Metabolic process engineering of Clostridium tyrobutyricum Δack-adhE2 for enhanced n-butanol production from glucose: effects of methyl viologen on NADH availability, flux distribution, and fermentation kinetics.

Butanol biosynthesis through aldehyde/alcohol dehydrogenase (adhE2) is usually limited by NADH availability, resulting in low butanol titer, yield, and productivity. To alleviate this limitation and improve n-butanol production by Clostridium tyrobutyricum Δack-adhE2 overexpressing adhE2, the NADH availability was increased by using methyl viologen (MV) as an artificial electron carrier to divert electrons from ferredoxin normally used for H2 production. In the batch fermentation with the addition of 500 µM MV, H2 , acetate, and butyrate production was reduced by more than 80-90%, while butanol production increased more than 40% to 14.5 g/L. Metabolic flux analysis revealed that butanol production increased in the fermentation with MV because of increased NADH availability as a result of reduced H2 production. Furthermore, continuous butanol production of ∼55 g/L with a high yield of ∼0.33 g/g glucose and extremely low ethanol, acetate, and butyrate production was obtained in fed-batch fermentation with gas stripping for in situ butanol recovery. This study demonstrated a stable and reliable process for high-yield and high-titer n-butanol production by metabolically engineered C. tyrobutyricum by applying MV as an electron carrier to increase butanol biosynthesis.

Effects of different replicons in conjugative plasmids on transformation efficiency, plasmid stability, gene expression and n-butanol biosynthesis in Clostridium tyrobutyricum.

Clostridium tyrobutyricum ATCC 25755 can produce butyric acid, acetic acid, and hydrogen as the main products from various carbon sources. In this study, C. tyrobutyricum was used as a host to produce n-butanol by expressing adhE2 gene under the control of a native thiolase promoter using four different conjugative plasmids (pMTL82151, 83151, 84151, and 85151) each with a different replicon (pBP1 from C. botulinum NCTC2916, pCB102 from C. butyricum, pCD6 from Clostridium difficile, and pIM13 from Bacillus subtilis). The effects of different replicons on transformation efficiency, plasmid stability, adhE2 expression and aldehyde/alcohol dehydrogenase activities, and butanol production by different mutants of C. tyrobutyricum were investigated. Among the four plasmids and replicons studied, pMTL82151 with pBP1 gave the highest transformation efficiency, plasmid stability, gene expression, and butanol biosynthesis. Butanol production from various substrates, including glucose, xylose, mannose, and mannitol were then investigated with the best mutant strain harboring adhE2 in pMTL82151. A high butanol titer of 20.5 g/L with 0.33 g/g yield and 0.32 g/L h productivity was obtained with mannitol as the substrate in batch fermentation with pH controlled at ~6.0.

Production of n-butanol from cassava bagasse hydrolysate by engineered Clostridium tyrobutyricum overexpressing adhE2: Kinetics and cost analysis.

The production of biofuels such as butanol is usually limited by the availability of inexpensive raw materials and high substrate cost. Using food crops as feedstock in the biorefinery industry has been criticized for its competition with food supply, causing food shortage and increased food prices. In this study, cassava bagasse as an abundant, renewable, and inexpensive byproduct from the cassava starch industry was used for n-butanol production. Cassava bagasse hydrolysate containing mainly glucose was obtained after treatments with dilute acid and enzymes (glucoamylases and cellulases) and then supplemented with corn steep liquor for use as substrate in repeated-batch fermentation with engineered Clostridium tyrobutyricum CtΔack-adhE2 in a fibrous-bed bioreactor. Stable butanol production with high titer (>15.0 g/L), yield (>0.30 g/g), and productivity (~0.3 g/L∙h) was achieved, demonstrating the feasibility of an economically competitive process for n-butanol production from cassava bagasse for industrial application.

n-Butanol production from lignocellulosic biomass hydrolysates without detoxification by Clostridium tyrobutyricum Δack-adhE2 in a fibrous-bed bioreactor.

Acetone-butanol-ethanol fermentation suffers from high substrate cost and low butanol titer and yield. In this study, engineered Clostridium tyrobutyricum CtΔack-adhE2 immobilized in a fibrous-bed bioreactor was used for butanol production from glucose and xylose present in the hydrolysates of low-cost lignocellulosic biomass including corn fiber, cotton stalk, soybean hull, and sugarcane bagasse. The biomass hydrolysates obtained after acid pretreatment and enzymatic hydrolysis were supplemented with corn steep liquor and used in repeated-batch fermentations. Butanol production with high titer (∼15 g/L), yield (∼0.3 g/g), and productivity (∼0.3 g/L∙h) was obtained from cotton stalk, soybean hull, and sugarcane bagasse hydrolysates, while corn fiber hydrolysate with higher inhibitor contents gave somewhat inferior results. The fermentation process was stable for long-term operation without any noticeable degeneration, demonstrating its potential for industrial application. A techno-economic analysis showed that n-butanol could be produced from lignocellulosic biomass using this novel fermentation process at ∼$2.5/gal for biofuel application.