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Title: Investigation of bacterial biosurfactant production for industrial use
Author: Perfumo, Amedea
ISNI:       0000 0004 2721 9483
Awarding Body: University of Ulster
Current Institution: Ulster University
Date of Award: 2009
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This study presents part of a multifaceted study of microbial biosurfactants and their industrial potential. The first step was the assembly of a microbial culture collection of a variety of biosurfactant-producing organisms with the choice of biosurfactant types informed by the industrial partner. The choice was based on two main criteria, the ease of product recovery and its final yield. Two groups of glycolipid biosurfactants were selected for further study: rhamnolipids produced by Pseudomonas aeruginosa and sophorolipids by Candida spp. Efforts to manipulate the biosurfactant chemical profiles by changing the cultivation media (carbon source in particular) and conditions in shake-flasks, demonstrated that there is only a limited possibility for changing the biosurfactant composition. This raised the question of the extent to which biosurfactant production is constrained by genetic determinants? To overcome the limitations of the flask-scale production, selected P. aeruginosa and Candida strains were used in bioreactors, and rhamnolipids and sophorolipids were synthesised in quantities sufficient for extraction and purification. The purified biosurfactants were used by the project partners for further characterisation and formulation in trial industrial products. Rhamnolipid yields were approximately 10 g/L whilst sophorolipid production exceeded 100 g/L. Detailed examination of the orcinol assay, which is widely used for the determination of rhamnolipid yields, showed that the method is flawed and provides an overestimate of yield when compared to quantification following extraction and purification. The culture approach had demonstrated the restricted possibilities for manipulating rhamnolipid production profile in P. aeruginosa and therefore a wider range of strains from different environmental niches were selected for genetic analysis. The aim of this part of the investigation was to establish the extent of natural gene variation which could be exploited for customised biosurfactant production. The comparative analysis of the rhl genes, coding the factors involved in rhamnolipid biosynthesis, was carried out on different P. aeruginosa strains isolated from water, soil and including pathogenic strains infecting cystic fibrosis patients. The extent of the gene sequence diversity resulted < 5%, which indicated that the rhl genes are conserved and are part of the core genome of P. aeruginosa. The single polymorphisms that occurred on the gene sequences, which gave rise to several variants, revealed no clear effect on the phenotype but appeared rather random. These variants showed also no specific correlation with different habitats. The analysis of the codon usage further supported the confidence in the highly conservative nature of the rhl genes. Most amino acids were encoded by highly preferred codons, some of which were selected upon the optimal translational efficiency, some others were instead determined by the high GC content of P. aeruginosa genome. On the whole, our data correlated well in showing that rhamnolipid biosurfactants produced by P. aeruginosa are conserved in their structure and profile as well in the genetic makeup. As involved in many important biological activities under a variety of environmental conditions, rhamnolipids developed as highly fit molecules. In the light of this, it appears that the likelihood that isolates of P. aeruginosa displaying unusual rhamnolipid profiles and features occurring naturally would be limited. Alternative routes for the development of industrially customised rhamnolipid biosurfactants are suggested as either the genetic manipulation of P. aeruginosa or the screening of rhamnolipid-producing organisms closely related.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available