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Applied and Environmental Microbiology, December 2005, p. 8925-8928, Vol. 71, No. 12
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.12.8925-8928.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

SHORT REPORT

Cooccurrence of Aerobic and Anaerobic Methane Oxidation in the Water Column of Lake Plußsee

Gundula Eller,1 Layla Känel,2 and Martin Krüger2,3*

Max Planck Institute for Limnology, August-Thienemann-Strasse 2, 24306 Plön, Germany,1 Max Planck Institute for Marine Microbiology, Celsiusstrasse 1, 28359 Bremen, Germany,2 Geomicrobiology, Federal Institute for Geosciences and Natural Resources, Stilleweg 2, 30655 Hannover, Germany3

Received 19 June 2005/ Accepted 6 September 2005


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ABSTRACT
 
Dissolved methane was investigated in the water column of eutrophic Lake Plußsee and compared to temperature, oxygen, and sulfide profiles. Methane concentrations and {delta}-13C signatures indicated a zone of aerobic methane oxidation and additionally a zone of anaerobic methane oxidation in the anoxic water body. The latter coincided with a peak in hydrogen sulfide concentration. High cell numbers of aerobic and anaerobic methane-oxidizing microorganisms were detected by fluorescence in situ hybridization (FISH) or the more sensitive catalyst-amplified reporter deposition-FISH, respectively, in these layers.


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INTRODUCTION
 
Methane emissions from lakes contribute 6 to 16% of global methane emissions (3). Consequently, methane oxidation in lakes is an important process for the mitigation of methane emissions. Hitherto, only aerobic methane oxidation has been described for freshwater systems, which preferentially occurs at oxic-anoxic interfaces (17), where methane and oxygen are available. The anaerobic oxidation of methane (AOM) has so far only been described in marine environments (7), even though indications for its occurrence in other habitats exist (6).

Lake Plußsee is well studied and has been described in detail elsewhere (14). It has a stable thermal stratification during the summer and regularly occurring anoxia in the hypolimnion, leading to high methane concentrations in the water column. Profiles of methane, oxygen, and hydrogen sulfide concentrations and {delta}-13C signatures of dissolved methane were measured to localize methane oxidation activity in the water column. Water samples for measurements of methane were taken as described by Bastviken et al. (3). Methane concentrations were determined by gas chromatography, and stable carbon isotopes using gas chromatograph-combustion-isotope ratio mass spectrometry (10). Temperature and oxygen were measured in situ with an EOT 190 oxygen probe (WTW Germany). These profiles revealed an anoxic hypolimnion for both sampling time points in June and September 2004 (Fig. 1A and B). Oxygen was not detectable below 8 m in June and 6 m in September. Methane concentrations (Fig. 1C and D) first increased below the oxocline but then showed a layer of decreasing concentrations in the anoxic hypolimnion, located between 12 and 16 m in June and between 8 and 12.5 m in September. Below, a strong increase in methane concentration towards the sediment was detected. Both methane and oxygen concentration profiles indicate a layer of aerobic methane oxidation in the 9-m depth in June and 6 to 7 m in September. The second decrease in methane concentration detected at both sampling time points was located in the anoxic water body and can therefore not be explained by aerobic methane oxidation. The maximum in methane concentration between the two layers of methane oxidation could be explained by high methane production rates in this layer. These might be caused by a high availability of substrates for methanogens. The sulfate originating from the sediment, reaching 300 µM in the bottom water in September, was most likely depleted below this zone by AOM.



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  		FIG. 1. Methane, oxygen, and temperature profiles (A and B) in the water column of Lake Plußsee in June (A and C) and September (B and D) 2004, compared to methane isotopic signatures and sulfide concentrations (C and D).

