MicrobiologyOpen. 2020;00:e1013.  | 1 of 24https://doi.org/10.1002/mbo3.1013

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Received:23October2019  |  Revised:3February2020  |  Accepted:3February2020DOI: 10.1002/mbo3.1013

O R I G I N A L A R T I C L E

DNA stable-isotope probing reveals potential key players for microbial decomposition and degradation of diatom-derived marine particulate matter

Ying Liu1  | Jiasong Fang2,3,4 | Zhongjun Jia5 | Songze Chen1 | Li Zhang6 | Wei Gao5

ThisisanopenaccessarticleunderthetermsoftheCreativeCommonsAttribution-NonCommercial-NoDerivsLicense,whichpermitsuseanddistributioninanymedium,providedtheoriginalworkisproperlycited,theuseisnon-commercialandnomodificationsoradaptationsaremade.©2020TheAuthors.MicrobiologyOpenpublishedbyJohnWiley&SonsLtd.

1StateKeyLaboratoryofMarineGeology,TongjiUniversity,Shanghai,China2ShanghaiEngineeringResearchCenterofHadalScienceandTechnology,ShanghaiOceanUniversity,Shanghai,China3LaboratoryforMarineBiologyandBiotechnology,QingdaoNationalLaboratoryforMarineScienceandTechnology,Qingdao,China4DepartmentofNaturalSciences,HawaiiPacificUniversity,Honolulu,HI,USA5StateKeyLaboratoryofSoilandSustainableAgriculture,InstituteofSoilScience,ChineseAcademyofSciences,Nanjing,China6StateKeyLaboratoryofGeologicalProcessandMineralResources,FacultyofEarthSciences,ChinaUniversityofGeosciences,Wuhan,China

CorrespondenceJiasongFang,ShanghaiEngineeringResearchCenterofHadalScienceandTechnology,ShanghaiOceanUniversity,Shanghai,China.Email:jsfang@shou.edu.cn

LiZhang,StateKeyLaboratoryofGeologicalProcessandMineralResources,FacultyofEarthSciences,ChinaUniversityofGeosciences,Wuhan,Hubei,China.Email:lizhang@cug.edu.cn

Funding informationTongjiUniversity;SIP;NationalNaturalScienceFoundationofChina,Grant/AwardNumber:41773069;KeyR&DProgramofChina,Grant/AwardNumber:2018YFC0310600

AbstractMicrobiallymediateddecompositionofparticulateorganiccarbon(POC)isacentralcomponentoftheoceaniccarboncycle,controllingthefluxoforganiccarbonfromthesurfaceoceantothedeepocean.Yet,thespecificmicrobialtaxaresponsibleforPOCdecompositionanddegradationinthedeepoceanarestillunknown.Totargettheactivemicrobiallineagesinvolvedintheseprocesses,13C-labeledparticulateor-ganicmatter(POM)wasusedasasubstratetoincubateparticle-attached(PAM)andfree-livingmicrobial(FLM)assemblagesfromtheepi-andbathypelagiczonesoftheNewBritainTrench (NBT).BycombiningDNAstable-isotopeprobingandIlluminaMiseqhigh-throughputsequencingofbacterial16SrRNAgene,weidentified14ac-tivebacterialtaxonomicgroupsthatimplicatedinthedecompositionof13C-labeledPOMat lowandhighpressuresunderthetemperatureof15°C.OurresultsshowthatbothPAMandFLMwereabletodecomposePOCandassimilatethereleasedDOC.However,similarbacterialtaxainboththePAMandFLMassemblageswereinvolvedinPOCdecompositionandDOCdegradation,suggestingthedecouplingbe-tweenmicrobiallifestylesandecologicalfunctions.MicrobialdecompositionofPOCanddegradationofDOCwereaccomplishedprimarilybyparticle-attachedbacteriaatatmosphericpressureandby free-livingbacteriaathighpressures.Overall, thePOCdegradationrateswerehigheratatmosphericpressure(0.1MPa)thanathighpressures(20and40MPa)under15°C.Ourresultsprovidedirectevidencelinkingthespecificparticle-attachedandfree-livingbacteriallineagestodecompositionanddegradationofdiatomicdetritusatlowandhighpressuresandidentifiedthepoten-tialmediatorsofPOCfluxesintheepi-andbathypelagiczones.

K E Y W O R D S

decomposition,free-livingmicroorganisms,NewBritainTrench,particle-attachedmicroorganisms,particulateorganiccarbon

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1  | INTRODUC TION

Organicmatterproducedbyphytoplanktoninthesurfaceoceanisexportedtothedeepoceanandtheseabedvia,mostly,sinkingpartic-ulateorganicmatter(POM)bytheprocessesofthebiologicalpump(Azam&Malfatti,2007;Eppley&Peterson,1979;Falkowski,1998;Volk&Hoffert,1985).Itisestimatedthatsinkingparticulateorganiccarbon(POC)(102–104 μmdiameter;Verdugoetal.,2004)istrans-portedtothedeepoceanwithaglobalrateof2–13PgCyear−1(Boyd&Trull,2007;Eppley&Peterson,1979;Lima,Lam,&Doney,2014).Duringdescent,thesinkingPOCfluxattenuatessharplywithdepthasaresultofdecompositionofPOCandreleaseofdissolvedorganiccarbon(DOC)fromabiotic (disaggregationandfragmentation)andbioticprocesses(zooplanktonconsumptionandmicrobialremineral-ization)(Burd&Jackson,2009;DeLong,Franks,&Alldredge,1993;Simon,Grossart,Schweitzer,&Ploug,2002;Steinbergetal.,2008;Turner,2015;Wilson,Steinberg,&Buesseler,2008).Approximately90%ofthesinkingPOCisremineralizedintheupper1,000m,andonly0.1%–0.3%ofthePOCcanreachtheseafloorandisultimatelyburiedinsediments(Arístegui,Agustí,Middelburg,&Duarte,2005;Arístegui,Gasol,Duarte,&Herndld,2009;Hedges,1992;Wakeham&Lee,1993).

Marine microorganisms play a critical role in the decomposi-tionandremineralizationofPOC(Anderson&Ryabchenko,2009;Anderson&Tang,2010;Azam&Malfatti,2007;Fangetal.,2015).It has beenhypothesized that the particulates act as carbon- andnutrient-rich hotspots, and the particle-attached microorganisms(PAM)secreteexoenzymestodecomposetheparticlesandreleasedissolvedorganiccarbon(Simonetal.,2002).PAMsolubilizePOMfaster than theyareable to takeup the releasedDOC, leading totheformationofcarbon-andnutrient-richDOCplumes,streamingbehindthePOCparticulates.ThereleasedDOCsupportsPAMandthe free-livingmicroorganisms (FLM) in the surroundingwater fortheirgrowthandrespiration(Azam&Long,2001;Fangetal.,2015;Kiørboe& Jackson, 2001; Li et al., 2015). Thus,microbial decom-positionand transformationofPOMcontrolsorganiccarbon (OC)fluxesdeliveredfromthesurfaceoceantothedeepocean,andhet-erotrophicmicroorganismsplayanessentialroleintheprocess(e.g.,Belcheretal.,2016;Karl,Knauer,&Martin,1988;Steinbergetal.,2008). Understanding POC decomposition and degradation pro-cessesiscriticaltoassessthesequestrationandstorageofcarbonintheoceanandoceaniccarboncycling.

Hydrostatic pressure is a critical parameter in affecting mi-crobial physiology (Bubendorfer et al., 2012;Marietou&Bartlett,2014), abundance (Grossart andGust, 2009;Marietou&Bartlett,2014),metabolicactivity(Tamburini,Garcin,Ragot,&Bianchi,2002;Tamburinietal.,2006;Tamburinietal.,2003;Turley,1993),diver-sity,andcommunitystructure(GrossartandGust,2009;Marietou& Bartlett, 2014; Tamburini et al., 2006, 2009). Piezophiles referto pressure-loving microorganisms, which grow preferentially athigh pressure than atmospheric pressure. Piezotolerant bacteriahad optimal growth at atmospheric pressure (Fang et al., 2010).For instance, deep-sea bacterium Photobacterium profundum SS9

increased the proportion of unsaturated fatty acids at high pres-sure (Allen et al., 1999), Shewanella putrefaciens CN-32 possessesdualflagellarsystem(Bubendorferetal.,2012)underhighpressureconditions.Highpressurereducedbacterialcellnumbers(Grossartand Gust, 2009; Marietou & Bartlett, 2014), decreased microbialmetabolic activity (Tamburini et al., 2006; Tamburini et al., 2002;Tamburini et al., 2003; Turley, 1993) and organicmatter degrada-tionrates(Tamburini,Boutrif,Garel,Colwell,&Deming,2013).HighpressureincreasedtherelativeabundanceofGammaproteobacteriaandCytophaga-Flavobacterclusterwhenprokaryotesincubatedwithdiatomdetritus (Tamburinietal.,2006)or fecalpellets (Tamburiniet al., 2009). Laboratory experiments showed that high pressurecausedanincreaseintherelativeproportionofAlphaproteobacteriainacoastalenvironment(Marietou&Bartlett,2014).

Despiteitsimportance,itremainsaprincipalchallengetoidentifyandquantifytherelativeimportanceofvariousmicrobesinvolvedintheseprocesses.Therehasbeennodirectevidencelinkingspecificbacterial lineages to POM decomposition and DOM degradation.Somestudies indicatethatbacterialactivityonsinkingparticles inthedeepocean is inadequate toaccount for theattenuatingPOCfluxwithdepth(Karletal.,1988).Otherssuggestthatthefree-liv-ingbacteria,ratherthanparticle-attachedbacteria,aretheprimarymediatorsofparticulatedecompositionintheocean'sinterior(Cho&Azam,1988;Karletal.,1988).Furthermore,therehavebeencon-trastingestimatesonthecontributionsofPAM inparticledecom-position and remineralization. Smith, Simon, Alldredge, and Azam(1992)estimatedthat97%ofthehydrolysatesproducedbybacteriainmarinesnowwerereleased,with3%beingutilizedbyPAMintheaggregates(Smithetal.,1992).Inanotherstudy,Belcheretal.(2016)showedthatPAMrespirationrepresentsaminorportion(3%–16%)ofPOCattenuationintheupperwatercolumn,butbecomesmoreimportant (10%–65%) in the deep, suggesting great variations inrates of the processeswith depth (Belcher et al., 2016). KnowingwhichtypesofmicroorganismsbreakdownPOManddegradeDOMmaybeusefulintheassessmentoffactorsthatcontrolOCfluxfromthesurfaceocean to thedeepocean.As thesuccessionofmicro-bialcommunitiesoftenoccurswithdepthintheocean,studyingtheresponseofmicrobialcommunitiestoPOMatdifferentdepthscanprovidecluesaboutlinkagesbetweenmicrobialnichespecializationandtheirresourceutilization(e.g.,Lauroetal.,2009).

