LiquidâLiquid Equilibrium and Molecular Toxicity of Active and Inert

Article Cite This: J. Chem. Eng. Data 2019, 64, 3028−3035

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Liquid−Liquid Equilibrium and Molecular Toxicity of Active and Inert Diluents of the Organic Mixture Tri-iso-octylamine/Dodecanol/ Dodecane as a Potential Liquid Membrane for Lactic Acid Removal

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Alan D. Pérez,† Verónica M. Gómez,‡ Sneyder Rodríguez-Barona,‡ and Javier Fontalvo*,† Facultad de Ingeniería y Arquitectura - Departamento de Ingeniería Química, Universidad Nacional de Colombia-Sede Manizales, Manizales 170003, Colombia ‡ Grupo de Investigación en Bacterias Á cido Lácticas y sus Aplicaciones Biotecnológicas Industriales, Laboratorio de Ciencia de Alimentos, Universidad Nacional de Colombia, Manizales, 170003, Colombia † Grupo de Investigación en Aplicación de Nuevas Tecnologías, Laboratorio de Intensificación de Procesos y Sistemas Híbridos, Universidad Nacional de Colombia, Manizales, 170003, Colombia

ABSTRACT: Lactic acid can be in situ removed from a fermentation broth through reactive liquid extraction or a liquid membrane to enhance the fermentation process. The organic mixture tri-iso-octylamine (TiOA)/dodecanol/dodecane at 10 vol % of the amine is a potential organic mixture for lactic acid removal. Liquid−liquid equilibria with lactic acid aqueous solutions and molecular toxicity on the bacteria Lactobacillus casei ATCC 393 were measured with several dodecanol proportions in dodecane (0 to 90 vol %) and 10 vol % TiOA as potential solvents or membrane phases for LA removal from a fermentation broth. Effects of the organic phase on the bacteria as cell growth, biomass production, glucose consumption, productivity, and product to biomass yield are analyzed. Dodecanol increases the lactic acid chemical equilibrium constant for the liquid−liquid equilibria, while increasing the molecular toxicity on the bacteria. However, for dodecanol concentrations from 30 to 40 vol % the value of the chemical equilibrium constant is high enough for lactic acid distribution between the phases and its toxicity is low enough on the bacteria, making a proper range of dodecanol concentrations for lactic acid removal. Also, the distribution coefficient and the chemical equilibrium constant are fitted as a function of the dodecanol concentration in the organic mixture.

1. INTRODUCTION Lactic acid (LA) is a commodity chemical1,2 which can be obtained by chemical synthesis or by carbohydrate fermentation.3−5 Almost 90% of LA is produced by the biotechnological route.4,6 However, in the conventional process for LA production, bacterial growth is inhibited on the one hand by LA, achieving low concentrations of LA, and on the other hand by substrate, limiting initial substrate concentration within the bioreactor.1,7,8 Besides, this process requires multiple separation steps for recovery and purification of the LA from the fermentation broth.1,2,9 The whole LA production process is expensive,1,2 and 50% of the total cost is due to the separation and final purification steps.1,2,9 LA biotechnological production requires an enhancement on its production process that avoids the common drawbacks of the conventional process, which are cell growth inhibition © 2019 American Chemical Society

(by product and substrate), the use of neutralizers, low yield, and productivity, and high overall costs of the process.2 Several separation technologies, such as solvent extraction, adsorption (with and without ion exchange), distillation, filtration, ultrafiltration, nanofiltration, electrodialysis, reverse osmosis, evaporation, crystallization, and liquid membranes have been tested for LA recovery from fermentation broths as promising alternatives over the conventional process.1,9,10 In situ LA removal using one or several of the above separation technologies is a potential strategy to overcome the typical drawbacks of LA production.1,10 In situ LA removal reduces end-product inhibition1 and contributes to the fedReceived: February 4, 2019 Accepted: June 11, 2019 Published: June 25, 2019 3028

DOI: 10.1021/acs.jced.9b00132 J. Chem. Eng. Data 2019, 64, 3028−3035

Journal of Chemical & Engineering Data

Article

Table 1. Chemicals Used for the Experimentsa name

CASRN

supplier

molecular weight

density (g·mL−1)

purity (wt %)

tri-iso-octylamine n-dodecane 1-dodecanol L(+)-lactic acid sodium hydroxide

25549-16-0 112-40-3 112-53-8 79-33-4 1310-73-2

Merck Merck Merck Scharlau Merck

353.68 170.34 186.33 90.08 40.00

0.8 0.75 0.83 1.206 2.13

95 99 98 88−92 99

a

Physicochemical properties were taken from the respective MSDS of the supplier (each one at room temperature).

