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1.
ISME J ; 2021 Nov 06.
Artículo en Inglés | MEDLINE | ID: mdl-34743174

RESUMEN

The advance of metagenomics in combination with intricate cultivation approaches has facilitated the discovery of novel ammonia-, methane-, and other short-chain alkane-oxidizing microorganisms, indicating that our understanding of the microbial biodiversity within the biogeochemical nitrogen and carbon cycles still is incomplete. The in situ detection and phylogenetic identification of novel ammonia- and alkane-oxidizing bacteria remain challenging due to their naturally low abundances and difficulties in obtaining new isolates from complex samples. Here, we describe an activity-based protein profiling protocol allowing cultivation-independent unveiling of ammonia- and alkane-oxidizing bacteria. In this protocol, 1,7-octadiyne is used as a bifunctional enzyme probe that, in combination with a highly specific alkyne-azide cycloaddition reaction, enables the fluorescent or biotin labeling of cells harboring active ammonia and alkane monooxygenases. Biotinylation of these enzymes in combination with immunogold labeling revealed the subcellular localization of the tagged proteins, which corroborated expected enzyme targets in model strains. In addition, fluorescent labeling of cells harboring active ammonia or alkane monooxygenases provided a direct link of these functional lifestyles to phylogenetic identification when combined with fluorescence in situ hybridization. Furthermore, we show that this activity-based labeling protocol can be successfully coupled with fluorescence-activated cell sorting for the enrichment of nitrifiers and alkane-oxidizing bacteria from complex environmental samples, enabling the recovery of high-quality metagenome-assembled genomes. In conclusion, this study demonstrates a novel, functional tagging technique for the reliable detection, identification, and enrichment of ammonia- and alkane-oxidizing bacteria present in complex microbial communities.

2.
mBio ; 12(5): e0170821, 2021 Oct 26.
Artículo en Inglés | MEDLINE | ID: mdl-34544276

RESUMEN

The methane-oxidizing bacterium Methylacidimicrobium thermophilum AP8 thrives in acidic geothermal ecosystems that are characterized by high degassing of methane (CH4), H2, H2S, and by relatively high lanthanide concentrations. Lanthanides (atomic numbers 57 to 71) are essential in a variety of high-tech devices, including mobile phones. Remarkably, the same elements are actively taken up by methanotrophs/methylotrophs in a range of environments, since their XoxF-type methanol dehydrogenases require lanthanides as a metal cofactor. Lanthanide-dependent enzymes seem to prefer the lighter lanthanides (lanthanum, cerium, praseodymium, and neodymium), as slower methanotrophic/methylotrophic growth is observed in medium supplemented with only heavier lanthanides. Here, we purified XoxF1 from the thermoacidophilic methanotroph Methylacidimicrobium thermophilum AP8, which was grown in medium supplemented with neodymium as the sole lanthanide. The neodymium occupancy of the enzyme is 94.5% ± 2.0%, and through X-ray crystallography, we reveal that the structure of the active site shows interesting differences from the active sites of other methanol dehydrogenases, such as an additional aspartate residue in close proximity to the lanthanide. Nd-XoxF1 oxidizes methanol at a maximum rate of metabolism (Vmax) of 0.15 ± 0.01 µmol · min-1 · mg protein-1 and an affinity constant (Km) of 1.4 ± 0.6 µM. The structural analysis of this neodymium-containing XoxF1-type methanol dehydrogenase will expand our knowledge in the exciting new field of lanthanide biochemistry. IMPORTANCE Lanthanides comprise a group of 15 elements with atomic numbers 57 to 71 that are essential in a variety of high-tech devices, such as mobile phones, but were considered biologically inert for a long time. The biological relevance of lanthanides became evident when the acidophilic methanotroph Methylacidiphilum fumariolicum SolV, isolated from a volcanic mud pot, could only grow when lanthanides were supplied to the growth medium. We expanded knowledge in the exciting and rapidly developing field of lanthanide biochemistry by the purification and characterization of a neodymium-containing methanol dehydrogenase from a thermoacidophilic methanotroph.

