Variations in culturable bacterial communities and biochemical properties in the foreland of the retreating Tianshan No. 1 glacier

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

Environmental

Microbiology

Variations

in

culturable

bacterial

communities

and

biochemical

properties

in

the

foreland

of

the

retreating

Tianshan

No.

1

glacier

Xiukun

Wu

_,

_Gaosen

_Zhang

_,

_Wei

_Zhang

_,

_Guangxiu

_Liu

_,

_Tuo

_Chen

_,

Yun

Wang

_,

_Haozhi

_Long

_,

_Xisheng

_Tai

_,

_Baogui

_Zhang

_,

_Zhongqin

_Li

a_{ChineseAcademyofSciences,NorthwestInstituteofEco-EnvironmentandResources,KeyLaboratoryofDesertandDesertification,}

Lanzhou,China

b_{KeyLaboratoryofExtremeEnvironmentalMicrobialResourcesandEngineering,Lanzhou,GansuProvince,China}

c_{ChineseAcademyofSciences,NorthwestInstituteofEco-EnvironmentandResources,StateKeyLaboratoryofCryosphericSciences,}

Lanzhou,China

a

r

t

i

c

l

e

i

n

f

o

Received4June2015 Accepted24October2016 Availableonline15February2018 AssociateEditor:IêdadeCarvalho Mendes

Keywords:

TianshanNo.1glacier Foreland

Culturablebacteria

Soilbiochemicalcharacteristics

a

b

s

t

r

a

c

t

Asaglacierretreats,barrenareasareexposed,andthesebarrenareasareidealsitestostudy microbialsuccession.Inthisstudy,wecharacterizedthesoilculturablebacterial commu-nitiesandbiochemicalparametersofearlysuccessionalsoilsfromarecedingglacierin theTianshanMountains.Thetotalnumberofculturablebacteriarangedfrom2.19×105_to

1.30×106_CFUg−1_{dwandfrom9.33×10}5_to2.53×106_CFUg−1_dwat4◦_Cand25◦_C,

respec-tively.Thenumberofculturablebacteriainthesoilincreasedat25◦Cbutdecreasedat4◦C alongthechronosequence.Thetotalorganiccarbon,totalnitrogencontent,andenzymatic activitywererelativelylowintheglacierforeland.Thenumberofculturablebacteria iso-latedat25◦CwassignificantlypositivelycorrelatedwiththeTOCandTNaswellasthe soilurease,protease,polyphenoloxidase,sucrase,catalase,anddehydrogenaseactivities. Weobtained358isolatesfromtheglacierforelandsoilsthatclusteredinto35groupsusing amplifiedribosomalDNArestrictionanalysis.Thesegroupsareaffiliatedwith20generathat belongtosixtaxa,namely,Alphaproteobacteria,Betaproteobacteria,Gammaproteobacteria, Actinobacteria,Bacteroides,andDeinococcus-Thermus,withapredominanceofmembers ofActinobacteriaandProteobacteriainallofthesamples.Aredundancyanalysisshowed thatthebacterialsuccessionwasdividedintothreeperiods,anearlystage(10a),a mid-dlestage(25–74a),andalatestage(100–130a),withthetotalnumberofculturablebacteria mainlybeingaffectedbythesoilenzymaticactivity,suggestingthatthemicrobialsuccession correlatedwiththesoilagealongtheforeland.

©2018PublishedbyElsevierEditoraLtda.onbehalfofSociedadeBrasileirade Microbiologia.ThisisanopenaccessarticleundertheCCBY-NC-NDlicense(http://

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

∗ _{Correspondingauthorat:KeyLaboratoryofDesertandDesertification,NorthwestInstituteofEco-EnvironmentandResources,Chinese}

AcademyofSciences,DonggangWestRoadNo.320,Lanzhou730000,China. E-mail:liugx@lzb.ac.cn(G.Liu).

https://doi.org/10.1016/j.bjm.2018.01.001

1517-8382/©2018PublishedbyElsevierEditoraLtda.onbehalfofSociedadeBrasileiradeMicrobiologia.Thisisanopenaccessarticle undertheCCBY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/4.0/).

Introduction

Over the past 100 years, the average global tempera-ture has increased by 0.74◦C,1 _{and one consequence of}

this temperature increase is that glaciers are retreating in many mountainous areas of the world. As the glaciers retreat, the newly exposed land is a new habitat for microorganisms2 _{derived from the air, clouds, snow, rain,}

and runoff waters from the glacier body in addition to autochthonousmicroorganisms.3_{Bacterialcommunitiesmay}

bekeydeterminantsofglacierforelandecosystemstability andfunctionbecauseoftheirimportantroles insoil devel-opment,biogeochemicalcyclesandheterotrophicactivities. Microbialcommunitieschangealongthesoilagegradientofa glacialforeland.Sigleretal.4_{foundthatthenumberof}

dom-inant organism typesand community evenness decreased withsuccession,butothersfoundthatthephylotypenumber, diversity,andevennessincreasedovertime.2,5_However,most

ofthosestudiesarefocusedonPolarandEuropeanmountain areas;therefore,studiesonthebacterialcommunity,soil bio-chemicalpropertiesandthecorrelationbetweenbacteriaand soilbiochemicalproperties along chronosequences insuch highAsianregionsastheTianshanMountainsarestillneeded. The Tianshan No. 1 glacier is located in the Eastern TianshanMountainsofCentralAsia,mountainsthatare sur-rounded by desert.6 _{Theclimate in this area isa classical}

continentalclimate,andwindisanimportantclimatic fac-torintheupperelevationsofthemountains.7_TheTianshan

No.1glacierhasbeenstudiedintensivelyfromaglaciological pointofviewsince1959,8–10 _{whentheTianshan}

Glaciologi-calStationwasbuilt.Becauseoftheavailabilityofextensive glaciologicaldataanddetailedglacierretreatdata,thisarea is anideal location for the study of microbial distribution andgrowthrelatedtobothclimaticandother environmen-talfactors.11,12_{Althoughthestudyofmicroorganismsinthis}

area is very important, few studies have been performed. Baietal.13 _{reportedthebacterialdiversityfrompermafrost}

in the Tianshan Mountains, and Yang et al.14 _{studied the}

permafrostbacterialand archaealcommunitystructuresin thesamearea.Shenget al.15 firstdescribedtheindigenous endophyticbacteriawithinsubnivalplantsofthe Tianshan Mountains.Wangetal.16_{reportedmicrobialbiomassandsoil}

enzymeactivity variationsalongchronosequences,andWu etal.11_{usedpyrosequencingtoanalyzethebacterialdiversity}

alongchronosequences.However,studiesregardingthe varia-tionoftheculturablebacterialcommunitiesandbiochemical characteristicsalongchronosequencesintheTianshan Moun-tainsarenotavailable,andthusfundamentalknowledgeon theculturablebacterialcommunitiesandbiochemical charac-teristicsintheTianshanNo.1glacierforelandsislacking.

