Grazing affects methanotroph activity and diversity in an alpine meadow soil

ArticleinEnvironmental Microbiology Reports 1(5):457-65 · October 2009with 440 Reads 
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Abstract
The role of methane-oxidizing bacteria (MOB) in alpine environments is poorly understood, but is of importance given the abundance of alpine environments and the role of MOB in the global carbon cycle. Using a combination of approaches we examined both seasonal and land usage effects on the ecology of microbial methane oxidation in an alpine meadow soil. Analysis of the abundance and diversity of MOB demonstrated that the abundance and diversity of the dominant type II MOB, predominantly Metylocystis and relatives, was only influenced by season. Conversely type Ia MOB abundance was significantly affected by season and land usage, while diversity changes were effected predominantly by land use. Assessment of methane oxidation potential and soil physical properties demonstrated a strong link between type Ia MOB abundance and methane oxidation potential as well as a complex series of relationships between soil moisture, pH and MOB abundance, changing with season. The results of this study suggest that, while type II MOB, unaffected by land use, represent the dominant MOB, Methylobacter-related type Ia MOB appear to be responsible for the majority of methane oxidation and are strongly affected by the grazing of cattle.

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  • ... Compared with the ungrazed control, the abundance of pmoA gene significantly decreased in G1 soil, while increased in G3 soil (Fig. 1a). Abell et al. (2009) found that the abundance of the predominant type I methanotrophs was positively affected by long-term cattle grazing in an alpine meadow soil. In contrast, no significant change of methanotroph abundance with grazing was observed in an alpine meadow (Zheng et al. 2012). ...
    ... Most of the active methanotrophs (> 90%) were closely related to the type I methanotroph, Methylobacter luteus (Fig. 5), a species originally isolated from a sewage (Bowman et al. 1993;Romanovskaia et al. 1978). Methylobacter-related type I methanotrophs have been reported to be responsible for the majority of methane oxidation in a long-term grazing site in Austria and also in six grazed grassland soils across New Zealand (Abell et al. 2009;Di et al. 2010). The mean annual temperature of 0.3°C and maximum monthly mean temperatures of 19°C in the studied field favored the growth of Methylobacter-related type I methanotrophs, as Methylobacter species have been reported to prefer cold environments such as the active layer of Arctic permafrost (Liebner et al. 2009), high Arctic wetlands (Graef et al. 2011), lake sediments (He et al. 2012), plateau wetlands (Deng and Dumont 2016), and rice fields from cold regions (Sultana et al. 2019). ...
    ... Animal grazing alters soil water and energy balance by reducing vegetation, increasing soil compaction, or reducing soil aeration by trampling and also soil chemical properties (e.g., pH and organic matter content), which would subsequently induce variation of microbial communities and activity Liu et al. 2019b;Lu et al. 2019;Pan et al. 2018c;Saggar et al. 2004;Steffens et al. 2008;Yu et al. 2018). Numerous studies have estimated the impact of grazing on either methanotroph communities or methanotroph activity (CH 4 uptake), even though the abundance and composition of methanotrophs may not necessarily reflect their activity (Abell et al. 2009;Savian et al. 2014;Van den Pol-van Dasselaar et al. 1999;Zheng et al. 2012). In this study, DNA-SIP was used to link the identity and function of methanotrophs in grazed grassland soils. ...
    Article
    The effect of grazing on the abundance, composition, and methane (CH 4) uptake of methanotrophs in grasslands has been well documented in the past few decades, but the dominant communities of active methanotrophs responsible for CH 4 oxidation activity in grazed soils are still poorly understood. In this study, we characterized the metabolically active, aerobic methanotrophs in grasslands with three different levels of grazing (light, medium, and heavy) by combining DNA-stable isotope probing (SIP) and quantitative PCR (qPCR) for methane monooxygenase (pmoA) gene-and 16S rRNA gene-based amplicon sequencing. The CH 4 oxidation potential was as low as 0.51 μmol g dry weight −1 day −1 in the ungrazed control, while it decreased as grazing intensity increased in grazed fields, ranging from 2.25 μmol g dry weight −1 day −1 in light grazed fields to 1.59 in heavily grazed fields. Increased CH 4 oxidation activity was paralleled by twofold increases in abundance of pmoA genes and relative abundance of methanotroph-affiliated 16S rRNA genes in the total microbial community in grazed soils. SIP and sequencing revealed that the genera Methylobacter and Methylosarcina (type I; Gammaproteobacteria) and Methylocystis (type II; Alphaproteobacteria) were active methanotrophs responsible for CH 4 oxidation in grazed soils. Light and intermediate grazing stimulated the growth and activity of methanotrophs, while heavy grazing decreased the abundance and diversity of the active methanotrophs in the typical steppe. Redundancy and correlation analysis further indicated that the variation of bulk density and soil C and N induced by grazing determined the abundance, diversity of active methanotrophs, and methane oxidation activity in the long-term grazed grassland soil.
  • ... For this reason, a comprehensive study of a novel environment may involve both approaches to best account for any new diversity. The method has been used for the study of a number of environments including landfill cover soil 17 , alpine meadow soil 28 , coal mine soil 29 , estuarine sediment 30 , peat 31 and peat moss 32 . ...
    ... Microarray data is the same as that used in Figure 3, and highlights the difference in methane-oxidizing bacterial community structure between alpine meadow samples taken during different seasons: autumn (A), winter (W), spring (SP) and summer (SU). Adapted from Abell et al. 28 . ...
    ... In our laboratory, the pmoA-based microarray technology has been evaluated and applied successfully for the analysis of methanotroph communities in a range of environmental studies 24,[28][29][30][31][45][46][47][48][49][50][51] . The microarray provides a high-resolution fingerprint of the methanotroph community structure. ...
    Article
    The analysis of methanotroph community composition is relevant to studies of methane oxidation in a number of environments where methane is a significant carbon source. The development and application of a microarray targeting the particulate methane monooxygenase gene (pmoA) have allowed a high-throughput, semiquantitative analysis of the major methanotroph groups in a number of different environments. Here we describe the use of a pmoA-based short oligo array for the analysis of methanotroph populations in sediment samples. The method is suitable for analysis of any type of environmental sample from which DNA can be extracted.
  • ... Methanotrophs [methane-oxidizing bacteria (MOB)] comprise a distinct group of highly specialized bacteria that are able to use methane as their sole carbon and energy source. They have been detected from diverse environments, including soils, sediments, freshwater and marine habitats Abell et al., 2009;Moussard et al., 2009;Antony et al., 2010;Semrau et al., 2010) and anthropogenically created habitats such as landfills (Stralis-Pavese et al., 2004;Hery et al., 2007;Gebert et al., 2009). MOB are important in the global carbon cycle as the largest known atmospheric biological methane sink. ...
    ... The ecological roles of different methanotrophic taxa are unclear, but there is some evidence that they occupy specific niches, with factors such as oxygen and methane concentrations, nitrogen, pH, carbon and overlying plant community influencing distribution (Bender & Conrad, 1995;Hanson & Hanson, 1996;Bodelier & Laanbroek, 2004;Bussmann et al., 2006;Noll et al., 2008;Abell et al., 2009;Tsutsumi et al., 2009). For example, (Henckel et al., 2001) suggested that type I methanotrophs react more quickly to changing methane availability, (Bull et al., 2000) suggested that type II methanotrophs may be responsible for high-affinity methane oxidation in welldrained soils, and (Tsutsumi et al., 2009) found that understory plant species composition influenced MOB community structure significantly. ...
    ... Perhaps, the most conspicuous result regarding the lower salinity soils in this study is the absence of type II phylotypes from two of these soils and lower numbers in the third. Representatives of this group, such as Methylocystis and Methylosinus, have been observed in soils, freshwater and brackish sediments (Bussmann et al., 2004;Abell et al., 2009;Moussard et al., 2009). They are very widely distributed and their apparent absence from some of the soils in this study is surprising, but not without precedent. ...
    Article
    Despite their large areas and potential importance as methane sinks, the role of methane-oxidizing bacteria (MOB) in native woodland soils is poorly understood. These environments are increasingly being altered by anthropogenic disturbances, which potentially alter ecosystem service provision. Dryland salinity is one such disturbance and is becoming increasingly prevalent in Australian soils. We used microarrays and analysis of soil physicochemical variables to investigate the methane-oxidizing communities of several Australian natural woodland soils affected to varying degrees by dryland salinity. Soils varied in terms of salinity, gravitational water content, NO(3)-N, SO(4)-S and Mg, all of which explained to a significant degree MOB community composition. Analysis of the relative abundance and diversity of the MOB communities also revealed significant differences between soils of different salinities. Type II and type Ib methanotrophs dominated the soils and differences in methanotroph communities existed between salinity groups. The low salinity soils possessed less diverse MOB communities, including most conspicuously, the low numbers or absence of type II Methylocystis phylotypes. The differences in MOB communities suggest niche separation of MOB across varying salinities, as has been observed in the closely related ammonia-oxidizing bacteria, and that anthropogenic disturbance, such as dryland salinity, has the potential to alter MOB community and therefore the methane uptake rates in soils in which disturbance occurs.
  • ... Oxidation of CH 4 in soil by the methane-oxidizing bacteria (methanotrophs) currently removes 30 Tg annually from the atmosphere, which equals 5.4% of the global CH 4 sink (IPCC 2007), and therefore play a critical role in the mitigation of global warming. Methanotrophs are widely distribute in various environments (e.g., McDonald et al. 2008; Op den Camp et al. 2009; Semrau et al. 2010), such as in paddy soils (Bodelier et al. 2000; Zheng et al. 2008), forest soils (Mohanty et al. 2007; Kolb 2009), landfill soils ( Einola et al. 2007; Semrau 2011), grassland soils (Zhou et al. 2008; Abell et al. 2009), oil field soil (), and extreme thermoacidophilic environments (Dunfield et al. 2007; Pol et al. 2007; Islam et al. 2008). Methanotrophs are traditionally classified into type I (aerobic γ-Proteobacteria) and type II (aerobic α- Proteobacteria) groups (Hanson and Hanson 1996). ...
    ... d utilization patterns in grassland, would result in alteration to soil properties, such as nitrogen input (Yamulki et al. 1998), and to the nitrifier community (Le Roux et al. 2008). Moreover, the different grazing intensities have conspicuous impact on the structure of methanotroph community in the Inner Mongolia steppe, China (Zhou et al. 2008). Abell et al. (2009) demonstrated that predominant type II methanotrophs in an alpine meadow soil in Austria were not influenced by grazing, while the CH 4 oxidation and the abundance of type I methanotrophs increased due to grazing. However, knowledge of the grazing effect on the community structure and activity of methanotrophs in alpine meadow soil of th ...
    ... hs and thus increased their activities. However, it is unclear why warming did not strengthen the activity under grazing in this study. Our results indicated that the sheep grazing had little effect on methanotrophic abundance. However, we observed that grazing significantly increased CH 4 oxidation potential under ambient temperature (no warming). Abell et al. (2009) also found that the abundance of the predominant type II methanotrophs was not significantly affected by cattle2 A neighbor-joining tree constructed based on the methanotrophic PmoA (amino acid) sequences. Bootstrap values were calculated on the basis of 1,000 data resamplings, and more than 50% are shown. Bold OTUs (01–64) were obtaine ...
    Article
    Full-text available
    Knowledge about methanotrophs and their activities is important to understand the microbial mediation of the greenhouse gas CH(4) under climate change and human activities in terrestrial ecosystems. The effects of simulated warming and sheep grazing on methanotrophic abundance, community composition, and activity were studied in an alpine meadow soil on the Tibetan Plateau. There was high abundance of methanotrophs (1.2-3.4 × 10(8) pmoA gene copies per gram of dry weight soil) assessed by real-time PCR, and warming significantly increased the abundance regardless of grazing. A total of 64 methanotrophic operational taxonomic units (OTUs) were obtained from 1,439 clone sequences, of these OTUs; 63 OTUs (98.4%) belonged to type I methanotrophs, and only one OTU was Methylocystis of type II methanotrophs. The methanotroph community composition and diversity were not apparently affected by the treatments. Warming and grazing significantly enhanced the potential CH(4) oxidation activity. There were significantly negative correlations between methanotrophic abundance and soil moisture and between methanotrophic abundance and NH(4)-N content. The study suggests that type I methanotrophs, as the dominance, may play a key role in CH(4) oxidation, and the alpine meadow has great potential to consume more CH(4) under future warmer and grazing conditions on the Tibetan Plateau.
  • ... Microorganisms in the CH 4 cycle are also active at low temperatures, with winter emissions contributing 12-28% of the annual CH 4 emissions from boreal littoral wetlands (Larmola et al., 2004). There are results indicating that type I methanotrophs contribute to CH 4 oxidation at low temperatures in biofilters, landfills and arctic wetlands (Gebert et al., 2003(Gebert et al., , 2004Börjesson et al., 2004;Graef et al., 2011), and variation in the occurrence of type I methanotrophs across seasons correlates with CH 4 oxidation in alpine meadows (Abell et al., 2009). Nevertheless, the community composition of methanotrophs in seasons with low temperatures is poorly known (Semrau et al., 2010). ...
    ... We examined the seasonal variation of methanotrophs and their activity across four seasons in three hydrologically different subsites of a boreal littoral wetland. We used in vitro bottle experiments to study the CH 4 oxidation potential of methanotroph communities, and a pmoA targeting microbial diagnostic microarray (Bodrossy et al., 2003(Bodrossy et al., , 2006Stralis-Pavese et al., 2004;Abell et al., 2009) to study the diversity of methanotrophs and gene expression of pmoA. ...
    ... Previously described PCR reaction mixtures and amplification conditions were used (Siljanen et al., 2011). Microarray construction and the set of oligonucleotide probes used in this study have been described by Bodrossy et al. (2003) and Abell et al. (2009). Target labelling, hybridization and scanning were performed as described previously (Stralis-Pavese et al., 2004). ...
    Article
    Full-text available
    The atmospheric concentration of methane (CH4) has increased by approximately 150% since pre-industrial times because of increased CH4 emissions and decreased CH4consumption. Methane oxidizing bacteria, methanotrophs, consume CH4 for their carbon and energy needs and thus play a significant role in reducing CH4 emissions and decelerating global warming. In wetlands, for example, methanotrophs can consume as much as 90% of the CH4 produced. In freshwater lakes, even 70% of the total release of CH4 from the lakes can originate from the littoral wetland. Globally, wetlands are responsible for 44% of methane emissions. In boreal region, where lakes are abundant in landscape, it is important to understand how different seasons affect CH4 oxidation activity and are there changes in the diversity of methanotrophs over seasons? This study belongs to the research consortium METHECO (Eurodiversity programme of European Science Foundation), where the activity and diversity of methane oxidising bacteria are studied in various European ecosystems. The activity and diversity of methanotrophs were studied in a littoral wetland of the shallow hyper-eutrophic Lake Kevätön in east-central Finland. The study area has a gradient with moisture and vegetation. Sediment sampling for CH4 oxidation activity and diversity studies of methanotrophs were performed over four seasons: autumn, winter, spring and summer. Samples were taken from three sampling points with different distance from the shoreline (2m; 8m; 17m) and sediment cores were separated to layers 0-2cm, 2-10cm, 10-20cm and 20-30cm. Methane oxidation potential was analyzed with flask experiments and diversity of methanotrophs with pmoA-microarray. Methane was oxidized in all seasons. In the wettest area (nearest to the shoreline) the highest activity occurred in autumn, while in drier areas CH4 oxidation was most active in spring time. In winter time the activity of methanotrophs was not significantly decreased. Methane oxidation activity was always the highest in the organic surface layer (0-10 cm) and the activity decreased with depth. According to the first pmoA-microarray results, methanotroph community structure is significantly changing over seasons.
