Archaeal communities in High Arctic wetlands at Spitsbergen, Norway (78 degrees N) as characterized by 16S rRNA gene fingerprinting

Emissions of the greenhouse gas methane from Arctic wetlands have been studied extensively, though little is known about the ecology and community structure of methanogenic archaea that catalyze the methane production. As part of a project addressing microbial transformations of methane in Arctic wetlands, we studied archaeal communities in two wetlands (Solvatnet and Stuphallet) at Spitsbergen, Norway (78 degrees N) during two summer seasons. Directly extracted peat community DNA and enrichment cultures of methanogenic archaea were analyzed by nested PCR combined with denaturing gradient gel electrophoresis and subsequent sequencing of 16S rRNA gene fragments. Sequences affiliated with Methanomicrobiales, Methanobacteriaceae, Methanosaeta and Group I.3b of the uncultured crenarchaeota were detected at both sites. Sequences affiliated with Methanosarcina were recovered only from the site Solvatnet, while sequences affiliated with the euryarchaeotal clusters Rice Cluster II and Sediment 1 were detected only at the site Stuphallet. The phylogenetic affiliation of the recovered sequences suggested a potential of both hydrogenotrophic and acetoclastic methanogenesis at both sites. At Solvatnet, there were clear temporal trends in the archaeal community structure over the Arctic summer season. The archaeal community composition was significantly affected by factors influencing the activity of the overall bacterial community, as measured by in situ emissions of CO2. Methane emissions at both sites were influenced more by peat temperatures and thaw depth than by the archaeal community structure. Enrichment cultures for methanogenic archaea determined that most of the methanogens detected directly in peat could grow in culture at 10 degrees C. Culture based biases were indicated in later enrichment steps by the abundant growth of a Methanosarcina strain that was not detected directly in peat samples.     

Diversity of methanotrophic bacteria in tropical upland soils under different land uses

Three upland soils from Thailand, a natural forest, a 16-year-old reforested site, and an agricultural field, were studied with regard to methane uptake and the community composition of methanotrophic bacteria (MB). The methane uptake rates were similar to rates described previously for forest and farmland soils of the temperate zone. The rates were lower at the agricultural site than at the native forest and reforested sites. The sites also differed in the MB community composition, which was characterized by denaturing gradient gel electrophoresis (DGGE) of pmoA gene fragments (coding for a subunit of particulate methane monooxygenase) that were PCR amplified from total soil DNA extracts. Cluster analysis based on the DGGE banding patterns indicated that the MB communities at the forested and reforested sites were similar to each other but different from that at the farmland site. Sequence analysis of excised DGGE bands indicated that Methylobacter spp. and Methylocystis spp. were present. Sequences of the "forest soil cluster" or "upland soil cluster alpha," which is postulated to represent organisms involved in atmospheric methane consumption in diverse soils, were detected only in samples from the native forest and reforested sites. Additional sequences that may represent uncultivated groups of MB in the Gammaproteobacteria were also detected.     

低温湿地甲烷古菌及其介导的甲烷产生途径

甲烷是重要的温室气体,低温湿地是大气甲烷的重要来源,因为湿地土壤中生活着大量的微生物包括甲烷古菌,它们将有机物降解转化为甲烷.本文总结了近年来低温湿地甲烷古菌群落组成、甲烷产生途径及其与环境的关系.研究显示,乙酸是低温湿地中主要的产甲烷物质,氢产甲烷过程主要发生在中温地区或酸性泥炭土中,而在盐碱水域中甲醇、甲胺是甲烷的重要底物.位于我国青藏高原的若尔盖湿地具有高海拔但低纬度的地理特征,我们的前期研究却显示甲醇在该湿地的甲烷排放中具有重要贡献.相应地,低温湿地中的甲烷古菌主要是利用甲基类化合物/乙酸的甲烷八叠球菌目和氢营养型的甲烷微球菌目.然而不同类型湿地甲烷排放途径及甲烷古菌的差异主要与环境的土壤类型、pH及植被类型相关,如刚毛荸荠生长的若尔盖湿地土壤中来源于甲醇的甲烷占全部甲烷的l7％;而木里苔草土壤中乙酸是产甲烷的主要前体物质.尽管已知冷适应的甲烷古菌在低温湿地的甲烷排放中发挥重要作用,但目前获得培养的嗜冷甲烷古菌却很少.冷响应的组学研究显示甲烷古菌的冷适应涉及到全局性生物学过程.     