Stable-isotopeprobing(SIP)isapowerfultechniquethatdirectlylinksspecificfunctionalmicroorganismstoaparticularbiogeochem-ical process (Radajewski, Ineson, Parekh, & Murrell, 2000). Thismethodisbasedonthepremisethatmetabolicallyactivemicrobesthatassimilate stable-isotope-labeledsubstrateswould incorporatethe rare isotope (e.g., 13C) into cellularmacromolecules (e.g.,DNA,phospholipid fatty acids, etc.), that can be detected by molecularmicrobiological techniques, thereby enabling the identification andquantificationofthefunctionallyactivemicroorganisms(Radajewskietal.,2000).Inthepresentstudy,weusedDNAstable-isotopeprob-ingtotargettheactivemicrobialtaxainvolvedindecompositionanddegradation of POM and DOM by incubating microorganisms en-richedfromshallow(75m)anddeepwaters(2,000and4,000m)of

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theNewBritainTrenchwith13C-labeledPOM.WehypothesizethatPAMandFLMperformdifferentfunctionsintheocean,thatis,POCdecompositionbytheformerandDOCdegradationbythelatter.WefurtherpostulatethatthedividedfunctionsbetweenPAMandFLMbecomeundividedwithincreasingpressureinthedeepocean,thatis,bothgroupsofmicrobescanperformthesamefunctions,POCde-compositionandDOCdegradation.Theobjectiveofthisstudywastogaininsightintothemicrobial-mediateddecompositionanddegrada-tionprocessesofdiatom-derivedmarineparticulateorganiccarbon(POC),bymimickingPOCsinkingfromthesurfaceoceantothedeepocean(increaseinhydrostaticpressure)andexaminingthechangesincommunitystructureofparticle-attachedandfree-livingmicroor-ganisms,theprocessestheyparticipatedin(decompositionanddeg-radation),andtheratesofcarbonutilizationbythesemicrobes.

2  | MATERIAL S AND METHODS

2.1 | Sampling

SeawatersampleswerecollectedfromtheEstation(06°02.1243′S,151°58.5042′E)(FigureA1)oftheNewBritainTrench(NBT)duringthecruiseofM/VZhangjianinSeptember2016.Samplesweretakenat threedepths,75-,2000-, and4000-m,byusingaSBE-911PlusCTDwithNiskinbottles(Sea-BirdInc.).Theseawatersampleswerenotkeptunder insitupressurepriorSIP incubations.Thephysicalandchemicalparametersoftheseawatersamplesinthewatercol-umnofNBTcanbeseeninTableA1.

About1Lseawatersamplefromeachdepthwasfilteredsequen-tiallythrougha47-mm3μmporesizepolycarbonate(PC)membrane(MerckMilliporeLtd.)tocollecttheparticle-attachedmicrobes,anda47-mm0.22μmporesizePCmembranetocollectthefree-livingbacterialassemblages (Eloeetal.,2011).Atotalofsix filterswereusedforcollectingPAMorFLMfromseawaterateachdepth.ThreefilterswereusedforthetotalDNAextractionandcommunitydiver-sityanalysis.TheotherthreefilterswereusedforsubsequentSIPincubations(seebelow).

2.2 | Stable-isotope probing experiments

2.2.1 | Production of 13C-labeled particulate organic matter (POM)

TheplanktonicdiatomspeciesThalassiosira weissflogii(strainCCMA-102),oneofthemostcommondiatomspeciesintheglobalsurfaceocean, was selected for the 13C-labeled tracer experiment. Thediatoms were cultured either with added 13C-labeled NaHCO3 or nonlabeledNaHCO3 (control), inthef/2mediaprepared ina5.5LErlenmeyerflask,with5Lsterileartificialseawater(ASW).TheASWcontained(w/v%)thefollowing:2.12NaCl,0.34Na2SO4,0.06KCl,0.008KBr,0.0058H3BO3,0.00028NaF,0.96MgCl2▪6H2O,0.13CaCl2 ▪ 2H2O, 0.0022 SrCl2 ▪ 6H2O, 0.029NaH

13CO3 in distilled

water(Berges,Franklin,&Harrison,2001),supplementwithNaSiO3 (Guillard,1975),250mlThalassiosira weissflogii suspension (precul-turedfor20days).TheNaH13CO3solutionwaspreparedbydissolv-ing1.5gNaH13CO3in50mlsteriledouble-distilledH2O(ddH2O)andfilteredthrougha0.22μmpolycarbonatefilter(Pasotti,Troch,Raes,&Vanreusel,2012).After30daysofgrowinginanilluminationincu-batorat20°Canda12-hr,12-hrlight/darkregime,diatomdetritus,13C-labeledandnonlabeled (control)werecollectedbycentrifuga-tion(BeckmanCoulter)at11,180gfor10min.ThecollecteddiatomdetrituswaslyophilizedinaLyophilizer(ChristCorp.)at−44°Cfor48hr.Thedrydiatomdetrituswasgroundwithliquidnitrogen,andautoclavedat121°Cfor20min,andthenstoredat−20°Cuntiluse.Thepercentageofthe13C-labeledand12C-controlfordiatomdetri-tuswasdeterminedtobe71.7%and1.3%,respectively.

2.2.2 | High pressure incubation

Particle-attachedmicrobialandFLMwereobtainedfromserial fil-trationsasdescribedabove,thethreefiltersforcollectingPAMorFLMviafiltrationofseawaterwereaddeddirectlyintothepouchesforSIPincubations.Thecollectedparticle-attachedandfree-livingmicrobes were incubated with the added bacteria-free detritalparticulate organic matter in separate cultures, designated as 75(2000/4000)-m-13C(12C)-PAM(FLM),atinsitupressure(0.1,20,and40megapascal,MPa) and15°Cunderdark condition for30days,usingair-tightpouches.Themediacontained60mlsterileartificialseawater,0.2g13C-labeleddiatomdetritus(≥0.7μmporesize),and20mlFluorinert™(3M™Corp.).Fluorinertwasaddedtothepouchestoserveasasourceofoxygentothecultures(Fang,Uhle,Billmark,Bartlett,&Kato,2006;Kato,Sato,&Horikoshi,1995).Priortouse,Fluorinertwassaturatedbybubblinghigh-purityoxygenfor8–10hrat4°Candthenfilteredthrougha0.22μmpolycarbonatefilter(Fangetal.,2006).Thecontrolwaspreparedusingnonlabeleddetritusasagrowthsubstrate.Allculturesweredoneintriplicate.GrowthofPAMandFLMplateauedafter30days.

Itmustbeemphasizedthattheinsitutemperaturewasaround2°Cat2,000and4,000mdepthsatoursamplingsites(TableA1),muchlowerthantheincubationtemperature(15°C).Forthistestofconceptstudy,weused15°C,insteadoftheinsitutemperature,inmicrobialcultivation,inordertogenerateenoughbiomassforsub-sequentmolecularmicrobiologicalstudy.

2.3 | Determining the concentration and δ13C of POC and DOC

Analiquotof10mlculturewasfilteredthrough25-mm0.7μm pore sizeglassfiberfilter(Whatman)whichwasprecombustedinamuf-fle furnace at 450°C for 5 hr. The collected filtermembrane andfiltratewereusedforPOCandDOCconcentrationmeasurements,respectively.InpreparationforPOCandDOCanalysis,thecollectedfilters were pickled with 12M HCl for 24 hr in fume cupboards

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and then dried at 50°C for 24 hr in an oven, and the filtratewasacidizedwith20μl12MHCl.TheconcentrationofPOCandDOCwas measured by a PE2400 Series II CHNS/O analyzer (PerkinElmer)andaTOC-VCHPanalyzer(ShimadzuCorp.),respectively,attheInstituteofSoilScience,ChineseAcademyofSciences,Nanjing,China. The δ13Cof POC andDOCwas determined by an IsotopeRatioMassSpectrometer (ThermoScientific)attheThirdInstituteofOceanography,Xiamen,China.

2.4 | DNA extraction and SIP gradient fractionation

After 30 days incubation, an aliquot of 10ml culturewas centri-fugedat10,000rpmfor5mintocollectcellbiomass.ThePCfiltersforPAMandFLMfiltrationsatday0werecutintopiecesandthentransferredto2mlsterilizedcentrifugetubes.DNAwasextractedwithFastDNA™SPINKitforSoil(MPBiomedical)accordingtothemanufacturer's instructions.ThetotalDNAconcentrationwasde-termined by NANO DROP 2000 (Thermo Scientific) at 260 nmwavelengthandtheDNAwasstoredat−80°Cuntilfurtheranalysis.

TheDNA-SIPgradient fractionationwasperformedbyfollow-ingtheprotocoldescribedpreviously(Jia&Conrad,2009;Neufeld,Vohra, et al., 2007). In brief, about 2.0μgof particle-attachedorfree-livingbacterialDNAatday30wasmixedwithGradientBuffer(GB)andCsCltoachieveabuoyantdensityof1.725g/ml.Aftersym-metry andbalance, themixtures in theultracentrifuge tubewerecentrifuged at 190,000 g at 20°C for 44 hr. After centrifugation,

eachgradientwasfractionatedfromthebottomofthetubeusingaperistalticpumpoperatingat380μl/minoncontinuousmodetocol-lect14fractions(~380μleach).Therefractiveindexofthe14DNAgradientfractionswasmeasuredusingAR200digitalrefractometer(Reichert, Inc.).EachDNAgradientfractionwaspurifiedandthendissolved in 30 μl sterile ddH2O(Jia&Conrad,2009;Neufeld,Vohra,etal.,2007),andpreservedat−20°Cuntilsubsequenttreatment.

2.5 | Real-time quantitative PCR

The copy number of bacterial 16S rRNA gene in the 3th-14th fractionated DNA of PAM and FLM was determinedon a CFX 96 Optical Real-Time Detection System (Bio-RadLaboratories Inc.). DNA samples were amplified using primerset Bac331F (5 -́TCCTACGGGAGGCAGCAGT-3´) and 797R(5 -́GGACTACCAGGGTCTAATCCTGTT-3´) (Nadkarni, Martin,Jacques,&Hunter,2002).Each20-μlreactionsystemcontained1μl DNAtemplate,0.3μlofeachprimer(0.5μM),10μlSYBRPremixExTaq™(TakaraBiotech,Dalian,China),and8.4μl sterile ddH2O.Eachsamplewasperformedintriplicate.TheqPCRamplificationcondi-tionwasasfollows:initialdenaturation(95°C)for30s;40cyclesofdenaturation(95°C)for5s,annealingat55°Cfor30s,andextensionat 72°C for 1min. The construction of standard curves (Ct valuevs.genecopynumber)wasperformedwithplasmidsDNAincludingampliconfragmentofBac27F/1492R.Theamplificationefficiencyofthebacterial16SrRNAgenewasaround100%andtheR2was<.99.

F I G U R E 1  VariationsofPOCandDOCconcentrationsinPAM(a)andFLM(b)cultures,andPOCdegradationrates(c)ofPAMandFLMincubatedwith13C-POCat0.1,20,and40MPaand15°Catday0andday30

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2.6 | Illumina MiSeq sequencing

Before Illumina high-throughput sequencing, DNA from PAMand FLM at day 0 and the 4th-10th layers of the DNA gra-dient fractions at day 30 were amplified using the primerpair 515F (5′-GTGCCAGCMGCCGCGG-3′) and 907R(5′-CCGTCAATTCMTTTRAGTTT-3′) (Lane,1991;Stubner,2002),targetingtheV4-V5regionsofthebacterial16SrRNAgene.The12bpuniquebarcodesequencesthatwereadheredtothe5′endof515Fwereusedtodemultiplexsequences.The50-μlPCRampli-ficationsystemwasconductedintriplicate,witheachcontaining2.5 μlofDNAtemplate,1μlofeachprimer(10μM),25μlPremixTaqTM (Takara),and20.5μl sterile ddH2O.ThePCRamplificationconditionswereasfollows:94°Cfor5min;32cyclesof94°Cfor30s,55°Cfor30s,and72°Cfor45s;72°Cfor8min.AllPCRam-pliconswerevisualizedon1%(w/v)agarosegels,thenpooledandpurified using E.Z.N.A Cycle Pure Kit (OMEGA bio-tek). Finally,thepurifiedDNAwasquantifiedbyQuantiFluorTM-ST(Promega).ThesequencingofpurifiedDNAsampleswasperformedwithanIlluminaMiSeqplatform(Illumina)attheInstituteofSoilScience,ChineseAcademyofSciences,Nanjing,China.

