Analysis of 16S rRNA and methane monooxygenase gene sequences reveals a novel group of thermotolerant and thermophilic methanotrophs, Methylocaldum gen. nov.

Two methanotrophic bacteria with optimum growth temperatures above 40° 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 (∼ 8% divergence) and, together with Methylomonas gracilis, they form a distinct, new genus within the γ-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-14L^T 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. Received: 2 April 1997 / Accepted: 23 July 1997     

Detection of Methanotroph Diversity on Roots of Submerged Rice Plants by Molecular Retrieval of pmoA, mmoX, mxaF, and 16S rRNA and Ribosomal DNA, Including pmoA-Based Terminal Restriction Fragment Length Polymorphism Profiling

The diversity of methanotrophic bacteria associated with roots of submerged rice plants was assessed using cultivation-independent techniques. The research focused mainly on the retrieval of pmoA, which encodes the α subunit of the particulate methane monooxygenase. A novel methanotroph-specific community-profiling method was established using the terminal restriction fragment length polymorphism (T-RFLP) technique. The T-RFLP profiles clearly revealed a more complex root-associated methanotrophic community than did banding patterns obtained by pmoA-based denaturing gradient gel electrophoresis. The comparison of pmoA-based T-RFLP profiles obtained from rice roots and bulk soil of flooded rice microcosms suggested that there was a substantially higher abundance of type I methanotrophs on rice roots than in the bulk soil. These were affiliated to the genera Methylomonas, Methylobacter, Methylococcus, and to a novel type I methanotroph sublineage. By contrast, type II methanotrophs of the Methylocystis-Methylosinus group could be detected with high relative signal intensity in both soil and root compartments. Phylogenetic treeing analyses and a set of substrate-diagnostic amino acid residues provided evidence that a novel pmoA lineage was detected. This branched distinctly from all currently known methanotrophs. To examine whether the retrieval of pmoA provided a complete view of root-associated methanotroph diversity, we also assessed the diversity detectable by recovery of genes coding for subunits of soluble methane monooxygenase (mmoX) and methanol dehydrogenase (mxaF). In addition, both 16S rRNA and 16S ribosomal DNA (rDNA) were retrieved using a PCR primer set specific to type I methanotrophs. The overall methanotroph diversity detected by recovery of mmoX, mxaF, and 16S rRNA and 16S rDNA corresponded well to the diversity detectable by retrieval of pmoA.     

Improved Method for Detection of Methanotrophic Bacteria in Forest Soils by PCR

A primer set was designed for the specific detection of methanotrophic bacteria in forest soils by PCR. The primer sequences were derived from highly conservative regions of the pmoA gene, encoding the α-subunit of the particulate methane monooxygenase present in all methanotrophs. In control experiments with genomic DNA from a collection of different type I, II, and X methanotrophs, it could be demonstrated that the new primers were specific for members of the genera Methylosinus, Methylocystis, Methylomonas, Methylobacter, and Methylococcus. To test the suitability of the new primers for the detection of particulate methane monooxygenase (pMMO) containing methanotrophs in environmental samples we used DNA extracts from an acid spruce forest soil. For simple and rapid purification of the DNA extracts, the samples were separated by electrophoresis on a low-melting-point agarose gel. This allowed us to efficiently separate the DNA from coextracted humic acids. The DNA from the melted agarose gel was ready for use in PCR reactions. In PCR reactions with DNA from the Ah soil layer, products of the correct size were amplified by PCR by use of the new primers. By sequencing of cloned PCR products, it could be confirmed that the PCR products represented partial sequences with strong similarity to the pmoA gene. The sequence was most related to the pmoA sequence of a type II methanotroph strain isolated from the Ah layer of the investigated soils. Received: 1 September 2000 / Accepted: 2 October 2000     

Comparison of Methanotrophic Community and Methane Oxidation between Rhizospheric and Non-Rhizospheric Soils

