Optimized anaerobic bacterial fermentation

Abstract

It provides a process for converting CO2 and CO from emissions into organic compounds. [Solution] The present invention provides a method for increasing the growth efficiency in anaerobic microbial fermentation of microorganisms capable of converting CO2, CO or a mixture thereof into at least one organic compound, the method comprising growing the microorganisms in a culture medium containing selenium at a concentration of at least 1.2 μM.

Description

[Technical Field]

[0001] Technical field of the invention The present invention relates to the fields of biotechnology, microbiology, fermentation, and the growth of microorganisms and the conversion of carbon dioxide or carbon monoxide to organic compounds. [Background technology]

[0002] Background of the Invention Carbon-releasing gases, such as carbon dioxide (CO2) and carbon monoxide (CO), form during many industrial processes, primarily during manufacturing, raw material extraction, energy, and transportation. While CO2 and CO have traditionally been released into the atmosphere as relatively harmless compounds that enter the global carbon cycle, environmental and health safety efforts have focused on limiting the release of other gases, including toxic ones such as NOx, SO2, and methane. Due to global population growth and ongoing global warming and climate change, there is an urgent need for industrial processes that are more circular in nature—that is, that leave little footprint and do not generate large emissions that disrupt global balance. Undoubtedly, this undertaking will require many diverse efforts to intervene in a timely manner to address ongoing developments that are far more rapid than the rate at which we fundamentally change humanity and the industrial processes that support our current population. Examples include replacing the combustion of fossil fuels with electricity generated from wind, sun, water, and nuclear power, as well as minimizing CO2 and CO emissions from industrial processes through alternative processes or by capturing CO2 and CO.

[0003] The capture of CO2 and CO from industrial processes can be done in two fundamentally different ways. First, CO2 can be captured in the industrial process and stored away from the atmosphere. Attempts have been made to pump CO2 into underground caverns, for example in areas where oil and gas exploration is taking place. There are also large-scale processes where CO2 is liquefied and transported to suitable locations for long-term storage. However, the concept of storing CO2 suggests that it may not be the optimal solution from a sustainability point of view. Second, CO2 can be captured from the industrial process in which it is produced and then used in another process or converted into some carbon-containing product with commercial value.

[0004] WO 98 / 37179 discloses chemically defined media for the fermentative production of valuable compounds on an industrial scale.

[0005] WO 2010 / 064932 discloses an optimized fermentation medium for the production of alcohol by microbial fermentation of a CO-containing substrate.

[0006] US Patent Application Publication No. 2015 / 0024449 discloses a process and medium for fermenting syngas in which selenium levels in the biomass are reduced.

[0007] Redl et al., Frontiers in Microbiology (2020), Vol. 10, pp. 1-15, disclose a medium for culturing Moorella species. Summary of the Invention [Problem to be solved by the invention]

[0008] Therefore, there is a great need for effective new processes that convert CO2 or CO emitted from industrial processes into products that have commercial value, thus serving the dual purpose of removing CO2 or CO from the exhaust and producing products that can ensure the process is commercially viable. [Means for solving the problem]

[0009] Summary of the Invention It is an object of the present invention to provide a process for converting CO and CO from effluents to organic compounds. In particular, it is an object of the present invention to provide an improved fermentation medium useful in microbial processes for converting CO or CO to at least one organic compound. It is a further object of the present invention to provide an improved fermentation medium that promotes a cost-effective process, e.g., low cost of the medium and low cooling requirements. It is also an object of the present invention to overcome some of the disadvantages of processes known in the art.

[0010] The inventors have unexpectedly found that media with high concentrations of selenium and molybdenum promote high growth rates and high biomass yields of butyric acid-producing microorganisms. In contrast to previous teachings, the inventors have also found that nickel and magnesium concentrations have novel and optimal intervals for growth rate and yield of acetogenic microorganisms.

[0011] In a first aspect, the present invention provides a method for increasing the growth efficiency in anaerobic microbial fermentation of a microorganism capable of converting CO, CO, or a mixture thereof to at least one organic compound, the method comprising growing the microorganism in a culture medium comprising selenium at a concentration of at least 1.2 μM.

[0012] In a second aspect, the present invention provides a method for increasing the growth efficiency in anaerobic microbial fermentation of microorganisms that convert CO, CO, or mixtures thereof to at least one organic compound, the method comprising growing the microorganisms in a medium containing nickel at a concentration in the range of 3.0 μM to 8.5 μM.

[0013] In a third aspect, the present invention provides a method for increasing the growth efficiency in anaerobic microbial fermentation of microorganisms that convert CO, CO, or mixtures thereof to at least one organic compound, the method comprising growing the microorganisms in a culture medium comprising molybdenum at a concentration of at least 1.5 μM.

[0014] In a fourth aspect, the present invention provides a method for switching from a phase of primarily biomass growth to a phase of primarily product formation for anaerobic microbial fermentation of a microorganism capable of converting CO, CO or a mixture thereof to at least one organic compound, wherein the product is said at least one organic compound, the method comprising growing the microorganism in a culture medium, wherein the concentrations of molybdenum and nickel remain substantially constant during the switchover, and the concentrations of both selenium and magnesium increase by 5 to 15 fold during the switchover.

[0015] In a fifth aspect, the present invention provides the use of a method as defined in any of the first to fourth aspects for the industrial production of at least one organic compound from CO, CO or mixtures thereof.

[0016] In an embodiment of the invention, the microorganism is an acetogen, i.e., a microorganism that metabolically converts CO2 (together with H2) or CO to acetyl-coenzyme A.

[0017] In another embodiment, the microorganism is selected from the group consisting of Clostridium, Moorella, Thermoanaerobacter, Thermoanaerobacterium, Acetogenium, Acetobacterium, Acetoanaerobium, Butyribacterium, Eubacterium, Pyrococcus, Desulfobacterium, and Carboxydothermus.

[0018] The invention is further summarized in the following list of items: 1. A method for increasing the growth efficiency in anaerobic microbial fermentation of microorganisms capable of converting CO2, CO or mixtures thereof to at least one organic compound, the method comprising growing the microorganisms in a culture medium containing selenium at a concentration of at least 1.2 μM.

[0019] 2. A method for increasing the growth efficiency in anaerobic microbial fermentation of microorganisms that convert CO2, CO, or mixtures thereof to at least one organic compound, the method comprising growing the microorganisms in a culture medium containing nickel at a concentration ranging from 3.0 μM to 8.5 μM.

[0020] 3. A method for increasing the growth efficiency in anaerobic microbial fermentation of microorganisms that convert CO2, CO or mixtures thereof to at least one organic compound, the method comprising growing the microorganisms in a culture medium containing molybdenum at a concentration of at least 1.5 μM.

[0021] 4. The method according to any of items 1 to 3, wherein the culture medium comprises selenium at a concentration that is at least 1.3 μM, at least 2 μM, at least 3 μM, or at least 4 μM.

[0022] 5. The method according to any of items 1 to 4, wherein the culture medium comprises selenium at a concentration in the range of 2.0 μM to 8 μM, in the range of 3.0 μM to 7 μM, in the range of 4 μM to 6 μM, or in the range of 4.4 μM to 5.6 μM.

[0023] 6. The method according to any of items 1 to 5, wherein the culture medium contains selenium at a concentration ranging from 4.4 μM to 5.6 μM.

[0024] 7. The method according to any of items 1 to 6, wherein the microorganism grows on a hexose as the main carbon source, such as fructose or glucose.

