FERMENTATION METHOD

Abstract

Describes is a fermentation method for producing a hydrocarbon compound comprising the culturing of an organism in a liquid fermentation medium, wherein said organism produces a desired hydrocarbon compound by an enzymatic pathway, said enzymatic pathway comprising an intermediate which evaporates into the gaseous phase and wherein said intermediate is recovered from the gaseous phase and is reintroduced into the liquid fermentation medium.

Claims

1. A fermentation method for producing a hydrocarbon compound, wherein said method comprises: (a) culturing an organism capable of producing a desired hydrocarbon compound by an enzymatic pathway in a liquid fermentation medium, (b) recovering an intermediate wherein said intermediate evaporates into the gaseous phase; and (c) reintroducing said intermediate into the liquid fermentation medium.

2. The fermentation method of claim 1, wherein when said intermediate is present as a pure compound and/or as a potential compound-water azeotrope the intermediate has an apparent boiling point of less than 120 C. at 1.013 bar or an apparent vapour pressure at infinite dilution in water at 25 C., which is defined as the pure component vapour pressure multiplied by the water-intermediate activity coefficient at infinite water dilution, is greater than 10 millibar.

3. The fermentation method of claim 1, wherein said intermediate is selected from an aldehyde, a ketone or an alcohol.

4. The fermentation method of claim 1, wherein said intermediate is selected from acetaldehyde, ethanol, acetone, propanal, propan-1-ol, propan-2-ol, butanal, butan-1-ol, butanone, butan-2-ol, 2-methyl-propanal, 2-methyl-propan-1-ol, 2-buten-1-ol (crotyl alcohol), 3-buten-2-ol, 3-methyl-2-buten-1-ol (prenol), 2-methyl-3-buten-2-ol, 3-methyl-3-buten-1-ol (isoprenol) or 3-methyl-3-buten-2-ol.

5. The fermentation method of claim 4, wherein the hydrocarbon is a C.sub.2-8 alkene of the following formula (I): ##STR00002## wherein the total number of carbon atoms in the compound of formula (I) is an integer of 2 to 8 and R.sup.1, R.sup.2, R.sup.3 and R.sup.4 are each independently selected from hydrogen, C.sub.1-6 alkyl or C2-6 alkenyl.

6. The fermentation method of claim 4, wherein the hydrocarbon is selected from isobutene, 1,3-butadiene, isoprene (2-methyl-1,3-butadiene) or propene.

7. The fermentation method of claim 1, wherein said intermediate is acetone.

8. The fermentation method of claim 7, wherein the hydrocarbon is isobutene and the enzymatic pathway for producing isobutene comprises acetone as an intermediate.

9. The fermentation method of claim 8, wherein the enzymatic pathway for producing isobutene comprises converting acetone and acetyl-CoA into 3-hydroxy-3-methylbutyric acid.

10. The fermentation method of claim 9, wherein the enzymatic conversion of acetone into 3-hydroxy-3-methylbutyric acid is achieved by the use of an enzyme having the activity of an HMG CoA synthase (EC 2.3.3.10) or an enzyme having the activity of a CC bond cleavage/condensation lyase, such as a HMG CoA lyase (EC 4.1.3.4), or a PksG protein.

11. The fermentation method of claim 7, wherein the hydrocarbon is propylene and the enzymatic pathway for producing propylene comprises acetone as an intermediate.

12. The fermentation method of claim 11, wherein the enzymatic pathway for producing propylene comprises converting acetone into propane-2-ol.

13. The fermentation method of claim 1, wherein the organism is a microorganism.

14. The fermentation method of claim 13, wherein the microorganism is a bacterium.

15. The fermentation method of claim 14, wherein the bacterium is E. coli.

16. The fermentation method of claim 1, wherein the intermediate is recovered from the gaseous phase by a. Physical absorption with or without distillation; b. Reactive absorption with or without distillation; c. Adsorption; d. Condensation; e. Cryogenic technologies; and f. Membrane based separations.

17. The fermentation method of claim 1, wherein said fermentation method is carried out as a batch method.

18. The fermentation method of 1, wherein said fermentation method is carried out under increased pressure.

Description

[0083] FIG. 1 shows schematically a possible fermentation arrangement to be used in the context of the present invention

[0084] FIG. 2 shows schematically an alternative possible fermentation arrangement to be used in the context of the present invention

[0085] FIG. 3 shows an overview of preferred intermediates and their physical properties.

[0086] FIG. 4 shows the positive impact of the volatile intermediate acetone on the formation of the product isobutene (IBN) and the key pathway compound 3-hydroxyisovalerate (HIV).

[0087] FIG. 5 shows the results of the experiments described in Example 10 concerning the effect of pressure during fermentation on the concentration of the volatile intermediate acetone.

[0088] It is to be understood that the present invention specifically relates to each and every combination of features and process parameters described herein, including any combination of general and/or preferred features/parameters. In particular, the invention specifically relates to all combinations of preferred features (including all degrees of preference) of the process provided herein.

[0089] In this specification, a number of documents including patent applications are cited. The disclosure of these documents, while not considered relevant for the patentability of this invention, is herewith incorporated by reference in its entirety. More specifically, all referenced documents are incorporated by reference to the same extent as if each individual document was specifically and individually indicated to be incorporated by reference.

[0090] The invention will now be described by reference to the following examples which are merely illustrative and are not to be construed as a limitation of the scope of the present invention.

