Palaeogeography, Palaeoclimatology, Palaeoecology 227 (2005) 6 – 17 www.elsevier.com/locate/palaeo
Microbial methane turnover in different marine habitats Martin Kru¨ger a,*, Tina Treude a,b, Heike Wolters a, Katja Nauhaus c, Antje Boetius a,d a Max Planck Institute for Marine Microbiology, Celsiusstrasse 1, D-28359 Bremen, Germany Alfred-Wegener-Institute for Polar and Marine Research, Columbusstrasse, 27568 Bremerhaven, Germany c LMU Munich, Department Biology I, Maria-Ward Strasse 1A, D-80638 Munich, Germany d International University Bremen, Campusring 1, D-28759 Bremen, Germany
b
Received 29 March 2004; received in revised form 28 August 2004; accepted 11 April 2005
Abstract Microbial methanogenesis in the subsurface seafloor is responsible for the formation of large and dynamic gas reservoirs like the recently discovered gas hydrate deposits. Gas seepage occurs wherever methane builds up an overpressure outside the hydrate stability field, illustrating the potential importance of ocean margins for the global methane budget. However, a variety of bacteria and archaea are capable of methane consumption, and control the emission of methane to the hydrosphere. Unfortunately, much less is known about the microbial methane turnover in the ocean than about methane turnover in freshwater or terrestrial habitats. This investigation compares rates of methane production, anaerobic and aerobic methane oxidation at different marine sites, combining radiotracer (on-site) and in vitro measurements. Samples were obtained from gas hydrate bearing sediments, cold seeps, organic-rich and organic-poor subsurface sediments. All investigated subsurface sediments had the potential for methanogenesis as well as for methanotrophy. The anaerobic oxidation of methane (AOM) was highest in samples from gas hydrate areas and cold seeps. AOM was strongly influenced by methane partial pressure and temperature, indicating a substantial underestimation of in situ activity with current ex situ measuring techniques. A potential for aerobic methane oxidation was detected at all sites where the sediment had contact with oxic bottom water. A first comparison of methane turnover rates in diverse marine habitats showed that microbial methane oxidation provides a very effective barrier for methane emissions from the subsurface seafloor. D 2005 Elsevier B.V. All rights reserved. Keywords: Methanogenesis; Methane oxidation; Methane seeps; Global methane budget; Microorganisms
1. Introduction
* Corresponding author. Current address: Federal Institute for Geosciences and Resources, Stilleweg 2, D-30655 Hannover, Germany. Tel.: +49 511 6433102; fax: +49 511 6433664. E-mail address:
[email protected] (M. Kru¨ger). 0031-0182/$ - see front matter D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.palaeo.2005.04.031
Around two-thirds of the world’s surface is covered by oceanic systems. However, the contribution of these large areas to the global CH4 emission to the atmosphere is extremely low with around 3–5% (IPCC, 1994). Reeburgh (1996) proposed that up to 80% of
M. Kru¨ger et al. / Palaeogeography, Palaeoclimatology, Palaeoecology 227 (2005) 6–17
the CH4 produced in the predominantly anoxic marine sediments is oxidized prior to its release into the hydrosphere. The most important temporary sinks for methane are gas hydrate deposits. Another important control mechanism is the process of sulfate-dependent anaerobic oxidation of methane (AOM) in anoxic subsurface sediments. Using biogeochemical (tracer measurements, modeling, lipid biomarker analysis) and molecular approaches, the importance of AOM could be demonstrated for a number of different sites (see Hinrichs and Boetius, 2002, and references therein). It was shown that AOM can be the dominant sulfate consuming process in a variety of marine environments, especially where methane is available in high concentrations and as the main carbon source (Boetius et al., 2000; Borowski et al., 2000; Treude et al., 2003). Still, the biochemical functioning of AOM as well as the physiology of the relevant microorganisms remain unknown. In a recent paper we reported on the in vitro investigation of AOM and its relation to methane concentration and temperature (Nauhaus et al., 2002). In aerobic surface sediments, oxygen-dependent methane oxidation also contributes to the reduction of CH4 emissions (King, 1992; Bussmann, 1994). However, oxygen penetration depth does usually not exceed 1 cm in coastal and margin sediments (Hensen and
7
Zabel, 2000). Exceptions are habitats with high bioturbation activities, e.g. through bivalves or worms, or with organic-poor coarse sediments. Therefore, aerobic oxidation of methane is generally restricted to a much narrower sediment horizon compared to AOM, because of the deeper penetration depth of sulfate. The present study aimed at determining the potential rates of anaerobic and aerobic methane oxidation in different marine sediments and to compare them with rates of methane production. A number of different methane-rich sites were investigated as well as control sites with low bbackgroundQ methane concentrations (Fig. 1). Extrapolations of these site-specific rates of methane turnover to regional and global scales will allow to estimate more accurately the importance of marine systems for the global methane budget.
2. Methodology 2.1. Origin and storage of sediment samples Sediment samples were taken from a variety of marine habitats on continental margins. The characteristics of the sampled sites are summarized in Table 1 and described in more detail in the following:
4
5,6 1 7
3 2
8
Fig. 1. Map showing the distribution of sampling sites considered in this study, (1) Hydrate Ridge, (2) Chile, (3) Gulf of Mexico, (4) Haakon Mosby Mud Volcano, (5) North Sea: Spiekeroog, (6) Baltic Sea: Bay of Eckernfo¨rde/Kattegat, (7) Black Sea, (8) Namibia.
8
Location
Site (position)
Cruise time
Water depth [m]
Sampling depth/depth of SO2 4 :CH4 transition [m]
CH4 source
Baltic Sea
Bay of Eckernfo¨rde (54830V N 10806V E) Kattegat (5881V N 9834V E) Spiekeroog (5681V N 9831V E) Crimean peninsula (44846V N 31860V E) Haakon Mosby Mud Volcano (728 N 14843V E) Namibia (23810V S 13831V E) Gulf of Mexico (27847V N 91830 E) Hydrate Ridge (44834V N 12588V W) Chile (44809V S 75809V W)
Sept-01
28
0.4/0–0.3
May-02
0.5
0.5/0–0.3
Jul-01
0–5
0.5/n. d.a
Jul-01
250
0.5/0–0.15
Organic matter degradation Organic matter degradation Organic matter degradation Fossil methane
Aug-01
1250
0.8/0–0.15
Fossil methane
Damm and Bude´us (2003)
Jul-02
25
1/0–0.1
Niewo¨hner et al. (1998)
Jul-01
650
0.5/0–0.15
Organic matter degradation Gas hydrates
2000 and 2002
700
1/0–0.15
Gas hydrates
2000
800–4600
5/1.5–4
Organic matter degradation
North Sea Black Sea Atlantic Ocean
Pacific Ocean
a b c d
n. d.: not determined. Estimated from methane profiles of Abegg and Anderson (1997). Calculated from methane profile. Calculated from methane consumption (Treude et al., 2003) and methane effluxes (Torres et al., 2002).
