Biotechnological method for producing butanol and butyric acid

Abstract

The present invention relates to a method for producing C4 bodies, preferably butyric acid and/or butanol, comprising the steps contacting an aqueous medium comprising an acetogenic bacterial cell in an aqueous medium with syngas and incubating the mixture obtained in step a) at a temperature between 0 and 100° C. for at least 30 minutes, wherein the aqueous medium comprises, in step b), ethanol and/or acetate at a total combined concentration is at least 0.1 gL.sup.−1.

Claims

1. A method for producing a C4 compound, the method comprising: contacting an acetogenic bacterial cell in an aqueous medium with syngas under anaerobic conditions in the absence of carbohydrates, wherein the aqueous medium comprises ethanol, an acetate, or both, in a total concentration of at least 0.1 g L.sup.−1, wherein at least a portion of the ethanol, acetate, or both, is present in the aqueous medium before the acetogenic bacterial cell is brought into contact with the aqueous medium, thereby obtaining a mixture; and incubating the mixture at a temperature between 0 and 100° C. for at least 30 minutes, to obtain a C4 compound.

2. The method according to claim 1, wherein the total concentration of the ethanol and the acetate is from 0.5 to 20 g L.sup.−1.

3. The method according to claim 1, wherein the syngas comprises from 40 to 100% of CO.

4. The method according to claim 1, wherein the syngas comprises less than 10% of CO.sub.2.

5. The method according to claim 1, wherein the syngas comprises less than 10% of CO.

6. The method according to claim 1, further comprising: separating and, optionally, recycling to the aqueous medium, the ethanol, the acetate, or both, from the mixture following said incubating.

7. The method according to claim 1, wherein the acetogenic bacterial cell is selected from the group consisting of Clostridium, Moorella and Carboxythermus.

8. The method according to claim 1, wherein a pH in said contacting and incubating is maintained between 3 and 7.

9. The method according to claim 1, wherein said incubating is carried out at a temperature between 15° C. and 45° C.

10. The method according to claim 1, wherein the syngas provides more than 80% of carbon present initially in said contacting.

11. The method according to claim 1, wherein the method is run in a continuous mode.

12. The method according to claim 1, wherein said incubating is carried out in the absence of carbohydrates.

13. A method for increasing a proportion of syngas converted by an acetogenic bacterial cell in an aqueous medium, the method comprising: introducing ethanol, an acetate, or both, into an aqueous medium comprising an acetogenic bacterial cell, where syngas is converted by the acetogenic bacterial cell under anaerobic conditions in the absence of carbohydrates to a C4 compound, wherein the ethanol, the acetate, or both, are exogenously added to the aqueous medium prior to accumulation of detectable quantities of ethanol and acetate produced endogenously by the cell.

14. The method according to claim 13, wherein the acetate and ethanol are present in the aqueous medium comprising the acetogenic bacterial cell at a total concentration of 0.5 to 5 g L.sup.−1.

15. The method according to claim 13, wherein the acetogenic bacterial cell is selected from the group consisting of Clostridium, Moorella and Carboxythermus.

16. The method according to claim 1, wherein the C4 compound is butyric acid.

17. The method according to claim 1, wherein the C4 compound is butanol.

18. The method according to claim 1, wherein the syngas comprises from 40 to 95% of CO.

19. The method according to claim 1, wherein the aqueous medium comprises ethanol.

20. The method according to claim 1, wherein the aqueous medium comprises the acetate.

Description

EXAMPLE 1: PRODUCTION OF BUTYRIC ACID IN THE ABSENCE OR PRESENCE OF ETHANOL

(1) A Clostridium carboxidivorans DSMZ 15243 preculture was grown in anaerobic 1 L bottles sealed using a butylseptum, comprising 200 mL of modified PETC according to Hurst, K. M., and Lewis, R. (2010), Biochemical Engineering Journal 2010, 48, 159-165, consisting of 1 g yeast extract, 19 g MES, 30 mL mineral salt solution, 10 mL of trace element solution and 10 mL of vitamin solution. The mineral salt solution comprises 80 g NaCl, 100 g ammonium chloride, 10 g potassium chloride, 10 g potassium monophosphate, 20 g magnesium sulfate and 4 g calcium chloride per liter. The vitamin solution consists of 0.01 g pyridoxin, 0.005 g thiamin, 0.005 g riboflavin, 0.005 g calcium pantothenate, 0.005 g thioctacid, 0.005 g p-aminobenzoic acid, 0.005 g nicotinic acid, 0.005 vitamin B12, 0.002 g folic acid and 0.01 g MESNA per liter. The trace element solution consists of 2 g nitriloacetic acid, 1 g MnSO.sub.4, 0.8 g iron ammonium sulfate, 0.2 g cobalt chloride, 0.2 g zinc sulfate, 0.02 g copper(II) chloride, 0.02 g nickel chloride, 0.02 g sodium molybdate, 0.02 g Na.sub.2SeO.sub.4, 0.02 Na.sub.2WO.sub.4 per liter. The pH was adjusted to 5.9.

