Microbial Community in a Biofilter for Removal of Low Load Nitrobenzene Waste Gas

12 Pages·2017·1.44 MB·English

RESEARCHARTICLE
Microbial Community in a Biofilter for
Removal of Low Load Nitrobenzene Waste
Gas
JianZhai1,2,ZhuWang1,PengShi1,ChaoLong1*
1 StateKeyLaboratoryofPollutionControlandResourcesReuse,SchooloftheEnvironment,Nanjing
University,Nanjing,PRChina,2 DepartmentofAppliedChemistry,NanjingPolytechnicInstitute,Nanjing,PR
China
*[email protected]
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Abstract
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Toimprovebiofilterperformance,themicrobialcommunityofabiofiltermustbeclearlydefined.
Inthisstudy,theperformanceofalab-scalepolyurethanebiofilterfortreatingwastegaswith
lowloadsofnitrobenzene(NB)(<20gm-3h-1)wasinvestigatedwhenusingdifferentempty
bedresidencetimes(EBRT)(64,55.4and34s,respectively).Inaddition,thevariationsofthe
OPENACCESS
bacterialcommunityinthebiofilmonthelongitudinaldistributionofthebiofilterswereanalysed
Citation:ZhaiJ,WangZ,ShiP,LongC(2017) byusingIlluminaMiSeqhigh-throughputsequencing.TheresultsshowedthatNBwastegas
MicrobialCommunityinaBiofilterforRemovalof
wassuccessfullydegradedinthebiofilter.High-throughputsequencingdatasuggestedthat
LowLoadNitrobenzeneWasteGas.PLoSONE12
(1):e0170417.doi:10.1371/journal.pone.0170417 thephylumActinobacteriaandgenusRhodococcusplayedimportantrolesinthedegradation
ofNB.Thevariationsofthemicrobialcommunitywereattributedtothedifferentintermediate
Editor:AndrewCSinger,NaturalEnvironment
ResearchCouncil,UNITEDKINGDOM degradationproductsofNBineachlayer.Thestrainsidentifiedinthisstudywerepotentialcan-
didatesforpurifyingwastegaseffluentscontainingNB.
Received:August24,2016
Accepted:January4,2017
Published:January23,2017
Copyright:©2017Zhaietal.Thisisanopen
accessarticledistributedunderthetermsofthe Introduction
CreativeCommonsAttributionLicense,which
Nitrobenzene(NB)isatypicalhydrophobic(Henry’slawconstant2.41×10−6MPam3mol-1and
permitsunrestricteduse,distribution,and
reproductioninanymedium,providedtheoriginal othersalientpropertiesontheS1Table)andrefractorybiodegradablevolatileorganiccompound.
authorandsourcearecredited. NBisemittedmainlyfromexplosives,aniline,dyes,pesticides,pharmaceuticalmanufacturing
processesandwastewatertreatmentplants.BecauseNBisrelativelytoxicandpersistentinthe
DataAvailabilityStatement:Theauthorsconfirm
thatalldataunderlyingthefindingsarefully environment,NBemissionsarefacingincreasinglystringentenvironmentalregulations.Biofil-
availablewithoutrestriction.Thehigh-throughput terswereoriginallydevelopedforodorpollutioncontrolduetotheirhighefficiency,lowenergy
sequencingdatasetshavebeendepositedintothe consumption,lowoperatingcosts,andenvironmental-friendliness[1]andhaverecentlybeen
NCBIShortReadsArchiveDatabase(accession
proventobeapromisingtechnologyforthetreatmentofvolatileorganiccompounds(VOCs)at
number:SRP095067).
moderatelyhighflowratesandlowconcentrations(<1000ppm)[2–3].
Funding:Thisstudywasfinanciallysupportedby BiofiltershavebeensuccessfullyappliedtotreathydrophilicVOCs,suchasstyrene[4],eth-
NaturalScienceFoundationofNanjingPolytechnic
anol[5],methanol[6],xyleneisomers[7],hexane[8],toluene[9],phenol[10],methylamine
Institute(GrantNo.NHKY-2016-02,PI:JZ,URL:
[11],triethylamine[12],dimethylsulfide[13],anddichloromethane[14].However,thelow
http://menhu.njcc.edu.cn/);JiangsuProvincial
masstransferofhydrophobicVOCsfromthegasphasetothebiofilmphase,whichlimitsthe
ScienceandTechnologyPlanProject(No.
