A novel strategy of bloom forming cyanobacteria Microcystis sp. in response to phosphorus deficiency: Using non-phosphorus lipids substitute phospholipids.

Despite significant reductions in phosphorus (P) loads, lakes still experience cyanobacterial blooms. Little is known regarding cellular P regulation in response to P deficiency in widely distributed bloom causing species such as Microcystis. In this study, we investigated changes in P containing and non-P lipids contents and their ratios concomitantly with the determinations of expression levels of genes encoding these lipids in cultural and field Microcystis samples. In the culture, the content of phosphatidylglycerol (PG) decreased from 2.1 µg g-1 in P replete control to 1.2 µg g-1 in P-deficient treatment, while non-P lipids, like sulfoquinovosyldiacylglycerol (SQDG) and monogalactosyldiacylglycerol (MGDG), increased dramatically from 13.6 µg g-1 to 142.3 µg g-1, and from 0.9 µg g-1 to 16.74 µg g-1, respectively. The expression of the MGDG synthesis gene, mgdE, also increased under low P conditions. Significant positive relationships between soluble reactive phosphorus (SRP) and ratios of P-containing lipids (PG) to non-P lipids, including SQDG, MGDG and digalactosyldiacylglycerol (DGDG) (P < 0.05) were observed in the field investigations. Both cultural and field data indicated that Microcystis sp. might increase non-P lipids proportion to lower P demand when suffering from P deficiency. Furthermore, despite lipid remodeling, photosynthetic activity remained stable, as indicated by comparable chlorophyll fluorescence and Fv/Fm ratios among cultural treatments. These findings suggested that Microcystis sp. may dominate in P-limited environments by substituting glycolipids and sulfolipids for phospholipids to reduce P demand without compromising the photosynthetic activity. This effective strategy in response to P deficiency meant a stricter P reduction threshold is needed in terms of Microcystis bloom control.

Comparison of Bacterial Assemblages Associated with Harmful Cyanobacteria under Different Light Conditions.

Harmful cyanobacterial blooms in freshwater ecosystems are closely associated with changes in the composition of symbiotic microbiomes, water quality, and environmental factors. In this work, the relationship between two representative harmful cyanobacterial species (Anabaena sp. and Microcystis sp.) and their associated bacterial assemblages were investigated using a 16S rRNA-based meta-amplicon sequencing analysis during a large-scale cultivation of cyanobacteria under different light conditions with limited wavelength ranges (natural light, blue-filtered light, green-filtered light, and dark conditions). During the cultivation periods, the growth pattern of cyanobacteria and bacterial composition of the phycosphere considerably varied in relation to light restrictions. Unlike other conditions, the cyanobacterial species exhibited significant growth during the cultivation period under both the natural and the blue light conditions. Analyses of the nitrogenous substances revealed that nitrogen assimilation by nitrate reductase for the growth of cyanobacteria occurred primarily under natural light conditions, whereas nitrogenase in symbiotic bacteria could also be activated under blue light conditions. Sphingobium sp., associated with nitrogen assimilation via nitrogenase, was particularly dominant when the cell density of Microcystis sp. increased under the blue light conditions. Thus, cyanobacteria could have symbiotic relationships with ammonium-assimilating bacteria under light-limited conditions, which aids the growth of cyanobacteria.

Modelling of threats that affect Cyano-HABs in an eutrophicated reservoir: First phase towards water security and environmental governance in watersheds.

