A novel method for extraction of a proteinous coagulant from Plantago ovata seeds for water treatment purposes
Bahman Ramavandi *, Seyedenayat Hashemi, Raheleh Kafaei
EnvironmentalHealthEngineeringDepartment,FacultyofHealth,BushehrUniversityofMedicalSciences, Bushehr7518759577,Iran
GRAPHICAL ABSTRACT
ABSTRACT
Severalchemicalshavebeenappliedintheprocessofcoagulantextractionfromherbalseeds,andthebest extractionhasbeenobtainedinthepresenceofKClorNaNO3[1–3],andNaCl[4].However,themainchallenge posedtothesemethodsofcoagulantextractionistheirrelativelylowefficiencyforwatertreatmentpurposesand theformationofdissolvedorganicmatterduringthetreatmentprocess.Inthesemethodsthesalts,whichhavea one-valancemetal(Na+andK+),aredepositedintheinternalstructureandtheporeofthecoagulant,andmaybe usefulforthecoagulation/flocculationprocess.Inthisresearch,wefoundthatmodifiedmethodsproducedmore denseprotein.Therefore,themodifiedprocedurewasbetterthantheolderoneforremovalofturbidityand harnessfromthecontaminatedwater.Herewedescribeamethodwhere:
AccordingtotheHardy–Schulzerule,weappliedtheFe3+ionsinsteadofNa+andK+fortheextractionofprotein fromPlantagoovataseeds.
Themethodwasnarrowedtoextractproteinbyethanol(defatting)andammoniumacetateandCM-Sepharose (proteinextraction).
Twoconsecutiveelutriationsofcrudeextractwasdirectlyperformedusing0.025-MFeCl3and0.05-MFeCl3
accordingtothebasisoftheion-exchangeprocesses.
*Correspondingauthor.Tel.:+989363311903;fax:+9877334550134.
E-mailaddresses:[email protected],[email protected](B.Ramavandi).
http://dx.doi.org/10.1016/j.mex.2015.05.006
2215-0161/ã2015TheAuthors.PublishedbyElsevierB.V.ThisisanopenaccessarticleundertheCCBYlicense(http://
creativecommons.org/licenses/by/4.0/).
ContentslistsavailableatScienceDirect
MethodsX
journalhomepage: www.elsevier.com/locate/mex
ã2015TheAuthors.PublishedbyElsevierB.V.ThisisanopenaccessarticleundertheCCBYlicense(http://
creativecommons.org/licenses/by/4.0/).
ARTICLE INFO
Methodname:WaterturbiditycoagulantextractedfromPlantagoovataseedsbyusinganFeCl3-inducedcrudeextract Keywords:Plantagoovataseeds,Protein,Extraction,Coagulant
Articlehistory:Received16December2014;Accepted20May2015;Availableonline26May2015
Methoddetails
1. DryPlantagoovataseeds 2. Ammoniumacetate 3. Sodiumhydroxide 4. Hydrochloricacid 5. Ferricchloride 6. Calciumchloride 7. Ethanol(98%) 8. CM-Sepharose 9. Distilledwater
10.Membranefilter(0.42
m
m)ExtractionandpurificationprocessesofcoagulantfromPlantagoovataseeds
ExtractionofthecrudecoagulantfromP.ovataseedswasdoneasfollows:
Theseedsweresoakedindistilledwaterfor1day.
Theobtainedgelatinousmaterialwaspassedthrough0.42
m
mmembranefiltersanddriedat70C inanovenandthenmilledinadomesticblender(VARING).Theobtainedpowderwasdefattedbymixingitwithethanol98%usingamagneticstirrerfor60min.
Then,thesupernatantwasseparatedbycentrifugation(3500rpm,40min),andthesettledpowder wasdriedovernightinanoven(at60C).
Thecrudecoagulantwasextractedfromtheoil-freepowderusing10-mMammoniumacetatein5%
w/w.Themixturewasstirredfor50minusingamagneticstirrer,andthesupernatant,namelythe crudeextract,wasseparatedbycentrifugation(3500rpm,40min).
Thepurificationofthecoagulantproteinfromthecrudeextractwascarriedoutasfollows:
CM-Sepharoseionexchangewasequilibratedusinga10-mMammoniumacetatesolution.
EquilibratedCM-Sepharoseionexchangewasaddedtothecrudeextractin10%(v/v)proportionand mixedusingabladestirrerfor50min.
Finally,theabsorbedcoagulantproteinwaselutedbydifferentconcentrationsofanFeCl3solution.
Thepurificationprocedurewasperformeddirectlywith0.025-MFeCl3firstandthenwith0.05-M FeCl3elutionaccordingtothebasisoftheion-exchangeprocesses[4,5].Inthefirstelution,proteins thatpresumablydidnotleadtoahighercoagulationperformance(efficiencylessthan 45%)and simplyaddeddissolvedorganiccarbon(DOC)(about3.5mg/L)inthetreatedwaterwereremoved.
