Effects of aeration induced turbulence on colonial morphology and microcystin release of the bloom-forming cyanoabcterium Microcystis

Aeration induced turbulence was considered as an important measure to control the occurrence of cyanobacterial blooms in many lakes.Different aeration intensities were set for the culture of Microcystis aeruginosa based on the formation of Microcystis colony at high iron concentrations.The turbulent dissipation rate was calculated using a computational fluid dynamics(CFD)model to evaluate the intensity of turbulence.The effects of turbulence on the formation of Microcystis colony and the release of microcystin were analyzed.Results show that turbulence produced by aeration promoted the growth of Microcystis compared to that in stagnant water.Low intensity turbulence(4×10^(-8)-1×10^(-7) m2/s3)promoted the formation of Microcystis colonies,but high intensity turbulence(1.28×10^(-6)-1.8×10^(-5) m^(2)/s^(3))did not.The increase in the number of cells per colony was slower than that in total biomass,indicating that the low intensity turbulence induced colony formation via cell division,while the high level turbulence disaggregated colonies formed by both cell division and cell adhesion.Low aeration intensity induced more production of reactive oxygen species(ROS)and malondialdehyde(MDA)in the cells of Microcystis than those in high aeration intensity.In addition,the content of microcystin(MC)-LR in the cells was positively correlated with turbulence intensity,showing that turbulence affected not only the growth and aggregation of Microcystis colonies but also their toxin production.These findings provide a better understanding of the cyanobacterial bloom formation mechanisms and help to propose feasible methods to prevent the formation of Microcystis colonies in a natural environment.

Plant allelochemicals inhibit the growth and colony formation of Microcystis

Genus Microcystis is the most common dominant species of bloom-forming cyanobacteria.Growth and colony formation are critical processes for the occurrence of Microcystis blooms.To identify allelochemicals that can inhibit the growth and colony formation of Microcystis,Microcystis aeruginosa was cultivated and treated with various concentrations of gramine,catechol,berberine,and sanguinarine in laboratory.Results indicate that the growth,colony formation,and extracellular polysaccharides(EPS)content of Microcystis were significantly inhibited when treated with gramine,catechol,and berberine in appropriate concentrations at high iron level(6.8 mg/L).Microcystis growth was inhibited and colony formation and EPS content were promoted when treated with sanguinarine at high iron level.These findings suggest that cyanobacterial blooms can be controlled by regulating the input of gramine,catechol,and berberine.Furthermore,this study revealed that plant allelochemicals could affect the growth and morphology of algae in aquatic ecological restoration,which may be potentially important for improving water quality.

Detoxification strategy of Microcystis aeruginosa to the toxicity of Cd(Ⅱ):role of EPS in alleviating toxicity

Although many studies have found that cadmium(Cd)can be toxic to microalgae,only a few reports focused on the role of extracellular polymeric substances(EPS)in Cd(Ⅱ)detoxification.The biochemical and physiological endpoints of Microcystis aeruginosa,including the composition and functional groups of soluble EPS(SL-EPS),loosely bound EPS(LB-EPS),and tightly bound EPS(TB-EPS),were detected to elucidate the toxicity and detoxification mechanisms of Cd(Ⅱ)for cyanobacteria.Toxicological and physiological assays on M.aeruginosa showed that the 0.25-mg/L Cd(Ⅱ)resulted in a larger inhibition on growth and F_(v)/F_(m).Nevertheless,Cd(Ⅱ)significantly induced much higher contents of superoxide dismutase(SOD),intracellular microcystin LR(MC-LR),extracellular MC-LR,and EPS.Scanning electron microscopy with energy dispersive X-ray spectroscopy confirmed that Cd(Ⅱ)was absorbed into the EPS layer.Fourier transform infrared spectrum analysis revealed that the functional groups bound with Cd(Ⅱ)of algae biomass,SL-EPS,LB-EPS,and TB-EPS were somewhat different.The C=O/C=N groups ofδ-lactam or protein were their prominent functional groups,suggesting that amide or proteins in the EPS played a key role in the adsorption in Cd(Ⅱ).The concentration of 0.25 mg/L of Cd(Ⅱ)may change the chemical structure of EPS by altering the production of protein-like substances containing tryptophan.This study indicated that M.aeruginosa could detoxify Cd(Ⅱ)stress via induction of antioxidant capacity(higher SOD activity and MC synthesis),EPS production,and modification in chemical structure of EPS.

