Diversity Of Methanogenic Archaea Research Articles

Impact of monoterpenes in the stability of the anaerobic digestion of Mediterranean Wholesale Market Waste

The stability and robustness of long-term anaerobic digestion process of fruit and vegetable waste (FVW) generated in Mediterranean wholesale markets were assessed, monitoring potential inhibitory volatile organic compounds (VOCs) such as volatile fatty acids (VFAs) and/or monoterpenes. Particle size (4 and 10 mm) and organic loading rate (OLR) were evaluated during the experimentation. Anaerobic digestion remained stable at OLR of 1.0 g VS L−1 d−1 (VS, total volatile solids) regardless of the feedstock particle size. At this OLR, methane yield was 254 ± 8 and 229 ± 11 mL CH4 g VS−1 for 4 and 10 mm, respectively. As the OLR increased above 1.0 g VS L−1 d−1, the system rapidly destabilized, and a high accumulation of VFAs was observed. In parallel of to the VFAs accumulation, other VOCs such as fenchone also reached concentrations that contributes to the destabilisation of the process. The destabilization process at an OLR of 3.0 g VS L−1 d−1 also resulted in a drastic reduction in the relative abundance and species diversity of methanogenic archaea in all digesters.

Bacterial species in the ruminal content of steers fed oilseeds in the diet.

The aim of this study was to evaluate bacterial species and diversity of methanogenic Archaea in the solid fraction of the ruminal content, through the gene sequences of the conserved 16S rDNA region, in response to the following diets: canola, cottonseed, sunflower, soybean, corn silage, and control diet. Six rumen-fistulated crossbred steers, with body weight (BW) of 416.33 ± 93.30kg, were distributed in a 6 × 6 Latin square design. Regardless of the diet provided, amylolytic, proteolytic, and lactic bacteria were identified in the rumen fluid. Cellulolytic bacteria were predominant for all diets, reaching 47.75% of operational taxonomic units (OTUs) in animals fed with the cottonseed diet. Amylolytic bacteria reach 62.51% of OTU in animal fed sunflower diet, while proteolytic bacteria correspond to 65.96% of OTU in this same diet. Also, Megasphaera elsdenii bacterium was identified for all diets, with a greater percentage of OTU in steers fed the cottonseed diet. The diversity analysis of the species identified the methanogenic Archaea Methanobrevibacter ruminantium in all diets. We conclude that the control and corn silage diets have the most similar bacterial flora; diets with oilseeds had 47.5% similarity in rumen flora bacteria species. Animals fed with soybean showed a reduced number of methanogenic Archaea in the rumen content, which could be an alternative feed for cattle due to their low potential for energy losses with the production of methane.

Archaeal community dynamics in biogas fermentation at various temperatures assessed by mcrA amplicon sequencing using different primer pairs.

In this study, the taxonomic and functional diversity of methanogenic archaea in two parallel 120l fermenters operated at different temperatures and fed with maize silage was estimated by mcrA metabarcoding analysis using two typical primer pairs (ML and MLA) amplifying part of the functional methyl coenzyme M reductase (mcrA) gene. The alpha diversity indices showed that the ML primer pair detected a higher Operational Taxonomic Unit (OTU) abundance compared to the MLA primer pair and methanogen diversity was significantly lower in the 60°C fermenters. The beta diversity analysis showed the methanogenic community clustered together at 50°C and 40° and was statistically different from the 60°C community. Similar, to alpha diversity, beta diversity was also significantly different between primer pairs. At all temperatures analysed, the primer pairs showed a different abundance of the different methanogenic OTUs, e.g. more OTUs relative to Methanoculleus sp. with the ML primer pair, and more OTUs corresponding to Methanobacterium sp. with the MLA primer pair. Moreover, OTUs corresponding to Methanosphaera sp. and Methanobrevibacter sp. were found only by using ML primer pair, while the MLA primer pair detected sequences corresponding to Methanothrix sp.

