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.     

Effect of Temperature on Carbon and Electron Flow and on the Archaeal Community in Methanogenic Rice Field Soil

Temperature is an important factor controlling CH₄ 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°C or in a temperature gradient block covering the same temperature range at intervals of 1°C. Methane production reached quasi-steady state after 60 to 90 days. Steady-state CH₄ production rates increased with temperature, with an apparent activation energy of 61 kJ mol⁻¹. Steady-state partial pressures of the methanogenic precursor H₂ also increased with increasing temperature from <0.5 to 3.5 Pa, so that the Gibbs free energy change of H₂ plus CO₂-dependent methanogenesis was kept at −20 to −25 kJ mol of CH₄⁻¹ 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 μM. Simultaneously, the relative contribution of H₂ as methanogenic precursor decreased, as determined by the conversion of radioactive bicarbonate to ¹⁴CH₄, so that the carbon and electron flow to CH₄ 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 Temperature on Structure and Function of the Methanogenic Archaeal Community in an Anoxic Rice Field Soil

Soil temperatures in Italian rice fields typically range between about 15 and 30°C. A change in the incubation temperature of anoxic methanogenic soil slurry from 30°C to 15°C typically resulted in a decrease in the CH₄ production rate, a decrease in the steady-state H₂ partial pressure, and a transient accumulation of acetate. Previous experiments have shown that these changes were due to an alteration of the carbon and electron flow in the methanogenic degradation pathway of organic matter caused by the temperature shift (K. J. Chin and R. Conrad, FEMS Microbiol. Ecol. 18:85–102, 1995). To investigate how temperature affects the structure of the methanogenic archaeal community, total DNA was extracted from soil slurries incubated at 30 and 15°C. The archaeal small-subunit (SSU) rRNA-encoding genes (rDNA) of these environmental DNA samples were amplified by PCR with an archaeal-specific primer system and used for the generation of clone libraries. Representative rDNA clones (n = 90) were characterized by terminal restriction fragment length polymorphism (T-RFLP) and sequence analysis. T-RFLP analysis produced for the clones terminally labeled fragments with a characteristic length of mostly 185, 284, or 392 bp. Sequence analysis allowed determination of the phylogenetic affiliation of the individual clones with their characteristic T-RFLP fragment lengths and showed that the archaeal community of the anoxic rice soil slurry was dominated by members of the families Methanosarcinaceae (185 bp) and Methanosaetaceae (284 bp), the kingdom Crenarchaeota (185 or 284 bp), and a novel, deeply branching lineage of the (probably methanogenic) kingdom Euryarchaeota (392 bp) that has recently been detected on rice roots (R. Großkopf, S. Stubner, and W. Liesack, Appl. Environ. Microbiol. 64:4983–4989, 1998). The structure of the archaeal community changed when the temperature was shifted from 30°C to 15°C. Before the temperature shift, the clones (n = 30) retrieved from the community were dominated by Crenarchaeota (70%), “novel Euryarchaeota” (23%), and Methanosarcinacaeae (7%). Further incubation at 30°C (n = 30 clones) resulted in a relative increase in members of the Methanosarcinaceae (77%), whereas further incubation at 15°C (n = 30 clones) resulted in a much more diverse community consisting of 33% Methanosarcinaceae, 23% Crenarchaeota, 20% Methanosaetaceae, and 17% novel Euryarchaeota. The appearance of Methanosaetaceae at 15°C was conspicuous. These results demonstrate that the structure of the archaeal community in anoxic rice field soil changed with time and incubation temperature.     

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.     

Shift from Acetoclastic to H2-Dependent Methanogenesis in a West Siberian Peat Bog at Low pH Values and Isolation of an Acidophilic Methanobacterium Strain

Methane production and archaeal community composition were studied in samples from an acidic peat bog incubated at different temperatures and pH values. H₂-dependent methanogenesis increased strongly at the lowest pH, 3.8, and Methanobacteriaceae became important except for Methanomicrobiaceae and Methanosarcinaceae. An acidophilic and psychrotolerant Methanobacterium sp. was isolated using H₂-plus-CO₂-supplemented medium at pH 4.5.     

