Bottom Phase Research Articles

Driving forces for chymosin partitioning on the macromolecule-salt aqueous two phase system

Aqueous two-phase systems (ATPSs) are strategic liquid–liquid systems for extraction and purification of compounds. However, only a few studies have evaluated the thermodynamic parameters that allow comprehension of the partition process of different molecules. Here, we investigated the chymosin (Chy) partitioning behavior in macromolecule+salt+water ATPSs by obtaining the partition coefficient (KChy), Gibbs free energy change of transference (ΔGtr,Chy∞), enthalpy change of transference (ΔHtr,Chy∞), and entropy change of transference (ΔStr,Chy∞), at infinite dilution, and their dependence on the ATPS properties. Chy transfer from the bottom to the top phase of the ATPS was enthalpically driven, with −4.84kJmol−1<ΔHtr,Chy∞<−170.34kJmol−1 and −11.69Jmol−1K−1<ΔStr,Chy∞<−558.95Jmol−1K−1 characterizing an enthalpy–entropy compensation process; −1.36kJmol−1<ΔGtr,Chy∞<−3.77kJmol−1. ΔHtr,Chy∞ became more negative as the tie-line length increased, showing that specific macromolecule–Chy interactions determine the enzyme concentration in the top phase. The nature of the cation/anion, hydrophobic/hydrophilic balance of the top phase, and macromolecule molar mass influence the intermolecular interaction between Chy and top phase components, changing the enzyme partition behavior. Negative ΔStr,Chy∞ parameters were attributed to the Chy transfer from a higher (bottom phase) to the lower (top phase) configurational entropy region.

Glycolysis of high resilience flexible polyurethane foams containing polyurethane dispersion polyol

In the last years, high resilience polyurethane foams (HR foams) production has experienced an intensive growth as a consequence of their wide application field, causing an important increment of the generated waste. HR foams containing PU dispersions polyols are the last tendency in the HR foam industry, due to the improvement of the flame retardant properties and the emissions reduction of volatile compounds in comparison to the traditional graft polymeric HR polyols. In this work the extension of the glycolysis process to the recycling of this kind of foams has been carried out. Diethylene glycol (DEG), glycerol 99% PS and crude glycerol, coming from the biodiesel production, have been assayed as glycolysis agents. All of these glycolysis agents provided a split phase product with an upper phase mainly constituted by a traditional HR polyol and a bottom phase consisting of the excess of glycolysis agent and several reaction byproducts. However, the polyol content in the final product has been greater with the employment of glycerol. Moreover, flexible and rigid foams have been synthesized by using the recycled polyols or the glycolysis bottom phase, respectively. This way, it is achieved a global and sustainable recycling process for the valorization of two waste substances: the HR PU foam and the crude glycerol.

Partitioning of diuron in a novel aqueous two-phase system based on polyols and tetrahydrofuran

Diuron is an herbicide widely used in agriculture as a soil sterilant to control a large variety of weeds. This compound is considered moderately toxic and dangerous to the environment. This study examined the formation of an aqueous two-phase system (ATPS) based on tetrahydrofuran (THF) and polyols (glycerol, erythritol, xylitol, sorbitol and maltitol) in order to promote a simple recovery method that is low cost since diuron can be found in the uptake of water for human consumption. The binodal curves were determined at 25 °C and atmospheric pressure. The ability of the polyols to promote the formation of ATPS with THF preferably occurred in the growing direction of hydroxyl groups, glycerol (3-OH) to maltitol (9-OH). To explore the differences induced by the presence of the distinct polyols, the partition behavior of diuron was monitored by determining of the partition coefficient (KDIU) and recovery in the top phase (RT). In all systems, THF was in the top phase and the polyols in the bottom phase. Preferably, diuron was best partitioned to the top phase (KDIU ≈ 20 and RT ≈ 93) due the effect salting-out caused by maltitol on the diuron molecules.

