Assessment of estrogenic potential from exudates of microcystin-producing and non-microcystin-producing Microcystis by metabolomics, machine learning and E-screen assay.

Cyanobacterial blooms, often dominated by Microcystis aeruginosa, are capable of producing estrogenic effects. It is important to identify specific estrogenic compounds produced by cyanobacteria, though this can prove challenging owing to the complexity of exudate mixtures. In this study, we used untargeted metabolomics to compare components of exudates from microcystin-producing and non-microcystin-producing M. aeruginosa strains that differed with respect to their ability to produce microcystins, and across two growth phases. We identified 416 chemicals and found that the two strains produced similar components, mainly organoheterocyclic compounds (20.2%), organic acids and derivatives (17.3%), phenylpropanoids and polyketides (12.7%), benzenoids (12.0%), lipids and lipid-like molecules (11.5%), and organic oxygen compounds (10.1%). We then predicted estrogenic compounds from this group using random forest machine learning. Six compounds (daidzin, biochanin A, phenylethylamine, rhein, o-Cresol, and arbutin) belonging to phenylpropanoids and polyketides (3), benzenoids (2), and organic oxygen compound (1) were tested and exhibited estrogenic potency based upon the E-screen assay. This study confirmed that both Microcystis strains produce exudates that contain compounds with estrogenic properties, a growing concern in cyanobacteria management.

Seaweed Essential Oils as a New Source of Bioactive Compounds for Cyanobacteria Growth Control: Innovative Ecological Biocontrol Approach.

The application of natural compounds extracted from seaweeds is a promising eco-friendly alternative solution for harmful algae control in aquatic ecosystems. In the present study, the anti-cyanobacterial activity of three Moroccan marine macroalgae essential oils (EOs) was tested and evaluated on unicellular Microcystis aeruginosa cyanobacterium. Additionally, the possible anti-cyanobacterial response mechanisms were investigated by analyzing the antioxidant enzyme activities of M. aeruginosa cells. The results of EOs GC-MS analyses revealed a complex chemical composition, allowing the identification of 91 constituents. Palmitic acid, palmitoleic acid, and eicosapentaenoic acid were the most predominant compounds in Cystoseira tamariscifolia, Sargassum muticum, and Ulva lactuca EOs, respectively. The highest anti-cyanobacterial activity was recorded for Cystoseira tamariscifolia EO (ZI = 46.33 mm, MIC = 7.81 µg mL-1, and MBC = 15.62 µg mL-1). The growth, chlorophyll-a and protein content of the tested cyanobacteria were significantly reduced by C. tamariscifolia EO at both used concentrations (inhibition rate >67% during the 6 days test period in liquid media). Furthermore, oxidative stress caused by C. tamariscifolia EO on cyanobacterium cells showed an increase of the activities of superoxide dismutase (SOD) and catalase (CAT), and malondialdehyde (MDA) concentration was significantly elevated after 2 days of exposure. Overall, these experimental findings can open a promising new natural pathway based on the use of seaweed essential oils to the fight against potent toxic harmful cyanobacterial blooms (HCBs).

Growth, physiological responses and microcystin-production/-release dynamics of Microcystis aeruginosa exposed to various luteolin doses.

By testing time-dependent IC50 of luteolin against Microcystis growth, this study revealed 6.5 mg/L as nearly IC50 value during prolonged stress until day 14, and explored chlorophyll-a (CLA) and phycobiliproteins (PBPs) contents, antioxidant responses and microcystin (MC)-production/-release dynamics at rising luteolin doses (0.5~2-fold IC50). Growth inhibition ratio (GIR) generally rose at rising luteolin dose, while at each dose GIR firstly increased and then leveled off or dropped. In early stage, CLA, allophycocyanin (APC), phycoerythrin (PE) and glutathione (GSH) contents, and superoxide dismutase (SOD) and catalase (CAT) activities, were increasingly stimulated at rising luteolin dose to enhance energy yield and antioxidant defense, but Microcystis was damaged more severely at rising dose, due to stress-repair imbalance. Such more severe damage in early stage, coupled with stronger PBPs-inhibition in mid-late stage, at rising dose could jointly account for rising GIR at rising dose. The CAT/GSH-stimulation persisting until late stage could alleviate cell damage in late stage, which explained for why GIR no longer increased in late stage at each luteolin dose. Besides, more MCs were produced and retained in cell to exert protective roles against luteolin-stress in early stage, but intracellular MCs decreased following inhibited MC-production by prolonged stress to decrease cell protectant. Extracellular MCs detection showed that less MCs amount existed in water phase than control along luteolin-stress, implying luteolin as eco-friendly algaecide with promising potential to remove MPM blooms and MC-risks. This is the first study to reveal the effect of various luteolin doses on MC-production/release and PBP-synthesis dynamics of Microcystis during prolonged stress. The findings shed novel views in anti-algal mechanisms of luteolin, and provided direct evidence for luteolin applied as safe agent to remediate Microcystis-dominant blooms.

Suppression of water-bloom cyanobacterium Microcystis aeruginosa by algaecide hydrogen peroxide maximized through programmed cell death.