Thermophilic methanogens in rice field soil

The soil temperature in flooded Italian rice fields is generally lower than 30°C. However, two temperature optima at ≈ 41°C and 50°C were found when soil slurries were anoxically incubated at a temperature range of 10–80°C. The second temperature optimum indicates the presence of thermophilic methanogens in the rice field soil. Experiments with ¹⁴C-labelled bicarbonate showed that the thermophilic CH₄ was exclusively produced from H₂/CO₂. Terminal restriction fragment length polymorphism (T-RFLP) of archaeal SSU rRNA gene fragments revealed a dramatic change in the archaeal community structure at temperatures > 37°C, with the euryarchaeotal rice cluster I becoming the dominant group (about 80%). A clone library of archaeal SSU rRNA gene fragments generated at 49°C was also dominated (10 out of 11 clones) by rice cluster I. Our results demonstrate that Italian rice field soil contains thermophilic methanogenic activity that was most probably a result of members of the as yet uncultivated euryarchaeotal rice cluster I.     

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.     

Methane Biogeochemistry and Methanogen Communities in Two Northern Peatland Ecosystems,New York State

Rates of methane (CH₄) production vary considerably among northern peat-forming wetlands, and it is not clear whether variability is caused by environmental factors affecting CH₄ production or differences in methanogen communities. We investigated CH₄ production and emission dynamics concomitantly with 16S rRNA gene sequence-based community analysis of Archaea in two contrasting peat-forming northern wetlands, an ombrotrophic bog and a minerotrophic conifer swamp. Individual measurements of CH₄ emissions to the atmosphere followed a lognormal distribution pattern in both sites, and mean rates were 30× greater in the bog site. Rates of CH₄ production measured in vitro were initially 3× greater in the bog than in the conifer swamp; although, after 30 days of incubation, production rates were similar suggesting that in situ environmental conditions limited production in the conifer swamp. Amplified ribosomal DNA restriction analysis (ARDRA) and rarefaction techniques indicated that both sites had similar levels of archaeal richness, with 27 unique taxa in the bog and 23 taxa in the conifer swamp. However, the bog had more pronounced dominance of a few taxa, whereas the conifer swamp had more even distribution among taxa. A 16S rRNA gene sequence-based phylogenetic analysis indicated high levels of diversity with similarity to known methanogenic families Methanosarcinaceae, Methanosaetaceae, Methanobacteriaceae, and likely Methanomicrobiaceae as well as two additional lineages previously characterized as groups of yet uncultivated Euryarchaeota commonly occurring in flooded rice soils. Therefore, sites with low and high rates of CH₄ production supported very diverse methanogenic communities.     

Molecular analyses of methyl-coenzyme M reductase alpha-subunit (mcrA) genes in rice field soil and enrichment cultures reveal the methanogenic phenotype of a novel archaeal lineage

The diversity of methanogen-specific methyl-coenzyme M reductase alpha-subunit (mcrA/mrtA) genes in Italian rice field soil was analysed using a combination of molecular techniques and enrichment cultures. From 75 mcrA/mrtA clones retrieved from rice field soil, 52 were related to members of the Methanosarcinaceae, Methanosaetaceae and Methanobacteriaceae. However, 19 and four clones formed two novel clusters of deeply branching mcrA sequences, respectively, which could not be affiliated to known methanogens. A new methanogen-specific fingerprinting assay based on terminal restriction fragment length polymorphism (T-RFLP) analysis of fluorescently labelled polymerase chain reaction (PCR) products allowed us to distinguish all environmental mcrA/mrtA sequences via group-specific Sau96I restriction sites. Even genes for the isoenzyme methyl-coenzyme M reductase two (mrtA) of Methanobacteriaceae present in rice field soil were represented by a unique 470 bp terminal restriction fragment (T-RF). Both cloning and T-RFLP analysis indicated a significant representation of novel environmental mcrA sequences in rice field soil (238 bp T-RF). To identify these mcrA sequences, methanogenic enrichment cultures with rice field soil as inoculum were established with H2/CO2 as substrates at a temperature of 50 degrees C, and these were monitored using molecular tools. In subsequent transfers of these enrichment cultures, cloning and T-RFLP analysis detected predominantly SSU rRNA genes of rice cluster I (RC-I), an uncultivated euryarchaeotal lineage discovered previously in anoxic rice field soil. In parallel, both mcrA cloning and T-RFLP analyses of the enrichment culture identified the more frequent cluster of novel environmental mcrA sequences as belonging to members of RC-I. Thus, we could demonstrate the genotype and phenotype of RC-I Archaea by the presence of a catabolic gene in a methanogenic enrichment culture before the isolation of pure cultures.     