The raw high-throughput sequencing data were processedusingQuantitativeInsightsIntoMicrobialEcology(QIIMEversion:1.9.1)tofiltrateeffectivereads(Caporasoetal.,2010;http://www.qiime.org) with default parameters. The optimizing reads wereclusteredtooperationaltaxonomicunit(OTU)withacutoffvalueof 97% similarity usingUPARSE (version7.1). The representativesequences of OTUs were aligned using the Ribosomal DatabaseProject (RDP) classifier (http://rdp.cme.msu.edu/) against theSilva16SrRNAdatabase(SSU123)(www.arb-silva.de/)usingacon-fidencethresholdof70%.

2.7 | Statistical analysis

To analyze the α-diversity (Shannon, Simpson, Coverage, Ace andChao)ofPAMandFLMcommunities,allsamplesintheOTUtableweresubsampled to the samenumberof sequencesbasedon the fewestsequences (n =4,099)usingQIIMEsoftware (Caporasoet al., 2010;http://www.qiime.org).ThevariationsofsharedanduniqueOTUs inPAMandFLMwerecalculatedbyusingVenny2.1 (http://bioinfogp.cnb.csic.es/tools/venny/index.html).Nonmetricmultidimensionalscal-ing(NMDS)analysiswasusedtoevaluatethedissimilarityofdifferentsamplesbasedontheBray-CurtissimilaritymatrixusingthesoftwareRprogram(v3.2.3)withveganpackage(Pinheiro,Bates,Debroy,Sarkar,&Team,2012).Theanalysisofsimilarity(ANOSIM)wasusedtotestthedissimilaritybetweensamples.TheheatmapplotwasperformedbyHeatmapIllustrator1.0software(http://hemi.biocuckoo.org/down.php).PhylogenetictreeoftheactiveOTUsinPAMandFLMwascon-structedthroughneighbor-joiningmethodswith1,000bootstrapval-uesusingMEGA7.0software(Kumar,Stecher,&Tamura,2016).

Inourstudy,theactiveOTUsaredefinedasthosewithrelativeabundance ≥1% that the abundance of an OTU in the 13C-heavyDNAfractionminusthecorrespondingabundanceinthe12C-heavyDNAfractioninPAMandFLMafterincubation(Orsietal.,2016).

3  | RESULTS

3.1 | POC decomposition and DOC degradation

MicrobialdecompositionanddegradationofPOCandDOCresultedindrasticchangesinconcentrationsofPOCandDOCinthecultures(Figure 1 andTableA2). For PAMcultures, the reduction of POC

F I G U R E 2  Thequantitativedistributionofthe16SrRNAgeneinthe3th-14thDNAfractionsacrossthedifferentbuoyantdensitygradientsofthePAMandFLMassemblagesincubatedwith13C-or12C-POCat0.1,20,and40MPaand15°Cfor30days.(a)0.1MPa-PAM;(b)0.1MPa-FLM;(c)20MPa-PAM;(d)20MPa-FLM;(e)40MPa-PAM;(f)40MPa-FLM.Thex-axisrepresentstheratioofthegenecopynumberineachDNAfractiontothemaximumgenecopiesineachtreatment,accordingtothepreviousmethod(Lueders,Manefield,&Friedrich,2004).They-axisrepresentsthevariationsofCsClbuoyantdensityinthe3th-14thDNAfractions.Thenumbers6and8indicatethe 13C-labeled“heavy”DNAfractionswhilethenumbers10and11showthe12C-labeled“light”DNAfractions

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concentrationsdecreasedwithpressure,averaging52%(from54.0to25.7) in the0.1-MPaculture,27% (55.6 to40.4)and24% (53.3to 40.5mg/L) in the 20- and 40-MPa-PAM cultures, respectively(Figure 1a). The FLM cultures showed a slightly different pattern,POCconcentrationsdecreasedby29%(0.1MPa),41%(20MPa),and46% (40MPa) (Figure1b).On theother hand, the concentrationsofDOC inPAMandFLMcultures decreased in the0.1-MPa, andincreasedinthe20-MPaand40-MPa(Figure1aandb).

Biodegradation rates byPAMandFLMassemblageswere cal-culatedbasedontheincorporationof13CintomicrobialbiomassasdescribedbySuroy,Panagiotopoulos,Boutorh,Goutx,andMoriceau(2015).ThePOCdegradation ratesbyPAMranged from0.009to0.025 μmolCL−1day−1atthethreepressures,withthemaximumrateatatmosphericpressure(Figure1c).POCdegradationratesbyFLMwererelativelyhigher,rangingfrom0.012to0.021μmolCL−1day−1,with the maximum rate at high pressure (40 MPa). Thus, POC

F I G U R E 3  VenndiagramsshowingthenumberandpercentageofsharedoruniqueactiveOTUsforPAMandFLMassemblagesincubatedat0.1,20,and40MPaand15°C.Abbreviation:a,active.ThenumbersinboldarethenumberoftotalOTUsinthePAMandFLMassemblagesatthethreepressures

F I G U R E 4  Nonmetricmultidimensionalscaling(NMDS)analysisofthetotal(a)andactive(b)PAMandFLMcommunitiesincubatedwith13C-POCat0.1,20,and40MPaand15°Catday0andday30basedonBray-CurtissimilaritymatrixattheOTUlevel.Differentsymbolsandcolorsindicatevarioussamples

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degradation rates by the PAM communities decreased markedlywithpressure,whereasPOCdegradationratesbytheFLMcommu-nitiesincreasedwithpressure(Figure1c).Overall,POCdegradationratesweremuchhigheratatmosphericpressure than thatathighpressureat15°C(Figure1c).

Furthermore,thevariationsinδ13CvaluesofPOCshowedthatthepercentageof 13C-labeled substrate (the atompercentof 13C)utilized by the PAM and FLMwere 4.2 and 2.6%, 2.1 and 2.2%,and1.2and3.5%at0.1,20,and40MPaunder15°C,respectively(TableA3).

TA B L E 1  Analysisofsimilarity(ANOSIM)betweenthePAMandFLMassemblagesat0.1,20,and40MPaand15°Catday0andday30

ANOSIM R value P value

Comparison1 TotalOTUs Day0 PAMvs.FLM .1481 .391

Comparison2 TotalOTUs Epipelagic(75m)vs.Bathypelagic(2,000–4,000m) .5 .075

Comparison3 TotalOTUs Day0vs.Day30 .6583 .003

Comparison4 TotalOTUs Day30 PAMvs.FLM .0741 .384

Comparison5 TotalOTUs 75mvs.2,000–4,000m .3214 .292

Comparison6 ActiveOTUs PAMvs.FLM 0 .529

Comparison7 ActiveOTUs 75mvs.2,000m .5 .338

Comparison8 ActiveOTUs 75mvs.4,000m .25 .32

Comparison9 ActiveOTUs 2,000mvs.4,000m .125 .346

Note: The Rvalueindicatesdifferencesbetweenthetwogroups,whilethepvalueshowsthelevelofsignificantdifference.

F I G U R E 5  TherelativeabundanceofthemostactivePAMandFLM(≥1%)atthephylum(class)levelforPOCdecompositionat0.1,20,and40MPaand15°Cfor30days.Thex-axisrepresentstheincreasedrelativeabundance,thatis,theabundanceofaphylum(class)inthe 13C-labeled“heavy”DNAfractionminusthecorrespondingabundanceinthe 12C-labeled“heavy”DNAfractionatday30

F I G U R E 6  Therelativeabundanceofthemostactivegenera(OTUs)(≥1%)inthePAMandFLMassemblagesforPOCdecompositionat0.1,20,and40MPaand15°Cfor30days

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3.2 | Identifying PAM and FLM that utilized POC

Results of real-time quantitative PCR showed that the maximumcopynumberofbacterial16SrRNAgeneappearedinthe6thor8th“heavy”DNAfractionsofthePAM-andFLM-13Ccultures(Figure2),with themaximumvalueof5×106 copies/ml in 13C-PAMculture

at 0.1MPa,while the bacterial abundance peaked in the 10th or11th “light” fractionatedDNA for 12C-control treatment,with thehighestvalueof1.2×107 copies/ml in 12C-FLMcultureat0.1MPa(Table A4). The bacterial copy number in 13C-PAM culture washigher than that of 13C-FLM culture at 0.1 and 40MPa,whereasthebacterialcopynumberofPAMwas lower thanthatofFLMat

F I G U R E 7  TherelativeabundanceofactiveOTUsforPOCdecompositioninthe4th-10thDNAfractionsacrossthedifferentbuoyantdensitygradientsfrom13C-labeled(green,red,andbluesolidlines)and12C-control(graysolidlines)groupsinthePAMandFLMassemblagesincubatedat0.1,20,and40MPaand15°Cfor30days

F I G U R E 8  PhylogenetictreeofactivePAMandFLMOTUsat0.1,20,and40MPaandthemostmatchedsequencesintheNCBIdatabaseafter30-dayincubation.Thetreewasconstructedbyneighbor-joiningmethodwithMega(7.0).TheBootstrapmethodis1,000Bootstrapreplicates,andvalueshigherthan50%areshownatthenodes.TherelativeabundanceofactiveOTUsinPAMandFLMat0.1,20,and40MPawasshownatright

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20MPa(TableA4).TheCsClbuoyantdensity(BD)shift,definedasthedifferenceinbuoyantdensitybetweenthe(13C-12C)peaklayers,ranged from0.013–0.02 g/ml in PAMandFLMat three differentpressures,withthehighestinthe13C-PAMcultureat0.1MPaand13C-FLMcultureat20MPa, and lowest in the 13C-FLMcultureat40MPa(TableA5).

By Illuminasequencing,atotalof2,112,639high-qualityreadsin90sampleswereobtainedwithanaveragelengthof421bp.Thereadswereclustered into3,533operational taxonomicunit (OTU)based on 97% sequence similarity. Alpha-diversity index showedthatthediversityindex(ShannonandSimpson)ofPAMandFLMde-creasedmarkedlyaftercultivation.However,themicrobialrichness(AceandChao)anddiversity(ShannonandSimpson)betweenPAMandFLMweresimilarinday30(TableA6andFigureA2).

TheVenndiagramsindicatethatbeforeincubation,atotalof771and244OTUswere identified in thePAMandFLMassemblages,respectively.Around452,118,and498OTUsinthePAMfractionweredistributedat75-,2,000-,and4,000-mdepths,respectively.Correspondingly, only 142, 92, and 58 OTUs in the FLM frac-tionwereidentifiedat75,2,000,and4,000mdepths(FigureA3).However,after30-dcultivation,only17and23activeOTUswereidentifiedinPAMandFLMfractions,with8,11,and6activeOTUsforPAM,and9,11,and4activeOTUsforFLMat0.1-,20-,and40-MPapressures,respectively(Figure3).Atday0,theOTUsofPAMonly,FLMonly,andthetransientform(occursinboththePAMandFLMfractions)were405,47,and95for75-mculture,respectively;theuniqueOTUsofPAM,theuniqueOTUsofFLM,andthesharedOTUsbetweenthePAMandFLMwere64,54,and38for2,000-mculture,respectively;andonlyPAMOTUs,onlyFLMOTUs,andthecommonOTUsofPAMandFLMwere456,42,and16for4,000-mculture,respectively(FigureA3).After30-daysincubation,thecor-respondingnumberof activeOTUswere reduced to5, 3, 6; 8, 3,8;and6,0,4 (Figure3).Among31OTUs,8and14OTUsexistedexclusivelyasPAMandFLM,respectively,and9astransientform(TableA7).