Inthisstudy,wepresentdataregardingthesoil biochem-icalpropertiesanddiverse bacteriaculturedusing samples fromtheTianshanNo.1glacierforelands.Theresultscould leadtoabetterunderstandingoftheinitialcolonizationand successionpatternsofmicroorganismsandsoildevelopment and the correlation between bacteria and soilbiochemical propertiesalongchronosequencesinahighAsianregion.Our aimswere(1)toinvestigatethesoilbiochemicalpropertiesand culturablebacterialabundancevariationsalonga

chronose-1-3_4-7 8-11 12-16 17-21 22-25 1760 1675 1911 1962 Sample location Moraine

Fig.1–Mapofsamplingposition.

quence,(2)todeterminetheculturablebacterialcommunity variationsbyusingalow-nutrientmediumculturedat4◦C and 25◦C,and (3)toexaminethecorrelations betweenthe abundance of culturable bacteria and the soil biochemical propertieswithincreasingsoilage.

Materials

and

methods

Studysiteandsampling

TheTianshanMountainsextendthroughChina,Kyrgyzstan andKazakhstaninCentralAsiaandhave15,953glacierswith atotalareaof15,416km2_.17_{Thesamplesiteswerelocatedat}

TianshanNo.1glacier(N43◦06,E86◦48),120kmsouthwest ofUrumchi,China(Fig.1).Thetopelevationatthisglacieris 4486m.Thesampleswerecollectedattheeastbranchof Tian-shanNo.1glacierforelandalongthechronosequenceinfront oftheretreatingglaciers.Twenty-fivesoilsampleswere col-lectedinAugust2010.Thesesoilsamplesrepresent6periods: sites1to3,sites4to7,sites8to11,sites12to16,sites17to21, andsites22to25represent10a,25a,60a,74a,100a,and130a, respectively.Thesuccessiontimeofeverysamplingsitewas determinedusingtheannualglacierretreatobservationdata (from1959to2010) fromtheTianshanGlaciologicalStation (Chinese AcademyofSciences) and lichenometric chronol-ogy data(from 1958to1538).18 _{Each soilsampleconsisted}

ofthree subsamplecores ata5cmdepth collected at ran-dominanareaapproximately 2m×2m;the sampleswere mixedafterthelargergravelhadbeenremoved.Pioneerplants appeared in the deglaciated soil within 10–100 years, and vegetationdevelopedafter100yearsofdeglaciation. Succes-sionalspeciesarrivingwithin10–100yearsincludedCancrinia tianschanica, Bryophytaspp., Poa tianshanica, Draba nemorosa, SaxifragahirculusL.,Melandriumapricum,Leontopodium lentopo-dioides,Saussureagnaphalodes,Crepisflexuosa,Rhodiolacoccinea, Oxyriadigyna,andSaussureainvolucrata,whereasSenecio thian-schanicus,Polygonumviviparum,andPedicularisspp.additionally appearedoutsidetheglacierforeland.Thesoilsampleswere placedinasterilesoilboxandkeptoniceduringtransportto thelaboratoryandthenanalyzedimmediately.

Biochemicalanalysesofthesoils

Thesoilwatercontentwasmeasuredbyaweightlossmethod after48hat80◦Cinadryingoven.ThesoilpHofsoil: double-distilledwater(1:1w/v)wasmeasuredusinganaciditymeter (SartoriusPT-10,Germany).19_{Afterair-dryingandgrindingto}

allowpassagethrougha100meshsieve,thesoilorganicCand totalNweredeterminedusinganelementalanalyzer (Elemen-tarVario-EL,Germany).

All of the enzymatic preparations were performed at 0–4◦C.Thesoilenzymesurease,sucrase,protease, polyphe-nol oxidase, catalase, and dehydrogenase were measured. Theactivityofureasewasmeasuredaccordingtoamodified methoddescribedbyTayloretal.,20_{andtheureaseactivitywas}

expressedas1mgammonia-N/gdryweight(dw)soilper24h. Thedehydrogenaseactivitywasdeterminedbythereduction of2-p-iodo-nitrophenyl-phenyltetrazoliumchloride (INT) to iodo-nitrophenylformazan(INTF)usingthemethodof Tay-loretal.,20_{andtheactivitywasexpressedas1␮gINTF/gdw}

soilper24h.Thecatalaseactivitywasdeterminedby measur-ingtheO2absorbedbyKMnO4aftertheadditionofH2O2to

thereactionmixture.21 _{Theproteaseactivitywasmeasured}

accordingtoNannipieri etal.22 _{and Garcıa-Gil et al.,}21 _and

theactivitywasexpressedas1mgN/gdwsoilper24h.The sucraseactivitywasdeterminedaccordingtothemethodby Schinner,23_{andtheactivitywasexpressedas1mgglucose/g}

dwsoilper24h.Thepolyphenoloxidaseactivitywas mea-suredaccordingtoSaiya-Corket al.,24 _{andthe activitywas}

expressedas1mgpyrogallol/gdwsoilper24h. Bacterialisolationandcultivation

Becauseacultivationstrategyusinganutrient-richmedium isratherselective,favoringfast-overslow-growingbacterial species,25_{alow-nutrientmedium,PYGV(DSMZmedium621,}

http://www.dsmz.de),was usedtoisolatethe bacteria. The

bacterialcultivationandisolationwereperformedaccording tothemethodofZhangetal.19_{Soilsamples(5g)wereplaced}

ina250mLflaskcontaining45mLofautoclaved0.85%NaCl solutionwithglassbeadsandshakenat150rpmat4◦Cfor 30min.Afterthistreatment,1mLofsuspendedcellandsoil particlesubsamplesweredilutedfrom 10-foldto10−4 with autoclaved0.85%NaClsolution.A100␮Laliquotofthe dilu-tionwasplatedonPYGVmediumandincubatedat25◦Cfor oneweekor4◦Cforthreeweeks.Theinoculationprocedure waspreparedintriplicateforeachdilution.Asterilizedsoil samplethathadbeenautoclaved(121◦Cfor2h)wasusedasa negativecontrol.Subsequently,thecolonyformingunits(CFU) werecalculatedastheaveragesofthetriplicateplates.Distinct coloniesontheplateswererestreakedontoPYGVagarplates andthenimmediatelypreservedat−70◦_{Cinliquidmedium}

with15%glycerol.

