Anaerobic methane oxidation in freshwater wetlands?

Methane emitted from wetlands plays a major role in the global climate – it is a powerful greenhouse gas, and it is produced by microbes. The methane cycle in wetlands is a complex process, as the methane is produced deeper down, in the anoxic part of the wetland sediment, and then rises up through its pores. During this process, it is also consumed by bacteria and thus methane emissions are attenuated. But why do microorganisms make it?

An introduction to redox chemistry

Microbes have many ways of sustaining themselves, but what is common to all of them is the basic chemistry behind them. The reactions they use are called reduction-oxidation reactions, or redox reactions. A simple example of a redox reaction occurs between hydrogen (H2) and oxygen (O2). This can be observed when you apply a spark to a balloon. When this occurs, the balloon will explode with a loud burst, as you have just provided “activation energy” to start the following reaction:

2H2 + O2  → 2H2O

Even though you needed a spark to start it, once it is ongoing, it releases much more energy than was need to start it. This is very common to many useful chemical redox reactions, such as the oxidation of organic matter with oxygen – also known as fire. In microbes and other forms of life, the ‘spark’ is created by enzymes, and the reaction can involve all kinds of organic and inorganic compounds. Animals oxidise organic matter with oxygen using the following reaction:

C6H12O6 + O2  → 6CO2 +6 H2

However, microbes can use all kinds of chemical compounds to oxidise organic matter when oxygen is no longer present. This requires a compound through which energy is produced when it receives electrons from organic matter. The energy that can be produced in chemical reactions, is defined as the redox potential.

In the natural environment, electron acceptors that produce the most energy when they react with organic matter are usually consumed first. The most reactive electron acceptor is oxygen. When all the oxygen has been used up, other less reactive compounds are consumed (e.g. nitrate, iron, sulphate, manganese; Figure 1). There is a lot of sulphate in seawater, so sulphate reduction is very common in marine settings. The hydrogen sulphide that is produced by it is the reason for the particular “estuarine smell” (= not very nice smell of rotten eggs). When no other electron acceptors are left over, but there is still organic matter, it is finally converted into methane by organisms called methanogenic archaea. Following this, the methane then diffuses upwards through the sediment (as it is a gas), and will eventually be oxidised when it interacts with other electron donors, either within the sediment or the overlying water column.

Redox

Fig. 1. The successive degradation of organic matter (OM) in the sediment

The consumption of methane: anaerobic vs aerobic?

One of the key questions in biogeochemistry concerns the fate of methane produced in the sediments. It is known that methane can be oxidised aerobically to CO2 by bacteria (Hanson and Hanson, 1996). These microbes are called aerobic methanotrophs and there are two groups of them – the Type I methanotrophs (γ-proteobacteria), and the Type II methanotrophs (α-proteobacteria). These organisms have been shown to make specific compounds called aminobacteriohopanetetrol and aminobacteriohopanepentol (van Winden et al., 2012), and those are used as biomarkers for aerobic methanotrophs. However, there are other microbes that use alternative electron acceptors, especially in anaerobic environments, to consume methane.

ANMEA

Figure 2. ANME consortium: Sulfate-reducing bacteria in green, anaerobic methane oxidisers in red. (Boetius et al. 2000).

Relatively recently (1999/2000), it was shown that archaea were able to oxidise methane anaerobically when working in a partnership with sulfate reducing bacteria. This is shown in the following reaction:

CH4 + SO42− → HCO3 + HS + H2O

This process is called anaerobic oxidation of methane (AOM) and the archaea involved in this reaction are closely related to those who produce methane. However, they were not mediating all of this themselves: they had outsourced the reduction process to sulphate reducing bacteria who they were living in close collaboration with (Boetius et al., 2000). Although they also use very similar enzymes to the methane-producing ones, the key difference is that they are operating the pathway in reverse (this is known as reverse methanogenesis). Biomarkers associated with anaerobic oxidation of methane include archaeols (diether lipids) and tetraether lipids (Niemann and Elvert, 2008; Pancost et al., 2000).

Structures

Figure 3. Biomarkers archaeol and GDGT-0, a tetraether lipid, which are both commonly found in anaerobic methane oxidizers.

