Autotrophy as a predominant mode of fixation in anaerobic methane-oxidizing microbial communities

Matthias Y. Kellermanna,1,2,3, Gunter Wegenerb,c,1, Marcus Elverta, Marcos Yukio Yoshinagaa, Yu-Shih Lina, Thomas Hollerc, Xavier Prieto Mollara, Katrin Knittelc, and Kai-Uwe Hinrichsa

aOrganic Geochemistry Group, MARUM-Center for Marine Environmental Sciences and Department of Geosciences, University of Bremen, D-28359 Bremen, Germany; bAlfred Wegener Institute for Polar and Marine Research, Research Group for Deep Sea Ecology and Technology, D-27515 Bremerhaven, Germany; and cMax Planck Institute for Marine Microbiology, D-28359 Bremen, Germany

Edited by Donald E. Canfield, University of Southern Denmark, Odense M, Denmark, and approved October 5, 2012 (received for review May 24, 2012) The methane-rich, hydrothermally heated sediments of the Guay- (20, 21). By contrast, Alperin and Hoehler (22) argued mas Basin are inhabited by thermophilic microorganisms, including on the basis of isotopic considerations that autotrophic metha- anaerobic methane-oxidizing (mainly ANME-1) and sulfate- nogens could equally be responsible for the observations of strongly reducing (e.g., HotSeep-1 cluster). We studied the microbial 13C-depleted and biomass at numerous cold seep sites. carbon flow in ANME-1/ HotSeep-1 enrichments in stable-isotope– Recently, dual SIP with simultaneous addition of deuterated 13 13 probing experiments with and without methane. The relative incor- water (D2O) and C-labeled inorganic carbon ( CDIC) was in- poration of 13C from either dissolved inorganic carbon or methane troduced as an assay for the quantification of rates of de novo into lipids revealed that methane-oxidizing archaea assimilated pri- synthesis and inorganic carbon assimilation into lipids (23). Fur- 13 marily inorganic carbon. This assimilation is strongly accelerated in thermore, via the relationship of incorporation of CDIC relative the presence of methane. Experiments with simultaneous amend- to D in microbial lipids, lipid biosynthesis by autotrophs and het- ments of both 13C-labeled dissolved inorganic carbon and deuter- erotrophs can be distinguished. To constrain mechanisms and ated water provided further insights into production rates of patterns of carbon flow in microbial communities mediating AOM individual lipids derived from members of the methane-oxidizing 13 we performed a series of SIP experiments with combined CDIC 13 community as well as their carbon sources used for lipid biosynthe- SCIENCES and D2O as well as CH4, using enrichments of AOM-mediating sis. In the presence of methane, all prominent lipids carried a dual microbial communities from the Guaymas Basin (cf. ref. 24). This ENVIRONMENTAL isotopic signal indicative of their origin from primarily autotrophic approach enabled us to differentiate which lipids were produced in microbes. In the absence of methane, archaeal lipid production the presence and absence of methane and to quantify the roles of ceased and bacterial lipid production dropped by 90%; the lipids methane and inorganic carbon as carbon sources for microbes produced by the residual fraction of the metabolically active bacte- involved in AOM. rial community predominantly carried a heterotrophic signal. Collec- tively our results strongly suggest that the studied ANME-1 archaea Results and Discussion oxidize methane but assimilate inorganic carbon and should thus be Activity and Microbial Composition of the Guaymas Basin Enrichments. fi classi ed as methane-oxidizing chemoorganoautotrophs. We incubated replicates of hot seep sediments naturally enriched in moderately thermophilic ANME-1 dominated communities at methanotrophy | biomarker | acetyl-CoA pathway | syntrophy suitable growth conditions (200 kPa CH4,37°C,andartificial seawater medium for sulfate reducers) (25). Samples were amen- 13 13 ethane is an important greenhouse gas and the most abun- ded either with CDIC [resulting C fraction was 9.6% of dissolved Mdant hydrocarbon