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PubMed1995

The phylogenetic position of the family Methylococcaceae.

The 16S ribosomal DNA-based phylogenetic positions of various members of the Methylococcaceae (group I methanotrophs) were investigated. The Methylococcaceae as a whole formed a distinct branch in the gamma subdivision of the Proteobacteria, and this branch had five distinct subbranches. On the basis of a number of phenotypic traits, phospholipid fatty acid patterns, and the results of a 16S ribosomal DNA analysis, we determined that the species belonging to one subbranch, Methylobacter albus, Methylobacter agilis, and Methylobacter pelagicus, formed a distinct group that could be differentiated from other members of the genus Methylobacter, which grouped in an adjacent subbranch. We propose that these species belong to a new taxon, Methylomicrobium gen. nov.

Base Sequence↗
PubMed2001

Changes in activity and community structure of methane-oxidizing bacteria over the growth period of rice.

The activity and community structure of methanotrophs in compartmented microcosms were investigated over the growth period of rice plants. In situ methane oxidation was important only during the vegetative growth phase of the plants and later became negligible. The in situ activity was not directly correlated with methanotrophic cell counts, which increased even after the decrease in in situ activity, possibly due to the presence of both vegetative cells and resting stages. By dividing the microcosms into two soil and two root compartments it was possible to locate methanotrophic growth and activity, which was greatest in the rhizoplane of the rice plants. Molecular analysis by denaturing gradient gel electrophoresis and fluorescent in situ hybridization (FISH) with family-specific probes revealed the presence of both families of methanotrophs in soil and root compartments over the whole season. Changes in community structure were detected only for members of the Methylococcaceae and could be associated only with changes in the genus Methylobacter and not with changes in the dominance of different genera in the family Methylococcaceae. For the family Methylocystaceae stable communities in all compartments for the whole season were observed. FISH analysis revealed evidence of in situ dominance of the Methylocystaceae in all compartments. The numbers of Methylococcaceae cells were relatively high only in the rhizoplane, demonstrating the importance of rice roots for growth and maintenance of methanotrophic diversity in the soil.

Colony Count, Microbial↗
PubMed2001

Family- and genus-level 16S rRNA-targeted oligonucleotide probes for ecological studies of methanotrophic bacteria.

Methanotrophic bacteria play a major role in the global carbon cycle, degrade xenobiotic pollutants, and have the potential for a variety of biotechnological applications. To facilitate ecological studies of these important organisms, we developed a suite of oligonucleotide probes for quantitative analysis of methanotroph-specific 16S rRNA from environmental samples. Two probes target methanotrophs in the family Methylocystaceae (type II methanotrophs) as a group. No oligonucleotide signatures that distinguish between the two genera in this family, Methylocystis and Methylosinus, were identified. Two other probes target, as a single group, a majority of the known methanotrophs belonging to the family Methylococcaceae (type I/X methanotrophs). The remaining probes target members of individual genera of the Methylococcaceae, including Methylobacter, Methylomonas, Methylomicrobium, Methylococcus, and Methylocaldum. One of the family-level probes also covers all methanotrophic endosymbionts of marine mollusks for which 16S rRNA sequences have been published. The two known species of the newly described genus Methylosarcina gen. nov. are covered by a probe that otherwise targets only members of the closely related genus Methylomicrobium. None of the probes covers strains of the newly proposed genera Methylocella and "Methylothermus," which are polyphyletic with respect to the recognized methanotrophic families. Empirically determined midpoint dissociation temperatures were 49 to 57 degrees C for all probes. In dot blot screening against RNA from positive- and negative-control strains, the probes were specific to their intended targets. The broad coverage and high degree of specificity of this new suite of probes will provide more detailed, quantitative information about the community structure of methanotrophs in environmental samples than was previously available.

Alphaproteobacteria↗
PubMed2001

Methylosarcina fibrata gen. nov., sp. nov. and Methylosarcina quisquiliarum sp.nov., novel type 1 methanotrophs.

