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Two-step salting-out extraction of 1,3-propanediol, butyric acid and acetic acid from fermentation broths
Accepted Manuscript Two-step salting-out extraction of 1, 3-propanediol, butyric acid and acetic acid from fermentation broths Zhen Li, Ling Yan, Jinjie Zhou, Xiaoli Wang, Yaqin Sun, Zhi-Long Xiu PII: DOI: Reference:
S1383-5866(18)30841-4 https://doi.org/10.1016/j.seppur.2018.07.021 SEPPUR 14752
To appear in:
Separation and Purification Technology
Received Date: Revised Date: Accepted Date:
13 March 2018 20 June 2018 9 July 2018
Please cite this article as: Z. Li, L. Yan, J. Zhou, X. Wang, Y. Sun, Z-L. Xiu, Two-step salting-out extraction of 1, 3-propanediol, butyric acid and acetic acid from fermentation broths, Separation and Purification Technology (2018), doi: https://doi.org/10.1016/j.seppur.2018.07.021
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Two-step salting-out extraction of 1, 3-propanediol, butyric acid and acetic acid from fermentation broths Zhen Li, Ling Yan, Jinjie Zhou, Xiaoli Wang, Yaqin Sun, Zhi-Long Xiu* School of Life Science and Biotechnology, Dalian University of Technology, Dalian, Liaoning 116024, P.R. China
*Correspondence: Prof. Zhilong Xiu ( Email: [email protected], Tel/Fax: +86-0411-84706369 ), School of Life Science and Biotechnology, Dalian University of Technology, No.2 Linggong Road, Dalian 116024, P. R. China
1
Abstract: The separation of 1, 3-propanediol (1, 3-PD), butyric acid (BA) and acetic acid (HAc) from the fermentation broth was studied by two-step salting-out extraction. In the first salting-out extraction, the partition coefficient and recovery of BA reached 42.21 and 96.42%, respectively, under the optimal condition of 25 wt% NaH2PO4/30 wt% n-butyl acetate. Subsequently, 91.28% of BA in the organic phase could be recovered through back-extraction when sodium carbonate solution was mixed with organic phase with initial phase ratio of n-butyl acetate solution to alkaline solution being 2: 1, in which the molar ratio of sodium carbonate to organic acids was 3: 5. Finally, 50% (v/v) ethanol (95%) was added to the bottom phase for the second step salting-out extraction. And the partition coefficient and recovery of 1, 3-PD were 9.40 and 95.50%, and those of HAc were 7.46 and 94.40%, respectively. All the cells and most of the proteins (97.16%) could be removed. Effective separation of 1, 3-PD from BA was realized by two-step salting-out extraction, which provides a potential method for separation of 1, 3-PD, BA and HAc on an industrial scale. Keywords: 1, 3-Propanediol; Butyric acid; Acetic acid; Salting-out extraction; Back-extraction
2
1.
Introduction 1, 3-Propanediol (1, 3-PD) is an important chemical material, which could be
used in multiple fields in virtue of versatility, such as solvent, adhesive, cosmetic, preservative and medicine. It could participate in a variety of condensation polymerization as monomer for production of excellent performance polymers such as polyethers, polyesters and polyurethanes due to its two symmetrical hydroxyl groups, in which polytrimethylene terephthalate (PTT) has shown tremendous application prospect in the fiber, textile and engineering thermoplastics industry because of its advantages, such as good tensile elastic recovery, low static generation, stain resistance and biodegradability [1-3]. Up to now, 1, 3-PD has been mainly manufactured by chemical synthesis with rigorous reactions, valuable catalysts and multitudinous by-products, in which down-streaming products from petrochemical industry are used as raw materials. As people have increasingly worried about fossil energy exhaustion and environmental pollution, biorefinery technology has been paid a great deal of attention, by which renewable biomass is used to produce bioenergy and chemicals. For example, microbial conversion of glycerol, a by-product from biodiesel industry, into 1, 3-PD is a superb application of biorefinery, which not only guarantees continuous supply of raw materials but also avoids resource waste
[4]
. Microorganisms used in 1, 3-PD
fermentation mainly include Klebsiella pneumoniae Clostridium butyricum
[7]
[5]
, Clostridium pasteurianum
, Clostridium beijerinckii 3
[8]
[6]
,
, Escherichia coli[9] and
Citrobacter freundii
[10]
, in which K. pneumonia and C. butyricum have been widely
used due to their high productivity. Compared with K. pneumonia, a few metabolites are produced from C. butyricum, i.e. 1, 3-PD, acetic acid (HAc) and butyric acid (BA). Recently, microbial consortia were investigated for 1, 3-PD production, in which K. pneumonia [11] or C. butyricum [12] was the main bacterium, respectively. Although microbial production of 1, 3-PD is of multiple advantages, the down-stream processing of fermentation broth has been beset with some difficulties. Due to its strong hydrophilicity and relatively high boiling point (214 ℃ at atmospheric pressure), it is a challenge to effectively separate 1, 3-PD from dilute fermentation broth (approximately 5-9% of 1, 3-PD for glycerol-based fermentation) [13]
. Furthermore, the fermentation broth always consists of pigments, residual
glycerol or glucose, biomacromolecules (nucleic acids, polysaccharides, and proteins), salts (organic salts formed in fermentation and residual inorganic salts in medium) and other by-products in addition to water and 1, 3-PD. The complexity of fermentation broth normally leads to inefficient separation and purification of 1, 3-PD for traditional methods, such as solvent extraction electrodialysis
[21, 22]
chromatography
[14, 15]
, reactive extraction
, alcohol precipitation and dilution crystallization
[24-26]
[16-20]
[23]
,
and
. The separation cost accounts for more than 50% of the total
cost of biologically produced 1, 3-PD, hindering further development of commercial application. Traditional liquid-liquid extraction is a desired method obtaining target product 4
from dilute aqueous solution. However it appears to be not good enough to make simple extraction efficient for distribution of 1, 3-PD into extraction solvent
[14]
.
Boonsongsawat et al. added ethanol into ethyl acetate as cosolvent to improve the hydrophilicity of extraction system, and the distribution coefficient of 1, 3-PD only increased from 0.22 to 0.31 because of strong hydrophilicity
[15]
. Therefore, reactive
extraction was developed, in which 1, 3-PD is converted into hydrophobic substance by chemical reaction such as esterification or acetalization, and then the product would be extracted by the common hydrophobic solvents with improved recovery [16-19]
. 1, 3-PD could be transformed to an ester by esterification reaction with caprylic
acid under lipase catalysis and is obtained by lipase-directed ester hydrolysis
[20]
.