The impact of grassland management on archaeal community structure in upland pasture rhizosphere soil

The community structure of rhizosphere soil Archaea from three grassland types, associated with different management practices, was examined at a site in the Borders region of Scotland, by analysis of 16S rRNA gene fragments amplified from 16S rDNA and from rRNA. Denaturing gradient gel electrophoresis (DGGE) and sequence analysis of amplified products indicated high relative abundance within the archaeal community of two distinct lineages of non-thermophilic (group 1) Crenarchaeota. Grassland management practices influenced archaeal community structure, as characterized by both 16S rRNA- and 16S rDNA-derived DGGE profiles. One band dominated DGGE profiles in all three grassland types examined, and reproducible differences in the presence and intensity of bands were observed between profiles from managed and natural grassland sites. Analysis of 16S rRNA-derived amplicons from managed and natural grasslands at sites in the north of England and the north of Wales also indicated high relative abundance of non-thermophilic crenarchaeotes within the archaeal community. The band dominating the Scottish grassland site also dominated DGGE profiles from the English and Welsh sites, and similar differences were seen between profiles derived from soils subjected to different management regimes. The study indicates that grassland archaeal communities are dominated by Crenarchaeota, with closely related members of this lineage ubiquitous in distribution in UK upland pasture, and indicate that management practices influence the nature of the crenarchaeotal community.     

Succession of methanogenic archaea in rice straw incorporated into a Japanese rice field: estimation by PCR-DGGE and sequence analyses

The succession and phylogenetic profiles of methanogenic archaeal communities associated with rice straw decomposition in rice-field soil were studied by polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) analysis followed by 16S rDNA sequencing. Nylon bags containing either leaf sheaths or blades were buried in the plowed layer of a Japanese rice field under drained conditions during the off-crop season and under flooded conditions after transplanting. In addition, rice straw samples that had been buried in the rice field under drained conditions during the off-crop season were temporarily removed during spring plowing and then re-buried in the same rice field under flooded conditions at transplanting. Populations of methanogenic archaea were examined by amplification of the 16S rRNA genes in the DNA extracted from the rice straw samples. No PCR product was produced for samples of leaf sheath or blade prior to burial or after burial under drained conditions, indicating that the methanogen population was very small during decomposition of rice straw under oxic conditions. Many common bands were observed in rice straw samples of leaf sheath and blade during decomposition of rice straw under flooded conditions. Cluster analysis based on DGGE patterns divided methanogenic archaeal communities into two groups before and after the mid-season drainage. Sequence analysis of DGGE bands that were commonly present were closely related to Methanomicrobiales and Rice cluster I. Methanomicrobiales, Rice cluster I and Methanosarcinales were major members before the mid-season drainage, whereas the DGGE bands that characterized methanogenic archaeal communities after the mid-season drainage were closely related to Methanomicrobiales. These results indicate that mid-season drainage affected the methanogenic archaeal communities irrespective of their location on rice straw (sheath and blade) and the previous history of decomposition during the off-crop season.     

Diversity of Methanotrophic Bacteria in Tropical Upland Soils under Different Land Uses

Three upland soils from Thailand, a natural forest, a 16-year-old reforested site, and an agricultural field, were studied with regard to methane uptake and the community composition of methanotrophic bacteria (MB). The methane uptake rates were similar to rates described previously for forest and farmland soils of the temperate zone. The rates were lower at the agricultural site than at the native forest and reforested sites. The sites also differed in the MB community composition, which was characterized by denaturing gradient gel electrophoresis (DGGE) of pmoA gene fragments (coding for a subunit of particulate methane monooxygenase) that were PCR amplified from total soil DNA extracts. Cluster analysis based on the DGGE banding patterns indicated that the MB communities at the forested and reforested sites were similar to each other but different from that at the farmland site. Sequence analysis of excised DGGE bands indicated that Methylobacter spp. and Methylocystis spp. were present. Sequences of the “forest soil cluster” or “upland soil cluster α,” which is postulated to represent organisms involved in atmospheric methane consumption in diverse soils, were detected only in samples from the native forest and reforested sites. Additional sequences that may represent uncultivated groups of MB in the Gammaproteobacteria were also detected.     

