Control on Rate and Pathway of Anaerobic Organic Carbon Degradation in the Seabed

Control on rate and pathway of anaerobic organic carbon degradation in the seabed

F. Beuliga,1, H. Røya, C. Glombitzaa,b, and B. B. Jørgensena,1

aCenter for Geomicrobiology, Department of Bioscience, Aarhus University, 8000 Aarhus C, Denmark; and bSpace Sciences Division, NASA Ames Research Center, Moffett Field, CA 94035-1000

Edited by David M. Karl, University of Hawaii, Honolulu, HI, and approved December 4, 2017 (received for review September 7, 2017) 14 The degradation of organic matter in the anoxic seabed proceeds (iii)MGRusing[2- C]-labeled acetate (MGRAC), and (iv)acetate through a complex microbial network in which the terminal steps oxidation rates using [2-14C]-labeled acetate (AOR). are dominated by oxidation with sulfate or conversion into

methane and CO2. The controls on pathway and rate of the deg- Results and Discussion radation process in different geochemical zones remain elusive. The sampling sites were located at a water depth of 88–96 m in Radiotracer techniques were used to perform measurements of the Bornholm Basin, SE Baltic Sea, which is characterized by sulfate reduction, methanogenesis, and acetate oxidation with un- seasonally hypoxic bottom water (stations BB01 to BB04; Table precedented sensitivity throughout Holocene sediment columns S1) (10). Guided by preceding seismoacoustic surveys and ex- from the Baltic Sea. We found that degradation rates transition ploratory coring campaigns, we have carefully chosen each sta- continuously from the sulfate to the methane zone, thereby dem- tion according to organic matter burial history. The shallow onstrating that terminal steps do not exert feedback control on depth of sulfate depletion (from 0.4 mbsf at BB03 to 4 mbsf at upstream hydrolytic and fermentative processes, as previously BB04) and the high rates of methane production within the top suspected. Acetate was a key intermediate for carbon mineraliza- −2 −1 few meters (up to 140 nmol CH4·cm ·d at BB03) depended tion in both zones. However, acetate was not directly converted primarily on the thickness of the organic-rich Holocene mud into methane. Instead, an additional subterminal step converted layer. The Holocene mud layer was formed by stable sedimen- acetate to CO2 and reducing equivalents, such as H2, which then tation during the past 7,000 y and reached 4–9 m thickness, fed autotrophic reduction of CO to methane. 2 depending on the topography of the underlying organic-poor, postglacial clay from >8,500 y B.P. (Fig. S1) (10). Thus, the marine sediment | organic matter mineralization | sulfate reduction | composition of the deposited mud was uniform across the flat methanogenesis | syntrophic acetate oxidation − basin floor, while only the sedimentation rate, 0.5–1mm·y 1, varied between stations. icrobial processes in most of the global seabed are directly SRR decreased in a log–log linear relationship with sediment age or indirectly coupled to the degradation of organic matter M at all stations in accordance with an expected control by organic SCIENCES (1). With increasing depth and age in the sediment, the remaining A matter age on the overall metabolic rate (Fig. 1 )(2).Thistrend ENVIRONMENTAL organic matter becomes increasingly recalcitrant to further deg- continued down toward the sulfate–methane transition (SMT), radation by microorganisms (2). Sulfate is quantitatively the most where sulfate dropped below 1 mM with a concomitant rise of important terminal electron acceptor for the anaerobic oxidation methane. Below the SMT, SRR dropped sharply away from the of organic carbon in ocean margin sediments (3). When sulfate is log–log linear depth trend. In the sulfate zone, above the SMT, depleted at depth, the terminal process of degradation shifts to methanogenesis with the production of methane and CO2.Mi- crobial sulfate reduction rates (SRR) and methanogenesis rates Significance (MGR) can be directly determined from experiments using ra- dioactively labeled tracers. Such measurements show that SRR The subsurface seabed is a great anaerobic bioreactor where drop with increasing sediment depth and age according to a re- organic matter deposited from the ocean’s water column is active continuum that may be simulated by a simple power law slowly being degraded by microorganisms. It is uncertain how function (3–5). This relationship reflects diagenetic alteration and rates of organic matter degradation progress through different increasing recalcitrance during aging of the organic matter, which geochemical zones, and the deep production of methane and limits the hydrolytic attack by extracellular enzymes and thereby CO2 in the global seabed therefore remains poorly constrained. the release of substrates that feed the microbial food chain (2, 6). With highly depth-resolved analyses of geochemistry and mi- In contrast to SRR, MGR have been difficult to determine ex- crobial activities, we demonstrate that rates of organic matter perimentally in sediments due to extremely low rates and due to degradation decrease continuously with sediment age, irre- outgassing of supersaturated methane. The deep production of spective of the prevailing redox zonation and associated changes methane in the global seabed and the associated organic matter in the degradation pathway. Moreover, our results show that degradation are therefore poorly constrained. the microbial food chain leading to methane does not proceed It has been questioned whether extracellular hydrolysis of the directly through the general key intermediate acetate, but rather organic matter is always limiting the rate of degradation (7, 8) or through an additional, as of yet unidentified, syntrophic step. if downstream fermentative or terminal respiratory steps may Author contributions: F.B., H.R., and B.B.J. designed research; B.B.J. and H.R. obtained the also affect the overall rates (9). If the terminal electron acceptor funding; F.B. performed research; F.B. analyzed data; C.G. performed acetate analysis; does not affect the kinetics of organic matter degradation, then and F.B. prepared the manuscript with comments from all authors. the trend of rate versus age determined in the sulfate zone The authors declare no conflict of interest. should continue down into the methane zone. Thus, by extrap- This article is a PNAS Direct Submission. olation of this trend, the distribution of organic matter degra- Published under the PNAS license. dation and methane production deep within the methane zone 1To whom correspondence may be addressed. Email: [email protected] or bo.barker@ could be estimated. To test this, we performed radiotracer-based bios.au.dk. i 35 ii experiments to measure ( ) SRR using [ S]-labeled sulfate, ( ) This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. 14 MGR using [ C]-labeled dissolved inorganic carbon (MGRDIC), 1073/pnas.1715789115/-/DCSupplemental.

