Relation between the relative abundance and collapse of Aphanizomenon flos-aquae and microbial antagonism in Upper Klamath Lake, Oregon.

Aphanizomenon flos-aquae (AFA) is the dominant filamentous cyanobacterium that develops into blooms in Upper Klamath Lake, Oregon, each year. During AFA bloom and collapse, ecosystem conditions for endangered Lost River and shortnose suckers deteriorate, thus motivating the need to identify processes that limit AFA abundance and decline. Here, we investigate the relations between AFA and other members of the microbial community (photosynthetic and nonphotosynthetic bacteria and archaea), how those relations impact abundance and collapse of AFA, and the types of microbial conditions that suppress AFA. We found significant spatial variation in AFA relative abundance during the 2016 bloom period using 16S rRNA sequencing. The Pelican Marina site had the lowest AFA relative abundance, and this was coincident with increased relative abundance of Candidatus Sericytochromatia, Flavobacterium, and Rheinheimera, some of which are known AFA antagonists. The AFA collapse coincided with phosphorus limitation relative to nitrogen and the increased relative abundance of Cyanobium and Candidatus Sericytochromatia, which outcompete AFA when dissolved inorganic nitrogen is available. The data collected in this study indicate the importance of dissolved inorganic nitrogen combined with microbial community structure in suppressing AFA abundance.

Spatial and Temporal Variability of Saxitoxin-Producing Cyanobacteria in U.S. Urban Lakes.

Harmful cyanobacterial blooms (HCBs) are of growing global concern due to their production of toxic compounds, which threaten ecosystems and human health. Saxitoxins (STXs), commonly known as paralytic shellfish poison, are a neurotoxic alkaloid produced by some cyanobacteria. Although many field studies indicate a widespread distribution of STX, it is understudied relative to other cyanotoxins such as microcystins (MCs). In this study, we assessed eleven U.S. urban lakes using qPCR, sxtA gene-targeting sequencing, and 16S rRNA gene sequencing to understand the spatio-temporal variations in cyanobacteria and their potential role in STX production. During the blooms, qPCR analysis confirmed the presence of the STX-encoding gene sxtA at all lakes. In particular, the abundance of the sxtA gene had a strong positive correlation with STX concentrations in Big 11 Lake in Kansas City, which was also the site with the highest quantified STX concentration. Sequencing analysis revealed that potential STX producers, such as Aphanizomenon, Dolichospermum, and Raphidiopsis, were present. Further analysis targeting amplicons of the sxtA gene identified that Aphanizomenon and/or Dolichospermum are the primary STX producer, showing a significant correlation with sxtA gene abundances and STX concentrations. In addition, Aphanizomenon was associated with environmental factors, such as conductivity, sulfate, and orthophosphate, whereas Dolichospermum was correlated with temperature and pH. Overall, the results herein enhance our understanding of the STX-producing cyanobacteria and aid in developing strategies to control HCBs.

Grazing preferences of three species of amoebae on cyanobacteria and green algae.

Twenty species/isolates of cyanobacteria and green algae were isolated from cyanobacterial bloom samples in lakes associated with the upper Qu'Appelle River drainage system in southern Saskatchewan, Canada. Three amoebae species (Cochliopodium sp., Vannella sp. and Vermamoeba vermiformis) were also isolated from one of these samples, and were subjected to grazing assays to determine which species of cyanobacteria or algae could potentially serve as a food source. Amoeba grazing rates were quantified based on the diameter of the plaque after 12 days on agar plate assays, and by estimation of the amoeba population growth rate from the rate of increase of plaque area. The common cyanobacterial bloom-formers Dolichospermum sp. and Aphanizomenon flos-aquae supported high growth rates for all three amoebae, while green algae, with the exception of one green alga/amoeba combination, did not support growth of the tested amoebae. Many of the cyanobacterial and algal isolates that did not support amoebae growth were ingested, suggesting that ingestion did not determine grazing success. Overall, while the cyanobacteria Dolichospermum sp. and Aphanizomenon flos-aquae were suitable food sources for the amoebae, the other cyanobacteria were grazed in an unpredictable manner, with some species/strains grazed by some amoebae and some species not grazed at all.

