Butanol Tolerance in a Selection of Microorganisms
Butanol tolerance is a critical factor affecting the ability of microorganisms to generate economically viable quantities of butanol. Current Clostridium strains are unable to tolerate greater than 2% 1-butanol thus membrane or gas stripping technologies to actively remove butanol during fermentation are advantageous. To evaluate the potential of alternative hosts for butanol production, we screened 24 different microorganisms for their tolerance to butanol. We found that in general, a barrier to growth exists between 1% and 2% butanol and few microorganisms can tolerate 2% butanol. Strains of Escherichia coli, Zymomonas mobilis, and non-Saccharomyces yeasts were unable to surmount the 2% butanol growth barrier. Several strains of Saccharomyces cerevisiae exhibit limited growth in 2% butanol, while two strains of Lactobacillus were able to tolerate and grow in up to 3% butanol.
KeywordsButanol Tolerance BioScreenC
As an alternative liquid fuel, butanol offers distinct advantages because of its high energy content, miscibility with gasoline, octane rating, and low volatility . With the increasing price of oil, there is renewed interest in producing butanol biologically [2, 3]. Butanol can be produced from anaerobic bacterial (Clostridia) fermentations in a process that also produces acetone and ethanol (“ABE” fermentation). These fermentations suffer from low yield, low productivity, and low titer. In batch fermentations, Clostridia are quite sensitive to butanol and are typically unable to produce [4, 5] or tolerate concentrations greater than 2% [6, 7, 8]. A couple of attempts have been made using mutagenesis or serial enrichment to increase butanol tolerance in Clostridium acetobutylicum ATCC824. In these experiments, an increase from tolerance to 0.5% butanol to 1.5% butanol was observed [6, 9, 10]. However, in one case, this mutagenic event resulting in increased tolerance did not also result in a higher butanol yield . A compounding factor in Clostridia fermentation is the complex regulatory pathways involved in switching from acidogenesis to solventogenesis [11, 12, 13, 14]. Thus, it has been difficult to make significant progress in engineering highly productive strains for ABE fermentations . Many attempts have been made to develop economically viable methods of concurrent butanol removal and all have their disadvantages . An alternative strategy would be to establish a butanol production pathway in an alternative host lacking these complex regulatory pathways. Recently, a non-fermentative pathway for production of butanol was described [16, 17]. An important consideration in selecting a host for butanol production is butanol tolerance. Reports of butanol tolerance in organisms other than Clostridia are few. In one report, Lactobacillus hilgardii was reported to be more tolerant to an ethanol challenge if pre-stressed by exposure to various solvents including butanol . Two additional reports show that Saccharomyces and other yeast species can tolerate butanol at 1% up to near 2% [19, 20].
To evaluate the potential of alternative hosts for butanol production, we conducted a screening of a variety of microorganisms which are amenable to genetic engineering, are tolerant to hydrolysate inhibitors or ethanol, or are capable of fermentation at higher temperatures. The results of this initial screening will be useful for considering which organisms to engineer for butanol production.
Materials and Methods
Media and Strains
Strains screened for 1-butanol tolerance.
Growth was monitored using the BioScreenC (GrowthCurvesUSA, NJ, USA). Strains were grown overnight and then inoculated into fresh media and incubated until early log phase with an optical density (OD600) of 0.1–0.3 optical density units (ODU)/ml. The cells were harvested, washed with sterile H2O, and inoculated into media containing various concentrations (v/v) of 1-butanol. YPD medium was used at 1/2 strength due its high background OD. Quadruplicates of each condition were aliquoted into wells in the honeycomb BioScreenC plate and wide-band OD (420–580 nm) was recorded. Growth temperatures were the same as those used to grow the cultures, 30 or 37 °C as appropriate. Shaking of the BioscreenC plate was performed for 5 s prior to each reading. Growth rates were calculated from the linear range of exponential growth. This typically occurred at an OD between 0.08 and 0.3 but varied for different species.
Results and Discussion
Tolerance to Butanol
Various strains were chosen to test the effect of 1-butanol on growth (Table 1). The growth rate was calculated from growth curves monitored using the BioScreenC. The results showed that 1-butanol is toxic at low levels to most of the selected microorganisms. With a few exceptions, in the presence of 1% butanol, the relative growth rates were about 60% as that in medium without butanol. Very few microorganisms, however, can tolerate and grow in 2% or higher butanol.
Yeast strains used for this study are able to ferment at increased temperatures and/or were tolerant to ethanol. Candida acidothermophilum is tolerant to a high concentration of ethanol (14%) at high temperature (40 °C). C. sonorensis, P. tannophilus, and P. guilliermondii have a fast growth rate based on our previous unpublished work and are tolerant to 40–42 °C. Strains of P. methanolica are tolerant to methanol and ethanol. The yeast strain S. cerevisiae D5A was tolerant to degradation products present in pre-treated hardwoods yet failed to grow in 2% butanol. The S. cerevisiae strain ATCC20252 was tolerant to 40 °C and 9.5% ethanol, whereas ATCC26602 was tolerant to 43 °C and 11% ethanol. Both strains grew somewhat in 2% butanol. Strain ATCC9763 was tolerant to 14% ethanol at 30 °C and 12% ethanol at 35 °C, and strain ATCC4126 was found to ferment optimally at 36 °C. The Fali yeast strain was used for commercial ethanol production. No correlation can be drawn about whether tolerance to hydrolysates, high temperature, or high ethanol concentrations in yeast provides any benefit to butanol tolerance.
