RESULTS

CHAPTER V Microbial ether lipid biogeochemistry in hydrothermal sediments

97 with the lack of vigorous hydrothermal circulation. In contrast, the hot hydrothermal sites at Ultra Mound and Cathedral Hill showed only partial sulfate depletion, most likely a consequence of hydrothermal circulation and seawater inmixing within the upper sediment layers (Teske et al., 2016) (Fig. V.1B).

Concentration and isotopic compositions of C1-C5 hydrocarbons and DIC. The gaseous hydrocarbon concentrations are affected by outgassing and bubble formation during core retrieval to the surface of the unpressurized cores, therefore here only residual porewater concentrations can be reported.

Methane was consistently the most abundant hydrocarbon at each site, followed by ethane, propane, butane and pentane, when detectable (Fig. V.1C). Methane concentration of Octopus Mound samples ranged between 0.01 and 0.1 mM, while they were slightly higher in the hydrothermally influenced sediments (Northern Tower Site 2, Site 3, Aceto Balsamico, Ultra Mound and Cathedral Hill), ranging from 0.1 to 1.5 mM with no apparent trend with depth (Fig. V.1C). Ethane concentrations ranged from 1 to 100 µM at the hydrothermally influenced sites, which are several orders of magnitude higher than those from the cold seep Octopus Mound. Concentrations of C3-C5 hydrocarbons were generally one to two orders of magnitude lower than those of ethane. The highest hydrocarbon contents were detected in oil impregnated sediments of Cathedral Hill (Fig. V.1C).

Isotopic compositions of methane (δ¹³C1) ranged from strongly ¹³C-depleted biogenic values (-74.9‰) towards ¹³C enrichment (-29.7‰) previously seen in sediments with active methane-oxidizing archaeal populations (McKay et al., 2016). The lightest methane (near and below -70‰) was found at the bottom and top of the cold seep Octopus Mound core, around a zone of ¹³C-enriched methane (up to -55.3‰) at approximately 7 cm depth (Fig. V.1D). Intermediate δ¹³C1 values ranging from -55.1 to -43.0‰, matching the previously reported range of hydrothermal methane at Guaymas Basin (Welhan and Lupton, 1987), were observed at the two warmer sites Northern Tower Site 2 and Aceto Balsamico, with the latter site exhibiting a sharp ¹³C-enrichement at the sediment surface. The least negative δ¹³C1 values that characterize methane impacted by microbial methane oxidation (-41.3 to -29.7‰) were observed at the hot site Ultra Mound, where ¹³C-enriched methane was observed at ca. 7 cm (Fig. V.1D). Similarly, δ¹³C1 values ranged from -44.0 to -39.7‰ for the remaining two hot sites Northern Tower Site 3 and Cathedral Hill (Fig. V.1D).

δ¹³C values for C2-C5 hydrocarbons ranged from -20 to -11‰ for ethane, -24 to -10‰ for propane, -24 to -21‰ for butane, and -24 to -18‰ for pentane (Fig. V.1D). In Ultra Mound sediments, δ¹³C values for ethane and propane were notably enriched in ¹³C within the upper 7 cm, roughly coinciding with the depths where ¹³C-enriched methane was observed. Similarly, ¹³C-enriched butane and particularly pentane were observed in Cathedral Hill sediments at depths between 5 to 11 cm.

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98

Figure V.1. Geochemical data of the six studied sites. The results are displayed from cool to warm to hot sites. A.

Extrapolated temperatures based on data from Alvin’s heat-flow probe; B. Sulfate and sulfide concentrations; C.

Concentrations of C1-C5 hydrocarbons; D. δ¹³C values of C1-C5 hydrocarbons; E. δ¹³C values of dissolved inorganic carbon (DIC).

δ¹³C values of DIC were particularly negative at Octopus Mound (-29.1±13.7‰; mean±SD; n=9), Ultra Mound (-10.7±6.5‰; mean±SD; n=9) and Cathedral Hill (-7.6±2.8‰; mean±SD; n=10). At Octopus Mound, δ¹³CDIC decreased with depth, whereas at Ultra Mound δ¹³CDIC first decreased (from -3‰ to -19‰) with depth until ~ 9 cm, then increased again to -2‰. At Cathedral Hill δ¹³CDIC increased gradually with depth (Fig. V.1E). In contrast, δ¹³CDIC values were comparably positive at Northern Tower Site 2 (5.8±1.3‰;

mean±SD; n=5), Aceto Balsamico (4.8±4.2‰; mean±SD; n=12) and Northern Tower Site 3 (3.8±1.1‰;

mean±SD; n=10), with values from the first two sites increasing with depth.

