Biomolecules produced by living organisms can perform vast array of functions and play an important role in the cell. Important biomolecules such as lysozyme, bovine serum albumin (BSA), and bromelain are often studied by researchers due to their beneficial properties. The application of reverse micelles is an effective tool for protein separation from their sources due to the special system structure. Mechanisms of transferring biomolecules and factors that influence the extraction of biomolecules are reviewed in this paper. The enhancement of biomolecule extraction could be achieved depending on the properties of reverse micelles. This paper provides an overall review on lysozyme, BSA, and bromelain extraction by reverse micelle for various applications.
Adsorption of lysozyme on the dye-affinity nanofiber membranes was investigated in batch and dynamic modes. The membrane matrix was made of electrospun polyacrylonitrile nanofibers that were grafted with ethylene diamine (EDA) and/or chitosan (CS) for the coupling of Reactive Blue 49 dye. The physicochemical properties of these dye-immobilized nanofiber membranes (P-EDA-Dye and P-CS-Dye) were characterized microscopically, spectroscopically and thermogravimetrically. The capacities of lysozyme adsorption by the dye-affinity nanofiber membranes were evaluated under various conditions, namely pH, dye immobilized density, and loading flow rate. The adsorption of lysozyme to the dye-affinity nanofiber membranes was well fitted by Langmuir isotherm and pseudo-second kinetic models. P-CS-Dye nanofiber membrane had a better performance in the dynamic adsorption of lysozyme from complex chicken egg white solution. It was observed that after five cycles of adsorption-desorption, the dye-affinity nanofiber membrane did not show a significant loss in its capacity for lysozyme adsorption. The robustness as well as high dynamic adsorption capability of P-CS-Dye nanofiber membrane are promising for the efficient recovery of lysozyme from complex feedstock via nanofiber membrane chromatography.
A stirred fluidized bed (SFB) ion exchange chromatography was successfully applied in the direct recovery of recombinant enhanced green fluorescent protein (EGFP) from the unclarified Escherichia coli homogenate. Optimal conditions for both adsorption and elution processes were determined from the packed-bed adsorption systems conducted at a small scale using the clarified cell homogenate. The maximal adsorption capacity and dissociation constant for EGFP-adsorbent complex were found to be 6.3 mg/mL and 1.3 × 10-3 mg/mL, respectively. In an optimal elution of EGFP with 0.2 M of NaCl solution (pH 9) and at 200 cm/h, the recovery percent of the EGFP was approximately 93%. The performances of SFB chromatography for direct recovery of EGFP was also evaluated under different loading volumes (50-200 mL) of crude cell homogenate. The single-step purification of EGFP by SFB recorded in a high yield (95-98%) and a satisfactory purification factor (~3 folds) of EGFP from the cell homogenate at 200 rpm of rotating speed.
Ionic liquids (ILs) have emerged as an alternative solvent used in the bioprocessing of microalgae for recovery of valuable biomolecules. The aim of this work is to extract fucoxanthin from Chaetoceros calcitrants (C. calcitrans) by using the readily distillable CO2-based alkyl carbamate ILs. The degree of cell permeabilization was analysed by the quantification of extracted fucoxanthin and the analyses of cell surface morphology. Among the tested CO2-based alkyl carbamate ILs, diallylammonium diallylcarbamate (DACARB) extraction system gave the maximal yield of fucoxanthin at 17.51 mg/g under the optimal extraction conditions [90% (v/v), 3 min and 25 °C]. Moreover, the extracted fucoxanthin fraction exhibited the satisfactory antioxidant activities. The recyclability of DACARB was demonstrated in the multiple batches of fucoxanthin extraction. Hence, CO2-based alkyl carbamate ILs can prospectively substitute conventional organic solvents in the downstream processing of bioactive compounds from microalgae.
