Scholarly article on topic 'Methanogenic microbial communities in sediment from the coastal area of puck bay (Southern Baltic)'

Methanogenic microbial communities in sediment from the coastal area of puck bay (Southern Baltic) Academic research paper on "Biological sciences"

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Academic research paper on topic "Methanogenic microbial communities in sediment from the coastal area of puck bay (Southern Baltic)"

Oceanological and Hydrobiological Studies

International Journal of Oceanography and Hy drobiology

Volume 41, Issue 3

ISSN 1730-413X elSSN 1897-3191

(33-39) 2012

VERS ITA

DOI: 10.2478/s13545-012-0025-z Original research paper

Received: Accepted:

November 22, 2011 April 02, 2012

Methanogenic Microbial Communities in Sediment From the Coastal Area of Puck Bay (Southern Baltic)

Andrzej R. Reindl*, Jerzy Bolalek

Dept. of Marine Chemistry and Environmental Protection, Faculty of Oceanography and Geography, University of Gdansk, Al. Mars%alka Pilsudskiego 48, 81-378 Gdynia, Poland

Key words: methanogens, Archaea, PCR, mcrA gene, Baltic Sea

Abstract

In this work, data on methanogenic Archaea communities in sediment from the coastal area of Puck Bay were investigated. Sediments were collected along the Hel Peninsula from areas characterized by the occurrence of gas bubbles. Based on the analysis of molecular markers, the presence of a specific methanogenic Archaea gene was detected at all stations. Further research involved the cloning and sequencing of methanogenic DNA. Based on the comparison of obtained genetic sequences with existing genetic databases, it was confirmed that all of the nucleotide sequences belonged to the domain Archaea. Furthermore, in the investigated sediment certain sequences had certain similarities to the sequences of organisms from the families Methanosarcinaceae, Methanospirillaceae and

Methanocorpusculaceae.

' Corresponding author: andrzej.reindl@ug.edu.pl

Introduction

One possible source of methane in the marine environment is thermogenic decomposition of organic fraction; it may also be produced as a result of biological transformation (Judd 2003, 2004; Reeburgh 2007). Methane emitted into the atmosphere absorbs infrared radiation from the Earth, thereby preventing heat escape and thus retaining the thermal energy in the atmosphere (Houghton et al. 1997; Gitay et al. 2002; Bates et al. 2008). This gas is one of the six significant greenhouse gases listed in the Kyoto Protocol on the reduction of the emission of greenhouse gases from anthropogenic sources. Since the beginning of the 20th century, a significant and abrupt increase in methane concentration has been observed in the atmosphere (Masse et al. 2003). Forster et al. (2007) reported a successive rise in methane concentration in the atmosphere and increased infrared radiation. A higher level of methane concentration in the atmosphere on a global scale has also been confirmed in the report published by IPCC (2007). However, the global capacity to produce methane is higher than the quantity of methane emitted into the atmosphere. It has been assumed that the remaining gas was consumed in the place of its origin (King 1990; Wegener 2008). Because of the increasing emission of greenhouse gases, including methane, it is crucial to identify both types of sources, i.e. anthropogenic and natural. Methane resulting from biological transformation is actually one of the biggest sources of emission of this gas from marine ecosystems (Bange 2006). Besides this methane-producing microorganisms are common used in the biochemical transformation of environmental organic pollutants (Liu et al. 2004; Busch et al. 2009).

Methane forming bacteria are a large and diverse group of microorganisms. They obtain energy for

growth from the conversion of substrates such as carbon dioxide and hydrogen, formate or acetate. In addition, some other compounds such as methanol, methylated amines, cyclopentanol, isobutanol, isopropanol or ethanol might be used as carbon sources for biochemical methane formation (Whitman et al. 2006). Methanogenic Archaea produce a number of coenzymes that participate in the biochemical synthesis of methane. The reduction of a methyl group to methane is the final step of metabolic transformation in each defined pathway of carbohydrate respiration accompanied by methane production. This reduction reaction is catalyzed by an enzyme called methyl coenzyme M reductase (MCR). It is a coenzyme present in the cells of all methanogens and therefore it can serve as a biomarker in the molecular analysis of these organisms. The designed methanogen-specific primers, i.e. mcrAF and mcrAR, are used to amplify the gene region responsible for methane production in the cells of methanogenic Archaea (Hales et al. 1996).