The global expansion and intensification of toxic cyanobacterial blooms require effective algaecides. Algaecides should be selective, effective, fast-acting, and ideally suppress cyanotoxin production. In this study, whether both maximum growth suppression and minimal toxin production can be simultaneously achieved was tested with a selective algaecide H2O2, through its ability to induce apoptosis-like programmed cell death (AL PCD) in a common bloom species Microcystis aeruginosa. Under doses of 1-15 mg L-1, non-monotonic dose-response suppression of H2O2 on M. aeruginosa were observed, where maximal cell death and minimal microcystin production both occurred at a moderate dose of 10 mg L-1 H2O2. Maximal cell death was indeed achieved through AL PCD, as revealed by integrated biochemical, structural, physiological and transcriptional evidence; transcriptional profile suggested AL PCD was mediated by mazEF and lexA systems. Higher H2O2 doses directly led to necrosis in M. aeruginosa, while lower doses only caused recoverable stress. The integrated data showed the choice between the two modes of cell death is determined by the intracellular energy state under stress. A model was proposed for suppressing M. aeruginosa with AL PCD or necrosis. H2O2 was demonstrated to simultaneously maximize the suppression of both growth and microcystin production through triggering AL PCD.

Effect of Zinc on Microcystis aeruginosa UTEX LB 2385 and Its Toxin Production.

Cyanobacteria harmful algal blooms (CHABs) are primarily caused by man-made eutrophication and increasing climate-change conditions. The presence of heavy metal runoff in affected water systems may result in CHABs alteration to their ecological interactions. Certain CHABs produce by-products, such as microcystin (MC) cyanotoxins, that have detrimentally affected humans through contact via recreation activities within implicated water bodies, directly drinking contaminated water, ingesting biomagnified cyanotoxins in seafood, and/or contact through miscellaneous water treatment. Metallothionein (MT) is a small, metal-sequestration cysteine rich protein often upregulated within the stress response mechanism. This study focused on zinc metal resistance and stress response in a toxigenic cyanobacterium, Microcystis aeruginosa UTEX LB 2385, by monitoring cells with (0, 0.1, 0.25, and 0.5 mg/L) ZnCl2 treatment. Flow cytometry and phase contrast microscopy were used to evaluate physiological responses in cultures. Molecular assays and an immunosorbent assay were used to characterize the expression of MT and MC under zinc stress. The results showed that the half maximal inhibitory concentration (IC50) was 0.25 mg/L ZnCl2. Flow cytometry and phase contrast microscopy showed morphological changes occurred in cultures exposed to 0.25 and 0.5 mg/L ZnCl2. Quantitative PCR (qPCR) analysis of selected cDNA samples showed significant upregulation of Mmt through all time points, significant upregulation of mcyC at a later time point. ELISA MC-LR analysis showed extracellular MC-LR (µg/L) and intracellular MC-LR (µg/cell) quota measurements persisted through 15 days, although 0.25 mg/L ZnCl2 treatment produced half the normal cell biomass and 0.5 mg/L treatment largely inhibited growth. The 0.25 and 0.5 mg/L ZnCl2 treated cells demonstrated a ~40% and 33% increase of extracellular MC-LR(µg/L) equivalents, respectively, as early as Day 5 compared to control cells. The 0.5 mg/L ZnCl2 treated cells showed higher total MC-LR (µg/cell) quota yield by Day 8 than both 0 mg/L ZnCl2 control cells and 0.1 mg/L ZnCl2 treated cells, indicating release of MCs upon cell lysis. This study showed this Microcystis aeruginosa strain is able to survive in 0.25 mg/L ZnCl2 concentration. Certain morphological zinc stress responses and the upregulation of mt and mcy genes, as well as periodical increased extracellular MC-LR concentration with ZnCl2 treatment were observed.

Antibiotics promoted the recovery of Microcystis aeruginosa after UV-B radiation at cellular and proteomic levels.

Elevated UV-B radiation due to ozone layer depletion may prevent the growth of bloom-forming cyanobacteria in aquatic environments, while antibiotic contaminants may cause effects opposite to that of UV-B due to hormesis. This study investigated the influence of a quaternary antibiotic mixture on Microcystis aeruginosa after UV-B radiation through a 15-day exposure test. UV-B radiation extended the lag phase of M. aeruginosa at doses of 600 and 900 mJ/cm2, and significantly (p < 0.05) reduced the growth rate and the Fv/Fm value at doses of 300-900 mJ/cm2. Although UV-B radiation significantly (p < 0.05) stimulated the microcystin production ability in each cyanobacterial cell, the total microcystin concentration still significantly (p < 0.05) decreased due to the reduction of cell density. Mixed antibiotics and UV-B regulated the proteomic expression profile of M. aeruginosa in different manners. UV-B radiation upregulated 19 proteins and downregulated 49 proteins in M. aeruginosa, while mixed antibiotics upregulated 45 proteins and downregulated 25 proteins in UV-B treated cells. Mixed antibiotics significantly (p < 0.05) stimulated growth and photosynthesis, increased cell density and microcystin concentration, and reduced oxidative stress in UV-B treated cells through the upregulation of proteins involved in photosynthesis, biosynthesis, cell division, oxidation-reduction, gene expression and microcystin synthesis. This study verified the hypothesis that antibiotics accelerated the recovery of M. aeruginosa from UV-B induced damage. A safe threshold of 20 ng/L was suggested for mixed antibiotics (5 ng/L for each antibiotic), in order to eliminate the stimulatory effects of antibiotics on bloom-forming cyanobacteria.

