Methane Production and Methanogenic Archaea in the Digestive Tracts of Millipedes (Diplopoda)

Methane production by intestinal methanogenic Archaea and their community structure were compared among phylogenetic lineages of millipedes. Tropical and temperate millipedes of 35 species and 17 families were investigated. Species that emitted methane were mostly in the juliform orders Julida, Spirobolida, and Spirostreptida. The irregular phylogenetic distribution of methane production correlated with the presence of the methanogen-specific mcrA gene. The study brings the first detailed survey of methanogens’ diversity in the digestive tract of millipedes. Sequences related to Methanosarcinales, Methanobacteriales, Methanomicrobiales and some unclassified Archaea were detected using molecular profiling (DGGE). The differences in substrate preferences of the main lineages of methanogenic Archaea found in different millipede orders indicate that the composition of methanogen communities may reflect the differences in available substrates for methanogenesis or the presence of symbiotic protozoa in the digestive tract. We conclude that differences in methane production in the millipede gut reflect differences in the activity and proliferation of intestinal methanogens rather than an absolute inability of some millipede taxa to host methanogens. This inference was supported by the general presence of methanogenic activity in millipede faecal pellets and the presence of the 16S rRNA gene of methanogens in all tested taxa in the two main groups of millipedes, the Helminthophora and the Pentazonia.     

Phylogenetic characterization of methanogenic assemblages in eutrophic and oligotrophic areas of the Florida Everglades

Agricultural activities have produced well-documented changes in the Florida Everglades, including establishment of a gradient in phosphorus concentrations in Water Conservation Area 2A (WCA-2A) of the northern Everglades. An effect of increased phosphorus concentrations is increased methanogenesis in the eutrophic regions compared to the oligotrophic regions of WCA-2A. The goal of this study was to identify relationships between eutrophication and composition and activity of methanogenic assemblages in WCA-2A soils. Distributions of two genes associated with methanogens were characterized in soils taken from WCA-2A: the archaeal 16S rRNA gene and the methyl coenzyme M reductase gene. The richness of methanogen phylotypes was greater in eutrophic than in oligotrophic sites, and sequences related to previously cultivated and uncultivated methanogens were found. A preferential selection for the order Methanomicrobiales was observed in mcrA clone libraries, suggesting primer bias for this group. A greater diversity within the Methanomicrobiales was observed in mcrA clone libraries than in 16S rRNA gene libraries. 16S rRNA phylogenetic analyses revealed a dominance of clones related to Methanosaeta spp., an acetoclastic methanogen dominant in environments with low acetate concentrations. A significant number of clones were related to Methanomicrobiales, an order characterized by species utilizing hydrogen and formate as methanogenic substrates. No representatives of the orders Methanobacteriales and Methanococcales were found in any 16S rRNA clone library, although some Methanobacteriales were found in mcrA libraries. Hydrogenotrophs are the dominant methanogens in WCA-2A, and acetoclastic methanogen genotypes that proliferate in low acetate concentrations outnumber those that typically dominate in higher acetate concentrations.     

Methanogen diversity evidenced by molecular characterization of methyl coenzyme M reductase A (mcrA) genes in hydrothermal sediments of the Guaymas Basin

The methanogenic community in hydrothermally active sediments of Guaymas Basin (Gulf of California, Mexico) was analyzed by PCR amplification, cloning, and sequencing of methyl coenzyme M reductase (mcrA) and 16S rRNA genes. Members of the Methanomicrobiales and Methanosarcinales dominated the mcrA and 16S rRNA clone libraries from the upper 15 cm of the sediments. Within the H2/CO2- and formate-utilizing family Methanomicrobiales, two mcrA and 16S rRNA lineages were closely affiliated with cultured species of the genera Methanoculleus and Methanocorpusculum. The most frequently recovered mcrA PCR amplicons within the Methanomicrobiales did not branch with any cultured genera. Within the nutritionally versatile family Methanosarcinales, one 16S rRNA amplicon and most of the mcrA PCR amplicons were affiliated with the obligately acetate utilizing species Methanosaeta concilii. The mcrA clone libraries also included phylotypes related to the methyl-disproportionating genus Methanococcoides. However, two mcrA and two 16S rRNA lineages within the Methanosarcinales were unrelated to any cultured genus. Overall, the clone libraries indicate a diversified methanogen community that uses H2/CO2, formate, acetate, and methylated substrates. Phylogenetic affiliations of mcrA and 16S rRNA clones with thermophilic and nonthermophilic cultured isolates indicate a mixed mesophilic and thermophilic methanogen community in the surficial Guaymas sediments.     

