Characterisation and development of bacterial biosurfactants for biotechnological applications.

Specific objectives of the project:
1. To first select a subset of bacterial strains that produce powerful surface-active agents (i.e. bioemulsifiers or biosurfactants).
2. Determine the growth kinetics and production of the SAs during growth of the strains in pre-optimised liquid medium.
3. To recover and partially purify small-scale quantities of the SAs and determine their composition by, for e.g., chemical assays, NMR etc.
4. To determine the functional properties of the SAs, such as their pH and temperature tolerances, interaction with different oil substrates, critical micelle concentration etc.
5. To optimise the production of the SAs by the strains using initially flask-based cultures followed by small-scale fermenter studies.
6. To explore protocols and fine-tune the experimental conditions of the SAs for subsequent testing to evaluate their potential for use in commercial formulations and/or biotechnological applications.

Methodology:
To screen for strains that produce SAs with emulsifying properties (i.e. bio-emulsifiers), sugar substrates (e.g. glucose, sucrose, malt extract) will be tested since these have been shown, from our experience, to be suitable feedstocks for yielding powerful bio-emulsifiers from marine bacterial strains. A standard emulsification assay, which involves emulsifying the spent culture medium against an oil substrate, will be used to identify strains that produce bio-emulsifiers that form oil-in-water (O/W) and/or water-in-oil (W/O) emulsions. High O/W and W/O emulsification activities, of at least 50% relative to uninoculated controls, will be used as the criterion for the selection of strains for further evaluation.
For strains that produce SAs with surfactant properties (i.e. bio-surfactants), oil substrates (e.g. rapeseed oil) will be used because they are recognized to favour the production of SAs with surfactant, rather than emulsifying, properties. During incubation of the strains on different oil substrates, the interfacial meniscus, which sits at the boundary between the lower aqueous and upper non-aqueous (oil) phases, will be monitored for any change to its curvature. Strains that completely straighten the interfacial meniscus - a positive indication of bio-surfactant production - will be selected for further evaluation using surface and interfacial measurements in the production medium.
Strains selected for producing SAs with putative powerful surface-active properties will be regrown under the same conditions that yielded these activities, this time to determine if the SAs are cell-bound or released extracellularly into the culture medium. This is important because the physical location (cell-bound or extracellularly released) of the SA molecules in the culture medium during their production, such as in a fermentation process, can have a significant impact to the processes involved in their recovery and, in turn, whether it is economically feasible. To determine this, the cells will be removed from the culture medium and thoroughly washed prior to testing if they possess emulsifying and/or surfactant activities. The same will be tested on the cell-free spent medium. Only strains that are found to produce extracellulary-released SAs exhibiting high emulsifying and/or surfactant qualities will be selected for further study.
Properties of the SAs will be determined for various conditions (i.e. tolerances to temperature, pH, salinity, etc.) and the oil substrates they are able to emulsify and/or reduce their surface tension will also be determined. Criteria that will be used for selecting candidate SAs for further development will be based on their ability to confer high stabilization and emulsification activities and/or surface tension reduction.

This PhD project aims to discover new types of SAs produced by novel marine bacterial strains, and provide new knowledge on the properties of the SAs the strains produce, including their potential biotechnological use.