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The enhancement of butanol production by in situ butanol removal using biodiesel extraction in the fermentation of ABE (acetone–butanol–ethanol)
Bioresource Technology xxx (2012) xxx–xxx
Contents lists available at SciVerse ScienceDirect
Bioresource Technology journal homepage: www.elsevier.com/locate/biortech
The enhancement of butanol production by in situ butanol removal using biodiesel extraction in the fermentation of ABE (acetone–butanol–ethanol) Hong-Wei Yen ⇑, Yi-Cheng Wang Department of Chemical and Materials Engineering, Tunghai University, Taichung, Taiwan, ROC
h i g h l i g h t s " Adding biodiesel as an extractant for butanol removal is an attractive idea to relieve butanol toxicity. " Butanol dissolved in biodiesel could act as an additive to enhance energy content. " No significant toxicity of biodiesel on cell growth was observed. " Adding biodiesel could greatly enhance butanol productivity.
a r t i c l e
i n f o
Article history: Available online xxxx Keywords: ABE Biodiesel Extraction Separation In situ removal
a b s t r a c t High butanol accumulation is due to feedback inhibition which leads to the low butanol productivity observed in acetone–butanol–ethanol (ABE) fermentation. The aim of this study is to use biodiesel as an extractant for the in situ removal of butanol from the broth. The results indicate that adding biodiesel as an extractant at the beginning of fermentation significantly enhances butanol production. No significant toxicity of biodiesel on the growth of Clostridium acetobutylicum is observed. In the fed-batch operation with glucose feeding, the maximum total butanol obtained is 31.44 g/L, as compared to the control batch (without the addition of biodiesel) at 9.85 g/L. Moreover, the productivity obtained is 0.295 g/L h in the fed-batch, which is higher than that of 0.185 g/L h for the control batch. The in situ butanol removal by the addition of biodiesel has great potential for commercial ABE production. Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction The rapid decline of petroleum reserves, the surge in population and the growing concern regarding the environmental impacts resulting from the over-consumption of petroleum-based products have initiated interest in the development of renewable biofuels. Among all biofuel alternatives, butanol, obtained through the acetone–butanol–ethanol (ABE) process through a biological approach, is considered one of the replacement fuels with the most potential (García et al., 2011; Jones and Woods, 1986). The production of butanol by an isolated/pure microorganism was one of the first large-scale industrial microbial processes for chemical production (Mariano et al., 2011; Volesky et al., 1981). ABE fermentation is subject to a strong end-product inhibition (mainly coming from butanol) which has adversely affected the economics of commercial production. The main limitation of ABE fermentation is the toxic effect of butanol on microorganisms ⇑ Corresponding author. Address: Department of Chemical and Materials Engineering, Tunghai University, 181, Taichung Harbor Rd., Taichung 407, Taiwan, ROC. Tel.: +886 4 23590262/209; fax: +886 4 23590009. E-mail address: [email protected] (H.-W. Yen).
which leads to the low solvent productivity (Zheng et al., 2009). Therefore, it has been necessary to search for high butanol-tolerant strains of microorganisms or to use in situ toxicity removal technologies (such as extraction, gas stripping, adsorption and pervaporation) to overcome the toxicity problem and increase the final concentration of butanol (Badr and Hamdy, 1992; Li et al., 2011; Mariano et al., 2012). Numerous process designs have been developed to incorporate in situ or ex situ auxiliary separation techniques into a fermentation system to achieve end-product removal, most of which rely on traditional mass transfer phenomena (e.g., absorption or adsorption) (Groot and Luyben, 1986; Lin et al., 2012; Nielsen and Prather, 2009; Yen et al., 2012; Yen and Li, 2011). In situ solvent extraction fermentation has been proposed as one approach for minimizing butanol inhibition and increasing product titer, such as using 1-dodecancol in a membrane-assisted extractive system (Tanaka et al., 2012). However, the market value of the extractant and the subsequent cost of extractant recycling have prevented their being applied on a large scale. An ideal in situ extractant would be one that has a direct end-use as a fuel, which would then eliminate the need for expensive butanol recovery and extractant recycling procedures.
