Microcystin Release Research Articles

Delayed Release of Intracellular Microcystin Following Partial Oxidation of Cultured and Naturally Occurring Cyanobacteria.

Oxidation processes can provide an effective barrier to eliminate cyanotoxins by damaging cyanobacteria cell membranes, releasing intracellular cyanotoxins, and subsequently oxidizing these toxins (now in extracellular form) based on published reaction kinetics. In this work, cyanobacteria cells from two natural blooms (from the United States and Canada) and a laboratory-cultured Microcystis aeruginosa strain were treated with chlorine, monochloramine, chlorine dioxide, ozone, and potassium permanganate. The release of microcystin was measured immediately after oxidation (t ≤ 20 min), and following oxidant residual quenching (stagnation times = 96 or 168 h). Oxidant exposures (CT) were determined resulting in complete release of intracellular microcystin following chlorine (21 mg-min/L), chloramine (72 mg-min/L), chlorine dioxide (58 mg-min/L), ozone (4.1 mg-min/L), and permanganate (391 mg-min/L). Required oxidant exposures using indigenous cells were greater than lab-cultured Microcystis. Following partial oxidation of cells (oxidant exposures ≤ CT values cited above), additional intracellular microcystin and dissolved organic carbon (DOC) were released while the samples remained stagnant in the absence of an oxidant (>96 h after quenching). The delayed release of microcystin from partially oxidized cells has implications for drinking water treatment as these cells may be retained on a filter surface or in solids and continue to slowly release cyanotoxins and other metabolites into the finished water.

Impact of Hydrogen Peroxide and Copper Sulfate on the Delayed Release of Microcystin

Algicides, like hydrogen peroxide and copper sulfate, are commonly applied to recreational waters and drinking water sources to mitigate cyanobacterial blooms. In this work, the effects of hydrogen peroxide and copper sulfate were evaluated in two natural bloom samples (collected from Canadian and American waterbodies) and one lab-cultured Microcystis aeruginosa suspended in Colorado River water. Five algicide to dissolved organic carbon (DOC) dose ratios were evaluated during an initial exposure period of 24 h. One dose ratio (0.4 H2O2:DOC or 0.25 CuSO4:DOC) was then evaluated during stagnation after quenching (hydrogen peroxide) or extended exposure (copper sulfate) for up to 96 or 168 h. During the initial hydrogen peroxide exposure, the CA bloom had no release of intracellular microcystins (MCs) and the USA bloom only released MC at 4 H2O2:DOC. The reverse occurred with copper sulfate, where the CA bloom released MCs at 0.6 CuSO4:DOC but the USA bloom had no detectable extracellular MCs. Extracellular MC was released from the lab-cultured Microcystis at the lowest hydrogen peroxide and copper sulfate doses. In the hydrogen peroxide stagnation experiment, intracellular MC decreased in the USA bloom after 168 h despite the low dose applied. Similarly, the extended copper sulfate exposure led to intracellular MC decreases in both bloom samples after 168 h, despite showing no impact during the initial 24 h monitoring period. The lab-cultured Microcystis was again less resistant to both algicides, with releases observed after less than 2 h of stagnation or exposure. The damage to cells as measured by pigments during these experiments did not match the MC data, indicating that blooms with depressed pigment levels can still be a risk to nearby drinking water sources or recreational activities. These results provide insight on the timeline (up to one week) required for monitoring the potential release of MCs after algicide application.

Salt Shock Responses of Microcystis Revealed through Physiological, Transcript, and Metabolomic Analyses.

