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phosphate system
Process Biochemistry 44 (2009) 112–117
Contents lists available at ScienceDirect
Process Biochemistry journal homepage: www.elsevier.com/locate/procbio
Aqueous two-phase extraction of 2,3-butanediol from fermentation broths using an ethanol/phosphate system Bo Jiang, Zhi-Gang Li, Jian-Ying Dai, Dai-Jia Zhang, Zhi-Long Xiu * Department of Bioscience and Biotechnology, School of Environmental and Biological Science and Technology, Dalian University of Technology, Linggong Road 2, Dalian 116024, PR China
A R T I C L E I N F O
A B S T R A C T
Article history: Received 25 June 2008 Received in revised form 17 September 2008 Accepted 29 September 2008
Separation of 2,3-butanediol from the complex fermentation broths is a difficult task and becomes a bottleneck in industrial production. Aqueous two-phase systems composed of hydrophilic solvents and inorganic salts could be used to extract 2,3-butanediol from fermentation broths. Aqueous two-phase extraction of 2,3-butanediol from fermentation broths was studied by ethanol and dipotassium hydrogen phosphate system. The influences of phase composition on partition of 2,3-butanediol, removal of cells and biomacromolecules were investigated. The partition coefficient and recovery of 2,3-butanediol reached up to 28.34 and 98.13%, respectively, and the selective coefficient of 2,3-butanediol to glucose was 615.87 when the system was composed of 24% (w/w) ethanol and 25% (w/w) dipotassium hydrogen phosphate. Simultaneously, cells and proteins could be removed from the fermentation broths and the removal ratio reached 99.63 and 85.9%, respectively. This process is convenient and economic, furthermore, the operation is easy to scale-up, that is, this method provides a new possibility for the separation and refining of 2,3-butanediol. ß 2008 Elsevier Ltd. All rights reserved.
Keywords: 2,3-Butanediol Fermentation Aqueous two-phase extraction Partition coefficient Recovery Ethanol/phosphate
1. Introduction 2,3-Butanediol (2,3-BD) is an important promising chemical which can be used as a variety of chemical feedstocks and liquid fuel. 2,3-BD has been shown to have potential applications in the manufacture of printing inks, perfumes, fumigants, moistening and softening agents, explosives and plasticizers, and as a carrier for pharmaceuticals. It can be readily dehydrated to methylethyl ketone (MEK, an excellent organic solvent for resins and lacquers), and to butadiene for manufacture of synthetic rubber. It can also be easily dehydrogenated into acetoin and diacetyl which are flavoring agents used in dairy products, margarines and cosmetics [1,2]. Acetoin is a precursor of 2,3-BD and is widely used in the world as a favorable flavor to concoct the fragrances of cream, yoghourt, strawberry jam and so on. So far, 2,3-BD is mainly manufactured by microbial route and has acquired considerable progresses in the fermentation [3–7]. However, the high boiling point (180–184 8C) and high affinity of water make 2,3-BD difficult to be separated from complicated fermentation broths, thus the cost of downstream processing can take a very high portion in the total production cost. The recovery
* Corresponding author. Tel.: +86 411 84706369; fax: +86 411 84706369. E-mail address: [email protected] (Z.-L. Xiu). 1359-5113/$ – see front matter ß 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.procbio.2008.09.019
becomes a bottle-neck for the development of a commercially available process [8]. In the last decade, few reports about separation of 2,3-BD have been published, and the previously reported separation techniques mainly include steam stripping [9], solvent extraction [10], reverse osmosis and pervaporation [11]. Steam stripping requires a large amount of energy and prevents its application today. Liquid–liquid extraction has been attracting much attention, including solvent extraction of 2,3-BD. But no effective extractants have been found so far for this purpose because of its hydrophilicity, except for adding a large amount of solvent into a concentrated broth. The reactive extraction of 2,3-BD is also reported. Anticorrosion of devices due to acidity is a main problem at a large scale for this method. The conventional vacuum distillation is inefficient due to its high boiling point. The dissolved substances would become viscous slurry before distillation temperature is attained. The difficulties in developing an efficient process to separate 2,3-BD from fermentation broths are associated with the hydrophilicity and high boiling point of the target product, and the complexity of the fermentation broths. All the separation methods and techniques mentioned above have some drawbacks or limitations. Most of them require pretreatment and high energy input, which increase the cost. For these problems, one novel purification method — aqueous two-phase extraction (ATPE) was developed to meet all these criteria.
B. Jiang et al. / Process Biochemistry 44 (2009) 112–117
ATPE has been widely applied in the separation of biomacromolecules, such as proteins and nucleic acids because of its mild conditions and high capacity [12–15]. Up to now, most aqueous twophase systems (ATPS) used for purification were based either on polyethylene glycol (PEG)/salt systems or polymer/polymer (e.g., PEG/dextran) systems. Due to the high cost of the polymers and difficulty in isolating the extracted molecules from the polymer phase by back extraction, these systems can not be used in a large scale, especially for production of bulk chemicals. Recently, short chain alcohol/salt system has been used as a novel aqueous twophase system to purify natural compounds because of its advantages such as low cost, easy recovery of alcohol by evaporation and simple scale-up [16]. Ethanol can form stable and adjustable aqueous twophase system with inorganic salt solutions, which might be due to salting-out and the low solubility of inorganic salt in ethanol. When aqueous two-phase system is formed, the top phase is rich in ethanol and the bottom phase is rich in inorganic salt. In this paper, a hydrophilic organic solvent/salt ATPS was formed by ethanol and dipotassium hydrogen phosphate. The influences of phase composition on partition of 2,3-BD, removal of cells and biomacromolecules were investigated. This study is the first report presenting a simple and effective method for the extraction 2,3-BD from fermentation broths directly by an aqueous two-phase system. 2. Materials and methods 2.1. Chemicals and materials 2,3-BD standard was purchased from Sigma Chemical Co. The strain used was Klebsella pneumoniae CICC 10011, purchased from China Center of Industrial Culture Collection (Beijing, China). The fermentation broths were gained by glucose-based fed-batch fermentation under micro-aerobic conditions in our lab [5]. The concentrations of 2,3-butandiol, acetoin and glucose in the fermentation broths were 65.23, 4.03 and 15.86 g/L, respectively. The OD650 of the fermentation broths was 10.25. Bovine serum albumin (BSA) was purchased from the Shanghai Institute of Bioproducts, Ministry of Health of China. Coomassic Brilliant Blue G250 was made in the Shanghai Boao Biotechnology Corp. The cellulose triacetate hollow fiber dialyzer with effective surface area of 1.5 m2 and cut-off molecular weight of 5000 was manufactured by NISSHO Corp., Osaka, Japan. All the other chemicals were of analytical grade. 2.2. Analytical methods The concentrations of 2,3-BD and acetoin were analyzed by gas chromatography (SHIMAZU GC-14B, FID-detector, 2 m w5 mm glass column packed with Chromosorb 101 and operated with N2 as carrier gas at flow rate of 50 mL/min, detector temperature 190 8C and column temperature 180 8C). The Coomassie Brilliant Blue method (Bradford, 1976) was used to measure the concentration of protein, using BAS as a standard protein. The biomass concentration was measured by the optical density at 650 nm using a spectrophotometer. Glucose was assayed by glucose analyzer (Biosensor SBA-50, Shandong Academy of Sciences, Shandong, China).