Cyano-HABs are proliferating around the world due to anthropogenic nutrient enrichment of freshwater bodies. This study seeks to obtain a holistic vision over the various threats that affect the Cyano-HABs of Umia basin and especially of A Baxe reservoir (Galicia, NW Spain), through the method of Partial least squares path modelling (PLS-PM). The A Baxe reservoirs is a fundamental source of drinking water supply to surrounding dwellings. This study identifies and quantify the variables that increase contaminant concentration and decrease ecological integrity, as well as how this scenario evolved over various hydrologic years. In this regard, the PLS-PM equations will be robust and powerful tools to predict changes in eutrophication and ecological integrity, as response to measures implemented in the basin that can improve water quality. The dependent latent variables are "Eutrophication" (chlorophyl-a, Microcystis sp.) and "Ecological Integrity" (METI Bioindicator). The independent latent variables are "SWP", which represents surface water parameters (phosphorus, nitrogen and pH) and "Climatic Conditions" (temperature, precipitation). The PLS-PM results revealed that 51.0% of "Eutrophication" is predicted by the independent variables. The connections between latent variables are quantified through path coefficients (ß). The "SWP" contributes by increasing "Eutrophication" (ß = 0.235), the same occurring with the "Climatic Conditions" (ß = -0.672). The variables "Eutrophication" (ß = -0.217) and "SWP" (ß = -0.483) lower the "Ecological Integrity". On the other hand, different trophic scenarios, adapted to the temperature increase predicted for the study area, were tested, and it was found that ecological integrity would improve by 46% if the oligotrophic state were reached. Therefore, it is recommended to prevent pollution by means of water control and governance plans, as well as corrective and preventive measures, which guarantee the water security of the river basins. Despite the complex mathematics behind the PLS-PM models, their user-friendly development and application through interactive graphical interfaces make them easily transposable to other eutrophic reservoirs, widening the readership of these studies focused on multiple-geosphere assessment of environmental impacts.

Effect of seasonal succession of algal communities on fouling of MF membrane.

An emerging threat to membrane application is the seasonal proliferation of algae in water sources such as rivers, reservoirs and lakes. This study investigated the link between feed parameters and the membrane performance of a pilot-scale microfiltration (MF) plant for 7 months. The seasonal succession of algae in relation to temperature dynamics was monitored. Temperature-dependent seasonal patterns for algae species were observed. The water temperatures during the dominance of cyanobacteria, especially Microcystis, were relatively higher (over 25 °C) than those during the dominance of diatoms. Diatoms did not much affect membrane performance (less than 0.2 kgf/cm2), however, under the cyanobacterial dominance condition, especially Microcystis sp., transmembrane pressure (TMP) reached up to the limited level (0.4 kgf/cm2) within one month. Concurrently UV absorbance at 254 nm wavelength and dissolved organic carbon values increased significantly during the Microcystis bloom and the build-up rate of TMP increased up to 0.005 kgf/cm2/day. Membrane autopsy also showed that during the dominance of diatom, application of cleaning agents can fully remove foulants on the membrane surface. However, during the dominance of cyanobacteria, there is a lot of Al, Si and organic complex on the fouled membrane, indicating the formation of Al-organic complexes that contributed to the residual membrane fouling. It is suggested that the irrecoverable fouling layer still contained some Al, mostly in complex with organics. Thus, organic matter originated from cyanobacteria may cause a serious impact on membrane fouling by forming the complex with metal ions originated from coagulant.

Dynamics of microcystins and saxitoxin in the Indian River Lagoon, Florida.

Harmful algal blooms that can produce toxins are common in the Indian River Lagoon (IRL), which covers ~250 km of Florida's east coast. The current study assessed the dynamics of microcystins and saxitoxin in six segments of the IRL: Banana River Lagoon (BRL), Mosquito Lagoon (ML), Northern IRL (NIRL), Central IRL (CIRL), Southern IRL (SIRL), and the St. Lucie Estuary (SLE). Surface water samples (n = 40) collected during the 2018 wet and 2019 dry season were analyzed to determine associations between toxins and temperature, salinity, pH, oxygen saturation, concentrations of dissolved nutrients and chlorophyll-a, presence of biosynthetic genes for toxins, relative abundance of planktonic species, and composition of the microbial community. The potential toxicity of samples was assessed using multiple mammalian cell lines. Enzyme-Linked Immunosorbent Assays were used to determine concentrations of microcystins and saxitoxin. Overall, the microcystins concentration ranged between 0.01-85.70 µg/L, and saxitoxin concentrations ranged between 0.01-2.43 µg/L across the IRL. Microcystins concentrations were 65% below the limit of quantification (0.05 µg/L), and saxitoxin concentrations were 85% below the limit of detection (0.02 µg/L). Microcystins concentrations were higher in the SLE, while saxitoxin was elevated in the NIRL and BRL. Cytotoxicity related to the presence of microcystins was seen in the SLE during the wet season. No significant patterns between cytotoxicity and saxitoxin were identified. Dissolved nutrients were identified as the most highly related parameters, explaining 53% of microcystin and 47% of saxitoxin variability. Multivariate models suggested cyanobacteria, flagellates, ciliates, and diatoms as the subset of microorganisms whose abundances were maximally correlated with saxitoxin and microcystins concentrations. Lastly, biosynthetic genes for microcystins were detected in the SLE and for saxitoxin in the BRL and NIRL. These results highlight the synergistic roles environmental and biological parameters play in influencing the dynamics of toxin production by harmful algae in the IRL.

