Metabolic and trophic interactions modulate methane production by Arctic peat microbiota in response to warming.

Arctic permafrost soils store large amounts of soil organic carbon (SOC) that could be released into the atmosphere as methane (CH4) in a future warmer climate. How warming affects the complex microbial network decomposing SOC is not understood. We studied CH4 production of Arctic peat soil microbiota in anoxic microcosms over a temperature gradient from 1 to 30 °C, combining metatranscriptomic, metagenomic, and targeted metabolic profiling. The CH4 production rate at 4 °C was 25% of that at 25 °C and increased rapidly with temperature, driven by fast adaptations of microbial community structure, metabolic network of SOC decomposition, and trophic interactions. Below 7 °C, syntrophic propionate oxidation was the rate-limiting step for CH4 production; above this threshold temperature, polysaccharide hydrolysis became rate limiting. This change was associated with a shift within the functional guild for syntrophic propionate oxidation, with Firmicutes being replaced by Bacteroidetes. Correspondingly, there was a shift from the formate- and H2-using Methanobacteriales to Methanomicrobiales and from the acetotrophic Methanosarcinaceae to Methanosaetaceae. Methanogenesis from methylamines, probably stemming from degradation of bacterial cells, became more important with increasing temperature and corresponded with an increased relative abundance of predatory protists of the phylum Cercozoa. We concluded that Arctic peat microbiota responds rapidly to increased temperatures by modulating metabolic and trophic interactions so that CH4 is always highly produced: The microbial community adapts through taxonomic shifts, and cascade effects of substrate availability cause replacement of functional guilds and functional changes within taxa.

Field-scale tracking of active methane-oxidizing communities in a landfill cover soil reveals spatial and seasonal variability.

Aerobic methane-oxidizing bacteria (MOB) in soils mitigate methane (CH4 ) emissions. We assessed spatial and seasonal differences in active MOB communities in a landfill cover soil characterized by highly variable environmental conditions. Field-based measurements of CH4 oxidation activity and stable-isotope probing of polar lipid-derived fatty acids (PLFA-SIP) were complemented by microarray analysis of pmoA genes and transcripts, linking diversity and function at the field scale. In situ CH4 oxidation rates varied between sites and were generally one order of magnitude lower in winter compared with summer. Results from PLFA-SIP and pmoA transcripts were largely congruent, revealing distinct spatial and seasonal clustering. Overall, active MOB communities were highly diverse. Type Ia MOB, specifically Methylomonas and Methylobacter, were key drivers for CH4 oxidation, particularly at a high-activity site. Type II MOB were mainly active at a site showing substantial fluctuations in CH4 loading and soil moisture content. Notably, Upland Soil Cluster-gamma-related pmoA transcripts were also detected, indicating concurrent oxidation of atmospheric CH4 . Spatial separation was less distinct in winter, with Methylobacter and uncultured MOB mediating CH4 oxidation. We propose that high diversity of active MOB communities in this soil is promoted by high variability in environmental conditions, facilitating substantial removal of CH4 generated in the waste body.

Macroecology of methane-oxidizing bacteria: the ß-diversity of pmoA genotypes in tropical and subtropical rice paddies.

Studies addressing microbial biogeography haveincreased during the past decade, but research onmicrobial distribution patterns is still in its infancies,and many aspects are only poorly understood. Here,we compared the methanotroph community in paddysoils sampled in Indonesia, Vietnam, China and Italy,focusing on the distance­decay relationship.We usedthe pmoA gene as marker for methanotroph diversityin terminal restriction fragment length polymorphism,microarray and pyrosequencing approaches. Wecould observe a significant increase of ß-diversity with geographical distance across continents (12 000 km).Measured environmental parameters explained only asmall amount of data variation, and we found no evidencefor dispersal limitation. Thus, we propose historicalcontingencies being responsible for theobserved patterns. Furthermore, we performed anin-depth analysis of type II methanotroph pmoA distributionat the sequence level. We used ordinationanalysis to project sequence dissimilarities into athree-dimensional space (multidimensional scaling).The ordination suggests that type II methanotrophs inpaddy fields can be divided into five major groups.However, these groups were found to be distributed inall soils independent of the geographic origin. Byincluding tropical field sites (Indonesia and Vietnam)into the analysis, we further observed the firstpaddy fields harbouring a methanotroph communitydepleted in type II methanotrophs.

