Biocatalysis and Agricultural Biotechnology 1 (2012) 5761

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Biocatalysis and Agricultural Biotechnology

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Adaptation of lactic acid bacteria to butanol$

Siqing Liu a,n, Kenneth M. Bischoff a, Timothy D. Leathers a, Nasib Qureshi b, Joseph O. Rich a, Stephen R. Hughes a
a b

Renewable Product Technology Research Unit, National Center for Agricultural Utilization Research, US Dept. of Agriculture, 1815 N. University St., Peoria, IL 61604, USA Bioenergy Research Unit, National Center for Agricultural Utilization Research, US Dept. of Agriculture, 1815 N. University St., Peoria, IL 61604, USA

a r t i c l e i n f o
Article history: Received 18 April 2011 Received in revised form 11 July 2011 Accepted 17 August 2011 Available online 30 August 2011 Keywords: Butanol Stress Tolerance Fermentation Biofuel Gram-positive bacteria

abstract
Butanol can be produced biologically through fermentation of various substrates by Gram-positive Clostridium species. However, to protably produce butanol at industrial scales, new microbial biocatalysts with increased tolerance to butanol are needed. In this study we report the isolation and selection of microbes to butanol. Ten strains, including Lactobacillus mucosae strains BR0605-3, BR0605-B15, BR071318, BR0713-20, BR0713-30, BR0713-33, Lactobacillus amylovorus strain NE-L 0206-19, Pediococcus parvulus NE-L 0206-31, Lactobacillus crispatus NE-L 0206-47, and Weissella confusa BR0216-18 were found capable of growth in 34% butanol after long term adaptation. These strains can be used to study tolerance mechanisms as well as identifying specic butanol stress response genes for development of butanol tolerant microbes. & 2011 Published by Elsevier Ltd.

1. Introduction Due to a limited supply of petroleum and increased environmental concerns, biological production of transportation fuels from renewable feedstocks has become a global priority (Atsumi et al., 2008; Ezeji et al., 2007; Jones and Woods, 1986). Butanol has been recognized as a superior biofuel for gasoline engines since it is energy dense than ethanol and it can be used directly as a drop-in fuel without engine modication (Antoni et al., 2007). Biologically, butanol is produced along with acetone and ethanol through anaerobic fermentation by Gram-positive bacteria, particularly Clostridium species. Previous studies have indicated that butanol can be produced from various agricultural feedstocks through microbial fermentation (Qureshi et al., 2007, 2008, 2010a, 2010b). However, the overall economics for commercial scale butanol fermentation remains uncertain as numerous barriers exist for protable production. Major hurdles include low fermentation efciency with cheaper lignocellulosic feedstocks and high toxicity of butanol to its producing Clostridia cultures (Fischer et al., 2008; Liu and Qureshi, 2009).

In a butanol fermentation bioreactor, the increased butanol concentrations, albeit desired for cost-effective product recovery, often lead to reduced growth and eventually death of the producing Clostridium species (Baer et al., 1987). The low (less than 2%) tolerance of butanol by producing Clostridium strains require an energy-efcient alternative butanol removal and recovery process (Borden and Papoutsakis, 2007; Qureshi et al., 2005). Suitable microbial hosts to be used for metabolic engineering strategies for butanol production must be able to tolerate higher titers of butanol in order to improve butanol yields (Fischer et al., 2008). Previous reports of butanol tolerance traits among industrially important microbes indicated that selected species of lactobacilli were able to grow in up to 3% butanol (Knoshaug and Zhang, 2008; Liu and Qureshi 2009). The goal of the present research was to evaluate butanol tolerance of a collection of lactic acid bacteria isolated from commercial ethanol plants (Liu et al., 2008; Skinner and Leathers, 2004). Furthermore, these strains were subjected to increasing butanol concentrations in the growth media to improve their butanol tolerance traits. Here we report our studies of screening, generating, and isolating butanol tolerant strains.

