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\begin{document}

\title{High-rate continuous n -butanol production by Clostridium acetobutylicum
from glucose and butyric acid in a single-pass fibrous bed bioreactor}



\author[1]{Wei-Lun Chang}%
\author[1]{Wenjie Hou}%
\author[1]{Mengmeng Xu}%
\author[1]{Shang-Tian Yang}%
\affil[1]{The Ohio State University William G Lowrie Department of Chemical and Biomolecular Engineering}%


\vspace{-1em}



  
  \date{May 12, 2024}


\begingroup
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\selectlanguage{english}
\begin{abstract}
Biobutanol produced in acetone-butanol-ethanol fermentation at batch
mode cannot compete with chemically derived butanol because of the low
reactor productivity. Continuous fermentation can dramatically enhance
productivity and lower capital and operating costs but are rarely used
in industrial fermentation because of increased risks in culture
degeneration, cell washout, and contamination. In this study, cells of
the asporogenous \emph{Clostridium acetobutylicum} ATCC55025 were
immobilized in a single-pass fibrous-bed bioreactor (FBB) for continuous
production of butanol from glucose and butyrate at various dilution
rates. Butyric acid in the feed medium helped maintaining cells in the
solventogenic phase for stable continuous butanol production. At the
dilution rate of 1.88 h \textsuperscript{-1}, butanol was produced at
9.55 g/L with a yield of 0.24 g/g and productivity of 16.8 g/L[?]h, which
was the highest ever achieved for biobutanol fermentation and an 80-fold
improvement over the conventional ABE fermentation. The extremely high
productivity was attributed to the high density of viable cells
(\textasciitilde{}100 g/L at \textgreater{}70\% viability) immobilized
in the fibrous matrix, which also enabled the cells to better tolerate
butanol and butyric acid. The FBB was stable for continuous operation
for an extended period of over one month.%
\end{abstract}%



\sloppy


\textbf{High-rate continuous \emph{n}-butanol production
by\emph{Clostridium acetobutylicum} from glucose and butyric acid in a
single-pass fibrous bed bioreactor}

Wei-Lun Chang \textsuperscript{a}, Wenjie Hou \textsuperscript{a,b},
Mengmeng Xu \textsuperscript{a}, Shang-Tian Yang \textsuperscript{a,}*

\textsuperscript{a} William G. Lowrie Department of Chemical and
Biomolecular Engineering, The Ohio State University, 151 West Woodruff
Avenue, Columbus, OH 43210, USA

\textsuperscript{b} College of Life Science, Northwest A\&F University,
Yangling, Shaanxi 712100, China

*Corresponding author: Phone: 1-614-2926611; Fax: 1-614-2923769; email:
yang.15@osu.edu

Submitted to \emph{Biotechnology \& Bioengineering}

\textbf{Abstract}

Biobutanol produced in acetone-butanol-ethanol fermentation at batch
mode cannot compete with chemically derived butanol because of the low
reactor productivity. Continuous fermentation can dramatically enhance
productivity and lower capital and operating costs but are rarely used
in industrial fermentation because of increased risks in culture
degeneration, cell washout, and contamination. In this study, cells of
the asporogenous \emph{Clostridium acetobutylicum} ATCC55025 were
immobilized in a single-pass fibrous-bed bioreactor (FBB) for continuous
production of butanol from glucose and butyrate at various dilution
rates. Butyric acid in the feed medium helped maintaining cells in the
solventogenic phase for stable continuous butanol production. At the
dilution rate of 1.88 h\textsuperscript{-1}, butanol was produced at
9.55 g/L with a yield of 0.24 g/g and productivity of 16.8 g/L[?]h, which
was the highest ever achieved for biobutanol fermentation and an 80-fold
improvement over the conventional ABE fermentation. The extremely high
productivity was attributed to the high density of viable cells
(\textasciitilde{}100 g/L at \textgreater{}70\% viability) immobilized
in the fibrous matrix, which also enabled the cells to better tolerate
butanol and butyric acid. The FBB was stable for continuous operation
for an extended period of over one month.

\textbf{Keywords} : Acetone-butanol-ethanol fermentation; continuous
fermentation; \emph{Clostridium acetobutylicum} ; fibrous bed bioreactor



\textbf{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). \emph{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 \emph{Clostridium acetobutylicum}
and\emph{Clostridium 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 for\emph{n} -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
\textasciitilde{}72 h with a productivity of \textasciitilde{}0.2 g/L[?]h
and a final butanol concentration of \textasciitilde{}12 g/L and yield
of \textasciitilde{}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
\textgreater{}2 g/L[?]h; however, the final solvent titer decreased to
\textasciitilde{}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 \emph{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 \textasciitilde{}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
the\emph{sol} operon) are among the major causes for low productivity
and short production duration associated with \emph{C. acetobutylicum}
(Long and Jones, 1984; L\selectlanguage{ngerman}ütke-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 \emph{C. acetobutylicum} ATCC55025
derived from the Weizmann strain ATCC4259 was used for
continuous\emph{n} -butanol production from glucose and butyrate in a
single-pass fibrous bed bioreactor (FBB). \emph{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 \textgreater{}16.5 g/L\selectlanguage{english}[?]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.

\textbf{2. Materials and Methods}

2.1 Cultures and Media

\emph{C. acetobutylicum} ATCC55025 was cultured in modified P2 medium
containing (per liter, at pH 5.5): 0.5 g
KH\textsubscript{2}PO\textsubscript{4}, 0.5 g
K\textsubscript{2}HPO\textsubscript{4}, 1 g yeast extract, 0.5 g
tryptone, 0.2 g MgSO\textsubscript{4}\selectlanguage{english}[?]7H\textsubscript{2}O, 0.01 g
MnSO\textsubscript{4}\selectlanguage{english}[?]H\textsubscript{2}O, 0.01 g NaCl, and 0.01 g
FeSO\textsubscript{4}\selectlanguage{english}[?]7H\textsubscript{2}O, 0.1 mg \emph{p}
-aminobenzoic acid, 0.1 mg thiamine, 1 \selectlanguage{greek}µ\selectlanguage{english}g biotin, and 50 g glucose as
the carbon source, unless otherwise noted (Xu et al., 2015).
Concentrated mineral (MgSO\textsubscript{4}[?]7H\textsubscript{2}O,
MnSO\textsubscript{4}[?]H\textsubscript{2}O, NaCl,
FeSO\textsubscript{4}[?]7H\textsubscript{2}O) and vitamin (\emph{p}
-aminobenzoic acid, thiamine, biotin) solutions were prepared
separately, filter-sterilized with 0.2 \selectlanguage{greek}µ\selectlanguage{english}m pore size filter and stored in
a refrigerator until use. The medium without glucose was sterilized by
autoclaving at 121 \selectlanguage{ngerman}ºC for 30 minutes. The main carbon source, glucose,
was sterilized separately in a concentrated solution and added
aseptically to the medium to a final concentration of
\textasciitilde{}50 g/L or as specified. Butyric acid at various amounts
(up to 10 g/L) was also added to the medium to study its effects on cell
growth and fermentation kinetics. The medium was adjusted to pH 5.5 with
NH\textsubscript{4}OH and purged with sterile N\textsubscript{2} for
\textasciitilde{}30 min to reach anaerobiosis before use.