  • ... Oxidation of CH 4 in soil by the methane-oxidizing bacteria (methanotrophs) currently removes 30 Tg annually from the atmosphere, which equals 5.4% of the global CH 4 sink (IPCC 2007), and therefore play a critical role in the mitigation of global warming. Methanotrophs are widely distribute in various environments (e.g., McDonald et al. 2008; Op den Camp et al. 2009; Semrau et al. 2010), such as in paddy soils (Bodelier et al. 2000; Zheng et al. 2008), forest soils (Mohanty et al. 2007; Kolb 2009), landfill soils ( Einola et al. 2007; Semrau 2011), grassland soils (Zhou et al. 2008; Abell et al. 2009), oil field soil (), and extreme thermoacidophilic environments (Dunfield et al. 2007; Pol et al. 2007; Islam et al. 2008). Methanotrophs are traditionally classified into type I (aerobic γ-Proteobacteria) and type II (aerobic α- Proteobacteria) groups (Hanson and Hanson 1996). ...
    ... d utilization patterns in grassland, would result in alteration to soil properties, such as nitrogen input (Yamulki et al. 1998), and to the nitrifier community (Le Roux et al. 2008). Moreover, the different grazing intensities have conspicuous impact on the structure of methanotroph community in the Inner Mongolia steppe, China (Zhou et al. 2008). Abell et al. (2009) demonstrated that predominant type II methanotrophs in an alpine meadow soil in Austria were not influenced by grazing, while the CH 4 oxidation and the abundance of type I methanotrophs increased due to grazing. However, knowledge of the grazing effect on the community structure and activity of methanotrophs in alpine meadow soil of th ...
    ... hs and thus increased their activities. However, it is unclear why warming did not strengthen the activity under grazing in this study. Our results indicated that the sheep grazing had little effect on methanotrophic abundance. However, we observed that grazing significantly increased CH 4 oxidation potential under ambient temperature (no warming). Abell et al. (2009) also found that the abundance of the predominant type II methanotrophs was not significantly affected by cattle2 A neighbor-joining tree constructed based on the methanotrophic PmoA (amino acid) sequences. Bootstrap values were calculated on the basis of 1,000 data resamplings, and more than 50% are shown. Bold OTUs (01–64) were obtaine ...
    Article
    Full-text available
    Knowledge about methanotrophs and their activ-ities is important to understand the microbial mediation of the greenhouse gas CH 4 under climate change and human activities in terrestrial ecosystems. The effects of simulated warming and sheep grazing on methanotrophic abundance, community composition, and activity were studied in an alpine meadow soil on the Tibetan Plateau. There was high abundance of methanotrophs (1.2–3.4×10 8 pmoA gene copies per gram of dry weight soil) assessed by real-time PCR, and warming significantly increased the abundance regardless of grazing. A total of 64 methanotrophic operational taxonomic units (OTUs) were obtained from 1,439 clone sequences, of these OTUs; 63 OTUs (98.4%) belonged to type I methanotrophs, and only one OTU was Methylocystis of type II methanotrophs. The methanotroph community composition and diversity were not apparently affected by the treatments. Warming and grazing significantly enhanced the potential CH 4 oxidation activity. There were significantly negative correlations between methanotrophic abundance and soil moisture and between methanotrophic abundance and NH 4 –N content. The study suggests that type I methanotrophs, as the dominance, may play a key role in CH 4 oxidation, and the alpine meadow has great potential to consume more CH 4 under future warmer and grazing conditions on the Tibetan Plateau.
  • ... Microorganisms in the CH 4 cycle are also active at low temperatures, with winter emissions contributing 12-28% of the annual CH 4 emissions from boreal littoral wetlands (Larmola et al., 2004). There are results indicating that type I methanotrophs contribute to CH 4 oxidation at low temperatures in biofilters, landfills and arctic wetlands (Gebert et al., 2003(Gebert et al., , 2004Börjesson et al., 2004;Graef et al., 2011), and variation in the occurrence of type I methanotrophs across seasons correlates with CH 4 oxidation in alpine meadows (Abell et al., 2009). Nevertheless, the community composition of methanotrophs in seasons with low temperatures is poorly known (Semrau et al., 2010). ...
    ... We examined the seasonal variation of methanotrophs and their activity across four seasons in three hydrologically different subsites of a boreal littoral wetland. We used in vitro bottle experiments to study the CH 4 oxidation potential of methanotroph communities, and a pmoA targeting microbial diagnostic microarray (Bodrossy et al., 2003(Bodrossy et al., , 2006Stralis-Pavese et al., 2004;Abell et al., 2009) to study the diversity of methanotrophs and gene expression of pmoA. ...
    ... Previously described PCR reaction mixtures and amplification conditions were used (Siljanen et al., 2011). Microarray construction and the set of oligonucleotide probes used in this study have been described by Bodrossy et al. (2003) and Abell et al. (2009). Target labelling, hybridization and scanning were performed as described previously (Stralis-Pavese et al., 2004). ...
    Article
    Littoral wetlands are responsible for most of the total methane (CH(4) ) emissions from lake ecosystems. We show that seasonally variable hydrological and temperature conditions in the littoral wetland of a eutrophic boreal lake affect the community composition and gene transcription of methanotrophs measured by a particulate methane monooxygenase (pmoA) gene-targeted microarray. Type Ib freshwater-cluster methanotrophs were favoured by the high water level, and CH(4) oxidation was positively correlated with their pmoA gene transcripts. In the dry subsite of the wetland, the more stagnant hydrological conditions in summer and autumn induced the dominance of type II methanotrophs over type I methanotrophs (community composition and pmoA gene transcripts). The relative abundance of type II methanotrophs increased in winter. The results provide new insight into the variation of methanotroph communities across seasons in littoral wetlands.
  • ... Many studies consider upland soils as methane sinks of atmospheric CH 4 . For example, Abell et al. [1] showed that grazing influences the activity and diversity of methanotrophic bacteria in alpine meadow soils. Furthermore, ammonium can competitively inhibit CH 4 oxidation, but the complex relationship between nitrifiers and methanotrophs is not clearly understood so far, and other ecological factors such as acidity have to be considered too [29]. ...
    ... High concentrations of commercially available methane can also contain traces of other gases like acetylene which can inhibit methane oxidation even at very low concentrations [8]. Several further (interactive) factors like temperature [35], N-fertilization [10,29], soil moisture [36], vegetation [31] or grazing [1] were shown to influence the activity and diversity of methanotrophs in soils. A study of landfill cover soils suggest that methane oxidation increases with temperature, has an optimum at 30 C, and decreases from that point onwards to a temperature maximum threshold of 55 C. In this study, it is also said that the optimal soil moisture potential for methane oxidation is around field capacity (50 kPa) [35]. ...
    Article
    Full-text available
    Three subalpine soils with similar properties and climate conditions, but with different land use were chosen for assessing the abundance and activity of aerobic and anaerobic microorganisms, particularly of methanotrophic Bacteria and methanogenic Archaea. Within these three soils, there was a distinct gradient in grazing and manure input that ranged from abandoned site (no grazing) to pasture (intense grazing) and included also a meadow (moderate grazing). Activities of dehydrogenase and ammonification indicated higher microbial activities in soil from the abandoned site than in the pasture and in the meadow soil. These differences in microbial activity between unmanaged and managed soils additionally increased during the growing season. Our results show that temperature and soil water content significantly influence methane production and that the grazing gradient is an additional important factor. At 50 °C and under water-saturated conditions, methane production reached 4.42 ± 0.09 ml g−1 dry weight in the pasture soil samples after 6 weeks. By contrast, low methane production was detected in soil from the abandoned site, whereas the meadow values were intermediate. Additionally, methanotrophic activities were investigated under an aerobic atmosphere with 5% CH4 and led to the surprising result that methane oxidation only occurred in undisturbed soils, whereas all sieved soil samples even produced further methane.
  • ... Greater deposits of urine may also have contributed to higher net CH 4 emissions from grazed compared to ungrazed pasture. Urine adds nitrogen to soils and can inhibit methanotrophic activity, decreasing net CH 4 uptake under drier soil conditions and increasing net CH 4 emissions at wetter soil conditions (Abell et al. 2009, Banger et al. 2012, Zheng et al. 2012. However, in our study, the inhibitory effect of nitrogen addition on methanotrophic bacteria activity was probably small because at similar soil moisture levels CH 4 emissions from soils between the grazed and ungrazed pastures were close in magnitude (Appendix S2: Fig. S3). ...
  • ... The governing role of soil plowing in determining the community structure of methanotrophs in agricultural soil is further supported by the dominance of T-RFs associated with Type 1a in this soil (Fig. 5). The presence of Type 1a methanotrophs is usually associated with disturbed environments (Abel et al, 2009;Dorr et al., 2010). ...
    Article
    Variations in the rates of atmospheric CH4 uptake in upland soils can arise from both abiotic and biotic factors. Among the less-studied biotic factors is the degree to which methanotroph activity and community composition interact with supply of CH4 to the soil. Here, we investigated whether the abundance of high affinity methanotrophs in a range of soils representing different land use types is substrate (CH4) dependent. Field replicates of three soils sampled from deciduous forest, spruce forest and agricultural sites were incubated in columns flushed continuously for 24 days with air at one of four CH4 concentrations: <1 ppm (starvation), 1.8 (ambient), 30 (low elevated) and 60 (high elevated) ppm. In all soils, CH4 oxidation rates increased linearly with CH4 supply. For all levels of CH4 supply, CH4 oxidation rates were the highest in deciduous forest soil followed by spruce forest and agricultural soils. Terminal restriction fragment length polymorphism (T-RFLP) analysis indicated that the agricultural soil had a distinct methanotrophic community compared to the two forest soils. In particular, the T-RFs (Terminal restriction fragments) associated with USCα and Type II methanotrophs (Methylocystis sp, Methylosinus sp.) were the most abundant in forest soils while Type 1a associated T-RFs dominated in agricultural soil. The agricultural and forest soils also differed in their fractionation of stable isotopes, 13C and 2H, during CH4 oxidation. Altering CH4 concentration in the inlet air did not change methanotroph abundance, as evidenced by three different assays, two qPCR and T-RFLP, that recorded no changes in the number of pmoA genes and/or the relative abundance of T-RFs. Altogether, it is proposed that intrinsic differences in CH4 oxidation rates between soils, particularly between temperate agricultural and forest soils, are driven by methanotroph community structure. The population size of methanotrophs in upland soils did not respond to CH4 availability and is most probably regulated by other factors, such as the availability of nitrogen, cross-feeding or other carbon sources.
  • ... Concentration of PCR products was quantified using a Qubit fluorometer (Invitrogen, Carlsbad, CA, USA). In vitro transcription and hybridization was performed as in Pavese et al. (2004) and the applied probe set was similar to that applied by Abell et al. (2009). One of three parallel original inactive moss samples from the 0 day time point could be successfully analyzed (see Larmola et al. (2010) that SAM inactive Sphagnum mosses host MOB DNA). ...
    Article
    Full-text available
    We studied methanogen activity—measured by in vitro methane production potential and by detection of the messenger RNA (mRNA) of a functional gene—in two boreal fens under high and deep water table (WT) level conditions resulting from a rainy growing season and a dry growing season. The depth of the highest CH4-producing layers differed between the years. In the wet year, the highest CH4 production rate was around 20cm below the mean WT. In the dry year, the highest rates were measured close to the peat surface, well above the mean WT. The distribution of activity in the peat profiles of the two fens appeared to be site specific. Under deep-WT conditions, CH4 production potential was generally lower than that under high-WT conditions. Detection of the mRNA of the methanogen-specific mcrA gene indicated in situ methanogenesis in both water-saturated peat (below the WT) and unsaturated peat (above the WT). Analyses of DNA-derived and mRNA-derived methanogen community structures showed greater similarity between those two in water-saturated peat than in unsaturated peat. This suggested that favorable conditions promoted the activity of most members in methanogen communities, but unfavorable conditions showed differences between distinct community members in adaptation to adverse conditions.
  • ... can utilize CH 4 as a sole carbon and energy source and currently remove 30 Tg annually from the atmosphere (IPCC, 2007), which suggests that they play a crucial role in the mitigation of global warming. Methanotrophs are ubiquitous in the environments including paddy field and wetland soils (Bodelier et al., 2000;Yun et al., 2010), upland and forest soils (Kolb, 2009), landfill soils Chang et al., 2010), and some other ecological niches (Abell et al., 2009;Zheng et al., 2012). ...
  • ... dominating the methanotrophic population, in spite of differences in location and physico-chemical properties of the soil covers (Wise et al., 1999;Uz et al., 2003;Cebron et al., 2007;Chen et al., 2007;Gebert et al., 2008;Kumaresan et al., 2009;Im et al., 2011). Type I and Type II methanotrophs are known to favour different ecological niches (Hanson and Hanson, 1996;Stralis-Pavese et al., 2004;Abell et al., 2009;Scheutz et al., 2009). For example, Type I methanotrophs are known to dominate methane oxidation under oxygenrich and methane-limited conditions, whereas Type II methanotrophs dominate in nitrogen-limited conditions (Amaral and Knowles, 1995;Henckel et al., 2000). ...
    Article
    Aggregates of different sizes and stability in soil create a composite of ecological niches differing in terms of physico-chemical and structural characteristics. The aim of this study was to identify, using DNA-SIP and mRNA-based microarray analysis, whether shifts in activity and community composition of methanotrophs occur when ecological niches created by soil structure are physically perturbed. Landfill cover soil was subject to three treatments termed: 'control' (minimal structural disruption), 'sieved' (sieved soil using 2 mm mesh) and 'ground' (grinding using mortar and pestle). 'Sieved' and 'ground' soil treatments exhibited higher methane oxidation potentials compared with the 'control' soil treatment. Analysis of the active community composition revealed an effect of physical disruption on active methanotrophs. Type I methanotrophs were the most active methanotrophs in 'sieved' and 'ground' soil treatments, whereas both Type I and Type II methanotrophs were active in the 'control' soil treatment. The result emphasize that changes to a particular ecological niche may not result in an immediate change to the active bacterial composition and change in composition will depend on the ability of the bacterial communities to respond to the perturbation.
  • ... The microarray construction and the set of oligonucleotide probes used in this study have been described by Bodrossy et al. (2003) and Abell et al. (2009). Targets were amplified with the same two-step semi-nested PCR protocol as used for clone library construction with the exception that the A682r and mb661r primers had a 5 0 T7 recognition site (5 0 - TAATACGACTCACTATAG-3 0 ). ...