Methanogenic bacteria are abundant in habitats where electron acceptors such as O2, NO3 , Fe3+ and SO2 are limiting. One of the most common methanogenic habitats are anoxic lake and marine sediments (Whitman et al. 2006). Even when the surrounding water column or sediment contains oxygen locally, micro-anaerobic conditions might form and in that habitat methane forming bacteria can exist. That situation has been documented in the Baltic ecosystem (Edlund 2007). The presence of methane in the Baltic was detected in benthic sediment and the near-bottom water (Jorgensen et al. 1990; Piker et al. 1998; Wilkens and Richardson 1998; Martens et al. 1999; Schlüter et al. 2004; Mathys et al. 2005). The emission of this greenhouse gas from the coastal water ecosystem in the Baltic Sea into the atmosphere has also been confirmed by earlier studies (Heyer and Berger 2000; Liikanen et al. 2009). Free gas bubbles were also detected in the Baltic sediment (Schmaljohann 1996; Jensen and Fossing 2005; Judd and Hovland 2007). Methanogenic bacteria have been isolated from Baltic sediment as well. Sediment from the east coast of Jutlant in Aarhus Bay consisted of non motile, hydrogenothropic, rod-shaped cells of Archaea from the Methanobacteriacea family strain Methanobacterium aarhusen (Shlimon et al. 2004). In sediment from Gotland Deep, the strain Methanosarcina baltica was detected. The catabolic substrate used by these

irregular coccid cells included methanol, methylated amines and acetate (von Klein et al. 2002).

In our research, samples of coastal sediment were taken from stations in which free gas bubbles were observed. To determine the possibility of methane-forming bacteria participation in gas production in the sediment, molecular biology techniques were employed.

Materials And Methods Research area

The study was conducted in 2010 in the coastal waters of Puck Bay. Samples were collected on September 5 at six sampling stations (Fig. 1). In the research area, we observed organic fraction in some parts of the benthic sediment. At station P7 (Wladyslawowo area), an approximately 10 cm thick layer of caked surface sediment was observed. Together with organic fraction, free gas bubbles were detected in the sediment. A sample of benthic sediment for molecular biology analysis was collected from each sampling station at the exact location of the release of free gas bubbles. The samples consisted of ca. 15-cm deep sediment cores. Water depth in the sampling points was about 40 to 60 cm.

Chemicals

For molecular analysis, we applied commercial kits from A&A Biotechnology. We used: "Genomic Mini AX Bacteria" for genomic DNA isolation, "2xPCR Master Mix Plus" for PCR reaction mixture, "Gel-Out" for mcrA gene isolation after agarose gel electrophoresis, and "Plasmid Mini AX" for plasmid DNA isolation. We used DNA size marker and DNA thermo stable polymerase WALK from A&A Biotechnology, too. For the DNA cloning procedure, we used the "CloneJET" PCR Cloning kit from Fermentas, which contains a cloning vector named pJET1.2. For this procedure, we used chemically competent E. coli cells (One Shot T0P10F') from Invitrogen. We used primers of the mcrA gene for polymerase chain reaction (PCR) as described previously by Luton et al. (2002), made to order by Sigma-Aldrich. The primers had the following genetic sequences:

5' -GGTGGTGTMGGATTCACACARTAYGCWACAGC-3' [mcrAF] 5' -TTCATTGCRTAGTTWGGRTAGTT-3' [mcrAR]

10°E IS'E 20° E 25°E 30°E

60° N

Fig. 1. Localization of sampling area in the Puck Bay and localization of sediment corer sampling points.

