Imidazolium ionic liquids-based salting-out extraction of 2,3-butanediol from fermentation broths

Imidazolium ionic liquids-based salting-out extraction of 2,3-butanediol from fermentation broths

Accepted Manuscript Title: Imidazolium ionic liquids-based salting-out extraction of 2,3-butanediol from fermentation broths Authors: Jianying Dai, Hu...

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Accepted Manuscript Title: Imidazolium ionic liquids-based salting-out extraction of 2,3-butanediol from fermentation broths Authors: Jianying Dai, Hui Wang, Yan Li, Zhilong Xiu PII: DOI: Reference:

S1359-5113(18)30142-9 https://doi.org/10.1016/j.procbio.2018.05.015 PRBI 11351

To appear in:

Process Biochemistry

Received date: Revised date: Accepted date:

25-1-2018 27-4-2018 21-5-2018

Please cite this article as: Dai J, Wang H, Li Y, Xiu Z, Imidazolium ionic liquids-based salting-out extraction of 2,3-butanediol from fermentation broths, Process Biochemistry (2018), https://doi.org/10.1016/j.procbio.2018.05.015 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Imidazolium ionic liquids-based salting-out extraction of 2,3-butanediol from fermentation broths Jianying Dai

Hui Wang Yan Li

Zhilong Xiu*

School of Life Science and Biotechnology, Dalian University of Technology, Dalian 116024, P. R.

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China

*Corresponding author.

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Zhilong Xiu

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E-mail: [email protected]

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Tel/Fax: 86-411-84706369

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GRAPHICAL ABSTRACT

Top phase: Most of 2,3-butanediol and ionic liquids Part of water, salt and impurities

K2HPO4

Mixing

Bottom phase: Most of water and salt Part of 2,3-butanediol and ionic liquids and impurities

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Fermentation broth of 2,3-butanediol

Standing

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Ionic liquids

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HIGHLIGHTS

Partition of 2, 3-butandiol in ionic liquid-based aqueous two-phase systems



[C2mim][CF3SO3]- and [C4mim][Cl]-K2HPO4 systems were studied



Different trends of partition coefficient variation were observed



Selectivity for glucose, succinic acid and lactic acid was investigated



95.7% 2,3-Butanediol and 99.5% [C2mim][CF3SO3] were distributed to the top phase

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Abstract

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Salting-out extraction is an efficient technique for the separation of bio-based chemicals from

fermentation broths. So far, salting-out extraction of 2,3-butanediol has mainly focused on systems composed of organic solvents and salts, while less attention has been given to the green solvent  ionic liquids. In this research, imidazolium ionic liquids were used to examine the recovery of 2,3-butanediol from fermentation broths. The influences of the temperature and concentration of 2

2,3-butanediol on phase formation were determined using two systems of 1-butyl-3-methylimidazolium chloride

([C4mim][Cl])-K2HPO4

and

1-ethyl-3-methylimidazolium

trifluoromethanesulfonate

([C2mim][CF3SO3])-K2HPO4. The partition behaviors of 2,3-butandiol and ionic liquids, and the selectivity of 2,3-butanediol over glucose and organic acids were investigated and compared at

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different concentrations of ionic liquids and K2HPO4. Most of 2,3-butanediol and ionic liquids were distributed to the top phase, whereas different trends of partition coefficient variation were observed.

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Under the condition of 25% [C2mim][CF3SO3]-30% K2HPO4 (w/w), the recovery of 2,3-butanediol and [C2mim][CF3SO3] was 95.7% and 99.5%, respectively, and selectivity for glucose, succinic acid and

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lactic acid was 2.59, 115 and 20.8, respectively.

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Keywords: salting-out extraction; 2,3-butanediol; ionic liquids; partition behavior; fermentation broth

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1.

Introduction 2,3-Butanediol (2,3-BD) is one of the promising platform chemicals, which could be transformed

to 1,3-butadiene, methyl ethyl ketone, potential gasoline blending agent acetone 2,3-butanediol ketal, etc. [1-3]. The biological production of 2,3-BD demonstrated special advantages such as low toxicity to

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bacteria and wide substrate spectrum, and high concentration of product was obtained from different carbon sources and strains [2, 4-7]. Nowadays, separation of 2,3-BD from fermentation broths has

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become the bottleneck for large-scale production due to its high hydrophilicity and boiling point. Many primary recovery methods have been attempted and developed, such as solvent extraction, reactive

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extraction, salting-out, and salting-out extraction (SOE) [8, 9]. Among these methods, SOE is of great

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prospect for its high recovery, easy operation and scale-up. The partition behaviors of 2,3-BD in

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organic solvent-based SOE systems have been well studied [9-11]. When hydrophilic solvents were

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used, the recovery of 2,3-BD in top phase was generally greater than 90% at optimal conditions, and

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most of the impurities (residual substrates, byproducts, coloring matters, etc.) were removed [9]. However, organic solvents are flammable and combustible. To minimize the risk to the process safety

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solvents.

