Fossil organic carbon utilization in marine Arctic fjord sediments by subsurface micro-organisms

Rock-derived or petrogenic organic carbon has traditionally been regarded as being non-bioavailable and bypassing the active carbon cycle when eroded. However, it has become apparent that this organic carbon might not be so inert, especially in fjord systems where petrogenic organic carbon influxes can be high, making its degradation another potential source of greenhouse gas emissions. The extent to which subsurface micro-organisms use this organic carbon is not well constrained, despite its potential impacts on global carbon cycling. Here, we performed compound-specific radiocarbon analyses on intact polar lipid–fatty acids of live micro-organisms from marine sediments in Hornsund Fjord, Svalbard. By this means, we estimate that local bacterial communities utilize between 5 ± 2% and 55 ± 6% (average of 25 ± 16%) of petrogenic organic carbon for their biosynthesis, providing evidence for the important role of petrogenic organic carbon as a substrate after sediment redeposition. We hypothesize that the lack of sufficient recently synthesized organic carbon from primary production forces micro-organisms into utilization of petrogenic organic carbon as an alternative energy source. The input of petrogenic organic carbon to marine sediments and subsequent utilization by subsurface micro-organisms represents a natural source of fossil greenhouse gas emissions over geological timescales. Ancient, rock-derived organic matter is consumed by micro-organisms in Arctic fjord sediments despite its presumed limited bioavailability, representing a potential source of greenhouse gas emissions, according to compound-specific radiocarbon analyses of lipids from living bacteria.