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Fig. 1. Rates of anaerobic carbon mineralization in Bornholm Basin sediments. Double logarithmic plots of rates versus age in the sediment for four stations

(BB01–BB04): (A) SRR, (B) total MGR (MGRDIC + MGRAc), (C) total anaerobic organic carbon oxidation rate (COR). Shaded zones indicate the SMT at each individual station using the same color coding for station number as the data symbols. The two gray lines in C depict the upper and lower 95% CI for the best fitted power law function to the data (Eq. 1). Detailed biogeochemical and prokaryotic activity profiles of individual stations are shown in Figs. S3–S10.

−1 −3 −1 MGRDIC were detectable but very low, <10 nmol CH4·cm ·d , between the neighboring stations, BB04 and BB02, triggered by a − or two to three orders of magnitude lower than SRR (Fig. 1B). The moderate increase in sedimentation rate from 0.051 cm·y 1 at · −1 MGRDIC rose sharply and peaked in the SMT with rates similar to BB04 to 0.055 cm y at BB02. Such an abrupt shift is due to a −3 −1 the SRR in that zone (up to ∼4nmolCH4·cm ·d at station positive feedback whereby a slight enhancement of the upwards BB03). Thus, the highest rates of MGRDIC occurred in the SMT diffusion of methane lifts up the SMT and causes a strong en- because the organic matter here had the youngest age and the hancement of methanogenesis in shallower and much more active highest reactivity. In the methane zone below, MGRDIC continued layers (5). to drop along the same log–log linear depth trend of organic carbon Degradation of sedimentary organic matter should lead to mineralization as was found for SRR in the sulfate zone. By con- fermentative production of acetate, as the main single end product, plus some H2 and CO2 (12, 13). Accordingly, it could be trast, MGRAC were extremely low throughout the sediment at all −3 −3 −1 expected that most of the methane production originates from investigated stations (<10 nmol CH4·cm ·d )(Figs. S7–S9). We calculated total anaerobic carbon oxidation rates (COR) acetoclastic methanogenesis (13). Indeed, we found that the rate from the sum of SRR and MGR based on their stoichiometry of acetate consumption corresponded to about one-third of the rate of sulfate reduction above the SMT and about half of the during organic carbon mineralization (Fig. S2) (11). The result- B ing vertical depth trend of COR (Fig. 1C) revealed that rates of rate of methanogenesis below the SMT (Fig. 2 ). Intriguingly, however, our incubations with [14C]-DIC showed that CO re- organic carbon mineralization decreased smoothly and continu- 2 duction accounted for more than 99% of total methanogenesis, ously with organic matter age throughout the sediment, irre- while incubations with [2-14C]-acetate showed no transfer of spective of the prevailing redox zonation and the associated 14 labeled methyl-carbon from acetate to methane. Instead, [2- C]- change in the terminal pathway of the microbial food chain. This acetate was converted to [14C]-CO in the methane zone at rates trend has previously been assumed but not experimentally 2 that closely followed CO2 reduction to methane (Fig. 2C). We demonstrated. The data thereby provide direct support for the attribute this to the only known alternative degradation pathway validity of commonly applied organic matter decay models, such for acetate under methanogenic conditions—a syntrophic cou- as the time-continuous and reactive-continuum approaches (2, pling where acetate is first oxidized to CO2 plus reducing 6), without the necessity to correct organic matter degradation equivalents (e.g., H2), followed by hydrogenotrophic methano- rate with pathway-specific inhibition factors (1). The depth genesis (Fig. 3) (14, 15). trend, based on activity measurements from all four stations in This two-step process was first proposed by Barker (1936) (14) the Bornholm Basin, corresponded to the following power law and was rediscovered half a century later by Zinder and Koch dependence on sediment age (in years B.P.): (1984) (15) in a thermophilic coculture isolated from an anaer- - À - - Á obic digestion reactor. Since then it has been primarily regarded COR = 1,640 × age 1.21 nmol C cm 3 d 1 . [1] as a niche process that only occurs at high temperature or under conditions inhibitory for acetoclastic methanogens, such as ex- + In a uniform and continuously deposited sediment, such a cessively high NH4 concentrations (16, 17) that are about relationship can be extrapolated to estimate organic carbon 100 times higher than those measured in Bornholm Basin sedi- − mineralization also in deeper sediment layers where process ments (<5 mmol·L 1; Figs. S3–S6). However, the detection of rates are difficult to quantify by radiotracer methods due to syntrophic acetate oxidation coupled to methanogenesis in batch exceedingly low microbial activity and due to supersaturation of incubations of lake sediment (18, 19) and oil reservoir fluids (20) methane. Total depth-integrated MGR contributed to the total has indicated broader environmental relevance. The process depth-integrated COR in the Holocene mud layer from 8% at corresponds to a reversal of acetogenesis (from H2/CO2), and BB02 to 40% at BB03—that is, a relatively low contribution, the enzymatic requirements—that is, variations of the Wood– although the methanogenic zone covered 74–94% of the Holo- Ljungdahl pathway—might therefore be widespread among an- cene mud layer (Figs. S1 and S3–S6). This low contribution of aerobic prokaryotes, including sulfate reducers and methanogens methanogenesis was a consequence of the steep decrease in (17, 21, 22). organoclastic activity with sediment depth and age. There was Consistent with our findings, incubation studies and investi- an abrupt shift from no methane to a well-developed methane zone gations of stable carbon isotopic compositions of CH4 in marine