    Article
    In lake ecosystems a major proportion of methane (CH(4) ) emissions originate from the littoral zone, which can have a great spatial variability in hydrology, soil quality and vegetation. Hitherto, spatial heterogeneity and the effects it has on functioning and diversity of methanotrophs in littoral wetlands have been poorly understood. A diagnostic microarray based on the particulate methane monooxygenase gene coupled with geostatistics was used to analyse spatial patterns of methanotrophs in the littoral wetland of a eutrophic boreal lake (Lake Kevätön, Eastern Finland). The wetland had a hydrology gradient with a mean water table varying from -8 to -25 cm. The wettest area, comprising the highest CH(4) oxidation, had the highest abundance and species richness of methanotrophs. A high water table favoured the occurrence of type Ib methanotrophs, whereas types Ia and II were found under all moisture conditions. Thus the spatial heterogeneity in functioning and diversity of methanotrophs in littoral wetlands is highly dependent on the water table, which in turn varies spatially in relation to the geomorphology of the wetland. We suggest that changes in water levels resulting from regulation of lakes and/or global change will affect the abundance, activity and diversity of methanotrophs, and consequently CH(4) emissions from such systems.
  • ... Greater deposits of urine may also have contributed to higher net CH 4 emissions from grazed compared to ungrazed pasture. Urine adds nitrogen to soils and can inhibit methanotrophic activity, decreasing net CH 4 uptake under drier soil conditions and increasing net CH 4 emissions at wetter soil conditions (Abell et al. 2009, Banger et al. 2012, Zheng et al. 2012. However, in our study, the inhibitory effect of nitrogen addition on methanotrophic bacteria activity was probably small because at similar soil moisture levels CH 4 emissions from soils between the grazed and ungrazed pastures were close in magnitude (Appendix S2: Fig. S3). ...
    Article
    The impact of grazing on C fluxes from pastures in subtropical and tropical regions, and on the environment is uncertain, although these systems account for a substantial portion of global C storage. We investigated how cattle grazing influences net ecosystem CO2 and CH4 exchange in subtropical pastures using the eddy covariance technique. Measurements were made over several wet-dry seasonal cycles in a grazed pasture, and in an adjacent pasture during the first three years of grazer exclusion. Grazing increased soil wetness but did not affect soil temperature. By removing aboveground biomass, grazing decreased ecosystem respiration (Reco) and Gross Primary Productivity (GPP). As the decrease in Reco was larger than the reduction in GPP, grazing consistently increased the net CO2 sink strength of subtropical pastures (55, 219 and 187 more C m−2 in 2013, 2014 and 2015). Enteric ruminant fermentation and increased soil wetness due to grazers, increased total net ecosystem CH4 emissions in grazed relative to ungrazed pasture (27% - 80%). Unlike temperate, arid, and semi-arid pastures, where differences in CH4 emissions between grazed and ungrazed pastures are mainly driven by enteric ruminant fermentation, our results showed that the effect of grazing on soil CH4 emissions can be greater than CH4 produced by cattle. Thus, our results suggest that the interactions between grazers and soil hydrology affecting soil CH4 emissions play an important role in determining the environmental impacts of this management practice in a subtropical pasture. Although grazing increased total net ecosystem CH4 emissions and removed aboveground biomass, it increased the net storage of C and decreased the global warming potential associated with C fluxes of pasture by increasing its net CO2 sink strength. This article is protected by copyright. All rights reserved.
  • ... It has been reported that Type I MOB preferentially grow at high O2 and low CH4 concentration environments, while Type II MOB preferentially grow at low O2 and high CH4 concentration environments [39]. Therefore, Type I and II MOB occupy different niches [40]. The results also demonstrated the uniqueness of each soil environment and the effects of ecological factors on MOB at the species level. ...
    Article
    Full-text available
    Ammonia oxidizing bacteria (AOB), Ammonia oxidizing archaea (AOA) and methane oxidizing bacteria (MOB) play cogent roles in oxidation and nitrification processes, and hence have important ecological functions in several ecosystems. However, their distribution and compositional differences in different long-term flooded paddy fields (FPFs) management at different soil depths remains under-investigated. Using qPCR and phylogenetic analysis, this study investigated the abundance, niches, and compositional differences of AOA, AOB, and MOB along with their potential nitrification and oxidation rate in three soil layers from three FPFs (ShaPingBa (SPB), HeChuan (HC), and JiDi (JD)) in Chongqing, China. In all the FPFs, CH4 oxidation occurred mainly in the surface (0–3 cm) and subsurface layers (3–5 cm). A significant difference in potential methane oxidation and nitrification rates was observed among the three FPFs, in which SPB had the highest. The higher amoA genes are the marker for abundance of AOA compared to AOB while pmoA genes, which is the marker for MOB abundance and diversity, indicated their significant role in the nitrification process across the three FPFs. The phylogenetic analysis revealed that AOA were mainly composed of Nitrososphaera, Nitrosospumilus, and Nitrosotalea, while the genus Nitrosomonas accounted for the greatest proportion of AOB in the three soil layers. MOB were mainly composed of Methylocaldum and Methylocystis genera. Overall, this finding pointed to niche differences as well as suitability of the surface and subsurface soil environments for the co-occurrence of ammonia oxidation and methane oxidation in FPFs.
  • ... (Berchidda) to 10.5±0.65 (Viggiano) and highly significant differences (P<0.01) could be observed in richness and diversity (Shannon-Weiner and Simpson indices) between soil sampled in the different experimental areas. Methanotrophs type II, resulted more abundant in our soils than type I, according to statements from Rastogi et al. (2009) andAbell et al. (2009). The mean number of bands of methanotrophs 16SrDNA-DGGE profiles ranged from 2.8±0.37 (Berchidda) to 11.67±1.67 ...
    Article
    Full-text available
    This paper addresses the diversity of two soil bacterial groups involved in the biogeochemical carbon cycle: bacteria implicated in chitin degradation and methanotrophs. To evaluate the influence of soil physico-chemical and anthropic characteristics on the diversity of these microbial groups, total DNA was directly extracted from soils differently managed and sampled in central and south Italy. PCR-Denaturing Gradient Gel Electrophoresis (DGGE) analyses targeting genes coding for chitinase (chiA), particulate methane monooxygenase (pmoA) and 16S rRNA from bacteria, actinomycetes and type I or II methanotrophs were used to fingerprint the soil bacterial communities. DGGE cluster analysis showed a clear separation of the bacterial communities on the basis of the sampling sites. The Canonical Corrispondance Analysis (CCA) suggests that the edaphic factors such as granulometry and pH, could be responsible for determining the composition of these bacterial groups.
  • ... It has been reported that Type I MOB preferentially grow at high O2 and low CH4 concentration environments, while Type II MOB preferentially grow at low O2 and high CH4 concentration environments [39]. Therefore, Type I and II MOB occupy different niches [40]. The results also demonstrated the uniqueness of each soil environment and the effects of ecological factors on MOB at the species level. ...
  • ... can utilize CH 4 as a sole carbon and energy source and currently remove 30 Tg annually from the atmosphere (IPCC, 2007), which suggests that they play a crucial role in the mitigation of global warming. Methanotrophs are ubiquitous in the environments including paddy field and wetland soils (Bodelier et al., 2000;Yun et al., 2010), upland and forest soils (Kolb, 2009), landfill soils Chang et al., 2010), and some other ecological niches (Abell et al., 2009;Zheng et al., 2012). ...
  • ... Several studies have put forward preferred conditions for growth and activity of type I and II MOB (see Conrad 2007; Semrau et al. 2010) of which high methane concentrations for growth of type II MOB was the most common one (e.g., Henckel et al. 2000). However, using activity proxies (i.e., mRNA; Chen et al. 2007; Abell et al. 2009) or SIP DNA or RNA (Nercessian et al. 2005); (Cebron et al. 2007; Dumont et al. 2011) or SIP–PLFA (Knoblauch et al. 2008) it has been demonstrated that type I MOB are the dominant active MOB in landfill c ...
    Article
    Full-text available
    Climate change will lead to more extreme precipitation and associated increase of flooding events of soils. This can turn these soils from a sink into a source of atmospheric methane. The latter will depend on the balance of microbial methane production and oxidation. In the present study, the structural and functional response of methane oxidizing microbial communities was investigated in a riparian flooding gradient. Four sites differing in flooding frequency were sampled and soil-physico-chemistry as well as methane oxidizing activities, numbers and community composition were assessed. Next to this, the active community members were determined by stable isotope probing of lipids. Methane consumption as well as population size distinctly increased with flooding frequency. All methane consumption parameters (activity, numbers, lipids) correlated with soil moisture, organic matter content, and conductivity. Methane oxidizing bacteria were present and activated quickly even in seldom flooded soils. However, the active species comprised only a few representatives belonging to the genera Methylobacter, Methylosarcina, and Methylocystis, the latter being active only in permanently or regularly flooded soils. This study demonstrates that soils exposed to irregular flooding harbor a very responsive methane oxidizing community that has the potential to mitigate methane produced in these soils. The number of active species is limited and dominated by one methane oxidizing lineage. Knowledge on the characteristics of these microbes is necessary to assess the effects of flooding of soils and subsequent methane cycling therein.
  • ... However, MOB needed >100 yr to reach an abundance in the range of the reference locations. When compared with other mature upland soil ecosystems (up to 10 6 copies (g soil d.w.) À1 ; Kolb et al., 2005;Abell et al., 2009), pmoA gene abundance detected in B and C soil-age classes was at least one order of magnitude smaller, suggesting that the establishment of MOB communities in the investigated soils is still an ongoing process. Due to the low concentration of CH 4 in the atmosphere, high-affinity MOB have been suggested to be relatively slow-growing organisms, primarily limited by CH 4 availability (Bender and Conrad, 1992;Priem e et al., 1996;Dunfield, 2007). ...
  • ... Concentration of PCR products was quantified using a Qubit fluorometer (Invitrogen, Carlsbad, CA, USA). In vitro transcription and hybridization was performed as in StralisPavese et al. (2004)and the applied probe set was similar to that applied byAbell et al. (2009). One of three parallel original inactive moss samples from the 0 day time point could be successfully analyzed (seeLarmola et al. (2010)that SAM inactive Sphagnum mosses host MOB DNA). ...
    Article
    Full-text available
    It is known that Sphagnum associated methanotrophy (SAM) changes in relation to the peatland water table (WT) level. After drought, rising WT is able to reactivate SAM. We aimed to reveal whether this reactivation is due to activation of indigenous methane (CH(4)) oxidizing bacteria (MOB) already present in the mosses or to MOB present in water. This was tested through two approaches: in a transplantation experiment, Sphagna lacking SAM activity were transplanted into flark water next to Sphagna oxidizing CH(4). Already after 3 days, most of the transplants showed CH(4) oxidation activity. Microarray showed that the MOB community compositions of the transplants and the original active mosses had become more similar within 28 days thus indicating MOB movement through water between mosses. Methylocystis-related type II MOB dominated the community. In a following experiment, SAM inactive mosses were bathed overnight in non-sterile and sterile-filtered SAM active site flark water. Only mosses bathed with non-sterile flark water became SAM active, which was also shown by the pmoA copy number increase of over 60 times. Thus, it was evident that MOB present in the water can colonize Sphagnum mosses. This colonization could act as a resilience mechanism for peatland CH(4) dynamics by allowing the re-emergence of CH(4) oxidation activity in Sphagnum.
  • ... 2014). The abundance and structure of soil MOB community can be influenced by a number of factors, such as land management (Abell et al. 2009), nitrogen fertilizer (Alam and Jia 2012;Dai et al. 2013), moisture (Shrestha et al. 2012), salinity (Bissett et al. 2012), and temperature (Martineau et al. 2010). It could be assumed that these multiple environmental factors might collectively regulate the MOB distribution in soil ecosystems. ...
    Article
    Full-text available
    Aerobic methane-oxidizing bacteria (MOB) play an important role in mitigating the methane emission in soil ecosystems to the atmosphere. However, the impact of plant species and plantation ways on the distribution of MOB remains unclear. The present study investigated MOB abundance and structure in plateau soils with different plant species and plantation ways (natural and managed). Soils were collected from unmanaged wild grassland and naturally forested sites, and managed farmland and afforested sites. A large variation in MOB abundance and structure was found in these studied soils. In addition, both type I MOB (Methylocaldum) and type II MOB (Methylocystis) were detected in these soils, while type II MOB usually outnumbered type I MOB. The distribution of soil MOB community was found to be collectively regulated by plantation way, plant species, the altitude of sampling site, and soil properties.
  • ... (Berchidda) to 10.5±0.65 (Viggiano) and highly significant differences (P<0.01) could be observed in richness and diversity (Shannon-Weiner and Simpson indices) between soil sampled in the different experimental areas. Methanotrophs type II, resulted more abundant in our soils than type I, according to statements from Rastogi et al. (2009) andAbell et al. (2009). The mean number of bands of methanotrophs 16SrDNA-DGGE profiles ranged from 2.8±0.37 (Berchidda) to 11.67±1.67 ...
    Article
    Full-text available
    This paper addresses the diversity of two soil bacterial groups involved in the biogeochemical carbon cycle: bacteria implicated in chitin degradation and methanotrophs. To evaluate the influence of soil physico-chemical and anthropic characteristics on the diversity of these microbial groups, total DNA was directly extracted from soils differently managed and sampled in central and south Italy. PCR-Denaturing Gradient Gel Electrophoresis (DGGE) analyses targeting genes coding for chitinase (chiA), particulate methane monooxygenase (pmoA) and 16S rRNA from bacteria, actinomycetes and type I or II methanotrophs were used to fingerprint the soil bacterial communities. DGGE cluster analysis showed a clear separation of the bacterial communities on the basis of the sampling sites. The Canonical Corrispondance Analysis (CCA) suggests that the edaphic factors such as granulometry and pH, could be responsible for determining the composition of these bacterial groups. Résumé Cet article concerne deux groupes microbiotiens clés impliqués dans le cycle du carbone: ceux qui dégradent de la chitine et les méthanotrophes. Les expériences, effectuées par l' extraction directe de l'ADN et par l'analyse DGGE (électrophorèse sur gel en gradient dénaturant) se sont déroulées sur divers systèmes de sols sujets à différents modes de gestion et répartis dans plusieurs zones de l'Italie centrale et
  • ... Therefore, MMO genes are widely used as a biological marker in molecular ecological studies of methanotrophs (McDonald et al., 2008). Methanotrophs are widely distributed in various environments: such as paddy soils (Bodelier et al., 2000), upland forest soils (Knief et al., 2006; Lau et al., 2007; Mohanty et al., 2007; Kolb, 2009), landfill soils, wetlands (Einola et al., 2007; Siljanen et al., 2011), alpine grassland soils (Abell et al., 2009), and extreme thermoacidophilic environments (Pol et al., 2007; Islam et al., 2008). Soil moisture is important for induction of CH 4 oxidation and regulation of CH 4 uptake in soil (Bender and Conrad, 1995; Shrestha et al., 2012). ...
