| Recently, eutrophication and harmful algae blooms (HABs) are the major environmental problems in China. Therefore, seeking effective methods to control the HABs has always been of great interest. The UV/H2O2 involves the production of reactive oxidative species, especially the hydroxyl radicals (·OH) which are able to effectively damage microorganisms and organic substances. Therefore, UV/H2O2 has broad application prospect for controlling HABs. In this study, it was systematically studied that the damage mechanism and effect of UV/H2O2 on Microcystis aeruginosa, the concentration variation of extracellular and intracellular microcystins cause by damaged algae, the degradation kinetics and mechanism of MCLR by UV/H2O2 treatment. The main contents and achievements of this study are as follows.1. Research of the inhibiton mechanism and growth inhibitioin effect of UV/H2O2 on Microcystis aeruginosaThe HPLC detection conditions for·OH through salicylic acid trapping were established: the mobile phrase was 20% methol and 80% water, in which 0.03M acetic acid and 0.03M citric acid was included; the flowrate was 1mL/min. Meanwhile, the produced·OH in UV/H2O2 system participated in oxidative damage of alga. Base on the gene transcription level of alga,MDA content and the SOD activity, the damage mechanism of UV/H2O2 to alga could be as follows:alga cell membrane was oxidative damaged through lipid peroxidation reaction and then the DNA and protein molecular was damaged and photosynthesis activity was inhibited. The transcription expression of microcystin synthesis related gene was also inhibited under UV/H2O2 treatment and the synthesis content of microcystin could be reduced by UV/H2O2.In this study, Microcystis aeruginosa was respectively treated with H2O2, UV254 and UV/H2O2, and then continued to culture for 10 days. It was found that the inhibition effect of H2O2 treatment and UV254 irradiation treatment on algae were both enhanced with the corresponding increase of dosage.UV/H2O2 had a synergic inhibition effect on the growth of algae. Moreover, it was indicated that the combined treatment of 0.2 mM H2O2 and 38 mJ/cm2 UV254 irradiation had the best algal inhibition activity in this study. The analysis with scanning electron microscope (SEM) showed that UV/H2O2 were able to damage the morphology of Microcystis aeruginosa. After algae was treated by UV254 irradiation (38 mJ/cm2) combined with H2O2 (0.2 mM) almost all of the algae became sag deformation on the 10th day of the culture period, with cell wall lysis and cell cytoplasm release.2. Research of the concentration variation of extracellular and intracellular microcystins of Microcystis aeruginosa treated by UV/H2O2Using HPLC and ESI-MS/MS analysis, the microcystins (MCs) produced by Microcystis aeruginosa 905 were determined. The result indicated that besides the production of MCLR, Microcystis aeruginosa 905 also has a demethylated MCLR (Demethyl MCLR), which can be assigned to [D-Asp3]mcyst-LR, [Dha7]mcyst-LR, [D-Asp3, (E)-Dhb7]mcyst-LR or [D-Asp3, (Z)-Dhb7]mcyst-LR.The extraction procedure, employed in solid-phase extraction on intracellular microcystin extraction, was studied and optimized. The optimized extraction procedure of MCLR was as follows. First, algae slurry was collected through centrifugation, and w (methanol)=75% was added as extraction solvent. Then the mixed liquid was treated with freeze-thawing for 3 times followed by ultrasonication for 0.5 h, and the supernatant was collected for solid phase extraction (SPE). C18 cartridge was eluted with w (methanol)=10% and then washed with w (methanol) =80%+w (TFA)=0.05%.ELISA kit which shows very good cross-reactivity with MCLR and Demethyl MCLR was chosen for detecting MCs. It was indicated that UV/H2O2 could effectively degrade the extracellular MCs and decrease the yield of MCs by inhibiting the growth of algae. After being treated with 0.2 mM H2O2 combined with 38 mJ/cm2 UV254 irradiation, the algae were inhibited and its intracellular MCs released gradually into water. The