Dynamic transition of a methanogenic population in response to the concentration of volatile fatty acids in a thermophilic anaerobic digester

In this study, the microbial community succession in a thermophilic methanogenic bioreactor under deteriorative and stable conditions that were induced by acidification and neutralization, respectively, was investigated using PCR-mediated single-strand conformation polymorphism (SSCP) based on the 16S rRNA gene, quantitative PCR, and fluorescence in situ hybridization (FISH). The SSCP analysis indicated that the archaeal community structure was closely correlated with the volatile fatty acid (VFA) concentration, while the bacterial population was impacted by pH. The archaeal community consisted mainly of two species of hydrogenotrophic methanogen (i.e., a Methanoculleus sp. and a Methanothermobacter sp.) and one species of aceticlastic methanogen (i.e., a Methanosarcina sp.). The quantitative PCR of the 16S rRNA gene from each methanogen revealed that the Methanoculleus sp. predominated among the methanogens during operation under stable conditions in the absence of VFAs. Accumulation of VFAs induced a dynamic transition of hydrogenotrophic methanogens, and in particular, a drastic change (i.e., an approximately 10,000-fold increase) in the amount of the 16S rRNA gene from the Methanothermobacter sp. The predominance of the one species of hydrogenotrophic methanogen was replaced by that of the other in response to the VFA concentration, suggesting that the dissolved hydrogen concentration played a decisive role in the predominance. The hydrogenotrophic methanogens existed close to bacteria in aggregates, and a transition of the associated bacteria was also observed by FISH analyses. The degradation of acetate accumulated during operation under deteriorative conditions was concomitant with the selective proliferation of the Methanosarcina sp., indicating effective acetate degradation by the aceticlastic methanogen. The simple methanogenic population in the thermophilic anaerobic digester significantly responded to the environmental conditions, especially to the concentration of VFAs.     

Methanosarcina as the dominant aceticlastic methanogens during mesophilic anaerobic digestion of putrescible waste

Taking into account isotope ¹³C value a mathematical model was developed to describe the dynamics of methanogenic population during mesophilic anaerobic digestion of putrescible solid waste and waste imitating Chinese municipal solid waste. Three groups of methanogens were considered in the model including unified hydrogenotrophic methanogens and two aceticlastic methanogens Methanosaeta sp. and Methanosarcina sp. It was assumed that Methanosaeta sp. and Methanosarcina sp. are inhibited by high volatile fatty acids concentration. The total organic and inorganic carbon concentrations, methane production, methane and carbon dioxide partial pressures as well as the isotope ¹³C incorporation in PSW and CMSW were used for the model calibration and validation. The model showed that in spite of the high initial biomass concentration of Methanosaeta sp. Methanosarcina sp. became the dominant aceticlastic methanogens in the system. This prediction was confirmed by FISH. It is concluded that Methanosarcina sp. forming multicellular aggregates may resist to inhibition by volatile fatty acids (VFAs) because a slow diffusion rate of the acids limits the VFA concentrations inside the Methanosarcina sp. aggregates.     

Effect of dilution rate on the microbial structure of a mesophilic butyrate-degrading methanogenic community during continuous cultivation

We constructed two mesophilic anaerobic chemostats that were continuously fed with synthetic wastewater containing butyrate as the sole source of carbon and energy. Steady-state conditions were achieved at dilution rates between 0.025 and 0.7 day⁻¹. Butyrate, fed into the chemostat, was almost completely mineralized to CH₄ and CO₂ at dilution rates below 0.5 day⁻¹. The butyrate-degrading methanogenic communities in the chemostats at dilution rates between 0.025 and 0.7 day⁻¹ were monitored based on the 16S rRNA gene, using molecular biological techniques including clone library analysis, denaturing gradient gel electrophoresis, and quantitative real-time polymerase chain reaction. The aceticlastic methanogen Methanosaeta and the hydrogenotrophic methanogen Methanoculleus dominated in methanogens at low dilution rates, whereas the aceticlastic methanogen Methanosaeta, Methanosarcina, the hydrogenotrophic methanogen Methanoculleus, and Methanospirillum dominated at high dilution rates. Bacteria affiliated with the family Syntrophaceae in the phylum Proteobacteria predominated at the low dilution rate of 0.025 day⁻¹, whereas bacteria affiliated with the phylum Firmicutes and Candidate division OP3 predominated at high dilution rates. A significant quantity of bacteria closely related to the genus Syntrophomonas was detected at high dilution rates. Dilution rate showed an apparent effect on archaeal and bacterial communities in the butyrate-fed chemostats.     

