Effect of culture conditions on growth and adhesion of Bacillus licheniformis

Adhesion to glass of actively growing cells of the thermophilic Bacillus licheniformis, isolated from the Medyaginskaya test borehole (Yaroslavl' oblast), was studied. The reversible adhesion (RA) manifests itself in a decline of cell density (short of cell lysis) in the liquid culture over the first 20-40 min of growth followed by normal exponential growth. The RA is minimal under favorable growth conditions but increases when cells are transferred to a new medium, especially one with a pH, temperature, salinity, or concentration of Ca2+ ions nonoptimal for the given species. Under unfavorable growth conditions, the adhesion becomes irreversible. The obtained data suggest that RA represents an adaptation mechanism important for population survival.     

Effect of bacitracin on the sporulation of Bacillus licheniformis 28 KA

The bacitracin effect on spore germination in Bacillus licheniformis 28 KA--the producer of this antibiotic on media of various composition has been studied. Different intensity of antibiotic synthesis has been revealed on various media. A high content of presynthesized bacitracin has an inhibitory effect on spore germination in B. licheniformis.     

Light-induced inhibition of sporulation in Bacillus licheniformis.

Sporulation of Bacillus licheniformis is inhibited by broad-spectrum light. This phenomenon is intensity dependent and is a near-ultraviolet and blue light effect.     

Motility of Bacillus subtilis during growth and sporulation.

The change of motility and the presence of flagella were followed throughout growth and sporulation in a standard sporulating strain and in 19 cacogenic sporulation mutants of Bacillus subtilis. For the standard strain, the fraction of motile cells decreased during the developmental period to less than 10% at T4. Motility was lost well before the cells lose their flagella. Conditions reducing the decrease of motility also reduced sporulation: motile cells never contained spores. The decrease of motility was not coupled with a decrease in the cellular concentration of adenosine 5'-triphosphate or a decline in oxygen consumption, but an uncoupling agent immediately destroyed motility at any time. Apparently, motility decreased during development because it became increasingly uncoupled from the energy generating systems of the cell. The motility of sporulation mutants decreased after the end of growth at the same time as or earlier than the motility of the standard strain; the early decrease of motility in an aconitase mutant, but not that in an alpha-ketoglurate dehydrogenase mutant, could be avoided by addition of L-glutamate. Sporulation or related events such as extracellular antibiotic or protease production were not needed for the motility decline.     

Aeration effects on metabolic events during sporulation of Bacillus thuringiensis

The metabolism of Bacillus thuringiensis during its sporulation process was investigated under different concentrations of oxygen. At the beginning of sporulation, the aeration conditions were regulated to obtain different oxygen transfer rates (OTR) in four separate fermentations, representing interrupted, limited, non-limited, and saturated oxygenation, respectively. A higher OTR resulted in a higher pH, up to about 9 in the case of saturated oxygenation, while the interrupted oxygenation resulted in a significantly acidic culture. In contrast, the absence of oxygen resulted in rapid sporangia lysis and caused acidification of the medium, indicating a distinctly different sporangia composition and different metabolism. The bacterium also showed different CO₂ production rates during sporulation, although amaximum point was observed in every case.With a higher OTR, the maximal value was observed after a longer time and at a lower value (40, 26, and 13 mmol/L/h for limited, non-limited, and saturated cases, respectively). Despite the exhaustion of glucose prior to the sporulation phase, the interrupted oxygenation resulted in acetate, lactate, and citrate in the medium with a maximum concentration of 4.8, 1.3, and 5.0 g/L, respectively. Notwithstanding, while the metabolic events differed visibly in the absence of oxygen, once sporulation was triggered, it was completed, even in the case of an interrupted oxygen supply.     

Bacitracin and protease production in relation to sporulation during exponential growth of Bacillus licheniformis on poorly utilized carbon and nitrogen sources.

Growth of Bacillus licheniformis in a chemically defined medium containing glucose and ammonium chloride yielded a doubling time of 1.00 h. Examination of the culture during exponential growth revealed a lack of heat-resistant spores together with a complete absence of detectable concentrations of bacitracin or extracellular serine protease. Replacement of glucose as the sole carbon source by glycerol, pyruvic acid, citric acid, or lactic acid resulted in doubling times of 1.13, 2.00, 3.16, and 3.95 h, respectively. Bacitracin, protease, and heat-resistant spores were produced during exponential growth in amounts related to these doubling times. A qualitatively similar pattern was observed when ammonium chloride was replaced by sodium nitrate, alanine, or glutamic acid which gave doubling times of 1.65, 1.77, and 1.90 h, respectively. Protease, but not bacitracin, concentrations were substantially higher when the growth rate was restricted by use of poor nitrogen rather than poor carbon sources. The relationships between bacitracin production, protease production, and the sporulation process are discussed.     

Effect of bioprocess conditions on growth and alkaline protease production by halotolerant Bacillus licheniformis BA17

The effect of bioprocess conditions (pH and temperature) on the growth and alkaline protease production of halotolerant Bacillus licheniformis BA17 bioreactor cultures have been systematically analyzed using response surface methodology in order to assess the importance of these generally disregarded parameters. Two models were proposed differing by the choice of response variable. Under optimized bioprocess conditions, whole alkaline protease activity was about 3 fold higher than the activities obtained in the preliminary studies. Results of this study not only highlight the importance of pH and temperature for further engineering purposes but also serve as basis for understanding the true mechanism lying under the relation between these process parameters and growth and whole alkaline protease production.     

