Screening Of Microcystis-inhibiting Bacteria And Elucidating Its Inhibitory Effects And Mechianism

In recent years,harmful algal-blooms outbreak frequently with the increasing eutrophication,which have been causing great harm to aquatic ecosystem and polluting water environment.Microcystis aeruginosa is the dominant algal-bloom species,producing algal toxins and endangering the health of human beings.So exploring effective methods to control the harmful Microcystis has become the most crucial subject.Algal-inhibition bacteria is a high efficient,economic and eco-friendly method and has become a research hotspot with promising application prospects.Bacillus is widely distributed and non-toxic,which can produce many antibacterial substance.According to the existing research,we screened algaecidal Bacillus from the existing Bacillus and water sample from Taihu.And then,the active substances were isolated from the algaecidal bacterium.The inhibitory effect and mechanism were also explored in this work.The research contents and results are shown as follows:1.Screening of Bacillus with highly algal-inhibition effects.Strain B15 and 9-13,which have highly inhibitory effect on Microcystis,were screened by algal-inhibition experiments.The fermentation broth of B15 and 9-13 had a high inhibitory effect on M.aeruginosa with an initial concentration of 1.0×106 cells/mL.During 10 days' treatment by B15,the cell density of M.aeruginosa kept decreasing,and the inhibitory rate was above 90%.9-13 could inhibit the growth of M.aeruginosa in the first six days,but the algal cells began to duplicate afterwards and the cell density overnumbered the initial density on the 10 day.B15 inhibits the growth of M.aeruginosa by secreting active substances,while the inhibitory mode of 9-13 is undefined.2.The inhibitory characteristics and effect of B15 cell-free filtrate on M.aeruginosa.The sterile filtrate with the added amount of 0.3%,0.7%,1%and 2%had an inhibitory rate of 51.89%,79.89%,89.47%and 86.94%,respectively.There was no significant differencebetween the inhibitory activity of 48 h and 72 h fermentation,both of which were better than that of 24 h fermentation.The inhibitory rate of 2%sterile filtrate on M.aeruginosa at 1.0?20×106cells/mL was more than 75%after 6 days'treatment.The inhibitory rate of 1%sterile filtrate on M.aeruginosa at 1.0?10×106 cells/mL was more than 70%,but on terms of 20 X 106 cells/mL of M.aeruginosa,the inhibitory rate was only 48%.1%B15 filtrate had an algal-inhibitory rate of nearly 100%on the 10th day of treatment,and the number of viable cells was still decreasing on the 24th day,which suggested that B15 had a stable and lasting inhibitory effect.Furthermore,B15 could control several other cyanobacteria(such as Anabaena flos-aquae and Synechococcus)efficiently.It could also inhibit the growth of some green algae(such as Scenedesmus obliquus),the inhibitory rate of which was less than that of cyanobacteria.After 6 days' treatment,Fv/Fm and ETRmax decreased significantly,which suggested the inhibition of photosynthesis.Under the laser scanning confocal microscope(LSCM),many cells were found to remain at the end of cell division by forming 2-or 4-cell aggregates.The proportion of 2-and 4-cell after treatment for 6 d by 1%of fermentation filtrate was 62.23%and 14.95%,respectively,showing the cell division was severely blocked.The result of transmission electron microscope was in consistent with that of LSCM.And more cells in treatment groups showed seriously damaged.,with the phenomenon of plasmolysis,the rupture of the thylakoid and cytomembrane.Flow cytometry with PI?FDA and DCFH-DA fluorescence and chlorophyll-a to investigate cell viability of algal cells.The results showed that the proportion of normal algal cells.treatment by 1%and 2%fermentation filtrate was 28.1%and 22.4%,respectively,-stained with PI.With the increase of the amount of sterile filtrate,the proportipn.of strong FDA fluorescent cells was decreased,but ROS damage cells was increased,and 20.7%and 35.9%,respectively,treatment by 2%fermentation filtrate,showing the cell metabolic activity decreased and the ROS level increased.3.Purification of the active substances from B15 cell-free filtrate.The molecular weight of the main active substances was speculated to be less than 3 kDa by ultrafiltration.After treatment for 6 h and 24 h at 4,25 and 37? respectively,the inhibitory activity had no significant difference from the control,but at 50 and 70?,the inhibitory activity decreased significantly,which showed that the active substances couldn't tolerate high temperatures.In addition,after treated in the solution at pH2?12,the active substances remained good inhibitory activity.The fermentation filtrate was not sensitive to trypsin or protease K.The active substances couldn't be precipitated by HCl.They could be isolated by ion exchange chromatography,but the resulting fratction couldn't combine to the C18 reversed column.It is inferred that the active substances were non-protein,alkaline,hydrophilic molecules.More work should be performed to isolate and identify the algicidal compounds.4.Proteomic analysis of algicide-induced changes in M.aeruginosa cells.The quantitative proteome analysis method iTRAQ was applied to investigate the changes in proteomic expression of M.aeruginosa cells.Cells were treated by active fractions for 6 d,and the total proteins was compared to untreated cells.A total of 1459 distinct proteins were identified with more than two unique peptides.69 differentially expressed proteins were identified,of which 23 were up-regulated and 46 were down-regulated.Proteins involved in photosynthesis,carbon and nitrate assimilation were among the most down-regulated,which resulted in growth suppression.