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. www.pnas.org/cgi/doi/10.1073/pnas.1715789115 PNAS | January 9, 2018 | vol. 115 | no. 2 | 367–372 Downloaded by guest on September 26, 2021 ABC 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.