Masters Degrees (Microbial, Biochemical and Food Biotechnology)
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Browsing Masters Degrees (Microbial, Biochemical and Food Biotechnology) by Advisor "Du Preez, J. C."
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Item Open Access ADH2 regulation in the yeast Saccharomyces cerevisiae(University of the Free State, 2004-05) Khoboko, Mojabatho Portia; Albertyn, J.; Du Preez, J. C.English: The aim of this study was to investigate the ability of ethanol to repress the expression of ADH2 in the genome of Saccharomyces cerevisiae. To achieve this, an expression cassette (ADH2::LacZ) using LacZ as a reporter gene was constructed using the YIp356R shuttle vector. A 1000 bp up and downstream region, flanking the open reading frame of ADH2, was fused to the 5’-end and 3’-end of the LacZ gene in the YIp356R shuttle vector. Numerous attempts were made to transform the expression cassette (ADH2::LacZ) into S. cerevisiae (strain W303) containing a deleted ADH2 (adh2Δ::URA3), to displace the deletion cassette through homologous recombination, thereby placing ADH2::LacZ in the place of the ADH2 in genome of S. cerevisiae. This was unfortunately not successful and it was decided to use an alternative approach. In this case the expression cassette was cloned into the integrative vector YIplac211 and transformed into S. cerevisiae. For initial confirmation, the yeast transformants were grown on selective plates containing X-gal, which allows for the detection of β-galactosidase activity through the production of blue coloured colonies. The detection of the blue colour confirmed that the expression cassette was successfully constructed and integrated into the genome. Two randomly selected transformants were cultivated on 20 g glucose l-1 as sole carbon source, to study glucose repression and on three different ethanol concentrations to study the effect of ethanol on the expression of ADH2. Selection was maintained by growing the yeast in a URA– chemically defined media (pH 5.5) at 30ºC. Samples were taken at appropriate intervals to perform β-galactosidase assay, assess utilization of substrate (ethanol and glucose), ethanol formation and biomass determination. During growth on 20 g glucose l-1 the production of β-galactosidase was apparent only when glucose concentrations were very low (2.3 g l-1), indicating that glucose levels have to decrease to a critical level before ADH2 expression can resume. The highest final biomass was produced during growth on 20 g glucose l-1. During growth on the three different ethanol concentrations the highest β-galactosidase maximum specific activity was obtained during growth on 20 g ethanol l-1 (3 643 U mg-1) and the lowest during growth on 5 g ethanol l-1 (2 533 U mg-1). Although the maximum specific activity obtained during growth on 30 g ethanol l-1 were higher than that obtained during growth on 5 g l-1, the production rate was the lowest (93 U mg-1h-1) during growth on 30 g ethanol l-1, suggesting that 30 g ethanol l-1 concentration has negative effect on the expression of ADH2. However this slow production might have been due to the slow growth during this cultivation and not due to ethanol repression. The possible repression of ADH2 is further disputed by the high β-galactosidase production on 30 g ethanol l-1.Item Open Access Dicarboxylic acid production by Yarrowia lipolytica strains(University of the Free State, 2003-05) Mokgoro, Masego Marjorie; Smit, M. S.; Du Preez, J. C.; Setati, M. E.English:Dicarboxylic acids are value added products, which can be prepared by diterminal oxidation of n-alkanes by bacteria or yeasts with a block in β-oxidation. Yarrowia lipolytica is one of the alkane-utilizing yeasts for which genetic tools have been developed. Y lipolytica has a complex set of five acyl Coenzyme A (CoA) oxidase isozymes (encoded by POXI through POX5 genes) with different substrate specificities. Abundant information is available on dioic acid accumulation from n-alkanes by C. maltosa and C. tropicalis strains deficient in β-oxidation. In many cases β-oxidation had been blocked at the level of the acyl CoA oxidases. In comparison very little information was available .on dioic acid accumulation by Yarrowia lipolytica. We had through our collaboration with Dr J-M Nicaud of the INRA-CNRS in France access to the above mentioned series of Y lipolytica strains with the acyl CoA oxidase