黑暗条件下不同氮源对铜绿微囊藻(Microcystis aeruginosa)生长和pH的影响

在黑暗条件下,利用不同形态的氮源(硝酸盐氮,氨氮,有机氮和硝酸盐氮,有机氮)培养蓝藻水华优势种铜绿微囊藻(Microcystis aeruginosa),分析其氮代谢和对水体pH的影响。研究结果表明,在不同氮源的培养液中铜绿微囊藻密度在最初的24h内出现波动,之后下降。培养液中pH值在试验最初的24h显著下降,之后趋于稳定,在硝态氮培养液中pH值下降最大,从8.18下降到7.19,其反硝化作用产生的NO2-浓度也最大。不同氮源培养液中总氮含量都有所下降,以混合氮源培养液中总氮减少量最大,说明化合态氮经过反硝化作用生成了氮气并溢出培养液,因此,在黑夜条件下藻华水体中存在反硝化作用。

Study on Induction of Microcystis aeruginosa Cell Apoptosis by Signal Molecule N-acetyl-homoserine Lactones(AHLs)

[Objective]The relationship between signal molecule N-acety-homoserine lactones（AHLs） and Microcystis aeruginosa cell apoptosis was studied.[Method]With M.aeruginosa as the test materials treated by 5 μmol/L N-acety-homoserine lactones（AHLs）,the morphology of cell apoptosis was observed through staining with DAPI.[Result]Microcystis aeruginosa cell apoptosis was induced by signal molecule N-acetyhomoserine lactones（AHLs） with the concentration of 1 μmol/L to inhibit the growth and proliferation of Microcystis aeruginosa.[Conclusion] The results provided the important scientific basis and new management ideas for the treatment of water bloom of Microcystis aeruginosa.

Study on the Phosphorus Metabolism of Microcystis aeruginosa and Adnascent Pseudomonas under Different pH Conditions

[Objective] The study aimed to discuss the effects of pH value on the growth metabolism of Microcystis aeruginosa and the phosphorus metabolism relationship with adnascent Pseudomonas.[Method] By the phosphorus uptake experiment of M.aeruginosa under different pH conditions(8.0-10.0) and the effect experiment on the phosphorus metabolism of M.aeruginosa and adnascent Pseudomonas under different pH conditions(7.0-9.0),the phosphorus uptake of M.aeruginosa in the short time and the growth curve of M.aeruginosa,the change of phosphorus concentration in the water,the change of total phosphorus content in M.aeruginosa in the longer time were measured.[Results] In the short time,pH value had the effects on the absorption phosphorus ability of M.aeruginosa.As pH value rose,the absorption phosphorus ability enhanced.During the longer time,the higher pH value was,the quicker the growth speed of M.aeruginosa was,and the better the growth situation was.M.aeruginosa had the ability of self regulation pH value and could use the phosphorus well in the water which was released from Pseudomonas.In the system of the algae,bacteria and water,the phosphorus in the bacteria played the role of phosphorus source which was released slowly.Though the phosphorus concentration was lower,it was favorable to the growth of algae.[Conclusions] pH value was the factor that affected the circle of the phosphorus element in the system of algae-bacteria-water.

盐京九号水稻种植水对铜绿微囊藻(Microcystis aeruginosa)抑制作用的研究

以培养盐京九号水稻幼苗20 d的种植水配置培养液,培养铜绿微囊藻(Microcystis aeruginqsa),追踪测定微囊藻生长量、藻细胞水溶性蛋白含量、超氧化物岐化酶(SOD)活性、叶绿素含量等相关生理生化指标的变化,研究水稻种植水对铜绿微囊藻生长的抑制作用及其机制.实验结果显示,藻细胞生长量和叶绿素含量显著降低;可溶性蛋白含量和SOD酶活性呈先升高后急剧下降的趋势.对水稻种植水抑藻物质作初步分离和鉴定,表明乙醚萃取液中有机酸和酚类组分抑藻效果明显.对有机酸组分进一步做GC/MS分析鉴定,可确定其中含有1,2-二甲基十三酸、棕榈酸和十八烷酸等组分.