Liquid-liquid equilibrium of poly (ethylene glycol) 4000 + sodium sulfate + urea/guanidine hydrochloride aqueous two-phase systems at different pH: Experimental results and thermodynamic modeling

Aqueous two-phase systems in the presence of denaturants (urea or guanidine hydrochloride) have been used for the initial recovery steps during protein refolding process. To investigate phase behavior of such systems, liquid-liquid equilibrium data for a poly (ethylene glycol) (PEG) 4000 + sodium sulfate + urea/guanidine hydrochloride (5% and 10% mass) aqueous two phase system was investigated at 25 °C and pH values of 5.3 and 9.2. The calibration plots for refractive index and conductivity were prepared to obtain the compositions of the top and bottom phase. It was observed that the heterogeneous region decreases with the increase in the denaturants concentration. The effect of pH, denaturants concentration, tie line length, and slope of tie line on the partition behavior of denaturants was examined. In this study, the modified UNIQUAC-FV and extended UNIQUAC models are used to calculate the phase equilibria of mention aqueous two phase systems. The values of average AAD% (partition coefficients) for modified UNIQUAC-FV and extended UNIQUAC are 3.48 and 13.80, respectively.

Enhanced extraction of oleoresin from ginger (Zingiber officinale) rhizome powder using enzyme-assisted three phase partitioning

Ginger (Zingiber officinale R.) is a popular spice used worldwide. The oleoresin consists of gingerols, shogaols and other non-volatiles as chief bioactive constituents. Three phase partitioning (TPP), a bioseparation technique, based on partitioning of polar constituents, proteins, and hydrophobic constituents in three phases comprising of water, ammonium sulphate and t-butanol, was explored for extraction of oleoresin and gingerols from dry powder. Parameters optimized for maximum recovery of gingerols and [6]-shogaol were ammonium sulphate concentration, ratio of t-butanol to slurry, solid loading and pH. Ultrasound and enzymatic pretreatments increased the yield of oleoresin and its phytoconstituents. Ultrasound pretreatment showed separation of starch in the bottom aqueous phase but is an additional step in extraction. Enzymatic pretreatment using accellerase increased the yield of [6]-, [8]-, [10]-gingerols and [6]-shogaol by 64.10, 87.8, 62.78 and 32.0% within 4h and is recommended. The efficacy of the enzymatic pretreatment was confirmed by SEM and FTIR.

Searching for prey in a three‐dimensional environment: hierarchical movements enhance foraging success in northern elephant seals

Summary Foraging theory predicts that predators adjust their movements according to the spatial distribution of prey. Since prey is often patchily distributed, area‐restricted search (ARS) behaviour, characterized by sinuous search paths of predators with increased turning frequency, should be effective in foraging. However, it remains unclear whether ARS behaviour actually enhances foraging success in free‐ranging animals, especially in marine animals that forage in a three‐dimensional (3D) environment. Here, we reconstructed 3D dive paths of a highly pelagic marine predator, the northern elephant seal (n = 3), with multisensor data loggers that recorded depth, tri‐axis acceleration, tri‐axis magnetism and swim speed. We identified spatial scales of volume‐restricted search (VRS, termed for 3D ARS) behaviour using spherical first‐passage time analysis on 3D dive paths, accompanied with quantifying feeding rates in VRS by using mandible accelerometers that recorded feeding events. Seals exhibited VRS behaviour at two spatial scales (radius of spheres): small‐VRS (8–10 m) and large‐VRS (17–19 m). Most feeding events occurred in VRS zones (78 and 86% for small and large‐VRS, respectively), although VRS accounted for a small proportion of bottom phase of dives in distance travelled. This suggests a strong link between VRS behaviour and foraging success. There was a hierarchical structure to the VRS; most small‐VRS (95%) were nested within large‐VRS (i.e. nested VRS). Importantly, nested VRS had significantly higher feeding rates than non‐nested VRS, because nested VRS contained small‐ and large‐VRS with higher and lower feeding rates, respectively. These results suggest that seals forage on mesopelagic prey in a hierarchical patch system where high‐density patches at small scales are nested within low‐density patches at larger scales. We demonstrated that seals employed scale‐dependent, hierarchical 3D movements and that underwater fine‐scale sinuous movements (i.e. VRS) were strongly linked to higher foraging success, particularly within nested VRS zones. We suggest that seals enhanced foraging success by employing hierarchical movements that possibly reflect the hierarchical property of prey distribution. Although recent studies advocate that optimal searching behaviour would be scale‐independent (e.g. Lévy walk), our study suggests that scale‐dependent processes are important components of successful foraging behaviour.

