METHOD FOR PRODUCING HIGHER LINEAR ALKANES

Abstract

The present invention relates to a method of producing higher linear alkanes using a combined biotechnological and chemical method. In particular, the present invention relates to producing linear alkanes comprising 7 to 28 carbon atoms, preferably undecane, via higher alkanones, i.e. linear alkanones comprising 7 to 28 carbon atoms, preferably 6-undecanone.

Claims

1. A method of producing linear alkanes comprising 7 to 28 carbon atoms, from ethanol and/or linear alkanoic acids comprising 2 to 5 carbon atoms, the method comprising (a) contacting ethanol and/or a linear alkanoic acid comprising 2 to 5 carbon atoms or any salts thereof with at least one microorganism capable of carrying out two-carbon chain elongation to produce as an intermediate a linear alkanoic acid comprising 4 to 7 carbon atoms and/or a salt thereof and/or an ester thereof; (b) extracting the intermediate, its salt and/or ester thereof from (a) using at least one extractant, wherein the extractant comprises at least one alkyl-phosphine oxide and optionally at least one alkane comprising at least 12 carbon atoms; or at least one trialkylamine and at least one alkane comprising at least 12 carbon atoms; or a branched higher alcohol like 2 octyl-dodecanol; (c) contacting the extracted intermediate and/or ester thereof from (b) and optionally a further linear alkanoic acid comprising 1 to 22 carbon atoms with at least one ketonization catalyst to a linear alkanone comprising 7 to 28 carbon atoms; (d) contacting the linear alkanone comprising 7 to 28 carbon atoms from step (c) with at least one hydrogenation metal catalyst for catalytic hydrogenation of the linear alkanone comprising 7 to 28 carbon atoms to a corresponding secondary linear alkanol comprising 7 to 28 carbon atoms; (e) dehydration of the secondary linear alkanol comprising 7 to 28 carbon atoms in the presence of an acidic heterogeneous catalyst to form a corresponding linear alkene comprising 7 to 28 carbon atoms; and (f) contacting the linear alkene comprising 7 to 28 carbon atoms obtained in step (e) with at least one hydrogenation metal catalyst for catalytic hydrogenation to a corresponding secondary linear alkane comprising 7 to 28 carbon atoms; or combining step (e) and (f) into one single step using a catalyst or catalyst mixture providing dehydrating and hydrogenating properties, thus hydrogenolysing the secondary linear alkanol comprising 7 to 28 carbon atoms obtained in step (d) directly to the corresponding linear alkane comprising 7 to 28 carbon atoms.

2. The method according to claim 1, wherein the ketonization catalyst of (c) is a metal oxide catalyst or mixtures thereof.

3. The method according to claim 2, wherein the metal oxide catalyst or mixtures thereof is selected from the group consisting of heteropoly acid (H.sub.3PW.sub.12O.sub.40) catalyst, titanium oxide (TiO.sub.2) catalyst, cerium oxide (CeO.sub.2) catalyst, zinc-chromium (ZnCr) mixed oxide catalyst, manganese oxide (MnO.sub.2) catalyst, lanthanum oxide (La.sub.2O.sub.3) catalyst, magnesium oxide (MgO) catalyst, iron oxide (FeO, FeO.sub.2, Fe.sub.2O.sub.3, Fe.sub.3O.sub.4, Fe.sub.4O.sub.5, Fe.sub.5O.sub.6, Fe.sub.5O.sub.7), silicon-aluminium (SiAl) mixed oxide catalyst and zirconia (ZrO.sub.2) catalyst.

4. The method according to claim 1, wherein the ketonization catalyst in step (c) is manganese oxide (MnO.sub.2) catalyst, or magnesium oxide (MgO) catalyst,.

5. The method according to claim 1, wherein the suitable reaction conditions of step (c) comprises reaction temperatures of 150? C.-350? C.

6. The method according to claim 1, wherein the microorganism in (a) is selected from the group consisting of Clostridium carboxidivorans and Clostridium kluyveri.

7. The method according to claim 1, wherein the alkyl-phosphine oxide is selected from the group consisting of trioctylphosphine oxide, octylphosphine oxide and mixtures thereof and the alkane is selected from the group consisting of pentadecane, hexadecane, heptadecane, octadecane, tetradecane and mixtures thereof.

8. The method according to claim 7, wherein the weight ratio of TOPO to mixture of alkanes is between 1:100 to 1:10.

9. The method according to claim 1, wherein the pH of the aqueous medium in (b) is maintained between 5.5 and 8.

10. The method according to claim 1, wherein the hydrogenation metal catalyst of step (d) is selected from the group consisting of ruthenium (Ru) catalyst, rhenium (Re) catalyst, nickel (Ni) catalyst, iron (Fe), cobalt (Co) and platinum (Pt) catalyst.

11. The method according to claim 1, wherein the acidic heterogeneous catalyst of step (e) is a catalyst comprising an aluminosilicate zeolite.

12. The method according of claim 11, wherein the acidic heterogeneous catalyst of step (e) comprises ZSM-5.

13. The method according to claim 1, wherein the catalyst of step (e) further comprises a transition metal.

14. The method of claim 13, wherein the transition metal is molybdenum.

Description

EXAMPLES

Example 1

Clostridium kluyveri Forming Hexanoic Acid From Acetate and Ethanol

[0092] For the biotransformation of ethanol and acetate to hexanoic acid the bacterium Clostridium kluyveri was used. All cultivation steps were carried out under anaerobic conditions in pressure-resistant glass bottles that can be closed airtight with a butyl rubber stopper.

