A metagenomic study of methanotrophic microorganisms in Coal Oil Point seep sediments
© Håvelsrud et al; licensee BioMed Central Ltd. 2011
Received: 8 June 2011
Accepted: 4 October 2011
Published: 4 October 2011
Methane oxidizing prokaryotes in marine sediments are believed to function as a methane filter reducing the oceanic contribution to the global methane emission. In the anoxic parts of the sediments, oxidation of methane is accomplished by anaerobic methanotrophic archaea (ANME) living in syntrophy with sulphate reducing bacteria. This anaerobic oxidation of methane is assumed to be a coupling of reversed methanogenesis and dissimilatory sulphate reduction. Where oxygen is available aerobic methanotrophs take part in methane oxidation. In this study, we used metagenomics to characterize the taxonomic and metabolic potential for methane oxidation at the Tonya seep in the Coal Oil Point area, California. Two metagenomes from different sediment depth horizons (0-4 cm and 10-15 cm below sea floor) were sequenced by 454 technology. The metagenomes were analysed to characterize the distribution of aerobic and anaerobic methanotrophic taxa at the two sediment depths. To gain insight into the metabolic potential the metagenomes were searched for marker genes associated with methane oxidation.
Blast searches followed by taxonomic binning in MEGAN revealed aerobic methanotrophs of the genus Methylococcus to be overrepresented in the 0-4 cm metagenome compared to the 10-15 cm metagenome. In the 10-15 cm metagenome, ANME of the ANME-1 clade, were identified as the most abundant methanotrophic taxon with 8.6% of the reads. Searches for particulate methane monooxygenase (pmoA) and methyl-coenzyme M reductase (mcrA), marker genes for aerobic and anaerobic oxidation of methane respectively, identified pmoA in the 0-4 cm metagenome as Methylococcaceae related. The mcrA reads from the 10-15 cm horizon were all classified as originating from the ANME-1 clade.
Most of the taxa detected were present in both metagenomes and differences in community structure and corresponding metabolic potential between the two samples were mainly due to abundance differences.
The results suggests that the Tonya Seep sediment is a robust methane filter, where taxa presently dominating this process could be replaced by less abundant methanotrophic taxa in case of changed environmental conditions.
The Coal Oil Point seep area (COP), located in the Santa Barbara Channel, California, is one of the most active seep areas in the world . Seepage of the greenhouse gas methane and other hydrocarbons has occurred in this area for over 500 000 years . The methane emitted from the COP is mainly of thermogenic origin and the daily emission has been estimated to be at least 40 metric tons [1, 3].
At a global scale, the oceans only make up about 2% of the global methane emission budget . This low level is explained by prokaryotic oxidation of methane in marine sediments and bedrocks before it reaches the water column .
The oxygen penetration level in marine sediments is shallow, so most of the methane oxidation takes place at anaerobic conditions. Anaerobic oxidation of methane (AOM) is assumed to be a coupling of reversed methanogenesis and sulphate reduction. This process is likely performed by the yet uncultured anaerobic methanotrophic archaea (ANME) in syntrophy with sulphate reducing bacteria (SRB). Based on phylogeny, ANME can be divided into three clades: ANME-1, ANME-2 and ANME-3 [6–9]. ANME-2 and ANME-3 are affiliated to the Methanosarcinales, while ANME-1 is only distantly related to the Methanosarcinales and Methanomicrobiales [7–9]. Both ANME-1 and ANME-2 are associated with sulphur reducing deltaproteobacteria of the Desulfosarcina/Desulfococcus-branch [7, 9, 10]. ANME-3 is mainly associated with SRB strains closely related to Desulfobulbus .
The reversed methanogenesis model for AOM has gained support by a metagenomic study on ANME at Eel River  and sequencing of an ANME-1 draft genome . In these studies sequence homologues of all enzymes needed for CO2-based methanogenesis with exception of N5, N10-methylene-tetrahydromethanopterin reductase (mer) were identified. Methyl-coenzyme M reductase (mcrA) is assumed to catalyze the first step of AOM and the last step of methanogenesis, and is therefore a marker gene for both processes. Similarly, dissimilatory sulphite reductase (dsrAB) is often used as a marker gene for SRB .
When oxygen is present, aerobic methanotrophs are active in methane oxidation. Known aerobic methanotrophs include representatives of Gammaproteobacteria, Alphaproteobacteria and Verrucomicrobia [14–18]. These organisms convert methane to methanol using the enzyme methane monooxygenase . The particulate, membrane bound version of methane monooxygenase (pmoA), found in all aerobic methanotrophs (with exception of Methanocella), is used as a marker gene for aerobic oxidation of methane . The methanol formed is converted to formaldehyde, which is assimilated by one of two known pathways. Type I and type II methanotrophs utilize the ribulose monophosphate pathway and the serine pathway respectively. Type × methanotrophs use primarily the ribulose monophosphate pathway, but possess the enzymes needed for the serine pathway as well .
Stable isotope probing and sequencing of 16S rDNA and pmoA, as well as lipid biomarker analysis, have detected type-I aerobic methanotrophs in sediments and biofilms at the COP Shane and Brian seeps [21, 22]. Recently, measurements of average δ13C of carbonates and lipid biomarkers associated with ANME and SRB also indicated occurrence of AOM at the Brian seep . Another survey at the Brian seep detected ANME-2 at 6-9 cm bsf (below sea floor) by FISH (Fluorescent in situ hybridization) .
In the present study, we have used metagenomics to characterize the taxonomic and metabolic potential for both aerobic and anaerobic methane oxidation in two sediment samples from different depths at the Tonya seep (COP). By avoiding PCR amplification and primer target specificity, the metagenomics approach offered further insight into the taxonomy and metabolic potential of the prokaryotic communities of the methane seep sediments.
Gas measurements and methane oxidation rate
The average methane oxidation rate based on 11 measurements in the top 15 cm of the seep sediments was 156 ± 64 nmol cm-3 day-1. Still, the gas emitted from the Tonya seep sediments into the water phase contained a large fraction of methane. Even after travelling 25 m through the water column, where dissolved O2 and N2 entered the bubbles, the two gas samples contained 80.4% (gas sample I) and 68.1% (gas sample II) methane. When O2 and N2 were excluded, and the hydrocarbon and CO2 content were normalized, methane accounted for 93.6% in both gas samples. The remainder consisted of CO2 and short chain hydrocarbons (C2, C3, i-C4 and n-C4).
