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.     

Some cultural and physiological aspects of methane-utilizing bacteria

A number of different methane-utilizing bacteria are described and compared with isolates of other investigators. The strains can be divided into three groups based on pigmentation, cell morphology and internal membrane structures. The oxidation of hydrocarbons, alcohols, aldehydes, fatty acids, methyl ethers and sugar phosphates by these bacteria was studied. There was much similarity between strains within the same group. Differences between groups as regards oxidative properties could be detected, but these were mainly quantitative and could not be used as taxonomical criteria. In addition, the inhibition of methane oxidation by metabolites and enzyme inhibitors was investigated. Formaldehyde proved to be the most active of the organic compounds tested. Iodoacetic acid inhibited both methane and methanol oxidation at concentrations of 0.03m or above. Of the inorganic compounds, KCN completely suppressed methane oxidation at 5×10^?4 m and to more than 90% at 5×10^?5 m.     

Some characteristics of Methylococcus mobilis sp. nov.

A methane-assimilating coccus was isolated from a continuous culture. Under certain conditions, in pure culture the cells grew in sarcina-like, refractile clusters.That these clusters resembled multiple-bodied cysts was confirmed by electron microscopy. In addition, tube-like structures, not previously reported in methane-oxidizing bacteria, were found. Motile cells, with one or, rarely, two flagella were occasionally observed. Methane and methanol were exclusively assimilated as the sole source of carbon and energy. Formaldehyde, formate, lower alkanes, alcohols and aldehydes, with the exception of acetaldehyde, were oxidized by cell suspensions. The presence of a number of key enzymes in cell-free extracts suggested that the ribulose-monophosphate pathway of carbon assimilation is active. This agreed with the internal membrane structure found (type I organism).Nitrate, ammonia, peptone, yeast extract, asparagine, aspartic acid and glutamic acid were assimilated as sources of nitrogen. No nitrogen fixation could be demonstrated.The cells contained 9.1–9.7% nitrogen and 7–10% lipid, dependent on the growth conditions. Poly--hydroxybutyric acid could not be detected. The guanine+cytosine (G+C) content of the DNA was found to be 56.3%. As a name for the organism Methylococcus mobilis sp. nov. is proposed.Abbreviations used G+C Guanine+Cytosine     

Growth of Thiobacillus ferrooxidans on Formic Acid

A variety of acidophilic microorganisms were shown to be capable of oxidizing formate. These included Thiobacillus ferrooxidans ATCC 21834, which, however, could not grow on formate in normal batch cultures. However, the organism could be grown on formate when the substrate supply was growth limiting, e.g., in formate-limited chemostat cultures. The cell densities achieved by the use of the latter cultivation method were higher than cell densities reported for growth of T. ferrooxidans on ferrous iron or reduced sulfur compounds. Inhibition of formate oxidation by cell suspensions, but not cell extracts, of formate-grown T. ferrooxidans occurred at formate concentrations above 100 μM. This observation explains the inability of the organism to grow on formate in batch cultures. Cells grown in formate-limited chemostat cultures retained the ability to oxidize ferrous iron at high rates. Ribulose 1,5-bisphosphate carboxylase activities in cell extracts indicated that T. ferrooxidans employs the Calvin cycle for carbon assimilation during growth on formate. Oxidation of formate by cell extracts was NAD(P) independent.     

The use of β-propiolactone for the sterilization of heat-labile materials

A short literature survey on chemical sterilization with -propiolactone is given. The auto-inactivatio 1 of this compound in aqueous solution appears to be one of its main advantages.Time-kill studies have been carried out with bacterial cells and spores, and with fungal spores. The compatibility of BPL with textiles and plastics was also studied to some extent. It is concluded that heat-sensitive materials can be sterilized with a 1.0% aqueous solution of BPL within one hour at room temperature. The compatibility of BPL with the materials studied does not appear to offer serious problems.     

Kinetics and energetics of reduced sulfur oxidation by chemostat cultures of Thiobacillus ferrooxidans

Thiobacillus ferrooxidans was grown in chemostat cultures with thiosulfate and tetrathionate as the limiting substrates. The yields at steady state on both substrates at different dilution rates were calculated. In a few experiments the air supply was supplemented with 2% CO₂ (v/v). This resulted in a slightly increased yield.Cells from the chemostat cultures were used to study the kinetics of thiosulfate, tetrathionate, sulfite and sulfide oxidation. With all substrates mentioned the Ks values were in the micromolar range. The values for thiosulfate and tetrathionate were 2 orders of magnitude lower that those published previously.     

Observations on the fine structure of a methane-oxidizing bacterium

Structural details of membranes, intracellular organelles, and the cell wall of a methane-utilizing bacterium identified as aMethylomonas sp. were studied by electron microscopy. The cell wall structure is similar to that found in other gram-negative bacteria. The cytoplasmic membrane shows invaginations, presumably forming the internal membrane bundles. Two types of polar organelles were encountered. In older cells myelin-like structures were observed. Under certain cultural conditions bleb-formation and possibly accumulation of reserve materials occurred. We wish to thank Mrs. M. H. Bakker-van der Velden, Mrs. W. H. Batenburgvan der Vegte and Miss J. C. de Bruyn for making the electron microscopical preparations and photographs. Thanks are due to Mr R. S. M. Revell of the Philips E. M. Application Laboratory, Eindhoven, for the photographs with the goniometer attachment.     

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.