Gene-centered metagenome analysis of Vulcano Island soil (Aeolian archipelago, Italy) reveals diverse microbial key players in methane, hydrogen and sulfur cycles.

The Aeolian archipelago is known worldwide for its volcanic activity and hydrothermal emissions, of mainly carbon dioxide and hydrogen sulfide. Hydrogen, methane, and carbon monoxide are minor components of these emissions which together can feed large quantities of bacteria and archaea that do contribute to the removal of these notorious greenhouse gases. Here we analyzed the metagenome of samples taken from the Levante bay on Vulcano Island, Italy. Using a gene-centric approach, the hydrothermal vent community appeared to be dominated by Proteobacteria, and Sulfurimonas was the most abundant genus. Metabolic reconstructions highlight a prominent role of formaldehyde oxidation and the reverse TCA cycle in carbon fixation. [NiFe]-hydrogenases seemed to constitute the preferred strategy to oxidize H2, indicating that besides H2S, H2 could be an essential electron donor in this system. Moreover, the sulfur cycle analysis showed a high abundance and diversity of sulfate reduction genes underpinning the H2S production. This study covers the diversity and metabolic potential of the microbial soil community in Levante bay and adds to our understanding of the biogeochemistry of volcanic ecosystems.

Active coexistence of the novel gammaproteobacterial methanotroph 'Ca. Methylocalor cossyra' CH1 and verrucomicrobial methanotrophs in acidic, hot geothermal soil.

Terrestrial geothermal ecosystems are hostile habitats, characterized by large emissions of environmentally relevant gases such as CO2 , CH4 , H2 S and H2 . These conditions provide a niche for chemolithoautotrophic microorganisms. Methanotrophs of the phylum Verrucomicrobia, which inhabit these ecosystems, can utilize these gases and grow at pH levels below 1 and temperatures up to 65°C. In contrast, methanotrophs of the phylum Proteobacteria are primarily found in various moderate environments. Previously, novel verrucomicrobial methanotrophs were detected and isolated from the geothermal soil of the Favara Grande on the island of Pantelleria, Italy. The detection of pmoA genes, specific for verrucomicrobial and proteobacterial methanotrophs in this environment, and the partially overlapping pH and temperature growth ranges of these isolates suggest that these distinct phylogenetic groups could coexist in the environment. In this report, we present the isolation and characterization of a thermophilic and acid-tolerant gammaproteobacterial methanotroph (family Methylococcaceae) from the Favara Grande. This isolate grows at pH values ranging from 3.5 to 7.0 and temperatures from 35°C to 55°C, and diazotrophic growth was demonstrated. Its genome contains genes encoding particulate and soluble methane monooxygenases, XoxF- and MxaFI-type methanol dehydrogenases, and all enzymes of the Calvin cycle. For this novel genus and species, we propose the name 'Candidatus Methylocalor cossyra' CH1.

Identification and characterisation of a major outer membrane protein from Methylacidiphilum fumariolicum SolV.

The outer membrane (OM) protects Gram-negative bacteria against a hostile environment. The proteins embedded in the OM fulfil a number of tasks that are crucial to the bacterial cell. In this study, we identified and characterised a major outer membrane protein (WP_009059494) from Methylacidiphilum fumariolicum SolV. PRED-TMBB and AlphaFold2 predicted this protein to form a porin with a ß-barrel structure consisting of ten antiparallel ß-sheets and with a small amphipathic N-terminal α-helix in the periplasm. We purified soluble recombinant protein WP_009059494 from E. coli using Tris-HCl buffer with SDS. Antibodies were raised against two peptides in the two large extracellular loops of protein WP_009059494 and immunogold localisation showed this protein to be mainly present in the OM of strain SolV. In addition, this protein is tightly associated with the OM, and is resistant to extraction. Only a small amount can be isolated from the cell envelope using harsh conditions (SDS and boiling). Despite this resistance to extraction, WP_009059494 most likely is an outer membrane protein. A regular lattice could not be detected by negative staining TEM of strain SolV and isolated protein WP_009059494. Considering the specific ecological niche of strain SolV living in a geothermal environment with low pH and high temperatures, this major protein WP_009059494 may act as barrier to resist the extreme conditions found in its natural environment. In addition, we found an absence of the BamB, BamC and BamE proteins of the canonical BAM complex, in Methylacidiphilum and Methylacidimicrobium species. This suggests that these bacteria use a simple BAM complex for folding and transport of OM proteins.

Learning from nature: recovery of rare earth elements by the extremophilic bacterium Methylacidiphilum fumariolicum.

