Groups Of Methanogens Research Articles

Simultaneous Amplicon Sequencing to Explore Co-Occurrence Patterns of Bacterial, Archaeal and Eukaryotic Microorganisms in Rumen Microbial Communities

Ruminants rely on a complex rumen microbial community to convert dietary plant material to energy-yielding products. Here we developed a method to simultaneously analyze the community's bacterial and archaeal 16S rRNA genes, ciliate 18S rRNA genes and anaerobic fungal internal transcribed spacer 1 genes using 12 DNA samples derived from 11 different rumen samples from three host species (Ovis aries, Bos taurus, Cervus elephas) and multiplex 454 Titanium pyrosequencing. We show that the mixing ratio of the group-specific DNA templates before emulsion PCR is crucial to compensate for differences in amplicon length. This method, in contrast to using a non-specific universal primer pair, avoids sequencing non-targeted DNA, such as plant- or endophyte-derived rRNA genes, and allows increased or decreased levels of community structure resolution for each microbial group as needed. Communities analyzed with different primers always grouped by sample origin rather than by the primers used. However, primer choice had a greater impact on apparent archaeal community structure than on bacterial community structure, and biases for certain methanogen groups were detected. Co-occurrence analysis of microbial taxa from all three domains of life suggested strong within- and between-domain correlations between different groups of microorganisms within the rumen. The approach used to simultaneously characterize bacterial, archaeal and eukaryotic components of a microbiota should be applicable to other communities occupying diverse habitats.

Evolution of Na+ and H+ bioenergetics in methanogenic archaea

Methanogenic archaea live at the thermodynamic limit of life and use sophisticated mechanisms for ATP synthesis and energy coupling. The group of methanogens without cytochromes use an Na(+) current across the membrane for ATP synthesis, whereas the cytochrome-containing methanogens have additional coupling sites that also translocate protons. The ATP synthase in this group is promiscuous and uses Na(+) and H(+) simultaneously.

Methane production by treating vinasses from hydrous ethanol using a modified UASB reactor

BackgroundA modified laboratory-scale upflow anaerobic sludge blanket (UASB) reactor was used to obtain methane by treating hydrous ethanol vinasse. Vinasses or stillage are waste materials with high organic loads, and a complex composition resulting from the process of alcohol distillation. They must initially be treated with anaerobic processes due to their high organic loads. Vinasses can be considered multipurpose waste for energy recovery and once treated they can be used in agriculture without the risk of polluting soil, underground water or crops. In this sense, treatment of vinasse combines the elimination of organic waste with the formation of methane. Biogas is considered as a promising renewable energy source. The aim of this study was to determine the optimum organic loading rate for operating a modified UASB reactor to treat vinasse generated in the production of hydrous ethanol from sugar cane molasses.ResultsThe study showed that chemical oxygen demand (COD) removal efficiency was 69% at an optimum organic loading rate (OLR) of 17.05 kg COD/m3-day, achieving a methane yield of 0.263 m3/kg CODadded and a biogas methane content of 84%. During this stage, effluent characterization presented lower values than the vinasse, except for potassium, sulfide and ammonia nitrogen. On the other hand, primers used to amplify the 16S-rDNA genes for the domains Archaea and Bacteria showed the presence of microorganisms which favor methane production at the optimum organic loading rate.ConclusionsThe modified UASB reactor proposed in this study provided a successful treatment of the vinasse obtained from hydrous ethanol production.Methanogen groups (Methanobacteriales and Methanosarcinales) detected by PCR during operational optimum OLR of the modified UASB reactor, favored methane production.

