Improving an aqueous two-phase process for C-phycocyanin extraction from Spirulina
Biotechnology and bioprocess engineering have made it possible to expand production of natural compounds, and markets have moved more towards this direction. An example of this is in pigments, where many synthetic pigments have been banned or pulled out of the market due to health concerns, while naturally-derived pigments are growing in popularity. The algal pigment, C-phycocyanin (C-PC) is a blue photosynthetic pigment of the phycobiliprotein family, found in cyanobacteria and red algae. Algal pigments like C-PC have been growing in demand and the market is expected to grow at 5-7 % annually. Phycocyanin has applications as a food and cosmetics dye, a health product with therapeutic uses, and as a diagnostic protein. Various processes have been studied to recover and purify C-PC from cyanobacteria such as Arthrospira platensis, commonly referred to as Spirulina, the most used organism for producing the pigment. The C-PC recovery process includes extraction, recovery and purification steps. One recovery and purification step reported in literature is aqueous twophase separation (ATPS), which is able to produce high purity C-PC with good recovery. At the University of Cape Town (UCT), the Centre for Bioprocess Engineering Research (CeBER) has patented a process for extracting and purifying C-PC from Spirulina using a polyethylene-glycol (PEG) and maltodextrin (MDX) ATPS. This is a less-studied form of ATPS, with most C-PC extraction studies using PEG – salt systems. The PEG – MDX system was studied due to challenges faced with C-PC recovery from the PEG phase. The patented CeBER process begins with cell disruption, a period of leaching into a buffered solution followed by cell debris removal. The C-PC is subsequently purified and recovered by the PEG – MDX ATPS and three ammonium sulfate precipitation stages, and finally dried to powder. The patented process requires optimisation and up-scaling before being commercially applied in industry, the ultimate aim of the greater project. As such, this project aimed to improve the understanding of the overall process for C-PC recovery from Spirulina and, in particular, the ATPS step involved, to improve the ATPS performance. The study also sought to produce cosmetic grade C-PC (purity number of > 1.5), and develop process options and simulations for this production based on a combination of literature and experimental results. The work was conducted in view of future up-scaling. This included study of the leaching step with the Spirulina used in this project as well as refinement and optimisation of the ATPS step. For the latter, phase diagrams for the PEG – MDX ATPS were produced to inform ATPS refinement and improvement before evaluating its performance. The phase diagrams give an understanding of how the phases partition in an ATPS, allowing prediction and optimisation of top and bottom phase compositions. Leaching experiments showed that a maximum C-PC concentration was found after 2 h with the Spirulina powder used without the need for cell disruption, and that the purity decreased slowly over time (using 100 g/L Spirulina powder in 5 g/L citrate buffer at pH 6). This recommended a relatively short leaching time be used compared to previous work using different starting material. The PEG – MDX ATPS phase diagrams produced corresponded well with similar ATPS data found in literature. The PEG molecular weight was tested for the best performance, finding that PEG 10000 performed slightly better than both PEG 6000 and PEG 20000, achieving a C-PC purification factor of 1.21 ± 0.01 at 9 wt% PEG 10000 and 20 wt% MDX, with a recovery of 95.1 ± 7.8 %. However, the PEG – MDX ATPS for C-PC purification gave lower purification factors compared to PEG – salt ATPS studies from literature. A two-stage ATPS was therefore considered, with a PEG – citrate ATPS used before the PEG – MDX ATPS. This aimed to take advantage of the good C-PC selective recovery reported in literature for PEG – salt ATPS systems while still using the PEG – MDX to separate the C-PC from the PEG phase. PEG – citrate phase diagrams were produced; these compared well with those found in literature studies. A screening of PEG molecular weights across both ATPS steps found PEG 4000 to be best, mainly due to the performance in the PEG – citrate stage. Using PEG 4000 in two factorial studies on the impact of PEG and citrate concentrations in the first ATPS, and PEG and MDX concentrations in the second ATPS, response curves for the purification, recovery and concentration of the C-PC in the desired phase were produced. A Statistica (version 13.5.0.17) model was used to predict a local