Browsing by Author "Johannes, Emile"
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- ItemKinetic evaluation of the production of Bacillus lipopeptides effective against Tuberculosis(Stellenbosch : Stellenbosch University, 2019-04) Johannes, Emile; Clarke, K. G.; Pott, Robert William M.; Stellenbosch University. Faculty of Engineering. Dept. Process Engineering.ENGLISH ABSTRACT:Tuberculosis (TB), caused by Mycobacterium Tuberculosis, is the second largest cause of death resulting from a single infectious agent globally. South Africa has one of the highest number of active TB cases globally and it was estimated that approximately 1% of South Africans develops active TB each year. Multi-drug resistant TB (MDR-TB) is of even greater concern due its low cure rate of only 50% for treated MDR-TB patients. The lipopeptide biosurfactant, surfactin, produced by various Bacillus species, offers a promising alternative antimicrobial agent against TB causing organisms due to its ability to lyse cell membranes and alter membrane permeability. The haemolytic activity of surfactin, however, limits its use as a medical drug to be ingested by humans, but does not limit its use in other applications such as detergents and disinfectants in the fight against TB. The large-scale production of surfactin is limited by low yields and high purification costs, hence economically attractive approaches needs to be developed to realise the commercial production of surfactin as an antimicrobial agent to be used in the fight against TB. Lipopeptide production greatly relies on factors such as medium composition, process conditions and environmental factors, thus by optimising these conditions the cost of both upstream processing, and downstream purification, can be reduced significantly. The overall aim of this study was to investigate the effect of medium composition and process conditions on the growth and lipopeptide production kinetics of B. subtilis in batch culture and advise on the conditions that will improve the upstream production of surfactin, for possible use as an antimicrobial agent against M. tuberculosis. Shake flasks were used to study the effect of distinct nitrogen sources (NH4+ and NO3-) on the process kinetics by supplying ammonium and nitrate at discrete nitrogen ratios (NH4-N:NO3-N). A rigorous kinetic analysis yielded the optimum nitrogen source ratio for surfactin production by B. subtilis to be 0.5:0.5. An NH4-N:NO3-N ratio of 0.5:0.5 yielded the highest surfactin concentration (1084 mg/L), the highest surfactin productivity (36.1 mg/L/h), the second highest specific surfactin production (Yp/x = 0.078), the highest surfactin yield on glucose (Yp/s = 0.031) and the third highest surfactin selectivity (5.11 gsurfactin / gfengycin). The effect of manganese concentration on the process kinetics were also studied in shake flasks and rigorous kinetic evaluation yielded the optimal manganese concentration for surfactin production to be 0.1 mM, however increasing the manganese concentration from 0.05 to 0.1 mM did not significantly improve the surfactin production kinetics. 0.1 mM manganese yielded the highest surfactin concentration (884 mg/L), the highest surfactin yield on glucose (Yp/s = 0.022), the highest surfactin productivity (46.5 mg/L/h), and the second highest the highest specific surfactin productivity (Yp/x = 0.089) and surfactin selectivity (5.9 g surfactin / g fengycin). The optimal nitrogen source ratio and manganese concentration from the shake flask studies were used to evaluate the process kinetics under controlled conditions in a batch bioreactor and were compared to the process kinetics obtained in the shake flasks. All bioreactor kinetic parameters (surfactin concentration – 891 mg/L; Yp/x – 0.113; Yp/s – 0.021) were almost identical to those in shake flasks (surfactin concentration – 854 mg/L; Yp/x – 0.087; Yp/s – 0.022), except for μmax (0.39 h-1 in the bioreactor and 0.5 h-1 in the shake flask culture) and surfactin productivity (18.56 mg/L/h in the bioreactor culture and 44.95 mg/L/h the shake flask culture). The differences were attributed to interference caused by antifoam addition in the bioreactor culture due to vigorous foaming, however further investigation is required. It was also recommended that alternative methods to handle foaming, such as foam fractionation, should be investigated in future work. A response surface methodology (CCD) design of shake flask experiments yielded a nitrogen source ratio (NH4-N:NO3-N) of 0.35:075, manganese concentration of 0.06 mM, and a relative filling volume (RFV) of 0.5 as optimal to achieve maximum surfactin production by B. subtilis. NH4-N:NO3-N ratio and oxygen availability (relative filling volume) were significant parameters (α = 0.05) affecting surfactin concentration, Yp/x, and surfactin selectivity, whilst manganese concentration did not have a significant effect on any of the responses. It was recommended that nitrogen source ratio and oxygen availability should be optimised under controlled conditions in a batch bioreactor as shake flasks offer limited control over oxygen availability Finally, the cell-free supernatant was used to test for antimicrobial activity against Mycobacterium aurum. The antimicrobial cell-free supernatant did not show any antimicrobial activity against M. aurum. It was recommended that the supernatant undergo further processing such as acid precipitation, solvent extraction and/or adsorption followed by antimicrobial testing against M. aurum after each purification step. Different methods for antimicrobial testing should also be investigated.