Analysis of methane-producing and metabolizing archaeal and bacterial communities in sediments of the northern South China Sea and coastal Mai Po Nature Reserve revealed by PCR amplification of mcrA and pmoA genes.
ABSTRACT: Communities of methanogens, anaerobic methanotrophic archaea and aerobic methanotrophic bacteria (MOB) were compared by profiling polymerase chain reaction (PCR)-amplified products of mcrA and pmoA genes encoded by methyl-coenzyme M reductase alpha subunit and particulate methane monooxygenase alpha subunit, respectively, in sediments of northern South China Sea (nSCS) and Mai Po mangrove wetland. Community structures representing by mcrA gene based on 12 clone libraries from nSCS showed separate clusters indicating niche specificity, while, Methanomicrobiales, Methanosarcinales clades 1,2, and Methanomassiliicoccus-like groups of methanogens were the most abundant groups in nSCS sediment samples. Novel clusters specific to the SCS were identified and the phylogeny of mcrA gene-harboring archaea was updated. Quantitative polymerase chain reaction was used to detect mcrA gene abundance in all samples: similar abundance of mcrA gene in the surface layers of mangrove (3.4?3.9 × 10(6) copies per gram dry weight) and of intertidal mudflat (5.5?5.8 × 10(6) copies per gram dry weight) was observed, but higher abundance (6.9 × 10(6) to 1.02 × 10(8) copies per gram dry weight) was found in subsurface samples of both sediment types. Aerobic MOB were more abundant in surface layers (6.7?11.1 × 10(5) copies per gram dry weight) than the subsurface layers (1.2?5.9 × 10(5) copies per gram dry weight) based on pmoA gene. Mangrove surface layers harbored more abundant pmoA gene than intertidal mudflat, but less pmoA genes in the subsurface layers. Meanwhile, it is also noted that in surface layers of all samples, more pmoA gene copies were detected than the subsurface layers. Reedbed rhizosphere exhibited the highest gene abundance of mcrA gene (8.51 × 10(8) copies per gram dry weight) and pmoA gene (1.56 × 10(7) copies per gram dry weight). This study investigated the prokaryotic communities responsible for methane cycling in both marine and coastal wetland ecosystems, showing the distribution characteristics of mcrA gene-harboring communities in nSCS and stratification of mcrA and pmoA gene diversity and abundance in the Mai Po Nature Reserve.
Project description: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.
Project description:Nitrite-dependent anaerobic methane oxidation (n-damo) is performed by "Candidatus Methylomirabilis oxyfera" (M. oxyfera), which connects the carbon and nitrogen global nutrient cycles. In the present study, M. oxyfera-like bacteria sequences were successfully recovered from Yellow River Estuary sediments using specific primers for 16S rRNA and pmoA genes. A M. oxyfera-like sequences analysis based on the 16S rRNA gene revealed greater diversity compared with the pmoA gene; the 16S rRNA gene sequences retrieved from the Yellow River Estuary sediments belong to groups A as well as B and were mainly found in freshwater habitats. Quantitative PCR showed that 16S rRNA gene abundance varied from 9.28±0.11×10(3) to 2.10±0.13×10(5) copies g(-1) (dry weight), and the pmoA gene abundance ranged from 8.63±0.50×10(3) to 1.83±0.18×10(5) copies g(-1) (dry weight). A correlation analysis showed that the total organic carbon (TOC) and ammonium (NH4(+)) as well as the ratio of total phosphorus to total nitrogen (TP/TN) influenced the M. oxyfera-like bacteria distribution in the Yellow River Estuary sediments. These findings will aid in understanding the n-damo bacterial distribution pattern as well as their correlation with surrounding environmental factors in temperate estuarine ecosystems.
