Diversity and ubiquity of thermophilic methanogenic archaea in temperate anoxic soils

Temperate rice field soil from Vercelli (Italy) contains moderately thermophilic methanogens of the yet uncultivated rice cluster I (RC-I), which become prevalent upon incubation at temperatures of 45-50 degrees C. We studied whether such thermophilic methanogens were ubiquitously present in anoxic soils. Incubation of different rice field soils (from Italy, China and the Philippines) and flooded riparian soils (from the Netherlands) at 45 degrees C resulted in vigorous CH(4) production after a lag phase of about 10 days. The archaeal community structure in the soils was analysed by terminal restriction fragment length polymorphism (T-RFLP) targeting the SSU rRNA genes retrieved from the soil, and by cloning and sequencing. Clones of RC-I methanogens mostly exhibited T-RF of 393 bp, but also terminal restriction fragment (T-RF) of 158 and 258 bp length, indicating a larger diversity than previously assumed. No RC-I methanogens were initially found in flooded riparian soils. However, these archaea became abundant upon incubation of the soil at 45 degrees C. Thermophilic RC-I methanogens were also found in the rice field soils from Pavia, Pila and Gapan. However, the archaeal communities in these soils also contained other methanogenic archaea at high temperature. Rice field soil from Buggalon, on the other hand, only contained thermophilic Methanomicrobiales rather than RC-I methanogens, and rice field soil from Jurong mostly Methanomicrobiales and only a few RC-I methanogens. The archaeal community of rice field soil from Zhenjiang almost exclusively consisted of Methanosarcinaceae when incubated at high temperature. Our results show that moderately thermophilic methanogens are common in temperate soils. However, RC-I methanogens are not always dominating or ubiquitous.     

Community analysis of methanogenic archaea within a riparian flooding gradient

Anoxic soils in river floodplains (or riparian soils) are a source of methane emission. However, little is known about the ecology and community structure of archaeal methanogenic microbes, which are a crucial component of methane flux in those habitats. We studied the archaeal community in the vertical profile of four different sites along the River Waal in the Netherlands. These sites differ in their annual flooding regime ranging from never or seldom to permanently flooded. The archaeal community structure has been characterized by terminal restriction fragment length polymorphism (T-RFLP) and comparative sequence analysis of the archaeal SSU rRNA gene and the mcrA gene. The latter gene codes for the alpha-subunit of methyl-coenzyme M reductase. Additionally, the potential methanogenic activity was determined by incubation of soil slurries under anoxic conditions. The community composition differed only slightly with the depth of the soil (0-20 cm). However, the diversity of archaeal SSU rRNA genes increased with the frequency of flooding. Terminal restriction fragment length polymorphism analysis of mcrA gene amplicons confirmed the results concerning methanogenic archaea. In the never and rarely flooded soils, crenarchaeotal sequences were the dominant group. In the frequently and permanently flooded soils, Methanomicrobiaceae, Methanobacteriaceae, Methanosarcinaceae and the uncultured Rice Clusters IV and VI (Crenarchaeota) were detectable independently from duration of anoxic conditions. Methanosaetaceae, on the other hand, were only found in the permanently and frequently flooded soils under conditions where concentrations of acetate were < 30 microM. The results indicate that methanogens as well as other archaea occupy characteristic niches according to the flooding conditions in the field. Methanosaetaceae, in particular, seem to be adapted (or proliferate at) to low acetate concentrations.     

Composition of Archaeal Community in a Paddy Field as Affected by Rice Cultivar and N Fertilizer

Methanogenesis in paddy fields is significantly influenced by environmental and field management factors such as rice cultivar and nitrogenous fertilizer. However, it has been unclear whether such effects are reflected in the structure of methanogenic archaeal populations. In the present study, molecular analyses including cloning and sequencing and terminal restriction fragment length polymorphism (T-RFLP) fingerprinting of archaeal 16S rRNA genes were used to characterize the methanogenic archaeal assemblages and to identify the effect of environmental variables including rice cultivar and N fertilizer on archaeal community compositions in a Chinese paddy field soil. The correlation between methanogenic archaeal composition and environmental variables was explored by correspondence analysis. The results showed that the spatial or niche factor (rice roots versus rhizosphere, surface, and the deeper layer soils) had the greatest influence on the archaeal community composition. There was an obvious enrichment or selection of hydrogenotrophic as opposed to acetoclastic methanogens by rice roots. The archaeal community also changed, though slightly, between the rhizosphere and bulk soils and between the surface soil and the deeper layer soil. However, rice cultivar and N fertilizer appear to have an effect only on methanogens tightly associated with rice roots.     

