Fermentation Technology

Lactic acid is produced by chemical synthesis and fermentation. This product and its derivatives are important industrial chemicals in the fields of food processing, pharmaceuticals and cosmetics [1], and they have attracted great interest as the raw materials of biodegradable polymer，polylactic acid [2]. In the fermentative production of lactic acid, traditionally, the fermentation broth was neutralized with lime and recovered as calcium lactate [1-5]. After adding sulfuric acid, lactic acid is concentrated by evaporation and purified by methods involving short-path distillation [3]. This recovery and purification of lactic acid from the fermentation broth is the most costly process [4]. Therefore, many recovery techniques such as diffusion dialysis, solvent extraction, adsorption, chromatographic methods, membrane use, drying and electrodialysis have been proposed [5]. Additionally, the in situ recovery technique during fermentation offers the potential to not only recover lactic acid but also relieve end-product (lactic acid) inhibition [5]. Among the proposed separation techniques, reactive extraction is a promising separation method that can be applied in situ and has the potential to provide a high recovery ratio [6]. The reactive extraction of organic acids with amines and ammonium salts has already been heavily investigated. Tri-n-octylamine (TOA) in 1-octanol is reported to be the extractant-diluent combination with the highest distribution ratio [6]. To apply a TOA/1-octanol system to in situ extractive fermentation, two critical problems remain. One is the toxicity of the diluents and extractant. Marinova and Yankov [7] studied the toxicity of solvents and extractants for Lactobacillus casei , and all of the alcohols investigated involving 1-octanol were found to show high phase toxicity. Therefore, we focus on Hiochi bacteria, Lactobacillus homohiochii and Lactobacillus fructivorans , which are known for their ability to spoil Japanese rice wine (sake) and for being the most alcohol-tolerant organisms [8]. On the other hand, extractant TOA has been reported to be highly toxic for Lactobacillus strains [7,9,10]. Studies on the alleviation of the toxic effect have been attempted, such as co-immobilization of soybean oil [11] or sunflower oil [12] with the cells and passing extracted broth through a column filled with organic solvent [13]. The other problem is the difference in optimum pH between fermentation and extraction. It is well known that the polar diluents and low pH (14]. Generally, fermentation pH is in the range of 6–8, while acid production acidifies the medium. Therefore, one method to overcome a difference in optimum pH is to develop an acid-tolerant organism1. The other method is to remove the acid in situ by a chemical or physical method without pH control [3,15].

In this study, the aim of this work is to establish the in situ extractive fermentation of lactic acid with alcohol-tolerant Hiochi bacteria using the co-encapsulation of the bacteria and calcium carbonate to depress the decrease in the pH in the capsules. First, the tolerance of Hiochi bacteria to aliphatic long-chain alcohols as diluents and TOA as an extractant was examined. Then the effects of pH and the components in the medium on the extraction of lactic acid with TOA in the selected diluent, 1-decanol, were investigated. Finally, an in situ extractive fermentation process of lactic acid was developed by co-immobilizing decanol-tolerant Lactobacillus fructivorans NRIC0224 cells and calcium carbonate into Ca-alginate capsules without pH control [6-9].

Organisms and cultivation

We used the four lactic acid-producing Hiochi bacteria listed in Table 1. All bacteria were provided by NODAI Culture Collection Center (NRIC, Japan). The GYP-CaCO₃ medium was used for the organisms NRIC 0119 and 0224. SI medium (commercial medium for Hiochi bacteria [16]) was used for NRIC 1814 and 1815. In the cases of organic solvents containing tri-n-octylamine, both the GYP-CaCO₃ and GYP media were used for the organisms NRIC 0224. Cultivations were performed at 30°C.

Table 1: Toxicity of long-chain aliphatic alcohols to Hiochi bacteria. +, Relative activity >0.5; relative activity <0.5. Relative activity is defined as the ratio of lactic acid concentration of the sample to that of the control.

Growth in presence of organic solvents

Growth in the presence of a second phase was determined based on a method described previously [17]. 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 were transferred to new culture bottles (20 mL), and 1% (v/v) of one of the organic solvents listed in Tables 1 and 2 was added. Added organic solvents formed the second phase. The culture bottles were closed with Teflon valves to prevent evaporation and then incubated for 48 h at 30°C. Then, the optical density (OD600 for NRIC 0199 and 0224, and OD550 for NRIC 1814 and 1815) and the concentrations of glucose and lactic acid were measured [10,11].