Effect of oxytetracycline on biogas production and active microbial populations during batch anaerobic digestion of cow manure

The aim of this study was to investigate the effect of a common veterinary antibiotic in biogas plants. 20mg/kg of oxytetracycline was intramuscularly injected into a cow and its concentration in manure, which was sampled daily during the following 20days, was measured. A total of 20% of the injected oxytetracycline was detected in manure. Collected manure samples on days 1, 2, 3, 5, 10, 15, and 20 were digested in triplicate serum bottles at 37°C for 30days. Control serum bottles produced 255±13mL biogas, whereas 50-60% inhibitions were obtained for the serum bottles operated with samples collected for the 5days after medication. Multivariate statistics used for the evaluation of FISH results showed that Methanomicrobiales were the main methanogenic group responsible for most of the biogas production. Numbers of active Bacteria and Methanomicrobiales were negatively correlated with the presence of oxytetracycline, whereas Methanosarcinales and Methanobacteriales were less affected.

Structures of free-living and protozoa-associated methanogen communities in the bovine rumen differ according to comparative analysis of 16S rRNA and mcrA genes

Structures of free-living and protozoa-associated methanogen (PAM) communities from forage-fed cattle were investigated by comparative sequence analysis of 16S rRNA and methyl coenzyme M reductase (mcrA) gene clone libraries. The free-living and protozoa-associated communities were composed of the same three genera [namely Methanobrevibacter, Methanomicrobium and rumen cluster C (RCC), which is distantly related to Thermoplasma]; however, the distribution of the methanogen genera differed between the two communities. Despite previous reports of potential bias for the degenerate mcrA primer set, the 16S rRNA (n = 100 clones) and mcrA (n = 92 clones) gene libraries exhibited a similar distribution pattern for the three methanogenic genera. RCC was more abundant in the free-living community and represented 72 and 42 % of the 16S rRNA and mrcA gene sequences, respectively, versus 54 and 13 % of the 16S rRNA and mrcA gene sequences from the PAM community, respectively. The majority of RCC sequences from the free-living and protozoa-associated communities belonged to different species-level operational taxonomic units. Methanobrevibacter species were more abundant in the PAM community and represented 42 and 79 % of clones for the 16S rRNA and mrcA gene libraries, respectively, versus 9 and 27 % of 16S rRNA and mrcA gene clones from the free-living community, respectively. Methanomicrobium species were predominantly free-living. Primers for quantitative PCR were designed to target specific methanogen groups and used to assess the effect of a high-grain diet on methanogen species composition. Switching the ruminant diet from forage to high-grain resulted in reduced protozoal diversity, along with a profound overall reduction in the relative abundance of RCC and an increase in the relative abundance of free-living Methanobrevibacter spp. It was unclear whether the reduced abundance of RCC in grain-fed animals was due to the loss of symbiotic protozoa species or due to broader changes in the rumen environment that affected both RCC and protozoa. Importantly, results from this study emphasize the need to consider the different methanogen communities when developing strategies for mitigating methane emissions in ruminants.

Community structure of methanogenic archaea and methane production associated with compost-treated tropical rice-field soil

The diversity and density of methanogenic archaea and methane production were investigated ex situ at different growth stages of rice plant cultivated in compost-treated tropical rice fields. The qPCR analysis revealed variation in methanogens population from 3.40×10(6) to 1.11×10(7) copiesg(-1) dws, in the year 2009 and 4.37×10(6) to 1.36×10(7) copiesg(-1) dws in the year 2010. Apart from methanogens, a large number of bacterial (9.60×10(9) -1.44×10(10) copiesg(-1) dws) and archaeal (7.13×10(7) -3.02×10(8) copiesg(-1) dws) communities were also associated with methanogenesis. Methanogen population size varied in the order: flowering > ripening > tillering > postharvest > preplantation stage. The RFLP-based 16S rRNA gene-targeted phylogenetic analysis showed that clones were closely related to diverse group of methanogens comprising members of Methanomicrobiaceae, Methanosarcinaceae, Methanosaetaceae and RC I. Laboratory incubation studies revealed higher amount of cumulative CH(4) at the flowering stage. The integration of methanogenic community structure and CH(4) production potential of soil resulted in a better understanding of the dynamics of CH(4) production in organically treated rice-field soil. The hypothesis that the stages of plant development influence the methanogenic community structure leading to temporal variation in the CH(4) production has been successfully tested.