Archaeal population dynamics during sequential reduction processes in rice field soil

The population dynamics of Archaea after flooding of an Italian rice field soil were studied over 17 days. Anoxically incubated rice field soil slurries exhibited a typical sequence of reduction processes characterized by reduction of nitrate, Fe(3+), and sulfate prior to the initiation of methane production. Archaeal population dynamics were followed using a dual approach involving molecular sequence retrieval and fingerprinting of small-subunit (SSU) rRNA genes. We retrieved archaeal sequences from four clone libraries (30 each) constructed for different time points (days 0, 1, 8, and 17) after flooding of the soil. The clones could be assigned to known methanogens (i.e., Methanosarcinaceae, Methanosaetaceae, Methanomicrobiaceae, and Methanobacteriaceae) and to novel euryarchaeotal (rice clusters I, II, and III) and crenarchaeotal (rice clusters IV and VI) lineages previously detected in anoxic rice field soil and on rice roots (R. Grosskopf, S. Stubner, and W. Liesack, Appl. Environ. Microbiol. 64:4983-4989, 1998). During the initiation of methanogenesis (days 0 to 17), we detected significant changes in the frequency of individual clones, especially of those affiliated with the Methanosaetaceae and Methanobacteriaceae. However, these findings could not be confirmed by terminal restriction fragment length polymorphism (T-RFLP) analysis of SSU rDNA amplicons. Most likely, the fluctuations in sequence composition of clone libraries resulted from cloning bias. Clonal SSU rRNA gene sequences were used to define operational taxonomic units (OTUs) for T-RFLP analysis, which were distinguished by group-specific TaqI restriction sites. Sequence analysis showed a high degree of conservation of TaqI restriction sites within the different archaeal lineages present in Italian rice field soil. Direct T-RFLP analysis of archaeal populations in rice field soil slurries revealed the presence of all archaeal lineages detected by cloning with a predominance of terminal restriction fragments characteristic of rice cluster I (389 bp), Methanosaetaceae (280 bp), and Methanosarcinaceae/rice cluster VI (182 bp). In general, the relative gene frequency of most detected OTUs remained rather constant over time during the first 17 days after flooding of the soil. Most minor OTUs (e.g., Methanomicrobiaceae and rice cluster III) and Methanosaetaceae did not change in relative frequency. Rice cluster I (37 to 30%) and to a lesser extent rice cluster IV as well as Methanobacteriaceae decreased over time. Only the relative abundance of Methanosarcinaceae (182 bp) increased, roughly doubling from 15 to 29% of total archaeal gene frequency within the first 11 days, which was positively correlated to the dynamics of acetate and formate concentrations. Our results indicate that a functionally dynamic ecosystem, a rice field soil after flooding, was linked to a relatively stable archaeal community structure.     

Evaluation of PCR amplification bias by terminal restriction fragment length polymorphism analysis of small-subunit rRNA and mcrA genes by using defined template mixtures of methanogenic pure cultures and soil DNA extracts