Positive Impacts of Aphanizomenon Flos Aquae Extract on Obesity-Related Dysmetabolism in Mice with Diet-Induced Obesity.

The present study evaluated the ability of KlamExtra®, an Aphanizomenon flos aquae (AFA) extract, to counteract metabolic dysfunctions due to a high fat diet (HFD) or to accelerate their reversion induced by switching an HFD to a normocaloric diet in mice with diet-induced obesity. A group of HFD mice was fed with an HFD supplemented with AFA (HFD-AFA) and another one was fed with regular chow (standard diet-STD) alone or supplemented with AFA (STD-AFA). AFA was able to significantly reduce body weight, hypertriglyceridemia, liver fat accumulation and adipocyte size in HFD mice. AFA also reduced hyperglycaemia, insulinaemia, HOMA-IR and ameliorated the glucose tolerance and the insulin response of obese mice. Furthermore, in obese mice AFA normalised the gene and the protein expression of factors involved in lipid metabolism (FAS, PPAR-γ, SREBP-1c and FAT-P mRNA), inflammation (TNF-α and IL-6 mRNA, NFkB and IL-10 proteins) and oxidative stress (ROS levels and SOD activity). Interestingly, AFA accelerated the STD-induced reversion of glucose dysmetabolism, hepatic and VAT inflammation and oxidative stress. In conclusion, AFA supplementation prevents HFD-induced dysmetabolism and accelerates the STD-dependent recovery of glucose dysmetabolism by positively modulating oxidative stress, inflammation and the expression of the genes linked to lipid metabolism.

The Role of Heterocysts in Cyanotoxin Production during Nitrogen Limitation.

Cyanobacteria harmful algal blooms (cyanoHABs) can have impacts on human health, aquatic ecosystems, and the economy. Nutrient management is an important mitigation and even remediation strategy. In this work, the paralytic shellfish toxin (PST)-producing Aphanizomenon (Aphan.) flos-aquae (Linnaeus) Ralfs ex Bornet & Flahault (now identified as Aphan. sp.) single filament isolate NH-5 was grown in P-depleted media, N-depleted media, and complete BG-11 media. Growth and heterocyst and vegetative cells were monitored using dry weight and cell counts. Ultrasonication was used to separate heterocysts from vegetative cells. HPLC-FLD with post-column derivatization was used to determine the saxitoxin (STX) and neosaxitoxin (NEOSTX) concentration per cell. Aphan. sp. NH-5 biomass was lower in the P-depleted media than in the N-depleted media and the control, though higher heterocyst counts were detected in the N-depleted media. The heterocyst toxin concentration was significantly higher compared to the vegetative cells for the N-depleted media, control, and P-depleted media. However, no significant differences were found among all preparations with regard to the STX-to-NEOSTX ratio. We conclude that N limitation induced higher heterocyst numbers and that N fixation activity is a factor behind the increase in the STX and NEOSTX production of Aphan. sp. NH-5.

Dynamic response of bacterial communities to Microcystis blooms: A three-year study.

Although nutrient availability is widely recognized as the driving force behind Microcystis blooms, identifying the microorganisms that play a pivotal role in their formation is a challenging task. Our understanding of the contribution of bacterial communities to the development of Microcystis blooms remains incomplete, despite the fact that the relationship between Microcystis and bacterial communities has been extensively investigated. Most studies have focused on their interaction for a single year rather than for multiple years. To determine key bacteria crucial for the formation of Microcystis blooms, we collected samples from three sites in the Daechung Reservoir (Chuso, Hoenam, and Janggye) over three years (2017, 2019, and 2020). Our results indicated that Microcystis bloom-associated bacterial communities were more conserved across stations than across years. Bacterial communities could be separated into modules corresponding to the different phases of Microcystis blooms. Dolichospermum and Aphanizomenon belonged to the same module, whereas the module of Microcystis was distinct. The microbial recurrent association network (MRAN) showed that amplicon sequence variants (ASVs) directly linked to Microcystis belonged to Pseudanabaena, Microscillaceae, Sutterellaceae, Flavobacterium, Candidatus Aquiluna, Bryobacter, and DSSD61. These ASVs were also identified as key indicators of the bloom stage, indicating that they were fundamental biological elements in the development of Microcystis blooms. Overall, our study highlights that, although bacterial communities change annually, they continue to share core ASVs that may be crucial for the formation and maintenance of Microcystis blooms.

