Design, synthesis and activity evaluation of pseudilin analogs against cyanobacteria as IspD inhibitors.

The discovery of safe, effective, and selective chemical algicides is the stringent need for the algicides development, and it is also one of the effective routes to control cyanobacteria harmful algal blooms and to meet the higher requirements of environmental and ecological. In this work, a series of novel bromo-N-phenyl-5-o-hydroxyphenylpyrazole-3-carboxyamides were rationally designed as pseudilin analogs by bioisosteric replacement and molecular hybridization strategies, in which the pyrrole unit of pseudilin was replaced with pyrazole and further combined with the dominant structural fragments of algicide diuron. The synthesis was carried out by a facile four-step routeincluding cyclization, amidation, transanulation, and halogenation. The biological activity evaluation on AtIspD, EcIspD, Synechocystis sp. PCC6803 and Microcystis aeruginosa FACHB905 revealed that most compounds had good EcIspD and excellent cyanobacteria inhibitory activity. In particular, compound 6bb exhibited potent algicidal activity against PCC6803 and FACHB905 with EC50 = 1.28 µM and 0.37 µM, respectively, 1.4-fold and 4.0-fold enhancement compared to copper sulfate (EC50 = 1.79 and 1.49 µM, respectively), and it also showed the best inhibitory activity of EcIspD. The binding of 6bb to EcIspD was explored by molecular docking, and it was confirmed that 6bb could bind to the EcIspD active site. Compound 6bb was proven to be a potential structure for the further development of novel algicides that targets IspD in the MEP pathway.

Decoding Microcystis aeruginosa quorum sensing through AHL-mediated transcriptomic molecular regulation mechanisms.

Acyl-homoserine lactone (AHL) serves as a key signaling molecule for quorum sensing (QS) in bacteria. QS-related genes and physiological processes in Microcystis aeruginosa remain elusive. In this study, we elucidated the regulatory role of AHL-mediated QS in M. aeruginosa. Using AHL activity extract and transcriptomic analysis, we revealed significant effects of the AHL on growth and photosynthesis. AHL significantly increased chlorophyll a (Chl-a) content and accelerated photosynthetic rate thereby promoting growth. Transcriptome analysis revealed that AHL stimulated the up-regulation of photosynthesis-related genes (apcABF, petE, psaBFK, psbUV, etc.) as well as nitrogen metabolism and ribosomal metabolism. In addition, AHL-regulated pathways are associated with lipopolysaccharide and phenazine synthesis. Our findings deepen the understanding of the QS system in M. aeruginosa and are important for gaining insights into the role of QS in Microcystis bloom formation. It also provides new insights into the prevalence of M. aeruginosa in water blooms.

CyanoStrainChip: A Novel DNA Microarray Tool for High-Throughput Detection of Environmental Cyanobacteria at the Strain Level.

Detecting cyanobacteria in environments is an important concern due to their crucial roles in ecosystems, and they can form blooms with the potential to harm humans and nonhuman entities. However, the most widely used methods for high-throughput detection of environmental cyanobacteria, such as 16S rRNA sequencing, typically provide above-species-level resolution, thereby disregarding intraspecific variation. To address this, we developed a novel DNA microarray tool, termed the CyanoStrainChip, that enables strain-level comprehensive profiling of environmental cyanobacteria. The CyanoStrainChip was designed to target 1277 strains; nearly all major groups of cyanobacteria are included by implementing 43,666 genome-wide, strain-specific probes. It demonstrated strong specificity by in vitro mock community experiments. The high correlation (Pearson's R > 0.97) between probe fluorescence intensities and the corresponding DNA amounts (ranging from 1-100 ng) indicated excellent quantitative capability. Consistent cyanobacterial profiles of field samples were observed by both the CyanoStrainChip and next-generation sequencing methods. Furthermore, CyanoStrainChip analysis of surface water samples in Lake Chaohu uncovered a high intraspecific variation of abundance change within the genus Microcystis between different severity levels of cyanobacterial blooms, highlighting two toxic Microcystis strains that are of critical concern for Lake Chaohu harmful blooms suppression. Overall, these results suggest a potential for CyanoStrainChip as a valuable tool for cyanobacterial ecological research and harmful bloom monitoring to supplement existing techniques.

