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RESEARCHARTICLEOpenAccessExperimentaldesignapproachtotheoptimizationofPAHsbioremediationfromartificiallycontaminatedsoil:applicationofvariablesscreeningdevelopmentMasoumehRavanipour1,RoshanakRezaeiKalantary2,AnoushiravanMohseni-Bandpi3*,AliEsrafili2,MahdiFarzadkia2andSamirehHashemi-Najafabadi4AbstractBackground:TheeffectivenessofbioremediationsystemsforPAH-contaminatedsoilmaybeconstrainedbyphysicochemicalpropertiesofcontaminantsandenvironmentalfactors.
Informationonwhatisthemosteffectivefactorinbioremediationprocessisessentialinthedecisionofwhatstimulationscanbetakentoassistthebiodegradationefficacy.
Methods:Inthisstudy,fourfactorsofsurfactant(Tween80),humicacid(HA),salinityandnutrientsina24fullfactorialdesignwerescreenedinbioremediationofphenanthrenecontaminatedsoilbyusingaconsortiumofbacteria.
Results:Betweentheemployedlevelsofthefactorsonlysalinityhadnotsignificanteffect.
Optimalconcentrationsofsurfactant,HAandnutrientwereobtainedbyaresponsesurfacedesign.
Forphenanthrenebiodegradation,acentralcompositefacecentreddesign(CCFD)showedthatnutrient,surfactantandHAconcentrationshadhighlysignificant,significantandinsignificanteffects,respectively.
Thebestconditionswith87.
1%phenanthrenebiodegradationwere150mgHA/Kgsoil,12.
68μg/Lsurfactant,andnutrientsasK2HPO4,0.
8;KH2PO4,0.
2andKNO3,1g/L.
Ahighsimilaritywasbetweenthemodelpredictionandexperimentalresults.
Conclusions:Thisstudyshowedthatnutrientwith81.
27%efficiencycouldbeconsideredasthemosteffectivefactorforpracticalimplicationsofbioremediationprocessforPAHscontaminatedsoilcleanupstrategies.
Keywords:Bioremediation,PolycyclicAromaticHydrocarbons,ResponseSurfaceMethod,Nutrient,Tween80BackgroundPetroleumderivativesareintroducedintotheenviron-mentthroughdifferentwayssuchasanthropogenicac-tivities,incompletecombustionofpetroleumproducts,woodandcoal,undesirabledischargingofoiltankers,spillsaroundpetroleumrefineriesandgasplantfacilities[1].
PolycyclicAromaticHydrocarbons(PAHs)areagroupofthesecompoundswithcarcinogenicandtoxicpotentially[2,3]whichcontributetoenvironmentalcontaminationandhealthhazards.
SoilandsedimentsarethemostimportantenvironmentalreservoirforPAHs.
Atpresent,employingbiologicaltreatmentisthemostpopularandcost–effectivestrategyamongthedifferentmethodstoremovethesepollutantsfromthesoil[4].
However,successfulapplicationofbioremediationisoftenlimitedbyenvironmental,physicalandchemicalfactors[5]suchasavailabilityofpollutantstoundergobiologicaltransformations[6]toxicityandcomplexstructuresofPAHsderivatives,limitationfornitrogen,phosphorusorothernutrients,pHandtemperature[7].
OneofthemainfactorslimitingbioavailabilityisthelowaqueoussolubilityofPAHs.
Additionofsurfactant*Correspondence:a.
mohseni8@yahoo.
com3DepartmentofEnvironmentalHealthEngineering,SchoolofPublicHealth,ShahidBeheshtiUniversityofMedicalSciences,Tehran,IranFulllistofauthorinformationisavailableattheendofthearticleJOURNALOFENVIRONMENTALHEALTHSCIENCE&ENGINEERING2015Ravanipouretal.
;licenseeBioMedCentral.
ThisisanOpenAccessarticledistributedunderthetermsoftheCreativeCommonsAttributionLicense(http://creativecommons.
org/licenses/by/4.
0),whichpermitsunrestricteduse,distribution,andreproductioninanymedium,providedtheoriginalworkisproperlycredited.
TheCreativeCommonsPublicDomainDedicationwaiver(http://creativecommons.
org/publicdomain/zero/1.
0/)appliestothedatamadeavailableinthisarticle,unlessotherwisestated.
Ravanipouretal.
JournalofEnvironmentalHealthScienceandEngineering(2015)13:22DOI10.
