Diversity of retrievable heterotrophic bacteria in Kongsfjorden, an Arctic fjord

h tt p : / / w w w . b j m i c r o b i o l . c o m . b r /

Environmental

Microbiology

Diversity

of

retrievable

heterotrophic

bacteria

in

Kongsfjorden,

an

Arctic

fjord

Rupesh

Kumar

Sinha

a,∗

_,

_Kottekkatu

_Padinchati

_Krishnan

a

_,

Ammanamveetil

Abdulla

Mohamed

Hatha

b

_,

_Mujeeb

_Rahiman

b

_,

Divya

David

Thresyamma

a

_,

_Savita

_Kerkar

c

a_{NationalCentreforAntarcticandOceanResearch,ESSO-NCAOR,HeadlandSada,Vasco-da-Gama,Goa,India}

b_{CochinUniversityofScienceandTechnology,MicrobiologyandBiochemistry,DepartmentofMarineBiology,Cochin,Kerala,India} c_{GoaUniversity,DepartmentofBiotechnology,TaleigaoPlateau,Goa,India}

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received10July2015 Accepted22December2015 Availableonline15October2016 AssociateEditor:ValeriaMaiade Oliveira

Keywords:

Kongsfjorden

Retrievableheterotrophicbacteria 16SrRNA

Arcticfjord

a

b

s

t

r

a

c

t

ThediversityandabundanceofretrievablepelagicheterotrophicbacteriainKongsfjorden, anArctic fjord,wasstudiedduringthesummerof2011(June,August,andSeptember). Retrievablebacterialloadrangedfrom103_to107_CFUL−1_{inJune,whileitwas10}4_–106_CFUL−1

inAugustandSeptember.Basedon16SrRNAgenesequencesimilarities,ahighernumber ofphylotypeswasobservedduringAugust(22phylotypes)comparedtothatduringJune (6phylotypes)andSeptember(12phylotypes).Thegroupswereclassifiedintofourphyla:

Firmicutes,Actinobacteria,Proteobacteria,andBacteroidetes.Bacteroideteswasrepresentedonly byasinglememberLeewenhoekiellaaequoreaduringthethreemonthsandwasdominant (40%)inJune.However,thisdominancechangedinAugusttoawell-knownphytopathogenic speciesRhodococcusfascians(32%),whichcouldbearesultofdecreaseinthephytoplankton biomassfollowingthesecondarybloom.ItisthefirstreportofHalomonastitanicaeisolation fromtheArcticwaters.ItshowedanincreaseinitsabundancewiththeintrusionofAtlantic waterintoKongsfjorden.IncreasedabundanceofPsychrobacterspeciesinthelatesummer monthscoincidedwiththepresenceofcoolerwaters.Thus,thecompositionandfunctionof heterotrophicbacterialcommunitywasfundamentallydifferentindifferentmonths.This couldbelinkedtothechangesinthewatermassesand/orphytoplanktonbloomdynamics occurringinArcticsummer.

©2016SociedadeBrasileiradeMicrobiologia.PublishedbyElsevierEditoraLtda.Thisis anopenaccessarticleundertheCCBY-NC-NDlicense(http://creativecommons.org/

licenses/by-nc-nd/4.0/).

Introduction

Arcticmarine ecosystemshaverecentlyreceived increased attention,astheyareconsidered tobesensitivetothe cli-matechange.1 _{Kongsfjorden, aglacialfjordinthe Svalbard}

∗ _{Correspondingauthor.}

E-mail:kineto.magnetic@gmail.com(R.K.Sinha).

archipelago, Spitsbergen 79◦N–12◦E, is a key site for the monitoring of Arctic biodiversity and also considered for modelinginclimatechangestudies.1_{Themarineecosystem}

ofKongsfjorden iswell explored withregards to hydrogra-phy, mesozooplankton,andhigher trophiclevels,whilethe knowledgeonitsbacterialdiversitystillremainsinsufficient.2

http://dx.doi.org/10.1016/j.bjm.2016.09.011

1517-8382/©2016SociedadeBrasileiradeMicrobiologia.PublishedbyElsevierEditoraLtda.ThisisanopenaccessarticleundertheCC BY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/4.0/).

