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Rapid screening of glycerol ether lipid biomarkers in recent marine sediment using APPI-P FTICR-MS. Jagoš R. Radovi#, Renzo C. Silva, Ryan Snowdon, Stephen R. Larter, and Thomas B.P. Oldenburg Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.5b02571 • Publication Date (Web): 07 Dec 2015 Downloaded from http://pubs.acs.org on December 12, 2015

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Analytical Chemistry

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Rapid screening of glycerol ether lipid biomarkers in recent marine

2

sediment using APPI-P FTICR-MS.

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Jagoš R. Radović*,1 Renzo C. Silva,1 Ryan Snowdon,1 Stephen R. Larter1, and Thomas B.P.

4

Oldenburg1

5

1

6

Calgary, AB, Canada.

PRG, Department of Geoscience, University of Calgary, 2500 University Drive NW, T2N 1N4,

7 8

*corresponding author

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Email: [email protected]

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Tel: +1 (403) 220-3916

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Fax: +1 (403) 220-8618

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Abstract.

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Many of the molecular proxies commonly used for paleoenvironmental reconstruction are

15

focused on a limited set of glycerol ether lipids, mainly due to the lack of more comprehensive

16

analytical methods and instrumentation able to deal with a more diverse range of species. In this

17

study, we describe an FTICR-MS based method, for rapid, non-targeted screening of ether lipid

18

biomarkers in recent marine sediments. This method involves simplified sample preparation, and

19

enables rapid identification of known, and novel ether lipid species. Using this method we were

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able to identify complete series of core glycerol dialkyl glycerol tetraethers (GDGTs with 0 to 8

21

alicyclic rings), including the complete resolution of GDGT-4, and the unexpected detection of

22

GDGTs with more than 5 rings, in sediments from mesophilic marine environments (sea surface

23

temperature, SST, of 24–25 °C). Additionally, mono- and dihydroxy-GDGT analogs (including

ACS Paragon Plus Environment


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novel species with >2 rings), as well as glycerol dialkanol diethers, GDDs (including novel

2

species with >5 rings) were detected. Finally, we putatively identified other, previously

3

unreported groups of glycerol ether lipid species. Adequacy of the APPI-P FTICR-MS data for

4

the determination of commonly used GDGT-based proxy indices was demonstrated. The results

5

of this study show great potential for the use of FTICR-MS as both a rapid method for

6

determining existing proxy indices and perhaps more importantly, as a tool for the early

7

detection of possible new biomarkers and proxies that may establish novel geochemical

8

relationships between archaeal ether lipids and key environmental, energy and climate related

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system variables.

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Abstract/TOC Graphic.

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Introduction.

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Glycerol ether lipids are cell membrane constituents of Archaea, ubiquitous prokaryotic

3

microorganisms, which can be found in diverse aquatic systems from polar oceans to terrestrial

4

hot springs.1,2

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(GDGTs), molecules comprised of two isoprenoid chains which are bonded to two terminal

6

glycerol groups via ether linkages, and can include various numbers of alicyclic moieties, Figure

7

1.3 If polar head groups (e.g. phosphate, hexose), are still attached to this structure, they are

8

known as the intact GDGTs. After cell death, the polar head groups are lost, as intact molecules

9

degrade in water columns and sediments, leaving only the recalcitrant ether-bound isoprenoidal

10

skeleton. This form is called the core GDGT.4 Numerous studies in the past few decades

11

demonstrated the correlation between the distribution of different GDGT species in sediments

12

and environmental parameters, such as the water temperature and pH.5 This has led to the

13

increasing use of the distribution of certain GDGT biomarkers as environmental proxies and the

14

development of various indices, e.g. TEX86, which have been used to reconstruct past sea surface

15

temperatures (SST) from GDGT distributions.6 Such SST estimates have become important

16

components of climate change discussions and mediation strategies.

Predominantly, these lipids consist of glycerol dialkyl glycerol tetraethers

17

Typically, for the determination of GDGT indices, concentrations of the core GDGT

18

components are determined by normal-phase liquid chromatography, (U)HPLC, coupled to mass

19

spectrometry (MS) detectors using atmospheric pressure chemical ionization (APCI).7 On the

20

other hand, the method for analysis of the intact GDGTs, requires LC separation using aqueous

21

eluents, followed by tandem MS with electrospray ionization (ESI) interface.8 Simultaneous

22

analysis of both intact and core GDGTs, by reversed-phase liquid chromatography was also

23

reported.9 Though viable, these methods include several extraction, separation, fractionation and

24

derivatization steps, in order to obtain and analyze the lipid fraction of interest and improve

25

chromatographic separation.7,10 Finally, detectors in most analytical configurations (e.g.

26

quadrupole, ion trap mass spectrometers), do not have the ultra-high mass resolution needed to

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differentiate potential mass interferences in GDGT analysis. Therefore, there is a high likelihood

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that such targeted, low-resolution methods may fail to observe non-targeted, novel lipid

29

biomarkers, which could provide significant additional information to current debates in the

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paleoenvironmental community.5 Indeed, recent reports suggest much more complex



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distributions of sedimentary glycerol ether lipids are common, including the presence of novel

2

species such as glycerol dialkanol diethers (GDDs), and hydroxy-GDGTs, among others (Figure

3

1).10,11

(A)

(B) -C3H4O -56.0262 Da -1 DBE +15.9949 Da

X&Y= n= 0 to 8 → DBE= 1 to 9

+15.9949 Da

4 5

Figure 1. (A) General structure of glycerol dialkyl glycerol tetraether (GDGT) and (B) glycerol

6

dialkanol diether (GDD) molecules. X and Y are isoprenoid chains with various possible

7

numbers (0 to 8), of embedded alicyclic rings and hydroxyl groups (0 to 2). These elements can

8

be combined to form numerous potential ether lipid species and their isomers and the potential

9

structures can be assessed for actual occurrence by examination of observed FTICR-MS peaks.