1186/s40201-015-0178-ycanbeusedforincreasingthephasepartitioningofor-ganiccompoundsandtheirbioavailability.
Ontheotherhandbioavailabilitymaybedecreasedbyuptakingofcontaminantsintothesurfactantmicelle[8].
Thus,theconcentrationofsurfactantplaysanimportantroleinbiodegradationofPAHs.
Manystudieshavebeenconductedtoovercomeprob-lemsrelatedtothepoorbioavailabilityofPAHsbyusingorganicmatter.
BorresenandRike[9]haveshownthathumicsubstances(HS)canincreasethesolubilityofPAHsleadsinincreasingthebioavailabilityofPAHsinsoil.
Nutrientsuchasnitrogenandphosphorusaretheotherimportantfactorsonbiodegradation.
Microorgan-ismsneedfornutrientssimilartotheircompositionofcells[10,11].
Betancur-GalvisLAetal.
[12]usedbiosolidandinorganicfertilizerinbioremediationofphenan-threne.
Theyfoundthattheremovalofphenanthreneinthesoilwithfertilizationwas25foldmorethantheothersoils.
Thereisarelationbetweenmineralizationratesofphenanthreneandtheinitialconcentrationsofnitrogenandphosphorusinthesoils[13].
Themarinesoilandsedimentisoneofthemostpet-roleumcontaminantssites,sothesalinityisanimport-antfactorwhichmustbeinvestigated.
Minai-TehraniD.
etal.
[14]showedthatincreasingsalinitycontentofsoilhaddecreasingeffectonthebiodegradationoftotalcrudeoilandPAHs.
Thebioremediationstrategyisdependentontheopti-mizingthefactorswhichaffectonthemicrobialgrowthandbiodegradationofpollutant[15].
Incombinationofseveralfactors,theeffectofanyonemaybeinfluencedbytheothersandinteractionsamongthemmaybeoc-curredtoo.
Theexperimentaldesigncanbeusedforoptimizingoperationalconditionsforthemultivariablesystem[16]andtheinteractionbetweenvariableswouldbeconsid-eredtoo[17].
Thenumberofexperiment'srunswouldbereducedbystatisticaldesignofexperiments[17,18].
Inthisstudynumerousfactors,involvingsurfactant(Tween80(Su.
)),salinity(Sal.
),soilnutrients(N,P(Nu.
))andorganicmatters(humicacid(HA)),individu-allyandincombination,havebeenstudiedtoremedyPAHartificiallycontaminatedsoils.
Theaimofthisstudywastoinvestigatetheeffectofphysicochemicalfactors;HA,surfactant(Tween80),salinityandnutrient(N,P)togetheronthebioremediationofphenanthreneathree-ringPAH,anappropriatemodelcompound,insoilslurry.
Inordertofindoutthemosteffectivefactorandthesequenceimportanceoftheminbiodegradationofphenanthreneathree-ringPAHinthesoilslurry,thestudywasconductedintwophase:1)Screeningthefactorsbyusingfullfactorialexperimentaldesign,and2)Optimizationofthephenanthrene-contaminatedsoilbiodegradationbyusingacentralcompositefacecentereddesign(CCFD)underresponsesurfacemeth-odology(RSM).
Thentheoptimizedconditionwasex-aminedinPAHsrealcontaminatedsoil.
MethodsChemicalsAcetoneinHPLCgradewaspurchasedfromROMILCompany.
Phenanthrene(Purity>98%),NaClandchem-icalmaterialsformineralsaltmedium(MSM)werepur-chasedfromMerckCompany.
HAandTween80weresuppliedbySigmaAldridgeandFluka,respectively.
TheformulaofHAwasC17H17BrN2O5.
NutrientBrothandR2AAgarwerepurchasedfromBIOMARKCompany.
PhenanthrenebiodegradationCleansoilwascollectedfromadepthof5–20cmofground'ssurface,Tehran,Iran.
Itwasairdriedandpassedthrougha2-mmsieve.
Thesoilwasclassifiedassand(consistedof89%sand,11.
9%siltand5%clay)bytheuseofstandardsieves.
Totalnitrogenandphosphoruswere0.
025%and0.
0012%,respectively.
Totalorganiccarbonwas0.
18%.
ThepHandelectricalconductivity(EC)were7.
4and3.
2ds/m,respectively.
Twogramsofdrysoilwasplacedinto50mLErlen-meyerflaskasnon-continuousbioreactors.
