Salting-out Extraction System Research Articles

Interfacial behavior and extraction kinetics of phages in a salting-out extraction system of ammonium citrate and ethyl acetate

To fight the increasing emergence and spread of antimicrobial resistance, phages have regained much more interest as the potential substitute in recent years. Separation and purification of phages were developed by salting-out extraction (SOE) with advantages of simpleness, high efficiency, high yield and the easiness to scale-up over the traditional means. In this study, SOE system composed of ammonium citrate and ethyl acetate was used to investigate the partition behavior of phages, phase separation, and extraction kinetics. With the increase of phase volume ratio, the phage phiAB9 was progressively transferred from the bottom phase to the middle phase. Accordingly, the solubility and aggregation of the middle phase increased, and the color got darker. Furthermore, the phase inversion point was kept at phase volume ratio between 1.0 and 1.1, at which the phase separation time was the shortest. The extraction kinetics revealed that the partition behavior could be finished in 10 min without centrifugation. Finally, the SOE system was also viable alternatives for the isolation and purification of phage λ, phiKP1 and phiSM1. The mechanism of SOE process is very important for its application and scale up in phage separation.

One-pot synthesis of fuel precursor from acetoin fermentation broth using ionic liquid-based salting-out extraction system

BackgroundThe development of biofuels, especially liquid hydrocarbon fuels, has been widely concerned due to the depletion of fossil resources. In order to obtain fuel precursors, the reaction of C–C bond formation is usually carried out with biomass derived ketones/aldehydes as reactants. Acetoin and 2,3-butanediol are two platform chemicals, which are co-existed in fermentation broth and traditionally separated by distillation, and then acetoin could be use as C4 building block to prepare hydrocarbon fuels. In order to mitigate the process complexity, direct aldol condensation reaction of acetoin in fermentation broth was studied in this work.ResultsA one-pot process of product separation and acetoin derivative synthesis was proposed based on salting-out extraction (SOE). Aldol condensation reaction of acetoin and 5-methyl furfural in different SOE systems was compared, and the results showed that the synthesis of C10 fuel precursors and separation of C10 products and 2,3-butanediol from fermentation broth were achieved in one-pot with ethanolammonium butyrate (EOAB) and K2HPO4 as SOE reagents and catalysts. The SOE and reaction conditions such as the concentrations of EOAB and K2HPO4, reaction temperature and time were optimized. When the system was composed of 6 wt% EOAB-44 wt% K2HPO4 and the mixture was stirred for 6 h at 200 rpm, 40 ℃, the yield of C10 products was 80.7%, and 95.5% 2,3-butanediol was distributed to the top EOAB-rich phase. The exploration of reaction mechanism showed that an imine intermediate was rapidly formed and the subsequent C10 product formation was the key step for aldol condensation reaction.ConclusionsWith EOAB and K2HPO4 as SOE reagents and catalysts, one-pot synthesis of fuel precursor from acetoin fermentation broth was achieved without prior purification. A yield of 80.7% for C10 products was obtained which was accumulated at the interface of two aqueous-phase, and 95.5% 2,3-BD was distributed to the top EOAB-rich phase. This work provides a new integration process of product separation and derivative synthesis from fermentation broth based on ionic liquid SOE.Graphical

Salting-out extraction of recombinant κ-carrageenase and phage T7 released from Escherichia coli cells.

Traditional technology of cell disruption has become one of the bottlenecks restricting the industrialization of genetic engineering products due to its high cost and low efficiency. In this study, a novel bioprocess of phage lysis coupled with salting-out extraction (SOE) was evaluated. The lysis effect of T7 phage on genetically engineered Escherichia coli expressing κ-carrageenase was investigated at different multiplicity of infection (MOI), meanwhile the phage and enzyme released into the lysate were separated by SOE. It was found that T7 phage could lyse 99.9% of host cells at MOI=1 and release more than 90.0% of enzyme within 90min. After phage lysis, 87.1% of T7 phage and 71.2% of κ-carrageenase could be distributed at the middle phase and the bottom phase, respectively, in the SOE system composed of 16% ammonium sulfate and 20% ethyl acetate (w/w). Furthermore, κ-carrageenase in the bottom phase could be salted out by ammonium sulfate with a yield of 40.1%. Phage lysis exhibits some advantages, such as mild operation conditions and low cost. While SOE can efficiently separate phage and intracellular products. Therefore, phage lysis coupled with SOE is expected to become a viable alternative to the classical cell disruption and intracellular product recovery.