EXAMPLES

Example 1: Biological Production of Acetone

[0091] A recombinant E. coli strain, derived from MG1655 (ATCC Number 700926; American Type Culture Collection (ATCC), Manassas, Va.), was used for the production of acetone. The strain, designated GBE2273, comprised a host cell (GBE1518) and a plasmid (pGBE1020). Strain GBE1518 comprised a modified central carbon metabolism network which employed a phosphoketolase pathway to more effectively channel carbon from a glucose feedstock to acetyl-coenzyme A (Ac-CoA), a key metabolite for the production of acetone. The plasmid pGBE1020 comprised genes encoding thiolase, coenzyme A transferase, and acetoacetate decarboxylase enzymes, which are responsible for catalyzing the conversion of two molecules of Ac-CoA into acetoacetyl-CoA, acetoacetyl-CoA into acetoacetate, and acetoacetate into acetone, respectively. Strain GBE2273 was inoculated at an initial OD.sub.600 of 0.1 absorbance units (AU) in a 250 mL screw cap flask containing 200 mL of medium. The medium was MS minimal medium (Richaud et al., J. Biol. Chem. (1998), 26827-26835), supplemented with 150 mM K.sub.2HPO.sub.4, 0.1 g/L yeast extract, 0.1 mg/L ampicillin, and 10 g/L glucose. The flask was incubated at 30 C. and 170 rpm for 6 days. Aliquots were withdrawn every 24 hr for 6 days. The flask was kept closed to avoid escape of acetone by evaporation, except for a brief period during each sampling. Glucose was determined by HPLC analysis and acetone was determined by GC. In particular, the acetone concentration was determined by gas chromatography using Gas chromatograph 450-GC (Bruker) and the following program:

[0092] Column: DB-WAX (123-7033, Agilent Technologies)

[0093] Injector Split/Splitless: T=250 C.

[0094] Oven: [0095] 80 C. for 6 minutes [0096] 10 C. per minutes until 220 C. [0097] 220 C. for 7 minutes [0098] Column flow: 1.5 ml/minute (Nitrogen)

[0099] Detector FID: T=300 C.

[0100] The yield of acetone was 0.51 mol acetone per mole glucose consumed.

Example 2: Fermentative Production of Isobutene

[0101] An E. coli strain, strain MG1655 transformed with a plasmid comprising genes encoding an HMG-CoA synthase for the conversion of acetone and acetyl-CoA into 3-hydroxy-3-methylbutyric acid, a mevalonate diphosphate decarboxylase for the conversion of 3-hydroxy-3-methylbutyric acid and ATP to 3-methyl-3-phosphonoxy-butyric acid (3-phosphonoxy-isovaleric acid), and a second mevalonate diphosphate decarboxylase for the conversion of 3-methyl-3-phosphonoxy-butyric acid to isobutene was constructed. Each of these genes had previously been mutated from their parental genes in order to more effectively perform the conversions described above, see WO 2010/001078 and WO 2012/052427.

[0102] The E. coli strain was inoculated into a fermenter equipped with temperature, pH and dO (dissolved oxygen) control and substrate feed pumps at an initial OD.sub.600 of 0.5 AU. The initial volume was 1.0 L of MS minimal medium (Richaud et al., J. Biol. Chem. (1998), 26827-26835) supplemented with 0.1 g/L yeast extract and 0.1 mg/mL ampicillin. The temperature was set for 30 C.; the pH was set to control at pH=6.8, with ammonium hydroxide addition; the gas (air) flow rate and initial stirrer rate was set at 0.25 VVM (volume per volume per minute) and 400 rpm (1200 rpm maximum), respectively; and the dO control was set for 50% with no back pressure. The fermentation was performed at atmospheric pressure. Glucose was maintained below 30 mM for 12 h by pump feed, at which time a bolus was added (approximately 45 g/L) and the fermentation was subsequently run in batch mode with respect to glucose. Acetone, initially present at approximately 100 mM, was subsequently provided by pump feed in order to make up for acetone lost by evaporation into the exit gases.

[0103] At various times, glucose, 3-hydroxy-3-methylbutyric acid and acetone in the liquid phase were determined by HPLC while acetone and isobutene in the gas phase were monitored by GC. The maximum specific isobutene productivity was 0.007 mmol/g dcw/hr with a concomitant specific 3-hydroxy-3-methylbutyric acid productivity of 0.08 mmol/g dcw/hr and a specific glucose consumption rate of 1.0 mmol/g dcw/hr, where dcw is dry cell weight.

Example 3: Fermentative Production of Acetone and Isobutene

[0104] A recombinant E. coli strain, derived from strain MG1655, is constructed. The recombinant strain comprises a modified central carbon metabolism network employing a phosphoketolase pathway to more effectively channel carbon from a glucose feedstock to acetyl-coenzyme A (Ac-CoA); thiolase, coenzyme A transferase, and acetoacetate decarboxylase enzymes to convert Ac-CoA into acetone; and a mutated HMG-CoA synthase enzyme and mutated mevalonate diphosphate decarboxylase enzymes to convert acetone and Ac-CoA into isobutene.

