Effects Of Extracellular Polymeric Substances On Arsenic Accumulation And Transformation By Phaeodactylum Tricornutum

Arsenic（As）pollution in water bodies affects the health of millions of people and is one of the global environmental issues that have attracted much attention.Arsenic has a carcinogenic effect,and its forms are mainly arsenate（As（Ⅴ））and arsenite（As（Ⅲ））.Microbial extracellular polymeric substances（EPS）are widely present in nature and affect the migration and transformation of arsenic in the aquatic environment.These macromolecules are important metabolites in the"water-algae"interface,and have good application prospects in the field of arsenic wastewater treatment.Microalgal EPS are rich in various oxidation/reduction organic ligands,which can affect the migration and transformation of heavy metals such as As.Diatoms are primary producers in the ocean,with high biodiversity and huge biomass.There are still few reports on the relationship between diatom EPS and arsenic accumulation and transformation.This thesis selected a model diatom—Phaeodactylum tricornutum.EDTA method and heating method were compared in terms of efficiency in EPS extraction.Attenuated Total Reflection Fourier Transform Infrared Spectroscopy（ATR-IR）and Three-Dimensional Fluorescence Spectroscopy（3D-EEM）were used to characterize the extracted EPS.Then,differences in the growth,cell morphology,and As accumulation,adsorption,absorption and morphological transformation of P.tricornutum with and without EPS under As（Ⅲ）and As（Ⅴ）treatments were compared.Finally,using ATR-IR and 3D-EEM technology,changes in the secretion of EPS under As（Ⅲ）and As（Ⅴ）treatments,and in the cell surface and the characteristics of the extracted EPS were evaluated,and the binding mechanism between As and EPS were discussed.The results of the study are as follows:1.Comparison of the extraction methods of P.tricornutum EPS and their characterizationEDTA method and heating method were used to extract P.tricornutum EPS.We compared and analyzed the content and composition of EPS obtained by each method,and observed the cell morphology and cell activity in combination with optical microscope,SEM and cell regrowth to determine the best extraction method of P.tricornutum.The results showed that the content of EPS obtained by different extraction methods had significant differences.In the EDTA method,as the concentration of the extractant increases,the amount of polysaccharide,protein and DNA extracted gradually increased,but when the concentration of EDTA exceeds 20 mmol·L^-1(The total amount of EPS extracted is 5.35 mg·g^-1 DW),the cells ruptured and died,and chlorophyll was dissolved outand the cells no longer grew.For the heating method,as the heating temperature increases,the amount of extracellular polysaccharide,protein and DNA extracted from P.tricornutum were also increasing;but when the extraction temperature was greater than40℃,the algae cells became greenish,the cell structure was broken,and the intracellular material flowed out,leading to cell death.For heating extraction,heating at 40℃was the most suitable,and the EPS extraction volume was 3.58 mg·g^-1 DW.In summary,20mmol·L^-1 EDTA was the best extraction method for P.tricornutum EPS,and the extraction amount was 5.35 mg·g^-1 DW,among them,polysaccharide content was the highest,accounting for 66.73%,followed by protein,accounting for 17.76%,DNA had the least proportion,accounting for 15.45%.ATR-IR results showed that the EPS extracted by EDTA contains absorption peaks of C—O—C,C—OH and other sugars and protein absorption peaks such as C—O,C=O,and—NH.In addition,EPS also contained some lipids and the absorption peaks of the salt of the amino acid.3D-EEM confirmed the presence of tryptophan and tyrosine protein peaks in P.tricornutum EPS.2.Effects of EPS on the growth,arsenic accumulation and morphological transformation of P.tricornutumDifferent concentrations of As（Ⅴ）/As（Ⅲ）were treated to P.tricornutum with and without EPS for 7 days.We observed the growth and cell morphology of the algae cells change,determined the total amount of cell accumulation,adsorption,absorption,and intracellular and extracellular As speciation.The results showed that P.tricornutum had certain tolerance to As（Ⅴ）and As（Ⅲ）.The EC₁₀ value of 72 h under As（Ⅴ）treatment was 1.75 mg·L^-1,and the EC₁₀ value was 1.00mg·L^-1 under As（Ⅲ）treatment.Therefore,P.tricornutum was more resistant to As（Ⅴ）.The presence of EPS reduced the changes in cell morphology caused by arsenic toxicity and improve the tolerance of P.tricornutum to arsenic.With the increased of arsenic concentration,the ability of P.tricornutum to accumulate,adsorbtion,absorbtion and form transformation of arsenic increased,but the removal rate of arsenic gradually decreased.At the same time,the presence of EPS increased the accumulation and adsorption of As by algal cells,and some of its own reducing compounds could be used to promote the transformation of intracellular arsenic form,but as a barrier,it weakened the efflux of arsenic.3.Preliminary study of interaction mechanism of P.tricornutum EPS and arsenicAfter treating with different concentrations of As（Ⅴ）/As（Ⅲ）for 7 days,we observed the changes of P.tricornutum EPS secretion and used ATR-IR observe the changes in the surface of algae cells and the functional groups of the EPS;at the same time,we added the EPS to different concentrations of As（Ⅴ）/As（Ⅲ）treatment,and analyzed the changes in the composition of the EPS combined with As using 3D-EEM.With the increase of As（Ⅴ）and As（Ⅲ）concentrations,the secretion of EPS showed an increasing trend.The secretion of EPS under the treatment of 2000μg·L^-1As（Ⅴ）and As（Ⅲ）was 1.47 and 1.23 times that of the control group,respectively.At the same time,ATR-IR spectroscopy analysis showed that functional groups（such as—OH,—NH₂,C—O—C）of protein and amino acid participate in the adsorption of EPS through surface adsorption/complexation mechanism or hydrophobic interaction,thereby limiting the transport of As across the membrane.3D-EEM determined that there were two protein peaks of tryptophan and tyrosine in P.tricornutum EPS.The interaction between tryptophan and As was stronger than that of tyrosine,and the fluorescence decreased more intensely.P.tricornutum EPS provided more binding sites for As（Ⅲ）,because As（Ⅲ）treatment EPS was more variable than As（Ⅴ）treatment.In summary,this thesis compared and clarified the best extraction method of P.tricornutum EPS,and explored the enrichment and morphology of P.tricornutum on As（Ⅴ）and As（Ⅲ）with and without EPS.The difference in transformation clarified the role of EPS in the metabolic process of P.tricornutum on As accumulation and the possible mechanisms of the combination of EPS and As.In short,EPS enhanced the tolerance of P.tricornutum to As and promoted the As accumulation and morphological transformation of algal cells.