Butanol Tolerance in a Selection of Microorganisms
Eric P. Knoshaug & Min Zhang
Received: 4 June 2008 /Accepted: 26 November 2008 /
Published online: 17 December 2008
# Humana Press 2008
Abstract 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
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% [6–8]. A couple of attempts have been made using mutagenesis or serial enrichment to
increase butanol tolerance in Clostridium acetobutylicum ATCC824. In these experiments,
Appl Biochem Biotechnol (2009) 153:13–20
E. P. Knoshaug (*):M. Zhang
National Renewable Energy Laboratory, National Bioenergy Center, Golden, CO 80401, USA
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
[11–14]. 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 used are listed in Table 1. Stock cultures of Lactobacillus were grown in MRS
medium (Difco, 0881-17-5) at 30 or 37 °C without shaking. Stock cultures of Escherichia
coli were grown in LB medium (Sigma, L-3022) at 30 or 37 °C with shaking at 220 rpm.
Yeast stock cultures were grown in yeast-peptone-dextrose (YPD) medium (Sigma, Y-1375)
at 30 °C with shaking at 220 rpm. Stock cultures of Zymomonas mobilis were grown in
RMG medium, consisting of 10 g/L yeast extract, 2 g/L KH2PO4, and 20 g/L glucose, at
30 °C with shaking at 100 rpm. Strains were acquired from the American Type Culture
Collection (ATCC), the National Center for Agricultural Utilization Research (NRRL), and
the Centraalbureau voor Schimmelcultures (CBS).
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.
14Appl Biochem Biotechnol (2009) 153:13–20
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.
Of the non-Saccharomyces yeast species, only one, Candida sonorensis, was able to
tolerate 2% butanol. However, C. sonorensis grew slowly and the initial OD did not double
(Fig. 1). Both strains of P. methanolica were extremely sensitive to butanol and were not
able to grow in 1% butanol. Pachysolen tannophilus and P. guilliermondii were also
sensitive to 1% butanol and reached only 40% of their relative growth rates (Fig. 1). Strains
of Saccharomyces cerevisiae, in general, were not able to tolerate and grow in 2% butanol
and the haploid strain GY5196 could not tolerate 1% butanol (Fig. 2). Only three out of the
ten strains grew in 2% butanol. The S. cerevisiae strains ATCC26602, ATCC20252, and
Fali grew but were severely inhibited by 2% butanol reaching growth rates 10–20% of
Table 1 Strains screened for 1-butanol tolerance.
aStrain is only designated if known
bReference is only given if species or strain has been previously referenced with regards to relevant biomass
to biofuels work. Unreferenced species and strains were already present in our laboratory from unknown
Appl Biochem Biotechnol (2009) 153:13–20 15
those in medium without butanol (Fig. 2). These strains reached cell densities of 2-, 3-, and
6-fold, respectively, of initial cell density in 24 h.
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%
Fig. 2 Growth rates in butanol relative to medium without butanol of Saccharomyces cerevisiae strains
Fig. 1 Growth rates in butanol relative to medium without butanol of non-Saccharomyces yeast species
16Appl Biochem Biotechnol (2009) 153:13–20
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.
Strains of Lactobacillus were more tolerant of butanol than yeast. Two strains, L.
delbrueckii and Lactobacillus brevis, were able to grow in 2% butanol with relative growth
rates of 55% and 58%, respectively (Fig. 3). These two strains were also able to grow in
2.5% butanol with relative growth rates of 30% and 44%, respectively (Fig. 3). L. brevis
was able to grow in 3% butanol with a 30% growth rate relative to the no butanol control
(Fig. 3). In 2% butanol, L. delbrueckii reached a cell density 90% of that of the cell density
in the control culture grown without butanol and reached a cell density 10-fold of the initial
cell density in 2.5% butanol in 24 h. Additionally, L. delbrueckii was grown at an increased
temperature of 37 °C. Similarly, in 2% butanol, L. brevis reached a cell density 80% of that
of the cell density in the control culture grown without butanol and reached a cell density 4-
fold of the initial cell density in both 2.5% and 3% butanol in 24 h. The Lactobacillus strain
MONT4 did not grow in 2% butanol.
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 [22–24].
Z. mobilis strains were sensitive to 2% butanol. Z. mobilis ATCC31821 grew slowly and
the initial OD did not double (Fig. 4). The Z. mobilis 8b strain is relatively tolerant to acetic
acid present in hydrolysates, while the parental ATCC31821 strain is tolerant to 13% ethanol.
Effects of Temperature
We investigated the effect of temperature on butanol tolerance in E. coli. The cultures grew
faster at 37 °C; however, they were not as tolerant to 1% butanol as when grown at 30 °C.
Fig. 3 Growth rates in butanol relative to medium without butanol of Lactobacillus strains
Appl Biochem Biotechnol (2009) 153:13–2017
Although a lower temperature improved growth in the presence of 1% butanol, it did not
allow E. coli to grow at the higher concentration of 2% butanol (Fig. 5). A similar effect
was observed for C. acetobutylicum ATCC824. The absolute growth rate was slower at the
lower temperatures and growth was observed in 1.5% butanol, while at the higher
temperature, the absolute growth rate was faster but the strain was not able to tolerate 1.5%
butanol . E. coli was included in the screening based on their increasingly being targeted
for development as a butanologen [16, 17].
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
Fig. 5 Growth rates in butanol relative to medium without butanol for Escherichia coli strains at 30 and 37 °C
Fig. 4 Growth rates in butanol relative to medium without butanol of Zymomonas mobilis strains
18Appl Biochem Biotechnol (2009) 153:13–20
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 [26–29]. 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, 31–34]. 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.
Acknowledgment The authors would like to thank the National Renewable Energy Laboratory’s
Laboratory Directed Research and Development program for funding this work.
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