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

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

Applied and Environmental Microbiology ◽

10.1128/aem.71.12.8925-8928.2005 ◽

2005 ◽

Vol 71 (12) ◽

pp. 8925-8928 ◽

Cited By ~ 101

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.

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

Microbial Ecology ◽

10.1007/s00248-006-9172-3 ◽

2007 ◽

Vol 53 (3) ◽

pp. 384-398 ◽

Cited By ~ 70

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

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Succession of Internal Sulfur Cycles and Sulfur-Oxidizing Bacterial Communities in Microaerophilic Wastewater Biofilms

Applied and Environmental Microbiology ◽

10.1128/aem.71.5.2520-2529.2005 ◽

2005 ◽

Vol 71 (5) ◽

pp. 2520-2529 ◽

Cited By ~ 55

Author(s):

Satoshi Okabe ◽

Tsukasa Ito ◽

Kenichi Sugita ◽

Hisashi Satoh

Keyword(s):

In Situ Hybridization ◽

Sulfur Cycle ◽

Rrna Gene ◽

Second Phase ◽

Sulfate Reducing ◽

Sulfur Oxidizing ◽

Reducing Activity ◽

Two Phases ◽

Microelectrode Measurements

ABSTRACT The succession of sulfur-oxidizing bacterial (SOB) community structure and the complex internal sulfur cycle occurring in wastewater biofilms growing under microaerophilic conditions was analyzed by using a polyphasic approach that employed 16S rRNA gene-cloning analysis combined with fluorescence in situ hybridization, microelectrode measurements, and standard batch and reactor experiments. A complete sulfur cycle was established via S0 accumulation within 80 days in the biofilms in replicate. This development was generally split into two phases, (i) a sulfur-accumulating phase and (ii) a sulfate-producing phase. In the first phase (until about 40 days), since the sulfide production rate (sulfate-reducing activity) exceeded the maximum sulfide-oxidizing capacity of SOB in the biofilms, H2S was only partially oxidized to S0 by mainly Thiomicrospira denitirificans with NO3 − as an electron acceptor, leading to significant accumulation of S0 in the biofilms. In the second phase, the SOB populations developed further and diversified with time. In particular, S0 accumulation promoted the growth of a novel strain, strain SO07, which predominantly carried out the oxidation of S0 to SO4 2− under oxic conditions, and Thiothrix sp. strain CT3. In situ hybridization analysis revealed that the dense populations of Thiothrix (ca. 109 cells cm−3) and strain SO07 (ca. 108 cells cm−3) were found at the sulfur-rich surface (100 μm), while the population of Thiomicrospira denitirificans was distributed throughout the biofilms with a density of ca. 107 to 108 cells cm−3. Microelectrode measurements revealed that active sulfide-oxidizing zones overlapped the spatial distributions of different phylogenetic SOB groups in the biofilms. As a consequence, the sulfide-oxidizing capacities of the biofilms became high enough to completely oxidize all H2S produced by SRB to SO4 2− in the second phase, indicating establishment of the complete sulfur cycle in the biofilms.

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Anaerobic methane oxidation coupled to nitrite reduction can be a potential methane sink in coastal environments

Applied Microbiology and Biotechnology ◽

10.1007/s00253-016-7627-0 ◽

2016 ◽

Vol 100 (16) ◽

pp. 7171-7180 ◽

Cited By ~ 32

Author(s):

Li-dong Shen ◽

Bao-lan Hu ◽

Shuai Liu ◽

Xiao-ping Chai ◽

Zhan-fei He ◽

...

Keyword(s):

Methane Oxidation ◽

Nitrite Reduction ◽

Anaerobic Methane Oxidation ◽

Coastal Environments ◽

Methane Sink

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Anaerobic Oxidation of Methane Coupled to Nitrite Reduction by Halophilic Marine NC10 Bacteria

Applied and Environmental Microbiology ◽

10.1128/aem.00984-15 ◽

2015 ◽

Vol 81 (16) ◽

pp. 5538-5545 ◽

Cited By ~ 42

Author(s):

Zhanfei He ◽

Sha Geng ◽

Chaoyang Cai ◽

Shuai Liu ◽

Yan Liu ◽

...