0960-8524/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.biortech.2012.11.039
Please cite this article in press as: 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. (2012), http://dx.doi.org/10.1016/j.biortech.2012.11.039
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H.-W. Yen, Y.-C. Wang / Bioresource Technology xxx (2012) xxx–xxx
The extractive acetone–butanol–ethanol (ABE) fermentation of Clostridium acetobutylicum has been evaluated using biodiesel as the in situ extractant. The fuel properties of the biodiesel–ABE mixture are comparable to that of No. 2 diesel, but with higher cetane numbers; therefore, it could serve as an efficient No. 2 diesel substitute (Crabbe et al., 2001). Blending butanol with biodiesel effected an improvement in the flow properties of butanol-enriched biodiesel. Ethanol is generally used to blend in diesohol; however, butanol is an alcohol which has higher solubility in diesel than ethanol and can improve the fuel properties of the blends (Chotwichien et al., 2009). To this end, microbial-produced butanol is the best choice for enriching and improving the fuel properties of biodiesel. The biodiesel preferentially extracted butanol minimized product inhibition and increased both the production of butanol (from 11.6 to 16.5 g/L) and the total solvents (from 20.0 to 29.9 g/L) by 42% and 50%, respectively. The fuel properties of the ABE-enriched biodiesel obtained by means of extractive fermentation were analyzed. The key quality indicators of diesel fuel, such as the cetane number (which increased from 48 to 54) and the cold filter plugging point (which decreased from 5.8 to 0.2 °C), were significantly improved in ABE-enriched biodiesel. Thus, the application of biodiesel as the extractant for ABE fermentation would increase ABE production, bypass the energy intensive butanol recovery process and result in an ABE-enriched biodiesel with improved fuel properties (Li et al., 2010). The aim of this study was to investigate the effects of using biodiesel as an extractant for butanol production in ABE fermentation using C. acetobutylicum. The influence of the ratio when adding biodiesel to broth and the point in time when biodiesel should be added in butanol production were evaluated, as well as the fed-batch operation by glucose addition compared to the batch operation. 2. Methods 2.1. Strain and medium The bacterial strain used in this study was C. acetobutylicum BCRC 10639 (the same as ATCC 824), which was purchased from the Bioresource Collection and Research Center (BCRC), Taiwan. The cells were cultured in glass tubes (diameter, 3 cm; length, 15 cm) containing 25 mL of fermentation medium and purged with 50% CO2 and 50% N2, to ensure 100% anaerobic conditions. After being removed from the freezer, the strain was heat-shocked at 70 °C for 2 min, and then inoculated into a glass tube (3 cm OD 15 cm high) containing 25 mL of reinforced clostridial medium (RCM), used as the seed medium, followed by an incubation period of 30 h at 37 °C. The fermentation medium was then inoculated with seed culture at a ratio of 10% (v/v). The fermentation medium contained the following components (per liter): 80 g glucose; 0.18 g Na2SO4; 0.175 g K2HPO4; 0.01 g biotin; 0.01 g p-aminobenzoic acid; 1.0 g tryptone; 5.0 g yeast extract; and 1 mL mineral salt solution. The mineral salt solution contained the following components in l liter of distilled water: 0.24 g of NaMoO42H2O; 0.24 g of CoCl26H2O; 1.5 g of CaCl22H2O; 16.203 g of FeCl3; 0.1598 g of CuSO4; 0.5164 g of ZnSO47H2O; 1.7 g of MnSO4H2O; 24.574 g of MgSO47H2O; and 28 mL of H2SO4 (6 M). The pH was adjusted to 4.8 before sterilization; the pH was not controlled during the entire fermentation process involved in this study. 2.2. Batch operation with addition of biodiesel The batch operation was performed in a glass tube, as described previously, with a 25 mL working volume. The ratios of biodiesel
added to the broth of 1:1 and 0.5:1 (v/v) were determined, respectively. Biodiesel is known to contain several solvents and chemicals which are potentially toxic to microorganisms; therefore, in addition to the examination of the influence of the ratio of added biodiesel, the effect of the time at which additions were made (12, 24 and 36 h after the inoculation) on the growth of C. acetobutylicum was also investigated. All periods of cultivation lasted for 72 h under anaerobic conditions in this study. 2.3. Fed-batch operation with glucose feeding The batch operation process was the same as described in the previous section. Biodiesel was added to the glass tube at the beginning at a ratio of 1:1 (25 mL biodiesel added to 25 mL broth). After 48 h of cultivation, the glucose was almost completely consumed in the fermentation broth. Therefore, 1 mL of concentrated glucose solution (500 g/L) was added to the broth to raise the glucose concentration to 20 g/L. After glucose feeding, cultivation lasted for 48 h to allow examination of the performance of in situ butanol removal by biodiesel extraction in the fed-batch operation. The purpose of feeding glucose here was to evaluate the performance of biodiesel addition on the enhancement of butanol production in the fed-batch operation mode. 2.4. Analytical methods Using optical density measurements, biomass concentration was determined at 620 nm by means of a Thermo Spectronic GENESYS™ 10 Spectrophotometer and by the gravimetric method (dry cell weight). Concentrations of butanol and other ingredients were measured by means of a gas chromatographer equipped with a flame-ionization detector. The samples were analyzed in a 25 m 0.22 mm stainless steel column, with a film thickness of 0.25 lm (SEG BP20). Nitrogen was used as the carrier gas. The oven temperature was held at 120 °C for 3 min and then increased by 16 °C/min to 200 °C with a 5-min final hold. The temperatures for the detector and injection port were 250 °C and 200 °C, respectively. Glucose concentration was determined using a YSI 1500 Analyzer (Yellow Springs, OH, USA) according to the enzymatic reaction of glucose oxidase. Total butanol concentration is defined as the sum of butanol in the water phase plus that in the oil phase divided by the broth volume. 3. Results and discussion 3.1. Effects of the amount of biodiesel added and the time of addition on ABE fermentation The toxicity of biodiesel to microorganisms can potentially inhibit the growth of cells during ABE fermentation. Therefore, the effects of the time of the biodiesel addition and the amounts added were first examined. A total of four different times (at 0, 12, 24 and 36 h) for biodiesel additions were set at the two different ratios of 1:1 and 1:0.5 (defined as the volume ratio of broth to biodiesel), respectively. The results indicated that adding biodiesel to extract butanol from the broth (water phase) to the biodiesel layer (oil phase) significantly enhanced the growth of C. acetobutylicum. The biomass obtained in the batches with added biodiesel was almost twice that of the control batch. The improvement in cell growth possibly came from the release of butanol toxicity due to the in situ removal of butanol by the biodiesel. No significant difference in biomass concentration was observed in the batches where biodiesel was added at different times. Even when biodiesel was added at the beginning of fermentation, there were no negative impacts on cell growth when compared to other batches.