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and environment, it is essential to improve SOE process based on more eco-friendly and biocompatible

Ionic liquids (ILs), a type of salts which are liquid below 100C, are typically composed of

unsymmetrical organic cations and organic or inorganic anions. Due to its near-zero vapor pressure,

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high solvation interactions with organic and inorganic compounds, excellent thermal and chemical stabilities, they are widely used as solvents, reaction media, and catalysts in various biological processes, such as extraction and separation of bioactive compounds [12, 13], processing of biopolymers [14, 15], enzymatic catalysis [16], and production of renewable energy [17]. In the 4

production of bio-based chemicals, ILs are mainly used in the pretreatment of lignocellulose. Hundreds of ILs have been explored, and the ILs based on imidazolium cations are the most successful in cellulose dissolution [18]. The efficiency of cellulose hydrolysis was improved by increasing the accessible surface area of the substrates to solvents and cellulase [18, 19].

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ILs can form aqueous two-phase systems (ATPS) with organic or inorganic salts, carbohydrates and amino acids [20-22]. The ATPS formed by ILs and salts, a type of SOE systems [9], showed great

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potential in primary recovery of biological products, including proteins [23], bioactive compounds

(nicotine, eugenol, etc.) [24-26], and antibiotics [27, 28]. The phase equilibrium, separation mechanism

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and application were well discussed in the reviews, especially on proteins [22, 23, 29], while less

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endeavor was made concerning the separation of bio-based chemicals with high hydrophilicity.

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Till date, only the separation of 1,3-propanediol (1,3-PD) [30-32] and succinic acid [33] from

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fermentation broths were attempted, and the partition behaviors of target products were studied. The

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effects of cations and anions on 1,3-PD separation were studied using SOE systems composed of ILs and phosphate [30, 31]. 1,3-PD was distributed to the IL-rich phase when hydrophilic ILs were used.

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The partition coefficient of 1,3-PD was mainly influenced by the polarity or hydrogen-bond accepting

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ability () of cations or anions, and anions showed greater influence than cations [30]. The partition coefficient of 1,3-PD was increased with increasing polarity of cations, and the sequence of partition coefficient for anions was [CF3SO3]ˉ < [N(CN)2]ˉ  [SCN]ˉ < [CH3SO4]ˉ [30, 31]. The ionic liquid

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1-butyl-3-methylimidazolium trifluoromethansulfonate ([C4mim][CF3SO3]) was successfully recovered from the top phase and re-used in the separation of 1,3-PD [31]. As for succinic acid, a hydrophilic compound with charge, there was no obvious trend of partition coefficient variation with the chain length of alkyl when 1-alkyl-3-methylimidazolium bromide and KH2PO4 was applied [33]. Therefore, 5

SOE based on ILs was more complicated than organic solvents due to the characteristics of target products and the variety of cations and anions, and the combination of both. Moreover, the research on SOE of bio-based chemicals using ILs mainly focused on the partition behavior of target products, little attention was paid on the partition of ILs simultaneously, which was critical in evaluating the loss of

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ILs during the downstream processing. It is widely recognized that the properties of ILs are usually dominated by anions. The comparison

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of imidazolium, morpholinium and pyrrolidinium ionic liquids showed that core structure of cations had little influence on 1,3-PD distribution [30, 31]. Among all the ILs, the system of

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1-butyl-3-methylimidazolium chloride ([C4mim]]Cl])-K3PO4 was the first ATPS with which attempts

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were made to study the distribution of short chain alcohols [34]. Presently, imidazolium ILs have been

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extensively studied in many biological processes [12, 18, 23, 31, 33, 35], and some microorganisms

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showed high tolerance level to them [36]. Therefore, in this study water-miscible imidazolium ILs/salt

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systems were selected to examine the primary recovery of 2,3-BD from fermentation broths. The partition behaviors of 2,3-BD, ILs, glucose and organic acids were studied at different concentrations

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of ILs and salts, especially the recovery of 2,3-BD and ILs. The results showed that 2,3-BD could be

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efficiently recovered from fermentation broth with little loss of ILs at appropriate condition.