The transfer of Microcystis aeruginosa from freshwater to estuaries has been described worldwide and salinity is reported as the main factor controlling the expansion of M. aeruginosa to coastal environments. Analyzing the expression levels of targeted genes and employing both targeted and non-targeted metabolomic approaches, this study investigated the effect of a sudden salt increase on the physiological and metabolic responses of two toxic M. aeruginosa strains separately isolated from fresh and brackish waters, respectively, PCC 7820 and 7806. Supported by differences in gene expressions and metabolic profiles, salt tolerance was found to be strain specific. An increase in salinity decreased the growth of M. aeruginosa with a lesser impact on the brackish strain. The production of intracellular microcystin variants in response to salt stress correlated well to the growth rate for both strains. Furthermore, the release of microcystins into the surrounding medium only occurred at the highest salinity treatment when cell lysis occurred. This study suggests that the physiological responses of M. aeruginosa involve the accumulation of common metabolites but that the intraspecific salt tolerance is based on the accumulation of specific metabolites. While one of these was determined to be sucrose, many others remain to be identified. Taken together, these results provide evidence that M. aeruginosa is relatively salt tolerant in the mesohaline zone and microcystin (MC) release only occurs when the capacity of the cells to deal with salt increase is exceeded.

The influence of extracellular polymeric substances on the coagulation process of cyanobacteria

The hydrophobicity and flocculation properties of cyanobacterial cells are closely related to their extracellular polymeric substances (EPS). During the treatment of drinking water, the coagulation and removal of EPS-wrapped cyanobacterial particles from natural water sources is very difficult. In this work, a series of surface characteristics of cyanobacterial cells with different EPS fractions were analyzed to evaluate their influences on the coagulation process. With the removal of EPS, the coagulation efficiency of cyanobacteria was gradually improved. The intracellular microcystin release showed that the cyanobacterial cells in each EPS removal phase were almost intact with few broken cells. The surface of cyanobacterial cells had higher hydrophobicity and lower zeta potentials with each step of the EPS extraction, which improved the ratio of particles that were in an unstable state. Furthermore, the deeper the EPS extraction phase, the larger the decreased in size of cyanobacterial particles, thus increasing their specific surface area for adsorption with coagulant. It was concluded that the coagulation mechanism of EPS-wrapped cyanobacterial particles was: the cyanobacterial cells were first peeled off through attraction by opposite charges from the coagulant, and then they were adsorbed before settling down. This study provides a scientific basis for the removal of cyanobacteria by enhancing coagulation.

Antibiotic contaminants reduced the treatment efficiency of UV-C on Microcystis aeruginosa through hormesis

Antibiotic contaminants exert stimulatory hormetic effects in cyanobacteria at low (ng L−1) concentrations, which may interfere with the control of cyanobacterial bloom in aquatic environments exhibiting combined pollution. This study investigated the influence of a mixture of four popular antibiotics (sulfamethoxazole, amoxicillin, ciprofloxacin, and tetracycline) during the application of UV-C irradiation for controlling the bloom of Microcystis aeruginosa. In the absence of antibiotics, 100–500 mJ cm−2 UV-C irradiation reduced cell density, growth rate, chlorophyll a content, Fv/Fm value and microcystin concentration in M. aeruginosa in a dose-dependent manner through the downregulation of proteins related to cell division, chlorophyll synthesis, photosynthesis and microcystin synthesis. UV-C irradiation stimulated microcystin release through the upregulation of the microcystin release regulatory protein (mcyH). The presence of 40 ng L−1 antibiotic mixture during UV-C treatment significantly reduced (p < 0.05) the treatment efficiency of 100–300 mJ cm−2 UV-C on microcystin concentration, while 80 and 160 ng L−1 antibiotic mixture significantly reduced (p < 0.05) the treatment efficiency of 100–500 mJ cm−2 UV-C on cell density and microcystin concentration. The antibiotic mixture alleviated the toxicity of UV-C on M. aeruginosa through a significant stimulation of photosynthetic activity (p < 0.05) and the upregulation of proteins involved in photosynthesis, biosynthesis, protein expression, and DNA repair. Microcystin release in UV-C-treated cyanobacterial cells was further stimulated by the antibiotic mixture through the upregulation of mcyH and four ATP-binding cassette transport proteins. The interference effects of antibiotic contaminants should be fully considered when UV-C is applied to control cyanobacterial bloom in antibiotic-polluted environments. In order to eliminate the interference effects of antibiotics, the concentration of each target antibiotic is suggested to be controlled below 5 ng L−1 before the application of UV-C irradiation.