2.2 Batch fermentation

Batch fermentation in serum bottles and bioreactors was conducted to
evaluate the effect of butyric acid on the growth of \emph{C.
acetobutylicum} ATCC55025. Each bottle containing 100 ml of P2 medium
with 20 g/L glucose and various amounts of butyric acid at an initial
concentration of 0, 2, 5, or 10 g/L was inoculated with 3 mL of 24-h
active culture and incubated at 37 \textsuperscript{o}C without shaking.
Liquid samples were withdrawn from the bottles periodically to monitor
changes in the cell density by measuring the optical density at 600 nm
(OD\textsubscript{600}). Batch fermentation was also conducted in a
500-mL stirred-tank bioreactor containing 250 mL P2 medium with
\textasciitilde{}50 g/L glucose and 4 g/L butyric acid as the carbon
sources. The reactor pH was controlled at \textasciitilde{}5.0 with
ammonium hydroxide (20\% w/v). Liquid samples were withdrawn
periodically for analyses of cell (optical density,
OD\textsubscript{600}), glucose, butyric and acetic acids, and solvents
(acetone, butanol, and ethanol).

2.3 Continuous Fermentation

Continuous fermentation was conducted with cells immobilized in a
fibrous bed bioreactor, which was made of a water-jacketed glass column.
The glass column with a working volume of 500 mL was packed with a
spirally wound cotton towel laminated with a stainless-steel wire mesh
as spacers between adjacent fibrous matrices (\textbf{Figure 1} ). The
FBB was autoclaved at 121 \textsuperscript{o}C for 30 min, left
overnight at room temperature to allow spore germination, and autoclaved
again for one hour. The FBB was purged with N\textsubscript{2} from the
bottom of the bioreactor for \textasciitilde{}1 h to remove air and then
aseptically connected to a 10-L feed medium bottle containing sterile P2
medium. The bioreactor was then partially filled with the medium while
continued being sparged with N\textsubscript{2} for 10 min and
inoculated with 60\textasciitilde{}100 mL of an overnight culture grown
in a serum bottle. The FBB was controlled at 35 \textsuperscript{o}C and
held static for 12\textasciitilde{}24 h to allow cells to grow and
attach/adsorb onto the fibrous bed. When significant gas production
(bubbles) was observed and the fermentation broth had reached
\textasciitilde{}1.0 OD\textsubscript{600}, continuous fermentation was
started by pumping the feed medium containing 50 g/L glucose and 4 g/L
butyric acid into the bottom of the FBB at a constant dilution rate of
0.06 h\textsuperscript{-1}. The continuous fermentation study was
operated for over 28 days or until reaching a pseudo-steady state. The
continuous fermentation was then studied in the same FBB fed with P2
medium containing 60 g/L glucose and 6.8 g/L butyric acid at 0.12
h\textsuperscript{-1} dilution rate. A smaller FBB with a 125-mL working
volume was also set up for studying the continuous fermentation at a
higher dilution rate of 1.88 h\textsuperscript{-1}. Fermentation broth
samples were taken periodically from the effluent stream throughout the
continuous fermentation studies and analyzed for
OD\textsubscript{600}and concentrations of glucose, butyric and acetic
acids, and solvents (acetone, butanol, and ethanol). At the end of the
continuous fermentation studies, the fibrous matrix with immobilized
cells was removed from the FBB and examined for cell density, viability,
and morphology.

2.4 Scanning Electron Microscopy

Scanning electron microscopy (SEM) was used to study cells immobilized
on the fibrous matrix. Small pieces of fibrous matrices with cells cut
from various locations in the FBB were examined at 20 kV with FEI Quanta
200 SEM. Samples were fixed in 2.5\% glutaraldehyde solution at 4 °C
overnight, dehydrated with ethanol progressively from 20\% to 100\% at
20\% increment for 30 min at each step, dried with hexamethyldisilazane
(HMDS), and spotter-coated with gold particles under argon gas. The
samples were scanned, photographed, and analyzed visually for cell
morphology.

2.5 Cell Viability Assay

The modified method of Glenner (Glenner, 1977) was used. Briefly, cells
were collected from 1 mL culture broth by centrifugation (16,000 rpm for
10 min) and resuspended in 1 mL 2,3,5-triphenyl-2H-tetrazolium chloride
(TTC) solution (1 g/L) by vigorously vortexing. After incubation in dark
at room temperature for 30 min, cells were collected by centrifugation
and mixed with 1 mL methanol to extract the pink pigment. The color
intensity in the supernatant was measured at 485 nm using a
spectrophotometer (UV-16-1, Shimazu, Columbia, MD). The viability of
tested cells was determined using the exponential-phase cells as the
control with 100\% viability and boiled (dead) cells as the negative
control with 0\% viability.

2.6 Analytical Methods

The optical density at 600 nm (OD\textsubscript{600}) was measured in a
1.5-mL cuvette (1 cm light path length) with a spectrophotometer
(UV-16-1, Shimazu). One unit of OD\textsubscript{600} was equivalent to
0.534 g/L cell dry weight. Glucose in the fermentation broth samples was
determined with a YSI 2300 glucose analyzer (Yellow Spring, OH). All
metabolites including acetone, butanol, ethanol, acetic acid, and
butyric acid in the fermentation broth samples were analyzed, after
removing cells by centrifugation or microfiltration using 0.2-\selectlanguage{greek}µ\selectlanguage{english}m syringe
filters and acidified with phosphoric acid (1\% v/v), with a gas
chromatograph (GC-2014, Shimadzu) following the method described
elsewhere (Xu et al., 2015).