encoding genes disrupted. It thus became the purpose of my studies to investigate dioic acid .accumulation by Y lipolytica strains with impaired β-oxidation. In order to study the growth of different Yarrowia lipolytica strains on or in the presence of a range of liquid and solid n-alkanes or alkane derivatives we required a rapid method using small samples to estimate biomass production. Insoluble hydrophobic substrates iriterfere with turbidimetric measurements. This problem is more severe if the hydrophobic substrate is a solid. It is not possible to efficiently separate Y lipolytica cells from a hydrophobic substrate by centrifugation in the presence of an organic solvent, because Y lipolytica cells are hydrophic and a large percentage of cells cling to the water/solvent interface. We established that pre-treatment of samples for turbidimetric analysis with 5 M NaOH, thus increasing the pH of samples to 14, abolished to a large extent the hydrophobicity of Y lipolytica cells. We also established that washing of the cells with cyclohexane and NaOH when cells were harvested by filtration, did not result in any significant loss in biomass. Based on these observations we developed a simple, cost effective sample preparation procedure for turbidimetric analysis, which gave accurate, repeatable turbidity measurements with no Interference from the hydrophobic substrates. This method involved the pre-treatment of small samples (500 µl)with 5 M NaOH plus cyclohexane, prior to harvesting the biomass by centrifugation. Sample preparation was carried out in microcentrifuge tubes and turbidimetric measurements were done with a microtitre plate reader. The newly developed procedure was used to investigate the toxicity of dodecane and hexadecane as well as their terminal and diterminal oxidation products to Y lipolytica wild type strain H222. The alkanes and dicarboxylic acids were never toxic to Y lipolytica. Dodecanol severely inhibited growth of Y lipolytica strain H222 in YP media with glucose whereas in a semi-synthetic YNB medium without additional carbon source dodecanal and dodecanoic acid were the most toxic. Hexadecanol and hexadecanoic acid did not inhibit growth of Y lipolytica in yP medium with glucose, but were toxic to Y. lipolytica in a semi-synthetic YNB medium without glucose. The results obtained in the first round of experiments indicated to us the possibility of preparing dodecanol-tolerant strains. Two dodecanol-tolerant strains were subsequently prepared. The first H222A was prepared by step-wise increasing dodecanol concentrations in YP broth supplemented with glucose to 7.5% (v/v). The second strain MTL Y35A was prepared on YP agar plates without glucose by step-wise increasing dodecanol concentrations to 8.5% (v/v). Dodecanedioic acid was not accumulated by the dodecanol-tolerant strains H222A or by the triple POX-deleted strain MTL Y35A, when grown on glucose in the presence of dodecanol. Two dodecanol concentration were tested 3 % and 10 % (v/v). Dioic acid accumulation from C 12 and C 16 alkanes and alkane degradation intermediates was investigated using Y lipolytica wild type strain W29 as well as the POX deleted strains MTLY21 (∆POX2, POX3), MTLY35 (∆POX2, POX3, POX5) and MTLY37 (∆POX2, POX3, POX4, POX5). The quadruple-deleted strain MTLY37 was the only strain that was able to accumulate dioic acids from alkanes, alkanols and monocarboxylic acids. Dodecane was the best alkane substrate for dioic acid accumulation yielding 7 mg/ml dodecanedioic acid after 144h (23% w/v conversion). Lauric acid did not yield any dioic acid (probably due to toxicity), ·but 5 mg/ml hexadecanedioic acid was accumulated from hexadecanoic acid after 48h. All the strains accumulated dodecanedioic acid from the diterminal functionalised 1,12 dodecane diol and eo-hydroxy dodecanoic acid. The quadruple-deleted strain MTL Y37 accumulated a maximum concentration of 20 mg/ml dodecanedioic acid after 48h from 1,12 dodecanediol, while the triple-deleted strain MTL Y35 accumulated 18 mg/ml dodecanedioic acid after 48h from 12-hydroxydodecanoic acid.Item Open Access Effect of cultivation conditions on the dimorphism of and heterologous protein production by Arxula adeninivorans(University of the Free State, 2007-11) Jansen, Arina Corli; Du Preez, J. C.; Albertyn, J.; Killian, S. G.Arxula adeninivorans strain LS3, a dimorphic yeast with potential for biotechnological applications, has