3.
Appl Microbiol Biotechnol ; 105(16-17): 6515-6527, 2021 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-34423412

RESUMEN

Pharmaceuticals find their way to the aquatic environment via wastewater treatment plants (WWTPs). Biotransformation plays an important role in mitigating environmental risks; however, a mechanistic understanding of involved processes is limited. The aim of this study was to evaluate potential relationships between first-order biotransformation rate constants (kb) of nine pharmaceuticals and initial concentration of the selected compounds, and sampling season of the used activated sludge inocula. Four-day bottle experiments were performed with activated sludge from WWTP Groesbeek (The Netherlands) of two different seasons, summer and winter, spiked with two environmentally relevant concentrations (3 and 30 nM) of pharmaceuticals. Concentrations of the compounds were measured by LC-MS/MS, microbial community composition was assessed by 16S rRNA gene amplicon sequencing, and kb values were calculated. The biodegradable pharmaceuticals were acetaminophen, metformin, metoprolol, terbutaline, and phenazone (ranked from high to low biotransformation rates). Carbamazepine, diatrizoic acid, diclofenac, and fluoxetine were not converted. Summer and winter inocula did not show significant differences in microbial community composition, but resulted in a slightly different kb for some pharmaceuticals. Likely microbial activity was responsible instead of community composition. In the same inoculum, different kb values were measured, depending on initial concentration. In general, biodegradable compounds had a higher kb when the initial concentration was higher. This demonstrates that Michealis-Menten kinetic theory has shortcomings for some pharmaceuticals at low, environmentally relevant concentrations and that the pharmaceutical concentration should be taken into account when measuring the kb in order to reliably predict the fate of pharmaceuticals in the WWTP. KEY POINTS: • Biotransformation and sorption of pharmaceuticals were assessed in activated sludge. • Higher initial concentrations resulted in higher biotransformation rate constants for biodegradable pharmaceuticals. • Summer and winter inocula produced slightly different biotransformation rate constants although microbial community composition did not significantly change.


Asunto(s)
Preparaciones Farmacéuticas , Contaminantes Químicos del Agua , Biotransformación , Cromatografía Liquida , ARN Ribosómico 16S/genética , Aguas del Alcantarillado , Espectrometría de Masas en Tándem , Contaminantes Químicos del Agua/análisis
4.
mSystems ; 6(4): e0017321, 2021 Aug 31.
Artículo en Inglés | MEDLINE | ID: mdl-34402644

RESUMEN

Nitrite-oxidizing bacteria belonging to the genus Nitrospira mediate a key step in nitrification and play important roles in the biogeochemical nitrogen cycle and wastewater treatment. While these organisms have recently been shown to exhibit metabolic flexibility beyond their chemolithoautotrophic lifestyle, including the use of simple organic compounds to fuel their energy metabolism, the metabolic networks controlling their autotrophic and mixotrophic growth remain poorly understood. Here, we reconstructed a genome-scale metabolic model for Nitrospira moscoviensis (iNmo686) and used flux balance analysis to evaluate the metabolic networks controlling autotrophic and formatotrophic growth on nitrite and formate, respectively. Subsequently, proteomic analysis and [13C]bicarbonate and [13C]formate tracer experiments coupled to metabolomic analysis were performed to experimentally validate model predictions. Our findings corroborate that N. moscoviensis uses the reductive tricarboxylic acid cycle for CO2 fixation, and we also show that N. moscoviensis can indirectly use formate as a carbon source by oxidizing it first to CO2 followed by reassimilation, rather than direct incorporation via the reductive glycine pathway. Our study offers the first measurements of Nitrospira's in vivo central carbon metabolism and provides a quantitative tool that can be used for understanding and predicting their metabolic processes. IMPORTANCE Nitrospira spp. are globally abundant nitrifying bacteria in soil and aquatic ecosystems and in wastewater treatment plants, where they control the oxidation of nitrite to nitrate. Despite their critical contribution to nitrogen cycling across diverse environments, detailed understanding of their metabolic network and prediction of their function under different environmental conditions remains a major challenge. Here, we provide the first constraint-based metabolic model of Nitrospira moscoviensis representing the ubiquitous Nitrospira lineage II and subsequently validate this model using proteomics and 13C-tracers combined with intracellular metabolomic analysis. The resulting genome-scale model will serve as a knowledge base of Nitrospira metabolism and lays the foundation for quantitative systems biology studies of these globally important nitrite-oxidizing bacteria.