\textbf{3. Results and Discussion}

3.1 Batch Fermentation Kinetics

Butyric acid is a strong growth inhibitor and plays a critical role in
regulating cell metabolism in ABE fermentation, which switches from
acidogenesis to solventogenesis when the butyric acid concentration
reaches a critical level in the culture medium (Bahl et al., 1982).
Previous studies have shown that adding a small amount of butyric acid
in the culture medium could induce early solventogenesis and increase
butanol production (Huang et al., 2004; Lee SM et al., 2008). The effect
of butyric acid on cell growth was first investigated in serum bottles
and the results are shown in \textbf{Figure 2} . As expected, cell
growth was strongly inhibited by butyric acid and no growth was observed
at 10 g/L of butyric acid. Then, batch fermentation kinetics with medium
initially containing 4 g/L butyric acid in a stirred-tank bioreactor
with pH controlled at \textasciitilde{}5.0 was investigated. After a
12-h lag phase, cells grew to reach the maximum OD of
\textasciitilde{}2.0 at 34 h and then decreased gradually, whereas both
butyric acid consumption and solvent production started when cell growth
ceased at 34 h (\textbf{Figure 3} ). About 13.1 g/L butanol, 5.6 g/L
acetone, and 0.9 g/L ethanol were produced from \textasciitilde{}50 g/L
glucose and \textasciitilde{}2 g/L butyric acid consumed in the
fermentation ended at \textasciitilde{}311 h, which was much longer than
the typical \textasciitilde{}72 h for batch ABE fermentation with
glucose but without butyric acid addition in the medium (\textbf{Figure
S1} ). The butanol yield of \textasciitilde{}0.26 g/g glucose obtained
in the fermentation with 4 g/L butyric acid was significantly higher
than that obtained in typical ABE fermentation (\textasciitilde{}0.20
g/g glucose), whereas the productivity was much lower at
\textasciitilde{}0.05 g/L[?]h (vs. \textasciitilde{}0.2 g/L[?]h) due to the
strong inhibition by butyric acid resulting in a much lower cell density
of \textless{}2.0 OD (vs. Max. OD of \textasciitilde{}14 reached in
batch fermentation without butyric acid added in the culture medium).
The results indicated that the addition of butyric acid could maintain
stable butanol production in ABE fermentation, but the optimal butyric
acid concentration must be explored in order to minimize its inhibition
effect on cell growth and reactor productivity.

3.2 Continuous Fermentation

Continuous ABE fermentation with cells immobilized in a FBB was studied
at different dilution rates and butyric acid concentrations in the feed
medium. \textbf{Figure 4} shows the time-course data on OD, and glucose,
butyrate, acetate, and ABE concentrations in the effluent from the
single-pass FBB fed with the medium containing 50 g/L glucose and 4 g/L
butyric acid at the dilution rate of 0.06 h\textsuperscript{-1}. Cell OD
and butanol production increased rapidly and reached
\textasciitilde{}3.0 and \textasciitilde{}5 g/L, respectively, at 181 h
but then decreased. The bioreactor was drained and refilled aseptically
with a fresh medium at 290 h, which restored and increased butanol
production to \textasciitilde{}8.4 g/L at 490 h. The continuous
fermentation maintained a pseudo-steady state for the next 160 h, with
butanol production fluctuated between 8.4 and 9.4 g/L. During this
period, about 25\% of the butyric acid (1 g/L) in the feed medium was
consumed and the average butanol yield and productivity were
\textasciitilde{}0.24 g/g and \textasciitilde{}0.52 g/L[?]h, respectively.
There was continuous cell bleeding from the FBB throughout the
continuous fermentation, which remained at \textasciitilde{}OD 1.0
during the pseudo-steady state. To investigate if more butyrate could be
consumed by cells, the feed butyrate concentration was raised to 8 g/L
at 700 h. Soon after increasing the feed butyric acid concentration to 8
g/L, butanol concentration in the outlet stream started to drop rapidly
to less than 1 g/L and both glucose and butyric acid concentrations
approached the feed concentrations at 890 h (see \textbf{Figure S2} ),
indicating cells were completely inhibited by butyric acid at 8 g/L.

The continuous fermentation was then studied at a higher dilution rate
of 0.12 h\textsuperscript{-1}, with the feed medium containing 60 g/L
glucose and 6.8 g/L butyric acid. As shown in \textbf{Figure 5} ,
butanol production increased to \textasciitilde{}10 g/L while glucose
concentration decreased to \textasciitilde{}20 g/L and butyric acid
decreased to \textasciitilde{}2.6 g/L when the fermentation reached a
pseudo-steady state at \textasciitilde{}480 h. Afterward, the continuous
fermentation produced butanol at the average yield of
\textasciitilde{}0.24 g/g and productivity of \textasciitilde{}1.2
g/L[?]h, which was 2.3-fold of that obtained at the lower dilution rate of
0.06 h\textsuperscript{-1}. It should be noted that butanol production
dropped from 9.1 g/L at 340 h to 7.3 g/L at 390 h, probably because
cells were strongly inhibited by butanol at \textgreater{}8 g/L.
However, the reactor was able to recover by itself and continue to
increase butanol production to reach \textasciitilde{}10.5 g/L at the
pseudo-steady state. The ability of the self-recovery from butanol
stress could be attributed to the adaptation of cells in the FBB, which
showed the resilience of the immobilized cell bioreactor. The effluent
cell OD reached the maximum value of \textasciitilde{}2.6 at 170 h and
then decreased to \textasciitilde{}0.8 during the pseudo-steady state.
To evaluate the reproducibility of the FBB performance, the reactor was
drained and refilled with fresh media at \textasciitilde{}610 h. The
effluent butanol concentration reached \textasciitilde{}10.5 g/L in 200
h and stayed in a pseudo-steady state for the next 200 h with average
butanol production of 10.8 g/L (see \textbf{Figure S3} ), similar to
that obtained in the previous pseudo-state state.

