Salting-out extraction of acetoin from fermentation broth using ethyl acetate and K2HPO4

Salting-out extraction of acetoin from fermentation broth using ethyl acetate and K2HPO4

Separation and Purification Technology 184 (2017) 275–279 Contents lists available at ScienceDirect Separation and Purification Technology journal h...

431KB Sizes 95 Downloads 269 Views

Separation and Purification Technology 184 (2017) 275–279

Contents lists available at ScienceDirect

Separation and Purification Technology journal homepage: www.elsevier.com/locate/seppur

Salting-out extraction of acetoin from fermentation broth using ethyl acetate and K2HPO4 Jianying Dai, Wentian Guan, Linhui Ma, Zhilong Xiu ⇑ School of Life Science and Biotechnology, Dalian University of Technology, Dalian 116024, PR China

a r t i c l e

i n f o

Article history: Received 10 January 2017 Received in revised form 4 May 2017 Accepted 5 May 2017 Available online 6 May 2017 Keywords: Separation Acetoin Salting-out extraction Mixed solvent Countercurrent extraction

a b s t r a c t As one of the hydrophilic platform chemicals, acetoin could be produced by microbial fermentation. Although it could be separated from fermentation broth at high recovery by salting-out extraction (SOE) using hydrophilic solvent and inorganic salt, some of impurities in fermentation broth were also distributed to the top phase, which was not favorable for the following distillation process. In this work, a system based on ethyl acetate and K2HPO4 was selected to improve this technique by adding ethanol as cosolvent in the single SOE process or using two-stage countercurrent extraction. The separation conditions such as ethanol and K2HPO4 concentration, stirring and standing time were explored and the reuse of recovered solvent and phosphate were tried. The acetoin recovery of 95.3% was obtained through a single SOE process using a mixed solvent containing 20% ethanol, or 91.3% via a two-stage countercurrent SOE process using ethyl acetate only under the condition of K2HPO4 concentration 50% (m/v) in fermentation broth and volume ratio 1:1 for fermentation broth to solvent. Organic acids, residual glucose and coloring matters were not detected in the top phase, and acetoin loss during solvent removal was greatly reduced. The results indicate that the methods are very prospective in industrial application. Ó 2017 Elsevier B.V. All rights reserved.

1. Introduction Acetoin (3-hydroxy-2-butone or acetyl methyl carbinol) is a flavor widely used to enhancing the fragrance of food, cosmetics, detergents, lotions, and so on [1]. It is also one of the bio-based platform chemicals utilized in the synthesis of diacetyl, acetylbutanediol, pyrazines, optically active a-hydroxyketone derivatives [1]. With the increasing demand of natural flavor and stereoisomers, the biological production of acetoin has attracted much attention in recent years. Many GRAS strains with high acetoin production were obtained and different carbon sources were tried. The acetoin concentration in fermentation broth was generally greater than 70 g/L with glucose or sucrose as carbon source [2– 4], and the highest could reach 100.1 g/L by an engineered strain of Saccharomyces cerevisiae [5]. When sugarcane molasses was used as carbon source, high acetoin production was greater than 60 g/L [2]. Acetoin is a liquid with high hydrophilicity and boiling point, so the separation of acetoin from fermentation broth became one of the key problems for acetoin production. Till now, several methods were tried to separate acetoin from aqueous solution or fermenta-

⇑ Corresponding author. E-mail address: [email protected] (Z. Xiu). http://dx.doi.org/10.1016/j.seppur.2017.05.012 1383-5866/Ó 2017 Elsevier B.V. All rights reserved.