Crystal structure of cyclo-bis-(µ4-2,2-di-allyl-malonato-κ(6) O (1),O (3):O (3):O (1'),O (3'):O (1'))tetra-kis-(triphenyl-phosphane-κP)tetra-silver(I).

In the tetra-nuclear mol-ecule of the title compound, [Ag4(C9H10O4)2(C18H15P)4], the Ag(I) ion is coordinated by one P and three O atoms in a considerably distorted tetra-hedral environment. The two 2,2-di-allyl-malonate anions bridge four Ag(I) ions in a µ4-(κ(6) O (1),O (3):O (3):O (1'),O (3'):O (1')) mode, setting up an Ag4O8P4 core (point group symmetry -4..) of corner-sharing tetra-hedra. The shortest intra-molecular Ag⋯Ag distance of 3.9510 (3) Šreveals that no direct d (10)⋯d (10) inter-actions are present. Four weak intra-molecular C-H⋯O hydrogen bonds are observed in the crystal structure of the title compound, which most likely stabilize the tetra-nuclear silver core.

Revisiting methanotrophic communities in sewage treatment plants.

The methanotrophic potential in sewage treatment sludge was investigated. We detected a diverse aerobic methanotrophic community that potentially plays a significant role in mitigating methane emission in this environment. The results suggest that community structure was determined by conditions specific to the processes in a sewage treatment plant.

Termites facilitate methane oxidation and shape the methanotrophic community.

Termite-derived methane contributes 3 to 4% to the total methane budget globally. Termites are not known to harbor methane-oxidizing microorganisms (methanotrophs). However, a considerable fraction of the methane produced can be consumed by methanotrophs that inhabit the mound material, yet the methanotroph ecology in these environments is virtually unknown. The potential for methane oxidation was determined using slurry incubations under conditions with high (12%) and in situ (∼0.004%) methane concentrations through a vertical profile of a termite (Macrotermes falciger) mound and a reference soil. Interestingly, the mound material showed higher methanotrophic activity. The methanotroph community structure was determined by means of a pmoA-based diagnostic microarray. Although the methanotrophs in the mound were derived from populations in the reference soil, it appears that termite activity selected for a distinct community. Applying an indicator species analysis revealed that putative atmospheric methane oxidizers (high-indicator-value probes specific for the JR3 cluster) were indicative of the active nest area, whereas methanotrophs belonging to both type I and type II were indicative of the reference soil. We conclude that termites modify their environment, resulting in higher methane oxidation and selecting and/or enriching for a distinct methanotroph population.

A prehistoric tsunami induced long-lasting ecosystem changes on a semi-arid tropical island--the case of Boka Bartol (Bonaire, Leeward Antilles).

The Caribbean is highly vulnerable to coastal hazards. Based on their short recurrence intervals over the intra-American seas, high-category tropical cyclones and their associated effects of elevated storm surge, heavy wave impacts, mudslides and floods represent the most serious threat. Given the abundance of historical accounts and trigger mechanisms (strike-slip motion and oblique collision at the northern and southern Caribbean plate boundaries, submarine and coastal landslides, volcanism), tsunamis must be considered as well. This paper presents interdisciplinary multi-proxy investigations of sediment cores (grain size distribution, carbonate content, loss-on-ignition, magnetic susceptibility, microfauna, macrofauna) from Washington-Slagbaai National Park, NW Bonaire (Leeward Antilles). No historical tsunami is recorded for this island. However, an allochthonous marine layer found in all cores at Boka Bartol reveals several sedimentary criteria typically linked with tsunami deposits. Calibrated (14)C data from these cores point to a palaeotsunami with a maximum age of 3,300 years. Alternative explanations for the creation of this layer, such as inland flooding during tropical cyclones, cannot entirely be ruled out, though in recent times even the strongest of these events on Bonaire did not deposit significant amounts of sediment onshore. The setting of Boka Bartol changed from an open mangrove-fringed embayment into a poly- to hyperhaline lagoon due to the establishment or closure of a barrier of coral rubble during or subsequent to the inferred event. The timing of the event is supported by further sedimentary evidence from other lagoonal and alluvial archives on Bonaire.

