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Seven new oleanane-type triterpene saponins, lysimaponins A-G, were isolated from aerial parts of Lysimachia laxa Baudo. Their chemical structures have been elucidated by analysis of spectroscopic and chemical methods. All compounds were evaluated for their antibacterial effects against Microcystis aeruginosa, Vibrio parahaemolyticus, V. harveyi, V. vulinificus, V. cholerae, and V. alginolyticus. All compounds showed potent antibacterial activities against the cyanobacteria M. aeruginosa with IC50 values ranging from 14.4 ± 1.2 to 35.3 ± 2.2 µg/mL. Compounds 1, 2, 4-7 inhibited V. parahaemolyticus with MIC values ranging from 64 to 256 µg/mL. The results suggested that saponins from L. laxa could be potential anti-cyanobacteria agents., (© The Author(s) 2024. Published by Oxford University Press on behalf of Japan Society for Bioscience, Biotechnology, and Agrochemistry.)

Aeruginosins are common metabolites of cyanobacteria. In the course of re-isolation of the known aeruginosins KT608A and KT608B for bioassay studies, we isolated three new sulfated aeruginosins, named aeruginosins KT688 ( 1 ), KT718 ( 2 ), and KT575 ( 3 ), from the extract of a Microcystis cell mass collected during the 2016 spring bloom event in Lake Kinneret, Israel. The structures of the new compounds were established on the basis of analyses of the 1D and 2D NMR, as well as HRESIMS data. Marfey's method, coupled with HR ESI LCMS and chiral HPLC, was used to establish the absolute configuration of the amino acid and hydroxyphenyl lactic acid residues, respectively. Compounds 1 - 3 were tested for inhibition of the serine protease trypsin, and compounds 1 and 2 were found to exhibit IC 50 values of 2.38 and 1.43 µM, respectively.

Microcystins (MCs) produced by Microcystis aeruginosa are harmful to animal and human health, and there is currently no effective method for their removal. Therefore, the development of biological approaches that inhibit cyanobacteria and remove MCs is needed. We identified strain MB1, confirmed as Morchella, using morphological and mole-cular evolution methods. To assess the impact of strain MB1 on M. aeruginosa, we conducted an experiment in which we inoculated M. aeruginosa with Morchella strain MB1. After their co-cultivation for 4‍ ‍d, the inoculation with 0.9696‍ ‍g MB1 completely inhibited and removed M. aeruginosa while concurrently removing up to 95% of the MC content. Moreover, within 3‍ ‍d of their co-cultivation, MB1 removed more than 50% of nitrogen and phosphorus from the M. aeruginosa solution. Therefore, the development of effective biological techniques for MC removal is paramount in safeguarding both the environment and human well-being. We herein successfully isolated MB1 from its natural habitat. This strain effectively inhibited and removed M. aeruginosa and also reduced the content of nitrogen and phosphorus in the M. aeruginosa solution. Most importantly, it exhibited a robust capability to eliminate MCs. The present results offer a new method and technical reference for mitigating harmful algal blooms.

Recently, harmful algal blooms (HABs) have become occurred with increasingly frequency worldwide. High nitrate content is one of the primary causes of eutrophication. Research has shown that photocatalytic materials enhance the effectiveness of microbial denitrification while removing other contaminants, despite some shortcomings. Based on this, we loaded TiO 2 /C 3 N 4 heterojunctions onto weaveable, flexible carbon fibers and established a novel photocatalytically enhanced microbial denitrification system for the simultaneous removal of harmful algae and Microcystin-LR. We found that 99.35% of Microcystis aeruginosa and 95.34% of MC-LR were simultaneously and effectively removed. Compared to existing denitrification systems, the nitrate removal capacity improved by 72.33%. The denitrifying enzyme activity and electron transport system activity of microorganisms were enhanced by 3.54-3.86 times. Furthermore, the microbial community structure was optimized by the regulation of photogenerated electrons, and the relative abundance of main denitrifying bacteria increased from 50.72% to 66.45%, including Proteobacteria and Bacteroidetes. More importantly, we found that the increased secretion of extracellular polymeric substances by microorganisms may be responsible for the persistence of the reinforcing effect caused by photogenerated electrons in darkness. The higher removal of Microcystis aeruginosa and Microcystin-LR (MC-LR) achieved by the proposed system would reduce the frequency of HAB outbreaks and prevent the associated secondary pollution., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Anabaenopeptins are common metabolites of cyanobacteria. In the course of reisolation of the known aeruginosins KT608A and KT608B for bioassay studies, we noticed the presence of some unknown anabaenopeptins in the extract of a Microcystis cell mass collected during the 2016 spring bloom event in Lake Kinneret, Israel. The 1 H NMR spectra of some of these compounds presented a significant difference in the appearance of the ureido bridge protons, and their molecular masses did not match any one of the 152 known anabaenopeptins. Analyses of the 1D and 2D NMR, HRMS, and MS/MS spectra of the new compounds revealed their structures as the hydantoin derivatives of anabaenopeptins A, B, F, and 1 [Dht]-anabaenopeptin A and oscillamide Y ( 1 , 2 , 3 , 6 , and 4 , respectively) and a new anabaenopeptin, 1 [Dht]-anabaenopeptin A ( 5 ). The known anabaenopeptins A, B, and F and oscillamide Y ( 7 , 8 , 9, and 10 , respectively) were present in the extract as well. We propose that 1 - 4 and 6 are the possible missing intermediates in the previously proposed partial biosynthesis route to the anabaenopeptins. Compounds 1 - 6 were tested for inhibition of the serine proteases trypsin and chymotrypsin and found inactive at a final concentration of ca. 54 μM.

While algal blooms occur frequently in lakes and reservoirs worldwide, the effects of dissolved organic matter (DOM) from lakeside and riparian zones on bloom formation are not well understood. In this study, we characterized the molecular composition of DOM from Cynodon dactylon (L.) Pers. (CD-DOM) and Xanthium sibiricum Patrin ex Widder (XS-DOM) and assessed their effects on the growth, physiology, volatile organic compounds (VOCs), and stable carbon isotope in four bloom-forming algae species (Microcystis aeruginosa, Anabaena sp., Chlamydomonas sp., and Peridiniopsis sp.). Stable carbon isotope analysis showed that the four species were affected by DOM. Both DOM types increased the cell biomass, polysaccharide and protein contents, chlorophyll fluorescence parameter values, and VOCs release of Anabaena sp., Chlamydomonas sp. and Microcystis aeruginosa, suggesting that DOM stimulated algal growth by increasing nutrient sources, photosynthetic efficiency, and stress tolerance. And in general, these three strains exhibited better growth at higher DOM concentrations. However, DOM treatment inhibited the growth of Peridiniopsis sp., as indicated by the increases in reactive oxygen species, damage in photosystem II reaction centers, and blockage in electron transport. Fluorescence analysis showed that tryptophan-like compounds were the main DOM components that affected algal growth. Molecular-level analysis suggested that unsaturated aliphatic compounds may be the most important DOM components. The findings indicate that CD-DOM and XS-DOM promote the blue-green algal blooms formation and thus should be considered in the management of natural water quality., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Algal blooms have a negative impact on the safety of drinking water. Ultrasonic radiation technology is an "environment-friendly" technology that is widely used in algae removal. However, this technology leads to the release of intracellular organic matter (IOM), which is an important precursor of disinfection by-products (DBPs). This study investigated the relationship between the release of IOM in Microcystis aeruginosa and the generation of DBPs after ultrasonic radiation as well as analyzed the generation mechanism of DBPs. Results showed that the content of extracellular organic matter (EOM) in M. aeruginosa increased in the order of 740 kHz >1120 kHz >20 kHz after 2 min of ultrasonic radiation. Organic matter with a molecular weight (MW) greater than 30 kDa increased the most, including protein-like, phycocyanin (PC) and chlorophyll a, followed by small-molecule organic matter less than 3 kDa, mainly humic-like substances and protein-like. DBPs with an organic MW range of less than 30 kDa were dominated by trichloroacetic acid (TCAA), while those with an MW greater than 30 kDa had the highest trichloromethane (TCM) content. Ultrasonic irradiation changed the organic structure of EOM, affected the amount and type of DBPs, and tended to generate TCM., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier Ltd.)

