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 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 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 Field-Based Stable Isotope Probing Reveals the Identities of Benzoic Acid-Metabolizing Microorganisms and Their In Situ Growth in Agricultural Soil

2008 ◽  
Vol 74 (13) ◽  
pp. 4111-4118 ◽  
Author(s):  
Graham M. Pumphrey ◽  
Eugene L. Madsen
Keyword(s):  
Stable Isotope ◽  
16S Rrna ◽  
Benzoic Acid ◽  
Stable Isotope Probing ◽  
Heavy Fraction ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
In Situ Growth ◽  
Probable Number

ABSTRACT We used a combination of stable isotope probing (SIP), gas chromatography-mass spectrometry-based respiration, isolation/cultivation, and quantitative PCR procedures to discover the identity and in situ growth of soil microorganisms that metabolize benzoic acid. We added [13C]benzoic acid or [12C]benzoic acid (100 μg) once, four times, or five times at 2-day intervals to agricultural field plots. After monitoring 13CO2 evolution from the benzoic acid-dosed soil, field soils were harvested and used for nucleic acid extraction and for cultivation of benzoate-degrading bacteria. Exposure of soil to benzoate increased the number of culturable benzoate degraders compared to unamended soil, and exposure to benzoate shifted the dominant culturable benzoate degraders from Pseudomonas species to Burkholderia species. Isopycnic separation of heavy [13C]DNA from the unlabeled fraction allowed terminal restriction fragment length polymorphism (T-RFLP) analyses to confirm that distinct 16S rRNA genes were localized in the heavy fraction. Phylogenetic analysis of sequenced 16S rRNA genes revealed a predominance (15 of 58 clones) of Burkholderia species in the heavy fraction. Burkholderia sp. strain EBA09 shared 99.5% 16S rRNA sequence similarity with a group of clones representing the dominant RFLP pattern, and the T-RFLP fragment for strain EBA09 and a clone from that cluster matched the fragment enriched in the [13C]DNA fraction. Growth of the population represented by EBA09 during the field-dosing experiment was demonstrated by using most-probable-number-PCR and primers targeting EBA09 and the closely related species Burkholderia hospita. Thus, the target population identified by SIP not only actively metabolized benzoic acid but reproduced in the field upon the addition of the substrate.

scholarly journals Biphenyl-Metabolizing Bacteria in the Rhizosphere of Horseradish and Bulk Soil Contaminated by Polychlorinated Biphenyls as Revealed by Stable Isotope Probing

2009 ◽  
Vol 75 (20) ◽  
pp. 6471-6477 ◽  
Author(s):  
Ondrej Uhlik ◽  
Katerina Jecna ◽  
Martina Mackova ◽  
Cestmir Vlcek ◽  
Miluse Hroudova ◽  
...  
Keyword(s):  
Microbial Community ◽  
Stable Isotope ◽  
Polychlorinated Biphenyls ◽  
Stable Isotope Probing ◽  
Amino Acid Sequences ◽  
16S Rrna Genes ◽  
Bulk Soil ◽  
Rrna Genes ◽  
Soil Environment ◽  
Α Subunits

ABSTRACT DNA-based stable isotope probing in combination with terminal restriction fragment length polymorphism was used in order to identify members of the microbial community that metabolize biphenyl in the rhizosphere of horseradish (Armoracia rusticana) cultivated in soil contaminated with polychlorinated biphenyls (PCBs) compared to members of the microbial community in initial, uncultivated bulk soil. On the basis of early and recurrent detection of their 16S rRNA genes in clone libraries constructed from [13C]DNA, Hydrogenophaga spp. appeared to dominate biphenyl catabolism in the horseradish rhizosphere soil, whereas Paenibacillus spp. were the predominant biphenyl-utilizing bacteria in the initial bulk soil. Other bacteria found to derive carbon from biphenyl in this nutrient-amended microcosm-based study belonged mostly to the class Betaproteobacteria and were identified as Achromobacter spp., Variovorax spp., Methylovorus spp., or Methylophilus spp. Some bacteria that were unclassified at the genus level were also detected, and these bacteria may be members of undescribed genera. The deduced amino acid sequences of the biphenyl dioxygenase α subunits (BphA) from bacteria that incorporated [13C]into DNA in 3-day incubations of the soils with [13C]biphenyl are almost identical to that of Pseudomonas alcaligenes B-357. This suggests that the spectrum of the PCB congeners that can be degraded by these enzymes may be similar to that of strain B-357. These results demonstrate that altering the soil environment can result in the participation of different bacteria in the metabolism of biphenyl.