Potential of pmoA amplicon pyrosequencing for methanotroph diversity studies.

We analyzed the potential of pmoA amplicon pyrosequencing compared to that of Sanger sequencing with paddy soils as a model environment. We defined operational taxonomic unit (OTU) cutoff values of 7% and 18%, reflecting methanotrophic species and major phylogenetic pmoA lineages, respectively. Major lineages were already well covered by clone libraries; nevertheless, pyrosequencing provided a higher level of diversity at the species level.

Spatial distribution and consequences of contaminants in harbour sediments - A case study from Richards Bay Harbour, South Africa.

Richards Bay Harbour (RBH) is situated in the industrialized area on the northeast coast of South Africa. To decipher recent human activities and accompanying environmental degradation, surface sediment was collected across RBH and analysed for granulometric and elemental composition, microfaunal assemblages, and microplastics. Microplastics occur most abundantly near recreational areas, whereas metal contamination relates to activities at bulk goods terminals from which they are imported or exported. In particular, Cr and Cu concentrations in surface sediment near bulk goods terminals exceed South African sediment quality guidelines. In metal contaminated sediment, bioindicators reflected stress and were noticeably impacted. A transect of short sediment cores reflects spatial and historical metal contamination and allows quantification of the load of metals within the sediment column. The volume of metal (Cr) contaminated sediment was estimated at almost 2 million m3.

Biogeography of wetland rice methanotrophs.

We focused on the functional guild of methane oxidizing bacteria (MOB) as model organisms to get deeper insights into microbial biogeography. The pmoA gene was used as a functional and phylogenetic marker for MOB in two approaches: (i) a pmoA database (> 4000 sequences) was evaluated to obtain insights into MOB diversity in Italian rice paddies, and paddy fields worldwide. The results show a wide geographical distribution of pmoA genotypes that seem to be specifically adapted to paddy fields (e.g. Rice Paddy Cluster 1 and Rice Paddy Cluster 2). (ii) On the smaller geographical scale, we designed a factorial experiment including three different locations, two rice varieties and two habitats (soil and roots) within each of three rice fields. Multivariate analysis of terminal restriction fragment analysis profiles revealed different community patterns at the three field sites, located 10-20 km apart. Root samples were characterized by high abundance of type I MOB whereas the rice variety had no effect. With the agronomical practice being nearly identical, historical contingencies might be responsible for the field site differences. Considering a large reservoir of viable yet inactive MOB cells acting as a microbial seed bank, environmental conditions might have selected and activated a different subset at a time thereby shaping the community.

Impacts of inter- and intralaboratory variations on the reproducibility of microbial community analyses.

With the advent of molecular biological techniques, especially next-generation sequencing and metagenomics, the number of microbial biogeography studies is rapidly increasing. However, these studies involve the synthesis of data generated by different laboratories using different protocols, chemicals, etc., all with inherent biases. The aim of this study was to assess inter- as well as intralaboratory variations in microbial community composition when standardized protocols are applied to a single soil sample. Aliquots from a homogenized soil sample from a rice field in Italy were sent to five participating laboratories. DNA was extracted by two investigators per laboratory using an identical protocol. All DNA samples were sent to one laboratory to perform DNA quantification, quantitative PCR (QPCR), and microarray and denaturing gradient gel electrophoresis (DGGE) analyses of methanotrophic communities. Yields, as well as purity of DNA, were significantly different between laboratories but in some cases also between investigators within the same laboratory. The differences in yield and quality of the extracted DNA were reflected in QPCR, microarray, and DGGE analysis results. Diversity indices (Shannon-Wiener, evenness, and richness) differed significantly between laboratories. The observed differences have implications for every project in which microbial communities are compared in different habitats, even if assessed within the same laboratory. To be able to make sensible comparisons leading to valid conclusions, intralaboratory variation should be assessed. Standardization of DNA extraction protocols and possible use of internal standards in interlaboratory comparisons may help in rendering a "quantifiable" bias.

Methylotetracoccus oryzae Strain C50C1 Is a Novel Type Ib Gammaproteobacterial Methanotroph Adapted to Freshwater Environments.