[0025] 8. The method according to any of items 1 to 7, wherein the culture medium comprises selenium at a concentration that is at least 5.0 μM, at least 10 μM, at least 20 μM, or at least 30 μM.

[0026] 9. The method according to any of items 1 to 8, wherein the culture medium comprises selenium at a concentration in the range of 5.0 μM to 70 μM, in the range of 10 μM to 60 μM, in the range of 20 μM to 50 μM, or in the range of 30 μM to 50 μM.

[0027] 10. The method according to any one of items 1 to 6 and 8 to 9, wherein the microorganism grows on a gas such as CO2 or CO as the main carbon source.

[0028] 11. The method according to any one of items 1 to 10, wherein the culture medium contains nickel at a concentration in the range of 4.0 μM to 8.0 μM, in the range of 5.0 μM to 7.0 μM, or in the range of 5.5 μM to 7.0 μM.

[0029] 12. The method according to any of items 1 to 11, wherein the culture medium comprises molybdenum at a concentration that is at least 1.7 μM, at least 2.0 μM, or at least 2.2 μM.

[0030] 13. The method according to any of items 1 to 12, wherein the culture medium comprises molybdenum at a concentration ranging from 1.5 μM to 20 μM, from 1.5 μM to 10 μM, or from 2.0 μM to 5 μM.

[0031] 14. The method according to any one of items 1 to 13, wherein the concentration of magnesium in the culture medium ranges from 0.05 mM to 40 mM.

[0032] 15. The method according to item 14, wherein the microorganism grows on hexose as the main carbon source and the magnesium concentration in the culture medium ranges from 0.05 mM to 1.3 mM, from 0.10 mM to 0.8 mM, or from 0.15 mM to 0.30 mM.

[0033] 16. The method according to item 14, wherein the microorganism grows on a gas such as CO or CO as the main carbon source, and the magnesium concentration in the culture medium ranges from 10 mM to 40 mM, 15 mM to 35 mM, or 18 mM to 27 mM.

[0034] 17. The method according to any one of items 1 to 16, wherein the microorganism is capable of converting CO2 into at least one organic compound.

[0035] 18. The method according to any one of items 1 to 17, wherein the microorganism is capable of converting CO into at least one organic compound.

[0036] 19. The method according to any one of items 1 to 18, wherein the at least one organic compound is a C1-6 compound.

[0037] 20. The method according to item 9a, wherein the C1-6 compound is a C1-6 alcohol, a C1-6 carboxylic acid, or a C1-6 ketone.

[0038] 21. The method according to any one of items 1 to 20, wherein the microorganism is an acetogen.

[0039] 22. The method according to any one of items 1 to 21, wherein the microorganism is selected from the group consisting of Clostridium, Moorella, Thermoanaerobacter, Thermoanaerobacterium, Acetogenium, Acetobacterium, Acetoanaerobium, Butyribacterium, Eubacterium, Pyrococcus, Desulfobacterium, and Carboxydothermus.

[0040] 23. The method according to item 22, wherein the microorganism is Moorella or Thermoanaerobacter.

[0041] 24. The method according to item 23, wherein the microorganism is Moorella thermoacetica or Moorella thermoautotrophica.

[0042] 25. The method according to any one of items 1 to 24, wherein the at least one organic compound comprises acetate.

[0043] 26. The method according to any one of items 1 to 25, wherein the at least one organic compound comprises ethanol.

[0044] 27. The method according to any one of items 1 to 26, wherein the anaerobic microbial fermentation is a biomass propagation process.

[0045] 28. The method according to any one of items 1 to 9 and 11 to 27, wherein the culture medium contains hexose as the main carbon source.

[0046] 29. The method according to item 28, wherein the hexose is fructose or glucose.

[0047] 30. The method according to any one of items 1 to 26, wherein the anaerobic microbial fermentation is the production of the at least one organic compound from the culture medium and a gas comprising CO2, CO or a mixture thereof.

[0048] 31. The method according to item 30, wherein the gas is synthesis gas, i.e., a mixture containing CO and H2.

[0049] 32. The method according to any one of items 1 to 31, wherein the culture medium comprises a nutrient selected from the group consisting of Al, Mn, Fe, Co, Zn, and Cu.

[0050] 33. The method according to any one of items 1 to 32, wherein the culture medium contains Mn, Fe, Co, Zn and Cu.

[0051] 34. The method according to any one of items 1 to 33, wherein the culture medium comprises a nutrient selected from the group consisting of biotin, folic acid, pyridoxine, thiamine, riboflavin, nicotinic acid, D-pantothenic acid, vitamin B12, para-amino-benzoic acid, and thiotic acid.

[0052] 35. The method according to any one of items 1 to 34, wherein the culture medium contains biotin, folic acid, pyridoxine, thiamine, riboflavin, nicotinic acid, D-pantothenic acid, vitamin B12, para-amino-benzoic acid, and thioctic acid.

[0053] 36. The method according to any one of items 1 to 35, wherein the proliferation efficiency is a specific proliferation rate.

[0054] 37. The method according to any one of items 1 to 36, wherein the growth efficiency is the biomass yield per carbon substrate provided in the culture medium.

[0055] 38. The method according to any one of items 1 to 37, wherein the microbial fermentation is a batch fermentation or a fed-batch fermentation.

[0056] 39. The method according to any one of items 1 to 38, wherein the fermentation of the microorganism is continuous fermentation.

[0057] 40. The method according to any one of items 1 to 39, wherein the microbial fermentation is an industrial-scale process.

[0058] 41. The industrial scale process is at least 5 m 3 Scale, at least 30m 3 Scale or at least 80m 3 Item 41. The method according to item 40, wherein the fermentation is carried out on a large scale.

[0059] 42. A method for switching from a phase of primarily biomass growth to a phase of primarily product formation for anaerobic microbial fermentation of a microorganism capable of converting CO2, CO or a mixture thereof to at least one organic compound, wherein the product is at least one organic compound, the method comprising growing the microorganism in a culture medium, wherein the concentrations of molybdenum and nickel remain substantially constant during the switchover, and the concentrations of both selenium and magnesium increase 5 to 15 times during the switchover.

[0060] 43. The method according to item 42, wherein the concentrations of both selenium and magnesium are increased 7 to 13 times during switching.

[0061] 44. The method according to any one of items 42 to 43, wherein the selenium concentration in the culture medium used before switching is in the range of 2.0 μM to 8 μM, and the magnesium concentration in the culture medium used before switching is in the range of 0.15 mM to 0.30 mM.

[0062] 45. The method according to any of items 42 to 44, wherein the concentration of molybdenum in the culture medium used before switching is at least 1.5 μM, and the concentration of nickel in the culture medium used before switching is in the range of 3.0 μM to 8.5 μM.

[0063] 46. ​​The method according to any one of items 42 to 45, wherein the main biomass growth phase is carried out using a culture medium in which one or more hexoses are the main carbon source.

[0064] 47. The method according to any of items 42 to 46, wherein the phase of main product formation is carried out using a culture medium in which a gas such as CO2 or CO is the main carbon source.

[0065] 48. The method according to any one of items 42 to 47, wherein the main biomass growth phase is carried out as a batch or fed-batch fermentation.

[0066] 49. The method according to any one of items 42 to 48, wherein the phase of main product formation is carried out as fed-batch fermentation or continuous fermentation.

[0067] 50. The method according to any of items 42 to 49, wherein the change of culture medium lasts less than 5 hours, less than 2 hours, less than 1 hour or less than 30 minutes.

