Research Article: Development of butanol-tolerant Bacillus subtilis strain GRSW2-B1 as a potential bioproduction host

Date Published: May 30, 2011

Publisher: Springer

Author(s): Naoya Kataoka, Takahisa Tajima, Junichi Kato, Wanitcha Rachadech, Alisa S Vangnai.


As alternative microbial hosts for butanol production with organic-solvent tolerant trait are in high demands, a butanol-tolerant bacterium, Bacillus subtilis GRSW2-B1, was thus isolated. Its tolerance covered a range of organic solvents at high concentration (5%v/v), with remarkable tolerance in particular to butanol and alcohol groups. It was susceptible for butanol acclimatization, which resulted in significant tolerance improvement. It has versatility for application in a variety of fermentation process because it has superior tolerance when cells were exposed to butanol either as high-density, late-exponential grown cells (up to 5%v/v) or under growing conditions (up to 2.25%v/v). Genetic transformation procedure was optimized, yielding the highest efficiency at 5.17 × 103 colony forming unit (μg DNA)-1. Gene expression could be effectively driven by several promoters with different levels, where as the highest expression was observed with a xylose promoter. The constructed vector was stably maintained in the transformants, in the presence or absence of butanol stress. Adverse effect of efflux-mediated tetracycline resistance determinant (TetL) to bacterial organic-solvent tolerance property was unexpectedly observed and thus discussed. Overall results indicate that B. subtilis GRSW2-B1 has potential to be engineered and further established as a genetic host for bioproduction of butanol.

Partial Text

n-Butanol (hereafter referred to as butanol) is an important industrial chemical, widely used as a solvent, a stabilizer and feedstock for the production of polymers and plastics. Recently, butanol has been considered as a potential advanced biofuel with several advantages over ethanol because it contains higher energy density, lower vapor pressure, less corrosive and less water solubility (Connor and Liao 2009). Due to a limited supply of petroleum oil, microbial production of butanol has gained more attentions in present years. However, major roadblocks of the current butanol fermentation are low yield, low productivity and, most importantly, low titer due to the toxicity of butanol to its producing strains (Liu and Qureshi 2009). Generally, butanol inhibits microbial growth, including growth of current butanol-producing Clostridium strains, when the concentration reaches 2%v/v (ca. 16 g L-1). Butanol sensitivity and complex regulatory pathways of Clostridium strains are the key restrictions to the progress of butanol fermentation in the native host. Therefore, an alternative approach for butanol production is to find and construct butanol biosynthesis pathway in a heterologous host, of which one of the crucial considerable characteristics is butanol tolerance (Liu and Qureshi 2009). So far, alternative hosts being engineered for butanol production are well-characterized, genetically-amenable microorganisms, such as Escherichia coli (Atsumi et al. 2008Inui et al. 2008; Nielsen et al. 2009), Saccharomyces cerevisiae (Steen et al. 2008), Clostridium ljungdahlii (Kopke et al. 2010) and organic-solvent tolerant bacteria (OSTB), such as Pseudomonas putida S12 and Bacillus subtilis KS438 (Nielsen et al. 2009). They were capable of producing butanol, although at relatively low yield, but the critical remaining problem was that they still severely suffer from butanol toxicity as their viability was significantly decreased at 0.75, 1.0, 1.25, 2.0%v/v butanol for P. putida, E. coli, B. subtilis, (Nielsen et al. 2009), S. cerevisiae (Liu and Qureshi 2009) and Clostridia (Ezeji et al. 2010), respectively. Therefore, it is obviously shown that butanol tolerance is one of the important traits, if not the most, in selecting host and thus several studies have been conducted to search for butanol-tolerant microorganisms (Fischer et al. 2008;Knoshaug and Zhang 2009). Nevertheless, to be suitable as a potential genetic engineered host for bioproduction of chemicals, other fundamental, but requisite, knowledge of the host regarding genetic competency, gene expression strength, etc. should be proven feasible.

The aim of this work was to search for and develop a butanol-tolerant bacterium as a genetic-recombinant host for further application in bioproduction of alcohol-biofuel, initially focusing on butanol. Because butanol is classified as an extremely toxic chemical to microorganisms, its toxicity becomes the primary problem for its production via microbial fermentation. Numerous studies have been conducted to find, modify and construct an optimal host with high tolerance to butanol. While the construction of a butanol biosynthesis pathway in several heterologous hosts has been reported, the major obstacle limiting their achievement is due to low tolerance of the host to butanol toxicity (Fischer et al. 2008).

HS buffer: (1 mM HEPES buffer containing 250 mM sucrose, pH 7.0); HSMG buffer: (HS buffer with 1 mM MgCl2 and 10% glycerol, pH 7.0); GRSW2-B1: (Bacillus subtilis strain GRSW2-B1); OSTB: (organic-solvent tolerant bacteria);

The authors declare that they have no competing interests.

NK participated in the design of the study, performed the experimental work and data interpretation. WR participated in bacterial screening. TT, JK and ASV participated in the design of the study and analysis of the data. ASV wrote the manuscript and all authors participated in commenting and revising it. All authors contributed to the scientific discussion throughout the work and have read and approved the final manuscript.