Keyword(s):

16S Rrna ◽

Methane Oxidation ◽

Nitrite Reduction ◽

Substrate Affinity ◽

Rrna Gene ◽

Anaerobic Oxidation Of Methane ◽

Anaerobic Oxidation ◽

Oxidation Activity ◽

Optimal Salinity ◽

Oxidation Of Methane

ABSTRACTAnaerobic oxidation of methane (AOM) coupled to nitrite reduction is a novel AOM process that is mediated by denitrifying methanotrophs. To date, enrichments of these denitrifying methanotrophs have been confined to freshwater systems; however, the recent findings of 16S rRNA andpmoAgene sequences in marine sediments suggest a possible occurrence of AOM coupled to nitrite reduction in marine systems. In this research, a marine denitrifying methanotrophic culture was obtained after 20 months of enrichment. Activity testing and quantitative PCR (qPCR) analysis were then conducted and showed that the methane oxidation activity and the number of NC10 bacteria increased correlatively during the enrichment period. 16S rRNA gene sequencing indicated that only bacteria in group A of the NC10 phylum were enriched and responsible for the resulting methane oxidation activity, although a diverse community of NC10 bacteria was harbored in the inoculum. Fluorescencein situhybridization showed that NC10 bacteria were dominant in the enrichment culture after 20 months. The effect of salinity on the marine denitrifying methanotrophic culture was investigated, and the apparent optimal salinity was 20.5‰, which suggested that halophilic bacterial AOM coupled to nitrite reduction was obtained. Moreover, the apparent substrate affinity coefficients of the halophilic denitrifying methanotrophs were determined to be 9.8 ± 2.2 μM for methane and 8.7 ± 1.5 μM for nitrite.

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Distribution and characteristic of nitrite-dependent anaerobic methane oxidation bacteria in wastewater treatment plants and agriculture fields of northern China

10.7287/peerj.preprints.2421 ◽

2016 ◽

Author(s):

Zhen Hu ◽

Ru Ma

Keyword(s):

Wastewater Treatment ◽

Methane Oxidation ◽

Paddy Field ◽

Wastewater Treatment Plants ◽

Enrichment Culture ◽

Treatment Plant ◽

Northern China ◽

Anaerobic Methane Oxidation ◽

Corn Field ◽

Geographical Regions

Nitrite-dependent anaerobic methane oxidation (n-damo) is a recently discovered biological process, which has been arousing global attention because of its potential in minimizing greenhouse gases emissions. In this study, molecular biological techniques and potential n-damo activity batch experiments were conducted to investigate the presence and diversity of M. oxyfera bacteria in paddy field, corn field, and wastewater treatment plant (WWTP) of northern China, as well as lab-scale n-damo enrichment culture. N-damo enrichment culture showed the highest abundance of M. oxyfera bacteria and positive correlation was observed between potential n-damo rate and abundance of M. oxyfera bacteria. Both paddy field and corn field were believed to be better inoculum than WWTP for the enrichment of M. oxyfera bacteria, due to their higher abundance and diversity of M. oxyfera bacteria. Comparative analysis revealed that long biomass retention time and optimum environment (low NH4+ and high NO2- content) were suitable for the growth of M. oxyfera bacteria. In addition, the distribution and diversity of M. oxyfera bacterial might be related to geographical regions.

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Re-evaluation of the phylogenetic diversity and global distribution of the genus Candidatus Accumulibacter

10.1101/2021.12.20.473458 ◽

2021 ◽

Author(s):

Francesca Petriglieri ◽

Caitlin Singleton ◽

Zivile Kondrotaite ◽

Morten K. D. Dueholm ◽

Elizabeth A. McDaniel ◽

...

