1. Introduction
Biobutanol with a 30% higher energy content and lower water miscibility, volatility, flammability, and corrosiveness than ethanol is an attractive drop-in biofuel that can fit with the existing fuel infrastructure and be used in car engines without modification (Zhao et al., 2013). n -Butanol can also be dehydrated to 1-butene and further converted to longer-chain aviation fuels. As one of the oldest industrial fermentations (Jones and Woods, 1986; Moon et al., 2016; Soni et al. 1987), acetone-butanol-ethanol (ABE) fermentation by solventogenic clostridia including Clostridium acetobutylicum andClostridium beijerinckii has been extensively studied in various process modes, including batch, fed-batch, and continuous (Ezeji et al., 2004; Huang et al., 2004; Jiang et al., 2014; Lu et al., 2012; Qureshi et al., 2008). However, commercial application of ABE fermentation forn -butanol and acetone production is hindered by the high production cost (Kumar et al., 2013; Lu et al., 2013; Li et al., 2019). Even after extensive efforts to develop engineered strains and novel process strategies, batch ABE fermentation suffers from low productivity and is unable to compete with solvents produced through petrochemical routes (Cheng et al., 2019; Lee SY et al., 2008; Wang et al., 2014; Xue et al., 2017; Zhao et al., 2013).
A typical batch ABE fermentation usually completes in ~72 h with a productivity of ~0.2 g/L∙h and a final butanol concentration of ~12 g/L and yield of ~0.2 g/g glucose (Xu et al., 2015). Compared to batch fermentation, continuous fermentation offers several advantages including higher productivity and little downtime. For ABE fermentation in a bioreactor with continuous supply of nutrients at a high dilution rate, solvent productivity was improved by over 10-fold to >2 g/L∙h; however, the final solvent titer decreased to ~5 g/L, which would drastically increase downstream processing costs (Pierrot et al., 1986; Qureshi and Maddox, 1991). In order to enhance butanol productivity, various techniques, including cell immobilization and in situ butanol recovery, have been incorporated into ABE fermentation to increase cell density, alleviate butanol toxicity, and increase overall productivity (Cai et al., 2016; Lu et al., 2012; Nguyen et al., 2018; Xue et al., 2016a; 2016b). Cell immobilization on solid support materials, such as brick, bonechar, chitosan, and corn stalk, increased cell density in fermentation and improved ABE productivity to as high as ~10 g/L∙h (Frick and Schugerl, 1986; Qureshi et al., 1988; Zhang et al. 2009). Notably, cell immobilization in a highly porous fibrous matrix not only greatly increased cell density in the bioreactor but also facilitated the adaptation of cells to better tolerate environmental stress with over 50% improvements in product titer, yield, and productivity in fermentations for organic acids (Suwannakham and Yang, 2005; Wei et al., 2013; Yang et al., 1994; Zhu et al., 2002; Zhu and Yang, 2003) as well as butanol production (Huang et al., 1998; Huang et al., 2019; Jiang et al., 2014; Li et al., 2019).
However, ABE fermentation is difficult to operate and control in a continuous fermentation process (Al-Shorgani et al., 2019) because of its complex life cycle involving acidogenesis, solventogenesis, and sporulation that are highly regulated by multiple gene regulators involving various kinases, transcription factors, and interlocking signal transduction pathways (Al-Hinai et al., 2015; Steiner et al., 2011; Yang et al., 2018). Acid crash (due to failed transition from acidogenesis to solventogenesis), sporulation (induced by environmental stresses such as butanol toxicity), and strain degeneration (lost solvent production due to cells losing the mega-plasmid carrying thesol operon) are among the major causes for low productivity and short production duration associated with C. acetobutylicum (Long and Jones, 1984; Lütke-Eversloh and Bahl, 2011). Under stress, clostridia sporulate and halt their metabolism (Diallo et al., 2021), limiting their ability to produce butanol at desirable titers, rates, and yields and longevity for continuous operation.
In this study, the asporogenous C. acetobutylicum ATCC55025 derived from the Weizmann strain ATCC4259 was used for continuousn -butanol production from glucose and butyrate in a single-pass fibrous bed bioreactor (FBB). C. acetobutylicum ATCC 55025 is deficient in forming endospores and has a high butyrate uptake rate and butanol productivity (Xu et al., 2017). With butyric acid in the feed medium and cells immobilized in the FBB, continuous ABE fermentation can be stably maintained in the solventogenic phase at a high rate with >16.5 g/L∙h butanol productivity, which was the highest productivity ever reported for a biobutanol fermentation process. The effects of feed butyrate concentration and dilution rate on the single-pass continuous bioreactor were studied and the results are reported in this paper.