in recent years has been studied for the production of heterologous proteins. The growth kinetics of this species and the effect of environmental conditions on its morphology under controlled cultivation conditions have not been well documented. Because genetic transformation might affect the yeast physiology, a comparative investigation of the growth characteristics of strains LS3, G1211 (LEU2+) (the auxotroph derived from strain LS3) and LS3/pXynA, derived from strain LS3 by transformation with the Thermomyces lanuginosus xylanase gene under the control of the TEF1 promoter, was conducted, focusing on the effect of temperature and dissolved oxygen tension (DOT) on the morphology and growth parameters of the strains. This xylanase gene (XynA) was integrated in the 25S rDNA locus and expressed under control of the strong constitutive Arxula-derived TEF1 promoter. The plasmid copy number integrated into the rDNA locus was unknown. Little to no activity was found with the A. adeninivorans LS3 transformants, namely only 5.86 nkat ml-1-1 (0.35 U ml) compared to the 4 418 nkat ml-1-1 (265 U ml) obtained with the T. lanuginosus strain SSBP positive control. The protein itself might have been defective and since it accumulated intracellularly, this could also have resulted in the diminished activity observed. Cultivation in a temperature gradient incubator over a wide temperature range revealed significant differences in the maximum specific growth rates of the strains LS3 and G1211(LEU2+), namely 0.48 and 0.52 h-1, respectively. The minimum and maximum temperatures were 18 and 46°C, respectively, with the optimum temperature in the range of 37 to 39°C. In accordance with the literature, in shake flask cultures the morphology of LS3 changed with an increase in temperature from predominantly budding cells at 30°C to pseudohyphae at 42°C and resembled a mycelial culture at 45°C. By contrast, the morphology of strain G1211 (LEU2+) remained pseudohyphal at 45°C. During batch cultivations no true hyphae were observed, but a change in morphology, from budding cells to pseudohyphae, was observed during cultivation at different dissolved oxygen concentrations at different temperatures for LS3 and LS3/pXynA. G1211 (LEU2+) remained a pseudohyphal culture. The critical dissolved oxygen concentration (Ccrit) value, determined from the intersection of tangent lines of a plot of specific rate of oxygen uptake as function of dissolved oxygen concentration, of strains LS3 and G1211 (LEU2+) was 0.016 mmol l-1-1 (9% of saturation) and 0.013 mmol l (7% of saturation), respectively. This revealed that A. adeninivorans strain G1211 (LEU2+) had a slight but significantly lower Ccrit than strain LS3. This study showed that insertion of plasmid pAL-ALEU2m has a significant effect on the specific growth rate and on the morphology of Arxula adeninivorans LS3. Insertion of plasmid pXynA had only a slight effect on the morphology but no effect on the specific growth rate.Item Open Access Ethanol production by yeast fermentation of an Opuntia ficus-indica biomass hydrolysate(University of the Free State, 2012-02) Kuloyo, Olukayode Olakunle; Du Preez, J. C.; Kilian, S. G.Opuntia ficus-indica, the prickly pear cactus, is well adapted for cultivation in arid and semi-arid regions, with a yield of 10 to 40 tonnes (dry wt) cladode biomass per ha. The cladodes (the “leaves”, which in fact are the stems) might serve as lignocellulosic biomass feedstock for second generation bioethanol production, without competing for agricultural land or replacing significant natural vegetation. The main objective of this study was to investigate the feasibility of bioethanol production from an enzymatic hydrolysate of O. ficus-indica cladodes. The potential of a Kluyveromyces marxianus isolate UOFS Y-2791, a yeast capable of utilising a wider range of carbon substrates and of ethanol production at higher temperatures than Saccharomyces cerevisiae, was investigated for bioethanol production using an O. ficus-indica cladode enzymatic hydrolysate as feedstock. S. cerevisiae UOFS Y-0528, a wine yeast strain, was used as benchmark. Compositional analysis of the cladode biomass indicated that it had a low lignin content of 8% (dry wt). The content of readily fermentable carbohydrates in the cladode, which was 34.3 g per 100 g dry biomass of which 23 g was glucose, was comparable to other conventional biomass feedstocks such as