BIOTECHNOLOGICAL PRODUCTION OF PROPANOL AND/OR PROPIONIC ACID

Abstract

The present invention relates to a method of producing propanol and/or propionic acid, the method comprising: (b) contacting at least one propionogen to carbon dioxide and to an aqueous medium comprising ethanol and acetate; wherein the acetate is maintained at a concentration of at least about 1 g/L in the aqueous medium.

Claims

1-15. (canceled)

16. A method of producing propanol and/or propionic acid, comprising contacting at least one propionogen with carbon dioxide and with an aqueous medium comprising ethanol and acetate, wherein the acetate is maintained at a concentration of at least about 1 g/L in the aqueous medium.

17. The method of claim 16, wherein the ethanol and acetate are exogenously produced.

18. The method of claim 16, wherein the propionogen is selected from the group consisting of: Clostridium neopropionicum, Clostridium propionicum, Pelobacter propionicus, Desulfobulbus propionicus, Syntrophobacter wolinii, Syntrophobacter pfennigii, Syntrophobacter fumaroxidans, Syntrophobacter sulfatireducens, Smithella propionica, Desulfotomaculum thermobenzoicum subspecies thermosyntrophicum, Pelotomaculum thermopropionicum, and Pelotomaculum schinkii.

19. The method of claim 16, wherein the propionogen is Clostridium neopropionicum.

20. The method of claim 16, wherein the acetate concentration in the aqueous medium is maintained at between 1 g/L to 10 g/L in the aqueous medium.

21. The method of claim 16, wherein the acetate concentration in the aqueous medium is maintained at between 1.5 g/L to 7 g/L in the aqueous medium.

22. The method of claim 16, wherein the acetate concentration in the aqueous medium is maintained at about 2 g/L in the aqueous medium.

23. The method of claim 20, wherein the ethanol and acetate are exogenously produced.

24. The method of claim 23, wherein the propionogen is selected from the group consisting of: Clostridium neopropionicum, Clostridium propionicum, Pelobacter propionicus, Desulfobulbus propionicus, Syntrophobacter wolinii, Syntrophobacter pfennigii, Syntrophobacter fumaroxidans, Syntrophobacter sulfatireducens, Smithella propionica, Desulfotomaculum thermobenzoicum subspecies thermosyntrophicum, Pelotomaculum thermopropionicum, and Pelotomaculum schinkii.

25. A method of producing propanol and/or propionic acid, the method comprising: (a) contacting at least one acetogenic bacteria with a carbon source comprising carbon dioxide and/or carbon monoxide to produce ethanol and acetate; (b) contacting at least one propionogen with carbon dioxide and an aqueous medium comprising said ethanol and acetate, wherein the acetate in the aqueous medium is maintained at a concentration of at least about 1 g/L.

26. The method of claim 25, wherein the acetogenic bacteria is selected from the group consisting of: Clostridium autothenogenum DSMZ 19630, Clostridium ragsdahlei ATCC no. BAA-622, Clostridium autoethanogenum, Moorella sp HUC22-1, Moorella thermoaceticum, Moorella thermoautotrophica, Rumicoccus productus, Acetoanaerobum, Oxobacter pfennigii, Methanosarcina barkeri, Methanosarcina acetivorans, Carboxydothermus, Desulfotomaculum kutznetsovii, Pyrococcus, Peptostreptococcus, Butyribacterium methylotrophicum ATCC 33266, Clostridium formicoaceticum, Clostridium butyricum, Lactobacillus delbrukii, Propionibacterium acidoproprionici, Proprionispera arboris, Anaerobierspirillum succiniproducens, Bacterioides amylophilus, Becterioides ruminicola, Thermoanaerobacter kivui, Acetobacterium woodii, Acetoanaerobium notera, Clostridium aceticum, Butyribacterium methylotrophicum, Moorella thermoacetica, Eubacterium limosum, Peptostreptococcus productus, Clostridium ljungdahlii, Clostridium ATCC 29797 and Clostridium carboxidivorans.

27. The method of claim 25, wherein steps (a) and (b) are carried out in a single fermenter.

28. The method of claim 27, wherein the acetate concentration in the aqueous medium is maintained at between 1.5 g/L to 7 g/L.

29. A method for increasing the proportion of ethanol converted to propanol and/or propionic acid by a propionogen in an aqueous medium, comprising maintaining a concentration of acetate in the aqueous medium of at least about 1 g/L.

30. The method of claim 29, wherein the ethanol and acetate are exogenously produced.

31. The method of claim 29, wherein the propionogen is selected from the group consisting of: Clostridium neopropionicum, Clostridium propionicum, Pelobacter propionicus, Desulfobulbus propionicus, Syntrophobacter wolinii, Syntrophobacter pfennigii, Syntrophobacter fumaroxidans, Syntrophobacter sulfatireducens, Smithella propionica, Desulfotomaculum thermobenzoicum subspecies thermosyntrophicum, Pelotomaculum thermopropionicum, and Pelotomaculum schinkii.

32. The method of claim 29, wherein the acetate concentration in the aqueous medium is maintained at between 1.5 g/L to 7 g/L.

33. The method of claim 29, wherein the acetate concentration of acetate in the aqueous medium is maintained at about 2 g/L.

34. The method of claim 31, wherein the ethanol and acetate are exogenously produced.

35. The method of claim 34, wherein the acetate concentration in the aqueous medium is maintained at between 1.5 g/L to 7 g/L.

Description

EXAMPLES

[0057] The foregoing describes preferred embodiments, which, as will be understood by those skilled in the art, may be subject to variations or modifications in design, construction or operation without departing from the scope of the claims. These variations, for instance, are intended to be covered by the scope of the claims.