batch or continuous operation of the fermentation.7 One of the most extensively studied separation technologies for LA removal has been reactive extraction.11−16 However, this process requires high amounts of solvent and the use of backextraction for the regeneration of the solvent.8,17 Perstraction is a liquid membrane process which can use the same solvent (diluent and extractant) of the reactive extraction as the membrane phase.18 It does not require high amounts of solvent, and the extraction and back-extraction processes are combined in a single device.17,18 This process is also called liquid membrane, and several studies using this membrane technology have been focused on LA removal.19−27 Usually, the selection of the solvent, for either reactive extraction or liquid membranes, is based on the ability of the solvent to enhance the LA removal from the fermentation broth and the toxic effect of the solvent on the specific microorganism in the fermentation broth.28,29 However, most of the solvents used for LA removal from fermentation broths are toxic on the specific microorganism.28 There are two kinds of toxicity of the solvent on the microorganism depending on the type of contact between the solvent and the microorganism. On the one hand, molecular toxicity is due to the soluble portion of the solvent in the fermentation broth. On the other hand, phase level toxicity is due to the presence of two phases in the fermentation process, solvent phase, and fermentation broth phase.30,31 For in situ LA removal using reactive extraction or liquid membranes, both levels of toxicity can affect the microorganism. However, filtration is usually used for removing biomass from the fermentation broth to avoid phase toxicity on the microorganism.32 However, performing biomass filtration and product removal externally to the bioreactor will leave molecular toxicity to occur on the microorganism.33 There are several cellular responses due to toxic organic solvents, such as stress,34 changes in cell morphology (filamentation),34,35 cell surface modification (hydrophobic shift),34 cell membrane adaptions (membrane fluidity),34,35 and solvent excretion through efflux pumps,34,35 among others. Most of these cellular responses are energy demanding.36 Single alcohols and tertiary amines mixed within alkanes or alcohols have been extensively studied for LA removal because they have shown high extraction capabilities.11−16,22,37−39 The liquid−liquid equilibrium of the mixture trioctylamine at 0.8 M (around 23 mol %) in 1-dodecanol with LA aqueous solutions have shown a high value of the chemical equilibrium constant,12 making it a potential solvent for reactive extraction or a potential membrane phase for liquid membrane processes. However, toxicity studies in an in situ LA removal process either by liquid−liquid extraction or liquid membrane processes are required for the selection of these organic compounds. The molecular toxicity of pure 1-dodecanol, n-dodecane, oleyl alcohol, Aliquiat 336, trioctylamine, and tri-iso-octyl-

amine (TiOA) was tested in a previous work,33 showing that ndodecane is nontoxic on the Lactobacillus casei ATCC 393 (probiotic and homofermentative bacteria in which the main metabolite is lactic acid). Depending on the concentration of oleyl alcohol within the fermentation broth, it can be classified as nontoxic or as medium toxic. 1-Dodecanol has medium toxicity, and the tertiary amines are toxic on the bacteria. However, tri-iso-octylamine at a low concentration within the fermentation broth (concentrations of TiOA 50% lower than the saturation point on the fermentation broth) has medium toxicity. Also, the toxicity of the mixture TiOA/dodecane on the same bacteria was tested at 10, 20, and 30 vol % of TiOA (5.40, 11.38, and 18.04 mol %) showing that the mixture at 5.40 mol % is nontoxic, at 11.38 mol % is low toxic, and at 18.04 mol % is medium toxic.33 Additionally, the liquid−liquid equilibria (LLE) of a mixture of TiOA within n-dodecane and another mixture of TiOA within 1-dodecanol, both at 10 vol % of TiOA (5.40 and 5.34 mol %, respectively) were previously carried out.40 The mixture TiOA/dodecane showed a low capability to LA removal because this mixture does not promote the chemical reaction between the amine and the LA, while the mixture TiOA/dodecanol enhances LA removal because dodecanol stabilizes the LA formed complex by solvation. Summarizing, if pure dodecane is used, the molecular toxicity is low but also the LA removal is poor. If dodecanol is used, there is a high LA removal, but also molecular toxicity is high. Thus, a mixture of dodecane and dodecanol with TiOA could be a good compromise between LLE and molecular toxicity. However, the toxicity of the mixture TiOA/ dodecanol/dodecane, at several proportions of dodecanol, on the Lactobacillus casei ATCC 393 bacteria and the LLE have been not tested. In this work, the LLE (organic/aqueous LA) and molecular toxicity on the Lactobacillus casei ATCC 393 at several ratios of dodecanol/dodecane with 10 vol % of TiOA (5.4 to 5.34 mol %) was measured. Dodecanol concentrations between 0 to 90 vol % (0 to 94.66 mol %) were used. Correlations for prediction of the distribution coefficient and the chemical equilibrium constant are proposed as a function of the dodecanol concentration within the TiOA/dodecanol/dodecane mixture.