Metagenome creation through filtering of reads
Estimated effective genome sizes (EGS) were 4.8 Mbp and 4.0 Mbp for the 0-4 cm and 10-15 cm sample respectively (Additional file 1, Table S1).
There was a significant difference in the proportion of reads assigned to Bacteria and Archaea for the two metagenomes (Figure 3). In the 0-4 cm metagenome 60.87% of the reads were assigned to Bacteria and 0.97% to Archaea, while in the 10-15 cm metagenome 47.14% of the reads were assigned to Bacteria and as much as 18.09% to Archaea. This shift in the prokaryotic community structure suggests that Archaea thrive better and thereby also are likely to contribute more to the metabolism in the 10-15 cm sediment horizon.
Reads assigned to bacterial and archaeal taxa at the phylum-level in MEGAN
0-4 cm metagenome
10-15 cm metagenome
Percent of reads
Percent of reads
Archaeal environmental samples
Among the Proteobacteria, Sulfurovum was the most abundant genus in the 0-4 cm metagenome (Additional file 2, Table S2). This sulphur oxidizing genus, with its versatile energy metabolism, is known to thrive in sediments related to hydrothermal seepage where reductive and oxidative states in the mixing zone often fluctuate . Sulfurovum was almost four times more abundant in the 0-4 cm metagenome compared to the 10-15 cm metagenome. This is consistent with oxidative zones being its preferred habitat .
Taxa potentially involved in methane oxidation
"Archaeal environmental samples"- reads assigned to ANME-sequences
0-4 cm metagenome
10-15 cm metagenome
Percent of reads
Percent of reads
ANME-1, Black Sea 
ANME-1b, Black Sea 
ANME-2, Eel River 
ANME-3, Hydrate Ridge 
The ANME-1 clade was by far the anaerobic methanotroph with most assigned reads, although ANME-2 and ANME-3 also were present in the 10-15 cm metagenome (Figure 4). ANME-1 and ANME-2 were detected with low abundance in the 0-4 cm metagenome. The high abundance of ANME in the 10-15 cm metagenome indicates that AOM caused the high methane oxidation rates measured at this depth.
ANME are assumed to live in syntrophy with SRB. The most abundant genera of SRB in the metagenomes from the Tonya seep were Desulfococcus, Desulfobacterium and Desulfatibacillum (Figure 4). These genera were abundant in both metagenomes, and Desulfococcus, a common partner of ANME [7, 9, 10], especially so in the 10-15 cm metagenome (Additional file 2, Table S2).
Reads assigned to the bacterial NC10 group were present in both metagenomes (Figure 4). It has been proposed that Candidatus Methylomirabilis oxyfera of the NC10 group can oxidize methane anaerobically without an archaeal partner [30, 31]. A pathway of "intra-aerobic" methane oxidation where an intracellular supply of oxygen is produced by metabolism of nitrite to oxygen and dinitrogen has been suggested. This intracellularly produced oxygen is then used for the oxidation of methane via pmoA . Reads assigned to NC10 were significantly overrepresented (99% confidence interval) in the 10-15 cm metagenome compared to the 0-4 cm metagenome. Still, there was far less reads (approximately 1:100) assigned to NC10 than to ANME-1 in the 10-15 cm metagenome.
Methane oxidation pathways
To gain insight into the metabolic pathways for methane oxidation at the Tonya Seep, we annotated the reads from each metagenome to KO and EC numbers and plotted them onto KEGG pathway maps. In this way, the methane monooxygenase gene (EC: 184.108.40.206) was identified in the 0-4 cm sample, supporting the idea of aerobic methane oxidation in this sediment horizon. This gene was not detected in the 10-15 cm metagenome.
Anaerobic oxidation of methane is usually associated with dissimilatory sulphate reduction, where adenylyl-sulphate reductase (EC: 220.127.116.11) first reduces sulphate to sulphite before dissimilatory sulphite reductase (EC: 18.104.22.168) reduces sulphite to sulphide . These genes were detected in both metagenomes.
The marker gene for AOM, mcrA, is also a key gene in methanogenesis, where it catalyzes the last step. The 0-4 cm sample contained only one mcrA read, assigned to the methanogenic genus Methanosarcina (Figure 6). In the 10-15 cm sample 28 reads matching mcrA were found, all assigned to ANME-1. Based on EGS and expected number of reads matching mcrA, the estimated fraction of the community in the 10-15 cm sample made up of ANME-1 was 77.4% (Additional file 1, Table S1).
In order to detect possible SRB partners of ANME, we compared the two metagenomes to a dsrAB library. Of 60 hits, 33 were assigned to the reversed form of dsrAB found in sulphur compound-oxidizing bacteria. Sixteen and eleven dsrAB reads from the possible SRB partners of ANME were detected in the 0-4 cm and 10-15 cm metagenomes respectively, estimations based on the probability of detecting this gene thereby indicate that 43.2% and 24.6% of the 0-4 cm and 10-15 cm community were made up by SRB respectively (Additional file 1, Table S1).
Most SRB dsrAB reads were assigned to "bacterial environmental samples" and the deltaproteobacterial genera Desulfotaela, Desulfobacula, Desulfobacterium, Desulfobacter, Desulfatibacillum and Bilophila (Figure 6). The reads assigned to "bacterial environmental samples" matched clones from a diverse range of sediments [33–41] and one clone from an acidic fan soil sample . Eight of the reads assigned to "bacterial environmental samples" (six from the 0-4 cm metagenome and two from the 10-15 cm metagenome) were most similar to dsrAB sequences from Deltaproteobacteria [33–39] (results not shown). None of the dsrAB reads were assigned to Desulfosarcina or Desulfococcus, the previously described syntrophic partners of ANME-1 [7, 9, 10].
Methane oxidation rate
Methane oxidation rates in our sediment cores were 156 ± 64 nmol cm-3 day-1. This is much higher than the methane oxidation rates at the nearby Brian seep (6-87 nmol cm-3 day-1)  and within the range of AOM at seeps with surface hydrates, mud volcanoes and gas chimneys ( and refs therein). It has been suggested that the relatively low methane oxidation rate at the Brian seep could be caused by the permeable, sandy sediments leading to low amounts of dissolved methane in the pore water . Conversely, the higher methane oxidation rate at the Tonya seep could be due to the less permeable, relatively oily tar containing sediments at this seep.