Methane-oxidizing microorganisms perform an important role in reducing emissions of the greenhouse gas methane to the atmosphere. To date, known bacterial methanotrophs belong to the Proteobacteria, Verrucomicrobia, and NC10 phyla. Within the Proteobacteria phylum, they can be divided into type Ia, type Ib, and type II methanotrophs. Type Ia and type II are well represented by isolates. Contrastingly, the vast majority of type Ib methanotrophs have not been able to be cultivated so far. Here, we compared the distributions of type Ib lineages in different environments. Whereas the cultivated type Ib methanotrophs (Methylococcus and Methylocaldum) are found in landfill and upland soils, lineages that are not represented by isolates are mostly dominant in freshwater environments, such as paddy fields and lake sediments. Thus, we observed a clear niche differentiation within type Ib methanotrophs. Our subsequent isolation attempts resulted in obtaining a pure culture of a novel type Ib methanotroph, tentatively named "Methylotetracoccus oryzae" C50C1. Strain C50C1 was further characterized to be an obligate methanotroph, containing C16:1ω9c as the major membrane phospholipid fatty acid, which has not been found in other methanotrophs. Genome analysis of strain C50C1 showed the presence of two pmoCAB operon copies and XoxF5-type methanol dehydrogenase in addition to MxaFI. The genome also contained genes involved in nitrogen and sulfur cycling, but it remains to be demonstrated if and how these help this type Ib methanotroph to adapt to fluctuating environmental conditions in freshwater ecosystems.IMPORTANCE Most of the methane produced on our planet gets naturally oxidized by a group of methanotrophic microorganisms before it reaches the atmosphere. These microorganisms are able to oxidize methane, both aerobically and anaerobically, and use it as their sole energy source. Although methanotrophs have been studied for more than a century, there are still many unknown and uncultivated groups prevalent in various ecosystems. This study focused on the diversity and adaptation of aerobic methane-oxidizing bacteria in different environments by comparing their phenotypic and genotypic properties. We used lab-scale microcosms to create a countergradient of oxygen and methane for preenrichment, followed by classical isolation techniques to obtain methane-oxidizing bacteria from a freshwater environment. This resulted in the discovery and isolation of a novel methanotroph with interesting physiological and genomic properties that could possibly make this bacterium able to cope with fluctuating environmental conditions.

Synthesis of Mg and Zn diolates and their use in metal oxide deposition.

The synthesis of complexes [M(OCHMeCH2NMeCH2)2] (5, M = Mg; 7, M = Zn) is described. Treatment of MeHNCH2CH2NMeH (1) with 2-methyloxirane (2) gave diol (HOCHMeCH2NMeCH2)2 (3), which upon reaction with equimolar amounts of MR2 (4, M = Mg, R = Bu; 6, M = Zn, R = Et) gave 5 and 7. The thermal behavior and vapor pressure of 5 and 7 were investigated to show whether they are suited as CVD (= chemical vapor deposition) and/or spin-coating precursors for MgO or ZnO layer formation. Thermogravimetric (TG) studies revealed that 5 and 7 decompose between 80-530 °C forming MgO and ZnO as evidenced by PXRD studies. In addition, TG-MS-coupled experiments were carried out with 7 proving that decomposition occurs by M-O, C-O, C-N and C-C bond cleavages, as evidenced from the detection of fragments such as CH4N+, C2H4N+, C2H5N+, CH2O+, C2H2O+ and C2H3O+. The vapor pressure of 7 was measured at 10.4 mbar at 160 °C, while 5 is non-volatile. The layers obtained by CVD are dense and conformal with a somewhat granulated surface morphology as evidenced by SEM studies. In addition, spin-coating experiments using 5 and 7 as precursors were applied. The corresponding MO layer thicknesses are between 7-140 nm (CVD) or 80 nm and 65 nm (5, 7; spin-coating). EDX and XPS measurements confirm the formation of MgO and ZnO films, however, containing 12-24 mol% (CVD) or 5-9 mol% (spin-coating) carbon. GIXRD studies verify the crystalline character of the deposited layers obtained by CVD and the spin-coating processes.

Selective grazing of methanotrophs by protozoa in a rice field soil.