Resistance of cyanobacteria Microcystis aeruginosa to erythromycin with multiple exposure

Here we report a set of experiments in which water blooming cyanobacteria Microcystis aeruginosa was repeatedly exposed to erythromycin. Growth inhibition increased with increasing erythromycin concentration (1–150 μg/L) upon first exposure. Maximum inhibition rate (76.06%), occurred under 150 μg/L erythromycin. Moreover, 96-h 50% effective concentration (EC50) was 22.97 μg/L, indicating that the growth of M. aeruginosa was affected by erythromycin under common environmental concentrations. Photosynthesis was hindered by chlorophyll and photosystem II limitations. Malondialdehyde, reactive oxygen species, and superoxide dismutase contents increased significantly under certain concentrations of erythromycin, but superoxide dismutase was suppressed by 150 μg/L erythromycin. Synthesis of intracellular and extracellular microcystins was promoted by 10–60 and by 20–60 μg/L erythromycin, respectively, but both were inhibited by 100–150 μg/L. Principal component analysis and Pearson’s correlation revealed the accumulation of reactive oxygen species as the dominant mechanism of erythromycin toxicity to cells. M. aeruginosa repeatedly subjected to erythromycin exposure showed obvious resistance against the antibiotic, especially when treated twice with 60 μg/L erythromycin. The 96-h EC50 was 81.29 μg/L. As compared to the first exposure to erythromycin, photosynthetic and antioxidant activities increased, while growth inhibition and oxidation stress decreased upon multiple exposures. Production and release of microcystins were enhanced by repeated exposure to the antibiotic. Thus, erythromycin persistence in water should be examined, as repeated exposure may lead to serious environmental and human health hazards.

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.

Impacts of antibiotic contaminants on Microcystis aeruginosa during potassium permanganate treatment

Application of KMnO4 for preventing the formation of cyanobacterial bloom at early growth stage has not been reported. Antibiotics generate hormesis effects in cyanobacteria at currently reported concentrations, which may negatively affect the control of cyanobacterial bloom. This study assessed the treatment performance of KMnO4 in Microcystis aeruginosa with and without the existence of the antibiotic mixture composed of four simultaneously detected antibiotics in aquatic environments (sulfamethoxazole, ciprofloxacin, amoxicillin and tetracycline). KMnO4 downregulated two chlorophyll a synthetases (chlG and chlM), 14 photosynthesis-related proteins and two microcystin synthetases (mcyB and mcyD) in M. aeruginosa, and reduced chlorophyll a content, photosynthetic activity and microcystin concentration in a dose-dependent manner. Inhibition of photosynthesis and biosynthesis resulted in extended lag phase and decreased growth rate in KMnO4-treated Microcystis aeruginosa. In contrast, mixed antibiotics upregulated 6 oxidation-reduction proteins, a cell division regulatory protein (MAE_37210), 14 photosynthesis-related proteins, 14 biosynthesis-related proteins (including microcystin synthetases mcyA and mcyB) and a microcystin transport protein (mcyH), which consequently reduced oxidative stress, shortened lag phase as well as significantly stimulated (p < 0.05) cyanobacterial growth, photosynthetic activity, microcystin synthesis and microcystin release in KMnO4-treated M. aeruginosa. An optimal dose of 3 mg L−1 was suggested for KMnO4 treatment. Mixed antibiotics should be controlled below a no-impact threshold of 20 ng L−1 (5 ng L-1 for each antibiotic) for eliminating their adverse effects during KMnO4 treatment of cyanobacteria in antibiotics polluted environments.

Nanoplastics Promote Microcystin Synthesis and Release from Cyanobacterial Microcystis aeruginosa.