Taxonomic richness and coverage
Taxonomic classification was based on a blastX query against the NCBI non-redundant Protein Database (ncbiP-nr). It has previously been shown that the prokaryotic representation in public sequence databases, such as the ncbiP-nr, is heavily biased towards taxa that are easily cultivable or of anthropogenic interest [43, 44]. Many of the taxa represented are further only partially sequenced . These issues may lead to false assignment of reads, especially if only the top hit is considered. By employing the LCA algorithm of MEGAN, most of these wrong assignments are avoided at the cost of more reads being assigned to taxa of low specificity or not being assigned at all [45, 46]. Short reads may also be a source of ambiguous taxonomic classification, especially if they are from a highly conserved region of the genome or from a region susceptible to horizontal gene transfer [44, 45, 47]. We therefore calculated the average read length for reads assigned to different taxonomic levels in MEGAN to see if it decreased with decreasing taxonomic specificity (Additional file 4, Table S4). This was not the case as average lengths of reads assigned to all taxonomic levels in MEGAN (including "not assigned") were in the same range (approximately 450 bases). Read with no hits against the ncbiP-nr were however considerably shorter (average read lengths of 263 ± 181 and 232 ± 175 bases in 0-4 cm and 10-15 cm metagenome respectively).
Rarefaction analyses indicated that the most abundant taxa of the Tonya Seep sediments were accounted for in our metagenomes. The taxonomic richness of prokaryotes, in combination with high EGS, does however lead to low coverage of most genomes represented in the metagenomes. Absence of a single marker gene assigned to a specific taxon might therefore be due to chance. Still, we detected more marker genes than expected based on the taxonomic binning of reads. This could be due to an overestimation of the EGS. It has previously been discussed that a bit score threshold of 60 (as used in this work according to the method developed by Raes et al. ) might discriminate against short reads, and that lowering of the threshold would result in decreased EGS . A decreased EGS would in turn result in a reduction of the estimated fraction of the community carrying the marker genes mcrA, pmoA and dsrAB. Differences in copy number for organisms carrying the gene might also affect the expected number of hits.
Aerobic methane oxidation
Due to limited oxygen penetration, active aerobic methane oxidation is probably limited to a thin surface layer. The maximum oxygen penetration at the nearby Brian seep sediments was measured to a depth of 1.4 cm . Due to high tar content, oxygen penetration in the sediments of the Tonya seep is expected to be more restricted than at the Brian seep.
Methane monooxygenase (EC: 22.214.171.124) was only detected in the 0-4 cm metagenome after plotting of KO and EC numbers onto KEGG pathway maps. Overrepresentation of aerobic methanotrophic genera and pmoA (based on library comparison) in the 0-4 cm metagenome compared to the 10-15 cm metagenome further support aerobic oxidation of methane in the 0-4 cm sediment sample (see Figures 4 and 6).
Both taxonomic binning of reads and marker gene classification point to type I methanotrophs of Methylococcaceae as the most important aerobic methane oxidizers in our samples. While Methylococcus was the aerobic methanotrophic genus with most reads assigned (see Figure 4), most of the detected pmoA reads were assigned to unclassified Methylococcaceae (see Figure 6). This indicates that uncultured type I methanotrophs might play an important role in aerobic methane oxidation at the Tonya Seep. Also in microbial mats and sediments of the nearby Shane and Brian seeps aerobic type I methanotrophs have been identified, while no type II methanotrophs were detected at either of these sites [21, 22]. This is consistent with type I methanotrophs dominating over type II methanotrophs in most marine settings (and refs therein).
Anaerobic methane oxidation
Genes for AOM were detected in both metagenomes (see Figure 5). The taxonomic binning of reads points to AMNE-1 as the predominant anaerobic oxidizer of methane in the Tonya seep sediment, especially in the 10-15 cm sediment sample. It is however, important to notice that ANME-1, due to the genome sequencing efforts , is the most sequenced ANME-clade, and therefore overrepresented in the database. This could skew our relative abundance results. However, the presence and dominance of ANME-1 was further supported by the mcrA reads in our metagenomes (see Figure 6). This gene is identified in all ANME-clades, still all reads matching mcrA in the 10-15 cm metagenome were assigned to ANME-1. Taken together, these results provide strong evidence of ANME-1 being the most important clade for anaerobic methane oxidation in the Tonya seep sediments. In contrast, only ANME-2 was detected at the nearby Brian Seep . ANME-1 and ANME-2 are known to co-occur in sediments, usually with one type more abundant than the other [7, 51, 52]. The environmental conditions that might regulate the relative abundance of the different ANME clades in marine sediments are still not known [7, 51]. Differences in permeability of the sediments at the Tonya and Brian seeps could be one factor selecting for different ANME clades at the two sites.
Sulphate reducing bacteria
Anaerobic oxidation of methane is assumed to be coupled to dissimilatory reduction of sulphate. Both metagenomes had reads assigned to SRB genera, predominantly Desulfococcus, Desulfobacterium and Desulfatibacillum (see Figure 4). The ratio of total reads assigned to ANME related to reads assigned to each of these SRB genera in the 10-15 cm metagenome were ANME: Desulfobacterium; 16: 1, ANME Desulfatibacillum; 20:1 and ANME: Desulfococcus; 24: 1. The total ratio ANME: SRB (including "Bacteria environmental samples") was 4: 1.
Reads assigned to dsrAB were detected in both metagenomes and classified to a diverse set of taxa (see Figure 6). Although the fraction of the community containing mcrA and dsrAB, calculated based on sampling probability of the specific marker genes, is likely to be overestimated it gives a similar ratio of 3: 1 of mcrA-containing organisms: dsrAB containing organisms as the taxonomic binning of reads. None of our dsrAB reads were assigned to the known ANME partner Desulfococcus, although this genus was one of the most abundant SRB genera in our metagenomes (see Figure 4). This does not imply absence of dsrAB among Desulfococcus in our samples; the gene was more likely missed by chance due to low coverage (see Additional file 2, Table S2).
ANME might also form syntrophic relationships to other bacteria than those most commonly recognized. ANME-2 has previously been detected to form physical associations to both Desulfobulbus and a member of the Betaproteobacteria, as well as their regular partners from the Desulfococcus/Desulfosarcina branch . The main bulk of dsrAB-reads in the 10-15 cm metagenome were assigned to "bacterial environmental samples" and the ANME partners might be found among these organisms. The "bacterial environmental samples" is however a diverse group and was also abundant in the 0-4 cm metagenome, where ANME were less abundant.