AmplifiedribosomalDNArestrictionanalysis(ARDRA)

ARDRA was used to group the 358 isolates. The genomic DNA was extracted and purified using an AxyPrep Bacte-rialGenomic MiniprepKit (AXYGEN,USA)accordingtothe manufacturer’sinstructions.Approximately20ngofDNAwas amplified with 27F (5-AGAGTTTGATCCTGGCTCAG-3) and

1492R(5-GGTTACCTTGTTACGACTT-3),asdescribedbyZhang etal.,19_{inatotalvolumeof50␮L.Thereactioncontained2}

unitsofTaqDNApolymerase(Fermentas),1×TaqBuffer,3mM MgCl2,0.2mMdNTP,and0.4␮Meachprimer.Afteraninitial

stepat94◦Cfor3min,30cyclesof94◦Cfor1min,58◦Cfor 1min,and72◦Cfor1.5minwereperformed,withaterminal 10minextensionat72◦C.

A10␮LaliquotofthePCRproductswasdoubledigested withrestrictionenzymesHaeIIIandAluI(MBIFermentas)by 1.5Uofeachrestrictionenzymeand1␮Lof10×TangoBuffer at37◦Cfor16h. Theenzymes were inactivatedbyheating thepreparationsat65◦Cfor20min,andtheproductswere separated using2.5%agarosegel(wt/vol)electrophoresisin TAEbuffercontaining0.5␮g/mLofethidiumbromide.A50bp ladderwasusedasamarker.

Sequenceandphylogeneticanalysis

Based on the amplified ribosomal DNA restriction

analysis (ARDRA) of the isolates, one representa-tive strain of each group was selected for sequence determination of the 16S rRNA gene. Three univer-sal primers, 27F (5-AGAGTTTGATCCTGGCTCAG-3), 517F (5-CCAGCAGCCGCGGTAAT-3), and 907F (5

-AAACTCAAATGAATTGACGGG-3), were utilized for

sequencing.19

The16SrRNAgenesequenceclassificationswere identi-fiedusingthe16SrRNAtrainingset 16referencedatabases withtheRDPClassifiermethod(http://rdp.cme.msu.edu/)and aligned against representative reference sequences of the mostclosely relatedmembersobtainedfrom the16S rRNA database inNCBI. CLUSTALW1.81 software wasthen used to perform the multiple alignment26 _{using the method of}

Jukes and Cantor to calculate the evolutionary distances. Thephylogenetic dendrograms were constructedusing the neighbor-joiningmethod,27_{andthetreetopologieswere}

eval-uatedbyperformingbootstrapanalysisof1000datasetsusing theMEGA4.1package.28

Dataanalysis

Thecorrelationcoefficients(R)andtheirpvalueswere cal-culated bythe Pearson methodusing SPSS 13.0 (SPSSInc., Chicago,IL, USA). Thesignificancelevels were within con-fidence limits of 0.05 or less. The data presented are the means ofat least threeindependent experiments and are expressedasthemean±SE.Comparisonsbetweenthemean valueswereperformedusingtheleastsignificantdifference (LSDtest)atp<0.05.Thenumber ofeach differenttypeof colonyappearingontheplateswascountedwhentheCFU werecalculated.CombiningtheARDRAandsequence anal-ysisresults,thebacterialtaxaabundancesweredetermined. Theanalysisofculturablebacterialtaxaabundancecombined withtheenvironmentparameters,suchaspH,C,N,andsoil enzymeactivity,wasperformedusingCANOCO(version4.5, MicrocomputerPower,Ithaca,NY,USA).Aninitialdetrended correspondence analysis (DCA) ofculturable bacterial taxa abundancerevealedthatthedataexhibitedalinear(Lengths ofgradient<3), ratherthan aunimodal,trend, which indi-catedthatredundancyanalysis(RDA)shouldbeused.29_The

Table1–Thenumberofculturablebacteriaandsoilbiochemicalparametersofthesuccessionalsitesalongthe chronosequences.

Samples Soilageafter

deglaciation(years)

4C 25C pH OrganicC(%dw) TotalN(%dw) C/Nratio(w/w)

1–3 10a 13.05±11.66a 9.33±0.44b 8.41±0.084a 0.438±0.018a 0.047±0.005a 9.60±1.03b

4–7 25a 8.39±3.61a 12.39±0.54ab 7.69±0.046bc 0.503±0.092a 0.055±0.010a 9.11±0.11b

8–11 60a 2.43±0.92a 14.86±0.41ab 7.86±0.034b 0.523±0.040a 0.040±0.005a 13.45±1.31a

12–16 74a 3.21±0.85a 15.44±0.25ab 7.54±0.039bcd 0.809±0.190a 0.068±0.016a 12.07.±0.85ab

17–21 100a 2.19±1.15a 17.46±0.59ab 7.20±0.052d 0.751±0.197a 0.062±0.012a 12.02±1.42ab

22–25 130a 4.57±0.53a 25.32±0.38a 7.32±0.060cd 1.600±0.110a 0.127±0.089a 13.30±1.22a

Resultsaregivenasthemeans±SE.Differentsuffixlettersindicatevaluesthataresignificantlydifferentfromoneanother(ANOVA,p<0.05). 4Cand25Cdenotethenumberofculturablebacteriaat4◦Cand25◦C(×105_{),respectively.}

differentagedsoilswereclusteredbyusingthehierarchical clustermethod(SPSS13.0).

Nucleotidesequenceaccessionnumbers

The16S rRNAgenesequencesofthe35representative

iso-latedstrainsweredepositedinGenBank.Thesequencesfrom thebacteriallibraries wereassigned toaccessionnumbers: JN662509-JN662543.

