Bioresource Technology

Bioresource Technology 100 (2009) 2878 2882 Contents lists available at ScienceDirect Bioresource Technology journal homepage: www.elsevier.com/locate/biortech Short Communication Study on solvent extraction of propionic acid from simulated discharged water in vitamin B 12 production by anaerobic fermentation Kang Wang a,b, Zhidong Chang a, *, Yinchen Ma a,b, Chao Lei a,b, Jing Wang a, Tingyu Zhu a, Huizhou Liu a, *, Yanjun Zuo a, Xin Li c a Laboratory of Green Process and Engineering, Laboratory of Separation Science and Engineering, Institute of Process Engineering, Chinese Academy of Sciences, Beijing 100190, China b Graduate University of the Chinese Academy of Sciences, Beijing 100049, China c Graduate School of Chinese Academy of Agricultural Sciences, Beijing 100081, China article info abstract Article history: Received 25 July 2008 Received in revised form 28 December 2008 Accepted 29 December 2008 Available online 6 February 2009 Keywords: Primary amine Propionic acid Extraction Wastewater The potential of recovering propionic acid from discharged water in vitamin B 12 production by anaerobic fermentation was investigated in this paper. A primary amine, N 1923, was used as the extractant, kerosene as diluter and n-octanol as modifier. The influences of the content of N 1923 in the organic phase, the phase ratio and the ph of aqueous phase on the extraction yield of propionic acid were studied. The organic phase composition with the volume ratio was proposed of N 1923 :kerosene:n-octanol as 45:35:20. Under conditions of the phase ratio (o/w) as 1:4, the ph of aqueous phase of 3.0 and after 5 min extraction, the extraction yield of propionic acid can be over 97%. Ó 2008 Elsevier Ltd. All rights reserved. 1. Introduction Propionic acid is a widely used carboxylic acid in the plastics, coatings, agricultural chemical, perfume industry. It also can replace chemical preservatives and is of substantial commercial importance in the sale of natural bakery products. The Na +,Ca 2+ and K + salts of propionic acid have also been listed as preservatives which are of the category known as generally recognized as safe (GRAS) food additives (Yang and Zhu, 1994). Together with anaerobic fermentation producing Vitamin B 12, propionic acid is also produced as a byproduct. Now after extraction of vitamin B 12, the water was treated as waste. The recovery of the propionic acid from wastewater will decrease the COD (chemical oxygen demand) value of the wastewater, which will reduce the difficulty of the wastewater treatment in the next step. Contemporaneously, the recovered propionic acid can increase economic benefit of the whole vitamin production process. Extractive recovery of carboxylic acids from dilute aqueous solutions, such as fermentation broth or wastewater, in which acid concentration is lower than 10% (w/w), has received increasing attention. Solvent extraction using conventional solvents such as alcohols, ketones, ethers or aliphatic hydrocarbons give low distribution coefficient because of the high affinity of the acids to water. * Corresponding authors. Tel.: +86 10 62642032; fax: +86 10 62554264. E-mail addresses: zdchang@home.ipe.ac.cn (Z. Chang), hzliu@home.ipe.ac.cn (H. Liu). Consequently, physical extraction with conventional solvent is not an efficient method for the recovery of these acids (Marinova and Albet, 2005; Vanura and Kuca, 1976; Kertes and King, 1986). However, reactive extraction with aliphatic amines has been proposed as a promising technique for separation of the acids. The strong interaction between the amine and the acid allows formation of acid amine complexes, which provides high distribution coefficients. In addition, the high affinity of amine to the acid gives high selectivity for the acid to non-acid components in the mixture (Yang et al., 1991). In the available literature, there is a significant amount of data on the reactive extraction of carboxylic acids using amine-based extractants. Various aspects of this subject were considered. Grinstead and Davis reported that the base strength decreased in the order primary > secondary > tertiary (Grinstead and Davis, 1968). Shan et al. (2006a,b) investigated the relationship between extraction equilibrium of monocarboxylic acid and the apparent basicity of extractant. The dependence of tartaric acid extraction on ph of the aqueous phase was explored by Tomovska et al. (1999). The spectroscopic studies of Yang et al. (1991) revealed that Alamine 336 binds the non-dissociated part of acid in the organic phase through reversible complexation. Other factors, such as the nature of acids, type of diluent, temperature on the extraction equilibrium has also been studied (Tamada and King, 1990; Tamada et al., 1990; Yang et al., 1991; Bizek et al., 1993; Malmary et al., 1993; Prochazka et al., 1994; Matsumoto et al., 2003; Morales et al., 2003; Qin et al., 2003; Senol, 2000, 2002, 2005; Shi et al., 2006; Uslu, 2007). 0960-8524/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2008.12.056

