Phenotypic and Genotypic Methods for the Identification and Quantification of Cyanobacteria in Lake Water.

Early monitoring of Microcystis, a cyanobacterium that produces microcystin, is paramount in order to confirm the presence of Microcystis spp. Both phenotypic and genotypic methods have been used. The phenotypic methods provide the presence of the microcystis but do not confirm its species type and toxin produced. Additionally, phenotypic methods cannot differentiate toxigenic from non-toxigenic Microcystis. Therefore, the current protocol also describes genetic methods based on PCR to detect toxigenic Microcystis spp. based on microcystin synthetase E (mcy E) gene and 16-23S RNA genes for species-specific identification, which can effectively comprehend distinct lineages and discrimination of potential complexity of microcystin populations. The presence of these microcystin toxins in blood, in most cases, indicates contamination of drinking water by cyanobacteria. The methods presented herein are used to identify microcystin toxins in drinking water and blood.

First Report on Microcystis as a Potential Microviridin Producer in Bulgarian Waterbodies.

Bulgaria, situated on the Balkan Peninsula, is rich in small and shallow, natural and man-made non-lotic waterbodies, which are threatened by blooms of Cyanoprokaryota/Cyanobacteria. Although cyanotoxins in Bulgarian surface waters are receiving increased attention, there is no information on microviridins and their producers. This paper presents results from a phytoplankton study, conducted in August 2019 in three lakes (Durankulak, Vaya, Uzungeren) and five reservoirs (Duvanli, Mandra, Poroy, Sinyata Reka, Zhrebchevo) in which a molecular-genetic analysis (PCR based on the precursor mdnA gene and subsequent translation to amino acid alignments), combined with conventional light microscopy and an HPLC analysis of marker pigments, were applied for the identification of potential microviridin producers. The results provide evidence that ten strains of the genus Microcystis, and of its most widespread species M. aeruginosa in particular, are potentially toxigenic in respect to microviridins. The mdnA sequences were obtained from all studied waterbodies and their translation to amino-acid alignments revealed the presence of five microviridin variants (types B/C, Izancya, CBJ55500.1 (Microcystis 199), and MC19, as well as a variant, which was very close to type A). This study adds to the general understanding of the microviridin occurrence, producers, and sequence diversity.

Potentially Toxic Planktic and Benthic Cyanobacteria in Slovenian Freshwater Bodies: Detection by Quantitative PCR.

Due to increased frequency of cyanobacterial blooms and emerging evidence of cyanotoxicity in biofilm, reliable methods for early cyanotoxin threat detection are of major importance for protection of human, animal and environmental health. To complement the current methods of risk assessment, this study aimed to evaluate selected qPCR assays for detection of potentially toxic cyanobacteria in environmental samples. In the course of one year, 25 plankton and 23 biofilm samples were collected from 15 water bodies in Slovenia. Three different analyses were performed and compared to each other; qPCR targeting mcyE, cyrJ and sxtA genes involved in cyanotoxin production, LC-MS/MS quantifying microcystin, cylindrospermopsin and saxitoxin concentration, and microscopic analyses identifying potentially toxic cyanobacterial taxa. qPCR analyses detected potentially toxic Microcystis in 10 lake plankton samples, and potentially toxic Planktothrix cells in 12 lake plankton and one lake biofilm sample. A positive correlation was observed between numbers of mcyE gene copies and microcystin concentrations. Potential cylindrospermopsin- and saxitoxin-producers were detected in three and seven lake biofilm samples, respectively. The study demonstrated a potential for cyanotoxin production that was left undetected by traditional methods in both plankton and biofilm samples. Thus, the qPCR method could be useful in regular monitoring of water bodies to improve risk assessment and enable timely measures.

Potential Impacts on Treated Water Quality of Recycling Dewatered Sludge Supernatant during Harmful Cyanobacterial Blooms.