tion broth, such as adsorption by a hyper-cross-linked resin [6], salting-out extraction (SOE) by hydrophilic solvent and inorganic salts [7,8], or converting it into its derivatives such as diacetyl [9], 2,3,5,6-tetramethylpyrazine [10]. Among these methods, SOE is preferred due to its easy operation, scale-up and lower energy consumption. Salting-out extraction of bio-based chemicals from fermentation broth or aqueous solution was proved to be an efficient way in the application of 1,3-propanediol [11,12], 2,3-butanediol [13,14], acetoin [7] and organic acids including acetic acid, propionic acid, lactic acid and succinic acid [15,16]. For example, over 90% acetoin was extracted to the top phase from fermentation broth in a range of 3–60 g/L [8], and the phosphate could be reused after recovery from bottom phase [7]. However, due to the hydrophilic solvent used in SOE large amount of water were also existed in the top phase [17,18]. Finally, some of inorganic salt used in SOE [17,18] and impurities in fermentation broth were also distributed to the top phase, including organic acids [11,19], residual sugars [7,14,20], proteins [7,14,20] and coloring matters [7]. After the removal of solvent and water from top phase, these concentrated impurities would increase the viscosity of concentrated solution and deposit in the tank during product distillation, thus other operation was required before distillation.

276

J. Dai et al. / Separation and Purification Technology 184 (2017) 275–279

During the acetoin purification from top phase after SOE, we found another problem except impurity enrichment. Because acetoin could form azeotropic mixture, a significant acetoin loss was observed during solvent removal by vacuum distillation when hydrophilic solvent was used. For example, acetoin concentration of 21 g/L was detected in the recovered solvent from ethanolK2HPO4 top phase [21]. Therefore, it was essential to improve the SOE technique to reduce impurity concentrations in top phase and decrease acetoin loss during solvent removal. To obtain high recovery of acetoin from fermentation broth and high removal of impurities from top phase, the SOE system based on ethyl acetate (EAC) and K2HPO4 was explored in this study. Two methods were tried. One was SOE using mixed solvent of EAC and ethanol to increase solvent polarity, the other was countercurrent SOE to enhance extraction efficiency. At appropriate conditions the acetoin recovery was above 90% using these two methods while organic acids, residual glucose and coloring matters were not detected in top phase.

2. Materials and methods

2.5. Separation of acetoin from fermentation broth by two-stage countercurrent SOE The effect of stirring and standing time on acetoin distribution was studied. The mixing experiment was carried out in a 1000 mL flask containing 100 mL fermentation broth, 100 mL EAC and 50 g K2HPO4 by stirring at 300 rpm for different minutes. After mixing, the mixture was transferred into a 500 mL separating funnel and stood at ambient temperature for various hours. Finally, the two-stage countercurrent SOE was performed under a stirring time of 15 min and standing time of 4 h. 2.6. Recovery of solvent in top phase and phosphate in bottom phase The solvent in top phase from SOE based on mixed solvent of EAC and ethanol was recovered by atmospheric distillation and the temperature of water bath was 85 °C. EAC in top phase from countercurrent SOE was recovered using a vacuum rotary evaporator at 20 °C and 0.095 MPa. The phosphate in bottom phase was recovered by pH adjustment (pH 4.0) [11].

2.1. Materials

2.7. Analytical method

The standard of racemic mixture of acetoin was purchased from Shanghai Nuotai Chemical Co. Ltd. (Shanghai, China). Ethyl acetate (EAC), ethanol and K2HPO4 were purchased from Sinopharm Chemical Reagent Co., Ltd (Shanghai, China).

Acetoin was analyzed by GC system (Agilent Technologies 7890 A GC systems) equipped with a capillary column BGB-174 (30 m  0.25 mm I.D. 0.25 lm df) and a FID detector. Organic acids and residual glucose in top phase were analyzed by HPLC equipped with an Aminex HPX-87H Column (300  7.8 mm) and a refractive index detector (Waters 2414). The concentration of coloring matters was represented by the absorption at 480 nm (OD480) detected by a spectrophotometer.

2.2. Preparation of fermentation broth The strain used was Bacillus subtilis DL01 and fermentation broth was prepared according to the published method [21]. The fermentation broth was pretreated by centrifugation at 6800g for 30 min, followed by membrane filtration (Superflux-150G, Nipro Corporation, Japan). The final clear solution was used for SOE experiments.