Traditional cattle manure application determines abundance, diversity and activity of methanogenic Archaea in arable European soil

Based on lipid analyses, 16S rRNA/rRNA gene single-strand conformation polymorphism fingerprints and methane flux measurements, influences of the fertilization regime on abundance and diversity of archaeal communities were investigated in soil samples from the long-term (103 years) field trial in Bad Lauchst?dt, Germany. The investigated plots followed a gradient of increasing fertilization beginning at no fertilization and ending at the 'cattle manure' itself. The archaeal phospholipid etherlipid (PLEL) concentration was used as an indicator for archaeal biomass and increased with the gradient of increasing fertilization, whereby the concentrations determined for organically fertilized soils were well above previously reported values. Methane emission, although at a low level, were occasionally only observed in organically fertilized soils, whereas the other treatments showed significant methane uptake. Euryarchaeotal organisms were abundant in all investigated samples but 16S rRNA analysis also demonstrated the presence of Crenarchaeota in fertilized soils. Lowest molecular archaeal diversity was found in highest fertilized treatments. Archaea phylogenetically most closely related to cultured methanogens were abundant in these fertilized soils, whereas Archaea with low relatedness to cultured microorganisms dominated in non-fertilized soils. Relatives of Methanoculleus spp. were found almost exclusively in organically fertilized soils or cattle manure. Methanosarcina-related microorganisms were detected in all soils as well as in the cattle manure, but soils with highest organic application rate were specifically dominated by a close phylogenetic relative of Methanosarcina thermophila. Our findings suggest that regular application of cattle manure increased archaeal biomass, but reduced archaeal diversity and selected for methanogenic Methanoculleus and Methanosarcina strains, leading to the circumstance that high organic fertilized soils did not function as a methane sink at the investigated site anymore.     

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.     

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.     

Heavy-machinery traffic impacts methane emissions as well as methanogen abundance and community structure in oxic forest soils

Temperate forest soils are usually efficient sinks for the greenhouse gas methane, at least in the absence of significant amounts of methanogens. We demonstrate here that trafficking with heavy harvesting machines caused a large reduction in CH(4) consumption and even turned well-aerated forest soils into net methane sources. In addition to studying methane fluxes, we investigated the responses of methanogens after trafficking in two different forest sites. Trafficking generated wheel tracks with different impact (low, moderate, severe, and unaffected). We found that machine passes decreased the soils' macropore space and lowered hydraulic conductivities in wheel tracks. Severely compacted soils yielded high methanogenic abundance, as demonstrated by quantitative PCR analyses of methyl coenzyme M reductase (mcrA) genes, whereas these sequences were undetectable in unaffected soils. Even after a year after traffic compression, methanogen abundance in compacted soils did not decline, indicating a stability of methanogens here over time. Compacted wheel tracks exhibited a relatively constant community structure, since we found several persisting mcrA sequence types continuously present at all sampling times. Phylogenetic analysis revealed a rather large methanogen diversity in the compacted soil, and most mcrA gene sequences were mostly similar to known sequences from wetlands. The majority of mcrA gene sequences belonged either to the order Methanosarcinales or Methanomicrobiales, whereas both sites were dominated by members of the families Methanomicrobiaceae Fencluster, with similar sequences obtained from peatland environments. The results show that compacting wet forest soils by heavy machinery causes increases in methane production and release.     

Molecular analysis of methanogenic archaeal communities in managed and natural upland pasture soils

Grassland management influences soil archaeal communities, which appear to be dominated by nonthermophilic crenarchaeotes. To determine whether methanogenic Archaea associated with the Euryarchaeota lineage are also present in grassland soils, anaerobic microcosms containing both managed (improved) and natural (unimproved) grassland rhizosphere soils were incubated for 28 days to encourage the growth of anaerobic Archaea. The contribution of potential methanogenic organisms to the archaeal community was assessed by the molecular analysis of RNA extracted from soil, using primers targeting all Archaea and Euryarchaeota. Archaeal RT‐PCR products were obtained from all anaerobic microcosms. However, euryarchaeal RT‐PCR products (of putative methanogen origin) were obtained only from anaerobic microcosms of improved soil, their presence coinciding with detectable methane production. Sequence analysis of excised denaturing gradient gel electrophoresis (DGGE) bands revealed the presence of euryarchaeal organisms that could not be detected before anaerobic enrichment. These data indicate that nonmethanogenic Crenarchaeota dominate archaeal communities in grassland soil and suggest that management practices encourage euryarchaeal methanogenic activity.     

