Elucidation of the electron transfer environment in the MMOR FAD-binding domain from Methylosinus sporium 5.

By facilitating electron transfer to the hydroxylase diiron center, MMOR-a reductase-serves as an essential component of the catalytic cycle of soluble methane monooxygenase. Here, the X-ray structure analysis of the FAD-binding domain of MMOR identified crucial residues and its influence on the catalytic cycle.

Characterization of a Copper-Chelating Natural Product from the Methanotroph Methylosinus sp. LW3.

Methanobactins (Mbns) are ribosomally produced, post-translationally modified peptidic natural products that bind copper with high affinity. Methanotrophic bacteria use Mbns to acquire copper needed for enzymatic methane oxidation. Despite the presence of Mbn operons in a range of methanotroph and other bacterial genomes, few Mbns have been isolated and structurally characterized. Here we report the isolation of a novel Mbn from the methanotroph Methylosinus (Ms.) sp. LW3. Mass spectrometric and nuclear magnetic resonance spectroscopic data indicate that this Mbn, the largest characterized to date, consists of a 13-amino acid backbone modified to include pyrazinedione/oxazolone rings and neighboring thioamide groups derived from cysteine residues. The pyrazinedione ring is more stable to acid hydrolysis than the oxazolone ring and likely protects the Mbn from degradation. The structure corresponds exactly to that predicted on the basis of the Ms. sp. LW3 Mbn operon content, providing support for the proposed role of an uncharacterized biosynthetic enzyme, MbnF, and expanding the diversity of known Mbns.

Isolation and Genomic Characterization of a Proteobacterial Methanotroph Requiring Lanthanides.

Although the bioavailability of rare earth elements (REEs, including scandium, yttrium, and 15 lanthanides) has not yet been examined in detail, methane-oxidizing bacteria (methanotrophs) were recently shown to harbor specific types of methanol dehydrogenases (XoxF-MDHs) that contain lanthanides in their active site, whereas their well-characterized counterparts (MxaF-MDHs) were Ca2+-dependent. However, lanthanide dependency in methanotrophs has not been demonstrated, except in acidic environments in which the solubility of lanthanides is high. We herein report the isolation of a lanthanide-dependent methanotroph from a circumneutral environment in which lanthanides only slightly dissolved. Methanotrophs were enriched and isolated from pond sediment using mineral medium supplemented with CaCl2 or REE chlorides. A methanotroph isolated from the cerium (Ce) chloride-supplemented culture, Methylosinus sp. strain Ce-a6, was clearly dependent on lanthanide. Strain Ce-a6 only required approximately 30 nM lanthanide chloride for its optimal growth and exhibited the ability to utilize insoluble lanthanide oxides, which may enable survival in circumneutral environments. Genome and gene expression analyses revealed that strain Ce-a6 lost the ability to produce functional MxaF-MDH, and this may have been due to a large-scale deletion around the mxa gene cluster. The present results provide evidence for lanthanide dependency as a novel survival strategy by methanotrophs in circumneutral environments.

Repurposed HisC Aminotransferases Complete the Biosynthesis of Some Methanobactins.

Methanobactins (Mbns) are ribosomally produced, post-translationally modified bacterial natural products with a high affinity for copper. MbnN, a pyridoxal 5'-phosphate-dependent aminotransferase, performs a transamination reaction that is the last step in the biosynthesis of Mbns produced by several Methylosinus species. Our bioinformatic analyses indicate that MbnNs likely derive from histidinol-phosphate aminotransferases (HisCs), which play a key role in histidine biosynthesis. A comparison of the HisC active site with the predicted MbnN structure suggests that MbnN's active site is altered to accommodate the larger and more hydrophobic substrates necessary for Mbn biosynthesis. Moreover, we have confirmed that MbnN is capable of catalyzing the final transamination step in Mbn biosynthesis in vitro and in vivo. We also demonstrate that without this final modification, Mbn exhibits significantly decreased stability under physiological conditions. An examination of other Mbns and Mbn operons suggests that N-terminal protection of this family of natural products is of critical importance and that several different means of N-terminal stabilization have evolved independently in Mbn subfamilies.

Methylmercury uptake and degradation by methanotrophs.

