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Continuous Butanol Production Using Suspended and Immobilized Clostridium beijerinckii NCIMB 8052 with Supplementary Butyrate Sun-Mi Lee,† Min Ok Cho,†,‡ Cheol Hee Park,§ Yun-Chul Chung,† Ji Hyeon Kim,‡ Byoung-In Sang,*,† and Youngsoon Um*,† Center for EnVironmental Technology Research, Korea Institute of Science and Technology, 39-1 Hawolgok-dong, Seongbuk-gu, Seoul 136-791, Korea, Department of Chemical and Biochemical Engineering, Dongguk UniVersity, 3-26 Pil-dong, Chung-gu, Seoul 100-715, Korea, and SK Corporation, 140-1, Wonchon-dong, Yuseong-gu, Daejon 305-712, Korea ReceiVed May 27, 2007. ReVised Manuscript ReceiVed June 10, 2008
Butanol production by Clostridium beijerinckii NCIMB 8052 was investigated using both batch and continuous cultures containing suspended or immobilized cells. In the batch reactor, the initial addition of acetate and butyrate into the culture media was found not only to enhance solvent production but also to affect the ratio of acetone/butanol, which might result from the metabolic changes in solvent production. Furthermore, the addition of butyrate to the medium prevented strain degeneration during an extended subculturing, significantly induced butanol production (11.2 g/L butanol versus 0.45 g/L butanol with and without 36 mM butyrate, respectively), and shifted the acetone/butanol ratio to 1:3, which resulted in a higher yield (0.45 g of butanol/g of glucose) when compared to other studies. The beneficial effects of butyrate were also observed in continuous reactor tests containing suspended cells as the solvent production was maintained over more than 300 h of continuous operation. During a continuous butanol production with immobilized cells, using porous hydrophilic media and a dilution rate of 0.04 h-1, the overall butanol productivity and yield were 0.40 g L-1 h-1 and 0.44 g of butanol/g of glucose, respectively, which are approximately twice the values seen in a continuous reactor with suspended cells. Moreover, the butanol production was maintained over 150 days without apparent degeneration, even in the presence of high butanol concentrations (10-13 g/L). These results validate the effectiveness of producing butanol with an immobilized cell system supplemented with butyrate.
Introduction Acetone-butanol-ethanol (ABE) fermentation was one of the largest industrial fermentation processes during the first half of the 20th century until the petroleum chemical industry produced ABE at competitive prices.1 Recently, however, ABE fermentation has again attracted the attention of many researchers because biobutanol could be one of the most promising biofuels, with the potential to meet the needs of sustainable and green energy systems.2 Considering the high price of petroleum oil and recent reports on the production of butanol with agricultural and domestic wastes,3–6 it is expected that biobutanol will become a more economically attractive biofuel. Furthermore, butanol offers several advantages over ethanol for gasoline-alcohol blending because of its high energy content, * To whom correspondence should be addressed. Telephone: +82-2958-5819. Fax: 82-2-958-6858. E-mail:
[email protected] (B.-I.S.);
[email protected] (Y.U.). † Korea Institute of Science and Technology. ‡ Dongguk University. § SK R&D Center. (1) Tashiro, Y.; Takeda, K.; Kobayashi, G.; Sonomoto, K. J. Biotechnol. 2005, 120, 197–206. (2) Fernando, S.; Adhikari, S.; Chandrapal, C.; Murali, N. Energy Fuels 2006, 20, 1727–1737. (3) Thaddeus Ezeji, N. Q. H. P. B. Biotechnol. Bioeng. 2007, 97, 1460– 1469. (4) Ezeji, T.; Qureshi, N.; Blaschek, H. P. Proc. Biochem. 2007, 42, 34–39. (5) Qureshi, N.; Karcher, P.; Cotta, M.; Blaschek, H. P. Appl. Biochem. Biotechnol. 2004, 114, 713–722. (6) Zverlov, V. V.; Berezina, O.; Velikodvorskaya, G. A.; Schwarz, W. H. Appl. Microbiol. Biotechnol. 2006, 71, 587–597.
