多种藻类对As(Ⅲ)的耐受性及吸附研究(英文)_李妍丽.pdf

Study on Tolerance and Biosorption of VariousMicroalgae Species to ArsenicYanli LI,Lin KE*College of Environmental Science and Engineering,South China University of Technology,Guangzhou 510006,ChinaSupported by Natural Science Foundation of Guangdong Province(10151064101000-041)；Ph.D.Funds from Ministry of Education of China(20090172120032).*Corresponding author.E-mail:Received:March 17,2012Accepted:March 30.2012AAgricultural Science&Technology,2012,13(6):1303-1308,1327Copyright 訫 2012,Information Institute of HAAS.All rights reservedResources and EnvironmentAbstractObjective This study aimed to select microalgae species which are capa-ble to effectively remove arsenic contamination from water under natural conditions.Method Four microalgae species Chlorella sp.(zfsaia),Chlorella minata,Chlorella vul-garis and Selenastrum capricormulum were used as experimental materials and cul-tured with six different concentrations of As(III)(0.5,1.0,2.0,5.0,10.0,20.0 mg/L).Biomass,chlorophyll a content and other physiological indicators were determined toinvestigate the arsenic tolerance and biosorption of four microalgae species.ResultChlorella sp.is sensitive to arsenic toxicity,its growth was inhibited when arsenicconcentration exceeded 10 mg/L,with an EC50of 17.32 mg/L;when the arsenicconcentration was 0-20 mg/L,growth of S.c,ww1 and C.v was not affected,whichshowed relatively high tolerance to arsenic,with arsenic removal rates of 77.02%,72.18%and 81.36%respectively after 24 h.Conclusion This study indicates thatmicroalgae have good application prospects for processing arsenic wastewater andbeing indicator plants of arsenic wastewater.Key words As(III);Microalgae;Toxicity effect;BiosorptionArsenic is ubiquitous in the soil,sediment,water,atmosphere,and even organisms,which isodorless and colorless,with strongtoxicity.Arsenic contamination in theenvironment poses a serious threat tohuman health,which has been recog-nized as a global public health prob-lem.High levels of arsenic in waterbodies are caused by natural or hu-man activities,such as the geothermalactivities or weathering of ore,mining,manufacturinganduseofarsenicpesticides.Under natural conditions,arsenic has four stable valence statesin different redox environments,in-cluding+5(arsenate),+3(arsenite),0(arsenic)and-3(arsine).The con-centration and existing form of arsenicdetermine its toxicity and removal effi-ciency in water treatment.Arseniccontamination of groundwater is themost serious in most basins of MeikeNa-Brahmaputra River in Bangladeshand India,where as many as 500 mil-lion people are exposed to arsenicthreat1.According to statistics,thereare at least three million people livingin arsenic contaminated areas in Chi-na,and more than 30 000 patientshave been confirmed arsenic poison-ing2.Specifically,Chia Nan,Kuitun,Hetao and Datong are typical arsenicpoisoning areas caused by drinkingwater3.According to reports,the ar-senic poisoning accident happened inHechi in October 2008 was caused byarsenic contamination of drinking wa-ter,resulting in about 450 victims.A large number of researches arereported on the acute toxicity tests ofheavy metals(Hg,Cu,Cd and As)byusingaquaticorganisms,includingtilapia,carp,protozoa,aquatic vascu-lar plants,etc4-7.However,few resea-rches are reported on the toxic effectsof arsenic on plant cells,especially onalgae.Algae are very sensitive to thetoxicity of many pollutants8-9.To bespecific,green algae is the simplestandmostprimitivealgaespecies,which plays very important roles forthe balance and stability of aquatic e-cosystems as the primary producers ofaquatic ecosystems.Therefore,it isvery necessary to study the feedbackof algae species to arsenic contamina-tion,including