BY2015060-03).Thefunderhadnoroleinstudy supplyofsubstratestothemicroorganisms,resultsinlowbiodegradationratesofthese
PLOSONE|DOI:10.1371/journal.pone.0170417 January23,2017 1/12
MicrobialCommunityinaBiofilterTreatingNitrobenzeneWasteGas
design,datacollectionandanalysis,decisionto compounds.Severalresearchershaveattemptedtoenhancetheremovalofhydrophobic
publish,orpreparationofthemanuscript. VOCsinbiofiltersbyaddingsurfactants,applyingfungalbiocatalysts,andbyusingbiofiltra-
CompetingInterests:Theauthorshavedeclared tionwithpretreatment,innovativebioreactors,andhydrophiliccompounds[15].
thatnocompetinginterestsexist. However,tothebestofourknowledge,onlyOhandBarthareportedanNB-elimination
capacity(EC)of50gm-3packingh-1inatricklingairbiofilter[16].Inaddition,fewstudies
haveinvestigatedtherelationbetweenbiofilterperformanceandthemicrobialcommunityin
NB-degradingbiofilters.Microbialcommunitiesaresensitivetochangesinenvironmental
conditions;therefore,understandingthemicrobialecologyofbiofilmsingasphasebiofilters
helpstooptimizethedesignandoperationofsuchbiologicaltreatmentsystems[17].
Culture-dependentconventionalisolationandidentificationmethodsonlyprovideinfor-
mationonrelativelyfewmicrobialcommunities.Itremainsunclearwhethertheisolateswere
ofanyquantitativeorfunctionalsignificanceinsitu[18].Withtherapiddevelopmentofanal-
ysistechnologies,someadvancedanalysismethods,suchasconfocallaserscanningmicros-
copy(CLSM),microautoradiography(MAR),polymerasechainreaction(PCR)techniques,
quantitativereversetranscriptasereal-timePCR(qrt-PCR),denaturinggradientgelelectro-
phoresis(DGGE),fluorescentinsituhybridization(FISH),environmentalscanningelectron
microscopy(ESEM),microprobesensortechniques,lasertriangulationsensor(LTC)tech-
niques,andBIOLOGECO-plates,havebeensuccessfullyusedforstudyingbiofilms[19].
Comparedwiththeabovemethods,high-throughputsequencingprovidesresearcherswitha
deeperunderstandingofthepopulationdynamicsorfunctionsofmicroorganismsinmicro-
bialcommunitiesandanewperspectiveforunderstandingthedegradationprocessesoccur-
ringinbioreactorsintheirnativehabitatwithouttheneedofisolation[20].
Inthisstudy,gas-phaseNBwastreatedinabiofilter,andthemicrobialcommunitywas
analysedbyhigh-throughputsequencingtechniques.Theprimaryobjectivewastoclearly
understandtheverticaldistributionofthebacterialcommunityinabiofilterforremovalof
lowloadnitrobenzenewastegas.Thepresentresearchwillhelpenhancethedegradationper-
formanceofbiofiltersfortreatingNBwastegas.
MaterialsandMethods
Biofiltersetup
AschematicoftheexperimentalsetupisillustratedinFig1.Thebiofilterreactor(innerdiame-
ter=7cm,effectivebedheight=60cm),whichwaspackedwith2.3-Lpolyurethanefoam
cubeswithlowheadlossandhighporosity,wasoperatedinaup-flowmode[21–22].The
packingparametersaresummarizedinTable1.NBwastegaspassedthroughthebiofilterata
flowrateof129.4,149.5and243.5Lh-1,respectively.Gassamplingportssealedwithrubber
septawereprovidedatequalintervalsalongthebiofilterheighttoestimatethechangesinthe
NBconcentrationsandmicrobialcommunities.