In general, the reactor productivity would increase with increasing the
dilution rate in the continuous fermentation. To explore the optimal
dilution rate for maximum productivity, the continuous fermentation was
further studied in a smaller FBB with a 150-mL working volume at a high
dilution rate of 1.88 h\textsuperscript{-1}. The feed medium contained
60 g/L glucose and 5 g/L butyric acid. Soon after the feeding started,
the outlet butanol concentration and cell OD rapidly increased and
reached 11.6 g/L and 6.47, respectively, while glucose dropped to 2.5
g/L in \textasciitilde{}140 h (\textbf{Figure 6} ). Then butanol
concentration dropped to less than 8 g/L with glucose back up to
\textgreater{}20 g/L in the next 20 h. The sudden drop in butanol
production could be attributed to butanol toxicity as well as glucose
starvation. An excessive amount or a high concentration of sugar is
essential for both onset and maintenance of solventogenesis in
clostridial fermentation (Jones and Woods, 1986). When the glucose was
back up to \textasciitilde{}20 g/L, butanol production was restored to
\textasciitilde{}9.5 g/L and then fluctuated between 8.5 and 10.5 g/L
for the next 200 h in the pseudo-steady state. The fluctuation in
butanol production could be attributed to the response of cells to
butanol stress. At the high dilution rate, butanol production in the FBB
reached \textasciitilde{}9.5 g/L at a high productivity of
\textasciitilde{}16.8 g/L[?]h and with an average yield of 0.24 g/g
glucose. About 3 g/L or 60\% of the butyric acid in the feed medium was
co-metabolized with glucose in the fermentation. The effluent cell OD
reached the maximum value of 6.47 at \textasciitilde{}140 h and then
decreased to \textasciitilde{}3.0 by \textasciitilde{}200 h and stayed
at that level during the pseudo-steady state period (200 -- 380 h).

\textbf{Table 1} summarizes and compares the results from batch and
continuous fermentations at different dilution rates and feed butyric
acid concentrations. In general, butanol yields were
\textasciitilde{}0.24 g/g in the continuous fermentation at all dilution
rates studied while the reactor productivity increased proportionally
with the dilution rate from 0.52 g/L[?]h at 0.06 h\textsuperscript{-1}to
16.8 g/L[?]h at 1.88 h\textsuperscript{-1}, which was much higher than the
maximum specific growth rate (0.2\textasciitilde{}0.45
h\textsuperscript{-1}) observed for \emph{C. acetobutylicum} . Cells
immobilization allowed the continuous fermentation to operate at a
dilution rate much higher than the specific growth rate without cell
washout. Cells had a higher activity and faster growth at the higher
dilution rate, as evidenced by the higher max. OD of 6.47 at 1.88
h\textsuperscript{-1} (vs. 3.0 at 0.06 h\textsuperscript{-1}) and
steady-state OD in the effluent (3.0 at 1.88 h\textsuperscript{-1} vs.
1.0 at 0.06 h\textsuperscript{-1}). The continuous immobilized-cell
bioreactor is thus advantageous to operate at a high dilution rate
although the final butanol titer and yield were slightly lower than
those in batch fermentation.

3.3 Effects of Butyric Acid

In a typical ABE fermentation, acetic and butyric acids produced in
acidogenesis were reassimilated via the CoA transferase, which transfers
the CoA from acetoacetyl-CoA to acetate and butyrate to form acetyl-CoA
and butyryl-CoA, respectively, with acetone, ethanol, and butanol as the
final products (Long and Jones, 1984; Zhao et al., 2013). Previous
studies have shown that overexpressing \emph{ctf} AB encoding the CoA
transferase could increase butanol production and the robustness of
solventogenic clostridia (Lu et al., 2017; Yu et al., 2015) and butyric
acid could upregulate \emph{ctf} AB (Bahl et al., 1982; Lee SM et al.,
2008). In this study, butyric acid was thus supplemented in the feed
medium to promote solventogenesis in clostridial fermentation, resulting
in \textasciitilde{}20\% higher butanol yield (0. 24 g/g vs.
\textasciitilde{}0.20 g/g) compared to without butyric acid addition.
The increased butanol yield could also be attributed to reduced cell
growth and acetone and ethanol production, which resulted in a much
higher ratio of butanol present in the total solvents
(\textasciitilde{}68\% vs. \textasciitilde{}60\% w/w in typical ABE
fermentation with C. \emph{acetobutylicum} (Xu et al., 2015). Similar
results were also reported for \emph{C. beijerinckii} (Lee SM et al.,
2008). The addition of butyric acid as a carbon source and precursor for
butanol biosynthesis inhibited the conversion of butyryl-CoA to butyric
acid and thus increased carbon flow toward butanol production. However,
butyric acid at \textgreater{}5 g/L strongly inhibited cell growth and
reduced cell viability and productivity in free cell fermentation (see
Figs. 2 and 3). Nevertheless, cells immobilized in the FBB were
resilient to butyric acid toxicity and could tolerate butyric acid at
\textasciitilde{}5 g/L, although butyric acid at 8 g/L completely halted
cell metabolism in the FBB (Fig. S2).

3.4 Cell Density, Viability, and Morphology in FBB

Cells in the fibrous matrices were collected from the FBB at the end of
the continuous fermentation study and analyzed for their density,
viability, and morphology. In general, more cells were observed at the
lower parts closer to the entrance of the reactor with higher glucose
and lower butanol concentrations since cell metabolism would be faster
with higher concentrations of nutrients and lower concentrations of
inhibiting end products. Biofilms with dense and thick layers of
elongated rod-shaped cells covering the fibers in the fibrous matrices
were observed throughout the fibrous bed, as can be seen in the scanning
electron micrographs (\textbf{Figure 7} ). The total cell density in the
FBB was approx. 100 \selectlanguage{ngerman}± 15 g cell dry weight/L reactor with
\textgreater{}70\% cell viability. No obvious sporulation nor autolysis
was observed even for cells from the top part of the bioreactor,
suggesting that cells in the FBB remained healthy and were highly active
throughout the entire process over a period of more than
400\textasciitilde{}600 h. It should be noted that there were continuous
cell bleedings (\textasciitilde{}1.0 OD at 0.06 h\textsuperscript{-1}and
0.12 h\textsuperscript{-1} and \textasciitilde{}3.0 OD at 1.88
h\textsuperscript{-1}) from the FBB throughout the continuous
fermentation, which allowed for continuous removal of old or stressed
cells to be replaced by new and more productive cells (Lewis and Yang,
1992). Furthermore, cells with the elongated morphology in the FBB were
adapted to better tolerate toxic metabolites such as butyric acid and
butanol. The elongated rod morphology with a higher specific surface
area could increase mass transfer and efflux for exchanges of nutrients
and metabolites between cells and the environment as previously reported
(Suwannakham and Yang, 2005; Zhu and Yang, 2003). The beneficial effects
of cell immobilization in the FBB including enhanced tolerance to toxic
metabolic end products and increased product titer, productivity and
yield have also been reported for other clostridia such as \emph{C.
tyrobutyricum} (Jiang et al., 2011) and \emph{C. formicoaceticum} (Huang
et al., 1998) and other bacteria such as propionibacteria (Zhang and
Yang, 2009).