2.3. Effects of ethanol and phosphate on acetoin distribution The salt K2HPO4 was dissolved in 4 mL fermentation broth (70.8 g/L acetoin), then 4 mL mixed solvent of ethanol and EAC was added. The mixture was mixed by a vortex mixer for 2 min, then stood at 24 °C for 12 h. Each experiment was carried-out in triplicate. The concentration of acetoin in top phase was determined by GC. The parameters including phase ratio (R), distribution coefficient (K) and recovery were calculated according to the published paper [22]. R was the volume ratio of top phase and bottom phase, K was the concentration ratio of acetoin in top phase and bottom phase, and recovery was the mass ratio of acetoin in the top phase and the total. The amount of K2HPO4 was 2 g and ethanol concentration was 0–100% in mixed solvent in the experiments for ethanol effect, while in the experiments for phosphate effect the ethanol concentration 20% and phosphate concentration was 50–150% in fermentation broth (m/v).

2.4. Separation of acetoin based on mixed solvent through a single SOE process Fermentation broth (100 mL, 58.0 g/L acetoin), ethanol (20%)EAC (100 mL) and K2HPO4 (50–125 g) were mixed in a 1000 mL flask by stirring at 300 rpm for 3 min, then transferred into a 500 mL separating funnel and stood at ambient temperature for 12 h.

3. Results and discussion 3.1. Separation of acetoin from fermentation broth through a single SOE process based on mixed solvent of EAC and ethanol Hydrophilic solvents such as methanol, ethanol, propanol and acetone could form aqueous two phases with inorganic salts at appropriate concentrations, and the distribution coefficient of acetoin (K) in these systems was generally greater than 5 [8]. If an immiscible solvent such as EAC, butyl acetate and butanol was used to extract acetoin from aqueous solution without addition of salts, the distribution coefficient was around 0.5 for butyl acetate [23] and 1-butanol [24], and 1 for EAC [25] indicating the low efficiency of extraction. However, the weak polarity of solvent meant low water content in top phase. For example, the water content in EAC phase was only 4–8% at 25 °C [25]. By adding salt, the water content would be lowered which was beneficial to reduce the concentrations of impurities in top phase. Therefore, in this study EAC was selected as extracting reagent, and K2HPO4 was selected as salting-out reagent for it showed a high recovery around 95–99% in previous SOE of acetoin based on acetone [7] and ethanol [14,26]. When EAC was used to extract acetoin during SOE, the K was 4.16 and recovery was 80.8% with K2HPO4 concentration of 50% (m/v) in fermentation broth (Fig. 1A). The value of K was much higher than that from EAC extraction [25], but still lower than that from SOE based on hydrophilic solvent [8]. By mixing ethanol with EAC to increase the polarity of solvent, K, phase ratio and recovery were increased with the increasing concentration of ethanol. When solvent was switched from EAC to ethanol, the K and recovery reached 19.9 and 97.9%, respectively. As shown in Fig. 1A, the effect of ethanol addition was very significant. The K value could be increased about 20% even at an etha-

J. Dai et al. / Separation and Purification Technology 184 (2017) 275–279

277

Fig. 1. Effect of the concentrations of ethanol and K2HPO4 on the partition behavior of acetoin. R, phase ratio; K, distribution coefficient of acetoin; recovery, recovery of acetoin; Vt, volume of top phase; Vb, volume of bottom phase. Composition of system: A, ethanol (0–100%)-EAC: K2HPO4: fermentation broth = 1 (v): 0.5 (m): 1 (v); B, ethanol (20%)-EAC: K2HPO4: fermentation broth = 1 (v): 0.5–1.5 (m): 1 (v).