Transcriptionally active nitrogen fixation and biosynthesis of diverse secondary metabolites by Dolichospermum and Aphanizomenon-like Cyanobacteria in western Lake Erie Microcystis blooms.

Cyanobacterial harmful algal blooms (cyanoHABs) in the western basin of Lake Erie are dominated by microcystin producing Microcystis spp., but other cyanobacterial taxa that coexist in these communities may play important roles in production of toxins and shaping bloom dynamics and community function. In this study, we used metagenomic and metatranscriptomic data from the 2014 western Lake Erie cyanoHAB to explore the genetic diversity and biosynthetic potential of cyanobacteria belonging to the Anabaena, Dolichospermum, Aphanizomenon (ADA) clade. We reconstructed two near-complete metagenome-assembled genomes from two distinct ADA clade species, each containing biosynthetic gene clusters that encode novel and known secondary metabolites, including those with toxic and/or known taste and odor properties, that were transcriptionally active. However, neither ADA metagenome-assembled genome contained genes encoding guanitoxins, anatoxins, or saxitoxins, which are known to be produced by ADA. The ADA cyanobacteria accounted for most of the metagenomic and metatranscriptomic reads from nitrogen fixation genes, suggesting they were the dominant N-fixers at the times and stations sampled. Despite their relatively low abundance, our results highlight the possibility that ADA taxa could influence the water quality and ecology of Microcystis blooms, although the extent of these impacts remains to be quantified.

Aphanizomenon flos-aquae (AFA) Extract Prevents Neurodegeneration in the HFD Mouse Model by Modulating Astrocytes and Microglia Activation.

Obesity and related metabolic dysfunctions are associated with neurodegenerative diseases, such as Alzheimer's disease. Aphanizomenon flos-aquae (AFA) is a cyanobacterium considered a suitable supplement for its nutritional profile and beneficial properties. The potential neuroprotective effect of an AFA extract, commercialized as KlamExtra®, including the two AFA extracts Klamin® and AphaMax®, in High-Fat Diet (HFD)-fed mice was explored. Three groups of mice were provided with a standard diet (Lean), HFD or HFD supplemented with AFA extract (HFD + AFA) for 28 weeks. Metabolic parameters, brain insulin resistance, expression of apoptosis biomarkers, modulation of astrocytes and microglia activation markers, and Aß deposition were analyzed and compared in the brains of different groups. AFA extract treatment attenuated HFD-induced neurodegeneration by reducing insulin resistance and loss of neurons. AFA supplementation improved the expression of synaptic proteins and reduced the HFD-induced astrocytes and microglia activation, and Aß plaques accumulation. Together, these outcomes indicate that regular intake of AFA extract could benefit the metabolic and neuronal dysfunction caused by HFD, decreasing neuroinflammation and promoting Aß plaques clearance.

Response of phytoplankton community composition to physicochemical and meteorological factors under different hydrological conditions in Lake Diefenbaker.

Changes in hydro-meteorological conditions due to warming climate and the operation of reservoirs may support algal blooms. Lake Diefenbaker is a large reservoir on the Canadian Prairies. Annual flow volume from its major tributary, the South Saskatchewan River (SSR), varies with precipitation and temperature in the Rocky Mountains. Furthermore, plans are underway to increase water abstraction from Lake Diefenbaker for irrigation. Therefore, we used a nine-year dataset that comprised a drought year (1984), four consecutive high flow years (2011 to 2014), and four subsequent low flow years (2015 to 2018) to investigate how these changes could affect the major phytoplankton groups and cyanobacterial community. Diatoms (38.5%) were the most abundant phytoplankton, followed by cryptomonads (28.9%) under low and high flow years. Diatoms were associated with greater mixing in late spring and fall, whereas the cryptomonads were related to the high nutrients from spring flow. Cyanobacteria (79.3%) contributed the greatest to the total phytoplankton biomass under drought; we hypothesized that the high abundance of cyanobacteria during drought was associated with thermocline deepening and subsequent internal loading of nutrients. Microcystis, a potential bloom-forming and toxin-producing genus, was dominant during the drought and correlated with reduced water level, increased air temperature, and moderate wind speed. Although its biomass was low, another potential bloom-forming and toxin-producing genus, Aphanizomenon, was present in low and high flow years. Aphanizomenon was correlated with decreased SSR flow and increased particulate carbon to particulate phosphorus ratios, which may be related to their ability to cope with P limitation. These results highlight that Lake Diefenbaker and other similar reservoirs are vulnerable to an increase in potential toxic cyanobacteria species with future expectations of climate warming and water abstraction.

