scholarly journals Alpha- and Gammaproteobacterial Methanotrophs Codominate the Active Methane-Oxidizing Communities in an Acidic Boreal Peat Bog

2016 ◽  
Vol 82 (8) ◽  
pp. 2363-2371 ◽  
Author(s):  
Kaitlin C. Esson ◽  
Xueju Lin ◽  
Deepak Kumaresan ◽  
Jeffrey P. Chanton ◽  
J. Colin Murrell ◽  
...  
Keyword(s):  
Dry Weight ◽  
Amplicon Sequencing ◽  
Stable Isotope Probing ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Type I ◽  
Type Ii ◽  
Content Type ◽  
Methane Consumption ◽  
Field Samples

ABSTRACTThe 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 μmol of CH4g dry weight soil−1day−1. Within DNA-SIP incubations, the relative abundance of methanotrophs increased from 4%in situto 25 to 36% after 8 to 14 days. Phylogenetic analysis of the13C-enriched DNA fractions revealed that the active methanotrophs were dominated by the generaMethylocystis(type II;Alphaproteobacteria),Methylomonas, andMethylovulum(both, type I;Gammaproteobacteria). In field samples, a transcript-to-gene ratio of 1 to 2 was observed forpmoAin 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 cDNApmoAamplicons from field samples confirmed that the dominant active methanotrophs wereMethylocystisandMethylomonas. Although type II methanotrophs have long been shown to mediate methane consumption in peatlands, our results indicate that members of the generaMethylomonasandMethylovulum(type I) can significantly contribute to aerobic methane oxidation in these ecosystems.

scholarly journals Shifts in Identity and Activity of Methanotrophs in Arctic Lake Sediments in Response to Temperature Changes

2012 ◽  
Vol 78 (13) ◽  
pp. 4715-4723 ◽  
Author(s):  
Ruo He ◽  
Matthew J. Wooller ◽  
John W. Pohlman ◽  
John Quensen ◽  
James M. Tiedje ◽  
...  
Keyword(s):  
16S Rrna ◽  
Lake Sediments ◽  
Incubation Temperature ◽  
Stable Isotope Probing ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Type I ◽  
Content Type ◽  
Arctic Lake ◽  
Methanotrophic Communities

ABSTRACTMethane (CH4) flux to the atmosphere is mitigated via microbial CH4oxidation in sediments and water. As arctic temperatures increase, understanding the effects of temperature on the activity and identity of methanotrophs in arctic lake sediments is important to predicting future CH4emissions. We used DNA-based stable-isotope probing (SIP), quantitative PCR (Q-PCR), and pyrosequencing analyses to identify and characterize methanotrophic communities active at a range of temperatures (4°C, 10°C, and 21°C) in sediments (to a depth of 25 cm) sampled from Lake Qalluuraq on the North Slope of Alaska. CH4oxidation activity was measured in microcosm incubations containing sediments at all temperatures, with the highest CH4oxidation potential of 37.5 μmol g−1day−1in the uppermost (depth, 0 to 1 cm) sediment at 21°C after 2 to 5 days of incubation. Q-PCR ofpmoAand of the 16S rRNA genes of type I and type II methanotrophs, and pyrosequencing of 16S rRNA genes in13C-labeled DNA obtained by SIP demonstrated that the type I methanotrophsMethylobacter,Methylomonas, andMethylosomadominated carbon acquisition from CH4in the sediments. The identity and relative abundance of active methanotrophs differed with the incubation temperature. Methylotrophs were also abundant in the microbial community that derived carbon from CH4, especially in the deeper sediments (depth, 15 to 20 cm) at low temperatures (4°C and 10°C), and showed a good linear relationship (R= 0.82) with the relative abundances of methanotrophs in pyrosequencing reads. This study describes for the first time how methanotrophic communities in arctic lake sediments respond to temperature variations.

scholarly journals Active Ammonia Oxidizers in an Acidic Soil Are Phylogenetically Closely Related to Neutrophilic Archaeon