optimum, where the combination of component concentrations produce C-PC at high purify, recovery and concentration. The PEG – citrate ATPS model predicted the best component concentrations to be 11 wt% PEG and 20 wt% citrate, which gave a C-PC purification factor of 1.63 ± 0.28, at a recovery of 95.6 ± 8.0 %. The PEG – MDX ATPS model predicted a 1.43 ± 0.09 C-PC purification factor and a recovery of 86.8 ± 4.7 %, using a composition of 11 wt% PEG and 22 wt% MDX. A combination of experimental results and literature data were used to underpin the simulation of five process configurations for C-PC production using SuperPro Designer (version 9.5), each targeting a cosmetic grade C-PC product or better. These sought to simplify and improve C-PC production using the PEG – MDX ATPS as the core unit procedure. The first simulation was of the original patented process (cell-disruption, leaching, ATPS, precipitation and finally freeze-drying), with the ATPS operation updated with the best case experimental results obtained in this work. The second and third simulations used the newly proposed two-stage ATPS. In the third option ultrafiltration replaced the precipitation steps. Spray-drying replaced freeze-drying as a faster and more cost-effective means of drying C-PC from the second simulation onward. The fourth simulation incorporated a pre-treatment step, using activated carbon and chitosan to adsorb contaminant proteins and purify the C-PC, before using a single PEG – MDX ATPS. The operation and performance of the pre-treatment step were based on literature information. This model used (NH4)2SO4 precipitation as in the original process and required two precipitation stages for final purification. The fifth simulation used the pre-treatment process as in simulation 4, with the second precipitation step replaced with filter-sterilisation, before spray-drying. The two-stage ATPS processes lead to slightly improved recoveries of 39.1 % and 39.5 % for the second and third of process recommendations, respectively. This is above the original process simulated to recover 38.7 % of the C-PC in the crude starting solution. The two-stage ATPS processes also have lower ammonium sulfate usage due to having fewer precipitation stages, in the case of the second process simulation, and due to replacing precipitation wit ultrafiltration in the third process. The fourth process showed a higher C-PC purity of > 4.00, compared to the 3.77 simulated for the original process, and a C-PC recovery of 42.7 % from the crude extract. The chemical consumption was similar to the original process, while decreased amounts of ammonium sulfate were required. This process showed the shortest path time of the five options. Of the process configurations presented, the fifth one is best recommended, since it leads to a short batch-time (24.8 h per 3 batches), fewer process units than the original and the lowest overall chemical usage of the five processes. It also produces higher C-PC purification and recovery with reduced complexity compared to the original process. A C-PC purity of up to or above 4.0 is estimated to be achievable with this process, at a recovery of 47.7 % (using the leached C-PC as the starting point). This process produces a C-PC quality well above cosmetic grade. The trade-off between recovery and purity could be explored to achieve a higher recovery of cosmetic grade C-PC. To summarise, phase diagrams were produced for the PEG – MDX ATPS, and the C-PC leaching was tested on the Spirulina used in this project. This ATPS was then tested before moving on to a two-stage ATPS, using a PEG – citrate stage, for which PEG – citrate phase diagrams were produced, before the PEG – MDX stage. This produced better results, comparable to PEG – salt ATPS studies found in literature. Results from the process simulations done on Superpro Designer then supported the use of a pre-treatment step before a single-phase PEG – MDX ATPS, followed by precipitation and spray drying, based on information from this study and other literature. This could lead to a feasible design for pilot-testing and a novel process for industrial C-PC production. It is recommended that the results of the simulations be tested experimentally in further studies. A thorough techno-economic analysis of the proposed processes is also required. The pre-treatment process based on adsorption requires experimental validation and pilot scale confirmation before being applied in industry. Due to the rapidly growing demand for C-PC, a commercial process capable of producing multiple grades of C-PC, from foodgrade, to cosmetic-and reagent-grade, could be lucrative for business interests involved.
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