in marine sediments. Its upward flux to the inorganic carbon (DIC)] and deuterated water (3% deuterium in – 13 13 sediment water interface is strongly reduced by sulfate-dependent total water) or with CH4 (15.9% C of methane carbon) in anaerobic oxidation of methane (AOM) (1, 2). AOM is performed a time series of 10, 17, and 24 d, with and without methane by syntrophic associations of anaerobic methane-oxidizing archaea headspace and in killed controls (Table 1). The incubations with (ANMEs) (3, 4) and their sulfate-reducing bacterial partners methane showed a strong increase of sulfide production in re- Desulfosarcina Desulfobulbus – 13 (SRBs) (mainly relatives of or )(5 8). sponse to stimulation of sulfate reduction. Incubations with CH4 The free yield of the AOM net reaction is one of the lowest revealed methane oxidation rates (SI Text, Eq. S1)thatwere – – known for catabolic reactions under environmental conditions (ΔG roughly 85% (3.4 μmol·d 1·g 1) of the sulfate reduction rate – dm ranges from –20 to –40 kJ·mol 1; e.g., refs. 9, 10). Consequently, (Table 1). In the absence of methane, sulfate reduction decreased activity and biomass doubling times determined under optimized by ∼90% (Table 1). Sequencing of 16S rRNA and fluorescence in laboratory conditions range from 2 to 5 mo (11) and growth yields situ hybridization revealed a dominance of aggregate-forming are extremely low, around 1% relative to oxidized methane (11, 12). The biomass of ANMEs and SRBs involved in AOM is usually 13 strongly depleted in C. For instance, at methane seep locations, Author contributions: M.Y.K., G.W., Y.-S.L., and K.-U.H. designed research; M.Y.K., G.W., the δ13Cvaluesofspecific bacterial fatty acids and archaeal ether M.E., M.Y.Y., and T.H. performed research; M.Y.K., M.E., M.Y.Y., X.P.M., and K.K. ana- lipids range from –60 to –100‰ and –70 to –130‰, respectively lyzed data; and M.Y.K., G.W., M.Y.Y., and K.-U.H. wrote the paper. (e.g., refs. 13–17). Such low values have been interpreted as evi- The authors declare no conflict of interest. dence for the incorporation of 13C-depleted methane into biomass This article is a PNAS Direct Submission. (e.g., refs. 3, 4, 18). Data deposition: The sequences reported in this paper have been deposited at One way to identify the carbon sources of microbial biomass is to the European Molecular Laboratory, GenBank, and the DNA Data Base of Japan (DDBJ) [accession nos. FR682479–FR682487 and HE817766–HE817767 (archaeal 16S rRNA perform stable–isotope–probing (SIP) experiments (19), followed genes); and FR682618–FR682643 (bacterial 16S rRNA genes)]. by analysis of such as membrane lipids (lipid-SIP 1M.Y.K. and G.W. contributed equally to this work. hereafter). Application of lipid-SIP to cold seep sediments and 2Present address: Department of Earth Science and Marine Science Institute, University of microbial mats indicated that archaeal communities dominated by California, Santa Barbara, CA 93106. ANME-2 use both methane-derived carbon and inorganic carbon 3To whom correspondence should be addressed. E-mail: [email protected]. for lipid biosynthesis, whereas their bacterial partners assimilate This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. only inorganic carbon to produce biomass and are thus complete 1073/pnas.1208795109/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1208795109 PNAS | November 20, 2012 | vol. 109 | no. 47 | 19321–19326 Downloaded by guest on September 26, 2021 Table 1. Overview of conditions in incubation experiments 13 –1 –1 Incubation time Substrate combination δ CDIC, ‰ δDH2O, ‰ H2S production, μmol·d ·gdm

Control

t0, no label CH4 NA NA NA 13 – t0, + CH4 CH4;D2O; H CO3 NA 244,000 NA 13 13 t0, + CH4 CH4 (∼16,000‰)NANANA 13 – t24, killed CH4;D2O; H CO3 ; ZnCl2 8,500 199,000 0.4

+ CH4 t10, + CH4 8,400 196,000 13 – t17, + CH4 CH4;D2O; H CO3 8,200 206,000 6.0 ± 0.4

t24, + CH4 8,000 206,000 13 13 t24, + CH4 CH4 950 NA 4.0 w/o CH4 13 – t24,w/oCH4 D2O; H CO3 8,700 210,000 0.7