3.5 Comparison to Other Studies

\textbf{Table 2} summarizes and compare the solvent titer, productivity,
and yield of ABE fermentation obtained in this study and other notable
continuous fermentation studies. In general, continuous fermentation
systems can dramatically enhance productivity and lower capital costs by
reducing the size of the fermentation system compared to batch
fermentation (Vees et al., 2020). Continuous systems can experience
little to no downtime between batches and have other processing
advantages leading to increased productivity. However, continuous
fermentation has rarely been used in industry because of increased risks
in culture degeneration, cell washout and contamination during operation
for an extended period that may result in catastrophic production lost.
Also, the high productivity obtained at a high dilution rate is usually
at the expense of incomplete or low substrate conversion. To overcome
these problems, continuous fermentation with cell recycling and/or
retention via immobilization in the bioreactor has been used to attain a
high cell density of \textasciitilde{}100 g DCW/L reactor volume with
greatly increased reactor productivity of \textgreater{}2 g/L\selectlanguage{english}[?]h (Huang
et al., 2004; Jang et al., 2013). Various solid support materials have
been applied for cell immobilization via adsorption and entrapment (Badr
et al., 2001; Bankar et al., 2012; Chang et al., 2016; Davison and
Thompson 1993; Frick and Schugerl, 1986; Gallazzi et al., 2015; Huang et
al., 2004; Kong et al., 2015; Qureshi and Maddox, 1988; Qureshi et al.,
2000; Survase et al., 2012; Zhang et al. 2009). However, immobilized
cell bioreactors such as packed-bed and membrane bioreactors often
suffer from lost/declined productivity during prolonged operation due to
the accumulation of dead, aging or non-viable cells, which also causes
clogging/fouling and limits reactor's operating life to less than a few
weeks (Qureshi and Maddox, 1988; Qureshi et al., 2000; Zhang et al.,
2009). For continuous fermentation with cell recycling through
microfiltration, cell bleeding is necessary to avoid over accumulation
of dead and inactive cells and to prolong the reactor life (Tashiro et
al., 2005).

In the present study, the ABE productivity of 24.2 g/L\selectlanguage{english}[?]h obtained in the
single-pass FBB with ATCC55025 at the dilution rate of 1.88
h\textsuperscript{-1} was the highest ever reported to date. In general,
a higher productivity can be obtained with a higher dilution rate in a
continuous fermentation process. However, a higher dilution rate usually
results in lower substrate conversion and butanol production, which will
increase production cost (Huang et al., 2019). Furthermore, a high
dilution rate usually favors cell growth and acid production in ABE
fermentation. To maintain a high productivity while also achieve a high
conversion with high butanol yield, continuous fermentation with stirred
tank reactors (STR) or recirculating packed bed reactors (PBR) may have
to be operated with multiple stages (Badr et al., 2001; Chang et al.,
2016; Frick and Schugerl, 1986), which increases the capital cost. In
this study, butyric acid was used in the feed medium as a co-substrate
with glucose to keep cells in the single-pass FBB in the solventogenesis
phase. It has also been reported that adding butyric acid in the feed
medium could improve butanol production in a continuous STR (Lee SM et
al., 2008).

A PBR with solid support particles like brick (Qureshi et al., 2000) and
ceramic beads (Badr et al., 2001) suffered from low void volume, high
pressure drop, and clogging and channeling due to the accumulation of
cell biomass, which impeded the reactor performance and operating life.
In this study, cells were immobilized in the highly porous fibrous
matrix spirally wound with gaps between the matrix layers as flow
channels to allow for free flow of fermentation broth, suspended solids
(cells), and gases (CO\textsubscript{2} and H\textsubscript{2}) through
the fibrous bed with a low pressure drop without clogging occurring to
conventional packed-bed bioreactors (Zhu et al., 2002). Consequently,
the FBB could have stable performance throughout the entire operation
period of over several months as demonstrated in our previous studies
(Lewis and Yang, 1992). Moreover, the FBB with greatly increased cell
density also facilitated in-process adaptation or evolutionary
engineering of cells to attain higher tolerance to toxic chemicals
(e.g., organic acids and butanol) and increase product titer, yield, and
productivity as demonstrated in previous studies (Huang et al., 1998; Li
et al., 2019; Suwannakham and Yang, 2005; Wei et al., 2013; Yang et al.,
1994; Zhu and Yang, 2003). Since butyric acid and butanol are highly
inhibitory to most microorganisms, no contamination was found throughout
the continuous fermentation study. The continuous FBB was operated for
over 30 days without encountering any performance issues.

The continuous fermentation process can be operated with \emph{in
situ}product separation to alleviate butanol toxicity and increase final
product titer and reactor productivity (Veza et al., 2021; Yang and Lu,
2013). Gas stripping (Lu et al., 2012; Xue et al., 2016b), adsorption
(Xue et al., 2016a), extraction (Bankar et al., 2012; Davison and
Thompson 1993), and pervaporation (Cai et al., 2016; Zhu et al., 2018)
are the most studied \emph{in situ} butanol separation methods. More
recently, vacuum distillation was applied to continuously recover ABE
from the fermentation broth in a separate tank, achieving a high final
butanol titer of 550 g/L and productivity of 14 g/L\selectlanguage{english}[?]h in a continuous
ABE fermentation with cell recycling operated at a dilute rate of 0.076
h\textsuperscript{-1} (Nguyen et al., 2018). The process maintained a
steady state for \textasciitilde{}170 h. The continuous single-pass FBB
can be integrated with gas stripping and pervaporation (or vapor
stripping-vapor permeation, VSVP) to further increase productivity and
product titer to higher than 600 g/L (Du et al., 2021; Lu et al., 2012).

\textbf{4. Conclusions}

Continuous fermentation with the asporogenous \emph{C.
acetobutylicum}ATCC55025 immobilized in a fibrous bed bioreactor was
studied for\emph{n} -butanol production from glucose and butyric acid.
At a high dilution rate of 1.88 h\textsuperscript{-1}, butanol
production at a high productivity of 16.8 g/L\selectlanguage{english}[?]h, more than 80 times of
that in batch free-cell fermentation, was obtained. Although butyric
acid strongly inhibited cell growth, butyric acid added in the feed
medium helped maintain stable butanol production and improved butanol
yield from glucose by \textasciitilde{}20\%. The risk of contamination
by commensal bacteria was reduced by butyric acid and butanol, allowing
the continuous process to operate for an extended period over 30 days.

\textbf{Acknowledgements}

This work was supported by the National Science Foundation STTR program
(IIP-0810568, IIP-1026648) and the Ohio Department of Development --
Third Frontier Advanced Energy Program (Tech 08-036).