Table 2: Toxicity of extractants to Hiochi bacteria. a: Cultivation in the presence of 10 gL-1 CaCO₃. b: not determined.

Extraction of lactic acid from fermentation broth

Tri-n-octylamine (TOA) was used as the extractant without further purification. An organic solution was prepared by dissolving 0.5 mol/L of TOA in 1-decanol. In the aqueous media, aqueous solutions were prepared by dissolving lactic acid in deionized water. Prior to the experiment, commercial lactic acid was diluted with distilled water and heated for at least 12 h to hydrolyze the lactic acid polymers. In the fermentative media, lactic acid fermentation was carried out using L. fructivorans NRIC0224 cells and lactic acid was produced from culture grown in 100 mL of GYP-CaCO₃ medium at 30°C for 48 h. The fermentation broth was centrifuged at 40,000 g for 30 min, and then the supernatant was used as an aqueous solution for the extraction experiment. The pH values of the aqueous solutions were adjusted by adding a 1 mol/L H₂SO₄ or NaOH aqueous solution. The aqueous and organic solutions of equal volume (10 mL) were mixed in a vial and shaken at 30°C to attain extraction equilibrium. After the two phases were separated, the concentration of lactic acid in the aqueous solution was analyzed. The concentration of lactic acid in the organic solution was calculated from the mass balance. The pH of the aqueous solution before and after equilibration was determined by a Horiba F-12 pH meter.

Immobilization of cells in Ca-alginate capsules

Cells of L. fructivorans NRIC0224 for encapsulation were obtained from culture grown in 20 mL of GYP-CaCO₃ medium at 30°C for 18 h. The cells were harvested from the fermentation broth by centrifugation at 40,000 g for 30 min and then washed with sterile saline solution. The cells were resuspended in 10 mL of a 2.0% (w/v) steric CaCl₂ solution containing 20 g/L CaCO₃. The suspended solution was mixed with 10 mL of a 2.0% (w/v) sterile sodium alginate solution. The mixture was added dropwise into a sterile 1.0 mol/L CaCl₂ solution at room temperature. The beads were hardened in this solution for 1 h. The beads were washed with sterile saline prior to use [12-16].

Fermentations

Batch fermentations were performed in 250-mL culture bottles containing 100 mL of GYP medium and 20 mL of alginate beads with cells. Samples were withdrawn periodically during fermentation and analyzed for glucose and lactic acid. We also carried out lactic acid production using immobilized cells in a packed bed reactor (dia. 15 mm, length 100 mm) with broth recycled to a stirring tank in a batch mode as illustrated in Figure 1. The stirring tank initially contained 175 mL of GYP medium including 25 g/L glucose. The broth in the tank was held at 30°C, 500 rpm agitation rate and an initial pH of 6.75. From this, the medium was pumped to the top of the packed bed. The medium emerging from the bed was returned to the tank. The flow rate of the medium in the bed was kept to 1.5 mL/min by the two pumps. In an extractive fermentation, 175 mL of 1-decanol solution containing 0.5 mol/dm³ TOA was placed on top of the medium solution. Samples were withdrawn periodically from the medium and the organic phase during fermentation and analyzed for glucose and lactic acid. Ca-alginate capsules were broken down by the homogenizer, and the number of viable cells (CFU/mL) in the cell suspension was counted before and after fermentation.

Figure 1: Schematic diagram of extractive fermentation with packed bed column.

Analysis

Optical density was determined using a spectrophotometer (Shimadzu UV-2500PC). Lactic acid in the organic phase was stripped with 1 mol/dm³ NaOH solution. The concentrations of lactic acid and glucose in the resultant aqueous solutions were determined by HPLC with a Shodex SH-1011 column and a 5 mM H₂SO₄ solution as the mobile phase. Lactic acid and glucose were detected with an RI detector (Shimadzu RID10A). All experiments were duplicated [17-19].

