PRODUCTION OF PROPANOL AND/OR PROPIONIC ACID

Abstract

The present invention relates to a method of producing propanol and/or propionic acid from a carbon source in aerobic conditions, the method comprising:

(a) step of producing ethanol and/or acetate from the carbon source in aerobic conditions, comprising
(i) contacting a reaction mixture comprising a first acetogenic microorganism in an exponential growth phase; free oxygen; and a second acetogenic microorganism in a stationary phase
wherein the first and second acetogenic microorganism is capable of converting the carbon source to the acetate and/or ethanol; and
(b) step of contacting the acetate and/or ethanol from step (a) with a third microorganism capable of converting the acetate and/or ethanol to propanol and/or propionic acid.

Claims

1. A method of producing propanol and/or propionic acid from a carbon source in aerobic conditions, the method comprising: (a) step of producing ethanol and/or acetate from the carbon source in aerobic conditions, comprising (i) contacting a reaction mixture comprising a first acetogenic microorganism in an exponential growth phase; free oxygen; and a second acetogenic microorganism in a stationary phase wherein the first and second acetogenic microorganism is capable of converting the carbon source to the acetate and/or ethanol; and (b) step of contacting the acetate and/or ethanol from step (a) with a third microorganism capable of converting the acetate and/or ethanol to propanol and/or propionic acid.

2. The method according to claim 1, wherein the third microorganism capable of converting the acetate and/or ethanol to propanol and/or propionic acid is a propionogen.

3. The method according to claim 2, wherein the propionogen uses the actate-acrylate pathway for the production of propanol and/or propionic acid.

4. The method according to claim 2, wherein the propionogen uses methylmalonyl-succinate pathway for the production of propanol and/or propionic acid.

5. The method according to claim 2, wherein the C3-producing microorganism 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.

6. The method according to claim 1, wherein the third microorganism is a genetically modified organism comprising increased expression relative to the wild type cell of propionate CoA-transferase (E.sub.1), lactoyl-CoA dehydratase (E.sub.2) and acryloyl-CoA reductase ((E.sub.3).

7. The method according to claim 1, wherein the first and second microorganism 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, Butyribacterlum methylotrophicum, Moorella thermoacetica, Eubacterium limosum, Peptostreptococcus productus, Clostridium Ijungdahlii, Clostridium ATCC 29797 and Clostridium carboxidivorans.

8. The method according to claim 1, wherein the first acetogenic microorganism in the exponential growth phase has a growth rate of 0.01 to 2 h.sup.1.

9. The method according to claim 1, wherein the first acetogenic microorganism in the exponential growth phase has an OD.sub.600 of 0.01 to 2.

10. The method according to claim 1, wherein the aerobic conditions is a result of oxygen being at a concentration of 0.000005-1% volume in the gas phase.

11. The method according to claim 1, wherein the third microorganism is Clostridium neopropionicum.

12. The method according to claim 1, wherein the first and second microorganism is Clostridium Ijungdahlii and the third microorganism is Clostridium neopropionicum.

13. The method according to claim 1, wherein the first and second microorganism is Clostridium autoethanogenum and the third microorganism is Clostridium neopropionicum.

14. The mixture according to claim 1, wherein the carbon source comprises CO.

15. The method according to claim 1, wherein steps (a) and (b) are carried out in a single fermenter.

Description

BRIEF DESCRIPTION OF FIGURES

[0068] FIG. 1 is an illustration of the methylmaionyl-succinate pathway used by anaerobic microorganisms for C3 (propionate) production (Source: WO2014/140336).

[0069] FIG. 2 is an illustration of the lactate-acrylate pathway used by anaerobic microorganisms for C3 (propionate/propanol) production (Source: WO2014/140336).

EXAMPLES

[0070] 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 I

[0071] Production of Propionic Acid and Propanol on Synthesis Gas with Oxygen with Clostridium autoethanogenum and Clostridium neopropionicum

[0072] For the biotransformation of hydrogen and carbon dioxide to propionic acid and propanol the homoacetogenic bacterium Clostridium autoethanogenum was cultivated on synthesis gas in combination with Clostridium neopropionicum in a co-cultivation phase. All cultivation steps were carried out under anaerobic or microaerophile conditions in pressure-resistant glass bottles that can be closed airtight with a butyl rubber stopper.

