Manganese- and Iron-Dependent Marine Methane Oxidation

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Science  10 Jul 2009:
Vol. 325, Issue 5937, pp. 184-187
DOI: 10.1126/science.1169984


Anaerobic methanotrophs help regulate Earth’s climate and may have been an important part of the microbial ecosystem on the early Earth. The anaerobic oxidation of methane (AOM) is often thought of as a sulfate-dependent process, despite the fact that other electron acceptors are more energetically favorable. Here, we show that microorganisms from marine methane-seep sediment in the Eel River Basin in California are capable of using manganese (birnessite) and iron (ferrihydrite) to oxidize methane, revealing that marine AOM is coupled, either directly or indirectly, to a larger variety of oxidants than previously thought. Large amounts of manganese and iron are provided to oceans from rivers, indicating that manganese- and iron-dependent AOM have the potential to be globally important.

Anaerobic oxidation of methane (AOM) occurs in freshwater samples in the absence of sulfate, provided nitrite or nitrate is present (1, 2). Incubation studies show that the addition of manganese (MnO2) or iron (FeCl2 and FeCl3) to anoxic sediments and digested sewage increases the ratio of methane oxidized to methane produced (3). However, there has been no direct evidence for AOM in the absence of sulfate in marine samples (4). Studies of pore-water geochemistry show manganese and iron reduction in areas where AOM occurs (5), and the highest AOM rates in marine sediment do not always correlate with the highest sulfate reduction rates (6). Furthermore, sediments of the uplifted Franciscan Complex, a paleo-analog of the Eel River Basin (ERB), show methane-derived 13C-depleted carbonate associated with rhodocrosite (MnCO3) (7). In addition, there is enrichment of manganese and other metals in methane seep–associated carbonates from the Black Sea (8).

Here, we show that birnessite (Fig. 1) and ferrihydrite (Fig. 2) can be used as electron acceptors in marine AOM. Large amounts of manganese [~19 Tg/year (9)] and iron [~730 Tg/year (10)] are provided to continental margins from rivers (11). Iron and manganese are provided to the ERB in this manner by high sediment discharge from the Eel River, which drains the northern California Coast Range (12). If the entire global flux of manganese and iron is used to oxidize methane, it could account for about one-fourth of present-day AOM consumption. Even if only a small percentage of the influx of manganese and iron is used for AOM, it still has the potential to be a large methane sink because both manganese and iron can be oxidized and reduced 100 to 300 times before burial (13).

Fig. 1

13C enrichment of CO2 reported in 13FCO2 (13C/13C+12C) values and converted to moles methane oxidized. The incubations with manganese (birnessite) oxidize about 3.5 times as much methane as the live control (sulfate free, no provided electron acceptor), indicating that manganese can be used as an electron acceptor in AOM. Error bars represent the range of the triplicate incubations. The standard deviations of the triplicate incubations for the birnessite and live controls are within the symbol for each data point. In addition, when more birnessite is injected into the cultures, the rate of AOM increases ~30%, from ~11 μmole/year/cm3sed (days 23 to 43) to ~14 μmole/year/cm3sed (days 43 to 57).

Fig. 2

13C enrichment of CO2 reported in 13FCO2 (13C/13C+12C) values and converted moles methane oxidized. The incubations with iron (ferrihydrite) oxidize about 5 times as much methane as the live control (sulfate free, no provided electron acceptor), indicating that iron can be used as an electron acceptor in AOM. Error bars represent the range of data from the triplicate incubations. The standard deviations of the triplicate incubations for the ferrihydrite and live controls are within the symbol for each data point.

Methane-seep sediment from the ERB was incubated with methane, 13C-labeled methane, CO2, and artificial sulfate-free seawater. Triplicate incubations were given either sulfate, birnessite, ferric oxyhydroxide, ferrihydrite, nitrate, nitrate and sulfate, or no electron acceptor (live control). The birnessite and ferrihydrite experiments were pre-incubated (14) to ensure that they were sulfate free. As methane is oxidized, the 13C-label is transferred from methane to CO2, and thus we can monitor AOM by measuring the 13C enrichment in the CO2 throughout the experiment. δ13CO2 values are then converted into the amount of methane oxidized (15, 16).

We measured 13C-enrichment of CO2 in cultures supplied with sulfate, birnessite, and ferrihydrite, indicating that AOM can proceed in the absence of sulfate if birnessite (Fig. 1) or ferrihydrite (Fig. 2) is present (17). Sulfate was measured (SulfaVer4 method; Hach, Loveland, CO) periodically in all live control, birnessite, and ferrihydrite incubations to show that they remained sulfate free (<30 μM sulfate) for the duration of the experiment. An autoclaved bottle (Figs. 1 and 2), containing either birnessite or ferrihydrite, shows that no abiotic isotopic exchange between methane and CO2 and no abiotic production of sulfate occurs. Incubations with just nitrate, as well as with nitrate and sulfate, appear to inhibit AOM (fig. S1). We also see no evidence for AOM in the presence of ferric oxyhydroxide (fig. S1).