their response,adsorp-tion and metabolism to the toxicity ofarsenic.Among heavy metals,toxic ef-fects of Cu on algae are the most in-vestigated10-11.Davies12reported thatDunaliella salina can form giant cellswithout splitting when the Hg concen-tration reaches 10 mg/L.In 1984,Hosea et al.13found that Chlorella vul-garishasahighaffinitytogold.Travieso et al.14reported that thegrowth of Chlorella is not affected un-der 45 mg/L of Cr(VI),while Scenede-smus acuminatus shows no growthunder Cr concentration higher than 15mg/L.Xiao and Yamaoka et al.15-17found that the toxicity threshold of As(V)is 10-41 mol/dm3to Phaeodactylumtricornutum Bohlin and 10-49 mol/dm3toDirateriainornate.Inaddition,Dunaliella sp.was found to have higharsenic removal efficiency17.Algae test is integral to the eco-toxicological studies,with short growthcycle,easy isolation and culture,directobservation of poisoning symptomsand other characteristics.Therefore,algae are ideal materials for toxicitytest.In this study,in accordance withthe methods prescribed by the Inter-national Standards Organization(ISO,2010)18,four microalgae species wereused as experimental biological mate-rials to investigate the toxicity effect ofAs(III)and the adsorption of microal-gae species with high tolerance to ar-senic based on a variety of indicators.Materials and MethodsMaterialsExperimental materialsMicroalgaeDOI:10.16175/ki.1009-4229.2012.06.002Agricultural Science&TechnologyAgricultural Science&TechnologyVol.13,No.6,20122012Table 1 Microalgae species used in this experimentNo.Microalgae speciesAbbreviation1Selenastrum capricormulumS.c2Chlorella vulgarisC.v3Chlorella minataww14Chlorella sp.(zfsaia)zfsaiaTable 2 Various As(III)concentrations for toxicity testTreatmentFinal concentrationof As(III)mg/LVolume of As(III)lVolume ofmediummlVolume of microalgaesolutionml10020220.512.520231.02520242.05020255.0125202610.0250202720.0500202species used in this experiment wereprovided by the City University of HongKong,and the specific species areshown in Table 1.Reagents and culture mediumTrivalent arsenic(As2O3)was purcha-sedfrom Shanghai Jingchun ReagentsCo.,Ltd.Experimental drugs used inthis experiment were all of analyticalgrade.Bristol medium(ISO,2004)was adopted in this experiment,con-taining 25 g/L of NaNO3,7.5 g/L ofK2HPO4,17.5 g/L of KH2PO4,11.8 g/Lof MgSO47H2O,2.5 g/L of NaCl,2.5 g/L of CaCl22H2O,0.5 g/L ofFeCl36H2O,0.03 g/L of MnCl24H2O,0.002 g/L of CoCl26H2O,0.001 g/L ofCuSO45H2O,0.004g/LofZnSO47H2O,0.002 g/L of NaMoO42H2O and 0.54g/L of EDTA.The cultivation liquid wasprepared by using pure water,with ini-tial pH of(6.50 0.2).Main equipments and instrumentsMultifunctional Microplate Reader(BiotekServices,Inc.);centrifuge(Heraeus,Germany);desktop freezethermostat oscillator(Shanghai Tai-cang);Vertical Pressure Steam Steril-izer(Shanghai Shenan Medical In-strument Factory);pH meter(OAK-TON,U.S.);hydride generation-atom-ic fluorescence spectrophotometer(Ji-tian AFS-830).To remove external ar-senic and other metal contamination,all glassware used in this experimentwere soaked with 10%nitric acid for24 h,rinsed with ultrapure water,andthen placed in the oven and dried at 80 before use18.MethodsToxicity testToxicant concentra-tions were set in accordance withTable 2,different volumes of As(III)solution was added to the preparedmedium as cultivation liquid for mi-croalgae.Liquid of microalgae at loga-rithmic growth phase was inoculated tocultivation liquid with different concen-trations of As(III).Sampling was con-ducted every day for 7 d to determinethe growth conditions of microalgae,microalgae liquid without arsenic un-der thesamecultivationconditionswas used as the control.The experiment was carried out in50 ml wide-mouth erlenmeyer