scholarly journals Experimentally-validated correlation analysis reveals new anaerobic methane oxidation partnerships with consortium-level heterogeneity in diazotrophy

2020 ◽  
Author(s):  
Kyle S. Metcalfe ◽  
Ranjani Murali ◽  
Sean W. Mullin ◽  
Stephanie A. Connon ◽  
Victoria J. Orphan
Keyword(s):  
16S Rrna ◽  
Methane Oxidation ◽  
Methane Flux ◽  
Anaerobic Methane Oxidation ◽  
Hybridization Chain Reaction ◽  
Strong Positive Correlation ◽  
Opposite Pattern ◽  
Sulfate Reducing ◽  
Active Expression

Abstract Archaeal anaerobic methanotrophs (“ANME”) and sulfate-reducing Deltaproteobacteria (“SRB”) form symbiotic multicellular consortia capable of anaerobic methane oxidation (AOM), and in so doing modulate methane flux from marine sediments. The specificity with which ANME associate with particular SRB partners in situ, however, is poorly understood. To characterize partnership specificity in ANME-SRB consortia, we applied the correlation inference technique SparCC to 310 16S rRNA amplicon libraries prepared from Costa Rica seep sediment samples, uncovering a strong positive correlation between ANME-2b and members of a clade of Deltaproteobacteria we termed SEEP-SRB1g. We confirmed this association by examining 16S rRNA diversity in individual ANME-SRB consortia sorted using flow cytometry and by imaging ANME-SRB consortia with fluorescence in situ hybridization (FISH) microscopy using newly-designed probes targeting the SEEP-SRB1g clade. Analysis of genome bins belonging to SEEP-SRB1g revealed the presence of a complete nifHDK operon required for diazotrophy, unusual in published genomes of ANME-associated SRB. Active expression of nifH in SEEP-SRB1g within ANME-2b—SEEP-SRB1g consortia was then demonstrated by microscopy using hybridization chain reaction (HCR-) FISH targeting nifH transcripts and diazotrophic activity was documented by FISH-nanoSIMS experiments. NanoSIMS analysis of ANME-2b—SEEP-SRB1g consortia incubated with a headspace containing CH4 and 15N2 revealed differences in cellular 15N-enrichment between the two partners that varied between individual consortia, with SEEP-SRB1g cells enriched in 15N relative to ANME-2b in one consortium and the opposite pattern observed in others, indicating both ANME-2b and SEEP-SRB1g are capable of nitrogen fixation, but with consortium-specific variation in whether the archaea or bacterial partner is the dominant diazotroph.

scholarly journals Experimentally-validated correlation analysis reveals new anaerobic methane oxidation partnerships with consortium-level heterogeneity in diazotrophy

2020 ◽  
Author(s):  
Kyle S. Metcalfe ◽  
Ranjani Murali ◽  
Sean W. Mullin ◽  
Stephanie A. Connon ◽  
Victoria J. Orphan
Keyword(s):  
16S Rrna ◽  
Methane Oxidation ◽  
Methane Flux ◽  
Anaerobic Methane Oxidation ◽  
Hybridization Chain Reaction ◽  
Strong Positive Correlation ◽  
Opposite Pattern ◽  
Sulfate Reducing ◽  
Active Expression

AbstractArchaeal anaerobic methanotrophs (‘ANME’) and sulfate-reducing Deltaproteobacteria (‘SRB’) form symbiotic multicellular consortia capable of anaerobic methane oxidation (AOM), and in so doing modulate methane flux from marine sediments. The specificity with which ANME associate with particular SRB partners in situ, however, is poorly understood. To characterize partnership specificity in ANME-SRB consortia, we applied the correlation inference technique SparCC to 310 16S rRNA Illumina iTag amplicon libraries prepared from Costa Rica sediment samples, uncovering a strong positive correlation between ANME-2b and members of a clade of Deltaproteobacteria we termed SEEP-SRB1g. We confirmed this association by examining 16S rRNA diversity in individual ANME-SRB consortia sorted using flow cytometry and by imaging ANME-SRB consortia with fluorescence in situ hybridization (FISH) microscopy using newly-designed probes targeting the SEEP-SRB1g clade. Analysis of genome bins belonging to SEEP-SRB1g revealed the presence of a complete nifHDK operon required for diazotrophy, unusual in published genomes of ANME-associated SRB. Active expression of nifH in SEEP-SRB1g and diazotrophic activity within ANME-2b/SEEP-SRB1g consortia was then demonstrated by microscopy using hybridization chain-reaction (HCR-) FISH targeting nifH transcripts and by FISH-nanoSIMS experiments. NanoSIMS analysis of ANME-2b/SEEP-SRB1g consortia incubated with a headspace containing CH4 and 15N2 revealed differences in cellular 15N-enrichment between the two partners that varied between individual consortia, with SEEP-SRB1g cells enriched in 15N relative to ANME-2b in one consortium and the opposite pattern observed in others, indicating both ANME-2b and SEEP-SRB1g are capable of nitrogen fixation, but with consortium-specific variation in whether the archaea or bacterial partner is the dominant diazotroph.