[0068] 51. Use of the process as defined in any of items 1 to 50 for the industrial production of at least one organic compound from CO2, CO or a mixture thereof.

[0069] 52. The use according to item 51, wherein the at least one organic compound comprises a C1-6 compound, such as a C1-6 alcohol, a C1-6 carboxylic acid, or a C1-6 ketone.

[0070] 53. The use according to item 52, wherein the at least one organic compound comprises acetate.

[0071] 54. The use according to any one of items 51 to 53, wherein the at least one organic compound comprises ethanol. [Brief explanation of the drawings]

[0072] BRIEF DESCRIPTION OF THE DRAWINGS [Figure 1] Significance of compounds tested using a Plackett-Burman design for the growth of Moorella thermoacetica on fructose. A t-value greater than 1.65 or less than -1.65 indicates a significant effect at the 90% confidence level. [Figure 2] A generated response surface plot showing the effect of yeast extract and MgCl2 concentration on biomass production after 24 hours of fermentation on fructose. [Figure 3] Significance of compounds tested using a Plackett-Burman design for the growth of Moorella thermoacetica on fructose. A t-value greater than 1.65 or less than -1.65 indicates a significant effect at the 90% confidence level. [Figure 4] Generated response surface plot showing the effect of Na2MoO4, NiCl2 and Na2SeO4 concentrations on biomass production after 24 hours of fermentation on fructose. [Figure 5] Growth patterns of Moorella thermoacetica in improved and original media using fructose as the carbon source. Cell density is measured using a real-time monitoring device. [Figure 6] Acetate concentrations achieved after 63 hours of fermentation with Moorella thermoacetica in improved and original media using fructose as the carbon source. [Figure 7]Significance of compounds tested using a Plackett-Burman design for the growth of Moorella thermoacetica on CO2. A t-value greater than 1.65 or less than -1.65 indicates a significant effect at the 90% confidence level. [Figure 8] Generated response surface plot showing the effect of MgCl2 concentration on biomass production after 24 hours of fermentation using CO2 as the carbon source. [Figure 9] Significance of compounds tested using a Plackett-Burman design for the growth of Moorella thermoacetica on CO2. A t-value greater than 1.65 or less than -1.65 indicates a significant effect at the 90% confidence level. [Figure 10] Response surface plots generated showing the effect of Na2SeO4 and Na2MoO4 concentrations on biomass production after 24 hours of fermentation using CO2 as the carbon source. [Figure 11] Absorbance representing biomass concentration in improved and original media after 24 hours of fermentation using CO2 as the carbon source. DETAILED DESCRIPTION OF THE INVENTION

[0073] Detailed Description of the Invention Unless otherwise defined herein, all technical and scientific terms used have the same meaning as commonly understood by one of ordinary skill in the art of biotechnology, microbiology, fermentation and microbial growth and product formation.

[0074] Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described herein. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. Furthermore, the materials, methods, and examples are illustrative only and, unless otherwise specified, are not intended to be limiting.

[0075] The practice of the present invention will employ, unless otherwise indicated, conventional techniques of biotechnology, microbiology and fermentation technology that are within the skill of the art and are explained fully in the literature.

[0076] Culture medium In a first aspect, the present invention provides a method for increasing the growth efficiency in anaerobic microbial fermentation of a microorganism capable of converting O, CO, or a mixture thereof to at least one organic compound, the method comprising growing the microorganism in a culture medium comprising selenium at a concentration of at least 1.2 μM.

[0077] In a second aspect, the present invention provides a method for increasing the growth efficiency in anaerobic microbial fermentation of a microorganism capable of converting O, CO, or a mixture thereof to at least one organic compound, the method comprising growing the microorganism in a culture medium comprising nickel at a concentration in the range of 3.0 μM to 8.5 μM.

[0078] In a third aspect, the present invention provides a method for increasing the growth efficiency in anaerobic microbial fermentation of a microorganism capable of converting O2, CO or a mixture thereof to at least one organic compound, the method comprising growing the microorganism in a culture medium comprising molybdenum at a concentration of at least 1.5 μM.

[0079] It is understood that a selenium concentration of at least 1.2 μM may be combined in the culture medium with nickel concentrations ranging from 3.0 μM to 8.5 μM and molybdenum concentrations of at least 1.5 μM, and indeed, a combination of these three metals is considered a preferred embodiment.

[0080] In one embodiment, the culture medium comprises selenium at a concentration of at least 1.3 μM, at least 2 μM, at least 3 μM, or at least 4 μM. In another embodiment, the culture medium comprises selenium at a concentration ranging from 2.0 μM to 8 μM, from 3.0 μM to 7 μM, from 4 μM to 6 μM, or from 4.4 μM to 5.6 μM. In another embodiment, the culture medium comprises selenium at a concentration ranging from 4.4 μM to 5.6 μM. In yet another embodiment, the culture medium comprises selenium at a concentration of at least 5.0 μM, at least 10 μM, at least 20 μM, or at least 30 μM. In yet another embodiment, the culture medium comprises selenium at a concentration ranging from 5.0 μM to 70 μM, from 10 μM to 60 μM, from 20 μM to 50 μM, or from 30 μM to 50 μM.

[0081] In another embodiment, the culture medium comprises nickel at a concentration ranging from 4.0 μM to 8.0 μM, from 5.0 μM to 7.0 μM, or from 5.5 μM to 7.0 μM.

[0082] In another embodiment, the culture medium comprises molybdenum at a concentration of at least 1.7 μM, at least 2.0 μM, or at least 2.2 μM, hi another embodiment, the culture medium comprises molybdenum at a concentration ranging from 1.5 μM to 20 μM, from 1.5 μM to 10 μM, or from 2.0 μM to 5 μM.

[0083] In another embodiment, the concentration of magnesium in the culture medium ranges from 0.05 mM to 40 mM. In another embodiment, the microorganism is grown on hexose as the primary carbon source and the concentration of magnesium in the culture medium ranges from 0.05 mM to 1.3 mM, 0.10 mM to 0.8 mM, or 0.15 mM to 0.30 mM. In yet another embodiment, the microorganism is grown on CO2 or CO as the primary carbon source and the concentration of magnesium in the culture medium ranges from 10 mM to 40 mM, 15 mM to 35 mM, or 18 mM to 27 mM.

[0084] A different carbon source may be used to grow the microorganisms prior to the product formation phase, and in some embodiments, such a carbon source may also be added during the product formation phase, where CO, CO, or a mixture thereof is converted to at least one organic compound.

[0085] In one embodiment, the microorganisms grow on a hexose, such as fructose or glucose, as the primary carbon source, hi another embodiment, the microorganisms grow on CO, CO, or a mixture thereof as the primary carbon source.

[0086] Other useful media components will be apparent from the examples and sections of this application.

[0087] microorganisms It will be appreciated that a variety of different microorganisms can be used in the present invention, so long as they are capable of converting CO, CO, or a mixture thereof into at least one organic compound. Generally, useful microorganisms are anaerobic microorganisms.

[0088] In one embodiment, the microorganism is an acetogen.

[0089] Different acetogens can be used in the present invention. In some embodiments, the microorganisms can convert CO2 into at least one organic compound. In some embodiments, the microorganisms can convert CO2 into at least one organic compound.

[0090] In one embodiment, the at least one organic compound is a C1-6 compound.

[0091] In another embodiment, the C1-6 compound is a C1-6 alcohol, a C1-6 carboxylic acid, or a C1-6 ketone.

