UV/H_2O_2对铜绿微囊藻抑制特性及其对微囊藻毒素降解机理研究
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摘要
水体富营养化及其引发的有害藻类水华是我国当前主要面临的环境问题之一‘。有关有害藻类污染治理的方法和技术的研究一直以来是水污染控制领域的热点。在UV/H2O2光氧化体系中,H202光解产生强氧化剂·OH,其具有高效杀灭微生物、快速降解有机化合物的特点,因而UV/H2O2在有害水华藻类防治领域的运用潜力巨大。本论文系统地研究了UV/H2O2对水华铜绿微囊藻抑活机理及效应、UV/H2O2处理后藻胞内／外毒素变化、UV/H2O2对微囊藻毒素的降解动力学和降解机理。
本论文的主要研究如下：
1. UV/H2O2抑藻机理及抑藻效应研究
建立了水杨酸捕获法测定·OH的HPLC检测条件：流动相为20%甲醇+80%水(含0.03’M乙酸和0.03 M柠檬酸),流速为1mL/min;同时证实了UV/H2O2体系中生成的·OH参与了藻细胞的氧化损伤过程。基于藻细胞基因转录水平和生理生化指标分析得出结论,UV/H2O2对藻细胞的损伤机理主要是通过·OH氧化损伤藻细胞膜,使其发生脂质过氧化反应,进而损伤藻细胞DNA及蛋白质等生物大分子和光合作用系统。藻毒素合成相关基因的转录表达量受到UV/H2O2处理的抑制,因而UV/H2O2抑制藻生长的同时可抑制胞内毒素的合成量,从而减少释放至水体的毒素含量。
本论文同时研究了铜绿微囊藻分别经UV254、H2O2及UV/H2O2处理后继续培养过程中的生长情况,研究结果表明：H202和UV254对铜绿微囊藻生长抑制效应均随作用剂量的增加而增强；UV/H2O2对铜绿微囊藻的抑制存在明显的协同效应,在实验室条件下,0.2 mM H2O2结合38 mJ/cm2 UV254辐照剂量是抑藻效应最强的组合；结合扫描电镜分析表明,UV/H2O2处理对铜绿微囊藻细胞形态结构造成破坏。藻液经0.2 mM H2O2结合38 mJ/cm2 UV254辐照剂量联合处理后第10d,几乎所有藻细胞凹陷变形,很多藻细胞细胞壁破裂,细胞质外溢而死亡。
2. UV/H2O2抑藻过程中铜绿微囊藻胞内／外毒素变化研究
本论文结合HPLC和ESI-MS/MS技术对铜绿微囊藻905胞内毒素鉴定结果表明,胞内毒素除MCLR以外,还存在去甲基MCLR (Demethyl MCLR)。根据双电荷分子离子(M+2H)2~+m/z=491.3的MS/MS图谱初步推测Demethyl MCLR的分子结构是：[D-Asp3]mcyst-LR、[Dha7]mcyst-LR、[D-Asp3,(E)-Dhb7]mcyst-LR或者[D-Asp3,(Z)-Dhb7]mcyst-LR。对结合固相萃取(SPE)技术的藻细胞的胞内毒素提取流程进行了优化。优化后的毒素提取流程如下：离心收集纯培养藻液,于藻浆中加入w(甲醇)=75%提取液,反复冻融3次,后辅以超声破碎0.5h,离心收集上清液并过C18固相萃取柱进行富集,然后用w(甲醇)=10%淋洗液对C18固相萃取柱进行淋洗以除去杂质,最后用w(甲醇)=80%+w(TFA)=0.05%洗脱液将毒素洗脱收集。
结合对MCLR和Demethyl MCLR具有良好交叉反应性的酶联免疫试剂盒,测定并分析UV/H2O2抑藻过程中胞内胞外毒素的变化。结果表明,UV/H2O2主要通过产生的·OH迅速降解藻细胞的胞外毒素,同时通过抑制藻细胞生物量和藻毒素合成相关基因转录表达量从而减少藻细胞的胞内毒素合成量。实验结果表明,藻液在经38 mJ/cm2 UV254辐照结合0.2 mM H2O2处理后的培养过程中,藻细胞生长受到抑制,在此过程中胞内毒素逐渐释放至水体,胞外毒素逐渐增加,藻液总毒素含量基本保持不变。
3. UV/H2O2对微囊藻毒素的降解动力学研究