Rock-derived or petrogenic organic carbon has traditionally been regarded as being non-bioavailable and bypassing the active carbon cycle when eroded. However, it has become apparent that this organic carbon might not be so inert, especially in fjord systems where petrogenic organic carbon influxes can be high, making its degradation another potential source of greenhouse gas emissions. The extent to which subsurface micro-organisms use this organic carbon is not well constrained, despite its potential impacts on global carbon cycling. Here, we performed compound-specific radiocarbon analyses on intact polar lipid-fatty acids of live micro-organisms from marine sediments in Hornsund Fjord, Svalbard. By this means, we estimate that local bacterial communities utilize between 5 ± 2% and 55 ± 6% (average of 25 ± 16%) of petrogenic organic carbon for their biosynthesis, providing evidence for the important role of petrogenic organic carbon as a substrate after sediment redeposition. We hypothesize that the lack of sufficient recently synthesized organic carbon from primary production forces micro-organisms into utilization of petrogenic organic carbon as an alternative energy source. The input of petrogenic organic carbon to marine sediments and subsequent utilization by subsurface micro-organisms represents a natural source of fossil greenhouse gas emissions over geological timescales.
Shales and other sedimentary deposits store around 90% of global organic carbon (OC) 1 . However, this fossil rock-derived or petrogenic OC (OC petro ) has been widely neglected as a potential microbial substrate and source of fossil greenhouse gases 2 (GHGs). Traditionally, OC petro has not been included in studies of the active carbon cycle as the majority of it was synthesized, deposited and degraded millions of years ago and is commonly regarded as non-bioavailable 2 . However, within the past two decades, several studies have investigated the availability of OC petro from different sources as a substrate for micro-organisms, painting a more diverse picture of its bioavailability [2][3][4][5][6] . Globally, OC petro oxidation is estimated to account for release of 40-100 × 10 6 tC annually 7 , opposing the effects of OC burial 8 and silicate weathering 9 . Thus, a proper assessment of OC petro bioavailability and the role of micro-organisms becomes increasingly important as more evidence of GHG release from OC petro into the atmosphere is discovered 6,[10][11][12][13] .
Previous work focused on dissolved OC from glacial runoff, showing it to be highly bioavailable, despite its old age 14,15 . Although microbial communities may play a crucial role in glacial nutrient and carbon Article https://doi.org/10.1038/s41561-023-01198-z core (27-cm-long core He519_2-3) was retrieved from the centre of the main basin at a depth of 202 m. It records the sedimentary history from approximately the 1950s to 2018. A gravity core was collected in the Brepollen basin centre (149-cm-long core HH14-897-GC-MF) at a water depth of 140 m, archiving the time span from the 1960s to 2014. The 23-cm-long core, He560_26-2-K1, was taken ~1 km from the glacier termini at a water depth of 46 m, covering the time period from about 2012 to 2020 (Methods).
The catchment geology of the Hornsund Fjord is very diverse 28 . The majority of sediments supplied to the fjord come from the eastern part of the drainage basin, built of OC-rich Palaeogene mudstones and sandstones formed in a continental shelf sea environment (Supplementary Information) 28,29 . The area is mainly glacier-covered 27 ; however, these strata extend northwards. The better exposure displays some low-to mid-grade coal seams; however, these represent only a minor portion of the rock volume 30 (<170-m-thick unit) 31 . Since the late nineteenth century, the local glaciers have been retreating rapidly at rates of several tens of metres to more than 100 m annually 27 , simultaneously shifting the sedimentary depocentre alongside the glacier termini position 32 . Sediment accumulation rates in the studied core locations varied from more than 10 cm to a few millimetres per year with respect to distance from the retreating glacier termini. Average total OC (TOC) contents range between 1.3 ± 0.1 and 1.9 ± 0.1 wt% (mean ± s.d.) and are constant throughout the individual cores, independent of glacial proximity (Fig. 2a). The origin of the OC was assessed using several geochemical parameters and biomarker indices, including bulk δ 13 C, cycling 16 , the extent to which the particulate OC supplied by glaciers can be utilized by micro-organisms after its redeposition is virtually unexplored. According to conservative estimates, fjords bury about 18 Mt of OC annually ( ~11% of marine carbon burial) 17 . Globally, about 11% of landmasses are covered by polar ice sheets and alpine glaciers 18 , eroding into the underlying bedrocks 19 , including OC-rich strata. Increasing temperatures at high latitudes 20 are expected to increase runoff and sediment exported from both polar glaciers 21 and ice sheets 22 to downstream depositional environments, thus increasing OC petro fluxes in the upcoming decades 23 . At marine-terminating glaciers, the bulk of this OC petro is deposited within a distance of several kilometres from glacier termini 24 , with a strong dominance of particulate OC over dissolved OC exported from ice sheets 25 . However, OC petro deposition is not limited to fjords but may supply 40-50% of OC buried in Arctic Ocean sediments 26 . It is therefore of interest whether this vast pool of remobilized OC petro can be microbially degraded, and a proper budget and assessment of its rates are necessary to understand impacts on the global carbon cycle.

OC dynamics in Hornsund Fjord
To investigate this process, we analysed three sediment cores, two short and one long, from Hornsund Fjord, Svalbard (Fig. 1). Hornsund's marine-influenced main basin is separated from the tidewater-glacier-dominated inner basin, Brepollen, by a shallow sill. The Brepollen basin was formed during the last century following the post Little Ice Age deglaciation 27   Article https://doi.org/10.1038/s41561-023-01198-z fatty-acid-based terrestrial aquatic ratio (TAR) 33 , the branched and isoprenoid tetraether (BIT) index as a soil OC marker 34 , the n-alkane carbon preference index (CPI) as an indicator for degradation/thermal maturity 35 (Fig. 2c-f and Methods) and bulk radiocarbon (F 14 C) signature ( Fig. 3b,d,f). Contributions to the OC pool by terrestrial plants and soils can be neglected based on both the low TAR and BIT index, which reflect exclusive input of fresh, soil-derived organic matter and are not sensitive to old, mature terrestrial OC from source rock 34 . Based on the above-mentioned biogeochemical parameters, all three cores show a homogenous OC composition consisting of a mixture of two types of material: (1) young, freshly synthesized, labile marine organic matter (OC marine ) from primary production; and (2) old, thermally very mature, supposedly non-bioavailable OC petro eroded from organic-rich sedimentary rocks in the fjord catchment 29 . Further evidence for a petrogenic origin of much of the organic matter is provided by the infinite compound-specific radiocarbon ages of long-chain n-alkanes extracted from the central Brepollen core (Supplementary Table 1). Even though primary production rates in Hornsund are similar 36 to other fjord systems with marine-terminating glaciers 37 , the relative abundance of sedimentary OC marine (f marine ) is rather low and increases with increasing distance to the glacier termini. The f marine value was estimated using an isotope mass balance based on F 14 C of the bulk TOC, with two endmembers: one modern OC marine (F 14 C ≈ 1 = modern) and one fossil OC petro (F 14 C = 0 = fossil; Methods). The short core in the vicinity of glacier termini and the long core in the centre of the Brepollen basin both have low f marine values of 2 ± 2 to 11 ± 2%. By contrast, in the short core (He519_2-3) from the fjord main basin, the f marine ranges from 42 ± 2% at the core top to 26 ± 6% at the bottom. Overall, the TOC age is primarily controlled by the input of OC marine as this input is the main difference between the OC deposited in the main basin versus the Brepollen basin.

