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An integrated in situ extraction-gas stripping process for Acetone–Butanol–Ethanol (ABE) fermentation
Journal of the Taiwan Institute of Chemical Engineers 45 (2014) 2106–2110
Contents lists available at ScienceDirect
Journal of the Taiwan Institute of Chemical Engineers journal homepage: www.elsevier.com/locate/jtice
An integrated in situ extraction-gas stripping process for Acetone–Butanol–Ethanol (ABE) fermentation Kuan-Ming Lu, Si-Yu Li * Department of Chemical Engineering, National Chung Hsing University, Taichung 402, Taiwan
A R T I C L E I N F O
A B S T R A C T
Article history: Received 17 March 2014 Received in revised form 24 June 2014 Accepted 29 June 2014 Available online 31 July 2014
A novel integrated in situ extraction-gas stripping process (Process 3 in this study) is proposed for running batch Acetone–Butanol–Ethanol (ABE) fermentation. The process involves two butanol separation processes in which butanol is first extracted by oleyl alcohol during ABE fermentation and gas stripping is carried out on butanol in the oleyl alcohol phase at the same time. The butanol productivity and yield of Process 3 is 0.28 0.01 g/L/h and 0.226 0.001 g-butanol/g-glucose. The ABE productivity and yield are 0.46 0.01 g/L/h and 0.374 0.002 g-ABE/g-glucose. Glucose utilization was 97% and initial glucose consumption was 121 2 g/L. Butanol and ABE concentrations of 93–113 and 166–204 g/L in the condensate can be achieved. This study demonstrates that Process 3 as described here enhances ABE fermentation and also results in the production of high purity solvents. ß 2014 Taiwan Institute of Chemical Engineers. Published by Elsevier B.V. All rights reserved.
Keywords: Clostridium acetobutylicum Acetone–Butanol–Ethanol (ABE) fermentation Extraction Gas stripping Oleyl alcohol
1. Introduction 1-Butanol has been proposed as an alternative fuel that is both renewable and sustainable and can be produced by traditional Acetone–Butanol–Ethanol fermentation using Clostridium acetobutylicum or Clostridium beijerinckii [1]. One obstacle for ABE fermentation is the toxicity of 1-butanol which results in low productivity and high recovery costs for conventional distillation [2–4]. One promising way to tackle butanol toxicity is by the use of in situ product recovery [5]. Many bio-compatible separation processes, such as gas stripping [6,7], extraction [8–10], and pervaporation [11,12] have been proposed and investigated for in situ butanol recovery during ABE fermentation. Gas stripping and extraction are two well known methods that are easy to maintain and scale-up. However, both gas stripping and extraction have low selectivity for butanol during the ABE fermentation process. Also, the low selectivity of the extraction further leads to the need for large volumes of solvent in the batch reactor to keep the butanol concentration in the aqueous phase below the threshold of around 10 g/L. In practice, the best ABE fermentation performance was achieved with a solvent to fermentation broth volume ratio of 1.75 [13]. However, this dramatically decreases the volume available for ABE fermentation in the batch reactor.
In this study, a combination of extraction and gas stripping as an integrated process is proposed for in situ butanol recovery in ABE fermentation as shown in Fig. 1. The non-volatile solvent oleyl alcohol acts as the extraction solvent and nitrogen is used for gas stripping. Oleyl alcohol is biocompatible and a feasible extraction agent for in situ butanol recovery under these conditions [13,14]. The performance of integrated in situ extraction-gas stripping during ABE fermentation is investigated and discussed. 2. Materials and methods 2.1. Strains, culture media, and growth conditions The bacterial strain used in this study was C. acetobutylicum ATCC 824. The culture was in spore form and stored at room temperature. Each liter of the fermentation medium used in this study contained: 38 g Reinforced Clostridial Medium (RCM), 0.11 g FeSO47H2O, 0.6 g MgSO47H2O, 0.008 g CaCl2, and 125 g glucose. For batch ABE fermentation, 32 mL of C. acetobutylicum spore stock solution was added aseptically to 300 mL of fresh medium without using a heat-induced germination process. Batch ABE fermentation was carried out at 37 8C with stirring at 150 rpm. 2.2. Integrated in situ extraction-gas stripping ABE fermentation
* Corresponding author. Tel.: +886 4 2284 0510x509. E-mail address: [email protected] (S.-Y. Li).
As shown in Fig. 1, the equipment used in the integrated in situ extraction-gas stripping process includes a 500-mL serum bottle, two
http://dx.doi.org/10.1016/j.jtice.2014.06.023 1876-1070/ß 2014 Taiwan Institute of Chemical Engineers. Published by Elsevier B.V. All rights reserved.
K.-M. Lu, S.-Y. Li / Journal of the Taiwan Institute of Chemical Engineers 45 (2014) 2106–2110
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Fig. 1. Schematic of integrated in situ extraction-gas stripping process for ABE fermentation.