We present the extremophilic bacterium Methylacidiphilum fumariolicum SolV as a platform for the recovery of rare earth elements (REE). Strain SolV is able to selectively extract the light REE from artificial industrial waste sources, natural REE-containing and post-mining waters. Upscaling, different media composition and accumulation over several cycles were successfully implemented, underlining the potential for bio-recovery of REE.

Identification and characterization of an abundant lipoprotein from Methylacidiphilum fumariolicum SolV.

Bacterial lipoproteins are characterized by the presence of a conserved N-terminal lipid-modified cysteine residue that allows the hydrophilic protein to anchor into bacterial cell membranes. These lipoproteins play essential roles in a wide variety of physiological processes. Based on transcriptome analysis of the verrucomicrobial methanotroph Methylacidiphilum fumariolicum SolV, we identified a highly expressed lipoprotein, WP_009060351 (139 amino acids), in its genome. The first 86 amino acids are specific for the methanotrophic genera Methylacidiphilum and Methylacidmicrobium, while the last 53 amino acids are present only in lipoproteins of members from the phylum Verrucomicrobiota (Hedlund). Heterologous expression of WP_009060351 in Escherichia coli revealed a 25-kDa dimeric protein and a 60-kDa tetrameric protein. Immunoblotting showed that WP_009060351 was present in the total membrane protein and peptidoglycan fractions of M. fumariolicum SolV. The results suggest an involvement of lipoprotein WP_009060351 in the linkage between the outer membrane and the peptidoglycan.

Simultaneous sulfide and methane oxidation by an extremophile.

Hydrogen sulfide (H2S) and methane (CH4) are produced in anoxic environments through sulfate reduction and organic matter decomposition. Both gases diffuse upwards into oxic zones where aerobic methanotrophs mitigate CH4 emissions by oxidizing this potent greenhouse gas. Although methanotrophs in myriad environments encounter toxic H2S, it is virtually unknown how they are affected. Here, through extensive chemostat culturing we show that a single microorganism can oxidize CH4 and H2S simultaneously at equally high rates. By oxidizing H2S to elemental sulfur, the thermoacidophilic methanotroph Methylacidiphilum fumariolicum SolV alleviates the inhibitory effects of H2S on methanotrophy. Strain SolV adapts to increasing H2S by expressing a sulfide-insensitive ba3-type terminal oxidase and grows as chemolithoautotroph using H2S as sole energy source. Genomic surveys revealed putative sulfide-oxidizing enzymes in numerous methanotrophs, suggesting that H2S oxidation is much more widespread in methanotrophs than previously assumed, enabling them to connect carbon and sulfur cycles in novel ways.

"Candidatus Hydrogenisulfobacillus filiaventi" strain R50 gen. nov. sp. nov., a highly efficient producer of extracellular organic compounds from H2 and CO2.

Production of organic molecules is largely depending on fossil fuels. A sustainable alternative would be the synthesis of these compounds from CO2 and a cheap energy source, such as H2, CH4, NH3, CO, sulfur compounds or iron(II). Volcanic and geothermal areas are rich in CO2 and reduced inorganic gasses and therefore habitats where novel chemolithoautotrophic microorganisms for the synthesis of organic compounds could be discovered. Here we describe "Candidatus Hydrogenisulfobacillus filiaventi" R50 gen. nov., sp. nov., a thermoacidophilic, autotrophic H2-oxidizing microorganism, that fixed CO2 and excreted no less than 0.54 mol organic carbon per mole fixed CO2. Extensive metabolomics and NMR analyses revealed that Val, Ala and Ile are the most dominant form of excreted organic carbon while the aromatic amino acids Tyr and Phe, and Glu and Lys were present at much lower concentrations. In addition to these proteinogenic amino acids, the excreted carbon consisted of homoserine lactone, homoserine and an unidentified amino acid. The biological role of the excretion remains uncertain. In the laboratory, we noticed the production under high growth rates (0.034 h-1, doubling time of 20 h) in combination with O2-limitation, which will most likely not occur in the natural habitat of this strain. Nevertheless, this large production of extracellular organic molecules from CO2 may open possibilities to use chemolithoautotrophic microorganisms for the sustainable production of important biomolecules.

Minor Actinides Can Replace Essential Lanthanides in Bacterial Life.

Certain f-block elements-the lanthanides-have biological relevance in the context of methylotrophic bacteria. The respective strains incorporate these 4 f elements into the active site of one of their key metabolic enzymes, a lanthanide-dependent methanol dehydrogenase. In this study, we investigated whether actinides, the radioactive 5 f elements, can replace the essential 4 f elements in lanthanide-dependent bacterial metabolism. Growth studies with Methylacidiphilum fumariolicum SolV and the Methylobacterium extorquens AM1 ΔmxaF mutant demonstrate that americium and curium support growth in the absence of lanthanides. Moreover, strain SolV favors these actinides over late lanthanides when presented with a mixture of equal amounts of lanthanides together with americium and curium. Our combined in vivo and in vitro results establish that methylotrophic bacteria can utilize actinides instead of lanthanides to sustain their one-carbon metabolism if they possess the correct size and a +III oxidation state.