McrA and 16S rRNA gene analysis suggests a novel lineage of Archaea phylogenetically affiliated with Thermoplasmatales in human subgingival plaque

Based on the molecular analysis of human subgingival plaque samples from 30 periodontitis patients a novel lineage of Archaea within the phylogenetic radiation of Thermoplasmatales was identified in 10% of cases. Co-occurrence of unique 16S rRNA gene and mcrA gene sequences suggests that this lineage corresponds to a hitherto unknown group of methanogens.

Temporal variation in methanogenic community structure and methane production potential of tropical rice ecosystem

Temporal variations in diversity of methanogenic community and CH4 production potential were analyzed in an Indian tropical rice ecosystem. Laboratory incubations showed that methane production varied from 20.86 to 134.11 μg CH4 g−1 d.w.s. during the two consecutive years, 2009 and 2010. CH4 production potential was high at the flowering stage of the rice crop followed by ripening, tillering, post-harvest and pre-plantation stage. Phylogenetic analysis of 16S rRNA genes of methanogenic community indicated that flowering and ripening stages comprised of Methanomicrobiaceae, Methanosarcinaceae, Methanosaetaceae and RC I methanogenic groups, while only the members of Methanomicrobiaceae and RC I were present in the remaining stages. Further, the dominance of RC I was observed in all stages. This study demonstrates that flowering and ripening stages of rice crop offer relatively favorable ecological niche for methanogenic community. The overall analyses suggest that the temporal change in diversity of methanogens regulates CH4 production potential in rice field soils.

Seasonal Changes in Methanogenesis and Methanogenic Community in Three Peatlands, New York State

Fluctuating environmental conditions can promote diversity and control dominance in community composition. In addition to seasonal temperature and moisture changes, seasonal supply of metabolic substrates selects populations temporally. Here we demonstrate cascading effects in the supply of metabolic substrates on methanogenesis and community composition of anaerobic methanogenic archaea in three contrasting peatlands in upstate New York. Fresh samples of peat soils, collected about every 3 months for 20 months and incubated at 22 ± 2°C regardless of the in situ temperature, exhibited potential rates of methane (CH4) production of 0.02–0.2 mmol L−1 day−1 [380–3800 nmol g−1 (dry) day−1). The addition of acetate stimulated rates of CH4 production in a fen peatland soil, whereas addition of hydrogen (H2), and simultaneous inhibition of H2-consuming acetogenic bacteria with rifampicin, stimulated CH4 production in two acidic bog soils, especially, in autumn and winter. The methanogenic community structure was characterized using T-RFLP analyses of SSU rRNA genes. The E2 group of methanogens (Methanoregulaceae) dominated in the two acidic bog peatlands with relatively greater abundance in winter. In the fen peatland, the E1 group (Methanoregulaceae) and members of the Methanosaetaceae were co-dominant, with E1 having a high relative abundance in spring. Change in relative abundance profiles among methanogenic groups in response to added metabolic substrates was as predicted. The acetate-amendment increased abundance of Methanosarcinaceae, and H2-amendment enhanced abundance of E2 group in all peat soils studied, respectively. Additionally, addition of acetate increased abundance of Methanosaetaceae only in the bog soils. Variation in the supply of metabolic substrates helps explain the moderate diversity of methanogens in peatlands.