Theinformationabout thebacterial assemblageata given time-pointcouldconveyvitalinformationpertainingtothe ecologicalaspectsoftheenvironment. Severalfactors have beenreportedtoaffectthecompositionofthemarinebacterial communities.3_{Phytoplanktoncompositionandthereby}

sub-stratecompositionandconcentrationhavebeenfoundtoplay importantrolesinthedynamicsofbacterialcommunities.4,5

Heterotrophicbacteriacansupportthegrowthof phytoplank-ton via recycling of nutrients, but atthe same time, they alsocompetewithphytoplanktonforessentialnutrients.Both aliveanddead(ordying)phytoplanktonreleaseorganic com-pounds that are consumed by heterotrophic bacteria; and these interactions vary with the bacterial species and the physiological status of the phytoplankton.6 _Indeed,

bacte-ria sustain at a high level immediately after the collapse of a bloom, as they continue to use the organic matter releasedfromthedyingphytoplankton.7_{Despitethevariation}

inphytoplanktoncompositionandenvironmentalconditions, alimitednumberoftaxaareconsistentlyfoundtodominate bloom-associatedbacterialcommunities.Themostfrequent bacteria, identified by 16S rRNA gene-based analyses, are themembersoftheclasses,Flavobacteriaand␣-Proteobacteria, andalsoincludeRhodobacteraceaeand -Proteobacteria.7,8 _An

insight,aboutrolesthatindividual bacterialspeciesplayin theformationofbloomsandtheireventualcollapse,will ulti-matelyunraveltheforcesthatcontrolenergyflowinthefjord aswellasthecyclingofcompounds,whichultimately influ-encetheclimatechange.

Culture-dependent and -independent methods using oligonucleotideprobesand/orthecloningofenvironmental DNAhaveindicatedoccurrenceofexceptionallydiverse bac-terialcommunitiesthrivinginArcticfjordhabitats.9,10_Inthe

recent studiesof Piquet et al.11,12 _{on the pelagic microbial}

communitiesofKongsfjorden,thediversityofnon-culturable communities of bacteria, phytoplankton, and protists was monitored.However,very fewattemptshavebeen madeto understandthediversityofretrievableheterotrophicbacteria ofthisecosystem.13,14

The present study attempts to understand the taxon-omy of retrievable heterotrophic bacteria in Kongsfjorden during the summer of 2011. We carried out an investiga-tion inJune–September 2011 to determine the changes in retrievablebacterialcommunity.Observationon phytoplank-ton blooming pattern using SAMS mooring for the year 2010(SupplementaryFig. 1)indicatedthatsampling during thesesummermonthsmaximizedthepossibilityofhigh het-erotrophicbacterialactivityanddiversityduetothecollapse of the spring bloom (June–July) and the secondary bloom (August–September).Inaddition,wealsoaddressthe ecolog-icalsignificanceofthevariousgroupsofbacteriapresentin Kongsfjorden.

Materials

and

methods

Samplingsite

Kongsfjorden (Fig. 1) is a polar fjord located between 78◦04N–79◦05N and 11◦03E–13◦03E on the west coast of Spitsbergen, Svalbard Archipelago. The fjord is

characterized by a weak tidalrange (∼2m) strongly influ-enced by the topography and adjacent ocean. Western Svalbardcoastalwatersareinfluencedbythenorthernmost extensionofthewarmNorth AtlanticCurrent.15

Kongsfjor-den,atitsinnerend,hasthreemainglaciers,viz.,Kongsbreen, Conwaybreen,andBlomstrandbreen,drainingintoitand pro-vidingthemajorsourceoffreshwater.Thus,Kongsfjordenis undertheinfluenceofbothmeltwaterofglacialoriginaswell asbymildtemperaturesmediatedbytheinflowofAtlantic water.

Samplingmethod

Duringtheyear2011,watersampleswerecollectedfrom16 locations(Fig.1)atvariousdepthsfrom5mtoamaximumof 100minthemonthofJune,August,andSeptember.Inorder tomonitortheeffectofphytoplanktonassemblageon retriev-ableheterotrophicbacteria,mostofoursamplingdepthswere restrictedtoupper40mwithfewexceptionsinAugustwhere weakfluorescencewasalsoobservedathigherdepths (Sup-plementary Table1).Thesampling wasdone followingthe hydrological observationswith a conductivity-temperature-depth (CTD) profiler(SBE 19 plus V2, Sea Bird Electronics, Bellevue,WA,USA)equippedwithafluorescencesensor(Wet Labs,Philomath,USA).Theaveragevolumeofthetransformed AtlanticwaterinsideKongsfjordenwas calculatedwiththe upper boundary (thickness)of the watermass, which was delineatedusingacombinationofitscharacteristic hydrogra-phyvalues16_{withupperlimittemperatureof3}◦_Candsalinity

of>34.65psu.Watersampleswerecollectedasepticallyin ster-ileglassbottles(DuranSchott,Stafford,UK).Thesampleswere immediatelysubjectedtoanalysisintheshorelaboratory.