13 13 13 Duration of the incubation, substrate combinations (DH2O and CDIC; CCH4), labeling strength (δ CDIC and 13 δ CCH4 of the headspace; δDH2O of the aqueous media), and the sulfide production rate of all analyzed samples incubated at 37 °C are shown. CO2 = 50 kPa; CH4 = 200 kPa; N2 = 200 kPa. + CH4, presence of methane headspace; killed, killed control (ZnCl2, final concentration 2% wt/vol); NA, not analyzed; w/o, without.

ANME-1 archaea and partner bacteria from the HotSeep-1 relative to time zero (Δδ) was most pronounced for incubations cluster (Fig. S1 and Table S1). with methane, and Δδ increased fairly linearly with time (Fig. 1 A and B). The extent of label uptake differed between individual Microbial Lipid Distribution and Natural Isotopic Compositions. compounds, with the highest change in ΔδDandΔδ13C found for Concentrations and relative abundances of lipids did not change archaeal phytane and bacterial lipids such as iC16:1ω6,iC18:1ω6, fi signi cantly during the incubations, an observation that is consis- and C18:1ω7 FAs (Table S3). For instance, in the 24-d incubation tent with low growth rates reported for this microbial community with methane, changes in phytane’s δ-values were remarkably (doubling time of 77 d at 37 °C) (24). Ether cleavage of archaeal higher than in biphytanes (Fig. 1 A and B and Table S3). The ΔδD lipids in the total lipid extract released mainly acyclic biphytane, of bacterial C14–C18 FAs was high (∼ +12,000‰) and similar to phytane, and bicyclic biphytane (Fig. S2 A and C and Table S2). that of phytane, whereas Δδ13C was significantly lower in the FAs High relative amounts of biphytanes are derived from (∼ +380‰;Fig.1A and B and Table S3). In the killed control, no dibiphytanyl glycerol tetraethers (GDGTs), which have been enrichment of D and 13C in lipids was detected (Table S2). In routinely found in methane-rich sediments and cold seeps domi- incubations without methane, archaeal lipids showed only traces nated by ANME-1 (26–28), whereas phytanes derived from the of D-label uptake but none of 13C; likewise the uptake into bac- cleavage of archaeols are less specific and likely produced by all terial lipids was strongly reduced compared with the incubations ANMEs (e.g., refs. 16, 17, 26). However, because archaeal 16S with methane (Fig. 1 A and B and Table S3). This observation rRNA gene libraries and microscopic analysis of this enrichment confirms the central role of methane as an energy source in the revealed dominance of ANME-1 archaea, this specific group is examined AOM system. 13 13 most likely the main source of both phytanes and biphytanes. Incubation with CH4 (24 d) resulted in relatively low Cas- Fatty acids (FAs) are composed of saturated even-numbered, similation into lipids. The Δδ13Cvaluesofaround30‰ for both saturated, and unsaturated terminally or subterminally branched bacterial and archaeal lipids (Table S3) corresponded to about 1/ 13 FAs (iso, i;andanteiso, ai); monounsaturated C16 and C18 FAs; 12th and 1/4th of the Δδ C values for the respective lipid pools in B C 13 and isoprenoidal FAs (Fig. S2 and and Table S2). This dis- the parallel experiment with CDIC but unlabeled methane. We tribution differs significantly from those found at cold seeps (14, assign this slight positive shift in δ13C values to indirect uptake via 17, 26), which is consistent with the distinct bacterial members in assimilation of inorganic carbon from the 13C-enriched DIC pool 13 13 our enrichment. The unusual methyl branched and unsaturated resulting from CH4 oxidation (after 24 d δ CDIC ∼ +950‰; FAs iC16:1ω6 (Fig. S3)andiC18:1ω6 have to our knowledge not Table 1). This indicates that not only SRBs, but also ANME-1 ar- been detected at cold seeps, but are known constituents of some chaea assimilate predominantly inorganic carbon. Our findings are sulfate-reducing bacteria (mostly in Desulfovibrio species) (29, in agreement with the result from Treude and coworkers (12), who fl 14 14 30). Phylogenetic as well as catalyzed reporter deposition uores- detected a predominance of CO2 over CH4 incorporation within cence in situ hybridization (CARD-FISH) analysis of the enrich- a microbial mat known to be dominated by ANME-1/SRB con- ment indicated that the dominant sulfate reducers belong to the sortia. However, this result differs from a previous labeling study HotSeep-1 cluster (Table S1 and Fig. S1), which has been re- with cold seep sediments by Wegener et al. (21), in which methane peatedly found at Guaymas Basin but nowhere else yet. contributed up to 50% during archaeal lipid carbon fixation for Both, archaeal isoprenoids and bacterial FAs displayed rela- mixed ANME-1/ANME-2 and ANME-2–dominated communities. tively low natural δ13C values indicative of AOM activity (13–17); phytanes and biphytanes ranged from –18 to –47‰ and bacterial Lipid Production, Inorganic Carbon Assimilation, and Methane-Dependent 13 FAs from –21 to –50‰, whereas δD values ranged from –147 to Carbon Fixation. We converted the incorporation of D and CDIC in –286‰ for archaeal isoprenoids and from –86 to –232‰ for microbial lipids into rates of lipid production (prodlipid)andin- FAs (Table S2). This large range is similar to earlier observations organic carbon assimilation (assimIC), respectively. The ratio of R of δD values in anoxic marine sediments (31). assimIC to prodlipid ( a/p) indicates the dominant mode of microbial carbon fixation, with Ra/p values ≤ 0.3 indicative of heterotrophic Assimilation of Stable Isotope Label into Microbial Lipids. Although and Ra/p values of close to 1 signaling autotrophic changes in lipid concentrations were not detectable, the in- metabolism (cf. ref. 23) (Materials and Methods). Intermediate Ra/p corporation of D and 13C from labeled substrates indicated values are interpreted to represent a mixture of auto- and hetero- freshly synthesized lipids (Fig. 1 A and B, respectively, and Table trophically produced lipids, e.g., via production of a single com- S3). The incorporation of both heavy isotopes in microbial lipids pound by multiple sources (23).