Vertical profiles of methanogenesis and methanogens in two contrasting acidic peatlands in central New York State, USA

Northern acidic peatlands are important sources of atmospheric methane, yet the methanogens in them are poorly characterized. We examined methanogenic activities and methanogen populations at different depths in two peatlands, McLean bog (MB) and Chicago bog (CB). Both have acidic (pH 3.5-4.5) peat soils, but the pH of the deeper layers of CB is near-neutral, reflecting its previous existence as a neutral-pH fen. Acetotrophic and hydrogenotrophic methanogenesis could be stimulated in upper samples from both bogs, and phylotypes of methanogens using H2/CO2 (Methanomicrobiales) or acetate (Methanosarcinales) were identified in 16S rRNA gene clone libraries and by terminal restriction fragment length polymorphism (T-RFLP) analyses using a novel primer/restriction enzyme set that we developed. Particularly dominant in the upper layers was a clade in the Methanomicrobiales, called E2 here and the R10 or fen group elsewhere, estimated by quantitative polymerase chain reaction to be present at approximately 10(8) cells per gram of dry peat. Methanogenic activity was considerably lower in deeper samples from both bogs. The methanogen populations detected by T-RFLP in deeper portions of MB were mainly E2 and the uncultured euryarchaeal rice cluster (RC)-II group, whereas populations in the less acidic CB deep layers were considerably different, and included a Methanomicrobiales clade we call E1-E1', as well as RC-I, RC-II, marine benthic group D, and a new cluster that we call the subaqueous cluster. E2 was barely detectable in the deeper samples from CB, further evidence for the associations of most organisms in this group with acidic habitats.     

Diversity of prokaryotes and methanogenesis in deep subsurface sediments from the Nankai Trough, Ocean Drilling Program Leg 190

Diversity of Bacteria and Archaea was studied in deep marine sediments by PCR amplification and sequence analysis of 16S rRNA and methyl co-enzyme M reductase (mcrA) genes. Samples analysed were from Ocean Drilling Program (ODP) Leg 190 deep subsurface sediments at three sites spanning the Nankai Trough in the Pacific Ocean off Shikoku Island, Japan. DNA was amplified, from three depths at site 1173 (4.15, 98.29 and 193.29 mbsf; metres below the sea floor), and phylogenetic analysis of clone libraries showed a wide variety of uncultured Bacteria and Archaea. Sequences of Bacteria were dominated by an uncultured and deeply branching 'deep sediment group' (53% of sequences). Archaeal 16S rRNA gene sequences were mainly within the uncultured clades of the Crenarchaeota. There was good agreement between sequences obtained independently by cloning and by denaturing gradient gel electrophoresis. These sequences were similar to others retrieved from marine sediment and other anoxic habitats, and so probably represent important indigenous bacteria. The mcrA gene analysis suggested limited methanogen diversity with only three gene clusters identified within the Methanosarcinales and Methanobacteriales. The cultivated members of the Methanobacteriales and some of the Methanosarcinales can use CO2 and H2 for methanogenesis. These substrates also gave the highest rates in 14C-radiotracer estimates of methanogenic activity, with rates comparable to those from other deep marine sediments. Thus, this research demonstrates the importance of the 'deep sediment group' of uncultured Bacteria and links limited diversity of methanogens to the dominance of CO2/H2 based methanogenesis in deep sub-seafloor sediments.     

Cultivation of methanogens from shallow marine sediments at Hydrate Ridge, Oregon

Little is known about the methanogenic degradation of acetate, the fate of molecular hydrogen and formate or the ability of methanogens to grow and produce methane in cold, anoxic marine sediments. The microbes that produce methane were examined in permanently cold, anoxic marine sediments at Hydrate Ridge (44 degrees 35' N, 125 degrees 10' W, depth 800 m). Sediment samples (15 to 35 cm deep) were collected from areas of active methane ebullition or areas where methane hydrates occurred. The samples were diluted into enrichment medium with formate, acetate or trimethylamine as catabolic substrate. After 2 years of incubation at 4 degrees C to 15 degrees C, enrichment cultures produced methane. PCR amplification and sequencing of the rRNA genes from the highest dilutions with growth suggested that each enrichment culture contained a single strain of methanogen. The level of sequence similarity (91 to 98%) to previously characterized prokaryotes suggested that these methanogens belonged to novel genera or species within the orders Methanomicrobiales and Methanosarcinales. Analysis of the 16S rRNA gene libraries from DNA extracted directly from the sediment samples revealed phylotypes that were either distantly related to cultivated methanogens or possible anaerobic methane oxidizers related to the ANME-1 and ANME-2 groups of the Archaea. However, no methanogenic sequences were detected, suggesting that methanogens represented only a small proportion of the archaeal community.     