Totalcounts,culture,andisolationofheterotrophic bacteria

Totalcellsinthewatersamples(10mLeach)collectedfrom all the discrete depths were stainedwith 4 ,6-diamidino-2-phenylindole(DAPI)17_{beforecounting.CellsstainedwithDAPI}

were first fixed with filter sterilized particle free buffered formaldehyde (final concentration, 3.7%) to preserve the cell morphology and improve the staining efficiency. The stainedcellswerefilteredthrough0.22␮blackpolycarbonate membranes (Nucleopore Track-Etch Membrane, Whatman, Maidstone,UK)andcountedunderanOlympus epifluoresce-ncemicroscope(BX51)withtheaidofanOlympusU-MWU2 filter(Excitation330–385nmandEmission420nm).Counting wasdoneonaWhipplegridwitha100×objective(Olympus UPLNFLN,Tokyo,Japan).

Watersampleswerespreadplatedusing100␮Laliquoton precooled(4◦C)quarterstrengthZobellMarine Agar(ZMA) andincubatedat4◦Cfor1–2weeks.Colonieswithunique mor-phologicalfeatureswereisolatedandsub-culturedtoobtain purecultures.

PCRamplificationofthe16SrDNA,sequencingand phylogeneticanalysis

Cell biomass forDNA extractionwas obtained by growing the pure cultures in quarter strength Zobell Marine Broth

11º 12º

79º

11º 12º

79º

Fig.1–BathymetricmapofKongsfjordenwithsamplinglocations.Sampleswerecollectedfrom16stations.Stations1–9 alongthefjordandstations3.1–3.4and7.1–7.3attheintersectionofstation3and7,respectively.

(ZMB). Total bacterial genomic DNA was extracted using ChargeSwitchgDNAmini bacteriakit (Invitrogen, Carlsbad, CA,USA).ThepurityoftheDNAextractswasverifiedbygel electrophoresison1%agarose.The16SrRNAgenewas ampli-fiedusingthe universalbacterial16Sprimers:forward(27f) 5-AGAGTT TGA TCM TGG CTC AG-3 and reverse (1492r) 5-GGTTAC CTT GTTACG ACTT-3.18 _TheDNA

amplifica-tioninafinalreactionvolumeof50␮Lcontaining0.3pM/␮L ofeach primer,1.5mMMgCl2, 200␮MdNTPsand2.5UTaq

DNApolymerase(Invitrogen,Carlsbad,CA,USA)wascarried out in a thermocycler (BioRad CFX 96) with the following conditions: initial denaturation at94◦C for 2min followed by 29 cycles of 30s at 95◦C, 30s at 45◦C and 2min at 72◦C, and a final extension of 10min at 72◦C. Amplifica-tion was confirmed by electrophoresis in 1% agarose gel. The amplicons were purified using ChargeSwitch Pro PCR cleanupkit(Invitrogen,Carlsbad,CA,USA).Theeluted frag-mentsweresequencedusinganautomatedDNAsequencing system(AppliedBiosystems,Carlsbad,CA,USA).Theobtained sequenceswerecheckedforchimerawiththeCHIMERA detec-tion program (http://rdp8.cme.msu.edu/cgis/chimera.cgi).19

The 16S rDNA sequences of the isolates (∼1200bp) were compared with the type strains belonging to the same phylogenetic group, using the Seqmatch tool of Ribo-somal Database Project (http://rdp.cme.msu.edu/).19 _SINA

v1.2.1120 _{software was used to align the 16S rRNA gene}

sequences. Thephylogenetic trees were constructed using maximum likelihood and neighbor-joining methods using MEGA Version 521 _{and 1000 subsamples were generated}

usingthebootstrapanalysis.The16SrRNAgenesequences of the isolated strains were deposited with accession numberHE800807-HE800839,HE815462-HE815463, HG795014-HG795016,HF569156-HF569159andHF913434-HF913440inthe EMBLdatabase.

Results

Physicalpropertiesofthesamplingsites

The details about the geographic location of the stations selectedforsamplingduringsummer2011aregiveninFig.1. Thewatercolumnwaswarmer(4◦C)duringJunecomparedto thatinAugust,wheretemperaturedecreasedgraduallyalong withthedepth,exceptatthesurface,whichwaswarmerinthe vicinityoftheglacierasdepictedinFig.2.Watertemperature wasuniform(4◦C)throughoutthecolumnduringSeptember. Salinityrangedfrom31to35psuduringsummer2011(Fig.2). ThesurfacewaterwaslesssalineduringJuneandSeptember (∼31psu),whilesalinityincreasedalong withthedepth up to∼35psu.Autotrophicbiomass(fluorescence)wasrestricted toshallowerdepthsduringSeptemberandwashigherduring JuneandAugustascomparedtothatinSeptember(Fig.2).The abundancewasconfinedinthevicinityofKongsbreenglacier duringAugust,whereas duringJuneandSeptember,it was moretowardthemouthofKongsfjorden(Fig.2).Theaverage volumeofthetransformedAtlanticwaterinside Kongsfjor-den was7.9km3_{, 27.2km}3 _{and 12km}3 _{duringJune,August,}

andSeptember,respectively.