Although the fate of nanoplastics (<100 nm) in freshwater systems is increasingly well studied, much less is known about its potential threats to cyanobacterial blooms, the ultimate phenomenon of eutrophication occurrence worldwide. Previous studies have evaluated the consequences of nanoplastics increasing the membrane permeability of microbes, however, there is no direct evidence for interactions between nanoplastics and microcystin; intracellular hepatotoxins are produced by some genera of cyanobacteria. Here, we show that the amino-modified polystyrene nanoplastics (PS-NH2) promote microcystin synthesis and release from Microcystis aeruginosa, a dominant species causing cyanobacterial blooms, even without the change of coloration. We demonstrate that PS-NH2 inhibits photosystem II efficiency, reduces organic substance synthesis, and induces oxidative stress, enhancing the synthesis of microcystin. Furthermore, PS-NH2 promotes the extracellular release of microcystin from M. aeruginosa via transporter protein upregulation and impaired cell membrane integrity. Our findings propose that the presence of nanoplastics in freshwater ecosystems might enhance the threat of eutrophication to aquatic ecology and human health.

Proteomic mechanisms for the stimulatory effects of antibiotics on Microcystis aeruginosa during hydrogen peroxide treatment

Application of low dosage of H2O2 at early stage of cyanobacterial life cycle is a promising route for cyanobacterial bloom mitigation, which could minimize adverse effects on non-target organisms. Besides, influence of co-existing contaminants on cyanobacterial bloom mitigation under combined pollution conditions remains unclear. This study assessed the influence of a mixture of four frequently detected antibiotics (tetracycline, sulfamethoxazole, ciprofloxacin and amoxicillin) during H2O2 treatment of Microcystis aeruginosa at early growth stage. H2O2 significantly (p < 0.05) inhibited growth rate, chlorophyll a content, Fv/Fm and rETRmax in a dose-dependent manner at low doses of 0.25–1 mg L−1, through downregulating proteins involved in cell division, cellular component organization, gene expression and photosynthesis. Although H2O2 increased microcystin content in each cyanobacterial cell through the upregulation of microcystin synthetases (mcyC and mcyF), total microcystin concentration in H2O2 treated groups was significantly (p < 0.05) reduced due to the decrease of cell density. Existence of 80 and 200 ng L−1 mixed antibiotics during H2O2 treatment facilitated the scavenging of ROS by antioxidant enzymes and significantly (p < 0.05) stimulated growth, photosynthesis, microcystin synthesis and microcystin release in H2O2 treated cells, through the upregulation of proteins involved in photosynthesis, oxidation-reduction process, biosynthesis, gene expression and transport. Mixed antibiotics increased the hazard of M. aeruginosa during H2O2 treatment, through the stimulation of microcystin synthesis and release at the proteomic level. Each target antibiotic should be controlled below 5 ng L−1 before the application of H2O2 for eliminating the interference of antibiotics on cyanobacterial bloom mitigation.

Assessment of Constructed Wetlands’ Potential for the Removal of Cyanobacteria and Microcystins (MC-LR)

