted by the added H2O2. In response to the treatments, the cyanotoxins microcystins and anabaenopeptins were released from the cells into the water column, but disappeared after a few days. We conclude that lake treatments with low concentrations of H2O2 can be a successful tool to suppress harmful cyanobacterial blooms, but may negatively affect some of the zooplankton taxa in lakes. We advise pre-tests prior to the treatment of lakes to define optimal treatment concentrations that kill the majority of the cyanobacteria and to minimize potential side effects on non-target organisms. In some cases, the pre-tests may discourage treatment of the lake.

The harmful cyanobacterium Microcystis aeruginosa PCC7806 is more resistant to hydrogen peroxide at elevated CO2.

Rising atmospheric CO2 can intensify harmful cyanobacterial blooms in eutrophic lakes. Worldwide, these blooms are an increasing environmental concern. Low concentrations of hydrogen peroxide (H2O2) have been proposed as a short-term but eco-friendly approach to selectively mitigate cyanobacterial blooms. However, sensitivity of cyanobacteria to H2O2 can vary depending on the available resources. To find out how cyanobacteria respond to H2O2 under elevated CO2, Microcystis aeruginosa PCC 7806 was cultured in chemostats with nutrient-replete medium under C-limiting and C-replete conditions (150 ppm and 1500 ppm CO2, respectively). Microcystis chemostats exposed to high CO2 showed higher cell densities, biovolumes, and microcystin contents, but a lower photosynthetic efficiency and pH compared to the cultures grown under low CO2. Subsamples of the chemostats were treated with different concentrations of H2O2 (0-10 mg·L-1 H2O2) in batch cultures under two different light intensities (15 and 100 µmol photons m-2·s-1) and the response in photosynthetic vitality was monitored during 24 h. Results showed that Microcystis was more resistant to H2O2 at elevated CO2 than under carbon-limited conditions. Both low and high CO2-adapted cells were more sensitive to H2O2 at high light than at low light. Microcystins (MCs) leaked out of the cells of cultures exposed to 2-10 mg·L-1 H2O2, while the sum of intra- and extracellular MCs decreased. Although both H2O2 and CO2 concentrations in lakes vary in response to many factors, these results imply that it may become more difficult to suppress cyanobacterial blooms in eutrophic lakes when atmospheric CO2 concentrations continue to rise.

Sensitivity of phytoplankton, zooplankton and macroinvertebrates to hydrogen peroxide treatments of cyanobacterial blooms.

Addition of hydrogen peroxide (H2O2) is a promising method to acutely suppress cyanobacterial blooms in lakes. However, a reliable H2O2 risk assessment to identify potential effects on non-target species is currently hampered by a lack of appropriate ecotoxicity data. The aim of the present study was therefore to quantify the responses of a wide diversity of freshwater phytoplankton, zooplankton and macroinvertebrates to H2O2 treatments of cyanobacterial blooms. To this end, we applied a multifaceted approach. First, we investigated the 24-h toxicity of H2O2 to three cyanobacteria (Planktothrix agardhii, Microcystis aeruginosa, Anabaena sp.) and 23 non-target species (six green algae, eight zooplankton and nine macroinvertebrate taxa), using EC50 values based on photosynthetic yield for phytoplankton and LC50 values based on mortality for the other organisms. The most sensitive species included all three cyanobacterial taxa, but also the rotifer Brachionus calyciflores and the cladocerans Ceriodaphnia dubia and Daphnia pulex. Next, the EC50 and LC50 values obtained from the laboratory toxicity tests were used to construct a species sensitivity distribution (SSD) for H2O2. Finally, the species predicted to be at risk by the SSD were compared with the responses of phytoplankton, zooplankton and macroinvertebrates to two whole-lake treatments with H2O2. The predictions of the laboratory-based SSD matched well with the responses of the different taxa to H2O2 in the lake. The first lake treatment, with a relatively low H2O2 concentration and short residence time, successfully suppressed cyanobacteria without major effects on non-target species. The second lake treatment had a higher H2O2 concentration with a longer residence time, which resulted in partial suppression of cyanobacteria, but also in a major collapse of rotifers and decreased abundance of small cladocerans. Our results thus revealed a trade-off between the successful suppression of cyanobacteria at the expense of adverse effects on part of the zooplankton community. This delicate balance strongly depends on the applied H2O2 dosage and may affect the decision whether to treat a lake or not.