2. EXPERIMENTAL SECTION 2.1. Materials. Tri-iso-octylamine (TiOA, water solubility < 1 g·L−1 at 20 °C), n-dodecane (assay 99%, insoluble at 25 °C), and 1-dodecanol (assay 98%, water solubility = 0.004 g· L−1 at 20 °C) were used. L(+)-lactic acid was supplied by ́ Panreac Quimica S.A.U. (assay 88.0−92.0%). The purity of L(+)-lactic acid (extra pure) was assessed by titration with NaOH (assay ≥ 99.0%) using Metrohm automatic titrator (702 SM Titrino, 703 TI Stand). A stock solution of lactic acid (150 g/L) was heated at 90 °C under total reflux between 8 3029



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and 10 h for dimer hydrolysis,15,41 and afterward, the lactic acid concentration was measured by titration. Type I water was used for all aqueous solutions (Barnstead Nanopure). All chemicals used are listed in Table 1. Lactobacillus casei ATCC 393 (Microbiologics) was used for fermentations, with MRS broth (Scharlau) used as culture media. For the liquid−liquid equilibria and toxicity tests, a TiOA concentration of 10 vol % (around 5.4 mol %) in all organic mixtures at each proportion of dodecane/dodecanol was used. This TiOA concentration was chosen according to previous experimental results of molecular toxicity. These results showed that using TiOA at 10% within dodecane has a nontoxic effect on the lactic acid bacteria Lactobacillus casei ATCC 393, while using TiOA at concentrations higher than 10 vol % within dodecane has toxic effects on the bacterial that increase with the concentration of dodecane within the organic phase.33 2.2. Liquid−Liquid Equilibria Experiments (LLE). LLE experiments for organic phases with 10 vol % (5.40 to 5.34 mol %) of TiOA (extractant or carrier) in n-dodecane (inert diluent) and 1-dodecanol (active diluent) with concentrations of 1-dodecanol of 10, 20, 30, and 50 vol % were performed (10.62, 21,22, 31.79, and 52.85 mol %). For the LLE experiments and each of the above organic phases, five vials of 10 mL were filled with 2 mL of an aqueous solution of LA, at concentrations between 1 and 38 g/L (0.0166 and 0.6465 mol %). Then, 2 mL of the organic phase was added to each vial. The experimental procedure was performed at a constant temperature (37 °C) and consists of the next three steps:12 agitation, decantation, and sampling. First, all vials were shaken at 180 rpm (Wiseshake Shot-1D, Wisd) with an incubator (Wisd, ± 0.6 K) during 72 h. Afterward, the agitation was stopped, and the vials were kept in the incubator (decantation step) during 72 h. One sample of the aqueous solution was taken from every single vial, and its lactic acid concentration was measured by high-performance liquid chromatography (HPLC ELITE LaChrom). For HPLC analysis, an ORH-801 column (Chrom Tech) with a solution of 0.01 N H2SO4 (Merck, assay 95−97%) for the mobile phase, and a RI detector at 45 °C were used. The total LA concentration, in equilibrium within the organic phase, was calculated by a material balance.12 The measured LA concentrations at equilibrium were used to fit the distribution coefficient (eq 1) and the chemical equilibrium constant (eq 2) based on a LLE model developed in previous works.12,40 This model is based on Nernst’s distribution law, mass action law, equilibrium equations, and material balances. The model can be used for LLE with stoichiometric ratios (amine/acid) 1:n, and it predicts the total LA concentration in the organic phase in equilibrium with the LA concentration in the aqueous phase. Brackets in eqs 1 and 2 represent molar concentrations.

