Masters Degrees (Chemical Engineering)
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Browsing Masters Degrees (Chemical Engineering) by browse.metadata.advisor "Clarke, K. G."
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- ItemBacterial production of antimicrobial biosurfactants(Stellenbosch : University of Stellenbosch, 2009-03) Ballot, Francis; Clarke, K. G.; University of Stellenbosch. Faculty of Engineering. Dept. of Process Engineering.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.
- ItemBacterial production of antimicrobial biosurfactants by Bacillus subtilis.(Stellenbosch : Stellenbosch University, 2011-12) Bence, Keenan; Clarke, K. G.; Stellenbosch University. Faculty of Engineering. Dept. of Process Engineering.ENGLISH ABSTRACT: Biosurfactants are microbially produced molecules that show excellent surface-active properties. Bacillus subtilis ATCC 21332 produces the biosurfactant, surfactin, which exhibits antimicrobial activity against bacteria as well as fungi. Although antimicrobial activity has been exhibited by a number of bacterially produced biosurfactants, notably the rhamnolipid from the pathogen Pseudomonas aeruginosa, the GRAS status B. subtilis makes the use of this organism preferable for large scale bioprocesses. The objectives of this study were to: (1) evaluate the effect of different nutrient conditions on growth and surfactin production; (2) evaluate the growth of B. subtilis ATCC 21332 and associated surfactin production on a hydrocarbon substrate; (3) evaluate the antimicrobial activity of surfactin against Mycobacterium aurum, and (4) to establish whether active growth of B. subtilis ATCC 21332 and associated surfactin production can be extended during fed-batch culture. B. subtilis ATCC 21332 was grown on low-nitrate; phosphate-limited and nutrient rich media with glucose as substrate during shake flask culture. Nitrate, phosphate, glucose and surfactin were quantified by HPLC analyses and growth via CDW and optical density measurements. Growth and surfactinproduction were further evaluated during shake flask cultureon a hydrocarbon substratereplacing the glucose in the nutrient rich medium with an equivalent amount of n-hexadecane. The antimicrobial activity was quantified by growth inhibition of M. aurum. Bioreactor batch and fed-batch studies were conducted to evaluate growth and surfactin production under controlled conditions. The fed-batch experiments included four constant dilution rate (D=0.40h-1; D=0.15h-1; D=0.10h-1 and D=0.05h-1) and two constant feed rate (F=0.40L/h and F=0.125L/h) fed-batch strategies. The nutrient rich medium was used for these experiments and also as the feed medium for fed-batch experiments. A CDW of 12.6 g/L was achieved in the nutrient rich medium during shake flask culture and was 2.5- and 1.6-fold higher than that achieved in the phosphate-limited medium and the lownitrate medium respectively. A surfactin concentration of 652 mg/L was achieved in the nutrient rich medium, while a maximum surfactin concentration of 730 mg/L was achieved in the phosphate-limited medium. A surfactin concentration of only 172 mg/L was achieved in the low-nitrate medium. Subsequently, growth and surfactin production were evaluated on n-hexadecane as sole carbon source. After inoculation, the CDW did not increase over a period of 119 h, which indicated that B. subtilis ATCC 21332 was unable to utilize n-hexadecane for growth and surfactin production. The maximum CDW (27 g/L) and maximum surfactin concentration (1737 mg/L) achieved in the bioreactor batch experiments were 2.1- and 2.6-fold higher respectively than that achieved in the nutrient rich medium during shake flask experiments. These results served as a benchmark for further fed-batch experiments. During the fed-batch phase of the D=0.40h-1 experiment, the biomass further increasedby 9 g/h, which was 3.5-, 3.1- and 5.3-fold higher compared to the fed-batch phases of the D=0.15h-1, D=0.10h-1 and D=0.05h-1 experiments respectively. Similarly, the biomass increased by 10.7 g/h during the fed-batch phase of the F=0.40L/h experiment, which was 4.6-fold higher than that of the F=0.125L/h experiment. The average rate of surfactin production was 633 mg/h during the fed-batch phase of the D=0.40h-1 experiment, 29.4-, 5.4- and 34.2-fold higher compared to the fed-batch phases of the D=0.15h-1, D=0.10h-1 and D=0.05h-1 experiments respectively. Analogously, the average rate of surfactin production (544 mg/h) of the F=0.40L/h experiment was 9.4 fold higher than that of the F=0.125L/h experiment. The antimicrobial assay showed that surfactin inhibits M. aurum growth. An inhibition zone diamater of 4mm was measured at a surfactin concentration of 208 mg/L, which linearly increased to 24mm at a surfactin concentration of 1662 mg/L. High feed flow rate strategies achieved higher rates of biomass increase and surfactin production and will thus decrease the production time required for large scale surfactin production.The antimicrobial activity of surfactin against M. aurum indicates that this biosurfactant has the potential to be used against M. tuberculosis, and as such has the potential to be used in the medical industry to reduce the spread of this, and other deadly diseases.