Genetic diversity of 16S rRNA and mcrA genes from methanogenic archaeas

Methanogenic archaeas are found in aquatic and terrestrial environments and are fundamental in the conversion of organic matter into methane, a gas that has a potential use as renewable source of energy, which is also considered as one of the main agents of the greenhouse effect. The vast majority of microbial genomes can be identified by a conservative molecular marker, the 16S ribosomal gene. However, the mcrA gene have been using in studies of methanogenic archaea diversity as an alternative marker, highly conserved and present only in methanogens. This gene allows the expression of the enzyme Methyl-coenzyme M reductase, the main agent in converting by-products of anaerobic digestion into methane. In this context, we aimed to study the genetic diversity of mcrA and 16S rRNA genes sequences available in databases. The nucleotide sequences were selected from the NCBI. The heterozygosity and molecular diversity indexes were calculated using the Arlequin 3.5 software, with plots generated by package R v3.0. The diversity and heterozygosity indices for both genes may have been influenced by the number and size of the sequences. Descriptive analysis of genetic diversity generated by sequences deposited in databases allowed a detailed study of these molecules. It is known that the organisms in a population are genetically distinct, and that, despite having similarities in their gene composition, the differences are essential for their adaptation to different environments.

Methane Production and Methanogens Diversity in in vitro Ruminal Fermentation with Mahogany Leaves Meal (Swietenia mahagoni) as Tannin Source

Swietenia mahagoni has been extensively studied and applied as feed additives, a strategy to manipulate rumen fermentation in reducing methanogenesis. The research aimed to determine the effect of using Swietenia mahagoni as tannin source on methane production, gas production kinetics and the diversity of methanogenic archaea by in vitro rumen fermentation. Swietenia mahagoni was given to substitute the elephant grass at levels of 0, 25, and 50%. Fermentation was carried out by in vitro gas production technique according to Menke and Steingass. Methane was analyzed using gas chromatography (GC) methode. Terminal restriction fragment length polymorphism (TRFLP) analysis was used to detect changes of rumen methanogenic archaea diversity. Result showed that Swietenia mahagoni leaves with level 50% reduced methane production by 39,26% (P=0,08). Gas production from insoluble fraction (b) decreased as much as 47.23% from control with using Swietenia mahagoni leaves 50%. The uncultured methanoculles was dominating community member 36.35% with MspI, while uncultured euryarchaeote clone is 80.24% MCR-F1SP-1 with TaqI. Inclusion of Swietenia mahagoni as much as 50% in diet could decrease methane production, gas production from potential degraded fractions and diversity of methanogenic archaea.

Effect of Cinnamon Bark Meal (Cinnamomun burmanni Ness ex Bl) on In Vitro Methane Production and Rumen Methanogens Diversity

Plant secondary metabolites such as cinnamaldehyde have been used to manipulate methane production in the rumen. The research aimed to investigate the effect of cinnamon bark meal as cinnamaldehyde source on methane production and methanogen diversity in the rumen. Substrate of fermentation consist of Pennisetum purpureum, wheat pollard and soybean meal. Cinnamon bark meal was added into feed sample equal to cinnamaldehyde level as much as 200, 400, 600, 800 mg based on dry matter (DM). Parameters recorded were dry matter digestibility (DMD), organic matter digestibility (OMD), methane production, number of protozoa and communities of methanogen. Fermentation parameters were measured by incubating the sample in a rumen liquor buffer that was taken from a rumen fistulated Bali cattle. In vitro gas production was analyzed using Menke and Steingass gas production technique. Methanogenic communities were observed using Terminal Restriction Fragment Length Polymorphism (T-RFLP) technique. The bacterial diversity (H’ index) and evenness (E index) were calculated from the peak value. Results showed that there was no significant (P>0.05) different among treatments on IVDMD, IVOMD, methane production and number of protozoa. Based on T-RFLP, methanogenic archaea diversity was not affected by the diet. This study shows that addition of cinnamon bark meal as cinnamaldehyde source may not viable CH4 mitigation strategies to reduce methanogenic activity in the rumen.