Effects of phosphorus availability and phosphorus utilization behavior of Microcystis aeruginosa on its adaptation capability to ultraviolet radiation.

Phosphorus (P) plays a critical role in eutrophication and algal growth; therefore, improving our understanding of the impact of P is essential to control harmful algal blooms. In this study, Microcystis aeruginosa was treated with 5-h ambient irradiation in the medium with different dissolved inorganic P (DIP) concentrations, DIP-free, moderate-DIP, and high-DIP, to explore its growth and other physiological responses. Compared to photosynthetically active radiation (PAR), UV-A (320-400 nm) and UV-B (280-320 nm) radiation had inhibitive effects on the photosynthesis and growth of M. aeruginosa, while high P availability could alleviate or eliminate the negative effects of UV radiation. The photosynthetic parameters had a minimum reduction and quickly recovered after re-inoculation under high-DIP conditions. Confirmed by SEM, photosynthetic pigments, the generation of reactive oxygen species (ROS), superoxide dismutase (SOD) activity and other methods, ambient UV radiation exerted oxidative stresses rather than direct lethal effects on M. aeruginosa. Photosynthetic parameters indicated that algal UV-adaptation processes could include decreasing photo-induced damages and increasing self-repair efficiency. The P acquired by M. aeruginosa cells can have two function, which included alleviating UV-induced negative effects and sustaining algal growth. Consequently, UV-adaptation processes of M. aeruginosa resulted in an elevated demand for DIP, which resulted to increased P uptake rates and cellular P quota under moderate and high-DIP conditions. Therefore, the production of carotenoid and phycocyanin, and SOD activity increased under UV stress, leading to a better adaptation capability of M. aeruginosa and decreased negative effects of UV radiation on its growth. Overall, our findings demonstrated the significant interactive effects of P enrichment and irradiation on typical cyanobacteria, and the strong adaptation capability of M. aeruginosa in the eutrophic UV-radiated waters.

Evaluación anual del fitoplancton y su respuesta a la calidad de agua en el lago de Amatitlán, Guatemala / Annual evaluation of phytoplankton and its answer to water quality in Amatitlán lake, Guatemala

Se realizó un estudio sobre la composición y abundancia del fitoplancton en el lago de Amatitlán, y el efecto de la calidad de agua sobre su biodiversidad. Para ello, se colectaron muestras de agua en cuatro puntos específicos del lago, en la superficie del agua y a profundidades de 5, 10 y 20 m, de manera mensual durante el 2017. Se midieron parámetros fisicoquímicos in situ como temperatura y pH. Igualmente, se identificaron y contabilizaron cianobacterias y microalgas. El índice de estado trófico (IETP) catalogó al lago como eutrófico e hipertrófico (IETP = 63.80-88.18). Se reportan 34 géneros de fitoplancton distribuidos en 30 familias, 17 órdenes y 10 clases. Los indicadores biológicos, tales como, floraciones algales de Microcystis (38.41%), baja diversidad de diatomeas (Nitzschia, Aulacoseira y Cyclotella), presencia de microalgas Nitzschia y Scenedesmusresistentes a procesos de eutrofización, y alta concentración de coliformes fecales, de hasta 24,000 NMP/100 ml, evidenciaron la baja calidad de agua que se presenta en el lago de Amatitlán. En época seca se encontró más diversidad de microalgas debido a la mayor incidencia de radiación solar, el poco recambio de agua y la acumulación de materia orgánica. Aunque esto varía con los cambios en la concentración de nitrógeno total (NT) y fosforo total (PT), que potencian la proliferación de cianobacterias tóxicas. La biodiversidad del lago fue baja debido al estado hipereutrófico en que se encuentra. Se recomienda poner en funcionamiento plantas de tratamiento de aguas residuales para evitar que esta problemática continúe.

A study was carried out on the composition and abundance of phytoplankton in Amatitlán lake, and the effect of water quality on its biodiversity. For this, water samples were collected at four specific points in the lake, on the water surface and at depths of 5, 10 and 20 m, in a monthly way during 2017. Physicochemical parameters were measured in situ such as temperature and pH. Likewise, cyanobacteria and microalgae were identified and accounted. The trophic state index (IETP) cataloged the lake as eutrophic and hypertrophic (IETP = 63.80-88.18). 34 genera of phytoplankton distributed in 30 families, 17 orders and 10 classes are reported. Biological indicators, such as Microcystis algal blooms (38.41%), low diatom diversity (Nitzschia, Aulacoseira and Cyclotella), presence of Nitzschia and Scenedesmus microalgae resistant to eutrophication processes, and high concentration of fecal coliforms, up to 24,000 NMP/100 ml, evidenced the low quality of water that occurs in lake Amatitlán. In the dry season, more microalgae diversity was found due to the higher incidence of solar radiation, little water change and the accumulation of organic matter. Even though this varies with changes in the concentration of total nitrogen (NT) and total phosphorus (PT), which enhance the proliferation of toxic cyanobacteria. The lake's biodiversity was low due to its hypereutrophic state. We recommend to put this wastewater treatment plants into operation to prevent this problem to continue.