Consequently,thesecondelutionproducedamorepurifiedcoagulantasitcontainedonlytheactive coagulantproteins.Theelutionstageswererepeatedthreetimestorecoverasmuchcoagulantas possible.Further,afterthebothpurificationofextractionbyFeCl3,theresultantcoagulantwasrinsed manytimeswithdoubledistilledwateruntilFeionsnotdetectableinrinsedwater.Thecoagulant
obtainedinthesecondelutionwasremoved99%ofwaterturbidityandaddedonly0.8mg/LofDOCto thetreatedwater.
Additionalinformation
Theextractedcoagulantwasappliedforremovingofturbidityfromwater.Here, thescanning electronmicroscope(SEM)micrographsofthesettledfloc(sludge)duringwatertreatmentbythe coagulantand the Fouriertransform infraredspectroscopy (FTIR)of the extractedcoagulantare discussed.FromtheSEMmicrographs(Fig.1),itisclearthatthesludgeafterthewatertreatmentwith extractedproteinarehardandsturdy.Thehoneycomb-likestructure,withsmallcolloidalparticles entrappedinit,mightindicatethecontributionofthesweepflocculationmechanismfortheremoval ofcolloidalparticlesinthewatermixture.TheFTIRspectraofextractedproteinpresentedinFig.2 indicatesseveralmainpeaksat3410,2920,1604,and1000cm1onthespectrawhichcorrespondsto OHgroups,CHgroups,C¼Obands,andOCH3groups[6,7].Theseactivegroupsonthesurfaceof extractedproteinouscoagulantmaybe involvedin turbidityremovalfromwaters.The chemical compositionofthesludgeandfreshextractedcoagulantisgiveninTable1.Amongvariouselements,
70 75 80 85 90 95 100
400 900 1400 1900 2400 2900 3400 3900
Trancemittance (%)
Wavenumbers (cm-1) C=O -CH
-OCH3
-OH
Fig.2.FTIRspectraofextractedproteinfromPlantagoovataseeds.
Fig.1.TheSEMimageofsludgeafterwaterturbidityremovalbythecoagulantextractedfromPlantagoovataseeds.
carbonwasfoundtobedominantinthesludge.Regardingtopolysaccharide[Cn(H2O)m],fattyacids [CH3(CH2)nCOOH],andproteingeneralformula[RCH(NH2)COOH],theobtainedcoagulantcould beneitherpolysaccharidenorfattyacidasitcontainedN.Also,regardingtomentionedformula,the polysaccharidescontainhighamountofoxygenratherthanproteinsandfattyacids,sothecoagulant structure could not bemade from polysaccharide. Further, someamino acids such asmet- and cys-aminoacidscontainS,thuspresenceofSinthestructureofcoagulantmightbeanotherreasonfor proteinousoftheextractedcoagulant.However,lowvaluesofPmaybeduetophospholipidsacidsin theextractedcoagulant.Theproteinsnamelymetallo-proteincontainmetalssuchasFe,howeverwe modifiedthecrudeextractwithFeCl3.Naandothersuchmetalisnaturallypresentedinplantseeds.
Thus,itcanbeconcludedthattheextractedcoagulantismajorityproteinandminorityphospholipids acids.
Thecoagulationprocessisusuallyasurfacephenomenon;therefore,theperformanceacoagulant canbesignificantlyinfluencedbythesurfacecharge duetothemassofthecoagulant.Thus,the optimizationofthecoagulantdoseandthebest-requiredmassofthecoagulantforthescale-upand designoflarge-scaleequipmentiseconomicallynecessary.Hence,theeffectofcoagulantdoseonthe Table1
TheEDAXanalysisofthecrudeextract,freshextractedprotein,andsludgeduringwatertreatment.
Element(wt%) Value
Crudeextract Freshextractedprotein Sludge
C 22.10 24.15 58.07
N 0.72 0.82 1.91
O 0.61 0.63 0.761
S 2.22 2.98 3.85
Al 0.16 0.170 0.132
P 0.77 0.52 0.61
Fe 0.64 4.77 4.01
Na 3.01 5.11 6.22
Cl 0.22 2.132 5.80
0 0.5 1 1.5 2 2.5 3 3.5 4
0 20 40 60 80 100 120
0.25 0.5 0.75 1 1.25 1.5 2 2.5 3 5
DOC change (mg/L)
Water Turbidity removal (%)
Dose of coagulant (mg/L) Turbidity removal DOC
Fig.3. EffectsofthecoagulantdoseonwaterturbidityremovalandDOCchangeintreatedwater.