cold traps (1 and 2) in parallel for the condensation of stripped solvents, and a cylinder of nitrogen gas. A 500-ml serum bottle was used as the reactor throughout the study. The serum bottle contained 300 mL of fresh medium and 100 mL of oleyl alcohol (80–85%, Alfa Aesar1). Cold trap 3 was used for security to ensure condensation of all stripped solvents. All cold traps were immersed in liquid nitrogen at 196 8C. In all the experiments the nitrogen gas for gas stripping was sterilized using a 0.2 mm Millipore membrane filter and delivered through a single 18-G syringe needle at a flow rate of 0.5 L/min. 0.2 mm filters were used to filter-sterilize the nitrogen gas stream throughout the experiment. Gas stripping was performed on the oleyl alcohol phase and was initiated after a fermentation time of 48 h. Condensates from the cold trap were analyzed by gas chromatography (GC) at 12 hourly intervals as described below. 2.3. Analytical methods The cell density was measured at 600 nm using a UV–vis spectrophotometer (GENESYS 10S, Thermo Scientific, USA). The concentration of residual sugars was determined by the DNS method [15]. The concentrations of acetone, butanol, ethanol, acetate, and butyrate in the fermentation culture solution were determined by GC (Hewlett Packard HP 5890 Series II) using a flame ionization detector and a capillary column DP-FFAP (30 m 0.32 mm 0.25 mm) as described earlier [16]. 3. Results and discussion 3.1. Performance of ABE fermentation using an in situ extraction-gas stripping separation process ABE fermentation was carried out using the in situ extractiongas stripping separation process described here, while batch ABE
fermentation as well as batch ABE fermentation with in situ extraction were used as controls. It can be seen in Fig. 2a that without the heat-induced germination treatment, a 36-h lag phase was observed for batch ABE fermentation. Despite this, the clostridial culture still reached a peak OD600 of 9.6 1.5 after 72 h. The glucose concentration in Process 1 remained steady for the first 36 h and then decreased strongly up to 72 h, see process 1 in Fig. 2b. The total glucose consumption in process 1 was 57 8 g/L in 72 h. The concentration of butanol in Process 1 (see Fig. 2c) started accumulating after 48 h when bacterial growth entered the exponential phase and the final butanol titer reached a concentration of 13.0 0.7 g/L. Note that the final acetone and ethanol titers were 9.0 0.4 and 2.1 0.3 g/L, respectively (data not shown). This data suggests that the direct use of spores is feasible and can be used for initiating ABE fermentation. Both bacterial growth and glucose consumption can be enhanced by incorporating in situ butanol extraction with oleyl alcohol. It can be seen in Fig. 2a that the peak OD600 in Process 2 reached 12.2 0.7 by 72 h and total glucose consumption reached 96 9 g/L in 96 h. Process 2 showed significant improvements in ABE fermentation over Process 1 and this can be attributed to the efficient in situ extraction of toxic butanol from the fermentation broth into the oleyl alcohol phase [8,13,14]. At 96 h the butanol concentrations in the aqueous and organic phases were 9 1 and 40 5 g/L, respectively. On the other hand, while butanol was selectively extracted into the oleyl alcohol, this was not the case with acetone and ethanol. It was found that the concentration of acetone and ethanol in the organic phase was lower than in the aqueous phase where their partition ratios were 0.4 and 0.2, respectively (data not shown). These are consistent with previous data [13,14]. For example, Ishizaki et al. reported that the partition ratios of acetone and ethanol were 0.3 and 0.2, respectively [14]. Although 60% of the butanol produced was extracted from the aqueous phase to the organic phase,
Table 1 Characteristics of ABE fermentation for Process 1, 2, and 3.a Characteristics
Process 1
Initial glucose (g/L) Final glucose (g/L) Glucose consumption (g/L) Glucose utilization (%) Glucose consumption rate (g/L/h)b Max. optical density (time) Butanol production (g/L) Total ABE production (g/L) Butanol productivity (g/L/h)b Total ABE productivity (g/L/h)b Butanol yield (g/g) Total ABE yield (g/g)
125 1 65 6 57 8 48 5 0.79 0.11 9.61 (72 h) 12.5 0.3 23.1 0.7 0.18 0.01 0.33 0.02 0.21 0.02 0.39 0.03
Process 2 115 2 19 10 96 9 83 9 1.00 0.09 12.17 (72 h) 22 3 34 4 0.23 0.03 0.36 0.04 0.23 0.02 0.36 0.03
Process 3 121 2 3.6 0.2 117 2 97.0 0.2 1.22 0.02 14.57 (96 h) 26.5 0.5 43.9 0.6 0.28 0.01 0.46 0.01 0.226 0.001 0.374 0.002
a Process 1: Batch ABE fermentation, Process 2: Batch ABE fermentation with in situ butanol extraction using oleyl alcohol, Process 3: Integrated in situ extraction-gas stripping ABE fermentation. Error bars represent standard deviation. b Fermentation time frames for calculating the glucose consumption rate and solvent productivities are 72, 96, and 96 h for Process 1, 2, and 3, respectively.
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Fig. 2. (a) Bacterial growth, (b) glucose consumption, (c) butanol production of three fermentation processes, and (d) partition ratios of butanol in the organic phase to butanol in the aqueous phase. Process 1: Batch ABE fermentation, Process 2: Batch ABE fermentation with in situ butanol extraction using oleyl alcohol, Process 3: Integrated in situ extraction-gas stripping ABE fermentation. Error bars represent standard deviations.