Molecular analysis of meso- and thermophilic microbiota associated with anaerobic biowaste degradation

BackgroundMicrobial anaerobic digestion (AD) is used as a waste treatment process to degrade complex organic compounds into methane. The archaeal and bacterial taxa involved in AD are well known, whereas composition of the fungal community in the process has been less studied. The present study aimed to reveal the composition of archaeal, bacterial and fungal communities in response to increasing organic loading in mesophilic and thermophilic AD processes by applying 454 amplicon sequencing technology. Furthermore, a DNA microarray method was evaluated in order to develop a tool for monitoring the microbiological status of AD.ResultsThe 454 sequencing showed that the diversity and number of bacterial taxa decreased with increasing organic load, while archaeal i.e. methanogenic taxa remained more constant. The number and diversity of fungal taxa increased during the process and varied less in composition with process temperature than bacterial and archaeal taxa, even though the fungal diversity increased with temperature as well. Evaluation of the microarray using AD sample DNA showed correlation of signal intensities with sequence read numbers of corresponding target groups. The sensitivity of the test was found to be about 1%.ConclusionsThe fungal community survives in anoxic conditions and grows with increasing organic loading, suggesting that Fungi may contribute to the digestion by metabolising organic nutrients for bacterial and methanogenic groups. The microarray proof of principle tests suggest that the method has the potential for semiquantitative detection of target microbial groups given that comprehensive sequence data is available for probe design.

Archaea in symbioses.

During the last few years, the analysis of microbial diversity in various habitats greatly increased our knowledge on the kingdom Archaea. At the same time, we became aware of the multiple ways in which Archaea may interact with each other and with organisms of other kingdoms. The large group of euryarchaeal methanogens and their methane oxidizing relatives, in particular, take part in essential steps of the global methane cycle. Both of these processes, which are in reverse to each other, are partially conducted in a symbiotic interaction with different partners, either ciliates and xylophagous animals or sulfate reducing bacteria. Other symbiotic interactions are mostly of unknown ecological significance but depend on highly specific mechanisms. This paper will give an overview on interactions between Archaea and other organisms and will point out the ecological relevance of these symbiotic processes, as long as these have been already recognized.

Diversity analysis of methanogens in rumen of Bubalus bubalis by 16S riboprinting and sequence analysis

The molecular diversity of rumen methanogens was investigated by 16S rDNA gene library prepared from the rumen contents obtained from Murrah buffaloes in India. Genomic DNA was isolated from adult male fistulated buffaloes and PCR conditions were set up using specific primers. Amplified product was cloned into a suitable vector, and the positive clones were selected assuming based on blue–white screening and sequenced. Positive clones were reamplified and the resulting PCR products were further subjected to Amplified Ribosomal DNA Restriction Analysis (ARDRA) by using HaeIII enzyme. A total of 108 clones were examined, and the analysis revealed 16 phylotypes. Out of sixteen phylotypes, nine phylotypes belong to the uncultured group of methanogens, and the rest of seven phylotypes belong to the order Methanomicrobiales, Methanococcales and Methanobacteriales. Out of the 108 rDNA clones, 66 clones which constitute 61.1% of the total clone representing 9 phylotypes, show less than 97% sequence similarity with any of the cultured strain of methanogens. The second largest group of clones (24 clones) represented by four phylotypes show a sequence similarity ranging from 91% to 99% with Methanomicrobium mobile strain of methanogens. The third group of 16S rDNA clones clustered along with M. burtonii strain of methanogens. This group consists of 6 clones and constitutes about 5.5% of the total clones and represented by only single phylotype. Fourth and fifth clusters of 16S rDNA clones consist of 5 and 7 clones respectively, and these were matched with Methanobrevibacter gottschalkii and Methanobrevibacter rumanatium strain of methanogens and constitute about 4.6% and 6.4% of the total clones.