Example 1

[0058] Production of Propionic Acid and Propanol with Clostridium neopropionicum on Acetate

[0059] For the biotransformation of ethanol and carbon dioxide to propionic acid and propanol the bacterium Clostridium neopropionicum was cultivated in mineral medium with ethanol, acetate and a gas atmosphere with carbon dioxide. All cultivation steps were carried out under anaerobic conditions in pressure-resistant glass bottles that can be closed airtight with a butyl rubber stopper. For the first preculture of C. neopropionicum 2100 ml DSMZ318 medium (pH 7.4; 0.61 g/l NaCl, 0.047 g/l MgCl.sub.2, 0.30 g/l KH.sub.2PO.sub.4, 1.00 g/l NH.sub.4Cl, 0.081 g/l CaCl.sub.22 H.sub.2O, 0.5 g/l yeast extract, 0.5 g/l BBL Trypticase Peptone, 4 g/L KHCO.sub.3, 1.026 g/L ethanol, 0.5 mg/l resazurin, 128 mg/L nitrilotriacetic acid, 135 mg/L FeCl.sub.36 H.sub.2O, 1 mg/L MnCl.sub.24 H.sub.2O, 0.24 mg/L CoCl.sub.26 H.sub.2O, 1 mg/L ZnC.sub.2, 0.25 mg/L CuCl.sub.22 H.sub.2O, 0.1 mg/L H.sub.3BO.sub.3, 0.24 mg/L Na.sub.2MoO.sub.42 H.sub.2O, 1.2 mg/L NiCl.sub.26 H.sub.2O, 0.26 mg/L Na.sub.2SeO.sub.35 H.sub.2O, 0.02 mg/L biotin, 0.02 mg/L folic acid, 0.1 mg/L pyridoxin-HCl, 0.05 mg/L thiamine-HClH.sub.2O, 0.05 mg/L riboflavin, 0.05 mg/L nicotinic acid, 0.05 mg/L D-Ca-pantothenate, 1 g/L vitamin B12, 0.05 mg/L p-aminobenzoate, 0.05 mg/L lipoic acid, 0.25 g/L cysteine-HClH.sub.2O) in a 250 ml bottle were inoculated with 5 ml of a frozen cryoculture and flushed with a premixed gas with 67% H.sub.2, 33% CO.sub.2 to an overpressure of 0.8 bar. The culture was incubated at 30 C. and 100 rpm in an open water bath shaker for 24 h till an OD.sub.600nm>0.15. For a second preculture of C. neopropionicum 3200 ml of fresh DSMZ318 medium in a 500 ml bottle were inoculated with centrifuged cells from the first preculture to an OD.sub.600nm of 0.03 and flushed with a premixed gas with 67% H.sub.2, 33% CO.sub.2 to an overpressure of 0.8 bar. This growing culture was incubated at 30 C. and 100 rpm in an open water bath shaker for 22 h till an OD.sub.600nm >0.23.

[0060] For the main culture, as many centrifuged cells from the second preculture of C. neopropionicum as necessary for an OD.sub.600nm of 0.2 were added to 200 ml of fresh LM33 mineral medium (pH=6.8, 10 g/L ethanol, 1 g/L NaOH, 0.5 g/L MgCl.sub.2, 0.21 g/L NaCl, 0.135 g/L CaCl.sub.22H.sub.2O, 2.65 g/L NaH.sub.2PO.sub.42H.sub.2O, 0.5 g/L KCl, 2.5 g/L NH.sub.4Cl, 15 mg/L nitrilotriacetic acid, 30 mg/L MgSO.sub.47 H.sub.2O, 5 mg/L MnSO.sub.4H.sub.2O, 1 mg/L FeSO.sub.47 H.sub.2O, 8 mg/L Fe(SO.sub.4).sub.2(NH.sub.4).sub.26 H.sub.2O, 2 mg/L CoCl.sub.26 H.sub.2O, 2 mg/L ZnSO.sub.47 H.sub.2O, 200 g/L CuCl.sub.22 H.sub.2O, 200 g/L KAl(SO.sub.4).sub.212 H.sub.2O, 3 mg/L H.sub.3BO.sub.3, 300 g/L Na.sub.2MoO.sub.42 H.sub.2O, 200 g/L Na.sub.2SeO.sub.3, 200 g/L NiCl.sub.26 H.sub.2O, 200 g/L Na.sub.2WO.sub.46 H.sub.2O, 200 g/L d-biotin, 200 g/L folic acid, 100 g/L pyridoxine-HCl, 500 g/L thiamine-HCl; 500 g/L riboflavin; 500 g/L nicotinic acid; 500 g/L Ca-pantothenate; 500 g/L vitamin B.sub.12; 500 g/L p-aminobenzoate; 500 g/L lipoic acid, 10 mg/L FeCl.sub.3, aerated for 30 min with a premixed gas with 67% H.sub.2 and 33% CO.sub.2) with additional 1.39 g/L sodium acetate. The cultivation was carried out in a 500 mL pressure-resistant glass bottle at 30 C., 100 rpm and an overpressure of 0.8 bar of a premixed gas with 67% H.sub.2, 33% CO.sub.2 in an open water bath shaker for 113 h. The pH was hold at 6.8 by automatic addition of NaOH solution (100 g/L).

[0061] During cultivation several 5 mL samples were taken to determinate OD.sub.600nm, pH and product formation. The determination of the product concentrations was performed by semi-quantitative .sup.1H-NMR spectroscopy. As an internal quantification standard sodium trimethylsilylpropionate (T(M)SP) was used.

[0062] During the cultivation the concentration of propionate increased from 0.05 g/L to 3.20 g/L, for propanol from 0.01 to 0.26 g/L, for butyrate from 0.01 to 0.33 g/L and for lactate from 0.00 g/L to 0.13 g/L. The concentration of ethanol decreased from 10.1 g/L to 5.2 g/L during this time. The selectivity of propionate/propanol formation was about 88.0%.