sugar cane bagasse and corn stover, whereas it had a low xylose content. By applying a statistical design experimental approach where acid concentration and contact time were varied, optimum conditions for dilute acid pretreatment of the dried and milled cladode were determined to be 1.5% (w/w) sulphuric acid for 50 min at a temperature of 120oC and a dry biomass loading of 30% (w/v). Enzymatic hydrolysis experiments were performed with varied enzyme loadings of cellulase and β-glucosidase with or without the addition of pectinase, and the enzyme loadings chosen were 15 FPU cellulase, 15 IU β-glucosidase and 100 IU pectinase per gram of dry biomass. These parameters yielded an O. ficus-indica hydrolysate containing (per litre) 45.5 g glucose, 6.3 g xylose, 9.1 g galactose, 10.8 g arabinose and 9.6 g fructose. Using a chemically-defined medium with a sugar composition similar to the hydrolysate as benchmark, K. marxianus and S. cerevisiae were grown in the O. ficus-indica hydrolysate at 40oC and 35oC, respectively, under non-aerated conditions, whereas the performance of K. marxianus was also investigated under oxygen-limited conditions where the DOT was controlled at less than 1% saturation. The fermentation profiles of both yeasts were compared using separate hydrolysis and fermentation (SHF) and simultaneous hydrolysis and fermentation (SSF) process configurations, at a water-insoluble solids (WIS) content of 14%. Both yeasts achieved comparable ethanol yields in SHF and SSF under nonaerated conditions, although K. marxianus exhibited a lower volumetric ethanol productivity than S. cerevisiae. K. marxianus, cultivated under oxygen-limited conditions, achieved a lower ethanol yield than both yeasts cultivated without aeration. However, K. marxianus exhibited the highest volumetric ethanol productivity of 2.3 g l-1 h-1 and 1.57 g l-1 h-1 in SHF and SSF, respectively, although the ethanol produced was assimilated upon hexose depletion. K. marxianus utilised galactose poorly in the absence of aeration, but completely consumed the sugar under oxygen-limited conditions. The overall ethanol productivity of SSF was double that of SHF. An ethanol concentration of 20.6 g l-1; the highest concentration achieved in this study, was an improvement on the 14 g l-1 previously reported elsewhere. This study provided more information on the chemical composition of the O. ficus-indica cladode, particularly regarding its constituent carbohydrates, and also highlighted the feasibility of ethanol production from the cladodes, albeit at low concentrations from an industrial point of view. K. marxianus demonstrated its potential as an alternative to S. cerevisiae for bioethanol production from lignocellulosic biomass.Item Open Access Evaluation of cell-wall modifying enzymes for improved refining of pulp from two Eucalyptus species(University of the Free State, 2010-11) Steel, Crystal Leone; Du Preez, J. C.; Wolfaardt, J. F.English: Refining of pulps requires a large amount of energy, capital input and maintenance costs. Wood fibres can be treated enzymatically prior to refining to reduce the refining energy requirement of the pulps and/or to improve drainage and strength properties. Cell-wall modifying enzymes such as the cellulases and hemicellulases can assist refining by opening up the crystalline and amorphous cellulose and hemicellulose in the fibre walls. This application requires cell-wall modifying enzymes to be matched to the specific pulp. Characterisation of commercially-available cellulases showed that the activities of the enzymes were suitable for paper mills. The protein content of the enzyme formulations were determined to establish levels for comparative dosing. Using this information the effects of over-dosing and the extended incubation with the enzymes were also determined. The pulp yield and fibre strength appeared to be at risk of being decreased with elevated enzyme dosages, since Ecopulp® Energy, Novozyme 613 and Celluclast 1.5L increased the release of reducing sugars. The critical period for fibre modification by enzyme treatment was the first 30 min of incubation. The most suitable enzyme for application to a paper mill appeared to be Novozyme 476 due to its ability to improve bonding strength with minimal loss in fibre strength. According to published reports, dried and never-dried Eucalyptus globulus pulps responded differently to enzyme treatments and it