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.     

Microbial community dynamics in anaerobic bioreactors and algal tanks treating piggery wastewater

Integrated biosystem is becoming a major aspect of wastewater management practice. Microbial communities in piggery wastewater sampled from anaerobic (thermophilic and mesophilic) and aerobic digesters (algal tanks) during waste remediation were analyzed by culture-independent techniques based on polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE). The use of Muyzer's 314F-GC, 518R bacterial primers, and archaeal A934F, 1309R primers followed by partial 16s rDNA sequence analysis of the main bands from DGGE revealed the presence of unknown and as yet uncultured microorganisms but also showed functional and ecologically significant denitrifying, acetogenic bacteria along with autotrophic, hydrogenotrophic, and acetoclastic methanogen archaea. Thermophilic digesters were dominated by γ-Proteobacteria, Methanothermobacter sp., while mesophilic digesters showed dominance by Firmicutes, uncultured bacteria, Methanosarcina, and Methanoculleus genera. Under aerobic conditions within algal tanks, pH rose from 7.17 to 9.32, with a significant decrease in total ammonia nitrogen, chemical oxygen demand, and soluble phosphorus levels. PCR-DGGE proved a useful tool for investigating the dynamics of microbial community in the bio-processing of piggery wastewater. Knowledge of the microbial communities involved in digestion of piggery wastewater will allow optimization of integrated biosystem by removing the main pollutants like inorganic ammonium-nitrogen, phosphorus, and pathogens from intensive farming system.     

Cultivation and biogeochemical analyses reveal insights into methanogenesis in deep subseafloor sediment at a biogenic gas hydrate site

Gas hydrates deposited in subseafloor sediments are considered to primarily consist of biogenic methane. However, little evidence for the occurrence of living methanogens in subseafloor sediments has been provided. This study investigated viable methanogen diversity, population, physiology and potential activity in hydrate-bearing sediments (1–307 m below the seafloor) from the eastern Nankai Trough. Radiotracer experiments, the quantification of coenzyme F430 and molecular sequencing analysis indicated the occurrence of potential methanogenic activity and living methanogens in the sediments and the predominance of hydrogenotrophic methanogens followed by methylotrophic methanogens. Ten isolates and nine representative culture clones of hydrogenotrophic, methylotrophic and acetoclastic methanogens were obtained from the batch incubation of sediments and accounted for 0.5–76% of the total methanogenic sequences directly recovered from each sediment. The hydrogenotrophic methanogen isolates of Methanocalculus and Methanoculleus that dominated the sediment methanogen communities produced methane at temperatures from 4 to 55 °C, with an abrupt decline in the methane production rate at temperatures above 40 °C, which is consistent with the depth profiles of potential methanogenic activity in the Nankai Trough sediments in this and previous studies. Our results reveal the previously overlooked phylogenetic and metabolic diversity of living methanogens, including methylotrophic methanogenesis.Subject terms: Biogeochemistry, Biodiversity, Environmental microbiology     

Co-cultivation of Thermoanaerobacter strains with a methanogenic partner enhances glycerol conversion