Biological methane oxidation is a key process in the methane cycle of wetland ecosystems. The methanotrophic biomass may be grazed by protozoa, thus linking the methane cycle to the soil microbial food web. In the present study, the edibility of different methanotrophs for soil protozoa was compared. The number of methanotroph-feeding protozoa in a rice field soil was estimated by determining the most-probable number (MPN) using methanotrophs as food bacteria; naked amoebae and flagellates were the dominant protozoa. Among ten methanotrophic strains examined as a food source, seven yielded a number of protozoa comparable with the yield with Escherichia coli [10(4) MPN (g soil dry weight)(-1)], and three out of four Methylocystis spp. yielded significantly fewer numbers [10(2)-10(3) MPN (g soil dry weight)(-1)]. The lower edibility of the Methylocystis spp. was not explained either by their growth phase or by harmful effects on protozoa. Incubation of the soil under methane resulted in a higher number of protozoa actively grazing on methanotrophs, especially on the less-edible group. Protozoa isolated from the soil demonstrated a grazing preference on the different methanotrophs consistent with the results of MPN counts. The results indicate that selective grazing by protozoa may be a biological factor affecting the methanotrophic community in a wetland soil.

Selective stimulation of type I methanotrophs in a rice paddy soil by urea fertilization revealed by RNA-based stable isotope probing.

Methane-oxidizing bacteria (MOB) in soil are not only controlled by their main substrates, methane and oxygen, but also by nitrogen availability. We compared an unfertilized control with a urea-fertilized treatment and applied RNA-stable-isotope-probing to follow activity changes upon fertilization as closely as possible. Nitrogen fertilization of an Italian rice field soil increased the CH4 oxidation rates sevenfold. In the fertilized treatment, isopycnic separation of 13C-enriched RNA became possible after 7 days when 300 micromol 13CH4 g(dry soil)(-1) had been consumed. Terminal-restriction fragment length polymorphism (T-RFLP) fingerprints and clone libraries documented that the type I methanotrophic genera Methylomicrobium and Methylocaldum assimilated 13CH4 nearly exclusively. Although previous studies had shown that the same soil contains a much larger diversity of MOB, including both type I and type II, nitrogen fertilization apparently activated only a small subset of the overall diversity of MOB, type I MOB in particular.

Synthesis of [{AgO2CCH2OMe(PPh3)} n ] and theoretical study of its use in focused electron beam induced deposition.

The synthesis, chemical and physical properties of [{AgO2CCH2OMe} n ] (1) and [{AgO2CCH2OMe(PPh3)} n ] (2) are reported. Consecutive reaction of AgNO3 with HO2CCH2OMe gave 1, which upon treatment with PPh3 produced 2. Coordination compound 2 forms a 1D coordination polymer in the solid state as evidenced by single crystal X-ray structure analysis. The coordination geometry at Ag+ is of the [3 + 1] type, whereby the carboxylate anions act as bridging ligands. The formation of PPh3-Ag(I) coordinative bonds results in distorted T-shaped AgPO2 units, which are stabilized further by an additional O-Ag dative bond. TG and TG-MS measurements show that 1 and 2 decompose at 190-250 °C (1) and 260-300 °C (2) via decarboxylation, involving Ag-P (2), C-C and C-O bond cleavages to give elemental silver as confirmed by PXRD studies. In order to verify if polymeric 2 is suitable as a FEBID precursor for silver deposition, its vapor pressure was determined (p170 °C = 5.318 mbar, ∆Hvap = 126.1 kJ mol-1), evincing little volatility. Also EI and ESI mass spectrometric studies were carried out. The dissociation of the silver(I) compound 2 under typical electron-driven FEBID conditions was studied by DFT (B3LYP) calculations on monomeric [AgO2CCH2OMe(PPh3)]. At an energy of the secondary electrons up to 0.8 eV elimination of PPh3 occurs, giving Ag+ and O2CCH2OMe-. Likewise, by release of PPh3 from [AgO2CCH2OMe(PPh3)] the fragment [AgO2CCH2OMe]- is formed from which Ag+ and O2CCH2OMe- is generated, further following the first fragmentation route. However, at 1.3 eV the initial step is decarboxylation giving [AgCH2OMe(PPh3)], followed by Ag-P and Ag-C bond cleavages.

Crystal structure of (µ-1,4-di-carb-oxy-butane-1,4-di-carboxyl-ato)bis-[bis-(tri-phenyl-phosphane)silver(I)] di-chloro-methane tris-olvate.