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Fig. 2. Acetate turnover in station BB03 sediment. (A) Down-core profile of acetate concentrations in the pore water. (B) AOR accounted for about one-third and half of the COR above and below the SMT (shaded zone), respectively, and they were about 1,000-fold higher than the rates of direct methanogenesis

from acetate. (C) Activity ratios indicate the electron/mass balance between SRR and AOR or MGRDIC and AOR, with the dashed line indicating a 1:1 ratio. (D) Gibbs energy yield (ΔGR) for acetate metabolism via sulfate reduction (blue line) or via methane production (green line) is shown relative to suggested minimum energy levels of sulfate-reducing prokaryotes (dashed blue line) (29) and methanogenic archaea (dashed green line) (26). Data for other stations are shown in Figs. S7–S9.

sediments suggested a predominance of methanogenesis via CO2 reported below the SMT (e.g., <10 nM in the Cape Lookout reduction in marine sediments (4, 13, 23, 24), thus indicating a Bight) (26). Consistent with our findings, that site was also global relevance of syntrophic methanogenesis from acetate. characterized by a conspicuously high contribution of methano- However, minor contributions of acetoclastic methanogenesis genesis via CO2 reduction (70–80% of total methane production) (<20% of total methane production) have also been detected in (25) and by similar energy yields for methane production from ac- marine sediments (4, 24, 25), thereby leading to the question of etate (27). what controls the relative contribution of the two acetate deg- Our results highlight the importance of a syntrophic re- radation pathways in methanogenic environments. There is no lationship between acetate oxidizers and methanogens to enable obvious thermodynamic rationale for a syntrophic coupling be- acetate degradation under sulfate-limiting conditions. Intrigu- SCIENCES tween acetate oxidation and hydrogenotrophic methanogenesis, ingly, at stations BB04 and BB02, where the methane zone is ENVIRONMENTAL as there is no difference in the overall energy yield of the sum of still in early development, very high acetate concentrations (up − these processes compared with that of direct acetoclastic meth- to >1 mmol·L 1; Figs. S5 and S6) indicate a non-steady state anogenesis. In Bornholm Basin sediments, methanogenesis from situation where syntrophic degradation is not yet able to balance, acetate was exergonic beneath the SMT (Fig. 2D) but was close at the energetic limit, the continuous acetate production by to the proposed energetic limit of methanogenic archaea (26), fermenters. −1 −10 kJ·mol .IfH2 is the exchanged reducing equivalent, then The predominance of acetate oxidizing prokaryotes over the syntrophic relationship would require a narrow concentration acetoclastic methanogens might be due to substrate kinetics and/ range of 1–4 nM to allow both reactions to be exergonic so that or other competitive factors, such as the ability to engage in al- the low overall energy yield can be shared between the partner ternative forms of energy metabolism like acetogenesis and fer- organisms. Such low H2 concentrations have, indeed, been mentation (17). Methanogens have a very narrow substrate

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Fig. 3. Pathways of acetate turnover. The experiments with radiotracers (red) show how the key intermediate, acetate, may be degraded in the sulfate zone

and the methane zone: (A) When sulfate was above a threshold concentration, acetate was oxidized to CO2, coupled with sulfate reduction to sulfide. (B) Depletion of sulfate did not lead to direct methane production from acetate. (C) Instead, acetate was completely oxidized to CO2.(D) Reducing equiv- − alents, symbolized by electrons [e ], from acetate were used for methane production from CO2.