2013 ◽  
Vol 80 (5) ◽  
pp. 1684-1691 ◽  
Author(s):  
Baozhan Wang ◽  
Yan Zheng ◽  
Rong Huang ◽  
Xue Zhou ◽  
Dongmei Wang ◽  
...  
Keyword(s):  
16S Rrna ◽  
Buoyant Density ◽  
Stable Isotope Probing ◽  
Acidic Soil ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Molecular Evidence ◽  
Ammonia Oxidizing Bacteria ◽  
Content Type ◽  
Metabolic Versatility

ABSTRACTAll cultivated ammonia-oxidizing archaea (AOA) within theNitrososphaeracluster (former soil group 1.1b) are neutrophilic. Molecular surveys also indicate the existence ofNitrososphaera-like phylotypes in acidic soil, but their ecological roles are poorly understood. In this study, we present molecular evidence for the chemolithoautotrophic growth ofNitrososphaera-like AOA in an acidic soil with pH 4.92 using DNA-based stable isotope probing (SIP). Soil microcosm incubations demonstrated that nitrification was stimulated by urea fertilization and accompanied by a significant increase in the abundance of AOA rather than ammonia-oxidizing bacteria (AOB). Real-time PCR analysis ofamoAgenes as a function of the buoyant density of the DNA gradient following the ultracentrifugation of the total DNA extracted from SIP microcosms indicated a substantial growth of soil AOA during nitrification. Pyrosequencing of the total 16S rRNA genes in the “heavy” DNA fractions suggested that archaeal communities were labeled to a much greater extent than soil AOB. Acetylene inhibition further showed that13CO2assimilation by nitrifying communities depended solely on ammonia oxidation activity, suggesting a chemolithoautotrophic lifestyle. Phylogenetic analysis of both13C-labeledamoAand 16S rRNA genes revealed that most of the active AOA were phylogenetically closely related to the neutrophilic strainsNitrososphaera viennensisEN76 and JG1 within theNitrososphaeracluster. Our results provide strong evidence for the adaptive growth ofNitrososphaera-like AOA in acidic soil, suggesting a greater metabolic versatility of soil AOA than previously appreciated.

scholarly journals Stable-Isotope Probing Identifies Uncultured Planctomycetes as Primary Degraders of a Complex Heteropolysaccharide in Soil

2015 ◽  
Vol 81 (14) ◽  
pp. 4607-4615 ◽  
Author(s):  
Xiaoqing Wang ◽  
Christine E. Sharp ◽  
Gareth M. Jones ◽  
Stephen E. Grasby ◽  
Allyson L. Brady ◽  
...  
Keyword(s):  
Stable Isotope ◽  
16S Rrna ◽  
Soil Bacteria ◽  
Stable Isotope Probing ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Rrna Gene ◽  
Content Type ◽  
Degrading Bacteria ◽  
Aerobic Soil

ABSTRACTThe exopolysaccharides (EPSs) produced by some bacteria are potential growth substrates for other bacteria in soil. We used stable-isotope probing (SIP) to identify aerobic soil bacteria that assimilated the cellulose produced byGluconacetobacter xylinusor the EPS produced byBeijerinckia indica. The latter is a heteropolysaccharide comprised primarily ofl-guluronic acid,d-glucose, andd-glycero-d-mannoheptose.13C-labeled EPS and13C-labeled cellulose were purified from bacterial cultures grown on [13C]glucose. Two soils were incubated with these substrates, and bacteria actively assimilating them were identified via pyrosequencing of 16S rRNA genes recovered from13C-labeled DNA. Cellulose C was assimilated primarily by soil bacteria closely related (93 to 100% 16S rRNA gene sequence identities) to known cellulose-degrading bacteria. However,B. indicaEPS was assimilated primarily by bacteria with low identities (80 to 95%) to known species, particularly by different members of the phylumPlanctomycetes. In one incubation, members of thePlanctomycetesmade up >60% of all reads in the labeled DNA and were only distantly related (<85% identity) to any described species. Although it is impossible with SIP to completely distinguish primary polysaccharide hydrolyzers from bacteria growing on produced oligo- or monosaccharides, the predominance ofPlanctomycetessuggested that they were primary degraders of EPS. Other bacteria assimilatingB. indicaEPS included members of theVerrucomicrobia, candidate division OD1, and theArmatimonadetes. The results indicate that some uncultured bacteria in soils may be adapted to using complex heteropolysaccharides for growth and suggest that the use of these substrates may provide a means for culturing new species.