Selective partition of caffeine from coffee bean and guaraná seed extracts using alcohol–salt aqueous two-phase systems

ABSTRACTThis work proposes the application of flexible alcoholic aqueous two-phase systems to manipulate the partition of caffeine. An optimization study was performed the caffeine partitioning towards top phase (60.0 wt% 2-propanol + 20.0 wt% K3PO4 at 303 K – Kcaf = 3.4 and RT = 89.1%) and bottom phase (60.0 wt% methanol + 17.5 wt% K2HPO4/KH2PO4 at 278 K – Kcaf = 0.06 and RB = 81.1%). These processes were evaluated by applying them to real biomass as guaraná seeds (purification factor (PF) = 6.59-fold) and coffee beans (PF = 3.24-fold) for the 2-propanol and K2HPO4/KH2PO4 system.

Partitioning of cerrena unicolor laccase activity in an aqueous two-phase system

Abstract Culture supernatant containing laccase produced by Cerrena unicolor strain was used to examine laccase partitioning between phases in an aqueous two-phase system. The investigated system consisted of polyethylene glycol 3000 and sodium phosphate buffer adjusted to pH = 7. Influence of several parameters on partitioning was measured, including phase forming components’ concentrations, tie line lengths, phase volume ratio, supernatant dilution, process temperature and halogen salt supplementation. Partitioning coefficients up to 78 in the bottom phase were achieved with yields of over 90%. Tie line length and phase volume ratio had significant effect on enzyme partitioning.

Characterization of partitioning behaviors of immunoglobulin G in polymer-salt aqueous two-phase systems

The partitioning behavior of immunoglobulin G (IgG) in the aqueous two-phase system (ATPS) composed of poly(ethylene glycol) (PEG) and phosphate was studied. The parameters of ATPS exhibiting the pronounced effects on the partitioning behavior of IgG include phase composition, PEG molecular weight, and the addition of sodium chloride (NaCl). The accumulation of IgG at the interface of the ATPS increased drastically as the tie-line length (TLL) was increased. This trend was correlated with a linear relationship relating the natural logarithm of interfacial partition coefficient (ln G) to the difference of PEG concentration between the top phase and the bottom phase (Δ[PEG]), and a good fit was obtained. An attempt was made to correlate the natural logarithm of partition coefficient (ln K) to the presence of NaCl with the proposed linear relationship, ln K=α″ ln [Cl-]+β″. The proposed relationship, which serves as a better description of the underlying mechanics of the protein partitioning behavior in the polymer-salt ATPS, provides a good fit (r2>0.95) for the data of IgG partitioning. An optimum recovery of 99.97% was achieved in an ATPS (pH 7.5) composed of 14.0% (w/w) PEG 1450, 12.5% (w/w) phosphate and 5.0% (w/w) NaCl.

Purification of a fibrinolytic protease from Mucor subtilissimus UCP 1262 by aqueous two-phase systems (PEG/sulfate).

A fibrinolytic protease from M. subtilissimus UCP 1262 was recovered and partially purified by polyethylene glycol (PEG)/sodium sulfate aqueous two-phase systems (ATPS). The simultaneous influence of PEG molar mass, PEG concentration and sulfate concentration on the enzyme recovery was first investigated using a 2(3) full factorial design, and the Response Surface Methodology used to identify the optimum conditions for enzyme extraction by ATPS. Once the best PEG molar mass for the process had been selected (6000g/mol), a two-factor central composite rotary design was applied to better evaluate the effects of the other two independent variables. The fibrinolytic enzyme was shown to preferentially partition to the bottom phase with a partition coefficient (K) ranging from 0.2 to 0.7. The best results in terms of enzyme purification were obtained with the system formed by 30.0% (w/w) PEG 6000g/mol and 13.2% (w/w) sodium sulfate, which ensured a purification factor of 10.0, K of 0.2 and activity yield of 102.0%. SDS-PAGE and fibrin zymography showed that the purified protease has a molecular mass of 97kDa and an apparent isoelectric point of 5.4. When submitted to assays with different substrates and inhibitors, it showed selectivity for succinyl-l-ala-ala-pro-l-phenylalanine-p-nitroanilide and was almost completely inhibited by phenylmethylsulfonyl fluoride, behaving as a chymotrypsin-like protease. At the optimum temperature of 37°C, the enzyme residual activity was 94 and 68% of the initial one after 120 and 150min of incubation, respectively. This study demonstrated that M. subtilissimus protease has potent fibrinolytic activity compared with similar enzymes produced by solid-state fermentation, therefore it may be used as an agent for the prevention and therapy of thrombosis. Furthermore, it appears to have the advantages of low cost production and simple purification.

