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Environmental
Microbiology
Cellulase
activity
and
dissolved
organic
carbon
release
from
lignocellulose
macrophyte-derived
in
four
trophic
conditions
Flávia
Bottino
∗,
Marcela
Bianchessi
Cunha-Santino,
Irineu
Bianchini
Jr.
UniversidadeFederaldeSãoCarlos,DepartamentodeHidrobiologia,SãoCarlos,SP,Brazila
r
t
i
c
l
e
i
n
f
o
Articlehistory:
Received2April2015 Accepted14October2015 Availableonline2March2016 AssociateEditor:SolangeInes Mussatto Keywords: Anaerobicdecomposition Cellulose Carboncycle Aquaticmacrophytes
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Consideringtheimportanceoflignocellulosemacrophyte-derivedfortheenergyfluxin aquaticecosystemsandthenutrientconcentrationsasafunctionofforcewhichinfluences thedecompositionprocess,thisstudyaimstorelatetheenzymaticactivityand lignocel-lulosehydrolysisindifferenttrophicstatuses.Watersamplesandtwomacrophytespecies werecollectedfromthelittoralzoneofasubtropicalBrazilianReservoir.Alignocellulosic matrixwasobtainedusingaqueousextractionofdriedplantmaterial(≈40◦C).Incubations fordecompositionofthelignocellulosicmatrixwerepreparedusinglignocelluloses, inocu-lumsandfilteredwatersimulatingdifferenttrophicstatuseswiththesameN:Pratio.The particulateorganiccarbonanddissolvedorganiccarbon(POCandDOC,respectively)were quantified,thecellulaseenzymaticactivitywasmeasuredbyreleasingreducingsugarsand immobilizedcarbonwasanalyzedbyfiltration.Duringthecellulosedegradationindicatedby thecellulaseactivity,thedissolvedorganiccarbondailyrateandenzymeactivityincreased. Itwasrelatedtoafasthydrolysablefractionofcellulosethatcontributedtoshort-term car-bonimmobilization(ca.10days).Afterapproximately20days,thedissolvedorganiccarbon andenzymeactivitywereinverselycorrelatedsuggestingthattherespirationof microor-ganismswasresponsibleforcarbonmineralization.Cellulosewasanimportantresourcein lownutrientconditions(oligotrophic).However,thedetritusqualityplayedamajorrolein thelignocellulosesdegradation(i.e.,enzymeactivity)andcarbonrelease.
©2016SociedadeBrasileiradeMicrobiologia.PublishedbyElsevierEditoraLtda.Thisis anopenaccessarticleundertheCCBY-NC-NDlicense
(http://creativecommons.org/licenses/by-nc-nd/4.0/).
Introduction
The macrophytes are, after death, an important detritus sourceduetotheirroleincarbonandnutrientcyclesinaquatic systems.Decompositionisakeyprocessforthemaintenance oftheheterotrophicactivity1releasingbothhydrosolubleand
∗ Correspondingauthor.
E-mail:flaviabottino@yahoo.com.br(F.Bottino).
non-solublecompounds.2Thereleaseofhydrosolublefraction
(i.e.,dissolvedmaterial)occursbyleachingpolarcompounds ofthedetritusandmayvaryfrom24hto15days3;the
non-soluble material (i.e., lignocellulosic material) is a fraction showingslowdecayconstantratescontributingwith diage-neticprocesses.
http://dx.doi.org/10.1016/j.bjm.2016.01.022
1517-8382/©2016SociedadeBrasileiradeMicrobiologia.PublishedbyElsevierEditoraLtda.ThisisanopenaccessarticleundertheCC BY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/4.0/).
Theroleofthedissolvedfractionasasourceofcarbonto themicrobialloopiswelldocumented.4–6Fromthisconcept,
heterotrophicbacteria are the maindissolvedcarbon recy-clerintheaquaticecosystemsandtheenergyischanneled tohighertrophiclevels.However,somestudies7–9 have
rec-ognizedthat the lignocelluloses(i.e., POC)also sustainthe aquaticfoodwebduetothe enzymaticactionofmicrobial community and DOC production. The lignocelluloses con-tributiontotheDOCpool may beconsidered animportant pathwayofcarboncycleinthedetritusfoodweb.