(2) Prior to inoculation the medium was boiled for 20 minutes and subsequently flushed with pure nitrogen for 20 minutes. Subsequently it was autoclaved at 121° C. for 20 minutes, followed by cooling down, then it was filled using process gas comprising 50% CO, 45% H.sub.2 and 5% CO.sub.2 at 1 bar gauge pressure. Subsequently, the pressure was adjusted to 0.8 bar gauge pressure.

(3) Also, prior to inoculation, 1.5 mL of a solution comprising 4% each of sodium sulfate and cystein hydrochloride as reducing agent was added under sterile anaerobic conditions.

(4) The culture was grown at 37° C. and 100 rpm. The culture was transferred to fresh medium every 72 hours.

(5) For the experiments the medium was prepared in the same way using process gas comprising 95% CO and 5% CO.sub.2. In addition, 0.6 g per liter ethanol was added to half the flasks under sterile anaerobic conditions.

(6) The solutions were inoculated under sterile anaerobic conditions using 10 vol. % of inoculum from a 48 hour culture. The flasks were shaken at 37° C. at 100 rpm for 160 hours. The dry biomass and the product concentration were determined at the beginning and at the end of the experiment.

(7) The concentrations of acetic acid, ethanol, butyric acid and butanol were determined using HPLC. A aminex HPX-87H column was used as a stationary phase. 5 mM sulfuric acid was used as an eluent at a constant flow rate of 0.6 mL/min. The temperature of the column was 40° C. Ethanol and butanol were detected using a refractive index detector. A diode array detector was used at a wave length of 210 nm to detect acetic acid and butyric acid. The concentrations of the compounds were calculated by integration of the peak using calibration graphs of the respective compound at defined concentrations.

(8) FIG. 1 shows the difference between product concentrations at the beginning and at the end of the cultivation with respect to the carbon content.

(9) In the presence of ethanol 105 nmolC/l acetic acid were formed compared to 97.17 mmolC/L formed in the absence of ethanol. 22.06 mmolC/L of butyric acid were formed in the presence of ethanol compared to 13.57 mmolC/L in the absence of ethanol, when equal amounts of dry biomass, more specifically 480 mg/L, were used.

(10) In summary, addition of ethanol leads to a significant increase in the amount of butyric acid formed.

EXAMPLE 2: PRODUCTION OF BUTYRIC ACID IN THE ABSENCE OR PRESENCE OF ACETATE

(11) The experimental protocol followed was as described in example 1, except for the fact that 2 g/L acetic acid was added to half the flask instead of 0.6 g/L ethanol and that the batch of syngas used comprised 50% carbon monoxide and 50% hydrogen.

(12) FIG. 2 shows the difference between product concentrations at the beginning and at the end of the cultivation with respect to the carbon content. In the presence of acetate 42.13 nmolC/l butyric acid was formed compared to 26.43 mmolC/L formed in the absence of acetate.

(13) In summary addition of acetate also leads to an increase in the amount of butyric acid formed.

EXAMPLE 3: PRODUCTION OF BUTYRIC ACID IN THE PRESENCE OF ACETIC OR ETHANOL USING ALTERNATIVE STRAINS AND GAS MIXTURES

(14) Media and Solutions Used:

(15) ATCC 1754 modified (PETC minimal medium)

(16) TABLE-US-00001 Amount Substance MES 10.00 g/l Solution trace elements 10 ml/l vitamins 141 10 ml/l fructose 250 g/L 20 ml/l reducing agent ATCC 10 ml/l

(17) The substance was mixed with vitamins and trace elements and the volume adjusted pH value and NaOH solution using deminerialized water (VE water). Subsequently the pH value was adjusted to 6.0 using NaOH solution, the medium was boiled and transferred to pressure-resistant 1 l glass bottles. Subsequently, the medium was cooled on ice and sparged using N.sub.2 in order to remove any remaining oxygen.

(18) Subsequently, the medium was autoclaved. Then the reducing agent was added to the medium and the volume was adjusted using anaerobic VE water. The reducing agent was sterilized separately and stored under anaerobic conditions.