Thebiofilterwasinoculatedusingaerobicactivatedsludgefromthesecondarysedimenta-
tiontankofanNBproductionwastewatertreatmentplant(WWTP).Themixedculturewas
re-circulatedthroughthepackedbedbioreactorusingaperistalticpump(WT600-1F,Baoding
longerPumpLtd.,CHN)atarateof0.5Lmin-1untilavisiblebiomasswasnoticedonthesup-
portmaterialsurface.Themoisturecontentofthebedwasmaintainedat75%-85%byperiodi-
callyadding(onceaday)freshmineralsaltmedia.Thetricklingdensitywasmaintainedat
0.52m3m-2h-1throughouttheexperiments.Themineralsaltnutrientsolutioncontainedthe
followingcomponents(perlitreindistilledwater):0.5gK HPO ,0.1gMgSO (cid:1)7H O,4.5g
2 4 4 2
KH PO ,2gNH Cl,and2mLofvitaminsandtraceminerals[23].TheoriginalpHofthe
2 4 4
nutrientsolutionwasadjustedto7.0.
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MicrobialCommunityinaBiofilterTreatingNitrobenzeneWasteGas
Fig1.Schematicrepresentationoftheexperimentalsetup.
doi:10.1371/journal.pone.0170417.g001
Analyticalmethods
TheNBconcentrationsinthegassamplesweremeasuredbygaschromatography(GC)(2014,
SHIMADZU,USA)usingaRtx-5column(30m,0.32mmID,0.5μm)andanFID.Theflow
rateswere30mLmin−1forH and300mLmin−1forair.Nitrogenwasusedasthecarriergas
2
ataflowrateof2mLmin−1.ThetemperatureslocatedattheGCinjection,ovenanddetection
portswere150˚C,220˚Cand250˚C,respectively.ThepHvalueofthefilterliquorwasmea-
suredbyapHmetre(FEP20,Mettler-Toledo,CHE).Thenitriteconcentrationwasmonitored
accordingtotheChineseNationalStandardGB7493–87waterquality-determinationofnitro-
gen(nitrite)-spectrophotometricmethod[24].
Abioticexperiment
TheNBabioticexperimentwasperformedwithabiofiltercontainingacleanpackingmedium
(2.3L)todeterminetheamountofNBremovedbyabioticmeans.
Table1. Thepackingparameters.
parameters value
shape cube
size perside4–6mm
bulkdensity 0.015g/cm3
waterholdingcapacity 55g-H O/g
2
porosity 95%
averageporesize 0.8mm
specificsurfacearea 73.65m2/g
Valuesprovidedbythemanufacturer.
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MicrobialCommunityinaBiofilterTreatingNitrobenzeneWasteGas
Samplecollection,DNAextractionandpolymerasechainreaction(PCR)
Therepresentativebiologicalsampleswerecollectedfromthesamplingportwithinthebiofil-
terattheendoftheexperiment(EBRT55.4s).Theperformanceofthebiofilterwasstabledur-
ingthisstage(ILR<20gm-3h-1inaccordancewithengineeringpractice).Eachsamplewas
immersedin20mLofsterilizedwaterandsonicatedfor20stostripthebiomassfromthe
packingmaterial.Atroomtemperature,2mLsampleswerethencentrifugedat13,000rpmfor
2minusinganEppendorfcentrifuge5417R(Eppendorf,Hamburg,GER).TheDNAtemplate
wasextractedusingaFastDNA1SpinKitforSoil(MPBiomedicals,CA,USA)accordingto
themanufacturer’sinstructions.
DNAsamples(10mgeach)wereemployedtogenerateampliconsthatcoveredtheV3-V5
hypervariableregionsofthebacterial16SrRNA.PCRswereperformedin20μLreactionmix-
turesthateachcontained1μLoftheDNAtemplatewithapproximately10ngofDNA,0.8μL
(5μM)oftheforward(338F(5'-ACTCCTACGGGAGGCAGCA-3'))andreverse(806R
(5'-GGACTACHVGGGTWTCTAAT-3'))primers,2μL(2.5mM)ofdeoxynucleosidetri-
phosphate(dNTPs),0.4μLofFastPfuPolymerase,4μLof5×FastPfuBufferandcomplement
ddH O.TheDNAwasamplifiedbyusingaGeneAmp1(9700,ABI,USA).PCRamplification
2
wasperformedusinginitialdenaturationat95˚Cfor3min,followedby27cyclesofdenatur-
ationat95˚Cfor30s,annealingat55˚Cfor30s,extensionat72˚Cfor45sandafinalexten-
sionat72˚Cfor10min.Ampliconswereextractedfrom2%agarosegelsandpurifiedusingan
AxyPrepDNAGelExtractionKit(AxygenBiosciences,UnionCity,CA,USA)accordingto
themanufacturer’sinstructionsandwerequantifiedusingQuantiFluor™-ST(Promega,USA).