was, therefore, likely that dried and never-dried E. grandis pulps would also respond differently. Ecopulp® Energy, Novozyme 476 and Celluclast 1.5L were applied to E. grandis pulps and refined on pilot scale. Overall, the dried E. grandis fibres responded differently to the enzyme treatments than never-dried fibres and enzymes should, therefore, be matched to the specific fibre used. It was proved that the enzymes can be used to save on refining energy usage as well as to improve strength properties of paper made from dried or never-dried E. grandis pulps. Ecopulp® Energy and Novozyme 476 were subsequently applied to the E. nitens pulps and refined on pilot scale. All of the enzyme applications had a similar effect on the never-dried pulp, where the properties were not changed as much as on the dried pulp. Novozyme 476 was the most suitable enzyme for application on the dried E. nitens pulps, because it saved on refining and drying energy due to the decreased freeness and water retention values. The Novozyme 476 also improved the physical properties of the pulps where tear and tensile indices and porosity were increased and bulk was reduced. This work demonstrated that enzymes must be selected to match the pulps being used and to suit product requirements. Novozyme 476 was selected for application on a fine-paper machine in order to improve the refining efficiency of E. nitens pulp. At the high dosage (100 ml/t) the refining current of the mixed stock refiner was reduced and the porosity and tear strength was decreased and tensile strength was increased. The reduction in tear strength was probably due to too much refining energy applied to enzyme treated pulp, thereby damaging the fibres. The damage to the fibres was confirmed by electron microscopy, showing excessive fibrillation of the fibres treated with the endoglucanase. The endoglucanase successfully improved the refining efficiency of the paper machine and should be considered for application on a permanent basis.Item Open Access Geobacillus thermoleovorans: growth and lipase production(University of the Free State, 2004-01) Knoesen, Shaun; Litthauer, D.; Van Heerden, E.; Du Preez, J. C.English: The aim of this study was to elucidate factors, specifically media composition and growth conditions that would improve the thermophilic lipase production by Geobacillus thermoleovorans. A semi-starved state where carbon and or nitrogen becomes limiting was found not to be conducive to optimum lipase production. Instead, the notion that lipase activity peaks at these limiting conditions, normally during stationary phase was disproved as we could get a 25% increase in biomass obtained under optimized conditions, yielding a 100% increase in lipase production. This showed that the availability of specific nutrients has a direct positive effect on the amount of enzyme produced. The occurrence of free fatty acids (products of lipolysis) under these conditions was found to be the main signal for the commencement of lipase production. This was shown by the almost immediate production of lipase once free fatty acids were present at the initial point of inoculation (stearic acid induction), showing that the presence of free fatty acids does not repress the production of inducible lipases by Geobacillus thermoleovorans but that it rather acts as a signal for lipase induction. The presence of glucose as a second utilizable carbon source at inoculation was not inhibitory to lipase production. The consumption of glucose and stearic acid occurred simultaneously, with the consumption of free fatty acids being enhanced by the presence of glucose. The pH of the media also had a noticeable effect on the physiology surrounding lipase production, specifically effecting the rate of uptake of free fatty acids. This indicated that not only the presence but also the rate of uptake of free fatty acids probably plays a direct role in the regulation and/or secretion of lipases. The optimum temperature for lipase production and the optimum for growth differed. The optimum temperature for lipase production was found to be 55ºC with little or no lipase production occurring at 65ºC, which is the optimum growth temperature. The addition of NaCl increased lipase yield and/or activity further. It is however not clear whether the presence of NaCl has an effect on lipase production or merely acts as a stabilizer of lipase activity. We propose that the presence