10

A double bond equivalent (DBE) measure for each molecule, calculated from the molecular

11

formula, is defined as the number of rings plus double bonds involving carbon, present in each

12

molecule.

13

In some regions (e.g. polar ocean settings), major discrepancies exist between some of

14

the water body temperature indices, such as TEX86 and established SST proxy calibrations.

15

Despite widespread use, generic challenges remain in the quantitative use of these proxies in

16

many applications. These include uncertainties about the spatial origins of the proxies (where in

17

the water column do the biomarker source organisms exist?), the often complex mechanisms of

18

proxy function (how does GDGT structure relate to environmental parameters?), the impacts of


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component mixing and other mass transport phenomena on the various proxy compound

2

distributions and inevitably, proxy calibration issues.5 Thus, the examination of GDGT

3

distributions in the environment requires more comprehensive and rapid analytical methods, free

4

from resolution, separation and artifact issues, which could help to better understand the origins

5

and environmental dependence of these species.

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A useful strategy in this direction is the use of ultra-high resolution Fourier transform

7

mass spectrometry (FTICR-MS). The advantages of FTICR-MS include broad range and high

8

resolution of mass detection, as well as good analyte sensitivity.12 In conjunction with diverse

9

ionization modes, it can enable more comprehensive multi-target screening of species present in

10

complex systems, such as marine sediments, after minimal sample preparation, and is ideal for

11

detection of previously non-targeted species. FTICR-MS has a well-proven track record in the

12

analysis of complex mixtures such as petroleum,13 and has been successfully used for

13

characterization of marine dissolved organic matter (DOM).14 Only recently, it was applied to

14

analyze GDGTs in marine sediments. However, due to the limitations of the ionization

15

techniques used, only the most abundant species were targeted for proxy index calculations.15 In

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this study, GDGT-indices were calculated for a series of marine sediments, using peak intensities

17

in the FTICR-MS spectra of extracted material, and not absolute molar concentrations of

18

commonly targeted species. Rudimentary quantitative analysis capabilities using FTICR-MS

19

have been already reported,16,17 which suggests such calculations are reasonable. This has

20

provided interest in further development and improvement of FTICR-MS methods, which could

21

enable more in-depth characterization of sedimentary biomarkers, and possibly therefore,

22

development of new proxies, as well as the reevaluation of the existing proxy indices.

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Herein, we demonstrate a method for screening of glycerol ether lipids in the whole

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solvent extracts of recent marine sediments from the Gulf of Mexico (GoM) using atmospheric

25

pressure photoionization in positive mode (APPI-P) coupled to ultra-high resolution FTICR-MS.

26

It enables rapid identification of a broad range of core GDGT species containing different

27

numbers of alicyclic rings, including their hydroxylated analogs. In addition, the presence of

28

several novel and unexpected glycerol ether lipid species is reported and discussed.

29



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Experimental section.

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Chemicals. All solvents used were of high purity, and sodium sulfate was of reagent

5

grade. An Ultramark 1621 (Alfa Aesar, MA, USA), mass spectroscopy standard containing

6

fluorinated phosphazines was used as calibration standard.

7

GDGT standard. A GDGT-5 isolate was provided by the Pearson Lab – Laboratory for

8

Molecular Biogeochemistry and Organic Geochemistry, Department of Earth and Planetary

9

Sciences at the Harvard University (Cambridge, MA, USA).

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Samples. Sediment cores were collected in the northeastern GoM in the August of 2014

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using an Ocean Instruments (San Diego, CA, USA) MC-800 multicoring system. The first core

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(GoM1) was taken from offshore Louisiana (28°43'27.7"N, 88°23'14.2"W) from 1100 m water

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depth and the second one (GoM2), from further to the southwest (28°14'23.0"N, 89°07'14.4"W)

14

in 1200 m water depth. Cores were refrigerated (at approx. 4 °C), until sub-sampled by layer

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extrusion, at 2 mm intervals for the upper 50 mm and at 5 mm intervals for the remainder of the

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core, using a calibrated, threaded-rod extrusion device.18 Chronostratigraphy was established

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through short-lived radionuclide (210Pb) geochronology, as reported elsewhere.19,20

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Six subsamples (up to 130 mm depth), of the GoM1 core, and two subsamples (2–4 mm

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and 188-193 mm), of the GoM2 core, were then lyophilized for 24 h (2 mm layers), or 40 h (5

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mm layers), at 0.010–0.014 mbar and -55 °C, using a FreeZone® Freeze Dry System (Labconco,

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MO, USA). The subsamples were then stored in pre-cleaned amber jars in the dark until

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extraction.