Methanogenic profiles by denaturing gradient gel electrophoresis using order-specific primers in anaerobic sludge digestion

In the present study, the diversity of methanogenic populations was monitored for 25 days, together with the process data for an anaerobic batch reactor treating waste-activated sludge. To understand this microbial diversity and dynamics, 16S rRNA-gene-targeted denaturing gradient gel electrophoresis (DGGE) fingerprinting was conducted at two different taxonomic levels: the domain and order levels. The DGGE profiles of the domain Archaea and the three orders Methanosarcinales, Methanomicrobiales, and Methanobacteriales were comparatively analyzed after each DGGE band was sequenced to enable identification. The DGGE profiles of the three orders showed methanogens belonging to each order that were not detected in the DGGE profile of the Archaea. This discrepancy may have resulted from PCR bias or differences in the abundances of the three microbial orders in the anaerobic bioreactor. In conclusion, to fully understand the detailed methanogenic diversity and dynamics in an anaerobic bioreactor, it is necessary to conduct DGGE analysis with 16S rRNA gene primers that target lower taxonomic groups.     

Methanogen community in Zoige wetland of Tibetan plateau and phenotypic characterization of a dominant uncultured methanogen cluster ZC-I

Zoige wetland of Tibetan plateau is characterized by being located at a low latitude (33°56'N, 102°52'E) region and under the annual temperature around 1°C. Previous studies indicated that Zoige wetland was one of the CH₄ emission centres in Qinghai-Tibetan plateau; in this study, the methanogen community in this low-latitude wetland was analysed based on the homology of 16S rRNA and mcrA genes retrieved from the soil. The results indicated that members of Methanosarcinales and Methanomicrobiales constituted the majority of methanogens, and a novel uncultured methanogen cluster, Zoige cluster I (ZC-I) affiliated to Methanosarcinales , could be dominant. Using quantitative polymerase chain reaction (qPCR) assay, ZC-I methanogens were estimated to be 10⁷ cells per gram of soil, accounting for about 30% of the total Archeae . By combining culturable enrichment with qPCR assay, the quantity of ZC-I methanogens in the methanogenic enrichment with acetate, H2/CO₂, methanol or trimethylamine was determined to increase to 10⁸ cells ml⁻¹, but not with formate, which indicated that ZC-I methanogens could use the four methanogenic substrates. The growth rates at 30°C and 15°C were not pronounced different, implying ZC-I to be the cold-adaptive methanogens. The broad substrate spectrum identified the ZC-I methanogens to be a member of Methanosarcinaceae , and could represent a novel sub-branch specifically inhabited in cold ecosystems. Fluorescence in situ hybridization (FISH) images also visualized ZC-I methanogens the sarcina-like aggregate of the spherical cells. The prevalence and flexibility in substrate utilization and growth temperature suggested ZC-I methanogens to be an important player in the methanogenesis of Zoige wetland.     

Archaea diversity within a commercial biogas plant utilizing herbal biomass determined by 16S rDNA and mcrA analysis

Aims: The Archaea diversity was evaluated in an agricultural biogas plant supplied with cattle liquid manure and maize silage under mesophilic conditions. Methods and Results: Two different genes (16S rRNA; methyl‐coenzyme‐M‐reductase, MCR) targeted by three different PCR primer sets were selected and used for the construction of three clone libraries comprising between 104 and 118 clones. The clone libraries were analysed by restriction fragment polymorphism (RFLP). Between 11 and 31 operational taxonomic units (OTUs) were detected and assigned to orders Methanomicrobiales, Methanosarcinales and Methanobacteriales. Over 70% of all Archaea OTUs belong to the order Methanomicrobiales which mostly include hydrogenotrophic methanogens. Acetotrophic methanogens were detected in minor rates. Similar relative values were obtained by a quantitative real‐time PCR analysis. Conclusions: The results implied that in this biogas plant the most of the methane formation resulted from the conversion of H₂ and CO₂. Significance and Impact of the Study: This study reports, for the first time, a molecular analysis of the archaeal community in this type of agricultural biogas plants. Therein the hydrogenotrophic methanogenesis seems to be the major pathway of methane formation. These results are in contrast with the common thesis that in biogas fermentations the primary substrate for methanogenesis is acetate.     