Example 2

[0063] Production of Propionic Acid and Propanol with Clostridium neopropionicum on Acetate

[0064] For the biotransformation of ethanol and carbon dioxide to propionic acid and propanol the bacterium Clostridium neopropionicum was cultivated in mineral medium with ethanol, acetate and a gas atmosphere with carbon dioxide. All cultivation steps were carried out under anaerobic conditions in pressure-resistant glass bottles that can be closed airtight with a butyl rubber stopper. For the first preculture of C. neopropionicum 2100 ml DSMZ318 medium (pH 7.4; 0.61 g/l NaCl, 0.047 g/l MgCl.sub.2, 0.30 g/l KH.sub.2PO.sub.4, 1.00 g/l NH.sub.4Cl, 0.081 g/l CaCl.sub.22 H.sub.2O, 0.5 g/l yeast extract, 0.5 g/l BBL Trypticase Peptone, 4 g/L KHCO.sub.3, 1.026 g/L ethanol, 0.5 mg/l resazurin, 128 mg/L nitrilotriacetic acid, 135 mg/L FeCl.sub.36 H.sub.2O, 1 mg/L MnCl.sub.24 H.sub.2O, 0.24 mg/L CoCl.sub.26 H.sub.2O, 1 mg/L ZnCl.sub.2, 0.25 mg/L CuCl.sub.22 H.sub.2O, 0.1 mg/L H.sub.3BO.sub.3, 0.24 mg/L Na.sub.2MoO.sub.42 H.sub.2O, 1.2 mg/L NiCl.sub.26 H.sub.2O, 0.26 mg/L Na.sub.2SeO.sub.35 H.sub.2O, 0.02 mg/L biotin, 0.02 mg/L folic acid, 0.1 mg/L pyridoxin-HCl, 0.05 mg/L thiamine-HClH.sub.2O, 0.05 mg/L riboflavin, 0.05 mg/L nicotinic acid, 0.05 mg/L D-Ca-pantothenate, 1 g/L vitamin B12, 0.05 mg/L p-aminobenzoate, 0.05 mg/L lipoic acid, 0.25 g/L cysteine-HClH.sub.2O) in a 250 ml bottle were inoculated with 5 ml of a frozen cryoculture and flushed with a premixed gas with 67% H.sub.2, 33% CO.sub.2 to an overpressure of 0.8 bar. The culture was incubated at 30 C. and 100 rpm in an open water bath shaker for 24 h till an OD.sub.600nm>0.15.

[0065] For a second preculture of C. neopropionicum 3200 ml of fresh DSMZ318 medium in a 500 ml bottle were inoculated with centrifuged cells from the first preculture to an OD.sub.600nm of 0.03 and flushed with a premixed gas with 67% H.sub.2, 33% CO.sub.2 to an overpressure of 0.8 bar. This growing culture was incubated at 30 C. and 100 rpm in an open water bath shaker for 22 h till an OD.sub.600nm>0.23.

[0066] For the main culture, as many centrifuged cells from the second preculture of C. neopropionicum as necessary for an OD.sub.600nm of 0.2 were added to 200 ml of fresh LM33 mineral medium (pH=6.8, 10 g/L ethanol, 1 g/L NaOH, 0.5 g/L MgCl.sub.2, 0.21 g/L NaCl, 0.135 g/L CaCl.sub.22H.sub.2O, 2.65 g/L NaH.sub.2PO.sub.42H.sub.2O, 0.5 g/L KCl, 2.5 g/L NH.sub.4Cl, 15 mg/L nitrilotriacetic acid, 30 mg/L MgSO.sub.47 H.sub.2O, 5 mg/L MnSO.sub.4H.sub.2O, 1 mg/L FeSO.sub.47 H.sub.2O, 8 mg/L Fe(SO.sub.4).sub.2(NH.sub.4).sub.26 H.sub.2O, 2 mg/L CoCl.sub.26 H.sub.2O, 2 mg/L ZnSO.sub.47 H.sub.2O, 200 g/L CuCl.sub.22 H.sub.2O, 200 g/L KAl(SO.sub.4).sub.212 H.sub.2O, 3 mg/L H.sub.3BO.sub.3, 300 g/L Na.sub.2MoO.sub.42 H.sub.2O, 200 g/L Na.sub.2SeO.sub.3, 200 g/L NiCl.sub.26 H.sub.2O, 200 g/L Na.sub.2WO.sub.46 H.sub.2O, 200 g/L d-biotin, 200 g/L folic acid, 100 g/L pyridoxine-HCl, 500 g/L thiamine-HCl; 500 g/L riboflavin; 500 g/L nicotinic acid; 500 g/L Ca-pantothenate; 500 g/L vitamin B.sub.12; 500 g/L p-aminobenzoate; 500 g/L lipoic acid, 10 mg/L FeCl.sub.3, aerated for 30 min with a premixed gas with 67% H.sub.2 and 33% CO.sub.2) with additional 6.95 g/L sodium acetate. The cultivation was carried out in a 500 mL pressure-resistant glass bottle at 30 C., 100 rpm and an overpressure of 0.8 bar of a premixed gas with 67% H.sub.2, 33% CO.sub.2 in an open water bath shaker for 113 h. The pH was hold at 6.8 by automatic addition of NaOH solution (100 g/L).

[0067] During cultivation several 5 mL samples were taken to determinate OD.sub.600nm, pH and product formation. The determination of the product concentrations was performed by semi-quantitative .sup.1H-NMR spectroscopy. As an internal quantification standard sodium trimethylsilylpropionate (T(M)SP) was used.

[0068] During the cultivation the concentration of propionate increased from 0.05 g/L to 3.35 g/L, for propanol from 0.01 to 0.19 g/L, for butyrate from 0.00 to 0.19 g/L and for lactate from 0.00 g/L to 0.16 g/L. The concentration of ethanol decreased from 10.0 g/L to 5.4 g/L during this time. The selectivity of propionate/propanol formation was about 91.0%.