A novel fingerprint method to assess the diversity of methanogens in microbial systems

Understanding the ecology of methanogens in natural and engineered environments is a prerequisite to predicting or managing methane emissions. In this study, a novel high-throughput fingerprint method was developed for determining methanogen diversity and relative abundance within environmental samples. The method described here, designated amplicon length heterogeneity PCR of the mcrA gene (LH-mcrA), is based on the natural length variation in the mcrA gene. The mcrA gene encodes the alpha-subunit of the methyl-coenzyme M reductase, which is involved in the terminal step of methane production by methanogens. The methanogenic communities from stored swine and dairy manures were distinct from each other. To validate the method, methanogenic communities in a plug flow-type bioreactor (PFBR) treating swine manure were characterized using LH-mcrA method and correlated to mcrA gene clone libraries. The diversity and relative abundance of the methanogenic groups were assessed. Methanobrevibacter, Methanosarcinaceae, Methanoculleus, Methanogenium, Methanocorpusculum and one unidentified group were assigned to particular LH-mcrA amplicons. Particular phylotypes related to Methanoculleus were predominant in the last compartment of the PFBR where the bulk of methane was produced. LH-mcrA method was found to be a reliable, fast and cost-effective alternative for diversity assessment of methanogenic communities in microbial systems.

Methyl coenzyme M reductase (mcrA) gene based phylogenetic analysis of methanogens population in Murrah buffaloes (Bubalus bubalis)

The aim of the present study was to decipher the diversity of methanogens in rumen of Murrah buffaloes so that effective strategies can be made in order to mitigate methane emission from these methanogens. In the present study diversity of rumen methanogens in Murrah buffaloes (Bubalus bubalis) from North India was evaluated by using mcr-A gene library obtained from the pooled PCR product from four animals and by using MEGA4 software. A total of 104 clones were examined, revealing 26 different mcr-A gene sequences or phylotypes. Of the 26 phylotypes, 16 (64 of 104 clones) were less than 97% similar to any of the cultured strain of methanogens. Seven clone sequences were clustered with Methanomicrobium mobile and three clone sequences were clustered with Methanobrevibacter gottschalkii during the phylogenetic analysis. Uncultured group of methanogens comes out to be the major component of the methanogens community structure in Murrah buffaloes. Methanomicrobium phylotype comes out to be major phylotype among cultured methanogens followed by Methanobrevibacter phylotype. These results help in making effective strategies to check the growth of dominant communities in the rumen of this animal which in turn help in the reduction of methane emission in the environment and ultimately helps us in fighting with the problem of global warming.

Exploring rumen methanogen genomes to identify targets for methane mitigation strategies

Methane emissions from ruminant livestock is generated by the action of methanogenic archaea, mainly in the rumen. A variety of methanogen genera are responsible for CH4 production, including a large group that lacks cultivated representatives. To be generally effective, technologies for reducing ruminant CH4 emissions must target all rumen methanogens to prevent any unaffected methanogen from expanding to occupy the vacated niche. Interventions must also be specific for methanogens so that other rumen microbes can continue normal digestive functions. Thus a detailed knowledge of the diversity and physiology of rumen methanogens is required to define conserved features that can be targeted for methanogen inhibition. Genome sequencing projects are underway in New Zealand and Australia on several ruminal methanogen groups, including representatives of the genera Methanobrevibacter, Methanobacterium, Methanosphaera, Methanosarcina, and the uncultured group, Rumen Cluster C. The completed Methanobrevibacter ruminantium genome and draft sequences from other rumen methanogen species are beginning to allow identification of underlying cellular processes that define these organisms, and is leading to a better understanding of their lifestyles within the rumen. Although the research is mainly at the explorative stage, two types of opportunities for inhibiting methanogens are emerging, being inactivation of conserved methanogen enzymes by screening for, or designing, small inhibitors via a chemogenomics approach, and identifying surface proteins shared among rumen methanogens that can be used as antigens in an anti-methanogen vaccine. Many of the conserved enzyme targets are involved in energy generation via the methanogenesis pathway, while the majority of the conserved surface protein targets are of unknown function. An understanding of the expression and accessibility of these targets within methanogen cells and/or microbial biofilms under ruminal conditions will be required for their development as CH4 production mitigations.This paper is part of the special issue entitled: Greenhouse Gases in Animal Agriculture – Finding a Balance Between Food and Emissions, Guest Edited by T.A. McAllister, Section Guest Editors: K.A. Beauchemin, X. Hao, S. McGinn and Editor for Animal Feed Science and Technology, P.H. Robinson.