However, the complex components in fermentation broths give rise to various side reactions and toxicity for expensive catalyst, resulting in reduction of the separation efficiency of 1, 3-PD as well as difficulty to reutilize catalyst. Electrodialysis could be used to remove inorganic or organic salts in the fermentation broth so that the other separation units are carried out smoothly
[21]
. Wu et al. developed a novel process,
including bipolar membrane electrodialysis (EDBM), acid crystallization and base recycling, to get high-added value by-products and separate 1, 3-PD
[22]
. But high
energy demanding and serious membrane pollution restrict its industrial application. Compared with electrodialysis, ethanol precipitation for biomacromolecules and dilution crystallization for salts seem to be simple and feasible by adding alcohol into the concentrated fermentation broth
[23]
. However, its imperfection is to demand a 5
great deal of volatile solvent and be difficult for complete removal of all impurities. Chromatography method has the advantages of good selectivity and relatively high purity
[24]
. Silica gel chromatography was employed to purify 1, 3-PD from
fermentation broth and the yield of 1, 3-PD reached 89%
[25]
. Nevertheless, low
adsorption capacity and frequent regeneration of the resin are also its disadvantages on a large scale. In addition, the concentration of 1, 3-PD could decrease after elution with the help of mobile phase, which increases the following workload to remove solvent and water [27]. Salting-out technology was also exploited to separate 1, 3-PD from aqueous solution without solvent
[28]
. Massive inorganic salts, such as K2CO3, K3PO4 and
K4P2O7, were added into the 1, 3-PD solution, resulting in liquid-liquid phase splitting. As a result, over 90% of 1, 3-PD could be recovered, but there were some problems about the recovery of salts when the concentrations of salts ranged from 375 g to 575 g per kg salt solutions. If salting-out effect of salt is combined with solvent extraction it will be more simple and effective to separate 1, 3-PD from fermentation broth. Salting-out extraction (SOE) is a separation method used to extract hydrophilic product from its aqueous solution with the aid of inorganic salt as the salting-out reagent and organic solvent as the extractant. When the solvent is hydrophilic, the SOE can be also called aqueous two-phase extraction (ATPE)
[29]
. It is well known
that SOE has been employed to obtain proteins [30-32], natural products chemicals
[35-40]
[33, 34]
and bulk
with the merits of low cost, rapid phase splitting, simple operation 6
and high efficiency. Certainly, it is easy to understand for ATPE to be used for separation of hydrophilic chemicals (e.g. 1, 3-PD [35-37], 2, 3-butanediol [38], lactic acid [37, 39]
and succinic acid
[40]
) from fermentation broths. However, separation of two
kinds of hydrophilic products occurring in fermentation broths, e.g. 1, 3-PD and lactic acid, is usually a difficult task using one-step ATPE because they would partition into the same phase, i.e. the hydrophilic solvent-rich phase or top phase. Two-step salting-out extraction was put forward to separate 1, 3-PD from lactic acid
[37]
. In the
first step of extraction, 92.4% of 1, 3-PD was recovered by an ATPE system of 30% isopropanol and 30% potassium carbonate. Subsequently, ethanol was added into the salt phase to perform the second step salting-out extraction at pH 6.5, resulting in a recovery of 73.8% for lactic acid. However, 11% of lactic acid would partition into the 1, 3-PD-rich phase in the first step of ATPE. Additionally, adjustment of pH from basic to acidic would lead to some loss of potassium carbonate and thereof recovery of lactic acid. In this study, a novel two-step salting-out extraction will be used to separate 1, 3-PD, BA and HAc from fermentation broth by an anaerobic microbial consortium in which C. butyricum was the main strain
[12]
. The solvent used in the first step
extraction will be not hydrophilic, but hydrophobic n-butyl acetate. An acidic inorganic salt, NaH2PO4, will be chosen without adjusting pH during the two-step extraction. Certainly, different parameters will be investigated in order to achieve the optimal partition coefficient and recovery, and reduce the cost of down-stream 7
processing. 2. Materials and methods 2.1 Materials 1, 3-PD standard was purchased from Shanghai Aladdin Bio-Chem Technology Co., Ltd. HAc and BA standard were purchased from Sinopharm Chemical Reagent Co., Ltd. The cellulose triacetate hollow fiber dialyzer with effective surface area of 1.5 m2 and cut-off molecular weight of 5000 Da was manufactured by NISSHO Corp., Osaka, Japan. All other chemicals were of analytical grade. 2.2 Fermentation The microbial consortium, screened from anaerobic activated sludge and mainly composed of Clostridium butylicum, was used to produce fermentation broths. Fed-batch fermentation with 10% inoculation volume was performed in a 5 L bioreactor with 2 L working volume. Nitrogen was sparged for 1 h before and after inoculation to keep the anaerobic environment in the bioreactor. The whole fermentation process was carried out at a temperature of 37 ℃ and a stirring rate of 250 rpm. The medium pH was maintained at 7.0 by automatic pumping of 5 M NaOH. Two-pulse feeding strategy was adopted in the fed-batch fermentation. The initial glycerol concentration was 80 g/L. When glycerol concentration was less than 15 g/L, it was increased to 80 g/L and 40 g/L, respectively. After the two-pulse feeding, the fermentation was finished when the glycerol was no longer consumed. The concentration of 1, 3-PDO, HAc, BA and residual glycerol in the fermentation broths 8
was 81.74 g/L, 8.65 g/L, 13.82 g/L and 11.22 g/L, respectively. Firstly, the fermentation broth was filtered by cellulose triacetate hollow fiber, which could remove almost all the bacteria and most of the proteins. Then the obtained filtrate was preserved in refrigerator for further study. 2.3 Two-step salting-out extraction of 1, 3-PD, HAc and BA Extraction experiments were performed in 25 mL graduated test tubes. The total mass of extraction system was 20 g. Different inorganic salts were added into the filtrate to acquire salt solutions, among which pH values of solutions containing NaCl, (NH4)2SO4 and NaH2PO4 were adjusted to 4.5 (below the pKa value of BA) and those of solutions containing Na2CO3 and K3PO4 were natural. Then, different organic solvents were added to the salt solution, forming different SOE systems composed of 10 wt% salt and 30 wt% solvent for the first step salting-out extraction. The mixture was drastically shaken for 1 min on a vortex mixer and placed in the condition of room temperature (about 20 ℃ ) for 10 h. Subsequently, effects of concentrations of salt and solvent on the partition of BA were studied for the optimal conditions with screened salt and solvent. After the first step salting-out extraction, the organic phase was withdrawn and sodium carbonate solution was then added into the organic phase for back-extraction of BA. Ethanol (purity 95%) was added into the salt phase from the first step salting-out extraction to perform the second step salting-out extraction in order to recover 1, 3-PD and HAc. 9
The volumes of top and bottom phase were recorded and samples from each phase were taken out for concentration analysis after the system reached phase equilibrium. Partition coefficient (K), phase ratio (P), recovery yield (Y) and removal ratio (R) were defined as follows: Ct Cb
Ki
(1) Pi
Yi
Vt Vb
(2)
PK i i 1 PK i i
(3)
Ri 100% - Yi
(4)
where i represents 1, 3-PD, HAc or BA. Ct and Cb are the concentrations of product in top phase and bottom phase, respectively. Vt and Vb are the volumes of top phase and bottom phase, respectively. Selectivity coefficient is the ratio of partition coefficient of BA to 1, 3-PD. The final recovery (Yf) of product was defined as follows through the entire separation procedure:
Yf ,1,3-PD RI YIII
(5)
Yf , HAc YI YII RI YIII
(6)
Y f , BA YI YII
(7)
where I, II and III represent the first step salting-out extraction, back-extraction and the second step salting-out extraction, respectively. 10
The two-step extraction could be carried out using real fermentation broths without filtration. The experiments were similar as described above, while the filtrate was replaced with the fermentation broth. During the salting-out extractions, the removal ratio of the proteins and cells in the fermentation broth was defined as follows: The first step salting-out extraction:
R1, s
Mm 100% M1
(8)
The second step salting-out extraction:
M R2, s 1 t 100% M2
(9)
where s represents proteins or cells. M m and Mt are mass of proteins or cells in the interphase and top phase, respectively. M1 and M2 are the total mass of proteins or cells in the extraction system. 2.4 Analytical methods 1, 3-PD, HAc and BA concentrations in the samples from organic phase and aqueous phase were determined by gas chromatography and HPLC as described previously [41]. The protein concentration was determined by Coomassie Brilliant Blue method
[42]
. The biomass concentration was measured by absorbance at 650 nm using
spectrophotometer. 2.5 Statistical analysis The experiments were repeated three times and the value stood for the average. The differences in mean values were evaluated using the analysis of variance (ANOVA) method, and standard deviations were calculated to verify the results reliability. 11