Using particulate methane monooxygenase (pMMO) encoding gene, pmoA-based terminal-restrict fragment length polymorphism (T-RFLP), the methanotrophic communities between rhizospheric soils (RSs) and non-rhizospheric soil (NRSs) of landfill cover (LC), riparian wetland (RW) and rice paddy (RP) were compared before and after pre-incubation of 90 days. The ultimate potential of methane oxidation rate (UPMOR) and gene copy number of pmoA were evaluated in the soil samples after pre-incubation. Compared to the methanotrophic community in the soil samples before pre-incubation, type II methanotrophs, the Methylocystis-Methylosinus group, was mostly increased after pre-incubation, regardless of the soil type. The UPMOR (11.82 ± 0.27 μmol-CH₄· g^?1 _soil-DW·h^?1) in the LC-RS was significantly higher than that (9.57 ± 0.14 μmol-CH₄· g^?1 _soil-DW·h^?1) in the LC-NRS. However, no significant difference was found between RSs and NRSs in the RW (15.28 ± 0.91 and 13.23 ± 0.69 μmol-CH₄· g^?1 _soil-DW·h^?1, respectively) and RP (13.81 ± 1.04 and 12.81 ± 2.40 μmol-CH₄· g^?1 _soil-DW·h^?1, respectively) soils. There was no significantly difference in the gene copy numbers of pmoA in the RSs compared with those in the NRSs at all of the sampling sites. This study provides basic metagenomic information about both rhizospheric and non-rhizospheric methanotrophs, which will be helpful in developing a better strategy of biological methane removal from both natural and anthropogenic major methane sources.     

Methanotrophic diversity in an agricultural soil as evaluated by denaturing gradient gel electrophoresis profiles of pmoA, mxaF and 16S rDNA sequences

Molecular methods were used to characterize the diversity of a methanotrophic population in an agricultural soil. For this purpose we have used DGGE analysis of functional and phylogenetic markers. Functional markers utilised comprised the pmoA-gene coding for the -subunit of the particulate methane monooxygenase (pMMO) present in all known methanotrophs and the mxaF-gene coding for the -subunit of methanol dehydrogenase (MDH) present in all Gram-negative methylotrophs. In addition, we have used 16S rDNA as a phylogenetic marker. DGGE patterns of an enrichment culture, and sequencing of major DGGE bands obtained with the bacterial specific primers showed that the community structure was dominated by methanotrophic populations related to Methylobacter sp. and Methylomicrobium sp. The PCR products amplified with the functional primer sets were related to both type I and type II methanotrophs. We also designed a new pmoA-targeting primer set which could be used in a nested protocol to amplify PCR-products from DNA extracted directly from the soil.     

Field application of nitrogen and phenylacetylene to mitigate greenhouse gas emissions from landfill cover soils: effects on microbial community structure

Landfills are large sources of CH₄, but a considerable amount of CH₄ can be removed in situ by methanotrophs if their activity can be stimulated through the addition of nitrogen. Nitrogen can, however, lead to increased N₂O production. To examine the effects of nitrogen and a selective inhibitor on CH₄ oxidation and N₂O production in situ, 0.5 M of NH₄Cl and 0.25 M of KNO₃, with and without 0.01% (w/v) phenylacetylene, were applied to test plots at a landfill in Kalamazoo, MI from 2007 November to 2009 July. Nitrogen amendments stimulated N₂O production but had no effect on CH₄ oxidation. The addition of phenylacetylene stimulated CH₄ oxidation while reducing N₂O production. Methanotrophs possessing particulate methane monooxygenase and archaeal ammonia-oxidizers (AOAs) were abundant. The addition of nitrogen reduced methanotrophic diversity, particularly for type I methanotrophs. The simultaneous addition of phenylacetylene increased methanotrophic diversity and the presence of type I methanotrophs. Clone libraries of the archaeal amoA gene showed that the addition of nitrogen increased AOAs affiliated with Crenarchaeal group 1.1b, while they decreased with the simultaneous addition of phenylacetylene. These results suggest that the addition of phenylacetylene with nitrogen reduces N₂O production by selectively inhibiting AOAs and/or type II methanotrophs.     

Rice roots select for type I methanotrophs in rice field soil

Methanotrophs are an important regulator for reducing methane (CH₄) emissions from rice field soils. The type I group of the proteobacterial methanotrophs are generally favored at low CH₄ concentration and high O₂ availability, while the type II group lives better under high CH₄ and limiting O₂ conditions. Such physiological differences are possibly reflected in their ecological preferences. In the present study, methanotrophic compositions were compared between rice-planted soil and non-planted soil and between the rhizosphere and rice roots by using terminal restriction fragment length polymorphism (T-RFLP) analysis of particulate methane monooxygenase (pmoA) genes. In addition, the effects of rice variety and nitrogen fertilizer were evaluated. The results showed that the terminal restriction fragments (T-RFs), which were characteristic for type I methanotrophs, substantially increased in the rhizosphere and on the roots compared with non-planted soils. Furthermore, the relative abundances of the type I methanotroph T-RFs were greater on roots than in the rhizosphere. Of type I methanotrophs, the 79 bp T-RF, which was characteristic for an unknown group or Methylococcus/Methylocaldum, markedly increased in field samples, while the 437 bp, which possibly represented Methylomonas, dominated in microcosm samples. These results suggested that type I methanotrophs were enriched or selected for by rice roots compared to type II methanotrophs. However, the members of type I methanotrophs are dynamic and sensitive to environmental change. Rice planting appeared to increase the copy number of pmoA genes relative to the non-planted soils. However, neither the rice variety nor the N fertilizer significantly influenced the dynamics of the methanotrophic community.     