Assessment of different mcy genes for detecting the toxic to non-toxic Microcystis ratio in the field by multiplex qPCR

Harmful cyanobacterial blooms, especially Microcystis blooms, occur worldwide and draw widespread attention. The dynamics of microcystin-producing Microcystis and competition between microcystin-producing Microcystis and non-microcystin-producing Microcystis are key to predicting and treating Microcystis blooms. Multiplex qPCR is a useful tool to assess such issues. In this study, we developed multiplex qPCR methods with newly-designed probes and primers for the microcystin-synthesis related genes mcyA and mcyE. We used seven toxic Microcystis strains and four non-toxic Microcystis strains to compare the differences in the ratios of toxic and non-toxic Microcystis in mixed cultures, which were calculated using abundances of the genes mcyA, mcyB, mcyD, mcy E and phycocyanin( PC). We also compared traditional cell counting and multiplex qPCR. Hierarchical clustering and principal component analysis indicated that mcyD was the most suitable mcy gene for quantification in laboratory experiments. mcyB abundances were always higher; we suggest that the amount of toxic Microcystis measured using mcyB might overestimate the actual percentages.

Study on the Inhibitory Effect of Sophora japonica Extracts against the Growth of Microcystis aeruginosa

[Objective] The research aimed to discuss the inhibitory effect of Sophora japonica extracts against the growth of Microcystis aeruginosa.[Method] The inhibitory effect of extracted liquid of Sophora japonica leaf against the growth of M.aeruginosa was measured.Moreover,the active component was studied and analyzed initially.[Result] The absolute alcohol extract of Sophora japonica leaf was separated by n-hexane,ethyl acetate,n-butanol and water phases in turn.The polar fractions were found being the majority （〉60%）.The non-polar fraction in n-nexane （about 25%） was found significantly inhibiting the growth of M.aeruginosa.The inhibition rates of fraction in n-hexane at the concentrations of 25 and 50 mg/L against M.aeruginosa in 7 d were higher than 75% and 90% respectively.In addition,chlorophyll a of M.aeruginosa was also destroyed in the presence of the hexane fraction.[Conclusion] The research provided the theoretical basis for preventing and controlling the water bloom of M.aeruginosa.

Seasonal variation of Microcystis in Lake Taihu and its relationships with environmental factors

In order to monitor the changes of Microcystis along with temporal and spatial variations, seasonal variation of Microcystis in Lake Taihu was investigated by 16S-23S rRNA internal transcribed spacer denaturing gradient gel electrophoresis （16S-23S rRNA-ITS DGGE） and microscopic evaluation. Samples were collected quarterly at four sites （River Mouth, Meiliang Bay, Cross Area, and Lake Center） from August 2006 to April 2007. Results showed that Microcystis dominated total phytoplankton abundance at the four sites in all seasons except winter. The average annual abundance of Microcystis was relatively high at River Mouth and Meiliang Bay, reaching 81.22×10^6 and 61.32×10^6 cells/L, respectively. For temporal variations, Shannon-Wiener diversity index （H＇） according to DGGE profile revealed the richness of Microcystis in summer （H＇ = 1.375±0.034） and winter （H＇ = 1.650 ±0.032） was lower than that in spring （H＇ = 2.078 ±0.031） and autumn （H＇ = 2.365 ±0.032） （P 〈 0.05）. While for spatial variations, the richness of Microcystis at River Mouth （H＇ = 2.015± 0.074） was higher than at other sites during four seasons （P 〈 0.01）. Very few differences of Microcystis diversity in the same season were observed among the other three sites （P 〉 0.05）. Canonical correspondence analysis （CCA） was performed to elucidate the relationships between Microcystis operational taxonomic units （OTUs） composition and the environmental factors. Results of CCA revealed that temperature was strongly positively correlated with the first axis （r = 0.963）, while TSS was negative correlated with the second axis （r = -0.716）. Phylogenetic tree based on the sequencing results of target bands on DGGE gel indicated that samples collected in summer and winter constituted two separated clusters.