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ethanol were added into the clarified filtrate to form aqueous two-phase systems consisting of 8%–30% (w/w) ethanol and 16–30% (w/w) K2HPO4. The mixture was held for 8 h at room temperature. The concentrations of compounds in the top and bottom phases were analyzed by gas chromatography (GC) system. The partition coefficient (K) is defined as the ratio of the concentration of compound in the top phase to that in the bottom phase. The recovery (Y) is the mass ratio of compound partitioned in the top phase to the total amount of compounds. 2.5. Aqueous two-phase extraction of 2,3-BD from fermentation broths The fermentation broths were used for aqueous two-phase extraction without any pretreatment. The other steps were the same as Section 2.4. The removal of cells and proteins were examined in ATPE. The partition behavior of glucose was also determined and the selective coefficient of 2,3-BD to glucose was defined as the ratio of the partition coefficient of 2,3-BD to that of glucose. The removal ratio (R) of cells and soluble proteins in top phase was defined as follows: 1 Mt R¼ 100% M where Mt and M were the mass in the top phase and the total mass, respectively.
3. Results and discussion 3.1. Phase diagram of ethanol and dipotassium hydrogen phosphate ATPS The phase diagram was determined in both de-ionized water and clarified filtrate respectively. As shown in Fig. 1, the area of aqueous two system made by clarified filtrate was larger than the area made by de-ionized water. The reason is that some products such as ethanol, 2,3-BD and the residual salts in the fermentation broths enhanced the forming of ATPS. 3.2. Partition behavior of 2,3-BD in ethanol/K2HPO4 ATPS The partition behavior of 2,3-BD in ethanol/K2HPO4 aqueous two-phase system was investigated by clarified filtrate, and the effects of ethanol and K2HPO4 concentration on the partitioning of 2,3-BD were determined. As shown in Fig. 2 and Fig. 3, 2,3-BD had a high partition coefficient and recovery, indicating that 2,3-BD tends to concentrate to the top phase. The partition coefficient and the recovery of 2,3-BD were enhanced when the concentration of phosphate or ethanol in the system was increased. But the recovery increased slowly after salt and ethanol reaching a certain concentration, as shown in Fig. 2. When the concentration of ethanol changed from 22 to 28%, the recovery increased only one percentage, while the volume of top phase increased from 5.4 to 6.3 mL. In order to obtain high recovery of 2,3-BD and reduce the cost, an appropriate concentration of salt and ethanol was adopted. At the dipotassium hydrogen phosphate concentration of 25% (w/w)
2.3. Phase diagram of ethanol and dipotassium hydrogen phosphate ATPS The phase diagram was obtained using a turbidity titration method. De-ionized water and dipotassium hydrogen phosphate (K2HPO4) were added into tubes. Ethanol was subsequently added drop by drop into each tube on an electrical balancer for measuring the added ethanol. After each droplet, the mixture was shaken for 2 min on a vortex mixer. When the mixture was turbid after mixing, one drop of water was added and the turbidity disappeared. Then one more drop of ethanol was added, turbidity appeared again. The point at which the mixture firstly became turbid was the turbid point. The total weight of added ethanol was measured exactly, the concentrations of ethanol and dipotassium hydrogen phosphate at different turbid points were calculated and the phase diagram curve was plotted. When the effects of 2,3-BD fermentation broths on the phase diagram were investigated, dipotassium hydrogen phosphate was dissolved into 2,3-BD fermentation broths. The other steps were the same as above. 2.4. Partition behavior of 2,3-BD in ethanol/K2HPO4 ATPS The fermentation broths were first filtered by a cellulose triacetate hollow fiber, which could remove all cells and the most of proteins, and then solid K2HPO4 and
Fig. 1. Effect of clarified filtrate on phase diagram of ethanol/dipotassium hydrogen phosphate aqueous two-phase system.
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Fig. 3. Effects of concentrations of dipotassium hydrogen phosphate on the partition coefficient (K) and recovery (Y) of 2,3-BD with samples filtered. The concentrations of ethanol (w/w) are 20% (&), 25% (*) and 30% (~). Fig. 2. Effects of ethanol concentrations on the partition coefficient (K) and recovery (Y) of 2,3-BD with samples filtered. The concentrations of dipotassium hydrogen phosphate (w/w) are 20% (&), 25% (*) and 30% (~).
and ethanol of 24% (w/w), the partition coefficient and recovery of 2,3-BD reached 27.82 and 98.06%, respectively, indicating that almost all the 2,3-BD was enriched in the top phase. Although 2,3-BD has great affinity to water, 2,3-BD is partitioned into the top ethanol phase with a high partition coefficient and recovery due to salting-out. When the concentration of phosphate or ethanol increased, the salting-out effect enhanced, resulting in the increase of partition coefficient and recovery of 2,3-BD. The distribution of acetoin in this system was also studied at constant phosphate concentration of 30%. Acetoin has similar partition coefficient and recovery with 2,3-BD, as shown in Table 1. It can be separated effectively from 2,3-BD by the subsequent distillation process. These results were better than those obtained by traditional ATPS for the recovery of 2,3-BD [17], e.g. the maximum partition coefficient of 2,3-BD was only 1.15 by PEG/ dextran ATPS. In the current contribution, the low-cost ethanol and phosphate were used, and higher partition coefficient and recovery were achieved.