Beulig et al. PNAS | January 9, 2018 | vol. 115 | no. 2 | 369 Downloaded by guest on September 26, 2021 spectrum (28), and the direct conversion of acetate to methane has measured from the headspace of the Exetainer by a DeltaV isotope ratio so far only been ascribed to two microbial groups, Methanosarcina mass spectrometer. Samples for acetate analysis were stored in combusted Methanosaeta glass vials at −80 °C until analysis by 2D ion chromatography–mass spec- and , of which only the latter is reported to grow at in + < trometry (29, 34). Pore water for ammonium (NH4 ) analysis was stored in situ acetate concentrations ( 0.2 mM) (29, 30). Thus, the conver- − + 14 14 2 mL Eppendorf vials at 20 °C, and NH4 was measured photometrically sion of high levels of [2- C]-acetate to [ C]-CO2 in batch incuba- according to the Danish Standard DS 224 protocol adapted for seawater tions of manure and wastewater sludge was only detected in the samples (35). The detailed pore-water geochemistry and the measurements absence of Methanosaeta (19). of microbial activity were derived from different cores. To ensure compa- Our results have important implications for the interpretation rable geochemistry between replicate cores, additional pore-water samples of organic matter degradation in the methane zone of marine for sulfate and acetate analysis were extracted from selected sediment sediments: (i) The terminal process of anaerobic organic matter samples before radiotracer incubation. For this, sediment aliquots (up to 1 g) mineralization does not control the overall rate of degradation, were centrifuged at 14,000 × g with an Eppendorf Minispin benchtop cen- (ii) fermentation produces acetate as the main potential sub- trifuge, and the supernatant was stored and processed as described above. strate for the terminal degradation step in both the sulfate and iii Diffusive Fluxes and Stoichiometric Ratios During Organic Matter Mineralization. the methane zones, and ( ) methanogenesis from acetate pro- Diffusive fluxes of methane were calculated from the concentration gradients ceeds through a syntrophic pathway via CO2 rather than by a using Fick’s First Law: simple conversion of the methyl-carbon in acetate to CH4. The = −φ × × = reasons for this indirect pathway and the key parameters con- J Ds dC dz, trolling it remain to be identified. In fact, it also remains to be where J is the diffusive flux, C is the concentration, z is depth (cm), D is the shown whether the oxidation of acetate, as the single most im- s whole-sediment diffusion coefficient, and φ is porosity. Ds was calculated portant substrate feeding sulfate reduction, may also partly take from porosity, φ, and molecular diffusion coefficients of individual species 2 −1 place in syntrophy. Similar to methanogenesis, a syntrophic ac- (36), Di, at 8 °C in situ temperature and a salinity of 15 (DCH4 = 1.08 cm ·d , 2 −1 2 −1 etate oxidation coupled to sulfate reduction would be exergonic DSO42− = 0.566 cm ·d ,DHCO3− = 0.609 cm ·d ), as described before [Ds = Di/ + − φ for both partners under the low H2 concentrations available. (1 3(1 ))] (37). 2− The concentration profiles of pore-water DIC and SO4 (Fig. S2) were used Materials and Methods to calculate the stoichiometry of organic carbon oxidation to sulfate re- Site Description and Sediment Coring. Cruises were undertaken with the R/V duction (COR:SRR, or rC:SO42−) in the sediment (11): Aurora to the Bornholm Basin (Stations BB01, BB02, BB03, and BB04; Table À Á = 2- × ð = Þ S1). After initial exploratory sampling campaigns in 2014 and 2015, stations rC : SO42- - dDIC dSO4 DHCO3- DSO42- ,