it was found that 9 1 g/L of butanol remained and had a negative effect on clostridial growth. The cell density in Process 2 decreased and there was a 19 10 g/L of glucose residue. Although a lower volume ratio of fermentation broth to oleyl alcohol could be achieved, the ABE fermentation capacity of a single fermentor would be less. The dilemma encountered in Process 2 can be resolved by using integrated in situ extraction-gas stripping, Process 3 in this study. While it can be seen from Fig. 2a that all three processes had similar specific growth rates during the early exponential phase, bacterial growth in Process 3 had an extended growth profile and reached an OD600 value of 14.6 0.7 at 96 h. An examination of Fig. 2b shows that bacterial culture consumed 97% of the available glucose in 96 h with a total glucose consumption of 117 2 g/L. This suggests that there was no obvious inhibition of bacterial growth and glucose consumption over the total fermentation time of 96 h. This finding is supported by the data in Fig. 2c which shows that the highest butanol concentration in the aqueous solution, during Process 3 fermentation, was only 6.4 0.6 g/L at 96 h. Also, the butanol concentrations in the organic phase reached 16.6 2.3 and 20.9 1.3 g/L at 72 and 96 h, respectively. These high butanol concentrations served as a strong driving force for the mass transfer of butanol from liquid to the gas bubbles during gas stripping. Note that the partition of butanol between the organic and aqueous phases dropped from 4.6–4.9 (Process 2) to 3.2–3.5 (Process 3) after the initiation of gas stripping at 48 h, see Fig. 2d. This suggested that the organic phase in Process 3 was not saturated with butanol during fermentation. Therefore, a mass transfer resistance for butanol extraction in Process 3 was noticeable [17]. Nevertheless, ABE fermentation using Process 3 is still significantly better than in Process 1 and 2. Table 1 shows that the glucose utilization in Process 3 is 102% and 17% higher than that in Process 1 and Process 2, respectively. It can be seen in Fig. 3 that final titers of butanol,
acetone, and ethanol in Process 3 are higher than that in Processes 1 and 2, where the final titers of butanol and ABE from Process 3 at 96 h are 26.5 0.5 and 43.9 0.6 g/L, respectively. Note the calculation of solvent titers for Process 3 is based on the total amount of solvents in the fermentation broth, oleyl alcohol, and condensates with respect to the volume of fermentation broth. While the three processes have similar butanol and ABE yields, the butanol and ABE productivity of Process 3 is significantly better. The butanol productivity in process 3
Fig. 3. Comparisons of ABE fermentation performance using processes 1, 2, and 3. Process 1: Batch ABE fermentation, Process 2: Batch ABE fermentation with in situ butanol extraction using oleyl alcohol, Process 3: Integrated in situ extraction-gas stripping ABE fermentation. Note: the total solvent concentration in Process 3 is the sum of that in the aqueous, organic, and condensate phases with respect to the volume of fermentation broth. Error bars represent standard deviation.
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is 0.276 0.005 g/L/h, indicating a 22% and 56% higher than that of Process 2 and 1, respectively. Also, the ABE productivity in Process 3 is 0.458 0.007 g/L/h, which is 28% and 39% higher than that of Process 2 and 1, respectively. The enhanced productivity achieved in Process 3 results from the fact that more glucose is consumed in a single batch because toxic butanol is continuously removed. Fermentation Process 3 as presented in this study is very competitive compared to others [6,7,18–20]. Table 1 summarizes the performance of Process 1, 2, and 3. 3.2. Separation efficiency of the integrated extraction-gas stripping process Performance of in situ gas stripping of butanol from oleyl alcohol during ABE fermentation is discussed. It can be seen in Fig. 4a that concentrations of acetone, ethanol, and butanol in the first 12 h condensate were 45 17, 12 1, and 82 14 g/L, respectively. During the stripping process the concentration of acetone, ethanol, and butanol reached peaks of 71 5, 19 1, and 113 5 g/L in the condensate of the third batch. This increase in solvent concentration simply reflects an increase in solvent concentrations in the oleyl alcohol layer during fermentation. A total ABE solvent concentration of 160–200 g/L in the condensate can be achieved with Process 3. It has been shown in previous studies that common ABE concentrations in the condensate spanned a range of between 20 and 80 g/L [21–24]. The findings of this study represent a great improvement in terms of the purity of the recovered solvents while the butanol flux was maintained. It is also notable that no oleyl alcohol, butyric acid, nor acetic acid is detected in the condensate, and this is an indication that short chain fatty acids are difficult to strip from either the aqueous phase [7,18,19,22] or the oleyl alcohol during ABE fermentation. It was interesting to see that there were 800–850 g/L of water in the condensate even though gas stripping of the solvents was performed directly in the organic phase layer. This can be attributed to out-gassing from bacterial growth where the released gas carries water from the fermentation broth to the condensate. Considering the final composition of the condensate, the average water removal rate was roughly four times higher than the average removal rate of ABE, see Fig. 4b. This suggests a way to further increase the purity of solvents using this process would be to increase the flux of solvents, where it has been shown previously that the solute removal rate (g-solute/h) is directly proportional to the stripping gas flow rate [25–27]. As shown in Fig. 4b, ABE removal rates of 0.14–0.17 g/h were achieved over 12–48 h with a stripping gas flow rate of 0.5 L/min. Clearly, the solvent removal rate could be improved by increasing the flow rate of the stripping gas. Furthermore, the solvent purity in the condensate can be improved by an increase in the amount of solvent in the condensate while the amount of water it contains remains steady. Two other techniques can be employed with Process 3 to further improve the degree of solvent purity in the condensate. It has been shown that a high ABE concentration of 195.9 g/L can be achieved by using the fractional condensation during recycled in situ gas stripping [6]. By using cooling water at 2 8C, the butanol, which has a relatively low vapor pressure, was condensed while water remained in the vapor [6]. Another promising technique that could be used with Process 3 is two-stage in situ gas stripping [7]. Xue et al. demonstrated that a twofold increase in ABE concentration in the condensate could be achieved [7]. The separation efficiency of each step of Process 3 was evaluated by using the separation factor. The separation factor for butanol extraction is defined as follows:
ab;ao ¼
yb;ao =ð1 yb;ao Þ xb;ao =ð1 xb;ao Þ
(1)
Fig. 4. Performance of butanol gas stripping from oleyl alcohol during ABE fermentation. (a) solvent concentrations in the condensate, (b) solvent removal rates, and (c) average butanol selectivity of extraction, gas stripping, and the combination of extraction and gas stripping. Gas stripping was initiated after 48 h of ABE fermentation. Error bars represent standard deviation.