3.3. Aqueous two-phase extraction of 2,3-BD from fermentation broths 3.3.1. Recovery of 2,3-BD from fermentation broths Based on the above results, the fermentation broths were directly used for ATPE without filtration. The results were shown in Table 2 and Table 3. The partition coefficient and the recovery of 2,3-BD increased dramatically as the concentration of phosphate and ethanol in the system increased, it was the same as using clarified filtrate. At the dipotassium hydrogen phosphate concentration of 25% (w/w) and ethanol 24% (w/w), the partition coefficient and recovery of 2,3-BD reached 28.34 and 98.13%, respectively. In order to compare expediently, the partition coefficient and recovery of 2,3-BD that obtained from samples filtered and without filtration were shown in Fig. 4. The two approaches had similar results, indicating that cells and biomacromolecules in broths had little effects on extraction efficiency. Compared with ATPE of the clarified filtrate, both partition coefficients and recoveries are slightly higher. With the ATPE of fermentation broths, almost all the 2,3-BD was enriched in the top ethanol phase. The mass fraction of ethanol in the top phase exceeded to 30% and the mass fraction of 2,3-BD was
Table 1 Effects of ethanol concentrations on partition of 2,3-butanediol and acetoin at a constant phosphate concentration of 30% (w/w). Concentration of ethanol (w/w, %)
8
10
12
14
16
18
K2,3-BD KAcetoin Y2,3-BD (%) YAcetoin (%)
4.69 0.05 4.18 0.08 73.5 0.5 71.2 0.6
7.49 0.2 6.39 0.1 84.8 0.8 82.7 0.9
10.71 0.3 8.96 0.2 90.5 0.2 88.8 0.2
14.69 0.2 11.91 0.3 93.3 0.4 91.9 0.5
18.99 0.6 14.88 0.4 95.2 0.3 93.9 0.3
23.73 0.4 17.55 0.1 96.5 0.1 95.0 0.3
Table 2 Effects of ethanol concentrations on partition of 2,3-BD with fermentation broths. Salt concentration (w/w, %)
Ethanol concentration (w/w, %)
30
K2,3-BD Y2,3-BD (%) K2,3-BD Y2,3-BD (%)
25
10
12
14
18
20
22
24
26
9.53 0.01 90.26 0.44 – –
13.36 0.39 93.44 0.70 – –
16.63 0.22 95.64 0.40 – –
24.67 0.15 97.15 0.58 15.79 0.24 95.24 0.22
27.62 0.10 98.41 0.40 19.76 0.08 97.11 0.22
– – 24.51 0.47 97.54 0.35
– – 28.34 0.36 98.13 0.21
– – 31.90 0.10 98.55 0.05
B. Jiang et al. / Process Biochemistry 44 (2009) 112–117
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Table 3 Effects of concentrations of dipotassium hydrogen phosphate on partition of 2,3-BD with fermentation broths. Ethanol concentration (w/w, %)
Salt concentration (w/w, %)
20
K2,3-BD Y2,3-BD (%) K2,3-BD Y2,3-BD (%)
25
19
20
21
22
23
24
25
26
– – 19.94 0.82 98.012 0.14
13.63 0.26 96.85 0.069 – –
– – 22.82 0.60 98.54567 0.13
15.72 0.30 97.16 0.081 – –
– – 26.31 0.41 98.68 0.024
19.60 0.54 97.33 0.043 – –
– – 27.75 0.050 99.14 0.050
22.61 0.069 97.40 0.016 – –
to 5.0 with phosphoric acid, and the concentration of ethanol adjusted to 17% (w/w), the recovery of K2HPO4 crystallized as KH2PO4 could reach up to 79.5%. Additionally, K2HPO4 is also an important component in fermentation medium, thus, it could be reused in fermentation. The recovery of salt will be further investigated.
Fig. 4. Comparison of partition coefficient and recovery of 2,3-BD between samples without filtration and samples filtered at constant dipotassium hydrogen phosphate concentrations of 25%. (samples without filtration (*) and filtered samples (&)).
8%. This progress can effectively save the cost of distillation because ethanol is more volatile than water, and the recycled ethanol can be used for ATPE again. On the other hand, it is also necessary to recover K2HPO4 after ATPE for the demand of industrialization. The concentration of K2HPO4 in the top phase was only 1–2% (w/w) and it could crystallize from solution in the distillation process consequently. Most of the salt used in this system enriched in the bottom phase. Some works have been done in this aspect. The 400 g/L K2HPO4 solution was used to simulate the bottom phase. If pH was adjusted
3.3.2. Removal of cells and proteins When the fermentation broths were directly used to conduct phase separation experiments without filtration, a third ‘middle phase’ was formed at the interface between the top and bottom phase. The removal ratios of cells and soluble proteins in the top phase were shown in Fig. 5 and Fig. 6. The removal ratios were enhanced when the concentration of phosphate or ethanol in the system was increased. At the dipotassium hydrogen phosphate concentration of 25% (w/w) and ethanol 24% (w/w), the removal ratios of cells and soluble proteins in the top phase reached 99.63 and 85.9%, respectively. The soluble proteins in the top phase were decreased from 0.700 g/L in broths to 0.046 g/L. This is much better than that using chitosan and polyacrylamide as flocculating agents [18]. The ATPE has been reported to be used to remove protein impurities and cell fragments, and irreversible protein denaturation was shown to occur in the phase partition studies [12]. Regardless of the differences of electric charge and density, the denatured proteins and cells concentrated at the interface between the top and bottom phase. The downstream processing of biologically produced 2,3-BD usually includes two main steps. The first step is the removal of microbial cells and impurities using membrane filtration or highspeed centrifugation [19], and the second step is the separation of 2,3-BD from the fermentation broths by distillation. When using ATPE, all of the above operations can be achieved at the same time economically. Moreover, more than 30% (w/w) of ethanol or salts is rich in the top phase or the bottom phase, respectively. In this
Fig. 5. Effects of concentrations of ethanol and dipotassium on the removal ratio of cells. (A) The concentrations of dipotassium hydrogen phosphate (w/w) are 25% (&) and 30% (*). (B) The concentrations of ethanol (w/w) are 25% (~) and 30% (!).
