Ethane oxidation by methane-oxidizing bacteria

The relationship between the rates of methane and ethane oxidation by washed suspensions of methane-oxidizing bacteria has been investigated. Considerable differences between bacterial strains were observed. Two closely related Methylomonas strains which differed in their oxidizing capacity were further investigated. The low ethane oxidation rate of one strain could be strongly stimulated by the addition of oxidizable co-substrates, and the presence of ethane stimulated formate oxidation. The other strain had a much higher ethane oxidation rate and stimulation by co-substrates was negligible.Differences between the levels of dissimilative enzymes in cell-free extracts could not be detected. Attempts to produce extracts with methane mono-oxygenase activity failed. When cells were made permeable with chitosan the results suggested that strains with a low ethane oxidizing capacity obtain the required reductant for the mono-oxygenase from endogenous respiration. In strains with a high ethane oxidation rate, the reductant appears to be derived from oxidation of ethanol or acetaldehyde.     

Microbial transformations of selenite by methane-oxidizing bacteria

Methane-oxidizing bacteria are well known for their role in the global methane cycle and their potential for microbial transformation of wide range of hydrocarbon and chlorinated hydrocarbon pollution. Recently, it has also emerged that methane-oxidizing bacteria interact with inorganic pollutants in the environment. Here, we report what we believe to be the first study of the interaction of pure strains of methane-oxidizing bacteria with selenite. Results indicate that the commonly used laboratory model strains of methane-oxidizing bacteria, Methylococcus capsulatus (Bath) and Methylosinus trichosporium OB3b, are both able to reduce the toxic selenite (SeO₃ ^2?) but not selenate (SeO₄ ^2?) to red spherical nanoparticulate elemental selenium (Se⁰), which was characterized via energy-dispersive X-ray spectroscopy (EDXS), X-ray absorption near-edge structure (XANES) and extended X-ray absorption fine structure (EXAFS). The cultures also produced volatile selenium-containing species, which suggests that both strains may have an additional activity that can transform either Se⁰ or selenite into volatile methylated forms of selenium. Transmission electron microscopy (TEM) measurements and experiments with the cell fractions cytoplasm, cell wall and cell membrane show that the nanoparticles are formed mainly on the cell wall. Collectively, these results are promising for the use of methane-oxidizing bacteria for bioremediation or suggest possible uses in the production of selenium nanoparticles for biotechnology.

     

Selection of resistant bacteria at very low antibiotic concentrations

The widespread use of antibiotics is selecting for a variety of resistance mechanisms that seriously challenge our ability to treat bacterial infections. Resistant bacteria can be selected at the high concentrations of antibiotics used therapeutically, but what role the much lower antibiotic concentrations present in many environments plays in selection remains largely unclear. Here we show using highly sensitive competition experiments that selection of resistant bacteria occurs at extremely low antibiotic concentrations. Thus, for three clinically important antibiotics, drug concentrations up to several hundred-fold below the minimal inhibitory concentration of susceptible bacteria could enrich for resistant bacteria, even when present at a very low initial fraction. We also show that de novo mutants can be selected at sub-MIC concentrations of antibiotics, and we provide a mathematical model predicting how rapidly such mutants would take over in a susceptible population. These results add another dimension to the evolution of resistance and suggest that the low antibiotic concentrations found in many natural environments are important for enrichment and maintenance of resistance in bacterial populations.     

Extraction of methane-oxidizing bacteria from soil particles

Abstract: We present a method for extraction of active methane (CH₄)-oxidizing bacteria from soil samples. The method is based on physical dispersion of bacteria from the soil particles followed by separation of bacteria and soil particles by floatation in the density media Nycodenz or Percoll. Separation on Nycodenz produced very pure bacterial suspensions while separation on Percoll produced rather impure suspensions. However, more than 60% of the methane-oxidizing activity was irreversibly inhibited in the procedure using Nycodenz compared to less than 10% irreversible inhibition when Percoll was employed. The bacterial suspensions extracted from soil can be used to study the physiology and ecology of soil bacteria that oxidize methane at atmospheric concentrations. Our data indicated that these bacteria are extremely difficult to dislodge from particles compared to the majority of bacteria in soil. Tentatively, we interpret the strong attachment to long residence time (i.e. slow turnover) of the methane-oxidizing bacteria. A slow turnover/growth rate would explain why soil disturbances, like cultivation, have a long lasting effect on the oxidation of atmospheric methane in soil.     