Algal extracellular organic matter (EOM) metabolites exert considerable impact on the carbon (C), nitrogen (N), and phosphorus (P) cycles mediated by attached bacteria. Field investigations were conducted in two ponds to explore the relationship among EOM metabolites from Microcystis and Dolichospermum, co-occurring microbes, and nutrient recycling from April 2021 to December 2021. Microcystis blooms primarily produced more complex bound EOM (bEOM) metabolites with many amino acid components, which facilitated bacterial colonization and provided sufficient substrates for ammonification. Meanwhile, high abundances of dissimilatory nitrate reduction to ammonium genes from co-occurring microbes such as Rhodobacter have demonstrated their strong N retention ability. Metabolic products of bEOM from Microcystis comprise a large number of organic acids that can solubilize non-bioavailable P. All these factors have collectively resulted in the increase of all fractions of N and P, except for nitrate (NO 3 - -N) in the water column. In contrast, the EOM metabolite from Dolichospermum was simple, coupled with high abundance of functional genes of α-glucosidase, and produced small molecular substances fueling denitrification. The metabolic products of EOM from Dolichospermum include abundant N-containing substances dominated by heterocyclic substances, suggesting that the metabolic products of Dolichospermum are not conducive to N regeneration and retention. Therefore, the metabolic products of EOM from Microcystis triggered a shift in the attached microbial community and function toward C, N, and P recycling with close mutual coupling. Acquisition of N and P in Dolichospermum is dependent on itself based on N fixation and organic P hydrolysis capacity. This study provides a new understanding of the contribution of algal EOM to the nutrient cycle., Competing Interests: Declaration of competing interest Here, I state that all the reported work is original; all coauthors fully participated in and accept responsibility for the work and agree to publish in Journal of “Science of the Total Environment”, if accepted. Also, all prevailing local, national and international regulations and conventions, and normal scientific ethical practices, have been respected and the manuscript presents original work that is not published or being considered for publication elsewhere, in whole or in part., (Copyright © 2022 Elsevier B.V. All rights reserved.)

In this work, a floating photocatalyst was constructed by loading g-C 3 N 4 @Bi 2 MoO 6 @AgI (GBA) nanocomposite on a modified polyurethane sponge via a simple dip-coating method and applied for the inactivation of Microcystis aeruginosa under visible light. GBA ternary photocatalyst was fabricated successfully and the morphology, structure, chemical state, and optical properties were characterized systematically. The floating catalyst achieved near 100% removal efficiency of algae cells under 6 h visible light irradiation and also could be retrieved and used at least three times repeatedly. The influences of various conditions on photocatalytic performance such as loading content of nanoparticles, algae density, and concentration of natural organic matters were also studied, which revealed that the GBA floating catalyst exhibited excellent photocatalytic performance of algae removal under different conditions. Furthermore, the physiological characteristics of algae cells during the photocatalytic process, including cell morphology, membrane permeability, Zeta potential, photosynthetic system, antioxidant system, and the metabolic activity were investigated. Results confirmed that the algae cells were severely damaged during the photocatalytic inactivation and the normal physiological functions were significantly affected, which resulted in the death of algae cells at last. Finally, a possible photocatalytic inactivation mechanism of algae cells was proposed. In summary, GBA floating catalyst can effectively inactivate Microcystis aeruginosa under visible light, which confirmed the high efficiency of the novel photocatalytic algae removal technology. Meanwhile, the recyclable floating material also makes the practical application in eutrophic waters of the algae removal technology possible., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Microginins are a large family of cyanobacterial lipopeptide protease inhibitors. A hybrid polyketide synthase/non-ribosomal peptide synthetase biosynthetic gene cluster (BGC) found in several microginin-producing strains─ mic ─was proposed to encode the production of microginins, based on bioinformatic analysis. Here, we explored a cyanobacterium, Microcystis aeruginosa LEGE 91341, which contains a mic BGC, to discover 12 new microginin variants. The new compounds contain uncommon amino acids, namely, homophenylalanine (Hphe), homotyrosine (Htyr), or methylproline, as well as a 3-aminodecanoic acid (Ada) residue, which in some variants was chlorinated at its terminal methyl group. We have used direct pathway cloning (DiPaC) to heterologously express the mic BGC from M. aeruginosa LEGE 91341 in Escherichia coli , which led to the production of several microginins. This proved that the mic BGC is, in fact, responsible for the biosynthesis of microginins and paves the way to accessing new variants from (meta)genome data or through pathway engineering.

Algal blooms caused by eutrophication are global phenomena that seriously threaten the sustainable use of freshwater resources. Traditional water treatment chemicals often typically lead to high levels of residue and cause damage to the morphology of algal cells. This study investigated an eco-friendly fungal bio-flocculant, Aspergillus oryzae, to remove the representative microalgae (Microcystis aeruginosa). Furthermore, it explored crucial flocculation parameters, adsorption kinetics, and thermodynamics of microalgae using A. oryzae. Accordingly, a flocculation efficiency of >95% was achieved when the fungus was cultured for six days, flocculant dosage was 11 g/L, rotation speed was 100 rpm, temperature was 25 °C, flocculation time was 5 h, and pH ranged between 4.0 and 9.0. KEGG analysis based on the genomic data, and chemical composition analysis revealed that proteins and polysaccharides were the major components of metabolites. Zeta potential analysis, scanning electron microscopy, three-dimensional fluorescence, X-ray spectroscopy, and infrared spectroscopy, electrostatic attraction revealed that electrostatic attraction promoted the destabilization and aggregation of microalgae. Additionally, hyphal surface adsorption and chemisorption from extracellular proteins and exopolysaccharides aided in the removal of microalgae. Therefore, fungi-based bio-flocculants have the potential to remove microalgae in a simple, effective, and eco-friendly manner without the complex extraction of extracellular metabolites., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier B.V. All rights reserved.)

The design of a photocatalyst for efficient algal inactivation under visible light is essential for the application of photocatalysis to the control of harmful algal blooms. In this study, a novel Z-scheme heterojunction tubular photocatalyst, Ag 2 O@PG, was synthesized by chemically depositing silver oxide compounded with P-doped hollow tubular graphitic carbon nitride for the photocatalytic inactivation of Microcystis aeruginosa (M. aeruginosa). The photocatalytic algal inactivation experiments showed that the photocatalytic activity of Ag 2 O@PG was influenced by the ratio of the composition of the obtained materials. The optimal algal inactivation efficiency was observed when using Ag 2 O@PG-0.4 at a dosage of 0.2 g/L. It was able to achieve a 99.1 % M. aeruginosa inactivation at an initial concentration of 4.5 × 10 6 cells/mL following 5 h' visible light irradiation. During the process, the cell membrane permeability and cell morphology changed. Furthermore, under the constant attack of superoxide radicals and holes caused by Ag 2 O@PG, the superoxide dismutase, glutathione and malondialdehyde of algae cells increased during the experiments to alleviate oxidative damage. Eventually, the antioxidant system of algae cells was destroyed. To further validate the potential application of Ag 2 O@PG-0.4 in real algal bloom environment, an experiment in real water samples was carried out. Overall, the Ag 2 O@PG-0.4 as an efficient photocatalyst has a promising potential for emergency treatment measures to alleviate algal blooms., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Inorganic and synthetic flocculants are widely investigated for removing harmful microalgae, such as Microcystis aeruginosa . However, their toxicity and non-biodegradability are shortcomings. Bioflocculants based on extracellular polysaccharides have attracted much attention as alternative flocculants. However, its high production cost is a limiting factor for applying bioflocculants. Here, we investigate the potential of the dead cells of a marine filamentous bacterium, Aureispira sp. CCB-QB1, as a novel flocculant on M. aeruginosa cells. The removal efficiency of M. aeruginosa cells by the dead cells was measured by mixing and shaking both components in a buffer with 5 mM CaCl 2 in different incubation times and concentrations of the dead cells. After that, the minimum effective concentration of CaCl 2 was determined. The combination effect of FeCl 3 and the dead cells on the removal efficiency was tested. The structure of cell aggregates consisted of the dead cells and M. aeruginosa cells were also observed using a scanning electron microscope. The maximum removal efficiency (75.39%) was reached within 3 min in the presence of CaCl 2 when 5 mg/ml of the dead cells (wet cells) were added. The optimal concentration of CaCl 2 was 5 mM. The combination of the dead cells and a low concentration of FeCl 3 (10 mg/L) with 5 mM of CaCl 2 significantly improved the removal efficiency by about 1.2 times ( P  < 0.05). This result indicates that the combination usage of the dead cells can reduce the use of FeCl 3 . These results indicated that the dead cells could potentially be a novel biolfocculant to remove M. aeruginosa cells., Competing Interests: The authors declare there are no competing interests., (©2022 Furusawa and Iwamoto.)