    Article
    Full-text available
    Cork oak woodlands (montado) are agroforestry systems distributed all over the Mediterranean basin with a very important social, economic and ecological value. A generalized cork oak decline has been occurring in the last decades jeopardizing its future sustainability. It is unknown how loss of tree cover affects microbial processes that are consuming greenhouse gases in the montado ecosystem. The study was conducted under two different conditions in the natural understory of a cork oak woodland in center Portugal: under tree canopy (UC) and open areas without trees (OA). Fluxes of methane and nitrous oxide were measured with a static chamber technique. In order to quantify methanotrophs and bacteria capable of nitrous oxide consumption, we used quantitative real-time PCR targeting the pmoA and nosZ genes encoding the subunit of particulate methane mono-oxygenase and catalytic subunit of the nitrous oxide reductase, respectively. A significant seasonal effect was found on CH 4 and N 2 O fluxes and pmoA and nosZ gene abundance. Tree cover had no effect on methane fluxes; conversely, whereas the UC plots were net emitters of nitrous oxide, the loss of tree cover resulted in a shift in the emission pattern such that the OA plots were a net sink for nitrous oxide. In a seasonal time scale, the UC had higher gene abundance of Type I methanotrophs. Methane flux correlated negatively with abundance of Type I methanotrophs in the UC plots. Nitrous oxide flux correlated negatively with nosZ gene abundance at the OA plots in contrast to that at the UC plots. In the UC soil, soil organic matter had a positive effect on soil extracellular enzyme activities, which correlated positively with the N 2 O flux. Our results demonstrated that tree cover affects soil properties, key enzyme activities and abundance of microorganisms and, consequently net CH 4 and N 2 O exchange.
  • ... The behavior of SF and AG soil to sub-oxic conditions indicate presence of Type 1 methanotrophs as CH 4 oxidation rates were increased or remain unchanged in these soils. The dominancy of Type 1 methanotrophs in agricultural soil is evident in other studies (Abell et al., 2009) however there is no report regarding SF land use. On other side, our results regarding DF soil are in consistent with previous reports that also reported significant decrease in CH 4 oxidation rates in a variety of unsaturated soils when exposed to low O 2 concentrations (Bender and Conrad, 1994;Czepiel et al., 1996;Teh et al., 2005). ...
  • ... Our results show that seasonal mean soil properties differed significantly in soil profile sections of 0-10, 10-20 and 20-30 cm depth, which showed similar patterns to other studies in alpine grasslands (Wang et al. 2007b;Abell et al. 2009;Feng et al. 2010). Soil properties differed significantly between areas with and without burrowing pikas, but there were no significant differences between medium-and high-density pika occupied areas. ...
    Article
    The foraging and burrowing activities of small mammalian herbivores may have either detrimental or beneficial effects on grassland ecosystems; the direction of the effect is determined by the species, population abundances and fluctuations. Twelve survey sites with active burrow of plateau pika were classified into four degrees of density: approximately zero-density, low-density, medium-density and high-density, to evaluate the impact of different pika densities on vegetation, plant biomass, soil organic carbon and nutrients in a whole growing season. We show that pika as a main supplement to livestock activities contributed to a decrease in the number of plant species, vegetation cover, plant height and seasonal mean biomass, while values at medium-density site except above-ground biomass were the lowest. With the exception of available potassium, soil organic carbon, nitrogen, total phosphorus and soil water content, zero-density areas were significantly higher than those of pika occupied areas. However, there were slight or no differences in vegetation characteristics and soil properties between medium-and high-density sites. Our study suggests pika activities with high-density made palatable forage less and soil carbon and nitrogen more than low-density, moreover, plateau pika had greater impacts on above-ground vegetation than on root system.
  • ... The present study collected data about methanotroph abundance to further compare them between grassland and other ecosystems ( Table 2). The comparison showed no great variation of methanotroph abundance among all ecosystems, where the order of magnitude for pmoA gene copies per gram of soil reached 10 7 -10 8 (Abell et al. 2009;Yun et al. 2012Yun et al. , 2014 Zheng et al. 2012;Deng et al. 2013). For forest and grassland, the soil is under well-aerated conditions (Wang et al. 2014). ...
    Article
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    Purpose Despite the great number of studies about methane uptake and its response to grazing in the Inner Mongolia grasslands, only a few focused on the methanotrophic composition. This study aimed to investigate the methanotrophic community structure and abundance, then to analyze the abiotic driving factors of methanotrophic community structure in different enclosed times in this area. Materials and methods In this study we chose typical grasslands in the Xilin River Basin of Inner Mongolia, China to investigate methanotrophic community structure and abundance under different enclosure treatments as follows: 79E (grassland enclosed since 1979), 99E (grassland enclosed since 1999), and G (freely grazed grassland). A clone library was used to reveal the methanotroph community structure, and their relationships with abiotic factors were analyzed by redundancy analysis. Methanotroph abundance was determined by real-time PCR. Results and discussion The OTUs of the three treatments mainly belonged to Type I methanotrophs, probably caused by the high pH value. Among all OTUs, only OTU1 belonged to upland soil cluster γ (USC-γ), whose abundance was the largest in all OTUs, indicating the USC-γ cluster was the main one to oxidize CH4 in the Inner Mongolia grasslands. Methanotrophic abundance (represented by the pmoA gene copies per gram of dry weight soil) decreased with the enclosure time as G (4.5 × 107) > 99E (2.8 × 107) > 79E (2.0 × 107), mainly caused by the lower soil moisture content in G. Lower soil moisture content facilitates more CH4 and O2 diffusive into soil thus leading to the proliferation of methanotrophs. Conclusions This study found a high abundance of methanotrophs in the soils of the Inner Mongolia grasslands, with the USC-γ cluster having the largest abundance, which may play a key role in oxidizing CH4 in the Inner Mongolia grasslands. Combined with those of previous studies, the results showed an obvious change of methanotrophic community composition with the increase of enclosure time.
  • ... The clusters are consistently found in forest soils, often as most abundant group, they are quite frequently detected in grassland soils, but rarely detected in intensively managed agricultural soils (Knief et al., 2006;Dunfield, 2007). It has been reported that populations decrease and become inactive when forest soils are converted into agricultural soils, or grasslands are subjected to grazing Abell et al., 2009;Dörr et al., 2010;Lima et al., 2014). They recover in afforested or reforested sites and grassland soil in which nitrogen fertilization is reduced (Nazaries et al., 2011;Shrestha et al., 2012). ...
    Article
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    Methane-oxidizing bacteria are characterized by their capability to grow on methane as sole source of carbon and energy. Cultivation-dependent and -independent methods have revealed that this functional guild of bacteria comprises a substantial diversity of organisms. In particular the use of cultivation-independent methods targeting a subunit of the particulate methane monooxygenase (pmoA) as functional marker for the detection of aerobic methanotrophs has resulted in thousands of sequences representing “unknown methanotrophic bacteria.” This limits data interpretation due to restricted information about these uncultured methanotrophs. A few groups of uncultivated methanotrophs are assumed to play important roles in methane oxidation in specific habitats, while the biology behind other sequence clusters remains still largely unknown. The discovery of evolutionary related monooxygenases in non-methanotrophic bacteria and of pmoA paralogs in methanotrophs requires that sequence clusters of uncultivated organisms have to be interpreted with care. This review article describes the present diversity of cultivated and uncultivated aerobic methanotrophic bacteria based on pmoA gene sequence diversity. It summarizes current knowledge about cultivated and major clusters of uncultivated methanotrophic bacteria and evaluates habitat specificity of these bacteria at different levels of taxonomic resolution. Habitat specificity exists for diverse lineages and at different taxonomic levels. Methanotrophic genera such as Methylocystis and Methylocaldum are identified as generalists, but they harbor habitat specific methanotrophs at species level. This finding implies that future studies should consider these diverging preferences at different taxonomic levels when analyzing methanotrophic communities.
  • ... The transcript of type Ia MOB (related to Methylobacter) clearly characterized the wet regime of the southern fen, whereas several transcripts related to, e.g., type II Methylocystis, type Ib Methylomicrobium album and Methylosarcina, and clones related to forest soil (RA14) and watershed and upland soil cluster 1 (Wsh1-566) characterized the drier regime of the southern fen. These forest and watershed and upland related clones have been found in temperate forest soil, wet meadow soils, watershed, and flooded forest soils exhibiting uptake of atmospheric methane (Holmes et al., 1999;Uz et al., 2003;Knief et al., 2006;Abell et al., 2009). Thus, we propose that conditions in the drier regime of the southern fen may favor a diverse MOB community that has ability for oxidation at atmospheric CH 4 concentrations if needed. ...
    Article
    Peatlands are one of the major sources of the powerful greenhouse gas methane (CH4). Our aim was to detect responses of methanogenic archaeal and methane-oxidizing bacterial (MOB) communities that control the methane (CH4) cycle to climatic warming. This study took place in two boreal fens three years after experimental warming in un-manipulated wet and drier regimes, thus simulating future climate scenarios. We determined active methanogen and MOB communities as transcripts of mcrA and pmoA genes, along with the abundance of these genes, CH4 production and oxidation potentials, and in situ CH4 fluxes. Methanogenic community remained similar, although methanogen abundance decreased after warming. In the wet regime, this decrease resulted in a small but significant reduction on the potential CH4 production in such peat layers where the average production potential was high. Drying alone, however, reduced the potential CH4 production more than warming, and this impact was strong enough to mask the small warming impact in the drier regime. Warming did not affect the MOB community or the potential CH4 oxidation in the wet regime; however, type Ib MOB abundance decreased and MOB related to genus Methylocapsa became typical after warming in the drier regime of the southern fen. The in situ measured CH4 fluxes indicated similar patterns as potential measurements; both warming and drying reduced methane emissions, drying more than warming. These results indicate that methanogens and MOB may have different controlling patterns on CH4 fluxes when facing global warming. These patterns may further differ not only between moisture regimes, but inside the same habitat type, here boreal fen. Irrespective of this variation, the in situ CH4 fluxes still seem to respond similarly across sites.
  • ... This corresponds to the quantification with cPCR/t-RFLP (Figure 3), which revealed a higher mRNA:DNA ratio in the anoxic zone than in the surface zone. The role of type II MOB and Methylobacter is surprisingly similar to that found in a seasonal study on an alpine meadow (Abell et al., 2009), in which type II MOB remained largely unaffected by season and environment but nevertheless represented the dominant MOB. Methylobacter-related MOB, however, were found to be responsible for the majority of methane oxidation. ...
    Article
    Full-text available
    Aerobic methane-oxidizing bacteria (MOB) use a restricted substrate range, yet >30 species-equivalent operational taxonomical units (OTUs) are found in one paddy soil. How these OTUs physically share their microhabitat is unknown. Here we highly resolved the vertical distribution of MOB and their activity. Using microcosms and cryosectioning, we sub-sampled the top 3-mm of a water-saturated soil at near in situ conditions in 100-μm steps. We assessed the community structure and activity using the particulate methane monooxygenase gene pmoA as a functional and phylogenetic marker by terminal restriction fragment length polymorphism (t-RFLP), a pmoA-specific diagnostic microarray, and cloning and sequencing. pmoA genes and transcripts were quantified using competitive reverse transcriptase PCR combined with t-RFLP. Only a subset of the methanotroph community was active. Oxygen microprofiles showed that 89% of total respiration was confined to a 0.67-mm-thick zone immediately above the oxic-anoxic interface, most probably driven by methane oxidation. In this zone, a Methylobacter-affiliated OTU was highly active with up to 18 pmoA transcripts per cell and seemed to be adapted to oxygen and methane concentrations in the micromolar range. Analysis of transcripts with a pmoA-specific microarray found a Methylosarcina-affiliated OTU associated with the surface zone. High oxygen but only nanomolar methane concentrations at the surface suggested an adaptation of this OTU to oligotrophic conditions. No transcripts of type II methanotrophs (Methylosinus, Methylocystis) were found, which indicated that this group was represented by resting stages only. Hence, different OTUs within a single guild shared the same microenvironment and exploited different niches.
  • Article
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    Microbial diagnostic microarrays (MDMs) are highly parallel hybridization platforms containing multiple sets of immobilized oligonucleotide probes used for parallel detection and identification of many different microorganisms in environmental and clinical samples. Each probe is approximately specific to a given group of organisms. Here we describe the protocol used to develop and validate an MDM method for the semiquantification of a range of functional genes--in this case, particulate methane monooxygenase (pmoA)--and we give an example of its application to the study of the community structure of methanotrophs and functionally related bacteria in the environment. The development and validation of an MDM, following this protocol, takes ∼6 months. The pmoA MDM described in detail comprises 199 probes and addresses ∼50 different species-level clades. An experiment comprising 24 samples can be completed, from DNA extraction to data acquisition, within 3 d (12-13 h bench work).
  • Article
    The microbial mechanisms of how different long-term fertilizations change methane oxidation of Chinese upland arable soil is unclear so far. In the present study, we attempted to investigate the "soil properties-community properties of methanotrophs-methane oxidation" relation of dark brown soil in Northeastern China under different long-term fertilization regimes. Community structure and abundance were monitored with PCR-DGGE and real time PCR, respectively. Methane oxidizing rate and soil properties were measured as well. The results show that combined use of mineral fertilizer and compost (MNP) reduce soil methane oxidation by 61.2%, whereas either mineral fertilizer (NP) or compost (M) shows no effect. Comparing with no fertilizer (CK), M and MNP increase the Shannon index of methanotrophs by 91.9% and 102.5%, respectively, whereas NP has no effect. Similarly, M ( M or MNP) significantly increases pmoA gene abundance by up to 12.7 folds compared with no M addition (CK or NP). Methane oxidizing rates are significantly correlated with community structure and specific activity of methanotrophs, with correlation coefficients of 0.363 and 0.684, respectively. However, methane oxidizing rates do not correlate with abundance and diversity of methanotrophs. In addition, community structures and specific activity of methanotrophs are significantly correlated with soil pH and content of total nitrogen and organic matter. Our results suggest that long-term different fertilizations may change soil properties (such as pH and content of total nitrogen and organic matter) and thereafter the community structure and specific activity of soil methanotrophs, by which long-term different fertilizations influence soil methane oxidizing rate. The opposite change of methane oxidation to methanotrophs diversity and abundance in MNP suggests that only parts of the methanotrophs are active, which needs further research.
  • Article
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    Sphagnum peatlands are important ecosystems in the methane cycle. Methane-oxidizing bacteria in these ecosystems serve as a methane filter and limit methane emissions. Yet little is known about the diversity and identity of the methanotrophs present in and on Sphagnum mosses of peatlands, and only a few isolates are known. The methanotrophic community in Sphagnum mosses, originating from a Dutch peat bog, was investigated using a pmoA microarray. A high biodiversity of both gamma- and alphaproteobacterial methanotrophs was found. With Sphagnum mosses as the inoculum, alpha- and gammaproteobacterial acidophilic methanotrophs were isolated using established and newly designed media. The 16S rRNA, pmoA, pxmA, and mmoX gene sequences showed that the alphaproteobacterial isolates belonged to the Methylocystis and Methylosinus genera. The Methylosinus species isolated are the first acid-tolerant members of this genus. Of the acidophilic gammaproteobacterial strains isolated, strain M5 was affiliated with the Methylomonas genus, and the other strain, M200, may represent a novel genus, most closely related to the genera Methylosoma and Methylovulum. So far, no acidophilic or acid-tolerant methanotrophs in the Gammaproteobacteria class are known. All strains showed the typical features of either type I or II methanotrophs and are, to the best of our knowledge, the first isolated (acidophilic or acid-tolerant) methanotrophs from Sphagnum mosses.
