Bacterial production of antimicrobial biosurfactants
Thesis (MScEng (Process Engineering))--University of Stellenbosch, 2009.
Surfactants are compounds that reduce interfacial surface tension, resulting in detergency, emulsifying, foaming and dispersing properties. Surfactants produced via biochemical processes (biosurfactants) form a niche market with their low toxicity, biodegradability and high specificity attributes. Biosurfactants have recently received considerable attention owing to their potential as biomedical molecules. In this study a knowledge base was established for the development of a process which produces biosurfactants for use as antimicrobial agents. Specifically, rhamnolipid biosurfactants were produced from Pseudomonas aeruginosa and tested for antimicrobial activity against target organisms. Accurate and reproducible analyses for the quantification of rhamnolipids and antimicrobial activity were developed. The amount of rhamnolipid was determined indirectly by measuring the rhamnose concentration. A novel HPLC method as well as an orcinol colorimetric method were developed for rhamnose measurement. In order to obtain accuracy with the orcinol method it was found that samples must be extracted at least three times prior to the analysis. An examination of literature on rhamnolipid production showed that many studies used colorimetric methods without extraction. Antibacterial activity was quantified by zone clearing around wells of supernatant in soft agar containing the target organism Mycobacterium aurum. This target organism is especially important in a South African context, since it is used to indicate possible susceptibility of tuberculosis to antibiotics. This method was developed for antibacterial testing, after a standard disk diffusion method proved to be ineffective. Antifungal activity of rhamnolipids was evaluated against the fungus Botrytis cinerea, by growing a lawn of fungus on a plate and adding rhamnolipid. The factors influencing rhamnolipid production were studied by growing different Pseudomonas aeruginosa strains from the ATCC culture collection, namely ATCC 9027 and ATCC 27853 as well as a locally isolated strain under different media conditions. The initial focus was on production of biosurfactants in media containing glucose as substrate. Alkanes were subsequently investigated as an alternative substrate, since they are readily available in South Africa as byproducts from the petrochemical industry. The rhamnolipids produced from the culture collection strains were evaluated for their antibacterial activity against Mycobacterium aurum. A number of key factors were identified which were important for the development of a rhamnolipid production process. Of critical importance were the media conditions. Good production was achieved on glucose media containing a phosphate limitation, pH buffering around neutral pH and a high carbon concentration (2 % carbon). When Pseudomonas aeruginosa ATCC 9027 was cultured on this medium (a minimal salts phosphate limited medium with a Tris buffer), it produced 1.31 g/l rhamnose, equivalent to 4.0 g/l rhamnolipid. This rhamnolipid concentration is 2.7-fold higher that of 1.47 g/l reported in the literature with the same strain (cultured on a different phosphate limited medium The particular strain also proved to be a factor which influenced the yield of rhamnolipids. A rhamnose concentration of 0.43 g/l was obtained with Pseudomonas aeruginosa ATCC 27853 grown on MSM+Tris medium, compared to 1.31 g/l produced by Pseudomonas aeruginosa ATCC 9027 on the same medium. The most promising strain and medium, Pseudomonas aeruginosa ATCC 9027 and MSM+Tris medium, were evaluated under controlled conditions in an instrumented bioreactor. Nearly double the rate of growth and production were obtained in the bioreactor, indicating that production time can be shortened considerably under controlled conditions. However, when compared to shake flask studies, only a 4 % increase in growth and a 5 % increase in rhamnolipid production were achieved in the bioreactor, indicating that the yield was limited by the media components or process conditions. With media containing hexadecane as sole carbon source, negligible rhamnolipid production was achieved. Slow growth was observed and the stationary phase had not been reached even after 2 weeks of growth. It was shown that in glucose media rhamnolipid production only commenced in the stationary phase. Since the stationary phase was not reached during growth on hexadecane, rhamnolipids, which are known to increase the availability of alkanes through emulsification and solubilisation, could not be produced. A strategy was devised to accelerate growth on alkane media. A dual substrate medium containing both glucose and hexadecane was investigated. It was hypothesised that growth would be promoted by glucose leading to rhamnolipid production, which would then increase the uptake of hexadecane. Rhamnolipid was produced in the dual substrate experiments, but the hexadecane uptake was still poor. This was suggested to be due to the exposure of the cells to glucose in the inoculum or test flask, which hampered the ability of the cells to utilise hexadecane. It was reasoned that the ability to utilise hexadecane was determined by the cell hydrophobicity, which was influenced by the exposure to hydrophilic or hydrophobic substrates. Rhamnolipids from Pseudomonas aeruginosa ATCC 9027 and ATCC 27853 were shown to have antibacterial activity against Mycobacterium aurum. The largest zone of clearing of 45 mm was obtained with 4 g/l rhamnolipid from Pseudomonas aeruginosa ATCC 9027. The activity was shown to be directly related to the rhamnolipid concentration, highlighting the importance of maximising the biosurfactant yield when developing a process for the production of rhamnolipids as antimicrobial agents. Antifungal activity tests against Botrytis cinerea were inconclusive. Future studies should expand the antimicrobial application of rhamnolipids by testing their activity against a larger range of target organisms. In order to maximise the rhamnolipid yield in future studies, a fed batch process is proposed which would increase the cell density thereby increasing rhamnolipid production and prolonging the stationary phase, which was found to be the phase associated with rhamnolipid production. Different feeding strategies should be investigated, depending on the kinetics of substrate consumption. It is desirable to feed the smallest volume of substrate that is necessary with a high concentration in order to keep the dilution rate low and maximise the product concentration. A factorial design is recommended for this purpose. Further studies with alkanes as carbon source should be conducted using strains that have been maintained and cultured on media containing alkanes as sole carbon source. Alternative biosurfactant producing strains should also be investigated, which have higher natural cell hydrophobicities.