Competing formate- and carbon dioxide-utilizing prokaryotes in an anoxic methane-emitting fen soil

Methanogenesis in wetlands is dependent on intermediary substrates derived from the degradation of biopolymers. Formate is one such substrate and is stimulatory to methanogenesis and acetogenesis in anoxic microcosms of soil from the fen Schlöppnerbrunnen. Formate dissimilation also yields CO₂ as a potential secondary substrate. The objective of this study was to resolve potential differences between anaerobic formate- and CO₂-utilizing prokaryotes of this fen by stable isotope probing. Anoxic soil microcosms were pulsed daily with low concentrations of [¹³C]formate or ¹³CO₂ (i.e., [¹³C]bicarbonate). Taxa were evaluated by assessment of 16S rRNA genes, mcrA (encoding the alpha-subunit of methyl-coenzyme M reductase), and fhs (encoding formyltetrahydrofolate synthetase). Methanogens, acetogens, and formate-hydrogen lyase-containing taxa appeared to compete for formate. Genes affiliated with Methanocellaceae, Methanobacteriaceae, Acetobacteraceae, and Rhodospirillaceae were ¹³C enriched (i.e., labeled) in [¹³C]formate treatments, whereas genes affiliated with Methanosarcinaceae, Conexibacteraceae, and Solirubrobacteraceae were labeled in ¹³CO₂ treatments. [¹³C]acetate was enriched in [¹³C]formate treatments, but labeling of known acetogenic taxa was not detected. However, several phylotypes were affiliated with acetogen-containing taxa (e.g., Sporomusa). Methanosaetaceae-affiliated methanogens appeared to participate in the consumption of acetate. Twelve and 58 family-level archaeal and bacterial 16S rRNA phylotypes, respectively, were detected, approximately half of which had no isolated representatives. Crenarchaeota constituted half of the detected archaeal 16S rRNA phylotypes. The results highlight the unresolved microbial diversity of the fen Schlöppnerbrunnen, suggest that differing taxa competed for the same substrate, and indicate that Methanocellaceae, Methanobacteriaceae, Methanosarcinaceae, and Methanosaetaceae were linked to the production of methane, but they do not clearly resolve the taxa responsible for the apparent conversion of formate to acetate.     

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.     

Effect of temperature on structure and function of the methanogenic archaeal community in an anoxic rice field soil.