Please cite this article in press as: 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. (2012), http://dx.doi.org/10.1016/j.biortech.2012.11.039
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H.-W. Yen, Y.-C. Wang / Bioresource Technology xxx (2012) xxx–xxx Table 1 Butanol concentrations of water and oil phase in the batches of biodiesel adding at the ratio of 1:1 and 1:0.5 for each different adding time point. Adding time (h)
Biodiesel adding ratio 1:1
0 12 24 36
1:0.5
Water phase (g butanol/L)
Oil phase (g butanol/L)
Water phase (g butanol/L)
Oil phase (g butanol/L)
6.95 5.99 6.06 6.35
7.81 8.09 9.46 8.93
8.88 6.98 5.90 7.06
10.19 10.47 10.90 10.85
The results revealed that the potential toxicity of biodiesel to microorganisms was outweighed by the benefits derived from the removal of butanol by the addition of biodiesel. In addition to the enhancement of cell growth, the increase of butanol production by biodiesel addition at ratios of 1:1 and 1:0.5 was also observed. The average totals of butanol (butanol in the broth plus butanol in the biodiesel divided by the fermentation volume) achieved were 14.8 ± 1.4 and 12.7 ± 1.7 g/L in the batches with biodiesel added at the ratios of 1:1 and 1:0.5, respectively, which was significantly higher than the butanol value of 8.1 ± 1.5 g/L in the control batch. As mentioned previously, biodiesel is not toxic in the growth of cells. Also, the inhibition of biodiesel on the required enzymes of ABE fermentation was not an issue in this study since no repression of butanol production was observed in the batches with biodiesel addition. In the paper published by Li et al. (2010), the suggested optimal adding time point was 48 h after the inoculation. Nevertheless, ABE production was not significantly different according to the data shown in the paper. Therefore, based on the consideration of industrial production, adding biodiesel at the beginning of fermentation might be more easily performed than adding it later in the process. The addition of hydrophobic biodiesel in the fermentation tubes efficiently divides the broth with air (the upper layer is the biodiesel), which might be helpful in achieving a completely anaerobic environment. It is well known that C. acetobutylicum is a strictly anaerobic microorganism which can only tolerate a slight amount of oxygen (Kashket and Cao, 1995). Therefore, adding biodiesel as an extractant can not only relieve butanol toxicity but also create a strictly anaerobic environment, which might be beneficial to the enhancement of cell growth. The participation coefficient of butyrate is known to be higher than 1 (Li et al., 2010). Therefore, it was a concern that most of the butyrate produced in the water phase probably diffused into the oil phase, leading to decreased butanol production. However, as shown in the discussion of this study and Li et al., even adding biodiesel at the beginning of fermentation did not reduce the final butanol production. The results suggested that the rate of butyrate going into the oil phase might be less than that assimilated by cells for butanol production. The butanol concentrations in both phases of oil and water, as shown in Table 1, indicated a higher butanol concentration in the oil phase than in the water phase. The average butanol participation coefficient for the oil phase over that of the water phase was 1.43 ± 0.24 according to the data, slightly higher than the 1.24 obtained by Li et al. (2010). It was obvious that adding biodiesel in the fermentation broth efficiently enhanced cell growth and more butanol was produced. Since biodiesel does not inhibit cell growth, adding biodiesel at the beginning at the ratio of 1:1 was selected for building up the profile of butanol concentration in the batch with biodiesel added as compared to the control batch (without biodiesel added).
Fig. 1. Time course of pH and biomass in the batch operation with biodiesel addition at a ratio of 1:1 as compared to the control (without the addition of biodiesel).
indicate that adding biodiesel as the extractant enhanced butanol production compared to the production of the control batch. Since some acids might be transmitted to the oil phase from the water phase, the pH value in the batch with added biodiesel was slightly higher than that of the control batch (Fig. 1). Keeping a low butanol concentration in the broth might account for the increase in total butanol production, which led to more glucose being consumed compared to the control batch. As shown in Fig. 2, the glucose consumption rate in the batch with added biodiesel was higher than that of the control batch after 24 h of cultivation, where butanol
3.2. Time course of biodiesel addition vs. control batch The time course results for biodiesel addition at the beginning and at a ratio of 1:1 are shown in Figs. 1 and 2. The results clearly
Fig. 2. Time course of total butanol produced and residual glucose in the batch with biodiesel added at a ratio of 1:1 as compared to the control batch (without the addition of biodiesel).