自然湿地土壤产甲烷菌和甲烷氧化菌多样性的分子检测

自然湿地是CH4排放的重要来源之一。产甲烷菌和甲烷氧化菌是介导自然湿地甲烷循环的重要功能菌群。开展产甲烷菌和甲烷氧化菌多样性的检测研究有助于揭示微生物介导的甲烷循环以及自然湿地甲烷排放的时空异质性。传统基于培养的检测方法已被证实无法充分描述产甲烷菌和甲烷氧化菌的多样性,而分子检测方法为自然湿地土壤产甲烷菌和甲烷氧化菌的多样性检测提供了一种更准确和科学的工具。本文综述了自然湿地土壤产甲烷菌和甲烷氧化菌的定性和定量分子检测方法,包括末端限制性片段长度多态性（T-RFLP）、变性梯度凝胶电泳（DGGE）、荧光原位杂交（FISH）和实时定量PCR（real-time qPCR）,重点分析了分子检测中两类重要的标记基因,总结了不同类型自然湿地产甲烷菌和甲烷氧化菌群落多样性的最新成果,提出了我国在该领域今后应深入研究探讨的一些问题及建议。     

Long-term effects of mineral versus organic fertilizers on activity and structure of the methanotrophic community in agricultural soils

Agricultural practices, such as mineral nitrogen fertilization, have an impact on the soil's ability to oxidize methane, but little is known about the shifts in the methanotrophic community composition associated with these practices. Therefore, the long-term effect of both mineral (NH4NO3) and organic (manure and GFT-compost) fertilizer applications on the soil methanotrophic community activity and structure were investigated. Both high and low affinity methane oxidation rates were lower in the soil treated with mineral fertilizer compared to the other soils. An enhanced nitrate concentration was observed in the mineral fertilized soil but nitrate did not show a direct affect on the high affinity methane oxidation. In contrast, the low affinity methane oxidation was slowed down by increased nitrate concentrations, which suggests a direct effect of nitrate on low affinity methane oxidation. Denaturing gradient gel electrophoresis (DGGE) analysis of 16S rRNA gene fragments specific for methanotrophs revealed a distinct community between the mineral and organic fertilized soils as extra Type I methanotrophic bands (phylotypes) became visible in the organic fertilized soils. These phylotypes were not visible in the patterns of the added organic fertilizers suggesting an indirect effect of the organic fertilizers on the methanotrophic community. Additionally, a molecular analysis was performed after the low affinity methane oxidation test. The enhanced methane concentrations used in the test enriched certain low affinity methanotrophs in the organic fertilized soils but not in the mineral fertilized soil. Supporting the molecular and functional observations, fatty acids characteristic for methanotrophs were less abundant in the soil treated with mineral fertilizer compared to the soil treated with compost. In conclusion, the function and molecular and chemical composition of the methanotrophic community are all altered in soil fertilized with mineral fertilizer.     

Archaeal Communities in Boreal Forest Tree Rhizospheres Respond to Changing Soil Temperatures

Temperature has generally great effects on both the activity and composition of microbial communities in different soils. We tested the impact of soil temperature and three different boreal forest tree species on the archaeal populations in the bulk soil, rhizosphere, and mycorrhizosphere. Scots pine, silver birch, and Norway spruce seedlings were grown in forest humus microcosms at three different temperatures, 7–11.5°C (night–day temperature), 12–16°C, and 16–22°C, of which 12–16°C represents the typical mid-summer soil temperature in Finnish forests. RNA and DNA were extracted from indigenous ectomycorrhiza, non-mycorrhizal long roots, and boreal forest humus and tested for the presence of archaea by nested PCR of the archaeal 16S rRNA gene followed by denaturing gradient gel electrophoresis (DGGE) profiling and sequencing. Methanogenic Euryarchaeota belonging to Methanolobus sp. and Methanosaeta sp. were detected on the roots and mycorrhiza. The most commonly detected archaeal 16S rRNA gene sequences belonged to group I.1c Crenarchaeota, which are typically found in boreal and alpine forest soils. Interestingly, also one sequence belonging to group I.1b Crenarchaeota was detected from Scots pine mycorrhiza although sequences of this group are usually found in agricultural and forest soils in temperate areas. Tree- and temperature-related shifts in the archaeal population structure were observed. A clear decrease in crenarchaeotal DGGE band number was seen with increasing temperature, and correspondingly, the number of euryarchaeotal DGGE bands, mostly methanogens, increased. The greatest diversity of archaeal DGGE bands was detected in Scots pine roots and mycorrhizas. No archaea were detected from humus samples from microcosms without tree seedling, indicating that the archaea found in the mycorrhizosphere and root systems were dependent on the plant host. The detection of archaeal 16S rRNA gene sequences from both RNA and DNA extractions show that the archaeal populations were living and that they may have significant contribution to the methane cycle in boreal forest soil, especially when soil temperatures rise.     

