Toxicity at the Edge of Life: A Review on Cyanobacterial Toxins from Extreme Environments.

Cyanotoxins are secondary metabolites produced by cyanobacteria, of varied chemical nature and toxic effects. Although cyanobacteria thrive in all kinds of ecosystems on Earth even under very harsh conditions, current knowledge on cyanotoxin distribution is almost restricted to freshwaters from temperate latitudes. In this review, we bring to the forefront the presence of cyanotoxins in extreme environments. Cyanotoxins have been reported especially in polar deserts (both from the Arctic and Antarctica) and alkaline lakes, but also in hot deserts, hypersaline environments, and hot springs. Cyanotoxins detected in these ecosystems include neurotoxins-anatoxin-a, anatoxin-a (S), paralytic shellfish toxins, ß-methylaminopropionic acid, N-(2-aminoethyl) glycine and 2,4-diaminobutyric acid- and hepatotoxins -cylindrospermopsins, microcystins and nodularins-with microcystins being the most frequently reported. Toxin production there has been linked to at least eleven cyanobacterial genera yet only three of these (Arthrospira, Synechococcus and Oscillatoria) have been confirmed as producers in culture. Beyond a comprehensive analysis of cyanotoxin presence in each of the extreme environments, this review also identifies the main knowledge gaps to overcome (e.g., scarcity of isolates and -omics data, among others) toward an initial assessment of ecological and human health risks in these amazing ecosystems developing at the very edge of life.

First use of the WAVE™ disposable rocking bioreactor for enhanced bioproduct synthesis by N2 -fixing cyanobacteria.

WAVE™ rocking disposable bioreactors have been successfully utilized for bioproduct development from bacteria, yeast, microalgae, and animal and plant cells but not from cyanobacteria so far. N2 -fixing cyanobacteria represent a prolific bioproducts source with reduced cultivation costs. In this study, 1 L cultures of the N2 -fixing cyanobacterium Anabaena siamensis grown diazotrophically in the WAVE™ bioreactor exhibited increased phosphate consumption and 37-70% higher CO2 fixation rates than those grown in conventional bubbled suspension (BS) batch cultures. This generated 40-80% increased biomass productivities in the WAVE™ bioreactor reaching 60 mg L(-1) day(-1) when supplemented with 10% CO2 . Consequently, WAVE™ generated 36-153% more protein, lipid, and carbohydrate than BS, including 47-100% increased productivity of phycocyanin and stearidonic acid (SA) with relevant biomedical applications. While the type of culture system (BS or WAVE(TM) ) did not affect the biochemical profile of cyanobacterial biomass, 10% CO2 supplementation induced a significant decrease in fatty acids and phycocyanin contents (mg g(-1) DW). Therefore, for commercial applications, the CO2 supplementation of WAVE™ should be optimized for each targeted bioproduct separately. This study opens possibilities for upgrading the WAVE™ systems to photobioreactors (PBRs) for bioproduct development from cyanobacteria, with opportunities and challenges critically evaluated herein.

Phylogeography of cylindrospermopsin and paralytic shellfish toxin-producing nostocales cyanobacteria from mediterranean europe (Spain).

Planktonic Nostocales cyanobacteria represent a challenge for microbiological research because of the wide range of cyanotoxins that they synthesize and their invasive behavior, which is presumably enhanced by global warming. To gain insight into the phylogeography of potentially toxic Nostocales from Mediterranean Europe, 31 strains of Anabaena (Anabaena crassa, A. lemmermannii, A. mendotae, and A. planctonica), Aphanizomenon (Aphanizomenon gracile, A. ovalisporum), and Cylindrospermopsis raciborskii were isolated from 14 freshwater bodies in Spain and polyphasically analyzed for their phylogeography, cyanotoxin production, and the presence of cyanotoxin biosynthesis genes. The potent cytotoxin cylindrospermopsin (CYN) was produced by all 6 Aphanizomenon ovalisporum strains at high levels (5.7 to 9.1 µg CYN mg(-1) [dry weight]) with low variation between strains (1.5 to 3.9-fold) and a marked extracellular release (19 to 41% dissolved CYN) during exponential growth. Paralytic shellfish poisoning (PSP) neurotoxins (saxitoxin, neosaxitoxin, and decarbamoylsaxitoxin) were detected in 2 Aphanizomenon gracile strains, both containing the sxtA gene. This gene was also amplified in non-PSP toxin-producing Aphanizomenon gracile and Aphanizomenon ovalisporum. Phylogenetic analyses supported the species identification and confirmed the high similarity of Spanish Anabaena and Aphanizomenon strains with other European strains. In contrast, Cylindrospermopsis raciborskii from Spain grouped together with American strains and was clearly separate from the rest of the European strains, raising questions about the current assumptions of the phylogeography and spreading routes of C. raciborskii. The present study confirms that the nostocalean genus Aphanizomenon is a major source of CYN and PSP toxins in Europe and demonstrates the presence of the sxtA gene in CYN-producing Aphanizomenon ovalisporum.