(19) ATCC 1754 modified (PETC)

(20) TABLE-US-00002 Amount Substance NH.sub.4Cl 1.00 g/l KCl 0.10 g/l MgSO.sub.4 × 7H.sub.2O 0.20 g/l NaCl 0.80 g/l KH.sub.2PO.sub.4 0.10 g/l CaCl.sub.2 × 2H.sub.2O 20.00 mg/l resazurine 1.00 mg/l yeast extract 1.00 g/l MES 20.00 g/l Solution trace elements ATCC 10 ml/l 1754 vitamins 141 10 ml/l fructose 250 g/L 20 ml/l reducing agent ATCC 10 ml/l

(21) The substance, vitamins and trace elements were mixed and the volume adjusted pH value and NaOH solution using deminerialized water (VE water). Subsequently, the pH value was adjusted to 6.0 using NaOH solution, the medium was boiled and transferred to pressure-resistant 1 l glass bottles. Subsequently the medium was cooled on ice and sparged using N.sub.2 in order to remove any remaining oxygen.

(22) Subsequently, the medium was autoclaved. Then the reducing agent was added to the medium and the volume was adjusted using anaerobic VE water. The reducing agent was sterilized separately and stored under anaerobic conditions.

(23) ATCC 1754 modified (PETC modified)

(24) TABLE-US-00003 Amount Substance yeast extract 1.00 g/l MES 10.00 g/l Solution trace elements ATCC 10 ml/l 1754 vitamins PETC mod 10 ml/l mineral solution PETC 30 ml/l mod reducing agent ATCC 7.5 ml/l

(25) The substance, vitamins and trace elements were mixed and the volume adjusted pH value and NaOH solution using deminerialized water (VE water). Subsequently, the pH value was adjusted to 6.0 using NaOH solution, the medium was boiled and transferred to pressure-resistant 1 l glass bottles. Subsequently the medium was cooled on ice and sparged using N.sub.2 in order to remove any remaining oxygen.

(26) Subsequently, the medium was autoclaved. Then the reducing agent was added to the medium and the volume was adjusted using anaerobic VE water. The reducing agent was sterilized separately and stored under anaerobic conditions.

(27) trace elements ATCC 1754

(28) TABLE-US-00004 Substance Amount nitrilotriacetic acid 2 g/l MnSO.sub.4 × H.sub.2O 1 g/l (NH.sub.4).sub.2Fe(SO.sub.4).sub.2 × 6H.sub.2O 0.8 g/l CoCl.sub.2 × 6H.sub.2O 0.2 g/l ZnSO.sub.4 × 7H.sub.2O 0.2 g/l CuCl.sub.2 × 2H.sub.2O 0.02 g/l Na.sub.2MoO.sub.4 × 2H.sub.2O 0.02 g/l NiCl.sub.2 × 6H.sub.2O 0.02 g/l Na.sub.2SeO.sub.4 0.02 g/l Na.sub.2WO.sub.4 × 2H.sub.2O 0.02 g/l

(29) First of all, the nitrilotriacetic acid was dissolved in 1 l demineralized water (VE water), and the pH value was adjusted to 6.0 using a KOH solution. Subsequently, all the other chemicals were added. The trace element solution was stored at 4° C. in the dark.

(30) Vitamins 141

(31) TABLE-US-00005 Substance Amount biotin 2 mg/l folic acid 2 mg/l pyridoxine-HCl 10 mg/l thiamine-HCl × H.sub.2O 5 mg/l riboflavine 5 mg/l nicotinic acid 5 mg/l D-Ca-pantothenate 5 mg/l vitamin B12 0.1 mg/l p-amino benzoic acid 5 mg/l liponic acid 5 mg/l

(32) The substances were solved in 1 l demineralized water (VE water) and frozen at −20° C. in sterile falcon tubes in 10 ml portions until use.

(33) Mineral Solution PETC Mod

(34) TABLE-US-00006 Substance Amount NaCl 80 g/l NH.sub.4Cl 100 g/l KCl 10 g/l KH.sub.2PO.sub.4 10 g/l MgSO.sub.4 × 7H.sub.2O 20 g/l CaCl.sub.2 × 2H.sub.2O 4 g/l

(35) The substances were dissolved in 1 l demineralized water (VE water). The mineral solution was stored at 4° C. in the dark.

(36) Reducing agent ATCC 1754

(37) TABLE-US-00007 Substance Amount NaOH 9 g/l L-Cysteine × HCl 40 g/l Na.sub.2S × 9H.sub.2O 40 g/l

(38) First of all NaOH was dissolved in 1 l demineralized water (VE water) and boiled. Subsequently the solution was transferred to a pressure-resistant 1 l glass bottle and spaged using N.sub.2 while cooling down on ice. The other substances were added while the solution cooled down. Subsequently, the reducing agent was autoclaved for 20 minutes.