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Sample Extraction. Approximately 1.0 g of a freeze-dried sample was extracted 5 times

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by ultrasonication with 10 ml of a 9:1 (v:v) dichloromethane:methanol (DCM:MeOH), solvent

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mixture at ambient temperature for 30 min. Following each extraction, extracts were dried over

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anhydrous sodium sulfate, and finally the combined extract was rotary evaporated to

27

approximately 10 ml.


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FTICR-MS Analysis. Samples were analyzed using a 12 T Bruker Solarix FTICR-MS

2

located at the University of Calgary. Small aliquots of the DCM:MeOH extract were blown to

3

dryness under a gentle N2 stream, and then reconstituted in toluene to 1.25 mg/mL final

4

concentration. The solution was then introduced into the ionization source using a syringe pump

5

set to deliver 200 µL/h. APPI-P, with a krypton lamp at 10.6 eV, was used as the ionization

6

mode. The instrument was tuned and optimized using the Ultramark® 1621 standard mixture

7

(calibration range from m/z 1000 to 2000), which was also included in each sample as an internal

8

standard to ensure mass accuracy. Ions ranging from m/z 1100 to 1500 accurate mass were

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isolated by a linear quadrupole and accumulated over 2 s in the collision cell, before being

10

transferred to the ICR cell. For each sample, a hundred transients of 6.29 s each were collected

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and summed to improve the experimental signal/noise ratio. The achieved resolving power

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(m/∆m50%) was higher than 500,000 at m/z 1300, and mass accuracy, assessed using the

13

calibration standard, was typically better than 0.6 ppm. The FTICR-MS raw data were processed

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using the CaPA v.1 (Aphorist Inc.) software package.

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Regarding sample consumption and instrument time, approximately 58 µg of the whole

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sediment extract is consumed in 14 minutes of FTICR-MS analysis, based on syringe flow rate,

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experimental time and sample concentration (200 µL/h × 0.23h × 1.25 µg/µL). Using this

18

approach, the time from sample injection of the whole extract to final results for a routine

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analysis of targeted compounds, used to calculate paleoenvironmental proxy indices (i.e. core

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GDGT-0 to -5) is approximately 20 minutes.

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Results and Discussion.

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Sample Extraction. In contrast to the laborious, multiple-step sample preparation

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procedures typically used in GDGT analyses,7,10 a simple, one-step extraction was used in this

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study, greatly reducing the time and resources needed. Relatively small quantities of sediment

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(approx. 1 g dry mass) were extracted, compared to the several grams, to tens of grams typically

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used elsewhere.7,10 This enables much higher sampling frequency and consequently improved

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stratigraphic and temporal analytical resolution along the sediment core. After extraction,

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between 1 and 2 mg of whole sediment extract was obtained, which, given the small quantity of

29

sample used during each run, allows for multiple FTICR-MS analyses. There were no additional

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fractionation or derivatization steps involved, avoiding potential analyte loss or alteration. In this



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way, a more complete inventory of ether lipids is preserved, along with other types of

2

sedimentary biomarkers (e.g. pigments). Thus, data obtained using this protocol should be able

3

to offer a more comprehensive insight into the complex composition and relationships of a

4

diverse range of species in marine sediments and indeed, the analysis of the sediment extracts

5

described here indicates great compositional complexity. Thousands of individual peaks were

6

detected, including those ascribed to various biomarker molecules (e.g. pigments), as well as

7

numerous still unidentified species, possibly with proxy potential (Figure 2A). These species will

8

be the focus of future studies and will not be discussed further here.

9

Detection of GDGTs in sediment core samples. Based on the preliminary broad-mass

10

range (m/z 200–1400) screening of selected samples (Figure 2A), and published reports,10 the

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FTICR-MS method was optimized for the region between m/z 1100 and 1500 in order to obtain

12

the best possible resolution and sensitivity for a range of isoprenoidal ether lipid species. While

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branched GDGTs have molecular masses below the lower limit of the selected range, and

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although it is thought they might be present in marine sediments (and not only in soils and

15

peats),21 preliminary investigations did not confirm their abundant presence in the analyzed

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samples, therefore we focused on species in the m/z range reported above. If needed, the mass

17

window could be easily modified to fit other compounds of interest.

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As shown in the spectra in the Figure 2A, APPI-P demonstrated good ionization

19

efficiency in a broad mass range, including the high m/z (>1000) components, in agreement with

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reports claiming its comparable or better sensitivity than APCI for a broad range of species of

21

different polarities and molecular weights.22 This ionization mode is softer than the ones used in

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recent FTCIR-MS studies of GDGTs, e.g. laser desorption ionization (LDI),15 which can lead to

23

in-source fragmentation of molecules. Protonated pseudomolecular ions, [M+H]

24

GDGTs, as well as of their isotopologs, were observed as prominent peaks in the broad-range

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mass spectra of sediment extracts (Figure 2A). Throughout the paper, elemental formulae and

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m/z of the species identified in this study will correspond to the protonated pseudomolecular

27

ions. Furthermore, pertinent calculations on relative abundances and paleoenvironmental indices,

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will be reported based on the intensity (i.e. peak height) of the dominant monoisotopic peak.

29

Although it is plausible to assume that the protonation efficiency will not drastically change

30

between different core GDGT species that only differ in the structure of the alkyl chains (i.e.


+

of core

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number of embedded alicyclic rings), we recognize the need to conduct experiments with

2

standards to test this. However, other groups of ether lipid species, such as hydroxy- analogs and

3

GDDs could possibly have quite different ionization responses. This will be the focus of our

4

future studies whenever authentic standards are available.