本论文对比研究了UV254、H2O2及UV/H2O2对MCLR的降解效率,结果表明：1)H202几乎不能氧化降解MCLR, UV254直接光解以及UV/H2O2都能较好的降解MCLRO当pH值为8.0、UV254辐照强度为1738μW/cm2、H202浓度为5mM时,第40 min三种处理对MCLR的降解效果存在明显差异,其中H202对MCLR的降解率仅为8.4%±2.1%、UV254直接光解去除率为73.6%±3.4%、UV/H2O2处理效果最好(去除率为98%±4.8%)。2)三个处理过程均能较好的拟合表观一级动力学方程,其表观一级动力学常数分别为kH2O2=0.0021 min-1、kUV=0.045 min-1和kUV/H2O2=0.082 min-1。kUV/H2O2值超过k_(H2O2)与Kuv之和,表明UV/H2O2具有明显的协同作用。
UV/H2O2对MCLR的降解过程受初始H202浓度、UV254辐照强度、MCLR初始浓度以及溶液pH值的影响。当H202在0-10mM浓度范围内,UV/H2O2对MCLR降解的表观一级动力学常数与H202浓度呈正相关关系k=-0.007×[H202]+0.0446,(R2=0.9918)；UV/H2O2对MCLR的降解效率随UV254辐照强度的提高而增加,随MCLR初始浓度的提高而降低,MCLR降解的表观一级动力学常数与MCLR初始浓度呈负相关关系：k=.0.0056×[MCLR]0+0.1771(R2=0.8528)。
此外,本论文选取硝基苯(NB)作为竞争参考物质,根据UV254光解和·OH氧化MCLR的原理和·OH稳态理论建立UV/H2O2降解MCLR的竞争动力学模型,并结合模型测定了·OH与MCLR的二级动力学常数(k=-2.8(±0.21)×1010 M-1s-1)。
4. UV/H2O2降解微囊藻毒素的中间产物及途径研究
结合LC-MS分析,基于总离子流图谱(TIC)及(M+H)~+m/z数据,结果表明UV/H2O2降解MCLR过程主要生成9种中间产物,其正离子质荷比分别为(M+H)+m/z 1029.5、(M+H)+ m+z 1011.5、(M+H)+ m/z795.4、(M+H)+ m/z835.4、(M+H)+ m/z 811.3、(M+H)+ m/z 1027.5、(M+H)+ m/z 1009.5、(M+H) + m/z 965.5和(M+H)+ m/z 1045.5.
UV/H2O2降解MCLR过程中主要涉及·OH降解、氧化降解以及UV254直接光解作用。·OH主要进攻MCLR分子Adda侧链上的共轭双键、苯环及甲氧基。根据中间产物的分子量信息及·OH与多肽和蛋白质反应机理等资料信息,推测UV/H2O2对MCLR降解途径主要有3条,分别如下：1)·OH进攻Adda侧链共轭双键,通过亲电加成反应生成4,5-二羟基衍生MCLR或者6,7-二羟基衍生MCLR,然后相应的Adda侧链C4-C5双羟基键或C6-C7双羟基键氧化断裂,生成醛或酮衍生中间产物,这些中间产物进一步被氧化生成相应的羧酸衍生物；2)·OH进攻Adda侧链苯环,通过亲电取代反应生成(ortho、meta或para)苯环羟基衍生MCLR或苯环双羟基衍生MCLR,然后进一步氧化生成醛或酮衍生中间产物；3)·OH进攻Adda侧链甲氧基,通过夺氢反应生成甲酸酯衍生MCLR,然后与·OH进一步反应,生成去甲氧基MCLR (DmADDA).结合中间产物含量的时间变化过程,表明·OH攻击共轭双键及苯环的反应为主要途径,其占总反应途径的90%以上。结合UV/H2O2降解MCLR的作用机理、MCLR降解中间产物结构和已有的文献资料分析,表明UV/H2O2降解MCLR的产物毒性基本消失。
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 aeruginosa
The 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/H2O2
Using 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 kinetics
In 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/H2O2
The 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.
