By using standard isolation procedures 55 microbial isolates were successfully obtained from a number of oil-contaminated sources. These microbial isolates, along with a further 17 obtained from the University culture collection were screened for their ability to degrade hexadecane. Of these, all but two strains were able to utilise hexadecane as their sole carbon and energy source. One isolate, JP1, demonstrated outstanding growth on liquid hexadecane media (final cell-density equal to 1.9 x 10 10 CFU ml -1). A number of techniques were implemented in the screening of the 70 microorganisms for the ability to produce biosurfactants. These methods were based upon the ability of a surfactant to emulsify hexadecane. However, all proved to be unsuccessful methods of detection due to limits of quantification. Surface-tension measurement of the cell-free broth of each isolate was deemed to be the most successful detection method of all those attempted. This technique was selected on the basis of its simplicity, rapidity and reproducibility. On the basis of surface-tension measurements, isolate JP1 demonstrated the greatest reduction to 30 dynes cm "1. On the basis of surface-tension measurements JP1 was deemed the best biosurfactant producer and was selected for further investigations. An absence of suitable quantitative surfactant assays led to the need for the development of such a technique. Two spectrophotometric assays were investigated, the first based upon a surfactants ability to disrupt a liposomal suspension and the second based upon the ability to induce blood haemolysis. Both assays were successfully developed using Triton X-100, a known biological surfactant. Limitations of the liposomal assay were identified when the cell-free broth of JP 1 was introduced into the system. The blood haemolysis assay was successfully implemented to determine biosurfactant production by JP I during a fourteen day fermentation period. The influence of media formulation on biosurfactant production by JP1, demonstrated that maximum production was achieved with a2% (v/v) hexadecane medium incorporating a nitrogen concentration of 0.351 g 1.1 and a phosphorus concentration of 1.218 g 1-1. These concentrations corresponded to a C: N ratio of 48: 1 and a C: P ratio of 14: 1. The biosurfactant exhibited some secondary metabolite characteristics and attained its maximum production during stationary phase. To enable a suitable downstream processing method to be selected for the harvest of the biosurfactant, the nature of the biosurfactant was elucidated. Analytical techniques identified the compound responsible for the surface-tension reducing abilities of the cell-free broth as a glycolipid (30 dynes cm "1). It was demonstrated that the lipid was not responsible for the emulsification abilities of the cell-free broth. Further analytical methods identified the bioemulsifier as a protein with a molecular weight of 14 KDa
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:308158 |
| Date | January 1996 |
| Creators | Perry, Lisa J. |
| Publisher | University of Wolverhampton |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
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