Rising of a single Taylor drop in a stagnant liquid—2D laminar flow and axisymmetry limits

A numerical (computational fluid dynamics (CFD)) study concerning the rise of individual liquid Taylor drops through vertical columns of stagnant heavier liquids is presented in this paper. CFD simulations were performed in Ansys Fluent, using its implementation of volume of fluid method, assuming the flow to be axisymmetric and laminar. Different physical conditions were tested, corresponding to different combinations of relevant dimensionless parameters and the numerical method was validated through experimental data available in the literature. The viscosity ratio between the lighter and the heavier liquid was within the range 0.01–40 and Eötvös number was between 8 and 30. Morton number was within the interval of 2.32 × 10−6–100. Froude number results were compared to data from a literature correlation. The accordance is acceptable for the ranges studied. Velocity profiles in significant regions are reported (drop nose, drop bottom and continuous phase liquid film). The influence of changing one dimensionless parameter alone was assessed. For small and large viscosity ratios, axisymmetric behavior is not a valid assumption.

Separation of α-Lactalbumin and β-Lactoglobulin in Whey Protein Isolate by Aqueous Two-phase System of Polymer/Phosphate

Abstract Poly(ethylene glycol-ran-propylene glycol)monobutyl ether/phosphate aqueous two-phase system (ATPS) coupled with HPLC was developed for the separation of α -lactalbumin and β -lactoglobulin in whey protein isolate (WPI). The main parameters relevant to this method were investigated and optimized. The effects of pH value, volume ratio of the solution of polymer and KH 2 PO 4 , concentration of NaCl and WPI on the partition of α -lactalbumin and β -lactoglobulin in whey protein isolate were investigated. The results showed that an efficient separation of both proteins in whey protein isolate was achieved by an ATPS comprising of 4 mL of poly(ethylene glycol-ran-propylene glycol) monobutyl ether solution (40%, w/w ) and 4 mL of 15.5% KH 2 PO 4 solution (15.5%, w/w ). When the addition of NaCl was 0.40 g per 10 mL, the concentration of WPI was 1 mg mL −1 and pH = 4.0, α -lactalbumin was separated to the top phase while β -lactoglobulin to the bottom phase with the yield of 98.23% and 96.56%, respectively.

Hydrodynamic modeling of an industrial turbulent fluidized bed reactor with FCC particles

This study presents a CFD modeling of the hydrodynamics in an industrial turbulent fluidized bed reactor with FCC particles. Considering the mesoscale structure of particle clusters and the impact of reactor temperatures, a gas-solid drag model based on the equivalent diameter of particle clusters is proposed. A CFD model is developed, the model parameters are evaluated, and the gas-solid flow behaviors are explored. Compared with the conventional drag models, the proposed model gives a reasonable hydrodynamic prediction in the industrial turbulent fluidized bed reactor. The hydrodynamics show more sensitive to the drag models than to the flow types, the kinetics theories and the restitution coefficients. There exist two coexisting flow regions in the industrial turbulent fluidized bed: a bottom dense, bubbling region and an upper dilute, dispersed flow region. Moreover, the bed density in the dense phase is much lower than those in the cold experiments. In the dilute phase, particles ascend in the center and descend near the wall, showing a core-annulus flow. In the bottom dense phase, some vortex flows appear, leading to the non-uniform distribution of particle velocity and solid holdup. With increasing the superficial gas velocity, the bed surface rises; and the bed density decreases in the bottom dense phase and increases in the upper dilute phase.