Please cite this article in press as: 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. (2012), http://dx.doi.org/10.1016/j.biortech.2012.11.039
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Fig. 3. Biomass, total butanol produced and residual glucose in the batch and in the fed-batch operations with biodiesel added at a ratio of 1:1.
began to accumulate fast, leading to the feedback inhibition. Glucose was completely consumed 48 h after inoculation, after which no further butanol production was observed. The butanol concentration in the broth in our study at this time was about 6–7 g/L, which was lower than the suggested butanol inhibition level of 12–15 g/L (Ezeji et al., 2007). Therefore, the stoppage in butanol production in the batch with added biodiesel might have resulted from the shortage of carbon source-glucose. To further explore the potential maximum butanol production in the batch with added biodiesel, a fed-batch operation with a single glucose feeding was performed. 3.3. Fed-batch vs. ABE fermentation batch in the biodiesel extraction system Since the carbon source was almost totally consumed after 48 h in the batch operation with biodiesel added as an extractant (Fig. 2), a fed-batch operation with glucose feeding was performed to explore the ability of continuous ABE fermentation with in situ removal by the addition of biodiesel. The results, as shown in Fig. 3, indicate that the shortage of glucose in the batch operation with added biodiesel was the main reason leading to the halt in butanol production. Therefore, when the glucose concentration was raised to 20 g/L after 48 h of cultivation, more butanol production was observed. The maximum total butanol obtained was 31.44 g/L in the fed-batch operation, as compared to the batch operation’s total of 16.97 g/L. However, it is noteworthy that a high butanol concentration in the water phase (fermentation broth) was also observed in the fed-batch operation. As shown in Fig. 4, butanol concentrations in both the water and oil phases were higher than 12 g/L, which is the suggested inhibitive level described in the literature (Ezeji et al., 2007; Kumar and Gayen, 2011). The high butanol concentration found in the broth suggested that butanol production could be totally inhibited under these operational conditions. Therefore, to achieve the goal of continuous ABE fermentation, a continuous biodiesel replacing process was required to keep butanol concentration lower than the suggested level. A further continuous operation was undertaken with controlled glucose concentration and continuous fresh biodiesel replacement. Comprehensive kinetic parameters of the control batch with biodiesel added and the fed-batch with biodiesel added were calculated, as shown in Table 2. These results indicate that the introduction of biodiesel in fermentation serving and as an in situ extractant efficiently enhanced the butanol productivity as
Fig. 4. Butanol concentrations of the water and oil phases in fed-batch operations with biodiesel added at the beginning at a ratio of 1:1.
Table 2 Comprehensive kinetic parameters. Control
Max biomass (g/L) Butanol productivity (g/L h) Yield (g/g) Max total butanol conc. (g/L)a
2.99 0.185 0.185 9.33
Biodiesel adding as extractant Batch
Fed-batch
3.08 0.236 0.212 16.97
3.08 0.295 0.306 31.44
a
Total butanol = (amounts of butanol in water + amounts of butanol in oil)/fermentation volume.
compared to that of the control batch. In the fed-batch operation with biodiesel added, the productivity value obtained was 0.295 g/L h, which was higher than that of 0.185 g/L h for the control batch. The yield of butanol produced per gram of glucose consumed was also improved by adding biodiesel, with the highest value of 0.306 obtained in the fed-batch operation. 4. Conclusion It is known that the feedback inhibition of butanol hinders commercial ABE production. All in situ butanol removal processes
Please cite this article in press as: 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. (2012), http://dx.doi.org/10.1016/j.biortech.2012.11.039
H.-W. Yen, Y.-C. Wang / Bioresource Technology xxx (2012) xxx–xxx
consume extra energy in order to enhance butanol production. Therefore, the proposed adding of biodiesel is a potential way to avoid that extra energy consumption. The results reveal that adding biodiesel at the beginning at a ratio of 1:1 successfully enhanced butanol production. The fed-batch operation with in situ butanol removal, accomplished by adding biodiesel, greatly enhanced butanol productivity as compared to that of a simple batch. Successful removal of butanol by adding biodiesel has great potential when applied to the scale-up of ABE fermentation. Acknowledgement The authors wish to thank the National Science Council of the ROC for financial supports. References Badr, H.R., Hamdy, M.K., 1992. Optimization of acetone–butanol production using response surface methodology. Biomass and Bioenergy 3, 49–55. Chotwichien, A., Luengnaruemitchai, A., Jai-In, S., 2009. Utilization of palm oil alkyl esters as an additive in ethanol–diesel and butanol–diesel blends. Fuel 88, 1618–1624. Crabbe, E., Nolasco-Hipolito, C., Kobayashi, G., Sonomoto, K., Ishizaki, A., 2001. Biodiesel production from crude palm oil and evaluation of butanol extraction and fuel properties. Process Biochemistry 37, 65–71. Ezeji, T.C., Qureshi, N., Blaschek, H.P., 2007. Bioproduction of butanol from biomass: from genes to bioreactors. Current Opinion in Biotechnology 18, 220–227. García, V., Päkkilä, J., Ojamo, H., Muurinen, E., Keiski, R.L., 2011. Challenges in biobutanol production: how to improve the efficiency? Renewable and Sustainable Energy Reviews 15, 964–980. Groot, W., Luyben, K., 1986. In situ product recovery by adsorption in the butanol– isopropanol batch fermentation. Applied Microbiology and Biotechnology 25, 29–31. Jones, D.T., Woods, D.R., 1986. Acetone–butanol fermentation revisited. Microbiological Reviews 50, 484–524.