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 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 Stable Isotope Probing for Microbial Iron Reduction in Chocolate Pots Hot Spring, Yellowstone National Park

2018 ◽  
Vol 84 (11) ◽  
Author(s):  
Nathaniel W. Fortney ◽  
Shaomei He ◽  
Ajinkya Kulkarni ◽  
Michael W. Friedrich ◽  
Charlotte Holz ◽  
...  
Keyword(s):  
Microbial Community ◽  
Stable Isotope ◽  
Yellowstone National Park ◽  
National Park ◽  
Hot Spring ◽  
Redox Cycling ◽  
Stable Isotope Probing ◽  
Content Type ◽  
Insight Into

ABSTRACTChocolate Pots hot springs (CP) is a circumneutral-pH Fe-rich geothermal feature located in Yellowstone National Park. Previous Fe(III)-reducing enrichment culture studies with CP sediments identified close relatives of known dissimilatory Fe(III)-reducing bacterial (FeRB) taxa, includingGeobacterandMelioribacter. However, the abundances and activities of such organisms in the native microbial community are unknown. Here, we used stable isotope probing experiments combined with 16S rRNA gene amplicon and shotgun metagenomic sequencing to gain an understanding of thein situFe(III)-reducing microbial community at CP. Fe-Si oxide precipitates collected near the hot spring vent were incubated with unlabeled and13C-labeled acetate to target active FeRB. We searched reconstructed genomes for homologs of genes involved in known extracellular electron transfer (EET) systems to identify the taxa involved in Fe redox transformations. Known FeRB taxa containing putative EET systems (Geobacter,Ignavibacteria) increased in abundance under acetate-amended conditions, whereas genomes related toIgnavibacteriumandThermodesulfovibriothat contained putative EET systems were recovered from incubations without electron donor. Our results suggest that FeRB play an active role in Fe redox cycling within Fe-Si oxide-rich deposits located at the hot spring vent.IMPORTANCEThe identification of past near-surface hydrothermal environments on Mars emphasizes the importance of using modern Earth environments, such as CP, to gain insight into potential Fe-based microbial life on other rocky worlds, as well as ancient Fe-rich Earth ecosystems. By combining stable carbon isotope probing techniques and DNA sequencing technology, we gained insight into the pathways of microbial Fe redox cycling at CP. The results suggest that microbial Fe(III) oxide reduction is prominentin situ, with important implications for the generation of geochemical and stable Fe isotopic signatures of microbial Fe redox metabolism within Fe-rich circumneutral-pH thermal spring environments on Earth and Mars.

scholarly journals Arctic tundra soil bacterial communities active at subzero temperatures detected by stable isotope probing

2019 ◽  
Vol 96 (2) ◽  
Author(s):  
Preshita S Gadkari ◽  
Lora R McGuinness ◽  
Minna K Männistö ◽  
Lee J Kerkhof ◽  
Max M Häggblom
Keyword(s):  
Stable Isotope ◽  
16S Rrna ◽  
Arctic Tundra ◽  
Stable Isotope Probing ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Subzero Temperature ◽  
Tundra Soil ◽  
Arctic Soils ◽  
Subzero Temperatures