CH4 flux [mmol m 2 d 1]
Reference
0.6–1.3b
Bussmann et al. (1999)
0.2–0.5c
Dando et al. (1994) Bo¨ttcher et al. (1998) Michaelis et al. (2002)
Joye et al. (2004) 56–200d
Boetius et al. (2000)
0.07–0.13c
Treude et al. (2005)
M. Kru¨ger et al. / Palaeogeography, Palaeoclimatology, Palaeoecology 227 (2005) 6–17
Table 1 Overview of sampling sites, sample collection and methane sources
M. Kru¨ger et al. / Palaeogeography, Palaeoclimatology, Palaeoecology 227 (2005) 6–17
The muddy, organic-rich sediments of Eckernfo¨rde Bay, German Baltic have very high methane concentrations that result in gas bubble development (Abegg and Anderson, 1997). The methane is of biogenic origin produced by organic matter degradation and accumulates only below the sulfate penetration depth due to AOM (Bussmann et al., 1999; Martens et al., 1999). Spiekeroog and Kattegat represent samples collected in shallow intertidal zones in the German Wadden Sea and in the transition zone between North and Baltic Sea, respectively. Both consisted of coarse sandy sediment with layers of attached black organic material (Dando et al., 1994). The site in the Kattegat is further characterised by the seepage of mainly biogenic methane from large underground reservoirs. This led to the formation of typical seep associated carbonate structures also in very shallow water depths. In the northwestern Black Sea, hundreds of active methane seeps occur along the shelf edge west of the Crimea peninsula at water depths between 35 and 800 m (Ivanov et al., 1991). In the present study, we investigated surface sediments within the anoxic zone, i.e. at 250 m water depth, overlying the methane seeps. The sediments were located close to recently described methanotrophic reefs (Michaelis et al., 2002). The Ha˚kon Mosby mud volcano (HMMV) is a cold methane seep situated at the Norwegian continental margin. The sediment is characterised by active fluid and gas seepage leading to disruptions of the glacial sediments. The temperature of the bottom water is 1.5 8C in a water depth of 1250 m. The HMMV has a central zone with gas saturated sediments, surrounded by an area with gas hydrates and microbial mats (Pimenov et al., 2000). The coastal upwelling system off central Namibia is one of the most productive regions of the oceans (Bru¨chert et al., 2003). The sediment consists of fluid, darkgreen diatom ooze with high organic carbon contents. Samples were collected on the inner continental shelf which is further characterised by anoxic, sulfidic bottom waters as well as a high rates of primary production and sulfate reduction (Baily, 1991). The sediments in the northern Gulf of Mexico overlie large reservoirs of liquid and gaseous hydrocarbons (Joye et al., 2004, and references therein). Oil, gas and brines rise to the sediment surface in conduits by salt-driven tectonics. Gas hydrates form at the sediment–water interface. These methane and oil-
9
rich sediments support methanotrophic and sulfatereducing communities as well as chemosynthesis. At Hydrate Ridge on the Cascadia convergent margin off Oregon (USA), fluids and methane ascend along faults from deep sediments to the surface due to tectonic activity (Suess et al., 1999). Under the prevailing conditions of low temperature and high hydrostatic pressure, gas hydrates form in the surface sediments at water depths between 600 and 800 m. The hydrate composition is dominated by methane (vol.% N 95, Suess et al., 1999). The high methane flow supports methanotrophic and chemosynthetic communities in the surface sediments (Boetius et al., 2000; Sahling et al., 2002; Treude et al., 2003). The sediments along the Chilean continental margin receive a continuous deposition of organic material produced by phytoplankton in the euphotic zone (Arntz and Fahrbach, 1991). The upwelling system off the Chilean coast belongs to the worldTs largest highproductivity area (up to 0.11 mol C m 3 d 1; Peterson et al., 1988). The investigated sediments consisted mainly of hemipelagic mud with a sharp sulfate– methane-transition at 1.5–4 m sediment depth (Hebbeln et al., 2001). Methane is produced in situ during organic matter degradation. Generally, two types of samples were obtained: (a) sediment for in vitro rate measurements, i.e. for incubations of sediment slurries under controlled laboratory conditions, (b) sediment for ex situ (on-site) rate measurements, i.e. for measurements with intact sediment cores at in situ temperature immediately after retrieval of the samples. For the in vitro analysis, sediment cores were sliced into horizons of 0–1, 1– 4 and 8–12 cm sediment depth, combined according to depth and site, and stored anoxically in glass bottles (250 ml) at in situ temperature until further processing in the laboratory. For ex situ rate measurements, samples were obtained by subsampling sediments either with 2.6 cm F push-cores (multicorer samples) or with 5 ml glass tubes (gravity corer samples). Pushcores as well as glass tubes were closed with rubber stoppers at both ends for anoxic incubations. 2.2. Anaerobic methane oxidation rates 2.2.1. In vitro rate measurements Potential rates of anaerobic oxidation of methane (AOM) were determined in vitro as described previ-
10
M. Kru¨ger et al. / Palaeogeography, Palaeoclimatology, Palaeoecology 227 (2005) 6–17
ously (Nauhaus et al., 2002). Sediment samples were mixed with artificial seawater medium (Widdel and Bak, 1992) to obtain homogenous slurries. Subsequently, 9 ml of medium were added to 3 ml of sediment slurry. The headspace of the incubations consisted of pure methane at atmospheric pressure (0.1 MPa) or of a N2–CO2-mixture (90 / 10, [vol/vol]) in controls. All incubations were done in triplicate. Samples for chemical analyses were collected using microliter syringes (pre-flushed with N2). For the determination of the temperature optimum of AOM, tubes were incubated at 1, 4, 8, 12, 16, 20, 24 and 28 8C. AOM activity was determined via the measurement of sulfide production in incubations with and without methane in the headspace. Generally, sulfate and methane concentrations were 28 and 1.5 mM, respectively. Higher partial pressures of methane were applied using a high-pressure incubation device as described in detail in Nauhaus et al. (2002). 2.2.2. Ex situ rate measurements Ex situ rates of AOM were determined measuring the turnover of injected 14CH4 into 14CO2. The tracer was injected in 1 cm intervals through pre-drilled holes in the push-cores, according to the whole-core injection method of Jørgensen (1978), or through the rubber stopper of the glass tubes, respectively. After an incubation time of 24 to 48 h, push-cores were sectioned into 1 cm intervals. The sediments were transferred into 50 ml glass bottles filled with 25 ml sodium hydroxide (2.5% w/w) to stop bacterial activity. The bottles were closed with rubber stoppers and shaken thoroughly.
