Response of methanogenic archaea to Late Pleistocene and Holocene climate changes in the ...

14 Pages·2013·1.57 MB·English

Originally published as: 

Bischoff, J., Mangelsdorf, K., Gattinger, A., Schloter, M., Kurchatova, A. N., Herzschuh, U., Wagner, D. 
(2013): Response of methanogenic archaea to late pleistocene and holocene climate changes in the 
siberian arctic. ‐ Global Biogeochemical Cycles, 27, 2, 305‐317  
DOI: 10.1029/2011GB004238
GLOBALBIOGEOCHEMICALCYCLES,VOL.27,305–317,doi:10.1029/2011GB004238,2013
Response of methanogenic archaea to Late Pleistocene
and Holocene climate changes in the Siberian Arctic
Juliane Bischoff,1,2,5 Kai Mangelsdorf,2 Andreas Gattinger,3,6 Michael Schloter,3
Anna N. Kurchatova,4 Ulrike Herzschuh,1 and Dirk Wagner1,7
Received19October2011;revised10October2012;accepted6November2012;published8April2013.
[1] In order to investigate the link between the methane dynamics in permafrost deposits
and climate changes in the past, we studied the abundance, composition, and methane
production of methanogenic communities in Late Pleistocene and Holocene sediments of
the Siberian Arctic. We detected intervals of increased methane concentrations in Late
Pleistocene and Holocene deposits along a 42ka old permafrost sequence from
KurungnakhIslandintheLenaDelta(northeastSiberia).Increasedamountsofarchaeallife
markers (intact phospholipid ethers) and a high variety in genetic fingerprints detected by
16S ribosomal ribonucleic acid gene analyses of methanogenic archaea suggest presently
living and presumably active methanogenic archaea in distinct layers predominantly in
Holocene deposits, but also in deep frozen ground at 17m depth. Potential methanogenic
activitywasconfirmed byincubation experiments. Bycomparing methaneconcentrations,
microbial incubation experiments, gene analysis of methanogens, and microbial life
markers(intactphospholipidestersandethers)toalreadypartlydegradedmembranelipids,
suchasarchaeolandisoprenoidglyceroldialkylglyceroltetraethers,wedemonstratedthat
archaeol likely represents a signal of past methanogenic archaea. The archaeol signal was
usedtoreconstructtheresponseofmethanogeniccommunitiestopasttemperaturechanges
in the Siberian Arctic, and the data suggest higher methane emissions occurred during
warm periods, particularly during an interval in the Late Pleistocene and during the
Holocene. This new data on present and past methanogenic communities in the Siberian
terrestrial permafrost imply that these microorganisms will respond to the predicted future
temperature rise in the Arctic with increasing methane production, as demonstrated in
previous warmer periods.
Citation: Bischoff,J.,K.Mangelsdorf,A.Gattinger,M.Schloter,A.N.Kurchatova,U.Herzschuh,andD.Wagner
(2013),ResponseofmethanogenicarchaeatoLatePleistoceneandHoloceneclimatechangesintheSiberianArctic,
GlobalBiochem.Cycles,27,305–317,doi:10.1029/2011GB004238.
1. Introduction pronouncedforthepolarregionsoftheNorthernHemisphere
(NH); both long-term measurements and future climate
[2] In recent years, the debate about climate change has models indicate a significant increase in atmospheric and
increasingly focused on Arctic regions. The predicted
ground temperature for this particular environment [ACIA,
temperatureriseassociatedwithclimatechangeisespecially 2005;IPCC,2007].Permafrost,definedas groundremain-
ing below 0(cid:1)C for 2 consecutive years [Washburn, 1980
and references therein], is widely distributed in the Arctic
1ResearchDepartmentPotsdam,AlfredWegenerInstituteforPolarand and covers about 25% of the exposed land surface of the
MarineResearch,Potsdam,Germany.
NH [Zhang et al., 2008]. Recently, a deepening of the
2Helmholtz Center Potsdam, GFZ German Research Center for
seasonally thawed layer (active layer) and an increase in
Geosciences,Section4.3OrganicGeochemistry,Potsdam,Germany.
3Helmholtz Zentrum München, Research Unit for Environmental ground temperatures of the permafrost were reported for
Genomics,Oberschleissheim,Germany. several sites in the Russian [Romanovsky et al., 2010] and
4TyumenStateOilandGasUniversity,Tyumen,Russia. North American Arctic [Smith et al., 2010]. In general,
5Now at Newcastle University, School of Civil Engineering and
increased permafrost thawing is assumed to be associated
Geosciences,NewcastleUponTyne,UnitedKingdom.
6Research Institute of Organic Agriculture (FiBL), Soil Sciences with enhanced carbon emission [Dutta et al., 2006] as
Division,Frick,Switzerland. carbon dioxide and methane, which are suspected to have
7NowatHelmholtzCenterPotsdam,GFZGermanResearchCenterfor a positive feedback on climate warming. Future modeling
Geosciences,Section4.5Geomicrobiology,Potsdam,Germany.
scenarios suggest that permafrost environments might
Corresponding author: J. Bischoff, Newcastle University, Newcastle change from a carbon sink to a carbon source [Oechel
UponTyne,NE17RU,UnitedKingdom.([email protected]) et al., 1993]. Therefore, Arctic regions are of enormous
importance for the global terrestrial carbon cycle [Zimov
©2012.AmericanGeophysicalUnion.AllRightsReserved.
0886-6236/13/2011GB004238 etal.,2006;Tarnocaietal.,2009].
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BISCHOFFETAL.:METHANOGENICRESPONSETOCLIMATECHANGES
[3] Methane isa significant trace gas that contributes with andHolocenedepositsfromtheLenaRiverDeltainSiberia,
~20% to the total greenhouse gases impacting on global Russia.Theaimofthisstudywastoanalyzetheabundance
warming[Denmanetal.,2007].Presently,methaneemission and composition of methanogenic communities in perma-
estimatesfromnorthern,highlatitude,terrestrialenvironments frostsedimentsdepositedunderdifferentclimaticconditions
rangefrom10to51Tga–1(1012g/yr),whichaccountsforup andtoreconstructtheimpactofpastclimatechangesonthe
to8.6%oftheglobal,naturalmethaneemissions[Bartlettand methane production.