One-step salting-out extraction of bacteriophage from its infection broth of Acinetobacter baumannii

Phages as a potential alternative antibiotic for multidrug-resistant bacterial infections are receiving great attention worldwide. However, the traditional separation and purification of phage are cumbersome, time-consuming, costly and inefficient. In this study, phage phiAB9 for multidrug-resistant Acinetobacter baumannii was separated and purified by a simple and cost-saving one-step salting-out extraction (SOE). Several kinds of salts and organic solvents without effect on phage survival were chosen to form the SOE systems, and the composition of SOE systems were optimized according to the phage recovery rate and impurity removal rate. After one-step SOE by an optimal system composed of 18% (w/w) ammonium citrate and 40% (w/w) ethyl acetate, the recovery rate of phage in the middle phase could reach to 90.82%, and most proteins (99.57%), cells (97.98%) and endotoxin (84.08%) were well removed, with a concentration factor of 210 and the purification factors of phage to proteins, cells and endotoxin were 303.64, 133.55 and 5.36, respectively. Comparing with two-step SOE and traditional aqueous two-phase extraction, one-step SOE may be an available method for the separation and purification of phages.

A novel salting-out extraction system for determination of ethylene glycol and diethylene glycol in liquid samples followed by GC-FID

In this work, a new high-efficient salting-out agent, potassium tripolyphosphate, assisted liquid–liquid extraction (SALLE) sample preparation method for the determination of the polar ethylene glycol and diethylene glycol in liquid samples (food stimulants and liquid foods) were developed. To obtain satisfactory extraction efficiencies for the studied analytes, several parameters affecting the SALLE procedure were optimized. The effects of type of salting-out reagent, type of extraction solvent, volumes of ethanol, amount of potassium triphosphate, vortexing and standing times on the extraction efficiency were investigated. Analytical performances of the method were satisfactory, showed good linearity in the selected range (R2＞0.9998), limits of detection (LOD) were 0.2 mg·L−1 in all cases. Average recoveries in the ranges of 81.2–109.3% (RSD, 0.03–7.0%) for EG, 88.1 ∼ 109.1% (RSD, 0.1–8.6%) for DEG were obtained. In relation to the green and low solvent consumption, the proposed methodology could be considered rapid and environment friendly. The developed method can be used not only for the determination of ethylene glycol and diethylene glycol in food simulation solution, but also for liquid foods with obtaining acceptable results.

Synthesis of Ammonium-Based Ionic Liquids for the Extraction Process of a Natural Pigment (Betanin)

The use of new synthesized ammonium-based ionic liquids was explored as an alternative to the current process implemented in the betanin extraction from red beet juice, resulting in high yields: 70% and 82%. Betanin is a vegetal pigment that has been applied to a large variety of products in the food industry, which is important, for it can work as a substitute for the red synthetic dyes used nowadays. Additionally, the use of the kosmotropic salt sodium acetate was explored in order to separate the complex formed by the ionic liquid and pigment of interest in a process that combined two techniques: ATPS (aqueous two-phase system) and SOES (salting-out extraction system). The results reveal that the studied techniques could work as a novel process for the extraction of betanin from red beet juice employing ionic liquids, which have not been tested for this purpose in other research.

Simultaneous solid-liquid separation and primary purification of clavulanic acid from fermentation broth of Streptomyces clavuligerus using salting-out extraction system.