KD =

KE =

bacteria Lactobacillus casei ATCC 393 as shown elsewhere.33 The MRS broth was prepared according to the manufacturer instructions (Scharlau). Three milliliters of the organic phase was added in a falcon tube with 30 mL of sterile MRS. The Falcon tube was shaken for 72 h at 180 rpm (Wiseshake Shot1D, Wisd) at 37 °C in a bod incubator (Wisd, ± 0.6 K). Afterward, the shaker was stopped, and the falcon tubes were kept in the incubator at the same temperature for 72 h to reach phase splitting (the aqueous phase from the organic phase). Then, the aqueous phases (culture media saturated with the organic phase) were separated from the organic phase by splitting. Each culture media within a new sterile falcon tube was inoculated at 5 vol % from a culture of 12 h. One control fermentation was prepared using fresh culture media (MRS without contact with the organic phase). The fermentation of each inoculated falcon tube was kept in a bod incubator (Wisd, ± 0.6 K) for 24 h at 37 °C. Samples from each falcon tube were taken at the beginning (0 h) and at the end (24 h) of each fermentation to measure the percentage of bacterial growth, LA production, and glucose consumption based on the control fermentation. Bacterial growth was measured by the dry cell weight method.33 LA production and glucose consumption were measured by HPLC using the same method of LA measurements in the LLE experiments. The aforementioned procedure also was used to test the molecular toxicity of the organic phases with 10 vol % of TiOA (5.40 to 5.34 mol %) in n-dodecane and at 1-dodecanol proportions of 10, 20, 30, 40, 50, and 90 vol % (0, 10.62, 21.22, 31.79, 42.33, 52.85, and 94.66 mol %, respectively). All experiments were performed in duplicate.

3. RESULTS AND DISCUSSION 3.1. Liquid−Liquid Equilibria. The distribution coefficient and the chemical equilibrium constant for the LLE of aqueous LA solutions in contact with tri-iso-octylamine/ndodecane/1-dodecanol at 37 °C increases as the concentration of dodecanol in the organic mixture rises (Figure 1). In the organic phase, there was always TiOA at 10 vol % (5.40 to 5.34 mol %) and the proportion of dodecane to dodecanol varied. Dodecane is an inert diluent and dodecanol is an active

[LA]org [LA]aq

(1)

[(LA)(TiOA)]org [LA]org [TiOA]org

(2) Figure 1. Fitted values of the distribution coefficient (squares) and the chemical equilibrium constants (triangles) with its respective correlations (continuous line) using eqs 3 and 4, respectively. Values of KD and KE at 0, 42.33, and 94.66 mol % of dodecanol were taken from a previous work.40

For the distribution coefficient, the LA concentration in the organic phase corresponds to the LA in its free form. 2.3. Molecular Toxicity Test. The toxicity of TiOA in 1dodecanol at 5.34 mol % of TiOA was measured on the 3030



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Table 3. Several Values of KE for Systems Diluent/Carrier for LA Removal

diluent.40 Dodecanol provides a solvation shell for the free LA and for the LA complex in the organic phase,40 which stabilizes the acid−amine complex. Therefore, the availability of the dodecanol molecules to solvate increases with its concentration in the organic phase. For the TiOA/dodecanol/dodecane systems at several dodecanol proportions, different values of the KD and KE according to the concentration of the solvents in the organic phase are expected. The activities of LA and TiOA depend on dodecane and dodecanol fractions within the organic phase, and what is important, the dodecanol provides a solvation shell to the LA−TiOA complex to stabilize it that dodecane does not. Thus, solvation highly enhances the formation of the LA− TiOA complex and the equilibrium constant, as a function of dodecanol concentration, takes into account this effect. The stoichiometric ratio (amine/acid) 1:1 is the most common for these systems where a tertiary amine is involved and especially when the LA concentration is low (lower than the stoichiometric concentration40). Therefore, a stoichiometric ratio of 1:1 was used for fitting the values of KD and KE for all dodecanol concentrations from 0 to 94.66 mol % (Figure 1) using a LLE model previously developed for this kind of systems.40 For the organic mixture without dodecanol, there is no complex formation, and the value of KE is zero.40 The increase of the KE with dodecanol concentration was monotonic, while for the KD, it does not show a proportional increase (Figure 1). For KD, ia third-grade polynomial fit was used (eq 3), while for the KE eq 4 was used. For both fits, molar concentrations of dodecanol were used. The corre-

0.9977 R2 0.9863

a3 (L·mol−1)3

a2 (L·mol−1)2

a1 (L·mol−1)

1.9987 −0.6186 0.1010 K1 (L·mol−1) K2 (L·mol−1) 485.18

carrier

T [K]

