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 and Sporulation of Clostridium perfringens

A chemically defined medium was developed that could support sporulation and growth of Clostridium perfringens strains ATCC 12916 and H9. This medium consisted of a modification of the basal medium of Boyd et al. plus 0.1% sodium thioglycolate and 0.5% monosodium glutamate. Five other strains grew, but did not sporulate, in this medium. With the addition of more vatamins into the medium, two more strains grew but did not sporulate. The effects of glucose, monosodium glutamate, ammonium glutamate, and sodium thioglycolate on growth and sporulation of C. perfringens ATCC 12916 in the defined medium was investigated.     

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.     

Sporulation of Clostridium cylindrosporum on a Defined, Low-Manganese Medium

Clostridium cylindrosporum HC-1 grew and sporulated well on a defined medium. This is the first demonstration of sporulation of a purinolytic clostridium on a defined medium; manganese levels were below those considered essential for sporulation of most Bacillus species. Sporulation appeared to be initiated before exhaustion of the purine substrate.     

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.     

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.     

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.     

Conditions Affecting Germination of Clostridium botulinum 62A Spores in a Chemically Defined Medium

Spores of Clostridium botulinum type 62A were germinated in a chemically defined medium (8 mm l-cysteine, 11.9 mm sodium bicarbonate, 4.4 mm sodium thioglycolate; buffered with 100 mm TES, pH 7.0). The rate and extent of germination were increased when an aqueous spore suspension was heated sublethally (80 C, 60 min) before addition to the germination medium. Neither sublethal nor lethal doses of gamma radiation had any marked effect on subsequent germination. Maximum germination (>90% in 2 hr) in the defined medium occurred in the pH range of 6.5 to 7.5, at 30 to 37 C, with an l-cysteine level of 8 mm. Increasing l-cysteine to 32 mm increased the rate (over that with 8 mm l-cysteine) but not the extent of germination. The rate and extent of germination increased with NaHCO(3) addition to 8.3 mm, but increasing levels to 11.9 mm had no further effect. For maximum germination, 2.2 mm sodium thioglycolate was required and higher levels (to 8.8 mm) had no further enhancing or inhibitory effect. Under optimal conditions for germination, 97% of the spores had become heat sensitive; 98% had become sensitive to radiation; 88 and 91% had become phase dark and stainable, respectively, and the spore suspension had lost 46% of its initial optical density by 2 hr. Loss of heat resistance preceded loss of radiation resistance, acquisition of stainability, and phase darkening by about 12 min.     

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.     

Inhibitor of Clostridium perfringens Formed by Heating Sodium Nitrite in a Chemically Defined Medium

An inhibitor of Clostridium perfringens formed when low levels of nitrite were autoclaved with a defined chemical medium. A systematic study of the medium revealed that only amino acids and mineral salts were involved in the production of this inhibitor, which was proven to be a toxic compound formed from cysteine, ferrous sulfate, and sodium nitrite. The inhibitor was compared to several known compounds. S-nitrosocysteine inhibited the test organism, but would not form in the test system in amounts large enough to explain the observed inhibition. Roussin red salt was unstable in the test system and therefore was not the inhibitor. Roussin black salt, which was also inhibitory, could form in sufficient amounts to explain the inhibition. A complex of cysteine, iron, and nitric oxide was detected in the autoclaved solution of cysteine, ferrous sulfate, and sodium nitrite; this cysteine complex did not appear to be inhibitory, however, at levels which could form in the autoclaved medium. The observed inhibition may have been due to the combined effects of sublethal concentrations of each compound.     

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.     

Effect of Arginine on Toxin Production by Clostridium difficile in Defined Medium

Twenty strains of Clostridium difficile were examined for the effect of arginine on toxin production in a defined medium. In three strains, the production of toxins A and B was greatly enhanced in the absence of arginine. These strains showed distinctively poorer growth in the absence of arginine in comparison with the remaining 17 strains, indicating that the presence of arginine is required for good growth among the three strains. From the present results, test strains were divided into two groups: a group in which arginine insufficiency caused distinctly poor growth and enhanced toxin production, and another group in which there was neither distinctly poor growth nor enhanced toxin production. The phenomenon is discussed in relation to the biosynthesis and catabolism of arginine.     

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.