of free fatty acids in the culture media is the primary signal for lipase production, possibly by inducing the transcription of an inducible lipase gene.Item Open Access The growth kinetic characterisation of Saccharomyces cerevisiae strains transformed with amylase genes(University of the Free State, 2002-03) Knox, Alison Margaret; Du Preez, J. C.; Kilian, S. G.English: The direct fermentation of starch to ethanol using an amylase-producing yeast is of interest as an alternative to the conventional fermentation processes, which utilise commercial amylases. Starch is an abundant renewable biopolymer, comprising two major components, namely amylose (α-I,4-linked D-glucose residues) and amylopectin (α-I,4 and α-I,6-linked D-glucose residues), typically constituting 80 % of cereal starches. Efficient starch degradation, therefore, necessitates the use of α- and glucoamylases, together with an α-I,6 debranching activity. Naturally amylolytic yeasts are not suited to fermentations, whereas Saccharomyces cerevisiae, known for its strong fermentation capacity, lacks amylolytic activity (with the exception of S. cerevisiae var. diastaticus, which has weak glucoamylase activity). Consequently, the diploid "Sigma" strain of S. cerevisiae was transformed with different combinations of amylase genes from Lipomyces spencermartinsiae (with the PGK] promoter), and Saccharomycopsis fibuligera (with its natural promoters) using an integrating plasmid in a single copy form. These recombinant strains, provided by the University of Stellenbosch, were evaluated in respect of their ability to ferment starch to ethanol. Recombinant strains of S. cerevisiae containing the S. fibuligera amylase genes with non-integrating plasmids in a multi-copy and a single copy form, provided by the University of the Free State, were also evaluated. Notable differences in the hydrolysis zones on starch agar plates indicated that the type of starch medium used and the amylase produced exerted a significant effect. The dimensions of these hydrolysis zones were a poor indicator of the performance of the strains in liquid starch media. Anoxic cultivations in shake flasks and in 2-1 bioreactors containing a 2 % starch medium yielded less than 109 ethanol.l-1 over a 200 h incubation period. Aerobic growth yielded more biomass and, therefore, higher amylase values and higher rates of starch hydrolysis, but with no detectable amounts of ethanol. Initial evaluations indicated poor amylase activity, particularly with strains containing the S. fibuligera amylase genes. The limited amylase production by a strain containing the S. fibuligera amylase genes was not due to proteolytic activity or intracellular enzyme . accumulation. Strains containing the L. spencermartinsiae amylase genes gave the best overall results. However, a strain containing a combination of both a-amylase and . glucoamylase yielded disappointing results. The curious multi-phasic growth profile obtained with this strain, accompanied by a delay in amylase production, suggested regulation of the PGKl promoter by glucose. However, Northern blot analyses indicated very low levels of glucoamylase mRNA, with no clear indication of induction of the PGKl promoter. A recombinant strain (strain steIl7) with both glucoamylase genes (LKAll from L. spencermartinsiae and GLUI from S. fibuligera) proved to be the most prormsmg. Further evaluation in 15-1 bioreactors resulted in the production of ea. 21 g ethanol.l-1 from 55 g starch.l-1 by strain stell7. Despite a relatively high α.-amylase activity of 500 U.l-1 after 150 h, the slow rate of enzyme production remained the rate-limiting step. Although some of these recombinant strains were capable of complete starch hydrolysis, the slow rate of starch saccharification and the concomitant low ethanol productivity rendered these strains unattractive for commercial application.Item Open Access Production of laccase by the white-rot fungus Pycnoporus sanguineus(University of the Free State, 2002-05) Van der Merwe, Johannes Jacobus; Wolfaardt, J. F.; Du Preez, J. C.English: White-rot fungi and their enzymes are receiving increasing attention for biotechnological applications in the pulp and paper industry as alternatives to conventional bleaching. Laccase has been identified as one of the enzymes that plays a major role in lignin degradation. Laccase only attacks phenolic subunits of lignin, but its substrate range