- ItemBioconversion of alkylbenzenes by Yarrowia lipolytica(Stellenbosch : University of Stellenbosch, 2009-03) Lind, Aingy Chantel; Clarke, K. G.; Smit, M. S.; University of Stellenbosch. Faculty of Engineering. Dept. of Process Engineering.The abundance of alkane by-products formed in South Africa presents a feedstock opportunity for the production of a wide range of commercially important products, such as long-chain dioic acids and alcohols. These compounds are formed as intermediates through the biological conversion of alkanes, a route which is particularly attractive when compared with chemical conversion due to its operation under milder process conditions. Furthermore, advances in genetic manipulation, which enable the accumulation of a range of metabolic intermediates, make the biological route remarkably flexible. From the literature review Yarrowia lipolytica was identified as a promising organism for use in studying alkane bioconversion because of its ability to produce large quantities of fatty acids when grown on n-paraffins as a sole carbon source. The bioconversion of alkanes will not only depend on the genetic modification but also on the process conditions to maximise growth and bioconversion. The overall objective of this project was therefore to investigate the potential of Y. lipolytica for alkane bioconversion by defining the conditions that maximise both cell growth and bioconversion. The Y. lipolytica strains supplied (TVN348, TVN493 and WT), however, were not yet modified to the extent that accumulation of metabolic intermediates was possible. Use was therefore made of a model system in which the alkane substrate was substituted with an even chain alkylbenzene. Since Y. lipolytica is unable to metabolise the benzene ring, the alkylbenzene is converted to the metabolic intermediate, phenyl acetic acid (PAA), and the potential for bioconversion assessed through measuring the accumulation of PAA. The specific objectives of the project were therefore 1) to define and quantify the parameters for the establishment of an effective model system in shake flasks with respect to trace elements, buffering, added nitrogen, oxygen supply, glucose concentration, alkylbenzene substrate and inducer requirements 2) to use the defined model system to identify the most promising strain of Y. lipolytica TVN348, TVN493 and WT 3) to use the defined model system and selected strain for evaluation of the influence of time of substrate addition and glucose concentration on cell growth and bioconversion of Y. lipolytica under controlled conditions in an instrumented bioreactor Furthermore, since poor reproducibility in cell growth and bioconversion had been prevalent in previous studies, it was also aimed to identify and statistically quantify the reproducibility between duplicate or triplicate samples in each experiment and between sets of different experiments with respect to PAA formation and cell concentrations. Studies were conducted in shake flask cultures to define and quantify the parameters for the model system. The parameters assessed included trace elements, buffering, nitrogen concentration, oxygen supply, glucose concentration, alkylbenzene substrate type and possible inducer requirements. Trace elements, phosphate buffering and added nitrogen did not significantly affect the cell growth of Y. lipolytica TVN348. The cell concentration of Y. lipolytica TVN348 and TVN493 was increased by 65% and 43% respectively for an increase in oxygen supply by decreasing the working volume from 150ml to 50ml, while the cell concentration of Y. lipolytica WT was increased by 41% when oxygen supply was increased by switching from non-baffled to baffled flasks in 50ml cultures. Bioconversion was also increased for an increase in oxygen supply: 2.4mM to 29.0mM PAA (Y. lipolytica TVN348) and 1.2mM to 21.7mM PAA (Y. lipolytica TVN493) for a decrease in working volume; 10.5mM to 46.6mM PAA (Y. lipolytica WT) when switching from non-baffled to baffled flasks. These results indicated that adequate oxygen supply is crucial to both growth and bioconversion, and that further study should be conducted in 50ml working volumes. Cell concentrations obtained in 1.6% (wt/v) and 3.2% (wt/v) glucose cultures (3.95x108cells/ml and 4.03x108cells/ml respectively) indicated that cell growth was neither enhanced nor inhibited by 3.2% (wt/v) glucose. Of the range of substrates examined (propylbenzene, butylbenzene, sec-butylbenzene, hexylbenzene, ethyltoluene and tert-butyltoluene for Y. lipolytica TVN348 and TVN493; octylbenzene and decylbenzene for Y. lipolytica WT), hexylbenzene was regarded as the best substrate for bioconversion (14.7mM and 14.1mM