[0105] The E. coli strain is inoculated into a fermenter equipped with temperature, pH and dO (dissolved oxygen) control and substrate feed pumps. The fermentation was performed at atmospheric pressure. Temperature is controlled at 32 C., pH is controlled at 6.8, and dO is controlled at 20% by air flow (initially 0.25 VVM) and stirrer speed (initially 400 rpm) with no back pressure. The medium initially contains: KH.sub.2PO.sub.4, 7.5 g/L; MgSO.sub.4.7H.sub.2O, 2.0 g/L; citric acid, 1.83 g/L; ferric ammonium citrate, 0.3 g/L; CaCl.sub.2.2H.sub.2O, 0.2 g/L; sulfuric acid (98%), 1.2 mL/L; yeast extract, 5 g/L; and glucose (10 g/L). After sterilization, 10 mL of a trace metal mixture and 1 mL of a thiamine solution (150 mM) is added and the medium is adjusted to pH 6.8 with 3 M NaOH. The trace metal mixture contains: MnSO.sub.4.H.sub.2O, 3 g/L; NaCl, 1 g/L; FeSO.sub.4.7H.sub.2O, 0.1 g/L; CoCl.sub.2.6H.sub.2O, 0.1 g/L; ZnSO.sub.4.7H.sub.2O, 0.1 g/L; CuSO.sub.4.5H.sub.2O, 0.0064 g/L; H.sub.3BO.sub.3, 0.01 g/L; and Na.sub.2MoO.sub.4.2H.sub.2O, 0.01 g/L.

[0106] The initial OD.sub.600 of the fermenter is approximately 0.1 AU. Glucose is added as necessary to maintain the concentration above zero but below 10 g/L. When the OD.sub.600 of the fermenter reaches approximate 5 AU, acetone is added to the fermenter to approximately 250 mM and this concentration is maintained throughout the course of the fermentation. Isobutene and acetone are produced. The amount of exogenously added acetone that is consumed to produce isobutene is roughly equal to the amount of acetone that is produced from glucose.

Example 4: Production of Isobutene From Glucose

[0107] This example is meant to be illustrative but not inclusive of the various means to convert glucose to isobutene via fermentation. Materials and methods suitable for the maintenance and growth of bacterial cultures are well known in the art and will not be described in detail in this example. Techniques suitable for use can be found in Thomas D. Brock in Biotechnology: A Textbook of Industrial Microbiology, 2.sup.nd Edition (1989), Sinnauer Associates, Inc, Sunderland, Mass.

[0108] Cultures are started from frozen stock in shaker flasks and grown in medium until an OD 550 of approximately 1.0 A.U. is reached at which time the contents are transferred to the 1,000 liter seed fermenter. The microbes are further grown in the seed fermenter until they reach an OD 550 of approximately 10 A.U. at which time they are transferred into the 10,000 liter production fermenter

[0109] Anti-foam, medium salts (containing phosphates, sulfate and citrates) and 6000 liters of water are sterilized in the production fermenter prior to the addition of the contents of the seed fermenter. pH is adjusted to 6.8 via ammonium hydroxide addition and enough concentrated glucose solution (60-70 wt %) is added to bring the glucose concentration to 10 grams/liter. Glucose concentration is maintained at 0 to 10 grams/liter throughout the fermentation. Temperature is controlled at 32 C.

[0110] The pressure in the head space of the fermenter is maintained at 4 bar absolute throughout the course of the fermentation. The air flow rate is set in a range of 1,400 to 1,700 standard liters/minute of air over the course of the fermentation cycle.

[0111] Agitation intensity is maintained such as to ensure that the utilization of the oxygen fed to the fermenter is greater than 35%. Typically this means that the agitation intensity will be above 0.7 KW during the early stage of the fermentation cycle (ie microbe growth phase) and will rise to greater than 7 KW by the end of the fermentation cycle.

[0112] Due to isobutene's low solubility in water (about 260 ppm at 1 bar and 20 C.), isobutene is continuously vaporized and continuously exits with the fermentation off-gases throughout the course of the fermentation. Isobutene does not accumulate to any appreciable extent in the fermentation liquid.

[0113] Due to acetone's higher boiling point, it will partly vent into the fermentation off-gases. Since the acetone conversion step is rather slow and a high rate depends on a high acetone concentration, acetone is recovered from the gaseous phase, i.e. the fermentation-off gas, and is continuously or semi-continuously reintroduced into the fermentation medium. The recovery of acetone from the fermentation-off gas can be achieved as shown in FIG. 1. During the course of the initial microbe growth phase, acetone will accumulate to a 20 to 50 grams/liter concentration. An equilibrium between accumulation and consumption/venting will be reached and the acetone level will be maintained in this range during the rest of the fermentation cycle. The fermentation will continue until either contamination or the build-up of inhibitory compounds causes a notable decrease in overall isobutene production rate. This typically occurs in the 60th to 80.sup.th hour of the cycle.

Example 5: Fermentative Production of Isobutene from Glucose (at 4 Bar)

[0114] This example is meant to be illustrative but not inclusive of the various means to convert glucose to isobutene via fermentation. Materials and methods suitable for the maintenance and growth of bacterial cultures are well known in the art and will not be described in detail in this example. Techniques suitable for use can be found in Thomas D. Brock in Biotechnology: A Textbook of Industrial Microbiology, 2nd Edition (1989), Sinnauer Associates, Inc, Sunderland, Mass. Cultures are started from frozen stock in shaker flasks and grown in medium until an OD.sub.550 of approximately 1.0 absorbance units (AU) is reached at which time the contents are transferred to a 1,000 liter seed fermenter. The microbes are further grown in the seed fermenter until they reach an OD.sub.550 of approximately 10 AU at which time they are transferred into the 10,000 liter production fermenter.