Project description:Forest and other upland soils are important sinks for atmospheric CH(4), consuming 20 to 60 Tg of CH(4) per year. Consumption of atmospheric CH(4) by soil is a microbiological process. However, little is known about the methanotrophic bacterial community in forest soils. We measured vertical profiles of atmospheric CH(4) oxidation rates in a German forest soil and characterized the methanotrophic populations by PCR and denaturing gradient gel electrophoresis (DGGE) with primer sets targeting the pmoA gene, coding for the alpha subunit of the particulate methane monooxygenase, and the small-subunit rRNA gene (SSU rDNA) of all life. The forest soil was a sink for atmospheric CH(4) in situ and in vitro at all times. In winter, atmospheric CH(4) was oxidized in a well-defined subsurface soil layer (6 to 14 cm deep), whereas in summer, the complete soil core was active (0 cm to 26 cm deep). The content of total extractable DNA was about 10-fold higher in summer than in winter. It decreased with soil depth (0 to 28 cm deep) from about 40 to 1 microg DNA per g (dry weight) of soil. The PCR product concentration of SSU rDNA of all life was constant both in winter and in summer. However, the PCR product concentration of pmoA changed with depth and season. pmoA was detected only in soil layers with active CH(4) oxidation, i.e., 6 to 16 cm deep in winter and throughout the soil core in summer. The same methanotrophic populations were present in winter and summer. Layers with high CH(4) consumption rates also exhibited more bands of pmoA in DGGE, indicating that high CH(4) oxidation activity was positively correlated with the number of methanotrophic populations present. The pmoA sequences derived from excised DGGE bands were only distantly related to those of known methanotrophs, indicating the existence of unknown methanotrophs involved in atmospheric CH(4) consumption.
Project description:Previous studies have suggested that protozoa prey on Fe(III)- and sulfate-reducing bacteria that are enriched when acetate is added to uranium contaminated subsurface sediments to stimulate U(VI) reduction. In order to determine whether protozoa continue to impact subsurface biogeochemistry after these acetate amendments have stopped, 18S rRNA and ß-tubulin sequences from this phase of an in situ uranium bioremediation field experiment were analyzed. Sequences most similar to Metopus species predominated, with the majority of sequences most closely related to M. palaeformis, a cilitated protozoan known to harbor methanogenic symbionts. Quantification of mcrA mRNA transcripts in the groundwater suggested that methanogens closely related to Metopus endosymbionts were metabolically active at this time. There was a strong correlation between the number of mcrA transcripts from the putative endosymbiotic methanogen and Metopus ß-tubulin mRNA transcripts during the course of the field experiment, suggesting that the activity of the methanogens was dependent upon the activity of the Metopus species. Addition of the eukaryotic inhibitors cyclohexamide and colchicine to laboratory incubations of acetate-amended subsurface sediments significantly inhibited methane production and there was a direct correlation between methane concentration and Metopus ß-tubulin and putative symbiont mcrA gene copies. These results suggest that, following the stimulation of subsurface microbial growth with acetate, protozoa harboring methanogenic endosymbionts become important members of the microbial community, feeding on moribund biomass and producing methane.
Project description:The nitrogen and methane cycles are important biogeochemical processes. Recently, 'Candidatus Methanoperedens nitroreducens,' archaea that catalyze nitrate-dependent anaerobic oxidation of methane (AOM), were enriched, and their genomes were analyzed. Diagnostic molecular tools for the sensitive detection of 'Candidatus M. nitroreducens' are not yet available. Here, we report the design of two novel mcrA primer combinations that specifically target the alpha sub-unit of the methyl-coenzyme M reductase (mcrA) gene of 'Candidatus M. nitroreducens'. The first primer pair produces a fragment of 186-bp that can be used to quantify 'Candidatus M. nitroreducens' cells, whereas the second primer pair yields an 1191-bp amplicon that is with sufficient length and well suited for more detailed phylogenetic analyses. Six different environmental samples were evaluated with the new qPCR primer pair, and the abundances were compared with those determined using primers for the 16S rRNA gene. The qPCR results indicated that the number of copies of the 'Candidatus M. nitroreducens' mcrA gene was highest in rice field soil, with 5.6 ± 0.8 × 106 copies g-1 wet weight, whereas Indonesian river sediment had only 4.6 ± 2.7 × 102 copies g-1 wet weight. In addition to freshwater environments, sequences were also detected in marine sediment of the North Sea, which contained approximately 2.5 ± 0.7 × 104 copies g-1 wet weight. Phylogenetic analysis revealed that the amplified 1191-bp mcrA gene sequences from the different environments all clustered together with available genome sequences of mcrA from known 'Candidatus M. nitroreducens' archaea. Taken together, these results demonstrate the validity and utility of the new primers for the quantitative and sensitive detection of the mcrA gene sequences of these important nitrate-dependent AOM archaea. Furthermore, the newly obtained mcrA sequences will contribute to greater phylogenetic resolution of 'Candidatus M. nitroreducens' sequences, which have been only poorly captured by general methanogenic mcrA primers.