Two novel species of obligate methane-oxidizing bacteria, isolated from landfill soil, were characterized. Both strains were unusual in that some members of the population grew in irregularly shaped, refractile cell packets that resembled sarcina-like clusters. Electron microscopy revealed that the cell packets were covered with a slime layer and the cells contained many large granular inclusion bodies. The individual cells of each strain were sometimes motile and had differing morphologies. Isolate AML-C10T was always coccoidal in shape, and the cells were covered with extracellular fibrils. Isolate AML-D4T was pleomorphic, changing from rod to coccal form, sometimes exhibiting an unusual fusiform morphology. AML-D4T lacked the extensive fibrillar matrix observed with AML-C10T. Both strains utilized only methane and methanol as carbon sources. In stationary phase, the cells of each strain swelled in size and formed cysts. Aside from morphological differences, strains could also be distinguished from each other by cellular protein patterns, as well as by temperature and pH tolerances. 16S rDNA phylogenetic analysis showed that these are type I methanotrophs (family: Methylococcaceae) most closely related to the Methylobacter/Methylomicrobium clade, although they form a monophyletic grouping supported by moderately high bootstrap values. By 16S rDNA database searches, the most similar species to both isolates were Methylobacter spp. However, partial particulate methane monooxygenase sequence analysis suggested that these bacteria might be more closely related to Methylomicrobium than Methylobacter. Furthermore, cellular fatty acid profiles of the strains more closely resemble those of Methylomicrobium, although the absence of significant levels of 16:1omega5c argues for the uniqueness of these two strains. On the basis of the results described here, it is proposed that a new genus should be created, Methylosarcina gen. nov., harbouring two species, Methylosarcina fibrata sp. nov. (type species) and Methylosarcina quisquiliarum sp. nov. The type strains are AML-C10T (= ATCC 700909T = DSM 13736T) and AML-D4T (= ATCC 700908T = DSM 13737T), respectively.

Bacterial Proteins↗
PubMed1997

Methylosphaera hansonii gen. nov., sp. nov., a psychrophilic, group I methanotroph from Antarctic marine-salinity, meromictic lakes.

Methanotrophic bacteria were enumerated and isolated from the chemocline and surface sediments of marine-salinity Antarctic meromictic lakes located in the Vestfold Hills, Antarctica (68 degrees S 78 degrees E). Most probable number (MPN) analysis indicated that at the chemocline of Ace Lake the methanotroph population made up only a small proportion of the total microbial population and was sharply stratified, with higher populations detected in the surface sediments collected at the edge of Ace Lake and Burton Lake. Methanotrophs were not detected in Pendant Lake. Only a single phenotypic group of methanotrophs was successfully enriched, enumerated and isolated into pure culture from the lake samples. Strains of this group were non-motile, coccoidal in morphology, did not form resting cells, reproduced by constriction, and required seawater for growth. The strains were also psychrophilic, with optimal growth occurring at 10-13 degrees C and maximum growth temperatures of 16-21 degrees C. The ribulose monophosphate pathway but not the serine pathway for incorporation of C1 compounds was detectable in the strains. The guanine plus cytosine (G + C) content of the genomic DNA was 43-46 mol%. Whole-cell fatty acid analysis indicated that 16:1 omega 8c (37-41%), 16:1 omega 6c (17-19%), 16:1 omega 7c (15-19%) and 16:0 (14-15%) were the major fatty acids in the strains. 16s rDNA sequence analysis revealed that the strains form a distinct line of descent in the family Methylococcaceae (group I methanotrophs), with the closest relative being the Louisiana Slope methanotrophic mytilid endosymbiont (91.8-92.3% sequence similarity). On the basis of polyphasic taxonomic characteristics the Antarctic lake isolates represent a novel group I methanotrophic genus with the proposed name Methylosphaera hansonii (type strain ACAM 549).

Antarctic Regions↗
PubMed2000

Sterols in a psychrophilic methanotroph, Methylosphaera hansonii.

A suite of six sterols, lanosterol, lanost-8(9)-en-3beta-ol, 4, 4-dimethylcholesta-8(14),24-dien-3beta-ol, 4, 4-dimethylcholest-8(14)-en-3beta-ol, 4-methylcholesta-8(14), 24-dien-3beta-ol and 4-methylcholest-8(14)-en-3beta-ol, were identified in the psychrophilic methanotrophic bacterium, Methylosphaera hansonii. Their presence suggests that the capacity for sterol biosynthesis in methanotrophic bacteria is limited to the family Methylococcaceae but which have widely different optimal growth temperatures.

Antarctic Regions↗
PubMed2001

Group-specific 16S rRNA targeted probes for the detection of type I and type II methanotrophs by fluorescence in situ hybridisation.