Methylmercury (CH3Hg+) is a potent neurotoxin produced by certain anaerobic microorganisms in natural environments. Although numerous studies have characterized the basis of mercury (Hg) methylation, no studies have examined CH3Hg+ degradation by methanotrophs, despite their ubiquitous presence in the environment. We report that some methanotrophs, such as Methylosinus trichosporium OB3b, can take up and degrade CH3Hg+ rapidly, whereas others, such as Methylococcus capsulatus Bath, can take up but not degrade CH3Hg+. Demethylation by M. trichosporium OB3b increases with increasing CH3Hg+ concentrations but was abolished in mutants deficient in the synthesis of methanobactin, a metal-binding compound used by some methanotrophs, such as M. trichosporium OB3b. Furthermore, addition of methanol (>5 mM) as a competing one-carbon (C1) substrate inhibits demethylation, suggesting that CH3Hg+ degradation by methanotrophs may involve an initial bonding of CH3Hg+ by methanobactin followed by cleavage of the C-Hg bond in CH3Hg+ by the methanol dehydrogenase. This new demethylation pathway by methanotrophs indicates possible broader involvement of C1-metabolizing aerobes in the degradation and cycling of toxic CH3Hg+ in the environment.

Enrichment culture and identification of endophytic methanotrophs isolated from peatland plants.

Aerobic methane-oxidizing bacteria (MOB) are an environmentally significant group of microorganisms due to their role in the global carbon cycle. Research conducted over the past few decades has increased the interest in discovering novel genera of methane-degrading bacteria, which efficiently utilize methane and decrease the global warming effect. Moreover, methanotrophs have more promising applications in environmental bioengineering, biotechnology, and pharmacy. The investigations were undertaken to recognize the variety of endophytic methanotrophic bacteria associated with Carex nigra, Vaccinium oxycoccus, and Eriophorum vaginatum originating from Moszne peatland (East Poland). Methanotrophic bacteria were isolated from plants by adding sterile fragments of different parts of plants (roots and stems) to agar mineral medium (nitrate mineral salts (NMS)) and incubated at different methane values (1-20% CH4). Single colonies were streaked on new NMS agar media and, after incubation, transferred to liquid NMS medium. Bacterial growth dynamics in the culture solution was studied by optical density-OD600 and methane consumption. Changes in the methane concentration during incubation were controlled by the gas chromatography technique. Characterization of methanotrophs was made by fluorescence in situ hybridization (FISH) with Mg705 and Mg84 for type I methanotrophs and Ma450 for type II methanotrophs. Identification of endophytes was performed after 16S ribosomal RNA (rRNA) and mmoX gene amplification. Our study confirmed the presence of both types of methanotrophic bacteria (types I and II) with the predominance of type I methanotrophs. Among cultivable methanotrophs, there were different strains of the genus Methylomonas and Methylosinus. Furthermore, we determined the potential of the examined bacteria for methane oxidation, which ranged from 0.463 ± 0.067 to 5.928 ± 0.169 µmol/L CH4/mL/day.

Ammonium conversion and its feedback effect on methane oxidation of Methylosinus sporium.

Ammonium (NH4+) is not only nitrogen source that can support methanotrophic growth, but also it can inhibit methane (CH4) oxidation by competing with CH4 for the active site of methane monooxygenase. NH4+ conversion and its feedback effect on the growth and activity of methanotrophs were evaluated with Methylosinus sporium used as a model methanotroph. Nitrogen sources could affect the CH4-derived carbon distribution, which varied with incubation time and nitrogen concentrations. More CH4-derived carbon was incorporated into biomass in the media with NH4+-N, compared to nitrate-nitrogen (NO3--N), as sole nitrogen source at the nitrogen concentrations of 10-18 mmol L-1. Although ammonia (NH3) oxidation activity of methanotrophs was considerably lower, only accounting for 0.01-0.06% of CH4 oxidation activity in the experimental cultures, NH4+ conversion could lead to the pH decrease and toxic intermediates accumulation in the their habits. Compared with NH4+, nitrite (NO2-) accumulation in the NH4+ conversion of methanotroph had stronger inhibition on its activity, especially the joint inhibition of NO2- accumulation and the pH decrease during the NH4+-N conversion. These results suggested that more attention should be paid to the feedback effects of NH4+ conversion by methanotrophs to understand effects of NH4+ on CH4 oxidation in the environments.

Production of Methanol from Methane by Encapsulated Methylosinus sporium.