Our results do not indicate only one predominant ANME partner, but rather that several syntrophic partners may be involved. Diverse dsrAB signatures with only weak coupling to AOM have previously been detected in ANME-1 dominated sediments in the Gulf of Mexico . This suggests that these seep environments have a high diversity of taxa involved in sulphate reduction.
By using 454 sequenced metagenomes we achieved an insight into the taxonomic richness of the seep sediments. Most of the taxa were present in both metagenomes and differences in community structure and corresponding metabolic potential between the two samples were due to abundance, indicating sliding boundaries between the different communities. Our approach provided strong evidence for the taxa responsible for methane oxidation. The Tonya Seep harboured several taxa potentially capable of methane oxidation under both aerobic and anaerobic conditions. This suggests that the sediment is a robust methane filter, where taxa presently dominating this important process could be replaced by less abundant taxa should the environmental conditions change.
Tonya Seep (34°24.043'N; 119°52.841'W) is located in the Coal Oil Point seep field offshore Santa Barbara, California, USA. Tonya Seep is primarily a single 2 m diameter pit with many vents inside that rapidly coalesce into a single plume. There was a high content of hydrocarbons and tar in the sediments. Four sediment cores, two for methane oxidation studies and two for metagenomic analysis, were collected at 25 m depth on July 16th 2008 by UC Santa Barbara Marine Operation divers. The polycarbonate liners used (30 cm length and 3.5 cm diameter) were treated with 70% ethanol and dried before sampling. The parallel cores (core I, II, III and IV) were sealed at the seafloor and kept on ice during transportation back to shore.
Gas Sample Collection
Two seep gas samples (Gas samples I and II) were collected in the surface waters above the seep. The samples were collected on two occasions from small vessels via an inverted funnel method in which seep gas bubbles were captured into 120 mL glass serum vials after rising through the water column. Bottles were capped underwater after filling to avoid contamination with atmospheric gases. Seep gases were analyzed by gas chromatography as previously described . Error associated with the concentration measurements was ±4%.
Methane oxidation rates
Cores III and IV designated for methane oxidation rate (MOR) measurements were injected with radiotracer 14C-CH4 (1 kBq 14CH4 dissolved in water, 20 μL injection volume) at 2 cm intervals and incubated at near in-situ temperature. After 18 hours the core was sub-sectioned and placed into vials with 1 M NaOH and quickly sealed, ending the incubation and trapping the CO2. A small sample of headspace (0.2 mL) was removed to determine CH4 concentration (which is not affected by the 14CH4 spike) by GC-FID (Shimadzu GC-4A, 6 ft length 80/100 mesh Molsieve 13X packed column run isothermally at 140°C with N2 carrier flow at 15 mL min-1). The remaining 14CH4 in the headspace of the vial was purged via a slow flow of air through a combustion tube filled with Cu(II)-oxide and maintained at 850°C. The resulting 14CO2 was trapped using a mixture of phenethylamine and 2-methoxyethanol. The remaining 14CO2, which was assumed to be microbially produced, was measured by first transferring the sediment into a 100 mL Erlenmeyer flask fitted with a small (7 mL) phenethylamine/NaOH-filled scintillation vial suspended beneath its rubber stopper. Six ml of hydrochloric acid (6 M) was injected through the rubber stopper to degas the CO2 from the sediment/NaOH slurry, and the flask was placed in a shaker for ~8 hrs to transfer the CO2 to the suspended scintillation vial. Radioactivity was quantified by scintillation counting (Beckman LSC 6500).
where 14CO2 is the activity of the microbially-produced CO2, CH4 is the amount of CH4 in the sample, 14CH4 is the activity of the injected CH4, v is the volume of the sediment and t is the incubation time.
For metagenomic analysis, cores I and II were pushed out from the liners and the 0-4 cm bsf and the 10-15 cm bsf horizons were removed for DNA extraction. Multiple parallel 0.5 g subsamples of the cores at each horizon were used for DNA extraction. Total genomic DNA was extracted with a FastDNA®SPIN for Soil Kit (MP Biomedicals) and cleaned using Wizard DNA Clean-Up (Promega) according to the manufacturer's instructions. The DNA quality was assessed by agarose gel electrophoresis and by optical density using a NanoDrop instrument (NanoDrop Products, Thermo Scientific). To get enough high quality DNA for the subsequent 454 sequencing DNA, subsamples from the same horizon were pooled. Of the total DNA isolated from the 0-4 cm horizon, 35% originated from core I and 65% from core II. For the 10-15 cm horizon, 38% was isolated from core I and 62% from core II.
For creation of the metagenomic libraries, 9.8 μg DNA of the 0-4 cm sample and 6.8 μg of the 10-15 cm sample were used. Sample preparation and sequencing of the extracted DNA were performed at the Norwegian High-Throughput Sequencing Centre (NSC) at CEES , University of Oslo according to standard GS FLX Titanium protocols, except that after the initial dsDNA immobilization, ssDNA was brought into solution by adding 50 μl 1 × TE to the beads, followed by 2 min at 90°C and rapid cooling on ice.
The samples were tagged (fusion primers with tag sequences were used to mark sample origin), mixed and sequenced on a 70 × 75 format PicoTiterPlate™ on a GS FLX titanium instrument.
The metagenomic reads have been submitted to the Genbank Sequence Read archive [GeneBank: SRP005641].
The average of the mean quality score per sequence was 33.1 (standard deviation: 3.6) and 32.9 (standard deviation: 3.5) for the 0-4 cm metagenome and 10-15 cm metagenome respectively.
Replicate reads were removed from the two metagenomes using the 454 Replicate filter [56, 57]. Standard settings of a sequence identity cut off of 0.9, a length difference requirement of 0 and a number of beginning base pairs to check of 3, were used. After removal of replicates, the 0-4 cm metagenome contained 525 reads with more than 2 ambiguous bases and 1222 reads with long homopolymers (> 10 nt), making a total of 1733 (0.65%) low quality reads. The 10-15 cm metagenome contained 395 reads with more than 2 ambiguous bases and 143 reads with long homopolymers (> 10 nt), making a total of 535 (0.28%) low quality reads.