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Kashket, E.R., Cao, Z.-Y., 1995. Clostridial strain degeneration. FEMS Microbiology Reviews 17, 307–315. Kumar, M., Gayen, K., 2011. Developments in biobutanol production: new insights. Applied Energy 88, 1999–2012. Li, Q., Cai, H., Hao, B., Zhang, C., Yu, Z., Zhou, S., Chenjuan, L., 2010. Enhancing clostridial acetone–butanol–ethanol (ABE) production and improving fuel properties of ABE-enriched biodiesel by extractive fermentation with biodiesel. Applied Biochemistry and Biotechnology 162, 2381–2386. Li, S.-Y., Srivastava, R., Suib, S.L., Li, Y., Parnas, R.S., 2011. Performance of batch, fedbatch, and continuous A–B–E fermentation with pH-control. Bioresource Technology 102, 4241–4250. Lin, X., Wu, J., Fan, J., Qian, W., Zhou, X., Qian, C., Jin, X., Wang, L., Bai, J., Ying, H., 2012. Adsorption of butanol from aqueous solution onto a new type of macroporous adsorption resin: studies of adsorption isotherms and kinetics simulation. Journal of Chemical Technology & Biotechnology 87 (7), 924–931. Mariano, A.P., Quresh, N., Filho, R.M., Ezeji, T.C., 2011. Bioproduction of butanol in bioreactors: new insights from simultaneous in situ butanol recovery to eliminate product toxicity. Biotechnology and Bioengineering 108, 1757–1765. Mariano, A.P., Qureshi, N., Filho, R.M., Ezejia, T.C., 2012. Assessment of in situ butanol recovery by vacuum during acetone butanol ethanol (ABE) fermentation. Journal of Chemical Technology and Biotechnology 87, 334–340. Nielsen, D.R., Prather, K.J., 2009. In situ product recovery of n-butanol using polymeric resins. Biotechnology and Bioengineering 102, 811–821. Tanaka, S., Tashiro, Y., Kobayashi, G., Ikegami, T., Negishi, H., Sakaki, K., 2012. Membrane-assisted extractive butanol fermentation by Clostridium saccharoperbutylacetonicum N1–4 with 1-dodecanol as the extractant. Bioresource Technology 116, 448–452. Volesky, B., Mulchandani, A., Williams, J., 1981. Biochemical production of industrial solvents (acetone–butanol–ethanol) from renewable resources. Annual New York Academy of Sciences, 205–218. Yen, H.-W., Chen, Z.-H., Yang, I.-K., 2012. Use of the composite membrane of poly(ether-block-amide) and carbon nanotubes (CNTs) in a pervaporation system incorporated with fermentation for butanol production by Clostridium acetobutylicum. Bioresource Technology 109, 105–109. Yen, H.-W., Li, R.-J., 2011. The effects of dilution rate and glucose concentration on continuous acetone–butanol–ethanol fermentation by Clostridium acetobutylicum immobilized on bricks. Journal of Chemical Technology and Biotechnology 86, 1399–1404. Zheng, Y.-N., Li, L.-Z., Xian, M., Ma, Y.-J., Yang, J.-M., Xu, X., He, D.-Z., 2009. Problems with the microbial production of butanol. Journal of Industrial Microbiology and Biotechnology 36, 1127–1138.
Please cite this article in press as: 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. (2012), http://dx.doi.org/10.1016/j.biortech.2012.11.039
Contents lists available at SciVerse ScienceDirect
Bioresource Technology journal homepage: www.elsevier.com/locate/biortech
The enhancement of butanol production by in situ butanol removal using biodiesel extraction in the fermentation of ABE (acetone–butanol–ethanol) Hong-Wei Yen ⇑, Yi-Cheng Wang Department of Chemical and Materials Engineering, Tunghai University, Taichung, Taiwan, ROC
h i g h l i g h t s " Adding biodiesel as an extractant for butanol removal is an attractive idea to relieve butanol toxicity. " Butanol dissolved in biodiesel could act as an additive to enhance energy content. " No significant toxicity of biodiesel on cell growth was observed. " Adding biodiesel could greatly enhance butanol productivity.
a r t i c l e
i n f o
Article history: Available online xxxx Keywords: ABE Biodiesel Extraction Separation In situ removal
a b s t r a c t High butanol accumulation is due to feedback inhibition which leads to the low butanol productivity observed in acetone–butanol–ethanol (ABE) fermentation. The aim of this study is to use biodiesel as an extractant for the in situ removal of butanol from the broth. The results indicate that adding biodiesel as an extractant at the beginning of fermentation significantly enhances butanol production. No significant toxicity of biodiesel on the growth of Clostridium acetobutylicum is observed. In the fed-batch operation with glucose feeding, the maximum total butanol obtained is 31.44 g/L, as compared to the control batch (without the addition of biodiesel) at 9.85 g/L. Moreover, the productivity obtained is 0.295 g/L h in the fed-batch, which is higher than that of 0.185 g/L h for the control batch. The in situ butanol removal by the addition of biodiesel has great potential for commercial ABE production. Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction The rapid decline of petroleum reserves, the surge in population and the growing concern regarding the environmental impacts resulting from the over-consumption of petroleum-based products have initiated interest in the development of renewable biofuels. Among all biofuel alternatives, butanol, obtained through the acetone–butanol–ethanol (ABE) process through a biological approach, is considered one of the replacement fuels with the most potential (García et al., 2011; Jones and Woods, 1986). The production of butanol by an isolated/pure microorganism was one of the first large-scale industrial microbial processes for chemical production (Mariano et al., 2011; Volesky et al., 1981). ABE fermentation is subject to a strong end-product inhibition (mainly coming from butanol) which has adversely affected the economics of commercial production. The main limitation of ABE fermentation is the toxic effect of butanol on microorganisms ⇑ Corresponding author. Address: Department of Chemical and Materials Engineering, Tunghai University, 181, Taichung Harbor Rd., Taichung 407, Taiwan, ROC. Tel.: +886 4 23590262/209; fax: +886 4 23590009. E-mail address: [email protected] (H.-W. Yen).
