Surfactants are chemical compounds that are able to alter interfacial properties, particularly surface tension. When they are biologically produced, the term biosurfactant is used. One of the most important groups of biosurfactants is a family of chemical compounds known as glycolipids, whose structure consists of a sugar group and a lipid tail. Glycolipids are subdivided into three main groups: rhamnolipids, sophorolipids and trehalolipids, named following their sugar moieties, respectively rhamnose, trehalose and sophorose. Biosurfactants exhibit attractive advantages over chemical surfactants. Examples of these are biodegradability, low toxicity, and effectiveness at extreme temperature, pH and salinity. The objective of the present research project was, first, to investigate the potential of liquid aliphatic hydrocarbons to induce biosurfactant production by the bacterium Ps. aeruginosa 2Bf isolated based on its ability to metabolise alkanes. The second objective was to optimise biosurfactant production using alkanes as sole carbon and energy source, through optimising the mixing & aeration conditions, media conditions as well as provision of alkane, in a stirred tank batch reactor system. The final objective was to describe the biosurfactant formed. Experiments were organised in three major series: the exploratory shake flask based experiments, the bioreactor-based experiments to optimise biosurfactant production and characterise biokinetics and performance, and the biosurfactant characterisation experiments. Following review of a number of methods, microbial cell counts were selected as the most reproducible measure of biomass formation in the presence of alkanes. The presence of biosurfactant was quantified functionally in terms of the emulsification index and alteration of surface tension. Using a shake flask-based study, nitrogen source was investigated in terms of biomass and biosurfactant synthesis. Four pre-selected nitrogen sources were tested in order to select the best for bioreactor based study. These nitrogen sources consisted of specific combinations of three nitrogen compounds, NH4NO3, NaNO3 and (NH4)2SO4. During the study, long chain liquid n-alkanes were used as sole carbon source and the C/N ratio maintained at the value of 18.6 in mass terms. Results confirmed that both a combination of NO3 ' and NH4+ ions or a nitrogen source composed solely of NH4+ ions were suitable for biomass growth and biosurfactant production. (NH4)SO4 was used as the N-source of choice in the remainder of the study. While the C14-C17 alkanes cut was the carbon source of interest in the study, two pure alkanes, n-C12 and n-C16 were tested and compared to the C14-C17 blend. The C14-C17 fraction, sourced as an industrial byproduct, compared favourably as a carbon source with respect to hexadecane and dodecane. ii Biosurfactant production was not observed in Ps. aeruginosa 2Bf cultures where glucose was the sole carbon source and the bacteria were not previously exposed to linear alkanes. Using a mixed carbon source of glucose and alkane, or on pre-exposure of the bacteria to alkane, biosurfactant production was induced. Induction was optimised where alkane was the sole carbon source over a period of four sub-culture steps. In the quantitative optimisation of biosurfactant production through the bioreactor based study, mixing and aeration were optimised; agitation and aeration proved to be equally important, the first at intermediate rates, the second at lower rates. Their interaction, when maximum biomass was used as the variable for response, was found to be important for agitation rates up to 500 rpm. Beyond this range of agitation speed, the interaction between aeration and agitation became negligible. In the case of Eindex as the variable for response, similar results were obtained with regard to the impact of the interaction between aeration and agitation on the process. It was significant from lower to intermediate agitation rates, and negligible from intermediate to higher rates of agitation. Lower aeration rate was found to enhance the oxygen utilisation rate, while mass transfer was relatively favoured by high aeration rate. Regarding the emulsification power of the product, quantitative tests were carried out on culture suspension, supernatant prepared by centrifugation and supernatant prepared by centrifugation and filtration at 0.22μm pore size filters. Results showed that some emulsification effect was lost through centrifugation and filtration. This loss of emulsification effect was more pronounced in the filtration case, thus showing that some biosurfactant was removed along some other material or substance through sticking on filter paper. Foam control was required, and two mechanical foam breakers were compared to anti-foam reagent. It was experimentally established that mechanical foam breakers are preferable to chemical anti-foam reagents. On comparing the two different mechanical foam breakers, the modified two blade paddle with three slits, FB-2, performed better than the simple two blade paddle foam breaker, FB-1. Further investigations showed that the interaction between type of foam control and agitation rate was negligible throughout the process. The Biosurfactant was characterised at the structural level and the antibiotic potential of Ps. aeruginosa 2Bf's biosurfactant was analysed. In addition to the thin layer chromatography, three different spectroscopic methods (mass, infrared & nuclear magnetic resonance) were used to study the chemical structure of the biosurfactant produced. Up to six rhamnolipid structures were tentatively identified with spectrometric analysis whereas only four to five structures could be detected with thin layer chromatography. Possession of an anti-microbial activity by the rhamnolipids produced was confirmed with the B. subtilis inhibition test.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:uct/oai:localhost:11427/5344 |
| Date | January 2009 |
| Creators | Bamara, Prosper |
| Contributors | Harrison, STL |
| Publisher | University of Cape Town, Faculty of Engineering and the Built Environment, Centre for Bioprocess Engineering Research |
| Source Sets | South African National ETD Portal |
| Language | English |
| Detected Language | English |
| Type | Master Thesis, Masters, MSc |
| Format | application/pdf |
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