bottles.Mouths of all the flasks were sealedwith sterile seal membrane.Threeparallel samples were set per concen-tration.The cultivation liquid was ster-ilized under high pressure at 121 for30 min,and then sterilized by ultravio-let radiation for 30 min in a cleanbench before inoculation.All experi-mental systems were cultured in themodifieddesktopfrozenconstant-temperatureoscillatoratshakingspeed of 150 r/min,culture tempera-ture of(25 2),light intensity of(3 000 5 00)lx),light-dark ratio of 12h:12 h under sterile conditions.Adsorption of As(III)by microalgaeAs2O3was prepared into 1.0 mg/LAs(III)solution with sterile deionizedwater.Liquid of microalgae with vigor-ous growth at logarithmic phase wasadded to the As(III)solution by 1:10.Two milliliters of the mixture was col-lected respectively after 0,10,20,30,60,90,120,240,360 and 720 min,todetermine the arsenic concentration inthe mixture.Meanwhile,thesameconcentration of As(III)solution with-out microalgae was set as the control.Determination methodsDetermi-nation of the growth of microalgae:samples were collected regularly everyday to determine the OD value at 680nm with a multifunctional microplatereader.Determination of chlorophyll acontent:2 ml of experimental microal-gae liquid was collected every 24 h,centrifuged at 10 000 r/min for 1 min,removed the supernatant,added with1 ml of 100%methanol,mixed fullywith shaking,placed in the dark for 2 h,mixed evenly,and then centrifuged at10 000 r/min for 1 min;200 l of su-pernatant was collected to measurethe OD value at 663 and 645 nm with amultifunctional microplate reader.De-termination of heavy metal ions:about2 ml of cultivation solution containingthe same concentration of As(III)wascollected and centrifuged at 10 000r/min for 1 min to determine the con-centration of As(III)in the supernatantby using a hydride generation-atomicfluorescence spectrophotometer.Data processing(1)Growth condi-tions of various microalgae specieswere compared based on the numberof cells,OD680,chlorophyll a concen-tration and growth inhibition rate(IR).Specifically,the chlorophyll a concen-tration and growth inhibition rate werecalculated according to the followingformula:Cholorophy II a(mg/L)=12.7A663-2.69A645;=(ln Nt-ln N0)/(t-t0);To be specific,indicates therelative maximum growth rate;N0indi-cates the initial biomass;Ntindicatesthe biomass at the 96thh.IR=(b-t0 x)/b 100;To be specific,IR indicates thegrowth inhibition rate;bindicates therelative maximum growth rate of thecontrol group;t0 xindicates the relativemaximum growth rate of microalgaewith addition of experimental material.R=(0-t)/0 100%;To be specific,R indicates the re-moval rate;0indicates the initial massconcentration of metal ions(mg/L);tindicates the mass concentration ofmetal ions(mg/L).(2)Experimental data were pro-cessed with Microsoft Excel,Origin 8.0and SPSS 13.0.Three parallel sam-ples were set for each treatment to re-ducetheexperimentalerror.Data1304Agricultural Science&TechnologyVol.13,No.6,2012Agricultural Science&Technology2012were processed by single-factor anal-ysis of variance,P0.05),compared with microalgae inthe control group;when the concen-tration of arsenic ions reached 20mg/L,microalgal cells maintained nor-mal growth.Data shown in Table 3 indicatedthat with the increase of arsenic con-centration,the growth rate of microal-gal cells showed an overall downwardtrend,while the microalgal cells show-ed significant growth after a slow lagphase,which might be due to that themetallothionein-like proteins in the mi-croalgal cells were induced by arsenicand conferred microalgal cells certainresistance against arsenic,thereby re-moving the toxicity of arsenic tem-porarily19.However,zfsaia had sensi-tivity to the toxicity of arsenic,the