The study of methane-oxidising bacteria (methanotrophs) is of special interest, because of their role in the natural reduction of methane emissions from many different sources. Therefore new probes were developed to detect specifically either type I (Methylococcaceae) or type II methanotrophs (Methylocystaceae). The probes have shown high specificity in fluorescence in situ hybridisations (FISH), as demonstrated by parallel hybridisation of target and reference strains as well as sequence data analysis. With these probes, methanotrophs were detected in soil and root samples from rice microcosms, demonstrating their applicability even in a complex environmental matrix.

Gram-Negative Aerobic Rods and Cocci↗
PubMed1989

Physiology, biochemistry, and specific inhibitors of CH4, NH4+, and CO oxidation by methanotrophs and nitrifiers.

Ammonia oxidizers (family Nitrobacteraceae) and methanotrophs (family Methylococcaceae) oxidize CO and CH4 to CO2 and NH4+ to NO2-. However, the relative contributions of the two groups of organisms to the metabolism of CO, CH4, and NH4+ in various environments are not known. In the ammonia oxidizers, ammonia monooxygenase, the enzyme responsible for the conversion of NH4+ to NH2OH, also catalyzes the oxidation of CH4 to CH3OH. Ammonia monooxygenase also mediates the transformation of CH3OH to CO2 and cell carbon, but the pathway by which this is done is not known. At least one species of ammonia oxidizer, Nitrosococcus oceanus, exhibits a Km for CH4 oxidation similar to that of methanotrophs. However, the highest rate of CH4 oxidation recorded in an ammonia oxidizer is still five times lower than rates in methanotrophs, and ammonia oxidizers are apparently unable to grow on CH4. Methanotrophs oxidize NH4+ to NH2OH via methane monooxygenase and NH4+ to NH2OH via methane monooxygenase and NH2OH to NO2- via an NH2OH oxidase which may resemble the enzyme found in ammonia oxidizers. Maximum rates of NH4+ oxidation are considerably lower than in ammonia oxidizers, and the affinity for NH4+ is generally lower than in ammonia oxidizers. NH4+ does not apparently support growth in methanotrophs. Both ammonia monooxygenase and methane monooxygenase oxidize CO to CO2, but CO cannot support growth in either ammonia oxidizers or methanotrophs. These organisms have affinities for CO which are comparable to those for their growth substrates and often higher than those in carboxydobacteria. The methane monooxygenases of methanotrophs exist in two forms: a soluble form and a particulate form. The soluble form is well characterized and appears unrelated to the particulate. Ammonia monooxygenase and the particulate methane monooxygenase share a number of similarities. Both enzymes contain copper and are membrane bound. They oxidize a variety of inorganic and organic compounds, and their inhibitor profiles are similar. Inhibitors thought to be specific to ammonia oxidizers have been used in environmental studies of nitrification. However, almost all of the numerous compounds found to inhibit ammonia oxidizers also inhibit methanotrophs, and most of the inhibitors act upon the monooxygenases. Many probably exert their effect by chelating copper, which is essential to the proper functioning of some monooxygenases. The lack of inhibitors specific for one or the other of the two groups of bacteria hampers the determination of their relative roles in nature.

Ammonia↗
PubMed2026

Elevated water levels drive greenhouse gas mitigation in the riparian zone profile.

Wetlands are critical for climate regulation, with their hyporheic zone serving as sensitive interfaces for groundwater-soil-atmosphere exchange. These zones are active hotspots for carbon-nitrogen cycling and greenhouse gas (GHG) emissions (CO2, CH4, N2O), yet the impact of water level fluctuations on these emissions and their microbial drivers in freshwater wetlands remains poorly understood. This study investigated the spatiotemporal dynamics of GHG emissions and carbon-nitrogen coupling processes along riparian soil profiles of Baiyangdian Lake during water level fluctuations. Employing static chamber measurements, microcosms, quantitative PCR, Metagenome-Assembled genome (MAG) analyses, and Structural Equation Modeling (SEM), we observed that GHG emissions were significantly affected by water level fluctuations. Specifically, CO2 and N2O fluxes, as well as CO2 production potential were significantly lower at high-water-level conditions. Water level also emerged as a key driver of microbial community structure, with Methylococcaceae and Methanosarcinaceae as key regulators of CH4 emission, and Anaeromyxobacteraceae as central to N2O dynamics. A high-quality Methylomirabilales-like MAG, possessing the complete pathway for coupled nitrate reduction and methane oxidation, was identified. Its abundance negatively correlated with water level, suggesting that these C-N coupling bacteria contribute to reducing GHG emissions. This study provides crucial theoretical insights and identifies microbial targets for mitigating wetland GHG emission through hydrological management.