waterturbidityremovalwasconsideredatthewaterpHof7;theobtainedresultsareplottedinFig.3 fromtheperspectiveoftheturbidityremovalefficiencyandchangeoftheDOCofthetreatedwater.As canbeinferredfromFig.3,forthetwolowestcoagulantdosages,i.e.,0.25–0.5mg/L,thefinalturbidity waslow,lessthan5NTU,themaximumadmissibleconcentrationaccordingtoEuropeandirective[8]
fordrinking water. Further,the resultsof theeffect of coagulantdose ontheturbidity removal exhibitedthevarioustrendsasfollow:First,theremovalefficiencyofwaterturbiditydecreasedfrom 99%to26%asthecoagulantdoseincreasedfrom0.25to1mg/L.Then,theturbidityremovalefficiency increasedwithanincreaseinthecoagulantdosefrom1to2mg/L.Finally,theturbidityremoval efficiencydecreasedwhenthecoagulantdosewasmorethan2mg/L(Fig.3).Thisphenomenoncould beexplainedbyacoupleofreasons.Ononehand,atalowercoagulantconcentration,itslongchain adsorbedonthesurfaceofonecolloidparticlewasadsorbedontothesurfacesoftheothers,and thereforetwoormoreparticlesaggregatedbybridgingflocculation.However,whenthecoagulant dosewasincreasedtoacertainvalue(here,1mg/L),theadsorbedaggregationcompletelycoveredthe particlesurfaceandpreventedtheparticlesfromflocculating.Ontheotherhand,underexperimental conditionsandpH=7<pHofzeropointchargeofthecoagulant(thatequalto7.9),thecoagulantwas positively charged as the functional groups bonded with hydrogen ions. Therefore, when the coagulantdosewasincreasedto2mg/Lthepositivelychargedcoagulantadsorbedonthesurfaceof thenegativelychargedcolloidparticlesbychargeneutralizationmechanismandresultingabetter coagulationeffect.
TheuseofnaturalcoagulantsmightincreaseDOCloadinwater,whichmayberesultedthein increasingofmicrobialactivityofthetreatedwater.Further,DOCmightconsumeadditionalchlorine inthewatertreatmentplantandactasaprecursoroftoxicbyproductsduringthedisinfectionprocess.
Thus,theDOCchangesduringthetestsweremonitored.AscanbeseenfromFig.3,theDOCchange duringtheturbidityremovalinthecaseofallthetestedcoagulantdoseswaslessthan3.6mg/L.
However,theDOCchangewasonly0.8mg/Lfortheoptimumcoagulantdoseof0.25mg/L,implying thatthebackgroundDOC19.9%wasincreased.TheDOCinducedtotreatedwaterascomparedtoother coagulants[1–4]usedforturbidityremovalwasverylow.
Acknowledgements
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References
[1]T.Okuda,A.U.Baes,W.Nishijima,M.Okada,Improvementofextractionmethodofcoagulationactivecomponentsfrom Moringaoleiferaseed,WaterRes.33(1999)3373–3378.
[2]T.Okuda,A.U.Baes,W.Nishijima,M.Okada,Coagulationmechanismofsaltsolution–extractedactivecomponentinMoringa oleiferaseeds,WaterRes.35(2001)830–834.
[3]G.Trefalt,I.Szilagyi,M.Borkovec,Poisson–Boltzmanndescriptionofinteractionforcesandaggregationratesinvolving chargedcolloidalparticlesinasymmetricelectrolytes,J.ColloidInterfaceSci.406(2013)111–120.
[4]J.Sanchez-Martin,K.Ghebremichael,J.Beltran-Heredia,Comparisonofsingle-stepandtwo-steppurifiedcoagulantsfrom MoringaoleiferaseedforturbidityandDOCremoval,Bioresour.Technol.101(2010)6259–6261.
[5]B.Ramavandi,TreatmentofwaterturbidityandbacteriabyusingacoagulantextractedfromPlantagoovate,WaterResour.
Ind.6(2014)36–50.
[6]G.Asgari,B.Ramavandi,S.Sahebi,RemovalofacationicdyefromwastewaterduringpurificationbyPhoenixdactylifera, Desalin.WaterTreat.(2013),doi:http://dx.doi.org/10.1080/19443994.2013.823358.
[7]B.Ramavandi,S.Farjadfard,Removalofchemicaloxygendemandfromtextilewastewaterusinganaturalcoagulant,Korean J.Chem.Eng.31(2014)81–87.
[8]M.G.Antov,M.B.Šciban,N.J.Petrovic,Proteinsfromcommonbean(Phaseolusvulgaris)seedasanaturalcoagulantfor potentialapplicationinwaterturbidityremoval,Bioresour.Technol.101(2010)2167–2172.
