Development of an efficient protein recovery system using liquid biphasic flotation

Extensive development of biotechnology over the past several decades has induced a great impact in the production of biological products in various industries. To date, major challenges in the biotechnology industry includes the production process of biomolecules with high purity and low cost, whilst retaining functionality. The conventional downstream processing of valuable bioproducts which are widely employed usually involves multi-processing steps, high energy and chemical consumption and often has a large influence on the cost of the finishing product. Therefore, the demand for cost-efficient and simple downstream processes has directed towards an intensive research for exploiting novel separation tool that can achieve high level of product purity with minimum number of processing stages and greener approach. The aim of this thesis is to develop a new separation method that has minimum number of steps, environmentally friendly and with the ability to achieve maximum level of product purity. Liquid biphasic flotation system is a novel technique which incorporates the principles of aqueous two-phase systems and mass transfer mode of solvent sublation. This system has been proposed as an ideal purification technique for separation, purification and concentration of biomolecules. Liquid biphasic flotation have been utilised previously to purify several biomolecules whilst maintaining their functionality. This thesis emphasised on extending the applications, improvising and to diversify liquid biphasic flotation technique as an efficient tool for downstream processing. This thesis has four objectives, in which all the objectives highlight on the usage of liquid biphasic flotation system for maximum biomolecules extraction. The initial part of liquid biphasic flotation application study is to investigate the effect of full and continuous recycling of alcohol and salt phase components in large scale liquid biphasic flotation system for lipase extraction. In this section, main focus was to optimize operating conditions for the recycling of both phase component and to investigate the competence of recycling phase components using liquid biphasic flotation system on a large scale. The liquid biphasic flotation system investigated is composed of 1-propanol and ammonium sulphate whereby both phase components went through complete recycling process. From the results obtained, it is exhibited that by reusing the bottom phase, separation efficiency of lipase was sustained beyond 77.33 % and yield with 80%. This study showed that the recovered phase components could be recycled effectively up to four cycles and able to produce a significantly high yield of lipase. Next study was on a novel approach of liquid biphasic flotation system for lipase recovery utilizing recycling phase components comprising surfactant and sorbitol. This novel method utilized Triton X-100 and xylitol for lipase extraction from Burkholderia cepacia. The scope of this study focuses on eliminating pollution and environmentally friendly process for enzyme extraction via liquid biphasic flotation. This scope is achieved by utilising phase forming components that have recovery and recycling abilities to minimize the use of chemicals for enzyme extraction. A set of optimum conditions were identified which provides a high yield of lipase with 87.49 % and separation efficiency of 86.46%. From the recycling study, it is revealed 97.20% and 98.67% of Triton X-100 and xylitol respectively were recovered after five times of recycling and 75.87% lipase separation efficiency was obtained. Third objective is on the study of the integration process of fermentation and separation of lipase from Burkholderia cepacia using liquid biphasic flotation. Integration process exhibited high lipase separation efficiency reaching 92.29% and a yield of 95.73%. This study has proven the diversification of liquid biphasic flotation system in integration of upstream and downstream processes. Since liquid biphasic flotation system can be utilised for various type of biomolecules, the final study was done to examine the integration process of sonication and protein extraction from microalgae using sugaring-out effect. Various operating conditions were assessed for high separation efficiency and yield of protein. Maximum protein separation efficiency of 86.38% and yield with 93.33% were attained from this integration process. This study demonstrated that liquid biphasic flotation system could be integrated with ultrasound for protein separation. This thesis demonstrates the importance and diverse applications of liquid biphasic flotation for biomolecules extraction. This study has led to several novel discoveries of liquid biphasic flotation applications with economic downstream processes on an industrial scale. Keywords: Downstream processing, liquid biphasic flotation, aqueous two-phase systems, solvent sublation, biomolecule

Structure–selectivity relationship of a zirconia-based heterogeneous acid catalyst in the production of green mono- and dioleate product