Cyanobacterial blooms and the associated release of cyanotoxins pose problems for many conventional water treatment plants due to their limited removal by typical unit operations. In this study, a conventional water treatment process consisting of coagulation, flocculation, sedimentation, filtration, and sludge dewatering was assessed in lab-scale experiments to measure the removal of microcystin-LR and Microcystis aeruginosa cells using liquid chromatography with mass spectrometer (LC-MS) and a hemacytometer, respectively. The overall goal was to determine the effect of recycling cyanotoxin-laden dewatered sludge supernatant on treated water quality. The lab-scale experimental system was able to maintain the effluent water quality below relevant the United States Environmental Protection Agency (US EPA) and World Health Organisation (WHO) standards for every parameter analyzed at influent concentrations of M. aeruginosa above 106 cells/mL. However, substantial increases of 0.171 NTU (Nephelometric Turbidity Unit), 7 × 104 cells/L, and 0.26 µg/L in turbidity, cyanobacteria cell counts, and microcystin-LR concentration were observed at the time of dewatered supernatant injection. Microcystin-LR concentrations of 1.55 µg/L and 0.25 µg/L were still observed in the dewatering process over 24 and 48 h, respectively, after the initial addition of M.aeruginosa cells, suggesting the possibility that a single cyanobacterial bloom may affect the filtered water quality long after the bloom has dissipated when sludge supernatant recycling is practiced.

Growth of Microcystisstrains isolated from environments with the presence and absence of submerged macrophytes in coexistence with Ceratophyllum demersum

Cyanobacterial blooms can cause severe ecological and health problems in drinking water reservoirs. To alleviate this problem, allelopathically active submerged macrophytes can be used to reduce cyanobacterial growth. Accordingly, this study aimed to evaluate the sensitivity of strains of the Microcystis aeruginosacomplex isolated from reservoirs with the presence and absence of submerged macrophytes to the allelochemicals of Ceratophyllum demersum.A coexistence experiment was carried out between the submerged macrophyte C. demersum and four Microcystisstrains, with two treatments for each strain, one in coexistence with the submerged macrophyte (7 g L-1) and control (in the absence of the macrophyte). Two strains of M. aeruginosa(BMIUFRPE-06 and BMIUFRPE-07) and two of M. panniformis(BMIUFRPE-08 and BMIUFRPE-09) were used, which were isolated from Cajueiro (with submerged macrophytes) and Tapacurá (without submerged macrophytes) reservoirs, respectively. The biomass of Microcystisstrains from the reservoir without macrophytes (BMIUFRPE-08 and BMIUFRPE-09) was significantly inhibited in 96% (T-test: p 0.05; growth rate -ANOVA: p > 0.05). These results suggest that strains isolated from environments with submerged macrophytes are less sensitive to allelochemicals of these plants,as these strains may be adapted to the coexistence with submerged macrophytes.

Isolation of axenic cyanobacterium and the promoting effect of associated bacterium on axenic cyanobacterium.

BACKGROUND: Harmful cyanobacterial blooms have attracted wide attention all over the world as they cause water quality deterioration and ecosystem health issues. Microcystis aeruginosa associated with a large number of bacteria is one of the most common and widespread bloom-forming cyanobacteria that secret toxins. These associated bacteria are considered to benefit from organic substrates released by the cyanobacterium. In order to avoid the influence of associated heterotrophic bacteria on the target cyanobacteria for physiological and molecular studies, it is urgent to obtain an axenic M. aeruginosa culture and further investigate the specific interaction between the heterotroph and the cyanobacterium. RESULTS: A traditional and reliable method based on solid-liquid alternate cultivation was carried out to purify the xenic cyanobacterium M. aeruginosa FACHB-905. On the basis of 16S rDNA gene sequences, two associated bacteria named strain B905-1 and strain B905-2, were identified as Pannonibacter sp. and Chryseobacterium sp. with a 99 and 97% similarity value, respectively. The axenic M. aeruginosa FACHB-905A (Microcystis 905A) was not able to form colonies on BG11 agar medium without the addition of strain B905-1, while it grew well in BG11 liquid medium. Although the presence of B905-1 was not indispensable for the growth of Microcystis 905A, B905-1 had a positive effect on promoting the growth of Microcystis 905A. CONCLUSIONS: The associated bacteria were eliminated by solid-liquid alternate cultivation method and the axenic Microcystis 905A was successfully purified. The associated bacterium B905-1 has the potentiality to promote the growth of Microcystis 905A. Moreover, the purification technique for cyanobacteria described in this study is potentially applicable to a wider range of unicellular cyanobacteria.