Effectiveness of enriched microbial culture under mesophilic and thermophilic condition in palm oil mill effluent for anaerobic digestion of Brachiaria mutica

In this study, we investigated the effectiveness of microbial enrichment in enhancing the anaerobic digestion of lignocellulosic biomass of anaerobically digested sewage sludge in a complex substrate under both mesophilic (305 K) and thermophilic (328 K) conditions. Palm oil mill effluent (POME) was used as the enrichment culture, and Brachiaria mutica (paragrass) as a representative of a lignocellulosic biomass with relatively low lignin content (ca. 10 wt%). The microbial enrichment was found to increase the hydrolytic activities of the sludge more than twofold, and to increase the bacterial diversity, especially under the mesophilic condition. The microbial enrichment was also found to increase the diversity of methanogenic archaea and the methanogenic activities of the sludge. When compared with the original sludge, the enriched microbial culture under the mesophilic condition increased the methane yield from 275 to 312 cm3 STP/g VS added. However, despite increased hydrolytic, acetogenic and methanogenic activities, the enriched microbial culture under the thermophilic condition failed to enhance methane production in a long-term anaerobic digestion of paragrass.

Effects of particle size of ground alfalfa hay on caecal bacteria and archaea populations of rabbits.

This work was aimed to investigate the effects of the different particle size of ground alfalfa hay on caecal microbial and archeal communities of rabbits. One hundred-twenty New Zealand rabbits (950.3 ± 8.82 g) were allocated into four treatments, with five replicates in each treatment and six rabbits in each replicate. The particle sizes of the alfalfa meal in the four treatment diets were 2,500, 1,000, 100 and 10 µm respectively, while the other ingredients were ground through a 2.5 mm sieve. High-throughput sequencing technology was applied to examine the differences in bacteria and methanogenic archaea diversity in the caecum of the four treatment groups of rabbits. A total of 745,946 bacterial sequences (a mean of 31,081 ± 13,901 sequences per sample) and 539,227 archaeal sequences (a mean of 22,468 ± 2,443 sequences per sample) were recovered from twenty-four caecal samples, and were clustered into 9,953 and 2,246 OTUs respectively. A total of 26 bacterial phyla with 465 genera and three archaeal phyla with 10 genera were identified after taxonomic summarization. Bioinformatic analyses illustrated that Firmicutes (58.69% ∼ 68.50%) and Bacteroidetes (23.96% ∼ 36.05%) were the two most predominant bacterial phyla and Euryarchaeota (over 99.9%) was the most predominant archaeal phyla in the caecum of all rabbits. At genus level, as the particle size of alfalfa decreased from 2,500 to 10 µm, the relative abundances of Ruminococcaceae UCG-014 (P < 0.001) and Lactobacillus (P = 0.043) were increased and Ruminococcaceae UCG-005 (P = 0.012) was increased first and then decreased when the alfalfa particle size decreased, while Lachnospiraceae NK4A136 group (P = 0.016), Ruminococcaceae NK4A214 (P = 0.044), Christensenellaceae R-7 group (P = 0.019), Lachnospiraceae other (Family) (P = 0.011) and Ruminococcaceae UCG-013 (P = 0.021) were decreased. The relative abundance of Methanobrevibacter was increased from 62.48% to 90.40% (P < 0.001), whereas the relative abundance of Methanosphaera was reduced from 35.47% to 8.62% (P < 0.001). In conclusion, as the particle size of alfalfa meal decreased, both the bacterial and archaeal population in the caecum of rabbit experienced alterations, however archaea response earlier than bacteria to the decrease of alfalfa meal particle size.

Metabolic Adaptation of Methanogens in Anaerobic Digesters Upon Trace Element Limitation.