2.4. Aerobic CH4 oxidation rates Potential aerobic methane oxidation rates in the sediment samples were determined in vitro as described in Kru¨ger et al. (2002). Slurries were prepared under oxic conditions with artificial seawater (1 : 1). Aliquots of these sediment slurries (20 ml) were transferred into sterile glass bottles (175 ml) and supplemented with 1% to 20% methane. The bottles were incubated at 4–12 8C in the dark and shaken once per day. Methane depletion was quantified by sampling the headspace of triplicate incubations after thorough shaking of the bottles for subsequent GCFID analysis. Sediment dry mass was determined after drying at 80 8C for 2 days. 2.5. Analytical procedures Methane was determined using a GC 14B gas chromatograph (Shimadzu) equipped with a Supel-Q Plot column (30 m 0.53 mm; Supelco) and a flame ionization detector (Nauhaus et al., 2002). Sulfide was determined colorimetrically using the methylene blue formation reaction in a miniaturized assay (Aeckersberg et al., 1991) or the formation of colloidal copper sulfide (Cord-Ruwisch, 1985). The ex situ AOM rate was determined by CH4 gas chromatography, 14CH4 combustion/trapping and 14CO2 acidification/trapping after Treude et al. (2003), which is a modified method of Iversen and Blackburn (1981). AOM rates were calculated by the following equation (Eq. (1)): 14
AOM ¼ 2.3. Methane production rates Potential methanogenesis was measured in vitro without the addition of substrates as described in Kru¨ger et al. (2001). Three milliliters of anoxic sediment slurries were transferred into glass tubes as described above. Nine milliliters of an artificial sulfate-free seawater medium were added and the tubes sealed with butyl stoppers. The headspace consisted of a N2/CO2-mixture (90 / 10). Incubation was the same as described for AOM. Headspace samples (0.1 ml) were taken with pressure lock syringes twice per day after shaking of the tubes by hand, and analyzed for CH4.
CO2 CH4 4vt
14 CH
ð1Þ
where 14CO2 is the activity of the produced radioactive carbon dioxide, CH4 is the amount of methane in the sample, 14CH4 is the activity of the radioactive methane, v is the volume of the sediment, and t is the incubation time.
3. Results and discussion Compared to terrestrial and freshwater environments, only little is known about the cycling of methane-derived carbon in marine environments (Reeburgh, 1996). Attempts to estimate the impor-
M. Kru¨ger et al. / Palaeogeography, Palaeoclimatology, Palaeoecology 227 (2005) 6–17
tance of microbial methane production and consumption in the global methane budget had to rely on a few biogeochemical investigations of ocean margin subsurface sediments. In a combined approach we now determined the potential rates of anaerobic and aerobic methane oxidation as well as methane production rates in a variety of marine sediments (Fig. 1). These new data will support future attempts to assess the role of the ocean in controlling the global atmospheric methane concentration. 3.1. The importance of methane production Three major sources for methane—microbial, thermogenic, and abiogenic—are found in the marine system (Schoell, 1988). Abiogenic methane is formed at hot vents by water–rock reactions with hydrogen and CO2 and during serpentinisation on large areas of the seafloor (Sherwood Lollar et al., 2002). Thermogenic methane stems from the geothermal transformation of organic substances buried in marine sediments (Tissot and Welte, 1984). Microbial methane production is predominantly found in organic-rich anoxic sediments, especially below the penetration depth of sulfate. Typically a zonation of the sediment is observed: In the lower, sulfate-depleted horizons methane is produced, while in the upper sulfate-containing horizon the methane is oxidized by anaerobic methanotrophs (Whiticar, 2002). The zone of anaerobic methanotrophy is usually characterized by a methane-sulfate transition (Iversen and Jørgensen, 1985). In the present study, potential methane production rates (MPR) were highest in organic-rich sediment horizons, especially if sulfate as electron donor was limiting. The highest MPR were found in samples from Namibia and Eckernfo¨rde Bay (Table 2), ranging from 0.04 to 0.5 Amol CH4 gdw 1 d 1. Both sites are characterised by an extremely high input of organic matter from the water column (Niewo¨hner et al., 1998; Whiticar, 2002), leading to the formation of up to several meters thick layers of fluffy sediments. In these sediments a rapid depletion of sulfate within the upper centimeters of sediment is observed, leading to ideal conditions for methanogenesis as a terminal process in organic matter degradation. The lowest MPR were determined in the rather sandy sediments from the North Sea and the Kattegat as well as from the Black Sea.
11
Table 2 Potential rates of aerobic methane oxidation and methane production in different sediment samples determined in vitro, the latter without the addition of substrates Sampling site
Rates of aerobic methane oxidation [Amol gdw 1 d 1]
Rates of methane production [Amol gdw 1 d 1]
Hydrate Ridge Black Sea Chile Haakon Mosby Mud Volcano Namibia Gulf of Mexico North Sea: Spiekeroog Baltic Sea: Kattegat Bay of Eckernfo¨rdeb
0.03–0.04 0.02–0.04 0.02–0.03 0.02–0.1
0.04–0.05 0.005–0.01 0.03–0.04 0.02–0.08
0.1–0.4 n. m.a 0.01–0.2 0.03–0.05 0.01–0.16
0.03–0.05 n. m.a 0.01–0.03 0.02–0.03 0.01–0.1
a b
n. m.: not measured. Treude et al. (in press).
