Browsing by Author "Tai, Siew"

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    Open Access
    Design of the aerobic hail reactor - towards improved energy efficiency
    (2022) Shaer, Gianluca Sasha Salvatore Ganter; Harrison, Susan; Tai, Siew; Fagan-Endres, Marijke
    This dissertation presents the results of an investigation into the design of a novel low aspect ratio reactor, dubbed the HAIL (horizontal air-injected loop) reactor. Current industrial high cell density aerobic reactors for cultivation of bacteria and yeast are typically either stirred tank reactors (STR's), bubble column reactors (BCR's) or airlift reactors (ALR's). These systems can attain high mass transfer rates and short mixing times; however, their energy efficiency remains a concern. Many studies have attempted to further optimise these reactors, but they are ultimately limited by their high aspect ratios. These lead to large pressure heads that the air compressor needs to overcome on sparging, contributing significantly to energy costs. Low aspect ratio (LAR) reactors, such as the wave bag, orbital shaker and raceway reactors offer an alternative to these systems, providing superior energy efficiency for both mixing and aeration. However, each has core issues preventing their usage in high cell density aerobic culture. Their maximum mass transfer coefficient is typically too low to support high cell density cultures. Additionally, these reactors tend to have poor scalability, making them unfeasible for large scale industrial usage. To overcome these challenges, the HAIL reactor makes use of a tubular loop design. The anticipated benefit of the loop design was that it forces the air to travel the length of the reactor before leaving the system, enabling significant surface aeration and residence time in the reactor. These both impact the mass transfer coefficient. Additionally, the loops can be stacked upon one another, overcoming the scalability issue. The reactor would also be energy efficient based on its LAR. To establish target performance ranges, a literature review on the gas-liquid mass transfer coefficient, mixing time and efficiency of current low and high aspect ratio (HAR) reactors was conducted. This was supplemented with experimental results (including mass transfer coefficients, cell density and viscosity) from the fed-batch STR cultivation of Saccharomyces cerevisiae, an easy to work with highly aerobic yeast. A fed-batch feeding profile was developed for this. To better compare reactor performance, a term was introduced called the mass transfer energy efficiency, with units m3 ∙h -1 ∙W-1 , obtained via the quotient of the kLa and the power input per unit volume. The literature mass transfer energy efficiency ranges for the STR, BCR and ALR were found to be 0.022-0.236 m3 ∙h -1 ∙W-1 , 0.084-0.317 m3 ∙h -1 ∙W-1 and 0.142-0.493 m3 ∙h -1 ∙W-1 respectively, with maximum kLa values ranging up to 1000 h-1 depending on the power input. Mixing times for these systems differ depending on scale and configuration, ranging from below a minute up to 20 minutes. Experimental fed-batch and sterile water systems had efficiency ranges of 0.044-0.245 m3 ∙h -1 ∙W-1 and 0.059-0.285 m3 ∙h -1 ∙W-1 respectively, with a maximum kLa of 240 h-1 and 226 h-1 . Based on cellular growth results, the theoretical minimum kLa required was calculated as 372 h-1 . The most notable literature efficiencies for LAR reactors were held by the travelling loop, raceway, and wave reactors with ranges of 0.286- 0.295 m3 ∙h -1 ∙W-1 , 0.034-0.867 m3 ∙h -1 ∙W-1 , and 0.112-0.742 m3 ∙h -1 ∙W-1 . For the wave and travelling loop reactors, mixing times below a minute were attainable. A 6.2 L proof-of-concept and 31.4 L laboratory-scale prototype of the HAIL reactor were developed. In the proof-of-concept prototype, preliminary studies were carried out on the impact of sparger depth and angle on circulation time. Using the laboratory-scale system a range of sparger designs, including different angled jets, outlet areas and a circular sparger design, were investigated. The circular sparger design was found to be the ideal sparger type. A mixing time of 7-19 minutes depending on the power input was found for the 31.4 L configuration. The power efficiency range determined was 0.120- 0.281 m3 ∙h -1 ∙W-1 ; however, the calculation used to determine this is an underapproximation. The maximum kLa of 13.84 h-1 is an order of magnitude (between 10 and 100) lower than the values that can be obtained in HAR reactors for industrial aerobic culture. It was found that HAIL reactor performance did not change substantially with an increase in viscosity from 1 to 1.4 cP. The HAIL reactor did not compete with existing low and high aspect ratio reactors in its current configuration in terms of mass transfer. Additional research on the design is recommended to enhance gas - liquid contacting and associated mass transfer. These ongoing studies will enable the potential relevance and application of the novel reactor to be determined.
