Two-phase System Research Articles

Use of genomics & proteomics in studying lipase producing microorganisms & its application

In biotechnological applications, lipases are recognized as the most widely utilized and versatile enzymes, pivotal in biocatalytic processes, predominantly produced by various microbial species. Utilizing omics technology, natural sources can be meticulously screened to find microbial flora which are responsible for oil production. Lipases are versatile biocatalysts. They are used in a variety of bioconversion reactions and are receiving a lot of attention because of the quick development of enzyme technology and its usefulness in industrial operations. This article offers recent insights into microbial lipase sources, including fungi, bacteria, and yeast, alongside traditional and modern methods of purification such as precipitation, immunopurification and chromatographic separation. Additionally, it explores innovative methods like the reversed micellar system, aqueous two-phase system (ATPS), and aqueous two-phase flotation (ATPF). The article deals with the use of microbial lipases in a variety of sectors, including the food, textile, leather, cosmetics, paper, detergent, while also critically analyzing lipase-producing microbes. Moreover, it highlights the role of lipases in biosensors, biodiesel production, tea processing, bioremediation, and racemization. This review provides the concept of the use of omics technique in the mechanism of screening of microbial species those are capable of producing lipase and also find the potential applications.

Direct hydrogenation of natural oils to fatty alcohols enabled by an alcoholysis/hydrogenation relay strategy and two-phase solvent system.

Direct hydrogenation of natural oils to fatty alcohols was achieved via a relay strategy involving alcoholysis of natural oils followed by hydrogenation of fatty acid esters. A two-phase system was used to avoid catalyst poisoning by glycerol. This protocol is suitable for plant oils, animal fats and waste cooking oil.

Obtaining Phenolic-Enriched Liquid Fractions and Compostable Pomace for Agriculture from Alperujo Using Standard Two-Phase Olive Oil Mill Equipment

Olive oil extraction by two-phase systems generates a by-product called “alperujo” which presents several difficulties for its valorization. The present work evaluated an industrial approach, based on the application of thermal treatments to alperujo followed by solid/liquid separation using standard two-phase olive oil mill equipment. Treatments consisted of the thermo-malaxation of alperujo at 70 °C for 45 or 90 min, with or without acid addition, followed by solid/liquid separation in an industrial decanter. The solid was characterized concerning subsequent use for composting, while total and hydrophilic phenolics were analyzed in liquid for their recovery. Additionally, a laboratory-scale trial to compare phenolic purification by ethylic acetate extraction with chromatographic procedures was also included. The static respiration index showed that solid fractions presented higher susceptibility to biodegradation processes than raw alperujo. The phenolic content of treated liquid fractions was higher than in raw alperujo. Total phenolics were maximum at the longest exposure time without acid addition, while hydrophilic phenolics were highest at the shortest exposure time in acidified samples. The use of non-ionic resins seemed attractive for obtaining highly concentrated phenolic fractions. The proposed thermal treatments can be applied in olive oil industries, allowing in situ pomace valorization and the recovery of phenolic-enriched liquid fractions.

Affinity-Controlled Partitioning of Biomolecules at Aqueous Interfaces and Their Bioanalytic Applications.

All-aqueous phase separation systems play essential roles in bioanalytical and biochemical applications. Compared to conventional oil and organic solvent-based systems, these systems are characterized by their rich bulk and interfacial properties, offering superior biocompatibility. In particular, phase separation in all-aqueous systems facilitates the creation of compartments with specific physicochemical properties, and therefore largely enhances the accessibility of the systems. In addition, the all-aqueous compartments have diverse affinities, with an important property known as partitioning, which can concentrate (bio)molecules toward distinct immiscible phases. This partitioning affinity imparts all-aqueous interfaces with selective permeability, enabling the controlled enrichment of target (bio)molecules. This review introduces the basic principles and applications of partitioning-induced interfacial phenomena in a typical all-aqueous system, namely aqueous two-phase systems (ATPSs); these applications include interfacial chemical reactions, bioprinting, and assembly, as well as bio-sensing and detection. The primary challenges associated with designing all-aqueous phase separation systems and several future directions are also discussed, such as the stabilization of aqueous interfaces, the handling of low-volume samples, and exploration of suitable ATPSs compositions with the efficient protocol.