[0073] For the cultivation of C. autoethanogenum 500 ml medium (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 and 400 mg/L Na.sub.2S9H.sub.2O were inoculated with 5 mL of a frozen cryo stock. The chemolithoautotrophic cultivation was carried out in a 1 L pressure-resistant glass bottle at 37 C., 100 rpm and a ventilation rate of 3 L/h with a premixed gas with 67% H.sub.2, 33% CO.sub.2 in an open water bath shaker for 64 h till OD.sub.600nm of 0.36. The gas was discharged into the medium through a sparger with a pore size of 10 m, which was mounted in the center of the reactors. Then the cell suspension was centrifuged, washed with fresh ATCC1754-medium and centrifuged again. For the main culture of C. autoethanogenum 500 ml ATCC1754-medium with additional 400 mg/L L-cysteine-hydrochlorid and 400 mg/L Na.sub.2S9H.sub.2O were inoculated with washed cells from the first preculture to an OD.sub.600nm of 0.1. The chemolithoautotrophic cultivation was carried out in a 1 L pressure-resistant glass bottle at 37 C., 100 rpm and a ventilation rate of 1 L/h with a premixed gas with 66.85% H.sub.2, 33% CO.sub.2, 0.15% O.sub.2 in an open water bath shaker for 51 h till OD.sub.600nm of 0.19 and pH 5.7. The gas was discharged into the medium through a sparger with a pore size of 10 m, which was mounted in the center of the reactors.

[0074] 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 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 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 19 h till an OD.sub.600nm>0.14.

[0075] For a second preculture of Clostridium neopropionicum 5200 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 0.02 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 an OD.sub.600nm>0.26. Then the cell suspension was centrifuged, washed with fresh ATCC1754-medium and centrifuged again.

[0076] For the co-cultivation culture, as many washed cells from the second preculture of C. neopropionicum as necessary for an OD.sub.600nm of 0.2 were added to the continuously aerated main culture of C. autoethanogenum after 51 h of cultivation. The cultivation was carried out in a 1 L pressure-resistant glass bottle at 37 C., 100 rpm and a ventilation rate of 1 L/h with a premixed gas with 66.85% H.sub.2, 33% CO.sub.2, 0.15% O.sub.2 in an open water bath shaker for another 41 h. At the beginning the pH was set to 6.7 with 140 g/l KOH and then the co-cultivation was carried out without pH control.

[0077] During cultivation several 5 mL samples were taken to determinate OD.sub.600nm, pH und product formation. The determination of the product concentrations was performed by LCMS and semiquantitative .sup.1H-NMR spectroscopy. As an internal quantification standard sodium trimethylsilylpropionate (T(M)SP) was used.

[0078] During the co-cultivation the concentration of acetate increased from 0.79 g/L to 1.83 g/L, for propionate from 0.00 g/L to 0.23 g/L, for propanol from 0 to 19 mg/L, for butyrate from 0 to 14 mg/L, and for formate from 32 mg/L to 335 mg/L. The concentration of ethanol decreased from 47 mg/L to 25 mg/L during this time.

Example 2

Culture of Clostridium Ijungdahlii in Log Phase in the Presence of Synthesis Gas Comprising CO.SUB.2 .and 0.15% Oxygen

[0079] C. jungdahlii was fed H.sub.2 and CO.sub.2 out of the feed-through gas phase and formed acetate and ethanol. For the cultivation, pressure-resistant glass bottle that can be sealed airtight with a butyl rubber stopper were used. All cultivation steps, where C. Ijungdahlii cells were involved were carried out under anaerobic conditions.

[0080] For cell culture of C. Ijungdahlii 5 mL Cryoculture was cultured anaerobically in 500 ml of medium (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.22H.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, approximately 67.5 mg/L NaOH) with about 400 mg/L L-cysteine hydrochloride and 400 mg/L Na.sub.2S9H.sub.2O. Cultivation was carried chemolithoautotrophically in a flameproof 1 L glass bottle with a premixed gas mixture composed of 67% H.sub.2, 33% CO.sub.2 in an open water bath shaker at 37 C., 100 rpm and a fumigation of 3 L/h for 72 h. The gas entry into the medium was carried out by a filter with a pore size of 10 microns, and was mounted in the middle of the reactor, at a gassing tube. The cells were centrifuged, washed with 10 ml ATCC medium and centrifuged again.