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    Open Access
    Development of a novel bioreactor and systems for suspension cell culture in biopharmaceutical production
    (2021) Sharma, Rajesh; Tai, Siew; Harrison, Susan
    Mammalian cells offer superior cellular machinery for the production of complex biological products. These cells provide proper post-translational processing machinery for recombinant protein expression to acquire the desired folding for optimal activity. With this advantage, mammalian cells have become the preferred choice for the production of biological products. These cells may grow either attached to a solid surface (adherent cells) or, where adapted, as suspension cultures. In order to grow these cells efficiently in suspension, a bioreactor is therefore required. Bioreactors play a key role in the production of biologicals. Due to the continuous advancement of medicine and the healthcare industry, the demand for biological drugs has increased in the last three decades. This has placed a significant pressure on the biopharmaceutical industry to meet this increasing demand and has become a key driving force behind the need to develop better, safer and more economical bioreactor designs and culture processes. Continuous stirred tank bioreactor is the norm for production of many bioproducts. However, these bioreactors exert high shear forces to cells due to the impeller speed, bubble disruption, and foam formation. In addition, at a large scale, improper mass transfer impairs the performance of cell lines and achieving high cell densities and prolonged viability with correct glycosylation of a secreted proteins is still a challenge during scale-up. Many cell lines, for example Vero cells, which are widely used to produce human vaccines are difficult to adapt into suspension culture. Fixed-bed bioreactors and the use of microcarriers provide an alternative platform for their growth to produce biologicals. However, a high surface area is required to achieve the high cell density which leading to an elevated cost of production (mainly from microcarriers) and ensuing a costly and technically challenging scaling-up of these systems. Other designs such as single-use bioreactors and novel bioreactors based on different operating principles have been explored, but their utilisation is limited from laboratory to pilot scale. Hence, a comprehensive bioreactor design which would be suitable for a large variety of cell lines to produce high-yielding products in suspension culture with the lowest cost and risk in the shortest span of time is still sought. In the current research, two approaches were investigated to address these challenges. Firstly, a horizontal tubular bioreactor (HTB) with a spiral impeller was designed and fabricated for the propagation of suspended mammalian cells with a focus to achieve middle to high cell density by improving mass transfer whilst reducing hydrodynamic shear and energy requirements through surface aeration. The second approach is to test the adaptation of adherent Vero cells into single-cell suspension culture in serum-free media by treating them with an anti-cancer drug, Puromycin amino nucleoside (PAN). The absence of a supporting surface for cell growth (e.g. microcarriers) and serum-free conditions are expected to reduce the cost of manufacturing and to achieve higher productivity of biological production per unit volume of bioreactor. In the first approach, the horizontal tubular vessel was designed to achieve the final volume of approximately 5.0 L. Design of the impeller is a key component that dictates the mixing patterns and mass transfer efficiency. Different geometric configurations were used to design the spiral impeller by considering various parameters such as impeller diameter, the pitch of the blade, pitch angle, height of the blade, the thickness of the blade, clearance efficiency and the position of the heating element. Another important aspect of the prototype design was incorporating an external magnetically-coupled motor drive which assisted in not only in aseptic handling but also reduction in mechanical stress and generation of fewer particles for cleanroom operations. The side plate was designed with the appropriate number of addition ports to allow execution of batches with minimum cross-contamination and for the ease of operation. Thereafter, the engineering characterisation of the HTB was carried out. The performance of the HTB was evaluated for (i) oxygen mass transfer (kLa) through the dynamic gassing-in method, (ii) mixing time and fluid flow by tracer and phenolphthalein method, (iii) minimum stirring speed (Njs) through alginate beads mimicking cell loading and modelling through modifying Zwietering equation, (iv) power consumption through heat calorimetry (temperature method) and (v) shear stress by determining specific death constant (kd) at different impeller speeds. The general characterisation profile of HTB has shown that at high agitation speed, homogeneity and mass transfer