5.
Environ Microbiol Rep ; 13(6): 841-851, 2021 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-34374217

RESUMEN

Denitrifying Betaproteobacteria play a key role in the anaerobic degradation of monoaromatic hydrocarbons. We performed a multi-omics study to better understand the metabolism of the representative organism Georgfuchsia toluolica strain G5G6 known as a strict anaerobe coupling toluene oxidation with dissimilatory nitrate and Fe(III) reduction. Despite the genomic potential for degradation of different carbon sources, we did not find sugar or organic acid transporters, in line with the inability of strain G5G6 to use these substrates. Using a proteomics analysis, we detected proteins of fumarate-dependent toluene activation, membrane-bound nitrate reductase, and key components of the metal-reducing (Mtr) pathway under both nitrate- and Fe(III)-reducing conditions. High abundance of the multiheme cytochrome MtrC implied that a porin-cytochrome complex was used for respiratory Fe(III) reduction. Remarkably, strain G5G6 contains a full set of genes for aerobic toluene degradation, and we detected enzymes of aerobic toluene degradation under both nitrate- and Fe(III)-reducing conditions. We further detected an ATP-dependent benzoyl-CoA reductase, reactive oxygen species detoxification proteins, and cytochrome c oxidase indicating a facultative anaerobic lifestyle of strain G5G6. Correspondingly, we found diffusion through the septa a substantial source of oxygen in the cultures enabling concurrent aerobic and anaerobic toluene degradation by strain G5G6.

6.
Nat Microbiol ; 6(9): 1129-1139, 2021 09.
Artículo en Inglés | MEDLINE | ID: mdl-34267357

RESUMEN

Nitrate is an abundant nutrient and electron acceptor throughout Earth's biosphere. Virtually all nitrate in nature is produced by the oxidation of nitrite by the nitrite oxidoreductase (NXR) multiprotein complex. NXR is a crucial enzyme in the global biological nitrogen cycle, and is found in nitrite-oxidizing bacteria (including comammox organisms), which generate the bulk of the nitrate in the environment, and in anaerobic ammonium-oxidizing (anammox) bacteria which produce half of the dinitrogen gas in our atmosphere. However, despite its central role in biology and decades of intense study, no structural information on NXR is available. Here, we present a structural and biochemical analysis of the NXR from the anammox bacterium Kuenenia stuttgartiensis, integrating X-ray crystallography, cryo-electron tomography, helical reconstruction cryo-electron microscopy, interaction and reconstitution studies and enzyme kinetics. We find that NXR catalyses both nitrite oxidation and nitrate reduction, and show that in the cell, NXR is arranged in tubules several hundred nanometres long. We reveal the tubule architecture and show that tubule formation is induced by a previously unidentified, haem-containing subunit, NXR-T. The results also reveal unexpected features in the active site of the enzyme, an unusual cofactor coordination in the protein's electron transport chain, and elucidate the electron transfer pathways within the complex.


Asunto(s)
Bacterias/enzimología , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Oxidorreductasas/química , Oxidorreductasas/metabolismo , Bacterias/química , Bacterias/genética , Proteínas Bacterianas/genética , Dominio Catalítico , Microscopía por Crioelectrón , Cristalografía por Rayos X , Cinética , Complejos Multiproteicos/química , Complejos Multiproteicos/genética , Complejos Multiproteicos/metabolismo , Nitratos/metabolismo , Nitritos/metabolismo , Oxidación-Reducción , Oxidorreductasas/genética
7.
ISME J ; 2021 Jun 18.
Artículo en Inglés | MEDLINE | ID: mdl-34145392

RESUMEN

Methane-generating archaea drive the final step in anaerobic organic compound mineralization and dictate the carbon flow of Earth's diverse anoxic ecosystems in the absence of inorganic electron acceptors. Although such Archaea were presumed to be restricted to life on simple compounds like hydrogen (H2), acetate or methanol, an archaeon, Methermicoccus shengliensis, was recently found to convert methoxylated aromatic compounds to methane. Methoxylated aromatic compounds are important components of lignin and coal, and are present in most subsurface sediments. Despite the novelty of such a methoxydotrophic archaeon its metabolism has not yet been explored. In this study, transcriptomics and proteomics reveal that under methoxydotrophic growth M. shengliensis expresses an O-demethylation/methyltransferase system related to the one used by acetogenic bacteria. Enzymatic assays provide evidence for a two step-mechanisms in which the methyl-group from the methoxy compound is (1) transferred on cobalamin and (2) further transferred on the C1-carrier tetrahydromethanopterin, a mechanism distinct from conventional methanogenic methyl-transfer systems which use coenzyme M as final acceptor. We further hypothesize that this likely leads to an atypical use of the methanogenesis pathway that derives cellular energy from methyl transfer (Mtr) rather than electron transfer (F420H2 re-oxidation) as found for methylotrophic methanogenesis.

