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Salting-out extraction of acetoin from fermentation broths using hydroxylammonium ionic liquids as extractants
Journal Pre-proofs Salting-out extraction of acetoin from fermentation broths using hydroxylammonium ionic liquids as extractants Yan Li, Jian-Ying Dai, Zhi-Long Xiu PII: DOI: Reference:
S1383-5866(19)34541-1 https://doi.org/10.1016/j.seppur.2020.116584 SEPPUR 116584
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Separation and Purification Technology
Received Date: Revised Date: Accepted Date:
4 October 2019 10 January 2020 16 January 2020
Please cite this article as: Y. Li, J-Y. Dai, Z-L. Xiu, Salting-out extraction of acetoin from fermentation broths using hydroxylammonium ionic liquids as extractants, Separation and Purification Technology (2020), doi: https://doi.org/10.1016/j.seppur.2020.116584
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Salting-out extraction of acetoin from fermentation broths using hydroxylammonium ionic liquids as extractants Yan Li
Jian-Ying Dai
Zhi-Long Xiu*
School of Bioengineering, Dalian University of Technology, Dalian 116024, P. R. China
*Corresponding author. E-mail: [email protected]
1
Abstract 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. Keywords: Hydroxylammonium ionic liquids; Salting-out extraction; Acetoin; Partition behaviors; Extraction mechanism
2
1.
Introduction
Salting-out extraction (SOE) is a separation method used to extract a hydrophilic product from an aqueous solution with the aid of solvent as the extractant and salt as a salting-out reagent [1]. SOE has been widely used in the separation of bio-chemicals, proteins, and natural active compounds. It shows significant advantages in recovering hydrophilic products from fermentation broths, such as an integrated bioprocess combining solid-liquid separation, impurity removal and product concentration into one step [2-4], easy scale-up and low energy consumption [1]. However, most of organic solvents are flammable and volatile. To lower the risk to process safety and reduce the environmental impacts during extraction process, ionic liquids (ILs) as green solvents have been used in separation processes [5]. ILs are molten salts with a melting point below 100 °C, typically composed of organic cations and inorganic or organic anions [6]. The ILs show great potential to replace traditional organic solvents due to their characteristics such as negligible vapor pressure, low melting point, great solvation ability, and flexibility to modify their physical properties by altering the combination of cations and anions [7]. ILs are widely used in various fields such as catalyst in organic reaction, pretreatment of lignocellulose, separation of biomolecules, and electrolyte materials for batteries [4, 8-10]. In 2003, the first discovered IL-based aqueous two-phase system (ATPS) of 1-butyl-3-methylimidazolium chloride ([C4mim]Cl) and K3PO4 was reported by Gutowski and co-authors [11]. Since then more and more studies about ILs-based
3
ATPSs were reported, especially ILs based on imidazolium cations. The phase equilibrium, influencing factors and separation mechanism were well studied [12]. In fact, IL-based ATPS is also a kind of SOE systems (SOESs). Generally, the formation of IL-based SOESs can be considered to be a competition of water between hydrophilic ILs and inorganic salts [13], which is affected by the structures of ILs and salts, as well as the temperature and pH [14-16]. The ILs-based SOE was widely studied in the primary recovery of biological products, such as proteins, natural products, and antibiotics [4, 17-19], while less attention was paid on the recovery of bio-based chemicals. Until now, only the recoveries of 1,3-propanediol (1,3-PD), 2,3-butanediol (2,3-BD) and succinic acid (SA) from fermentation broths have been tried using imidazolium ILs-based SOESs [5, 14, 16, 20-22]. The results showed that the partition behavior of target product was more complicated than that in organic solvent-based SOESs. For example, hydrogen bond of the molecular interaction was the main force affecting the distribution of 1,3-PD and 2,3-BD [14, 21]. Therefore, the distribution coefficient was related to hydrogen-bond accepting ability. Increasing the hydrogen bond accepting ability of the anion or cation, the distribution coefficient of 1,3-PD increases, and the effect of anion is greater than that of cation [21]. Moreover, 1,3-PD and 2,3-BD take part in phase formation of SOESs. The addition of 1,3-PD and 2,3-BD changes the location of bimodal curves, depending on the type of IL and concentration of 1,3-PD and 2,3-BD [14, 20]. SA, a hydrophilic compound with charge, was efficiently extracted at lower pH with alcohol-based SOESs. When the pH value was over pH 6,
4
ILs-based SOESs had higher extractabilities than alcohol-based [16]. However, the high cost of ILs is not conducive to the industrial application. Moreover, the toxicity and biodegradability of ILs need to be taken into account. Toxicity tests showed that imidazolium-based ILs had antimicrobial activity against bacteria, fungi and microalgae, inhibiting cell growth and destroying cell integrity [23-27], while ILs based on hydroxylamines and short-chain acids were less toxic due to the similar structures with choline-based ILs which are easily biodegradable [28-30]. In recent years, the application of ILs containing hydroxylammonium has caused much attention due to the lower cost and toxicity. Now hydroxylammonium ILs are widely studied in the dissolution of zein, catalysis of the reaction, separation of sulphur components from diesel, gas absorbents for CO2 and SO2 [10, 31-33]. For example, ethanolammonium acetate (EOAA) and ethanolammonium lactate (EOAL) were attempted as catalysts in the aldol condensation reaction of acetoin with lignocellulose derived aldehydes, and the reaction achieved 83% conversion rate and 78% yield with 5-methyl furfural (5-MF) under the catalysis of EOAA [34]. The product of condensation is a type of fuel precursor which could be converted into long chain alkane by hydrodeoxygenation [34]. At present, there are few reports of hydroxylammonium ILs application in the separation of bio-based chemicals by SOES. When hydroxylammonium ILs was used as adjuvants (5 wt%) in PEG-based ATPS, the biphasic area did not extend in the phase diagram, but the purification factor of peroxidase was increased by 7.6 times [35]. If SOE of acetoin from fermentation broths using hydroxylammonium ILs achieves a high yield, further
5
purification process of acetoin from the top phase might be not required by coupling the separation with aldol condensation reaction to produce fuel precursor. Based on this idea, the application of hydroxylammonium-based ILs in the separation of bio-based chemicals was investigated in this work. Five ILs with different structures were prepared, and the abilities of SOES formation and acetoin separation were compared and studied. The extraction mechanism was explained by analyzing the formation and breakage of hydrogen bonds via ATR-IR spectra. As far as we know, there are no studies yet on SOE of acetoin by ILs-based systems. It is the first time that hydroxylammonium ILs have been used as extractants to separate bio-based chemicals by SOE.
2. Materials and method 2.1 Materials The raw materials of ionic liquid preparation and other chemicals were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Acetoin and K3PO4 were purchased from Aladdin (Shanghai, China), and meso-2,3-BD was purchased from Sigma-Aldrich.