Harriss,1993;Caoetal.,1998].Withthethawingofperma-
frost, large amounts of carbon stored in permafrost succes- 2. Materials and Methods
sionsmightbecomeavailableagainformicrobialdegradation.
2.1. Study Site
Therefore,thiscarbonisapotentialsourceofcarbondioxide
and methane release to the atmosphere due to intensified [7] The Lena RiverDelta (Figure 1a) isthe largest Arctic
microbialrespirationofrecentandoldercarbonpools. delta, with an area of about 29(cid:3)103km2 [Schneider et al.,
[4] Microbially mediated methane cycling includes pro- 2009].TheentireLenaDeltaislocatedinthezoneofcontin-
cesses ofmethaneproduction(methanogenesis)andmethane uouspermafrost,wherethepermafrostreachesathicknessof
oxidation (methanotrophy); these processes are spatially about500–600m[Romanovskii andHubberten,2001]. The
separated.Methanogenesisisdrivenbymethanogenicarchaea fieldwork for this study was conducted on Kurungnakh
(methanogens), a strictly anaerobic group belonging to the Island, which is located in the southern part of the Lena
kingdom Euryarchaeota [Hedderich and Whitman, 2006]. RiverDeltaclosetotheOlenyekskayaChannel(Figure1b).
Subsequently, methane is released to the atmosphere by KurungnakhIslandiscomposedofLateQuaternarysediments
ebullition, plant-mediatedtransportbyaerenchyma, or diffu- belonging to the oldest Lena River terrace, and is partly
sionthroughtheactivelayerofthepermafrost.Whenmethane exposedupto40mabovetheriverlevel.Thisso-called“third
diffusesthroughnear-surfaceaerobiclayers,itcanbeoxidized terrace”wasformedduringtheLatePleistoceneandischarac-
by aerobic methanotrophic Proteobacteria [Hanson and terizedbyitslowestformationofsandydepositscoveredbya
Hanson,1996]. hugecharacteristic“ice complex”(IC)formationabout20m
[5] ThefirststudiesofHolocenepermafrostdepositsfrom thick [Schwamborn et al., 2002]. The IC is characterized by
the Lena Delta (Siberia) by Wagner et al. [2007] revealed ice-rich, silty deposits with peat block inclusions of less
significant amounts of methane, which could be attributed decomposed material (Yedoma suite). Furthermore, on top
toinsituactivityofmethanogenicarchaea.Theactivelayer oftheIC,Holocenelayersabout2–3mthickandcontaining
of permafrost containsthe highest cell counts (up to 3(cid:3)108 icewedges3–5mwidearewidelydistributed[Schirrmeister
cellsg–1)ofmethanogenicarchaea[Kobabeetal.,2004],with etal.,2003].Thedepthoftheactivelayermeasuredatthedrill
ahighdiversityofspeciesbelongingtothefamiliesMethano- siteinJuly2002was32cm.
microbiaceae, Methanosarcinaceae, and Methanosaetaceae [8] DuringtheLatePleistoceneandHolocene,Kurungnakh
[Ganzertetal.,2007].AstudyontheLaptevSeaShelfsubma- Island had undergone several climatic stages. This was indi-
rinepermafrost[Kochetal.,2009]demonstratedthatatdepths catedbythepaleoclimaticand-environmentalreconstruction
wheremethane concentrations wereatmaximum,the lowest presented in Wetterich et al. [2008] for this site. This recon-
carbon isotopic values for methane (up to (cid:4)72.2% V-PDB) structionwasbasedonastratigraphicanalysisusingacombi-
were measured. This indicated active methanogenesis in the nation of several fossil bioindicators, such as pollen, plant
frozensediments.Sampleswithhighmethaneconcentrations macrofossils,ostracodes,insectremains,andmammalbones,
were dominated by sequences affiliated with the methylo- aswellasgeochronologybasedonisochronuranium-thorium
trophicgenera,MethanosarcinaandMethanococcoides.Koch disequilibriatechniqueand14Cradiocarbondating.Wetterich
et al. [2009] also demonstrated that intact deoxyribonucleic et al. [2008] defined six climatic stages for the last 100ka
acid (DNA) was extractable from Late Pleistocene perma- BP. Following this classification, our Kurungnakh perma-
frostdepositsupto111kaold.OnEllesmereIsland,located frost core was sectioned into five units (Figure 2). Late
in the Canadian Arctic Archipelago, Steven et al. [2007] Pleistocene deposits were divided into a cold and dry Unit
reported an archaeal community composed of 61% II (2400–2476cm; Early Weichselian Stadial, Zyryan), a
Euryarchaeota and 39% Crenarchaeota, displaying a warm and wet Unit III (1400–2400cm; Middle Weichselian
diversearchaealcommunityintheperenniallyfrozen sedi- Interstadial, Kargin), and a cold and dry Unit IVa (300–
ments. Recently, several studies used lipid biomarkers to 1400cm; Late Weichselian Stadial, Sartan). Holocene
detectandquantifymethanogenicarchaeainanAntarcticlake deposits were sectioned into a warm and dry Unit IVb
[Coolen et al., 2004], permafrost soils [Wagner et al., 2005, (100–300cm; Early Holocene) and a warm and wet Unit
2007], and peatlands [Weijers et al., 2004; Pancost et al., V(0–100cm;MiddleandLateHolocene).