Indole-3-acetic acid promotes growth in bloom-forming Microcystis via an antioxidant response.

Interactions between bacteria and phytoplankton in the phycosphere facilitate and constrain biogeochemical cycling in aquatic ecosystems. Indole-3-acetic acid (IAA) is a bacterially produced chemical signal that promotes growth of phytoplankton and plants. Here, we explored the impact of IAA on bloom-forming cyanobacteria and their associated bacteria. Exposure to IAA and its precursor, tryptophan, resulted in a strong growth response in a bloom of the freshwater cyanobacterium, Microcystis. Metatranscriptome analysis revealed the induction of an antioxidant response in Microcystis upon exposure to IAA, potentially allowing populations to increase photosynthetic rate and overcome internally generated reactive oxygen. Our data reveal that co-occurring bacteria within the phycosphere microbiome exhibit a division of labor for supportive functions, such as nutrient mineralization and transport, vitamin synthesis, and reactive oxygen neutralization. These complex dynamics within the Microcystis phycosphere microbiome are an example of interactions within a microenvironment that can have ecosystem-scale consequences.

Bacterial community shifts induced by high concentration hydrogen peroxide treatment of Microcystis bloom in a mesocosm study.

Hydrogen peroxide has gained popularity as an environmentally friendly treatment for cyanobacterial harmful algal blooms (cHABs) that takes advantage of oxidative stress sensitivity in cyanobacteria at controlled concentrations. Higher concentrations of hydrogen peroxide treatments may seem appealing for more severe cHABs but there is currently little understanding of the environmental impacts of this approach. Of specific concern is the associated microbial community, which may play key roles in the succession/recovery process post-treatment. To better understand impacts of a high concentration treatment on non-target microbial communities, we applied a hydrogen peroxide spray equating to a total volume concentration of 14 mM (473 mg/L, 0.04%) to 250 L mesocosms containing Microcystis bloom biomass, monitoring treatment and control mesocosms for 4 days. Cyanobacteria dominated control mesocosms throughout the experiment while treatment mesocosms experienced a 99% reduction, as determined by bacterial amplicon sequencing, and a 92% reduction in bacterial cell density within 1 day post-treatment. Only the bacterial community exhibited signs of regrowth, with a fold change of 9.2 bacterial cell density from day 1 to day 2. Recovery consisted of succession by Planctomycetota (47%) and Gammaproteobacteria (17%), which were likely resilient due to passive cell component compartmentalization and rapid upregulation of dnaK and groEL oxidative stress genes, respectively. The altered microbiome retained beneficial functionality of microcystin degradation through a currently recognized but unidentified pathway in Gammaproteobacteria, resulting in a 70% reduction coinciding with bacterial regrowth. There was also an 81% reduction of both total nitrogen and phosphorus, as compared to 91 and 93% in the control, respectively, due to high expressions of genes related to nitrogen (argH, carB, glts, glnA) and phosphorus (pntAB, phoB, pstSCB) cycling. Overall, we found a portion of the bacterial community was resilient to the high-concentration hydrogen peroxide treatment, resulting in Planctomycetota and Gammaproteobacteria dominance. This high-concentration treatment may be suitable to rapidly end cHABs which have already negatively impacted the aquatic environment rather than allow them to persist.

Mechanisms of harmful effects of Microcystis aeruginosa on a brackish water organism Moina mongolica based on physiological and transcriptomic responses.