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Materials and methods

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2.1. Chemicals and materials Standard

2,3-BD

was

(1-butyl-3-methylimidazolium trifluoromethansulfonate),

purchased chloride),

from

Sigma.

[C2mim][CF3SO3]

[C2OHmim][Ser]

The

ILs

of

[C4mim][Cl]

(1-ethyl-3-methylimidazolium

(1-hydroxyethyl-3-methylimidazolium 6

serine),

[C4mim][CF3SO3], [C4mim][BF4], [C2mim][BF4], and [C2OHmim][BF4] were purchased from Shanghai Cheng Jie Chemical Co. Ltd., China. 2,3-BD for screening experiments was purchased from Hongtai Biological Co. Ltd., China. Other chemicals were purchased from Sinopharm Chemical

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Reagent Co., Ltd (Shanghai, China).

2.2. Preparation of fermentation broth

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Enterobacter cloacae CGMCC 6053 was used to produce 2,3-BD. The fermentation broth was

prepared according to the published method, with glucose used as the carbon source [37]. The

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fermentation broth was centrifuged for 10 min at 8000 rpm, and temperature of 4C, then the

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supernatant was kept at -20 C for the following SOE experiments. The concentrations of components

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succinic acid 5.4 g/L and lactic acid 2.9 g/L.

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in supernatant were as follows: 2,3-BD 90.6 g/L, glucose 17.8 g/L, ethanol 6.1 g/L, citric acid 8.2 g/L,

2.3. Phase diagrams of ionic liquid and K2HPO4 aqueous two-phase systems

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The phase diagrams were obtained using a turbidity titration method according to the published

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method [38]. Upon investigating the effects of 2,3-BD and the components in fermentation broths, K2HPO4 was dissolved in 2,3-BD solution or fermentation broths, respectively. The mass fraction of K2HPO4 (w1) and ionic liquid (w2) was calculated according to the following equations: 𝑚1 × 100 𝑚1 + 𝑚2 + 𝑚3

𝑤2 (%) =

𝑚2 × 100 𝑚1 + 𝑚2 + 𝑚3

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𝑤1 (%) =

where m1, m2 and m3 represents the amount of added K2HPO4, ionic liquid, and 2,3-BD solution (or fermentation broths), respectively.

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2.4. Salting-out extraction of 2,3-butanediol using imidazolium ionic liquids The salt was dissolved in the fermentation broths, then ionic liquid was added and mixed. The mixture was left to stand overnight at room temperature (18 ~ 25 C) for phase separation. The samples

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from top phases were diluted at different folds for concentration detection using high performance liquid chromatography (HPLC). The phase ratio (R), partition coefficient (K), recovery (Y) and

𝐶𝑗𝑡 𝐶𝑗𝑏

𝑌𝑗 (%) =

𝐶𝑗𝑡 𝑉𝑡 × 100 𝐶𝑗0 𝑉0

𝑌𝐼𝐿 (%) =

𝐶𝐼𝐿𝑡 𝑉𝑡 × 100 𝑚𝐼𝐿

𝐾𝐵𝐷 𝐾𝑗

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𝑆𝑗 =

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𝐾𝑗 =

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𝑉𝑡 𝑉𝑏

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𝑅=

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selectivity (S) were defined as follows:

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where Vt , Vb and V0 represents the volume of top phase, bottom phase, and fermentation broth added, respectively; Cjt, Cjb and Cj0 represents the concentrations of chemical j in the top phase, bottom phase,

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and fermentation broth, respectively; KBD is the partition coefficient of 2,3-BD; YIL, CILt and mIL

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represents the recovery of ionic liquid, concentration of IL in top phase, and the mass of ionic liquid added to the system, respectively.

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2.5. Analytical method 2,3-BD, ILs, glucose and organic acids were analyzed by HPLC equipped with an Aminex HPX-87H column (300 × 7.8 mm) and a refractive index detector (Waters 2414). Peak overlap of citric acid and phosphate was observed upon detecting samples from [C4mim][Cl]-K2HPO4 system. The 8

peaks of glucose, citric acid and phosphate were overlapped upon detecting samples from [C2mim][CF3SO3]-K2HPO4 system. Thus the distribution of citric acid in the two systems and glucose in

[C2mim][CF3SO3]

system

were

not

obtained.

The

distribution

of

glucose

at

25%

[C2mim][CF3SO3]-30% K2HPO4 (w/w) was obtained by detecting the concentration of glucose in the

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bottom phase using a glucose analyzer (Biosensor SBA-50, Shandong Academy of Sciences, China).