2. Materials and methods

Mention of trade names, proprietary product, or specic equipment does not constitute a guarantee or warranty by the United States Department of Agriculture and does not imply its approval to the exclusion of other products that may be suitable. USDA is an equal opportunity provider and employer. n Corresponding author. Tel.: 1 309 681 6566. E-mail address: Siqing.liu@ars.usda.gov (S. Liu). 1878-8181/$ - see front matter & 2011 Published by Elsevier Ltd. doi:10.1016/j.bcab.2011.08.008
$

2.1. Bacterial strains and growth conditions Bacteria were isolated from commercial wet mill and dry-grind ethanol facilities as described previously (Liu et al., 2008; Skinner

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and Leathers, 2004). Cultures were maintained on MRS agar plates (Becton Dickinson, Sparks, MD) at 37 1C, and single isolates were grown in MRS broth and glycerol stocks were stored in 80 1C freezer. Liquid cultures were started by inoculating single colonies into 3 ml MRS broth and incubated overnight 37 1C without shaking. Strains showing no growth aerobically were re-inoculated and placed into a sealed anaerobic jar with an AnaeroGenTM sachet (OXOID Ltd., Basingstoke, England). Growth was monitored by measuring the optical absorbance at 600 nm. For butanol tolerance studies, 24% (v/v) of 1-butanol (SigmaAldrich) was added to MRS broth in a nal volume of 5 ml in a 14 ml culture tube. The tubes were vortexed briey and inoculated with fresh overnight cultures in MRS at 5% ratio. The cultures growing to A600 2 or above were transferred again with 5% inocula into new media with the same or higher butanol concentration. 2.2. Phenotype identication Individual cultures were subjected to substrate utilization prole analyses using the API 50 CHL system (bioMerieux, Marcy IEtoile, France) as instructed by the manufacturer. Based on the API CHL tests, specic bacterial strains were identied as described previously (Liu et al., 2008; Skinner and Leathers, 2004). 2.3. 16S rDNA sequencing Genomic DNAs were isolated from overnight cultures using the Gram-positive DNA purication kit (Epicentre, Madison, Wisconsin). PCR fragments obtained from genomic PCRs with 16 rDNA primers (Whitehead and Cotta, 2001) were sequenced with the ABI Prism Dye Terminator Cycle Sequencing Ready Reaction Kit and the ABI Prism 310 DNA sequencer (Perkin-Elmer, Foster City, CA). Sequence analyses were performed at the San Diego Supercomputer Center Biology workbench (http://workbench. sdsc.edu/) and bacterial strains were identied via blast search against the NCBI data base (http://www.ncbi.nlm.nih.gov/). 2.4. Long term butanol adaptation The long term adaptation studies were carried out under strict anaerobic conditions. Bacterial cultures were inoculated in MRS supplemented with 2%, 3% and 4% butanol and incubated at 37 1C in an anaerobic jar. Growth was monitored by measuring the optical density of each culture. The cultures were transferred

when A600 reached 2.0 (35 days) and above. Once the cultures demonstrated growth with one level of butanol concentration, they were subsequently subcultured into fresh nutrient media supplemented with the same or higher level of butanol. The initial screening for tolerance started with 2% butanol, after 20 passages/ serial transfers, the selected cultures were inoculated into MRS with 3% butanol, and the ones showing growth in 3% butanol were transferred for 20 passages and then moved to 4% butanol. 2.5. Short term butanol stress Selected strains from the original isolated cultures NE-L 0206-19, BR 0216-18, BR 0315-B4, BR 0713-30, and BR 0713-33 were plated out on MRS and single colonies were inoculated in MRS broth supplemented with 4% butanol. The cultures were maintained for one week and OD600 were recorded and plotted against time remained in the same culture broth. Three replicates were carried out.