  • Article
    Little is understood about the relationship between microbial assemblage history, the composition and function of specific functional guilds and the ecosystem functions they provide. To learn more about this relationship we used methane oxidizing bacteria (MOB) as model organisms and performed soil microcosm experiments comprised of identical soil substrates, hosting distinct overall microbial diversities (i.e., full, reduced and zero total microbial and MOB diversities). After inoculation with undisturbed soil, the recovery of MOB activity, MOB diversity and total bacterial diversity were followed over 3 months by methane oxidation potential measurements and analyses targeting pmoA and 16S rRNA genes. Measurement of methane oxidation potential demonstrated different recovery rates across the different treatments. Despite different starting microbial diversities, the recovery and succession of the MOB communities followed a similar pattern across the different treatment microcosms. In this study we found that edaphic parameters were the dominant factor shaping microbial communities over time and that the starting microbial community played only a minor role in shaping MOB microbial community.
  • Article
    A simple freeze-coring method was developed to obtain structurally intact cores from wetland soils. A copper tube was inserted into the wetland and filled with ethanol and dry ice to freeze the surrounding soil. Biological structure and function could be analyzed, and labile compounds such as mRNA were recovered.
  • Article
    This special issue highlights several recent discoveries in the microbial methane cycle, including the diversity and activity of methanotrophic bacteria in special habitats, distribution and contribution of the newly discovered Verrucomicrobia, metabolism of methane and related one-carbon compounds such as methanol and methylamine in freshwater and marine environments, methanol and methane-dependent nitrate reduction, the relationships of methane cycle microorganisms with plants and animals, and the environmental factors that regulate microbial processes of the methane cycle. These articles also highlight the plethora of new organisms and metabolism relating to the methane cycle that have been discovered in recent years and outline the many questions in the methane cycle that still need to be addressed. It is clear that despite a tremendous amount of research on the biology of the methane cycle, the microbes involved in catalysing methane production and consumption harbour many secrets that need to be disclosed in order for us to fully understand how the biogeochemical methane cycle is regulated in the environment, and for us to make future predictions about the global sources and sinks of methane and how anthropogenic changes impact on the cycling of this important greenhouse gas.
  • Article
    In the context of the current debate on climate change, studies on methane emission and methane consumption are of particular interest, and information about methane-producing and -consuming microorganisms and the parameters that influence their abundance and activity are urgently required. Hence, over a period of six months we conducted a laboratory experiment to test the effects of temperature, water content and fertilisation on soil microbiology, methane production and methane consumption of soil from fallow land. Soil treatments with soil from the fallow land were created in order to test the individual and/or the combined effects of the aforementioned parameters. These soil treatments were analysed for physical and chemical parameters, microbial activity in terms of enzyme activities and microbial biomass, methanogenic community composition and the potential to produce and consume methane. Overall, under water-saturated conditions and over time, ammonification rate, dehydrogenase activity and microbial biomass decreased. Potentials to produce methane were determined at psychro-, meso- and thermophilic conditions, and results indicated that the highest methane emission occurred under mesophilic conditions, meaning 37 °C. Water content was observed as a major influencing factor regarding both methane production and methane consumption potentials. Regardless of the incubation temperature, fertilisation with cattle manure led to an increase in the formation of methane. Ammonium concentration promoted both potentials to produce and oxidize methane. Molecular analysis revealed differences in methanogenic community compositions within the soil treatments. We found a representative of Methanosarcinaceae to be present in all soil treatments. Furthermore, Methanobrevibacter woesei, a typical gut organism of herbivores, was identified in all fertilised soil treatments. This indicates that this methanogen has survived in the microcosms for 6 months, which emphasised the survival potential of methanogens in soils.
  • Chapter
    During cycling from host to nonhost environments, Salmonella encounter a variety of environmental insults ranging from temperature fluctuations, nutrient availability, and changes in osmolarity to oxidative stress and desiccation. The ability of Salmonella to withstand desiccation stress enables this pathogen to persist in agricultural and in food industry settings for long times and to pose an important food safety risk. Desiccated Salmonella cells develop cross-tolerance to a range of other stresses the pathogen might encounter in agro-industrial environments with important food safety consequences. This chapter provides an overview of desiccation stress response in Salmonella, reviews the current literature on the development of cross-tolerance in desiccated Salmonella cells, and discusses stress response mechanisms potentially responsible for the development of cross-tolerance. The chapter also highlights the potential implications of the cross-tolerance phenomenon to the safety of the food supply chain.
  • Chapter
    Full-text available
    Methane is the second most important greenhouse gas in terms of amounts and effect in the atmosphere. Upland soils of the European Russia are important participants in the global carbon budget, but their role as a sink for atmospheric methane is poorly documented, and little information on biodiversity of methanotrophic microorganisms is available. We have found that managed soils from different climatic regions showed decreased methane oxidation rates in both field and laboratory experiments. Large fluctuations were revealed in CH4 uptake process at different time scales (monthly, daily, hourly), and soil organic matter, water content, and temperature were seen as the main environmental controlling factors. Methanotrophic populations of unmanaged soils turned out to be much low diverse and dominated by uncultivated methanotrophs. In Podzoluvisol, Luvisol, and Meadow Kastanozem, we have identified deeply branching pmoA sequences of Alphaproteobacteria referred as NSUC (natural soil uncultivated cluster), formed novel monophyletic cluster with other uncultured methanotrophs. Pronounced shift to cultured methanotrophs was observed in the same soils after agricultural loading.
  • Article
    Recent dynamics and uncertainties in global methane budgets necessitate a dissemination of the current knowledge about the controls of sources and sinks of atmospheric methane.Forest soils are considered to be efficient methane sinks; however, as they are microbially mediated they are sensitive to anthropogenic influences and tend to switch from being sinks to being CH4 sources. Regarding global changes in land use, the present study aimed at (i) investigating the influence of grazing on flux rates of methane in forest soils (ii) deducing possible (a)biotic factors regulating these fluxes, and (iii) gaining an insight into the complex interactions between CH4-cycling microorganisms and ecosystem functioning. Here we show that even very extensive grazing significantly mitigated the soils sink strength for atmospheric methane through alterations of both microbial activity and community composition. In situ flux measurements revealed that all native, non-grazed areas were net methane consumers, while the adjacent, grazed areas were net methane producers. Whereas neither parent material nor soil properties including moisture and organic matter showed any correlation to the ascertained fluxes, significantly higher archaeal abundances at the grazed study sites indicated that small inputs of methanogens associated with cattle grazing may be sufficient to sustainably increase methane emissions.
  • Article
    Full-text available
    Methan ist neben CO2 das wichtigste Treibhausgas, dessen relatives Treibhauspotential ungefähr ein drittel höher liegt als das von CO2. Der Großteil atmosphärischen Methans wird dabei aus biogenen Methanquellen freigesetzt, zum Beispiel renaturierte Mülldeponien, Feuchtgebiete oder Reisfelder. Methanotrophe Bakterien (MOB) können die Methanemission hier um bis zu 80 % reduzieren. Infolgedessen ist ihre Physiologie, Diversität und Ökologie in zahlreichen Studien untersucht worden. Es fehlen jedoch grundlegende Studien über die räumliche Verteilung von MOB in ihrer Umwelt. Des Weiteren sind die Populationsdynamiken von MOB und die Beteiligung spezifischer Taxa an der Methanoxidation bisher wenig verstanden. Zudem beginnt man erst jetzt zu erkennen, dass Umweltstörungen einen signifikanten Effekt auf die Stabilität und Funktion mikrobieller Lebensgemeinschaften haben. Die Zusammenhänge von Diversität und Funktion und die Regulation der MOB durch natürliche und/oder anthropogene Umweltfaktoren sind bisher jedoch kaum untersucht worden. In dieser Arbeit wurde das pmoA Gen als phylogenetischer und funktioneller Marker verwendet, um MOB in Umweltproben zu detektieren. Während es speziell an das Reisfeld adaptierte pmoA Genotypen zu geben scheint, können sich methanotrophe Lebensgemeinschaften in Reisfeldern derselben Region deutlich unterscheiden. Der Einfluss von Umweltgradienten variiert in Agrar- und natürlichen Ökosystemen und muss bei der Planung von Experimenten berücksichtigt werden. Am Beispiel von Reisfeldern konnte gezeigt werden, dass MOB keine großskalige räumliche Strukturierung aufwiesen und sowohl eine systematische als auch eine Zufallsprobennahme repräsentativ ist. Zudem konnten Populationdynamiken nach der Flutung eines Reisfeldes nachgewiesen werden, obwohl die Methanoxidationrate konstant blieb. Eine artenreiche mikrobielle „seed bank“ scheint für die Erhaltung der Funktion in solchen dynamischen Ökosystemen eine große Rolle zu spielen. Betrachtet man sich die methanotrophe Lebensgemeinschaft unter verschieden Energieflüssen und dem Effekt von Stickstoffdüngung, so hat die Düngung keinen Effekt auf die methanotrophen Lebensgemeinschaften. Es werden jedoch unter verschiedenen Energieflüssen aus der „seed bank“ unterschiedliche MOB aktiviert. Es scheint, dass Arten der Gattung Methylobacter und Arten deren pmoA Sequenzen zu einem Cluster mit Umweltsequenzen aus Reisfeldern gehören, speziell an Habitate mit hoher Methankonzentration adaptiert sind. MOB scheinen sehr widerstandsfähig zu sein und Änderungen in Energieflüssen scheinen einen größeren Effekt auf die methanotrophe Lebensgemeinschaft zu haben. Methane is the second most important greenhouse gas after CO2 exerting a radiative forcing about a third of that of CO2. Most of the atmospheric methane is released from biogenic sources such as landfills, natural wetlands and rice fields. Methane emission from these sources would be significantly higher without the activity of methanotrophs that oxidize the biogenically produced methane, thus reducing the methane emissions up to 80 %. Consequently, the physiology, diversity and ecology of methanotrophs have been studied. However, influences of biogeographical patterns and spatial hetero-geneities on the methanotrophic community are poorly investigated. Furthermore, little is known about population dynamics and contribution of specific taxa to methane oxidation. The effect of environmental disturbances on the stability and function of microbial communities has just begun to be realized. However, a link between diversity and function and the regulation of methanotrophic communities by natural and/or anthropogenic factors are not known in detail. In this thesis the pmoA gene was used as a functional and phylogenetic marker for the identification of methanotrophs from environmental samples. On a global scale certain pmoA genotypes seem to be specifically adapted to paddy fields while at closely geographically located field sites methanotrophic communities revealed different community patterns. The influence of environmental gradients varies between different habitats and has to be considered when designing experimental studies. In the studied agroecosystem, population structure showed no spatial pattern implying that both a systematic and random sampling design would be adequate. We observed a succession of methanotrophs, however, the oxidation performance stayed relatively stable. Hence, a diverse microbial seed bank of methanotrophs seems to play an important role in maintaining the function in such a dynamic ecosystem. From this seed bank different methanotrophs are activated under high and low energy fluxes. We identified species of the genus Methylobacter and an environmental cluster strictly affiliated with paddy soils that seem to be adapted to high methane environments. Methanotrophic community was not significantly affected by nitrogen fertilization under different energy flows. We suggest that methanotrophs are quite resilient, and that changes in the energy flow have major effects for the community structure.
  • Article
    Methane-oxidizing bacteria (methanotrophs) were isolated from sediments of estuaries. The representative strains, GM-1 and NM-11, are gram negative motile rods and exhibit characteristics typical of Type I methanotrophs. Both strains possessed cytomembranes arranged in stacked lamellae, and their predominant fatty acids were C 16:1 and C 16:0. The DNA base ratios of the strains, GM-1 and NM11, were 51.0, 52.9% mol G+C, respectively. The strains require NaCl, grew well on methane or methanol, but not on any other compounds as a cabon source, and their growth was inhibited in natural light.
  • Chapter
    IntroductionMethanotrophs are a group of bacteria possessing a highly specialized metabolism restricted to the utilization of methane and methanol and are a subset of the methylotrophs, bacteria and archaea able to utilize C1 compounds. Methanotrophs are by definition obligately methylotrophic and do not have the ability to grow on organic compounds possessing carbon-carbon bonds. Besides methane, the only other substrate generally utilized by methanotrophs for growth is methanol; however, a few strains can utilize methylamine and a narrow selection of other C1 compounds. Methanotrophs are an integral part of the natural ecosystem, consuming much of the methane that is biogenically (through methanogenesis) and non-biogenically (e.g., from hydrocarbon seeps, natural gas fields and coal mines) derived. This interception of methane helps maintain a balance of atmospheric methane. Methanotrophs can utilize methane as they possess an enzyme called methane monooxygenase (MMO) whi ...
  • Article
    The atmospheric concentrations of the greenhouse gases nitrous oxide and methane continue to increase. Since both gases are derived primarily from the soil, soil source/sink relationships in a variety of ecosystems must be understood before complete global source/sink budgets can be accurately made. A transect of flux measurement sites was established across a subalpine meadow and adjacent forest in southeastern Wyoming, and CH4 and N2O fluxes were measured at least weekly from snow melt in June until the winter snow covered the meadow in October during 1991 and 1992. Nitrous oxide emissions from the meadow were small both years, averaging only 2.5 and 1.3 ug N m-2h-1 in 1991 and 1992, respectively. Fluxes were larger during the wetter year and varied from point to point, generally in response to soil moisture conditions. Methane flux varied across the meadow and over the season, also in response to changing soil moisture conditions. As a whole, the meadow served as a sink for atmospheric methane, but sites within the meadow served as a net source of CH4 during the snow-free part of the year. -from Authors
  • Article
    Full-text available
    The bacterial endosymbionts of 3 hydrothermal vent mussels from Japanese waters were characterized by transmission electron microscopic (TEM) observation and phylogenetic analyses of 16S ribosomal RNA gene sequences. Endosymbionts of Bathymodiolus septemdierum were related to sulfur-oxidizing bacteria (thioautotrophs), while endosymbionts of B. platifrons and B. japonicus were related to methane-oxidizing bacteria (methanotrophs). This is the first report of deep-sea mussels containing only methanotrophs (lacking thioautotrophs) from hydrothermal vents. Comparison of methane and hydrogen sulfide concentrations in end-member fluids from deep-sea hydrothermal vents indicated that methane concentrations were much higher in habitats containing Bathymodiolus spp, which harbored only methanotrophs than in other habitats of hydrothermal vent mussels. The known distribution of other mussels containing only methanotrophs has thus far been limited to cold-seep environments with high methane concentrations from the interstitial water. These results suggest that the distribution of methanotrophic symbioses between deep-sea mussels and methanotrophs is strongly influenced by the methane or hydrocarbon concentrations provided from hydrothermal vent and cold-seep activities (or that methane concentration is a possible limiting factor that restricts the distribution of methanotrophy-dependent symbioses in the deep sea).