A novel catalytic technique is vital to produce mono- and dioleate (GMO and GDO) from bioglycerol: a renewable resource and by-product of biodiesel. The advantage of this invention is the direct production of GMO and GDO through catalytic approach compared to the conventional method that requires transesterification and distillation processes. In this paper, glycerol esterification with oleic acid using a catalyst was experimented. The process was carried out over a hydrophobic mesoporous zirconia–silica heterogeneous acid catalyst (ZrO2–SiO2–Me&Et–PhSO3H) with three types of sulphated zirconia catalysts (SO42−/ZrO2) to produce high-selectivity GMO and GDO products. The catalytic performance of the hydrophobic ZrO2–SiO2–Me&Et–PhSO3H catalyst was benchmarked with that of SO42−/ZrO2 which was developed from three zirconium precursors. Results showed that the pore volume and hydrophobicity of the designed catalyst greatly could influence the product selectivity, thus enabling smaller substrates GMO and GDO to be dominated in the synthesis. This finding was supported by characterisation data obtained through N2 adsorption–desorption, X-ray diffraction and scanning electron microscopy. In addition, a good correlation was found between pore volume (pore size) and product selectivity. High pore volume catalyst favoured GDO production under identical reaction conditions. Pore volume and size can be used to control product sensitivity. The hydrophobicity of the catalyst was found to improve the initial reaction rate effectively

Reverse micellar system in protein recovery - a review of the latest developments

Reversed micellar system (RMS) is an innovative technique used for the isolation, extraction and purification of proteins and enzymes. Studies have demonstrated that RMS is an efficient purification technology for extracting proteins and enzymes from natural plant materials or fermentation broth. Lately, reverse micelles have wider biological applications and the ease of scaling up and the possibility for the continuous process have made RMS a vital purification technique in various fields. In this study, an extensive review of RMS with the current application in biotechnology is examined. This review provides insights into the fundamental principles, key variables and parameters of RMS. In addition, a comparative study of RMS with other liquid-liquid extraction techniques is also included. The present review aims to provide a general overview of RMS by summarising the research works, since the introduction of the technology to current development

Development of an efficient protein recovery system using liquid biphasic flotation

Extensive development of biotechnology over the past several decades has induced a great impact in the production of biological products in various industries. To date, major challenges in the biotechnology industry includes the production process of biomolecules with high purity and low cost, whilst retaining functionality. The conventional downstream processing of valuable bioproducts which are widely employed usually involves multi-processing steps, high energy and chemical consumption and often has a large influence on the cost of the finishing product. Therefore, the demand for cost-efficient and simple downstream processes has directed towards an intensive research for exploiting novel separation tool that can achieve high level of product purity with minimum number of processing stages and greener approach. The aim of this thesis is to develop a new separation method that has minimum number of steps, environmentally friendly and with the ability to achieve maximum level of product purity. Liquid biphasic flotation system is a novel technique which incorporates the principles of aqueous two-phase systems and mass transfer mode of solvent sublation. This system has been proposed as an ideal purification technique for separation, purification and concentration of biomolecules. Liquid biphasic flotation have been utilised previously to purify several biomolecules whilst maintaining their functionality. This thesis emphasised on extending the applications, improvising and to diversify liquid biphasic flotation technique as an efficient tool for downstream processing. This thesis has four objectives, in which all the objectives highlight on the usage of liquid biphasic flotation system for maximum biomolecules extraction. The initial part of liquid biphasic flotation application study is to investigate the effect of full and continuous recycling of alcohol and salt phase components in large scale liquid biphasic flotation system for lipase extraction. In this section, main focus was to optimize operating conditions for the recycling of both phase component and to investigate the competence of recycling phase components using liquid biphasic flotation system on a large scale. The liquid biphasic flotation system investigated is composed of 1-propanol and ammonium sulphate whereby both phase components went through complete recycling process. From the results obtained, it is exhibited that by reusing the bottom phase, separation efficiency of lipase was sustained beyond 77.33 % and yield with 80%. This study showed that the recovered phase components could be recycled effectively up to four cycles and able to produce a significantly high yield of lipase. Next study was on a novel approach of liquid biphasic flotation system for lipase recovery utilizing recycling phase components comprising surfactant and sorbitol. This novel method utilized Triton X-100 and xylitol for lipase extraction from Burkholderia cepacia. The scope of this study focuses on eliminating pollution and environmentally friendly process for enzyme extraction via liquid biphasic flotation. This scope is achieved by utilising phase forming components that have recovery and recycling abilities to minimize the use of chemicals for enzyme extraction. A set of optimum conditions were identified which provides a high yield of lipase with 87.49 % and separation efficiency of 86.46%. From the recycling study, it is revealed 97.20% and 98.67% of Triton X-100 and xylitol respectively were recovered after five times of recycling and 75.87% lipase separation efficiency was obtained. Third objective is on the study of the integration process of fermentation and separation of lipase from Burkholderia cepacia using liquid biphasic flotation. Integration process exhibited high lipase separation efficiency reaching 92.29% and a yield of 95.73%. This study has proven the diversification of liquid biphasic flotation system in integration of upstream and downstream processes. Since liquid biphasic flotation system can be utilised for various type of biomolecules, the final study was done to examine the integration process of sonication and protein extraction from microalgae using sugaring-out effect. Various operating conditions were assessed for high separation efficiency and yield of protein. Maximum protein separation efficiency of 86.38% and yield with 93.33% were attained from this integration process. This study demonstrated that liquid biphasic flotation system could be integrated with ultrasound for protein separation. This thesis demonstrates the importance and diverse applications of liquid biphasic flotation for biomolecules extraction. This study has led to several novel discoveries of liquid biphasic flotation applications with economic downstream processes on an industrial scale. Keywords: Downstream processing, liquid biphasic flotation, aqueous two-phase systems, solvent sublation, biomolecule

