ContentslistsavailableatScienceDirect
Journal of Hazardous Materials
j ou rn a l h om epa ge :w w w . e l s e v i e r . c o m / l o c a t e / j h a z m a t
Allochthonous bioaugmentation in ex situ treatment of crude oil-polluted sediments in the presence of an effective degrading indigenous microbiome
S. Fodelianakis
a,b, E. Antoniou
a, F. Mapelli
c, M. Magagnini
d, M. Nikolopoulou
a, R. Marasco
b,c, M. Barbato
c, A. Tsiola
e, I. Tsikopoulou
e,f, L. Giaccaglia
d, M. Mahjoubi
g, A. Jaouani
h, R. Amer
i, E. Hussein
j, F.A. Al-Horani
k, F. Benzha
l, M. Blaghen
l, H.I.
Malkawi
j,m, Y. Abdel-Fattah
i, A. Cherif
g,h, D. Daffonchio
b,c, N. Kalogerakis
a,∗aSchoolofEnvironmentalEngineering,TechnicalUniversityofCrete,Chania,Greece
bKingAbdullahUniversityofScienceandTechnology,Thuwal,SaudiArabia
cDepartmentofFood,EnvironmentandNutritionalSciences,UniversityofMilan,Italy
dEcoTechSystemsS.r.l.,Italy
eHellenicCenterforMarineResearch,Heraklion,Crete,Greece
fDepartmentofBiology,UniversityofCrete,Heraklion,Crete,Greece
gLR11-ES31BiotechnologyandBio-GeoResourcesValorization,HigherInstituteforBiotechnology,BiotechpoleSidiThabet,UniversityofManouba,2020 Ariana,Tunisia
hLaboratoryofMicroorganismsandActiveBiomolecules,FacultyofSciencesofTunis,UniversityofTunisElManar,2092Tunis,Tunisia
iGeneticEngineeringandBiotechnologyResearchInstitute,CityforScientificResearchandTechnologyApplications(SRTA-City),Alexandria,Egypt
jDepartmentofBiologicalSciences,YarmoukUniversity,211-63Irbid,Jordan
kFacultyofMarineSciences,TheUniversityofJordan-Aqaba,7110Aqaba,Jordan
lLaboratoryofMicrobiolgy,BiotechnologyandEnvironmrent,UniversityHassanIICasablanca,FacultyofSciencesaîn-chock,B.P.5366Morocco
mHamdanBinMohammedSmartUniversity,AcademicCity,Dubai,UnitedArabEmirates
h i g h l i g h t s
•Allochthonousbioaugmentation,incontrasttobiostimulation,wasineffective.
•Theappliedconsortia/strainswerenotcapabletocolonizethesediment.
•Theindigenousmicrobiomewasveryeffectivewhensupplementedwithnutrients.
•Thereasonsfortheineffectivenessofbioaugementationhavebeenidentified.
a r t i c l e i n f o
Articlehistory:
Received29August2014 Receivedinrevisedform 12December2014 Accepted13January2015 Availableonline14January2015
Keywords:
Bioremediation Biostimulation Bioaugmentation Autochthonousdegraders Allochthonousdegraders Landfarming
Petroleum
a b s t r a c t
Oil-pollutedsedimentbioremediationdependsonbothphysicochemicalandbiologicalparameters,but theeffectofthelattercannotbeevaluatedwithouttheoptimizationoftheformer.Weaimedinopti- mizingthephysicochemicalparametersrelatedtobiodegradationbyapplyinganex-situlandfarming set-upcombinedwithbiostimulationtooil-pollutedsediment,inordertodeterminetheaddedeffectof bioaugmentationbyfourallochthonousoil-degradingbacterialconsortiainrelationtothedegradation efficiencyoftheindigenouscommunity.Wemonitoredhydrocarbondegradation,sedimentecotoxic- ityandhydrolyticactivity,bacterialpopulationsizesandbacterialcommunitydynamics,characterizing thedominanttaxathroughtimeandateachtreatment.Weobservednosignificantdifferencesintotal degradation,butincreasedecotoxicitybetweenthedifferenttreatmentsreceivingbothbiostimulation andbioaugmentationandthebiostimulated-onlycontrol.Moreover,theaddedallochthonousbacte- riaquicklyperishedandwererarelydetected,theiradditioninducingminimalshiftsincommunity structurealthoughitalteredthedistributionoftheresidualhydrocarbonsintwotreatments.There- fore,weconcludedthatbiodegradationwasmostlyperformedbytheautochthonouspopulationswhile
∗Correspondingauthorat:SchoolofEnvironmentalEngineering,TechnicalUniversityofCrete,Polytechneioupolis,Chania73100,Greece.
Tel.:+302821037794;fax:+302821037852.
E-mailaddress:nicolas.kalogerakis@enveng.tuc.gr(N.Kalogerakis).
http://dx.doi.org/10.1016/j.jhazmat.2015.01.038 0304-3894/©2015ElsevierB.V.Allrightsreserved.
bioaugmentation,incontrasttobiostimulation,didnotenhancetheremediationprocess.Ourresults indicatethatwhenenvironmentalconditionsareoptimized,theindigenousmicrobiomeatapolluted sitewilllikelyoutperformanyallochthonousconsortium.