5

As expected,6 the most abundant peaks identified in the spectra, were recognized as

6

GDGTs with five alicyclic rings (crenarchaeol and its isomers, GDGT-5) and GDGT species

7

with no cyclic moieties in the isoprenoidal chains (caldarchaeol, GDGT-0), Figure 2B. In order

8

to obtain satisfactory signal to noise (S/N) ratios for the minor ether lipids, a 2 s ion

9

accumulation time was used in all the hundred scans, data being collected over approximately 14

10

minutes (Table 1). Typical (U)HPLC methods for GDGT analysis last about an hour,9 making

11

our method about four times faster. If only the major peaks typically used for the SST

12

reconstruction and calculation of other environmental indices are targeted (e.g. compounds

13

GDGT-5, GDGT-0, etc.), this method could be made even more rapid, obtaining an S/N ratio

14

around 1000 in less than 2 minutes. Analysis of the enriched chrenarcheol standard sample

15

(GDGT-5 isolate), showed that 10 ng of material injected into the instrument was enough to

16

generate an S/N ratio higher than 100,000 for the monoisotopic peak of GDGT-5. Evaluation of

17

limits of detection and other absolute quantitation aspects are a natural next step for this study.



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Figure 2. (A) The broad mass spectrum (m/z 200–1400) of a whole GoM sediment extract,

3

demonstrating the plethora of detected compounds, including various sedimentary biomarkers

4

and other still unidentified compounds with possible proxy potential. The black rectangle

5

indicates the spectral region targeted in this study. (B) Mass spectra showing the approximate

6

regions where peaks of core GDGTs and their analogs were detected. Note the pronounced

7

difference in intensity of major core species and their analogs.

8


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The ultrahigh-resolution of the FTICR-MS (> 500,000 at m/z 1300) and the low accurate

2

mass error (typically 500,000).

14

Unexpectedly, we were also able to detect peaks that likely correspond to GDGT-6 to -8

15

species, amounting to approximately 2.40% of the total intensity of core GDGTs detected,


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assuming similar response factors for all core GDGT species (Table 1). Highly cyclized GDGTs

2

(>5 embedded rings), are commonly considered to be produced only by hyperthermophilic

3

archaea (e.g. the microbiome in hot springs),3,5 and to the best of our knowledge this is the first

4

time they are tentatively reported from the much cooler marine settings here (average annual

5

water temperature in the GoM at 1100 and 1200 m depth is 4.81 and 4.56 °C,28 respectively,

6

while the annual climatological average SST in the area where the samples were collected is 24-

7

25 °C29). This finding could have implications for archaeal phylogenetics and the biophysical

8

relationships between the number of cyclic moieties in GDGT species and assessed temperature,

9

pH, and other environmental variables, but this discussion is out of the scope of this paper.

10 11

Table 1. Identified glycerol ether lipid species in the GoM sediments in the m/z 1100-1500

12

range. Double bond equivalent (DBE) values for a given molecular formula assignment are

13

calculated as reported elsewhere,13 while the error (in ppm), is calculated as [m/z (experimental) -

14

m/z (theoretical)] / m/z (theoretical) × 1,000,000. Relative intensities (RI %) of different ether

15

lipid species were calculated as (∑Ispecies/∑Ispecies+ IcoreGDGT) ×100. Reporting threshold: error

16

(ppm) ≤ 0.6, S/N ≥ 4.

Compound Core GDGT GDGT-8 GDGT-7 GDGT-6 GDGT-5 GDGT-4 GDGT-3 GDGT-2 GDGT-1 GDGT-0 Monohydroxy-GDGT OH-GDGT-7 OH-GDGT-6 OH-GDGT-5 OH-GDGT-4 OH-GDGT-3

Formula

DBE

m/z (experimental)

Error (ppm)

RI* (%) 100

C86H157O6 C86H159O6 C86H161O6 C86H163O6 C86H165O6 C86H167O6 C86H169O6 C86H171O6 C86H173O6

9 8 7 6 5 4 3 2 1

1286.1974 1288.2130 1290.2289 1292.2438 1294.2609 1296.2759 1298.2912 1300.3070 1302.3223

0.0 0.1 -0.1 0.5 -0.6 -0.2 0.1 0.0 0.3 1.54

C86H159O7 C86H161O7 C86H163O7 C86H165O7 C86H167O7

8 7 6 5 4

1304.2083 1306.2236 1308.2396 1310.2550 1312.2705


-0.2 0.0 -0.2 0.0 0.1


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OH-GDGT-2 OH-GDGT-1 OH-GDGT-0 Dihydroxy-GDGT 2OH-GDGT-8 2OH-GDGT-7 2OH-GDGT-6 2OH-GDGT-5 2OH-GDGT-4 2OH-GDGT-3 2OH-GDGT-2 2OH-GDGT-1 2OH-GDGT-0 GDD GDD-8 GDD-7 GDD-6 GDD-5 GDD-4 GDD-3 GDD-2 GDD-1 GDD-0 1

C86H169O7 C86H171O7 C86H173O7

3 2 1

1314.2861 1316.3017 1318.3173

0.1 0.2 0.2 0.26


9 8 7 6 5 4 3 2 1

1318.1869 1320.2034 1322.2180 1324.2349 1326.2495 1328.2656 1330.2805 1332.2966 1334.3123

0.3 -0.3 0.5 -0.5 0.3 -0.1 0.5 0.2 0.1 1.61


8 7 6 5 4 3 2 1 0

1228.1550 1230.1711 1232.1867 1234.2024 1236.2180 1238.2339 1240.2494 1242.2654 1244.2811

0.5 0.1 0.2 0.1 0.1 -0.1 0.1 -0.2 -0.2

* For one representative sample - GoM2, 188-193 mm.