Results

Theabundanceofculturablebacteriainthesoils

The total number of the culturable bacteria ranged from

2.19×105 _{to 1.30×10}6_CFUg−1_{dw and from 9.33×10}5 _to 2.53×106_CFUg−1_dwat4◦_Cand25◦_{C,respectively.Thetotal} numberofculturablebacteriaisolatedat4◦Cdecreasedalong thechronosequence,butnotsignificantly(p=0.09),whereas thenumberofbacteriaisolatedat25◦Csignificantlyincreased alongthechronosequence(p=0.003).Thenumberof cultur-ablebacteriainthe10asoilisolateat4◦Cwas1.4timesthatat 25◦C;atlaterages,thenumberisolatedat4◦Cwaslowerthan at25◦C.Atthe130aage,thenumberisolatedat25◦Cwas5.5 timesthatisolatedat4◦C(Table1).

Biochemicalpropertiesofthesoils

ThesoilpHvaluesdecreasedsignificantly(p=0.04)from8.41 to7.32withthechronosequence.ThesoilorganicCandtotal Ncontentwereverylowatallagesoftheretreatingglacier, ranging from0.438% to1.600%and from0.040% to0.127%, respectively.ThesoilorganicCsignificantlyincreased(p=0.03) alongthechronosequence,andthesoiltotalNalsoincreased alongthechronosequence,butnotsignificantly(p=0.07).The soilC/Nratioshowedanincreasingtrendwiththesoilage (p=0.06).

Soilenzymeactivitiesintegrateinformationabout micro-bial status and soil physicochemical conditions30 _{and are}

often used as an indicator of the functioning of soil ecosystems.31_{Thesoilenzymeactivitysignificantlyincreased}

along the soil age gradient, including catalase, dehydro-genase, polyphenoloxidase, protease, sucrase and urease (p=0.015, 0.004, 0.017, 0.045, 0.027, and 0.023, respectively)

(Tables1and2).

ARDRAandphylogeneticanalyses

Using ARDRA, all 358 isolates cultured at 25◦C and 4◦C wereclusteredinto35groups.Aftercomparingthe16SrRNA sequence of a representative strain from each group, the recoveredbacteriaclusteredintosixgroups: Alphaproteobac-teria, Betaproteobacteria,Gammaproteobacteria, Actinobac-teria,Bacteroides,andDeinococcus-Thermus(Fig.2).

Table2–Thechangesinenzymeactivitiesinthesoilsofthesuccessionalsitesalongthechronosequences.

Samples Soilenzyme

activity Catalase(mg N/gsoilh) Dehydrogenase (␮gINTF/g soil24h) Polyphenoloxidase (mgpyrogallol/gsoil 24h) Protease(mg N/gsoil24h) Sucrase(mg glucose/gsoil 24h) Urease(mg N/gsoil24h) 1–3 2.07±0.108b 36.8±7.08cd 0.403±0.058c 0.233±0.035a 1.04±0.138bc 0.191±0.036b 4–7 1.88±0.501b 30.2±1.25d 0.558±0.045b 0.305±0.046a 0.786±0.187c 0.252±0.029ab 8–11 2.73±0.615b 42.8±1.37bc 0.546± ±0.059b 0.233±0.042a 1.76±0.150bc 0.283±0.019ab

12–16 2.92±0.274b 43.1±4.03bc 0.589±0.027b 0.346±0.030a 2.24±0.659ab 0.319±0.020ab

17–21 2.82±0.457b 51.5±5.82ab 0.598±0.031b 0.355±0.074a 1.57±0.513bc 0.273±0.022ab

22–25 4.71±0.320a 59.3±2.25a 0.815±0.068a 0.405±0.068a 3.26±0.299a 0.374±0.100a

Actinobacteria Deinococcus-thermus Gammaproteobacteria Betaproteobacteria Alphaproteobacteria Bacteroidetes alpine soil 0.02

Permafrost in Qinghai-Tibet Platuea Subnival plants in Tianshan Mountains

Arctic lichen sterocaulon ZhaDang Snow Pit glacier foreland Paddy Field Soil Arctic rhizosphere Arctic

Antarctica snow

Snowpack from the Tibetan Plateau Indian cold deserts high Arctic glacier Suraj Tal Lake Chandra Tal Lake

glacier foreland Friesland farmland soil Antarctica Sor Rondane Mountains

Qinghai-Tibet Plateau Origin 98 B1.7 (JN662533.1) Arthrobacter sp. c138 (AB167248.1) Arthrobacter sp. R-39621 (FR691392.1) Arthrobacter sp. RKS6-4 (GQ477171.1) Arthrobacter sp. Asd M3-2 (FM955860.1) Arthrobacter sp. V12 (JF313090.1) Arthrobacter sp. JSPB6 (JQ308619.1) Arthrobacter sp. JHBB9940 (KR085877.1) Arthrobacter sp. R-43110 (FR691390.1) Phycicocus sp. s23430 (D84624.2) Cryobacterium sp. hp36 (JN637331.1) Leifsonia sp. ZD5-4 (JKJ095109.1) Agresia sp. Enf63 (DQ33957.1) Microbacterium sp. R-36360 (FR682685.1) Microbacterium sp. IHBB 11136(KR085857.1) Microbacterium sp. 28 (KF923438.1) Deinococus sp. R-36590 (FR682759.1) Lysobacter sp. 3.2.11 (FR600120.1) Pseudoxanthomonas sp. C2603 (JX097007.1) Pseudomonas sp. IHBB 11130 (KR085942.1) Pseudomonas sp. PSAA4(4) (DQ628969.1) Sphingomonas sp. MSCB-3 (EF103200.1) Sphingomonas sp. Asd M4-14 (FM955867.1) Aurantimonas sp. R-36516 (FM682697.1) Rhizobium sp. MN6-12 (JQ396566.1) Bosea sp. GR060219 (EU448290.1)

Bradyrhizobium sp. S32010-a (AB64899.1)

