The physiology of Clostridium sporogenes NCIB 8053 growing in defined media

The physiology of Clostridium sporogenes was investigated in defined, minimal media. In batch culture, the major end products of glucose dissimilation were acetate, ethanol and formate. When L-proline was present as an electron acceptor, acetate production was strongly enhanced at the expense of ethanol. As judged by assay of the relevant enzymes, glucose was metabolized via the Embden-Meyerhof-Parnas pathway. The growth energetics of Cl. sporogenes were investigated in glucose- or L-valine-limited chemostat cultures. In the former case, the addition of L-proline to the medium caused a significant increase in the molar growth yield (as calculated by extrapolation to infinite dilution rate). This finding adds weight to the view that the reduction of L-proline by Cl. sporogenes is coupled to the conservation of free energy.     

Identity of proline dehydrogenase and delta1-pyrroline-5-carboxylic acid reductase in Clostridium sporogenes.

Proline dehydrogenase and delta1-pyrroline-5-carboxylic acid (PCA) reductase activities were copurified 60- and 130-fold, respectively, from extracts of Clostridium sporogenes. The primary change in the ratio of activites was the result of a loss of proline dehydrogenase activity during dialysis. Both activities were eluted in single peaks from diethylaminoethyl-cellulose, hydroxylapatite, and Sephadex G-200 columns. They had identical sedimentation coefficients (10.3S), as determined in linear sucrose gradients, and identical isoelectric points (4.95 to 5.12) based on isoelectric focusing. The proline dehydrogenase activity was dependent on nicotinamide adenine dinucleotide and L-proline, and the PCA reductase required L-PCA and reduced nicotinamide adenine dinucleotide. The optimum pH for the assay of proline dehydrogenase was approximately 10.2, whereas that for PCA reductase was 6.5 to 7.5. An increase in pH from 8.0 to 10.2 greatly decreased the apparent Michaelis constant observed for L-proline, and an increase from pH 8.3 to 8.6 resulted in a large shift in the reaction equilibrium toward PCA. Both the dehydrogenase and reductase activities were stabilized to heating at 65 degrees C for 5 min by solutes of high ionic strength and were inactivated in a similar fashion when dissolved in low-ionic-strength buffer. The specific activities for both were reduced by about 50% when glucose was added to the growth medium. The data support the conclusion that L-proline and L-PCA are interconverted by either a single enzyme or an enzyme complex in extracts of C. sporogenes cells.     

Development of a Selective Medium for the Isolation of Clostridium sporogenes and Related Organisms

An attempt was made to develop a selective isolation medium for Clostridium sporogenes and related organisms based on the ability of these organisms to obtain their energy for growth by means of coupled oxidation-reduction reactions between appropriate pairs of amino acids (Stickland reaction). Using a semi-defined basal medium containing various combinations of amino acids, it was found that Cl. sporogenes utilized a wider range of amino acid pairs than strains of five other species of clostridia known to carry out a Stickland-type fermentation.
With alanine and proline as the principal energy sources and the medium solidified with agar. it was shown that reference strains of Cl. sporogenes and proteolytic Cl. botulinum types A, B and F could be recovered almost quantitatively, with or without prior heating at 80 °C for 10 min. By contrast, growth of test strains of Streptococcus faecalis, Strep. faecium , 'saccharolytic' Cl. botulinum types B, C, D, E and F and 'proteolytic' strains of types C and D was suppressed on this medium, as were strains of 26 other species of clostridia.
Addition of 50 μg/ml of polymyxin to the agar medium had no detectable effect on the recovery of Cl. sporogenes or Cl. botulinum. When samples of soil and mud were plated on the antibiotic-containing medium, 63.1% of 225 isolates thus obtained were identified as Cl. sporogenes/botulinum.     