\textbf{Appendix A. Supplementary material}

Supplementary data associated with this article can be found in the
online version. All other relevant data that support the findings are
available from the corresponding author upon request.

\textbf{Conflict of Interests Statement} : The authors declare no
competing financial interests.

\textbf{Author Contribution Statement} : Chang and Yang contributed to
the initial conceptual and experimental design. Chang performed all
experiments and data analysis, and wrote the initial draft of the
manuscript, which was reviewed with inputs from Xu and Hou. Yang revised
and prepared the final version of the manuscript, which was read and
approved by all authors.

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\textbf{Figure Captions}

\textbf{Figure 1} . Experimental setup for the continuous fermentation
with clostridial cells immobilized in a fibrous bed bioreactor (FBB).
Inset diagram shows the construction of the spirally wound fibrous
matrices with stainless steel wire mesh as the mechanical support with
gaps between adjacent layers as flow channels for liquid, gas, and
solids (cells) to move freely. The highly porous fibrous matrix has a
large surface area and void volume (\textgreater{}90\% reactor volume)
to allow high densities of viable cells (up to 100 g/L cell dry weight)
to be immobilized by attachment to fiber surfaces and entrapment within
the matrix.

\textbf{Figure 2} . Effect of butyric acid on cell growth. Cells
of\emph{C. acetobutylicum} ATCC 55025 were cultured in P2 medium
containing 20 g/L glucose and various amounts of butyric acid (0, 2, 5,
or 10 g/L) in serum bottles at 37 \textsuperscript{o}C.

\textbf{Figure 3} . Kinetics of batch fermentation with free cells
cultured in medium containing \textasciitilde{}50 g/L glucose and 4 g/L
butyric acid as carbon sources in a stirred-tank bioreactor at
35\textsuperscript{o}C and pH controlled at \textasciitilde{}5.0.

\textbf{Figure 4} . Kinetics of continuous fermentation with 50 g/L
glucose and 4 g/L butyric acid at the dilution rate of 0.06
h\textsuperscript{-1}. (a) Time course data of cell density (OD),
glucose, solvents (acetone, butanol, and ethanol), and acids (acetic
acid and butyric acid) in the reactor outlet stream; (b) Butanol and ABE
yields and butanol productivity.

\textbf{Figure 5} . Kinetics of continuous fermentation with 60 g/L
glucose and 6.8 g/L butyric acid at the dilution rate of 0.12
h\textsuperscript{-1}. (a) Time course data of cell density (OD),
glucose, solvents (acetone, butanol, and ethanol), and acids (acetic
acid and butyric acid) in the reactor outlet stream; (b) Butanol and ABE
yields and butanol productivity.

\textbf{Figure 6} . Kinetics of continuous fermentation with 60 g/L
glucose and 5.0 g/L butyric acid at the dilution rate of 1.88
h\textsuperscript{-1}. (a) Time course data of cell density (OD),
glucose, solvents (acetone, butanol, and ethanol), and acids (acetic
acid and butyric acid) in the reactor outlet stream; (b) Butanol and ABE
yields and butanol productivity.

\textbf{Figure 7.} Scanning electron micrographs of cells immobilized in
the fibrous matrix in the fibrous bed bioreactor. Cells adsorbed on the
fiber surface formed a thick layer of biofilm. Most of cells had an
elongated rod shape between 3 to 5 \selectlanguage{greek}µ\selectlanguage{english}m.

\textbf{Table 1.} Comparison of fermentation kinetics of \emph{C.
acetobutylicum} ATCC55025 in batch and continuous fermentations at
different dilution rates.\selectlanguage{english}
\begin{longtable}[]{@{}llllll@{}}
\toprule
& & \textbf{Batch} & \textbf{Continuous} & \textbf{Continuous} &
\textbf{Continuous}\tabularnewline
\midrule
\endhead
& & & 0.06 h\textsuperscript{-1} & 0.12 h\textsuperscript{-1} & 1.88
h\textsuperscript{-1}\tabularnewline
\textbf{Glucose} & Feed Conc. (g/L) & 52 & 50 & 60 & 60\tabularnewline
& Outlet Conc. (g/L) & 1.0 \selectlanguage{ngerman}± 0.2 & 16.77 \selectlanguage{ngerman}± 1.24 & 15.87 \selectlanguage{ngerman}± 1.81 & 13.70 \selectlanguage{ngerman}±
1.62\tabularnewline
\textbf{Butyric Acid} & Feed Conc. (g/L) & 4.0 & 4.0 & 6.8 &
5.0\tabularnewline
& Outlet Conc. (g/L) & 2.35 \selectlanguage{ngerman}± 0.11 & 3.01 \selectlanguage{ngerman}± 0.61 & 2.59 \selectlanguage{ngerman}± 0.19 & 1.89 \selectlanguage{ngerman}±
0.28\tabularnewline
\textbf{Acetic Acid} & Titer (g/L) & 1.83 \selectlanguage{ngerman}± 0.076 & 3.26 \selectlanguage{ngerman}± 0.23 & 2.27 \selectlanguage{ngerman}±
0.08 & 1.85 \selectlanguage{ngerman}± 0.63\tabularnewline
& Yield (g/g) & 0.032 \selectlanguage{ngerman}± 0.002 & 0.090 \selectlanguage{ngerman}± 0.016 & 0.053 \selectlanguage{ngerman}± 0.005 & 0.042 \selectlanguage{ngerman}±
0.003\tabularnewline
& Productivity (g/L[?]h) & 0.007 \selectlanguage{ngerman}± 0.001 & 0.20 \selectlanguage{ngerman}± 0.02 & 0.27 \selectlanguage{ngerman}± 0.01 &
3.38 \selectlanguage{ngerman}± 0.10\tabularnewline
\textbf{Acetone} & Titer (g/L) & 5.58 \selectlanguage{ngerman}± 0.14 & 3.19 \selectlanguage{ngerman}± 0.24 & 3.86 \selectlanguage{ngerman}± 0.20
& 3.38 \selectlanguage{ngerman}± 0.55\tabularnewline
& Yield (g/g) & 0.11 \selectlanguage{ngerman}± 0.02 & 0.09 \selectlanguage{ngerman}± 0.01 & 0.09 \selectlanguage{ngerman}± 0.01 & 0.09 \selectlanguage{ngerman}±
0.01\tabularnewline
& Productivity (g/L[?]h) & 0.02 \selectlanguage{ngerman}± 0.002 & 0.19 \selectlanguage{ngerman}±0.01 & 0.46 \selectlanguage{ngerman}± 0.02 & 7.03
\selectlanguage{ngerman}± 0.36\tabularnewline
\textbf{Butanol} & Titer (g/L) & 13.08 \selectlanguage{ngerman}± 0.23 & 8.72 \selectlanguage{ngerman}± 0.51 & 10.37 \selectlanguage{ngerman}±
0.29 & 9.55 \selectlanguage{ngerman}± 0.75\tabularnewline
& Yield (g/g) & 0.25 \selectlanguage{ngerman}± 0.01 & 0.24 \selectlanguage{ngerman}± 0.02 & 0.24 \selectlanguage{ngerman}± 0.02 & 0.24 \selectlanguage{ngerman}±
0.02\tabularnewline
& Productivity (g/L[?]h) & 0.05 \selectlanguage{ngerman}± 0.01 & 0.52 \selectlanguage{ngerman}± 0.03 & 1.24 \selectlanguage{ngerman}± 0.04 & 16.75
\selectlanguage{ngerman}± 1.68\tabularnewline
\textbf{Ethanol} & Titer (g/L) & 0.87 \selectlanguage{ngerman}± 0.02 & 0.62 \selectlanguage{ngerman}± 0.18 & 0.72 \selectlanguage{ngerman}± 0.12
& 0.77 \selectlanguage{ngerman}± 0.20\tabularnewline
& Yield (g/g) & 0.014 \selectlanguage{ngerman}±0.001 & 0.017 \selectlanguage{ngerman}± 0.006 & 0.017 \selectlanguage{ngerman}± 0.002 & 0.02 \selectlanguage{ngerman}±
0.001\tabularnewline
& Productivity (g/L[?]h) & 0.003 \selectlanguage{ngerman}±0.0002 & 0.038 \selectlanguage{ngerman}± 0.013 & 0.083 \selectlanguage{ngerman}± 0.014 &
1.23 \selectlanguage{ngerman}± 0.07\tabularnewline
\textbf{ABE} & Titer (g/L) & 19.23 \selectlanguage{ngerman}± 0.38 & 12.52 \selectlanguage{ngerman}± 0.28 & 14.98 \selectlanguage{ngerman}± 0.55
& 13.70 \selectlanguage{ngerman}± 0.32\tabularnewline
& Yield (g/g) & 0.38 \selectlanguage{ngerman}± 0.02 & 0.35 \selectlanguage{ngerman}± 0.02 & 0.35 \selectlanguage{ngerman}± 0.04 & 0.35 \selectlanguage{ngerman}±
0.04\tabularnewline
& Productivity (g/L[?]h) & 0.07 \selectlanguage{ngerman}± 0.01 & 0.72 \selectlanguage{ngerman}± 0.09 & 1.79 \selectlanguage{ngerman}± 0.19 & 24.15
\selectlanguage{ngerman}± 2.75\tabularnewline
\bottomrule
\end{longtable}\selectlanguage{ngerman}