Brevundimonas sp. TMT2-42-1 (JX950098.1) Brevundimonas sp. TP-snow-C31 (JHQ327140.1) Chryseobacterium sp. BBCT4 (DQ337556.1) Muciladinibacter sp. PAMC 26640 (JX847597.1) Pedobacter sp. Axs12 (JQ977410.1) Pedobacter sp. HRB3 (GQ294578.1) Pedobacter sp. S8-2 (GQ294578.1) Sphingomonas sp. TP-snow-C72 (KC987002.1) Janthinobacterium sp. IARI-R-50 (JX429043.1) A3.2 (JN662514.1) A1.2 (JN662510.1) B10.6 (JN662538.1) A4.3 (JN662516.1) A5.1 (JN662517.1) B10.3 (JN662537.1) B6.3 (JN662535.1) A2.2 (JN6625137.1) A6.3 (JN662519.1) A15.2 (JN662524.1) A9.2 (JN662520.1) A15.4 (JN662525.1) A12.2 (JN662522.1) A5.3 (JN662518.1) A23.3 (JN662530.1) A20.4 (JN662527.1) A1.4 (JN662512.1) A1.1 (JN662509.1) B1.2 (JN662532.1) B18.6 (JN662541.1) A21.1 (JN662528.1) A3.4 (JN662515.1) B16.14 (JN662539.1) A12.1 (JN662521.1) A27.4 (JN662531.1) A22.1 (JN662529.1) A1.3 (JN662511.1) A16.2 (JN662526.1) A12.4 (JN662523.1) B20.9 (JN662542.1) B17.2 (JN662540.1) B8.2 (JN662536.1) B5.3 (JN662534.1) B26.7 (JN66253.1) 57 44 49 98 65 96 51 57 57 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 99 54 51 98 94 94 100 100 67 47 100 100 100 100 96 99 99 99 96 89 99 98 62 51 52 Antarctica

Antarctica Sor Rondane Mountains alpine subnival plants Dongkemadi glacier forefield

Zhadang Glacier snow pit Antarctica

Roopkund Glacier in Himalayas Arctic glacier

Arctic permafrost

Suraj Tal Lake

Fig.2–Phylogeneticdendrogrambasedonacomparisonofthe16SrRNAgenesequencesofthebacterialisolatesfromthe TianshanNo.1glacierforelandandsomeoftheirclosestphylogeneticrelatives.Thenumbersonthetreeindicatethe percentagesofbootstrapsamplingderivedfrom1000replications.Theisolationsourcecolumnliststheenvironmentsfrom whichtheclosestphylogeneticrelativescome.

The 35 studied isolates belonged to the following 20 genera: Agreia,Arthrobacter,Aurantimonas, Bosea, Bradyrhizo-bium,Brevundimonas,Chryseobacterium,Cryobacterium, Deinococ-cus, Janthinobacterium, Leifsonia, Lysobacter, Microbacterium, Mucilaginibacter,Pedobacter,Phycicoccus,Pseudomonas, Pseudox-anthomonas, Rhizobium, and Sphingomonas. A total of eight isolatesbelongedtoArthrobacter,andthreeisolatesbelonged eachtoMicrobacterium,Pedobacter,andSphingomonas. Brevundi-monasandPseudomonaseachhadtwoisolates,andtheother

generawererepresentedbyoneisolateeach.Asrevealedby thephylogenetictreeconstruction,thesebacteriawereclosely relatedtoothercold-environmentbacteria(Fig.2).

Actinobacteriawasthe dominanttaxonforthesamples incubated at 4◦C, and the abundance remained constant along the chronosequence, whereas the abundance of Alphaproteobacteria and Bacteroides were relatively lower than Actinobacteria. The Deinococcus-Thermus group was only found in the 100a soil, and Betaproteobacteria and

100 80 60 40 20 0 100 80 60 40 20 0

10a 25a 60a

Years since glacier retreat

Years since glacier retreat

74a 100a 130a

10a 25a 60a 74a 100a 130a

Relativ e a vundance ph ylum (%) Relativ e a vundance ph ylum (%)

a

b

Fig.3–Variationsofthetaxaabundancesoftheculturablebacteriainsoilsamplesalongthechronosequences((a) incubatedat4◦C,(b)incubatedat25◦C).

Gammaproteobacteriawerenotfoundinallofthesamples (Fig.3a).However,thespeciesrichness at25◦C washigher thanat4◦C,includingActinobacteria,Bacteroides,and Alpha-, Beta- and Gammaproteobacteria.The Actinobacteria and Proteobacteriawerethedominanttaxainthesoilscultured at25◦C,withtheabundanceofProteobacteria significantly decreasingalongthechronosequence(p=0.02)andthe abun-dance of Actinobacteria significantly increasing along the chronosequence(p=0.01).TheabundanceofBacteroideswas relativelystableinallofthesoilsamples.The Betaproteobac-teriawereonlyfoundintheoldestsoils,andtheabundance wasverylowcomparedtotheothertaxa(Fig.3b).

Thecorrelationsbetweentheabundanceofsoilculturable bacteriaandsoilbiochemicalparameters

The total number of culturable bacteria in the soils cul-tured at25◦C were significantly positively correlated with the soiltotalN(p<0.05),organic C (p<0.01), and soil cata-lase (p<0.01), dehydrogenase (p<0.05), polyphenoloxidase (p<0.01), protease (p<0.05), sucrase (p<0.05), and urease (p<0.05) activities. The soilpH value negatively correlated withthenumberofculturablebacteriabutwasnotsignificant. Thisresultindicatedthatthebacterialabundanceincreased with increasing soil N and organic C and increasing soil

enzymeactivitiesandwithdecreasingsoilpHvaluesalong thesoilagegradient(Table3).RDAaxes1and2werefoundto explain91.5%and7.2%,respectively,oftheoverallvariance, withthebacterialabundanceanddiversitydatacorrelating withtheenvironmentaldata.Thenumberofculturable bac-teriahadapositiverelationshipwiththesoilenzymeactivity, whichwasingoodagreementwiththeSPSScorrelation anal-ysis.Inaddition,thebacterialcommunitiesofthe 10asoils and100–130asoilswereseparatedfrom25–74a,indicatingthat thebacterialsuccessionintheTianshanNo.1glacierforeland shouldbedividedintothreestages:anearlystage(10a),a mid-dlestage(25–74a)andalatestage(100–130a)(Fig.4).Thisresult isconsistentwiththeclusteranalysisresults(Fig.5).