[0093] For the preculture 100 ml of DMSZ52 medium (pH=7.0; 10 g/L K-acetate, 0.31 g/L K.sub.2HPO.sub.4, 0.23 g/L KH.sub.2PO.sub.4, 0.25 g/l NH.sub.4Cl, 0.20 g/l MgSO.sub.4?7 H.sub.2O, 1 g/L yeast extract, 0.50 mg/L resazurin, 10 ?l/I HCl (25%, 7.7 M), 1.5 mg/L FeCl.sub.2?4H.sub.2O, 70 ?g/L ZnCl.sub.2?7H.sub.2O, 100 ?g/L MnCl.sub.2?4H.sub.2O, 6 ?g/L H.sub.3BO.sub.3, 190 ?g/L CoCl.sub.2?6H.sub.2O, 2 ?g/L CuCl.sub.2?6H.sub.2O, 24 ?g/L NiCl.sub.2?6H.sub.2O, 36 ?g/L Na.sub.2MO.sub.4?2H.sub.2O, 0.5 mg/L NaOH, 3 ?g/L Na.sub.2SeO.sub.3?5H.sub.2O, 4 ?g/L Na.sub.2WO.sub.4?2H.sub.2O, 100 ?g/L vitamin B12, 80 ?g/L p-aminobenzoic acid, 20 ?g/L D(+) Biotin, 200 ?g/L nicotinic acid, 100 ?g/L D-Ca-pantothenate, 300 ?g/L pyridoxine hydrochloride, 200 ?g/l thiamine HCl?2H.sub.2O, 20 ml/L ethanol, 2.5 g/L NaHCO.sub.3, 0.25 g/L cysteine-HCl?H.sub.2O, 0.25 g/L Na.sub.2S?9H.sub.2O) in a 250 ml bottle were inoculated with 5 ml of a frozen cryoculture of Clostridium kluyveri and incubated at 37? C. for 144 h to an OD.sub.600nm>0.2.

[0094] For the main culture 200 ml of fresh DMSZ52 medium in a 500 ml bottle were inoculated with centrifuged cells from the preculture to an OD.sub.600nm of 0.1. This growing culture was incubated at 37? C. for 27 h to an OD.sub.600nm>0.6. Then the cell suspension was centrifuged, washed with production buffer (pH 6.0; 0.832 g/L K-acetate, 5.0 g/l ethanol) and centrifuged again.

[0095] For the production culture, 200 ml of production buffer in a 500 ml bottle was inoculated with the washed cells from the main culture to an OD.sub.600nm of 0.2. The culture was capped with a butyl rubber stopper and incubated for 71 h at 37? C. and 100 rpm in an open water shaking bath. At the start and end of the culturing period, samples were taken. These were tested for optical density, pH and the different analytes (tested by NMR).

[0096] The results showed that in the production phase the amount of acetate decreased from 0.54 g/l to 0.03 g/l and the amount of ethanol decreased from 5.6 g/l to 4.9 g/l. Also, the concentration of butyric acid was increased from 0.05 g/l to 0.28 g/l and the concentration of hexanoic acid was increased from 0.03 g/l to 0.79 g/l.

Example 2

Clostridium kluyveri Forming Hexanoic Acid From Butyric Acid and Ethanol

[0097] For the biotransformation of ethanol and butyric acid to hexanoic acid the bacterium Clostridium kluyveri was used. 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 100 ml of DMSZ52 medium (pH=7.0; 10 g/L K-acetate, 0.31 g/L K.sub.2HPO.sub.4, 0.23 g/L KH.sub.2PO.sub.4, 0.25 g/l NH.sub.4Cl, 0.20 g/l MgSO.sub.4?7 H.sub.2O, 1 g/L yeast extract, 0.50 mg/L resazurin, 10 ?l/l HCl (25%, 7.7 M), 1.5 mg/L FeCl.sub.2?4H.sub.2O, 70 ?g/L ZnCl.sub.2?7H.sub.2O, 100 ?g/L MnCl.sub.2?4H.sub.2O, 6 ?g/L H.sub.3BO.sub.3, 190 ?g/L CoCl.sub.2?6H.sub.2O, 2 ?g/L CuCl.sub.2?6H.sub.2O, 24 ?g/L NiCl.sub.2?6H.sub.2O, 36 ?g/L Na.sub.2MO.sub.4?2H.sub.2O, 0.5 mg/L NaOH, 3 ?g/L Na.sub.2SeO.sub.3?5H.sub.2O, 4 ?g/L Na.sub.2WO.sub.4?2H.sub.2O, 100 ?g/L vitamin B12, 80 ?g/L p-aminobenzoic acid, 20 ?g/L D(+) Biotin, 200 ?g/L nicotinic acid, 100 ?g/L D-Ca-pantothenate, 300 ?g/L pyridoxine hydrochloride, 200 ?g/l thiamine HCl?2H.sub.2O, 20 ml/L ethanol, 2.5 g/L NaHCO.sub.3, 0.25 g/L cysteine-HCl?H.sub.2O, 0.25 g/L Na.sub.2S?9H.sub.2O) in a 250 ml bottle were inoculated with 5 ml of a frozen cryoculture of Clostridium kluyveri and incubated at 37? C. for 144 h to an OD.sub.600nm>0.3.

[0099] For the main culture 200 ml of fresh DMSZ52 medium in a 500 ml bottle were inoculated with centrifuged cells from the preculture to an OD.sub.600nm of 0.1. This growing culture was incubated at 37? C. for 25 h to an OD.sub.600nm>0.4. Then the cell suspension was centrifuged, washed with production buffer (pH 6.16; 4.16 g/L K-acetate, 10.0 g/l ethanol) and centrifuged again.

[0100] For the production cultures, 200 ml of production buffer in a 500 ml bottle was inoculated with the washed cells from the main culture to an OD.sub.600nm of 0.2. In a first culture, at the beginning 1.0 g/l butyric acid was added to the production buffer, in a second culture, no butyric acid was added to the production buffer. The cultures were capped with a butyl rubber stopper and incubated for 71 h at 37? C. and 100 rpm in an open water shaking bath. At the start and end of the culturing period, samples were taken. These were tested for optical density, pH and the different analytes (tested by NMR).

[0101] The results showed that in the production phase of the butyric acid supplemented culture the amount of acetate decreased from 3.1 g/l to 1.1 g/l and the amount of ethanol decreased from 10.6 g/l to 7.5 g/l. Also, the concentration of butyric acid was increased from 1.2 g/l to 2.2 g/l and the concentration of hexanoic acid was increased from 0.04 g/l to 2.30 g/l.