©2015ElsevierB.V.Allrightsreserved.
1. Introduction
Marinesandsandsedimentsthatareexposedtocrudeoilcon- taminationareofmajorconcernworldwide[1,2].Althoughboth physicochemicaland biologicaltreatmentmethodsexistforthe remediationofsuchpollutedenvironments,theuseofthelatteris continuouslyincreasingduetotheirmuchlowercostsandenvi- ronmentalfriendlynature[3–5].
Theremediationofoil-pollutedsedimentsthroughbiological means(bioremediation)involvestheuseofmicroorganismsthat are able to degrade the pollutants. Currently, studies focus on enhancingthedegradationpotentialoftheindigenous microor- ganismsby alteringthephysicochemical parameters that could potentiallystimulatethecommunity’smetaboliccapacity(bios- timulation), increasingthe number of microorganisms that are capableofdegradingthepollutants(bioaugmentation),oracom- binationofthetwo.Manystudieshaveexaminedtheeffectiveness ofeachapproachwithcontrastingresults,andanongoingscien- tificdebateofhowphysicochemical(e.g.,temperature,humidity, sedimentoxygenationandgrainsizeororganicmattercontent)or biological(e.g.,longtermsurvival oftheaddedmicroorganisms, antagonismwiththeindigenouspopulations,originoftheadded strains) parameters determine the efficiency of each approach [3,6–10].
Oneofthemajorbottlenecksofanyappliedmethodthatisbased ondegradationbyaerobicmicroorganismsinsitu,istheoften-poor oxygenavailabilitywithinthesedimentsthatcanseverelylimit degradation.Thesimplestmethodusedtoovercomeoxygenlimi- tationisexsitutreatmentbylandfarming,atechniquethatinvolves spreadingofcontaminatedsoils(includingbeachsand),petroleum sludgesorsedimentsinathinlayer(typicallylessthan25–30cm) andregulartilling,combinedwiththeadditionofnutrientsand water tomaintainmoisture(typically about20%).Thismethod hasbeenwidelyusedinsoilremediationforoveracenturydue toitslowcost,simplicityinuse,compliancewithgovernmental regulationsandpotentialapplicationinavarietyofenvironments [1].
Theaimofthepresentstudywastoevaluatetheeffectiveness of bioaugmentation with allochthonous hydrocarbon-degrading bacterialconsortiainthebioremediationofoil-contaminatedsed- iments treated ex situ by landfarming, in the presence of the indigenous microbiome and when the relevant physicochemi- calparametersareoptimized.Wesetupanexperimentaldesign to investigate the real advantage of bioaugmentation during landfarmingpracticesthroughtheevaluationofdegradationper- formance between the different tested consortia and between allochthonous and indigenous degrading populations. An inte- grated approach was adopted, allowing the monitoring of the degradationofdifferentcrudeoilcompounds,andthedynamicsin termsofenzymaticactivity,communitystructureandpopulation sizesofthebacterialcommunitiesineachtreatment.
2. Experimental
2.1. Sedimentsamplingandexperimentalsetup
Pollutedsediment(c.a.30L)wascollectedonJanuary14,2013, from a coast adjacent to an oil refinery (Elefsina bay, Greece, 38◦216.28N,23◦3045.85E),wherehydrocarbonreleasesoccur
intermittently,andwastransferredtothelabovernight,ina50L plasticbarrel.Adetailedphysicochemicalcharacterizationofthe sedimentisgiveninTableS1.Theinitialsedimentcontamination wasestimatedat5000ppmofcrudeoilpergramofsediment,using soxhletextraction(TableS1).Uponarrivaltothelabonthenext day,sedimentwasfilteredthrougha5mmsieveinordertoremove largeparticlesthatcouldinterferewithalldownstreamanalyses, andwasair-drieddownto20%humidity(w/w).Sedimenttemper- aturewasmonitoredconstantly,remainingwithinthe10–20◦C range.
Whensedimentreachedthedesiredhumidity(20%),totalhet- erotrophic bacterial colony forming units (CFUs) per gram of sedimentweredetermined(seebelow)andsedimentwasplaced withinnineglassmicrocosms(1.4L,19cm×18.5cm×4cm,open top) with 1.5kg of sediment in each. Each microcosm repre- sented a differenttreatment in terms of biostimulation and/or bioaugmentation. Biostimulation wasperformedwiththeaddi- tion of N/P in the form of KNO3/KH2PO4 to a final ratio of C:N:P (100:10:1) while bioaugmentation with the addition of allochthonous bacteria to a final ratio of 10:1 (in terms of CFUs)tothe indigenous.The additionofallochthonous consor- tia was performed by centrifuging the appropriate amount of liquid cultureand then dilutingtheresulting pelletin10mlof sterileseawater.Theresultingdilutionwasfinallysprinkledover eachtreatment,subsequentlymixedthoroughlyforhomogeniza- tion. The treatment codes and the actual treatments were as follows: C1:sediment withbiostimulation withoutbioaugmen- tation. C2: sediment with the addition of killed allochthonous bacteria to a final ratio as in bioaugmented treatments. Bac- teria were killed by means of UV-C irradiation, using a TUV PL-S11W/2P1CTlamp(Philips,Amsterdam,Netherlands–radi- ation wavelength 253.7nm, exposure time 1h). One hundred microlitersof the irradiatedinoculum wereplated in nutrient- rich agar in triplicate asa viabilitytest. Colony formation was negative in all three plates. This control treatment was per- formedinordertomonitordegradationandbacterialcommunity dynamics in the sediment where the added dead biomass is availableas a sourceofnutrients.C3: sedimentacidified topH 2 by means of HCl 37% (Sigma–Aldrich) plus biostimulation.