DNA 5-methylcytosine modification plays an important role in the regulation of a variety of biological functions in both prokaryotic and eukaryotic organisms. Previous studies show that DNA Cytosine-5-methylation is predominantly associated with restriction-modification system in bacteria. IPF4390 is deduced to be a putative type II DNA Cytosine-5 methyltransferase from a fresh water cyanobacterium, Microcystis aeruginosa. Both its substrate sequence specificity and catalytic mechanism need to be revealed. In this study, the cloning, expression, purification, DNA binding assays and crystallization of IPF4390 are reported. Results of DNA binding assays demonstrate that IPF4390 can specifically recognize and bind two double-stranded DNAs containing GGNCC (N = A, T, C or G) sequences (HgiBI: 5'-ATAAGGACCAATA-3'; TdeIII: 5'-ATAAGGGCCAATA-3'). Therefore, IPF4390 is probably capable of blocking endonuclease cleavage once restriction sites containing these sequences. Moreover, the crystal of IPF4390 in the presence of TdeIII was obtained, and its X-ray diffraction data were collected and scaled to a maximum resolution of 2.46 Å., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Cyanobacteria are photosynthetic microorganisms that are able to produce a large number of secondary metabolites. In freshwaters, under favorable conditions, they can rapidly multiply, forming blooms, and can release their toxic/bioactive metabolites in water. Among them, anabaenopeptins (APs) are a less studied class of cyclic bioactive cyanopeptides. The occurrence and structural variety of APs in cyanobacterial blooms and cultured strains from Greek freshwaters were investigated. Cyanobacterial extracts were analyzed with LC-qTRAP MS/MS using information-dependent acquisition in enhanced ion product mode in order to obtain the fragmentation mass spectra of APs. Thirteen APs were detected, and their possible structures were annotated based on the elucidation of fragmentation spectra, including three novel ones. APs were present in the majority of bloom samples (91%) collected from nine Greek lakes during different time periods. A large variety of APs was observed, with up to eight congeners co-occurring in the same sample. AP F (87%), Oscillamide Y (87%) and AP B (65%) were the most frequently detected congeners. Thirty cyanobacterial strain cultures were also analyzed. APs were only detected in one strain ( Microcystis ichtyoblabe ). The results contribute to a better understanding of APs produced by freshwater cyanobacteria and expand the range of structurally characterized APs.

Cyanobacteria are photosynthetic microorganisms that compose phytoplankton and therefore have a trophic relationship with zooplankton, which represent an important link for energy flux in aquatic food webs. Several species can form blooms and produce bioactive metabolites known as cyanotoxins. However, the ecological and adaptative role of these toxins are still under debate. Many studies have addressed the cyanotoxins' function in defense against herbivory when grazing pressure by zooplankton plays a role in phytoplankton top-down control. Thus, the present study evaluated the ecophysiological responses of the cyanobacterial strain Microcystis aeruginosa NPLJ-4 underlying the chemical induced defense against the cladoceran Daphnia gessneri. Exposure to predator infochemicals consisted of cultures established in ASM-1 medium prepared in a filtrate from a culture of adults of D. gessneri at an environmentally relevant density. Daphnia infochemicals promoted a significant increase in toxin production by M. aeruginosa. However, no differences in growth were observed, despite a significant increase in both maximum photosynthetic efficiency and electron transport rate in response to zooplankton. Additionally, there was no significant variation in the production of exopolysaccharides. Overall, although a grazer-induced defense response was demonstrated, there were no effects on M. aeruginosa fitness, which maintained its growth in the presence of Daphnia alarm cues., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

The Songkhla Lake Basin (SLB) located in Southern Thailand, has been increasingly polluted by urban and industrial wastewater, while the lake water has been intensively used. Here, we aimed to investigate cyanobacteria and cyanotoxins in the SLB. Ten cyanobacteria isolates were identified as Microcystis genus based on16S rDNA analysis. All isolates harbored microcystin genes, while five of them carried saxitoxin genes. On day 15 of culturing, the specific growth rate and Chl-a content were 0.2-0.3 per day and 4 µg/mL. The total extracellular polymeric substances (EPS) content was 0.37-0.49 µg/mL. The concentration of soluble EPS (sEPS) was 2 times higher than that of bound EPS (bEPS). The protein proportion in both sEPS and bEPS was higher than the carbohydrate proportion. The average of intracellular microcystins (IMCs) was 0.47 pg/cell on day 15 of culturing, while extracellular microcystins (EMCs) were undetectable. The IMCs were dramatically produced at the exponential phase, followed by EMCs release at the late exponential phase. On day 30, the total microcystins (MCs) production reached 2.67 pg/cell. Based on liquid chromatograph-quadrupole time-of-flight mass spectrometry, three new MCs variants were proposed. This study is the first report of both decarbamoylsaxitoxin (dcSTX) and new MCs congeners synthesized by Microcystis .

Fish are exposed to numerous stressors in the environment including pollution, bacterial and viral agents, and toxic substances. Our study with common carps leveraged an integrated approach (i.e., histology, biochemical and hematological measurements, and analytical chemistry) to understand how cyanobacteria interfere with the impact of a model viral agent, Carp sprivivirus (SVCV), on fish. In addition to the specific effects of a single stressor (SVCV or cyanobacteria), the combination of both stressors worsens markers related to the immune system and liver health. Solely combined exposure resulted in the rise in the production of immunoglobulins, changes in glucose and cholesterol levels, and an elevated marker of impaired liver, alanine aminotransferase (ALT). Analytical determination of the cyanobacterial toxin microcystin-LR (MC-LR) and its structurally similar congener MC-RR and their conjugates showed that SVCV affects neither the levels of MC in the liver nor the detoxification capacity of the liver. MC-LR and MC-RR were depurated from liver mostly in the form of cysteine conjugates (MC-LR-Cys, MC-RR-Cys) in comparison to glutathione conjugates (LR-GSH, RR-GSH). Our study brought new evidence that cyanobacteria worsen the effect of viral agents. Such inclusion of multiple stressor concept helps us to understand how and to what extent the relevant environmental stressors co-influence the health of the fish population.

Standardization and validation of alternative cell lysis methods used for quantifying total cyanotoxins is needed to improve laboratory response time goals for total cyanotoxin analysis. In this study, five cell lysis methods (i.e., probe sonication, microwave, freeze-thaw, chemical lysis with Abraxis QuikLyse TM , and chemical lysis with copper sulfate) were assessed using laboratory-cultured Microcystis aeruginosa ( M. aeruginosa ) cells. Methods were evaluated for destruction of cells (as determined by optical density of the sample) and recovery of total microcystin-LR (MC-LR) using three M. aeruginosa cell densities (i.e., 1 × 10 5 cells/mL (low-density), 1 × 10 6 cells/mL (medium-density), and 1 × 10 7 cells/mL (high-density)). Of the physical lysis methods, both freeze-thaw (1 to 5 cycles) and pulsed probe sonication (2 to 10 min) resulted in >80% destruction of cells and consistent (>80%) release and recovery of intracellular MC-LR. Microwave (3 to 5 min) did not demonstrate the same decrease in optical density (<50%), although it provided effective release and recovery of >80% intracellular MC-LR. Abraxis QuikLyse TM was similarly effective for intracellular MC-LR recovery across the different M. aeruginosa cell densities. Copper sulfate (up to 500 mg/L Cu 2+ ) did not lyse cells nor release intracellular MC-LR within 20 min. None of the methods appeared to cause degradation of MC-LR. Probe sonication, microwave, and Abraxis QuikLyse TM served as rapid lysis methods (within minutes) with varying associated costs, while freeze-thaw provided a viable, low-cost alternative if time permits.