scholarly journals Suspension Array Analysis of 16S rRNA from Fe- and SO42-Reducing Bacteria in Uranium-Contaminated Sediments Undergoing Bioremediation

2006 ◽  
Vol 72 (7) ◽  
pp. 4672-4687 ◽  
Author(s):  
Darrell P. Chandler ◽  
Ann E. Jarrell ◽  
Eric R. Roden ◽  
Julia Golova ◽  
Boris Chernov ◽  
...  
Keyword(s):  
16S Rrna ◽  
Total Rna ◽  
Iron Reducing Bacteria ◽  
Suspension Array ◽  
Sulfate Reducing ◽  
Subsurface Sediments ◽  
Reducing Bacteria ◽  
Bead Arrays ◽  
Microbiological Analyses

ABSTRACT A 16S rRNA-targeted tunable bead array was developed and used in a retrospective analysis of metal- and sulfate-reducing bacteria in contaminated subsurface sediments undergoing in situ U(VI) bioremediation. Total RNA was extracted from subsurface sediments and interrogated directly, without a PCR step. Bead array validation studies with total RNA derived from 24 isolates indicated that the behavior and response of the 16S rRNA-targeted oligonucleotide probes could not be predicted based on the primary nucleic acid sequence. Likewise, signal intensity (absolute or normalized) could not be used to assess the abundance of one organism (or rRNA) relative to the abundance of another organism (or rRNA). Nevertheless, the microbial community structure and dynamics through time and space and as measured by the rRNA-targeted bead array were consistent with previous data acquired at the site, where indigenous sulfate- and iron-reducing bacteria and near neighbors of Desulfotomaculum were the organisms that were most responsive to a change in injected acetate concentrations. Bead array data were best interpreted by analyzing the relative changes in the probe responses for spatially and temporally related samples and by considering only the response of one probe to itself in relation to a background (reference) environmental sample. By limiting the interpretation of the data in this manner and placing it in the context of supporting geochemical and microbiological analyses, we concluded that ecologically relevant and meaningful information can be derived from direct microarray analysis of rRNA in uncharacterized environmental samples, even with the current analytical uncertainty surrounding the behavior of individual probes on tunable bead arrays.

scholarly journals Cooccurrence of Aerobic and Anaerobic Methane Oxidation in the Water Column of Lake Plußsee

2005 ◽  
Vol 71 (12) ◽  
pp. 8925-8928 ◽  
Author(s):  
Gundula Eller ◽  
Layla Känel ◽  
Martin Krüger
Keyword(s):  
Water Column ◽  
Methane Oxidation ◽  
Eutrophic Lake ◽  
Anaerobic Methane Oxidation ◽  
Sulfide Concentration ◽  
High Cell ◽  
Cell Numbers ◽  
Aerobic And Anaerobic ◽  
Hydrogen Sulfide Concentration

ABSTRACT Dissolved methane was investigated in the water column of eutrophic Lake Plußsee and compared to temperature, oxygen, and sulfide profiles. Methane concentrations and δ-13C signatures indicated a zone of aerobic methane oxidation and additionally a zone of anaerobic methane oxidation in the anoxic water body. The latter coincided with a peak in hydrogen sulfide concentration. High cell numbers of aerobic and anaerobic methane-oxidizing microorganisms were detected by fluorescence in situ hybridization (FISH) or the more sensitive catalyst-amplified reporter deposition-FISH, respectively, in these layers.