[0115] Anti-foam, medium salts (containing phosphates, sulfate and citrates) and 6000 liters of water are sterilized in the production fermenter prior to the addition of the contents of the seed fermenter. pH is adjusted to 6.8 via ammonium hydroxide addition and enough concentrated glucose solution (60-70 wt %) is added to bring the glucose concentration to 10 grams/liter. Glucose concentration is maintained at 0 to 10 grams/liter throughout the fermentation. Temperature is controlled at 32 C. The pressure in the head space of the fermenter is maintained at 4 bar absolute throughout the course of the fermentation.

[0116] The air flow rate is set at such a rate that the exit oxygen concentration is always less than the about 13% level that is the flammable minimum oxygen concentration (MOC) value for isobutene in the presence of CO.sub.2 and N.sub.2 mixtures (Zabetakis MG, Flammability Characteristics of Combustible Gases and Vapors, U.S. Department of the Interior, Bureau of Mines, Bulletin 627, 1965, p. 52). Typically the exit concentration is maintained below 8%. Based on the chemistry of the reaction this corresponds to an air flow rate of less than 1,400 to 1,700 standard liters/minute of air at the end of the fermentation cycle. Agitation intensity is maintained such as to ensure that the utilization of the oxygen fed to the fermenter is greater than 35%. Typically this means that the agitation intensity will be about 0.7 KW during the early stage of the fermentation cycle (i.e., microbe growth phase) and will rise to 7 KW by the end of the fermentation cycle.

[0117] Due to isobutene's low solubility in water (about 260 ppm at 1 bar and 20 C.), isobutene is continuously vaporized and continuously exits with the fermentation off-gases throughout the course of the fermentation. Isobutene does not accumulate to any appreciable extent in the fermentation liquid.

[0118] Due to acetone's higher boiling point, while it is also continuously venting into the fermentation off-gases, it also accumulates in the fermentation broth. Since the acetone conversion step is slow and a high rate depends on a high acetone concentration, this is advantageous to overall fermenter productivity. During the course of the initial microbe growth phase, acetone will accumulate to a 20 to 50 grams/liter concentration. An equilibrium between accumulation and consumption/venting will be reached and the acetone level will be maintained in this range during the rest of the fermentation cycle. The fermentation will continue until either contamination or the build-up of inhibitory compounds causes a notable decrease in overall isobutene production rate. This typically occurs in the 60th to 80th hour of the cycle.

[0119] The amount of acetone vented during the course of the fermentation cycle will depend on such factors as gas feed rate, fermenter temperature, fermenter absolute pressure and concentration of acetone in the broth. Typically 10 to 30% of the acetone produced will be vented or remain in the fermentation broth at the end of the fermentation cycle depending on the value of the aforementioned parameters. Clearly such a loss is uneconomical and so the vaporized acetone is recovered from the fermenter off-gases by well documented and understood technologies and continuously or semi-continuously returned to the fermenter during the course of the fermentation cycle to maintain a consistent acetone concentration in the broth. Any residual acetone in the broth at the end of fermentation cycle may be easily recovered by well documented and understood technologies for use in the next fermentation batch.

Example 6: Fermentative Production of 1,3-Butadiene from Glucose (at 3 Bar)

[0120] This example is meant to be illustrative but not inclusive of the various means to convert glucose to 1,3-butadiene via fermentation. Materials and methods suitable for the maintenance and growth of bacterial cultures are well known in the art and will not be described in detail in this example. Techniques suitable for use can be found in Thomas D. Brock in Biotechnology: A Textbook of Industrial Microbiology, 2nd Edition (1989), Sinnauer Associates, Inc, Sunderland, Mass. Cultures are started from frozen stock in shaker flasks and grown in medium until an OD.sub.550 of approximately 1.0 AU is reached at which time the contents are transferred to a 1,000 liter seed fermenter. The microbes are further grown in the seed fermenter until they reach an OD.sub.550 of approximately 10 AU at which time they are transferred into the 10,000 liter production fermenter.

[0121] Anti-foam, medium salts (containing phosphates, sulfate and citrates) and 6000 liters of water are sterilized in the production fermenter prior to the addition of the contents of the seed fermenter. pH is adjusted to 6.8 via ammonium hydroxide addition and enough concentrated glucose solution (60-70 wt %) is added to bring the glucose concentration to 10 grams/liter. Glucose concentration is maintained at 0 to 10 grams/liter throughout the fermentation. Temperature is controlled at 32 C. The pressure in the head space of the fermenter is maintained at 3 bar absolute throughout the course of the fermentation.

[0122] The air flow rate is set at such a rate that the exit oxygen concentration is always less than the about 10.5% level that is the flammable minimum oxygen concentration (MOC) value for 1,3-butadiene in the presence of CO.sub.2 and N.sub.2 mixtures (Zabetakis MG, Flammability Characteristics of Combustible Gases and Vapors, U.S. Department of the Interior, Bureau of Mines, Bulletin 627, 1965, p. 55). Typically the exit concentration is maintained below 8%. Based on the chemistry of the reaction this corresponds to an air flow rate of less than 1,100 standard liters/minute of air at the end of the fermentation cycle. Agitation intensity is maintained such as to ensure that the utilization of the oxygen fed to the fermenter is greater than 45%. Typically this means that the agitation intensity will be about 0.4 KW during the early stage of the fermentation cycle (i.e., microbe growth phase) and will rise to 6 KW by the end of the fermentation cycle.