The net reaction for the AOM is often framed asCH4 + SO42− → HCO3 + HS + H2O (1)AOM with sulfate (1) provides organisms with a potential Gibbs free energy of ΔG = –14 kJ/mole for our in situ conditions. All of the sulfate incubations reported here oxidize methane at an approximate rate of 52 μmole/year/cm3sed (Fig. 1), corresponding to a potential energy gain of 0.7 J/year/cm3sed (Table 1). However, the oxidation of methane with birnessite (simplified to MnO2) yields ΔG = –556 kJ/mole at our in situ conditions (2). CH4 + 4MnO2 + 7H+ → HCO3 + 4Mn2+ + 5H2O (2)The observed rate of birnessite-dependent AOM is 14 μmole/year/cm3sed (Fig. 1), which equals a potential energy gain of 7.8 J/year/cm3sed (Table 1). Therefore, although the rate of sulfate-dependent AOM is about four times as fast as birnessite-dependent AOM, the birnessite incubations have the potential to gain 10 times as much energy as the sulfate incubations.

Table 1

Rates and potential energy gain from AOM with different electron acceptors.

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AOM coupled to ferrihydrite [simplified as Fe(OH)3] reduction (3) yields a potential free energy of ΔG = –270.3 kJ/mol at our in situ conditions.CH4 + 8 Fe(OH)3 + 15H+ → HCO3 + 8Fe2+ + 21H20 (3)The incubations with ferrihydrite oxidize methane at an average rate of 6 μmole/year/cm3sed (Fig. 2), corresponding to a potential energy gain of 1.6 J/year/cm3sed (Table 1). This shows that the microorganisms responsible for ferrihydrite-dependent AOM have the potential to receive energy at about twice the rate of sulfate-dependent AOM, despite the fact that they are oxidizing methane at about one-tenth the rate.

Previous culture studies have found that microorganisms from the Black Sea can reduce manganese oxides more efficiently than ferrihydrite (18). This result is consistent with our experiment, where we see that manganese-dependent AOM occurs at a faster rate than iron-dependent AOM. Both manganese- and iron-dependent AOM occur at much slower rates than sulfate-dependent AOM, although they are substantially more energetically favorable. This can be explained by considering that manganese and iron oxides are both solids, and thus less accessible than sulfate. Despite the slower methane oxidation rates of manganese and iron-dependent AOM, it is likely that they are an important part of biogeochemical methane cycling.

There are three known archaeal groups responsible for AOM: ANME-1 and ANME-2 (19) and ANME-3 (20). ANME commonly have sulfate-reducing bacterial partners, often related to Desulfosarcinales and Desulfobulbus (2124). However, ANME-1 and some ANME-2 have been found to live independently, suggesting that they may not need a physically associated sulfate-reducing bacteria to perform AOM (19, 22). The fact that ANME are often found with sulfate reducers does not necessitate that sulfate is needed for AOM to proceed. Some sulfate-reducing bacteria can facultatively use electron acceptors other than sulfate (2527). In fact, one species of Desulfobulbus is capable of iron reduction (28). The presence of greigite magnetosomes in sulfate-reducing bacteria associated with ANME-2 from the Black Sea further suggests a role in iron cycling (8, 29).

To study the microbial communities responsible for manganese-dependent AOM, we sampled one incubation from each set of conditions at the end of the experiment and determined changes in the microbial assemblage based on 16S rRNA and methyl coenzyme M reductase (mcrA) gene diversity. Over the course of the 10-month incubation (14), a shift was observed in the archaeal diversity relative to the starting sediment. The proportion of phylotypes associated with the crenarchaeota increased in both the manganese incubation and the live control (sulfate-free, no added electron acceptor), whereas the sulfate incubation supported an increase in euryarchaeota, in particular phylotypes belonging to ANME 2b and 2c (fig. S2). Uncultured phylotypes belonging to Marine Benthic Group D (MBGD) were the most abundant in the starting sediment and remained a substantial component of the archaeal diversity in all treatments, representing 35% or more of the total clones (fig. S2). The metabolic potential of MBGD is not currently known; however, it is interesting to note that the closest cultured relatives of many of the recovered phylotypes are methanogens (80% identity), and their potential role in methane cycling warrants further investigation. An increase in phylotypes associated with the Crenarchaeota Marine Benthic Group C (MBGC), which is absent in the sulfate incubations, was observed in both the manganese and live control incubations (fig. S2).

16S rRNA phylotypes belonging to the known methanotrophic ANME groups made up a relatively small proportion of all sediment incubations, with ANME-1 representing no more than 5% of the total archaeal diversity. However, analysis of the mcrA gene (specific for methanogens and methanotrophic archaea) indicated a greater diversity of the methanotrophic ANME than was recovered by the initial 16S rRNA screen. Specifically, with the exception of the sulfate incubations, the most common mcrA gene came from the ANME-1 (~85%) (fig. S2). In the sulfate incubations, ANME-1 represented 42% of the recovered mcrA genes, with ANME-2 representing 46%. The manganese and sulfate incubations revealed an increase in diversity supporting a small percentage of ANME-3, not observed in the original sediment or live control (fig. S2).

