Effect of Culture Medium and Carbon Dioxide Concentration on Growth of Anaerobic Bacteria Commonly Encountered in Clinical Specimens

Representative strains of anaerobic bacteria from human infections were used to evaluate broth media, gas mixtures, and inocula for use in developing a procedure for performing minimal inhibitory concentration antimicrobic susceptibility tests. Nine commercially available media, including two that were chemically defined, were tested. Tests were performed in atmospheres with carbon dioxide concentrations between 2.5 and 10% and also in the GasPak system (BBL) that had a disposable hydrogen-carbon dioxide generator. Growth curves on each organism grown in schaedler broth and a 5% carbon dioxide atmosphere were used to determine growth characteristics, equate time of the particular growth phases to turbidity readings, and determine the numbers of viable organisms present in the culture. Schaedler broth proved to be most advantageous in combination with an atmosphere of 5% carbon dioxide, 10% hydrogen, and 85% nitrogen. The growth curve studies yielded valuable data on the rapidity and quantity of growth under these conditions. We believe these data have provided information which can be used as the basis for developing a standardized procedure for antimicrobic susceptibility testing for anaerobic bacteria.     

Some aspects on the role of bacteria in deep water ecosystems

Summary Some remarks on the role of bacteria in deep lakes will be presented with an emphasis on their functioning in the carbon cycle in such ecosystems.The metabolic intensity of most lakes is regulated to a large extent at the primary producer level. Aerobic and anaerobic decomposition by heterotrophic bacteria of the unstable ultimate products of photosynthesis results in the production of carbon dioxide and methane. Bacterial growth occurs at the expense of energy released by the flow of electrons from donors to acceptors. Typical electron acceptors for bacterially mediated reactions are oxygen, nitrate, sulphate and carbon dioxide. When oxygen is used as electron acceptor, the highest amount of energy is released, while the lowest is released when carbon dioxide is used. These reactions are mediated biologically, and the chemical reaction sequence is paralleled by an ecological succession of microorganisms: aerobic heterotrophs, denitrifiers, fermenting bacteria, sulphate reducers, and methane producers. The presence of oxygen is inhibitory to the organisms mediating the last reactions (STUMM, 1966; McCARTY, 1972), and this explains the succession of micro-organisms concomittant with the decrease in redox potential.Both labile and refractory fractions of the pelagial dissolved organic matter can directly be utilized largely by heterotrophic bacteria. At the onset of the summer stratification of Lake Vechten a high concentration (numbers 10⁹ bact/L) of heterotrophic bacteria has been detected in the lower water layers (CAPPENBERG, 1972). The reason for this increase may be a release of nutrients from the mud. During stratification the water layers above the mud become anaerobic owing to the metabolic activity of the bacterial flora and the chemical oxygen demand of the mud. In summer time the highest numbers of heterotrophic bacteria are found in the metalimnion, where organic matter accumulates due to a lower relative rate of sedimentation caused by an increase in viscosity and density.Furthermore it was found that sulphate-reducing bacteria which are capable of reducing sulphates to sulphides using sulphate as terminal electron acceptor, were observed in the hypolimnion only at the time of maximal stratification. During stagnation, gradually decreasing sulphate concentrations are found in the hypolimnion, correlated with increaseing cell numbers of sulphate reducers. As no sulphate could be detected in the mud of Lake Vechten as well, we may conclude that the sulphate concentration limits the number of these bacteria, as can be predicted for similar aquatic environments.Usually the sulphate reducers are found in bottom deposits and are an important group of bacteria of the non-methanogenic populations in mud. Summarizing the biological methane production and its subsequent oxidation by methane-oxidizing bacteria, we may conclude that these processes can be important factors functioning in the carbon cycle in deep fresh-water ecosystems.     

