DOI: https://doi.org/10.61435/ijred.2026.62506

Optimization of Fermentation Condition for Isobutanol Production in Escherichia coli BL21 (DE3) using Box-Behnken

1Institut Teknologi Bandung, Indonesia

2Muhammadiyah University of Bandung, Indonesia

Received: 14 Mar 2026; Published: 15 Oct 2026.
Editor(s): H Hadiyanto
Open Access Copyright (c) 2025 The Author(s). Published by Centre of Biomass and Renewable Energy (CBIORE)
Creative Commons License This work is licensed under a Creative Commons Attribution-ShareAlike 4.0 International License.

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Abstract
Isobutanol is a promising biofuel candidate as an alternative to conventional gasoline. In previous studies, isobutanol biosynthetic genes were constructed under the control of the lac promoter in Escherichia coli (E. coli) DH5a; in the present study, the lac promoter was replaced with the T7 promoter to enhance protein expression in E. coli BL21(DE3). Beside of protein expression, culture condition also has pivotal effect for fermentation. This study aimed to increase isobutanol production through enhancing the expression of responsible genes for isobutanol pathway and find the optimal condition for fermentation process using response surface methodology (RSM). The result showed that the expression with T7 promoter resulting in a 1.5-fold increase in isobutanol production.  For optimizing fermentation condition, a Box–Behnken experimental design was employed to evaluate the effects of temperature, pH, and fermentation time using Minitab Statistical Software 22. Fifteen experimental runs, including three center-point replicates, were conducted. The results of the RSM optimization were verified under optimal conditions and analysed using a one-sample t-test at a 95% confidence level. The optimal conditions were identified as 30 °C, pH 4.5, and 72 h of fermentation. Analysis of variance (ANOVA) showed that temperature being the most significant factor affecting isobutanol yield (p=0,011), with R2=83,91%. The validation of optimum condition yielded 0.157 ± 0,003 with 95% Cl ranged between 0,1449-0,4599. This result indicate that lower temperature is favorable for isobutanol production
Keywords: Isobutanol; Box-Behnken; Biofuel; Escherichia coli; T7 Promoter

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Section: Original Research Article
Language : EN
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