scholarly journals Identification of Methanoculleus spp. as Active Methanogens during Anoxic Incubations of Swine Manure Storage Tank Samples

2012 ◽  
Vol 79 (2) ◽  
pp. 424-433 ◽  
Author(s):  
Maialen Barret ◽  
Nathalie Gagnon ◽  
Martin L. Kalmokoff ◽  
Edward Topp ◽  
Yris Verastegui ◽  
...  
Keyword(s):  
Storage Tank ◽  
Swine Manure ◽  
Carbon Assimilation ◽  
Methanogenic Archaea ◽  
Methane Emissions ◽  
Stable Isotope Probing ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Content Type ◽  
Manure Storage

ABSTRACTMethane emissions represent a major environmental concern associated with manure management in the livestock industry. A more thorough understanding of how microbial communities function in manure storage tanks is a prerequisite for mitigating methane emissions. Identifying the microorganisms that are metabolically active is an important first step. Methanogenic archaea are major contributors to methanogenesis in stored swine manure, and we investigated active methanogenic populations by DNA stable isotope probing (DNA-SIP). Following a preincubation of manure samples under anoxic conditions to induce substrate starvation, [U-13C]acetate was added as a labeled substrate. Fingerprint analysis of density-fractionated DNA, using length-heterogeneity analysis of PCR-amplifiedmcrAgenes (encoding the alpha subunit of methyl coenzyme M reductase), showed that the incorporation of13C into DNA was detectable atin situacetate concentrations (∼7 g/liter). Fingerprints of DNA retrieved from heavy fractions of the13C treatment were primarily enriched in a 483-bp amplicon and, to a lesser extent, in a 481-bp amplicon. Analyses based on clone libraries of themcrAand 16S rRNA genes revealed that both of these heavy DNA amplicons corresponded toMethanoculleusspp. Our results demonstrate that uncultivated methanogenic archaea related toMethanoculleusspp. were major contributors to acetate-C assimilation during the anoxic incubation of swine manure storage tank samples. Carbon assimilation and dissimilation rate estimations suggested thatMethanoculleusspp. were also major contributors to methane emissions and that the hydrogenotrophic pathway predominated during methanogenesis.

scholarly journals Active Soil Nitrifying Communities Revealed by In Situ Transcriptomics and Microcosm-Based Stable-Isotope Probing

2020 ◽  
Vol 86 (23) ◽  
Author(s):  
Wei-Wei Xia ◽  
Jun Zhao ◽  
Yan Zheng ◽  
Hui-Min Zhang ◽  
Jia-Bao Zhang ◽  
...  
Keyword(s):  
Stable Isotope ◽  
Stable Isotope Probing ◽  
Field Conditions ◽  
Nitrogen Fertilizers ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Content Type ◽  
Nitrite Oxidizing Bacteria

