Characterization And Establishment Of Early Warning System Of Microcystin

More and more industrial effluents and domestic sewage have been discharged into source water with the development of industrial and agricultural production and the growth of population, which has induced pollution of source water unceasing uninterrupted. The rapid increasing of nitrogen and phosphours has offered redundant nutrition to algae for their generous propagation, which induced the outbreak of bloom all over the world. Till now the tendency of the frequency and graveness of bloom in freshwater lake seem to increase quickly. Many countries have reported explosion of bloom in lake or reservoir such as American, Japan, India, Canada, south African, Finland. The eutrophication has been more and more severe in our country ever since 1990s and the main river of Yangtze river, Yellow river, Preserved egg river and the main lake of the Tai lake, Nest lake, Boyang lake show to be in different eutrophication. Bloom has not only demolished the view character of lake and reservoir but taken threaten to the health of people. Bloom has been a serious problem and has provoked attention and reconstruction of the whole world.The main contamination material of bloom are all kinds of algae-toxin. Microcystin is one kind of intracellular toxin. Its frequency of occurrence is the highest and threaten to humen is the severest, which is generated by microcystis, anabena, oscillatoria, nostoc. Microcystin can cause all kinds of health lesion, espeialy liver cancer. The problem of environmental contamination of microcystin should be reconstructed by us and it is important to establish warning system and monitor system of microcystin. The study site of this research were Xiliu lake and Huayuankou pool . The forcasting model of microcystin was established by means of analysis of physical chemistry data with con-algae cell density, chlorophyl concentration and cyanobacteria cell density and the structural of microcystin was analyzed and the gene order was detected after the microcystis cell was isolated culture, which had provided scientific base and means for research of prevention and cure and effectiveness and removal of microcystis.Materials and methods1. Spot and samplingThe study site were Xiliu lake and Huayuankou pool. There were two sampling spots and No.l was the ingress of water plant Shiyuan and No.2 was the extent outside 1000 meters. Sampling was collected from march to October in 2004. there were two sites in Huayuankou pool and the water source ingress spot was No.1 and water reciped spot was No.2 and the sampling was made from march in 2005 to January in 2006. Rainy days should be avoided from sampling and the time should be at 8:00 to 9:00 in mornings. 1200ml water sample was collected by one 2500ml water sampler from the water plane under the water surface 0.5meters. Then the water sampling was put into one 2500ml polyethylene bottle and 1000ml glass bottle for measuring of physical chemic data and the con-algae cell density,chlorophy1 concentration and cyanobacteria cell density. The meteorological data such as water temperature, air temperature, illuminance and clarity should be recorded during the sampling.No.25 plankton net with the mesh diameter 0.064mm was used to sample. The sampling was made on water surface or under 0.5m from surface by means of circulating sampling with the type inverse 8 and the velocity 20cm to 30cm per second. The sampling time should be made according to algae cellar density and the normal time was from 3 minutes to 5 minutes. The floating algae was gathered in the net head after the water was filtered by means of raising the net. A 100ml plastic bottle was used to collect the floating algae. 30ml to 40ml sample was collected per sample point for isolating culture of microcystis and preparation and identification of microcystin.2. Items and methods of measurementLight illuminance (LI) was determined using luminometer; Water temperature (WT) using deep water thermometry; Secchi-depth (SD) using Secchi disc; Chemical oxygen demand (COD_Mn) using acidic potassium permanganate method; Total nitrogen (TN) using alkaline potassium persulphate digestion-UV spectrophotometric method; Total phosphorus (TP) using ammonium molybdate spectrophotometric method; Chlorophyll-a (Chla) using spectrophotometric method; Algae cell density (ACD) and cyanobacteria cell density (CCD) using blood cell counter; phycocyanin intergenic spacer region(PC-IGS) and microcystin synthetase gene B (mcyB) using the whole cell PCR method; blue-green gene order using gene cloing method with T bearer and direct sequencing of PCR product method; total Microcystin (MC) by ELISA kit method; isolation and depuration of eribble microcystin using solid pole of extraction(SPE); analysis and density measure of microcystin using high performance liquid chromatography(HPLC).3. Isolation culture of algae cellCyanobacteria was isolated and purified by 96-well microplates with ultimate dilution. The nutrient medium was BG-II; illumination 2500LUX; ratio of light and gloom 12h:10h; temperature 25±1℃; the medium should be shaken once every morning and afternoon.4. Statistical treatment4.1 Statistical analyzing softwareDNAssist 1.0 and DNATools 5.1 were used to analyze the sequence and comparation; BLAST to refer the gene sequence to GenBank; Excell to trimming and incoming data; SPSS 11.0 to model multiple regression forcasting model and calculate microcystin density; MATLAB 5.3 to model artificial neural network forcasting model.4.2 Statistical methodsGrading points method and comprehensive trophic State index method were used to evaluate the trophic state of xiliu lake and Huayuankou conservation pool; Backpropagation neural network with Levenberg-Marquardt algorithm and Stepwise multiple regressionwere were used to analyze the relationship among algae cell density, cyanobacteria cell density, microcystin concentration and environmental factors and to establish the forcasting model.Results1. Results of assessment of nutritional status of xiliu lake and huayuankou poolThe results of evaluation