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The concentrations of proteins were determined by Bradford method.

Results and discussion

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3.1. Screening of SOE systems based on the recovery of 2,3-butanediol and ionic liquids

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There are no studies yet on salting-out extraction of 2,3-BD by IL-based systems, as such,

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appropriate systems were first determined. In previous studies of organic solvent-based SOE systems,

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some inorganic salts demonstrated high efficiency in the recovery of 2,3-BD and 1,3-PD, such as

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K2HPO4, (NH4)2SO4, Na2CO3, K2CO3, (NH4)2HPO4 and NaH2PO4 [9, 38, 39]. [C4mim][Cl] is a common IL used in lignocellulose pretreatment [40, 41] to produce 2,3-BD [35], acetoin [35] and

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biobutanol [42], and separation of proteins [43] and alcohols [34]. Therefore, for the purpose of this

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study [C4mim][Cl] was selected for salt screening experiments based on the recovery of 2,3-BD and ILs. Under present conditions, aqueous two phases were formed when the system contained K2HPO4, NaH2PO4 or K2CO3 (Table 1). The recovery of 2,3-BD varied from 73% to 90% with different salts,

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while the recovery of [C4mim][Cl] was greater than 90%. The high recovery of [C4mim][Cl] indicated that the salt-outing of [C4mim][Cl] was mainly affected by the characteristics of IL. Based on the recovery of 2,3-BD and [C4mim][Cl], K2HPO4 was selected for the following studies. Due to its wide application, [C4mim][Cl] was first selected for SOE experiment. The anions of

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[Cl]ˉ and [BF4]ˉ were the most used in the extraction and separation of small organic compounds from biomass [12], and dihydroxy alcohols displayed a greater miscibility in [C4mim][BF4] than those containing [Tf2N]ˉ and [PF6]ˉ [44]. The anion [CF3SO3]ˉ formed stable ILs, and [C4mim][CF3SO3] showed good efficiency in recovering 1,3-PD from fermentation broth [31]. When amino acid was used

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as anions, Ser showed much lower toxicity to bacteria than Pro, GLy and Ala in water-miscible imidazolium ILs [45]. Other ILs containing [C2OHmim]+ and [C2mim]+ were also selected due to their

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higher hydrophilicity and lower toxicity than [C4mim]+ [45]. Thus, a total of 7 ILs formed by 3 cations and 4 anions were selected to investigate the effects of cations and anions on phase formation and

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distribution of 2,3-BD and ILs.

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The systems containing [C4mim][BF4], [C2mim][BF4] and [C2OHmim][BF4] appeared as

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liquid-solid two phases, and [C2OHmim][Ser]-K2HPO4 system was homogeneous. This result

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demonstrated the strong influence of anion on phase separation. In the ATPS of [C4mim][Cl]-,

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[C2mim][CF3SO3]- or [C4mim][CF3SO3]-K2HPO4, most of the 2,3-BD and ILs were distributed to the top phase (Table 1). The distribution of 2,3-BD was influenced by the hydrogen bond forming ability

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of anions and cations of ILs, which was in agreement with the result obtained in 1,3-PD SOE study [30,

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31]. Higher recovery of 2,3-BD was obtained from [C4mim][Cl], which possessed a larger hydrogen bond accepting ability  (0.87) than [C4mim][CF3SO3] (0.46) [46]. The hydrogen bond donating ability

 of an IL was dominated by the cation, which depended very slightly on the anion [47]. The  value

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of [C2mim]+ was larger than that of [C4mim]+ [48]. Thus, the recovery of 2,3-BD obtained from [C2mim][CF3SO3] was greater than that from [C4mim][CF3SO3]. The system of [C4mim][Cl]-K2HPO4 showed a recovery of IL of about 98% and 2,3-BD of about 90%. Although the recovery of [C2mim][CF3SO3] was lower than that of [C4mim][Cl], the phase ratio of 0.91 was smaller than that of 10

[C4mim][Cl] (1.35). It was quite difficult to determine which system was more practical according to present data of [C4mim][Cl] and [C2mim][CF3SO3]. Therefore, both systems were selected for the purpose of this study.