2011]. Microbial lipid markers indicating living microorgan-
isms (phospholipids) and low numbers of methanogens were 2.2. Permafrost Drilling and Sample Preparation
found at more than 1000m depth in the Mallik gas hydrate [9] A23mlongpermafrostcorewasdrilledinthedepression
production research well, drilled into the Canadian terrestrial of a low-centered ice-wedge polygon during the Russian-
permafrostatthenorthernedgeoftheMackenzieRiverDelta GermanLENA2002expeditioninJuly2002onKurungnakh
[Colwelletal.,2005;Mangelsdorfetal.,2005].However,lipid Island(72(cid:1)20’N,126(cid:1)17’E;Figure1c)[Grigorievetal.,2003].
biomarkerstudiesintheSiberianArcticonmethanogeniccom- The drilling was carried out with a portable gasoline-powered
munitiesinolderpermafrostdepositsarecurrentlymissing. permafrost corer. Drilling fluids were not used to avoid
[6] Here we present a combined stratigraphic analysis contaminating the permafrost samples with surface micro-
usingmicrobiallipidmarkersandribosomalribonucleicacid organisms, as described previously [Wagner et al., 2007].
(rRNA)genesonapermafrostsequenceofLatePleistocene Theindividualcore segments,upto50cmlong,werestored
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BISCHOFFETAL.:METHANOGENICRESPONSETOCLIMATECHANGES
Figure1. LocationoftheLenaRiverDeltaintheRussianArctic(a).KurungnakhIslandstudysiteinthe
LenaRiverDelta(b),andlocationofthedrillsite,markedwithsymbol(c).(ESRIdata,LandsatETM+).
at about –8(cid:1)C in the permafrost cellar of the Samoylov of each subsample was thawed at 4(cid:1)C and homogenized
ResearchStationandweretransportedunderfrozenconditions under anoxic and sterile conditions for analysis of the sedi-
toPotsdam,Germany(storagetemperatureinPotsdam–22(cid:1)C). ment properties, microbial activity, and lipid biomarkers.
[10] Core segments were split along their axes into two Thesecondhalfofthecoreiskeptasanarchiveintheicecore
halves under aseptic conditions with a diamond saw in an repository at the Alfred Wegener Institute for Polar and
icelaboratoryat–10(cid:1)C.Afterwards,thesurfaceofonehalf MarineResearchinBremerhaven(Bremerhaven,Germany).
of the core was cleaned with a sterile knife for lithological [11] Overall, 73 samples were analyzed for their sediment
and geocryological descriptions. Subsequently, this half was properties (elemental analyses of carbon, grain size, and
cut into segments about 10–30cm long according to the methane concentration). According to the depth and the
lithology and the geocryology. Small pieces (approximately physicochemicalcharacterization(low/hightotalorganiccar-
5g)ofthesesubsampleswereusedforanalyzingthemethane bon (TOC), low/high methane concentration), we selected
concentrationinthefrozensediments.Theremainingmaterial 31samplesformolecularandbiomarkerstudies.
307
BISCHOFFETAL.:METHANOGENICRESPONSETOCLIMATECHANGES
Figure2.AbioticandbioticparametersofthepermafrostdepositsfromKurungnakhIsland,LenaDelta(SiberianArctic),presentedwithrespecttosedimentdepth(leftaxis),sedimentageincalendaryearsBP(rightaxis),andthefiunderliningpaleoclimatereconstructionprovidedbyWetterichetal.[2008].Theverticalproleshows:a.grainsizedis-–1tributioninwt%ofclay(black),silt(grey),andsand(white);b.TOCcontentinwt%;c.methanecontentinnmolCHg4–––m111sediment;d.abundanceofPLFAsinnmolgsedimentdwandggsedimentdw;e.abundanceofPLELsinnmolg–m1sedimentdwandggsedimentdw;f.potentialmethanogenicactivitywithoutsubstrate(dots)andafteradditionof––11hydrogen(triangles);g.abundanceofarchaeolinnggsedimentdw;h.totalisoprenoidGDGTsinnggsedimentfidw;andi.DGGEanalysisprolewithdepthof16SRNAgenesofmethanogenicarchaea(sequencedbandsmarkedwithblackdotswereusedforphylogenticanalysis,bandsmarkedwithopencircleswereexcludedduetosequencequalityorlength),respectivesampleIDofdepthandconsecutivebandnumber.
308
BISCHOFFETAL.:METHANOGENICRESPONSETOCLIMATECHANGES
2.3. Sediment Properties 2.5. Lipid Biomarker Analysis
[12] TheTOCandtotalcarboncontentsofthefreeze-dried [18] Aliquotsofthefreeze-driedandgroundsampleswere
and homogenized permafrost samples were determined as extracted using a flow blending system with a mixture of
described previously in Wagneret al.[2007]. methanol/dichloromethane/ammonium acetate buffer (pH
[13] Pyrolysis of 10 selected freeze-dried homogenized 7.6, 2:1:0.8, v/v), modified after Bligh and Dyer [1959].
sampleswas conductedbyAPT AS(Kjeller,Norway)using Forcompoundquantification,internalphospholipidstandards
aRock-Eval6instrumenttoobtaininformationonthecharac- (1-palmitoyl(D )-2-hydroxy-glycero-3-phosphocholine and
31
teristicsoftheorganicmatterinthepermafrostdeposits. 1,2-distearoyl(D )-glycero-3-phosphatidylcholine)wereadded.
70
[14] Accelerator mass spectrometry (AMS) 14C radiocar- In a second step, the solvent extracts were collected in a
bondating of bulkorganic matter was carried out for seven separationfunnel,andforphaseseparation,dichloromethane
selectedsamplesfromtheKurungnakhpermafrostsequence and water were added to achieve a ratio of methanol/
(Figure2)intheAMSfacilityattheLeibnizLaboratoryfor dichloromethane/ammonium acetate buffer (pH 7.6) of
RadiometricDatingandStableIsotopeResearch,University 1:1:0.9 (v/v). Afterwards, the organic phase was removed and
ofKiel(Kiel,Germany),asdescribedpreviouslyinWagner thewaterphasewasreextractedtwotimeswithdichloromethane.
et al.[2007]. Fordetailsonthemethod,seeZinkandMangelsdorf[2004].