Dissolved organic phosphorus enhances arsenate bioaccumulation and biotransformation in Microcystis aeruginosa.

Only limited information is available on the effects of dissolved organic phosphorus (DOP) on arsenate (As(V)) bioaccumulation and biotransformation in organisms. In this study, we examined the influence of three different DOP forms (ß-sodium glycerophosphate (ßP), adenosine 5'-triphosphate (ATP), and D-Glucose-6-phosphate disodium (GP) salts) and inorganic phosphate (IP) on As(V) toxicity, accumulation, and biotransformation in Microcystis aeruginosa. Results showed that M. aeruginosa utilized the three DOP forms to sustain its growth. At a subcellular level, the higher phosphorus (P) distribution in metal-sensitive fractions (MSF) observed in the IP treatments could explain the comparatively lower toxic stress of algae compared to the DOP treatments. Meanwhile, the higher MSF distribution of arsenic (As) in M. aeruginosa in the presence of DOP could explain the higher toxicity with lower 96-h half maximal effective concentration (EC50) values. Although we observed As(V) and P discrimination in M. aeruginosa under IP treatments with high intracellular P/As, we did not find this discrimination under the DOP treatments. As accumulation in algal cells was therefore greatly enhanced by DOP, especially ßP, given its lower transformation rate to phosphate compared to ATP and GP in media. Additionally, As(V) reduction and, subsequently, As(III) methylation were greatly facilitated in M. aeruginosa by the presence of DOP, particularly GP, which was confirmed by the higher relative expression of its two functional genes (arsC and arsM). Our findings indicate that As(V) accumulation and its subsequent biotransformation were enhanced by organic P forms, which provides new insight into how DOP modulates As metabolism in algae.

Relationship between Photosynthetic Capacity and Microcystin Production in Toxic Microcystis Aeruginosa under Different Iron Regimes.

Blooms of harmful cyanobacteria have been observed in various water bodies across the world and some of them can produce intracellular toxins, such as microcystins (MCs), which negatively impact aquatic organisms and human health. Iron participates significantly in cyanobacterial photosynthesis and is proposed to be linked to MC production. Here, the cyanobacteria Microcystis aeruginosa was cultivated under different iron regimes to investigate the relationship between photosynthetic capacity and MC production. The results showed that iron addition increased cell density, cellular protein concentration and the Chl-a (chlorophyll-a) content. Similarly, it can also up⁻regulate photosynthetic capacity and promote MC⁻leucine⁻arginine (MC⁻LR) production, but not in a dose⁻dependent manner. Moreover, a significant positive correlation between photosynthetic capacity and MC production was observed, and electron transport parameters were the most important parameters contributing to the variation of intracellular MC⁻LR concentration revealed by Generalized Additive Model analysis. As the electron transport chain was affected by iron variation, adenosine triphosphate production was inhibited, leading to the alteration of MC synthetase gene expression. Therefore, it is demonstrated that MC production greatly relies on redox status and energy metabolism of photosynthesis in M. aeruginosa. In consequence, more attention should be paid to the involvement of photosynthesis in the regulation of MC production by iron variation in the future.

Branched Poly(ethylene imine)s as Anti-algal and Anti-cyanobacterial Agents with Selective Flocculation Behavior to Cyanobacteria over Algae.

Poly(ethylene imine)s (PEIs) have been widely studied for biomedical applications, including antimicrobial agents against potential human pathogens. The interactions of branched PEIs (B-PEIs) with environmentally relevant microorganisms whose uncontrolled growth in natural or engineered environments causes health, economic, and technical issues in many sectors of water management are studied. B-PEIs are shown to be potent antimicrobials effective in controlling the growth of environmentally relevant algae and cyanobacteria with dual-functionality and selectivity. Not only did they effectively inhibit growth of both algae and cyanobacteria, mostly without causing cell death (static activity), but they also selectively flocculated cyanobacteria over algae. Thus, unmodified B-PEIs provide a cost-effective and chemically facile framework for the further development of effective and selective antimicrobial agents useful for control of growth and separation of algae and cyanobacteria in natural or engineered environments.

Comprehensive assessment of three typical antibiotics on cyanobacteria (Microcystis aeruginosa): The impact and recovery capability.

This innovative study provided a comprehensive evaluation of the effects of three typical antibiotics exposures (cefradine, norfloxacin and amoxicillin) on Microcystis aeruginosa in two periods (exposure and post-exposure) at a new perspective. The results indicated that the irreversible growth inhibition of M. aeruginosa attributed to the norfloxacin in the exposure and the re-exposure stages. In contrast, although the algal cell size recovered to the control level after the exposure of 20 mg/L of cefradine, the significant stimulation on glutathione (GSH) still persisted even if the contaminants were removed. On the other hand, amoxicillin inhibited the activities of superoxide dismutase (SOD), GSH contents and the algal cell size in the exposure period while malonaldehyde (MDA) contents increased significantly in two periods.

Mathematical modeling of Microcystis aeruginosa growth and [D-Leu1] microcystin-LR production in culture media at different temperatures.