A rapid and cost-effective system is vital for the detection of harmful algae that causes environmental problems in terms of water quality. The approach for algae detection was to capture images based on hyperspectral fluorescence imaging microscope by detecting specific fluorescence signatures. With the high degree of overlapping spectra of algae, the distribution of pigment in the region of interest was unknown according to a previous report. We propose an optimization method of multivariate curve resolution (MCR) to improve the performance of pigment analysis. The reconstruction image described location and concentration of the microalgae pigments. This result indicated the cyanobacterial pigment distribution and mapped the relative pigment content. In conclusion, with the advantage of acquiring two-dimensional images across a range of spectra, HSI conjoining spectral features with spatial information efficiently estimated specific features of harmful microalgae in MCR models., Competing Interests: The authors have declared that no competing interests exist.

Microcystins (MCs), produced by Microcystis sp, are the most commonly detected cyanotoxins in freshwater, and due to their toxicity, worldwide distribution, and persistence in water, an improvement in the monitoring programs for their early detection and removal from water is necessary. To this end, we investigate the performance of three covalent organic frameworks (COFs), TpBD-(CF 3 ) 2 , TpBD-(NO 2 ) 2 , and TpBD-(NH 2 ) 2 , for the adsorption of the most common and/or toxic MC derivatives, MC-LR, MC-RR, MC-LA, and MC-YR, from water. While MC-LR and MC-YR can be efficiently adsorbed using all three COF derivatives, high adsorption efficiencies were found for the most lipophilic toxin, MC-LA, with TpBD-(NH 2 ) 2 , and the most hydrophilic one, MC-RR, with TpBD-(NO 2 ). Theoretical calculations revealed that MC-LA and MC-RR have a tendency to be located mainly on the COF surface, interacting through hydrogen bonds with the amino and nitro functional groups of TpBD-(NH 2 ) 2 and TpBD-(NO 2 ) 2 , respectively. TpBD-(NO 2 ) 2 outperforms the adsorbent materials reported for the capture of MC-RR, resulting in an increase in the maximum adsorption capacity by one order of magnitude. TpBD-(NH 2 ) 2 is reported as the first efficient adsorbent material for the capture of MC-LA. Large differences in desorption efficiencies were observed for the MCs with different COFs, highlighting the importance of COF-adsorbate interactions in the material recovery. Herein we show that efficient capture of these toxins from water can be achieved through the proper selection of the COF material. More importantly, this study demonstrates that by careful choice of COF functionalities, specific compounds can be targeted or excluded from a group of analogues, providing insight into the design of more efficient and selective adsorbent materials.

The aim of this study was to investigate the potential interference of cyanobacterial metabolites, in particular microcystins (MCs), with steroid hormone biosynthesis. Steroid hormones control many fundamental processes in an organism, thus alteration of their tissue concentrations may affect normal homeostasis. We used liquid chromatography-tandem mass spectrometry (LC-MS/MS) to investigate the modulation of 14 hormones involved in the adrenal steroid biosynthesis pathway using forskolin-treated H295R cells, following exposure with either microcystin-LR (MC-LR) alone, a mixture made up of MC-LR together with eight other MCs and nodularin-R (NOD-R), or extracts from the MC-LR-producing Microcystis aeruginosa PCC7806 strain or its MC-deficient mutant PCC7806mcyB-. Production of 17-hydroxypregnenolone and dehydroepiandrosterone (DHEA) was increased in the presence of MC-LR in a dose-dependent manner, indicating an inhibitory effect on 3β-hydroxysteroid dehydrogenase (3β-HSD). This effect was not observed following exposure with a MCs/NOD-R mixture, and thus the effect of MC-LR on 3β-HSD appears to be stronger than for other congeners. Exposure to extracts from both M. aeruginosa PCC7806 and M. aeruginosa PCC7806mcyB- had an opposite effect on 3β-HSD, i.e. concentrations of pregnenolone, 17-hydroxypregnenolone and DHEA were significantly decreased, showing that there are other cyanobacterial metabolites that outcompete the effect of MC-LR, and possibly result instead in net-induction. Another finding was a possible concentration-dependent inhibition of CYP21A2 or CYP11β1, which catalyse oxidation reactions leading to cortisol and cortisone, by MC-LR and the MCs/NOD-R mixture. However, both M. aeruginosa PCC7806 and M. aeruginosa PCC7806mcyB- extracts had an opposite effect resulting in a substantial increase in cortisol levels. Our results suggest that MCs can modulate steroidogenesis, but the net effect of the M. aeruginosa metabolome on steroidogenesis is different from that of pure MC-LR and independent of MC production., Competing Interests: The authors have declared that no competing interests exist.

Chitosan has been tested as a coagulant to remove cyanobacterial nuisance. While its coagulation efficiency is well studied, little is known about its effect on the viability of the cyanobacterial cells. This study aimed to test eight strains of the most frequent bloom-forming cyanobacterium, Microcystis aeruginosa , exposed to a realistic concentration range of chitosan used in lake restoration management (0 to 8 mg chitosan L -1 ). We found that after 1 h of contact with chitosan, in seven of the eight strains tested, photosystem II efficiency was decreased, and after 24 h, all the strains tested were affected. EC 50 values varied from 0.47 to > 8 mg chitosan L -1 between the strains, which might be related to the amount of extracellular polymeric substances. Nucleic acid staining (Sytox-Green ® ) illustrated the loss of membrane integrity in all the strains tested, and subsequent leakage of pigments was observed, as well as the release of intracellular microcystin. Our results indicate that strain variability hampers generalization about species response to chitosan exposure. Hence, when used as a coagulant to manage cyanobacterial nuisance, chitosan should be first tested on the natural site-specific biota on cyanobacteria removal efficiency, as well as on cell integrity aspects.

Man-made organic matter acting as carbon source for oligotrophic aerobic denitrification has been studied extensively, while less attention has been paid to the actual organic matter derived from drinking water reservoir. In this study, the effect of extracellular organic matter (EOM) released from Microcystis aeruginosa and Chlorella sp. and organic matter in actual reservoir water on aerobic denitrification performance of Acinetobacter johnsonii strain WGX-9 has been investigated, by measuring nitrogen removal and determining changes in the properties of organic matter. Results indicated that the Acinetobacter johnsonii strain WGX-9 showed effective nitrogen removal efficiency when cultural conditions were low C/N of 5, pH of 5-11, and low temperature of 8 °C. The nitrate removal efficiency with EOM as the sole carbon source was relatively higher than that with intracellular organic matter or natural organic matter as the sole carbon source. This is probably due to that EOM exerts a lower molecular weight and better ability of donating electrons. Besides, the findings can elucidate that nitrate and total organic matter removal efficiency with actual high-density algal water as the carbon source was higher than that with actual low-density algal water as the carbon source. This was attributed to that more EOM was released in high-density algal water, which highlighted the aerobic denitrification performance of Acinetobacter johnsonii strain WGX-9. This study will provide a reference for the application of aerobic denitrifier in drinking water reservoirs.

Microcystins are toxins produced by cyanobacteria, notorious for negatively affecting a wide range of living organisms, among which several plant species. Although microtubules are a well-established target of microcystin toxicity, its effect on filamentous actin (F-actin) in plant cells has not yet been studied. Τhe effects of microcystin-LR (MC-LR) and an extract of a microcystin-producing freshwater cyanobacterial strain (Microcystis flos-aquae TAU-MAC 1510) on the cytoskeleton (F-actin and microtubules) of Oryza sativa (rice) root cells were studied with light, confocal, and transmission electron microscopy. Considering the role of F-actin in endomembrane system distribution, the endoplasmic reticulum and the Golgi apparatus in extract-treated cells were also examined. F-actin in both MC-LR- and extract-treated meristematic and differentiating root cells exhibited time-dependent alterations, ranging from disorientation and bundling to the formation of ring-like structures, eventually resulting in a collapse of the F-actin network after longer treatments. Disorganization and eventual depolymerization of microtubules, as well as abnormal chromatin condensation were observed following treatment with the extract, effects which could be attributed to microcystins and other bioactive compounds. Moreover, cell cycle progression was inhibited in extract-treated roots, specifically affecting the mitotic events. As a consequence of F-actin network disorganization, endoplasmic reticulum elements appeared stacked and diminished, while Golgi dictyosomes appeared aggregated. These results support that F-actin is a prominent target of MC-LR, both in pure form and as an extract ingredient. Endomembrane system alterations can also be attributed to the effects of cyanobacterial bioactive compounds (including microcystins) on the F-actin cytoskeleton., (© 2020 German Society for Plant Sciences and The Royal Botanical Society of the Netherlands.)