Fermentation of isoleucine and arginine by pure and syntrophic cultures of Clostridium sporogenes

Abstract The fermentation of isoleucine, arginine and isoleucine + arginine by pure and syntrophic cultures of Clostridium sporogenes was investigated. Growth of C. sporogenes on isoleucine, if any, was poor, but some isoleucine was fermented to 2-methylbutyrate and hydrogen. In syntrophic cultures with Methanobacterium formicicum or Methanosarcina barkeri growth was better, and isoleucine was completely fermented, the hydrogen being used for methane production. Pure cultures of C. sporogenes grew on arginine and produced 5-aminovalerate, ornithine and acetate. The reducing equivalents for 5-aminovalerate production from intermediarily formed proline were provided by oxidative conversion of arginine to acetate and by oxidative metabolism of some amino acids present in the yeast extract. However, when isoleucine was available together with arginine in syntrophic cultures of C. sporogenes and M. formicicum , the reducing equivalents for arginine fermentation came mainly from the oxidation of isoleucine (Stickland reaction), and the hydrogen produced in excess served for the reduction of CO₂ to methane.     

Rhizomorph Formation in Fungi I. Stimulation by Ethanol and Acetate and Inhibition by Disul-firam of Growth and Rhizomorph Formation in Armillaria mellea

Rhizomorph formation in Armillaria mellea (Vahl ex Fr.) Quél. has been studied on a synthetic glucose medium with ethanol and acetate as inducers. Disulfiram (antabuse) inhibited growth and rhizomorph formation when ethanol was the inducer, while the stimulating effect of acetate was unaffected. This indicates that ethanol is metabolised by the normal pathway via acetate. Growth, ethanol- and glucose uptake were measured as a function of time. Ethanol was used up in 12 days; after that, growth and glucose uptake declined. A further supply of ethanol gave a significant increase in the growth rate. The results show that ethanol is a limiting growth factor during the entire growth period.     

Ester formation from ethanol by Candida pseudotropicalis

The production of ethanol, acetate ion and ethyl acetate from glucose by the yeast Candida pseudotropicalis NCYC 143 was investigated under aerobic and anaerobic growth conditions. Acetate and ethyl acetate only accumulated under aerobic conditions, whereas production of the alcohol was favoured by anaerobic conditions. Ester production during aerobic growth was enhanced substantially by growth in iron-deficient media. Possible conditions for optimising ester production from ethanol in dilute product streams were characterised.     

Balance between aerobic and anaerobic metabolites production of Amycolatopsis orientalis depending on initial glucose concentration

The effect of glucose concentration as a carbon source in the range of 5-20 g/L on the fermentative productions of intra-and extra-cellular ethanol, acetate, formate, oxalate, lactate, and pyruvate, as well as pyruvate decarboxylase in A. orientalis were investigated, depending on the incubation period. Intra-and extra-cellular pyruvate levels increased with rising glucose concentrations up to 15 and 20 g/L of glucose, respectively. In addition, intra-cellular pyruvate levels reached their maximum on the 48th hour in the range of 12.5-20 g/L of glucose, except for 5 and 10 g/L while extra-cellular pyruvate were at the 48th and 60th hours. As a fermentative end product, intra-and extra-cellular ethanol levels increased with increasing glucose concentrations of the growth medium and with incubation period. Activity of pyruvate decarboxylase, one of the key enzymes of the alcoholic fermentation, increased significantly with increasing glucose concentrations up to the 48th hour. Intra-and extra-cellular acetate levels increased significantly with increasing glucose concentrations of the growth medium and reached their maximums on the 48th hour, as was the case also for pyruvate. Intra-cellular formate levels increased up to 15 g/L, while extra-cellular levels increased with increasing glucose concentration. The maximum intra-and extra-cellular lactate levels were determined at 12.5 g/L and 20 g/L of glucose on the 48th hour, respectively. The results suggest that elevated ethanol production suppressed lactate and formate production, supported via possibly formed CO(2). In addition, pyruvate, as well as acetate, were used as carbon sources due to the depletion of glucose contents in the growth medium.     