[0081] For the main culture many washed cells from the growth culture of C. Ijungdahlii were transferred into 500 mL of ATCC medium with about 400 mg/L L-cysteine hydrochloride and grown to an OD.sub.600 of 0.1. Cultivation was carried out in a pressure-resistant 1 L glass bottle with a premixed gas mixture composed of 66.85% H.sub.2, 33% CO.sub.2, 0.15% O.sub.2 in an open water bath shaker at 37 C., 150 rpm and with aeration of 1 L/h for 45 h. The gas entry into the medium was carried out by a filter with a pore size of 10 microns, which was placed in the middle of the reactors. When sampling each 5 ml sample was removed for determination of OD.sub.600 nm, pH and the product range. The determination of the product concentration was performed by semi-quantitative 1H-NMR spectroscopy. As an internal quantification standard sodium trimethylsilylpropionate served (T (M) SP).

[0082] There was significant cell growth shown during the cultivation period, evidenced by an increase in OD.sub.600 nm of 0.10 to 0.54, corresponding to a growth rate p=0.037 h.sup.1. The concentration of acetate increased at the same time from 9.6 mg/L to 3,304 mg/L and the concentration of ethanol from 2.2 mg/L to 399 mg/L.

Example 3

Culture of Clostridium Jungdahlii in Log Phase in the Presence of Synthesis Gas Comprising CO and 0.1% Oxygen

[0083] C. Ijungdahlii was autotrophically cultivated in complex medium with synthesis gas, consisting of CO, H.sub.2 and CO.sub.2 in the presence of oxygen in order to produce acetate and ethanol.

[0084] A complex medium was used consisting of 1 g/L NH.sub.4Cl, 0.1 g/L KCl, 0.2 g/L MgSO.sub.47 H.sub.2O, 0.8 g/L NaCl, 0.1 g/L KH.sub.2PO.sub.4, 20 mg/L CaCl.sub.22 H.sub.2O, 20 g/L MES, 1 g/L yeast extract, 0.4 g/L L-cysteine-HCl, 0.4 g/L Na.sub.2S9H.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 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-pantothenoic acid, 1 g/L vitamine B12, 50 g/L p-aminobenzoic acid, 50 g/L lipoic acid.

[0085] The autotrophic cultivation was performed in 500 mL medium in a 1 L serum bottle that was continuously gassed with synthesis gas consisting of 67.7% CO, 3.5% H.sub.2 and 15.6% CO.sub.2 at a rate of 3.6 L/h. The gas was introduced into the liquid phase by a microbubble disperser with a pore diameter of 10 m. The serum bottle was continuously shaken in an open water bath Innova 3100 from New Brunswick Scientific at 37 C. and a shaking rate of 120 min.sup.1.

[0086] pH was not controlled.

[0087] At the beginning of the experiment, C. Ijungdahlii was inoculated with an OD.sub.600 of 0.1 with autotrophically grown cells on H.sub.2/CO.sub.2. Therefore, C. Ijungdahlii was grown in complex medium under continuous gassing with synthesis gas consisting of 67% H.sub.2 and 33% CO.sub.2 at a rate of 3 L/h in 1 L serum bottles with 500 mL complex medium. Above described medium was also used for this cultivation. The gas was introduced into the liquid phase by a microbubble disperser with a pore diameter of 10 m. The serum bottle was continuously shaken in an open water bath Innova 3100 from New Brunswick Scientific at 37 C. and a shaking rate of 150 min.sup.1. The cells were harvested in the logarithmic phase with an OD.sub.600 of 0.49 and a pH of 5.03 by anaerobic centrifugation (4500 min.sup.1, 4300 g, 20 C., 10 min). The supernatant was discarded and the pellet was resuspended in 10 mL of above described medium. This cell suspension was then used to inoculate the cultivation experiment. Gas phase concentration of carbon monoxide was measured sampling of the gas phase and offline analysis by an gas chromatograph GC 6890N of Agilent Technologies Inc. with an thermal conductivity detector. Gas phase concentration of oxygen was measured by a trace oxygen dipping probe from PreSens Precision Sensing GmbH. Oxygen concentration was measured by fluorescence quenching, whereas the degree of quenching correlates to the partial pressure of oxygen in the gas phase. Oxygen measurement indicated a concentration of 0.1% vol of O.sub.2 in the used synthesis gas.

