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High-flux isobutanol production using engineered Escherichia coli: a bioreactor study with in situ product removal.

Baez A, Cho KM, Liao JC - Appl. Microbiol. Biotechnol. (2011)

Bottom Line: Promising approaches to produce higher alcohols, e.g., isobutanol, using Escherichia coli have been developed with successful results.Here, we translated the isobutanol process from shake flasks to a 1-L bioreactor in order to characterize three E. coli strains.The isobutanol productivity was approximately twofold and the titer was 9% higher than n-butanol produced by Clostridium in a similar integrated system.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemical and Biomolecular Engineering, University of California-Los Angeles, 420 Westwood Plaza, Los Angeles, CA 90095, USA.

ABSTRACT
Promising approaches to produce higher alcohols, e.g., isobutanol, using Escherichia coli have been developed with successful results. Here, we translated the isobutanol process from shake flasks to a 1-L bioreactor in order to characterize three E. coli strains. With in situ isobutanol removal from the bioreactor using gas stripping, the engineered E. coli strain (JCL260) produced more than 50 g/L in 72 h. In addition, the isobutanol production by the parental strain (JCL16) and the high isobutanol-tolerant mutant (SA481) were compared with JCL260. Interestingly, we found that the isobutanol-tolerant strain in fact produced worse than either JCL16 or JCL260. This result suggests that in situ product removal can properly overcome isobutanol toxicity in E. coli cultures. The isobutanol productivity was approximately twofold and the titer was 9% higher than n-butanol produced by Clostridium in a similar integrated system.

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Typical kinetics of isobutanol production by JCL260, JCL16, and SA481 strains harboring pSA65/pSA69. a Total isobutanol production (calculated as sum of isobutanol quantities determined in receivers B, D, and broth culture considering a working volume of 0.35 L). b Isobutanol concentration in fermentation broth. c Cell growth. d, f Glucose consumption. e Acetate production. High isobutanol producer (JCL260) at 30°C (closed squares), parental (JCL16) at 30°C (closed circle), high isobutanol producer (JCL260) at 37°C (closed triangles) and high isobutanol-tolerant strain (closed diamond). Error bars correspond to the difference between duplicate cultures (a–c, e). For clarity, one set of data has been plotted in d and f
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Fig2: Typical kinetics of isobutanol production by JCL260, JCL16, and SA481 strains harboring pSA65/pSA69. a Total isobutanol production (calculated as sum of isobutanol quantities determined in receivers B, D, and broth culture considering a working volume of 0.35 L). b Isobutanol concentration in fermentation broth. c Cell growth. d, f Glucose consumption. e Acetate production. High isobutanol producer (JCL260) at 30°C (closed squares), parental (JCL16) at 30°C (closed circle), high isobutanol producer (JCL260) at 37°C (closed triangles) and high isobutanol-tolerant strain (closed diamond). Error bars correspond to the difference between duplicate cultures (a–c, e). For clarity, one set of data has been plotted in d and f

Mentions: Cultures of E. coli were performed in 1-L stirred tank bioreactor (Applikon Biotechnology, Schiedam, the Netherlands) using a working volume of 0.35 L. The bioreactor was inoculated with 2% of overnight pre-culture and the cells grown at 37°C. After 2 h, 0.1 mM IPTG was added and the temperature was decreased to 30°C to start isobutanol production. The induction time represents the beginning of fermentation described in Fig. 2. Isobutanol production was tested at two temperatures, 30°C and 37°C, for JCL260. The pH was controlled at 6.8 by automatic addition of 2 M NaOH solution. Dissolved oxygen (DO) was maintained above 20% with respect to air saturation by raising stirrer speed (from 200 to 600 rpm). Air was bubbled in the bioreactor with two goals: (1) provide oxygen and (2) in situ isobutanol stripping out. Air flow rate was maintained at 0.5 vvm the first 3 h of isobutanol production and then was increased to 1.2 vvm in order to remove isobutanol from the culture broth. The evaporated isobutanol was condensed using two Graham condensers connected in series (Fig. 1). The exhaust gases from the fermentor were bubbled in a trap (picker B containing 800 mL of water; Fig. 1) cooled with ice and then circulated through condenser 1 (L 300 mm and cooling coil surface area 400 cm2; Fig. 1) maintained at 4°C. After that, gas continued circulating through a second equal loop (D receiver and condenser 2; Fig. 1). Fermentation was allowed to proceed in batch mode until entry into the stationary phase (between 9 and 10 h), and then an intermittent manual feeding was started to avoid glucose depletion. Fermentation samples were collected to determinate growth, isobutanol production, and organic acid and glucose concentrations.Fig. 1


High-flux isobutanol production using engineered Escherichia coli: a bioreactor study with in situ product removal.