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B. Jiang et al. / Process Biochemistry 44 (2009) 112–117
Fig. 6. Effects of concentrations of ethanol and dipotassium on the removal ratio of proteins. Symbols are the same as those shown in Fig. 5.
Fig. 7. Effects of concentrations of ethanol and dipotassium hydrogen phosphate on partition of glucose. Symbols are the same as those shown in Fig. 5.
extreme environment, cells are impossible to grow. It is worthwhile pointing out that this method can remove impurities effectively, which made it simple to vaporize the 2,3-BD from the top phase and viscous slurry was avoided. 3.3.3. Partition behavior of glucose in ethanol/K2HPO4 ATPS The partition behavior of glucose was also determined and the result was shown in Fig. 7. Partition coefficients of glucose decreased obviously with the increasing concentrations of phosphate and ethanol, which indicated a preference of glucose for the bottom salt-rich phase. At the dipotassium hydrogen phosphate concentration of 25% (w/w) and ethanol 24% (w/w), the partition coefficient was only 0.046, i.e. 86.60% of glucose distributed in the bottom phase. The selective partition coefficient of 2,3-BD to glucose was 615.87, indicating that the substrate and the product have been separated efficiently. It is helpful that the glucose enriched in the bottom salt phase. Because it can prevent forming viscous slurry in the distillation process of top phase and the glucose in the bottom phase may be recovered as part of medium for subsequent fermentation. 3.3.4. Effect of 2,3-BD concentration on partition coefficient and recovery In the fermentation process, the concentration of 2,3-BD was different in each batch in a range of 6–12%. 2,3-BD can form ATPS
Fig. 8. Effects of concentrations of 2,3-BD on the partition coefficient (K) and recovery (Y) of 2,3-BD. The concentrations of dipotassium hydrogen phosphate (w/ w) and ethanol (w/w) are 24 and 25%.
B. Jiang et al. / Process Biochemistry 44 (2009) 112–117
with phosphate when its concentration is enough high. So the 2,3BD solution with different concentrations was used to study the effect of 2,3-BD on K and Y. The result was shown in Fig. 8. The partition coefficient and recovery of 2,3-BD were enhanced as the concentration of 2,3-BD in the system increased. 2,3-BD alone with K2HPO4 could form an ATPS in an appropriate range, that is, with the increasing concentration of 2,3-BD, the ability of separation could be enhanced. It is advantaged for this method when applied in the industry for higher concentration of 2,3-BD broths. The results above show that it is feasible to separate 2,3-BD from fermentation broths with ethanol/phosphate aqueous twophase system. ATPE can extract 2,3-BD effectively, removing cells, protein impurities and glucose simultaneously. This process is convenient and economic, furthermore, the operation is easy to scale-up. 4. Conclusion A hydrophilic organic solvent/salt aqueous two-phase system was formed by ethanol and dipotassium hydrogen phosphate. As indicated by the results, 2,3-BD can be extracted efficiently to the top phase. The partition coefficient and recovery of 2,3-BD reached up to 28.34 and 98.13%, respectively, and the selective coefficient of 2,3-BD to glucose was 615.87 when the system was composed of 24% (w/w) ethanol and 25% (w/w) dipotassium hydrogen phosphate. Simultaneously, cells and proteins could be removed from the fermentation broths and the removal ratio reached 99.63 and 85.9%, respectively. Ethanol/phosphate aqueous two-phase system is effective to the separation of 2,3-BD, with advantages of low cost and convenient operation. This method provides a new possibility for the separation and refining of 2,3-BD. Acknowledgements This work was supported by the grant from the Major State Basic Research Development Program of China (973 Program) (No. 2007CB714306) and Teaching and Research Award Program for Outstanding Young Teachers in High Education Institutions of Ministry of Education of the People’s Republic of China.