[0123] Due to 1,3-butadiene's low solubility in water (about 735 ppm at 1 bar and 20 C.), 1,3-butadiene is continuously vaporized and continuously exits with the fermentation off-gases throughout the course of the fermentation. 1,3-butadiene does not accumulate to any appreciable extent in the fermentation liquid.

[0124] Due to crotyl alcohol's higher boiling point, while it is also continuously venting into the fermentation off-gases, it also accumulates in the fermentation broth. Since the crotyl alcohol conversion step is slow and a high rate depends on a high crotyl alcohol concentration, this is advantageous to overall fermenter productivity. During the course of the initial microbe growth phase, crotyl alcohol will accumulate to about 5 to 10 grams/liter concentration. An equilibrium between accumulation and consumption/venting will be reached and the crotyl alcohol level will be maintained in this range during the rest of the fermentation cycle. The fermentation will continue until either contamination or the build-up of inhibitory compounds causes a notable decrease in overall 1,3-butadiene production rate. This typically occurs in the 60th to 80th hour of the cycle.

[0125] The amount of crotyl alcohol vented during the course of the fermentation cycle will depend on such factors as gas feed rate, fermenter temperature, fermenter absolute pressure and concentration of crotyl alcohol in the broth. Typically 5 to 10% of the crotyl alcohol produced will be vented or remain in the fermentation broth at the end of the fermentation cycle depending on the value of the aforementioned parameters. Clearly such a loss is uneconomical and so the vaporized crotyl alcohol is recovered from the fermenter off-gases by well documented and understood technologies and continuously or semi-continuously returned to the fermenter during the course of the fermentation cycle to maintain a consistent crotyl alcohol concentration in the broth. Any residual crotyl alcohol in the broth at the end of fermentation cycle may be easily recovered by well documented and understood technologies for use in the next fermentation batch.

Example 7: Fermentative Production of 1,3-Butadiene from Glucose (at 2 Bar)

[0126] This example is meant to be illustrative but not inclusive of the various means to convert glucose to 1,3-butadiene via fermentation. Materials and methods suitable for the maintenance and growth of bacterial cultures are well known in the art and will not be described in detail in this example. Techniques suitable for use can be found in Thomas D. Brock in Biotechnology: A Textbook of Industrial Microbiology, 2nd Edition (1989), Sinnauer Associates, Inc, Sunderland, Mass. Cultures are started from frozen stock in shaker flasks and grown in medium until an OD.sub.550 of approximately 1.0 absorbance units (AU) is reached at which time the contents are transferred to a 1,000 liter seed fermenter. The microbes are further grown in the seed fermenter until they reach an OD.sub.550 of approximately 10 AU at which time they are transferred into the 10,000 liter production fermenter.

[0127] Anti-foam, medium salts (containing phosphates, sulfate and citrates) and 6000 liters of water are sterilized in the production fermenter prior to the addition of the contents of the seed fermenter. pH is adjusted to 6.8 via ammonium hydroxide and/or caustic addition and enough concentrated glucose solution (60-70 wt %) is added to bring the glucose concentration to 10 grams/liter. Glucose concentration is maintained at 0 to 10 grams/liter throughout the fermentation. Temperature is controlled at 32 C. The pressure in the head space of the fermenter is maintained at 2 bar absolute throughout the course of the fermentation.

[0128] The air flow rate is set at such a rate that the exit oxygen concentration is always less than the about 10.5% level that is the flammable minimum oxygen concentration (MOC) value for 1,3-butadiene in the presence of CO.sub.2 and N.sub.2 mixtures (Zabetakis MG, Flammability Characteristics of Combustible Gases and Vapors, U.S. Department of the Interior, Bureau of Mines, Bulletin 627, 1965, p. 55). Typically the exit concentration is maintained below 8%. Based on the chemistry of the reaction this corresponds to an air flow rate of less than 1,100 standard liters/minute of air at the end of the fermentation cycle. Agitation intensity is maintained such as to ensure that the utilization of the oxygen fed to the fermenter is greater than 45%. Typically this means that the agitation intensity will be about 0.4 KW during the early stage of the fermentation cycle (i.e., microbe growth phase) and will rise to 14 KW by the end of the fermentation cycle.

[0129] Due to 1,3-butadiene's low solubility in water (about 735 ppm at 1 bar and 20 C.), 1,3-butadiene is continuously vaporized and continuously exits with the fermentation off-gases throughout the course of the fermentation. 1,3-butadiene does not accumulate to any appreciable extent in the fermentation liquid.

[0130] Due to crotyl alcohol's higher boiling point, while it is also continuously venting into the fermentation off-gases, it also accumulates in the fermentation broth. Since the crotyl alcohol conversion step is slow and a high rate depends on a high crotyl alcohol concentration, this is advantageous to overall fermenter productivity. During the course of the initial microbe growth phase, crotyl alcohol will accumulate to about 5 to 10 grams/liter concentration. An equilibrium between accumulation and consumption/venting will be reached and the crotyl alcohol level will be maintained in this range during the rest of the fermentation cycle. The fermentation will continue until either contamination or the build-up of inhibitory compounds causes a notable decrease in overall 1,3-butadiene production rate. This typically occurs in the 60th to 80th hour of the cycle.