KE [L/mol]n

n

ref

decane decane/decanol dodecanol octanol octanol dodecane dodecane octanol

TBP TOA TOA DDAP TOA TBP TBP Alamine 336 TOA Alamine 336 Alamine 336

293.15 293.15 310.15 310 298.15 298.15 293.15 298.15 293 298.15 298.15

0.355 13.9 49.2 350 297 50 4.18 75 1.31 20.417 371.535

1.27 1.94 1 1 1 1 1.32 1 1 1 1

42 42 12 13 39 43 43 1 16 44 44

MIBK chloroform

diluents, such as TOA in octanol, DDAP in octanol, and Alamine 336 in chloroform. The KE values of the aforementioned carrier/diluent systems are higher than the KE values achieved of the system TiOA/ dodecanol/dodecane, which was studied in this work. However, a high KE value does not mean that it is suitable for an integrated fermentation and LA removal process. Also, toxicity studies have to be taken into account on the specific microorganism to be used in the hybrid fermentation. Thus, in this work, both studies were combined for selecting a suitable mixture for LA removal from a fermentation broth by Lactobacillus casei ATCC 393. 3.2. Molecular Toxicity Test. Molecular toxicity of the mixtures TiOA/dodecanol/dodecane at concentrations of dodecanol from 0 to 94.66 mol % on the bacteria Lactobacillus casei ATCC 393 is shown in Figure 2. The level of toxicity was

Table 2. Fitted Parameters of eqs 3 and 4 with the Corresponding Correlation Coefficients R2

diluent

a0 0.0194

2.3060

sponding fitted parameters are presented in Table 2, with their corresponding correlation coefficients. KD = a3[DOH]3 + a 2[DOH]2 + a1[DOH] + a0 KE = K1

K 2[DOH] 1 + K 2[DOH]

(3)

(4)

Generally, in systems that used tertiary amines, most of the organic acid within the organic phase is due to a LA-amine complex that it is formed with the tertiary amine.12,40 Therefore, the amount of free LA within the organic phase in equilibrium is very small as compared to the LA−TiOA complex.40 Thus, KE provides more valuable information to determine which organic mixture provides a better capacity to remove the LA than does KD. According to several LLE studies of diluent/carrier systems for LA removal (shown in Table 3), the single tertiary amine (without diluent) achieves low values of KE because the LA− amine complex does not have diluent polar molecules that stabilize it. The system studied in this work achieves KE values (Figure 1) higher than most of the systems showed in Table 3. However, high values of KE were achieved for TOA, DDAP, and Alamine 336 when these carriers are mixed with proper

Figure 2. Relative cell growth, lactic acid production, and glucose consumption as compared to a control fermentation of the saturated media with TiOA/dodecanol/dodecane at 10 vol % of TiOA (5.4 to 5.34 mol %) and at proportions of dodecanol of 0, 10.62, 21.22, 31.79, 42.33, 52.85, and 94.66 mol % at 37 °C. Molecular toxicity for a dodecanol concentration of 0 mol % was previously reported.33

classified according to Marták et al.,29 for which the toxicity level is divided into three categories. Nontoxic, when the production rate is beyond 75%, medium toxicity when the production rate is between the 25% and 75%, and toxic when the production rate is less than 25% as compared with control fermentation. 3031