Interactions between Ciliate Species and Aphanizomenon flos-aquae Vary Depending on the Morphological Form and Biomass of the Diazotrophic Cyanobacterium.

Aphanizomenon flos-aquae can form extensive blooms from freshwater to the brackish environment and, being a diazotrophic species, contribute significantly to the nitrogen and carbon cycle. It occurs as single filaments or aggregates and could be used as an alternative nutrients source for bacteria and ciliates. Ciliates are a group of organisms playing a crucial role in the transfer of nitrogen from primary producers to higher trophic levels in aquatic food webs. The aim of the experiment was to study the effects of the cyanobacterium A. flos-aquae on the community of five ciliate species (Spirostomum minus, Euplotes aediculatus, Strobilidium sp., Vorticella sp. and Paramecium tetraurelia). The response of each species to the presence of a low/high cyanobacterial biomass and to the different morphological forms of A. flos-aquae (single filaments or aggregates) was demonstrated. The results of the experiment showed the variability of interactions between the cyanobacterium A. flos-aquae and ciliates and pointed out the possible benefits that A. flos-aquae provides to the ciliates (e.g., a substrate for the development of bacteria as food for ciliates or as a source of nitrogen and carbon).

Temporal and functional interrelationships between bacterioplankton communities and the development of a toxigenic Microcystis bloom in a lowland European reservoir.

The cyanobacteria-associated microbiome is constantly reshaped by bloom development. However, the synergistic-antagonistic nature of the relationships between Microcystis and its microbiome still remains unclear. Therefore, temporal changes of bacterioplankton communities and their functional potential through different developing stages of a Microcystis toxigenic bloom were investigated, considering bacterioplankton assemblages as particle-attached (PAB) and free-living (FLB) bacteria. 16S rRNA sequencing revealed that PAB were represented by Proteobacteria and Cyanobacteria, while FLB by Proteobacteria and Actinobacteria. Network and ordination analyses indicated that PAB inter-relationships were more complex-numerous connections between taxa with stronger correlations, than FLB-rather influenced by physico-chemical parameters. PAB in pre-summer was diverse with Proteobacteria containing potential taxa involved in nitrogen-transforming processes. In mid-summer, PAB presented a mix-bloom dominated by Snowella, Aphanizomenon, and Microcystis, which were succeeded by toxigenic Microcystis in post-summer. Both periods were associated to potential taxa with parasitic/predatory lifestyles against cyanobacteria. In post-summer, Sutterellaceae were recognized as poor water quality indicators, and their strong association with Microcystis could have represented an increased threat for that period. Microcystis was a major factor significantly reducing PAB diversity and evenness, suggesting that it negatively influenced bacterioplankton assemblages, probably also altering the overall community functional potential.

Quantitative prediction of toxin-producing Aphanizomenon cyanobacteria in freshwaters using Sentinel-2 satellite imagery.

This study aimed to develop an empirical model to predict the spatial distribution of Aphanizomenon using the Ridiyagama reservoir in Sri Lanka with a dual-model strategy. In December 2020, a bloom was detected with a high density of Aphanizomenon and chlorophyll-a concentration. We generated a set of algorithms using in situ chlorophyll-a data with surface reflectance of Sentinel-2 bands on the same day using linear regression analysis. The in situ chlorophyll-a concentration was better regressed to the reflectance ratio of (1 + R665)/(1-R705) derived from B4 and B5 bands of Sentinel-2 with high reliability (R2 = 0.81, p < 0.001). The second regression model was developed to predict Aphanizomenon cell density using chlorophyll-a as the proxy and the relationship was strong and significant (R2 = 0.75, p<0.001). Coupling the former regression models, an empirical model was derived to predict Aphanizomenon cell density in the same reservoir with high reliability (R2 = 0.71, p<0.001). Furthermore, the predicted and observed spatial distribution of Aphanizomenon was fairly agreed. Our results highlight that the present empirical model has a high capability for an accurate prediction of Aphanizomenon cell density and their spatial distribution in freshwaters, which helps in the management of toxic algal blooms and associated health impacts.

