Growth of mesophilic methanotrophs at low temperatures

The optimal growth of mesophilic methanotrophic bacteria (collection strains of the genera Methylocystis, Methylomonas, Methylosinus, and Methylobacter) occurred within temperature ranges of 31-34 degrees C and 23-25 degrees C. None of the strains studied were able to grow at 1.5 or 4 degrees C. Representatives of six methanotrophic species (strains Mcs. echinoides 2, Mm. methanica 12, Mb. bovis 89, Mcs. pyriformis 14, Mb. chroococcum 90, and Mb. vinelandii 87) could grow at 10 degrees C (with a low specific growth rate). The results obtained suggest that some mesophilic methane-oxidizing bacteria display psychrotolerant (psychrotrophic) but not psychrophilic properties. In general, the Rosso model, which describes bacterial growth rate as a function of temperature, fits well the experimental data, although, for most methanotrophs, with symmetrical approximations for optimal temperature.     

Growth of methanotrophs in methane and oxygen counter gradients

Abstract A gel-stabilized system with counter gradients of CH₄ and O₂ was used to grow methanotrophs from wetland, agricultural and forest soils and lake sediment. Columns of semi-solid nitrate- or ammonium-minerai salts medium were continuously flushed at opposite ends with CH₄ and O₂ to create opposing concentration gradients of the two gases. Methanotrophs grew from all samples except forest soil, and were visible as thin bands after 5 to 15 days of incubation. The position of growth was CH₄ and O₂ concentration-dependent and occurred at the point of maximum possible CH₄ oxidation, where both substrates were completely consumed. Evidence was obtained for denitrification and nitrification activities concomitant with CH₄ oxidation. This approach may be useful to isolate methanotrophs with different CH₄ and O₂ requirements and to study their interactions with other groups of bacteria in nature.     

Growth yields of bacteria on selected organic compounds

Cell yields were determined for two bacterial soil isolants grown aerobically in minimal media on a variety of synthetic organic compounds. 1-Dodecanol, benzoic acid, phenylacetic acid, phenylglyoxylic acid, and diethylene, triethylene, and tetraethylene glycols were tested. Two “biochemicals,” succinate and acetate, were also tested for comparison. Yields were calculated on the basis of grams of cells obtained per mole of substrate utilized, gram atom of carbon utilized, mole of oxygen consumed, and equivalent of “available electrons” in the substrates. This latter value appears to be nearly constant at 3 g of cells per equivalent of “available electrons.” Yields predicted on this basis for other bacteria and for yeasts on other substrates are in fair agreement with reported values.     

Growth yields in bacterial denitrification and nitrate ammonification

Denitrification and nitrate ammonification are considered the highest-energy-yielding respiration systems in anoxic environments after oxygen has been consumed. The corresponding free energy changes are 7 and 35% lower than that of aerobic respiration, respectively. Growth yield determinations with pure cultures of Paracoccus denitrificans and Pseudomonas stutzeri revealed that far less energy is converted via ATP into cell mass than expected from the above calculations. Denitrification with formate or hydrogen as electron donor yielded about 2.4 to 3.0 g dry matter per mol formate or hydrogen and 15 to 18 g dry matter per mol acetate. Similar yields with acetate were obtained with Pseudomonas stutzeri. Wolinella succinogenes and Sulfurospirillum deleyianum, which reduce nitrate to ammonia, both exhibited similar yield values with formate or H2 plus nitrate. The results indicate that ATP synthesis in denitrification is far lower than expected from the free energy changes and even lower than in nitrate ammonification. The results are discussed against the background of our present understanding of electron flow in denitrification and with respect to the importance of denitrification and nitrate ammonification in the environment.     

Biogeography of wetland rice methanotrophs

We focused on the functional guild of methane oxidizing bacteria (MOB) as model organisms to get deeper insights into microbial biogeography. The pmoA gene was used as a functional and phylogenetic marker for MOB in two approaches: (i) a pmoA database (> 4000 sequences) was evaluated to obtain insights into MOB diversity in Italian rice paddies, and paddy fields worldwide. The results show a wide geographical distribution of pmoA genotypes that seem to be specifically adapted to paddy fields (e.g. Rice Paddy Cluster 1 and Rice Paddy Cluster 2). (ii) On the smaller geographical scale, we designed a factorial experiment including three different locations, two rice varieties and two habitats (soil and roots) within each of three rice fields. Multivariate analysis of terminal restriction fragment analysis profiles revealed different community patterns at the three field sites, located 10–20 km apart. Root samples were characterized by high abundance of type I MOB whereas the rice variety had no effect. With the agronomical practice being nearly identical, historical contingencies might be responsible for the field site differences. Considering a large reservoir of viable yet inactive MOB cells acting as a microbial seed bank, environmental conditions might have selected and activated a different subset at a time thereby shaping the community.     