The reads were taxonomically classified by BlastX query against the NCBI non-redundant Protein Database (ncbiP-nr) . The computation was performed at the freely available Bioportal computer service . Maximum expectation-value was set to 10.0 and maximum 25 alignments were reported per hit. The BlastX output files were analysed according to NCBI taxonomy in the program MEGAN, version 3.9  with default LCA-parameters (Min Score: 35, Top Percent: 10.0 and Min Support: 5). We used the option "enable all taxa" in MEGAN in order to account for reads with hits to the artificial taxa archaeal and bacterial "environmental samples".
The species richness was estimated by rarefaction analysis performed in MEGAN . The MEGAN program uses an LCA-algorithm to bin reads to taxa based on their blast-hits. This results in a rooted tree where each node represents a taxon. The leaves in this tree are then used as OTUs in the rarefaction analysis. The program randomly chooses 10%, 20% ... 100% of the total number of reads as subsets. For each of these random subsets the number of leaves (hit with at least 5 reads (Min Support) is determined. This sub sampling is repeated 20 times and then the average value is used for each percentage. We did the analysis at the most resolved level of the NCBI taxonomy to capture as much of the richness as possible. At this level, the leaves are mostly strains and species but also some sequences like fosmids and plasmids are included. In cases were no reads are assigned to species the most detailed taxonomic level with 5 reads or more assigned are used.
The analysis was performed for total taxa in the metagenomes (including Bacteria, Archaea, Eukaryota, Viruses and Environmental sequences), and separately for archaeal and bacterial taxa.
Comparison of metagenomes
The metagenomes were compared at the phylum, class and genus level in MEGAN using absolute read counts . Tabulated text files for each level were extracted from MEGAN and analyzed in the following manner: The metagenomes were normalized to the size of the smallest metagenome. Taxa without matches in one metagenome, or with less than 20 reads in both metagenomes, were removed from the comparison since they (due to their low abundance) could have been identified by chance and thereby represent uninformative data. The resulting normalized comparison was analyzed for overrepresented taxa using XIPE-totec with 20.000 samplings and with a confidence cut-off of 0.95, 0.98 and 0.99 .
Reads were annotated to KEGG Orthologe (KO)-identifiers using KEGG Automatic Annotation Server (KAAS) [60, 61]. Parameters used were: single-directional best hit, default bit score (60) and 40 manually selected reference genomes (Additional file 5, Table S5). Reference genomes were chosen from the most abundant species present in the metagenomes based on annotation in MEGAN.
Lists of unique EC and KO numbers (when no EC-number was obtained) were created for each metagenome. These lists were then used to plot metabolic pathways for the two metagenomes onto metabolic pathway maps using KEGG Mapper: Colour Objects in KEGG Pathways [62–65].
Signature genes for methane oxidation
The reads were compared to protein sequence libraries for methyl-coenzyme M reductase (mcrA), particulate methane monooxygenase (pmoA) and dissimilatory sulphite reductase (dsrAB) on the freely available Bioportal computer service . The reference library for each enzyme was downloaded from Fungene (Functional gene pipeline & repository) version v6.1 . We limited the libraries by selecting only the sequences with a score (bits saved) of 100 or more from the HMMER Hidden Markov Model search against NCBIs non-redundant protein database. We used blastX against the protein sequences of each enzyme library with a maximum expectation value of 1.0E-20 . Maximum one alignment was reported.
BlastX output files were further analyzed using NCBI-taxonomy in MEGAN, version 3.9 . The LCA-parameters were set to: Min Score: 35, Top Percent: 10.0 and Min Support: 1. All taxa were enabled.
Estimates of effective genome sizes (EGS) and sampling probabilities of individual genes
EGS was calculated according to the method developed by Raes et al  using the parameters a = 18.26, b = 3650 and c = 0.733. Blast against a subset of the STRING database (v9.0), containing the COGs concerned, were conducted at the freely available Bioportal computer service [59, 67].
Sampling probability of the individual marker genes and expected number of sequences detected was calculated according to Beszteri et al . We calculated with an average copy number of two for pmoA  and one for mcrA and dsrAB [70–72]. Average marker gene length was based on the reads present in the respective marker gene databases.
The project was granted by VISTA/Statoil. OEH and the analytical costs were financed by project 6151 to AGR and THAH was financed by project 6503 to KSJ. The project was also supported by Norwegian Geotechnical Institutes education fund. We thank UC Santa Barbara Marine Operation divers in cooperation with David Valentine and Frank Kinnaman at UCSB for the core samples. We acknowledge David Valentine for valuable comments on the manuscript. The methane oxidation rate data of the cores and the seep gas analysis were generated by Frank Kinnaman and Blair Paul (UCSB) and kindly provided to our metagenomic project.