To investigate the detrimental impacts of cyanobacterial bloom, specifically Microcystis aeruginosa, on brackish water ecosystems, the study used Moina mongolica, a cladoceran species, as the test organism. In a chronic toxicology experiment, the survival and reproductive rates of M. mongolica were assessed under M. aeruginosa stress. It was observed that the survival rate of M. mongolica fed with M. aeruginosa significantly decreased with time and their reproduction rate dropped to zero, while the control group remained maintained stable and normal reproduction. To further explore the underlying molecular mechanisms of the effects of M. aeruginosa on M. mongolica, we conducted a transcriptomic analysis on newly hatched M. mongolica cultured under different food conditions for 24 h. The results revealed significant expression differences in 572 genes, with 233 genes significantly up-regulated and 339 genes significantly down-regulated. Functional analysis of these differentially expressed genes identified six categories of physiological functional changes, including nutrition and metabolism, oxidative phosphorylation, neuroimmunology, cuticle and molting, reproduction, and programmed cell death. Based on these findings, we outlined the basic mechanisms of microcystin toxicity. The discovery provides critical insights into the mechanisms of Microcystis toxicity on organisms and explores the response mechanisms of cladocerans under the stress of Microcystis.

The synchronicity of bloom-forming cyanobacteria transcription patterns and hydrogen peroxide dynamics.

Hydrogen peroxide is a reactive oxygen species (ROS) naturally occurring at low levels in aquatic environments and production varies widely across different ecosystems. Oxygenic photosynthesis generates hydrogen peroxide as a byproduct, of which some portion can be released to ambient water. However, few studies have examined hydrogen peroxide dynamics in relation to cyanobacterial harmful algal blooms (cHABs). A year-long investigation of algal succession and hydrogen peroxide dynamics was conducted at the Caloosahatchee River, Florida, USA. We aimed to identify potential biological mechanisms responsible for elevated hydrogen peroxide production during cHAB events through the exploration of the freshwater microbial metatranscriptome. Hydrogen peroxide concentrations were elevated from February to September of 2021 when cyanobacteria were active and abundant. We observed one Microcystis cHAB event in spring and one in winter. Both had distinct nutrient uptake and cyanotoxin gene expression patterns. While meaningful levels of microcystin were only detected during periods of elevated hydrogen peroxide, cyanopeptolin was by far the most expressed cyanotoxin during the spring bloom when hydrogen peroxide was at its yearly maxima. Gene expressions of five microbial enzymes (Rubisco, superoxide dismutase, cytochrome b559, pyruvate oxidase, and NADH dehydrogenase) positively correlated to hydrogen peroxide concentrations. Additionally, there was higher nitrogen-fixing gene (nifDKH) expression by filamentous cyanobacteria after the spring bloom but no secondary bloom formation occurred. Overall, elevated environmental hydrogen peroxide concentrations were linked to cyanobacterial dominance and greater expression of specific enzymes in the photosynthesis of cyanobacteria. This implicates cyanobacterial photosynthesis and growth results in increased hydrogen peroxide generation as reflected in measured environmental concentrations.

Evaluating microcystinase A-based approach on microcystins degradation during harvested cyanobacterial blooms.

Addressing notorious and worldwide Microcystis blooms, mechanical algae harvesting is an effective emergency technology for bloom mitigation and removal of nutrient loads in waterbodies. However, the absence of effective methods for removal of cyanobacterial toxins, e.g., microcystins (MCs), poses a challenge to recycle the harvested Microcystis biomass. In this study, we therefore introduced a novel approach, the "captured biomass-MlrA enzymatic MC degradation", by enriching microcystinase A (MlrA) via fermentation and spraying it onto salvaged Microcystis slurry to degrade all MCs. After storing the harvested Microcystis slurry, a rapid release of extracellular MCs occurred within the initial 8 h, reaching a peak concentration of 5.33 µg/mL at 48 h during the composting process. Upon spraying the recombinant MlrA crude extract (about 3.36 U) onto the Microcystis slurry in a ratio of 0.1% (v/v), over 95% of total MCs were degraded within a 24-h period. Importantly, we evaluated the reliability and safety of using MlrA extracts to degrade MCs. Results showed that organic matter/nutrient contents, e.g. soluble proteins, polysaccharides, phycocyanin and carotenoids, were not significantly altered. Furthermore, the addition of MlrA extracts did not significantly change the bacterial community composition and diversity in the Microcystis slurry, indicating that the MlrA extracts did not increase the risk of pathogenic bacteria. Our study provides an effective and promising method for the pre-treatment of harvested Microcystis biomass, highlighting an ecologically sustainable framework for addressing Microcystis blooms.