Thenonmetricmultidimensionalscaling (NMDS)analysiswasperformed to compare the similarities between PAM and FLMat 0.1, 20, and 40 MPa and 15°C before and after incubation(Figure4a).Twoclusterswereformed:thePAMandFLMinday0(I)andday30 (II),suggestingsignificantdissimilarity inmicrobialcommunitiesbeforeandafter incubation (Figure4a).Analysisofsimilarity(ANOSIM)alsorevealedsignificantdifferencesinmicro-bialcommunitiesbetweenday0andday30(R=.6583,p=.003,Table1).TheactivePAMandFLMforPOCdecompositionat0.1,20,and40MPaand15°Cinday30alsoformedtwoclusters,ac-tive PAM (I), and active FLM (II) (Figure 4b).However, ANOSIManalysis (R=0,p= .529,Table1)revealedmuch lessdifferencesbetween the two.

Before incubation, the PAM assemblage was dominatedby Gammaproteobacteria (genus Pseudoalteromonas) andAlphaproteobacteria (genus Erythrobacter), Alphaproteobacteria(Rhodobiaceae) and Gammaproteobacteria (genus Marinobacter),and Gammaproteobacteria (genus Amphritea) and Actinobacteria

(genusPropionibacterium) at 75-, 2,000-, and 4,000-m depths, re-spectively(FiguresA4andA5).ThepreincubationFLMassemblageswere dominated by Gammaproteobacteria (genus Alcanivorax)and Alphaproteobacteria (genus Surface 1), Bacteroidetes (genusPolaribacter 4) and Gammaproteobacteria (genus Alcanivorax),andGammaproteobacteria (genusAlcanivorax) at 75-, 2,000-, and4,000-m depths, respectively (Figures A4 and A5). Archaea hadan abundance of <1% (data not shown) andwill not be discussedthereafter.

Afterthe30-dayincubation,thePAMcommunitiesresponsiblefor POC utilizing were dominated by Alphaproteobacteria (genusRuegeria) and Gammaproteobacteria (genera Pseudomonas andHalomonas) in the0.1-MPa culture,Gammaproteobacteria (generaPseudomonas,MarinobacterandHalomonas)andAlphaproteobacteria(genusLabrenzia)inthe20-MPaculture,andGammaproteobacteria(Halomonas and Marinobacter genera) in the 40-MPa communi-ties (Figures5and6).Ontheotherhand,theactiveFLMcommu-nity was dominated by Gammaproteobacteria (Marinobacter) andAlphaproteobacteria(unclassifiedRhodobacteraceaeandRuegeria),Gammaproteobacteria (Pseudomonas) and Alphaproteobacteria(Labrenzia), and Gammaproteobacteria (Pseudoalteromonas) andBacteroidetes(Zunongwangia)inthe0.1-,20-,and40-MPacultures,respectively(Figures5and6).

Thirty-one active OTUs responsible for POC decomposi-tionwere identified inPAMandFLMassemblagesafter30days(Figures 7 and 8 and Table A8). These active OTUs were affil-iated with 14 genera in three classes: Gammaproteobacteria,Alphaproteobacteria, and Flavobacteria (Table A8). The relativeabundanceofdominantactiveOTUsinthe4th-10thfractionatedDNAalong thedifferent buoyant density gradients canbe seenin Figure 7. This result indicates that the relative abundance ofeachOTUwasmuchhigher in the “heavy” fractionatedDNAofthe 13C-labeled treatment than the corresponding abundance inthe 12C-controltreatment(Figure7).Italsorevealedthattheseac-tivemicroorganismshadsufficientcelldivisionand incorporatedenough13C-labeledPOCintocellbiomass,fordetectionbyDNA-SIP.Phylogeneticanalysis(Figure8)showedthatthesimilarityofeachOTUsequencecould reachup to99%–100%whenalignedintheNCBIdatabase,which impliesthatmostofthemarinemi-crobesinvolvedinPOCdecompositionandutilizationwereknownmicrobialstrains.

As shown in the hierarchically clustered heatmap, the activeOTUs decomposingPOC in PAMandFLM incubated at three dif-ferent pressures and 15°C formed two distinct clusters; one forPAMandFLMincubatedatatmosphericpressure(0.1MPa),andtheotherforPAMandFLMincubatedathighpressures(20and40MPa)(Figure A6). We found that members of Alphaproteobacteria andGammaproteobacteria were mainly responsible for POC decom-positionatatmosphericpressure,whereasmicrobial taxaaffiliatedwith Gammaproteobacteria predominated POC decomposition athighpressures(Figure5).TherelativelyhighproportionofRuegeria (Alphaproteobacteria) appeared at atmospheric pressure in par-ticle-associated fraction (42.9% of the total sequences), and their

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abundancedecreasedsharplywithpressure.Similarly,Marinobacter showedmuchhigherrelativeabundanceat0.1MPathanathighpres-sures,withthemaximumvalueof24.7%inthefree-livingfraction.Pseudomonaswas themostabundant taxa forPOCdecompositionat20MPapressureintheparticle-attached(63.5%)andfree-living(50.7%)fractions.Inaddition,Labrenzia,Nitratireductor,Erythrobacter werethedominantgeneraat20MPainthefree-livingfraction,withthe relative abundances of 22.7%, 3.3%, and 2.3%, respectively(Figure9).At40MPa,Pseudoalteromonas(76.2%)andZunongwangia (12.7%)werethemostdominantfree-livingtaxautilizingPOCwhileHalomonas(53.6%)werethemostabundantbacterialtaxonassimi-latingPOCinparticle-attachedfraction(Figure9).

4  | DISCUSSION

Diatom-deriveddetritalparticlesformaverticalPOM-DOMcontin-uum(Azam,1998;Fangetal.,2015;Grossart,2010;Verdugoetal.,2004)andprovideimportantnutrient-andcarbon-richhotspotsforthegrowthandmetabolismofheterotrophicprokaryotes(Azam&Long,2001), fromthesurfacewaterstothebathypelagiczones intheocean.Here,weinvestigatedthespecificbacteriallineagesac-tivelyparticipatedinthePOCdecompositionandDOCdegradationatlowandhighpressuresunder15°Ccondition,anddemonstratedthat most of the active microbial taxa were those previously de-tectedinorganicparticledecompositionandutilization.

4.1 | POC decomposers, PAM or FLM?

OurresultsrevealedthatnotonlyPAM,butalsoFLMdecomposeddiatom-derivedPOCandassimilatedthereleasedDOCat lowandhighpressures under15°C conditions (Figures1, 5, and6). These

resultsfurthersupportaprevioushypothesisthatPAMalmostim-mediatelyattachedtoorganicparticlesandsecretedexoenzymetohydrolyzePOCandproduceDOC(Azam&Long,2001;Fangetal.,2015;Grossart,Tang,Kiørboe,&Ploug,2007;Kiørboe&Jackson,2001;Smithetal.,1992).Itisimportanttonotethat,duetomotility(Grossart,2010;Grossart&Ploug,2001)andchemotaxis(Kiørboe,Grossart,Ploug,&Tang,2002),theFLMassemblageswerealsoabletoattachtodiatomdetritalparticles,andproduceextracellularen-zyme(Grossartetal.,2007)todecomposePOCandtakeadvantageofthereleasedDOCwhenPAMwasabsent(Figure1).Theseresultssuggest that microbial lifestyles (i.e., particle-attached and free-living)were not strictly correlatedwith their ecological functions,differentfromtheoriginalhypothesis:PAMfordecomposingPOCandFLMfordegradingthereleasedDOC(Azam&Long,2001;Fanget al., 2015;Kiørboe& Jackson,2001).These findingsalsoaddedtoourcurrentunderstandingthateitherparticle-associatedmicro-organisms (Karl et al., 1988) or free-livingmicroorganisms (Cho&Azam,1988)wereresponsibleforPOCdecomposition.

Our DNA-SIP results revealed that POC decomposi-tion was accomplished by members of Gammaproteobacteria,Alphaproteobacteria,andBacteroidetesat lowandhighpressures(Figure5),inagreementwithfindingsinpreviousstudiesthatthesemicrobial taxa had the specialized capability in decomposition ofphytoplankton-derivedorganicmatter(e.g.,Buchan,LeCleir,Gulvik,&González, 2014;Mayali, Stewart,Mabery,&Weber, 2016;Orsietal.,2016;Tamburinietal.,2006;Teelingetal.,2012).

EventhoughtheoriginalPAMcommunitiesdifferedgreatlyfromtheFLMcommunitiesatphylum-orgenus-level(FiguresA4andA5),muchmore similarmicrobial taxa,ofbothPAMandFLM,activelyparticipatedinPOCdecomposition(Figure4bandTable1).Forin-stance,inthePAMfraction,themicrobialPOCdecomposersweremainlyaffiliatedwithRuegeria (43%,0.1MPa),Pseudomonas (63%,20MPa),andHalomonas(53%,40MPa)(Figure6).Similarly,theFLM

F I G U R E 9  Therelativeabundanceofactiveparticle-attachedandfree-livingmicrobialtaxaforPOCdecompositionincubatedat0.1,20,and40MPaand15°Cfor30days.(a–e)membersofGammaproteobacteria;(f)memberofBacteroidetes;(g–k)membersofAlphaproteobacteria.Thecontrolindicatestherelativeabundanceofthesamegenusintheoriginalseawater(day0)

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microbial taxa thatmediatedPOCdecompositionanddegradationweredominatedbyMarinobacter (28%,0.1MPa),byPseudomonas (49%,20MPa),andbyPseudoalteromonas(77%,40MPa)(Figure6).This resultsuggests the functionalcommonalityofmicrobialcom-munities of the two lifestyles (i.e., PAMandFLM)under the con-ditionsof thiswork (15°Ctemperatureandhighconcentrationsofsubstrates)(Ghiglione,Conan,&Pujopay,2009;Hollibaugh,Wong,&Murrell,2000;Liu,Wang,etal.,2018;Riemann&winding,2001).

Ourresults indicatedthatmostoftheactivemicrobialtaxa in-volved inPOCdecompositionweredominated ineither theparti-cle-attached or free-livingmode (Table A7).We hypothesize thatswitching of lifestyles (particle-attached vs. free-living) reflectsmicrobial adaptation to the changing environmental conditions(e.g., hydrostatic pressure; Ghiglione et al., 2007; Moeseneder,Winter, &Herndl, 2001) and/or seeking and utilization of growthsubstrates (e.g., Lauroetal.,2009).For instance, thecopiotrophicMarinobacteroccurredinbothPAMandFLMfractionsandchangedtheir lifestyle from the free-living state at atmospheric pressureto particle-attached at high pressure, and became undetectablein theFLMfractionat40MPa (Figure9).This resultsupports thenotion that a particle-attached lifestyle may be preferentially se-lectedbydeep-seamicroorganisms(DeLongetal.,2006;Herndl&Reinthaler, 2013; Liu,Wang, et al., 2018).On theother hand, thefouractiveAlphaproteobacterialtaxa,Labrenzia,Rhodobacteraceae,Nitratireductor, andErythrobacter thatassimilated13C-labeledPOCseemtobepresentednearlyexclusivelyinfree-livinglifestyle,asdoPseudoalteromonasandZunongwangia(Figure9).

