Terrestrial and oceanic geothermal areas emit substantial amounts of hydrocarbons in the form of methane and the short-chain alkanes ethane and propane. Under hydrothermal conditions, these alkanes can also be oxidised to their respective alcohols and ketones, with a preference for the 2-position. The thermoacidophilic verrucomicrobial methanotroph Methylacidiphilum fumariolicum SolV, isolated from the Solfatara volcano, was previously shown to oxidise methane as well as the short-chain hydrocarbons propane and ethane. Here, we show the growth of strain SolV on the C3 compounds 2-propanol and acetone with growth rates of 0.054 h-1 and 0.042 h-1, respectively. In contrast to methanotrophic growth (rate 0.07 h-1), growth was not dependent on CO2 or lanthanides. Respiration experiments on steady-state continuous cultures showed an apparent affinity of 0.4 μM acetone and 5.4 μM 2-propanol. Transcriptomic analysis of these cultures showed that a gene cluster including a novel acetone monooxygenase (PMO3), previously identified in the closely related species Methylacidiphilum caldifontis, was highly upregulated under growth on C3 substrates. These results support the versatile metabolism of verrucomicrobial methanotrophs. The conversion of other compounds besides methane can be important in view of the ecological relevance of methanotrophs.

Understanding stable isotopic fractionation of dissolved O2 in aquatic environments is crucial to constrain and accurately model the processes responsible for biological O2 consumption, which are closely linked to the overall health of an ecosystem. This study aimed to investigate whether O2 consumption by microbial methane and ammonia oxidation may contribute to the observed discrepancy in O2 isotopic fractionation (18ϵ) between heterotrophic O2 respiration in laboratory incubations (-18 to -24 ‰) and in situ measurements of O2 consumption in lakes and oceans (-10 to -18 ‰). To estimate the in vivo 18ϵ values of soluble methane monooxygenase (sMMO), particulate methane monooxygenase (pMMO), and ammonia monooxygenase (AMO), which are the first enzymes required for the oxidation of methane and ammonia, experiments were performed with three methanotrophic bacteria and one comammox (complete-ammonia-oxidizing) bacterium. The resulting 18ϵ values for pMMO and AMO ranged from -18 ± 12 to -24 ± 5 ‰, not significantly different from 18ϵ values typical for heterotrophic respiration. The 18ϵ value determined for sMMO (-22 ± 2 ‰) was in the same range, yet more negative than the previously reported 18ϵ value for the isolated enzyme. Our results provide insights into the potential reaction mechanisms of pMMO and AMO and indicate that O2 consumption by sMMO, pMMO, or AMO cannot explain the observed discrepancy between in situ and laboratory 18ϵ values for "community" O2 consumption in aquatic environments. Instead, the apparent difference may be attributed to aspects involving substrate diffusion limitation.

Acidophilic microorganisms thrive in environments where the external pH is orders of magnitude lower than their intracellular pH. Verrucomicrobial methanotrophs of the family Methylacidiphilaceae, including Methylacidiphilum and Methylacidimicrobium, inhabit extremely acidic geothermal environments and can grow at a pH < 1.0 and temperatures up to 65 °C. We analyzed and compared their membrane fatty acid compositions at pH 3.0 across strains with different temperature optima. Thermophilic Methylacidiphilum strains almost exclusively contain saturated fatty acids, while the mesophilic Methylacidimicrobium strains we studied incorporate 16–47

Hydrogen (H2) and methane (CH4) are produced in the anoxic layers of wetlands and sediments. In the overlaying oxygenated surface layers, these gases become available for oxidation by aerobic hydrogenotrophic and methanotrophic microorganisms. While H2 oxidation by verrucomicrobial methane-oxidising bacteria (MOB) is extensively studied, less is known about this metabolism in MOB from the class Alphaproteobacteria, which frequently inhabit wetlands. We show that Methylocystis bryophila H2sT, Methylocapsa aurea KYGT, and "Methylosinus acidophilus" 29 encode diverse hydrogenases, instantly oxidise H2 when cultivated under CH4-limited and low-oxygen conditions, under which hydrogenase transcription is upregulated compared to CH4-replete conditions. H2 exposure accelerated the maximum H2 oxidation rates but caused no upregulation of hydrogenases. Furthermore, while CH4 oxidation activity was affected by substrate-limited growth conditions, H2 oxidation rates remained unaffected, and H2 supply to CH4-limited chemostats caused increased biomass yield. Moreover, CH4 oxidation was severely inhibited by sulfide (H2S), while H2 and methanol oxidation rates were only moderately affected. In summary, the ability to conserve energy from H2 oxidation increases resilience and enhances growth of alphaproteobacterial methanotrophs in CH4-limited environments, which revises the ecological role of these MOB in ecosystems with naturally fluctuating CH4 and H2 concentrations.

