1. A zinc-binding ligand of the following formula (III)
A-B-C-D-X\u2003\u2003(III)
wherein:
A is a chemical group of the following formula which reversibly binds to a HisB10 Zn2+ site of an insulin hexamer;
wherein A1 is a valence bond;
AR1 is the group
where C1 is attached to the nitrogen;
C1 is CH2;
AR2 is phenyl;
B is a linker selected from the group consisting of
A valence bond and
A chemical group GB of the formula \u2014B1\u2014B2\u2014C(O)\u2014, \u2014B1\u2014B2\u2014SO2\u2014, \u2014B1\u2014B2\u2014CH2\u2014, or \u2014B1\u2014B2\u2014NH\u2014; wherein B1 is a valence bond, \u2014O\u2014, \u2014S\u2014, or \u2014NR6\u2014,
B2 is a valence bond, C1-C18-alkylene, C2-C18-alkenylene, C2-C18-alkynylene, arylene, heteroarylene, \u2014C1-C18-alkyl-aryl-, \u2014C2-C18-alkenyl-aryl-, \u2014C2-C18-alkynyl-aryl-, \u2014C(\u2550O)\u2014C1-C18-alkyl-C(\u2550O)\u2014, \u2014C(\u2550O)\u2014C1-C18-alkenyl-C(\u2550O)\u2014, \u2014C(\u2550O)\u2014C1-C18-alkyl-O\u2014C1-C18-alkyl-C(\u2550O)\u2014, \u2014C(\u2550O)\u2014C1-C18-alkyl-S\u2014C1-C18-alkyl-C(\u2550O)\u2014, \u2014C(\u2550O)\u2014C1-C18-alkyl-NR6\u2014C1-C18-alkyl-C(\u2550O)\u2014, \u2014C(\u2550O)-aryl-C(\u2550O)\u2014, \u2014C(\u2550O)-heteroaryl-C(\u2550O)\u2014;
wherein the alkylene, alkenylene, and alkynylene moieties are optionally substituted with \u2014CN, \u2014CF3, \u2014OCF3, \u2014OR6, or \u2014NR6R7 and the arylene and heteroarylene moieties are optionally substituted with halogen, \u2014C(O)OR6, \u2014C(O)H, OCOR6, \u2014SO2, \u2014CN, \u2014CF3, \u2014OCF3, \u2014NO2, \u2014OR6, \u2014NR6R7, C1-C18-alkyl, or C1-C18-alkanoyl;
R6 and R7 are independently H, C1-C4-alkyl;
C is a fragment consisting of 0 to 5 neutral amino acids, wherein the individual neutral amino acids are the same or different
D is a fragment consisting of 1 to 20 basic amino acids independently selected from the group consisting of Lys and Arg or D-isomers thereof; and
X is \u2014OH, \u2014NH2 or a diamino group,
or a salt thereof with a pharmaceutically acceptable acid or base, or any optical isomer or mixture of optical isomers, racemic mixture, or tautomeric forms thereof.
2. A zinc-binding ligand according to claim 1 wherein A1 is a valence bond, C1-C6-alkylene, \u2014NH\u2014C(\u2550O)-A2-, \u2014C1-C6-alkyl-S\u2014, \u2014C1-C6-alkyl-O\u2014-, or \u2014C(\u2550O)\u2014, wherein any C1-C6-alkyl moiety is optionally substituted by R1A.
3. A zinc-binding ligand according to claim 2 wherein A1 is a valence bond, C1-C6-alkylene, \u2014NH\u2014C(\u2550O)-A2-, \u2014C1-C6-alkyl-S\u2014, or \u2014C1-C6-alkyl-O, wherein any C1-C6-alkyl moiety is optionally substituted by R1A.
4. A zinc-binding ligand according to claim 3 wherein A1 is a valence bond, C1-C6-alkylene, or \u2014NH\u2014C(\u2550O)-A2, wherein any C1-C6-alkyl moiety is optionally substituted by R1A.
5. A zinc-binding ligand according to claim 4 wherein A1 is a valence bond or C1-C6-alkylene, wherein any C1-C6-alkyl moiety is optionally substituted by R1A.