The utilization of Microcystis biomass is an urgent issue in the mitigation of cyanobacterial bloom. In this study, Microcystis-derived biochar (MB) and Fe 3 O 4 -modified biochar (Fe 3 O 4 /MB) were fabricated for the U(VI) elimination. The results showed that U(VI) sorption process by either MB or Fe 3 O 4 /MB was pH-dependent and ionic strength-independent. The maximum sorption capacity of MB was higher than that of Fe 3 O 4 /MB. According to the analysis of X-ray photoelectron spectroscopy, U(VI) sorption on both MB and Fe 3 O 4 /MB was mainly ascribed to the surface complexation between U(VI) and oxygen-containing functional groups on the surface of MB. Fe 3 O 4 particles on the surface of MB didn't provide extra active sites for the sorption of U(VI), but it enabled the adsorbent to be magnetically separated. Five consecutive sorption/desorption cycles verified the good reusability of Fe 3 O 4 /MB in this study. Therefore, the investigation is not only meaningful for the utilization of nuisance biomass from cyanobacterial blooms, but also provides novel adsorbents for the U(VI) removal from aqueous solutions., Competing Interests: Declaration of competing interest We declare that we have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

An abnormal growth of cyanobacteria in eutrophicated freshwaters can cause various environmental problems. In particular, Microcystis producing hepatotoxic cyclic heptapeptides microcystins (MCs) has been globally observed. Recent studies have demonstrated that matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) offers a rapid classification of cyanobacteria; however, they have not fully considered the toxicity yet. In this study, we have performed MALDI-TOF MS for intact cyanobacterial cells using Biotyper software and optimized their conditions to achieve cyanobacterial classification with the toxicity. The detection mass range used for Biotyper was extended to cover small molecules, but their intense ions were suppressed as a function of the used instrument Autoflex Speed, which enabled simultaneous observations of large molecular fingerprints and small MCs with comparable ion intensity. Hierarchical clustering of mass spectra obtained under the optimized conditions differentiated toxic and non-toxic clusters of Microcystis strains and furthermore formed a tight cluster of non-toxic strains possessing the MC biosynthesis gene mcyG . Spectral libraries were expanded to >30 genera (>80 strains) under the default and optimized conditions to improve the confidence of cyanobacterial classification. Consequently, spectral library searching allowed for characterization of cyanobacteria from a field sample as mixed toxic and non-toxic Microcystis cells, without isolating those cells.

Microcystins, cyclic nonribosomal heptapeptides, are the most well-known cyanobacterial toxins. They are exceptionally well studied, but open questions remain concerning their physiological role for the producing microorganism or their suitability as lead compounds for anticancer drug development. One means to study specialized metabolites in more detail is the introduction of functional groups that make a compound amenable for bioorthogonal, so-called click reactions. Although it was reported that microcystins cannot be derivatized by precursor-directed biosynthesis, we successfully used this approach to prepare clickable microcystins. Supplementing different azide- or terminal alkyne containing amino acid analogues into the cultivation medium of microcystin-producing cyanobacteria strains, we found that these strains differ strongly in their substrate acceptance. Exploiting this flexibility, we generated more than 40 different clickable microcystins. We conjugated one of these derivatives with a fluorogenic dye and showed that neither incorporation of the unnatural amino acid analogue nor attachment of the fluorescent label significantly affects the cytotoxicity against cell lines expressing the human organic anion transporting polypeptides 1B1 or 1B3. Using time-lapse microscopy, we observed that the fluorescent microcystin is rapidly taken up into eukaryotic cells expressing these transporters.

Microcystins (MCs) are potent toxins produced by environmental cyanobacterial blooms. The present study evaluated the effects of a Microcystis aeruginosa cyanobacterial lysate containing 0.1, 1, and 10 μg L -1 MC-LR equivalent in the C. elegans Bristol N2 wild-type and the effects caused by equivalent concentrations of a MC-LR standard. The lysate was prepared from a culture of toxic strain (RST9501) originated from the Patos Lagoon Estuary (RS, Brazil). The minimal concentration necessary to cause significant effects in C. elegans under exposure to M. aeruginosa lysate or to MC-LR standard were, respectively, 10 and 0.1 μg L -1 MC-LR equivalent for growth and 10 and 1 μg L -1 MC-LR equivalent for fertility. Reproduction (ie, brood size) was only affected by the exposure to 10 μg L -1 MC-LR standard and was not affected by the lysate. The nematodes that were exposed to lysate containing 1 μg L -1 MC-LR equivalent or MC-LR were also analyzed for pharyngeal pumping and gene expression using RT-qPCR. The worms' rhythmic contractions of the pharynx were similarly affected by the lysate containing 1 μg L -1 of MC-LR equivalent and the MC-LR standard. The MC-LR standard caused down-regulation of genes related to growth (daf-16), fertility (spe-10), and biotransformation (gst-2). This is the first study to evaluate the effects of a toxic cyanobacterial lysate using the C. elegans model. This study suggests the organism as a potential biotest to evaluate toxicity of natural waters containing M. aeruginosa cells and to environmental risk assessment associated to cyanobacterial bloom events., (© 2020 Wiley Periodicals, Inc.)

Eutrophication and warming lead to frequent occurrence of cyanobacterial blooms, which significantly impact on zooplankton. Freshwater zooplankton Daphnia adopts two distinct ways of reproduction: asexual (parthenogenetic) reproduction for rapidly reproducing many offspring in favorable environment and sexual reproduction for producing resting eggs as seed bank to survive in harsh environments. Daphnia pulex has worse performance in growth and reproduction under the exposure to toxic cyanobacteria Microcystis aeruginosa and tends to allocate less energy to reproduction in the case of insufficient food. However, the relative reproductive allocation strategy (energy allocation) of D. pulex individuals exposed to toxic M. aeruginosa is still unclear. Here we tested the relative reproductive performance of D. pulex fed on solely Chlorella pyrenoidosa (high quality food) or Chlorella mixed with toxic M. aeruginosa (low quality food), based on the parthenogenetic reproduction (life-history experiments) and sexual reproduction (population experiments). The results showed that under low quality food conditions, D. pulex reproduced fewer offspring which were also smaller and thus led to a reduced absolute output in parthenogenetic reproduction, but produced ephippia in the same size and quantity compared to those cultured under high quality food conditions. However, as the body size of maternal D. pulex cultured under low quality food conditions decreased, the relative reproductive allocation significantly increased in both parthenogenetic and sexual reproduction, compared to those cultured under high quality food conditions. In conclusion, D. pulex tend to allocate relatively more energy to reproduction under Microcystis conditions, which is a reasonable strategy for it to decentralize the risks from low-quality food., Competing Interests: Declaration of competing interest None., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Cyanobacteria always massively grow and even occur blooms in summer, with releasing amount of β-cyclocitral. To uncover the effects of summer high irradiance and temperature on cyanobacterial growth and β-cyclocitral emission, the cell growth, reactive oxygen species (ROS) levels, photosynthetic pigment content, chlorophyll fluorescence and β-cyclocitral emission were investigated in Microcystis aeruginosa under high light and temperature. Compared to the control under 50 μmol m -2 ·s -1 , the cell growth was promoted under 100 μmol m -2 ·s -1 , but inhibited under 500 and 1000 μmol m -2 ·s -1 . The inhibition was also detected under high temperature at 30 and 35 °C in contrast to the control at 25 °C. Under high light and high temperature, M. aeruginosa increased ROS levels and reduced photosynthetic pigment content and photosystem II (PSII) efficiency, which resulted in the inhibition on cell growth. With increasing the light intensity and temperature, 1 O 2 levels gradually increased, while β-carotene content gradually decreased by quenching 1 O 2 , with increasing β-cyclocitral emission. In summer, high irradiance and temperature not benefited the growth of cyanobacteria, but the emission of β-cyclocitral derived from β-carotene quenching 1 O 2 may offset the disadvantages by poisoning other algae., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Seasonal algal bloom, a water supply issue worldwide, can be efficiently solved by membrane technology. However, membranes typically suffer from serious fouling, which hinders the wide application of this technology. In this study, the feasibility of adding Ca 2+ to control membrane fouling in coagulation-membrane treatment of algal-rich water was investigated. According to the results obtained, the normalized membrane flux decreased by a lower extent upon increasing the concentration of Ca 2+ from 0 to 10 mmol/L. Simultaneously, the floc particle size increased significantly with the concentration of Ca 2+ , which leads to a lower hydraulic resistance. The coagulation performance is also enhanced with the concentration of Ca 2+ , inducing a slight osmotic pressure-induced resistance. The formation of Ca 2+ coagulation flocs resulted in a looser, thin, and permeable cake layer on the membrane surface. This cake layer rejected organic pollutants and could be easily removed by physical and chemical cleaning treatments, as revealed by scanning electron microscopy images. The hydraulic irreversible membrane resistance was significantly reduced upon addition of Ca 2+ . All these findings suggest that the addition of Ca 2+ may provide a simple-operation, cost-effective, and environmentally friendly technology for controlling membrane fouling during coagulation-membrane process for algal-rich water treatment.

