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.