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This article was submitted to Microbial Physiology and Metabolism, a section of the journal Frontiers in Microbiology
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The anaerobic oxidation of methane (AOM) is a key biogeochemical process regulating methane emission from marine sediments into the hydrosphere. AOM is largely mediated by consortia of anaerobic methanotrophic archaea (ANME) and sulfate-reducing bacteria (SRB), and has mainly been investigated in deep-sea sediments. Here we studied methane seepage at four spots located at 12 m water depth in coastal, organic carbon depleted permeable sands off the Island of Elba (Italy). We combined biogeochemical measurements, sequencing-based community analyses and
Methane seeps are widespread features of the seafloor along continental margins, where methane ascends from subsurface reservoirs and fuels methanotrophic communities or is emitted to the hydrosphere. The anaerobic oxidation of methane (AOM) is a key biogeochemical process regulating methane emission from marine sediments and is mediated by anaerobic methane-oxidizing archaea (ANME) and sulfate-reducing bacteria (SRB) (
Shallow-water coastal methane seeps can be found at continental margins of all oceans, e.g., in the North Sea at 75–170 m water depth (
The coastal seafloor is exposed to strong hydrodynamic forces caused by waves and tides. These high energies allow for the settlement of only larger particles of the sand fraction forming permeable sediments. Wave-driven advection furthermore greatly impacts the habitats of benthic microorganisms by the enhanced supply of electron donors, electron acceptors and nutrients (
Here, we investigated shallow-water methane seepage off the coast of the Tuscan Island Elba (Italy). Elba is located in the Northern Tyrrhenian Sea, a relatively young (<15 Ma) back-arc basin formed by the roll-back of the Adriatic and Ionian subducting plates. The region is underlain by very thin continental crust and is tectonically very active (
We focused this first investigation of the site on the detailed analysis of the biogeochemistry and microbial community structure. We chose four methane emission spots situated in these permeable sands and performed biogeochemical measurements, 16S rRNA gene sequencing and whole cell hybridizations. The study was based on three hypotheses: Methane seeps located in shallow, permeable sands (i) have characteristic biogeochemical profiles that are shaped by the profound hydrodynamic forces, (ii) harbor similar anaerobic methanotrophic communities than seeps found in the deep sea due to the strong selective pressure of methane as the predominant energy source, and (iii) have a higher diversity than deep-sea seeps, due to the greater number of niches available in coastal sands.
The investigated Pomonte methane seep site is part of the larger Tuscan Island seep area that is situated between the islands of Elba and Montecristo (
The total organic carbon (TOC) content of the sediment was determined using a Carlo Erba NA-1500 CNS (Carbon, nitrogen, sulfur) analyzer with a precision of 0.2 wt% TC. Pore water was retrieved by
We performed radiotracer incubations at standardized conditions in artificial seawater medium with 28 mM sulfate (pH 7). Approximately 2 g of wet sediment was transferred into 5 ml Hungate tubes that were filled with medium as described above equilibrated with a 1.5 atmosphere CH4:CO2 (90:10) gas phase to study methane oxidation and methane-dependent (SR), or with a N2:CO2 gas phase (1.5 atm) to study methane-independent SR. AOM and SR rates were determined from replicate incubations (
The AOM enrichment culture was started with sands from emission spot 1. The sands were diluted 1:1 with artificial seawater medium (
Total nucleic acids were extracted from 2 ml sediment (in duplicates) using a chloroform-based method (
We prepared 16S rRNA gene libraries from sediments of emission spot 1a (30–40 cm) and emission spot 3 (10–20 cm). Both sediment horizons showed the highest microbial activity and cell density of the respective seeps and thus were chosen to compare the most active seep communities. 16S rRNA genes were amplified by polymerase chain reaction (PCR) using ∼20 ng of environmental DNA, 30 cycles and the primer pairs GM3F/GM4R (
Taxonomic classification of the sequences was carried out using ARB (
Samples were amplified for pyrosequencing using forward and reverse fusion primers. The forward fusion primer was constructed with the Roche A linker (5′-CCATCTCATCCCTGCGTGTCTCCGACTCAG-3′), an 8–10 bp barcode, and the forward primer 340F (5′-CCCTAYGGGGYGCASCAG-3′) for archaea (
16S rRNA partial gene sequences derived from Sanger-sequenced gene libraries were deposited under the accession numbers KT907894–KT908003. 16S rRNA amplicon sequences were deposited in the sequence read archive under SRA BioProject accession number SRP064784.