  • Article
    Four different soils (meadow cambisol, forest luvisol, cultivated cambisol, paddy soil) were incubated under different CH4 mixing ratios (1.7 μl CH4 l−1 to 20% CH4) in air (20% O2, rest N2) and the CH4 oxidation activities as well as the numbers of methanotrophic bacteria were determined. All soils showed an increase of the microbial CH4 oxidation activity and the numbers of methanotrophic bacteria only if incubated at CH4 mixing ratios exceeding about 100–1000 μl CH4 l−1. The induction of the CH4 oxidation activity was strictly O2-dependent and was inhibited by acetylene or autoclaving, demonstrating that CH4 oxidation in the soil was due to methanotrophic bacteria. The induction process was influenced by physico-chemical soil variables such as soil moisture, pH, temperature, NH4+ concentration, Cu2+ concentration and aggregate size. Optimum ranges for the induction process were: soil water content of 20–35% H2O; pH 6.7–8.1; temperatures of 25–35°C; and NH4+ concentrations in the soil water phase of 12–61 mm. Copper concentrations >4.3 mm Cu2+ inhibited the induction of CH4 oxidation activity. Increasing soil aggregate sizes between 50 μm and >2 mm dia resulted in a slight but steady stimulation of the induction. Our results demonstrate that the development of an active methanotrophic population in oxic soils requires not only sufficient CH4 but also soil physico-chemical conditions that are suitable for growth or activation of methanotrophic bacteria.
  • Article
    Soil from the zone of maximal methanotrophic activity (approximately 5–8 cm depth) in a mixed coniferous–hardwood forest consumed atmospheric methane over a wide pH range (3.5–7.5) with a broad optimum between 4.8 and 6.0. Methane uptake at native soil pH values (4.4–4.8) was only slightly less rapid than rates at optimal pH values. Addition of mineral acids to intact soil cores in pulsed applications decreased atmospheric methane consumption. The extent of inhibition varied with the type, concentration and volume of acid added: nitric acid was more inhibitory than sulfuric acid at an equivalent soil pH, and methane uptake decreased with increasing volumes and concentrations of added acid. Although ammonium chloride at 1 μmol g fresh weight (gfw) soil−1 inhibited methane uptake, the extent of inhibition did not vary significantly with decreasing soil pH below values of 4.4.
  • Article
    Full-text available
    Rates of methane emission from a Swedish landfill, measured by chamber technique and permanent frames, ranged between 0.034 and 20 mmol CH4m−2. h−1on average. The emissions followed a seasonal pattern, with the highest fluxes occurring between September and May. Methane concentrations in soil also followed a seasonal pattern, with a marked decrease during summers. Using the means of methane emission rates from frost-free periods, a stepwise regression model was made, that could explain 95% of the variation. Soil temperature turned out to be the dominating factor, explaining 85% when transformed to a second-degree function. Methane emissions were negatively correlated with soil temperature, which strongly suggests that biological methane oxidation is an important regulating factor. The activity of methane-oxidizing microorganisms was greatest around 0.5–0.6 m depth in the soil profile, and moisture at this level enhanced emissions. The tendency for methane emissions to be higher at night was probably due to the inhibitory influence of low soil temperatures on methane-oxidizing microorganisms.
  • Article
    Crop production on acid soils can be improved greatly by adjusting the pH to near neutrality. While soil acidity is commonly corrected by liming, there is evidence that animal manure amendments can increase the pH of acid soils. The effect of fresh cattle manure on soil acidity and nutrient availability was determined in the laboratory for two acid soils from Beaverlodge and Fort Vermillion in the Peace River region of Alberta, Canada. The effect of manure on soil pH was immediate and persisted during an 8-wk incubation. Manure-amended soil had significantly higher pH than unamended soil, and the highest rate (40 g manure kg-1, dry weight basis) increased the pH of Beaverlodge and Fort Vermillion soils from 4.8 to 6.0 and 5.5 to 6.3, respectively. The higher pH in manure-amended than unamended soils was attributed to buffering from bicarbonates and organic acids in cattle manure. Mineral N (NH4-N + NO3-N), available P, K, Ca, and Mg increased immediately after manure application, and available P and K remained significantly higher in manure-amended than unamended soil after the 8-wk incubation. Soils amended with 40 g manure kg-1 had three to four times more plant-available P and K than unamended soils after incubation. Available S concentrations did not differ significantly in manure-amended and unamended soils. Extractable Al and Fe declined slightly after manure application, but did not differ in manure-amended or unamended soils after incubation. No change in the cation-exchange capacity (CEC) of manure-amended soils compared to unamended soils was observed in this study, and it appears that appreciable changes in Al, Fe, and CEC from manure application do not occur in the short-term (weeks). Our results indicate that, in the short-term, cattle manure amendments can increase the pH and the quantity of plant-available P and K in acid soils.
  • Article
    A novel thermophilic methane-oxidising bacterium was isolated from underground hot springs in Hungary. Strain HB grew on methane at up to 72°C, the highest recorded growth temperature for a methanotroph. 16S rDNA phylogenetic analysis showed that strain HB was the first known representative of a novel, deep-branching group of the γ-Proteobacteria quite distinct from extant methanotrophs. The nucleotide sequence of pmoA, encoding particulate methane monooxygenase, was determined for this novel thermophile. Sequence comparison showed that the methane monooxygenase of this strain was most closely related to that of Methylocaldum and Methylococcus species. Particulate methane monooxygenase gene fragments having a high degree of identity to that of pmoA from strain HB were amplified by PCR from DNA isolated from thermophilic methane-oxidising enrichments inoculated with hot spring samples (55–70°C) from Japan, suggesting that this novel genus, for which we informally suggest the name ‘Methylothermus’, is widespread in thermophilic environments.
  • Article
    The activity and distribution of methanotrophs in soil depend on the availability of CH4 and O2. Therefore, we investigated the activity and structure of the methanotrophic community in rice field soil under four factorial combinations of high and low CH4 and O2 concentrations. The methanotrophic population structure was resolved by denaturant gradient gel electrophoresis (DGGE) with different PCR primer sets targeting the 16S rRNA gene, and two functional genes coding for key enzymes in methanotrophs, i.e. the particulate methane monooxygenase (pmoA) and the methanol dehydrogenase (mxaF). Changes in the biomass of type I and II methanotrophic bacteria in the rice soil were determined by analysis of phospholipid-ester-linked fatty acid (PLFA) biomarkers. The relative contribution of type I and II methanotrophs to the measured methane oxidation activity was determined by labelling of soil samples with 14CH4 followed by analysis of [14C]-PLFAs. CH4 oxidation was repressed by high O2 (20.5%), and enhanced by low O2 (1%). Depending on the CH4 and O2 mixing ratios, different methanotrophic communities developed with a higher diversity at low than at high CH4 concentration as revealed by PCR-DGGE. However, a prevalence of type I or II populations was not detected. The [14C]-PLFA fingerprints, on the other hand, revealed that CH4 oxidation activity was dominated by type I methanotrophs in incubations with low CH4 mixing ratios (1000 p.p.m.v.) and during initiation of CH4 consumption regardless of O2 or CH4 mixing ratio. At high methane mixing ratios (10 000 p.p.m.v.), type I and II methanotrophs contributed equally to the measured CH4 metabolism. Collectively, type I methanotrophs responded fast and with pronounced shifts in population structure and dominated the activity under all four gas mixtures. Type II methanotrophs, on the other hand, although apparently more abundant, always present and showing a largely stable population structure, became active later and contributed to CH4 oxidation activity mainly under high CH4 mixing ratios.
  • Article
    Full-text available
    Experiments were done to test the hypothesis that atmospheric CH4 oxidizers in a well-drained alpine tundra soil are supported by CH4 production from anaerobic microsites in the soil. Soil was subjected to 22 days of anaerobic conditions with elevated H2 and CO2 in order to stimulate methanogenesis. This treatment stimulated subsequent atmospheric CH4 consumption, probably by increasing soil methanogenesis. After removal from anaerobic conditions, soils emitted CH4 for up to 6 h, then oxidized atmospheric CH4 at 111 (&#455.7) pmol (g dry weight)-1 h-1, which was more than 3 times the rate of control soils. Further supporting our hypothesis, additions of lumazine, a highly specific inhibitor of methanogenesis, prevented the stimulation of atmospheric CH4 oxidation by the anaerobic treatment. The method used to create anaerobic conditions with elevated H2 and CO2 also elevated headspace CH4 concentrations. However, elevated CH4 concentrations under aerobic conditions did not stimulate CH4 oxidation as much as preexposure to H2 and CO2 under anaerobic conditions. Anaerobic conditions created by N2 flushing did not stimulate atmospheric CH4 oxidation, probably because N2 flushing inhibited methanogenesis by removing necessary precursors for methane production. We conclude that anaerobic conditions with elevated H2 and CO2 stimulate atmospheric CH4 oxidation in this dry alpine tundra soil by increasing endogenous CH4 production. This effect was prevented by inhibiting methanogenesis, indicating the importance of endogenous CH4 production in a CH4-consuming soil.
  • Article
    CH4 emission from irrigated rice field is one of the major sources in the global budget of atmoshperic CH4. Rates of CH4 emission depend on both CH4 production in anoxic parts of the soil and on CH4 oxidation at oxic-anoxic interfaces. In the present study we used planted and unplanted rice microcosms and characterized them by numbers of CH4-oxidizing bacteria (MOB), porewater CH4 and O2 concentrations and CH4 fluxes. Plant roots had a stimulating effect on both the number of total soil bacteria and CH4-oxidizing bacteria as determined by fluorescein isothiocyanate fluorescent staining and the most probable number technique, respectively. In the rhizosphere and on the root surface CH4-oxidizing bacteria were enriched during the growth period of tice, while their numbers remained constant in unplanted soils. In the presence of rice plants, the porewater CH4 concentration was significantly lower, with 0.1–0.4mM CH4, than in unplanted microcosms, with 0.5–0.7mM CH4. O2 was detected at depths of up to 16 mm in planted microcosms, whereas it had disappeared at a depth of 2 mm in the unplanted experiments. CH4 oxidation was determined as the difference between the CH4 emission rates under oxic (air) and anoxic (N2) headspace, and by inhibition experiments with C2H2. Flux measurements showed varying oxic emission rates of between 2.5 and 29.0 mmol CH4m-2 day-1. An average of 34% of the anoxically emitted CH4 was oxidized in the planted microcosms, which was surprisingly constant. The rice rhizosphere appeared to be an important oxic-anoxic interface, significantly reducing CH4 emission.
  • Article
    Of the 17 elements known to be essential for plants, 7 are required in such small amounts as to be called micronutrients. Micronutrient elements include boron (B), manganese (Mn), iron (Fe), copper (Cu), Zinc (Zn), molybdenum (Mo), and chlorine (Cl). This text will be limited to B, Mn, Fe, Cu, Zn, and Mo since natural deficiencies of Cl are essentially nonexistent.
  • Article
    We studied methane oxidation capacity in soil profiles of Dutch and Finnish coniferous forests. The Finnish sites (n = 9) had nitrogen depositions from 3 to 36 kg N ha−1 a−1. The deposition of N on the Dutch sites (n = 13) was higher ranging from 50 to 92 kg N ha−1 a−1. The Dutch sites had also limed counterparts. Methane oxidation rates were determined by incubating soil samples in the laboratory at + 15°C with 10 μl CH4 l−1 (10 ppmv CH4). In general, CH4 oxidation rates were highest in the uppermost mineral layers. The average CH4 oxidation rate in the Finnish mineral soils was three times higher than that in the Dutch soils. The litter layers did not oxidize CH4. In the Netherlands all organic horizons had a negligible capacity to oxidize CH4. However, some Finnish organic horizons showed high CH4 oxidation capacity. In the Netherlands, in contrast to Finland, there were some soil profiles lacking CH4 oxidation. Higher contents of nitrate and ammonium, as well as greater production of nitrous oxide (N2O) and lower production of carbon dioxide in the Dutch than in the Finnish forest soils reflected the high N deposition rate in the Netherlands. Not only the N deposition, but also the highly sorted soil texture (fine sand) with low amounts of both coarse and fine particles is an important reason for the low CH4 oxidation in the Dutch soils. The proportions of fine and coarse particles, both well represented in moraine soils typical in northern Europe, correlated positively with the CH4 oxidation. Fine particles provide a good surface for microbial growth. Coarse particles, on the other hand, enhance diffusion of CH4 and oxygen into the soil. Methane oxidation in the Dutch mineral soils was slightly enhanced by liming.
  • Methane-oxidizing bacteria (methanotrophs) attenuate methane emission from major sources, such as wetlands, rice paddies, and landfills, and constitute the only biological sink for atmospheric methane in upland soils. Their key enzyme is particulate methane monooxygenase (pMMO), which converts methane to methanol. It has long been believed that methane at the trace atmospheric mixing ratio of 1.75 parts per million by volume (ppmv) is not oxidized by the methanotrophs cultured to date, but rather only by some uncultured methanotrophs, and that type I and type II methanotrophs contain a single type of pMMO. Here, we show that the type II methanotroph Methylocystis sp. strain SC2 possesses two pMMO isozymes with different methane oxidation kinetics. The pmoCAB1 genes encoding the known type of pMMO (pMMO1) are expressed and pMMO1 oxidizes methane only at mixing ratios >600 ppmv. The pmoCAB2 genes encoding pMMO2, in contrast, are constitutively expressed, and pMMO2 oxidizes methane at lower mixing ratios, even at the trace level of atmospheric methane. Wild-type strain SC2 and mutants expressing pmoCAB2 but defective in pmoCAB1 consumed atmospheric methane for >3 months. Growth occurred at 10–100 ppmv methane. Most type II but no type I methanotrophs possess the pmoCAB2 genes. The apparent Km of pMMO2 (0.11 μM) in strain SC2 corresponds well with the Km(app) values for methane oxidation measured in soils that consume atmospheric methane, thereby explaining why these soils are dominated by type II methanotrophs, and some by Methylocystis spp., in particular. These findings change our concept of methanotroph ecology. • atmospheric methane • methanotrophs • pmoA
  • Article
    Peatlands represent an enormous carbon reservoir and have a potential impact on the global climate because of the active methanogenesis and methanotrophy in these soils. Uncultivated methanotrophs from seven European peatlands were studied using a combination of molecular methods. Screening for methanotroph diversity using a particulate methane monooxygenase-based diagnostic gene array revealed that Methylocystis-related species were dominant in six of the seven peatlands studied. The abundance and methane oxidation activity of Methylocystis spp. were further confirmed by DNA stable-isotope probing analysis of a sample taken from the Moor House peatland (England). After ultracentrifugation, (13)C-labelled DNA, containing genomic DNA of these Methylocystis spp., was separated from (12)C DNA and subjected to multiple displacement amplification (MDA) to generate sufficient DNA for the preparation of a fosmid metagenomic library. Potential bias of MDA was detected by fingerprint analysis of 16S rRNA using denaturing gradient gel electrophoresis for low-template amplification (0.01 ng template). Sufficient template (1-5 ng) was used in MDA to circumvent this bias and chimeric artefacts were minimized by using an enzymatic treatment of MDA-generated DNA with S1 nuclease and DNA polymerase I. Screening of the metagenomic library revealed one fosmid containing methanol dehydrogenase and two fosmids containing 16S rRNA genes from these Methylocystis-related species as well as one fosmid containing a 16S rRNA gene related to that of Methylocella/Methylocapsa. Sequencing of the 14 kb methanol dehydrogenase-containing fosmid allowed the assembly of a gene cluster encoding polypeptides involved in bacterial methanol utilization (mxaFJGIRSAC). This combination of DNA stable-isotope probing, MDA and metagenomics provided access to genomic information of a relatively large DNA fragment of these thus far uncultivated, predominant and active methanotrophs in peatland soil.