Our data also showed that only 7% of PAM and 10% of FLMOTUswere actively involved in POC decomposition and reminer-alizationprocesses(TableA7).However,onaverage,34%and52%of the total sequences appeared in active PAM and FLM OTUs,respectively,with theoverlap fractions reachingup to31% in thetwosizefractions(TableA7),suggestingthatparticle-attachedandfree-living microbial communities were interacting assemblages,with active succession of lifestyles between the two (Ghiglioneet al., 2009; Riemann &Winding, 2001). Our results accordwithpreviousstudiesthatshowedhighproportionsofsharedOTUsbe-tweenPAMandFLMintheeuphoticwaters(25%,Crespo,Pommier,Fernández-Gómez,&Pedrós-Alió,2013),mesopelagicwaters(30%,Moeseneder et al., 2001), and bathy- and abyssopelagic waters(82%,Liu,Wang,etal.,2018).

At theOTU level, therepresentativeOTU1508affiliatedwithRuegeriagenusassimilatedmore13C-POCthanother7OTUsinthePAMfractionat0.1MPa,withtherelativeabundanceof43%andBDshiftof0.02g/ml(Figures7and8),implyingthatRuegeriaprobablyplayinganimportantroleinPOCdecomposition(Reischetal.,2013;Reisch,Moran,&Whitman,2011). Incontrast, themostabundantactiveOTU3391,affiliatedwithMarinobacterandknownforbeingabletodegradealgaeexudate(Nelson&Carlson,2012),wasrespon-sible for POCdecomposition in the FLM fraction at 0.1MPa andaccountedfor25%ofthetotalsequences,withBDshiftof0.015g/ml(Figures7and8).OTU1159andOTU2166fromthemarinebac-teriaPseudmonaswerethemostdominantandactivetaxaforPOC

incorporatedinbothparticle-attachedandfree-livingbacterialcom-munitiesat20MPa,withtherelativeproportionof27%and42%,respectively(Figures7and8).OurresultsareapparentlydifferentfromapreviousreportwhichshowedthatthePseudomonas species havelimitedabilitytodegradehighmolecularweightorganicmatter(Fukami,Simidu,&Taga,1981).OTU3229affiliatedwithHalomonas genuswasthemostdominanttaxonforutilizingPOCinparticle-as-sociated fraction at 40MPa, constituting 49% of the total reads,whereas OTU2664 affiliated to Pseudoalteromonas was the mostabundantgroupfordecomposingPOCexclusivelyinthefree-livingfraction,withapercentageof73%(Figures7and8).TheBDshiftsofactiveOTU3229andOTU2664were0.015and0.013g/ml forparticle-associatedandfree-livingbacteriaat40MPa,respectively,muchlessthanthatat0.1and20MPa(Figure7).ByNCBIblast,wefoundthattheOTU2664had100%similaritytoPseudoalteromonas sp. strainDZ-05-01-aga (MK577382.1) (Figure8),an isolatewhichwasidentifiedasamacroalgae–polysaccharide-degradingbacterium(Martinetal.,2015).IthasbeenshownthatPseudoalteromonascanproducea largenumberofectoenzymes tohydrolyzePOC (Chen,Zhang, Gao, & Luan, 2003; Qin et al., 2011) and high molecularweightDOC (Alonso& Pernthaler, 2005; Covert &Moran, 2001;Nelson&Carlson,2012).

Itseems likelythatPOCdecompositionwasmediatedmainlyby members of the “rare biosphere” (Sogin et al., 2006), thosethat were of a rather low abundance in the epi- and bathype-lagic waters of the NBT (Ghiglione et al., 2007, 2009). For in-stance, thebacterial taxonRuegeria (0%of the total communityinday0vs43%of theactivecommunity inday30)at0.1MPa,Pseudomonas (2% vs. 63%) at 20MPa, and Halomonas (0% vs.53%) andPseudoalteromonas (7%vs. 77%) at40MPa (Figure6andFigureA5). It is alsopossible thatour incubationconditionsfavored microbes that prefer nutrient-rich environments (i.e.,copiotrophs).

4.2 | Effects of pressure and temperature on POC decomposition rates

ParticulateorganiccarbondegradationratesofPAMandFLMwereremarkably affectedbyhydrostaticpressure, that is,POCdecom-position was accomplished primarily by PAM communities at at-mosphericpressureandbyFLMathighpressures(20and40MPa)and temperatureof 15°C (Figure1 andTableA2). It has been as-sumed that thePAMassemblageshadhighermetabolic rates andextracellularenzymeactivitiesthanFLMinorganiccarbonreminer-alizationatatmosphericpressure,whichhadalsobeenobservedinpreviousstudies inthesurfaceocean(Fandino,Riemann,Steward,Long, & Azam, 2001; Grossart et al., 2007; Herndl & Reinthaler,2013;Lapoussière,Michel,Starr,Gosselin,&Poulin,2011).Recentreports claimed that the particle-attached microorganisms in thedeepoceanoriginated from the surfacewater by adhering to thesinkingparticles(Duret,Lampitt,&Lam,2019;Mestreetal.,2018;Thiele,Fuchs,Amann,&Iversen,2015).Whentheseheterotrophic

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prokaryotes reached to the deep ocean along with sinking parti-cles, their extracellular enzymeactivitydeclined significantlywithelevated pressure (Liu, Fang, et al., 2018; Tamburini et al., 2006);therefore,POCdegradation ratebyPAMatatmosphericpressurewasusuallyhigherthanthatathighpressure(Tamburinietal.,2013).Ontheotherhand,free-livingbacteria,theinsitucolonizersofthedeepocean,mayshowoptimalmetabolicactivityforPOCdecom-position and remineralization under high pressure (Dang& Lovell,2016; Lauro & Bartlett, 2008). Thus, POC degradation rates bydeep-seaFLMweregenerallyhigherthanbyPAM.Inaddition,somepreviousstudiesnotedthatparticle-attachedbacteriaweretypicalcopiotrophswhichcangrowrapidlyunderhighnutrientconditionssuchasnutrient-enrichedparticles in thesurfacewaters,whereasfree-livingbacteriawereoligotrophs thatmayadaptbetter to thebathypelagicrealm(Herndl&Reinthaler,2013;Lauroetal.,2009).

Furthermore, variations of DOC concentrations showed thatDOC concentrations decreased at low pressure (0.1MPa) and in-creasedathighpressures(20and40MPa)(Figure1),suggestingthatDOCutilizationbyPAMandFLMwaslikelylessefficientathigherpressuresthanatatmosphericpressureunder15°Ccondition.TheseresultsalsorevealedthatmicrobialdecompositionofPOCwasac-companiedbythereleaseofDOC,andthattherewasatightcou-pling between POC decomposition and DOC utilization (Azam &Long,2001;Fangetal.,2015).

Itmustbeemphasizedthatinthepresentstudy,weset15°Castheincubationtemperature,muchhigherthantheinsitutemperatureofthedeepsea(around2°C), inordertoproduceenoughbiomassforsubse-quent analysis. Previous studies also reported that increasing incuba-tiontemperaturegenerallyincreasedbacterialactivityandmetabolism,andcouldsignificantly improvebacterialgrowthandcarbonutilization(Middelboe&Lundsgaard,2003;Pomeroy&Wiebe,2001).Otherfind-ings showed that increasing temperaturewouldenhance theadhesivepropertiesofmarinemicrobes(Garrett,Bhakoo,&Zhang,2008),facili-tatingbacteriaattachmenttoparticles.Elevatedtemperaturemayalsoincreasetherateofenzymaticreactions,thus,thehydrolyticactivityofectoenzymesthatsecretedmainlybyparticle-attachedbacteriacanbeenhanced(seee.g.,Garrettetal.,2008),furtherincreasingtheefficiencyofmicrobialdecompositionofPOCanddegradationofDOC(Middelboe&Lundsgaard,2003).Thus,itislikelythatthemeasuredPOCdegradationrateswerehigherthantheactualratesinthebathypelagiczoneoftheNBT.Futurestudiesshouldfocusonhowtemperatureaffectsmicrobi-al-mediatedPOCdegradationratesandmicrobialexoenzymaticactivities.

4.3 | Pressure effects on active PAM and FLM communities involved in POC decomposition

Hydrostatic pressure significantly affected particle-attached andfree-livingmicrobialcommunitiesresponsibleforPOCdecomposi-tionandDOCdegradation(seeFigures5,6and9,andFigureA6).The microbial communities incubated at atmospheric pressure(0.1MPa)andhighpressures(20and40MPa)formedtwodistinctclusters(FigureA6).

The members of Gammaproteobacteria (e.g., copiotrophic gen-era Halomonas, Marinobacter, and Pseudoalteromonas) were mainlyPOCdecomposersathighpressures (20and40MPa) (Figures5,6,and9).Similarly,aseriesofthelatestfindingsdemonstratedthatthePAMandFLMcommunitiesweredominatedbyGammaproteobacteria(e.g., Colwellia, Moritella, Shewanella, Alteromonas, Halomonas,Pseudoalteromonas,andMarinobacter)inthebathypelagiczoneofthePacificOcean(Boeufetal.,2019),theNewBritainTrench(Liu,Wang,etal.,2018)andtheglobalocean(Salazaretal.,2016),theabyssope-lagic zoneof thePuertoRicoTrench (Eloeet al., 2011), thebottomwaters of the Mariana Trench and the Kermadec Trench (Peoplesetal.,2018).Otherstudiesalsorevealedthathighpressureledtotheincrease in the relative abundance ofGammaproteobacteria in pro-karyoticassemblageswhenincubatedwithdiatomdetritus(Tamburinietal.,2006)orsinkingfecalpellets(Tamburinietal.,2009),whichwereconsistentwithourstudy.

On the other hand, members of Alphaproteobacteria (genusRuegeria) andGammaproteobacteria (genusMarinobacter) dominatedthediatom-associatedandfree-livingPOCdecomposersatatmosphericpressure(Figures5,6,and9).However,previousreportsindicatedthatparticle-attached and free-livingbacteriaweremainly affiliatedwithBacteroidetes,Gammaproteobacteria,andAlphaproteobacteriaduringdiatomordinoflagellateblooms (Bidle&Azam,2001;Fandinoetal.,2001;Grossart,Levold,Allgaier,Simon,&Brinkhoff,2005).

Nevertheless, Marietou and Bartlett (2014) investigated theeffects of pressure on coastal bacterial community, and reportedthat the relative abundanceofAlphaproteobacteria increased sig-nificantlywithpressurewhileGammaproteobacteriadominatedthemicrobial communities at atmospheric pressure, in opposition toourobservations in this study.Themicrobial communitiesmaybeoriginatedfromthewatercolumnoftheopenocean(theNBT),sig-nificantlydifferentfromthoseintheCaliforniacoastalwater.Othercontributingfactorsmayincludetheincubationconditions,suchastemperature(15°Cinourstudy)andpressure.

TheeffectsofincubationtemperatureonPAMandFLMcommu-nitiesforPOCdecompositionmustbeconsideredhere.Ithasbeendemonstratedthattemperatureplaysan importantrole inshapingthemicrobial community structure in the ocean. For instance, aninvestigation showed that the shallow coast-watermicrobial com-munityformedtwodistinctclusterswhenincubatedat3and16°Catatmosphericpressure(Marietou&Bartlett,2014).Therefore,thehighertemperature(thaninsitu)incubationsmaychangethecom-munitystructureofPAMandFLM.FuturestudiesshouldinvestigatehowtemperatureaffectstheactivemicrobialcommunitystructureinvolvedinPOCdecomposition.