The mol-ecular structure of the tetra-kis(tri-phenyl-phosphan-yl)disilver salt of butane-1,1,4,4-tetra-carb-oxy-lic acid, [Ag2(C8H8O8)(C18H15P)4]·3CH2Cl2, crystallizes with one and a half mol-ecules of di-chloro-methane in the asymmetric unit. The coordination complex exhibits an inversion centre through the central CH2-CH2 bond. The Ag(I) atom has a distorted trigonal-planar P2O coordination environment. The packing is characterized by inter-molecular T-shaped π-π inter-actions between the phenyl rings of the PPh3 substituents in neighbouring mol-ecules, forming a ladder-type superstructure parallel to [010]. These ladders are arranged in layers parallel to (101). Intra-molecular hydrogen bonds between the OH group and one O atom of the Ag-bonded carboxyl-ate group results in an asymmetric bidendate coordination of the carboxyl-ate moiety to the Ag(I) ion.

Comparing field and microcosm experiments: a case study on methano- and methylo-trophic bacteria in paddy soil.

Methane-oxidising bacteria (MOB) play an important role in the reduction of methane emissions from rice agriculture. In rice fields, they are subjected to many environmental and field management parameters, which may have a significant impact on their community composition. To study this in greater detail, the community structure of methano- and methylo-trophic bacteria was investigated in a rice field in northern Italy during the summer 1999 and compared to a microcosm study described previously. We used PCR-based denaturing gradient gel electrophoresis applying 16S rDNA (9alpha and 10gamma) and mxaF (methanol-dehydrogenase) primer sets. In parallel, population size and activity of MOB were determined. This study provides the first comprehensive investigation of different compartments (bulk soil, rhizosphere, rhizoplane, and homogenate) throughout an entire rice-growing season in the field. Lower cell numbers of MOB were detected in the field compared to the microcosms, possibly due to lower CH4 concentrations in the soil pore water. In both studies, growth of MOB occurred predominantly at the root surface (rhizoplane) and in the root (homogenate), whereas cell numbers in bulk soil showed only minor changes throughout the season. Molecular analysis detected only few changes in alpha-proteobacterial methylotrophs during the season, whereas a higher variability was detected in gamma-proteobacteria. Nevertheless, the sequences of electrophoretic bands showed that the diversity in the field study and in the microcosms was comparable. Activity patterns of MOB and the population structure of methylotrophic bacteria agreed well between both studies, even though the detected quantities differed. Extrapolations of microcosm data to the field scale are thus possible, but should be used carefully when concerning quantitative changes.

Methanogenic symbionts of anaerobic ciliates and their contribution to methanogenesis in an anoxic rice field soil.

Methanogenesis in rice field soils starts soon after flooding while potentially competing processes like reduction of sulphate and iron take place. Early methanogenesis is mainly driven by hydrogen, while later in the season acetate tends to become more important. Anaerobic ciliates are abundant during this period, and their endosymbionts use hydrogen produced by the ciliates to reduce carbon dioxide to methane. These endosymbiotic methanogens are protected from the competition for substrates with other bacteria that may control methanogenesis outside the protozoan cells. Thus, we focussed on early methanogenesis and on the potential contribution from ciliates and their endosymbionts. Only ciliates of the genus Metopus were found to harbour methanogens, as identified by the F(420)-fluorescence of the endosymbionts. We followed the population dynamics of the ciliates with time, and calculated the ratio of symbiotic methane production to overall methanogenesis. Symbiotic methane production was calculated from the species-specific numbers of methanogenic endosymbionts times the cell-specific methane production of the symbionts. According to this calculation, the symbionts' contribution to overall methane production was only 6.4% at the beginning and decreased with time. In a second experiment, colchicine and cycloheximide were used to inhibit all eukaryotes, comparing the remaining methane production rate to a control without inhibitors. In the inhibition experiment, the contribution from symbionts decreased from 40% to 6% during the first days after flooding, and dropped to near zero within 2 weeks. However, nearly all methane produced from H(2)/CO(2) could be attributed to the ciliates' symbionts between days 5 and 10 after flooding. Both experiments showed that the contribution of methanogenic symbionts to overall methane production is a transient phenomenon, restricted to the first 2 weeks.

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.