Assessment of water quality in eutrophized water bodies through the application of indexes and toxicity.

It is essential to have tools that can be used to diagnose water resources. For this reason, this study sets out on the one hand to assess water quality in three reservoirs in Spain (Salas, A Baxe and Conchas) with Cyano-Habs problems through the application of water quality indexes: The National Sanitation Foundation Water Quality Index (NFSWQI), the General Quality Index (GQI), the Trophic State Index (TSI), and the Trophic Contamination Index (ICOTRO). On the other hand, it seeks to learn how parameters such as nitrogen and phosphorus influence the presence of cyanotoxin, specifically Microcystis aeruginosa. To that end, samples from the three reservoirs are cultured and physical-chemical parameters and the toxicity of the water are measured. The results show that Salas reservoir has the worst water quality rating (GQI is bad and NFSWQI medium), while As Conchas and A Baxe obtain very good figures for NFSWQI. This contrasts with the data obtained via the TSI of moderately eutrophic conditions for all three reservoirs, and hypereutrophic levels for As Conchas and A Baxe downstream. On the other hand, the toxicity analysis shows levels of 1.12 ±â€¯0.06 µg/l microcystin-LR (MC-LR) for As Conchas, 0.64 ±â€¯0.04 µg/l MC-LR for Salas, and 1.24 ±â€¯0.05 µg/l MC-LR for A Baxe, of which 20% corresponds to free MC-LR. This study finds that nitrogen is the parameter that most favors the production of MC-LR. We conclude that the eutrophication indexes are more reliable when studying the presence of cyanobacteria. Furthermore, nitrogen and phosphorous are the most significant parameters in this regard. They are taken into account in the quality indices (GQI, NFSWI), but they are not sufficiently representative. It is recommended as a future line of research that water quality indices be adapted or designed to incorporate eutrophication levels and even water toxicity.

Citricoccus lacusdiani sp. nov., an actinobacterium promoting Microcystis growth with limited soluble phosphorus.

A novel actinobacterium, designated strain JXJ CY 21T, was isolated from the culture mass of Microcystis sp. FACHB-905 collected from Lake Dianchi, South-west China. Polyphasic taxonomic study revealed that the isolate should be a member of the genus Citricoccus. Comparison of the 16S rRNA gene sequence of strain JXJ CY 21T with the available sequences in the GenBank database showed that the strain is closely related to Citricoccus zhacaiensis FS24T (97.8 % similarity), Citricoccus parietis 02-Je-010T (97.7 %), Citricoccus terreus V3M1T (97.6 %), Citricoccus nitrophenolicus PNP1T (97.2 %), Citricoccus alkalitolerans YIM 70010T (97.2 %) and Citricoccus muralis 4-0T (97.0 %). The DNA-DNA hybridization values between strain JXJ CY 21T and the related type strains C. zhacaiensis FS24T and C. parietis 02-Je-010T were 16.0 ± 2.6 and 5.4 ± 1.7 %, respectively. The peptidoglycan in the cell wall was A4α type containing lysine-glutamic acid-glycine. The major respiratory menaquinone was found to be MK-8 (H2) (98.5 %), while the major cellular fatty acids (>10 %) were anteiso-C15:0, iso-C16:0, iso-C15:0 and iso-C14:0. The polar lipids detected were diphosphatidylglycerol, phosphatidylinositol, phosphatidylglycerol, an unidentified phospholipid and an unidentified glycolipid. The DNA G + C content was determined to be 62.7 mol%. Strain JXJ CY 21T can solubilize both insoluble inorganic and organic phosphates up to 24.7 and 1.7 mg/l respectively. This property of the novel actinobacterium acts as a modulator for enhancement of growth of Microcystis sp. FACHB-905 in the lake ecosystem where the amount of soluble phosphate is limited. On the basis of the above taxonomic data, strain JXJ CY 21T represents a novel species of the genus Citricoccus, for which the name Citricoccus lacusdiani sp. nov. is proposed. The type strain is JXJ CY 21T (=KCTC 29653T = DSM 29160T).