were revisited in 2016 with a keen focus on the transition in mineralization 2− where dDIC/dSO is the slope of the linear regression. Because r − is processes across the SMT. If not indicated otherwise, each site was sampled 4 C:SO42 determined by the oxidation state of the organic matter (ox), we can use r − with a gravity corer of up to 9 m in length and complementary Rumohr cores C:SO42 to calculate the stoichiometry of organic carbon oxidation to methanogenesis (31). All cores at each station were sampled with the vessel positioned within (COR:MGR, or r )(11): 5 m diameter. The Rumohr corer allows nearly undisturbed sampling of the C:CH4 top meter of sediment below the seafloor. The extent of the unavoidable rC : SO42- = 8=ð4 − oxÞ = rC : CH4. sediment loss from the top of the gravity cores (ca. 10–20 cm) was determined by aligning the sulfate concentration profiles of parallel gravity and Rumohr cores. All cores were processed on board the R/V Aurora, with Process Rate Measurements Using Radiotracers. As processing under strictly high priority given to samples for methane and microbial activity analysis to temperature-controlled conditions was not possible on board, the temper- ensure that samples could be taken within minutes after core retrieval. ature of the cores was monitored continuously. Over the course of the entire Gravity cores were cut into 1-m or 2-m subsections, capped, and processed sampling procedure (1–2 h), the temperature in the cores increased by immediately depending on the type of analysis, as described below. In only 1–3°C. Rumohr cores, sediment material was sampled by stepwise extrusion from SRR were determined in subcores collected in butyl rubber-stoppered 5-mL the core liner with a piston, while sediment material in gravity-core sub- sterile cutoff polycarbonate syringes, as described before (33). Each syringe sections was sampled, starting from the bottom, by drilling holes (for subcore was injected with 5 μL carrier-free [35S]-sulfate solution containing methane samples) or by cutting windows in the core liner (for microbial up to 140 kBq of radioactivity, depending on the expected microbial activity. activity samples). The outermost part of the sediment that had been in MGRDIC or MGRAc, as well as AOR, were determined in subcores collected with contact with the core liner was avoided during sampling. butyl rubber-stoppered glass tubes equipped with acrylic plungers similar to the cut syringes. Each subcore was injected with 5 μL[14C]-DIC solution, containing Methane Analysis. A 2.5-mL sediment sample was taken with a cutoff plastic up to 170 kBq of 14C radioactivity, or 5 μL[2-14C]-acetate solution, containing up syringe, immediately transferred to 20-mL preweighed glass vials containing to 10 kBq of 14C radioactivity. The addition of [2-14C]-acetate led to an increase 4 mL of saturated NaCl solution, and crimp-capped with butyl rubber stop- of the acetate concentration by only ∼20%. All samples for measurement of pers. Samples were then shaken thoroughly and stored upside down at microbial activity were incubated at the in situ temperature of 8 °C (281 K) −20 °C until analysis. Before analysis, the samples were weighed, thawed, within gas-tight plastic bags containing an Oxoid AnaeroGenTM pad (Oxoid A/S).

and shaken again to equilibrate methane between slurry and headspace of This oxygen scrubber effectively removes O2 but does not generate H2.The the glass vial. We injected 100–500 μL of the headspace with a glass syringe incubation times were chosen according to the expected microbial into a gas chromatograph equipped with a 0.9 m packed silica gel column activity and differed from 12 h to 30 h. For example, in samples from the shallow (3.1 mm inner diameter) and a flame ionization detector (GC-FID; SRI 310C; Rumohr cores, we expected a fast turnover of the typically small acetate pool, SRI Instruments). and therefore, incubations were kept relatively short (∼12 h). SRR incubations in cut syringes were terminated by freezing the intact Pore-Water Analyses. Rhizon samplers (32) for pore-water analysis were samples at −20 °C, without opening the bags. After thawing, the total re- 0 flushed with at least 50 mL Milli-Q water, dried, and stored in gas-tight foil duced inorganic sulfur (TRIS = H2S, S , FeS, and FeS2) was separated from the 35 2− bags until use. In Rumohr and gravity cores, small holes were drilled into the SO4 by a single-step cold chromium distillation (33, 38). The radioactiv- core liner of undisturbed sections to collect pore water with the Rhizon ities of [35S]-sulfate and 35S-TRIS were counted in 15 mL scintillation mixture samplers by creating a vacuum with a 20-mL syringe. The first 1 mL of the (Gold Star, Meridian Biotechnologies) on a TriCarb 2900TR liquid scintillation extracted pore water was discarded, and then up to ∼10 mL was collected analyzer (Packard Instrument Company). before the samples were aliquoted for analysis of individual pore-water To terminate [14C]-DIC and [2-14C]-acetate incubations, the entire glass parameters. For sulfate analysis, the extracted pore water was acidified syringe was transferred into glass vials, containing 5 mL NaOH (2.5%), and stripped of hydrogen sulfide (H2S) by bubbling with a stream of moist without extruding the mud, and immediately crimp-capped with butyl CO2. Sulfate samples were stored at 4 °C until analysis as described before rubber stoppers. The glass vials were then shaken thoroughly to wash the (33). DIC samples were stored in 2-mL glass vials without headspace at 4 °C. sample out of the glass syringe and stored upside down at −20 °C until 14 Samples were transferred to sealed Exetainers, acidified with 85% (v:v) analysis. CH4 in these vials was determined by flushing the headspace with −1 14 phosphoric acid, and after 24 h of equilibration time, the produced CO2 was CO2-free air at 25 mL·min for 15 min and oxidation of the CH4 in the

