Extraction of organic acids using imidazolium-based ionic liquids and their toxicity to Lactobacillus rhamnosus

Extraction of organic acids using imidazolium-based ionic liquids and their toxicity to Lactobacillus rhamnosus

Separation and Purification Technology 40 (2004) 97–101 Extraction of organic acids using imidazolium-based ionic liquids and their toxicity to Lacto...

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Separation and Purification Technology 40 (2004) 97–101

Extraction of organic acids using imidazolium-based ionic liquids and their toxicity to Lactobacillus rhamnosus Michiaki Matsumoto∗ , Kenji Mochiduki, Kei Fukunishi, Kazuo Kondo Department of Chemical Engineering and Materials Science, Doshisha University, Kyotanabe, Kyoto 610-0321, Japan Received in revised form 13 January 2004; accepted 31 January 2004

Abstract In situ extractive fermentation of lactic acid using organic solvents has already been heavily investigated. Now ionic liquids are emerging as alternative solvents for volatile organic compounds traditionally used in liquid–liquid extraction. In this paper, we examine whether imidazolium-based ionic liquids can replace conventional organic solvents in the extractive fermentation of lactate by investigating their extraction behaviors and solvent toxicity. The extractions of organic acid using imidazolium-based ionic liquids without any extractant are controlled by the hydrophobicity of the acids, and in all cases the extractability is very low. The extraction behaviors of lactic acid with imidazolium-based ionic liquids containing tri-n-butylphosphate extractant are similar to those of conventional organic solvents. Lactic acid producing bacterium, Lactobacillus rhamnosus NBRC 3863, grew, consumed glucose and produced lactate in the presence of imidazolium-based ionic liquids. These findings suggest that ionic liquids could be used in an in situ extractive fermentation process. © 2004 Elsevier B.V. All rights reserved. Keywords: Ionic liquids; Extraction; Organic acid; Tri-n-butylphosphate; Green solvent

1. Introduction A typical example of a bioproduction process using whole-cell and organic solvents is the extractive fermentation of ethanol, acetone–butanol or organic acid. Particularly, lactic acid production by fermentation has gained interest because optical pure lactic acid is the raw material of a biodegradable polymer. The economics of the process, however, depend on the development of an effective recovery method for lactic acid from the broth because the separation and purification steps account for up to 50% of the production costs [1]. Solvent extraction has been proposed as a promising recovery technique and as an alternative to the conventional precipitation process [2]. However, whole bacterial cells pose far greater problems in operating bioproduction processes when organic solvents are present as a second phase, with the most critical problem being the inherent toxicity to living organisms [3]. Moreover, use of volatile organic compounds is also undesirable for environmental reasons.

The suitability of ionic liquids as green solvents for chemical processes has been extensively recognized due to their having effectively no vapor pressure, which lends them to be replacements for volatile, conventional organic solvents. In the field of bioprocessing, it has been reported that the use of ionic liquids as an alternative to organic solvents improved the selectivity and yield in enzymatic reactions [4]. However, few studies have been conducted on whole-cell processes [5,6]. In this paper, we examine whether imidazolium-based ionic liquids can replace conventional organic solvents in the extractive fermentation of lactate by investigating their extraction behaviors and solvent toxicity. The use of the ionic liquids has been studied only in relation to the extractions of alkali metals [7–9], heavy metals [10] and lanthanide [11].

2. Experimental 2.1. Preparation of ionic liquids



Corresponding author. Tel.: +81-774-65-6655; fax: +81-774-65-6655. E-mail address: [email protected] (M. Matsumoto). 1383-5866/$ – see front matter © 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.seppur.2004.01.009

The hexafluorophosphates of 1-butyl-, 1-hexyl- and 1-octyl-3-methyl imidazolium, [Bmim][PF6 ], [Hmim][PF6 ]

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Nomenclature D distribution ratio of organic acid between aqueous phase and ionic liquids [ j] concentration of species j (mol/dm3 ) Kd distribution constant of organic acid between aqueous phase and ionic liquids K1 equilibrium constant of Eq. (2) (dm3 /mol) K2 equilibrium constant of Eq. (3) (dm3 /mol)2 Subscripts aq aqueous phase IL ionic liquids

