Search Results (59)

The development of bioplastics, such as poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV), as sustainable alternatives to petroleum-based plastics, requires efforts to reduce their economic and environmental impacts. Aqueous Two-Phase System (ATPS) represents a sustainable alternative to isolate PHBV, as it is water-based. A polyethylene glycol (PEG8000)/phosphate salts-based ATPS was employed as a sustainable approach to isolate and purify PHBV produced by the haloarchaeon Haloferax mediterranei. The Taguchi design method was used to optimise an ATPS, integrating variables such as the concentration of commercial PEG8000 and phosphate salts, extraction temperature, system pH, and biomass-to-system volume ratio. Results revealed a maximum PHBV recovery of 80% with a purity of 93% under the following conditions: 20% of PEG8000, 20% of phosphate salts, pH of 7, 50 °C, and a 1:100 ratio. Furthermore, the potential recycling of ATPS components was studied to reduce the overall cost of the biopolymer isolation procedure. However, a significant decrease in the PHBV recovery was observed (52% when using recycled components). Finally, the use of PEG8000 from ethylene glycol (EG) polymerisation, aimed at the valorisation of EG obtained from other industrial processes, yielded comparable recovery and purity of PHBV (78% and 89%, respectively). Full article

Photobioreactor-based microalgae cultivation offers an integrated approach for nutrient-rich wastewater treatment while producing valuable biomass. One of the main microalgae components is proteins, making them a biotechnological target. In this work, to develop efficient and greener extraction methodologies, aqueous two-phase systems (ATPSs) based on natural deep eutectic solvents (NADESs) were evaluated for one-step protein extraction from microalgae cultivated in swine wastewater. Six ATPSs combining two NADES—betaine:levulinic acid (Bet:2LA) and choline chloride:urea (ChCl:2Urea)—and their individual components (Bet or ChCl) with phosphate salts were compared. Systems {NADES + K₃PO₄ + water} were characterized and reported for the first time. Protein recovery yield (PRY) and selectivity (protein-to-carbohydrate mass ratio, R) were assessed for three extraction times and at room temperature. The ATPS {Bet:2LA + K₃PO₄ + H₂O} achieved a PRY of 16.4% and remarkable selectivity after 30 min (R = 2.17 g·g⁻¹), with proteins concentrated in the NADES-rich phase, and negligible recovery in the salt-rich phase. Although the maximum PRY (18.2% at 120 min) was achieved with the precursor betaine, the ATPS with Bet:2LA at 30 min offered an optimal balance between efficiency and process time. With a water content of up to 50%, these systems underscore the potential of NADES-based ATPSs as sustainable platforms for protein recovery. Full article

Extracellular vesicles (EVs) secreted by Fusarium oxysporum play an important role in the process of its infestation of the host, but the in vitro research system for EVs of F. oxysporum (Fo-EVs) has not yet been improved, and the mechanism of its action remains unclear. In this study, particle size distribution, particle concentration, number of particles per unit of protein, number of particles per unit of mycelial biomass, and concentration of contaminated proteins were used as indicators to evaluate the yield and purity of Fo-EVs. The optimal method for Fo-EV preparation and extraction was screened by comparing liquid culture, solid culture, and solid culture with enzymatic cell wall hydrolysis. The optimal system for Fo-EVs separation and purification was screened by a pairwise combination of three primary methods (Ultracentrifugation (UC), Ultrafiltration (UF), and Polyethylene glycol precipitation method (PEG)) and two secondary methods (Size-exclusion chromatography (SEC) and Aqueous two-phase system (ATPS)), respectively. The protein composition was identified via mass spectrometry technology, followed by GO annotation and GO enrichment analysis using whole-genome proteins as the background. Based on these steps, a Fo-EV protein library was constructed to reveal Fo-EV’s most active biological functions. The results showed that solid culture combined with the UC-SEC method could effectively enrich Fo-EVs with a typical cup-shaped membrane structure. The obtained Fo-EVs had an average particle size of 253.50 nm, a main peak value of 200.60 nm, a particle concentration of 2.04 × 10¹⁰ particles/mL, and a particle number per unit protein of 1.09 × 10⁸ particles/μg, which were significantly superior to those of other combined methods. Through proteomic analysis, 1931 proteins enriched in Fo-EVs were identified, among which 350 contained signal peptides and 375 had transmembrane domains. GO enrichment analysis revealed that these proteins were mainly involved in cell wall synthesis, vesicle transport, and pathogenicity-related metabolic pathways. Additionally, 9 potential fungal EV markers, including Hsp70, Rho GTPase family, and SNARE proteins, were screened. This study constructed an isolation system and a marker database for Fo-EVs, providing a methodological and theoretical basis for in-depth analysis of the biological functions of Fo-EVs. Full article