The toxicity of the cyanobacterium Microcystis aeruginosa EAWAG 127a was evaluated against the sensitive grazer Thamnocephalus platyurus , and the extract possessed strong activity. To investigate the compounds responsible for cytotoxicity, a series of peptides from this cyanobacterium were studied using a combined genomic and molecular networking approach. The results led to the isolation, structure elucidation, and biological evaluation of microviridin 1777, which represents the most potent chymotrypsin inhibitor characterized from this family of peptides to date. Furthermore, the biosynthetic gene clusters of microviridin, anabaenopeptin, aeruginosin, and piricyclamide were located in the producing organism, and six additional natural products were identified by tandem mass spectrometry analyses. These results highlight the potential of modern techniques for the identification of natural products, demonstrate the ecological role of protease inhibitors produced by cyanobacteria, and raise ramifications concerning the presence of novel, yet uncharacterized, toxin families in cyanobacteria beyond microcystin.

β-Cyclocitral, specifically produced by Microcystis , is one of the volatile organic compounds (VOCs) derived from cyanobacteria and has a lytic activity. It is postulated that β-cyclocitral is a key compound for regulating the occurrence of cyanobacteria and related microorganisms in an aquatic environment. β-Cyclocitral is sensitively detected when a high density of the cells is achieved from late summer to autumn. Moreover, it is expected to be involved in changes in the species composition of cyanobacteria in a lake. Although several analysis methods for β-cyclocitral have already been reported, β-cyclocitral could be detected using only solid phase micro-extraction (SPME), whereas it could not be found at all using the solvent extraction method in a previous study. In this study, we investigated why β-cyclocitral was detected using only SPME GC/MS. Particularly, three operations in SPME, i.e., extraction temperature, sample stirring rate, and the effect of salt, were examined for the production of β-cyclocitral. Among these, heating (60 °C) was critical for the β-cyclocitral formation. Furthermore, acidification with a 1-h storage was more effective than heating when comparing the obtained amounts. The present results indicated that β-cyclocitral did not exist as the intact form in cells, because it was formed by heating or acidification of the resulting intermediates during the analysis by SPME. The obtained results would be helpful to understand the formation and role of β-cyclocitral in an aquatic environment., Competing Interests: The authors declare no conflict of interest.

In this work, Metal-organic Frameworks (MOFs) were applied to inactivate algae under visible light with low doses. Five MOFs with different compositions (Zn and Fe; carboxylates or imidazolates) were successfully synthesized and characterized by XRD, SEM and UV-vis. The effects of MOFs on Microcystis aeruginosa were evaluated with regard to morphology characteristics, physiological activity, cell integrity and pigment degradation. The results indicated that Ag/AgCl@ZIF-8 outperformed MOF-235, ZIF-8, Bi 2 WO 6 /MIL-100(Fe) and BiOBr/MOF-5 in the degradation of chlorophyll a at the dose of 10 mg L -1 . After 6 h of irradiation, 93.1% of Microcystis aeruginosa died and was unable to regrow and reproduce, which was demonstrated by changes in cell morphology, damage of cell membrane integrity and antioxidant enzyme system. Besides, the intracellular organic matter (IOM) and extracellular organic matter (EOM) were proven to be efficiently removed by MOF-assisted photocatalytic inactivation. Superoxide radical (O 2 · - ) was demonstrated to be the major reactive oxygen species. A probable mechanism was proposed that the electrons in the valence band of Ag/AgCl@ZIF-8 transfer into the conduction band under irradiation to produce O 2 · - which inactivated the algae cells. Furthermore, Ag/AgCl@ZIF-8 can effectively remove Microcystis aeruginosa under sunlight and is of great application prospects for algae removal in real water bodies., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

The 2-carboxy-6-hydroxyoctahydroindole (Choi) moiety is an essential residue for the antithrombotic activities of aeruginosins, which are a class of cyanobacterial derived bioactive linear tetrapeptides. Biosynthetic pathway of Choi is still elusive. AerF was suggested to be involved in the biosynthesis of Choi, and can be assigned to the short-chain dehydrogenase/reductase (SDR) superfamily. However, both the exact role and the catalytic mechanism of AerF have not been elucidated. In this study, functional and mechanistic analyses of AerF from Microcystis aeruginosa were performed. Observation of enzymatic assay demonstrates that AerF is a NADPH-dependent alkenal double bond reductase that catalyzes the reduction of dihydro-4-hydroxyphenylpyruvate (H 2 HPP) to generate tetrahydro-4-hydroxyphenylpyruvate (H 4 HPP), which is the third step of the biosynthetic pathway from prephenate to Choi. Comparative structural analysis indicates that ligand binding-induced conformational change of AerF is different from that of the other SDR superfamily reductase using H 2 HPP as a substrate. Analyses of NADPH and substrate analogue binding sites combined with the results of mutagenesis analyses suggest that a particular serine residue mainly involves in the initiation of the proton transfer between the substrate and the residues of AerF, which is an uncommon feature in SDR superfamily reductase. Furthermore, based on the observations of structural and mutagenesis analyses, the catalytic mechanism of AerF is proposed and a proton transfer pathway in AerF is deduced., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Surface water contaminated with algae and with enteric viruses is a global problem. When surface water is used as a drinking water source, it is important to know the influence of algal organic matter on the disinfection of viruses. In this work, we studied the disinfection efficacy of ferrate and the influence of algal organic matter on the disinfection. We determined the MS2 inactivation kinetics by ferrate under three pH conditions (7, 8, and 8.7). The experimental results and pH-dependent calculation suggest that H 2 FeO 4 and HFeO 4 - are 1935 and 8 times as effective as FeO 4 2- in MS2 inactivation. We also found that intracellular algal organic matter (IAOM) had a stronger effect on MS2 inactivation kinetics than extracellular algal organic matter (EAOM) suggesting that IAOM led to higher consumption of Fe(VI) compared to EAOM. At pH 8.7, while significant consumption of FeO 4 2- by as low as 8 mg C/L of EAOM and 2 mg C/L of IAOM was detected, MS2 inactivation was negatively influenced only when 13 mg C/L of IAOM present. This study showed that it is important to control pH and to determine the concentration of algal organic matter if ferrate is used for disinfection of surface water contaminated with algae., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Aminopeptidase M (AMP) inhibition is of interest for several diseases, such as highly vascularized cancer types. AMP can be inhibited by linear pentapeptides isolated from Microcystis aeruginosa LTPNA08 (MG7XX). Porcine AMP inhibition-a model for human AMP-activity was spectrophotometrically measured by the formation of p-nitroanilide from L-leucine-p-nitroanilide substrate by AMP. AMP inhibition by MG770 exhibited comparable inhibition levels to amastatin (IC 50 values: 1.20 ± 0.1 μM and 0.98 ± 0.1 μM, respectively), while MG756 was slightly less potent (with IC 50 values of 3.26 ± 0.5 μM). Molecular modelling suggests a potential binding mode, based on the interaction with the Zn 2+ cofactor, where MG770's extra methyl group contributes to the disturbance of the Zn 2+ cofactor complex and highlights the importance of hydrophobicity for the site.