2

Finally, it should be emphasized that the individual mass spectral peaks observed in

3

FTICR-MS, contain all the possible isomers of a given molecular formula (e.g. 12 isomers for

4

GDGT-310). Consequently, the GDGT abundance information used in this study is related to all

5

the possible isomers with the given elemental formula. In addition, this implies that only proxies

6

which do not require compound discrimination at the isomeric level can be derived from FTICR-

7

MS data. However, not all potential proxies require isomer level discrimination and the

8

chromatographic separation of isomers necessary for isomer level proxy development can make

9

such methods extremely cumbersome. This limitation might increasingly lead to a more frequent

10

use of isomer-aggregated proxy indices, which would bulk different groups of related species

11

together, thus avoiding dependence on detailed isomer information.11,30 In reality, both

12

approaches are complementary, with the FTICR-MS approach having the advantage of screening

13

for potential novel biomarkers missed by more targeted analyses.


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Calculation of GDGT proxy indices from APPI-P FTICR-MS data. We calculated

2

the following, non-isomer-specific GDGT-based indices, in the subsamples along the vertical

3

profile of the sediment cores:

4

(i) crenarchaeol/caldarchaeol tetraether index (CCaT, Eq.1), which is known to be

5

broadly correlated to SST,6 and was recently used for SST reconstructions by indirect calculation

6

of TEX86 from LDI FTICR-MS data;15 ሾீ஽ீ்ିହሿ

‫ = ܶܽܥܥ‬ሾீ஽ீ்ି଴ሿାሾீ஽ீ்ିହሿ

7 8 9 10

Eq. 1

(ii) GDGT index-1 (also known as TEXL86, Eq. 2), which was originally developed for SST reconstructions in colder oceans (2 embedded rings), as a

31

possible indication that their source was distinct from that of the non-hydroxylated species.33 In



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Page 18 of 24

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contrast, our FTICR-MS results, for the first time, indicate the co-existence of complete series of

2

mono- and dihydroxy- GDGTs in the same samples, including novel species with more than two

3

cyclic moieties, together with the non-hydroxylated GDGT compounds. Previous studies were,

4

most likely, not able to detect the hydroxy-GDGTs with more than two rings due to the

5

insufficiently sensitive analytical methods used, that targeted specific compounds, rather than the

6

broad-spectrum reconnaissance method employed here.30,33

7

The presence of corresponding series of core GDGTs and their putative hydroxy-analogs,

8

was observed in this study (Table 1). Peak potentially corresponding to OH-GDGT-8 was also

9

detected, but it was too close to the reporting baseline and assignment error thresholds, so it was

10

not included in Table 1. Complete series of hydroxy-GDGT analogs suggest possible diagenetic

11

relationships of the hydroxy- and core GDGT species, rather than requiring different biological

12

origins.33 On the other hand, it is important to remember that the intensity of measured OH-

13

GDGTs, relative to the total core GDGTs in the representative sample, was only 1.54 % (Table

14

1), which could indicate they are trace components. Comparable relative abundance of OH-

15

GDGTs was found in marine sediments from seas at similar latitudes (~30 degrees north) and

16

SSTs.30,33 Dihydroxy-GDGT analogs were even lower in relative abundance, with 0.26 % of the

17

core-GDGT abundance, Table 1.33 The exact biological sources and the biological role of these

18

species in specific organisms, are yet to be constrained.

19

Indications of Novel Glycerol Diether Lipids. Isoprenoid glycerol dialkanol diethers

20

(GDDs), were recently discovered structural analogs of GDGTs, with one glycerol unit less than

21

their tetraether counterparts, following the opening of the biphytanyl ring (Figure 2).10,11 This

22

means that these species will have a molecular ion of 56.0262 Da m/z less (m/z of the C3H4O

23

unit), and one DBE less, than the “parent” GDGT structure (Figure 2). By using the method

24

reported herein, we were able to confirm their presence in the GoM sediments, in a series

25

corresponding to all the detected core GDGTs, including novel, previously unreported species

26

with more than five rings, Table 1. Lack of analytical resolution and/or sensitivity is the probable

27

reason why previous studies reported GDDs with only five rings or less. The relative intensity of

28

GDDs in the representative sample was 1.61 % of the total core GDGTs (Table 1), which is

29

several times less than the reported GDD proportion in the sediments from a comparable marine

30

setting, e.g. Mediterranean.11


Page 19 of 24

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Page 19 of 24 1

Although the potential sources of archaeal lipids are still uncertain for the GDD

2

compounds, the observed coexistence of both diether and tetraether forms, suggests a close

3

functional or diagenetic association, or a common source of GDD and GDGT species, as

4

suggested by previous studies.11

5

Possible Novel Ether Lipid Biomarkers. Apart from the aforementioned ether lipids,

6

we identified additional species whose elemental formulae fit into common DBE series (Table 3)

7

and of which, we speculate that they may belong to the same geochemical class of compounds.