Project description:Intermittent drainage can substantially reduce methane emission from rice fields, but the microbial mechanisms remain poorly understood. In the present study, we determined the rates of methane production and emission, the dynamics of ferric iron and sulfate, and the abundance of methanogen mcrA genes (encoding the alpha subunit of methyl coenzyme M reductase) and their transcripts in response to alternate dry/wet cycles in paddy field soil. We found that intermittent drainage did not affect the growth of rice plants but significantly reduced the rates of both methane production and emission. The dry/wet cycles also resulted in shifts of soil redox conditions, increasing the concentrations of ferric iron and sulfate in the soil. Quantitative PCR analysis revealed that both mcrA gene copies and mcrA transcripts significantly decreased after dry/wet alternation compared to continuous flooding. Correlation and regression analyses showed that the abundance of mcrA genes and transcripts positively correlated with methane production potential and soil water content and negatively correlated with the concentrations of ferric iron and sulfate in the soil. However, the transcription of mcrA genes was reduced to a greater extent than the abundance of mcrA genes, resulting in very low mcrA transcript/gene ratios after intermittent drainage. Furthermore, terminal restriction fragment length polymorphism analysis revealed that the composition of methanogenic community remained stable under dry/wet cycles, whereas that of metabolically active methanogens strongly changed. Collectively, our study demonstrated a stronger effect of intermittent drainage on the abundance of mcrA transcripts than of mcrA genes in rice field soil.
Project description:Ecological processes regulating soil carbon (C) and nitrogen (N) cycles are still poorly understood, especially in the world's largest agricultural frontier in Southern Amazonia. We analyzed soil parameters in samples from pristine rainforest and after land use change to pasture and crop fields, and correlated them with abundance of functional and phylogenetic marker genes (amoA, nirK, nirS, norB, nosZ, nifH, mcrA, pmoA, and 16S/18S rRNA). Additionally, we integrated these parameters using path analysis and multiple regressions. Following forest removal, concentrations of soil C and N declined, and pH and nutrient levels increased, which influenced microbial abundances and biogeochemical processes. A seasonal trend was observed, suggesting that abundances of microbial groups were restored to near native levels after the dry winter fallow. Integration of the marker gene abundances with soil parameters using path analysis and multiple regressions provided good predictions of biogeochemical processes, such as the fluxes of NO3, N2O, CO2, and CH4. In the wet season, agricultural soil showed the highest abundance of nitrifiers (amoA) and Archaea, however, forest soils showed the highest abundances of denitrifiers (nirK, nosZ) and high N, which correlated with increased N2O emissions. Methanogens (mcrA) and methanotrophs (pmoA) were more abundant in forest soil, but methane flux was highest in pasture sites, which was related to soil compaction. Rather than analyzing direct correlations, the data integration using multivariate tools provided a better overview of biogeochemical processes. Overall, in the wet season, land use change from forest to agriculture reduced the abundance of different functional microbial groups related to the soil C and N cycles; integrating the gene abundance data and soil parameters provided a comprehensive overview of these interactions. Path analysis and multiple regressions addressed the need for more comprehensive approaches to improve our mechanistic understanding of biogeochemical cycles.