This treatment was performed in order to monitor hydrocar- bonlossesbyanymeansotherthanbiodegradation.Allreported degradation rates have beencorrected taking intoaccount this controltreatment.C4:sedimentwithoutbiostimulationorbioaug- mentation.MUCSATB:sedimentwithbioaugmentationwiththe MUCSATBconsortiumplusbiostimulation,UH2C2:sedimentwith bioaugmentationwiththeUH2C2consortiumplusbiostimulation, UTUNB:sedimentwithbioaugmentationwiththeUTUNBconsor- tiumplusbiostimulation,YUOW:sedimentwithbioaugmentation with the YUOW consortium plus biostimulation (Table 1). For additionalinformation regardingtheorigin,isolationand initial acclimationoftheappliedbacterialconsortiaseeSupplementary material.
Theoverallperiodoftheexperimentalwas56days.Sediment humiditywasmonitoredconstantlyandwaskeptbetween8–20%
bytheadditionofsteriledistilledwaterwhenneeded.Sediment wastilledeveryoneortwodaysthroughouttheexperimentfor aerationbymeansofsterileplasticspatulas.Airtemperaturedur- ingtheexperimentwas10–15◦Candsedimenttemperaturewas 12–13◦C. Sediment for thedetermination of thesaturated and
Table1
Samplecodesandtreatmentdescription.N&PadditionreferstotheadditionofNandPintheformofKNO3andKH2PO4toafinalratioofC:N:P(100:10:1).Sediment acidificationreferstosedimentpHfixationtopH2.0byHCl37%.Added/indigenousratioreferstotheCFUsratioofallochthonoustoindigenousbacteria.
Samplecode Allochthonousconsortiumaddition N&Paddition Sedimentacidification Added:indigenousratio(inCFUs)
C1 No Yes No n/a
C2 Yes,killedbyUVirradiation Yes No 10:1
C3 No Yes Yes n/a
C4 No No No n/a
MUCSATB Yes Yes No 10:1
UH2C2 Yes Yes No 10:1
UTUNB Yes Yes No 10:1
YUOW Yes Yes No 10:1
aromatichydrocarbonsdegradationrates,thehumidity,andthe totalbacterialpopulationsizeswerecollectedondays0(7–8hafter theadditionoftheallochthonousconsortia),7,14,28,42and56.
Sediment for total hydrolyticactivity monitoringwascollected with1-daylagperiod,i.e.,ondays1,8,15,29,43and57.Sedi- mentforbacterialcommunitycharacterizationanddynamicswas collectedondays0,7,28and42,intriplicates.Sedimentforeco- toxicityanalyseswascollectedatthebeginning(Day0)andatthe end(Day56)oftheexperiment.
2.2. Degradationofpetroleumhydrocarbonsmonitoring
Sedimentsampleswereinitiallytreatedwithsoxhletextrac- tiontoextractthetotalpetroleumhydrocarbons(TPH)fromthe sediment,thentheextractswereseparatedinsaturatedandaro- matichydrocarbonsfractionswithsolidphaseextraction(SPE),and thealiphaticandaromaticcomponentsofthefractionsweresep- aratedandquantifiedbyGasChromatography–MassSpectroscopy (GC–MS).FormoredetailspleaserefertoSupplementarymaterial.
2.3. Totalbacterialpopulations
Total heterotrophic bacterial CFUs at the beginning of the experiment were determined by plate counts on nutrient-rich agar(seeSupplementarymaterial)andduringtheexperimentby flowcytometry.For flowcytometry,triplicatesedimentaliquots (0.25ml)werefixedbeforeproceedingtoextraction,by adding glutaraldehyde(4%finalconcentration)andkeepingatroomtem- peraturefor30min.Bacterialcellsweredetachedfromsediment particleswithacombinationofchemicalandphysicaltreatments, accordingtoAmalfitanoandFazi[11]withsomemodifications(see Supplementarymaterial).
2.4. Sedimenttotalhydrolyticactivity
Total hydrolytic activity in the sediment was measured by fluoresceindiacetate (FDA)hydrolysis using a slightly modified protocoldescribedelsewhere[12](seeSupplementarymaterial).