where yb,ao is the mass fraction of butanol in the organic phase while xb,ao is the mass fraction of butanol in the aqueous phase. The separation factor for gas stripping of butanol is defined as follows:
ab;oc ¼
yb;oc =ð1 yb;oc Þ xb;oc =ð1 xb;oc Þ
(2)
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K.-M. Lu, S.-Y. Li / Journal of the Taiwan Institute of Chemical Engineers 45 (2014) 2106–2110
where yb,oc is the mass fraction of butanol in the condensate while xb,oc is the mass fraction of butanol in the organic phase. It can be seen in Fig. 4c that in the first 24 h stripping period, the butanol selectivity for extraction and gas stripping were 5.48 and 6.45, respectively. The overall selectivity of Process 3 was 35.31. In the second 24 h-stripping period, the butanol selectivity for extraction and gas stripping dropped to 3.96 and 5.06, respectively. The high separation factor of 35.31 suggests that the integrated in situ extraction-gas stripping process has a good separation factor that can be compared to pervaporative butanol separation where the separation factor typically lies in a range between 30 and 120 [12].
4. Conclusions The butanol productivity and yield of 0.28 0.01 g/L/h and 0.226 0.001 g-butanol/g-glucose were achieved with a novel integrated in situ extraction-gas stripping process. The ABE productivity and yield were 0.46 0.01 g/L/h and 0.374 0.002 g-ABE/gglucose. The glucose utilization was 97% where the initial glucose consumption was 121 2 g/L. Butanol and ABE concentrations of 93– 113 and 166–204 g/L in the condensate can be achieved. This study demonstrated that an enhanced ABE fermentation can be achieved with Process 3 while a high purity of solvents is simultaneously obtained. Acknowledgement This work was funded by National Science Council Taiwan, NSC102-2221-E-005-064. References [1] Branduardi P, de Ferra F, Longo V, Porro D. Microbial n-butanol production from Clostridia to non-Clostridial hosts. Eng Life Sci 2014;14:16–26. [2] Li S-Y, Srivastava R, Parnas RS. Study of in situ 1-butanol pervaporation from AB-E fermentation using a PDMS composite membrane: validity of solutiondiffusion model for pervaporative A-B-E fermentation. Biotechnol Progr 2011;27:111–20. [3] Chen B-Y, Chuang F-Y, Lin C-L, Chang J-S. Deciphering butanol inhibition to Clostridial species in acclimatized sludge for improving biobutanol production. Biochem Eng J 2012;69:100–5. [4] Hwang K-J, Ku C-Y. Model development for estimating microfiltration performance of bio-ethanol fermentation broth. J Taiwan Instit Chem Eng 2014;45:1233–40. [5] Vane LM. Separation technologies for the recovery and dehydration of alcohols from fermentation broths. Biofuels Bioprod Biorefining 2008;2:553–88. [6] Xue C, Zhao J, Lu C, Yang S-T, Bai F, Tang IC. High-titer n-butanol production by Clostridium acetobutylicum JB200 in fed-batch fermentation with intermittent gas stripping. Biotechnol Bioeng 2012;109:2746–56.
[7] Xue C, Zhao J, Liu F, Lu C, Yang S-T, Bai F-W. Two-stage in situ gas stripping for enhanced butanol fermentation and energy-saving product recovery. Bioresour Technol 2013;135:396–402. [8] Yen H-W, Wang Y-C. The enhancement of butanol production by in situ butanol removal using biodiesel extraction in the fermentation of ABE (acetone–butanol–ethanol). Bioresour Technol 2013;145:224–8. [9] Kraemer K, Harwardt A, Bronneberg R, Marquardt W. Separation of butanol from acetone–butanol–ethanol fermentation by a hybrid extraction–distillation process. Comput Chem Eng 2011;35:949–63. [10] Yeh C-Y, Lan JC-W. Direct recovery of polyhydroxyalkanoates synthase from recombinant Escherichia coli feedstock by using aqueous two-phase systems. J Taiwan Instit Chem Eng 2014;45:1119–25. [11] Yen H-W, Lin S-F, Yang IK. Use of poly(ether-block-amide) in pervaporation coupling with a fermentor to enhance butanol production in the cultivation of Clostridium acetobutylicum. J Biosci Bioeng 2012;113:372–7. [12] Li S-Y, Srivastava R, Parnas RS. Separation of 1-butanol by pervaporation using a novel tri-layer PDMS composite membrane. J Membr Sci 2010;363:287–94. [13] Roffler SR, Blanch HW, Wilke CR. In-situ recovery of butanol during fermentation. Bioprocess Eng 1987;2:181–90. [14] Ishizaki A, Michiwaki S, Crabbe E, Kobayashi G, Sonomoto K, Yoshino S. Extractive acetone–butanol–ethanol fermentation using methylated crude palm oil as extractant in batch culture of Clostridium saccharoperbutylacetonicum N1-4 (ATCC 13564). J Biosci Bioeng 1999;87:352–6. [15] Miller GL. Use of dinitrosalicylic acid reagent for determination of reducing sugar. Anal Chem 1959;31:426–8. [16] Chen S-K, Chin W-C, Tsuge K, Huang C-C, Li S-Y. Fermentation approach for enhancing 1-butanol production using engineered butanologenic Escherichia coli. Bioresour Technol 2013;145:204–9. [17] Saien J, Daliri S. Improving performance of liquid–liquid extraction with temperature for mass transfer resistance in both phases. J Taiwan Instit Chem Eng 2014;45:808–14. [18] Lu C, Zhao J, Yang