Discriminating bloom-forming cyanobacteria using lab-based hyperspectral imagery and machine learning: Validation with toxic species under environmental ranges.

Cyanobacteria are major contributors to algal blooms in inland waters, threatening ecosystem function and water uses, especially when toxin-producing strains dominate. Here, we examine 140 hyperspectral (HS) images of five representatives of the widespread, potentially toxin-producing and bloom-forming genera Microcystis, Planktothrix, Aphanizomenon, Chrysosporum and Dolichospermum, to determine the potential of utilizing visible and near-infrared (VIS/NIR) reflectance for their discrimination. Cultures were grown under various light and nutrient conditions to induce a wide range of pigment and spectral variability, mimicking variations potentially found in natural environments. Importantly, we assumed a simplified scenario where all spectral variability was derived from cyanobacteria. Throughout the cyanobacterial life cycle, multiple HS images were acquired along with extractions of chlorophyll a and phycocyanin. Images were calibrated and average spectra from the region of interest were extracted using k-means algorithm. The spectral data were pre-processed with seven methods for subsequent integration into Random Forest models, whose performances were evaluated with different metrics on the training, validation and testing sets. Successful classification rates close to 90 % were achieved using either the first or second derivative along with spectral smoothing, identifying important wavelengths in both the VIS and NIR. Microcystis and Chrysosporum were the genera achieving the highest accuracy (>95 %), followed by Planktothrix (79 %), and finally Dolichospermum and Aphanizomenon (>50 %). The potential of HS imagery to discriminate among toxic cyanobacteria is discussed in the context of advanced monitoring, aiming to enhance remote sensing capabilities and risk predictions for water bodies affected by cyanobacterial harmful algal blooms.

Effect of salinity on scytonemin yield in endolithic cyanobacteria from the Atacama Desert.

Cyanobacteria inhabiting extreme environments constitute a promising source for natural products with biotechnological applications. However, they have not been studied in-depth for this purpose due to the difficulties in their isolation and mass culturing. The Atacama Desert suffers one of the highest solar irradiances that limits the presence of life on its hyperarid core to endolithic microbial communities supported by cyanobacteria as primary producers. Some of these cyanobacteria are known to produce scytonemin, a UV-screening liposoluble pigment with varied biotechnological applications in cosmetics and other industries. In this work we carried out a strain selection based on growth performance among 8 endolithic cyanobacteria of the genera Chroococcidiopsis, Gloeocapsa and Gloeocapsopsis isolated from non-saline rocks of the Atacama Desert. Then we investigated the influence of NaCl exposure on scytonemin production yield. Results in the selected strain (Chroococcidiopsis sp. UAM571) showed that rising concentrations of NaCl lead to a growth decrease while triggering a remarkable increase in the scytonemin content, reaching maximum values at 20 g L-1 of NaCl over 50-fold higher scytonemin contents than those obtained without NaCl. Altogether, these findings point out to cyanobacteria from the Atacama Desert as potentially suitable candidates for pilot-scale cultivation with biotechnological purposes, particularly to obtain scytonemin.

LSTM networks provide efficient cyanobacterial blooms forecasting even with incomplete spatio-temporal data.