Foraging segregation of two congeneric diving seabird species breeding on St. George Island, Bering Sea

Abstract. Subarctic environmental changes are expected to affect the foraging ecology of marine top predators, but the response to such changes may vary among species if they use food resources differently. We examined the characteristics of foraging behavior of two sympatric congeneric diving seabird: common (Uria aalge: hereafter COMUs) and thick-billed (U. lomvia: hereafter TBMUs) murres breeding on St. George Island, located in the seasonal sea-ice region of the Bering Sea. We investigated their foraging trip and flight durations, diel patterns of dive depth, and underwater wing strokes, along with wing morphology and blood stable isotope signatures and stress hormones. Acceleration–temperature–depth loggers were attached to chick-guarding birds, and data were obtained from 7 COMUs and 12 TBMUs. Both species showed similar mean trip duration (13.2 h for COMUs and 10.5 h for TBMUs) and similar diurnal patterns of diving (frequent dives to various depths in the daytime and less frequent dives to shallow depths in the nighttime). During the daytime, the dive depths of COMUs had two peaks in shallow (18.1 m) and deep (74.2 m) depths, while those of TBMUs were 20.2 m and 59.7 m. COMUs showed more frequent wing strokes during the bottom phase of dives (1.90 s−1) than TBMUs (1.66 s−1). Fish occurred more frequently in the bill loads of COMUs (85 %) than those of TBMUs (56 %). The δ15N value of blood was significantly higher in COMUs (14.5 ‰) than in TBMUs (13.1 ‰). The relatively small wing area (0.053 m2) of COMUs compared to TBMUs (0.067 m2) may facilitate their increased agility while foraging and allow them to capture more mobile prey such as larger fishes that inhabit deeper depths. These differences in food resource use may lead to the differential responses of the two murre species to marine environmental changes in the Bering Sea.

Liquid-liquid partition of perchlorate ion in the aqueous two-phase system formed by NaNO3 + Poly(ethylene glycol) + H2O

The partitioning behavior of the perchlorate ion on the aqueous two phase system (ATPS) NaNO3 + poly(ethylene glycol) 4000 (PEG 4000) + H2O was studied at T=(288.15, 298.15 and 308.15) K. Two salts with different cation were used, NaClO4 and KClO4. The partition coefficient for the perchlorate ion in the ATPS was investigated as a function of temperature, concentration gradient of the PEG between the phases and perchlorate concentration. It was found that as temperature increases the partition coefficient values increases, similar behaviours were observed when the concentration gradient of PEG increases and when the perchlorate concentration increases. The thermodynamic parameters obtained by the thermodynamic relations show that the transfer of anions from the bottom phase to the top phase is entropic driven and that the partition process is endothermic. In addition, the Chen-NRTL model was applied to correlate the experimental data of the partition coefficients, for this purpose, the Flory-Huggins and Pitzer-Debye-Hückel expressions were used to take into account the entropy of mixing molecules and the long-range electrostatic interactions of the ions, respectively. A good agreement between experimental data and correlated results was obtained.

Aqueous two-phase system purification for superoxide dismutase induced by menadione from Phanerochaete chrysosporium

In the present work, the partitioning behavior of menadione-induced superoxide dismutase (SOD; EC 1.15.1.1), an antioxidant enzyme that has various applications in the medical and cosmetic industries, from the white rot fungus Phanerochaete chrysosporium has been characterized on different types of aqueous two-phase systems (ATPSs) (poly(ethylene glycol)/polypropylene glycol (PEG/PPG)-dextran, PEG-salt and PPG-salt). PEG-salt combinations were found most optimal systems for the purification of SOD. The best partition conditions were found using the PEG-3350 24% and K2HPO4 5% (w/w) with pH 7.0 at 25 °C. The partition coefficient of total SOD activity and total protein concentration observed in this system were 0.17 and 6.65, respectively, with the recovery percentage as 78.90% in the bottom phase and 13.17% in the top phase. The highest purification fold for SOD from P. chrysosporium was found as 6.04 in the bottom phase of PEG 3350%24 – K2HPO4%5 (w/w) system with pH 7.0. SOD purified from P. chrysosporium was determined to be a homodimer in its native state with a molecular weight of 60 ± 4 kDa. Consequently, simple and only one step PEG-salt ATPS system was developed for SOD purification from P. chrysosporium.