6. A zinc-binding ligand according to claim 5 wherein A1 is a valence bond.
7. A zinc-binding ligand according to claim 1 wherein A2 is a valence bond or \u2014C1-C6-alkyl-O\u2014.
8. A zinc-binding ligand according to claim 7 wherein A2 is a valence bond.
9. A zinc-binding ligand according to claim 1 wherein AR1 is arylene or heteroarylene, wherein the aryl or heteroaryl moieties are optionally substituted by one or more R1B independently.
10. A zinc-binding ligand according to claim 9 wherein AR1 is selected from the group of compounds consisting of phenylene, biphenylylene, naphthylene, anthra-cenylene, phenanthrenylene, fluorenylene, indenylene, azulenylene, furylene, thienylene, pyrrolylene, oxazolylene, thiazolylene, imidazolylene, isoxazolylene, isothiazolylene, 1,2,3-triazolylene, 1,2,4-triazolylene, pyranylene, pyridylene, pyridazinylene, pyrimidinylene, pyrazinylene, 1,2,3-triazinylene, 1,2,4-triazinylene, 1,3,5-triazinylene, 1,2,3-oxadiazolylene, 1,2,4-oxadiazolylene, 1,2,5-oxadiazolylene, 1,3,4-oxadiazolylene, 1,2,3-thiadiazolylene, 1,2,4-thiadiazolylene, 1,2,5-thiadiazolylene, 1,3,4-thiadiazolylene, tetrazolylene, thiadiazinylene, indolylene, isoindolylene, benzofurylene, benzothienylene, indazolylene, benzimidazolylene, benzthiazolylene, benzisothiazolylene, benzoxazolylene, benzisoxazolylene, purinylene, quinazolinylene, quinolizinylene, quinolinylene, isoquinolinylene, quinoxalinylene, naphthyridinylene, pteridinylene, carbazolylene, azepinylene, diazepinylene, and acridinylene, optionally substituted by one or more R1B independently.
11. A zinc-binding ligand according to claim 10 wherein AR1 is selected from the group of compounds consisting of phenylene, biphenylylene, naphthylene, pyridinylene, fyrylene, indolylene and carbazolylene, optionally substituted by one or more R1B independently.
12. A zinc-binding ligand according to claim 11 wherein AR1 is selected from the group of compounds consisting of phenylene, indolylene and carbazolylene, optionally substituted by one or more R1B independently.
13. A zinc-binding ligand according to claim 12 wherein AR1 is phenylene optionally substituted by one or more R1B independently.
14. A zinc-binding ligand according to claim 12 wherein AR1 is indolylene optionally substituted by one or more R1B independently.
15. A zinc-binding ligand according to claim 14 wherein AR1 is
16. A zinc-binding ligand according to claim 12 wherein AR1 is carbazolylene optionally substituted by one or more R1B independently.
17. A zinc-binding ligand according to claim 16 wherein AR1 is
18. A zinc-binding ligand according to claim 1 wherein R1B is selected from the group consisting of
hydrogen, halogen, \u2014CN, \u2014CF3, \u2014OCF3, \u2014NO2, \u2014OR1C, \u2014NR1CR1D, \u2014SR1C, \u2014S(O)2R1C, \u2014NR1CC(O)R1D, \u2014OC1-C6-alkyl-C(O)NR1CR1D, \u2014C2-C6-alkenyl-C(\u2550O)OR1C, \u2014C(O)OR1C, \u2550O, \u2014NH\u2014C(\u2550O)\u2014O\u2014C1-C6-alkyl, or \u2014NH\u2014C(\u2550O)\u2014C(\u2550O)\u2014O\u2014C1-C6-alkyl
C1-C6-alkyl or C2-C6-alkenyl
which is optionally substituted with one or more substituents selected from the group consisting of halogen, \u2014CN, \u2014CF3, \u2014OCF3, \u2014OR1C, and \u2014NR1CR1D and
aryl, aryloxy, aryl-C1-C6-alkoxy, aryl-C1-C6-alkyl, aryl-C2-C6-alkenyl, heteroaryl, heteroaryl-C1-C6-alkyl, or heteroaryl-C2-C6-alkenyl
of which the cyclic moieties are optionally substituted with one or more substituents selected from halogen, \u2014C(O)OR1C, \u2014CN, \u2014CF3, \u2014OCF3, \u2014NO2, \u2014OR1C, \u2014NR1CR1D and C1-C6-alkyl.