Crystal structures of E. coli laccase CueO at different copper concentrations

CueO protein is a hypothetical bacterial laccase and a good laccase candidate for large scale industrial application. Four CueO crystal structures were determined at different copper concentrations. Low copper occupancy in apo-CueO and slow copper reconstitution process in CueO with exogenous copper were demonstrated. These observations well explain the copper dependence of CueO oxidase activity. Structural comparison between CueO and other three fungal laccase proteins indicates that Glu106 in CueO constitutes the primary counter-work for reconstitution of the trinuclear copper site. Mutation of Glu106 to a Phe enhanced CueO oxidation activity and supported this hypothesis. In addition, an extra alpha-helix from Leu351 to Gly378 covers substrate biding pocket of CueO and might compromises the electron transfer from substrate to type I copper.     

Comparative characterization of cultured methane-oxidizing bacteria by serological and molecular methods

Three stable methane-oxidizing enrichment cultures, SB26, SB31, and SB31A were analyzed by transmission electron microscopy and by serological and molecular techniques. Electron microscopy revealed the presence of both type I and type II methanotrophs in SB31 and SB31A enrichments; only type II methanotrophs were found in SB26 enrichment. Methylosinus trichosporium was detected in all three enrichments by the application of species-specific antibodies. Additionally, Methylocystis echinoides was found in SB26 culture; Methylococcus capsulatus, in SB31 and SB31A; and Methylomonas methanica, in SB31. The analysis with pmoA and nifH gene sequences as phylogenetic markers revealed the presence of Methylosinus/Methylocystis group in all communities. Moreover, the analysis of pmoA sequences revealed the presence of Methylomonas in SB31. Methylocella was detected in SB31 and SB31A enrichments only by nifH analysis. It was concluded that the simultaneous application of different approaches reveals more reliable information on the diversity of methanotrophs.     

甲烷氧化混合菌的保藏方法研究

【目的】甲烷氧化混合菌是自然界中吸收甲烷的关键微生物,在甲烷氧化混合菌的研究和应用中,首先要解决其长期稳定保藏的问题,保藏方法应能有效保持菌群结构和功能的完整性、稳定性。【方法】以从煤矿土壤富集得到的两种结构稳定的甲烷氧化混合菌为实验体系,研究对比了冷藏法、低温冷冻法、石蜡油冷冻法、甘油冷冻法4种保藏方法,考察保藏前后混合菌的生长状况、MMO活性、菌群结构等。【结果】保藏6个月后,除甘油冷冻法以外,经其它3种方法保藏的混合菌,都具有与保藏前相当的细胞密度、甲烷氧化能力、MMO酶活以及传代稳定性,且DGGE图谱显示保藏前后的菌群结构变化不大。【结论】这3种保藏方法都可以有效的保持甲烷氧化混合菌功能和菌群结构的稳定性。     

Atmospheric methane removal by methane-oxidizing bacteria immobilized on porous building materials

Biological treatment using methane-oxidizing bacteria (MOB) immobilized on six porous carrier materials have been used to mitigate methane emission. Experiments were performed with different MOB inoculated in building materials at high (~20 % (v/v)) and low (~100 ppmv) methane mixing ratios. Methylocystis parvus in autoclaved aerated concrete (AAC) exhibited the highest methane removal rate at high (28.5?±?3.8 μg CH₄ g^?1 building material h^?1) and low (1.7?±?0.4 μg CH₄ g^?1 building material h^?1) methane mixing ratio. Due to the higher volume of pores with diameter >5 μm compared to other materials tested, AAC was able to adsorb more bacteria which might explain for the higher methane removal observed. The total methane and carbon dioxide-carbon in the headspace was decreased for 65.2?±?10.9 % when M. parvus in Ytong was incubated for 100 h. This study showed that immobilized MOB on building materials could be used to remove methane from the air and also act as carbon sink.     