Discussion

ThesoilorganicC,totalNand enzymeactivitiesincreased along the exposure time gradient, while the soil pH decreased in the foreland of Tianshan No. 1 glacier. Sim-ilar results were reported at Dongkemadi glacier and Damma glacier.32,33 _{The total number of culturable}

bac-teria recovered from the Tianshan No. 1 glacier foreland was higher than that of the permafrost in the same area (2.5–6.0×105_CFUg−1₎13_{butsimilartothenumberrecovered}

Table3–Thecorrelationsbetweenthenumbersofculturablebacteriaat25◦Candthesoilbiochemicalparametersalong

thechronosequences.

pH TotalN OrganicC Protease Polyphenoloxidase Catalase Urease Dehydrogenase Sucrase

25C −0.769 0.889b _0.947a _0.820b _0.972a _0.956a _0.914b _0.900b _0.895b pH −0.571 −0.620 −0.864b _{−0.794 −0.579 −0.746 −0.646}b _−0.537 TotalN 0.983a _0.845b _0.901b _0.907b _{0.802 0.754 0.842}b OrganicC 0.834b _0.933a _0.967a _0.869b _0.845b _0.917b Protease 0.859b _{0.724 0.779 0.688 0.681} Polyphenoloxidase 0.900b _0.937a _{0.776 0.845}b Catalase 0.884b _0.913b _0.969b Urease 0.734 0.914b Dehydrogenase 0.861b

25Cdenotesthenumberofculturablebacteriaat25◦C.

a _{Thecorrelationissignificantatthe0.01level.} b _{Thecorrelationissignificantat0.05(2-tailed).}

100a PR PO CFU N UR C DE 74a _10a 60a pH 130a SU CA Actin Bact 25a prot -1.0 -1.0 1.0 1.0

Fig.4–RDAbiplotofthecorrelationbetweenthetaxa abundanceofbacteriaculturedat25◦Candsoil

biochemicalparameters.Thetaxaabundanceofbacteria culturedat25◦C(seeFig.3b);soilbiochemicalparameters (seeTable2).Dashlinearrowsindicatetheenvironmental variables(N,TN(%);C,OC(%);UR,urease;DE,

dehydrogenase;SU,sucrase;CA,catalase;PO,polyphenol oxidase,PR,protease).Solidlinearrowsindicatethe abundanceofbacteria(Bact,bacteroides(%);Prot, proteobacteria(%);Actin,actinobacteria(%)).

Rescaled distance CA S E Label Num 0 100a 130a 25a 60a 74a 10a 5 10 15 20 25

Fig.5–Hierarchicalclusteranalysisofculturablebacterial abundanceandcommunitiesinsoilsofdifferentages.

fromtheDongkemadiglacierforelandintheCentralTanggula Mountains(1.29×105_–2.54×106_CFUg−1_).32_{ThesoilorganicC}

andtotalNhavepositiverelationshipswiththesoilenzyme

activities and microbial abundance in the Tianshan No. 1 glacier foreland. Zumsteg et al.33 _{had findings similar to}

ours,reportingthatthemicrobialcommunitystructureand enzymatic activitypatterns arestronglyconditionedbythe successional stage in addition tothe C and Ncontents of theglacierforelandsoils.Furthermore,theincubation tem-peratures alsoaffected theabundance anddiversity ofthe bacteria.ThisfindingisinaccordancewiththeLapanjeetal.25

studyattheDammaglacierandLiuetal.32_{attheDongkemadi}

glacier.Thereasonforthedifferencemaybethat,although theywerenotthedominantbacteriaattheearlyage(10a), psychrotolerant bacteriabecome dominantwithincreasing soilage.34

Thebacterialtaxaanddominantphylaisolatedfromthe TianshanNo.1glacierforelandweresimilartothe Qinghai-Tibet PlateauJadang glacierforeland,35 _{Dongkemadiglacier}

foreland in the Central Tanggula Mountains,32 _Himalayan

Pindariglacier foreland,36 _{high Articpermafrost soil,}37 _and

Antarcticpermafrost soil.38 _{Thisresultindicatedthat}

simi-larcoldenvironmentsaccommodatesimilarmicrobes,which couldbeevidencetosupportthe“environmentselect”theory. SomeofthebacterialisolatesfromtheTianshanNo.1glacier forelandmayoriginatefromtheglacierandglaciersediment, whileothersmayderivefromtheatmosphericdustfromother environments.39_{ThebacterialisolatesfromTianshanNo.1}

glacier foreland belong to sixphyla. Bai et al.13 _examined

thephylogeneticdiversityofbacteriafrompermafrostinthe TianshanMountainsusingaculture-dependentmethodand found 4 phyla: Actinobacteria, Bacteroides, Firmicutes and Proteobacteria. Yang et al.14 _{studiedthe permafrost}

bacte-rial communitystructuresanddiversityusingadenaturing gradientgelelectrophoresismethodandfound7phylaof bac-teria, including the Acidobacteria,Gemmatimonadetes and ChloroflexiphylawhichwerenotfoundbyBaietal.13

Com-paredwiththepreviousreportsinthisstudyarea,thepresent study isthe firstto recoverbacterialstrains fromthe phy-lum Deinococcus-Thermus. Deinococcus-Thermus consists mainlyofthermophiles,40_{andfindingDeinococcus-Thermus}

inthiscoldandhigherUVexposureenvironmentis interest-ingand shouldbefurtherstudiedforitscold-adaptionand UV-resistancemechanisms.

The dominate actinobacteria isolates in this study belonged to the genus Arthrobacter, which was the most

highlydiversegroupamongourisolatesandaretypicallythe predominantgenus incold environments.19,25,32,37,38,41

Pro-teobacteriaareafavoredtaxoninthisinitialecosystemwith alownutrientcontent,becausemanyofthemhavearange ofmetabolism.42_{Someofthegenerainourstudyhavebeen}

reportedbyotherstohaveparticularweatheringcapabilities, suchasSphingomonassp.,43_{Pseudomonassp.}44_and Janthinobac-teriumsp.25 _{Thesegenerashouldbefurtherinvestigatedfor}

thesecapabilities.Studiesshowapositivecorrelationbetween Proteobacteriaand thesoilpH; incontrast,anegative rela-tionshipbetweenActinobacteriaandsoilpHwasreportedby Philippotetal.45

Apreviousstudyfoundthatbacterialnumbersdepended mostlyonthesoilageafterdeglaciationintheDammaglacier foreland46_{andintheLymanglacierforeland.}47_Liuetal.32_also

foundsimilarresults.Theseresultssuggestthatthenumber andactivityofmicrobialpopulationsincreasewithsoil devel-opment after glacial retreat.46 _{Other studies also reported}

thatmicrobialabundancehassignificantcorrelationswithsoil enzymeactivitiesinthemountainvalleywetland,48_reclaimed

soilonasurfacecoalmine,49_{andpaddysoil.}50_TheRDAand

hierarchical cluster analysis showed the same result, that thebacterialsuccessionintheTianshanNo.1glaciercanbe dividedintothreesuccessionalperiods:anearlystage(10a), amiddlestage(25–74a),andalatestage(100–130a).Similar resultswerereportedbyHuangetal.51_{forcoppermine}

tail-ings.Theseresultsindicatethatsoilmicrobesplayarolein thedevelopmentofthebaresoilintheglacierforeland, sug-gestingthatmicrobialsuccessioncorrelateswiththesoilage alongtheforeland.