Nowadays, downstream bioprocessing industries inclines towards the development of a green and high efficient bioseparation technology. Betacyanins are presently gaining higher interest in the food science as driven by their high tinctorial strength and health promoting functional properties. In this study, a novel green integration process of liquid biphasic electric partitioning system (LBEPS) was proposed for betacyanins extraction from peel and flesh of red-purple pitaya. Initially, the betacyanins extraction using LBEPS with initial settings was compared with that of liquid biphasic partitioning system (LBPS), and the results revealed that both systems demonstrated a comparable betacyanins extraction. This was followed by further optimizing the LBEPS for better betacyanins extraction. Several operating parameters including operation time, voltage applied, and position of graphitic electrodes in the system were investigated. Moreover, comparison between optimized LBEPS and LBPS with optimized conditions of electric system (as post-treatment) as well as color characterization and antioxidant properties assessment were conducted. Overall, the betacyanins extraction employing the optimized LBEPS showed the significant highest values of betacyanins concentration in alcohol-rich top phase (C t ) and partition coefficient (K) of betacyanins from peel (99.256 ± 0.014% and 133.433 ± 2.566) and flesh (97.189 ± 0.172% and 34.665 ± 2.253) of red-purple pitaya. These results inferred that an optimal betacyanins extraction was successfully achieved by this approach. Also, the LBEPS with the peel and flesh showed phase volume ratio (V r ) values of 1.667 and 2.167, respectively, and this indicated that they have a clear biphasic separation. In addition, the peel and flesh extract obtained from the optimized LBEPS demonstrated different variations of red color as well as their antioxidant properties were well-retained. This article introduces a new, reliable, and effective bioseparation approach for the extraction of biomolecules, which is definitely worth to explore further as a bioseparation tool in the downstream bioprocessing.
Pursuing the current trend, the "green-polymers", polyhydroxyalkanoates (PHAs) which are degradable and made from renewable sources have been a potential substitute for synthetic plastics. Due to the increasing concern towards escalating crude oil price, depleting petroleum resource and environmental damages done by plastics, PHAs have gained more and more attractions, both from industry and research. From the view point of Escherichia coli, a microorganism that used in the biopolymer large scale production, this paper describes the backgrounds of PHA and summarizes the current advances in PHA developments. In the short-chain-length (scl) PHAs section, the study of poly[(R)-3-hydroxybutyrate] [P(3HB)] as model polymer, ultra-high-molecular-weight P(3HB) which rarely discussed, and P(3HB-co-3HV), another commercialized PHA polymer are included. Other than that, this review also shed some light on the new members of PHA family, lactate-based PHAs and P(3HP) with topics such as block copolymers and invention of novel biopolymers. Flexibility of microorganisms in utilizing different carbon sources to accumulate medium-chain-length (mcl) PHAs and lastly, the promising scl-mcl-PHAs with interesting properties are also discussed.
Cylinder-shaped NaY zeolite was used as an adsorbent for eradicating both heavy metal ions (Cu2+, Zn2+, Ni2+, and Co2+) and proteins from the waste streams. As a pseudo-metal ion affinity adsorbent, NaY zeolite was used in the capture of heavy metal ions in the first stage. The amount (molar basis) of metal ions adsorbed onto NaY zeolite decreased in the order of Cu2+ > Zn2+ > Co2+ > Ni2+. Bovine serum albumin (BSA) was utilized as a model of proteins used in the waste adsorption process by NaY zeolite. The adsorption capacities of NaY zeolite and Cu/NaY zeolite for BSA were 14.90 mg BSA/g zeolite and 84.61 mg BSA/g zeolite, respectively. Moreover, Cu/NaY zeolite was highly stable in the solutions made of 2 M NaCl, 500 mM imidazole or 125 mM EDTA solutions. These conditions indicated that the minimal probability of secondary contamination caused by metal ions and soluble proteins in the waste stream. This study demonstrates the potential of Cu/NaY zeolite complex as an efficient pseudo-metal chelate adsorbent that could remove metal ions and water-soluble proteins from wastewater concurrently.
The presence of reactive species in plasma-activated water is known to induce oxidative stresses in bacterial species, which can result in their inactivation. By integrating a microfludic chipscale nebulizer driven by surface acoustic waves (SAWs) with a low-temperature atmospheric plasma source, we demonstrate an efficient technique for in situ production and application of plasma-activated aerosols for surface disinfection. Unlike bulk conventional systems wherein the water is separately batch-treated within a container, we show in this work the first demonstration of continuous plasma-treatment of water as it is transported through a paper strip from a reservoir onto the chipscale SAW device. The significantly larger surface area to volume ratio of the water within the paper strip leads to a significant reduction in the duration of the plasma-treatment, while maintaining the concentration of the reactive species. The subsequent nebulization of the plasma-activated water by the SAW then allows the generation of plasma-activated aerosols, which can be directly sprayed onto the contaminated surface, therefore eliminating the storage of the plasma-activated water and hence circumventing the typical limitation in conventional systems wherein the concentration of the reactive species diminishes over time during storage, resulting in a reduction in the efficacy of bacterial inactivation. In particular, we show up to 96% reduction in Escherichia coli colonies through direct spraying with the plasma-activated aerosols. This novel, low-cost, portable and energy-efficient hybrid system necessitates only minimal maintenance as it only requires the supply of tap water and battery power for operation, and is thus suitable for decontamination in home environments.