Identification of novel laccase from cyanobacterium Microcystis flos-aquae and enhanced azo dye bioremediation potential.

Textile industries discharge up to 280,000 tons of dye waste annually, resulting in global pollution and health risks. In Nigeria and other African countries, persistent dyes threaten aquatic life and human health. This study introduces a cost-effective, enzyme-mediated bioremediation alternative using a novel laccase from the cyanobacteriumMicrocystis flos-aquae. This purified enzyme yielded 0.55 % (w/w)with significant activity at 40 °C and pH 4.00. Kinetic studies showed the dependence of M. flos-aquae laccase on Cu2+and its inhibition by EDTA and Fe2+. The efficacy of the enzyme was demonstrated through rapid decolorization of the azo dye Cibacron Brilliant Blue over a wide temperature and pH range. As this enzyme effectively decolorizes dyes across a broad temperature and pH range, it offers a promising solution for bioremediation of textile effluents.

ß-cyclocitral induced rapid cell death of Microcystis aeruginosa.

ß-cyclocitral (BCC) is an odorous compound that can be produced by bloom-forming cyanobacteria, for example, Microcystis aeruginosa. BCC has been proposed to explain the rapid decline of cyanobacterial blooms in natural water bodies due to its lytic effects on cyanobacteria cells. However, few insights have been gained regarding the mechanisms of its lethality on cyanobacteria. In this study, M. aeruginosa was exposed to 0-300 mg/L BCC, and the physiological responses were comprehensively studied at the cellular, molecular, and transcriptomic levels. The result indicated that the lethal effect was concentration-dependent; 100 mg/L BCC only caused recoverable stress, while 150-300 mg/L BCC caused rapid rupture of cyanobacterial cells. Scanning electron microscope images suggested two typical morphological changes exposed to above 150 mg/LBCC: wrinkled/shrank with limited holes on the surface at 150 and 200 mg/L BCC exposure; no apparent shrinkage at the surface but with cell perforation at 250 and 300 mg/L BCC exposure. BCC can rapidly inhibit the photosynthetic activity of M. aeruginosa cells (40%∼100% decreases for 100-300 mg/L BCC) and significantly down-regulate photosynthetic system Ⅰ-related genes. Also, chlorophyll a (by 30%∼90%) and ATP (by ∼80%) contents severely decreased, suggesting overwhelming pressure on the energy metabolism in cells. Glutathione levels increased significantly, and stress response-related genes were upregulated, indicating the perturbation of intracellular redox homeostasis. Two cell death pathways were proposed to explain the lethal effect: apoptosis-like death as revealed by the upregulation of SOS response genes when exposed to 200 mg/L BCC and mazEF-mediated death as revealed by the upregulation of mazEF system genes when exposed to 300 mg/L BCC. Results of the current work not only provide insights into the potential role of BCC in inducing programmed cell death during bloom demise but also indicate the potential of using BCC for harmful algal control.

Phthalocyanine blue leaching and exposure effects on Microcystis aeruginosa (cyanobacteria) of photoaged microplastics.