本论文的主要研究如下：
1. UV/H2O2抑藻机理及抑藻效应研究
建立了水杨酸捕获法测定·OH的HPLC检测条件：流动相为20%甲醇+80%水(含0.03’M乙酸和0.03 M柠檬酸),流速为1mL/min;同时证实了UV/H2O2体系中生成的·OH参与了藻细胞的氧化损伤过程。基于藻细胞基因转录水平和生理生化指标分析得出结论,UV/H2O2对藻细胞的损伤机理主要是通过·OH氧化损伤藻细胞膜,使其发生脂质过氧化反应,进而损伤藻细胞DNA及蛋白质等生物大分子和光合作用系统。藻毒素合成相关基因的转录表达量受到UV/H2O2处理的抑制,因而UV/H2O2抑制藻生长的同时可抑制胞内毒素的合成量,从而减少释放至水体的毒素含量。
本论文同时研究了铜绿微囊藻分别经UV254、H2O2及UV/H2O2处理后继续培养过程中的生长情况,研究结果表明：H202和UV254对铜绿微囊藻生长抑制效应均随作用剂量的增加而增强；UV/H2O2对铜绿微囊藻的抑制存在明显的协同效应,在实验室条件下,0.2 mM H2O2结合38 mJ/cm2 UV254辐照剂量是抑藻效应最强的组合；结合扫描电镜分析表明,UV/H2O2处理对铜绿微囊藻细胞形态结构造成破坏。藻液经0.2 mM H2O2结合38 mJ/cm2 UV254辐照剂量联合处理后第10d,几乎所有藻细胞凹陷变形,很多藻细胞细胞壁破裂,细胞质外溢而死亡。
2. UV/H2O2抑藻过程中铜绿微囊藻胞内／外毒素变化研究
本论文结合HPLC和ESI-MS/MS技术对铜绿微囊藻905胞内毒素鉴定结果表明,胞内毒素除MCLR以外,还存在去甲基MCLR (Demethyl MCLR)。根据双电荷分子离子(M+2H)2~+m/z=491.3的MS/MS图谱初步推测Demethyl MCLR的分子结构是：[D-Asp3]mcyst-LR、[Dha7]mcyst-LR、[D-Asp3,(E)-Dhb7]mcyst-LR或者[D-Asp3,(Z)-Dhb7]mcyst-LR。对结合固相萃取(SPE)技术的藻细胞的胞内毒素提取流程进行了优化。优化后的毒素提取流程如下：离心收集纯培养藻液,于藻浆中加入w(甲醇)=75%提取液,反复冻融3次,后辅以超声破碎0.5h,离心收集上清液并过C18固相萃取柱进行富集,然后用w(甲醇)=10%淋洗液对C18固相萃取柱进行淋洗以除去杂质,最后用w(甲醇)=80%+w(TFA)=0.05%洗脱液将毒素洗脱收集。
结合对MCLR和Demethyl MCLR具有良好交叉反应性的酶联免疫试剂盒,测定并分析UV/H2O2抑藻过程中胞内胞外毒素的变化。结果表明,UV/H2O2主要通过产生的·OH迅速降解藻细胞的胞外毒素,同时通过抑制藻细胞生物量和藻毒素合成相关基因转录表达量从而减少藻细胞的胞内毒素合成量。实验结果表明,藻液在经38 mJ/cm2 UV254辐照结合0.2 mM H2O2处理后的培养过程中,藻细胞生长受到抑制,在此过程中胞内毒素逐渐释放至水体,胞外毒素逐渐增加,藻液总毒素含量基本保持不变。
3. UV/H2O2对微囊藻毒素的降解动力学研究
本论文对比研究了UV254、H2O2及UV/H2O2对MCLR的降解效率,结果表明：1)H202几乎不能氧化降解MCLR, UV254直接光解以及UV/H2O2都能较好的降解MCLRO当pH值为8.0、UV254辐照强度为1738μW/cm2、H202浓度为5mM时,第40 min三种处理对MCLR的降解效果存在明显差异,其中H202对MCLR的降解率仅为8.4%±2.1%、UV254直接光解去除率为73.6%±3.4%、UV/H2O2处理效果最好(去除率为98%±4.8%)。2)三个处理过程均能较好的拟合表观一级动力学方程,其表观一级动力学常数分别为kH2O2=0.0021 min-1、kUV=0.045 min-1和kUV/H2O2=0.082 min-1。kUV/H2O2值超过k_(H2O2)与Kuv之和,表明UV/H2O2具有明显的协同作用。
