Avaliação da ultraestrutura e morte celular em Cylindrospermopsis raciborskii (Woloszynska) Seenayya & Subba Raju (Cianobacteria) sob efeito da radiação ultravioleta

Solar radiation is essential for life on Earth, providing energy for photosynthesis and heat. The total radiation that reaches the Earth surface, 9% is represented by the ultraviolet radiation (UV). Studies with cyanobacterial groups show negative effects of UV radiation on physiological processes such as growth, survival and enzymes of nitrogen metabolism and CO2 fixation. Cylindrospermopsis raciborskii is a filamentous nitrogen fixing cyanobacteria that has become one of the species of cyanobacteria of interest to researchers because of its potential toxicity and its high ecophysiological adaptability. In this work, we investigated the effect of UV-A, UV-B and UV (A + B) on the density, cell viability and occurrence of morphological changes by transmission electron microscopy. Natural intensities were used on strain of C. racirborskii in a period of 6 hours of treatment. The treatments UV-A and UV had significant negative effects on the density and integrity of membrane (99.3 and 95% of dead cells, respectively), while UV-B showed no significant difference in density, but induced alteration of membrane integrity in 57% of the cells at the end of treatment. Ultrastructural analysis showed morphological changes in all treatments characterized mainly by reducing the proportion of thylakoid structures and pigments accessories storage. Our results demonstrate the induction of cell death in strains of C. racirborskii treatments by UV-A and UV. However, the UV-B treatment seems to be less lethal to this strain in the intensity used. The understanding of processes of cell death in phytoplanktoic populations induced by UV radiation opens new perspectives on the influence of such radiation on aquatic ecosystems and its consequences on the persistence of species, energy flow and biogeochemical cycles.A radiação solar é essencial para a vida na Terra, fornecendo energia para a fotossíntese e calor. Do total da radiação que tinge o planeta, 9% é representada pela a radiação ultravioleta (UV). Estudos com grupos de cianobactérias mostram efeitos negativos de radiação UV em processos fisiológicos, como crescimento e sobrevivência e alterações de enzimas relacionadas ao metabolismo de nitrogênio e fixação de CO2. Cylindrospermopsis raciborskii é uma cianobactéria filamentosa fixadora de nitrogênio que vem se tornando uma das espécies de cianobactérias de maior interesse de pesquisadores devido a sua potencial toxicidade e sua elevada adaptabilidade ecofisiológica. Neste trabalho, investigou-se o efeito da radiação UV A, UV-B e UV (A+B) na densidade, viabilidade celular e ocorrência de alterações morfológicas por microscopia eletrônica de transmissão. Foram usadas intensidades naturais sobre a cepa de C. racirborskii no período de 6 horas de tratamento. Os tratamentos UV-A e UV (A+B) apresentaram efeitos negativos significativos na densidade e integridade de membrana (99,3 e 95% de células mortas, respectivamente), enquanto UV-B não apresentou diferença significativa na densidade, mas induziu alteração de integridade da membrana em 57% das células no final do tratamento. Análises ultraestruturais demonstraram modificações morfológicas em todos os tratamentos caracterizadas principalmente por redução da proporção de tilacóides e estruturas de armazenamento de pigmentos acessórios. Nossos resultados demonstram a indução de morte celular significativa em cepa de C. racirborskii pelos tratamentos UV-A e UV (A+B). Entretanto, o tratamento UV-B parece ser menos letal para esta cepa na intensidade usada. O entendimento de processos de morte celular em populações fitoplactônicas induzida por radiação UV (A+B) abre novas perspectivas sobre a influência deste tipo de radiação em ecossistemas aquáticos e suas conseqüências na persistência de espécies, fluxo energético e ciclos biogeoquímicos

Increasing Temperature Counteracts the Negative Effect of UV Radiation on Growth and Photosynthetic Efficiency of Microcystis aeruginosa and Raphidiopsis raciborskii