[0131] The amount of crotyl alcohol vented during the course of the fermentation cycle will depend on such factors as gas feed rate, fermenter temperature, fermenter absolute pressure and concentration of crotyl alcohol in the broth. Typically 5 to 10% of the crotyl alcohol produced will be vented or remain in the fermentation broth at the end of the fermentation cycle depending on the value of the aforementioned parameters. Clearly such a loss is uneconomical and so the vaporized crotyl alcohol is recovered from the fermenter off-gases by well documented and understood technologies and continuously or semi-continuously returned to the fermenter during the course of the fermentation cycle to maintain a consistent crotyl alcohol concentration in the broth. Any residual crotyl alcohol in the broth at the end of fermentation cycle may be easily recovered by well documented and understood technologies for use in the next fermentation batch.

Example 8: Fermentative Production of Propene from Glucose (at 3 Bar)

[0132] This example is meant to be illustrative but not inclusive of the various means to convert glucose to propene via fermentation. Materials and methods suitable for the maintenance and growth of bacterial cultures are well known in the art and will not be described in detail in this example. Techniques suitable for use can be found in Thomas D. Brock in Biotechnology: A Textbook of Industrial Microbiology, 2nd Edition (1989), Sinnauer Associates, Inc, Sunderland, Mass. Cultures are started from frozen stock in shaker flasks and grown in medium until an OD.sub.550 of approximately 1.0 absorbance units (AU) is reached at which time the contents are transferred to a 1,000 liter seed fermenter. The microbes are further grown in the seed fermenter until they reach an OD.sub.550 of approximately 10 AU at which time they are transferred into the 10,000 liter production fermenter.

[0133] Anti-foam, medium salts (containing phosphates, sulfate and citrates) and 6000 liters of water are sterilized in the production fermenter prior to the addition of the contents of the seed fermenter. pH is adjusted to 6.8 via ammonium hydroxide and/or caustic addition and enough concentrated glucose solution (60-70 wt %) is added to bring the glucose concentration to 10 grams/liter. Glucose concentration is maintained at 0 to 10 grams/liter throughout the fermentation. Temperature is controlled at 32 C. The pressure in the head space of the fermenter is maintained at 3 bar absolute throughout the course of the fermentation.

[0134] The air flow rate is set at such a rate that the exit oxygen concentration is always less than the about 13% level that is the flammable minimum oxygen concentration (MOC) value for propylene in the presence of CO.sub.2 and N.sub.2 mixtures (Zabetakis MG, Flammability Characteristics of Combustible Gases and Vapors, U.S. Department of the Interior, Bureau of Mines, Bulletin 627, 1965, p. 51). Typically the exit concentration is maintained below 4%. Based on the chemistry of the reaction this corresponds to an air flow rate of less than 560 standard liters/minute of air at the end of the fermentation cycle. Agitation intensity is maintained such as to ensure that the utilization of the oxygen fed to the fermenter is greater than 55%. Typically this means that the agitation intensity will be about 0.4 KW during the early stage of the fermentation cycle (i.e., microbe growth phase) and will rise to 7.5 KW by the end of the fermentation cycle.

[0135] Due to propylene's low solubility in water (about 400 ppm at 1 bar and 20 C.), propylene is continuously vaporized and continuously exits with the fermentation off-gases throughout the course of the fermentation. Propylene does not accumulate to any appreciable extent in the fermentation liquid.

[0136] Due to acetone's and isopropanol's higher boiling points, while they are also continuously venting into the fermentation off-gases, they also accumulate in the fermentation broth. Since the acetone and isopropanol conversion steps are not instantaneous, a high rate depends on high acetone and isopropanol concentrations; this is advantageous to overall fermenter productivity. During the course of the initial microbe growth and early production phases, acetone will accumulate to about 10-15 grams/liter concentration and isopropanol will accumulate to about 20-30 grams/liter concentration. An equilibrium between accumulation and consumption/venting will be reached and the acetone and isopropanol levels will be maintained in this range during the rest of the fermentation cycle.

[0137] The amount of acetone and isopropanol vented during the course of the fermentation cycle will depend on such factors as gas feed rate, fermenter temperature, fermenter absolute pressure and concentration of acetone and isopropanol in the broth. Typically 5 to 8% of the acetone and 8 to 12% of the isopropanol produced will be vented or remain in the fermentation broth at the end of the fermentation cycle depending on the value of the aforementioned parameters. Clearly such a loss is uneconomical and so the vaporized acetone and isopropanol are recovered from the fermenter off-gases by well documented and understood technologies and continuously or semi-continuously returned to the fermenter during the course of the fermentation cycle to maintain consistent acetone and isopropanol concentrations in the broth. Any residual acetone and isopropanol in the broth at the end of fermentation cycle may be easily recovered by well documented and understood technologies for use in the next fermentation batch.

Example 9: Fermentative Production of Propene from Glucose (at 10 Bar)

[0138] This example is meant to be illustrative but not inclusive of the various means to convert glucose to propene via fermentation. Materials and methods suitable for the maintenance and growth of bacterial cultures are well known in the art and will not be described in detail in this example. Techniques suitable for use can be found in Thomas D. Brock in Biotechnology: A Textbook of Industrial Microbiology, 2nd Edition (1989), Sinnauer Associates, Inc, Sunderland, Mass. Cultures are started from frozen stock in shaker flasks and grown in medium until an OD.sub.550 of approximately 1.0 absorbance units (AU) is reached at which time the contents are transferred to a 1,000 liter seed fermenter. The microbes are further grown in the seed fermenter until they reach an OD.sub.550 of approximately 10 AU at which time they are transferred into the 10,000 liter production fermenter.