A high-performance polyacid ion exchange (IEX) nanofiber membrane was used in membrane chromatography for the recovery of lysozyme from chicken egg white (CEW). The polyacid IEX nanofiber membrane (P-BrA) was prepared by the functionalization of polyacrylonitrile (PAN) nanofiber membrane with ethylene diamine (EDA) and bromoacetic acid (BrA). The adsorption performance of P-BrA was evaluated under various operating conditions using Pall filter holder. The results showed that optimal conditions of IEX membrane chromatography for lysozyme adsorption were 10% (w/v) of CEW, pH 9 and 0.1 mL/min. The purification factor and yield of lysozyme were 402 and 91%, respectively. The adsorption process was further scaled up to a larger loading volume, and the purification performance was found to be consistent. Furthermore, the regeneration of IEX nanofiber membrane was achieved under mild conditions. The adsorption process was repeated for five times and the adsorption capacity of adsorber was found to be unaffected.
This study aimed to develop a novel electrospun polyacrylonitrile (PAN) nanofiber membrane with the enhanced antibacterial property. The PAN nanofiber membrane was first subjected to alkaline hydrolysis treatment, and the treated membrane was subsequently grafted with chitosan (CS) to obtain a CS-modified nanofiber membrane (P-COOH-CS). The modified membrane was then coupled with different dye molecules to form P-COOH-CS-Dye membranes. Lastly, poly(hexamethylene biguanide) hydrochloride (PHMB) was immobilized on the modified membrane to produce P-COOH-CS-Dye-PHMB. Physical characterization studies were conducted on all the synthesized nanofiber membranes. The antibacterial efficacies of nanofiber membranes prepared under different synthesis conditions were evaluated systematically. Under the optimum synthesis conditions, P-COOH-CS-Dye-PHMB was highly effective in disinfecting a high concentration of Escherichia coli, with an antibacterial efficacy of approximately 100%. Additionally, the P-COOH-CS-Dye-PHMB exhibited an outstanding wash durability as its antibacterial efficacy was only reduced in the range of 5%-7% even after 5 repeated cycles of treatment. Overall, the experimental results of this study suggested that the P-COOH-CS-Dye-PHMB is a promising antibacterial nanofiber membrane that can be adopted in the food, pharmaceutical, and textile industries.
Over the past few decades, Escherichia coli (E. coli) remains the most favorable host among the microbial cell factories for the production of soluble recombinant proteins. Recombinant protein production (RPP) via E. coli is optimized at the level of gene expression (expression level) and the process condition of fermentation (process level). Presently, the reported studies do not give a clear view on the selection of methods employed in the optimization of RPP. Here, we have reviewed various optimization methods and their preferences with respect to the factors at expression and process levels to achieve the optimal levels of soluble RPP. With a greater understanding of these optimization methods, we proposed a stepwise methodology linking the factors from both levels for optimizing the production of soluble recombinant protein in E. coli. The proposed methodology is further explained through five sets of examples demonstrating the optimization of RPP at both expression and process levels.Key Points• Stepwise methodology of optimizing recombinant protein production is proposed.• In silico tools can facilitate the optimization of gene- and protein-based factors.• Optimization of gene- and protein-based factors aids host-vector selection.• Statistical optimization is preferred for achieving optimal levels of process factors.
Polyhydroxyalkanoates (PHAs), a class of renewable and biodegradable green polymers, have gained attraction as a potential substitute for the conventional plastics due to the increasing concern towards environmental pollution as well as the rapidly depleting petroleum reserve. Nevertheless, the high cost of downstream processing of PHA has been a bottleneck for the wide adoption of PHAs. Among the options of PHAs recovery techniques, aqueous two-phase extraction (ATPE) outshines the others by having the advantages of providing a mild environment for bioseparation, being green and non-toxic, the capability to handle a large operating volume and easily scaled-up. Utilizing unique properties of thermo-responsive polymer which has decreasing solubility in its aqueous solution as the temperature rises, cloud point extraction (CPE) is an ATPE technique that allows its phase-forming component to be recycled and reused. A thorough literature review has shown that this is the first time isolation and recovery of PHAs from Cupriavidus necator H16 via CPE was reported. The optimum condition for PHAs extraction (recovery yield of 94.8% and purification factor of 1.42 fold) was achieved under the conditions of 20 wt/wt % ethylene oxide-propylene oxide (EOPO) with molecular weight of 3900 g/mol and 10 mM of sodium chloride addition at thermoseparating temperature of 60°C with crude feedstock limit of 37.5 wt/wt %. Recycling and reutilization of EOPO 3900 can be done at least twice with satisfying yield and PF. CPE has been demonstrated as an effective technique for the extraction of PHAs from microbial crude culture.
