Studies On The Algicidal Characteristics And Mechanisms Of Algicidal Bacteria Against Microcystis Aeruginosa

With the rapid development of the society,the frequency and influence of harmful algal blooms,specifically harmful cyanobacteria algal blooms(Cyano HABs),have been increased year by year,and cause severe environmental problems,economic losses and threaten human health seriously.The development of efficient and economical techniques to prevent and manage the negative effects of Cyano HABs is in urgent need.Using microbes to control algal blooms has attracted great attention and been considered to be an effective strategy,because it is economical,environment-friendly and simple to be operated.In the present study,several actinomycetes and a bacterial strain with high algicidal activity against Microcystis aeruginosa were isolated from purple soil of Chongqing.Streptomyces sp.G3,G9 and Bacillus sp.AF-1,which showed the highest algicidal activity,were chosen to study the interaction between cyanobacteria and bacteria,algicidal characteristics and algicidal mechanisms.The main results are listed as follows:1.Isolation and identification of algicidal actinomycetes.In this part of study,10 strains of actinomycetes with high algicidal activity against Microcystis aeruginosa were isolated from purple soil.Among them,strains G3 and G9 showed the highest algicidal activity(Ae=84 %,87%,t=5 d).G3 and G9 were identified as Streptomyces sp.by the observation of their cell morphology and the analysis of 16 S r RNA gene sequences.2.Study on the algicidal charicteristics of Streptomyces sp.G3 and G9.G3 and G9 exhibited high algicidal activity against M.aeruginosa,and also significantly inhibited the growth of other cyanobacterial strains causing Cyano HABs(Anabaena flos-aquae,Nostoc sp.,Phormidium sp.)with algicidal ratio more than 60%(t=5 d),and led to the rupture of filamentous cyanobacteria.The algicidal activity of strain G3 and G9 was negatively correlated to the cell concentration and growth phase of M.aeruginosa;however,as culture time being prolonged they kill the algal cells finally.G3 and G9 killed cyanobacterial cells indirectly by releasing algicidal substances,and physical contact of cyanobacteria-bacteria was not necessary for release of the algicidal substance(s).3.Isolation and characterization of a multifunctional algicidal bacterial strain.A bacterial strain,designated as AF-1,exhibiting high antagonistic activity against F. graminearum,was isolated by plate confrontation test from purple soil.A large growth inhibition zone of F.graminearum appeared around the colony of AF-1,and obvious morphological changes of the fungal hyphae were observed.AF-1 also showed strong algicidal activity against M.aeruginosa(Ae=93%,t=5 d).It was identified as Bacillus sp.by 16 S r RNA gene sequenc analysis and morphology of cells and colony.The effective cell density threshold of Bacillus sp.AF-1 to kill the cyanobacterium was very low(1.56 × 103 cfu/m L),moreover,AF-1 could grow on the nutrients released by M.aeruginosa cells.Algicidal efficiency was related to the physiological state of M.aeruginosa,rather than its own physiological state.The algicidal activity of strain AF-1 was species-specific,it could selectively kill M.aeruginosa and had no effect on the growth of filamentous cyanobacteria and eukaryotic green algae.4.Studied on the algicidal materials and mechanisms of Bacillus sp.AF-1.AF-1 exhibited algicidal activity mainly by excreting algicides that were heat-tolerant and p H-resistant;Proteinase-K digestion did not affect the algicidal activity of AF-1 on M.aeruginosa,suggesting that the algicidal substances were not proteins or peptides.Hydrophilic substances excreted by strain AF-1 played a more important role in killing M.aeruginosa.The results of cell morphological observation and the intracellular ROS levels measurement of M.aeruginosa treated by cell-free supernantant of AF-1 suggest that AF-1 killed M.aeruginosa by inducing high levels of ROS in cyanobacterial cells,which led to oxidative stress of cyanovacterial cells,the damage of cell integrity.RT-q PCR analysis showed that AF-1 also caused DNA damage of the algal cells and showed impacts on the photosynthesis system and microcystins synthesis of M.aeruginosa.