19322 | www.pnas.org/cgi/doi/10.1073/pnas.1208795109 Kellermann et al. Downloaded by guest on September 26, 2021 ABC 12,000 600 30 bacterial lipids archaeal lipids total FAs prodlipid 10,000 Phy 500 25 assimIC total BPs ] –1

8,000 400 yr. 20 = 0.7 –1 R a/p dm = 1.0 R 6,000 C [‰] 300 15 a/p 13 ΔδD [‰] Δδ 4,000 200 10 [μg lipid g = 0.2 2,000 100 5 R a/p = b.d. R

N.A. a/p 0 0 0 + + + + + + t10 t17 t24 t24 t24 t10 t17 t24 t24 t24 average t24 average t24 13 13 + + t10-24 t10-24 CH4 w/o CH4 CH4 w/o CH4 w/o CH4 w/o CH4

Fig. 1. Results of lipid stable-isotope probing. (A and B) Isotopic shifts of (A) and (B) carbon, expressed as weighted-average Δδ of total fatty acids (FA, light gray bars), archaeols as phytanes (Phy, dark gray bars), and glycerol dibiphytanyl glycerol tetraethers (GDGTs) as biphytanes (BPs, black bars) during

the experiment. (C) Production of lipids (prodlipid, green bars) and assimilation of inorganic carbon (assimIC, blue bars) into bacterial (Left) and archaeal (Right) lipids. Time series experiments in C are presented as average values (n = 3, error bar = SD). The ratio of assimIC to prodlipid (Ra/p) gives a measure for the dominance of a heterotrophic (Ra/p < 0.3) or autotrophic (Ra/p ~ 1) mode of carbon fixation. In the absence of methane, Ra/p values are not presented because 13 13 of low values detected for archaeal inorganic carbon lipid assimilation. The symbol “+” indicates incubations with CDIC ∼ 9.6% C, D ∼ 3%, and unlabeled 13 13 methane. Incubations with CH4 contained ∼15.9% C.