nol concentration of 10%. The main reason was that ethanol contains a hydroxyl group which has stronger ability to form hydrogen bond with acetoin and water than EAC. Thus more acetoin and water were distributed to the top phase. Similar phenomenon was also observed in the EAC extraction of 1,3-propanediol by adding ethanol as cosolvent [27]. However, the increased water content in top phase was unfavorable for the removal of organic acids and residual sugar. Moreover, the color in top phase was gradually varied from colorless to light orange. At the point of 20% ethanol the top phase was near colorless, so the mixed solvent containing 20% ethanol was selected to check the effect of K2HPO4 addition. When the concentration of K2HPO4 in fermentation broth was increased, more water was trapped into the bottom phase, and more acetoin and solvent were repelled to the top phase, so the volume of top phase was increased firstly (Fig. 1B). The largest volume of 4.65 mL for top phase was obtained at the K2HPO4 concentration of 75–125% (m/v) in fermentation broth, and the highest K and recovery were obtained in the range of 100–125% K2HPO4 with a value of 22.5 and 95.5%, respectively. The composition of top phase was sensitive to the salt concentration, and a tiny concentration increment of salt in the total composition would cause a large content variation of solvent and water in the top phase [17,18,28]. For example, in the systems of 31.13% ethanol-15.97% K3PO4 and 31.08% ethanol-17.98% K3PO4, the ethanol content in top phase was 56.87% and 61.35% and water content was 42.50% and 38.38%, respectively [28]. The water content in top phase was gradually decreased with the increasing concentration of salt, thus affected the solubility of acetoin in top phase. As a result, the K and recovery were decreased to 13.7 and 91.5%, respectively, when K2HPO4 in fermentation broth was near saturation (150%, m/v). When the treated volume of fermentation broth was amplified from 4 mL to 100 mL, the mixing mode was changed from vortexing to mechanical agitation. Unlike the SOE using hydrophilic solvent where the distribution of the components was not affected by amplification because a complete mixing was easily achieved [7], the partition behavior of acetoin was greatly affected by the mixing conditions due to the immiscibility of EAC. As shown in Table 1, similar changing tendency of K and recovery was observed, but the highest recovery was obtained at lower salt concentration than that in tube due to the intensified mixing. For example, the recovery was increased from 86.4% to 95.3% when the K2HPO4 concentration in fermentation broth was 50%, while decreased from 91.5% to 75.7% when K2HPO4 concentration was 125%. It was notable that the impurities such as residual glucose, organic acids, and coloring matters were not detected in the top

phase when the mixed solvent of 20% ethanol was applied, while a high acetoin recovery about 95% was obtained. In this experiment, the fermentation broth contained 58.0 g/L acetoin, 13.5 g/L glucose, 4.2 g/L formic acid, 7.7 g/L acetic acid, and 4.4 g/L lactic acid, and OD480 was 0.66. The detection limit for HPLC was 0.1 g/ L and 0.002 of OD480 for spectrophotometer, which meant at least 99% residual glucose, 97.5% organic acids and 99% coloring matters were removed from top phase. If a system of ethanol-K2HPO4 was used, the glucose removal was about 87% [14]. Changing ethanol to acetone, the removal of residual sucrose and prodigiosin was about 85% [7]. If methanol was used, the removal of acetic, lactic and succinic acid was around 75–90% [11]. Compared with SOE based on hydrophilic solvent, SOE based on EAC-20% ethanol was improved in impurity removal but kept the characteristic of high recovery, thus its applicability was greatly increased from the view of acetoin distillation. 3.2. Two-stage countercurrent SOE of acetoin from fermentation broth using EAC and K2HPO4 The above results showed high recovery of acetoin could be achieved by vigorous mixing with 50% K2HPO4 addition, so the amount of 50% addition was selected for two-stage countercurrent. Before carrying out this experiment the effect of standing time and stirring time on acetoin distribution were explored. As shown in Table 2, the phase ratio was not affected by acetoin concentration, stirring time and standing time once the composition of SOE system was fixed, while the equilibrium of acetoin distribution was influenced. Longer time of standing and stirring was beneficial for acetoin recovery. The K value increased 46.4% when standing time was elongated from 2 h to 4 h, while changed little when elongated from 4 h to 8 h, which meant it took about 4 h for acetoin distribution to achieve equilibrium. Therefore, the least of 4 h was required for standing in practical application. The mixing efficiency was affected by the design of stirring paddle, agitation speed and time. In this experiment a regular polytetrafluoroethylene stirring paddle and agitation speed of 300 rpm were used to test the effect of stirring time. Unlike the mixed solvent of EAC and ethanol (20%) where the stirring time was only 3 min (Table 1), a stirring time of 5 min was too short for mixing thoroughly and acetoin recovery was only 73.4%. Elongating time from 15 min to 30 min, the increasing of recovery was slowing down. At present condition a stirring time of 15–30 min could be applied in the countercurrent SOE. Under the stirring time of 15 min and standing time of 4 h, an acetoin recovery of 91.3% was obtained after two-stage counter-