2.2 Synthesis of hydroxylammonium-based ILs The ILs used in this work included the following: ethanolammonium acetate (EOAA), ethanolammonium propionate (EOAP), ethanolammonium butyrate (EOAB), isopropanolammonium acetate (IPOAA), ethanolammonium lactate (EOAL). All ILs
6
were synthesized by direct neutralization of hydroxylammonium with different carboxylic acids. Briefly, a certain amount of hydroxylammonium was added into a 500 ml three-necked flask which was placed in an ice water bath. Then, carboxylic acid was slowly dropped into the flask under mechanical stirring. After finishing the reaction, a small amount of water was removed by rotary evaporation. All synthesized ILs was stored in a dryer [36, 37].
2.3. Preparation of fermentation broth Bacillus subtilis CGMCC 13141 was used to produce acetoin. The fermentation broth was prepared according to the published method [38]. The fermentation broth was centrifuged for 10 minutes at 10000 rpm, and then the supernatant was kept at -20 °C for the following SOE experiments. The concentrations of components in supernatant were as follows: acetoin 57.7 g/L, 2,3-BD 30.9 g/L, acetic acid 5.1 g/L and lactic acid 5.3 g/L.
2.4. Phase diagram The phase diagrams were obtained using a turbidity titration method of hydroxylammonium ionic liquids and K3PO4 [5]. Firstly, K3PO4 and deionized water were added to the tube and mixed by a vortex mixer until the salt was thoroughly dissolved. Then ionic liquid was added drop by drop. After each addition, the solution was mixed for 2 minutes, standing for 2 minutes to ensure the stable phenomena. The molarity of K3PO4 (M1) and ionic liquid (M2) were calculated according to the
7
following equations: 𝑀1 =
𝑀2 =
𝑛1 𝑚1 + 𝑚2 + 𝑚3 𝑛2 𝑚1 + 𝑚2 + 𝑚3
where m1, m2 and m3 represent the mass (kg) of added K3PO4, ionic liquid, and deionized water, respectively, n1 and n2 represent the mole of added K3PO4 and ionic liquid, respectively.
2.5. Salting-out extraction of acetoin The K3PO4 was dissolved in the fermentation broth, and the ionic liquid was then added and vortexed for 2 minutes. After standing for 2 hours for phase balancing, the concentration of sample from top phase was detected using high performance liquid chromatography (HPLC). The phase ratio (R), partition coefficient (K), recovery (Y) and selectivity (S) were defined as follows: Vt
Phase ratio: R = Vb Cjt
Partition coefficient: Kj = Cjb Vt × Cjt
Recovery: Yj (%) = V0 × Recovery: YIL (%) = Selectivity: Sj =
Cj0
Vt × CILt mIL
Kac Kj
where Vt, Vb and V0 represent the volume of top phase, bottom phase, and fermentation broth added, respectively; Cjt, Cjb and Cj0 represent the concentration of chemical j in the top phase, bottom phase, and fermentation broth, respectively; Kac is
8
the partition coefficient of acetoin; YIL, CILt and mIL represent the recovery of ionic liquid, concentration of ionic liquid in the top phase, and the mass of ionic liquid added to the system, respectively. Kj is the partition coefficient of chemical j.
2.6. Analytical methods The structures of ILs were identified by Fourier transform infrared (Nicolet 6700, Thermo Fisher) and water content was determined by Karl Fischer titration (EasyPlus ET08, Mettler Toledo). Acetoin, ILs, 2,3-BD and organic acids in top phase were analyzed by HPLC with an Aminex HPX-87H column (300 × 7.8 mm) and a refractive index detector (Waters 2414), using 5 mM sulfuric acid as mobile phase at a flow rate of 0.6 mL/min. Due to peak overlap of acetoin and EOAP from EOAP– K3PO4 system, the content of acetoin from this system was analyzed by gas chromatography (GC-2010, Shimadzu, Japan) equipped with a capillary column BGB-174 (30 m × 0.25 mm, ID 0.25 µm df) and a FID detector. Before GC analysis, acetoin in the sample was extracted with ethyl acetate. The obtained ethyl acetate supernatant was diluted to suitable concentration with absolute ethyl acetate for GC analysis. Acetoin, ILs, 2,3-BD, and organic acid of bottom phase were deduced from the mass balance. Fourier-transform infrared spectrometer (Nicolet 6700, Thermo Fisher, USA) was used to characterize the extraction mechanism and diamond as an ATR crystal and spectra were evaluated between wave numbers of 400 to 4000 cm-1.
9
2.7. Statistics Each experiment was carried out in triplicate. The mean experimental values with standard deviations were given in the tables and figures.
3. Results and discussion 3.1. Effects of ILs structures and temperature on phase diagram In
this
work,
five
ILs
containing
two
cations
(ethanolammonium
and
isopropanolamonium) and 4 anions (acetate, propionate, butyrate and lactate) were synthesized for SOE study, and the structures of ILs were identified by FT-IR. The water content of ILs was about 1.3% except EOAL, in which it was 6.5% (data shown in the supplementary data). In previous works inorganic salts of (NH4)2SO4, K2HPO4, K2CO3, Na2CO3, (NH4)2HPO4, and K3PO4 have achieved high SOE efficiency [4, 14, 16, 17]. Considering the high hydrophilicity of ILs with groups of -NH3+, -OH and -COO[32], five inorganic salts (Na2CO3, (NH4)2SO4, K2HPO4, K3PO4, and CH3COONa) with good solubility were selected to investigate the ability of phase formation with ILs. It was found that the five ILs could not form ATPSs with saturated solutions of K2HPO4, Na2CO3, (NH4)2SO4 and CH3COONa at 25 C, except K3PO4. K3PO4 is one of the most investigated inorganic salts as a salting-out reagent due to the high kosmotropocity [20, 39, 40]. The ability of ILs to form SOES with salts can be related to the Gibbs free energy of hydration of the salt ions (∆Ghyd) [41]. Among the five selected salts, K3PO4 showed the highest hydration ability (∆Ghyd, kJ/mol: K3PO4,
10
-3650; K2HPO4, -2379; Na2CO3: -2045, (NH4)2SO4: -1650, CH3COONa: -730) [41, 42]. The phase diagrams for ILs and K3PO4 system were presented in Fig. 1. The influence of anion on the area of two-phase region was shown in the following order: EOAB > EOAP > EOAA > EOAL. The different phase-forming abilities were attributed to the polarities and hydrogen-bond accepting strengths of the anions [21]. The logarithm of the octanol–water distribution coefficients (log D) shows the hydrophobicity of organic compounds. The order of area of two-phase region was consistent with the decreasing order of log D of anions (butyric acid, -1.75; propionic acid, -2.16; acetic acid, -2.65; lactic acid, -4.10) [43]. Similar phenomenon was also observed when hydroxylammonium ILs were used as adjuvants in PEG-based ATPSs to increase the two-phase area [30]. IPOAA system had the larger two-phase region than EOAA. The increase in the cation alkyl chain length led to an increase in the hydrophobicity of fluid (an intrinsic result of the aliphatic part extension), and thus to a poorer solubility of the ionic liquid in water [12]. In general, ILs with lower water affinity require less salt to promote separation of the two phases, resulting in biphasic region enlargement [44].
11
Fig. 1. Phase diagrams of ILs-K3PO4-H2O systems at 25 C.