Heterologous protein expression underpins the production of therapeutics, industrial enzymes, and diagnostic reagents, yet persistent challenges remain in enhancing yields, achieving correct folding, and reducing the costs and environmental burdens of downstream processing. Natural Deep Eutectic Solvents (NADESs)—a class of biocompatible, sustainable, and highly tunable solvents—have recently emerged as promising tools to overcome these limitations. This review systematically examines the intersection of recombinant protein production and NADES technology, assessing their applications across the full workflow, from host strain expression to purification and final formulation. Literature analysis highlights the potential of NADESs as media additives that mitigate cellular stress and improve soluble protein yields, as gentle solubilizing and refolding agents for inclusion bodies, as phase-forming components in aqueous two-phase systems for green purification, and as stabilizing excipients for long-term storage. Key constituents such as betaine, proline, urea, and arginine are identified as functional agents whose eutectic mixtures often deliver synergistic benefits that differ mechanistically from the action of the individual components. The integration of NADESs into recombinant protein production offers a path toward more sustainable and economically viable biomanufacturing. Critical gaps remain, including in vivo validation and techno-economic assessment. Future opportunities include high-throughput NADES screening and computational design of application-specific solvents. Full article

Aqueous two-phase systems (ATPSs) based on two incompatible polymers have recently garnered considerable attention due to the promising characteristics of all-aqueous emulsions for a range of applications. Recent investigations have indicated strong potential for interfacial assemblies of oppositely charged components in the stabilization of these emulsions. The formation of these confined assemblies is likely to depend on the size of the ATPS-constituting polymers; however, the role of this parameter remains to be elucidated. The primary objective of this study was to examine the effect of polyethylene oxide (PEO) molecular weight on the aging processes of PEO/dextran emulsions that are stabilized by the interfacial association of oppositely charged silica particles and polycations. It has been demonstrated that the stability of emulsions containing one high-molecular-weight dextran is significantly enhanced by increasing the size of the PEO molecules. Furthermore, a compression-induced bijel formation was observed in the ATPS with the largest molecular weight PEO sample. The observations were explained by the impact of the rheology of the aqueous phases on the aggregation, adsorption, and network formation capabilities of polycation/silica assemblies. These findings may facilitate the design of stable all-aqueous emulsions with optimal molecular weights for the ATPS-forming polymers. Full article