8.
Microb Biotechnol ; 14(4): 1707-1721, 2021 07.
Artículo en Inglés | MEDLINE | ID: mdl-34132479

RESUMEN

Pharmaceuticals are often not fully removed in wastewater treatment plants (WWTPs) and are thus being detected at trace levels in water bodies all over the world posing a risk to numerous organisms. These organic micropollutants (OMPs) reach WWTPs at concentrations sometimes too low to serve as growth substrate for microorganisms; thus, co-metabolism is thought to be the main conversion mechanism. In this study, the microbial removal of six pharmaceuticals was investigated in a membrane bioreactor at increasing concentrations (4-800 nM) of the compounds and using three different hydraulic retention times (HRT; 1, 3.5 and 5 days). The bioreactor was inoculated with activated sludge from a municipal WWTP and fed with ammonium, acetate and methanol as main growth substrates to mimic co-metabolism. Each pharmaceutical had a different average removal efficiency: acetaminophen (100%) > fluoxetine (50%) > metoprolol (25%) > diclofenac (20%) > metformin (15%) > carbamazepine (10%). Higher pharmaceutical influent concentrations proportionally increased the removal rate of each compound, but surprisingly not the removal percentage. Furthermore, only metformin removal improved to 80-100% when HRT or biomass concentration was increased. Microbial community changes were followed with 16S rRNA gene amplicon sequencing in response to the increment of pharmaceutical concentration: Nitrospirae and Planctomycetes 16S rRNA relative gene abundance decreased, whereas Acidobacteria and Bacteroidetes increased. Remarkably, the Dokdonella genus, previously implicated in acetaminophen metabolism, showed a 30-fold increase in abundance at the highest concentration of pharmaceuticals applied. Taken together, these results suggest that the incomplete removal of most pharmaceutical compounds in WWTPs is dependent on neither concentration nor reaction time. Accordingly, we propose a chemical equilibrium or a growth substrate limitation as the responsible mechanisms of the incomplete removal. Finally, Dokdonella could be the main acetaminophen degrader under activated sludge conditions, and non-antibiotic pharmaceuticals might still be toxic to relevant WWTP bacteria.


Asunto(s)
Preparaciones Farmacéuticas , Contaminantes Químicos del Agua , Reactores Biológicos , ARN Ribosómico 16S/genética , Tiempo de Reacción , Aguas del Alcantarillado , Eliminación de Residuos Líquidos , Aguas Residuales/análisis , Contaminantes Químicos del Agua/análisis
9.
Front Microbiol ; 12: 666929, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-34093485

RESUMEN

Verrucomicrobial methanotrophs are a group of aerobic bacteria isolated from volcanic environments. They are acidophiles, characterized by the presence of a particulate methane monooxygenase (pMMO) and a XoxF-type methanol dehydrogenase (MDH). Metagenomic analysis of DNA extracted from the soil of Favara Grande, a geothermal area on Pantelleria Island, Italy, revealed the presence of two verrucomicrobial Metagenome Assembled Genomes (MAGs). One of these MAGs did not phylogenetically classify within any existing genus. After extensive analysis of the MAG, we propose the name of "Candidatus Methylacidithermus pantelleriae" PQ17 gen. nov. sp. nov. The MAG consisted of 2,466,655 bp, 71 contigs and 3,127 predicted coding sequences. Completeness was found at 98.6% and contamination at 1.3%. Genes encoding the pMMO and XoxF-MDH were identified. Inorganic carbon fixation might use the Calvin-Benson-Bassham cycle since all genes were identified. The serine and ribulose monophosphate pathways were incomplete. The detoxification of formaldehyde could follow the tetrahydrofolate pathway. Furthermore, "Ca. Methylacidithermus pantelleriae" might be capable of nitric oxide reduction but genes for dissimilatory nitrate reduction and nitrogen fixation were not identified. Unlike other verrucomicrobial methanotrophs, genes encoding for enzymes involved in hydrogen oxidation could not be found. In conclusion, the discovery of this new MAG expands the diversity and metabolism of verrucomicrobial methanotrophs.

10.
Front Microbiol ; 12: 652486, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33981290

RESUMEN

Degraded peatlands are often rewetted to prevent oxidation of the peat, which reduces CO2 emission. However, the created anoxic conditions will boost methane (CH4) production and thus emission. Here, we show that submerged Sphagnum peat mosses in rewetted-submerged peatlands can reduce CH4 emission from peatlands with 93%. We were able to mimic the field situation in the laboratory by using a novel mesocosm set-up. By combining these with 16S rRNA gene amplicon sequencing and qPCR analysis of the pmoA and mmoX genes, we showed that submerged Sphagnum mosses act as a niche for CH4 oxidizing bacteria. The tight association between Sphagnum peat mosses and methane oxidizing bacteria (MOB) significantly reduces CH4 emissions by peatlands and can be studied in more detail in the mesocosm setup developed in this study.