2.5. Sedimentecotoxicity
Sediment elutriates were prepared as described elsewhere [13].Twodifferenttargetspecieswereusedfor thedetermina- tionofsedimenttoxicity:VibriofischeriandParacentrotuslividus.
Toxicityassays onV. fischeri wereperformed throughstandard Microtox® tests(UNIENISO11348-3),using aModel 500ana- lyzer. Toxicityassays onP. lividus wereperformed considering theinhibition potentialof sediment elutriates on egg fertiliza- tionrates(spermiotoxicity),followingthestandardmethodology describedelsewhere[14].Bothtestswereperformedontheinitially collected,untreated,sedimentandontreatmentsC1,UTUNB,MUC- SATBandUH2C2attheendoftheincubation(Day56).Resultsof
MicrotoxtestwereexpressedasToxicUnits50(TU50),calculated as100/EC50,whereEC50istheelutriatedilution(%)resultingin a50%decreaseinbioluminescenceafter5and15minofincuba- tion.ResultsofspermiotoxicityassaysonP.lividusgametswere expressedas%ofunfertilizedeggs.Furtherdetailsforeachecotox- icitytestaregivenattheSupplementarymaterial.
2.6. Bacterialcommunitycharacterizationanddynamics
DNAwasextractedfrom0.5gof sedimentusing the“Power Soil” kit (MoBio LaboratoriesInc., Carlsbad, CA, USA) following the manufacturer’s instructions. Bacterial 16S rRNA gene frag- ments(∼550bp)wereamplifiedwithPolymeraseChainReaction (PCR)usingprimers907R(3-CCGTCAATTCCTTTGAGTTT-5)and GC-357F(3-CCTACGGGAGGCAGCAG-5witha5-endGC-clamp) targetingaportionofthe16SrRNAgenethatincludesthehyper- variable V3–V5 regions [15]. PCR reactionswere performed as previouslydescribed [16]. Presenceand lengthofPCR products wereverifiedby electrophoresisin 1%w/vagarosegel priorto DenaturingGradientGelElectrophoresis(DGGE)analysis.
DGGEwasperformedaccordingtoMuyzeretal.(1993)[15], and DGGE bands wereexcisedand sequenced. ARISA wasper- formedfor all samplesupto Week6 and wasconducted ona standard amount of DNA on each sample by using the primer setITSF,5-GTCGTAACAAGGTAGGCCGTA-3andITSReub,5- GCCAAGGCATCCACC-3,aspreviouslyreported[17].Forfurther detailsonDGGEandARISAseeSupplementaryMaterial.Nucleotide sequencesoftheexcisedbandswereeditedinChromasLite2.01 (http://www.technelysium.com.au)andsubjectedtoBLASTsearch (http://blast.ncbi.nlm.nih.gov/Blast.cgi).Thepartial16SrRNAgene sequenceshavebeendepositedintheEMBLunderaccessionnum- bersLK022102–LK022293,LM643751andLM643752.
2.7. Statisticalanalyses
Non-parametric t-tests were performed in PAST 2.17c [18].
For ecotoxicity results, the responses of each end point were corrected for effects in controls through the Abbott’s formula (http://www.astm.org/Standards/E724.htm).Differencesbetween samplesandcontrolaswellasandamongsampleswereanalyzed byone-wayanalysisofvariance(ANOVA).Whensignificanteffects occurred,Tukeys’spost-hoctestwasperformed.Inordertostudy thestructureofthewholebacterialcommunity,avoidingthelimi- tationduetotheexclusivephylogeneticanalysesofthecutDGGE bands,theimagesoftheDGGEgelswereconvertedinanumerical matrixandstatisticallyanalyzedbyPrincipalComponentAnalysis (PCA)andANOVAonthemainaxes.
3. Resultsanddiscussion
TheaveragenumberofheterotrophicbacterialCFUspergram ofsedimentatDay0,was(1±0.2)×105.Thus,thetotalCFUsper
treatment(1.5kg)wereestimatedat 1.5×108.Hence,1.5×109 CFUs wereadded tothe MUCSATB,UH2C2, UTUNB and YUOW treatmentsfromtherespectiveconsortiaatDay0,sothattheinitial ratiototheindigenousmicrobiomewas10:1.
3.1. Bioaugmentationdidnotimproveoveralldegradation
GC–MS analysis results revealed a quick decrease of the saturatedcompounds(intotal,C10–C35)withinthefirsttwoexper- imentalweeks, with70–90%of thecompounds beingdegraded by Day 7 and up to 95% by Day 56 for all treatments except C4 where degradation occurred after Day 28 (Fig. 1). Overall, the light (C10–C20) and medium chain (C20–C27) alkanes were degraded faster than the heavier (C27–C35) alkanes. After the second experimental week, the degradation of the saturated compoundsdecreasedexceptfromthecaseofC4wherealowper- centage(10–20%)ofdegradationwasobservedwithindays28–56.
