Growth of Methylacidiphilum fumariolicum SolV, an extremely acidophilic methanotrophic microbe isolated from an Italian volcanic mudpot, is shown to be strictly dependent on the presence of lanthanides, a group of rare earth elements (REEs) such as lanthanum (Ln), cerium (Ce), praseodymium (Pr) and neodymium (Nd). After fractionation of the bacterial cells and crystallization of the methanol dehydrogenase (MDH), it was shown that lanthanides were essential as cofactor in a homodimeric MDH comparable with one of the MDHs of Methylobacterium extorquens AM1. We hypothesize that the lanthanides provide superior catalytic properties to pyrroloquinoline quinone (PQQ)-dependent MDH, which is a key enzyme for both methanotrophs and methylotrophs. Thus far, all isolated MxaF-type MDHs contain calcium as a catalytic cofactor. The gene encoding the MDH of strain SolV was identified to be a xoxF-ortholog, phylogenetically closely related to mxaF. Analysis of the protein structure and alignment of amino acids showed potential REE-binding motifs in XoxF enzymes of many methylotrophs, suggesting that these may also be lanthanide-dependent MDHs. Our findings will have major environmental implications as metagenome studies showed (lanthanide-containing) XoxF-type MDH is much more prominent in nature than MxaF-type enzymes.

Genome data of the extreme acidophilic verrucomicrobial methanotroph Methylacidiphilum fumariolicum strain SolV indicated the ability of autotrophic growth. This was further validated by transcriptome analysis, which showed that all genes required for a functional Calvin-Benson-Bassham (CBB) cycle were transcribed. Experiments with 13 Methanotrophs are a unique group of microorganisms within the methylotrophs that oxidize methane (CH 4 ) to carbon dioxide (CO 2 ). Until 2007, the phylogenetic distribution of the aerobic methanotrophs was limited to the ␣ and ␥ subclasses of the proteobacteria (16). In 2007, novel thermoacidophilic aerobic methanotrophs were discovered in geothermal areas in New Zealand, Russia, and Italy (9,18,23). These methanotrophs represented a distinct phylogenetic lineage within the Verrucomicrobia, for which the genus name Methylacidiphilum was proposed (22).Recently, methanotrophy was discovered in a member of the NC10 phylum. It was shown that "Candidatus Methylomirabilis oxyfera," enriched under strict anoxic conditions, produces its own oxygen from nitrite (12). This oxygen is then used for CH 4 oxidation in a biochemical pathway comparable to those of aerobic methanotrophs.During the aerobic oxidation of CH 4 and methanol by proteobacterial methanotrophs, formaldehyde is produced. This central metabolite can be further oxidized to CO 2 or directly assimilated via intermediates of the central metabolism. Based on the pathway used for formaldehyde assimilation, methanotrophs were divided into type I and type II. Type II methanotrophs use the serine pathway, in which formaldehyde and CO 2 are utilized in a one-to-one ratio to produce acetyl coenzyme A (acetyl-CoA) for biosynthesis (8), while type I methanotrophs use the ribulose monophosphate pathway for the assimilation of formaldehyde to form glyceraldehyde-3-phosphate as an intermediate of central metabolism (16). In the latter pathway, all cellular carbon is assimilated at the oxidation level of formaldehyde. Genome data of some proteobacterial methanotrophs (Methylococcus capsulatus Bath, Methylocella silvestris BL2 [7,31]) and nonproteobacterial aerobic methanotrophs (Methylacidiphilum infernorum V4, Methylacidiphilum fumariolicum SolV, and "Candidatus Methylomirabilis oxyfera" [12,17,22]) revealed the presence of ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO), the key enzyme of the Calvin-Benson-Bassham (CBB) cycle. M. capsulatus Bath was found to contain RuBisCO in an active form (27), and genome analysis suggested that a variant of the CBB cycle may operate (19,31). Although hydrogen seems to support moderate growth with CO 2 on agar plates for M. capsulatus Bath and some other methanotrophs (3), autotrophic growth in liquid cultures has not been reported. Marker exchange mutagenesis deleting the genes encoding RuBisCO may give definite answers on the exact role of RuBisCO, but unfortunately, a good genetic system for manipulation of these bacteria is lacking.Analyses of the complete genome sequence of M. infernorum...

In recent years, biogas production from complex biomass has received great interest. Therefore, many studies have been conducted to understand the anaerobic digestion process and to characterise responsible microbes for the biochemical conversions. Although our knowledge about biogas production in general is rapidly increasing, less information is available about hydrolytic microbes within anaerobic bioreactors. Here, we pinpoint the urgent need for solid fundamental knowledge about hydrolytic bacteria within biogas plants. In this review, current knowledge about anaerobic hydrolytic microbes is presented, including their abundance in biogas plants, and the factors impacting their activity.