278

J. Dai et al. / Separation and Purification Technology 184 (2017) 275–279

Table 1 Separation of acetoin through a single SOE process. K2HPO4 (%, m/v)

50 75 100 125

Volume (mL) Top phase

Bottom phase

107.0 ± 0.2 108.0 ± 0.1 103.0 ± 0.1 98.0 ± 0.2

97.0 ± 0.1 106.0 ± 0.2 111.0 ± 0.1 125.0 ± 0.2

Distribution coefficient

Recovery (%)

Impurities in top phase Glucose

Organic acids

Coloring matters

18.5 ± 0.2 21.2 ± 0.3 4.45 ± 0.14 3.98 ± 0.03

95.3 ± 0.3 95.6 ± 0.4 80.5 ± 0.2 75.7 ± 0.7

ND ND ND ND

ND ND ND ND

ND ND ND ND

*ND, not detected.

Table 2 Acetoin distribution under different standing and stirring time. Acetoin in fermentation broth

Time

Phase ratio

Distribution coefficient

Recovery (%)

2h 4h 8h

1.01 ± 0.01 1.00 ± 0.01 1.01 ± 0.01

2.22 ± 0.02 3.25 ± 0.07 3.49 ± 0.45

69.3 ± 0.3 76.4 ± 0.4 77.4 ± 2.5

4h 4h 4h

0.99 ± 0.01 0.99 ± 0.01 1.00 ± 0.01

4.67 ± 0.07 3.90 ± 0.27 2.80 ± 0.28

82.2 ± 0.2 79.4 ± 1.0 73.4 ± 1.9

Stirring

Standing

79.0 g/L

30 min 30 min 30 min

51.2 g/L

30 min 15 min 5 min

current SOE, and glucose, organic acids and coloring matters were not detected in the top phase (Table 3). Compared with the single SOE process (Table 2), countercurrent extraction improved the extraction efficiency of 15% for EAC-K2HPO4 system. The recovery from two-stage countercurrent extraction was a little lower than that based mixed on mixed solvent (Table 1). The above results clearly showed that the polarity of solvent was the key factor influencing the separation efficiency. When the top phase obtained from above two methods was treated by distillation to remove solvent, only 8–9 g/L acetoin was detected in the recovered solvent. Compared with ethanolK2HPO4 SOE system in which acetoin concentration was 21 g/L in recovered ethanol [21], the acetoin loss during concentrating process was reduced about 70% by using EAC-based solvent and the concentrated acetoin solution could be used for distillation directly. 3.3. Reuse of solvent and phosphate The solvent recovery of top phase was about 85% by distillation, and the recovery of phosphate was 86.4% by pH adjustment. When the recovered solvent from the top phase based on mixed solvent was used directly to perform SOE, the recovery of acetoin was decreased from 95.3% to 84.3% due to the changed ratio of two solvents (Table 4). So it was essential to analyze the solvent ratio in recovered solvent and re-prepare it in the recycling of mixed solvent. The work on phosphate reuse in acetoin recovery was tried before using an acetone-K2HPO4 system, and acetoin recovery was only decreased 1% after three times of recovery and reusing [7]. However, the decreasing of acetoin recovery was much greater in this work when phosphate was reused once no matter which solvent was used (Table 4). After two times of phosphate recovery