Clavulanic acid (CA) is usually used together with other β‐lactam antibiotics as combination drugs to inhibit bacterial β‐lactamases, which is mainly produced from the fermentation of microorganism such as Streptomyces clavuligerus. Recently, it is still a challenge for downstream processing of low concentration and unstable CA from fermentation broth with high solid content, high viscosity, and small cell size. In this study, an integrated process was developed for simultaneous solid–liquid separation and primary purification of CA from real fermentation broth of S. clavuligerus using salting‐out extraction system (SOES). First, different SOESs were investigated, and a suitable SOES composed of ethanol/phosphate was chosen and further optimized using the pretreated fermentation broth. Then, the optimal system composed of 20% ethanol/15% K2HPO4 and 10% KH2PO4 w/w was used to direct separation of CA from untreated fermentation broth. The result showed that the partition coefficient (K) and recovery yield (Y) of CA from untreated fermentation broth were 29.13 and 96.8%, respectively. Simultaneously, the removal rates of the cells and proteins were 99.8% and 63.3%, respectively. Compared with the traditional method of membrane filtration or liquid–liquid extraction system, this developed SOES showed the advantages of simple operation, shorter operation time, lower process cost and higher recovery yield of CA. These results demonstrated that the developed SOES could be used as an attractive alternative for the downstream processing of CA from real fermentation broth.

Salting-out extraction of acetoin from fermentation broths using hydroxylammonium ionic liquids as extractants

The salting-out extraction (SOE) systems based on ionic liquids (ILs) have attracted extensive attention in the separation of bio-based products, in which imidazolium ILs were widely studied. However, the high cost and toxicity have hindered their further industrial application. Hydroxylammonium ILs have the characteristics of cheap raw material, simple synthesis process and low toxicity, but are rarely used in the SOE systems. In this work, five hydroxylammonium ILs (2 cations and 4 carboxylate anions) were synthesized and used in SOE of bio-chemicals. The phase forming abilities of ILs with K3PO4 and H2O were affected by the hydrophilicity of anions and cations. With the increase of carbon chain length, phase forming abilities of ILs increased, while the temperature had little influence. The partition behaviors of acetoin, ILs and 2,3-butandiol, and the selectivity of acetoin to organic acids were investigated and compared at different concentrations of ethanolammonium butyrate (EOAB) and K3PO4. In a SOE system consisted of 6% EOAB-38% K3PO4 (w/w), the recovery of acetoin, IL and 2,3-butandiol was 92.7%, 76.0% and 86.0%, respectively, and the selectivity of acetoin to lactic acid and acetic acid was 16.46 and 3.85, respectively. The ATR-IR spectra showed the hydrogen bonds formed between acetoin and O-H, N-H, –COO– of hydroxylammonium IL played an important role in the efficient extraction of acetoin from fermentation broths.

Recovery of Lactic Acid from Corn Stover Hemicellulose-Derived Liquor.

Lactic acid is an attractive target platform chemical obtained from biorefinery processes based on renewable resources. This study focuses on the recovery of lactic acid from corn stover hemicellulose-derived liquor. Two processes were investigated comparatively. In process I, under the optimized conditions of 10 wt % trioctylamine/octanol, the extraction efficiency and distribution coefficient of lactic acid were 50.8% and 1.03, respectively. In process II, a salting-out extraction step was introduced, which had significant impacts on the following reactive extraction process. The extraction efficiency and distribution coefficient of lactic acid were about 32.2% and 3.85 times higher than that of process I, respectively. All residual sugars and most of the salts (82.8%) could be removed by the salting-out extraction system used. Additionally, five extraction cycles with back-extraction and solvent regeneration were performed, and the results showed that the extraction system still exhibited stable performance. Effective extraction of lactic acid from crude reaction liquor of corn stover was realized by first salting-out and consecutive reactive extraction, which provided a potential method for recovery of lactic acid from actual biomass-derived liquor.