  • Article
    The particulate methane monooxygenase gene pmoA, encoding the 27 kDa polypeptide of the membrane-bound particulate methane monooxygenase, was amplified by PCR from DNA isolated from a blanket peat bog and from enrichment cultures established, from the same environment, using methane as sole carbon and energy source. The resulting 525 bp PCR products were cloned and a representative number of clones were sequenced. Phylogenetic analysis of the derived amino acid sequences of the pmoA clones retrieved directly from environmental DNA samples revealed that they form a distinct cluster within representative PmoA sequences from type II methanotrophs and may originate from a novel group of acidophilic methanotrophs. The study also demonstrated the utility of the pmoA gene as a phylogenetic marker for identifying methanotroph-specific DNA sequences in the environment.
  • Article
    Most widely used medium for cultivation of methanotrophic bacteria from various environments is that proposed in 1970 by Whittenbury. In order to adapt and optimize medium for culturing of methanotrophs from freshwater sediment, media with varying concentrations of substrates, phosphate, nitrate, and other mineral salts were used to enumerate methanotrophs by the most probable number method. High concentrations (>1 mM) of magnesium and sulfate, and high concentrations of nitrate (>500 microM) significantly reduced the number of cultured methanotrophs, whereas phosphate in the range of 15-1500 microM had no influence. Also oxygen and carbon dioxide influenced the culturing efficiency, with an optimal mixing ratio of 17% O(2) and 3% CO(2); the mixing ratio of methane (6-32%) had no effect. A clone library of pmoA genes amplified by PCR from DNA extracted from sediment revealed the presence of both type I and type II methanotrophs. Nonetheless, the cultivation of methanotrophs, also with the improved medium, clearly favored growth of type II methanotrophs of the Methylosinus/Methylocystis group. Although significantly more methanotrophs could be cultured with the modified medium, their diversity did not mirror the diversity of methanotrophs in the sediment sample detected by molecular biology method.
  • Article
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    The oxidation of methane by methane-oxidising microorganisms is an important link in the global methane budget. Oxic soils are a net sink while wetland soils are a net source of atmospheric methane. It has generally been accepted that the consumption of methane in upland as well as lowland systems is inhibited by nitrogenous fertiliser additions. Hence, mineral nitrogen (i.e. ammonium/nitrate) has conceptually been treated as a component with the potential to enhance emission of methane from soils and sediments to the atmosphere, and results from numerous studies have been interpreted as such. Recently, ammonium-based fertilisation was demonstrated to stimulate methane consumption in rice paddies. Growth and activity of methane-consuming bacteria in microcosms as well as in natural rice paddies was N limited. Analysing the available literature revealed that indications for N limitation of methane consumption have been reported in a variety of lowland soils, upland soils, and sediments. Obviously, depriving methane-oxidising bacteria of a suitable source of N hampers their growth and activity. However, an almost instantaneous link between the presence of mineral nitrogen (i.e. ammonium, nitrate) and methane-oxidising activity, as found in rice soils and culture experiments, requires an alternative explanation. We propose that switching from mineral N assimilation to the fixation of molecular nitrogen may explain this phenomenon. However, there is as yet no experimental evidence for any mechanism of instantaneous stimulation, since most studies have assumed that nitrogenous fertiliser is inhibitory of methane oxidation in soils and have focused only on this aspect. Nitrogen as essential factor on the sink side of the global methane budget has been neglected, leading to erroneous interpretation of methane emission dynamics, especially from wetland environments. The purpose of this minireview is to summarise and balance the data on the regulatory role of nitrogen in the consumption of methane by soils and sediments, and thereby stimulate the scientific community to embark on experiments to close the existing gap in knowledge.
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    The temporal and spatial snow cover dynamics is the primary factor controlling the plant communities' composition and biogeochemical cycles in arctic and alpine tundra. However, the relationships between the distribution of snow and the diversity of soil microbial communities remain largely unexplored. Over a period of 2 years, we monitored soil microbial communities at three sites, including contiguous alpine meadows of late and early snowmelt locations (LSM and ESM, respectively). Bacterial and fungal communities were characterized by using molecular fingerprinting and cloning/sequencing of microbial ribosomal DNA extracted from the soil. Herein, we show that the spatial and temporal distribution of snow strongly correlates with microbial community composition. High seasonal contrast in ESM is associated with marked seasonal shifts for bacterial communities; whereas less contrasted seasons because of long-lasting snowpack in LSM is associated with increased fungal diversity. Finally, our results indicate that, similar to plant communities, microbial communities exhibit important shifts in composition at two extremes of the snow cover gradient. However, winter conditions lead to the convergence of microbial communities independently of snow cover presence. This study provides new insights into the distribution of microbial communities in alpine tundra in relation to snow cover dynamics, and may be helpful in predicting the future of microbial communities and biogeochemical cycles in arctic and alpine tundra in the context of a warmer climate.
  • Article
    Soil samples were collected along two slopes (south and north) at subalpine (1500-1900 m, under closed vegetation, up to the forest line) and alpine altitudes (2300-2530, under scattered vegetation, above the forest line) in the Grossglockner mountain area (Austrian central Alps). Soils were analyzed for a number of properties, including physical and chemical soil properties, microbial activity and microbial communities that were investigated using culture-dependent (viable heterotrophic bacteria) and culture-independent methods (phospholipid fatty acid analysis, FISH). Alpine soils were characterized by significantly (P<0.01) colder climate conditions, i.e. lower mean annual air and soil temperatures, more frost and ice days and higher precipitation, compared with subalpine soils. Microbial activity (soil dehydrogenase activity) decreased with altitude; however, dehydrogenase activity was better adapted to cold in alpine soils compared with subalpine soils, as shown by the lower apparent optimum temperature for activity (30 vs. 37 degrees C) and the significantly (P<0.01-0.001) higher relative activity in the low-temperature range. With increasing altitude, i.e. in alpine soils, a significant (P<0.05-0.01) increase in the relative amount of culturable psychrophilic heterotrophic bacteria, in the relative amount of the fungal population and in the relative amount of Gram-negative bacteria was found, which indicates shifts in microbial community composition with altitude.
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    A major limitation of rRNA-targeted group-specific probes is that they may cross-react with organisms of other physiological, or even phylogenetic groups when applied to environmental samples containing unknown sequences. We have exploited the restricted physiology of methane-oxidizing bacteria to assess the specificity and efficiency of probes for this physiological type which target the 16S rRNA or genes involved in methanotroph physiology. Seawater samples were enriched for methanotrophs by addition of methane and essential nutrients. The changes in composition of the bacterial population were monitored by analysis of 16S rRNA gene libraries. Methanotroph group-specific probes failed to give a signal with samples from these enrichments even though a methanol dehydrogenase structural gene was detected. A 16S rDNA sequence that was abundant only after methane addition was recovered and found to show a close phylogenetic relationship to Methylomonas. Organisms containing this sequence were observed in enrichments by in situ hybridization. The combination of enrichment on methane and screening with the broad specificity methanol dehydrogenase probe allowed detection of novel methanotrophs that were not detected with the original suite of methanotroph group-specific probes.
  • Article
    Genes encoding particulate methane monooxygenase and ammonia monooxygenase share high sequence identity. Degenerate oligonucleotide primers were designed, based on regions of shared amino acid sequence between the 27-kDa polypeptides, which are believed to contain the active sites, of particulate methane monooxygenase and ammonia monooxygenase. A 525-bp internal DNA fragment of the genes encoding these polypeptides (pmoA and amoA) from a variety of methanotrophic and nitrifying bacteria was amplified by PCR, cloned and sequenced. Representatives of each of the phylogenetic groups of both methanotrophs (alpha- and gamma-Proteobacteria) and ammonia-oxidizing nitrifying bacteria (beta- and gamma-Proteobacteria) were included. Analysis of the predicted amino acid sequences of these genes revealed strong conservation of both primary and secondary structure. Nitrosococcus oceanus AmoA showed higher identity to PmoA sequences from other members of the gamma-Proteobacteria than to AmoA sequences. These results suggest that the particulate methane monooxygenase and ammonia monooxygenase are evolutionarily related enzymes despite their different physiological roles in these bacteria.
  • Article
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    Methane-utilizing bacteria (methanotrophs) are a diverse group of gram-negative bacteria that are related to other members of the Proteobacteria. These bacteria are classified into three groups based on the pathways used for assimilation of formaldehyde, the major source of cell carbon, and other physiological and morphological features. The type I and type X methanotrophs are found within the gamma subdivision of the Proteobacteria and employ the ribulose monophosphate pathway for formaldehyde assimilation, whereas type II methanotrophs, which employ the serine pathway for formaldehyde assimilation, form a coherent cluster within the beta subdivision of the Proteobacteria. Methanotrophic bacteria are ubiquitous. The growth of type II bacteria appears to be favored in environments that contain relatively high levels of methane, low levels of dissolved oxygen, and limiting concentrations of combined nitrogen and/or copper. Type I methanotrophs appear to be dominant in environments in which methane is limiting and combined nitrogen and copper levels are relatively high. These bacteria serve as biofilters for the oxidation of methane produced in anaerobic environments, and when oxygen is present in soils, atmospheric methane is oxidized. Their activities in nature are greatly influenced by agricultural practices and other human activities. Recent evidence indicates that naturally occurring, uncultured methanotrophs represent new genera. Methanotrophs that are capable of oxidizing methane at atmospheric levels exhibit methane oxidation kinetics different from those of methanotrophs available in pure cultures. A limited number of methanotrophs have the genetic capacity to synthesize a soluble methane monooxygenase which catalyzes the rapid oxidation of environmental pollutants including trichloroethylene.
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    Methanotrophic bacteria were enumerated and isolated from the chemocline and surface sediments of marine-salinity Antarctic meromictic lakes located in the Vestfold Hills, Antarctica (68 degrees S 78 degrees E). Most probable number (MPN) analysis indicated that at the chemocline of Ace Lake the methanotroph population made up only a small proportion of the total microbial population and was sharply stratified, with higher populations detected in the surface sediments collected at the edge of Ace Lake and Burton Lake. Methanotrophs were not detected in Pendant Lake. Only a single phenotypic group of methanotrophs was successfully enriched, enumerated and isolated into pure culture from the lake samples. Strains of this group were non-motile, coccoidal in morphology, did not form resting cells, reproduced by constriction, and required seawater for growth. The strains were also psychrophilic, with optimal growth occurring at 10-13 degrees C and maximum growth temperatures of 16-21 degrees C. The ribulose monophosphate pathway but not the serine pathway for incorporation of C1 compounds was detectable in the strains. The guanine plus cytosine (G + C) content of the genomic DNA was 43-46 mol%. Whole-cell fatty acid analysis indicated that 16:1 omega 8c (37-41%), 16:1 omega 6c (17-19%), 16:1 omega 7c (15-19%) and 16:0 (14-15%) were the major fatty acids in the strains. 16s rDNA sequence analysis revealed that the strains form a distinct line of descent in the family Methylococcaceae (group I methanotrophs), with the closest relative being the Louisiana Slope methanotrophic mytilid endosymbiont (91.8-92.3% sequence similarity). On the basis of polyphasic taxonomic characteristics the Antarctic lake isolates represent a novel group I methanotrophic genus with the proposed name Methylosphaera hansonii (type strain ACAM 549).
  • Article
    Two methanotrophic bacteria with optimum growth temperatures above 40 degrees C were isolated. Thermotolerant strain LK6 was isolated from agricultural soil, and the moderately thermophilic strain OR2 was isolated from the effluent of an underground hot spring. When compared to the described thermophilic methanotrophs Methylococcus capsulatus and Methylococcus thermophilus, these strains are phenotypically similar to Methylococcus thermophilus. However, their 16S rRNA gene sequences are markedly different from the sequence of Methylococcus thermophilus ( approximately 8% divergence) and, together with Methylomonas gracilis, they form a distinct, new genus within the gamma-subgroup of the Proteobacteria related to extant Type I methanotrophs. Further phenotypic characterisation showed that the isolates possess particulate methane monooxygenase (pMMO) but do not contain soluble methane monooxygenase. The nucleotide sequence of a gene encoding pMMO (pmoA) was determined for both isolates and for Methylomonas gracilis. PmoA sequence comparisons confirmed the monophyletic nature of this newly recognised group of thermophilic methanotrophs and their relationship to previously described Type I methanotrophs. We propose that strains OR2 and LK6, together with the misclassified thermophilic strains Methylomonas gracilis VKM-14LT and Methylococcus thermophilus IMV-B3122, comprise a new genus of thermophilic methanotrophs, Methylocaldum gen. nov., containing three new species: Methylocaldum szegediense, Methylocaldum tepidum and Methylocaldum gracile.
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    The global methane cycle includes both terrestrial and atmospheric processes and may contribute to feedback regulation of the climate. Most oxic soils are a net sink for methane, and these soils consume approximately 20 to 60 Tg of methane per year. The soil sink for atmospheric methane is microbially mediated and sensitive to disturbance. A decrease in the capacity of this sink may have contributed to the approximately 1%. year(-1) increase in the atmospheric methane level in this century. The organisms responsible for methane uptake by soils (the atmospheric methane sink) are not known, and factors that influence the activity of these organisms are poorly understood. In this study the soil methane-oxidizing population was characterized by both labelling soil microbiota with (14)CH(4) and analyzing a total soil monooxygenase gene library. Comparative analyses of [(14)C]phospholipid ester-linked fatty acid profiles performed with representative methane-oxidizing bacteria revealed that the soil sink for atmospheric methane consists of an unknown group of methanotrophic bacteria that exhibit some similarity to type II methanotrophs. An analysis of monooxygenase gene libraries from the same soil samples indicated that an unknown group of bacteria belonging to the alpha subclass of the class Proteobacteria was present; these organisms were only distantly related to extant methane-oxidizing strains. Studies on factors that affect the activity, population dynamics, and contribution to global methane flux of "atmospheric methane oxidizers" should be greatly facilitated by use of biomarkers identified in this study.
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    Methane is involved in a number of chemical and physical processes in the Earth's atmosphere, including global warming. Atmospheric methane originates mainly from biogenic sources, such as rice paddies and natural wetlands; the former account for at least 30% of the global annual emission of methane to the atmosphere. As an increase of rice production by 60% is the most appropriate way to sustain the estimated increase of the human population during the next three decades, intensified global fertilizer application will be necessary: but it is known that an increase of the commonly used ammonium-based fertilizers can enhance methane emission from rice agriculture. Approximately 10-30% of the methane produced by methanogens in rice paddies is consumed by methane-oxidizing bacteria associated with the roots of rice; these bacteria are generally thought to be inhibited by ammonium-based fertilizers, as was demonstrated for soils and sediments. In contrast, we show here that the activity and growth of such bacteria in the root zone of rice plants are stimulated after fertilization. Using a combination of radioactive fingerprinting and molecular biology techniques, we identify the bacteria responsible for this effect. We expect that our results will make necessary a re-evaluation of the link between fertilizer use and methane emissions, with effects on global warming studies.