High temperature can promote cyanobacterial blooms, whereas ultraviolet radiation (UVR) can potentially depress cyanobacterial growth by damaging their photosynthetic apparatus. Although the damaging effect of UVR has been well documented, reports on the interactive effects of UV radiation exposure and warming on cyanobacteria remain scarce. To better understand the combined effects of temperature and UVR on cyanobacteria, two strains of nuisance species, Microcystis aeruginosa (MIRF) and Raphidiopsis raciborskii (formerly Cylindrospermopsis raciborskii, CYRF), were grown at 24°C and 28°C and were daily exposed to UVA + UVB (PAR + UVA+UVB) or only UVA (PAR + UVA) radiation. MIRF and CYRF growth rates were most affected by PAR + UVA+UVB treatment and to a lesser extent by the PAR + UVA treatment. Negative UVR effects on growth, Photosystem II (PSII) efficiency and photosynthesis were pronounced at 24°C when compared to that at 28°C. Our results showed a cumulative negative effect on PSII efficiency in MIRF, but not in CYRF. Hence, although higher temperature ameliorates UVR damage, interspecific differences may lead to deviating impacts on different species, and combined elevated temperature and UVR stress could influence species competition.</p

Potential effects of UV radiation on photosynthetic structures of the bloom-forming cyanobacterium Cylindrospermopsis raciborskii CYRF-01

Cyanobacteria are aquatic photosynthetic microorganisms. While of enormous ecological importance, they have also been linked to human and animal illnesses around the world as a consequence of toxin production by some species. Cylindrospermopsis raciborskii, a filamentous nitrogen-fixing cyanobacterium, has attracted considerable attention due to its potential toxicity and ecophysiological adaptability. We investigated whether C. raciborskii could be affected by ultraviolet (UV) radiation. Non-axenic cultures of C. raciborskii were exposed to three UV treatments (UVA, UVB or UVA + UVB) over a 6 h period, during which cell concentration, viability and ultrastructure were analyzed. UVA and UVA + UVB treatments showed significant negative effects on cell concentration (decreases of 56.4% and 64.3%, respectively). This decrease was directly associated with cell death as revealed by a cell viability fluorescent probe. Over 90% of UVA + UVB- and UVA-treated cells died. UVB did not alter cell concentration, but reduced cell viability in almost 50% of organisms. Transmission electron microscopy (TEM) revealed a drastic loss of thylakoids, membranes in which cyanobacteria photosystems are localized, after all treatments. Moreover, other photosynthetic- and metabolic-related structures, such as accessory pigments and polyphosphate granules, were damaged. Quantitative TEM analyses revealed a 95.8 % reduction in cell area occupied by thylakoids after UVA treatment, and reduction of 77.6 % and 81.3 % after UVB and UVA + UVB treatments, respectively. Results demonstrated clear alterations in viability and photosynthetic structures of C. raciborskii induced by various UV radiation fractions. This study facilitates our understanding of the subcellular organization of this cyanobacterium species, identifies specific intracellular targets of UVA and UVB radiation and reinforces the importance of UV radiation as an environmental stressor

Lanthanum in Water, Sediment, Macrophytes and chironomid larvae following application of Lanthanum modified bentonite to lake Rauwbraken (The Netherlands)

Lanthanum Modified Bentonite (LMB; Phoslock®) is used to mitigate eutrophication by binding phosphate released from sediments. This study investigated the fate of lanthanum (La) from LMB in water, sediment, macrophytes, and chironomid larvae in Lake Rauwbraken (The Netherlands). Before the LMB application, water column filterable La (FLa) was 0.02 µg L−1, total La (TLa) was 0.22 µg L−1. In sediment the total La ranged 0.03–1.86 g m−2. The day after the application the maximum FLa concentration in the water column was 44 µg L−1, TLa was 528 µg L−1, exceeding the Dutch Maximum Permissible Concentrations (MPC) of 10.1 µg L−1 by three to fourfold. TLa declined below the MPC after 15 days, FLa after 75 days. After ten years, FLa was 0.4 µg L−1 and TLa was 0.7 µg L−1. Over the post-application years, FLa and TLa showed statistically significant downward trends. While the LMB settled homogeneously on sediment, after 3 years it redistributed to 0.2–5.4 g La m−2 within shallow zones, and 30.7 g m−2 to 40.0 g La m−2 in deeper zones. In the upper 20 cm of sediment, La concentrations were 7–6702 mg kg −1 dry weight (DW) compared to 0.5–7.0 mg kg−1 before application. Pre-application anaerobic sediment release of FLa was 0.006 mg m−2 day−1. Three months after the application it was 1.02 mg m−2 day−1. Three years later it was 0.063 mg m−2 day−1. Before application La in plants was 0.8–5.1 mg La kg−1 DW, post-application values were up to 2925 mg La kg−1 DW. In chironomid larvae, La increased from 1.7 µg g−1 DW before application to 1421 µg g−1 DW after one month, 3 years later it was 277 µg g−1 DW. Filtration experiments indicate FLa is not truly dissolved free La3+ cations.</p