Terminal restriction fragment length polymorphism (T-RFLP) analysis is a widely used method for profiling microbial community structure in different habitats by targeting small-subunit (SSU) rRNA and also functional marker genes. It is not known, however, whether relative gene frequencies of individual community members are adequately represented in post-PCR amplicon frequencies as shown by T-RFLP. In this study, precisely defined artificial template mixtures containing genomic DNA of four different methanogens in various ratios were prepared for subsequent T-RFLP analysis. PCR amplicons were generated from defined mixtures targeting not only the SSU rRNA but also the methyl-coenzyme M reductase (mcrA/mrtA) genes of methanogens. Relative amplicon frequencies of microorganisms were quantified by comparing fluorescence intensities of characteristic terminal restriction fragments. SSU ribosomal DNA (rDNA) template ratios in defined template mixtures of the four-membered community were recovered absolutely by PCR-T-RFLP analysis, which demonstrates that the T-RFLP analysis evaluated can give a quantitative view of the template pool. SSU rDNA-targeted T-RFLP analysis of a natural community was found to be highly reproducible, independent of PCR annealing temperature, and unaffected by increasing PCR cycle numbers. Ratios of mcrA-targeted T-RFLP analysis were biased, most likely by PCR selection due to the degeneracy of the primers used. Consequently, for microbial community analyses, each primer system used should be evaluated carefully for possible PCR bias. In fact, such bias can be detected by using T-RFLP analysis as a tool for the precise quantification of the PCR product pool.     

Distribution and stability of sulfate-reducing prokaryotic and hydrogenotrophic methanogenic assemblages in nutrient-impacted regions of the Florida Everglades

Although the influence of phosphorus loading on the Everglades ecosystem has received a great deal of attention, most research has targeted macro indicators, such as those based on vegetation or fauna, or chemical and physical parameters involved in biogeochemical cycles. Fewer studies have addressed the role of microorganisms, and these have mainly targeted gross informative parameters such as microbial biomass, enzymatic activities, and microbial enumerations. The objectives of this study were to characterize the dynamics of sulfate-reducing and methanogenic assemblages using terminal restriction fragment length polymorphism (T-RFLP) targeting the dissimilatory sulfite reductase (dsrA) and methyl coenzyme M reductase (mcrA) genes, respectively, and assess the impact of nutrient enrichment on microbial assemblages in the northern Everglades. T-RFLP combined with principal component analysis was a powerful technique to discriminate between soils from sites with eutrophic, transitional, and oligotrophic nutrient concentrations. dsrA T-RFLP provided a higher level of discrimination between the three sites. mcrA was a relatively weaker system to distinguish between sites, since it could not categorically discriminate between eutrophic and transition soil samples, but may be useful as an early indicator of phosphorus loading which is altering hydrogenotrophic methanogenic community in the transition zones, making them more similar to eutrophic zones. Clearly, targeting a combination of different microbial communities provides greater insight into the functioning of this ecosystem and provides useful information for understanding the relationship between eutrophication effects and microbial assemblages.     

Methanogen communities and Bacteria along an ecohydrological gradient in a northern raised bog complex

Mires forming an ecohydrological gradient from nutrient-rich, groundwater-fed mesotrophic and oligotrophic fens to a nutrient-poor ombrotrophic bog were studied by comparing potential methane (CH(4)) production and methanogenic microbial communities. Methane production was measured from different depths of anoxic peat and methanogen communities were detected by detailed restriction fragment length polymorphism (RFLP) analysis of clone libraries, sequencing and phylogenetic analysis. Potential CH(4) production changed along the ecohydrological gradient with the fens displaying much higher production than the ombrotrophic bog. Methanogen diversity also decreased along the gradient. The two fens had very similar diversity of methanogenic methyl-coenzyme M reductase gene (mcrA), but in the upper layer of the bog the methanogen diversity was strikingly lower, and only one type of mcrA sequence was retrieved. It was related to the Fen cluster, a group of novel methanogenic sequences found earlier in Finnish mires. Bacterial 16S rDNA sequences from the fens fell into at least nine phyla, but only four phyla were retrieved from the bog. The most common bacterial groups were Deltaproteobacteria, Verrucomicrobia and Acidobacteria.     