which leads to the low solvent productivity (Zheng et al., 2009). Therefore, it has been necessary to search for high butanol-tolerant strains of microorganisms or to use in situ toxicity removal technologies (such as extraction, gas stripping, adsorption and pervaporation) to overcome the toxicity problem and increase the final concentration of butanol (Badr and Hamdy, 1992; Li et al., 2011; Mariano et al., 2012). Numerous process designs have been developed to incorporate in situ or ex situ auxiliary separation techniques into a fermentation system to achieve end-product removal, most of which rely on traditional mass transfer phenomena (e.g., absorption or adsorption) (Groot and Luyben, 1986; Lin et al., 2012; Nielsen and Prather, 2009; Yen et al., 2012; Yen and Li, 2011). In situ solvent extraction fermentation has been proposed as one approach for minimizing butanol inhibition and increasing product titer, such as using 1-dodecancol in a membrane-assisted extractive system (Tanaka et al., 2012). However, the market value of the extractant and the subsequent cost of extractant recycling have prevented their being applied on a large scale. An ideal in situ extractant would be one that has a direct end-use as a fuel, which would then eliminate the need for expensive butanol recovery and extractant recycling procedures.
0960-8524/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.biortech.2012.11.039
Please cite this article in press as: 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. (2012), http://dx.doi.org/10.1016/j.biortech.2012.11.039
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The extractive acetone–butanol–ethanol (ABE) fermentation of Clostridium acetobutylicum has been evaluated using biodiesel as the in situ extractant. The fuel properties of the biodiesel–ABE mixture are comparable to that of No. 2 diesel, but with higher cetane numbers; therefore, it could serve as an efficient No. 2 diesel substitute (Crabbe et al., 2001). Blending butanol with biodiesel effected an improvement in the flow properties of butanol-enriched biodiesel. Ethanol is generally used to blend in diesohol; however, butanol is an alcohol which has higher solubility in diesel than ethanol and can improve the fuel properties of the blends (Chotwichien et al., 2009). To this end, microbial-produced butanol is the best choice for enriching and improving the fuel properties of biodiesel. The biodiesel preferentially extracted butanol minimized product inhibition and increased both the production of butanol (from 11.6 to 16.5 g/L) and the total solvents (from 20.0 to 29.9 g/L) by 42% and 50%, respectively. The fuel properties of the ABE-enriched biodiesel obtained by means of extractive fermentation were analyzed. The key quality indicators of diesel fuel, such as the cetane number (which increased from 48 to 54) and the cold filter plugging point (which decreased from 5.8 to 0.2 °C), were significantly improved in ABE-enriched biodiesel. Thus, the application of biodiesel as the extractant for ABE fermentation would increase ABE production, bypass the energy intensive butanol recovery process and result in an ABE-enriched biodiesel with improved fuel properties (Li et al., 2010). The aim of this study was to investigate the effects of using biodiesel as an extractant for butanol production in ABE fermentation using C. acetobutylicum. The influence of the ratio when adding biodiesel to broth and the point in time when biodiesel should be added in butanol production were evaluated, as well as the fed-batch operation by glucose addition compared to the batch operation. 2. Methods 2.1. Strain and medium The bacterial strain used in this study was C. acetobutylicum BCRC 10639 (the same as ATCC 824), which was purchased from the Bioresource Collection and Research Center (BCRC), Taiwan. The cells were cultured in glass tubes (diameter, 3 cm; length, 15 cm) containing 25 mL of fermentation medium and purged with 50% CO2 and 50% N2, to ensure 100% anaerobic conditions. After being removed from the freezer, the strain was heat-shocked at 70 °C for 2 min, and then inoculated into a glass tube (3 cm OD 15 cm high) containing 25 mL of reinforced clostridial medium (RCM), used as the seed medium, followed by an incubation period of 30 h at 37 °C. The fermentation medium was then inoculated with seed culture at a ratio of 10% (v/v). The fermentation medium contained the following components (per liter): 80 g glucose; 0.18 g Na2SO4; 0.175 g K2HPO4; 0.01 g biotin; 0.01 g p-aminobenzoic acid; 1.0 g tryptone; 5.0 g yeast extract; and 1 mL mineral salt solution. The mineral salt solution contained the following components in l liter of distilled water: 0.24 g of NaMoO42H2O; 0.24 g of CoCl26H2O; 1.5 g of CaCl22H2O; 16.203 g of FeCl3; 0.1598 g of CuSO4; 0.5164 g of ZnSO47H2O; 1.7 g of MnSO4H2O; 24.574 g of MgSO47H2O; and 28 mL of H2SO4 (6 M). The pH was adjusted to 4.8 before sterilization; the pH was not controlled during the entire fermentation process involved in this study. 2.2. Batch operation with addition of biodiesel The batch operation was performed in a glass tube, as described previously, with a 25 mL working volume. The ratios of biodiesel
added to the broth of 1:1 and 0.5:1 (v/v) were determined, respectively. Biodiesel is known to contain several solvents and chemicals which are potentially toxic to microorganisms; therefore, in addition to the examination of the influence of the ratio of added biodiesel, the effect of the time at which additions were made (12, 24 and 36 h after the inoculation) on the growth of C. acetobutylicum was also investigated. All periods of cultivation lasted for 72 h under anaerobic conditions in this study. 