Detection of methanotrophs with highly divergent pmoA genes from Arctic soils

Tundra soil samples from the Canadian Arctic community, Kuujjuaq, were analyzed for the presence of the soluble (sMMO) and particulate (pMMO) methane monooxygenase genes. Total genomic DNA extracted from these soils was used as template for PCR using sMMO- and pMMO-specific primers, mmoX1-mmoX2 and A189-A682, respectively. pMMO and sMMO genes were detected in the Kuujjuaq soil samples. Isolation of sMMO-possessing methanotrophic microorganisms from the three soils, as determined by the colony naphthalene oxidation assay, was carried out using direct plating (5 degrees C) and methane enrichment studies (5 degrees C and 25 degrees C). Direct plating did not yield sMMO-possessing methanotrophic bacteria, whereas methane enrichments yielded isolates possessing and expressing sMMO activity. Analysis of derived amino acid sequences of pmoA genes and partial 16S rRNA genes obtained by PCR, using DNA isolated directly from this environment and from isolates, revealed the presence of highly divergent PmoA/AmoA sequences and 16S rRNA sequences that cluster closely with but are distinct from the genes from the genera Methylosinus and Methylocystis.     

The particulate methane monooxygenase gene pmoA and its use as a functional gene probe for methanotrophs

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.     

A newly designed degenerate PCR primer based on pmoA gene for detection of nitrite-dependent anaerobic methane-oxidizing bacteria from different ecological niches

A new pmoA gene-based PCR primer set was designed for detection of nitrite-dependent anaerobic oxidation of methane (n-damo) bacteria from four different ecosystems, namely rice paddy soil, freshwater reservoir, reed bed, and sludge from wastewater treatment plant. This primer set showed high specificity and efficiency in recovering n-damo bacteria from these diverse samples. The obtained sequences showed 88–94 and 90–96 % similarity to nucleotide and amino acid sequences, respectively, with the known NC10 phylum bacterium. According to the UniFrac principal coordinates analysis (PCoA), DNA sequences retrieved by the new PCR primer set in this study formed a separate group from the reported sequences, indicating higher diversity of n-damo in the environment. This newly designed PCR primer is capable of amplifying not only the currently known n-damo bacteria but also those that have not been reported, providing new information on the ecological diversity and distribution of this group of microorganisms in the ecosystem.     

Aerobic and anaerobic methanotrophs in the Black Sea water column

Inputs of CH(4) from sediments, including methane seeps on the continental margin and methane-rich mud volcanoes on the abyssal plain, make the Black Sea the world's largest surface water reservoir of dissolved methane and drive a high rate of aerobic and anaerobic oxidation of methane in the water column. Here we present the first combined organic geochemical and molecular ecology data on a water column profile of the western Black Sea. We show that aerobic methanotrophs type I are responsible for methane oxidation in the oxic water column and ANME-1- and ANME-2-related organisms for anaerobic methane oxidation. The occurrence of methanotrophs type I cells in the anoxic zone suggests that inactive cells settle to deeper waters. Molecular and biomarker results suggest that a clear distinction between the occurrence of ANME-1- and ANME-2-related lineages exists, i.e. ANME-1-related organisms are responsible for anaerobic methane oxidation below 600 m water depth, whereas ANME-2-related organisms are responsible for this process in the anoxic water column above approximately 600 m water depth.     

New insight into stratification of anaerobic methanotrophs in cold seep sediments

Methane seepages typically harbor communities of anaerobic methane oxidizers (ANME); however, knowledge about fine-scale vertical variation of ANME in response to geochemical gradients is limited. We investigated microbial communities in sediments below a white microbial mat in the G11 pockmark at Nyegga by 16S rRNA gene tag pyrosequencing and real-time quantitative PCR. A vertical stratification of dominating ANME communities was observed at 4 cmbsf (cm below seafloor) and below in the following order: ANME-2a/b, ANME-1 and ANME-2c. The ANME-1 community was most numerous and comprised single or chains of cells with typical rectangular morphology, accounting up to 89.2% of the retrieved 16S rRNA gene sequences. Detection rates for sulfate-reducing Deltaproteobacteria possibly involved in anaerobic oxidation of methane were low throughout the core. However, a correlation in the abundance of Candidate division JS-1 with ANME-2 was observed, indicating involvement in metabolisms occurring in ANME-2-dominated horizons. The white microbial mat and shallow sediments were dominated by organisms affiliated with Sulfurovum (Epsilonproteobacteria) and Methylococcales (Gammaproteobacteria), suggesting that aerobic oxidation of sulfur and methane is taking place. In intermediate horizons, typical microbial groups associated with methane seeps were recovered. The data are discussed with respect to co-occurring microbial assemblages and interspecies interactions.     