3. Results and discussion 3.1 Screening of SOE systems for separation of BA Compared with the metabolites by K. pneumonia, i.e. 1, 3-PD, HAc, ethanol, 2,3-butanediol, lactic acid, and succinic acid, a few metabolites are produced by C. butylicum, e.g. 1, 3-PD, BA and HAc. The fermentation broth used in this study was formed by a microbial consortium mainly composed of C. butylicum, in which the metabolites were target product 1, 3-PD and by-products BA and HAc. Among the three products, the hydrophilicity of 1, 3-PD is the strongest, then HAc, and BA the [35-38]
weakest. According to the previous works
, it is difficult for diols (1, 3-PD, 2,
3-butanediol) and organic acids (lactic acid, HAc) to separate from each other using ATPE systems composed of hydrophilic organic solvents and inorganic salts. Additionally, salting out was not effective for separation of BA at low concentration in fermentation broth by C. tyrobutyricum
[43]
. Whereas the hydrophobicity of BA is
stronger than that of 1, 3-PD and HAc, BA could be considered to be extracted using SOE system consisting of salt and hydrophobic solvent. The partitions of 1, 3-PD, HAc and BA in different SOE systems are shown in Table 1. Table 1 Partitions of 1, 3-PD, HAc and BA in the different SOE systems (system composition:10 wt% salt/30 wt% solvent; the pH values of SOE systems comprising of NaCl, (NH4)2SO4 and NaH2PO4 being 4.5 and the pH values of the others being natural). SOE systems
1, 3-PD K
Y（%） 12
HAc K
Y（%）
BA K
Y（%）
NaCl-n-propanol
0.92
41.40
0.40
23.42
6.48
83.24
NaCl-isobutanol
0.42
22.27
0.34
18.87
6.88
82.35
NaCl-n-butanol
0.48
24.58
0.35
19.18
7.47
83.67
NaCl-ethyl acetate
0.051
2.82
0.29
14.20
5.35
75.13
NaCl-n-butyl acetate
0.021
1.26
0.18
10.26
3.84
70.25
NaCl-methyl isobutyl ketone
0.065
4.72
0.21
14.02
4.61
77.73
(NH4)2SO4-n-propanol
1.35
60.465
0.97
52.33
6.96
88.73
(NH4)2SO4-isobutanol
0.60
29.85
0.55
28.17
9.26
86.82
(NH4)2SO4-n-butanol
0.65
31.59
0.58
28.96
7.40
83.89
(NH4)2SO4-ethyl acetate
0.061
3.23
0.38
16.96
4.34
70.29
(NH4)2SO4-n-butyl acetate
0.023
1.28
0.22
10.84
3.39
65.30
0.069
4.82
0.35
20.56
5.24
79.32
NaH2PO4-n-propanol
1.42
66.58
1.12
61.12
11.36
94.10
NaH2PO4-isobutanol
0.54
29.15
0.56
29.83
9.78
88.09
NaH2PO4-n-butanol
0.56
29.44
0.62
31.54
9.95
88.04
NaH2PO4-ethyl acetate
0.066
3.59
0.44
19.97
6.45
78.44
NaH2PO4-n-butyl acetate
0.022
1.33
0.21
11.41
3.53
68.60
NaH2PO4-methyl isobutyl ketone
0.067
4.90
0.40
23.63
6.36
83.06
Na2CO3-n-propanol
2.51
77.88
0.40
35.73
2.60
78.51
Na2CO3-isobutanol
0.88
41.50
0.072
5.46
0.28
18.38
Na2CO3-n-butanol
0.10
45.20
0.061
4.83
0.40
24.93
Na2CO3-ethyl acetate
0.068
3.65
-
-
0.053
2.86
Na2CO3-n-butyl acetate
0.027
1.57
0.014
0.81
0.062
3.50
Na2CO3-methyl isobutyl ketone
0.074
5.46
0.053
3.98
0.28
18.23
K3PO4-n-propanol
1.67
72.83
0.49
44.11
1.66
72.68
K3PO4-isobutanol
0.52
27.83
0.072
5.01
0.12
8.34
K3PO4-n-butanol
0.636
32.93
0.066
4.86
0.21
13.86
K3PO4-ethyl acetate
0.049
2.43
-
-
0.011
0.53
K3PO4-n-butyl acetate
0.021
1.16
-
-
0.032
1.80
K3PO4-methyl isobutyl ketone
0.68
5.25
0.051
4.02
0.21
14.88
(NH4)2SO4-methyl isobutyl ketone
“-” represented HAc was excessive for ester hydrolysis. The hydrophilicity or hydrophobicity of solvents in the SOE systems listed in Table 1 played an important role in the partition of 1, 3-PD, HAc, and BA. 1, 3-PD was likely to partition into the top phase of hydrophilic solvent, which was similar to the previous reports
[35-37]
. The more the hydrophilicity of solvent, the more the 13
partition coefficient and recovery of HAc when using the same salt. Acidic salts, e.g. (NH4)2SO4 and NaH2PO4, could obtain higher partition coefficient and recovery than alkaline salts, e. g. Na2CO3 and K3PO4, when using the same solvent. A similar partition was observed for BA with recoveries ranging from 65.3% to 94.1%, which was also consistent to the previous reports
[43]
. It is worth noticing that pH values of
solutions containing NaCl, (NH4)2SO4 and NaH2PO4 were adjusted to 4.5, which was below the pKa value of BA, 4.82. In another word, BA was extracted more easily into the organic phase in undissociated form, whereas it was partitioned in the salt phase with the pH being higher than the pKa [44, 45]. An interesting phenomenon is that a better extraction efficacy was also obtained using hydrophobic solvent as extractant under acidic conditions. This phenomenon was found in SOE of acetoin [41]. Apparently, 1, 3-PD and HAc were likely to partition into the salt phase in the hydrophobic solvent-based SOE systems. This is undoubtedly favorable for separation of BA from 1, 3-PD and HAc. For example, in the system composed of (NH4)2SO4 or NaH2PO4/n-butyl acetate the recovery of 1, 3-PD was below 1.33%, whereas that of BA was over 65.30%, which showed wonderful selectivity. As NaH2PO4 is a stronger acidic salt than (NH4)2SO4 with the pH value of 4.2-4.6 at 50 g/L, so NaH2PO4 was chosen as salting-out reagent in the subsequent experiments, avoiding addition of acid for extraction system.
14
Fig. 1 Effects of salting-out extraction systems composed of NaH2PO4 and different solvents on selectivity coefficients of BA, HAc and 1, 3-PD in the filtered fermentation broth (system composition:10 wt% salt/30 wt% solvent). The selectivity coefficients of BA to 1, 3-PD and HAc are shown in Fig. 1. Compared with SOE systems of hydrophilic solvents and NaH2PO4, i. e. n-propanol, isobutanol and n-butanol, the hydrophobic solvent-based SOE systems were more favorable for separation of BA from 1, 3-PD and HAc. Ethyl acetate, n-butyl acetate and methyl isobutyl ketone were of stronger hydrophobicity, but the largest selectivity coefficient of BA to 1, 3-PD was obtained using an SOE system of n-butyl acetate and NaH2PO4. Therefore, this system was further investigated in subsequent experiments. 3.2 The first step SOE of BA using NaH2PO4 and n-butyl acetate Salt and solvent concentrations play rather important roles in partition of product in SOE system. The influence of NaH2PO4 concentration on partition coefficients and recoveries of BA, HAc and 1, 3-PD in SOE system with settled n-butyl acetate 15
concentrations of 25 wt%, 30 wt% and 35 wt% is shown in Fig. 2. The pH value of system decreased and salting-out effect was enhanced with an increase in the salt concentration. Thus, even though the phase ratio of system changed a little, the partition coefficients of BA, HAc and 1, 3-PD were improved apparently so that the recoveries of three products were enhanced as NaH2PO4 was increased. BA had a much higher partition coefficient than HAc and 1, 3-PD because its hydrophobicity was the strongest with four carbon atoms in molecule.
16
Fig. 2 Effect of NaH2PO4 concentration on partition coefficients and recoveries of BA, HAc and 1, 3-PD from the filtered fermentation broth. BA (A); HAc (B); 1, 3-PD (C) (natural pH). 17
The influence of n-butyl acetate concentration on partition coefficients and recoveries of BA, HAc and 1, 3-PD in SOE system with a settled salt concentration of 20 wt% as well as 25 wt% is shown in Fig. 3. Taken as a whole, partition coefficients and recoveries of BA, HAc and 1, 3-PD were improved when solvent concentration was increased with an exception for partition coefficient of 1, 3-PD. The reason is that the solubility of 1, 3-PD is low in the n-butyl acetate due to its high hydrophilicity. When the concentration of n-butyl acetate was too high, the concentration of 1, 3-PD in the top phase would decreased, which resulted in a low partition coefficient. In view of high recovery of BA and low loss of 1, 3-PD, the SOE system composed of 25 wt% NaH2PO4/30 wt% n-butyl acetate was adopted. Under this optimum condition, the partition coefficient and recovery of BA were 42.21 and 96.42%, respectively. At the same time, 33.21% of HAc, 3.71% of 1, 3-PD and 2.41% of glycerol were also extracted.
18
Fig. 3 Effect of n-butyl acetate concentration on partition coefficients and recoveries of BA, HAc and 1, 3-PD from the filtered fermentation broth. BA (A); HAc (B); 1, 3-PD (C) (natural pH). 19
3.3 Back-extraction of BA from n-butyl acetate solution More than 90% of BA was extracted into organic phase after the first step salting-out extraction, and the next question faced with was how to recover BA from n-butyl acetate solution. It was inadvisable to achieve BA by means of distillation for industrial scale, owing to high boiling points of BA and n-butyl acetate, i.e. 163.5 ℃ and 126.5 ℃ at atmospheric pressure, respectively. As discussed above, the dissociated form of BA was mainly distributed in the aqueous solution. BA should be recovered by back-extraction with alkaline solution. Organic phase was obtained from the first step salting-out extraction with concentrations of BA, HAc and 1, 3-PD being 16.24 g/L, 3.96 g/L and 3.64 g/L, respectively. The back-extraction experiment was initiated in the condition where the phase ratio of n-butyl acetate solution to alkaline solution was 2: 1 for concentration effect of BA. Firstly, NaOH solution was introduced as stripping solution because of its strong alkalinity. However, an interesting phenomenon was observed that n-butanol appeared in the bottom phase (Fig. 4), and the recovery of HAc exceeded the theoretical value. This was attributed to that n-butyl acetate was hydrolyzed by NaOH as catalyst. In order to avoid the loss of extractant (n-butyl acetate), Na2CO3 was used for recovery of BA instead of NaOH.