Methanogenic activity and diversity in the centre of the Amsterdam Mud Volcano, Eastern Mediterranean Sea

Marine mud volcanoes are geological structures emitting large amounts of methane from their active centres. The Amsterdam mud volcano (AMV), located in the Anaximander Mountains south of Turkey, is characterized by intense active methane seepage produced in part by methanogens. To date, information about the diversity or the metabolic pathways used by the methanogens in active centres of marine mud volcanoes is limited. (14)C-radiotracer measurements showed that methylamines/methanol, H(2)/CO(2) and acetate were used for methanogenesis in the AMV. Methylotrophic methanogenesis was measured all along the sediment core, Methanosarcinales affiliated sequences were detected using archaeal 16S PCR-DGGE and mcrA gene libraries, and enrichments of methanogens showed the presence of Methanococcoides in the shallow sediment layers. Overall acetoclastic methanogenesis was higher than hydrogenotrophic methanogenesis, which is unusual for cold seep sediments. Interestingly, acetate porewater concentrations were extremely high in the AMV sediments. This might be the result of organic matter cracking in deeper hotter sediment layers. Methane was also produced from hexadecanes. For the most part, the methanogenic community diversity was in accordance with the depth distribution of the H(2)/CO(2) and acetate methanogenesis. These results demonstrate the importance of methanogenic communities in the centres of marine mud volcanoes.     

Primer evaluation and adaption for cost-efficient SYBR Green-based qPCR and its applicability for specific quantification of methanogens

In the present study nine promising primer sets, targeting Archaea and methanogenic Archaea in particular, were evaluated in silico, in vitro and in situ concerning specificity, accuracy and applicability in end-point (ep-) and especially quantitative (q-)PCR research. The main goal was to adapt and evaluate already adapted primer sets, which were partially designed in combination with TaqMan probes, in substantially cheaper SYBR Green-based qPCR applications. An initial 16S rRNA gene bank-based in silico evaluation revealed high coverage potentials for all primers within targeted groups, ranging from 71 to 90 %, except the Methanosaeta specific set showing a low potential of 37 %. Mentionable cross-reacting potentials could be detected for the Methanothermobacter, Methanomicrobiales and Methanoculleus sets. The in vitro evaluation with selected reference organisms revealed a specific behavior for most primer sets, while the Methanosarcina and Methanothermobacter sets showed most problematic cross-reactions in epPCR application. We were able to show that primers for detecting the total archaeal community, methanogenic orders Methanosarcinales, Methanobacteriales, Methanococcales and the genus Methanoculleus performed in a highly specific way and allowed an accurate quantification of targeted organisms without the use of expensive TaqMan probes. However, primer pairs designed for detecting Methanomicrobiales, Methanothermobacter, Methanosarcina and Methanosaeta are not suitable for SYBR Green applications. The reliability of in situ quantifications was assessed for a typical methanogenic community, derived from a thermophilic fermenter, and confirmed via denaturing gradient gel band quantification and sequencing. Thereby, we revealed high abundances of methanogenic Archaea, mainly comprising Methanoculleus and Methanosarcinales, while Methanobacteriales only formed a minor fraction.     

Phylogenetic Characterization of Methanogenic Assemblages in Eutrophic and Oligotrophic Areas of the Florida Everglades

Agricultural activities have produced well-documented changes in the Florida Everglades, including establishment of a gradient in phosphorus concentrations in Water Conservation Area 2A (WCA-2A) of the northern Everglades. An effect of increased phosphorus concentrations is increased methanogenesis in the eutrophic regions compared to the oligotrophic regions of WCA-2A. The goal of this study was to identify relationships between eutrophication and composition and activity of methanogenic assemblages in WCA-2A soils. Distributions of two genes associated with methanogens were characterized in soils taken from WCA-2A: the archaeal 16S rRNA gene and the methyl coenzyme M reductase gene. The richness of methanogen phylotypes was greater in eutrophic than in oligotrophic sites, and sequences related to previously cultivated and uncultivated methanogens were found. A preferential selection for the order Methanomicrobiales was observed in mcrA clone libraries, suggesting primer bias for this group. A greater diversity within the Methanomicrobiales was observed in mcrA clone libraries than in 16S rRNA gene libraries. 16S rRNA phylogenetic analyses revealed a dominance of clones related to Methanosaeta spp., an acetoclastic methanogen dominant in environments with low acetate concentrations. A significant number of clones were related to Methanomicrobiales, an order characterized by species utilizing hydrogen and formate as methanogenic substrates. No representatives of the orders Methanobacteriales and Methanococcales were found in any 16S rRNA clone library, although some Methanobacteriales were found in mcrA libraries. Hydrogenotrophs are the dominant methanogens in WCA-2A, and acetoclastic methanogen genotypes that proliferate in low acetate concentrations outnumber those that typically dominate in higher acetate concentrations.     