[0102] In the production phase of the non-supplemented culture the amount of acetate decreased from 3.0 g/l to 1.3 g/l and the amount of ethanol decreased from 10.2 g/l to 8.2 g/l. Also, the concentration of butyric acid was increased from 0.1 g/l to 1.7 g/l and the concentration of hexanoic acid was increased from 0.01 g/l to 1.40 g/l.

Example 3

Cultivation of Clostridium kluyveri in Presence of Decane and TOPO

[0103] The bacterium Clostridium kluyveri was cultivated for the biotransformation of ethanol and acetate to hexanoic acid. For the inSitu extraction of the produced hexanoic acid a mixture of decane with trioctylphosphineoxide (TOPO) was added to the cultivation. All cultivation steps were carried out under anaerobic conditions in pressure-resistant glass bottles that can be closed airtight with a butyl rubber stopper.

[0104] For the preculture 250 ml of Veri01 medium (pH 7.0; 10 g/L potassium acetate, 0.31 g/L K.sub.2HPO.sub.4, 0.23 g/L KH.sub.2PO.sub.4, 0.25 g/L NH.sub.4Cl, 0.20 g/L MgSO.sub.4?7 H.sub.2O, 10 ?l/L HCl (7.7 M), 1.5 mg/L FeCl.sub.2?4 H.sub.2O, 36 ?g/L ZnCl.sub.2, 64 ?g/L MnCl.sub.2?4 H.sub.2O, 6 ?g/L H.sub.3BO.sub.3, 190 ?g/L CoCl.sub.2?6 H.sub.2O, 1.2 ?g/L CuCl.sub.2?6 H.sub.2O, 24 ?g/L NiCl.sub.2?6 H.sub.2O, 36 ?g/L Na.sub.2MO.sub.4?2 H.sub.2O, 0.5 mg/L NaOH, 3 ?g/L Na.sub.2SeO.sub.3?5 H.sub.2O, 4 ?g/L Na.sub.2WO.sub.4?2 H.sub.2O, 100 ?g/L vitamin B12, 80 ?g/L p-aminobenzoic acid, 20 ?g/L D(+) Biotin, 200 ?g/L nicotinic acid, 100 ?g/L D-Ca-pantothenate, 300 ?g/L pyridoxine hydrochloride, 200 ?g/l thiamine-HCl?2H.sub.2O, 20 ml/L ethanol, 2.5 g/L NaHCO.sub.3, 65 mg/L glycine, 24 mg/L histidine, 64.6 mg/L isoleucine, 93.8 mg/L leucine, 103 mg/L lysine, 60.4 mg/L arginine, 21.64 mg/L L-cysteine-HCl, 21 mg/L methionine, 52 mg/L proline, 56.8 mg/L serine, 59 mg/L threonine, 75.8 mg/L valine) were inoculated with 10 ml of a living culture of Clostridium kluyveri to a start OD.sub.600nm of 0.1.

[0105] The cultivation was carried out in a 1000 mL pressure-resistant glass bottle at 37? C., 150 rpm and a ventilation rate of 1 L/h with 100% CO.sub.2 in an open water bath shaker for 671 h. The gas was discharged into the headspace of the reactor. The pH was hold at 6.2 by automatic addition of 100 g/L NaOH solution. Fresh medium was continuously fed to the reactor with a dilution rate of 2.0 d.sup.?1 and fermentation broth continuously removed from the reactor through a KrosFlo? hollow fibre polyethersulfone membrane with a pore size of 0.2 ?m (Spectrumlabs, Rancho Dominguez, USA) to retain the cells in the reactor.

[0106] For the main culture 100 ml of fresh Veri01 medium in a 250 ml bottle was inoculated with centrifuged cells from the preculture to an OD.sub.600nm of 0.1. Additional 1 ml of a mixture of 6% (w/w) TOPO in decane was added. The culture was capped with a butyl rubber stopper and incubated at 37? C. and 150 rpm in an open water bath shaker for 43 h under 100% CO.sub.2 atmosphere. 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 1H-NMR spectroscopy. As an internal quantification standard sodium trimethylsilylpropionate (T(M)SP) was used.

[0107] During the main cultivation the concentration of butyrate increased from 0.14 g/L to 2.12 g/L and the concentration of hexanoate increased from 0.22 g/L to 0.91 g/L, whereas the concentration of ethanol decreased from 15.04 to 11.98 g/l and the concentration of acetate decreased from 6.01 to 4.23 g/L.

[0108] The OD.sub.600nm decreased during this time from 0.111 to 0.076.

Example 4

Cultivation of Clostridium kluyveri in Presence of Tetradecane and TOPO

[0109] The bacterium Clostridium kluyveri was cultivated for the biotransformation of ethanol and acetate to hexanoic acid. For the inSitu extraction of the produced hexanoic acid a mixture of tetradecane with trioctylphosphineoxide (TOPO) was added to the cultivation. All cultivation steps were carried out under anaerobic conditions in pressure-resistant glass bottles that can be closed airtight with a butyl rubber stopper.

[0110] The precultivation of Clostridium kluyveri was carried out in a 1000 mL pressure-resistant glass bottle in 250 ml of EvoDM24 medium (pH 5.5; 0.429 g/L Mg-acetate, 0.164 g/l Na-acetate, 0.016 g/L Ca-acetate, 2.454 g/l K-acetate, 0.107 mL/L H.sub.3PO.sub.4 (8.5%), 0.7 g/L NH.sub.4acetate, 0.35 mg/L Co-acetate, 1.245 mg/L Ni-acetate, 20 ?g/L d-biotin, 20 ?g/L folic acid, 10 ?g/L pyridoxine-HCl, 50 ?g/L thiamine-HCl, 50 ?g/L Riboflavin, 50 ?g/L nicotinic acid, 50 ?g/L Ca-pantothenate, 50 ?g/L Vitamin B12, 50 ?g/L p-aminobenzoate, 50 ?g/L lipoic acid, 0.702 mg/L (NH4).sub.2Fe(SO.sub.4).sub.2?4 H.sub.2O, 1 ml/L KS-acetate (93.5 mM), 20 mL/L ethanol, 0.37 g/L acetic acid) at 37? C., 150 rpm and a ventilation rate of 1 L/h with a mixture of 25% CO.sub.2 and 75% N.sub.2 in an open water bath shake. The gas was discharged into the headspace of the reactor. The pH was hold at 5.5 by automatic addition of 2.5 M NH.sub.3 solution. Fresh medium was continuously feeded to the reactor with a dilution rate of 2.0 d.sup.?1 and fermentation broth continuously removed from the reactor through a KrosFlo? hollow fibre polyethersulfone membrane with a pore size of 0.2 ?m (Spectrumlabs, Rancho Dominguez, USA) to retain the cells in the reactor and hold an OD.sub.600nm of ?1.5.