The effect of methane and methanol on the terrestrial ammonia-oxidizing archaeon ‘Candidatus Nitrosocosmicus franklandus C13’

The ammonia monooxygenase (AMO) is a key enzyme in ammonia-oxidizing archaea, which are abundant and ubiquitous in soil environments. The AMO belongs to the copper-containing membrane monooxygenase (CuMMO) enzyme superfamily, which also contains particulate methane monooxygenase (pMMO). Enzymes in the CuMMO superfamily are promiscuous, which results in co-oxidation of alternative substrates. The phylogenetic and structural similarity between the pMMO and the archaeal AMO is well-established, but there is surprisingly little information on the influence of methane and methanol on the archaeal AMO and terrestrial nitrification. The aim of this study was to examine the effects of methane and methanol on the soil ammonia-oxidizing archaeon ‘Candidatus Nitrosocosmicus franklandus C13’. We demonstrate that both methane and methanol are competitive inhibitors of the archaeal AMO. The inhibition constants (K_i) for methane and methanol were 2.2 and 20 μM, respectively, concentrations which are environmentally relevant and orders of magnitude lower than those previously reported for ammonia-oxidizing bacteria. Furthermore, we demonstrate that a specific suite of proteins is upregulated and downregulated in ‘Ca. Nitrosocosmicus franklandus C13’ in the presence of methane or methanol, which provides a foundation for future studies into metabolism of one-carbon (C1) compounds in ammonia-oxidizing archaea.     

Methanotrophic community structure and activity under warming and grazing of alpine meadow on the Tibetan Plateau

Knowledge about methanotrophs and their activities is important to understand the microbial mediation of the greenhouse gas CH₄ 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 × 10⁸ 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 CH₄ oxidation activity. There were significantly negative correlations between methanotrophic abundance and soil moisture and between methanotrophic abundance and NH₄–N content. The study suggests that type I methanotrophs, as the dominance, may play a key role in CH₄ oxidation, and the alpine meadow has great potential to consume more CH₄ under future warmer and grazing conditions on the Tibetan Plateau.     

Activity of a Methanotrophic Consortium Isolated from Landfill Cover Soil: Response to Temperature,pH, CO2, and Porous Adsorbent

A robust, naturally evolving methanotrophic community in landfill cover soil (LFCS) can be the simplest way to mitigate landfill methane emission. In this study, bacterial community composition in LFCS and methane oxidation potential of enriched methanotrophic consortium, in comparison to that of axenic Methylosinus sporium, was investigated. Growth and methane oxidation of the consortium was studied in liquid phase batch experiments under varying temperature (20–40°C), pH (5–10), headspace CO2, and in presence of porous adsorbent (1.3 cm³ sponge cubes). The 16S rRNA gene analysis revealed presence of both type-I and type-II methanotrophs along with few obligate methylotroph in LFCS. Though the optimal growth condition of the consortium was at 30°C and pH 7, it was more resilient in comparison to M. sporium. With increasing availability of porous adsorbent, methane consumption by the consortium was significantly improved (p < 0.001) reaching a maximum specific methane oxidation rate of 11.4 μmol mg^?1 biomass h^?1. Thus, inducing naturally thriving methanotrophs in LFCS is a better alternative to axenic methanotrophic culture in methane emission management.     

Methanotrophic Communities in the Soils of the Russian Northern Taiga and Subarctic Tundra

The PCR analysis of DNA extracted from soil samples taken in the Russian northern taiga and subarctic tundra showed that the DNA extracts contain genes specific to methanotrophic bacteria, i.e., the mmoX gene encoding the conserved -subunit of the hydroxylase component of soluble methane monooxygenase, the pmoA gene encoding the -subunit of particulate methane monooxygenase, and the mxaFgene encoding the -subunit of methanol dehydrogenase. PCR analysis with group-specific primers also showed that methanotrophic bacteria in the northern taiga and subarctic tundra soils are essentially represented by the type I genera Methylobacter, Methylomonas, Methylosphaera, and Methylomicrobium and that some soil samples contain type II methanotrophs close to members of the genera Methylosinus and Methylocystis. The electron microscopic examination of enrichment cultures obtained from the soil samples confirmed the presence of methanotrophic bacteria in the ecosystems studied and showed that the methanotrophs contain only small amounts of intracytoplasmic membranes.     