19. A zinc-binding ligand according to claim 18 wherein R1B is selected from the group consisting of
hydrogen, halogen, \u2014CF3, \u2014NO2, \u2014OR1C, \u2014NR1CR1D, \u2014C(O)OR1C, \u2550O, \u2014NH\u2014C(\u2550O)\u2014O\u2014C1-C6-alkyl, or \u2014NH\u2014C(\u2550O)\u2014C(\u2550O)\u2014O\u2014C1-C6-alkyl and
C1-C6-alkyl.
20. A zinc-binding ligand according to claim 1 wherein R1C and R1D independently are hydrogen, C1-C6-alkyl, or aryl, wherein the aryl moieties may optionally be substituted by halogen or \u2014COOH.
21. A zinc-binding ligand according to claim 20 wherein R1C and R1D independently are hydrogen, methyl, ethyl, or phenyl, wherein the phenyl moieties may optionally be substituted by halogen or \u2014COOH.
22. A zinc-binding ligand according to claim 1 wherein C1 is a valence bond, C1-C6-alkylene, \u2014NH\u2014C(\u2550O)\u2014, \u2014C(\u2550O)\u2014NH\u2014, \u2014C(\u2550O)\u2014, or \u2014C1-C6-alkyl-C(\u2550O)\u2014N(R1E)\u2014 wherein the alkyl moieties are optionally substituted by one or more R1F independently.
23. A zinc-binding ligand according to claim 22 wherein C1 is a valence bond, \u2014CH2\u2014, \u2014CH2\u2014CH2\u2014, \u2014CH2\u2014O\u2014, \u2014CH2\u2014CH2\u2014O\u2014, \u2014CH2\u2014NH\u2014, \u2014CH2\u2014CH2\u2014NH\u2014, \u2014NH\u2014CH2\u2014, \u2014NH\u2014CH2\u2014CH2\u2014, \u2014NH\u2014C(\u2550O)\u2014, \u2014C(\u2550O)\u2014NH\u2014, \u2014O\u2014CH2\u2014, \u2014O\u2014CH2\u2014CH2\u2014, or \u2014C(\u2550O)\u2014.
24. A zinc-binding ligand according to claim 1 wherein R1E and R1F are independently selected from C1-C6-alkyl.
25. A zinc-binding ligand according to claim 1 wherein AR2 is
a valence bond
C1-C6-alkylene, wherein the alkyl is optionally substituted by one or more R2A independently
arylene, aryl-C1-C6-alkyl, heteroarylene, wherein the arylene and heteroarylene moieties are optionally substituted by one or more R2A independently.
26. A zinc-binding ligand according to claim 25 wherein AR2 is
a valence bond
C1-C6-alkylene, wherein the alkyl is optionally substituted by one or more R2A independently
phenyl, phenyl-C1-C6-alkyl, wherein the phenyl moieties are optionally substituted by one or more R2A independently.
27. A zinc-binding ligand according to claim 1 wherein R2A is C1-C6-alkyl, C1-C6-alkoxy, aryl, aryloxy, heteroaryl, \u2014C1-C6-alkyl-COOH, \u2014O\u2014C1-C6-alkyl-COOH, \u2014S(O)2R2B, \u2014C2-C6-alkenyl-COOH, \u2014OR2B, \u2014NO2, halogen, \u2014COOH, \u2014CF3, \u2014CN, \u2014N(R2BR2C), wherein the aryl or heteroaryl moieties are optionally substituted by one or more C1-C6-alkyl, C1-C6-alkoxy, \u2014C1-C6-alkyl-COOH, \u2014C2-C6-alkenyl-COOH, \u2014OR2B, \u2014NO2, halogen, \u2014COOH, \u2014CF3, \u2014CN, or \u2014N(R2BR2C).
28. A zinc-binding ligand according to claim 27 wherein R2A is C1-C6-alkyl, C1-C6-alkoxy, aryl, \u2014OR2B, \u2014NO2, halogen, \u2014COOH, \u2014CF3, \u2014CN, \u2014N(R2BR2C), wherein the aryl is optionally substituted by one or more C1-C6-alkyl, C1-C6-alkoxy, \u2014OR2B, \u2014NO2, halogen, \u2014COOH, \u2014CF3, \u2014CN, or \u2014N(R2BR2C).