[0111] For the main culture 100 ml of Veri01 medium (pH 6.5; 10 g/L potassium acetate, 0.31 g/L K.sub.2HPO.sub.4, 0.23 g/L KH.sub.2PO.sub.4, 0.25 g/L NH.sub.4Cl, 0.20 g/L MgSO.sub.4?7 H.sub.2O, 10 ?l/L HCl (7.7 M), 1.5 mg/L FeCl.sub.2?4 H.sub.2O, 36 ?g/L ZnCl.sub.2, 64 ?g/L MnCl.sub.2?4 H.sub.2O, 6 ?g/L H.sub.3BO.sub.3, 190 ?g/L CoCl.sub.2?6 H.sub.2O, 1.2 ?g/L CuCl.sub.2?6 H.sub.2O, 24 ?g/L NiCl.sub.2?6 H.sub.2O, 36 ?g/L Na.sub.2MO.sub.4?2 H.sub.2O, 0.5 mg/L NaOH, 3 ?g/L Na.sub.2SeO.sub.3?5 H.sub.2O, 4 g/L Na.sub.2WO.sub.4?2 H.sub.2O, 100 ?g/L vitamin B12, 80 ?g/L p-aminobenzoic acid, 20 ?g/L D(+) Biotin, 200 g/L nicotinic acid, 100 ?g/L D-Ca-pantothenate, 300 ?g/L pyridoxine hydrochloride, 200 ?g/l thiamine-HCl?2H.sub.2O, 20 ml/L ethanol, 2.5 g/L NaHCO.sub.3, 65 mg/L glycine, 24 mg/L histidine, 64.6 mg/L isoleucine, 93.8 mg/L leucine, 103 mg/L lysine, 60.4 mg/L arginine, 21.64 mg/L L-cysteine-HCl, 21 mg/L methionine, 52 mg/L proline, 56.8 mg/L serine, 59 mg/L threonine, 75.8 mg/L valine, 2.5 mL/L HCL 25%) in a 250 ml bottle were inoculated with centrifuged cells from the preculture to an OD.sub.600nm of 0.1. Additional 1 ml of a mixture of 6% (w/w) TOPO in tetradecane was added. The culture was capped with a butyl rubber stopper and incubated at 37? C. and 150 rpm in an open water bath shaker for 47 h under 100% CO.sub.2 atmosphere.

[0112] 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.

[0113] During the main cultivation the concentration of butyrate increased from 0.05 g/L to 3.78 g/L and the concentration of hexanoate increased from 0.09 g/L to 4.93 g/L, whereas the concentration of ethanol decreased from 15.52 to 9.36 g/l and the concentration of acetate decreased from 6.36 to 2.49 g/L.

[0114] The OD.sub.600nm increased during this time from 0.095 to 0.685.

Example 5

Cultivation of Clostridium kluyveri in Presence of Hexadecane and TOPO

[0115] The bacterium Clostridium kluyveri was cultivated for the biotransformation of ethanol and acetate to hexanoic acid. For the inSitu extraction of the produced hexanoic acid a mixture of hexadecane with trioctylphosphineoxide (TOPO) was added to the cultivation. All cultivation steps were carried out under anaerobic conditions in pressure-resistant glass bottles that can be closed airtight with a butyl rubber stopper.

[0116] For the preculture 250 ml of Veri01 medium (pH 7.0; 10 g/L potassium acetate, 0.31 g/L K.sub.2HPO.sub.4, 0.23 g/L KH.sub.2PO.sub.4, 0.25 g/L NH.sub.4Cl, 0.20 g/L MgSO.sub.4?7 H.sub.2O, 10 ?l/L HCl (7.7 M), 1.5 mg/L FeCl.sub.2?4 H.sub.2O, 36 ?g/L ZnCl.sub.2, 64 ?g/L MnCl.sub.2?4 H.sub.2O, 6 ?g/L H.sub.3BO.sub.3, 190 ?g/L CoCl.sub.2?6 H.sub.2O, 1.2 ?g/L CuCl.sub.2?6 H.sub.2O, 24 ?g/L NiCl.sub.2?6 H.sub.2O, 36 ?g/L Na.sub.2MO.sub.4?2 H.sub.2O, 0.5 mg/L NaOH, 3 ?g/L Na.sub.2SeO.sub.3?5 H.sub.2O, 4 ?g/L Na.sub.2WO.sub.4?2 H.sub.2O, 100 ?g/L vitamin B12, 80 ?g/L p-aminobenzoic acid, 20 ?g/L D(+) Biotin, 200 ?g/L nicotinic acid, 100 ?g/L D-Ca-pantothenate, 300 ?g/L pyridoxine hydrochloride, 200 ?g/l thiamine-HCl?2H.sub.2O, 20 ml/L ethanol, 2.5 g/L NaHCO.sub.3, 65 mg/L glycine, 24 mg/L histidine, 64.6 mg/L isoleucine, 93.8 mg/L leucine, 103 mg/L lysine, 60.4 mg/L arginine, 21.64 mg/L L-cysteine-HCl, 21 mg/L methionine, 52 mg/L proline, 56.8 mg/L serine, 59 mg/L threonine, 75.8 mg/L valine) were inoculated with 10 ml of a living culture of Clostridium kluyveri to a start OD.sub.600nm of 0.1.

[0117] The cultivation was carried out in a 1000 mL pressure-resistant glass bottle at 37? C., 150 rpm and a ventilation rate of 1 L/h with 100% CO.sub.2 in an open water bath shaker for 671 h. The gas was discharged into the headspace of the reactor. The pH was hold at 6.2 by automatic addition of 100 g/L NaOH solution. Fresh medium was continuously fed to the reactor with a dilution rate of 2.0 d.sup.?1 and fermentation broth continuously removed from the reactor through a KrosFlo? hollow fibre polyethersulfone membrane with a pore size of 0.2 ?m (Spectrumlabs, Rancho Dominguez, USA) to retain the cells in the reactor.