The effect of temperature (26°C, 28°C, 30°C and 35°C) on the growth of native CAAT-3-2005 Microcystis aeruginosa and the production of Chlorophyll-a (Chl-a) and Microcystin-LR (MC-LR) were examined through laboratory studies. Kinetic parameters such as specific growth rate (µ), lag phase duration (LPD) and maximum population density (MPD) were determined by fitting the modified Gompertz equation to the M. aeruginosa strain cell count (cellsmL-1). A 4.8-fold increase in µ values and a 10.8-fold decrease in the LPD values were found for M. aeruginosa growth when the temperature changed from 15°C to 35°C. The activation energy of the specific growth rate (Eµ) and of the adaptation rate (E1/LPD) were significantly correlated (R2=0.86). The cardinal temperatures estimated by the modified Ratkowsky model were minimum temperature=8.58±2.34°C, maximum temperature=45.04±1.35°C and optimum temperature=33.39±0.55°C. Maximum MC-LR production decreased 9.5-fold when the temperature was increased from 26°C to 35°C. The maximum production values were obtained at 26°C and the maximum depletion rate of intracellular MC-LR was observed at 30-35°C. The MC-LR cell quota was higher at 26 and 28°C (83 and 80fgcell-1, respectively) and the MC-LR Chl-a quota was similar at all the different temperatures (0.5-1.5fgng-1). The Gompertz equation and dynamic model were found to be the most appropriate approaches to calculate M. aeruginosa growth and production of MC-LR, respectively. Given that toxin production decreased with increasing temperatures but growth increased, this study demonstrates that growth and toxin production processes are uncoupled in M. aeruginosa. These data and models may be useful to predict M. aeruginosa bloom formation in the environment.

Impacts of Rac- and S-metolachlor on cyanobacterial cell integrity and release of microcystins at different nitrogen levels.

Pesticide residues and nitrogen overload (which caused cyanobacteria blooms) have been two serious environmental concerns. In particular, chiral pesticides with different structures may have various impacts on cyanobacteria. Nitrogen may affect the behavior between pesticides and cyanobacteria (e.g., increase the adverse effects of pesticides on cyanobacteria). This study evaluated the impacts of Rac- and S-metolachlor on the cell integrity and toxin release of Microcystis aeruginosa cells at different nitrogen levels. The results showed that (both of the configurations: Rac-, S-) metolachlor could inhibit M. aeruginosa cell growth under most conditions, and the inhibition rates were increased with the growing concentrations of nitrogen and metolachlor. However, cyanobacterial growth was promoted in 48 h under environmental relevant condition (1 mg/L metolachlor and 0.15 mg/L nitrogen). Therefore, the water authorities should adjust the treatment parameters to remove possible larger numbers of cyaonbacteria under that condition. On the other hand, the inhibition degree of M. aeruginosa cell growth by S-metolachlor treatments was obviously larger than Rac-metolachlor treatments. S-metolachlor also had a stronger ability in compromising M. aeruginosa cells than Rac-metolachlor treatments. Compared to control samples, more extracellular toxins (12%-86% increases) were detected after 5 mg/L S-metolachlor treatment for 72 h at different nitrogen levels, but the variations of extracellular toxins caused by 5 mg/L Rac-metolachlor addition could be neglected. Consequently, higher concentrations of metolachlor in source waters are harmful to humans, but it may prevent cyanobacterial blooms. However, the potential risks (e.g. build-up of extracellular toxins) should be considered.

Response of Microcystis aeruginosa BCCUSP 232 to barley (Hordeum vulgare L.) straw degradation extract and fractions.

The eutrophication of aquatic ecosystems is a serious environmental problem that leads to increased frequency of cyanobacterial blooms and concentrations of cyanotoxins. These changes in aquatic chemistry can negatively affect animal and human health. Environment-friendly methods are needed to control bloom forming cyanobacteria. We investigated the effect of Hordeum vulgare L. (barley) straw degradation extract and its fractions on the growth, oxidative stress, antioxidant enzyme activities, and microcystins content of Microcystis aeruginosa (Kützing) Kützing BCCUSP232. Exposure to the extract significantly (p<0.05) inhibited the growth of M. aeruginosa throughout the study, whereas only the highest concentration of fractions 1 and 2 significantly (p<0.05) reduced the growth of the cyanobacterium on day 10 of the experiment. The production of reactive oxygen species (ROS), lipid peroxidation and antioxidant enzyme activities were significantly (p<0.05) altered by the extract and fractions 1 and 2. Phytochemical profiling of the extract and its fractions revealed that the barley straw degradation process yielded predominantly phenolic acids. These results demonstrate that barley straw extract and its fractions can efficiently interfere with the growth and development of M. aeruginosa under laboratory conditions.

Transcriptional and Physiological Responses to Nutrient Loading on Toxin Formation and Photosynthesis in Microcystis Aeruginosa FACHB-905.