Light-stabilizing additives may contribute to the overall pollution load of microplastics (MPs) and potentially enter the food chain, severely threatening aquatic life and human health. This study investigated the variation between polystyrene (PS) MPs and phthalocyanine blue (CuPC)-containing MPs before and after photoaging, as well as their effects on Microcystis aeruginosa. The presence of PS-MPs increased cell mortality, antioxidant enzyme activity, and the variation in extracellular components, while the presence of CuPC exacerbated these variations. CuPC-containing MPs caused different increasing trends in superoxide dismutase and malondialdehyde activities due to electron transfer across the membrane. Transcriptomic analysis revealed that the MPs and CuPC affected various cellular processes, with the greatest impact being on cell membranes. Compared with MPs, CuPC negatively affected ribosome and polysaccharide formation. These findings provide insights into the molecular mechanisms underlying the cellular response to MPs and their associated light-stabilizer pollution and imply the necessity for mitigating the pollution of both MPs and light-stabilizers.

Integrated physio-biochemical and transcriptomic analysis reveals the joint toxicity mechanisms of two typical antidepressants fluoxetine and sertraline on Microcystis aeruginosa.

Selective serotonin reuptake inhibitor (SSRI) antidepressants are of increasing concern worldwide due to their ubiquitous occurrence and detrimental effects on aquatic organisms. However, little is known regarding their effects on the dominant bloom-forming cyanobacterium, Microcystis aeruginosa. Here, we investigated the individual and joint effects of two typical SSRIs fluoxetine (FLX) and sertraline (SER) on M. aeruginosa at physio-biochemical and molecular levels. Results showed that FLX and SER had strong growth inhibitory effects on M. aeruginosa with the 96-h median effect concentrations (EC50s) of 362 and 225 µg/L, respectively. Besides, the mixtures showed an additive effect on microalgal growth. Meanwhile, both individual SSRIs and their mixtures can inhibit photosynthetic pigment synthesis, cause oxidative damage, destroy cell membrane, and promote microcystin-leucine-arginine (MC-LR) synthesis and release. Moreover, the mixtures enhanced the damage to photosynthesis, antioxidant system, and cell membrane and facilitated MC-LR synthesis and release compared to individuals. Furthermore, transcriptomic analysis revealed that the dysregulation of the key genes related to transport, photosystem, protein synthesis, and non-ribosomal peptide structures was the fundamental molecular mechanism underlying the physio-biochemical responses of M. aeruginosa. These findings provide a better understanding of the toxicity mechanisms of SSRIs to microalgae and their risks to aquatic ecosystems.

Discovery of two novel bioactive algicidal substances from Brevibacillus sp. via metabolomics profiling and back-validation.

Identifying potent bacterial algicidal agents is essential for the development of effective, safe, and economically viable algaecides. Challenges in isolating and purifying these substances from complex secretions have impeded progress in this field. Metabolomics profiling, an efficient strategy for identifying metabolites, was pioneered in identifying bacterial algicidal substances in this study. Extracellular secretions from different generations of the algicidal bacterium Brevibacillus sp. were isolated for comprehensive analysis. Specifically, a higher algicidal efficacy was observed in the secretion from Generation 3 (G3) of Brevibacillus sp. compared to Generation 1 (G1). Subsequent metabolomics profiling comparing G3 and 1 revealed 83 significantly up-regulated metabolites, of which 9 were identified as potential algicidal candidates. Back-validation highlighted the potency of 4-acetamidobutanoic acid (4-ABC) and 8-hydroxyquinoline (8-HQL), which exhibited robust algicidal activity with 3d-EC50 values of 6.40 mg/L and 92.90 µg/L, respectively. These substances disrupted photosynthetic activity in M. aeruginosa by ceasing electron transfer in PSⅡ, like the impact exerted by Brevibacillus sp. secretion. These findings confirmed that 4-ABC and 8-HQL were the main algicidal components derived from Brevibacillus sp.. Thus, this study presents a streamlined strategy for identifying bacterial algicidal substances and unveils two novel and highly active algicidal substances. ENVIRONMENTAL IMPLICATION: Harmful cyanobacterial blooms (HCBs) pose significant environmental problems and health effects to humans and other organisms. The increasing frequency of HCBs has emerged as a pressing global concern. Bacterial-derived algicidal substances are expected to serve as effective, safe, and economically viable algaecides against HCBs. This study presents a streamlined strategy for identifying bacterial algicidal substances and unveils two novel substances (4-ABC and 8-HQL). These two substances demonstrate remarkable algicidal activity and disrupt the photosynthetic system in M. aeruginosa. They hold potential as prospective algaecides for addressing HCBs.

Changes of characteristics and disinfection by-products formation potential of intracellular organic matter with different molecular weight in metalimnetic oxygen minimum.

Metalimnetic oxygen minimum (MOM) occurs in reservoirs or lakes due to stratification and algal blooms, which has low dissolved oxygen (DO) levels and leads to the deterioration of water quality. The transformation mechanism and the impact on the water quality of intracellular organic matter (IOM) derived from algae are poorly understood under MOM conditions. In this study, IOM extracted by Microcystis aeruginosa was divided into five components according to molecular weight (MW), and the changes of characteristics and correlated disinfection by-products formation potential (DBPFP) were analyzed and compared under MOM conditions. The removal efficiency of dissolved organic carbon (DOC) in the <5 kDa fraction (66.6%) was higher than that in the >100 kDa fraction (41.8%) after a 14-day incubation under MOM conditions. The same tendency also occurred in Fmax and DBPFP. The decrease in Fmax was mainly due to the decline in tryptophan-like and tyrosine-like for all IOM fractions. The diversity of microorganisms degrading the MW > 100 kDa fraction was lower than others. Besides low MW fractions, these findings indicated that more attention should be paid to high MW fractions which were resistant to biodegradation under MOM conditions during water treatment.

The Microcystis-microbiome interactions: origins of the colonial lifestyle.

Species of the Microcystis genus are the most common bloom-forming toxic cyanobacteria worldwide. They belong to a clade of unicellular cyanobacteria whose ability to reach high biomasses during blooms is linked to the formation of colonies. Colonial lifestyle provides several advantages under stressing conditions of light intensity, ultraviolet light, toxic substances and grazing. The progression from a single-celled organism to multicellularity in Microcystis has usually been interpreted as individual phenotypic responses of the cyanobacterial cells to the environment. Here, we synthesize current knowledge about Microcystis colonial lifestyle and its role in the organism ecology. We then briefly review the available information on Microcystis microbiome and propose that changes leading from single cells to colonies are the consequence of specific and tightly regulated signals between the cyanobacterium and its microbiome through a biofilm-like mechanism. The resulting colony is a multi-specific community of interdependent microorganisms.

Selection of photosynthetic traits by turbulent mixing governs formation of cyanobacterial blooms in shallow eutrophic lakes.

Prediction of the complex cyanobacteria-environment interactions is vital for understanding harmful bloom formation. Most previous studies on these interactions considered specific properties of cyanobacterial cells as representative for the entire population (e.g. growth rate, mortality, and photosynthetic capacity (Pmax)), and assumed that they remained spatiotemporally unchanged. Although, at the population level, the alteration of such traits can be driven by intraspecific competition, little is known about how traits and their plasticity change in response to environmental conditions and affect the bloom formation. Here we test the hypothesis that intraspecific variations in Pmax of cyanobacteria (Microcystis spp.) play an important role in its population dynamics. We coupled a one-dimensional hydrodynamic model with a trait-based phytoplankton model to simulate the effects of physical drivers (turbulence and turbidity) on the Pmax of Microcystis populations for a range of dynamic conditions typical for shallow eutrophic lakes. Our results revealed that turbulence acts as a directional selective driver for changes in Pmax. Depending on the intensity of daily-periodic turbulence, representing wind-driven mixing, a shift in population-averaged phenotypes occurred toward either low Pmax, allowing the population to capture additional light in the upper layers, or high Pmax, enhancing the efficiency of light utilization. Moreover, we observed that a high intraspecific diversity in Pmax accelerated the formation of surface scum by up to more than four times compared to a lower diversity. This study offers insights into mechanisms by which cyanobacteria populations respond to turbulence and underscores the significance of intraspecific variations in cyanobacterial bloom formation.

Biological activity and molecular mechanism of inactivation of Microcystis aeruginosa by ultrasound irradiation.

Harmful algal blooms (HABs) significantly impact on water quality and ecological balance. Ultrasound irradiation has proven to be an effective method for algal control. Nevertheless, the molecular mechanisms underlying the inactivation of M. aeruginosa by ultrasound are still unknown. In this study, the physiological activity and molecular mechanism of algal cells exposed to different frequencies of ultrasound were studied. The results indicated a pronounced inhibition of algal cell growth by high-frequency, high-dose ultrasound. Moreover, with increasing ultrasound dosage, there was a higher percentage of algal cell membrane ruptures. SEM and TEM observed obvious disruptions in membrane structure and internal matrix. Hydroxyl radicals generated by high-frequency ultrasound inflicted substantial cell membrane damage, while increased antioxidant enzyme activities fortified cells against oxidative stress. Following 2 min of ultrasound irradiation at 740 kHz, significant differential gene expression occurred in various aspects, including energy metabolism, carbohydrate metabolism, and environmental information processing pathways. Moreover, ultrasound irradiation influenced DNA repair and cellular apoptosis, suggesting that the algal cells underwent biological stress to counteract the damage caused by ultrasound. These findings reveal that ultrasound irradiation inactivates algae by destroying their cell structures and metabolic pathways, thereby achieving the purpose of algal suppression.

Colonial Microcystis' biomass affects its shift to diatom aggregates under aeration mixing.

The effect of hydrodynamic mixing on controlling Microcystis blooms or changing the algal community to diatom dominance has been widely studied; however, the effects of colonial Microcystis biomass on the development of the algal community are poorly known. Here, in order to study the changes in Microcystis blooms under continuous aeration mixing, an experiment was carried out in a greenhouse with factors of varying biomass of Microcystis and inorganic nitrogen and phosphorus enrichment in summer. There were three chlorophyll a (Chl-a) levels in six treatments: low Chl-a level of 68.4 µg L-1 (treatments L, L-E), medium Chl-a level of 468.7 µg L-1 (treatments M, M-E), and high Chl-a level of 924.1 µg L-1 (treatments H, H-E). Treatments L-E, M-E and H-E were enriched with the same inorganic nitrogen and phosphorus nutrients. During the experiment of 30 days, the concentration of Microcystis and Chl-a decreased, and diatom Nitzschia palea cells appeared in all the treatments, which became dominant in treatments M, M-E, H and H-E, with the highest biomass of 9.41 ± 1.96 mg L-1 Nitzschia in treatment H-E on day 30. The rank order of the biomass of Nitzschia from low to high was (L = L-E) < (M = M-E) < H < H-E (P < 0.05). In addition, Nitzschia cells were aggregates attached to Microcystis colonies in all the treatments. The results showed that the initial biomass of colonial Microcystis affected the algal shift from Microcystis dominance to Nitzschia dominance. However, the enriched inorganic nitrogen and phosphorus was beneficial for the Nitzschia increase in the high biomass treatment alone. The shift from Microcystis dominance to diatom dominance under continuous aeration mixing may be caused by low light conditions as well as the nutrients released from Microcystis decay. Moreover, the aerobic condition caused by aeration mixing maintained the colonial mucilaginous sheath to support the growth of Nitzschia cells in aggregation. This study found for the first time that Microcystis blooms could shift to diatom Nitzschia dominance in aggregates. It provided a method to control and manipulate Microcystis blooms to diatom dominance through continuous aeration mixing to proper biomass of Microcystis colonies. The shift to diatoms dominance would provide more high quality food organisms for aquaculture and be beneficial to the material cycling and energy flowing in food web dynamics.