About 40% of the bacteria found in the birnessite incubation are possible manganese reducers (Fig. 3 and fig. S3). These include clones related to microorganisms found in heavy-metal contaminated sites or from hydrothermal systems. Specifically, the groups Bacteriodes, Proteobacteria (including Geobacter), Acidobacteria, and Verrucomicrobia contain representatives likely capable of metal reduction (Fig. 3) (3033). Bacteriodes are only present in the manganese and control incubations, whereas Acidobacteria are only present in the manganese incubations (Fig. 3). The clones related to sulfur cycling in the birnessite incubations are almost all sulfur oxidizers, such as the ε-Proteobacteria Sulfurovumales. The bacteria in the sulfate incubations are dominated by sulfate reducers, mainly Desulfobulbus.

Fig. 3

Percent distribution of recovered bacterial clones based on 16S rRNA genes in the starting sediment (Other includes clades OP3 and Marine Group A), live control, manganese (Other includes clades Elusimicrobia and KSB3), and sulfate incubations (Other includes clades Marine Group A, KSB3, GN02, and TM6). Sulfur metabolism indicates phylotypes putatively involved in sulfur cycling. Metal associated represent phylotypes that are possible manganese reducers. Metal/Sulfur are the phylotypes that have the potential to partake in sulfur and/or metal cycling. The starting sediment was stored anaerobically for ~1 year before use and therefore does not reflect the proportions of bacteria when it was sampled.

The large change toward manganese reducers observed in the bacterial community from the birnessite incubation suggests that bacteria are playing a vital role in manganese-dependent AOM and that archaea are not solely responsible (Fig. 3). In the birnessite incubation, the relative proportion of ANME-2 decreases, whereas Methanococcoides/ANME-3 increases and ANME-1 stays relatively constant (fig. S2). Overall, our data imply either that manganese-dependent AOM is carried out by ANME-1 and/or Methanococcoides/ANME-3 with a bacterial partner, or that manganese-dependent AOM in this case is not performed by archaea but rather solely by bacteria. If bacteria are indeed solely responsible for manganese-dependent AOM, it is likely that they do not contain the mcrA gene, as recently observed for nitrite-dependent AOM (1).

Abiotic and biotic processes can oxidize sulfide to sulfur in the presence of metal oxides (34, 35). In principle, sulfur disproportionation producing transient sulfate, mediated perhaps by Desulfobulbus (36) or ε-Proteobacteria, could be the underlying process observed, indirectly linking AOM to metal reduction. Although the shift in the bacterial community from known sulfate-reducing bacteria to putative metal-reducing microorganisms in the birnessite incubations supports the idea that the AOM is directly linked to metal reduction, the observed shift in microbial community could also be a result of the stimulation of heterotrophic metal reduction. If metal reduction is indirectly linked to AOM in marine sediments, then the realized energy gain for the microorganisms directly catalyzing AOM would be much lower than that suggested in Table 1. Regardless of mechanism, the stimulation of AOM with Mn and Fe has important implications for capacity of CH4 oxidation.

It is estimated that AOM consumes most methane released in marine settings, equaling 5 to 20% of today’s total global methane flux (37), making this process an important part of the global carbon cycle today. However, before Earth became oxygenated, growth of methanotrophs was limited by their ability to find electron acceptors. Based on the column-integrated photooxidation rates of 5 mg/cm2/year of manganese and 200 mg/cm2/year of iron (38), on the order of 10,000 Tg/year of methane could be oxidized during this time period by manganese- and iron-dependent AOM, irrespective of whether the processes directly link metal reduction to methane oxidation. Estimates of the methane flux to the atmosphere during the Proterozoic are on the order of 1,000 to 10,000 Tg/year (39), meaning that manganese- and iron-dependent AOM had the oxidative potential to oxidize the entire early Earth methane flux. Thus, manganese- and iron-dependent AOM could have been extremely important methane sinks, as well as energy sources, for the early biosphere.

Supporting Online Material

Materials and Methods

SOM Text

Figs. S1 to S3


References and Notes

  1. There are minor contributions of manganese and iron from hydrothermal systems and aeolian input. See SOM text for further discussion.

  2. Materials and methods are available as supporting material on Science Online.

  3. See relevant SOM text for discussion regarding dissolved manganese and iron concentrations.

  4. We would like to thank M. Arthur for the use of his mass spectrometer, Z. Zhang and S. Goffredi for laboratory assistance, D. Walizer for technical assistance, and D. Jones for help with phylogenetics. We also thank the shipboard scientists, crew, and pilots of R/V Atlantis and R/V Western Flyer. Funding for this project has come from the National Science Foundation (MCB-0348492), National Aeronautics and Space Administration (NASA) Astrobiology Institute under NASA-Ames Cooperative Agreement NNA04CC06A, and the Penn State Biogeochemical Research Initiative for Education (BRIE) funded by NSF (IGERT) grant DGE-9972759. Sequences were submitted to GenBank and have accession numbers FJ264513 to FJ264602 and FJ264604 to FJ264884.
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