Microcystis blooms and the subsequent release of hepatotoxic microcystins (MCs) pose a serious threat to the safety of water for human and livestock consumption, agriculture irrigation, and aquaculture worldwide. Microcystin-LR (MC-LR), the most toxic variant of MCs, has been widely detected in a variety of environments such as water, sediments, plants, and many aquatic organisms. Conventional solutions of water treatment are costly, requiring specific infrastructure, as well as specialized personnel and equipment. Therefore, these solutions are not feasible in many rural areas or in the treatment of large reservoirs. In this regard, low-cost and low-technology solutions, such as constructed wetlands (CWs), are attractive solutions to treat surface waters contaminated with toxic cyanobacteria blooms from lakes, ponds, reservoirs, and irrigation systems. In line with this, the main aim of this work was to evaluate the potential of CWs for the treatment of water contaminated with MC-LR produced by Microcystis aeruginosa—LEGE 91094. For that, microcosms (0.4 × 0.3 × 0.3 m) simulating CWs were assembled with Phragmites australis to treat lake water contaminated with Microcystis aeruginosa cells and MCs. Results showed removal percentages of M. aeruginosa cells above 94% and about 99% removal of MC-LR during 1 week treatment cycles. CWs maintained their functions, regardless the presence of MC-LR in the system, and also showed significant removal of nutrients (ammonium ion removal up to 86%) and organic matter (removal reaching 98%). The present work indicates that CWs have the potential for removal of cyanobacterial cells and cyanotoxins, which can be useful for the treatment of eutrophic waters and provide water of sufficient quality to be used, for instance, in agriculture.

Programmed Cell Death-Like and Accompanying Release of Microcystin in Freshwater Bloom-Forming Cyanobacterium Microcystis: From Identification to Ecological Relevance.

Microcystis is the most common freshwater bloom-forming cyanobacterium. Its massive blooms not only adversely affect the functionality of aquatic ecosystems, but are also associated with the production of microcystins (MCs), a group of potent toxins that become a threat to public health when cell-bound MCs are significantly released from the dying Microcystis into the water column. Managing Microcystis blooms thus requires sufficient knowledge regarding both the cell death modes and the release of toxins. Recently, more and more studies have demonstrated the occurrence of programmed cell death-like (or apoptosis-like) events in laboratory and field samples of Microcystis. Apoptosis is a genetically controlled process that is essential for the development and survival of metazoa; however, it has been gradually realized to be an existing phenomenon playing important ecological roles in unicellular microorganisms. Here, we review the current progress and the existing knowledge gap regarding apoptosis-like death in Microcystis. Specifically, we focus first on the tools utilized to characterize the apoptosis-related biochemical and morphological features in Microcystis. We further outline various stressful stimuli that trigger the occurrence of apoptosis and discuss the potential mechanisms of apoptosis in Microcystis. We then propose a conceptual model to describe the functional coupling of apoptosis and MC in Microcystis. This model could be useful for understanding both roles of MC and apoptosis in this species. Lastly, we conclude the review by highlighting the current knowledge gap and considering the direction of future research. Overall, this review provides a recent update with respect to the knowledge of apoptosis in Microcystis and also offers a guide for future investigations of its ecology and survival strategies.

Release of Microcystin from Microcystis sp. by Pre-Oxidation and Removal of Microcystin by Advanced Drinking Water Treatment

Objectives The objectives of this study were to evaluate the characteristics of microcystins (MCs) release form Microcystis sp. in surface water during pre-oxidation process using chlorine and ozone and it’s removal by advanced water treatment process as post process when raw water containing high Microcystis sp. is introduced into the water treatment plant. Methods Raw water which had contained over 400,000 cells/mL of Microcystis sp. were used to evaluate the characteristics of MCs released from Microcyctis sp. and chlorination (Cl2) and ozonation (O3) were used as pre-oxidation process and O3, O3/H2O2, UV/H2O2 and biological activated carbon (BAC) were used as post oxidation process. Results and Discussion As the concentration of chlorine increased, the concentration of released MCs in the water increased, but decreased when higher than 5 mg/L of chlorine dosage. Released MCs were consisted of almost MC-RR (57∼86%) and MC–LR (11∼29%). In the ozone treatment after prechlorination process, the MCs concentration was increased by several fold to dozen fold to compare before ozonation. However, when chlorine dosage was over 3 mg/L, MCs were rapidly removed as the ozone concentration increased. The MCs concentration of flocculation/sedimentation treated sample after the pre-oxidation was increased more than two times before treatment, the MCs was continuously released during the coagulation/sedimentation process from the damaged Microcystis sp. due to the prior oxidation process. In the post-oxidation process, O3 and O3/H2O2 processes were more effective for removing MCs than the UV/H2O2 process, and O3 and O3/H2O2 processes removed 100% of the MCs when O3 was added above 1 mg/L. 100% and 93% were removed respectively at 15 minutes of EBCT (empty bed contact time) in the BAC process, when the MCs concentrations of influent were 0.8 µg/L and 2.8 µg/L. The biodegradability of MC-LR was lower than that of MC-RR. It was necessary to increase the EBCT of the BAC process when the concentrations of MCs or the ratio of MC-LR were high. Conclusions Although, pre-oxidation treatment to the raw water containing high concentration of Microcystis release higher MCs concentration in the oxidized water, that can be removed by post O3, O3/H2O2, UV/H2O2 and BAC processes. The removal rate of MCs by O3 process was higher than that of UV-AOP process, and 93∼99% of MCs removed with 15 min of EBCT in BAC process. In the drinking water treatment plant where the pre-oxidation process is applied to improve the efficiency of the flocculation/sedimentation process during the blooming season of Microcystis sp., O3/BAC process after the flocculation/sedimentation process is recommendable to MCs remove. Key words: Microcystis sp., Microcystin, Pre-Oxidation, Release, Advanced Oxidation Process (AOP), Biological Activated Carbon (BAC)