Keyword(s):

Phosphorus Removal ◽

Marker Gene ◽

Genome Mining ◽

Niche Differentiation ◽

Nitrite Reduction ◽

Rrna Gene ◽

Taxonomic Framework ◽

Taxonomic Assignments ◽

Species Specific

Candidatus Accumulibacter was the first microorganism identified as a polyphosphate-accumulating organism (PAO), important for phosphorus removal from wastewater. This genus is diverse, and the current phylogeny and taxonomic framework appears complicated, with the majority of publicly available genomes classified as Candidatus Accumulibacter phosphatis, despite notable phylogenetic divergence. The ppk1 marker gene allows for a finer scale differentiation into different types and clades, nevertheless taxonomic assignments remain confusing and inconsistent across studies. Therefore, a comprehensive re-evaluation is needed to establish a common understanding of this genus, both in terms of naming and basic conserved physiological traits. Here, we provide this re-assessment using a comparison of genome, ppk1, and 16S rRNA gene-based approaches from comprehensive datasets. We identified 15 novel species, along with the well-known Ca. A. phosphatis, Ca. A. deltensis and Ca. A. aalborgensis. To compare the species in situ, we designed new species-specific FISH probes and revealed their morphology and arrangement in activated sludge. Based on the MiDAS global survey, Ca. Accumulibacter species were widespread in WWTPs with phosphorus removal, indicating the process design as a major driver for their abundance. Genome mining for PAO related pathways and FISH-Raman microspectroscopy confirmed the potential for the PAO metabolism in all Ca. Accumulibacter species, with detection in situ of the typical PAO storage polymers. Genome annotation further revealed fine-scale differences in the nitrate/nitrite reduction pathways. This provides insights into the niche differentiation of these lineages, potentially explaining their coexistence in the same ecosystem while contributing to overall phosphorus and nitrogen removal.

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Structure and distribution of nitrite-dependent anaerobic methane oxidation bacteria vary with water tables in Zoige peatlands

FEMS Microbiology Ecology ◽

10.1093/femsec/fiaa039 ◽

2020 ◽

Vol 96 (5) ◽

Cited By ~ 1

Author(s):

Qiuping Zhong ◽

Dan Xue ◽

Huai Chen ◽

Liangfeng Liu ◽

Yixin He ◽

...

Keyword(s):

Global Change ◽

Methane Oxidation ◽

Bacterial Communities ◽

High Throughput Sequencing ◽

Anaerobic Methane Oxidation ◽

Rrna Gene ◽

Dominant Group ◽

Group A ◽

Group B ◽

Damo Bacteria

ABSTRACT The recently discovered nitrite-dependent anaerobic methane oxidation (n-damo) is an important methane sink in natural ecosystems performed by NC10 phylum bacteria. However, the effect of water table (WT) gradient due to global change on n-damo bacterial communities is not well studied in peatlands. Here, we analysed the vertical distribution (0–100 cm) of n-damo bacterial communities at three sites with different WTs of the Zoige peatlands in the Qinghai-Tibetan Plateau. Using an n-damo bacterial specific 16S rRNA gene clone library, we obtained 25 operational taxonomic units (OTUs) that could be divided into Groups A, B, C, D and E (dominated by A and B). The dominant group was Group B at the high (OTU14 and OTU20) and intermediate (OTU7 and OTU8) WT sites and Group A was dominant at the low WT site (OTU6 and OTU5). Using high-throughput sequencing, we observed that n-damo bacteria mainly distributed in subsurface soils (50–60 and 20–30 cm), and their relative abundances were higher at the low WT site than at the other two sites. In addition, we found that pH and nitrate were positively correlated with Group A, while total organic carbon, total nitrogen and ammonia were positively associated with Group B. Our study provides new insights into our understanding of the response of n-damo bacteria to WT gradient in peatlands, with important implications for global change.

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