Optimized fermentation of anaerobic bacteria

Abstract

The present invention provides a method for increasing the growth efficiency of a microorganism capable of converting CO2, CO or a mixture thereof to at least one organic compound in anaerobic microbial fermentation, the method comprising growing the microorganism in a culture medium comprising selenium at a concentration of at least 1.2 [mu] M.

Description

Technical Field

[0001] The present invention relates to the fields of biotechnology, microbiology, fermentation, and the conversion of microbial growth and carbon dioxide or carbon monoxide into organic compounds. Background Art

[0002] During many industrial processes (primarily manufacturing, raw material extraction, energy, and transportation), carbon emission gases such as carbon dioxide (CO 2 ) and carbon monoxide (CO) are formed. CO 2 and CO have traditionally been emitted into the atmosphere as relatively harmless compounds that enter the global carbon cycle, while the focus of environmental and health safety has been on restricting the emissions of other gases (including toxic gases such as NOx, SO 2 , methane, etc.). Due to the increasing global population, persistent global warming, and climate change, there is an urgent need for industrial processes that are inherently more circular (i.e., leaving little footprint and not causing large emissions that disrupt the global balance). Without a doubt, this task will require many different efforts in order to intervene in ongoing developments in a timely manner, which are very fast compared to the rate of fundamental changes to the industrial processes that support humanity and the existing population. Examples are the replacement of the combustion of fossil fuels with wind, solar, hydro, and nuclear power generation, and the minimization of CO 2 and CO emissions from industrial processes through alternative processes or by capturing CO 2 and CO.

[0003] Capturing CO 2 and CO from industrial processes can be carried out essentially in two different ways. First, the CO of the industrial process can be collected 2 and stored so that it does not enter the atmosphere. Attempts have been made to pump CO 2 into underground chambers, such as in areas where oil and gas exploration has been carried out. There are also large-scale processes where CO 2 is liquefied and transported to a suitable location for long-term storage. However, from the perspective of sustainability, the concept of storing CO 2 is indeed a less than ideal solution. Second, CO 2 can be collected from the industrial process in which it is produced and then used in another process to convert it into some commercially valuable carbon-containing products.

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

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

[0006] US 2015 / 0024449 discloses methods and media for fermenting syngas, in which the selenium level in biomass is reduced.

[0007] Redl et al., Frontiers in Microbiology (2020), Volume 10, Pages 1-15 disclose media for culturing species of the genus Moorella.

[0008] Therefore, there is a great need for new and effective methods for converting CO 2 or CO emissions from industrial processes into commercially valuable products, thereby achieving the dual purpose of eliminating CO from emissions 2 or CO and manufacturing products that can ensure the commercial viability of the method. SUMMARY OF THE INVENTION

[0009] The object of the present invention is to provide a method for converting CO from emissions 2 and CO into organic compounds. In particular, the object of the present invention is to provide an improved fermentation medium useful in a microbial process for converting CO 2 or CO into at least one organic compound. Another object of the present invention is to provide an improved fermentation medium that promotes a cost-effective process (e.g., low medium cost and low cooling requirements). The object of the present invention is also to overcome some of the disadvantages of the methods known in the art.

[0010] The inventors unexpectedly found that a medium with high concentrations of selenium and molybdenum promotes a high growth rate and high biomass yield of acetic acid-producing microorganisms. The inventors also found that, compared with some previous teachings, the nickel concentration and magnesium concentration have novel and optimal ranges in terms of the growth rate and yield of acetic acid-producing microorganisms.

[0011] In a first aspect, the present invention provides a method for increasing the growth efficiency of a microorganism capable of converting CO 2 , CO or a mixture thereof into at least one organic compound in anaerobic microbial fermentation, the method comprising growing the microorganism in a 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 of a microorganism for converting CO 2 , CO or a mixture thereof into at least one organic compound in anaerobic microbial fermentation, the method comprising growing the microorganism in a medium comprising 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 enhancing the growth efficiency of a microorganism capable of converting CO 2 , CO or a mixture thereof into at least one organic compound in anaerobic microbial fermentation, the method comprising growing the microorganism in a culture medium containing molybdenum at a concentration of at least 1.5 μM.