[15] Grain-sizedistributionwasanalyzedforbulksediment [19] The obtained sediment extract was separated into
samples every 30cm. The sediments were oxidized using a fractions of different polarity (low polar lipids, free fatty
5% hydrogen peroxide (H O ) solution to remove organic acids, glycolipids, and phospholipids) using a pure silica
2 2
matter from each sample. The sand (0.063–2.0mm) fraction column (1g silica gel, 63–200mm) and a florisil column
was determined by wet sieving. The remaining silt (0.002– (1g magnesium silica gel, 150–250mm) in sequence; for
0.063mm) and clay (<0.002mm) materials were separated details,seeZinkandMangelsdorf[2004].Blanks(preheated
by sedimentation in ammonia solution (10mL 25% NH in sea sand) were measured regularly and no phospholipids or
3
100Ldeionizedwater). glyceroldialkylglyceroltetraethers(GDGTs)weredetected.
[16] In order to obtain a vertical distribution of methane
withdepth,methaneconcentrationswereanalyzedbyputting 2.6. Detectionof Archaeol and Isoprenoid GDGTs
aslice(approximately5g)offrozensedimentinaglassbottle [20] Thelowpolarlipidfractionwasdissolvedinn-hexane
and immediately sealing the bottle gas-tight with a butyl- toprecipitateasphaltenes,whichwereremovedfromthesolu-
rubber stopper. After the addition of 20mL saturated saline ble fraction byfiltrationover Na SO . The n-hexane-soluble
2 4
(NaCl) solution, samples were shaken until thawed. Head- fractionwasseparatedbymedium-pressureliquidchromatog-
space gas samples were taken using a gas-tight syringe raphy[Radkeetal.,1980]intofractionsofaliphatic/alicyclic
(Hamilton; Hamilton Bonaduz AG, Bonaduz, Switzerland). hydrocarbons,aromatichydrocarbons,andnitrogen,sulphur,
Gas analysis was performed using a gas chromatograph andoxygen(NSO)-containingcompounds.
(Agilent6890;AgilentTechnologies,SantaClara,California, [21] The NSO fraction was investigated for tetraether
USA) equipped with a bonded polystyrene-divinylbenzene– lipids using an high performance liquid chromatography/
basedcolumn(HP-Plot-Q15m,0.53mm,40mm)andaflame atmospheric pressure chemical ionization (APCI) mass
ionization detector at operating conditions described previ- spectrometry (MS) method modified after Hopmans et al.
ously[Kochetal.,2009]. [2000] and Schouten et al. [2007]. Chromatographic separa-
tion was achieved on an Econosphere Amino column
(4.6(cid:3)250mm, 5mm; Altech). The mobile phase consisted
2.4. Potential Methane Production Rates ofn-hexaneAandisopropanolB(5minutes,99%A,1%B)
[17] The potential methane production rates of permafrost lineargradientto1.4%Bwithin22.5minutes,in1minuteto
sedimentswereanalyzedfor25selectedsamples,bothwithout 10% B, holding 5minutes to clean the column and back to
anyadditionalsubstrateandwithhydrogen,asasubstratefor initialconditionsin1minute,heldfor6minutesforequilibra-
methanogenic archaea. Freshly thawed sediments (1g) were tion. The flow rate was set to 200ml/min, and injection was
weighedin25mLglassjars,closedgas-tightwithabutyl-rub- performed by an autosampler with a 5ml loop. APCI source
ber stopper, and secured using aluminum crimp caps. The conditions were as follows: corona current 5mA giving a
samples were evacuated and flushed with ultrapure N /CO . voltageofaround4kV;vaporizertemperature350(cid:1)C;capil-
Afterwards,thesamplesweresupplementedwith0.1m2Lster2- lary temperature 200(cid:1)C and voltage 7.5V; nitrogen sheath
ileandanoxictapwater(tapwaterflushedwithultrapureN / gasat60psi;withoutauxiliarygas.Massspectraweregener-
2
CO and subsequently autoclaved) for analyzing methane ated by selected (single) ion monitoring, in the positive ion
2
productionwithoutsubstrateaddition.Inthecaseofpotential mode. The multiplier voltage was 1500V, and the scan rate
hydrogen-driven methane production, sterile and anoxic tap was1second.Forsemiquantitativedeterminationoftetraether
water in combination with H /CO (80:20, v/v, pressurized compounds, an external synthetic archaeol standard (Avanti
2 2
to 150 kPA) was added as substrate. Three replicates were Polar Lipids, Inc., Alabaster, Alabama, USA) was regularly
usedforeachsegmentandsubstrate.Theincubationtempera- measuredinparallel.BlanksdidnotcontainanyGDGTs.
ture was 10(cid:1)C. Methane production was measured semi-
2.7. Detectionof Phospholipid Fatty Acid
weeklyoveraperiodof3monthsbysamplingtheheadspace
and Phospholipid Ether Lipids
usingaHamiltongas-tightsyringeandanalyzedasdescribed
insection2.3.Methaneproductionrateswerecalculatedfrom [22] Lipids were extracted from the freshly homogenized
thelinearincreaseinmethaneconcentration. material (ca. 30g dw) from selected segments, according
309
BISCHOFFETAL.:METHANOGENICRESPONSETOCLIMATECHANGES
to the Bligh-Dyer method described in Zelles and Bai 1minute at 95(cid:1)C, 1minute at 56 or 53(cid:1)C, 1minute at
[1993]. The resulting lipid material was fractionated into 72(cid:1)C, and a final elongation of 72(cid:1)C for 10minutes.
