Biorefinery of Chlorella sp. using integrated multiphasic systems for biofuel, feed and wound healing application

Microalgae have been explored as a sustainable alternative to fuel and feed on natural resources. Microalgae possess numerous advantages over their renewable counterparts such as soybean and palm oil. It does not compete with agricultural land or freshwater for food crop production, making it a potential biofuel source. However, commercialisation of microalgae biodiesel is yet to make a presence in the billion-dollar biofuel industry due to the bottlenecks. These include rigid microalgae cell wall, low biomass concentration in the harvested culture and high downstream costs. Therefore, a fossil fuel-derived concept of refinery can be introduced to microalgae to overcome as aforementioned challenges. This project aims to focus on the algae downstream process for biorefinery applications. First, a novel biocomponent extraction method, named sugaring-out assisted liquid biphasic electric flotation (LBEF) system, for protein separation from Chlorella vulgaris was developed. High yield of proteins (69.66±0.86 %) was extracted from microalgae with a rapid and single-step process. Following this, a multiphase integrated system that focused on the extraction of two or more biomolecules in microalgae was introduced. This system focused on simultaneous component extraction rather than conventional cascade approach. The system were incorporated in two different studies. First study aimed to extract two biomolecules (protein and lipid), whereas second study focused on a concurrent three biomolecules extraction approach. The parameters of this system such as volume ratio of ammonium sulphate and t-butanol, flotation air flowrate, flotation time, ultrasound pulse settings and pH were optimised to achieve a high recovery of biomolecules. Highest yield of protein, lipids and carbohydrates were observed at 96.59±8.15 %, 61.02±0.91 % and 52.69±1.90 %, respectively. Control run without flotation technique resulted in lower yield of proteins, lipids and carbohydrates at 25.33±3.50 %, 52.96±4.59 % and 32.44±0.29 %, respectively. Whereas, control run without flotation and cell-disruption technique had lowest yield of proteins, lipids and carbohydrates at 16.73±1.26 %, 51.13±6.27 % and 26.21±0.23 %, respectively. Besides, a large-scale set-up up to 10-15 times was tested out. Recycling ability of the chemicals involved in the extraction were presented. Up to 90 % of the alcohols and salt used in the experiment were recycled. Lastly, the extracted proteins from the multiphase integrated system were purified and its application in wound healing of human keratinocyte cells was investigated. Proteins were adsorbed on a gelatine-glutaraldehyde membrane. This membrane system was used to observe the wound healing of keratinocytes. The biocompatibility, cell adhesion, proliferation and wound scratch of human keratinocyte cells were studied and presented. Overall, multiphase integrated system presented in this project serves as a successful demonstration of microalgae biorefinery concept. The improved yield of biomolecules provide potential applications of microalgae in biofuel, food and medicine field industry. Future studies should focus on analysis of life-cycle cost and optimising the operational cost required for this whole biorefinery set up. The project presented in this thesis offers a platform for future biorefinery research and possible commercial large-scale utilisation

Biorefinery of Chlorella sp. using integrated multiphasic systems for biofuel, feed and wound healing application