Thebioreac-torscontainingcleansoilwereautoclaved.
Then,itwasspikedwithdissolvedphenanthreneinacetonetohave500mgphenanthrene/kgdrysoil.
Thebioreactorscon-tainingspikedsoilwereplacedinashaker(Heidolph,ProMax2020model)atthevelocityof180rpminroomtemperatureanddarkconditiontohaveauniformdis-persionofphenanthreneandevaporationofacetone.
ThesoilwasinoculatedwithaconsortiumofbacteriaindifferentconcentrationofMSMwithanopticaldens-ityof1at630nm[5]usingCECILUV/Visspectropho-tometer(model7100).
ThebacterialconsortiumwasconsistedofBacillussporogenes,Bacilluslicheniformis,Capnocytophagaochracea(presumably),AcinetobactersporogenesandStaphylococcusxylosus.
Enrichmentoftheconsortiumandthepotentialofitinbioremediationofphenanthrenecontaminatedsoilshadbeenprovedinourpreviousstudy[6].
ThebaseofMSMwascontainedofthefollowing(perliter):0.
2gMgSO4.
7H2O,0.
1gCaCl2.
2H2O,0.
1gNaCl,0.
01gFeCl3.
6H2Oand1mLtraceelementsolution.
Thetraceelementsolutioncon-tainedthefollowing(perliter):23mgMnCl2.
2H2O,30mgMnCl4,32mgH3BO3,39mgCoCl2.
2H2O,50mgZnCl2,30mgNaMnO4.
2H2Oand20mgNiCl2[5].
ThentheamountofK2HPO4,KH2PO4,KNO3,HA,Sur-factantandNaClwereaddedaccordingtoTables1and2forphase1and2respectively.
ThepHwasadjustedto6.
8±0.
2usingapHmeter(HACH40dmodel).
Attheend,theslurryofsoilliquidratiowas10%w:v[6].
AllthesamplesandtheirsimilarblankswereputintheRavanipouretal.
JournalofEnvironmentalHealthScience&Engineering(2015)13:22Page2of10Table1Actualvaluescodedandofvariablesusedinthefullfactorial(24)designRunActualvalue/codedLevelsSalinity(Sal.
)%W/VSurfactant(Su.
)L/LμHumicAcid(HA)mg/KgNutrient(N,P)(Nu.
)g/LKNO3KH2PO4K2HPO4R10(1)0(1)0(1)0.
000132(1)0.
000103(1)0.
0017(1)R2200(1)0(1)0(1)0.
000132(1)0.
000103(1)0.
0017(1)R30(1)13(1)0(1)0.
000132(1)0.
000103(1)0.
0017(1)R4200(1)13(1)0(1)0.
000132(1)0.
000103(1)0.
0017(1)R50(1)0(1)2(1)0.
000132(1)0.
000103(1)0.
0017(1)R6200(1)0(1)2(1)0.
000132(1)0.
000103(1)0.
0017(1)R70(1)13(1)2(1)0.
000132(1)0.
000103(1)0.
0017(1)R8200(1)13(1)2(1)0.
000132(1)0.
000103(1)0.
0017(1)R90(1)0(1)0(1)0.
8(1)0.
2(1)1(1)R10200(1)0(1)0(1)0.
8(1)0.
2(1)1(1)R110(1)13(1)0(1)0.
8(1)0.
2(1)1(1)R12200(1)13(1)0(1)0.
8(1)0.
2(1)1(1)R130(1)0(1)2(1)0.
8(1)0.
2(1)1(1)R14200(1)0(1)2(1)0.
8(1)0.
2(1)1(1)R150(1)13(1)2(1)0.
8(1)0.
2(1)1(1)R16200(1)13(1)2(1)0.
8(1)0.
2(1)1(1)Table2ExperimentalmatrixforcentralcompositedesignforoptimizationRunActualvalue/codedlevelsRemovedamountofphenanthrene(mg/Kg)HA(mg/Kg)Su.
(μg/L)Nu.
(N,P)(g/L)K2HPO4KH2PO4KNO3ExperimentedvaluePredictedvalueR10(1)*5(1)0.
4(1)0.
1(1)0.
5(1)196.
4190.
67R2150(+1)*5(1)0.
4(1)0.
1(1)0.
5(1)222213.
63R30(1)13(+1)0.
4(1)0.
1(1)0.
5(1)208.
4214.
23R4150(+1)13(+1)0.
4(1)0.