Fructose metabolism of the purple non-sulfur bacterium Rhodospirillum rubrum: effect of carbon dioxide on growth, and production of bacteriochlorophyll and organic acids

During fermentative metabolism, carbon dioxide fixation plays a key role in many bacteria regarding growth and production of organic acids. The present contribution, dealing with the facultative photosynthetic bacterium Rhodospirillum rubrum, reveals not only the strong influence of ambient carbon dioxide on the fermentative break-down of fructose but also a high impact on aerobic growth with fructose as sole carbon source. Both growth rates and biomass yield increased with increasing carbon dioxide supply in chemoheterotrophic aerobic cultures. Furthermore, intracellular metabolite concentration measurements showed almost negligible concentrations of the tricarboxylic acid cycle intermediates succinate, fumarate and malate under aerobic growth, in contrast to several metabolites of the glycolysis. In addition, we present a dual phase fed-batch process, where an aerobic growth phase is followed by an anaerobic production phase. The biosynthesis of bacteriochlorophyll and the secretion of organic acids were both affected by the carbon dioxide supply, the pH value and by the cell density at the time of switching from aerobic to anaerobic conditions. The formation of pigmented photosynthetic membranes and the amount of bacteriochlorophyll were inversely correlated to the secretion of succinate. Accounting the high biotechnological potential of R. rubrum, optimization of carbon dioxide supply is important because of the favored application of fructose-containing fermentable feedstock solutions in bio-industrial processes.     

On culturing Escherichia coli on a mineral salts medium during anaerobic conditions

The substrate and products of the hydrogenlyase complex, formic acid, carbon dioxide, and molecular hydrogen, are co-operatively implicated in maintaining growth of E. coli under anaerobic conditions. Growth is observed in the presence of a combination of carbon dioxide + molecular hydrogen, or carbon dioxide + formic acid in the medium. The study shows that it is possible to culture E. coli under anaerobic conditions while sparging with nitrogen, without supplementing exogenous carbon dioxide, formic acid or molecular hydrogen. This condition occurs when the strain is allowed an appropriate induction period and is present at a sufficiently high cell density, since the cell density affects the rate of e.g. CO₂ production. In a system sparged with nitrogen gas, the removal of CO₂ due to this sparging must be balanced with a cell density dependent production rate of CO₂. It is concluded that the formic hydrogenlyase complex should be considered as an integral part of the general maintenance of the anabolism of E. coli during anaerobic conditions on a mineral salts medium, as well as being a net producer of end products in E. coli metabolism.This work was supported by the Swedish National Board for Industrial and Technical Development. A. Askendahl is acknowledged for valuable assistance in the preparation of figures and P. Warkentin for language editing.     

硝酸盐和硫酸盐厌氧氧化甲烷途径及氧化菌群

甲烷属于温室气体,厌氧氧化甲烷有效地减少了大气环境中甲烷的含量。依据吉布斯自由能变,以SO42、Mn4+、Fe3+、NO3等作为电子受体,厌氧条件下甲烷可以转化为CO2。重点阐述以SO42和NO3为电子受体时甲烷厌氧氧化的机理、反应发生的环境条件以及甲烷厌氧氧化菌的特点。针对目前研究存在的主要问题,提出了今后的发展方向。SO42为电子受体时,甲烷厌氧氧化的可能途径包括:逆甲烷生成途径、乙酰生成途径以及甲基生成途径。甲烷的好氧或厌氧氧化协同反硝化是以NO3为电子受体的甲烷氧化的可能途径。环境中的甲烷、硫酸盐或硝酸盐的浓度,有机质的数量,以及环境条件对甲烷的厌氧氧化有显著影响。     

Catechol and phenol degradation by a methanogenic population of bacteria

An anaerobic population of bacteria became acclimated to catechol and phenol in 32 and 18 days, respectively. Evidence from carbon balance measurements indicates that the aromatic ring is cleaved and that the products are stoichiometrically fermentable to methane and carbon dioxide.     

Commensalism of methane-producing and motile aerobic bacteria in certain freshwater wetlands

It is pointed out that the methane flux measured experimentally for certain ponds and swamps is quantitatively consistent with a commensal dependence of Methanobacteria on O₂-chemotactic motile aerobic bacteria. The Methano species is thereby shielded from oxygen and provided with carbon dioxide for the anaerobic production of methane.     