Interestingly, MPR in Hydrate Ridge sediments were not affected by sulfate concentrations ranging from 0 to 100 mM (data not shown). Major substrates for methanogenesis in sulfate-rich zones are non-competitive compounds like methylamines or methylthiols (Oremland et al., 1982). Usually, methanogens in these layers are successfully outcompeted by sulfatereducing bacteria for hydrogen and acetate, as the latter have a much lower threshold for these compounds (Oremland et al., 1982). In deeper sediment layers, depleted of sulfate, these substrates become available for methane production. In our study we did not test for different methanogenic substrates. However, the production of methane in Hydrate Ridge sediments despite high concentrations of sulfate suggests the presence of substrates that are non-competitive to sulfate reducers. Also in less organic-rich sediments methane production has been detected. The ubiquitous detection of MPR in surface to shallow subsurface sediments from different cold-water marine habitats (see map in Fig. 1) indicates an important role of microbial methanogenesis in the marine carbon cycle. 3.2. The anaerobic oxidation of methane in marine sediments Several geological structures like gas hydrates, mud volcanoes and hydrothermal vents exhibit high methane fluxes (Judd et al., 2002). All upwelling
12
M. Kru¨ger et al. / Palaeogeography, Palaeoclimatology, Palaeoecology 227 (2005) 6–17
CH4 oxidised [ mol gdw-1 d-1]
3.0 on-site (with radiotracer)
2.5
in-vitro
2.0 1.5
0.10 0.05 0.00
n.d. Hydrate Ridge Eckernförde
Chile
Fig. 2. Comparison of potential rates of anaerobic oxidation of CH4 measured in samples from Hydrate Ridge (high-methane), Bay of Eckernfo¨rde (Baltic Sea, low-methane) and Chile, either determined on-site (ex situ) using 14C-labeled CH4 or in vitro via sulfide production (mean F SD, n = 3–5). n. d.: not detectable.
areas and most coastal areas harbor organic-rich sediments with a high methane production. Although there might be a high variability in the emission of methane from the oceans in space and time (Suess et al., 1999; Torres et al., 2002), the global contribution of the ocean to the annual methane emission is estimated to be only in the range of 3–5% in current times (IPCC, 1994; Reeburgh, 1996). It has been suggested that the major sink for methane in the marine environment is the microbially mediated anaerobic oxidation of methane (AOM) in sulfate penetrated anoxic sediments (Whiticar, 2002). Recently, we were able to demonstrate that methane-driven sulfate reduction follows a 1 : 1 stoichiometry, i. e. one mole sulfide being produced for each mole of methane consumed (Michaelis et al., 2002; Nauhaus et al., 2002; Treude et al., 2003). This stoichiometry is also represented in pore water gradients in field samples and in concurrent radiotracer measurements of AOM and sulphate reduction (Martens and Berner, 1974; Iversen and Jørgensen, 1985). In the present study, the rates of AOM were measured either in vitro in slurries of sediment diluted with defined artificial media, or using freshly sampled intact sediment cores injected with radio labelled CH4 (ex situ). For sediments with high in situ methane concentrations these different types of measurements resulted in similar values at atmospheric pressure, because generally 1.5 mM of methane was available to the methanotrophic community during rate mea-
surements (Fig. 2). However, for samples from less methane-rich sites like Eckernfo¨rde Bay, the potential rates measured in vitro were much higher than those of the ex situ set-ups, because the latter were exposed to generally lower methane concentrations during incubation. On the other hand, the radiotracer method is more sensitive. In Chile samples for example no AOM could be detected after more than 12 weeks using the in vitro approach. At this site, AOM turned out to be sometimes restricted to a narrow zone of a few cm in sediment depths between 1.5 and 4 mbsf (Treude et al., 2005). Using radiotracers, very low rates of AOM are detectable, because the detection limit of our method is around 0.01–0.1 nmol gdw 1 d 1 (Fig. 2). In contrast, the detection limit of the in vitro method is around 10 nmol gdw 1 d 1. For both, ex situ and some in vitro measurements, several cores were analyzed separately to account for possible small-scale heterogeneities. Consequently, the rates provided here represent the range of activities found at a site. It was found that even within one site the rates of both, AOM and methane formation, may vary by up to an order of magnitude (Tables 2 and 3). AOM was highest in sediments recovered from above gas hydrates at Hydrate Ridge, Gulf of Mexico and from a seep site in the Black Sea ranging from 1 to 13 Amol CH4 gdw 1 d 1 (Table 3). Nevertheless, AOM was also measured in non-seep samples from Table 3 Range of anaerobic methane oxidation rates in different sediment samples determined either via the conversion of 14CH4 to 14CO2 or in vitro following the CH4-dependent formation of sulfide Sampling site
AOM determined with radiotracers [Amol gdw 1 d 1]
AOM determined in vitro [Amol gdw 1 d 1]
Hydrate Ridge Black Sea Chile Haakon Mosby Mud Volcano Gulf of Mexico North Sea: Spiekeroog Baltic Sea: Kattegat Bay of Eckernfo¨rdeb
0.3–6c 0.2–7.5 0.001–0.07d n. d.a
2–8 0.5–3.5 n. m.e 0.1–1
n. d.a n. d.a 0.05–0.2 0.03–0.06
1–13 0.01–0.2 0.05–1 0.1–0.3
a b c d e
n.d.: not determined in this study. Treude et al. (in press). Treude et al. (2003). Treude et al. (2005). n. m.: not measurable.