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3.2 Phase diagrams of [C4mim][Cl]-K2HPO4 and [C2mim][CF3SO3]-K2HPO4 systems To examine the effect of 2,3-BD on phase formation, the phase diagrams were made with 2,3-BD

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solution and fermentation broths at different temperatures. As shown in Fig. 1A and 1C, in the presence of 2,3-BD, the location of the curve shifted from that of water, depending on the concentration of

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2,3-BD and IL structure. In [C4mim][Cl]-K2HPO4 system the phase formation was very sensitive to

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2,3-BD concentration, and shift could be observed even at a low concentration of 8.6 g/L. Obviously,

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the higher the 2,3-BD concentration, the larger the shift. This phenomenon meant that at the same

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concentration of K2HPO4 less [C4mim][Cl] was required to form ATPS when 2,3-BD was present.

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While in [C2mim][CF3SO3] system, only a small shift was observed at 88.8 g/L 2,3-BD. The formation of ATPS mainly resulted from salting-out effect and the hydration ability of ILs [22, 34]. The properties

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of ILs were dominated by anions, while the ability of IL anion to induce ATPS was closely correlated

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with the hydrogen-bonding accepting ability  [49]. Anions with lower  value present lower abilities to form coordinative bonds and to create hydration complexes, and are therefore more easily salted-out by conventional salts. [C4mim][Cl] has a larger  value, indicating weaker phase forming ability, so it

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was more sensitive when 2,3-BD was present. In fermentation broths, other components existed, such as byproducts of organic acids, residual

glucose and inorganic salts from medium, which also influenced the phase formation [9]. Upon application of fermentation broths (Fig. 1B and 1C), the shift of curves was observed, indicating that

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fermentation broths were beneficial for phase formation. The comparison of two curves obtained at 20 C and 35 C showed that temperature had little impact on phase formation. Thus the fermentation broths could be used for separation without lowering temperature, since most of the fermentation process was carried out at 30 ~ 37 C [5, 50-52]. For operation convenience, the separation in this work

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was carried out at room temperature.

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3.3 Effects of the concentrations of ILs and K2HPO4 on phase ratio

Phase ratio (R) is a very important parameter for practical application. When ATPS was utilized,

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large R usually meant large amount of water in top phase, which would increase energy consumption of

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water removal in the following process. As shown in Fig. 2, with increasing concentration of K2HPO4

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different phenomenon of R variation was observed. In [C4mim][Cl]-K2HPO4 system R decreased faster

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at lower K2HPO4 concentration, then gradually decreased to a constant level with increasing

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concentration of K2HPO4. According to the result, [C4mim][Cl] concentration had slight impact on phase ratio at high concentration of K2HPO4. Based on the results of [C4mim][Cl] system, the

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concentrations of 15% and 25% [C2mim][CF3SO3] (w/w) were selected. At 25% [C2mim][CF3SO3]

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(w/w) R demonstrated a similar decreasing trend to that in [C4mim][Cl] system, while at 15% [C2mim][CF3SO3] (w/w) R increased nearly to 0.55. The theoretical R values for these systems were

different and less than 1 due to the IL density and system composition. For operation convenience,

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R value less than 1 was preferred. Thus, the appropriate concentration of K2HPO4 should be greater than 22% for [C4mim][Cl], or 18% for [C2mim][CF3SO3].

3.4 Partition behaviors of 2,3-BD and ionic liquids The partition behaviors of 2,3-BD and ILs in the systems of [C2mim][CF3SO3]-K2HPO4 and 12

[C4mim][Cl]-K2HPO4 are presented in Fig. 3 and Fig. 4, respectively. In general, the partition coefficients of 2,3-BD (KBD) and ILs (KIL) were all greater than 1, indicating that 2,3-BD and ILs were preferred to be in the top phase. In a 10 ~ 20% (w/w) concentration of [C4mim][Cl], KBD was increased first to a highest titer then decreased with increasing concentration of K2HPO4, while only a decreasing

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trend was observed at 30% [C4mim][Cl] (w/w) (Fig. 3A). The highest KBD of 14 was obtained at 10% [C4mim][Cl]-32% K2HPO4 (w/w). The law for KBD in [C4mim][Cl]-K2HPO4 system was not

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consistent with that in organic solvent-based SOE systems, in which KBD only showed increasing

trend with increasing concentration of salt [9, 53-55]. In [C2mim][CF3SO3] system, KBD showed an

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increasing trend with increasing concentration of K2HPO4.