Cyanobacteria harmful algal blooms are of global concern, but all currently available algicides in the market are nonselective and have potential side effects on nontarget species. In the present work, two series of compounds ( 4 and 6 ) comprising 16 novel 1,2,3-triazole aminopyrimidines were rationally designed and synthesized as control agent for cyanobacteria. Our design focus was the inhibiting cyanobacteria by inhibition against pyruvate dehydrogenase complex E1 (PDHc-E1). Compounds 4 and 6 showed potent inhibition against Escherichia coli PDHc-E1 (IC 50 = 4.13-23.76 μM) and also strong algicidal activities against Synechocystis sp. PCC 6803 (EC 50 = 1.7-8.1 μM) and Microcystis sp. FACHB905 (EC 50 = 2.1-11.8 μM). In particular, the algicidal activities of 6d against four algal species were not only higher than that of prometryn; they were also comparable to or higher than that of copper sulfate. The analogues 4c , 4d , 6d , and 6e displayed potent algicidal activities and inhibition of E. coli PDHc-E1 but exhibited negligible inhibition of porcine PDHc-E1. As revealed by molecular docking, site-directed mutagenesis, enzymatic assays, and an inhibition kinetic analysis, 4c and 6d inhibited PDHc-E1 in a competitive manner. Our results suggest that highly selective, effective algicides can be developed by rationally designing competitive PDHc-E1 inhibitors.

Microcystin Leucine-Arginine (MC-LR) is a toxin produced by the cyanobacteria Microcystis aeruginosa. It is the most encountered and toxic type of cyanotoxins. Oxidative stress was shown to play a role in the pathogenesis of microcystin LR by the induction of intracellular reactive oxygen species (ROS) formation that oxidize and damage cellular macromolecules. In the present study we examined the effect of acute MC-LR dose on the cardiac muscle of BALB/c mice. Afterwards, melatonin and N-acetyl cysteine (NAC) were assayed and evaluated as potential protective and antioxidant agents against damages generated by MC-LR. For this purpose, thirty mice were assigned into six groups of five mice each. The effect of MC-LR was first compared to the control group supplied with distilled water, then compared to the other groups supplied with melatonin and NAC. The experiment lasted 10 days after which animals were euthanized. Biomarkers of toxicity such as alkaline phosphatase activity, lipid peroxidation, protein carbonyl content, reduced glutathione content, serum lactate dehydrogenase and serum sorbitol dehydrogenase were assayed. Results showed that toxin treated mice have experienced significant oxidative damage in their myocardial tissue as revealed by noticeable levels of oxidative stress biomarkers and by the reduction in alkaline phosphatase activity. Whereas, melatonin and NAC treated mice manifested lesser oxidative damages. Our findings suggest a potential therapeutic use of melatonin and N-acetyl cysteine as antioxidant protective agents against oxidative damage induced by MC-LR., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Toxin-producing cyanobacteria are responsible for the presence of hundreds of bioactive compounds in aquatic environments undergoing increasing eutrophication. The identification of cyanotoxins is still emerging, due to the great diversity of potential congeners, yet high-resolution mass spectrometry (HRMS) has the potential to deepen this knowledge in aquatic environments. In this study, high-throughput and sensitive on-line solid-phase extraction ultra-high performance liquid chromatography (SPE-UHPLC) coupled to HRMS was applied to a data-independent acquisition (DIA) workflow for the suspect screening of cyanopeptides, including microcystin and anabaenopeptin toxin classes. The unambiguous characterization of 11 uncommon cyanopeptides was possible using a characterization workflow through extensive analysis of fragmentation patterns. This method also allowed the characterization of four unknown cyanotoxins ([Leu 1 , Ser 7 ] MC-HtyR, [Asp 3 ]MC-RHar, AP731, and AP803). The quantification of 17 common cyanotoxins along with the semi-quantification of the characterized uncommon cyanopeptides resulted with the identification of 23 different cyanotoxins in 12 lakes in Canada, United Kingdom and France. The concentrations of the compounds varied between 39 and 41,000 ng L -1 . To our knowledge, this is the first DIA method applied for the suspect screening of two families of cyanopeptides simultaneously. Moreover, this study shows the great diversity of cyanotoxins in lake water cyanobacterial blooms, a growing concern in aquatic systems.

This study was carried out with a view of understanding the temporal dynamics of microcystin concentrations in both algal seston and water samples and the associated public health risk. All the major MC variants, namely MC-LR, MC-YR, and MC-RR, were detected in both the algal seston and water samples. In the majority of the samples, the most potent variant, MC-LR, constituted the greatest proportion of the total MC concentration suggesting extremely high potential public health risk. The exceptionally high concentrations (μg L -1 ) of all the variants, MC-LR (815), MC-YR (466.6) and MC-RR (265.68), were observed in May. Although the extracellular MCs were relatively less concentrated and less frequently detected, concentrations (μg L -1 ) of up to 20 of MC-LR, 6.13 of MC-YR, and 1.27 MC-RR were encountered. The strong and significant association between Microcystis abundance and concentration of nitrate (Spearman Rank Order Correlation r = 0.793, p < 0.001) may suggest that nitrate is the key dictating factor in the dynamics of Microcystis, and may have consequently influenced the MC levels in the reservoir., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Microcystins are the most worldwide extended and common toxins produced by cyanobacteria in freshwater. Microcystin-leucine arginine (MC-LR), associated with the most toxic incidents involving microcystins, are within the cyanobacteria (intracellular) until released into the surrounding waters (extracellular) during cell lysis. Therefore, the relationship between intracellular and extracellular cyanotoxins will allow a comprehensive risk of cyanobacteria-containing waters, preventing disease and improving human safety. In this work, we present the development of a novel portable microfluidic sensing platform for the simultaneous detection of free (extracellular) and total MC-LR (intracellular and extracellular). The integrated system contains the sample processing and detection modules capable of performing the chemical lysis, filtration, sample mixing with antibodies, and electrochemical detection of MC-LR based on an indirect strategy. The performance of the immunosensors was evaluated by electrochemical impedance spectroscopy, showing a linear dynamic range between 3.3 × 10 -4 and 10 -7  g L -1 and a limit of detection of 5.7 × 10 -10  g L -1 . The results demonstrate the potential of the developed portable biosensor platform and its suitable application for the analysis of MC-LR at regulated levels for drinking water. Finally, the integrated system was able to simultaneously detect the free and total MC-LR on a Microcystis aeruginosa culture. To the best of our knowledge this is the first described system that can differentiate between intracellular and extracellular concentration of MC-LR. This novel electrochemical sensing platform avoids the multiple processing steps typically needed for standard MC-LR analysis in the laboratory and provides an early warning system for MC-LR remote monitoring in water., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Rise in cyanobacterial blooms and massive discharge of nanoparticles (NPs) in aquatic ecosystems cause zooplankton to be exposed in toxic food and NPs simultaneously, which may impact on zooplankton interactively. Therefore, the present study focused on assessing the combined effects of different ZnO NPs levels (0, 0.10, 0.15, 0.20 mg L -1 ) and different proportions of toxic Microcystis (0%, 10%, 20%, 30%) in the food on a model zooplankton, Daphnia magna. The results showed that both toxic Microcystis and ZnO NPs significantly delayed the development of D. magna to maturation, but there was no significant interaction between the two factors on the times to maturation except the body length at maturation. Both ZnO NPs and toxic Microcystis also significantly decreased the number of neonates in the first brood, total offspring, and number of broods per female, and there was a significant interaction between ZnO NPs and food composition on the reproductive performance of D. magna. Specifically, presence of toxic Microcystis reduced the gap among the effects of different ZnO NPs concentrations on the reproductive performance of D. magna. When the ZnO NPs concentration was at 0.15 mg L -1 , the gap of the reproductive performance among different proportions of toxic Microcystis also tended to be narrow. Similar phenomenon also occurred in mortality. Such results suggested that low concentration of ZnO NPs and toxic Microcystis can mutually attenuate their harmful effects on D. magna, which has significantly implications in appropriately assessing the ecotoxicological effects of emerging pollutants in a complex food conditions., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Background: The decomposition of Microcystis can dramatically change the physicochemical properties of freshwater ecosystems. Bacteria play an important role in decomposing organic matters and accelerating the cycling of materials within freshwater lakes. However, actions of the bacterial community are greatly influenced by temperature and the amount of organic matter available to decompose during a bloom. Therefore, it is vital to understand how different temperatures and biomass levels affect the bacterial community during the decomposition of Microcystis., Results: Microcystis addition reduced the diversity of bacterial community. The composition of bacterial community differed markedly between samples with different biomass of Microcystis (no addition, low biomass addition: 0.17 g/L, and high biomass addition: 0.33 g/L). In contrast, temperature factor did not contribute much to the different bacterial community composition. Total nitrogen (TN), total phosphorus (TP), total organic carbon (TOC), ammonia nitrogen (NH 4 + -N) and oxidation-reduction potential (ORP) were the key measured environmental variables shaping the composition of bacterial community., Conclusions: Decomposition of Microcystis changed the physicochemical characteristics of the water and controlled the diversity and composition of the bacterial community. Microcystis biomass rather than temperature was the dominant factor affecting the diversity and composition of the bacterial community.