S-T, Wei D. Fed-batch fermentation for n-butanol production from cassava bagasse hydrolysate in a fibrous bed bioreactor with continuous gas stripping. Bioresour Technol 2012;104:380–7. [19] Ezeji TC, Qureshi N, Blaschek HP. Acetone butanol ethanol (ABE) production from concentrated substrate: reduction in substrate inhibition by fed-batch technique and product inhibition by gas stripping. Appl Microbiol Biotechnol 2004;63:653–8. [20] Yen H-W, Li R-J, Ma T-W. The development process for a continuous acetone– butanol–ethanol (ABE) fermentation by immobilized Clostridium acetobutylicum. J Taiwan Instit Chem Eng 2011;42:902–7. [21] Ennis BM, Marshall CT, Maddox IS, Paterson AHJ. Continuous product recovery by in-situ gas stripping/condensation during solvent production from whey permeate using Clostridium acetobutylicum. Biotechnol Lett 1986;8:725–30. [22] Ezeji TC, Qureshi N, Blaschek HP. Production of acetone, butanol and ethanol by Clostridium beijerinckii BA101 and in situ recovery by gas stripping. World J Microbiol Biotechnol 2003;19:595–603. [23] Qureshi N, Saha BC, Hector RE, Hughes SR, Cotta MA. Butanol production from wheat straw by simultaneous saccharification and fermentation using Clostridium beijerinckii: part I—Batch fermentation. Biomass Bioenergy 2008;32: 168–175. [24] Mariano AP, Qureshi N, Filho RM, Ezeji TC. Bioproduction of butanol in bioreactors: new insights from simultaneous in situ butanol recovery to eliminate product toxicity. Biotechnol Bioeng 2011;108:1757–65. [25] Truong KN, Blackburn JW. The stripping of organic chemicals in biological treatment processes. Environ Prog 1984;3:143–52. [26] Ezeji T, Karcher P, Qureshi N, Blaschek H. Improving performance of a gas stripping-based recovery system to remove butanol from Clostridium beijerinckii fermentation. Bioprocess Biosyst Eng 2005;27:207–14. [27] Liao Y-C, Lu K-M, Li S-Y. Process parameters for operating 1-butanol gas stripping in a fermentor. J Biosci Bioeng 2014. http://dx.doi.org/10.1016/ j.jbiosc.2014.04.020 [in press].
Contents lists available at ScienceDirect
Journal of the Taiwan Institute of Chemical Engineers journal homepage: www.elsevier.com/locate/jtice
An integrated in situ extraction-gas stripping process for Acetone–Butanol–Ethanol (ABE) fermentation Kuan-Ming Lu, Si-Yu Li * Department of Chemical Engineering, National Chung Hsing University, Taichung 402, Taiwan
A R T I C L E I N F O
A B S T R A C T
Article history: Received 17 March 2014 Received in revised form 24 June 2014 Accepted 29 June 2014 Available online 31 July 2014
A novel integrated in situ extraction-gas stripping process (Process 3 in this study) is proposed for running batch Acetone–Butanol–Ethanol (ABE) fermentation. The process involves two butanol separation processes in which butanol is first extracted by oleyl alcohol during ABE fermentation and gas stripping is carried out on butanol in the oleyl alcohol phase at the same time. The butanol productivity and yield of Process 3 is 0.28 0.01 g/L/h and 0.226 0.001 g-butanol/g-glucose. The ABE productivity and yield are 0.46 0.01 g/L/h and 0.374 0.002 g-ABE/g-glucose. Glucose utilization was 97% and initial glucose consumption was 121 2 g/L. Butanol and ABE concentrations of 93–113 and 166–204 g/L in the condensate can be achieved. This study demonstrates that Process 3 as described here enhances ABE fermentation and also results in the production of high purity solvents. ß 2014 Taiwan Institute of Chemical Engineers. Published by Elsevier B.V. All rights reserved.
Keywords: Clostridium acetobutylicum Acetone–Butanol–Ethanol (ABE) fermentation Extraction Gas stripping Oleyl alcohol
1. Introduction 1-Butanol has been proposed as an alternative fuel that is both renewable and sustainable and can be produced by traditional Acetone–Butanol–Ethanol fermentation using Clostridium acetobutylicum or Clostridium beijerinckii [1]. One obstacle for ABE fermentation is the toxicity of 1-butanol which results in low productivity and high recovery costs for conventional distillation [2–4]. One promising way to tackle butanol toxicity is by the use of in situ product recovery [5]. Many bio-compatible separation processes, such as gas stripping [6,7], extraction [8–10], and pervaporation [11,12] have been proposed and investigated for in situ butanol recovery during ABE fermentation. Gas stripping and extraction are two well known methods that are easy to maintain and scale-up. However, both gas stripping and extraction have low selectivity for butanol during the ABE fermentation process. Also, the low selectivity of the extraction further leads to the need for large volumes of solvent in the batch reactor to keep the butanol concentration in the aqueous phase below the threshold of around 10 g/L. In practice, the best ABE fermentation performance was achieved with a solvent to fermentation broth volume ratio of 1.75 [13]. However, this dramatically decreases the volume available for ABE fermentation in the batch reactor.