[0118] For the main culture 100 ml of fresh Veri01 medium in a 250 ml bottle was inoculated with centrifuged cells from the preculture to an OD.sub.600nm of 0.1. Additional 1 ml of a mixture of 6% (w/w) TOPO in hexadecane was added. The culture was capped with a butyl rubber stopper and incubated at 37? C. and 150 rpm in an open water bath shaker for 43 h under 100% CO.sub.2 atmosphere.

[0119] 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 1H-NMR spectroscopy. As an internal quantification standard sodium trimethylsilylpropionate (T(M)SP) was used.

[0120] During the main cultivation the concentration of butyrate increased from 0.14 g/L to 2.86 g/L and the concentration of hexanoate increased from 0.20 g/L to 2.37 g/L, whereas the concentration of ethanol decreased from 14.59 to 10.24 g/l and the concentration of acetate decreased from 5.87 to 3.32 g/L.

[0121] The OD.sub.600nm increased during this time from 0.091 to 0.256.

Example 6

Cultivation of Clostridium kluyveri in Presence of Heptadecane and TOPO

[0122] The bacterium Clostridium kluyveri was cultivated for the biotransformation of ethanol and acetate to hexanoic acid. For the inSitu extraction of the produced hexanoic acid a mixture of heptadecane with trioctylphosphineoxide (TOPO) was added to the cultivation. All cultivation steps were carried out under anaerobic conditions in pressure-resistant glass bottles that can be closed airtight with a butyl rubber stopper.

[0123] For the preculture 250 ml of Veri01 medium (pH 7.0; 10 g/L potassium acetate, 0.31 g/L K.sub.2HPO.sub.4, 0.23 g/L KH.sub.2PO.sub.4, 0.25 g/L NH.sub.4Cl, 0.20 g/L MgSO.sub.4?7 H.sub.2O, 10 ?l/L HCl (7.7 M), 1.5 mg/L FeCl.sub.2?4 H.sub.2O, 36 ?g/L ZnCl.sub.2, 64 ?g/L MnCl.sub.2?4 H.sub.2O, 6 ?g/L H.sub.3BO.sub.3, 190 ?g/L CoCl.sub.2?6 H.sub.2O, 1.2 ?g/L CuCl.sub.2?6 H.sub.2O, 24 ?g/L NiCl.sub.2?6 H.sub.2O, 36 ?g/L Na.sub.2MO.sub.4?2 H.sub.2O, 0.5 mg/L NaOH, 3 ?g/L Na.sub.2SeO.sub.3?5 H.sub.2O, 4 ?g/L Na.sub.2WO.sub.4?2 H.sub.2O, 100 ?g/L vitamin B12, 80 ?g/L p-aminobenzoic acid, 20 ?g/L D(+) Biotin, 200 ?g/L nicotinic acid, 100 ?g/L D-Ca-pantothenate, 300 ?g/L pyridoxine hydrochloride, 200 ?g/l thiamine-HCl?2H.sub.2O, 20 ml/L ethanol, 2.5 g/L NaHCO.sub.3, 65 mg/L glycine, 24 mg/L histidine, 64.6 mg/L isoleucine, 93.8 mg/L leucine, 103 mg/L lysine, 60.4 mg/L arginine, 21.64 mg/L L-cysteine-HCl, 21 mg/L methionine, 52 mg/L proline, 56.8 mg/L serine, 59 mg/L threonine, 75.8 mg/L valine) were inoculated with 10 ml of a living culture of Clostridium kluyveri to a start OD.sub.600nm of 0.1.

[0124] The cultivation was carried out in a 1000 mL pressure-resistant glass bottle at 37? C., 150 rpm and a ventilation rate of 1 L/h with 100% CO.sub.2 in an open water bath shaker for 671 h. The gas was discharged into the headspace of the reactor. The pH was hold at 6.2 by automatic addition of 100 g/L NaOH solution. Fresh medium was continuously feeded to the reactor with a dilution rate of 2.0 d.sup.?1 and fermentation broth continuously removed from the reactor through a KrosFlo? hollow fibre polyethersulfone membrane with a pore size of 0.2 ?m (Spectrumlabs, Rancho Dominguez, USA) to retain the cells in the reactor.

[0125] For the main culture 100 ml of fresh Veri01 medium in a 250 ml bottle were inoculated with centrifuged cells from the preculture to an OD.sub.600nm of 0.1. Additional 1 ml of a mixture of 6% (w/w) TOPO in heptadecane was added. The culture was capped with a butyl rubber stopper and incubated at 37? C. and 150 rpm in an open water bath shaker for 43 h under 100% CO.sub.2 atmosphere.

[0126] 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.

[0127] During the main cultivation the concentration of butyrate increased from 0.15 g/L to 2.82 g/L and the concentration of hexanoate increased from 0.19 g/L to 2.85 g/L, whereas the concentration of ethanol decreased from 14.34 to 9.58 g/l and the concentration of acetate decreased from 5.88 to 3.20 g/L.

[0128] The OD.sub.600nm increased during this time from 0.083 to 0.363.

Example 7

Cultivation of Clostridium kluyveri in Presence of Dodecane and TOPO

[0129] The bacterium Clostridium kluyveri was cultivated for the biotransformation of ethanol and acetate to hexanoic acid. For the inSitu extraction of the produced hexanoic acid a mixture of dodecane with trioctylphosphineoxide (TOPO) was added to the cultivation. All cultivation steps were carried out under anaerobic conditions in pressure-resistant glass bottles that can be closed airtight with a butyl rubber stopper.