IlluminaMiSeqhigh-throughputsequencing
Purifiedampliconswerepooledinequimolarandpaired-endsequences(2×250)onanIllu-
minaMiSeqplatform(PE250,Illumina,USA)accordingtothestandardprotocols.Corre-
spondingsequenceswithspecificbarcodesinsideeachsamplewereselectedusingMothur
(http://www.mothur.org/).Then,datadenoisingwasconductedasdescribedinaprevious
study[25].Afternoisereduction,23,600readsweredrawnrandomlyfromthesamplesto
compareallthesamplesatthesamesequencingdepth.Downstreamtaxonomicassignment
wasperformedusingtheRibosomalDatabaseProject(RDP)(http://rdp.cme.msu.edu/)classi-
fierawithconfidencethresholdof50%.Mothurwasappliedtocalculatetherichnessand
diversityindices,includingtheoperationaltaxonomicunits(OTUs),Chaosindex,andShan-
nonindex[26].AclusteranalysisofthemicrobialcommunitywasconductedusingPAleonto-
logicalSTatistics(PAST,v.3.01)softwarewithanunweightedpair-groupaveragemethod.
Calculations
Theemptybedresidencetime(EBRT,s),removalefficiencyofNB(RE,%),inletloadingrate
ofNB(ILR,gm–3h–1)andtheeliminationcapacityofNB(EC,gm–3h–1)ofthereactorwere
estimatedbyusingthefollowingequations:
Emptybedresidencetime; EBRT ¼V=Q s ð1Þ
Removalefficiency; RE¼ðC(cid:0) C Þ=C (cid:3)100% % ð2Þ
i o i
Inletloadingrate; ILR¼QC=V gm(cid:0) 3 h(cid:0) 1 ð3Þ
i
Eliminationcapacity; EC¼QðC(cid:0) C Þ=V gm(cid:0) 3 h(cid:0) 1 ð4Þ
i o
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MicrobialCommunityinaBiofilterTreatingNitrobenzeneWasteGas
whereVisthevolume(m3)ofthepackedbedsection,Qisthegasflowrate(m3h-1),andC
i
andC aretheinletandoutletconcentrations(mgm-3)ofNB,respectively.
o
ResultsandDiscussion
NBremovalbyabioticmeans
Duringtheabioticexperiment,100mgm-3gas-phaseNBwasintroducedtothebiofilterata
flowrateof149.5Lh-1foraperiodof7days,andtheNBremovalefficiencywasmeasured
underthetricklingdensityofamineralsaltnutrientsolutionof0.52m3m-2h-1.Thepacking
mediumremovedlessthan2.47%oftheinletNBfromthegasstream(datanotshown),which
showedthattheeffectoftheNBabioticdegradationwasnegligibleinthebiofilter.
NBremovalbythebiofilter
Generally,mostlaboratory-scalebiofilterswerefedwithonlylowconcentrationsofpollutants
duringacclimation.Inthisstudy,20–30mgm-3gas-phaseNBwasintroducedtothebiofilter
withanemptybedresidencetime(EBRT)of64s.ThebiofilterachievedanearlystableRE
valueof98.8%after14days(Fig2A),whichindicatedthatthebiofilterinoculatedwithaerobic
activatedsludgefromtheNBwastewatertreatmentplantrequiredashorteracclimationtime.
Aftertheacclimationperiod,steady-stateexperimentswerecarriedoutatdifferentEBRTsand
inletNBconcentrations,whichareimportantparametersthataffecttheefficiencyofbiofiltra-
tionunits.AsdepictedinFig2A,whentheinletconcentrationsofNBgraduallyincreased
from43.0to174.2mgm-3withanEBRTof64sduringdays15–53,theREvaluesofNBfluctu-
atedbetween93.5%and100%.TheEBRTdecreasedfrom64sto55.4sduringthenextperiod
fromthe54thdaytothe88thday(Fig3),theinletconcentrationsofNBweremaintainedin
therangeof62.0–138.4mgm-3andtheoutletconcentrationsofNBinthebottomlayer,mid-
dlelayerandtoplayerwere25.6–64.9mgm-3,11.1–41.1mgm-3,and0–14.3mgm-3,respec-
tively.REvaluesof49.5%-60.3%,68.5%-82.7%,and85.1–100%wereachievedinthebottom
layer,middlelayerandtoplayer,respectively.Finally,thebiofilterwassubjectedtoamuch
lowerEBRT(34s)andREvaluesof89.4%-100%weremaintainedduringdays89–113(Fig
2A).ThesedataindicatethatthebiofilterperformedwellandthattheREwashardlyrelevant
totheEBRTwithlowNBconcentrations.