efficiency improved while power consumption increases with an increase in agitation speed. The bioreactor operated well at 2 L and 3 L capacity when the impeller is 40 - 90 % immersed in the liquid. The maximum mass transfer coefficient (kLa) of 16 h-1 was measured with a 3 L volume with an impeller speed of 500 rpm. These results are comparable with the other culture systems of the same scale. The HTB was also tested for suitability to grow mammalian cells. Three batches were carried out, of which one was with the Chinese hamster ovary (CHO) cells expressing the somatic angiotensinconverting enzyme (sACE) and the two with plain CHO cells without expressing any recombinant protein. The maximum cell density achieved was of 5.48 x 106 cells mL-1 with plain CHO cells and 4.14 x 106 cells mL-1 with CHO cells expressing sACE with a maximum protein productivity of 465 mg mL-1 . The specific death rate constant of 0.025 (h-1 ) was obtained when impeller speed was increase from 150 rpm (normal) to 300 rpm (induced shear) for 72 h. In this study, CHO cells have been successfully adapted to suspension in serum-free conditions using the slow weaning of serum method and propagated in the HTB whereas Vero cells have been adapted successfully to serum-free media in adherent conditions. Attempt to suspend Vero cells based on literature using the weaning method remains timeous. Therefore, an alternative approach was explored using an anti-cancer drug (PAN) which is known to suppress the expression of integrin (cell adhesion receptors). The expectations from this approach were that the suppression of integrin would allow cells to detach and grow as a suspended culture (Krishnamurti et al., 2001). The results indicated that the anti-cancerous drug may have modulated the structure and function of the integrin which resulted in dislodging of the cells from the surface and form clumps which were viable for a week in suspension culture without increase in cell density. The viability of the cell clumps and few suspended cells were tested by re-seeding of these cells back to tissue culture (TC) flasks in serum-containing media without the presence of PAN. The culture in the TC flask regained confluency in the 2-3 day which confirms the viability of the cells and the likeliness of integrin re-modulating itself in the absence of PAN. As the suspended Vero cells did not grow, they were not tested for growth in the HTB. To investigate the biological activity of these Vero cells, Isothermal microcalorimetry was used to evaluate the heat generation profile of the Vero cells quantitatively before and after drug treatment. The heat flow data (metabolic heat) from the treated and normal cells showed a distinct decrease in the heat generation profile which indicated that the treated cells were viable but not as active as the normal (non-treated) cells. It was evident from the heat flow data obtained for the PAN-treated Vero cells (-0.13 µW) from that of non-treated cells (13.12 µW) and thereafter when PAN-treated Vero cells regrown in serum-containing media, they regain their metabolic activities which were indicated by their heat flow values as positive control (9.30 µW), 100 µg mL-1 (10.12 µW), 200 µg mL-1 (10.18 µW), and 250 µg mL1 (9.15 µW). It is recommended that dielectric spectroscopy and total DNA in the culture from the lysed cells could also be used to measure the bioactivity of the pre and post treated cells and data can be compared with IMC for more insight into the behaviour of the cells It has been concluded that the horizontal tubular bioreactor (HTB) can sustain the middle to high cell density by imparting desired mixing and mass and heat transfer requirements whilst exerting minimum hydrodynamic shear. For the improvement of the design, it is recommended that more batches at different agitation speeds in combination with different airflow rate would further unravel the suitability of HTB to grow mammalian cells and stringently decode the optimum process conditions to achieve high cell densities with extended longevity. Additionally, changes in the pitch of the impeller blades could result in the improved fluid flow profile, mixing and mass transfer while drawing low power input. Subsequently, different modes of operation, e.g. fed-batch or continuous operation are suggested to investigate the suitability of the HTB for integrity, sterility, and possible higher productivities of products. In suspending Vero cells, it has been concluded that the presence of serum-containing media reversibly stimulates the re-modulation of the integrin which poses hurdles in suspending Vero cells by reattaching the cells to the TC flasks. Therefore, it is recommended that a thorough investigation of the drug-treated cell integrin profile is examined through fluorescence-activated cell sorting (FACS) which would give details of the inhibition of the different integrin subunits. This information could form the basis of adapting cell-lines into suspension in a single step, which is otherwise difficult to adapt.
