Role of extracellular polymeric substances in resistance to allelochemical stress on Microcystis aeruginsosa and its mechanism

Using allelochemicals to suppress cyanobacteria growth is a prospective method for its high efficiency and ecological safety.However,the suppression efficiency is affected inevitably by the extracellular polymeric substances(EPS)produced by cyanobacteria,and the knowledge about the roles of EPS in resistance to allelochemical stress is scarce.For the study,two typical anti-cyanobacterial allelochemicals were adopted to investigate the role of EPS in resistance to allelochemical stress on Microcystis aeruginosa.Results show that EPS was crucial in alleviating the toxicity of allelochemicals to algae,especially in stabilizing the metabolism and photosynthetic activity of algal cells.The aggregation rate of algal cells increased with the increase of EPS secretion,which alleviated the stress of allelopathy.Tryptophan proteins and humic acids in EPS provided a binding site for allelochemicals,and the EPS-allelochemicals complex were formed by chemical bonding.This study improved our comprehension of the role of EPS in algal inhibition by allelochemicals.

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。

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.

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.

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.

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 .

Effects of Iron and Phosphorus on Microcystis Physiological Reactions

Objective To observe the effects of iron and phosphorus on Microcystis physiological reactions. Methods The experimental conditions were chosen as the light dark cycles of 16 h 8 h, 12 h 12 h, and 8 h 16 h. The cell change of morphology and life history, cell number, cell color, and cell area of Microcystis were analyzed quantitatively. According to the resource competition and Monod equation, Microcystis kinetics of phosphorus and iron were also examined. Results The longer light time caused more special cell division, slower growth rate, and easier change of bigger cell area. The color of alga was changed from green to brown. Ks and μmax of phosphorus absorption were 0.0352 μmol·L^-1and 0.493 d^-1, respectively. Those of iron absorption were 0.00323 μmol.L^-1and 0.483 d^-1. Conclusion Microcystis bloom is more dominant than other algae.

Toxicity Evaluation of Freshwater Cyanobacterium Microcystis aeruginosa PCC 7806: Ⅱ Nephrotoxicity in Rats

Nephrotoxic potential of laboratory cultures of freshwater cyanobacterium (blue-green al ga) Microcystis aeruginosa PCC 7806 (Pasteur Institute) was assessed in male rats. The ani mals were injected intraperitoneally with 0. 5, 1. 0 and 2. 0 LD50 doses of lyophilized cell ex tract. Elevated plasma urea and creatinine levels were accompanied by decrease in protein and albumin levels, followed by hematuria, proteinuria and bilirubinuria. Also decrease in kidney lactate dehydrogenase and glutamic oxaloacetic transaminase indicated possible nephrotoxic po tential of the cyanobacteria. The extract also produced various hematological changes associat ed with stagnant type of hypoxia. High perfomance liquid chromatography of the culture I dentified the active principle (toxin) as Microcystin-LR

Algicidal effect of bacterial isolates of Pedobacter sp. against cyanobacterium Microcystis aeruginosa

The aim of this study was to isolate algicidal bacteria so as to control harmful cyanobacterium Microcystis aeruginosa （M. aeruginosa） blooms using biological methods. Nine bacterial strains were isolated to inhibit the growth ofM. aeruginosa, among which the Mail 1-5 bacterial strain exhibited remarkable algicidal activity against M. aeruginosa cells during the test. Based on the 16S rDNA analysis, the isolated Mail 1-5 was identified as Pedobacter sp. through morphology and homology research. The results of cocultivation of the cyanobacteria with MaI 11-5 algicidal isolates showed obvious algicidal activity against cyanobacterial cells. The algicidal effect of Mail 1-5 exceeded 50% after two days, exceeded 70% after four days, and reached 80% after seven days. The observation results with a scanning electron microscope showed that the cyanobacterial cells aggregated and produced mucous-like substances when cocultivated with the algicidal bacteria. The results indicated that the Mail 1-5 bacterial strain may possess a novel function for controlling harmful blooms and further studies will provide new insights into its role in water environment.

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.