Compound-specific radiocarbon analysis
Owing to the characteristic F 14 C signature of the two pools, we were able to use 14 C as an inverse tracer (absence of 14 C) under the assumption that the isotopic signature of the substrate (that is, in sediments) will be passed on through the heterotrophic utilization into the synthesized biomass 3 . Following the approach of ref. 3, we assessed the bioavailability of these two OC pools in the sediment cores by radiocarbon analyses of the fatty acid (FA) side chains of intact polar lipids (IPLs; IPL-FAs), extracted with a modified ref. 38 approach. Bacterial IPLs have been reported to decay within days to weeks after cell lysis and are therefore regarded as indicators for living microbiota 39 . Bacterially produced FAs C br-15:0 and C 16:1 n-7 5 were purified into single-compound fractions and subsequently radiocarbon dated. With this approach, we were able to identify the average F 14 C signature of the substrate utilized by bacteria in the sediment 5 . To ensure bacterial FA origin, precursor lipids were determined by high-pressure liquid chromatography coupled to mass spectrometry (HPLC-MS).
Using HPLC-MS, the dated C br-15:0 and C 16:1 n-7 FAs were found to derive from a diverse group of phospholipid precursors: mainly phosphatidylglycerol and phosphatidylethanolamine in the glacier termini and Brepollen long core, and additionally phosphatidylcholine in the main basin core ( Supplementary Fig. 1). While most of these lipids can be assigned to sulfate-reducing bacteria 40 or other sedimentary marine bacteria 41 , minor contributions of potentially algae-derived betaine lipids and phosphatidylcholine ( <10%) could potentially lead to an increase in the measured F 14 C FA values and hence an underestimation of OC petro degradation (Supplementary Information).
In the marine-influenced main basin core (He519_2-3), compoundspecific F 14 C values for IPL-FAs within the topmost part of the core ( <15 cm; F 14 C = 0.939 ± 0.008 to 1.002 ± 0.009) agree closely with modelled surface dissolved inorganic carbon (DIC) values (F 14 C = 1.013 ± 0.015 to 1.116 ± 0.020), indicating an exclusive or at least strong preferential utilization of recently synthesized OC marine (Fig. 3a). Further downcore (17-21 and 21-24 cm), the FAs diverge from modelled DIC signatures towards lower F 14 C values (F 14 C < 1.000 ± 0.007), indicating an increase in OC petro utilization. Interestingly, this shift mirrors a decrease of f marine from 30 to 42% in the topmost 15 cm to less than 30% below. Nevertheless, OC marine is the primary, but not exclusive, substrate utilized by the sedimentary microbiome in sediment core He519_2-3, while an apparent shift towards increasing OC petro utilization occurs downcore.
A different picture emerges at the glacier termini core (He560_26-2-K1; Fig. 3e). The C 16:1 n-7 F 14 C values range between 0.767 ± 0.011 and 0.697 ± 0.016, which is far lower and outside the 2σ uncertainty of the modelled surface DIC F 14 C (ranging between 1.009 ± 0.015 and 1.023 ± 0.015). This indicates the substantial uptake of OC petro into the bacterial membrane lipids. Unfortunately, sedimentary contents of C br-15:0 were too low to perform compound-specific radiocarbon dating. IPL-FA data from the Brepollen long core (HH14-897-GC-MF; Fig. 3c) show F 14 C values similar to those from the He560 glacier termini core  The percentage of ancient carbon used for the microbial biosynthesis (Fig. 3b,d,f) was estimated with an isotope mass balance model, using a radiocarbon-free fossil endmember for OC petro (F 14 C = 0) and modern OC marine endmember according to the reservoir age modelled at the respective depth intervals (Methods). A pronounced difference between the two Brepollen cores and the main basin core is evident from this mass balance estimate. Within the top 15 cm of the main basin core, OC petro accounts for 5 ± 2 to 9 ± 2% of the utilized carbon, whereas in the Brepollen cores, OC petro contributes up to 37 ± 2% in the topmost intervals. The most proximal core at the glacier termini is characterized by extremely high sedimentation rates, f marine values consistently below 6 ± 2% throughout the core and fairly constant OC petro utilization (24 ± 2 to 32 ± 2%). On the contrary, in both the marine-influenced main basin short core and the central Brepollen basin long core, we can observe an increased utilization of OC petro with increasing depth and decreasing f marine . The highest estimate of OC petro utilization reached 55 ± 6% in the central Brepollen core in the depth interval of 86-89 cm, compared with the lowest OC petro of only 5 ± 6% in the marine-influenced main basin core (see above). Here, we show that even over short distances within one fjord system the microbial utilization of OC petro can vary widely, suggesting both low and substantial fossil GHG emission potential from increasing glacial erosion.
Although we cannot directly identify the mechanisms for OC petro utilization, we hypothesize that with decreasing abundance of fresh, labile OC marine , micro-organisms are forced to utilize OC petro for their biosynthesis. For example, in the interval with the highest percentage of OC petro utilized for lipid synthesis (HH14, 86-89 cm) the mass balance suggests that 55 ± 6% of utilized carbon originates from OC petro when the abundance of labile OC marine in the sediment is low (f marine = 5 ± 6%). In the topmost three dated intervals of the main basin core, OC petro utilization is much lower, but still accounts for 5 ± 2 to 9 ± 2% when f marine is above 30%.
Under the assumption that sedimentary micro-organisms use the same substrate for both their anabolic and catabolic pathways 42 , we estimate that heterotrophic remineralization of OC petro accounts for between 5 ± 2 and 55 ± 6% of local microbiota's overall energy consumption. This remineralization leads to the conclusion that CO 2 (and CH 4 ) emitted from sediments as metabolic end-products originates in some part from fossil sources, which might be enhanced with increased mobilization of ancient organic-rich deposits in a warming climate.