Lignocellulosematerialishighlyresistanttothemicrobial communityand otheranimalsandisgenerallynotusedby grazers.Theenzymatic activity ofmicroorganisms reduces themolecularweightofthepolymerbyhydrolysisand/or sol-ubilizationduringdecomposition,enabling ittobeusedby themicrobialcells.Celluloseisthemostabundantcomponent intheplanttissueanditsdepolymerizationmainlyinvolves three enzymes: 1,4--exoglucanases, 1,4--endoglucanases and1,4--glucosidades.10Thispolymerisrelatively
suscep-tibletodegradation11,12yieldingby-productsreadilyavailable
tomicrobialmetabolism.10
Function forces, including temperature and nutrient availabilitymay influencethe enzymatic activity and, con-sequently, the rate of DOC production. High temperatures play an important role in enzyme metabolism,13 but the
influenceofnutrientsisstillunclear.14,15Thedegradationof
celluloseindifferenttrophic statusescontributesto under-standing the biochemical aspects of carbon turnover in aquaticecosystems. In this study, we address (i) the rela-tionshipbetweenDOC releaseduring macrophyte-cellulose decompositionandenzymaticactivity;(ii)themainaspects oftheDOCrelease/use;(iii)theroleofthetrophicstatusin thecarbonturnover.WesupposedthattheDOCrelease con-trolstheenzymaticactivityandbotharehigherinabundant nutrientconditions(i.e.,hypereutrophic).
Materials
and
methods
Samplingarea
WaterandmacrophytesampleswerecollectedinBarraBonita Reservoir (22◦29–22◦32 S and 48◦29–48◦34 W), located in SãoPauloState.BarraBonita(325km2ofarea)isthefirstof sixcascade reservoirs along the Tiete Riverand is consid-eredeutrophicduetointenseanthropicactivity.16Thelittoral
zoneofthereservoirismainlycolonizedbyPaspalumrepens
P.J. Bergius (Poaceae) and Pistiastratiotes L. (Araceae). The mainlimnologicalcharacteristicsofBarraBonitaReservoir17
arehighconcentrationsoftotalphosphorus(89–143gL−1), lowconcentrationsofdissolvedoxygen(4.5–5.0mgL−1),total nitrogenconcentrationrangingfrom 3.0–3.5mgL−1, neutral pH (7.0–7.5) and total organic carbon varying from 4.8 to 24mgL−1.
Waterandmacrophytesampling
Water samples (40L) were collected from the littoral zone of the reservoir at an eutrophic site (N:P ratio: 55) with
P. repens (22◦2819.27 S and 48◦2920.41 W); and at a
hypereutrophicsite(N:Pratio:79)withP.stratiotes(22◦2620.1 S and48◦3118.2 W)witha5.0LVan Dornbottleat differ-entdepths(surface,middleandbottom).Thewatersamples were mixedinapolyethylenecontainertoobtain vertically integratedsamples.Matureplantsamplesweremanually col-lectedinthelittoralzoneofthereservoir.Inthelaboratory,the watersampleswerefilteredthroughacelluloseester mem-brane(Ф=0.45m)andtheplantswerewashedwithtapwater toremovethe adheredcoarsematerial.Theplantmaterial wasovendried(40◦C),andthePOCwasobtainedaftercold aqueousextractionofthehydrosolublefractions(4◦C,24h)18
frompreviouslysterilizedplants(120◦C,1atm,15min).The POCwasconsideredthelignocellulosematrixandwasused inthedecompositionassays.Thedetritusqualityisindicated byC:NC:PmolecularratiosofinitialPOC(P.repens:C:N=1:1.5; C:P1:0.8;P.stratiotes:C:N:1:0.37;C:P1:0.6)
Experimentaldesign
DOCandcellulosedetermination
Anaerobic decomposition assays were carried out using 180 (n=90 per species) decomposition chambers (400mL) containing POC and filtered water sample (proportion of 10.0gDML−1).Thechambersweremaintainedinthedarkat 25◦C(averagewatertemperaturemeasuredinthereservoir) and 1mLofindigenousinoculum (i.e.,waterand sediment fromreservoir)wasaddedtoeachchamber.Theinitialtrophic conditionsofbothsamplingstationswereadoptedasnutrient backgroundstopreparethedecompositionchambers simulat-ingfourtrophicregimes19bydilutionorbyaddingsolutionsof
nitrogenandphosphorus.TheN:Pratioofsamplingstations wasmaintainedintheincubations.