Glycerol-rich waste streams produced by the biodiesel, bioethanol and oleochemical industries can be treated and valorized by anaerobic microbial communities to produce methane. As current knowledge of the microorganisms involved in thermophilic glycerol conversion to methane is scarce, thermophilic glycerol-degrading methanogenic communities were enriched. A co-culture of Thermoanaerobacter and Methanothermobacter species was obtained, pointing to a non-obligately syntrophic glycerol degradation. This hypothesis was further studied by incubating Thermoanaerobacter brockii subsp. finnii and T. wiegelii with glycerol (10 mM) in pure culture and with different hydrogenotrophic methanogens. The presence of the methanogen accelerated glycerol fermentation by the two Thermoanaerobacter strains up to 3.3 mM day⁻¹, corresponding to 12 times higher volumetric glycerol depletion rates in the methanogenic co-cultures than in the pure bacterial cultures. The catabolic pathways of glycerol conversion were identified by genome analysis of the two Thermoanaerobacter strains. NADH and reduced ferredoxin formed in the pathway are linked to proton reduction, which becomes thermodynamically favourable when the hydrogen partial pressure is kept low by the hydrogenotrophic methanogenic partner.     

Bacteria and archaea involved in anaerobic digestion of distillers grains with solubles

Cereal distillers grains, a by-product from bioethanol industry, proved to be a suitable feedstock for biogas production in laboratory scale anaerobic digesters. Five continuously stirred tank reactors were run under constant conditions and monitored for biogas production and composition along with other process parameters. Iron additives for sulfide precipitation significantly improved the process stability and efficiency, whereas aerobic pretreatment of the grains had no effect. The microbial communities in the reactors were investigated for their phylogenetic composition by terminal restriction fragment length polymorphism analysis and sequencing of 16S rRNA genes. The bacterial subcommunities were highly diverse, and their composition did not show any correlation with reactor performance. The dominant phylotypes were affiliated to the Bacteroidetes. The archaeal subcommunities were less diverse and correlated with the reactor performance. The well-performing reactors operated at lower organic loading rates and amended with iron chloride were dominated by aceticlastic methanogens of the genus Methanosaeta. The well-performing reactor operated at a high organic loading rate and supplemented with iron hydroxide was dominated by Methanosarcina ssp. The reactor without iron additives was characterized by propionate and acetate accumulation and high hydrogen sulfide content and was dominated by hydrogenotrophic methanogens of the genus Methanoculleus.     

Use of a Hierarchical Oligonucleotide Primer Extension Approach for Multiplexed Relative Abundance Analysis of Methanogens in Anaerobic Digestion Systems

In this study, we established a rapid multiplex method to detect the relative abundances of amplified 16S rRNA genes from known cultivatable methanogens at hierarchical specificities in anaerobic digestion systems treating industrial wastewater and sewage sludge. The method was based on the hierarchical oligonucleotide primer extension (HOPE) technique and combined with a set of 27 primers designed to target the total archaeal populations and methanogens from 22 genera within 4 taxonomic orders. After optimization for their specificities and detection sensitivity under the conditions of multiple single-nucleotide primer extension reactions, the HOPE approach was applied to analyze the methanogens in 19 consortium samples from 7 anaerobic treatment systems (i.e., 513 reactions). Among the samples, the methanogen populations detected with order-level primers accounted for >77.2% of the PCR-amplified 16S rRNA genes detected using an Archaea-specific primer. The archaeal communities typically consisted of 2 to 7 known methanogen genera within the Methanobacteriales, Methanomicrobiales, and Methanosarcinales and displayed population dynamic and spatial distributions in anaerobic reactor operations. Principal component analysis of the HOPE data further showed that the methanogen communities could be clustered into 3 distinctive groups, in accordance with the distribution of the Methanosaeta, Methanolinea, and Methanomethylovorans, respectively. This finding suggested that in addition to acetotrophic and hydrogenotrophic methanogens, the methylotrophic methanogens might play a key role in the anaerobic treatment of industrial wastewater. Overall, the results demonstrated that the HOPE approach is a specific, rapid, and multiplexing platform to determine the relative abundances of targeted methanogens in PCR-amplified 16S rRNA gene products.     