Resilience of Microbial Communities after Hydrogen Peroxide Treatment of a Eutrophic Lake to Suppress Harmful Cyanobacterial Blooms.

Applying low concentrations of hydrogen peroxide (H2O2) to lakes is an emerging method to mitigate harmful cyanobacterial blooms. While cyanobacteria are very sensitive to H2O2, little is known about the impacts of these H2O2 treatments on other members of the microbial community. In this study, we investigated changes in microbial community composition during two lake treatments with low H2O2 concentrations (target: 2.5 mg L-1) and in two series of controlled lake incubations. The results show that the H2O2 treatments effectively suppressed the dominant cyanobacteria Aphanizomenon klebahnii, Dolichospermum sp. and, to a lesser extent, Planktothrix agardhii. Microbial community analysis revealed that several Proteobacteria (e.g., Alteromonadales, Pseudomonadales, Rhodobacterales) profited from the treatments, whereas some bacterial taxa declined (e.g., Verrucomicrobia). In particular, the taxa known to be resistant to oxidative stress (e.g., Rheinheimera) strongly increased in relative abundance during the first 24 h after H2O2 addition, but subsequently declined again. Alpha and beta diversity showed a temporary decline but recovered within a few days, demonstrating resilience of the microbial community. The predicted functionality of the microbial community revealed a temporary increase of anti-ROS defenses and glycoside hydrolases but otherwise remained stable throughout the treatments. We conclude that the use of low concentrations of H2O2 to suppress cyanobacterial blooms provides a short-term pulse disturbance but is not detrimental to lake microbial communities and their ecosystem functioning.

Sensitivity to hydrogen peroxide of the bloom-forming cyanobacterium Microcystis PCC 7806 depends on nutrient availability.

Application of low concentrations of hydrogen peroxide (H2O2) is a relatively new and promising method to selectively suppress harmful cyanobacterial blooms, while minimizing effects on eukaryotic organisms. However, it is still unknown how nutrient limitation affects the sensitivity of cyanobacteria to H2O2. In this study, we compare effects of H2O2 on the microcystin-producing cyanobacterium Microcystis PCC 7806 under light-limited but nutrient-replete conditions, nitrogen (N) limitation and phosphorus (P) limitation. Microcystis was first grown in chemostats to acclimate to these different experimental conditions, and subsequently transferred to batch cultures where they were treated with a range of H2O2 concentrations (0-10 mg L-1) while exposed to high light (100 µmol photons m-2 s-1) or low light (15 µmol photons m-2 s-1). Our results show that, at low light, N- and P-limited Microcystis were less sensitive to H2O2 than light-limited but nutrient-replete Microcystis. A significantly higher expression of the genes encoding for anti-oxidative stress enzymes (2-cys-peroxiredoxin, thioredoxin A and type II peroxiredoxin) was observed prior to and after the H2O2 treatment for both N- and P-limited Microcystis, which may explain their increased resistance against H2O2. At high light, Microcystis was more sensitive to H2O2 than at low light, and differences in the decline of the photosynthetic yield between nutrient-replete and nutrient-limited Microcystis exposed to H2O2 were less pronounced. Leakage of microcystin was stronger and faster from nutrient-replete than from N- and P-limited Microcystis. Overall, this study provides insight in the sensitivity of harmful cyanobacteria to H2O2 under various environmental conditions.

Suppressing Cyanobacteria with Hydrogen Peroxide Is More Effective at High Light Intensities.