[0139] Anti-foam, medium salts (containing phosphates, sulfate and citrates) and 6000 liters of water are sterilized in the production fermenter prior to the addition of the contents of the seed fermenter. pH is adjusted to 6.8 via ammonium hydroxide and/or caustic addition and enough concentrated glucose solution (60-70 wt %) is added to bring the glucose concentration to 10 grams/liter. Glucose concentration is maintained at 0 to 10 grams/liter throughout the fermentation. Temperature is controlled at 32 C. The pressure in the head space of the fermenter is maintained at 10 bar absolute throughout the course of the fermentation.

[0140] The air flow rate is set at such a rate that the exit oxygen concentration is always less than the about 13% level that is the flammable minimum oxygen concentration (MOC) value for propylene in the presence of CO.sub.2 and N.sub.2 mixtures (Zabetakis MG, Flammability Characteristics of Combustible Gases and Vapors, U.S. Department of the Interior, Bureau of Mines, Bulletin 627, 1965, p. 51). Typically the exit concentration is maintained below 1%. Based on the chemistry of the reaction this corresponds to an air flow rate of less than 380 standard liters/minute of air at the end of the fermentation cycle. Agitation intensity is maintained such as to ensure that the utilization of the oxygen fed to the fermenter is greater than 85%. Typically this means that the agitation intensity will be about 0.4 KW during the early stage of the fermentation cycle (i.e., microbe growth phase) and will rise to 6.6 KW by the end the end of the fermentation cycle.

[0141] Due to propylene's low solubility in water (about 400 ppm at 1 bar and 20 C.), propylene is continuously vaporized and continuously exits with the fermentation off-gases throughout the course of the fermentation. Propylene does not accumulate to any appreciable extent in the fermentation liquid.

[0142] Due to acetone's and isopropanol's higher boiling points, while they are also continuously venting into the fermentation off-gases, they also accumulate in the fermentation broth. Since the acetone and isopropanol conversion steps are not instantaneous, a high rate depends on high acetone and isopropanol concentrations; this is advantageous to overall fermenter productivity. During the course of the initial microbe growth and early production phases, acetone will accumulate to about 10-15 grams/liter concentration and isopropanol will accumulate to about 20-30 grams/liter concentration. An equilibrium between accumulation and consumption/venting will be reached and the acetone and isopropanol levels will be maintained in this range during the rest of the fermentation cycle.

[0143] The amount of acetone and isopropanol vented during the course of the fermentation cycle will depend on such factors as gas feed rate, fermenter temperature, fermenter absolute pressure and concentration of acetone and isopropanol in the broth. Typically 4 to 6% of the acetone and 7 to 10% of the isopropanol produced will be vented or remain in the fermentation broth at the end of the fermentation cycle depending on the value of the aforementioned parameters. Clearly such a loss is uneconomical and so the vaporized acetone and isopropanol are recovered from the fermenter off-gases by well documented and understood technologies and continuously or semi-continuously returned to the fermenter during the course of the fermentation cycle to maintain consistent acetone and isopropanol concentrations in the broth. Any residual acetone and isopropanol in the broth at the end of fermentation cycle may be easily recovered by well documented and understood technologies for use in the next fermentation batch.

Example 10: Fermentative Production of Propene from Glucose (at 15 Bar)

[0144] This example is meant to be illustrative but not inclusive of the various means to convert glucose to propene via fermentation. Materials and methods suitable for the maintenance and growth of bacterial cultures are well known in the art and will not be described in detail in this example. Techniques suitable for use can be found in Thomas D. Brock in Biotechnology: A Textbook of Industrial Microbiology, 2nd Edition (1989), Sinnauer Associates, Inc, Sunderland, Mass. Cultures are started from frozen stock in shaker flasks and grown in medium until an OD.sub.550 of approximately 1.0 absorbance units (AU) is reached at which time the contents are transferred to a 1,000 liter seed fermenter. The microbes are further grown in the seed fermenter until they reach an OD.sub.550 of approximately 10 AU at which time they are transferred into the 10,000 liter production fermenter.

[0145] Anti-foam, medium salts (containing phosphates, sulfate and citrates) and 6000 liters of water are sterilized in the production fermenter prior to the addition of the contents of the seed fermenter. pH is adjusted to 6.8 via ammonium hydroxide and/or caustic addition and enough concentrated glucose solution (60-70 wt %) is added to bring the glucose concentration to 10 grams/liter. Glucose concentration is maintained at 0 to 10 grams/liter throughout the fermentation. Temperature is controlled at 32 C. The pressure in the head space of the fermenter is maintained at 15 bar absolute throughout the course of the fermentation.

[0146] The air flow rate is set at such a rate that the exit oxygen concentration is always less than the about 13% level that is the flammable minimum oxygen concentration (MOC) value for propylene in the presence of CO.sub.2 and N.sub.2 mixtures (Zabetakis MG, Flammability Characteristics of Combustible Gases and Vapors, U.S. Department of the Interior, Bureau of Mines, Bulletin 627, 1965, p. 51). Typically the exit concentration is maintained below 1%. Based on the chemistry of the reaction this corresponds to an air flow rate of less than 370 standard liters/minute of air at the end of the fermentation cycle. Agitation intensity is maintained such as to ensure that the utilization of the oxygen fed to the fermenter is greater than 85%. Typically this means that the agitation intensity will be about 0.4 KW during the early stage of the fermentation cycle (i.e., microbe growth phase) and will rise to 4.2 KW by the end of the fermentation cycle.