Baez A, Cho KM, Liao JC - Appl. Microbiol. Biotechnol. (2011)

Typical kinetics of isobutanol production by JCL260, JCL16, and SA481 strains harboring pSA65/pSA69. a Total isobutanol production (calculated as sum of isobutanol quantities determined in receivers B, D, and broth culture considering a working volume of 0.35 L). b Isobutanol concentration in fermentation broth. c Cell growth. d, f Glucose consumption. e Acetate production. High isobutanol producer (JCL260) at 30°C (closed squares), parental (JCL16) at 30°C (closed circle), high isobutanol producer (JCL260) at 37°C (closed triangles) and high isobutanol-tolerant strain (closed diamond). Error bars correspond to the difference between duplicate cultures (a–c, e). For clarity, one set of data has been plotted in d and f
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3094657&req=5

Fig2: Typical kinetics of isobutanol production by JCL260, JCL16, and SA481 strains harboring pSA65/pSA69. a Total isobutanol production (calculated as sum of isobutanol quantities determined in receivers B, D, and broth culture considering a working volume of 0.35 L). b Isobutanol concentration in fermentation broth. c Cell growth. d, f Glucose consumption. e Acetate production. High isobutanol producer (JCL260) at 30°C (closed squares), parental (JCL16) at 30°C (closed circle), high isobutanol producer (JCL260) at 37°C (closed triangles) and high isobutanol-tolerant strain (closed diamond). Error bars correspond to the difference between duplicate cultures (a–c, e). For clarity, one set of data has been plotted in d and f
Mentions: Cultures of E. coli were performed in 1-L stirred tank bioreactor (Applikon Biotechnology, Schiedam, the Netherlands) using a working volume of 0.35 L. The bioreactor was inoculated with 2% of overnight pre-culture and the cells grown at 37°C. After 2 h, 0.1 mM IPTG was added and the temperature was decreased to 30°C to start isobutanol production. The induction time represents the beginning of fermentation described in Fig. 2. Isobutanol production was tested at two temperatures, 30°C and 37°C, for JCL260. The pH was controlled at 6.8 by automatic addition of 2 M NaOH solution. Dissolved oxygen (DO) was maintained above 20% with respect to air saturation by raising stirrer speed (from 200 to 600 rpm). Air was bubbled in the bioreactor with two goals: (1) provide oxygen and (2) in situ isobutanol stripping out. Air flow rate was maintained at 0.5 vvm the first 3 h of isobutanol production and then was increased to 1.2 vvm in order to remove isobutanol from the culture broth. The evaporated isobutanol was condensed using two Graham condensers connected in series (Fig. 1). The exhaust gases from the fermentor were bubbled in a trap (picker B containing 800 mL of water; Fig. 1) cooled with ice and then circulated through condenser 1 (L 300 mm and cooling coil surface area 400 cm2; Fig. 1) maintained at 4°C. After that, gas continued circulating through a second equal loop (D receiver and condenser 2; Fig. 1). Fermentation was allowed to proceed in batch mode until entry into the stationary phase (between 9 and 10 h), and then an intermittent manual feeding was started to avoid glucose depletion. Fermentation samples were collected to determinate growth, isobutanol production, and organic acid and glucose concentrations.Fig. 1

Bottom Line: Promising approaches to produce higher alcohols, e.g., isobutanol, using Escherichia coli have been developed with successful results.Here, we translated the isobutanol process from shake flasks to a 1-L bioreactor in order to characterize three E. coli strains.The isobutanol productivity was approximately twofold and the titer was 9% higher than n-butanol produced by Clostridium in a similar integrated system.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemical and Biomolecular Engineering, University of California-Los Angeles, 420 Westwood Plaza, Los Angeles, CA 90095, USA.

ABSTRACT
Promising approaches to produce higher alcohols, e.g., isobutanol, using Escherichia coli have been developed with successful results. Here, we translated the isobutanol process from shake flasks to a 1-L bioreactor in order to characterize three E. coli strains. With in situ isobutanol removal from the bioreactor using gas stripping, the engineered E. coli strain (JCL260) produced more than 50 g/L in 72 h. In addition, the isobutanol production by the parental strain (JCL16) and the high isobutanol-tolerant mutant (SA481) were compared with JCL260. Interestingly, we found that the isobutanol-tolerant strain in fact produced worse than either JCL16 or JCL260. This result suggests that in situ product removal can properly overcome isobutanol toxicity in E. coli cultures. The isobutanol productivity was approximately twofold and the titer was 9% higher than n-butanol produced by Clostridium in a similar integrated system.

Show MeSH
Related in: MedlinePlus