4.4 | Limiting factors for the DNA-SIP approach and caveats of this study

The success of the DNA-SIP method depends on several factorsamongwhichincorporationoftheisotopicallylabeledsubstrateintotargetedmacromolecules(e.g.DNA,RNA)iscritical(Liu,Wawrik,&

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Liu,2017;Radajewski,Mcdonald,&Murrell,2003).Oneofthechal-lengesistheincubationtimesandconditionsthatmirrortheinsituenvironmental conditions (Neufeld,Wagner, &Murrell, 2007). AsmicrobialDNAreplicationrequirescelldivision,unlikeusingPLFA-orRNA-SIP,asuccessfulDNA-SIPexperimentrequiressufficientcelldivisioninthepresenceofisotopicallylabeledsubstrateforsepara-tionoflabeledDNAfromnonlabeledDNA(Neufeld,Dumont,Vohra,&Murrell,2007;Neufeld,Wagner,etal.,2007).Ontheotherhand,piezophilicandpiezotolerantbacterialgrowthatinsitutemperatureandpressureconditions is typically ratherslow (Fangetal.,2003,2006).Forthesereasons,weaddedrelativelyhighconcentrationsofgrowthsubstrateandincubatedthePAMandFLMfor30daysatinsitupressure,butat15°C,untilearlystationaryphase,togeneratehighenoughbiomass.

Our results showed that sufficient isotopic labeling of nucleicacidsrelativetounlabeledsubstrateshasbeenachieved.However,the long incubation timecouldhave likely led to thecross-feedingofmetabolicbyproducts andmicrobial community shifts (Neufeld,Dumont, et al., 2007;Wang et al., 2015). Cross-feeding is one ofthe intercellular interactions amongmicrobes tomaintain the sta-bility and ecological functions of microbial communities and canexist in any environment (Pande et al., 2016; Seth & Taga, 2014).Additionally,longincubationtimeoftenprovidesgreaterisotopein-corporation, but at the same time,may result in thebottle effect.Previousworkhasshownthatwithinafewhours,thebottleeffectis seen for microbes (Lee & Fuhrman, 1991). In essence, this andothercloselyrelatedmicrobiologicalinteractions(e.g.,producersandcheatersinmicrobialextracellularenzymeproductionandsubstrateutilization,Allison&Vitousek,2005)warrantfurtherexploitationinfutureDNA-orRNA-SIPstudies.

Inourexperiment,weinitiallyseparatedthetotalmicrobesintothePAMandFLMfractionsandincubatedtheseparatedmicrobeswith the 13C-labeledandunlabeleddiatomicparticulates.However,PAMandFLMassemblageswerenotseparatedafter30-daysincu-bation.Indeed,therecanbemicrobialsuccessions(attachmentandde-attachmenttoandfromtheparticles)duringtheincubation.Thus,thespecificrolesofPAMandFLMinPOCdecompositionandDOCutilizationcannotbeexclusivelyspecifiedinourstudy.Nevertheless,weusedthesensitiveSIPmethodtotargetbothPAMandFLMcom-munities simultaneously andovercome the limitationof their slowgrowth, todetect thepotentialprimaryplayers inPOMdecompo-sitionandDOMdegradationatlowandhighpressuresunder15°Ccondition.Thehighdegreeof13C-enrichment,evidencedbytheBDshift(0.013–0.02g/ml)ofthefractionatedDNA(TableA5)andthe13C-labeledPOCutilizationbyPAMandFLMcommunities(TableA3),indicatestheactive13CincorporationintobacterialDNA.

5  | CONCLUSIONS

ByusingtheDNA-SIPapproach,weprovideddirectevidencethatlinkedspecificbacterialtaxatoPOMdecompositionandDOMdeg-radationintheepi-andbathypelagiczoneswheretheseprocesses

primarilytakeplaceintheocean.Majorconclusionsandimplicationsareasfollows:

1. A totalof14activeparticle-attachedand free-livingbacterialtaxonomic groups that actively participated in decompositionandassimilationofdiatom-derivedPOMwereidentified.Boththeparticle-attachedandfree-livingmicroorganismswereableto decompose POC and assimilate the released DOC.

2. POM decomposition process appears to be significantly influ-enced by hydrostatic pressure, accomplished primarily by par-ticle-attached microorganisms at atmospheric pressure and byfree-living microorganisms at high pressure. Overall, the POCdegradationrateswerehigheratlowpressurethanathighpres-sure.However, similarbacterial taxa inboth thePAMandFLMassemblageswereinvolvedinPOCdecompositionandDOCdeg-radation,suggestingthedecouplingbetweenmicrobiallifestylesandecologicalfunctions.

3. ItappearsthatthemicrobialtaxaactivelyinvolvedinPOCdecom-positionanddegradationweretypicalofrelativelylowabundanceamongthose thatcanperformthesamefunction inprocessingorganicmatterinthepelagiczones,reflectingthefunctionalre-dundancyofmicrobialcommunitiesinboththesurfaceanddeepwaters of the ocean.Our study highlights the connectivity be-tweenmicrobial lifestyles (i.e., PA or FL) and POC degradationprocessesintheocean.

ACKNOWLEDG EMENTSThisworkwassupportedbytheNationalNaturalScienceFoundationof China (Nos. 91951210, 41673085, 41773069), by the NationalKeyR&DProgramofChina (GrantNo.2018YFC0310600),andbythe InternationalExchangeProgramforGraduateStudents,TongjiUniversity (No.20171115).Wethank thecaptainandcrewof theM/VZhangJianforassistanceinsampling.WealsothankDongmeiWangforhelpintheexecutionoftheSIPexperiments.

CONFLIC T OF INTERE S TNonedeclared.

AUTHOR CONTRIBUTIONSYing Liu: Data curation (lead); formal analysis (lead); investiga-tion (lead); methodology (lead); software (lead); writing-originaldraft (lead); writing-review & editing (equal); Jiasong Fang:Conceptualization (lead); funding acquisition (lead); project admin-istration (lead); resources (lead); writing-review & editing (equal);ZhongjunJia:formalanalysis(equal);methodology(equal);software(equal);review&editing(equal);SongzeChen:formalanalysis(equal);methodology (equal); software (equal); validation (equal); writing-review&editing (equal); LiZhang: conceptualization (equal); fund-ingacquisition(equal);projectadministration(equal);WeiGao:datacuration(equal);software(equal);writing-review&editing(equal).

E THIC S S TATEMENTNonerequired.

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DATA AVAIL ABILIT Y S TATEMENTThe raw sequence datasets of the bacterial 16S rRNA gene fromthis project have been deposited at the National Center forBiotechnology Information (NCBI) Sequence Read Archive (SRA)with accessionnumbersSRR6027268 toSRR6027351 fromDNA-SIP.Thenucleotidesequencesof16SrRNAgeneofPAMandFLMinthewatercolumnoftheNBThavebeendepositedattheNCBIdatabasewithaccessionnumbersSRR6031465toSRR6031470.

ORCIDYing Liu https://orcid.org/0000-0003-0375-8303

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APPENDIX

F I G U R E A 1  LocationsoftheseawatersamplingsitesintheNewBritainTrench(Estation)

18 of 24  |     LIU et aL.

F I G U R E A 2  RarefactioncurvesofthePAMandFLMat0.1,20,and40MPaand15°Catday0andday30atacutoffvalueof97%sequencesimilarity

F I G U R E A 3  VenndiagramsshowingchangesinsharedanduniquetotalOTUsbetweenthePAMandFLMassemblagesincubatedat0.1,20,and40MPaand15°Catday0andday30

F I G U R E A 4  Therelativeabundanceofthemostabundantphylum(class)(≥1%)inPAMandFLMcommunitiesat75,2,000,and4,000mthreedepthsintheNBTatday0

     |  19 of 24LIU et aL.

F I G U R E A 5  Therelativeabundanceofthemostabundantgenera(OTUs)(≥1%)inPAMandFLMcommunitiesat75(a),2,000(b),and4,000m(c)intheNBTatday0

F I G U R E A 6  HierarchicallyclusteredheatmapoftherepresentiveOTUsinPAMandFLMforPOCdecompositionat0.1,20,and40MPaunder15°Cafter30-dayincubation

20 of 24  |     LIU et aL.

TA B L E A 1  ThephysicalandchemicalparametersinthewatercolumnoftheNewBritainTrench

Depth (m) Temperature (°C) Salinity (PSU) Oxygen (mg/L) POC (mg/L) Bacterial abundance (cells/ml)

75 27.2 35.1 5.6 0.02 1×105

2,000 2.3 34.6 3.8 0.02 1×104

4,000 2.0 34.7 4.7 0.01 1×104

TA B L E A 2  POCandDOCconcentrations,andPOCdegradationratesforthe13C-labeledand12C-controltreatmentsinPAMandFLMincubationsat0.1,20,and40MPaand15°Catday0andday30

Pressure (MPa)

Time (day)

The concentration of 13C-POC The concentration of 12C-POC

PAM (mg/L) FLM (mg/L) PAM (mg/L) FLM (mg/L)

Mean

POC Degradation Rate (μmol C L−1 day−1) Mean

POC Degradation Rate (μmol C L−1 day−1) Mean

POC Degradation Rate (μmol C L−1 day−1) Mean

POC Degradation Rate (μmol C L−1 day−1)

0.1 0 54 0.025 47.5 0.012 65.1 0.028 55.7 0.028

30 25.7 33.5 28.4 23.9

20 0 55.6 0.011 53.2 0.018 50.4 0.016 51.0 0.011

30 40.4 31.3 31.5 36.5

40 0 53.3 0.009 71.2 0.021 55.4 0.006 57.3 0.030

30 40.5 38.1 46.8 23.4

Pressure (MPa) Time (day)

The concentration of 13C-DOC The concentration of 12C-DOC

PAM (mg/L) FLM (mg/L) PAM (mg/L) FLM (mg/L)

0.1 0 18.3 15.0 103 81

30 10.9 12.4 55 50

20 0 17.8 15.2 77 81

30 29.9 28.2 161 142

40 0 16.2 18.6 78 80

30 31.0 25.8 132 158

TA B L E A 3   The δ13CvaluesofPOCandDOCinthePAMandFLMculturesat0.1,20,and40MPaatday30(a)and13Catom%thatutilizedbyPAMandFLM(b)

(a)

TreatmentsDepth (m)

The δ13C value of POC and DOC in PAM culture system at day 30

The δ13C value of POC and DOC in FLM culture system at day 30

POC DOC POC DOC

δ13C (‰) 13C atom (%) δ13C (‰) 13C atom (%) δ13C (‰) 13C atom (%) δ13C (‰) 13C atom (%)

13C-labeled 75 170,437.2 67.49 125,864.8 60.58 183,508.9 69.09 124,270.8 60.27

2,000 187,652.4 69.56 122,270.6 59.89 187,534.9 69.54 122,710.8 59.97

4,000 196,041.6 70.47 129,673.6 61.28 175,908.1 68.18 109,681.8 57.2812C-control 75 873.8 2.22 528.3 1.82 67.1 1.28 284.8 1.53

2,000 197.6 1.43 746.3 2.07 231.0 1.47 676.1 1.99

4,000 392.1 1.66 652.8 1.96 115.9 1.33 623.0 1.93

(b)

Treatments Depth (m)

The 13C atom% of DOC in PAM culture system (%) The 13C atom% of DOC in FLM culture system (%)

Day 0 Day 30 Utilization by PAM Day 0 Day 30 Utilization by FLM

13C 75 71.7 60.58 11.1 71.7 60.27 11.4

2,000 71.7 59.89 11.8 71.7 59.97 11.7

4,000 71.7 61.28 10.4 71.7 57.28 14.4

     |  21 of 24LIU et aL.