An important goal of understanding harmful algae blooms is to determine how environmental factors affect the growth and toxin formation of toxin-producing species. In this study, we investigated the transcriptional responses of toxin formation gene (mcyB) and key photosynthesis genes (psaB, psbD and rbcL) of Microcystis aeruginosa FACHB-905 in different nutrient loading conditions using real-time reverse transcription quantitative polymerase chain reaction (RT-qPCR). Three physio-biochemical parameters (malondialdehyde (MDA), superoxide dismutase (SOD) and glutathione (GSH)) were also evaluated to provide insight into the physiological responses of Microcystis cells. We observed an upregulation of mcyB gene in nutrient-deficient conditions, especially in nitrogen (N) limitation condition, and the transcript abundance declined after the nutrient were resupplied. Differently, high transcription levels were seen in phosphorus (P) deficient treatments for key photosynthesis genes throughout the culture period, while those in N-deficient cells varied with time, suggesting an adaptive regulation of Microsystis cells to nutrient stress. Increased contents of antioxidant enzymes (SOD and GSH) were seen in both N and P-deficient conditions, suggesting the presence of excess amount of free radical generation caused by nutrient stress. The amount of SOD and GSH continued to increase even after the nutrient was reintroduced and a strong correlation was seen between the MDA and enzyme activities, indicating the robust effort of rebalancing the redox system in Microcystis cells. Based on these transcriptional and physiological responses of M. aeruginosa to nutrient loading, these results could provide more insight into Microcystis blooms management and toxin formation regulation.

Enhanced toxicity to the cyanobacterium Microcystis aeruginosa by low-dosage repeated exposure to the allelochemical N-phenyl-1-naphthylamine.

It has been puzzling whether and how a plant could exert a strong allelopathic inhibition to the target organisms by releasing low concentrations of allelochemicals. Plant allelochemicals have been proposed to be released continuously, however, direct evidence from specific allelochemicals is urgently required. In the present study, the toxicity of allelochemical N-phenyl-1-naphthylamine (NPN) towards the cyanobacterium Microcystis aeruginosa by two different exposure patterns was compared. One was low-dosage repeated exposure (LRE), in which 50  µg L-1 NPN was repeatedly dosed to simulate the continual release of allelochemicals, and the other one was high-dosage single exposure (HSE) as per the routine toxicity assay. The results showed a significant growth inhibition to M. aeruginosa in the LRE group, where the inhibition rate reached above 90% from day 6 to day 9. The cell-membrane damage ratio increased from 64.05% on day 5 up to 96.60% on day 9. PSII photosynthesis activity expressed as Fv/Fm, ΦPSII, NPQ and ETRmax was also thoroughly inhibited in this group. Whereas the growth and PSII photosynthesis activity of M. aeruginosa in the HSE group were inhibited initially, but recovered gradually from day 4 or 5, which was accompanied by a continuous reduction of NPN content in culture solutions. Although NPN content in the LRE group was relatively lower, it remained at a more stable level throughout the experiment. These results indicate that continual release of low-dosage allelochemicals by aquatic plants plays crucial roles in their potent inhibition against cyanobacteria. Low-dosage continual exposure pattern needs to be investigated further.

[Selective Inhibition of Rice Straw Extract on Growth of Cyanobacteria and Chlorophyta].

Rice straw is supposed to be an environment-friendly biomaterial for inhibiting the growth of harmful blooms of the cyanobacterium Microcystis aeruginosa. The effects of rice straw extract(RSE) on algal growth, morphologic parameters(cell size), and physiological parameters(in vivo Chl-a fluorescence) were investigated using flow cytometry. We examined the selective inhibitory potential of rice straw on four cyanobacterial strains(toxic and non-toxic Microcystis aeruginosa, toxic Anabaena flos-aquae, and Microcystis ichthyoblabe), in comparison with inhibitory effects on three common freshwater green algae(Selenastrum capricornutum, Chlorella pyrenoidosa, and Scenedesmus obliqnus). Concentrations from 2.0 to 10.0 g·L-1 of RSE were found to efficiently inhibit the growth of cyanobacteria in a dose-dependent manner, simultaneously modifying the in vivo Chl-a fluorescence and cell size. The 50% growth-inhibition concentration(7 d) of A. flos-aquae, M. ichthyoblabe, M. aeruginosa(toxic strain), M. aeruginosa(non-toxic strain) was 1.72, 2.21, 2.92 and 5.72 g·L-1, respectively. Interestingly, the growth and cell size of C. pyrenoidosa and S. obliqnus increased with the addition of RSE and colony formation was observed. In the case of S. capricornutum, the inhibitory effect of RSE on growth and in vivo Chl-a fluorescence occurred at 1.0-4.0 g·L-1, while RSE induced a stimulatory effect on algal growth at 8.0-10.0 g·L-1. Taken together, the sensitivity of cyanobacteria to RSE was significantly higher than that of S. capricornutum, C. pyrenoidosa and S. obliqnus. The higher sensitivity of PSⅡ reaction center of cyanobacteria and the ability to form colonies of green algae may have important implications for the species-specific allelopathic antialgal activity of rice straw.