[0130] For the preculture 250 ml of Veri01 medium (pH 7.0; 10 g/L potassium acetate, 0.31 g/L K.sub.2HPO.sub.4, 0.23 g/L KH.sub.2PO.sub.4, 0.25 g/L NH.sub.4Cl, 0.20 g/L MgSO.sub.4?7 H.sub.2O, 10 ?l/L HCl (7.7 M), 1.5 mg/L FeCl.sub.2?4 H.sub.2O, 36 ?g/L ZnCl.sub.2, 64 ?g/L MnCl.sub.2?4 H.sub.2O, 6 ?g/L H.sub.3BO.sub.3, 190 ?g/L CoCl.sub.2?6 H.sub.2O, 1.2 ?g/L CuCl.sub.2?6 H.sub.2O, 24 ?g/L NiCl.sub.2?6 H.sub.2O, 36 ?g/L Na.sub.2MO.sub.4?2 H.sub.2O, 0.5 mg/L NaOH, 3 ?g/L Na.sub.2SeO.sub.3?5 H.sub.2O, 4 ?g/L Na.sub.2WO.sub.4?2 H.sub.2O, 100 ?g/L vitamin B12, 80 ?g/L p-aminobenzoic acid, 20 ?g/L D(+) Biotin, 200 ?g/L nicotinic acid, 100 ?g/L D-Ca-pantothenate, 300 ?g/L pyridoxine hydrochloride, 200 ?g/l thiamine-HCl?2H.sub.2O, 20 ml/L ethanol, 2.5 g/L NaHCO.sub.3, 65 mg/L glycine, 24 mg/L histidine, 64.6 mg/L isoleucine, 93.8 mg/L leucine, 103 mg/L lysine, 60.4 mg/L arginine, 21.64 mg/L L-cysteine-HCl, 21 mg/L methionine, 52 mg/L proline, 56.8 mg/L serine, 59 mg/L threonine, 75.8 mg/L valine) were inoculated with 10 ml of a living culture of Clostridium kluyveri to a start OD.sub.600nm of 0.1.

[0131] The cultivation was carried out in a 1000 mL pressure-resistant glass bottle at 37? C., 150 rpm and a ventilation rate of 1 L/h with 100% CO.sub.2 in an open water bath shaker for 671 h. The gas was discharged into the headspace of the reactor. The pH was hold at 6.2 by automatic addition of 100 g/L NaOH solution. Fresh medium was continuously feeded to the reactor with a dilution rate of 2.0 d.sup.?1 and fermentation broth continuously removed from the reactor through a KrosFlo? hollow fibre polyethersulfone membrane with a pore size of 0.2 ?m (Spectrumlabs, Rancho Dominguez, USA) to retain the cells in the reactor.

[0132] For the main culture 100 ml of fresh Veri01 medium in a 250 ml bottle were inoculated with centrifuged cells from the preculture to an OD.sub.600nm of 0.1. Additional 1 ml of a mixture of 6% (w/w) TOPO in dodecane was added. The culture was capped with a butyl rubber stopper and incubated at 37? C. and 150 rpm in an open water bath shaker for 43 h under 100% CO.sub.2 atmosphere. 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.

[0133] During the main cultivation the concentration of butyrate increased from 0.14 g/L to 2.62 g/L and the concentration of hexanoate increased from 0.22 g/L to 2.05 g/L, whereas the concentration of ethanol decreased from 14.62 to 10.64 g/l and the concentration of acetate decreased from 5.92 to 3.54 g/L.

[0134] The OD.sub.600nm increased during this time from 0.091 to 0.259.

Example 8

Determination of the Distribution Coefficient for Hexanoic Acid Between Water and a Mixture of Hexadecane and TOPO

[0135] During all stages of the experiment, samples from both phases were taken for determination of pH and concentration of hexanoic acid by high performance liquid chromatography (HPLC). 100 g of an aqueous solution of 5 g/kg hexanoic acid and 33 g of a mixture of 6% trioctylphosphinoxide (TOPO) in hexadecane were filled in a separatory funnel and mixed for 1 minute at 37? C. Then the funnel was placed in a tripod ring and the emulsion was left to stand to separate spontaneously. The pH of the aqueous phase was measured. Then 1M NaOH solution was added to the funnel and mixed. The step of separation and sampling was repeated until a pH of 6.2 in the aqueous phase was reached. Samples from both phases were taken for later analysis at this point. The aqueous phase could be analyzed directly by HPLC. For the analysis of the organic phase the diluted hexanoic acid was first re-extracted to water (pH 12.0 by addition of 1 M NaOH) and then analyzed by HPLC. The distribution coefficient K.sub.D of hexanoic acid in the system of water and 6% TOPO in hexadecane was calculated from the concentrations of hexanoic acid in both phases.

[00001]$K\left(D\right)=\frac{c\left(\mathrm{Hex},\mathrm{organic}\mathrm{phase}\right)}{c\left(\mathrm{Hex},\mathrm{aqueous}\mathrm{phase}\right)}$

[0136] The K.sub.D for hexanoic acid in the system of water and 6% TOPO in hexadecane at pH 6.2 was 4.7.

Example 9

Determination of the Distribution Coefficient for Hexanoic Acid Between Water and a Mixture of Heptadecane and TOPO

[0137] During all stages of the experiment, samples from both phases were taken for determination of pH and concentration of hexanoic acid by high performance liquid chromatography (HPLC). 100 g of an aqueous solution of 5 g/kg hexanoic acid and 33 g of a mixture of 6% trioctylphosphinoxide (TOPO) in heptadecane were filled in a separatory funnel and mixed for 1 minute at 37? C. Then the funnel was placed in a tripod ring and the emulsion was left to stand to separate spontaneously. The pH of the aqueous phase was measured. 1M NaOH solution was added to the funnel and mixed. The step of separation and sampling was repeated until a pH of 6.2 in the aqueous phase was reached. Samples from both phases were taken for later analysis at this point. The aqueous phase could be analyzed directly by HPLC. For the analysis of the organic phase the diluted hexanoic acid was first re-extracted to water (pH 12.0 by addition of 1 M NaOH) and then analyzed by HPLC. The distribution coefficient K.sub.D of hexanoic acid in the system of water and 6% TOPO in heptadecane was calculated from the concentrations of hexanoic acid in both phases.