Totalandretrievablebacterialcountandphylogenetic identificationofbacterialisolates

TotalbacterialcountduringJune–Septemberrangedfrom107

to109_cellsL−1_{.DuringJuneandAugust,thecellcountswere}

intherangeof107_–108_cellsL−1_{,whileinSeptemberthe}

bac-terialloadwashigher(107_–109_cellsL−1_{).Thelowestbacterial}

count(2.3×107_cellsL−1_{)wasrecordedinthevicinityof}

Fig.2–PhysiochemicalpropertiesofwatersamplescollectedfromKongsfjordeninJune(A),August(B),andSeptember(C) 2011.

Fig.3–PercentagecompositionandabundanceofheterotrophicbacterialspeciesbelongingtothephylumFirmicutes( ),

Actinobacteria( ),˛-Proteobacteria( ),-Proteobacteria( )andBacteroidetes( )isolatedfromKongsfjordenwatersamples duringJune,August,andSeptember2011.

inAugust.Thehighestbacterialload(2.7×109_cellsL−1_)was

observedduringSeptembertowardthemouthof Kongsfjor-den.TheretrievablebacterialloadduringJunerangedfrom 103 _{to 10}6_CFUL−1_{, while during August and September, it}

wasintherangeof104_–106_CFUL−1_{.Atotalof117,332,and}

107 bacterial isolates were recovered in June, August, and September, respectively, from the water samples collected atvariousdepths.Theisolateshaving>97%16SrRNAgene sequence similarity with the type strain were considered asphylotypes. Using the representative phylotypes,all the bacterialisolatesrecoveredfromsamplescollected inJune, August,andSeptember2011werecategorizedinto6,22,and 12phylotypes, respectively;and the obtainedresultsabout abundanceofeachspeciesaregiveninFig.3.The phyloge-netictreesillustratingtheevolutionaryrelationshipsamong thebacterialisolatesarepresentedinFig.4A–C.Twobacterial strains,Paenibacillussp.(Kongs–47,HG795014)and Alterery-throbactersp.(Kongs– 48,HG795015)recovered from water samplesofAugustandonestrain,Brevundimonassp.(Kongs –49,HG795016),recoveredfromSeptembercouldnotbe iden-tifiedasanyknownspeciesduetolowsequencesimilarity (≤97%) withany typestrain, and probablymight represent newspecies. Theclosest phylogenetic relativeofKongs–47 wastypestrainofPaenibacillusphyllosphaerae(96%)andthose

ofKongs–48and Kongs–49were Altererythrobacter gangjinen-sis (96%)and Brevundimonas subvibrioides(97%), respectively (Fig.4A).

BacterialdiversityatKongsfjorden

The bacterial isolates retrieved from the water sam-ples belonged to five phyla: Firmicutes, Actinobacteria, ˛-Proteobacteria, -Proteobacteria and Bacteroidetes (Fig. 3). The phylum Bacteroidetes was the most dominant (40%) during June and was entirelyrepresented bythe species Leeuwen-hoekiella aequorea. Itwas followed by ˛-Proteobacteria (27%),

Actinobacteria (17%),-Proteobacteria(9%)and Firmicutes(7%).

Sphingopyxisbaekryungensis(25%)andErythrobactercitreus(2%) constituted˛-ProteobacterialpopulationwhileAgrococcusbaldri, Psychrobacter nivimaris, and Bacillus subtilis represented the phyla Actinobacteria, -Proteobacteria, and Firmicutes, respec-tively.

The retrievable bacterial diversity was higher during August;it was representedby22 phylotypesincluding two unclassified sequences (Paenibacillussp.and Altererythrobac-ter sp.). The population was dominated by Actinobacteria

(39%) and -Proteobacteria (23%), while Firmicutes (14%),

Kongs-25HE800831 Kongs-38HF569158 Kongs-1HE800807 Kongs-2HE800808 Kongs-12HE800818 Kongs-21HE800827 Kongs-23HE800829 Kongs-45HF913439 Kongs-6HE800812 Kongs-42HF913436 Kongs-8HE800814 Kongs-19HE800825 Kongs-4HE800810 Kongs-5HE800811 100 100 57 98 100 97 100 86 95 0.05 100 100 62 100 87