11.
Appl Environ Microbiol ; 87(13): e0004321, 2021 06 11.
Artículo en Inglés | MEDLINE | ID: mdl-33893122

RESUMEN

Methane and ammonia have to be removed from wastewater treatment effluent in order to discharge it to receiving water bodies. A potential solution for this is a combination of simultaneous ammonia and methane oxidation by anaerobic ammonia oxidation (anammox) bacteria and nitrite/nitrate-dependent anaerobic methane oxidation (N-damo) microorganisms. When applied, these microorganisms will be exposed to oxygen, but little is known about the effect of a low concentration of oxygen on a culture containing these microorganisms. In this study, a stable coculture containing anammox and N-damo microorganisms in a laboratory scale bioreactor was established under oxygen limitation. Membrane inlet mass spectrometry (MIMS) was used to directly measure the in situ simultaneous activity of N-damo, anammox, and aerobic ammonia-oxidizing microorganisms. In addition, batch tests revealed that the bioreactor also harbored aerobic methanotrophs and anaerobic methanogens. Together with fluorescence in situ hybridization (FISH) analysis and metagenomics, these results indicate that the combination of N-damo and anammox activity under the continuous supply of limiting oxygen concentrations is feasible and can be implemented for the removal of methane and ammonia from anaerobic digester effluents. IMPORTANCE Nitrogen in wastewater leads to eutrophication of the receiving water bodies, and methane is a potent greenhouse gas; it is therefore important that these are removed from wastewater. A potential solution for the simultaneous removal of nitrogenous compounds and methane is the application of a combination of nitrite/nitrate-dependent methane oxidation (N-damo) and anaerobic ammonia oxidation (annamox). In order to do so, it is important to investigate the effect of oxygen on these two anaerobic processes. In this study, we investigate the effect of a continuous oxygen supply on the activity of an anaerobic methane- and ammonia-oxidizing coculture. The findings presented in this study are important for the potential application of these two microbial processes in wastewater treatment.


Asunto(s)
Amoníaco/metabolismo , Metano/metabolismo , Oxígeno , Contaminantes Químicos del Agua/metabolismo , Purificación del Agua/métodos , Aerobiosis , Anaerobiosis , Archaea/metabolismo , Bacterias/metabolismo , Reactores Biológicos , Oxidación-Reducción
12.
Environ Microbiol ; 23(7): 4017-4033, 2021 07.
Artículo en Inglés | MEDLINE | ID: mdl-33913565

RESUMEN

Methoxylated aromatic compounds (MACs) are important components of lignin found in significant amounts in the subsurface. Recently, the methanogenic archaeon Methermicoccus shengliensis was shown to be able to use a variety of MACs during methoxydotrophic growth. After a molecular survey, we found that the hyperthermophilic non-methanogenic archaeon Archaeoglobus fulgidus also encodes genes for a bacterial-like demethoxylation system. In this study, we performed growth and metabolite analysis, and used transcriptomics to investigate the response of A. fulgidus during growth on MACs in comparison to growth on lactate. We observed that A. fulgidus converts MACs to their hydroxylated derivatives with CO2 as the main product and sulfate as electron acceptor. Furthermore, we could show that MACs improve the growth of A. fulgidus in the presence of organic substrates such as lactate. We also found evidence that other archaea such as Bathyarchaeota, Lokiarchaeota, Verstraetearchaeota, Korarchaeota, Helarchaeota and Nezhaarchaeota encode a demethoxylation system. In summary, we here describe the first non-methanogenic archaeon with the ability to grow on MACs indicating that methoxydotrophic archaea might play a so far underestimated role in the global carbon cycle.


Asunto(s)
Archaea , Archaeoglobus fulgidus , Methanosarcinales , Oxidación-Reducción , Sulfatos
13.
J Biol Chem ; 296: 100476, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33652023

RESUMEN

The hydroxylamine oxidoreductase (HAO) family consists of octaheme proteins that harbor seven bis-His ligated electron-transferring hemes and one 5-coordinate catalytic heme with His axial ligation. Oxidative HAOs have a homotrimeric configuration with the monomers covalently attached to each other via a unique double cross-link between a Tyr residue and the catalytic heme moiety of an adjacent subunit. This cross-linked active site heme, termed the P460 cofactor, has been hypothesized to modulate enzyme reactivity toward oxidative catalysis. Conversely, the absence of this cross-link is predicted to favor reductive catalysis. However, this prediction has not been directly tested. In this study, an HAO homolog that lacks the heme-Tyr cross-link (HAOr) was purified to homogeneity from the nitrite-dependent anaerobic ammonium-oxidizing (anammox) bacterium Kuenenia stuttgartiensis, and its catalytic and spectroscopic properties were assessed. We show that HAOr reduced nitrite to nitric oxide and also reduced nitric oxide and hydroxylamine as nonphysiological substrates. In contrast, HAOr was not able to oxidize hydroxylamine or hydrazine supporting the notion that cross-link-deficient HAO enzymes are reductases. Compared with oxidative HAOs, we found that HAOr harbors an active site heme with a higher (at least 80 mV) midpoint potential and a much lower degree of porphyrin ruffling. Based on the physiology of anammox bacteria and our results, we propose that HAOr reduces nitrite to nitric oxide in vivo, providing anammox bacteria with NO, which they use to activate ammonium in the absence of oxygen.