The effects of temperature on phase diagrams were examined at 25, 35, and 45 °C. As shown in Fig. 2, the temperature had little impact on phase diagrams. This temperature-insensitive behavior has been reported in other ILs-salt systems, such as [C4mim]Cl-K2HPO4,
[C2mim][CF3SO3]-K2HPO4,
[C4mim]Br-K2HPO4,
[Emim]DMP-acetate, and [Bpy]BF4-NaH2PO4 [14, 32, 45, 46]. The characteristic of temperature-insensitivity is beneficial for the application of these ILs-based SOE systems, because most of the fermentation processes of bio-chemicals were carried out at 30~37 °C [38, 47, 48]. For the convenience of operation, the following experiments were carried out in room without temperature control.
12
Fig. 2. Effects of temperature on phase diagram.
3.2 Screening of SOE systems based on the recovery of acetoin According to the phase diagram, more K3PO4 was required for EOAL to form SOES at the same concentration of ILs. The system composition molar ratio of n(K3PO4): n(IL): n(acetoin)=8:5:2 was selected to compare the extraction abilities of different ILs, in which EOAL system was homogeneous (Table 1). The partition coefficient showed that acetoin and ILs were preferred to distribute into the top phase. With the increase of carbon chain length of anions, the phase ratio and partition coefficient increased. The recovery of acetoin varied from 67% to 93% with different ILs. The highest recovery of acetoin and IL was obtained from EOAB, which carbon chain was 13
the longest among all anions. The structure of cation also demonstrated its influence. The hydrophilicity of isopropanolammonium was a little lower than that of ethanolammonium, thus the values of K and R increased a little. The hydrophobicity of solvent played a very important role in influencing the distribution of bio-chemicals [49]. As reported before, the structural differences between anions were greater than those between cations [50-52]. In terms of the recovery of acetoin and ILs, EOAB was selected for the following studies. Table 1 Screening of IL-K3PO4 SOES based on the recovery of acetoin and IL Phase
Partition coefficient
Recovery (%)
ILs ratio
Acetoin
ILs
Acetoin
ILs
EOAA
0.27 ± 0.01
7.85 ± 0.08
1.80 ± 0.04
67.9 ± 0.6
32.4 ± 0.5
EOAP
0.90 ± 0.05
5.71 ± 0.40
3.40 ± 0.13
84.5 ± 1.0
75.3 ± 0.5
EOAB
1.09 ± 0.03
12.2 ± 1.1
10.2 ± 0.6
93.0 ± 0.5
91.8 ± 0.3
IPOAA
0.37 ± 0.03
15.8 ± 1.3
1.92 ± 0.02
84.2 ± 1.7
41.5 ± 2.0
*The molar composition of system: n(K3PO4): n(IL): n(acetoin) = 8:5:2.
3.3 Salting-out extraction of acetoin from fermentation broth with EOAB-K3PO4 system 3.3.1 Effects of the EOAB and K3PO4 concentrations on phase ratio Phase ratio is an important parameter for practical application. Large volume of top phase means high solvents content, resulting in a cost increment of following concentration of target product and solvent removal. As shown in Fig. 3, with the 14
increasing concentration of ionic liquid and decreasing concentration of K3PO4, the phase ratio increased. This tendency was similar with the systems of [C4mim]Cl-K2HPO4
and
[EMPip][Br]-K2HPO4
[14,
22].
The
higher
the
concentrations of K3PO4, the slower the increase rate of phase ratio. In general, to facilitate organic reagent recovery, lower R value was preferred.
Fig. 3. Phase ratio variation at different concentrations of EOAB and K3PO4.
3.3.2 Partition behaviors of acetoin and ionic liquids The influences of EOAB and K3PO4 concentration on partition coefficient and recovery of acetoin and EOAB were shown in Fig. 4 and Fig. 5. The partition coefficient of acetoin was increased first to a highest value then decreased with increasing concentration of EOAB when K3PO4 concentration was 38% (w/w), while the decreasing trend were observed in the range of 28~34% (w/w) K3PO4 (Fig. 4a). The highest partition coefficient of acetoin (40.54) was obtained at 6% EOAB-38% K3PO4 (w/w). In a 28~34% (w/w) concentration of K3PO4, the recovery of acetoin increased firstly and decreased later, while a decreasing trend was observed at concentration of 38% (Fig. 4b). The highest recovery of acetoin (94.9%) was obtained 15
at 6% EOAB-34% K3PO4 (w/w). The concentration of K3PO4 had slight impact on partition coefficient and recovery of acetoin when the concentration of EOAB was over 18%. The variation of acetoin partition coefficient in EOAB-K3PO4 system was more complex than that in organic solvent-based SOESs, in which the partition coefficient increased with the increase of salt concentration [53]. As the concentration of K3PO4 varied from 28~34%, the recovery of acetoin increased due to the strong salting-out effect at lower IL concentration [54].
Fig. 4. Partition coefficient and recovery of acetoin in EOAB-K3PO4 system.
The variation of partition coefficient of EOAB is similar to acetoin. With the increase of EOAB concentration, the distribution coefficient showed a decreasing trend, while the recovery of EOAB was gradually increased (Fig. 5). The highest recovery of EOAB (86.3%) was obtained at 18% EOAB-34% K3PO4 (w/w).
16
Fig. 5. Partition coefficient and recovery of EOAB in EOAB-K3PO4 system.
The production of acetoin by microorganisms is usually accompanied by another product of 2,3-BD, which is also a promising platform chemical [14, 38, 53]. It is worthwhile to study the distribution behavior of 2,3-BD for its further recovery. With increasing concentration of EOAB, the partition coefficient of 2,3-BD showed a decreasing trend (Fig. 6a). Because of the difference in structure of 2,3-BD and acetoin, their distribution behaviors were not completely consistent. The recovery of 2,3-BD gradually increased as the concentration of EOAB increased (Fig. 6b). The highest recovery of 2,3-BD was 95.4% obtained at 18% EOAB-31% K3PO4. Further separation of 2,3-BD and acetoin in the top phase can be performed by vacuum distillation [1]. Previous work of imidazolium ILs showed high efficiency in SOE of 2,3-BD from fermentation broth [14], indicating acetoin also could be efficiently extracted by imidazolium ILs. Imidazolium IL of [C4mim][[OH] displayed catalytic activity in the reaction of acetoin and 5-MF, but the conversion rate and yield were much
lower
than
those
from
hydroxylammonium
ILs
[34].
Moreover,
hydroxylammonium ILs are easier to be prepared with lower cost and biological 17
toxicity [14]. Therefore, hydroxylammonium ILs have more potential as extractants in SOE and catalysts in aldol condensation reaction of acetoin.
Fig. 6. Partition coefficient and recovery of 2,3-butanediol in EOAB-K3PO4 system.