Example 3

[0069] Production of Propionic Acid and Propanol with Clostridium neopropionicum

[0070] For the biotransformation of ethanol and carbon dioxide to propionic acid and propanol the bacterium Clostridium neopropionicum was cultivated in mineral medium with ethanol and a gas atmosphere with carbon dioxide. All cultivation steps were carried out under anaerobic conditions in pressure-resistant glass bottles that can be closed airtight with a butyl rubber stopper. For the first preculture of C. neopropionicum 2100 ml DSMZ318 medium (pH 7.4; 0.61 g/l NaCl, 0.047 g/l MgCl.sub.2, 0.30 g/l KH.sub.2PO.sub.4, 1.00 g/l NH.sub.4Cl, 0.081 g/l CaCl.sub.22 H.sub.2O, 0.5 g/l yeast extract, 0.5 g/l BBL Trypticase Peptone, 4 g/L KHCO.sub.3, 1.026 g/L ethanol, 0.5 mg/l resazurin, 128 mg/L nitrilotriacetic acid, 135 mg/L FeCl.sub.36 H.sub.2O, 1 mg/L MnCl.sub.24 H.sub.2O, 0.24 mg/L CoCl.sub.26 H.sub.2O, 1 mg/L ZnCl.sub.2, 0.25 mg/L CuCl.sub.22 H.sub.2O, 0.1 mg/L H.sub.3BO.sub.3, 0.24 mg/L Na.sub.2MoO.sub.42 H.sub.2O, 1.2 mg/L NiCl.sub.26 H.sub.2O, 0.26 mg/L Na.sub.2SeO.sub.35 H.sub.2O, 0.02 mg/L biotin, 0.02 mg/L folic acid, 0.1 mg/L pyridoxin-HCl, 0.05 mg/L thiamine-HClH.sub.2O, 0.05 mg/L riboflavin, 0.05 mg/L nicotinic acid, 0.05 mg/L D-Ca-pantothenate, 1 g/L vitamin B12, 0.05 mg/L p-aminobenzoate, 0.05 mg/L lipoic acid, 0.25 g/L cysteine-HClH.sub.2O) in a 250 ml bottle were inoculated with 5 ml of a frozen cryoculture and flushed with a premixed gas with 67% Hz, 33% CO.sub.2 to an overpressure of 0.8 bar. The culture was incubated at 30 C. and 100 rpm in an open water bath shaker for 24 h till an OD.sub.600nm>0.15. For a second preculture of C. neopropionicum 3200 ml of fresh DSMZ318 medium in a 500 ml bottle were inoculated with centrifuged cells from the first preculture to an OD.sub.600nm of 0.03 and flushed with a premixed gas with 67% H.sub.2, 33% CO.sub.2 to an overpressure of 0.8 bar. This growing culture was incubated at 30 C. and 100 rpm in an open water bath shaker for 22 h till an OD.sub.600nm>0.23.

[0071] For the main culture, as many centrifuged cells from the second preculture of C. neopropionicum as necessary for an OD.sub.600nm of 0.2 were added to 200 ml of fresh LM33 mineral medium (pH=6.8, 10 g/L ethanol, 1 g/L NaOH, 0.5 g/L MgCl.sub.2, 0.21 g/L NaCl, 0.135 g/L CaCl.sub.22H.sub.2O, 2.65 g/L NaH.sub.2PO.sub.42H.sub.2O, 0.5 g/L KCl, 2.5 g/L NH.sub.4Cl, 15 mg/L nitrilotriacetic acid, 30 mg/L MgSO.sub.47 H.sub.2O, 5 mg/L MnSO.sub.4H.sub.2O, 1 mg/L FeSO.sub.47 H.sub.2O, 8 mg/L Fe(SO.sub.4).sub.2(NH.sub.4).sub.26 H.sub.2O, 2 mg/L CoCl.sub.26 H.sub.2O, 2 mg/L ZnSO.sub.47 H.sub.2O, 200 g/L CuCl.sub.22 H.sub.2O, 200 g/L KAl(SO.sub.4).sub.212 H.sub.2O, 3 mg/L H.sub.3BO.sub.3, 300 g/L Na.sub.2MoO.sub.42 H.sub.2O, 200 g/L Na.sub.2SeO.sub.3, 200 g/L NiCl.sub.26 H.sub.2O, 200 g/L Na.sub.2WO.sub.46 H.sub.2O, 200 g/L d-biotin, 200 g/L folic acid, 100 g/L pyridoxine-HCl, 500 g/L thiamine-HCl; 500 g/L riboflavin; 500 g/L nicotinic acid; 500 g/L Ca-pantothenate; 500 g/L vitamin B.sub.12; 500 g/L p-aminobenzoate; 500 g/L lipoic acid, 10 mg/L FeCl.sub.3, aerated for 30 min with a premixed gas with 67% H.sub.2 and 33% CO.sub.2). The cultivation was carried out in a 500 mL pressure-resistant glass bottle at 30 C., 100 rpm and an overpressure of 0.8 bar of a premixed gas with 67% Hz, 33% CO.sub.2 in an open water bath shaker for 113 h. The pH was hold at 6.8 by automatic addition of NaOH solution (100 g/L).

[0072] During cultivation several 5 mL samples were taken to determinate OD.sub.600nm, pH and product formation. The determination of the product concentrations was performed by semi-quantitative .sup.1H-NMR spectroscopy. As an internal quantification standard sodium trimethylsilylpropionate (T(M)SP) was used.

[0073] During the cultivation the concentration of propionate increased from 0.05 g/L to 2.45 g/L, for propanol from 0.01 to 0.45 g/L, for butyrate from 0.01 to 0.32 g/L and for lactate from 0.00 g/L to 0.13 g/L. The concentration of ethanol decreased from 10.5 g/L to 5.4 g/L during this time. The selectivity of propionate/propanol formation was about 86.5%.