370 | www.pnas.org/cgi/doi/10.1073/pnas.1715789115 Beulig et al. Downloaded by guest on September 26, 2021 14 evolving gas stream to CO2 in a quartz glass tube containing CuO pellets, MGRAc = ATR × ACH4=½ACH4 + ADIC , heated to 900 °C. The efficiency of methane oxidation was tested by adding known amounts of CH4 to a reaction vessel and following its conversion to and CO2 in the exhaust gas by GC-FID (see Methane Analysis). Conversion effi- 14 = × =½ + ciencies were always >99%. CO2 from the oven exhaust gas was trapped in AOR ATR ADIC ACH4 ADIC . 5 mL Carbosorb (Perkin-Elmer). In contrast to our earlier application of 14C for determination of methanogenesis, the gas stream was subjected to a COR were calculated from SRR and MGR based on the estimated stoichi- wash step in 1 M NaOH before combustion to prevent trace amounts of ometry of organic matter mineralization during sulfate reduction and = = ± labeled DIC to penetrate into the oven. In addition, the entire gas line was methanogenesis (rC:SO42− rC:CH4 1.37 0.16; see Diffusive Fluxes and

made of glass, which does not absorb CO2. These precautions lowered Stoichiometric Ratios During Organic Matter Mineralization and Fig. S2) (11): the background by at least two orders of magnitude and thus enhanced the = ½ × + ½ð + Þ × sensitivity of the method by a similar factor. This improvement of the COR SRR rC : SO42- MGRDIC MGRAc rC : CH4 . method was a prerequisite for the precise measurement of extremely low The power law correlation lines describing the age or depth trend of COR rates of methanogenesis in the deep part of the sediment. were generally calculated under omission of elevated rates within the SMT, To determine the [14C]-DIC remaining in the glass vial, after extraction of fed by upwards diffusing methane, and those from 0 to 10 cmbsf (0–100 y CH , a 0.5-mL subsample of sediment slurry was transferred into a new glass 4 sediment age) where sulfate might not be the main electron acceptor. The vial, crimp-capped with butyl rubber stoppers, and acidified with 2 mL of HCl 14 linear regression after log–log transformation was corrected for logarithmic (6 M). All CO2 produced was flushed out of the vial headspace with N2 at − 25 mL·min 1 for 35 min and trapped in 5 mL Carbosorb. The radioactivity of skewing as described before (5). 14 CO2 was counted in a 5 mL scintillation mixture (Permafluor, PerkinElmer) Δ 0 on a TriCarb 2900TR liquid scintillation analyzer (PerkinElmer). To determine Calculation of Gibbs Free Energy. The standard Gibbs energy ( G insitu)of − − − − 14 14 2 + → + the residual C-acetate in [2- C]-acetate incubations, an additional 1-mL sub- sulfate reduction from acetate (SO4 CH3COO HS 2HCO3 )and − − sample of the acidified and flushed supernatant was counted in 10 mL Gold Star methanogenesis from acetate (direct: CH3COO + H2O → CH4 + HCO3 ;or − − + − scintillation mixture on a TriCarb 2900TR liquid scintillation analyzer. In control syntrophic: CH3COO + 4H2O → 4H2 + 2HCO3 + H , coupled to 4H2 + HCO3 + + experiments to determine abiotic radiotracer conversion and/or carryover during H → CH4 + 3H2O) was calculated using the SUPCRT92 software package (41) sample processing, radiotracer was added to sediments, immediately frozen, and and reported thermodynamic data (42–44) for a mean pressure of 1.1 MPa and processed in the same way as the incubated samples. 8 °C in situ temperature. The energy of reactions at nonstandard conditions The SRR were calculated as described before (33, 39): (ΔGR) was calculated according to Â Ã 2- SRR = SO × ATRIS × 1.06 × φ=ðt × ½ATRIS + ASO42Þ, Δ = Δ 0 + × × 4 GR Ginsitu R T lnQ,

2− where [SO4 ] is the sulfate concentration, ATRIS is the radioactivity of TRIS at where R is the ideal gas constant, T is the in situ temperature, and Q denotes 2− the end of the incubation, ASO42− is the radioactivity of SO4 at the end of the activity quotient of the reactants and reaction products. Activities in the the incubation, and t is the length of the incubation. Similarly, the MGRDIC saline pore fluids were estimated by multiplying the measured concentration were calculated as described before (25, 40): 2− − of the species with activity coefficients (0.245 for SO4 , 0.725 for HCO3 , − − 0.699 for HS , 0.725 for CH3COO , 1.08 for CH4, and 1 for H2O) calculated MGRDIC = ½DIC × ACH4 × 1.08 × φ=ðt × ½ACH4 + ADIC Þ, from an extended version of the Debye–Hückel equation (45) for an ionic = where [DIC] is the DIC concentration, ACH4 is the radioactivity of CH4 at the strength of I 0.28 and at 8 °C using the Geochemists Workbench Software SCIENCES end of the incubation, and ADIC is the radioactivity of DIC at the end of the (www.gwb.com). incubation. In contrast to incubations with [14C]-DIC, a significant fraction of ENVIRONMENTAL the [2-14C]-acetate tracer was consumed during the incubation of samples ACKNOWLEDGMENTS. The authors thank captain Torben Vang and the close to the sediment surface, where activities were high and acetate con- crew of the R/V Aurora for sampling; Susanne Nielsen, Jeanette Pedersen, centrations were low. To account for this consumption, we assumed a first- Karina Bomholt Oest, and Julie Rotschi (Aarhus University) for analytical order decrease in the specific radioactivity of the acetate pool (25, 40). The work and technical support; André Pellerin, Alexander Brice Olson Michaud, acetate turnover rate (ATR) was therefore calculated as Karen-Marie Hilligsøe (Aarhus University), and Gilad Antler (Cambridge Uni- versity) for help during sampling; and Julia Beulig for constructive comments

ATR = ð½Ac × 1.08 × φ=tÞ × lnð½AAc + ACH4 + ADIC =AAcÞ, on the text. The research was funded by the Danish National Research Foun- dation Grant DNRF104 and FP7 European Research Council (ERC) Advanced where [Ac] is the acetate concentration and AAc is the radioactivity of ace- Grant 294200 (to B.B.J.) and Danish Center for Marine Research Grant “Cryp- tate at the end of the incubation. MGRAc and AOR were then calculated as tic Biogeochemistry in the Bornholm Basin” (to H.R.).

