Isolation And Preliminary Identification Of Algicidal Substances From Bacteria F5-2

Background: Numerous studies have been carried out to develop strategies for controlling HABs.These strategies can be summarized as physical methods(e.g.,clays and flocculants),chemical methods(e.g.,copper sulfate surfactants,and sodium hypochlorite),and biological methods(e.g.,algicidal bacteria,algicidal viruses,and plankton grazers).However,the use of physical and chemical methods in aquatic ecosystems is costly,inconvenient operation,limited effect,and some even may lead to the potentially dangerous.Biological methods have received particular interest in recent years because of their potential effectiveness,species specificity,and eco-friendliness,so they are the main development of algae control.The purpose and significance: On the one hand,under the premise of maintaining algicidal activity,the method is explored that effectively removed the total nitrogen and total organic carbon in the broth for creating conditions for the downstream processing and the practical application of fermentation products;on the other hand,pure algicidal substance is obtained and its chemical structure is preliminary identification to determine its chemical structure,that is the base of further study of its algicidal mechanism,improving the efficiency of fermentation and synthetic modification of more efficient algaecide.Methods and results: Our laboratory isolated an algicidal bacteria,strain F5-2,the supernatant has algicidal activity against Heterosigma akashiwo.We find that alga cells have swum to the bottom,then it become xanthic and be killed.Strain F5-2,that produced some extracellular products have algicidal activity against the Heterosigma akashiwo.In order to explore the nature and the structure of the substance,we optimized the carbon source of fermentation medium in order to improve the yield of the active substance algicidal firstly,and then we cruded the active substance algicidal through freeze-drying,extraction and stability tests,then separated by both column chromatography and the HPLC methods,and preliminary identified by Fourier transform infrared spectrometer and LC-MS analysis.The results show when the concentration of glutamic acid was 1% in the MSI medium,the cost decreases 87% and the algicidal effect increases more than five times than optimized medium;the active component has algcidal activity after freeze-drying;we can use ethanol as preliminary purification and the active substance is basically stable,the influence of the light,dissolved oxygen and pH is few;The stationary phase of column chromatography is 200~300 mesh silica through the static adsorption,and the mobile phase of column chromatography is ethanol through the desorption experiments;After the column chromatography conditions are determined,the column packing methods were explored,and the results show that wet packed column can achieve the separation and save the elution time;After algicidal activity and TOC testing,the TN and TOC of fine extracting were reduced 98.45% and 92.9% than fermentation liquid and the algicidal activity was not significantly lowered;The optimal separation contitions of HPLC: The chromatogram was detected at Inertsil Diol column,detection wavelength at310 nm,25 ?,water and methanol in a linear gradient elution:0~1min(85:15),1~10min(15:85),10~15min(15:85),15~16min(85:15),the flow is1mL/min(analyti-cal HPLC)or 2mL/min(semi-preparative HPLC),the algcidal substance that need collect is the 9.9min fraction;then mass production of active fractions were collected according to the HPLC separation conditions that determined;After the LC-MS detection,algicidal active substance molecular weight is about 271 Da,after the Fourier transform infrared spectrometer detection,we could know its structure does not contain aldehyde group and alkyne,may contain at least of Ar-and-CH2-,have C=N unsaturated bonds,and it may amine compounds;These results not only laid the foundation of preparation for pure algicidal substance of bacteria F5-2 and determining its structure using NMR,but also provided a reference for operating procedures for purifying of other allelopathic algicidal substances.