Utility of a PCR-based method for rapid and specific detection of toxigenic Microcystis spp. in farm ponds.

Microcystis is a widespread freshwater cyanobacterium that can produce microcystin, a potent hepatotoxin harmful to animals and humans. Therefore, it is crucial to monitor for the presence of toxigenic Microcystis spp. to provide early warning of potential microcystin contamination. Microscopy, which has been used traditionally to identify Microcystis spp., cannot differentiate toxigenic from non-toxigenic Microcystis. We developed a PCR-based method to detect toxigenic Microcystis spp. based on detection of the microcystin synthetase C (mcyC) gene and 16S rRNA gene. Specificity was validated against toxic and nontoxic M. aeruginosa strains, as well as 4 intergeneric freshwater cyanobacterial strains. Analytical sensitivity was as low as 747 fg/µL genomic DNA (or 3 cells/µL) for toxic M. aeruginosa. Furthermore, we tested 60 water samples from 4 farm ponds providing drinking water to swine facilities in the midwestern United States using this method. Although all water samples were positive for Microcystis spp. (i.e., 16S rRNA gene), toxigenic Microcystis spp. were detected in only 34 samples (57%). Seventeen water samples contained microcystin (0.1-9.1 µg/L) determined with liquid chromatography-mass spectrometry, of which 14 samples (82%) were positive for mcyC. A significant correlation was found between the presence of toxigenic Microcystis spp. and microcystin in water samples (p = 0.0004). Our PCR method can be a low-cost molecular tool for rapid and specific identification of toxigenic Microcystis spp. in farm ponds, improving detection of microcystin contamination, and ensuring water safety for farm animals.

Analytical Technique Optimization on the Detection of ß-cyclocitral in Microcystis Species.

ß-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 ß-cyclocitral is a key compound for regulating the occurrence of cyanobacteria and related microorganisms in an aquatic environment. ß-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 ß-cyclocitral have already been reported, ß-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 ß-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 ß-cyclocitral. Among these, heating (60 °C) was critical for the ß-cyclocitral formation. Furthermore, acidification with a 1-h storage was more effective than heating when comparing the obtained amounts. The present results indicated that ß-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 ß-cyclocitral in an aquatic environment.

High Diversity of Microcystin Chemotypes within a Summer Bloom of the Cyanobacterium Microcystis botrys.

The fresh-water cyanobacterium Microcystis is known to form blooms world-wide, and is often responsible for the production of microcystins found in lake water. Microcystins are non-ribosomal peptides with toxic effects, e.g. on vertebrates, but their function remains largely unresolved. Moreover, not all strains produce microcystins, and many different microcystin variants have been described. Here we explored the diversity of microcystin variants within Microcystis botrys, a common bloom-former in Sweden. We isolated a total of 130 strains through the duration of a bloom in eutrophic Lake Vomb, and analyzed their microcystin profiles with tandem mass spectrometry (LC-MS/MS). We found that microcystin producing (28.5%) and non-producing (71.5%) M. botrys strains, co-existed throughout the bloom. However, microcystin producing strains were more prevalent towards the end of the sampling period. Overall, 26 unique M. botrys chemotypes were identified, and while some chemotypes re-occurred, others were found only once. The M. botrys chemotypes showed considerable variation both in terms of number of microcystin variants, as well as in what combinations the variants occurred. To our knowledge, this is the first report on microcystin chemotype variation and dynamics in M. botrys. In addition, our study verifies the co-existence of microcystin and non-microcystin producing strains, and we propose that environmental conditions may be implicated in determining their composition.