[0092] Other useful microorganisms are selected from the group consisting of Clostridium, Moorella, Thermoanaerobacter, Thermoanaerobacterium, Acetogenium, Acetobacterium, Acetoanaerobium, Butyribacterium, Eubacterium, Pyrococcus, Desulfobacterium, and Carboxydothermus.

[0093] In one embodiment, the microorganism is Moorella or Thermoanaerobacter.

[0094] In another embodiment, the microorganism is Moorella thermoacetica or Moorella thermoautotrophica.

[0095] The microorganism used in the present invention can be a naturally occurring microorganism, it can be a microorganism that has been optimized by selection, and it can be a microorganism that has been genetically engineered, for example to increase product formation or to enable it to synthesize a particular organic compound of interest.

[0096] In one embodiment, the at least one organic compound comprises acetate, hi another embodiment, the at least one organic compound is predominantly acetate.

[0097] In another embodiment, the at least one organic compound comprises ethanol, hi another embodiment, the at least one organic compound is predominantly ethanol.

[0098] The growth efficiency of a microorganism indicates how well it will grow under specific lighting conditions. In one embodiment, the growth efficiency is the specific growth efficiency. In another embodiment, the growth efficiency is the biomass yield per carbon substrate provided in the culture medium. [Example]

[0099] Example 1 This experiment describes the enhancement of Moorella thermoacetica growth on fructose achieved by modifying the growth medium composition using a statistical approach. First, a Plackett-Burman design is used to identify which components have a significant effect on cell growth. Then, response surface methodology (RSM) is used to determine the enhanced concentrations of these components. The factors tested in this experiment are the following eight components: yeast extract, NH4Cl, trace elements, vitamins, MgCl2, CaCl2, KH2PO4, and NaCl.

[0100] The following stock solutions are prepared and sterilized: fructose (180 g / L), yeast extract (100 g / L), NH4Cl (40 g / L), MgCl2·6H2O (20 g / L), CaCl2·2H2O (10 g / L), KH2PO4 (20 g / L), NaCl (20 g / L), MES monohydrate (200 g / L, pH 6.5), cysteine ​​HCl (100 mM), and resazurin (0.01 g / L). The vitamin solution is filter sterilized and contains biotin (2 mg / L), folic acid (2 mg / L), pyridoxine hydrochloride (10 mg / L), thiamine HCl (5 mg / L), riboflavin (5 mg / L), nicotinic acid (5 mg / L), calcium-D-(+)-pantothenate (5 mg / L), vitamin B12 (0.5 mg / L), p-aminobenzoic acid (5 mg / L), and thioctic acid (5 mg / L). A trace element solution is prepared by dissolving nitrilotriacetic acid (2 g / L) in water while increasing the pH to 6.0 with 2 M KOH, followed by the addition of MnSO4·H2O (1 g / L), Fe(SO4)2(NH4)2·6H2O (0.8 g / L), CoCl2·6H2O (0.2 g / L), ZnSO4·7H2O (0.2 g / L), CuCl2·2H2O (20 mg / L), NiCl2·6H2O (20 mg / L), Na2MoO4·2H2O (20 mg / L), Na2SeO4 (20 mg / L), and Na2WO4·2H2O (20 mg / L).

[0101] Each of the eight components is screened at two levels (Table 1). A Plackett-Burman matrix describing the final concentration of each factor in the 12 experiments performed is created with the statistical program JMP (Table 2). The experimental procedure is carried out in an anaerobic chamber to ensure an anaerobic environment. The anaerobic and sterile stock solutions are mixed with MES monohydrate (final concentration 20 g / L; pH 6.5), resazurin (0.001 g / L), fructose (10.8 g / L), and cysteine ​​HCl (1 mM) to reach the final concentrations shown in Table 2. 4.9 mL of each medium mix is ​​divided into three wells of a 24-well deep-well plate to prepare the inoculum (OD ). 600Add 0.1 mL of ...

[0102] [Table 1]

[0103] [Table 2]

[0104] [Table 3]

[0105] The results of the Plackett-Burman experiment are analyzed in JMP (Figure 1). Yeast extract and MgCl2 are the only components with significant effects on cell growth. MgCl2 has a significantly negative effect on cell growth, while yeast extract has a significantly positive effect. Based on these results, a response surface methodology (RSM) experiment is performed to optimize the concentrations of the two components.

[0106] The response surface experiment was conducted as a central composite design with two center points and an axis value of 1.414. Each matrix was generated using JMP (Table 4). The high and low values ​​were selected based on the Plackett-Burman experiment, and the center and axis points were calculated using JMP. The axis and center points were used to determine the curvature of the response surface. In the response matrix, -, +, 0, a, and A represent the low, high, center, lower, and upper axis points, respectively. Yeast extract has a positive effect; therefore, the low value of RSM is selected as the high value in the Plackett-Burman experiment (2 g / L). The high value is arbitrarily selected as 10 g / L. Conversely, MgCl₂·6H₂O has a negative effect; therefore, the low value in the Plackett-Burman experiment is selected as the high value in the response surface experiment (0.1 g / L). The low value is selected as 0.02 g / L.

[0107] [Table 4]

[0108] The entire procedure for the response surface experiment is carried out in an anaerobic chamber. Ten medium solutions are prepared according to the response surface matrix (Table 4). The concentrations of yeast extract and MgCl 6H O are varied by adding stock solutions to a medium containing MES monohydrate (final concentration 20 g / L; pH 6.5), NH Cl (0.4 g / L), KH PO (0.5 g / L), trace elements (1% v / v), vitamins (1% v / v), CaCl 2H O (0.05 g / L), NaCl (0.4 g / L), fructose (5.4 g / L), resazurin (0.001 g / L), and cysteine ​​HCl (1 mM). 4.9 mL of each solution is divided into three wells of a 24-well deep-well plate to prepare the inoculum (OD ). 600Add 0.1 mL of (=0.343) to all wells. Place the plate in an anoxic box flushed with N2 / CO2 (80 / 20) and with a final pressure of 0.8 bar. Incubate the box at 60 °C without shaking. After 24 hours, remove the plate from the incubator and transfer each solution to a 96-well microtiter plate. Read the absorbance at 630 nm in a spectrophotometer (Table 5).

[0109] [Table 5]

[0110] The results were analyzed using JMP, which generated a second-order polynomial equation (Equation 1) and a corresponding response surface plot (Figure 2). The analysis showed that when Moorella thermoacetica was grown on fructose, biomass concentration increased with increasing yeast extract and decreasing MgCl concentration.

[0111] 0.738734065270186+ +0.119117490895689 * ((yeast extract-6) / 4)+ +-0.00856188505625192 * ((MgCl2-0.06) / 0.04)+ +((yeast extract-6) / 4) * (((MgCl2-0.06) / 0.04) * -0.04625)+ +((yeast extract-6) / 4) * (((yeast extract-6) / 4) * -0.0595951861901537)+ +((MgCl2-0.06) / 0.04) * (((MgCl2-0.06) / 0.04) * -0.0161111425222695) Equation 1. Second-order polynomial obtained from RSM experiments with yeast extract and MgCl

[0112] Example 2 This experiment describes the statistical optimization of selected trace elements for enhanced growth of Moorella thermoacetica on fructose. A Plackett-Burman design is used to identify which elements have a significant effect on cell growth, and response surface methodology (RSM) is used to determine the enhanced concentrations of those elements. The following 12 elements are tested in this experiment: KAl(SO4)2, CoCl2, CuCl2, H3BO3, FeSO4, MnSO4, NiCl2, nitrilotriacetic acid, Na2SeO4, Na2MoO4, Na2WO4, and ZnSO4.