Moreover,thearomaticcomponentsfluorene,phenanthreneand dibenzothiopheneweredegradedupto50%by Day14, andup to90%byDay42(Fig.S1).Theheavypolyaromaticcomponents monitored(i.e.,pyrene,fluoranthene, chrysene,benzo[a]pyrene, benzo[k]fluoranthene,benzo[b]fluoranthene,benzo[g,h,i]perylene and indeno[1.2.3-cd]pyrene) were not degraded significantly withinthe56-dayexperimentaltime(resultsnotshown).
Flowcytometryresultsindicatedthatatthebeginningofthe treatment(Day0),totalbacterialcellnumberswerehigherinthe bioaugmentedtreatments(averagecountsof4.8107cellspergof drysediment)thaninthecontrolsamples(averagecountsof2.8107 cellspergofdrysediment)(non-parametrict-test,p=0.0108,aver- age increase 71.4%) (Fig. 2).On thecontrary, the bacterialcell numberswerehigherinthecontroltreatmentsattheendofthefirst experimentalweek(Day7,non-parametrict-test,p=0.01421,aver- agecountsof4.6107cellspergofdrysedimentincontrolsvs.2.4107 cellspergofdrysedimentinbioaugmentedtreatments)anddid notdiffersignificantlyattheendofthesecondexperimentalweek (Day14,non-parametrict-test,p=0.241)(Fig.2).Cellcountsdid notdiffersignificantlybetweenC1andC2,oramonganybioaug- mentatedtreatmentsatanypointintime(non-parametrict-tests, p>0.05).Bacterialcellcountsinthenon-biostimulatedtreatment (C4)werelessthan2107cellspergofdrysedimentateachtime point.Afterthesecondexperimentalweek,bacterialcellnumbers inalltreatments,exceptfromC4,remainedrelativelyconstantuntil theendoftheexperiment(resultsnotshown).
3.2. Ecotoxicityanalysis
At the end of the incubations, results obtained through Microtox®assaysshowedasignificantreductionoftoxicityinall treatmentscomparedtotheuntreatedsediment(initial)(Fig.3a).
Inbioaugmentedtreatments,thebestperformancewasobtainedin MUSCATB,wheretheoriginaltoxicityof16.08TU50and9.33TU50 wasreduced,ontheaverage,by37.62%.Theaveragereductionof toxicitywasgreaterthan20%inallthebioaugmentedtreatments, withtheonlyexception ofUTUNB showinganaveragetoxicity reductionof7.98%.Thesamplescollectedfromthenegativecontrol (C1)showedagreatertoxicityreductionattheendofincubations comparedwiththebioaugmentatedtreatments(6.92and2.76TU50 for5minand 15min,respectively).Theattenuationof biolumi- nescencewas moreevident after5min incubationthan 15min incubationinallsamples.
Thespermiotoxicity assaysongamets ofP.lividus showeda similarpatternwiththeMicrotox®assays(Fig.3b).Theuntreated (Initial)elutriatesresultedin24.31%ofunfertilizedeggs,whereasin treatedsedimentsthepercentageofunfertilizedeggsrangedfrom 9.00%(MUSCATB)to19.67%(UH2C2).Amongthebioaugmented
treatments,theUH2C2displayedasignificantlyhigherpercentage ofunfertilizedeggsthanMUSCATBandYUOW(p<0.01).Thesetwo treatmentsdisplayedthebestperformancesintermsoffertiliza- tionratesamongthebioaugmentedones,withanaverageof9.83%
ofunfertilizedeggs.However,thebiostimulatedcontroltreatment (C1)resultedinthelowestpercentageofunfertilizedeggs(9.33%
averagevalue).Takentogether,bothassaysdemonstratedthattox- icityattheendoftheexperimentinallbioaugmentedtreatments waslowercomparedtotheuntreatedsediment;however,itwas higherthaninthebiostimulatedcontrolC1.
3.3. Hydrolyticactivity
Sedimenthydrolyticactivity(Fig.S2)showedasimilartrendin alltreatmentsexceptC4whereitremainedminimalthroughout theexperiment,andC2.Thehighestactivitywasobservedatthe beginningoftheexperiment(Day0)andthenagradualdecrease wasevidentuntilDay14.Afterthat,theactivitygraduallyincreased untilDay28whenasecondpeakwasobserved.Finally,theactiv- ityconstantlydecreaseduntiltheendoftheexperiment.Theonly exceptiontotheoveralltrendwasrecordedinthecaseofC2,for whichthehydrolyticactivitypeakwasobservedoneweekbefore alltheothertreatments.Thedifferenceintheobservedenzymatic activitybetweenC2andthebioaugmentedtreatmentsindicates thatunlikeintreatmentC2,theaddedallochthonousbacteriain thebioaugmentedtreatmentswerenotdeadatthepointoftheir additiontothesediment,nordidtheyperishimmediatelyafterthat point.Thedoubleactivitypeakthatwasobservedmaybeindicative ofdifferentbacterialpopulationsbeingactivatedatdifferentpoints intime,possiblyduetotheaccumulationofsecondarymetabolites inthesedimentovertime,combinedwiththedepletionofthealka- nesandthepersistenceofPAHsinthesediment.Suchacommunity turnoverwasindeeddetected(seeSection3.4).