The strains of Lactobacillus used were tolerant to the inhibitors present in hydrolysates  and are known to be acid- and bile-tolerant. Several reports in the literature show, however, that although tolerant to acid and bile, Lactobacillus strains are not tolerant to more than 1% (v/v) butanol [22, 23, 24].
Effects of Temperature
The results of current research show that microorganisms can be naturally tolerant to relatively high concentrations of butanol. These results suggest that tolerance to inhibitors present in hydrolysates, high temperatures, or high ethanol concentrations may not be indicative of a strain’s ability to tolerate butanol. It has been demonstrated, however, that solvent tolerance leads to tolerance of heavy metals and antibiotics .
Butanol is toxic due to inhibition of membrane transport systems and enzymes and membrane disruption [26, 27, 28, 29]. Additionally, in S. cerevisiae, butanol has been shown to negatively impact initiation of translation . Cellular responses to butanol and other toxic solvents vary by alteration of the cell membrane lipid composition, expulsion by efflux pumps, and adjustments in cellular membrane protein content [6, 8, 25, 31, 32, 33, 34]. It appears that solvent tolerance is a complex mechanism and requires a robust general stress response.
In general, the majority of the 24 strains screened were not able to tolerate 2% butanol. The barrier to growth in the presence of butanol was between 1% and 2% butanol. These results are consistent with the reported growth barrier to strains of Clostridium. Typically, growth in the presence of 1% butanol resulted in a decrease of growth rate to about 60% of that in the absence of butanol. Two strains of Lactobacillus were able to grow in 2% butanol and reach a final OD 80–90% of that reached in the medium without butanol and may be a promising host for butanol production. Additionally, these strains were able to sustain limited growth in 2.5% and 3% butanol. Temperature affects the toxicity of butanol. A lower temperature allows better growth in limiting concentrations of butanol but does not allow growth in higher concentrations. Future experiments will broaden the range of microorganisms tested for butanol tolerance and further explore the detrimental effect of increased temperature on butanol tolerance.
The authors would like to thank the National Renewable Energy Laboratory’s Laboratory Directed Research and Development program for funding this work.
- 1.Schwarz, W. H., & Gapes, R. (2006). BioWorld Europe, 1, 16–19.Google Scholar
- 6.Baer, S. H., Blaschek, H. P., & Smith, T. L. (1987). Applied and Environmental Microbiology, 53(12), 2854–2861.Google Scholar
- 8.Vollherbst-Schneck, K., Sands, J. A., & Montenecourt, B. S. (1984). Applied and Environmental Microbiology, 47(1), 193–194.Google Scholar
- 9.Lin, Y. L., & Blaschek, H. P. (1983). Applied and Environmental Microbiology, 45(3), 966–973.Google Scholar
- 16.Atsumi, S., et al. . Metabolic engineering of Escherichia coli for 1-butanol production. Metab. Eng. (2008), doi:10.1016/j.ymben.2007.08.003.
- 17.Atsumi, S., Hanai, T., & Liao, J. C. (2008). Nature, 451(3), 87–90.Google Scholar
- 19.Antoce, O. A., et al. (1997). American Journal of Enology and Viticulture, 48(4), 413–422.Google Scholar
- 24.Matsumoto, M., Mochiduki, K., & Kondo, K. (2004). Journal of Bioscience and Bioengineering, 98(5), 344–347.Google Scholar
- 27.Moreira, A. R., Ulmer, D. C., & Linden, J. C. (1981). Biotechnology and Bioengineering Symposium, 11, 567–579.Google Scholar
- 28.Bowles, L. K., & Ellefson, W. L. (1985). Applied and Environmental Microbiology, 50, 1165–1170.Google Scholar
- 29.Ingram, L. O. (1976). Journal of Bacteriology, 125, 670–678.Google Scholar
- 32.Weber, F. J., & de Bont, J. A. (1996). Biochimica et Biophysica Acta, 1286, 225–245.Google Scholar
- 34.Isken, S., & Heipieper, H. J. (2002). Toxicity of organic solvents to microorganisms. In G. Bitton (Ed.), encyclopedia of environmental microbiology (vol. 6, (pp. 3147–3155)). New York: Wiley.Google Scholar
- 36.Barnett, J., Payne, R., & Yarrow, D. (2000). Yeasts: Characteristics and identification. Cambridge, UK: Cambridge University Press.Google Scholar
- 39.Slapack, G. E., Russell, I., & STewart, G. G. (1987). Thermophilic microbes in ethanol production. Boca Raton, FL: CRC.Google Scholar
- 41.Yamada, K., Ito, T., & Kobayashi, T. (1951). In H. Kyokai-Shi (Ed.), Selection of yeast by reuse, method., 9 pp. 176–179. Tokyo: Tokyo University.Google Scholar
- 43.Kunduru, M. R., & Pometto, A. (1996). Journal of Industrial Microbiology & Biotechnology, 16(4), 249–256.Google Scholar
- 45.Stevnsborg, N., & Lawford, H. G. (1986). Applied Microbiology and Biotechnology, 25(2), 106–115.Google Scholar