Sorting and isolating specific cells from heterogeneous populations are crucial for many biomedical applications, including drug discovery and medical diagnostics. Conventional methods such as Fluorescent Activated Cell Sorting (FACS) and Magnetic Activated Cell Sorting (MACS) face limitations in throughput, cost, and the ability to separate subtly different cells. Cell partitioning in Aqueous Two-Phase Systems (ATPSs) offers a biocompatible and cost-effective alternative, particularly when combined with continuous-flow microfluidics. However, it remains challenging to rationally design microfluidic ATPS devices and operation to separate cells with similar origin but different phenotypes. In this paper, using a model ATPS, polyethylene glycol (PEG)—Dextran (Dex) system, and model cells, human chondrocytes (hChs), and carboxylated polystyrene (PS) microparticles, we systematically characterized the material properties affecting cell partitioning in ATPSs, such as surface energies of the solutions and cells and solution viscosities. We developed an energy balance approach between interfacial energy and viscous dissipation to estimate the interface translocation dynamic of cells partitioning into the preferred phase. Combining the experimental measurement and the energy balance model, our calculation reveals that the time required for complete cell partitioning at the ATPS interface can be exploited in microfluidic ATPS devices to separate hChs with different phenotypes (healthy and diseased). We expect our dynamic energy approach to provide a basis and a design strategy for optimizing microfluidic ATPS devices to achieve the efficient separation of phenotypically similar cell populations and further expand the potential of microfluidic cell separation. Full article

Food waste remains a critical global concern, with approximately one third of all food produced being ultimately discarded. Therefore, it is urgent to develop new techniques for the effective repurpose of waste. Aqueous two-phase systems (ATPSs) stand out as a simple and biocompatible liquid–liquid extraction technique for the recovery of bioactive substances from food waste. In ATPSs, the target species partition between two liquid phases, according to affinity, which facilitates its extraction. This work aimed at extracting three polyphenols—chlorogenic acid (CA), ferulic acid (FA), and resveratrol (RV)—through the application of eco-friendly ATPSs composed of water, ethyl lactate (EL), and organic salts, namely disodium succinate (Na₂Succinate) and disodium tartrate (Na₂Tartrate), for future application in the valorisation of food waste. All partitions presented successful results, with values of partition coefficients (K) higher than 1 and extraction efficiencies (E) higher than 50%, indicating a preferential migration of the polyphenols to the top phase. The extraction of FA using the ATPS based on Na₂Tartrate presented the most promising results, with K = 19 ± 6 and E = (94.2 ± 0.9)% for the longest tie-line. Additionally, a comparison with previous works of the research group was drawn, with the extraction of RV exhibiting outstanding performance across all studied ATPSs. Therefore, the assessed ATPSs were shown to hold immense potential for the recovery of polyphenols. Full article

Aqueous two-phase systems (ATPSs) have long been used for the facile and rapid extraction of biomolecules of interest. Selective partitioning of DNA is useful for nucleic acid purification and in the design of novel sensing technologies. This paper investigates the partitioning of a plasmid within a poorly understood ATPS comprising the polymers poly(ethylene glycol) (PEG) 35 kDa and Ficoll 400 kDa. The focus is placed on dissecting the compositional effects of the ATPS—that is, whether set concentrations of physiological ions or the polymers themselves can tune DNA phase preference and strength of partitioning. The work here uncovers the antagonistic effects of magnesium and ammonium ions, as well as the role that phase-forming polymer partitioning plays in plasmid enrichment. Testing the ions in conjunction with different ATPS formulations highlights the complexity of the system at hand, prompting the exploration of DNA’s conformational changes in response to polymer and salt presence. The work presented here offers multiple optimization parameters for downstream applications of PEG–Ficoll ATPSs, such as in vitro transcription/translation-based biosensing, in which performance is heavily dependent upon nucleic acid partitioning. Full article

Pesticides used in agriculture can contaminate foods like fruits and vegetables, posing health risks to consumers and highlighting the need for effective residue monitoring. This study explores aqueous two-phase systems (ATPSs) comprising phosphonium or ammonium ionic liquids (ILs) combined with ammonium sulfate as an alternative pre-treatment method for extracting and concentrating the pesticides clomazone, pyraclostrobin, and deltamethrin from strawberry samples. Liquid–liquid equilibrium measurements for each ATPS were conducted, followed by extraction experiments to determine the most efficient systems for pesticide extraction. Results showed that all three pesticides migrated effectively to the IL-rich phase across the tested ATPSs. For the most promising system, tetrabutylphosphonium salicylate ([TBP][Sal]) with ammonium sulfate, extraction efficiencies for each pesticide exceeded 98% under optimized conditions for parameters such as pH, temperature, and ATPS composition. Application of this ATPS to strawberries resulted in significant pesticide preconcentration, reaching mg/L levels suitable for detection by liquid chromatography. The method’s sustainability was supported by green chemistry metrics, with AGREEprep and AGREE scores of 0.68 and 0.55, respectively, underscoring its alignment with eco-friendly practices. Full article