PAA for TVN348 and TVN493 respectively; 42.6mM PAA for WT). Lastly, the absence of a requirement for an additional inducer such as ethanol or oleic acid was confirmed when PAA was formed from hexylbenzene in the culture containing additional glucose (25.0mM). This suggested that when using hexylbenzene as substrate, bioconversion was induced provided sufficient glucose was available for cell maintenance. Results from duplicate or triplicate flasks in each individual shake flask experiment were reproducible and conclusions were based solely on results which showed 95% confidence intervals. However, reproducibility problems were experienced with results between different sets of experiments carried out under the same conditions. The model system was therefore defined by: 1) no addition of trace elements, additional buffering or added nitrogen, 2) cultures grown in 50ml volumes to supply an adequate amount of oxygen crucial for growth and bioconversion, 3) 3.2% (wt/v) glucose and 4) addition of 1% (v/v) hexylbenzene at 24h with no inducer requirements. Use of the model system in shake flask cultures to identify the most promising of the three strains of Y. lipolytica supplied demonstrated that there was no significant difference in cell growth or bioconversion between these strains. Y. lipolytica WT (which has no genetic modifications) was therefore used for further investigation until an appropriate strain could be substituted when it became available. The growth and bioconversion of Y. lipolytica WT was further investigated under controlled conditions in a bioreactor. The influence of time of substrate addition (11h, 24h, 48h) and glucose concentration (3.2% and 6.4% (wt/v)) on growth and bioconversion was examined. When hexylbenzene was added at 48h, cell growth was increased (8.90x108cells/ml) when compared to two of the triplicate cultures with hexylbenzene addition at 24h (4.74x108cells/ml and 3.92x108cells/ml) and the culture with hexylbenzene addition at 11h (2.82x108cells/ml). The third of the triplicate cultures with hexylbenzene addition at 24h, on the other hand, exhibited the strongest growth (2.23x109cells/ml). The poor reproducibility between the triplicate cultures with hexylbenzene addition as 24h made it difficult to determine whether hexylbenzene addition at 24h or 48h maximised cell growth. Furthermore, the cell growth was not significantly improved when the glucose concentration was increased from 3.2% (wt/v) to 6.4% (wt/v) (7.47x108cells/ml for 6.4% glucose culture), however it was also not inhibited. The highest amount of specific PAA formed by Y. lipolytica WT was found when hexylbenzene was added at 11h (7.4x10-11mmol PAA/cell), however the highest accumulated PAA was produced in the culture that exhibited the strongest growth with hexylbenzene addition at 24h (41.4mM). This suggested that the bioconversion of hexylbenzene was maximised when it was added during the active growth phase. It is therefore recommended to conduct fed-batch experiments in future to maintain the active growth phase. Accumulated PAA was increased in 6.4% (wt/v) glucose culture (15.2mM PAA) when compared with two of the 3.2% (wt/v) glucose cultures (5.4mM and 4.3mM PAA). These results indicated that the increased glucose concentration did not inhibit the bioconversion. Furthermore, PAA was formed when 5% (wt/v) residual glucose was observed in the culture, suggesting that the bioconversion of hexylbenzene was not inhibited at glucose concentrations as high as 5.0% (wt/v). If future work were to be conducted in bioreactor culture where glucose is added in fed-batch operation, glucose concentrations in cultures of up to 5% (wt/v) could be considered for initial studies. During bioconversion by Y. lipolytica, the PAA measured after hexylbenzene exhaustion did not, however, correspond to 100% conversion. Further, poor reproducibility was found in the bioreactor cultures. The disappearance of hexylbenzene without a corresponding accumulation of PAA and poor reproducibility was investigated by determining whether PAA was further degraded or alternatively, whether other metabolic intermediates were being formed and accumulated from the hexylbenzene. However, substitution of the hexylbenzene with PAA as substrate confirmed that PAA could not be metabolised. Further, NMR analyses of both the aqueous and organic phases of the culture did not identify any additional metabolic intermediates. It is recommended that additional analyses be conducted on the aqueous and organic phases to further assess the possible accumulation of intermediates. The development of the model system in shake flask cultures demonstrated the importance of adequate oxygen