3.3.3 Selectivity of acetoin to organic acids It can be seen from the above results that EOAB-K3PO4 system can achieve the separation of acetoin and the recovery of IL. However, except target products the fermentation broth also contained some byproducts such as lactic acid and acetic acid. Thus, selectivity of acetoin to byproducts was also an important parameter to evaluate the applicability of SOES except recovery of acetoin and ILs. As shown in Fig. 7, high concentration of EOAB was not conducive to the separation of acetic acid and lactic acid. The selectivity to lactic acid was greater than 5 under the condition of the highest recovery of acetoin, indicating that acetoin and lactic acid could be separated well. The highest selectivity to lactic acid was obtained at 6% EOAB and 38% K3PO4, which was16.46 (Fig. 7b). Most of the selectivity values for acetic acid were less than 1, illustrating the difficulty in separating acetoin and acetic acids (Fig. 7a). The highest selectivity to acetic acid of 3.85 was obtained at 6% 18
EOAB-38% K3PO4, indicating acetoin was much more preferred to distribute into the top phase than acetic acid at this condition. Due to the higher hydrophilicity of lactic acid, it was much easier to separate acetoin with lactic acid than acetic acid. Although the highest recovery of acetoin was obtained in the system of 6% EOAB-34% K3PO4 (w/w), the recovery of EOAB was not the highest. In a word, the system of 6% EOAB-34% K3PO4 (w/w) was the most efficient for the direct recovery of acetoin from fermentation broth, but not for EOAB recovery and the removals of lactic and acetic acid. Concerning all these factors, 6% EOAB-38% K3PO4 (w/w) owed more application values, as the recoveries of acetoin and EOAB were similar to those obtained from 6% EOAB-34% K3PO4 while 2,3-BD recovery was a little lower, which were 92.7%, 76.0% and 86.0%, respectively. It has been reported that EOAA and EOAL are excellent catalysts for aldol condensation reaction of acetoin with 5-MF, and the catalyst EOAA could be recycled five times with no apparent loss of catalytic activity [34]. Acetoin in the top phase can react with added 5-MF to form liquid hydrocarbon fuel precursor under the catalysis of basic ILs. Thus, the following operation of acetoin purification was not required, and the separation of acetoin from fermentation broth and derivative production was integrated.
19
Fig. 7. Selectivity of acetoin to organic acids in SOES of EOAB-K3PO4. (a), acetic acid; (b), lactic acid.
3.4. Extraction mechanism The interaction of molecules is a critical factor for SOE, especially hydrogen bonds. The extraction ability in SOE is affected by the polarity of organic solvents [3]. Salting-out effect is the basis for the formation of SOESs consisting of ILs and salts. For ILs with a fixed anions and different cations, steric and entropic contributions are two main factors for the formation of ILs-based SOESs [12]. The ability of ILs to induce SOESs is closely related to the decrease of hydrogen bond accepting ability or the increase of hydrogen bond acidity of the ILs anion [21]. Compared with Kamlet-Taft parameters (polarisability π*, hydrogen-bond donating ability α, and hydrogen-bond accepting ability β), IR analysis is a more intuitive method to explore the hydrogen bond, which is the main interaction force in the SOE of acetoin. As shown in Fig. 8, when EOAB was added into H2O solution, there was a great shift of the stretching vibration of O-H in H2O (3331 cm-1) to lower wavenumber (3144 cm-1), reflecting the formation of hydrogen bond between water molecules and 20
EOAB. The ν (-COO-), centered at around 1528 cm-1 and 1397 cm-1, were red-shifted by about 9 cm-1 and 4 cm-1, respectively, further suggesting that the breakage of intermolecular hydrogen bonding of H2O. The stretching vibration of C=O in the carboxylic acid can result in a strong absorption peak around 1700 cm-1. The symmetrical stretching vibration and asymmetric stretching vibration of -COO(between about 1300-1600 cm-1) appeared after carboxylic acid reacted with base to form carboxylate, where the strong absorption peak around 1700 cm-1 of C=O disappeared [55]. With the addition of acetoin to H2O-EOAB system, there was an obvious shift from 3144 cm-1 to 3337 cm-1, reflecting the breakage of intermolecular hydrogen bond between water and EOAB and demonstrating the interaction of acetoin and H2O-EOAB system, which was also proved by the red shift of symmetric and asymmetric stretching vibrations of -NH2 in H2O-EOAB system. It can be seen that the structure of acetoin was not changed in the IL phase due to negligible shift of the stretching vibration of C=O in acetoin and no new chemical bonds generated. And there was a bigger shift of the symmetrical stretching vibration (1528 cm-1) to higher wavenumber (1548 cm-1) of -COO- in EOAB with the addition of acetoin into EOAB, implying the breakage of intermolecular hydrogen bond of EOAB and proving the presence of interaction between EOAB and acetoin. Although H2O and ionic liquids, as well as acetoin, exist interactions in SOES, the interaction between EOAB and acetoin was much stronger than those because EOAB is able to form more hydrogen bonds with acetoin. Furthermore, the stretching vibration of N-H, O-H and -COO- in hydroxylammonium IL illustrated the formation of intermolecular hydrogen bonds
21
with
acetoin.
These
results
clearly
showed
the
interaction
between
hydroxylammonium ILs and acetoin, explaining the high extraction efficiency of the SOESs.
Fig. 8. ATR-IR spectra of H2O (a), the mixture of acetoin and water (b), acetoin (c), the mixture of EOAB and water (d), EOAB (e), the mixture of acetoin and EOAB (f),
22
the mixture of acetoin, EOAB and water (g). 4. Conclusion In order to find a cheap and environmentally friendly SOES for separation of bio-chemicals, five hydroxylammonium ILs of EOAA, EOAP, EOAB, EOAL, and IPOAA were synthesized. The phase diagram showed the IL phase-forming ability was closely related to the carbon chain length of anions and cations. The temperature had little effect on the phase formation in the range of 25 ~ 45 °C, which was beneficial to separate of target substances in fermentation broths. The distribution behaviors of acetoin, EOAB and 2,3-BD, and selectivity of acetoin to lactic acid and acetic acid were investigated under different concentrations of EOAB and K3PO4. Most of acetoin, EOAB and 2,3-BD were partitioned to the top phase and the selectivity to lactic acid were larger than acetic acid. Under the condition of 6% EOAB-38% K3PO4 (w/w) the recovery of acetoin, IL and 2,3-butandiol was 92.7%, 76.0% and 95.0%, respectively, and the selectivity to lactic acid and acetic acid was 5.35 and 1.41, respectively. The AIR-IR sepectra showed the hydrogen bond formed between acetoin and O-H, N-H, -COO- of hydroxylamine IL, which played an important role in the efficient extraction of acetoin from fermentation broth. It is the first time that hydroxylammonium IL was used as salting-out agent to separate acetoin, demonstrating its potential in the separation of bio-chemicals. Acknowledgement This work was supported by the National Natural Science Foundation of China (Grant No. 21978038).
23
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CRediT authorship contribution statement Yan Li: Conceptualization, Methodology, Investigation, Writing - original draft, Data curation, Formal analysis. Jian-Ying Dai: Conceptualization, Writing – review & editing, Resources, Project administration, Supervision, Funding acquisition. Zhi-Long Xiu: Conceptualization, Writing – review & editing, Resources, Supervision.
Declaration of Competing Interest The authors declared that there is no Conflict of Interest.