First Report of Microcystis Strains Producing MC-FR and -WR Toxins in Japan.

Microcystins (MCs) are a group of cyclic heptapeptide hepatotoxins produced by Microcystis and several other genera of cyanobacteria. Many structural variants have been characterized using various methods such as liquid chromatography-mass spectrometry (LC-MS) analysis, enzyme-linked immunosorbent assay (ELISA) and protein phosphatase 2A (PP2A) inhibition assay. The representative MC, MC-LR, and related cyanobacterial toxins strongly inhibit PP2A activity and can therefore be assayed by measuring the extent of PP2A inhibition. However, these methods require reference toxin standards for the quantification and identification of known MCs. To obtain various MC-producing cyanobacterial strains, we surveyed and collected MC-producing cyanobacteria from environmental sources of water in Okinawa, Japan. Using a dual assay (LC-MS analysis and PP2A inhibition assay), we identified and isolated Microcystis strains producing five MC variants (MC-LR, -RR, -LA, -FR and -WR). Approximately 4 mg of MC-WR and -FR toxins were purified from the laboratory culture of the Microcystis isolate NIES-4344. Pure MC-WR and -FR variants were prepared for future use as toxin standards in LC-MS analysis. Phylogenetic analysis based on ftsZ revealed that the NIES-4344 strain belongs to the identified groups in Microcystis aeruginosa. This is the first report of Microcystis strains producing mainly MC-WR and -FR toxins in Japan.

Coherence of Microcystis species revealed through population genomics.

Microcystis is a genus of freshwater cyanobacteria, which causes harmful blooms in ecosystems worldwide. Some Microcystis strains produce harmful toxins such as microcystin, impacting drinking water quality. Microcystis colony morphology, rather than genetic similarity, is often used to classify Microcystis into morphospecies. Yet colony morphology is a plastic trait, which can change depending on environmental and laboratory culture conditions, and is thus an inadequate criterion for species delineation. Furthermore, Microcystis populations are thought to disperse globally and constitute a homogeneous gene pool. However, this assertion is based on relatively incomplete characterization of Microcystis genomic diversity. To better understand these issues, we performed a population genomic analysis of 33 newly sequenced genomes mainly from Canada and Brazil. We identified 17 Microcystis clusters of genomic similarity, five of which correspond to monophyletic clades containing at least three newly sequenced genomes. Four out of these five clades match to named morphospecies. Notably, M. aeruginosa is paraphyletic, distributed across 12 genomic clusters, suggesting it is not a coherent species. A few clades of closely related isolates are specific to a unique geographic location, suggesting biogeographic structure over relatively short evolutionary time scales. Higher homologous recombination rates within than between clades further suggest that monophyletic groups might adhere to a Biological Species-like concept, in which barriers to gene flow maintain species distinctness. However, certain genes-including some involved in microcystin and micropeptin biosynthesis-are recombined between monophyletic groups in the same geographic location, suggesting local adaptation.

Genome evolution and host-microbiome shifts correspond with intraspecific niche divergence within harmful algal bloom-forming Microcystis aeruginosa.