Inthisstudy,maximumECvaluesof17.5,12.4and13.9gm-3h-1wereachievedwithEBRT
valuesof64,55.4and34sandILRvaluesof18.7,13.8and15.7gm-3h-1,respectively(Fig2B).
TheslopeofthelinearfittingequationofNBdegradationwithanEBRTof64s(0.921)was
Fig2. PerformanceofthebiofiltersfortheremovalofNB:effectoftheEBRT(a)andtheeliminationcapacityvs.
theinletloadingrate(b).
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Fig3.OutletconcentrationsofNBandREsindifferentlayerswithanEBRTof55.4s.
doi:10.1371/journal.pone.0170417.g003
higherthanthatwithanEBRTof34s(0.792)andwassimilartothatwithanEBRTof55.4s
(0.938),whichwasnear1.0underlowNBloads.Inthisregion,theECofthebiofilterishighly
dependentontheEBRT.IncreasingthegivenEBRTenhancedthetransferrateofNBfromthe
gas-phasetothebiofilmandimprovedtheEC[27].
Overallmicrobialcommunitydiversity
Sampleswerecollectedfromtheinitialinoculuminthetop,middleandbottomlayersofthe
biofilterwithanEBRTof55.4s,respectively.Themicrobialcommunitydiversitiesandphylo-
geneticstructuresofthesesampleswereanalysedbyusingIlluminaMiSeqhigh-throughput
sequencing.Over20000sequenceswereobtainedforeachsample.TheGood’scoveragesof
thesesamplesrangedfrom99.76%to99.89%,indicatingthatthesequencesgeneratedatthis
sequencingdepthrepresentedthebacterialcommunitiesofthesesampleswell[26].Forfair
comparison,thelibrarysizeofeachsamplewasnormalizedtothesamesequencingdepth
(23,600reads)byrandomlyremovingtheredundantreads.RelativelyhigherOTUswere
foundintheinitialinoculum(366)thanthesamplesofthetop(360),middle(309)andbottom
layers(289).TheestimatedspeciesrichnessChao1foreachsamplewasalsoconsistentwith
thistrend(Fig4).BasedontheShannonindex,theinitialinoculum(4.60)alsohadarelatively
higherdiversitythanthesampleofthetoplayer(4.12),followedbythesamplesofthemiddle
(3.84)andbottomlayer(3.43).ThebiodiversityvariationtrendswereattributedtotheNB
wastegasactingasthesolecarbonandenergysourcetothebiofilter,whichdecreasedthebio-
diversityofeachlayerinthebiofilter.DuringthecontinuousbiodegradationofNB,theincre-
mentofintermediateproducttypesmayhaveresultedintheincrementofbiodiversityfrom
thebottomlayertothetoplayerinthebiofilter.
Shiftsinmicrobialcommunitystructureandcomposition
Thesmalldifferencesbetweenthemicrobialcommunitystructuresinthethreelayersofthe
biofilterweredistinguishedbyusingprincipalcomponentanalysis(PCA)andfromtheabun-
danceofmajorgenera(Fig5).PCArevealedthatthebacterialcommunitystructurevaried
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Fig4.Rarefactioncurvesbasedon16SrRNAgenesequencingofthesamples(fromtop,middleand
bottomlayers)andtheinitialinoculum.TheChao1indexisalsoshown.TheOTUsweredefinedbya97%
cutoff.