Isolation and characterization of two vibrio-shaped methane-oxidizing bacteria

An enrichment method for, and the isolation of two related vibrio-shaped methane-oxidizing bacteria are described. Their morphological and physiological characteristics are given. As a name for the genus of the organisms Methy lovibrio is proposed.We wish to thank Miss W. E. de Boer and J. van der Toorn of this laboratory for making the photographs. One of the authors (P. J. Steennis) is indebted to the Royal Netherlands Fermentation Industries Ltd., Delft for a grant.     

Activity of methane-oxidizing bacteria in the adsorbed state]

Adsorption of pure cultures of methane oxidizing bacteria, Methylosinus trichosporium 20 and Methylococcus ucrainicus 21, on glass and coal was studied; the former strain was sorbed on both sorbents, the latter strain was sorbed on coal but not on glass. The rate of methane oxidation by the cells of adsorbed microorganisms was higher than in the case of free cells, and increased with a decrease in dimensions of the sorbent particles.     

Accumulation of intermediates in a denitrifying process at different copper and high nitrate concentrations

NO2- accumulation occurred in an upflow anaerobic sludge blanket reactor at 500, 1000 and 1500 mg NO3- -N/l, but not after copper in the medium was increased to 28 g Cu2/l. N2O release was not avoided even with 56 g Cu2/l, at 1500 and 2500 mg NO3- -N/l and reached 251 mg N2O-N/ld. © Rapid Science Ltd. 1998     

Chemical and structural status of copper associated with oxygenic and anoxygenic phototrophs and heterotrophs: possible evolutionary consequences

Copper adsorption on the surface and intracellular uptake inside the cells of four representative taxons of soil and aquatic micro‐organisms: aerobic rhizospheric heterotrophs (Pseudomonas aureofaciens), anoxygenic (Rhodovulum steppense) and oxygenic (cyanobacteria Gloeocapsa sp. and freshwater diatoms Navicula minima) phototrophs were studied in a wide range of pH, copper concentration, and time of exposure. Chemical status of adsorbed and assimilated Cu was investigated using in situ X‐ray absorption spectroscopy. In case of adsorbed copper, XANES spectra demonstrated significant fractions of Cu(I) likely in the form of tri‐coordinate complexes with O/N and/or S ligands. Upon short‐term reversible adsorption at all four studied micro‐organisms’ cell surface, Cu(II) is coordinated by 4.0 ± 0.5 planar oxygens at an average distance of 1.97 ± 0.02 Å, which is tentatively assigned to the carboxylate groups. The atomic environment of copper incorporated into diatoms and cyanobacteria during long‐term growth is similar to that of the adsorbed metal with slightly shorter distances to the first O/N neighbor (1.95 Å). In contrast to the common view of Cu status in phototrophic micro‐organisms, XAFS failed to detect sulfur in the nearest atomic environment of Cu assimilated by freshwater plankton (cyanobacteria) and periphyton (diatoms). The appearance of S in Cu 1st coordination shell at 2.27–2.32 Å was revealed only after long‐term interaction of Cu with anoxygenic phototrophs (and Cu uptake by soil heterotrophs), suggesting Cu scavenging in the form of sulfhydryl, histidine/carboxyl or a mixture of carboxylate and sulfhydryl complexes. These new structural constraints suggest that adsorbed Cu(II) is partially reduced to Cu(I) already at the cell surface, where as intracellular Cu uptake and storage occur in the form of both Cu(I)‐S linked proteins and Cu(II) carboxylates. Obtained results allow to better understand how, in the course of biological evolution, micro‐organisms elaborated various mechanisms of Cu uptake and storage, from passive adsorption and uptake to active, protein‐controlled surface reduction, and intracellular storage.