Nitrogen-fixing cyanobacteria not only cause severe blooms but also play an important role in the nitrogen input processes of lakes. The production of extracellular polymeric substances (EPS) and the ability to fix nitrogen from the atmosphere provide nitrogen-fixing cyanobacteria with a competitive advantage over other organisms. Temperature and nitrogen availability are key environmental factors in regulating the growth of cyanobacteria. In this study, Dolichospermum (formerly known as Anabaena) was cultivated at three different temperatures (10 °C, 20 °C, and 30 °C) to examine the impact of temperature and nitrogen availability on nitrogen fixation capacity and the release of EPS. Initially, confocal laser scanning microscopy (CLSM) and the quantification of heterocysts at different temperatures revealed that lower temperatures (10 °C) hindered the differentiation of heterocysts under nitrogen-deprived conditions. Additionally, while heterocysts inhibited the photosynthetic activity of Dolichospermum, the secretion of EPS was notably affected by nitrogen limitation, particularly at 30 °C. Finally, real-time quantitative polymerase chain reaction (qPCR) was used to measure the expression of nitrogen-utilizing genes (ntcA and nifH) and EPS synthesis-related genes (wzb and wzc). The results indicated that under nitrogen-deprived conditions, the expression of each gene was upregulated, and there was a significant correlation between the upregulation of nitrogen-utilizing and EPS synthesis genes (P < 0.05). Our findings suggested that Dolichospermum responded to temperature variation by affecting the formation of heterocysts, impacting its potential nitrogen fixation capacity. Furthermore, the quantity of EPS released was more influenced by nitrogen availability than temperature. This research enhances our comprehension of interconnections between nitrogen deprivation and EPS production under the different temperatures.

Cyanobacteria can reach high densities in eutrophic lakes, which may cause problems due to their potential toxin production. Several methods are in use to prevent, control or mitigate harmful cyanobacterial blooms. Treatment of blooms with low concentrations of hydrogen peroxide (H2O2) is a promising emergency method. However, effects of H2O2 on cyanobacteria, eukaryotic phytoplankton and zooplankton have mainly been studied in controlled cultures and mesocosm experiments, while much less is known about the effectiveness and potential side effects of H2O2 treatments on entire lake ecosystems. In this study, we report on three different lakes in the Netherlands that were treated with average H2O2 concentrations ranging from 2 to 5 mg L-1 to suppress cyanobacterial blooms. Effects on phytoplankton and zooplankton communities, on cyanotoxin concentrations, and on nutrient availability in the lakes were assessed. After every H2O2 treatment, cyanobacteria drastically declined, sometimes by more than 99%, although blooms of Dolichospermum sp., Aphanizomenon sp., and Planktothrix rubescens were more strongly suppressed than a Planktothrix agardhii bloom. Eukaryotic phytoplankton were not significantly affected by the H2O2 additions and had an initial advantage over cyanobacteria after the treatment, when ample nutrients and light were available. In all three lakes, a new cyanobacterial bloom developed within several weeks after the first H2O2 treatment, and in two lakes a second H2O2 treatment was therefore applied to again suppress the cyanobacterial population. Rotifers strongly declined after most H2O2 treatments except when the H2O2 concentration was ≤ 2 mg L-1, whereas cladocerans were only mildly affected and copepods were least impacted by the added H2O2. In response to the treatments, the cyanotoxins microcystins and anabaenopeptins were released from the cells into the water column, but disappeared after a few days. We conclude that lake treatments with low concentrations of H2O2 can be a successful tool to suppress harmful cyanobacterial blooms, but may negatively affect some of the zooplankton taxa in lakes. We advise pre-tests prior to the treatment of lakes to define optimal treatment concentrations that kill the majority of the cyanobacteria and to minimize potential side effects on non-target organisms. In some cases, the pre-tests may discourage treatment of the lake.