Separation and purification of wortmannilactone analogues by three-liquid-phase salting-out extraction coupled with column chromatography

Separation and purification of secondary metabolites from wild microorganisms remains a challenge due to the analogues existing in the fermentation broth. In this study, a three-liquid-phase salting-out extraction system (TLP-SOES) coupled with column chromatography was developed to purify wortmannilactone F and its dehydrated derivative wortmannilactone L. The TLP-SOES composed of 25.5% petroleum ether/20.0% ethanol/12.9% ammonium sulfate (w/w) at pH 5.6 and room temperature was found to separate wortmannilactone F in the middle (ethanol-rich) phase and wortmannilactone L in the top (petroleum ether-rich) phase with the recovery rates of 88.9% and 83.3%, respectively. The two compounds were further purified by ODS column and semi-preparative HPLC. The total recovery rates of the two compounds were over 2-fold higher than that of the conventional method, which included ethyl acetate/hexane extraction and silica gel column chromatography. Additionally, the calculated logP (ClogP) value of wortmannilactones showed a good linear correlation with their natural logarithm of partition coefficient according to the tie line length (TLL), and that the critical ClogP value might be useful for the selection of TLL or the composition of the extraction system. In summary, our study provided an effective approach for the separation and purification of analogues of natural products.

Ionic liquid-based sugaring-out and salting-out extraction of succinic acid

In this study, ionic liquids-based salting-out extraction and sugaring-out extraction were developed to extract succinic acid. Among these five investigated ionic liquids, the ionic liquid with BF4- and OTF− anion showed the ability to form two phases not only with glucose but also with salts. However, the ionic liquid with Br− anion cannot form aqueous two-phase system with glucose, but with salts. Relatively high extractabilities were obtained by ILs–based salting-out extraction systems compared to sugaring-out systems. The extraction efficiency values ranging between 47% and 86% were obtained when succinic acid concentration increased from 5 g/L to 70 g/L in [Bmim]BF4/(NH4)2SO4 systems. It was implied that succinic acid concentration was a key factor in ILs-based salting-out extraction system. Compared to salting-out extraction, sugaring-out extraction was more suitable for succinic acid separation due to the bottom phase reutilization to achieve coupling of fermentation and separation. The distribution behavior of succinic acid in [Bmim]BF4/glucose sugaring-out extraction system depends on not only the concentrations of ionic liquid and glucose, but also the pH. The partition coefficient (2.53) and recovery (75.96%) of succinic acid were obtained by a sugaring-out system consisting of 45% (w/w) [Bmim]BF4 and 30% (w/w) glucose at pH 2.0. Moreover, to support the applicability of ILs-based sugaring-out extraction for SA separation, the most prominent systems were further tested with actual fermentation broth. The extraction efficiencies were 73.71% and 72.53% for filtered and unfiltered fermentation broth, respectively. The remarkable results obtained in this work support the establishment of IL-based sugaring-out extraction as a sound basis of greener cost-effective strategies with a substantial reduction in the environmental footprint and economical issues.

Partition behavior of monocarboxylic acids in salting-out extraction systems of monohydric alcohols and dipotassium phosphate

Abstract Extraction of organic acids has been attracted much attention due to their hydrophilicity, pH-dependence and structural diversity. Partition behavior of seven monocarboxylic acids with different chemical structures, including formic acid, acetic acid, n-propionic acid, n-butyric acid, n-valeric acid, glycollic acid and lactic acid, have been investigated in salting-out extraction systems (SOES) composed of monohydric alcohol (ethanol, isopropanol, n-propanol, or tert-butanol)/K2HPO4. The relative hydrophobicity of the equilibrium phases in the SOES has been characterized by the Gibbs energy of transfer for methylene group from the salt-rich bottom phase to the solvent-rich top phase. The effects of the structures of target products and organic extractants, SOES composition, system pH and temperature on partition behavior were investigated. The results showed that natural logarithm of partition coefficient (ln K) of target products increased linearly with their calculated 1-octanol/water partition coefficient (Clog P), but decreased with the calculated 1-octanol/water partition coefficient of organic extractant. The partition coefficient of target products increased or decreased linearly as tie line length, which depends on their hydrophobicity. The partition coefficient and recovery decreased with the increasing of system pH values. In addition, the increasing of temperature will decreased the extraction efficiency of monocarboxylic acids, because the salting-out extraction is an exothermic process.