Example 4

[0074] Production of Propionate with Desulfobulbus propionicus

[0075] For the biotransformation of ethanol and carbon dioxide to propionic acid the bacterium Desulfobulbus propionicus was cultivated in medium with ethanol and a gas atmosphere with carbon dioxide. All cultivation steps were carried out under anaerobic conditions in pressure-resistant glass bottles that can be closed airtight with a butyl rubber stopper. For the first preculture of Desulfobulbus propionicus, 50 ml DSMZ194-medium (3 g/L Na.sub.2SO.sub.4; 0.2 g/L KH.sub.2PO.sub.4; 0.3 g/L NH.sub.4Cl; 1 g/L NaCl; 0.4 g/L MgCl.sub.26 H.sub.2O; 0.5 g/L KCl; 0.15 g/L CaCl.sub.22 H.sub.2O; 0.5 mg/L NaOH; 0.003 mg/L Na.sub.2SeO.sub.35 H.sub.2O; 0.004 mg/L Na.sub.2WO.sub.42 H.sub.2O; 1 mg/L resazurin; 5 g/L NaHCO.sub.3; 1.5 g/L Na-propionate; 0.4 g/L Na.sub.2S9 H.sub.2O; 2.8 mg/L HCl; 1.5 mg/L FeCl.sub.24 H.sub.2O; 0.07 mg/L ZnCl.sub.27 H.sub.2O; 0.1 mg/L MnCl.sub.24 H.sub.2O; 0.006 mg/L H.sub.3BO.sub.3; 0.19 mg/L CoCl.sub.26 H.sub.2O; 0.002 mg/L CuCl.sub.26 H.sub.2O; 0.024 mg/L NiCl.sub.26 H.sub.2O; 0.036 mg/L Na.sub.2MO.sub.42 H.sub.2O; 0.02 mg/L biotin; 0.02 mg/L folic acid; 0.1 mg/L pyridoxine-HCl; 0.05 mg/L thiamine-HCl; 0.05 mg/L riboflavin; 0.05 mg/L nicotinic acid; 0.05 mg/L D-Ca-pantothenate; 0.001 mg/L vitamin B.sub.12; 0.05 mg/L p-aminobenzoic acid; 0.05 mg/L lipoic acid) with additional 400 mg/L Na.sub.2S and 420 mg/L KOH were inoculated with 5 mL of a frozen cryo stock. The cultivation was carried out in a 250 mL pressure-resistant glass bottle at 37 C., and an overpressure of 0.8 bar of a premixed gas with 67% N.sub.2 and 33% CO.sub.2 in a heat cabinet without shaking for 72 h till OD.sub.600nm of 0.18. For the second preculture 2100 ml DSMZ194-medium with additional 400 mg/L Na.sub.2S and 420 mg/L KOH were inoculated with centrifuged cells from the first preculture to an OD.sub.600nm of 0.038 and 0.034, respectively. The cultivation was carried out in 2250 mL pressure-resistant glass bottles at 37 C., and an overpressure of 0.8 bar of a premixed gas with 67% N.sub.2 and 33% CO.sub.2 for 47 h, one in a heat cabinet without shaking, the other in an open water bath shaker at 100 rpm till OD.sub.600nm of 0.2 and 0.18, respectively.

[0076] For the main culture 100 ml ATCC1754-medium (pH=6.0; 20 g/L MES; 1 g/L yeast extract, 0.8 g/L NaCl; 1 g/L NH.sub.4Cl; 0.1 g/L KCl; 0.1 g/L KH.sub.2PO.sub.4; 0.2 g/L MgSO.sub.47 H.sub.2O; 0.02 g/L CaCl22 H.sub.2O; 20 mg/L nitrilotriacetic acid; 10 mg/L MnSO.sub.4H.sub.2O; 8 mg/L (NH.sub.4).sub.2Fe(SO.sub.4).sub.26 H.sub.2O; 2 mg/L CoCl.sub.26 H.sub.2O; 2 mg/L ZnSO.sub.47 H.sub.2O; 0.2 mg/L CuCl.sub.22 H.sub.2O; 0.2 mg/L Na.sub.2MoO.sub.42 H.sub.2O; 0.2 mg/L NiCl.sub.26 H.sub.2O; 0.2 mg/L Na.sub.2SeO.sub.4; 0.2 mg/L Na.sub.2WO.sub.42 H.sub.2O; 20 g/L d-biotin; 20 g/L folic acid; 100 g/L pyridoxine-HCl; 50 g/L thiamine-HClH.sub.2O; 50 g/L riboflavin; 50 g/L nicotinic acid; 50 g/L Ca-pantothenate; 1 g/L vitamin B.sub.12; 50 g/L p-aminobenzoate; 50 g/L lipoic acid; approx. 67.5 mg/L NaOH) with additional 400 mg/L L-cysteine-hydrochloride, 5 mL/L ethanol and 5.74 g/L KOH, were inoculated with centrifuged cells from both second precultures to an OD.sub.600nm of 0.09. The cultivation was started at a pH of 7.0.

[0077] The cultivation was carried out in a 500 mL pressure-resistant glass bottle at 37 C., 100 rpm and an overpressure of 0.8 bar of a premixed gas with 67% N.sub.2 and 33% CO.sub.2 in an open water bath shaker for 170 h till OD.sub.600nm of 0.07 and pH 6.8.

[0078] During cultivation several 5 mL samples were taken to determinate OD.sub.600nm, pH and product formation. The determination of the product concentrations was performed by semi-quantitative .sup.1H-NMR spectroscopy. As an internal quantification standard sodium trimethylsilylpropionate (T(M)SP) was used.

[0079] During the main cultivation the concentration of acetate increased from 0.01 g/L to 0.45 g/L, for propionate from 0.00 g/L to 0.3 g/L, and for propanol from 0.00 g/L to 0.16 g/L. The concentration of ethanol decreased from 4.85 g/L to 3.95 g/L.

Example 5

[0080] Production of Propionate with Clostridium propionicum

[0081] For the biotransformation of ethanol and carbon dioxide to propionic acid the bacterium Clostridium propionicum was cultivated in medium with ethanol and a gas atmosphere with carbon dioxide. All cultivation steps were carried out under anaerobic conditions in pressure-resistant glass bottles that can be closed airtight with a butyl rubber stopper.