Algal extracellular organic matter (EOM) metabolites exert considerable impact on the carbon (C), nitrogen (N), and phosphorus (P) cycles mediated by attached bacteria. Field investigations were conducted in two ponds to explore the relationship among EOM metabolites from Microcystis and Dolichospermum, co-occurring microbes, and nutrient recycling from April 2021 to December 2021. Microcystis blooms primarily produced more complex bound EOM (bEOM) metabolites with many amino acid components, which facilitated bacterial colonization and provided sufficient substrates for ammonification. Meanwhile, high abundances of dissimilatory nitrate reduction to ammonium genes from co-occurring microbes such as Rhodobacter have demonstrated their strong N retention ability. Metabolic products of bEOM from Microcystis comprise a large number of organic acids that can solubilize non-bioavailable P. All these factors have collectively resulted in the increase of all fractions of N and P, except for nitrate (NO3--N) in the water column. In contrast, the EOM metabolite from Dolichospermum was simple, coupled with high abundance of functional genes of α-glucosidase, and produced small molecular substances fueling denitrification. The metabolic products of EOM from Dolichospermum include abundant N-containing substances dominated by heterocyclic substances, suggesting that the metabolic products of Dolichospermum are not conducive to N regeneration and retention. Therefore, the metabolic products of EOM from Microcystis triggered a shift in the attached microbial community and function toward C, N, and P recycling with close mutual coupling. Acquisition of N and P in Dolichospermum is dependent on itself based on N fixation and organic P hydrolysis capacity. This study provides a new understanding of the contribution of algal EOM to the nutrient cycle.

The evaluation about the relative distribution of cyanobacterial biomass between the sediment and water column would be indispensable to understand if benthic cyanobacteria are important to cyanobacterial biomass in the water column. A separation method for the rapid quantification of benthic cyanobacteria in Lake Chaohu was developed by density-gradient centrifugation. A 24 full factorial design and response surface methodology was employed to optimize the extraction protocol. Under the optimal operating parameters including 29% Percoll solution, 30 min centrifugation time, 7200 r/min centrifugation speed, and a 1:10 ratio between the volume of sediment and Percoll solution, the recovery rate of cyanobacteria in sediment was 96.73%. Temporal and spatial variations in cyanobacterial biomass in water and sediment were investigated monthly throughout a whole year. In general, cyanobacterial biomass per square centimeter in the water column showed high spatial-temporal changes, tending to increase in February and reaching a peak in April at some sites due to the growth of Dolichospermum. The second peak arrived in July and September and was caused by the rapid growth of Microcystis. Concurrently, cyanobacteria biomass per unit area in sediment showed a clear temporal change pattern, increasing from October and reaching a peak level in February at all the sampling sites. The average ratio of cyanobacterial biomass in water to that in sediment was lowest in January at 1.48 and increased to the highest level in July at 318.61. Although Microcystis and Dolichospermum were dominant species in the water column, only Microcystis was observed in the Percoll solution extraction from sediment. Microscopic observation revealed that a very small fraction of Microcystis cells could survive in sediment, and most of the cells decomposed when the water temperature increased after June. Therefore, the contribution of the recruitment of cyanobacteria could be negligible in Lake Chaohu.

Harmful algal blooms caused by cyanobacteria have been increasing in frequency worldwide. However, the main environmental drivers of this change are often difficult to identify because of the effects of the interaction between eutrophication and climate change. Recently, filamentous N2-fixing cyanobacteria and non-diazotrophic Microcystis have been observed to be co-existing and undergoing succession in some eutrophic lakes. However, the succession patterns of dominant cyanobacteria and the factors driving this in mesotrophic lakes are not well understood. We hypothesized that the changes in cyanobacterial assemblages in mesotrophic lakes could result in a relatively high risks of toxic blooms, and that these changes are associated with the global climatic changes. We tested these hypotheses using data from the subtropical mesotrophic Lake Erhai. We found that the high spatiotemporal variability in the cyanobacterial community, and the increase in biomass were driven primarily by the growth of bloom-forming cyanobacterial taxa. Species-specific biomasses were related to a different environmental stressor; increases in dissolved organic carbon (DOC) concentrations were statistically associated with an increase of Microcystis biomass, whereas increases in surface water temperature favored higher biomass of Pseudanabaena at low transparency and high concentration of phosphorus. In addition, low nitrogen- to- phosphorus ratios were identified as potential determinants of the abundance of N2-fixing Dolichospermum. Furthermore, changes in the concentration of DOC, total nitrogen, pH and water transparency levels were found to affect the composition of Microcystis morphotypes and genotypes mostly. This study highlights that the toxic to non-toxic Microcystis ratio might increase with the water darkening and browning (which occurs in many subtropical plateau lakes). Lake management strategies, therefore, need to consider the toxicity of cyanobacterial assemblages in mesotrophic lakes over the intensity of the cyanobacterial blooms.