Massive reserves of methane (CH4) remain unexplored as a feedstock for the production of liquid fuels and chemicals, mainly because of the lack of economically suitable and sustainable strategies for selective oxidation of CH4 to methanol. The present study demonstrates the bioconversion of CH4 to methanol mediated by Type I methanotrophs, such as Methylomicrobium album and Methylomicrobium alcaliphilum. Furthermore, immobilization of a Type II methanotroph, Methylosinus sporium, was carried out using different encapsulation methods, employing sodium-alginate (Na-alginate) and silica gel. The encapsulated cells demonstrated higher stability for methanol production. The optimal pH, temperature, and agitation rate were determined to be pH 7.0, 30°C, and 175 rpm, respectively, using inoculum (1.5 mg of dry cell mass/ml) and 20% of CH4 as a feed. Under these conditions, maximum methanol production (3.43 and 3.73 mM) by the encapsulated cells was recorded. Even after six cycles of reuse, the Na-alginate and silica gel encapsulated cells retained 61.8% and 51.6% of their initial efficiency for methanol production, respectively, in comparison with the efficiency of 11.5% observed in the case of free cells. These results suggest that encapsulation of methanotrophs is a promising approach to improve the stability of methanol production.

Characterization of Methanobactin from Methylosinus sp. LW4.

Methanobactins (Mbns) are a growing family of ribosomally produced, post-translationally modified natural products. Characteristic nitrogen-containing heterocycles and neighboring thioamides allow these compounds to bind copper with high affinity. Genome mining has enabled the identification of Mbn operons in bacterial genomes and the prediction of diverse Mbn structures from operon content and precursor peptide sequence. Here we report the characterization of Mbn from Methylosinus (Ms.) species (sp.) LW4. The peptide backbone is distinct from all previously characterized Mbns, and the post-translational modifications correspond precisely to those predicted on the basis of the Ms. sp. LW4 Mbn operon. Thus, prediction based on genome analysis combined with isolation and structural characterization represents a phylogenetic approach to finding diverse Mbns and elucidating their biosynthetic pathways.

Non-linear dynamics of stable carbon and hydrogen isotope signatures based on a biological kinetic model of aerobic enzymatic methane oxidation.

The non-linear dynamics of stable carbon and hydrogen isotope signatures during methane oxidation by the methanotrophic bacteria Methylosinus sporium strain 5 (NCIMB 11126) and Methylocaldum gracile strain 14 L (NCIMB 11912) under copper-rich (8.9 µM Cu(2+)), copper-limited (0.3 µM Cu(2+)) or copper-regular (1.1 µM Cu(2+)) conditions has been described mathematically. The model was calibrated by experimental data of methane quantities and carbon and hydrogen isotope signatures of methane measured previously in laboratory microcosms reported by Feisthauer et al. [ 1 ] M. gracile initially oxidizes methane by a particulate methane monooxygenase and assimilates formaldehyde via the ribulose monophosphate pathway, whereas M. sporium expresses a soluble methane monooxygenase under copper-limited conditions and uses the serine pathway for carbon assimilation. The model shows that during methane solubilization dominant carbon and hydrogen isotope fractionation occurs. An increase of biomass due to growth of methanotrophs causes an increase of particulate or soluble monooxygenase that, in turn, decreases soluble methane concentration intensifying methane solubilization. The specific maximum rate of methane oxidation υm was proved to be equal to 4.0 and 1.3 mM mM(-1) h(-1) for M. sporium under copper-rich and copper-limited conditions, respectively, and 0.5 mM mM(-1) h(-1) for M. gracile. The model shows that methane oxidation cannot be described by traditional first-order kinetics. The kinetic isotope fractionation ceases when methane concentrations decrease close to the threshold value. Applicability of the non-linear model was confirmed by dynamics of carbon isotope signature for carbon dioxide that was depleted and later enriched in (13)C. Contrasting to the common Rayleigh linear graph, the dynamic curves allow identifying inappropriate isotope data due to inaccurate substrate concentration analyses. The non-linear model pretty adequately described experimental data presented in the two-dimensional plot of hydrogen versus carbon stable isotope signatures.

Assessment of methane biodegradation kinetics in two-phase partitioning bioreactors by pulse respirometry.

Biological methane biodegradation is a promising treatment alternative when the methane produced in waste management facilities cannot be used for energy generation. Two-phase partitioning bioreactors (TPPBs), provided with a non-aqueous phase (NAP) with high affinity for the target pollutant, are particularly suitable for the treatment of poorly water-soluble compounds such as methane. Nevertheless, little is known about the influence of the presence of the NAP on the resulting biodegradation kinetics in TPPBs. In this study, an experimental framework based on the in situ pulse respirometry technique was developed to assess the impact of NAP addition on the methane biodegradation kinetics using Methylosinus sporium as a model methane-degrading microorganism. A comprehensive mass transfer characterization was performed in order to avoid mass transfer limiting scenarios and ensure a correct kinetic parameter characterization. The presence of the NAP mediated significant changes in the apparent kinetic parameters of M. sporium during methane biodegradation, with variations of 60, 120, and 150% in the maximum oxygen uptake rate, half-saturation constant and maximum specific growth rate, respectively, compared with the intrinsic kinetic parameters retrieved from a control without NAP. These significant changes in the kinetic parameters mediated by the NAP must be considered for the design, operation and modeling of TPPBs devoted to air pollution control.

