| Lake eutrophication is one of the main ecological problems in the world.Human factors,including rapid urbanization,industrialization and excessive agricultural cultivation,lead to nutrient content rise in water body,further increasing the frequency of cyanobacteria bloom.Cyanobacteria produce a variety of toxins and odor compounds,which pose a direct threat to the supply water,especially drinking water.However,the existing water evaluation indicators focus on physical property indicators,yet the chemical indicators are relatively general,which cannot fully and accurately reflect the specific content of toxic and harmful substances.In this study,metabolomics research technology was used to analyze the secondary metabolites produced by cyanobacteria bloom and their impact on water quality,which is applied to water quality evaluation.Firstly,in order to solve the problem of incomplete and inaccurate water evaluation indexes,the volatile and water-soluble metabolites in Microcystis samples at different periods were studied by metabolomics and statistical methods,and the statistically different metabolites were screened out.A novel headspace solid phase microextraction Arrow method combined with gas chromatography-mass spectrometry was developed for extraction and analysis of volatile organic compounds of microcystis under simulated natural conditions.By selecting suitable extraction fibers,210 kinds of volatile metabolites were identified by full scanning qualitative analysis from the samples of Microcystis in producing period.The metabolic changes of volatile molecules during the growth of Microcystis were analyzed by multivariate statistical analysis and clustering Analysis.Chemometric analysis based on principal component analysis,partial least squares-discriminant analysis and heatmap associated with hierarchical cluster analysis provided a suitable tool to differentiate volatile organic compounds in algal material.And 10metabolites in statistical significance were identified,including cyclohexanol,dimethyl trisulfide,benzenemethanol,camphor,2-methoxyphenol,3-hexene-1-ol,2,4-decadienal,3-methylindole,citral and 1-nonanol.Parameters affecting the extraction,such as extraction temperature,time and salt were optimized.In addition,the analysis conditions,including desorption temperature and time as well as gas chromatographic parameters,were optimized.The calibration curves showed a good linearity（correlation coefficient>0.998）in the concentration range of 0.050ng/L～1000 ng/L.The detection limits of this method were 0.010 ng/L～0.030 ng/L,and the recoveries at the concentration level of 100 ng/L were 76.3%～93.0%.The method was satisfactorily precise,with the RSD values of less than 12.7%.The new method is simple,quick,stable,and applicable to complex matrices.It is suitable to the determination of volatile metabolites in natural water at the early stage of blue-green algae bloom.UPLC Q-TOF MS non-target metabolomics analysis was established for four groups of Microcystis samples in different growth periods.The original data was analyzed with various metabolomics tools.The comprehensive evaluation of different metabolisms of Microcystis in different growth periods and different environments showed that MC-RR and MC-LR decreased significantly from the peak to the aging period.Using the latest metabolomics software,the author analyzed the collected sample data and got 5896 components with detailed fragment and peak area information.The screening parameters were set:parent ion matching error,fragment matching error,isotopic similarity,fragment ion fraction,total score of compound identification and ANOVA（p）value.Matched with the information of the compounds in the whole natural product library of Chemsipider,26potential markers with significant difference and qualitative reliability were obtained.In the correlation analysis of the different metabolites,the author found that there was a high degree of consistency between the algal toxin and pyropheophyll A and calendula.The target analysis of MCs was carried out and the solid phase extraction conditions were optimized.The detection methods of 13 microcystins toxins in cyanobacteria were established.Results show that the matrix has little interference to the method,which can be used for the determination of algae samples.Nine suspected microcystin compounds were discovered by self-built microcystin library.Secondly,in order to improve the sensitivity and stability of metabolite determination,based on the selected statistically different metabolites,the 3-MI Molecularly Imprinted Electrochemical sensor was prepared by electropolymerization.The characteristic compound 3-methylindole was selected as the template molecule and o-phenylene was used as the monomer.The electrochemical sensor greatly reduced the detection limit and analysis cost of 3-MI.3-MI is a recalcitrant and toxic substance that can cause damages to human and animal lung cells and may have a pronounced effect on mood and stress levels.Rapid and sensitive detection of 3-MI is therefore of great important for its environmental safety evaluation.A novel electrochemical sensor for the determination of 3-MI was fabricated based on electropolymerization of molecular imprinted polymer film on a glassy carbon electrode.Rational selection of the suitable monomers was conducted by evaluation of the interaction between each monomer and template using the density functional theory method.The results indicated that the stabilization energy of 3-MI-o-Phenylenediamine complex is greater than that obtained from other evaluated monomers,hence o-PD was used to prepare the MIP sensor.The imprinted layer was systematically characterized by cyclic voltammetry,electrochemical impendence spectroscopy scanning electron microscopy and atomic force microscopy.Under optimal experimental conditions,a linear relationship was observed between the peak current change of the electrochemical probe[Fe（CN）6]3-and the concentration of 3-MI over the range0.010μM～1.2μM,with a detection limit of 4 n M.This sensor also exhibited superb selectivity to 3-MI as minimum interferences were observed from other structurally similar compounds,thus it was successfully applied for the determination of 3-MI in real samples.Thirdly,in order to verify the consistency and superiority of differential metabolites with traditional evaluation factors,the analysis of differential metabolites was applied to the analysis of actual water samples,and the production and temporal and spatial variation trend of cyanobacteria bloom metabolites were discussed.The results show that through the analysis of differential metabolites,the analyst can obtain information about the algae growth or pollution of the water body,which preliminarily proves that thistechnology can be used to evaluate the water quality of natural lakes.During the sampling and monitoring period from March to November 2019,it was found that there was a significant positive correlation between 3-methylindole and water quality parameters such as chlorophyll a,algal density,water temperature.There was a significant positive correlation between indole,β-cyclocitral and Chl-a,algal density.Free and intracellular microcystins were positively correlated with total phosphorus,chlorophyll and algae density.Finally,the analysis method based on differential metabolites was applied to the actual samples（fish flesh and cyanobacteria landfill leachate）,and the pretreatment method was optimized.The experimental results showed good quantitative analysis ability.A method for the detection of cyanobacteria odor metabolites in fish was established.By optimizing the extraction and enrichment conditions such as microwave distillation and solid-phase microextraction,the complete separation of eight common odor compounds in cyanobacteria was effectively realized by full two-dimensional gas chromatography,and the internal standard method was used for quantitative analysis with the accurate mass number of high-resolution characteristic ions.Eight odor compounds in the range of 0.010μg/L～100μg/L showed good linearity.The detection limit of the method is 0.070μg/kg～3.3μg/kg。Using divinylbenzene,porous activated carbon powder and polydimethylsiloxane as raw materials,SPME materials were prepared by one pot method.The home-made SPME fiber coating was characterized and extraction conditions were optimized.Compared with imported commercial products,the home-made SPME fiber can not only extract VOCs,but also enhance the extraction efficiency of sulfur-containing odor substances.Combined with GC-MS,the water quality of leachate from cyanobacteria landfill was analyzed to investigate the removal efficiency of odor substances in leachate treatment station. |