[0014] In a fourth aspect, the present invention provides a method for converting the anaerobic microbial fermentation of a microorganism capable of converting CO 2 , CO or a mixture thereof into at least one organic compound from a major biomass growth phase to a major product formation phase, wherein the product is the at least one organic compound, and the method comprises growing the microorganism in a culture medium in which the concentrations of molybdenum and nickel remain substantially constant during the conversion, and the concentrations of selenium and magnesium both increase by 5 - 15 times during the conversion.

[0015] In a fifth aspect, the present invention provides the use of the method as defined in any one of the first to fourth aspects for the industrial manufacture of at least one organic compound from CO 2 , CO or a mixture thereof.

[0016] In an embodiment of the present invention, the microorganism is an acetogen, i.e., a microorganism having a metabolism of converting CO 2 (utilizing H 2 ) or CO into acetyl-CoA.

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

[0018] The present invention is further summarized in the following list of items:

[0019] 1. A method for enhancing the growth efficiency of a microorganism capable of converting CO 2 , CO or a mixture thereof into at least one organic compound in anaerobic microbial fermentation, the method comprising growing the microorganism in a culture medium containing selenium at a concentration of at least 1.2 μM.

[0020] 2. A method for improving the growth efficiency of microorganisms that convert CO 2 , CO or a mixture thereof into at least one organic compound in anaerobic microbial fermentation, the method comprising growing the microorganisms in a culture medium comprising nickel at a concentration in the range of 3.0 μM to 8.5 μM.

[0021] 3. A method for improving the growth efficiency of microorganisms that convert CO 2 , CO or a mixture thereof into at least one organic compound in anaerobic microbial fermentation, the method comprising growing the microorganisms in a culture medium comprising molybdenum at a concentration of at least 1.5 μM.

[0022] 4. The method according to any one of the preceding items, 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.

[0023] 5. The method according to any one of the preceding items, 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.

[0024] 6. The method according to any one of the preceding items, wherein the culture medium comprises selenium at a concentration in the range of 4.4 μM to 5.6 μM.

[0025] 7. The method according to any one of the preceding items, wherein the microorganisms grow using hexoses such as fructose or glucose as the main carbon source.

[0026] 8. The method according to any one of the preceding items, wherein 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.

[0027] 9. The method according to any one of the preceding items, 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.

[0028] 10. The method according to any one of items 1-6 and 8-9, wherein the microorganisms grow using gases such as CO 2 or CO as the main carbon source.

[0029] 11. The method according to any one of the preceding items, 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.

[0030] 12. A method according to any one of the preceding items, wherein the culture medium contains molybdenum at a concentration of at least 1.7 μM, at least 2.0 μM or at least 2.2 μM.

[0031] 13. A method according to any one of the preceding items, wherein the culture medium contains molybdenum at a concentration in the range of 1.5 μM to 20 μM, in the range of 1.5 μM to 10 μM or in the range of 2.0 μM to 5 μM.

[0032] 14. A method according to any one of the preceding items, wherein the concentration of magnesium in the culture medium is in the range of 0.05 mM to 40 mM.

[0033] 15. A method according to item 14, wherein the microorganism grows using hexose as the main carbon source, and the concentration of magnesium in the culture medium is in the range of 0.05 mM to 1.3 mM, 0.10 mM to 0.8 mM or 0.15 mM to 0.30 mM.

[0034] 16. A method according to item 14, wherein the microorganism grows using a gas such as CO 2 or CO as the main carbon source, and the concentration of magnesium in the culture medium is in the range of 10 mM to 40 mM, 15 mM to 35 mM or 18 mM to 27 mM.

[0035] 17. A method according to any one of the preceding items, wherein the microorganism is capable of converting CO 2 into at least one organic compound.

[0036] 18. A method according to any one of the preceding items, wherein the microorganism is capable of converting CO into at least one organic compound.

[0037] 19. A method according to any one of the preceding items, wherein the at least one organic compound is a C 1-6 compound.

[0038] 20. A method according to item 9a, wherein the C 1-6 compound is a C 1-6 alcohol, a C 1-6 carboxylic acid or a C 1-6 ketone.

[0039] 21. A method according to any one of the preceding items, wherein the microorganism is an acetogen.

[0040] 22. The method according to any one of the preceding items, wherein the microorganism is selected from the group consisting of Clostridium, Moorella, Thermoanaerobacterium, Thermoanaerobacter, Acetogenium, Acetobacterium, Anaerovibrio, Butyribacterium, Eubacterium, Pyrococcus, Desulfobacterium, and Thermophilaceae.