AOM in non-marine settings?

In the absence of sulphate, other electron acceptors might be used to drive anaerobic oxidation of methane. These settings might include lakes or peatlands (in other words, where there is an absence of sulphate-rich seawater – the typical “wetland-smell” is, ulike the “estuarine smell”, not of rotten eggs). However, the isolation of the microorganisms, or conclusive evidence for freshwater AOM is still lacking in some cases. To date, two organisms were found – Ettwig et al. (2010) found a methane-consuming, nitrite reducing bacterium and Haroon et al. (2013) found a nitrate-reducing bacterium. Other possible electron acceptors include Fe and Mn reduction (which happens a lot with standard organic matter, but there are only indications for it being coupled with methane at the moment).

Given the lack of sulphate in wetlands, anaerobic methanotrophy was not expected to occur in freshwater environments. However, some new research challenges this idea:

The paper: High rates of anaerobic methane oxidation in freshwater wetlands reduce potential atmospheric methane emissions

In the paper, Segarra et al. (2015) investigate the rate and impact of AOM in three freshwater wetlands located in Florida, Georgia and Maine, respectively. To quantify the rate of AOM and sulphate reduction (SR), they utilised in vitro radiotracer assays (in this case, the addition of 14C-labelled methane). Using this approach, they conclude that AOM occurs at all depths and is sustained over a relatively large temperature range (25°C). Intriguingly, the rates of AOM are comparable to those observed in gas hydrate-related methane seeps. Sulphate reduction rates were also high, yet the concentration of sulphate itself was low. This has been interpreted to indicate very rapid turnover of sulphur species (2-5 days).

Profiles

Figure 4. The downcore profiles of sulfate and methane in the three freshwater wetlands (after Segarra et al. 2015).

The key electron acceptor?

It remains unclear whether AOM is coupled to sulphate reduction in terrestrial settings. To explore this further, Segarra et al. (2015, 2013) compared the downcore geochemical profiles of SR and AOM in each site. In most settings, SR rates were sufficient to support AOM. This suggests that the primary electron acceptor is sulphate (analogous to deep-marine settings). However, in Georgia, the rate of AOM exceeds SR and implies the use of an alternative electron acceptor such as nitrate, nitrite, iron oxides or manganese oxides. Although a high correlation was observed between reduced iron and AOM rates in Florida, previous studies have shown that the addition of sulphate is more likely to lead to higher AOM rates than the addition of iron or other electron acceptors. Evidence for nitrate-dependant AOM (n-damo) was also limited, with no significant correlation between nitrate and AOM in either Florida or Georgia. This is consistent with the carbon isotopic composition of a biomarker associated with n-damo, which was not significantly depleted in 13C, something that would be expected when AOM is involved. The authors also speculate that the role of humic acids may be important, however evidence for this process remains poorly constrained.

Impact upon microbial biogeochemistry?

To explore the impact of AOM upon the microbial community, this paper also reconstructs the carbon isotopic composition of methane, dissolved inorganic carbon and a suite of microbial lipids. In this study, both archaeal and bacterial membrane lipids were analysed. In cold-seep settings, archaea produce a suite of glycerol dialkyl glycerol tetraethers (GDGTs) with 0-2 cyclopentane moieties. However, in order to analyse the carbon isotopic value of these compounds, the GDGTs must be cleaved. This produces biphytane chains with 0 to 2 rings (or in the case of this study, up to 3 rings).

In the FWW, the carbon isotopic composition of the archaeal-derived biphytanes range from ~-37 to -61‰; while these values are 13C depleted, they are much heavier than observed in marine cold-seep settings where values can be as low as ~120‰. The can potentially be explained by invoking additional biological sources of biphytanes (e.g. Thaumarchaeota), the incorporation of dissolved inorganic carbon (rather than isotopically-light methane) into the lipids. Alternatively, these organisms may be completely distinct from those found in marine settings and use unknown metabolic pathways. Segarra et al. (2015) also analysed a range of bacterial-derived fatty acids which typically co-occur with sulphate-reducing bacteria in marine cold-seeps. These lipids are isotopically depleted (at least -50‰) and support a methanotrophic source. Notably, the authors do not report other diagnostic lipids associated with anaerobic methanotrophs (e.g. PMI) or sulphate-reducing bacteria (non-isoprenoidal dialkyl glycerol diethers).