Evaluation of disruption/permeabilization methodologies for Microcystis aeruginosa as alternatives to obtain high yields of microcystin release

The extraction of the wide range of useful bioactive compounds produced by cyanobacteria is still a major bottleneck at industrial scale. In addition to the high costs, extraction efficiencies are also commonly low, with low cell disruption efficiencies playing a particularly significant role in intracellular compounds' release. To increase the chances of an extended use of the cyanobacteria toxin microcystin in several biotechnological fields, we aimed to optimize five different disruption techniques: bead milling, microwave, freeze-thaw cycles, high-speed homogenization, and sonication. For each of the methods tested, the conditions that maximized the intracellular organic matter release were: i) 20% of beads and treatment time of 7 min (bead milling); ii) 800 W for 1.5 min (microwave); iii) three 12-h freeze-thaw cycles at −20 °C; iv) 15,000 rpm for 7 min (high-speed homogenization); and v) 40 kHz for 10 min (sonication).Sonication and freeze-thaw cycles followed by sonication revealed to be the most effective methodologies to ensure a maximum intracellular organic matter release and, consequently, microcystin availability for being extracted. The decrease of cells' viability was however more evident in freeze-thaw cycles, freeze-thaw cycles followed by sonication, and microwave where only 0.3, 0.05 and 0.9% of the initial cells, respectively, maintained their viability after being treated. On the other hand, sonication and bead milling reduced the viability of the original culture to 5 and 15.5%, respectively, while high-speed homogenization did not show any significant differences compared to control.According to the results obtained in this study, the most suitable methodology to maximize the release of microcystin was therefore the use of sonication (40 kHz) during 10 min.

Long-term exposure to antibiotic mixtures favors microcystin synthesis and release in Microcystis aeruginosa with different morphologies

The ecological risks of antibiotics in aquatic environments have raised great concerns worldwide, but the chronic effect of antibiotic contaminants on cyanotoxin production and release remains unclear. This study investigated the long-term combined effects of spiramycin (SP) and ampicillin (AMP) on microcystin (MC) production and release in both unicellular and colonial Microcystis aeruginosa (MA) through semi-continuous exposure test. At exposure concentration of 300 ng L−1, MA growth rates were stimulated till the end of exponential phase accompanied with the up-regulation of photosynthesis-related gene. The exponential growth phases of unicellular and colonial MA were prolonged for 2 and 4 days, respectively. The stimulation rate of growth rate and MC content in unicellular MA were significantly higher than that in colonial MA. The highest concentrations of intracellular MC (IMC) and extracellular MC (EMC) were observed in the binary mixture at equivalent SP/AMP ratio (1:1). The promotion of IMC concentration was in consistent with the stimulated expression of MC-synthesis-related gene and nitrogen-transport-related gene. The malondialdehyde content and activities of superoxide dismutase and catalase in unicellular MA were significantly higher than those in colonial MA. The EMC concentration and the antioxidant responses of both unicellular and colonial MA significantly increased with exposure time. Long-term exposure to mixture of SA and AMP at environmentally relevant concentrations would aggravate the disturbance to aquatic ecosystem balance through the stimulation of MA proliferation as well as the promotion of MC production and release.