Asunto(s)
Oxidorreductasas/química , Oxidorreductasas/metabolismo , Planctomycetales/metabolismo , Compuestos de Amonio/metabolismo , Bacterias/metabolismo , Catálisis , Dominio Catalítico , Transporte de Electrón/fisiología , Hemo/metabolismo , Hidrazinas/química , Hidroxilamina/química , Hidroxilaminas/química , Óxido Nítrico/metabolismo , Nitritos/metabolismo , Oxidación-Reducción , Tirosina/química , Tirosina/metabolismo
14.
Microbiologyopen ; 10(1): e1175, 2021 01.
Artículo en Inglés | MEDLINE | ID: mdl-33650794

RESUMEN

Microbial methane oxidation is a major biofilter preventing larger emissions of this powerful greenhouse gas from marine coastal areas into the atmosphere. In these zones, various electron acceptors such as sulfate, metal oxides, nitrate, or oxygen can be used. However, the key microbial players and mechanisms of methane oxidation are poorly understood. In this study, we inoculated a bioreactor with methane- and iron-rich sediments from the Bothnian Sea to investigate microbial methane and iron cycling under low oxygen concentrations. Using metagenomics, we investigated shifts in microbial community composition after approximately 2.5 years of bioreactor operation. Marker genes for methane and iron cycling, as well as respiratory and fermentative metabolism, were identified and used to infer putative microbial metabolism. Metagenome-assembled genomes representing novel Verrucomicrobia, Bacteroidetes, and Krumholzibacteria were recovered and revealed a potential for methane oxidation, organic matter degradation, and iron cycling, respectively. This work brings new hypotheses on the identity and metabolic versatility of microorganisms that may be members of such functional guilds in coastal marine sediments and highlights that microorganisms potentially composing the methane biofilter in these sediments may be more diverse than previously appreciated.


Asunto(s)
Bacteroidetes/metabolismo , Reactores Biológicos/microbiología , Sedimentos Geológicos/microbiología , Hierro/metabolismo , Metano/metabolismo , Verrucomicrobia/metabolismo , Anaerobiosis/fisiología , Bacteroidetes/crecimiento & desarrollo , Finlandia , Microbiota , Océanos y Mares , Oxidación-Reducción , Oxígeno/metabolismo , Suecia , Verrucomicrobia/crecimiento & desarrollo
15.
Front Microbiol ; 12: 637762, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33643272

RESUMEN

The Favara Grande is a geothermal area located on Pantelleria Island, Italy. The area is characterized high temperatures in the top layer of the soil (60°C), low pH (3-5) and hydrothermal gas emissions mainly composed of carbon dioxide (CO2), methane (CH4), and hydrogen (H2). These geothermal features may provide a suitable niche for the growth of chemolithotrophic thermoacidophiles, including the lanthanide-dependent methanotrophs of the phylum Verrucomicrobia. In this study, we started enrichment cultures inoculated with soil of the Favara Grande at 50 and 60°C with CH4 as energy source and medium containing sufficient lanthanides at pH 3 and 5. From these cultures, a verrucomicrobial methanotroph could be isolated via serial dilution and floating filters techniques. The genome of strain AP8 was sequenced and based on phylogenetic analysis we propose to name this new species Methylacidimicrobium thermophilum AP8. The transcriptome data at µmax (0.051 ± 0.001 h-1, doubling time ~14 h) of the new strain showed a high expression of the pmoCAB2 operon encoding the membrane-bound methane monooxygenase and of the gene xoxF1, encoding the lanthanide-dependent methanol dehydrogenase. A second pmoCAB operon and xoxF2 gene were not expressed. The physiology of strain AP8 was further investigated and revealed an optimal growth in a pH range of 3-5 at 50°C, representing the first thermophilic strain of the genus Methylacidimicrobium. Moreover, strain AP8 had a KS(app) for methane of 8 ± 1 µM. Beside methane, a type 1b [NiFe] hydrogenase enabled hydrogen oxidation at oxygen concentrations up to 1%. Taken together, our results expand the knowledge on the characteristics and adaptations of verrucomicrobial methanotrophs in hydrothermal environments and add a new thermophilic strain to the genus Methylacidimicrobium.

