Production of Clostridium pasteurianum in a Defined Medium

A method for the growth of Clostridium pasteurianum in a 140-liter (total capacity) stainless-steel vessel is described. By preventing the pH value from falling below 5.6, the growth of cultures was prolonged. Larger amounts of the carbon source (sucrose) and the nitrogen source (ammonium ion) were supplied and consumed, and cell yields of up to 5.56 g (dry weight) per liter were obtained. The highest cell yield previously reported was 1.7 g (dry weight) per liter obtained under nitrogen-fixing conditions in 500-ml cultures. The ferredoxin content of the cells was comparable with that obtained by earlier workers.     

Chemically Defined Medium for the Growth of Clostridium perfringens

A defined medium which supports growth of Clostridium perfringens at low inoculum levels was developed. Generation time for strain 8797 was 1.5 times greater than previously reported for growth in purged fluid thioglycolate medium.     

Chemically Defined Medium for Growth of Clostridium thermocellum, a Cellulolytic Thermophilic Anaerobe

Nutritional requirements of Clostridium thermocellum were examined, and a defined medium was formulated which supported reproducible growth through 10 serial subcultures.     

Sporulation of Clostridium acetobutylicum P262 in a Defined Medium

A defined minimal sporulation medium for Clostridium acetobutylicum P262, which produces high levels of solvents, is described. The overall sporulation sequence was similar to that of other endospore-forming bacteria. However, we observed a presporulation stage, during which swollen phase-bright cells which contained large amounts of granulose formed. During sporulation, the initiation of spore coat formation occurred before the onset of cortex formation. Other Clostridium strains tested showed marked variations in ability to grow and sporulate in various minimal media.     

Chemically Defined Minimal Medium for Growth of the Anaerobic Cellulolytic Thermophile Clostridium thermocellum

A minimal chemically defined medium has been developed for Clostridium thermocellum. The growth factors required are biotin, pyridoxamine, vitamin B₁₂, and p-aminobenzoic acid.     

Growth of Clostridium tertium and Clostridium septicum in Chemically Defined Media

Defined media for the growth of Clostridium tertium and Clostridium septicum are described. The requirements for growth of these two species are compared with each other and with those of Clostridium perfringens.     

Defined Medium for Growth of Haemophilus influenzae

Adenosine-3',5'-cyclic phosphate (cyclic AMP) is absolutely required for flagella formation and, hence, motility in cyclic AMP-deficient mutants of Escherichia coli and Salmonella typhimurium.     

Hydrogenase I of Clostridium pasteurianum functions as a novel selenite reductase

Clostridium pasteurianum's hydrogenase I, an important constitutive metabolic enzyme, has been shown to function as a 'novel selenite reductase'. Selenite reductase activity was found to co-purify with hydrogenase I activity; the fold purification and specific activities for these two activities paralleled each other throughout the purification steps. The highly purified hydrogenase I apparent K(m) for the selenite substrate was 0.2 mM. The stoichiometry for the enzymatic reduction of SeO3(2-) to Se(0) via H2 oxidation, was determined to be 2.3:1 (H2:Se(0)), very close to the theoretical ratio of 2:1 for this reduction reaction. Known electron carriers required for hydrogenase I activity were also found to couple its selenite reductase activity, the most efficient one being ferredoxin. The purified hydrogenase I not only reduced selenite but also tellurite, and its selenite activity was completely inhibited by O2 and CuSO4, potent inhibitors of hydrogenase I activity.     

Growth of Phytomonas serpens in a Defined Medium; Nutritional Requirements

ABSTRACT. Three strains of Phytomonas serpens two from tomatoes, Lycopersicon esculentum one from the insect Phtia picta (Hemiptera, Coreidae), were cultivated in a chemically defined medium developed from a defined medium for cultivating insect flagellates. Besides organic growth factors required by other insect trypanosomatids this flagellate requires, serine and inositol. Glutamine stimulates growth, and, surprisingly, does not require heme.     