low miscibility with water, and low volatility. In addition, butanol can replace gasoline without any modification of the current vehicle and engine technologies. Despite the remarkable advantages of butanol as a fuel, biobutanol production has limitations, including low values for the yield, productivity, and final product concentration, as well as degeneration of the butanol-producing strains, which refers to the loss of solvent production and spore formation capabilities in solvent-producing clostridia.7 To overcome some of these, continuous ABE production using immobilized cell systems with various Clostridium species have been studied with the hope of enhancing butanol productivity by achieving a high cell density.1,8–10 It has also been demonstrated that butanol productivity and strain degeneration are significantly dependent upon the pH and acetate, butyrate, and phosphate concentrations.7,11–14 (7) Chen, C.-K.; Blaschek, H. P. Appl. EnViron. Microbiol. 1999, 65, 499–505. (8) Qureshi, N.; Maddox, I. S. J. Ferment. Bioeng. 1995, 80, 185–189. (9) Badr, H. R.; Toledo, R.; Hamdy, M. K. Biomass Bioenergy 2001, 20, 119–132. (10) Huang, W.-C.; Ramey, D. E.; Yang, S.-T. Appl. Biochem. Biotechnol. 2004, 115, 887–898. (11) Matta-El-Ammouri, G.; Janati-Idrissi, R.; Junelles, A.-M.; Petitdemange, H.; Gay, R. Biochimie 1987, 69, 109–115. (12) Husemann, M. H.; Papoutsakis, E. T. Appl. EnViron. Microbiol. 1990, 56, 1497–1500. (13) Assobhei, O.; Kanouni, A. E.; Ismaili, M.; Loutfi, M.; Petitdemange, H. J. Ferment. Bioeng. 1998, 85, 209–212. (14) Chen, C. K.; Blaschek, H. P. Appl. Microbiol. Biotechnol. 1999, 52, 170–173.
10.1021/ef800076j CCC: $40.75 2008 American Chemical Society Published on Web 07/29/2008
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Clostridium beijerinckii NCIMB 8052 is one of the most common solventogenic clostridia studied15–21 and the parental strain of C. beijerinckii BA101, a solvent-hyperproducing mutant.14 C. beijerinckii NCIMB 8052 has distinctly different physiological and genetic characteristics from those of Clostridium acetobutylicum ATCC 824. For example, C. acetobutylicum ATCC 824 has a 210 kb plasmid (pSOL1) encoding several solventogenic genes, and the loss of this plasmid causes degeneration, whereas C. beijerinckii NCIMB 8052 has a single circular chromosome but no plasmid, while degeneration results from genetic alternations to the chromosome.18 Although C. beijerinckii NCIMB 8052 has attracted attention for butanol production and this strain has been studied for several decades, the effects of acetate and butyrate on butanol production have not been clearly evaluated. Chen and Blaschek found that the addition of acetate (20-80 mM) to a defined medium increased solvent production and prevented strain degeneration in C. beijerinckii NCIMB 8052.7 Holt et al., however, reported that the addition of 20 mM acetate caused a decrease in butanol concentration by half in cultures of C. beijerinckii NCIMB 8052 as compared to those grown without acetate addition.21 Furthermore, the addition of 10 mM butyrate to C. beijerinckii NCIMB 8052 cultures was found to initiate solvent production during the early exponential growth phase of C. beijerinckii NCIMB 8052, but there was no increase in the final solvent concentration.21 Unlike C. beijerinckii NCIMB 8052, C. beijerinckii BA101 showed an enhanced butanol production when butyrate was added to the media in a continuous Biofilm reactor.5 The objective of this study, therefore, was first to investigate the effect of acetate and butyrate on butanol production in a batch culture of C. beijerinckii NCIMB 8052 and, second, to enhance the butanol productivity by conducting a continuous butanol fermentation with an immobilized culture. Butyrate was added to the feed of the continuous bioreactor because it was found to increase the butanol production and prevent degeneration based on the results from the batch fermentation. We also compared the butanol production of freely suspended cells to that of immobilized cells in the continuous fermentor. To the best of our knowledge, no study has been reported on the effects of butyrate on strain degeneration or a continuous butanol fermentation with immobilized C. beijerinckii NCIMB 8052. Consequently, the results presented herein provide a foundation for developing immobilized cell systems that do not exhibit strain degeneration and offer a more cost-effective butanol production with a variety of carbon sources. Materials and Methods Bacterial Culture and Medium. C. beijerinckii NCIMB 8052 (the same strain as C. beijerinckii ATCC 51743) was purchased from the Korean Collection for Type Cultures and grown in modified CAB medium (M-CAB medium). M-CAB medium contained the following components per liter of distilled water: 4 g of yeast extract, 1 g of tryptone, 1.5 g of K2HPO4, 1.5 g of KH2PO4, (15) Wang, F.; Kashket, S.; Kashket, E. R. Microbiology 2005, 151, 607–613. (16) Lee, J.; Mitchell, W. J.; Tangney, M.; Blaschek, H. P. Appl. EnViron. Microbiol. 2005, 71, 3384–3387. (17) Evans, V. J.; Liyanage, H.; Ravagnani, A.; Young, M.; Kashket, E. R. Appl. EnViron. Microbiol. 1998, 64, 1780–1785. (18) Wilkinson, S. R.; Young, M. J. Bacteriol. 1995, 177, 439–448. (19) Wilkinson, S. R.; Young, D. I.; Gareth Morris, J.; Young, M. FEMS Microbiol. ReV. 1995, 17, 275–285. (20) Kashket, E. R.; Cao, Z.-Y. Appl. EnViron. Microbiol. 1993, 59, 4198–4202. (21) Holt, R. A.; Stephens, G. M.; Morris, J. G. Appl. EnViron. Microbiol. 1984, 48, 1166–1170.