Molecular characterization of microbial communities in deep coal seam groundwater of northern Japan

We investigated microbial methanogenesis and community structure based on 16S rRNA gene sequences from a coal seam aquifer located 843–907 m below ground level in northern Japan; additionally, we studied the δ¹³C and δ²H (δD) of coal‐bed gases and other physicochemical parameters. Although isotopic analysis suggested a thermocatalytic origin for the gases, the microbial activity and community structure strongly implied the existence of methanogenic microbial communities in situ. Methane was generated in the enrichment cultures of the hydrogenotrophic and methylotrophic microorganisms obtained from coal seam groundwater. Methanogen clones dominated the archaeal 16S rRNA gene libraries and were mostly related to the hydrogenotrophic genus Methanoculleus and the methylotrophic genus Methanolobus. Bacterial 16S rRNA gene libraries were dominated by the clones related to the genera Acetobacterium and Syntrophus which have a symbiotic association with methanogens. LIBSHUFF analysis revealed that N₂ gas injected into the coal seam (for enhanced methane production) does not affect the coverage of archaeal and bacterial populations. However, amova analysis does provide evidence for a change in the genetic diversity of archaeal populations that are dominated by methanogens. Therefore, N₂ injection into the coal seam might affect the cycling of matter by methanogens in situ.     

Effects of Amendment with Ferrihydrite and Gypsum on the Structure and Activity of Methanogenic Populations in Rice Field Soil

Methane emission from paddy fields may be reduced by the addition of electron acceptors to stimulate microbial populations competitive to methanogens. We have studied the effects of ferrihydrite and gypsum (CaSO₄·2H₂O) amendment on methanogenesis and population dynamics of methanogens after flooding of Italian rice field soil slurries. Changes in methanogen community structure were followed by archaeal small subunit (SSU) ribosomal DNA (rDNA)- and rRNA-based terminal restriction fragment length polymorphism analysis and by quantitative SSU rRNA hybridization probing. Under ferrihydrite amendment, acetate was consumed efficiently (<60 μM) and a rapid but incomplete inhibition of methanogenesis occurred after 3 days. In contrast to unamended controls, the dynamics of Methanosarcina populations were largely suppressed as indicated by rDNA and rRNA analysis. However, the low acetate availability was still sufficient for activation of Methanosaeta spp., as indicated by a strong increase of SSU rRNA but not of relative rDNA frequencies. Unexpectedly, rRNA amounts of the novel rice cluster I (RC-I) methanogens increased significantly, while methanogenesis was low, which may be indicative of transient energy conservation coupled to Fe(III) reduction by these methanogens. Under gypsum addition, hydrogen was rapidly consumed to low levels (~0.4 Pa), indicating the presence of a competitive population of hydrogenotrophic sulfate-reducing bacteria (SRB). This was paralleled by a suppressed activity of the hydrogenotrophic RC-I methanogens as indicated by the lowest SSU rRNA quantities detected in all experiments. Full inhibition of methanogenesis only became apparent when acetate was depleted to nonpermissive thresholds (<5 μM) after 10 days. Apparently, a competitive, acetotrophic population of SRB was not present initially, and hence, acetotrophic methanosarcinal populations were less suppressed than under ferrihydrite amendment. In conclusion, although methane production was inhibited effectively under both mitigation regimens, different methanogenic populations were either suppressed or stimulated, which demonstrates that functionally similar disturbances of an ecosystem may result in distinct responses of the populations involved.     