3. Results and discussion 3.1. Butanol tolerance Of 138 individual isolates screened in the collection, 86 strains (62%) were found capable of growth in MRS with 23% butanol (Tables 1 and 2). Interestingly, for the rst batch of nineteen isolates screened against MRS supplemented with 2% butanol as shown in Table 1, almost all of the 2% butanol tolerant cultures except NE-L 0206-18 were also capable of growing in 3% butanol. Thus, the rest of the cultures were screened directly with 3% butanol in the media (Table 2). Overall, a total of 85 isolates (61% of the collection) were found to be tolerant to 3% butanol, and 44 isolates (31%) showed tolerance to 4% butanol (Tables 1 and 2). Among the 44 tolerant cultures, 10 isolates showed strong growth in 4% butanol ( ): OD600 is equal to or greater than 3.0; a total of 12 strains showed moderate growth ( ): OD600 is equal or greater than 2.0 and 18 isolates showed slow growth ( ): OD600 is equal or greater than 1.0, and 4 strains showed poor growth ( ): OD600 is less than 1.0 in 2, 3, or 4% butanol. 3.2. Identication of butanol tolerant strains Among the most tolerant cultures from the long term butanol adaptation procedure, the NE-L 0206-31 and NE-L 0206-47 were

Table 1 Long term adaptation and selection for tolerance of 24% butanol among LAB isolated from ethanol plants. Original isolate NE-L 0206-2 NE-L 0206-5 NE-L 0206-22 NE-L 0206-10 NE-L 0206-19 NE-L 0206-20 NE-L 0206-26 NE-L 0206-13 NE-L 0206-3 NE-L 0206-21 NE-L 0206-18 NE-L 0206-24 NE-L 0206-34 NE-L 0206-32 NE-L 0206-31 NE-L 0206-25 NE-L 0206-6 NE-L 0206-35 NE-L 0206-38 Strain identication (ID) Fusobacterium nucleatum ss nucleatum Fusobacterium nucleatum ss polymorphum Fusobacterium nucleatum ss nucleatum Fusobacterium nucleatum ss nucleatum Lactobacillus amylovorus Lactobacillus amylovorus Lactobacillus crispatus Lactobacillus delbrueckii ss delbrueckii Lactobacillus delbrueckii ss lactis Lactobacillus fermentum Lactobacillus kitasontonis Lactobacillus kitasontonis Lactobacillus pontis Lactobacillus vaginalis Pediococcus parvulus Weissella viridescens Methods used for ID API CHL API CHL API CHL API CHL 16S rDNA 16S rDNA API CHL API CHL API CHL API CHL 16S rDNA 16S rDNA 16S rDNA API CHL API CHL API CHL 2% Butanol OD600 11 11 11 1 3% Butanol OD600 1 11 1 11 4% Butanol OD600

sequencing sequencing

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sequencing sequencing sequencing