Intraspecific niche divergence is an important driver of species range, population abundance and impacts on ecosystem functions. Genetic changes are the primary focus when studying intraspecific divergence; however, the role of ecological interactions, particularly host-microbiome symbioses, is receiving increased attention. The relative importance of these evolutionary and ecological mechanisms has seen only limited evaluation. To address this question, we used Microcystis aeruginosa, the globally distributed cyanobacterium that dominates freshwater harmful algal blooms. These blooms have been increasing in occurrence and intensity worldwide, causing major economic and ecological damages. We evaluated 46 isolates of M. aeruginosa and their microbiomes, collected from 14 lakes in Michigan, USA, that vary over 20-fold in phosphorus levels, the primary limiting nutrient in freshwater systems. Genomes of M. aeruginosa diverged along this phosphorus gradient in genomic architecture and protein functions. Fitness in low-phosphorus lakes corresponded with additional shifts within M. aeruginosa including genome-wide reductions in nitrogen use, an expansion of phosphorus assimilation genes and an alternative life history strategy of nonclonal colony formation. In addition to host shifts, despite culturing in common-garden conditions, host-microbiomes diverged along the gradient in taxonomy, but converged in function with evidence of metabolic interdependence between the host and its microbiome. Divergence corresponded with a physiological trade-off between fitness in low-phosphorus environments and growth rate in phosphorus-rich conditions. Co-occurrence of genotypes adapted to different nutrient environments in phosphorus-rich lakes may have critical implications for understanding how M. aeruginosa blooms persist after initial nutrient depletion. Ultimately, we demonstrate that the intertwined effects of genome evolution, host life history strategy and ecological interactions between a host and its microbiome correspond with an intraspecific niche shift with important implications for whole ecosystem function.

In Situ Collection and Preservation of Intact Microcystis Colonies to Assess Population Diversity and Microcystin Quotas.

Understanding of colony specific properties of cyanobacteria in the natural environment has been challenging because sampling methods disaggregate colonies and there are often delays before they can be isolated and preserved. Microcystis is a ubiquitous cyanobacteria that forms large colonies in situ and often produces microcystins, a potent hepatotoxin. In the present study a new cryo-sampling technique was used to collect intact Microcystis colonies in situ by embedding them in a sheet of ice. Thirty-two of these Microcystis colonies were investigated with image analysis, liquid chromatography-mass spectrometry, quantitative polymerase chain reaction and high-throughput sequencing to assess their volume, microcystin quota and internal transcribed spacer (ITS) genotype diversity. Microcystin quotas were positively correlated to colony volume (R2 = 0.32; p = 0.004). Individual colonies had low Microcystis ITS genotype diversity and one ITS operational taxonomic unit predominated in all samples. This study demonstrates the utility of the cryo-sampling method to enhance the understanding of colony-specific properties of cyanobacteria with higher precision than previously possible.

Removal of Microcystis aeruginosa by ultrasound: Inactivation mechanism and release of algal organic matter.

The efficacy of ultrasonic irradiation for removal of Microcystis aeruginosa and release of algal organic matter (AOM) was investigated under different ultrasound conditions, including ultrasonic frequency, power density, and time. Laboratory results suggested that the ultrasonic efficiency and the release of AOM were influenced by frequency, power density, and time. The mechanism of AOM algae removal by ultrasound was systematically explored. The inactivation of algae resulted from mechanical and chemical effects caused by ultrasound. Mechanical destruction and free-radical oxidation considerably affected the structure and physiological function of algal cells. The SEM and TEM images indicated that ultrasound could damage the cell membrane, wall, and organelle. Flow cell cytometry results showed decreases in the size, internal granularity, integrity, and activity of algal cells, revealing that ultrasound exerted severe damage to the structure and function of algal cells. The activity of the antioxidant system of algal cells was then studied by investigating changes in MDA, SOD, and CAT concentration after ultrasound to confirm the inactivation of the cells. The release of AOM was explored by determining changes in water quality indices (UV254, DOC, and SUVA) at 10 min and 48 h after ultrasound. This study provides information about the safety of ultrasound usage on algae removal and references for ultrasonic parameters to be selected to ensure effective and safe algae removal.

Microcystis Chemotype Diversity in the Alimentary Tract of Bigheaded Carp.