[0041] 23. The method according to item 22, wherein the microorganism is Moorella or Thermoanaerobacterium.

[0042] 24. The method according to item 23, wherein the microorganism is Moorella thermoacetica or Moorella thermoautotrophica.

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

[0044] 26. The method according to any one of the preceding items, wherein the at least one organic compound comprises ethanol.

[0045] 27. The method according to any one of the preceding items, wherein the anaerobic microbial fermentation is a biomass proliferation step.

[0046] 28. The method according to any one of items 1-9 and 11-27, wherein the culture medium contains hexose as the main carbon source.

[0047] 29. The method according to item 28, wherein the hexose is fructose or glucose.

[0048] 30. The method according to any one of items 1-26, wherein the anaerobic microbial fermentation is to produce the at least one organic compound from the culture medium and a gas comprising CO 2 , CO or a mixture thereof.

[0049] 31. The method according to item 30, wherein the gas is syngas, i.e., a mixture comprising CO and H 2 .

[0050] 32. The method according to any one of the preceding items, wherein the culture medium contains nutrients selected from the following: Al, Mn, Fe, Co, Zn, and Cu.

[0051] 33. The method according to any one of the preceding items, wherein the culture medium contains Mn, Fe, Co, Zn, and Cu.

[0052] 34. The method according to any one of the preceding items, wherein the culture medium comprises nutrients selected from the following: biotin, folic acid, pyridoxine, thiamine, riboflavin, niacin, D-pantothenate, vitamin B12, p-aminobenzoic acid, and lipoic acid.

[0053] 35. The method according to any one of the preceding items, wherein the culture medium comprises biotin, folic acid, pyridoxine, thiamine, riboflavin, niacin, D-pantothenate, vitamin B12, p-aminobenzoic acid, and lipoic acid.

[0054] 36. The method according to any one of the preceding items, wherein the growth efficiency is the specific growth rate.

[0055] 37. The method according to any one of the preceding items, wherein the growth efficiency is the biomass yield per carbon substrate provided in the culture medium.

[0056] 38. The method according to any one of the preceding items, wherein the microbial fermentation is batch fermentation or fed-batch fermentation.

[0057] 39. The method according to any one of the preceding items, wherein the microbial fermentation is continuous fermentation.

[0058] 40. The method according to any one of the preceding items, wherein the microbial fermentation is an industrial-scale process.

[0059] 41. The method according to item 40, wherein the industrial-scale process is at least 5 m 3 scale, at least 30 m 3 scale or at least 80 m 3 scale of fermentation.

[0060] 42. A method for converting the anaerobic microbial fermentation of a microorganism capable of converting CO 2 , CO or a mixture thereof into at least one organic compound from the main biomass growth stage to the main product formation stage, wherein the product is the at least one organic compound, and the method comprises growing the microorganism in a culture medium in which the concentrations of molybdenum and nickel remain substantially constant during the conversion, and the concentrations of selenium and magnesium both increase by 5 - 15 times during the conversion.

[0061] 43. The method according to item 42, wherein the concentrations of selenium and magnesium both increase by 7 - 13 times during the conversion.

[0062] 44. The method according to any one of items 42 - 43, wherein the concentration of selenium in the culture medium used before the conversion is in the range of 2.0 μM to 8 μM, and the concentration of magnesium in the culture medium used before the conversion is in the range of 0.15 mM to 0.30 mM.

[0063] 45. The method according to any one of items 42 - 44, wherein the concentration of molybdenum in the culture medium used before the conversion is at least 1.5 μM, and the concentration of nickel in the culture medium used before the conversion is in the range of 3.0 μM to 8.5 μM.

[0064] 46. The method according to any one of items 42 - 45, wherein the main biomass growth stage occurs in a culture medium in which one or more hexoses are the main carbon source.

[0065] 47. The method according to any one of items 42 - 46, wherein the main product formation stage occurs in a culture medium in which gases such as CO 2 or CO are the main carbon source.

[0066] 48. The method according to any one of items 42 - 47, wherein the main biomass growth stage is carried out by batch or fed - batch fermentation.

[0067] 49. The method according to any one of items 42 - 48, wherein the main product formation stage is carried out by fed - batch fermentation or continuous fermentation.

[0068] 50. The method according to any one of items 42 - 49, wherein the culture medium conversion lasts less than 5 hours, less than 2 hours, less than 1 hour or less than 30 minutes.