Cyanobacteria are the most frequent dominant species of algal blooms in inland waters, threatening ecosystem function and water quality, especially when toxin-producing strains predominate. Enhanced by anthropogenic activities and global warming, cyanobacterial blooms are expected to increase in frequency and global distribution. Early Warning Systems (EWS) for cyanobacterial blooms development allow timely implementation of management measures, reducing the risks associated to these blooms. In this paper, we propose an effective EWS for cyanobacterial bloom forecasting, which uses 6 years of incomplete high-frequency spatio-temporal data from multiparametric probes, including phycocyanin (PC) fluorescence as a proxy for cyanobacteria. A probe agnostic and replicable method is proposed to pre-process the data and to generate time series specific for cyanobacterial bloom forecasting. Using these pre-processed data, six different non-site/species-specific predictive models were compared including the autoregressive and multivariate versions of Linear Regression, Random Forest, and Long-Term Short-Term (LSTM) neural networks. Results were analyzed for seven forecasting time horizons ranging from 4 to 28 days evaluated with a hybrid system that combined regression metrics (MSE, R2, MAPE) for PC values, classification metrics (Accuracy, F1, Kappa) for a proposed alarm level of 10 µg PC/L, and a forecasting-specific metric to measure prediction improvement over the displaced signal (skill). The multivariate version of LSTM showed the best and most consistent results across all forecasting horizons and metrics, achieving accuracies of up to 90 % in predicting the proposed PC alarm level. Additionally, positive skill values indicated its outstanding effectiveness to forecast cyanobacterial blooms from 16 to 28 days in advance.

Temperature-dependent dispersal strategies of Aphanizomenon ovalisporum (Nostocales, Cyanobacteria): implications for the annual life cycle.

Aphanizomenon ovalisporum is a planktonic nostocalean cyanobacterium with increasing research interest due to its ability to produce the potent cytotoxin cylindrospermopsin and its potential invasiveness under the global warming scenario. The present study provides novel data on the potential dispersal strategies of A. ovalisporum by analyzing the influence of temperature (10-40 °C) on akinete differentiation and cell morphometry in cultures of A. ovalisporum UAM 290 isolated from a Spanish pond. Our results confirmed a temperature-dependent akinete differentiation, with the maximum akinete production reached at 20 °C (15 % of the cells), a low basal production at 25-30 °C (<0.4 % of the cells) and no detectable production at 35 °C. Furthermore, we reported the fragmentation of A. ovalisporum filaments at temperatures of 25 °C and above. Additionally, we observed that the morphology of vegetative cells varied under different temperature scenarios. Indeed, a strong negative correlation was found between temperature and the width, length and biovolume of vegetative cells, whereas akinete dimensions remained stable along the temperature gradient. Therefore, linear regressions between temperature and the cell size parameters are herein presented aiming to facilitate the identification of A. ovalisporum in the field throughout the course of the year. This is the first study evidencing that akinete production is triggered by temperatures between 20 and 25 °C in A. ovalisporum and reporting the existence of filament fragmentation as a potential dispersal strategy of this species. The importance of these findings for understanding the annual life cycle and invasive potential of A. ovalisporum is further discussed herein.

Photo-Fenton oxidation of cylindrospermopsin at neutral pH with LEDs.

Cylindrospermopsin (CYN) is a potent cyanobacterial toxin found in freshwaters worldwide. In this work, the feasibility of the photo-Fenton process under neutral pH using light emitting diodes as irradiation source for the removal of this hazardous cyanotoxin from freshwater was investigated. The impact of the kind of iron chelating agent (ethylenediamine-N, N'-disuccinic acid vs. ethylenedinitrilotetraacetic acid) as well as the effect of the main operating conditions viz. H2O2 dose, Fe(III) load, initial CYN concentration, and Fe(III):EDDS molar ratio on the performance of the process was systematically evaluated. EDDS was selected as the most appropriate iron chelating agent considering the kinetics of the process and the environmental impact (Vibrio fischeri and Artemia salina). Under optimized conditions ([H2O2] = 30 mg L-1; [Fe(III)] = 5 mg L-1; Fe(III):ligand = 1:0.5 (molar ratio)), complete removal of CYN was achieved in 15-min reaction time. Furthermore, the catalytic system showed to be effective in real water matrices (river and reservoir waters) spiked with CYN. Although the presence of inorganic ions (mainly HCO3-/CO32-) and dissolved organic carbon decreased the oxidation rate of CYN due to scavenging reactions and iron coordination, respectively, complete elimination of the cyanotoxin was achieved in all cases. The fate of EDDS along the process was also evaluated to demonstrate that the catalytic system investigated, apart from its effectiveness, warrants the complete absence of residues after reaction. Therefore, the proposed system constitutes a promising method for cyanotoxin treatment either as a drinking water treatment step in conventional plants or as a potential remediation strategy in the natural environment.