CHAPTER V Microbial ether lipid biogeochemistry in hydrothermal sediments

99 V.3.2. Distribution of microbial lipids

The highest archaeal IPL concentration was observed at the cold seep site Octopus Mound (2.4 µg/g dry weight sediment; DW) at ~ 6 cm, followed by hot sites Ultra Mound (0.4 µg/g DW at ~ 9 cm) and Cathedral Hill (0.3 µg/g DW at ~ 5 cm) and warm site Aceto Balsamico (0.2 µg/g DW at ~ 10 cm). IPL concentrations at the hot Northern Tower Site 3 and cool Site 2 were overall low (< 0.1 and < 0.01 µg/g DW, respectively) (Fig. V.2A). In comparison, total archaeal core lipids (CLs) concentrations were one to three orders of magnitude higher than IPL concentrations in the same samples. The highest CLs concentration was also found at Octopus Mound at ~ 6 cm, whereas CLs concentrations generally decreased with depth at the other sites (Fig. V.2A).

Archaeal intact polar lipids. Among the studied sites, monoglycosidic glycerol dialkyl glycerol tetraether (1G-GDGT) was predominant in most of the sediment layers under cold to hot conditions (Fig.

V.2B). 2G-GDGT accounted < 10% of total IPLs at the cold seep Octopus Mound. At the other sites it was only detected in some sediment layers, predominantly in the first 5 cm (10-20% of total IPLs) (Fig. V.2B).

In both of the 1G-GDGT and 2G-GDGT pools (Fig. V.3A, B), GDGT-2 and GDGT-1 dominated at the cold seep site Octopus Mound, whereas the contribution of GDGT-4 increased significantly with elevated temperatures in Northern Tower Site 3 sediments and was consistently abundant in Ultra Mound sediments and dominated the surficial sediments of Cathedral Hill. Only very few sediment layers contained detectable amounts of IPLs at Cathedral Hill. We assign this lack in detectability to the high oil content at this site, which probably increased ion suppression during analysis. At the less hydrothermally influenced sites Northern Tower Site 2 and Aceto Balsamico, ring distribution in the 1G-GDGT and 2G-GDGT pools was distinct, at both sites 1G-GDGT pool was dominated by GDGT-0 and GDGT-5 (we expect GDGT-5 to be crenarchaeol according to the retention time reported by Zhu et al., 2014b, however, we have not confirmed this with IPL-derived core lipid measurements), while 2G-GDGT was dominated by 2 and GDGT-1 (Fig. V.3A, B).

Notable amounts of phosphatidylglycerol (PG-) GDGT were detected in near surface sediments (< 10 cm) of both cold (Octopus Mound, Northern Tower Site 2) and hot sites (Ultra Mound, Cathedral Hill).

PG-GDGT also displayed different ring distribution in sediments from cold and hot sites, with GDGT-2 and 3 dominating in sediments from Octopus Mound and Northern Tower Site 2, whereas GDGT-4 was detected in surface sediments of Cathedral Hill and its relative abundance increased with depth at Ultra Mound (Fig. V.3C).

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100

Figure V.2. Distribution of archaeal lipids at studied sites. A. Absolute total concentrations of intact polar lipids (IPLs) and core lipids (CLs), note the different scales between sites; B. Relative abundance of IPLs; C. Relative abundance of CLs among the six studied sites. nd, not detected.

Hydroxylated GDGT (OH-GDGT and 2OH-GDGT) with 1G and 2G head groups were minor lipids detected at some of the sites. While 1G-2OH-GDGT was exclusively present at Octopus Mound, 1G-OH-GDGT was also detected in Aceto Balsamico sediments and found with higher amounts at Northern Tower Site 3; 2G-OH-GDGT was found abundantly in sediments where temperatures exceeded 15 °C with the exception of Ultra Mound. The minor lipid isoprenoid glycerol dibiphytanol diether (GDD) with 1G head group was only detected in Cathedral Hill sediments at 5 cm (Fig. V.2B).

Intact AR and hydroxy (OH-) AR with 1G and 2G head groups were only detected in surface sediments where in situ temperatures were below 10 °C. Unsaturated GDGT (unsGDGT) with 1G and 2G head groups were exclusively detected at Octopus Mound. Furthermore, in surface layer of Aceto Balsamico low abundance of PG-AR and PG-OH-AR (together < 3% of total IPLs) were observed.

1G-GMGTs were predominantly detected in sediments from hot sites, and were barely detectable at greater depth of warm site Aceto Balsamico (Fig. V.2B). 1G-GMGT comprised around 20-40% in the deeper layers of Northern Tower Site 3 and overwhelmingly dominated the subsurface sediments of Ultra Mound and Cathedral Hill, where it comprised > 80% of the total IPL pool (Fig. V.2B). 1G-GMGT was detected with up to 4-5 rings, while relative abundance of 1G-GMGT-0 increased with depth at Northern Tower Site 3, 1G-GMGT-4 comprised > 20% at most depths at Northern Tower Site 3 and Ultra Mound and dominated at Cathedral Hill, where it decreased with depth (Fig. V.4A). 1G-GMGT-5 was only detected

CHAPTER V Microbial ether lipid biogeochemistry in hydrothermal sediments

101 in Cathedral Hill at 17 cm. 2G-GMGT (accounting 15% of total IPLs) with up to 3 rings was only detected at 5 cm at Northern Tower Site 3.