Soil temperatures in Italian rice fields typically range between about 15 and 30 degrees C. A change in the incubation temperature of anoxic methanogenic soil slurry from 30 degrees C to 15 degrees C typically resulted in a decrease in the CH4 production rate, a decrease in the steady-state H2 partial pressure, and a transient accumulation of acetate. Previous experiments have shown that these changes were due to an alteration of the carbon and electron flow in the methanogenic degradation pathway of organic matter caused by the temperature shift (K. J. Chin and R. Conrad, FEMS Microbiol. Ecol. 18:85-102, 1995). To investigate how temperature affects the structure of the methanogenic archaeal community, total DNA was extracted from soil slurries incubated at 30 and 15 degrees C. The archaeal small-subunit (SSU) rRNA-encoding genes (rDNA) of these environmental DNA samples were amplified by PCR with an archaeal-specific primer system and used for the generation of clone libraries. Representative rDNA clones (n = 90) were characterized by terminal restriction fragment length polymorphism (T-RFLP) and sequence analysis. T-RFLP analysis produced for the clones terminally labeled fragments with a characteristic length of mostly 185, 284, or 392 bp. Sequence analysis allowed determination of the phylogenetic affiliation of the individual clones with their characteristic T-RFLP fragment lengths and showed that the archaeal community of the anoxic rice soil slurry was dominated by members of the families Methanosarcinaceae (185 bp) and Methanosaetaceae (284 bp), the kingdom Crenarchaeota (185 or 284 bp), and a novel, deeply branching lineage of the (probably methanogenic) kingdom Euryarchaeota (392 bp) that has recently been detected on rice roots (R. Grosskopf, S. Stubner, and W. Liesack, Appl. Environ. Microbiol. 64:4983-4989, 1998). The structure of the archaeal community changed when the temperature was shifted from 30 degrees C to 15 degrees C. Before the temperature shift, the clones (n = 30) retrieved from the community were dominated by Crenarchaeota (70%), "novel Euryarchaeota" (23%), and Methanosarcinacaeae (7%). Further incubation at 30 degrees C (n = 30 clones) resulted in a relative increase in members of the Methanosarcinaceae (77%), whereas further incubation at 15 degrees C (n = 30 clones) resulted in a much more diverse community consisting of 33% Methanosarcinaceae, 23% Crenarchaeota, 20% Methanosaetaceae, and 17% novel Euryarchaeota. The appearance of Methanosaetaceae at 15 degrees C was conspicuous. These results demonstrate that the structure of the archaeal community in anoxic rice field soil changed with time and incubation temperature.     

Impact of Virioplankton on Archaeal and Bacterial Community Richness as Assessed in Seawater Batch Cultures

During cruises in the tropical Atlantic Ocean (January to February 2000) and the southern North Sea (December 2000), experiments were conducted to monitor the impact of virioplankton on archaeal and bacterial community richness. Prokaryotic cells equivalent to 10 to 100% of the in situ abundance were inoculated into virus-free seawater, and viruses equivalent to 35 to 360% of the in situ abundance were added. Batch cultures with microwave-inactivated viruses and without viruses served as controls. The apparent richness of archaeal and bacterial communities was determined by terminal restriction fragment length polymorphism (T-RFLP) analysis of PCR-amplified 16S rRNA gene fragments. Although the estimated richness of the prokaryotic communities generally was greatly reduced within the first 24 h of incubation due to confinement, the effects of virus amendment were detected at the level of individual operational taxonomic units (OTUs) in the T-RFLP patterns of both groups, Archaea and Bacteria. One group of OTUs was detected in the control samples but was absent from the virus-treated samples. This negative response of OTUs to virus amendment probably was caused by viral lysis. Additionally, we found OTUs not responding to the amendments, and several OTUs exhibited variable responses to the addition of inactive or active viruses. Therefore, we conclude that individual members of pelagic archaeal and bacterial communities can be differently affected by the presence of virioplankton.     

Mariana Forearc Serpentinite Mud Volcanoes Harbor Novel Communities of Extremophilic Archaea

Extremophilic archaeal communities living in serpentinized muds influenced by pH 12.5 deep-slab derived fluids were detected and their richness and relatedness assessed from across seven serpentinite mud volcanoes located along the Mariana forearc. In addition, samples from two near surface core sections (Holes D and E) at ODP Site 1200 from South Chamorro were subjected to SSU rDNA clone library and phylogenetic analysis resulting in the discovery of several novel operational taxonomic units (OTUs). Five dominant OTUs of Archaea from Hole 1200D and six dominant OTUs of Archaea from Hole 1200E were determined by groups having three or more clones. Terminal-restriction fragment length polymorphism (T-RFLP) analysis revealed all of the dominant OTUs were detected within both clone libraries. Cluster analysis of the T-RFLP data revealed archaeal community structures from sites on Big Blue and Blue Moon to be analogous to the South Chamorro Hole 1200E site. These unique archaeal community fingerprints resulted from an abundance of potential methane-oxidizing and sulfate-reducing phylotypes. This study used deep-sea sediment coring techniques across seven different mud volcanoes along the entire Mariana forearc system. The discovery and detection of both novel Euryarchaeota and Marine Benthic Group B Crenarcheaota phylotypes could be efficacious archaeal indicator populations involved with anaerobic methane oxidation (AMO) and sulfate reduction fueled by deep subsurface serpentinization reactions.     