and reusing, the recovery was below 80%. So it was not feasible for phosphate recycling in this study. The decreasing of acetoin recovery occurred in phosphate reuse was resulted from the composition of fermentation broth where different types of strains were applied. It was found that B. subtilis could produce an extracellular polymer – poly(c-glutamic acid) (cPGA) [29]. Based on the reaction of c-PGA and hexadecyltrimethylammonium bromide [30], c-PGA was detected in fermentation broth by wavelength scan between 200 and 400 nm. The phosphate was recovered by precipitation, so part of c-PGA might be smuggled with phosphate. In the SOE experiment of reusing phosphate, the solution became very viscous after adding recovered phosphate thus affected the partition behavior of acetoin. If other fermentation broth used, such a recovery decrease might not be happened. In fact, the recycling times of salt were limited during the SOE because a decreasing trend was always existed [7]. Therefore, all the recovered salts had to find a final destination. Potassium and phosphate are two types of fundamental fertilizer, maybe the recovered phosphate or bottom phase could be used to prepare fertilizer. Compared with hydrophilic solvent by which the removal of organic acids, residual glucose and coloring matters were around 85% [7,11,14], high recovery was still obtained while the impurities in top phase were reduced to 3–2% based on EAC SOE system. Thus, the two methods tried in this work greatly improved, and the top phase could be directly applied for product distillation after solvent removal. Of course, the two methods were not perfect. When mixed solvent was used, the operation of a single SOE process was enough to obtain high recovery, just like SOE using hydrophilic solvent [7,14,31], but the recovered solvent had to be re-prepared to obtain appropriate ratio. When only EAC was used for SOE, a twostage countercurrent SOE process at least was required to obtain high recovery and the recovered solvent could be used directly,

Table 3 Results of two-stage countercurrent SOE of acetoin from fermentation broth using EAC and K2HPO4. Phase ratio

First stage Second stage *

ND, not detected.

1.01 ± 0.01 0.933 ± 0.03

Distribution coefficient

1.23 ± 0.07 5.47 ± 0.09

Recovery (%)

54.5 ± 0.9 91.3 ± 0.6

Impurities in top phase Glucose

Organic acids

Coloring matters

– ND*

– ND

– ND

279

J. Dai et al. / Separation and Purification Technology 184 (2017) 275–279 Table 4 Acetoin separation from fermentation broth based on recovered solvent and phosphate. Experiment condition Extraction mode