Alltheaboveresultsindicatethattheremediationprocesswas rapidin allbiostimulatedtreatmentsand wasnot enhancedby bioaugmentation. Most of the degradation occurredwithin the firstexperimentalweekwithminordifferencesamongthetreat- ments.Thetotalbacterialpopulationsofbioaugmentedtreatments werelowercomparedtothecontrolsandthetrendinsediment hydrolytic activity was similar among all treatments after the firstexperimentalweek,whilesedimentecotoxicityattheendof theexperimentwashigherinthebioaugmentedtreatmentscom- paredtothebiostimulatedcontrolC1.Otherstudiesperformed inpollutedsedimentandsoilshavealsoreportedalimitedeffect of bioaugmentation on theremoval of hydrocarbons, sediment metabolic activity and bacterial population densities [3,19,20].
Moreover,inthepresentstudyweobservedanincreasedecotoxic- ityofthebiostimulatedandbioaugmentedtreatmentscomparedto thebiostimulated-onlycontrolC1.Thetoxiccompoundspresentin thebioaugmentedtreatmentsattheendoftheexperimenthaveto bedifferentfromtheresidualhydrocarbons,sincetheirconcentra- tionwassimilarwithinallmicrocosms.Thereis,toourknowledge, nopreviousreportwithsimilarfindings.Wehypothesizethatthese compoundsmayhadbeenreleasedasaresultofantagonismamong theallochthonousandindigenousbacteria.
3.4. Biodegradationwasmostlyperformedbytheautochthonous populations
For monitoring bacterial community structure dynamics through time and partially identify the bacteria inhabiting the sediments, DGGE and ARISA were performed on the bioaug- mentedtreatments(YUOW,UH2C2,MUCSATBandUTUNB),theN/P biostimulatedcontrol(C1)andthecontrolcorrespondingtothe originalsediments thatwere neitherbiostimulatednorbioaug- mented(C4).
Fig.1.Totaldegradationofalkanes(C10–C35)duringeachsamplingperiodforeachtreatment.TreatmentcodesaredescribedinTable1.Totaldegradationpersampling periodisdefinedasthepercentdecreaseinthesumofthealkaneconcentrationsattheendofasamplingperiod(attimetn)inrelationtotheconcentrationsatthebeginning ofthesamplingperiod(attimetn−1).Barsrepresentthestandarderror,derivedfromfourtechnicalreplicates.
TheclusteranalysisofARISAprofilesidentifiedfourgroupsof samplesshowinglessthan40%ofsimilarity(Fig.4a).Thefirstclus- tercomprisedallthesedimentsamplescollectedatDay0,after theadditionof theselectedconsortia, and theC4samplescol- lectedafterthefirstweekoftreatment.Excludingthosebelonging totreatmentC4,allthesamplescollectedatWeek1formedasec- ondcluster.Thethirdgroupofsampleswasrepresentedbyallthe samplesanalyzedafterWeek4andWeek6excludingtreatment C4,thelatterformingthefourthidentifiedcluster.Thebacterial communityofthesedimentscollectedatWeek4and6wereonly partiallydistinguishablewithinthesetwogroups.Overall,ARISA fingerprintsclearlyshowedthatthebacterialcommunitiesclus- teredaccordingtotheexperimentaltimeratherthantotheaddition or the identity of allochthonous consortia (Fig. 4a). The only exceptionfromtheobservedtrendwasrepresentedbythecom- munityinthenon-biostimulatedtreatmentC4.TheARISAresults impliedthatbiostimulation,ratherthanbioaugmentation,signif- icantlyinfluencedthetemporalchangesinbacterialcommunities (Fig.4a).
TheDGGEanalysiswasperformeduptoWeek6for control treatmentsC1and C4and uptoWeek4 forthebioaugmented treatments.Weaimedtodirectlycomparethepossibleeffectof theallochthonousinoculum additionontheoverallstructure of thebacterialmicrobiome,byvisualizingonthesameDGGE gel
theprofileoftheinoculumbeforetheaddition,theprofileofthe bioaugmentedsedimentaftertheadditionandthatofC1atDay 0.ThePCAoftheDGGEprofilesofthesedimentscollectedateach experimentaltime foreachtreatment, showedthat,overall,the resultsobtainedfromtheanalyticalreplicatesclusteredtogether, demonstratingthereliabilityoftheadoptedtechnique(Fig.4b).