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    Open Access
    Feasibility for value addition to sucrose in South Africa through conversion to platform chemicals
    (2018) Jegede, Kemi; Harrison, Susan T L; Tai, Siew
    The world sugar price is constantly changing in response to supply and demand and is currently very low as compared to the prices it is sold at domestically in South Africa. The drop in the worldwide price of sugar is due to its oversupply as yields of sugar production have increased in recent years and subsidies and protection measures in other producing countries. The low prices also mean imports are cheaper than local sugar. This pushes down the average sugar price and leads to a low profit margin. Further, sugar production in South Africa is facing a number of challenges. The industrialization of the sugar belt in KwaZulu-Natal has resulted in less plantations and challenging topography for these. Incentivisation of small, medium and micro-scale commercial operations has increased the number of smaller scaled operations, with less economy of scale and less capital backing. Climatic factors have impacted crop yields. Production costs have increased in accordance with South Africa’s consumer price index whereas selling price has moved with the less inflationary global platform. Together, these have made the industry less economically viable. This has led to a need for value addition to sucrose and to eliminate the dependency on a single commodity. Re-positioning of sugar into value-added products has potential to boost the country’s economy by introducing other sources of revenue. Moreover there is a worldwide need to find alternative means to produce petroleum-based fuels and chemicals and bio-based products are being targeted to meet some of this need. A review of the global status shows that there has been value addition in the sugar industry producing mostly ethanol and other commodity chemicals such as surfactants, organic acids and polyols. It is therefore imperative to find sustainable ways of generating value added platform chemicals from sucrose. The quantitative and qualitative study of this project looks at determining the chemicals that should be considered as having the highest potential for value addition from sucrose in a South African context. The project was scoped to focus on chemicals and fuels that can be produced by biological conversions of sucrose. For the quantitative study, a set of 39 chemicals was selected from major studies performed globally on potential bio-based platform chemicals and these catalogued according to a set of criteria. The decision of the chemical/fuel to be studied was based on the gap in the chemical industry. This list comprised of chemicals that were selected in the US department of energy top 10 list in 2004 and 2010 and top 15 chemicals in the EU list in 2015. In addition to these, chemicals that are currently of interest (which were mostly chemicals that can be used as polymers and biofuels) were included to make up the list of 39 chemicals. The selected chemicals then went through a knock out selection where chemicals that cannot be produced with current technology from sugar or via a biological route were eliminated from the list. A quantitative analysis was then done on the remaining chemicals from the knock out stage. A weighting method which considered a series of factors was used to determine the top platform chemicals. The factors used were to identify platform chemicals that are at a high demand (both in South Africa and internationally), chemicals that showed great potential for profitability based on cost, technology readiness level and product yield. The quantitative analysis allowed seven chemicals to be selected. Finally a qualitative study based on interviews with experts in the field was done. Most of this information provided by the experts was supported by several literatures (Taylor, et al., 2015; Villadsen, et al., 2011; Choi, et al., 2015; Jansen & van Gulik, 2014). The qualitative study identified Succinic acid, Lactic acid and Citric acid as the top three chemicals. A techno-economic study was done on succinic acid, one of the most promising platform chemicals identified. The reasons for its selection was because it has a higher performance and it generates less carbon footprint than petroleum based succinic acid, competiveness for niche market, multiple application via BDO and PBS and its overall favourable environmental process that uses up carbon dioxide from the environment. Firstly, the succinic acid process was designed to be produced using Saccharomyces cerevisiae in a dual phase fed batch fermentation process. The overall design for the succinic acid process was based on the design proposed by Efe, et al (2013). A cost evaluation was then done on the design for an economic analysis. The economic analysis was done on the process to ascertain that there is indeed value addition of sucrose to the platform chemicals chosen. This was done in the form of profitability analysis of the process. An economic analysis of the design shows that the plant is profitable after the first year of operation. The total investment on the plant is R 22.3 billion and the start-up expense is R 1.05 billion. This project serves as a preliminary paper based overview of the general background for the selected platform chemicals that will be researched further in subsequent research.
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    Open Access
    Impact of cryoprotectants during freeze drying on Lactobacillus plantarum viability and their role in enhancing probiotic storage stability
    (2021) Oluwatosin, Olasumbo O; Fagan-Endres, Marijke; Tai, Siew