[0113] The following stock solutions are prepared and sterilized: fructose (500 g / L), yeast extract (100 g / L), NH4Cl (40 g / L), MgCl2·6H2O (20 g / L), CaCl2·2H2O (10 g / L), KH2PO4 (20 g / L), NaCl (20 g / L), MES monohydrate (200 g / L; pH 6.5), cysteine ​​HCl (100 mM), resazurin (0.01 g / L), nitrilotriacetic acid (1 g / L; K pH adjusted with OH6), MnSO4·H2O(20g / L), Fe(SO4)·7H2O(14g / L), CoCl2·6H2O(10g / L), ZnSO4·7H2O(4g / L), KAl(SO4)2·1 2H2O (1g / L), CuCl2·2H2O (1g / L), NiCl2·6H20 (1g / L), Na2MoO4·2H2O (1g / L), Na2SeO4 (1g / L) and Na2WO4·2H2O (1g / L). The vitamin solution is filter sterilized and contains biotin (2 mg / L), folic acid (2 mg / L), pyridoxine hydrochloride (10 mg / L), thiamine HCl (5 mg / L), riboflavin (5 mg / L), nicotinic acid (5 mg / L), calcium-D-(+)-pantothenate (5 mg / L), vitamin B12 (0.5 mg / L), p-aminobenzoic acid (5 mg / L), and thioctic acid (5 mg / L).

[0114] Initial screening was performed at two levels (Table 6). A Plackett-Burman matrix describing the final concentrations of each factor in the 20 experiments performed was created using the statistical program JMP (Table 7). The experimental procedure was performed in an anaerobic chamber to ensure an anaerobic environment. The anaerobic and sterile stock solutions were mixed with MES monohydrate (final concentration 20 g / L; pH 6.5), fructose (5 g / L), yeast extract (0.5 g / L), NH4Cl (0.4 g / L), MgCl2·6H2O (0.33 g / L), CaCl2·2H2O (0.05 g / L), KH2PO4 (0.5 g / L), NaCl (0.4 g / L), vitamin solution (1% v / v), resazurin (0.001 g / L), and cysteine ​​HCl (1 mM) to reach the final concentrations shown in Table 7. Divide 490 µL of each medium mix into three wells of a 96-well deep-well plate to obtain the inoculum (OD 600 Add 10 μL of PBS (=0.6) to all wells. Place the plate in an anoxic box flushed with N2 / CO2 (80 / 20) and with a final pressure of 0.8 bar and incubate at 60 °C without shaking. After 24 h, remove the plate from the incubator and transfer the culture from each well to a 96-well microtiter plate. Read the absorbance at 630 nm in a spectrophotometer (Table 8).

[0115] [Table 6]

[0116] [Table 7-1]

[0117] [Table 7-2]

[0118] [Table 7-3]

[0119] [Table 8]

[0120] The results of the Plackett-Burman experiment are analyzed in JMP (Figure 3). Components containing selenium, nickel, molybdenum, and cobalt have a significant positive effect on cell growth, while copper, aluminum, boric acid, and manganese have a significant negative effect on cell growth. The five components with the strongest effects (Na2SeO4, CuCl2, NiCl2, KAl(SO4)2, and Na2MoO4) are further tested using response surface methodology to optimize their concentrations in the growth medium.

[0121] The response surface experiment was conducted as a central composite design, with three center points and two axis values ​​selected. Each matrix was generated using JMP (Table 9). The high and low values ​​were selected based on the Plackett-Burman experiment, and the center and axis points were calculated using JMP. CuCl2·2H2O and KAl(SO4)2·12H2O had negative effects; therefore, the high values ​​in the RSM were selected as low values ​​in the Plackett-Burman experiment, i.e., 0.05 mg / L for both. The low values ​​were arbitrarily selected as 0.01 mg / L for both. Na2MoO4·2H2O, NiCl2·6H2O, and Na2SeO4 had positive effects; therefore, the low concentrations in the RSM were selected as high concentrations in the Plackett-Burman experiment, i.e., 0.2, 0.5, and 0.2 mg / L, respectively. The high levels were arbitrarily selected as 0.6, 1.5, and 1 mg / L, respectively.

[0122] [Table 9]

[0123] The entire procedure of the response surface experiment was carried out in an anaerobic chamber. The 29 medium solutions were prepared according to the response surface matrix (Table 9). The concentrations of Na2SeO4, CuCl2·2H2O, NiCl2·6H2O, KAl(SO4)2·12H2O, and Na2MoO4·2H2O were adjusted to the following concentrations: MES monohydrate (final concentration 20 g / L; pH 6.5), NH4Cl (0.4 g / L), KH2PO4 (0.5 g / L), CaCl2·2H2O (0.05 g / L), MgCl2·6H2O (0.33 g / L), NaCl (0.4 g / L), fructose (5 g / L), yeast extract (0.5 g / L), vitamins (1%), and Calcium Carbonate (CuCl4). The stock solutions are varied by adding them to a medium containing (v / v), CoCl 6H O (2 mg / L), H BO (0.1 mg / L), FeSO 7H O (2.24 mg / L), MnSO 7H O (10 mg / L), nitrilotriacetic acid (20 mg / L), Na WO 2H O (0.2 mg / L), ZnSO 7H O (2 mg / L), resazurin (0.001 g / L), and cysteine ​​HCl (1 mM). Divide 490 μL of each solution into three wells of a 96-well deep-well plate to prepare the inoculum (OD). 600 Add 10 μL of (=0.948) to all wells. Place the plate in an anoxic box flushed with N2 / CO2 (80 / 20) and with a final pressure of 0.8 bar. Incubate the box at 60 °C without shaking. After 24 hours, remove the plate from the incubator and transfer each solution to a 96-well microtiter plate. Read the absorbance at 630 nm in a spectrophotometer (Table 10).

[0124] [Table 10-1]

[0125] [Table 10-2]

[0126] The results were analyzed using JMP, which produced a second-order polynomial equation (Equation 2) and a corresponding response surface plot (Figure 4). The analysis shows that, within the boundaries tested, biomass concentration increases as Na2SeO4 concentration increases up to a critical level, after which it begins to decrease. At the same time, the highest biomass concentration is achieved at the highest Na2MoO4 concentration tested. With respect to NiCl2, the model suggests that the highest biomass concentration is achieved with nickel concentrations close to the two boundaries tested.