Solvent

K2HPO4

Single SOE process

Recovered EAC-ethanol Fresh EAC-ethanol Recovered EAC

Fresh Recovered Recovered

Two-stage countercurrent SOE

but one more set of extraction equipment was required compared with single process. 4. Conclusion The SOE of acetoin from fermentation broth based on EAC and K2HPO4 was explored to reduce the impurities in top phase and acetoin loss occurred in the solvent removal process of top phase. An acetoin recovery of 95.3% was achieved through a single SOE process using mixed solvent of ethanol (20%)–EAC, and recovery of 91.3% by two-stage countercurrent SOE using EAC only when the SOE system was composed of 100 mL fermentation broth, 50 g K2HPO4 and 100 mL solvent. Moreover, the impurities of organic acids, residual glucose and coloring matters were not detected in the top phase, and the acetoin loss during solvent removal were greatly reduced. This study provided two methods with good prospect in industrial application. Acknowledgement This work was supported by the National Natural Science Foundation of China (Grant No. 21476042). References [1] Z. Xiao, J.R. Lu, Strategies for enhancing fermentative production of acetoin: a review, Biotechnol. Adv. 32 (2014) 492–503. [2] J.-Y. Dai, L. Cheng, Q.-F. He, Z.-L. Xiu, High acetoin production by a newly isolated marine Bacillus subtilis strain with low requirement of oxygen supply, Process Biochem. 50 (2015) 1730–1734. [3] X. Zhang, T. Bao, Z. Rao, T. Yang, Z. Xu, S. Yang, H. Li, Two-stage pH control strategy based on the pH preference of acetoin reductase regulates acetoin and 2,3-butanediol distribution in Bacillus subtilis, PLoS ONE 9 (2014) e91187. [4] Q. Luo, J. Wu, M. Wu, Enhanced acetoin production by Bacillus amyloliquefaciens through improved acetoin tolerance, Process Biochem. 49 (2014) 1223–1230. [5] S.-J. Bae, S. Kim, J.-S. Hahn, Efficient production of acetoin in Saccharomyces cerevisiae by disruption of 2,3-butanediol dehydrogenase and expression of NADH oxidase, Sci. Rep. 6 (2016) 27667. [6] J. Wu, L. Wang, J. Zhou, X. Zhang, Y. Liu, X. Zhao, J. Wu, W. Zhuang, J. Xie, X. He, H. Ying, Recovery of acetoin from the aqueous solution by means of a novel hyper-cross-linked resin: equilibrium and kinetics, J. Food Eng. 119 (2013) 714–723. [7] J. Sun, B. Rao, L. Zhang, Y. Shen, D. Wei, Extraction of acetoin from fermentation broth using an acetone/phosphate aqueous two-phase system, Chem. Eng. Comm. 199 (2012) 1492–1503. [8] J.-Y. Dai, Y.-Q. Sun, Z.-L. Xiu, Separation of bio-based chemicals from fermentation broths by salting-out extraction, Eng. Life Sci. 14 (2014) 108– 117. [9] H. Chen, Y. Du, Q. Ma, Z. Wei, Z. Xiao, P. Xu, Y. Zeng, Z. Zhang, A preparation method of butadione by gas-phase oxidating 3-hydroxy-butanone, European patent (2007) EP1826194. [10] B.-F. Zhu, Y. Xu, Production of tetramethylpyrazine by batch culture of Bacillus subtilis with optimal pH control strategy, J. Ind. Microbiol. Biotechnol. 37 (2010) 815–821.

Phase ratio

Distribution coefficient

Recovery (%)