M. Kru¨ger et al. / Palaeogeography, Palaeoclimatology, Palaeoecology 227 (2005) 6–17 Table 4 Influence of elevated methane partial pressure (1.1 MPa) on potential rates of AOM measured in vitro (mean F SE, n = 3) Sampling site
AOM rate at 0.1 MPa [Amol gdw 1 d 1]
AOM rate at 1.1 MPa [Amol gdw 1 d 1]
Hydrate Ridge 2000a Hydrate Ridge 2002 Hydrate Ridge 2002 reference site Gulf of Mexico Baltic Sea: Bay of Eckernfo¨rde Baltic Sea: Kattegat
1.13 F 0.24 3.14 F 0.43 n. d.b
5.65 F 1.32 10.21 F1.59 0.45 F 0.34
6.54 F 0.09 0.22 F 0.09
16.88 F 0.75 0.37 F 0.08
0.12 F 0.03
0.24 F 0.15
a b
Modified after Nauhaus et al. (2002). n. d.: not detectable.
the North and Baltic Sea. Here, rates were on average 10- to 100-fold lower compared to seep sites. This difference in AOM activity was confirmed by the in vitro incubations, which were carried out at 1.5 mM methane concentrations. Hence, the lower rates are most likely due to a difference in the biomass of the AOM-community (Treude et al., submitted for publication). Cell numbers of AOM microorganisms were 3 orders of magnitude higher at Hydrate Ridge compared to the Baltic Sea site (Treude et al., 2003; Treude et al., in press). However, an increase of the methane concentration from 1 to 15 mM significantly enhanced the in vitro rates of AOM in Hydrate Ridge sediments by 4- to 5fold (Table 4). A similar effect was observed for other sediments with AOM activity from methane-rich sites like Gulf of Mexico. Samples from sites with lower in situ methane concentrations, including the North Sea and Baltic Sea, were less inducible by increased methane concentrations. In these samples the increase from 1.5 to 15 mM CH4 led to an increase in AOM between 50% and 100%. It was also possible to induce AOM in formerly inactive sediments from a Hydrate Ridge reference site by increasing the methane availability. After 40 days of incubation with 15 mM methane, rates increased from zero to 0.5 Amol CH4 gdw 1 d 1. The induction of AOM in inactive sediments has also been reported for samples recovered close to a seep in the Monterey Canon, CA, in incubations at atmospheric pressure (Girguis et al., 2003). At cold seeps with their high methane flux, concentrations of dissolved methane depend on water
13
depth, due to the higher solubility of methane at higher hydrostatic pressure. Here, the rates of AOM measured ex situ at atmospheric pressure substantially underestimate in situ AOM rates. With the in vitro incubation set-up used in this investigation a maximum of 15 mM methane solution can be reached, which corresponds to the maximum methane solubility at a water depth around 100 m. Hence, even such high rates as 13 Amol CH4 gdw 1 d 1 (ca 10 mM CH4 d 1) are underestimates of the microbial methane consumption in situ. Future studies will have to aim at sample collection and subsequent investigation under in situ conditions of pressure and methane concentration. Besides methane availability, AOM was also strongly influenced by temperature. In Nauhaus et al. (2002) we could show that AOM at Hydrate Ridge is a psychrophilic process with a temperature optimum (12 8C) relatively close to the in situ values of 4 8C (Table 5). Temperature optima ranged from approximately 4 8C at Haakon Mosby (in situ 1.5 8C) to 25 8C in the Baltic Sea (Kattegat samples). So far, the different temperature optima of AOM at the different sites reflected the different in situ temperatures at the seafloor (Table 5). Especially at shallow sites seasonal temperature changes might also cause changes in AOM activity. Indeed, such seasonality of AOM activity was reported from studies in Eckernfo¨rde Bay (Treude et al., in press) and Cape Lookout Bight (USA; Hoehler et al., 1994). Hinrichs and Boetius (2002) provided global estimates of AOM in different ocean zones, and discussed the relevance of AOM as a sink for methane. For this calculation, only few measurements of AOM were
Table 5 Temperature optima for AOM activity in different samples determined in vitro Sampling site
Temperature optima for AOM [8C]
In situ temperature (maximum) [8C]
Hydrate Ridge 2000a Haakon Mosby Mud Volcano Gulf of Mexico Baltic Sea: Bay of Eckernfo¨rdeb Baltic Sea: Kattegat
12–16 4–8 16–20 20
2–6 1.5 8–10 12–15
20–24
20–25
a b
Data from Nauhaus et al. (2002). Data from Treude et al. (2005).
14
M. Kru¨ger et al. / Palaeogeography, Palaeoclimatology, Palaeoecology 227 (2005) 6–17
available. On the basis of the data reported here, the estimates of Hinrichs and Boetius (2002) are on the low side, especially for the seep sites. The amount of methane oxidized in the anoxic zones of sediments reduces the emission of methane from the ocean by probably more than 80% (Reeburgh et al., 1993). We propose that the main escape route for methane is ebullition of free gas or gas hydrates floating from the seafloor. Wherever sulfate is available, AOM communities control methane emission. Without this mechanism, the emission of methane from the world oceans would approximately equal the amount of methane emanating from ruminants, one of the biggest sources of today’s methane emission into the atmosphere (Reeburgh, 1996). 