On eachsampling day(days10,20,30,60,90,and 120), threechambersofeachplantandtrophicconditionwere fil-tered (Ф=0.45m) and fractioned into POC and DOC. The DOC was filteredsuccessively(Ф=0.45mand 0.2m) and thecarbonconcentrationwasmeasuredusingacarbon ana-lyzer(ShimadzuTOC-LCPH,Japan).TheDOCdailyratewas calculatedaccordingtoEq.(1).TheDOCdailyratewas con-sideredasmineralizationorimmobilization.Thedifference betweentheconcentrationsoffiltereddissolvedcarbon(0.45 and0.2m)wasassumedasformationoforganiccompounds includingmicrobialbiomass(immobilization).Cellulose con-tentofremainingPOCwasmeasuredbygravimetricanalysis withaprioraciddigestion(H2SO4−72%,3h).20
DOC=[DOC]
t , (1)
whereDOC,DOCdailyrate(mgL−1d−1);[DOC],DOC concen-tration(mgL−1);t,time(days).
Enzymeassay–cellulolyticactivitydetermination
From each decomposition chamber, enzymeextracts were preparedusing20.0mLofDOCand1gofPOC(freshmass). Thesampleswerehomogenized(Ultra-TurraxmodelT10, Ger-many),sonicatedwithanultrasound(modelUnique,Brazil) andcentrifuged(3000×g,15min,4◦C;HeraeusInstruments, Megafuge3.0R, Germany).Thecellulase activity was deter-minedspectrophotometrically(Ultrospec2100Pro,Sweden)
Table1–Cellulose(%)ontheremainingParticulateOrganicCarbon(POC)(after120days)inthedetritusofPaspalum repensandPistiastratiotes.N:PratiosofsamplingstationswithPaspalumrepensandPistiastratiotes:55and79, respectively.C:N:PratiosofPaspalumrepensandPistiastratiotesdetritus:1:1.5:0.8and1:0.3:0.6,respectively.
Trophicstatusdetritus Oligotrophic Mesotrophic Eutrophic Hypereutrophic
P.repens 19.0 45.0 65.0 44.8
P.stratiotes 26.5 38.0 28.0 26.5
bymeasuringtheconcentrationofreleasedreducingsugar21
actingonspecificsubstrates22(apurecellulosefilter–
What-manno.1).Themeasurementof1molofglucoseperminute ofreactionpermillilitercorrespondsto1internationalunit permilliliter.
Statisticalanalysis
ThenonparametrictestofKruskal–Wallis(significancelevel
p<0.05)followedbytheDunn’smultiplecomparisontestwere appliedtotheDOCandtothecellulasedatatoevaluate dif-ferencesinthenutrienttreatments.Thedifferencesbetween theplantswereassessedusingKruskal–Wallis.
Results
TheremainingcontentofcellulosefromdetritusofP.repens
and P. stratiotes at the end of the decomposition experi-mentsare presentedinTable1. Theoligotrophiccondition showedthe majorcellulosedecay fortheP. repensdetritus anditdecreasedaccordingtoanincreaseinthetrophic
sta-tus(Table1).For P. stratiotes,thehighestdecayoccurred in
oligotrophicandhypereutrophicenvironmentsthatshowed similarcellulosecontentafter120days(Table1).
Cellulase activity onP. repens detritusincreasedover 60 daysforalltrophicstatuses(Fig.1).Intheeutrophicand hyper-eutrophicconditions, the enzyme activity showed a slight decreasefromday20today30(0.62–0.47;0.64–0.54IUmL−1, respectively).Thehighestcellulaseactivityoccurredinthe oli-gotrophicstatusonday60(0.81IUmL−1).Thevaluesshowed asharpdecreaseuntilday120(0.25IUmL−1).Mesotrophicand eutrophicstatusesshowedasimilarvariationpatternof enzy-maticactivity;forthehypereutrophiccondition,thecellulase activity decreased from day 90 (0.7–0.3IUmL−1). Statistical analysisoftemporalenzymeactivitiespointedoutno signifi-cantdifferencesamongthetrophicstates(p<0.05).