16.
Front Microbiol ; 12: 631621, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33679659

RESUMEN

Large amounts of methane, a potent greenhouse gas, are produced in anoxic sediments by methanogenic archaea. Nonetheless, over 90% of the produced methane is oxidized via sulfate-dependent anaerobic oxidation of methane (S-AOM) in the sulfate-methane transition zone (SMTZ) by consortia of anaerobic methane-oxidizing archaea (ANME) and sulfate-reducing bacteria (SRB). Coastal systems account for the majority of total marine methane emissions and typically have lower sulfate concentrations, hence S-AOM is less significant. However, alternative electron acceptors such as metal oxides or nitrate could be used for AOM instead of sulfate. The availability of electron acceptors is determined by the redox zonation in the sediment, which may vary due to changes in oxygen availability and the type and rate of organic matter inputs. Additionally, eutrophication and climate change can affect the microbiome, biogeochemical zonation, and methane cycling in coastal sediments. This review summarizes the current knowledge on the processes and microorganisms involved in methane cycling in coastal sediments and the factors influencing methane emissions from these systems. In eutrophic coastal areas, organic matter inputs are a key driver of bottom water hypoxia. Global warming can reduce the solubility of oxygen in surface waters, enhancing water column stratification, increasing primary production, and favoring methanogenesis. ANME are notoriously slow growers and may not be able to effectively oxidize methane upon rapid sedimentation and shoaling of the SMTZ. In such settings, ANME-2d (Methanoperedenaceae) and ANME-2a may couple iron- and/or manganese reduction to AOM, while ANME-2d and NC10 bacteria (Methylomirabilota) could couple AOM to nitrate or nitrite reduction. Ultimately, methane may be oxidized by aerobic methanotrophs in the upper millimeters of the sediment or in the water column. The role of these processes in mitigating methane emissions from eutrophic coastal sediments, including the exact pathways and microorganisms involved, are still underexplored, and factors controlling these processes are unclear. Further studies are needed in order to understand the factors driving methane-cycling pathways and to identify the responsible microorganisms. Integration of the knowledge on microbial pathways and geochemical processes is expected to lead to more accurate predictions of methane emissions from coastal zones in the future.

17.
Environ Sci Technol ; 55(8): 4573-4584, 2021 04 20.
Artículo en Inglés | MEDLINE | ID: mdl-33733744

RESUMEN

The discovery of complete ammonia oxidation (comammox) has altered our understanding of nitrification, which is the rate-limiting process in the global nitrogen cycle. However, understanding the ecological role of comammox or its contribution to nitrification in both natural and artificial ecosystems is still in its infancy. Here, we investigated the community distribution and function of comammox bacteria in riparian ecosystems and analyzed interactions between comammox and other nitrogen cycling microorganisms. The comammox bacterial abundance and rate were higher in summer than in winter and higher in nonrhizosphere soils than in the rhizosphere. Fringe soils in the riparian zone comprise a comammox hotspot, where the abundance (2.58 × 108 copies g-1) and rate (0.86 mg N kg-1 d-1) of comammox were not only higher than at other sampling sites but also higher than those of other ammonia oxidation processes. The comammox rate correlated significantly positively with relative abundance of the comammox species Candidatus Nitrospira nitrificans but not with that of the species Candidatus Nitrospira nitrosa. Analysis of comammox interaction with other ammonia-oxidizing processes revealed ammonia-oxidizing archaea to dominate interface soils, comammox to dominate in fringe soils, and anaerobic ammonium oxidation (anammox) to dominate in interface sediments of the riparian zone. These results indicate that comammox may constitute an important and currently underestimated process of microbial nitrification in riparian zone ecosystems.


Asunto(s)
Amoníaco , Ecosistema , Archaea , Nitrificación , Oxidación-Reducción , Filogenia
18.
FEMS Microbiol Rev ; 45(5)2021 Sep 08.
Artículo en Inglés | MEDLINE | ID: mdl-33524112