P6 / PS ' P4

Puck Bay

Sampling points

• P4 - Kuznica (2)

A Gdynia VB • P6 - Chalupy (2) • P7 ■ Wtadystawowo • P8 -Puck • P9 - Rzucewo • P10 - Ostonirio

Samples collection

Samples of benthic sediment from the coastal area of Puck Bay were collected in sterile vials made of synthetic material, ranging in volume from 100 cm3 to 120 cm3. The vials filled with sediment were put in a protective bag made of synthetic material, and then immediately placed in a refrigerator. The sediment samples were kept at a temperature not exceeding 5°C until molecular analysis, which began no later than 12 hours after sampling.

Molecular analysis of sediment from Puck Bay

In order to study the occurrence of methanogenic communities in benthic sediments in the coastal area of Puck Bay, the isolation of genomic DNA followed by PCR amplification of the methanogenic archeon gene from template DNA was performed. In this study the mcrA gene, which constitutes a part of an a unit of coenzyme mcr, was amplified via PCR. A thermostable DNA polymerase, taq was used in all reactions (Nunoura et al. 2008; Steinberg, Regan 2009).

Preliminary molecular analysis consisted of the following steps: 1) isolation of the total genomic DNA from sediment, and 2) PCR amplification of the methanogen-specific gene region. Further molecular analysis included the cloning of the mcrA gene and sequencing of the obtained plasmid DNA

from these clones. The final analytical step involved a comparison between the obtained genomic sequences and the sequences of methanogenic Archaea from the database that have been previously taxonomically identified. General techniques for cloning, described previously by Sambrook et al. (1989), were applied.

Genomic DNA was isolated from ca. 2 g of each sediment sample by means of a "Genomic Mini AX Bacteria" kit and used in further analysis. For the PCR mixture we applied chemicals from a "2xPCR Master Mix Plus" kit and 0.1 [g of the mcrAF and mcrAR primers and 1 [g of the previously isolated template DNA. The amplification program described by Innis and Gelfand (1990) was used. The mcrA gene fragments amplified with mcrAF and mcrAR primers were reacted with the DNA polymerase WALK. The obtained product was separated electrophoretically on an agarose gel. The mcrA gene fragment was extracted and purified from an agarose gel by means of a "Gel-Out" kit. Purified mcrA gene was transferred into pJET1.2 vector to form recombinant plasmid (pJET1.2./mcrA). In the next step, recombinant plasmids were ligated into competent E. coli cells (T0P10F') for mcrA gene cloning. Isolation of potential recombinant plasmids was accomplished by means of a 'Tlasmid Mini AX" kit for plasmid DNA isolation. The presence of the mcrA gene in plasmids was confirmed by PCR under the conditions described above. Plasmids were

automatically sequenced by a commercial firm — MacroGen Europe (Holland).

Based on the sequencing data from the clones containing the gene responsible for methane production in methanogens, the corresponding nucleotide sequence was obtained and later compared with the previously identified nucleotide sequences by means of the NCBI BLASTN 2.2.25 application (Mori et al. 2000). Similarity between the obtained sequences was explored using this tool, which resulted in the elimination of identical nucleotide sequences. The differing DNA sequences of the mcrA gene, obtained from the preliminary selection, are presented in the phylogenetic tree formed using the neighbor-joining method (Saitou and Nei 1987).

Results And Discussion

The obtained DNA was electrophoresed, in order to confirm its presence in the sediment samples, and visualized by UV light. Genetic material was present in all samples, but it was degraded. PCR amplification of the mcrA gene from the obtained template DNA confirmed the presence of genomes belonging to sediment-dwelling methanogenic Archaea. A ca. 500 bp DNA fragment visualized by UV light after agarose gel electrophoresis suggests the presence of methane forming bacteria in the PCR products (Fig. 2).

Based on the Neighbor-Joining method (Saitou and Nei 1987), a phylogenetic tree was constructed that illustrates sequence similarities between the clones of methanogens dwelling in the Puck Bay sediments and the previously identified methanogenic sequences (Fig. 3). Eight different methanogen sequences were detected in sediments from six sampling stations. All nucleotide sequences belonged to the domain Archaea. Based on a preliminary analysis, all organisms were mcrA clones, and therefore methanogenic Archaea.