Decomposition and phosphorus release from four different size fractions of Microcystis spp.taken from Lake Taihu,China

Decomposition of Microcystis is accompanied by the release of phosphorus,during bacteria play an important role.A series of experiments were undertaken to evaluate the effect of bacteria on the decomposition of Microcystis taken from Lake Taihu,China,a lake that is suffering from dense Microcystis blooms.The 16 experiments involved four size fractions of colonial Microcystis with or without the addition of lake sediment and Gram-negative bacterial inhibitor NAN_3.The highest decomposition rates were recorde...

Isolation and Characterization of an Algicidal Bacterium Indigenous to Lake Taihu with a Red Pigment able to Lyse Microcystis aeruginosa

Objective To isolate and characterize indigenous algicidal bacteria and their algae-lysing compounds active against Microcystis aeruginosa, strains TH1, TH2, and FACHB 905. Methods The bacteria were identified using the Biolog automated microbial identification system and 16S rDNA sequence analysis. The algae-lysing compounds were isolated and purified by silica gel column chromatography and reverse-phase high performance liquid chromatography. Their structures were confirmed by Nuclear Magnetic Resonance （NMR） and Fourier Transform infrared （FT-IR） spectroscopy. Algae-lysing activity was observed using microscopy. Results The algae-lysing bacterium LTH-2 isolated from Lake Taihu was identified as Serratia marcescens. Strain LTH-2 secreted a red pigment identified as prodigiosin （C20H25N30）, which showed strong lytic activity with algal strains M. aeruginoso TH1, TH2, and FACHB 905 in a concentration-dependent manner. The 50% inhibitory concentration （ICs0） of prodigiosin with the algal strains was 4.8 （±0.4）×10^-2 μg/mL, 8.9 （±1.1）×10^-2μg/mL, and 1.7 （±0.1）×10^-1 μg/mL in 24 h, respectively. Conclusion The bacterium LTH-2 and its pigment related to damage of cell membranes. The bacterium for regulating blooms of harmful M. aeruginosa. had strong Microcystis-lysing activity probably LTH-2 and its red pigment are potentially useful

Phylogenetic diversity and specificity of bacteria associated with Microcystis aeruginosa and other cyanobacteria

Interactions between bacteria and cyanobacteria have been suggested to have a potential to influence harmful algal bloom dynamics; however, little information on these interactions has been reported. In this study, the bacterial communities associated with five strains of Microcystis aeruginosa, three species of other Microcystis spp., and four representative species of non-Microcystis cyanobacteria were compared. Bacterial 16S rDNA fragments were amplified and separated by denaturing gradient gel electrophoresis （DGGE） followed by DNA sequence analysis. The similarities among bacterial communities associated with these cyanobacteria were compared to the digitized DGGE profiles using the cluster analyses. The bacterial community structure of all cyanobacterial cultures differed. Cluster analysis showed that the similarity values among M. aeruginosa cultures were higher than those of other cyanobacterial cultures. Sequence analysis of DGGE fragments indicated the presence of bacteria including, Alphaproteobacteria, Betaproteobacteria, Gammaproteobacteria, Bacteroidetes and Actinobacteria in the cyanobacterial cultures. Members of the Sphingomonadales were the prevalent group among the Microcystis-associated bacteria. The results provided further evidence for species-specific associations between cyanoabcteria and heterotrophic bacteria, which are useful for understanding interactions between Microcystis and their associated bacteria.