Diversity and Community Structure of Archaea Inhabiting the Rhizoplane of Two Contrasting Plants from an Acidic Bog

Plant root exudates increase nutrient availability and influence microbial communities including archaeal members. We examined the archaeal community inhabiting the rhizoplane of two contrasting vascular plants, Dulichium arundinaceum and Sarracenia purpurea, from an acidic bog in upstate NY. Multiple archaeal 16S rRNA gene libraries showed that methanogenic Archaea were dominant in the rhizoplane of both plants. In addition, the community structure (evenness) of the rhizoplane was found markedly different from the bulk peat. The archaeal community in peat from the same site has been found dominated by the E2 group, meanwhile the rhizoplane communities on both plants were co-dominated by Methanosarcinaceae (MS), rice cluster (RC)-I, and E2. Complementary T-RFLP analysis confirmed the difference between bulk peat and rhizoplane, and further characterized the dominance pattern of MS, RC-I, and E2. In the rhizoplane, MS was dominant on both plants although as a less variable fraction in S. purpurea. RC-I was significantly more abundant than E2 on S. purpurea, while the opposite was observed on D. arundinaceum, suggesting a plant-specific enrichment. Also, the statistical analyses of T-RFLP data showed that although both plants overlap in their community structure, factors such as plant type, patch location, and time could explain nearly a third of the variability in the dataset. Other factors such as water table, plant replicate, and root depth had a low contribution to the observed variance. The results of this study illustrate the general effects of roots and the specific effects of plant types on their nearby archaeal communities which in bog-inhabiting plants were mainly composed by methanogenic groups.     

Archaeal and Bacterial Communities Associated with the Surface Mucus of Caribbean Corals Differ in Their Degree of Host Specificity and Community Turnover Over Reefs

Comparative studies on the distribution of archaeal versus bacterial communities associated with the surface mucus layer of corals have rarely taken place. It has therefore remained enigmatic whether mucus-associated archaeal and bacterial communities exhibit a similar specificity towards coral hosts and whether they vary in the same fashion over spatial gradients and between reef locations. We used microbial community profiling (terminal-restriction fragment length polymorphism, T-RFLP) and clone library sequencing of the 16S rRNA gene to compare the diversity and community structure of dominant archaeal and bacterial communities associating with the mucus of three common reef-building coral species (Porites astreoides, Siderastrea siderea and Orbicella annularis) over different spatial scales on a Caribbean fringing reef. Sampling locations included three reef sites, three reef patches within each site and two depths. Reference sediment samples and ambient water were also taken for each of the 18 sampling locations resulting in a total of 239 samples. While only 41% of the bacterial operational taxonomic units (OTUs) characterized by T-RFLP were shared between mucus and the ambient water or sediment, for archaeal OTUs this percentage was 2-fold higher (78%). About half of the mucus-associated OTUs (44% and 58% of bacterial and archaeal OTUs, respectively) were shared between the three coral species. Our multivariate statistical analysis (ANOSIM, PERMANOVA and CCA) showed that while the bacterial community composition was determined by habitat (mucus, sediment or seawater), host coral species, location and spatial distance, the archaeal community composition was solely determined by the habitat. This study highlights that mucus-associated archaeal and bacterial communities differ in their degree of community turnover over reefs and in their host-specificity.     