20
Fig. 4 Concentration of n-butanol in the bottom phase during back-extraction of BA with sodium hydroxide solution (the initial phase ratio of n-butyl acetate solution to sodium hydroxide solution being 2:1). Effect of Na2CO3 concentration on the recoveries of BA, HAc and 1, 3-PD is shown in Fig. 5. The recoveries of products were improved when Na2CO3 concentration was increased with differential extent in the order of BA > HAc > 1, 3-PD. The back-extraction yield of BA increased from 9.98% to 91.28% with an increase of Na2CO3 concentration from 0 to 0.3 mol/L and almost kept constant afterwards. The recovery of HAc could reach 93.96% with Na 2CO3 concentration of 0.3 mol/L, similar to back-extraction of BA. However, the recovery of 1, 3-PD (about 90%) changed a little when Na2CO3 concentration varied, as it did not dissociate in the solution. Therefore, an efficient back-extraction could be achieved with 0.3 mol/L of Na2CO3 solution as stripping reagent, in which the molar ratio of Na2CO3 to BA and HAc in the initial n-butyl acetate solution was 3: 5.
21
Fig. 5 Effect of sodium carbonate concentration on the recoveries of BA, HAc and 1, 3-PD (the initial phase ratio of n-butyl acetate solution to sodium carbonate solution being 2:1). Subsequently, effect of initial phase ratio of n-butyl acetate solution to Na2CO3 solution was studied with the above-mentioned molar ratio. As shown in Table 2, the concentrations of three products in the bottom phase were enhanced with initial phase ratio increasing after back-extraction, exhibiting good condensed effect. However, their recoveries decreased, because larger phase ratio impaired contact and mass transfer between two phases. The initial phase ratio of 2: 1 was suitable for higher recovery of BA. A mixture of sodium butyrate and sodium acetate would be obtained after condensing and drying the bottom phase in back-extraction. It could be used as feed additive on account of important physiological effect of BA and HAc in animal gut.
22
Table 2 Effect of initial back-extraction phase ratio on the distribution behaviors of BA, HAc and 1, 3-PD (the molar ratio of Na2CO3 to organic acids in the initial n-butyl acetate solution being 3: 5). Phase
BA
HAc
1, 3-PD
ratio
CBA（g/L）
Y（%）
CHAc（g/L）
Y（%）
C1, 3-PD（g/L）
Y（%）
2:1
27.59±0.10
91.28±0.33
5.67±0.027
93.96±0.45
7.32±0.10
91.20±1.14
2.4:1
33.16±0.58
88.26±2.08
6.82±0.074
92.07±1.00
7.41±0.11
80.19±2.45
3:1
39.71±0.24
87.23±3.83
8.39±0.062
91.06±2.16
9.03±0.00069
79.78±3.14
4:1
51.33±0.78
86.70±3.26
10.86±0.14
89.50±2.21
11.50±0.0089
79.34±2.34
3.4 The second step SOE of 1, 3-PD and HAc The second step salting-out extraction experiment was conducted using synthetic fermentation broth, in which the components containing NaH2PO4 were similar to the bottom phase in the first step SOE. After adding and mixing 95% ethanol, the effect of ethanol volume on partition coefficients and recoveries of HAc and 1, 3-PD was exhibited in Fig. 6. The recoveries of HAc and 1, 3-PD were enhanced with volume fraction of ethanol due to increasing partition coefficient and phase ratio. Nevertheless, if ethanol content continued to increase, the salt would precipitate out for dilution crystallization of solvent. At 40% (v/v) of ethanol the recoveries of HAc and 1, 3-PD reached 89.59% and 92.32%, respectively.
23
Fig. 6 Effect of ethanol volume on partition coefficients and recoveries of HAc and 1, 3-PD from the simulated solution (natural pH). Subsequently, the experiment was carried out using the filtered fermentation broth. According to above result, different ethanol volume fractions, ranging from 40%-52% (v/v), were selected for investigating effect of ethanol volume on partitions of HAc and 1, 3-PD. And the second step SOE system was formed with addition of ethanol (95%) to the bottom phase in the first step SOE. As shown in Fig. 7, similar trend and higher partition coefficients were observed using filtered fermentation broth compared with the simulated solution as shown in Fig.6. The partition coefficient of HAc was kept between 6.71-7.74 and that of 1, 3-PD about 9.3. In the SOE system consisting of 50% (v/v) ethanol the recoveries of HAc, 1, 3-PD, and glycerol were 94.40%, 95.50%, and 76.19%, respectively.
24
Fig. 7 Effect of ethanol volume on partition coefficients and recoveries of HAc and 1, 3-PD from the filtered fermentation broth (natural pH). 3.5 Two-step salting-out extraction of 1, 3-PD, HAc and BA from fermentation broth Based on all above results, two-step salting-out extraction experiment was applied to unfiltered fermentation broth under optimized conditions. As indicated in Fig.8, in the first step salting-out extraction partition coefficient and recovery of BA reached 23.34 and 93.78%, respectively, and the removal ratios of 1, 3-PD, HAc and glycerol were 95.20%, 69.04% and 93.82%, respectively. At the same time, 99.63% of the cells and 95.78% of the proteins were accumulated in the interphase between the top and bottom phase. Then, 88.72% of BA was recovered by back-extraction using Na2CO3 solution. Finally, 96.29% of 1, 3-PD, 97.26% of HAc, and 68.48% of glycerol were partitioned into the organic phase during the second step salting-out extraction. In addition, all the cells and 77.89% of proteins were removed. After two-step SOE and back extraction, the total recoveries of 1, 3-PD, HAc, BA and glycerol reached 91.67%, 92.75%, 83.20% and 64.25%, respectively, with almost all 25
the cells and 97.16% of the proteins being removed. It is feasible to effectively separate 1, 3-PD and organic acids by adopting two-step salting-out extraction with simple operations, avoiding energy-intensive process such as electrodialysis
[22]
. Furthermore, cells and proteins could be removed
simultaneously from fermentation broths, which would be used for feed because of nontoxicity of C. butylicum. However, there are still some questions that need to be solved such as the recycle of salt, which is an important factor imposing restrictions on the large-scale application of salting-out extraction technology. In the first step salting-out extraction 25 wt% NaH2PO4 was added and most of NaH2PO4 was enriched in the bottom phase at last. Although salt recycle was studied in some researches [35, 36], the reuse of NaH2PO4 needs to be further investigated.
Fig. 8 Two-step salting-out extraction of 1, 3-PD, HAc and BA from fermentation broth (the total mass of the first step SOE system being 500 g; back-extraction condition: initial phase ratio of n-butyl acetate solution to Na2CO3 solution being 2: 1 and molar ratio of sodium carbonate to organic acids being 3: 5). 4. Conclusion It is simple and feasible to separate 1, 3-PD and HAc from BA efficiently in 26
fermentation broths with removal of cells and proteins through two-step salting-out extraction. In the first step salting-out extraction, partition coefficient and recovery of BA could reach 42.21 and 96.42%, and furthermore, 33.21% of HAc and 3.71% of 1, 3-PD were extracted at 25 wt% NaH2PO4/30 wt% n-butyl acetate. In the back-extraction, the recoveries of BA, HAc and 1, 3-PD were 91.28%, 93.96% and 91.20%, respectively, with initial phase ratio of n-butyl acetate solution to Na2CO3 solution being 2: 1 when molar ratio of sodium carbonate to organic acids was 3: 5. In the second step salting-out extraction, the recoveries of HAc and 1, 3-PD were 94.40% and 95.50%, respectively, at 50% (v/v) ethanol. In a scale-up experiment on 500 g, the recoveries of 1, 3-PD, HAc and BA reached 91.67%, 92.75% and 83.20%, respectively, with the removal of almost all the cells and 97.16% of the proteins after two-step salting-out extraction. The recycle of salt needs to be researched further. Acknowledgements This work was supported by the National Natural Science Foundation of China (Grant No. 21476042).
27
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31
Highlight:
A novel two-step salting-out extraction was used to separate 1, 3-propanediol, butyric acid and acetic acid from fermentation broth by an anaerobic microbial consortium in which Clostridium butyricum was the main strain.