Greenhouse Gases↗
PubMed1999

Demonstration of efficient trichloroethylene biodegradation in a hollow-fiber membrane bioreactor.

Rapid cometabolism of trichloroethylene (TCE) by pure cultures of Methylosinus trichosporium OB3b PP358 was demonstrated in a two-stage hollow-fiber membrane bioreactor over the course of 3 weeks. PP358 was grown in a continuous-flow chemostat and circulated through the shell of a hollow-fiber membrane module (HFMM), while TCE contaminated water (160 to 1450 micrograms/L) was pumped through the fiber lumen (fiber interior). In parallel-flow HFMM biological experiments, 82% to 89% of the influent TCE was removed from the lumen (5.1-min residence time) with 99% of the transferred TCE undergoing biodegradation. Biological experiments in a larger capacity baffled radial-flow HFMM resulted in 66% to 99% TCE transferred and 93% to 96% TCE biodegradation at lumen residence times of between 1.5 and 3.7 min. Biodegradation was maintained throughout the experiments at pseudo-first-order biodegradation rate constants of 0.41 to 2.8 L/mg TSS/day. Best-fit computer modeling of the baffled radial-flow biological process estimated mass transfer coefficients as large as 2.7 x 10(-2) cm/min. The computer model was also shown to simulate the experimental results quite well.

Biodegradation, Environmental↗
PubMed1999

Trichloroethene degradation in a two-step system by methylosinus trichosporium OB3b. Optimization of system performance: use of formate and methane.

The breakdown of dissolved TCE in a two-step bioremediation system is described. In the first reactor, the organism Methylosinus trichosporium OB3b is grown; in the second reactor, consisting of three 17-L column reactors in series, the cells degrade TCE. A special design allowed both for the addition of air (uG,s = 0.01-0. 04 mm s-1) in the conversion reactor to prevent oxygen limitation while minimizing stripping of TCE, and for the use of methane as exogenous electron donor. In two-step systems presented thus far, only formate was used (excess, 20 mM). We found formate additions could be reduced by 75% (15 degrees C), whereas small amounts of methane (0.02-0.04 mol CH4/g cells) could replace formate and led to equally optimal results. Example calculations show that up to 90% reduction in operating cost of chemicals can be obtained by using methane instead of formate. A model was developed to describe each of the conditions studied: excess formate and optimal methane addition, suboptimal formate addition and suboptimal methane addition. Using parameters obtained from independent batch experiments, the model gives a very good description of the overall TCE conversion in the two-step system. The system presented is flexible (oxygen/methane addition) and can easily be scaled up for field application. The model provides a tool for the design of an effective and low-cost treatment system based on methane addition in the conversion reactor.

Biodegradation, Environmental↗
PubMed1999

Biomass accumulation and clogging in biotrickling filters for waste gas treatment. Evaluation of a dynamic model using dichloromethane as a model pollutant.

A dynamic model is developed that describes the degradation of volatile acidifying pollutants in biotrickling filters (BTFs) for waste gas purification. Dynamic modelling enables the engineer to predict the clogging rate of a filter bed and the time it takes the BTF to adapt to changes in its inlet concentration. The most important mechanisms that govern the behaviour of the BTF are incorporated in the model. The time scale of the accumulation of biomass in a filter is investigated, and an approach is presented that can be used to estimate how long a BTF can be operated before its packing has to be cleaned. A three-month experiment was carried out to validate the model, using dichloromethane (DCM) as a model acidifying pollutant. Valuable experimental data about biomass accumulation and liquid hold-up in the reactor were obtained with an experimental set-up that allows the continuous registration of the weight of the BTF. The results show that in BTFs eliminating DCM from a waste gas, clogging is not to be expected up to concentrations of several g/m3. Model calculations based on the measurements also suggest that the maximum carbon load that can safely be applied per unit void packing volume should not exceed 0.5-1.6 C mol/(m3. h), depending on the density of the biofilm formed. The model is a good predictor of the elimination of the pollutant in the system, the axial gas and liquid concentration profiles, the axial biomass distribution, and the response of the system upon a stepwise increase in the DCM inlet concentration. The influence of the buffer concentrations in the liquid phase upon the performance of the BTF is investigated.