[0082] For the first preculture 50 ml DSMZ156-medium (3.0 g/L L-Alanine; 3.0 g/L peptone; 4.0 g/L yeast extract; 0.1 g/L MgSO.sub.47 H.sub.2O; 18.0 mg/l FeSO.sub.47 H.sub.2O; 5.0 mmol/L phosphate buffered saline pH 7.1; 2.5 mL/L saturated calcium sulfate solution; 1.0 mg/L resazurin; 1 g/L NaHCO.sub.3) with additional 400 mg/L L-cysteine-hydrochlorid were inoculated with 5 mL of a frozen cryo stock. The cultivation was carried out in a 250 mL pressure-resistant glass bottle at 37 C., 100 rpm and an overpressure of 0.8 bar of a premixed gas with 67% N.sub.2 and 33% CO.sub.2 in an open water bath shaker for 21 h till OD.sub.600nm of 0.64.

[0083] For the second preculture 100 ml DSMZ156-medium with additional 400 mg/L L-cysteine-hydrochlorid were inoculated with centrifuged cells from the first preculture to an OD.sub.600nm of 0.07. The cultivation was carried out in a 250 mL pressure-resistant glass bottle at 37 C., 100 rpm and an overpressure of 0.8 bar of a premixed gas with 67% N.sub.2 and 33% CO.sub.2 in an open water bath shaker for 47 h till OD.sub.600nm of 0.69.

[0084] For the main culture 100 ml ATCC1754-medium (pH=6.0; 20 g/L MES; 1 g/L yeast extract, 0.8 g/L NaCl; 1 g/L NH.sub.4Cl; 0.1 g/L KCl; 0.1 g/L KH.sub.2PO.sub.4; 0.2 g/L MgSO.sub.47 H.sub.2O; 0.02 g/L CaCl.sub.22 H.sub.2O; 20 mg/L nitrilotriacetic acid; 10 mg/L MnSO.sub.4H.sub.2O; 8 mg/L (NH.sub.4).sub.2Fe(SO.sub.4).sub.26 H.sub.2O; 2 mg/L CoCl.sub.26 H.sub.2O; 2 mg/L ZnSO.sub.47 H.sub.2O; 0.2 mg/L CuCl.sub.22 H.sub.2O; 0.2 mg/L Na.sub.2MoO.sub.42 H.sub.2O; 0.2 mg/L NiCl.sub.26 H.sub.2O; 0.2 mg/L Na.sub.2SeO.sub.4; 0.2 mg/L Na.sub.2WO.sub.42 H.sub.2O; 20 g/L d-biotin; 20 g/L folic acid; 100 g/L pyridoxine-HCl; 50 g/L thiamine-HClH.sub.2O; 50 g/L riboflavin; 50 g/L nicotinic acid; 50 g/L Ca-pantothenate; 1 g/L vitamin B.sub.12; 50 g/L p-aminobenzoate; 50 g/L lipoic acid; approx. 67.5 mg/L NaOH) with additional 400 mg/L L-cysteine-hydrochlorid, 5 mL/L ethanol and 2.35 g/L KOH, were inoculated with centrifuged cells from the second preculture to an OD.sub.600nm of 0.166. The cultivation was started at a pH of 6.6.

[0085] The cultivation was carried out in a 500 mL pressure-resistant glass bottle at 37 C., 100 rpm and an overpressure of 0.8 bar of a premixed gas with 67% N.sub.2 and 33% CO.sub.2 in an open water bath shaker for 165 h till OD.sub.600nm of 0.21 and pH 6.0.

[0086] During cultivation several 5 mL samples were taken to determinate OD.sub.600nm, pH and product formation. The determination of the product concentrations was performed by semiquantitative .sup.1H-NMR spectroscopy. As an internal quantification standard sodium trimethylsilylpropionate (T(M)SP) was used.

[0087] During the main cultivation the concentration of acetate increased from 0.01 g/L to 1.15 g/L, for propionate from 0.02 g/L to 1.05 g/L, for propanol from 0.00 to 0.39 g/L, and for butyrate from 0 to 110 mg/L. The concentration of ethanol decreased from 4.40 g/L to 2.75 g/L and for alanine from 34 mg/L to 0 mg/L during this time.

Example 6

[0088] Production of Propionate with Pelobacter propionicus

[0089] For the biotransformation of ethanol and carbon dioxide to propionic acid the bacterium Pelobacter propionicus was cultivated in medium with ethanol and a gas atmosphere with carbon dioxide. All cultivation steps were carried out under anaerobic conditions in pressure-resistant glass bottles that can be closed airtight with a butyl rubber stopper.

[0090] For the first preculture 50 ml DSMZ295-medium (0.2 g/L KH.sub.2PO.sub.4; 0.25 g/L NH.sub.4Cl; 1 g/L NaCl; 0.4 g/L MgCl.sub.26 H.sub.2O; 0.5 g/L KCl; 0.15 g/L CaCl.sub.22 H.sub.2O; 2.8 mg/L HCl; 1.5 mg/L FeCl.sub.24 H.sub.2O; 0.07 mg/L ZnCl.sub.27 H.sub.2O; 0.1 mg/L MnCl.sub.24 H.sub.2O; 0.006 mg/L H.sub.3BO.sub.3; 0.19 mg/L CoCl.sub.26 H.sub.2O; 0.002 mg/L CuCl.sub.26 H.sub.2O; 0.024 mg/L NiCl.sub.26 H.sub.2O; 0.036 mg/L Na.sub.2MO.sub.42 H.sub.2O; 1 mg/L resazurin; 2.5 g/L NaHCO.sub.3; 0.9 g/L 2,3-butanediol) with additional 360 mg/L Na.sub.2S were inoculated with 5 mL of a frozen cryo stock. The cultivation was carried out in a 250 mL pressure-resistant glass bottle, at 30 C., 100 rpm in an open water bath shaker and an overpressure of 0.8 bar of a premixed gas with 67% N.sub.2 and 33% CO.sub.2 for 69 h till OD.sub.600nm of 0.19 and pH 6.2.