Tolerance of Hiochi bacteria to long-chain aliphatic alcohols

In a previous study [17], we investigated the tolerance of lactic acid producing bacteria, including L. homohiochii NRIC0119, 1815, L. fructivorans NRIC0224, 1814, to organic solvents. The long-chain aliphatic alcohols, such as octanol and decanol, were reported to be active diluents for lactic acid extraction [18]. Consequently, we examined the tolerance of Hiochi bacteria to long-chain aliphatic alcohols. Table 1 shows the relationship between the bacterial activity retained by cells exposed to a long-chain aliphatic alcohol and their log PO/W values for four Hiochi bacteria. Microbial activity in the presence of organic solvents was determined on the basis of the concentration of lactic acid produced after adding alcohol, with relative activity being defined as the ratio of the lactic acid concentration of the sample to that of a control. As pointed out by Laane et al. [19], solvents having a log PO/W lower than 2 are not suitable for biocatalytic systems due to the high solubility of the solvent in the water phase. Unfortunately, 1- octanol was toxic to all Hiochi bacteria investigated. L. fructivorans NRIC0224 had a tolerance to 1-decanol, which is an active diluent for lactic acid extraction. In the following experiment, we selected L. fructivorans NRIC0224 as the Hiochi bacterium and 1-decanol as the diluent.

Tolerance of Hiochi bacteria to tri-n-octylamine extractant

As described above, TOA has been reported to be highly toxic to Lactobacillus strains [7,9,10]. In this study, we examined the toxicity of extractants to Hiochi bacteria and their relative activities in the presence and absence of CaCO₃ (10 g/L) are summarized in Table 2. In the absence of CaCO₃, TOA depressed the microbial activities of Hiochi bacteria. It was found that the presence of CaCO₃ in the medium alleviated the effect of TOA on the Hiochi bacteria, but CaCO₃ had no effect on the other extractants, tri-n-butylphosphate and tri-n-octylmethylammonium chloride, for lactic acid extraction. And then it was found that other carbonates and calcium salts had also no effect, suggesting that the effect of calcium carbonate was specific to extractants. There have been reports on the mechanism of calcium alleviation for mineral toxicities, where a specific effect is considered to be the collective ameliorative effect of Ca²⁺ based on the Ca²⁺-toxicant interactions at the cell surface [20]. A small amount of dissolved Ca²⁺ from slightly soluble calcium carbonate may interact with TOA. Figure 2 shows the effect of CaCO₃ concentration in the medium on the microbial activity. The presence of CaCO₃ at more than 2.5 g/L caused an alleviation effect for TOA.

Figure 2: Effect of CaCO₃ in the medium on relative activity in the presence of TOA as a second phase

Generally, a viscous extractant such as TOA is diluted by the appropriate diluents, and the TOA solution is used as an extract. As described above, 1-decanol was an active diluent for lactic acid extraction. Therefore, the toxicity of a TOA solution diluted by 1- decanol was also examined as shown in Table 1. From these experimental results, a 1-decanol solution containing TOA is promising for the extractive fermentation of lactic acid with Hiochi bacteria in the presence of CaCO₃.

Extraction of lactic acid from fermentation broth

The distribution ratio, D, of lactic acid, HA, is defined by Eq. (1).

Figure 3 shows the distribution ratios of lactic acid in an aqueous medium and the fermentative medium at pH 3.5. Evidently, the distribution ratio dramatically decreased from 15 in the aqueous medium to 2 in the fermentative medium. In order to specify the causative component in the medium, the effects of glucose, yeast extract and peptone on the distribution ratio are also shown in Figure 3. The causative components were found to be yeast extract and peptone, although it was reported that peptone, yeast extract and lactose did not affect the lactic acid extraction with TOA in a mixture of dodecane and 1-decanol [18]. However, the concentration ranges investigated were unclear in the previous paper. It is because the relative high concentrations of yeast extract and peptone were used in this fermentation. Probably, the organic acids involved in yeast extract and peptone, and lactic acid completed for TOA and the organic acids interfered with the extraction of lactic acid using TOA. In the future, the optimum medium composition must be considered for both fermentation and extraction.

Figure 3: Effects of components in the aqueous medium on distribution ratio of lactic acid at pH 3.5; Fermentation medium contains 10 g/L glucose, 5 g/L yeast extract and 5 g/L peptone.

As described above, in the extractive fermentation of lactic acid with a chemical reaction using tertiary amines, one of the critical problems to be solved was the difference in the optimum pH between the fermentation and extraction. Therefore, the pH effect on the distribution ratio was examined, and the results are shown in Figure 4. As described in previous papers [13,18,21] the distribution ratio decreased with increasing pH, suggesting that undissociated lactic acid was the extracted species. In this study, even under the non-optimized medium composition, the distribution ratio of lactic acid from the fermentation broth was larger than unity at pH 5.

Figure 4: pH effects on distribution ratio of lactic acid in fermentative medium and aqueous medium.