Microalgae contribute up to 60% of the oxygen content in the Earth's atmosphere by absorbing carbon dioxide and releasing oxygen during photosynthesis. Microalgae are abundantly available in the natural environment, thanks to their ability to survive and grow rapidly under harsh and inhospitable conditions. Microalgal cultivation is environmentally friendly because the microalgal biomass can be utilized for the productions of biofuels, food and feed supplements, pharmaceuticals, nutraceuticals, and cosmetics. The cultivation of microalgal also can complement approaches like carbon dioxide sequestration and bioremediation of wastewaters, thereby addressing the serious environmental concerns. This review focuses on the factors affecting microalgal cultures, techniques adapted to obtain high-density microalgal cultures in photobioreactors, and the conversion of microalgal biomass into biofuels. The applications of microalgae in carbon dioxide sequestration and phycoremediation of wastewater are also discussed.
The formation of aqueous two-phase system (ATPS) with the environmentally friendly and recyclable ionic liquid has been gaining popularity in the field of protein separation. In this study, the ATPSs comprising N,N-dimethylammonium N',N'-dimethylcarbamate (DIMCARB) and thermo-responsive poly(propylene) glycol (PPG) were applied for the recovery of recombinant green fluorescent protein (GFP) derived from Escherichia coli. The partition behavior of GFP in the PPG + DIMCARB + water system was investigated systematically by varying the molecular weight of PPG and the total composition of ATPS. Overall, GFP was found to be preferentially partitioned to the hydrophilic DIMCARB-rich phase. An ATPS composed of 42% (w/w) PPG 1000 and 4.4% (w/w) DIMCARB gave the optimum performance in terms of GFP selectivity (1,237) and yield (98.8%). The optimal system was also successfully scaled up by 50 times without compromising the purification performance. The bottom phase containing GFP was subjected to rotary evaporation of DIMCARB. The stability of GFP was not affected by the distillation of DIMCARB, and the DIMCARB was successfully recycled in three successive rounds of GFP purification. The potential of PPG + DIMCARB + water system as a sustainable protein purification tool is promising.
Nanofiber membrane chromatography integrates liquid membrane chromatography and nanofiber filtration into a single-step purification process. Nanofiber membrane can be functionalised with affinity ligands for promoting binding specificity of membrane. Dye molecules are a good affinity ligand for nanofiber membrane due to their low cost and high binding affinity. In this study, a dye-affinity nanofiber membrane (P-Chitosan-Dye membrane) was prepared by using polyacrylonitrile nanofiber membrane modified with chitosan molecules and immobilized with dye molecules. Reactive Orange 4, commercially known as Procion Orange MX2R, was found to be the best dye ligand for membrane chromatography. The binding capacity of P-Chitosan-Dye membrane for lysozyme was investigated under different operating conditions in batch mode. Furthermore, desorption of lysozyme using the P-Chitosan-Dye membrane was evaluated systematically. The recovery percentage of lysozyme was found to be ~100%. The optimal conditions obtained from batch-mode study were adopted to develop a purification process to separate lysozyme from chicken egg white. The process was operated continuously using the membrane chromatography and the characteristic of the breakthrough curve was evaluated. At a lower flow rate (i.e., 0.1 mL/min), the total recovery of lysozyme and purification factor of lysozyme were 98.59% and 56.89 folds, respectively.
In this study, polyacrylonitrile (PAN) nanofiber membrane was prepared by an electrospinning technique. After alkaline hydrolysis, the ion-exchange nanofiber membrane (P-COOH) was grafted with chitosan molecules to form a chitosan-modified nanofiber membrane (P-COOH-CS). Poly(hexamethylene biguanide) (PHMB) was then covalently immobilized on P-COOH and P-COOH-CS to form P-COOH-PHMB and P-COOH-CS-PHMB, respectively. The nanofiber membranes were subjected to various surface analyses as well as to the evaluations of antibacterial activity against Escherichia coli. The optimal modification conditions for P-COOH-CS-PHMB were attained by water-soluble chitosan at 50 kDa of molecular weight, coupling pH at 7, and 0.05% (w/w) of PHMB. Within 10 min of treatment, the antibacterial rate was close to 100%. Under the similar conditions of antibacterial treatment, the P-COOH-CS-PHMB exhibited a better antibacterial efficacy than the P-COOH-PHMB. When the number of bacterial cells was increased by 2000 folds, both types of nanofiber membranes still maintained the antibacterial rate close to 100%. After five cycles of repeated antibacterial treatment, the antibacterial efficacy of P-COOH-PHMB was 96%, which was higher than that of P-COOH-CS-PHMB (83%). The experimental results revealed that the PHMB-modified nanofiber membranes can be suitably applied in water treatment such as water disinfection and biofouling control.
