Rates and Microbial Players of Iron-Driven Anaerobic Oxidation of Methane in Methanic Marine Sediments

Abstract

The flux of methane, a potent greenhouse gas, from the seabed is largely controlled by anaerobic oxidation of methane (AOM) coupled to sulfate reduction (S-AOM) in the sulfate methane transition (SMT). S-AOM is estimated to oxidize 90% of the methane produced in marine sediments and is mediated by a consortium of anaerobic methanotrophic archaea (ANME) and sulfate reducing bacteria. An additional methane sink, i.e., iron oxide coupled AOM (Fe-AOM), has been suggested to be active in the methanic zone of marine sediments. Geochemical signatures below the SMT such as high dissolved iron, low to undetectable sulfate and high methane concentrations, together with the presence of iron oxides are taken as prerequisites for this process. So far, Fe-AOM has neither been proven in marine sediments nor have the governing key microorganisms been identified. Here, using a multidisciplinary approach, we show that Fe-AOM occurs in iron oxide-rich methanic sediments of the Helgoland Mud Area (North Sea). When sulfate reduction was inhibited, different iron oxides facilitated AOM in long-term sediment slurry incubations but manganese oxide did not. Especially magnetite triggered substantial Fe-AOM activity and caused an enrichment of ANME-2a archaea. Methane oxidation rates of 0.095 ± 0.03 nmol cm^-3 d^-1 attributable to Fe-AOM were obtained in short-term radiotracer experiments. The decoupling of AOM from sulfate reduction in the methanic zone further corroborated that AOM was iron oxide-driven below the SMT. Thus, our findings prove that Fe-AOM occurs in methanic marine sediments containing mineral-bound ferric iron and is a previously overlooked but likely important component in the global methane budget. This process has the potential to sustain microbial life in the deep biosphere.

Keywords: ANME-2a; anaerobic methanotrophs; anaerobic oxidation of methane; iron oxides; marine sediment; microbial community analysis; radiotracer; stable isotope probing.

FIGURE 1

Global map of marine environments with elevated dissolved iron concentrations in the methanic zone. This has been found in both shallow (e.g., Bothnian Sea) and deep-sea environments (e.g., Argentine Basin) of several meters below sea surface (mbss). Presence of elevated dissolved iron concentrations in the methanic zone is currently hypothesized to be primarily driven by Fe-AOM. Absolute dissolved iron concentrations and references are listed in Supplementary Table S1.

FIGURE 2

Geochemical profiles reflecting the existence of the geochemical prerequisites for Fe-AOM in the methanic sediments of Helgoland Mud Area. (A) Pore-water profiles of sulfate, sulfide, methane, dissolved iron and dissolved manganese in the sediments. (B) Solid-phase determination of total Fe contents. (C) Distribution of operationally defined iron phases within the sediments (FeCarb: sodium acetate extractable, FeOX1: hydroxylamine-HCl extractable, FeOX2: dithionite extractable and FeMag: oxalate extractable iron oxide phases). Gray area represents the SMT. Sulfate zone, SMT and methanic zones were identified using pore-water profiles in (A).

FIGURE 3

Distribution and abundance of microorganisms in the Helgoland Mud Area sediment core (HE443-010-3). Relative abundances of (A) mcrA genes and (B) mcrA gene copies of ANME. Error bars represent 1 s.d. of technical qPCR triplicates. (C) Cell counts of potentially active bacteria and archaea based on CARD-FISH. Gray bars within the profiles depict the SMT.

FIGURE 4

Direct indication for ¹³CH₄ and ¹⁴CH₄ turnover to CO₂ in incubations with Helgoland Mud Area sediments. (A) Change in δ¹³C-DIC values over 250 days in slurry incubation experiments with sediments from the sulfate zone (0–25 cm) and the methanic zone (347–372 cm) with ¹³CH₄ tracer, n = 3, error bars represent 1 s.d. of biological replicates. The ¹³C-increase in the DIC pool serves as proxy for AOM. DIC isotope values in control incubations are provided in Supplementary Figure S11. (B) Rates of methane turnover based on ¹⁴CH₄ in samples from the SMT (50–75 cm), n = 3, and the methanic zone (200–225 cm, 300–325 cm, 400–425 cm), n = 2, error bar represents 1 s.d. of biological replicates. INSET: scale adjusted activity rates in the methanic zone. Using ¹⁴CH₄, rates of methane turnover were below abiotic control samples in ¹⁴CH₄ and molybdate treatment from the SMT and in the methanic zone samples from depths 300–325 cm and 400–425 cm after 8 days. “n.d.”: rates not detected above abiotic controls.

FIGURE 5

Molecular fingerprints providing insights into microbial activity during ¹³CH₄ oxidation and potential key players involved in Fe-AOM. (A) Abundance of mcrA gene copies assigned to different ANME clades in the methanic zone after 120 and 250 days of incubation in molybdate amended incubations from the methanic zone. Gray line in each plot represents estimates of gene copies of the different ANME phylotypes at respective incubation depths (see Figure 3B) as indication for increasing gene copies during the 250-day incubation experiment. INSET: log scaled, adjusted mcrA gene copies of ANME-2a. mcrA gene copies across all incubations from the sulfate zone and methanic zone are provided in Supplementary Figure S5. Error bars represent 1 s.d. of technical qPCR replicates. (B) Relative abundances of ANME and Deltaproteobacteria based on 16S rRNA gene sequencing in the Fe-AOM incubations from the methanic zone after 120 and 250 days. Relative abundance based on total sum scaling of archaeal and bacterial 16S rRNA genes is provided in Supplementary Figures S3, S4, S7, S9.

FIGURE 6

Uptake of ¹³CH₄ label into bacterial polar lipid fatty acids during AOM. Development of carbon isotopic composition of dominant bacterial fatty acids (‰ VPDB) over time during S-AOM in the sulfate zone (supplemented with sulfate) and Fe-AOM in the methanic zone (supplemented with molybdate and lepidocrocite) is presented. Complete list of δ¹³C values of fatty acids and total uptake by each fatty acid is given in Supplementary Tables S3, S4.