Co-metabolism of citrate and glucose by Leuconostoc spp.: effects on growth, substrates and products

Growth, substrate utilization and product formation from glucose, citrate and a mixture of both substrates were studied in four strains of Leuconostoc spp. Citrate was not used as an energy source but was rapidly metabolized when glucose was present. The predictable amounts of D-lactate and ethanol were produced from glucose, although strains X2 and 7–1 gave lower yields of ethanol. In strains NCW1, S3 and X2, co-metabolism of both glucose and citrate resulted in stimulation of growth, decreased uptake of glucose, increased acetate and D-lactate production and lack of ethanol production compared with that obtained with glucose alone. Strain 7–1 showed only growth stimulation and increased acetate production. Diacetyl, acetoin or 2, 3-butylene glycol were not detected. In strain NCW1 citrate had a slightly inhibitory effect on the enzymes of the 'ethanol' leg of glucose metabolism. Except for strain 7–1, these observations are consistent with a switch in glucose metabolism from ethanol to acetate production.     

Influence of residual ethanol concentration on the growth of Gluconacetobacter xylinus I 2281

The influence of residual ethanol on metabolism of food grade Gluconacetobacter xylinus I 2281 was investigated during controlled cultivations on 35 g/l glucose and 5 g/l ethanol. Bacterial growth was strongly reduced in the presence of ethanol, which is unusual for acetic acid bacteria. Biomass accumulated only after complete oxidation of ethanol to acetate and carbon dioxide. In contrast, bacterial growth initiated without delay on 35 g/l glucose and 5 g/l acetate. It was found that acetyl CoA was activated by the acetyl coenzyme A synthetase (Acs) pathway in parallel with the phosphotransacetylase (Pta)-acetate kinase (Ack) pathway. The presence of ethanol in the culture medium strongly reduced Pta activity while Acs and Ack remained active. A carbon balance calculation showed that the overall catabolism could be divided into two independent parts: upper glycolysis linked to glucose catabolism and lower glycolysis liked to ethanol catabolism. This calculation showed that the carbon flux through the tricarboxylic cycle is lower on ethanol than on acetate. This corroborated the diminution of carbon flux through the Pta-Ack pathway due to the inhibition of Pta activity on ethanol.     

Oxygen- and glucose-dependent regulation of central carbon metabolism in Pichia anomala

We investigated the regulation of the central aerobic and hypoxic metabolism of the biocontrol and non-Saccharomyces wine yeast Pichia anomala. In aerobic batch culture, P. anomala grows in the respiratory mode with a high biomass yield (0.59 g [dry weight] of cells g of glucose(-1)) and marginal ethanol, glycerol, acetate, and ethyl acetate production. Oxygen limitation, but not glucose pulse, induced fermentation with substantial ethanol production and 10-fold-increased ethyl acetate production. Despite low or absent ethanol formation, the activities of pyruvate decarboxylase and alcohol dehydrogenase were high during aerobic growth on glucose or succinate. No activation of these enzyme activities was observed after a glucose pulse. However, after the shift to oxygen limitation, both enzymes were activated threefold. Metabolic flux analysis revealed that the tricarboxylic acid pathway operates as a cycle during aerobic batch culture and as a two-branched pathway under oxygen limitation. Glucose catabolism through the pentose phosphate pathway was lower during oxygen limitation than under aerobic growth. Overall, our results demonstrate that P. anomala exhibits a Pasteur effect and not a Crabtree effect, i.e., oxygen availability, but not glucose concentration, is the main stimulus for the regulation of the central carbon metabolism.     

Microcalorimetric investigations of the metabolism of yeasts VII. Flow-calorimetry of aerobic batch cultures

Summary The heat evolution of aerobic batch cultures of growing yeast (Saccharomyces cerevisiae) in glucose media was investigated by a combination of a flow-microcalorimeter with a fermentor vessel. The course of heat production, cell production and the rate of oxygen consumption were qualitatively the same for all glucose concentrations between 10 mM and 100 mM. Under optimal aerobic conditions a triphasic growth was observed due to the fermentation of glucose to ethanol, respiration of ethanol to CO₂ and acetate, and respiration of acetate to C0₂. Energy and carbon were found to be in balance for all glucose concentrations.     