1(1)0.
5(1)275.
7274.
69R50(1)5(1)0.
8(+1)0.
2(+1)1(+1)332.
1331.
61R6150(+1)5(1)0.
8(+1)0.
2(+1)1(+1)342.
8335.
47R70(1)13(+1)0.
8(+1)0.
2(+1)1(+1)380.
8387.
67R8150(+1)13(+1)0.
8(+1)0.
2(+1)1(+1)424.
8429.
03R90(1)9(0)0.
6(0)0.
15(0)0.
75(0)247.
6281.
05R10150(+1)9(0)0.
6(0)0.
15(0)0.
75(0)260.
8313.
21R1175(0)*5(1)0.
6(0)0.
15(0)0.
75(0)283.
6267.
85R1275(0)13(+1)0.
6(0)0.
15(0)0.
75(0)380326.
41R1375(0)9(0)0.
4(1)0.
1(1)0.
5(1)234.
2223.
31R1475(0)9(0)0.
8(+1)0.
2(+1)1(+1)394.
41370.
95R1575(0)9(0)0.
6(0)0.
15(0)0.
75(0)340297.
13R1675(0)9(0)0.
6(0)0.
15(0)0.
75(0)279297.
13R1775(0)9(0)0.
6(0)0.
15(0)0.
75(0)285.
2297.
13R1875(0)9(0)0.
6(0)0.
15(0)0.
75(0)260.
5297.
13*LowLevel:(1);Middle:(0);HighLevel:(1).
Ravanipouretal.
JournalofEnvironmentalHealthScience&Engineering(2015)13:22Page3of10shakeratthevelocityof180rpminroomtemperature(22±3°C)for8weeks.
ExperimentaldesignsTheexperimentwasaccomplishedintwophases;screeningofimportantvariablesandthelevelsofthemthatsignificantlyinfluencedphenanthrenedegradation,followedbyoptimizationofvariableslevelsbyusingre-sponsesurfacemethodology.
ScreeningofvariablesScreeningstepwasusedforidentifyingtheimportantoffourfactorsbasedonfullfactorialdesign(24).
TheserelevantfactorswereTween80,asanon-ionicsurfac-tant(Su),HA,NutrientandSalinityintwolevelsofhigh(+1)andlow(1).
Theimportanceofthefactorswasonthebaseofthelargesteffectonthebiodegradationofphenanthreneincontaminatedsoil.
Inthisphase16-runwasappliedtoevaluatefactors(variables).
Table1illus-tratesthevariablesandtheircorrespondinglevels.
ThelevelsofthefactorswereonthebaseofpreviousstudiesinliteratureforPAHsbioremediation[14,19-21].
Alltheexperimentrunswereperformedintriplicatesandtheaverageofthemwastakenastheresult.
Eachofexperimentrunhadthesimilarchemicalcontrolwithoutanyinoculation.
ThestatisticalsoftwareDesignExpertV.
7,(Stat-Ease,USA)wasusedtoevaluatetheanalysisofvariance(PSurfactant>Humicacid>Salinity.
Thebiodeg-radationrateatoptimumconditionwas7.
78mgphenan-threne/kgsoil/day.
CompetinginterestsTheauthorsdeclarethattheyhavenocompetinginterests.
Authors'contributionsMR,RRK,AM,AE,MFandSHNcarriedoutthearticlewiththetitleofExperimentaldesignapproachtotheoptimizationofPAHsbioremediationfromartificiallycontaminatedsoil:Applicationofvariablesscreeningdevelopmentparticipatedinthesequencealignmentanddraftedthemanuscript.
Allauthorsreadandapprovedthefinalmanuscript.
AcknowledgmentTheauthorssincerelythanktheDepartmentofEnvironmentalHealthEngineering,TehranUniversityofmedicalSciencesforfinancialsupportofthisproject.
Authordetails1DepartmentofEnvironmentalHealthEngineering,SchoolofPublicHealth,TehranUniversityofMedicalSciences,Tehran,Iran.
2DepartmentofEnvironmentalHealthEngineering,SchoolofPublicHealth,IranUniversityofMedicalSciences,Tehran,Iran.
3DepartmentofEnvironmentalHealthEngineering,SchoolofPublicHealth,ShahidBeheshtiUniversityofMedicalSciences,Tehran,Iran.
4DepartmentofChemicalEngineering,BiotechnologyGroup,TarbiatModaresUniversity,Tehran,Iran.
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