Hydrogen peroxide (H2O2) can be used as an emergency method to selectively suppress cyanobacterial blooms in lakes and drinking water reservoirs. However, it is largely unknown how environmental parameters alter the effectiveness of H2O2 treatments. In this study, the toxic cyanobacterial strain Microcystis aeruginosa PCC 7806 was treated with a range of H2O2 concentrations (0 to 10 mg/L), while being exposed to different light intensities and light colors. H2O2 treatments caused a stronger decline of the photosynthetic yield in high light than in low light or in the dark, and also a stronger decline in orange than in blue light. Our results are consistent with the hypothesis that H2O2 causes major damage at photosystem II (PSII) and interferes with PSII repair, which makes cells more sensitive to photoinhibition. Furthermore, H2O2 treatments caused a decrease in cell size and an increase in extracellular microcystin concentrations, indicative of leakage from disrupted cells. Our findings imply that even low H2O2 concentrations of 1-2 mg/L can be highly effective, if cyanobacteria are exposed to high light intensities. We therefore recommend performing lake treatments during sunny days, when a low H2O2 dosage is sufficient to suppress cyanobacteria, and may help to minimize impacts on non-target organisms.

Rapid adaptation of harmful cyanobacteria to rising CO2.

Rising atmospheric CO2 concentrations are likely to affect many ecosystems worldwide. However, to what extent elevated CO2 will induce evolutionary changes in photosynthetic organisms is still a major open question. Here, we show rapid microevolutionary adaptation of a harmful cyanobacterium to changes in inorganic carbon (Ci) availability. We studied the cyanobacterium Microcystis, a notorious genus that can develop toxic cyanobacterial blooms in many eutrophic lakes and reservoirs worldwide. Microcystis displays genetic variation in the Ci uptake systems BicA and SbtA, where BicA has a low affinity for bicarbonate but high flux rate, and SbtA has a high affinity but low flux rate. Our laboratory competition experiments show that bicA + sbtA genotypes were favored by natural selection at low CO2 levels, but were partially replaced by the bicA genotype at elevated CO2 Similarly, in a eutrophic lake, bicA + sbtA strains were dominant when Ci concentrations were depleted during a dense cyanobacterial bloom, but were replaced by strains with only the high-flux bicA gene when Ci concentrations increased later in the season. Hence, our results provide both laboratory and field evidence that increasing carbon concentrations induce rapid adaptive changes in the genotype composition of harmful cyanobacterial blooms.

Diel Variation in Gene Expression of the CO2-Concentrating Mechanism during a Harmful Cyanobacterial Bloom.

Dense phytoplankton blooms in eutrophic waters often experience large daily fluctuations in environmental conditions. We investigated how this diel variation affects in situ gene expression of the CO2-concentrating mechanism (CCM) and other selected genes of the harmful cyanobacterium Microcystis aeruginosa. Photosynthetic activity of the cyanobacterial bloom depleted the dissolved CO2 concentration, raised pH to 10, and caused large diel fluctuations in the bicarbonate and O2 concentration. The Microcystis population consisted of three Ci uptake genotypes that differed in the presence of the low-affinity and high-affinity bicarbonate uptake genes bicA and sbtA. Expression of the bicarbonate uptake genes bicA, sbtA, and cmpA (encoding a subunit of the high-affinity bicarbonate uptake system BCT1), the CCM transcriptional regulator gene ccmR and the photoprotection gene flv4 increased at first daylight and was negatively correlated with the bicarbonate concentration. In contrast, genes of the two CO2 uptake systems were constitutively expressed, whereas expression of the RuBisCO chaperone gene rbcX, the carboxysome gene ccmM, and the photoprotection gene isiA was highest at night and down-regulated during daytime. In total, our results show that the harmful cyanobacterium Microcystis is very responsive to the large diel variations in carbon and light availability often encountered in dense cyanobacterial blooms.

How rising CO2 and global warming may stimulate harmful cyanobacterial blooms.