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References [1] Garg SK, Jain A. Fermentative production of 2,3-butanediol: a review. Bioresour Technol 1995;51:103–9. [2] Syu MJ. Biological production of 2,3-butanediol. Appl Microbiol Biotechnol 2001;55:10–8. [3] Clark JH. Green chemistry for the second generation biorefinery-sustainable chemical manufacturing based on biomass. J Chem Technol Biotechnol 2007;82:603–9. [4] Zeng AP, Biebl H, Deckwer WD. 2,3-Butanediol production by Enterobacter aerogenes in continuous culture: role of oxygen supply. Appl Microbiol Biotechnol 1990;33:264–8. [5] Qin JY, Xiao ZJ, Ma CQ, Xie NZ, Liu PH, Xu P. Production of 2,3-butanediol by Klebsiella pneumoniae using glucose and ammonium phosphate. Chin J Chem Eng 2006;14(1):132–6. [6] Zeng AP, Biebl H, Deckwer WD. Production of 2,3-butanediol in a membrane bioreactor with cell recycle. Appl Microbiol Biotechnol 1991;34:463–8. [7] Nakashimada Y, Marwoto B, Kashiwamura T, Kakizono T, Nishio N. Enhanced 2,3-butanediol production by addition of acetic acid in Paenibacillus polymyxa. J Biosci Bioeng 2000;90:661–4. [8] Xiu ZL, Zeng AP. Present state and perspective of downstream processing of biologically produced 1,3-propanediol and 2,3-butanediol. Appl Microbiol Biotechnol 2008;78:917–26. [9] Wheat JA, Leslie JD, Tomkins RV, Mitton HE, Scott DS, Ledingham GA. Production and properties of 2,3-butanediol. XXVIII. Pilot plant recovery of levo-2,3butanediol from whole wheat mashes fermented by Aerobacillus polymyxa. Can J Res 1948;26F:469–96. [10] Tsao GT. Conversion of biomass from agriculture into useful products. Final Report, USDDE Contact No. EG-77-S-02-4298. [11] Qureshi N, Meagher MM, Hutjins RW. Recovery of 2,3-butanediol by vacuum membrane distillation. Sep Sci Technol 1994;29(13):1733–48. [12] Louwrier A. Model phase separations of proteins using aqueous/ethanol components. Biotechnol Tech 1998;12(5):363–5. [13] Zhi WB, Deng QY. Purification of salvianolic acid B from the crude extract of Salvia miltiorrhiza with hydrophilic organic/salt-containing aqueous two-phase system by counter-current chromatography. J Chromatogr A 2006;1116:149–52. [14] Tan TW, Huo Q, Ling Q. Purification of glycyrrhizin from Glycyrrhiza uralensis Fisch with ethanol/phosphate aqueous two phase system. Biotechnol Lett 2002;24:1417–20. [15] Aguilar O, Rito-Palomares M. Processing of soybean (Glycine max) extracts in aqueous two-phase systems as a first step for the potential recovery of recombinant proteins. J Chem Technol Biotechnol 2008;83:286–93. [16] Greve A, Kula MR. Cost structure and estimation for the recycling of salt in a protein extraction process system. Bioproc Biosyst Eng 1990;6:173–7. [17] Ghosh S, Swaminathan. Optimization of the phase system composition of aqueous two-phase system for extraction of 2,3-butanediol by theoretical formulation and using response surface methodology. Chem Biochem Eng 2004;Q18(3):263–71. [18] Hao J, Xu F, Liu HJ, Liu DH. Downstream processing of 1,3-propanediol fermentation broth. J Chem Technol Biotechnol 2006;81:102–8. [19] Ames TT. Process for the isolation of 1,3-propanediol from fermentation broth. US Patent 6,361,983 (2006).
Contents lists available at ScienceDirect
Process Biochemistry journal homepage: www.elsevier.com/locate/procbio
Aqueous two-phase extraction of 2,3-butanediol from fermentation broths using an ethanol/phosphate system Bo Jiang, Zhi-Gang Li, Jian-Ying Dai, Dai-Jia Zhang, Zhi-Long Xiu * Department of Bioscience and Biotechnology, School of Environmental and Biological Science and Technology, Dalian University of Technology, Linggong Road 2, Dalian 116024, PR China
A R T I C L E I N F O
A B S T R A C T
Article history: Received 25 June 2008 Received in revised form 17 September 2008 Accepted 29 September 2008
Separation of 2,3-butanediol from the complex fermentation broths is a difficult task and becomes a bottleneck in industrial production. Aqueous two-phase systems composed of hydrophilic solvents and inorganic salts could be used to extract 2,3-butanediol from fermentation broths. Aqueous two-phase extraction of 2,3-butanediol from fermentation broths was studied by ethanol and dipotassium hydrogen phosphate system. The influences of phase composition on partition of 2,3-butanediol, removal of cells and biomacromolecules were investigated. The partition coefficient and recovery of 2,3-butanediol reached up to 28.34 and 98.13%, respectively, and the selective coefficient of 2,3-butanediol to glucose was 615.87 when the system was composed of 24% (w/w) ethanol and 25% (w/w) dipotassium hydrogen phosphate. Simultaneously, cells and proteins could be removed from the fermentation broths and the removal ratio reached 99.63 and 85.9%, respectively. This process is convenient and economic, furthermore, the operation is easy to scale-up, that is, this method provides a new possibility for the separation and refining of 2,3-butanediol. ß 2008 Elsevier Ltd. All rights reserved.
Keywords: 2,3-Butanediol Fermentation Aqueous two-phase extraction Partition coefficient Recovery Ethanol/phosphate
1. Introduction 2,3-Butanediol (2,3-BD) is an important promising chemical which can be used as a variety of chemical feedstocks and liquid fuel. 2,3-BD has been shown to have potential applications in the manufacture of printing inks, perfumes, fumigants, moistening and softening agents, explosives and plasticizers, and as a carrier for pharmaceuticals. It can be readily dehydrated to methylethyl ketone (MEK, an excellent organic solvent for resins and lacquers), and to butadiene for manufacture of synthetic rubber. It can also be easily dehydrogenated into acetoin and diacetyl which are flavoring agents used in dairy products, margarines and cosmetics [1,2]. Acetoin is a precursor of 2,3-BD and is widely used in the world as a favorable flavor to concoct the fragrances of cream, yoghourt, strawberry jam and so on. So far, 2,3-BD is mainly manufactured by microbial route and has acquired considerable progresses in the fermentation [3–7]. However, the high boiling point (180–184 8C) and high affinity of water make 2,3-BD difficult to be separated from complicated fermentation broths, thus the cost of downstream processing can take a very high portion in the total production cost. The recovery
* Corresponding author. Tel.: +86 411 84706369; fax: +86 411 84706369. E-mail address: [email protected] (Z.-L. Xiu). 1359-5113/$ – see front matter ß 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.procbio.2008.09.019
becomes a bottle-neck for the development of a commercially available process [8]. In the last decade, few reports about separation of 2,3-BD have been published, and the previously reported separation techniques mainly include steam stripping [9], solvent extraction [10], reverse osmosis and pervaporation [11]. Steam stripping requires a large amount of energy and prevents its application today. Liquid–liquid extraction has been attracting much attention, including solvent extraction of 2,3-BD. But no effective extractants have been found so far for this purpose because of its hydrophilicity, except for adding a large amount of solvent into a concentrated broth. The reactive extraction of 2,3-BD is also reported. Anticorrosion of devices due to acidity is a main problem at a large scale for this method. The conventional vacuum distillation is inefficient due to its high boiling point. The dissolved substances would become viscous slurry before distillation temperature is attained. The difficulties in developing an efficient process to separate 2,3-BD from fermentation broths are associated with the hydrophilicity and high boiling point of the target product, and the complexity of the fermentation broths. All the separation methods and techniques mentioned above have some drawbacks or limitations. Most of them require pretreatment and high energy input, which increase the cost. For these problems, one novel purification method — aqueous two-phase extraction (ATPE) was developed to meet all these criteria.