[00002]$K\left(D\right)=\frac{c\left(\mathrm{Hex},\mathrm{organic}\mathrm{phase}\right)}{c\left(\mathrm{Hex},\mathrm{aqueous}\mathrm{phase}\right)}$

[0138] The K.sub.D for hexanoic acid in the system water and 6% TOPO in heptadecane at pH 6.2 was 5.0.

Example 10

Determination of the Distribution Coefficient for Hexanoic Acid Between Water and a Mixture of Tetradecane and TOPO

[0139] During all stages of the experiment, samples from both phases were taken for determination of pH and concentration of hexanoic acid by high performance liquid chromatography (HPLC). 130 g of an aqueous solution of 5 g/kg hexanoic acid plus 0.5 g/kg acetic acid and 15 g of a mixture of 6% trioctylphosphinoxid (TOPO) in tetradecane were filled in a separatory funnel and mixed for 1 minute at 37? C. Then the funnel was placed in a tripod ring and the emulsion was led stand to separate spontaneously. The pH of the aqueous phase was measured. 1M NaOH solution was added to the funnel and mixed. The step of separation and sampling was repeated until a pH of 6.2 in the aqueous phase was reached. Samples from both phases were taken for later analysis at this point. The aqueous phase could be analyzed directly by HPLC. For the analysis of the organic phase the diluted hexanoic acid was first re-extracted to water (pH 12.0 by addition of 1 M NaOH) and then analyzed by HPLC. The distribution coefficient K.sub.D of hexanoic acid in the system water and 6% TOPO in tetradecane was calculated from the concentrations of hexanoic acid in both phases.

[00003]$K\left(D\right)=\frac{c\left(\mathrm{Hex},\mathrm{organic}\mathrm{phase}\right)}{c\left(\mathrm{Hex},\mathrm{aqueous}\mathrm{phase}\right)}$

[0140] The K.sub.D for hexanoic acid in the system water and 6% TOPO in tetradecane at pH 6.9 was 1.3.

Example 11

Cultivation of Clostridium kluyveri and Extraction of Hexanoic Acid

[0141] The bacterium Clostridium kluyveri was cultivated for the biotransformation of ethanol and acetate to hexanoic acid. For the inSitu extraction of the produced hexanoic acid a mixture of tetradecane with trioctylphosphineoxide (TOPO) was continuously passed through the cultivation. All cultivation steps were carried out under anaerobic conditions in pressure-resistant glass bottles that can be closed airtight with a butyl rubber stopper.

[0142] The precultivation of Clostridium kluyveri was carried out in a 1000 mL pressure-resistant glass bottle in 250 ml of EvoDM45 medium (pH 5.5; 0.004 g/L Mg-acetate, 0.164 g/l Na-acetate, 0.016 g/L Ca-acetate, 0.25 g/l K-acetate, 0.107 mL/L H.sub.3PO.sub.4 (8.5%), 2.92 g/L NH.sub.4acetate, 0.35 mg/L Co-acetate, 1.245 mg/L Ni-acetate, 20 ?g/L d-biotin, 20 ?g/L folic acid, 10 ?g/L pyridoxine-HCl, 50 ?g/L thiamine-HCl, 50 ?g/L Riboflavin, 50 ?g/L nicotinic acid, 50 ?g/L Ca-pantothenate, 50 ?g/L Vitamin B12, 50 ?g/L p-aminobenzoate, 50 ?g/L lipoic acid, 0.702 mg/L (NH4).sub.2Fe(SO.sub.4).sub.2?4 H.sub.2O, 1 ml/L KS-acetate (93.5 mM), 20 mL/L ethanol, 0.37 g/L acetic acid) at 37? C., 150 rpm and a ventilation rate of 1 L/h with a mixture of 25% CO.sub.2 and 75% N.sub.2 in an open water bath shaker. The gas was discharged into the headspace of the reactor. The pH was hold at 5.5 by automatic addition of 2.5 M NH.sub.3 solution. Fresh medium was continuously feeded to the reactor with a dilution rate of 2.0 d.sup.?1 and fermentation broth continuously removed from the reactor through a KrosFlo? hollow fibre polyethersulfone membrane with a pore size of 0.2 ?m (Spectrumlabs, Rancho Dominguez, USA) to retain the cells in the reactor and hold an OD.sub.600nm of ?1.5.

[0143] For the main culture 150 ml of EvoDM39 medium (pH 5.8; 0.429 g/L Mg-acetate, 0.164 g/l Na-acetate, 0.016 g/L Ca-acetate, 2.454 g/l K-acetate, 0.107 mL/L H.sub.3PO.sub.4 (8.5%), 1.01 mL/L acetic acid, 0.35 mg/L Co-acetate, 1.245 mg/L Ni-acetate, 20 ?g/L d-biotin, 20 ?g/L folic acid, 10 ?g/L pyridoxine-HCl, 50 ?g/L thiamine-HCl, 50 ?g/L Riboflavin, 50 ?g/L nicotinic acid, 50 ?g/L Ca-pantothenate, 50 ?g/L Vitamin B12, 50 ?g/L p-aminobenzoate, 50 ?g/L lipoic acid, 0.702 mg/L (NH4).sub.2Fe(SO.sub.4).sub.2?4 H.sub.2O, 1 ml/L KS-acetate (93.5 mM), 20 mL/L ethanol, 8.8 mL NH.sub.3 solution (2.5 mol/L), 27.75 ml/L acetic acid (144 g/L))

[0144] in a 1000 ml bottle were inoculated with 100 ml cell broth from the preculture to an OD.sub.600nm of 0.71.

[0145] The cultivation was carried out at 37? C., 150 rpm and a ventilation rate of 1 L/h with a mixture of 25% CO.sub.2 and 75% N.sub.2 in an open water bath shaker for 65 h. The gas was discharged into the headspace of the reactor. The pH was hold at 5.8 by automatic addition of 2.5 M NH.sub.3 solution. Fresh medium was continuously feeded to the reactor with a dilution rate of 0.5 d.sup.?1 and fermentation broth continuously removed from the reactor by holding an OD.sub.600nm of ?0.5. Additional 120 g of a mixture of 6% (w/w) TOPO in tetradecane was added to the fermentation broth. Then this organic mixture was continuously feeded to the reactor and the organic phase also continuously removed from the reactor with a dilution rate of 1 d.sup.?1.