K. Wang et al. / Bioresource Technology 100 (2009) 2878 2882 2879 It is worth mentioning that the third phase or emulsification was observed in the extraction process while amine was used as extractant (Bizek et al., 1993; Heyberger et al., 1998; Jeong et al., 2001; Song et al., 2002). Hong and Hong (1999) have stated that the use of a mixture of a short chain amine with a long chain one prevents the third phase from forming. Han et al. (2000) and Jung et al. (2000) have studied the extraction of lactic acid with tertiary amines with different chain length in different diluents. They have mentioned the importance of organic phase composition for the successful extraction. Han et al. have found triotylamine (TOA) to be the most effective amine to prevent the third phase from forming. While Jung et al. have suggested the use of trihexylamine in isobutanol to avoid the formation of the third phase. The formation of the third phase or emulsification will not only affect the extraction efficiency, but also increase the extractant loss, which will decrease the economic benefit of the whole process. In order to overcome problems concerned with the third phase formation or emulsification, a second diluent (modifier) is used. Polar solvents can help to solvate the acid amine complex, which will help to avoid the formation of the third phase or emulsification. This paper tries to investigate the potential of using amine to recover propionic acid from wastewater by solvent extraction. In our experiments, a primary amine, N 1923, was chosen as the extractant because it can provide high extraction yield of propionic acid, low solubility in water (0.00625%) and good regenerability by back-extract with sodium hydroxide solution. In the first step, the effect of n-octanol on suppressing the emulsification trend was investigated. Then the influence of the content of N 1923, the phase ratio, the ph of aqueous phase and operation time on the extraction yield of propionic acid were investigated, so that the production process could be optimally designed. 2. Methods 2.1. Materials The extractant N 1923 is a primary amine, having two carbon chains branched (its structure is R 1 R 2 CHNH 2 and total number of carbon atoms is 19 23). It was obtained from Shanghai Institute of Organic Chemistry, China. N 1923 was used as the extractant without further purification. Kerosene (commercial grade) was purchased from Tianjin Damao Chemical Reagent Factory (Tianjin, China). Propionic acid, n-octanol and sodium hydroxide were of analytical grade, and were purchased from Beijing Chemical Reagents Company (Beijing, China). The organic phase was prepared by mixing N 1923, kerosene, n-octanol together at certain fraction, and the aqueous solution was prepared by dissolving propionic acid in deionized water, the ph value of aqueous solution was adjusted by adding sodium hydroxide. The initial concentration of propionic acid was 0.2703 mol/l, which was the same concentration as the real discharged water. 2.2. Effect of n-octanol on suppressing the emulsification trend Experiments were conducted to investigate the addition of n- octanol on suppressing the emulsification trend. The component of the organic phase was shown in Table 1. Twenty millilitre of organic phase and twenty millilitre of aqueous phase were added into Erlenmeyer flask, then the erlenmeyer flask was put in water bath at 30 C and shaken at a frequency of 160 rpm for 1 h, at last, the mixture was kept in separatory funnel for phase separation, then the absorbance of light of the aqueous was measured. The transmittance of light of the aqueous phase was calculated using Lambert Beer Equation. The emulsification of the aqueous phase after extraction was investigated according to the transmittance of light of the aqueous phase. Table 1 Experiment condition for testing effect of n-octanol on suppressing the emulsification trend. Compose of organic phase (v/v%) n-octanol N 1923 Kerosene 0 20 80 0.2703 10 20 70 0.2703 20 20 60 0.2703 30 20 50 0.2703 0 30 70 0.2703 10 30 60 0.2703 20 30 50 0.2703 30 30 40 0.2703 0 50 50 0.2703 10 50 40 0.2703 20 50 30 0.2703 30 50 20 0.2703 2.3. Batch extraction experiment Concentration of propionic acid in aqueous phase, mol/l Batch experiments were conducted to investigate the influence of the content of N 1923, the phase ratio, the ph of aqueous phase and operation time on the extraction efficiency. The operation