Browsing by Author "Fagan, Marijke"

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    Open Access
    Purification of C-phycocyanin from Spirulina - adsorption pretreatment options
    (2023) Payne, Eric; Fagan, Marijke; Harrison Susan
    C-phycocyanin (C-PC) is an attractive blue pigment obtained from the cyanobacterium Spirulina and certain other species of microalgae. In recent years, C-PC has been used as a naturally derived colourant in cosmetic and food products due to the toxic effects of synthetic pigments. Cultivation of microalgae, like Spirulina, is well established, but there is scope to find new and more efficient ways of recovering the products they possess, including C-PC. There are many techniques which can be used to purify C-PC from Spirulina, where the choice of which is dependent on the desired C-PC application. Different applications require different degrees of C-PC purity, where this is measured as a ratio of the absorbance of C-PC in a sample relative to the total absorbance protein. Work was done at the Centre for Bioprocess Engineering Research (CeBER) to develop a process using aqueous two-phase separation (ATPS) and ammonium sulfate precipitation to generate C-PC for cosmetic use, which has a minimum purity requirement of 1.5. A patent was obtained before further work was conducted on this process to optimise certain aspects of it. Following on from this research, it was recommended that an adsorption pretreatment be incorporated into the process prior to the ATPS and precipitation to improve the C-PC purity entering these stages. This adsorption pretreatment was proposed to use chitosan and activated charcoal due to the potential they have shown in C-PC purification processes. They are both easy to use and are known to be efficient as adsorbents – chitosan due to its two distinct hydroxyl and carbonyl functional groups, and activated charcoal because of its high surface area. The goal of this research project was to test the pretreatment and understand what impacts it has on the ability of the overall process to generate cosmetic-grade C-PC reliably and efficiently. Leaching tests of the Carbocraft Spirulina used in this research project yielded an optimal period of 17 to 24 hours for the C-PC extraction from the Spirulina and showed that a cell disruption step was not necessary for this specific powder. Testing the adsorbents individually indicated that chitosan can selectively remove unwanted proteins while activated charcoal tends to adsorb indiscriminately. However, a significant improvement in purity is observed when they are used in combination. An attempt was made to optimise the concentrations of chitosan and activated charcoal as well as the adsorption pH and contact time through a central composite design. However, this does not provide any clear indication of optimal values, due to the high degree of experimental error and narrow range of purities. Running an adsorption prior to a polyethylene glycol (PEG) and citrate ATPS generated C-PC purities of approximately 1.5. However, the ATPS required a significant dilution to be effective and the C-PC recovery of this step was lower than 40%, making it potentially inefficient to scale up and impractical economically due to the high C-PC loss. A different process configuration was thus suggested, excluding ATPS but using ammonium sulfate precipitation followed by microfiltration and ultrafiltration. These two steps are included to reduce the levels of unwanted microorganisms and salts for cosmetic-grade C-PC. When feeding a crude extract with its concentration reduced with buffer by 50%, this purification train was able to consistently generate purities greater than 1.5. Additionally, by conducting activated charcoal adsorption in a packed column, the C-PC recovery of this stage of the process was improved and the low recovery as a consequence of using powdered activated charcoal in suspension and dead-end centrifugation can be avoided. C-PC powder was obtained by freeze drying samples which had been purified using the proposed process. These powdered samples were tested and found to have E-values (measure of colour intensity) competitive with commercially available products. The process was also shown to be robust and can handle variations in Spirulina feed. Ultimately, this confirmed that an adsorption step coupled with other standard purification techniques can be used to reliably generate cosmetic-grade C-PC with recoveries of 80% and above.