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Highlights
Phase forming abilities of hydroxylammonium ionic liquids with K3PO4 were studied
Acetoin partition behavior in ionic liquid-K3PO4 system was studied
93% acetoin and 76% ethanolammonium butyrate were distributed into the top phase
The selectivity of acetoin over lactic acid and acetic acid was 16.46 and 3.85
IR spectra showed the intermolecular hydrogen bonds between acetoin and ILs
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S1383-5866(19)34541-1 https://doi.org/10.1016/j.seppur.2020.116584 SEPPUR 116584
To appear in:
Separation and Purification Technology
Received Date: Revised Date: Accepted Date:
4 October 2019 10 January 2020 16 January 2020
Please cite this article as: Y. Li, J-Y. Dai, Z-L. Xiu, Salting-out extraction of acetoin from fermentation broths using hydroxylammonium ionic liquids as extractants, Separation and Purification Technology (2020), doi: https://doi.org/10.1016/j.seppur.2020.116584
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Salting-out extraction of acetoin from fermentation broths using hydroxylammonium ionic liquids as extractants Yan Li
Jian-Ying Dai
Zhi-Long Xiu*
School of Bioengineering, Dalian University of Technology, Dalian 116024, P. R. China
*Corresponding author. E-mail: [email protected]
1
Abstract 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. Keywords: Hydroxylammonium ionic liquids; Salting-out extraction; Acetoin; Partition behaviors; Extraction mechanism
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1.
Introduction
Salting-out extraction (SOE) is a separation method used to extract a hydrophilic product from an aqueous solution with the aid of solvent as the extractant and salt as a salting-out reagent [1]. SOE has been widely used in the separation of bio-chemicals, proteins, and natural active compounds. It shows significant advantages in recovering hydrophilic products from fermentation broths, such as an integrated bioprocess combining solid-liquid separation, impurity removal and product concentration into one step [2-4], easy scale-up and low energy consumption [1]. However, most of organic solvents are flammable and volatile. To lower the risk to process safety and reduce the environmental impacts during extraction process, ionic liquids (ILs) as green solvents have been used in separation processes [5]. ILs are molten salts with a melting point below 100 °C, typically composed of organic cations and inorganic or organic anions [6]. The ILs show great potential to replace traditional organic solvents due to their characteristics such as negligible vapor pressure, low melting point, great solvation ability, and flexibility to modify their physical properties by altering the combination of cations and anions [7]. ILs are widely used in various fields such as catalyst in organic reaction, pretreatment of lignocellulose, separation of biomolecules, and electrolyte materials for batteries [4, 8-10]. In 2003, the first discovered IL-based aqueous two-phase system (ATPS) of 1-butyl-3-methylimidazolium chloride ([C4mim]Cl) and K3PO4 was reported by Gutowski and co-authors [11]. Since then more and more studies about ILs-based
3
ATPSs were reported, especially ILs based on imidazolium cations. The phase equilibrium, influencing factors and separation mechanism were well studied [12]. In fact, IL-based ATPS is also a kind of SOE systems (SOESs). Generally, the formation of IL-based SOESs can be considered to be a competition of water between hydrophilic ILs and inorganic salts [13], which is affected by the structures of ILs and salts, as well as the temperature and pH [14-16]. The ILs-based SOE was widely studied in the primary recovery of biological products, such as proteins, natural products, and antibiotics [4, 17-19], while less attention was paid on the recovery of bio-based chemicals. Until now, only the recoveries of 1,3-propanediol (1,3-PD), 2,3-butanediol (2,3-BD) and succinic acid (SA) from fermentation broths have been tried using imidazolium ILs-based SOESs [5, 14, 16, 20-22]. The results showed that the partition behavior of target product was more complicated than that in organic solvent-based SOESs. For example, hydrogen bond of the molecular interaction was the main force affecting the distribution of 1,3-PD and 2,3-BD [14, 21]. Therefore, the distribution coefficient was related to hydrogen-bond accepting ability. Increasing the hydrogen bond accepting ability of the anion or cation, the distribution coefficient of 1,3-PD increases, and the effect of anion is greater than that of cation [21]. Moreover, 1,3-PD and 2,3-BD take part in phase formation of SOESs. The addition of 1,3-PD and 2,3-BD changes the location of bimodal curves, depending on the type of IL and concentration of 1,3-PD and 2,3-BD [14, 20]. SA, a hydrophilic compound with charge, was efficiently extracted at lower pH with alcohol-based SOESs. When the pH value was over pH 6,
4
ILs-based SOESs had higher extractabilities than alcohol-based [16]. However, the high cost of ILs is not conducive to the industrial application. Moreover, the toxicity and biodegradability of ILs need to be taken into account. Toxicity tests showed that imidazolium-based ILs had antimicrobial activity against bacteria, fungi and microalgae, inhibiting cell growth and destroying cell integrity [23-27], while ILs based on hydroxylamines and short-chain acids were less toxic due to the similar structures with choline-based ILs which are easily biodegradable [28-30]. In recent years, the application of ILs containing hydroxylammonium has caused much attention due to the lower cost and toxicity. Now hydroxylammonium ILs are widely studied in the dissolution of zein, catalysis of the reaction, separation of sulphur components from diesel, gas absorbents for CO2 and SO2 [10, 31-33]. For example, ethanolammonium acetate (EOAA) and ethanolammonium lactate (EOAL) were attempted as catalysts in the aldol condensation reaction of acetoin with lignocellulose derived aldehydes, and the reaction achieved 83% conversion rate and 78% yield with 5-methyl furfural (5-MF) under the catalysis of EOAA [34]. The product of condensation is a type of fuel precursor which could be converted into long chain alkane by hydrodeoxygenation [34]. At present, there are few reports of hydroxylammonium ILs application in the separation of bio-based chemicals by SOES. When hydroxylammonium ILs was used as adjuvants (5 wt%) in PEG-based ATPS, the biphasic area did not extend in the phase diagram, but the purification factor of peroxidase was increased by 7.6 times [35]. If SOE of acetoin from fermentation broths using hydroxylammonium ILs achieves a high yield, further
5
purification process of acetoin from the top phase might be not required by coupling the separation with aldol condensation reaction to produce fuel precursor. Based on this idea, the application of hydroxylammonium-based ILs in the separation of bio-based chemicals was investigated in this work. Five ILs with different structures were prepared, and the abilities of SOES formation and acetoin separation were compared and studied. The extraction mechanism was explained by analyzing the formation and breakage of hydrogen bonds via ATR-IR spectra. As far as we know, there are no studies yet on SOE of acetoin by ILs-based systems. It is the first time that hydroxylammonium ILs have been used as extractants to separate bio-based chemicals by SOE.
2. Materials and method 2.1 Materials The raw materials of ionic liquid preparation and other chemicals were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Acetoin and K3PO4 were purchased from Aladdin (Shanghai, China), and meso-2,3-BD was purchased from Sigma-Aldrich.
2.2 Synthesis of hydroxylammonium-based ILs The ILs used in this work included the following: ethanolammonium acetate (EOAA), ethanolammonium propionate (EOAP), ethanolammonium butyrate (EOAB), isopropanolammonium acetate (IPOAA), ethanolammonium lactate (EOAL). All ILs
6
were synthesized by direct neutralization of hydroxylammonium with different carboxylic acids. Briefly, a certain amount of hydroxylammonium was added into a 500 ml three-necked flask which was placed in an ice water bath. Then, carboxylic acid was slowly dropped into the flask under mechanical stirring. After finishing the reaction, a small amount of water was removed by rotary evaporation. All synthesized ILs was stored in a dryer [36, 37].
