The Adsorption And Uptake Of Inorganic Arsenic By Chlorella Pyrenoidosa And The Interaction Mechanism

The role of extracellular polymeric substances(EPS)which are produced from a variety of microalgae is well-known to affect the process of arsenic bio-purification in aquatic ecosystem.However,the bioaccumulation patterns of inorganic As(Ⅲ)and As(Ⅴ)in As-tolerant green algae and EPS influences on adsorption and uptake remain to explore.The aim of this study is to better understand the EPS influences and mechanism on toxicity,adsorption and uptake of As(Ⅲ)and As(Ⅴ)in freshwater green algae of Chlorella pyrenoidosa(C.pyrenoidosa).The major conclusions were obtained as followed:(1)The presence of EPS enhanced the tolerance ability of C.pyrenoidosa to As(Ⅲ)and As(Ⅴ).The density of algae cells after exposure to As(Ⅲ)and As(Ⅳ)of 0-1500 μmol L-1 were measured by ultrviolet spectrophotometer and calculated the IC50 values(Inhibitory concentration of 50%growth).Results showed that As(Ⅲ)and As(Ⅴ)higher than 300 μmol L-1 both inhibited the growth of C.pyrenoidosa,and the values of 96 h IC50 were 613.0 μmol L-1 and 707.2 μmol L-1,respectively.In addition,after treatment of 1000 μmol L-1 As(Ⅲ)and As(Ⅴ),the concentrations of intracellular reactive oxygen species(ROS),superoxide dismutase(SOD),chla and the chlorophyll fluorescence parameters of photosynthetic system Ⅱ(PS Ⅱ)were also determined with flow cytometry,microplate reader and chlorophyll fluorometer.Compared to the blank.As(Ⅲ)and As(Ⅴ)increased ROS levels and SOD enzyme activity,while reduced the content of Chla significantly(P<0.05),and the values of maximum quantum yields(Fv/Fm),maximum electron transport rate(ETRmax),initial slope of rapid light curve(α)and half-saturation light intensity(Ik)of PSII also obviously decreased after 6 h exposure.These results verified arsenic oxidative stress and photosynthesis inhibition towards the C.pyrenoidosa,then SEM-TEM also observed the fold and fracture on cell surface and intracellular disordered pyrenoid and thylakoid.Compared to intact cells,the toxic effects of As(Ⅲ)and As(Ⅴ)to EPS-removed algae cells(-EPS)were further promoted,because the values of 96 h IC50 and chla further decreased to 522.0.640.6 μmol L-1 and 0.71、0.84 mg L-1,while ROS and SOD levels further increased to 296%、247%and 25.53、21.44 unites mg-1 protein.The presence of EPS can effectively enhance algal tolerance ability to inorganic arsenic stress.(2)The intracellular uptake of As(Ⅲ)and As(Ⅴ)both agreed with the Bio-dynamic model and algal EPS increased inorganic arsenic bioaccumulation capacity.After 24 h-exposure to As(Ⅲ)and As(Ⅴ)in the concentration range of 0-1000 μmol L-1,the ICP-MS was used to detect concentration of total arsenic in C.pyrenoidosa.Results suggested that intracellular arsenic uptake capacity increased with the rising exposure levels,and the obtained maximum uptake for As(V)reached to 1.23 μmol g-1,significantly higher than As(Ⅲ)(1.08 μmol g-1)(P<0.05).The results of short-term exposure experiment(0-8 h)showed the uptake process of As(Ⅲ)and As(Ⅴ)can be well described with Bio-dynamic model(R2>0.95),and the obtained values of uptake rate contants Ku for As(Ⅴ)were in the range of 10.89-22.23 L g-1h-1 at different concentrations,which were all larger than that of As(Ⅲ)in the range of 3.45-14.12 L g-1 h-1.The result implied that the intracellular arsenic accumulation capacity were positively related to its uptake rates when exposed to As(Ⅲ)and As(Ⅴ).During 24 h-exposure to As(Ⅲ)and As(Ⅴ),the intracellular and extracellular arsenic concentrations of algae cells with and without EPS were determined with ICP-MS,respectively.The obtained maximum arsenic uptake capacity were three orders magnitude lower than its maximum extracellular adsorption capacity.Compared to algae cells without EPS,the maximum arsenic adsorption capacity of intact cell under As(Ⅲ)and As(Ⅴ)exposure increased by 39.4%and 42.2%,while the absorption capacity significantly reduced by 43.1%and 20.5%(P<0.05),respectively.Results showed EPS can limit arsenic uptake to improve algal tolerance,and enhance surface adsorption to increase arsenic bioaccumulation capacity in C.pyrenoidosa.(3)The major functional groups in algal EPS for binding As(Ⅲ)and As(Ⅴ)were C-O-C,C-O-H and-NH2.The 3D-EEM spectra of algal EPS indicated the fluorescent substances in EPS were tyrosine-like and tryptophan-like substances,but only the tyrosine-like substances involved in As-EPS interactions.In addition,based on fluorescence quenching titration data(0-10.0 mmol L-1)at four different temperatures of 288K,293K,298K and 303K,together with model fittings of Stern-Volmer and van’t Hoff equations,we finally found the EPS-As fluorescence quenching was a spontaneous static quenching process because of negtive ΔG values and the higher Kq value than 1.0×1010 L mol-1 S-1.Combined qualitative and quantitative analysis of FTIR and XPS spectra further identified three kinds of functional groups of C-O-C,C-O-H and-NH2 in algal EPS as the active binding sites for As(Ⅲ)and As(Ⅴ).