Duringthecellulosedegradationindicatedspecificallyby cellulase activity,theDOC dailyrate derivedfrom P. repens
detritusincreasedalmostproportionallytotheenzyme activ-ity. After approximately 20 days, the DOC daily rate and enzymeactivitywereinverselycorrelated(Fig.1)showingthe predominanceoftheconsumptionprocess(i.e., mineraliza-tionor immobilization).TheDOCdailyratedecreasedover timein all trophicstatuses. Nostatisticaldifferences were observedamongthetreatments(p>0.05).
ForP.stratiotesdetritus,thecellulaseactivityincreaseduntil day10foralltrophicconditions(0.78,0.63,0.69,0.5IUmL−1 foroligotrophic,mesotrophic,eutrophicandhypereutrophic, respectively) (Fig. 2). The values declined until day 30 for mesotrophicandhypereutrophicstatuseswhileahighpeak ofcellulaseoccurredforoligotrophicandeutrophic(0.57and 0.36IUmL−1,respectively).Onday60,thehighestenzymatic
activitywasrecordedforthemesotrophicstatus(0.41IUmL−1) decreasinguntilday120formesotrophicandhypereutrophic conditions. Oligotrophicandeutrophicstatuses showedthe highestcellulaseactivityonday90(0.35IUmL−1).Statistical analysisoftemporalenzymeactivitiespointedoutno signifi-cantdifferencesamongthetreatments(p<0.05).
TheincreaseintheDOCdailyratewascloselyrelatedto thecellulaseactivityuntilday10.Afterthatthepatternwas opposite(i.e.,increaseofDOCconsumption)andtheDOCdaily ratedecayincreasedovertime,exceptfortheeutrophic condi-tion(highdailyrateonday60:0.19%).AraiseofDOCoccurred onday90foroligotrophic,mesotrophicandhypereutrophic statuses (0.09%, 0.07%,0.02%, respectively). In general, the increaseinDOCwasthedominantprocessonthemesotophic statuswhileitsconsumptionduetomineralizationor immo-bilizationwasthemainroutetoahypereutrophiccondition. StatisticalanalysisoftheDOCdailyrateshowednosignificant differencesamongthetrophicstatuses(p<0.05).
In general, the immobilization of DOC (i.e., microbial biomass formation) was higher for P. repens (Fig. 3A). The maximumcarbonimmobilizationoccurredfromday20to60, mainlyforthehypereutrophiccondition.ForP.stratiotes,the highestbiomassformationwasonday60foreutrophicand hypereutrophicstatuses(Fig.3B).
Discussion
After120daysofdecomposition,ourresultsshowedahigh loss of cellulose content in P. repens detritus mainly in poor-nutrient conditions (i.e., oligotrophic) suggesting that the particulate detritus is an important carbon resource for microorganisms in nutrient-limited environments. The lignin–celullose–hemicellulose matrix interactions and also the detritus quality (indicated by C:N:P ratios) may reflect the major decay of cellulose from P. stratiotes detritus in eutrophicconditions. Theaccess ofmicroorganismsto the fibers depends on their structural arrangement, which is differentforeachplantandcanalsobedifferentforeach struc-ture(e.g.leaves,stem)ofthesameplant.11Furthermore,the
biochemicaldegradationofanorganicresourceisimproved inanutrient-richenvironmentduetothehighextracellular enzymeactivity.23Thesignificantdifferencesbetweenthe
cel-lulosedecayofbothplantsshowtheroleofthedetritusquality (indicatedherebyC:NC:Pratio)inthedegradationoffibers.
In general, the enzymatic activity in P. repens detritus increasedovertimewithaslightdecreaseover30dayswhen the DOC immobilizationwasthe predominantprocess.We supposethattheDOCdailyrateincrease(untilday10)was faster than the microbial uptake and the microorganisms responsible forthis process produced the highestcellulase enzymatic activity inthe first days ofincubation. After 10
120 100 80 60 40 20 0 –1.0 –0.5 0.0 0.5 1.0 1.5 2.0 Time (d) DOC (mg.L -1.d -1) DOC (mg.L -1.d -1) DOC (mg.L -1.d -1) DOC (mg.L -1.d -1) 0.0 0.2 0.4 0.6 0.8 1.0 Cellulase (IU.ml -1 ) Cellulase (IU.ml -1 ) Cellulase (IU.ml -1 ) Cellulase (IU.ml -1 ) 120 100 80 60 40 20 0 –1.0 –0.5 0.0 0.5 1.0 1.5 2.0 Time (d) 0.0 0.2 0.4 0.6 0.8 1.0 120 100 80 60 40 20 0 –1.0 –0.5 0.0 0.5 1.0 1.5 2.0 Time (d) 0.0 0.2 0.4 0.6 0.8 1.0 120 100 80 60 40 20 0 –1.0 –0.5 0.0 0.5 1.0 1.5 2.0 Time (d) 0.0 0.2 0.4 0.6 0.8 1.0
A
B
C
D
Fig.1–TemporalvariationofDissolvedOrganicCarbon(DOC)dailyrate(black)andcellulaseactivity(gray)ofPaspalum repensinfourtrophicconditions:(A)oligotrophic,(B)mesotrophic,(C)eutrophic,and(D)hypereutrophic.