Exchange of Thiol Ligands on CuInS2 Quantum Dots in High Boiling Solvents.

As-prepared quantum dots are covered with long-chain ligands to prevent aggregation. When quantum dots are used in optoelectronic devices such as solar cells and QD-LED, ligand exchange is necessary to replace long-chain ligands with short-chain ones to increase the efficiency of charge transfer from the quantum dots to the electrode. In this study, we successfully exchanged 1-dodecanethiol (DDT) ligands on CuInS2 quantum dots with mercaptopropionic acid (MPA) ligands by using a two-phase system of high-boiling hydrophilic and hydrophobic solvents. The ligand exchange to MPA was achieved by using diethylene glycol (DEG) or ethylene glycol (EG) as the hydrophilic phase and tetradecane as the hydrophobic phase. The ligand exchange rate increased with increasing ligand exchange temperature. When quantum dot sensitized solar cells (QDSSCs) were fabricated using the ligand-exchanged quantum dots, a positive correlation was observed between the progress of ligand exchange and short-circuit current density. This is because charge transfer efficiency from the quantum dots to the TiO2 electrode was improved by the ligand exchange. This work has shown that QDs synthesized using DDT can be applied to optoelectronic devices.

Anti-cancer activity of secreted aspartyl proteinase protein from Candida tropicalis on human cervical cancer HeLa cells

Cervical cancer is the fourth leading cause of cancer-related death in women worldwide. Microbial products are valuable sources of anti-cancer drugs. The aim of this study was to isolate secreted aspartyl proteinase protein from Candida tropicalis, investigate its inhibitory effect on human cervical cancer HeLa cells, and analyze the expression profiling of selected nuclear stem cell-associated transcription factors. The presence of secreted aspartyl proteinase protein was confirmed by the expression of SAP2 and SAP4 genes in C. tropicalis during the yeast-hyphae transition phase. The enzyme was purified and characterized using the aqueous two-phase system purification method, as well as proteolytic activity and the Bradford and micro-Kjeldahl methods, respectively. The in vitro anti-cancer properties of secreted aspartyl proteinase protein were evaluated by MTT assay, microscopic image analysis, nitric oxide (NO) scavenging activity assay, intracellular reactive oxygen species (ROS) production assay, and RT-qPCR. The isolated C. tropicalis secreted aspartyl proteinase protein exhibited proteinase activity with values ranging from 93.72 to 130.70 μg/mL and 89.88–127.72 μg/mL according to the Bradford and micro-Kjeldahl methods, respectively. Secreted aspartyl proteinase showed effective cytotoxicity in HeLa cell line leading to significant morphological changes. Additionally, it exhibited increased free radical scavenging activity compared to the untreated control group, as evidenced by nitrite inhibition. ROS production increased in HeLa cells exposed to secreted aspartyl proteinase. The expression levels of the nuclear stem cell-associated transcription factors octamer-binding transcription factor 4 (OCT4), sex determining region Y-box 2 (SOX2), and Nanog homeobox (NANOG) were significantly downregulated in the HeLa cells treated with secreted aspartyl proteinase. Secreted aspartyl proteinase protein may be a promising anti-cancer agent, as it effectively affects gene expression and may ultimately reduce the development and progression of cervical cancer. Targeting the genes related to nuclear stem cell-associated transcription factors may provide a novel amenable to cancer treatment.