\textbf{Table 2} . Comparison of continuous ABE fermentation in product
titer, yield, and productivity.\selectlanguage{english}
\begin{longtable}[]{@{}llllllllll@{}}
\toprule
\begin{minipage}[b]{0.07\columnwidth}\raggedright\strut
Process and Immobilization media\strut
\end{minipage} & \begin{minipage}[b]{0.07\columnwidth}\raggedright\strut
Dilution rate (h\textsuperscript{-1})\strut
\end{minipage} & \begin{minipage}[b]{0.07\columnwidth}\raggedright\strut
Operation duration\strut
\end{minipage} & \begin{minipage}[b]{0.07\columnwidth}\raggedright\strut
Butanol\strut
\end{minipage} & \begin{minipage}[b]{0.07\columnwidth}\raggedright\strut
Butanol\strut
\end{minipage} & \begin{minipage}[b]{0.07\columnwidth}\raggedright\strut
Butanol\strut
\end{minipage} & \begin{minipage}[b]{0.07\columnwidth}\raggedright\strut
ABE\strut
\end{minipage} & \begin{minipage}[b]{0.07\columnwidth}\raggedright\strut
ABE\strut
\end{minipage} & \begin{minipage}[b]{0.07\columnwidth}\raggedright\strut
ABE\strut
\end{minipage} & \begin{minipage}[b]{0.07\columnwidth}\raggedright\strut
Reference\strut
\end{minipage}\tabularnewline
\midrule
\endhead
\begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
Titer (g/L)\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
Yield (g/g)\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
Productivity (g/L\selectlanguage{ngerman}·h)\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
Titer (g/L)\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
Yield (g/g)\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
Productivity (g/L\selectlanguage{ngerman}·h)\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
STR, free cells\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
0.02 0.05\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
200 h 200 h\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
6.9 9.3\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
0.24 0.27\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
0.14 0.46\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
12.4 16.5\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
0.43 0.48\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
0.25 0.82\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
Al-Shorgani et al., 2019\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
STR, free cells STR, corn stover\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
0.01 0.13 0.44\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
200 h 100 h 200 h\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
7.6 10.1 9.5\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
0.4 0.35 0.33\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
0.1 1.3 4.2\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
NA\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
NA\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
NA\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
Gallazzi et al., 2015\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
STR, free cells STR, polyvinyl alcohol\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
0.04\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
300 h 140 h\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
7.1 13.4\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
0.24 0.44\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
0.22 0.4\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
9.4 22.1\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
0.32 0.73\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
0.29 0.66\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
Lee SM et al., 2008\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
STR with cell recycling\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
0.85\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
200 h\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
NA\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
NA\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
NA\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
12.9\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
NA\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
11.0\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
Tashiro et al., 2005\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
STR with cell recycling\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
0.86\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
40 h\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
11.9\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
0.17\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
10.7\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
23.5\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
0.34\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
21.1\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
Jang et al., 2013\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
STR with cell recycling and \emph{in situ} product separation\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
0.076\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
170 h\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
550\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
0.35\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
14.0\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
NA\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
NA\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
NA\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
Nguyen et al., 2018\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
2-stage STR, Ca-alginate\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
1.0\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
727 h\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
NA\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
NA\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
NA\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
3.9\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
0.21\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
4.0\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
Frick and Schugerl, 1986\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
PBR with brick\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
2.0\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
450 h\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
4.5\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
0.23\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
9.0\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
7.6\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
0.38\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
15.2\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
Qureshi et al., 2000\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
PBR with sugarcane bagasse\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
2.0\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
NA\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
3.8\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
0.20\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
7.5\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
5.7\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
0.31\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
11.3\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
Kong et al., 2015\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
PBR with wood pulp fiber\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
1.5\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
NA\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
4.6\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
0.16\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
6.9\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
8.1\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
0.28\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
12.1\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
Survase et al., 2012\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
3-stage PBR with corn stalk\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
0.04\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
750 h\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
12.6\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
0.24\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
0.51\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
19.9\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
0.38\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
0.8\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
Chang et al., 2016\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
4-stage PBR with ceramic beads\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
0.13\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
96\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
5.5\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
0.14\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
0.71\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
7.3\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
0.20\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
1.0\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
Badr et al., 2001\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
Recirculating PBR, corn stalk\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
0.2--1.0\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
20 days\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
2.9-5.5\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
0.18\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
0.9-2.9\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
5.5-9\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
0.32\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
1.5-5.1\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
Zhang et al., 2009\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
Recirculating FBB, cotton towel\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
0.9\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
600 h\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
5.1\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
0.42\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
4.6\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
6.8\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
0.51\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
6.1\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
Huang et al., 2004\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
FBB, cotton towel\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
0.12 1.88\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
600 h 300 h\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
10.4 9.6\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
0.24\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
1.24 \textbf{16.8}\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
15.0 13.7\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
0.35\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
1.8 \textbf{24.2}\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
\textbf{This work}\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
2-stage PBR with extraction, sugarcane bagasse\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
0.2\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
30 days\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
16.9\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
0.23\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
1.67\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
25.3\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
0.35\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
2.5\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
Bankar et al., 2012\strut
\end{minipage}\tabularnewline
\bottomrule
\end{longtable}