conditions were summarized in Table 2. The experiments were carried out in Erlenmeyer flask as follows: (1) twenty millilitre of organic phase with certain fraction of N 1923, kerosene, n-octanol was added into Erlenmeyer flask; (2) certain amount of aqueous solution with certain ph was added into the Erlenmeyer flask; (3) Erlenmeyer flask was put in water bath at 30 C and shaken at a frequency of 160 rpm for a certain time for extraction; (4) the mixture was kept in separatory funnel for phase separation and the concentration of propionic acid in the aqueous phase was determined by GC. The concentration of organic acid in the organic solution was calculated by mass balance. Table 2 Batch experiment conditions to investigate to influence of the operation parameters on extraction efficiency of propionic acid. Batch Content of n- octanol (v/v%) Content of N 1923 (v/v%) Phase ratio (o/ w) ph of aqueous phase A 1 20 10 1:4 2.83 60 A 2 20 20 1:4 2.83 60 A 3 20 30 1:4 2.83 60 A 4 20 40 1:4 2.83 60 A 5 20 45 1:4 2.83 60 A 6 20 50 1:4 2.83 60 A 7 20 70 1:4 2.83 60 B 1 20 45 1:7 2.83 60 B 2 20 45 1:6 2.83 60 B 3 20 45 1:5 2.83 60 B 4 20 45 1:4 2.83 60 B 5 20 45 1:3 2.83 60 C 1 20 45 1:4 2.83 60 C 2 20 45 1:4 3.68 60 C 3 20 45 1:4 4.66 60 C 4 20 45 1:4 5.62 60 C 5 20 45 1:4 6.4 60 D 1 20 45 1:4 2.83 5 D 2 20 45 1:4 2.83 10 D 3 20 45 1:4 2.83 20 D 4 20 45 1:4 2.83 30 D 5 20 45 1:4 2.83 40 D 6 20 45 1:4 2.83 60 Operation time (min)

2880 K. Wang et al. / Bioresource Technology 100 (2009) 2878 2882 2.4. Analytical methods The absorbance of light of the aqueous phase after extraction was measured by UV/vis Spectrophotometry (Perkin Elmer, Lambda Bio40, USA) at the wave of 680 nm (Xin, 2001). The ph of aqueous solution was measured by PH211 ph-meter. The concentration of propionic acid in the aqueous solution was determined by HP6890 GC equipped with FID detector and a 30 m 0.53 mm DB-FFAP capillary column (Agilent Technologies, USA). The GC conditions are as follow: hydrogen is used as the carrier gas, the injection port temperature is 200 C and the temperature of detector is 300 C. 2.5. Theoretical The extraction of propionic acid with N 1923 can be described by the reaction mha þ RNH 2 () RNH 2 mha where HA represents the propionic acid present in the aqueous phase and organic phase species are marked with (). Reaction (1) can be characterized by the overall equilibrium constant, K K ¼ ½RNH 2 mhaš ½RNH 2 Š½HAŠ m where [HA] denotes concentration (Tamada et al., 1990). The loading of the extractant, Z, is defined as the total concentration of acid in the organic phase, divided by the total concentration of amine in the organic phase. The expression for the loading, Z, can be written in the form Z ¼ C a;org =C e;org The efficiency of extraction, E is expressed as E ¼ð1 C a =C ao Þ100 where C a is the concentration of acid in the aqueous phase after extraction and C a0 is the initial concentration of acid in the aqueous phase, mol/l. An E value of 100% means that all of the acid in the aqueous phase has been removed and is present in the organic phase. ð1þ ð2þ ð3þ ð4þ Table 3 shows the transmittance of the aqueous phase after extraction at the wave of 680 nm versus the component of organic solvent. Form this table we can see that, the component of the organic phase had a significant influence on the emulsification trend. With the absence of n-octanol in the organic phase, the transmittance of the aqueous phase after extraction decreased with the increasing of N 1923, when the content of N 1923 reached 50%, the transmittance of the aqueous phase after extraction had an obvious decrease. When n-octanol was added into the organic phase, the transmittance of the aqueous phase after extraction increased with the increasing of the content of n-octanol at first, however, when the content of n-octanol was larger than 20%, the transmittance of the aqueous phase after extraction decreased, the emulsification was best controlled around 20% of n-octanol. This may be explained that the pure primary amine (N 1923 ) had a high viscosity and poor solubility, which easily produced emulsification. So the emulsification trend increased with the increasing of the content of N 1923. Adding certain amount n-octanol into organic phase would provide a better dissolving capacity for N 1923, which would help to decrease the emulsification trend. However, with the further increasing of the content of n-octanol, the interfacial tension of the oil water interface would be further decreased, which would be favorable for the forming of emulsion. 