Implications of OC petro utilization
Our data indicate that OC petro is indeed microbially utilized after deposition in Hornsund Fjord. These findings are in line with previous studies 3 Article https://doi.org/10.1038/s41561-023-01198-z part of the active carbon cycle, and that these may be affected by microbial processing and consumption. Glaciated fjord ecosystems similar to the Hornsund Fjord with often OC-rich (including coal-bearing) bedrock in their drainage areas are fairly widespread and can be found in Svalbard 43 , Alaska 44 , Greenland 45 , Franz Josef Land 46 and Antarctica 47 . These ecosystems may likewise supply suitable substrates for microbial degradation to marine sediments. Recent studies of other glacial environments based on modern glacial sediments 48 , watershed analysis 12 and palaeo CO 2 isotopic compositions 10 indicate that similar utilization of old, previously 'locked up' OC may also occur onshore, indicating the geographical pervasiveness of OC petro utilization. Microbial OC petro utilization has also been reported from terrestrial shales 3 .
These findings indicate that OC petro utilization at the rock interface, after erosion and redeposition, is likely to occur globally. The resulting fossil GHG emissions may be substantial on a geological timescale-even if only a fraction of the OC petro becomes remineralized after deposition or exposure. Based on our data, we cannot estimate GHG fluxes resulting from OC petro utilization in marine sediments. However, considering the size of the global OC petro reservoir 1 , further quantitative research into this topic seems to be mandated, both in terms of a global OC petro flux from rivers, ice sheets and glaciers, and OC petro utilization dynamics in sediments, soils and the water column. High-latitude temperatures continue to rise up to four times more rapidly than in the rest of the world 49 , and sediment export rates are expected to increase from both glaciers 21 and ice sheets 22 to downstream depositional environments. Next to oxidation of OC petro , increases in fertilization of primary production 25 and turbidity 24 are just two of the consequent manifold associated environmental changes impacting carbon cycling in the glacial environment. Considering a recent estimate of global atmospheric CO 2 concentrations increasing by 50 ppm due to fjord sediment mobilization during the Last Glacial Maximum 50 , a potential climate impact on decadal to centennial timescales seems worth investigating. Therefore, to fully grasp the impact of glacial retreat on global carbon budgets, studying these processes in both marine and terrestrial settings may be needed, given the Intergovernmental Panel on Climate Change projections based on the low-emission Representative Concentration Pathway 2.6 scenario, which predict global glacial mass loss of 18% in 2100 relative to 2015, suggesting long-lasting effects even in the event of zero anthropogenic GHG emissions 20 .

Online content
Any methods, additional references, Nature Portfolio reporting summaries, source data, extended data, supplementary information, acknowledgements, peer review information; details of author contributions and competing interests; and statements of data and code availability are available at https://doi.org/10.1038/s41561-023-01198-z.
Publisher's note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Sampling
The sediment cores analysed in this study were taken on three separate expeditions in Hornsund Fjord, Svalbard. Gravity core HH14-897-MF-GC was taken in October 2014 onboard the Norwegian RV Helmer Hanssen in the central Brepollen basin. The two short cores were taken on the German RV Heincke during cruises He519 in September 2018 and He560 in August 2020. Core He519_2-3 was taken at the central main basin, whereas core He560_26-2-K1 was retrieved in the inner Brepollen basin (Supplementary Table 2).
Both short cores were sliced onboard RV Heincke, transferred into glass containers and frozen at −20 °C immediately after coring until analysis. The archive half of gravity core HH14-897-MF-GC was stored at 4 °C in the core repository at the Department of Geosciences, UiT The Arctic University of Norway, prior to sampling in January 2019. After sampling, sediments were transferred into glass containers and stored at −20 °C. Even though the long sediment core was not frozen immediately after coring, biomarkers, bulk parameters, compound-specific radiocarbon data and IPL data show similar patterns to the second Brepollen basin core He560_26-2-K1. In particular, the matching IPL (Supplementary Information) and compound-specific radiocarbon data provide confidence that the data obtained from the Brepollen long core accurately reflect in-situ information and allow for OC petro utilization estimates in the deeper core sections. Any potential storage effects would be expected to result in increased IPL concentrations and F 14 C values of IPL biased towards modern atmospheric values, which was not observed.
All glassware used was combusted at 450 °C for 6 hours and equipment cleaned with solvents before usage for both sampling and laboratory activities.

Age models
The age models were established using the short-lived isotopes 210 Pb and 137 Cs. The 210 Pb in recent marine sediments is of twofold origin. The supported 210 Pb ( 210 Pb sup ) is continuously produced within the sediments by the decay of parent isotopes, while excess 210 Pb ( 210 Pb ex ) is delivered to the sediment from above, produced by 222 Rn decay in the atmosphere and the water column overlying the sediment. Sediment cores He519_2-3 and He560_26-2-K1 were analysed at the Alfred Wegener Institute Bremerhaven, Germany, using a planar-type high-purity germanium (HPGe) gamma spectrometer. Core HH14-897-GC-MF was measured at the Institute of Geology at Adam Mickiewicz University in Poznań, Poland, using a gamma detector Canberra BE3830. The age models of the three cores were generated based on 210 Pb ex using the constant flux-constant sedimentation (CFCS) model and verified with penetration depth and peaks in 137 Cs isotope and historical information on the fjord deglaciation 27 . However, alternative models were also considered and the resulting accumulation rates should be regarded as approximates as the particular assumptions behind each model were not fully met. The analysis was conducted with the help of the R-based serac code 51 (Supplementary Figs. 4-6).

Surface DIC age model
Dissolved inorganic radiocarbon concentrations of surface water are simulated using the Finite-volumE Sea ice-Ocean Model (FESOM2) 52 equipped with radiocarbon 53 . Radiocarbon is implemented in terms of F 14 C, neglecting marine biological processes, which play a minor role compared with circulation and radioactive decay 54,55 . Air-sea exchange fluxes of 14 CO 2 in FESOM2 depend on wind speed and CO 2 solubility 56 , and assume a surface water global mean DIC concentration of 2.0 mol m −3 . The model was spun up in a previous simulation to quasi steady-state conditions typical of 1850 53 . We continued the simulation to 2015, using periodic climate forcing 57 60,61 . FESOM2 employs unstructured meshes with variable resolution, here featuring about 127,000 surface nodes and 47 layers. After the simulation, the model results were remapped to regular geographical coordinates and evaluated at the surface level considering the grid cell nearest to Hornsund.

TOC and stable carbon isotope ratios
TOC concentrations of core HH14-897-MF-GC were measured at the Department of Quaternary Geology and Palaeogeography of the Adam Mickiewicz University. The analyses were performed with a vario MAX CNS elemental analyser (Elementar). To determine the OC content, prior to the analyses, samples were treated with 1 M liquid hydrochloric acid (HCl) at room temperature for over a week (until no sign of reaction is visible) to remove carbonates. The δ 13 C of bulk OC in sediment was obtained using a Flash EA 1112 HT elemental analyser combined with a Thermo DELTA V Advantage isotopic ratio mass spectrometer in a continuous-flow mode. Results are expressed relative to Vienna PeeDee Belemnite. Methods are described in detail in ref. 62. The preliminary results were presented by ref. 32.
Both sediment cores He519_2-3 and He560_26-2-K1 were analysed for TOC and δ 13 C by continuous-flow elemental analyser-isotope ratio mass spectrometer using a Thermo Finnigan Flash EA 2000 connected to a Delta V Plus isotope ratio mass spectrometer at MARUM, Bremen, Germany, following the protocols of ref. 63 and ref. 64. Pre-treatment involved sample homogenization and carbonate removal overnight with 10% HCl or until no further gas development was visible. Afterwards the sample was neutralized with deionized water, freeze-dried and weighed for analysis.

Bulk radiocarbon dating
Radiocarbon ages of the TOC were determined by accelerator mass spectrometry at the MICADAS facility of the Alfred Wegener Institute. Accelerator mass spectrometry dating was performed on graphite targets of 1 mgC, and sediment masses were chosen according to TOC concentrations. As a pre-treatment, samples were homogenized and carbonates were removed three times with 6 M HCl at 60 °C. Methodology and blank determination were performed as described in ref. 65.

Lipid biomarkers
Lipid biomarkers were extracted from about 3 g of sediment using the method by ref. 66 at the Alfred Wegener Institute and subsequently separated into four subfractions for alkanes, ketones, alcohols (containing glycerol dialkyl glycerol tetraethers: GDGTs) and FAs, as described in ref. 67. The subfractions of alkanes and FAs were quantified on a GC-FID on a setup as in ref. 67. GDGTs were quantified on a HPLC-MS setup as described in ref. 67, after the protocol of ref. 68. Known amounts of the internal standards squalane, C 46 -GDGT and 19-methylarachidic acid were added to the sediments before the extraction for the quantification of alkanes, GDGTs and FAs, respectively.