Chelatometric salting-out extraction and characteristics of flavonoids from Folium nelumbinis based on an ethanol/K2HPO4 system

ABSTRACTA new salting-out extraction method, namely chelatometric salting-out extraction, was established for rapidly extracting flavonoids from Folium nelumbinis. Borax was used as a chelating agent and an ethanol/K2HPO4 system as the salting-out extraction system. The maximum flavonoid concentration in the top phase was observed at an ethanol concentration of 43.0% and an amount of salt of 2.45 g under the experimental conditions. The pH value of the system and extraction time had no obvious influence on the extraction efficiency of flavonoids. The results also showed that the extraction efficiency of flavonoids exceeds 90.4% at the first time extraction. Moreover, the antioxidant activities of flavonoids were evaluated by DPPH▪ radical assays.

Salting-out extraction systems of ethanol and water induced by high-solubility inorganic electrolytes

Abstract The salting-out extraction systems of ethanol and water induced by the high-solubility inorganic electrolytes were investigated at 25 °C. More than 99.90% of ethanol was salted out into the organic phase. The salt content of the organic phase is only about 0.01 wt%, which is negligible for the application of the ethanol–salt salting-out extraction. More than 90% of water can be retained in the aqueous phase. It is concluded that the salting-out effects of four salting-out agents on the aqueous solution of ethanol are ordered in the sequence: K4P2O7 > K3PO4 > K2HPO4 > K2CO3.

Salting‐out extraction of sinomenine from Sinomenium acutum by an alcohol/salt aqueous two‐phase system using ionic liquids as additives

AbstractBACKGROUNDThe aqueous two‐phase extraction (ATPE) based on organic solvent and inorganic salt was considered a promising method in the extraction, enrichment, and purification field. Ionic liquids (ILs) used as additives to the aqueous two‐phase system (ATPS) can improve the extraction yield.RESULTSIn this study, seven ILs were added to the ethanol/ammonium sulfate ATPS and used for the salting‐out extraction (SOE) of sinomenine from Sinomenium acutum. The IL [C2OHmim]FeCl4 was screened as the optimal additive, then the influencing factors IL amount, ethanol concentration, salt concentration, and temperature were investigated. The maximum extraction efficiency of 96.4% was obtained under the following conditions: 23.06 wt% ethanol concentration, 27.28 wt% (NH4)2SO4 concentration, 4.0 wt% [C2OHmim]FeCl4 and 40°C temperature. Moreover, the components in the SOE system can be recycled after extraction.CONCLUSIONThus, this ATPS had lower cost and higher extraction ability, and it is feasible to scale up using a small amount of ILs. This system can also be used in the extraction of some other important active compounds from natural plant resources. © 2017 Society of Chemical Industry

Effects of salting-out and salting-out extraction on the separation of butyric acid

Abstract The effects of salting-out and salting-out extraction on the separation of butyric acid from the simulated solution were investigated. Ammonium sulfate, monosodium phosphate and calcium chloride were screened out and showed outstanding salting-out ability under acidic conditions, resulting in a butyric acid-rich top phase with a concentration multiple of 6.5–18.7 and an increased selectivity of butyric acid to acetic acid from 4 to 13.8–50.0. However, the salting-out effect of inorganic salt decreased with the acid concentration, indicating that this method was not effective for separation of butyric acid at low concentration. In contrast, salting-out extraction system composed of monosodium phosphate and ethanol exhibited excellent extraction efficiency for both butyric acid (∼99%) and acetic acid (∼90%). The partition coefficient and recovery yield of butyric acid increased with the acid concentration and system composition, while decreased with the hydrophobicity of extractant. Finally, two-step salting-out and salting-out extraction was employed, as a result, the total recovery yield of butyric acid and acetic acid were 99.4–99.5% and 88.5–88.9%, respectively.

Salting-out extraction of allicin from garlic (Allium sativum L.) based on ethanol/ammonium sulfate in laboratory and pilot scale

Salting-out extraction (SOE) based on lower molecular organic solvent and inorganic salt was considered as a good substitute for conventional polymers aqueous two-phase extraction (ATPE) used for the extraction of some bioactive compounds from natural plants resources. In this study, the ethanol/ammonium sulfate was screened as the optimal SOE system for the extraction and preliminary purification of allicin from garlic. Response surface methodology (RSM) was developed to optimize the major conditions. The maximum extraction efficiency of 94.17% was obtained at the optimized conditions for routine use: 23% (w/w) ethanol concentration and 24% (w/w) salt concentration, 31g/L loaded sample at 25°C with pH being not adjusted. The extraction efficiency had no obvious decrease after amplification of the extraction. This ethanol/ammonium sulfate SOE is much simpler, cheaper, and effective, which has the potentiality of scale-up production for the extraction and purification of other compounds from plant resources.