UV/H2O2对MCLR的降解过程受初始H202浓度、UV254辐照强度、MCLR初始浓度以及溶液pH值的影响。当H202在0-10mM浓度范围内,UV/H2O2对MCLR降解的表观一级动力学常数与H202浓度呈正相关关系k=-0.007×[H202]+0.0446,(R2=0.9918)；UV/H2O2对MCLR的降解效率随UV254辐照强度的提高而增加,随MCLR初始浓度的提高而降低,MCLR降解的表观一级动力学常数与MCLR初始浓度呈负相关关系：k=.0.0056×[MCLR]0+0.1771(R2=0.8528)。
此外,本论文选取硝基苯(NB)作为竞争参考物质,根据UV254光解和·OH氧化MCLR的原理和·OH稳态理论建立UV/H2O2降解MCLR的竞争动力学模型,并结合模型测定了·OH与MCLR的二级动力学常数(k=-2.8(±0.21)×1010 M-1s-1)。
4. UV/H2O2降解微囊藻毒素的中间产物及途径研究
结合LC-MS分析,基于总离子流图谱(TIC)及(M+H)~+m/z数据,结果表明UV/H2O2降解MCLR过程主要生成9种中间产物,其正离子质荷比分别为(M+H)+m/z 1029.5、(M+H)+ m+z 1011.5、(M+H)+ m/z795.4、(M+H)+ m/z835.4、(M+H)+ m/z 811.3、(M+H)+ m/z 1027.5、(M+H)+ m/z 1009.5、(M+H) + m/z 965.5和(M+H)+ m/z 1045.5.
UV/H2O2降解MCLR过程中主要涉及·OH降解、氧化降解以及UV254直接光解作用。·OH主要进攻MCLR分子Adda侧链上的共轭双键、苯环及甲氧基。根据中间产物的分子量信息及·OH与多肽和蛋白质反应机理等资料信息,推测UV/H2O2对MCLR降解途径主要有3条,分别如下：1)·OH进攻Adda侧链共轭双键,通过亲电加成反应生成4,5-二羟基衍生MCLR或者6,7-二羟基衍生MCLR,然后相应的Adda侧链C4-C5双羟基键或C6-C7双羟基键氧化断裂,生成醛或酮衍生中间产物,这些中间产物进一步被氧化生成相应的羧酸衍生物；2)·OH进攻Adda侧链苯环,通过亲电取代反应生成(ortho、meta或para)苯环羟基衍生MCLR或苯环双羟基衍生MCLR,然后进一步氧化生成醛或酮衍生中间产物；3)·OH进攻Adda侧链甲氧基,通过夺氢反应生成甲酸酯衍生MCLR,然后与·OH进一步反应,生成去甲氧基MCLR (DmADDA).结合中间产物含量的时间变化过程,表明·OH攻击共轭双键及苯环的反应为主要途径,其占总反应途径的90%以上。结合UV/H2O2降解MCLR的作用机理、MCLR降解中间产物结构和已有的文献资料分析,表明UV/H2O2降解MCLR的产物毒性基本消失。
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 aeruginosa
The 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/H2O2
Using 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 kinetics
In 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/H2O2
The 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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