Liquid-liquid equilibrium of the 1-butyl-3-methylimidazolium-based ionic liquids + sodium citrate + water systems at 298.15 K

Liquid-liquid extraction has long been the preferred option for process engineers to develop separation processes and it is used to separate and purify various biological products, metallic ions, colorants, pharmaceutical molecules, and small organic species. Ionic liquids (ILs) are special ecological solvents that can be used as possible substitutes for traditional organic solvents used for extraction and separation processes, becoming them more sustainable and safer. In combination with salts or inorganic polymers, they can form a liquid system of two phases, advantageous due to its wide polarity and viscosity. In this scenario, liquid–liquid equilibrium (LLE) data and phase diagrams for new aqueous two-phase systems containing ionic liquid ([BMIM][OH], [BMIM][Cl], [BMIM][Br]) (1) + sodium citrate (2) + water (3) were determined experimentally at 298.15 K. The ionic liquids were synthesized and characterized in the first part of the study. COSMO-RS was used as a tool to calculate polarity graphs of study molecules. In the second part, liquid–liquid equilibrium was studied using experimental measurements of the binodal curve and tie-lines, testing the quality of experimental data by mass balance, thermodynamic modeling using the NRTL model to correlate liquid–liquid equilibrium data and to do their phase stability. The accuracy of the model data results was verified by Gibbs energy topological mixing surface analysis and σ profiles. The experimental results indicated that the studied systems had type I liquid–liquid phase behavior. It was demonstrated that the ionic liquid [BMIM][OH] demonstrated better behavior to form Aqueous Two-Phase Systems mixed with sodium citrate and water. The results obtained by the thermodynamic modeling and stability with NRTL model weren’t satisfactory, because, besides the low deviations for the liquid phases compositions, the majority of calculated tie-lines was not tangent with the Gibbs surface, showing the limitations of the model for systems with electrolytes.

Thermodynamics and simulation of 3D crystals and phase transitions under external fields.

A field-supported multiphase kinetic Monte Carlo method previously applied to self-assembled trimesic acid molecular layers [Ustinov et al., Phys. Chem. Chem. Phys. 24, 26111 (2022)] was generalized to three-dimensional gas-liquid and gas-solid systems. This method allows us to calculate the thermodynamic potentials of the liquid and solid phases and then determine the parameters of the liquid-solid phase transition. In this study, the requirement that the gas phase be ideal was introduced as an additional condition. It was shown that in a two-phase system, the sum of the analytical expression for the chemical potential of an ideal gas and the external potential imposed on the gas phase exactly equals the chemical potential of the equilibrium crystal or liquid phase. For example, the coexistence of crystalline/liquid krypton and ideal gas has been considered. A comparison with previously published data has shown that the proposed approach provides the most accurate results for determining the parameters of phase transitions and fully satisfies the Gibbs-Duhem equation. This method does not impose any restrictions on the complexity or hardness of dense phases.

Microscopic insights into the aggregation dynamics behavior and tribological properties of graphene

The stable dispersion mechanism of graphene nanoparticles in solid-liquid two-phase lubrication systems still lacks in-depth insights into the dynamic aggregation process at the microscale. Herein, the effects of size difference and defect degree on the agglomeration dynamics behavior and tribological properties of graphene nanosheets were revealed by aggregation, pull-out, and friction molecular dynamics simulations. The results showed that for suspended graphene smaller than 100 nm, the increase in size and defect degree effectively improved the dispersion stability of nanosheets in base oil. Attributed to the rise in size improved solid-liquid chemical affinity and promoted out-of-plane deformation of nanosheets to enhance the steric hindrance effect. The increase in defect degree induced spontaneous wrinkling of nanosheets, leading to enhanced interlayer sliding resistance and thus inhibiting agglomeration behavior. Meanwhile, the rise in nanosheet size and degree of defects sufficiently bear the normal load, effectively avoiding the direct contact of asperities and thus improving the wear and temperature rise at the friction interface. However, friction-induced defective graphene curled to form a “carbon nanotube-like” structure, which reduced the oil film strength and led to deterioration of lubrication performance. The research results will provide certain microscale insights into the dispersion and friction of graphene in base oil.