In June, the {delta}-13C of dissolved methane was around –62{per thousand} above the sediment and increased slightly to –61{per thousand} at 17 m depth (Fig. 1C). Between 16.5 m and 13 m, in the same anoxic water layer where a decrease in methane concentrations was detected, a maximum in methane {delta}-13C was measured, with –52{per thousand} in 16 m, indicating a zone with AOM activity. Above 13 m, {delta}-13C signatures increased to values of –47{per thousand} due to aerobic methane oxidation, cooccurring with a decrease in methane concentrations to about 1 µM just below the oxocline. In September, changes in methane {delta}-13C were less pronounced than in June, but again a maximum in {delta}-13C values at 10 m was detected in the anoxic hypolimnion and thus below the increase originating from aerobic methane oxidation from 8.5 m upwards (Fig. 1D). Interestingly, also hydrogen sulfide concentrations (determined photometrically after conversion to methylene blue) formed a distinct maximum at 10 m, supporting the assumption of AOM activity.

Total cell counts (with 4',6'-diamidino-2-phenylindole [DAPI]) and fluorescence in situ hybridization (FISH) were carried out in water samples to localize the microorganisms involved in methane oxidation. These samples (10 or 30 ml) were taken with a Ruttner sampler, preserved with 2% formaldehyde, filtered onto GTTP membrane filters (0.2 µm; Millipore), and stored at –20°C. Methane-oxidizing bacteria (MOB) were detected by applying probes M{gamma}84/M{gamma}705 for type I MOB, M{alpha}450 for type II MOB (5), and eubacterial probe Eub388 (2) as a control.

With FISH, no type II MOB cells were detected. Additionally, the 16S rRNA gene of type II MOB could not be amplified from water samples (data not shown). In June, type I cells were only found at 10 m and below, thus explaining the decrease in methane concentrations just below the oxocline. In the oxic epilimnion, MOB cell numbers were most probably reduced by grazing, which did not occur in the anoxic hypolimnion. Therefore, MOB cell numbers seem to remain high in the hypolimnion after the complete mixture of the water body during spring overturn, even though MOB are not oxidizing methane but are instead using endogenous substrates (16).

With 1.6 x 105 cells ml–1, MOB represented 3% of DAPI-stained cells in the hypolimnion in June (Fig. 2A). In September, the pattern differed completely, with a distinct maximum in MOB cell numbers between 6 m and 8 m depth (6.6 x 104 MOB ml–1 in 8 m, representing 1.2% of DAPI counts) (Fig. 2B) and lower MOB numbers than detected in June. Thus, at the end of the summer stratification period, MOB seem to have changed position to the oxic-anoxic boundary layer, following changes of oxygen and CH4 concentrations (Fig. 1).



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  		FIG. 2. Total cell numbers as determined by DAPI staining compared to cell numbers of type I methanotrophs (MOB) as determined by FISH in June (A) and September (B) 2004.

To detect anaerobic methane-oxidizing archaea (ANME) in the water column, catalyst-amplified reporter deposition-FISH (CARD-FISH) (15) with probes ANME-1-350 (ANME-I) and EelMS932 (ANME-II) was carried out in parallel with probes Arch915 (Archaea) and DSS658 (Desulfosarcina/Desulfococcus) (4). Parallel hybridization of our samples without probes (negative control) and hybridization of ANME-containing marine samples (positive control) were carried out to check for specificity. Due to lack of samples in June and the expected low ANME cell numbers in the remaining water body, only water samples from 8, 10, and 15 m in depth in September were analyzed.

In these samples, ANME-I were detected in the zone of AOM activity, either as single cells (Fig. 3B) or as short chains of up to five cells, comprising 0.1 to 1% of DAPI-stained cells (non-ANME I cells). Additionally, ANME-II were discovered in the same water depths in small aggregates (Fig. 3D) ranging from 0.1 to 0.5% of all cells. Furthermore, AOM activity was evident from the pronounced shifts in concentration as well as stable isotopic signatures of dissolved methane in the water column (Fig. 1C and D).



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  		FIG. 3. CARD-FISH pictures compared to DAPI staining of water samples collected in September 2004 from 8 m (A and B) and 15 m (C and D) depth. ANME-I cells were detected with the probe ANME-1-350 (B), and ANME-II cells were detected with the probe EelMS932 (D). White arrows indicate probe-positive cells, and orange arrows indicate non-ANME cells.