Eutrophication leads to frequent outbreaks of cyanobacterial blooms, however, the effect of heterotrophic bacteria attached to cyanobacterial cells is unclear. Field investigations were carried out to gain a deeper understanding of the community composition and functional role of heterotrophic bacteria attached to Dolichospermum and Microcystins cells. The significantly positive relationships between Dolichospermum density and total nitrogen (TN) and between Microcystins density and particle nitrogen (PN) indicated the strong nitrogen (N) demand of these two species. The lack of functional genes that mediate the nitrification process in bacteria attached to both Microcystins and Dolichospermum cells indicated that these two genera preferred ammonium (NH4+-N). Dolichospermum cells obtained more available N through N2 fixation, which was expressed by high nitrogenase gene abundance. Bacteria attached to Microcystins cells showed a higher activity of leucine aminopeptidase and a significantly higher abundance of functional genes that mediate dissimilatory nitrate reduction to ammonium (DNRA) than those attached to Dolichospermum cells. The significantly higher abundance of carbon degradation genes and β-glucosidase activity of bacteria attached to Microcystins cells compared with those of bacteria attached to Dolichospermum cells suggested that abundant organic carbon was bound to Microcystins cells, which is a prerequisite for DNRA. In addition, Microcystins cells exhibited a great advantage in soluble reactive phosphorus (SRP) production through high levels of organic phosphorus (P) hydrolysis associated with high levels of phosphatase genes of attached bacteria. In conclusion, bacteria attached to Microcystins cells performed more important functions on NH4+-N and SRP production through ammonification and DNRA, as well as phosphatase hydrolysis respectively, compared to those attached to Dolichospermum. Thus, algal growth is the result of different variables such as nutrient concentration, their ratio and the microbial ability.

Freshwater lakes are threatened by harmful blooms characterized by Cyanobacterial Aggregates (CAs) that are normally aggregated with extracellular polysaccharides released by cyanobacteria to form a phycosphere. It is possible that mutualistic relationships exist between bacteria and cyanobacteria in these CAs wherein bacterial products supplement cyanobacterial growth, and cyanobacterial exudates, in turn, serve as substrates for bacteria, thus augmenting the stability of each constituent. However, little is known about the exact interaction between cyanobacteria and their attached bacteria in CAs. Therefore, in this study, we collected 26 CA samples from Lake Taihu, a large freshwater lake in China from March of 2015 to February of 2016. We then sequenced both the V4 regions of 16S rRNA genes and full metagenomes, resulting in 610 Mb of 16S rRNA gene data and 198.98 Gb of high-quality metagenomic data. We observed that two cyanobacteria genera (Microcystis and Dolichospermum) alternately dominated CAs along the sampling time and specific bacterial genera attached to different cyanobacteria genera dominated CAs. More specifically, Dolichospermum dominates CAs when water temperature is low and total nitrogen is high, while Microcystis dominates CAs when water temperature is high and total nitrogen is low. Moreover, we found specific bacterial genera attached to different cyanobacteria genera dominated CAs. The cyanobacteria-bacteria related pairs Dolichospermum-Burkholderia and Microcystis-Hyphomonas were detected by ecological networks construction. Bacterial communities in CAs were found to be more correlated with the cyanobacterial community (Mantel\'s r = 0.76, P = 0.001) than with environmental factors (Mantel\'s r = 0.27, P = 0.017). A potential codependent nitrogen-cycling pathway between cyanobacteria and their attached bacteria was constructed, indicating their functional link. Overall, these results demonstrated that mutualistic relationships do, indeed, exist between cyanobacteria and bacteria in CAs at both taxonomic and gene levels, providing biological clues potentially leading to the control of blooms by interventional strategies to disrupt bacteria-cyanobacteria relationships and co-pathways.

Non-diazotrophic Microcystis and filamentous N2-fixing Aphanizomenon and Dolichospermum (formerly Anabaena) co-occur or successively dominate freshwaters globally. Previous studies indicate that dual nitrogen (N) and phosphorus (P) reduction is needed to control cyanobacterial blooms; however, N limitation may cause replacement of non-N2-fixing by N2-fixing taxa. To evaluate potentially counterproductive scenarios, the effects of temperature, nutrients, and zooplankton on the spatio-temporal variations of cyanobacteria were investigated in three large, shallow eutrophic lakes in China. The results illustrate that the community composition of cyanobacteria is primarily driven by physical factors and the zooplankton community, and their interactions. Niche differentiation between Microcystis and two N2-fixing taxa in Lake Taihu and Lake Chaohu was observed, whereas small temperature fluctuations in Lake Dianchi supported co-dominance. Through structural equation modelling, predictor variables were aggregated into \'composites\' representing their combined effects on species-specific biomass. The model results showed that Microcystis biomass was affected by water temperature and P concentrations across the studied lakes. The biomass of two filamentous taxa, by contrast, exhibited lake-specific responses. Understanding of driving forces of the succession and competition among bloom-forming cyanobacteria will help to guide lake restoration in the context of climate warming and N:P stoichiometry imbalances.