The sequences coded ZP.04.1, ZP.06.1 and ZP.09.1 were obtained from the sediments collected at stations P4 (Kuznica area), P6 (Chalupy 2 area) and P9 (Rzucewo area), respectively. Based on phylogenetic analysis of taxonomic relatedness, the similarity of these sequences to the total genomic sequence of Methanospirillum hungatei JF-1 strain was 90%, 90% and 89%, respectively. In addition, some sequences on the phylogenetic tree were located at a distance from all the other sequences. That suggests the possibility of a new organism form in the domain

M P4 P6 P7 PS P9 P10 K* K'

£_______

Fig. 2. Result of electrophoretic separation of PCR products. Red arrowhead indicates 500 bp marker.

M - DNA size marker (1000, 900, 800, 700, 600, 500, 400, 300, 200, 100 bp); P4, P6, P7, P8, P9, P10 - PCR products from genomic DNA isolated from sediments points P4, P6, P7, P8, P9, P10 respectively; K+ - positive control; K- -negative control

of Archaea. That possibility requires further detailed analyses of the ecosystem for confirmation. The clone sequence from station P7 (Wladyslawowo area), coded ZP.07.1, showed 87% similarity to the total genomic sequence of Methanospirillum hungatei JF-1. The wild-type archaeon clone of the methyl coenzyme M reductase alpha subunit (mcrA) gene from station P8 (Puck area), coded ZP.08.1, had a 98% similarity to Methanocorpusculum sp., which has been taxonomically classified as incertae sedis. Two other sequences of wild-type clones of mcrA gene were detected in sediments from station P8 (Puck area), and coded ZP.08.2 and ZP.08.3. Both sequences showed similarity to the organisms belonging to the genus Methanosarcina. ZP.08.2 showed 93% similarity to the Methanosarcina sp. T-36 strain, while ZP.08.3 showed 95% similarity to Methanosarcina sp. WH-1 strain. The sequence coded ZP.10.1 was obtained from sediments collected at station P10 (Oslonino area); it showed 94% similarity to Methanolobus taylori which, in taxonomic classification, belongs to the family Methanosarcinaceae. It has been established that Archaea from the order Methanosarcinales from the family Methanosarcinaceae and the order Methanomicrobiales from the family Methanospirillaceae and the family Methanocorpusculaceae might exist in the Puck Bay sediments.

Based on the obtained molecular biology results, it should be stated that in the analyzed ecosystem, marine organic matter might have decomposed under anaerobic conditions in the microenvironment of the benthic sediment. Oxygenated water in the coastal zone and oxygen diffusion into the surgical sediment

Methanocorpuscutum sp, (0.0184)

ZP0S.1 (0.0035)

f/fethanospirillum hungatei JF-1 (0.0585) -ZP.07.1 (0,0922)

i— i

tfi 0] ai ra

ZP.04.1 (0.0005)

ZP.09.1 (0.0057) ZP.06,1 (C.0040)

td = = =

¡5 ro

L. Ü-

/Vieifiano/obus iayiora (0 .04S1i

1- 2P.10.1 (0.0142)

— Methanosarcina sp. T36 (0.0345) ■ ZP.08.2 (0.0263)

-Methanosarcina sp. WH-1 (0.0284)

■ ZP 08.3 (0.0000)

01 -= ■D c

Fig. 3. Neighbor - joining method phylogenetic tree of methane forming archeons clones from the Puck Bay sediment.

layer by the waves seem to exclude the possibility of the development of strictly anaerobic organisms, such as methane-producing Archaea. However, the wave action causes vertical displacement of organic matter into the deeper sediment layers. Such a mechanism might result in local, micro-scale anaerobic conditions and, consequently, create the possibility of the development of methanogens. The phenomenon of localized anaerobic conditions in oxygenated sediment or the seawater column, which facilitated the development of anaerobic microorganisms and methane production from decomposing organic matter in the Baltic, have been previously reported (Edlund 2007). In the case of the sediment from the coastal zone of Puck Bay, the occurrence of local anaerobic conditions has also been confirmed previously (Witkowski 1993).