Coal-packed methane biofilter for mitigation of green house gas emissions from coal mine ventilation air.

Methane emitted by coal mine ventilation air (MVA) is a significant greenhouse gas. A mitigation strategy is the oxidation of methane to carbon dioxide, which is approximately twenty-one times less effective at global warming than methane on a mass-basis. The low non-combustible methane concentrations at high MVA flow rates call for a catalytic strategy of oxidation. A laboratory-scale coal-packed biofilter was designed and partially removed methane from humidified air at flow rates between 0.2 and 2.4 L min-1 at 30°C with nutrient solution added every three days. Methane oxidation was catalysed by a complex community of naturally-occurring microorganisms, with the most abundant member being identified by 16S rRNA gene sequence as belonging to the methanotrophic genus Methylocystis. Additional inoculation with a laboratory-grown culture of Methylosinus sporium, as investigated in a parallel run, only enhanced methane consumption during the initial 12 weeks. The greatest level of methane removal of 27.2±0.66 g methane m-3 empty bed h-1 was attained for the non-inoculated system, which was equivalent to removing 19.7±2.9% methane from an inlet concentration of 1% v/v at an inlet gas flow rate of 1.6 L min-1 (2.4 min empty bed residence time). These results show that low-cost coal packing holds promising potential as a suitable growth surface and contains methanotrophic microorganisms for the catalytic oxidative removal of methane.

Methane emission and dynamics of methanotrophic and methanogenic communities in a flooded rice field ecosystem.

Methane emissions, along with methanotrophs and methanogens and soil chemical properties, were investigated in a flooded rice ecosystem. Methane emission increased after rice transplantation (from 7.2 to 552 mg day(-1) m(-2) ) and was positively and significantly correlated with transcripts of pmoA and mcrA genes, transcript/gene ratios of mcrA, temperature and total organic carbon. Methane flux was negatively correlated with sulfate concentration. Methanotrophs represented only a small proportion (0.79-1.75%) of the total bacterial 16S rRNA gene reads: Methylocystis (type II methanotroph) decreased rapidly after rice transplantation, while Methylosinus and unclassified Methylocystaceae (type II) were relatively constant throughout rice cultivation. Methylocaldum, Methylobacter, Methylomonas and Methylosarcina (type I) were sparse during the early period, but they increased after 60 days, and their maximum abundances were observed at 90-120 days. Of 33 218 archaeal reads, 68.3-86.6% were classified as methanogens. Methanosaeta, Methanocella, Methanosarcina and Methanobacterium were dominant methanogens, and their maximum abundances were observed at days 60-90. Only four reads were characteristic of anaerobic methanotrophs, suggesting that anaerobic methane metabolism is negligible in this rice paddy system. After completing a multivariate canonical correspondence analysis of our integrated data set, we found normalized mcrA/pmoA transcript ratios to be a promising parameter for predicting net methane fluxes emitted from rice paddy soils.

Stress resistance and C1 metabolism involved in plant colonization of a methanotroph Methylosinus sp. B4S.

Methanotrophs are widespread and have been isolated from various environments including the phyllosphere. In this study, we characterized the plant colonization by Methylosinus sp. B4S, an α-proteobacterial methanotroph isolated from plant leaf. The gfp-tagged Methylosinus sp. B4S cells were observed to colonize Arabidopsis leaf surfaces by forming aggregates. We cloned and sequenced the general stress response genes, phyR, nepR and ecfG, from Methylosinus sp. B4S. In vitro analysis showed that the phyR expression level was increased after heat shock challenge, and phyR was shown to be involved in resistance to heat shock and UV light. In the phyllospheric condition, the gene expression level of phyR as well as mmoX and mxaF was found to be relatively high, compared with methane-grown liquid cultures. The phyR-deletion strain as well as the wild-type strain inoculated on Arabidopsis leaves proliferated at the initial phase and then gradually decreased during plant colonization. These results have shed light firstly on the importance of general stress resistance and C1 metabolism in methanotroph living in the phyllosphere.

Single cell methods for methane oxidation analysis.