Effects of copper on expression of methane monooxygenases,trichloroethylene degradation,and community structure in methanotrophic consortia

Copper plays a key role in regulating the expression of enzymes that promote biodegradation of contaminants in methanotrophic consortia (MC). Here, we utilized MC isolated from landfill cover to investigate cometabolic degradation of trichloroethylene (TCE) at nine different copper (Cu²⁺) concentrations. The results demonstrated that an increase in Cu²⁺ concentration from 0 to 15 μM altered the specific first‐order rate constant k_1,TCE, the expression levels of methane monooxygenase (pmoA and mmoX) genes, and the specific activity of soluble methane monooxygenase (sMMO). High efficiency TCE degradation (95%) and the expression levels of methane monooxygenase (MMO) were detected at a Cu²⁺ concentration of 0.03 μM. Notably, sMMO‐specific activity ranged from 74.41 nmol/(mg_cell h) in 15 μM Cu²⁺ to 654.99 nmol/(mg_cell h) in 0.03 μM Cu²⁺, which contrasts with cultures of pure methanotrophs in which sMMO activity is depressed at high Cu²⁺ concentrations, indicating a special regulatory role for Cu²⁺ in MC. The results of MiSeq pyrosequencing indicated that higher Cu²⁺ concentrations stimulated the growth of methanotrophic microorganisms in MC. These findings have important implications for the elucidation of copper‐mediated regulatory mechanisms in MC.     

不同放牧强度下土壤氨氧化和反硝化微生物的变化特征

土壤硝化及反硝化功能微生物在氮素可利用性、硝酸盐淋溶和氧化亚氮温室气体排放等方面起着关键作用,在指示不同放牧强度对生态系统的影响及预测草地生态系统退化状况等方面具有重要意义。以内蒙古干旱半干旱草原不同放牧强度(轻度、中度和重度)的长期试验样地为对象,应用定量PCR和限制性末端片段长度多态性(Terminal restriction fragment length polymorphism,T-RFLP)的方法,研究土壤氨氧化古菌(ammonia-oxidizing archaea,AOA)、氨氧化细菌(ammonia-oxidizing bacteria,AOB)和反硝化细菌的丰度、群落结构和多样性对不同放牧强度的响应。结果表明,土壤p H和铵态氮含量分别在7.90—8.18和6.37—35.92 mg/kg之间,中度放牧处理显著增高了土壤pH(P=0.03),而铵态氮含量在重度放牧处理中最高(P=0.02)。不同放牧强度下土壤异养呼吸相比未放牧处理均显著降低(P=0.02)。土壤AOA-amoA和AOB-amoA基因丰度范围分别为每克干土(4.94—7.60)×10~9个拷贝数和(0.68—3.75)×10~6个拷贝数,放牧处理对AOA-amoA基因丰度无显著影响,中度放牧处理显著降低了AOB-amoA基因丰度(P=0.04);反硝化微生物nosZ基因丰度随在轻度放牧处理中最低(P=0.03)。土壤铵态氮含量是影响AOA-amoA和AOB-amoA基因丰度的主要因子,而nosZ基因丰度主要受反硝化底物含量及土壤通气状况的影响。冗余分析表明由放牧所引起的可利用性氮含量的变化是导致氨氧化和反硝化微生物群落结构显著变化的主要因素。     

Recovery of methanotrophs from disturbance: population dynamics,evenness and functioning

Biodiversity is claimed to be essential for ecosystem functioning, but is threatened by anthropogenic disturbances. Prokaryotes have been assumed to be functionally redundant and virtually inextinguishable. However, recent work indicates that microbes may well be sensitive to environmental disturbance. Focusing on methane-oxidizing bacteria as model organisms, we simulated disturbance-induced mortality by mixing native with sterilized paddy soil in two ratios, 1:4 and 1:40, representing moderate and severe die-offs. Disturbed microcosms were compared with an untreated control. Recovery of activity and populations was followed over 4 months by methane uptake measurements, pmoA-qPCR, pmoA-based terminal restriction fragment length polymorphism and a pmoA-based diagnostic microarray. Diversity and evenness of methanotrophs decreased in disturbed microcosms, but functioning was not compromised. We consistently observed distinctive temporal shifts between type I and type II methanotrophs, and a rapid population growth leading to even higher cell numbers comparing disturbed microcosms with the control. Overcompensating mortality suggested that population size in the control was limited by competition with other bacteria. Overall, methanotrophs showed a remarkable ability to compensate for die-offs.     