Effects of titanium dioxide nanoparticles on Microcystis aeruginosa and microcystins production and release.

Due to growing production and use, release of nanoparticles (NPs) into the aquatic environment may pose a hazard to ecosystem. In this study, Microcystis aeruginosa was exposed to different concentrations (0.1, 1, 10, 50, 100 mg/L) of titanium dioxide (TiO2) NPs to assess their impact on algae. Meanwhile, the production and release of microcystins (MCs) was determined. Results showed that TiO2 NPs significantly decreased the maximal photochemical efficiency of photosystem II, and thus inhibited the photosynthetic activity of M. aeruginosa. They also increased the content of reactive oxygen species (ROS) and malondialdehyde (MDA), indicating their oxidative damage on algae. Besides, TiO2 NPs at high concentrations (50 and 100 mg/L) aggregated on the algal surface and block the light, herein inhibited algae growth (16.03%±2.50% and 54.13%±0.93%) but induced the production (25.02%±1.23% and 114.43%±2.96%) and release (20.96%±13.30% and 12.10%±8.80%) of MCs. These results indicated that high concentrations of TiO2 NPs increased MCs concentration in water system, which may be harmful to aquatic ecosystem.

Impacts of Identified Bacterium Ensifer adhaerens on Microcystis aeruginosa and Subsequent Microcystin Release

The release of microcystin and dissolved organic matter (DOM) during algae-lysing process draws much attention. In this study, Ensifer adhaerens (bacterium r23) was screened for algicidal activity against Microcystis aeruginosa (MA). The effects of dosage (2.04 × 106, 3.90 × 106, and 7.15 × 106 CFU ml−1) and dosing modes (single, double, and quadruple treatments totaling 7.15 × 106 CFU ml−1) on the release of microcystin-LR (MC-LR) and DOM were investigated. Besides cell density, intra/extra-cellular, and total MC-LR were measured during the lysing treatment. The DOM components were analyzed by parallel factor analysis (PARAFAC). Results show that the lowest dosage (2.04 × 106 CFU ml−1) not only stimulated MA growth during the initial 4 days but also triggered the production of more toxins, resulting in higher total MC-LR than the controls on 2–16 days. The higher dosages suppressed MA growth and MC-LR production simultaneously, and the total MC-LR content was substantially lower than those of the controls during the whole experimental period. The total MC-LR for the double and quadruple treatments were lower than the controls after 30-day treatment, but still higher than the single dosage. Both intracellular and extracellular DOM (IDOM, EDOM) of MA had five components, namely tyrosine-like (C1), tryptophan-like (C2 and C3), fulvic acid-like (C4), and humic acid-like (C5) substances. C1 and C3 in the EDOM resulted from MA lysis and increased along with the treatment. C2 in the EDOM came from the nutrient broth and could be consumed by r23. C4 and C5 in the EDOM mainly resulted from the growth of r23.

Influence of mixed antibiotics on Microcystis aeruginosa during the application of glyphosate and hydrogen peroxide algaecides.