Variations in methanogenic population structure under overloading of pre-acidified high-strength organic wastewaters

Methanogenic populations were quantitatively investigated in two separate anaerobic reactors, which received effluents from acidogenesis of either food waste-recycling wastewater or swine wastewater. Each set of the anaerobic digesters was operated under high organic loading rate (OLR) conditions (8.3–39.8 g chemical oxygen demand (COD)/L/d) with four different hydraulic retention times (10.4–3.0 days). Real-time PCR analysis demonstrated that the order Methanosarcinales was the most abundant (>55%) methanogenic group in the six runs with >50% COD removal, while the dominance (>70%) of Methanobacteriales was observed in the other two runs (<20% COD removal). Methanomicrobiales constituted a minor (<30%) population in all eight runs. The two reactors with relatively low performance were the only trials which contained less than 5 × 10 7 copies/mL of 16S rRNA gene of Methanosarcinales. These results imply that a minimum level of Methanosarcinales is important for achieving even moderate levels of process efficiency under high OLR.

Molecular identification of methanogenic archaea from surti buffaloes (bubalus bubalis), reveals more hydrogenotrophic methanogens phylotypes.

Methane emissions from ruminant livestock are considered to be one of the more potent forms of greenhouses gases contributing to global warming. Many strategies to reduce emissions are targeting the methanogens that inhabit the rumen, but such an approach can only be successful if it targets all the major groups of ruminant methanogens. Therefore, a thorough knowledge of the diversity of these microbes in breeds of buffaloes, as well as in response to geographical location and different diets, is required. Therefore, molecular diversity of rumen methanogens in Surti buffaloes was investigated using 16S rRNA gene libraries prepared from pooled rumen contents from three Surti buffaloes. A total of 171 clones were identified revealing 23 different sequences (phylotypes). Of these 23 sequences, twelve sequences (12 OTUs, 83 clones) and 10 sequences (10 OTUs, 83 clones) were similar to methanogens belonging to the orders Methanomicrobiales and Methanobacteriales, and the remaining 1 phylotype (5 clones) were similar to Methanosarcina barkeri. These unique sequences clustered within a distinct and strongly supported phylogenetic group. Further studies and effective strategies can be made to inhibit the growth of Methanomicrobiales and Methanobacteriales phylotypes to reduce the methane emission from rumen and thus help in preventing global warming.

The role of different methanogen groups evaluated by Real-Time qPCR as high-efficiency bioindicators of wet anaerobic co-digestion of organic waste

Methanogen populations and their domains are poorly understood; however, in recent years, research on this topic has emerged. The relevance of this field has also been enhanced by the growing economic interest in methanogen skills, particularly the production of methane from organic substrates. Management attention turned to anaerobic wastes digestion because the volume and environmental impact reductions. Methanogenesis is the biochemically limiting step of the process and the industrially interesting phase because it connects to the amount of biogas production. For this reason, several studies have evaluated the structure of methanogen communities during this process. Currently, it is clear that the methanogen load and diversity depend on the feeding characteristics and the process conditions, but not much data is available. In this study, we apply a Real-Time Polymerase Chain Reaction (RT-PCR) method based on mcrA target to evaluate, by specific probes, some subgroups of methanogens during the mesophilic anaerobic digestion process fed wastewater sludge and organic fraction of the municipal solid waste with two different pre-treatments. The obtained data showed the prevalence of Methanomicrobiales and significantly positive correlation between Methanosarcina and Methanosaetae and the biogas production rate (0.744 p < 0.01 and 0.641 p < 0.05). Methanosarcina detected levels are different during the process after the two pre-treatment of the input materials (T-test p < 0.05). Moreover, a role as diagnostic tool could be suggested in digestion optimisation.