This work studies the partition of phenolic compounds, namely caffeic acid, syringic acid, vanillic acid, ferulic acid, and vanillin, in aqueous two-phase systems (ATPSs) formed by acetonitrile and deep eutectic solvents (DESs) based on choline chloride ([Ch]Cl) and carbohydrates (sucrose, d-glucose, d-mannose, arabinose, and d-xylose). The binodal curves built at 25 °C and 0.1 MPa using DES were compared with ATPS composed of [Ch]Cl and the same carbohydrates. The ability to form ATPS depends on the number and kind of hydroxyl groups in DES’s hydrogen-bond donor compound (carbohydrates). ATPS based on DES showed biphasic regions larger than the systems based on [Ch]Cl and carbohydrates alone due to the larger hydrophilicity of DES. The ATPS were used to study the partition of the phenolic compounds. For all the systems, the biomolecules preferentially partitioned to the acetonitrile-rich phase (K > 1), and the best recovery in the top phase ranged between 53.36% (caffeic acid) and 90.09% (vanillin). According to the remarkable results, DES-based ATPS can selectively separate ferulic acid and vanillin for the top phase and syringic, caffeic, and vanillic acids for the bottom phase, achieving a selectivity higher than two. Full article

In recent years, the increasing need for energy conservation and environmental protection has driven industries to explore more efficient and sustainable processes. Liquid–liquid extraction (LLE) is a common method used in various sectors for separating components of liquid mixtures. However, the traditional use of toxic solvents poses significant health and environmental risks, prompting the shift toward green solvents. This review deals with the principles, applications, and advantages of aqueous two-phase systems (ATPS) as an alternative to conventional LLE. ATPS, which typically utilize water and nontoxic components, offer significant benefits such as high purity and single-step biomolecule extraction. This paper explores the thermodynamic principles of ATPS, factors influencing enzyme partitioning, and recent advancements in the field. Specific emphasis is placed on the use of ATPS for enzyme extraction, showcasing its potential in improving yields and purity while minimizing environmental impact. The review also highlights the role of ionic liquids and deep eutectic solvents in enhancing the efficiency of ATPS, making them viable for industrial applications. The discussion extends to the challenges of integrating ATPS into biotransformation processes, including enzyme stability and process optimization. Through comprehensive analysis, this paper aims to provide insights into the future prospects of ATPS in sustainable industrial practices and biotechnological applications. Full article

Aqueous two-phase systems (ATPSs) are formed when two nonionic polymers, or a single polymer and salt, are mixed in water above a specific concentration, resulting in the emergence of phase separation and the formation of two immiscible aqueous phases. The solvent properties of the aqueous media within the phases of ATPSs rely on the specific composition of the co-solutes and the arrangement of the hydrogen bond network within each phase. Here, we investigate the anion effect of various sodium salts on the enhancement or destabilization of polyethylene glycol (PEG)–salt ATPS formation. Relatively small changes in ATPS ionic composition were shown to result in significant changes in solute partitioning. Additionally, we previously established that the arrangement of hydrogen bonds within the coexisting phases of ATPSs is different, as evidenced by Attenuated Total Reflection—Fourier Transform Infrared (ATR-FTIR) spectroscopic analysis of OH-stretch bands. The hydrogen bond arrangement was shown to abruptly change at concentrations below the threshold of macroscopic phase separation in the ATPSs. Using dynamic light scattering (DLS), we observed a correlation between these abrupt changes in H-bond arrangement and the detection of agglomerate formation in both polymer–polymer and polymer–salt systems. Full article