3.3. Aerobic methane oxidation Several studies showed that the occurrence of oxygen at seep and methane-rich sites is limited to the uppermost millimeters of the sediment (Wallmann et al., 1997; Suess et al., 1999; Sauter et al., 2001). However, information about rates and microorganisms involved in the aerobic methane oxidation (MO) in the ocean are scarce. So far only few studies were published dealing mainly with pelagic methane oxidizing bacteria (MOB) (e. g. Lidstrom, 1988; Holmes et al., 1996; Valentine et al., 2001). In the present study we show the potential for MO at most of the sites considered (Table 2). In some surface sediments, the potential MO rates were comparable to those determined for AOM under the respective in vitro conditions (Table 3). A potential for MO was also measured in aerobic sediment slurries from deeper sediment layers (data not shown). This might be explained by the ability of MOB to survive long periods of anoxia. It was found that MOB might handle anoxia even better than starvation due to methane limitation under aerobic conditions (Roslev and King, 1995). Aerobic methane oxidation rates (MOR) were highest in samples from Namibia and sandy sediments from the Bay of Eckernfo¨rde (Baltic Sea) and Spiekeroog (North Sea) with up to 0.5 and 0.2 Amol CH4 gdw 1 d 1, respectively (Table 2). Sandy sediments generally enable a deeper penetration of oxygen into the sediment (Booij et al., 1991). At most other sites MOR were similar, ranging from 0.01 to 0.2 Amol CH4 gdw 1 d 1. High methane
additions (N 10%) were necessary to induce MOR activity. At lower concentrations MOR only started after a lag-phase of 5–20 d, indicating a need for adaptation in the responsible microbial community. As was observed for AOM, an increase in the methane partial pressure also stimulated rates of MO (data not shown; De Angelis et al., 1991). Our data indicate that MOR in the sediment also contributes to the reduction of methane emissions from marine systems. However, we can assume that MO plays a minor role in marine sediments compared to AOM, as oxygen is available in much lower concentrations compared to sulfate ´ Hondt et al., 2002). Moreover, methane concentra(D tions are often already strongly reduced by AOM before methane is transported into the oxic layer of the sediment (Martens and Berner, 1974; Iversen and Jørgensen, 1985). It is still unknown if MO in the water column could possibly act as an important methane sink in the ocean, especially above methane seeps in deeper waters. Here, oxygen supply is not limiting and dissolved methane could be used as a carbon source for methylotrophs. However, in a recent study on the fate of seep-derived methane above the Haakon Mosby mud volcano, Damm and Bude´us (2003) concluded from isotopic data that the methane is primarily diluted out and not microbially oxidized. 3.4. Consequences for the global methane budget Previous investigations and calculations on marine methane budgets left no doubt about the importance of AOM in the regulation of methane emission from the world’s oceans (Reeburgh et al., 1993; Reeburgh, 1996; Valentine and Reeburgh, 2000; Hinrichs and Boetius, 2002). The question is not any longer how important AOM is but rather how much methane is retained or transformed to carbonate in marine sediments by this process, and what defines the conditions for methane escape via ebullition. Our investigations demonstrated that rates of AOM are most likely underestimated when determined under ex situ conditions, i.e. after decompression from the ambient hydrostatic pressure, due to the positive correlation between methane solubility and hydrostatic pressure. The only solution to these methodological problems is the development of appropriate incubation devices and in situ methane sensors. High-pressure incubators could be an alternative to gain data through in vitro
M. Kru¨ger et al. / Palaeogeography, Palaeoclimatology, Palaeoecology 227 (2005) 6–17
incubations simulating in situ conditions, however, this is always difficult due to the complexity of natural sediment systems and their hydrodynamics. In addition, proper methods of determining in situ methane turnover are useless for global budget calculations if the collected data cannot be extrapolated to larger scales. The sore spot in calculating marine methane budgets is the missing knowledge on the hot spots of AOM habitats—the cold seeps. We know today that highest methane turnover rates occur at cold seeps (this study; Boetius et al., 2000; Michaelis et al., 2002; Nauhaus et al., 2002; Treude et al., 2003; Joye et al., 2004). In order to close the gaps in our understanding of the marine methane budget, mapping, monitoring and sampling of these hot spots of methane turnover have to be continued and intensified. Additionally, the regulation of methanogenesis and AOM in diffusive sediment systems has to be understood, since this type of environment accounts for the majority of the ocean seafloor. Acknowledgements
We especially thank Ramona Appel, Anita Eppelin, Friederike Heinrich, Imke Mu¨ller and Thomas Holler for technical assistance as well as John Hayes and one anonymous reviewer for their helpful comments. This study was part of the GEOTECHNOLOGIEN program MUMM and obtained further support by the programs TECFLUX II, OMEGA; LOTUS, GHOSTDABS and PUCK also funded by the Bundesministerium fu¨r Bildung und Forschung (BMBF, Germany). Further support was from the Max Planck Society. This is publication GEOTECH-80 of the program GEOTECHNOLOGIEN of the BMBF and the DFG as well as GHOSTDABS-7. References Abegg, F., Anderson, A.L., 1997. The acoustic turbid layer in muddy sediments of Eckernfoerde Bay, western Baltic: methane concentration, saturation and bubble characteristics. Mar. Geol. 137, 137 – 147. Aeckersberg, F., Bak, F., Widdel, F., 1991. Anaerobic oxidation of saturated hydrocarbons to CO2 by a new type of sulfate-reducing bacterium. Arch. Microbiol. 156, 5 – 14.
15
