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Dublin, OH, United States

Agency: National Science Foundation | Branch: | Program: STTR | Phase: Phase II | Award Amount: 500.00K | Year: 2010

This STTR Phase II project will develop novel engineered Clostridia strains for fermentation and economically produce butanol as a biofuel from sugars derived from starchy and lignocellulosic biomass. The conventional acetone-butanol-ethanol (ABE) fermentation has low butanol yield (

Yu M.,Ohio State University | Du Y.,Ohio State University | Jiang W.,Ohio State University | Chang W.-L.,Ohio State University | And 2 more authors.
Applied Microbiology and Biotechnology | Year: 2012

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. © 2011 Springer-Verlag. Source

Xue C.,Dalian University of Technology | Xue C.,Ohio State University | Zhao J.,Ohio State University | Lu C.,Ohio State University | And 3 more authors.
Biotechnology and Bioengineering | Year: 2012

Acetone-butanol-ethanol (ABE) fermentation with a hyper-butanol producing Clostridium acetobutylicum JB200 was studied for its potential to produce a high titer of butanol that can be readily recovered with gas stripping. In batch fermentation without gas stripping, a final butanol concentration of 19.1g/L was produced from 86.4g/L glucose consumed in 78h, and butanol productivity and yield were 0.24g/Lh and 0.21g/g, respectively. In contrast, when gas stripping was applied intermittently in fed-batch fermentation, 172g/L ABE (113.3g/L butanol, 49.2g/L acetone, 9.7g/L ethanol) were produced from 474.9g/L glucose in six feeding cycles over 326h. The overall productivity and yield were 0.53g/Lh and 0.36g/g for ABE and 0.35g/Lh and 0.24g/g for butanol, respectively. The higher productivity was attributed to the reduced butanol concentration in the fermentation broth by gas stripping that alleviated butanol inhibition, whereas the increased butanol yield could be attributed to the reduced acids accumulation as most acids produced in acidogenesis were reassimilated by cells for ABE production. The intermittent gas stripping produced a highly concentrated condensate containing 195.9g/L ABE or 150.5g/L butanol that far exceeded butanol solubility in water. After liquid-liquid demixing or phase separation, a final product containing ∼610g/L butanol, ∼40g/L acetone, ∼10g/L ethanol, and no acids was obtained. Compared to conventional ABE fermentation, the fed-batch fermentation with intermittent gas stripping has the potential to reduce at least 90% of energy consumption and water usage in n-butanol production from glucose. © 2012 Wiley Periodicals, Inc. Source

Yu M.,Ohio State University | Zhang Y.,Ohio State University | Tang I.-C.,Bioprocessing Innovative C | Yang S.-T.,Ohio State University
Metabolic Engineering | Year: 2011

Clostridium tyrobutyricum ATCC 25755, a butyric acid producing bacterium, has been engineered to overexpress aldehyde/alcohol dehydrogenase 2 (adhE2, Genebank no. AF321779) from Clostridium acetobutylicum ATCC 824, which converts butyryl-CoA to butanol, under the control of native thiolase (thl) promoter. Butanol titer of 1.1. g/L was obtained in C. tyrobutyricum overexpressing adhE2. The effects of inactivating acetate kinase (ack) and phosphotransbutyrylase (ptb) genes in the host on butanol production were then studied. A high C4/C2 product ratio of 10.6 (mol/mol) was obtained in ack knockout mutant, whereas a low C4/C2 product ratio of 1.4 (mol/mol) was obtained in ptb knockout mutant, confirming that ack and ptb genes play important roles in controlling metabolic flux distribution in C. tyrobutyricum. The highest butanol titer of 10.0. g/L and butanol yield of 27.0% (w/w, 66% of theoretical yield) were achieved from glucose in the ack knockout mutant overexpressing adhE2. When a more reduced substrate mannitol was used, the butanol titer reached 16.0. g/L with 30.6% (w/w) yield (75% theoretical yield). Moreover, C. tyrobutyricum showed good butanol tolerance, with >80% and ~60% relative growth rate at 1.0% and 1.5% (v/v) butanol. These results suggest that C. tyrobutyricum is a promising heterologous host for n-butanol production from renewable biomass. © 2011 Elsevier Inc. Source

Yu L.,Ohio State University | Xu M.,Ohio State University | Tang I.-C.,Bioprocessing Innovative C | Yang S.-T.,Ohio State University
Applied Microbiology and Biotechnology | Year: 2015

Clostridium tyrobutyricum does not have the enzymes needed for using maltose or starch. Two extracellular α-glucosidases encoded by agluI and agluII from Clostridium acetobutylicum ATCC 824 catalyzing the hydrolysis of α-1,4-glycosidic bonds in maltose and starch from the non-reducing end were cloned and expressed in C. tyrobutyricum (Δack, adhE2), and their effects on n-butanol production from maltose and soluble starch in batch fermentations were studied. Compared to the parental strain grown on glucose, mutants expressing agluI showed robust activity in breaking down maltose and produced more butanol (17.2 vs. 9.5 g/L) with a higher butanol yield (0.20 vs. 0.10 g/g) and productivity (0.29 vs. 0.16 g/L h). The mutant was also able to use soluble starch as substrate, although at a slower rate compared to maltose. Compared to C. acetobutylicum ATCC 824, the mutant produced more butanol from maltose (17.2 vs. 11.2 g/L) and soluble starch (16.2 vs. 8.8 g/L) in batch fermentations. The mutant was stable in batch fermentation without adding antibiotics, achieving a high butanol productivity of 0.40 g/L h. This mutant strain thus can be used in industrial production of n-butanol from maltose and soluble starch. © 2015, Springer-Verlag Berlin Heidelberg. Source

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