Aqueous two-phase extraction (APTE) stands out as an environmentally friendly technique for the separation of metal ions. The separation of Re (VII) and Mo (VI) in an aqueous solution was investigated using a novel aqueous two-phase system (ATPS) consisting of isodecanol polyoxyethylene ether (E-1006), ammonium sulfate, and water. A phase diagram of this system was developed, and the effects of pH, temperature, extraction time, the concentrations of E-1006 and (NH₄)₂SO₄, and metal ions on the separation of Re (VII) and Mo (VI) were examined. The results show that at pH 7.0, Mo (VI) had almost transformed into the (NH₄)₂SO₄-rich phase, while Re (VI) was extracted into the E-1006-rich phase. The increase in temperature induces a transition of Mo (VI) to the salt-rich phase, which is unfavorable for the extraction of Re (VII). The increase in the concentrations of E-1006 and (NH₄)₂SO₄ has a positive effect on the separation of rhenium and molybdenum. Overall, the ATPS consisting of 200 g/L of E-1006, 200 g/L of (NH₄)₂SO₄, and water yields an extraction efficiency of 97.2% for Re and a high separation factor of 2700 for Re (VII) and Mo (VI) from a mixture of 0.1 g/L of Re (VII) and 5 g/L of Mo (VI) at pH 7.0 and 323.15 K. Separation studies of the simulated leaching solution show that the extraction efficiency for Re (VI) is 99.1% and the separation factor of Re (VII) and Mo (VI) is 5100. Full article

Extractive fermentation is an in situ method for the production and recovery of biomolecules of interest. Aqueous two-phase systems (ATPS) allow the product to be recovered in one phase of the system, reducing unit operations in the bioprocess. Thermosensitive polymers such as EOPOs are an interesting alternative to be applied in ATPS. In this work, different EOPOs were tested in an extractive fermentation strategy with the green microalgae Neochloris oleoabundans to provide a basis for future implementations of these systems in microalgae bioprocesses. Extractive fermentations were carried out with two EOPOs of different molecular weights (3900 and 12,000 g/mol) at concentrations of 10% and 15% (w/v). The microalga was incubated axenically under two different sets of conditions for 21 and 45 days, respectively. Cell counts were performed, and cell growth curves were obtained. Additionally, a semi-continuous and batch extractive fermentation assay was performed. The extractive fermentation with EOPO showed lower cell growth and a longer adaptation time of the microalgae in the fermentation, and EPS production yields of up to 8–23 g/L were obtained. Extractive fermentation is an interesting method to be implemented in microalgae cultures; however, further conditions need to be explored to achieve an appropriate bioprocess. Full article

Bacteriocins may be used as natural preservatives and antibiotic substitutes in various foods. However, the multistep purification process of bacteriocins results in high production costs, which is an obstacle to their commercial use and consumer accessibility. In this study, a bacteriocin-like inhibitory substance (BLIS) from Bacillus spp. isolated from Korean fermented foods was partially purified using the aqueous two-phase system (ATPS). The maximum activity of the BLIS was achieved for ATPS composed of PEG 1000 (15% [w/w])/ammonium sulfate (20% [w/w])/sodium chloride (2% [w/w]), which caused BLIS activity to increase by 3 times with a 99% recovery rate. In particular, B. amyloliquefaciens Y138-6 BLIS exhibited broad antibacterial activity, high resistance to acid-base stress, and excellent thermal stability. This antibacterial substance inhibited the growth of aerobic bacteria and fungi on the walls of cheese and ripening rooms. These antibacterial properties have been shown to increase food safety and have the potential for use as biopreservatives. Moreover, considering that the execution of the ATPS requires only salts and PEG, it is a simple, environmentally friendly, and cost-effective process and may have industrial applications in the recovery of BLIS from fermentation broth. Full article