Lee et al. 0.5 g of asparagines, 0.1 g of MgSO4 · 7H2O, 0.1 g of MnSO4 · H2O, 15 mg of FeSO4 · 7H2O, 0.1 g of NaCl, 30 or 60 g of glucose, and 0.25 g of cysteine-HCl · H2O. Batch Fermentation. To investigate the effects of acetate and butyrate on butanol production, batch experiments were carried out in 250 mL serum bottles containing 100 mL of M-CAB medium and 0-36 mM sodium butyrate or sodium acetate. The medium was purged with nitrogen gas to remove dissolved oxygen, and then the serum bottles were sealed with septa and aluminum crimp seals. After the serum bottles were autoclaved, C. beijerinckii NCIMB 8052, grown overnight (37 °C) in the same media, was inoculated (10% v/v) and incubated at 37 °C with shaking (200 rpm). All batch experiments were performed in duplicate or triplicate. For pH-controlled experiments, 2-(N-morpholino)ethanesulfonic acid (MES) (100 mM) (Sigma Chemical Co.) was used to prevent over-acidification (pH < 4.7) and to exclude any interference caused by the buffering effects of butyrate.7 Continuous Fermentation. A continuous production of butanol using freely suspended cells was conducted in a 5 L bioreactor with a working volume of 2 L (Figure 1). M-CAB medium supplemented with 2 g/L of sodium butyrate was used during the continuous culture to improve butanol production. After autoclaving the reactor and purging it with filtered oxygen-free argon gas, 400 mL of C. beijerinckii NCIMB 8052 culture (grown overnight) was inoculated (20% v/v). The reactor was then operated at 37 ( 0.5 °C and stirred at 100 rpm. Initially, the reactor was operated for 23 h in a batch mode to allow for an appropriate cell growth, and then the medium was continuously fed by a peristaltic pump with a hydraulic retention time (HRT) of 24 h, which is equivalent to a dilution rate (D) of 0.04 h-1. Likewise, the continuous production of butanol with an immobilized culture was conducted under the same conditions (Figure 1). To do this, porous polyvinyl alcohol media was used for the immobilization of the cells. The density of the media was 0.16 g/cm3, and the surface area was 4.6 m2/g. The pore size was in the range of 0.2-0.5 mm. The total dry weight of the media was 16 g, and the dimension of each media particle was 2.5 × 2.5 × 2.5 mm. Culture broth was drawn through a metal membrane to prevent the loss of the immobilization media. The reactor was operated at 37 ( 0.5 °C and stirred at 125 rpm to ensure a complete mixing and the free motion of the media. Initially, the reactor was operated for 25 h in a batch mode to allow for cell growth and immobilization. During the continuous mode, the HRT was set at 24 h. Antifoam was added as needed. After 42 h, the pH of the reactor was adjusted to 5.00 ( 0.05 and thereon automatically monitored and adjusted with 5 N HCl and 5 N NaOH to ensure a high butanol production and to prevent over-acidification.21 Analytical Methods. Acetone, butanol, ethanol, acetic acid, and butyric acid were analyzed with a gas chromatograph (Agilent technology 6890N Network GC System) equipped with a HPINNOWax column (30 m × 250 µm × 0.25 µm, Agilent Technologies) and a flame-ionized detector. The oven temperature was programmed to increase from 50 to 170 °C at the rate of 1 °C/min. The injector and detector temperatures were set to 250 °C.22 Helium was used as a carrier gas with a flow rate of 1 mL/min. The glucose concentrations were analyzed using a Reflect Quant Strip (Glucose Test Strip, Merck Co., Ltd.), and the cell concentration was estimated by measuring the optical density (OD) at 600 nm with a spectrophotometer (UV-1700, SHIMAZU). The gas composition was analyzed by gas chromatography (Agilent Technology 6890N Network GC System) equipped with a thermal conductivity detector. The temperatures of the oven, injector, and detector were 50, 100, and 200 °C, (22) Tashiro, Y.; Takeda, K.; Kobayashi, G.; Sonomoto, K.; Ishizaki, A.; Yoshino, S. J. Biosci. Bioeng. 2004, 98, 263–268.