The Cyanobacterium Cylindrospermopsis raciborskii (CYRF-01) Responds to Environmental Stresses with Increased Vesiculation Detected at Single-Cell Resolution

Secretion of membrane-limited vesicles, collectively termed extracellular vesicles (EVs), is an important biological process of both eukaryotic and prokaryotic cells. This process has been observed in bacteria, but remains to be better characterized at high resolution in cyanobacteria. In the present work, we address the release of EVs by Cylindrospermopsis raciborskii (CYRF-01), a filamentous bloom-forming cyanobacterium, exposed to environmental stressors. First, non-axenic cultures of C. raciborskii (CYRF-01) were exposed to ultraviolet radiation (UVA + UVB) over a 6 h period, which is known to induce structural damage to this species. Second, C. raciborskii was co-cultured in interaction with another cyanobacterium species, Microcystis aeruginosa (MIRF-01), over a 24 h period. After the incubation times, cell density and viability were analyzed, and samples were processed for transmission electron microscopy (TEM). Our ultrastructural analyses revealed that C. raciborskii constitutively releases EVs from the outer membrane during its normal growth and amplifies such ability in response to environmental stressors. Both situations induced significant formation of outer membrane vesicles (OMVs) by C. raciborskii compared to control cells. Quantitative TEM revealed an increase of 48% (UV) and 60% (interaction) in the OMV numbers compared to control groups. Considering all groups, the OMVs ranged in size from 20 to 300 nm in diameter, with most OMVs showing diameters between 20 and 140 nm. Additionally, we detected that OMV formation is accompanied by phosphatidylserine exposure, a molecular event also observed in EV-secreting eukaryotic cells. Altogether, we identified for the first time that C. raciborskii has the competence to secrete OMVs and that under different stress situations the genesis of these vesicles is increased. The amplified ability of cyanobacteria to release OMVs may be associated with adaptive responses to changes in environmental conditions and interspecies cell communication

‘Floc and Sink’ Technique Removes Cyanobacteria and Microcystins from Tropical Reservoir Water

Combining coagulants with ballast (natural soil or modified clay) to remove cyanobacteria from the water column is a promising tool to mitigate nuisance blooms. Nevertheless, the possible effects of this technique on different toxin-producing cyanobacteria species have not been thoroughly investigated. This laboratory study evaluated the potential effects of the “Floc and Sink” technique on releasing microcystins (MC) from the precipitated biomass. A combined treatment of polyaluminium chloride (PAC) with lanthanum modified bentonite (LMB) and/or local red soil (LRS) was applied to the bloom material (mainly Dolichospermum circinalis and Microcystis aeruginosa) of a tropical reservoir. Intra and extracellular MC and biomass removal were evaluated. PAC alone was not efficient to remove the biomass, while PAC + LMB + LRS was the most efficient and removed 4.3–7.5 times more biomass than other treatments. Intracellular MC concentrations ranged between 12 and 2.180 µg L−1 independent from the biomass. PAC treatment increased extracellular MC concentrations from 3.5 to 6 times. However, when combined with ballast, extracellular MC was up to 4.2 times lower in the top of the test tubes. Nevertheless, PAC + LRS and PAC + LMB + LRS treatments showed extracellular MC concentration eight times higher than controls in the bottom. Our results showed that Floc and Sink appears to be more promising in removing cyanobacteria and extracellular MC from the water column than a sole coagulant (PAC).</p

Temporal and spatial variation in the efficiency of a Floc & Sink technique for controlling cyanobacterial blooms in a tropical reservoir