2.3. Fed-batch operation with glucose feeding The batch operation process was the same as described in the previous section. Biodiesel was added to the glass tube at the beginning at a ratio of 1:1 (25 mL biodiesel added to 25 mL broth). After 48 h of cultivation, the glucose was almost completely consumed in the fermentation broth. Therefore, 1 mL of concentrated glucose solution (500 g/L) was added to the broth to raise the glucose concentration to 20 g/L. After glucose feeding, cultivation lasted for 48 h to allow examination of the performance of in situ butanol removal by biodiesel extraction in the fed-batch operation. The purpose of feeding glucose here was to evaluate the performance of biodiesel addition on the enhancement of butanol production in the fed-batch operation mode. 2.4. Analytical methods Using optical density measurements, biomass concentration was determined at 620 nm by means of a Thermo Spectronic GENESYS™ 10 Spectrophotometer and by the gravimetric method (dry cell weight). Concentrations of butanol and other ingredients were measured by means of a gas chromatographer equipped with a flame-ionization detector. The samples were analyzed in a 25 m 0.22 mm stainless steel column, with a film thickness of 0.25 lm (SEG BP20). Nitrogen was used as the carrier gas. The oven temperature was held at 120 °C for 3 min and then increased by 16 °C/min to 200 °C with a 5-min final hold. The temperatures for the detector and injection port were 250 °C and 200 °C, respectively. Glucose concentration was determined using a YSI 1500 Analyzer (Yellow Springs, OH, USA) according to the enzymatic reaction of glucose oxidase. Total butanol concentration is defined as the sum of butanol in the water phase plus that in the oil phase divided by the broth volume. 3. Results and discussion 3.1. Effects of the amount of biodiesel added and the time of addition on ABE fermentation The toxicity of biodiesel to microorganisms can potentially inhibit the growth of cells during ABE fermentation. Therefore, the effects of the time of the biodiesel addition and the amounts added were first examined. A total of four different times (at 0, 12, 24 and 36 h) for biodiesel additions were set at the two different ratios of 1:1 and 1:0.5 (defined as the volume ratio of broth to biodiesel), respectively. The results indicated that adding biodiesel to extract butanol from the broth (water phase) to the biodiesel layer (oil phase) significantly enhanced the growth of C. acetobutylicum. The biomass obtained in the batches with added biodiesel was almost twice that of the control batch. The improvement in cell growth possibly came from the release of butanol toxicity due to the in situ removal of butanol by the biodiesel. No significant difference in biomass concentration was observed in the batches where biodiesel was added at different times. Even when biodiesel was added at the beginning of fermentation, there were no negative impacts on cell growth when compared to other batches.
Please cite this article in press as: 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. (2012), http://dx.doi.org/10.1016/j.biortech.2012.11.039
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H.-W. Yen, Y.-C. Wang / Bioresource Technology xxx (2012) xxx–xxx Table 1 Butanol concentrations of water and oil phase in the batches of biodiesel adding at the ratio of 1:1 and 1:0.5 for each different adding time point. Adding time (h)
Biodiesel adding ratio 1:1
0 12 24 36
1:0.5
Water phase (g butanol/L)
Oil phase (g butanol/L)
Water phase (g butanol/L)
Oil phase (g butanol/L)
6.95 5.99 6.06 6.35
7.81 8.09 9.46 8.93
8.88 6.98 5.90 7.06
10.19 10.47 10.90 10.85
The results revealed that the potential toxicity of biodiesel to microorganisms was outweighed by the benefits derived from the removal of butanol by the addition of biodiesel. In addition to the enhancement of cell growth, the increase of butanol production by biodiesel addition at ratios of 1:1 and 1:0.5 was also observed. The average totals of butanol (butanol in the broth plus butanol in the biodiesel divided by the fermentation volume) achieved were 14.8 ± 1.4 and 12.7 ± 1.7 g/L in the batches with biodiesel added at the ratios of 1:1 and 1:0.5, respectively, which was significantly higher than the butanol value of 8.1 ± 1.5 g/L in the control batch. As mentioned previously, biodiesel is not toxic in the growth of cells. Also, the inhibition of biodiesel on the required enzymes of ABE fermentation was not an issue in this study since no repression of butanol production was observed in the batches with biodiesel addition. In the paper published by Li et al. (2010), the suggested optimal adding time point was 48 h after the inoculation. Nevertheless, ABE production was not significantly different according to the data shown in the paper. Therefore, based on the consideration of industrial production, adding biodiesel at the beginning of fermentation might be more easily performed than adding it later in the process. The addition of hydrophobic biodiesel in the fermentation tubes efficiently divides the broth with air (the upper layer is the biodiesel), which might be helpful in achieving a completely anaerobic environment. It is well known that C. acetobutylicum is a strictly anaerobic microorganism which can only tolerate a slight amount of oxygen (Kashket and Cao, 1995). Therefore, adding biodiesel as an extractant can not only relieve butanol toxicity but also create a strictly anaerobic environment, which might be beneficial to the enhancement of cell growth. The participation coefficient of butyrate is known to be higher than 1 (Li et al., 2010). Therefore, it was a concern that most of the butyrate produced in the water phase probably diffused into the oil phase, leading to decreased butanol production. However, as shown in the discussion of this study and Li et al., even adding biodiesel at the beginning of fermentation did not reduce the final butanol production. The results suggested that the rate of butyrate going into the oil phase might be less than that assimilated by cells for butanol production. The butanol concentrations in both phases of oil and water, as shown in Table 1, indicated a higher butanol concentration in the oil phase than in the water phase. The average butanol participation coefficient for the oil phase over that of the water phase was 1.43 ± 0.24 according to the data, slightly higher than the 1.24 obtained by Li et al. (2010). It was obvious that adding biodiesel in the fermentation broth efficiently enhanced cell growth and more butanol was produced. Since biodiesel does not inhibit cell growth, adding biodiesel at the beginning at the ratio of 1:1 was selected for building up the profile of butanol concentration in the batch with biodiesel added as compared to the control batch (without biodiesel added).