Microalgae have been explored as a sustainable alternative to fuel and feed on natural resources. Microalgae possess numerous advantages over their renewable counterparts such as soybean and palm oil. It does not compete with agricultural land or freshwater for food crop production, making it a potential biofuel source. However, commercialisation of microalgae biodiesel is yet to make a presence in the billion-dollar biofuel industry due to the bottlenecks. These include rigid microalgae cell wall, low biomass concentration in the harvested culture and high downstream costs. Therefore, a fossil fuel-derived concept of refinery can be introduced to microalgae to overcome as aforementioned challenges. This project aims to focus on the algae downstream process for biorefinery applications. First, a novel biocomponent extraction method, named sugaring-out assisted liquid biphasic electric flotation (LBEF) system, for protein separation from Chlorella vulgaris was developed. High yield of proteins (69.66±0.86 %) was extracted from microalgae with a rapid and single-step process. Following this, a multiphase integrated system that focused on the extraction of two or more biomolecules in microalgae was introduced. This system focused on simultaneous component extraction rather than conventional cascade approach. The system were incorporated in two different studies. First study aimed to extract two biomolecules (protein and lipid), whereas second study focused on a concurrent three biomolecules extraction approach. The parameters of this system such as volume ratio of ammonium sulphate and t-butanol, flotation air flowrate, flotation time, ultrasound pulse settings and pH were optimised to achieve a high recovery of biomolecules. Highest yield of protein, lipids and carbohydrates were observed at 96.59±8.15 %, 61.02±0.91 % and 52.69±1.90 %, respectively. Control run without flotation technique resulted in lower yield of proteins, lipids and carbohydrates at 25.33±3.50 %, 52.96±4.59 % and 32.44±0.29 %, respectively. Whereas, control run without flotation and cell-disruption technique had lowest yield of proteins, lipids and carbohydrates at 16.73±1.26 %, 51.13±6.27 % and 26.21±0.23 %, respectively. Besides, a large-scale set-up up to 10-15 times was tested out. Recycling ability of the chemicals involved in the extraction were presented. Up to 90 % of the alcohols and salt used in the experiment were recycled. Lastly, the extracted proteins from the multiphase integrated system were purified and its application in wound healing of human keratinocyte cells was investigated. Proteins were adsorbed on a gelatine-glutaraldehyde membrane. This membrane system was used to observe the wound healing of keratinocytes. The biocompatibility, cell adhesion, proliferation and wound scratch of human keratinocyte cells were studied and presented. Overall, multiphase integrated system presented in this project serves as a successful demonstration of microalgae biorefinery concept. The improved yield of biomolecules provide potential applications of microalgae in biofuel, food and medicine field industry. Future studies should focus on analysis of life-cycle cost and optimising the operational cost required for this whole biorefinery set up. The project presented in this thesis offers a platform for future biorefinery research and possible commercial large-scale utilisation

Treatment of palm oil mill effluent (POME) by coagulation flocculation process using peanut–okra and wheat germ–okra

Coagulation–flocculation has been proven as one of the effective processes in treating palm oil mill effluent (POME), which is a highly polluted wastewater generated from palm oil milling process. Two pairs of natural coagulant–flocculant were studied and evaluated: peanut–okra and wheat germ–okra. This research aims to optimize the operating parameters of the coagulation flocculation process in removing turbidity, total suspended solid and chemical oxygen demand (COD) from POME by using a central composite design in the Design Expert® software. Important parameters such as operating pH, coagulant and flocculant dosages were empirically determined using jar test experiment and optimized using response surface methodology module. Significant quadratic polynomial models were obtained via regression analyses (R2) for peanut–okra (0.9355, 0.9534 and 0.8586 for turbidity, total suspended solids and COD removal, respectively) and wheat germ–okra (0.9638, 0.9578 and 0.7691 for turbidity, total suspended solids and COD removal, respectively). The highest observed removal efficiencies of turbidity, total suspended solids and COD (92.5, 86.6 and 34.8%, respectively, for peanut–okra; 86.6, 87.5 and 43.6%, respectively, for wheat germ–okra) were obtained at optimum pH, coagulant and flocculant dosages (pH 11.6, 1000.1 mg/L and 135.5 mg/L, respectively, for peanut–okra; pH 12, 1170.5 mg/L and 100 mg/L, respectively, for wheat germ–okra). The coagulation flocculation performance of peanut–okra and wheat germ–okra were comparable to each other. Characterizations of the natural coagulant–flocculant, as well as the sludge produced, were performed using Fourier transform infrared, energy-dispersive X-ray spectroscopy and field emission scanning electron microscope. More than 98% of water was removed from POME sludge by using centrifuge and drying methods, indicating that a significant reduction in sludge volume was achieved.</p