29. A zinc-binding ligand according to claim 28 wherein R2A is C1-C6-alkyl, C1-C6-alkoxy, aryl, halogen, \u2014CF3, wherein the aryl is optionally substituted by one or more C1-C6-alkyl, halogen, \u2014COOH, \u2014CF3, or \u2014CN.
30. A zinc-binding ligand according to claim 29 wherein R2A is C1-C6-alkyl, C1-C6-alkoxy, phenyl, halogen, \u2014CF3, wherein the phenyl is optionally substituted by one or more C1-C6-alkyl, halogen, \u2014COOH, \u2014CF3, or \u2014CN.
31. A zinc-binding ligand according to claim 1, wherein GB is of the formula B1\u2014B2\u2014C(O)\u2014, B1\u2014B2\u2014SO2\u2014 or \u2014B1\u2014B2\u2014CH2\u2014.
32. A zinc-binding ligand according to claim 1, wherein GB is of the formula B1\u2014B2\u2014C(O)\u2014, B1\u2014B2\u2014SO2\u2014 or B1\u2014B2\u2014NH\u2014.
33. A zinc-binding ligand according to claim 1, wherein GB is of the formula B1\u2014B2\u2014C(O)\u2014, \u2014B1\u2014B2\u2014CH2\u2014 or B1\u2014B2\u2014NH.
34. A zinc-binding ligand according to claim 31, wherein GB is of the formula B1\u2014B2\u2014C(O)\u2014 or B1\u2014B2\u2014SO2.
35. A zinc-binding ligand according to claim 31, wherein GB is of the formula B1\u2014B2\u2014C(O)\u2014 or \u2014B1\u2014B2\u2014CH2\u2014.
36. A zinc-binding ligand according to claim 32 wherein GB is of the formula B1\u2014B2\u2014C(O)\u2014 or B1\u2014B2\u2014NH\u2014.
37. A zinc-binding ligand according to claim 34, wherein GB is of the formula B1\u2014B2\u2014C(O)\u2014.
38. A zinc-binding ligand according to claim 1 wherein B1 is a valence bond, \u2014O\u2014, or \u2014S\u2014.
39. A zinc-binding ligand according to claim 1 wherein B1 is a valence bond, \u2014O\u2014, or \u2014N(R6)\u2014.
40. A zinc-binding ligand according to claim 1 wherein B1 is a valence bond, \u2014S\u2014, or \u2014N(R6)\u2014.
41. A zinc-binding ligand according to claim 1 wherein B1 is \u2014O\u2014, \u2014S\u2014 or \u2014N(R6)\u2014.
42. A zinc-binding ligand according to claim 38 wherein B1 is a valence bond or \u2014O\u2014.
43. A zinc-binding ligand according to claim 38 wherein B1 is \u2014O\u2014 or \u2014S\u2014.
44. A zinc-binding ligand according to claim 39 wherein B1 is \u2014O\u2014 or \u2014N(R6)\u2014.
45. A zinc-binding ligand according to claim 42, wherein B1 is \u2014O\u2014.
46. A zinc-binding ligand according to claim 1, wherein B2 is a valence bond, C1-C18-alkylene, C2-C18-alkenylene, C2-C18-alkynylene, arylene, heteroarylene, \u2014C1-C18-alkyl-aryl-, \u2014C(\u2550O)\u2014C1-C18-alkyl-C(\u2550O)\u2014, \u2014C(\u2550O)\u2014C1-C18-alkyl-NR6\u2014C1-C18-alkyl-C(\u2550O)\u2014; and the alkylene moieties are optionally substituted with \u2014CN, \u2014CF3, \u2014OCF3, \u2014OR6, or \u2014NR6R7 and the arylene moieties are optionally substituted with halogen, \u2014C(O)OR6, \u2014C(O)H, OCOR6, \u2014SO2, \u2014CN, \u2014CF3, \u2014OCF3, \u2014NO2, \u2014OR6, \u2014NR6R7, C1-C18-alkyl, or C1-C18-alkanoyl.
