# US 9,040,268 B2 · Method for controlling undesirable byproducts formation caused by contaminating organisms in the production of ethanol from syngas

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- Number: US 9,040,268 B2
- Document ID: US9040268B2
- Status: Granted
- Kind: B2
- Application number: 13/619,547
- Filed: 2012-09-14
- Published: 2013-03-21
- Granted: 2015-05-26
- Earliest claimed: 2011-09-21
- Assignees: Coskata, Inc.
- Applicants: Rathin Datta; Andrew Reeves; Laura T. Kliman
- Inventors: Rathin Datta; Andrew Reeves; Laura T. Kliman
- Web page: https://patentgrep.com/patent/US9040268B2

## CPC

- C12P 7/04: acyclic
- C12P 7/065: {with microorganisms other than yeasts}
- C12P 7/14: Multiple stages of fermentation; Multiple types of microorganisms or re-use of microorganisms
- C12P 7/52: Propionic acid; Butyric acids
- C12P 7/54: Acetic acid (vinegar C12J)
- C12P 39/00: Processes involving microorganisms of different genera in the same process, simultaneously
- Y02E 50/10: Biofuels, e.g. bio-diesel
- C12P: FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE {(brewing of beer C12C; producing vinegar C12J; producing specific peptides or proteins C07K; producing enzymes C12N9/00; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification C12N15/00; measuring or testing processes involving enzymes or microorganisms C12Q; measuring or testing processes involving nucleic acid amplification reactions C12Q1/6844; fermentation processes to form a food composition, A21 or A23; compounds in general, see the relevant compound class, e.g. C01, C07)}
- Y02E: REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION

## Abstract

A method of operating a fermentation zone for the production of ethanol from syngas uses a crotonate-like compound to prevent or reverse the effects of butyrogen contamination. The crotonate-like compound works in continuous fermentation processes to reduce or eliminate contamination from butyrate and butanol in the syngas derived ethanol product.

## Claims

### Claim 1

A process for the metabolic conversion of substrate to light oxygenates comprising:
a. contacting in a fermentation liquid containing said substrate with microorganisms capable of bioconverting the substrate to light oxygenates under fermentation conditions, said fermentation liquid being susceptible to a presence of contaminating bacteria capable of producing at least one of butyrate or butanol from said substrate or from said light oxygenates through a butyryl CoA enzyme; and
b. providing in said fermentation liquid a sufficient amount of a crotonate-like compound to restrict the production of said at least one of butyrate or butanol by a butyryl-CoA dehydrogenase, wherein the crotonate-like compound is represented by the structural formula:
R2(R3)C═C(H)C(O)R1 I.
or
R2-A-C(O)R1 II.
wherein:
R1 is —OH, —OR5, or —N(R4)2, wherein R5 is hydrocarbyl and each R4 may be the same or different and is hydrogen or hydrocarbyl;
R2 is hydrogen, —NH2, —OH or —CX3 wherein each X may be the same or different and is hydrogen or halogen;
R3 is hydrocarbyl of 1 to 18 carbons which may be unsubstituted or substituted with —OH, alkoxyl of 1 to about 6 carbons, or halogen;
A is an aromatic moiety having 5 or 6 ring atoms which may be all carbon atoms or may be heterocyclic with one hetero atom selected from the group consisting of oxygen and nitrogen which aromatic moiety has —R2 at the 2 position wherein —R2 is as defined above and which aromatic moiety may be unsubstituted or substituted at one or more of the higher carbon positions with (i) hydrocarbyl of 1 to 18 carbons which may be substituted with —OH, alkoxyl of 1 to about 6 carbons, or halogen, (ii) —OR6 wherein R6 is lower alkyl, (iii) —N(R7)2 wherein each R7 is the same or different and is hydrogen or hydrocarbyl or (iv) halogen;
with the proviso that in structural formula I, at least one of R2 and R3 is or is substituted with haloalkyl and that in formula II at least the 2 or 3 carbon position of the aryl moiety is substituted with an electron withdrawing group selected from the group consisting of halogen and haloalkyl.