Detecting active methanogenic populations on rice roots using stable isotope probing

Methane is formed on rice roots mainly by CO2 reduction. The present study aimed to identify the active methanogenic populations responsible for this process. Soil-free rice roots were incubated anaerobically under an atmosphere of H2/(13CO2) or N2/(13CO2) with phosphate or carbonate (marble) as buffer medium. Nucleic acids were extracted and fractionated by caesium trifluoroacetate equilibrium density gradient centrifugation after 16-day incubation. Community analyses were performed for gradient fractions using terminal restriction fragment polymorphism analysis (T-RFLP) and sequencing of the 16S rRNA genes. In addition, rRNA was extracted and analysed at different time points to trace the community change during the 16-day incubation. The Methanosarcinaceae and the yet-uncultured archaeal lineage Rice Cluster-I (RC-I) were predominant in the root incubations when carbonate buffer and N2 headspace were used. The analysis of [13C]DNA showed that the relative 16S rRNA gene abundance of RC-I increased whereas that of the Methanosarcinaceae decreased with increasing DNA buoyant density, indicating that members of RC-I were more active than the Methanosarcinaceae. However, an unexpected finding was that RC-I was suppressed in the presence of high H2 concentrations (80%, v/v), which during the early incubation period caused a lower CH4 production compared with that with N2 in the headspace. Eventually, however, CH4 production increased, probably because of the activity of Methanosarcinaceae, which became prevalent. Phosphate buffer appeared to inhibit the activity of the Methanosarcinaceae, resulting in lower CH4 production as compared with carbonate buffer. Under these conditions, Methanobacteriaceae were the prevalent methanogens. Our study suggests that the active methanogenic populations on rice roots change in correspondence to the presence of H2 (80%, v/v) and the type of buffer used in the system.     

Investigation of the methanogen population structure and activity in a brackish lake sediment

The methanogen community in sediment from the edge of a small brackish lake connected to the Beaulieu Estuary (Hampshire, UK) was investigated by analysis of 16S rRNA gene diversity using new methanogen-specific primers plus Archaea-specific primers. 16S rRNA gene primers previously used for polymerase chain reaction (PCR) detection of methanogenic Archaea from a variety of environments were evaluated by in silico testing. The primers displayed variable coverage of the four main orders of methanogens, highlighting the importance of this type of primer evaluation. Three PCR primer sets were designed using novel reverse primers to facilitate specific amplification of the orders Methanomicrobiales/Methanosarcinales, Methanobacteriales and Methanococcales. Diversity of the methanogen functional gene, methyl coenzyme M reductase (mcrA), was also studied. All gene libraries constructed from this sediment indicated that Methanomicrobiales and Methanosarcinales were the only methanogens detected. There was good agreement between the relative sequence abundances in the methanogen-specific 16S rRNA gene library and terminal restriction fragment length polymorphism (T-RFLP) profiling, suggesting that the population was dominated by putative H2 CO2 utilizing Methanomicrobiales, although acetate-utilizing methanogens were also present. The methanogen population analyses were in agreement with methanogenic activity measurements, which indicated that bicarbonate methanogenesis was higher than acetate methanogenesis at all depths measured and overall there was a significant difference (P = 0.001) between the rates of the two pathways. This study demonstrates the utility of new 16S rRNA gene PCR primers targeting specific methanogenic orders, and the combined results suggest that the CO2 reduction pathway dominates methanogenesis in the brackish sediment investigated.     

Modified T-RFLP methods for taxonomic interpretation of T-RF

Terminal restriction fragment length polymorphism (TRFLP) is a method that has been frequently used to survey the microbial diversity of environmental samples and to monitor changes in microbial communities. T-RFLP is a highly sensitive and reproducible procedure that combines a PCR with a labeled primer, restriction digestion of the amplified DNA, and separation of the terminal restriction fragment (T-RF). The reliable identification of T-RF requires the information of nucleotide sequences as well as the size of T-RF. However, it is difficult to obtain the information of nucleotide sequences because the T-RFs are fragmented and lack a priming site of 3'-end for efficient cloning and sequence analysis. Here, we improved on the T-RFLP method in order to analyze the nucleotide sequences of the distinct TRFs. The first method is to selectively amplify the portion of T-RF ligated with specific oligonucleotide adapters. In the second method, the termini of T-RFs were tailed with deoxynucleotides using terminal deoxynucleotidyl transferase (TdT) and amplified by a second round of PCR. The major T-RFs generated from reference strains and from T-RFLP profiles of activated sludge samples were efficiently isolated and identified by using two modified T-RFLP methods. These methods are less time consuming and labor-intensive when compared with other methods. The T-RFLP method using TdT has the advantages of being a simple process and having no limit of restriction enzymes. Our results suggest that these methods could be useful tools for the taxonomic interpretation of T-RFs.     