47. A zinc-binding ligand according to claim 46, wherein B2 is a valence bond, C1-C18-alkylene, C2-C18-alkenylene, C2-C18-alkynylene, arylene, heteroarylene, \u2014C1-C18-alkyl-aryl-, \u2014C(\u2550O)\u2014C1-C18-alkyl-C(\u2550O)\u2014, \u2014C(\u2550O)\u2014C1-C18-alkyl-O\u2014C1-C18-alkyl-C(\u2550O)\u2014, and the alkylene moieties are optionally substituted with \u2014CN, \u2014CF3, \u2014OCF3, \u2014OR6, or \u2014NR6R7 and the arylene moieties are optionally substituted with halogen, \u2014C(O)OR6, \u2014C(O)H, OCOR6, \u2014SO2, \u2014CN, \u2014CF3, \u2014OCF3, \u2014NO2, \u2014OR6, \u2014NR6R7, C1-C18-alkyl, or C1-C18-alkanoyl.
48. A zinc-binding ligand according to claim 47, wherein B2 is a valence bond, C1-C18-alkylene, C2-C18-alkenylene, C2-C18-alkynylene, arylene, heteroarylene, \u2014C1-C18-alkyl-aryl-, \u2014C(\u2550O)\u2014C1-C18-alkyl-C(\u2550O)\u2014, and the alkylene moieties are optionally substituted with \u2014CN, \u2014CF3, \u2014OCF3, \u2014OR6, or \u2014NR6R7 and the arylene moieties are optionally substituted with halogen, \u2014C(O)OR6, \u2014C(O)H, OCOR6, \u2014SO2, \u2014CN, \u2014CF3, \u2014OCF3, \u2014NO2, \u2014OR6, \u2014NR6R7, C1-C18-alkyl, or C1-C18-alkanoyl.
49. A zinc-binding ligand according to claim 48, wherein B2 is a valence bond, C1-C18-alkylene, arylene, heteroarylene, \u2014C1-C18-alkyl-aryl-, \u2014C(\u2550O)\u2014C1-C18-alkyl-C(\u2550O)\u2014, and the alkylene moieties are optionally substituted with \u2014CN, \u2014CF3, \u2014OCF3, \u2014OR6, or \u2014NR6R7 and the arylene moieties are optionally substituted with halogen, \u2014C(O)OR6, \u2014C(O)H, OCOR6, \u2014SO2, \u2014CN, \u2014CF3, \u2014OCF3, \u2014NO2, \u2014OR6, \u2014NR6R7, C1-C18-alkyl, or C1-C18-alkanoyl.
50. A zinc-binding ligand according to claim 49 wherein B2 is a valence bond, C1-C18-alkylene, arylene, heteroarylene, \u2014C1-C18-alkyl-aryl-, and the alkylene moieties are optionally substituted with \u2014CN, \u2014CF3, \u2014OCF3, \u2014OR6, or \u2014NR6R7 and the arylene moieties are optionally substituted with halogen, \u2014C(O)OR6, \u2014C(O)H, OCOR6, \u2014SO2, \u2014CN, \u2014CF3, \u2014OCF3, \u2014NO2, \u2014OR6, \u2014NR6R7, C1-C18-alkyl, or C1-C18-alkanoyl.
51. A zinc-binding ligand according to claim 50, wherein B2 is a valence bond, C1-C18-alkylene, arylene, \u2014C1-C18-alkyl-aryl-, and the alkylene moieties are optionally substituted with \u2014CN, \u2014CF3, \u2014OCF3, \u2014OR6, or \u2014NR6R7 and the arylene moieties are optionally substituted with halogen, \u2014C(O)OR6, \u2014C(O)H, OCOR6, \u2014SO2, \u2014CN, \u2014CF3, \u2014OCF3, \u2014NO2, \u2014OR6, \u2014NR6R7, C1-C18-alkyl, or C1-C18-alkanoyl.
52. A zinc-binding ligand according to claim 51, wherein B2 is a valence bond or \u2014C1-C18-alkylene, and the alkylene moieties are optionally substituted moieties are optionally substituted with \u2014CN, \u2014CF3, \u2014OCF3, \u2014OR6, or \u2014NR6R7 and the arylene moieties are optionally substituted with halogen, \u2014C(O)OR6, \u2014C(O)H, OCOR6, \u2014SO2, \u2014CN, \u2014CF3, \u2014OCF3, \u2014NO2, \u2014OR6, \u2014NR6R7, C1-C18-alkyl, or C1-C18-alkanoyl.