Figure V.3. Relative abundance of intact and core GDGTs among the six studied sites. nd, not detected. A. 1G-GDGTs; B. 2G-1G-GDGTs; C. PG-1G-GDGTs; D. C-GDGTs. E. Cren’ and GDGT-5 to GDGT-8.

Archaeal core lipids. Mirroring the IPL distribution, with a shift from intact GDGT to intact GMGT in sediments from low to higher temperatures, core lipids exhibited a similar dominance of GDGT in cold sediments, while GMGT increased with temperature and were particularly abundant at hot sites Ultra Mound and Cathedral Hill (Fig. V.2C). Similarly, the ring distribution of GDGT was also distinct in sediments from cold to hot sites, where GDGT-0 and crenarchaeol (composed of cyclopentane and one cyclohexane rings) dominated the cooler sites Octopus Mound, Northern Tower Site 2 and Aceto Balsamico, and GDGT-4 increased with temperature and became dominant at Ultra Mound and Cathedral

V.3. RESULTS

102

Hill. Notably, relative abundances of GDGT-1 to GDGT-3 were elevated in sediments of both the cold seep site Octopus Mound and the hot sites Ultra Mound and Cathedral Hill. Minor amounts of GDGT-5 (with five cyclopentane rings) were detected in Ultra Mound (< 0.3% of core GDGTs), whereas up to GDGT-8 were detected in sediments of Cathedral Hill and Northern Tower Site 3 (0.2-2% of total GDGTs; Fig.

V.3C).

Another abundant core lipid was AR, which varied from 1 to 30% in the core lipid pool with no apparent trend linked to temperature (Fig. V.2C). Additionally, OH-AR and unsGDGT were particularly abundant (up to 20-30% of core lipid pool) at Octopus Mound (Fig. V.2C). Some other detectable minor core lipids include OH-GDGT and 2OH-GDGT, which had relatively high abundances at Octopus Mound and Aceto Balsamico, and were barely detectable at Northern Tower Site 2 and 3. GDD was detected at all sites with low abundance, while OH-GDD was only detected in deeper sediments of Octopus Mound and surficial sediments of Northern Tower Site 3 (Fig. V.2C).

Relative abundance of core GMGT, together with its mono- and di-methylated analogs (Me-GMGT and 2Me-GMGT) increased significantly in sediments from hot sites Ultra Mound and Cathedral Hill, while only minor amounts of these compounds were detected in the third hot site Northern Tower Site 3 (Fig.

V.2C). Core GMGT with 0-8 rings were detected at all of the three hot sites (Fig. V.4B), with GMGT-4 (accounting for 30-60% of the GMGT pool) predominating most depths. Relative abundance of GMGT-5 to GMGT-8 were elevated in Cathedral Hill and Northern Tower Site 3 sediments, accounting for 10-40%

of the GMGT pool (Fig. V.4B). Analogous to GMGT, Me-GMGT and 2Me-GMGT were also dominated by four rings, whereas contribution of 5-8 rings were more pronounced (Fig. V.4C, D).

Meanwhile, H-shaped glycerol monoalkyl diether (GMD) and H-tetrols with 80 to 82 carbon numbers were detected at these hot sites, and small amount of H-shaped acids (H-acids) were detected at the Ultra Mound where in situ temperature was around 60 °C (Fig. V.2C). GMD with up to 8 rings were detected and were mostly dominated by GMD-4 and GMD-0 in Ultra Mound sediments, whereas GMD with 5-8 rings were more abundant at Cathedral Hill (Fig. V.4E). While H-C80-tetrol was dominated by 0-4 rings, the contribution of 5-8 rings increased significantly in H-C81-tetrol and particularly in H-C82-tetrol (Fig.

V.4F-H).

CHAPTER V Microbial ether lipid biogeochemistry in hydrothermal sediments

103 Figure V.4. Relative abundance of H-shaped tetraethers, diether and H-tetrols (A-H) in sediments from the three hot sites Ultra Mound, Cathedral Hill (Marker 24) and Northern Tower Site 3.

Branched GDGTs. The brGDGT and its analogs with higher or lower degrees of methylation, i.e., OB-GDGT and SB-OB-GDGT, as well as IB-OB-GDGT were also detected at all six sites. The total concentrations of these compounds ranged from 0.01 to 1.0 µg/g DW (Fig. V.5A); thus the total branched GDGTs pool equates in size to 1-3% of the archaeal core lipid pool. While brGDGT and IB-GDGT predominated the most of the investigated sites (Octopus Mound, Northern Tower site 2, Aceto Balsamico and Northern