Identification of methyl coenzyme M reductase A (mcrA) genes associated with methane-oxidizing archaea

Phylogenetic and stable-isotope analyses implicated two methanogen-like archaeal groups, ANME-1 and ANME-2, as key participants in the process of anaerobic methane oxidation. Although nothing is known about anaerobic methane oxidation at the molecular level, the evolutionary relationship between methane-oxidizing archaea (MOA) and methanogenic archaea raises the possibility that MOA have co-opted key elements of the methanogenic pathway, reversing many of its steps to oxidize methane anaerobically. In order to explore this hypothesis, the existence and genomic conservation of methyl coenzyme M reductase (MCR), the enzyme catalyzing the terminal step in methanogenesis, was studied in ANME-1 and ANME-2 archaea isolated from various marine environments. Clone libraries targeting a conserved region of the alpha subunit of MCR (mcrA) were generated and compared from environmental samples, laboratory-incubated microcosms, and fosmid libraries. Four out of five novel mcrA types identified from these sources were associated with ANME-1 or ANME-2 group members. Assignment of mcrA types to specific phylogenetic groups was based on environmental clone recoveries, selective enrichment of specific MOA and mcrA types in a microcosm, phylogenetic congruence between mcrA and small-subunit rRNA tree topologies, and genomic context derived from fosmid sequences. Analysis of the ANME-1 and ANME-2 mcrA sequences suggested the potential for catalytic activity based on conservation of active-site amino acids. These results provide a basis for identifying methanotrophic archaea with mcrA sequences and define a functional genomic link between methanogenic and methanotrophic archaea.     

A novel strategy to extract specific phylogenetic sequence information from community T-RFLP

Cultivation-independent analyses of soil microbial community structures are frequently used to describe microbiological soil characteristics. Semi-automated terminal restriction fragment length polymorphism (T-RFLP) analyses yield high-resolution genetic profiles of highly diverse soil microbial communities and hold great potential for use in routine soil quality monitoring. A serious limitation of T-RFLP analyses has been the inability to reliably affiliate observed terminal restriction fragments (T-RF) to phylogenetic groups. In the study presented here, we were able to overcome this limitation of T-RFLP. With a combination of adapter ligation, fragment size selection, and re-amplification with adapter site specific PCR, we were able to isolate a T-RF-fraction of a narrow size-range containing a T-RF that was significantly more abundant in heavy metal amended soils. Cloning the size-selected T-RF fraction allowed for the efficient isolation of clones containing this specific T-RF. Sequence determination and phylogenetic inference in RDP-II affiliated the sequence to unclassified cyanobacteria. Specific primer design and PCR amplification from bulk soil DNA allowed for independent confirmation of the results from bacterial T-RFLP and T-RF cloning. Our results show that specific T-RFs can be efficiently isolated and identified, and that the adapter ligation approach holds great potential for genetic profiling and for identification of community components of interest.     

Phylogenetic analysis of methyl coenzyme-M reductase detected from the bovine rumen

AIMS: The object of the present study is isolation of methyl coenzyme-M reductase (MCR) genes (mcrA) from the bovine rumen fluid and determination of phylogenetical placements of the genes to investigate mechanisms of methanogenesis in the rumen from a point of view of mcrA genes. METHODS: Genes for methanogen-specific MCR were isolated from the bovine rumen by PCR amplification. The deduced amino acid sequences were fitted to the alignments of mcrA gene products from the referred sequences. SIGNIFICANCE AND IMPACT OF THE STUDY: Although the deduced amino acid sequences of mcrA genes, isolated from the bovine rumen in the present study, were close to that of Methanobrevibacter ruminantium, these amino acid sequences did not fall into known clusters of MCR. The findings suggest that methanogenesis in the rumen would be partially carried out by unknown methanogens.     