concentration of extracellular MCs increased gradually, while the sum of the MCs almost kept constant.3. Research of the effect of operation parameters on the degradation of MCLR and degradation kineticsIn this study, the degradation of MCLR in aqueous solutions by UV254,H2O2 and combined UV/H2O2 processes was investigated. The result was as follows. H2O2 had little effect on degrading MCLR while UV254 direct photolysis and UV/H2O2 could both degrade MCLR effectively. On the condition of pH 8.0,1738μW/cm2 UV254 and 5 mM H2O2, at 40 min, the MCLR degradation ratio was 8.4%±2.1% by H2O2 treatment,73.6%±3.4% by UV254 treatment and 98%±4.8% by UV/H2O2 treatment. The degradation process by UV254, H2O2 and combined UV/H2O2 treatment could be well fitted with pseudo-first-order kinetic, respectively. The three pseudo-first-order kinetic constant (k) was kH2O2(0.0021 min-1),kUV(0.045 min-1) and kUV/H2O2(0.082 min-1) respectively.kUV/H2O2 was higher than the sum of kH2O2 Plus KUV, suggesting a synergetic effect between UV254 radiation and H2O2 oxidation. The effect of initial concentrations of H2O2, UV254 irradiation intensity, initial concentrations of MCLR and solution pH values on the degradation efficiency of MCLR was studies respectively. The results showed that H2O2 and UV254 indeed facilitated MCLR degradation in a dose and intensity dependent manner respectively. The relationship between pseudo-first-order kinetic constant (k) and H2O2 concentration could be expressed as follows: k=0.007×[H2O2]+0.0446, (R2=0.9918). The reaction rate constant of MCLR degradation was found to be negatively correlated with the initial concentration of MCLR as follows: k=-0.0056×[MCLR]0+0.1771(R2=0.8528). The optimum range of pH was found to be between 7 and 8.In addition, on the basis of nitrobenzene (NB) as competition reference compound, the competition kinetic model for the degradation of MCLR by UV/H2O2 was developed using the pseudo-first-order equation and steady-state approximation. With the help of the competition kinetic model, the second-order rate constant of the reaction between MCLR and·OH was determined to be 2.8 (±0.21)×1010 (M-1s-1).4. Research of intermediates and pathways of MCLR degradation by UV/H2O2The degradation products of MCLR were studied in detail by employing liquid chromatography-mass spectrometry (LC-MS) analysis and 9 intermediates products were found. They are as follows:(M+H)+at m/z 1029.5, (M+H)+at m/z 1011.5, (M+H)+at m/z 795.4, (M+H)+at m/z 835.4, (M+H)+at m/z 811.3, (M+H)+at m/z 1027.5, (M+H)+at m/z 1009.5, (M+H)+at m/z 965.5 and (M+H)+at m/z 1045.5 respectively.The main process of MCLR degradation by UV/H2O2 treatment involved·OH attack, oxidation and UV254 direct photolysis. The main sites of MCLR molecule attacked by·OH were the conjugated diene bond, benzene ring and methoxy group of the Adda side chain.Based on the molecular weight of the products and the reaction mechanism between·OH and pepetides or protein, three main degradation pathways were proposed as follows:1)·OH attacked on the conjugated diene bond of Adda side chain through electrophilic addition reaction and produced dihydroxylated-MCLR, and then the hydroxylated C4-C5 or C6-C7 bond of Adda was cleaved through further oxidation to form aldehyde or ketone peptide residues which were subsequently oxidized into the corresponding carboxylic acids; 2)·OH attacked on the benzene ring and formed benzene hydroxylation and/or benzene dihydroxylation through electrophilic substitution reaction, followed by further oxidation to form aldehyde or ketone peptide residues. 3)·OH attack on the methoxy group of the Adda side chain through hydrogen abstraction reaction to form formic acid-(MCLR), and then experienced complete removal of the methoxy group. The pathway 1 and pathway 2 probably accounted for over 90% of total degradation reactions. Meanwhile, on the basis of previous research, it was inferred that the toxicity of intermediates products almost disappeared.
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