Air Pollutants↗
PubMed1998

Purification and properties of methanol dehydrogenase from Methylosinus sp. WI 14.

Similarly to the recently described methanol dehydrogenase (MDH) from Methylocystis sp. GB 25 (Grosse et al. 1997) MDH from Methylosinus sp. WI 14 is able to catalyse the oxidation of methanol to formate directly. The enzyme was purified about 9-fold to electrophoretic homogeneity and is localised in the soluble fraction. The relative molecular mass of the native enzyme has been determined to be 140 kDa. It is composed of two identical subunits of relative molecular mass 70 kDa. The amino terminal sequence shows a strong similarity (a match of 80% over the first 20 amino acids) to the MDH from Methylocystis sp. GB 25. PQQ could be detected as the prosthetic group of MDH in the purified enzyme fraction by using the apoenzyme of a membrane-bound glucose dehydrogenase from Pseudomonas aeruginosa. A PQQ ratio of 1.3 per mole MDH was estimated. The purified enzyme has an optimum activity at pH 9.0 and at 57 degrees C. MDH from Methylosinus sp. WI 14 oxidises only primary alcohols up to octanol and several aldehydes. The estimated K(m)-values vary between 0.18 mM for the sorbic alcohol and 6.3 mM for butanol and show no dependence upon the chain length.

Alcohol Oxidoreductases↗
PubMed1990

Effect of fixation-resin combinations and ruthenium red on elucidating outer envelope structure and surface morphology of two methanotrophic bacteria.

We examined the ultrastructure of the cell envelope in Type I, Methylomonas albus (BG8), and Type II, Methylosinus trichosporium (OB3b), methane-oxidizing bacteria by using different fixatives, ruthenium red (RR) combinations and resins. We compared LR White and Spurr embedments with the following fixations: glutaraldehyde/OsO4, two glutaraldehyde-paraformaldehyde, and two different en bloc ruthenium red procedures, one utilizing OsO4 and the other with glutaraldehyde/OsO4 in sequential fixation. These fixations were also studied by scanning electron microscopy (SEM). Unfixed cells prepared by freeze etch were used for comparison. Transmission electron microscopy of BG8 embedded in LR White resin (with or without red0 preserved a layer of cup-like structures that were not seen in Spurr resin-embedded cells unless ruthenium red was used. For OB3b, the second RR method preserved beads and filaments where only "spike-like" structures were seen in all other fixations in both resins. By SEM, all fixations preserved a capsular slime layer of BG8 that was removed from some cells by both RR methods. In all SEM fixations, a bead layer was preserved in OB3b that was enhanced by RR. Filaments seen by freeze-etch and thin-section techniques were not seen in SEM. Presence or absence of particular envelope structures in these methanotrophs is dependent on the combination of fixatives and/or resins employed and is species-specific. The chemical preparation methods used resulted in enhanced understanding of the structure and composition of the cell envelope.

Cell Membrane↗
PubMed1992

Comparison of alcian blue and ruthenium red effects on preservation of outer envelope ultrastructure in methanotrophic bacteria.

Alcian blue (AB) and ruthenium red (RR) effects on ultrastructural preservation of the bacterial cell envelope of methanotrophs are compared. A previous successful method with RR that enhanced preservation of outer envelope layers in two representative methanotroph species is applied to other genera and species of methanotropic bacteria. Alcian blue is substituted for RR in this en bloc protocol. The effect of AB on preservation of these layers is assessed at the ultrastructural level and compared to RR for all species examined. Further, comparison with freeze etch and a fixation in the absence of either RR or AB is made. Both RR and AB are found to aid preservation and help visualize additional components of the cell envelope which are lost or minimized in a standard fixation not employing these cationic reagents. For some species, images obtained are similar between RR and AB procedures and agree with images seen by freeze etch. For other species, AB preserves extended filamentous material that is partially condensed even with the use of RR. Thus, use of AB improves the preservation of outer envelope structure in these organisms equally or more effectively than use of RR.

Alcian Blue↗