and [Omim][PF6 ], respectively, were prepared following the method of Laszlo and Compton [12]. The structure of the ionic liquids is shown in Fig. 1. 2.2. Extraction of organic acids Unless otherwise stated, an organic solvent or ionic liquid did not contain any extractant, but in some cases it contained the extractant tri-n-butylphosphate (TBP). Aqueous solutions were prepared by dissolving an acid of G.R. grade in deionized water, with its concentration adjusted to 0.1 mol/dm3 . The molecular structures of the organic acids (acetic, glycolic, propionic, lactic, butyric and pyruvic) studied are shown in Table 1. The experiments were performed without pH adjustment because we confirmed that from the pH value after equilibration the predominant species of organic acid in the aqueous solution was its undissociated form. Equal volumes (20 cm3 ) of the aqueous and organic solutions were mixed in an Erlenmeyer flask and shaken at 303 K to attain extraction equilibrium. After the two phases were separated, organic acid in the samples taken from the organic solution was stripped by 2 M sodium hydroxide solution. The concentrations of organic acid in both phases were determined by HPLC as described in the previous paper [13]. The pHs of the aqueous solutions before and after equilibration were determined by a Horiba F-12 pH meter.

Fig. 1. Imidazolium-based ionic liquids. R = C4 H9 : [Bmim][PF6 ]; R = C6 H13 : [Hmim][PF6 ]; R = C8 H17 : [Omim][PF6 ].

2.3. Growth of microbes in the presence of ionic liquids We used Lactobacillus rhamnosus NBRC 3863 as a lactic acid-producing bacterium, previously used in our studies on extractive fermentation of lactic acid [14,15]. The MRS medium (Difco, USA) was applied to the organism. Cultivation was performed at 303 K. Growth in the presence of a second phase was determined based on a method described previously [16]. An inoculum of 2.5% (v/v) from an overnight culture was transferred to a fresh medium. When the culture reached the exponential growth phase (OD560 ≈ 0.3), 2 ml was transferred to new culture bottles (20 ml) and 5% (v/v) of one of the ionic liquids was added to each. These added organic solvent or ionic liquids formed the second phase. The culture bottles were closed with Teflon valves and were then incubated for 48 h at 30 ◦ C. Then, the optical density (OD560 ), and the concentrations of glucose and lactic acid were measured. In addition, the number of colony-forming units (CFU) per milliliter of culture was calculated from the number of cells forming colonies on the agar plates before and after contact with the ionic liquids.

3. Results and discussion 3.1. Extraction of organic acids into imidazolium-based ionic liquids Table 1 shows the distribution constant, Kd , of organic acid (HA) between aqueous solution and ionic liquids. The distribution constant is defined by Eq. (1). Kd =

[HA]IL [HA]aq

(1)

Table 1 Characteristics and distribution ratio of organic acids investigated Acid

Chemical formula

pKa a

log Pb

Kd (C4)c

Kd (C6)c

Kd (C8)c

Acetic Glycolic Propionic Lactic Pyruvic Butyric

CH3 COOH CH2 OHCOOH C2 H5 COOH CH3 CHOHCOOH CH3 COCOOH C3 H7 COOH

4.56 3.63 4.67 3.66 2.26 4.94

−0.17 −1.11 0.33 −0.72 −0.11 0.88

0.024 0 0.42 0.024 0.11 1.03

0.35 0 0.32 0.040 0.15 1.06

0.080 0 0.16 0.025 0.059 0.54

a b c

At 25 ◦ C and ionic strength = 0.1 mol/dm3 [21]. Logarithm value of partition coefficient between octanol and water [19]. C4, C6 and C8 denote [Bmim][PF6 ], [Hmim][PF6 ] and [Omim][PF6 ], respectively.

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Fig. 3. Extraction of lactic acid with tri-n-butylphosphate diluted in Bmim (䊉), Hmim (䊊), Omim (䉲), hexane () and toluene (䊏). Fig. 2. Relationship between log Kd and log P of organic acids. The solid line is a reference line with slope = 1. (䊉) [Bmim][PF6 ] (䊊) [Hmim][PF6 ] (䉲) [Omim][PF6 ].