Optical Oxygen Sensors Show Reversible Cross-Talk and/or Degradation in the Presence of Nitrogen Dioxide.

A variety of luminescent dyes including the most common indicators for optical oxygen sensors were investigated in regard to their stability and photophysical properties in the presence of nitrogen dioxide. The dyes were immobilized in polystyrene and subjected to NO2 concentrations from 40 to 5500 ppm. The majority of dyes show fast degradation of optical properties due to the reaction with NO2. The class of phosphorescent metalloporphyrins shows the highest resistance against nitrogen dioxide. Among them, palladium(II) and platinum(II) complexes of octasubstituted sulfonylated benzoporphyrins are identified as the most stable dyes with almost no decomposition in the presence of NO2. The phosphorescence of these dyes is reversibly quenched by nitrogen dioxide. Immobilized in various polymeric matrices, the sulfonylated Pt(II) benzoporphyrin demonstrates about one order of magnitude more efficient quenching by NO2 than by molecular oxygen. Our study demonstrates that virtually all commercially available and reported optical oxygen sensors are likely to show either irreversible decomposition in the presence of nitrogen dioxide or reversible luminescence quenching. They should be used with extreme caution if NO2 is present in relatively high concentrations or it may be generated from other species such as nitric oxide. As an important consequence of nearly anoxic systems, production of nitrogen dioxide or nitric oxide may be therefore erroneously interpreted as an increase in oxygen concentration.

A perspective on the role of lanthanides in biology: Discovery, open questions and possible applications.

Because of their use in high technologies like computers, smartphones and renewable energy applications, lanthanides (belonging to the group of rare earth elements) are essential for our daily lives. A range of applications in medicine and biochemical research made use of their photo-physical properties. The discovery of a biological role for lanthanides has boosted research in this new field. Several methanotrophs and methylotrophs are strictly dependent on the presence of lanthanides in the growth medium while others show a regulatory response. After the first demonstration of a lanthanide in the active site of the XoxF-type pyrroloquinoline quinone methanol dehydrogenases, follow-up studies showed the same for other pyrroloquinoline quinone-containing enzymes. In addition, research focused on the effect of lanthanides on regulation of gene expression and uptake mechanism into bacterial cells. This review briefly describes the discovery of the role of lanthanides in biology and focuses on open questions in biological lanthanide research and possible application of lanthanide-containing bacteria and enzymes in recovery of these special elements.

Studies of pyrroloquinoline quinone species in solution and in lanthanide-dependent methanol dehydrogenases.

Pyrroloquinoline quinone (PQQ) is a redox cofactor in calcium- and lanthanide-dependent alcohol dehydrogenases that has been known and studied for over 40 years. Despite its long history, many questions regarding its fluorescence properties, speciation in solution and in the active site of alcohol dehydrogenase remain open. Here we investigate the effects of pH and temperature on the distribution of different PQQ species (H3PQQ to PQQ3- in addition to water adducts and in complex with lanthanides) with NMR and UV-Vis spectroscopy as well as time-resolved laser-induced fluorescence spectroscopy (TRLFS). Using a europium derivative from a new, recently-discovered class of lanthanide-dependent methanol dehydrogenase (MDH) enzymes, we utilized two techniques to monitor Ln binding to the active sites of these enzymes. Employing TRLFS, we were able to follow Eu(III) binding directly to the active site of MDH using its luminescence and could quantify three Eu(III) states: Eu(III) in the active site of MDH, but also in solution as PQQ-bound Eu(III) and in the aquo-ion form. Additionally, we used the antenna effect to study PQQ and simultaneously Eu(III) in the active site.

Methanethiol Consumption and Hydrogen Sulfide Production by the Thermoacidophilic Methanotroph Methylacidiphilum fumariolicum SolV.