Biomass pre-treatments of the N2-fixing cyanobacterium Tolypothrix for co-production of methane.

Tolypothrix, a self-flocculating, fast growing, CO2 and nitrogen-fixing cyanobacterium, can be cultivated in nutrient-poor ash dam waters of coal-fired power stations, converting CO2 emissions into organic biomass. Therefore, the biomass of Tolypothrix sp. is a promising source for bio-fertiliser production, providing micro- and macronutrients. Energy requirements for production could potentially be offset via anaerobic digestion (AD) of the produced biomass, which may further improve the efficiency of the resulting biofertilizer. The aim of this study was to evaluate the effectiveness of pre-treatment conditions and subsequent methane (CH4) production of Tolypothrix under out-door cultivation conditions. Pre-treatments on biogas and methane production for Tolypothrix sp. biomass investigated were: (1) thermal at 95 °C for 10 h, (2) hydrothermal by autoclave at 121 °C at 1013.25 hPa for 20 min, using a standard moisture-heat procedure, (3) microwave at an output power of 900 W and an exposure time of 3 min, (4) sonication at an output power of 10 W for 3.5 h at 10 min intervals with 20 s breaks and (5) freeze-thaw cycles at -80 °C for 24 h followed by thawing at room temperature. Thermal, hydrothermal and sonication pre-treatments supported high solubilization of organic compounds up to 24.40 g L-1. However, higher specific CH4 production of 0.012 and 0.01 L CH4 g-1 volatile solidsadded. was achieved for thermal and sonic pre-treatments, respectively. High N- and low C-content of the Tolypothrix biomass affected CH4 recovery, while pre-treatment accelerated production of volatile acids (15.90 g L-1) and ammonia-N-accumulation (1.41 g L-1), leading to poor CH4 yields. Calculated theoretical CH4 yields based on the elemental composition of the biomass were ~55% higher than actual yields. This highlights the complexity of interactions during AD which are not adequately represented by elemental composition.

Overview of toxic cyanobacteria and cyanotoxins in Ibero-American freshwaters: Challenges for risk management and opportunities for removal by advanced technologies.

The increasing occurrence of cyanobacterial blooms worldwide represents an important threat for both the environment and public health. In this context, the development of risk analysis and management tools as well as sustainable and cost-effective treatment processes is essential. The research project TALGENTOX, funded by the Ibero-American Science and Technology Program for Development (CYTED-2019), aims to address this ambitious challenge in countries with different environmental and social conditions within the Ibero-American context. It is based on a multidisciplinary approach that combines ecology, water management and technology fields, and includes research groups from Chile, Colombia, Mexico, Peru and Spain. In this review, the occurrence of toxic cyanobacteria and cyanotoxins in freshwaters from these countries are summarized. The presence of cyanotoxins has been confirmed in all countries but the information is still scarce and further monitoring is required. In this regard, remote sensing or metagenomics are good alternatives at reasonable cost. The risk management of freshwaters from those countries considering the most frequent uses (consumption and recreation) has been also evaluated. Only Spain and Peru include cyanotoxins in its drinking water legislation (only MC-LR) and thus, there is a need for regulatory improvements. The development of preventive strategies like diminishing nutrient loads to aquatic systems is also required. In the same line, corrective measures are urgently needed especially in drinking waters. Advanced Oxidation Processes (AOPs) have the potential to play a major role in this scenario as they are effective for the elimination of most cyanotoxins classes. The research on the field of AOPs is herein summarized considering the cost-effectiveness, environmental character and technical applicability of such technologies. Fenton-based processes and photocatalysis using solar irradiation or LED light represent very promising alternatives given their high cost-efficiency. Further research should focus on developing stable long-term operation systems, addressing their scale-up.

Bioproduct Potential of Outdoor Cultures of Tolypothrix sp.: Effect of Carbon Dioxide and Metal-Rich Wastewater.