Extractive fermentation by immobilized L. fructivorans in Ca-alginate capsules

As described above, an in situ extractive fermentation process with a chemical reaction using TOA has two critical problems: solvent toxicity and pH. In the previous sections, we found that the presence of CaCO₃ in the medium considerably alleviated the toxicity of TOA in 1-decanol for the lactic acid fermentation with L. fructivorans NRIC0224, and lower pH values caused the high extractability of lactic acid with TOA. Based on these results, we proposed the coimmobilization of cells and CaCO₃ powders in Ca-alginate capsules. In such a scenario, the cells do not receive the phase toxicity, that is, there is no direct contact of the cells with organic solvent, and the presence of CaCO₃ in the vicinity of the cells alleviated the molecular toxicity of TOA dissolved in the aqueous solution. Although initial fermentation pH is 6.8, the medium pH gradually decreases as the fermented product, lactic acid, is produced if the pH is not kept at the initial value. However, the medium pH within the Ca-alginate capsules can be maintained due to the neutralizing effect of CaCO₃ [22]. On the other hand, the pH of bulk medium may be lowered due to the release of lactic acid from the capsules. Furthermore, a lower pH of the medium is preferable for extraction with TOA. Based on this notion, we constructed an in situ extractive fermentation system with a chemical reaction using TOA as shown in Figure 1.

Prior to the extractive fermentation with the packed bed, we examined the effect of co-immobilization of L. fructivorans NRIC0224 cells and CaCO₃ in Ca-alginate capsules on the lactic acid fermentation in a batch operation. Table 3 gives a summary of the lactic acid produced in the medium under various conditions. While TOA was toxic in the GYP medium, co-immobilization of CaCO₃ in Ca-alginate capsules had an alleviation effect on the toxicity of TOA. In all cases, the presence of CaCO₃ caused higher lactic acid production, suggesting that CaCO₃ depresses the decrease in pH because CaCO₃ neutralizes lactic acid fermentation.

Table 3: Summary of batch fermentation of lactic acid using Caalginate capsules including L. fructivorans NRIC 0224. Initial glucose concentration was 9.44 g L^-1. Values are concentrations of lactic acid in the medium after 48 h.

Figure 5 shows time courses of glucose and lactic acid concentrations in the extractive fermentation of lactic acid using a packed bed column without pH control. In the control fermentation in the absence of an organic phase (Figure 5A), the rates of glucose consumption and lactate production gradually decreased due to the decrease in the medium pH. When pH was kept at the initial value, glucose was completely consumed. The extractive fermentation (Figure 5B) proceeded successfully. Table 4 shows a summary of the fermentation, along with the viability of the cells. Productivity of the extractive fermentation was larger than that of the control at 30 h, and the yield of the extractive fermentation was improved. In the presence of an organic solution, encapsulated cells survived, but the vial cells slightly decreased due to the toxicity of the organic solution. Therefore, improvement of productivity was found to be depressed by the molecular toxicity of TOA. More hydrophobic amines with less water solubility should be developed. Recovery of lactic acid may be enhanced by continuous stripping of lactate from the loaded organic phase [20-22].

Table 4: Summary of extractive fermentation with packed bed and viability of L. fructivorans NRIC 0224 during the fermentation *1) glactate produced per g, glucose consumed; *2) g-lactate produced L^-1h^-1; *3) CFU mL^-1.

Figure 5: Time courses of glucose and lactic acid concentrations, and pH in control (A) and extractive (B) fermentations using packed bed column.

The extractive fermentation of lactic acid with Hiochi bacteria was carried out. We selected L. fructivorans NRIC0224 as the Hiochi bacterium and 1-decanol as a diluent from the viewpoints of the tolerance of organic solvents to microbes and the extraction capacity. Although TOA has been reported to be highly toxic to Lactobacillus strains, we found that the presence of powdered CaCO₃ in the medium considerably alleviated the toxicity of TOA. We also found that yeast extract and peptone affected the extraction of lactic acid with TOA in 1-decanol. Then, we immobilized both L. fructivorans NRIC0224 cells and CaCO₃ in Ca-alginate capsules and constructed an in situ extractive fermentation system with a chemical reaction using TOA and a packed bed of Ca-alginate capsules without pH control. This system worked successfully, and the yield and productivity were improved over those of the control fermentation.

This work was supported by JSPS KAKENHI Grant Number 25420813 and MEXT-Supported Program for the Strategic Research Foundation at Private Universities.