[0088] During the experiment samples of 5 mL were taken for the determination of OD.sub.600, pH and product concentrations. The latter were determined by quantitative .sup.1H-NMR-spectroscopy.

[0089] After inoculation of C. Ijungdahlii, cells began to grow with a growth rate of 0.062 h.sup.1 and continuously produced acetate up to a concentration of 6.2 g/L after 94.5 hours. Concomitant to the production of acetate, ethanol was produced in a lower rate compared to the production of acetate up to a concentration of 1 g/L after 94.5 hours.

TABLE-US-00001 TABLE 1 results of example 4 NMR-analytics Process Acetate, Ethanol, time, h pH OD600 mg/L mg/L 0.0 6.15 0.10 18 n.d. 18.0 5.97 0.69 973 97 42.5 5.20 1.50 66.0 4.67 1.95 5368 966 94.5 4.54 1.77 6187 1070 (n.d. = not detected)

Example 4

[0090] Growth and Acetate Production by Clostridium Ijungdahlii on Synthesis Gas with 2% Oxygen

[0091] For the biotransformation of hydrogen and carbon dioxide to acetic acid the homoacetogenic bacterium Clostridium Ijungdahlii was cultivated on synthesis gas with oxygen. All cultivation steps were carried out under anaerobic conditions in pressure-resistant glass bottles that can be closed airtight with a butyl rubber stopper.

[0092] For the preculture 500 ml medium (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 and 400 mg/L Na.sub.2S9H.sub.2O were inoculated with 5 mL of a frozen cryo stock of C. Ijungdahlii. The chemolithoautotrophic cultivation was carried out in a 1 L pressure-resistant glass bottle at 37 C., 100 rpm and a ventilation rate of 3 L/h with a premixed gas with 67% H.sub.2, 33% CO.sub.2 in an open water bath shaker for 72 h. The gas was discharged into the medium through a sparger with a pore size of 10 m, which was mounted in the center of the reactors. Culturing was carried out with no pH control.

[0093] After the precultivation, the cell suspension was centrifuged (10 min, 4200 rpm) and the pellet was washed with 10 ml medium and centrifuged again. For the main culture, as many washed cells from the preculture as necessary for an OD.sub.600nm of 0.1 were transferred in 200 mL medium with additional 400 mg/L L-cysteine-hydrochlorid. The chemolithoautotrophic cultivation was carried out in a 250 mL pressure-resistant glass bottles at 37 C., 150 rpm and a ventilation rate of 1 L/h with a premixed gas with 65% H.sub.2, 33% CO.sub.2, 2% O.sub.2 in an open water bath shaker for 47 h. The gas was discharged into the medium through a sparger with a pore size of 10 m, which was mounted in the center of the reactors. Culturing was carried out with no pH control. During cultivation several 5 mL samples were taken to determinate OD.sub.600nm, pH und product formation. The determination of the product concentrations was performed by semiquantitative 1H-NMR spectroscopy. As an internal quantification standard sodium trimethylsilylpropionate (T(M)SP) was used. Also the dissolved oxygen in the cultivation medium was measured online by oxygen dipping probes (PSt6 with Oxy4Trace, Presens, Germany).

[0094] During the cultivation period cell growth was observed by an increase of the OD.sub.600nm from 0.11 to 0.32, which correlates with a growth rate of p=0.022 h.sup.1. The concentration of acetate increased from 8 mg/L to 91 mg/L, an increase of the ethanol concentration was not observed. Over the cultivation period the dissolved oxygen concentration varied between 0.06 and 0.15 mg/L.

[0095] In a similar technical setting with the same parameters (medium composition, volume, bottle, gas, ventilation rate, temperature, shaking frequency), but without cells in the medium, a dissolved oxygen concentration of 0.50 mg/L was measured.

Example 5

[0096] Growth and Production of Acetate and Other Compounds by Clostridium carboxidivorans on Synthesis Gas with 0.05% Oxygen

[0097] For the biotransformation of hydrogen and carbon dioxide to acetic acid and other compounds the homoacetogenic bacterium Clostridium carboxidivorans was cultivated on synthesis gas with oxygen. All cultivation steps were carried out under anaerobic conditions in pressure-resistant glass bottles that can be closed airtight with a butyl rubber stopper.