In this study, a combination of extraction and gas stripping as an integrated process is proposed for in situ butanol recovery in ABE fermentation as shown in Fig. 1. The non-volatile solvent oleyl alcohol acts as the extraction solvent and nitrogen is used for gas stripping. Oleyl alcohol is biocompatible and a feasible extraction agent for in situ butanol recovery under these conditions [13,14]. The performance of integrated in situ extraction-gas stripping during ABE fermentation is investigated and discussed. 2. Materials and methods 2.1. Strains, culture media, and growth conditions The bacterial strain used in this study was C. acetobutylicum ATCC 824. The culture was in spore form and stored at room temperature. Each liter of the fermentation medium used in this study contained: 38 g Reinforced Clostridial Medium (RCM), 0.11 g FeSO47H2O, 0.6 g MgSO47H2O, 0.008 g CaCl2, and 125 g glucose. For batch ABE fermentation, 32 mL of C. acetobutylicum spore stock solution was added aseptically to 300 mL of fresh medium without using a heat-induced germination process. Batch ABE fermentation was carried out at 37 8C with stirring at 150 rpm. 2.2. Integrated in situ extraction-gas stripping ABE fermentation
* Corresponding author. Tel.: +886 4 2284 0510x509. E-mail address: [email protected] (S.-Y. Li).
As shown in Fig. 1, the equipment used in the integrated in situ extraction-gas stripping process includes a 500-mL serum bottle, two
http://dx.doi.org/10.1016/j.jtice.2014.06.023 1876-1070/ß 2014 Taiwan Institute of Chemical Engineers. Published by Elsevier B.V. All rights reserved.
K.-M. Lu, S.-Y. Li / Journal of the Taiwan Institute of Chemical Engineers 45 (2014) 2106–2110
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Fig. 1. Schematic of integrated in situ extraction-gas stripping process for ABE fermentation.
cold traps (1 and 2) in parallel for the condensation of stripped solvents, and a cylinder of nitrogen gas. A 500-ml serum bottle was used as the reactor throughout the study. The serum bottle contained 300 mL of fresh medium and 100 mL of oleyl alcohol (80–85%, Alfa Aesar1). Cold trap 3 was used for security to ensure condensation of all stripped solvents. All cold traps were immersed in liquid nitrogen at 196 8C. In all the experiments the nitrogen gas for gas stripping was sterilized using a 0.2 mm Millipore membrane filter and delivered through a single 18-G syringe needle at a flow rate of 0.5 L/min. 0.2 mm filters were used to filter-sterilize the nitrogen gas stream throughout the experiment. Gas stripping was performed on the oleyl alcohol phase and was initiated after a fermentation time of 48 h. Condensates from the cold trap were analyzed by gas chromatography (GC) at 12 hourly intervals as described below. 2.3. Analytical methods The cell density was measured at 600 nm using a UV–vis spectrophotometer (GENESYS 10S, Thermo Scientific, USA). The concentration of residual sugars was determined by the DNS method [15]. The concentrations of acetone, butanol, ethanol, acetate, and butyrate in the fermentation culture solution were determined by GC (Hewlett Packard HP 5890 Series II) using a flame ionization detector and a capillary column DP-FFAP (30 m 0.32 mm 0.25 mm) as described earlier [16]. 3. Results and discussion 3.1. Performance of ABE fermentation using an in situ extraction-gas stripping separation process ABE fermentation was carried out using the in situ extractiongas stripping separation process described here, while batch ABE
fermentation as well as batch ABE fermentation with in situ extraction were used as controls. It can be seen in Fig. 2a that without the heat-induced germination treatment, a 36-h lag phase was observed for batch ABE fermentation. Despite this, the clostridial culture still reached a peak OD600 of 9.6 1.5 after 72 h. The glucose concentration in Process 1 remained steady for the first 36 h and then decreased strongly up to 72 h, see process 1 in Fig. 2b. The total glucose consumption in process 1 was 57 8 g/L in 72 h. The concentration of butanol in Process 1 (see Fig. 2c) started accumulating after 48 h when bacterial growth entered the exponential phase and the final butanol titer reached a concentration of 13.0 0.7 g/L. Note that the final acetone and ethanol titers were 9.0 0.4 and 2.1 0.3 g/L, respectively (data not shown). This data suggests that the direct use of spores is feasible and can be used for initiating ABE fermentation. Both bacterial growth and glucose consumption can be enhanced by incorporating in situ butanol extraction with oleyl alcohol. It can be seen in Fig. 2a that the peak OD600 in Process 2 reached 12.2 0.7 by 72 h and total glucose consumption reached 96 9 g/L in 96 h. Process 2 showed significant improvements in ABE fermentation over Process 1 and this can be attributed to the efficient in situ extraction of toxic butanol from the fermentation broth into the oleyl alcohol phase [8,13,14]. At 96 h the butanol concentrations in the aqueous and organic phases were 9 1 and 40 5 g/L, respectively. On the other hand, while butanol was selectively extracted into the oleyl alcohol, this was not the case with acetone and ethanol. It was found that the concentration of acetone and ethanol in the organic phase was lower than in the aqueous phase where their partition ratios were 0.4 and 0.2, respectively (data not shown). These are consistent with previous data [13,14]. For example, Ishizaki et al. reported that the partition ratios of acetone and ethanol were 0.3 and 0.2, respectively [14]. Although 60% of the butanol produced was extracted from the aqueous phase to the organic phase,
Table 1 Characteristics of ABE fermentation for Process 1, 2, and 3.a Characteristics
Process 1
Initial glucose (g/L) Final glucose (g/L) Glucose consumption (g/L) Glucose utilization (%) Glucose consumption rate (g/L/h)b Max. optical density (time) Butanol production (g/L) Total ABE production (g/L) Butanol productivity (g/L/h)b Total ABE productivity (g/L/h)b Butanol yield (g/g) Total ABE yield (g/g)
125 1 65 6 57 8 48 5 0.79 0.11 9.61 (72 h) 12.5 0.3 23.1 0.7 0.18 0.01 0.33 0.02 0.21 0.02 0.39 0.03
Process 2 115 2 19 10 96 9 83 9 1.00 0.09 12.17 (72 h) 22 3 34 4 0.23 0.03 0.36 0.04 0.23 0.02 0.36 0.03
Process 3 121 2 3.6 0.2 117 2 97.0 0.2 1.22 0.02 14.57 (96 h) 26.5 0.5 43.9 0.6 0.28 0.01 0.46 0.01 0.226 0.001 0.374 0.002
a Process 1: Batch ABE fermentation, Process 2: Batch ABE fermentation with in situ butanol extraction using oleyl alcohol, Process 3: Integrated in situ extraction-gas stripping ABE fermentation. Error bars represent standard deviation. b Fermentation time frames for calculating the glucose consumption rate and solvent productivities are 72, 96, and 96 h for Process 1, 2, and 3, respectively.