In the presence of methane as an energy source the values of indicates that the lipid distribution is a cumulative record affected SCIENCES ± μ · –1· –1 archaeal prodlipid reached 10 3 g lipid gdm y , indicating ar- by the temporally varying rates of production and degradation of ENVIRONMENTAL chaeal cell growth (Fig. 1C and Table S4). The assimIC value was individual lipids, whereas the prodlipid records the metabolic nearly identical and thus indicated strictly autotrophic carbon fix- activity of those community members stimulated by the culture ation by the archaeal community (Ra/p values of 1.03 ± 0.05; Fig. conditions. In the absence of methane, production of all lipids R 1C). In the absence of methane, the values of archaeal prodlipid and decreased, and a/p values dropped toward heterotrophic sig- assimIC were marginal (Table S4), demonstrating that the presence natures. This decline was most pronounced in lipids with auto- of methane is mandatory to sustain lipid production and thus the trophic signatures in the presence of methane (e.g., C18:1ω7, archaeal community. iC16:1ω6,andiC18:1ω6; Figs. 2B and 3, Fig. S4,andTable S4). We In all incubations with methane, prodlipid of phytane exceeded cannot resolve to what degree the drastic change in both pro- that of biphytanes by at least one order of magnitude (Fig. 2A, Fig. duction and carbon metabolism expressed in dual isotopic sig- S4,andTable S4), whereas Ra/p values of around 1 for both phytane natures of individual lipids is due to metabolic adaptation or and biphytanes indicated a strictly autotrophic carbon metabolism simply to the shutoff of SRBs affiliated with AOM. The perma- of these lipids’ microbial producers. Detection of assimIC in biphy- nently low Ra/p values observed for some i- and ai-FAs, specifically tane was hindered by the large background of fossil core-GDGTs in iC15:0 and iC17:0 (Fig. 3 and Table S4), are best explained by other, the total lipid extract (Fig. S5), which reduces the SIP sensitivity and less active, heterotrophic bacterial community members not in- prevents the use of biphytane-derived Ra/p values as a gauge for volved in AOM, such as relatives of the genus Anaerolinea within distinguishing hetero- or autotrophic producers of lipids (Table S4). subphylum I of Chloroflexi, which are abundant in the 16S rRNA To increase the sensitivity of the SIP experiment, we performed gene library from this Guaymas Basin enrichment (Table S1). a similar isotopic assay on biphytanes from intact polar GDGTs Fermenters usually show low heterotrophic CO2 fixation (33). purified from the total lipid extract by preparative liquid chroma- We demonstrated that both archaea and bacteria mediating tography (cf. ref. 32) and obtained an Ra/p value of 1.2 in support of AOM in hydrothermal sediments in the Guaymas Basin assimilate –1 –1 autotrophic production (254 and 217 ng lipid·gdm ·y for assimIC preferentially inorganic carbon. This was unexpected for the ar- and prodlipid, respectively). chaeal community members. Our results indicate that the archaea As observed for archaeal lipids, the prodlipid and assimIC values do not assimilate methane but instead act as autotrophic methane of bulk bacterial FAs were highest in the presence of methane (24 ± oxidizers. The carbon assimilation of many ,inpar- –1 –1 2and16± 1 μg lipid·gdm ·y , respectively; Fig. 1C and Table S4), ticular , is performed via the reductive acetyl-CoA resulting in an Ra/p value of 0.70 ± 0.01. Without methane, both pathway (34). This pathway combines two one-carbon-atom moie- + prodlipid and assimIC of bulk bacterial FAs were strongly reduced; ties of the oxidation state of II (equivalent of carbon monoxide) – the resulting Ra/p value of 0.2 signaled predominant production by and III (methyl group) to form acetyl-CoA. It was speculated that heterotrophic microbes (Fig. 1C and Table S4). This contrast ANME archaea shuttle methyl groups from the acetyl-CoA path- emphasizes the importance of bacterial CO2 assimilation in con- way into their assimilatory system (21, 35). We have shown that in nection with methane oxidation. ANME-1 archaea from the Guaymas Basin growing at 37 °C, The bacterial lipids with the highest production in the pres- methane oxidation is decoupled from the assimilatory system. ence of methane were iC16:1ω6,iC16:0,C16:0,iC18:1ω6, and C18:1ω7. Hence these ANME-1 archaea qualify as chemoorganoautotrophs. Most of these lipids, including iC16:1ω6 and iC18:1ω6, which are The lacking transfer of methane carbon into the assimilatory system both specific to HotSeep-1 phylotype, and C18:1ω7, show com- may appear counterintuitive. However, autotrophic carbon fixation parable prodlipid and assimIC values that are indicative of pre- during growth on organic substrates has been previously demon- dominant production by autotrophs (Figs. 2A and 3, Fig. S4, and strated (36, 37). Particularly under the strongly negative Table S4). The production intensity of these lipids does not cor- potentials in sulfidic environments, microorganisms are able to fix respond to the distribution of FA concentrations (Fig. S5). This inorganic carbon with relatively low input of energy, using the