Most cyanobacterial organisms included in the genus Microcystis can produce a wide repertoire of secondary metabolites. In the mid-2010s, summer cyanobacterial blooms of Microcystis sp. occurred regularly in Lake Balaton. During this period, we investigated how the alimentary tract of filter-feeding bigheaded carps could deliver different chemotypes of viable cyanobacteria with specific peptide patterns. Twenty-five Microcystis strains were isolated from pelagic plankton samples (14 samples) and the hindguts of bigheaded carp (11 samples), and three bloom samples were collected from the scums of cyanobacterial blooms. An LC-MS/MS-based untargeted approach was used to analyze peptide patterns, which identified 36 anabaenopeptin, 17 microginin, and 13 microcystin variants. Heat map clustering visualization was used to compare the identified chemotypes. A lack of separation was observed in peptide patterns of Microcystis that originated from hindguts, water samples, and bloom-samples. Except for 13 peptides, all other congeners were detected from the viable and cultivated chemotypes of bigheaded carp. This finding suggests that the alimentary tract of bigheaded carps is not simply an extreme habitat, but may also supply the cyanobacterial strains that represent the pelagic chemotypes.

A laboratory based exposure of Microcystis and Oscillatoria cyanobacterial isolates to heterotrophic bacteria.

Biological control of cyanobacteria is a viable means of controlling nuisance bloom occurrences; however the majority of studies done are against Microcystis sp., with a commonly lytic effect caused. Filamentous cyanobacteria such as Oscillatoria are not as extensively studied in this area of biological control and are often part of Microcystis dominated blooms. This study employed heterotrophic bacterial isolates selected from bloom waters that indicated potential predatory behaviour against both filamentous and colonial cyanobacterial isolates. In comparison to a known Bacillus isolate, which is often reported among bacterial control agents, three other bacteria isolates were tested as control agents against non-axenic Oscillatoria and Microcystis cyanobacterial cultures. Assessments of cyanobacterial cell responses to the bacteria were conducted through water chemistry, chlorophyll a, alkaline phosphatase activity, microscopy and cyanotoxin measurements. The changes in these parameters were compared to untreated cyanobacterial cultures where no bacteria were added. The study found that at ratios of bacteria half that of Microcystis, minimal changes in chlorophyll a were observed, whilst Oscillatoria showed a decreased chlorophyll a more in the presence of isolates 1 and 3w. The assessment of alkaline phosphatase activity showed decreased activity in both cyanobacterial isolates exposed to the bacteria, relative to the untreated control sample. Microscopy analysis through fluorescence indicated that the attachment of the bacteria to the surface of the cyanobacteria hampered with the fluorescence and scanning electron microscopy indicated that the cells were damaged by the addition of the bacterial isolates. Cyanotoxin detection through the ELISA kit testing indicated that there was toxin reduction in samples treated with the bacterial isolates, with the highest reduction being close to 60% in the case of Microcystis sp. treated with isolate 3w. Similar reductions were noted in the filamentous cyanobacterium Oscillatoria, in the presence of isolate 1.

Enhanced coagulation by high-frequency ultrasound in Microcystis aeruginosa-laden water: Strategies and mechanisms.

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%.

Cyanobacterial removal by a red soil-based flocculant and its effect on zooplankton: an experiment with deep enclosures in a tropical reservoir in China.

As one kind of cheap, environmentally-friendly and efficient treatment materials for direct control of cyanobacterial blooms, modified clays have been widely concerned. The present study evaluated cyanobaterial removal by a red soil-based flocculant (RSBF) with a large enclosure experiment in a tropical mesotrophic reservoir, in which phytoplankton community was dominated by Microcystis spp. and Anabaena spp. The flocculant was composed of red soil, chitosan and FeCl3. Twelve enclosures were used in the experiment: three replicates for each of one control and three treatments RSBF15 (15 mg FeCl3 l-1), RSBF25 (25 mg FeCl3 l-1), and RSBF35 (35 mg FeCl3 l-1). The results showed that the red soil-based flocculant can significantly remove cyanobacterial biomass and reduce concentrations of nutrients including total nitrogen, nitrate, ammonia, total phosphorus, and orthophosphate. Biomass of Microcystis spp. and Anabaena spp. was reduced more efficiently (95%) than other filamentous cyanobacteria (50%). In the RSBF15 treatment, phytoplankton biomass recovered to the level of the control group after 12 days and cyanobacteria quickly dominated. Phytoplankton biomass in the RSBF25 treatment also recovered after 12 days, but green algae co-dominated with cyanobacteria. A much later recovery of phytoplankton until the day of 28 was observed under RSBF35 treatment, and cyanobacteria did no longer dominate the phytoplankton community. The application of red soil-based flocculant greatly reduces zooplankton, especially rotifers, however, Copepods and Cladocera recovered fast. Generally, the red soil-based flocculant can be effective for urgent treatments at local scales in cyanobacteria dominating systems.