TAB

LE A

4 The16SrRNAgenecopynumberofthe3th-14thDNAgradientfractionsinthePAMandFLMassemblagesat0.1,20,and40MPabyquantitativePCRafter30-dincubation.(a)

13C-PAM;(b)12C-PAM;(c)13C-FLM;and(d)12C-FLM

(a)

DN

A

frac

tion

CsCl

bu

oyan

t de

nsity

(g/

ml)

13C-

PAM

0.1

MPa

20 M

Pa40

MPa

12

3M

ean

12

3M

ean

12

3M

ean

31.7479

6.29E+03

6.29E+03

6.29E+03

6.29E+03

6.37E+03

6.30E+03

7.32E+03

6.66E+03

3.11E+03

3.14E+03

4.33E+03

3.53E+03

41.7445

5.83E+03

5.83E+03

5.83E+03

5.83E+03

3.01E+04

2.53E+04

2.76E+04

2.77E+04

1.12E+04

1.11E+04

1.13E+04

1.12E+04

51.7399

5.13E+05

5.13E+05

5.13E+05

5.13E+05

3.93E+05

4.21E+05

4.24E+05

4.13E+05

4.05E+05

4.08E+05

3.81E+05

3.98E+05

61.7365

5.15E+06

5.15E+06

5.15E+06

5.15E+06

2.04E+06

2.13E+06

2.12E+06

2.10E+06

2.39E+06

2.56E+06

2.63E+06

2.53E+06

71.7331

1.97E+06

1.97E+06

1.97E+06

1.97E+06

9.63E+05

9.39E+05

1.01E+06

9.72E+05

6.71E+05

6.28E+05

7.02E+05

6.67E+05

81.7296

6.24E+06

6.24E+06

6.24E+06

6.24E+06

7.36E+05

6.26E+05

6.60E+05

6.74E+05

2.21E+05

2.08E+05

2.15E+05

2.15E+05

91.7250

5.54E+05

5.54E+05

5.54E+05

5.54E+05

1.06E+05

9.70E+04

1.03E+05

1.02E+05

5.54E+04

5.76E+04

5.34E+04

5.55E+04

101.7216

2.55E+04

2.55E+04

2.55E+04

2.55E+04

1.02E+04

9.79E+03

8.76E+03

9.58E+03

2.79E+03

2.61E+03

2.32E+03

2.57E+03

111.7170

1.16E+04

1.16E+04

1.16E+04

1.16E+04

2.33E+04

2.42E+04

2.45E+04

2.40E+04

1.94E+03

1.91E+03

2.04E+03

1.96E+03

121.7135

1.40E+04

1.40E+04

1.40E+04

1.40E+04

2.58E+04

3.27E+04

3.16E+04

3.00E+04

3.12E+03

2.48E+03

1.28E+04

6.14E+03

131.7088

1.65E+03

1.65E+03

1.65E+03

1.65E+03

2.81E+03

1.80E+03

2.29E+03

2.30E+03

3.43E+02

2.42E+02

2.11E+02

2.66E+02

141.7065

3.26E+02

3.26E+02

3.26E+02

3.26E+02

4.59E+02

4.76E+02

5.71E+02

5.02E+02

2.88E+02

3.03E+02

3.32E+02

3.07E+02

(b)

DN

A

frac

tion

CsCl

bu

oyan

t de

nsity

(g/

ml)

12C-

PAM

0.1

MPa

20 M

Pa40

MPa

12

3M

ean

12

3M

ean

12

3M

ean

31.7479

2.98E+02

2.98E+02

2.98E+02

2.98E+02

3.61E+03

3.75E+03

4.57E+03

3.98E+03

3.31E+02

4.14E+02

4.85E+02

4.10E+02

41.7445

1.83E+03

1.83E+03

1.83E+03

1.83E+03

5.44E+03

5.38E+03

4.90E+03

5.24E+03

2.20E+03

2.05E+03

2.19E+03

2.15E+03

51.7399

8.72E+02

8.72E+02

8.72E+02

8.72E+02

8.36E+03

6.90E+03

8.06E+03

7.77E+03

2.83E+03

2.57E+03

2.55E+03

2.65E+03

61.7365

7.41E+02

7.41E+02

7.41E+02

7.41E+02

1.19E+04

1.09E+04

1.06E+04

1.11E+04

3.13E+03

2.91E+03

2.25E+03

2.76E+03

71.7331

6.09E+03

6.09E+03

6.09E+03

6.09E+03

2.83E+04

2.81E+04

2.56E+04

2.73E+04

2.88E+03

2.78E+03

5.00E+04

1.86E+04

81.7296

7.98E+03

7.98E+03

7.98E+03

7.98E+03

1.90E+05

1.93E+05

2.08E+05

1.97E+05

7.49E+03

8.01E+03

8.46E+03

7.99E+03

91.7250

1.28E+05

1.28E+05

1.28E+05

1.28E+05

1.96E+06

1.79E+06

1.68E+06

1.81E+06

1.02E+05

7.95E+04

7.64E+04

8.58E+04

101.7216

2.58E+06

2.58E+06

2.58E+06

2.58E+06

4.67E+06

4.55E+06

4.41E+06

4.55E+06

5.67E+05

5.73E+05

5.94E+05

5.78E+05

111.7170

4.64E+06

4.64E+06

4.64E+06

4.64E+06

2.22E+06

2.38E+06

2.55E+06

2.38E+06

3.14E+05

3.30E+05

3.22E+05

3.22E+05

121.7135

9.24E+05

9.24E+05

9.24E+05

9.24E+05

4.13E+06

4.22E+06

3.54E+06

3.96E+06

4.33E+04

3.95E+04

3.92E+04

4.07E+04

131.7088

3.60E+05

3.60E+05

3.60E+05

3.60E+05

2.18E+05

2.07E+05

2.17E+05

2.14E+05

7.85E+03

8.23E+03

5.43E+03

7.17E+03

141.7065

4.32E+03

4.32E+03

4.32E+03

4.32E+03

2.31E+03

2.67E+03

2.33E+03

2.44E+03

4.11E+02

4.45E+02

4.13E+02

4.23E+02

(Continues)

22 of 24  |     LIU et aL.

(C)

DN

A

frac

tion

CsCl

bu

oyan

t de

nsity

(g/

ml)

13C-

FLM

0.1

MPa

20 M

Pa40

MPa

12

3M

ean

12

3M

ean

12

3M

ean

31.7479

4.39E+02

4.88E+02

4.57E+02

4.61E+02

1.58E+03

1.79E+03

1.77E+03

1.71E+03

1.49E+03

1.72E+03

1.66E+03

1.62E+03

41.7445

4.96E+03

5.05E+03

5.29E+03

5.10E+03

3.53E+03

3.07E+03

3.76E+03

3.45E+03

3.40E+03

3.71E+03

3.27E+03

3.46E+03

51.7399

5.30E+05

5.83E+05

5.83E+05

5.65E+05

7.21E+05

8.40E+05

8.19E+05

7.93E+05

2.37E+04

2.40E+04

2.43E+04

2.40E+04

61.7365

2.44E+06

2.53E+06

2.74E+06

2.57E+06

5.02E+06

5.31E+06

5.10E+06

5.14E+06

2.03E+05

1.77E+05

1.87E+05

1.89E+05

71.7331

5.40E+05

5.53E+05

5.19E+05

5.37E+05

1.10E+06

9.51E+05

1.06E+06

1.04E+06

2.60E+05

2.36E+05

2.28E+05

2.41E+05

81.7296

4.04E+05

3.56E+05

3.51E+05

3.71E+05

3.82E+06

3.55E+06

3.70E+06

3.69E+06

3.34E+05

3.23E+05

3.18E+05

3.25E+05

91.7250

3.79E+04

2.38E+04

3.56E+04

3.25E+04

1.42E+06

1.56E+06

1.35E+06

1.44E+06

7.18E+04

8.54E+04

8.52E+04

8.08E+04

101.7216

5.30E+03

5.54E+03

5.60E+03

5.48E+03

1.50E+05

1.58E+05

1.46E+05

1.52E+05

2.64E+03

2.57E+03

2.34E+03

2.52E+03

111.7170

7.03E+03

6.31E+03

6.77E+03

6.70E+03

7.33E+04

7.18E+04

6.91E+04

7.14E+04

1.93E+03

1.92E+03

1.87E+03

1.91E+03

121.7135

2.90E+03

3.00E+03

2.77E+03

2.89E+03

3.57E+04

3.29E+04

3.00E+04

3.29E+04

2.70E+03

2.53E+03

2.57E+03

2.60E+03

131.7088

6.64E+01

8.49E+01

1.15E+02

8.88E+01

3.30E+03

3.03E+03

3.15E+03

3.16E+03

2.19E+02

1.50E+02

2.03E+02

1.91E+02

141.7065

2.60E+02

1.58E+02

1.74E+02

1.98E+02

1.67E+03

1.25E+03

1.24E+03

1.39E+03

1.40E+02

1.42E+02

1.11E+02

1.31E+02

(d)

DN

A

frac

tion

CsCl

bu

oyan

t de

nsity

(g/

ml)

12C-

FLM

0.1

MPa

20 M

Pa40

MPa

12

3M

ean

12

3M

ean

12

3M

ean

31.7479

3.31E+02

2.72E+02

3.06E+02

3.03E+02

1.35E+02

4.49E+01

7.16E+01

8.38E+01

7.63E+02

1.01E+03

7.02E+02

8.24E+02

41.7445

3.27E+02

3.19E+02

4.92E+02

3.79E+02

1.69E+02

8.32E+01

7.58E+01

1.09E+02

2.39E+02

2.15E+02

2.03E+02

2.19E+02

51.7399

6.92E+02

6.31E+02

7.06E+02

6.76E+02

1.30E+02

1.12E+02

1.52E+02

1.31E+02

1.18E+03

1.17E+03

1.27E+03

1.21E+03

61.7365

4.58E+03

4.65E+03

2.11E+04

1.01E+04

2.11E+02

1.61E+02

9.43E+01

1.56E+02

1.11E+03

8.85E+02

1.14E+03

1.05E+03

71.7331

8.57E+03

8.40E+03

7.75E+03

8.24E+03

1.07E+02

8.82E+01

7.61E+01

9.03E+01

2.11E+03

1.69E+03

1.64E+03

1.81E+03

81.7296

3.15E+04

2.94E+04

2.57E+04

2.89E+04

1.42E+02

6.37E+01

7.43E+01

9.33E+01

2.66E+03

2.53E+03

2.44E+03

2.54E+03

91.7250

8.83E+05

8.41E+05

7.15E+05

8.13E+05

8.14E+02

8.25E+02

6.70E+02

7.70E+02

9.85E+04

8.67E+04

9.08E+04

9.20E+04

101.7216

1.31E+07

1.25E+07

9.90E+06

1.19E+07

1.10E+03

9.84E+02

1.01E+03

1.03E+03

1.17E+06

1.11E+06

1.04E+06

1.11E+06

111.7170

4.54E+06

4.76E+06

4.12E+06

4.47E+06

1.33E+04

1.32E+04

1.25E+04

1.30E+04

3.39E+06

3.34E+06

3.42E+06

3.38E+06

121.7135

4.22E+06

4.58E+06

4.48E+06

4.43E+06

5.42E+03

4.81E+03

4.88E+03

5.04E+03

1.64E+06

1.41E+06

1.55E+06

1.53E+06

131.7088

1.58E+06

1.32E+06

1.31E+06

1.40E+06

1.66E+03

1.66E+03

1.38E+03

1.57E+03

2.77E+05

2.71E+05

2.47E+05

2.65E+05

141.7065

3.14E+04

2.20E+04

2.31E+04

2.55E+04

2.67E+02

2.36E+02

1.80E+02

2.28E+02

5.00E+03

4.43E+03

3.62E+03

4.35E+03

TAB

LE A

4 (Continued)

     |  23 of 24LIU et aL.