Extraction and purification of IgG by hydrophilic organic solvent salting-out extraction.

The distribution of IgG in different salting-out extraction systems (SOESs), including ethanol/K2HPO4, Na2CO3, and trisodium citrate, was investigated. The SOESs that were composed of 20% K2HPO4 (w/w)-14% ethanol (w/w), 19% Na2CO3 (w/w)-13% ethanol (w/w) and 25% trisodium citrate (w/w)-19% ethanol (w/w) showed satisfactory results, with IgG recovery rates as high as 97.4%, 93.1%, and 90.2%, respectively. ELISA, CD and IR analyses confirmed the active retention and structural constant of IgG in the K2HPO4-ethanol system. An optimized system that consisted of 14% ethanol (w/w)-20% K2HPO4 (w/w) (pH 7.0) was selected for extracting active plasma proteins directly from pig plasma. As a result, recovery rates as high as 95.7% for IgG and 93.0% for albumin were achieved. In addition, some contaminating proteins were also removed by this system, which may provide a new alternative method for the separation and purification of immunoglobulin.

Three-liquid-phase salting-out extraction of effective components from waste liquor of processing sea cucumber

A three-liquid-phase (TLP) salting-out extraction system composed of n-hexane/ethanol/sodium carbonate/water was investigated to extract oils, saponins, proteins and polysaccharides simultaneously from waste liquor of processing sea cucumber. The effects of the ratio of ethanol to sodium carbonate, n-hexane concentration and extraction time were investigated. The results showed that 86.7% of oils were distributed in the top n-hexane phase, 82.9% of saponins in the middle ethanol phase, 93.2% of proteins and 92.9% of polysaccharides in the interface between the middle and the bottom salt phase when the system composed of 28% (w/w) n-hexane/11.52% (w/w) ethanol/8.64% (w/w) sodium carbonate was used at 37°C for 0.5h. When the system was progressively enlarged from 30g to 25kg, the yield of polysaccharides, proteins, oils and saponins was decreased only by 1.0%, 2.8%, 1.0% and 2.6%, respectively. The recycle of salt and solvents in three-liquid-phase system was also studied, and the results showed that the recovery of n-hexane, ethanol and salt were 81.4%, 80.8% and 72.0%, respectively. Recycling materials for the extraction, the yield for proteins and oils decreased by 2.0% and 5.4%, respectively, comparing with the pure system.

Phase separation in a salting-out extraction system of ethanol–ammonium sulfate

The effects of tie line length, phase volume ratio, phase continuity, degree of mixing and equipment geometry size on the kinetics of phase separation in the ethanol–ammonium sulfate salting-out extraction system were studied with tie line lengths of 42.5%, 51.4% and 56.6% and phase volume ratio in the range of 1:9–9:1. The time for product distribution and phase equilibrium was also investigated. It was found that the rate of phase separation increased as the tie line length increased and the shortest separation time was obtained at the phase inversion point for all the tie lines at a constant phase volume ratio. The phase continuity was determined not only by the phase volume ratio, but also by the degree of mixing. An ambiguity area where gentle vortex produced a top-continuous system and intense mixing produced a bottom-continuous system was observed and its scope was extended as the mixing intensity decreased. Additionally, increasing the cross-sectional area of equipment had a significant effect on the rate of phase separation because the coalescence process determined the separation rate in the salting-out extraction system. By comparing the concentration of target product (butanol) and phase-forming component (ethanol) in the top phase at different time intervals, it was concluded that this system can achieve complete phase equilibrium within 10min. The knowledge of kinetics of phase separation is important to the deep understanding of this system and beneficial to process scale up for industrialization.