Response of microbial community structure to natural and accelerated hydrarch successions in the boreal wetlands in northern Hokkaido, Japan

We estimated the effect of invading Sasa vegetation and accelerated terrestrialization on the microbial community structure in Sarobetsu-genya wetland (SGW) and Nakanominedaira wetland (NW) (original vegetation, Sphagnum). All examined peat-pore water samples were acidic. Electrical conductivity significantly differed between SGW and NW. Nonmetric multidimensional scaling (NMDS) and analysis of similarity based on denaturing gradient gel electrophoresis (DGGE) band patterns revealed differences in the bacterial community structure between the Sasa and Sphagnum vegetations at a depth of 10 cm in NW. In contrast, the bacterial NMDS profiles at all depths differed between the 2 wetlands rather than between the 2 vegetations. The archaeal community structure significantly differed between the wetlands at depths of 30 and 50 cm. The bacterial diversity index derived from the DGGE profiles significantly differed between the wetlands at all depths. The archaeal diversity index significantly differed between the wetlands at a depth of 50 cm. Sasa invasion affected the microbial community structure in the rhizosphere, up to a depth of 10 cm; this effect differed with the terrestrialization speed. These results suggest that in peat bogs subjected to artificially accelerated terrestrialization, the microbial community changes before the occurrence of the natural hydrarch ecological succession involving ground vegetation.     

Influence of effluent irrigation on community composition and function of ammonia-oxidizing bacteria in soil

The effect of effluent irrigation on community composition and function of ammonia-oxidizing bacteria (AOB) in soil was evaluated, using techniques of molecular biology and analytical soil chemistry. Analyses were conducted on soil sampled from lysimeters and from a grapefruit orchard which had been irrigated with wastewater effluent or fertilizer-amended water (FAW). Specifically, comparisons of AOB community composition were conducted using denaturing gradient gel electrophoresis (DGGE) of PCR-amplified fragments of the gene encoding the alpha-subunit of the ammonia monooxygenase gene (amoA) recovered from soil samples and subsequent sequencing of relevant bands. A significant and consistent shift in the population composition of AOB was detected in soil irrigated with effluent. This shift was absent in soils irrigated with FAW, despite the fact that the ammonium concentration in the FAW was similar. At the end of the irrigation period, Nitrosospira-like populations were dominant in soils irrigated with FAW, while Nitrosomonas-like populations were dominant in effluent-irrigated soils. Furthermore, DGGE analysis of the amoA gene proved to be a powerful tool in evaluating the soil AOB community population and population shifts therein.     

Arbuscular mycorrhizal colonization of semi-aquatic grasses along a wide hydrologic gradient

The effect of soil flooding on arbuscular-mycorrhizal (AM) fungal colonization of wetland plants was investigated using Panicum hemitomon and Leersia hexandra , two semi-aquatic grasses (Graminaceae) that grow along a wide hydrologic gradient in Carolina bay wetlands of the southeastern US coastal plain. Three related investigations were conducted along the dry-to-wet gradient in these wetlands; a field survey of AM fungal root colonization in eight wetlands, monthly monitoring of colonization patterns in P. hemitomon over a growing season, and an inoculum potential bioassay of soils collected along the gradient. The field survey showed that AM fungal colonization was strongly negatively correlated with water depth, but colonization was present in most root samples. The monthly assessment indicated that AM fungal colonization was lowest in plots that were consistently wet but rose as some plots underwent seasonal drying. The inoculum potential assay of dry, intermediate, and wet soils performed under both dry and saturated conditions showed that soils that were wet for >1 yr had the same ability to form mycorrhizas in bait plants as those that had remained dry. These findings suggested that the lower degree of colonization in wet areas observed in the field survey was because of the presence of surface water rather than low numbers of mycorrhizal propagules in the soil. Overall, the results of these investigations show that flooding is partially but not totally inhibitory to AM fungal colonization of wetland grasses.