Leeuwenhoekiella aequorea (T) AJ278780

Psychrobacter nivimaris (T) AJ313425

Erythrobacter citreus (T) AF118020

Sphingopyxis baekryungensis (T) AY608604

Bacillus subtilis (T) AJ276351

Aquifex pyrophilus (T) M83548

Agrococcus baldri (T) AB279548

A

Fig.4–Phylogenetictreeconstructedonthebasisof16SrRNAgenesequencesshowingtherelationshipamongthe bacterialstrains(prefixwithKongs),obtainedfromthewatersamplescollectedduringJune(A),August(B)andSeptember (C)2011fromKongsfjorden,withtheirnearestphylogenetictypestrains.Theaccessionnumberof16srRNAgenesequence ofthestrainsisdenotednexttoitsname.Phylogenetictreewasconstructedbymaximumlikelihoodmethod.Numbers shownatnodesarebootstrapvalues.Thebarrepresents0.02substitutionsperalignmentposition.

abundance.TheabundanceofFirmicuteswasalmosttwiceof thatinJune,andwasmainlyconstitutedbythegenus Staphy-lococcus,whichwasrepresentedbyStaphylococcushaemolyticus

andStaphylococcus pasteuri(5%ofeach).Awell-known phy-topathogenic bacterium Rhodococcus fascians constituted to the most (32%) of the Actinobacterial population, which increasedby22%ascomparedtothatinJune.Atotaloffive genera(Brevundimonas,Erythrobacter,Phenylobacterium, Sphin-gopyxis,andAltererythrobacter)wererecoveredwithintheclass ˛-Proteobacteria,wherePhenylobacteriumfalsumwasmajor con-tributor(4%). However,thetotal abundanceofthis phylum decreasedby15% ascomparedtoJune.-Proteobacteriawas representedbyfourgeneraandsevenspecies.Themajor frac-tionwasconstitutedbythegenusPsychrobacter(17%),which wasrepresentedbyPsychrobacterfozii(2%),Psychrobacter glac-incola(6%),P.nivimaris(4%),andPsychrobacterokhotskensis(4%). OthergeneraincludedAcinetobacter(2%),Halomonas(4%),and

Janibacter(1%).InJune,L.aequoreawastheonlyspecies recov-eredunderthephylumBacteroidetes;however,itsabundance decreasedby28%duringAugust.

During September, Proteobacteria was the major phylum with ˛-Proteobacteria (31%) and -Proteobacteria (33%) being dominant,followedbyActinobacteria(21%),Bacteroidetes(12%), and Firmicutes (3%). Halomonas titanicae (16%) represented the major fraction of -Proteobacteria, while Brevundimonas variabilis(15%)wasdominant-Proteobacteria.Firmicuteswas

representedbytwobacterialspecies,Bacillusidriensis(2%)and

Paenibacillusurinalis(1%).ThepopulationofActinobacteriawas reducedby18%inSeptemberascomparedtothatinAugust and it wasrepresentedbyNocardioides basaltis(12%)andR. fascians(9%).L.aequoreawastheonlyspeciesrepresenting Bac-teroideteseveninSeptemberwithanalmostequalabundance asinAugust.

Discussion

Thephysico-chemicalconditionsintheKongsfjordenwater columnarehighlydynamic,especiallyinthesummer,when thereisalargescalefluxofcoldArcticwaterfromthe east-ernsideandintrusionofwarmAtlanticwater(AW) onthe westernside.1_{Kongsfjordenisoneofthewesternfjordsthose}

receiveAW.Accordingtothemooredobservatories,thefjord normallyreceivesthemaximumcontentofAWinlate sum-mer,whilethroughthefallandwinter,thefjordwatermasses aregraduallyreplacedbyfresherandcoldArcticwater.16_In

thepresentstudy,thefjordwasfoundtoreceivehighest vol-umeoftransformedAtlanticwater(27.2km3_{)duringAugust}

ascomparedtoJune(7.9km3_{)andSeptember (12km}3_).The

complexdynamicsofthefjordwatermassesmayberegarded asthemajordriving forceforthevariabilityin phytoplank-tonassemblages.DuringJuneandSeptember,theautotrophic

83 94 57 100 98 100 80 97 100 100 100 100 100 100 100 100 100 22 38 65 81 99 100 89 100 100 100 100 100 100 100 100 100 99 91 97 0.05 100 99 Kongs-24HE800830

B

Kongs-17HE800823 Kongs-26HE800832 Kongs-37HF569157 Kongs-3HE800809 Kongs-16HE800822 Kongs-39HF569159 Kongs-7HE800813 Kongs-9HE800815 Kongs-36HF569156 Kongs-40HF913434 Kongs-34HE815462 Kongs-48HG795015 Kongs-15HE800821 Kongs-20HE800826 Kongs-10HE800816 Kongs-14HE800820 Kongs-11HE800817 Kongs-47HG795014 Kongs-13HE800819 Kongs-22HE800828 Kongs-18HE800824