[0069] 51. Use of the method according to any one of items 1 - 50 for the industrial manufacture of at least one organic compound from CO 2 , CO or a mixture thereof.

[0070] 52. The use according to item 51, wherein the at least one organic compound comprises C 1-6 compounds, such as C 1-6 alcohols, C 1-6 carboxylic acids or C 1-6 ketones.

[0071] 53. The use according to item 52, wherein the at least one organic compound comprises acetate.

[0072] 54. The use according to any one of items 51 - 53, wherein the at least one organic compound comprises ethanol. BRIEF DESCRIPTION OF THE DRAWINGS

[0073] Figure 1 . Significance of the compounds tested using the Plackett - Burman design on the growth of Moorella thermoacetica with fructose. A t - value above 1.65 or below - 1.65 indicates a significant effect at the 90% confidence level.

[0074] Figure 2 . Generated response surface plots showing the effect of yeast extract and MgCl 2 concentration on biomass production after 24 - hour fermentation with fructose.

[0075] Figure 3 . Significance of the compounds tested using the Plackett - Burman design on the growth of Moorella thermoacetica with fructose. A t - value above 1.65 or below - 1.65 indicates a significant effect at the 90% confidence level.

[0076] Figure 4 . Generated response surface plots showing the effect of Na 2 MoO 4 , NiCl 2 and Na 2 SeO 4 concentration on biomass production after 24 - hour fermentation with fructose.

[0077] Figure 5 . Growth patterns of Moorella thermoacetica in modified and original media using fructose as a carbon source. Cell density was measured using a real - time monitoring device.

[0078] Figure 6 . Acetate concentrations reached by Moorella thermoacetica after 63 - hour fermentation in modified and original media using fructose as a carbon source.

[0079] Figure 7 . Significance of the compounds tested using the Plackett - Burman design on the growth of Moorella thermoacetica with CO 2 growth. A t - value above 1.65 or below - 1.65 indicates a significant effect at the 90% confidence level.

[0080] Figure 8 . Generated response surface plots showing the effect of MgCl 2 concentration on biomass production after 24 - hour fermentation using CO 2 as a carbon source.

[0081] Figure 9 . Significance of the compounds tested using the Plackett - Burman design on the growth of Moorella thermoacetica with CO 2Significance of growth. A t-value above 1.65 or below -1.65 indicates a significant effect at the 90% confidence level.

[0082] Figure 10 .The generated response surface plot showing the effect of the concentration of Na 2 SeO 4 and Na 2 MoO 4 on the biomass yield after 24 hours of fermentation using CO 2 as a carbon source.

[0083] Figure 11 .Absorbance, which indicates the biomass concentration in the modified medium and the original medium after 24 hours of fermentation using CO 2 as a carbon source. Detailed Description

[0084] Unless specifically defined herein, all technical and scientific terms used have the same meaning as commonly understood by one of ordinary skill in the art in the fields of biotechnology, microbiology, fermentation, and microbial growth and product formation.

[0085] All methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, where 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. In addition, unless otherwise noted, the materials, methods, and examples are illustrative only and not intended to be limiting.

[0086] Unless otherwise indicated, the practice of the present invention will employ conventional techniques of biotechnology, microbiology, and fermentation technology within the skill of the art. Such techniques are well explained in the literature.

[0087] culture medium

[0088] In a first aspect, the present invention provides a method for improving the growth efficiency of a microorganism capable of converting CO 2 , CO, or a mixture thereof into at least one organic compound in anaerobic microbial fermentation, the method comprising growing the microorganism in a medium comprising selenium at a concentration of at least 1.2 μM.

[0089] In a second aspect, the present invention provides a method for improving the growth efficiency of a microorganism capable of converting CO 2 , CO, or a mixture thereof into at least one organic compound in anaerobic microbial fermentation, the method comprising growing the microorganism in a medium comprising nickel at a concentration in the range of 3.0 μM to 8.5 μM.

[0090] In a third aspect, the present invention provides a method for increasing the growth efficiency of a microorganism that converts CO 2 , CO or a mixture thereof into at least one organic compound in anaerobic microbial fermentation, the method comprising growing the microorganism in a culture medium comprising molybdenum at a concentration of at least 1.5 μM.

[0091] It should be understood that in the culture medium, a selenium concentration of at least 1.2 μM can be combined with nickel at a concentration in the range of 3.0 μM to 8.5 μM and molybdenum at a concentration of at least 1.5 μM. In fact, this combination of the three metals is considered to be a preferred embodiment.