B. Jiang et al. / Process Biochemistry 44 (2009) 112–117
ATPE has been widely applied in the separation of biomacromolecules, such as proteins and nucleic acids because of its mild conditions and high capacity [12–15]. Up to now, most aqueous twophase systems (ATPS) used for purification were based either on polyethylene glycol (PEG)/salt systems or polymer/polymer (e.g., PEG/dextran) systems. Due to the high cost of the polymers and difficulty in isolating the extracted molecules from the polymer phase by back extraction, these systems can not be used in a large scale, especially for production of bulk chemicals. Recently, short chain alcohol/salt system has been used as a novel aqueous twophase system to purify natural compounds because of its advantages such as low cost, easy recovery of alcohol by evaporation and simple scale-up [16]. Ethanol can form stable and adjustable aqueous twophase system with inorganic salt solutions, which might be due to salting-out and the low solubility of inorganic salt in ethanol. When aqueous two-phase system is formed, the top phase is rich in ethanol and the bottom phase is rich in inorganic salt. In this paper, a hydrophilic organic solvent/salt ATPS was formed by ethanol and dipotassium hydrogen phosphate. The influences of phase composition on partition of 2,3-BD, removal of cells and biomacromolecules were investigated. This study is the first report presenting a simple and effective method for the extraction 2,3-BD from fermentation broths directly by an aqueous two-phase system. 2. Materials and methods 2.1. Chemicals and materials 2,3-BD standard was purchased from Sigma Chemical Co. The strain used was Klebsella pneumoniae CICC 10011, purchased from China Center of Industrial Culture Collection (Beijing, China). The fermentation broths were gained by glucose-based fed-batch fermentation under micro-aerobic conditions in our lab [5]. The concentrations of 2,3-butandiol, acetoin and glucose in the fermentation broths were 65.23, 4.03 and 15.86 g/L, respectively. The OD650 of the fermentation broths was 10.25. Bovine serum albumin (BSA) was purchased from the Shanghai Institute of Bioproducts, Ministry of Health of China. Coomassic Brilliant Blue G250 was made in the Shanghai Boao Biotechnology Corp. The cellulose triacetate hollow fiber dialyzer with effective surface area of 1.5 m2 and cut-off molecular weight of 5000 was manufactured by NISSHO Corp., Osaka, Japan. All the other chemicals were of analytical grade. 2.2. Analytical methods The concentrations of 2,3-BD and acetoin were analyzed by gas chromatography (SHIMAZU GC-14B, FID-detector, 2 m w5 mm glass column packed with Chromosorb 101 and operated with N2 as carrier gas at flow rate of 50 mL/min, detector temperature 190 8C and column temperature 180 8C). The Coomassie Brilliant Blue method (Bradford, 1976) was used to measure the concentration of protein, using BAS as a standard protein. The biomass concentration was measured by the optical density at 650 nm using a spectrophotometer. Glucose was assayed by glucose analyzer (Biosensor SBA-50, Shandong Academy of Sciences, Shandong, China).
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ethanol were added into the clarified filtrate to form aqueous two-phase systems consisting of 8%–30% (w/w) ethanol and 16–30% (w/w) K2HPO4. The mixture was held for 8 h at room temperature. The concentrations of compounds in the top and bottom phases were analyzed by gas chromatography (GC) system. The partition coefficient (K) is defined as the ratio of the concentration of compound in the top phase to that in the bottom phase. The recovery (Y) is the mass ratio of compound partitioned in the top phase to the total amount of compounds. 2.5. Aqueous two-phase extraction of 2,3-BD from fermentation broths The fermentation broths were used for aqueous two-phase extraction without any pretreatment. The other steps were the same as Section 2.4. The removal of cells and proteins were examined in ATPE. The partition behavior of glucose was also determined and the selective coefficient of 2,3-BD to glucose was defined as the ratio of the partition coefficient of 2,3-BD to that of glucose. The removal ratio (R) of cells and soluble proteins in top phase was defined as follows: 1 Mt R¼ 100% M where Mt and M were the mass in the top phase and the total mass, respectively.
3. Results and discussion 3.1. Phase diagram of ethanol and dipotassium hydrogen phosphate ATPS The phase diagram was determined in both de-ionized water and clarified filtrate respectively. As shown in Fig. 1, the area of aqueous two system made by clarified filtrate was larger than the area made by de-ionized water. The reason is that some products such as ethanol, 2,3-BD and the residual salts in the fermentation broths enhanced the forming of ATPS. 3.2. Partition behavior of 2,3-BD in ethanol/K2HPO4 ATPS The partition behavior of 2,3-BD in ethanol/K2HPO4 aqueous two-phase system was investigated by clarified filtrate, and the effects of ethanol and K2HPO4 concentration on the partitioning of 2,3-BD were determined. As shown in Fig. 2 and Fig. 3, 2,3-BD had a high partition coefficient and recovery, indicating that 2,3-BD tends to concentrate to the top phase. The partition coefficient and the recovery of 2,3-BD were enhanced when the concentration of phosphate or ethanol in the system was increased. But the recovery increased slowly after salt and ethanol reaching a certain concentration, as shown in Fig. 2. When the concentration of ethanol changed from 22 to 28%, the recovery increased only one percentage, while the volume of top phase increased from 5.4 to 6.3 mL. In order to obtain high recovery of 2,3-BD and reduce the cost, an appropriate concentration of salt and ethanol was adopted. At the dipotassium hydrogen phosphate concentration of 25% (w/w)
2.3. Phase diagram of ethanol and dipotassium hydrogen phosphate ATPS The phase diagram was obtained using a turbidity titration method. De-ionized water and dipotassium hydrogen phosphate (K2HPO4) were added into tubes. Ethanol was subsequently added drop by drop into each tube on an electrical balancer for measuring the added ethanol. After each droplet, the mixture was shaken for 2 min on a vortex mixer. When the mixture was turbid after mixing, one drop of water was added and the turbidity disappeared. Then one more drop of ethanol was added, turbidity appeared again. The point at which the mixture firstly became turbid was the turbid point. The total weight of added ethanol was measured exactly, the concentrations of ethanol and dipotassium hydrogen phosphate at different turbid points were calculated and the phase diagram curve was plotted. When the effects of 2,3-BD fermentation broths on the phase diagram were investigated, dipotassium hydrogen phosphate was dissolved into 2,3-BD fermentation broths. The other steps were the same as above. 2.4. Partition behavior of 2,3-BD in ethanol/K2HPO4 ATPS The fermentation broths were first filtered by a cellulose triacetate hollow fiber, which could remove all cells and the most of proteins, and then solid K2HPO4 and
Fig. 1. Effect of clarified filtrate on phase diagram of ethanol/dipotassium hydrogen phosphate aqueous two-phase system.