Characterization of the bacterial and archaeal communities in rice field soils subjected to long-term fertilization practices

The bacterial and archaeal communities in rice field soils subjected to different fertilization regimes for 57 years were investigated in two different seasons, a non-planted, drained season (April) and a rice-growing, flooded season (August), by performing soil dehydrogenase assay, real-time PCR assay and pyrosequencing analysis. All fertilization regimes increased the soil dehydrogenase activity while the abundances of bacteria and archaea increased in the plots receiving inorganic fertilizers plus compost and not in those receiving inorganic fertilizers only. Rice-growing and flooding decreased the soil dehydrogenase activity while they increased the bacterial diversity in rice field soils. The bacterial communities were dominated by Chloroflexi, Proteobacteria, and Actinobacteria and the archaeal communities by Crenarchaeota at the phylum level. In principal coordinates analysis based on the weighted Fast UniFrac metric, the bacterial and archaeal communities were separated primarily by season, and generally distributed along with soil pH, the variation of which had been caused by long-term fertilization. Variations in the relative abundance according to the season or soil pH were observed for many bacterial and archaeal groups. In conclusion, the microbial activity, prokaryotic abundance and diversity, and prokaryotic community structure in the rice field soils were changed by season and long-term fertilization.     

Activity, structure and dynamics of the methanogenic archaeal community in a flooded Italian rice field

Methane production was studied in an Italian rice field over two consecutive years (1998, 1999) by measuring the rates of total and acetate-dependent methanogenesis in soil and root samples. Population dynamics of methanogens were followed by terminal restriction fragment length polymorphism and real-time PCR targeting archaeal SSU rRNA genes. Rates of total and acetate-dependent methanogenesis in soil increased during the season, reached a maximum at about 70-80 days after flooding and then decreased again. In contrast, the size of the archaeal community remained relatively constant. Therefore, the seasonal changes in the methanogenic processes were probably not caused by changes in the size of the methanogenic community but in its activity. During the 1998/1999 winter period, a slight decrease in archaeal cell numbers was found. In both years, the dominant groups were methanogens affiliated with Rice cluster I, Methanosaetaceae, Methanosarcinaceae and Methanobacteriaceae. Correspondence analysis showed, however, that the archaeal community structure was different in 1998 and 1999. Methanogens with potential acetoclastic activity made up a larger fraction of the total archaeal community in 1999 (32-53%) than in 1998 (20-32%). Furthermore, the frequency of Methanosaetaceae relative to Methanosarcinaceae was significantly higher in 1999 than in 1998. This difference could be explained by the much lower soil acetate concentrations in 1999, to which Methanosaetaceae are physiologically better adapted than Methanosarcinaceae. Over the season, however, the composition of the archaeal community remained relatively constant and thus did not reflect the observed seasonal change in CH(4) production activity. The analysis of rice root samples in 1999 showed that the archaeal community structure on the roots was similar to that in soil but with acetoclastic methanogens being relatively less common. This observation is in agreement with domination of CH(4) production by H(2)/CO(2)-dependent methanogenesis on roots. Our study provided a link between size, structure and function of the methanogenic community in an Italian rice field.     

Application of New Primer-Enzyme Combinations to Terminal Restriction Fragment Length Polymorphism Profiling of Bacterial Populations in Human Feces

New primer-enzyme combinations for terminal restriction fragment length polymorphism (T-RFLP) targeting of the 16S rRNA gene were constructed by using the T-RFLP analysis program (designated TAP T-RFLP) located at the Ribosomal Database Project website, and their performance was examined empirically. By using the fluorescently labeled 516f primer (Escherichia coli positions 516 to 532) and 1510r primer (positions 1510 to 1492), the 16S rRNA gene was amplified from human fecal DNA. The resulting amplified product was digested with RsaI plus BfaI or with BslI. When the T-RFLP was carried out with fecal DNAs from eight individuals, eight predominant operational taxonomic units (OTUs) were detected with RsaI and BfaI digestion and 14 predominant OTUs were detected with BslI digestion. The distribution of the OTUs was consistent with the results of the computer simulations with TAP T-RFLP. The T-RFLP analyses of the fecal DNAs from individuals gave characteristic profiles, while the variability of the T-RFLP profiles between duplicate DNA preparations from the same samples were minimal. This new T-RFLP method made it easy to predict what kind of intestinal bacterial group corresponded to each OTU on the basis of the terminal restriction fragment length compared with the conventional T-RFLP and, moreover, made it possible to identify the bacterial species that an OTU represents by cloning and sequencing.     

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.