The purpose of this investigation is to evaluate the implementation of ultrasound-assisted liquid biphasic flotation (LBF) system for the recovery of natural astaxanthin from Haematococcus pluvialis microalgae. Various operating conditions of ultrasound-assisted LBF systems such as the position of ultrasound horn, mode of ultrasonication (pulse and continuous), amplitude of ultrasonication, air flowrate, duration of air flotation, and mass of H. pluvialis microalgae were evaluated. The effect of ultrasonication on the cellular morphology of microalgae was also assessed using microscopic analysis. Under the optimized operating conditions of UALBF, the maximum recovery yield, extraction efficiency, and partition coefficient of astaxanthin were 95.08 ± 3.02%, 99.74 ± 0.05%, and 185.09 ± 4.78, respectively. In addition, the successful scale-up operation of ultrasound-assisted LBF system verified the practicability of this integrated approach for an effective extraction of natural astaxanthin.
Haematococcus pluvialis is one of the most abundant sources of natural astaxanthin as compared to others microorganism. Therefore, it is important to understand the biorefinery of astaxanthin from H. pluvialis, starting from the cultivation stage to the downstream processing of astaxanthin. The present review begins with an introduction of cellular morphologies and life cycle of H. pluvialis from green vegetative motile stage to red non-motile haematocyst stage. Subsequently, the conventional biorefinery methods (e.g., mechanical disruption, solvent extraction, direct extraction using vegetable oils, and enhanced solvent extraction) and recent advanced biorefinery techniques (e.g., supercritical CO2 extraction, magnetic-assisted extraction, ionic liquids extraction, and supramolecular solvent extraction) were presented and evaluated. Moreover, future prospect and challenges were highlighted to provide a useful guide for future development of biorefinery of astaxanthin from H. pluvialis. The review aims to serve as a present knowledge for researchers dealing with the bioproduction of astaxanthin from H. pluvialis.
Polyacrylonitrile nanofiber membrane functionalized with tris(hydroxymethyl)aminomethane (P-Tris) was used in affinity membrane chromatography for lysozyme adsorption. The effects of pH and protein concentration on lysozyme adsorption were investigated. Based on Langmuir model, the adsorption capacity of P-Tris nanofiber membrane was estimated to be 345.83 mg/g. For the operation of dynamic membrane chromatography with three-layer P-Tris nanofiber membranes, the optimal operating conditions were at pH 9, 1.0 mL/min of feed flow rate, and 2 mg/mL of feed concentration. Chicken egg white (CEW) was applied as the crude feedstock of lysozyme in the optimized dynamic membrane chromatography. The percent recovery and purification factor of lysozyme obtained from the chromatography were 93.28% and 103.98 folds, respectively. Our findings demonstrated the effectiveness of P-Tris affinity nanofiber membrane for the recovery of lysozyme from complex CEW solution.
The polyacrylonitrile (PAN) nanofiber membrane was prepared by the electrospinning technique. The nitrile group on the PAN nanofiber surface was oxidized to carboxyl group by alkaline hydrolysis. The carboxylic group on the membrane surface was then converted to dye affinity membrane through reaction with ethylenediamine (EDA) and Cibacron Blue F3GA, sequentially. The adsorption characteristics of lysozyme onto the dye ligand affinity nanofiber membrane (namely P-EDA-Dye) were investigated under various conditions (e.g., adsorption pH, EDA coupling concentration, lysozyme concentration, ionic strength, and temperature). Optimum experimental parameters were determined to be pH 7.5, a coupling concentration of EDA 40 μmol/mL, and an immobilization density of dye 267.19 mg/g membrane. To understand the mechanism of adsorption and possible rate controlling steps, a pseudo first-order, a pseudo second-order, and the Elovich models were first used to describe the experimental kinetic data. Equilibrium isotherms for the adsorption of lysozyme onto P-EDA-Dye nanofiber membrane were determined experimentally in this work. Our kinetic analysis on the adsorption of lysozyme onto P-EDA-Dye nanofiber membranes revealed that the pseudo second-order rate equation was favorable. The experimental data were satisfactorily fitted by the Langmuir isotherm model, and the thermodynamic parameters including the free energy change, enthalpy change, and entropy change of adsorption were also determined accordingly. Our results indicated that the free energy change had a negative value, suggesting that the adsorption process occurred spontaneously. Moreover, after five cycles of reuse, P-EDA-Dye nanofiber membranes still showed promising efficiency of lysozyme adsorption.