[0091] For the second preculture 2100 ml DSMZ295-medium with additional 360 mg/L Na.sub.2S and 420 mg/L KOH were inoculated with centrifuged cells from the first preculture to an OD.sub.600nm of 0.055 and 0.053, respectively. The cultivation was carried out in 2250 mL pressure-resistant glass bottles at 30 C., 100 rpm in an open water bath shaker and an overpressure of 0.8 bar of a premixed gas with 67% N.sub.2 and 33% CO.sub.2 for 72 h till OD.sub.600nm of 0.24 and pH 6.6 and OD.sub.600nm of 0.23 and pH 6.4, respectively.

[0092] For the main culture 100 ml modified DSMZ318-medium (pH 7.4; 0.61 g/L NaCl, 0.047 g/L MgCl.sub.2, 0.30 g/L KH.sub.2PO.sub.4, 1.00 g/L NH.sub.4Cl, 0.081 g/L CaCl.sub.22 H.sub.2O, 0.5 g/L yeast extract, 0.5 g/L BBL Trypticase Peptone, 4 g/L KHCO.sub.3, 5 g/L ethanol, 0.5 mg/L resazurin, 128 mg/L nitrilotriacetic acid, 135 mg/L FeCl.sub.36 H.sub.2O, 1 mg/L MnCl.sub.24 H.sub.2O, 0.24 mg/L CoCl.sub.26 H.sub.2O, 1 mg/L ZnCl.sub.2, 0.25 mg/L CuCl.sub.22 H.sub.2O, 0.1 mg/L H.sub.3BO.sub.3, 0.24 mg/L Na.sub.2MoO.sub.42 H.sub.2O, 1.2 mg/L NiCl.sub.26 H.sub.2O, 0.26 mg/L Na.sub.2SeO.sub.35 H.sub.2O, 0.02 mg/L biotin, 0.02 mg/L folic acid, 0.1 mg/L pyridoxin-HCl, 0.05 mg/L thiamine-HClH.sub.2O, 0.05 mg/L riboflavin, 0.05 mg/L nicotinic acid, 0.05 mg/L D-Ca-pantothenate, 1 g/L vitamin B12, 0.05 mg/L p-aminobenzoate, 0.05 mg/L lipoic acid) with additional 0.4 g/L L-cysteine-hydrochlorid, 0.7 g/L KOH, and 11.2 mg/L HCl were inoculated with centrifuged cells from both second precultures to an OD.sub.600nm of 0.10. The cultivation was started at a pH of 6.8. The cultivation was carried out in a 500 mL pressure-resistant glass bottle at 30 C., 100 rpm and an overpressure of 0.8 bar of a premixed gas with 67% N.sub.2 and 33% CO.sub.2 in an open water bath shaker for 162 h till OD.sub.600nm of 0.13 and pH 6.5.

[0093] During cultivation several 5 mL samples were taken to determinate OD.sub.600nm, pH and product formation. The determination of the product concentrations was performed by semiquantitative .sup.1H-NMR spectroscopy. As an internal quantification standard sodium trimethylsilylpropionate (T(M)SP) was used.

[0094] During the main cultivation the concentration of acetate increased from 0.02 g/L to 0.45 g/L, for propionate from 0.02 to 0.27 g/L, and for propanol from 0.00 to 0.18 g/L. The concentration of ethanol decreased from 4.6 g/L to 4.00 g/L.

Example 7

[0095] Production of Propionic Acid and Propanol with Clostridium neopropionicum on Acetate

[0096] For the biotransformation of ethanol and carbon dioxide to propionic acid and propanol the bacterium Clostridium neopropionicum was cultivated in mineral medium with ethanol, acetate and a gas atmosphere with carbon dioxide. All cultivation steps were carried out under anaerobic conditions in pressure-resistant glass bottles that can be closed airtight with a butyl rubber stopper. For the first preculture of C. neopropionicum 500 mL DSMZ318 medium (pH 7.4; 0.61 g/L NaCl; 0.047 g/L MgCl.sub.2; 0.30 g/L KH.sub.2PO.sub.4; 1.00 g/L NH.sub.4Cl; 0.081 g/L CaCl.sub.22 H.sub.2O; 0.5 g/L yeast extract; 0.5 g/L BBL Trypticase Peptone; 4 g/L KHCO.sub.3; 10 g/L ethanol; 0.5 mg/L resazurin; 128 mg/L nitrilotriacetic acid; 135 mg/L FeCl.sub.36 H.sub.2O; 1 mg/L MnCl.sub.24 H.sub.2O; 0.24 mg/L CoCl.sub.26 H.sub.2O; 1 mg/L ZnCl.sub.2; 0.25 mg/L CuCl.sub.22 H.sub.2O; 0.1 mg/L H.sub.3BO.sub.3; 0.24 mg/L Na.sub.2MoO.sub.42 H.sub.2O; 1.2 mg/L NiCl.sub.26 H.sub.2O; 0.26 mg/L Na.sub.2SeO.sub.35 H.sub.2O; 0.02 mg/L biotin; 0.02 mg/L folic acid; 0.1 mg/L pyridoxin-HCl; 0.05 mg/L thiamine-HClH.sub.2O; 0.05 mg/L riboflavin; 0.05 mg/L nicotinic acid; 0.05 mg/L D-Ca-pantothenate; 1 g/L vitamin B.sub.12; 0.05 mg/L p-aminobenzoate; 0.05 mg/L lipoic acid; 0.25 g/L cysteine-HClH.sub.2O) in a 1 L bottle were inoculated with 5 ml of a frozen cryoculture and flushed with a premixed gas with 67% H.sub.2, 33% CO.sub.2 to an overpressure of 0.8 bar. The culture was incubated at 30 C. and 100 rpm in an open water bath shaker for 67 h till an OD.sub.600nm 0.17.