Effect of flow patterns on recovery of invertases from Saccharomyces cerevisiae using polyethylene glycol/magnesium sulfate system in microchannels

The use of microdevices in extraction processes in aqueous two-phase systems (ATPS) has been studied for years in the literature. However, the effects of flow patterns still need to be adequately explored, especially for real samples. In this sense, the present study investigated the performance of microdevices in the recovery and partial purification of invertases with varying flow patterns. The invertases from Saccharomyces cerevisiae and the polyethylene glycol 1500/magnesium sulfate system were used in the experiments. Beyond the flow pattern, the study sought to maximize recovery and purification factor (PF) values by varying other operating conditions, including residence time, volumetric ratio, and channel diameter. It was observed that the short slug and long slug patterns showed better separation performance than the parallel pattern regardless of residence time and volumetric ratio. Increasing the residence time from 15 to 35 min increased the top recovery and PF values by two times, indicating that S. cerevisiae invertase partition is slow to the top phase. The maximum values of recovery (57.01 % ± 2.67 %) and PF (4.16 ± 0.06) were reached in the condition with long slug pattern, 35 min of operation, volumetric ratio of 2:1, and a channel diameter of 0.51 mm. The present study provides valuable information for integrating microdevices in a protocol for purifying invertase from S. cerevisiae and other proteins in real systems.

IMIDAZOLIUM-BASED IONIC LIQUIDS FOR THE SELECTIVE SEPARATION OF TOCOTRIENOL HOMOLOGUES FROM PALM FATTY ACID DISTILLATE VIA LIQUID-LIQUID EXTRACTION

Objective: Tocotrienols have now stepped into the limelight of vitamin E research and have proven to have some exceptional benefits that are not shared by their “older” tocopherol siblings. Unlike tocopherols, tocotrienols are able to inhibit cholesterol biosynthesis, have specific neuroprotective activities stronger antioxidant effects, antihypertensive and anti-cancer. The purpose of this study was to carry out selective separation of tocotrienols homologues from Palm Fatty Acid Distillate (PFAD) by liquid-liquid extraction, using Ionic liquids (ILs) as extractants in the presence of diluent. Methods: Four kinds of imidazolium-based ILs that used are 1-butyl-3-methylimidazolium chloride ([Bmim]Cl), 1-Butyl-3-methylimidazolium acetate ([Bmim]Ac), 1-Hexyl-3-methylimidazolium chloride ([Hmim]Cl) and 1-Ethyl-3-methylimidazolium chloride ([Emim]C]). The extraction is carried out by creating a two-phase system between ILs and PFAD in n-hexane. Quantification of extracted tocotrienols was performed using High-Performance liquid Chromatography (HPLC) with a C18 column, mobile phase methanol: water (97.5:2.5), flow rate 1 ml/min and Ultraviolet (UV) detector at 295 nm. Results: The results showed that the concentration (extraction efficiency) of tocotrienols extracted using ILs in order were [Bmim] Ac 1611.09 mg/Kg (75.41%) and [Hmim][Cl] 1603.39 mg/Kg (75.05%), [Bmim]Cl 1523.60 mg/Kg (71.32%) and [Emim]Cl 1174.24 mg/Kg (54.96%). Conclusion: [Bmim]Ac yielded the highest tocotrienols concentration and extraction efficiency.

Vibration-induced wall–bubble interactions under zero-gravity conditions

This work is devoted to a theoretical and numerical study of the dynamics of a two-phase system vapour bubble in equilibrium with its liquid phase under translational vibrations in the absence of gravity. The bubble is initially located in the container centre. The liquid and vapour phases are considered as viscous and incompressible. Analysis focuses on the vibrational conditions used in experiments with the two-phase system SF $_6$ in the MIR space station and with the two-phase system para-Hydrogen (p-H $_2$ ) under magnetic compensation of Earth's gravity. These conditions correspond to small-amplitude high-frequency vibrations. Under vibrations, additionally to the forced oscillations, an average displacement of the bubble to the wall is observed due to an average vibrational attraction force related to the Bernoulli effect. Vibrational conditions for SF $_6$ correspond to much smaller average vibrational force (weak vibrations) than for p-H $_2$ (strong vibrations). For weak vibrations, the role of the initial vibration phase is crucial. The difference in the behaviour at different initial phases is explained using a simple mechanical model. For strong vibrations, the average displacement to the wall stops when the bubble reaches a quasi-equilibrium position where the resulting average force is zero. At large vibration velocity amplitudes this position is near the wall where the bubble performs only forced oscillations. At moderate vibration velocity amplitudes the bubble average displacement stops at a finite distance from the wall, then large-scale damped oscillations around this position accompanied by forced oscillations are observed. Bubble shape oscillations and the parametric resonance of forced oscillations are also studied.