Diversity of Archaea in marine sediments from Skan Bay, Alaska, including cultivated methanogens, and description of Methanogenium boonei sp. nov

Methanogenesis in cold marine sediments is a globally important process leading to methane hydrate deposits, cold seeps, physical instability of sediment, and atmospheric methane emissions. We employed a multidisciplinary approach that combined culture-dependent and -independent analyses with geochemical measurements in the sediments of Skan Bay, Alaska (53 degrees N, 167 degrees W), to investigate methanogenesis there. Cultivation-independent analyses of the archaeal community revealed that uncultivated microbes of the kingdoms Euryarchaeota and Crenarchaeota are present at Skan Bay and that methanogens constituted a small proportion of the archaeal community. Methanogens were cultivated from depths of 0 to 60 cm in the sediments, and several strains related to the orders Methanomicrobiales and Methanosarcinales were isolated. Isolates were psychrotolerant marine-adapted strains and included an aceticlastic methanogen, strain AK-6, as well as three strains of CO(2)-reducing methanogens: AK-3, AK7, and AK-8. The phylogenetic positions and physiological characteristics of these strains are described. We propose a new species, Methanogenium boonei, with strain AK-7 as the type strain.     

Phylogenetic relationships of symbiotic methanogens in diverse termites

Termites harbor symbiotic microorganisms in their gut which emit methane. The phylogeny of the termite methanogens was inferred without cultivation based on nucleotide sequences of PCR-amplified 16S ribosomal RNA genes. Seven methanogen sequences from four termite species were newly isolated, and together with those previously published, these sequences were phylogenetically compared. The termite methanogen sequences were divided into three clusters. Two clusters of sequences, derived from the gut DNA of so-called higher termites, were related to methanogens in the orders Methanosarcinales or Methanomicrobiales. All of the sequences in the case of lower termites were closely related to the genus Methanobrevibacter. However, most of the termite symbionts were found to be distinct from known methanogens. They are not dispersed among diverse methanogen species, but rather formed unique lineages in the phylogenetic trees.     

Quantification of mcrA by fluorescent PCR in methanogenic and methanotrophic microbial communities

A quantitative fluorogenic PCR method for detecting methanogenic and methanotrophic orders was established using a refined primer set for the methyl coenzyme M reductase subunit A gene (mcrA). The method developed was applied to several microbial communities in which diversity and abundance of methanogens or anaerobic methanotrophs (ANMEs) was identified by 16S rRNA gene clone analysis, and strong correlations between the copy numbers of mcrA with those of archaeal 16S rRNA genes in the communities were observed. The assay can be applied to detecting and assessing the abundance of methanogens and/or ANMEs in anoxic environments that could not be detected by 16S rRNA gene sequence analyses.     

Characterization of the methanogenic Archaea within two-phase biogas reactor systems operated with plant biomass

The two-phase leach-bed system is a biogas reactor system optimized for the utilization of energy crop silages at maximized loading rates under maintenance of an optimal microbial activity. In this study, a characterization of the methanogenic microbial community within this reactor system was conducted for the first time. Accordingly, effluent samples from the anaerobic filter and the silage digesting leach-bed reactors of both a laboratory-scale two-phase biogas reactor system and a scaled-up commercial on-farm pilot plant were investigated. In total, five Archaea-specific 16S rDNA libraries were constructed and analyzed by amplified rDNA restriction analysis (ARDRA), with subsequent phylogenetic analysis of nucleotide sequences for individual ARDRA patterns. A quantification of major methanogenic Archaea groups was conducted by real-time PCR. A total of 663 clones were analyzed and 45 operational taxonomic units (OTUs) related to methanogenic Archaea were detected. These OTUs were related to the orders Methanosarcinales, Methanomicrobiales and Methanobacteriales, as well as the hitherto uncultured CA-11 and ARC-I groups, and most of them occurred throughout all the compartments of both two-phase biogas reactors. The proportion of acetotrophic to hydrogenotrophic methanogens differed between the laboratory and the pilot scale system. A total of 56% of the clones from the 16S rDNA library derived from the laboratory biogas system were assigned to presumably acetotrophic members of Methanosarcinales. In contrast, these OTUs were less abundant in the 16S rDNA library derived from samples of the pilot plant. Therein, the most dominant OTUs were Methanoculleus-related OTUs, which presumably indicated the predominant presence of hydrogenotrophic methanogens. These findings were confirmed by group-specific quantitative real-time PCR assays. The results indicated that the fraction of acetotrophic and hydrogenotrophic methanogens within a biogas reactor caused certain variations, which may reflect varying substrate utilization during methanogenesis.     