We used the original and subsampled sequence abundance tables to calculate diversity indices and Chao1 richness (
The sediment was sonicated (Sonoplus HD70 sonication probe, Bandelin, Berlin) seven times on ice (20 cycles, 30 s, 30% intensity). After each sonication step, 1 ml of supernatant was replaced with 1 ml 1:1 phosphate buffered saline (PBS)/ethanol and the supernatants combined. Depending on the sample we filtered 10–20 μl of supernatant onto a polycarbonate filter (0.2 μm pore size) and embedded the filter in 0.2% low-melting agarose to prevent detachment of cells. Filter sections were used for catalyzed reporter deposition fluorescence
The investigated Pomonte methane seep site is located 200 m off the coast of Elba (Italy) at 12 m water depth. Within the 400 m2 study area (
The emitted gas contained up to 85% methane (and not further quantified proportions of ethane, propane, and CO2), with an unusual carbon isotopic signature of around ∂13C = -16aaaa vs. the Vienna Pee Dee Belemnite (VPDB) standard. This indicated abiogenic origin, which was further supported by the basement of this site consisting of fractured magmatic rock and the low organic carbon content of the sediment. We measured methane concentrations between 50 and 550 μM in the pore water (
In the three investigated emission spots (ES1-3) the reactants and products of AOM were elevated. DIC, alkalinity and sulfide values, which also derived from
Anaerobic oxidation of methane and methane-dependent SR was measured in three horizons of emission spot 1 (Supplementary Figure
To characterize the microbial processes involved in AOM it is desirable to have sediment-free microbial enrichments. Due to the slow growth of AOM-mediating organisms (
The sediment horizons of ES1a (30–40 cm) and ES3 (10–20 cm) that showed the highest AOM activity were used for the construction of archaeal and bacterial 16S rRNA gene libraries. Despite the proximity and the observed geochemical similarity of the seep sites we found striking differences in their microbial richness and community composition (
Diversity parameters based on pyrosequencing the V3–V5 region of sediment samples of the emission spots ES1a, ES1b, ES3, and the reference spots Ref1–3.
Sample | Total reads | OTU0.02(S)∗ | Chao1 richness (Chao1)∗ | Inverse simpson diversity (D)∗ | |
---|---|---|---|---|---|
ES 1a | 14419 | 99 | 140 | 5.3 | |
ES 1b | 3756 | 145 | 194 | 16 | |
ES 3 | 12103 | 255 | 463 | 3.1 | |
Ref 1 | 10462 | 150 | 270 | 5.6 | |
Ref 2 | 8601 | 40 | 64 | 3.4 | |
Ref 3 | 14296 | 257 | 428 | 6.0 | |
ES 1a | 3887 | 306 | 500 | 11 | |
ES 1b | 4310 | 576 | 1057 | 202 | |
ES 3 | 6511 | 493 | 890 | 77 | |
Ref 1 | 6779 | 564 | 1166 | 177 | |
Ref 2 | 4254 | 469 | 766 | 141 | |
Ref 3 | 1444 | 558 | 1079 | 194 |
The relative cell abundance of ANME-1, ANME-2, SEEP-SRB1a, and SEEP-SRB2 as determined by CARD–FISH varied substantially between seep sites and sediment layers. At all seeps the layer with the highest total cell abundance coincided with the highest relative abundance of anaerobic methanotrophs and sulfate reducers. These layers were between 10 and 40 cmbsf (cm below sea floor) and were highly dominated by ANME and SEEP-SRB (
The morphology of these aggregates varied greatly ranging from consortia, in which ANME and SRB were interwoven (
In sediments of ES1 we found the highest morphological diversity. The analysis of 366 aggregates of ES1, ES3 and ES4 revealed that filamentous chain-type aggregates were the most relative abundant type of consortia (18%), followed by bubble- and mixed-type (each 12%) and by shell-type aggregates (11%), whereas woven-type aggregates were rare (1%) (Supplementary Figure
Most of the so far studied methane seeps are located in muddy, silty deep-sea sediments that are less affected by hydrodynamic forces and temperature changes, and are thus very stable and permanently cold. The microbial communities at these deep-sea ecosystems develop over long periods of time and are predominantly shaped by faunal activity (
In deep-sea methane seep sediments the sulfate-methane transition zone (SMTZ) and hence the zone of highest activity is usually only a few centimeters thick. Often the SMTZs are located close to the sediment surface and harbor between 109 and 1010 microbial cells per milliliter sediment (