Catechol and phenol degradation by a methanogenic population of bacteria.

An anaerobic population of bacteria became acclimated to catechol and phenol in 32 and 18 days, respectively. Evidence from carbon balance measurements indicates that the aromatic ring is cleaved and that the products are stoichiometrically fermentable to methane and carbon dioxide.     

Improved Isolation of Anaerobic Bacteria From the Gingival Crevice Area of Man

A roll tube technique (Hungate method) was employed in an attempt to cultivate a maximal portion of the organisms in the gingival crevice area of man. This technique achieves an anaerobic state by flushing the local environment with oxygen-free gas. Once collected, the crevicular debris was immediately placed into sterile oxygen-free test tubes which were flushed out by the oxygen-free gas. In this manner, the samples were weighed, dispersed, diluted, and cultured in roll tubes and plates. The medium for control (Brewer Jar technique) and Hungate techniques was Heart Infusion Agar fortified with 10% defibrinated horse blood. When the Hungate technique was used, the recovery of viable bacteria, as a percentage of the direct microscopic count, was significantly greater than plates incubated aerobically or utilizing the Brewer Anaerobic technique. Cultural counts by using the Hungate method averaged 41.3% for six samples when 90% nitrogen and 10% hydrogen were used, 70.4% for eight samples when 85% nitrogen, 10% hydrogen, and 5% carbon dioxide were used, and 63.4% for eight samples when 100% carbon dioxide was the gaseous atmosphere. At no time were cultural counts, by using anaerobic plates (Brewer Jar), more than 24% of the direct microscopic count. This suggests that exclusion of oxygen and the presence of carbon dioxide maximized recovery of gingival crevice bacteria.     

Methane production and sulfate reduction in two Appalachian peatlands

Anaerobic carbon mineralization was evaluated over a 1-year period in two Sphagnum-dominated peatlands, Big Run Bog, West Virginia, and Buckle's Bog, Maryland. In the top 35 cm of peat, mean rates of methane production, anaerobic carbon dioxide production, and sulfate reduction at Big Run Bog were 63,406 and 146 mol L^-1 d^-1, respectively, and at Buckle's Bog were 18, 486 and 104 mol L^-1 d^-1. Annual anaerobic carbon mineralization to methane and carbon dioxide at Big Run Bog and Buckle's Bog was 52.8 and 57.2 mol m^-2, respectively. Rates of methane production were similar to rates reported for other freshwater peatlands, but methane production accounted for only 11.7 and 2.8%, respectively, of the total anaerobic carbon mineralization at these two sites. Carbon dioxide production, resulting substantially from sulfate reduction, dominated anaerobic carbon mineralization. Considerable sulfate reduction despite low instantaneous dissolved sulfate concentrations (typically < 300 mol L^-1 of substrate) was apparently fueled by oxidation and rapid turnover of the reduced inorganic sulfur pool.The coincidence of high sulfate inputs to the Big Run Bog and Buckle's Bog watersheds through acid precipitation with the unexpected importance of sulfate reduction leads us to suggest a new hypothesis: peatlands not receiving high sulfate loading should exhibit low rates of anaerobic decomposition, and a predominance of methane production over sulfate reduction; however, if such peatlands become subjected to high rates of sulfur deposition, sulfate reduction may be enhanced as an anaerobic mineralization pathway with attendant effects on carbon balance and peat accumulation.     

Energy conservation under extreme energy limitation: the role of cytochromes and quinones in acetogenic bacteria

Extremophiles - Acetogenic bacteria are a polyphyletic group of organisms that fix carbon dioxide under anaerobic, non-phototrophic conditions by reduction of two mol of CO2 to acetyl-CoA via the...     