The molecular diversity of the methanogenic community in a hypereutrophic freshwater lake determined by PCR-RFLP

AIMS: To combine database-held sequence information with a programme of experimental molecular ecology to define the methanogenic community of a hypereutrophic lake by a PCR-restriction fragment length polymorphism (RFLP) analysis. METHODS AND RESULTS: Methanogen diversity in a hypereutrophic freshwater lake was analysed using 16S rDNA PCR-RFLP. Database-held 16S rRNA gene sequences for 76 diverse methanogens were analysed for specific restriction sites that permitted unequivocal differentiation of methanogens. Restriction digestion and agarose gel electrophoresis of the 16S rDNA from selected methanogen pure cultures generated observed restriction profiles that corroborated the expected patterns. This method was then tested by analysing methanogen diversity in samples obtained over 1 year from sediment and water samples taken from the same sampling site. CONCLUSIONS: Restriction analysis of the 16S rRNA gene sequences from 157 methanogen clones generated from lakewater and sediment samples showed that over 50% were similar to Methanoculleus spp. Furthermore, a total of 16 RFLP types (1-16) were identified, eight of which contained no cultured representative archaeal 16S rRNA gene sequences. SIGNIFICANCE AND IMPACT OF THE STUDY: This RFLP strategy provides a robust and reliable means to rapidly identify methanogens in the environment.     

Field and laboratory studies on the bioconversion of coal to methane in the San Juan Basin

The bioconversion of coal to methane in the San Juan Basin, New Mexico, was investigated. Production waters were analyzed via enrichment studies, metabolite-profiling, and culture-independent methods. Analysis of 16S rRNA gene sequences indicated the presence of methanogens potentially capable of acetoclastic, hydrogenotrophic, and methylotrophic metabolisms, predominantly belonging to the Methanosarcinales and Methanomicrobiales. Incubations of produced water and coal readily produced methane, but there was no correlation between the thermal maturity and methanogenesis. Coal methanogenesis was greater when samples with a greater richness of Firmicutes were utilized. A greater archaeal diversity was observed in the presence of several aromatic and short-chain fatty acid metabolites. Incubations amended with lactate, hydrogen, formate, and short-chain alcohols produced methane above un-amended controls. Methanogenesis from acetate was not observed. Metabolite profiling showed the widespread occurrence of putative aromatic ring intermediates including benzoate, toluic acids, phthalic acids, and cresols. The detection of saturated and unsaturated alkylsuccinic acids indicated n-alkane and cyclic alkane/alkene metabolism. Microarray analysis complemented observations based on hybridization to functional genes related to the anaerobic metabolism of aromatic and aliphatic substrates. These data suggest that coal methanogenesis is unlikely to be limited by methanogen biomass, but rather the activation and degradation of coal constituents.     

Dynamic transition of microbial communities in response to acidification in fixed-bed anaerobic baffled reactors (FABR) of two different flow directions

Two five-compartment fixed-bed anaerobic baffled reactors (FABRs) were operated under deteriorative and stable conditions. The FABRs were identical except for flow direction: one was horizontal (H-reactor) and the other was vertical (V-reactor). The microbial community dynamics in 1, 3 and 5 compartments were analyzed using denaturing gradient gel electrophoresis (DGGE), 16S rRNA gene clone library screening and quantitative PCR. After start-up, the Methanomicrobiales were typically dominant in adhering sludge of 5th compartments of two reactors. Because methanogenesis mainly occurred in the latter compartment, Methanomicrobiales were likely to play important roles in FABRs. FABRs recovered from performance deterioration very quickly. Meanwhile, methanogens and dominant methanogens greatly increased in every compartment of two reactors. Our results indicated that 16S rRNA levels of methanogens in the adhering sludge were higher than those in the deposited sludge and the adhering fraction of V-reactor held up more acid-resistant bacteria and methanogens than that of H-reactor.