Characteristic microbial community of a dry thermophilic methanogenic digester: its long-term stability and change with feeding

Thermophilic dry anaerobic digestion of sludge for cellulose methanization was acclimated at 53 °C for nearly 5 years using a waste paper-based medium. The stability of the microbial community structure and the microbial community responsible for the cellulose methanization were studied by 16S rRNA gene-based clone library analysis. The microbial community structure remained stable during the long-term acclimation period. Hydrogenotrophic methanogens dominated in methanogens and Methanothermobacter, Methanobacterium, Methanoculleus, and Methanosarcina were responsible for the methane production. Bacteria showed relatively high diversity and distributed mainly in the phyla Firmicutes, Bacteroidetes, and Synergistetes. Ninety percent of operational taxonomic units (OTUs) were affiliated with the phylum Firmicutes, indicating the crucial roles of this phylum in the digestion. Relatives of Clostridium stercorarium, Clostridium thermocellum, and Halocella cellulosilytica were dominant cellulose degraders. The acclimated stable sludge was used to treat garbage stillage discharged from a fuel ethanol production process, and the shift of microbial communities with the change of feed was analyzed. Both archaeal and bacterial communities had obviously changed: Methanoculleus spp. and Methanothermobacter spp. and the protein- and fatty acid-degrading bacteria became dominant. Accumulation of ammonia as well as volatile fatty acids led to the inhibition of microbial activity and finally resulted in the deterioration of methane fermentation of the garbage stillage.     

Illumination enhances methane production from thermophilic anaerobic digestion

Incandescent lamp illumination enhanced methane production from a thermophilic anaerobic digestion reactor (55°C) supplied with glucose. After 10 days of operation, the volume of methane produced from light reactors was approximately 2.5 times higher than that from dark reactors. A comparison of the carbon balance between light and dark conditions showed that methane produced from hydrogen and carbon dioxide in the light reactors was higher than that from the dark reactors. When hydrogen or acetate was fed into the reactors, methane production with added hydrogen was faster and higher under light conditions than under dark conditions. The use of blue light-emitting diodes also enhanced methane production over that under dark conditions. The 16S rRNA gene copy numbers for Methanothermobacter spp. in the light reactor and in the dark reactor were at the same level. The copy number for Methanosarcina spp. in the light reactors was approximately double than that in the dark reactors. These results suggest that blue light enhances the methanogenic activity of hydrogenotrophic methanogens.     

Methanogenic archaea are globally ubiquitous in aerated soils and become active under wet anoxic conditions

The prototypical representatives of the Euryarchaeota—the methanogens—are oxygen sensitive and are thought to occur only in highly reduced, anoxic environments. However, we found methanogens of the genera Methanosarcina and Methanocella to be present in many types of upland soils (including dryland soils) sampled globally. These methanogens could be readily activated by incubating the soils as slurry under anoxic conditions, as seen by rapid methane production within a few weeks, without any additional carbon source. Analysis of the archaeal 16S ribosomal RNA gene community profile in the incubated samples through terminal restriction fragment length polymorphism and quantification through quantitative PCR indicated dominance of Methanosarcina, whose gene copy numbers also correlated with methane production rates. Analysis of the δ¹³C of the methane further supported this, as the dominant methanogenic pathway was in most cases aceticlastic, which Methanocella cannot perform. Sequences of the key methanogenic enzyme methyl coenzyme M reductase retrieved from the soil samples before incubation confirmed that Methanosarcina and Methanocella are the dominant methanogens, though some sequences of Methanobrevibacter and Methanobacterium were also detected. The global occurrence of only two active methanogenic archaea supports the hypothesis that these are autochthonous members of the upland soil biome and are well adapted to their environment.     

Polyphasic Analyses of Methanogenic Archaeal Communities in Agricultural Biogas Plants