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Figure 1. Schematic diagram of the continuous reactor.
respectively. A packed column (Porepack Q) was used, and the column flow rate was 15 mL/min.
Results and Discussion Effect of Acetate and Butyrate on Butanol Production. Butanol production by C. beijerinckii NCIMB 8052 was investigated with initial sodium acetate concentrations of 9, 18, and 36 mM. Figure 2A clearly shows that the butanol and acetone productions were only enhanced with 36 mM acetate, when compared to those of the control cultures. This is different from the results by Chen and Blaschek,7 where they found that 20 mM sodium acetate enhanced butanol concentration with C. beijerinckii NCIMB 8052. On the basis of the knowledge that acetone, butanol, and ethanol are produced by solventogenic clostridia in the ratio 3:6:1,23 it is of interest that the ratio of acetone/butanol was about 2:3 with 36 mM acetate (Figure 2A). This result suggests that the additional acetate might shift the metabolic pathway toward acetone production. It was reported that the addition of acetate enhanced CoA-transferase activity within C. beijerinckii NCIMB 8052.7,13 Because a higher CoAtransferase activity would accelerate the conversion of acetate to acetyl-CoA and this reaction is coupled with the production of acetoacetate and, subsequently, acetone according to the metabolic pathway,24,25 the presence of additional acetate likely leads to a shift toward acetone production. (23) Qureshi, N.; Li, X.-L.; Hughes, S.; Saha, B. C.; Cotta, M. A. Biotechnol. Prog. 2006, 22, 673–680. (24) Grupe, H.; Gottschalk, G. Appl. EnViron. Microbiol. 1992, 58, 3896–3902. (25) Nolling, J.; Breton, G.; Omelchenko, M. V.; Makarova, K. S.; Zeng, Q.; Gibson, R.; Lee, H. M.; Dubois, J.; Qiu, D.; Hitti, J.; Production, G. T. C. S. C.; Finishing, a. B. T.; Wolf, Y. I.; Tatusov, R. L.; Sabathe, F.; Doucette-Stamm, L.; Soucaille, P.; Daly, M. J.; Bennett, G. N.; Koonin, E. V.; Smith, D. R. J. Bacteriol. 2001, 183, 4823–4838.
Unlike the enhanced butanol production seen with 36 mM acetate, little butanol was produced with 0, 9, and 18 mM acetate. One possible reason for this could be rapid acid production and, consequently, a low pH (pH 4.0-4.5), causing a loss in the viability before the culture has a chance to switch to the solventogenesis phase. On the contrary, with 36 mM acetate, the pH was always greater than 5 during the fermentation, despite the acid production. This is likely due to the buffering capacity of the acetate that was added. Under these conditions, the culture successfully switched to solventogenesis and the acetic and butyric acids were rapidly utilized. The residual acetate concentrations were 0.10, 0.46, 0.35, and 0.24 g/L, with the additional acetate 0, 9, 18, and 36 mM, respectively. The glucose was completely used by 30 h in cultures that had 36 mM acetate added to them, with 9.4 g/L butanol produced and a yield of 0.32 g of butanol/g of glucose. When sodium butyrate (9, 18, or 36 mM) was added to cultures of C. beijerinckii NCIMB 8052, the butanol production increased significantly (Figure 2B). Interestingly, only small differences in the amount of butanol produced were seen for the various concentrations tested. Furthermore, an increased butanol production was seen during the early exponential phase (data not shown), presumably because the butyrate concentrations were high enough to initiate butanol production. The residual butyrate concentrations in the batch fermentations were 0.9 g/L without any additional butyrate and 0 g/L when butyrate was added to 9, 18, and 36 mM. To investigate the effects of lower initial butyrate concentrations on butanol production, butyrate was added to 0.9, 1.8, or 3.6 mM. However, the production of butanol was not fully initiated at these concentrations, and the final butanol concentrations seen for all three tests were less than 1.2 g/L. Whereas glucose was only mildly consumed in cultures without butyrate addition, the concentration of glucose decreased
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Figure 3. Comparison of butanol production levels in batch cultures with butyrate addition and the pH controlled: (b) butanol concentration with 0 mM butyrate, (2) butanol concentration with 18 mM butyrate, and (×) butanol concentration calculated on the basis of 100% conversion of 18 mM butyrate to butanol.