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activation of efflux pumps, change of the morphology, and surface adaptations, among others, which are energy demanding.36 Thus, it is probable that this additional energy requirement of the cells, as a mechanism to adapt or survive to toxic compounds, directly affects the cell growth. The energy is used by the cell to activate one of several mechanisms to tolerate the toxic compounds within the fermentation broth and instead use it entirely for cell growth. The mixture TiOA/dodecanol/dodecane at 94.66 mol % of dodecanol is toxic for cell growth, LA production, and glucose consumption according to the results shown in Figure 2. For this mixture, glucose consumption is reduced as compared to control fermentation. Glucose consumption is affected in the same proportion as LA production (around 19%). However, the cell growth is affected in a higher proportion achieving a bacterial growing of 4% of the one in the control fermentation. The low biomass production perhaps occurs because the bacteria kept the consumed glucose for maintenance instead of biomass production. Thus, the bacteria requires additional energy to use the efflux pumps to take out the toxic solvent from the bacteria, increasing the energy requirement on the bacteria. Hence, the cells reduce the energy consumption for biomass production. In Figure 2, it can be observed that for a dodecanol concentration of 94.66 mol % (which means 0 mol % of dodecane) in the mixture TiOA/dodecanol/dodecane, the cell growth is lower than the LA production, while for a dodecanol proportion of 0 mol % (which corresponds to 94.6 mol % of dodecane) the cell growth is higher than the LA production. Therefore, the effect that the LA production is promoted instead of the cell growth can be attributed to the presence of dodecanol within the fermentation broth, while the effect that cell growth is promoted instead of the LA production can be attributed to the presence of dodecane within the fermentation broth. Additionally, it is observed that for dodecanol concentrations from 10.62 to 52.85 mol %, the LA production is promoted instead of the cell growth despite that dodecane is also present within the fermentation broth (Figure 2). This means that the dodecanol effect on the bacteria is stronger than the effect of dodecane on the bacteria. In the studied system at dodecanol proportions from 10.62 to 94.66 mol % (where dodecanol is present), there was a decrease in glucose consumption as compared to that of the control fermentation. The bacteria can respond to the presence of the organic solvents within the fermentation broth in several ways.34,35 These include a hydrophobic shift on the cell surface to repel hydrophobic compounds34 and homeoviscous adaptation that increases membrane rigidity because the degree of membrane fatty acid saturation also increases.34 Perhaps these cellular processes may affect the mechanism of glucose transport through the membrane cell or phosphotransferase system for sugars (PTS), making the glucose consumption of the bacteria lower than the control fermentation. The bacteria probably do not have enough ATP to support this mechanism. Most of the published literature on molecular toxicity of organic compounds on specific bacteria have shown the toxic effect on cell growth. Only a few works have taken into account both the main metabolite production and the substrate consumption. In this work, it is observed that evaluating these three parameters is important to determine whether cell growth or main metabolite production is promoted. However, despite these results, it is still unclear how these organic

Surface tension and water solubility are properties that can be related to toxicity levels of the solvent on a specific microorganism.33 The toxicity of solvents increases as its surface tension rises because a high surface tension leads to a rapid loss of cellular activity.28,45 On the other hand, the higher is the water solubility of the solvent, the higher is the probability of contact between the bacteria and the organic solvent.33 The surface tension of dodecanol and dodecane are 31.01 and 22.53 dyn/cm (at 25 °C), respectively, and its water solubility for dodecanol is 0.004 g/L (at 25 °C), and dodecane is practically insoluble. Both physical properties have higher values for dodecanol than for dodecane. Therefore, increased toxicity is expected as the dodecanol concentration within the organic phase increases. Although the surface tension of the organic solvents affects the toxicity on specific microorganisms, the solubility of the organic solvent on the culture media can have a higher impact on the toxicity than the surface tension. The organic solvents size is in the order of angstroms, while the mean size of the lactic acid bacteria is in the order of micrometers. If the solubility of the organic solvent is low, there will be few molecules of the organic solvent within the culture media, and due to the small size, as compared to the bacteria size, and the small number of molecules, they cannot affect the bacteria by surface tension. On the other hand, if the solubility of the organic solvent is high enough, there will be enough molecules of solvent within the culture media to provide a toxic effect because the surface tension of the set of the molecules of solvent affect the flux exchange in the cellular wall of the bacteria. Although, the surface tension of organic solvents has been related to the toxicity on bacteria, because it can cause changes in the fluidity of the membrane cell, there are other physical properties of the solvents, such as, viscosity, functional groups, molecular size (related with the long-chain), electron density (related to the polarity), that also can influence toxicity on the bacteria.32,46−48 Moreover, these effects on the bacteria can increase as the number of molecules of the solvent within the culture media (solubility) rises. Hence, the solubility of the organic solvents must be considered as one of the most important features to take into account for analyzing toxicity. However, it is important to mention that the solubility by itself does not provide enough information to classify an organic solvent as toxic or nontoxic. It is necessary to consider other physicochemical properties of the organic solvent molecules. The mixture TiOA/dodecanol/dodecane at 0 mol % of dodecanol (Figure 2) is nontoxic, taking into account cell growth, lactic acid production, and glucose consumption. Cell growth is not affected by the presence of the solubilized organic compounds of the organic phase into the culture media. However, LA production and glucose consumption show a slight decrease compared with control fermentation. Glucose consumption is 84.31%, while LA production is 89.54% of the values of the control fermentation. The mixture TiOA/dodecanol/dodecane with 10.62 mol % of dodecanol is nontoxic, and mixtures with concentrations of dodecanol from 21.22 to 52.85 mol % are medium toxicity. For the organic mixtures TiOA/dodecanol/dodecane with dodecanol proportions from 10.62 to 52.86 mol %, the lactic acid production is around 5% higher than glucose consumption, while the biomass production is lower (between 6 and 25% below) than the LA production and the glucose consumption. Some responses of the cells to the toxic compounds are 3032