Fig. 1. Time course of pH and biomass in the batch operation with biodiesel addition at a ratio of 1:1 as compared to the control (without the addition of biodiesel).
indicate that adding biodiesel as the extractant enhanced butanol production compared to the production of the control batch. Since some acids might be transmitted to the oil phase from the water phase, the pH value in the batch with added biodiesel was slightly higher than that of the control batch (Fig. 1). Keeping a low butanol concentration in the broth might account for the increase in total butanol production, which led to more glucose being consumed compared to the control batch. As shown in Fig. 2, the glucose consumption rate in the batch with added biodiesel was higher than that of the control batch after 24 h of cultivation, where butanol
3.2. Time course of biodiesel addition vs. control batch The time course results for biodiesel addition at the beginning and at a ratio of 1:1 are shown in Figs. 1 and 2. The results clearly
Fig. 2. Time course of total butanol produced and residual glucose in the batch with biodiesel added at a ratio of 1:1 as compared to the control batch (without the addition of biodiesel).
Please cite this article in press as: 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. (2012), http://dx.doi.org/10.1016/j.biortech.2012.11.039
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Fig. 3. Biomass, total butanol produced and residual glucose in the batch and in the fed-batch operations with biodiesel added at a ratio of 1:1.
began to accumulate fast, leading to the feedback inhibition. Glucose was completely consumed 48 h after inoculation, after which no further butanol production was observed. The butanol concentration in the broth in our study at this time was about 6–7 g/L, which was lower than the suggested butanol inhibition level of 12–15 g/L (Ezeji et al., 2007). Therefore, the stoppage in butanol production in the batch with added biodiesel might have resulted from the shortage of carbon source-glucose. To further explore the potential maximum butanol production in the batch with added biodiesel, a fed-batch operation with a single glucose feeding was performed. 3.3. Fed-batch vs. ABE fermentation batch in the biodiesel extraction system Since the carbon source was almost totally consumed after 48 h in the batch operation with biodiesel added as an extractant (Fig. 2), a fed-batch operation with glucose feeding was performed to explore the ability of continuous ABE fermentation with in situ removal by the addition of biodiesel. The results, as shown in Fig. 3, indicate that the shortage of glucose in the batch operation with added biodiesel was the main reason leading to the halt in butanol production. Therefore, when the glucose concentration was raised to 20 g/L after 48 h of cultivation, more butanol production was observed. The maximum total butanol obtained was 31.44 g/L in the fed-batch operation, as compared to the batch operation’s total of 16.97 g/L. However, it is noteworthy that a high butanol concentration in the water phase (fermentation broth) was also observed in the fed-batch operation. As shown in Fig. 4, butanol concentrations in both the water and oil phases were higher than 12 g/L, which is the suggested inhibitive level described in the literature (Ezeji et al., 2007; Kumar and Gayen, 2011). The high butanol concentration found in the broth suggested that butanol production could be totally inhibited under these operational conditions. Therefore, to achieve the goal of continuous ABE fermentation, a continuous biodiesel replacing process was required to keep butanol concentration lower than the suggested level. A further continuous operation was undertaken with controlled glucose concentration and continuous fresh biodiesel replacement. Comprehensive kinetic parameters of the control batch with biodiesel added and the fed-batch with biodiesel added were calculated, as shown in Table 2. These results indicate that the introduction of biodiesel in fermentation serving and as an in situ extractant efficiently enhanced the butanol productivity as
Fig. 4. Butanol concentrations of the water and oil phases in fed-batch operations with biodiesel added at the beginning at a ratio of 1:1.
Table 2 Comprehensive kinetic parameters. Control
Max biomass (g/L) Butanol productivity (g/L h) Yield (g/g) Max total butanol conc. (g/L)a
2.99 0.185 0.185 9.33
Biodiesel adding as extractant Batch
Fed-batch
3.08 0.236 0.212 16.97
3.08 0.295 0.306 31.44
a
Total butanol = (amounts of butanol in water + amounts of butanol in oil)/fermentation volume.
compared to that of the control batch. In the fed-batch operation with biodiesel added, the productivity value obtained was 0.295 g/L h, which was higher than that of 0.185 g/L h for the control batch. The yield of butanol produced per gram of glucose consumed was also improved by adding biodiesel, with the highest value of 0.306 obtained in the fed-batch operation. 4. Conclusion It is known that the feedback inhibition of butanol hinders commercial ABE production. All in situ butanol removal processes
Please cite this article in press as: 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. (2012), http://dx.doi.org/10.1016/j.biortech.2012.11.039
H.-W. Yen, Y.-C. Wang / Bioresource Technology xxx (2012) xxx–xxx
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Please cite this article in press as: 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. (2012), http://dx.doi.org/10.1016/j.biortech.2012.11.039