2.3. Preparation of fermentation broth Bacillus subtilis CGMCC 13141 was used to produce acetoin. The fermentation broth was prepared according to the published method [38]. The fermentation broth was centrifuged for 10 minutes at 10000 rpm, and then the supernatant was kept at -20 °C for the following SOE experiments. The concentrations of components in supernatant were as follows: acetoin 57.7 g/L, 2,3-BD 30.9 g/L, acetic acid 5.1 g/L and lactic acid 5.3 g/L.
2.4. Phase diagram The phase diagrams were obtained using a turbidity titration method of hydroxylammonium ionic liquids and K3PO4 [5]. Firstly, K3PO4 and deionized water were added to the tube and mixed by a vortex mixer until the salt was thoroughly dissolved. Then ionic liquid was added drop by drop. After each addition, the solution was mixed for 2 minutes, standing for 2 minutes to ensure the stable phenomena. The molarity of K3PO4 (M1) and ionic liquid (M2) were calculated according to the
7
following equations: 𝑀1 =
𝑀2 =
𝑛1 𝑚1 + 𝑚2 + 𝑚3 𝑛2 𝑚1 + 𝑚2 + 𝑚3
where m1, m2 and m3 represent the mass (kg) of added K3PO4, ionic liquid, and deionized water, respectively, n1 and n2 represent the mole of added K3PO4 and ionic liquid, respectively.
2.5. Salting-out extraction of acetoin The K3PO4 was dissolved in the fermentation broth, and the ionic liquid was then added and vortexed for 2 minutes. After standing for 2 hours for phase balancing, the concentration of sample from top phase was detected using high performance liquid chromatography (HPLC). The phase ratio (R), partition coefficient (K), recovery (Y) and selectivity (S) were defined as follows: Vt
Phase ratio: R = Vb Cjt
Partition coefficient: Kj = Cjb Vt × Cjt
Recovery: Yj (%) = V0 × Recovery: YIL (%) = Selectivity: Sj =
Cj0
Vt × CILt mIL
Kac Kj
where Vt, Vb and V0 represent the volume of top phase, bottom phase, and fermentation broth added, respectively; Cjt, Cjb and Cj0 represent the concentration of chemical j in the top phase, bottom phase, and fermentation broth, respectively; Kac is
8
the partition coefficient of acetoin; YIL, CILt and mIL represent the recovery of ionic liquid, concentration of ionic liquid in the top phase, and the mass of ionic liquid added to the system, respectively. Kj is the partition coefficient of chemical j.
2.6. Analytical methods The structures of ILs were identified by Fourier transform infrared (Nicolet 6700, Thermo Fisher) and water content was determined by Karl Fischer titration (EasyPlus ET08, Mettler Toledo). Acetoin, ILs, 2,3-BD and organic acids in top phase were analyzed by HPLC with an Aminex HPX-87H column (300 × 7.8 mm) and a refractive index detector (Waters 2414), using 5 mM sulfuric acid as mobile phase at a flow rate of 0.6 mL/min. Due to peak overlap of acetoin and EOAP from EOAP– K3PO4 system, the content of acetoin from this system was analyzed by gas chromatography (GC-2010, Shimadzu, Japan) equipped with a capillary column BGB-174 (30 m × 0.25 mm, ID 0.25 µm df) and a FID detector. Before GC analysis, acetoin in the sample was extracted with ethyl acetate. The obtained ethyl acetate supernatant was diluted to suitable concentration with absolute ethyl acetate for GC analysis. Acetoin, ILs, 2,3-BD, and organic acid of bottom phase were deduced from the mass balance. Fourier-transform infrared spectrometer (Nicolet 6700, Thermo Fisher, USA) was used to characterize the extraction mechanism and diamond as an ATR crystal and spectra were evaluated between wave numbers of 400 to 4000 cm-1.
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2.7. Statistics Each experiment was carried out in triplicate. The mean experimental values with standard deviations were given in the tables and figures.
3. Results and discussion 3.1. Effects of ILs structures and temperature on phase diagram In
this
work,
five
ILs
containing
two
cations
(ethanolammonium
and
isopropanolamonium) and 4 anions (acetate, propionate, butyrate and lactate) were synthesized for SOE study, and the structures of ILs were identified by FT-IR. The water content of ILs was about 1.3% except EOAL, in which it was 6.5% (data shown in the supplementary data). In previous works inorganic salts of (NH4)2SO4, K2HPO4, K2CO3, Na2CO3, (NH4)2HPO4, and K3PO4 have achieved high SOE efficiency [4, 14, 16, 17]. Considering the high hydrophilicity of ILs with groups of -NH3+, -OH and -COO[32], five inorganic salts (Na2CO3, (NH4)2SO4, K2HPO4, K3PO4, and CH3COONa) with good solubility were selected to investigate the ability of phase formation with ILs. It was found that the five ILs could not form ATPSs with saturated solutions of K2HPO4, Na2CO3, (NH4)2SO4 and CH3COONa at 25 C, except K3PO4. K3PO4 is one of the most investigated inorganic salts as a salting-out reagent due to the high kosmotropocity [20, 39, 40]. The ability of ILs to form SOES with salts can be related to the Gibbs free energy of hydration of the salt ions (∆Ghyd) [41]. Among the five selected salts, K3PO4 showed the highest hydration ability (∆Ghyd, kJ/mol: K3PO4,
10
-3650; K2HPO4, -2379; Na2CO3: -2045, (NH4)2SO4: -1650, CH3COONa: -730) [41, 42]. The phase diagrams for ILs and K3PO4 system were presented in Fig. 1. The influence of anion on the area of two-phase region was shown in the following order: EOAB > EOAP > EOAA > EOAL. The different phase-forming abilities were attributed to the polarities and hydrogen-bond accepting strengths of the anions [21]. The logarithm of the octanol–water distribution coefficients (log D) shows the hydrophobicity of organic compounds. The order of area of two-phase region was consistent with the decreasing order of log D of anions (butyric acid, -1.75; propionic acid, -2.16; acetic acid, -2.65; lactic acid, -4.10) [43]. Similar phenomenon was also observed when hydroxylammonium ILs were used as adjuvants in PEG-based ATPSs to increase the two-phase area [30]. IPOAA system had the larger two-phase region than EOAA. The increase in the cation alkyl chain length led to an increase in the hydrophobicity of fluid (an intrinsic result of the aliphatic part extension), and thus to a poorer solubility of the ionic liquid in water [12]. In general, ILs with lower water affinity require less salt to promote separation of the two phases, resulting in biphasic region enlargement [44].
11
Fig. 1. Phase diagrams of ILs-K3PO4-H2O systems at 25 C.