Capsular polysaccharides facilitate enhanced iron acquisition by the colonial cyanobacterium Microcystis sp. isolated from a freshwater lake.

Microcystis sp., especially in its colonial form, is a common dominant species during cyanobacterial blooms in many iron-deficient water bodies. It is still not entirely clear, however, how the colonial forms of Microcystis acclimate to iron-deficient habitats, and the responses of unicellular and colonial forms to iron-replete and iron-deficient conditions were examined here. Growth rates and levels of photosynthetic pigments declined to a greater extent in cultures of unicellular Microcystis than in cultures of the colonial form in response to decreasing iron concentrations, resulting in the impaired photosynthetic performance of unicellular Microcystis as compared to colonial forms as measured by variable fluorescence and photosynthetic oxygen evolution. These results indicate that the light-harvesting ability and photosynthetic capacity of colonial Microcystis was less affected by iron deficiency than the unicellular form. The carotenoid contents and nonphotochemical quenching of colonial Microcystis were less reduced than those of the unicellular form under decreasing iron concentrations, indicating that the colonial morphology enhanced photoprotection and acclimation to iron-deficient conditions. Furthermore, large amounts of iron were detected in the capsular polysaccharides (CPS) of the colonies, and more iron was found to be attached to the colonial Microcystis CPS under decreasing iron conditions as compared to unicellular cultures. These results demonstrated that colonial Microcystis can acclimate to iron deficiencies better than the unicellular form, and that CPS plays an important role in their acclimation advantage in iron-deficient waters.

Harvesting green algae from eutrophic reservoir by electroflocculation and post-use for biodiesel production.

Each year there are more frequent blooms of green algae and cyanobacteria, representing a serious environmental problem of eutrophication. Electroflocculation (EF) was studied to harvest the algae which are present in reservoirs, as well as different factors which may influence on the effectiveness of the process: the voltage applied to the culture medium, run times, electrodes separation and natural sedimentation. Finally, the viability of its use to obtain biodiesel was studied by direct transesterification. The EF process carried out at 10V for 1min, with an electrode separation of 5.5cm and a height of 4cm in culture vessel, obtained a recovery efficiency greater than 95%, and octadecenoic and palmitic acids were obtained as the fatty acid methyl esters (FAMEs). EF is an effective method to harvest green algae during the blooms, obtaining the greatest amount of biomass for subsequent use as a source of biodiesel.

Growth and microcystin production of a Brazilian Microcystis aeruginosa strain (LTPNA 02) under different nutrient conditions

Cyanobacteria are prokaryotic and photosynthetic organisms, which can produce a wide range of bioactive compounds with different properties; including a variety of toxic compounds, also known as cyanotoxins. In this work, we describe the isolation of seven cyanobacterial strains from two reservoirs in São Paulo State, Brazil. Seven different chemical variants of microcystins (MC-RR, MC-LR, MC-YR, MC-LF, MC-LW, and two demethylated variants, dm-MC-RR and dm-MC-LR) were detected in three of the ten isolated strains. One particular Microcystis aeruginosa strain (LTPNA 02) was chosen to evaluate its growth by cell count, and its toxin production under seven different nutritional regimes. We observed different growth behaviors in the logarithmic growth period for only three experiments (p < 0.05). The total growth analysis identified four experiments as different from the control (p < 0.01). Three microcystin variants (MC-RR, MC-LR and MC-YR) were quantified by liquid chromatography-tandem mass spectrometry. At the experimental end, the toxin content was unchanged when comparing cell growth in ASM-1 (N:P = 1), MLA and BG-11 (N:P = 10) medium. In all other experiments, the lowest microcystin production was observed from cells grown in Bold 3N medium during the exponential growth phase. The highest microcystin content was observed in cultures using BG-11(N:P = 100) medium.