A Review of Cyanophage-Host Relationships: Highlighting Cyanophages as a Potential Cyanobacteria Control Strategy.

Harmful algal blooms (HABs) are naturally occurring phenomena, and cyanobacteria are the most commonly occurring HABs in freshwater systems. Cyanobacteria HABs (cyanoHABs) negatively affect ecosystems and drinking water resources through the production of potent toxins. Furthermore, the frequency, duration, and distribution of cyanoHABs are increasing, and conditions that favor cyanobacteria growth are predicted to increase in the coming years. Current methods for mitigating cyanoHABs are generally short-lived and resource-intensive, and have negative impacts on non-target species. Cyanophages (viruses that specifically target cyanobacteria) have the potential to provide a highly specific control strategy with minimal impacts on non-target species and propagation in the environment. A detailed review (primarily up to 2020) of cyanophage lifecycle, diversity, and factors influencing infectivity is provided in this paper, along with a discussion of cyanophage and host cyanobacteria relationships for seven prominent cyanoHAB-forming genera in North America, including: Synechococcus, Microcystis, Dolichospermum, Aphanizomenon, Cylindrospermopsis, Planktothrix, and Lyngbya. Lastly, factors affecting the potential application of cyanophages as a cyanoHAB control strategy are discussed, including efficacy considerations, optimization, and scalability for large-scale applications.

Cyanophage infections reduce photosynthetic activity and expression of CO2 fixation genes in the freshwater bloom-forming cyanobacterium Aphanizomenon flos-aquae.

Cyanobacteria play a significant role in ecosystem functioning as photosynthetic and CO2 fixing microorganisms. Whether and to what extent cyanophages alter these carbon and energy cycles in their cyanobacterial hosts is still poorly understood. In this study, we investigated changes in photosynthetic activity (PSII), expression of genes associated with the light phase of photosynthesis (psbA, petA, ndhK) and carbon metabolism (rbcL, zwf) as well as intracellular ATP and NADHP concentrations in freshwater bloom-forming filamentous cyanobacterium Aphanizomenon flos-aquae infected by cyanophage vB_AphaS-CL131. We found that PSII activity and expression level of rbcL genes, indicating potential for CO2 fixation, had decreased in response to cyanophage adsorption and DNA injection. During the period of viral DNA replication and assembly, PSII performance and gene expression remained at this decreased level and did not change significantly, indicating lack of transcriptional shutdown by the cyanophage. Combined, these observations suggest that although there is little to no interference between cyanophage DNA replication, host transcription and cellular metabolism, A. flos-aquae underwent a physiological state-shift toward lower efficiency of carbon and energy cycling. This further suggest potential cascading effect for co-occurring non-infected members of the microbial community.

7-epi-cylindrospermopsin and microcystin producers among diverse Anabaena/Dolichospermum/Aphanizomenon CyanoHABs in Oregon, USA.

Several genomes of Nostocales ADA clade members from the US Pacific Northwest were recently sequenced. Biosynthetic genes for microcystin, cylindrospermopsin or anatoxin-a were present in 7 of the 15 Dolichospermum/Anabaena strains and none of the 5 Aphanizomenon flos-aquae (AFA) strains. Toxin analyses (ELISA and LC-MS/MS) were conducted to quantitate and identify microcystin (MC) and cylindrospermopsin (CYN) congeners/analogs in samples dominated by Dolichospermum spp. of known genome sequence. MC-LR was the main congener produced by Dolichospermum spp. from Junipers Reservoir, Lake Billy Chinook and Odell Lake, while a congener provisionally identified as [Dha7]MC-HtyR was produced by a Dolichospermum sp. in Detroit Reservoir. A second Dolichospermum sp. from Detroit Reservoir was found to produce 7-epi-CYN, with 7-deoxy-CYN also present, but no CYN. The monitoring history of each of these lakes indicates the capacity for high levels of cyanotoxins during periods when Dolichospermum spp. are the dominant cyanobacteria. The diversity of ADA strains found in the US Pacific NW emphasizes the importance of these cyanobacteria as potentially toxic HAB formers in this temperate climatic region. Our results linking congener and genetic identity add data points that will help guide development of improved tools for predicting congener specificity from cyanotoxin gene sequences.