[0127] 1.01783470999122+-0.000929736362079477 * ((KAl(SO4)2-0.03) / 0.02)+ -0.0123591196301995 * ((CuCl2-0.03) / 0.02)+0.00973611111111112 * ((Na2MoO4-0.4) / 0.2)+0.00254166666666666 * ((NiCl2-1) / 0.5)+0.0833694065482988 * ((Na2SeO4-0.6) / 0.4)+(KAl(SO4)2-0.03) / 0.02 * (CuCl2-0.03) / 0.02 * -0.0106458333333333+(KAl(SO4)2-0.03) / 0.02 * (Na2MoO4-0.4) / 0.2 * -0.00589583333333332+(CuCl2-0.03) / 0.02 * (Na2MoO4-0.4) / 0.2 * -0.00389583333333333+(KAl(SO4)2-0.03) / 0.02 * (NiCl2-1) / 0.5 * 0.00177083333333332+(CuCl2-0.03) / 0.02 * (NiCl2-1) / 0.5* 0.00135416666666668+(Na2MoO4-0.4) / 0.2 * (NiCl2-1) / 0.5 * -0.0174791666666667+(KAl(SO4)2-0.03) / 0.02 * (Na2SeO4-0.6) / 0.4 * 0.00422916666666669+(CuCl2-0.03) / 0.02 * (Na2SeO4-0.6) / 0.4 * 0.00706249999999999+(Na2MoO4-0.4) / 0.2 * (Na2SeO4-0.6) / 0.4 * 0.0138125+(NiCl2-1) / 0.5 * (Na2SeO4-0.6) / 0.4 * -0.0046875+(KAl(SO4)2-0.03) / 0.02 * (KAl(SO4)2-0.03) / 0.02 * 0.071733898175278+(CuCl2-0.03) / 0.02 * (CuCl2-0.03) / 0.02 * 0.0817094897534409+(Na2MoO4-0.4) / 0.2 * (Na2MoO4-0.4) / 0.2 * 0.0576734726195067+(NiCl2-1) / 0.5 * (NiCl2-1) / 0.5 * 0.0594651392861734+(Na2SeO4-0.6) / 0.4 * (Na2SeO4-0.6) / 0.4 * -0.048660176726902 Equation 2. Second-order polynomials obtained from RSM experiments on KAl(SO4)2, CuCl2, Na2MoO4, NiCl2, and Na2SeO4

[0128] Example 3. This experiment describes improved biomass acid production by Moorella thermoacetica in growth media containing fructose as a carbon source and novel concentrations of specific nutrients.

[0129] To ensure an anoxic environment, the procedure is carried out in an anaerobic chamber. Sterile and anaerobic stock solutions of each component are prepared according to Examples 1 and 2. Two media, referred to as "original" and "improved" media, are prepared by mixing the stock solutions to reach the concentrations shown in Table 11. 49 mL of each medium is then divided into three 125 mL serum bottles and used to inoculate Moorella thermoacetica (OD ). 600 = 0.86) growing culture was inoculated and the initial OD 600 The OD reaches 0.02. The vials are incubated at 60°C with stirring at 200 rpm using a magnetic stirrer. The biomass concentration is monitored using a real-time cell density monitor. The growth patterns in the two media are shown in Figure 5, which shows that the improved medium supports more rapid growth and higher biomass density. 600 According to the measurements, the biomass concentration is 63% higher in the improved medium than in the original medium after 63 hours of fermentation. At the same time, Figure 6 shows that acetate production increased by an average of 60% in the improved medium.

[0130] [Table 11-1]

[0131] [Table 11-2]

[0132] Example 4 This example describes the optimization of medium composition, specifically for growth on CO2 and H2, with respect to five macronutrients, using Plackett-Burman designs and response surface methodology.

[0133] The following stock solutions are prepared and sterilized: yeast extract (100 g / L), NH4Cl (40 g / L), MgCl2·6H2O (20 g / L), CaCl2·2H2O (10 g / L), KH2PO4 (20 g / L), NaCl (20 g / L), MES monohydrate (200 g / L, pH 6.5), cysteine ​​HCl (100 mM), and resazurin (0.01 g / L). The trace element solution contained nitrilotriacetic acid (1 g / L; pH adjusted to 6 with KOH), MnSO4·H2O (20 g / L), Fe(SO4)·7H2O (14 g / L), CoCl2·6H2O (10 g / L), ZnSO4·7H2O (4 g / L), CuCl2·2H2O (1 g / L), NiCl2·6H2O (1 g / L), Na2MoO4·2H2O (1 g / L), Na2SeO4 (1 g / L), and Na2WO4·2H2O (1 g / L). The vitamin solution is filter-sterilized and contains biotin (2 mg / L), folic acid (2 mg / L), pyridoxine hydrochloride (10 mg / L), thiamine HCl (5 mg / L), riboflavin (5 mg / L), nicotinic acid (5 mg / L), calcium-D-(+)-pantothenate (5 mg / L), vitamin B12 (0.5 mg / L), p-aminobenzoic acid (5 mg / L), and thioctic acid (5 mg / L).

[0134] Two levels are selected for each factor tested (Table 12). A Plackett-Burman matrix describing the final concentrations of each factor in the 20 experiments conducted is created in the statistical program JMP (Table 13). The experimental procedure is carried out in an anaerobic chamber to ensure an anaerobic environment. The anaerobic and sterile stock solutions are mixed with MES monohydrate (final concentration 20 g / L; pH 6.5), yeast extract (0.5 g / L), vitamin solution (1% v / v), trace element solution (1% v / v), resazurin (0.001 g / L), and cysteine ​​HCl (1 mM) to reach the final concentrations shown in Table 13. 490 μL of each medium mix is ​​divided into three wells of a 96-well deep-well plate, and 10 μL of inoculum is added to all wells. The plates are placed in an anaerobic box flushed with H2 / CO2 (80 / 20) for 10 minutes at a final pressure of 0.6 bar and incubated at 60°C without shaking. After 24 hours, the plates are removed from the incubator and the cultures from each well are transferred to a 96-well microtiter plate. The absorbance is read at 630 nm in a spectrophotometer (Table 14).

[0135] [Table 12]

[0136] [Table 13]

[0137] [Table 14]

[0138] The results of the Plackett-Burman experiment are analyzed in JMP (Figure 7). The analysis shows that when CO2 is used as the carbon source, NH4Cl and KH2PO4 have a significant negative effect on growth, while MgCl2 and NaCl have a significant positive effect. CaCl2 has no significant effect. NH4Cl, KH2PO4, MgCl2, and NaCl are further tested using response surface methodology.

[0139] The response surface experiment was conducted as a central composite design, with three center points and one axis value selected. Each matrix was generated using JMP (Table 15). The high and low values ​​were selected based on the Plackett-Burman experiment, and the center and axis points were calculated using JMP. NH4Cl and KH2PO4 have a negative effect on Plackett-Burman, so the high concentrations in the RSM are equivalent to the low concentrations in Plackett-Burman, 0.2 g / L and 0.1 g / L, respectively. The low values ​​were selected as 0.02 g / L and 0.01 g / L, respectively. MgCl2 and NaCl have a positive effect on growth, so the low values ​​in the RSM are selected as 0.5 and 1 g / L, respectively, and the high values ​​are selected as 5 g / L for both compounds.

[0140] [Table 15-1]

[0141] [Table 15-2]

[0142] The entire procedure for the response surface experiment was carried out in an anaerobic chamber. Twenty-seven medium solutions were prepared according to the response surface matrix (Table 15). The concentrations of NH₄Cl, KH₂PO₄, MgCl₂·6H₂O, and NaCl were varied by adding stock solutions to a medium containing MES monohydrate (final concentration 20 g / L; pH 6.5), CaCl₂·2H₂O (0.05 g / L), yeast extract (0.5 g / L), vitamins (1% v / v), trace element solution (1% v / v), resazurin (0.001 g / L), and cysteine ​​HCl (1 mM). 490 μL of each solution was divided into three wells of a 96-well deep-well plate, and 10 μL of inoculum was added to each well. The plate was placed in an anaerobic chamber flushed with N₂ / CO₂ (80 / 20) for 10 min at a final pressure of 0.8 bar. The chamber was then incubated at 60 °C without shaking. After 24 hours, remove the plate from the incubator and transfer each solution to a 96-well microtiter plate. Read the absorbance at 630 nm in a spectrophotometer (Table 16).