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    Well-drained non-agricultural soils mediate the oxidation of methane directly from the atmosphere, contributing 5 to 10% towards the global methane sink. Studies of methane oxidation kinetics in soil infer the activity of two methanotrophic populations: one that is only active at high methane concentrations (low affinity) and another that tolerates atmospheric levels of methane (high affinity). The activity of the latter has not been demonstrated by cultured laboratory strains of methanotrophs, leaving the microbiology of methane oxidation at atmospheric concentrations unclear. Here we describe a new pulse-chase experiment using long-term enrichment with 12CH4 followed by short-term exposure to 13CH4 to isotopically label methanotrophs in a soil from a temperate forest. Analysis of labelled phospholipid fatty acids (PLFAs) provided unambiguous evidence of methane assimilation at true atmospheric concentrations (1.8-3.6 p.p.m.v.). High proportions of 13C-labelled C18 fatty acids and the co-occurrence of a labelled, branched C17 fatty acid indicated that a new methanotroph, similar at the PLFA level to known type II methanotrophs, was the predominant soil micro-organism responsible for atmospheric methane oxidation.
  • Article
    Soil from the zone of maximal methanotrophic activity (approximately 5-8 cm depth) in a mixed coniferous-hardwood forest consumed atmospheric methane over a wide pH range (3.5-7.5) with a broad optimum between 4.8 and 6.0. Methane uptake at native soil pH values (4.4-4.8) was only slightly less rapid than rates at optimal pH values. Addition of mineral acids to intact soil cores in pulsed applications decreased atmospheric methane consumption. The extent of inhibition varied with the type, concentration and volume of acid added: nitric acid was more inhibitory than sulfuric acid at an equivalent soil pH, and methane uptake decreased with increasing volumes and concentrations of added acid. Although ammonium chloride at 1 µmol g fresh weight (gfw) soil(-1) inhibited methane uptake, the extent of inhibition did not vary significantly with decreasing soil pH below values of 4.4.
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    Two novel species of obligate methane-oxidizing bacteria, isolated from landfill soil, were characterized. Both strains were unusual in that some members of the population grew in irregularly shaped, refractile cell packets that resembled sarcina-like clusters. Electron microscopy revealed that the cell packets were covered with a slime layer and the cells contained many large granular inclusion bodies. The individual cells of each strain were sometimes motile and had differing morphologies. Isolate AML-C10T was always coccoidal in shape, and the cells were covered with extracellular fibrils. Isolate AML-D4T was pleomorphic, changing from rod to coccal form, sometimes exhibiting an unusual fusiform morphology. AML-D4T lacked the extensive fibrillar matrix observed with AML-C10T. Both strains utilized only methane and methanol as carbon sources. In stationary phase, the cells of each strain swelled in size and formed cysts. Aside from morphological differences, strains could also be distinguished from each other by cellular protein patterns, as well as by temperature and pH tolerances. 16S rDNA phylogenetic analysis showed that these are type I methanotrophs (family: Methylococcaceae) most closely related to the Methylobacter/Methylomicrobium clade, although they form a monophyletic grouping supported by moderately high bootstrap values. By 16S rDNA database searches, the most similar species to both isolates were Methylobacter spp. However, partial particulate methane monooxygenase sequence analysis suggested that these bacteria might be more closely related to Methylomicrobium than Methylobacter. Furthermore, cellular fatty acid profiles of the strains more closely resemble those of Methylomicrobium, although the absence of significant levels of 16:1omega5c argues for the uniqueness of these two strains. On the basis of the results described here, it is proposed that a new genus should be created, Methylosarcina gen. nov., harbouring two species, Methylosarcina fibrata sp. nov. (type species) and Methylosarcina quisquiliarum sp. nov. The type strains are AML-C10T (= ATCC 700909T = DSM 13736T) and AML-D4T (= ATCC 700908T = DSM 13737T), respectively.
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    A novel genus and species, Methylocapsa acidiphila gen. nov., sp. nov., are proposed for a methane-oxidizing bacterium isolated from an acidic Sphagnum peat bog. This bacterium, designated strain B2T, represents aerobic, gram-negative, colourless, non-motile, curved coccoids that form conglomerates covered by an extracellular polysaccharide matrix. The cells use methane and methanol as sole sources of carbon and energy and utilize the serine pathway for carbon assimilation. Strain B2T is a moderately acidophilic organism with growth between pH 4.2 and 7.2 and at temperatures from 10 to 30 degrees C. The cells possess a well-developed system of intracytoplasmic membranes (ICM) packed in parallel on only one side of the cell membrane. This type of ICM structure represents a novel arrangement, which was termed type III. The resting cells are Azotobacter-type cysts. Strain B2T is capable of atmospheric nitrogen fixation; it possesses particulate methane monooxygenase and does not express soluble methane monooxygenase. The major phospholipid fatty acid is 18:1omega7c and the major phospholipids are phosphatidylglycerols. The G+C content of the DNA is 63.1 mol%. This bacterium belongs to the alpha-subclass of the Proteobacteria and is most closely related to the acidophilic methanotroph Methylocella palustris KT (97.3% 16S rDNA sequence similarity). However, the DNA-DNA hybridization value between strain B2T and Methylocella palustris K(T) is only 7%. Thus, strain B2T is proposed to comprise a novel genus and species, Methylocapsa acidiphila gen. nov., sp. nov. Strain B2T (= DSM 13967T = NCIMB 13765T) is the type strain.
  • Article
    Methane-oxidizing bacteria (methanotrophs) containing soluble methane monooxygenase (sMMO) are of interest in natural environments due to the high co-metabolic activity of this enzyme with contaminants such as trichloroethylene. We have analysed sMMO-containing methanotrophs in sediment from a freshwater lake. Environmental clone banks for a gene encoding a diagnostic sMMO subunit (mmoX) were generated using DNA extracted from Lake Washington sediment and subjected to RFLP analysis. Representatives from the six RFLP groups were cloned and sequenced, and all were found to group with Type I Methylomonas mmoX, although a majority were divergent from known Methylomonas mmoX sequences. Direct hybridization of Lake Washington sediment DNA was carried out using a series of sMMO- and Methylomonas-specific probes to assess the significance of these sMMO-containing Methylomonas-like strains in the sediment. The total sMMO-containing population and the sMMO-containing Methylomonas-like population were estimated to be similar to previous estimates for total methanotrophs and Type I methanotrophs. These results suggest that the major methanotrophic population in Lake Washington sediment consists of sMMO-containing Methylomonas-like (Type I) methanotrophs. The whole-cell TCE degradation kinetics of such a strain, LW15, isolated from this environment, were determined and found to be similar to values reported for other sMMO-containing methanotrophs. The numerical significance of sMMO-containing Methylomonas-like methanotrophs in a mesotrophic lake environment suggests that these methanotrophs may play an important role in methanotroph-mediated transformations, including co-metabolism of halogenated solvents, in natural environments.
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    Methane oxidation in soils is mostly accomplished by methanotrophic bacteria. Little is known about the abundance of methanotrophs in soils, since quantification by cultivation and microscopic techniques is cumbersome. Comparison of 16S ribosomal DNA and pmoA (α subunit of the particulate methane monooxygenase) phylogenetic trees showed good correlation and revealed five distinct groups of methanotrophs within the α and γ subclasses of Proteobacteria: the Methylococcus group, the Methylobacter/Methylosarcina group, the Methylosinus group, the Methylocapsa group, and the forest clones group (a cluster of pmoA sequences retrieved from forest soils). We developed quantitative real-time PCR assays with SybrGreen for each of these five groups and for all methanotrophic bacteria by targeting the pmoA gene. Detection limits were between 101 and 102 target molecules per reaction for all assays. Real-time PCR analysis of soil samples spiked with cells of Methylococcus capsulatus, Methylomicrobium album, and Methylosinus trichosporium recovered almost all the added bacteria. Only the Methylosinus-specific assay recovered only 20% of added cells, possibly due to a lower lysis efficiency of type II methanotrophs. Analysis of the methanotrophic community structure in a flooded rice field soil showed (5.0 ± 1.4) × 106 pmoA molecules g−1 for all methanotrophs. The Methylosinus group was predominant (2.7 × 106 ± 1.1 × 106 target molecules g−1). In addition, bacteria of the Methylobacter/Methylosarcina group were abundant (2.0 × 106 ± 0.9 × 106 target molecules g of soil−1). On the other hand, pmoA affiliated with the forest clones and the Methylocapsa group was below the detection limit of 1.9 × 104 target molecules g of soil−1. Our results showed that pmoA-targeted real-time PCR allowed fast and sensitive quantification of the five major groups of methanotrophs in soil. This approach will thus be useful for quantitative analysis of the community structure of methanotrophs in nature.
  • Article
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    Amplification of DNA from soil is often inhibited by co-purified contaminants. A rapid, inexpensive, large-scale DNA extraction method involving minimal purification has been developed that is applicable to various soil types (1). DNA is also suitable for PCR amplification using various DNA targets. DNA was extracted from 100g of soil using direct lysis with glass beads and SDS followed by potassium acetate precipitation, polyethylene glycol precipitation, phenol extraction and isopropanol precipitation. This method was compared to other DNA extraction methods with regard to DNA purity and size.
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    A greater understanding of the tightly linked trophic groups of anaerobic and aerobic bacteria residing in municipal solid waste landfills will increase our ability to control methane emissions and pollutant fate in these environments. To this end, we characterized the composition of methanogenic and methanotrophic bacteria in samples taken from two regions of a municipal solid waste landfill that varied in age. A method combining polymerase chain reaction amplification, restriction fragment length polymorphism analysis and phylogenetic analysis was used for this purpose. 16S rDNA sequence analysis revealed a rich assemblage of methanogens in both samples, including acetoclasts, H2/CO2-users and formate-users in the newer samples and H2/CO2-users and formate-users in the older samples, with closely related genera including Methanoculleus, Methanofollis, Methanosaeta and Methanosarcina. Fewer phylotypes of type 1 methanotrophs were observed relative to type 2 methanotrophs. Most type 1 sequences clustered within a clade related to Methylobacter, whereas type 2 sequences were broadly distributed among clades associated with Methylocystis and Methylosinus species. This genetic characterization tool promises rapid screening of landfill samples for genotypes and, therefore, degradation potentials.
  • Article
    Soda lakes are an environment with an unusually high pH and often high salinity. To identify the active methanotrophs in the Soda lake sediments, sediment slurries were incubated with a 10% (v/v) (13)CH(4) headspace and the (13)C-labelled DNA was subsequently extracted from these sediments following CsCl density gradient centrifugation. This DNA was then used as a template for PCR amplification of 16S rRNA genes and genes encoding PmoA and MmoX of methane monooxygenase, key enzymes in the methane oxidation pathway. Phylogenetic analysis of 16S rRNA genes, PmoA and MmoX identified that strains of Methylomicrobium, Methylobacter, Methylomonas and 'Methylothermus' had assimilated the (13)CH(4). Phylogenetic analysis of PmoA sequences amplified from DNA extracted from Soda lake sediments before Stable Isotope Probing (SIP) treatment showed that a much wider diversity of both type I and type II methanotroph sequences are present in this alkaline environment. The majority of methanotroph sequences detected in the (13)C-DNA studies were from type I methanotrophs, with 50% of 16S rRNA clones and 100% of pmoA clones from both Lake Suduntuiskii Torom and Lake Gorbunka suggesting that the type I methanotrophs are probably responsible for the majority of methane oxidation in this environment.
  • Article
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    Samples from three submerged sites (MC, a core obtained in the methane seep area; MR, a reference core obtained at a distance from the methane seep; and HC, a gas-bubbling carbonate sample) at the Kuroshima Knoll in the southern Ryuku arc were analyzed to gain insight into the organisms present and the processes involved in this oxic-anoxic methane seep environment. 16S rRNA gene analyses by quantitative real-time PCR and clone library sequencing revealed that the MC core sediments contained abundant archaea (approximately 34% of the total prokaryotes), including both mesophilic methanogens related to the genus Methanolobus and ANME-2 members of the Methanosarcinales, as well as members of the delta-Proteobacteria, suggesting that both anaerobic methane oxidation and methanogenesis occurred at this site. In addition, several functional genes connected with methane metabolism were analyzed by quantitative competitive-PCR, including the genes encoding particulate methane monooxygenase (pmoA), soluble methane monooxygenase (mmoX), methanol dehydrogenese (mxaF), and methyl coenzyme M reductase (mcrA). In the MC core sediments, the most abundant gene was mcrA (2.5 x 10(6) copies/g [wet weight]), while the pmoA gene of the type I methanotrophs (5.9 x 10(6) copies/g [wet weight]) was most abundant at the surface of the MC core. These results indicate that there is a very complex environment in which methane production, anaerobic methane oxidation, and aerobic methane oxidation all occur in close proximity. The HC carbonate site was rich in gamma-Proteobacteria and had a high copy number of mxaF (7.1 x 10(6) copies/g [wet weight]) and a much lower copy number of the pmoA gene (3.2 x 10(2) copies/g [wet weight]). The mmoX gene was never detected. In contrast, the reference core contained familiar sequences of marine sedimentary archaeal and bacterial groups but not groups specific to C1 metabolism. Geochemical characterization of the amounts and isotopic composition of pore water methane and sulfate strongly supported the notion that in this zone both aerobic methane oxidation and anaerobic methane oxidation, as well as methanogenesis, occur.
  • Article
    A novel moderately thermophilic methanotroph, strain MYHT(T), was isolated from a hot spring in Japan. The isolate grew on methane or methanol at 37-67 degrees C, and optimally at 57-59 degrees C. It was found to be a Gram-negative aerobe, with colourless colonies of non-motile coccoid cells, possessing type I intracytoplasmic membranes and regularly arranged surface layers of linear (p2) symmetry. Strain MYHT(T) expressed only the particulate methane monooxygenase and employed the ribulose monophosphate pathway for formaldehyde assimilation. It is a neutrophilic and halotolerant organism capable of growth at pH 6.5-7.5 (optimum pH 6.8) and in up to 3% NaCl (optimum 0.5-1% NaCl). Phylogenetic analysis based on 16S rRNA gene sequence analysis indicated that strain MYHT(T) is most closely related to the thermophilic undescribed methanotroph 'Methylothermus' HB (91% identity) and the novel halophilic methanotroph Methylohalobius crimeensis 10Ki(T) (90% identity). Comparative sequence analysis of particulate methane monooxygenase (pmoA) genes also confirmed the clustering of strain MYHT(T) with 'Methylothermus' HB and Methylohalobius crimeensis 10Ki(T) (98 and 92% derived amino acid sequence identity, respectively). The DNA G+C content was 62.5 mol%. The major cellular fatty acids were C(16:0) (37.2%) and C(18:1)omega9c (35.2%) and the major polar lipids were phosphatidylethanolamine and phosphatidylglycerol. The major ubiquinone was Q-8. On the basis of comparative phenotypic and genotypic characteristics, a new genus and species, Methylothermus thermalis gen. nov., sp. nov., is proposed, with MYHT(T) as the type strain (=VKM B-2345(T)=IPOD FERM P-19714(T)).
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