Toxicity thresholds for juvenile freshwater mussels Echyridella menziesii and crayfish Paranephrops planifrons, after acute or chronic exposure to Microcystis sp.

Survival of juvenile freshwater mussels (Echyridella menziesii (Gray, 1843) formerly known as Hyridella menziesi) and crayfish (Paranephrops planifrons, White, 1842) decreased after four days exposure to microcystin-containing cell-free extracts (MCFE) of Microcystis sp. at concentrations typical of severe cyanobacterial blooms. Crayfish survival was 100, 80, and 50% in microcystin concentrations of 1339, 2426, and 11146 µg L(-1) respectively, and shade- and shelter-seeking behavior was negatively affected when concentrations were ≥2426 µg L(-1) . Mussel survival decreased to 92% and reburial rates decreased to 16% after exposure for 96 h to MCFE containing microcystins at concentrations of 5300 µg L(-1) . Crayfish survival was 100% when fed freeze-dried Microcystis sp. incorporated into an artificial diet (6-100 µg microcystin kg(-1) ww) at dietary doses from 0.03 to 0.55 µg g(-1) body weight d(-1) for 27 days. Specific growth rate was significantly lower in crayfish fed ≥0.15 µg g(-1) body weight day(-1) compared with controls, but not compared with a diet incorporating nontoxic cyanobacteria. Microcystins accumulated preferentially in crayfish hepatopancreas and mussel digesta as MCFE or dietary concentrations increased. These laboratory data indicate that, assuming dissolved oxygen concentrations remain adequate, and no simultaneous exposure to live Microcystis sp. cells, cell-free microcystins will only be a significant stressor to juvenile crayfish and mussels in severe Microcystis sp. blooms. In contrast, crayfish were negatively affected by relatively low concentrations of microcystins in artificial diets compared with those measured locally in benthic cyanobacterial mats.

Effect of nutrient limitation of cyanobacteria on protease inhibitor production and fitness of Daphnia magna.

Herbivore-plant interactions have been well studied in both terrestrial and aquatic ecosystems as they are crucial for the trophic transfer of energy and matter. In nutrient-rich freshwater ecosystems, the interaction between primary producers and herbivores is to a large extent represented by Daphnia and cyanobacteria. The occurrence of cyanobacterial blooms in lakes and ponds has, at least partly, been attributed to cyanotoxins, which negatively affect the major grazer of planktonic cyanobacteria, i.e. Daphnia. Among these cyanotoxins are the widespread protease inhibitors. These inhibitors have been shown (both in vitro and in situ) to inhibit the most important group of digestive proteases in the gut of Daphnia, i.e. trypsins and chymotrypsins, and to reduce Daphnia growth. In this study we grew cultures of the cyanobacterium Microcystis sp. strain BM25 on nutrient-replete, N-depleted or P-depleted medium. We identified three different micropeptins to be the cause for the inhibitory activity of BM25 against chymotrypsins. The micropeptin content depended on nutrient availability: whereas N limitation led to a lower concentration of micropeptins per biomass, P limitation resulted in a higher production of these chymotrypsin inhibitors. The altered micropeptin content of BM25 was accompanied by changed effects on the fitness of Daphnia magna: a higher content of micropeptins led to lower IC50 values for D. magna gut proteases and vice versa. Following expectations, the lower micropeptin content in the N-depleted BM25 caused higher somatic growth of D. magna. Therefore, protease inhibitors can be regarded as a nutrient-dependent defence against grazers. Interestingly, although the P limitation of the cyanobacterium led to a higher micropeptin content, high growth of D. magna was observed when they were fed with P-depleted BM25. This might be due to reduced digestibility of P-depleted cells with putatively thick mucilaginous sheaths. These findings indicate that both the grazer and the cyanobacterium benefit from P reduction in terms of digestibility and growth inhibition, which is an interesting starting point for further studies.