A comparative analysis of the obtained nucleotide sequences indicates that methane-producing Archaea DNA are present within the sampling stations. Nucleotide sequences of the clones of the organisms obtained based on DNA from sampled sediments showed similarity to previously described nucleotide sequences of methane-forming bacteria from the Archaea domain. These are sequences of organisms from the order Methanosarcinales and Methanomicro biales.

Archaea from the genus Methanosarcina are able to use all three metabolic pathways (hydrogenothropic, acetothropic and methylothropic) of methane biosynthesis. The genus Methanolobus, on the other

hand, is represented by methylotrophic organisms that use, inter alia, methylamines to produce methane. The substrate for methanogenesis for organisms belonging to the genus Methanospirillum classify these organisms as hydrogentrophic. However some Methanospirillum species can also use simple organic acids to synthesize methane (Doerfert et al. 2009; Hanako et al. 2009, Takao et al. 2010). It might be said that in the analyzed coastal sediment, methane-forming bacteria produced methane via all known metabolic pathways.

Summary

The mcrA region has been successfully used as a molecular marker for identifying methanogens. The conducted molecular analysis confirmed that the primers, described by Luton et al. (2002), were best suited for amplifying the mcrA gene from template DNA isolated from sediment samples collected in transitional waters. The employed methodology for isolating genomic DNA from marine sediments, cloning, and isolating plasmids (all procedures based on A&A Biotechnology kits) allowed the isolation of genetic template material, PCR amplification and the clones of DNA gene fragments of organisms responsible for methane production in the analyzed environmental samples. Molecular analyses suggest the possibility of strict anaerobic microbial activity and anaerobic decomposition of organic matter in the coastal area of Puck Bay. Oxygen conditions that

are determined, inter alia, by the vertical distribution of organic matter along the sediment profile might be a decisive factor in the presence or absence of methane forming Archaea. Based on the conducted research, it has been proven that the environmental conditions in the coastal zone of Puck Bay are suitable to sustain the life functions of strict anaerobic microorganisms.

References

Bange, H.W. (2006). Nitrous oxide and methane in European coastal waters. Estuar. Coast. Shel. Sci. 70, 361—374. DOI:10.1016/j.ecss.2006.05.042. Bates, B.C. Kundzewicz Z.W. & Wu S. (2008). Climate Change and Water. Palutikof, J.P. (Eds.). Technical Paper of the Intergovernmental Panel on Climate Change, IPCC, Geneva. Busch, G. Großmann J. Sieber M. & Burkhardt M. (2009). A new and sound technology for biogas from solid waste and biomass. Water, Air, & Soil Pollution: Focus 9, 89-97. Doerfert, S.N. Reichlen M. Iyer P. Wang M. & Ferry J.G. (2009). Methanolobus zinderi sp. nov., a methylotrophic methanogen isolated from a deep subsurface coal seam. Int. J. Syst. Evol. Microbiol 59, 1064-1069. Edlund A. (2007). Microbial diversity in Baltic Sea sediment. Doctoral dissertation. Swedish Univwersity of Agricultural Science, Uppsala.

Forster, P. Ramaswamy V. Artaxo P. Berntsen T. Betts R. Fahey D.W. Haywood J. Lean J. Lowe D.C. Myhre G. Nganga J. Prinn R. Raga G. Schulz M. & Van Dorland M. (2007). Changes in Atmospheric Constituents and in Radiative Forcing. In: Climate Change, 2007. The Physical Science Basis. Contribution of Working Group I to the Fourth Assessment Report of the Intergovernmental Panel on Climate Change [Solomon, S., D. Qin, M. Manning, Z. Chen, M. Marquis, K.B. Averyt, M.Tignor and H.L. Miller (eds.)]. Cambridge University Press, Cambridge, United Kingdom and New York, NY, USA. Gitay, H. Suarez A. Watson R.T. & Dooken D.J. (2002). Climate change and biodiversity. Technical Paper of the Intergovernmental Panel on Climate Change, IPCC, Geneva. Hales, B.A. Edwards C. Ritchie D.A. Hall G. Pickup R.W. & Saunders J.R. (1996). Isolation and identification of methanogen-specific DNA from blanket bog peat by PCR amplification and sequence analysis. Appl. Environ. Microbiol. 62, 668-675.

