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Supporting Information
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Intramolecular stable carbon isotope probing of archaeal diglycosyl tetraether lipids, a globally
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prominent group of intact polar lipids in marine subsurface sediment
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by Lin et al.
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Correspondence: yushih@uni-bremen.de
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The Supporting Information file includes:
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Supporting Figure S1
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Supporting Table S1
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Tests with the TLE of [13C]S. platensis and Supporting Table S2
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IPL purification
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Stable isotopic analysis of glycerol from ether lipids
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Tests with the TLE of [13C]S. platensis
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To verify whether the positive isotopic values of glucose, galactose, and glycerol in the S.
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plantensis-added sediment are actually derived from 2G-GDGTs, we performed a series of tests
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to evaluate the effectiveness of our preparative HPLC protocols in separating sugar- and
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glycerol-bearing compounds from the TLE of S. platensis. The TLE of a sediment sample taken
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from the Peru Margin subsurface during Ocean Drilling Program (ODP) Leg 201 (ODP
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201-1229A-6H-2, 109-119 cm, 42.5 m below seafloor) was split into two aliquots. One was
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spiked with the TLE of [99%-13C]S. platensis and the other not spiked. The samples were
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subjected to the orthogonal method developed in this study. We collected the lipid fraction in the
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time window of 2G-GDGT elution and performed acid hydrolysis and ether cleavage for sugar
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and glycerol analysis, respectively.
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The results (Table S2) showed that galactose in the spiked sample was extraordinarily
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glycerol lipids in the 2G-GDGT fraction. Accordingly, we disregarded the 13C-rich galactose in
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the S. platensis incubation as a labeling signal (Fig. 3). However, the orthogonal preparative
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HPLC protocol successfully separates glucose- and glycerol-containing compounds from the
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TLE of [13C]S. platensis: the 13C values of glucose and glycerol in the spiked sample were
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indistinguishable from those of the non-spiked sample. Based on these results, we considered the
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positive 13C values of glucose and glycerol in the S. platensis-added sediment (Fig. 3) to be
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indicators of label uptake into 2G-GDGTs.
C-enriched, suggesting the presence of galactose-bearing compounds other than galactosyl
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IPL purification
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The TLE was initially purified following the published preparative HPLC protocol that
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employs a normal-phase LiChrospher Si-60 column (250×10 mm, 5 µm particle size; Alltech
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Associates Inc., Deerfield, IL, USA; Biddle et al., 2006). However, this purification protocol
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failed to efficiently separate 2G-GDGTs from other co-eluting IPLs, such as 2G-OH-GDGT and
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MGDGs, and from the polar organic matrix, which influences the subsequent isotopic analysis of
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IPL-derived glycerol and sugars. To solve these problems, we developed an orthogonal
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preparative HPLC method comprising a normal-phase preparative column followed by a
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reversed-phase column. Samples were purified first by a LiChrospher Si-60 column following
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published procedures (Biddle et al., 2006). The fraction containing 2G-(GDGTs+OH-GDGTs)
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was subjected to a second step of purification using a Zorbax Eclipse XDB-C18 column (150×4.6
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mm, 5 µm particle size; Agilent Technologies Deutschland GmbH, Böblingen, Germany). The
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Zorbax Eclipse XDB-C18 column was operated at room temperature with a flow rate of 1 mL
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min–1. The gradient was from 100% methanol to 100% isopropanol in 45 min, hold at 100%
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isopropanol for 20 min, followed by column re-conditioning with 100% methanol for 15 min. To
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establish the retention times of IPLs, a buffer solution (methanol/formic acid/14.8 M NH3(aq) =
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100:0.48:0.16 v/v/v) was added post-column through a T-piece using a second HPLC pump to
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assist ionization of the compounds by ESI.