TA B L E A 5  Themaximumnumberof16SrRNAgenecopiesandCsClbuoyantdensityshiftinthePAMandFLMassemblagesat0.1,20,and40MPaunder15°Cconditionin13C-labeledand12C-controltreatmentsafter30-dincubation

Sample ID

13C-labeled treatment 12C-control treatment

CsCl buoyant density (BD) shift from 12C-control to 13C-labeled (g/ml)

The maximum 16S rRNA gene copy number

The 16S rRNA gene copy number

CsCl buoyant density (g/ml)

The maximum 16S rRNA gene copy number

The16S rRNA gene copy number

CsCl buoyant density (g/ml)

30d-0.1MPa-PAM The6thfraction 5.15E+06 1.7365 the11thfraction 4.64E+06 1.7170 0.020

30d-20MPa-PAM The6thfraction 2.10E+06 1.7365 the10thfraction 4.55E+06 1.7216 0.015

30d-40MPa-PAM The6thfraction 2.53E+06 1.7365 the10thfraction 5.78E+05 1.7216 0.015

30d-0.1MPa-FLM The6thfraction 2.57E+06 1.7365 the10thfraction 1.19E+07 1.7216 0.015

30d-20MPa-FLM The6thfraction 5.14E+06 1.7365 the11thfraction 1.30E+04 1.7170 0.020

30d-40MPa-FLM The8thfraction 3.25E+05 1.7296 the11thfraction 3.38E+06 1.7170 0.013

TA B L E A 6   The α-diversityindicesofthePAMandFLMassemblagesat0.1,20,and40MPaunder15°Cconditionatday0andday30at97%sequencesimilarity(Allsequenceshadbeennormalizedbeforebeingusedfordiversityanalysisinthisstudy)

Time (day) PAM/FLM Depth (m) Reads OTUs Shannon Simpson Ace Chao Coverage (%)

0 PAM 75 4,099 452 4.65 0.03 585.65 598.99 98.6

2,000 4,099 118 2.66 0.20 512.64 418.50 99.7

4,000 4,099 498 5.26 0.01 620.64 598.56 96.6

FLM 75 4,099 142 2.80 0.13 548.46 495.04 99.8

2,000 4,099 92 2.76 0.12 433.15 345.59 99.7

4,000 4,099 58 2.10 0.19 411.76 266.73 99.7

30 PAM 75 4,099 83 2.11 0.28 694.88 440.89 99.8

2,000 4,099 68 2.37 0.15 174.47 120.00 99.6

4,000 4,099 69 1.71 0.32 554.81 336.56 99.6

FLM 75 4,099 70 2.26 0.17 517.29 346.15 99.7

2,000 4,099 70 2.19 0.23 229.91 157.11 99.6

4,000 4,099 65 1.19 0.55 729.83 482.80 99.6

TA B L E A 7  ThenumberandrelativeabundanceofsharedanduniqueactiveOTUsbetweenthePAMandFLMassemblagesat0.1,20,and40MPaunder15°Cconditionafterincubation

Active OTUs in three depths

The number of active OTUs

The total OTUs in day 30

The proportion of active OTUs (%)

The number of reads in active OTUs

The total reads in day 30

The relative abundance of active OTUs (%)

OnlyPAM 8 229 3% 6,676 228,121 3%

OnlyFLM 14 6% 47,886 21%

SharedPAMandFLM

9 4% 70,817 31%

SUM 31 125,379 55%

24 of 24  |     LIU et aL.

TAB

LE A

8 ThephylogeneticclassificationandincreasedrelativeabundanceofthemostactiveOTUsforPOCdecompositionat0.1,20,and40MPaunder15°Cconditionafterincubation

Dom

ain

Kin

gdom

Phy

lum

Cla

sses

Ord

ers

Fam

ily A

ctiv

e G

ener

a S

peci

es A

ctiv

e O

TUs

0.1

MPa

20 M

Pa40

MPa

PAM

FLM

PAM

FLM

PAM

FLM

Bacteria

norank

Proteobacteria

Gammaproteobacteria

Pseudomonadales

Pseudomonadaceae

Pseudomonas

unclassified_g__Pseudomonas

OTU

2158

7.5

1.6

18.0

0.0

0.0

0.0

Bacteria

norank

Proteobacteria

Gammaproteobacteria

Pseudomonadales

Pseudomonadaceae

Pseudomonas

unclassified_g__Pseudomonas

OTU

1159

5.2

0.0

26.6

0.0

0.0

0.0

Bacteria

norank

Proteobacteria

Gammaproteobacteria

Pseudomonadales

Pseudomonadaceae

Pseudomonas

Pseudomonas_pachastrellae

OTU

1304

1.0

0.0

5.8

2.8

0.0

0.0

Bacteria

norank

Proteobacteria

Gammaproteobacteria

Pseudomonadales

Pseudomonadaceae

Pseudomonas

unclassified_g__Pseudomonas

OTU

2165

1.0

0.0

0.0

2.2

0.0

0.0

Bacteria

norank

Proteobacteria

Gammaproteobacteria

Pseudomonadales

Pseudomonadaceae

Pseudomonas

unclassified_g__Pseudomonas

OTU

158

0.0

0.0

11.2

0.0

0.0

0.0

Bacteria

norank

Proteobacteria

Gammaproteobacteria

Pseudomonadales

Pseudomonadaceae

Pseudomonas

unclassified_g__Pseudomonas

OTU

3398

0.0

0.0

1.2

0.0

0.0

0.0

Bacteria

norank

Proteobacteria

Gammaproteobacteria

Pseudomonadales

Pseudomonadaceae

Pseudomonas

unclassified_g__Pseudomonas

OTU

2166

0.0

0.0

0.0

41.7

0.0

0.0

Bacteria

norank

Proteobacteria

Gammaproteobacteria

Pseudomonadales

Pseudomonadaceae

Pseudomonas

unclassified_g__Pseudomonas

OTU

153

0.0

0.0

0.0

2.4

0.0

0.0

Bacteria

norank

Proteobacteria

Gammaproteobacteria

Alteromonadales

Alteromonadaceae

Marinobacter

unclassified_g__Marinobacter

OTU

3391

3.0

25.1

1.3

0.0

0.0

0.0

Bacteria

norank

Proteobacteria

Gammaproteobacteria

Alteromonadales

Alteromonadaceae

Marinobacter

Marinobacter_sediminum

OTU754

1.4

0.0

7.9

2.9

0.0

0.0

Bacteria

norank

Proteobacteria

Gammaproteobacteria

Alteromonadales

Alteromonadaceae

Marinobacter

Marinobacter_adhaerens_HP15

OTU

2651

0.0

3.0

0.0

0.0

5.1

0.0

Bacteria

norank

Proteobacteria

Gammaproteobacteria

Alteromonadales

Alteromonadaceae

Marinobacter

Marinobacter_algicola

OTU

2186

0.0

0.0

0.0

1.9

0.0

0.0

Bacteria

norank

Proteobacteria

Gammaproteobacteria

Alteromonadales

Alteromonadaceae

Alteromonas

unclassified_g__Alteromonas

OTU703

0.0

0.0

1.2

0.0

0.0

0.0

Bacteria

norank

Proteobacteria

Gammaproteobacteria

Alteromonadales

Pseudoalteromonadaceae

Pseudoalteromonas

Pseudoalteromonas_

shioyasakiensis

OTU

610

0.0

0.0

1.8

0.0

0.0

4.0

Bacteria

norank

Proteobacteria

Gammaproteobacteria

Alteromonadales

Pseudoalteromonadaceae

Pseudoalteromonas

unclassified_g__

Pseudoalteromonas

OTU

2664

0.0

0.0

0.0

0.0

0.0

72.8

Bacteria

norank

Proteobacteria

Gammaproteobacteria

Oceanospirillales

Halomonadaceae

Halomonas

unclassified_g__Halomonas

OTU

3229

7.4

0.0

6.5

4.9

48.4

0.0

Bacteria

norank

Proteobacteria

Gammaproteobacteria

Oceanospirillales

Halomonadaceae

Halomonas

unclassified_g__Halomonas

OTU

1814

0.0

0.0

0.0

0.0

1.5

0.0

Bacteria

norank

Proteobacteria

Gammaproteobacteria

Oceanospirillales

Halomonadaceae

Halomonas

unclassified_g__Halomonas

OTU

1313

0.0

0.0

0.0

0.0

2.9

0.0

Bacteria

norank

Proteobacteria

Gammaproteobacteria

Oceanospirillales

Alcanivoracaceae

Alcanivorax

Alcanivorax_venustensis

OTU

3383

0.0

0.0

0.0

0.0

1.8

0.0

Bacteria

norank

Proteobacteria

Gammaproteobacteria

Vibrionales

Vibrionaceae

Photobacterium

Photobacterium_profundum

OTU1176

0.0

0.0

0.0

0.0

0.0

2.4

Bacteria

norank

Proteobacteria

Alphaproteobacteria

Rhodobacterales

Rhodobacteraceae

Ruegeria

Ruegeria_mobilis

OTU

1508

42.7

7.5

0.0

0.0

2.7

0.0

Bacteria

norank

Proteobacteria

Alphaproteobacteria

Rhodobacterales

Rhodobacteraceae

Labrenzia

Labrenzia_aggregata

OTU

141

0.0

2.5

0.0

0.0

0.0

0.0

Bacteria

norank

Proteobacteria

Alphaproteobacteria

Rhodobacterales

Rhodobacteraceae

Labrenzia

Labrenzia_aggregata

OTU157

0.0

0.0

5.6

0.0

0.0

0.0

Bacteria

norank

Proteobacteria

Alphaproteobacteria

Rhodobacterales

Rhodobacteraceae

Labrenzia

Labrenzia_aggregata

OTU

2164

0.0

0.0

0.0

22.1

0.0

0.0

Bacteria

norank

Proteobacteria

Alphaproteobacteria

Rhodobacterales

Rhodobacteraceae

Loktanella

unclassified_g__Loktanella

OTU1574

0.0

1.9

0.0

0.0

0.0

0.0

Bacteria

norank

Proteobacteria

Alphaproteobacteria

Rhodobacterales

Rhodobacteraceae

unclassified

Rhodobacteraceae

unclassified_f__

Rhodobacteraceae

OTU

3432

0.0

16.2

0.0

0.0

0.0

0.0

Bacteria

norank

Proteobacteria

Alphaproteobacteria

Rhodobacterales

Rhodobacteraceae

unclassified

Rhodobacteraceae

unclassified_f__

Rhodobacteraceae

OTU

1564

0.0

1.7

0.0

0.0

0.0

0.0

Bacteria

norank

Proteobacteria

Alphaproteobacteria

Rhodobacterales

Rhodobacteraceae

unclassified

Rhodobacteraceae

unclassified_f__

Rhodobacteraceae

OTU

2109

0.0

0.0

0.0

1.3

0.0

0.0

Bacteria

norank

Proteobacteria

Alphaproteobacteria

Sphingomonadales

Erythrobacteraceae

Erythrobacter

Erythrobacter_citreus

OTU

2102

0.0

2.9

0.0

2.3

0.0

0.0

Bacteria

norank

Proteobacteria

Alphaproteobacteria

Rhizobiales

Phyllobacteriaceae

Nitratireductor

uncultured_bacterium_g__

Nitratireductor

OTU

2146

0.0

0.0

0.0

3.3

0.0

0.0

Bacteria

norank

Bacteroidetes

Flavobacteriia

Flavobacteriales

Flavobacteriaceae

Zunongwangia

Zunongwangia_profunda_SM-

A87

OTU2673

0.0

0.0

0.0

0.0

0.0

12.6