Psychrobacter glacincincola (T) AJ312213

Psychrobacter okhotskensis (T) AB094794

Psychrobacter fozii (T) AJ430827

Acinetobacter lwoffii (T) X81665

Halomonas titanicae (T) FN433898

Leeuwenhoekiella aequurea (T) AJ278780

Brevundimonas mediterranea (T) AJ227801

Phenylobacterium falsum (T) AJ717391

Sphingopyxis flavimaris (T) AY554010

Sphingopyxis baekryungensis (T) AY608604

Erythrobacter citreus (T) AF118020

Alterythrobacter gangjinensis (T) JF751048

Agrococus baldri (T) AB279548

Janibacter limosus (T) Y08539

Mocrococcus yunnanensis (T) FJ214355

Rhodococcus fascians (T) X79186

Aquifex pyrophilus (T) M83548

Paenibacillus barcinonensis (T) AJ716019

Paenibacillus phyllosphaerae (T) AY598818

Bacillus subtilis (T) AJ276351

Staphylococcus haemolyticus (T) X66100

Staphylococcus pasteuri (T) AB009944

Psychrobacter nivimaris (T) AJ313425

Fig.4–(Continued)

biomasswasconfinedtowardthemouthofthefjord,while duringAugustitwashigherintheproximityofglacier. Dur-ingJuneandSeptember,theglacialmeltwaterinputcould behigherasevidentbythelowersalinityvaluesintheupper watercolumn.Highsedimentconcentrationsderivedfromthe inputofmeltedglacialwatercanlimitthelightavailabilityfor phytoplanktongrowthneartheglaciers.12_{Theeuphoticzone}

canberestrictedtothe upper0.3mclosetotheglaciers,22

leadingtohighly unfavorableconditions forphytoplankton growth.23

The bacteria–phytoplankton interactions during bloom events arecomplex and change throughoutthe lifetimeof thebloom.AnearlierstudybyJankowskaetal.,24_conducted

duringsummer 1999,reportedbacterioplanktonabundance intherangeof108_–109_cellsL−1_{inKongsfjordenandthe}

adja-cent Krossfjorden, while in our study the total cell count variedfrom107 _to109_cellsL−1_{.Thisvariable distributionof}

thebacteriaduringtheobservationperiodcouldbeprimarily attributedtothevariationinthewatermassesand/or phyto-planktondistribution.DuringAugust,thehigherbacterialcell counttowardheadofthefjordcoincidedwithahigher phyto-planktondensity.Thelocalizedandtransientincreaseinthe abundanceofphytoplanktoncouldalterthelevelsofdissolved organicmatter,particularlyduringthecollapseofthe phyto-planktonbloom.3_{Asphytoplanktonbloomsareoftenseasonal}

48 45 100 100 100 69 100 100 100 100 100 100 100 100 0.05 89 51 99 99 99 96 51 Kongs-41HF913435

C

Kongs-31HE800837 Kongs-32HE800838 Kongs-30HE800836 Kongs-43HF913437 Kongs-33HE800839 Kongs-44HF913438 Kongs-49HG795016 Kongs-46HF913440 Kongs-35HE815463 Kongs-28HE800834 Kongs-29HE800835 Kongs-27HE800833

Psychrobacter nivimaris (T) AJ313425

Psychrobacter piscatorii (T) AB453700

Halomonas titanicae (T) FN433898

Paracoccus marinus (T) AB185957

Brevundimonas mediterranea (T) AJ227801

Brevundimonas subvibrioides (T) AJ227784

Brevundimonas VAriabilis (T) AJ227783

Paenibacillus urinalis (T) EF212892

Bacillus diriensis (T) AY904033

Aquifex pyrophilus (T) M83548

Nocardioides basaltis (T) EU143365

Rhodococcus fascians (T) X79186

Leeuwenhoekiella qequorea (T) AJ278780

Fig.4–(Continued)

ofheterotrophicbacteria vary accordingly.Theseprocesses arepartlybalancedbyasubsequentincreaseintheactivity ofheterotrophic bacteria, which transform phytoplankton-derivedorganicmatter.3_{Theheterotrophicretrievablecounts}

inKongsfjordenwaterweremarkedlydynamicrangingfrom 103 _{to 10}7_CFUL−1 _{during summer 2011. Previous studies}

from Kongsfjorden have reported similar findings13,14 _with

lowcounts,thatcanbeattributedtotheculture-dependent approachusingonlyonemedium(ZMA).14_Theuseof

differ-entmediaand/orcultureconditionscanperhapsimprovethe viableheterotrophicbacterialcounts.

