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 × 10
6
copies g
−1
wet weight, whereas Indonesian river sediment had only 4.6 ± 2.7 × 10
2
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 × 10
4
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.
Nitrous oxide (N
2O), a potent greenhouse gas, can be emitted during wastewater treatment, significantly contributing to the greenhouse gas footprint. Measurements at lab-scale and full-scale ...wastewater treatment plants (WWTPs) have demonstrated that N
2O can be emitted in substantial amounts during nitrogen removal in WWTPs, however, a large variation in reported emission values exists. Analysis of literature data enabled the identification of the most important operational parameters leading to N
2O emission in WWTPs: (i) low dissolved oxygen concentration in the nitrification and denitrification stages, (ii) increased nitrite concentrations in both nitrification and denitrification stages, and (iii) low COD/N ratio in the denitrification stage. From the literature it remains unclear whether nitrifying or denitrifying microorganisms are the main source of N
2O emissions. Operational strategies to prevent N
2O emission from WWTPs are discussed and areas in which further research is urgently required are identified.
Nitric oxide (NO) has important functions in biology and atmospheric chemistry as a toxin, signaling molecule, ozone depleting agent and the precursor of the greenhouse gas nitrous oxide (N
O). ...Although NO is a potent oxidant, and was available on Earth earlier than oxygen, it is unclear whether NO can be used by microorganisms for growth. Anaerobic ammonium-oxidizing (anammox) bacteria couple nitrite reduction to ammonium oxidation with NO and hydrazine as intermediates, and produce N
and nitrate. Here, we show that the anammox bacterium Kuenenia stuttgartiensis is able to grow in the absence of nitrite by coupling ammonium oxidation to NO reduction, and produce only N
. Under these growth conditions, the transcription of proteins necessary for NO generation is downregulated. Our work has potential implications in the control of N
O and NO emissions from natural and manmade ecosystems, where anammox bacteria contribute significantly to N
release to the atmosphere. We hypothesize that microbial NO-dependent ammonium oxidation may have existed on early Earth.
Paddy fields are a significant source of methane and contribute up to 20% of total methane emissions from wetland ecosystems. These inundated, anoxic soils featuring abundant nitrogen compounds and ...methane are an ideal niche for nitrate-dependent anaerobic methanotrophs. After 2 years of enrichment with a continuous supply of methane and nitrate as the sole electron donor and acceptor, a stable enrichment dominated by ‘
Candidatus
Methanoperedens nitroreducens’ archaea and ‘
Candidatus
Methylomirabilis oxyfera’ NC10 phylum bacteria was achieved. In this community, the methanotrophic archaea supplied the NC10 phylum bacteria with the necessary nitrite through nitrate reduction coupled to methane oxidation. The results of qPCR quantification of 16S ribosomal RNA (rRNA) gene copies, analysis of metagenomic 16S rRNA reads, and fluorescence in situ hybridization (FISH) correlated well and showed that after 2 years, ‘
Candidatus
Methanoperedens nitroreducens’ had the highest abundance of (2.2 ± 0.4 × 10
8
) 16S rRNA copies per milliliter and constituted approximately 22% of the total microbial community. Phylogenetic analysis showed that the 16S rRNA genes of the dominant microorganisms clustered with previously described ‘
Candidatus
Methanoperedens nitroreducens ANME2D’ (96% identity) and ‘
Candidatus
Methylomirabilis oxyfera’ (99% identity) strains. The pooled metagenomic sequences resulted in a high-quality draft genome assembly of ‘
Candidatus
Methanoperedens nitroreducens Vercelli’ that contained all key functional genes for the reverse methanogenesis pathway and nitrate reduction. The diagnostic
mcrA
gene was 96% similar to ‘
Candidatus
Methanoperedens nitroreducens ANME2D’ (WP_048089615.1) at the protein level. The ‘
Candidatus
Methylomirabilis oxyfera’ draft genome contained the marker genes
pmoCAB
,
mdh
, and
nirS
and putative NO dismutase genes. Whole-reactor anaerobic activity measurements with methane and nitrate revealed an average methane oxidation rate of 0.012 mmol/h/L, with cell-specific methane oxidation rates up to 0.57 fmol/cell/day for ‘
Candidatus
Methanoperedens nitroreducens’. In summary, this study describes the first enrichment and draft genome of methanotrophic archaea from paddy field soil, where these organisms can contribute significantly to the mitigation of methane emissions.