Establishing spatial and temporal patterns in Microcystis sediment seed stock viability and their relationship to subsequent bloom development in Western Lake Erie.

This study assessed the distribution, abundance, and viability of pre- and post-overwintering Microcystis sediment seed stocks in Western Lake Erie and how these variables are potentially related to past and subsequent bloom formation. We conducted a two-year spatiotemporal survey of vegetative seed stocks in Western Lake Erie, the region where annual algal blooms generally develop. Sediment was collected from 16 sites covering an area of 375 km2 and water column depths ranging from 3-9 meters. Sample collection occurred in November 2014, April 2015, November 2015, and April 2016. The abundance of total and potentially-toxic Microcystis cell equivalents were determined using quantitative polymerase chain reaction. A series of laboratory experiments using lake sediment were conducted to assess the viability of Microcystis vegetative seed stocks. Across all sampling periods, the abundance of total Microcystis in the sediment ranged from 6.6 x 10(4) to 1.7 x 10(9) cell equivalents g-1, and potentially-toxic Microcystis ranged from 1.4 x 10(3) to 4.7 x 10(6) cell equivalents g-1. The percent potentially-toxic Microcystis in the sediment ranged from <1% to 68% across all samples. Total Microcystis abundance diminished significantly over winter with densities in spring nearly 10 times less than the previous fall. However, despite cell loss from fall to spring, lab experiments demonstrated that remaining non-toxic and potentially-toxic cells were viable after the overwintering period. Further, lab grow-out experiments indicate that potentially-toxic strains recruited at a slightly higher rate than non-toxic strains, and may in part, contribute to the pattern of higher relative toxicity during early stages of the blooms. The abundance and distribution of overwintering cells did not correlate strongly to areas in the lake where subsequent summer blooms were most persistent. However, numerical analysis suggests that recruitment of benthic overwintering populations could help explain a portion of the initial rapid increase in bloom biomass and the spatial extent of this bloom initiation, particularly when recruitment is paired with subsequent growth in appropriate water column conditions.

Isolation, molecular identification, and characterization of a unique toxic cyanobacterium Microcystis sp. found in Hunan Province, China.

Global proliferation of cyanobacterial blooms associated with climate change and eutrophication constitutes a serious environmental threat. In Hunan Province a freshwater pond located in Changsha City was found to contain high concentrations of cyanobacteria, however, the characteristics of these cyanobacteria at present are not known. This study thus aimed to isolate, identify the most common bloom-forming cyanobacteria in this region and determine the toxigenic characteristics of the predominant cyanobacteria. The cyanobacteria were isolated by serial dilution and identified using polymerase chain reaction (PCR). The cyanotoxins generated by the cyanobacterium were detected using high-performance liquid chromatography with an ultra-high resolution LTQ Orbitrap Velos Pro ETD mass spectrometry equipped with electrospray ionization interface (HPLC-ESI-MS). One  species of cyanobacterium was isolated and identified as Microcystis sp. YFM1 according to the sequence of the 16S ribosome deoxyribonucleic acid (16S rDNA). It was found that this cyanobacterium contained microcystin synthetase B gene (mcyB) and produced three types of cyanotoxins including microcystin-LR, RR and YR. Our findings indicate that the Microcystis sp. YFM1 isolated from the freshwater pond in Hunan Province exhibits unique characteristics distinguishable from other known cyanobacteria.