Conflicts

of

interest

Theauthorsdeclarenoconflictsofinterest.

Acknowledgements

ThisworkwasfundedbytheNaturalScienceFoundationof China(Nos.31500429,31170465),theNationalBasicResearch Program(973)ofChina(No.2012CB026105),the China Post-doctoralScience Fund(2014M562477), and the Scienceand TechnologyProjectsinGansuProvince(1506RJZA291)China.

r

e

f

e

r

e

n

c

e

s

1. IPCC.ClimateChange2007:SynthesisReport.Contributionof

WorkingGroupsI,IIandIIItotheFourthAssessmentReportofthe IntergovernmentalPanelonClimateChange.CoreWritingTeam, PachauriRK,ReisingerA.Geneva:IPCC;2007.

2. SchütteUME,AbdoZ,FosterJ,etal.Bacterialdiversityina

glacierforelandofthehighArctic.MolEcol.2010;19:54–66.

3. BauerH,Kasper-GieblA,LöflundM,etal.Thecontributionof

bacteriaandfungalsporestotheorganiccarboncontentof

cloudwater,precipitationandaerosols.AtmRe.

2002;64:109–119.

4. SiglerWV,CriviiS,ZeyerJ.Bacterialsuccessioninglacial

forefieldsoilscharacterizedbycommunitystructure,activity

andopportunisticgrowthdynamics.MicrobEcol.

2002;44:306–316.

5.NemergutDR,AndersonSP,ClevelandCC,etal.Microbial

communitysuccessioninanunvegetated,recently

deglaciatedsoil.MicrobEcol.2007;53:110–122.

6.LeeX,QinD,JiangG,DuanK,ZhouH.Atmosphericpollution

ofaremoteareaofTianshanMountain:icecorerecord.J

GeophysRes.2003;108:4406.

7.WilliamsMW,TonnessenKA,MelackJM,DaqingY.Sources

andspatialvariationofthechemicalcompositionofsnowin

theTienShan,China.ChinaAnnGlaciol.1992;16:25–32.

8.BolchT.ClimatechangeandglacierretreatinnorthernTien

Shan(Kazakhstan/Kyrgyzstan)usingremotesensingdata.

GlobPlanetChange.2007;56:1–12.

9.LiB,ZhuA,YiC.Glacierchangeoverthepastfourdecades

inthemiddleChineseTienShan.JGlaciol.2006;52:425–432.

10.NaramaC,KääbA,DuishonakunovM,AbdrakhmatovK.

SpatialvariabilityofrecentglacierareachangesintheTien

ShanMountains,CentralAsia,usingCorona(1970),Landsat

(2000),andALOS(2007)satellitedata.GlobPlanetChange.

2010;71:42–54.

11.WuX,ZhangW,LiuG,etal.Bacterialdiversityinthe

forelandoftheTianshanNo.1glacier,China.EnvironResLett.

2012;7:014038.

12.ZhangW,ZhangG,LiuG,LiZ,ChenT,AnL.Diversityof

bacterialcommunitiesinthesnowcoveratTianshan

Number1Glacieranditsrelationtoclimateand

environment.GeomicrobiolJ.2012;29:459–469.

13.BaiY,YangD,WangJ,XuS,WangX,AnL.Phylogenetic

diversityofculturablebacteriafromalpinepermafrostinthe

TianshanMountains,northwesternChina.ResMicrobiol.

2006;157:741–751.

14.YangD,WangJ,BaiY,XuS,AnL.Diversityanddistribution

oftheprokaryoticcommunityinnear-surfacepermafrost

sedimentsintheTianshanMountains,China.CanJMicrobiol.

2008;54:270–280.

15.ShengH,GaoH,XueL,etal.Analysisofthecompositionand

characteristicsofculturableendophyticbacteriawithin

subnivalplantsoftheTianshanMountains,Northwestern

China.CurrMicrobiol.2011;62:923–932.

16.WangX,ZhangT,SunJ,ZhangX,LiZ,LouK.Ecological

characterizationofsoilmicroflorainprimarysuccession

acrossglacierforefield:acasestudyofGlacierNo.1atthe

HeadwatersofUrumqiRiver.ActaEcolSin.2010;30:6563–6570

(inChinesewithEnglishabstract).

17.WangS,ZhangM,LiZ,etal.Glacierareavariationand

climatechangeintheChineseTianshanMountainssince

1960.JGeogrSci.2011;21:263–273.

18.ChenJ.Preliminaryresearchesonlichenometricchronology

ofHoloceneglacialfluctuationsandonothertopicsinthe

headwaterofUrumqiRiver,TianshanMountains.SciChina

SerB.1989;32:1487–1500.

19.ZhangG,MaX,NiuF,etal.Diversityanddistributionof

alkaliphilicpsychrotolerantbacteriaintheQinghai–Tibet

Plateaupermafrostregion.Extremophiles.2007;11:415–424.

20.TaylorJP,WilsonB,MillsMS,BurnsRG.Comparisonof

microbialnumbersandenzymaticactivitiesinsurfacesoils

andsubsoilsusingvarioustechniques.SoilBiolBiochem.

2002;34:387–401.

21.Garcıa-GilJC,PlazaC,Soler-RoviraP,PoloA.Long-term

effectsofmunicipalsolidwastecompostapplicationonsoil

enzymeactivitiesandmicrobialbiomass.SoilBiolBiochem.

2000;32:1907–1913.

22.NannipieriP,PedrazziniF,ArcaraPG,PiovanelliC.Changes

inaminoacids,enzymeactivities,andbiomassesduringsoil

microbialgrowth.SoilSci.1979;127:26–34.

23.SchinnerF,vonMersiW.Xylanase-,CM-cellulase-and

invertaseactivityinsoil:animprovedmethod.SoilBiol

Biochem.1990;22:511–515.