Summary
Growth of Methylacidiphilum fumariolicum SolV, an extremely acidophilic methanotrophic microbe isolated from an Italian volcanic mudpot, is shown to be strictly dependent on the presence of ...lanthanides, a group of rare earth elements (REEs) such as lanthanum (Ln), cerium (Ce), praseodymium (Pr) and neodymium (Nd). After fractionation of the bacterial cells and crystallization of the methanol dehydrogenase (MDH), it was shown that lanthanides were essential as cofactor in a homodimeric MDH comparable with one of the MDHs of Methylobacterium extorquens AM1. We hypothesize that the lanthanides provide superior catalytic properties to pyrroloquinoline quinone (PQQ)‐dependent MDH, which is a key enzyme for both methanotrophs and methylotrophs. Thus far, all isolated MxaF‐type MDHs contain calcium as a catalytic cofactor. The gene encoding the MDH of strain SolV was identified to be a xoxF‐ortholog, phylogenetically closely related to mxaF. Analysis of the protein structure and alignment of amino acids showed potential REE‐binding motifs in XoxF enzymes of many methylotrophs, suggesting that these may also be lanthanide‐dependent MDHs. Our findings will have major environmental implications as metagenome studies showed (lanthanide‐containing) XoxF‐type MDH is much more prominent in nature than MxaF‐type enzymes.
Anaerobic oxidation of methane (AOM) is crucial for controlling the emission of this potent greenhouse gas to the atmosphere. Nitrite-, nitrate-, and sulfate-dependent methane oxidation is ...well-documented, but AOM coupled to the reduction of oxidized metals has so far been demonstrated only in environmental samples. Here, using a freshwater enrichment culture, we show that archaea of the order Methanosarcinales, related to “Candidatus Methanoperedens nitroreducens,” couple the reduction of environmentally relevant forms of Fe3+ and Mn4+ to the oxidation of methane. We obtained an enrichment culture of these archaea under anaerobic, nitrate-reducing conditions with a continuous supply of methane. Via batch incubations using 13Cmethane, we demonstrated that soluble ferric iron (Fe3+, as Fe-citrate) and nanoparticulate forms of Fe3+ and Mn4+ supported methane-oxidizing activity. CO₂ and ferrous iron (Fe2+) were produced in stoichiometric amounts. Our study connects the previous finding of iron-dependent AOM to microorganisms detected in numerous habitats worldwide. Consequently, it enables a better understanding of the interaction between the biogeochemical cycles of iron and methane.
The hypothesis of an anammox hotspot in river riparian zones was put forward based on our investigation on freshwater ecotones for over 25 years and previous anammox research. Here we used a ...complementary array of methods including isotope-pairing technique, quantitative PCR assays, and 16S rRNA and hydrazine synthase gene (hzsB) clone libraries to document the spatiotemporal evidence for a high abundance zone of anammox bacteria in river riparian sediment with observed abundance of 1.3–12 × 106 (summer) and 1.4–20 × 108 (winter) hydrazine synthase gene copies g–1, which is the highest abundance in natural environments recorded so far. Meanwhile high anammox bacterial biodiversity were detected with ‘Brocadia’ and ‘Kuenenia’ dominating. However, the high anammox bacterial abundances were not related with high activities and contributions for nitrogen gas generation. The anammox activities ranged from 0.07 to 0.15 nmol N cm–3 h–1 (summer) to 1.0–2.6 nmol N cm–3 h–1 (winter) with high temporal heterogeneity. The retrieval of archaeal and bacterial amoA sequences indicated that nitrifying microbes might be the major source of nitrite for anammox bacteria in winter, while in summer the anaerobic nitrate reduction is more likely the main source. On the basis of 15N tracing technology, it was estimated that a total loss of 0.67–9.62 g N m–2 yr–1 is linked to anammox in the riparian zone while denitrification contributed 96.2–170.3 g N m–2 yr–1 in Pearl River riparian sediments.
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.
Anaerobic ammonium oxidation (anammox) bacteria contribute significantly to the global nitrogen cycle and play a major role in sustainable wastewater treatment. Anammox bacteria convert ammonium (NH
...) to dinitrogen gas (N
) using intracellular electron acceptors such as nitrite (NO
) or nitric oxide (NO). However, it is still unknown whether anammox bacteria have extracellular electron transfer (EET) capability with transfer of electrons to insoluble extracellular electron acceptors. Here we show that freshwater and marine anammox bacteria couple the oxidation of NH
with transfer of electrons to insoluble extracellular electron acceptors such as graphene oxide or electrodes in microbial electrolysis cells.
N-labeling experiments revealed that NH
was oxidized to N
via hydroxylamine (NH
OH) as intermediate, and comparative transcriptomics analysis revealed an alternative pathway for NH
oxidation with electrode as electron acceptor. Complete NH
oxidation to N
without accumulation of NO
and NO
was achieved in EET-dependent anammox. These findings are promising in the context of implementing EET-dependent anammox process for energy-efficient treatment of nitrogen.
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