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Generally, the product partitioning was commanded by solute–solvent interactions, such as

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hydrogen-bonding, van der Waals, and electrostatic forces, and steric and conformational effects. The

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difference of product distribution in the two phases depended on the properties of the two aqueous

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phases as well as those of the solute. When the same salt was used, the different properties of these aqueous phases in the systems of [C4mim][Cl]-K2HPO4 and [C2mim][CF3SO3]-K2HPO4 were resulted

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from the different structures of ILs, especially the hydrogen bond forming ability. The hydrogen bond

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donating ability () of [C4mim][Cl] was 0.47, which was much smaller than hydrogen bond accepting ability  (0.87) [46]. Therefore the hydrogen bond accepting ability of [C4mim][Cl] played more important role. The  and  of [C2mim][CF3SO3] was not obtained. The  value of [C2mim]+ was

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predicted to be 0.75 [48], while the  of [C4mim][CF3SO3] was 0.46 [46]. It could be inferred that hydrogen bond donating ability of [C2mim][CF3SO3] was much stronger. The  and  of H2O was 1.17 and 0.14, respectively, while  and  of 2,3-BD were about 0.8 inferred from the data of 1,3-propanediol and 1,2-propandiol, respectively [46]. The main interaction of 2,3-BD with H2O and 13

ILs is hydrogen-bonding. Due to the different composition in the two aqueous phases of [C2mim][CF3SO3] and [C4mim][Cl] systems and the hydrogen bond forming ability of ILs, the interaction of 2,3-BD were very complex and different in these systems. Finally, different interactions made the KBD curves to display different trends in the [C2mim][CF3SO3] and [C4mim][Cl] systems.

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As a result of R and K, YBD showed a similar increasing-titer-decreasing trend with increasing concentration of K2HPO4 when [C4mim][Cl] concentration was 10% (w/w), while this trend decreased

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in [C4mim][Cl] concentration of 15 ~ 30% (w/w). More than 90% 2,3-BD could be recovered when

[C4mim][Cl] concentration was greater than 20% (w/w) and K2HPO4 concentration was less than 14%

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(w/w). In [C2mim][CF3SO3] system, YBD was gradually increased as a convex curve but no titer was

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obtained, similar to those in organic solvent-based SOE systems [53, 56]. Over 95% 2,3-BD was

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higher than 25% and 30% (w/w), respectively.

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extracted from fermentation broth when the concentrations of [C2mim][CF3SO3] and K2HPO4 were

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The partition behavior of ILs was a little similar to that of 2,3-BD in [C4mim][Cl]-K2HPO4 and [C2mim][CF3SO3]-K2HPO4 systems (Fig. 4). The KIL value of [C4mim][Cl] was increased with

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increasing concentration of K2HPO4 at [C4mim][Cl] concentration of 10 ~ 20% (w/w), while it

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decreased at 30% [C4mim][Cl] (w/w). Thus, recovery of [C4mim][Cl] was increased with increasing K2HPO4 concentration at 10% [C4mim][Cl] (w/w), while it decreased at higher IL concentration. The highest IL recovery of 97.5% was obtained at 30% [C4mim][Cl]-8% K2HPO4 (w/w), where 2,3-BD

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recovery was 96.3%. However, the large phase ratio of 5 indicated that this point was not a good operation condition. At [C4mim][Cl] concentration of 10% (w/w) and K2HPO4 concentration of 32% (w/w), the highest KIL of 21.7 was obtained when YIL was 92.3% and YBD was 89.5%, but the small phase ratio of 0.61 was beneficial for water removal of top phase. In [C2mim][CF3SO3] system KIL was 14

increased with increasing concentration of K2HPO4, and the recovery of IL was gradually increased, tending to a maximum value due to salting-out effect. When K2HPO4 concentration was greater than 26% for 25% IL or 34% for 15% IL, YIL was greater than 98% where YBD was greater than 90%. At 25% [C2mim][CF3SO3]-30% K2HPO4 (w/w) the recovery of 2,3-BD and IL was 95.7% and 99.9%,

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respectively.

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3.5 Selectivity of 2,3-butanediol over glucose and organic acids

The above results showed that [C2mim][CF3SO3] was more preferred for 2,3-BD separation and

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IL recovery. However, fermentation broths contained many impurities such as residual glucose and

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organic acid byproducts. Thus, selectivity of 2,3-BD over impurities was also an important parameter

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to evaluate the applicability of SOE systems except recovery of 2,3-BD and ILs.