- Hornafius JS, Quigley D, Luyendyk BP: The world's most spectacular marine hydrocarbon seeps (Coal Oil Point, Santa Barbara Channel, California): Quantification of emissions. J Geophys Res. 1999, 104 (C9): 20703-20711. 10.1029/1999JC900148.View ArticleGoogle Scholar
- Boles JR, Eichhubl P, Garven G, Chen J: Evolution of a hydrocarbon migration pathway along basin-bounding faults: Evidence from fault cement. Am Assoc Pet Geol Bull. 2004, 88 (7): 947-970.Google Scholar
- Luyendyk B, Kennett J, Clark JF: Hypothesis for increased atmospheric methane input from hydrocarbon seeps on exposed continental shelves during glacial low sea level. Marine and Petroleum Geology. 2005, 22 (4): 591-596. 10.1016/j.marpetgeo.2004.08.005.View ArticleGoogle Scholar
- Reeburgh WS: Oceanic methane biogeochemistry. Chem Rev. 2007, 107 (2): 486-513. 10.1021/cr050362v.PubMedView ArticleGoogle Scholar
- Reeburgh WS: ''Soft spots'' in the global methane budget. Microbial Growth on C1 Compounds. 1996, 334-342.View ArticleGoogle Scholar
- Niemann H, Lösekann T, de Beer D, Elvert M, Nadalig T, Knittel K, Amann R, Sauter EJ, Schlüter M, Klages M, et al: Novel microbial communities of the Haakon Mosby mud volcano and their role as a methane sink. Nature. 2006, 443 (7113): 854-858. 10.1038/nature05227.PubMedView ArticleGoogle Scholar
- Knittel K, Lösekann T, Boetius A, Kort R, Amann R: Diversity and distribution of methanotrophic archaea at cold seeps. Appl Environ Microbiol. 2005, 71 (1): 467-479. 10.1128/AEM.71.1.467-479.2005.PubMedPubMed CentralView ArticleGoogle Scholar
- Hinrichs KU, Hayes JM, Sylva SP, Brewer PG, DeLong EF: Methane-consuming archaebacteria in marine sediments. Nature. 1999, 398 (6730): 802-805. 10.1038/19751.PubMedView ArticleGoogle Scholar
- Orphan VJ, Hinrichs KU, Ussler W, Paull CK, Taylor LT, Sylva SP, Hayes JM, Delong EF: Comparative analysis of methane-oxidizing archaea and sulfate-reducing bacteria in anoxic marine sediments. Appl Environ Microbiol. 2001, 67 (4): 1922-1934. 10.1128/AEM.67.4.1922-1934.2001.PubMedPubMed CentralView ArticleGoogle Scholar
- Boetius A, Ravenschlag K, Schubert CJ, Rickert D, Widdel F, Gieseke A, Amann R, Jørgensen BB, Witte U, Pfannkuche O: A marine microbial consortium apparently mediating anaerobic oxidation of methane. Nature. 2000, 407 (6804): 623-626. 10.1038/35036572.PubMedView ArticleGoogle Scholar
- Hallam SJ, Putnam N, Preston CM, Detter JC, Rokhsar D, Richardson PM, DeLong EF: Reverse methanogenesis: Testing the hypothesis with environmental genomics. Science. 2004, 305 (5689): 1457-1462. 10.1126/science.1100025.PubMedView ArticleGoogle Scholar
- Meyerdierks A, Kube M, Kostadinov I, Teeling H, Glöckner FO, Reinhardt R, Amann R: Metagenome and mRNA expression analyses of anaerobic methanotrophic archaea of the ANME-1 group. Environ Microbiol. 2010, 12 (2): 422-439. 10.1111/j.1462-2920.2009.02083.x.PubMedView ArticleGoogle Scholar
- Knittel K, Boetius A: Anaerobic oxidation of methane: Progress with an unknown process. Annu Rev Microbiol. 2009, 63: 311-334. 10.1146/annurev.micro.61.080706.093130.PubMedView ArticleGoogle Scholar
- Dunfield PF, Yuryev A, Senin P, Smirnova AV, Stott MB, Hou SB, Ly B, Saw JH, Zhou ZM, Ren Y, et al: Methane oxidation by an extremely acidophilic bacterium of the phylum Verrucomicrobia. Nature. 2007, 450 (7171): 879-882. 10.1038/nature06411.PubMedView ArticleGoogle Scholar
- Islam T, Jensen S, Reigstad LJ, Larsen Ø, Birkeland NK: Methane oxidation at 55°C and pH 2 by a thermoacidophilic bacterium belonging to the Verrucomicrobia phylum. Proc Natl Acad Sci USA. 2008, 105 (1): 300-304. 10.1073/pnas.0704162105.PubMedPubMed CentralView ArticleGoogle Scholar
- Pol A, Heijmans K, Harhangi HR, Tedesco D, Jetten MSM, Op den Camp HJM: Methanotrophy below pH1 by a new Verrucomicrobia species. Nature. 2007, 450: 874-878. 10.1038/nature06222.PubMedView ArticleGoogle Scholar
- Dumont MG, Murrell JC: Community-level analysis: Key genes of aerobic methane oxidation. Environ Microbiol. 2005, 397: 413-427.Google Scholar
- Stoecker K, Bendinger B, Schöning B, Nielsen PH, Nielsen JL, Baranyi C, Toenshoff ER, Daims H, Wagner M: Cohn's Crenothrix is a filamentous methane oxidizer with an unusual methane monooxygenase. Proc Natl Acad Sci USA. 2006, 103 (7): 2363-2367. 10.1073/pnas.0506361103.PubMedPubMed CentralView ArticleGoogle Scholar
- Conrad R: The global methane cycle: recent advances in understanding the microbial processes involved. Environ Microbiol Rep. 2009, 1 (5): 285-292. 10.1111/j.1758-2229.2009.00038.x.PubMedView ArticleGoogle Scholar
- Hanson RS, Hanson TE: Methanotrophic bacteria. Microbiol Rev. 1996, 60 (2): 439-471.PubMedPubMed CentralGoogle Scholar
- Ding H, Valentine DL: Methanotrophic bacteria occupy benthic microbial mats in shallow marine hydrocarbon seeps, Coal Oil Point, California. J Geophys Res. 2008, 113 (G1):
- Redmond MC, Valentine DL, Sessions AL: Identification of novel methane-, ethane-, and propane-oxidizing bacteria at marine hydrocarbon seeps by stable isotope probing. Appl Environ Microbiol. 2010, 76 (19): 6412-6422. 10.1128/AEM.00271-10.PubMedPubMed CentralView ArticleGoogle Scholar
- Kinnaman FS, Kimball JB, Busso L, Birgel D, Ding HB, Hinrichs KU, Valentine DL: Gas flux and carbonate occurrence at a shallow seep of thermogenic natural gas. Geo-Mar Lett. 2010, 30 (3-4): 355-365. 10.1007/s00367-010-0184-0.View ArticleGoogle Scholar