Carbonic acid from decarboxylation by "malic" enzyme in lactic acid bacteria

Carbonic anhydrase studies were used to determine the primary form of carbonic acid produced from decarboxylation of l-malic acid by "malic" enzyme in malolactic strains of five different species of lactic acid bacteria. Addition of carbonic anhydrase to the reaction mixture containing crude bacterial extract and l-malic acid, at pH 7, in all five cases resulted in an increase (13 to 23%) in the rate of carbon dioxide evolution over the control. The results indicated that the primary form of carbonic acid released from "malic" enzyme was not anhydrous carbon dioxide as previously supposed and as has been shown for other decarboxylating enzymes. The standard free-energy changes of the malo-lactic reaction with the various forms of carbonic acid as the primary decarboxylation product were calculated. The reaction is less exergonic when carbonic acid, bicarbonate ion, or carbonate ion is the primary decarboxylation product compared to anhydrous carbon dioxide. The free-energy of the reaction is not biologically available to the bacteria; with carbon dioxide not the primary decarboxylation product, the potential energy lost in a malo-lactic fermentation is not as great as previously considered. Endogenous carbonic anhydrase activity was not found.     

Microbial acetate conversion to methane: kinetics,yields and pathways in a two-step digestion process

Summary Organic waste is converted in a two-stage process to methane and carbon dioxide by mixed cultures of microorganisms. Acetate, a product of acidogenic and acetogenic bacteria and the main substrate for methanogenic bacteria, is an important intermediate of the anaerobic degradation process, which results in the generation of methane. It was shown by labelling experiments using (U-¹⁴C) acetate that as much as 65%–96% of the total methane produced came from the acetate. The first order utilization rate for acetate in the methanogenic stages of a two-stage digestion process was between 0.17 h^-1 and 0.5 h^-1. The kinetics as well as the mass flow and yields of acetate and the methyl group of acetate were determined by pulse-labelling experiments with (U-¹⁴C) acetate and (2-¹⁴C) acetate without a significant rise of the total concentrations. Up to 58% of the acetate carbon was transformed to methane, and about 30% to carbon dioxide; only 4%–15% was incorporated into the biomass. There are at least two parallel degradation mechanisms in the metabolic transformation of acetate to methane: acetate is cleaved either to form methane and carbon dioxide or to form hydrogen and carbon dioxide, which can be transformed by an additional reaction to methane. Labelling experiments with (2-¹⁴C) acetate show that both mechanisms took place at similar order.     

Heat tolerance of Salmonella typhimurium DT104 isolates attached to muscle tissue

Aerobic and anaerobic plate counts were compared for routine monitoring of the microflora, dominated by lactic acid bacteria, developing on vacuum- and carbon dioxide-packaged raw meat during chilled storage. No statistical differences were observed between aerobic and anaerobic enumerations, made on plate count and blood agar plates, of the microflora developing on beef striploins packaged under vacuum or carbon dioxide during 14 weeks' storage at 0°C. With both techniques the spoilage microflora development differed between the two packaging regimes. The results indicate that there is no necessity for aerobic plate counts to be replaced by anaerobic plate counts in the routine microbiological examination of the spoilage microflora developing on chilled meats packaged under anoxic modified atmospheres.     

Growth of methanogenic and sulfate-reducing bacteria with cathodic hydrogen

Summary Bacteria contribute to corrosion in various ways. Therefore the consumption of cathodic hydrogen as well as the sulfide production of sulfate-reducing bacteria may influence the anaerobic corrosion of iron. Also methanogenic bacteria are able to use elemental iron as a source of electrons for carbon dioxide reduction. We have studied both processes and have got evidence that cathodic depolarisation does not play a dominant role in methanogenic habitats.     

An Evaluation of the 'GasKit'Disposable Hydrogen and Carbon Dioxide Generator for the Culture of Anaerobic Bacteria

The new GasKit disposable hydrogen and carbon dioxide generator is a reliable and convenient method for producing anaerobiosis in standard anaerobic jars when compared with a conventional procedure. The generator produces an atmosphere containing 10–12% carbon dioxide by volume which is satisfactory for the culture of exacting anaerobes.     