IPLs
IPLs were extracted with a ref. 69  Aliquots of 1% of the polar lipid fractions were analysed on a Bruker maXis Plus ultra-high-resolution quadrupole time-of-flight mass spectrometer with an electrospray ionization source coupled to Dionex Ultimate 3000RS ultra-high-pressure liquid chromatography at MARUM, Bremen. The analyses were carried out using hydrophilic interaction chromatography in positive mode to check the separation of phospholipids with improved chromatographic separation and detection as described in ref. 71.

Compound-specific radiocarbon analysis
Compound-specific radiocarbon analysis (CSRA) was performed on purified IPL-FA and n-alkanes from aliquots obtained by modified Bligh and Dyer extraction 69 as described above. IPL-FA CSRA was performed of all extracted depth intervals. CSRA of n-alkanes purified from the neutral fraction was limited to three depth intervals (0-3, 86-89 and 133-136 cm) of core HH14-897-MF-GC. The n-alkane separation for CSRA was achieved following methods described by Meyer et al. 72 .
The polar lipid fractions were saponified at 80 °C with 1 ml of KOH (0.1 M) in MeOH:H 2 O (9:1, v/v) for 2 h. Neutral lipids were removed with a liquid-liquid phase separation using hexane. The remaining solution was acidified and FAs were extracted with a liquid-liquid phase separation using dichlormethan. The FAs were converted into fatty acid methyl esters (FAMEs) overnight at 50 °C in MeOH at a pH of 1 under a N 2 atmosphere. Subsequently, the FAMEs were separated from the MeOH phase by liquid-liquid phase separation using hexane and purified via passage through an activated (1% H 2 O) silica column, eluting FAMEs with 4 ml dichlormethan:hexan (2:1, v/v).
From both of the purified n-alkane and IPL-FA methyl ester fractions, single compounds were isolated using a gas chromatograph coupled to a preparative fraction collector (PFC) with the setup described in ref. 73. CSRA was performed as gas measurements at the MICADAS facility of the Alfred Wegener Institute following the protocol described in ref. 65.
Blank determination for CSRA was achieved in a two-step process. (1) Procedural blanks were run alongside the samples to ensure that no contamination from glassware, solvents or reagents occurred during the extraction and wet chemical preparation. All blanks were free of those FA and n-alkane homologues that were subsequently isolated with PFC. (2) Procedural blanks for PFC and subsequent radiocarbon analysis were determined using FAs and n-alkanes extracted from recent (apple peel) and fossil (Eocene Messel shale) laboratory internal standard materials, followed by subsequent radiocarbon age correction with according blanks as described in ref. 74 and ref. 75.

Isotope mass balance
The isotope mass balance calculations used a fossil, F 14 C fossil , and a modern endmember, F 14 C modelled DIC . The fossil endmember was set to a constant F 14 C value of 0, as the OC petro is expected to be radiocarbon-free, as organic-rich rocks outcropping the hinterland of Hornsund were deposited in the Tertiary 76,77 . Further, compound-specific radiocarbon analyses of isolated n-alkanes yielded F 14 C values near the detection limit, supporting the radiocarbon-free endmember definition (Supplementary Information). The modern endmember was defined as equivalent to the modelled surface DIC radiocarbon signature based on the biomarker data. The biomarker data indicated that the organic matter originated exclusively from the fixation of DIC during photosynthesis and OC from primary production is assumed to have the same radiocarbon signature. F 14 C modelled DIC values changed over time due to the rapid decrease in the F 14 C of the modelled surface DIC after the peak in atmospheric radiocarbon content resulting from above-ground nuclear weapons tests in the 1960s (Supplementary Fig. 7). Therefore, for the calculations, the F 14 C modelled DIC was adjusted according to the estimated year of sediment deposition, based on 210 Pb + 137 Cs age models as described above.
The isotopic mass balances were used to estimate the relative contribution of OC marine (f marine ) to the bulk sedimentary OC and to calculate the percentage of OC marine used for bacterial membrane lipid synthesis (%OC marine-synt ) based on the F 14 C signatures of the bulk TOC (F 14 C bulk ) and the dated single-compound IPL-FAs (F 14 C IPL-FA ). The general equations used for the calculations are: