Lethal Biological Effects Of Hydroxyl Radical On Microcystis Aeruginosa

In view of the major national people's livelihood issues that the frequent occurrence of blooms in coastal waters has been threating the safety of drinking water.Studying a rapid,efficient and safe method of bloom algae inactivation has become an international hotspot.The key point of advanced oxidant technology is high efficient generation of hydroxyl radicals(·OH),which can rapidly inactivate harmful micro-organisms and degrade organic pollutants.Traditional free radical biology mainly focuses on the damage of endogenous·OH to biological macromolecules.However,the acute lethal mechanism of exogenous high concentration ·OH to unicellular algae has not been studied yet.In this study based on the new method of·OH efficient generation by high-pressure ionization discharge combined with water jet cavitation,to carry out the biological effects of hydroxyl radicals(·OH)inactivation for Microcystis aeruginosa,such as morphological analysis and DNA damage detection.The main achievements are as follows:(1)High concentration of oxygen active species(OAS)was produced by oxygen dissociation and ionization using atmospheric pressure ionization discharge.Then,OAS were injected into the algae liquid by jet-flow,which instantly generated·OH and attacked algae rapidly,and inactivated the algae in the transporting bloom water within 3 s.The relationship of "dose-effect" and "time-effect" for algae inactivation were studied.SYTOX Green fluorescence staining combined with microscopic counting method and flow cytometry method were used to accurately quantify the CT thresholds of·OH inactivation for Microcystis aeruginosa,Scenedesmus quadricauda and Synedra sp.The CT thresholds of·OH inactivation for Microcystis aeruginosa,Scenedesmus quadricauda and Synedra sp.were 0.069 mg min/L,0.069 mg min/L and 0.139 mg·min/L,respectively,which were 1/200 of ClO2 method.(2)Conventional chemicals such as ClO2 could easily lead to cell rupture during inactivation,causing overflow of intracellular contents and toxins,which are biological toxicity.Therefore,at lethal threshold and 2 times lethal threshold,the morphological changes of cells were analyzed by scanning and transmission electron microscopies,and the cell membrane integrity,photosynthetic structure destruction and DNA damage were determined at·OH lethal threshold;cell rupture at 2 times lethal threshold.With·OH treatment,the concentrations of dissolved organic carbon,protein and DNA in extracellular water increased by 0.3 and 0.8 times respectively,while with ClO2 treatment increased by 1.1 and 1.8 times,which proved that there was not a large number of intracellular material overflowed from·OH inactivated cells.Based on this,it was determined that the cells did not rupture during·OH inactivation,but the internal structure of the cells was damaged.(3)Fluorescence probe HPF was used to detect·OH in Microcystis aeruginosa cells.·OH content gradually increased with the increase of total reactive oxidant(TRO)concentration.When TRO was 1.37 mg/L,the·OH content increased to 2.5 times,and when TRO was 2.78 mg/L,the·OH content increased to 3 times,which proved the direct biological function of·OH.Using chlorophyll fluorescence parameters detection to proved that the photosynthetic potential and electron transport capacity of algae with·OH inactivation were completely lost,and the photosynthetic system was severely damaged by·OH.(4)Using agarose gel electrophoresis and DNA quantitative detection determined the DNA content of·OH inactivated cells,which was reduced by 0.12 times;using single cell gel electrophoresis detected the DNA fragments forming tails,whichconfirmed that·OH caused significant DNA breakage(P<0.001);using TdT-mediated dUTP nick-end labeling detected the fluorescence of 96.42%cells were enhanced,which proved that the phosphodiester linkages of DNA chain were seriously broken;using enzyme-linked immunosorbent assay(ELISA)detected the content of 8-hydroxy-2 deoxyguanosine increased to 1.43 times,which proved that·OH reacted with guanine of DNA.Therefore,it was the first time to reveal that·OH produced by atmospheric pressure ionization discharge caused irreparable DNA damage in cells,impeding protein synthesis and activating cell death/apoptosis signaling pathway,which was the main cause of cell death.In conclusion,this study uses atmospheric pressure ionization discharge combined with water jet cavitation to generate·OH efficiently,realizing algae·OH inactivation of algae in 3 s during transporting bloom water.It was revealed that the main cause of·OH inactivation was the destruction of photosynthetic system and the irreparable damage of DNA.Explored biological effects of·OH inactivation for Microcystis aeruginosa,which is of great significance to promote the development of free radical biology,algal physiology and algae control technology.