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Fig. 2. (a) Bacterial growth, (b) glucose consumption, (c) butanol production of three fermentation processes, and (d) partition ratios of butanol in the organic phase to butanol in the aqueous phase. Process 1: Batch ABE fermentation, Process 2: Batch ABE fermentation with in situ butanol extraction using oleyl alcohol, Process 3: Integrated in situ extraction-gas stripping ABE fermentation. Error bars represent standard deviations.
it was found that 9 1 g/L of butanol remained and had a negative effect on clostridial growth. The cell density in Process 2 decreased and there was a 19 10 g/L of glucose residue. Although a lower volume ratio of fermentation broth to oleyl alcohol could be achieved, the ABE fermentation capacity of a single fermentor would be less. The dilemma encountered in Process 2 can be resolved by using integrated in situ extraction-gas stripping, Process 3 in this study. While it can be seen from Fig. 2a that all three processes had similar specific growth rates during the early exponential phase, bacterial growth in Process 3 had an extended growth profile and reached an OD600 value of 14.6 0.7 at 96 h. An examination of Fig. 2b shows that bacterial culture consumed 97% of the available glucose in 96 h with a total glucose consumption of 117 2 g/L. This suggests that there was no obvious inhibition of bacterial growth and glucose consumption over the total fermentation time of 96 h. This finding is supported by the data in Fig. 2c which shows that the highest butanol concentration in the aqueous solution, during Process 3 fermentation, was only 6.4 0.6 g/L at 96 h. Also, the butanol concentrations in the organic phase reached 16.6 2.3 and 20.9 1.3 g/L at 72 and 96 h, respectively. These high butanol concentrations served as a strong driving force for the mass transfer of butanol from liquid to the gas bubbles during gas stripping. Note that the partition of butanol between the organic and aqueous phases dropped from 4.6–4.9 (Process 2) to 3.2–3.5 (Process 3) after the initiation of gas stripping at 48 h, see Fig. 2d. This suggested that the organic phase in Process 3 was not saturated with butanol during fermentation. Therefore, a mass transfer resistance for butanol extraction in Process 3 was noticeable [17]. Nevertheless, ABE fermentation using Process 3 is still significantly better than in Process 1 and 2. Table 1 shows that the glucose utilization in Process 3 is 102% and 17% higher than that in Process 1 and Process 2, respectively. It can be seen in Fig. 3 that final titers of butanol,
acetone, and ethanol in Process 3 are higher than that in Processes 1 and 2, where the final titers of butanol and ABE from Process 3 at 96 h are 26.5 0.5 and 43.9 0.6 g/L, respectively. Note the calculation of solvent titers for Process 3 is based on the total amount of solvents in the fermentation broth, oleyl alcohol, and condensates with respect to the volume of fermentation broth. While the three processes have similar butanol and ABE yields, the butanol and ABE productivity of Process 3 is significantly better. The butanol productivity in process 3
Fig. 3. Comparisons of ABE fermentation performance using processes 1, 2, and 3. Process 1: Batch ABE fermentation, Process 2: Batch ABE fermentation with in situ butanol extraction using oleyl alcohol, Process 3: Integrated in situ extraction-gas stripping ABE fermentation. Note: the total solvent concentration in Process 3 is the sum of that in the aqueous, organic, and condensate phases with respect to the volume of fermentation broth. Error bars represent standard deviation.