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As earlier discussed, high recovery of 2,3-BD and ILs were obtained at IL concentrations of 10%

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and 30% [C4mim][Cl], and 25% [C2mim][CF3SO3] (w/w), as such the three concentrations were selected to examine the selectivity of 2,3-BD over glucose, lactic acid and succinic acid (Fig. 5). In

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[C4mim][Cl] SOE system, most of the selectivity values for glucose were greater than 10, indicating

CC E

2,3-BD as being more preferred than the glucose to be extracted into the top phase. At the condition of 10% [C4mim][Cl]-32% K2HPO4 (w/w), 84.2% of glucose was removed from the top phase. The distribution of organic acid was affected by [C4mim][Cl] concentration. At 10% [C4mim][Cl] (w/w), the

A

selectivity was usually less than 1 indicating a difficulty in separating 2,3-BD and organic acids. When [C4mim][Cl] concentration was increased to 30%, the selectivity was greater than 1.5 and decreased with increasing concentration of K2HPO4. When [C2mim][CF3SO3] was applied, most of succinic acid and lactic acid were distributed into

15

the bottom phase, and the selectivity was increased with increasing concentration of K2HPO4 (Fig. 5B). Under the condition of 25% [C2mim][CF3SO3]-30% K2HPO4 (w/w), the removal of lactic acid and succinic acid was 63.3% and 89.7%, respectively. As shown in Fig. 5B, it was much easier to separate 2,3-BD with succinic acid than lactic acid due to the higher hydrophilicity of succinic acid. The

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selectivity difference between the two ILs indicated organic acids as being easily extracted by ILs with large  and small , as presented by SOE of succinic acid in a research by Pratiwi et al [33, 46].

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The conditions to obtain highest KBD or YBD in the two systems are listed in Table 2. Although the

highest 2,3-BD recovery was obtained at 30% [C4mim][Cl]-8% K2HPO4 (w/w), the removal of organic

U

acids was less effective than [C2mim][CF3SO3]. Although the selectivity for glucose at 25%

N

[C2mim][CF3SO3]-30% K2HPO4 (w/w) was smaller than that from [C4mim][Cl]- K2HPO4, the value of

A

2.59 still meant that 2,3-BD was extracted more efficiently than glucose from fermentation broth.

M

Moreover, protein accumulation between the top phase and bottom phase was observed when

ED

[C2mim][CF3SO3]-K2HPO4 was applied. At 25% [C2mim][CF3SO3]-30% K2HPO4 (w/w), no soluble proteins were detected in top phase. When [C4mim][Cl]-K2HPO4 was applied, the accumulation of

PT

proteins was not observed. About 65% soluble proteins were reserved in the top phase at 10%

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[C4mim][Cl]-32% K2HPO4 (w/w), and almost 100% was reserved at 30% [C4mim][Cl]-8% K2HPO4 (w/w). At present, the system of 25% [C2mim][CF3SO3]-30% K2HPO4 (w/w) was the most efficient for the primary recovery of 2,3-BD from fermentation broth, but it was not perfect. More ILs should be

A

applied to improve the recovery and selectivity of 2,3-BD over impurities in future work.

4.

Conclusion The SOE systems of [C4mim][Cl]-K2HPO4 and [C2mim][CF3SO3]-K2HPO4 were screened from 7

16

ionic liquid-inorganic salt systems for the distribution investigation of 2,3-BD, ILs, glucose, succinic acid and lactic acid in fermentation broths. Phase diagrams showed that 2,3-BD participated in the phase formation, and higher 2,3-BD concentration in fermentation broths was beneficial for 2,3-BD recovery. Temperature had little impact on phase formation in the range of 20 ~ 35 C, so fermentation

IP T

broths could be directly used for SOE without lowering the temperature. Different partition behaviors of 2,3-BD were observed in the two systems, which were also not similar with those displayed in

SC R

organic solvent-inorganic salt systems. Most of 2,3-BD and ILs were partitioned to the top phase, while

the selectivity of 2,3-BD over organic acids and glucose displayed different trends in the two systems.

U

The protein accumulation between top phase and bottom phase was observed when

N

[C2mim][CF3SO3]-K2HPO4 system was applied, while most of the soluble proteins were extracted to

A

the top phase in [C4mim][Cl]-K2HPO4 system. Based on the recovery of 2,3-BD and ILs and the

M

removal of impurities, the system of 25% [C2mim][CF3SO3]-30% K2HPO4 (w/w) was selected for the

ED

separation of 2,3-BD from fermentation broths, where the recovery of 2,3-BD and IL was 95.7% and 99.5%, respectively, and the selectivity for glucose, succinic acid and lactic acid was 2.59, 115 and 20.8,

PT

respectively. This work provides the first investigation of ILs in the separation of 2,3-BD which

CC E

showed a promising application of ILs in the separation of bio-based chemicals.

A

Acknowledgements

This work was supported by the National Natural Science Foundation of China (Grant No.

21476042).