Continuous Culture of Ruminal Microorganisms in Chemically Defined Medium: II. Culture Medium Studies

Ruminal ciliates have been grown in continuous culture in chemically defined media and in the absence of viable bacteria. Oligotrichic ruminal ciliates seem to require insoluble carbohydrates for growth; the holotrichic ciliates require soluble carbohydrates, but at low concentrations. Both groups of ciliates utilize amino acids as their principal nitrogen source when these are supplied in micromolar concentrations; at millimolar concentrations, amino acids are toxic, possibly from excessive ammonia formation arising from ciliate deaminase activity. Holotrichic ruminal ciliates are destroyed by overdeposition of amylopectin when glucose is present above 0.1% concentration in the medium. Ecological requirements of ruminal ciliates are also described.     

Flux Analysis of the Metabolism of Clostridium cellulolyticum Grown in Cellulose-Fed Continuous Culture on a Chemically Defined Medium under Ammonium-Limited Conditions

An investigation of cellulose degradation by the nonruminal, cellulolytic, mesophilic bacterium Clostridium cellulolyticum was performed in cellulose-fed chemostat cultures with ammonium as the growth-limiting nutrient. At any dilution rate (D), acetate was always the main product of the catabolism, with a yield of product from substrate ranging between 37.7 and 51.5 g per mol of hexose equivalent fermented and an acetate/ethanol ratio always higher than 1. As D rose, the acetyl coenzyme A was rerouted in favor of ethanol pathways, and ethanol production could represent up to 17.7% of the carbon consumed. Lactate was significantly produced, but with increasing D, the specific lactate production rate declined, as did the specific rate of production of extracellular pyruvate. The proportion of the original carbon directed towards phosphoglucomutase remained constant, and the carbon surplus was balanced mainly by exopolysaccharide and glycogen biosyntheses at high D values, while cellodextrin excretion occurred mainly at lower ones. With increasing D, the specific rate of carbon flowing down catabolites increased as well, but when expressed as a percentage of carbon it declined, while the percentage of carbon directed through biosynthesis pathways was enhanced. The maximum growth and energetic yields were lower than those obtained in cellulose-limited chemostats and were related to an uncoupling between catabolism and anabolism leading to an excess of energy. Compared to growth on cellobiose in ammonium-limited chemostats (E. Guedon, M. Desvaux, and H. Petitdemange, J. Bacteriol. 182:2010–2017, 2000), (i) a specific consumption rate of carbon of as high as 26.72 mmol of hexose equivalent g of cells⁻¹ h⁻¹ could not be reached and (ii) the proportions of carbon directed towards cellodextrin, glycogen, and exopolysaccharide pathways were not as high as first determined on cellobiose. While the use of cellobiose allows highlighting of metabolic limitation and regulation of C. cellulolyticum under ammonium-limited conditions, some of these events should then rather be interpreted as distortions of the metabolism. Growth of cellulolytic bacteria on easily available carbon and nitrogen sources represents conditions far different from those of the natural lignocellulosic compounds.     

Development of a Defined Medium for Growth of Cercospora rosicola Passerini

A simple synthetic liquid medium containing a single amino acid, glucose, salts, trace metals, and thiamine was developed for cultivation of Cercospora rosicola Passerini. Thiamine was shown to be important to growth. Culture of C. rosicola Passerini in a chemically defined medium makes possible studies of (+)-abscisic acid biosynthesis and regulation.     

Insights into Actinobacillus succinogenes Fermentative Metabolism in a Chemically Defined Growth Medium

Chemically defined media allow for a variety of metabolic studies that are not possible with undefined media. A defined medium, AM3, was created to expand the experimental opportunities for investigating the fermentative metabolism of succinate-producing Actinobacillus succinogenes. AM3 is a phosphate-buffered medium containing vitamins, minerals, NH₄Cl as the main nitrogen source, and glutamate, cysteine, and methionine as required amino acids. A. succinogenes growth trends and end product distributions in AM3 and rich medium fermentations were compared. The effects of NaHCO₃ concentration in AM3 on end product distribution, growth rate, and metabolic rates were also examined. The A. succinogenes growth rate was 1.3 to 1.4 times higher at an NaHCO₃ concentration of 25 mM than at any other NaHCO₃ concentration, likely because both energy-producing metabolic branches (i.e., the succinate-producing branch and the formate-, acetate-, and ethanol-producing branch) were functioning at relatively high rates in the presence of 25 mM bicarbonate. To improve the accuracy of the A. succinogenes metabolic map, the reasons for A. succinogenes glutamate auxotrophy were examined by enzyme assays and by testing the ability of glutamate precursors to support growth. Enzyme activities were detected for glutamate synthesis that required glutamine or α-ketoglutarate. The inability to synthesize α-ketoglutarate from glucose indicates that at least two tricarboxylic acid cycle-associated enzyme activities are absent in A. succinogenes.     