[0098] For the preculture 500 ml medium (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-hydrochloride and 400 mg/L Na.sub.2S9H.sub.2O were inoculated with 5 mL of a frozen cryo stock of C. carboxidivorans. The chemolithoautotrophic cultivation was carried out in a 1 L pressure-resistant glass bottle at 37 C., 100 rpm and a ventilation rate of 3 L/h with a premixed gas with 60% H.sub.2, 20% CO.sub.2, and 20% CO in an open water bath shaker for 71 h. The gas was discharged into the medium through a sparger with a pore size of 10 m, which was mounted in the center of the reactors. Culturing was carried out with no pH control.

[0099] After the precultivation, the cell suspension was centrifuged (10 min, 4200 rpm) and the pellet was resuspended in fresh medium. For the main culture, as many cells from the preculture as necessary for an OD.sub.600nm of 0.2 were transferred in 200 mL complex medium (ATCC1754) and parallel in 200 ml mineral medium (DM4-medium: pH=6.00, 0.5 g/L MgCl.sub.26 H.sub.2O, 0.2 g/L CaCl.sub.22 H.sub.2O, 15 mg/L FeCl.sub.24 H.sub.2O, 2 g/L (NH.sub.4)H.sub.2PO.sub.4, 0.2 g/L NaCl, 0.15 g/L KCl, 3 mg/L H.sub.3BO.sub.3, 2 mg/L CoCl.sub.26 H.sub.2O, 1 mg/L ZnSO.sub.47 H.sub.2O, 300 g/L Na.sub.2MoO.sub.42 H.sub.2O, 300 g/L MnSO.sub.4H.sub.2O, 200 g/L NiCl.sub.26 H.sub.2O, 100 g/L CuCl.sub.22 H.sub.2O, 100 g/L Na.sub.2SeO.sub.3, 106 g/L d-biotin, 5 g/L folic acid, 2.5 g/L pyridoxine-HCl, 266 g/L thiamine-HCl, 12.5 g/L riboflavin, 12.5 g/L nicotinic acid, 413 g/L Ca-pantothenate, 12.5 g/L vitamin B.sub.12, 12.5 g/L p-aminobenzoate, 15.0 g/L lipoic acid, approx. 1.3 g/L KOH), with additional 400 mg/L L-cysteine-hydrochloride each. The chemolithoautotrophic cultivation was carried out in a 1 L pressure-resistant glass bottle at 37 C., 150 rpm and a ventilation rate of 1 L/h with a premixed gas with 66.95% H.sub.2, 33% CO.sub.2, and 0.05% O.sub.2 in an open water bath shaker for 40 h. The gas was discharged into the head space through a sparger with a pore size of 10 m, which was mounted in the center of the reactors. Culturing was carried out with no pH control. During cultivation several 5 mL samples were taken to determinate OD.sub.600nm, pH und product formation. The determination of the product concentrations was performed by semiquantitative 1H-NMR spectroscopy. As an internal quantification standard sodium trimethylsilylpropionate (T(M)SP) was used. Also the dissolved oxygen in the cultivation medium was measured online by oxygen dipping probes (PSt6 with Oxy4Trace, Presens, Germany).

[0100] During the cultivation period cell growth was observed in complex medium by an increase of the OD.sub.600nm from 0.20 to 0.36, which correlates with a growth rate of p=0.015 h.sup.1. In mineral medium, the OD.sub.600nm decreased from 0.20 to 0.19. In complex medium the concentration of acetate increased from 29 mg/L to 280 mg/L, for ethanol from 3 mg/L to 82 mg/L, for butyrate from 0 mg/L to 29 mg/L and for butanol from 0 mg/L to 10 mg/L. In mineral medium the concentration of acetate increased from 25 mg/L to 110 mg/L, for ethanol from 3 mg/L to 5 mg/L and for butyrate from 0 mg/L to 2 mg/L. Over the whole cultivation period the dissolved oxygen concentration in both cultures was 0.00 mg/L. In a similar technical setting with the same parameters (medium composition, volume, bottle, gas, ventilation rate, temperature, shaking frequency), but without cells in the medium, a dissolved oxygen concentration of 0.01 mg/L was measured in both media.