Figure 2. Solvent concentrations after 48 h of batch fermentation with supplemented (A) acetate or (B) butyrate. The experiments were performed in duplicate.
dramatically when butyrate was added (data not shown). Such an increase in both the butanol production and glucose consumption could be due to an enhanced buffering capacity of the medium by butyrate, which would aid in growth of the culture by preventing extremely acidic conditions.12 Of interest was the finding that the ratio of acetone/butanol was about 1:3 (Figure 2B), which is higher than the common ratio of 1:2.23 Furthermore, under these conditions, the butanol production was 11.2 g/L and the yield was 0.45 g of butanol/g of glucose when 36 mM of butyrate was added. To evaluate the effect of butyrate further and to minimize any inference caused by the buffering capacity of butyrate, batch experiments were performed under pH-controlled conditions. In contrast to the results shown in Figure 2B, butanol was produced when the pH was controlled even without the addition of butyrate to the media. However, the addition of butyrate enhanced the butanol production of the cultures (8.0, 8.5, 10.2, and 10.6 g/L with 0, 9, 18, and 36 mM butyrate, respectively). It is noteworthy that the increase in the butanol concentration (2.22 g/L or 30 mM butanol) in cultures with 18 mM butyrate added is much higher than the maximum butanol concentration possible when all of the additional butyrate is converted to butanol (i.e., 18 mM) (Figure 3), suggesting a shift in the metabolism toward butanol production. As well, the addition of 18 mM butyrate accelerated the production of butanol when compared to the control culture. It has been reported that adding
Figure 4. Solvent production during repeated subculturings of C. beijerinckii NCIMB 8052 in media containing 18 mM of sodium butyrate: (b) acetone, (9) ethanol, and (2) butanol.
organic acid, such as butyric acid or acetic acid, trigger a metabolic shift from acetogenesis to solventogenesis with Clostridium sp.7,14,22 On the basis of these results, therefore, it seems likely that the enhanced butanol production when butyrate was added to the media is due to both the buffering of the media as well as a shift in the metabolic pathways toward butanol production. In addition, as the butanol production pathway was induced by the added butyrate, acetoacetyl-CoA would tend to be converted into butyl-CoA instead of acetoacetate, consequently resulting in a lower acetone production. For further reference, a detailed metabolic pathway for butanol production can be found elsewhere.25 A further study involving an analysis of the individual enzyme activities would be required to verify such a metabolic shift with the addition of butyrate. There have been several reports about the effects of butyrate or acetate on butanol production with several solvent-producing clostridia,5,12,14,23 but the results are still inconclusive. With regard to C. beijerinckii NCIMB 8052, the addition of 20 mM acetate was found to decrease the final butanol concentration by half but there was no change in the acetone concentration, resulting in an acetone/butanol ratio of about 1:1.21 On the
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contrary, Chen and Blaschek7 reported on an enhanced butanol production with 20 mM acetate when compared to that of the control (without acetate added), but there was no mention of the acetone/butanol ratio. In this study, we found that the addition of 36 mM acetate or butyrate to concentrations greater than 9 mM not only enhanced solvent production but also affected the ratio of acetone/butanol (Figure 2). These differences with previous studies might be the result of varying culture conditions, such as the concentrations of glucose, the temperature, the composition of the media, etc. However, it should be noted that our butanol fermentation conditions resulted in a more impressive butanol production (yield 0.45 g/g glucose with 30 g/L glucose and 36 mM butyrate) when compared to Chen and Blaschek’s work7 (yield 0.39 g/g glucose with 60 g/L glucose and 60 mM acetate), Holt et al.’s result (yield 0.19 g/g glucose with 20 g/L glucose and 20 mM butyrate),21 and other studies.17,20,26 Butyrate Addition Prevents Degeneration of C. beijerinckii NCIMB 8052. One of the limitations in ABE fermentation is its unstable productivity because of strain degeneration. C. beijerinckii NCIMB 8052 is known to degenerate during the repeated subculturing of batch cultures or continuous fermentations.7 Meanwhile, it has been shown that the addition of acetate enhanced butanol production and prevented degeneration of C. beijerinckii NCIMB 8052.7 Because a butyrate addition also