ABSTRACT Long-term nitrogen field fertilization often results in significant changes in nitrifying communities that catalyze a key step in the global N cycle. However, whether microcosm studies are able to inform the dynamic changes in communities of ammonia-oxidizing bacteria (AOB) and archaea (AOA) under field conditions remains poorly understood. This study aimed to evaluate the transcriptional activities of nitrifying communities under in situ conditions, and we found that they were largely similar to those of 13C-labeled nitrifying communities in the urea-amended microcosms of soils that had received different N fertilization regimens for 22 years. High-throughput sequencing of 16S rRNA genes and transcripts suggested that Nitrosospira cluster 3-like AOB and Nitrososphaera viennensis-like AOA were significantly stimulated in N-fertilized fresh soils. Real-time quantitative PCR demonstrated that the significant increase of AOA and AOB in fresh soils upon nitrogen fertilization could be preserved in the air-dried soils. DNA-based stable-isotope probing (SIP) further revealed the greatest labeling of Nitrosospira cluster 3-like AOB and Nitrosospira viennensis-like AOA, despite the strong advantage of AOB over AOA in the N-fertilized soils. Nitrobacter-like nitrite-oxidizing bacteria (NOB) played more important roles than Nitrospira-like NOB in urea-amended SIP microcosms, while the situation was the opposite under field conditions. Our results suggest that long-term fertilization selected for physiologically versatile AOB and AOA that could have been adapted to a wide range of substrate ammonium concentrations. It also provides compelling evidence that the dominant communities of transcriptionally active nitrifiers under field conditions were largely similar to those revealed in 13C-labeled microcosms. IMPORTANCE The role of manipulated microcosms in microbial ecology has been much debated, because they cannot entirely represent the in situ situation. We collected soil samples from 20 field plots, including 5 different treatments with and without nitrogen fertilizers for 22 years, in order to assess active nitrifying communities by in situ transcriptomics and microcosm-based stable-isotope probing. The results showed that chronic N enrichment led to competitive advantages of Nitrosospira cluster 3-like AOB over N. viennensis-like AOA in soils under field conditions. Microcosm labeling revealed similar results for active AOA and AOB, although an apparent discrepancy was observed for nitrite-oxidizing bacteria. This study suggests that the soil microbiome represents a relatively stable community resulting from complex evolutionary processes over a large time scale, and microcosms can serve as powerful tools to test the theory of environmental filtering on the key functional microbial guilds.

scholarly journals Identification of the Functionally Active Methanotroph Population in a Peat Soil Microcosm by Stable-Isotope Probing

2002 ◽  
Vol 68 (3) ◽  
pp. 1446-1453 ◽  
Author(s):  
Samantha A. Morris ◽  
Stefan Radajewski ◽  
Toby W. Willison ◽  
J. Colin Murrell
Keyword(s):  
Stable Isotope ◽  
Gradient Centrifugation ◽  
Peat Soil ◽  
Pcr Amplification ◽  
Cesium Chloride ◽  
Stable Isotope Probing ◽  
Soil Microcosm ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Type I

ABSTRACT The active population of low-affinity methanotrophs in a peat soil microcosm was characterized by stable-isotope probing. “Heavy” 13C-labeled DNA, produced after microbial growth on 13CH4, was separated from naturally abundant 12C-DNA by cesium chloride density gradient centrifugation and used as a template for the PCR. Amplification products of 16S rRNA genes and pmoA, mxaF, and mmoX, which encode key enzymes in the CH4 oxidation pathway, were analyzed. Sequences related to extant type I and type II methanotrophs were identified, indicating that these methanotrophs were active in peat exposed to 8% (vol/vol) CH4. The 13C-DNA libraries also contained clones that were related to β-subclass Proteobacteria, suggesting that novel groups of bacteria may also be involved in CH4 cycling in this soil.

scholarly journals RNA–Stable-Isotope Probing Shows Utilization of Carbon from Inulin by Specific Bacterial Populations in the Rat Large Bowel

2014 ◽  
Vol 80 (7) ◽  
pp. 2240-2247 ◽  
Author(s):  
Gerald W. Tannock ◽  
Blair Lawley ◽  
Karen Munro ◽  
Ian M. Sims ◽  
Julian Lee ◽  
...  
Keyword(s):  
Stable Isotope ◽  
Bacterial Species ◽  
Buoyant Density ◽  
Stable Isotope Probing ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Bacterial Cells ◽  
Bacterial Populations ◽  
Content Type ◽  
And Function