Althoughthephytoplanktoncompositionvarieswiththe environmental conditions, a limited number of taxa are consistently found to dominate bloom-associated bacterial communities. The 16S rRNA gene sequences correspond-ing to ˛-Proteobacteria, Firmicutes and -Proteobacteria were earlier reported from freshwater as well as marine water samples of the Kongsfjorden.11 _{In the current study, the}

phyla, Bacteroidetes and Actinobacteria, were also retrieved along with ˛-Proteobacteria, Firmicutes, and -Proteobacteria. Thepredominance ofActinobacteria,Proteobacteria, and Bac-teroideteshasalreadybeenreportedinArcticwater,ice,and sediments.11,14,25,26 _{DuringJunearemarkabledominanceof}

Bacteroidetes represented entirely by L. aequorea was seen. The second most abundant species, during June, was S. baekryungensis–ayellow-pigmented˛-Proteobacteria.Specific associationsbetween phytoplanktonandcertain speciesof

Bacteroidetesand˛-Proteobacteriahavebeenfoundinlaboratory conditions.7,8,27 _{The metabolic properties ofthese bacteria}

enablethem torespondpromptlytothe transient nutrient pulses,whichareahallmarkofphytoplanktonblooms.3

Culture-independent studies have shown that Arctic waters harbor diverse taxa including Acidobacteria, Planc-tomycetes, Lentisphaerae, and Verrucomicrobiae9,10,28,29 _which

couldbeduetotherecalcitranceofthesebacterialgroupson culturemedia.Incontrasttoseveralothermajormarine bac-teriallineages,suchastheSAR86andSAR116clades,which areeitherdifficulttocultureorhavenotyetbeenbroughtinto culture,thecultivablerepresentativesareavailableforseveral importantgroupsincludingBacteroidetesand˛-Proteobacteria.30 Indeed,cultivatedrepresentativesofthesetwogroupshave frequentlybeenisolatedfrombloomsandinvitroenrichment culturesofdifferentphytoplankton31–33 _{andhaveevenbeen}

foundtobedirectlyattachedtothephytoplanktoncellsduring abloom.34_{Infact,thenatureofthesebacterial–phytoplankton}

interactionsrangesfrommutualistictoparasitic.Some bacte-riaprovidetheirhostswithessentialvitaminsandnutrients and bestow resistance againsttoxic metabolic byproducts, whereasotherscompetewiththeirhostsfornutrientsor pro-ducealgicidalcompounds.3

TheshiftofbacterialcommunityfromJunetoAugust2011 couldbeattributedtochangeintheenvironmentalconditions anddifferencesinthedepthofisolationofbacterialspecies (Supplementary Table 1), atleast in part as the effects of physicochemicalproperties.Forinstance,theincreased phy-toplanktonabundanceinAugustcouldpromotethegrowth ofR.fascians(anActinobacteria),awell-knownphytopathogen, which constituted 32% of the total retrievable bacterial

diversityduringthisperiod.However,itsassociationhasonly beenreportedwithhigherplants35,36_{andthesameneedsto}

beexaminedinthiscontext.Mergaertetal.37_observedthat

alargeproportionoffacultativeand psychrotrophicstrains isolatedfromArcticandAntarcticseawaterscanbegrouped intotheR.fascianscluster.Thestudiesbasedonstable car-bonisotopeprobinghavedemonstrated thatinaddition to themembersof˛-ProteobacteriaandBacteroidetes, Actinobacte-riaand-Proteobacteriaassimilatealgalextractsquickly;these phylacanexpressabroadrangeofhydrolasesinimmediate responsetotheavailabilityofphytoplanktondetritus.38_Thus,

oscillationsinthebacterialdiversityperhapsprovideaclue forbiochemicalprocess andrelatedchangesfunctioning in theecosystem.

InSeptember2011,themostabundantspeciesrecovered fromthewatersamplewasH.titanicae.Thisisthefirstreport ontheoccurrenceofH.titanicaeinKongsfjordenwater.The typestrainofthisspecieshasbeenisolatedfromthe sam-plesofrusticles collected from the RMSTitanic wrecksite ata depthof∼3000minAtlanticOceanandwas foundto beassociatedwithcorrosion ofironwrecks.39 _Arapidand

overwhelmingintrusionofAtlanticwateracrosstheshelfand intothefjordoccursduringmid-summer,andthefjordwater switchesfrombeingArcticdominanttoAtlanticdominant.16

Similarobservationswererecordedduringourstudy where the volume of the transformed Atlantic water was found toincreaseduringlatesummer(August andSeptember). A recentstudy fromNorthAtlanticOceanshowsthatdistinct waterbodieshostdifferentbacterialpopulations,whichmay serveasbiologicalmarkersforoceanicprovinces.40_Similarly,

Fuetal.41_{foundwatermasstobethemostimportantfactor}

forthedistributionofmarineRhodobacteralescommunitiesin theArcticmarinesystem.Thus,theincreasedpopulationof

H.titanicaecouldbeduetotheincreasedintrusionofAtlantic waterintoKongsfjordenduringthisperiod.