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Lactobacilli

API CHL

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Table 2 Screening and adaptation of LAB isolates from ethanol plants after being challenged to 3 and 4% butanol.
Original isolate NE-H 0206-10 NE-L 0206-45 NE-L 0206-42 NE-L 0206-46 NE-L 0206-48 NE-L 0206-49 NE-H 0206-7 NE-H 0206-44 BR0315-B7 BR0605-5 BR0605-8 BR0713-19 BR0216-39 BR0315-L5 BR0315-S1 BR0315-S2 BR0315-S3 BR0315-S4 BR0315-S5 BR0315-S6 BR0605-E4 BR0605-E5 BR0605-E8 NE-H 0206-17 NE-L 0206-47 NE-H 0206-12 NE-L 0206-43 BR0216-20 BR0216-29 BR0315-B1 BR0315-B5 BR0315-B6 BR0315-B8 BR0315-B9 BR0315-B10 BR0605 B10 BR0605 B 21 BR0315-1 BR0315-L2 BR0315-L3 BR0315-L6 BR0713-17 BR0713-21 BR0713-25 BR0713-27 BR0713-31 BR0315-B4 NE-L 0206-41 NE-H 0206-6 NE-H 0206-29 NE-H 0206-35 BR0605-1 BR0605-3 BR0605-4 BR0605-6 BR0605-7 BR0605-19 BR0605 B4 BR0650 B15 BR0713-16 BR0713-18 BR0713-20 BR0713-22 BR0713-23 BR0713-24 BR0713-26 BR0713-28 BR0713-30 BR0713-32 BR0713-33 BR0713-34 BR0713-15 BR0713-29 BR0605-2 BR0605-E6 NE-L 0206-40 Strain identication (ID) Fusobacterium nucleatum ss nucleatum Fusobacterium nucleatum ss nucleatum Lactobacillus amylovorus Lactobacillus amylovorus Lactobacillus amylovorus Lactobacillus amylovorus Lactobacillus amylovorus Lactobacillus amylovorus Lactobacillus amylovorus Lactobacillus amylovorus Lactobacillus amylovorus Lactobacillus amylovorus Lactobacillus brevis Lactobacillus brevis Lactobacillus brevis Lactobacillus brevis Lactobacillus brevis Lactobacillus brevis Lactobacillus brevis Lactobacillus brevis Lactobacillus brevis Lactobacillus brevis Lactobacillus brevis Lactobacillus crispatus Lactobacillus crispatus Lactobacillus delbrueckii ss. lactis Lactobacillus delbrueckii ss. lactis Lactobacillus fermentum Lactobacillus fermentum Lactobacillus fermentum Lactobacillus fermentum Lactobacillus fermentum Lactobacillus fermentum Lactobacillus fermentum Lactobacillus fermentum Lactobacillus fermentum Lactobacillus fermentum Lactobacillus fermentum Lactobacillus fermentum Lactobacillus fermentum Lactobacillus fermentum Lactobacillus fermentum Lactobacillus fermentum Lactobacillus fermentum Lactobacillus fermentum Lactobacillus fermentum Lactobacillus johnsonii Lactobacillus kitasontonis Lactobacillus kitasontonis Lactobacillus kitasontonis Lactobacillus kitasontonis Lactobacillus mucosae Lactobacillus mucosae Lactobacillus mucosae Lactobacillus mucosae Lactobacillus mucosae Lactobacillus mucosae Lactobacillus mucosae Lactobacillus mucosae Lactobacillus mucosae Lactobacillus mucosae Lactobacillus mucosae Lactobacillus mucosae Lactobacillus mucosae Lactobacillus mucosae Lactobacillus mucosae Lactobacillus mucosae Lactobacillus mucosae Lactobacillus mucosae Lactobacillus mucosae Lactobacillus mucosae Lactobacillus paracasei Lactobacillus paracasei Lactobacillus paracasei Lactobacillus paracasei Lactobacillus pontis Methods of ID API CHL API CHL 16S rDNA 16S rDNA API CHL 16S rDNA API CHL 16S rDNA 16S rDNA 16S rDNA 16S rDNA 16S rDNA 16S rDNA API CHL API CHL API CHL API CHL API CHL API CHL API CHL API CHL API CHL API CHL API CHL API CHL API CHL API CHL 16S rDNA 16S rDNA 16S rDNA 16S rDNA 16S rDNA 16S rDNA 16S rDNA 16S rDNA API CHL API CHL 16S rDNA API CHL API CHL API CHL 16S rDNA 16S rDNA 16S rDNA 16S rDNA 16S rDNA 16S rDNA 16S rDNA 16S rDNA 16S rDNA 16S rDNA 16S rDNA 16S rDNA 16S rDNA 16S rDNA 16S rDNA 16S rDNA API CHL API CHL 16S rDNA 16S rDNA 16S rDNA 16S rDNA 16S rDNA 16S rDNA 16S rDNA 16S rDNA 16S rDNA 16S rDNA 16S rDNA 16S rDNA 16S rDNA 16S rDNA 16S rDNA API CHL 16S rDNA 3% Butanol OD600 1 11 11 11 1 11 1 11 1 1 1 11 1 1 4% Butanol OD600

sequencing sequencing sequencing sequencing sequencing sequencing sequencing sequencing sequencing

sequencing sequencing sequencing sequencing sequencing sequencing sequencing sequencing

sequencing

sequencing sequencing sequencing sequencing sequencing sequencing sequencing sequencing sequencing sequencing sequencing sequencing sequencing sequencing sequencing sequencing