### Claim 2

A method of restricting the production of butyrate and/or butanol in an anaerobic fermentation of a gas substrate comprising at least one of CO and a mixture of CO2 with hydrogen, the method comprising:
a. passing the gas stream to an anaerobic fermentation zone containing at least one species of anaerobic microorganism capable of producing an liquid product comprising light oxygenates;
b. converting at least a portion of the gas stream to the liquid product by contact of the microorganism in the fermentation zone with the gas stream;
c. providing at least one crotonate-like compound in the fermentation liquid in an amount effective to restrict production of butyrate and/or butanol by butyryl-CoA dehydrogenase, said crotonate-like compound represented by the structural formula
R2(R3)C═C(H)C(O)R1 I.
or
R2-A-C(O)R1 II.
wherein:
R1 is —OH, —OR5, or —N(R4)2, wherein R5 is hydrocarbyl and each R4 may be the same or different and is hydrogen or hydrocarbyl;
R2 is hydrogen, —NH2, —OH or —CX3 wherein each X may be the same or different and is hydrogen or halogen;
R3 is hydrocarbyl of 1 to 18 carbons which may be unsubstituted or substituted with —OH, alkoxyl of 1 to about 6 carbons, or halogen;
A is an aromatic moiety having 5 or 6 ring atoms which may be all carbon atoms or may be heterocyclic with one hetero atom selected from the group consisting of oxygen and nitrogen which aromatic moiety has —R2 at the 2 position wherein —R2 is as defined above and which may be unsubstituted or substituted at one or more of the higher carbon positions with (i) hydrocarbyl of 1 to 18 carbons which may be substituted with —OH, alkoxyl of 1 to about 6 carbons, or halogen, (ii) —OR6 wherein R6 is lower alkyl, (iii) —N(R7)2 wherein each R7 is the same or different and is hydrogen or hydrocarbyl or (iv) halogen;
with the proviso that in structural formula I, at least one of R2 and R3 is or is substituted with haloalkyl and that in formula II at least the 2 or 3 carbon position of the aryl moiety is substituted with an electron withdrawing group selected from the group consisting of halogen and haloalkyl;
d. withdrawing fermentation liquid containing the liquid product from the fermentation zone; and
e. recovering the liquid product from the fermentation liquid.

### Claim 3 (depends on 2)

The method of claim 2 wherein the fermentation zone contains at least one of a homoacetogenic or heteroacetogenic microorganism for the production of a liquid product comprising ethanol or acetate and is contaminated with a butyrogenic microorganism that produces at least one of butyrate or butanol.

### Claim 4 (depends on 3)

The method of claim 3 wherein the fermentation zone contains a homoacetogenic microorganism that comprises at least one of Clostridium autoethanogenum, Clostridium ljungdahlii, Clostridium ragsdalei, and Clostridium coskatii and the fermentation zone produces a liquid product comprising at least one of acetate and ethanol.

### Claim 5 (depends on 2)

The method of claim 2 wherein the fermentation zone comprises a suspended cell bioreactor that suspends the anaerobic microorganism in the fermentation liquid and the at least one crotonate-like compound enters the bioreactor with a media mixture that flows into the fermentation liquid.

### Claim 6 (depends on 2)

The method of claim 2 wherein the fermentation zone comprises a membrane supported bioreactor.

### Claim 7 (depends on 2)

The method of claim 2 wherein the crotonate-like compound is represented by the formula:
R2(R3)C═C(H)C(O)R1 III.
or
R2-A-C(O)R1 IV.
wherein:
in formula III, R2 is —OH, —NH2 or —CX3 wherein X is halogen and R3 is —CX3 wherein X is halogen, and
in formula IV, the aryl is phenyl and at least one of the 2 and 3 position carbons of the phenyl moiety is —CX3 wherein X is halogen.

### Claim 8 (depends on 7)

The method of claim 7 wherein the at least one crotonate-like compound comprises at least one of alkyl 4,4,4-trifluoro-3-(trifluoromethyl)crotonate; 4,4,4-trifluoro-3-(trifluoromethyl)crotonic acid; 4,4,4-trifluoro-3-(trifluoromethyl)crotonamide; 2-trifluoromethylbenzoic acid; 2-trifluoromethylbenzamide; alkyl 2-trifluoromethylbenzoate; 3-trifluoromethylbenzoic acid; 3-trifluoromethylbenzamide; alkyl 3-trifluoromethylbenzoate; 2-amino-3-trifluoromethylbenzoic acid; 2-amino-3-trifluoromethylbenzamide; alkyl 2-amino-3-trifluoromethylbenzoate; 3-trifluoro-4-methoxybenzoic acid; 3-trifluoro-4-methoxybenzamide; alkyl 3-trifluoro-4-methoxybenzoate; 3-trifluoromethyl-4-fluorobenzoic acid; 3-trifluoromethyl-4-fluorobenzamide; alkyl 3-trifluoromethyl-4-fluorobenzoate; 3-trifluoromethyl-5-trifluoromethylbenzoic acid; 3-trifluoromethyl-5-trifluoromethylbenzamide; alkyl 3-trifluoromethyl-5-trifluoromethylbenzoate; 3-amino substituted crotonates such as 4,4,4-trifluoro-3-aminocrotonic acid; 4,4,4-trifluoro-3-aminocrotonamide; and alkyl 4,4,4-trifluoro-3-aminocrotonate wherein the alkyl is 1 to 4 carbons.

### Claim 9 (depends on 2)

The method of claim 2 wherein the anaerobic microorganism comprises a heteroacetogen that produces liquid products having at least 2 to 3 carbon atoms and the liquid product comprises at least one of acetic acid, ethanol, propanol, and propionic acid.