RESUMEN

Methanotrophs are an important group of microorganisms that counteract methane emissions to the atmosphere. Methane-oxidising bacteria of the Alpha- and Gammaproteobacteria have been studied for over a century, while methanotrophs of the phylum Verrucomicrobia are a more recent discovery. Verrucomicrobial methanotrophs are extremophiles that live in very acidic geothermal ecosystems. Currently, more than a dozen strains have been isolated, belonging to the genera Methylacidiphilum and Methylacidimicrobium. Initially, these methanotrophs were thought to be metabolically confined. However, genomic analyses and physiological and biochemical experiments over the past years revealed that verrucomicrobial methanotrophs, as well as proteobacterial methanotrophs, are much more metabolically versatile than previously assumed. Several inorganic gases and other molecules present in acidic geothermal ecosystems can be utilised, such as methane, hydrogen gas, carbon dioxide, ammonium, nitrogen gas and perhaps also hydrogen sulfide. Verrucomicrobial methanotrophs could therefore represent key players in multiple volcanic nutrient cycles and in the mitigation of greenhouse gas emissions from geothermal ecosystems. Here, we summarise the current knowledge on verrucomicrobial methanotrophs with respect to their metabolic versatility and discuss the factors that determine their diversity in their natural environment. In addition, key metabolic, morphological and ecological characteristics of verrucomicrobial and proteobacterial methanotrophs are reviewed.

19.
Antonie Van Leeuwenhoek ; 114(3): 313-324, 2021 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-33566237

RESUMEN

The genus Methylobacter is considered an important and often dominant group of aerobic methane-oxidizing bacteria in many oxic ecosystems, where members of this genus contribute to the reduction of CH4 emissions. Metagenomic studies of the upper oxic layers of geothermal soils of the Favara Grande, Pantelleria, Italy, revealed the presence of various methane-oxidizing bacteria, and resulted in a near complete metagenome assembled genome (MAG) of an aerobic methanotroph, which was classified as a Methylobacter species. In this study, the Methylobacter sp. B2 MAG was used to investigate its metabolic potential and phylogenetic affiliation. The MAG has a size of 4,086,539 bp, consists of 134 contigs and 3955 genes were found, of which 3902 were protein coding genes. All genes for CH4 oxidation to CO2 were detected, including pmoCAB encoding particulate methane monooxygenase (pMMO) and xoxF encoding a methanol dehydrogenase. No gene encoding a formaldehyde dehydrogenase was present and the formaldehyde to formate conversion follows the tetrahydromethanopterin (H4MPT) pathway. "Ca. Methylobacter favarea" B2 uses the Ribulose-Mono-Phosphate (RuMP) pathway for carbon fixation. Analysis of the MAG indicates that Na+/H+ antiporters and the urease system might be important in the maintenance of pH homeostasis of this strain to cope with acidic conditions. So far, thermoacidophilic Methylobacter species have not been isolated, however this study indicates that members of the genus Methylobacter can be found in distinct ecosystems and their presence is not restricted to freshwater or marine sediments.


Asunto(s)
Methylococcaceae , Suelo , ADN Bacteriano , Ecosistema , Metano , Methylococcaceae/genética , Filogenia , ARN Ribosómico 16S/genética
20.
ISME J ; 15(3): 673-687, 2021 03.
Artículo en Inglés | MEDLINE | ID: mdl-33082573

RESUMEN

Anaerobic ammonium-oxidizing (anammox) bacteria mediate a key step in the biogeochemical nitrogen cycle and have been applied worldwide for the energy-efficient removal of nitrogen from wastewater. However, outside their core energy metabolism, little is known about the metabolic networks driving anammox bacterial anabolism and use of different carbon and energy substrates beyond genome-based predictions. Here, we experimentally resolved the central carbon metabolism of the anammox bacterium Candidatus 'Kuenenia stuttgartiensis' using time-series 13C and 2H isotope tracing, metabolomics, and isotopically nonstationary metabolic flux analysis. Our findings confirm predicted metabolic pathways used for CO2 fixation, central metabolism, and amino acid biosynthesis in K. stuttgartiensis, and reveal several instances where genomic predictions are not supported by in vivo metabolic fluxes. This includes the use of the oxidative branch of an incomplete tricarboxylic acid cycle for alpha-ketoglutarate biosynthesis, despite the genome not having an annotated citrate synthase. We also demonstrate that K. stuttgartiensis is able to directly assimilate extracellular formate via the Wood-Ljungdahl pathway instead of oxidizing it completely to CO2 followed by reassimilation. In contrast, our data suggest that K. stuttgartiensis is not capable of using acetate as a carbon or energy source in situ and that acetate oxidation occurred via the metabolic activity of a low-abundance microorganism in the bioreactor's side population. Together, these findings provide a foundation for understanding the carbon metabolism of anammox bacteria at a systems-level and will inform future studies aimed at elucidating factors governing their function and niche differentiation in natural and engineered ecosystems.


Asunto(s)
Crecimiento Quimioautotrófico , Ecosistema , Anaerobiosis , Procesos Autotróficos , Bacterias , Reactores Biológicos , Redes y Vías Metabólicas , Nitrógeno , Oxidación-Reducción
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