One of the main symptoms of eutrophication is the proliferation of phytoplankton biomass, including nuisance cyanobacteria. Reduction of the external nutrient load is essential to control eutrophication, and in-lake interventions are suggested for mitigating cyanobacterial blooms to accelerate ecosystem recovery. Floc & Sink (F&S) is one such intervention technique that consists of applying a low dose of coagulants in combination with ballasts for removing cyanobacteria biomass. It is especially suitable for deep lakes with an external nutrient load that is higher than the internal load and suffers from perennial cyanobacterial bloom events. Studies showing the efficacy of the F&S technique have been published, but those testing its variation in efficacy with changes in the environmental conditions are still scarce. Therefore, we evaluated the efficiency of the F&S technique to remove cyanobacteria from water samples collected monthly from two different sites in a deep tropical reservoir (Funil Reservoir, Brazil) in the laboratory. We tested the efficacy of two coagulants, chitosan (CHI) and poly-aluminum chloride (PAC), alone and in combination with lanthanum-modified bentonite (LMB) in settling phytoplankton biomass. We hypothesized that: ⅰ) the combined treatments are more effective in removing the algal biomass and ⅱ) the efficiency of F&S treatments varies spatially and monthly due to changes in environmental conditions. The combined treatments (PAC + LMB or CHI + LMB) removed up to seven times more biomass than single treatments (PAC, CHI, or LMB). Only the treatments CHI and LMB + CHI differed in efficiency between the sites, although all treatments showed significant variation in efficiency over the months at both the sampling sites. The combined treatments exhibited lower removal efficacy during the warm-rainy months (October–March) than during the mild-cold dry months (April–September). At high pH (pH > 10), the efficiency of the CHI and LMB + CHI treatments decreased. CHI had lower removal efficiency when single-cell cyanobacteria were abundant, while the combined treatments were equally efficient regardless of the morphology of the cyanobacteria. Hence, the combination of PAC as a coagulant with a ballast LMB is the most effective technique to precipitate cyanobacteria under the conditions that are encountered around the year in this tropical reservoir

Efficacy of Coagulants and Ballast Compounds in Removal of Cyanobacteria (Microcystis) from Water of the Tropical Lagoon Jacarepaguá (Rio de Janeiro, Brazil)

Eutrophication is considered the most important water quality problem in freshwaters and coastal waters worldwide promoting frequent occurrence of blooms of potentially toxic cyanobacteria. Removal of cyanobacteria from the water column using a combination of coagulant and ballast is a promising technique for mitigation and an alternative to the use of algaecides. In laboratory, we tested experimentally the efficiency of two coagulants, polyaluminium chloride (PAC) and chitosan (made of shrimp shells), alone and combined with two ballasts: red soil (RS) and the own lagoon sediment, to remove natural populations of cyanobacteria, from an urban brackish coastal lagoon. PAC was a very effective coagulant when applied at low doses (≤8 mg Al L−1) and settled the cyanobacteria, while at high doses (≥16 mg Al L−1) large flocks aggregated in the top of test tubes. In contrast, chitosan was not able to form flocks, even in high doses (>16 mg L−1) and did not efficiently settle down cyanobacteria when combined with ballast. The RS itself removed 33–47 % of the cyanobacteria. This removal was strongly enhanced when combined with PAC in a dose-dependent matter; 8 mg Al L−1 was considered the best dose to be applied. The lagoon sediment alone did not promote any settling of cyanobacteria but removal was high when combined with PAC. Combined coagulant and ballast seems a very efficient, cheap, fast and safe curative measure to lessen the harmful cyanobacteria bloom nuisance in periods when particularly needed, such as around the 2016 Olympics in Jacarepaguá Lagoon

The efficiency of combined coagulant and ballast to remove harmful cyanobacterial blooms in a tropical shallow system

We tested the hypothesis that a combination of coagulant and ballast could be efficient for removal of positively buoyant harmful cyanobacteria in shallow tropical waterbodies, and will not promote the release of cyanotoxins. This laboratory study examined the efficacy of coagulants [polyaluminium chloride (PAC) and chitosan (made of shrimp shells)] alone, and combined with ballast (lanthanum modified bentonite, red soil or gravel) to remove the natural populations of cyanobacteria collected from a shallow eutrophic urban reservoir with alternating blooms of Cylindrospermopsis and Microcystis. PAC combined with ballast was effective in settling blooms dominated by Microcystis or Cylindrospermopsis. Contrary to our expectation, chitosan combined with ballast was only effective in settling Cylindrospermopsis-dominated blooms at low pH, whereas at pH ≥ 8 no effective flocculation and settling could be evoked. Chitosan also had a detrimental effect on Cylindrospermopsis causing the release of saxitoxins. In contrast, no detrimental effect on Microcystis was observed and all coagulant-ballast treatments were effective in not only settling the Microcystis dominated bloom, but also lowering dissolved microcystin concentrations. Our data show that the best procedure for biomass reduction also depends on the dominant species