Butyric acid could be separated from 1, 3-propanediol and acetic acid by the salting-out extraction system comprised of acidic inorganic salt and hydrophobic solvent.
Effect of sodium carbonate concentration on the back-extraction of butyric acid was investigated.
32
S1383-5866(18)30841-4 https://doi.org/10.1016/j.seppur.2018.07.021 SEPPUR 14752
To appear in:
Separation and Purification Technology
Received Date: Revised Date: Accepted Date:
13 March 2018 20 June 2018 9 July 2018
Please cite this article as: Z. Li, L. Yan, J. Zhou, X. Wang, Y. Sun, Z-L. Xiu, Two-step salting-out extraction of 1, 3-propanediol, butyric acid and acetic acid from fermentation broths, Separation and Purification Technology (2018), doi: https://doi.org/10.1016/j.seppur.2018.07.021
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Two-step salting-out extraction of 1, 3-propanediol, butyric acid and acetic acid from fermentation broths Zhen Li, Ling Yan, Jinjie Zhou, Xiaoli Wang, Yaqin Sun, Zhi-Long Xiu* School of Life Science and Biotechnology, Dalian University of Technology, Dalian, Liaoning 116024, P.R. China
*Correspondence: Prof. Zhilong Xiu ( Email: [email protected], Tel/Fax: +86-0411-84706369 ), School of Life Science and Biotechnology, Dalian University of Technology, No.2 Linggong Road, Dalian 116024, P. R. China
1
Abstract: The separation of 1, 3-propanediol (1, 3-PD), butyric acid (BA) and acetic acid (HAc) from the fermentation broth was studied by two-step salting-out extraction. In the first salting-out extraction, the partition coefficient and recovery of BA reached 42.21 and 96.42%, respectively, under the optimal condition of 25 wt% NaH2PO4/30 wt% n-butyl acetate. Subsequently, 91.28% of BA in the organic phase could be recovered through back-extraction when sodium carbonate solution was mixed with organic phase with initial phase ratio of n-butyl acetate solution to alkaline solution being 2: 1, in which the molar ratio of sodium carbonate to organic acids was 3: 5. Finally, 50% (v/v) ethanol (95%) was added to the bottom phase for the second step salting-out extraction. And the partition coefficient and recovery of 1, 3-PD were 9.40 and 95.50%, and those of HAc were 7.46 and 94.40%, respectively. All the cells and most of the proteins (97.16%) could be removed. Effective separation of 1, 3-PD from BA was realized by two-step salting-out extraction, which provides a potential method for separation of 1, 3-PD, BA and HAc on an industrial scale. Keywords: 1, 3-Propanediol; Butyric acid; Acetic acid; Salting-out extraction; Back-extraction
2
1.
Introduction 1, 3-Propanediol (1, 3-PD) is an important chemical material, which could be
used in multiple fields in virtue of versatility, such as solvent, adhesive, cosmetic, preservative and medicine. It could participate in a variety of condensation polymerization as monomer for production of excellent performance polymers such as polyethers, polyesters and polyurethanes due to its two symmetrical hydroxyl groups, in which polytrimethylene terephthalate (PTT) has shown tremendous application prospect in the fiber, textile and engineering thermoplastics industry because of its advantages, such as good tensile elastic recovery, low static generation, stain resistance and biodegradability [1-3]. Up to now, 1, 3-PD has been mainly manufactured by chemical synthesis with rigorous reactions, valuable catalysts and multitudinous by-products, in which down-streaming products from petrochemical industry are used as raw materials. As people have increasingly worried about fossil energy exhaustion and environmental pollution, biorefinery technology has been paid a great deal of attention, by which renewable biomass is used to produce bioenergy and chemicals. For example, microbial conversion of glycerol, a by-product from biodiesel industry, into 1, 3-PD is a superb application of biorefinery, which not only guarantees continuous supply of raw materials but also avoids resource waste
[4]
. Microorganisms used in 1, 3-PD
fermentation mainly include Klebsiella pneumoniae Clostridium butyricum
[7]
[5]
, Clostridium pasteurianum
, Clostridium beijerinckii 3
[8]
[6]
,
, Escherichia coli[9] and
Citrobacter freundii
[10]
, in which K. pneumonia and C. butyricum have been widely
used due to their high productivity. Compared with K. pneumonia, a few metabolites are produced from C. butyricum, i.e. 1, 3-PD, acetic acid (HAc) and butyric acid (BA). Recently, microbial consortia were investigated for 1, 3-PD production, in which K. pneumonia [11] or C. butyricum [12] was the main bacterium, respectively. Although microbial production of 1, 3-PD is of multiple advantages, the down-stream processing of fermentation broth has been beset with some difficulties. Due to its strong hydrophilicity and relatively high boiling point (214 ℃ at atmospheric pressure), it is a challenge to effectively separate 1, 3-PD from dilute fermentation broth (approximately 5-9% of 1, 3-PD for glycerol-based fermentation) [13]
. Furthermore, the fermentation broth always consists of pigments, residual
glycerol or glucose, biomacromolecules (nucleic acids, polysaccharides, and proteins), salts (organic salts formed in fermentation and residual inorganic salts in medium) and other by-products in addition to water and 1, 3-PD. The complexity of fermentation broth normally leads to inefficient separation and purification of 1, 3-PD for traditional methods, such as solvent extraction electrodialysis
[21, 22]
chromatography
[14, 15]
, reactive extraction
, alcohol precipitation and dilution crystallization
[24-26]
[16-20]
[23]
,
and
. The separation cost accounts for more than 50% of the total
cost of biologically produced 1, 3-PD, hindering further development of commercial application. Traditional liquid-liquid extraction is a desired method obtaining target product 4
from dilute aqueous solution. However it appears to be not good enough to make simple extraction efficient for distribution of 1, 3-PD into extraction solvent
[14]
.
Boonsongsawat et al. added ethanol into ethyl acetate as cosolvent to improve the hydrophilicity of extraction system, and the distribution coefficient of 1, 3-PD only increased from 0.22 to 0.31 because of strong hydrophilicity
[15]
. Therefore, reactive
extraction was developed, in which 1, 3-PD is converted into hydrophobic substance by chemical reaction such as esterification or acetalization, and then the product would be extracted by the common hydrophobic solvents with improved recovery [16-19]
. 1, 3-PD could be transformed to an ester by esterification reaction with caprylic
acid under lipase catalysis and is obtained by lipase-directed ester hydrolysis
[20]
.