53. A zinc-binding ligand according to claim 1, wherein C consists of 0 to 5 neutral amino acids independently selected from the group consisting of Abz, Gly, Ala, Thr, and Ser.
54. A zinc-binding ligand according to claim 53, wherein C consists of 0 to 5 Gly.
55. A zinc-binding ligand according to claim 54, wherein C consists of 1 Gly.
56. A zinc-binding ligand according to claim 54, wherein C consists of 2 Gly.
57. A zinc-binding ligand according to claim 54, wherein C consists of 3 Gly.
58. A zinc-binding ligand according to claim 54 wherein C consists of 4 Gly.
59. A zinc-binding ligand according to claim 54, wherein C consists of 5 Gly.
60. A zinc-binding ligand according to claim 1, wherein the positively charged groups of D number from 1 to 16.
61. A zinc-binding ligand according to claim 60, wherein the positively charged groups of D number from 1 to 12.
62. A zinc-binding ligand according to claim 61, wherein the positively charged groups of D number from 1 to 10.
63. A zinc-binding ligand according to claim 1, wherein the basic amino acid is Arg.
64. A zinc-binding ligand according to claim 1, wherein X is \u2014OH or \u2014NH2.
65. A zinc-binding ligand according to claim 64, wherein X is \u2014NH2.
66. Method of prolonging the action of an insulin preparation which comprises adding a zinc-binding ligand according to claim 1 to the insulin preparation.
67. A method of preparing a zinc-binding ligand according to claim 1 comprising the steps of
Identifying starter compounds that are able to displace a ligand from the R-state HisB10-Zn2+ site
optionally attaching a fragment consisting of 0 to 5 neutral \u03b1- or \u03b2-amino acids attaching a fragment comprising 1 to 20 positively charged groups independently selected from amino or guanidino groups.
The claims below are in addition to those above.
All refrences to claim(s) which appear below refer to the numbering after this setence.
1. A stable continuous method for producing ethanol from the anaerobic bacterial fermentation of a gaseous substrate, the method comprising:
culturing in a fermentation bioreactor anaerobic, acetogenic bacteria in a liquid nutrient medium and supplying to said bioreactor said gaseous substrate comprising at least one reducing gas selected from the group consisting of carbon monoxide and hydrogen; and
manipulating said bacteria in said bioreactor by reducing the redox potential, or increasing the NAD(P)H TO NAD(P) ratio, in the fermentation broth after said bacteria achieves a stable cell concentration in said bioreactor, wherein said free acetic acid concentration in said bioreactor is less than 5 gL free acid,
said culturing and manipulating steps causing said bacteria in said bioreactor to produce ethanol in a fermentation broth at a productivity greater than 10 gL per day and wherein both ethanol and acetate are produced in said fermentation broth in a ratio of ethanol to acetate ranging from 1:1 to 20:1.
2. The method according to claim 1, wherein said fermentation bioreactor comprises a growth reactor which feeds said fermentation broth to a second fermentation bioreactor in which most of said ethanol is produced.
3. The method according to claim 1, further comprising the steps of removing said fermentation broth from said bioreactor, distilling ethanol from said broth and recovering said ethanol.
4. The method according to claim 3, further comprising the steps of recycling water containing acetate from said distilling step back to the bioreactor.
5. The method according to claim 1, wherein said bacteria is selected from the group consisting of: Acetobacterium woodii, Butyribacterium methylotrophicum, Clostridium aceticum, C. acetobutylicium, C. thermoaceticum, Eubacterium limosum, Clostridium ljungdahlii, and Peptostreptococcus productus.
6. The method according to claim 5, wherein said Clostridium ljungdahlii is selected from the strains consisting of PETC, ERI2, O-52 and C-01.
7. The method according to claim 1, wherein said gaseous substrate is selected from the group consisting of (a) carbon monoxide, (b) carbon monoxide and hydrogen, (c) carbon dioxide and hydrogen, and (d) carbon monoxide, carbon dioxide and hydrogen.