1.00 ± 0.01 1.10 ± 0.02 0.910 ± 0.01

3.81 ± 0.03 7.16 ± 0.08 7.16 ± 0.18

84.3 ± 0.3 86.4 ± 0.5 86.7 ± 0.3

[11] Z. Li, H. Teng, Z. Xiu, Extraction of 1,3-propanediol from glycerol-based fermentation broths with methanol/phosphate aqueous two-phase system, Process Biochem. 46 (2011) 586–591. [12] H. Fu, J. Dai, Y. Sun, D. Zhang, Z. Xiu, Partition behavior of hydrophilic diols in an ethanol/ammonium sulfate salting-out extraction system, Eng. Life Sci. 15 (2015) 797–803. [13] S.D. Birajdar, S. Rajagopalan, J.S. Sawant, S. Padmanabhan, Continuous countercurrent liquid–liquid extraction method for the separation of 2,3butanediol from fermentation broth using n-butanol and phosphate salt, Process Biochem. 50 (2015) 1449–1458. [14] B. Jiang, Z.-G. Li, J.-Y. Dai, D.-J. Zhang, Z.-L. Xiu, Aqueous two-phase extraction of 2,3-butanediol from fermentation broths using an ethanol/phosphate system, Process Biochem. 45 (2009) 112–117. [15] H. Fu, Y. Sun, H. Teng, D. Zhang, Z. Xiu, Salting-out extraction of carboxylic acids, Sep. Purif. Technol. 139 (2015) 36–42. [16] Y. Sun, L. Yan, H. Fu, Z. Xiu, Salting-out extraction and crystallization of succinic acid from fermentation broths, Process Biochem. 49 (2014) 506–511. [17] Y. Wang, Y. Yan, S. Hu, J. Han, X. Xu, Phase diagrams of ammonium sulfate + ethanol/1-propanol/2-propanol + water aqueous two-phase systems at 298.15 K and correlation, J. Chem. Eng. Data 55 (2010) 876–881. [18] Y. Wang, S. Hu, Y. Yan, W. Guan, Liquid-liquid equilibrium of potassium/sodium carbonate + 2-propanol/ethanol + water aqueous twophase systems and correlation at 298.15 K, CALPHAD 33 (2009) 726–731. [19] G. Liu, B. Jiang, Y. Wang, J. Dai, Z. Xiu, Aqueous two-phase extraction of 2, 3butanediol by ethanol/potassium carbonate system from Dioscorea zingiberensis fermentative broths (in Chinese) CIESC J. 60 (2009) 2798–2804. [20] Z. Li, H. Teng, Z. Xiu, Aqueous two-phase extraction of 2,3-butanediol from fermentation broths using an ethanol/ammonium sulfate system, Process Biochem. 45 (2010) 731–737. [21] J.-Y. Dai, L.-H. Ma, Z.-F. Wang, W.-T. Guan, Z.-L. Xiu, Sugaring-out extraction of acetoin from fermentation broth by coupling with fermentation, Bioprocess Biosyst. Eng. 40 (2017) 423–429. [22] J.-Y. Dai, C.-J. Liu, Z.-L. Xiu, Sugaring-out extraction of 2,3-butanediol from fermentation broths, Process Biochem. 50 (2015) 1951–1957. [23] Y.-Y. Wu, K. Chen, D.-T. Pan, J.-W. Zhu, B. Wu, Y.-L. Shen, Liquid-liquid equilibria of water + 3-hydroxy-2-butanone + butyl ethanoate at several temperatures, J. Chem. Eng. Data 56 (2011) 910–914. [24] Y.-Y. Wu, K. Chen, D.-T. Pan, J.-W. Zhu, B. Wu, Y.-L. Shen, Liquid-liquid equilibria of water + 3-hydroxy-2-butanone + 1-butanol, J. Chem. Eng. Data 56 (2011) 2641–2646. [25] Y.-Y. Wu, K. Chen, D.-T. Pan, J.-W. Zhu, B. Wu, Y.-L. Shen, Liquid–liquid equilibria of water + 3-hydroxy-2-butanone + ethyl ethanoate, Fluid Phase Equilib. 305 (2011) 101–105. [26] J. Dai, Y. Zhang, Z. Xiu, Salting-out extraction of 2,3-butanediol from Jerusalem artichoke-based fermentation broth, Chin. J. Chem. Eng. 19 (2011) 682–686. [27] T. Boonsongsawat, A. Shotipruk, V. Tantayakom, P. Prasitchoke, C. Chandavasu, P. Boonnoun, C. Muangnapoh, Solvent extraction of biologically derived 1,3propanediol with ethyl acetate and ethanol cosolvent, Sep. Sci. Technol. 45 (2010) 541–547. [28] Y. Yun Wang, J. Mao, Y. Han, Y. Liu, Yan, Liquid-liquid equilibrium of potassium phosphate/potassium citrate/sodium citrate + ethanol aqueous two-phase systems at (298.15 and 313.15) K and correlation, J. Chem. Eng. Data 55 (2010) 5621–5626. [29] Q. Wu, H. Xu, H. Ying, P. Ouyang, Kinetic analysis and pH-shift control strategy for poly(c-glutamic acid) production with Bacillus subtilis CGMCC 0833, Biochem. Eng. J. 50 (2010) 24–28. [30] Q.-Q. Zhang, X.-Q. Jin, J.-X. Chen, A.-B. Zhao, H.-J. Li, Study on efficient determination method of poly-c-glutamic acid in fermentation broth (in Chinese) Sci. Technol. Food Ind. 33 (2012) 294–300. [31] L.-H. Sun, B. Jiang, Z.-L. Xiu, Aqueous two-phase extraction of 2,3-butanediol from fermentation broths by isopropanol/ammonium sulfate system, Biotechnol. Lett. 31 (2009) 371–376.