内陆湿地与水体甲烷厌氧氧化功能微生物研究进展

内陆湿地与水体(如湖泊、河流、水库等)是温室气体甲烷的重要排放源。微生物介导的甲烷厌氧氧化(anaerobic oxidation of methane,AOM)反应在控制内陆湿地与水体甲烷排放中起着不可忽视的作用,对缓解全球温室效应具有重要意义。内陆湿地与水体易形成缺氧环境,且电子受体的种类和数量繁多,是发生AOM反应的理想生境。近年来,不断有研究表明,内陆湿地与水体中存在多种电子受体(NO₂^-、NO₃^-、SO₄^2-、Fe (III)等)驱动的AOM途径。NC10门细菌和甲烷厌氧氧化古菌(anaerobic methanotrophic archaea,ANME)的一新分支ANME-2d主导了湿地和水体环境中的AOM反应,其中ANME-2d具有根据环境条件选择不同电子受体的潜力。研究系统综述了内陆湿地与水体中不同电子受体驱动的AOM途径及其参与的主要功能微生物类群;分析了AOM反应在控制温室气体甲烷排放中的作用及其环境影响因素;总结了相关功能微生物的分子生物学检测方法及甲烷厌氧氧化活性测定的同位素示踪技术。最后,对未来相关研究方向进行了展望。     

Identification of the dominant sulfate-reducing bacterial partner of anaerobic methanotrophs of the ANME-2 clade

The anaerobic oxidation of methane (AOM) with sulfate as terminal electron acceptor is mediated by consortia of methanotrophic archaea (ANME) and sulfate-reducing bacteria (SRB). Whereas three clades of ANME have been repeatedly studied with respect to phylogeny, key genes and genomic capabilities, little is known about their sulfate-reducing partner. In order to identify the partner of anaerobic methanotrophs of the ANME-2 clade, bacterial 16S rRNA gene libraries were constructed from cultures highly enriched for ANME-2a and ANME-2c in consortia with Deltaproteobacteria of the Desulfosarcina/Desulfococcus group (DSS). Phylogenetic analysis of those and publicly available sequences from AOM sites supported the hypothesis by Knittel and colleagues that the DSS partner belongs to the diverse SEEP-SRB1 cluster. Six subclusters of SEEP-SRB1, SEEP-SRB1a to SEEP-SRB1f, were proposed and specific oligonucleotide probes were designed. Using fluorescence in situ hybridization on samples from six different AOM sites, SEEP-SRB1a was identified as sulfate-reducing partner in up to 95% of total ANME-2 consortia. SEEP-SRB1a cells exhibited a rod-shaped, vibrioid, or coccoid morphology and were found to be associated with subgroups ANME-2a and ANME-2c. Moreover, SEEP-SRB1a was also detected in 8% to 23% of ANME-3 consortia in Haakon Mosby Mud Volcano sediments, previously described to be predominantly associated with SRB of the Desulfobulbus group. SEEP-SRB1a contributed to only 0.3% to 0.7% of all single cells in almost all samples indicating that these bacteria are highly adapted to a symbiotic relationship with ANME-2.     

Potential of pmoA Amplicon Pyrosequencing for Methanotroph Diversity Studies

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

Quantitative detection of methanotrophs in soil by novel pmoA-targeted real-time PCR assays

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 (alpha subunit of the particulate methane monooxygenase) phylogenetic trees showed good correlation and revealed five distinct groups of methanotrophs within the alpha and gamma 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 10(1) and 10(2) 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) x 10(6) pmoA molecules g(-1) for all methanotrophs. The Methylosinus group was predominant (2.7 x 10(6) +/- 1.1 x 10(6) target molecules g(-1)). In addition, bacteria of the Methylobacter/Methylosarcina group were abundant (2.0 x 10(6) +/- 0.9 x 10(6) 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 x 10(4) 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.     