- Treude T, Ziebis W: Methane oxidation in permeable sediments at hydrocarbon seeps in the Santa Barbara Channel, California. Biogeosciences. 2010, 7 (10): 3095-3108. 10.5194/bg-7-3095-2010.View ArticleGoogle Scholar
- Rodriguez-Brito B, Rohwer F, Edwards RA: An application of statistics to comparative metagenomics. BMC Bioinformatics. 2006, 7:Google Scholar
- Yamamoto M, Nakagawa S, Shimamura S, Takai K, Horikoshi K: Molecular characterization of inorganic sulfur-compound metabolism in the deep-sea epsilonproteobacterium Sulfurovum sp NBC37-1. Environ Microbiol. 2010, 12 (5): 1144-1152. 10.1111/j.1462-2920.2010.02155.x.PubMedView ArticleGoogle Scholar
- Hallam SJ, Girguis PR, Preston CM, Richardson PM, DeLong EF: Identification of methyl coenzyme M reductase A (mcrA) genes associated with methane-oxidizing archaea. Appl Environ Microbiol. 2003, 69 (9): 5483-5491. 10.1128/AEM.69.9.5483-5491.2003.PubMedPubMed CentralView ArticleGoogle Scholar
- Meyerdierks A, Kube M, Lombardot T, Knittel K, Bauer M, Glöckner FO, Reinhardt R, Amann R: Insights into the genomes of archaea mediating the anaerobic oxidation of methane. Environ Microbiol. 2005, 7 (12): 1937-1951. 10.1111/j.1462-2920.2005.00844.x.PubMedView ArticleGoogle Scholar
- Miyazaki J, Higa R, Toki T, Ashi J, Tsunogai U, Nunoura T, Imachi H, Takai K: Molecular characterization of potential nitrogen fixation by anaerobic methane-oxidizing archaea in the methane seep sediments at the number 8 Kumano Knoll in the Kumano Basin, offshore of Japan. Appl Environ Microbiol. 2009, 75 (22): 7153-7162. 10.1128/AEM.01184-09.PubMedPubMed CentralView ArticleGoogle Scholar
- Ettwig KF, Shima S, van de Pas-Schoonen KT, Kahnt J, Medema MH, op den Camp HJM, Jetten MSM, Strous M: Denitrifying bacteria anaerobically oxidize methane in the absence of Archaea. Environ Microbiol. 2008, 10 (11): 3164-3173. 10.1111/j.1462-2920.2008.01724.x.PubMedView ArticleGoogle Scholar
- Ettwig KF, van Alen T, van de Pas-Schoonen KT, Jetten MSM, Strous M: Enrichment and molecular detection of denitrifying methanotrophic bacteria of the NC10 phylum. Appl Environ Microbiol. 2009, 75 (11): 3656-3662. 10.1128/AEM.00067-09.PubMedPubMed CentralView ArticleGoogle Scholar
- Ettwig KF, Butler MK, Le Paslier D, Pelletier E, Mangenot S, Kuypers MMM, Schreiber F, Dutilh BE, Zedelius J, de Beer D, et al: Nitrite-driven anaerobic methane oxidation by oxygenic bacteria. Nature. 2010, 464 (7288): 543-548. 10.1038/nature08883.PubMedView ArticleGoogle Scholar
- Bahr M, Crump BC, Klepac-Ceraj V, Teske A, Sogin ML, Hobbie JE: Molecular characterization of sulfate-reducing bacteria in a New England salt marsh. Environ Microbiol. 2005, 7 (8): 1175-1185. 10.1111/j.1462-2920.2005.00796.x.PubMedView ArticleGoogle Scholar
- Giloteaux L, Goñi-Urriza M, Duran R: Nested PCR and New Primers for analysis of sulfate-reducing bacteria in low-cell-biomass environments. Appl Environ Microbiol. 2010, 76 (9): 2856-2865. 10.1128/AEM.02023-09.PubMedPubMed CentralView ArticleGoogle Scholar
- Kaneko R, Hayashi T, Tanahashi M, Naganuma T: Phylogenetic diversity and distribution of dissimilatory sulfite reductase genes from deep-sea sediment cores. Mar Biotechnol. 2007, 9 (4): 429-436. 10.1007/s10126-007-9003-7.PubMedView ArticleGoogle Scholar
- Madrid VM, Aller RC, Aller JY, Chistoserdov AY: Evidence of the activity of dissimilatory sulfate-reducing prokaryotes in nonsulfidogenic tropical mobile muds. FEMS Microbiol Ecol. 2006, 57 (2): 169-181. 10.1111/j.1574-6941.2006.00123.x.PubMedView ArticleGoogle Scholar
- Nakagawa T, Nakagawa S, Inagaki F, Takai K, Horikoshi K: Phylogenetic diversity of sulfate-reducing prokaryotes in active deep-sea hydrothermal vent chimney structures. FEMS Microbiol Lett. 2004, 232 (2): 145-152. 10.1016/S0378-1097(04)00044-8.PubMedView ArticleGoogle Scholar
- Smith AC, Kostka JE, Devereux R, Yates DF: Seasonal composition and activity of sulfate-reducing prokaryotic communities in seagrass bed sediments. Aquat Microb Ecol. 2004, 37 (2): 183-195.View ArticleGoogle Scholar
- Lloyd KG, Lapham L, Teske A: An anaerobic methane-oxidizing community of ANME-1b archaea in hypersaline Gulf of Mexico sediments. Appl Environ Microbiol. 2006, 72 (11): 7218-7230. 10.1128/AEM.00886-06.PubMedPubMed CentralView ArticleGoogle Scholar
- Jiang LJ, Zheng YP, Peng XT, Zhou HY, Zhang CL, Xiao X, Wang FP: Vertical distribution and diversity of sulfate-reducing prokaryotes in the Pearl River estuarine sediments, Southern China. FEMS Microbiol Ecol. 2009, 70 (2): 249-262. 10.1111/j.1574-6941.2009.00758.x.View ArticleGoogle Scholar
- Mussmann M, Richter M, Lombardot T, Meyerdierks A, Kuever J, Kube M, Glöckner FO, Amann R: Clustered genes related to sulfate respiration in uncultured prokaryotes support the theory of their concomitant horizontal transfer. J Bacteriol. 2005, 187 (20): 7126-7137. 10.1128/JB.187.20.7126-7137.2005.PubMedPubMed CentralView ArticleGoogle Scholar
- Schmalenberger A, Drake HL, Küsel K: High unique diversity of sulfate-reducing prokaryotes characterized in a depth gradient in an acidic fen. Environ Microbiol. 2007, 9 (5): 1317-1328. 10.1111/j.1462-2920.2007.01251.x.PubMedView ArticleGoogle Scholar
- Hugenholtz P: Exploring prokaryotic diversity in the genomic era. Genome Biology. 2002, 3 (2):
- Huson DH, Auch AF, Qi J, Schuster SC: MEGAN analysis of metagenomic data. Genome Res. 2007, 17 (3): 377-386. 10.1101/gr.5969107.PubMedPubMed CentralView ArticleGoogle Scholar
