Methods for the economical production of biofuel from biomass
View Patent ↗Methods for producing a biofuel are provided. Also provided are biocatalysts that convert a feedstock to a biofuel.
1. A method of making isobutanol, the method comprising:
a) providing a feedstock comprising a sugar selected from the group consisting of:
i) a six-carbon sugar;
ii) a six-carbon sugar oligomer;
iii) a five-carbon sugar; and
iv) any combination of i) through iii);
b) contacting the feedstock of a) with an engineered microorganism under suitable fermentation conditions such that said engineered microorganism converts the feedstock into isobutanol at:
i) a yield of at least 68 percent theoretical yield;
ii) a productivity of at least 0.75 grams isobutanol per liter per hour; and
iii) a total titer of at least 1.6% (w/v) of isobutanol in water, wherein said engineered microorganism expresses exogenous genes encoding an acetolactate synthase that catalyzes the conversion of pyruvate to acetolactate, a ketol-acid reductoisomerase that catalyzes the conversion of acetolactate to 2,3-dihydroxyisovalerate, a dihydroxy acid dehydratase that catalyzes the conversion of 2,3-dihydroxyisovalerate to α-ketoisovalerate, a ketoisovalerate decarboxylase that catalyzes the conversion of α-ketoisovalerate to isobutyraldehyde, and an isobutyraldehyde dehydrogenase that catalyzes the conversion of isobutyraldehyde to isobutanol; and
c) recovering the isobutanol.
2. The method of claim 1 , wherein the six-carbon sugar is glucose, galactose, or mannose.
3. The method of claim 2 , wherein the sugar is glucose.
4. The method of claim 1 , wherein the five-carbon sugar is arabinose or xylose.
5. The method of claim 1 , wherein the six-carbon sugar oligomer is sucrose or lactose.
6. The method of claim 1 , wherein the microorganism is converted to a co-product and wherein the co-product is feed grade.
7. The method of claim 1 , wherein the microorganism is operable to produce the isobutanol free of byproducts that would require additional processing steps for removal from the isobutanol.
8. The method of claim 1 , wherein byproducts account for less than or about 5% of the total product.
9. The method of claim 8 , wherein the byproducts are selected from the group consisting of acetate, lactate, butyrate, isobutyrate, acetolactate, acetoin, ethanol, isopentanol, and isobutyraldehyde.
10. The method of claim 1 , wherein the microorganism contains no DNA markers.
11. The method of claim 1 , wherein the microorganism provided includes DNA consisting of natural DNA.
12. The method of claim 1 , wherein the microorganism is operable at dissolved oxygen concentrations from about 0% to about 0.01% air saturation.
13. The method of claim 1 , wherein the microorganism converts a feedstock into the isobutanol under dissolved oxygen concentrations from about 0% to about 0.01% air saturation at essentially the same yield, productivity, and titer as under dissolved-oxygen concentrations greater than 0.01%.
14. The method of claim 1 , wherein the microorganism is selected to convert the feedstock into the isobutanol at a yield of at least 85 percent theoretical yield.
15. The method of claim 1 , wherein the microorganism is selected to convert the feedstock into the isobutanol at a yield of at least 90 percent theoretical yield.
16. The method of claim 1 , wherein the microorganism is selected to convert the feedstock into the isobutanol at a yield of at least 95 percent theoretical yield.
17. The method of claim 1 , wherein the microorganism is selected to convert the feedstock into the isobutanol at a yield of at least 99 percent theoretical yield.
18. The method of claim 1 , wherein the microorganism is selected to have the productivity of at least 1.5 grams isobutanol per gram cell dry weight per hour.
19. The method of claim 1 , wherein the microorganism is selected to have the productivity of at least 2 grams isobutanol per gram cell dry weight per hour.
20. The method of claim 1 , wherein the microorganism is selected to have the productivity of at least 2.5 grams isobutanol per liter per hour.
21. The method of claim 1 , wherein the microorganism is selected to have the productivity of at least 3 grams isobutanol per liter per hour.
22. The method of claim 1 , wherein the microorganism is selected to have the productivity of at least 3.5 grams isobutanol per liter per hour.
23. The method of claim 1 , wherein the microorganism is operable at a pH value between about 2 and 10 to produce the isobutanol.
24. The method of claim 23 , wherein the microorganism is operable at a pH value between about 6 and 8 to produce the isobutanol.
25. The method of claim 24 , wherein the microorganism is operable at a pH value of about 6.5.
26. The method of claim 1 , wherein the microorganism retains 85% productivity in the presence of a second pH value, wherein the second pH value represents an increase or decrease of about 1 pH unit from a first pH value.
27. The method of claim 26 , wherein the microorganism retains 95% productivity in the presence of a second pH value, wherein the second pH value represents an increase or decrease of about 1 pH unit from a first pH value.
28. The method of claim 27 , wherein the microorganism retains 99% productivity in the presence of a second pH value, wherein the second pH value represents an increase or decrease of about 1 pH unit from a first pH value.
29. The method of claim 26 , wherein the change in pH is a sudden change.
30. The method of claim 1 , wherein the microorganism is operable within a temperature range of about 20° C. to 80° C.
31. The method of claim 1 , wherein the microorganism is operable within a temperature range of about 25° C. to 60° C.
32. The method of claim 31 , wherein the microorganism is operable within a temperature range of about 30° C. to 40° C.
33. The method of claim 1 , wherein the microorganism retains 85% productivity in the presence of a second temperature value, wherein the second temperature value represents an increase or decrease of about 5° C. from a first temperature value.
34. The method of claim 33 , wherein the microorganism retains 95% productivity in the presence of a second temperature value, wherein the second temperature value represents an increase or decrease of about 5° C. from a first temperature value.
35. The method of claim 1 , wherein the microorganism retains 85% productivity in the presence of a second temperature value, wherein the second temperature value represents an increase or decrease of about 10° C. from a first temperature value.
36. The method of claim 35 , wherein the microorganism retains 95% productivity in the presence of a second temperature value, wherein the second temperature value represents an increase or decrease of about 10° C. from a first temperature value.
37. The method of claim 1 , wherein the microorganism is operable in a medium consisting of:
a) mineral salts comprised of major and minor bioelements;
b) vitamins; and
c) feedstock.
38. The method of claim 1 , wherein the microorganism is operable in a medium consisting of:
a) mineral salts comprised of major and minor bioelements; and
b) feedstock.
39. The method of claim 1 , wherein the microorganism is operable in a medium consisting of feedstock.
40. The method of claim 1 , wherein the microorganism comprises a growth rate of at least 0.2 generations per hour.
41. The method of claim 1 , wherein the microorganism is a facultative anaerobic microorganism operable at dissolved oxygen concentrations from about 0% to 0.01% air saturation.
42. The method of claim 41 , wherein the facultative anaerobic microorganism retains 85% productivity in the presence of dissolved oxygen concentrations of more than 1% air saturation.
43. The method of claim 42 , wherein the facultative anaerobic biocatalyst microorganism retains 95% productivity in the presence of dissolved oxygen concentrations of more than 1% air saturation.