(39) Strains Used:

(40) COX-Cdr-001 (Clostridium drakei)

(41) COX-Clj-001 (Clostridium ljungdahlii)

(42) Pre-Cultures:

(43) The strains used were transferred to pre-cultures using working cryocultures prepared immediately (prior to cultures using 4 ml culture in the exponential phase and 1 ml 50% glycerol solution for conservation, storage at −80° C.). The pre-cultures each consisted of 5 ml ATCC 1754 mod (PETC)-medium and a fixed inoculum of the strains. The ideal inoculum density was determined in pilot experiments for the respective strain.

(44) COX-Cdr-001: 0.1% in 5 ml medium

(45) COX-Clj-001: 10% in 5 ml medium

(46) Cultures:

(47) 50 ml each of any medium used was transferred into sterile anaerobic (oxygen-free) pressure-resistant 250 ml glass bottles and inoculated using 10% (5 ml) of the strains used taken from the freshly prepared 3 days old pre-cultures.

(48) The culture flasks were closed using a sterile butyl bung and a red lid (comprising three drilled wholes). Hollow needles (company Sterican, Ø0.90×40 mm) were pierced through all three wholes. A manometer for controlling pressure was attached to one of the hollow needles (ideally the one in the middle). Valves were attached to the other hollow needles, so gas added and gas removed from the culture flask could be controlled independent from each other.

(49) Each of the strains used were cultivated in duplicate for direct reproducible results.

(50) Preparation of Cultures:

(51) Prior to starting the actual cultivations the bottles were each sparged using syngas thrice. This was done by attaching to one of the valves a tube for pumping gas into the bottle and to the other valve a tube for releasing the gas removed under the hood in line with regulations.

(52) By opening the valve with the tube for adding gas syngas was pumped into the culture flask until the needle of the manometer displayed a pressure value of 0.8 bar. Subsequently the valve for adding gas was closed and the valve for removing gas was opened until the needle displayed a pressure value of 0 bar. This procedure was repeated thrice for each culture flask. When the procedure was repeated for the fourth time the pressure in the bottle was maintained, i. e. the cultures were over laid with syngas.

(53) The culture samples were then ready for cultivation at 35° C. and 100 rpm in a rocking water bath.

(54) Taking Samples:

(55) One or twice per day approximately 1 ml of culture was removed for determining the optical density at 600 nm (recording the growth of a culture).

(56) Initially and at the end of the experiments two samples of 1.5 ml each were transferred to 2 ml Eppendorf-tubes and used for NMR-analysis.

(57) Addition of Gas:

(58) In regular intervals (several times a day) the pressure inside the culture flask was checked in order to ensure that it was constant. The growth of the cultures and their metabolism consumed varying amounts of syngas. In case the pressure decreased further gas was added as described above. The same procedure was carried out if gas was removed from the culture flask before taking a sample.

(59) End of the Experiment:

(60) The experiment was terminated after approximately one week by sparging continuously for 10 minutes nitrogen through every culture flask before removing the hollow needles for increased safety. The cultures were transferred to sterile 50 ml falcon tubes under a sterile hood and spun down for 30 minutes at 4500 g. There was no need to work under anaerobic conditions any more. The cell pellets recovered were discarded and the supernatants of the cultures were transferred to sterile 50 ml syringes under the sterile hood. They were transferred to a new sterile 50 ml falcon tube via a 0.2 μm sterile filter and frozen at −20° C. as back up samples.

(61) Experiment 1: Cultivation Using Yeast Extract and 0.6 g/l Ethanol (BF-DM-12-COX-038)

(62) The experiments were carried out using modified ATCC 1754 (PETC)-medium comprising 1 g/I yeast extract. In addition 0.6 g/I ethanol were used as a carbon source. The gas mixture used comprised 30% CO.sub.2 and 70% H.sub.2.

(63) Experiment 2: Cultivation Using Yeast Extract and 2 g/l Acetic Acid (BF-DM-12-COX-041)

(64) The experiment was carried out as described in experiment 1 except that acetic acid was added to the medium rather than ethanol.

(65) Experiment 3: Cultivation Using Yeast Extract (BF-DM-12-COX-042) (Comparative Experiment)

(66) For direct comparison the same medium was used but without addition of ethanol or acetic acid.

(67) The results are depicted in FIGS. 3 and 4. In summary, the effect shown in Example 1 could be reproduced using alternative gas mixtures and other strains of acetogenic bacteria.