Ethanol production by thermophilic bacteria: metabolic control of end product formation in Thermoanaerobium brockii.

Specific changes in the chemical and microbial composition of Thermoanaerobium brockii fermentations were compared and related to alterations of process rates, end product yields, and growth parameters. Fermentation of starch as compared with glucose was associated with significant decreases in growth rate and intracellular fructose-1,6-bisphosphate concentration and with a dramatic increase in the ethanol/lactate product ratio. Glucose or pyruvate fermentation in the presence of acetone was correlated with increased substrate consumption, growth (both rate and yield), acetate yield, and quantitative reduction of acetone to isopropanol in lieu of normal reduced fermentation products (i.e., H2, ethanol, lactate). Acetone altered pyruvate phosphoroclastic activity of cell extracts in that H2, lactate, and ethanol levels decreased, whereas the acetate concentration increased. Glucose fermentation in the presence of exogenous hydrogen was associated with inhibition of endogenous H2 production and either increased ethanol/acetate product ratios and decreased growth at less than 0.5 atm (51 kPa) of H2 or total growth inhibition at 1.0 atm (102 kPA). The effects of exogenous hydrogen on glucose fermentation were totally reversed by the addition of acetone. Glucose fermentation in coculture with Methanobacterium thermoautotrophicum correlated with increased growth (both rate and yield), acetate yield, and the formation of methane in lieu of monoculture reduced products. In coculture, but not monoculture, T. brockii grew on ethanol as the energy source, and acetate and methane were the end products as a direct consequence of hydrogen consumption by the methanogen.     

Kinetics of growth of Lactobacillus plantarum with glucose,organic acids (malate,citrate, acetate) and ethanol

Summary L. plantarum was grown on glucose and organic acids, i.e. malate, citrate, and acetate, frequently jointly encountered in wine and cider fermentation. The effect on fermentation patterns of different mixtures of acids as well as ethanol was studied. Specific growth rates and apparent biomass yields on glucose increased when adding citrate or malate. Acetate and ethanol were not consummed by the lactobacillus. The presence of acetate, but not ethanol, slightly decreased citrate consumption rates, but did not significantly influence glucose or malate fermentations.     

Evaluation of the use of the bioMerieux Rapid ID32 A for the identification of Clostridium botulinum

The neurotoxins produced by Clostridium botulinum are amongst the most potent known to man. Toxin production is detected by a mouse bioassay, which requires several days for a result and is not acceptable for routine use unless there is a high level of suspicion. The Rapid ID32 A kit produced by bioMerieux gives an identification of an isolate within 4 h. The aim of this study was to examine the efficiency of the identification of Cl. botulinum using the Rapid ID32 A. Forty-two strains of Cl. botulinum , one strain each of botulinum toxin-producing Cl. butyricum and Cl. baratii , and four strains of Cl. sporogenes , were tested. One strain of Group I Cl. botulinum gave a presumptive identification of Group II Cl. botulinum , six strains of Cl. botulinum were identified as 50–98% Cl. botulinum in some tests, and 17 strains of Cl. botulinum were identified as <50% Cl. botulinum. Thirteen strains of Cl. botulinum were identified as >99% Cl. sporogenes or 86% Cl. histolyticum , and five strains gave a combination of these results. All strains of Cl. sporogenes were correctly identified. These results show that some strains of Cl. botulinum may not be correctly identified using the Rapid ID32 A.     

End Products of Glucose Fermentation by Brochothrix thermosphacta

Anaerobically, Brochothrix thermosphacta fermented glucose primarily to l-lactate, acetate, formate, and ethanol. The ratio of these end products varied with growth conditions. Both the presence of acetate and formate and a pH below about 6 increased l-lactate production from glucose. Small amounts of butane-2,3-diol were also produced when the pH of the culture was low (相似文献   

Does Giardia intestinalis need glucose as an energy source?

