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. 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-1. Cell OD
and butanol production increased rapidly and reached
~3.0 and ~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 ~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
~0.24 g/g and ~0.52 g/L∙h, respectively.
There was continuous cell bleeding from the FBB throughout the
continuous fermentation, which remained at ~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 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-1, with the feed medium containing 60 g/L
glucose and 6.8 g/L butyric acid. As shown in Figure 5 , butanol
production increased to ~10 g/L while glucose
concentration decreased to ~20 g/L and butyric acid
decreased to ~2.6 g/L when the fermentation reached a
pseudo-steady state at ~480 h. Afterward, the continuous
fermentation produced butanol at the average yield of
~0.24 g/g and productivity of ~1.2
g/L∙h, which was 2.3-fold of that obtained at the lower dilution rate of
0.06 h-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 >8 g/L.
However, the reactor was able to recover by itself and continue to
increase butanol production to reach ~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 ~2.6 at 170 h and
then decreased to ~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 ~610 h. The
effluent butanol concentration reached ~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 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-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 ~140 h (Figure 6 ). Then butanol
concentration dropped to less than 8 g/L with glucose back up to
>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 ~20 g/L, butanol production was restored to
~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 ~9.5 g/L at a high productivity of
~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 ~140 h and then
decreased to ~3.0 by ~200 h and stayed
at that level during the pseudo-steady state period (200 – 380 h).
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
~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-1to
16.8 g/L∙h at 1.88 h-1, which was much higher than the
maximum specific growth rate (0.2~0.45
h-1) observed for 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-1 (vs. 3.0 at 0.06 h-1) and
steady-state OD in the effluent (3.0 at 1.88 h-1 vs.
1.0 at 0.06 h-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.