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productivity slightly decreases, while the LA to biomass yield slightly increases as compared with those of the control fermentation (Figure 3). Additionally, as it is shown in Figure 2, the presence of dodecanol within the mixture promotes the glucose consumption instead cell growth, which could cause a reduction of the LA to glucose and the biomass to glucose yields during the fermentation for in situ LA removal. However, the productivity is expected to increase when the LA is in situ removed. Single tertiary amines such as Alamine 336, TOA, and TiOA have been shown to be toxic on several lactic acid bacteria. If they are combined with some diluents such as octanol, nheptane, kerosene, and dodecane, they have shown medium or nontoxicity (Table 4). Tributyl phosphate (TBP) is a carrier

compounds interact within the bacteria or on the bacteria metabolism, which could be explored in the future. The the ratio of the LA productivity for the fermentation saturated with the organic phase over that of the control fermentation is approximately one for dodecanol concentrations between 0 to 21.22 mol %. However, the fermentation saturated with the organic phase at 10.62 mol % of dodecanol leads to the maximum relative LA productivity, being around 1.4 (Figure 3). This effect occurs because dodecanol at this

Table 4. Molecular Toxicity of Several Carriers and Diluents for LA Removal on Specific LA Bacteria diluent

carrier

concentration promotes LA production instead of cell growth. Also, the toxic effects on the bacteria are low, and thus, cell growth, LA production, and glucose consumption are similar to that of the control fermentation. At this dodecanol concentration, the cell growth is reduced approximately 18% as compared with that of the control fermentation (Figure 2), while the LA production is increased around 7% (Figure 2). For dodecanol concentrations between 21.22 to 94.66 mol %, the ratio of LA productivity is lower than one and decreases as dodecanol concentration rises due to the toxic effect of the dodecanol on the bacteria (Figure 3). The ratio of the LA to biomass yield is higher than one at all dodecanol concentrations (Figure 3), except for a dodecanol concentration of 0 mol % (which was 0.98 ± 0.17), where the cell growth is slightly promoted instead LA production. The ratio of LA to biomass yield changes between 1 and 1.6 for dodecanol concentrations between 0 to 52.85 mol % (within of the organic phase TiOA/dodecanol/dodecane). A maximum ratio of the LA to biomass yield of 6.6 is reached for a dodecanol concentration of 94.66 mol %, at which the LA production was promoted instead of cell growth. The ratio of LA to biomass yields higher than one is due to biomass growth being always lower than the LA produced, except when the dodecanol concentration is 0 mol %. However, a high ratio of the LA to biomass yield does not mean high LA productivity as compared to that of the control fermentation. At the highest relative LA to biomass yield, the lowest LA and biomass concentrations (Figure 2) are obtained due to the high toxicity level. For the mixtures TiOA/dodecane/dodecanol at proportions of dodecanol between 31.79 and 52.85 mol %, the relative

LA bacteria

ref 30

toxic

oleyl alcohol oleyl alcohol dodecane

Alamine 336 TOA

L. rhamnosus NRRL B445 L. rhamnosus NRRL B445 L. rhamnosus NRRL B445 L. delbrueckii

nontoxicb

L. casei

50

TdDA

nontoxic

L. casei

50

TBP

toxic

31

dodecane

TOA

medium toxicity

n-heptane

medium toxicity

n-heptane

Alamine 336 TOA

kerosene

TBP

toxic

kerosene

TOA

medium toxicity

hexane

Alamine 304 TOA TiOA

nontoxic

L. rhamnosus ATCC N443 L. rhamnosus ATCC N443 L. rhamnosus ATCC N443 L. rhamnosus ATCC N443 L. rhamnosus ATCC N443 L. rhamnosus ATCC N443 L. rhamnosus ATCC N443 L. casei ATCC 393 L. casei ATCC 393 L. casei ATCC 393 L. casei ATCC 393 L. casei ATCC 393 L. rhamnosus NBRC 3863 L. rhamnosus NBRC 3863 L. rhamnosus NBRC 3863

TOA

Figure 3. Effect of the dodecanol concentration in the organic phase (TiOA/dodecanol/dodecane) on the relative productivity (squares) and yield of lactic acid to biomass ratio (triangles). Both are the ratio of the fermentations saturated with the organic phase to the control fermentation.