increased butanol production according to our batch study results, the effects of butyrate on degeneration were examined over a long period of time, where a subculturing was performed every 24 h for 16 days (Figure 4). Interestingly, the addition of 18 mM sodium butyrate was able to prevent degeneration, as the butanol production level was maintained at a high level (7-11 g/L). To our knowledge, this is the first report to find that the addition of butyrate prevents strain degeneration for C. beijerinckii NCIMB 8052. Continuous Butanol Production with Suspended Cells. The butanol production with the suspended cells was conducted in a continuous reactor with M-CAB medium containing sodium butyrate. Butyrate was selected, instead of acetate, for the continuous studies because of the high acetone concentrations seen when acetate was added, which could make it difficult to purify butanol for use as a fuel. Furthermore, the addition of butyrate was expected to both prevent strain degeneration and enhance the butanol production based on the results presented above. Besides, considering the batch results with 18 mM butyrate in this study and the report that found that butanol production was initiated when the butyrate concentration was 14 mM,21 a concentration of 18 mM butyrate was selected for use in the continuous reactor. Once it was confirmed that butanol was produced after 23 h in the batch operation, the dilution rate was set to 0.04 h-1 by feeding in fresh medium containing 18 mM sodium butyrate. A low dilution rate (0.04 h-1) was chosen because a low growth rate is preferable to enhance solvent production,17 while a high dilution rate can cause the wash-out of the cells. Figure 5 shows the ABE production, glucose concentration, and OD600 results for the continuous reactor. During the operation, the maximum butanol production and total ABE production were 7.1 and 9.4 g/L, respectively. The overall productivity9 and yield were 0.22 g L-1 h-1 and 0.24 g of butanol/g of glucose, respectively (Table 1). In the continuous reactor, the production of butanol varied from 3.8 to 7.1 g/L (Figure 5). Fluctuations in butanol production are typical of continuous butanol fermentations4,10 because of the metabolic shift between acidogenesis (associated with cell (26) Gottschal, J. C.; Morris, J. G. FEMS Microbiol. Lett. 1981, 12, 385–389.
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Figure 5. (A) Solvent and acid concentrations and (B) OD and glucose concentrations in the suspended cell continuous reactor: (b) acetone, (9) ethanol, (2) butanol, (O) acetic acid, (4) butyric acid, (×) glucose, and (1) OD. Table 1. Comparison of Butanol Production in the Suspended and Immobilized Cell Reactors
butanol production (g/L) total ABE production (g/L) yield (g/g) productivity (g L-1 h-1) glucose conversion (%) acetone/butanol
free cell (at 309 h)
immobilized cell (at 154 h)
7.1 9.4 0.24 0.22 81.3 1:3
13.4 22.1 0.44 0.40 83.7 1:1.9
growth) and solventogenesis (nongrowing cells) in solventogenic clostridia cells.4 It is noteworthy that a high butanol production was successfully achieved in the continuous reactor for a long operational time without the apparent degeneration of the cells. To examine the degeneracy more closely, some cells were taken from the reactor at the end of the operation and subcultured in the same media more than 50 times (subcultures) over 50 days to evaluate their butanol producing capabilities. As expected from the batch results with the supplemented butyrate, the butanol production was maintained (8-10 g/L) during the repeated subculturings. This result demonstrates that the addition of butyrate prevented degeneration of the cells. In contrast to Qureshi et al.,5 who found that the addition of acetate enhanced ABE production and prevented strain degeneration but did not improve the performance of a continuous Biofilm reactor with C. beijerinckii BA101, the results of this study found that C. beijerinckii NCIMB 8052 was positively affected by the
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et al.’s result (yield 0.42 g/g glucose with 54 g/L glucose and 3.6 g/L butyrate with a fibrous bed bioreactor),10 our butanol production was more efficient even though only 30 g/L glucose and 2 g/L (18 mM) butyrate was supplied. Furthermore, by immobilizing the cells, solvent production is possible without the need for frequent cell regeneration.10 While acid production is associated with cell growth, solvent production is not, and therefore, immobilization of the cells can lead to a higher butanol production via the lower growth rate of the cells that are immobilized on the porous media. As shown in Figure 6A, a high level of butanol production was maintained