Thermophilic and thermotolerant aerobic methanotrophs

The review generalizes the modern data on the taxonomic, structural, and functional diversity of aerobic methanotrophs growing at 25–50°C (Methylococcus capsulatus), 30–62°C (Methylocaldum szegediense, Methylocaldum gracile, and Methylocaldum tepidum), and 50–65°C (Methylothermus thermalis), which belong mainly to the Gammaproteobacteria. The specific features of adaptation of these methanotrophs to the temperature influences are considered on the metabolic and genetic levels. The recent sensational reports on the discovery and primary characterization of thermoacidophilic methanotrophs of the phylum Verrucomicrobia surviving at extreme pH (1–2) and temperature (65°C) values, corresponding to extremely low levels of CH₄ and O₂ solubility, are analyzed. The possibilities of implementation of the biotechnological potential of thermophilic and thermotolerant methanotrophs are discussed.     

Nitrogen metabolism in marine methanotrophs

Two new methane-oxidizing bacteria have been isolated from seawater samples from Plymouth Sound. These marine methanotrophs have an obligate requirement for NaCl and exhibit many properties of typical Type I methanotrophs previously isolated from freshwater environments. However, they are different from all other methanotrophs thus far described in that they failed to grow on all solid media tested. The nitrogen metabolism of both strains was investigated. They were not N₂-fixers nor would they use ammonia as nitrogen source. They appeared to utilize the glutamate dehydrogenase pathway for the assimilation of ammonia under all growth conditions tested.     

Isolation and characterization of marine methanotrophs

Four new methane-oxidizing bacteria have been isolated from marine samples taken at the Hyperion sewage outfall, near Los Angeles, CA. These bacteria require NaCl for growth. All exhibit characteristics typical of Type I methanotrophs, except they contain enzyme activities of both the ribulose monophosphate pathway and the serine cycle. All four strains are characterized by rapid growth in liquid culture and on agar plates, and all have temperature optima above 35° C. One strain, chosen for further study, has been shown to maintain broadhost range cloning vectors and is currently being used for genetic studies.     

Distribution of methanotrophs in the phyllosphere

Plants have been reported to emit methane as well as methanol originating in their cell-wall constituents. We investigated methanotrophs in the phyllosphere by the enrichment culture method with methane as sole carbon source. We enriched methanotrophs from the leaves, flowers, bark, and roots of various plants. Analysis of the pmoA and mxaF genes retrieved from the enrichment cultures revealed that methanotrophs closely related to the genera Methylomonas, Methylosinus, and Methylocystis inhabit not only the rhizosphere but also the phyllosphere, together with methanol-utilizing bacteria.     

Growth yields of Desulfotomaculum orientis with hydrogen in chemostat culture

Desulfotomaculum orientis (strain Singapore 1) was grown autotrophically with H₂+CO₂ and sulfate, thiosulfate or sulfite as electron acceptor in sulfide- and pH-controlled continuous culture. Under sulfate-limiting conditions real growth yields of up to 9.7 g cell dry mass per mol sulfate were obtained. Electron acceptor limitation resulted in the excretion of up to 14.5 mmol acetate per liter, formed by reduction of CO₂ with H₂. Acetate production was not coupled to an increase of growth yields: under hydrogen-limiting conditions only 1.6 mmol acetate per liter was produced, and even higher growth yields of up to 12,4 g cell dry mass per mol sulfate were obtained. With thiosulfate or sulfite as electron acceptor growth yields increased up to 17.9 g cell dry mass per mol electron acceptor. Growth yields were not simply correlated with the growth rate, and did not allow the determination of maintenance coefficients and the extrapolation to maximal yields at infinite growth rate (Y ^max). The maximal growth rates (_max) with sulfate and thiosulfate were 0.090 and 0.109 h^-1, respectively, if cells were grown continuously in sulfidostat culture under nonlimiting conditions.The net energy yield of sulfate reduction and the energy requirement for the activation of sulfate by Desulfotomaculum orientis are discussed.     

Growth yields and saturation constant of Desulfovibrio vulgaris in chemostat culture

Desulfovibrio vulgaris (strain Marburg) was grown on H₂ and sulfate as sole energy source in a chemostat limited by the sulfate supply. The biomass concentration and the sulfate concentration in the culture were determined as a function of the dilution rate. From the data a K _S (saturation constant) for sulfate of 10 M, a _max of 0.23 h^–1, and a of 13 g/mol were calculated. The organism was also grown in chemostat culture on H₂ and sulfite, H₂ and thiosulfate, and pyruvate (without sulfate). was found to be 35 g/mol, 36 g/mol, and Y _pyr ^max 10 g/mol. The growth yields are discussed with respect to ATP gains in dissimilatory sulfate reduction.     

Bacteriophages of methanotrophs isolated from fish

Bacteriophages of methanotrophic bacteria were isolated from 67 fish. Only two phages isolated from two fish species specifically lysed Methylocystis sp. and Flavobacterium gasotypicum. The phages lysing these species were designated 63-F and CMF-1-F, respectively. The isolated phages differed greatly in the fine structure of the virion, plaque morphology, spectrum of lytic action, serological properties, and UV sensitivity. At the same time, they had identical one-step growth characteristics: their latent period equalled 5 h, lysis time was 3 to 4 h, and burst size was about 240 virions. The phages had guanine- and cytosine-rich double-stranded DNAs consisting of common nitrogen bases. The molecular masses of the DNAs as determined by the sums of restriction endonuclease cleavage fragments were 28 X 10(6) daltons for phage 63-F and 31 X 10(6) daltons for phage CMF-1-F.     