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    Open Access
    Techno-Economic Evaluation of a Commercial c-Phycocyanin Extraction Process
    (2024) Williams, Aqeel; Fagan, Marijke; Harrison, Susan
    Growing health concerns over synthetically produced pigments have led to the development of natural pigment products from phycobiliproteins, a family of proteins found in cyanobacteria. One such phycobiliprotein is c-phycocyanin (cPC), a bright blue pigment that has already found widespread application in the food and pharmaceutical industries. It can also be used in the cosmetics industry, an application which has not been as extensively explored. Spirulina platensis (Spirulina), a cyanobacterium known for its relatively economical and easy cultivation, is a popular source for the production of cPC. The Centre for Bioprocess Engineering Research (CeBER) at the University of Cape Town (UCT) has patented a novel process for extracting and purifying cPC from Spirulina (Fagan-Endres et al., 2019). A manufacturer of raw materials for the cosmetics and consumer products industries in South Africa (anonymously referred to as ‘Company A' in this dissertation), possesses the necessary resources, expertise, and market access to establish a cPC extraction and purification process. The global market for cPC for cosmetics use, valued at $27 million in 2020 with a projected compound annual growth rate (CAGR) of 10%, presents a lucrative opportunity. Therefore, a detailed techno-economic analysis was conducted on three cPC extraction and purification processes to assess their commercial viability. Process 1 was the example process presented in the UCT patent and thus considered the base design. In this process, following liberation of the cPC into a buffer solution and separation of the cell debris, the cPC was purified using a PEG-MDX aqueous two-phase separation (ATPS) and an ammonium sulphate precipitation train before the product was dried. Process 2 was an extension of the base process design based on work done by Hockey (2022) and included an additional citrate-PEG ATPS at the start of the purification unit operations. Process 3 was another extension on the base process design based on further work done by Kadir (n.d.), Payne (2023) and Dunnett (n.d.). In this instance, an adsorption step using activated carbon and chitosan was used to purify the cPC leachate instead of an ATPS. As part of this research, process flow diagrams (PFDs) and mass balances were compiled for each process. The mass balance used specific unit operation performance parameters drawn from literature, previous and concurrent work to this research by Hockey (2022), Kadir (n.d.), Payne (2023) and Dunnett (n.d.), equipment technical data sheets, correspondence with equipment suppliers, and laboratory work. For the techno-economic analysis, equipment and raw material quotations were collected, and widely applied cost correlations from literature were used in a discounted cash flow analysis to calculate common economic indicators: the payback period (PB), net present value (NPV) and internal rate of return (IRR). The primary indication for profitability was taken as when the IRR is equal to or exceeds the investor's weighted average cost of capital (WACC). Centre for Bioprocess Engineering Research The baseline case techno-economic analysis was conducted at nominal values for input parameters, the most critical of which were a pilot-scale Spirulina input of 20 kg per batch, a Spirulina cPC content of 8 wt%, a cPC product price of 820 $/kg, a discount rate (WACC) of 19.5% and an operation period of four years. Baseline case results from the techno-economic analysis showed that only Process 2 demonstrated profitability, with Process 1, 2 and 3 reporting an IRR of 6.7%, 19.6%, and 15.3%, respectively. A sensitivity analysis revealed interesting process dynamics and highlighted the significance of batch time in profitability. A Monte Carlo analysis was conducted at a production scale of 200 kg per batch Spirulina input to understand further process dynamics in response to inherent variation in critical input parameters. Both of these analyses confirmed the profitability of Process 2, and showed how the profitability of Process 1 and 3 could be improved by increasing the scale and the cPC content in Spirulina, in particular. Furthermore, Process 3 was found to be the most resilient to variation in input parameters. Process 2 was excluded from further optimisation due to its high direct capital investment, raw material requirement and processability risk. Therefore, for Process 1 and 3, an optimisation opportunity was identified wherein batch times were reduced by staggering batches, thereby maximising equipment utilisation. This enhanced the profitability of Process 1 and 3, and revealed that Process 3 was more profitable than Process 1. Further analysis found the minimum profitable scale for Process 3 at a cPC price of 820 $/kg to be 65 kg Spirulina input per batch, producing 245 batches per year, and this scale would serve the estimated market opportunity of 1 120 kg cPC in the first year of operation. The minimum selling price at the production scale of 200 kg Spirulina input was found to be 680 $/kg, 17% less than the assumed market price. It is recommended that Process 3 is implemented at Company A for cPC production, due to the process' robustness and profitability, at a production scale of 65 kg Spirulina input per batch, to match the market opportunity of 1 120 kg cPC per year. Implementing Process 3 at Company A will enable the company to tap into the growing cosmetics cPC market, leveraging its competitive advantage and maintaining profitability. Finally, this dissertation recommends the need for future research into the synthesis and performance of a granular activated carbon-chitosan adsorbent, the scale-up of the adsorption column, and the evaluation of larger-scale centrifuges. These research areas are suggested as the adsorption step is a crucial part of the recommended Process 3, and it was assumed that granular activated carbon-chitosan was used, which is not yet widely available. Furthermore, performance of the adsorption column was assumed to remain constant during scale-up, which is not realistic over time. Additionally, it is imperative to confirm the performance of larger scale centrifuges using the materials used in Process 3, as the nature of the streams exiting these units was found to be sensitive to these units' performance.