Optimization of cutting parameters in manufacturing of polymeric materials for flexible two-phase thermal management systems

Abstract This research study with an extensive literature review represents a comprehensive multi-criteria analysis for optimizing the cutting parameters in the manufacturing of flexible two-phase passive thermal management systems (FTP-TMS) using thermoplastic materials. Recognizing the critical role of thermoplastics in FTP-TMS due to their inherent flexibility and lightweight properties, this research focuses on the precision cutting of polypropylene, polyethylene, and polyvinyl chloride using CO2 laser technology. The study is structured into three distinct phases. Initially, an experimental setup was conducted to cut 2 mm thick thermoplastic materials with varying power and cutting speed parameters. Subsequently, the SWARA method was employed to weight the criteria, followed by the application of seven different multi-criteria decision-making (MCDM) methods for optimization. The final phase involved a detailed analysis of the outputs, including ranking, correlation, and sensitivity analyses. The findings indicate that cutting polypropylene with a 90 W power setting and a speed of 15 mm s−1 yields the most optimal results. This study fills a significant gap in the existing literature by providing a dedicated analysis for thermoplastics in FTP-TMS manufacturing. The insights gained are pivotal for standardizing manufacturing practices and enhancing the design and fabrication of flexible thermal management solutions, offering substantial benefits to sectors like electronics, aerospace, and automotive industries.

Advancements in Aqueous Two-Phase Systems for Enzyme Extraction, Purification, and Biotransformation.

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.

Thermo-responsive aqueous two-phase system for two-level compartmentalization

Hierarchical compartmentalization responding to changes in intracellular and extracellular environments is ubiquitous in living eukaryotic cells but remains a formidable task in synthetic systems. Here we report a two-level compartmentalization approach based on a thermo-responsive aqueous two-phase system (TR-ATPS) comprising poly(N-isopropylacrylamide) (PNIPAM) and dextran (DEX). Liquid membraneless compartments enriched in PNIPAM are phase-separated from the continuous DEX solution via liquid-liquid phase separation at 25 °C and shrink dramatically with small second-level compartments generated at the interface, resembling the structure of colloidosome, by increasing the temperature to 35 °C. The TR-ATPS can store biomolecules, program the spatial distribution of enzymes, and accelerate the overall biochemical reaction efficiency by nearly 7-fold. The TR-ATPS inspires on-demand, stimulus-triggered spatiotemporal enrichment of biomolecules via two-level compartmentalization, creating opportunities in synthetic biology and biochemical engineering.

A Concept of Local Coordination Number for the Characterization of Solute Clusters within Atom Probe Tomography Data.

Identifying clusters of solute atoms in a matrix of solvent atoms helps to understand precipitation phenomena in alloys, for example, during the age hardening of certain aluminum alloys. Atom probe tomography datasets can deliver such information, provided that appropriate cluster identification routines are available. We investigate algorithms based on the local composition of the neighborhood of solute atoms and compare them with traditional approaches based on the local solute number density, such as the maximum separation distance method. For an ideal solid solution, the pair correlation functions of the kth nearest solute atom in the coordination number representation are derived, and the percolation threshold and the size distribution of clusters are studied. A criterion for selecting optimal control parameters based on maximizing the phase separation by the degree of clustering is proposed for a two-phase system. A map of phase compositions accessible for cluster analysis is constructed. The coordination number approach reduces the influence of density variations commonly observed in atom probe tomography data. Finally, a practical cluster analysis technique applied to the early stages of aluminum alloy aging is described.

Langevin Equation in Heterogeneous Landscapes: How to Choose the Interpretation.