Soil type links microbial colonization of rice roots to methane emission

Most of the methane (CH₄) emission from rice fields is derived from plant photosynthates, which are converted to CH₄. Rice cluster I (RC-1) archaea colonizing the rhizosphere were found to be the methanogens responsible for this process. Hence, RC-1 methanogens seem to play a crucial role in emission of the greenhouse gas CH₄. We determined the community composition and activity of methanogens colonizing the roots of eight different rice cultivars after growth on both Italian rice soil and river bank soil, which contained different communities of methanogenic archaea. The community composition was analyzed by terminal restriction fragment length polymorphism and cloning/sequencing of the archaeal 16S rRNA gene and the mcrA gene coding for a subunit of the methyl coenzyme M reductase. When grown on rice field soil, the methanogenic community of the different rice cultivars was always dominated by RC-1 methanogens. In contrast, roots were colonized by Methanomicrobiales when grown on river bank soil, in which RC-1 methanogens were initially not detectable. Roots colonized with Methanomicrobiales compared with RC-1 exhibited lower CH₄ production and CH₄ emission rates. The results show that the type of methanogens colonizing rice roots has a potentially important impact on the global CH₄ cycle.     

Structure and function of methanogens along a short-term restoration chronosequence in the Florida Everglades

The removal of plants and soil to bedrock to eradicate exotic invasive plants within the Hole-in-the-Donut (HID) region, part of the Everglades National Park (Florida), presented a unique opportunity to study the redevelopment of soil and the associated microbial communities in the context of short-term primary succession and ecosystem restoration. The goal of this study was to identify relationships between soil redevelopment and activity and composition of methanogenic assemblages in HID soils. Methane production potentials indicated a general decline in methanogenic activity with restoration age. Microcosm incubations strongly suggested hydrogenotrophic methanogenesis as the most favorable pathway for methane formation in HID soils from all sites. Culture-independent techniques targeting methyl coenzyme M reductase genes (mcrA) were used to assess the dynamics of methanogenic assemblages. Clone libraries were dominated by sequences related to hydrogenotrophic methanogens of the orders Methanobacteriales and Methanococcales and suggested a general decline in the relative abundance of Methanobacteriales mcrA with time since restoration. Terminal restriction fragment length polymorphism analysis indicated methanogenic assemblages remain relatively stable between wet and dry seasons. Interestingly, analysis of soils across the restoration chronosequence indicated a shift in Methanobacteriales populations with restoration age, suggesting genotypic shifts due to site-specific factors.     

Axial differences in community structure of Crenarchaeota and Euryarchaeota in the highly compartmentalized gut of the soil-feeding termite Cubitermes orthognathus

Methanogenesis represents an important electron sink reaction in the hindgut of soil-feeding termites. This is the first comprehensive analysis of the archaeal community structure within the highly compartmentalized intestinal tract of a humivorous insect, combining clonal analysis and terminal restriction fragment (T-RF) length polymorphism (T-RFLP) fingerprinting of the archaeal communities in the different gut compartments of Cubitermes orthognathus. We found that the morphological and physicochemical heterogeneity of the gut is reflected in a large phylogenetic diversity and pronounced axial differences in the composition of the archaeal gut microbiota, notably among those clones or ribotypes that could be assigned to methanogenic taxa. Comparative analysis of the relative frequencies of different archaeal lineages among the small-subunit rRNA gene (SSU rDNA) clones and their corresponding T-RF indicated that the archaeal community in the anterior, extremely alkaline hindgut compartment (P1) consists mainly of members of the Methanosarcinaceae, whereas Methanobacteriaceae and Methanomicrobiales predominate in the subsequent, more posterior compartments (P3/4a and P4b). The relative abundance of Thermoplasmales increased towards the rectum (P5). SSU rDNA sequences representing Crenarchaeota, which have not yet been reported to occur in the intestinal tracts of arthropods, were detected in all gut sections. We discuss how the spatial distribution of methanogenic populations may be linked to axial heterogeneity in the physicochemical gut conditions and to functional adaptations to their respective ecological niches.     