days,themainrouteoftheDOCwasimmobilizationandthe increaseincellulaseactivitymayberelatedtothehigh het-erotrophicactivityduetotheincreaseinbiomass.LowDOC availabilityinthesystemprobablystimulatedthemicrobial communitytoenzymaticallyattackthelignocellulosic detri-tustoobtaincarbon.24
The input of carbon in a system increases microbial respiration, enzyme activity production and changes the carbonavailability,and therefore,themicrobialcommunity structure.25 The degradation of less recalcitrant resources
(ca. hemicelluloses glycosidic bonds) in the first days of decomposition decreases the carbon limitation supporting the heterotrophic metabolism and the attack to the cellu-losefibrils.10,26 Thewholecelluloseactivatestheenzymatic
chainofcellulase productiondecreasing theDOC availabil-ityduetotheformationofbiomassinano-limitingnutrient environment.25,27 The input of soluble cellulose improved
the DOC formation (day 10) and its rapid assimilation, mainlybybacteria(biomassformation),increasedthe cellu-lolyticactivity.Itismoreevidentinnutrient-richincubations (frommesotrophictohypereutrophic).Thus,thepresenceof phosphorusand nitrogeninthe mediummayimprove the microbialactivityincreasingthecarbondemandreflectingin theenzymaticproduction.
ForP.stratiotes,theenzymaticactivityshowedan increas-ing pattern from day 20 to day 30 and a decrease in the DOCdailyrateafter10days.ThesharpdecreaseintheDOC
dailyrateindicatesitsmineralizationand lossoffiber con-tent (approximately 82% of cellulose lost to the eutrophic condition). The high enzymatic activity,mainly in the oli-gotrophic status, suggests that even in a nutrient-limited environment, the microbial community uses resources to depolymerizeextensivecarbonmolecules,probablyusingthe detritusasaresourceofcarbon.Thenutrient-richcondition supportedthemicroorganismsenergydemandsandthe car-bon immobilization.24 Thisassumptionconfirmsthat forP.
stratiotes,boththetrophicstatusand detritusquality influ-encedthecarbonflow.
Thedegradationofthecellulosefrommacrophytesdetritus dependsontheproductionofextracellularenzymesby bacte-riaandfungiactingsynergistically(CellulosomeConcept28).
In anaerobic conditions, microorganismsutilize a complex system ofenzymes tohydrolyzecellulosereleasing readily degradable compounds (monosaccharides and oligosaccha-rides)andhighmolecular-weightcompounds.29Bacteriaand fungiactinginthecellulosesubstratumincreasethe degra-dationofthecellulose.Fungiattackrecalcitrantsubstances (i.e.,high molecular weightcompounds)releasing interme-diate by-productstothebacteriauptakeand,consequently, increasingthecellulaseactivity.30Therelationshipbetween
enzymeactivityandDOCimmobilizationandmineralization presented here show the importance ofa recalcitrant car-bonsource(i.e.,cellulose)fortheheterotrophicmetabolism. Somestudies7,31,32haveshowntheroleofthelignocellulosic
120 100 80 60 40 20 0 –1.0 –0.5 0.0 0.5 1.0 1.5 2.0 Time 0.0 0.2 0.4 0.6 0.8 1.0 120 100 80 60 40 20 0 –1.0 –0.5 0.0 0.5 1.0 1.5 2.0 Time 0.0 0.2 0.4 0.6 0.8 1.0 120 100 80 60 40 20 0 –1.0 –0.5 0.0 0.5 1.0 1.5 2.0 Time 0.0 0.2 0.4 0.6 0.8 1.0 120 100 80 60 40 20 0 –1.0 –0.5 0.0 0.5 1.0 1.5 2.0 Time 0.0 0.2 0.4 0.6 0.8 1.0 DOC (mg.L -1.d -1) DOC (mg.L -1.d -1) Cellulase (IU.ml -1 ) Cellulase (IU.ml -1 ) DOC (mg.L -1.d -1) DOC (mg.L -1.d -1) Cellulase (IU.ml -1 ) Cellulase (IU.ml -1 )
D
C
B
A
Fig.2–TemporalvariationofDissolvedOrganicCarbon(DOC)dailyrate(black)andcellulaseactivity(gray)ofPistiastratiotes
infourtrophicconditions:(A)oligotrophic,(B)mesotrophic,(C)eutrophic,and(D)hypereutrophic.