Toxicity Evaluation of in vitro Cultures of Freshwater Cyanobacterium Microcystis aeruginosa:Ⅰ.Hepatotoxic and Histopathological Effects in Rats

Laboratory cultures of freshwater cyanobacterium (blue-green alga) Microcystis aeruginosa PCC 7806 was cvaluated for its hepatotoxic effects in rats. The lyophilized cell extract injected intraperitoneally at 1 and 2 LD50 (15.8 and 31.6 mg/kg, respectively) produced significant increase in liver-specific enzymes viz. plasma alkaline phosphatase,γ-glutamyl transferase, lactate dehydrogenase with a concomitant decrease in hepatic glutamic pyruvic transaminase. A corresponding increase in liver body weight index and histopathological changes in liver (degeneration of hepatocytes, congestion and hemorrhage etc.) are indicative of a dose and time dependent hepatotoxic nature of the algal extract

Integrated transcriptome and physiology analysis of Microcystis aeruginosa after exposure to copper sulfate

Copper sulfate(CuSO4)is widely used in controlling the Microcystis aeruginosa blooms.Many studies reported the toxicity mechanisms of Cu^2+to M.aeruginosa at the physiological level,but little is known about a transcriptomic basis of these mechanisms.In the present study,M.aeruginosa was treated by 0.5 mg/L Cu^2+(half of the 96-h EC50)for 72 h.The results show that CH2+content in M.aeruginosa increased after 72 h Cu^2+exposure,whereas the Fv/Fm chlorophyll fluorescence value and chlorophyll a content in M.aeruginosa sharply decreased.Reactive oxygen species concentration and activity of antioxidant enzymes(superoxide dismutase,catalase and peroxidase)were all increased.These physiological data confirmed toxicity of Cu^2+to m.aeruginosa.The RNA-seq analysis showed that 6646725 and 7880291 clean reads were obtained for the Cu-treated and control libraries,respectively.The 595 genes(252 downward trend and 343 upward trend)with the Gene Ontology(GO)annotations were divided into three main functional categories:cellular component,molecular function,and biological process.In the Cluster of Orthologous Groups(COG)annotation,418 differentially expressed genes with 25 functional definitions were obtained.Among them,‘replication,recombination and repair’,‘energy production and conversion’,and‘general function prediction only’were the largest three groups of transcripts.In the Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway analysis,genes involved in photosynthesis and oxidative phosphorylation were present at the highest percentages.In addition,the genes involved in photosynthesis and oxidative phosphorylation were identified,and then confirmed using real-time PCR.This study reported the first transcriptome of M.aeruginosa.Photosynthesis and oxidative phosphorylation were severely affected by Cu^2+toxicity,which may have contributed to cell death.These data provide the potential mechanism to explain the CuSO4 effect on the harmful M.aeruginosa.

Algae-lysing Effect of Algicidal Bacteria on Microcystis in Bloom Water

The Microcystis sample was selected from natural bloom water,then the algicidal effects of the bacterial strain fluid of the two algicidal bacteria,supernatant and bacterial liquid were studied. Moreover,the changes of microalgae colony and morphology in the process of algae-lysing were observed using neutral red staining and scanning electron microscope. It showed that AA06 bacteria had certain algicidal effect while AA10 bacteria had no effect. All the supernatants of the two bacterial strains had obvious algicidal effects,and the algicidal effects between the bacterial liquid and supernatant had no significant differences( P < 0. 05). The effects of algae-lysing mainly owed to the chemical substances secreted by algicidal bacteria. During the algae-lysing process,the algae colony did not disperse into individual cells. The segments of the dead cells still aggregated together.

Ecological damage of submerged macrophyte Myriophyllum spicatum by cell extracts from microcystin(MC)-and non-MC-producing cyanobacteria,Microcystis

To explore how decomposed Microcystis-dominant cyanobacterial blooms affect submerged macrophytes,the submerged plant Myriophyllum spicatum was exposed to cell extracts from microcystin(MC)-and non-MC-producing Microcystis strains in a laboratory experiment.Results showed that both Mcracystis cell extracts exerted obvious damages to plant biomass,photosynthesis,primary and secondary metabolism measures,and resistance of plant antioxidant systems,with MC-producing Microcystis having stronger effects due to the presence of MCs.Cyanotoxins other than MCs responsible for the negative effects from both strains needs further identification.The Shannon diversity and Chao1 indices of epiphytic and planktonic bacteria were decreased by the cell extracts from both Microcystis strains.However,epiphytic and planktonic bacterial communities responded differently to cell extracts at the genus level.The dominant genera of planktonic bacteria including Enterobacter,Pseudomonas,and Novosphingobium from phylum Proteobacteria,Chryseobacterium from phylum Bacteroidetes,and Microbacterium from Actinobacteriota in the treatments with cell extracts were previously reported to have strains with algicidal and MC-degrading capabilities.B acterial genes associated with energy production and conversion,amino acid transport and metabolism,and inorganic ion transport and metabolism,were more abundant in both treatments than the control for planktonic bacteria,but less abundant for epiphytic bacteria.We speculate that planktonic bacterial communities have the potential to use and degrade substances derived from Microcystis cell extracts,which may be beneficial for M.spicatum to alleviate damages from Microcystis.Further research is needed to verify the structure and function dynamics of epiphytic and planktonic bacteria in the interaction between cyanobacteria and submerged macrophytes.