Hanako, M. HideyukiT. Satoshi H. Hiroyuki I. Kohei N. Susumu S. & Yoichi K (2009). Methanolobus profundi sp. nov., a methylotrophic methanogen isolated from deep subsurface sediments in a natural gas field. Int. J. Syst. Evol. Microbiol. 59, 714-718. DOI 10.1099/ijs.0.001677-0. Heyer, J. & Berger U. (2000). Methane emission from the Coastal Area in the Southern Baltic Sea. Estuar. Coast. Shelf. Sci. 51, 13-30. DOI:10.1006/ecss.2000.0616. Houghton, J.T. Filho L.G.M. & Griggs D.J. (1997). Stabilization of atmospheric Greenhouse Gases: Physical, biological and socio-economic implication. Maskell (Eds.). Technical Paper of the Intergovernmental Panel on Climate Change, IPCC, Geneva.

Innis, M. A. & Gelfand D.H. (1990). Optimization of PCRs. [in:] PCR Protocols: a Guide to Methods and Applications. Innis,

M. A. Gelfand D.H. Sninisky J.J. & White T.J. (eds.). Academic Press, 3-12, San Diego, CA.

IPCC. (2007): Climate Change 2007: Synthesis Report. Contribution of Working Groups I, II and III to the Fourth Assessment Report of the Intergovernmental Panel on Climate Change [Core Writing Team, Pachauri, R.K & Reisinger, A. (eds.)], IPCC, Geneva, Switzerland.

Jensen, J.B. & Fossing H. (2005). Methane in the seabed sediments of the south-western Baltic Sea. Geophysical Research Abstract. 7.

Jorgensen, B.B. Bang M. & Blackburn T.H. (1990). Anaerobic mineralization in marine sediments from the Baltic Sea-North Sea transition. Marine Ecology Progress Series. 59, 39-54.

Judd, A.G. (2003). The global importance and context of methane escape from the seabed. Geo-Mar Lett. 23, 147-154. DOI: 10.1007/s00367-003-0136-z.

Judd, A.G. (2004). Natural seabed gas seeps as sources of atmospheric methane. Environ. Geol. 46, 988-996. DOI: 10.1007/ s00254-004-1083-3.

Judd, A.G. & Hovland M. (2007). Seabed fluid flow: the impact of geology, biology and the marine environment. Cambridge University Press. 20, pp.475.

King, G.M. (1990). Dynamics and controls of methane oxidation in a Danish wetland sediment. FEMS Microbiol. Ecol. 74, 309323. DOI:10.1016/0378-1097(90)90684-I.

Liikanen, A. Silvennoinen H. Karvo A. Rantakokko P. & Martikainen P.J. (2009). Methane and nitrous oxide fluxes in two coastal wetlands in the northeastern Gulf of Bothnia, Baltic Sea. BorealEnv. Res. 14, 351-368.

Liu, H. Ramnarayanan R. & Logan B.E. (2004). Production of electricity during wastewater treatment using a single chamber microbial fuel cell. Environ. Sci. Technol. 38, 22812285. DOI: 10.1021/es034923g.

Luton, P.E. Wayne J.M. Sharp R.J. & Riley P.W. (2002). The mcrA gene as an alternative to 16S rRNA in the phylogenetic analysis of methanogen populations in landfill. Microbiol. 148, 3521-3530.

Martens, Ch.S. Albert D.B. & Alperin M.J. (1999). Stable isotope trading of anaerobic methane oxidation in the Gassy sediments of Eckernforde Bay, German Baltic Sea. American J. Sci. 299, 589 - 610.

Masse, D.I. Croteau F. Patni N.K. & Masse L. (2003). Methane emissions from dairy cow and swine manure slurries stored at 10°C and 15°C. Canadian Biosystems Engineering. 45, 6.1-6.6.

Mathys, M. Thiessen O. Theilen F. & Schmidt M. (2005). Seismic characterization of gas-rich near surface sediments in the Arkona Basin, Baltic Sea. Marine Geoph.Res. 26, 207-224.