Arntz, W.E., Fahrbach, E., 1991. El Nin˜o. Klimaexperiment der NaturBirkha¨user Verlag, Basel. Baily, G.W., 1991. Organic carbon flux and development of oxygen deficiency on the modern Benguela continental shelf south of 22 8C: spatial and temporal variability. In: Tyson, R.V., Pearson, T.H. (Eds.), Modern and Ancient Continental Shelf Anoxia, vol. 58, pp. 171 – 183. Boetius, A., Ravenschlag, K., Schubert, C.J., Rickert, D., Widdel, F., Giesecke, A., Amann, R., Jørgensen, B.B., Witte, U., Pfannkuche, O., 2000. A marine microbial consortium apparently mediating anaerobic oxidation of methane. Nature 407, 623 – 626. Booij, K., Helder, W., Sundby, B., 1991. Rapid redestruction of oxygen in a sandy sediment induced by changes in the flow velocity of overlying waters. Neth. J. Sea Res. 28, 149 – 165. Borowski, W.S., Hoehler, T.M., Alperin, M.J., Rodriguez, N.M., Paull, C.K., 2000. Significance of anaerobic methane oxidation in methane-rich sediments overlying the Blake Ridge gas hydrates. In: Paull, C.K., Matsumoto, R., Wallace, P.J., Dillon, W.P. (Eds.), Proceedings of the Ocean Drilling Program, Scientific Results, pp. 87 – 99. Bo¨ttcher, M.E., Oelschla¨ger, B., Ho¨pner, T., Brumsack, H.J., Rullko¨tter, J., 1998. Sulfate reduction related to the early diagenetic degradation of organic matter and ddblack spotTT formation in tidal sandflats of the German Wadden Sea (southern North Sea): stable isotope (13C, 34S, 18O) and other geochemical results. Org. Geochem. 29, 1517 – 1530. Bru¨chert, V., Jørgensen, B.B., Neumann, K., Riechmann, D., Schlo¨sser, M., Schulz, H., 2003. Regulation of bacterial sulfate reduction and hydrogen sulfide fluxes in the central Namibian coastal upwelling zone. Geochim. Cosmochim. Acta 67, 4505 – 4518. Bussmann, I., 1994. Verteilung und Steuergro¨ßen der Aktivita¨t Methan-oxidierender Bakterien in Randmeeren des Nordatlantiks, Sonderforschungsbereich, vol. 313. Christian-AlbrechtsUniversita¨t zu Kiel, Kiel, p. 128. Bussmann, I., Dando, P.R., Niven, S.J., Suess, E., 1999. Groundwater seepage in the marine environment: role for mass flux and bacterial activity. Mar. Ecol., Prog. Ser. 178, 169 – 177. Cord-Ruwisch, R., 1985. A quick method for the determination of dissolved and precipitated sulfides in cultures of sulfate-reducing bacteria. J. Microbiol. Methods 4, 33 – 36. Damm, E., Bude´us, G., 2003. Fate of vent derived methane in seawater above the Haakon Mosby mud volcano (Norwegian Sea). Mar. Chem. 82, 1 – 11. ´ Hara, S.C.M., Schuster, U., Taylor, L.J., Clayton, Dando, P.R., O C.J., Baylis, S., Laier, T., 1994. Gas seepage from carbonatecemented sandstone reef on the Kattegat coast of Denmark. Mar. Pet. Geol. 11, 182 – 189. D’Hondt, S., Rutherford, S., Spivack, A.J., 2002. Metabolic activity of subsurface life in deep-sea sediments. Science 295, 2067 – 2070. De Angelis, M.A., Baross, J.A., Lilley, M.D., 1991. Enhanced microbial methane oxidation in water from a deep-sea hydrothermal vent field at simulated in situ hydrostatic pressures. Limnol. Oceanogr. 36, 565 – 570. Girguis, P.R., Orphan, V.J., Hallam, S.J., DeLong, E.F., 2003. Growth and methane oxidation rates of anaerobic methano-
16
M. Kru¨ger et al. / Palaeogeography, Palaeoclimatology, Palaeoecology 227 (2005) 6–17
trophic Archaea in a continuous-flow bioreactor. Appl. Environ. Microbiol. 69, 5472 – 5482. Hebbeln, D. and participants, 2001. PUCK: report and preliminary results of R/V Sonne Cruise SO 156, Valparaiso (Chile)– Talcahuano (Chile), March 29–May 14, 2001. Berichte aus dem Fachbereich Geowissenschaften der Universita¨t Bremen: 182. Hensen, C., Zabel, M., 2000. Early diagenesis at the benthic boundary layer: oxygen and nitrate in marine sediments. In: Schulz, H.D., Zabel, M. (Eds.), Marine Geochemistry. Springer Verlag, Berlin, pp. 209 – 231. Hinrichs, K.-U., Boetius, A., 2002. The anaerobic oxidation of methane: new insights in microbial ecology and biogeochemistry. In: Wefer, G., Billett, D., Hebbeln, D., et al. (Eds.), Ocean Margin Systems. Springer-Verlag, Berlin, pp. 457 – 477. Hoehler, T.M., Alperin, M.J., Albert, D.B., Martens, C.S., 1994. Field and laboratory studies of methane oxidation in an anoxic marine sediments: evidence for methanogen–sulphate reducer consortium. Global Biochem. Cycles 8, 451 – 463. Holmes, A.J., Owens, J.C., Murrell, N.J.P., 1996. Molecular analysis of enrichment cultures of marine methane oxidising bacteria. J. Exp. Mar. Biol. Ecol. 203, 27 – 28. IPCC, 1994. Radiative Forcing of Climate Change and Evaluation of the IPCC IS 92 Emission Scenarios. Cambridge Univ. Press, New York. Ivanov, M.V., Polikarpov, G.G., Lein, A.Y., 1991. Biogeochemistry of the carbon cycle in the zone of Black Sea methane seeps. Dokl. Akad. Nauk SSSR 320, 1235 – 1240. Iversen, N., Blackburn, T.H., 1981. Seasonal rates of methane oxidation in anoxic marine sediments. Appl. Environ. Microbiol. 41, 1295 – 1300. Iversen, N., Jørgensen, B.B., 1985. Anaerobic methane oxidation rates at the sulphate–methane transition in marine sediments from Kattegat and Skagerrak (Denmark). Limnol. Oceanogr. 30, 944 – 955. Jørgensen, B.B., 1978. A comparison of methods for the quantification of bacterial sulphate reduction in coastal marine sediments: I. Measurements with radiotracer techniques. Geomicrobiol. J. 1, 11 – 27. Joye, S.B., Boetius, A., Orcutt, B.N., Montoya, J.P., Schulz, H.N., Erickson, M.J., Logo, S.K., 2004. The anaerobic oxidation of methane and sulfate reduction in sediments from Gulf of Mexico cold seeps. Chem. Geol. 205, 219 – 238. Judd, A.G., Hovland, M., Dimitrov, L.I., Garcı´a Gil, S., Jukes, V., 2002. The geological methane budget at continental margins and its influence on climate change. Geofluids 2, 109 – 126. King, G.M., 1992. Ecological aspects of methane oxidation, a key determinant of global methane dynamics. Adv. Microb. Ecol. 12, 431 – 468. Kru¨ger, M., Frenzel, P., Conrad, R., 2001. Microbial processes influencing methane emission from rice fields. Glob. Chang. Biol. 7, 49 – 61. Kru¨ger, M., Eller, G., Conrad, R., Frenzel, P., 2002. Seasonal variations in CH4 oxidation and pathways of CH4 production in rice fields determined by stable carbon isotopes and specific inhibitors. Glob. Chang. Biol. 8, 265 – 280.