Rising CO2 levels, associated climatic instability, freshwater scarcity and diminishing arable land exacerbate the challenge to maintain food security for the fast growing human population. Although coal-fired power plants generate large amounts of CO2 emissions and wastewater, containing environmentally unsafe concentrations of metals, they ensure energy security. Nitrogen (N2)-fixation by cyanobacteria eliminate nitrogen fertilization costs, making them promising candidates for remediation of waste CO2 and metals from macronutrient-poor ash dam water and the biomass is suitable for phycocyanin and biofertilizer product development. Here, the effects of CO2 and metal mixtures on growth, bioproduct and metal removal potential were investigated for the self-flocculating, N2-fixing freshwater cyanobacterium Tolypothrix sp. Tolypothrix sp. was grown outdoors in simulated ash dam wastewater (SADW) in 500 L vertical bag suspension cultures and as biofilms in modified algal-turf scrubbers. The cultivation systems were aerated with air containing either 15% CO2 (v/v) or not. CO2-fertilization resulted in ∼1.25- and 1.45-fold higher biomass productivities and ∼40 and 27% increased phycocyanin and phycoerythrin contents for biofilm and suspension cultures, respectively. CO2 had no effect on removal of Al, As, Cu, Fe, Sr, and Zn, while Mo removal increased by 37% in both systems. In contrast, Ni removal was reduced in biofilm systems, while Se removal increased by 73% in suspension cultures. Based on biomass yields and biochemical data obtained, net present value (NPV) and sensitivities analyses used four bioproduct scenarios: (1) phycocyanin sole product, (2) biofertilizer sole product, (3) 50% phycocyanin and 50% biofertilizer, and (4) 100% phycocyanin and 100% biofertilizer (residual biomass) for power station co-located and not co-located 10 ha facilities over a 20-year period. Economic feasibility for the production of food-grade phycocyanin either as a sole product or with co-production of biofertilizer was demonstrated for CO2-enriched vertical and raceway suspension cultures raised without nitrogen-fertilization and co-location with power stations significantly increased profit margins.

Ecotoxicity assessment of microcystins from freshwater samples using a bioluminescent cyanobacterial bioassay.

The hepatotoxic cyanotoxins microcystins (MCs) are emerging contaminants naturally produced by cyanobacteria. Yet their ecological role remains unsolved, previous research suggests that MCs have allelopathic effects on competing photosynthetic microorganisms, even eliciting toxic effects on other freshwater cyanobacteria. In this context, the bioluminescent recombinant cyanobacterium Anabaena sp. PCC7120 CPB4337 (hereinafter Anabaena) was exposed to extracts of MCs. These were obtained from eight natural samples from freshwater reservoirs that contained MCs with a concentration range of 0.04-11.9 µg MCs L-1. MCs extracts included the three most common MCs variants (MC-LR, MC-RR, MC-YR) in different proportions (MC-LR: 100-0%; MC-RR: 100-0%; MC-YR: 14.2-0%). The Anabaena bioassay based on bioluminescence inhibition has been successfully used to test the toxicity of many emerging contaminants (e.g., pharmaceuticals) but never for cyanotoxins prior to this study. Exposure of Anabaena to MCs extracts induced a decrease in its bioluminescence with effective concentration decreasing bioluminescence by 50% ranging from 0.4 to 50.5 µg MC L-1 in the different samples. Bioluminescence responses suggested an interaction between MCs variants which was analyzed via the Additive Index method (AI), indicating an antagonistic effect (AI < 0) of MC-LR and MC-RR present in the samples. Additionally, MC extracts exposure triggered an increase of intracellular free Ca2+ in Anabaena. In short, this study supports the use of the Anabaena bioassay as a sensitive tool to assess the presence of MCs at environmentally relevant concentrations and opens interesting avenues regarding the interactions between MCs variants and the possible implication of Ca2+ in the mode of action of MCs towards cyanobacteria.

Effect of CO2 and metal-rich waste water on bioproduct potential of the diazotrophic freshwater cyanobacterium, Tolypothrix sp.