However, the complex components in fermentation broths give rise to various side reactions and toxicity for expensive catalyst, resulting in reduction of the separation efficiency of 1, 3-PD as well as difficulty to reutilize catalyst. Electrodialysis could be used to remove inorganic or organic salts in the fermentation broth so that the other separation units are carried out smoothly
[21]
. Wu et al. developed a novel process,
including bipolar membrane electrodialysis (EDBM), acid crystallization and base recycling, to get high-added value by-products and separate 1, 3-PD
[22]
. But high
energy demanding and serious membrane pollution restrict its industrial application. Compared with electrodialysis, ethanol precipitation for biomacromolecules and dilution crystallization for salts seem to be simple and feasible by adding alcohol into the concentrated fermentation broth
[23]
. However, its imperfection is to demand a 5
great deal of volatile solvent and be difficult for complete removal of all impurities. Chromatography method has the advantages of good selectivity and relatively high purity
[24]
. Silica gel chromatography was employed to purify 1, 3-PD from
fermentation broth and the yield of 1, 3-PD reached 89%
[25]
. Nevertheless, low
adsorption capacity and frequent regeneration of the resin are also its disadvantages on a large scale. In addition, the concentration of 1, 3-PD could decrease after elution with the help of mobile phase, which increases the following workload to remove solvent and water [27]. Salting-out technology was also exploited to separate 1, 3-PD from aqueous solution without solvent
[28]
. Massive inorganic salts, such as K2CO3, K3PO4 and
K4P2O7, were added into the 1, 3-PD solution, resulting in liquid-liquid phase splitting. As a result, over 90% of 1, 3-PD could be recovered, but there were some problems about the recovery of salts when the concentrations of salts ranged from 375 g to 575 g per kg salt solutions. If salting-out effect of salt is combined with solvent extraction it will be more simple and effective to separate 1, 3-PD from fermentation broth. Salting-out extraction (SOE) is a separation method used to extract hydrophilic product from its aqueous solution with the aid of inorganic salt as the salting-out reagent and organic solvent as the extractant. When the solvent is hydrophilic, the SOE can be also called aqueous two-phase extraction (ATPE)
[29]
. It is well known
that SOE has been employed to obtain proteins [30-32], natural products chemicals
[35-40]
[33, 34]
and bulk
with the merits of low cost, rapid phase splitting, simple operation 6
and high efficiency. Certainly, it is easy to understand for ATPE to be used for separation of hydrophilic chemicals (e.g. 1, 3-PD [35-37], 2, 3-butanediol [38], lactic acid [37, 39]
and succinic acid
[40]
) from fermentation broths. However, separation of two
kinds of hydrophilic products occurring in fermentation broths, e.g. 1, 3-PD and lactic acid, is usually a difficult task using one-step ATPE because they would partition into the same phase, i.e. the hydrophilic solvent-rich phase or top phase. Two-step salting-out extraction was put forward to separate 1, 3-PD from lactic acid
[37]
. In the
first step of extraction, 92.4% of 1, 3-PD was recovered by an ATPE system of 30% isopropanol and 30% potassium carbonate. Subsequently, ethanol was added into the salt phase to perform the second step salting-out extraction at pH 6.5, resulting in a recovery of 73.8% for lactic acid. However, 11% of lactic acid would partition into the 1, 3-PD-rich phase in the first step of ATPE. Additionally, adjustment of pH from basic to acidic would lead to some loss of potassium carbonate and thereof recovery of lactic acid. In this study, a novel two-step salting-out extraction will be used to separate 1, 3-PD, BA and HAc from fermentation broth by an anaerobic microbial consortium in which C. butyricum was the main strain
[12]
. The solvent used in the first step
extraction will be not hydrophilic, but hydrophobic n-butyl acetate. An acidic inorganic salt, NaH2PO4, will be chosen without adjusting pH during the two-step extraction. Certainly, different parameters will be investigated in order to achieve the optimal partition coefficient and recovery, and reduce the cost of down-stream 7
processing. 2. Materials and methods 2.1 Materials 1, 3-PD standard was purchased from Shanghai Aladdin Bio-Chem Technology Co., Ltd. HAc and BA standard were purchased from Sinopharm Chemical Reagent Co., Ltd. The cellulose triacetate hollow fiber dialyzer with effective surface area of 1.5 m2 and cut-off molecular weight of 5000 Da was manufactured by NISSHO Corp., Osaka, Japan. All other chemicals were of analytical grade. 2.2 Fermentation The microbial consortium, screened from anaerobic activated sludge and mainly composed of Clostridium butylicum, was used to produce fermentation broths. Fed-batch fermentation with 10% inoculation volume was performed in a 5 L bioreactor with 2 L working volume. Nitrogen was sparged for 1 h before and after inoculation to keep the anaerobic environment in the bioreactor. The whole fermentation process was carried out at a temperature of 37 ℃ and a stirring rate of 250 rpm. The medium pH was maintained at 7.0 by automatic pumping of 5 M NaOH. Two-pulse feeding strategy was adopted in the fed-batch fermentation. The initial glycerol concentration was 80 g/L. When glycerol concentration was less than 15 g/L, it was increased to 80 g/L and 40 g/L, respectively. After the two-pulse feeding, the fermentation was finished when the glycerol was no longer consumed. The concentration of 1, 3-PDO, HAc, BA and residual glycerol in the fermentation broths 8
was 81.74 g/L, 8.65 g/L, 13.82 g/L and 11.22 g/L, respectively. Firstly, the fermentation broth was filtered by cellulose triacetate hollow fiber, which could remove almost all the bacteria and most of the proteins. Then the obtained filtrate was preserved in refrigerator for further study. 2.3 Two-step salting-out extraction of 1, 3-PD, HAc and BA Extraction experiments were performed in 25 mL graduated test tubes. The total mass of extraction system was 20 g. Different inorganic salts were added into the filtrate to acquire salt solutions, among which pH values of solutions containing NaCl, (NH4)2SO4 and NaH2PO4 were adjusted to 4.5 (below the pKa value of BA) and those of solutions containing Na2CO3 and K3PO4 were natural. Then, different organic solvents were added to the salt solution, forming different SOE systems composed of 10 wt% salt and 30 wt% solvent for the first step salting-out extraction. The mixture was drastically shaken for 1 min on a vortex mixer and placed in the condition of room temperature (about 20 ℃ ) for 10 h. Subsequently, effects of concentrations of salt and solvent on the partition of BA were studied for the optimal conditions with screened salt and solvent. After the first step salting-out extraction, the organic phase was withdrawn and sodium carbonate solution was then added into the organic phase for back-extraction of BA. Ethanol (purity 95%) was added into the salt phase from the first step salting-out extraction to perform the second step salting-out extraction in order to recover 1, 3-PD and HAc. 9
The volumes of top and bottom phase were recorded and samples from each phase were taken out for concentration analysis after the system reached phase equilibrium. Partition coefficient (K), phase ratio (P), recovery yield (Y) and removal ratio (R) were defined as follows: Ct Cb
Ki
(1) Pi
Yi
Vt Vb
(2)
PK i i 1 PK i i
(3)
Ri 100% - Yi
(4)
where i represents 1, 3-PD, HAc or BA. Ct and Cb are the concentrations of product in top phase and bottom phase, respectively. Vt and Vb are the volumes of top phase and bottom phase, respectively. Selectivity coefficient is the ratio of partition coefficient of BA to 1, 3-PD. The final recovery (Yf) of product was defined as follows through the entire separation procedure:
Yf ,1,3-PD RI YIII
(5)
Yf , HAc YI YII RI YIII
(6)
Y f , BA YI YII
(7)
where I, II and III represent the first step salting-out extraction, back-extraction and the second step salting-out extraction, respectively. 10
The two-step extraction could be carried out using real fermentation broths without filtration. The experiments were similar as described above, while the filtrate was replaced with the fermentation broth. During the salting-out extractions, the removal ratio of the proteins and cells in the fermentation broth was defined as follows: The first step salting-out extraction:
R1, s
Mm 100% M1
(8)
The second step salting-out extraction:
M R2, s 1 t 100% M2
(9)
where s represents proteins or cells. M m and Mt are mass of proteins or cells in the interphase and top phase, respectively. M1 and M2 are the total mass of proteins or cells in the extraction system. 2.4 Analytical methods 1, 3-PD, HAc and BA concentrations in the samples from organic phase and aqueous phase were determined by gas chromatography and HPLC as described previously [41]. The protein concentration was determined by Coomassie Brilliant Blue method
[42]
. The biomass concentration was measured by absorbance at 650 nm using
spectrophotometer. 2.5 Statistical analysis The experiments were repeated three times and the value stood for the average. The differences in mean values were evaluated using the analysis of variance (ANOVA) method, and standard deviations were calculated to verify the results reliability. 11