Biochemical identification of an alkalophilic lactic acid bacterium and testing its fermentative capacity

A newly isolated lactic acid bacteria (LAB) from laboratory of Biofuel R&D (UNIMAS) that has the capability to produce lactic acid (LA) using hydrolysed sago starch as the only carbon source by fermentation process was identified biochemically and it’s fermentative capacity was studied by using batch fermentation. The strain was tested to ferment for some carbohydrates contained biochemical kit test API 20E. The isolated LAB was used for LA fermentation production. The production biomass and LA production of the LAB strain were studied with two conditions which were at pH7 and pH8 at temperature of 37°C. From this study, the strain was identified as Enterococcus faecalis that is able to produce high concentration of LA at pH 8 which is very useful for industrial application. Fermentation at pH 8 produced a maximum dry cell weight of about 8.48g/L and 130.03g/L of LA, while less production was shown at pH 7 with 5.83g/L and 69g/L. It has been found that the growth of biomass and LA production for Enterococcus faecalis are influenced by the pH and the optimum pH for this strain is found to be at pH8

Integration process of fermentation and liquid biphasic flotation for lipase separation from Burkholderia cepacia

Liquid Biphasic Flotation (LBF) is an advanced recovery method that has been effectively applied for biomolecules extraction. The objective of this investigation is to incorporate the fermentation and extraction process of lipase from Burkholderia cepacia using flotation system. Initial study was conducted to compare the performance of bacteria growth and lipase production using flotation and shaker system. From the results obtained, bacteria shows quicker growth and high lipase yield via flotation system. Integration process for lipase separation was investigated and the result showed high efficiency reaching 92.29% and yield of 95.73%. Upscaling of the flotation system exhibited consistent result with the lab-scale which are 89.53% efficiency and 93.82% yield. The combination of upstream and downstream processes in a single system enables the acceleration of product formation, improves the product yield and facilitates downstream processing. This integration system demonstrated its potential for biomolecules fermentation and separation that possibly open new opportunities for industrial production

Green technology of liquid biphasic flotation for enzyme recovery utilizing recycling surfactant and sorbitol

Liquid biphasic flotation (LBF) system has been recognized as an efficient, green, economically sustainable and biocompatible technique for biomolecules separation and purification. The main drawbacks of the conventional process of biomolecules separation are expensive production cost, utilization of phase components that are inefficiently recycled and global pollution due to high chemical consumption and wastage. In this paper, a novel approach of LBF system for lipase recovery utilizing recycling phase components comprising surfactant and xylitol was investigated. The scope of this study focuses on pollution prevention as well as clean and environmentally friendly process for enzyme extraction via LBF. The green process proposed in this study uses phase-forming components that have recovery and recycling abilities for minimal use of chemicals for enzyme extraction. This novel method utilized Triton X-100 and xylitol for lipase extraction from Burkholderia cepacia. A few parameters were optimized to obtain high lipase separation efficiency and yield. Based on the ideal conditions of LBF, the average lipase separation efficiency and yield are 86.46 and 87.49%, correspondingly. Phase components recycling were proposed in order to reduce the chemicals consumption in LBF system. Upscaling of the recycling study exhibited consistent result with the laboratory scale. It was found that 97.20 and 98.67% of Triton X-100 and xylitol were recovered after five times of recycling and that a total of 75.87% of lipase separation efficiency was obtained. Recovery and recycling of phase components in the extraction process are established as the principal green chemistry method, which yields high separation efficiency and is economically feasible on an industrial scale