Prospecção de compostos orgânicos voláteis e seus efeitos na auto-regulação fisiológica em cianobactérias / Prospection of volatile organic compounds and their effects on physiological self-regulation in cyanobacteria

Apesar dos diversos estudos sobre a presença de cianobactérias e a correlação entre fatores ambientais que influenciam ou desencadeiam florações, é ainda incipiente a informação sobre o controle fisiológico e bioquímico da produção de metabólitos secundários, cianotoxinas e compostos orgânicos voláteis (COVs) nestes organismos. Os COVs mais comumente encontrados em cianobactérias são a geosmina e o 2- metil-isoborneol, compostos que resistem ao tratamento convencional da água, causam mau cheiro e alteram seu gosto, além de bioacumular em peixes e moluscos. Estudos sobre possíveis sistemas de competição (alelopatia) entre linhagens de cianobactérias, ou entre elas e outros organismos, podem contribuir para elucidação do papel da produção de COVs por cianobactérias. Dessa forma, os objetivos deste projeto foram (i) prospectar a produção de COVs e seus efeitos na auto-regulação fisiológica em cianobactérias mantidas em laboratório; e (ii) desenvolver um método analítico, por microextração em fase sólida (SPME) e cromatografia em fase gasosa com detecção por espectrometria de massas (GC-MS), para a determinação destes compostos. Foram realizados ensaios para avaliar os perfis de produção dos COVs em duas linhagens de M. aeruginosa em diferentes fases de crescimento, sob diferentes intensidades luminosas (50, 150 e 250 ?µmol.fótons.m-2.s-1) e também ao longo do ritmo circadiano, avaliando a influência dos períodos claro e escuro. Para avaliar efeitos alelopáticos, exsudatos de uma linhagem de M. aeruginosa produtora de microcistinas foram testados em culturas de outra linhagem de M. aeruginosa não produtora de toxinas por meio de técnicas tradicionais de cultivo com monitoramento do crescimento. Na análise da produção de COVs, por GC-MS, observou-se que se destacam, majoritariamente, os compostos α-ciclocitral, ß-ciclocitral e ß-ionona, sendo o ß-ciclocitral o mais abundante, em todas as condições testadas, para as ambas as linhagens estudadas. A linhagem não toxigênica, no entanto, apresentou produção mais elevada de todos os compostos identificados. Dentre as intensidades luminosas testadas, a intensidade de 250 µmol.fótons.m-2s-1 foi a que apresentou a maior taxa de crescimento para a linhagem LTPNA 08 e relação negativa entre o aumento da irradiância e a produção de ß-ciclocitral. Foram identificadas, também, variações na produção dos compostos α-ciclocitral, ß-ciclocitral e ß-ionona nas linhagens ao longo do ritmo circadiano, sendo as maiores concentrações encontradas no período escuro. Observou-se morte celular e redução na produção de COVs 24 horas após adição de exsudatos pertencentes à linhagem de M. aeruginosa toxigênica em cultivos da linhagem não-toxigênica. Sendo assim, pode-se inferir que a produção dos COVs pode sofrer alterações qualitativas e quantitativas dependendo do estímulo ambiental presente, tanto por interações bióticas (com outros organismos e ritmo circadiano), quanto por fatores abióticos (intensidade luminosa)

There are several studies on the presence of cyanobacteria and the correlation between environmental factors that may influence or trigger blooms. However, information concerning the physiological and biochemical control of the production of secondary metabolites, toxins and volatile organic compounds (VOC) by cyanobacteria is poorly understood. Geosmin and 2-methyl-isoborneolare are commonly found VOC in cyanobacteria, they resist to conventional water treatment and can cause bad smell and taste in the final water. In addition, VOC can bioaccumulate in fish and shellfish. Studies on possible competition systems (allelopathy) either among strains of cyanobacteria or among them and other organisms such as green microalgae, may help to elucidate the role of VOC production by cyanobacteria. Thus, the main objectives of this study are: (i) prospect the production of VOCs and their effects on physiological self-regulation in cyanocrobacteria kept in the laboratory; and (ii) to develop an analytical method, by solid phase microextraction (SPME) and gas chromatography with mass spectrometry detection (GC-MS), for the determination of these compounds. The assays were carried out to evaluate the production profiles of VOCs in two strains of M. aeruginosa at different growth stages under different light intensities (50, 150 and 250 µmol.fótons.m-2.s-1) and also along of the circadian rhythm, evaluating the influence of light and dark periods. To assess allelopathic effects, exudates from a microcystin-producing strain of M. aeruginosa were tested on cultures of another non-toxin producing M. aeruginosa strain by traditional growth monitoring culture techniques. In the analysis of VOC production by GC-MS, it was observed that α-cyclocyclal, ß-cyclocyclal and ß-ionone compounds were the most prominent, with ß-cyclocitral being the most abundant in all conditions tested, for both strains studied. The non-toxigenic lineage, however, showed higher production of all the identified compounds. Among the light intensities tested, the intensity of 250 µmol.fótons.m-2s-1 was the one with the highest growth rate and positive relation between the irradiance increase and the ß-cyclocitral production. Variations in the production of the α-cyclocyclal, ß-cyclocyclal and ß-ionone compounds were also identified in the lines along the circadian rhythm, being the highest concentrations found in the dark period. Cell death and reduction in VOC production were observed 24 hours after addition of exudates belonging to the toxigenic M. aeruginosa lineage in cultures of the non-toxigenic lineage. Thus, it can be inferred that the production of VOCs can undergo qualitative and quantitative changes depending on the environmental stimulus present, both by biotic interactions (with other organisms and circadian rhythm) and by abiotic factors (luminous intensity)

Determinação de fármacos em mananciais do estado de São Paulo e estudo da sua ecotoxicidade sobre a cianobactéria Microcystis aeruginosa / Pharmaceuticals determination in São Paulo stare springs and evaluation of their toxicity in cyanobacterium Microcystis aeruginosa