molecular toxicity levela toxic

MIBK

medium toxicity

octanol

medium toxicity

dodecane dodecane dodecanol dodecane

toxic

TiOA Bmind

medium toxicc toxic nontoxic medium toxic medium toxic medium toxic

Hmind

medium toxic

Omind

medium toxic

30 30 49

31 31 31 31 31 31 33 33 33 33 33 47 47 47

a

According to Marták et al.29 classification of toxicity level. bThe LA production is promoted instead of cell growth. cToxic for TOA proportions higher than 20 vol %. dIonic liquids.

that is toxic on several lactic acid bacteria independently of the diluent used (Table 4). Dodecane mixed with tertiary amines can reduce the toxicity of the tertiary amines on different lactic acid bacteria (Table 4). Additionally, dodecane is nontoxic on the lactic acid bacteria used in this work. In this work, the system TiOA/dodecanol/dodecane at dodecanol proportions from 31.79 to 52.85 mol % is medium 3033



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toxic on the Lactobacillus casei ATCC 393. However, some diluent/carrier systems (Table 4) are nontoxic on several lactic acid bacteria (TOA in oleyl alcohol, TdDA in oleyl alcohol, and Alamine 304 in hexane), and perhaps these systems could provide low toxicity on the Lactobacillus casei ATCC 393. The system TOA in oleyl alcohol promotes the LA production instead of the cell growth (Table 4) as occurs in this work in the system TiOA/dodecanol/dodecane at dodecanol proportion between 10.62 to 94.66 mol %. Perhaps, this effect is common to all alcohols used as diluents for LA removal from fermentation broths.

This work was financially supported by Colciencias under calls 617, 645 and 647, and by the National University of Colombia under projects 28046 and 31011. Notes

The authors declare no competing financial interest.

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ABBREVIATIONS ai = Fitted parameters in eq 3, K1 = Fitted parameter eq 4 [L·mol−1] K2 = Fitted parameter 4 [L·mol−1] KD = Distribution coefficient KE = Chemical equilibrium constant [L·mol−1] DOH = Dodecanol HPLC = High performance liquid chromatography LA = Lactic acid LA−TiOA = The complex between lactic acid and tri-isooctylamine LLE = Liquid−liquid equilibria

4. CONCLUSIONS Molecular toxicity on the Lactobacillus casei ATCC 393 and liquid−liquid equilibria within aqueous lactic acid, for mixtures of TiOA/dodecane, TiOA/dodecanol, both at 10 vol % (5.4 mol %) of TiOA, and TiOA/dodecanol/dodecane at a TiOA concentration of 10 vol % (5.4 to 5.34 mol %) and dodecanol concentrations of 0, 10.62, 21.22, 31.79, 42.33, 52.85, and 94.66 mol % were measured. Both the distribution coefficient and the chemical equilibrium constant are higher for the mixture TiOA/ dodecanol than for the mixture TiOA/dodecane. However, the mixture TiOA/dodecanol was toxic on the bacteria, while the mixture TiOA/dodecane was nontoxic on the bacteria. For the systems containing TiOA/dodecane/dodecanol in the organic phase, at several ratios of dodecanol to dodecane, the distribution coefficient and the chemical equilibrium constant (KE) increase as dodecanol concentration rises. However, the organic mixture at a dodecanol concentration of 10.62 mol % is nontoxic on the bacteria, while at dodecanol concentrations from 21.22 to 52.85 mol % the organic mixture was medium toxic. Cell growth is promoted instead of glucose consumption and LA production when the culture media is saturated with a TiOA/dodecane mixture. Glucose consumption and LA production are promoted instead of cell growth when the culture media is saturated with a TiOA/dodecanol mixture. This behavior also occurs for all organic phases with dodecanol, regardless of the presence of dodecane in these organic mixtures. The highest value of KE is achieved for the mixture TiOA/ dodecanol, but the organic mixture is toxic on the bacteria. Similar values of KE (around 150 L·mol−1) are obtained at dodecanol concentrations of 31.78, 42.33, and 52.85 mol %, at which the organic mixtures are medium toxicity. Among the used diluents, the ability for LA removal in the mixture TiOA/ dodecane/dodecanol is provided by the dodecanol, and the low toxicity is provided by the dodecane. The mixtures of TiOA/dodecanol/dodecane at dodecanol concentrations between of 31.79 and 42.33 mol % have a good compromise between a high value of KE and a relatively low molecular toxicity.

■

Subscripts

aq = Aqueous phase org = Organic phase

■

REFERENCES

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel. +57 68879300, ext. 55410. ORCID

Alan D. Pérez: 0000-0001-7896-0130 Javier Fontalvo: 0000-0002-7868-8736 3034



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