8. The method according to claim 7, wherein said substrate additionally comprises nitrogen or methane.
9. The method according to claim 1, wherein said manipulating step further comprises altering at least one parameter selected from the group consisting of nutrient medium contents, nutrient feed rate, aqueous feed rate, operating pressure, operating pH, gaseous substrate contents, gas feed rate, fermentation broth agitation rate, product inhibition step, cell density, substrate inhibition and combinations thereof.
10. The method according to claim 9, wherein said manipulating step comprises raising the pH of said culture above 4.5.
11. The method according to claim 9, wherein said manipulating step comprises periodically purging bacterial cells from said bioreactor to a cell concentration less than said stable concentration that utilizes all reducing gas or nutrient substrates in said bioreactor.
12. The method according to claim 9, comprising increasing the aqueous feed rate when the free acetic acid portion of the acetate present in the fermentation broth exceeds 2 gL, thereby inhibiting any unwanted increase in the concentration of said free acetic acid.
13. The method according to claim 9, comprising reducing said gaseous substrate feed rate to eliminate excess carbon monoxide, to relieve substrate inhibition and maintain said productivity.
14. The method according to claim 9, wherein said agitation rate is reduced to eliminate excess carbon monoxide, to relieve substrate inhibition and maintain said productivity.
15. The method according to claim 9, wherein said manipulating step comprises supplying to said bioreactor said gaseous substrate comprising the reducing gas carbon monoxide at a rate of from 0.3 to 2 mmol COgram of dry cell of bacteria in said bioreactorminute.
16. The method according to claim 15, wherein said the amount of CO present in said bioreactor is greater than the amount required to maintain said bacteria at a stable bacterial concentration that would fully utilize the CO provided.
17. The method according to claim 15, wherein said the amount of CO present in said bioreactor maintains ethanol production in preference to acetate production.
18. The method according to claim 15, wherein said rate is in the range of 0.5 to 1.5 mmol COgram of dry cell of bacteria in said bioreactorminute.
19. The method according to claim 15, wherein said gaseous substrate further comprises an excess of hydrogen reducing gas relative to said carbon monoxide, wherein said excess hydrogen causes said bacteria to produce a high ethanol to acetate ratio in said fermentation broth.
20. The method according to claim 9, wherein said manipulating step comprises feeding into said fermentation bioreactor said nutrient medium comprising an amount of calcium pantothenate in a range of from 0.5 to 50 ggrams of dry cell of bacteria produced in said bioreactor.
21. The method according to claim 20, wherein said amount of calcium pantothenate is less than required to maintain said bacteria at a stable bacterial concentration that would fully utilize the calcium pantothenate provided.
22. The method according to claim 20, wherein said amount of calcium pantothenate maintains ethanol production in preference to acetate production.
23. The method according to claim 20, wherein said amount is from 1 to 25 g calcium pantothenategrams of dry cell of bacteria produced.
24. The method according to claim 20, wherein said amount is from 2 to 25 g calcium pantothenategrains of dry cell of bacteria produced.
25. The method according to claim 20, further comprising the step of preventing acclimation of said bacteria in said bioreactor to said calcium pantothenate amount by maintaining a constant calcium pantothenate concentration and adjusting the parameters selected from the group consisting of gas rate, liquid rate, agitation rate and hydrogen gas partial pressure.
26. The method according to claim 20, further comprising supplying excess hydrogen reducing gas to said bioreactor prior to providing said amount of calcium pantothenate.
27. The method according to claim 9, wherein said manipulating step comprises feeding into said fermentation bioreactor said nutrient medium comprising an amount of cobalt in a range of from 5 to 100 g cobaltgrams of dry cell of bacteria produced in said bioreactor.
28. The method according to claim 27, wherein said amount of cobalt is less than required to maintain said bacteria at a stable bacterial concentration that would fully utilize the cobalt provided.
29. The method according to claim 27, wherein said amount of cobalt maintains ethanol production in preference to acetate
30. The method according to claim 27, wherein said amount is from 20 to 50 g calcium pantothenategrams of dry cell of bacteria produced.
31. The method according to claim 27, further comprising the step of preventing acclimation of said bacteria in said bioreactor to said amount of cobalt by maintaining a constant cobalt concentration and adjusting the parameters selected from the group consisting of gas rate, liquid rate, agitation rate and hydrogen gas partial pressure.