supply for both cell growth and bioconversion. It was also shown that no inducer was needed because hexylbenzene acted as its own substrate inducer. Furthermore, comparison of Y. lipolytica strains TVN348, TVN493 and WT under the defined conditions of the model system revealed that the genetically modified strains (TVN348, TVN493) did not exhibit enhanced bioconversion. Bioreactor cultures using the model system under controlled conditions further showed that bioconversion was not inhibited at a 5% (wt/v) residual glucose concentration and suggested that bioconversion was maximised when hexylbenzene was added during active growth phase. This informs on future work, suggesting fed-batch operation in order to extend the active growth phase, where glucose concentrations in the bioreactor of up to 5% (wt/v) can be considered.
- ItemDevelopment and evaluation of an alkane bioconversion process using genetically modified Escherichia coli(Stellenbosch : Stellenbosch University, 2014-04) Roux, Philipp Francois; Clarke, K. G.; Callanan, L. H.; Smit, M. S.; Stellenbosch University. Faculty of Engineering. Dept. of Process Engineering.ENGLISH ABSTRACT: Alkanes can be used as an inexpensive feedstock to produce more valuable alcohols. The biotransformation of alkanes to alcohols provides an alternative to conventional chemical procedures. The scope of this research was to develop a process utilising a biocatalyst to catalyse the oxidation of an alkane to its corresponding alcohol on a larger scale than had been reported on in previous research. The research utilised a recombinant E. coli BL21(DE3) cell, containing the CYP153A6 operon in pET 28 vector, as the biocatalyst. The CYP153A6 enzyme catalyses the oxidation of octane to 1-octanol. The principle objective of the research was to determine the amount of 1-octanol that can be produced by a system utilising this strain of recombinant E. coli as a biocatalyst on a three orders of magnitude larger scale than what had previously been reported on for this reaction system. An additional objective was to model the 1-octanol production performance in the bioreactor. Bioconversion batch reactions, with excess octane used as a substrate, were conducted in 30ml McCartney bottles and in a 7.5L BioFlo 110 Modular Benchtop Fermentor (New Brunswick). The McCartney bottles were not equipped to actively control process conditions.The bioreactor was equipped to control process conditions such as temperature, pH and dissolved oxygen concentration. Experiments in the bioreactor were therefore described as being performed under controlled conditions. The procedures used to grow, maintain and harvest the biocatalyst cells were based on those developed by the Department of Microbial, Biochemical and Food Biotechnology at the University of the Free State. The product and substrate concentrations were determined through gas chromatography (GC) analysis. The McCartney bottle bioconversion reactions, with a 1.33ml reaction volume, produced 1.88 mg 1-octanol per gram of dry cell weight per hour. The bioreactor under controlled conditions, with a 2L reaction volume, produced 14.89 mg 1-octanol per gram of dry cell weight per hour. The formation of a secondary product, octanoic acid, was observed for the bioreactor under controlled conditions experiment at a production of 1.12 mg per gram of dry cell weight per hour. The McCartney bottle experiments did not produce any by-products. The 1-octanol production performance in the bioreactor experiments was empirically modelled. The empirical rate law was based on the form of the Monod equation, with the addition of a product inhibition term. The model achieved an average Root Mean Square Error of less than 5% when compared to experimental data, and was therefore concluded to be accurate within the range of experimental data and conditions tested for. The principal finding of the research is that the cells produced an order of magnitude more product in the bioreactor than in the McCartney bottles. The literature on this reaction system, however, reports only on smaller scale research than that performed in the bioreactor. The improved production results in the bioreactor therefore give the first insight into the potential that this technology has for being scaled up. Of equal significance is the finding that a secondary product developed during the biotransformations performed in the bioreactor. This refutes the assumption that the biocatalyst cells are unable to catalyse any secondary reactions. This aspect of the cells’ performance must be addressed before the biocatalyst cell strain can be considered to be a viable option for utilisation in large-scale processes.