Effect of temperature on carbon and electron flow and on the archaeal community in methanogenic rice field soil

Temperature is an important factor controlling CH(4) production in anoxic rice soils. Soil slurries, prepared from Italian rice field soil, were incubated anaerobically in the dark at six temperatures of between 10 to 37 degrees C or in a temperature gradient block covering the same temperature range at intervals of 1 degrees C. Methane production reached quasi-steady state after 60 to 90 days. Steady-state CH(4) production rates increased with temperature, with an apparent activation energy of 61 kJ mol(-1). Steady-state partial pressures of the methanogenic precursor H(2) also increased with increasing temperature from <0.5 to 3.5 Pa, so that the Gibbs free energy change of H(2) plus CO(2)-dependent methanogenesis was kept at -20 to -25 kJ mol of CH(4)(-1) over the whole temperature range. Steady-state concentrations of the methanogenic precursor acetate, on the other hand, increased with decreasing temperature from <5 to 50 microM. Simultaneously, the relative contribution of H(2) as methanogenic precursor decreased, as determined by the conversion of radioactive bicarbonate to (14)CH(4), so that the carbon and electron flow to CH(4) was increasingly dominated by acetate, indicating that psychrotolerant homoacetogenesis was important. The relative composition of the archaeal community was determined by terminal restriction fragment length polymorphism (T-RFLP) analysis of the 16S rRNA genes (16S rDNA). T-RFLP analysis differentiated the archaeal Methanobacteriaceae, Methanomicrobiaceae, Methanosaetaceae, Methanosarcinaceae, and Rice clusters I, III, IV, V, and VI, which were all present in the rice field soil incubated at different temperatures. The 16S rRNA genes of Rice cluster I and Methanosaetaceae were the most frequent methanogenic groups. The relative abundance of Rice cluster I decreased with temperature. The substrates used by this microbial cluster, and thus its function in the microbial community, are unknown. The relative abundance of acetoclastic methanogens, on the other hand, was consistent with their physiology and the acetate concentrations observed at the different temperatures, i.e., the high-acetate-requiring Methanosarcinaceae decreased and the more modest Methanosaetaceae increased with increasing temperature. Our results demonstrate that temperature not only affected the activity but also changed the structure and the function (carbon and electron flow) of a complex methanogenic system.     

Effect of hydrogen concentration on the hydrogenotrophic methanogenic community structure studied by T-RELP analysis of 16S rRNA gene amplicons

Analysis of length polymorphism of 16S rRNA genes terminal restriction fragments (T-RFLP analysis) was used to monitor the changes in the composition of the population of methanogens in enrichment cultures under high and low hydrogen concentrations. Hydrogen concentration was shown to determine the structure of a methanogenic community. High hydrogen concentration probably favors the hydrogen- and acetate-utilizing representatives of Methanosarcinaceae, while a more diverse methanogenic community is favored by low hydrogen concentrations.     

Enrichment of lignocellulose-degrading microbial communities from natural and engineered methanogenic environments

The aim of this study was to develop an effective bioaugmentation concept for anaerobic digesters treating lignocellulosic biomass such as straw. For that purpose, lignocellulose-degrading methanogenic communities were enriched on wheat straw from cow and goat rumen fluid as well as from a biogas reactor acclimated to lignocellulosic biomass (sorghum as mono-substrate). The bacterial communities of the enriched cultures and the different inocula were examined by 454 amplicon sequencing of 16S rRNA genes while the methanogenic archaeal communities were analyzed by terminal restriction fragment length polymorphism (T-RFLP) fingerprinting of the mcrA gene. Bacteroidetes was the most abundant phylum in all samples. Within the Bacteroidetes phylum, Bacteroidaceae was the most abundant family in the rumen-derived enrichment cultures, whereas Porphyromonadaceae was the predominant one in the reactor-derived culture. Additionally, the enrichment procedure increased the relative abundance of Ruminococcaceae (phylum: Firmicutes) in all cultures. T-RFLP profiles of the mcrA gene amplicons highlighted that the ruminal methanogenic communities were composed of hydrogenotrophic methanogens dominated by the order Methanobacteriales regardless of the host species. The methanogenic communities changed significantly during the enrichment procedure, but still the strict hydrogenotrophic Methanobacteriales and Methanomicrobiales were the predominant orders in the enrichment cultures. The bioaugmentation potential of the enriched methanogenic cultures will be evaluated in further studies.