[0143] [Table 16-1]

[0144] [Table 16-2]

[0145] The results were analyzed by JMP, which generated a second-order polynomial equation (Equation 3). The corresponding response surface plot showing the MgCl2 trend is shown in Figure 7. When the carbon source is CO2, the biomass concentration increases with MgCl2 concentration until a critical point is reached, after which the biomass concentration plateaus even as the MgCl2 concentration is further increased.

[0146] 0.0969428571428572+0.00151851851851852 * ((NH4Cl-0.11) / 0.09) +0.00124074074074074* ((MgCl2-2.75) / 2.25)+-0.00322222222222222 * ((KH2PO4-0.055) / 0.045)+-0.00566666666666667 * ((NaCl-3) / 2)+(NH4Cl-0.11) / 0.09 * (MgCl2-2.75) / 2.25 * 0.00239583333333333+(NH4Cl-0.11) / 0.09 * (KH2PO4-0.055) / 0.045 * -0.00147916666666667+(MgCl2-2.75) / 2.25 * (KH2PO4-0.055) / 0.045 * -0.00185416666666667+(NH4Cl-0.11) / 0.09 * (NaCl-3) / 2 * 0.000770833333333332+(MgCl2-2.75) / 2.25 * (NaCl-3) / 2 * -0.000104166666666667+(KH2PO4-0.055) / 0.045 * (NaCl-3) / 2 * 0.000770833333333333+(NH4Cl-0.11) / 0.09 * (NH4Cl-0.11) / 0.09 * -0.000257142857142861+(MgCl2-2.75) / 2.25 * (MgCl2-2.75) / 2.25 * -0.00275714285714286+(KH2PO4-0.055) / 0.045 * (KH2PO4-0.055) / 0.045 * 0.00307619047619048+(NaCl-3) / 2 * (NaCl-3) / 2 *-0.00259047619047619 Equation 3. Second-order polynomials obtained from RSM experiments with NH4Cl, MgCl2, KH2PO4, and NaCl

[0147] Example 5 This experiment describes the statistical optimization of selected trace elements and vitamins for enhanced growth of Moorella thermoacetica on H2 / CO2. Plackett-Burman designs are used to identify which components have significant effects on cell growth, and response surface methodology (RSM) is used to determine improved concentrations of those components. The following 14 factors are tested in this experiment: biotin, thiamine HCl, calcium d-(+)-pantothenate, vitamin B12, nicotinic acid, thioctic acid, CoCl2, CuCl2, FeSO4, MnSO4, NiCl2, Na2SeO4, Na2WO4, and Na2MoO4.

[0148] The following stock solutions are prepared and sterilized: yeast extract (100 g / L), NH4Cl (40 g / L), MgCl2·6H2O (20 g / L), CaCl2·2H2O (10 g / L), KH2PO4 (20 g / L), NaCl (20 g / L), MES monohydrate (200 g / L, pH 6.5), cysteine ​​HCl (100 mM), resazurin (0.01 g / L), nitrilotriacetic acid (1 g / L; pH 6 adjusted with KOH), MnSO4·H2O (20 g / L), Fe(SO4)·7H2O (14 g / L), CoCl2·6H2O (10 g / L), Zn SO4·7H2O (4 g / L), CuCl2·2H2O (1 g / L), NiCl2·6H2O (1 g / L), Na2MoO4·2H2O (1 g / L), Na2SeO4 (1 g / L), Na2WO4·2H2O (1 g / L), biotin (0.5 g / L), folic acid (1 g / L), pyridoxine hydrochloride (1 g / L), thiamine HCl (1 g / L), riboflavin (0.1 g / L), nicotinic acid (1 g / L), calcium-D-(+)-pantothenate (1 g / L), vitamin B12 (1 g / L), p-aminobenzoic acid (1 g / L), and thioctic acid (1 g / L).

[0149] An initial screening was performed at two levels (Table 17). A Plackett-Burman matrix was created with the statistical program JMP, describing the final concentrations of each factor in the 23 experiments performed (Table 18). The experimental procedure was carried out in an anaerobic chamber to ensure an anoxic environment. Mix the anoxic and sterile stock solution with MES monohydrate (final concentration 20 g / L; pH 6.5), yeast extract (0.5 g / L), NH4Cl (0.4 g / L), MgCl2·6H2O (0.33 g / L), CaCl2·2H2O (0.05 g / L), KH2PO4 (0.5 g / L), NaCl (0.4 g / L), folic acid (0.02 mg / L), pyridoxine HCl (0.1 mg / L), riboflavin (0.05 mg / L), p-aminobenzoic acid (0.05 mg / L), ZnSO4·7H2O (2 mg / L), nitrilotriacetic acid (20 mg / L), resazurin (0.001 g / L), and cysteine ​​HCl (1 mM) to reach the final concentrations shown in Table 18. Divide 490 μL of each medium mix into three wells of a 96-well deep-well plate, and add 10 μL of inoculum to every well. Place the plate in an anaerobic box flushed with H2 / CO2 (80 / 20) for 10 minutes at a final pressure of 0.8 bar and incubate at 60°C without shaking. After 24 hours, remove the plate from the incubator and transfer the culture from each well to a 96-well microtiter plate. Read the absorbance at 630 nm in a spectrophotometer (Table 19).

[0150] [Table 17]

[0151] [Table 18-1]

[0152] [Table 18-2]

[0153] [Table 18-3]

[0154] [Table 18-4]

[0155] [Table 18-5]

[0156] [Table 19]

[0157] The results of the Plackett-Burman experiment are analyzed in JMP (Figure 9). Within the concentrations tested, components containing selenium, molybdenum, nicotinic acid, calcium-D-(+)-pantothenate, and tungsten have a significant positive effect on cell proliferation, while copper, vitamin B12, cobalt, manganese, and thiamine have a significant negative effect. Several response surface experiments are performed based on these results, including analysis of four components: Na2SeO4, Na2MoO4, nicotinic acid, and calcium-D-(+)-pantothenate.

[0158] The response surface experiment was performed as a central composite design, with three center points and two axis values ​​selected. Each matrix was generated using JMP (Table 20). The high and low values ​​were selected based on the Plackett-Burman experiment, and the center and axis points were calculated using JMP. Because all factors had a positive effect in the Plackett-Burman experiment, the low values ​​in the RSM were selected as the high values ​​from the Plackett-Burman experiment: 1, 0.6, 0.1, and 0.1 mg / L for Na2SeO4, Na2MoO4·2H2O, nicotinic acid, and calcium-D-(+)-pantothenate, respectively. The high levels were 10 times higher than the low levels for each factor, resulting in 10, 6, 1, and 1 mg / L for Na2SeO4, Na2MoO4·2H2O, nicotinic acid, and calcium-D-(+)-pantothenate, respectively.

