A Study Of The Isolation And Identification,Algicidal Characteristics And Mechanism Of An Algicidal Bacteria Of Enterobacter

In recent years,the acceleration of artificial eutrophication and global warming have intensified the spread and outbreak of cyanobacteria blooms,posing serious threats to domestic and recreational water and human health.It is extremely urgent to find efficient and eco-friendly cyanobacteria control methods.At present,microbial control technology,especially algicidal bacteria,has proven to be the most promising method.Microcystis and Oscillatoria are the dominant algae species in cyanobacteria blooms.Among them,Microcystis has the highest incidence,the widest spread,the longest duration,and the greatest harm.Oscillatoria is notorious for secreting typical odor compounds and algal toxins.In recent years,it has received widespread attention due to frequent outbreaks in aquaculture water bodies.However,the current research on the microbial lysis of Oscillatoria is scarce and the algicidal mechanism on Oscillatoria remains unclear.In this study,a highly effective algicidal strain was isolated from soil samples collected from Liuhua Lake Park in Guangzhou,China.Taking Oscillatoria and Microcystis aeruginosa as algae control objects(mainly Oscillatoria),this study explored the algicidal properties and mechanism of Enterobacter sp.EA-1 and conducted a preliminary study on the properties of algicidal substances.The main findings were as follows:(1)The 16S r DNA sequence analysis and Gram staining results showed that the strain EA-1 belongs to the genus Enterobacter.The 16S r DNA sequence of the strain has been submitted to the Gen Bank database with the accession number JX983654.(2)EA-1 in the lag phase,logarithmic phase,stable phase and decay phase all showed significant algicidal activity and the EA-1 secreted algicidal substances mainly in the logarithmic phase.When the Chl-a content in the initial system of Oscillatoria was 1.43 mg/L,5%dosage can significantly inhibit the growth of Oscillatoria,and 10%dosage(cell density=2.3×10⁸ CFU/m L)can completely lyse Oscillatoria within 2 d.When the Chl-a content of the initial system of Microcystis aeruginosa was 1.52 mg/L,5%dosage can completely lyse Microcystis aeruginosa.Microcystis aeruginosa required a lower algicidal threshold density than Oscillatoria.The algicidal activity of EA-1 on Oscillatoria at different growth phases was sorted from high to low:lag phase>logarithmic phase>stable phase>decay phase.Chl-a content(algae density)is the main influencing factor of algicidal activity.When the Chl-a content was less than 1.74 mg/L,10%dosage can achieve complete algae lysis in 3 d.When the Chl-a content was less than 2.62 mg/L,10%dosage can basically achieve complete algae lysis in 6 d.(3)The effect of light on the algicidal activity depended on the effect of light on the growth of Oscillatoria.Under the conditions of full light,light-dark cycle and total darkness,the algicidal activity was not significantly different and highest under total dark conditions.The dark conditions were not conducive to the growth of Oscillatoria and obtained the highest algicidal activity.Temperature and p H affected the algicidal activity by inhibiting the growth of Oscillatoria and the physiological activity of EA-1.When the temperature was 25 and 35?,EA-1 behaved significant algicidal activity on Oscillatoria,and when the temperature was18?,the algicidal activity decreased.At p H 6 and 8,EA-1 behaved significant algicidal activity on Oscillatoria,while the growth of Oscillatoria and the algicidal activity of EA-1were significantly inhibited at p H 4.The p H of 4 was not conducive to the growth of Oscillatoria and the physiological activity of EA-1 and obtained the lowest algicidal activity.(4)EA-1 showed high-efficiency algicidal activity against harmful Oscillatoria and Microcystis,but behaved poor algicidal activity against beneficial algae Chlorella and Navicula.EA-1 lysed Oscillatoria and Microcystis aeruginosa cells mainly by secreting extracellular algicidal substances.The algicidal substance secreted by EA-1 retained great activity at the temperature of-20-60?and the p H of 4-10,which was conducive to the application of algicidal substances in actual water bodies.(5)Under the stress of algicidal substances secreted by EA-1,the lysis of Oscillatoria 528began with the rupture of the transverse cell wall between cells or cell and cell chains,followed by the destruction of photosynthetic lamella and nucleoid region,and finally the outflow of intracellular contents.The results of DAPI staining and the transcription abundance of the DNA repair gene rec A further revealed the destruction mechanism of the nucleoid region of Oscillatoria.The destruction of Microcystis aeruginosa 905 began with the separation of the colloidal layer from the cell wall,followed by the damage of the photosynthetic lamella,the degradation of DNA nuclear material and nutrient particles,and finally the complete destruction of the internal structure.Only the empty cell membrane of the algae was observed.(6)Under the stress of algicidal substances secreted by EA-1,excessive ROS was accumulated in the algae cells,the synthesis and degradation of protein and carbohydrates in Oscillatoria cells were significantly affected.In addition,ROS attacked the membrane system,resulting in severe lipid peroxidation damage of Oscillatoria.At the same time,the defense of Oscillatoria was stimulated and continuously strengthened,and the activities of T-AOC,SOD,CAT,POD and APX antioxidant enzymes increased significantly to eliminate ROS avoiding oxidative damage.As the antioxidant enzyme activity was insufficient to resist oxidative stress,the cell defense system of Oscillatoria was severely damaged,and the activities of T-AOC,SOD,CAT,POD and APX decreased,and finally,the algae cells died.(7)Under the stress of algicidal substances secreted by EA-1,the Chl-a content decreased significantly,the Chl-a structure was destroyed,the Chl-a fluorescence was significantly reduced,meanwhile,the maximum photochemical efficiency Fv/Fm value continued to decline,and the photosynthetic efficiency and photosynthetic capacity of the algal cells decreased significantly.In addition,the transcription abundance of the gene rbc L encoding the large subunit of CO₂ fixed carboxylase was significantly down-regulated,resulting in a decrease in the ability of algal cells to fix CO₂ and the dark response was blocked;The transcription abundance of the gene psb A1 encoding D1 protein was significantly down-regulated,and the transcription abundance of gene fts H regulating the turnover of D1protein was significantly up-regulated first and then down-regulated.The synthesis and turnover of D1 protein in Oscillatoria were inhibited,resulting in the destruction of the PSII response center structure and function.The above results all indicated that the photosynthetic system of the algae cells was severely damaged,which promoted the death of the algae cells.