scholarly journals Use of 16S rRNA Gene Based Clone Libraries to Assess Microbial Communities Potentially Involved in Anaerobic Methane Oxidation in a Mediterranean Cold Seep

2007 ◽  
Vol 53 (3) ◽  
pp. 384-398 ◽  
Author(s):  
Sander K. Heijs ◽  
Ralf R. Haese ◽  
Paul W. J. J. van der Wielen ◽  
Larry J. Forney ◽  
Jan Dirk van Elsas
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Microbial Communities ◽  
Methane Oxidation ◽  
Anaerobic Methane Oxidation ◽  
Cold Seep ◽  
Rrna Gene ◽  
Clone Libraries

scholarly journals Growth and Methane Oxidation Rates of Anaerobic Methanotrophic Archaea in a Continuous-Flow Bioreactor

2003 ◽  
Vol 69 (9) ◽  
pp. 5472-5482 ◽  
Author(s):  
Peter R. Girguis ◽  
Victoria J. Orphan ◽  
Steven J. Hallam ◽  
Edward F. DeLong
Keyword(s):  
Methane Oxidation ◽  
Continuous Flow ◽  
Molecular Techniques ◽  
Anaerobic Methane Oxidation ◽  
Cold Seep ◽  
Microbial Consortia ◽  
Sulfate Reducing ◽  
Oxidation Rates ◽  
In Situ Conditions ◽  
Pure Cultures

ABSTRACT Anaerobic methanotrophic archaea have recently been identified in anoxic marine sediments, but have not yet been recovered in pure culture. Physiological studies on freshly collected samples containing archaea and their sulfate-reducing syntrophic partners have been conducted, but sample availability and viability can limit the scope of these experiments. To better study microbial anaerobic methane oxidation, we developed a novel continuous-flow anaerobic methane incubation system (AMIS) that simulates the majority of in situ conditions and supports the metabolism and growth of anaerobic methanotrophic archaea. We incubated sediments collected from within and outside a methane cold seep in Monterey Canyon, Calif., for 24 weeks on the AMIS system. Anaerobic methane oxidation was measured in all sediments after incubation on AMIS, and quantitative molecular techniques verified the increases in methane-oxidizing archaeal populations in both seep and nonseep sediments. Our results demonstrate that the AMIS system stimulated the maintenance and growth of anaerobic methanotrophic archaea, and possibly their syntrophic, sulfate-reducing partners. Our data demonstrate the utility of combining physiological and molecular techniques to quantify the growth and metabolic activity of anaerobic microbial consortia. Further experiments with the AMIS system should provide a better understanding of the biological mechanisms of methane oxidation in anoxic marine environments. The AMIS may also enable the enrichment, purification, and isolation of methanotrophic archaea as pure cultures or defined syntrophic consortia.

A new intra-aerobic metabolism in the nitrite-dependent anaerobic methane-oxidizing bacterium Candidatus ‘Methylomirabilis oxyfera’

2011 ◽  
Vol 39 (1) ◽  
pp. 243-248 ◽  
Author(s):  
Ming L. Wu ◽  
Katharina F. Ettwig ◽  
Mike S.M. Jetten ◽  
Marc Strous ◽  
Jan T. Keltjens ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Methane Oxidation ◽  
Current Knowledge ◽  
Methanogenic Archaea ◽  
Anaerobic Methane Oxidation ◽  
Oxidation Pathway ◽  
Sulfate Reducing ◽  
Oxidation Of Methane ◽  
Aerobic Methanotrophs ◽  
Reverse Methanogenesis

Biological methane oxidation proceeds either through aerobic or anaerobic pathways. The newly discovered bacterium Candidatus ‘Methylomirabilis oxyfera’ challenges this dichotomy. This bacterium performs anaerobic methane oxidation coupled to denitrification, but does so in a peculiar way. Instead of scavenging oxygen from the environment, like the aerobic methanotrophs, or driving methane oxidation by reverse methanogenesis, like the methanogenic archaea in sulfate-reducing systems, it produces its own supply of oxygen by metabolizing nitrite via nitric oxide into oxygen and dinitrogen gas. The intracellularly produced oxygen is then used for the oxidation of methane by the classical aerobic methane oxidation pathway involving methane mono-oxygenase. The present mini-review summarizes the current knowledge about this process and the micro-organism responsible for it.