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artichoke-based fermentation broth, Chinese J. Chem. Eng. 19(4) (2011) 682-686.

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Figure legends:

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Fig. 1. Phase diagrams of aqueous two-phase systems: [C4mim][Cl]-K2HPO4 (A and B) and

A

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[C2mim][CF3SO3]-K2HPO4 (C).

22

IP T SC R U

N

Fig. 2. Phase ratio (R) variation at different concentrations of ILs and K2HPO4 in aqueous two-phase

A

CC E

PT

ED

M

A

systems of [C4mim][Cl]-K2HPO4 (A) and [C2mim][CF3SO3]-K2HPO4 (B) .

23

IP T SC R U N A M

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Fig. 3. Partition coefficient (KBD) and recovery (YBD) of 2,3-butanediol in aqueous two-phase systems

A

CC E

PT

of [C4mim][Cl]-K2HPO4 (A) and [C2mim][CF3SO3]-K2HPO4 (B).

24

IP T SC R U N A

M

Fig. 4. Partition coefficient (KIL) and recovery (YIL) of ionic liquids in aqueous two-phase systems of

A

CC E

PT

ED

[C4mim][Cl]-K2HPO4 (A) and [C2mim][CF3SO3]-K2HPO4 (B).

25

IP T SC R U

N

Fig. 5. Selectivity of 2,3-butanediol over impurities in aqueous two-phase systems of

A

[C4mim][Cl]-K2HPO4 (A) and [C2mim][CF3SO3]-K2HPO4 (B). Selectivity of 2,3-butanediol over

M

glucose (▇ and □), lactic acid (● and ○),and succinic acid (▲ and △). A, 10% (w/w)

A

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PT

ED

[C4mim][Cl], filled; 30% (w/w) [C4mim][Cl], unfilled. B, 25% (w/w) [C2mim][CF3SO3].

26

Table 1 Screening of IL-salt SOE systems based on the recovery of 2,3-BD and ILs System composition*

Recovery, % Phase formation

Inorganic salt (2 g)

[C4mim][Cl]

(NH4)2SO4

Solid-liquid two phases

ND

ND

(NH4)2HPO4

Solid-liquid two phases

ND

ND

Na2CO3

Solid-liquid two phases

ND

NaH2PO4

Aqueous two phases

K2CO3

Aqueous two phases

K2HPO4

Aqueous two phases

K2HPO4

Aqueous two phases

[C4mim][BF4]

SC R

73.4  0.1

89.6  1.0

88.5  2.6

82.7  1.3

80.7  1.2

89.9  1.8

One liquid phase

ND

ND

Solid-liquid two phases

ND

ND

Solid-liquid two phases

ND

ND

Solid-liquid two phases

ND

ND

U

98.6  0.1

M ED

PT

[C2mim][BF4]

ND

82.7  1.3

Aqueous two phases

[C2OHmim][Ser]

2,3-BD

95.7  0.5

A

[C2mim][CF3SO3]

[C2OHmim][BF4]

97.9  0.1

N

[C4mim][CF3SO3]

Ionic liquid

IP T

Ionic liquid (2 mL)

CC E

*The system was composed of 2 mL ionic liquid, 2 g salt and 4 mL 70.1 g/L 2,3-BD solution. After mixing, the

A

SOE system was stood overnight for phase separation at 20 C. ND, not determined.

27

Table

2

Comparison

of

the

partition

behaviors

in

[C4mim][Cl]-K2HPO4

Ionic liquid

Selectivity

and

[C2mim][CF3SO3]-K2HPO4 systems 2,3-BD Phase ratio YBD, %

KIL

YIL, %

0.86  0.01

25.8  0.8

95.7  0.1

ND*

0.61  0.01

14.1  2.4

89.5  2.0

21.7  2.5

92.3  0.2

5.0  0.2

5.2  0.9

96.3  0.5

8.2  2.2

97.5  0.5

SG

99.9  0.1 2.59  0.09

30% K2HPO4 (w/w) 10% [C4mim][Cl]-32%

43.1  1.5

30% [C4mim][Cl]-8% K2HPO4 (w/w)

115 1 0

11.2  0.7

SLA 20.8  0.5

0.28  0.06 1.16  0.08

SC R

K2HPO4 (w/w)

SSA

IP T

25% [C2mim][CF3SO3]-

KBD

4.94  0.18 4.76  0.15

A

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PT

ED

M

A

N

U

*ND, not determined. Selectivity of 2,3-butanediol over glucose (SG), succinic acid (SSA), and lactic acid (SLA).

28