- ItemDevelopment of a membrane immobilised amidase bioreactor system(Stellenbosch : Stellenbosch University, 2008-12) Du Preez, Ryne; Clarke, K. G.; Stellenbosch University. Faculty of Engineering. Dept. of Process Engineering.Nitriles are precursors of important amides and organic acids (e.g. acrylamide, nicotinamide, mandelic acid and acrylic acid) which are used, inter alia, as food additives, in plasticisers, detergents, make-up, medicine and as chemical intermediates in the production of various important polymers. Traditionally, chemical processes are used to convert nitriles to amides and organic acids but these processes are non-specific causing various by-products to form. Chemical processes are also environmentally unfriendly and require harsh conditions. Nitrile conversions through an enzymatic route, on the other hand, have the distinct advantages of excellent chemo-, regio- and stereo selectivities, mild process conditions and reduced downstream processing costs. The enzymatic process is mediated via an initial nitrilase catalysed conversion to amide, followed by an amidase catalysed conversion to acid. This research focused on the latter part of the enzymatic transformation of nitriles, which is the amidase catalysed biotransformation of an amide to an acid, specifically with respect to the development of a membrane immobilised amidase continuous process which has the major advantage of enzyme retention coupled with product separation. The research was conducted in three parts namely the characterisation of the free amidase, the development of the experimental bioreactor system and the quantification of the membrane immobilised amidase process.
- 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.
- ItemMeasurement and behavior of the overall volumetric oxygen transfer coefficient in aerated agitated alkane based multiphase systems(Stellenbosch : University of Stellenbosch, 2010-12) Manyuchi, Musaida Mercy; Clarke, K. G.; University of Stellenbosch. Faculty of Engineering. Dept. of Process Engineering.ENGLISH ABSTRACT: Hydrocarbons provide excellent feed stocks for bioconversion processes to produce value added products using various micro-organisms. However, hydrocarbon-based aerobic bioprocesses may exhibit transport problems where the bioconversion is limited by oxygen supply rather than reaction kinetics. Consequently, the overall volumetric oxygen transfer coefficient (KLa) becomes critical in designing, operating and scaling up of these processes. In view of KLa importance in hydrocarbon-based processes, this work evaluated KLa measurement methodologies as well as quantification of KLa behavior in aerated agitated alkane-solid-aqueous dispersions. A widely used KLa measurement methodology, the gassing out procedure (GOP) was improved. This improvement was done to account for the dissolved oxygen (DO) transfer resistances associated with probe. These resistances result in a lag in DO response during KLa measurement. The DO probe response lag time, was incorporated into the GOP resulting in the GOP (lag) methodology. The GOP (lag) compared well with the pressure step procedure (PSP), as documented in literature, which also incorporated the probe response lag time. Using the GOP (lag), KLa was quantified in alkane-solid-aqueous dispersions, using either inert compounds (corn flour and CaCO3) or inactive yeast cells as solids to represent the micro-organisms in a hydrocarbon bioprocess. Influences of agitation, alkane concentration, solids loading and solids particle sizes and their interactions on KLa behavior in these systems were quantified. In the application of an accurate KLa measurement methodology, the DO probe response lag time was investigated. Factors affecting the lag, which included process conditions such as agitation (600-1200rpm), alkane concentration (2.5-20% (v/v), alkane chain length (n-C10-13 and n-C14-20), inert solids loading (1-10g/L) and solids particle sizes (3-14μm) as well as probe characteristics such as membrane age and electrolyte age (5 day usage), were investigated. Kp, the oxygen transfer coefficient of the probe, was determined experimentally as the inverse of the time taken for the DO to reach 63.2% of saturation after a step change in DO concentration. Kp dependence on these factors was defined using 22 factorial design experiments. Kp decreased on increased membrane age with an effect double that of Kp decrease due to electrolyte age. Additionally, increased alkane concentration decreased Kp with an effect 7 times higher compared to that of Kp decrease due to increased alkane chain length. This was in accordance to Pareto charts quantification. KLa was then calculated, using the GOP (lag), according to equation [1] which incorporates the influence of Kp. Equation 1 is derived from