Driving forces for the growth of MIB-producing Planktothricoides raciborskii in a low-latitude reservoir.

In comparison with the middle- and high-latitude regions, the low-latitude regions are less associated with the occurrence of 2-methylisoborneol (MIB) episodes, since most of the previously identified MIB producers favor moderate/low light/temperature conditions. Here, we report a serious MIB outbreak over the period from Jul. 2018 to Jun. 2019 in a low-latitude reservoir with a mean annual water temperature of 25.6 °C. The MIB episode lasted for a long period, from Jul. 2018 to Jan. 2019, and Planktothricoides raciborskii was confirmed to be the main MIB producer. The growth characteristics of P. raciborskii were explored through both laboratory culturing and on-site verification experiments. The results indicated that this strain was not nutrient-sensitive at TN > 800 µg L-1 and TP > 10 µg L-1, but favored moderate light intensity (54 µmol photon m-2·s-1) and high temperature (30 °C). The two bloom-forming genera, Limnothrix and Aphanizomenon, favoring lower temperature and similar or relatively higher light intensity, showed much greater proliferation, about 13 folds (Limnothrix) and 58 folds (Aphanizomenon), from Dec. to Jun.; by contrast, the high water temperature (29.9 ± 2.8 °C) and light intensity (189.1 ± 87.6 µmol photon m-2·s-1) from Jul. to Nov. were not favorable to Limnothrix or Aphanizomenon, which might have created an opportunity for the growth of MIB-producing P. raciborskii. In addition, we also found that high temperature could promote the release of MIB from P. raciborskii cells, therefore exerting increased pressure on drinking water treatment processes.

Assessment of the Appearance and Toxin Production Potential of Invasive Nostocalean Cyanobacteria Using Quantitative Gene Analysis in Nakdong River, Korea.

Invasive nostocalean cyanobacteria (INC) were first reported in tropical regions and are now globally spreading rapidly due to climate change, appearing in temperate regions. INC require continuous monitoring for water resource management because of their high toxin production potential. However, it is difficult to analyze INC under a microscope because of their morphological similarity to nostocalean cyanobacteria such as the genus Aphanizomenon. This study calculates the gene copy number per cell for each target gene through quantitative gene analysis on the basis of genus-specific primers of genera Cylindrospermopsis, Sphaerospermopsis, and Cuspidothrix, and the toxin primers of anatoxin-a, saxitoxin, and cylindrospermopsin. In addition, quantitative gene analysis was performed at eight sites in the Nakdong River to assess the appearance of INC and their toxin production potential. Genera Cylindrospermopsis and Sphaerospermopsis did not exceed 100 cells mL-1 at the maximum, with a low likelihood of related toxin occurrence. The genus Cuspidothrix showed the highest cell density (1759 cells mL-1) among the INC. Nakdong River has potential for the occurrence of anatoxin-a through biosynthesis by genus Cuspidothrix because the appearance of this genus coincided with that of the anatoxin-a synthesis gene (anaF) and the detection of the toxin by ELISA.

Occurrence of Mycosporine-like Amino Acids (MAAs) from the Bloom-Forming Cyanobacteria Aphanizomenon Strains.