Knowledge of the microbial consortia participating in the generation of biogas, especially in methane formation, is still limited. To overcome this limitation, the methanogenic archaeal communities in six full-scale biogas plants supplied with different liquid manures and renewable raw materials as substrates were analyzed by a polyphasic approach. Fluorescence in situ hybridization (FISH) was carried out to quantify the methanogenic Archaea in the reactor samples. In addition, quantitative real-time PCR (Q-PCR) was used to support and complete the FISH analysis. Five of the six biogas reactors were dominated by hydrogenotrophic Methanomicrobiales. The average values were between 60 to 63% of archaeal cell counts (FISH) and 61 to 99% of archaeal 16S rRNA gene copies (Q-PCR). Within this order, Methanoculleus was found to be the predominant genus as determined by amplified rRNA gene restriction analysis. The aceticlastic family Methanosaetaceae was determined to be the dominant methanogenic group in only one biogas reactor, with average values for Q-PCR and FISH between 64% and 72%. Additionally, in three biogas reactors hitherto uncharacterized but potentially methanogenic species were detected. They showed closest accordance with nucleotide sequences of the hitherto unclassified CA-11 (85%) and ARC-I (98%) clusters. These results point to hydrogenotrophic methanogenesis as a predominant pathway for methane synthesis in five of the six analyzed biogas plants. In addition, a correlation between the absence of Methanosaetaceae in the biogas reactors and high concentrations of total ammonia (sum of NH₃ and NH₄⁺) was observed.During the last decade the production of biogas from organic materials and residues has increased continuously in order to reduce the greenhouse gas emission resulting from the use of fossil energy sources. The energy-bearing substance of biogas is methane, which is produced as an end product of microbial anaerobic degradation of organic substrates, such as energy crops like maize, grains, grasses, or beets. Research for optimization of biogas production from renewable materials was initially focused on the evaluation of substrate eligibility and on the development and optimization of technical systems. However, biogas formation primarily depends on the structure and activity of the microbial community (28).The key microorganisms in the biogas formation process are the methane-generating microorganisms (methanogens). The capacity for methanogenesis is limited to members of the domain Archaea and, within this domain, on the phylum Euryarchaeota. With respect to the main metabolic precursors used, methanogens are usually divided into two groups: the aceticlastic methanogens that strictly metabolize acetate and the hydrogenotrophic methanogens that use H₂ or formate as an electron donor and CO₂ as a carbon source for their metabolism. Besides these major groups, certain methanogens are also able to convert methyl groups, methylamines, or methanol to methane (23, 40). The substrates for the methanogens are provided by several physiological groups of bacteria which degrade organic matter, sometimes in close syntrophic interaction with the methanogens (1).Several studies on the microbial diversity present in lab-scale biogas reactors supplied with renewable raw material (7, 57) have been recently published. However, analyses under laboratory conditions do not necessarily reflect conditions in full-scale reactors (35). Therefore, further research on the methanogenic community in full-scale biogas reactors is crucial.Generally, studies regarding the microbial community structure in full-scale biogas reactors have focused on different systems for wastewater treatment or classical biogas plants based on manure digestion (32, 38, 43). In most systems, approximately 70% of the carbon fixed in methane was derived from acetate. Only minor amounts, up to approximately 30%, were deduced from CO₂ (1, 42). Together with the presence of huge assemblages of Methanosarcina sp., it was assumed by some authors that aceticlastic methanogenesis was the predominant pathway for methane formation. Moreover, as shown by other studies, the relative contribution of H₂/CO₂ versus acetate as metabolic precursors for methanogens can be quite different in other anaerobic environments (10, 33, 37). However, the methanogenic microfloras in full-scale biogas reactors supplied with energy crops as a primary or sole substrate have rarely been studied (35, 37, 45).The aim of this study was to gain insight into the diversity of methane-producing Archaea in six full-scale biogas plants supplied with renewable raw material and different types of liquid manure as substrates. Therefore, a polyphasic approach with three different culture-independent techniques (fluorescence in situ hybridization [FISH], quantitative PCR [Q-PCR], and 16S rRNA gene analysis) to analyze methanogen diversity was carried out to overcome the known limitations of each single approach (15, 46). To analyze potential effects of different process parameters on the methanogenic archaeal community, the reactor performances were correlated with the apparent archaeal diversity.     