In contrast to marine systems (4, 11), neither ANME-I nor -II seemed to be physically associated with sulfate-reducing bacteria. Nevertheless, CARD-FISH with the DSS658 probe revealed the presence of this group in the same depths in which the ANME were detected (up to 1% of total cells). Similar observations of "single" ANME have been reported previously (19, 13). It is still under debate whether AOM indeed depends on syntrophic action of two partners or if one type of organism might be able to carry out the entire process (18).

Sediment from 28 m depth incubated under anaerobic conditions (as described in reference 12) exhibited the potential for AOM. However, compared to aerobic methane oxidation rates of 3.6 ± 1.9 µmol gdw–1 h–1 in June, AOM rates (0.11 ± 0.023 µmol gdw–1 h–1) were relatively low. Nevertheless, AOM rates were comparable to those detected in diffusive marine systems (7, 9).

In conclusion, the relatively long anoxic periods during the summer together with a high organic load in Lake Plußsee favor anaerobic processes like methanogenesis in the sediment and even in the largely anoxic water column. Our data strongly indicate also the presence of AOM in a distinct layer of the water column. So far, the microorganisms responsible for sulfate-dependent AOM have only been discovered in marine systems (e.g., references 4 and 18). Indirect indications for AOM in nonmarine systems have been reported from landfills (6) or terrestrial mud volcanoes (1). Here we show for the first time the presence and activity of ANME in a freshwater lake. Nevertheless, further investigations are needed to investigate the organisms involved in AOM (e.g., DNA analysis).

Compared to MOB, the number of ANME cells and the concentration decrease of dissolved methane in the water indicate that AOM plays an important role in the overall methane oxidation in this lake. With up to 1 and 3% of total cells as ANME and MOB, respectively, both types of methanotrophs seem to represent a prominent part of the microbial community in the respective depth layers. Consequently, they might act as a conduit to transfer methane-derived carbon to higher trophic levels, like zooplankton (8). Further analyses of seasonal distribution and activity will allow us to estimate the proportions of methane oxidized by either of the two processes and their possible role in the lake food web.


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ACKNOWLEDGMENTS
 
We especially acknowledge Ines Schultz, Claudia Schleker, Ramona Appel, Dieter Albrecht, H.-H. Richnow, Mathias Gehre, and David Bastviken for help with experimental setup and field and laboratory work.

Financial support came from the Max Planck Society (MPG, Germany).


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FOOTNOTES
 
* Corresponding author. Mailing address: Geomicrobiology, Federal Institute for Geosciences and Natural Resources, Stilleweg 2, 30655 Hannover, Germany. Phone: 49-(0)511-6433102. Fax: 49-(0)511-6433664. E-mail: M.Krueger{at}bgr.de. Back


