Pulsed high-field gradient in vivo NMR spectroscopy to measure diffusional water permeability in Corynebacterium glutamicum.

Pulsed high-field gradient in vivo NMR spectroscopy was used to measure diffusional water permeability in cell suspensions of the Gram-positive bacterium Corynebacterium glutamicum. Two different regions of H2O mobility were detected. One was characterized by the apparent coefficient of self-diffusion, D(1 app) = (4.6-12.7)x10(-8) cm(2) s(-1), depending on the observation time t. The other region was characterized by D(2) = 1.4x10(-5) cm(2) s(-1). The value of D(2) was similar to the diffusion coefficient of H2O in free water and in extracellular biological fluids. Restricted diffusion could be demonstrated for the slower process (D(1)). It was attributed to the cytoplasm of the cells. The membrane permeability, P(d H2O), for C. glutamicum was (4.8+/-0.4)x10(-3) cm s(-1). It compared favorably with values reported for human erythrocytes and was higher by a factor of about 100 compared to the diffusional permeability for ethanol, P(d ethanol), in Zymomonas mobilis. Addition of HgCl2, a water channel inhibitor in eukaryotes, decreased P(d H2O) in C. glutamicum by a factor of approximately 8. To our knowledge, these are the first functional studies of water transport in prokaryotes that yielded quantitative data, viz., transmembrane water permeability expressed through D(H2O) and P(d H2O).

Ethanol transport in Zymomonas mobilis measured by using in vivo nuclear magnetic resonance spin transfer.

For the first time, unidirectional rate constants of ethanol diffusion through the lipid membrane of a microorganism, the bacterium Zymomonas mobilis, were determined, thus replacing indirect inferences with direct kinetic data. The rate constants k1 (in to out) were 6.8 +/- 0.4s(-1) at 29 degrees C and 2.7 +/- 0.3s(-1) at 20 degrees C. They were determined by using 1H selective nuclear magnetic resonance spin magnetization transfer. The measurements were done on l-ml cell suspensions. No addition of radiotracers, withdrawing of aliquots, physical separation methods, or chemical manipulations were required. Until now, the rate constants of ethanol transport in microorganisms have been unknown because ethanol diffuses through the cytoplasmic membrane too quickly for radiolabel approaches. Net velocities of ethanol exchange were calculated from unidirectional rate constants and cytoplasmic volume, which was also determined with the same nuclear magnetic resonance experiments. The results (i) confirmed that ethanol would not be rate limiting during the conversion of glucose by Z. mobilis and (ii) indicated that ethanol can serve as an in vivo marker of cytoplasmic volume changes. This was verified by monitoring for the first time the changes of both cytoplasmic volume and extracytoplasmic and cytoplasmic concentrations of alpha and beta anomers of D-glucose in cell suspensions of a microorganism. These findings may open up new possibilities for kinetic studies of ethanol and sugar transport in Z. mobilis and other organisms.

Use of in vivo 13C nuclear magnetic resonance spectroscopy to follow sugar uptake in Zymomonas mobilis.

A noninvasive, in situ, in vivo, and anomer-specific method for studying membrane transport of sugars in bacteria is presented. High-resolution 13C NMR was used to measure the distribution of alpha- and beta-xylose, maltose, Mes buffer, and ethanol in the extracellular and the cytoplasmic compartments in dense cell suspensions of Zymomonas mobilis, an aerotolerant bacterium that transports xylose but does not further metabolize it. The method relied on a difference in the magnetic susceptibility of the media inside and outside cells, induced with Dy-diethylenetriaminepentaacetic acid. The applicability of this method was demonstrated: (a) by showing that xylose and ethanol crossed the inner membrane of Z. mobilis while maltose and Mes buffer did not and (b) by a kinetic study of xylose uptake in this organism. After addition of xylose, both the extracellular decrease in the alpha- and beta-anomers in the medium and their intracellular accumulation could be followed.

Quantitative microbiological analysis of bacterial community shifts in a high-rate anaerobic bioreactor treating sulfite evaporator condensate.

The bacterial population of a high-rate, anaerobic, fixed-bed loop reactor treating sulfite evaporator condensate from the pulp industry was studied over a 14-month period. This period was divided into seven cycles that included a startup at the beginning of each cycle. Some 82% of the total biomass was immobilized on and between the porous glass rings filling the reactor. The range of the total number of microorganisms in these biofilms was 2 x 10 to 7 x 10 cells per ml. Enumeration and characterization by microbiological methods and by phase-contrast, epifluorescence, and electron microscopy showed that the samples consisted mainly of the following methanogens: a Methanobacterium sp., a Methanosarcina sp., a Methanobrevibacter sp., and a Methanothrix sp., as well as furfural-degrading sulfate-reducing bacteria resembling Desulfovibrio furfuralis. Viable counts of hydrogenotrophic methanogens were relatively stable (mostly within the range of 3.2 x 10 to 7.5 x 10 cells per ml), but Methanobrevibacter cells increased from <5 to 30% of the total hydrogenotrophic count after transfer of the fixed bed into a second reactor vessel. Acetotrophic methanogens reached their highest numbers of 1.3 x 10 to 2.6 x 10 cells per ml in the last fermentation cycles. They showed a morphological shift from sarcinalike packets in early samples to single coccoid forms in later phases of the fermentation. Furfural-degrading sulfate reducers reached counts of 1 x 10 to 5.8 x 10 cells per ml. The distribution of the chief metabolic groups between free fluid and biofilms was analyzed in the fifth fermentation cycle: 4.5 times more furfural degraders were found in the free fluid than in the biofilms. In contrast, 5.8 times more acetotrophic and 16.6 times more hydrogenotrophic methanogens were found in the biofilms than in the free liquid. The data concerning time shifts of morphotypes among the trophic groups of methanogens corroborated the trends observed by using immunological assays on the same samples.