Anaerobic digestion (AD) is a complex multi-stage process relying on the activity of highly diverse microbial communities including hydrolytic, acidogenic and syntrophic acetogenic bacteria as well as methanogenic archaea. The lower diversity of methanogenic archaea compared to the bacterial groups involved in AD and the corresponding lack of functional redundancy cause a stronger susceptibility of methanogenesis to unfavorable process conditions such as trace element (TE) deprivation, thus controlling the stability of the overall process. Here, we investigated the effects of a slowly increasing TE deficit on the methanogenic community function in a semi-continuous biogas process. The aim of the study was to understand how methanogens in digester communities cope with TE limitation and sustain their growth and metabolic activity. Two lab-scale biogas reactors fed with distillers grains and supplemented with TEs were operated in parallel for 76 weeks before one of the reactors was subjected to TE deprivation, leading to a decline of cobalt and molybdenum concentrations from 0.9 to 0.2 mg/L, nickel concentrations from 2.9 to 0.8 mg/L, manganese concentrations from 38 to 18 mg/L, and tungsten concentrations from 1.4 to 0.2 mg/L. Amplicon sequencing of mcrA genes revealed Methanosarcina (72%) and Methanoculleus (23%) as the predominant methanogens in the undisturbed reactors. With increasing TE limitation, the relative abundance of Methanosarcina dropped to 67% and a slight decrease of acetoclastic methanogenic activity was observed in batch tests with 13C-methyl-labeled acetate, suggesting a shift toward syntrophic acetate oxidation coupled to hydrogenotrophic methanogenesis. Metaproteome analysis revealed abundance shifts of the enzymes involved in methanogenic pathways. Proteins involved in methylotrophic and acetoclastic methanogenesis decreased in abundance while formylmethanofuran dehydrogenase from Methanosarcinaceae increased, confirming our hypothesis of a shift from acetoclastic to hydrogenotrophic methanogenesis by Methanosarcina. Both Methanosarcina and Methanoculleus increased the abundance of N5-methyltetrahydromethanopterin-coenzyme M methyltransferase and methyl-coenzyme M reductase. However, these efforts to preserve the ion motive force for energy conservation were seemingly more successful in Methanoculleus. We conclude that both methanogenic genera use different strategies to stabilize their energy balance under TE limitation. Methanosarcina switched from TE expensive pathways (methylotrophic and acetoclastic methanogenesis) to hydrogenotrophic methanogenesis. Methanoculleus showed a higher robustness and was favored over the more fastidious Methanosarcina, thus stabilizing reactor performance under TE limitation.

Diversity of methanogenic archaea in freshwater sediments of lacustrine ecosystems.

About half of the global methane (CH4 ) emission is contributed by the methanogenic archaeal communities leading to a significant increase in global warming. This unprecedented situation has increased the ever growing necessity of evaluating the control measures for limiting CH4 emission to the atmosphere. Unfortunately, research endeavors on the diversity and functional interactions of methanogens are not extensive till date. We anticipate that the study of the diversity of methanogenic community is paramount for understanding the metabolic processes in freshwater lake ecosystems. Although there are several disadvantages of conventional culture-based methods for determining the diversity of methanogenic archaeal communities, in order to understand their ecological roles in natural environments it is required to culture the microbes. Recently different molecular techniques have been developed for determining the structure of methanogenic archaeal communities thriving in freshwater lake ecosystem. The two gene based cloning techniques required for this purpose are 16S rRNA and methyl coenzyme M reductase (mcrA) in addition to the recently developed metagenomics approaches and high throughput next generation sequencing efforts. This review discusses the various methods of culture-dependent and -independent measures of determining the diversity of methanogen communities in lake sediments in lieu of the different molecular approaches and inter-relationships of diversity of methanogenic archaea.

Is the methanogenic community reflecting the methane emissions of river sediments?-comparison of two study sites.

Studies on methanogenesis from freshwater sediments have so far primarily focused on lake sediments. To expand our knowledge on the community composition of methanogenic archaea in river sediments, we studied the abundance and diversity of methanogenic archaea at two localities along a vertical profile (top 50 cm) obtained from sediment samples from Sitka stream (the Czech Republic). In this study, we compare two sites which previously have been shown to have a 10‐fold different methane emission. Archaeal and methanogen abundance were analyzed by real‐time PCR and T‐RFLP. Our results show that the absolute numbers for the methanogenic community (qPCR) are relatively stable along a vertical profile as well as for both study sites. This was also true for the archaeal community and for the three major methanogenic orders in our samples (Methanosarcinales, Methanomicrobiales, and Methanobacteriales). However, the underlying community structure (T‐RFLP) reveals different community compositions of the methanogens for both locations as well as for different depth layers and over different sampling times. In general, our data confirm that Methanosarcinales together with Methanomicrobiales are the two dominant methanogenic orders in river sediments, while members of Methanobacteriales contribute a smaller community and Methanocellales are only rarely present in this sediment. Our results show that the previously observed 10‐fold difference in methane emission of the two sites could not be explained by molecular methods alone.