The effects of temperature on phase diagrams were examined at 25, 35, and 45 °C. As shown in Fig. 2, the temperature had little impact on phase diagrams. This temperature-insensitive behavior has been reported in other ILs-salt systems, such as [C4mim]Cl-K2HPO4,
[C2mim][CF3SO3]-K2HPO4,
[C4mim]Br-K2HPO4,
[Emim]DMP-acetate, and [Bpy]BF4-NaH2PO4 [14, 32, 45, 46]. The characteristic of temperature-insensitivity is beneficial for the application of these ILs-based SOE systems, because most of the fermentation processes of bio-chemicals were carried out at 30~37 °C [38, 47, 48]. For the convenience of operation, the following experiments were carried out in room without temperature control.
12
Fig. 2. Effects of temperature on phase diagram.
3.2 Screening of SOE systems based on the recovery of acetoin According to the phase diagram, more K3PO4 was required for EOAL to form SOES at the same concentration of ILs. The system composition molar ratio of n(K3PO4): n(IL): n(acetoin)=8:5:2 was selected to compare the extraction abilities of different ILs, in which EOAL system was homogeneous (Table 1). The partition coefficient showed that acetoin and ILs were preferred to distribute into the top phase. With the increase of carbon chain length of anions, the phase ratio and partition coefficient increased. The recovery of acetoin varied from 67% to 93% with different ILs. The highest recovery of acetoin and IL was obtained from EOAB, which carbon chain was 13
the longest among all anions. The structure of cation also demonstrated its influence. The hydrophilicity of isopropanolammonium was a little lower than that of ethanolammonium, thus the values of K and R increased a little. The hydrophobicity of solvent played a very important role in influencing the distribution of bio-chemicals [49]. As reported before, the structural differences between anions were greater than those between cations [50-52]. In terms of the recovery of acetoin and ILs, EOAB was selected for the following studies. Table 1 Screening of IL-K3PO4 SOES based on the recovery of acetoin and IL Phase
Partition coefficient
Recovery (%)
ILs ratio
Acetoin
ILs
Acetoin
ILs
EOAA
0.27 ± 0.01
7.85 ± 0.08
1.80 ± 0.04
67.9 ± 0.6
32.4 ± 0.5
EOAP
0.90 ± 0.05
5.71 ± 0.40
3.40 ± 0.13
84.5 ± 1.0
75.3 ± 0.5
EOAB
1.09 ± 0.03
12.2 ± 1.1
10.2 ± 0.6
93.0 ± 0.5
91.8 ± 0.3
IPOAA
0.37 ± 0.03
15.8 ± 1.3
1.92 ± 0.02
84.2 ± 1.7
41.5 ± 2.0
*The molar composition of system: n(K3PO4): n(IL): n(acetoin) = 8:5:2.
3.3 Salting-out extraction of acetoin from fermentation broth with EOAB-K3PO4 system 3.3.1 Effects of the EOAB and K3PO4 concentrations on phase ratio Phase ratio is an important parameter for practical application. Large volume of top phase means high solvents content, resulting in a cost increment of following concentration of target product and solvent removal. As shown in Fig. 3, with the 14
increasing concentration of ionic liquid and decreasing concentration of K3PO4, the phase ratio increased. This tendency was similar with the systems of [C4mim]Cl-K2HPO4
and
[EMPip][Br]-K2HPO4
[14,
22].
The
higher
the
concentrations of K3PO4, the slower the increase rate of phase ratio. In general, to facilitate organic reagent recovery, lower R value was preferred.
Fig. 3. Phase ratio variation at different concentrations of EOAB and K3PO4.
3.3.2 Partition behaviors of acetoin and ionic liquids The influences of EOAB and K3PO4 concentration on partition coefficient and recovery of acetoin and EOAB were shown in Fig. 4 and Fig. 5. The partition coefficient of acetoin was increased first to a highest value then decreased with increasing concentration of EOAB when K3PO4 concentration was 38% (w/w), while the decreasing trend were observed in the range of 28~34% (w/w) K3PO4 (Fig. 4a). The highest partition coefficient of acetoin (40.54) was obtained at 6% EOAB-38% K3PO4 (w/w). In a 28~34% (w/w) concentration of K3PO4, the recovery of acetoin increased firstly and decreased later, while a decreasing trend was observed at concentration of 38% (Fig. 4b). The highest recovery of acetoin (94.9%) was obtained 15
at 6% EOAB-34% K3PO4 (w/w). The concentration of K3PO4 had slight impact on partition coefficient and recovery of acetoin when the concentration of EOAB was over 18%. The variation of acetoin partition coefficient in EOAB-K3PO4 system was more complex than that in organic solvent-based SOESs, in which the partition coefficient increased with the increase of salt concentration [53]. As the concentration of K3PO4 varied from 28~34%, the recovery of acetoin increased due to the strong salting-out effect at lower IL concentration [54].
Fig. 4. Partition coefficient and recovery of acetoin in EOAB-K3PO4 system.
The variation of partition coefficient of EOAB is similar to acetoin. With the increase of EOAB concentration, the distribution coefficient showed a decreasing trend, while the recovery of EOAB was gradually increased (Fig. 5). The highest recovery of EOAB (86.3%) was obtained at 18% EOAB-34% K3PO4 (w/w).
16
Fig. 5. Partition coefficient and recovery of EOAB in EOAB-K3PO4 system.
The production of acetoin by microorganisms is usually accompanied by another product of 2,3-BD, which is also a promising platform chemical [14, 38, 53]. It is worthwhile to study the distribution behavior of 2,3-BD for its further recovery. With increasing concentration of EOAB, the partition coefficient of 2,3-BD showed a decreasing trend (Fig. 6a). Because of the difference in structure of 2,3-BD and acetoin, their distribution behaviors were not completely consistent. The recovery of 2,3-BD gradually increased as the concentration of EOAB increased (Fig. 6b). The highest recovery of 2,3-BD was 95.4% obtained at 18% EOAB-31% K3PO4. Further separation of 2,3-BD and acetoin in the top phase can be performed by vacuum distillation [1]. Previous work of imidazolium ILs showed high efficiency in SOE of 2,3-BD from fermentation broth [14], indicating acetoin also could be efficiently extracted by imidazolium ILs. Imidazolium IL of [C4mim][[OH] displayed catalytic activity in the reaction of acetoin and 5-MF, but the conversion rate and yield were much
lower
than
those
from
hydroxylammonium
ILs
[34].
Moreover,
hydroxylammonium ILs are easier to be prepared with lower cost and biological 17
toxicity [14]. Therefore, hydroxylammonium ILs have more potential as extractants in SOE and catalysts in aldol condensation reaction of acetoin.
Fig. 6. Partition coefficient and recovery of 2,3-butanediol in EOAB-K3PO4 system.