Project description:The objective of this study was to characterize metabolically active, aerobic methanotrophs in an ombrotrophic peatland in the Marcell Experimental Forest, in Minnesota. Methanotrophs were investigated in the field and in laboratory incubations using DNA-stable isotope probing (SIP), expression studies on particulate methane monooxygenase (pmoA) genes, and amplicon sequencing of 16S rRNA genes. Potential rates of oxidation ranged from 14 to 17 ?mol of CH4g dry weight soil(-1)day(-1) Within DNA-SIP incubations, the relative abundance of methanotrophs increased from 4% in situ to 25 to 36% after 8 to 14 days. Phylogenetic analysis of the(13)C-enriched DNA fractions revealed that the active methanotrophs were dominated by the genera Methylocystis(type II;Alphaproteobacteria),Methylomonas, and Methylovulum(both, type I;Gammaproteobacteria). In field samples, a transcript-to-gene ratio of 1 to 2 was observed for pmoA in surface peat layers, which attenuated rapidly with depth, indicating that the highest methane consumption was associated with a depth of 0 to 10 cm. Metagenomes and sequencing of cDNA pmoA amplicons from field samples confirmed that the dominant active methanotrophs were Methylocystis and Methylomonas Although type II methanotrophs have long been shown to mediate methane consumption in peatlands, our results indicate that members of the genera Methylomonas and Methylovulum(type I) can significantly contribute to aerobic methane oxidation in these ecosystems.
Project description:Anaerobic ammonium oxidation (anammox) has been recognized as an important process for the global nitrogen cycle. In this study, the occurrence and diversity of anammox bacteria in the deep-sea subsurface sediments of the South China Sea (SCS) were investigated. Results indicated that the anammox bacterial sequences recovered from this habitat by amplifying both 16S rRNA gene and hydrazine oxidoreductase encoding hzo gene were all closely related to the Candidatus Scalindua genus. A total of 96 16S rRNA gene sequences from 346 clones were grouped into five subclusters: two subclusters affiliated with the brodae and arabica species, while three new subclusters named zhenghei-I, -II, and -III showed ? 97.4% nucleic acid sequence identity with other known Candidatus Scalindua species. Meanwhile, 88 hzo gene sequences from the sediments also formed five distant subclusters within hzo cluster 1c. Through fluorescent real-time PCR analysis, the abundance of anammox bacteria in deep-sea subsurface sediment was quantified by hzo genes, which ranged from 1.19 × 10(4) to 7.17 × 10(4) copies per gram of dry sediments. Combining all the information from this study, diverse Candidatus Scalindua anammox bacteria were found in the deep-sea subsurface sediments of the SCS, and they could be involved in the nitrogen loss from the fixed inventory in the habitat.
Project description:Straw returns to the soil is an effective way to improve soil organic carbon and reduce air pollution by straw burning, but this may increase CH4 and N2O emissions risks in paddy soils. Biochar has been used as a soil amendment to improve soil fertility and mitigate CH4 and N2O emissions. However, little is known about their interactive effect on CH4 and N2O emissions and the underlying microbial mechanisms. In this study, a 2-year pot experiment was conducted on two paddy soil types (an acidic Utisol, TY, and an alkaline Inceptisol, BH) to evaluate the influence of straw and biochar applications on CH4 and N2O emissions, and on related microbial functional genes. Results showed that straw addition markedly increased the cumulative CH4 emissions in both soils by 4.7- to 9.1-fold and 23.8- to 72.4-fold at low (S1) and high (S2) straw input rate, respectively, and significantly increased mcrA gene abundance. Biochar amendment under the high straw input (BS2) significantly decreased CH4 emissions by more than 50% in both soils, and increased both mcrA gene and pmoA gene abundances, with greatly enhanced pmoA gene and a decreased mcrA/pmoA gene ratio. Moreover, methanotrophs community changed distinctly in response to straw and biochar amendment in the alkaline BH soil, but showed slight change in the acidic TY soil. Straw had little effect on N2O emissions at low input rate (S1) but significantly increased N2O emissions at the high input rate (S2). Biochar amendment showed inconsistent effect on N2O emissions, with a decreasing trend in the BH soil but an increasing trend in the TY soil in which high ammonia existed. Correspondingly, increased nirS and nosZ gene abundances and obvious community changes in nosZ gene containing denitrifiers in response to biochar amendment were observed in the BH soil but not in the TY soil. Overall, our results suggested that biochar amendment could markedly mitigate the CH4 and N2O emissions risks under a straw return practice via regulating functional microbes and soil physicochemical properties, while the performance of this practice will vary depending on soil parent material characteristics.