Effects of dietary supplementation of active dried yeast on fecal methanogenic archaea diversity in dairy cows

This study aimed to investigate the effects of dietary supplementation of different dosages of active dried yeast (ADY) on the fecal methanogenic archaea community of dairy cattle. Twelve multiparous, healthy, mid-lactating Holstein dairy cows (body weight: 584 ± 23.2 kg, milk produced: 26.3 ± 1.22 kg/d) were randomly assigned to one of three treatments (control, ADY2, and ADY4) according to body weight with four replicates per treatment. Cows in the control group were fed conventional rations without ADY supplementation, while cows in the ADY2 and ADY4 group were fed rations supplemented with ADY at 2 or 4 g/d/head. Real-time PCR analysis showed the populations of total methanogens in the feces were significantly decreased (P < 0.05) in the ADY4 group compared with control. High-throughput sequencing technology was applied to examine the differences in methanogenic archaea diversity in the feces of the three treatment groups. A total of 155,609 sequences were recovered (a mean of 12,967 sequences per sample) from the twelve fecal samples, which consisted of a number of operational taxonomic units (OTUs) ranging from 1451 to 1,733, were assigned to two phyla, four classes, five orders, five families and six genera. Bioinformatic analyses illustrated that the natural fecal archaeal community of the control group was predominated by Methanobrevibacter (86.9% of the total sequence reads) and Methanocorpusculum (10.4%), while the relative abundance of the remaining four genera were below 1% with Methanosphaera comprising 0.8%, Thermoplasma composing 0.4%, and the relative abundance of Candidatus Nitrososphaera and Halalkalicoccus being close to zero. At the genus level, the relative abundances of Methanocorpusculum and Thermoplasma were increased (P < 0.05) with increasing dosage of ADY. Conversely, the predominant methanogen genus Methanobrevibacter was decreased with ADY dosage (P < 0.05). Dietary supplementation of ADY had no significant effect (P > 0.05) on the abundances of genera unclassified, Candidatus Nitrososphaera, and Halalkalicoccus. In conclusion, supplementation of ADY to the rations of dairy cattle could alter the population sizes and composition of fecal methanogenic archaea in the feces of dairy cattle. The decrease in Methanobrevibacter happened with a commensurate increase in the genera Methanocorpusculum and Thermoplasma.

Archaeal communities of Arctic methane-containing permafrost

In the present study, we used culture-independent methods to investigate the diversity of methanogenic archaea and their distribution in five permafrost samples collected from a borehole in the Kolyma River Lowland (north-east of Russia). Total DNA was extracted from methane-containing permafrost samples of different age and amplified by PCR. The resulting DNA fragments were cloned. Phylogenetic analysis of the sequences showed the presence of archaea in all studied samples; 60%-95% of sequences belonged to the Euryarchaeota. Methanogenic archaea were novel representatives of Methanosarcinales, Methanomicrobiales, Methanobacteriales and Methanocellales orders. Bathyarchaeota (Miscellaneous Crenarchaeota Group) representatives were found among nonmethanogenic archaea in all the samples studied. The Thaumarchaeota representatives were not found in the upper sample, whereas Woesearchaeota (formerly DHVEG-6) were found in the three deepest samples. Unexpectedly, the greatest diversity of archaea was observed at a depth of 22.3 m, probably due to the availability of the labile organic carbon and/or due to the migration of the microbial cells during the freezing front towards the bottom.