Effect of Afforestation and Reforestation of Pastures on the Activity and Population Dynamics of Methanotrophic Bacteria

We investigated the effect of afforestation and reforestation of pastures on methane oxidation and the methanotrophic communities in soils from three different New Zealand sites. Methane oxidation was measured in soils from two pine (Pinus radiata) forests and one shrubland (mainly Kunzea ericoides var. ericoides) and three adjacent permanent pastures. The methane oxidation rate was consistently higher in the pine forest or shrubland soils than in the adjacent pasture soils. A combination of phospholipid fatty acid (PLFA) and stable isotope probing (SIP) analyses of these soils revealed that different methanotrophic communities were active in soils under the different vegetations. The C₁₈ PLFAs (signature of type II methanotrophs) predominated under pine and shrublands, and C₁₆ PLFAs (type I methanotrophs) predominated under pastures. Analysis of the methanotrophs by molecular methods revealed further differences in methanotrophic community structure under the different vegetation types. Cloning and sequencing and terminal-restriction fragment length polymorphism analysis of the particulate methane oxygenase gene (pmoA) from different samples confirmed the PLFA-SIP results that methanotrophic bacteria related to type II methanotrophs were dominant in pine forest and shrubland, and type I methanotrophs (related to Methylococcus capsulatus) were dominant in all pasture soils. We report that afforestation and reforestation of pastures caused changes in methane oxidation by altering the community structure of methanotrophic bacteria in these soils.     

Nutrient Amendments in Soil DNA Stable Isotope Probing Experiments Reduce the Observed Methanotroph Diversity

Stable isotope probing (SIP) can be used to analyze the active bacterial populations involved in a process by incorporating ¹³C-labeled substrate into cellular components such as DNA. Relatively long incubation times are often used with laboratory microcosms in order to incorporate sufficient ¹³C into the DNA of the target organisms. Addition of nutrients can be used to accelerate the processes. However, unnatural concentrations of nutrients may artificially change bacterial diversity and activity. In this study, methanotroph activity and diversity in soil was examined during the consumption of ¹³CH₄ with three DNA-SIP experiments, using microcosms with natural field soil water conditions, the addition of water, and the addition of mineral salts solution. Methanotroph population diversity was studied by targeting 16S rRNA and pmoA genes. Clone library analyses, denaturing gradient gel electrophoresis fingerprinting, and pmoA microarray hybridization analyses were carried out. Most methanotroph diversity (type I and type II methanotrophs) was observed in nonamended SIP microcosms. Although this treatment probably best reflected the in situ environmental conditions, one major disadvantage of this incubation was that the incorporation of ¹³CH₄ was slow and some cross-feeding of ¹³C occurred, thereby leading to labeling of nonmethanotroph microorganisms. Conversely, microcosms supplemented with mineral salts medium exhibited rapid consumption of ¹³CH₄, resulting in the labeling of a less diverse population of only type I methanotrophs. DNA-SIP incubations using water-amended microcosms yielded faster incorporation of ¹³C into active methanotrophs while avoiding the cross-feeding of ¹³C.     

Differential Effects of Nitrogenous Fertilizers on Methane-Consuming Microbes in Rice Field and Forest Soils

The impact of environmental perturbation (e.g., nitrogenous fertilizers) on the dynamics of methane fluxes from soils and wetland systems is poorly understood. Results of fertilizer studies are often contradictory, even within similar ecosystems. In the present study the hypothesis of whether these contradictory results may be explained by the composition of the methane-consuming microbial community and hence whether methanotrophic diversity affects methane fluxes was investigated. To this end, rice field and forest soils were incubated in microcosms and supplemented with different nitrogenous fertilizers and methane concentrations. By labeling the methane with ¹³C, diversity and function could be coupled by analyses of phospholipid-derived fatty acids (PLFA) extracted from the soils at different time points during incubation. In both rice field and forest soils, the activity as well as the growth rate of methane-consuming bacteria was affected differentially. For type I methanotrophs, fertilizer application stimulated the consumption of methane and the subsequent growth, while type II methanotrophs were generally inhibited. Terminal restriction fragment length polymorphism analyses of the pmoA gene supported the PLFA results. Multivariate analyses of stable-isotope-probing PLFA profiles indicated that in forest and rice field soils, Methylocystis (type II) species were affected by fertilization. The type I methanotrophs active in forest soils (Methylomicrobium/Methylosarcina related) differed from the active species in rice field soils (Methylobacter/Methylomonas related). Our results provide a case example showing that microbial community structure indeed matters, especially when assessing and predicting the impact of environmental change on biodiversity loss and ecosystem functioning.