AspreviouslyobservedbyARISAresults,thesedimentscollected atDay0andWeek1fromthecontroltreatmentC4werenotsig- nificantlydifferent,similarlytothosecollectedatWeek4and6 (Fig.S3a;Fig.S3g).Onthecontrary,theDGGEprofilesofthebac- terialcommunitiesdwellingthebiostimulatedcontrolC1showed thatthecommunitystructuresignificantlychangedbetweenDay 0andWeek1(Fig.S3b),aresultconfirmedbyANOVA(Fig.S3h) andsharedbyallthebioaugmentedtreatments,irrespectivelyof theappliedconsortium(Fig.S3i–n).Themicrobiologicaldataset supportsthechemical andactivitydata,accordingtowhichthe mainhydrocarbondegradationactivityoccurredwithinWeek1, incorrespondencewiththedetectedchangeofthebacterialcom- munitycomposition.Moreover,ANOVAresultsshowedthattheC1 andthebioaugmentedtreatmentswerenotsignificantlydifferent intermsofcommunitystructureatDay0(Fig.S3i–n).Accordingly, theDGGEbandspresentintheDNAextractedfromthebacterial consortiausedforbioaugmentationwereonlyrarelydetectedin thecorrespondingtreatedsamplesatDay0(Fig.4c),suggestinga
Fig.2. Boxplots(25–75%percentiles)oftotalheterotrophicbacterialcellcountsatthebeginningoftheexperiment(Day0)andafterthefirst(Day7)andsecond(Day14) experimentalweekfortreatmentsC1andC2(“Controls”)andtreatmentsUTUNB,UH2C2,MUCSATBandYUOW(“Bioaugmentation”).Barsrepresenttheupperandlower valueswhilethehorizontallinewithineachboxrepresentsthemedianvalue.
Fig.3.Ecotoxicityassayresultsfortheuntreatedsedimentatthebeginningoftheexperiment(“initial”)andforthesedimentwithintreatmentsC1,MUCSATB,UH2C2, UTUNBandYUOWattheendoftheexperiment(experimentalDay56).(A)Microtox®assay,(B)P.lividusspermiotoxicityassay.Barsrepresentthestandarddeviation.
lowcapabilityoftheselectedbacteriatoefficientlycompetewith theautochthonousbacterialpopulationsforsedimentcolonization.
ThephylogeneticaffiliationofDNAsequences(obtainedfrom 192DGGEbands–sixDGGEgels)wasidentifiedinordertoexamine thebacteriainhabitingthepollutedsedimentsateachtreatment and time point (Table S2, Fig. 4c). The number of sequences retrievedforeachtreatmentrangedbetween17(UH2C2)and55 (controlC1)(Fig.4c).Thevastmajorityofthem(122outof192 sequences)belongtothefamilyRhodobacteraceae(TableS2).The secondandthirdmostabundanttaxonomicgroupswereFlavobac- teria and Halomonadaceae, respectively. Most of the detected sequencesare widely spread in marinesediments whilemem- bersofwell-knownhydrocarbonoclasticbacteria(e.g.,Alcanivorax genus)wererarelyretrieved(TableS2).However,Rhodobacteraceae has been reported as key player of hydrocarbon bioremedia- tioninbeachsandsimpactedbytherecentDeepwaterHorizon Spill by Kostka et al. [21] who proposedthis family as a tax- onomic sentinel for the second step of oil remediation, when recalcitrantcompoundsare theprimary oilconstituents.More- over,amongtheRhodobacteraceaegeneradetectedbyDGGEinthe analyzedsediments,Roseobactercomprises bacteriaforwhich a relevantroleinbiodegradationhasbeenreported[22].Similarly theHalomonadaceae family encompassesoil-degrading bacteria [21,23],includingmembersoftheHalomonas[24]andCobetia[23]
genera,alsoretrievedinthepresentstudy(TableS2).Inaddition, Flavobacteria,widelydetectedbyDGGE,havebeenpreviouslyindi- catedtoincludesecondaryusersinvolvedinthemethaneandoil degradation[25].Takentogether,theDGGEphylogeneticdatasug- gestedtheinvolvementinhydrocarbondegradation,ofarichand diversebacterialcommunity comprisingspeciesthat arepoorly characterizedcomparedtothegenerallyabundantobligatehydro- carbonoclastic genera(e.g., Alcanivoraxsp.,Cycloclasticus sp.). It shouldbenoted,however,thatonlythemostabundantphylotypes
aredetectableusingDGGE.Otherdegradersthatmayhavebeen presentinrelativelylownumberswereprobablynotdetectedusing theadoptedapproach.Ontheotherhand,theabsenceof“classic”
bacterialhydrocarbondegradershintstowardthepresenceofa fungalhydrocarbonoclasticcommunitythatmayhavebeenactive duringtheexperiment.Eventhoughwedidnotexaminethepres- enceoffungiinourexperiment,evidenceoffungalhydrocarbon degradingcommunitiesexistfromsimilarexperiments[26–28].
Wefurtherexaminedthesimilarityinthedistributionofthe oilconstituentsthatwerepresentwithineachbiostimulatedtreat- ment(i.e.,C1,C2,UTUNB,MUCSATB,UH2C2andYUOW)afterWeek 1,whenmostofthedegradationhadoccurred.Acomparisonofthe distributionoftheremaininghydrocarboncompoundsinthedif- ferenttreatmentscanexplainifthedegradationwasperformed bythesamemicroorganisms.Weobservedthatfouroutofthesix treatmentsshowedasimilarcompounddistribution,withhigher concentrationsatthemedium-chained alkanes(C22–C25,Fig.5).