Lidstrom, M.E., 1988. Isolation and characterization of marine methanotrophs. Antonie van Leeuwenhoek. Antonie van Leeuwenhoek 54, 189 – 199. Martens, C.S., Berner, R.A., 1974. Methane production in the interstitial waters of sulphate-depleted marine sediments. Science 185, 1167 – 1169. Martens, C.S., Albert, D.B., Alperin, M.J., 1999. Stable isotope tracing of anaerobic methane oxidation in the gassy sediments of Eckernfo¨rde Bay, German Baltic Sea. Am. J. Sci. 299, 589 – 610. Michaelis, W., Seifert, R., Nauhaus, K., Treude, T., Thiel, V., Blumenberg, M., Knittel, K., Gieseke, A., Peterknecht, K., Pape, T., Boetius, A., Aman, A., Jørgensen, B.B., Widdel, F., Peckmann, J., Pimenov, N.V., Gulin, M., 2002. Microbial reefs in the Black Sea fueled by anaerobic oxidation of methane. Science 297, 1013 – 1015. Nauhaus, K., Boetius, A., Kru¨ger, M., Widdel, F., 2002. In vitro demonstration of anaerobic oxidation of methane coupled to sulphate reduction in sediment from marine gas hydrate area. Environ. Microbiol. 4, 298 – 305. Niewo¨hner, C., Hensen, C., Kasten, S., Zabel, M., Schulz, H.D., 1998. Deep sulfate reduction completely mediated by anaerobic methane oxidation in sediments of the upwelling area off Namibia. Geochim. Cosmochim. Acta 62, 455 – 464. Oremland, R.S., Marsh, L.M., Polcin, S., 1982. Methane production and simultaneous sulphate reduction in anoxic, salt marsh sediments. Nature 296, 143 – 145. Peterson, W.T., Tarcos, D.F., McManos, G.B., Dam, H., Bellantoni, D., Johnson, T., Tiselius, P., 1988. The nearshore zone during coastal upwelling—daily variability and coupling between primary and secondary production off central Chile. Prog. Oceanogr. 20, 1 – 40. Pimenov, N.V., Savvichev, A.S., Rusanov, I.I., Lein, A.Y., Ivanov, M.V., 2000. Microbiological processes of the carbon and sulfur cycles at cold methane seeps of the North Atlantic. Microbiology 69, 709 – 720. Reeburgh, W.S., 1996. bSoft spotsQ in the global methane budget. In: Lidstrom, M.E., Tabita, F.R. (Eds.), Microbial Growth on C1 Compounds. Kluwer Academic Publishers, Andover, UK, pp. 334 – 342 (Intercept). Reeburgh, W.S., Whalen, S.C., Alperin, M.J., 1993. The role of methylotrophy in the global methane budget. In: Murell, J.C., Kelly, D.P. (Eds.), Microbial Growth on C1 Compounds. Proceedings of the 7th International Symposium. American Society for Microbiology, Washington, D.C., pp. 1 – 14. Roslev, P., King, G.M., 1995. Aerobic and anaerobic starvation metabolism in methanotrophic bacteria. Appl. Environ. Microbiol. 61, 1563 – 1570. Sahling, H., Rickert, D., Raymond, W.L., Linke, P., Suess, E., 2002. Macrofaunal community structure and sulfide flux at gas hydrate deposits from the Cascadia convergent margin, NE Pacific. Mar. Ecol., Prog. Ser. 231, 121 – 138. Sauter, E.J., Schlu¨ter, M., Suess, E., 2001. Organic carbon flux and remineralisation in surface sediments from the northern North Atlantic derived from pore-water oxygen microprofiles. Deep-Sea Res., Part 1, Oceanogr. Res. Pap. 48, 529 – 553.
M. Kru¨ger et al. / Palaeogeography, Palaeoclimatology, Palaeoecology 227 (2005) 6–17 Schoell, M., 1988. Multiple origins of methane in the earth. Chem. Geol. 71, 1 – 10. Sherwood Lollar, B., Westgate, T.D., Ward, J.A., Slater, G.F., Lacrampe-Couloume, G., 2002. Abiogenic formation of alkanes in the earth’s crust as a minor source for global hydrocarbon reservoirs. Nature 416, 522 – 524. Suess, E., Torres, M.E., Bohrmann, G., Collier, R.W., Greinert, J., Linke, P., Rehder, G., Trehu, A., Wallmann, K., Winckler, G., Zuleger, E., 1999. Gas hydrate destabilization: enhanced dewatering, benthic material turnover and large methane plumes at the Cascadia convergent margin. Earth Planet. Sci. Lett. 170, 1 – 15. Tissot, B.P., Welte, D.H., 1984. Petroleum Formation and Occurrence. Springer Verlag, Berlin. Torres, M.E., McManus, J., Hammond, D., Angelis de, M.A., Heeschen, K.U., Colbert, S.L., Tryon, M.D., Brown, K.M., Suess, E., 2002. Fluid and chemical fluxes in and out of sediments hosting methane hydrate deposits on Hydrate Ridge, OR: I. Hydrological provinces. Earth Planet. Sci. Lett. 201, 525 – 540. Treude, T., Boetius, A., Knittel, K., Wallmann, K., Jørgensen, B.B., 2003. Anaerobic oxidation of methane above gas hydrates at Hydrate Ridge, NE Pacific Ocean. Mar. Ecol., Prog. Ser. 264, 1 – 14. Treude, T., Kru¨ger, M., Boetius, A., Jørgensen, B.B., in press. Environmental control on anaerobic oxidation of methane in the gassy sediments of Eckernfo¨rde Bay (German Baltic). Limnol. Oceanogr.
17
Treude, T., Niggemann, J., Kallmeyer, J., Wintersteller, P., Schubert, C.J., Boetius, A., Jørgensen, B.B., 2005. Anaerobic oxidation of methane in the sulfate–methane transition along the Chilean continental margin. Geochim. Cosmochim. Acta 69, 2767–2779. Valentine, D.L., Reeburgh, W.S., 2000. New perspectives on anaerobic methane oxidation. Environ. Microbiol. 2, 477 – 484. Valentine, D.L., Blanton, D.C., Reeburgh, W.S., Kastner, M., 2001. Water column methane oxidation adjacent to an area of active gas hydrate dissociation, Eel River Basin. Geochim. Cosmochim. Acta 65, 2633 – 2640. Wallmann, K., Linke, P., Suess, E., Bohrmann, G., Sahling, H., Schlu¨ter, M., Da¨hlmann, A., Lammers, S., Greinert, J., Von Mirbach, N., 1997. Quantifying fluid flow, solute mixing, and biogeochemical turnover at cold vents of the eastern Aleutian subduction zone. Geochim. Cosmochim. Acta 61, 5209 – 5219. Whiticar, M.J., 2002. Diagenetic relationships of methanogenesis, nutrients, acoustic turbidity, pockmarks and freshwater seepages in Eckernfo¨rde Bay. Mar. Geol. 182, 29 – 53. Widdel, F., Bak, F., 1992. Gram-negative mesophilic sulfate-reducing bacteria. In: Balows, A., Tru¨per, H.G., Dworking, M., Harder, W., Schleifer, K.-H. (Eds.), The Prokaryotes. Springer, New York, pp. 3352 – 3378.