Investigação da biossíntese de toxinas produzidas por cepas de cianobactérias / Investigation on the cyanobacterial strains toxins biossinthesys

A demanda crescente de água doce de boa qualidade são problemas atuais e mundiais, além do descaso com os dejetos lançados nos ambientes aquáticos que comprometem a qualidade dos recursos hídricos. Um dos parâmetros que atesta a potabilidade da água é a presença de cianobactérias e cianotoxinas. Cianobactérias são microrganismos procariontes aeróbicos fotoautróficos que sintetizam as cianotoxinas. Estes compostos podem ser classificados de acordo com seus mecanismos de ação em hepatotóxicos, neurotóxicos e dermatotóxicos. Por sua diversidade, representam diferentes riscos não só ao ecossistema e a outros organismos dos ambientes aquáticos, como também aos seres humanos. Esse projeto visou o isolamento e cultivo de cepas de cianobactérias produtoras de toxinas para a investigação da biossíntese desses compostos. Com este intuito, foram realizadas coletas de água em três reservatórios no estado de São Paulo e um no Paraná. Cepas de cianobactérais foram isoladas, identificadas e analisadas quanto à produção de toxinas. Uma cepa de Microcystis aeruginosa (LTPNA 02) produtora de microcistinas (MC-LR, MC-RR, MC-YR, MC-LF, MC-LW e desm-MC-LR e desm- MC-RR) foi escolhida para ser estudada frente diferentes condições de cultivo e ter o seu crescimento, produção de toxinas e expressão gênica estudados. Foram utilizados os meios de cultura já referidos na literatura: ASM-1 (N:P=1, 10 e 20), MLA (N:P=10), Bold 3N (N:P=16) e BG-11 (N:P=10 e 100). Para acompanhar o crescimento, dois métodos foram utilizados: contagem de células e espectrofotometria. As toxinas foram quantificadas por LC-MS - QTrap. A análise da expressão gênica foi realizada por reação de PCR em tempo real pelo método de quantificação relativa ΔΔCt. Foi observada diferença no crescimento da cepa estudada nos diferentes meios de cultivo empregados. A contagem das células permitiu a identificação das fases logarítmica e total de crescimento. Durante a fase logarítmica, três experimentos demonstraram...

There is a great concern these days about potable and good quality water due to the increase of the population needs and also to the arising problems with contamination caused by anthropogenic sources. The presence of cyanobacteria and cyanotoxins are some parameters that attest water potability. Cyanobacteria are prokaryotic aerobic photoautotrophic microorganisms that may synthesize cyanotoxins. These compounds can be classified as hepatotoxic, neurotoxic and dermatotoxic according to their action mechanisms. Because of their diversity, they may represent different risks, not only to their ecosystem and other aquatic living organisms, but also to human beings. The aim of this project was the isolation and cultivation of cyanotoxin-producing cyanobacteria for further investigation on the biosynthesis of these compounds. Water samples from three different reservoirs in São Paulo state and one in Paraná state were collected in order to isolate cyanobacteria strains and accomplish their identification and to evaluate the toxin production. The Microcystis aeruginosa (LTPNA 02) microcystin producer strain (MCLR, MC-RR, MC-YR, MC-LF, MC-LW, desm-MC-LR and desm-MC-RR) was chosen to be grown in different cultivation conditions and later analyzed for its growth rate, toxin production and gene expression. All culture media used in this research were chosen according to the literature: ASM-1 (N:P=1, 10 and 20), MLA (N:P=10), Bold 3N (N:P=16) and BG-11 (N:P=10 and 100). To evaluate growth rate, two techniques were used: cell counting and absorbance determination in two different wavelengths (680 nm and 750 nm). Toxins were quantified by LC-MS in a hybrid triple-quadrupole instrument (Qtrap). Gene expression was assessed by real time PCR, using the ΔΔCt relative quantification method. Cell counting allowed total growth and logarithmic phase identification. During the last, three experiments showed statistical difference from control group (p<0,05). Four experiments...