[0147] Due to propylene's low solubility in water (about 400 ppm at 1 bar and 20 C.), propylene is continuously vaporized and continuously exits with the fermentation off-gases throughout the course of the fermentation. Propylene does not accumulate to any appreciable extent in the fermentation liquid.

[0148] Due to acetone's and isopropanol's higher boiling points, while they are also continuously venting into the fermentation off-gases, they also accumulate in the fermentation broth. Since the acetone and isopropanol conversion steps are not instantaneous, a high rate depends on high acetone and isopropanol concentrations; this is advantageous to overall fermenter productivity. During the course of the initial microbe growth and early production phases, acetone will accumulate to about 10-15 grams/liter concentration and isopropanol will accumulate to about 20-30 grams/liter concentration. An equilibrium between accumulation and consumption/venting will be reached and the acetone and isopropanol levels will be maintained in this range during the rest of the fermentation cycle.

[0149] The amount of acetone and isopropanol vented during the course of the fermentation cycle will depend on such factors as gas feed rate, fermenter temperature, fermenter absolute pressure and concentration of acetone and isopropanol in the broth. Typically 4 to 6% of the acetone and 7 to 10% of the isopropanol produced will be vented or remain in the fermentation broth at the end of the fermentation cycle depending on the value of the aforementioned parameters. Clearly such a loss is uneconomical and so the vaporized acetone and isopropanol are recovered from the fermenter off-gases by well documented and understood technologies and continuously or semi-continuously returned to the fermenter during the course of the fermentation cycle to maintain consistent acetone and isopropanol concentrations in the broth. Any residual acetone and isopropanol in the broth at the end of fermentation cycle may be easily recovered by well documented and understood technologies for use in the next fermentation batch.

Example 11: Assessment of the Optimal Concentration of Acetone for Isobutene (IBN) Production

Media

[0150] LB is Luria Bertani medium made of tryptone 10 g/l, yeast extract 5 g/l and NaCl 10 g/l ZYM-5052 medium is described in F W Studier (2005) Prot. Exp. Pur. 41, 207-234. MS medium is described in C. Richaud et al. (1993) J. Biol. Chem. Vol. 268, No 36, pp 26827-26835.

[0151] MSP medium is a MS medium with 200 mM di-potassium phosphate instead of 50 mM.

[0152] MSP 45 medium is an MSP medium which has been adjusted to pH 8.5 and contains 45 g/l glucose instead of 2.

[0153] Recombinant E. coli strains expressing genes for isobutene production (WO 2010/001078 A2, WO 2011/032934 A1) are isolated on LB plate+100 mg/l ampicillin. One colony is grown in 10 ml LB medium+100 mg/l ampicillin for one night at 30 C. Then 300 ml of ZYM-5052 medium+100 mg/l ampicillin are inoculated with 1 ml of the previous culture and grown at 30 C. for 24 h. Such cells are collected by centrifugation at 4000 g, then suspended in MSP45 medium+100 mg/l ampicillin to reach an optical density at 600 nm around 25. Finally 30 ml of the suspension are introduced in a 160 ml bottle and just after acetone (0.1 to 1 M final concentration) is added. The bottle is then sealed and incubated on a shaker at 30 C. After 6 h incubation period, a sample is taken to measure 3-hydroxy-isovaleric acid (HIV) content by HPLC and pH is adjusted back to 8.5 with ammonia. Samples of gas are taken after 2, 4, 6 or 24 hours of incubation to measure the IBN production by Gas Chromatography.

[0154] The following tables gather the results of the production kinetics of HIV and isobutene at various acetone concentrations in sealed bottles.

TABLE-US-00001 TABLE 1 [acetone] mM 0 100 200 400 600 800 1000 HIV at 6 h 0 + ++ +++ +++/++++ ++++ ++++ IBN at 6 h 0 ++ +++ +++/++++ +++++ +++++++ +++ HIV: 3-hydroxy-isovaleric acid IBN: Isobutene

TABLE-US-00002 TABLE 2 [acetone] mM 0 100 200 400 600 800 1000 IBN at 6 h 0 +/ + + +/++ ++ + IBN at 24 h 0 +/++ +++ ++++ ++++++ +++++ ++++++ HIV: 3-hydroxy-isovaleric acid IBN: Isobutene

[0155] The results are also illustrated in FIG. 4. First, it is noticeable that there is no or only traces of production of either HIV or IBN in the absence of acetone. Second, both HIV and IBN productions increase with the acetone concentration up to an optimal level around 600 mM. The advantage of the sealed bottle is that the acetone concentration decrease is only due to the consumption rate for HIV and IBN production and it is rather negligible in the experiment described.

[0156] The situation is quite different in an aerated bioreactor since a more significant portion of acetone will be removed by stripping, depending of the temperature, the pressure and the aeration rate.

[0157] For instance, in a 900 ml culture run at 30 C. and with an aeration rate of 0.3 L/min under standard pressure, the 600 mM initial acetone concentration decreases steadily and there is almost no acetone remaining in the bioreactor after 2 days.

[0158] An E. coli culture has been run in the presence of 100 mM acetone at 30 C. with an aeration rate of 7 L/min either at standard pressure or at 2 bars absolute pressure. The acetone content has been measured either in the culture by HPLC or in the off gas stream with on line gas chromatography. The results are shown in FIG. 5. There is a very significant increase in the amount of acetone stripped in the off gas when the pressure is decreased from 2 bars to atmospheric pressure at 32 hours.