ABSTRACTKnowledge of the trophisms that underpin bowel microbiota composition is required in order to understand its complex phylogeny and function. Stable-isotope (13C)-labeled inulin was added to the diet of rats on a single occasion in order to detect utilization of inulin-derived substrates by particular members of the cecal microbiota. Cecal digesta from Fibruline-inulin-fed rats was collected prior to (0 h) and at 6, 12, 18 and 24 h following provision of the [13C]inulin diet. RNA was extracted from these cecal specimens and fractionated in isopycnic buoyant density gradients in order to detect13C-labeled nucleic acid originating in bacterial cells that had metabolized the labeled dietary constituent. RNA extracted from specimens collected after provision of the labeled diet was more dense than 0-h RNA. Sequencing of 16S rRNA genes amplified from cDNA obtained from these fractions showed thatBacteroides uniformis,Blautia glucerasea,Clostridium indolis, andBifidobacterium animaliswere the main users of the13C-labeled substrate. Culture-based studies of strains of these bacterial species enabled trophisms associated with inulin and its hydrolysis products to be identified.B. uniformisutilized Fibruline-inulin for growth, whereas the other species used fructo-oligosaccharide and monosaccharides. Thus, RNA–stable-isotope probing (RNA-SIP) provided new information about the use of carbon from inulin in microbiota metabolism.

scholarly journals Long-Term Enrichment on Cellulose or Xylan Causes Functional and Taxonomic Convergence of Microbial Communities from Anaerobic Digesters

2015 ◽  
Vol 82 (5) ◽  
pp. 1519-1529 ◽  
Author(s):  
Yangyang Jia ◽  
David Wilkins ◽  
Hongyuan Lu ◽  
Mingwei Cai ◽  
Patrick K. H. Lee
Keyword(s):  
Anaerobic Digestion ◽  
Amplicon Sequencing ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Anaerobic Digesters ◽  
Unifrac Distance ◽  
Content Type ◽  
Term Enrichment ◽  
Unweighted Unifrac ◽  
Microbial System

ABSTRACTCellulose and xylan are two major components of lignocellulosic biomass, which represents a potentially important energy source, as it is abundant and can be converted to methane by microbial action. However, it is recalcitrant to hydrolysis, and the establishment of a complete anaerobic digestion system requires a specific repertoire of microbial functions. In this study, we maintained 2-year enrichment cultures of anaerobic digestion sludge amended with cellulose or xylan to investigate whether a cellulose- or xylan-digesting microbial system could be assembled from sludge previously used to treat neither of them. While efficient methane-producing communities developed under mesophilic (35°C) incubation, they did not under thermophilic (55°C) conditions. Illumina amplicon sequencing results of the archaeal and bacterial 16S rRNA genes revealed that the mature cultures were much lower in richness than the inocula and were dominated by single archaeal (genusMethanobacterium) and bacterial (orderClostridiales) groups, although at finer taxonomic levels the bacteria were differentiated by substrates. Methanogenesis was primarily via the hydrogenotrophic pathway under all conditions, although the identity and growth requirements of syntrophic acetate-oxidizing bacteria were unclear. Incubation conditions (substrate and temperature) had a much greater effect than inoculum source in shaping the mature microbial community, although analysis based on unweighted UniFrac distance found that the inoculum still determined the pool from which microbes could be enriched. Overall, this study confirmed that anaerobic digestion sludge treating nonlignocellulosic material is a potential source of microbial cellulose- and xylan-digesting functions given appropriate enrichment conditions.

scholarly journals Competing Formate- and Carbon Dioxide-Utilizing Prokaryotes in an Anoxic Methane-Emitting Fen Soil

2011 ◽  
Vol 77 (11) ◽  
pp. 3773-3785 ◽  
Author(s):  
Sindy Hunger ◽  
Oliver Schmidt ◽  
Maik Hilgarth ◽  
Marcus A. Horn ◽  
Steffen Kolb ◽  
...  
Keyword(s):  
16S Rrna ◽  
Stable Isotope Probing ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Soil Microcosms ◽  
Content Type ◽  
Formyltetrahydrofolate Synthetase ◽  
Low Concentrations ◽  
Family Level ◽  
Methyl Coenzyme M Reductase