[0146] During cultivation several 5 mL samples from both, the aqueous and the organic phase, 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.

[0147] During the main cultivation in the aqueous phase a steady state concentration of 8.18 g/L ethanol, 3.20 g/L acetate, 1.81 g/L butyrate and 0.81 g/L hexanoate was reached. The OD.sub.600nm remained stable at 0.5. In the organic phase a steady state concentration of 0.43 g/kg ethanol, 0.08 g/kg acetate, 1.13 g/kg butyrate and 8.09 g/kg hexanoate was reached. After the experiment the cells remained viable while transferred to further cultivations.

[0148] The distribution coefficient K.sub.D of the substrates and products in the system aqueous medium and 6% TOPO in tetradecane was calculated from the concentrations in both phases.

[00004]$K\left(D\right)=\frac{c\left(\mathrm{organic}\mathrm{phase}\right)}{c\left(\mathrm{aqueous}\mathrm{phase}\right)}$

[0149] The K.sub.D in the steady state was 0.05 for ethanol, 0.03 for acetic acid, 0.62 for butyric acid and 9.99 for hexanoic acid.

Example 12

Ketonization of Hexanoic Acid

[0150] The ketonization was conducted in a heated continuous flow-bed reactor. At first, the reactor was charged with magnesium oxide on silica (50 wt. %, 14.00 g) and heated under an argon flow (54 mL/min) at 330? ? C. for one hour. The temperature was raised to 360? C. Than a mixture of hexanoic acid in tetradecane (v/v: 3/1) was continuously fed to the reactor with a rate of 3.3 mL/h. The gaseous out stream was collected by two cooling traps, which were cooled with water and a mixture of dry ice and isopropanol. The collected fractions were weighted and analyzed by gas chromatography (GC) for their composition. In total, 370.65 g of hexanoic acid was fed to the reactor, which equals to a maximum theoretical yield of 271.70 g of 6-undecanone and 28.75 g of water and 70.21 g of carbon dioxide as by-products. The obtained amount of 6-undecanone was 267.67 g and the amount of water was 28.32 g. This corresponds to a 99% mass recovery at full conversion. The high productivity and selectivity were confirmed by regular GC measurements, as only traces of hexanoic acid and no side-products were detected.

Example 13

Cross Ketonization of Hexanoic Acid With Palmitic Acid

[0151] The technical procedure of the cross ketonization is identical to the sole ketonization of hexanoic acid (example 12) except the composition of the substrate feed. The feed consists of hexanoic acid (116.16 g, 1.00 mol) and palmitic acid (256.43 g, 1.00 mol) as substrates and tetradecane (124.20 g) as internal standard. The substrate feed is added with a rate of 3.3 mL/h and reacted at a temperature of 360? C. The presence of two alkanoic acids leads two a product mixture of three ketones: 6-undecanone, 6-henicosanone and 16-hentriacontanone. At full conversion, the amounts obtained are 42.58 g of 6-undecanone, 155.29 g of 6-henicosanone and 112.71 g of 16-hentriacontanone.

Example 14

Cross Ketonization of Hexanoic Acid With Acetic Acid

[0152] The technical procedure of the cross ketonization is identical to the sole ketonization of hexanoic acid (example 12) except the composition of the substrate feed. The feed consists of hexanoic acid 232.32 g, 2.00 mol) and acetic acid (120.10 g, 2.00 mol) as substrates and tetradecane (117.47 g) as internal standard. The substrate feed is added with a rate of 3.3 mL/h and reacted at a temperature of 360? C. The presence of two alkanoic acids leads two a product mixture of three ketones: 2-propanone, 2-heptanone and 6-undecanone. At full conversion, the amounts obtained are 29.04 g of 2-propanone, 114.19 g of 2-heptanone and 85.15 g of 6-undecanone.

Example 15

Hydrogenation of the Higher Linear Alkanone to a Corresponding Higher Linear Alkane

[0153] The hydrogenation reaction of the C.sub.11 ketone (6-undecanone) to the linear alcane (undecane) was performed in a 300 ml autoclave reactor (PARR Instrument Company). The reactor was placed in an aluminum block and the temperature was controlled by a thermocouple placed inside the reactor. Typically, 30 mg of solid catalyst, 170.3 mg, 1.0 mmol of substrate was added to a 4 ml glass vial having an oven dried magnetic stirrer. 2.0 ml of dry Toluene was used as solvent, vial was fitted with a screw cap and a needle was inserted through the septum. (The vial is placed in the reactor.) The reactor was purged three times with 10 bar of H.sub.2 and then the pressure was increased to 20 bar. The reactor was heated to the desired temperature (120? C.) for 20 h. After the reaction, the reactor was cooled down to 5? C. using an ice bath, the gas phase was slowly released and the remaining liquid was carefully separated from the solid catalyst and was analyzed separately using an internal standard (100 ?L n-hexadecane).

##STR00001##

[0154] The ketone hydrogenation was investigated on a supported heterogeneous catalyst, 3.0Co@?-Al.sub.2O.sub.3, and added H-ZSM5 zeolith. This mixture showed 99% ketone conversion and an alcane yield of 98%.

[0155] The metal catalyst preparation method is as follows:

[0156] 3 wt %Co@?-Al.sub.2O.sub.3, using Ascorbic acid as reductant and glucose as capping agent in H.sub.2O, Pyrolysis at 800? C. for 2 h, Co salt is Cobalt(II) nitrate hexahydrate. In a typical synthesis, 149 mg, 0.51 mmol of Co(NO.sub.3).sub.2. 6H.sub.2O was dissolved in 20 ml D.I H.sub.2O followed by the stepwise addition of aqueous solutions of 265 mg, 3.0 mmol Ascorbic acid and 92 mg, 1 mmol of D-(+)-Glucose. The contents were stirred at 90? C. for 2-3 h. Next, 1.0 g of ?-Al.sub.2O.sub.3 support was added and the slurry was stirred overnight at R.T. Excess water was removed through centrifugation and the solids were dried in oven at 120? C. for 10 h and then pyrolyzed at 800? C. for 2 h under argon atmosphere.