### Claim 10 (depends on 2)

The method of claim 2 wherein the at least one crotonate-like compound is added in an amount sufficient to produce a concentration of at least 10 ppm by mass in the fermentation liquid.

### Claim 11 (depends on 2)

The method of claim 2 wherein the at least one crotonate-like compound is added to the fermentation zone as single dose in an amount of at least 50 ppm by mass.

### Claim 12 (depends on 2)

The method of claim 2 wherein the fermentation zone comprises a membrane supported bioreactor and the at least one crotonate-like compound is added to the fermentation liquid.

### Claim 13 (depends on 2)

The method of claim 2 wherein the fermentation zone contains more than one species of anaerobic microorganisms.

### Claim 14 (depends on 13)

The method of claim 13 wherein the crotonate-like compound is represented by the formula:
R2(R3)C═C(H)C(O)R1 III.
or
R2-A-C(O)R1 IV.
wherein:
in formula III, R2 is —OH, —NH2 or —CX3 wherein X is fluorine, and R3 is —CX3 wherein X is fluorine, and
in formula IV, the aryl is phenyl and at least one of the 2 and 3 position carbons of the phenyl moiety is —CX3 wherein X is fluorine.

### Claim 15 (depends on 13)

The method of claim 13 wherein the fermentation zone contains a homoacetogenic microorganism for the production of a liquid product comprising ethanol and a butyrogenic microorganism that produces at least one of butyrate or butanol.

### Claim 16 (depends on 13)

The method of claim 13 wherein the anaerobic microorganism comprises a heteroacetogen that produces liquid products having at least 2 to 3 carbon atoms and the liquid product comprises at least one of acetic acid, ethanol, propanol, and propionic acid.

### Claim 17 (depends on 13)

The method of claim 13 wherein the at least one crotonate-like compound is added to the fermentation liquid at a concentration of at least 10 ppm.

### Claim 18

A method of producing a product comprising a C2 oxygenate by the fermentation of a gas substrate comprising at least one of CO and a mixture of CO2 with hydrogen using a homoacetogenic microorganism to convert the gas stream to the product, the method comprising:
a. passing the gas substrate stream to a fermentation zone containing an anaerobic microorganism and a fermentation liquid;
b. converting the gas substrate stream to the product by contact of the anaerobic microorganism in the fermentation zone with the gas substrate stream;
c. providing at least one crotonate-like compound to the fermentation liquid in an amount sufficient to inhibit a production of butyrate and/or butanol by butyryl-CoA dehydrogenase by at least one species of microorganism in the fermentation zone, said crotonate-like compound represented by the structural formula
R2(R3)C═C(H)C(O)R1 I.
or
R2-A-C(O)R1 II.
wherein:
R1 is —OH, —OR5, or —N(R4)2, wherein R5 is hydrocarbyl and each R4 may be the same or different and is hydrogen or hydrocarbyl;
R2 is hydrogen, —NH2, —OH or —CX3 wherein each X may be the same or different and is hydrogen or halogen;
R3 is hydrocarbyl of 1 to 18 carbons which may be unsubstituted or substituted with —OH, alkoxyl of 1 to about 6 carbons, or halogen;
A is an aromatic moiety having 5 or 6 ring atoms which may be all carbon atoms or may be heterocyclic with one hetero atom selected from the group consisting of oxygen and nitrogen which aromatic moiety has —R2 at the 2 position wherein —R2 is as defined above and which aromatic moiety may be unsubstituted or substituted at one or more of the higher carbon positions with (i) hydrocarbyl of 1 to 18 carbons which may be substituted with —OH, alkoxyl of 1 to about 6 carbons, or halogen, (ii) —OR6 wherein R6 is lower alkyl, (iii) —N(R7)2 wherein each R7 is the same or different and is hydrogen or hydrocarbyl or (iv) halogen;
with the proviso that in structural formula I, at least one of R2 and R3 is or is substituted with haloalkyl and that in formula II at least the 2 or 3 carbon position of the aryl moiety is substituted with an electron withdrawing group selected from the group consisting of halogen and haloalkyl;
d. withdrawing the product containing fermentation liquid from the fermentation zone; and
e. recovering the product from the fermentation liquid.

### Claim 19 (depends on 18)

The method of claim 18 wherein the crotonate-like compound is represented by the formula:
R2(R3)C═C(H)C(O)R1 III.
or
R2-A-C(O)R1 IV.
wherein:
in formula III, R2 is —OH, —NH2 or —CX3 wherein X is fluorine, and R3 is —CX3 wherein X is fluorine, and
in formula IV, the aryl is phenyl and at least one of the 2 and 3 position carbons of the phenyl moiety is —CX3 wherein X is fluorine.