- Kunin V, Copeland A, Lapidus A, Mavromatis K, Hugenholtz P: A Bioinformatician's Guide to Metagenomics. Microbiol Mol Biol Rev. 2008, 72 (4): 557-578. 10.1128/MMBR.00009-08.PubMedPubMed CentralView ArticleGoogle Scholar
- Mitra S, Schubach M, Huson DH: Short clones or long clones? A simulation study on the use of paired reads in metagenomics. BMC Bioinformatics. 2010, 11:Google Scholar
- Jain R, Rivera MC, Lake JA: Horizontal gene transfer among genomes: The complexity hypothesis. Nov 08-09. 1998, Natl Acad Sciences, 3801-3806. ; Irvine, CaliforniaGoogle Scholar
- Raes J, Korbel JO, Lercher MJ, von Mering C, Bork P: Prediction of effective genome size in metagenomic samples. Genome Biol. 2007, 8 (1): R10-10.1186/gb-2007-8-1-r10.PubMedPubMed CentralView ArticleGoogle Scholar
- Quaiser A, Zivanovic Y, Moreira D, López-García P: Comparative metagenomics of bathypelagic plankton and bottom sediment from the Sea of Marmara. ISME J. 2011, 5 (2): 285-304. 10.1038/ismej.2010.113.PubMedPubMed CentralView ArticleGoogle Scholar
- Valentine DL: Emerging topics in marine methane biogeochemistry. Ann Rev Mar Sci. 2011, 3 (1): 147-171. 10.1146/annurev-marine-120709-142734.PubMedView ArticleGoogle Scholar
- Nauhaus K, Treude T, Boetius A, Krüger M: Environmental regulation of the anaerobic oxidation of methane: a comparison of ANME-I and ANME-II communities. Environ Microbiol. 2005, 7 (1): 98-106. 10.1111/j.1462-2920.2004.00669.x.PubMedView ArticleGoogle Scholar
- Merkel A, Chernykh N, Kanapatskii T, Pimenov N: Detection of methanotrophic archaea in pockmark sediments (Gdansk Deep, Baltic Sea) by sequence analysis of the gene encoding the α subunit of methyl-coenzyme M reductase. Microbiology. 2010, 79 (6): 849-852. 10.1134/S0026261710060196.View ArticleGoogle Scholar
- Pernthaler A, Dekas AE, Brown CT, Goffredi SK, Embaye T, Orphan VJ: Diverse syntrophic partnerships from deep-sea methane vents revealed by direct cell capture and metagenomics. Proc Natl Acad Sci USA. 2008, 105 (19): 7052-7057. 10.1073/pnas.0711303105.PubMedPubMed CentralView ArticleGoogle Scholar
- Duffy M, Kinnaman F, Valentine DL, Keller E, Clark JF: Gaseous emission rates from natural petroleum seeps in the Upper Ojai Valley, California. Environmental Geosciences. 2007, 14 (4): 197-207. 10.1306/eg.07110707010.View ArticleGoogle Scholar
- Norwegian High-Throughput Sequencing Centre (NSC). [http://www.sequencing.uio.no/]
- Gomez-Alvarez V, Teal TK, Schmidt TM: Systematic artifacts in metagenomes from complex microbial communities. ISME J. 2009, 3: 1314-1317. 10.1038/ismej.2009.72.PubMedView ArticleGoogle Scholar
- 454 Replicate Filter. [http://microbiomes.msu.edu/replicates/]
- Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ: Basic local alignment search tool. J Mol Biol. 1990, 215 (3): 403-410.PubMedView ArticleGoogle Scholar
- Bioportal. [http://www.bioportal.uio.no]
- Moriya Y, Itoh M, Okuda S, Yoshizawa AC, Kanehisa M: KAAS: an automatic genome annotation and pathway reconstruction server. Nucleic Acids Res. 2007, 35: W182-W185. 10.1093/nar/gkm321.PubMedPubMed CentralView ArticleGoogle Scholar
- KAAS - KEGG Automatic Annotation Server. [http://www.genome.ad.jp/tools/kaas/]
- Kanehisa M, Goto S: KEGG: Kyoto Encyclopedia of Genes and Genomes. Nucleic Acids Res. 2000, 28 (1): 27-30. 10.1093/nar/28.1.27.PubMedPubMed CentralView ArticleGoogle Scholar
- Kanehisa M, Goto S, Furumichi M, Tanabe M, Hirakawa M: KEGG for representation and analysis of molecular networks involving diseases and drugs. Nucleic Acids Res. 2010, 38: D355-D360. 10.1093/nar/gkp896.PubMedPubMed CentralView ArticleGoogle Scholar
- Kanehisa M, Goto S, Hattori M, Aoki-Kinoshita KF, Itoh M, Kawashima S, Katayama T, Araki M, Hirakawa M: From genomics to chemical genomics: new developments in KEGG. Nucleic Acids Res. 2006, 34: D354-D357. 10.1093/nar/gkj102.PubMedPubMed CentralView ArticleGoogle Scholar
- KEGG: Kyoto Encyclopedia of Genes and Genomes. [http://www.genome.jp/kegg/]
- Functional gene pipeline & repository. [http://fungene.cme.msu.edu/index.spr]
- STRING - Known and Predicted Protein-Protein Interactions. [http://string-db.org/newstring_cgi/show_input_page.pl?UserId=Frnr4khlceg0&sessionId=t73cGlIGN8OV]
- Beszteri B, Temperton B, Frickenhaus S, Giovannoni SJ: Average genome size: a potential source of bias in comparative metagenomics. ISME J. 2010, 4 (8): 1075-1077. 10.1038/ismej.2010.29.PubMedView ArticleGoogle Scholar
- Murrell JC, Gilbert B, McDonald IR: Molecular biology and regulation of methane monooxygenase. Arch Microbiol. 2000, 173 (5-6): 325-332. 10.1007/s002030000158.PubMedView ArticleGoogle Scholar
- Klein M, Friedrich M, Roger AJ, Hugenholtz P, Fishbain S, Abicht H, Blackall LL, Stahl DA, Wagner M: Multiple lateral transfers of dissimilatory sulfite reductase genes between major lineages of sulfate-reducing prokaryotes. J Bacteriol. 2001, 183 (20): 6028-6035. 10.1128/JB.183.20.6028-6035.2001.PubMedPubMed CentralView ArticleGoogle Scholar
- Thauer RK: Biochemistry of methanogenesis: a tribute to Marjory Stephenson. Microbiology-Uk. 1998, 144: 2377-2406. 10.1099/00221287-144-9-2377.View ArticleGoogle Scholar
- Juottonen H: Archaea, Bacteria, and methane production along environmental gradients in fens and bogs. PhD thesis. 2008, University of HelsinkiGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.