1. Arndt S, et al. (2013) Quantifying the degradation of organic matter in marine sed- 12. Sørensen J, Christensen D, Jørgensen BB (1981) Volatile fatty acids and hydrogen as iments: A review and synthesis. Earth Sci Rev 123:53–86. substrates for sulfate-reducing bacteria in anaerobic marine sediment. Appl Environ 2. Middelburg JJ (1989) A simple rate model for organic matter decomposition in marine Microbiol 42:5–11. sediments. Geochim Cosmochim Acta 53:1577–1581. 13. Conrad R (1999) Contribution of hydrogen to methane production and control of 3. Jørgensen BB (1982) Mineralization of organic matter in the sea bed—The role of hydrogen concentrations in methanogenic soils and sediments. FEMS Microbiol Ecol sulphate reduction. Nature 296:643–645. 28:193–202. 4. Jørgensen BB, Parkes RJ (2010) Role of sulfate reduction and methane production by 14. Barker HA (1936) On the biochemistry of the methane fermentation. Arch Mikrobiol organic carbon degradation in eutrophic fjord sediments (Limfjorden, Denmark). 7:404–419. Limnol Oceanogr 55:1338–1352. 15. Zinder SH, Koch M (1984) Non-aceticlastic methanogenesis from acetate: Acetate 5. Flury S, et al. (2016) Controls on subsurface methane fluxes and shallow gas forma- oxidation by a thermophilic syntrophic coculture. Arch Microbiol 138:263–272. tion in Baltic Sea sediment (Aarhus Bay, Denmark). Geochim Cosmochim Acta 188: 16. Schnürer A, Zellner G, Svensson BH (1999) Mesophilic syntrophic acetate oxidation 297–309. during methane formation in biogas reactors. FEMS Microbiol Ecol 29:249–261. 6. Boudreau BP, Ruddick BR (1991) On a reactive continuum representation of organic 17. Hattori S (2008) Syntrophic acetate-oxidizing microbes in methanogenic environ- matter diagenesis. Am J Sci 291:507–538. ments. Microbes Environ 23:118–127. 7. Arnosti C (2004) Speed bumps and barricades in the carbon cycle: Substrate structural 18. Nüsslein B, Chin K-J, Eckert W, Conrad R (2001) Evidence for anaerobic syntrophic effects on carbon cycling. Mar Chem 92:263–273. acetate oxidation during methane production in the profundal sediment of sub- 8. Abell J, Laverman AM, Cappellen PV (2009) Bioavailability of organic matter in a tropical Lake Kinneret (Israel). Environ Microbiol 3:460–470. freshwater estuarine sediment: Long-term degradation experiments with and with- 19. Karakashev D, Batstone DJ, Trably E, Angelidaki I (2006) Acetate oxidation is the out nitrate supply. Biogeochemistry 94:13–28. dominant methanogenic pathway from acetate in the absence of Methanosaetaceae. 9. Burdige DJ, Gardner KG (1998) Molecular weight distribution of dissolved organic Appl Environ Microbiol 72:5138–5141. carbon in marine sediment pore waters. Mar Chem 62:45–64. 20. Mayumi D, et al. (2013) Carbon dioxide concentration dictates alternative meth- 10. Andrén E, Andrén T, Sohlenius G (2000) The Holocene history of the southwestern anogenic pathways in oil reservoirs. Nat Commun 4:1998. Baltic Sea as reflected in a sediment core from the Bornholm Basin. Boreas 29: 21. Ozuolmez D, et al. (2015) Methanogenic archaea and sulfate reducing bacteria co- 233–250. cultured on acetate: Teamwork or coexistence? Front Microbiol 6:492. 11. Burdige DJ (2006) Geochemistry of Marine Sediments (Princeton Univ Press, 22. Lever MA (2012) Acetogenesis in the energy-starved deep biosphere–A paradox? Princeton). Front Microbiol 2:284.