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sequencing sequencing sequencing sequencing sequencing sequencing sequencing sequencing sequencing sequencing sequencing sequencing sequencing sequencing sequencing sequencing

11 11 11 11 11 11 11 11 11 11 11 11

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Table 2 (continued ) Original isolate BR0605-E1 BR0605 B5 BR0605-E7 NE-H 0206-14 NE-H 0206-15 NE-H 0206-16 NE-H 0206-24 NE-H 0206-28 NE-H 0206-9 BR0216-1 BR0216-2 BR0216-3 BR0216-4 BR0216-5 BR0216-6 BR0216-7 BR0216-8 BR0216-9 BR0216-10 BR0216-11 BR0216-12 BR0216-13 BR0216-14 BR0216-15 BR0216-16 BR0216-17 BR0216-18 BR0216-19 BR0216-21 BR0216-22 BR0216-23 BR0216-24 BR0216-25 BR0216-26 BR0216-27 BR0216-28 BR0216-30 BR0216-31 BR0216-32 BR0216-33 BR0216-34 BR0216-35 NE-L 0206-44 Strain identication (ID) Lactobacillus rhamnosus Lactobacillus rhamnosus Lactobacillus rhamnosus Lactobacillus sp. Lactobacillus sp. Lactobacillus sp Lactobacillus sorbrius Leuconostoc mesenteroides ssp. cremoris Propionibacterium propionicus BGB Weissella confusa Weissella confusa Weissella confusa Weissella confusa Weissella confusa Weissella confusa Weissella confusa Weissella confusa Weissella confusa Weissella confusa Weissella confusa Weissella confusa Weissella confusa Weissella confusa Weissella confusa Weissella confusa Weissella confusa Weissella confusa Weissella confusa Weissella confusa Weissella confusa Weissella confusa Weissella confusa Weissella confusa Weissella confusa Weissella confusa Weissella confusa Weissella confusa Weissella confusa Weissella confusa Weissella confusa Weissella confusa Weissella confusa Weissella viridescens Methods of ID API CHL API CHL API CHL API CHL 16S rDNA API CHL 16S rDNA 16S rDNA API CHL 16S rDNA 16S rDNA 16S rDNA 16S rDNA 16S rDNA 16S rDNA 16S rDNA 16S rDNA 16S rDNA 16S rDNA 16S rDNA 16S rDNA 16S rDNA 16S rDNA 16S rDNA 16S rDNA 16S rDNA 16S rDNA 16S rDNA 16S rDNA 16S rDNA 16S rDNA 16S rDNA 16S rDNA 16S rDNA 16S rDNA 16S rDNA 16S rDNA 16S rDNA 16S rDNA 16S rDNA 16S rDNA 16S rDNA API CHL 3% Butanol OD600 11 11 4% Butanol OD600

sequencing sequencing sequencing sequencing sequencing sequencing sequencing sequencing sequencing sequencing sequencing sequencing sequencing sequencing sequencing sequencing sequencing sequencing sequencing sequencing sequencing sequencing sequencing sequencing sequencing sequencing sequencing sequencing sequencing sequencing sequencing sequencing sequencing sequencing sequencing sequencing

11 11

identied by the API 50 CHL system as Pediococcus parvulus and Lactobacillus crispatus, respectively. The other 8 butanol tolerant isolates were identied by 16 rDNA sequencing: NE-L 0206-19 was identied as Lactobacillus amylovorus, BR0216-18 as Weissella confusa, and the remaining six cultures BR0605-3, BR0605-B15, BR0713-18, BR0713-20, BR0713-30, and BR0713-33 were identied as Lactobacillus mucosae (Tables 1 and 2). It is interesting that Lactobacillus brevis and Lactobacillus delbrueckii were not among the 5 most tolerant species identied in the present study, although butanol-tolerant strains of these species were identied by others (Knoshaug and Zhang, 2008). This might be due to the different culture and selection conditions. We initially started with aerobic cultures, but found no strains could grow at 2% butanol. Only upon switching to strictly anaerobic conditions were we able to identify tolerant strains. The fact that the tolerant traits were found in species of lactobacilli suggested that the tolerant strains might possess specic pathways to compensate for the butanol stress, or there are unique membrane/cell wall structures to protect from cellular damage caused by butanol. It is possible that specic butanol stressinduced proteins work together to confer butanol tolerance. 3.3. Butanol stress In order to determine whether butanol tolerance was the result of long term adaptation to butanol, six original isolated strains (prior