Kellermann et al. PNAS | November 20, 2012 | vol. 109 | no. 47 | 19323 Downloaded by guest on September 26, 2021 A 10 strictly coupled to methane oxidation rather than . CH4 incubation The much larger range of isotopic compositions in putative lipids of methane-oxidizing archaea compared with methane at cold Bacterial lipids seeps (39) could be the result of a large variation of δ-values of CO ) 8 2 –1 Archaeal lipids Phy in conjunction with a more widespread distribution of ANME ar-

yr chaea that predominantly assimilate CO2. –1

dm 6 Materials and Methods Sample Collection, Genetic Analyses, and Stable Isotope-Labeling Experiments. C18:1ω7 Sediments derived from the gas-rich, -covered hydrothermal site in 50 % 4 the Guaymas Basin (27°00.437 N, 111°24.548 W) were retrieved during the R/

(μg lipid g –

IC V Atlantis cruise AT 15 56 (Alvin dive 4,570, 2009). Samples were diluted 1:1 iC16:1ω6 with anoxic artificial seawater medium supplemented with trace elements iC16:0 and for sulfate-reducing bacteria (25) and incubated at 37 °C with

assim iC 2 18:1ω6 a methane headspace at 100 kPa until further processing (24). After 90 d of C16:0 preincubation, DNA was extracted from a replicate sediment batch in- cubated following established protocols. rRNA (16S) was amplified, cloned, 0 and sequenced. The phylogenetic affiliation was inferred with the ARB 024 6810 software package (SI Text). For the SIP experiments, enrichments were equally distributed into 256-mL culture vials [∼4 g dry mass (gdm) per vial], B 0.6 filled up with seawater medium as described above. Samples were amended w/o CH4 incubation 13 13 with labeled substrates (D2O, CDIC, CH4) in different combinations and Bacterial lipids 0.5 autotroph )

–1 Archaeal lipids 100 yr

–1 0.4 Phy dm

i-18:1ω6 0.3 i-16:1ω6 C18:1ω9 50 % BP(1) Bulk (μg lipid g )] 4 IC 0.2 aiC15:0 iC16:1ω6

assim iC 0.1 18:1ω6 Phy C heterotrophC BP(0) 18:1ω7 16:0 w/o CH Bulk 18:1ω7 lipid iC15:0 iC16:0 0 0 0.1 0.2 0.3 0.4 0.5 0.6 –1 –1

prodlipid (μg lipid gdm yr ) ) / (prod 10 4 18:0 Fig. 2. Carbon assimilation and lipid production of microbial lipids. Compared 16:1ω7 are carbon assimilation (assim ) and production (prod ) of bacterial fatty + CH IC lipid acids (diamonds) and archaeal ether lipid-derived isoprenoids (circles) in the i-16:0 lipid (A) presence and (B) absence of methane after 24 d of incubation (see Fig. S4 for more details). 16:0 ai-17:0 18:1ω9 14:0 14:0 acetyl-CoA pathway (38). This is especially true for methane-oxi- 16:1ω5