Pelodictyon phaeoclathratiforme sp. nov., a new brown-colored member of the Chlorobiaceae forming net-like colonies

A new strain of the green sulfur bacteria was isolated from the monimolimnion of Buchensee (near Radolfzell, Lake Constance region, FRG). Single cells were rod-shaped, nonmotile and contained gas vacuoles. Typical net-like colonies were formed by ternary fission of the cells. As photosynthetic pigments bacteriochlorophylls a, e, isorenieratene and -isorenieratene were present. Sulfide, sulfur and thiosulfate were used as electron donors during anaerobic phototrophic growth. Besides carbon dioxide, acetate and propionate could serve as carbon sources under mixotrophic conditions in the light. Like all other members of the green sulfur bacteria, the new bacterium is strictly anaerobic and obligately phototrophic. The possession of gas vacuoles and the formation of net-like colonies and the guanine plus cytosine content of the DNA (47.9 mol% G+C) are typical characteristics of the genus Pelodictyon. Because of its photosynthetic pigments which differ from those of Pelodictyon clathratiforme, strain BU 1 represents a new species, P. Phaeoclathratiforme sp. nov.     

Oxidation of methane in the absence of oxygen in lake water samples.

Methane was oxidized to carbon dioxide in the absence of oxygen by water samples from Lake Mendota, Madison, Wis. The anaerobic oxidation of methane did not result in the assimilation of carbon from methane into material precipitable by cold 10% trichloracetic acid. Only samples taken at the suface of the sediment of Lake Mendota were capable of catalyzine the anaerobic oxidation of methane. The rate of methane oxidation in the presence of oxygen was highest in samples taken from near the thermocline. Of the radioactive methane oxidized, 30 to 60% was assimilated into material precipitable by cold 10% trichloroacetic acid during aerobic incubation of the samples. These data support the conclusion that two distinct groups of methane-oxidizing organisms occur in stratifield lakes. Enrichments with acetate and methane as the sole sources of carbon and energy and sulfate as the electron acceptor resulted in the growth of bacteria that oxidize methane. Sulfate, acetate, and methane were all required for growth of enrichments. Acetate was not oxidized to carbon dioxide but was assimilated by cells. Methane was not assimilated but was oxidized to carbon dioxide in the absence of air.     

Wireless bioreactor for anaerobic cultivation of bacteria

Anaerobic cultivation methods of bacteria are indispensable in microbiology. One methodology is to cultivate the microbes in anaerobic enclosure with oxygen-adosrbing chemicals. Here, we report an electronic extension of such strategy for facultative anaerobic bacteria. The technique is based a bioreactor with entire operation including turbidity measurement, fluidic mixing, and gas delivery in an anaerobic enclosure. Wireless data transmission is employed and the anaerobic condition is achieved with gas pack. Although the technique is not meant to completely replace the anaerobic chamber for strict anaerobic bacteria, it provides a convenient way to bypass the cumbersome operation in anaerobic chamber for facultative anaerobic bacteria. Such a cultivation strategy is demonstrated with Escherichia coli with different carbon sources and hydrogen as energy source.     

Oxidation of methane in the absence of oxygen in lake water samples.

Methane was oxidized to carbon dioxide in the absence of oxygen by water samples from Lake Mendota, Madison, Wis. The anaerobic oxidation of methane did not result in the assimilation of carbon from methane into material precipitable by cold 10% trichloracetic acid. Only samples taken at the suface of the sediment of Lake Mendota were capable of catalyzine the anaerobic oxidation of methane. The rate of methane oxidation in the presence of oxygen was highest in samples taken from near the thermocline. Of the radioactive methane oxidized, 30 to 60% was assimilated into material precipitable by cold 10% trichloroacetic acid during aerobic incubation of the samples. These data support the conclusion that two distinct groups of methane-oxidizing organisms occur in stratifield lakes. Enrichments with acetate and methane as the sole sources of carbon and energy and sulfate as the electron acceptor resulted in the growth of bacteria that oxidize methane. Sulfate, acetate, and methane were all required for growth of enrichments. Acetate was not oxidized to carbon dioxide but was assimilated by cells. Methane was not assimilated but was oxidized to carbon dioxide in the absence of air.