自然湿地土壤产甲烷菌和甲烷氧化菌多样性的分子检测

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

Growth yields of methanotrophs

Summary The growth yield ofMethylococcus capsulatus (Bath) on methane was dependent on the availability of copper in the growth medium. In nitrate mineral salts medium the carbon conversion efficiency increased by 38%, concomitant with the transition from soluble to particulate methane monooxygenase, after transfer from low to high copper medium. An increase in growth efficiency was also observed with ammonia as nitrogen source but not when methanol replaced methane as carbon source. The high growth efficiency is attributed to a reduced NADH requirement for methane oxidation. This could only arise if methanol dehydrogenase was capable of electron transfer, either directly or indirectly to the particulate methane monooxygenase (MMO). The carbon conversion efficiency from methanol with nitrate as nitrogen source was as high as theoretically predicted. It is suggested that the previously low yields of methanotrophs grown on methanol resulted from the use, as nitrogen source, of ammonia which was oxidised by the MMO still present under these growth conditions. The term ‘methanotroph’ is used throughout to distinguish those organisms capable of growth on methane from ‘methylotrophs’ capable of growth on reduced C, compounds other than methane     

Fault detection filter design for an anaerobic digestion process

In this paper, a Fault Detection and Isolation observer based method has been applied to biological wastewater treatment process. The method is designed with a dynamic model and the observer is determined using the eigenstructure assignment approach. The efficiency of the method is demonstrated for both detection and isolation of an actuator and a sensor failure using experimental data from a pilot scale anaerobic digestion process for the treatment of an industrial wine distillery vinasses.     

Biogeography of wetland rice methanotrophs

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

A rapid method for the detection of tryptophanase in anaerobic bacteria

A total of 633 anaerobic bacteria were examined for tryptophanase production using a rapid method which distinguishes within 5 to 180 minutes between anaerobes that contain tryptophanase and those that do not. Of the 196 tryptophanase-positive isolates tested, 99% showed tryptophanase activity within 2 hours as compared with 94.4% in 24 hours by a conventional method. A total of 299 tryptophanase-negative organisms were tested. Ninety three percent of these remained negative after 24 hours as compared with 95.3% when tested with a 24-h conventional method. Additional information was obtained on the sensitivity of this test and the time-dependent production of indole by tryptophanase.     

Development and validation of a diagnostic microbial microarray for methanotrophs

The potential of DNA microarray technology in high-throughput detection of bacteria and quantitative assessment of their community structures is widely acknowledged but has not been fully realised yet. A generally applicable set of techniques, based on readily available technologies and materials, was developed for the design, production and application of diagnostic microbial microarrays. A microarray targeting the particulate methane monooxygenase (pmoA) gene was developed for the detection and quantification of methanotrophs and functionally related bacteria. A microarray consisting of a set of 59 probes that covers the whole known diversity of these bacteria was validated with a representative set of extant strains and environmental clones. The potential of the pmoA microarray was tested with environmental samples. The results were in good agreement with those of clone library sequence analyses. The approach can currently detect less dominant bacteria down to 5% of the total community targeted. Initial tests assessing the quantification potential of this system with artificial PCR mixtures showed very good correlation with the expected results with standard deviations in the range of 0.4-17.2%. Quantification of environmental samples with this method requires the design of a reference mixture consisting of very close relatives of the strains within the sample and is currently limited by biases inherent in environmental DNA extraction and universal PCR amplification.     

Isolation and characterization of marine methanotrophs

Four new methane-oxidizing bacteria have been isolated from marine samples taken at the Hyperion sewage outfall, near Los Angeles, CA. These bacteria require NaCl for growth. All exhibit characteristics typical of Type I methanotrophs, except they contain enzyme activities of both the ribulose monophosphate pathway and the serine cycle. All four strains are characterized by rapid growth in liquid culture and on agar plates, and all have temperature optima above 35° C. One strain, chosen for further study, has been shown to maintain broadhost range cloning vectors and is currently being used for genetic studies.     

Distribution of methanotrophs in the phyllosphere

Plants have been reported to emit methane as well as methanol originating in their cell-wall constituents. We investigated methanotrophs in the phyllosphere by the enrichment culture method with methane as sole carbon source. We enriched methanotrophs from the leaves, flowers, bark, and roots of various plants. Analysis of the pmoA and mxaF genes retrieved from the enrichment cultures revealed that methanotrophs closely related to the genera Methylomonas, Methylosinus, and Methylocystis inhabit not only the rhizosphere but also the phyllosphere, together with methanol-utilizing bacteria.     

ARChip epoxy and ARChip UV for covalent on-chip immobilization of pmoA gene-specific oligonucleotides

ARChip Epoxy and ARChip UV are presented as novel chip platforms for oligonucleotide immobilization. ARChip Epoxy is made of reactive epoxy resin available commercially. ARChip UV consists of photoactivatable poly(styrene-co-4-vinylbenzylthiocyanate). Both ARChip surfaces are tested in a model assay based on oligonucleotide probes from a real-life genotyping project and are evaluated in comparison with five commercial chip surfaces based on nitrocellulose, epoxy, and aldehyde polymer, and two different aminosilanes. Optimum print buffer, spotter compatibility, and data normalization are discussed.