neutral lipids, glycolipids, and phospholipids on a silica- The amplified samples and positive and negative controls
bonded phase column (SPE-SI; Bond Elute; Analytichem were checked by electrophoresis on a 1.5% agarose gel in
International,HarborCity,California,USA)byelutionwith 0.5(cid:3)TAE (diluted from 50(cid:3)TAE; 5 PRIME, Hamburg,
™
chloroform,acetone,andmethanol,respectively.Onehalfof Germany), which was prestained with GelRed (Biotium,
the phospholipid fraction was used for phospholipid fatty Inc.,Hayward,California,USA).ThePCRproductswerepu-
acid (PLFA) analysis following procedures described in rified using a HiYield™PCR Clean-up/Gel Extraction Kit
Zelles and Bai [1993], who also checked the composition (Süd-Laborbedarf, Gauting, Germany). Subsequently,
of the polar lipid fraction and found no contamination with DGGE analyses were conducted as described in Ganzert
glycolipids under the elution conditions described. How- et al. [2007], and sequence analyses were conducted by
ever, we are not sure if this holds true for all kinds of envi- GATCBiotechAG(Konstanz,Germany).
ronmentalsamplesandsamplematrices.Aftermildalkaline
2.9. Phylogenetic Analysis
hydrolysis, the resulting fatty acid methylesters were mea-
suredbygaschromatography(GC)/MS(5973MSD;Agilent [26] Sequence data obtained after DGGE analyses were
W
Technologies, Palo Alto, California, USA) at operating checkedforqualityusingtheSequencher software(Version
conditions described elsewhere [Zelles and Bai, 1993]. The 4.7; Gene Codes Corporation, Ann Arbor, Michigan, USA).
detected PLFAs comprise several saturated (iso-C , n-C , When indicated, sequences were cut or edited. Sequences
14 14
iso-C , anteiso-C , n-C , iso-C , branched-C , n-C , were aligned with full-length, quality-controlled sequences
15 15 15 16 17 17
cyclo-C ,n-C ,branched-C ,cyclo-C ),monounsaturated providedbythe“Silva rRNA database project”(www.arb-
17 18 19 19
(n-C ,n-C Δ9,n-C Δ7,n-C Δ9,cyclo- Δ7,n-C Δ11, silva.de; Pruesse et al. [2007]). The phylogenetic analysis
15:1 15 16 16 16:1 16:1
n-C Δ9, n-C Δ11, n-C Δ9 n-C Δ11; n-C , n- of partial 16S rRNA gene sequences was performed using
17:1 17:1 18:1 18:1 18:1
C Δ11), andtwopolyunsaturated(n-C Δ9,12,18:3) fatty the ARB software package (www.arb-home.de; Ludwig
20:1 18:2
acids. The sum of all detected PLFAs resulted in a PLFA et al. [2004]). A phylogenetic tree (maximum likelihood)
parameter. was constructed using the pos_var_ssuref:archaea and
[23] Theotherhalfofthephospholipidextractsweresub- terminifilterprovidedbyARB.Forabetteroverview,only
jected to phospholipid ether lipid (PLEL) analysis, accord- selecteddataareshownintheresultingtree.Allsequences
ingtothemethoddescribedbyGattingeretal.[2003].After determined in this study were deposited in the GenBank
the formation of ether core lipids, ether-linked isoprenoids database(JN635597–JN635629).
were released following cleavage of ether bonds with HI
andreductive dehalogenationwithZninglacial aceticacid. 3. Results and Discussion
The resulting isoprenoid hydrocarbons were dissolved in
3.1. Methane Profile of the Kurungnakh
100ml internal standard solution (nonadecanoic methyl
Permafrost Sequence
ester) and subjected to GC/MS analysis at operating condi-
tions described elsewhere [Gattinger et al., 2003]. The [27] MethanewasdetectedatalldepthsfromtheKurung-
detected ether cleavage products comprise phytane and nakh permafrost sequence, but the vertical profile shows a
biphytane, and the sum of these compounds resulted in a large variability from 4to3600nmol methane g–1sediment
PLEL parameter. (Figure 2c).Threeintervalsofincreasedmethaneconcentra-
tion were identified in sediments at (i) 2000–2300cm, (ii)
2.8. DNA Extraction and Polymerase Chain around 1200cm, and (iii) 100–500cm depth. Overall, the
Reaction Amplification
highestmethaneconcentrationsweredeterminedinHolocene
[24] Total genomic DNA was extracted using a Power deposits(100–300cmdepth).
™
Soil DNAIsolationKit(MoBioLaboratoriesInc.,Carlsbad, [28] High methane concentrations during the Holocene
California, USA), according to the manufacturer’s protocol. are comparable with a methane profile described for a
Amplifications were driven with a nested approach using a permafrostsequenceonSamoylovIsland, whichislocated
universalarchaealprimerArUn4F[Hershbergeretal.,1996] close to Kurungnakh Island (Figure 1c). However, the
plus Ar958R [DeLong, 1992], subsequently followed by methaneconcentrationsreportedforSamoylovIslandwere
amplification with the methanogenic archaea specific primer 10-fold lower than in the Kurungnakh sequence [Wagner
set 0357F and 0691R [Watanabe et al., 2004]. DNA sam- etal.,2007].Toourknowledge,enhancedmethaneconcen-
ples that were destined for denaturing gradient gel electro- trations in core material from terrestrial Late Pleistocene
phoresis (DGGE) analysis were amplified using forward permafrost deposits have not been demonstrated before.
primerGC_0357Fthatwasequalinsequence,butenlarged Rivkina and Gilichinsky [1996] reported the occurrence
bya20-merGCclamp. of methane in Holocene deposits and in Early to Middle
[25] The polymerase chain reaction (PCR) reaction mix Pleistocenesediments(0.78–1.8MaBP),butwerenotable
contained 12.5ml of premixed SAHARA-Mix™, including todetectmethaneinLatePleistoceneicecomplexdeposits
a PCR buffer, deoxyucleoside triphosphates, and polymer- (11.5–130kaBP).
ase (Bioline GmbH, Luckenwalde, Germany), 0.25ml of [29] TheobservedmethaneprofileintheKurungnakhcore
each primer (20mM), and 1ml template, and was filled up correspondstothedistributionofthesedimentaryorganicmat-
to 25ml with PCR-clean water (MO BIO Laboratories, ter (TOC; Figure 2b). This implies that sedimentary organic
Inc.,Carlsbad,California,USA).PCRwasperformedusing carboncanbemetabolizedinacascadeofmicrobialprocesses
an iCycler (Bio-Rad, Hercules, California, USA) and the byabroadconsortiumofheterotrophicbacterialcommunities.
following PCR cycle: 95(cid:1)C for 7minutes, 30cycles of This provides appropriate substrates (e.g., hydrogen, carbon
310
BISCHOFFETAL.:METHANOGENICRESPONSETOCLIMATECHANGES
reflected in the TOC data and that the higher TOC contents
found in the middle part of Unit III did not result in higher
methanevalues.Thisindicatesthatthereareadditionalfactors
responsiblefortheobservedmethaneprofile.