doi:10.1371/journal.pone.0170417.g004
significantlyamongthelayersofthebiofilter.Principlecomponent1(PC1)andprinciple
component2(PC2)accountedfor54.76%and26.67%ofthetotalvariance,respectively.As
demonstratedbyPCA(Fig5A),theinitialinoculumwaslocatedinthefirstquadrant,samples
ofthetopandmiddlelayerwerelocatedinthesecondquadrant,andsamplesofthebottom
layerwerelocatedinthethirdquadrant.Interestingly,thesamplesofthetoplayerwerenot
nearthesamplesofthemiddleandbottomlayersortheinitialinoculum.Theheatmapshows
thatthemajorgeneraofeachlayerweredifferentfromeachother(Fig5B).Inthisstudy,
nitritewasdetectedinthefilterliquor(Fig6).Inaddition,nitriteandcatecholwere
Fig5. Principalcomponentanalysis(PCA)ofbacterialcommunitiesfromtheinitialinoculumandsamples
ofthetop,middleandbottomlayersbasedontheclassifiedOTUs(a).Heatmapillustratingtheabundances
ofallthemajorgenera(witharelativeabundanceofmorethan1%inatleastonesample).Thecolorintensity
(logscale)ineachpanelindicatestherelativeabundanceofthegenusineachsample(b).
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Fig6.Concentrationsofnitritereleased.
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characterizedasthemetabolicproductsofNBoxidationunderaerobicconditions[28].This
resultsuggeststhatthesubtlevariationsofthebacterialcommunitystructureareattributedto
theaccumulationofNBmetabolites.
ThetotalclassifiednumberofOTUsinallfourbacterialcommunitieswas659,and69
OTUs(10.47%)oftheOTUsweresharedbythefourcommunities.Morethan71.01%ofthe
sharedOTUsbelongedtoProteobacteriaaccordingtothephylum-leveltaxonomicdistribu-
tionofthefoursamples(inoculumandtop,middleandbottomlayers),andtheOTUsbelong-
ingtoBacteroidetes(8.70%),Actinobacteria(5.80%),Chloroflexi(5.80%)andAcidobacteria
(4.35%)weresharedatsimilarlevels(Fig7).Thedatashowedthatthemicrobesinthephylum
ProteobacteriawerekeyduringtheremovalprocessofNBfromwastegaswithlowamountsof
NB.
Atotalof14phylawereidentified,amongwhich14,12,12and12weredetectedfromthe
initialinoculumandthesamplesofthetop,middleandbottomlayers,respectively.Proteobac-
teriawereadominantpartofthebacterialcommunityintheinitialinoculum(25.73%),top
layer(52.36%),middlelayer(60.16%)andbottomlayer(59.45%),respectively.Therelative
abundanceofBacteroideteswas29.52%inthetoplayer,whichwashigherthanthoseinthe
middlelayer(17.23%),bottomlayer(10.46%)andtheinitialinoculum(15.86%).Therelative
abundanceofActinobacteriadecreasedfrom23.24%(bottomlayer)to9.18%(middlelayer),
Fig7.Venndiagramillustratingtheoverlapofthefourbacterialcommunitiesfromtheinitial
inoculumandthesamplesofthetop,middleandbottomlayers.ThesharedOTUs(69)wereanalysedat
thephylumlevel.
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Fig8.Abundancesanddistributionsofthedifferentphylainthefourbiofilmsamples.
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6.69%(toplayer)and1.63%(theinitialinoculum)(Fig8).FromFig3,NBwasremoved
mainlyinthebottomlayer(49.5%-60.3%)andmiddlelayer(68.5%-82.7%);therefore,itcanbe
concludedthatthephylumActinobacteriaandProteobacteriahadmajorcontributionstothe
degradationofNB,andthemainfunctionofthephylumBacteroideteswastodegradetheNB
metabolicintermediates.
Potentialfunctionsandrelativeabundancesofthedominantgenera
Atotalof89generawereclassifiedamongthetestsamples.Twelvegenerawitharelativeabun-
dance>1%intheinitialinoculumandsamplesofthetop,middleandbottomlayercommuni-
tiesareshowninTable2.Nitrospira (12.56%),Flexibacter(7.08%)andBlastocatella(5.05%)
wereobviouslydominantintheinitialinoculum.
Inthebottomlayer,genusRhodococcus(22.18%),Rhizobium(12.86%)andRhodanobacter
(8.36%)hadobviouslyhigherrelativeabundancethanintheinitialinoculum(Table2).The
Table2. Genera(relativeabundance>1%atgenuslevels)intheinitialinoculumandthesamplesofthetop,middleandbottomlayers. Thedomi-
nantgenerainthedifferentlayersareshowninbold.