Biology of extremophilic and extremotolerant methanotrophs

This review summarizes recent findings on the biology of obligate methanotrophic bacteria living in various extreme environments. By using molecular ecology techniques, it has become clear that obligate methanotrophs are ubiquitous in nature and well adapted to high or low temperature, pH and salinity. The isolation and characterization of pure cultures has led to the discovery of several new genera and species of extremophilic/tolerant methanotrophs. Their major physiological role is participation in the methane cycle and supplying C(1) intermediates and various metabolites to other members of microbial communities in extreme ecosystems. To survive under extreme conditions, methanotrophs have developed diverse structure-function adaptive mechanisms including cell-surface layer formation, changes in cellular phospholipid composition and de novo synthesis of organic osmolytes such as ectoine, 5-oxoproline and sucrose. However, despite the above advances, basic knowledge of other stress protectants, as well as bioenergetic and genetic aspects of methanotroph adaptation, is still lacking. This information is necessary for better understanding the molecular mechanisms underlying the versatility of methanotrophs and for the development of novel biotechnological processes.     

Genetics and molecular biology of methanotrophs

Abstract Over the last 20 years or so, the obligate methane-oxidizing bacteria (methanotrophs) have attracted considerable interest. As they grow on a relatively cheap and abundant carbon source, they appeared ideal organisms for the production of bulk chemicals, single-cell protein and for use in biotransformations. More recently their cooxidation properties have been investigated for bioremediation, including the removal of chlorinated compounds such as trichloroethylene from polluted groundwaters. These studies have resulted in a great deal of information on the physiology and biochemistry of methanotrophs but sadly the molecular biology and genetic studies of these organisms have lagged behind. This has been in part due to the obligate nature of the methanotrophs and the refractory nature of such organisms to conventional genetic analysis. However, the more recent availability of broad-host range plasmids coupled with improvements in molecular biology methods have allowed the development of molecular genetic techniques for methanotrophs. The purpose of this review is to summarize what is known about the genetics and molecular biology of methanotrophs and how this information can be used to complement previous and current biochemical studies on the unique property of these bacteria, i.e. the ability to oxidize methane to methanol. Recent developments in molecular ecology techniques that may be applied to these apparently ubiquitous organism are also considered.     

Characterization of two new facultative methanotrophs

Two new facultative methane-oxidizing bacteria have been isolated from lake water enrichments. The organisms have been characterized in terms of colony types, growth characteristics, the guanine plus cytosine content of their deoxyribonucleic acid, thin sections, oxidation rates, and carbon assimilation pathways. Methane-grown cells of both organisms contained intracytoplasmic membranes similar to those described as type II in other methanotrophic bacteria. Neither organism had such membranes when grown heterotrophically. Both organisms assimilated methane by way of the isocitrate lyase-negative serine pathway for formaldehyde incorporation. The enzymes of this pathway were high in specific activity in cells grown on methane and were at low levels in cells grown either on heterotrophic substrates or on heterotrophic substrates plus methane. It is proposed that both organisms be classified in the genus Methylobacterium as two new species, Methylobacterium ethanolicum and Methylobacterium hypolimneticum.     

Studies on phosphate metabolism in obligate methanotrophs

Abstract Studies on three representative species of methane-utilizing bacteria revealed high intracellular levels of inorganic pyrophosphate (5 mM) in contrast to low levels of ATP (0.5 mM) as well as the differences in the sets of enzymes utilizing pyro- and polyphosphates as phosphate and metabolic energy donors.     

Synthesis of osmoprotectors by halophilic and alkalophilic methanotrophs

The 1H-NMR analysis of methanol extracts of halophilic and halotolerant alkaliphilic methanotrophs isolated from the soda lakes of Southern Transbaikal and Tuva showed that bacterial cells grown at an optimum salinity accumulated mainly sucrose and 5-oxo-1-proline, whereas cells adapted to 0.5-1.0 M NaCl additionally synthesized ectoine. A more detailed study showed that nitrogen deficiency in the growth medium of Methylobacter alcaliphilus 20Z decreased the synthesis of nitrogen-containing osmoprotectants, ectoine and 5-oxo-1-proline. M. alcaliphilus 20Z cells exhibited activities of UDP-glucose pyrophosphorylase and sucrose-phosphate synthase involved in sucrose synthesis. Glutamine synthetase in vitro did not require NH4+ ions, which implies that this enzyme is involved in 5-oxo-1-proline synthesis. Cells grown at high salinity exhibited elevated levels of aspartate kinase, aspartate-semialdehyde dehydrogenase, and ectoine synthase. This suggests that ectoine is synthesized via aspartate and aspartate-semialdehyde, i.e., via the route earlier established for extremely halophilic bacteria.