ARTICLE High-Titer n-Butanol Production by Clostridium acetobutylicum JB200 in Fed-Batch Fermentation With Intermittent Gas Stripping Chuang Xue,1,2 Jingbo Zhao,2 Congcong Lu,2 Shang-Tian Yang,2 Fengwu Bai,1 I.-Ching Tang3 1
Department of Life Science and Biotechnology, Dalian University of Technology, Dalian, China 2 William G. Lowrie Department of Chemical and Biomolecular Engineering, The Ohio State University, Columbus, Ohio 43210; telephone: 614-292-6611; fax: 614-292-3769; e-mail:
[email protected] 3 Bioprocessing Innovative Company, Dublin, Ohio
ABSTRACT: Acetone–butanol–ethanol (ABE) fermentation with a hyper-butanol producing Clostridium acetobutylicum JB200 was studied for its potential to produce a high titer of butanol that can be readily recovered with gas stripping. In batch fermentation without gas stripping, a final butanol concentration of 19.1 g/L was produced from 86.4 g/L glucose consumed in 78 h, and butanol productivity and yield were 0.24 g/L h and 0.21 g/g, respectively. In contrast, when gas stripping was applied intermittently in fed-batch fermentation, 172 g/L ABE (113.3 g/L butanol, 49.2 g/L acetone, 9.7 g/L ethanol) were produced from 474.9 g/L glucose in six feeding cycles over 326 h. The overall productivity and yield were 0.53 g/L h and 0.36 g/g for ABE and 0.35 g/L h and 0.24 g/g for butanol, respectively. The higher productivity was attributed to the reduced butanol concentration in the fermentation broth by gas stripping that alleviated butanol inhibition, whereas the increased butanol yield could be attributed to the reduced acids accumulation as most acids produced in acidogenesis were reassimilated by cells for ABE production. The intermittent gas stripping produced a highly concentrated condensate containing 195.9 g/L ABE or 150.5 g/L butanol that far exceeded butanol
Correspondence to: S.-T. Yang Contract grant sponsor: Ohio Department of Development-Third Frontier Advanced Energy Program Contract grant number: Tech 08-036 Contract grant sponsor: National Science Foundation STTR Program Contract grant number: IIP-0810568; IIP-1026648 Contract grant sponsor: Advanced Research Projects Agency-Energy Contract grant number: DE-AR0000095 Contract grant sponsor: Fundamental Research Funds for the Central Universities Contract grant number: DUT11RC(3)77 Contract grant sponsor: China Postdoctoral Science Foundation Contract grant number: 20110491527 Contract grant sponsor: China Scholarship Council Contract grant number: 2009100607 Received 28 December 2011; Revision revised 7 May 2012; Accepted 14 May 2012 Accepted manuscript online 24 May 2012; Article first published online 8 June 2012 in Wiley Online Library (http://onlinelibrary.wiley.com/doi/10.1002/bit.24563/abstract) DOI 10.1002/bit.24563
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Biotechnology and Bioengineering, Vol. 109, No. 11, November, 2012
solubility in water. After liquid–liquid demixing or phase separation, a final product containing 610 g/L butanol, 40 g/L acetone, 10 g/L ethanol, and no acids was obtained. Compared to conventional ABE fermentation, the fed-batch fermentation with intermittent gas stripping has the potential to reduce at least 90% of energy consumption and water usage in n-butanol production from glucose. Biotechnol. Bioeng. 2012;109: 2746–2756. ß 2012 Wiley Periodicals, Inc. KEYWORDS: acetone–butanol–ethanol fermentation butanol; Clostridium acetobutylicum; fed-batch fermentation; gas stripping
Introduction Acetone–butanol–ethanol (ABE) fermentation by Clostridium acetobutylicum and related species was the second largest industrial fermentation before the rise of the petroleum refinery industry (Jones and Keis, 1995; Jones and Woods, 1986). Concerns on the eventual depletion of crude oils and escalating prices in petroleum-derived products have generated renewed interests in producing n-butanol from renewable bioresources (Du¨rre, 1998, 2007; Ezeji et al., 2007a; Lee et al., 2008). However, conventional ABE fermentation is limited by its low product titer, yield, and productivity due to severe inhibition caused by butanol (Ezeji et al., 2010; Nicolaou et al., 2010). Over the past three decades, extensive research and development efforts have been focused on finding solutions for overcoming the butanol toxicity problem. One approach is to increase the butanol tolerance of butanol-producing clostridia through mutagenesis and metabolic engineering (Papoutsakis, 2008; ß 2012 Wiley Periodicals, Inc.
Zheng et al., 2009), which has resulted in mutants that can produce up to 20 g/L of butanol (Chen and Blaschek, 1999). However, compared to 10% (w/v) of ethanol obtained in yeast fermentation, the butanol level at 2% (w/v) is still low and uneconomical because of intensive energy consumption in its recovery by distillation. Another approach is to integrate product recovery with fermentation to remove butanol in situ and thus reduce its inhibition effect and increase fermentation productivity (Ezeji et al., 2007b; Groot et al., 1992; Qureshi et al., 1992; Roffler et al., 1988), which has been demonstrated using several online butanol separation methods, including adsorption (Nielsen and Prather, 2009; Qureshi et al., 2005), liquid–liquid extraction (Barton and Daugulis, 1992; Evans and Wang, 1988; Roffler et al., 1987a), pervaporation (Matsumura et al., 1988; Qureshi and Blaschek, 1999), and gas stripping (Qureshi and Blaschek, 2001). Among them, gas stripping has been studied the most because it is simple to operate and scale up, is not harmful to cells, and does not require additional materials (Ennis et al., 1986; Ezeji et al., 2003, 2007a; Qureshi and Blaschek, 2001; Qureshi et al., 1992). However, gas stripping usually also removes a large amount of water with butanol and requires a higher energy input because of its lower butanol selectivity as compared to other separation techniques (Oudshoorn et al., 2009; Qureshi et al., 2005; Vane, 2008). To improve the performance of the integrated fermentation-gas stripping process for butanol production, optimization of gas stripping conditions, and a better understanding of its effects on the ABE fermentation are needed. All previous studies on integrated ABE fermentation with gas stripping were conducted at a relatively low butanol concentration of 8 g/L can produce a condensate with more than 150 g/L of butanol and a product of more than 640 g/L of butanol after phase separation, which would give a selectivity of more than 75 and should reduce the energy consumption by at least 2/3 or to