without degeneration by feeding in media containing butyrate. Interestingly, despite a successful cell immobilization and the addition of butyrate, the butanol concentration rarely exceeded 13 g/L. This is likely due to the toxicity and product inhibition brought on by the presence of high concentrations of butanol. It was reported that 13 g/L of butanol is the maximum possible concentration during ABE fermentation.15 Unexpectedly, the acetone concentration was much higher than that seen in either the batch cultures or the continuous reactor with the suspended cells (Figures 2 and 5) and tended to increase slightly with time (Figure 6A). This is likely the result of the high butanol concentrations, which lead to a partial inhibition in the butanol production and a metabolic pathway shift toward acetone production.25 To overcome such inhibitory effects during butanol fermentations, further studies into the development of a novel process in which the production and separation of butanol is accomplished concurrently are required. Conclusions
Figure 6. (A) Solvent and acid concentrations and (B) OD and glucose concentrations in the immobilized cell continuous reactor: (b) acetone, (9) ethanol, (2) butanol, (O) acetic acid, (4) butyric acid, (×) glucose, and (1) OD.
additional butyrate in both batch and continuous fermentations. The ratio of acetone/butanol in the continuous culture was about 1:3 (Figure 5A), which is similar to the results from batch cultures performed with additional butyrate. Butanol Production in a Continuous Reactor with Immobilized Cells. To enhance the productivity and yield during butanol fermentation, the C. beijerinckii NCIMB 8052 cells were immobilized using a hydrophilic media in a continuous reactor and grown in M-CAB supplemented with 18 mM butyrate. A porous and soft polymeric medium was chosen because it ensured a high surface area and the free movement of the media when stirring. In a previous study from our laboratory that studied hydrogen production, the same media were used to immobilize hydrogenproducing Clostridium sp. and found to provide a excellent immobilization matrix during a long-term operation.27 As shown in Figure 6, a relatively stable butanol production was observed after 40 h of continuous operation. During this operation, the maximum butanol concentration was 13.4 g/L and the overall yield (0.44 g of butanol/g of glucose) and productivity (0.40 g L-1 h-1) were also approximately 2 times higher than those obtained in a continuous reactor with the freely suspended cells, demonstrating that a high cell concentration and, consequently, higher butanol production was achieved through immobilization of the cells. In comparison to Huang (27) Jeon, B.-S.; Um, Y. S.; Lee, S.-M.; Lee, S.-Y.; Kim, H.-J.; Kim, Y. H.; Gu, M. B.; Sang, B.-I. Energy Fuels 2008, 22, 83–86.
Through the addition of butyrate and acetate to the media, the butanol production in batch cultures of C. beijerinckii NCIMB 8052 was significantly enhanced. More importantly, the addition of butyrate was found to both prevent strain degeneration and enhance butanol production. In a continuous reactor with freely suspended cells, butanol production was successfully achieved without degeneration for more than 300 h of operation when butyrate was present in the media. Furthermore, with immobilized cells in a continuous reactor system, the maximum butanol production seen was 13.4 g/L, which is just above to the reported maximum butanol concentration (13 g/L) at which solvent-producing clostridia are likely to be inhibited. Through the study presented here, it was demonstrated that the addition of butyrate to the media prevents degeneration of C. beijerinckii NCIMB 8052 in both batch and continuous cultures and that far improved butanol productivities and yields can be achieved by immobilizing cells in continuous cultures when compared to those with freely suspended cells. However, because of product inhibition, further studies that investigate the separation of butanol from the reactor during its production need to be conducted. In addition, the positive effects of butyrate on strain degeneration during butanol production need to be studied in detail using metabolic and enzymatic analyses. By combining our present study and the results from these further studies, it is feasible that more stable and productive butanol fermentation processes can be developed. Acknowledgment. This work was supported by the research programs of The Korea Research Council of Fundamental Science and Technology and the R&BD Support Center of the Seoul Development Institute. We are also grateful to Dr. Robert J. Mitchell for his critical review and editing of the paper. EF800076J