[0159] [Table 20]

[0160] The entire procedure for the response surface experiment was carried out in an anaerobic chamber. The 27 medium solutions were prepared according to the response surface matrix (Table 20). The concentrations of Na2SeO4, Na2MoO4·2H2O, nicotinic acid, and calcium-D-(+)-pantothenate were adjusted to MES monohydrate (final concentration 20 g / L; pH 6.5), NH4Cl (0.4 g / L), KH2PO4 (0.5 g / L), CaCl2·2H2O (0.05 g / L), MgCl2·6H2O (0.33 g / L), NaCl (0.4 g / L), yeast extract (0.5 g / L), biotin (0.12 mg / L), folic acid (0.02 mg / L), pyridoxine HCl (0.1 mg / L), thiamine HCl (0.1 mg / L), riboflavin (0.05 mg / L), and thiamine HCl (0.1 mg / L). The medium was varied by adding stock solutions to a medium containing niacin (0.0005 mg / L), p-aminobenzoic acid (0.05 mg / L), thioctic acid (0.3 mg / L), vitamin B12 (0.0005 mg / L), KAl(SO4)2·12H2O (0.01 mg / L), CoCl2·6H2O (2 mg / L), CuCl2·2H2O (0.01 mg / L), MnSO4·H2O (7.5 mg / L), FeSO4·7H2O (5.6 mg / L), nitrilotriacetic acid (20 mg / L), Na2WO4·2H2O (0.2 mg / L), ZnSO4·7H2O (2 mg / L), resazurin (0.001 g / L), and cysteine ​​HCl (1 mM). Divide 490 μL of each solution into three wells of a 96-well deep-well plate and add 10 μL of inoculum to all wells. Place the plate in an anoxic box flushed with H2 / CO2 (80 / 20) for 10 minutes and with a final pressure of 0.8 bar. Incubate the box at 60°C without shaking. After 24 hours, remove the plate from the incubator and transfer each solution to a 96-well microtiter plate. Read the absorbance at 630 nm in a spectrophotometer (Table 21).

[0161] [Table 21]

[0162] The results were analyzed using JMP, which generated a second-order polynomial equation (Equation 4) and corresponding response surface plot (Figure 10). The analysis shows that within the concentrations tested and when the carbon source is CO, biomass concentration increases with decreasing NaMoO. Concurrently, biomass concentration increases with increasing NaSeO until a critical level is reached, after which it begins to decrease.

[0163] 0.1182222222222222+0.00352777777777778 * ((Na2SeO4-5.5) / 4.5)+ -0.000611111111111111 * ((Na2MoO4-3.3) / 2.7)+-0.00252777777777778 * ((nicotinic acid -0.55) / 0.45) +-0.00138888888888889 * ((Ca-D-pantothenic acid-0.55) / 0.45)+(Na2SeO4-5.5) / 4.5 * (Na2MoO4-3.3) / 2.7 * 0.00241666666666667 +(Na2SeO4-5.5) / 4.5 * (nicotinic acid-0.55) / 0.45 * -0.000083333333333332 +(Na2MoO4-3.3) / 2.7 * (nicotinic acid-0.55) / 0.45 * 0.000583333333333334+(Na2SeO4-5.5) / 4.5 * (Calcium-D-pantothenate-0.55) / 0.45 * 8.10795832051072e-19 +(Na2MoO4-3.3) / 2.7 * (Ca-D-pantothenic acid-0.55) / 0.45 * 0.0003333333333333333+(nicotinic acid-0.55) / 0.45 * (Ca-D-pantothenic acid-0.55) / 0.45* 0.0015+(Na2SeO4-5.5) / 4.5 * (Na2SeO4-5.5) / 4.5 * -0.00401388888888889+(Na2MoO4-3.3) / 2.7 * (Na2MoO4-3.3) / 2.7 * -0.000472222222222224+(nicotinic acid-0.55) / 0.45 * (nicotinic acid-0.55) / 0.45 * 0.00023611111111111 + (Ca-D-pantothenic acid - 0.55) / 0.45 * (Ca-D-pantothenic acid-0.55) / 0.45 * -0.000805555555555556 Equation 4. Second-order polynomials obtained from RSM experiments with Na2SeO4, Na2MoO4, nicotinic acid, and calcium-D-pantothenate.

[0164] Example 6 This experiment describes the enhanced biomass production of Moorella thermoacetica in growth media containing novel concentrations of specific nutrients when CO2 was used as the carbon source.

[0165] The experimental procedure was carried out in an anaerobic chamber to ensure an anoxic environment. Sterile and anaerobic stock solutions of each component were prepared according to Examples 4 and 5. Two media, the "original" medium and the "improved" medium, were prepared to reach the concentrations shown in Table 22. 490 μL of each solution was divided into three wells of a 96-well deep-well plate, and 10 μL of inoculum was added to all wells. The plate was placed in an anoxic box flushed with H2 / CO2 (80 / 20) for 10 minutes and with a final pressure of 0.8 bar. The box was incubated at 60 °C without shaking. After 24 hours, the plate was removed from the incubator, and each solution was transferred to a 96-well microtiter plate. The absorbance was read at 630 nm in a spectrophotometer (Table 11). Thus, the improved medium showed an average 27% increase in biomass concentration compared to the original medium.

[0166] [Table 22]

Claims

1. CO 2 1. A method for increasing the growth efficiency in anaerobic microbial fermentation of microorganisms capable of converting selenium, CO, or mixtures thereof to at least one organic compound, the method comprising growing the microorganisms in a culture medium containing selenium at a concentration of at least 1.2 μM.

2. CO 2 1. A method for increasing the growth efficiency in anaerobic microbial fermentation of microorganisms capable of converting HCl, CO, or mixtures thereof to at least one organic compound, the method comprising growing the microorganisms in a culture medium containing nickel at a concentration ranging from 3.0 μM to 8.5 μM.

3. CO 2 1. A method for increasing the growth efficiency in anaerobic microbial fermentation of microorganisms capable of converting HCl, CO, or mixtures thereof to at least one organic compound, the method comprising growing the microorganisms in a culture medium containing molybdenum at a concentration of at least 1.5 μM.

4. 4. The method of claim 1, wherein the culture medium comprises selenium at a concentration of at least 1.3 μM, at least 2 μM, at least 3 μM, or at least 4 μM.

5. 5. The method of claim 1, wherein the culture medium comprises selenium at a concentration in the range of 2.0 μM to 8 μM, in the range of 3.0 μM to 7 μM, in the range of 4 μM to 6 μM, or in the range of 4.4 μM to 5.6 μM.

6. 6. The method of claim 1, wherein the culture medium comprises nickel at a concentration in the range of 4.0 μM to 8.0 μM, in the range of 5.0 μM to 7.0 μM, or in the range of 5.5 μM to 7.0 μM.

7. 7. The method of any one of claims 1 to 6, wherein the culture medium comprises molybdenum at a concentration of at least 1.7 μM, at least 2.0 μM, or at least 2.2 μM.

8. The microorganism is CO 2 The method according to any one of claims 1 to 7, wherein the compound (I) is capable of converting the compound (I) into at least one organic compound.

9. 9. The method according to any one of claims 1 to 8, wherein the microorganism is capable of converting CO into at least one organic compound.

10. The method according to any one of claims 1 to 9, wherein the microorganism is an acetogen.

11. 11. The method of any one of claims 1 to 10, wherein the microorganism is selected from the group consisting of Clostridium, Moorella, Thermoanaerobacter, Thermoanaerobacterium, Acetogenium, Acetobacterium, Acetoanaerobium, Butyribacterium, Eubacterium, Pyrococcus, Desulfobacterium and Carboxydothermus.

12. The method of any one of claims 1 to 11, wherein the at least one organic compound comprises acetate.

13. but, The anaerobic microbial fermentation comprises the culture medium and CO 2 13. The method according to any one of claims 1 to 12, wherein the production of said at least one organic compound is from a gas comprising CO, CO or mixtures thereof.

14. CO 2 14. Use of the method according to any one of claims 1 to 13 for the industrial production of at least one organic compound from , CO or mixtures thereof.

15. 15. The use of claim 14, wherein the at least one organic compound comprises acetate.

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