Cellulosic n-butanol from renewable lignocellulosic biomass has gained increased interest. Previously, we have engineered Clostridium cellulovorans, a cellulolytic acidogen, to overexpress the bifunctional butyraldehyde/butanol dehydrogenase gene adhE2 from C. acetobutylicum for n-butanol production from crystalline cellulose. However, butanol production by this engineered strain had a relatively low yield of approximately 0.22 g/g cellulose due to the coproduction of ethanol and acids. We hypothesized that strengthening the carbon flux through the central butyryl-CoA biosynthesis pathway and increasing intracellular NADH availability in C. cellulovorans adhE2 would enhance n-butanol production. In this study, thiolase (thlA CA ) from C. acetobutylicum and 3-hydroxybutyryl-CoA dehydrogenase (hbd CT ) from C. tyrobutyricum were overexpressed in C. cellulovorans adhE2 to increase the flux from acetyl-CoA to butyryl-CoA. In addition, ferredoxin-NAD(P) + oxidoreductase (fnr), which can regenerate the intracellular NAD(P)H and thus increase butanol biosynthesis, was also overexpressed. Metabolic flux analyses showed that mutants overexpressing these genes had a significantly increased carbon flux toward butyryl-CoA, which resulted in increased production of butyrate and butanol.The addition of methyl viologen as an electron carrier in batch fermentation further directed more carbon flux towards n-butanol biosynthesis due to increased reducing equivalent or NADH. The engineered strain C. cellulovorans adhE2-fnr CA -thlA CAhbd CT produced n-butanol from cellulose at a 50% higher yield (0.34 g/g), the highest ever obtained in batch fermentation by any known bacterial strain. The engineered C. cellulovorans is thus a promising host for n-butanol production from cellulosic biomass in consolidated bioprocessing.

Biobutanol produced in acetone–butanol–ethanol (ABE) 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 of culture degeneration, cell washout, and contamination. In this study, cells of the asporogenous 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 a dilution rate of 1.88 h−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 productivity 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 (~100 g/L at >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 1 month.

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 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 , 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 (~100 g/L at >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.