1 Supporting Information 2 3 Intramolecular stable carbon isotope probing of archaeal diglycosyl tetraether lipids, a globally 4 prominent group of intact polar lipids in marine subsurface sediment 5 by Lin et al. 6 7 8 Correspondence: yushih@uni-bremen.de 9 10 11 The Supporting Information file includes: 12 - Supporting Figure S1 13 - Supporting Table S1 14 - Tests with the TLE of [13C]S. platensis and Supporting Table S2 15 - IPL purification 16 - Stable isotopic analysis of glycerol from ether lipids 17 1 Tests with the TLE of [13C]S. platensis 2 To verify whether the positive isotopic values of glucose, galactose, and glycerol in the S. 3 plantensis-added sediment are actually derived from 2G-GDGTs, we performed a series of tests 4 to evaluate the effectiveness of our preparative HPLC protocols in separating sugar- and 5 glycerol-bearing compounds from the TLE of S. platensis. The TLE of a sediment sample taken 6 from the Peru Margin subsurface during Ocean Drilling Program (ODP) Leg 201 (ODP 7 201-1229A-6H-2, 109-119 cm, 42.5 m below seafloor) was split into two aliquots. One was 8 spiked with the TLE of [99%-13C]S. platensis and the other not spiked. The samples were 9 subjected to the orthogonal method developed in this study. We collected the lipid fraction in the 10 time window of 2G-GDGT elution and performed acid hydrolysis and ether cleavage for sugar 11 and glycerol analysis, respectively. 12 The results (Table S2) showed that galactose in the spiked sample was extraordinarily 13 13 14 glycerol lipids in the 2G-GDGT fraction. Accordingly, we disregarded the 13C-rich galactose in 15 the S. platensis incubation as a labeling signal (Fig. 3). However, the orthogonal preparative 16 HPLC protocol successfully separates glucose- and glycerol-containing compounds from the 17 TLE of [13C]S. platensis: the 13C values of glucose and glycerol in the spiked sample were 18 indistinguishable from those of the non-spiked sample. Based on these results, we considered the 19 positive 13C values of glucose and glycerol in the S. platensis-added sediment (Fig. 3) to be 20 indicators of label uptake into 2G-GDGTs. C-enriched, suggesting the presence of galactose-bearing compounds other than galactosyl 21 22 IPL purification 1 The TLE was initially purified following the published preparative HPLC protocol that 2 employs a normal-phase LiChrospher Si-60 column (250×10 mm, 5 µm particle size; Alltech 3 Associates Inc., Deerfield, IL, USA; Biddle et al., 2006). However, this purification protocol 4 failed to efficiently separate 2G-GDGTs from other co-eluting IPLs, such as 2G-OH-GDGT and 5 MGDGs, and from the polar organic matrix, which influences the subsequent isotopic analysis of 6 IPL-derived glycerol and sugars. To solve these problems, we developed an orthogonal 7 preparative HPLC method comprising a normal-phase preparative column followed by a 8 reversed-phase column. Samples were purified first by a LiChrospher Si-60 column following 9 published procedures (Biddle et al., 2006). The fraction containing 2G-(GDGTs+OH-GDGTs) 10 was subjected to a second step of purification using a Zorbax Eclipse XDB-C18 column (150×4.6 11 mm, 5 µm particle size; Agilent Technologies Deutschland GmbH, Böblingen, Germany). The 12 Zorbax Eclipse XDB-C18 column was operated at room temperature with a flow rate of 1 mL 13 min–1. The gradient was from 100% methanol to 100% isopropanol in 45 min, hold at 100% 14 isopropanol for 20 min, followed by column re-conditioning with 100% methanol for 15 min. To 15 establish the retention times of IPLs, a buffer solution (methanol/formic acid/14.8 M NH3(aq) = 16 100:0.48:0.16 v/v/v) was added post-column through a T-piece using a second HPLC pump to 17 assist ionization of the compounds by ESI. 18 19 20 Stable isotopic analysis of glycerol from ether lipids We first evaluated the method described in Takano et al. (2010) for detection of 21 lipid-derived glycerol as 1,2,3-tribromopropane. In short, standards of glycerol, 22 1,2-di-O-hexadecyl-rac-glycerol (Sigma-Aldrich GmbH, Munich, Germany) or 23 ß-L-gulosyl-phosphoglycerol dibiphytanyl glycerol tetraether (Gul-GDGT-PG; Matreya, LLC, 24 Pleasant