the simultaneous solution of the models which describe the response of the system and of the probe to a step change in DO. 1 1 * L p p p K at K t L p p La C K e K ae C K K = - - - - - [1] The KLa values documented in literature from the PSP and KLa calculated by the GOP (lag) showed only a 1.6% difference. However KLa values calculated by the GOP (lag) were more accurate than KLa calculated by the GOP, with up to >40% error observed in the latter according to t-tests analyses. These results demonstrated that incorporating Kp markedly improved KLa accuracy. Consequently, the GOP (lag) was chosen as the preferred KLa measurement methodology. KLa was determined in n-C14-20-inert solid-aqueous dispersions. Experiments were conducted in a stirred tank reactor with a 5L working volume at constant aeration of 0.8vvm, 22ºC and 101.3kPa. KLa behavior across a range of agitations (600- 1200rpm), alkane concentrations (2.5-20% (v/v)), inert solids loadings (1-10g/L) and solids particle sizes (3-14μm) was defined using a 24 factorial design experiment. In these dispersions, KLa increased significantly on increased agitation with an effect 5 times higher compared to that of KLa increase due to interaction of increased alkane concentration and inert solids loading. Additionally, KLa decreased significantly on increased alkane concentration with an effect 4 times higher compared to both that of increased solids particle sizes and the interaction of increased agitation and solids particle size. In n-C14-20-yeast-aqueous dispersions, KLa was determined under narrowed process conditions better representing typical bioprocess conditions. KLa behavior across a range of agitations (600-900rpm), alkane concentrations (2.5-11.25% (v/v)) and yeast loadings (1-5.5g/L) using a 5μm-yeast cell was defined using a 23 factorial design experiment. In these dispersions, KLa increased significantly on increased agitation. Additionally, KLa decreased significantly on increased yeast loading with an effect 1.2 times higher compared to that of KLa decrease due to interaction of increased alkane concentration and yeast loading. In this study, the importance of Kp for accurate KLa measurement in alkane based systems has been quantified and an accurate and less complex methodology for its measurement applied. Further, KLa behavior in aerated alkane-solid-aqueous dispersions was quantified, demonstrating KLa enhancement on increased agitation and KLa depression on increased alkane concentration, solids loading and solids particle sizes.
- ItemOxygen transfer in hydrocarbon-aqueous dispersions and its applicability to alkane-based bioprocesses(Stellenbosch : University of Stellenbosch, 2007-12) Correia, Leslie Daniel Camara; Clarke, K. G.; University of Stellenbosch. Faculty of Engineering. Dept. of Process Engineering.Adequate provision of oxygen to aerobic bioprocesses is essential for the optimisation of process kinetics. In bioprocesses in which the feedstock is an alkane, the supply of sufficient oxygen is of particular concern because the alkane molecular structure is deficient in oxygen. As a result, the oxygen demand has to be met solely by transfer of oxygen to the culture, necessitating a proportionately higher requirement for oxygen transfer. Maximisation of the rate of oxygen transfer is therefore of key importance in optimising the potential for alkane bioconversion, with respect to both operation and scale up. Nevertheless, the oxygen transfer rate (OTR), and its dependence on the overall volumetric mass transfer coefficient (KLa) in alkane-aqueous dispersions is not yet well understood. In view of the importance of an adequate OTR in the optimisation of alkane bioconversion, this study has focused on the identification and elucidation of the factors which underpin the behaviour of KLa in an alkane-aqueous dispersion. KLa behaviour was quantified in terms of the pressures imposed by turbulence and alkane fluid properties, through their influence on the Sauter mean diameter (D32), gas hold up, gas-liquid interface rigidity and gas-liquid interfacial area per unit volume. These properties were correlated with KLa over a wide range of agitation rates and alkane concentrations in alkane-aqueous dispersions. Experiments were conducted in a 5 litre aerated and agitated bioreactor at agitation rates of 600, 800, 1000 and 1200 rpm and alkane (n-C10-C13 cut) concentrations of 0, 2.5, 5, 10, and 20% (v/v). KLa determination was executed using both the gassing out and pressure step methods. The accuracy and reliability of these methods were compared under the full range of agitation rates and alkane concentrations. The pressure step method was conclusively shown to be superior provided that probe response was taken into account, and was therefore used in the correlations. The