[0097] For the second preculture of C. neopropionicum 500 mL of fresh DSMZ318 medium in a 1 L bottle were inoculated with centrifuged cells from the first preculture to an OD.sub.600nm of 0.05 and flushed with a premixed gas with 67% H.sub.2, 33% CO.sub.2 to an overpressure of 0.8 bar. This growing culture was incubated at 30 C. and 100 rpm in an open water bath shaker for 24 h till OD.sub.600nm 0.13.

[0098] For the third preculture of C. neopropionicum 2500 mL of fresh DSMZ318 medium in 1 L bottles were inoculated with centrifuged cells from the second preculture to an OD.sub.600nm of 0.05 and flushed with a premixed gas with 67% H.sub.2, 33% CO.sub.2 to an overpressure of 0.8 bar. These growing cultures were incubated at 30 C. and 100 rpm in an open water bath shaker for 18 h till OD.sub.600nm 0.26 and 0.25, respectively.

[0099] For the fourth preculture of C. neopropionicum 10500 mL of fresh DSMZ318 medium in 1 L bottles were inoculated with centrifuged cells from the third preculture to an OD.sub.600nm of 0.03-0.04 and flushed with a premixed gas with 67% H.sub.2, 33% CO.sub.2 to an overpressure of 0.8 bar. These growing cultures were incubated at 30 C. and 100 rpm in an open water bath shaker for 22 h till OD.sub.600nm>0.23

[0100] For the main culture, as many centrifuged cells from the fourth preculture of C. neopropionicum as necessary for an OD.sub.600nm of 0.75 were added to 50 ml of fresh LM33 mineral medium (pH=6.8, 10 g/L ethanol; 1 g/L NaOH; 0.5 g/L MgCl.sub.2; 0.21 g/L NaCl; 0.135 g/L CaCl.sub.22H.sub.2O; 2.65 g/L NaH.sub.2PO.sub.42 H.sub.2O; 0.5 g/L KCl; 2.5 g/L NH.sub.4Cl; 15 mg/L nitrilotriacetic acid; 30 mg/L MgSO.sub.47 H.sub.2O; 5 mg/L MnSO.sub.4H.sub.2O; 1 mg/L FeSO.sub.47 H.sub.2O; 8 mg/L Fe(SO.sub.4).sub.2(NH.sub.4).sub.26 H.sub.2O; 2 mg/L CoCl.sub.26 H.sub.2O; 2 mg/L ZnSO.sub.47 H.sub.2O; 200 g/L CuCl.sub.22 H.sub.2O; 200 g/L KAl(SO.sub.4).sub.212 H.sub.2O; 3 mg/L H31303; 300 g/L Na.sub.2MoO.sub.42 H.sub.2O; 200 g/L Na.sub.2SeO.sub.3; 200 g/L NiCl.sub.26 H.sub.2O; 200 g/L Na.sub.2WO.sub.46 H.sub.2O; 200 g/L d-biotin; 200 g/L folic acid; 100 g/L pyridoxine-HCl; 500 g/L thiamine-HCl; 500 g/L riboflavin; 500 g/L nicotinic acid; 500 g/L Ca-pantothenate; 500 g/L vitamin B.sub.12; 500 g/L p-aminobenzoate; 500 g/L lipoic acid; 10 mg/L FeCl.sub.3, aerated for 30 min with CO.sub.2) with additional 13.9 g/L sodium acetate. The cultivation was carried out in a 250 mL pressure-resistant glass bottle at 30 C., 150 rpm and an overpressure of 0.3 bar of CO.sub.2 in an open water bath shaker for 5 h. The pH decreased during this time from 7.0 to 6.8. The OD.sub.600nm decreased from 0.75 to 0.71.

[0101] During cultivation several 5 mL samples were taken to determinate OD.sub.600nm, pH and product formation. The determination of the product concentrations was performed by semiquantitative .sup.1H-NMR spectroscopy. As an internal quantification standard sodium trimethylsilylpropionate (T(M)SP) was used.

[0102] During the cultivation the concentration of propionate increased from 0.03 g/L to 0.77 g/L, for propanol from 0.01 to 0.02 g/L and for lactate from 0.00 g/L to 0.08 g/L. No butyrate was formed. The concentration of ethanol decreased from 9.25 g/L to 8.04 g/L during this time. The selectivity of propionate/propanol formation was about 92.1%.

REFERENCES

[0103] Drake et al., 2004. Strict and Facultative Anaerobes: Medical and Environmental Aspects. pp. 251-281, Horizon Scientific Press, United Kingdom

[0104] Drake & Kusel, 2005 Acetogenic clostridia. In: Drre, P. (ed.), Handbook on Clostridia, pp. 719-746. CRC Press, Boca Raton, Fla.

[0105] Drake et al., 2006, Acetogenic prokaryotes. In: Balows A, Trper H G, Dworkin M, Harder W and Gerhardt, P et al (ed) American Society for Microbiology, Washington, DC. p. 248-277

[0106] Fuchs G., Schlegel H.-G. (2007) Allgemeine Mikrobiologie, Georg Thieme Verlag, Stuttgart.

[0107] Galaction, A.-I. et al., (2012) Chemical Engineering & Technology, 35(9), 1657-1663

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[0109] Keshav, Amit et al., Desalination (2009), 244(1-3), 12-23

[0110] Sakai et al., 2004 Biotechnol. Let., Vol. 29, p. 1607-1612

[0111] Wood, 1991 Life with CO or CO2 and H2 as a source of carbon and energy. FASEB J. 5:156-163

[0112] Zhang Y, 2013, Bioprocess Biosyst Eng; 36(12):1897-1904

[0113] U.S. 2007/0275447, U.S. 2008/0057554, WO 98/00558, WO 00/68407