3. Results and discussion 3.1 Screening of SOE systems for separation of BA Compared with the metabolites by K. pneumonia, i.e. 1, 3-PD, HAc, ethanol, 2,3-butanediol, lactic acid, and succinic acid, a few metabolites are produced by C. butylicum, e.g. 1, 3-PD, BA and HAc. The fermentation broth used in this study was formed by a microbial consortium mainly composed of C. butylicum, in which the metabolites were target product 1, 3-PD and by-products BA and HAc. Among the three products, the hydrophilicity of 1, 3-PD is the strongest, then HAc, and BA the [35-38]
weakest. According to the previous works
, it is difficult for diols (1, 3-PD, 2,
3-butanediol) and organic acids (lactic acid, HAc) to separate from each other using ATPE systems composed of hydrophilic organic solvents and inorganic salts. Additionally, salting out was not effective for separation of BA at low concentration in fermentation broth by C. tyrobutyricum
[43]
. Whereas the hydrophobicity of BA is
stronger than that of 1, 3-PD and HAc, BA could be considered to be extracted using SOE system consisting of salt and hydrophobic solvent. The partitions of 1, 3-PD, HAc and BA in different SOE systems are shown in Table 1. Table 1 Partitions of 1, 3-PD, HAc and BA in the different SOE systems (system composition:10 wt% salt/30 wt% solvent; the pH values of SOE systems comprising of NaCl, (NH4)2SO4 and NaH2PO4 being 4.5 and the pH values of the others being natural). SOE systems
1, 3-PD K
Y（%） 12
HAc K
Y（%）
BA K
Y（%）
NaCl-n-propanol
0.92
41.40
0.40
23.42
6.48
83.24
NaCl-isobutanol
0.42
22.27
0.34
18.87
6.88
82.35
NaCl-n-butanol
0.48
24.58
0.35
19.18
7.47
83.67
NaCl-ethyl acetate
0.051
2.82
0.29
14.20
5.35
75.13
NaCl-n-butyl acetate
0.021
1.26
0.18
10.26
3.84
70.25
NaCl-methyl isobutyl ketone
0.065
4.72
0.21
14.02
4.61
77.73
(NH4)2SO4-n-propanol
1.35
60.465
0.97
52.33
6.96
88.73
(NH4)2SO4-isobutanol
0.60
29.85
0.55
28.17
9.26
86.82
(NH4)2SO4-n-butanol
0.65
31.59
0.58
28.96
7.40
83.89
(NH4)2SO4-ethyl acetate
0.061
3.23
0.38
16.96
4.34
70.29
(NH4)2SO4-n-butyl acetate
0.023
1.28
0.22
10.84
3.39
65.30
0.069
4.82
0.35
20.56
5.24
79.32
NaH2PO4-n-propanol
1.42
66.58
1.12
61.12
11.36
94.10
NaH2PO4-isobutanol
0.54
29.15
0.56
29.83
9.78
88.09
NaH2PO4-n-butanol
0.56
29.44
0.62
31.54
9.95
88.04
NaH2PO4-ethyl acetate
0.066
3.59
0.44
19.97
6.45
78.44
NaH2PO4-n-butyl acetate
0.022
1.33
0.21
11.41
3.53
68.60
NaH2PO4-methyl isobutyl ketone
0.067
4.90
0.40
23.63
6.36
83.06
Na2CO3-n-propanol
2.51
77.88
0.40
35.73
2.60
78.51
Na2CO3-isobutanol
0.88
41.50
0.072
5.46
0.28
18.38
Na2CO3-n-butanol
0.10
45.20
0.061
4.83
0.40
24.93
Na2CO3-ethyl acetate
0.068
3.65
-
-
0.053
2.86
Na2CO3-n-butyl acetate
0.027
1.57
0.014
0.81
0.062
3.50
Na2CO3-methyl isobutyl ketone
0.074
5.46
0.053
3.98
0.28
18.23
K3PO4-n-propanol
1.67
72.83
0.49
44.11
1.66
72.68
K3PO4-isobutanol
0.52
27.83
0.072
5.01
0.12
8.34
K3PO4-n-butanol
0.636
32.93
0.066
4.86
0.21
13.86
K3PO4-ethyl acetate
0.049
2.43
-
-
0.011
0.53
K3PO4-n-butyl acetate
0.021
1.16
-
-
0.032
1.80
K3PO4-methyl isobutyl ketone
0.68
5.25
0.051
4.02
0.21
14.88
(NH4)2SO4-methyl isobutyl ketone
“-” represented HAc was excessive for ester hydrolysis. The hydrophilicity or hydrophobicity of solvents in the SOE systems listed in Table 1 played an important role in the partition of 1, 3-PD, HAc, and BA. 1, 3-PD was likely to partition into the top phase of hydrophilic solvent, which was similar to the previous reports
[35-37]
. The more the hydrophilicity of solvent, the more the 13
partition coefficient and recovery of HAc when using the same salt. Acidic salts, e.g. (NH4)2SO4 and NaH2PO4, could obtain higher partition coefficient and recovery than alkaline salts, e. g. Na2CO3 and K3PO4, when using the same solvent. A similar partition was observed for BA with recoveries ranging from 65.3% to 94.1%, which was also consistent to the previous reports
[43]
. It is worth noticing that pH values of
solutions containing NaCl, (NH4)2SO4 and NaH2PO4 were adjusted to 4.5, which was below the pKa value of BA, 4.82. In another word, BA was extracted more easily into the organic phase in undissociated form, whereas it was partitioned in the salt phase with the pH being higher than the pKa [44, 45]. An interesting phenomenon is that a better extraction efficacy was also obtained using hydrophobic solvent as extractant under acidic conditions. This phenomenon was found in SOE of acetoin [41]. Apparently, 1, 3-PD and HAc were likely to partition into the salt phase in the hydrophobic solvent-based SOE systems. This is undoubtedly favorable for separation of BA from 1, 3-PD and HAc. For example, in the system composed of (NH4)2SO4 or NaH2PO4/n-butyl acetate the recovery of 1, 3-PD was below 1.33%, whereas that of BA was over 65.30%, which showed wonderful selectivity. As NaH2PO4 is a stronger acidic salt than (NH4)2SO4 with the pH value of 4.2-4.6 at 50 g/L, so NaH2PO4 was chosen as salting-out reagent in the subsequent experiments, avoiding addition of acid for extraction system.
14
Fig. 1 Effects of salting-out extraction systems composed of NaH2PO4 and different solvents on selectivity coefficients of BA, HAc and 1, 3-PD in the filtered fermentation broth (system composition:10 wt% salt/30 wt% solvent). The selectivity coefficients of BA to 1, 3-PD and HAc are shown in Fig. 1. Compared with SOE systems of hydrophilic solvents and NaH2PO4, i. e. n-propanol, isobutanol and n-butanol, the hydrophobic solvent-based SOE systems were more favorable for separation of BA from 1, 3-PD and HAc. Ethyl acetate, n-butyl acetate and methyl isobutyl ketone were of stronger hydrophobicity, but the largest selectivity coefficient of BA to 1, 3-PD was obtained using an SOE system of n-butyl acetate and NaH2PO4. Therefore, this system was further investigated in subsequent experiments. 3.2 The first step SOE of BA using NaH2PO4 and n-butyl acetate Salt and solvent concentrations play rather important roles in partition of product in SOE system. The influence of NaH2PO4 concentration on partition coefficients and recoveries of BA, HAc and 1, 3-PD in SOE system with settled n-butyl acetate 15
concentrations of 25 wt%, 30 wt% and 35 wt% is shown in Fig. 2. The pH value of system decreased and salting-out effect was enhanced with an increase in the salt concentration. Thus, even though the phase ratio of system changed a little, the partition coefficients of BA, HAc and 1, 3-PD were improved apparently so that the recoveries of three products were enhanced as NaH2PO4 was increased. BA had a much higher partition coefficient than HAc and 1, 3-PD because its hydrophobicity was the strongest with four carbon atoms in molecule.
16
Fig. 2 Effect of NaH2PO4 concentration on partition coefficients and recoveries of BA, HAc and 1, 3-PD from the filtered fermentation broth. BA (A); HAc (B); 1, 3-PD (C) (natural pH). 17
The influence of n-butyl acetate concentration on partition coefficients and recoveries of BA, HAc and 1, 3-PD in SOE system with a settled salt concentration of 20 wt% as well as 25 wt% is shown in Fig. 3. Taken as a whole, partition coefficients and recoveries of BA, HAc and 1, 3-PD were improved when solvent concentration was increased with an exception for partition coefficient of 1, 3-PD. The reason is that the solubility of 1, 3-PD is low in the n-butyl acetate due to its high hydrophilicity. When the concentration of n-butyl acetate was too high, the concentration of 1, 3-PD in the top phase would decreased, which resulted in a low partition coefficient. In view of high recovery of BA and low loss of 1, 3-PD, the SOE system composed of 25 wt% NaH2PO4/30 wt% n-butyl acetate was adopted. Under this optimum condition, the partition coefficient and recovery of BA were 42.21 and 96.42%, respectively. At the same time, 33.21% of HAc, 3.71% of 1, 3-PD and 2.41% of glycerol were also extracted.
18
Fig. 3 Effect of n-butyl acetate concentration on partition coefficients and recoveries of BA, HAc and 1, 3-PD from the filtered fermentation broth. BA (A); HAc (B); 1, 3-PD (C) (natural pH). 19
3.3 Back-extraction of BA from n-butyl acetate solution More than 90% of BA was extracted into organic phase after the first step salting-out extraction, and the next question faced with was how to recover BA from n-butyl acetate solution. It was inadvisable to achieve BA by means of distillation for industrial scale, owing to high boiling points of BA and n-butyl acetate, i.e. 163.5 ℃ and 126.5 ℃ at atmospheric pressure, respectively. As discussed above, the dissociated form of BA was mainly distributed in the aqueous solution. BA should be recovered by back-extraction with alkaline solution. Organic phase was obtained from the first step salting-out extraction with concentrations of BA, HAc and 1, 3-PD being 16.24 g/L, 3.96 g/L and 3.64 g/L, respectively. The back-extraction experiment was initiated in the condition where the phase ratio of n-butyl acetate solution to alkaline solution was 2: 1 for concentration effect of BA. Firstly, NaOH solution was introduced as stripping solution because of its strong alkalinity. However, an interesting phenomenon was observed that n-butanol appeared in the bottom phase (Fig. 4), and the recovery of HAc exceeded the theoretical value. This was attributed to that n-butyl acetate was hydrolyzed by NaOH as catalyst. In order to avoid the loss of extractant (n-butyl acetate), Na2CO3 was used for recovery of BA instead of NaOH.