interfacial areas corresponding to the KLa values were calculated from the combined effects of D32 and gas hold up. D32 was determined from the measurement of the dispersed air bubble diameters by means of a photographic technique and image analysis. Image analysis was performed by a program that was developed in Matlab® using image acquisition and image processing techniques. This program used these techniques to extract information of the gas bubbles in the image. The gas hold up was determined using the dispersion height technique. The behaviour of KLa was shown to be dependent on both agitation and alkane concentration. Increasing agitation from 600 to 1200 rpm increased KLa for each of the alkane concentrations. The influence of agitation on the interfacial area was evaluated over the same range of agitation rates and the relationship between the corresponding KLa values and interfacial areas assessed. Increasing agitation rate similarly enhanced the interfacial area available for transfer for each of the alkane concentrations, resulting in the concomitant increase in KLa. This increase in interfacial area was related directly to a shear-induced decease in D32 and indirectly to an increased gas holdup as a result of the lower rise velocity of the smaller bubbles. In addition to the agitation, the presence of alkane markedly influenced KLa behaviour, but in different ways, depending on the alkane concentration. Alkane concentration between 2.5 and 5% (v/v) reduced D32 at constant agitation of 800, 1000 and 1200 rpm, a likely consequence of decreased surface tension and retarded coalescence conferred by the alkane. The smaller D32 and the consequential enhanced gas hold up served to amplify KLa through increased interfacial area. However, as alkane concentration was increased above 5% (v/v), the gas hold up decreased despite a continued decrease in D32, resulting in a corresponding decrease in both the interfacial area and KLa. This suggests that at the higher alkane concentrations, the influence of viscosity predominated, exerting multiple negative influences on the interfacial area and oxygen transfer coefficient. The trends were however, not observed at the low agitation of 600 rpm, where turbulence was significantly reduced and KLa was repressed for all alkane concentrations. The pressures imposed by turbulence and alkane properties on the interfacial area defined locales of KLa behaviour and three distinct KLa behavioural trends were identified, depending on the agitation rate and alkane concentration. Regime 1 was constrained between 2.5 and 5% (v/v) for agitation rates of 800 rpm and above. Here KLa enhancement was directly associated with increased interfacial area which was the major factor defining KLa in this regime. Regime 2 was constrained by alkane concentrations higher than 5% (v/v) for agitation rates of 800 rpm and above. In this regime, the KLa depression was observed with increasing alkane concentration suggesting a predominant influence of viscosity which would be likely to exert multiple negative influences on KLa, through both the interfacial area and KL. The interfacial area in this regime decreased mainly due to the negative effect of viscosity on gas holdup. Regime 3, characterised by a decline in KLa irrespective of the alkane concentration, occurred at agitation rates smaller than 800 rpm. It is likely that at low agitation rates, the contribution of turbulence was insufficient to exert a positive influence on the interfacial area In this regime, the interfacial decreased through the combined negative effect of increased D32 and decreased gas holdup. The resultant variation in OTR depended directly on the relative magnitudes of the KLa and oxygen solubility and indirectly on the process conditions which defined these magnitudes. Under conditions of enhanced KLa, OTR benefited from the combined increases in KLa and oxygen solubility. However, under conditions of KLa depression, the elevated oxygen solubility did not invariably outweigh the influence of KLa depression on OTR. Consequently, despite the considerably increased solubility of oxygen in alkane-based bioprocesses a potential decrease in OTR through depressed KLa underlines the critical importance of the quantification of this parameter in alkane-aqueous dispersions and the necessity for a definition of the locales of optimal KLa. Through the identification of the parameters which underpin the behaviour of KLa in alkane-aqueous dispersions and the quantification of the effect of process conditions on these parameters, a fundamental understanding of the KLa and OTR in alkane-aqueous dispersions has been developed. This provides a knowledge base for the prediction of optimal KLa in these systems and has wide application across all alkane-based bioprocesses.