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.