Example 6

[0101] Growth and Production of Acetate by Acetobacterium Woodii on Synthesis Gas with Oxygen

[0102] For the biotransformation of hydrogen and carbon dioxide to acetic acid the homoacetogenic bacterium Acetobacterium woodii is cultivated on synthesis gas with oxygen. All cultivation steps are carried out under anaerobic conditions in pressure-resistant glass bottles that can be closed airtight with a butyl rubber stopper. For the preculture 500 ml medium (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-hydrochloride and 400 mg/L Na.sub.2S9H.sub.2O are inoculated with 5 mL of a frozen cryo stock of A. woodii. The chemolithoautotrophic cultivation is carried out in a 1 L pressure-resistant glass bottle at 37 C., 100 rpm and a ventilation rate of 3 L/h with a premixed gas with 67% H.sub.2, 33% CO.sub.2 in an open water bath shaker for 72 h. The gas is discharged into the medium through a sparger with a pore size of 10 m, which is mounted in the center of the reactors. Culturing is carried out with no pH control.

[0103] After the precultivation, the cell suspension is centrifuged (10 min, 4200 rpm) and the pellet is resuspended in fresh medium. For the main culture, as many cells from the preculture as necessary for an OD.sub.600nm of 0.1 are transferred in 500 mL medium with additional 400 mg/L L-cysteine-hydrochlorid. The chemolithoautotrophic cultivation is carried out in a 1 L pressure-resistant glass bottle at 37 C., 150 rpm and a ventilation rate of 1 L/h with a premixed gas with 66.95% H.sub.2, 33% CO.sub.2, 0.05% O.sub.2 in an open water bath shaker for 41 h. The gas is discharged into the medium through a sparger with a pore size of 10 m, which is mounted in the center of the reactors. Culturing is carried out with no pH control. During cultivation several 5 mL samples are taken to determinate OD.sub.600nm, pH und product formation. The determination of the product concentrations is performed by semiquantitative 1H-NMR spectroscopy. As an internal quantification standard sodium trimethylsilylpropionate (T(M)SP) is used. Also the dissolved oxygen in the cultivation medium is measured online by oxygen dipping probes (PSt6 with Oxy4Trace, Presens, Germany).

[0104] During the cultivation period cell growth is observed by an increase of the OD.sub.600nm. Also the concentration of acetate increases.

[0105] In a similar technical setting with the same parameters (medium composition, volume, bottle, gas, ventilation rate, temperature, shaking frequency), but without cells in the medium, a dissolved oxygen concentration of 0.01 mg/L is measured.

Example 7

[0106] Production of Propionic Acid and Propanol with Clostridium neopropionicum on Synthesis Gas

[0107] For the biotransformation of ethanol and carbon dioxide to propionic acid and propanol the bacterium Clostridium neopropionicum was cultivated 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.

[0108] 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 250 ml pressure resistant bottles 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 cultures were incubated at 30 C. and 100 rpm in an open water bath shaker for 24 h to an OD.sub.600nm 0.14-0.16.

[0109] For a second preculture of C. neopropionicum 3200 ml of fresh DSMZ318 medium in 500 ml pressure resistant bottles 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. These growing culture were incubated at 30 C. and 100 rpm in an open water bath shaker for 22 h to an OD.sub.600nm 0.24.

[0110] 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 KAI(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% H.sub.2, 33% CO.sub.2 in an open water bath shaker for 114 h. The pH was held at 6.8 by automatic addition of NaOH solution (100 g/L).

[0111] During cultivation several 5 mL samples were taken to determinate OD.sub.600nm, pH und 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.

[0112] During the cultivation the concentration of propionate increased from 0.03 g/L to 2.65 g/L, for propanol from 0.007 to 0.45 g/L, for butyrate from 0.003 to 0.38 g/L and for lactate from 0 g/L to 0.17 g/L. The concentration of ethanol decreased from 10.4 g/L to 5.7 g/L during this time.

Example 8

[0113] Production of Propionic Acid and Propanol with Clostridium neopropionicum on Synthesis Gas with 0.05% Oxygen

[0114] For the biotransformation of ethanol and carbon dioxide to propionic acid and propanol the bacterium Clostridium neopropionicum was cultivated 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.

[0115] 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 250 ml pressure resistant bottles 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 cultures were incubated at 30 C. and 100 rpm in an open water bath shaker for 24 h to an OD.sub.600nm 0.14-0.16.