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is 0.276 0.005 g/L/h, indicating a 22% and 56% higher than that of Process 2 and 1, respectively. Also, the ABE productivity in Process 3 is 0.458 0.007 g/L/h, which is 28% and 39% higher than that of Process 2 and 1, respectively. The enhanced productivity achieved in Process 3 results from the fact that more glucose is consumed in a single batch because toxic butanol is continuously removed. Fermentation Process 3 as presented in this study is very competitive compared to others [6,7,18–20]. Table 1 summarizes the performance of Process 1, 2, and 3. 3.2. Separation efficiency of the integrated extraction-gas stripping process Performance of in situ gas stripping of butanol from oleyl alcohol during ABE fermentation is discussed. It can be seen in Fig. 4a that concentrations of acetone, ethanol, and butanol in the first 12 h condensate were 45 17, 12 1, and 82 14 g/L, respectively. During the stripping process the concentration of acetone, ethanol, and butanol reached peaks of 71 5, 19 1, and 113 5 g/L in the condensate of the third batch. This increase in solvent concentration simply reflects an increase in solvent concentrations in the oleyl alcohol layer during fermentation. A total ABE solvent concentration of 160–200 g/L in the condensate can be achieved with Process 3. It has been shown in previous studies that common ABE concentrations in the condensate spanned a range of between 20 and 80 g/L [21–24]. The findings of this study represent a great improvement in terms of the purity of the recovered solvents while the butanol flux was maintained. It is also notable that no oleyl alcohol, butyric acid, nor acetic acid is detected in the condensate, and this is an indication that short chain fatty acids are difficult to strip from either the aqueous phase [7,18,19,22] or the oleyl alcohol during ABE fermentation. It was interesting to see that there were 800–850 g/L of water in the condensate even though gas stripping of the solvents was performed directly in the organic phase layer. This can be attributed to out-gassing from bacterial growth where the released gas carries water from the fermentation broth to the condensate. Considering the final composition of the condensate, the average water removal rate was roughly four times higher than the average removal rate of ABE, see Fig. 4b. This suggests a way to further increase the purity of solvents using this process would be to increase the flux of solvents, where it has been shown previously that the solute removal rate (g-solute/h) is directly proportional to the stripping gas flow rate [25–27]. As shown in Fig. 4b, ABE removal rates of 0.14–0.17 g/h were achieved over 12–48 h with a stripping gas flow rate of 0.5 L/min. Clearly, the solvent removal rate could be improved by increasing the flow rate of the stripping gas. Furthermore, the solvent purity in the condensate can be improved by an increase in the amount of solvent in the condensate while the amount of water it contains remains steady. Two other techniques can be employed with Process 3 to further improve the degree of solvent purity in the condensate. It has been shown that a high ABE concentration of 195.9 g/L can be achieved by using the fractional condensation during recycled in situ gas stripping [6]. By using cooling water at 2 8C, the butanol, which has a relatively low vapor pressure, was condensed while water remained in the vapor [6]. Another promising technique that could be used with Process 3 is two-stage in situ gas stripping [7]. Xue et al. demonstrated that a twofold increase in ABE concentration in the condensate could be achieved [7]. The separation efficiency of each step of Process 3 was evaluated by using the separation factor. The separation factor for butanol extraction is defined as follows:
ab;ao ¼
yb;ao =ð1 yb;ao Þ xb;ao =ð1 xb;ao Þ
(1)
Fig. 4. Performance of butanol gas stripping from oleyl alcohol during ABE fermentation. (a) solvent concentrations in the condensate, (b) solvent removal rates, and (c) average butanol selectivity of extraction, gas stripping, and the combination of extraction and gas stripping. Gas stripping was initiated after 48 h of ABE fermentation. Error bars represent standard deviation.
where yb,ao is the mass fraction of butanol in the organic phase while xb,ao is the mass fraction of butanol in the aqueous phase. The separation factor for gas stripping of butanol is defined as follows:
ab;oc ¼
yb;oc =ð1 yb;oc Þ xb;oc =ð1 xb;oc Þ
(2)
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where yb,oc is the mass fraction of butanol in the condensate while xb,oc is the mass fraction of butanol in the organic phase. It can be seen in Fig. 4c that in the first 24 h stripping period, the butanol selectivity for extraction and gas stripping were 5.48 and 6.45, respectively. The overall selectivity of Process 3 was 35.31. In the second 24 h-stripping period, the butanol selectivity for extraction and gas stripping dropped to 3.96 and 5.06, respectively. The high separation factor of 35.31 suggests that the integrated in situ extraction-gas stripping process has a good separation factor that can be compared to pervaporative butanol separation where the separation factor typically lies in a range between 30 and 120 [12].
4. Conclusions The butanol productivity and yield of 0.28 0.01 g/L/h and 0.226 0.001 g-butanol/g-glucose were achieved with a novel integrated in situ extraction-gas stripping process. The ABE productivity and yield were 0.46 0.01 g/L/h and 0.374 0.002 g-ABE/gglucose. The glucose utilization was 97% where the initial glucose consumption was 121 2 g/L. Butanol and ABE concentrations of 93– 113 and 166–204 g/L in the condensate can be achieved. This study demonstrated that an enhanced ABE fermentation can be achieved with Process 3 while a high purity of solvents is simultaneously obtained. Acknowledgement This work was funded by National Science Council Taiwan, NSC102-2221-E-005-064. References [1] Branduardi P, de Ferra F, Longo V, Porro D. Microbial n-butanol production from Clostridia to non-Clostridial hosts. Eng Life Sci 2014;14:16–26. [2] Li S-Y, Srivastava R, Parnas RS. Study of in situ 1-butanol pervaporation from AB-E fermentation using a PDMS composite membrane: validity of solutiondiffusion model for pervaporative A-B-E fermentation. Biotechnol Progr 2011;27:111–20. [3] Chen B-Y, Chuang F-Y, Lin C-L, Chang J-S. Deciphering butanol inhibition to Clostridial species in acclimatized sludge for improving biobutanol production. Biochem Eng J 2012;69:100–5. [4] Hwang K-J, Ku C-Y. Model development for estimating microfiltration performance of bio-ethanol fermentation broth. J Taiwan Instit Chem Eng 2014;45:1233–40. [5] Vane LM. Separation technologies for the recovery and dehydration of alcohols from fermentation broths. Biofuels Bioprod Biorefining 2008;2:553–88. [6] Xue C, Zhao J, Lu C, Yang S-T, Bai F, Tang IC. High-titer n-butanol production by Clostridium acetobutylicum JB200 in fed-batch fermentation with intermittent gas stripping. Biotechnol Bioeng 2012;109:2746–56.
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