Gap, PA, USA) were treated with 500 µL of 1 M BBr3 in dichloromethane (DCM) and 1 kept at 60°C for 2 h. After quenching the reaction with a few drops of Milli-Q water, another 500 2 µL DCM and 1 mL of Milli-Q water were added to the vials. The vials were vortexed, the DCM 3 phase was collected with a glass syringe, and the remaining mixture was washed four times with 4 1 mL DCM. All the DCM phases were combined in one vial and blown down gently to <100 µL 5 at 70°C. The volume of the remaining solvent was measured with a glass syringe and brought to a 6 defined volume with DCM before injection. However, only one of our multiple attempts was 7 successful in detecting glycerol as 1,2,3-tribromopropane. 8 Aiming for a more reproducible solution for glycerol detection, we developed a new 9 protocol that detects glycerol as 1,2,3-tris(trimethylsilanyloxy)propane (XVII in Fig. S1). Like 10 the protocol of Takano et al. (2010), standards of glycerol or ether lipids were treated with 1 M 11 BBr3 in DCM. After quenching the reaction with a few drops of Milli-Q water and adding DCM 12 (500 µL) and Milli-Q water (1 mL), liquid-liquid extraction was performed by washing the 13 aqueous phase with 1 mL DCM for a total of five times. While the organic phase was transferred 14 to another vial, the vial containing the aqueous phase and the remaining DCM were left open and 15 placed on a hot plate (70°C) to remove the DCM completely. The DCM-free aqueous phase was 16 then neutralized with silver carbonate (Sigma-Aldrich GmbH) and centrifuged at 800 g for 3 min, 17 and the supernatant was collected in a syringe and filtered through a Rotilabo PTFE syringe filter 18 (0.45 µm, 13 mm diameter; Carl Roth GmbH, Karlsruhe, Germany). The precipitates were 19 washed twice with 500 µL of Milli-Q water, and all the filtered supernatants were combined. The 20 aqueous solution was blown down mildly with N2 to <100 µL at 70°C and evaporated to dryness 21 at 70°C without a N2 stream. After addition of 500 µL cold (4°C) ethanol to the residue, the vials 22 were vortexed and centrifuged at 800 g for 3 min, and the supernatant was transferred to another 23 vial using a glass syringe. The washing step with cold ethanol was repeated for a total of three 24 times. The pooled ethanol, spiked with the derivatization standard 1-hexadecanol, was blown 1 down mildly with N2 to <100 µL at 70°C and evaporated to dryness by heat. The dried residue, 2 usually brownish red in color, was treated with 200 µL of pyridine and 100 µL 3 N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA) and kept at 70°C for 1 h. The derivatives 4 were purified using a self-packed silica gel column (0.4 g; Kieselgel, 0.06-0.2 mm, Carl Roth 5 GmbH). The silica gel was activated at 110°C for at least 2 h and deactivated with water (final 6 content = 5 mL H2O per 100 g silica gel) before use. The column was eluted with 5 mL of 7 hexane:ethyl acetate = 1:1 (v/v). The eluates were blown down mildly with N2 and, when a small 8 sample volume was needed, evaporated only passively at 70°C. The samples were brought to a 9 defined volume of hexane for injection. The recovery of glycerol from Gul-GDGT-PG was 14%, 10 and the isotopic value differed from offline-determined values of glycerol by 1-2‰ with a 11 standard error of 2‰. The accuracy of this method may not be sufficient to resolve small isotopic 12 differences (within 5‰) but is adequate for SIP work, in which the label uptake should result in a 13 large, positive shift of 13C values. 14 15 References 16 Biddle, J.F., Lipp, J.S., Lever, M.A., Lloyd, K.G., Sørensen, K.B., Anderson, R., et al. (2006) 17 Heterotrophic Archaea dominate sedimentary subsurface ecosystems off Peru. Proc Natl 18 Acad Sci USA 103: 3846-3851. 19 Takano, Y., Chikaraishi, Y., Ogawa, N.O., Nomaki, H., Morono, Y., Inagaki, F., et al. (2010) 20 Sedimentary membrane lipids recycled by deep-sea benthic archaea. Nature Geosci 3: 21 858-861. 22 1 Figure legends 2 3 Fig. S1. Molecular structures of major compounds mentioned in this work. The number in the 4 parentheses indicates the number of rings (both cyclopentane and cyclohexane rings) in the 5 molecule. 6