Giardia intestinalis was grown in Diamond's TYI-S-33 medium containing either 50 mM-glucose or no added glucose to assess its dependence on glucose availability as an energy source. The parameters monitored included cell growth, glucose utilization and the accumulation of end products in the medium. In the medium containing no added glucose, G. intestinalis trophozoites achieved a cell density of about half that of the control, and produced the same end products, alanine, ethanol and acetate. Decreased amounts of both ethanol and alanine were observed (10 and 33% of controls, respectively after 4 days) while there was no change in acetate production. These observations indicate that G. intestinalis can utilize carbon sources other than glucose, and is not absolutely dependent upon glucose as an energy source.     

Evaluation of a monoclonal antibody-based immunoassay for detecting type A Clostridium botulinum toxin produced in pure culture and an inoculated model cured meat system

A monoclonal antibody-based amplified enzyme-linked immunosorbent assay (ELISA) method for detecting Clostridium botulinum type A toxin was evaluated for its ability to detect the toxin in the supernatant fluid of pure cultures and after growth from Cl. botulinum spores inoculated into pork slurries. Slurries containing NaCl (1.5-4.5% w/v) and polyphosphate (0.3% w/v) were either unheated or heated, 80 degrees C/5 min + 70 degrees C/2 h, before storage at 15 degrees, 20 degrees or 27 degrees C. The presence of specific toxin was confirmed by mouse bioassay and results compared with those of the amplified ELISA method. A total of 49 strains, 39 Cl. botulinum and 10 Cl. sporogenes (putrefactive anaerobes), and 95 slurry samples were tested. Fourteen of 15 strains of type A Cl. botulinum and 34 of 36 slurry samples containing type A toxin were positive by ELISA. No false positive reactions occurred with Cl. botulinum types B, C, D, E and F, or with the 10 strains of Cl. sporogenes. However, toxin produced by one strain of Cl. botulinum type A (NCTC 2012) was not detected by the amplified ELISA.     

Thiaminase Activity Amongst Strains of Clostridium sporogenes

All the 28 strains of Clostridium sporogenes type I tested produced thiaminase. Only 2 of the 16 strains of Cl. sporogenes type II tested were positive for the enzyme; these gave a weak positive reaction. The single strain of Cl. sporogenes type III behaved in a manner similar to the strains of type I, giving a strong positive thiaminase reaction. Thiaminase production amongst the strains of Cl. sporogenes does in the main support the cultural, biochemical and immunological properties described earlier.     

Oxygen- and Glucose-Dependent Regulation of Central Carbon Metabolism in Pichia anomala

We investigated the regulation of the central aerobic and hypoxic metabolism of the biocontrol and non-Saccharomyces wine yeast Pichia anomala. In aerobic batch culture, P. anomala grows in the respiratory mode with a high biomass yield (0.59 g [dry weight] of cells g of glucose⁻¹) and marginal ethanol, glycerol, acetate, and ethyl acetate production. Oxygen limitation, but not glucose pulse, induced fermentation with substantial ethanol production and 10-fold-increased ethyl acetate production. Despite low or absent ethanol formation, the activities of pyruvate decarboxylase and alcohol dehydrogenase were high during aerobic growth on glucose or succinate. No activation of these enzyme activities was observed after a glucose pulse. However, after the shift to oxygen limitation, both enzymes were activated threefold. Metabolic flux analysis revealed that the tricarboxylic acid pathway operates as a cycle during aerobic batch culture and as a two-branched pathway under oxygen limitation. Glucose catabolism through the pentose phosphate pathway was lower during oxygen limitation than under aerobic growth. Overall, our results demonstrate that P. anomala exhibits a Pasteur effect and not a Crabtree effect, i.e., oxygen availability, but not glucose concentration, is the main stimulus for the regulation of the central carbon metabolism.