ABSTRACT Arctic soils store vast amounts of carbon and are subject to intense climate change. While the effects of thaw on the composition and activities of Arctic tundra microorganisms has been examined extensively, little is known about the consequences of temperature fluctuations within the subzero range in seasonally frozen or permafrost soils. This study identified tundra soil bacteria active at subzero temperatures using stable isotope probing (SIP). Soils from Kilpisjärvi, Finland, were amended with 13C-cellobiose and incubated at 0, −4 and −16°C for up to 40 weeks. 16S rRNA gene sequence analysis of 13C-labelled DNA revealed distinct subzero-active bacterial taxa. The SIP experiments demonstrated that diverse bacteria, including members of Candidatus Saccharibacteria, Melioribacteraceae, Verrucomicrobiaceae, Burkholderiaceae, Acetobacteraceae, Armatimonadaceae and Planctomycetaceae, were capable of synthesising 13C-DNA at subzero temperatures. Differences in subzero temperature optima were observed, for example, with members of Oxalobacteraceae and Rhizobiaceae found to be more active at 0°C than at −4°C or −16°C, whereas Melioribacteriaceae were active at all subzero temperatures tested. Phylogeny of 13C-labelled 16S rRNA genes from the Melioribacteriaceae, Verrucomicrobiaceae and Candidatus Saccharibacteria suggested that these taxa formed subzero-active clusters closely related to members from other cryo-environments. This study demonstrates that subzero temperatures impact active bacterial community composition and activity, which may influence biogeochemical cycles.

scholarly journals Impact of plants on the diversity and activity of methylotrophs in soil

Microbiome ◽  
2020 ◽  
Vol 8 (1) ◽  
Author(s):  
Michael C. Macey ◽  
Jennifer Pratscher ◽  
Andrew T. Crombie ◽  
J. Colin Murrell
Keyword(s):  
Stable Isotope ◽  
Plant Growth ◽  
Molecular Probes ◽  
Stable Isotope Probing ◽  
16S Rrna Genes ◽  
Bulk Soil ◽  
Rrna Genes ◽  
Methylotrophic Bacteria ◽  
Volatile Organic ◽  
Plant Exudates

Abstract Background Methanol is the second most abundant volatile organic compound in the atmosphere, with the majority produced as a metabolic by-product during plant growth. There is a large disparity between the estimated amount of methanol produced by plants and the amount which escapes to the atmosphere. This may be due to utilisation of methanol by plant-associated methanol-consuming bacteria (methylotrophs). The use of molecular probes has previously been effective in characterising the diversity of methylotrophs within the environment. Here, we developed and applied molecular probes in combination with stable isotope probing to identify the diversity, abundance and activity of methylotrophs in bulk and in plant-associated soils. Results Application of probes for methanol dehydrogenase genes (mxaF, xoxF, mdh2) in bulk and plant-associated soils revealed high levels of diversity of methylotrophic bacteria within the bulk soil, including Hyphomicrobium, Methylobacterium and members of the Comamonadaceae. The community of methylotrophic bacteria captured by this sequencing approach changed following plant growth. This shift in methylotrophic diversity was corroborated by identification of the active methylotrophs present in the soils by DNA stable isotope probing using 13C-labelled methanol. Sequencing of the 16S rRNA genes and construction of metagenomes from the 13C-labelled DNA revealed members of the Methylophilaceae as highly abundant and active in all soils examined. There was greater diversity of active members of the Methylophilaceae and Comamonadaceae and of the genus Methylobacterium in plant-associated soils compared to the bulk soil. Incubating growing pea plants in a 13CO2 atmosphere revealed that several genera of methylotrophs, as well as heterotrophic genera within the Actinomycetales, assimilated plant exudates in the pea rhizosphere. Conclusion In this study, we show that plant growth has a major impact on both the diversity and the activity of methanol-utilising methylotrophs in the soil environment, and thus, the study contributes significantly to efforts to balance the terrestrial methanol and carbon cycle.

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.

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