Beulig et al. PNAS | January 9, 2018 | vol. 115 | no. 2 | 371 Downloaded by guest on September 26, 2021 23. Whiticar MJ, Faber E, Schoell M (1986) Biogenic methane formation in marine and 36. Boudreau BP (1997) Diagenetic Models and Their Implementation: Modelling freshwater environments: CO2 reduction vs. acetate fermentation—Isotope evidence. Transport and Reactions in Aquatic Sediments (Springer, Berlin). Geochim Cosmochim Acta 50:693–709. 37. Iversen N, Jørgensen BB (1993) Diffusion coefficients of sulfate and methane in ma- 24. Parkes RJ, et al. (2007) Biogeochemistry and biodiversity of methane cycling in sub- rine sediments: Influence of porosity. Geochim Cosmochim Acta 57:571–578. – surface marine sediments (Skagerrak, Denmark). Environ Microbiol 9:1146 1161. 38. Kallmeyer J, Ferdelman TG, Weber A, Fossing H, Jørgensen BB (2004) A cold chro- 25. Crill PM, Martens CS (1986) Methane production from bicarbonate and acetate in an mium distillation procedure for radiolabeled sulfide applied to sulfate reduction anoxic marine sediment. Geochim Cosmochim Acta 50:2089–2097. measurements. Limnol Oceanogr Methods 2:171–180. 26. Hoehler TM, Alperin MJ, Albert DB, Martens CS (1998) Thermodynamic control on 39. Jørgensen BB (1978) A comparison of methods for the quantification of bacterial hydrogen concentrations in anoxic sediments. Geochim Cosmochim Acta 62: sulfate reduction in coastal marine sediments. Geomicrobiol J 1:11–27. 1745–1756. 40. Hansen LK, Jakobsen R, Postma D (2001) Methanogenesis in a shallow sandy aquifer, 27. Hoehler TM, Alperin MJ, Albert DB, Martens CS (1994) Field and laboratory studies of – methane oxidation in an anoxic marine sediment: Evidence for a methanogen-sulfate Rømø, Denmark. Geochim Cosmochim Acta 65:2925 2935. reducer consortium. Global Biogeochem Cycles 8:451–463. 41. Johnson JW, Oelkers EH, Helgeson HC (1992) SUPCRT92: A software package for 28. Ferry JG, Lessner DJ (2008) Methanogenesis in marine sediments. Ann N Y Acad Sci calculating the standard molal thermodynamic properties of minerals, gases, aqueous 1125:147–157. species, and reactions from 1 to 5000 bar and 0 to 1000°C. Comput Geosci 18:899–947. 29. Glombitza C, et al. (2015) Formate, acetate, and propionate as substrates for sulfate 42. Shock EL, Helgeson HC (1990) Calculation of the thermodynamic and transport reduction in sub-arctic sediments of Southwest Greenland. Front Microbiol 6:846. properties of aqueous species at high pressures and temperatures: Standard partial 30. Jetten MSM, Stams AJM, Zehnder AJB (1992) Methanogenesis from acetate: A com- molal properties of organic species. Geochim Cosmochim Acta 54:915–945. parison of the acetate metabolism in Methanothrix soehngenii and Methanosarcina 43. Shock EL, Helgeson HC (1988) Calculation of the thermodynamic and transport spp. FEMS Microbiol Rev 8:181–197. properties of aqueous species at high pressures and temperatures: Correlation al- 31. Meischner D, Rumohr J (1974) A light-weight, high-momentum gravity corer for gorithms for ionic species and equation of state predictions to 5 kb and 1000°C. – subaqueous sediments. Senckenb Marit 6:105 117. Geochim Cosmochim Acta 52:2009–2036. 32. Seeberg-Elverfeldt J, Schlüter M, Feseker T, Kölling M (2005) Rhizon sampling of 44. Shock EL (1995) Organic acids in hydrothermal solutions: Standard molal thermody- porewaters near the sediment-water interface of aquatic systems. Limnol Oceanogr namic properties of carboxylic acids and estimates of dissociation constants at high Methods 3:361–371. temperatures and pressures. Am J Sci 295:496–580. 33. Røy H, Weber HS, Tarpgaard IH, Ferdelman TG, Jørgensen BB (2014) Determination of 45. Helgeson HC (1969) Thermodynamics of hydrothermal systems at elevated temper- dissimilatory sulfate reduction rates in marine sediment via radioactive 35S tracer. – Limnol Oceanogr Methods 12:196–211. atures and pressures. Am J Sci 267:729 804. 34. Glombitza C, Pedersen J, Røy H, Jørgensen BB (2014) Direct analysis of volatile fatty 46. Jensen JB, Moros M, Endler R, IODP Expedition 347 Members (2016) The Bornholm acids in marine sediment porewater by two-dimensional ion chromatography-mass Basin, southern Scandinavia: A complex history from late Cretaceous structural de- spectrometry. Limnol Oceanogr Methods 12:455–468. velopments to recent sedimentation. Boreas 46:3–17. 35. Bower CE, Holm-Hansen T (1980) A salicylate–Hypochlorite method for determining 47. Andrén T, Barker Jorgensen B, Cotterill C, Green S, Slomp C (2015) IODP expedition ammonia in seawater. Can J Fish Aquat Sci 37:794–798. 347: Baltic Sea basin paleoenvironment and biosphere. Sci Drill 20:1–12.

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