Upstream nitrogen availability determines the Microcystis salt tolerance and influences microcystins release in brackish water.

The occurrence of large Microcystis biomass in brackish waters is primarily caused by its downward transportation from the upstream freshwater lakes and reservoirs through rivers rather than due to in situ bloom formation. Factors that determine the survival of freshwater cyanobacteria in brackish waters have not been well investigated. Here, we studied the spatiotemporal variability of inorganic nitrogen in an upstream lake and conducted laboratory and in-situ experiments to assess the role of nitrogen availability on the salt tolerance of Microcystis and the release of microcystins. A series of field experiments were carried out during bloom seasons to evaluate the salt tolerance of natural Microcystis colonies. The salt tolerance threshold varied from 7 to 17 and showed a positive relationship with intracellular carbohydrate content and a negative relationship with nitrogen availability in water. In August when upstream nitrogen availability was lower, the Microcystis colonies could maintain their biomass even after a sudden increase in salinity from 4 to 10. Laboratory-cultivated Microcystis that accumulated higher carbohydrate content at lower nitrogen availability showed better cell survival at higher salinity. The sharp release of microcystins into the surrounding water occurred when salinity exceeded the salt tolerance threshold of the Microcystis. Thus, Microcystis with higher salt tolerance can accumulate more toxins in cells. The obtained results suggest that the cell survival and toxin concentration in brackish waters depend on the physiological properties of Microcystis formed in the upstream waters. Thus, the life history of Microcystis in upstream waters could have a significant impact on its salt tolerance in downstream brackish waters, where the ecological risk of the salt-tolerant Microcystis requires special and careful management in summer at low nitrogen availability.

Quorum sensing regulation in Microcystis aeruginosa: Insights into AHL-mediated physiological processes and MC-LR production.

Quorum sensing (QS) is a widespread regulatory mechanism in Gram-negative bacteria, primarily involving the secretion of N-acyl homoserine lactone (AHL) to facilitate population density sensing. However, the existence of QS in blue-green algae, a subset of photoautotrophic Gram-negative bacteria forming high-density communities in water blooms, remains elusive. This study delves into the unexplored realm of QS in Microcystis aeruginosa (M. aeruginosa) by investigating AHL-related regulatory mechanisms and their impact on various physiological processes. Utilizing high-performance liquid chromatography coupled with mass spectrometry (HPLC-MS) and biosensors, a hitherto unknown long-chain AHL exhibiting a mass-to-charge ratio of 318 was identified in sterile M. aeruginosa cultures. Our investigation focused on discerning correlations between AHL activity fluctuations and key parameters such as microcystin (MC-LR) production, algal density, photosynthesis, buoyancy, and aggregation. Furthermore, the AHL extract was introduced during the logarithmic stage of M. aeruginosa cultures to observe the response in physiological processes. The results revealed that AHL, functioning as an autoinducer (AI), positively influenced algal growth and photosynthesis, as evidenced by the upregulated photosynthetic conversion efficiency of PSI and chlorophyll synthesis gene (psbA). AI also played a crucial role in altering surface characteristics through the synthesis of polysaccharides and proteins in EPS, subsequently promoting cell aggregation. Concomitantly, AI upregulated mcyD, enhancing the synthesis of MC-LR. Notably, our investigation pinpointed the initiation of QS in Microcystis at a density of approximately 1.22 × 10^7 cells/mL. This groundbreaking evidence underscores the regulatory role of AI in governing the physiological processes of growth, aggregation, buoyancy, and MC-LR production by activating pertinent gene expressions. This study significantly expands the understanding of QS in AHL, providing crucial insights into the regulatory networks operating in blue-green algae.