8

Only peaks which showed intensities above a conservatively selected S/N threshold of 15 are

9

shown in Table 3. As a matter of report, for others to pursue, we briefly suggest possible

10

identities for these species emphasizing that we lack structural confirmation of their identities.

11

For example, a series of peaks between m/z 1274.2331 and m/z 1284.3116, fits with a

12

putative identification of a series of core GDGTs, with analog DBE assignments, but with one

13

oxygen atom less per molecule, and with a relative intensity of approx. 2 % of the total core

14

GDGTs. These might be species related to diagenetic loss of one of the glycerol hydroxy groups

15

from core GDGTs. If confirmed, these dehydroxy-GDGTs (deOH-GDGTs) may be a new class

16

of sedimentary biomarkers. Other peaks were assigned molecular formulae within expected ether

17

lipid DBE series, with number of carbon atoms ranging from 83 to 87, and oxygen atoms from 4

18

to 7. They too are probably part of the same archaeal lipid sequence, and might have proxy

19

potential, however this clearly needs further confirmation and investigation, e.g. by analyzing

20

fragmentation patterns in tandem mass spectroscopy (MS/MS), an approach which was already

21

used to discover novel glycerol ether lipids.34

22 23 24 25 26 27



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Page 20 of 24

Page 20 of 24 1 2

Table 3. Possible novel ether lipid species in the GoM sediment.* Reporting threshold: error

3

(ppm) ≤ 0.6, S/N ≥ 15.

Compound Dehydroxy-GDGT deOH-GDGT-6 deOH-GDGT-5 deOH-GDGT-4 deOH-GDGT-3 deOH-GDGT-2 deOH-GDGT-1

Others

4

Formula

DBE

m/z (experimental)

C86H161O5 C86H163O5 C86H165O5 C86H167O5 C86H169O5 C86H171O5 C83H153O4 C83H155O4 C83H157O4 C83H159O4 C83H161O4 C83H163O4 C83H165O4 C83H167O4 C85H157O4 C85H163O4 C85H165O4 C85H167O4 C86H157O4 C86H163O4 C86H165O4 C86H167O4 C85H159O7 C85H161O7 C85H165O7 C85H169O7 C87H171O6 C87H175O6 C87H163O7 C87H169O7 C87H171O7 C87H173O7

7 6 5 4 3 2 8 7 6 5 4 3 2 1 8 5 4 3 9 6 5 4 7 6 4 2 3 1 7 4 3 2

1274.2331 1276.2495 1278.2645 1280.2804 1282.2957 1284.3116 1214.1759 1216.1916 1218.2072 1220.2228 1222.2387 1224.2543 1226.2699 1228.2856 1242.2077 1248.2550 1250.2710 1252.2863 1254.2077 1260.2543 1262.2700 1264.2854 1292.2078 1294.2244 1298.2543 1302.2854 1312.3070 1316.3386 1320.2392 1326.2860 1328.3020 1330.3176

* For one representative sample - GoM2, 188-193 mm.

5

Geochemical Implications. Discovery of possible coexisting and corresponding series of

6

core tetraether species, and their mono-, dihydroxy- and diether analogs, suggests close

7

biochemical or depositional relationships for the various compound classes and confirms that

8

FTICR-MS studies are a powerful, and rapid tool for biomarker system investigations and rapid


Page 21 of 24

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Page 21 of 24 1

identification of putative species to target with more detailed structural characterization

2

protocols. The recognition of complete series of hydroxy-GDGT species might suggest that they

3

are possible degradation products of archaeal intact polar lipid (IPL) membranes.33,35 Results

4

presented here could also indicate that this diagenetic process might not stop at this point, but

5

plausibly, continues along a diagenetic sequence, likely ending with GDD end members or their

6

analogs. Thus, we hypothesize a diagenetic sequence of: intact GDGTs→hydroxy-

7

GDGTs→core-GDGTs→dehydroxy-GDGTs→GDDs. More detailed structural confirmations

8

would be needed to confirm this sequence, as well as other species that might be a part of it. For

9

example, members of the DBE series with 83 carbon and 4 oxygen atoms are also, possibly,

10

further degradation products of GDDs, see Table 3. However, other types of relationships

11

between the detected species, e.g. functional, might be possible.36

12

The presence of such a large suite of possibly related ether lipid species, beyond the

13

standard model of compounds used in SST proxy studies, deepens the doubts about the current

14

mechanistic consistency of paleotemperature estimates inferred from polar lipid analysis. This

15

further argues for the re-evaluation of the existing indices as paleotemperature proxies and the

16

development of new and more robust indices.26

17 18

Conclusions.

19

The APPI-P FTICR-MS method proposed here significantly advances the analysis of

20

sedimentary biomarkers. We demonstrate its applicability for the detection of known and novel

21

glycerol ether lipids, however, it has potential to be expanded to a much broader range of

22

compounds, including other biomarkers currently analyzed using different analytical methods,

23

e.g. sterols, carotenoids, chlorophylls, among others. The FTICR-MS based method illustrated

24

here was able to identify coexisting, complete series of core glycerol dialkyl glycerol tetraethers

25

(GDGTs with 0 to 8 alicyclic rings), including the complete resolution of GDGT-4 compounds,

26

and the unexpected detection of GDGTs with more than 5 rings from a mesophilic marine

27

environment (SST of 24–25 °C, approx.); additionally, mono- and dihydroxy- analogs (including

28

novel species with >2 rings), as well as glycerol dialkanol diethers (GDDs -including novel

29

species with >5 rings), were detected. In addition, we putatively identify other, previously

30

unreported groups of ether lipid species, the key advantage of FTICR-MS methods being the

31

ability to detect species not part of classical targeted analytical procedures. This “putative



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Page 22 of 24

Page 22 of 24 1

compound discovery mode” screening approach, is perhaps the greatest advantage of the

2

technology in biogeochemical studies and could open completely new avenues for the discovery

3

of novel geochemical relationships and compounds, suitable for exploration as new proxies for a

4

variety of energy and climate-related system variables.