3.2. Influence of the operation parameters on extraction of propionic acid The influences of the content of N 1923, phase ratio, ph of aqueous phase, operation time on extraction efficiency of propionic acid are shown in Table 4. It can be seen that the extraction efficiency of propionic acid increased rapidly with increasing of content of N 1923 in the organic phase at first, however, when the content of N 1923 reached about 45%, the extraction efficiency remained constant with increasing of the content of N 1923. This may be explained that increasing of the content of N 1923 is favorable for extraction. However, higher content of N 1923 increases the viscosity of the organic phase, which is not favorable for extraction. In addition, the influence of the content of N 1923 on loading factor is shown in Fig. 1. The overloading, with the loading factor greater than unity, can be observed at low N 1923 content, which indicated that the formed acid amine complexes include more than one acid per complex (Yerger and Barrow, 1955). With the increasing of amine concentration in the organic phase, the loading Table 3 Influence of the compose of organic phase on transmittance of the aqueous phase after extraction. Compose of organic phase (v/v%) n-octanol N 1923 Kerosene 0 20 80 40.8 10 20 70 64.6 20 20 60 72.6 30 20 50 38.9 0 30 70 42.7 10 30 60 17.7 20 30 50 35.8 30 30 40 12.9 0 50 50 18.0 10 50 40 11.8 20 50 30 45.5 30 50 20 42.0 Transmittance of the aqueous phase after extraction (T%) 3. Results and discussion 3.1. Effect of n-octanol on suppressing the emulsification trend Table 4 Influence of the operation parameters on extraction efficiency of propionic acid. Influence factors Extraction efficiency (%) Content of N 1923 (v/v%) 10 52.35 20 74.46 30 92.56 40 94.80 45 96.50 50 97.10 70 97.34 Phase ratio (o/w) 1:7 67.80 1:6 79.90 1:5 88.70 1:4 97.50 1:3 98.60 ph of aqueous phase 2.83 96.17 3.68 93.76 4.66 62.94 5.62 19.98 6.40 13.55 Operation time (min) 5 97.00 10 97.20 20 97.20 30 97.10 40 97.40 60 97.50

K. Wang et al. / Bioresource Technology 100 (2009) 2878 2882 2881 Z 2.0 1.8 1.6 1.4 1.2 1.0 0.8 0.6 0.4 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2 C e,org /(mol/l) rapidly increase from 20% with ph of 5.6 to about 94% with ph of 3.6. As the extraction behaviours of propionic acid with N 1923 were investigated, the optimum recovery process was established. An organic phase composition was proposed with the volume ratio of N 1923 :kerosene:n-octanol as 45:35:20, and under conditions of the phase ratio (o/w) as 1:4, the ph of aqueous phase of 3.0 and 5 min of extraction, the extraction yield of propionic acid can be over 97%. Acknowledgements The authors thank the National Natural Science Foundation of China (No.2040602 and 20876157) and 973 program (No.2007CB714301) for financial support of this research. References factor of the extractant decreased. Considering both the extraction efficiency and the loading factor, 45% was chosen as the suitable content of N 1923 in the organic phase for extraction. The Influence of phase ratio on extraction of propionic acid was investigated. The extraction efficiency of propionic acid increased sharply at first and changed little later when the phase ratio (o/ w) increased. The optimum phase ratio (o/w) obtained was about 1:4. At high phase ratio (o/w), which means that there is relatively little water phase in the system, the extraction efficiency is high for good contact of the oil and water phase. With the decreasing of the phase ratio (o/w), the oil phase is saturated gradually by the water phase, making the extraction efficiency decrease. The influence of ph value on the extraction efficiency of propionic acid is also shown in Table 4. When the ph of aqueous phase decreased, the extraction yield of propionic acid had a rapidly increase from 20% with ph of 5.6 to about 94% with ph of 3.6. Usually propionic acid was extracted into the organic phase as molecule which was not dissociated. The ph value of aqueous phase has significant influence on the dissociation of propionic acid, which will influence the extraction efficiency of propionic acid. With the increasing of ph, the dissociation of propionic acid increases, and the fraction of molecule of propionic acid decreases, which results in a decrease of extraction efficiency of propionic acid. Operation time is one of the important variables in the extraction process. From the results we can see that the extraction can quickly reach equilibrium. After 5 min, the extraction reached equilibrium. This because the extraction process of acid using amine as extractant is a extraction accompanied by a fast general order chemical reaction (Wasewar et al, 2003), and the strong amine interaction with the acid provides a high rate constant for the reaction, which makes the extraction reaches equilibrium quickly. 4. 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