can be extended to non-phenolic subunits by the inclusion of a mediator. The use of this enzyme was, therefore, not successful in pulp bleaching trials until the discovery of mediators. Although the existence of natural mediators has not been confirmed, various components have been identified that are able to act as mediators. Improved methods of laccase production could benefit the industrial utilisation of the enzyme. White-rot fungi constitutively produce low concentrations of laccase, but higher concentrations can be obtained with the inclusion of inducers in the cultivation media. The enzyme is mainly produced during the stationary growth phase of the fungi, but various factors such as glucose, nitrogen and pH can influence levels of laccase production. The enzyme does not only hold potential for biological pulp bleaching operations, but also has application in bioremediation, the textile dye industry as well as the food and beverage industries.Item Open Access The production of potentially prebiotic oligosaccharides by Leucosporidium scottii Y - 1450(University of the Free State, 2016-11) Lum Nde, Adeline; Kilian, S. G.; Du Preez, J. C.English: Prebiotic oligosaccharides are gaining interest in the food, poultry and pharmaceutical industries due to their health benefits. These include prevention of specific allergies, improved calcium absorption, reduction in the duration, incidence, and symptoms of traveller’s diarrhoea, alleviation of irritable bowel syndrome (IBS) symptoms, increased satiety and reduced appetite. In the food industry they are responsible for modifying food flavour, they are considered as low energy ingredients alleviating obesity and have a high water-holding capacity, low sweetness and an anti-staling effect. The main objectives of this study were to produce, purify and identify oligosaccharides from Leucosporidium scottii Y-1450. Leucosporidium scottii Y-1450 produced neofructooligosaccharides during growth on sucrose. Two trisaccharides and a tetrasaccharide were produced. The highest oligosaccharide concentration obtained was 33.7 g l-¹ after 22 h of cultivation. A maximum specific growth rate, maximum volumetric rate of oligosaccharide production, and maximum yield coefficient for the production of oligosaccharides (oligosaccharides produced/sucrose assimilated) of 0.28 h-¹, 1.53 g (l h)-¹ and 0.58 were obtained respectively. In addition to the products of interest, glucose, fructose and sucrose were also present in the supernatant. Purification of the supernatant containing mono-, di and oligosaccharides was done by preparative HPLC. A total oligosaccharide concentration of 2.9 g l-¹ was obtained after purification from 7.8 ml of supernatant. The first unknown trisaccharide was not successfully purified as it overlapped with the second trisaccharide. The second trisaccharide and the tetrasaccharide were successfully purified with percentage recoveries of 0.12 % and 15.3 % respectively. LCMS identification using Electrospray Mass Spectrometry (ESMS) of the supernatant showed the presence of three oligosaccharides with m/z of 503.4, 539.3 and 665.5 Da, respectively. This was later followed by structural identification of the tetrasaccharide by NMR. Results from MALDI-TOF and NMR analysis confirmed the presence of two oligosaccharides present in the tetrasaccharide fraction, a trisaccharide (from the breakdown of the tetrasaccharide) identified as 1-kestose (α-Glcp-1-2-β-Fruf-1-2-β-Fruf) and a tetrasaccharide identified as neonystose (β-Fruf-2-6-α-Glcp-1-2-β-Fruf-1-2-β-Fruf). A two level fractional factorial screening design was used to investigate oligosaccharide production, with sucrose concentration, cell concentration, pH and temperature as the factors and maximum oligosaccharide concentration, yield coefficient and productivity as the responses. The highest concentration, yield coefficient and productivity of oligosaccharides were obtained at 200 g l-¹ sucrose, 8 g l-¹ cell concentration, pH 7 and 20 °C. ANOVA analysis indicated sucrose concentration as a highly significant main effect for all three responses while the interaction sucrose concentration*cell concentration was identified as a significant interaction for the responses. Temperature was an insignificant factor for all three responses. This study highlighted the feasibility of the production of neoFOS with possible prebiotic potential from sucrose by Leucosporidium scottii.