Quantitative and qualitative analyses of methanogenic community development in high-rate anaerobic bioreactors

Methanogenic community structure and population dynamics were investigated in two anaerobic reactors treating a dairy wastewater, an Inverted Fluidized Bed (IFB) and Expanded Granular Sludge Bed (EGSB). A combination of real-time PCR, denaturing gradient gel electrophoresis and statistical techniques was employed. Distinct methanogenic communities developed in the IFB and EGSB reactors reflecting step-wise reductions in the applied hydraulic retention time from 72 to 12 h during the 200-day trial. The aceticlastic family Methanosarcinaceae was only detected in the IFB and the order Methanomicrobiales was also much more abundant in this reactor, while the aceticlastic family Methanosaetaceae was more abundant in the EGSB. The hydrogenotrophic order, Methanobacteriales, predominated in both reactors under all applied operational conditions. Non-metric multidimensional scaling (NMS) and moving-window analyses, based on absolute and relative abundance quantification data, demonstrated that the methanogenic communities developed in a different manner in the IFB, compared to the EGSB reactor. In our study, relative abundance-based quantification by NMS and moving-window analysis appeared to be a valuable molecular approach that was more applicable to reflect the changes in the anaerobic digestion process than approaches based either on qualitative analysis, or solely on absolute quantification of the various methanogenic groups. The overall results and findings provided a comparative, quantitative and qualitative insight into anaerobic digestion processes, which could be helpful for better future reactor design and process control.

Revealing archaeal diversity patterns and methane fluxes in Admiralty Bay, King George Island, and their association to Brazilian Antarctic Station activities

The study of Antarctic archaeal communities adds information on the biogeography of this group and helps understanding the dynamics of biogenic methane production in such extreme habitats. Molecular methods were combined to methane flux determinations in Martel Inlet, Admiralty Bay, to assess archaeal diversity, to obtain information about contribution of the area to atmospheric methane budget and to detect possible interferences of the Antarctic Brazilian Station Comandante Ferraz (EACF) wastewater discharge on local archaeal communities and methane emissions. Methane fluxes in Martel Inlet ranged from 3.2 to 117.9 μmol CH 4 m −2 d −1, with an average of 51.3±8.5 μmol CH 4 m −2 d −1 and a median of 57.6 μmol CH 4 m −2d −1. However, three negative fluxes averaging −11.3 μmol CH 4 m −2 d −1 were detected in MacKellar Inlet, indicating that Admiralty Bay can be either a source or sink of atmospheric methane. Denaturing gradient gel electrophoresis (DGGE) showed that archaeal communities at EACF varied with depth and formed a group separated from the reference sites. Granulometric analysis indicated that differences observed may be mostly related to sediment type. However, an influence of wastewater input could not be discarded, since higher methane fluxes were found at CF site, suggesting stimulation of local methanogenesis. DGGE profile of the wastewater sample grouped separated from all other samples, suggesting that methanogenesis stimulation may be due to changes in environmental conditions rather than to the input of allochtonous species from the wastewater. 16S ribosomal DNA clone libraries analysis showed that all wastewater sequences were related to known methanogenic groups belonging to the hydrogenotrophic genera Methanobacterium and Methanobrevibacter and the aceticlastic genus Methanosaeta. EACF and Botany Point sediment clone libraries retrieved only groups of uncultivated Archaea, with predominance of Crenarchaeota representatives (MCG, MG1, MBG-B, MBG-C and MHVG groups). Euryarchaeota sequences found were mostly related to the LDS and RC-V groups, but MBG-D and DHVE-5 were also present. No representatives of cultivated methanogenic groups were found, but coverage estimates suggest that a higher number of clones would have to be analyzed in order to cover the greater archaeal diversity of Martel Inlet sediment. Nevertheless, the analysis of the libraries revealed groups not commonly found by other authors in Antarctic habitats and also indicated the presence of groups of uncultivated archaea previously associated to methane rich environments or to the methane cycle.