Both methane and sulfate occurred in excess throughout the sediment and were not depleted, indicating a low efficiency of the benthic filter in permeable, low-biomass sands. A large part of the methane that passes through the sand without being oxidized also passes through the shallow water column, making its way to the atmosphere, where it may act as a potent greenhouse gas (
Many studies in recent years have tried to elucidate the niche differentiation and ecophysiology of populations that are directly or indirectly involved in the anaerobic oxidation of methane and/or hydrocarbons. Although, evidence is accumulating that microbial populations differentiate based on the availability of electron acceptors (
Diversity and turnover of the microbial communities at the Pomonte site were described and compared to those found at deep-sea methane seeps. The microbial communities of Elba shallow seeps comprised organisms that were closely related to those found at other seep ecosystems worldwide (
Permeable sands are much more heterogeneous than soft deep-sea sediments and provide a large number of niches to microorganisms (
Phylogenetic diversity was paralleled by an unprecedented morphological diversity, including several different forms of spherical and filamentous consortia, monospecific aggregates and even indications for AOM consortia that are comprised of two ANME clades and one SRB (
Disturbance caused by the strong hydrodynamics may also explain the high microbial turnover between the sites. Other factors may include the fluctuations in immigrating and emigrating microbial populations, which is a stochastic process that is especially important in dynamic habitats (
Coastal sandy sediments have a higher permeability and lower porosity than the silty clays that constitute deep-sea sediments, which in turn results in a lesser interstitial volume and lesser overall particle surface. These sediment properties combined with the prevalent hydrodynamics due to wave action, currents and gas ebullition create microbial habitats in shallow methane seeps that are very different from those found at methane seeps in the deep sea. To distinguish these habitats we think that a standardized and detailed ontology of methane-fuelled ecosystems is needed. Our findings suggest that the high phylogenetic and morphological diversity of anaerobic methanotrophs, and the apparently low-efficient methane filter at the Pomonte seep site are linked to the sediment characteristics of the ecosystem. Yet, the underlying environmental processes that shape microbial diversity, abundance and function remain unclear and are promising objectives of further research. The study underlines that our understanding of shallow-water methane seeps is still incomplete, despite their widespread occurrence on active and passive continental margins and importance for the global methane budget. It is crucial to further investigate the microbial ecology and efficiency of methane removal as most of the emitted methane at shallow seeps is released directly to the atmosphere.
All authors were involved in the design and the writing of the study. CL, MW, HK, and JW sampled in the field. HK, JW, and GW processed the samples in the lab. ER, HK, GW, AR, JW analyzed the data.
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
We thank the HYDRA team for supporting the field sampling campaigns, Duygu Sevgi Sevilgen, and Eskil Salis Gross for measuring the sedimentological parameters, and Christian Deusner for measuring the methane gas content and its isotopic signature. We thank Nicole Rödiger, Kathrin Büttner, and Erika Weiz for technical support in cloning and sequencing. Sampling campaigns and laboratory analyses were financially supported by the HYDRA Institute for Marine Sciences and the Department of Molecular Ecology of the Max Planck Institute for Marine Microbiology in Bremen, Germany. Sequencing was financially supported by the HGF MPG Bridge Group for Deep Sea Ecology and Technology. SR and GW were supported by the Leibniz program of the DFG awarded to Antje Boetius. Further support was given by the Max Planck Society, Germany.
The Supplementary Material for this article can be found online at: