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Continuous butanol fermentation from inexpensive sugar-based feedstocks by Clostridium saccharobutylicum DSM 13864
Bioresource Technology 129 (2013) 680–685
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Bioresource Technology journal homepage: www.elsevier.com/locate/biortech
Short Communication
Continuous butanol fermentation from inexpensive sugar-based feedstocks by Clostridium saccharobutylicum DSM 13864 Ye Ni ⇑, Ziyi Xia, Yun Wang, Zhihao Sun The Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, 1800 Lihu Rd., Wuxi 214122, China
h i g h l i g h t s " Butanol can be produced from cane molasses and corn stover hydrolysate by C. saccharobutylicum. " A four-stage continuous butanol fermentation using CSH was steadily operated for 220 h. " A high solvent productivity of 0.429 g/L/h and solvent titer of 11.43 g/L were attained.
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Article history: Received 30 July 2012 Received in revised form 21 November 2012 Accepted 23 November 2012 Available online 8 December 2012 Keywords: Butanol Continuous fermentation Corn stover hydrolysate (CSH) Cane molasses Dilution rate
a b s t r a c t Corn stover hydrolysate (CSH) and cane molasses were studied for butanol fermentation by Clostridium saccharobutylicum DSM 13864 in continuous fermentation. Using cane molasses as substrate, solvent of 13.75 g/L (butanol 8.65 g/L) and productivity of 0.439 g/L/h were achieved in a four-stage continuous fermentation at a gradient dilution mode of 0.15–0.15–0.125–0.1 h 1. In continuous fermentation using CSH as substrate, total solvent titer of 11.43 g/L (butanol 7.81 g/L) and productivity of 0.429 g/L/h were reached at a dilution rate of 0.15 h 1, and the steady process was continuously operated for 220 h without compromise in solvent titer. Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction With an ever-growing global depletion of natural oil and gas resources, and various environmental issues resulting from the rapid consumption of petroleum fuels, the development of alternative fuel resources has been getting significant attentions for decades (Bankar et al., 2012). Butanol, an important C4 platform compounds, is considered as one of the most promising biofuels. Compared with traditional biofuel ethanol, butanol provides many advantages such as higher energy content, less hygroscopy, less volatile, less hazardous to handle, and lower vapor pressure (Qureshi and Ezeji, 2008). In addition, this chemical is also an excellent fuel extender as it contains 22% oxygen (Qureshi et al., 2010). Importantly, butanol can be used directly or blended with gasoline or diesel at any ratio without retrofit of vehicle engines, and can be supplied and stored through the existing gasoline pipeline (Ni and Sun, 2009). Butanol is a main product of acetone-butanol-ethanol (ABE) fermentation by Clostridia under anaerobic conditions, which was one ⇑ Corresponding author. Tel./fax: +86 510 85329265. E-mail address: [email protected] (Y. Ni). 0960-8524/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.biortech.2012.11.142
of the largest industrial fermentation processes in the early 20th century. Compared with continuous fermentation, the commercial development of batch ABE fermentation has been hampered by a number of deficiencies including lower productivity, and stronger end-product inhibition, etc. The productivity of a traditional ABE batch fermentation, usually less than 0.5 g/L/h, is obviously undesirable for industrial production (Qureshi and Ezeji, 2008). While in continuous fermentation, the solvent productivity could be enhanced to 0.5–1.0 g/L/h (Maddox, 1989) . An average overall solvent concentration of 15 g/L and an overall solvent productivity of 0.27 g/L/h were achieved using a two-stage continuous cultivation with Clostridium beijerinckii NRRL B592 (Mutschlechner et al., 2000). Furthermore, higher solvent productivity and concentration could also be achieved in single-stage continuous process. In a single-stage continuous fermentation with wood pulp as a cell holding material, maximum solvent productivity of 13.66 g/L/h was obtained at a dilution rate of 1.9 h 1 (Survase et al., 2012). Recently, using a cell recycling continuous fermentation with glycerol as substrate, a solvent productivity of 8.3 g/L/h was reached at a dilution rate of 0.9 h 1 (Malaviya et al., 2012). Although a number of studies on continuous ABE fermentation have been carried out,
Y. Ni et al. / Bioresource Technology 129 (2013) 680–685
glucose or corn starch were mostly utilized as major feedstock. As a matter of fact, the cost of fermentation substrate, which account for 60–70% of total production cost, is the most influential factor in the butanol fermentation (Qureshi and Blaschek, 2000). It is therefore necessary to explore cheaper feedstocks for making ABE fermentation economically more attractive. Cane molasses, a by-product of sugar industry, contains approximately 45–50% (w/w) total sugars (sucrose, glucose and fructose), vitamins, and nitrogenous compounds, etc. (Najafpour and Shan, 2003). It has therefore been regarded as an inexpensive and appropriate substrate for ABE fermentation (Ni et al., 2012). In the last decade, cellulosic hydrolysates of various agricultural residues and other biomasses have been investigated for the biofuel production, including degermed corn, corn stover (CS), wheat straw (WS), barley straw (BS), and energy crops like switchgrass (SG) and miscanthus (Qureshi and Ezeji, 2008). In China, the annual production of cane molasses and agricultural residues were about three million tons and one billion tons, respectively. To date, there is no Chinese solvent plant using corn stover hydrolysate (CSH) and cane molasses as sole substrate to produce butanol, especially in a continuous fermentation. In this work, the effect of dilution rate on solvent production in continuous ABE fermentation using corn stover hydrolysate (CSH) and cane molasses as substrates were investigated. 2. Methods 2.1. Microorganism, maintenance and inoculum preparation Clostridium saccharobutylicum DSM 13864 used in this study was purchased from DSMZ (German Collection of Microorganisms and Cell Cultures). It was cultivated in Reinforced Clostridia Medium (RCM) at 37 °C for 7 days to induce sporulation, followed by storage at room temperature. A 1.2 mL spore culture was inoculated into a glass tube (£1.5 cm 15 cm) containing 12 mL sterile RCM. It was then placed in a desiccator pumped to a vacuum level of 0.065 MPa for providing an anaerobic condition. Afterwards, the culture was maintained at 37 °C for 12–16 h and supplied as the seed medium. An inoculum size of 8% (v/v) was used in all fermentation in this study. 2.2. Fermentation medium Cane molasses was purchased from Xinyue chemical industry Co. Ltd., (Changshu, China). The fermentation medium reported by Ni et al. (2012) was used, which contains (per liter): cane molasses, 60 g; CaCO3, 3.2 g; (NH4)2SO4, 2 g; K2HPO4, 0.5 g; MnSO4H2O, 0.01 g; corn steep liquor, 10 g. The pH of medium was adjusted to 6.1 with 2 M NaOH or 2 M HCl prior to sterilization, and then the medium was autoclaved at 121 °C for 20 min. Cane molasses mainly consists of 270.4 g/l sucrose, 58.2 g/l glucose and 78.6 g/l fructose, with a total sugar of 407.20 g/L. The corn stover pretreatment method reported by Lin et al. (2011) was used with slight modification. The dried corn stover was pulverized and passed through a 40 mesh sieve. For pretreatment, 112 g of corn stover was added into 1.6 L of 1% (w/w) sodium hydroxide in a 3-L erlenmeyer flask, and autoclaved at 120 °C for 2 h. After cooling to room temperature, the pH of pretreated corn stover was adjusted to 4.8 using 250 g/L H2SO4 solution. Then cellulase solution of 11.2 mL (filter paper enzyme activity: 51.70 FPIU/mL), which was purchased from Zesheng bioengineering technology Co. Ltd., (Shandong, China), was added followed by incubation at 50 °C and 150 rpm for 40 h. Afterwards, the pretreated corn stover mixture was centrifuged at 8000 rpm for 10 min to obtain corn stover hydrolysate. The sugar composition
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(g/L) of CSH includes: glucose 29.48, xylose 11.51, arabinose 2.44, and cellobiose 4.19. The fermentation medium contains the following components (per liter CSH): CaCO3, 2.0 g; (NH4)2SO4, 1.0 g; K2HPO4, 0.5 g; MnSO4H2O, 0.01 g; corn steep liquor, 15 g (based on our previous optimization study, unpublished data). Prior to autoclaving, glucose was supplemented into CSH to raise the total sugar to 60 g/L. The medium was adjusted to pH6.1, and was then autoclaved at 115 °C for 20 min. After sterilization, the medium was sparged with carbon dioxide for 5–10 min to remove dissolved oxygen. 2.3. Continuous fermentation Continuous fermentations using cane molasses as substrate were also performed in 3-L stirred tank bioreactors. The experimental setup consists of four stirred tank bioreactors with working volume of 1-L. Four tanks were arranged sequentially and designated as stage one (R1), stage two (R2), stage three (R3) and stage four (R4), respectively. In order to investigate the effect of dilution rate on fermentation performance, continuous fermentations were conducted without pH control. For cane molasses substrate, to achieve a better sugar utilization in the later two stages of continuous fermentation, R3 and R4 were inoculated first and then incubated for 10–20 h, after which R1 and R2 were inoculated, and the continuous fermentation was started when R1 arrived at a desired stage (3–4 g/L total solvent). The dilution rates of R1, R2, R3 and R4 were controlled at 0.1–0.1–0.1–0.1 h 1 and 0.15–0.15–0.125–0.1 h 1, respectively. Sterilized fresh fermentation medium was stored in a feeding tank with a total volume of 20 L. The culture volume in each tanks was kept constant by using a peristaltic pump. First, R3 and R4 were started by inoculating 8% (v/v) seed culture, following by incubation for 14 h. Then R1 and R2 were inoculated and fermented for 16 h. Thereafter, continuous fermentations were started by feeding fresh fermentation medium at certain flow rates. It is assumed that the continuous fermentation process enters into a steady phase when a stable solvent titer was observed during the following period (for example: 100–200 h). Samples of 2 mL were taken at a regular time interval to monitor the progress of fermentation. Continuous fermentations using CSH as substrate were carried out in four 500 mL serum bottles with a working volume of 300 mL. The four bioreactors were inoculated with 8% (v/v) of seed culture simultaneously and proceed in batch fermentation for 16 h, after which continuous fermentation was started by feeding fresh fermentation medium using a peristaltic pump. The dilution rates were controlled at 0.1 and 0.15 h 1, separately. 2.4. Analytical procedure The cell concentration was measured at 660 nm (OD660) using an ultraviolet spectrophotometer (INESA, China). Sterilized fresh cane molasses or corn steep liquor medium was diluted 5–10 times with 2% HCl and used as blank control to eliminate the influence of their color in the detection. The total sugar in cane molasses fermentation medium was determined by 3,5-dinitrosalicylic acid (DNS) method (Tesun and Dhese, 1970). The sugar compositions of CSH were analyzed by HPLC (Waters 1525, USA) equipped with a refractive index detector and an Aminex HPX-87H column. The temperatures of column and detector were controlled at 55 °C and 35 °C, respectively. The injection volume was 10 lL. The concentrations of solvent and organic acids (acetic and butyric acid) were analyzed by GC (Varian cp 3900, USA) equipped with the flame ionization detector (FID) and an capillary column PEG-20 M (30 m0.32 mm0.5 lm, JK, China) using nitrogen as the carrier gas. The oven temperature was maintained at 60 °C for 0.5 min and then programmed with the increment of 10 °C to 120 °C, held for 0.5 min, and subsequently
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increased to 190 °C with the increment of 15 °C with 1 min final hold. The temperature of the injector and detector was held at 180 °C and 210 °C, respectively. The injection volume was 1 lL. 3. Results and Discussion 3.1. Continuous fermentation with 6% cane molasses Continuous fermentation is usually adopted in industrial ABE fermentation to improve solvent productivity, relieve end-product inhibition, and reduce downtime, and the main feedstocks used were simple sugars and starchy materials. (Maddox, 1989). Recently, various substrates such as wheat bran (Liu et al., 2010), barley straw hydrolysate (Qureshi et al., 2010), sugar maple wood
(Sun and Liu, 2012), and cassava bagasse hydrolysate (Lu et al., 2012) have been used in the ABE fermentation. Based on our preliminary studies on batch and semi-continuous fermentation (Ni et al., 2012), we achieved 17.88 g/L total solvent at 36 h, including acetone 4.60 g/L, butanol 11.86 g/L, ethanol 1.42 g/L, and the productivity and yield were 0.50 g/L/h and 0.33 g/g, respectively. In this study, four-stage continuous fermentations using 6% cane molasses at two sets of dilution rates (constant mode: 0.1–0.1– 0.1–0.1 h 1 and gradient mode: 0.15–0.15–0.125–0.1 h 1) were carried out. The gradient mode was attempted to increase the retention time of R3 and R4, and also improve the sugar consumption. The concentrations of residual sugar, solvents, organic acids and cell density (OD660) were shown in Fig. 1A–F. Under the constant mode when the dilution rate in all four stages were main-
Fig. 1. Time courses of continuous ABE fermentation by C. saccharobutylicum DSM 13864 using 6% cane molasses.(A) OD600 and residual sugar, (B) butanol and total solvent, (C) organic acids at constant dilution rates of 0.1–0.1–0.1–0.1 h 1. (D) OD600 and residual sugar, (E) butanol and total solvent, (F) organic acids at gradient dilution rates of 0.15–0.15–0.125–0.1 h 1. R1, R3 and R4 represent the first-stage, third-stage and fourth-stage bioreactor tanks, respectively.
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Y. Ni et al. / Bioresource Technology 129 (2013) 680–685 Table 1 Steady state parameters of four-stage ABE continuous fermentation using cane molasses and corn stover hydrolysate (CSH) as substrates. Cane molasses Parameters
OD660 ABE/(g/L) Butanol/(g/L) Acetone/(g/L) Ethanol/(g/L) Acetic acid/(g/L) Butyric acid/(g/L) Initial sugar/(g/L) Residual sugar/(g/L) Consumed sugar/(g/L) Yield of Butanol a/(g/g) Yield of ABE a/(g/g) Productivity of Butanol b/(g/L/h) Productivity of ABE b/(g/L/h) a b
CSH
D = 0.1–0.1–0.1–0.1
D = 0.1
D = 0.15
R1
R3
R4
R1
D = 0.15–0.15–0.125–0.1 R3
R4
R4
R4
4.57 7.71 4.68 2.26 0.77 2.17 1.33 56.62 33.69 22.93 0.204 0.336 0.468 0.771
3.12 11.37 6.93 3.16 1.28 1.77 0.72 58.22 24.77 33.45 0.207 0.340 0.231 0.379
3.06 11.74 7.18 3.32 1.25 1.78 0.70 57.06 20.27 36.79 0.195 0.319 0.180 0.294
4.72 7.15 4.04 1.86 1.26 3.00 2.32 59.81 35.14 24.67 0.164 0.290 0.606 1.072
4.56 12.24 7.52 3.68 1.03 2.67 2.13 59.09 18.26 40.83 0.184 0.300 0.353 0.574
4.17 13.75 8.26 3.99 1.49 2.55 2.10 58.07 15.78 42.29 0.195 0.325 0.264 0.439
1.37 13.44 9.29 3.85 0.30 1.24 1.01 51.31 14.98 36.33 0.256 0.370 0.232 0.336
3.79 11.43 7.81 3.32 0.30 0.46 1.80 51.58 23.63 27.95 0.279 0.409 0.293 0.429
Yield of ABE or butanol: concentration of ABE or butanol divided by concentration of sugar consumed. Productivity of ABE or butanol: concentration of ABE or butanol divided by fermentation time.
tained at 0.1 h 1, continuous fermentation could be stably operated during 30–130 h of fermentation where the average total solvent of R1, R3 and R4 were 7.71, 11.37 and 11.74 g/L, respectively (Table 1). Consequently, the solvent productivity in R1 was calculated to be 0.771 g/L/h, representing a 54.2% increase compared with that of batch fermentation. While productivities in R3 (0.379 g/L/h) and R4 (0.294 g/L/h) were slightly reduced due to the extended retention time. In continuous fermentation under gradient dilution mode, where the dilution rates in each stage were increased to 0.15, 0.15, 0.125 and 0.1 h 1, the average ABE titers of 7.15, 12.24 and 13.75 g/L were obtained for R1, R3, and R4, respectively (Fig. 1E). As a result, the solvent productivities of R1, R3 and R4 were 1.072, 0.574 and 0.439 g/L/h, respectively. Our results are comparable with those reported by Liew and coworkers, in which total solvent of 9.10 g/L and overall productivity of 0.46 g/L/h were attained using a single-stage continuous fermentation by Clostridium saccharobutylicum DSM 13864 at a dilution rate of 0.05 h 1 (Liew et al., 2006). Clearly, the butanol toxicity to cell culture could be alleviated by continuous fermentation, thereby enhancing solvent productivity in this study. Unexpectedly, solvent production began to reduce after 140 h of continuous fermentation, likely due to a drop in pH to around 4.5 (data not shown) which resulted in strain degeneration. As shown in Fig. 1A and D, average residual sugars in two R4 tanks were maintained at 20.27 and 15.78 g/L respectively, which would lead to a compromised solvent yield. Under the gradient mode, the accumulation of acetic acid and butyric acid in R4 were 2.55 and 2.10 g/L, respectively, which were slightly higher than 1.78 and 0.70 g/L at constant dilution rates. It is known that the production of organic acids is often accompanied by the ATP regeneration system driven by several enzymes including acetate kinase (AcK) in ABE fermentation (Jiang et al., 2009). Dilution rate is one major reason for the different cell density observed under gradient mode (4.17 OD660) and constant mode (3.06 OD660). Also, ATP regeneration rate could somewhat affect the cell growth. As demonstrated above, a continuous fermentation at a gradient dilution rate of 0.15–0.15–0.125–0.1 h 1 was advantageous to improve solvent productivity. 3.2. Continuous fermentation with CSH To exploit the utilization of sustainable sugar-based feedstock, ABE fermentation from various lignocellulosic hydrolysates of agricultural residues has been extensively investigated during the
last few years (Qureshi et al., 2010; Liu et al., 2010; Lin et al., 2011). Inhibitors including furfural, hydroxymethyl furfurl, ferulic, glucuronic, and phenolic compounds, are usually generated during the pretreatment of cellulosic materials, and are deleterious for cell growth and solvent production (Qureshi et al., 2010). A number of approaches have been developed to remove these inhibitors, including overliming, adsorbent resin, dilution of hydrolysate, and development of inhibitor-tolerant strains, etc. (Qureshi et al., 2008, 2010; Liu et al., 2010). Based on our previous work (unpublished data), 1% NaOH was used for the pretreatment of corn stover, which was autoclaved at 120 °C for 2 h to remove the inhibitors and followed by enzymatic hydrolysis. CSH was then obtained and used in four-stage continuous ABE fermentation in this study. A control batch fermentation with CSH as substrate was conducted, maximum total solvent of 16.09 g/L (10.59 g/L butanol, 4.04 g/L acetone, and 1.46 g/L ethanol) was attained at 40 h, and the ABE yield and productivity were 0.334 g/g and 0.402 g/L/h, respectively (data not show). The four-stage continuous fermentation was operated at two constant dilution rates of 0.1 and 0.15 h 1, respectively. The concentrations of residual sugar, solvent, organic acids and OD660 are shown in Table 1. Our results indicate that CSH is well suited to solvent production. At both dilution rates, continuous fermentation reached a steady state after 60 h of fermentation (Fig. 2A–B), where cell densities of R4 tanks were maintained at 1.37 and 3.79 OD660, respectively. The steady fermentation process could be continuously operated for 220 h at 0.15 h 1. At dilution rate of 0.1 h 1, the average total solvent concentration of 13.44 g/ L (9.29 g/L butanol) and total solvent productivity of 0.336 g/L/h were achieved. For 0.15 h 1, a relatively lower average total solvent titer of 11.43 g/L (7.81 g/L butanol) was obtained, accompanied by a higher total solvent productivity of 0.429 g/L/h. The accumulation of total organic acid (2.25 g/L) was almost equivalent at two dilution rates. The glucose was completely consumed at the dilution rate of 0.1 h 1, and the residual sugars consisted of xylose 7.71 g/L, arabinose 4.84 g/L, and cellobiose 2.43 g/L. Higher total residual sugars (23.63 g/L) was however detected at 0.15 h 1 dilution rate, which contained glucose 12.77 g/L, xylose 5.04 g/L, arabinose 4.44 g/L, and cellobiose 1.38 g/L, indicating dilution rate of 0.15 h 1 is inappropriate for efficient sugar utilization. It has been reported that higher dilution rate was beneficial to cell growth whereas lower dilution rate favored solvent production by C. saccharobutylicum DSM 13864 in a single-stage continuous culture using gelatinized sago starch as substrate. (Liew et al., 2006), and our results are in accordance with it. In comparison with other
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Y. Ni et al. / Bioresource Technology 129 (2013) 680–685
Fig. 2. Time courses of ABE fermentation in the final stage of four-stage continuous fermentation by C. saccharobutylicum DSM 13864 using CSH as substrate. (A) Dilution rate of 0.1 h 1. (B) Dilution rate of 0.15 h 1. OD660 (open upright triangle), residual sugar (filled square), butanol (filled inverted triangle), total solvent (filled circle), acetic acid (open inverted triangle), butyric acid (open circle).
Table 2 Comparison of ABE continuous fermentation parameters in the literatures and this study. Operation mode
Strain
Immobilized cell C. acetobutylicum DSM 792 Membrane cell bioreactor C. pasteurianum ATCC 6103 C. acetobutylicum ATCC 824 Free cell C. saccharobutylicum DSM 13864 C. beijerinckiiNRRL B592 C. saccharobutylicum DSM 13864
Substrate
Dilution rate (h
Glucose Glycerol Glucose Gelatinised sago starch Glucose Corn stover hydrolysate
1.9 0.9 0.017 0.05 0.022 (R2) 0.15
literatures (Table 2), total solvent concentration in this study is the highest although the productivity is not greatly improved. Therefore, further efforts should be dedicated to enhance the productivity while maintaining high solvent titer, e.g.: continuous fermentation using immobilized cells, integrated solvent removal strategy. It was presumed that the cost of solvent recovery would be reduced by half if the butanol titer could be enhanced from 1.2 to 2.0%.
4. Conclusion Butanol can be produced from cane molasses and CSH by C. saccharobutylicum DSM 13864 in continuous fermentation. In a four-stage continuous fermentation using cane molasses, a steady solvent production of 13.75 g/L and productivity of 0.439 g/L/h were obtained under a gradient dilution mode. In continuous fermentation using CSH, a high solvent productivity of 0.429 g/L/h was maintained for an extended period of 220 h. Therefore, the continuous fermentation using inexpensive sugar-based feedstocks could potentially help to drive butanol fermentation towards an economically viable process.
Acknowledgements The research was financially supported by the National High Technology Research and Development Program of China (2011CB710800), New Century Excellent Talents in University (NCET-11-0658), Natural Science Foundation of Jiangsu Province (BK2011150), the Program of Introducing Talents of Discipline to Universities No. 111-2-06, the Priority Academic Program Development of Jiangsu Higher Education Institutions.
1
) Productivity Total solvent Reference (g/L/h) (g/L) 13.66 8.3 0.37 0.46 0.27 0.429
7.19 9.2 124.4 9.10 15.00 11.43
Survase et al. 2012 Malaviya et al. (2012) Wouter et al. (2012) Liew et al. (2006) Mutschlechner et al. (2000) This study
References Bankar, S.B., Survase, S.A., Singhal, R.S., Granström, T., 2012. Continuous two stage acetone-butanol-ethanol fermentation with integrated solvent removal using Clostridium acetobutylicum B 5313. Bioresour. Technol. 106, 110–116. Jiang, Y., Xu, C., Dong, F., Yang, Y., Jiang, W.H., Yang, S., 2009. Disruption of the acetoacetate decarboxylase gene in solvent-producing Clostridium acetobutylicum increases the butanol ratio. Metab. Eng. 11, 284–291. Liew, S., Arbakariya, A., Rosfrizan, M., Raha, A.R., 2006. Production of solvent (acetone-butanol-ethanol) in continuous fermentation by Clostridium saccharobutylicum DSM 13864 using gelatinised sago starch as a carbon source. Malaysian J. Microbiol. 2, 42–50. Lin, Y.S., Wang, J., Wang, X.M., Sun, X.H., 2011. Optimization of butanol production from corn straw hydrolysate by Clostridium acetobutylicum using response surface method. Chin Sci. Bull. 56, 1422–1428. Liu, Z.Y., Ying, Y., Li, F.L., Ma, C.Q., Xu, P., 2010. Butanol production by clostridium beijerinckii ATCC 55025 from wheat bran. J. Ind. Microbiol. Biotechnol. 37, 495– 501. Lu, C., Zhao, J.B., Yang, S.T., Wei, D., 2012. Fed-batch fermentation for n-butanol production from cassava bagasse hydrolysate in a fibrous bed bioreactor with continuous gas stripping. Bioresour. Technol. 104, 380–387. Maddox, I.S., 1989. The acetone-butanol-ethanol fermentation: recent progress in technology. Biotechnol. Genet. Eng. Rev. 7, 189–220. Malaviya, A., Jang, Y.S., Lee, S.Y., 2012. Continuous butanol production with reduced byproducts formation from glycerol by a hyper producing mutant of Clostridium pasteurianum. Appl. Microbiol. Biotechnol. 93, 1485–1494. Mutschlechner, O., Swoboda, H., Gapes, J.R., 2000. Continuous two-stage ABEfermentation using Clostridium beijerinckii NRRL B 592 operating with a growth rate in the first stage vessel close to its maximal value. J. Mol. Microbiol. Biotechnol. 2, 101–105. Najafpour, G.D., Shan, C.P., 2003. Enzymatic hydrolysis of molasses. Bioresour. Technol. 86, 91–94. Ni, Y., Sun, Z.H., 2009. Recent Progress on Industrial Fermentative Production of Acetone–Butanol–Ethanol by Clostridium acetobutylicum in China. Appl Microbiol Biotechnol. 83, 415–423. Ni, Y., Wang, Y., Sun, Z.H., 2012. Butanol Production from Cane Molasses by Clostridium saccharobutylicum DSM 13864: Batch and Semi-continuous fermentation. Appl. Biochem. Biotechnol. 166, 1896–1907. Qureshi, N., Blaschek, H., 2000. Economics of Butanol Fermentation using Hyper-Butanol Producing Clostridium Beijerinckii BA101. Food Bioprod. Process. 78, 139–144. Qureshi, N., Ezeji, T.C., 2008. Butanol, ‘a superior biofuel’ production from agricultural residues (renewable biomass): recent progress in technology. Biofuels. Bioprod. Bioref. 2, 319–330.
Y. Ni et al. / Bioresource Technology 129 (2013) 680–685 Qureshi, N., Ezeji, T.C., Ebener, J., Dien, B., Cotta, M., Blaschek, H.P., 2008. Butanol production by Clostridium Beijerinckii. Part I: Use of acid and enzyme hydrolyzed corn fiber. Bioresour. Technol. 99, 5915–5922. Qureshi, N., Saha, B.C., Dien, B., Hector, E.H., Cotta, M.A., 2010. Production of butanol (a biofuel) from agricultural residues: Part I-Use of barley straw hydrolysate. Biomass Bioenergy. 34, 559–565. Sun, Z.J., Liu, S.J., 2012. Production of n-butanol from concentrated sugar maple hemicellulosic hydrolysate by Clostridia acetobutylicum ATCC824. Biomass Bioenerg. 39, 39–47.
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Survase, S.A., Heiningen, V.A., Granström, T., 2012. Continuous bio-catalytic conversion of sugar mixture to acetone-butanol-ethanol by immobilized Clostridium acetobutylicum DSM 792. Appl. Microbiol. Biotechnol. 93, 1–8. Tesun, K., Dhese, P., 1970. Sugar determination of DNS method. Biotechnol. Bioeng. 12, 921–922. Wouter, V.H., Vandezande, P., Claes, S., Vangeel, S., Beckers, H., Diels, L., Wever, H.D., 2012. Integrated bioprocess for long-term continuous cultivation of Clostridium acetobutylicum coupled to pervaporation with PDMS composite membranes. Bioresour. Technol. 111, 368–377.
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Short Communication
Continuous butanol fermentation from inexpensive sugar-based feedstocks by Clostridium saccharobutylicum DSM 13864 Ye Ni ⇑, Ziyi Xia, Yun Wang, Zhihao Sun The Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, 1800 Lihu Rd., Wuxi 214122, China
h i g h l i g h t s " Butanol can be produced from cane molasses and corn stover hydrolysate by C. saccharobutylicum. " A four-stage continuous butanol fermentation using CSH was steadily operated for 220 h. " A high solvent productivity of 0.429 g/L/h and solvent titer of 11.43 g/L were attained.
a r t i c l e
i n f o
Article history: Received 30 July 2012 Received in revised form 21 November 2012 Accepted 23 November 2012 Available online 8 December 2012 Keywords: Butanol Continuous fermentation Corn stover hydrolysate (CSH) Cane molasses Dilution rate
a b s t r a c t Corn stover hydrolysate (CSH) and cane molasses were studied for butanol fermentation by Clostridium saccharobutylicum DSM 13864 in continuous fermentation. Using cane molasses as substrate, solvent of 13.75 g/L (butanol 8.65 g/L) and productivity of 0.439 g/L/h were achieved in a four-stage continuous fermentation at a gradient dilution mode of 0.15–0.15–0.125–0.1 h 1. In continuous fermentation using CSH as substrate, total solvent titer of 11.43 g/L (butanol 7.81 g/L) and productivity of 0.429 g/L/h were reached at a dilution rate of 0.15 h 1, and the steady process was continuously operated for 220 h without compromise in solvent titer. Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction With an ever-growing global depletion of natural oil and gas resources, and various environmental issues resulting from the rapid consumption of petroleum fuels, the development of alternative fuel resources has been getting significant attentions for decades (Bankar et al., 2012). Butanol, an important C4 platform compounds, is considered as one of the most promising biofuels. Compared with traditional biofuel ethanol, butanol provides many advantages such as higher energy content, less hygroscopy, less volatile, less hazardous to handle, and lower vapor pressure (Qureshi and Ezeji, 2008). In addition, this chemical is also an excellent fuel extender as it contains 22% oxygen (Qureshi et al., 2010). Importantly, butanol can be used directly or blended with gasoline or diesel at any ratio without retrofit of vehicle engines, and can be supplied and stored through the existing gasoline pipeline (Ni and Sun, 2009). Butanol is a main product of acetone-butanol-ethanol (ABE) fermentation by Clostridia under anaerobic conditions, which was one ⇑ Corresponding author. Tel./fax: +86 510 85329265. E-mail address: [email protected] (Y. Ni). 0960-8524/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.biortech.2012.11.142
of the largest industrial fermentation processes in the early 20th century. Compared with continuous fermentation, the commercial development of batch ABE fermentation has been hampered by a number of deficiencies including lower productivity, and stronger end-product inhibition, etc. The productivity of a traditional ABE batch fermentation, usually less than 0.5 g/L/h, is obviously undesirable for industrial production (Qureshi and Ezeji, 2008). While in continuous fermentation, the solvent productivity could be enhanced to 0.5–1.0 g/L/h (Maddox, 1989) . An average overall solvent concentration of 15 g/L and an overall solvent productivity of 0.27 g/L/h were achieved using a two-stage continuous cultivation with Clostridium beijerinckii NRRL B592 (Mutschlechner et al., 2000). Furthermore, higher solvent productivity and concentration could also be achieved in single-stage continuous process. In a single-stage continuous fermentation with wood pulp as a cell holding material, maximum solvent productivity of 13.66 g/L/h was obtained at a dilution rate of 1.9 h 1 (Survase et al., 2012). Recently, using a cell recycling continuous fermentation with glycerol as substrate, a solvent productivity of 8.3 g/L/h was reached at a dilution rate of 0.9 h 1 (Malaviya et al., 2012). Although a number of studies on continuous ABE fermentation have been carried out,
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glucose or corn starch were mostly utilized as major feedstock. As a matter of fact, the cost of fermentation substrate, which account for 60–70% of total production cost, is the most influential factor in the butanol fermentation (Qureshi and Blaschek, 2000). It is therefore necessary to explore cheaper feedstocks for making ABE fermentation economically more attractive. Cane molasses, a by-product of sugar industry, contains approximately 45–50% (w/w) total sugars (sucrose, glucose and fructose), vitamins, and nitrogenous compounds, etc. (Najafpour and Shan, 2003). It has therefore been regarded as an inexpensive and appropriate substrate for ABE fermentation (Ni et al., 2012). In the last decade, cellulosic hydrolysates of various agricultural residues and other biomasses have been investigated for the biofuel production, including degermed corn, corn stover (CS), wheat straw (WS), barley straw (BS), and energy crops like switchgrass (SG) and miscanthus (Qureshi and Ezeji, 2008). In China, the annual production of cane molasses and agricultural residues were about three million tons and one billion tons, respectively. To date, there is no Chinese solvent plant using corn stover hydrolysate (CSH) and cane molasses as sole substrate to produce butanol, especially in a continuous fermentation. In this work, the effect of dilution rate on solvent production in continuous ABE fermentation using corn stover hydrolysate (CSH) and cane molasses as substrates were investigated. 2. Methods 2.1. Microorganism, maintenance and inoculum preparation Clostridium saccharobutylicum DSM 13864 used in this study was purchased from DSMZ (German Collection of Microorganisms and Cell Cultures). It was cultivated in Reinforced Clostridia Medium (RCM) at 37 °C for 7 days to induce sporulation, followed by storage at room temperature. A 1.2 mL spore culture was inoculated into a glass tube (£1.5 cm 15 cm) containing 12 mL sterile RCM. It was then placed in a desiccator pumped to a vacuum level of 0.065 MPa for providing an anaerobic condition. Afterwards, the culture was maintained at 37 °C for 12–16 h and supplied as the seed medium. An inoculum size of 8% (v/v) was used in all fermentation in this study. 2.2. Fermentation medium Cane molasses was purchased from Xinyue chemical industry Co. Ltd., (Changshu, China). The fermentation medium reported by Ni et al. (2012) was used, which contains (per liter): cane molasses, 60 g; CaCO3, 3.2 g; (NH4)2SO4, 2 g; K2HPO4, 0.5 g; MnSO4H2O, 0.01 g; corn steep liquor, 10 g. The pH of medium was adjusted to 6.1 with 2 M NaOH or 2 M HCl prior to sterilization, and then the medium was autoclaved at 121 °C for 20 min. Cane molasses mainly consists of 270.4 g/l sucrose, 58.2 g/l glucose and 78.6 g/l fructose, with a total sugar of 407.20 g/L. The corn stover pretreatment method reported by Lin et al. (2011) was used with slight modification. The dried corn stover was pulverized and passed through a 40 mesh sieve. For pretreatment, 112 g of corn stover was added into 1.6 L of 1% (w/w) sodium hydroxide in a 3-L erlenmeyer flask, and autoclaved at 120 °C for 2 h. After cooling to room temperature, the pH of pretreated corn stover was adjusted to 4.8 using 250 g/L H2SO4 solution. Then cellulase solution of 11.2 mL (filter paper enzyme activity: 51.70 FPIU/mL), which was purchased from Zesheng bioengineering technology Co. Ltd., (Shandong, China), was added followed by incubation at 50 °C and 150 rpm for 40 h. Afterwards, the pretreated corn stover mixture was centrifuged at 8000 rpm for 10 min to obtain corn stover hydrolysate. The sugar composition
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(g/L) of CSH includes: glucose 29.48, xylose 11.51, arabinose 2.44, and cellobiose 4.19. The fermentation medium contains the following components (per liter CSH): CaCO3, 2.0 g; (NH4)2SO4, 1.0 g; K2HPO4, 0.5 g; MnSO4H2O, 0.01 g; corn steep liquor, 15 g (based on our previous optimization study, unpublished data). Prior to autoclaving, glucose was supplemented into CSH to raise the total sugar to 60 g/L. The medium was adjusted to pH6.1, and was then autoclaved at 115 °C for 20 min. After sterilization, the medium was sparged with carbon dioxide for 5–10 min to remove dissolved oxygen. 2.3. Continuous fermentation Continuous fermentations using cane molasses as substrate were also performed in 3-L stirred tank bioreactors. The experimental setup consists of four stirred tank bioreactors with working volume of 1-L. Four tanks were arranged sequentially and designated as stage one (R1), stage two (R2), stage three (R3) and stage four (R4), respectively. In order to investigate the effect of dilution rate on fermentation performance, continuous fermentations were conducted without pH control. For cane molasses substrate, to achieve a better sugar utilization in the later two stages of continuous fermentation, R3 and R4 were inoculated first and then incubated for 10–20 h, after which R1 and R2 were inoculated, and the continuous fermentation was started when R1 arrived at a desired stage (3–4 g/L total solvent). The dilution rates of R1, R2, R3 and R4 were controlled at 0.1–0.1–0.1–0.1 h 1 and 0.15–0.15–0.125–0.1 h 1, respectively. Sterilized fresh fermentation medium was stored in a feeding tank with a total volume of 20 L. The culture volume in each tanks was kept constant by using a peristaltic pump. First, R3 and R4 were started by inoculating 8% (v/v) seed culture, following by incubation for 14 h. Then R1 and R2 were inoculated and fermented for 16 h. Thereafter, continuous fermentations were started by feeding fresh fermentation medium at certain flow rates. It is assumed that the continuous fermentation process enters into a steady phase when a stable solvent titer was observed during the following period (for example: 100–200 h). Samples of 2 mL were taken at a regular time interval to monitor the progress of fermentation. Continuous fermentations using CSH as substrate were carried out in four 500 mL serum bottles with a working volume of 300 mL. The four bioreactors were inoculated with 8% (v/v) of seed culture simultaneously and proceed in batch fermentation for 16 h, after which continuous fermentation was started by feeding fresh fermentation medium using a peristaltic pump. The dilution rates were controlled at 0.1 and 0.15 h 1, separately. 2.4. Analytical procedure The cell concentration was measured at 660 nm (OD660) using an ultraviolet spectrophotometer (INESA, China). Sterilized fresh cane molasses or corn steep liquor medium was diluted 5–10 times with 2% HCl and used as blank control to eliminate the influence of their color in the detection. The total sugar in cane molasses fermentation medium was determined by 3,5-dinitrosalicylic acid (DNS) method (Tesun and Dhese, 1970). The sugar compositions of CSH were analyzed by HPLC (Waters 1525, USA) equipped with a refractive index detector and an Aminex HPX-87H column. The temperatures of column and detector were controlled at 55 °C and 35 °C, respectively. The injection volume was 10 lL. The concentrations of solvent and organic acids (acetic and butyric acid) were analyzed by GC (Varian cp 3900, USA) equipped with the flame ionization detector (FID) and an capillary column PEG-20 M (30 m0.32 mm0.5 lm, JK, China) using nitrogen as the carrier gas. The oven temperature was maintained at 60 °C for 0.5 min and then programmed with the increment of 10 °C to 120 °C, held for 0.5 min, and subsequently
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increased to 190 °C with the increment of 15 °C with 1 min final hold. The temperature of the injector and detector was held at 180 °C and 210 °C, respectively. The injection volume was 1 lL. 3. Results and Discussion 3.1. Continuous fermentation with 6% cane molasses Continuous fermentation is usually adopted in industrial ABE fermentation to improve solvent productivity, relieve end-product inhibition, and reduce downtime, and the main feedstocks used were simple sugars and starchy materials. (Maddox, 1989). Recently, various substrates such as wheat bran (Liu et al., 2010), barley straw hydrolysate (Qureshi et al., 2010), sugar maple wood
(Sun and Liu, 2012), and cassava bagasse hydrolysate (Lu et al., 2012) have been used in the ABE fermentation. Based on our preliminary studies on batch and semi-continuous fermentation (Ni et al., 2012), we achieved 17.88 g/L total solvent at 36 h, including acetone 4.60 g/L, butanol 11.86 g/L, ethanol 1.42 g/L, and the productivity and yield were 0.50 g/L/h and 0.33 g/g, respectively. In this study, four-stage continuous fermentations using 6% cane molasses at two sets of dilution rates (constant mode: 0.1–0.1– 0.1–0.1 h 1 and gradient mode: 0.15–0.15–0.125–0.1 h 1) were carried out. The gradient mode was attempted to increase the retention time of R3 and R4, and also improve the sugar consumption. The concentrations of residual sugar, solvents, organic acids and cell density (OD660) were shown in Fig. 1A–F. Under the constant mode when the dilution rate in all four stages were main-
Fig. 1. Time courses of continuous ABE fermentation by C. saccharobutylicum DSM 13864 using 6% cane molasses.(A) OD600 and residual sugar, (B) butanol and total solvent, (C) organic acids at constant dilution rates of 0.1–0.1–0.1–0.1 h 1. (D) OD600 and residual sugar, (E) butanol and total solvent, (F) organic acids at gradient dilution rates of 0.15–0.15–0.125–0.1 h 1. R1, R3 and R4 represent the first-stage, third-stage and fourth-stage bioreactor tanks, respectively.
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Y. Ni et al. / Bioresource Technology 129 (2013) 680–685 Table 1 Steady state parameters of four-stage ABE continuous fermentation using cane molasses and corn stover hydrolysate (CSH) as substrates. Cane molasses Parameters
OD660 ABE/(g/L) Butanol/(g/L) Acetone/(g/L) Ethanol/(g/L) Acetic acid/(g/L) Butyric acid/(g/L) Initial sugar/(g/L) Residual sugar/(g/L) Consumed sugar/(g/L) Yield of Butanol a/(g/g) Yield of ABE a/(g/g) Productivity of Butanol b/(g/L/h) Productivity of ABE b/(g/L/h) a b
CSH
D = 0.1–0.1–0.1–0.1
D = 0.1
D = 0.15
R1
R3
R4
R1
D = 0.15–0.15–0.125–0.1 R3
R4
R4
R4
4.57 7.71 4.68 2.26 0.77 2.17 1.33 56.62 33.69 22.93 0.204 0.336 0.468 0.771
3.12 11.37 6.93 3.16 1.28 1.77 0.72 58.22 24.77 33.45 0.207 0.340 0.231 0.379
3.06 11.74 7.18 3.32 1.25 1.78 0.70 57.06 20.27 36.79 0.195 0.319 0.180 0.294
4.72 7.15 4.04 1.86 1.26 3.00 2.32 59.81 35.14 24.67 0.164 0.290 0.606 1.072
4.56 12.24 7.52 3.68 1.03 2.67 2.13 59.09 18.26 40.83 0.184 0.300 0.353 0.574
4.17 13.75 8.26 3.99 1.49 2.55 2.10 58.07 15.78 42.29 0.195 0.325 0.264 0.439
1.37 13.44 9.29 3.85 0.30 1.24 1.01 51.31 14.98 36.33 0.256 0.370 0.232 0.336
3.79 11.43 7.81 3.32 0.30 0.46 1.80 51.58 23.63 27.95 0.279 0.409 0.293 0.429
Yield of ABE or butanol: concentration of ABE or butanol divided by concentration of sugar consumed. Productivity of ABE or butanol: concentration of ABE or butanol divided by fermentation time.
tained at 0.1 h 1, continuous fermentation could be stably operated during 30–130 h of fermentation where the average total solvent of R1, R3 and R4 were 7.71, 11.37 and 11.74 g/L, respectively (Table 1). Consequently, the solvent productivity in R1 was calculated to be 0.771 g/L/h, representing a 54.2% increase compared with that of batch fermentation. While productivities in R3 (0.379 g/L/h) and R4 (0.294 g/L/h) were slightly reduced due to the extended retention time. In continuous fermentation under gradient dilution mode, where the dilution rates in each stage were increased to 0.15, 0.15, 0.125 and 0.1 h 1, the average ABE titers of 7.15, 12.24 and 13.75 g/L were obtained for R1, R3, and R4, respectively (Fig. 1E). As a result, the solvent productivities of R1, R3 and R4 were 1.072, 0.574 and 0.439 g/L/h, respectively. Our results are comparable with those reported by Liew and coworkers, in which total solvent of 9.10 g/L and overall productivity of 0.46 g/L/h were attained using a single-stage continuous fermentation by Clostridium saccharobutylicum DSM 13864 at a dilution rate of 0.05 h 1 (Liew et al., 2006). Clearly, the butanol toxicity to cell culture could be alleviated by continuous fermentation, thereby enhancing solvent productivity in this study. Unexpectedly, solvent production began to reduce after 140 h of continuous fermentation, likely due to a drop in pH to around 4.5 (data not shown) which resulted in strain degeneration. As shown in Fig. 1A and D, average residual sugars in two R4 tanks were maintained at 20.27 and 15.78 g/L respectively, which would lead to a compromised solvent yield. Under the gradient mode, the accumulation of acetic acid and butyric acid in R4 were 2.55 and 2.10 g/L, respectively, which were slightly higher than 1.78 and 0.70 g/L at constant dilution rates. It is known that the production of organic acids is often accompanied by the ATP regeneration system driven by several enzymes including acetate kinase (AcK) in ABE fermentation (Jiang et al., 2009). Dilution rate is one major reason for the different cell density observed under gradient mode (4.17 OD660) and constant mode (3.06 OD660). Also, ATP regeneration rate could somewhat affect the cell growth. As demonstrated above, a continuous fermentation at a gradient dilution rate of 0.15–0.15–0.125–0.1 h 1 was advantageous to improve solvent productivity. 3.2. Continuous fermentation with CSH To exploit the utilization of sustainable sugar-based feedstock, ABE fermentation from various lignocellulosic hydrolysates of agricultural residues has been extensively investigated during the
last few years (Qureshi et al., 2010; Liu et al., 2010; Lin et al., 2011). Inhibitors including furfural, hydroxymethyl furfurl, ferulic, glucuronic, and phenolic compounds, are usually generated during the pretreatment of cellulosic materials, and are deleterious for cell growth and solvent production (Qureshi et al., 2010). A number of approaches have been developed to remove these inhibitors, including overliming, adsorbent resin, dilution of hydrolysate, and development of inhibitor-tolerant strains, etc. (Qureshi et al., 2008, 2010; Liu et al., 2010). Based on our previous work (unpublished data), 1% NaOH was used for the pretreatment of corn stover, which was autoclaved at 120 °C for 2 h to remove the inhibitors and followed by enzymatic hydrolysis. CSH was then obtained and used in four-stage continuous ABE fermentation in this study. A control batch fermentation with CSH as substrate was conducted, maximum total solvent of 16.09 g/L (10.59 g/L butanol, 4.04 g/L acetone, and 1.46 g/L ethanol) was attained at 40 h, and the ABE yield and productivity were 0.334 g/g and 0.402 g/L/h, respectively (data not show). The four-stage continuous fermentation was operated at two constant dilution rates of 0.1 and 0.15 h 1, respectively. The concentrations of residual sugar, solvent, organic acids and OD660 are shown in Table 1. Our results indicate that CSH is well suited to solvent production. At both dilution rates, continuous fermentation reached a steady state after 60 h of fermentation (Fig. 2A–B), where cell densities of R4 tanks were maintained at 1.37 and 3.79 OD660, respectively. The steady fermentation process could be continuously operated for 220 h at 0.15 h 1. At dilution rate of 0.1 h 1, the average total solvent concentration of 13.44 g/ L (9.29 g/L butanol) and total solvent productivity of 0.336 g/L/h were achieved. For 0.15 h 1, a relatively lower average total solvent titer of 11.43 g/L (7.81 g/L butanol) was obtained, accompanied by a higher total solvent productivity of 0.429 g/L/h. The accumulation of total organic acid (2.25 g/L) was almost equivalent at two dilution rates. The glucose was completely consumed at the dilution rate of 0.1 h 1, and the residual sugars consisted of xylose 7.71 g/L, arabinose 4.84 g/L, and cellobiose 2.43 g/L. Higher total residual sugars (23.63 g/L) was however detected at 0.15 h 1 dilution rate, which contained glucose 12.77 g/L, xylose 5.04 g/L, arabinose 4.44 g/L, and cellobiose 1.38 g/L, indicating dilution rate of 0.15 h 1 is inappropriate for efficient sugar utilization. It has been reported that higher dilution rate was beneficial to cell growth whereas lower dilution rate favored solvent production by C. saccharobutylicum DSM 13864 in a single-stage continuous culture using gelatinized sago starch as substrate. (Liew et al., 2006), and our results are in accordance with it. In comparison with other
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Fig. 2. Time courses of ABE fermentation in the final stage of four-stage continuous fermentation by C. saccharobutylicum DSM 13864 using CSH as substrate. (A) Dilution rate of 0.1 h 1. (B) Dilution rate of 0.15 h 1. OD660 (open upright triangle), residual sugar (filled square), butanol (filled inverted triangle), total solvent (filled circle), acetic acid (open inverted triangle), butyric acid (open circle).
Table 2 Comparison of ABE continuous fermentation parameters in the literatures and this study. Operation mode
Strain
Immobilized cell C. acetobutylicum DSM 792 Membrane cell bioreactor C. pasteurianum ATCC 6103 C. acetobutylicum ATCC 824 Free cell C. saccharobutylicum DSM 13864 C. beijerinckiiNRRL B592 C. saccharobutylicum DSM 13864
Substrate
Dilution rate (h
Glucose Glycerol Glucose Gelatinised sago starch Glucose Corn stover hydrolysate
1.9 0.9 0.017 0.05 0.022 (R2) 0.15
literatures (Table 2), total solvent concentration in this study is the highest although the productivity is not greatly improved. Therefore, further efforts should be dedicated to enhance the productivity while maintaining high solvent titer, e.g.: continuous fermentation using immobilized cells, integrated solvent removal strategy. It was presumed that the cost of solvent recovery would be reduced by half if the butanol titer could be enhanced from 1.2 to 2.0%.
4. Conclusion Butanol can be produced from cane molasses and CSH by C. saccharobutylicum DSM 13864 in continuous fermentation. In a four-stage continuous fermentation using cane molasses, a steady solvent production of 13.75 g/L and productivity of 0.439 g/L/h were obtained under a gradient dilution mode. In continuous fermentation using CSH, a high solvent productivity of 0.429 g/L/h was maintained for an extended period of 220 h. Therefore, the continuous fermentation using inexpensive sugar-based feedstocks could potentially help to drive butanol fermentation towards an economically viable process.
Acknowledgements The research was financially supported by the National High Technology Research and Development Program of China (2011CB710800), New Century Excellent Talents in University (NCET-11-0658), Natural Science Foundation of Jiangsu Province (BK2011150), the Program of Introducing Talents of Discipline to Universities No. 111-2-06, the Priority Academic Program Development of Jiangsu Higher Education Institutions.
1
) Productivity Total solvent Reference (g/L/h) (g/L) 13.66 8.3 0.37 0.46 0.27 0.429
7.19 9.2 124.4 9.10 15.00 11.43
Survase et al. 2012 Malaviya et al. (2012) Wouter et al. (2012) Liew et al. (2006) Mutschlechner et al. (2000) This study
References Bankar, S.B., Survase, S.A., Singhal, R.S., Granström, T., 2012. Continuous two stage acetone-butanol-ethanol fermentation with integrated solvent removal using Clostridium acetobutylicum B 5313. Bioresour. Technol. 106, 110–116. Jiang, Y., Xu, C., Dong, F., Yang, Y., Jiang, W.H., Yang, S., 2009. Disruption of the acetoacetate decarboxylase gene in solvent-producing Clostridium acetobutylicum increases the butanol ratio. Metab. Eng. 11, 284–291. Liew, S., Arbakariya, A., Rosfrizan, M., Raha, A.R., 2006. Production of solvent (acetone-butanol-ethanol) in continuous fermentation by Clostridium saccharobutylicum DSM 13864 using gelatinised sago starch as a carbon source. Malaysian J. Microbiol. 2, 42–50. Lin, Y.S., Wang, J., Wang, X.M., Sun, X.H., 2011. Optimization of butanol production from corn straw hydrolysate by Clostridium acetobutylicum using response surface method. Chin Sci. Bull. 56, 1422–1428. Liu, Z.Y., Ying, Y., Li, F.L., Ma, C.Q., Xu, P., 2010. Butanol production by clostridium beijerinckii ATCC 55025 from wheat bran. J. Ind. Microbiol. Biotechnol. 37, 495– 501. Lu, C., Zhao, J.B., Yang, S.T., Wei, D., 2012. Fed-batch fermentation for n-butanol production from cassava bagasse hydrolysate in a fibrous bed bioreactor with continuous gas stripping. Bioresour. Technol. 104, 380–387. Maddox, I.S., 1989. The acetone-butanol-ethanol fermentation: recent progress in technology. Biotechnol. Genet. Eng. Rev. 7, 189–220. Malaviya, A., Jang, Y.S., Lee, S.Y., 2012. Continuous butanol production with reduced byproducts formation from glycerol by a hyper producing mutant of Clostridium pasteurianum. Appl. Microbiol. Biotechnol. 93, 1485–1494. Mutschlechner, O., Swoboda, H., Gapes, J.R., 2000. Continuous two-stage ABEfermentation using Clostridium beijerinckii NRRL B 592 operating with a growth rate in the first stage vessel close to its maximal value. J. Mol. Microbiol. Biotechnol. 2, 101–105. Najafpour, G.D., Shan, C.P., 2003. Enzymatic hydrolysis of molasses. Bioresour. Technol. 86, 91–94. Ni, Y., Sun, Z.H., 2009. Recent Progress on Industrial Fermentative Production of Acetone–Butanol–Ethanol by Clostridium acetobutylicum in China. Appl Microbiol Biotechnol. 83, 415–423. Ni, Y., Wang, Y., Sun, Z.H., 2012. Butanol Production from Cane Molasses by Clostridium saccharobutylicum DSM 13864: Batch and Semi-continuous fermentation. Appl. Biochem. Biotechnol. 166, 1896–1907. Qureshi, N., Blaschek, H., 2000. Economics of Butanol Fermentation using Hyper-Butanol Producing Clostridium Beijerinckii BA101. Food Bioprod. Process. 78, 139–144. Qureshi, N., Ezeji, T.C., 2008. Butanol, ‘a superior biofuel’ production from agricultural residues (renewable biomass): recent progress in technology. Biofuels. Bioprod. Bioref. 2, 319–330.
Y. Ni et al. / Bioresource Technology 129 (2013) 680–685 Qureshi, N., Ezeji, T.C., Ebener, J., Dien, B., Cotta, M., Blaschek, H.P., 2008. Butanol production by Clostridium Beijerinckii. Part I: Use of acid and enzyme hydrolyzed corn fiber. Bioresour. Technol. 99, 5915–5922. Qureshi, N., Saha, B.C., Dien, B., Hector, E.H., Cotta, M.A., 2010. Production of butanol (a biofuel) from agricultural residues: Part I-Use of barley straw hydrolysate. Biomass Bioenergy. 34, 559–565. Sun, Z.J., Liu, S.J., 2012. Production of n-butanol from concentrated sugar maple hemicellulosic hydrolysate by Clostridia acetobutylicum ATCC824. Biomass Bioenerg. 39, 39–47.
685
Survase, S.A., Heiningen, V.A., Granström, T., 2012. Continuous bio-catalytic conversion of sugar mixture to acetone-butanol-ethanol by immobilized Clostridium acetobutylicum DSM 792. Appl. Microbiol. Biotechnol. 93, 1–8. Tesun, K., Dhese, P., 1970. Sugar determination of DNS method. Biotechnol. Bioeng. 12, 921–922. Wouter, V.H., Vandezande, P., Claes, S., Vangeel, S., Beckers, H., Diels, L., Wever, H.D., 2012. Integrated bioprocess for long-term continuous cultivation of Clostridium acetobutylicum coupled to pervaporation with PDMS composite membranes. Bioresour. Technol. 111, 368–377.