Continued economic growth is reliant on stable, affordable energy, requiring at present fossil fuel-derived energy production. Coal-fired power stations produce metal-rich but macro-nutrient-poor waste waters and emit flue gas, containing ∼10% CO2. Algae and cyanobacteria remediate metals and CO2, but use of N2-fixing (diazotrophic) cyanobacteria can reduce nitrogen-fertilization costs. The resulting biomass represents a promising source for biofuel and bio-product development. This study investigated the effect of CO2- and trace metals on growth performance, biochemical profiles and metal content of the freshwater diazotrophic cyanobacterium Tolypothrix sp. to assess bioproduct potential. Aerated 2 L batch cultures were grown in simulated ash-dam water (SADW) and BG11 without nitrogen (BG11(-N) controls). Supplied air was supplemented with either 15% CO2 or not (non-CO2 controls). CO2 supplementation resulted in 2.4 and 3.3-fold higher biomass productivities and 1.3 and 1.2-fold higher phycocyanin and phycoerythrin contents, whilst metals (media) had no effect. Al, Cu, Ni and V were more efficiently removed (50-90%) with CO2-addition, while As, Mo, Se and Sr removal was higher (30-87%) for non-CO2 controls. No significant effect on Zn and Fe removal was evident. Calculated biomass metal concentrations, at quantities required to meet N-requirements of wheat, suggests no metal toxicity when applied as a mineral-nitrogen biofertilizer. With a carbohydrate content of 50%, the biomass is also suitable for bioethanol production. In summary, Tolypothrix sp. raised in ash dam waste water supplemented with flue gas CO2 could yield high-value phycobiliproteins, bioethanol or biogas, and mineral-rich nitrogen fertilizer which would offset remediation costs and improve agricultural productivity.

Unmasking the identity of toxigenic cyanobacteria driving a multi-toxin bloom by high-throughput sequencing of cyanotoxins genes and 16S rRNA metabarcoding.

Cyanobacterial harmful algal blooms (CyanoHABs) are complex communities that include coexisting toxic and non-toxic strains only distinguishable by genetic methods. This study shows a water-management oriented use of next generation sequencing (NGS) to specifically pinpoint toxigenic cyanobacteria within a bloom simultaneously containing three of the most widespread cyanotoxins (the hepatotoxins microcystins, MCs; and the neurotoxins anatoxin-a, ATX, and saxitoxins, STXs). The 2013 summer bloom in Rosarito reservoir (Spain) comprised 33 cyanobacterial OTUs based on 16S rRNA metabarcoding, 7 of which accounted for as much as 96.6% of the community. Cyanotoxins and their respective biosynthesis genes were concurrently present throughout the entire bloom event including: MCs and mcyE gene; ATX and anaF gene; and STXs and sxtI gene. NGS applied to amplicons of cyanotoxin-biosynthesis genes unveiled 6 toxigenic OTUs, comprising 3 involved in MCs production (Planktothrix agardhii and 2 Microcystis spp.), 2 in ATX production (Cuspidothrix issatschenkoi and Phormidium/Tychonema spp.) and 1 in STXs production (Aphanizomenon gracile). These toxigenic taxa were also present in 16S rRNA OTUs list and their relative abundance was positively correlated with the respective toxin concentrations. Our results point at MC-producing P. agardhii and ATX-producing C. issatschenkoi as the main contributors to the moderate toxin concentrations observed, and suggest that their distribution in Southern Europe is broader than previously thought. Our findings also stress the need for monitoring low-abundance cyanobacteria (<1% relative abundance) in cyanotoxicity studies, and provide novel data on the presence of picocyanobacteria and potentially ATX-producing benthic taxa (e.g., Phormidium) in deep thermally-stratified water bodies. This study showcases a straightforward use of amplicon metagenomics of cyanotoxin biosynthesis genes in a multi-toxin bloom thus illustrating the broad applicability of NGS for water management in risk-oriented monitoring of CyanoHABs.

Degradation of widespread cyanotoxins with high impact in drinking water (microcystins, cylindrospermopsin, anatoxin-a and saxitoxin) by CWPO.