    The human microbiome has recently garnered the interest of scientists and biopharma industries as studies have revealed the potential use of live bacteria known as probiotics as potential therapeutics for restoring and maintaining human health. These probiotic-biopharma formulations must contain the right strain(s) in sufficient numbers when administered to confer the desired health benefit. Cell dehydration is used to keep the probiotic microbes in an inactivated form during storage, thereby ensuring that there are enough viable cells still present when the probiotic is taken. However, the drying process itself is detrimental to the probiotic cells and can result in reduced viability and stability of cells over storage. In this study, various cryoprotectants were assessed for their ability to maintain cell integrity and improve yield during the freeze drying dehydration of Lactobacillus plantarum towards a potential topical pharmabiotic formulation. Inulin, sucrose, maltodextrin, and skimmed milk at 10% m/v concentration of the drying media were tested for their ability to protect bacterial cells during freeze drying and over a storage period of 12 weeks at 4oC and room temperature. Furthermore glucose, inulin, sucrose, and maltodextrin as sole carbon substrate were investigated as prebiotics in concentrations of 0.5% m/v, 2% m/v, and 4% m/v of the fermentation media by in vitro fermentation of L. plantarum in glucose-free MRS-free media. The influence of these cryoprotectants and prebiotics on L. plantarum was measured against cell viability, growth kinetic parameters (growth rate, lag phase, and maximum cell density), and pH reduction potential of L. plantarum. Improved survival of L. plantarum during freeze drying and over 12-weeks of storage was observed with all cryoprotectants. Skimmed milk demonstrated the highest protection after freeze drying, with a survival rate of 91% and viable cell counts of 9.1 × 108 ( CFU ml ) from an initial cell count prior to drying of 1.0 × 109 ( CFU ml ). Inulin demonstrated high protective efficiency, with 85% viability maintained during freeze drying which resulted in final cell counts of 1.1 × 109 ( CFU ml ) from an initial cell count of 1.3 × 109 ( CFU ml ). However, inulin provided the least protection over the 12 week storage period compared to cells dried in the presence of maltodextrin, sucrose, and skimmed milk, with cell counts of only 1.2 × 106 ( CFU ml ) at 4oC and 6.3 × 103 ( CFU ml ) at room temperature recorded at the end of the period. Following skimmed milk, which also demonstrated the highest stability of cells over storage, sucrose performed second best in maintaining the stability of cells at 4 oC at the end of the 12 weeks storage, with viability of 33% which resulted in final cell counts of 3.4 × 108 ( CFU ml ). Overall, the presence of cryoprotectants and prebiotics demonstrated a significant influence on propagation and viability. The presence of each of the various prebiotics as the sole carbon substrate in the fermentation media promoted proliferation of L. plantarum. An increase in cryoprotectant concentrations led to increased biomass yield but with no significant change in the growth rate and lag phase. Cells showed improved stability when stored at 4oC compared to room temperature. A delay in propagation up to 10 hours was observed upon rehydration of stored probiotic cells across all cases except for skimmed milk that resulted in a maximum delay in propagation of 2 hours at both storage temperatures.
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    Investigating process stresses on Saccharomyces cerevisiae using isothermal microcalorimetry
    (2017) Myers, Matthew; Harrison, Susan TL; Tai, Siew; Huddy, Robert; Fagan-Endres, Marijke
    Maximising performance of microbial processes, including yeast-based processes, in an industrial setting requires understanding of the impact of process stresses. These may be the result of process configuration, dilution, temperature changes, hydrodynamic conditions or process perturbations. Methods to determine the microbial metabolic response to such stresses have long been sought, but are typically limited, often requiring the use of a suite of methods to assess the physiological status and state. The recent technical advances in microcalorimetry suggest potential for the use of isothermal microcalorimetry (IMC) to determine yeast viability and vitality and is investigated here. IMC is a laboratory method whereby the real-time heat produced by a chemical, biological or physical process is measured in the micro to nano watt range. It is proposed that this heat production may be correlated to the physiological state of the microbial catalyst and can be used to measure the impact of different stresses. In this study, the potential of IMC as a method for exploring process stress is investigated using Saccharomyces cerevisiae and its application in the beer brewing industry as a case study. Here, it is well known that yeast viability and vitality have commercial significance. IMC is sufficiently sensitive to detect the heat given off by 1000 yeast cells. However, IMC cannot distinguish between different heat flows within a system i.e. it is non-specific. The literature demonstrates how IMC has been used in the study of numerous microbiological fields, including the growth and metabolism of yeast. Previous studies have successfully derived the specific growth rate and cell numbers of a growing yeast population from analysing power and heat curves. The specific growth activity and specific growth retardation of yeast and how these parameters relate to bactericidal and bacteriostatic effects has also been examined by a number of authors. The key objectives of this study were to determine the viability and vitality of Saccharomyces cerevisiae using IMC and to assess the impact of stresses on yeast viability and vitality. This was achieved by measuring the thermal power produced by a growing yeast suspension as a function of its overall growth and metabolism. Two industrially relevant stresses were examined: cold shock and ethanol shock. The effect of these stresses has yet to be studied using microcalorimetry. The growth of Saccharomyces cerevisiae under ethanol stress was used as an inhibition study to isolate its effects on the growth thermogram. Following the generation of thermograms under control and stress conditions using IMC, a method for their quantitative analysis was developed. Curves were fitted to the heat data using an exponential growth equation and the time for the heat flow curve to peak was determined. From the exponential curve, the specific growth rate of the yeast was determined with a high degree of repeatability. The coefficient of the exponential term in the growth equation gave highly reproducible and distinguishable results relating to the viability and vitality of the initial yeast population. The time of peak heat flow was also affected by the initial viability and vitality of the yeast and was used to estimate the initial active cell population size.