As is evident from Table 1, the distribution constants of organic acids were relatively small and glycolic acid, the most hydrophilic acid, was not extracted at all. These results suggest that extraction of organic acids with imidazolium-based ionic liquids controls the hydrophobicities of organic acids as well as the extraction of organic acids with trioctylphosphine oxide or trioctylamine [17,18]. Fig. 2 shows the relationship between logarithms of the distribution constants and log P, where P is the partition coefficient of the acid between octanol and water [19]. The values of log Kd were roughly correlated with values of log P. From Fig. 2, we also observed that with minor exceptions, generally the order of extractability is as follows; Omim < Bmim < Hmim, however, difference in the extraction capacities of the ionic liquids is relatively small. Recently, Anderson et al. reported characterization of ionic liquids on the basis of a multiple solvation interaction because while the ionic liquids have similar polarities they are nevertheless capable of behaving quite differently [20]. In their report, a small change of alkyl length in the imidazolium cation had little influence on the interaction parameters. However, it should be noted that the imidazolium compounds used in their report are different from those in this study. At present, although we cannot explain the order of extraction capacity, we expect that the interactions between the imidazolium-based ionic liquids and organic acids are similar. However, the values of the distribution ratios of organic acids with ionic liquids alone were not over unity, that is, the extraction capacities were very low. Next, we examined the capacity of the ionic liquids to act as diluents of extractants. 3.2. Suitability of ionic liquids as diluents We extracted lactic acid with an extractant diluted in the ionic liquids. We used typical extractants for the extraction of organic acids, tri-n-butylphosphate, tri-n-octylphophine oxide (TOPO) and tri-n-octylamine (TOA). Unfortunately,

TOA was immiscible with imidazolium-based ionic liquids and the extractability of lactic acid with TOPO diluted in the ionic liquids was poor. However, TBP mixed well with the ionic liquids and the extractability was good, as shown in Fig. 3, which shows the extractabilities in the ionic liquids compared to those in some conventional organic solvents [18]. Because the plots in Fig. 3 describe a parabola, lactic acid (HA) forms the complexes (HA·TBP and HA·2TBP) with TBP as well as in the conventional organic solvents reported previously. HA + TBP  HA · TBP : K1

(2)

HA + 2TBP  HA · 2TBP : K2

(3)

From the above equations, the distribution ratio, D, is expressed as Eq. (4). D = Kd + K1 [TBP] + K2 [TBP]2

(4)

The extraction equilibrium constants, K1 and K2 , are evaluated from Eq. (4) and the mass-balance equation of TBP by the trial and error method to minimize summation of the square of deviation between observed and calculated distribution ratios. The equilibrium constants obtained are listed in Table 2. 3.3. Tolerance of Lactobacillus rhamnosus NBRC 3863 to imidazolium-based ionic liquids The toxicity of organic solvents to microbes has been studied, but there are few studies on the toxicity of ionic liquids. In order to apply ionic liquids to bioprocessing, this point must of course be clarified. Batch culture experiments were performed to examine the solvent toxicity to

Table 2 Effect of diluents on extraction of lactic acid with TBP Diluent (dm3 /mol)

Bmim Hmim Omim Hexane 1-Octanol Toluene

K1 0 K2 ((dm3 /mol)2 ) 0.058

0.062 0.030

0.014 0.056

0.074 0.054

0.007 0.052

0.146 0.037

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on the extractability of organic acids and the survival of the cells.

Acknowledgements The present work was supported by a Grant-in-Aid for Scientific Research (C) (No. 13650833) from the Japan Society for the Promotion of Science.

References Fig. 4. Toxicity of ionic liquids to Lactobacillus rhamnosus. Black and gray bars denote relative activity on the basis of glucose consumption and CFU values, respectively.

L. rhamnosus. Fig. 4 shows the relative activity based on the amount of glucose consumed and CFU values, which are a measure of the survival rate of microbes. In the figure, control denotes the experiments conducted without ionic liquids and organic solvents. Activity based on CFU values corresponded to that of glucose consumption, suggesting that the viable cells produced lactate. L. rhamnosus could not survive in the presence of toluene. Although addition of ionic liquids to the culture results in relatively low activity, it still grew, consumed glucose and produced lactate in the presence of imidazolium-based ionic liquids. This relatively low toxicity is favorable for in situ extractive fermentation. A change of alkyl length in the imidazolium cation had little influence on the survival of the cells.

4. Conclusion In this paper, we examine whether imidazolium-based ionic liquids could replace conventional organic solvents in the extractive fermentation of lactate by investigating their extraction behaviors and solvent toxicity. The extractability of organic acid using imidazolium-based ionic liquids without any extractant is very low. The extraction behaviors of lactic acid with imidazolium-based ionic liquids containing tri-n-butylphosphate extractant are similar to those of conventional organic solvents. However, the solubility of alkylamines, which are frequently used in lactic acid extraction, in the ionic solvents was limited. Therefore, design of an ionic liquid solvent is needed that will enhance the solubility of alkylamines. Lactic acid producing bacterium, L. rhamnosus NBRC 3863, grew, consumed glucose and produced lactate in the presence of imidazolium-based ionic liquids. Therefore, it should be possible to apply ionic liquids to an in situ extractive fermentation process. A change of alkyl length in the imidazolium cation had little influence

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