Diversity of methanogenic archaea from the 2012 terrestrial hot spring (Valley of Geysers, Kamchatka)

Archaeal diversity in the 2012 terrestrial hot spring (Valley of Geysers, Kronotsky Nature Reserve, Kamchatka, Russia) was investigated using molecular and cultivation-based approaches. Analysis of the 16S rRNA gene sequences revealed predominance among archaea of uncultured microorganisms of the pSL12 and THSCG clusters. Analysis of the mcrA genes revealed that members of the order Methanomassiliicoccales were predominant (68%) among methanogens; the latter constituted 0.15% of the total number of archaea. Five stable thermophilic methanogenic associations utilizing hydrogen, formate, acetate, or methanol as substrates were obtained from the sediments of spring 2012. The diversity of cultured methanogens was limited to members of the genera Methanothermobacter, Methanothrix, and Methanomethylovorans. The association growing at 65°C and producing methane from methanol contained two components, which probably formed a syntrophic relationship: a Methanothermobacter methanogenic archaeon and a bacterium representing an separate cluster within the Firmicutes phylum, which was phylogenetically related to the genera Thermacetogenium and Syntrophaceticus. These data indicate high diversity of methanogens, notwithstanding their low abundance among archaea. The group of thermophilic Methanomassiliicoccales, which predominated among methanogens, is of special interest.

Analysis of the rumen bacteria and methanogenic archaea of yak (Bos grunniens) steers grazing on the Qinghai-Tibetan Plateau

Yak is an important domesticated ruminant on the Qinghai-Tibet Plateau in China. The prokaryotic community of yak remains largely uncharacterized when compared to that of other livestock species. In the present study, high-throughput sequencing of 16S rRNA genes (targeting bacterial and archaeal) and clone library of mcrA gene (targeting methanogenic archaea) were applied to investigate the rumen prokaryotic community structure. High-throughput sequencing results indicated that the rumen prokaryotic community consisted of 29 phyla, 40 classes, 63 orders, 77 families, and 79 genera. Bacteroidetes (59.1%) was the most abundant phylum, followed by Firmicutes, Proteobacteria, Fibrobacteres and Euryarchaeota. Prevotella was the predominant genus, averaging 28.5% of all rumen prokaryotic genera. Archaea accounted for 2.26% of the total prokaryotic community, with their community dominated by Methanobacteriaceae (82%), followed by Methanomassiliicoccaceae, and Methanosarcinaceae. Compared with the clone library of mcrA gene, high-throughput sequencing of 16S rRNA genes yielded a greater coverage of methanogenic archaea diversity. However, both molecular techniques showed that Methanobrevibacter is the predominant archaea of rumen microbiota in yaks grazing natural pastures. Our results should facilitate understanding of the complex rumen ecosystem and the main process of ruminal methanogenesis, which may help to further mitigate CH4 emissions from ruminants.

Effect of domestication on microorganism diversity and anaerobic digestion of food waste.

To accomplish the rapid start-up and stable operation of biogas digesters, an efficient inoculum is required. To obtain such an inoculum for food waste anaerobic digestion, we domesticated dairy manure anaerobic digestion residue by adding food waste every day. After 36 days, the pH and biogas yield stabilized signifying the completion of domestication. During domestication, the microbial communities in the inocula were investigated by constructing 16S rDNA clone libraries. We evaluated the effect of the domesticated inoculum by testing batch food waste anaerobic digestion with a non-domesticated inoculum as a control. The pH and methane yield of the digestion systems were determined as measurement indices. Domestication changed the composition and proportion of bacteria and archaea in the inocula. Of the bacteria, Clostridia (49.3%), Bacteroidales (19.5%), and Anaerolinaceae (8.1%) species were dominant in the seed sludge; Anaerolinaceae (49.0%), Clostridia (28.4%), and Bacteroidales (9.1%), in domestication sludge. Methanosaeta was the dominant genus in both of the seed (94.3%) and domestication (74.3%) sludge. However, the diversity of methanogenic archaea was higher in the domestication than in seed sludge. Methanoculleus, which was absent from the seed sludge, appeared in the domestication sludge (21.7%). When the domesticated inoculum was used, the digestion system worked stably (organic loading rate: 20 gVS/L; methane yield: 292.2 ± 9.8 mL/gVS; VS = volatile solids), whereas the digestion system inoculated with seed sludge failed to generate biogas. The results indicate that inoculum domestication ensures efficient and stable anaerobic digestion by enriching the methanogenic strains.

