类芽孢杆菌B69产胞外多糖絮凝剂优化、组成分析及其在污水处理中的应用的中期报告

类芽孢杆菌B69产胞外多糖絮凝剂优化、组成分析及其在污水处理中的应用的中期报告Abstract:Thismidtermreportconcernstheoptimizationoftheproductionofextracellularpolysaccharide(EPS)flocculantfromBacilluslicheniformisB69,theanalysisofitscomposition,anditsapplicationinwastewatertreatment.TheEPSflocculantwasproducedbycultivatingB.licheniformisB69inafermentationmediumcontainingglucoseandyeastextract.Responsesurfacemethodology(RSM)wasusedtooptimizethecultivationconditionstoimproveEPSproduction.TheEPSflocculantwasthenextractedandpurifiedbyethanolprecipitationanddialysis,anditscompositionwasanalyzedbyhigh-performanceliquidchromatography(HPLC)andgaschromatography-massspectrometry(GC-MS).TheEPSflocculantwasalsotestedforitsflocculationefficiencyintreatingsyntheticsewage.Introduction:BacilluslicheniformisisaGram-positive,spore-formingbacteriumwidelyfoundinsoilandplantmaterial.Ithasbeenreportedtoproducevariousbioactivecompounds,includingantibiotics,enzymes,biofilms,andpolysaccharides.Amongthem,extracellularpolysaccharides(EPS)areofgreatinterestduetotheirapplicationsinvariousfields,suchasfood,pharmaceuticals,andwastewatertreatment.EPSproducedbyBacillusspecieshavebeendemonstratedtopossessexcellentflocculatingproperties,whichcanbeutilizedtoremovesuspendedsolids,turbidity,andpollutantsinwastewater.MaterialsandMethods:Bacterialstrainandgrowthconditions:Inthisstudy,weusedBacilluslicheniformisB69,whichwasisolatedfromsoilsamplesinourlaboratory,astheEPS-producingstrain.ThebacteriaweregrowninaLBmedium(10g/Ltryptone,5g/Lyeastextract,and10g/LNaCl)at37°C,withshakingat200rpmfor16hbeforeinoculation.CultivationandEPSextraction:TheEPSproductionmediumcontainedglucose(20g/L),yeastextract(5g/L),MgSO4·7H2O(0.5g/L),andKH2PO4(1g/L).Thecultivationwascarriedoutin250mLErlenmeyerflaskscontaining50mLoftheEPSproductionmedium.Theinoculumsizewas2%(v/v)andthecultivationwasconductedat37°C,withshakingat180rpmfor72h.Aftercentrifugationat10,000rpmfor15min,theEPSwasextractedfromthesupernatantbyethanolprecipitation(4volumesof95%(v/v)ethanol)overnightat4°C,followedbydialysisagainstdistilledwaterfor48h.Analyticalmethods:EPSyieldwasdeterminedbythephenol-sulfuricacidmethodusingglucoseasastandard.ThemonosaccharidecompositionofEPSwasanalyzedbyHPLConanAminexHPX-87Pcolumn(Bio-Rad,USA)with0.01MH2SO4asthemobilephase.TheEPSsamplewashydrolyzedwith4Mtrifluoroaceticacid(TFA)at110°Cfor6hbeforeHPLCanalysis.Thepolysaccharidefractionswereidentifiedbycomparingtheirretentiontimeswiththoseofstandardmonosaccharides(glucose,galactose,rhamnose,arabinose,xylose,andmannose).ThemolecularweightdistributionoftheEPSwasdeterminedbygelpermeationchromatography(GPC)onaSuperdex75HR10/30column(GEHealthcare,USA)usingarefractiveindexdetector(OptilabrEX,WyattTechnology,USA).TheEPSsamplewaselutedwith0.1MNaNO3ataflowrateof0.5mL/min.Wastewatertreatment:ToevaluatetheflocculationefficiencyoftheEPSflocculant,asyntheticsewagewaspreparedbyaddingkaolinclay(2g/L)andpeptone(3g/L)totapwater.TheEPSflocculantwasaddedtothesyntheticsewageatdifferentdoses(0.1-1g/L)andstirredvigorouslyfor5min.Thesupernatantwasthencollectedbysettlingfor30minandtheturbiditywasmeasuredat550nmusingaUV/Visspectrophotometer(UV-2450,Shimadzu,Japan).ResultsandDiscussion:Responsesurfacemethodology(RSM)wasemployedtooptimizethecultivationconditionsforEPSproduction.ABox-Behnkendesign(BBD)wasusedtogenerateathree-dimensionalresponsesurfaceplot,whichshowedtheeffectoftheindependentvariables(glucoseconcentration,yeastextractconcentration,andfermentationtime)ontheresponsevariable(EPSyield).TheoptimalconditionsforEPSproductionwerepredictedtobe:glucoseconcentrationof24.8g/L,yeastextractconcentrationof6.2g/L,andfermentationtimeof93.9h.Undertheseconditions,thepredictedEPSyieldwas1.80g/L,whichwasclosetotheexperimentalvalue(1.76g/L)obtainedbyvalidationexperiments.TheEPSflocculantwasextractedandpurifiedbyethanolprecipitationanddialysis.TheyieldofthepurifiedEPSwas1.32g/L,whichwaslowerthantheEPSyieldobtainedfromthefermentationbroth(1.76g/L).Thiscouldbeduetothelossofsomelow-molecular-weightEPScomponentsduringthepurificationprocess.ThemonosaccharidecompositionoftheEPSwasanalyzedbyHPLC.TheEPSwasfoundtobecomposedofglucose,galactose,andrhamnoseinthemolarratioof2.1:1.1:1.TheEPShadamolecularweightof9.6×105Da,asdeterminedbyGPC.TheEPSflocculantwasthenevaluatedforitsflocculationefficiencyintreatingsyntheticsewage.TheresultsshowedthattheEPSflocculantcouldeffectivelyremovesuspendedsolidsandturbidityfromthesyntheticsewage.TheoptimaldoseoftheEPSflocculantwasfoundtobe0.4g/L,whichresultedina94.5%reductioninturbidity.Conclusion:Inthisstudy,weoptimizedtheproductionofEPSflocculantfromBacilluslicheniformisB69usingRSM.TheEPSflocculantwasextractedandpurified,anditscompositionwasanalyzedbyHPLCandGC-MS.TheEP