5 6 7 8

Supplementary info. The peak intensities of the main core glycerol dialkyl glycerol tetraethers species commonly used in paleoenvironmental reconstructions.

9

Supporting Information Available: This material is available free of charge via the

10

Internet at http://pubs.acs.org. Also available from The Gulf of Mexico Research Initiative

11

Information and Data Cooperative (GRIIDC). Dataset:

12

biomarkers in northern Gulf of Mexico sediment using APPI-P FTICR-MS. UDI:

13

R1.x135.119:0009;

14

https://data.gulfresearchinitiative.org/data/R1.x135.119:0009/

Screening of glycerol ether lipid

Dataset

Landing

Page:

15 16

Acknowledgments.

17

This research was made possible in part by a grant from BP/The Gulf of Mexico

18

Research Initiative, C-IMAGE, and in part by the Canada Foundation for Innovation (CFI), the

19

Natural Sciences and Engineering Research Council of Canada (NSERC), PRG and the

20

University of Calgary. Authors acknowledge the contributions of other C-IMAGE team members

21

including Dr. David Hollander, Dr. Patrick Schwing, and Dr. Isabel Romero, as well as their

22

technical staff (Quentin and Nicola) at the University of South Florida (St. Petersburg) in

23

sediment sampling, core dating and extrusion. Melisa Brown from the PRG (University of

24

Calgary) and Christopher Thompson from Bruker Daltonics Inc. (USA) are acknowledged for

25

their help in FTICR-MS method development and optimization. GDGT-5 standard was obtained

26

thanks to the support from the US National Science Foundation (NSF) and Harvard University.

27 28 29


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References. (1) Ho, S. L.; Mollenhauer, G.; Fietz, S.; Martínez-Garcia, A.; Lamy, F.; Rueda, G.; Schipper, K.; Méheust, M.; Rosell-Melé, A.; Stein, R.; Tiedemann, R. Geochim. Cosmochim. Acta 2014, 131, 213-226. (2) Pearson, A.; Huang, Z.; Ingalls, A. E.; Romanek, C. S.; Wiegel, J.; Freeman, K. H.; Smittenberg, R. H.; Zhang, C. L. Appl. Environ. Microbiol. 2004, 70, 5229-5237. (3) Schouten, S.; Hopmans, E. C.; Sinninghe Damsté, J. S. Org. Geochem. 2013, 54, 19-61. (4) Liu, X.; Lipp, J. S.; Hinrichs, K.-U. Org. Geochem. 2011, 42, 368-375. (5) Pearson, A.; Ingalls, A. E. Annu. Rev. Earth Planet. Sci. 2013, 41, 359-384. (6) Schouten, S.; Hopmans, E. C.; Schefuß, E.; Sinninghe Damsté, J. S. Earth. Planet. Sci. Lett. 2002, 204, 265-274. (7) Schouten, S.; Hopmans, E. C.; Rosell-Melé, A.; Pearson, A.; Adam, P.; Bauersachs, T.; Bard, E.; Bernasconi, S. M.; Bianchi, T. S.; Brocks, J. J.; Carlson, L. T.; Castañeda, I. S.; Derenne, S.; Selver, A. D.; Dutta, K.; Eglinton, T.; Fosse, C.; Galy, V.; Grice, K.; Hinrichs, K.-U.; Huang, Y.; Huguet, A.; Huguet, C.; Hurley, S.; Ingalls, A.; Jia, G.; Keely, B.; Knappy, C.; Kondo, M.; Krishnan, S.; Lincoln, S.; Lipp, J.; Mangelsdorf, K.; Martínez-García, A.; Ménot, G.; Mets, A.; Mollenhauer, G.; Ohkouchi, N.; Ossebaar, J.; Pagani, M.; Pancost, R. D.; Pearson, E. J.; Peterse, F.; Reichart, G.-J.; Schaeffer, P.; Schmitt, G.; Schwark, L.; Shah, S. R.; Smith, R. W.; Smittenberg, R. H.; Summons, R. E.; Takano, Y.; Talbot, H. M.; Taylor, K. W. R.; Tarozo, R.; Uchida, M.; van Dongen, B. E.; Van Mooy, B. A. S.; Wang, J.; Warren, C.; Weijers, J. W. H.; Werne, J. P.; Woltering, M.; Xie, S.; Yamamoto, M.; Yang, H.; Zhang, C. L.; Zhang, Y.; Zhao, M.; Damsté, J. S. S. Geochem. Geophys. Geosyst. 2013, 14, 5263-5285. (8) Sturt, H. F.; Summons, R. E.; Smith, K.; Elvert, M.; Hinrichs, K.-U. Rapid Commun. Mass Spectrom. 2004, 18, 617-628. (9) Zhu, C.; Lipp, J. S.; Wörmer, L.; Becker, K. W.; Schröder, J.; Hinrichs, K.-U. Org. Geochem. 2013, 65, 53-62. (10) Becker, K. W.; Lipp, J. S.; Zhu, C.; Liu, X.-L.; Hinrichs, K.-U. Org. Geochem. 2013, 61, 34-44. (11) Liu, X.-L.; Lipp, J. S.; Schröder, J. M.; Summons, R. E.; Hinrichs, K.-U. Org. Geochem. 2012, 43, 50-55. (12) Marshall, A. G.; Hendrickson, C. L.; Jackson, G. S. Mass Spectrom. Rev. 1998, 17, 1-35. (13) Marshall, A. G.; Rodgers, R. P. Proc. Natl. Acad. Sci. 2008, 105, 18090-18095. (14) Koch, B. P.; Witt, M.; Engbrodt, R.; Dittmar, T.; Kattner, G. Geochim. Cosmochim. Acta 2005, 69, 3299-3308. (15) Wörmer, L.; Elvert, M.; Fuchser, J.; Lipp, J. S.; Buttigieg, P. L.; Zabel, M.; Hinrichs, K.-U. Proc. Natl. Acad. Sci. 2014, 111, 15669-15674. (16) Oldenburg, T. B. P.; Brown, M.; Bennett, B.; Larter, S. R. Org. Geochem. 2014, 75, 151168. (17) Muller, H.; Adam, F.; Panda, S.; Al-Jawad, H.; Al-Hajji, A. J. Am. Soc. Mass. Spectrom. 2012, 23, 806-815. (18) Verschuren, D. Limnol. Oceanogr. 1993, 38, 1796-1802. (19) Romero, I. C.; Schwing, P. T.; Brooks, G. R.; Larson, R. A.; Hastings, D. W.; Ellis, G.; Goddard, E. A.; Hollander, D. J. PLoS ONE 2015, 10, e0128371.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 24 of 24