Antibiotics regulate various physiological functions in cyanobacteria and may interfere with the control of cyanobacterial blooms during the application of algaecides. In this study, Microcystis aeruginosa was exposed to H2 O2 and glyphosate for 7d in the presence of coexisting mixed antibiotics (amoxicillin, spiramycin, tetracycline, ciprofloxacin, and sulfamethoxazole) at an environmentally relevant concentration of 100ng·L-1 . The mixed antibiotics significantly (P<0.05) alleviated the growth inhibition effect of 15-45μM H2 O2 and 40-60mg·L-1 glyphosate. According to the increased contents of chlorophyll a and protein, decreased content of malondialdehyde, and decreased activities of superoxide dismutase and glutathione S-transferase, antibiotics may reduce the toxicity of the two algaecides through the stimulation of photosynthesis and the reduction in oxidative stress. The presence of coexisting antibiotics stimulated the production and release of microcystins in the M.aeruginosa exposed to low concentrations of algaecides and posed an increased threat to aquatic environments. To eliminate the secondary pollution caused by microcystins, high algaecide doses that are ≥45μM for H2 O2 and ≥60mg·L-1 for glyphosate are recommended. This study provides insights into the ecological hazards of antibiotic contaminants and the best management practices for cyanobacterial removal under combined antibiotic pollution conditions.

Interactions between Microcystis aeruginosa and coexisting bisphenol A at different nitrogen levels.

Microcystis aeruginosa is known as a main contributor of cyanobacterial bloom. However, factors that drive its formation and dispersion remain poorly understood. The cellular-level responses to nutrient drivers of eutrophication were investigated. The results showed that growth rate of M. aeruginosa was significantly enhanced with the increasing bisphenol A (BPA) and nitrogen (N) level. Stress of BPA significantly inhibited cellular density, chlorophyll-a content across all the nutrient conditions, while Fv/Fm and rETRmax value were promoted by BPA. Responses of reactive oxygen species (ROS) value, superoxide dismutase (SOD) activity and malodialdehyde (MDA) content indicated that nitrogen deficiency and BPA caused oxidative stress to M. aeruginosa. Besides, nitrogen and BPA regulated the production and release of microcystins (MCs). M. aeruginosa exposed to BPA caused 95 up-regulated proteins, which was primarily associated with photosynthesis, nitrogen metabolism, glycolysis/glyconeogenesis and carbon fixation in photosynthetic organisms. The 91 down-regulated proteins were related to quorum sensing, longevity regulating and cell cycle-caulobacter, confirming that the driving force of regulating the change of cellular density and genes expression weakened. These findings provide important clues to elucidate the combined regulatory mechanisms of cyanobacterial blooms triggered by endocrine-disrupting compounds and environmental factors and help to effectively prevent and reduce cyanobacterial blooms.

Microcystin Levels in Selected Cyanobacteria Exposed to Varying Salinity.

Microcystins produced by cyanobacteria pose a great threat to human health by releasing toxins upon cell death. In the present study, we studied microcystin production in the cyanobacterial strains Anabaena cylindrica (B629 and 2949) and Fremyella diplosiphon (SF33) exposed to 1, 2 and 4 g/L sodium chloride (NaCl). Cultures grown for 7 days in BG11/HEPES medium were pelleted, re-grown in the corresponding NaCl levels, and enzyme linked immunosorbent assay (ELISA) performed. ELISA assays revealed enhanced microcystin production in A. cylindrica B629 exposed to 4 g/L NaCl and A. cylindrica 29414 exposed to 2 and 4 g/L NaCl, after growth in the corresponding NaCl levels for 14 days. We observed a significant decrease (p >0.05) in microcystin levels in the control strains after exposure to NaCl for 5 days. After exposure to 1, 2, or 4 g/L NaCl for 10 days, no microcystin release was observed in A. cylindrica B629, A. cylindrica 29414 or F. diplosiphon SF33. Sodium dodecyl sulfate polyacrylamide gel electrophoresis identified the presence of an additional band at 120 – 130 kDa in A. cylindrica B629 exposed to 2 and 4 g/L NaCl, and at 14 kDa in cultures amended with 1 and 2 g/L NaCl as well as the untreated control, indicating that exposure to salinity induces alterations in protein expression.