log [(prod i-17:0 dizing archaea, which demand a sink for the four electron pairs per 15:0 17:0 fi molecule methane oxidized. Our ndings strongly suggest that in ai-15:0 ANME-1, as in many methanogens, lipid biosynthesis is linked to i-15:0 autotrophic carbon fixation. Because methane utilization appears to be limited to the dissimilatory pathway, the term methanotrophy should be used with caution, as “trophy” involves the assimilation of 1 a compound. 0 0.2 3.0 0.4 0.6 0.8 0.1 1.2 The unexpected chemoorganoautotrophic nature of ANME-1 Ra/p values demands a reevaluation of isotopic signatures of archaeal lipid autotrophy

biomarkers in marine sediments with active AOM communities. +CH4 Archaeal lipids Strongly 13C-depleted archaeal lipids are established as important heterotrophy w/o CH4 Bacterial lipids proxies for tracking the activity of methane-oxidizing archaea in such settings (e.g., refs. 3, 13–18); this isotopic depletion has been Fig. 3. Relative increase in individual lipid production in incubations with attributed to the direct transfer of 13C-depleted methane carbon methane compared with incubations without methane. Increases are plot- 13 into lipid biosynthesis. As suggested recently (22), the C-depleted ted against the ratio of carbon assimilation to lipid production (Ra/p)asan lipids could equally be products of autotrophic methanogens that indicator for auto- or heterotrophic source of the lipid. Archaeal use 13C-depleted CO . Although lipid products of CO -assimilat- ether lipid-derived hydrocarbons (circles) and bacterial fatty acids (dia- 2 2 monds) were produced in either the presence (red symbols) or the absence ing ANME archaea could probably not be distinguished easily (white symbols) of methane. Ra/p values in the absence of methane were not from those of such putative methanogens on the basis of their determined for iC ω ,C ω , and iC ω and for BP(0) and BP(1) due to fi 16:1 6 16:1 7 18:1 6 isotopic composition. However, our study identi ed close relatives assimIC values close to the detection limit. Shaded bars indicate ranges pri- fl of ubiquitous cold-seep archaea, in which CO2 assimilation is marily interpreted to re ect heterotrophy (brown) or autotrophy (blue).