[31] One reason influencing the vertical distribution of
methane is the lithological composition of the permafrost
deposits. Kurungnakh permafrost deposits are heteroge-
neous, characterized by continuously changing proportions
ofthesand,silt,andclayfractions(Figure 2a).Largestvar-
iationsinthesedimentcompositionscanbeobservedamong
the silt and sand fractions. In general, silt is the dominant
fraction,buttherearedistinctlayerswheresandsignificantly
increases and becomes dominant. The intervals of low
methane concentration found in Units III (1400–1800cm)
andIVa(400–1000cm)containgenerallyhigherproportions
of sand. In contrast to soils made up of finer-grained mate-
rial, which can trap gases more easily because of smaller
pore sizes [Wang et al., 1993], the coarser-grained sandy
deposits are more permeable for gases, which might be an
explanation for their low methane contents.
[32] Carbon isotopic analyses of methane [Rivkina et al.,
2007; Koch et al., 2009] as well as several microbiological
studiesonmethanogenesisinarcticenvironments[Højetal.,
Figure 3. Hydrogen index versus oxygen index plot from
2005; Wagner et al., 2005; Metje and Frenzel, 2007] report
RockEvalpyrolysisdatacharacterizingthesedimentorganic
thatmethanestoredinshallowpermafrostenvironmentsisof
matterofterrestrialpermafrostdeposits.Theplotindicatesdif-
microbialorigin.Inthiscontext,thequestionstobediscussed
ferences in the quality of organic matter. Holocene samples
furtherarewhethertheobservedmethanefoundintheterres-
(marked with “H”) cluster separately from Late Pleistocene
trialpermafrostdepositsonKurungnakhIsland(i)isrecently
(marked with “LP”) samples due to their higher hydrogen
produced by currently active methanogenic archaea within
index.
the permanently frozen ground or (ii) was produced and
trapped during the deposition of the Late Pleistocene and
dioxide, acetate, formate, and methanol) for methanogenic Holocenesediments?Asakeytothesequestions,tracesofpast
archaea and the final process of methanogenesis. The TOC andlivingmicroorganismsinthesedimentswereinvestigated.
contentvariedwidelybetween1.5and26.5wt%.Thedeeper,
oldersedimentsofUnitIIIarecharacterizedbyrelativelyhigh 3.2. Signals of LivingMicrobial Communities
TOC (up to 6%), followed by a decrease to almost constant in theKurungnakh Permafrost Sequence
values of about 2% inthe younger sedimentsof UnitIII and [33] Severalstudieshavereportedthatalthoughpermafrost
Unit IVa, with the exception of a small interval at ca. environments represent an extreme habitat, microorganisms
1200cmdepthofhigherTOC(around4%).Withthetransition (bacteriaandarchaea)notonlysurvive,butcanalsobemeta-
from the Late Pleistocene to the Holocene (Unit IVa to Unit bolically active [Rivkina et al., 2004; Wagner et al., 2005;
IVb) the TOC increases significantly, to an average value of PanikovandSizova,2007;Kochetal.,2009].Phospholipids
10%.Generally,theintervalsofhighermethaneconcentrations (intact polar lipids) are considered as suitable life markers
correspondtohigherTOC(Figures2band2c).Interestingly, for microbial communities because the degradation of these
theLatePleistoceneandHoloceneorganicmattershowdiffer- abundant membrane compounds takes place rapidly after
entcompositionsrevealedbyRock-Evalpyrolysis(Figure3). the death of the source microorganisms [White et al., 1979;
The Holocene sediments have significantly higher hydrogen Harvey et al., 1986]. PLFAs are used as markers for viable
index (HI) values. Higher HI values are often interpreted to Bacteria andEukarya[Ringelberg etal.,1997; Zelles,1999],
represent a larger proportion of algal material in the organic andPLELsarecharacteristicmarkersforlivingarchaealcom-
matter.Thisalgalmaterialmighthave beenderivedbyocca- munitiesinsediments[ZellesandBai,1993].
sional river flooding of Kurungnakh Island and, as a conse- [34] In the Late Pleistocene, deposits in the amounts of
quence, the formation of small algal-containing pools during PLFA and PLEL are comparatively lower than in Holocene
the warmer Holocene period.Thus,inadditiontohigherand deposits (Figures 2d and 2e), with the exception of a small
presumablylessalteredTOC,qualitativedifferencesintheor- interval at 1500–1700cm depth in Unit III, where slightly
ganicmattercompositioncouldexplainthesignificantlyhigher higher amounts of PLELs (up to 2nmol PLEL g–1 sediment
methanecontentsintheHolocenedeposits. dw)weredetected.AtthetransitionfromtheLatePleistocene
[30] Overall,thecorrespondingTOCandmethaneconcen- totheHolocene,thePLFAandPLELconcentrationssignifi-
tration suggest that the TOC acted as a substrate source for cantly increase, with the highest amounts occurring in the
the indigenous methanogenic communities. The fact that Holocene(UnitV).ComparableresultsontheverticalPLEL
methanecanbeattachedtoanorganicmatrixmightbeanother distributioninHolocenedepositswerereportedforpermafrost
reason for the observed correlation [Chalmers and Bustin, sediments located close to our study site by Wagner et al.