The occurrence of harmful cyanobacterial blooms has unabated increased over the last few decades, posing a significant risk for public health. In this work, we investigate the feasibility of catalytic wet peroxide oxidation (CWPO) promoted by modified natural magnetite (Fe3O4-R400/H2O2), as an inexpensive, simple-operation and environmentally-friendly process for the removal of the cyanotoxins that show the major impact on drinking water: microcystins (MC-LR and MC-RR), cylindrospermopsin (CYN), anatoxin-a (ATX) and saxitoxin (STX). The performance of the system was evaluated under ambient conditions and circumneutral pH (pH0 = 5) using relevant cyanotoxin concentrations (100-500 µg L-1). The nature of the cyanotoxins determined their reactivity towards CWPO, which decreased in the following order: MC-RR > CYN > MC-LR ≫ ATX > STX. In this sense, microcystins and CYN were completely removed in short reaction times (1-1.5 h) with a low catalyst concentration (0.2 g L-1) and the stoichiometric amount of H2O2 (2-2.6 mg L-1), while only 60-80% conversion was achieved with ATX and STX in 5 h. In these cases, an intensification of the operating conditions (1 g L-1 catalyst and up to 30 mg H2O2 L-1) was required to remove both toxins in 1 h. The impact of the main components of freshwaters i.e. natural organic matter (NOM) and several inorganic ions (HCO3-, HPO42-, SO42-) on the performance of the process was also investigated. Although the former led to a partial inhibition of the reaction due to HO· scavenging and catalyst coating, the latter did not show any remarkably effect, and the versatility of the process was finally confirmed in a real surface water. To further demonstrate the effectiveness of the catalytic system, the toxicity of both the initial cyanotoxins and the resulting CWPO effluents was measured with the brine shrimp Artemia salina. Remarkably, all CWPO effluents were non-toxic at the end of the treatment.

Temperature Influences the Production and Transport of Saxitoxin and the Expression of sxt Genes in the Cyanobacterium Aphanizomenon gracile.

The cyanobacterium Aphanizomenon gracile is the most widely distributed producer of the potent neurotoxin saxitoxin in freshwaters. In this work, total and extracellular saxitoxin and the transcriptional response of three genes linked to saxitoxin biosynthesis (sxtA) and transport (sxtM, sxtPer) were assessed in Aphanizomenon gracile UAM529 cultures under temperatures covering its annual cycle (12 °C, 23 °C, and 30 °C). Temperature influenced saxitoxin production being maximum at high temperatures (30 °C) above the growth optimum (23 °C), concurring with a 4.3-fold increased sxtA expression at 30 °C. Extracellular saxitoxin transport was temperature-dependent, with maxima at extremes of temperature (12 °C with 16.9% extracellular saxitoxin; and especially 30 °C with 53.8%) outside the growth optimum (23 °C), coinciding with a clear upregulation of sxtM at both 12 °C and 30 °C (3.8-4.1 fold respectively), and yet with just a slight upregulation of sxtPer at 30 °C (2.1-fold). Nitrate depletion also induced a high extracellular saxitoxin release (51.2%), although without variations of sxtM and sxtPer transcription, and showing evidence of membrane damage. This is the first study analysing the transcriptional response of sxtPer under environmental gradients, as well as the effect of temperature on putative saxitoxin transporters (sxtM and sxtPer) in cyanobacteria in general.

Culture scale-up and immobilisation of a mixed methanotrophic consortium for methane remediation in pilot-scale bio-filters.

Robust methanotrophic consortia for methane (CH4) remediation and by-product development are presently not readily available for industrial use. In this study, a mixed methanotrophic consortium (MMC), sequentially enriched from a marine sediment, was assessed for CH4 removal efficiency and potential biomass-generated by-product development. Suitable packing material for bio-filters to support MMC biofilm establishment and growth was also evaluated. The enriched MMC removed ∼7-13% CH4 under a very high gas flow rate (2.5 L min-1; 20-25% CH4) in continuous-stirred tank reactors (∼10 L working volume) and the biomass contained long-chain fatty acids (i.e. C16 and C18). Cultivation of the MMC on plastic bio-balls abated ∼95-97% CH4 in pilot-scale non-sterile outdoor-operated bio-filters (0.1 L min-1; 1% CH4). Contamination by cyanobacteria had beneficial effects on treating low-level CH4, by providing additional oxygen for methane oxidation by MMC, suggesting that the co-cultivation of MMC with cyanobacterial mats does not interfere with and may actually be beneficial for remediation of CH4 and CO2 at industrial scale.

A review of the phylogeny, ecology and toxin production of bloom-forming Aphanizomenon spp. and related species within the Nostocales (cyanobacteria).