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REFERENCES
 
    1
  1. Alain, K., T. Holler, F. Musat, and M. Krueger. Microbiological analyses of methane- and hydrocarbon-seeping mud volcanoes in the sub-Carpathes, Romania. Environ. Microbiol., in press.
    2. 2
  2. Amann, R. I., B. J. Binder, R. J. Olson, S. W. Chisholm, R. Devereux, and D. A. Stahl. 1990. Combination of 16S rRNA-targeted oligonucleotide probes with flow cytometry for analyzing mixed microbial populations. Appl. Environ. Microbiol. 56:1919-1925.[Abstract/Free Full Text]
    3. 3
  3. Bastviken, D., J. Cole, M. Pace, and L. Tranvik. 2004. Methane emissions from lakes: dependence of lake characteristics, two regional assessments, and a global estimate. Global Biogeochem. Cycles 18:B4009.
    4. 4
  4. Boetius, A., K. Ravenschlag, C. J. Schubert, D. Rickert, F. Widdel, A. Giesecke, R. Amann, B. B. Jorgensen, U. Witte, and O. Pfannkuche. 2000. A marine microbial consortium apparently mediating anaerobic oxidation of methane. Nature 407:623-626.
    5. 5
  5. Eller, G., S. Stubner, and P. Frenzel. 2001. Group-specific 16S rRNA targeted probes for the detection of type I and type II methanotrophs by fluorescence in situ hybridisation. FEMS Microbiol. Lett. 198:91-97.[CrossRef][Medline]
    6. 6
  6. Grossman, E. L., L. A. Cifuentes, and I. M. Cozzarelli. 2002. Anaerobic methane oxidation in a landfill-leachate plume. Environ. Sci. Technol. 36:2436-2442.[Medline]
    7. 7
  7. Hinrichs, K. U., and A. Boetius. 2002. The anaerobic oxidation of methane: new insights in microbial ecology and biogeochemistry, p. 457-477. In G. Wefer, D. Billett, D. Hebbeln, B. B. Jørgensen, M. Schluter, and T. van Weering (ed.), Ocean margin systems. Springer-Verlag, Heidelberg, Germany.
    8. 8
  8. Jones, R. I., J. Grey, D. Sleep, and L. Arvola. 1999. Stable isotope analysis of zooplankton carbon nutrition in humic lakes. Oikos 86:97-104.
    9. 9
  9. Krüger, M., T. Treude, H. Wolters, K. Nauhaus, and A. Boetius. Microbial methane turnover in different marine habitats. Palaeogeogr. Palaeoclimatol. Palaeoecol., in press.
    10. 10
  10. Krüger, M., P. Frenzel, and R. Conrad. 2001. Microbial processes influencing methane emission from rice fields. Global Change Biol. 7:49-61.
    11. 11
  11. Michaelis, W., R. Seifert, K. Nauhaus, T. Treude, V. Thiel, M. Blumenberg, K. Knittel, A. Gieseke, K. Peterknecht, T. Pape, A. Boetius, R. Amann, B. B. Jorgensen, F. Widdel, J. Peckmann, N. V. Pimenov, and M. B. Gulin. 2002. Microbial reefs in the Black Sea fueled by anaerobic oxidation of methane. Science 297:1013-1015.[Abstract/Free Full Text]
    12. 12
  12. Nauhaus, K., A. Boetius, M. Krüger, and F. Widdel. 2002. In-vitro demonstration of anaerobic methane oxidation coupled to sulfate reduction in a methane-rich marine sediment. Environ. Microbiol. 4:296-305.[CrossRef][Medline]
    13. 13
  13. Orphan, V. J., C. H. House, K.-U. Hinrichs, K. D. McKeegan, and E. F. DeLong. 2002. Multiple archaeal groups mediate methane oxidation in anoxic cold seep sediments. Proc. Natl. Acad. Sci. USA 99:7663-7668.[Abstract/Free Full Text]
    14. 14
  14. Overbeck, J., and R. J. Chróst (ed.). 1994. Microbial ecology of Lake Plußsee. Ecol. Studies 105:1-44.
    15. 15
  15. Pernthaler, A., J. Pernthaler, and R. Amann. 2002. Fluorescence in situ hybridization and catalyzed reporter deposition for the identification of marine bacteria. Appl. Environ. Microbiol. 68:3094-3101.[Abstract/Free Full Text]
    16. 16
  16. Roslev, P., and G. M. King. 1995. Aerobic and anaerobic starvation metabolism in methanotrophic bacteria. Appl. Environ. Microbiol. 61:1563-1570.[Abstract]
    17. 17
  17. Segers, R. 1998. Methane production and methane consumption—a review of processes underlying wetland methane fluxes. Biogeochem. 41:23-51.[CrossRef]
    18. 18
  18. Strous, M., and M. S. M. Jetten. 2004. Anaerobic oxidation of methane and ammonium. Annu. Rev. Microbiol. 58:99-117.[CrossRef][Medline]
    19. 19
  19. Treude, T., M. Krüger, A. Boetius, and B. B. Jørgensen. Environmental control on anaerobic oxidation of methane in the gassy sediments of Eckernförde Bay (German Baltic). Limnol. Oceanogr., in press.

Applied and Environmental Microbiology, December 2005, p. 8925-8928, Vol. 71, No. 12
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.12.8925-8928.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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