NA: not available; FBB: fibrous bed bioreactor; PBR: packed bed reactor;
STR: stirred-tank reactor

\textbf{Hosted file}

\verb`image1.emf`  available at \url{https://authorea.com/users/493818/articles/576120-high-rate-continuous-n-butanol-production-by-clostridium-acetobutylicum-from-glucose-and-butyric-acid-in-a-single-pass-fibrous-bed-bioreactor}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image2/image2}

\end{center}
\end{figure}

\textbf{Figure 1} . Experimental setup for the continuous fermentation
with clostridial cells immobilized in a fibrous bed bioreactor (FBB).
Inset diagram shows the construction of the spirally wound fibrous
matrices with stainless steel wire mesh as the mechanical support with
gaps between adjacent layers as flow channels for liquid, gas, and
solids (cells) to move freely. The highly porous fibrous matrix has a
large surface area and void volume (\textgreater{}90\% reactor volume)
to allow high densities of viable cells (up to 100 g/L cell dry weight)
to be immobilized by attachment to fiber surfaces and entrapment within
the matrix.

\textbf{Hosted file}

\verb`image3.emf`  available at \url{https://authorea.com/users/493818/articles/576120-high-rate-continuous-n-butanol-production-by-clostridium-acetobutylicum-from-glucose-and-butyric-acid-in-a-single-pass-fibrous-bed-bioreactor}

\textbf{Figure 2} . Effect of butyric acid on cell growth. Cells
of\emph{C. acetobutylicum} ATCC 55025 were cultured in P2 medium
containing 20 g/L glucose and various amounts of butyric acid (0, 2, 5,
or 10 g/L) in serum bottles at 37 \textsuperscript{o}C.

\textbf{Hosted file}

\verb`image4.emf`  available at \url{https://authorea.com/users/493818/articles/576120-high-rate-continuous-n-butanol-production-by-clostridium-acetobutylicum-from-glucose-and-butyric-acid-in-a-single-pass-fibrous-bed-bioreactor}

\textbf{Figure 3} . Kinetics of batch fermentation with free cells
cultured in medium containing \textasciitilde{}50 g/L glucose and 4 g/L
butyric acid as carbon sources in a stirred-tank bioreactor at 35
\textsuperscript{o}C and pH controlled at \textasciitilde{}5.0.

\textbf{Hosted file}

\verb`image5.emf`  available at \url{https://authorea.com/users/493818/articles/576120-high-rate-continuous-n-butanol-production-by-clostridium-acetobutylicum-from-glucose-and-butyric-acid-in-a-single-pass-fibrous-bed-bioreactor}

\textbf{Figure 4} . Kinetics of continuous fermentation with 50 g/L
glucose and 4 g/L butyric acid at the dilution rate of 0.06
h\textsuperscript{-1}. (a) Time course data of cell density (OD),
glucose, solvents (acetone, butanol, and ethanol), and acids (acetic
acid and butyric acid) in the reactor outlet stream; (b) Butanol and ABE
yields and butanol productivity.

\par\null

\textbf{Hosted file}

\verb`image6.emf`  available at \url{https://authorea.com/users/493818/articles/576120-high-rate-continuous-n-butanol-production-by-clostridium-acetobutylicum-from-glucose-and-butyric-acid-in-a-single-pass-fibrous-bed-bioreactor}

\textbf{Figure 5} . Kinetics of continuous fermentation with 60 g/L
glucose and 6.8 g/L butyric acid at the dilution rate of 0.12
h\textsuperscript{-1}. (a) Time course data of cell density (OD),
glucose, solvents (acetone, butanol, and ethanol), and acids (acetic
acid and butyric acid) in the reactor outlet stream; (b) Butanol and ABE
yields and butanol productivity.

\textbf{Hosted file}

\verb`image7.emf`  available at \url{https://authorea.com/users/493818/articles/576120-high-rate-continuous-n-butanol-production-by-clostridium-acetobutylicum-from-glucose-and-butyric-acid-in-a-single-pass-fibrous-bed-bioreactor}

\textbf{Figure 6} . Kinetics of continuous fermentation with 60 g/L
glucose and 5.0 g/L butyric acid at the dilution rate of 1.88
h\textsuperscript{-1}. (a) Time course data of cell density (OD),
glucose, solvents (acetone, butanol, and ethanol), and acids (acetic
acid and butyric acid) in the reactor outlet stream; (b) Butanol and ABE
yields and butanol productivity.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image8/image8}

\end{center}
\end{figure}

\textbf{Figure 7.} Scanning electron micrographs of cells immobilized in
the fibrous matrix in the fibrous bed bioreactor. Cells adsorbed on the
fiber surface formed a thick layer of biofilm. Most of cells had an
elongated rod shape between 3 to 5 \selectlanguage{greek}µ\selectlanguage{english}m.

\selectlanguage{english}
\FloatBarrier
\end{document}