Cultivation and Characterization of the MaMV-DC Cyanophage that Infects Bloom-forming Cyanobacterium Microcystis aeruginosa

The MaMV-DC cyanophage,which infects the bloom-forming cyanobacterium Microcystis aeruginosa,was isolated from Lake Dianchi,Kunming,China.Twenty-one cyanobacterial strains were used to detect the host range of MaMV-DC.Microcystic aeruginosa FACHB-524 and plaque purification were used to isolate individual cyanophages,and culturing MaMV-DC with cyanobacteria allowed us to prepare purified cyanophages for further analysis.Electron microscopy demonstrated that the negatively stained viral particles are tadpole-shaped with an icosahedral head approximately 70 nm in diameter and a contractile tail approximately 160 nm in length.Using one-step growth experiments,the latent period and burst size of MaMV-DC were estimated to be 24–48 hours and approximately 80infectious units per cell,respectively.Restriction endonuclease digestion and agarose gel electrophoresis were performed using purified MaMV-DC genomic DNA,and the genome size was estimated to be approximately 160 kb.Sodium dodecyl sulfate polyacrylamide gel electrophoresis(SDS-PAGE)analysis revealed four major structural proteins.These results support the growing interest in using freshwater cyanophages to control bloom-forming cyanobacterium.

Growth and phosphate uptake kinetics of Microcystis aeruginosa under various environmental conditions

Growth and uptake of exogenous phosphate by Microcystis aeruginosa in batch culture under different temperature, photoperiod, and turbulence were studied by the method of phosphate isotope tracer. Relatively high temperature, long photoperiod and strong turbulence increased the cell density of M. aeruginosa in these batch cultures. The initial rapid uptake of phosphate by M. aeruginosa was independent of the temperature, photoperiod, and turbulence. Similarly, maximum exogenous phosphate uptake was not related to these environmental factors. However, elevated temperature and turbulence shortened the time, required to obtain maximum P accumulation. The growth of M. aeruginosa could alleviate the phosphorous leakage. Total amounts of exogenous phosphate uptake to M. aeruginosa and the phosphorus leakage of M. aeruginosa were significantly influenced by the growth state of M. aeruginosa closely correlated with the environmental factors. The maximum volume of exogenous phosphate uptake to M. aeruginosa was 46% of added exogenous phosphate in water with 16 hours of photoperiod. Thus, total amounts of exogenous phosphate uptake to M. aeruginosa were more strongly affected by the photoperiod length than temperature and turbulence.

Effects of rice straw on the cell viability, photosynthesis, and growth of Microcystis aeruginosa

Rice straw is supposed to be an environment-friendly biomaterial for inhibiting the growth of harmful blooms of the cyanobacterium Microcystis aeruginosa. However, its potential mechanism is not well known. To explore this mechanism, the growth, cell viability(esterase activity, membrane potential, and membrane integrity), photosynthesis, and cell size of M. aeruginosa were determined using fl ow cytometry and Phyto-PAM after exposure to rice straw extracts(RSE). The results show that doses from 2.0 to 10.0 g/L of RSE effi ciently inhibited the alga for 15 days, while the physiologic and morphologic responses of the cyanobacteria were time-dependent. RSE interfered with the cell membrane potential, cell size, and in vivo chlorophyll- a fl uorescence on the fi rst day. After 7 days of exposure, RSE was transported into the cytosol, which disrupted enzyme activity and photosynthesis. The cyanobacteria then started to repair its physiology(enzyme activity, photosynthesis) and remained viable, suggesting that rice straw act as an algistatic agent.

Effect of fluid motion on colony formation in Microcystis aeruginosa

Microcystis aeruginosa, generally occurring in large colonies under natural conditions, mainly exists as single cells in laboratory cultures. The mechanisms involved in colony formation in Microcystis aeruginosa and their roles in algal blooms remain unknown. In this study, based on previous research findings that fluid motion may stimulate the colony formation in green algae, cul^are experiments were conducted under axenic conditions in a circular water chamber where the flow rate, temperature, light, and nutrients were controlled. The number of cells of Microcystis aeruginosa, the number of cells per colony, and the colonial characteristics in various growth phases were observed and measured. The results indicated that the colony formation in Microcystis aeruginosa, which was not observed under stagnant conditions, was evident when there was fluid motion, with the number of cells per largest colony reaching 120 and the proportion of the number of cells in colonial form to the total number of cells and the mean number of cells per colony reaching their peak values at a flow rate of 35 crn/s. Based on the analysis of colony formation process, fluid motion stimulates the colony formation in Microcystis aeruginosa in the lag growth phase, while flushes and disaggregates the colonies in the exponential growth phase. The stimulation effect in the lag growth phase may be attributable to the involvement of fluid motion in a series of physiological processes, including the uptake of trace elements and the synthesis and secretion of polysaccharides. In addition, the experimental groups exhibiting typical colonial characteristics in the lag growth phase were found to have higher cell biomass in the later phase.