Although microcystins (MCs) are the most commonly studied cyanotoxins, their significance to the producing organisms remains unclear. MCs are known as endotoxins, but they can be found in the surrounding environment due to cell lysis, designated as extracellular MCs. In the present study, the interactions between MC producing and the non-producing strains of Microcystis aeruginosa, PCC 7806 and PCC 7005, respectively, and a green alga, Desmodesmus subspicatus, were studied to better understand the probable ecological importance of MCs at the collapse phase of cyanobacterial blooms. We applied a dialysis co-cultivation system where M. aeruginosa was grown inside dialysis tubing for one month. Then, D. subspicatus was added to the culture system on the outside of the membrane. Consequently, the growth of D. subspicatus and MC contents were measured over a 14-day co-exposure period. The results showed that Microcystis negatively affected the green alga as the growth of D. subspicatus was significantly inhibited in co-cultivation with both the MC-producing and -deficient strains. However, the inhibitory effect of the MC-producing strain was greater and observed earlier compared to the MC-deficient strain. Thus, MCs might be considered as an assistant factor that, in combination with other secondary metabolites of Microcystis, reinforce the ability to outcompete co-existing species.

Hydrogen peroxide (HP) is a feasible algicide to control cyanobacterial blooms, but its application in the waters with strong reductive power is still a problem. The rapid decomposition rate of HP results in a short residence time in the waters, which renders the failure of bloom mitigation. In this study, the damage of Microcystis aeruginosa (M. aeruginosa) by HP, the optimization method for Microcystis bloom control and its field effects were investigated. Results of microcosm experiments indicated M. aeruginosa was vulnerable to HP. The HP-induced damage was mainly attributed to the impairments of HP detoxification pathways and photosystem. Repetitive additions of HP, which could prolong the residence time, were conducted in the mesocosm experiments. HP concentration ranged from 96 μM to 165 μM for 2 h could successfully mitigate Microcystis bloom, even though HP decomposition rate reached 109 μM per h. Besides the removal of M. aeruginosa, contents of total dissolved nitrogen, total dissolved phosphate, dissolved organic carbon and chromophoric dissolved organic matter in water column increased significantly (p < 0.05). The enrichment of nutrients promoted the growth of chlorophytes but the growth of M. aeruginosa couldn't be observed. The dominant species thrived in the HP-treated waters was Chlamydomonas sp. Results in this study confirmed that HP was a promising algicide for cyanobacterial blooms control. The optimization method further demonstrated that repetitive additions of HP could favor the mitigation of cyanobacterial blooms., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

The roles dissolved organic matter play when managing watersheds and controlling cyanobacteria blooms have been overlooked. We assessed the effects of dissolved organic matter extracted from biochar, paddy soil, pectin, and rice husks, at carbon concentrations of 0, 1, 3, 5, and 10 mg L -1 on Microcystis aeruginosa growth, photosynthesis, and physiological characteristics. The dissolved organic matter derived from paddy soil and rice husks increased M. aeruginosa growth by promoting photosynthesis. Biochar at low carbon concentrations (1, 3, and 5 mg L -1 ) also improved M. aeruginosa growth by increasing the maximum photosynthesis II quantum yield. However, biochar at a high concentration decreased the protein and RNA concentrations in M. aeruginosa and therefore inhibited the increase in M. aeruginosa biomass. Pectin did not affect M. aeruginosa photosynthesis, protein concentration, RNA concentration, or growth. The results suggested that M. aeruginosa growth was improved by the amino acids tryptophan and tyrosine, decreased by abundant humic-acid-like substances, and unaffected by polysaccharides., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Ultrasonic treatment has attracted much attention because of its physical and chemical effects that are distinct from those of chemical agents. In particularly, high-frequency ultrasound is known as an effective method because the theoretical resonance frequency of the gas vesicles in Microcystis aeruginosa is in the high frequency range (>100 kHz), which causes gas vesicles collapse and changes the settleability of the algal cells. In this work, the effects of the ultrasonic frequency, acoustic power density and duration on enhancing coagulation to remove turbidity in algae-laden water were studied. In order to explain the mechanism, the morphology of algae cells, the changes in extracellular organic substances, the zeta potential and the formation of hydroxyl radicals were analyzed systematically. Finally, Zeta potentials and flocs morphology after adding PAC were investigated to verify the mechanism. The results showed that the frequency exhibited fewer effects than power and duration on coagulation. SEM images showed that there were more severe cellular damages at 430 and 740 kHz than other frequencies. Sonication could cause the collapse of gas vesicle inside the cell, which was due to the instantaneous high pressure generated by the ultrasonic cavitation instead of the resonance. Furthermore, sonication would result in an increase in proteins in extracellular organic matter (EOM) with continuous ultrasonic irradiation, indicating that a small amount of proteins could promote coagulation and that the accumulation of proteins would inhibit coagulation. Free radical content testing showed that the production of excessive free radicals was often accompanied by a deterioration of the coagulation. The proper mechanical effects were the main mechanism of ultrasonic enhanced coagulation. Thus, it was recommended that the appropriate ultrasonic condition was the one that resulted in a small amount of protein leakage and little generation of free radicals, which occurred at 740 kHz and 0.02 W/mL in approximately 5 min, and would significantly enhance the turbidity removal rate in algae-containing water from approximately 80-90%., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Nowadays, eutrophication is a very popular environmental problem in numerous waters around the world. The main reason of eutrophication is the enrichment of the nutrient, which results in the excessive growth of phytoplankton and some of them are toxic and harmful. Fortunately, some studies have shown that some bivalves can filter the overgrown phytoplankton in water, which may alleviate water eutrophication. However, the physiological effects of toxic cyanobacteria on filter feeding animal have not been clarified very well. In this experiment, digestive enzyme activities in Hyriopsis cumingii exposed to different concentrations of the toxic Microcystis aeruginosa (0, 5 * 10 5 and 5 *10 6 cell ml -1 ) at two dissolved oxygen (DO) levels (6 and 2 mg l -1 ) for 14 days were investigated. Toxic M. aeruginosa significantly affected all digestive enzyme activities throughout the experiment. At high toxic M. aeruginosa concentration, the activities of cellulase, amylase and lipase in digestive gland and stomach were significantly increased (P<0.05). However, hypoxia reduced the activities of cellulase, amylase and lipase in digestive gland and stomach. Conflicting effects were observed between toxic M. aeruginosa and DO in most digestive enzyme activities during the exposure period. Therefore, it is not conducive for the digestion and absorption of M. aeruginosa in H. cumingii under hypoxic conditions. H. cumingii is tolerant to toxic M. aeruginosa and may remove toxic cyanobacteria from waters under normal DO conditions., (Copyright © 2019 Elsevier B.V. All rights reserved.)