matrixintheDOCproductionanditsfunctionintheenergy flow.
Cellulosebreakdownresultsinfermentativesugarsandthe fungigrowthimprovesthisprocessduetotheprocessingof lignocellulosicdetritus.33,34Duringcellulosedegradation,the
microbialcommunityshiftstoahighlyspecialized commu-nityproducingarangeof11enzymesactingsynergistically onthesubstratuminananaerobiccondition.35Theenzyme
activity supports the release of the soluble carbon from
the cellulosehigh molecular-weight carbon (but not recal-citrant) enhancing the heterotrophic metabolism.24 In this
regard, this study assumes that the hydrolysable fraction of the cellulose fiber was responsible for the high DOC dailyrates andincreasing biomass,mainlyduringP. repens
degradation. The high amount of proteins in that macro-phyte species36 is closely related to the complex enzyme
activation as it binds proteins and nitrogen compounds together. 120 100 80 60 40 20 0 1 10 100 1000 10 000 TOC (m g. L-1 ) Time (d) 120 100 80 60 40 20 0 1 10 100 1000 10 000 TO C ( m g .L-1) Time (d) Oligotrophic Mesotrophic Eutrophic Hypereutrophic
A
B
Fig.3–TotalOrganicCarbon(TOC)variationrepresentingthebiomassformationfromDissolvedOrganicCarbon(DOC)(in termsofcarbonconcentration)ofPaspalumrepens(A)andPistiastratiotes(B).
Accessibilityandhydrolysabilityarethemainfactors con-trollingthecellulosebreakdown.34Fromthispointofview,the
cellulosehydrolysisdecreaseduntiltherewasadegreeof con-versionofcelluloseof30%andacloggingsystemdiminished theaffinitybetweencelluloseandcellulase.Theresults sug-gestthatthedetritusquality,includingitsrecalcitrance(i.e., amountoffibers)wereimportanttopredicttherelationship betweencellulaseandcelluloseanditsconversiontoDOC.The highercellulosecontentinP.repensdetritus17limitedits
con-versiontoDOC(lowerratesthanP.stratiotes),butthecellulase activitymaybeduetotheenzymes’cloggingsystem.
Insummary,thecellulosemacrophyte-derivedshoweda fast hydrolysable fraction (ca. 10 days), which contributed tocarbonimmobilization.Mineralizationwasthelong-term predominant process mainly due to the respiratory activ-ityofmicroorganisms.Moreover,thehydrolysablefractionof celluloseimproved the immobilization process,which was stimulated byboththe nutrientcondition and thedetritus quality. High nutrient concentrations increased the respi-ratory activity of microorganisms and, therefore, the CO2 formation.Thetrophicstatusshowedminorimportancefor cellulosebreakdownandtheavailabilityofresourcesinthe environmentlimitstheattackofmicroorganismstothe detri-tus. The intrinsic characteristics of detritus (i.e., C:N C:P ratios) were important for the degradation of fibers. The outcomesshow analternative route ofthecarbon cycle in aquaticecosystemsandcontributetounderstandingthe pro-cessoccurringwithinthesediments.
Conflicts
of
interest
Noconflictsofinterest.
Acknowledgements
WewouldliketothankFundac¸ãodeAmparoàPesquisado EstadodeSãoPaulo(FAPESP)fortheirfinancialsupport(São PauloResearchFoundation,Processnumber2012/21829-0).
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