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    Open Access
    Troubleshooting, optimization and robustness testing of CeBER process for c-phycocyanin extraction and purification from spirulina
    (2025) Kadir, Uzair; Fagan, Marijke; Harrison, Susan
    c-Phycocyanin is a natural pigment, found as an intracellular protein in various microalgae. One of the most common sources of c-phycocyanin is Arthrospira platensis, more commonly known as Spirulina. Spirulina is a cyanobacterium, widely renowned for its many health benefits which, experts say, are a direct result of c-phycocyanin (Vernes, et al., 2015). Previous researchers in CeBER have demonstrated and patented a process to produce cosmetic grade c-phycocyanin (> 1.5 purity ratio) from Spirulina. The focus of this study was to optimize operating conditions for the key unit procedures posed previously for the CeBER patented process. The experimental work was performed in two main steps. The first considered the individual unit procedures (cell disruption via bead-milling, c-phycocyanin leaching rom biomass, cell debris removal via centrifugation, c-phycocyanin purification via polyethylene glycol-citrate and polyethylene glycol-maltodextrin aqueous two-phase separation, c-phycocyanin purification via ammonium sulfate precipitation), where the main aim was to optimize the operating conditions of the units and to mitigate redundancies in the pool of unit procedures. The second step involved assessing the performance of the overall process operating at the newly found operating conditions. Robustness of the process was qualified through the use of three different dried Spirulina powder samples (sourced from different suppliers) for the experiments. Bead-milling was demonstrated to narrow out the particle size distributions as expected, however a shift towards smaller cut sizes was only observed for the larger dried powders tested. In subsequent leaching, bead-milling did increase the initial extraction rate of c-phycocyanin from dried Spirulina powder. However, it was found that bead-milling may have no effect in the overall leaching time taken to reach an equilibrium concentration of c-phycocyanin in the crude extract. Bead-milling was also shown to have either little to no benefit in the recovery of product containing supernatant over the cell debris removal step. It was found that the c-phycocyanin leaching kinetics from the dried Spirulina powders could not be reliably predicting using standard models in literature. The highest R2 obtained was 0.95 for the fit of a 2nd order kinetic model to a Carbocraft milled sample. Most other samples tested did not yield a good correlation to first or second order leaching kinetics. It was found that to reach an equilibrium concentration across all samples, the minimum leaching time required was 19 hours. The crude extract purity ratios achieved were approximately 0.46, 0.41 and 0.12 in the Brenntag, Carbocraft and Indian Spirulina powder samples' crude extracts, respectively. As predicted, an increase in the g force used for cell debris removal via centrifugation yielded an increase in recovery of the product-containing supernatant. The highest recoveries were achieved at 10 000 g of operation. It was shown that a two-stage cell debris removal with an inter-stage rinsing step improved c-phycocyanin recovery overall. It was found that both of the ATPS systems investigated by previous CeBER researchers did not perform sufficiently robustly when required to handle a variation in the Spirulina source and hence crude extract feed. As such, the ATPS steps was deemed unsuitable for use in a commercialisation process and were removed from the process testing scope. Owing to the removal of the ATPS steps, microfiltration (0.22 m aperture) was added to the end of the process as an additional c-phycocyanin purification step. The ammonium sulfate precipitation train was optimized by identifying the ammonium sulfate concentrations that trigger selective c-phycocyanin precipitation and tracking how purity changes over concentration changes too. It was shown that a two-stage ammonium sulfate precipitation will yield a purer c-phycocyanin product than a single stage, producing a product stream with a higher final purity ratio and with less cell debris content. The ammonium sulfate saturation fraction recommended for the first stage is 0.22 where the supernatant is sequestered and treated with additional ammonium sulfate to achieve 0.45 saturation for the second stage. The c-phycocyanin is precipitated out in the second stage. The full runs of the process at the newly established “best” operating conditions yielded maximum purity ratios of 0.90 ± 0.02, 0.79 ± 0.03, 0.46 ± 0.03 for the Brenntag, Carbocraft and Indian dried Spirulina powders respectively. Thus, cosmetic grade c-phycocyanin was not achieved by any of the samples and food grade (> 0.7) only achieved using the Brenntag and Carbocraft samples. The recoveries were 24 ± 8%, 66 ± 8% and 65 ± 14% for the Brenntag, Carbocraft and Indian samples, respectively. It appears that, despite the low recovery obtained in the runs using the Brenntag powder, the amended process is robust enough to extract and purify c-phycocyanin from Spirulina, albeit without achieving the overall aim of cosmetic grade. The overall purification factors from the crude extract were approximately 2.6, 2.2 and 4.4 for the Brenntag, Carbocraft and Indian samples, respectively. These purification factors are not far off from those reported in literature for c-phycocyanin purification processes. It is, therefore, recommended that the process put more emphasis on increasing the purity ratio in the crude extract through means of using fresh Spirulina and chitosan/activated-charcoal adsorption techniques (as recommended by Hockey (2020) and Payne (2023)). With these changes it is probable that the amended process will achieve cosmetic grade purity. Further work should also be performed on increasing the recovery, especially over the precipitation stages, without compromising purity.