2007].However,inthiscontext,ithastobehighlightedthat [2005], but the abundance of total PLELs was considerably
themethaneincreaseattheendoftheLatePleistoceneisnot higher in the current study, indicating a greater number of
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BISCHOFFETAL.:METHANOGENICRESPONSETOCLIMATECHANGES
living archaea in the Kurungnakh permafrost sequence. For andtheGDGTsrepresentdeadmicrobialbiomass.Although
the intervals of increased PLEL concentrations (Holocene itisverylikelythatthesebiomarkerslargelyrepresentpastmi-
andinUnitIII),geneticfingerprint analysesofthe methano- crobialcommunities,inparticular,inoldersediments,thebio-
genic communities (Figure 2i) reveal a higher variability, markers per se provide no information about whether these
compared with other core sections. This indicates a diverse remnants derived from past or presently living microbial
methanogeniccommunityandimpliesthepossibilityofliving communities. To get a better insight into the source of the
methanogenicarchaea. biomarker signals, we compared the archaeol and GDGT
[35] Additionally,methanogenicactivityincubationexperi- distributions (Figures 2g and 2h) with the previously dis-
ments with hydrogen and without any additional substrate cussedlifemarkerprofiles(Figures2dand2e).
(Figure 2f) showed significant methane production in the [40] Archaeol was detected throughout the whole Kur-
Holocene deposits of upto 0.09nmol CH h–1g–1 sediment. ungnakh permafrost sequence and varied between 0.3 and
4
DuringtheLatePleistocene,thepotentialmethaneproduction 66.7ngg–1sedimentdw(Figure2g).Thearchaeolconcen-
wasusuallylowandcouldonlybestimulatedintwointervals trationincreasesatthetransitionfromLatePleistoceneUnit
representingtheendoftheLatePleistocene(350–377cm)and II to Unit III. It remains relatively high in Unit III and
in the middle of Unit III (1927–1985cm). Surprisingly, the decreases slowly to low values at the beginning of Unit
additionofhydrogenasasubstrateformethanogenicarchaea IVa. The signal remains low until the end of the Late
did not lead to a significant increase or spatial shift in the Pleistocene sequence. At the transition to the Holocene,
potentialmethaneproductionalongthecore,aswasobserved the archaeol signal increases again and shows the highest
byWagneretal.[2007]. amounts of the whole Kurungnakh permafrost core in the
[36] Overall, in the Holocene sequence, the PLFA and upper Holocene sequence (Unit V). The comparison with
PLEL life-marker profiles (Figures 2d and 2e) and the thelifemarkersimpliesthathigherarchaeolconcentrations
methanogenic activity experiments (Figure 2f) indicate the inLate Pleistocene Unit III mainlyrepresent increased mi-
presenceoflivingbacteriaandarchaeaaswellaspotentially crobialbiomassofpastarchaealcommunities.Thearchaeol
activemethanogenicarchaea.Theseresultsshowthatpoten- signalduringtheHoloceneismostlikelytheresultofboth
tialsubstratesformethanogenesisarepresent,eitherdirectly past and presently living archaea. Thus, especially in the
stored in the sediments or released due to alteration of the Late Pleistocene deposits, the archaeol profile seems to
organic matter by psychrotolerant heterotrophic bacterial reflect the past distribution of the archaeal community in
communities, whose presence is indicated by the PLFA theKurungnakhpermafrostsequence.
profile (see section 2.7). Furthermore, the data suggest that [41] Isoprenoid GDGTs (Figure 2h) are comparatively low
at least part of the methane detected in the Holocene duringtheHolocene;theirhighestconcentrationsoccurinLate
sequence (Figure 2c) might have been recently produced. PleistoceneUnitsIIIandIVa.AcomparisonwiththePLELlife
[37] In the Late Pleistocene Unit III, neither the enhanced markeralsosuggeststhattheGDGTsaccumulatingduringthe
PLEL concentrations at 1700–1500cm depth nor the LatePleistocenemainlyrepresentpastmicrobialbiomass.
increased potential methanogenic activity at 1927–1985cm [42] An interesting variability in the composition of the
depth correlate with the methane profile, suggesting living, archaealcommunityisindicatedbythearchaeol/crenarchaeol
but inactive, methanogenic communities at these depths ratio(Figure4).Archaeolisthedominatingcompoundthrough-
(Figures 2c, 2e, and 2f). In contrast, at the end of the Late out the whole permafrost sequence. However, the archaeol/
Pleistocene, higher potential methanogenic activity and at crenarchaeolratioshowsthatenhancedamountsofcrenarch-
least a small increase of PLELs coincidewith an increaseof aeol (Figure 4) are detected in the Late Pleistocene deposits
methane, indicating the possibility of active methanogens at of Unit IVa. This indicates a significant shift in the archaeal
thetransitionbetweentheLatePleistoceneandtheHolocene. communityduringthistime.Recently,crenarchaeolwascon-
[38] The higher methane concentrations detected in the sidered to be characteristicfor ammonium-oxidizing archaea
older sediments of the Late Pleistocene Unit III (Figure 2c) (AOA) [Pitcher et al., 2010]. AOA have been identified in
and at a depth of about 1200cm in Unit IVa do not various soils and in arctic environments [Leininger et al.,
correspond with any life-marker signal, indicating that this 2006;Yergeauetal.,2010].
methane was produced andtrapped in the past.