24.Saiya-CorkKR,SinsabaughRL,ZakDR.Theeffectsoflong

anAcersaccharumforestsoil.SoilBiolBiochem.

2002;34:1309–1315.

25.LapanjeA,WimmersbergerC,FurrerG,BrunnerI,FreyB.

Patternofelementalreleaseduringthegranitedissolution

canbechangedbyaerobicheterotrophicbacterialstrains

isolatedfromDammaGlacier(centralAlps)deglaciated

granitesand.MicrobEcol.2012;63:865–882.

26.ThompsonJD,GibsonTJ,PlewniakF,JeanmouginF,Higgins

DG.TheCLUSTALXwindowsinterface:flexiblestrategiesfor

multiplesequencealignmentaidedbyqualityanalysistools.

NucleicAcidsRes.1997;25:4876–4882.

27.SaitouN,NeiM.Theneighbor-joiningmethod:anew

methodforreconstructingphylogenetictrees.MolBiolEvol.

1987;4:406–425.

28.TamuraK,DudleyJ,NeiM,KumarS.MEGA4:molecular

evolutionarygeneticsanalysis(MEGA)softwareversion4.0.

MolBiolEvol.2007;24:1596–1599.

29.BraakCJFt,SmilauerP.CANOCOReferenceManualand CanoDrawforWindowsUser’sGuide:SoftwareforCanonical CommunityOrdination(Version4.5);2002.www.canoco.

com.

30.SinsabaughRL,HillBH,ShahJJF.Ecoenzymatic

stoichiometryofmicrobialorganicnutrientacquisitionin

soilandsediment.Nature.2009;462:795–798.

31.CiarkowskaK,Sołek-PodwikaK,WieczorekJ.Enzyme

activityasanindicatorofsoil-rehabilitationprocessesata

zincandleadoreminingandprocessingarea.JEnviron

Manag.2014;132:250–256.

32.LiuG,HuP,ZhangW,etal.Variationsinsoilculturable

bacteriacommunitiesandbiochemicalcharacteristicsinthe

Dongkemadiglacierforefieldalongachronosequence.Folia

Microbiol.2012;57:485–494.

33.ZumstegA,LusterJ,GöranssonH,etal.Bacterial,archaeal

andfungalsuccessionintheforefieldofarecedingglacier.

MicrobEcol.2012;63:552–564.

34.ZhangG,ZhangW,LiuG,AnL,ChenT,LiZ.Distributionof

aerobicheterotrophicbacteriamanagedbyenvironmental

factorsinglacierforeland.JGlaciolGeocryol.2012;34:965–971

(inChinesewithEnglishabstract).

35.YueJ,LiuG,ZhangG,ZhangW,XuS.Changesinsoil

propertiesandculturablebacteriadiversityinZhadang

glacierforeland.JGlaciolGeocryol.2010;32:1180–1185(in

ChinesewithEnglishabstract).

36.ShivajiS,PratibhaMS,SailajaB,etal.Bacterialdiversityof

soilinthevicinityofPindariglacier,Himalayanmountain

ranges,India,usingculturablebacteriaandsoil16SrRNA

geneclones.Extremophiles.2011;15:1–22.

37.HansenAA,HerbertRA,MikkelsenK,etal.Viability,

diversityandcompositionofthebacterialcommunityina

highArcticpermafrostsoilfromSpitsbergen,Northern

Norway.EnvironMicrobiol.2007;9:2870–2884.

38.AislabieJM,ChhourK-L,SaulDJ,etal.Dominantbacteriain

soilsofMarblePointandWrightValley,VictoriaLand,

Antarctica.SoilBiolBiochem.2006;38:3041–3056.

39.HodsonA,AnesioAM,TranterM,etal.Glacialecosystems.

EcolMonogr.2008;78:41–67.

40.GriffithsE,GuptaRS.Identificationofsignatureproteinsthat

aredistinctiveoftheDeinococcus-Thermusphylum.Int

Microbiol.2007;10:201–208.

41.Hinsa-LeasureSM,BhavarajuL,RodriguesJLM,Bakermans

C,GilichinskyDA,TiedjeJM.Characterizationofabacterial

communityfromaNortheastSiberianseacoastpermafrost

sample.FEMSMicrobiolEcol.2010;74:103–113.

42.HodkinsonID,WebbNR,CoulsonSJ.Primarycommunity

assemblyonland–themissingstages:whyarethe

heterotrophicorganismsalwaystherefirst?JEcol.

2002;90:569–577.

43.UrozS,CalvarusoC,TurpaultMP,PierratJC,MustinC,

Frey-KlettP.Effectofthemycorrhizosphereonthegenotypic

andmetabolicdiversityofthebacterialcommunities

involvedinmineralweatheringinaforestsoil.ApplEnviron

Microbiol.2007;73:3019–3027.

44.GorbushinaAA.Lifeontherocks.EnvironMicrobiol.

2007;9:1613–1631.

45.PhilippotL,TscherkoD,BruD,KandelerE.Distributionof

highbacterialtaxaacrossthechronosequenceoftwoalpine

glacierforelands.MicrobEcol.2011;61:303–312.

46.SiglerWV,ZeyerJ.Microbialdiversityandactivityalongthe

forefieldsoftworecedingglaciers.MicrobEcol.

2002;43:397–407.

47.OhtonenR,FritzeH,PennanenT,JumpponenA,TrappeJ.

Ecosystempropertiesandmicrobialcommunitychangesin

primarysuccessiononaglacierforefront.Oecologia.

1999;119:239–246.

48.BaiJ,XuH,XiaoR,GaoH,ZhangK,DingQ.Pathanalysisfor

soilureaseactivitiesandnutrientcontentsinaMountain

ValleyWetland,China.CleanSoilAirWater.2014;42:324–330.

49.LiJ,ZhouX,YanJ,LiH,HeJ.Effectsofregenerating

vegetationonsoilenzymeactivityandmicrobialstructurein

reclaimedsoilsonasurfacecoalminesite.ApplSoilEcol.

2015;87:56–62.

50.SuJ,DingL,XueK,etal.Long-termbalancedfertilization

increasesthesoilmicrobialfunctionaldiversityina

phosphorus-limitedpaddysoil.MolEcol.2015;24:136–150.

51.HuangL,TangF,SongY,etal.Biodiversity,abundance,and

activityofnitrogen-fixingbacteriaduringprimary

successiononacopperminetailings.FEMSMicrobiolEcol.