Author: Renao Bai
Bai, Renao, 2022 Advanced process development toward biorefinery of Spirulina biomass for the production of functional proteins and peptides, Flinders University, College of Medicine and Public Health
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The rapid global population growth to 9.5 billion by 2050 places unprecedented pressure on sustainable protein supply. It is predicted that the protein demand is expected to increase sharply by 80% from 202 million tons in 2019 to 361 million tons by 2050. Conventional plant and meat proteins cannot address the gap between the protein supply and demand in the future. Therefore, it is urgent to identify and develop some new and alternative protein sources. As a potential protein source, microalgae have two main advantages compared with conventional protein sources: substantially higher per-unit area yield of protein (from 4 to 15 tonnes·ha-1·year-1), and low dependence on freshwater resources (100 times lower than beef protein production) and arable lands. While microalgae-derived protein production is very promising, effective downstream biorefinery processes for the extraction and value-addition of proteins from microalgal biomass are still required for the development of the microalgal protein industry. In this study, we used the most commonly produced Spirulina (Arthrospira maxima) as the model microalgae and developed a few advanced processing technologies in the biorefinery context toward fully utilization of Spirulina biomass to produce multiple products including total proteins, C-phycocyanin (C-PC), other proteins, protein hydrolysates, and bioactive peptides. Furthermore, functional and nutritional properties of Spirulina protein-based and protein-derived products were investigated to identify their potential applications. Lastly, a techno-economic assessment was done to analyze the economic feasibility of the processing technologies focusing on C-PC production developed in this study.
As C-PC is among the most valuable and fragile protein, this study has started the process development to extract and purify C-PC as the first step in a biorefinery process. The ultrasonication-assisted method was used firstly for the disruption of cell walls to obtain the C-PC crude extract which was further subjected to a novel integrated process of pH-shifting and activated charcoal adsorption. This process could rapidly purify and recover C-PC at high purity. The purity of C-PC increased from 0.45 to 3.31 in this one integrated purification step, which is suitable for the cosmetic industry. The mechanism of this integrated purification of C-PC with pH-shifting and activated charcoal was investigated and elucidated.
The second step of this biorefinery process development focused on the efficient recovery of other total proteins from the residual biomass after C-PC was extracted. After the extraction of C-PC, the residual biomass contains protein, lipids, and carbohydrates of 62.3%, 17.8%, and 15.8% (dry weight), respectively. A defatting process was applied to remove most of the lipids and pigments (chlorophyll and carotenoid), by which the protein content of the defatted pellet-A (DPA) increased to 72.8%, with that of lipids and carbohydrates at 2.0% and 15.1%, respectively. The resulting DPA was further subjected to an extraction process with NaOH and urea mixed-solvent, and around 85.4% of the total protein was isolated in this step. The highly purified other proteins that was protein extract of defatted pellet-A (PE-DPA) have balanced amino acids composition and content with 39.2% essential amino acid content. Furthermore, the PE-DPA showed high water solubility at pH ranging from 6.0-12.0, suitable for applications in food formulations. An alternative process has also been developed for recovering other total proteins from the DPA via proteinase hydrolysis to produce the protein hydrolysates as the products. A sequential hydrolysis strategy with two proteinases of first hydrolyzing with Alcalase and then followed by papain has recovered 84.2% of other total protein from the DPA. The protein hydrolysates produced by this sequential hydrolysis process have been characterized with comparable properties to egg white proteins.
Focusing on the discovery and development of antioxidant peptides, the DPA has been subjected to hydrolysis using five different commercial proteases including Papain, Trypsin, Alcalase, Alcalase 2.4 L, and Protamex 1.6. The results showed that the hydrolysate using Alcalase 2.4 L had the highest antioxidative activities including scavenging 2,2-Diphenyl-1-picrylhydrazyl (DPPH), superoxide anion, hydroxyl radicals, and 2,2’-Azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) in vitro than the hydrolysates derived from other proteases. After purification by ethanol precipitation, ultrafiltration, and gel column chromatography, four bioactive peptides have been identified from the Alcalase 2.4 L hydrolysate. Their amino acid sequences are: (A)VLNGLK; (B)KIEDLK; (C)LTGMAFR; and (D)KAGKAGK.
Finally, a techno-economic assessment using industry software, SuperPro Designer was conducted by simulating the processes developed in this study for the production of C-PC and protein hydrolysates on an industrial scale. This assessment allows us to understand how the processing technologies developed in this study could impact on the economics of Spirulina biorefinery. The results demonstrated that the production of cosmetic-grade of C-PC using our process is much more profitable at lower investment risk than the production of food-grade of C-PC. A commercial plant of processing 300 metric tonnes dried Spirulina per year in 20 years of project lifespan could achieve its net present value and return on investment time of 120 million US dollars, and 4.3 years, respectively. In a biorefinery context, all the technology improvements developed in this study demonstrated its economic values to a certain extent, and the research and investment bottlenecks have also been identified for future directions.
In conclusion, this study has developed a few advanced processing technologies by focusing on the production of specific high-value protein (C-PC), other total proteins, protein hydrolysates, and antioxidative peptides toward the biorefinery of microalgae Spirulina. These product properties have demonstrated either superior purity, high yield or comparable quality to have potential commercial applications. The advances in both process and product developments are evidenced by techno-economic assessment with improved profitability and commercial viability, hence the significance of the results from this study. Further advances are still required to comprehensive utilisation of the microalgae biomass toward a full biorefinery process with zero waste, less water/solvent use, and optimised development of multiple products fit for market.
Keywords: Spirulina, proteins, C-phycocyanin, peptides, microalgae, cyanobacteria
Subject: Biotechnology thesis
Thesis type: Doctor of Philosophy
Completed: 2022
School: College of Medicine and Public Health
Supervisor: Wei Zhang