Methanotrophs aerobically oxidize methane to carbon dioxide to make a living and are known to degrade various other short chain carbon compounds as well. Volatile organic sulfur compounds such as methanethiol (CH3SH) are important intermediates in the sulfur cycle. Although volatile organic sulfur compounds co-occur with methane in various environments, little is known about how these compounds affect methanotrophy. The enzyme methanethiol oxidase catalyzing the oxidation of methanethiol has been known for decades, but only recently the mtoX gene encoding this enzyme was identified in a methylotrophic bacterium. The presence of a homologous gene in verrucomicrobial methanotrophs prompted us to examine how methanotrophs cope with methanethiol. Here, we show that the verrucomicrobial methanotroph Methylacidiphilum fumariolicum SolV consumes methanethiol and produces H2S, which is concurrently oxidized. Consumption of methanethiol is required since methanethiol inhibits methane oxidation. Cells incubated with ∼15 µM methanethiol from the start clearly showed inhibition of growth. After depletion of methanethiol, growth resumed within 1 day. Genes encoding a putative methanethiol oxidase were found in a variety of methanotrophs. Therefore, we hypothesize that methanethiol degradation is a widespread detoxification mechanism in methanotrophs in a range of environments.

Neodymium as Metal Cofactor for Biological Methanol Oxidation: Structure and Kinetics of an XoxF1-Type Methanol Dehydrogenase.

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.

Metagenome Assembled Genome of a Novel Verrucomicrobial Methanotroph From Pantelleria Island.

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.

Simultaneous Anaerobic and Aerobic Ammonia and Methane Oxidation under Oxygen Limitation Conditions.

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.

Methylacidimicrobium thermophilum AP8, a Novel Methane- and Hydrogen-Oxidizing Bacterium Isolated From Volcanic Soil on Pantelleria Island, Italy.

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.

Verrucomicrobial methanotrophs: ecophysiology of metabolically versatile acidophiles.

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.

Draft genome of a novel methanotrophic Methylobacter sp. from the volcanic soils of Pantelleria Island.

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.

Ammonia oxidation at pH 2.5 by a new gammaproteobacterial ammonia-oxidizing bacterium.

Ammonia oxidation was considered impossible under highly acidic conditions, as the protonation of ammonia leads to decreased substrate availability and formation of toxic nitrogenous compounds. Recently, some studies described archaeal and bacterial ammonia oxidizers growing at pH as low as 4, while environmental studies observed nitrification at even lower pH values. In this work, we report on the discovery, cultivation, and physiological, genomic, and transcriptomic characterization of a novel gammaproteobacterial ammonia-oxidizing bacterium enriched via continuous bioreactor cultivation from an acidic air biofilter that was able to grow and oxidize ammonia at pH 2.5. This microorganism has a chemolithoautotrophic lifestyle, using ammonia as energy source. The observed growth rate on ammonia was 0.196 day-1, with a doubling time of 3.5 days. The strain also displayed ureolytic activity and cultivation with urea as ammonia source resulted in a growth rate of 0.104 day-1 and a doubling time of 6.7 days. A high ammonia affinity (Km(app) = 147 ± 14 nM) and high tolerance to toxic nitric oxide could represent an adaptation to acidic environments. Electron microscopic analysis showed coccoid cell morphology with a large amount of intracytoplasmic membrane stacks, typical of gammaproteobacterial ammonia oxidizers. Furthermore, genome and transcriptome analysis showed the presence and expression of diagnostic genes for nitrifiers (amoCAB, hao, nor, ure, cbbLS), but no nirK was identified. Phylogenetic analysis revealed that this strain belonged to a novel bacterial genus, for which we propose the name "Candidatus Nitrosacidococcus tergens" sp. RJ19.

More Than a Methanotroph: A Broader Substrate Spectrum for Methylacidiphilum fumariolicum SolV.

Volcanic areas emit a number of gases including methane and other short chain alkanes, that may serve as energy source for the prevailing microorganisms. The verrucomicrobial methanotroph Methylacidiphilum fumariolicum SolV was isolated from a volcanic mud pot, and is able to grow under thermoacidophilic conditions on different gaseous substrates. Its genome contains three operons encoding a particulate methane monooxygenase (pMMO), the enzyme that converts methane to methanol. The expression of two of these pmo operons is subjected to oxygen-dependent regulation, whereas the expression of the third copy (pmoCAB3) has, so far, never been reported. In this study we investigated the ability of strain SolV to utilize short-chain alkanes and monitored the expression of the pmo operons under different conditions. In batch cultures and in carbon-limited continuous cultures, strain SolV was able to oxidize and grow on C1-C3 compounds. Oxidation of ethane did occur simultaneously with methane, while propane consumption only started once methane and ethane became limited. Butane oxidation was not observed. Transcriptome data showed that pmoCAB1 and pmoCAB3 were induced in the absence of methane and the expression of pmoCAB3 increased upon propane addition. Together the results of our study unprecedently show that a pMMO-containing methanotroph is able to co-metabolize other gaseous hydrocarbons, beside methane. Moreover, it expands the substrate spectrum of verrucomicrobial methanotrophs, supporting their high metabolic flexibility and adaptation to the harsh and dynamic conditions in volcanic ecosystems.