Occurrence, diversity, and abundance of methanogenic archaea in terrestrial hot springs of Kamchatka and Saõ Miguel Island

Detection and analysis of the mcrA gene encoding methyl-coenzyme M reductase, the key enzyme of methanogenesis, was used to assess occurrence and diversity of methanogenic archaea in terrestrial hot springs of Kamchatka and Sa~o Miguel Island (the Azores). For this analysis, phylogeny of methanogens was initially reconstructed based on available sequences of the mcrA gene, which is a common functional and phylogenetic marker for this physiological group of prokaryotes. Methanogens were revealed in most of the studied terrestrial hot springs with temperatures from 51 to 89°C, although they constituted an insignificant portion of the microbial population. The mcrA gene sequences revealed in the samples belonged to members of the genera Methanothermobacter, Methanothermus, and Methanothrix, previously detected in hot springs, as well as to methanogens not found earlier in these environments. The latter belonged to Methanomassiliicoccales, Methanocellales, and Methanomethylovorans, as well as to MCR-2a, the new deep phylogenetic cluster of uncultured methanogenic archaea; its phylotypes were present in all springs where the mcrA gene was detected. Our results indicate high diversity of the thermophilic methanogens inhabiting terrestrial hot springs and the presence among them of new groups with yet unknown substrate specificity.

The repertoire of archaea cultivated from severe periodontitis.

In previous studies, the abundance and diversity of methanogenic archaea in the dental microbiota have been analysed by the detection of specific DNA sequences by PCR-based investigations and metagenomic studies. Few data issued regarding methanogens actually living in dental plaque. We collected dental plaque specimens in 15 control individuals and 65 periodontitis patients. Dental plaque specimens were cultured in an anoxic liquid medium for methanogens in the presence of negative control tubes. Dental plaque methanogens were cultured from 1/15 (6.67%) control and 36/65 (55.38%) periodontitis patient samples (p<0.001). The cultures yielded Methanobrevibacter oralis in one control and thirty-one patients, Methanobrevibacter smithii in two patients and a potential new species named Methanobrevibacter sp. strain N13 in three patients with severe periodontitis. Our observations of living methanogens, strengthen previous observations made on DNA-based studies regarding the role of methanogens, in periodontitis.

Methanogenic archaea diversity in hyporheic sediments of a small lowland stream

Abundance and diversity of methanogenic archaea were studied at five localities along a longitudinal profile of a Sitka stream (Czech Republic). Samples of hyporheic sediments were collected from two sediment depths (0–25 cm and 25–50 cm) by freeze-core method. Methanogen community was analyzed by fluorescence in situ hybridization (FISH), denaturing gradient gel electrophoresis (DGGE) and sequencing method. The proportion of methanogens to the DAPI-stained cells varied among all localities and depths with an average value 2.08 × 105 per g of dry sediment with the range from 0.37 to 4.96 × 105 cells per g of dry sediment. A total of 73 bands were detected at 19 different positions on the DGGE gel and the highest methanogen diversity was found at the downstream located sites. There was no relationship between methanogen diversity and sediment depth. Cluster analysis of DGGE image showed three main clusters consisting of localities that differed in the number and similarity of the DGGE bands. Sequencing analysis of representative DGGE bands revealed phylotypes affiliated with members belonging to the orders Methanosarcinales, Methanomicrobiales and Methanocellales. The knowledge about occurrence and diversity of methanogenic archaea in freshwater ecosystems are essential for methane dynamics in river sediments and can contribute to the understanding of global warming process.

Methanogenic archaea diversity in Hanwoo (Bos taurus coreanae) rumen fluid, rectal dung, and barn floor manure using a culture-independent method based on mcrA gene sequences

The diversity of methanogenic archaea associated with Korean Hanwoo cattle was analyzed using mcrA gene sequences from samples of rumen fluid (RF), rectal dung (RD), and barn floor manure (BFM). The predominant species were Methanobrevibacter ruminantium in RF and BFM(63.6% and 62.4%, respectively) and Methanocorpusculum labreanum in RD (53.2%).