ABSTRACTMethanogenesis in wetlands is dependent on intermediary substrates derived from the degradation of biopolymers. Formate is one such substrate and is stimulatory to methanogenesis and acetogenesis in anoxic microcosms of soil from the fen Schlöppnerbrunnen. Formate dissimilation also yields CO2as a potential secondary substrate. The objective of this study was to resolve potential differences between anaerobic formate- and CO2-utilizing prokaryotes of this fen by stable isotope probing. Anoxic soil microcosms were pulsed daily with low concentrations of [13C]formate or13CO2(i.e., [13C]bicarbonate). Taxa were evaluated by assessment of 16S rRNA genes,mcrA(encoding the alpha-subunit of methyl-coenzyme M reductase), andfhs(encoding formyltetrahydrofolate synthetase). Methanogens, acetogens, and formate-hydrogen lyase-containing taxa appeared to compete for formate. Genes affiliated withMethanocellaceae,Methanobacteriaceae,Acetobacteraceae, andRhodospirillaceaewere13C enriched (i.e., labeled) in [13C]formate treatments, whereas genes affiliated withMethanosarcinaceae,Conexibacteraceae, andSolirubrobacteraceaewere labeled in13CO2treatments. [13C]acetate was enriched in [13C]formate treatments, but labeling of known acetogenic taxa was not detected. However, several phylotypes were affiliated with acetogen-containing taxa (e.g.,Sporomusa).Methanosaetaceae-affiliated methanogens appeared to participate in the consumption of acetate. Twelve and 58 family-level archaeal and bacterial 16S rRNA phylotypes, respectively, were detected, approximately half of which had no isolated representatives.Crenarchaeotaconstituted half of the detected archaeal 16S rRNA phylotypes. The results highlight the unresolved microbial diversity of the fen Schlöppnerbrunnen, suggest that differing taxa competed for the same substrate, and indicate thatMethanocellaceae,Methanobacteriaceae,Methanosarcinaceae, andMethanosaetaceaewere linked to the production of methane, but they do not clearly resolve the taxa responsible for the apparent conversion of formate to acetate.

scholarly journals Phylogenetically Distinct Phylotypes Modulate Nitrification in a Paddy Soil

2015 ◽  
Vol 81 (9) ◽  
pp. 3218-3227 ◽  
Author(s):  
Jun Zhao ◽  
Baozhan Wang ◽  
Zhongjun Jia
Keyword(s):  
High Throughput ◽  
Paddy Soil ◽  
Stable Isotope Probing ◽  
Paddy Fields ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Molecular Evidence ◽  
Ammonia Oxidizing Bacteria ◽  
Soil Nitrification ◽  
Content Type

ABSTRACTPaddy fields represent a unique ecosystem in which regular flooding occurs, allowing for rice cultivation. However, the taxonomic identity of the microbial functional guilds that catalyze soil nitrification remains poorly understood. In this study, we provide molecular evidence for distinctly different phylotypes of nitrifying communities in a neutral paddy soil using high-throughput pyrosequencing and DNA-based stable isotope probing (SIP). Following urea addition, the levels of soil nitrate increased significantly, accompanied by an increase in the abundance of the bacterial and archaealamoAgene in microcosms subjected to SIP (SIP microcosms) during a 56-day incubation period. High-throughput fingerprints of the total 16S rRNA genes in SIP microcosms indicated that nitrification activity positively correlated with the abundance ofNitrosospira-like ammonia-oxidizing bacteria (AOB), soil group 1.1b-like ammonia-oxidizing archaea (AOA), andNitrospira-like nitrite-oxidizing bacteria (NOB). Pyrosequencing of13C-labeled DNA further revealed that13CO2was assimilated by these functional groups to a much greater extent than by marine group 1.1a-associated AOA andNitrobacter-like NOB. Phylogenetic analysis demonstrated that active AOB communities were closely affiliated withNitrosospirasp. strain L115 and theNitrosospira multiformislineage and that the13C-labeled AOA were related to phylogenetically distinct groups, including the moderately thermophilic “CandidatusNitrososphaera gargensis,” uncultured fosmid 29i4, and acidophilic “CandidatusNitrosotalea devanaterra” lineages. These results suggest that a wide variety of microorganisms were involved in soil nitrification, implying physiological diversification of soil nitrifying communities that are constantly exposed to environmental fluctuations in paddy fields.

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