During the seasonal shift, along with the phytoplank-tonbloomdynamics,temperaturecouldbeoneofthemost importantparametersthatdeterminethedistributionofthe bacterialcommunitiesandaffectcellstructureandfunction. Lowtemperaturesreducebiochemicalreactionratesand sub-stratetransport.Notsurprisingly,anorganism’scompatibility withthetemperatureofitshabitatisultimatelydetermined by its underlying genetic structure. The genus Psychrobac-ter,under-Proteobacteria,includesagroupofGram-negative, heterotrophicbacteria, andamong them,many Psychrobac-ter species are capable to grow at low temperatures. The members ofthis genus can evengrow attemperatures as lowas−20◦_{Cand theyhavefrequentlybeenisolated from}

various cold environments, including Antarctic and Arctic seaice.42–46 _{Inthepresentstudy,weobservedahigh}

occur-renceofPsychrobacterspecieswithadecreaseintemperature fromJunetoSeptember.DuringAugustand September,the watercolumnwascolderthanthatinJune,whichcoincides withthemaximumretrievalofPsychrobacterspeciesinthese months.P. glacincola,P. fozii,and P. nivimarisretrieved from Kongsfjordenbelongtothesame typestrains reported ear-lier from Antarctic regimes.44,47,48 _{Several similaritiesexist}

betweenthemarineregimesofArcticandAntarctica; how-ever,therearealsofundamentaldifferencesinwatermasses andwatercurrents.Whetherthesedifferencesinfluencethe

distributionanddevelopmentofbacterialcommunityisstill anopenquestion.InastudybyBrinkmeyeretal.,42_the

anal-ysis of16S rRNA gene clone libraries from multipleArctic andAntarcticsamplesrevealedahighoccurrenceofclosely overlapping16SrRNAgenecloneandtheisolates’sequences. Approximately 50and 36% sequenceswere identifiedas

-Proteobacteria in Arcticand Antarctic samples, respectively. ThegeneralsimilarityofbacterialphylotypesinArcticand Antarctic samples implies that probably similar selective mechanismsoccuratboththepoles.However,theanalysis attheconservativegenelevelof16SrRNAisnotsufficientto determineifthesamespeciesarepresentatbothpoles.Other analyticalmethods,e.g.,DNA–DNAhybridization,might elu-cidatethediversitythatgoesundetectableby16SrRNAgene sequencing.

Conclusion

Theheterotrophicretrievable countsinKongsfjordenwater weremarkedlydynamicrangingfrom103_to107_CFUL−1

dur-ingsummer2011.Thevariabledistributionoftheretrievable heterotrophic bacteria during the observationperiod could primarilybeattributedtothevariationinthewatermasses and/orphytoplanktondistribution.Increasedphytoplankton concentrationduringAugustcouldpromotethegrowthofR. fascians,awell-knownphytopathogen.TheoccurrenceofH. titanicaefromKongsfjordenwaterhasbeenreportedfirsttime. Theincreasedpopulation ofH.titanicae duringAugust and September couldbeduetothehigherintrusion ofAtlantic water into Kongsfjorden during this period. Similarly, an increasedoccurrenceofthememberofthegenusPsychrobacter

inthelatesummerindicatesashiftintheheterotrophic bac-terialcommunitytowardtheonethatismorecapabletothrive at lower temperatures. Thus, changes in the environmen-talparameterscouldstronglyalterthespeciescomposition; therefore,itisworthwhiletomonitorthefjordecosystemfor alongtermtogetadeeperinsightofthissensitiveecosystem.

Conflicts

of

interest

Theauthorsdeclarenoconflictsofinterest.

Acknowledgements

TheauthorswishtoexpresstheirgratitudetoDr.S.Rajan, Director, NationalCentre forAntarctic andOceanResearch (ESSO-NCAOR). Thanksare duetoDr.FinloCottier,Headof Physics,SeaIceandTechnologyDepartment,Scottish Asso-ciationofMarineSciences,forfacilitatingtheSAMSmooring datacollectionfortheperiod2010.Thisworkwasundertaken asapartoftheproject‘Longtermmonitoringof Kongsfjor-densystemofArcticregionforclimatechangestudies’.This isESSO-NCAORcontributionnumber32/2016.

Appendix

A.

Supplementary

data

Supplementarydataassociatedwiththisarticlecanbefound, intheonlineversion,atdoi:10.1016/j.bjm.2016.09.011.

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