The Langevin equation is a common tool to model diffusion at a single-particle level. In nonhomogeneous environments, such as aqueous two-phase systems or biological condensates with different diffusion coefficients in different phases, the solution to a Langevin equation is not unique unless the interpretation of stochastic integrals involved is selected. We analyze the diffusion of particles in such systems and evaluate the mean, the mean square displacement, and the distribution of particles, as well as the variance of the time-averaged mean-square displacements. Our analytical results provide a method to choose the interpretation parameter from single-particle tracking experiments.

Enhancing experimental image quality in two-phase bubbly systems with super-resolution using generative adversarial networks

Fluid Dynamics is a key scientific field to multitudes of engineering applications. Experimental work in this field requires careful set-up and expensive image-capturing equipment, particularly when considering the finer details of complex phenomena. In this work, we study the application of super-resolution Generative Adversarial Networks (GANs) to achieve high-resolution results by upscaling lower-resolution experimental images.We train GANs proposed for natural images on a bubbly flow experimental Fluid Dynamics dataset and compare common super-resolution evaluation metrics to domain expert assessments of the upscaled images. We find that these models achieve promising results, as evaluated by experts, and transfer learning from natural images translates to better performance overall. Attention mechanisms are found to be particularly useful in recreating sharper details. On the other hand, traditional super-resolution evaluation metrics are found to align poorly with expert perception of quality, signaling the need for better systematic evaluation methodologies in this domain.

Acidic deep eutectic solvent as a greener medium for highly efficient extraction of anthocyanins from blackberry fruit: Optimization, stability and purification with two-aqueous phase method

Natural deep eutectic solvents (NADESs) are a sustainable and green solution for the recovery of food ingredients such as pigments. In this work, the extraction conditions to obtain the maximum anthocyanins from blackberry fruit were investigated using NADES composed of choline chloride-acetic acid (CHAC) as the green solvent. The central composite design was used conseidering molar ratio, water content, solvent ratio and extraction time as independent variables and total anthocyanin content (TAC), cyanidin-3-glucoside, cyanidin-3-rutinoside, pelargonidin-3-glucoside and cyanidin chloride as responses. The results showed that the independent variables significantly affected the responses. The optimum conditions for CHAC were 1:3.5 M, 40 %, 30 g and 15 min for molar ratio, water content, solvent ratio and extraction time, respectively. Under these conditions, the experimental responses were 131.54 ± 7.89 mg CGE/100 g, 1208.08 ± 13.76 mg/kg, 163.43 ± 1.72 mg/kg, 53.78 ± 1.37 mg/kg and 91.98 ± 0.62 mg/kg for TAC, cyanidin-3-glucoside, cyanidin-3-rutinoside, pelargonidin-3-glucoside and cyanidin chloride, respectively. The pH, viscosity and electric conductivity of CHAC were determined as 1.24 ± 0.03, 1.21 ± 0.17 and 7.50 ± 0.55 mPa and 4675.33 ± 57.74 µS.cm−1, respectively. After storing the NADES extract for 28 days in the dark, cyanidin-3-glucoside, cyanidin-3-rutinoside, pelargonidin-3-glucoside and cyanidin chloride remained 71.57 ± 0.64 %, 75.00 ± 0.26 %, 33.42 ± 0.41 % and 71.45 ± 0.21 %, respectively. Over the 28-day exposure of the NADES extract to light, the cyanidin-3-glucoside, cyanidin-3-rutinoside, pelargonidin-3-glucoside and cyanidin chloride remained 19.09 ± 0.10 %, 34.89 ± 2.28, 13.28 ± 0.25 and 30.97 ± 1.10, respectively. The application of the aqueous two-phase system resulted in the preliminary purification of the NADES extract, providing the highest purity in the top phase for cyanidin-3-glucoside (68.85 ± 1.05 %). The findings revealed that CHAC is a promising green medium for the industrial extraction of anthocyanins, and the aqueous two-phase is suitable for the purification of the anthocyanin-rich extract obtained with NADES.

A modified phase field method for the simulation of two-phase system in complex geometries

A modified phase field method for the simulation of two-phase system in complex geometries