19324 | www.pnas.org/cgi/doi/10.1073/pnas.1208795109 Kellermann et al. Downloaded by guest on September 26, 2021 incubated at 37 °C for 0, 10, 17, and 24 d (t0, t10, t17, t24) with either CH4 or Sample Analyses: GC-FID, GC-MS, and GC-irMS. Lipids were quantified by gas fi N2 atmosphere at 200 kPa + 50 kPa CO2. The pH in all incubations was chromatography coupled to a FID (Trace GC Ultra; Thermo Scienti c). Carbon 13 7.2. Samples amended with 3% D2Oand9.6% CDIC had δDH2O values of and hydrogen isotopic composition was determined using GC-irMS at least in 13 ∼ +200,000‰ vs. Vienna Standard Mean Ocean Water (VSMOW) and δ CDIC duplicate measurements (Trace GC Ultra coupled to a GC-IsoLink/ConFlow IV values of ∼ +8,400‰ vs. Vienna PeeDee Belemnite (VPDB) (VPDB = 9.6% interface and a Delta V Plus irMS; all from Thermo Scientific). Compounds 13C). The carbon isotopic composition of the methane headspace in the were oxidized in a combustion reactor at 940 °C, and molecular hydrogen 13 13 13 was produced from eluting lipids in a pyrolysis reactor at 1,420 °C. The an- CH4 (15.9% C) experiments had δ C values of ∼ +16,000‰ vs. VPDB alytical error was <0.5‰ and <10‰ for nonlabeled δ13C and δD values, re- (detailed information in Table 1). Killed controls were sterilized with ZnCl2 spectively. δ13C and δD values were corrected for additional carbon and [2% (wt/vol) final concentration] before label addition. Sulfide production hydrogen introduced during derivatization. was constantly monitored using a spectrophotometric method described by Cord-Ruwisch (40). For cell hybridization, a sediment aliquot was fixed (in – Calculations of Total Lipid Production Rate (Prod ), Assimilation Rate of formaldehyde; 30 g·L 1, final concentration, at room temperature) and lipid Inorganic Carbon (Assim ), and the Assim /Prod Ratio (R ). The stable blotted on a membrane filter and CARD-FISH was performed with estab- IC IC lipid a/p hydrogen and carbon isotope values are expressed in the δ-notation in per mill lished probes for ANME-1 and ANME-2. Subsequently samples were stained (‰) as deviation of the isotope ratio from the reference standards. Prod with DAPI and cells and/or microbial phyla were identified with fluorescence lipid and assimIC are calculated by multiplying lipid concentration (conclipid)bythe microscopy. D 13 13C increase of the fraction of either D (ΔF lipid)or C(ΔF lipid) relative to the D fraction of nonlabeled samples, divided by the fraction of D (F medium)and Medium Isotopic Compositions. Hydrogen isotopic composition of the in- 13 13C C(F medium) in the incubated medium (Eqs. 1 and 2)(23): cubation medium was determined using cavity ring-down laser spectroscopy (Liquid Water Isotope Analyzer DLT-100; Los Gatos Research) after dilution Δ D Flipid prod = conclipid × [1] with pure water (1:100). The δ-values of DIC and CH4 were measured from the lipid D × Fmedium t headspace of acidified medium by gas chromatography coupled to isotope- ratio mass spectrometry (irMS) (VG Optima, Fisons; and Trace GC ultra + ΔF13C DeltaPlus XP isotope-ratio MS, ThermoFinnigan, respectively). assim = conc × lipid : [2] IC lipid 13C × Fmedium t Lipid Extraction and Sample Treatment. Lipids were extracted from enrich- D 13C ment slurries (∼4gdm), using a modified Bligh and Dyer protocol (41). The fractions of F and F in individual lipids and incubation medium are Bacterial membrane-derived FAs were retrieved by saponification and calculated from the isotope ratios FD = RD/H/(RD/H + 1); F13C = R13C/12C/(R13C/12C + 1),

δ SCIENCES analyzed as methyl esters after derivatization with BF3 and MeOH. Dou- where R derives from the -notations (Eqs. S2 and S3 and SI Text). Both prodlipid μ · –1· –1 ble-bond positions of FAs were identified by examining mass spectra and assimIC are expressed in glipidgdm y . ENVIRONMENTAL of their pyrrolidide derivatives (42) (Fig. S3). Ether-bound archaeal iso- prenoids were released using boron tribromide treatment to cleave ethers, ACKNOWLEDGMENTS. We thank the shipboard scientific crew and pilots of followed by reduction of the resulting alkyl bromides with superhydride the R/V Atlantis and Research Submersible Alvin. We thank Jenny Wendt and Arne Leider for technical support and Jan Hoffmann for the water (Aldrich) (43). The products were purified on a silica gel column. FAs and iso- isotopic composition analysis. This study was supported by the Deutsche fi prenoidal hydrocarbons were quanti ed by gas chromatography coupled to Forschungsgemeinschaft (through the Research Center/Excellence Cluster a flame ionization detector (GC-FID) (ThermoFinnigan), using squalane as an MARUM–Center for Marine Environmental Sciences and the graduate injection standard. school GLOMAR-Globale Change in the Marine Realm), the European Re- ’ The analysis of isoprenoidal hydrocarbons from intact polar lipids was search Council under the European Union s Seventh Framework Pro- gramme–“Ideas” Specific Programme, ERC grant agreement No. 247153 performed after purification by an orthogonal preparative high-perfor- (to K.-U.H.), the Alexander von Humboldt Foundation (to M.Y.Y.), the Gott- mance liquid chromatography method in normal and reversed mode (cf. ref. fried Wilhelm Leibniz Prize awarded to Antje Boetius (to G.W.), and the 32) (SI Text). Subsequently, intact polar tetraether fractions were subjected Max Planck Society (to G.W., T.H. and K.K.). Sample acquisition was sup- to ether cleavage as described above. ported by Grant NSF OCE-0647633 to Andreas Teske.

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