Thermal analysis of nigerian oil palm biomass with sachet-water plasticwastes for sustainable production of biofuel

Nigeria, being the world's largest importer of diesel-powered gen-sets, is expected to invest in bio-fuels in the future. Hence, it is important to examine the thermal properties and synergy of wastes for potential downstream resource utilization. In this study, thermal conversion as a route to reduce the exploding volume of wastes from sachet-water plastic (SWP) and oil palm empty fruit bunch (OPEFB) biomass was studied. Thermogravimetric (TGA) and subsequent differential scanning calorimeter (DSC) was used for the analysis. The effect of heating rate at 20 °C min-1 causes the increase of activation energy of the decomposition in the first-stage across all the blends (0.96 and 16.29 kJ mol-1). A similar phenomenon was seen when the heating rate was increased from 10 to 20 °C min-1 in the second-stage of decomposition. Overall, based on this study on the synergistic effects during the process, it can be deduced that co-pyrolysis can be an effective waste for energy platform. © 2019 by the authors

Extraction of proteins from microalgae using integrated method of sugaring-out assisted liquid biphasic flotation (LBF) and ultrasound

In this study, a simple sugaring-out supported by liquid biphasic flotation technique combined with ultrasonication was introduced for the extraction of proteins from microalgae. Sugaring-out as a phase separation method is novel and has been used in the extraction of metal ions, biomolecules and drugs. But, its functioning in protein separation from microalgae is still unknown. In this work, the feasibility of sugaring-out coupled with ultrasound for the extraction of protein was investigated. Primary studies were carried out to examine the effect of sonication on the microalgae cell as well as the separation efficiency of the integrated method. Effect of various operating parameters such as the concentration of microalgae biomass, the location of sonication probe, sonication time, ultrasonic pulse mode (includes varying ON and OFF duration of sonication), concentration of glucose, types of sugar, concentration of acetonitrile and the flow rate in the flotation system for achieving a higher separation efficiency and yield of protein were assessed. Besides, a large-scale study of the integration method was conducted to verify the consistency of the followed technique. A maximum efficiency (86.38%) and yield (93.33%) were attained at the following optimized conditions: 0.6% biomass concentration, 200 g/L of glucose concentration, 100% acetonitrile concentration with 5 min of 5 s ON/10 s OFF pulse mode and at a flow rate of 100 cc/min. The results obtained for large scale were 85.25% and 92.24% for efficiency and yield respectively. The proposed liquid biphasic flotation assisted with ultrasound for protein separation employing sugaring-out demonstrates a high production and separation efficiency and is a cost-effective solution. More importantly, this method provides the possibility of extending its application for the extraction of other important biomolecules

Hygro-Thermo-Mechanical Responses of Balsa Wood Core Sandwich Composite Beam Exposed to Fire

In this study, the hygro-thermo-mechanical responses of balsa core sandwich structured composite was investigated by using experimental, analytical and numerical results. These investigations were performed on two types of specimen conditions: dry and moisture saturation sandwich composite specimens that are composed of E-glass/polyester skins bonded to a balsa core. The wet specimens were immersed in distilled water at 40 °C until saturated with water. The both dry and wet sandwich composite specimens were heated by fire. The mass loss kinetic and the mechanical properties were investigated by using a cone calorimeter following the ISO 5660 standard and three-point bending mechanical test device. Experimental data show that the permeability and fire resistance of the sandwich structure are controlled by two composite skins. Obtained results allow us to understand the Hygro-Thermo-Mechanical Responses of the sandwich structured composite under application conditions. © 2019 by the authors