Respiration is widely used for evaluation of the metabolic capabilities or physiological state of the microbial culture. This chapter describes novel approaches for characterization of respiration at a single cell level: (1) flow cytometry-based redox sensing (FCRS) of actively metabolizing microbes; (2) respiration response imaging (RRI) for real-time detection of substrate stimulated redox responses of individual cells; (3) respiration detection system: microobservation chamber (RDS: MC), a single cell analysis system for carrying out the physiological and genomic profiling of cells capable of respiring C(1) compounds. The techniques are suitable for description of physiological heterogeneity among cells in a single microbial population and could be used to characterize distribution of methylotrophic ability among microbial cells in the natural environmental samples.

[Effect of plant-associated methanolic bacteria on methane and methanol concentration dynamics in atmosphere of pressurized chamber].

Stability of Chinese cabbage crop colonization by methanolic bacteria Methylovorus mays, Methylomonas methanica and Methylosinus trichosporium inoculated using a space-applicable method was evaluated. Besides, trends of methane and methanol concentrations in the pressurized chamber with inoculated and uninoculated crops were calculated. Methylovorus mays and Methylosinus trichosporium were shown to establish more stable colonization as compared to Methylomonas methanica. Also, stable association of methanolic bacteria with plants reduced airborne methanol 75% faster owing to its uptake by bacteria. Therefore, inoculation of these microorganisms can be viewed as a promising method of controlling volatile pollutants in space vehicle atmosphere. Methane drop after 6-hour exposure to inoculated control and test crops was not significant.

Analysis of methanotrophic communities in landfill biofilters using diagnostic microarray.

Biofilters operated for the microbial oxidation of landfill methane at two sites in Northern Germany were analysed for the composition of their methanotrophic community by means of diagnostic microarray targeting the pmoA gene of methanotrophs. The gas emitted from site Francop (FR) contained the typical principal components (CH4, CO2, N2) only, while the gas at the second site Müggenburger Strasse (MU) was additionally charged with non-methane volatile organic compounds (NMVOCs). Methane oxidation activity measured at 22 degrees C varied between 7 and 103 microg CH4 (g dw)(-1) h(-1) at site FR and between 0.9 and 21 microg CH4 (g dw)(-1) h(-1) at site MU, depending on the depth considered. The calculated size of the active methanotrophic population varied between 3 x 10(9) and 5 x 10(11) cells (g dw)(-1) for biofilter FR and 4 x 10(8) to 1 x 10(10) cells (g dw)(-1) for biofilter MU. The methanotrophic community in both biofilters as well as the methanotrophs present in the landfill gas at site FR was strongly dominated by type II organisms, presumably as a result of high methane loads, low copper concentration and low nitrogen availability. Within each biofilter, community composition differed markedly with depth, reflecting either the different conditions of diffusive oxygen supply or the properties of the two layers of materials used in the filters or both. The two biofilter communities differed significantly. Type I methanotrophs were detected in biofilter FR but not in biofilter MU. The type II community in biofilter FR was dominated by Methylocystis species, whereas the biofilter at site MU hosted a high abundance of Methylosinus species while showing less overall methanotroph diversity. It is speculated that the differing composition of the type II population at site MU is driven by the presence of NMVOCs in the landfill gas fed to the biofilter, selecting for organisms capable of co-oxidative degradation of these compounds.

[Study on the structure and function of a stable methane-oxidizing mixed microbial consortium].

From an agricultural sample taken in Chongqing, a stable methane-oxidizing mixed microbial consortium was established by enrichment culture with methane as a sole source of carbon and energy. The mixed consortium showed high capability of phenol degradation and 1,2-epoxypropane production from propene. More than 99% of phenol at an initial concentration of 600mg/L could be degraded by the mixed microbial consortium after 11 h of cultivation. The productivity of 1, 2-epoxypropane could be increased with the decrease of phosphate concentration. The concentration of 1, 2-epoxypropane produced could reach to 5.0mmol/L. The bacterial structure of the methane-oxidizing mixed microbial consortium was analyzed by pure culture isolation combining with 16S rRNA and PCR of the related MMO functional genes. The results showed that the methane-oxidizing mixed microbial consortium was composed of a type 1U methanotroph identified as Methylosinus trichosporium and at least 4 kinds of heterotrophs ( Comamonas testosteroni, Cupriavidus metallidurans, Acinetobacter junii and Stenotrophomonas maltophilia ). M. trichosporium Y9, isolated from the mixed consortium, harbored both sMMO and pMMO genes.