The traditional genus Aphanizomenon comprises a group of filamentous nitrogen-fixing cyanobacteria of which several memebers are able to develop blooms and to produce toxic metabolites (cyanotoxins), including hepatotoxins (microcystins), neurotoxins (anatoxins and saxitoxins) and cytotoxins (cylindrospermopsin). This genus, representing geographically widespread and extensively studied cyanobacteria, is in fact heterogeneous and composed of at least five phylogenetically distant groups (Aphanizomenon, Anabaena/Aphanizomenon like cluster A, Cuspidothrix, Sphaerospermopsis and Chrysosporum) whose taxonomy is still under revision. This review provides a thorough insight into the phylogeny, ecology, biogeography and toxicogenomics (cyr, sxt, and ana genes) of the five best documented "Aphanizomenon" species with special relevance for water risk assessment: Aphanizomenon flos-aquae, Aphanizomenon gracile, Cuspidothrix issatschenkoi, Sphaerospermopsis aphanizomenoides and Chrysosporum ovalisporum. Aph. flos-aquae, Aph. gracile and C. issatschenkoi have been reported from temperate areas only whereas S. aphanizomenoides shows the widest distribution from the tropics to temperate areas. Ch. ovalisporum is found in tropical, subtropical and Mediterranean areas. While all five species show moderate growth rates (0.1-0.4day-1) within a wide range of temperatures (15-30°C), Aph. gracile and A. flos-aquae can grow from around (or below) 10°C, whereas Ch. ovalisporum and S. aphanizomenoides are much better competitors at high temperatures over 30°C or even close to 35°C. A. gracile has been confirmed as the producer of saxitoxins and cylindrospermopsin, C. issatschenkoi of anatoxins and saxitoxins and Ch. ovalisporum of cylindrospermopsin. The suspected cylindrospermopsin or anatoxin-a production of A. flos-aquae or microcystin production of S. aphanizomenoides is still uncertain. This review includes a critical discussion on the the reliability of toxicity reports and on the invasive potential of "Aphanizomenon" species in a climate change scenario, together with derived knowledge gaps and research needs. As a whole, this work is intended to represent a key reference for scientists and water managers involved in the major challenges of identifying, preventing and mitigating toxic Aphanizomenon blooms.

Variability in the sxt Gene Clusters of PSP Toxin Producing Aphanizomenon gracile Strains from Norway, Spain, Germany and North America.

Paralytic shellfish poisoning (PSP) toxin production has been detected worldwide in the cyanobacterial genera Anabaena, Lyngbya, Scytonema, Cuspidothrix and Aphanizomenon. In Europe Aphanizomenon gracile and Cuspidothrix issatschenkoi are the only known producers of PSP toxins and are found in Southwest and Central European freshwater bodies. In this study the PSP toxin producing Aphanizomenon sp. strain NIVA-CYA 851 was isolated from the Norwegian Lake Hillestadvannet. In a polyphasic approach NIVA-CYA 851 was morphologically and phylogenetically classified, and investigated for toxin production. The strain NIVA-CYA 851 was identified as A. gracile using 16S rRNA gene phylogeny and was confirmed to produce neosaxitoxin, saxitoxin and gonyautoxin 5 by LC-MS. The whole sxt gene clusters (circa 27.3 kb) of four A. gracile strains: NIVA-CYA 851 (Norway); NIVA-CYA 655 & NIVA-CYA 676 (Germany); and UAM 529 (Spain), all from latitudes between 40° and 59° North were sequenced and compared with the sxt gene cluster of reference strain A. gracile NH-5 from the USA. All five sxt gene clusters are highly conserved with similarities exceeding 99.4%, but they differ slightly in the number and presence of single nucleotide polymorphisms (SNPs) and insertions/deletions (In/Dels). Altogether 178 variable sites (44 SNPs and 4 In/Dels, comprising 134 nucleotides) were found in the sxt gene clusters of the Norwegian, German and Spanish strains compared to the reference strain. Thirty-nine SNPs were located in 16 of the 27 coding regions. The sxt gene clusters of NIVA-CYA 851, NIVA-CYA 655, NIVA-CYA 676 and UAM 529, were characterized by 15, 16, 19 and 23 SNPs respectively. Only the Norwegian strain NIVA-CYA 851 possessed an insertion of 126 base pairs (bp) in the noncoding area between the sxtA and sxtE genes and a deletion of 6 nucleotides in the sxtN gene. The sxtI gene showed the highest variability and is recommended as the best genetic marker for further phylogenetic studies of the sxt gene cluster of A. gracile. This study confirms for the first time the role of A. gracile as a PSP toxin producer in Norwegian waters, representing the northernmost occurrence of PSP toxin producing A. gracile in Europe known so far.