Survival,recovery and microcystin release of Microcystis aeruginosa in cold or dark condition

Microcystis often dominates phytoplankton in eutrophic lakes and must survive a long period of cold or dark conditions. However, the survival strategies of Microcystis to withstand cold or dark stress are less well known. In this study, we conducted experiments on the responses of two toxic Microcystis aeruginosa strains （FACHB-905 and FACHB-915） and their microcystin release in conditions of low temperature （15℃ or 4℃, with illumination） or darkness, and subsequent recovery in standard conditions （25℃ with illumination）. On exposure to 15℃, a small decrease in cell viability was observed, but the cell number increased gradually, suggesting that M. aeruginosa FACHB-905 and FACHB-915 cells seem in general tolerant in 15℃. Interestingly, our results show that a higher carotenoid content and microcystin release potentially enhance the fitness of surviving cells at 15℃. M. aeruginosa cells exposed to lower temperature light stress （4℃） did not completely lose viability and retained the ability to reinitiate growth. In darkness, the maximum quantum yield （Fv/Fm） and the maximum electron transport rate （ETRmax） values and cell viability of M. aeruginosa cells gradually decreased with time. During the recovery period, the photosynthetic efficiency of M. aeruginosa reverted to the normal level. Additionally, M. aeruginosa FACHB-905 and FACHB-915 exposed to low temperature had increased caspase-3-1ike activity and DNA fragmentation, which suggests the occurrence of a type of cell death in M. aeruginosa cells under cold stress similar to programmed cell death. Overall, our findings could confer certain advantages on the Microcystis for surviving cold or dark conditions encountered in the annual cycle, and help explain its repeated occurrence in water blooms in large and shallow lakes.

Differentially Expressed Proteome of Microcystis under Short-time Ultrasonic Stress

This study was conducted to explore the regulation mechanism for key protein expression. The Microcystis treated by short-time ultrasonic wave was select-ed to analyze the total protein based on 2-DE. The results showed that there were 71 up-regulated protein spots, 56 down-regulated protein spots, 54 new protein spots and 21 protein spots disappeared under short-time ultrasonic stress. Eight dif-ferential proteins were chosen for further MALDI-TOFTOF/MS analysis, and the re-sults showed that 2 unknown proteins and 6 functional proteins were detected. These proteins were relevant to some physiological processes, such as antioxidation and anti-inflammatory process, phosphate synthesis and electron transfer, which is beneficial to the metabolic balance and self-protection under short-time ultrasonic stress.

Influence of phosphorus on Microcystis growth and the changes of other environmental factors

The growth processes of Microcystis aeruginosa （FACHB-41） in simulated Taihu Lake water with different phosphorus concentrations were investigated using laboratory microcosms. The algal biomass increased with the increase of phosphorus concentration when it was lower than 0.445 mg/L, while the dissolved oxygen （DO） and pH increased, dissolved inorganic nitrogen （DIN） and light intensity underwater（I） decreased. Responding to the changes of the “environmental factors”, the cellular carbohydrate and its ratio to cellular protein decreased generally as phosphorus increased. However, when phosphorus concentration was higher than l. 645 mg/L, the biomass, the “environmental factors”, the cellular carbohydrate and its ratio to cellular protein did not change likewise. Since the environmental factors and the physiological and biochemical responses are important factors, the change of environmental factors and cell physiology and biochemistry induced by phosphorus may become the key factors that steer the growth and dominance of Microcystis under certain conditions. To sum up, phosphorus not only stimulate the growth of Microcystis directly by supplying nutrient element, but also has complex interactions with other “environmental factors” and play important roles in the growth processes of Microcystis .