3.4. Climate Impact onthe Distributionof Microbial
3.3. Reconstruction of Past Microbial Communities Communities in the Kurungnakh Permafrost Sequence
in theKurungnakh Permafrost Sequence
[43] As shown above (section 3.3), archaeol is an appro-
[39] Biomarker analyses were conducted on the low polar priateindicatortotracepastvariabilityofthearchaeal,likely
lipid fraction. The abundance of archaeol was measured the methanogenic community, in the Kurungnakh perma-
throughout the permafrost sequences, as it was recently frost sequence. Thus, in the following section, the archaeol
successfullyusedtoreconstructchangesintheabundanceof profile is compared to the Kurungnakh paleoclimate recon-
methanogenic archaea in a variety of ombotrophic peatland struction byWetterich et al.[2008].
settings [Pancost et al., 2011]. Additionally, isoprenoid [44] The Late Pleistocene Unit III (Middle Weichselian
GDGTsweredetectedandquantifiedascharacteristicmarkers (Kargin)interstadial)wascharacterizedbyachangetorela-
for archaea [De Rosa and Gambacorta, 1988; Koga and tivelywarmandwetsummerconditionsbyadistinctclimate
Morii, 2006]. The GDGTs and archaeol are already partly warming between 32and40kaBP [Wetterichet al.,2008].
degraded, as indicated by the loss of their head groups. The Thischangeintheclimatic conditions wasaccompanied by
remainingcorelipidstructuresarelessaffectedbydegradation ahigherconcentrationofarchaeolandariseinthemethane
processes and are stable over geological time scales [Pease content (Figures 2c and 2g), indicating an increase in
et al., 1998]. Thus, in contrast to the life markers, archaeol the methanogenic community during this period. Thus, an
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BISCHOFFETAL.:METHANOGENICRESPONSETOCLIMATECHANGES
and at the end of the Late Pleistocene (300–500cm).
The interval at 1200cm is characterized by relatively high
TOCandarchaeolvalues,indicatingpastmethaneproduction
andtrapping.Incontrast,thesmallincreaseofmethaneatthe
end of the Late Pleistocene, despite low archaeol concentra-
tions,isdifficulttoexplain.Asmentionedabove(section3.2),
the PLEL data and methanogenic activity experiments sug-
gest the possibility of recent methane production in this
interval. Another hypothesis is that the TOC and methane-
rich Holocene sediments might have acted as a seal for gas
percolatingupwardsfromdepth,leadingtotheaccumulation
ofmethane,whichwasproducedatalowrateovergeological
timesintheLatePleistocenesediments.
[47] During the early Holocene (Unit IVb), methane and
archaeol contents increased (Figures 2c and 2g), indicating
thatthetemperatureriseduringthisperiodcausedanincrease
intheabundanceofmethanogenicarchaeaagain.Theenviron-
ment during this time has been described as warm and dry
[Wetterichetal.,2008].
[48] The Middle to Late Holocene deposits (Unit V) are
characterizedbywarmerwintertemperatureswithdryhabi-
tats disappearing and the formation of peatlands [Wetterich
et al., 2008]. These changes to warm and wet conditions
triggered a dramatic increase in the abundance of methano-
genicarchaea,asamountsofarchaeolincreasedremarkably,
in comparison to Late Pleistocene and Early Holocene
deposits. This is in accordance with the highest concentra-
Figure4. Ratiooftherelativeabundancesofarchaeoland
tions of methane in these deposits (Figure 2c).
crenarchaeol alongthe Kurungnakh permafrost sequence.
[49] Overall, our results show a climate-driven response
of methanogenic archaea in terrestrial permafrost environ-
increaseinthepaleoproductionofmethaneunderwarmand ments during the Late Pleistocene and Holocene. Warm
wet conditions is suggested. Part of this generated methane and especially wet climatic conditions caused an increase
wassurelyemittedtotheatmosphere.However,anotherpart intheabundanceofmethanogenicarchaea;thisisreflected
was trapped and stored in the frozen ground during the by high amounts of the molecular marker, archaeol, being
timeofdeposition,whichisa reasonableexplanationfor the incorporated into the terrestrial permafrost deposits. A
relativelyhighmethaneconcentrationsmeasuredintheolder reason for this might be that the warm and wet climate
permafrostdeposits(1900–2200cm)ofUnitIII. conditions favored a landscape with flooded lowlands,
[45] In contrast, in the younger sediments of Unit III causing water-saturated sediments and therefore suitable
(1400–1900cm) the methane concentrations are generally anaerobicconditionsforthegrowthandactivityofmethano-
low, although the archaeol concentrations remain relatively genic archaea. Consequently, higher methane emissions to
high (Figures 2c and 2g), implying that methanogens were the atmosphere are suggested for these periods, as reported
still abundant and methane was still produced during that for northern peatlands in the early Holocene [MacDonald
time. As discussed above (section 3.1), a change in the soil etal.,2006].Furthermore,weshowthatacoldanddryclimate
lithologyinthisintervalmighthavepresentedlessfavorable causes a decline of the methanogenic archaeal community,
conditions for trapping methane. Generally, this interval implying lower methane emissions for this interval. These
(1400–1900cm depth) contains higher proportions of sand interpretations are also supported by Fischer et al. [2008],
(more permeable for gases), lower proportions of clay, and whodemonstratedthatborealwetlandscontributeenormously
partly also lower amounts of TOC, compared to the older tomethaneemissionsduringwarmingevents,whereasduring
sediments in Unit III (Figures 2a and b). The lower TOC coldclimateperiods,theseemissionsdecreaseconsiderably.
contents in the upper deposits of Unit III might also have
contributed to lower the capability of the deposits to store 3.5. Climatic Impacton the Composition of
methane because gases are reported to be attached to the Methanogenic Communities in theKurungnakh
organic matrixes [Chalmers and Bustin,2007]. Permafrost Sequence
[46] Climate conditions during the formation of Unit IVa [50] The biomarker data show a strong variability in the
havebeeninterpretedascoldandratherdry(LateWeichselian abundance of the archaeal community in the Kurungnakh
(Sartan) Stadial; Wetterich et al. [2008]). The decreasing permafrost sequence with respect to the stratigraphic units
amounts of archaeol (Figure 2g) in this interval reflect a and their associated climatic conditions. Additionally,
response of the archaeal communities to constant climate ecomolecular characterizationofthemethanogeniccommu-
cooling and dry conditions. The data imply a less abundant nities based on 16S rRNA gene sequence analysis revealed
communityofmethanogenicarchaeaandsmalleramountsof changes in the community composition of methanogens
methane produced and trapped during this time (Figure 2c). along the different stratigraphic units (Figure 2i). DNA
Two exceptions are found in the small interval at 1200cm was successfully extracted from samples throughout the
313