A contaminação de corpos d'água por fármacos é um tema de extrema relevância, tendo em vista problemas como a escassez de água, florações de cianobactérias tóxicas e lançamentos clandestinos de efluentes domésticos. Sendo assim, este trabalho teve como objetivo determinar a presença de cafeína (CAF), fluoxetina (FLX), levotiroxina (LVX) e bezafibrato (BZF) em mananciais do estado de São Paulo, bem como avaliar a toxicidade desses compostos à cianobactéria Microcystis aeruginosa LTPNA 08. Um método por LC-MS/MS foi desenvolvido e validado, de acordo com a RDC nº 166 da ANVISA, para a detecção de CAF, FLX, LVX e BZF em amostras ambientais. As represas Guarapiranga e Billings, bem como os rios Taiçupeba, Sorocaba, Baixo Cotia, Grande e Paraíba foram monitorados de abril a setembro de 2017. A toxicidade dos fármacos foi avaliada por meio do monitoramento do crescimento, produção de microcistinas e viabilidade celular da cianobactéria M. aeruginosa LTPNA 08. CAF foi detectada em todas as amostras analisadas, com concentrações que variaram de 6,6 ng.L-1 a 16,47 µg.L-1. No Rio Cotia foram verificadas as maiores concentrações de CAF, FLX e BZF (16,47 µg.L-1; 3,5 ng.L-1 e 322 ng.L-1, respectivamente). A LVX, cujos produtos de biotransformação não foram monitorados, não foi detectada em nenhuma amostra analisada. A concentração de 50 µg.L-1 de FLX inibiu o crescimento da cianobactéria em 82,3% (CE50: 31,4 µg.L-1). Em relação à produção de microcistinas totais, os fármacos inibiram a liberação da fração extracelular para a maior concentração testada ao longo do tempo de monitoramento, embora não tenham demonstrado efeito sobre a viabilidade celular. Sendo assim, considerando-se que fármacos estão presentes nos mananciais monitorados no estado de São Paulo e que a FLX pode causar efeito sobre a M. aeruginosa, os efeitos decorrentes da exposição a concentrações ambientais contínuas e cumulativas de fármacos em corpos d'água devem ser estudados. Além disso, uma vez que a ocorrência destas substâncias e outros contaminantes antropogênicos no ambiente aquático natural é uma questão emergente devido aos efeitos adversos potenciais que estes compostos representam para a vida aquática e os seres humanos, os tipos e níveis destes compostos, que têm um impacto maior na qualidade da água, deve ser constantemente monitorada. Práticas de gestão que investem em saneamento e na redução da descarga de efluentes não tratados, e um plano de proteção de recursos hídricos com o objetivo de garantir a segurança da água seriam medidas essenciais para reduzir o aporte de contaminantes nos corpos d'água do estado de São Paulo

Contamination of water bodies by drugs is a subject of extreme relevance considering related problems such as water scarcity, harmful cyanobacterial blooms and discharge of untreated domestic effluents. Therefore, the aim of this work was to determine the presence of caffeine (CAF), fluoxetine (FLX), levothyroxine (LVX) and bezafibrate (BZF) in springs in the State of São Paulo, and to evaluate the toxicity of these compounds in cyanobacteria Microcystis aeruginosa LTPNA 08. A LC-MS/MS method was developed and validated according to RDC nº 166 of ANVISA to assess the concentration of CAF, FLX, LVX and BZF in environmental samples. Guarapiranga and Billings reservoirs, as well as the Taiçupeba, Sorocaba, Baixo Cotia, Grande and Paraíba rivers were monitored from April to September 2017.The drugs toxicity in M. aeruginosa LTPNA 08 was assessed by monitoring their effects on cyanobacterial growth, microcystins production and cell viabilityby flow cytometry. CAF was detected in all analyzed samples at concentrations ranging from 6.6 ng to 16.47 µg.L-1.Among studied sites, Cotia river showed the highest concentrations of CAF, FLX and BZF (16.47 µg.L-1, 3.5 ng.L-1 and 322 ng.L-1, respectively). LVX, which biotransformation products were not monitored, was not detected in any of the analyzed samples. Regarding the drugs toxicity, 50 µg.L-1 of FLX inhibited the cyanobacterial grow thin 82.3% (EC50 of 31.4 µg.L-1). Although no effect on cell viability was seen by flow cytometry, the highest concentrations of all compounds tested were able to inhibit the release of microcystins. Therefore, considering that some of the drugs monitored showed to be present in water sources in São Paulo State and that FLX affects cyanobacteria M. aeruginosa growth, the effects of continuous and cumulative exposure at environmental drug concentrations of in water bodies should be evaluated. Also, since the occurrence of these substances and other anthropogenic contaminants in the natural aquatic environment is an emerging issue due to the potential adverse effects these compounds pose to aquatic life and humans, thet ypes and levels of these compounds, which have a greater impact on water quality, should be constantly monitored. Management practices investing in sanitation and in reducing discharge of untreated effluents, as well as a plan for water resources protection with the goal of ensuring water security would be essential measures in reducing drugs loading into water bodies situated in São Paulo State