Heat-shock proteins during growth and sporulation of Bacillus subtilis

Four major heat-shock proteins (hsps) with apparent molecular masses of 84, 69, 32 and 22 kDa were detected in exponentially growing stationary phase and sporulating cells of Bacillus subtilis heat-shocked from 30 to 43 degrees C. The most abundant, hsp69, is probably analogous to the E. coli groEL protein. These proteins were transiently inducible by heat-shock. Partial purification of RNA polymerase revealed several other minor hsps. One of these, a 48 kDa polypeptide probably corresponds to sigma 43. The synthesis of this polypeptide and at least two other proteins appeared to be under sporulation and heat-shock regulation and was affected by the SpoOA mutation.     

Regulation of dihydrodipicolinate synthase during growth and sporulation of Bacillus cereus.

A four- to sixfold increase in specific activity of dihydrodipicolinic acid synthase was observed during sporulation of Bacillus cereus. The enzyme from cells harvested before and after the increase in specific activity appeared to be very similar as judged by pH optima, heat denaturation kinetics, apparent Michaelis constants, chromatography on diethylaminoethyl-cellulose and Sephadex G-200, and polyacrylamide gel electrophoresis. Studies with various combinations of amino acids and one of the enzyme substrates, pyruvate, failed to give evidence for control of the enzyme by activation, inhibition, repression, induction, or stabilization. Omission of calcium from the sporulation medium had no significant effect on the specific activity pattern of the enzyme as a function of age of culture.     

Effect of temperature and aeration on Bacillus thuringiensis growth and sporulation

The growth of Bacillus thuringiensis was studied as a function of temperature and aeration. The vegetative growth, the yield of viable spores and their thermoresistance did not depend, for all practical purposes, on the rate of aeration within the range of 25 to 60 mg O2 per litre per minute. A rise of temperature from 20 to 35 degrees C doubled the titre of spores and increased their thermoresistance. When the temperature of cultivation was increased to 40 degrees C, the process of spore formation was inhibited.     

Fermentation during growth and sporulation of Bacillus thuringiensis HD-1

During growth in a glucose-tryptone-mineral salts broth, B. thuringiensis HD-1 Produced lactate, pyruvated, acetate, acetoin, and poly-beta-hydroxybutyrate (PHB). Based on the type and sequence of products found, all or part of the acetate appears to have been produced via the 2,3-butanediol cycle. PHB was produced and utilized during spore formation and may have provided energy for that process.     

Glutamine synthetase and glutamate synthase activities during growth and sporulation in Bacillus subtilis.

Glutamine synthetase in Bacillus subtilis 168 was repressed to a greater extent by L-glutamine or L-arginine than by ammonia when each was used as sole nitrogen source. It was derepressed when either L-glutamate or nitrate was used as nitrogen source. Glutamate synthase was repressed by L-glutamate or L-arginine and, to a lesser extent, by L-glutamine but was derepressed during growth with ammonia or nitrate. Glutamine synthetase activity was unaltered during the onset of sporulation. Glutamate synthase activity, however, underwent a small and apparently transient increase in bacteria induced to sporulate by nitrogen limitation.     

Biochemical changes during sporulation of Bacillus stearothermophilus.

The process of sporulation was studied in Bacillus stearothermophilus. A medium is described that supports good growth and sporulation of the organism. In this medium, which contains glucose, salts, and amino acids, acetate starts to accumulate before any of the glucose is catabolized. Enzymes of the tricarboxylic acid cycle are present at all times during growth and sporulation and are found in dormant spores. As the glucose in the culture is consumed, acetate rapidly increases and the pH of the medium drops. The acetate rapidly disappears during sporulation and the pH rises. Dipicolinic acid appears during sporulation and several key-enzyme activities fluctuate in a characteristic pattern.     

Inhibition of Bacillus subtilis growth and sporulation by threonine.

A 1-mg/ml amount of threonine (8.4 mM) inhibited growth and sporulation of Bacillus subtilis 168. Inhibition of sporulation was efficiently reversed by valine and less efficiently by pyruvate, arginine, glutamine, and isoleucine. Inhibition of vegetative growth was reversed by asparate and glutamate as well as by valine, arginine, or glutamine. Cells in minimal growth medium were inhibited only transiently by very high concentrations of threonine, whereas inhibition of sporulation was permanent. Addition of threonine prevented the normal increase in alkaline phosphatase and reduced the production of extracellular protease by about 50%, suggesting that threonine blocked the sporulation process relatively early. 2-Ketobutyrate was able to mimic the effect of threonine on sporulation. Sporulation in a strain selected for resistance to azaleucine was partially resistant. Seventy-five percent of the mutants selected for the ability to grow vegetatively in the presence of high threonine concentrations were found to be simultaneously isoleucine auxotrophs. In at least one of these mutants, the threonine resistance phenotpye could not be dissociated from the isoleucine requirement by transformation. This mutation was closely linked to a known ilvA mutation (recombination index, 0.16). This strain also had reduced intracellular threonine deaminase activity. These results suggest that threonine inhibits B. subtilis by causing valine starvation.     

Effect of sporulation conditions on the resistance of Bacillus subtilis spores to heat and high pressure

Bacillus subtilis(B. subtilis) cells were placed in various environmental conditions to study the effects of aeration, water activity of the medium, temperature, pH, and calcium content on spore formation and the resulting properties. Modification of the sporulation conditions lengthened the growth period of B. subtilis and its sporulation. In some cases, it reduced the final spore concentration. The sporulation conditions significantly affected the spore properties, including germination capacity and resistance to heat treatment in water (30 min at 97°C) or to high pressure (60 min at 350 MPa and 40°C). The relationship between the modifications of these spore properties and the change in the spore structure induced by different sporulation conditions is also considered. According to this study, sporulation conditions must be carefully taken into account during settling sterilization processes applied in the food industry.