[0116] For a second preculture of C. neopropionicum 3200 ml of fresh DSMZ318 medium in 500 ml pressure resistant bottles 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. These growing culture were incubated at 30 C. and 100 rpm in an open water bath shaker for 22 h to an OD.sub.600nm 0.24.

[0117] 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 KAI(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, 33% CO.sub.2, 0.05% O.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% H.sub.2, 33% CO.sub.2, 0.05% O.sub.2 in an open water bath shaker for 114 h. The pH was held at 6.8 by automatic addition of NaOH solution (100 g/L).

[0118] During cultivation several 5 mL samples were taken to determinate OD.sub.600nm pH und 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.

[0119] During the cultivation the concentration of propionate increased from 0.03 g/L to 3 g/L, for propanol from 0.009 to 0.4 g/L, for butyrate from 0.003 to 0.44 g/L and for lactate from 0 g/L to 0.19 g/L. The concentration of ethanol decreased from 10.8 g/L to 5.2 g/L during this time.

Example 9

[0120] Production of Propionic Acid and Propanol with Clostridium neopropionicum on Synthesis Gas with Spent Medium from a Gas Fermentation with C. autoethanogenum

[0121] For the biotransformation of hydrogen and carbon dioxide to propanol the homoacetogenic bacterium Clostridium autoethanogenum was cultivated on synthesis gas with a subsequent cultivation step with Clostridium neopropionicum. All cultivation steps were carried out under anaerobic conditions in pressure-resistant glass bottles that can be closed airtight with a butyl rubber stopper.

[0122] For the cultivation of C. autoethanogenum 500 ml medium (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 and 400 mg/L Na.sub.2S9H.sub.2O were inoculated with 5 mL of a frozen cryo stock. The chemolithoautotrophic cultivation was carried out in 1 L pressure-resistant glass bottle at 37 C., 100 rpm and a ventilation rate of 1 L/h with a premixed gas with 67% H.sub.2 and 33% CO.sub.2 in an open water bath shaker for 70 h till OD.sub.600nm of 0.56. The gas was discharged into the medium through a sparger with a pore size of 10 m, which was mounted in the center of the reactors. Then the cell suspension was centrifuged.

[0123] For the main culture of C. autoethanogenum 500 ml LM33 mineral medium (pH=5.9, 1.3 g/L KOH, 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 KAI(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 500 mg/L L-cysteine-hydrochlorid and 0.5 mg/L resazurin) were inoculated with cells from the first preculture to an OD.sub.600nm of 0.09. The cultivation was carried out in a 1 L pressure-resistant glass bottle at 37 C., 150 rpm in a water bath shaker, with manual pH adjustment to pH 5.8-5.9 at 19, 42, 67 and 93 h of incubation using 100 g/L NaOH. Premixed gas with 67% H.sub.2, 33% CO.sub.2 was discharged at 1 L/h into the medium through a sparger with a pore size of 10 m, which was mounted in the center of the reactor. During 187 h 3.3 g/L ethanol and 7.9 g/L acetate were produced. After cultivation, the medium was sterile-filtered and used for the main culture of Clostridium neopropionicum.

[0124] For the biotransformation of ethanol and carbon dioxide to propionic acid and propanol the bacterium Clostridium neopropionicum was cultivated 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.

[0125] 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 250 ml pressure resistant bottles 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 cultures were incubated at 30 C. and 100 rpm in an open water bath shaker for 24 h to an OD.sub.600nm 0.14-0.16.

[0126] For a second preculture of C. neopropionicum 3200 ml of fresh DSMZ318 medium in 500 ml pressure resistant bottles 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. These growing cultures were incubated at 30 C. and 100 rpm in an open water bath shaker for 22 h to an OD.sub.600nm 0.24.

[0127] 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 spent medium from the main culture of C. autoethanogenum. 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 114 h. The pH was held at 6.8 by automatic addition of NaOH solution (100 g/L).

[0128] During cultivation several 5 mL samples were taken to determinate OD.sub.600nm, pH und 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.

[0129] During the cultivation the concentration of propionate increased from 0.03 g/L to 2.65 g/L, for propanol from 0.006 to 0.07 g/L, for butyrate from 0.003 to 0.16 g/L and for lactate from 0 g/L to 0.035 g/L. The concentration of ethanol decreased from 3.3 g/L to 0.15 g/L during this time.

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