Shifts in methanogenic subpopulations measured with antibody probes in a fixed-bed loop anaerobic bioreactor treating sulfite evaporator condensate.

A fixed-bed loop, high-rate anaerobic bioreactor treating sulfite evaporator condensate was sampled when it reached steady state and afterwards following perturbations during a 14-month period. By using immunotechnology, it was observed that shifts in methanogenic subpopulations occurred in association with perturbations, such as restarting and relocating the biomass into a different tank. Methanogens related to Methanobacterium bryantii MoHG and Methanobrevibacter smithii ALI were numerous throughout the observation period, while Methanosarcina mazei S6 and Methanosarcina thermophila TM1 were found in the early and late samples, respectively. Also, Methanobacterium formicicum was more numerous at the top portion of the bioreactor, while Methanobrevibacter arboriphilus AZ and DC were at the bottom. Sample formalinization required for prolonged storage proved suitable for antigen preservation.

Relationship of Intracellular Coenzyme F(420) Content to Growth and Metabolic Activity of Methanobacterium bryantii and Methanosarcina barkeri.

The use of F(420) as a parameter for growth or metabolic activity of methanogenic bacteria was investigated. Two representative species of methanogens were grown in batch culture: Methanobacterium bryantii (strain M.o.H.G.) on H(2) and CO(2), and Methanosarcina barkeri (strain Fusaro) on methanol or acetate. The total intracellular content of coenzyme F(420) was followed by high-resolution fluorescence spectroscopy. F(420) concentration in M. bryantii ranged from 1.84 to 3.65 mumol . g of protein; and in M. barkeri grown with methanol it ranged from 0.84 to 1.54 mumol . g depending on growth conditions. The content of F(420) in M. barkeri was influenced by a factor of 2 depending on the composition of the medium (minimal or complex) and by a factor of 3 to 4 depending on whether methanol or acetate was used as the carbon source. A comparison of F(420) content with protein, cell dry weight, optical density, and specific methane production rate showed that the intracellular content of F(420) approximately followed the increase in biomass in both strains. In contrast, no correlation was found between specific methane production rate and intracellular F(420) content. However, qCH(4)(F(420)), calculated by dividing the methane production rate by the coenzyme F(420) concentration, almost paralleled qCH(4)(protein). These results suggest that F(420) may be used as a specific parameter for estimating the biomass, but not the metabolic activity, of methanogens; hence qCH(4)(F(420)) determined in mixed populations with complex carbon substrates must be considered as measure of the actual methanogenic activity and not as a measure of potential activity.

Anaerobic degradation of halogenated aromatic compounds.

Recent microbiological findings show how compounds, regarded hitherto as unusual substrates for anaerobic bacteria, are degraded under anaerobic conditions. The complete conversion of halobenzoic acids and halophenolic compounds to methane by lake sediment and sewage sludge microorganisms has been demonstrated. Since haloaromatic compounds are widely used and may be found in such effluents as those from the forest industry, these studies could stimulate a broader interest in anaerobic treatment of industrial waste waters which contain unusual organic compounds.

Formation of pyrrolidine by anaerobic polyamine degradation.

High-resolution mass spectrometry was used to identify pyrrolidine as a product of anaerobic polyamine degradation. Due to the widespread occurrence of polyamines in the living world this process could contribute considerably to the presence of pyrrolidine in the environment.

Growth of a strictly anaerobic bacterium on furfural (2-furaldehyde).

A strictly anaerobic bacterium was isolated from a continuous fermentor culture which converted the organic constituents of sulfite evaporator condensate to methane and carbon dioxide. Furfural is one of the major components of this condensate. This furfural isolate could degrade furfural as the sole source of carbon and energy in a defined mineral-vitamin-sulfate medium. Acetic acid was the major fermentation product. This organism could also use ethanol, lactate, pyruvate, or fumarate and contained cytochrome c(3) and desulfoviridin. Except for furfural degradation, the characteristics of the furfural isolate were remarkably similar to those of the sulfate reducer Desulfovibrio gigas. The furfural isolate has been tentatively identified as Desulfovibrio sp. strain F-1.

Enumeration of bacteria forming acetate from H2 and CO2 in anaerobic habitats.

A method has been worked out that allows the detection and isolation of bacteria fermenting molecular hydrogen and carbon dioxide to acetic acid. The ratio of methanogenic to acetogenic bacteria in sludge and lake sediment samples has been found to be approximately 100 to 1. Acetogenic bacteria could not be detected in rumen samples.

Electron microscopic investigation of the hydrogen-oxidizing acetate-forming anaerobic bacterium Acetobacterium woodii.

Acetobacterium woodii is a Gram-positive anaerobic nonsporeforming bacterium able to grow on H2 and CO2 as sole sources of energy. The product of fermentation is acetic acid. Fine structural analysis showed rod-shaped flagellated cells, and coccoid cells without flagella arranged predominantly in pairs and chains. The cell wall was found to be composed of three layers. The cell surface exhibited a periodic array of particles consisting of subunits. The cytoplasmic membrane showed particles either in random distribution or in a hexagonal pattern. Intracytoplasmic membranes were rarely observed, whereas inclusion bodies of varying shapes, predominantly in an uncommon disc-shape, could frequently be observed. Their content was dissolved in ultrathin sections indicating hydrophobic nature.