using grade method revealed that it was light nutritional state in spring and eutrophic state in autumn and summer of xiliu lake and it was eutrophic state in all the four seasons of huayuankou pool; the tendency of year grade index was from the lower critical value to eutrophic state to higher critical value of eutrophication of xiliu lake; the tendency of huayuankou pool was gradually increasing first and then gradually depressing.The results of comprehensive trophic state index revealed that it was almost eutrophic state in spring and autumn and almost midrange eutrophic state in summer; it was light eutrophic state in all the seasons of huayuankou pool. The change tendency of comprehensive trophic state index of xiliu lake was from the higher critical value of midrange eutrophic to and the higher critical value of light eutrophic to the critical value of midrange eutrophic and the tendency of huayuankou pool was decreasing first and then increasing and decreasing in the end.2. Results of the calculating of different taxon algae cellsThe main polluting algaes of xiliu lake and huayuankou pool were diatom,chlorella,cyanobac--teria and euglenophyta. Ascendant taxon algae and the cell density showed visible change with the seasonal variation. There was no obvious ascendant taxon with so many kinds algae in spring with low ACD and CCD; There were less kinds of algae in summer and autumn and the ascendant taxon cyanobacteria with high ACD and CCD; The kind and density were the lowest in the whole year in autumn.3. The result of establishing forcasting models of Chla,ACD and CCD with the physical chemistry items3.1 The result of establishing model using multiple regression analysisThe result of scatterplot between the physical chemistry items and Chla, ACD and CCD revealed that there was no linear correlation between TP, Li and Chla and CCD , which showed that TP, Li couldn't be analyzed using multiple regression. The result of establishing model was as follow:(1)ln(Chla+1)=0.626-0.717TN+0.644COD (R=0.855,R²=0.730,F=96.213,P=0.0);(2)ln(ACD+1)=14.090+0.206WT-2.771SD-0.824Li-0.289TN+4.608TP(R=0.873,R²=0.762,F=43.555,P=0.000) (3)ln(CCD+1)=6.154-1.712TN+0.881COD (R=0.854,R2=0.730,F=95.849,P=0.000);As we can see from the result that positive correlation was significant between Chla and COD_Mn; ACD and WT and TP; CCD and COD_Mn; Negative correlation was significant between Chla and TN; ACD and SD and Li and TN; CCD and TN. The coefficient correlation of the regression equation between predictive value and kernel value was more than 0.7, which improved that the equation could be used to forcast the microcystin contamination. 3.2 Result of establishing forcasting model using backpropagation neural network with Levenberg-Marquardt algorithmAll of the physical chemistry factors were brought into the model. The training errors of Chla, ACD and CCD were 1e-11, 1e-3 and 1e-13, respectively. The training errors of training set, check set and test set reached perigee almost in the same time and the fitness results between predictiv and practical as follow:(1) A=0.082T+4.48 (r=0.871)(2)A=0.556T+(3.16e+003) (r=0.838)(3)A=0.608T+798 (r=0.869)The result indicated that the fitness of the model was better than the model of regression and all the main factors were analyzed in the model, so the model was bettet to consistent with actual state and it was one good method for establishing forcasting model(table 1 and 2).4. The results of isolation culture of microcystisOne strain of Microcystis, Microcystis XLH from xiliu lake and two strains of microcystis, microcystis BM1 and BM2 and one strain Oscillatoria from huayuankou pool were isolated successfully by 96-well microplates with ultimate dilution.5. Result of gene sequencing and analysis of PC-IGS and mcyBThe results of the whole cell PCR revealed that amplification of PC-IGS and mcyB of XLH,BM1 and BM2 were masculine and the amplification of PC-IGS was masculine and mcyB negative of Oscillatoria.The PCR amplifying product of mcyB was cloned with T bearer and identified using PCR consensus primer. There was one strap in 800bp which revealed that the aim strain was cloned successfully. 1ml bacterium liquid capsulated was send to Shanghai bioengineering limited company for sequencing, then the sequence was refered to GenBank for sequence compare and the homology was almost 99.15%. the sequences of the three strains mocrocystis were refered to GenBank and were given a serial-number EF216872,EF216873 and EF216874,respectively.6. Results of extraction and depuration and detection of microcystinMicrocystin was measured using HPLC with the standare preparation toxin MC-LR and MC-RR and the result revealed that the proportionality of MC-LR and MC-RR to microcystin was 47.1,130.9 and 142.8,respectively. the multiple of MC-LR to MC-RR was almost 150, which improved that the main microcystin in the source water of xiliu lake and huayuankou pool was MC-LR. Total microcystin density was measured using ELISA kit and the result indicated that the producing toxin quantity of XLH, BM1 and BM2 was 471.4μg,1068.4 and 4697.2 per gram powder,respectively.Conclutions:1. The two source water had been polluted by microcystin.2. 96-well microplates with ultimate silution method could simplify the isolation process of algae cells and improve the efficiency.3. Whole cell PCR could be used to identify toxigenic cyanobacteria from natural water bodies and isolated strains. The three microcystin strains isolated were cyanobacteria and could produce microcystin.4. The main toxin isomeride of microcystin extracted was MC-LR and the microcystin extracted was homologized to the that in water, which revealed that it was the main pollution resource of the source water.5. Microcystin forcasting model could be established using artificial neural network and multiple refression analysis and the artificial neural network was better than multiple regression analysis.6. Provide science base to safe water supply and water source health protection of Zhengzhou city and basical data and methods to deep research of toxin potency mechanism and removal of microcystin.