Mycosporine-like amino acids (MAAs) are widespread in various microbes and protect them against harsh environments. Here, four different Aphanizomenon species were isolated from severely eutrophic waterbodies, Lake Dianchi and the Guanqiao fishpond. Morphological characters and molecular phylogenetic analysis verified that the CHAB5919, 5921, and 5926 strains belonged to the Aphanizomenon flos-aquae clade while Guanqiao01 belonged to the Aphanizomenon gracile clade. Full wavelength scanning proved that there was obvious maximal absorption at 334 nm through purified methanol extraction, and these substances were further analyzed by HPLC and UPLC-MS-MS. The results showed that two kinds of MAAs were discovered in the cultured Aphanizomenon strains. One molecular weight was 333.28 and the other was 347.25, and the daughter fragment patterns were in accordance with the previously articles reported shinorine and porphyra-334 ion characters. The concentration of the MAAs was calibrated from semi-prepared MAAs standards from dry cells of Microcystis aeruginosa PCC7806 algal powder, and the purity of shinorine and porphyra-334 were 90.2% and 85.4%, respectively. The average concentrations of shinorine and porphyra-334 were 0.307−0.385 µg/mg and 0.111−0.136 µg/mg in Aphanizomenon flos-aquae species, respectively. And there was only one kind of MAAs (shinorine) in Aphanizomenon gracile species.,with a content of 0.003−0.049 µg/mg dry weight among all Aphanizomenon gracile strains. The shinorine concentration in Aphanizomenon flos-aquae was higher than that in Aphanizomenon gracile strains. The total MAAs production can be ranked as Aphanizomenon flos-aquae > Aphanizomenon gracile.

Non-targeted metabolomic profiling of filamentous cyanobacteria Aphanizomenon flos-aquae exposed to a concentrated culture filtrate of Microcystis aeruginosa.

Microcystis and Aphanizomenon are two toxic cyanobacteria genera, which frequently cause blooms in freshwater lakes. In some cases, succession of these two genera was observed in natural water bodies. Among the diverse factors contributing to such succession of dominant cyanobacterial genera, an allelopathic effect was proposed to be involved after the growth inhibitory effect of several Microcystis species on A. flos-aquae was investigated. However, the response of target species exposed to Microcystis are poorly described. In the present study, we used two toxic cyanobacteria strains, Aphanizomenon flos-aquae (Aph1395) and Microcystis aeruginosa strain 905 (Ma905) as research subjects. Aph1395 was inhibited with a necessarily concentrated culture filtrate of Ma905 (MA905-SPE), and the response of the inhibited Aph1395 cells was explored via non-targeted metabolomic profiling. In total, 3735 features were significantly different in the Aph1395 treated with Ma905-SPE vs. those treated with BG11 medium. Among them, the annotations of 146 differential features were considered to be confident via MS/MS spectrum matching analysis. Based on the reported physiological functions of the annotated differential features, we proposed a putative model that in the growth-inhibited Aph1395, a suite of increased or decreased features with activities in apoptosis, growth inhibition, and stress response processes contributed to, or defended against, the allelopathic effect caused by Ma905. Our findings provide insights into the interaction between the bloom forming cyanobacterial species that share the same ecological environment.

Purification and refolding protocol for cold-active recombinant esterase AaSGNH1 from Aphanizomenon flos-aquae expressed as insoluble inclusion bodies.

Microbial esterases are a highly desirable tool for numerous biosynthetic and biotechnological applications requiring ester bond cleavage. Once identified, microbial esterases are often produced recombinantly in Escherichia coli to enhance yield and ease of purification. In this study a polyhistidine-tagged SGNH esterase gene (AaSGNH1), originating from the cyanobacterium Aphanizomenon flos-aquae, was cloned into an over-expression plasmid and expressed in BL21(DE3) cells. The recombinant esterase enzyme was produced as inactive inclusion bodies which were insoluble in 8 M urea but readily solubilized by the detergent Empigen BB®. Crucially, the procurement of active enzyme required controlled removal of detergent during column chromatography and dialysis steps. The refolded esterase was characterized with respect to its ability to catalyze the cleavage of p-nitrophenol esters of different chain lengths (C2, C8, C16). In addition, the temperature and pH optima were determined and it was found that the enzyme was most active at low temperatures (5-15 °C) and under alkaline conditions (pH 8-10). It was found that the kinetic properties of AaSGNH1 were remarkably similar to other SGNH esterases described thereby validating that the protein was effectively refolded. Overall, this study provides a simple strategy for isolating cold-active recombinant esterase enzyme when expressed as inclusion bodies.