On theotherhand,thedistributionofthecompoundsin treat- mentsUH2C2andYUOWwasmoreuniform,withoutsignificant differencesamongdifferentalkanes(Fig.5).Thisobservationsug- geststhat,mostprobably,theaddedconsortiaintreatmentsUH2C2 andYUOWcontributedsignificantlytotheoildegradation,altering thedistributionoftheremainingcompoundsascomparedtothe restofthetreatments,butnotthetotalamountofoildegraded.
Thus,theallochthonousdegradersmusthaveperformedandsub- sequently perishedwithinthe firstweek oftreatment, as their populationswerenotdetectedattheendoftheweek.Thisisalso supportedbythedetectionofphylotypesrelatedtosecond-step remediationbytheendofthefirstweekatalltreatments.Several studieshavedemonstratedthehighdegradationpotentialofthe indigenouscommunitiesinhabitingpollutedsedimentsandsoils, whenprovidedwithadditionalnutrientsandafterregulartilling or othermeansofaeration [1,3,20,29],whilesomeothers have
Fig.4. Bacterialcommunityfingerprintingresults.(a)ClusteranalysisbasedontheARISAfingerprints,(b)principalcomponentanalysisbasedontheDGGEprofilesofthe 16SrRNAamplifiedfromthemetagenomeextractedfromthesedimentsatdifferenttimepointsduringthelandfarmingexperiment.Foreachtimepoint,thethreeanalyzed replicatesareindicatedasR1,R2andR3,(c)taxonomicidentificationofbacterial16SrRNAsequencesexcisedandamplifiedfromDGGEbands.
Fig.5.Thedistributionoftheremainingalkanesattheendofthefirstexperimentalweekforeachtreatment.Pr:pristane,Ph:phytane.
suggestedthattheefficiencyofbioaugmentationdependsonthe phylogeneticrelationamongtheappliedandindigenousconsor- tia[5]andtheinitialcommunityrichnessoftheimpactedhabitat, withrichercommunitiesreceivinglimitedbenefitsfrombioaug- mentation[12].Thestrainsusedforbioaugmentationinthisstudy showedalimitedabilitytocolonizethesediment,atraitproba- blyrelatedtoboththeoriginoftheallochthonousconsortia,that werepreviouslyisolatedfromdifferentoilcontaminatedsediments collectedfromNorthAfrica(Morocco,Tunisia,Egypt)andRedSea (Aqaba,Jordan)shorelines,henceadaptedtodifferentenvironmen- talconditions,andtothehighinitialrichnessoftheautochthonous community.
3.5. Engineeringramifications
The presented experimental results and the reached con- clusions have significant ramifications in the management of oil-contaminatedsediments.If thesediment originatesfroman areaofchronicpollutionorwithintermittent/continuousseepage ofevensmallquantitiesofpetroleumhydrocarbons,theindigenous populationswithsuitablebiostimulationshouldsufficefortheex situtreatmentbylandfarming.Itshouldbenotedthatinsuchtreat- ments,biostimulationisessentiallymandatoryastheN&Ppresent withinthesedimentsisnotsufficient;itcannotbereplenishedby theseaasit occursintheinsitutreatmentofsedimentswhere oxygenistheprimarylimitingsubstrate[30].Theaboveapproach canreadilybeadoptedforpollutedsitesacrosstheMediterranean Seaasrecentstudieshavereportedthepresenceofrichhydrocar- bondegradingcommunitiesinthewatercolumnofevenseemingly pristineareasinthisregion(seeReferencesin[31]).
4. Conclusions
Ourresultsindicatethatbiodegradationwasmostlyperformed bytheautochthonousdegraders,whilebioaugmentationdidnot enhance the remediation process. The limited added effect of bioaugmentationlikely reflectstheinabilityof theappliedcon- sortiatoeffectivelycolonizethesediment.That,inturn,couldbe attributedtotheoriginoftheconsortiaandtothepresenceofarich
autochthonouscommunityatthecontaminatedsite.Accordingto ourdata,whenenvironmentalconditionsareoptimizedusingan efficientex-situtreatmentsuchaslandfarmingcombinedwithbios- timulation,theindigenousmicrobiomeatapollutedsitewilllikely outperformanyallochthonousconsortium.
Acknowledgements
ThisworkwasfundedbyFP-7PROJECTNo.266473,“Unrav- ellingand exploitingMediterraneanSeamicrobial diversityand ecologyforxenobiotics’andpollutants’cleanup”–ULIXES.The authorswouldliketothankProf.NicoBoonforhisvaluableadvice and thefive anonymous reviewers for their constructive com- ments.FrancescaMapelliwassupportedbyUniversitàdegliStudi diMilano,DeFENS,EuropeanSocialFound(FSE)andRegioneLom- bardia(contract“DoteRicerca”).
AppendixA. Supplementarydata
Supplementarydataassociatedwiththisarticlecanbefound, in the online version, at http://dx.doi.org/10.1016/j.jhazmat.
2015.01.038.
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