3.3.3 Selectivity of acetoin to organic acids It can be seen from the above results that EOAB-K3PO4 system can achieve the separation of acetoin and the recovery of IL. However, except target products the fermentation broth also contained some byproducts such as lactic acid and acetic acid. Thus, selectivity of acetoin to byproducts was also an important parameter to evaluate the applicability of SOES except recovery of acetoin and ILs. As shown in Fig. 7, high concentration of EOAB was not conducive to the separation of acetic acid and lactic acid. The selectivity to lactic acid was greater than 5 under the condition of the highest recovery of acetoin, indicating that acetoin and lactic acid could be separated well. The highest selectivity to lactic acid was obtained at 6% EOAB and 38% K3PO4, which was16.46 (Fig. 7b). Most of the selectivity values for acetic acid were less than 1, illustrating the difficulty in separating acetoin and acetic acids (Fig. 7a). The highest selectivity to acetic acid of 3.85 was obtained at 6% 18
EOAB-38% K3PO4, indicating acetoin was much more preferred to distribute into the top phase than acetic acid at this condition. Due to the higher hydrophilicity of lactic acid, it was much easier to separate acetoin with lactic acid than acetic acid. Although the highest recovery of acetoin was obtained in the system of 6% EOAB-34% K3PO4 (w/w), the recovery of EOAB was not the highest. In a word, the system of 6% EOAB-34% K3PO4 (w/w) was the most efficient for the direct recovery of acetoin from fermentation broth, but not for EOAB recovery and the removals of lactic and acetic acid. Concerning all these factors, 6% EOAB-38% K3PO4 (w/w) owed more application values, as the recoveries of acetoin and EOAB were similar to those obtained from 6% EOAB-34% K3PO4 while 2,3-BD recovery was a little lower, which were 92.7%, 76.0% and 86.0%, respectively. It has been reported that EOAA and EOAL are excellent catalysts for aldol condensation reaction of acetoin with 5-MF, and the catalyst EOAA could be recycled five times with no apparent loss of catalytic activity [34]. Acetoin in the top phase can react with added 5-MF to form liquid hydrocarbon fuel precursor under the catalysis of basic ILs. Thus, the following operation of acetoin purification was not required, and the separation of acetoin from fermentation broth and derivative production was integrated.
19
Fig. 7. Selectivity of acetoin to organic acids in SOES of EOAB-K3PO4. (a), acetic acid; (b), lactic acid.
3.4. Extraction mechanism The interaction of molecules is a critical factor for SOE, especially hydrogen bonds. The extraction ability in SOE is affected by the polarity of organic solvents [3]. Salting-out effect is the basis for the formation of SOESs consisting of ILs and salts. For ILs with a fixed anions and different cations, steric and entropic contributions are two main factors for the formation of ILs-based SOESs [12]. The ability of ILs to induce SOESs is closely related to the decrease of hydrogen bond accepting ability or the increase of hydrogen bond acidity of the ILs anion [21]. Compared with Kamlet-Taft parameters (polarisability π*, hydrogen-bond donating ability α, and hydrogen-bond accepting ability β), IR analysis is a more intuitive method to explore the hydrogen bond, which is the main interaction force in the SOE of acetoin. As shown in Fig. 8, when EOAB was added into H2O solution, there was a great shift of the stretching vibration of O-H in H2O (3331 cm-1) to lower wavenumber (3144 cm-1), reflecting the formation of hydrogen bond between water molecules and 20
EOAB. The ν (-COO-), centered at around 1528 cm-1 and 1397 cm-1, were red-shifted by about 9 cm-1 and 4 cm-1, respectively, further suggesting that the breakage of intermolecular hydrogen bonding of H2O. The stretching vibration of C=O in the carboxylic acid can result in a strong absorption peak around 1700 cm-1. The symmetrical stretching vibration and asymmetric stretching vibration of -COO(between about 1300-1600 cm-1) appeared after carboxylic acid reacted with base to form carboxylate, where the strong absorption peak around 1700 cm-1 of C=O disappeared [55]. With the addition of acetoin to H2O-EOAB system, there was an obvious shift from 3144 cm-1 to 3337 cm-1, reflecting the breakage of intermolecular hydrogen bond between water and EOAB and demonstrating the interaction of acetoin and H2O-EOAB system, which was also proved by the red shift of symmetric and asymmetric stretching vibrations of -NH2 in H2O-EOAB system. It can be seen that the structure of acetoin was not changed in the IL phase due to negligible shift of the stretching vibration of C=O in acetoin and no new chemical bonds generated. And there was a bigger shift of the symmetrical stretching vibration (1528 cm-1) to higher wavenumber (1548 cm-1) of -COO- in EOAB with the addition of acetoin into EOAB, implying the breakage of intermolecular hydrogen bond of EOAB and proving the presence of interaction between EOAB and acetoin. Although H2O and ionic liquids, as well as acetoin, exist interactions in SOES, the interaction between EOAB and acetoin was much stronger than those because EOAB is able to form more hydrogen bonds with acetoin. Furthermore, the stretching vibration of N-H, O-H and -COO- in hydroxylammonium IL illustrated the formation of intermolecular hydrogen bonds
21
with
acetoin.
These
results
clearly
showed
the
interaction
between
hydroxylammonium ILs and acetoin, explaining the high extraction efficiency of the SOESs.
Fig. 8. ATR-IR spectra of H2O (a), the mixture of acetoin and water (b), acetoin (c), the mixture of EOAB and water (d), EOAB (e), the mixture of acetoin and EOAB (f),
22
the mixture of acetoin, EOAB and water (g). 4. Conclusion In order to find a cheap and environmentally friendly SOES for separation of bio-chemicals, five hydroxylammonium ILs of EOAA, EOAP, EOAB, EOAL, and IPOAA were synthesized. The phase diagram showed the IL phase-forming ability was closely related to the carbon chain length of anions and cations. The temperature had little effect on the phase formation in the range of 25 ~ 45 °C, which was beneficial to separate of target substances in fermentation broths. The distribution behaviors of acetoin, EOAB and 2,3-BD, and selectivity of acetoin to lactic acid and acetic acid were investigated under different concentrations of EOAB and K3PO4. Most of acetoin, EOAB and 2,3-BD were partitioned to the top phase and the selectivity to lactic acid were larger than acetic acid. Under the condition of 6% EOAB-38% K3PO4 (w/w) the recovery of acetoin, IL and 2,3-butandiol was 92.7%, 76.0% and 95.0%, respectively, and the selectivity to lactic acid and acetic acid was 5.35 and 1.41, respectively. The AIR-IR sepectra showed the hydrogen bond formed between acetoin and O-H, N-H, -COO- of hydroxylamine IL, which played an important role in the efficient extraction of acetoin from fermentation broth. It is the first time that hydroxylammonium IL was used as salting-out agent to separate acetoin, demonstrating its potential in the separation of bio-chemicals. Acknowledgement This work was supported by the National Natural Science Foundation of China (Grant No. 21978038).
23
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CRediT authorship contribution statement Yan Li: Conceptualization, Methodology, Investigation, Writing - original draft, Data curation, Formal analysis. Jian-Ying Dai: Conceptualization, Writing – review & editing, Resources, Project administration, Supervision, Funding acquisition. Zhi-Long Xiu: Conceptualization, Writing – review & editing, Resources, Supervision.
Declaration of Competing Interest The authors declared that there is no Conflict of Interest.
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Highlights
Phase forming abilities of hydroxylammonium ionic liquids with K3PO4 were studied
Acetoin partition behavior in ionic liquid-K3PO4 system was studied
93% acetoin and 76% ethanolammonium butyrate were distributed into the top phase
The selectivity of acetoin over lactic acid and acetic acid was 16.46 and 3.85
IR spectra showed the intermolecular hydrogen bonds between acetoin and ILs
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