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Stable isotopic analysis of glycerol from ether lipids
We first evaluated the method described in Takano et al. (2010) for detection of
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lipid-derived glycerol as 1,2,3-tribromopropane. In short, standards of glycerol,
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1,2-di-O-hexadecyl-rac-glycerol (Sigma-Aldrich GmbH, Munich, Germany) or
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ß-L-gulosyl-phosphoglycerol dibiphytanyl glycerol tetraether (Gul-GDGT-PG; Matreya, LLC,
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Pleasant Gap, PA, USA) were treated with 500 µL of 1 M BBr3 in dichloromethane (DCM) and
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kept at 60°C for 2 h. After quenching the reaction with a few drops of Milli-Q water, another 500
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µL DCM and 1 mL of Milli-Q water were added to the vials. The vials were vortexed, the DCM
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phase was collected with a glass syringe, and the remaining mixture was washed four times with
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1 mL DCM. All the DCM phases were combined in one vial and blown down gently to <100 µL
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at 70°C. The volume of the remaining solvent was measured with a glass syringe and brought to a
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defined volume with DCM before injection. However, only one of our multiple attempts was
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successful in detecting glycerol as 1,2,3-tribromopropane.
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Aiming for a more reproducible solution for glycerol detection, we developed a new
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protocol that detects glycerol as 1,2,3-tris(trimethylsilanyloxy)propane (XVII in Fig. S1). Like
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the protocol of Takano et al. (2010), standards of glycerol or ether lipids were treated with 1 M
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BBr3 in DCM. After quenching the reaction with a few drops of Milli-Q water and adding DCM
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(500 µL) and Milli-Q water (1 mL), liquid-liquid extraction was performed by washing the
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aqueous phase with 1 mL DCM for a total of five times. While the organic phase was transferred
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to another vial, the vial containing the aqueous phase and the remaining DCM were left open and
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placed on a hot plate (70°C) to remove the DCM completely. The DCM-free aqueous phase was
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then neutralized with silver carbonate (Sigma-Aldrich GmbH) and centrifuged at 800 g for 3 min,
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and the supernatant was collected in a syringe and filtered through a Rotilabo PTFE syringe filter
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(0.45 µm, 13 mm diameter; Carl Roth GmbH, Karlsruhe, Germany). The precipitates were
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washed twice with 500 µL of Milli-Q water, and all the filtered supernatants were combined. The
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aqueous solution was blown down mildly with N2 to <100 µL at 70°C and evaporated to dryness
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at 70°C without a N2 stream. After addition of 500 µL cold (4°C) ethanol to the residue, the vials
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were vortexed and centrifuged at 800 g for 3 min, and the supernatant was transferred to another
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vial using a glass syringe. The washing step with cold ethanol was repeated for a total of three
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times. The pooled ethanol, spiked with the derivatization standard 1-hexadecanol, was blown
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down mildly with N2 to <100 µL at 70°C and evaporated to dryness by heat. The dried residue,
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usually brownish red in color, was treated with 200 µL of pyridine and 100 µL
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N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA) and kept at 70°C for 1 h. The derivatives
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were purified using a self-packed silica gel column (0.4 g; Kieselgel, 0.06-0.2 mm, Carl Roth
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GmbH). The silica gel was activated at 110°C for at least 2 h and deactivated with water (final
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content = 5 mL H2O per 100 g silica gel) before use. The column was eluted with 5 mL of
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hexane:ethyl acetate = 1:1 (v/v). The eluates were blown down mildly with N2 and, when a small
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sample volume was needed, evaporated only passively at 70°C. The samples were brought to a
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defined volume of hexane for injection. The recovery of glycerol from Gul-GDGT-PG was 14%,
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and the isotopic value differed from offline-determined values of glycerol by 1-2‰ with a
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standard error of 2‰. The accuracy of this method may not be sufficient to resolve small isotopic
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differences (within 5‰) but is adequate for SIP work, in which the label uptake should result in a
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large, positive shift of 13C values.
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References
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Biddle, J.F., Lipp, J.S., Lever, M.A., Lloyd, K.G., Sørensen, K.B., Anderson, R., et al. (2006)
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Heterotrophic Archaea dominate sedimentary subsurface ecosystems off Peru. Proc Natl
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Acad Sci USA 103: 3846-3851.
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Takano, Y., Chikaraishi, Y., Ogawa, N.O., Nomaki, H., Morono, Y., Inagaki, F., et al. (2010)
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Sedimentary membrane lipids recycled by deep-sea benthic archaea. Nature Geosci 3:
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858-861.
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Figure legends
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Fig. S1. Molecular structures of major compounds mentioned in this work. The number in the
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parentheses indicates the number of rings (both cyclopentane and cyclohexane rings) in the
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molecule.
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