Growth of an Insect Trypanosomatid at 37 C in a Defined Medium*

SYNOPSIS. A defined medium for an insect trypanosomatid, “Leptomonas pessoai” (probably a member of the genus Herpetomonas) isolated from the reduviid Zelus leucogrammus, allows growth up to 37 C. No marked differences were evident for growth at 37 C. The requirements for amino acids, vitamins, purine, and hemin, and pH range were like those established for Crithidia fasciculata. Again like C. fasciculata, it remains alive at 4 C for at least 3 months in a çlycerolated defined medium.     

Factors Influencing the Growth of Glaucoma chattoni in a Chemically Defined Medium*

SYNOPSIS. Glaucoma chattoni strain A has been grown in the chemically defined medium previously used for Colpidium campylum and Tetrahymena pyriformis. It was necessary to modify the amino acid, nucleotide and other components of the medium in order to obtain optimum growth. The modified medium contained 17 amino acids, nucleic acid components, fatty acids, stigmasterol, sodium acetate, a mixture of B-vitamins and several inorganic salts.     

Development of a Defined Minimal Medium for the Growth of Edwardsiella ictaluri

In this report, a complete defined medium and a minimally defined medium are described for Edwardsiella ictaluri. The complete defined medium consists of 46 individual components, including a basal salt solution, glucose, magnesium sulfate, iron sulfate, six trace metals, four nucleotides, 10 vitamins, and 19 amino acids. This medium supports growth in broth and on solid media. Optimal growth at 30(deg)C was obtained at pH 7.0, and at an osmolality of 390 mosmol/kg of H(inf2)O, with a glucose concentration of 4 g/liter. The defined minimal medium reduces the 46 components of the complete medium to eight essential components, including the basal salt solution, glucose, magnesium sulfate, pantothenic acid, and niacinamide. In addition, specific amino acids that depend on the specific requirements of the individual strains of E. ictaluri are added.     

Development of a Chemically Defined Medium for the Growth of Leuconostoc mesenteroides

A chemically defined medium for the growth of Leuconostoc mesenteroides was developed. This medium contained lactose, Mn(sup2+), Mg(sup2+), 12 amino acids, eight vitamins, adenine, uracil, and Tween 80. We showed the beneficial effect of aerobic conditions on growth and that potassium phosphate (135 mM) is a suitable buffer. The growth rate in this medium was 0.85 (plusmn) 0.10 h(sup-1) for the six strains examined, and cell densities up to 3.5 x 10(sup9) CFU/ml were attained.     

Biosynthesis of amino acids in Clostridium pasteurianum

1. Clostridium pasteurianum was grown on a synthetic medium with the following carbon sources: (a) (14)C-labelled glucose, alone or with unlabelled aspartate or glutamate, or (b) unlabelled glucose plus (14)C-labelled aspartate, glutamate, threonine, serine or glycine. The incorporation of (14)C into the amino acids of the cell protein was examined. 2. In both series of experiments carbon from exogenous glutamate was incorporated into proline and arginine; carbon from aspartate was incorporated into glutamate, proline, arginine, lysine, methionine, threonine, isoleucine, glycine and serine. Incorporations from the other exogenous amino acids indicated the metabolic sequence: aspartate --> threonine --> glycine right harpoon over left harpoon serine. 3. The following activities were demonstrated in cell-free extracts of the organism: (a) the formation of aspartate by carboxylation of phosphoenolpyruvate or pyruvate, followed by transamination; (b) the individual reactions of the tricarboxylic acid route to 2-oxoglutarate from oxaloacetate; glutamate dehydrogenase was not detected; (c) the conversion of aspartate into threonine via homoserine; (d) the conversion of threonine into glycine by a constitutive threonine aldolase; (e) serine transaminase, phosphoserine transaminase, glycerate dehydrogenase and phosphoglycerate dehydrogenase. This last activity was abnormally high. 4. The combined evidence indicates that in C. pasteurianum the biosynthetic role of aspartate and glutamate is generally similar to that in aerobic and facultatively aerobic organisms, but that glycine is synthesized from glucose via aspartate and threonine.