Effects of nutrients on Microcystis growth more easily forming bloom

Different nutrient media experimentally were N, P and Fe-limited conditions and a serial of diluted BG11 media. The cell change of morphology and life history, cell number, cell color and cell area of Microcystis were analyzed quantitatively. First, the effects of nitrogen, phosphorus and iron depletion were distinctively different. Phosphorus and iron depletion caused more special division cells, slowly growth increasing, the easier change of bigger cell area. Second, the nitrogen and iron depletion could make the color of alga from green to brown. Finally, according to the resource competition and Monod equation, Microcystis kinetics of phosphorus and iron were also examined. K s and μ max of phosphorus absorption were 0.0352 μmol/L, 0.493 d -1 respectively; iron absorption: 0.00323 μmol/L, 0.483 d -1. In a word, some evidences of the Microcystis bloom dominance in certain nutrient conditions were indicated in the experiments. The dominances were determined as the reviving under the adverse circumstances through the special division, the various nitrogen resources, and the lower kinetics of phosphorus and iron than that of most of other algae. The conclusions provided the scientific basis for preventing and managing Microcystis bloom in freshwater.

Degradation characteristics of two Bacillus strains on the Microcystis aeruginosa

The degradation kinetics of strains P05 and P07 and the degradation effects of mixed strain on Microcystis aeruginosa were studied. The results showed that: (1) The degradation processes of strains P05 and P07 on Microcystis aeruginosa accorded with the first-order reaction model when the range of Chl-a concentration was from 0 to 1500 μg/L. (2) The initial bacterium densities had a strong influence on the degradation velocity. The greater the initial bacterium density was, the faster the degradation was. The degradation velocity constants of P05 were 0.1913, 0.2175 and 0.3092 respectively, when bacterium densities were 4.8×10 5, 4.8×10 6, 2.4×10 7 cells/ml. For strain P07, they were 0.1509, 0.1647 and 0.2708. The degradation velocity constant of strain P05 was higher than that of P07 when the bacterium density was under 4.8×10 5 cells/ml, but the constant increasing of P07 was quicker than that of P05. (3) The degradation effects of P05 and P07 strains did not antagonize. When the concentration of Chl-a was high, the degradation effects of mixed strain excelled that of any single strains. But with the decrease of the Chl-a concentration, this advantage was not clear. When the concentration was less than 180 μg/L, the degradation effects of mixed were consistent with that of strain P07.

Competitiveness of alga Microcystis aeruginosa co-cultivated with cyanobacterium Raphidiopsis raciborskii confirms its dominating position

Microcystis aeruginosa has always been regarded as the main culprit of cyanobacterial blooms in freshwater.However,in recent years,Raphidiopsis raciborskii has gradually replaced M.aeruginosa as the culprit of cyanobacterial blooms in some tropical and subtropical shallow lakes.To reveal which one plays a more dominant role,interactions between cylindrospermospin(CYN)-producing R.raciborskii and microcystins(MCs)-producing or non-MCs-producing M.aeruginosa strains were studied using bialgal cultures at different initial ratios of biomasses of the two species at 25℃.During the co-cultivation,the M.aeruginosa strains inhibited the growth and heterocyst formation of R.raciborskii filaments,and thus occupied a dominant position during the co-cultivation regardless of the initial biomass ratios in the cultures.In addition,the MCs-producing M aeruginosa strain contributed to a higher portion of the total biomass and exerted a stronger inhibitory effect on R.raciborskii compared with the non-MCs-producing strain.However,the growth of both MCs-producing and non-MCs-producing M.aeruginosa strains was stimulated by R.raciborskii in the co-cultures compared with M.aeruginosa monoculture,indicating that M.aeruginosa could outcompete R.raciborskii if given enough time,enabling it to develop into the dominant species even in very low initial concentration.To our best knowledge,this is the first report on the loss of heterocyst formation by a species of cyanobacteria that resulted from interactions between two different species of cyanobacteria.These findings indicate that it is difficult for R.raciborskii to replace the dominant position of M.aeruginosa under the experimental environmental condition,and the allelopathic effects of M.aeruginosa on R.raciborskii could significantly contribute to the success of M.aeruginosa.