Mori, K. Yamamoto H. Kamagata Y. Hatsu M. & Takamizawa K (2000). Methanocalculus pumilus sp. nov., a heavymetal-tolerant methanogen isolated from a waste-disposal site. Int. J. Syst. Evolut. Microb. 50, 1723 - 1729.

Nunoura, T. Oida H. Miyazaki J. Miyashita A. Imachi H. & Takai K (2008). Quantification of mcrA by fluorescent PCR in methanogenic and methanotrophic microbial communities.

Microbiol. Ecology. 64, 240-247. DOI: 10.1111/j.1574-6941.2008.00451.x.

Piker, L. Schmaljohann R. & Imhoff J.F. (1998). Dissimilatory sulfate reduction and methane production in Gotland Deep sediments (Baltic Sea) during a transition period from oxic to anoxic bottom water (1993- 1996). Aquatic Microbial Ecology. 14, 183-193.

Reeburgh, W.S. (2007). Oceanic methane biogeochemistry. Chem. Rev.. 107, 486-513. DOI: 10.1021/cr050362v.

Saitou, N. & Nei M. (1987). The Neighbor-joining Method: A New Method for Reconstructing Phylogenetic Trees. Mol. Biol. Evol. 4, 406-425.

Sambrook, J. Fritsch E.F. Maniatis T. (1989). Molecular cloning: a laboratory manual. 2. ed., Cold Spring Harbor Laboratory Press, New York, 253pp.

Schlüter, M. Sauter E.J. Anderson C.E. Dahlgaard H. & Dando P.R. (2004). Spatial distribution and budget for submarine groundwater discharge in Eckenförde Bay (Western Baltic Sea). Limnol. Oceanogr. 49, 157-167.

Schmaljohann, R. (1996). Methane Dynamics in the sediment and water column of Kiel Harbour (Baltic Sea). Mar. Ecol. Prog. Ser. 131, 263-273.

Shlimon, A.G. Friedrich M.W. Niemann H. Ramsing N.B. & Finster K. (2004). Methanobacterium aarhusense sp. nov., a novel methanogen isolated from a marine sediment (Aarhus Bay, Denmark). Int. J. Syst. Evolut. Microb. 54, 759-763.

Sowers, K.R. Johnson J.L. & Ferry J.G. (1984). Phylogenic relationships among the methylotrophic methane-producing bacteria and emendation of the family Methanosarcinaceae. Int. J. Syst. Bacterial. 34, 444-450. DOI:10.1099/00207713-34-4-444.

Steinberg, L.M. & Regan J.M. (2009). mcrA-Targeted Real-Time Quantitative PCR Method to Examine Methanogen Communities. Appl. Environ. Microbiol. 75, 4435-4442.

Takao, I. Koji M. & Ken-ichiro S. (2010). Methanospirillum lacunae sp. nov., a methane-producing archaeon isolated from a puddly soil, and emended descriptions of the genus Methanospirillum and Methanospirillum hungatei. Int. J. Syst. Evol. Microbiol. 60, 2563-2566. DOI 10.1099/ijs.0.020131-0.

von Klein, D. Arab H. Völker H. & Thomm M. (2002). Methanosarcina baltica, sp. nov., a novel methanogen isolated from the Gotland Deep of the Baltic Sea. Extremophiles. 6(2), 103-110.

Wegener, G. (2008). Methane oxidation and carbon assimilation in marine sediments. Doctoral dissertation. Bremen University.

Whitman, W.B. Bowen T.L. Boone D.R. (2006). The methanogenic bacteria. In Dworkin M. (eds.), The Procaryotes: archaea. Bacteria: Firmicutes, Actinomycetes. (165-207). Springer.

Wilkens, R. H. & Richardson M.D. (1998). The influence of gas bubbles on sediment acoustic properties: in situ, laboratory, and theoretical results from Eckenförde Bay, Baltic Sea. Continental Shelf Research. 18, 1859-1892.

Witkowski, A. (1993). Mikrofitobentos. In Korzeniewski K. (eds.), Zatoka Pucka. Foundation of Gdansk University Development, Gdansk, 395-415.