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Fig. 3. Effects of concentrations of dipotassium hydrogen phosphate on the partition coefficient (K) and recovery (Y) of 2,3-BD with samples filtered. The concentrations of ethanol (w/w) are 20% (&), 25% (*) and 30% (~). Fig. 2. Effects of ethanol concentrations on the partition coefficient (K) and recovery (Y) of 2,3-BD with samples filtered. The concentrations of dipotassium hydrogen phosphate (w/w) are 20% (&), 25% (*) and 30% (~).
and ethanol of 24% (w/w), the partition coefficient and recovery of 2,3-BD reached 27.82 and 98.06%, respectively, indicating that almost all the 2,3-BD was enriched in the top phase. Although 2,3-BD has great affinity to water, 2,3-BD is partitioned into the top ethanol phase with a high partition coefficient and recovery due to salting-out. When the concentration of phosphate or ethanol increased, the salting-out effect enhanced, resulting in the increase of partition coefficient and recovery of 2,3-BD. The distribution of acetoin in this system was also studied at constant phosphate concentration of 30%. Acetoin has similar partition coefficient and recovery with 2,3-BD, as shown in Table 1. It can be separated effectively from 2,3-BD by the subsequent distillation process. These results were better than those obtained by traditional ATPS for the recovery of 2,3-BD [17], e.g. the maximum partition coefficient of 2,3-BD was only 1.15 by PEG/ dextran ATPS. In the current contribution, the low-cost ethanol and phosphate were used, and higher partition coefficient and recovery were achieved.
3.3. Aqueous two-phase extraction of 2,3-BD from fermentation broths 3.3.1. Recovery of 2,3-BD from fermentation broths Based on the above results, the fermentation broths were directly used for ATPE without filtration. The results were shown in Table 2 and Table 3. The partition coefficient and the recovery of 2,3-BD increased dramatically as the concentration of phosphate and ethanol in the system increased, it was the same as using clarified filtrate. At the dipotassium hydrogen phosphate concentration of 25% (w/w) and ethanol 24% (w/w), the partition coefficient and recovery of 2,3-BD reached 28.34 and 98.13%, respectively. In order to compare expediently, the partition coefficient and recovery of 2,3-BD that obtained from samples filtered and without filtration were shown in Fig. 4. The two approaches had similar results, indicating that cells and biomacromolecules in broths had little effects on extraction efficiency. Compared with ATPE of the clarified filtrate, both partition coefficients and recoveries are slightly higher. With the ATPE of fermentation broths, almost all the 2,3-BD was enriched in the top ethanol phase. The mass fraction of ethanol in the top phase exceeded to 30% and the mass fraction of 2,3-BD was
Table 1 Effects of ethanol concentrations on partition of 2,3-butanediol and acetoin at a constant phosphate concentration of 30% (w/w). Concentration of ethanol (w/w, %)
8
10
12
14
16
18
K2,3-BD KAcetoin Y2,3-BD (%) YAcetoin (%)
4.69 0.05 4.18 0.08 73.5 0.5 71.2 0.6
7.49 0.2 6.39 0.1 84.8 0.8 82.7 0.9
10.71 0.3 8.96 0.2 90.5 0.2 88.8 0.2
14.69 0.2 11.91 0.3 93.3 0.4 91.9 0.5
18.99 0.6 14.88 0.4 95.2 0.3 93.9 0.3
23.73 0.4 17.55 0.1 96.5 0.1 95.0 0.3
Table 2 Effects of ethanol concentrations on partition of 2,3-BD with fermentation broths. Salt concentration (w/w, %)
Ethanol concentration (w/w, %)
30
K2,3-BD Y2,3-BD (%) K2,3-BD Y2,3-BD (%)
25
10
12
14
18
20
22
24
26
9.53 0.01 90.26 0.44 – –
13.36 0.39 93.44 0.70 – –
16.63 0.22 95.64 0.40 – –
24.67 0.15 97.15 0.58 15.79 0.24 95.24 0.22
27.62 0.10 98.41 0.40 19.76 0.08 97.11 0.22
– – 24.51 0.47 97.54 0.35
– – 28.34 0.36 98.13 0.21
– – 31.90 0.10 98.55 0.05
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Table 3 Effects of concentrations of dipotassium hydrogen phosphate on partition of 2,3-BD with fermentation broths. Ethanol concentration (w/w, %)
Salt concentration (w/w, %)
20
K2,3-BD Y2,3-BD (%) K2,3-BD Y2,3-BD (%)
25
19
20
21
22
23
24
25
26
– – 19.94 0.82 98.012 0.14
13.63 0.26 96.85 0.069 – –
– – 22.82 0.60 98.54567 0.13
15.72 0.30 97.16 0.081 – –
– – 26.31 0.41 98.68 0.024
19.60 0.54 97.33 0.043 – –
– – 27.75 0.050 99.14 0.050
22.61 0.069 97.40 0.016 – –
to 5.0 with phosphoric acid, and the concentration of ethanol adjusted to 17% (w/w), the recovery of K2HPO4 crystallized as KH2PO4 could reach up to 79.5%. Additionally, K2HPO4 is also an important component in fermentation medium, thus, it could be reused in fermentation. The recovery of salt will be further investigated.
