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Preeti R.,Indian National Institute for Interdisciplinary Science and Technology | Pandey A.,Indian National Institute for Interdisciplinary Science and Technology | Pandey A.,Bioprocessing Innovative C | Binod P.,Indian National Institute for Interdisciplinary Science and Technology
Bioresource Technology | Year: 2017

An assay method for detection of enantiospecific chiral alcohol was developed based on ketoreductase, enantio-selective alcohol oxidase and 2,4-dinitrophenyl hydrazine (DNPH) reagent. The assay method was developed to check the conversion of 1-Acetonapthone to either (S) or (R) specific 1-(1-napthyl) ethanol or its racemic mixture using ketoreductases. Further, estimation was done with the help of 2,4-DNPH method. The resulting orange coloured chromogen showed a maximum absorbance at 560. nm. The assay was performed in 96 well microtiter plates and had a linear detection range from 0.05. mM to 4. mM. The method is found to be suitable for the detection of large numbers of crude samples and screening of ketoreductase producing strains in high-throughput manner. © 2017 Elsevier Ltd.


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.


Xu M.,Ohio State University | Zhao J.,Ohio State University | Yu L.,Ohio State University | Tang I.-C.,Bioprocessing Innovative C | And 2 more authors.
Applied Microbiology and Biotechnology | Year: 2014

Clostridium acetobutylicum JB200, a mutant strain of C. acetobutylicum ATCC 55025 obtained through strain evolution in a fibrous bed bioreactor, had high butanol tolerance and produced up to ~21 g/L butanol from glucose in batch fermentation, an improvement of ~67 % over the parental strain (~12.6 g/L). Comparative genomic analysis revealed a single-base deletion in the cac3319 gene leading to C-terminal truncation in its encoding histidine kinase (HK) in JB200. To study the effects of cac3319 mutation on cell growth and fermentation, the cac3319 gene in ATCC 55025 was disrupted using the ClosTron group II intron-based gene inactivation system. Compared to ATCC 55025, the cac3319 HK knockout mutant, HKKO, produced 44.4 % more butanol (18.2 ± 1.3 vs. 12.6 ± 0.2 g/L) with a 90 % higher productivity (0.38 ± 0.03 vs. 0.20 ± 0.02 g/L h) due to increased butanol tolerance, confirming, for the first time, that cac3319 plays an important role in regulating solvent production and tolerance in C. acetobutylicum. This work also provides a novel metabolic engineering strategy for generating high-butanol-tolerant and high-butanol-producing strains for industrial applications. © 2014, Springer-Verlag Berlin Heidelberg.


Du Y.,Ohio State University | Jiang W.,Ohio State University | Yu M.,Bioprocessing Innovative C | Tang I.-C.,Bioprocessing Innovative C | Yang S.-T.,Ohio State University
Biotechnology and Bioengineering | Year: 2015

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 500μM MV, H2, acetate, and butyrate production was reduced by more than 80-90%, while butanol production increased more than 40% to 14.5g/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 ∼55g/L with a high yield of ∼0.33g/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. Biotechnol. Bioeng. 2015;112: 705-715. © 2014 Wiley Periodicals, Inc.


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.


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.


Mishra B.,CSIR - Central Electrochemical Research Institute | Sangwan R.S.,Bioprocessing Innovative C | Mishra S.,CSIR - Central Electrochemical Research Institute | Jadaun J.S.,CSIR - Central Electrochemical Research Institute | And 2 more authors.
Protoplasma | Year: 2014

Withania somnifera is one of the most important medicinal plant and is credited with various pharmacological activities. In this study, in vitro multiple shoot cultures were exposed to different concentrations (5-300 μM) of cadmium (Cd) as cadmium sulphate to explore its ability to accumulate the heavy metal ion and its impact on the metabolic status and adaptive responses. The results showed that supplemental exposure to Cd interfered with N, P, and K uptake creating N, P, and K deficiency at higher doses of Cd that also caused stunting of growth, chlorosis, and necrosis. The study showed that in vitro shoots could markedly accumulate Cd in a concentration-dependent manner. Enzymatic activities and isozymic pattern of catalase, ascorbate peroxidase, guaiacol peroxidase, peroxidase, glutathione-S-transferase, glutathione peroxidase, monodehydroascorbate reductase, and dehydroascorbate reductase were altered substantially under Cd exposure. Sugar metabolism was also markedly modulated under Cd stress. Various other parameters including contents of photosynthetic pigments, phenolics, tocopherol, flavonoids, reduced glutathione, nonprotein thiol, ascorbate, and proline displayed major inductive responses reflecting their protective role. The results showed that interplay of enzymatic as well as nonenzymatic responses constituted a system endeavor of tolerance of Cd accumulation and an efficient scavenging strategy of its stress implications. © 2014 Springer-Verlag Wien.


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.


Grant
Agency: NSF | Branch: Standard Grant | Program: | Phase: | Award Amount: 512.00K | 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 (<25%), butanol concentration (<16 g/L), and reactor productivity (<0.5 g/L?h) due to a strong butanol inhibition, and the fermentation process is difficult to improve due to the complicated metabolic pathways and gene regulation involved in the production microorganisms, mainly Clostridium acetobutylicum.

The broader impact/commercial potential of the project is to produce butanol as a biofuel from sugars derived from starchy and lignocellulosic biomass. Biobutanol has great value as an alternative transportation fuel. There is a huge potential commercial and societal impact in improving yields and reducing costs of butanol production. The research and other activity proposed could lead directly to a marketable product and process and leads to several enabling technologies, including better manipulation of C. tyrobutylicum, further demonstration of strain improvements using the FBB, and others.


Grant
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 (

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