20
Fig. 4 Concentration of n-butanol in the bottom phase during back-extraction of BA with sodium hydroxide solution (the initial phase ratio of n-butyl acetate solution to sodium hydroxide solution being 2:1). Effect of Na2CO3 concentration on the recoveries of BA, HAc and 1, 3-PD is shown in Fig. 5. The recoveries of products were improved when Na2CO3 concentration was increased with differential extent in the order of BA > HAc > 1, 3-PD. The back-extraction yield of BA increased from 9.98% to 91.28% with an increase of Na2CO3 concentration from 0 to 0.3 mol/L and almost kept constant afterwards. The recovery of HAc could reach 93.96% with Na 2CO3 concentration of 0.3 mol/L, similar to back-extraction of BA. However, the recovery of 1, 3-PD (about 90%) changed a little when Na2CO3 concentration varied, as it did not dissociate in the solution. Therefore, an efficient back-extraction could be achieved with 0.3 mol/L of Na2CO3 solution as stripping reagent, in which the molar ratio of Na2CO3 to BA and HAc in the initial n-butyl acetate solution was 3: 5.
21
Fig. 5 Effect of sodium carbonate concentration on the recoveries of BA, HAc and 1, 3-PD (the initial phase ratio of n-butyl acetate solution to sodium carbonate solution being 2:1). Subsequently, effect of initial phase ratio of n-butyl acetate solution to Na2CO3 solution was studied with the above-mentioned molar ratio. As shown in Table 2, the concentrations of three products in the bottom phase were enhanced with initial phase ratio increasing after back-extraction, exhibiting good condensed effect. However, their recoveries decreased, because larger phase ratio impaired contact and mass transfer between two phases. The initial phase ratio of 2: 1 was suitable for higher recovery of BA. A mixture of sodium butyrate and sodium acetate would be obtained after condensing and drying the bottom phase in back-extraction. It could be used as feed additive on account of important physiological effect of BA and HAc in animal gut.
22
Table 2 Effect of initial back-extraction phase ratio on the distribution behaviors of BA, HAc and 1, 3-PD (the molar ratio of Na2CO3 to organic acids in the initial n-butyl acetate solution being 3: 5). Phase
BA
HAc
1, 3-PD
ratio
CBA（g/L）
Y（%）
CHAc（g/L）
Y（%）
C1, 3-PD（g/L）
Y（%）
2:1
27.59±0.10
91.28±0.33
5.67±0.027
93.96±0.45
7.32±0.10
91.20±1.14
2.4:1
33.16±0.58
88.26±2.08
6.82±0.074
92.07±1.00
7.41±0.11
80.19±2.45
3:1
39.71±0.24
87.23±3.83
8.39±0.062
91.06±2.16
9.03±0.00069
79.78±3.14
4:1
51.33±0.78
86.70±3.26
10.86±0.14
89.50±2.21
11.50±0.0089
79.34±2.34
3.4 The second step SOE of 1, 3-PD and HAc The second step salting-out extraction experiment was conducted using synthetic fermentation broth, in which the components containing NaH2PO4 were similar to the bottom phase in the first step SOE. After adding and mixing 95% ethanol, the effect of ethanol volume on partition coefficients and recoveries of HAc and 1, 3-PD was exhibited in Fig. 6. The recoveries of HAc and 1, 3-PD were enhanced with volume fraction of ethanol due to increasing partition coefficient and phase ratio. Nevertheless, if ethanol content continued to increase, the salt would precipitate out for dilution crystallization of solvent. At 40% (v/v) of ethanol the recoveries of HAc and 1, 3-PD reached 89.59% and 92.32%, respectively.
23
Fig. 6 Effect of ethanol volume on partition coefficients and recoveries of HAc and 1, 3-PD from the simulated solution (natural pH). Subsequently, the experiment was carried out using the filtered fermentation broth. According to above result, different ethanol volume fractions, ranging from 40%-52% (v/v), were selected for investigating effect of ethanol volume on partitions of HAc and 1, 3-PD. And the second step SOE system was formed with addition of ethanol (95%) to the bottom phase in the first step SOE. As shown in Fig. 7, similar trend and higher partition coefficients were observed using filtered fermentation broth compared with the simulated solution as shown in Fig.6. The partition coefficient of HAc was kept between 6.71-7.74 and that of 1, 3-PD about 9.3. In the SOE system consisting of 50% (v/v) ethanol the recoveries of HAc, 1, 3-PD, and glycerol were 94.40%, 95.50%, and 76.19%, respectively.
24
Fig. 7 Effect of ethanol volume on partition coefficients and recoveries of HAc and 1, 3-PD from the filtered fermentation broth (natural pH). 3.5 Two-step salting-out extraction of 1, 3-PD, HAc and BA from fermentation broth Based on all above results, two-step salting-out extraction experiment was applied to unfiltered fermentation broth under optimized conditions. As indicated in Fig.8, in the first step salting-out extraction partition coefficient and recovery of BA reached 23.34 and 93.78%, respectively, and the removal ratios of 1, 3-PD, HAc and glycerol were 95.20%, 69.04% and 93.82%, respectively. At the same time, 99.63% of the cells and 95.78% of the proteins were accumulated in the interphase between the top and bottom phase. Then, 88.72% of BA was recovered by back-extraction using Na2CO3 solution. Finally, 96.29% of 1, 3-PD, 97.26% of HAc, and 68.48% of glycerol were partitioned into the organic phase during the second step salting-out extraction. In addition, all the cells and 77.89% of proteins were removed. After two-step SOE and back extraction, the total recoveries of 1, 3-PD, HAc, BA and glycerol reached 91.67%, 92.75%, 83.20% and 64.25%, respectively, with almost all 25
the cells and 97.16% of the proteins being removed. It is feasible to effectively separate 1, 3-PD and organic acids by adopting two-step salting-out extraction with simple operations, avoiding energy-intensive process such as electrodialysis
[22]
. Furthermore, cells and proteins could be removed
simultaneously from fermentation broths, which would be used for feed because of nontoxicity of C. butylicum. However, there are still some questions that need to be solved such as the recycle of salt, which is an important factor imposing restrictions on the large-scale application of salting-out extraction technology. In the first step salting-out extraction 25 wt% NaH2PO4 was added and most of NaH2PO4 was enriched in the bottom phase at last. Although salt recycle was studied in some researches [35, 36], the reuse of NaH2PO4 needs to be further investigated.
Fig. 8 Two-step salting-out extraction of 1, 3-PD, HAc and BA from fermentation broth (the total mass of the first step SOE system being 500 g; back-extraction condition: initial phase ratio of n-butyl acetate solution to Na2CO3 solution being 2: 1 and molar ratio of sodium carbonate to organic acids being 3: 5). 4. Conclusion It is simple and feasible to separate 1, 3-PD and HAc from BA efficiently in 26
fermentation broths with removal of cells and proteins through two-step salting-out extraction. In the first step salting-out extraction, partition coefficient and recovery of BA could reach 42.21 and 96.42%, and furthermore, 33.21% of HAc and 3.71% of 1, 3-PD were extracted at 25 wt% NaH2PO4/30 wt% n-butyl acetate. In the back-extraction, the recoveries of BA, HAc and 1, 3-PD were 91.28%, 93.96% and 91.20%, respectively, with initial phase ratio of n-butyl acetate solution to Na2CO3 solution being 2: 1 when molar ratio of sodium carbonate to organic acids was 3: 5. In the second step salting-out extraction, the recoveries of HAc and 1, 3-PD were 94.40% and 95.50%, respectively, at 50% (v/v) ethanol. In a scale-up experiment on 500 g, the recoveries of 1, 3-PD, HAc and BA reached 91.67%, 92.75% and 83.20%, respectively, with the removal of almost all the cells and 97.16% of the proteins after two-step salting-out extraction. The recycle of salt needs to be researched further. Acknowledgements This work was supported by the National Natural Science Foundation of China (Grant No. 21476042).
27
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Highlight:
A novel two-step salting-out extraction was used to separate 1, 3-propanediol, butyric acid and acetic acid from fermentation broth by an anaerobic microbial consortium in which Clostridium butyricum was the main strain.
Butyric acid could be separated from 1, 3-propanediol and acetic acid by the salting-out extraction system comprised of acidic inorganic salt and hydrophobic solvent.
Effect of sodium carbonate concentration on the back-extraction of butyric acid was investigated.
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