Combined process of reaction, extraction, and purification of lutein in marigold flower by isopropanol–KOH aqueous two-phase system

ABSTRACTThis paper established a viable process for the simultaneous reaction, extraction, and primary purification of lutein in marigold flower by the isopropanol–KOH aqueous two-phase system (ATPS). In this process, the liposoluble lutein esters were translated into water-soluble lutein by saponification reaction in ATPS and distributed to the top phase. Meanwhile, the water-soluble flavonoids in marigold were also extracted and distributed to the bottom phase. The concentrations of phase-forming isopropanol and KOH, the added mass of marigold flower powder, temperature, and time were investigated to evaluate their effects on the extraction and partition behaviour of lutein and flavoniods in ATPS. Under the optimal conditions (38% isopropanol, 18% KOH, 0.05 g of marigold flower powder, 500°C and 1.5 h extraction time), the leaching efficiency, extraction efficiency, and partition coefficient of lutein were 84.64%, 86.73%, and 5.115, respectively, and at this point, the saponification rate of lutein reached 93.98%. Meanwhile, the leaching efficiency, extraction efficiency, and partition coefficient of flavoniods were 96.68%, 78.31%, and 0.2167, respectively. This method shows the superiority to the conventional one for it can not only efficiently extract lutein in marigold flower, but also achieve a high saponification rate of lutein esters and simultaneously separate two major pigments.

Stabilization mechanisms of oil-in-water emulsions by Saccharomyces cerevisiae

A multiphase system is commonly formed during the oil production by microbial route, which can lead to stable emulsions hindering product recovery. Thus, this study aimed to investigate the mechanisms of emulsion stabilization by the yeast Saccharomyces cerevisiae in order to contribute with processes development of oil production by fermentation. A model system using hexadecane as oil phase and yeast suspension as aqueous phase was used to prepare O/W emulsions. The yeast was subjected to different treatments as inactivation (autoclaving) and washing before to be resuspended in water. The washing water (water from the first washing) and suspension of commercial yeast (active) were also used as aqueous phase. After 24h of preparation, the emulsions separated into three phases: top (cream), intermediate, and bottom phase. The top or cream phase was a concentrated emulsion that kept stable during seven days, except for those prepared from washed yeast that were stable only for a short period of time. Emulsions prepared with washed yeast showed higher cell adhesion to the droplets interface, which implied in a higher amount of yeast into the cream phase in comparison to other formulations. Therefore, yeast cells adhesion plays a role on emulsion stability, but the greater contribution was provided by cell material dispersed into the aqueous phase, regardless of cell viability.

Partitioning and purification of menadione induced NAD(P)H oxidase from Phanerochaete chrysosporium in aqueous two-phase systems

Abstract The partitioning behavior of menadione-induced NAD(P)H oxidase from the white rot fungus Phanerochaete chrysosporium has been studied in various poly(ethylene glycol) (PEG)-salt aqueous two-phase systems (ATPSs). The effects of the molecular weight of the PEG, system composition, and pH of the system on NAD(P)H oxidase partitioning were investigated at 25 °C. The best partition parameters were observed in the PEG 2000 18%–K 2 HPO 4 13%–NaCl 0.5% (w/w, pH 7.0) system; the partition coefficient of total NAD(P)H oxidase activity ( K e ) of the system was 0.232. While the recovery in the bottom phase ( R b ) was 87.644%, this value was 20.350% in the top phase of the optimized system. The purification fold was found as 8.331 in the bottom phase. NAD(P)H oxidase purified from P. chrysosporium by the PEG–K 2 HPO 4 two-phase system was determined to be a homodimer in its native state with a molecular weight of 116 kDa.

An integrated practical implementation of continuous aqueous two-phase systems for the recovery of human IgG: From the microdevice to a multistage bench-scale mixer-settler device.

Aqueous two-phase systems (ATPS) are a liquid-liquid extraction technology with clear process benefits; however, its lack of industrial embracement is still a challenge to overcome. Antibodies are a potential product to be recovered by ATPS in a commercial context. The objective of this work is to present a more integral approach of the different isolated strategies that have arisen in order to enable a practical, generic implementation of ATPS, using human immunoglobulin G (IgG) as experimental model. A microfluidic device is used for ATPS parameters preselection for product recovery. ATPS were continuously operated in a mixer-settler device in one stage, multistage and multistage with recirculation configuration. Single-stage pure IgG extraction with a polyethylene glycol (PEG) 3350-phophates ATPS within continuous operation allowed a 65% recovery. Further implementation of a multistage platform promoted a higher particle partitioning reaching a 90% recovery. The processing of IgG from a cell supernatant culture harvest in a multistage system with top phase recirculation resulted in 78% IgG recovery in bottom phase. This work conjugates three not widely spread methodologies for ATPS: microfluidics, continuous and multistage operation.