Climate change is likely to stimulate the development of harmful cyanobacterial blooms in eutrophic waters, with negative consequences for water quality of many lakes, reservoirs and brackish ecosystems across the globe. In addition to effects of temperature and eutrophication, recent research has shed new light on the possible implications of rising atmospheric CO2 concentrations. Depletion of dissolved CO2 by dense cyanobacterial blooms creates a concentration gradient across the air-water interface. A steeper gradient at elevated atmospheric CO2 concentrations will lead to a greater influx of CO2, which can be intercepted by surface-dwelling blooms, thus intensifying cyanobacterial blooms in eutrophic waters. Bloom-forming cyanobacteria display an unexpected diversity in CO2 responses, because different strains combine their uptake systems for CO2 and bicarbonate in different ways. The genetic composition of cyanobacterial blooms may therefore shift. In particular, strains with high-flux carbon uptake systems may benefit from the anticipated rise in inorganic carbon availability. Increasing temperatures also stimulate cyanobacterial growth. Many bloom-forming cyanobacteria and also green algae have temperature optima above 25°C, often exceeding the temperature optima of diatoms and dinoflagellates. Analysis of published data suggests that the temperature dependence of the growth rate of cyanobacteria exceeds that of green algae. Indirect effects of elevated temperature, like an earlier onset and longer duration of thermal stratification, may also shift the competitive balance in favor of buoyant cyanobacteria while eukaryotic algae are impaired by higher sedimentation losses. Furthermore, cyanobacteria differ from eukaryotic algae in that they can fix dinitrogen, and new insights show that the nitrogen-fixation activity of heterocystous cyanobacteria can be strongly stimulated at elevated temperatures. Models and lake studies indicate that the response of cyanobacterial growth to rising CO2 concentrations and elevated temperatures can be suppressed by nutrient limitation. The greatest response of cyanobacterial blooms to climate change is therefore expected to occur in eutrophic and hypertrophic lakes.

Strains of the Harmful Cyanobacterium Microcystis aeruginosa Differ in Gene Expression and Activity of Inorganic Carbon Uptake Systems at Elevated CO2 Levels.

Cyanobacteria are generally assumed to be effective competitors at low CO2 levels because of their efficient CO2-concentrating mechanism (CCM), and yet how bloom-forming cyanobacteria respond to rising CO2 concentrations is less clear. Here, we investigate changes in CCM gene expression at ambient CO2 (400 ppm) and elevated CO2 (1,100 ppm) in six strains of the harmful cyanobacterium Microcystis. All strains downregulated cmpA encoding the high-affinity bicarbonate uptake system BCT1, whereas both the low- and high-affinity CO2 uptake genes were expressed constitutively. Four strains downregulated the bicarbonate uptake genes bicA and/or sbtA, whereas two strains showed constitutive expression of the bicA-sbtA operon. In one of the latter strains, a transposon insert in bicA caused low bicA and sbtA transcript levels, which made this strain solely dependent on BCT1 for bicarbonate uptake. Activity measurements of the inorganic carbon (Ci) uptake systems confirmed the CCM gene expression results. Interestingly, genes encoding the RuBisCO enzyme, structural carboxysome components, and carbonic anhydrases were not regulated. Hence, Microcystis mainly regulates the initial uptake of inorganic carbon, which might be an effective strategy for a species experiencing strongly fluctuating Ci concentrations. Our results show that CCM gene regulation of Microcystis varies among strains. The observed genetic and phenotypic variation in CCM responses may offer an important template for natural selection, leading to major changes in the genetic composition of harmful cyanobacterial blooms at elevated CO2.

Potassium sensitivity differs among strains of the harmful cyanobacterium Microcystis and correlates with the presence of salt tolerance genes.

Microcystis aeruginosa is a ubiquitous harmful cyanobacterium that causes problems in eutrophic lakes. Potassium ion (K(+)) addition is one of the suggested methods to combat harmful cyanobacterial blooms. To investigate the effectiveness of this method, we compared the potassium ion sensitivity of four Microcystis strains. Microcystis strains PCC 7005 and NIES-843 were very susceptible to potassium ion concentrations of ∼ 12 mmol L(-1), whereas strain PCC 7806 and its non-toxic mutant PCC 7806 ΔmcyB were not affected by added potassium ions. The origin of the strain appears to be of importance. Strain PCC 7806 originates from brackish water and possesses genes for the synthesis of the compatible solute sucrose, the water channel protein gene aqpZ and the sodium influx gene nhaS2, whereas strains PCC 7005 and NIES-843 have a freshwater origin and lack these genes. We conclude that potassium ion addition will not be a successful mitigation strategy in brackish waters, but may temporarily suppress Microcystis blooms in freshwater lakes. However, in the long run other Microcystis strains or other cyanobacteria with a higher salt tolerance will likely take over. In addition, our results also have implications for the potassium ion concentrations of mineral media used in laboratory studies with cyanobacteria.