A pilot scale two-stage anaerobic digester treating food waste leachate (FWL): Performance and microbial structure analysis using pyrosequencing

Food waste leachate (FWL) from the food waste recycling facilities in Korea is a serious environmental problem. Much research was done on anaerobic digestion of FWL in a lab-scale; however, there is little information on a large scale anaerobic digestion system (ADS). In this study, a two-phase ADS in a pilot scale was operated using FWL and the ADS performance and microbial structure dynamics using pyrosequencing were investigated. The ADS was operated for 136 days using FWL containing a high concentration of volatile fatty acid (12,435 ± 2203 mg/L), exhibiting volatile acid (VS) removal efficiency of 74–89% and CH₄ yield of 0.39–0.85 Nm³/kg of reduced VS. The microbial structure at 76, 101, and 132 days indicated the methanogen population shift from acetoclastic methanogens (Methanosarcina and Methanosaeta) to hydrogenotrophic methanogens (Methanobacterium and Methanoculleus). The bacterial community also shifted to the taxa syntrophically related with hydrogenotrophic methanogens (Clostridia). The statistical analysis revealed the positive correlation of VS removal efficiency with Methanosarcina, but the negative correlation with Methanobacterium. The results presented here suggest that acetoclastic methanogens and their associated bacteria were more efficient for VS removal in the pilot scale ADS system, providing useful information for FWL treatment in a large scale ADS.     

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 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.     

Molecular detection and direct enumeration of methanogenic Archaea and methanotrophic Bacteria in domestic solid waste landfill soils

Methane oxidizing and producing activities of cover soil (10, 30 cm depth) and burial waste (1, 3 m depth) were evaluated: top cover soil (10 cm) had the highest methane oxidizing activity, while 1 m depth buried waste showed the highest methane producing potential. All the sequences of the 1 m sample were found to be closely related to 16S rDNAs of mainly hydrogenotrophic methanogens known, such as genera Methanosarcina, Methanoculleus, and Methanobacterium. We developed a modified fluorescence in situ hybridization (FISH) direct counting method for landfill samples, resulting in the detection of approx. 1% of total cells as archaeal cells (presumably methanogens). However, probe-positive cells could not be found with probes for methanotrophs by the methods.     

Diversity of rumen microbiota using metagenome sequencing and methane yield in Indian sheep fed on straw and concentrate diet

An in vivo study aiming to investigate the rumen methanogens community structure was conducted in Mandya sheep fed on straw and concentrate diet. The ruminal fluid samples were collected and processed for unravelling the rumen microbiota and methanogens diversity. Further, the daily enteric methane emission and methane yield was also quantified using the SF₆ tracer technique. Results indicated that the Bacteroidetes (～57%) and Firmicutes (25%) were two prominent affiliates of the bacterial community. Archaea represented about 2.5% of the ruminal microbiota. Methanobacteriales affiliated methanogens were the most prevalent in sheep rumen. The study inveterate that the ruminal archaea community in sheep is composed of 9 genera and 18 species. Methanobrevibacter represented the largest genus of the archaeome, while methylotrophs genera constituted only 13% of the community. Methanobrevibacter gottschalkii was the prominent methanogen, and Methaobrevibacter ruminantium distributed at a lower frequency (～2.5%). Among Methanomassiliicoccales, Group 12 sp. ISO4-H5 constituted the most considerable fraction (～11%). KEGG reference pathway for methane metabolism indicated the formation of methane through hydrogenotrophic and methylotrophic pathways, whereas the acetoclastic pathway was not functional in sheep. The enteric methane emission and methane yield was 19.7 g/d and 20.8 g/kg DMI, respectively. Various species of Methanobrevibacter were differently correlated, and the distribution of hydrogenotrophic methanogens mainly explained the variability in methane yield between the individual sheep. It can be inferred from the study that the hydrogenotrophic methanogens dominate the rumen archaeal community in sheep and methylotrophic/aceticlastic methanogens represent a minor fraction of the community. Further studies are warranted for establishing the metabolic association between the prevalent hydrogenotrophs and methylotrophs to identify the key reaction for reducing methane emission.