Dolichospermum (formerly Anabaena) and Microcystis cause harmful cyanobacterial blooms in freshwater ecosystems worldwide. Input reduction of both nitrogen (N) and phosphorus (P) are commonly recognized as basic ways of controlling blooms, but little is known about the roles of nutrients and their using strategy among cyanobacteria in triggering the succession of diazotrophic to non-diazotrophic cyanobacteria. In this study, we investigated in situ responses of cyanobactria to ambient P status during the transition from Dolichospermum flos-aquae to Microcystis spp. in Lake Taihu and Lake Chaohu. While dominant in phytoplankton community, D. flos-aquae experienced P deficiency as evidenced by qualitative detection of extracellular phosphatase via enzyme labeled fluorescence (ELF). The percentage of ELF-labelled D. flos-aquae cells was 33% when it dominated the phytoplankton community, and was 78% when it co-dominated with Microcystis spp., indicating an increase in P deficiency. Meanwhile, no ELF-labelled Microcystis cells were observed while polyphosphate body (PPB) were present, suggesting that Microcystis spp. were not P deficient. Additionally, the percentages of Microcystis cells containing PPB showed an inverted \"U-shaped\" relationship with concentrations on soluble reactive phosphorus (SRP). To validate the field observation, a laboratory study of the monocultures of the dominant cyanobacteria was conducted. Extracellular alkaline phosphatase activity (APA) and PPB accumulation were regulated by P availability in monocultures of D. flos-aquae. Interestingly, no cell bound extracellular phosphatase was found on Microcystis aeruginasa even in the culture without P supply. Consistently, the expressions of phosphatase encoding gene phoX showed no differences among the treatments. The way in which PPB accumulation occurred in Microcystis spp. in response to P availability in the cultures was similar to that observed in the field, demonstrating a strategy of energy conservation over P accumulation. The competitive advantage of Microcystis spp. was displayed at low P concentrations: where it could rapidly uptake and store inorganic P, which also increased the P deficiency of the coexisting phytoplankton species. Responses of P-transport gene pstS confirmed this hypothesis. The physiological and molecular mechanisms mentioned above enable Microcystis to survive and proliferate in environment with low available P supply more efficiently. In conclusion, different cyanobacterial species have distinct ways of responding to P availability, suggesting that the control of cyanobacterial blooms by targeted nutrient reduction is largely dependent upon the dominant species. P reduction is more effective in controlling diazotrophic cyanobacteria than non-diazotrophic cyanobacteria.

Cyanobacterial blooms are intensifying global ecological hazards. The fine structure and dynamics of bloom community are critical to understanding bloom development but little understood. Here, the questions whether dominant bloomers have high diversity and whether dominant OTUs (operational taxonomical units) compete with one another were addressed. 16S rRNA gene amplicons from an annual bloom at five locations in Harsha Lake (Ohio, USA) showed cyanobacteria were the dominant phylum, and co-existing major bacterial phyla included Proteobacteria, Bacteroidetes, Actinoacteria, and Verrucomicrobia. On the genus level, the initial dominance by Dolichospermum in June yielded to Planktothrix in July, which were replaced by Microcystis and Cylindrospermopsis in August throughout the bloom. Based on the number of verified unique OTUs (a within-genus biodiversity metric), dominant genera tended to have high within-genus diversity. For example, Dolichospermum had 57 unique OTUs, Planktothrix had 36, Microcystis had 12, and Cylindrospermopsis had 4 unique OTUs. Interestingly, these different OTUs showed different dynamics and association with other OTUs. First, no between-OTU competitions were observed during the bloom cycle, and dominant OTUs were abundant throughout the bloom. Such biodiversity of OTUs and their dynamics were verified in Microcystis aeruginosa with two microcystin synthetase genes (mcyA and mcyG): the relative abundance of both genes varied during the bloom based on quantitative PCR. Two Dolichospermum circinale OTUs and one P. rubescens OTU were most abundant and persistently present throughout the entire bloom. Second, these OTUs differed in the OTUs they were associated with. Third, these OTUs tended to have different levels of association with the environmental factors, even they belonged to the same genera. These findings suggest the structure and dynamics of a cyanobacterial bloom community is complex, with only few OTUs dominating the bloom. Thus, high-resolution molecular characterization will be necessary to understand bloom development.