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.

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.

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.

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.

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 β-barrel structure consisting of ten antiparallel β-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.

Exploring new fields: Lanthanide-using bacteria that turn over methane or methanol in their metabolism can replace essential lanthanides by certain actinides. In their Research Article (e202303669), Lena J. Daumann et al. show that an extremophile isolated from a volcanic mud-pot as well as a methylotrophic laboratory strain can use americium and curium. In vivo growth experiments as well as in vitro studies with the key metabolic enzyme methanol dehydrogenase support that these actinides can sustain the one-carbon metabolism of these bacteria.

AbstractCertain 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.

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.

Hydrogen sulfide (H 2 S) and methane (CH 4 ) are produced in anoxic environments through sulfate reduction and organic matter decomposition. Both gases diffuse upwards into oxic zones where aerobic methanotrophs mitigate CH 4 emissions by oxidizing this potent greenhouse gas. Although methanotrophs in myriad environments encounter toxic H 2 S, it is virtually unknown how they are affected. Here, through extensive chemostat culturing we show that a single microorganism can oxidize CH 4 and H 2 S simultaneously at equally high rates. By oxidizing H 2 S to elemental sulfur, the thermoacidophilic methanotroph Methylacidiphilum fumariolicum SolV alleviates the inhibitory effects of H 2 S on methanotrophy. Strain SolV adapts to increasing H 2 S by expressing a sulfide-insensitive ba 3 -type terminal oxidase and grows as chemolithoautotroph using H 2 S as sole energy source. Genomic surveys revealed putative sulfide-oxidizing enzymes in numerous methanotrophs, suggesting that H 2 S oxidation is much more widespread in methanotrophs than previously assumed, enabling them to connect carbon and sulfur cycles in novel ways.

The biological relevance of early lanthanides, such as neodymium, remained undiscovered until methylotrophic bacteria with lanthanide-dependent metabolism were identified. The respective strains incorporate these elements into the active site of their key metabolic enzyme methanol dehydrogenase (MDH). Growth studies with the strictly lanthanide-dependent thermoacidophile Methylacidiphilum fumariolicum SolV and the mesophilic Methylorubrum extorquens AM1 ΔmxaF mutant demonstrate that the trivalent actinides americium and curium support growth in the absence of the essential lanthanides. In fact, the bacteria make no distinction between lanthanide and actinide ions if they have the correct size and oxidation state. Time-resolved laser-induced fluorescence spectroscopy, liquid scintillation counting, and inductively coupled plasma mass spectrometry confirm the bacterial uptake. The interchangeability of f-block elements is supported by very similar enzymatic activities of recombinant methanol dehydrogenase reconstituted with different metal ions. Our combined in vivo and in vitro results firmly establish that actinides support growth of methylotrophic bacteria, suggesting a potential biological role for these radioactive elements. Importantly, bacteria capable of utilizing actinides will also be able to mobilize these elements in the environment. This may lead to applications for bioremediation and recycling/separation of lanthanides and actinides.

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.

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.

Mangrove forest sediments produce significant amounts of methane, but the diversity of methanogenic archaea is not well known at present. Therefore, 16S rRNA gene libraries were made using archaea-specific primers and DNA extracted directly from Tanzanian mangrove sediment samples as a template. Analysis of sequence data showed phylotypes closely related to cultivated methylotrophic methanogenic archaea from the marine environment, or distantly related to acetoclastic and hydrogenotrophic methanogenic archaea. In an attempt to isolate relevant methanogenic archaea, we succeeded in obtaining a new mesophilic methylotrophic methanogenic archaeon (strain MM1) capable of utilizing methanol and methylated amines as the only substrates. Under optimum conditions, the cells of strain MM1 exhibited a high specific growth rate (mu) of 0.21+/-0.03 (i.e. doubling time of 3.2 h) on both methanol and trimethylamine. The 16S rRNA gene sequence of strain MM1 clustered with five environmental clones, indicating that MM1 is an important methanogenic methylotroph in mangrove sediments. Based on physiological and phylogenetic analyses, strain MM1 is proposed to be included in the species of Methanococcoides methylutens.

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.