Elicitation, a successful strategy for overproduction of secondary metabolites has been reported in plant, fungal and filamentous bacteria. Based on this evidence, elicitation was assessed for its potential to enhance antibiotic production in Bacillus cultures. This work reports, for the first time, the effect of oligosaccharide elicitors, concentration and addition time through optimisation studies for the enhancement of bacitracin A production by Bacillus licheniformis cultures. The optimal elicitor type, addition time and concentration was 100 mg L-1 of oligoguluronate (OG) at 24 hours. Different carbohydrate- based elicitors were used to investigate their effect on reactive oxygen species (ROS) and catalase production in B. licheniformis. Changes in ROS levels were observed when cells were challenged with OG and MO (mannan oligosaccharides) at 24 h and 72 h. Catalase activities were 50% and 43% higher in OG and MO supplemented cultures respectively compared to the control cultures. The effect of optimal single (OG: 100 mg L-1 , 24 h) and multiple (OG: 100 mg L-1 , 0 h; MO: 200 mg L-1 , 24 h) elicitor addition to enhance bacitracin A production in B. licheniformis cultures was studied. HPLC results demonstrated an increase of 37% and 23% in bacitracin A production in multiple and single elicitor addition respectively compared to control cultures. The effect of elicitors was also investigated at the transcriptional level for bacitracin biosynthetic bacABC and ABC transporter bcrABC genes. Absolute real-time PCR results revealed higher transcript levels of bacABC and bcrABC in elicitor-added cultures compared to control cultures. A relationship between the enhanced bacitracin A production and the increased bacABC and bcrABC transcript copy numbers was found. Stirred tank reactor fermentation with controlled pH (7.0), increased the production of bacitracin A by 30% compared to fermentations without pH regulation. The addition of the oligosaccharides to the cultures caused increases in carbon dioxide evolution rate, oxygen uptake rate, ATP levels, L-glutamic acid consumption rate and bacitracin A production compared to the control cultures. Intracellular calcium levels in bacterial cultures were measured by the use of aequorin technology. Addition of OG and MO caused 11 and 7 fold increases in cytosolic calcium levels in Escherichia coli. Fold increases of 10 and 3 were also observed with OG and MO supplementation to B. subtilis cultures, respectively. Addition of different elicitors also resulted in different calcium signatures which could be used to transmit specific signals inside the cell. Measurement of intracellular calcium levels in B. licheniformis cultures was unsuccessful and awaits further studies. Events involved in the elicitation of B. licheniformis cultures were presented and put together to bring forward the elucidation of the mechanism of elicitation in bacterial cultures. Understanding of the mechanism of elicitation would help in its application in other microbial systems, potentially providing economical benefits to biotechnological industries.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:502244 |
| Date | January 2008 |
| Creators | Murphy, Tania Marcela |
| Publisher | University of Westminster |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
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