Fig. 4. Comparison of partition coefficient and recovery of 2,3-BD between samples without filtration and samples filtered at constant dipotassium hydrogen phosphate concentrations of 25%. (samples without filtration (*) and filtered samples (&)).
8%. This progress can effectively save the cost of distillation because ethanol is more volatile than water, and the recycled ethanol can be used for ATPE again. On the other hand, it is also necessary to recover K2HPO4 after ATPE for the demand of industrialization. The concentration of K2HPO4 in the top phase was only 1–2% (w/w) and it could crystallize from solution in the distillation process consequently. Most of the salt used in this system enriched in the bottom phase. Some works have been done in this aspect. The 400 g/L K2HPO4 solution was used to simulate the bottom phase. If pH was adjusted
3.3.2. Removal of cells and proteins When the fermentation broths were directly used to conduct phase separation experiments without filtration, a third ‘middle phase’ was formed at the interface between the top and bottom phase. The removal ratios of cells and soluble proteins in the top phase were shown in Fig. 5 and Fig. 6. The removal ratios were enhanced when the concentration of phosphate or ethanol in the system was increased. At the dipotassium hydrogen phosphate concentration of 25% (w/w) and ethanol 24% (w/w), the removal ratios of cells and soluble proteins in the top phase reached 99.63 and 85.9%, respectively. The soluble proteins in the top phase were decreased from 0.700 g/L in broths to 0.046 g/L. This is much better than that using chitosan and polyacrylamide as flocculating agents [18]. The ATPE has been reported to be used to remove protein impurities and cell fragments, and irreversible protein denaturation was shown to occur in the phase partition studies [12]. Regardless of the differences of electric charge and density, the denatured proteins and cells concentrated at the interface between the top and bottom phase. The downstream processing of biologically produced 2,3-BD usually includes two main steps. The first step is the removal of microbial cells and impurities using membrane filtration or highspeed centrifugation [19], and the second step is the separation of 2,3-BD from the fermentation broths by distillation. When using ATPE, all of the above operations can be achieved at the same time economically. Moreover, more than 30% (w/w) of ethanol or salts is rich in the top phase or the bottom phase, respectively. In this
Fig. 5. Effects of concentrations of ethanol and dipotassium on the removal ratio of cells. (A) The concentrations of dipotassium hydrogen phosphate (w/w) are 25% (&) and 30% (*). (B) The concentrations of ethanol (w/w) are 25% (~) and 30% (!).
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Fig. 6. Effects of concentrations of ethanol and dipotassium on the removal ratio of proteins. Symbols are the same as those shown in Fig. 5.
Fig. 7. Effects of concentrations of ethanol and dipotassium hydrogen phosphate on partition of glucose. Symbols are the same as those shown in Fig. 5.
extreme environment, cells are impossible to grow. It is worthwhile pointing out that this method can remove impurities effectively, which made it simple to vaporize the 2,3-BD from the top phase and viscous slurry was avoided. 3.3.3. Partition behavior of glucose in ethanol/K2HPO4 ATPS The partition behavior of glucose was also determined and the result was shown in Fig. 7. Partition coefficients of glucose decreased obviously with the increasing concentrations of phosphate and ethanol, which indicated a preference of glucose for the bottom salt-rich phase. At the dipotassium hydrogen phosphate concentration of 25% (w/w) and ethanol 24% (w/w), the partition coefficient was only 0.046, i.e. 86.60% of glucose distributed in the bottom phase. The selective partition coefficient of 2,3-BD to glucose was 615.87, indicating that the substrate and the product have been separated efficiently. It is helpful that the glucose enriched in the bottom salt phase. Because it can prevent forming viscous slurry in the distillation process of top phase and the glucose in the bottom phase may be recovered as part of medium for subsequent fermentation. 3.3.4. Effect of 2,3-BD concentration on partition coefficient and recovery In the fermentation process, the concentration of 2,3-BD was different in each batch in a range of 6–12%. 2,3-BD can form ATPS
Fig. 8. Effects of concentrations of 2,3-BD on the partition coefficient (K) and recovery (Y) of 2,3-BD. The concentrations of dipotassium hydrogen phosphate (w/ w) and ethanol (w/w) are 24 and 25%.
B. Jiang et al. / Process Biochemistry 44 (2009) 112–117
with phosphate when its concentration is enough high. So the 2,3BD solution with different concentrations was used to study the effect of 2,3-BD on K and Y. The result was shown in Fig. 8. The partition coefficient and recovery of 2,3-BD were enhanced as the concentration of 2,3-BD in the system increased. 2,3-BD alone with K2HPO4 could form an ATPS in an appropriate range, that is, with the increasing concentration of 2,3-BD, the ability of separation could be enhanced. It is advantaged for this method when applied in the industry for higher concentration of 2,3-BD broths. The results above show that it is feasible to separate 2,3-BD from fermentation broths with ethanol/phosphate aqueous twophase system. ATPE can extract 2,3-BD effectively, removing cells, protein impurities and glucose simultaneously. This process is convenient and economic, furthermore, the operation is easy to scale-up. 4. Conclusion A hydrophilic organic solvent/salt aqueous two-phase system was formed by ethanol and dipotassium hydrogen phosphate. As indicated by the results, 2,3-BD can be extracted efficiently to the top phase. The partition coefficient and recovery of 2,3-BD reached up to 28.34 and 98.13%, respectively, and the selective coefficient of 2,3-BD to glucose was 615.87 when the system was composed of 24% (w/w) ethanol and 25% (w/w) dipotassium hydrogen phosphate. Simultaneously, cells and proteins could be removed from the fermentation broths and the removal ratio reached 99.63 and 85.9%, respectively. Ethanol/phosphate aqueous two-phase system is effective to the separation of 2,3-BD, with advantages of low cost and convenient operation. This method provides a new possibility for the separation and refining of 2,3-BD. Acknowledgements This work was supported by the grant from the Major State Basic Research Development Program of China (973 Program) (No. 2007CB714306) and Teaching and Research Award Program for Outstanding Young Teachers in High Education Institutions of Ministry of Education of the People’s Republic of China.
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