Microalgae: A potential alternative to health supplementation for humans

Microalgae has been consumed in human diet for thousands of years. It is an under-exploited crop for production of dietary foods. Microalgae cultivation does not compete with land and resources required for traditional crops and has a superior yield compared to terrestrial crops. Its high protein content has exhibited a huge potential to meet the dietary requirements of growing population. Apart from being a source of protein, presence of various bio-active components in microalgae provide an added health benefit. This review describes various microalgal sources of proteins and other bio-active components. One of the heavily studied group of bio-active components are pigments due to their anticarcenogenic, antioxidative and antihypertensive properties. Compared to various plant and floral species, microalgae contain higher amounts of pigments. Microalgal derived proteins have complete Essential Amino Acids (EAA) profiles and their protein content is higher than conventional sources such as meat, poultry and dairy products. However, microalgal based functional foods have not flooded the market. The lack of awareness coupled with scarce incentives for producers result in under-exploitation of microalgal potential. Application of microalgal derived components as dietary and nutraceutical supplements is discussed comprehensively. Keywords: Health, Human, Microalgae, Protein, Supplemen

Microalgae as a potential sustainable solution to environment health

Cyanobacteria such as Spirulina platensis secretes numerous biomolecules while consuming CO2 for photosynthesis which can reduce the environmental pollution as it can also be grown in wastewater. These biomolecules can be further processed in numerous pathways such as feed, fuel, pharmaceuticals, and nutraceuticals. This study aims to screen the potential molecular mechanisms of pigments from cyanobacteria as antidiabetic type-2 candidates through molecular docking. The activities of the test compounds were compared to commercial diabetic drugs, such as acarbose, linagliptin and polydatin. The results indicated that the binding affinity of pheophytin, β-carotene, and phycocyanobilin to α-amylase were 0.4, 2, and 2.6 kcal/mol higher than that of acarbose with α-amylase. Binding affinity between pheophytin, β-carotene, and phycocyanobilin with α-glucosidase were found to be comparable, which resulted 1.2, and 1.6 kcal/mol higher than that of acarbose with α-glucosidase. Meanwhile, binding activity of β-carotene and phycocyanobilin with DPP-IV were 0.5 and 0.3 kcal/mol higher than that of linagliptin with DPP-IV, whereas pheophytin, β-carotene, and phycocyanobilin with Glucose-6-phosphate dehydrogenase (G6PD) were 0.2, 1, and 1.4 kcal/mol higher from that of polydatin with G6PD. Moreover, pheophytin, β-carotene and phycocyanobilin were likely to inhibit α-amylase, α-glucosidase, and DPP-IV competitively, while uncompetitively for G6PD. Thus, the integration of molecular docking and experimental approach, such as in vitro and in vivo studies may greatly improve the discovery of true bioactive compounds in cyanobacteria for type 2 diabetes mellitus drugs and treatments. © 2022 Elsevier Lt

In silico proteolysis and molecular interaction of tilapia (Oreochromis niloticus) skin collagen-derived peptides for environmental remediation

Fish skin collagen hydrolyzate has demonstrated the potent inhibition of dipeptidyl peptidase-IV (DPP-IV), one of the treatments for type-2 diabetes mellitus (type-2 DM), but the precise mechanism is still unclear. This study used in silico method to evaluate the potential of the active peptides from tilapia skin collagen (Oreochromis niloticus) for DPP-IV inhibitor. The methodology includes collagen hydrolysis using BIOPEP, which is the data-base of bioactive peptides; active peptide selection; toxicity, allergenicity, sensory analysis of active peptides; and binding of active peptides to DPP-IV compared with linagliptin. The result indicated that in silico enzymatic hydrolysis of collagen produced active peptides with better prediction of biological activity than intact collagen. There are 13 active peptides were predicted as non-toxic and non-allergenic, some of which have a bitter, salty, and undetectable taste. Docking simulations showed all active peptides interacted with DPP-IV through hydrogen bonds, van der Waals force, hydrophobic interaction, electrostatic force, π-sulfur, and unfavorable interaction, where WF (Trp-Phe), VW (Val-Trp), WY (Trp-Tyr), and WG (Trp-Gly) displayed higher binding affinities of 0.8; 0.5; 0.4; and 0.3 kcal/mol compared with linagliptin. In this study, we successfully demonstrated antidiabetic type-2 DM potential of the active peptides from tilapia skin collagen. The obtained data provided preliminary data for further research in the utilization of fish skin waste as a functional compound to treat the type-2 DM patients. Alternatively, this treatment can be synergistically combined with the available antidiabetic drugs to improve the insulin secretion of the type-2 DM patients