scholarly journals Phylogenetic Identification and Substrate Uptake Patterns of Sulfate-Reducing Bacteria Inhabiting an Oxic-Anoxic Sewer Biofilm Determined by Combining Microautoradiography and Fluorescent In Situ Hybridization

2002 ◽  
Vol 68 (1) ◽  
pp. 356-364 ◽  
Author(s):  
Tsukasa Ito ◽  
Jeppe L. Nielsen ◽  
Satoshi Okabe ◽  
Yoshimasa Watanabe ◽  
Per H. Nielsen
Keyword(s):  
In Situ Hybridization ◽  
16S Rrna ◽  
Electron Acceptor ◽  
Fluorescent In Situ Hybridization ◽  
Sulfate Reducing Bacteria ◽  
Substrate Uptake ◽  
Sulfate Reducing ◽  
Phylogenetic Identification ◽  
Reducing Bacteria

ABSTRACT We simultaneously determined the phylogenetic identification and substrate uptake patterns of sulfate-reducing bacteria (SRB) inhabiting a sewer biofilm with oxygen, nitrate, or sulfate as an electron acceptor by combining microautoradiography and fluorescent in situ hybridization (MAR-FISH) with family- and genus-specific 16S rRNA probes. The MAR-FISH analysis revealed that Desulfobulbus hybridized with probe 660 was a dominant SRB subgroup in this sewer biofilm, accounting for 23% of the total SRB. Approximately 9 and 27% of Desulfobulbus cells detected with probe 660 could take up [14C]propionate with oxygen and nitrate, respectively, as an electron acceptor, which might explain the high abundance of this species in various oxic environments. Furthermore, more than 40% of Desulfobulbus cells incorporated acetate under anoxic conditions. SRB were also numerically important members of H2-utilizing and 14CO2-fixing microbial populations in this sewer biofilm, accounting for roughly 42% of total H2-utilizing bacteria hybridized with probe EUB338. A comparative 16S ribosomal DNA analysis revealed that two SRB populations, related to the Desulfomicrobium hypogeium and the Desulfovibrio desulfuricans MB lineages, were found to be important H2 utilizers in this biofilm. The substrate uptake characteristics of different phylogenetic SRB subgroups were compared with the characteristics described to date. These results provide further insight into the correlation between the 16S rRNA phylogenetic diversity and the physiological diversity of SRB populations inhabiting sewer biofilms.

scholarly journals Enrichment and Molecular Detection of Denitrifying Methanotrophic Bacteria of the NC10 Phylum

2009 ◽  
Vol 75 (11) ◽  
pp. 3656-3662 ◽  
Author(s):  
Katharina F. Ettwig ◽  
Theo van Alen ◽  
Katinka T. van de Pas-Schoonen ◽  
Mike S. M. Jetten ◽  
Marc Strous
Keyword(s):  
Methane Oxidation ◽  
Enrichment Culture ◽  
Nitrite Reduction ◽  
Anaerobic Methane Oxidation ◽  
Rrna Gene ◽  
Total Biomass ◽  
Environmental Distribution ◽  
Diverse Community ◽  
Reducing Activity

ABSTRACT Anaerobic methane oxidation coupled to denitrification was recently assigned to bacteria belonging to the uncultured phylum NC10. In this study, we incubated sediment from a eutrophic ditch harboring a diverse community of NC10 bacteria in a bioreactor with a constant supply of methane and nitrite. After 6 months, fluorescence in situ hybridization showed that NC10 bacteria dominated the resulting population. The enrichment culture oxidized methane and reduced nitrite to dinitrogen gas. We assessed NC10 phylum diversity in the inoculum and the enrichment culture, compiled the sequences currently available for this bacterial phylum, and showed that of the initial diversity, only members of one subgroup had been enriched. The growth of this subgroup was monitored by quantitative PCR and correlated to nitrite-reducing activity and the total biomass of the culture. Together, the results indicate that the enriched subgroup of NC10 bacteria is responsible for anaerobic methane oxidation coupled to nitrite reduction. Due to methodological limitations (a strong bias against NC10 bacteria in 16S rRNA gene clone libraries and inhibition by commonly used stopper material) the environmental distribution and importance of these bacteria could be largely underestimated at present.

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