Extremophilic organisms require specialized enzymes for their exotic metabolisms. Acid-loving thermophilic Archaea that live in the mudpots of volcanic solfataras obtain their energy from reduced sulphur compounds such as hydrogen sulphide (H(2)S) and carbon disulphide (CS(2)). The oxidation of these compounds into sulphuric acid creates the extremely acidic environment that characterizes solfataras. The hyperthermophilic Acidianus strain A1-3, which was isolated from the fumarolic, ancient sauna building at the Solfatara volcano (Naples, Italy), was shown to rapidly convert CS(2) into H(2)S and carbon dioxide (CO(2)), but nothing has been known about the modes of action and the evolution of the enzyme(s) involved. Here we describe the structure, the proposed mechanism and evolution of a CS(2) hydrolase from Acidianus A1-3. The enzyme monomer displays a typical β-carbonic anhydrase fold and active site, yet CO(2) is not one of its substrates. Owing to large carboxy- and amino-terminal arms, an unusual hexadecameric catenane oligomer has evolved. This structure results in the blocking of the entrance to the active site that is found in canonical β-carbonic anhydrases and the formation of a single 15-Å-long, highly hydrophobic tunnel that functions as a specificity filter. The tunnel determines the enzyme's substrate specificity for CS(2), which is hydrophobic. The transposon sequences that surround the gene encoding this CS(2) hydrolase point to horizontal gene transfer as a mechanism for its acquisition during evolution. Our results show how the ancient β-carbonic anhydrase, which is central to global carbon metabolism, was transformed by divergent evolution into a crucial enzyme in CS(2) metabolism.

The ability to utilize atmospheric nitrogen (N2) as a sole nitrogen source is an important trait for prokaryotes. Knowledge of N2 fixation by methanotrophs is needed to understand their role in nitrogen cycling in different environments. The verrucomicrobial methanotroph ‘Methylacidiphilum fumariolicum’ strain SolV was investigated for its ability to fix N2. Physiological studies were combined with nitrogenase activity measurements and phylogenetic analysis of the nifDHK genes, encoding the subunits of the nitrogenase. ‘M. fumariolicum’ SolV was able to fix N2 at low oxygen (O2) concentration (0.5 %, v/v) in chemostat cultures. This low oxygen concentration was also required for an optimal nitrogenase activity [47.4 nmol ethylene h−1 (mg cell dry weight)−1]. Based on acetylene reduction assay and growth experiments, the nitrogenase of strain SolV seems to be extremely oxygen sensitive compared to most proteobacterial methanotrophs. The activity of the nitrogenase was not inhibited by ammonium concentrations up to 94 mM. This is believed to be the first report on the physiology of N2 fixation within the phylum Verrucomicrobia.

The draft genome of Methylacidiphilum fumariolicum SolV, a thermoacidophilic methanotroph of the phylum Verrucomicrobia, is presented. Annotation revealed pathways for one-carbon, nitrogen, and hydrogen catabolism and respiration together with central metabolic pathways. The genome encodes three orthologues of particulate methane monooxygenases. Sequencing of this genome will help in the understanding of methane cycling in volcanic environments.

“Candidatus Methylacidiphilum fumariolicum” SolV is a verrucomicrobial methanotroph that can grow in extremely acidic environments at high temperature. Strain SolV fixes carbon dioxide (CO2) via the Calvin–Benson–Bassham cycle with methane as energy source, a trait so far very unusual in methanotrophs. In this study, the ability of “Ca. M. fumariolicum” to store carbon was explored by genome analysis, physiological studies, and electron microscopy. When cell cultures were depleted for nitrogen, glycogen storage was clearly observed in cytoplasmic storage vesicles by electron microscopy. After cessation of growth, the dry weight kept increasing and the bacteria were filled up almost entirely by glycogen. This was confirmed by biochemical analysis, which showed that glycogen accumulated to 36% of the total dry weight of the cells. When methane was removed from the culture, this glycogen was consumed within 47 days. During the period of glycogen consumption, the bacteria kept their viability high when compared to bacteria without glycogen (from cultures growing exponentially). The latter bacteria lost viability already after a few days when starved for methane. Analysis of the draft genome of “Ca. M. fumariolicum” SolV demonstrated that all known genes for glycogen storage and degradation were present and also transcribed. Phylogenetic analysis of these genes showed that they form a separate cluster with “Ca. M. infernorum” V4, and the most closely related other sequences only have an identity of 40%. This study presents the first physiological evidence of glycogen storage in the phylum Verrucomicrobia and indicates that carbon storage is important for survival at times of methane starvation.

Aerobic methanotrophic bacteria can use methane as their sole energy source. The discovery of “Ca. Methylacidiphilum fumariolicum” strain SolV and other verrucomicrobial methanotrophs has revealed that the ability of bacteria to oxidize CH4 is much more diverse than has previously been assumed in terms of ecology, phylogeny, and physiology. A remarkable characteristic of the methane-oxidizing Verrucomicrobia is their extremely acidophilic phenotype, growing even below pH 1. In this study we used RNA-Seq to analyze the metabolic regulation of “Ca. M. fumariolicum” SolV cells growing at μmax in batch culture or under nitrogen fixing or oxygen limited conditions in chemostats, all at pH 2. The analysis showed that two of the three pmoCAB operons each encoding particulate methane monoxygenases were differentially expressed, probably regulated by the available oxygen. The hydrogen produced during N2 fixation is apparently recycled as demonstrated by the upregulation of the genes encoding a Ni/Fe-dependent hydrogenase. These hydrogenase genes were also upregulated under low oxygen conditions. Handling of nitrosative stress was shown by the expression of the nitric oxide reductase encoding genes norB and norC under all conditions tested, the upregulation of nitrite reductase nirK under oxygen limitation and of hydroxylamine oxidoreductase hao in the presence of ammonium. Unraveling the gene regulation of carbon and nitrogen metabolism helps to understand the underlying physiological adaptations of strain SolV in view of the harsh conditions of its natural ecosystem.