The big picture:

Segarra et al. (2015) conclude by extrapolating their data that AOM may consume 200 Tg methane per year. This is roughly equal to the quantity of methane emitted from FWW each year; as such, this process may play a significant role in limiting methane production. However, this is based upon a small dataset and further studies are required to constrain this estimate.

Summary:

In summary, it is likely that the majority of AOM can be explained by coupling to sulphate reduction; however, alternative electron acceptors cannot be ruled out. Although the carbon isotopic composition of archaeal and bacterial lipids indicate a methanotrophic source, these lipids are not as 13C-depleted as expected. This can potentially be explain via a number of reasons (e.g. additional sources, different substrates and/or different metabolic pathways). On a global scale, the role of AOM in dampening methane emissions from freshwater wetlands may be more significant than previously acknowledged.

Boetius, A., Ravenschlag, K., Schubert, C.J., Rickert, D., Widdel, F., Gieseke, A., Amann, R., Barker Jørgensen, B., Witte, U., Pfannkuche, O., 2000. A marine microbial consortium apparently mediating anaerobic oxidation of methane. Nature 407, 623–626. doi:10.1038/35036572

Ettwig, K.F., Butler, M.K., Le Paslier, D., Pelletier, E., Mangenot, S., Kuypers, M.M.M., Schreiber, F., Dutilh, B.E., Zedelius, J., de Beer, D., Gloerich, J., Wessels, H.J.C.T., van Alen, T., Luesken, F., Wu, M.L., van de Pas-Schoonen, K.T., Op den Camp, H.J.M., Janssen-Megens, E.M., Francoijs, K.-J., Stunnenberg, H., Weissenbach, J., Jetten, M.S.M., Strous, M., 2010. Nitrite-driven anaerobic methane oxidation by oxygenic bacteria. Nature 464, 543–548. doi:10.1038/nature08883

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Haroon, M.F., Hu, S., Shi, Y., Imelfort, M., Keller, J., Hugenholtz, P., Yuan, Z., Tyson, G.W., 2013. Anaerobic oxidation of methane coupled to nitrate reduction in a novel archaeal lineage. Nature 500, 567–570. doi:10.1038/nature12375

Niemann, H., Elvert, M., 2008. Diagnostic lipid biomarker and stable carbon isotope signatures of microbial communities mediating the anaerobic oxidation of methane with sulphate. Org. Geochem. 39, 1668–1677. doi:10.1016/j.orggeochem.2007.11.003

Pancost, R.D., van Geel, B., Baas, M., Sinninghe Damsté, J.S., 2000. δ13C values and radiocarbon dates of microbial biomarkers as tracers for carbon recycling in peat deposits. Geology 28, 663. doi:10.1130/0091-7613(2000)28<663:CVARDO>2.0.CO;2

Segarra, K.E.A., Comerford, C., Slaughter, J., Joye, S.B., 2013. Impact of electron acceptor availability on the anaerobic oxidation of methane in coastal freshwater and brackish wetland sediments. Geochim. Cosmochim. Acta 115, 15–30. doi:10.1016/j.gca.2013.03.029

Segarra, K.E.A., Schubotz, F., Samarkin, V., Yoshinaga, M.Y., Hinrichs, K.-U., Joye, S.B., 2015. High rates of anaerobic methane oxidation in freshwater wetlands reduce potential atmospheric methane emissions. Nat. Commun. 6, 7477. doi:10.1038/ncomms8477

van Winden, J.F., Talbot, H.M., Kip, N., Reichart, G.-J., Pol, A., McNamara, N.P., Jetten, M.S.M., Op den Camp, H.J.M., Sinninghe Damsté, J.S., 2012. Bacteriohopanepolyol signatures as markers for methanotrophic bacteria in peat moss. Geochim. Cosmochim. Acta 77, 52–61. doi:10.1016/j.gca.2011.10.026

This post was written by Gordon Inglis and Sabine Lengger

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