Phylum Class Genus(%) Initial Top Middle Bottom
Nitrospirae Nitrospira Nitrospira 12.56 0.00 0.00 0.00
Bacteroidetes Sphingobacteria Flexibacter 7.08 0.17 0.02 0.02
Proteobacteria α-proteobacteria Blastocatella 5.05 0.00 0.01 0.04
Deinococcus-Thermus Deinococci Truepera 0.65 0.83 1.87 2.16
Proteobacteria α-proteobacteria Devosia 0.08 2.59 1.46 0.72
Proteobacteria α-proteobacteria Rhizomicrobium 0.04 3.09 3.22 2.32
Proteobacteria α-proteobacteria Rhodopseudomonas 0.02 3.62 1.35 1.21
Proteobacteria γ-proteobacteria Pseudomonas 0.02 1.92 0.93 1.58
Proteobacteria α-proteobacteria Ferruginibacter 0.01 14.74 2.98 3.22
Proteobacteria γ-proteobacteria Rhodanobacter 0.00 7.70 10.35 8.36
Proteobacteria α-proteobacteria Rhizobium 0.00 8.01 2.45 12.86
Actinobacteria Actinobacteria Rhodococcus 0.00 1.43 4.48 22.18
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genusRhodococcusbelongedtothephylumActinobacteria,whichisknowntobecapableof
NBbiodegradation[29–31].AccordingtoFig3,theREofNBwas49.5%-60.3%inthebottom
layer;thus,itwasconcludedthatthegenusRhodococcuswasthemajorNB-degradinggenus.
ThisfindingmayexplainwhyRhodococcuswasdominantintheinletofthebiofilter.Inthe
middlelayer,thegenusRhodanobacterwasobviouslyenriched(10.35%);however,therelative
abundanceofRhodococcusintheinitialinoculumwaslow(0.004%).Rhodococcusbelongsto
thephylumProteobacteria,whichiscapableofdenitrification[32–33].Nitriteisatypeof
metabolitethatisusedforNBoxidation,whichexplainsthepredominanceofRhodanobacter
inthislayer[34].Inthetoplayer,thegeneraFerruginibacter(14.74%),Rhizobium(8.01%)and
Rhodanobacter(7.70%)exhibitedexceedinglyhigherabundancethanthoseintheinitialinocu-
lum.ThegenusFerruginibacterbelongstothephylumBacteroidetes,whichhasfrequently
beendetectedatwastewatertreatmentplants[35–36].Apreviousstudyindicatedthatmem-
bersofFerruginibacterarecapableofhydrolysingsomeorganicmatter[37].Itwasasserted
thatthegenusFerruginibacterpredominatedinthetoplayerbecauseofthedegradationofthe
NBmetabolicintermediates.AlthoughPseudomonaspossessedafairlylowerabundanceinthe
initialinoculum(0.02%)andtop(1.92%),middle(0.93%)andbottom(1.58%)layers,respec-
tively,Pseudomonashasalsobeenreportedtohavetheabilitytodegradehighconcentrations
ofNB[38].
Conclusions
ThisstudydemonstratedanefficientsystemforNBremovalatlowILRvalues.TheREswere
alwayshigherthan93%atdifferentphases,andtheslopesofEC/ILRwerecloseto1.0.Illu-
minaMiSeqhigh-throughputsequencingtechnologywassuccessfullyappliedtoanalysethe
variationsofthebiodiversityandmicrobialcommunitystructureinthesebiofilmsalongthe
longitudinaldirectionofthebiofilter.Theincreaseofbiodiversityfromthebottomupwas
attributedtotheincreaseofNBmetabolicintermediatetypes.Themicrobialcommunity
structureaffectedtheecologicalfunctionsignificantlyandfurtherinfluencedtheperformance
ofthebiofilter.ThesequencingdatashowedthatthephylumActinobacteriaandgenusRhodo-
coccusplayedkeyrolesinthedegradationofNBinthebottomlayer.
SupportingInformation
S1Table.Nitrobenzeneproperties.
(DOCX)
AuthorContributions
Conceptualization:CLJZ.
Datacuration:JZZW.
Formalanalysis:JZPSCL.
Fundingacquisition:JZ.
Investigation:JZZWCL.
Methodology:CLJZ.
Projectadministration:CL.
Resources:CLJZ.
PLOSONE|DOI:10.1371/journal.pone.0170417 January23,2017 10/12