Solvent tolerant microorganisms and methods of isolation
View Patent ↗Lactobacillus bacteria having enhanced tolerance to butanols have been isolated. The bacteria are useful for the fermentive production of butanol. New methods for the isolation of butanol tolerant Lactobacillus are also provided.
1. A method for the isolation of a butanol tolerant Lactobacillus microorganism comprising:
a) providing a microbial sample comprising a microbial consortium;
b) contacting the microbial consortium with a growth medium comprising a fermentable carbon source until the members of the microbial consortium are growing;
c) contacting the growing microbial consortium of step (b) with butanol; and
d) isolating the viable members of step (c) wherein a butanol tolerant microorganism is isolated
wherein the viable members of step (d) are tolerant to at least one of the following:
i) 2.5% w/v 1-butanol when grown on a solid medium at 37.degree. C.;
ii) 3.9% w/v 2-butanol when grown on a solid medium at 37.degree. C.;
iii) 2.7% w/v isobutanol when grown on a solid medium at 37.degree. C.;
iv) 5.0% w/v 2-butanone when grown on a solid medium at 37.degree. C.; or
v) 9.0% w/v ethanol I when grown on a solid medium at 37.degree. C.
2. A method according to claim 1 wherein the growing consortium of step (b) are growing in log phase.
3. A method according to claim 1 wherein after step (c) the consortium is plated on solid medium.
4. A method according to claim 3 wherein the solid medium contains butanol.
5. A method according to claim 1 wherein the contacting of step (c) is repeated one or more times.
6. A method according to claim 1 wherein the butanol concentration of the contacting step (c) is from about 0.8% w/v to about 3.0% w/v.
7. A method according to claim 1 wherein the isolating of step (d) comprises the steps of:
i) growing the viable members of step (d) in a liquid medium in the absence of butanol; whereby the viable members multiply;
ii) growing the cells of step (i) in the presence of butanol; and
iii) collecting the cells of step (ii) that grow in the presence of butanol wherein a butanol tolerant microorganism is isolated.
8. A method according to claim 1 wherein the fermentable carbon source is selected from the group consisting of sucrose, fructose, glucose, butyric acid, valeric acid and mixtures thereof.
9. A method according to claim 1 wherein the butanol is predominantly 1-butanol.
10. A method according to claim 1 wherein the butanol is predominantly 2-butanol.
11. A method according to claim 1 wherein the butanol is predominantly isobutanol.
12. A method according to claim 1 wherein the consortium is grown under anaerobic conditions.
13. A method according to claim 1 wherein the consortium is grown under microaerophilic conditions.
14. A method according to claim 1 wherein the consortium is grown under aerobic conditions.
15. A method according to claim 1 wherein the microbial sample is an environmental sample.
16. A method for the isolation of a butanol tolerant Lactobacillus comprising:
a) providing a microbial sample comprising a microbial consortium;
b) enriching the microbial consortium for the presence of Lactobacillus in a medium containing a fermentable carbon source to generate a Lactobacillus enriched culture in which members of the Lactobacillus enriched culture are growing;
c) contacting the growing Lactobacillus enriched culture of step (b) with butanol; and
d) isolating the viable members of step (c) wherein a butanol tolerant Lactobacillus is isolated
wherein the viable members of sten (d) are tolerant to at least one of the following:
i) 2.5% w/v 1-butanol when grown on a solid medium at 37.degree. C.;
ii) 3.9% w/v 2-butanol when grown on a solid medium at 37.degree. C.;
iii) 2.7% w/v isobutanol when grown on a solid medium at 37.degree. C.;
iv) 5.0% w/v 2-butanone when grown on a solid medium at 37.degree. C.; or
v) 9.0% w/v ethanol I when grown on a solid medium at 37.degree. C.
17. A method according to claim 16 wherein the growing Lactobacillus of step (c) are growing in log phase.
18. A method according to claim 16 wherein the microbial sample is an environmental sample.
19. A method according to claim 16 wherein after step (c) the members of the consortium are plated on solid medium.
20. A method according to claim 19 wherein the solid medium contains butanol.
21. A method according to claim 20 wherein the solid medium is a lactic acid bacteria medium containing butanol.
22. A method according to claim 16 wherein the contacting of step (c) is repeated one or more times.
23. A method according to claim 16 wherein the contacting of step (c) is done with the butanol at a concentration between about 0.8% w/v to about 3.0% w/v.
24. A method according to claim 16 wherein the isolating of step (d) comprises the steps of:
i) growing the viable members of step (d) in a liquid medium in the absence of butanol whereby the viable members multiply;
ii) growing the cells of step (I) in the presence of butanol; and
iii) collecting the cells of step (ii) that grow in the presence of butanol wherein a butanol tolerant microorganism is isolated.
25. A method according to claim 16 wherein the fermentable carbon source is selected from the group consisting of sucrose, fructose, glucose, butyric acid, valeric acid and mixtures thereof.
26. A method according to claim 16 wherein the butanol is predominantly 1-butanol.
27. A method according to claim 16 wherein the butanol is predominantly 2-butanol.
28. A method according to claim 16 wherein the butanol is predominantly isobutanol.