high-er the concentration of arsenic ionswas,the stronger the inhibition ofgrowth would be;when the concentra-tion of arsenic ions reached 10 mg/L,growth of zfsaia was inhibited.Accord-ing to the probability and statisticsmethod20,50%efficiency concentra-tion(EC50)of As(III)to zfsaia was cal-culated as 17.32 mg/L in the 96thh.Kang et al.19studied the effect of dif-ferent concentrations of As2O3on thegrowth and photosynthesis of Cyano-bacteria,result showed that when As(III)concentration in the water reached4 mol/L,arsenic would lead to thedeath of Cyanobacteria cells.Effect of different concentrations ofAs(III)on chlorophyll a contentAs can be seen from Fig.3,aftercultured for 96 h,chlorophyll a con-tents of S.c and C.v showed the samevariation trend with the increase of ar-senic ion concentration and reachedthe maximum when the concentrationof arsenic ions was 5.0 mg/L,whichwere 1.96 and 1.53 mg/L,respectively;however,chlorophyllacontentofzfsaia was reduced with the increase ofarsenic ion concentration,showing asignificant negative correlation,andthe reduction of chlorophyll a contentwas relatively greater with the increaseof arsenic ion concentration,whichmight be due to that excessive arsenicions destroyed the chloroplast struc-ture of microalgae and inhibited thesynthesis of chlorophyll,thereby lead-ing tothesignificantdecreaseofchlorophyll a content and the inhibitionof cell growth by arsenic.Comparedwiththeotherthreemicroalgaespecies,ww1was quite different,thechlorophyll a content decreased to theminimum when the concentration of ar-senic ions was 5.0 mg/L,and then con-tinued increasing tothemaximumwhen the concentration of arsenic ionswas20 mg/L,which reached 1.27 mg/L,indicating that ww1has very high re-sistance against arsenic.According to the effect of differentAs(III)concentrations on the growth offour microalgae species and the con-tent of chlorophyll a,the tolerance offour microalgae species to arsenitewas different.Specifically,the toler-ance of S.c,ww1and C.v is thestrongest,while zfsaia is relatively sen-sitive to the toxicity of As(III).Previousstudies showed that Chlorella sp.12has high tolerance to As(III),with anEC50of 25.2 mg/L;C.v can grow nor-Fig.1 Growth curves of four microalgae speciesFig.2 Effect of different concentrations of As(III)on the growth of various microalgaespecies1305Agricultural Science&TechnologyAgricultural Science&TechnologyVol.13,No.6,20122012Table 3 Growth rate of four microalgae cultured for 96h under different concentrations ofAs(III)Concentra-tionmg/LGrowth rate/hS.cww1C.vzfsaia00.009 10.009 00.006 90.012 80.50.010 10.006 60.006 90.011 410.010 40.007 70.006 50.008 520.010 60.007 40.006 40.001 350.010 60.008 20.005 70.010 2100.009 50.008 40.006 50.010 8200.008 70.007 50.007 00.006 1mally with As(III)concentration of 40mg/L21-22;Selenastrum capricornutum,Scendesmus quadricauda and Chlam-ydomonas reinhardtii also have veryhigh tolerance to arsenic,with EC50of31,61 and 202 mg/L23-25.In this study,result shows that various microalgaespecies have relatively high toleranceto heavy metals;therefore,the ad-sorption of As(III)by S.c,ww1and C.vwas investigated.Adsorption of As(III)by microalgaeArsenic removal capabilities ofthree microalgae species were deter-mined in this study.Variations of As(III)absorption and removal rate R byvarious microalgae species with theinitial mass concentrations of 1.0 mg/Lwere shown in Fig.4 and Fig.5.As can be seen directly from Fig.4,ww1,C.v and S.c showed similar varia-tion rules of adsorption to arsenic ions,which all showed the adsorption-des-orption alternate phenomenon but nolinear relationship.Within the initial 20min of the experiment,the concentra-tion of arsenic ions rapidly declinedand then increased after 30 min