Changes in gene expression, cell physiology and toxicity of the harmful cyanobacterium Microcystis aeruginosa at elevated CO2.

Rising CO2 concentrations may have large effects on aquatic microorganisms. In this study, we investigated how elevated pCO2 affects the harmful freshwater cyanobacterium Microcystis aeruginosa. This species is capable of producing dense blooms and hepatotoxins called microcystins. Strain PCC 7806 was cultured in chemostats that were shifted from low to high pCO2 conditions. This resulted in a transition from a C-limited to a light-limited steady state, with a ~2.7-fold increase of the cyanobacterial biomass and ~2.5-fold more microcystin per cell. Cells increased their chlorophyll a and phycocyanin content, and raised their PSI/PSII ratio at high pCO2. Surprisingly, cells had a lower dry weight and contained less carbohydrates, which might be an adaptation to improve the buoyancy of Microcystis when light becomes more limiting at high pCO2. Only 234 of the 4691 genes responded to elevated pCO2. For instance, expression of the carboxysome, RuBisCO, photosystem and C metabolism genes did not change significantly, and only a few N assimilation genes were expressed differently. The lack of large-scale changes in the transcriptome could suit a buoyant species that lives in eutrophic lakes with strong CO2 fluctuations very well. However, we found major responses in inorganic carbon uptake. At low pCO2, cells were mainly dependent on bicarbonate uptake, whereas at high pCO2 gene expression of the bicarbonate uptake systems was down-regulated and cells shifted to CO2 and low-affinity bicarbonate uptake. These results show that the need for high-affinity bicarbonate uptake systems ceases at elevated CO2. Moreover, the combination of an increased cyanobacterial abundance, improved buoyancy, and higher toxin content per cell indicates that rising atmospheric CO2 levels may increase the problems associated with the harmful cyanobacterium Microcystis in eutrophic lakes.

Genetic diversity of inorganic carbon uptake systems causes variation in CO2 response of the cyanobacterium Microcystis.

Rising CO2 levels may act as an important selective factor on the CO2-concentrating mechanism (CCM) of cyanobacteria. We investigated genetic diversity in the CCM of Microcystis aeruginosa, a species producing harmful cyanobacterial blooms in many lakes worldwide. All 20 investigated Microcystis strains contained complete genes for two CO2 uptake systems, the ATP-dependent bicarbonate uptake system BCT1 and several carbonic anhydrases (CAs). However, 12 strains lacked either the high-flux bicarbonate transporter BicA or the high-affinity bicarbonate transporter SbtA. Both genes, bicA and sbtA, were located on the same operon, and the expression of this operon is most likely regulated by an additional LysR-type transcriptional regulator (CcmR2). Strains with only a small bicA fragment clustered together in the phylogenetic tree of sbtAB, and the bicA fragments were similar in strains isolated from different continents. This indicates that a common ancestor may first have lost most of its bicA gene and subsequently spread over the world. Growth experiments showed that strains with sbtA performed better at low inorganic carbon (Ci) conditions, whereas strains with bicA performed better at high Ci conditions. This offers an alternative explanation of previous competition experiments, as our results reveal that the competition at low CO2 levels was won by a specialist with only sbtA, whereas a generalist with both bicA and sbtA won at high CO2 levels. Hence, genetic and phenotypic variation in Ci uptake systems provide Microcystis with the potential for microevolutionary adaptation to changing CO2 conditions, with a selective advantage for bicA-containing strains in a high-CO2 world.