to adaptation) were grown on MRS plates in the absence of butanol and subsequently tested for butanol tolerance. L. amylovorus strain NE-L 0206-19, L. mucosae strains BR0713-30 and BR0713-33, and W. confusa strain BR0216-18 were chosen for this study because they were among the most butanol tolerant strains identied; on long-term culture, these strains were adapted and grew in the presence of 4% butanol to an OD600Z3.0 (Tables 1 and 2). L. brevis strain BR0315-L5 and Lactobacillus johnsonii strain BR0315-B4 were chosen as representatives of less tolerant strains, since they grew to an OD 600 of Z2.0 in the presence of 3% butanol, but failed to grow on 4% butanol (Table 2). These six strains were plated onto MRS plates in the absence of butanol, and isolated colonies were used to directly inoculate MRS broth containing 4% butanol (Fig. 1). Only highly butanol tolerant L. mucosae strains BR0713-30 and BR0713-33 showed growth within 7 days, suggesting that these strains possess genetic resistance to butanol. Notice that the short term butanol stress test (Fig. 1) differs with the long term adaptation (Tables 1 and 2), in (a) the initial inoculums, from single colonies versus 5% of overnight cultures, and b) initial butanol content from 4% butanol versus gradual transfer of cultures from 2% or 3% to 4% butanol. Thus it is not surprising that the growth of the L. mucosae strains BR0713-30 and BR0713-33 (highly butanol tolerant after adaptation; Tables 1 and 2) was relatively slow, and none of the remaining strains exhibited growth in the presence of 4% butanol after 7 days (Fig. 1), suggesting that their tolerance is the result of adaptation to butanol stress selection

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Acknowledgements We would like to thank Jacqueline Zane, Eric Hoecker, Tiffany Bone, and Melinda Nunnally for their excellent technical support.

References
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Fig. 1. Short term butanol stress test. Selected strains from the original isolates from ethanol plants NE-L 0206-19 (Lactobacillus amylovorus), BR 0216-18 (Weissella confusa), BR 0315-B4 (Lactobacillus johnsonii), BR 0713-30 (Lactobacillus mucosae), and BR 0713-33 (Lactobacillus mucosae) were plated out on MRS agar plates and single colonies were inoculated in MRS broth supplemented with 4% butanol. The cultures were maintained for one week and OD600 values were plotted against time when OD600 readings were taken in the same culture broth. Triplicate experiments were carried out and the average values were presented.

after being exposed to butanol and serially transferred for long time period. These results suggested that for selected species, including L. amylovorus, L. mucosae, and W. confusa, long term adaptation is necessary to acquire butanol tolerance and the acquired butanol tolerance trait appears to be associated with individual strains. Butanol stress and tolerance studies are of signicant importance for commercial production of butanol. Previous studies showed that during butanol stress, bacterial cells responded with changes of membrane fatty acid composition, structure, and membrane uidity (Baer et al., 1987; Liu and Qureshi, 2009; Vollherbst Schneck et al., 1984). In addition, general stress-responsive proteins such as heat shock proteins may be involved in tolerance. It is necessary to understand the tolerance mechanisms and to identify proteins/genes in lactobacilli strains that are responsible for butanol tolerance. Additional biochemical or proteomics studies comparing the selected strains before and after adaptations will be necessary to identify proteins/genes associated with butanol tolerance and stress. This study is the rst step in facilitating our understanding of the molecular mechanisms that microbes use to deal with butanol stress. These butanol tolerant microbes do not produce butanol naturally and we aim to introduce butanol production pathways by genetic engineering. This will benet researchers in developing efcient biofuel producing strains with increased butanol tolerance traits.