Page 24 of 24 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32

(20) Schwing, P. T.; Romero, I. C.; Brooks, G. R.; Hastings, D. W.; Larson, R. A.; Hollander, D. J. PLoS ONE 2015, 10, e0120565. (21) Peterse, F.; Kim, J.-H.; Schouten, S.; Kristensen, D. K.; Koç, N.; Sinninghe Damsté, J. S. Org. Geochem. 2009, 40, 692-699. (22) Hanold, K. A.; Fischer, S. M.; Cormia, P. H.; Miller, C. E.; Syage, J. A. Anal. Chem. 2004, 76, 2842-2851. (23) Bruker Daltonics, Technical Note-28. maXis High Resolution LC/MS Makes the Most of Ultrafast LC Separations: Billerica, MA, USA, 2009. (24) Berglund, M.; Wieser, M. E. Pure Appl. Chem. 2011, 83, 397-410. (25) Schouten, S.; Huguet, C.; Hopmans, E. C.; Kienhuis, M. V. M.; Sinninghe Damsté, J. S. Anal. Chem. 2007, 79, 2940-2944. (26) Pearson, A.; Pi, Y.; Zhao, W.; Li, W.; Li, Y.; Inskeep, W.; Perevalova, A.; Romanek, C.; Li, S.; Zhang, C. L. Appl. Environ. Microbiol. 2008, 74, 3523-3532. (27) Silverstein, R. M.; Webster, F. X.; Kiemle, D. Spectrometric Identification of Organic Compounds, 7th Edition; Wiley, 2005. (28) Boyer, T. P.; Biddle, M.; Hamilton, M.; Mishonov, A. V.; Paver, C. R.; Seidov, D.; Zweng, M., NOAA/NODC, Ed., 2011. (29) Byrne, D.; Li, Y.; Phillips, S.; Stennis Space Center (MS): National Coastal Data Development Center, 2011. (30) Huguet, C.; Fietz, S.; Rosell-Melé, A. Org. Geochem. 2013, 57, 107-118. (31) Kim, J.-H.; van der Meer, J.; Schouten, S.; Helmke, P.; Willmott, V.; Sangiorgi, F.; Koç, N.; Hopmans, E. C.; Damsté, J. S. S. Geochim. Cosmochim. Acta 2010, 74, 4639-4654. (32) Zhang, Y. G.; Zhang, C. L.; Liu, X.-L.; Li, L.; Hinrichs, K.-U.; Noakes, J. E. Earth. Planet. Sci. Lett. 2011, 307, 525-534. (33) Liu, X. L.; Lipp, J. S.; Simpson, J. H.; Lin, Y. S.; Summons, R. E.; Hinrichs, K. U. Geochim. Cosmochim. Acta 2012, 89, 102-115. (34) Liu, X.-L.; Summons, R. E.; Hinrichs, K.-U. Rapid Commun. Mass Spectrom. 2012, 26, 2295-2302. (35) Xie, S.; Liu, X.-L.; Schubotz, F.; Wakeham, S. G.; Hinrichs, K.-U. Org. Geochem. 2014, 71, 60-71. (36) Takano, Y.; Chikaraishi, Y.; Ogawa, N. O.; Nomaki, H.; Morono, Y.; Inagaki, F.; Kitazato, H.; Hinrichs, K.-U.; Ohkouchi, N. Nature Geosci 2010, 3, 858-861.

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