| dc.description.abstract | Bacillus cereus ATCC 14579 was grown under different conditions, in different growth media, and the following parameters were investigated; cell number, pH,oxygen levels, glucose levels, cell morphology, proportions of live/dead bacteria and the expression of selected genes involved in energy metabolism. Flow cytometry was
used in the determination of cell number and was validated by comparison to viable counts. B. cereus ATCC 14579 was grown with shaking in 1 L Erlenmeyer flask in seven different growth media (a defined media, 20%, 50%, 100% Luria bertani and 25%, 50%, 100% Tryptic soy broth), which contained different carbon and nitrogen
levels. The composition of the growth media was shown to have a significant effect on the pH of the media and cell morphology. The pH started to decrease from approximately 7.0 to 5.5, five hours after inoculation. This could be due to the formation of acidic fermentation products. Glucose was depleted after 8-9 hours of
growth and this was followed by an increase in pH, which was proportional to the amount of organic nitrogen in the media. This could be due to the utilization of the acidic fermentation products, an increase in ammonia levels from amino acid catabolism or a combination of these factors. A growth media, 50% LB that gave
satisfactory growth, minimal changes in pH and no detectable changes in cell morphology was identified and used in all further experiments. B. cereus was also grown in 50% LB media in 1 L Erlenmeyer flask (surface area 113 cm2) and in a large surface area (530 cm2) culture with and without shaking. Cultures grown with shaking reaches a 10-fold higher cell density in the stationary phase, than cultures grown without shaking. The pH in the cultures grown without shaking start to decrease five hours after inoculation but continued to remain low (pH 4.5-5.5) during further growth and throughout the stationary phase. Surface area does not have a significant influence on growth and pH.The expression levels of genes involved in fermentation, amino acid metabolism, the TCA cycle and ATP synthesis, in cultures grown with and without shaking, were
measured using real-time reverse transcription PCR, 5 and 12 hours after inoculation.The following genes were selected: Fructose-6-phosphatekinase (Fru), Lactate dehydrogenase (Lac1), Acetyl-CoA synthetase (Acco1), Glutaminase (Glu2), á-ketoglutarate dehydrogenase E1 (Oxde), Citrate synthase (Cisy) and Atp synthase å-chain (Atp). The results show different expression of these metabolic genes in
bacteria grown with and without shaking. “Atp” and “Fru” are slightly down regulated in the cultures grown with shaking compare to the cultures grown with shaking, at time 5 and 12. “ Lac1 “ was upregulated 12 hours after inoculation in both cultures but to a lesser degree in the cultures grown without shaking.“Acco1” and
“Glu2” were upregulated 12 hours after inoculation in the cultures grown with shaking and down regulated in the cultures grown without shaking. “Oxde” are upregulated at time 12 in both cultures. “Cisy1” are upregulated at time 12 in both cultures but to a greater degree in the cultures grown with shaking. There appears to be a correlation between the glucose exhaustion, a requirement of alternative carbon
and energy sources and the upregulation of “Lac1”, “Acco1”, “Glu2”, “Oxde” and “Cisy1” at time 12 in the shaking culture. | nor |