Culture conditions that allow Zymomonas mobilis to assimilate N2 gas as a nitrogen source during bio-ethanol production
Chemically defined culture medium and culture conditions that allow bacteria to assimilate dinitrogen gas (N 2 ) as a nitrogen source during bio-ethanol production are disclosed herein. Methods of bioethanol production using the chemically defined culture medium and culture conditions are also disclosed.
1. A composition for producing a cellulosic bioalcohol, the composition comprising:
a bioalcohol producing, N 2 gas-fixing bacteria in genus Zymomonas that assimilates N 2 gas as its sole nitrogen source during bioalcohol production after nitrogen in a cellulosic feedstock is depleted;
an anaerobic culture medium that lacks a soluble nitrogen compound supplement, the medium comprising a hydrolysate of the cellulosic feedstock, iron, and molybdenum; and,
a N 2 gas in fluid communication with the anaerobic culture medium, where the sole nitrogen source for the bioalcohol producing, N 2 gas-fixing bacteria in the genus Zymomonas during cellulosic bioalcohol production after the nitrogen in the cellulosic feedstock is depleted is the N 2 gas.
2. The composition for producing a cellulosic bioalcohol of claim 1 , where the anaerobic culture medium comprises: Na 2 HPO 4 , KH 2 PO 4 , and NaCl in a molar ratio of 1:1.2:1.5.
3. The composition for producing a cellulosic bioalcohol of claim 1 , where the anaerobic culture medium comprises: Na 2 HPO 4 , KH 2 PO 4 , NaCl, and trace elements, where the trace elements comprise: nitrilotriacetic acid, MgSO 4 , CaCl 2 ·2H 2 O, (NH 4 ) 6 Mo 7 O 24 ·4H 2 O, FeSO 4 ·7H 2 O, ethylenediaminetetraacetic acid, ZnSO 4 ·7H 2 O, MnSO 4 ·H 2 O, CuSO 4 ·5H 2 O, Co(NO 3 ) 2 ·6H 2 O, and Na 2 B 4 O 7 ·10H 2 O.
4. The composition for producing a cellulosic bioalcohol of claim 1 , where the bioalcohol production is production of a bioalcohol selected from the group consisting of bioethanol, biopropanol, and biobutanol.
5. The composition for producing a cellulosic bioalcohol of claim 1 , where the bioalcohol producing, N 2 gas-fixing bacteria in the genus Zymomonas that assimilates N 2 gas as its sole nitrogen source during bioalcohol production is Zymomonas mobilis.
6. The composition for producing a cellulosic bioalcohol of claim 1 , where the bioalcohol producing, N 2 gas-fixing bacteria in the genus Zymomonas that assimilates N 2 gas as its sole nitrogen source during bioalcohol production is Zymomonas mobilis ZM4.
7. The composition for producing a cellulosic bioalcohol of claim 1 , where the anaerobic culture medium that lacks a soluble nitrogen compound supplement further comprises a phosphate source.
8. The composition for producing a cellulosic bioalcohol of claim 1 , where the anaerobic culture medium that lacks a soluble nitrogen compound supplement further comprises NaCl.
9. The composition for producing a cellulosic bioalcohol of claim 1 , where the anaerobic culture medium that lacks a soluble nitrogen compound supplement lacks a soluble nitrogen compound selected from the group consisting of ammonium, corn steep liquor (CSL) diammonium phosphate (DAP), ammonium hydroxide and other ammonium salts.
10. The composition for producing a cellulosic bioalcohol of claim 1 , where the composition has a temperature from 20° C. to 40° C.
11. The composition for producing a cellulosic bioalcohol of claim 1 , where the composition has a pH ranging from 4 to 9.2.
12. The composition for producing a cellulosic bioalcohol of claim 1 , where a concentration of the N 2 gas is from 0.45 mM to 76 mM per liter of the composition.
13. The composition for producing a cellulosic bioalcohol of claim 1 , where the anaerobic culture medium comprises: Na 2 HPO 4 , KH 2 PO 4 , NaCl, and trace elements, wherein the trace elements comprise: nitrilotriacetic acid, MgSO 4 , CaCl 2 ·2H 2 O, (NH 4 ) 6 Mo 7 O 24 ·4H 2 O, FeSO 4 ·7H 2 O, ethylenediaminetetraacetic acid, ZnSO 4 ·7H 2 O, MnSO 4 ·H 2 O, CuSO 4 ·5H 2 O, Co(NO 3 ) 2 ·6H 2 O, and Na 2 B 4 O 7 ·10H 2 O.
14. The composition for producing a cellulosic bioalcohol of claim 1 , where the N 2 gas is present at a concentration of 0.45 mM to 76 mM per liter of the anaerobic culture medium.
15. The composition for producing a cellulosic bioalcohol of claim 1 , where the Na 2 HPO 4 and said KH 2 PO 4 are present in a molar ratio of 1:1.2.
16. The composition for producing a cellulosic bioalcohol of claim 1 , where the bioalcohol production is bioethanol production.
17. The composition for producing a cellulosic bioalcohol of claim 1 , where the N 2 gas-fixing bacteria comprises a nitrogenase enzyme.
18. A composition for producing a cellulosic bioethanol, the composition consisting essentially of:
a bioalcohol producing, N 2 gas-fixing bacteria in genus Zymomonas that assimilates N 2 gas as its sole nitrogen source during bioalcohol production after nitrogen in a cellulosic feedstock is depleted;
an anaerobic culture medium that lacks a soluble nitrogen compound supplement, the medium consisting essentially of a hydrolysate of the cellulosic feedstock, iron, and molybdenum; and,
a N 2 gas in fluid communication with the anaerobic culture medium, where the sole nitrogen source for the bioalcohol producing, N 2 gas-fixing bacteria in the genus Zymomonas during cellulosic bioalcohol production is the N 2 gas after the nitrogen in the cellulosic feedstock is depleted.
19. A composition for producing a cellulosic ethanol, the composition consisting of:
a bioalcohol producing, N 2 gas-fixing bacteria in genus Zymomonas that assimilates N 2 gas as its sole nitrogen source during bioalcohol production after nitrogen in a cellulosic feedstock is depleted;
an anaerobic culture medium that lacks a soluble nitrogen compound supplement, the medium consisting of a hydrolysate of the cellulosic feedstock, iron, and molybdenum; and,
a N 2 gas in fluid communication with the anaerobic culture medium, where the sole nitrogen source for the bioalcohol producing, N 2 gas-fixing bacteria in the genus Zymomonas during cellulosic bioalcohol production is the N 2 gas after the nitrogen in the cellulosic feedstock is depleted.