[0092] In an 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 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. In another embodiment, the culture medium comprises selenium at a concentration in the range of 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 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.

[0093] In another embodiment, 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.

[0094] 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. In another embodiment, the culture medium comprises molybdenum at a concentration in the range of 1.5 μM to 20 μM, in the range of 1.5 μM to 10 μM or in the range of 2.0 μM to 5 μM.

[0095] In another embodiment, the concentration of magnesium in the culture medium is in the range of 0.05 mM to 40 mM. In another embodiment, the microorganism grows using hexose as the main carbon source, and the concentration of magnesium in the culture medium is in the range of 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 uses a gas such as CO 2grow using C0 as the main carbon source, and the concentration of magnesium in the medium ranges from 10 mM to 40 mM, 15 mM to 35 mM, or 18 mM to 27 mM.

[0096] Different carbon sources can be used to propagate the microorganism before the product formation stage, and in some embodiments, such carbon sources can also be added during the product formation stage to convert CO 2 , CO, or a mixture thereof into at least one organic compound.

[0097] In an embodiment, the microorganism grows using hexose such as fructose or glucose as the main carbon source. In another embodiment, the microorganism grows using CO 2 , CO, or a mixture thereof as the main carbon source.

[0098] Other useful medium components are clear from the examples and items of the present application.

[0099] microorganism

[0100] It should be understood that a series of different microorganisms can be used in the present invention as long as they can convert CO 2 , CO, or a mixture thereof into at least one organic compound. Typically useful microorganisms are anaerobic microorganisms.

[0101] In an embodiment, the microorganism is an acetogen.

[0102] Different acetogens can be used in the present invention. In some embodiments, the microorganism is capable of converting CO 2 into at least one organic compound. In some embodiments, the microorganism is capable of converting CO into at least one organic compound.

[0103] In one embodiment, at least one organic compound is a C 1-6 compound.

[0104] In another embodiment, the C 1-6 compound is a C 1-6 alcohol, a C 1-6 carboxylic acid, or a C 1-6 ketone.

[0105] Other useful microorganisms are selected from the genera Clostridium, Moorella, Thermoanaerobacter, Thermoanaerobacterium, Acetobacterium, Acetobacter, Anaerovibrio, Butyribacterium, Eubacterium, Pyrococcus, Desulfobacterium, and Carboxydothermus.

[0106] In an embodiment, the microorganism is of the genus Moorella or Thermoanaerobacter.

[0107] In another embodiment, the microorganism is Moorella thermoacetica or Moorella thermoautotrophica.

[0108] The microorganism used in the present invention can be a naturally occurring microorganism, it can be a microorganism optimized by selection, and it can be a genetically engineered microorganism, for example, for increasing product formation or for enabling it to synthesize specific organic compounds of interest.

[0109] In an embodiment, at least one organic compound includes acetate. In another embodiment, at least one organic compound is mainly acetate.

[0110] In another embodiment, at least one organic compound includes ethanol. In another embodiment, at least one organic compound is mainly ethanol.

[0111] The growth efficiency of the microorganism indicates how well it grows under certain fermentation conditions. In one embodiment, the growth efficiency is the specific growth rate. In another embodiment, the growth efficiency is the biomass yield per carbon substrate provided in the culture medium.

[0112] example

[0113] Example 1

[0114] This experiment describes the growth improvement of Moorella thermoacetica growing on fructose achieved by changing the composition of the growth medium using statistical methods. Initially, the Plackett - Burman design was used to identify which components had a significant effect on cell growth. Then, the response surface method (RSM) was used to determine the improved concentrations of these components. The factors tested in this experiment were the following eight components: yeast extract, NH 4 Cl, trace elements, vitamins, MgCl 2 、CaCl 2 、KH 2 PO 4 and NaCl.

[0115] The following stock solutions were prepared and sterilized: fructose (180 g / L), yeast extract (100 g / L), NH 4 Cl (40 g / L), MgCl 2 ·6H 2 O (20 g / L), CaCl 2 ·2H 2 O (10 g / L), KH 2 PO 4(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 was filter-sterilized and contained 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), D-(+)-calcium pantothenate (5 mg / L), vitamin B12 (0.5 mg / L), p-aminobenzoic acid (5 mg / L), and lipoic acid (5 mg / L). The trace element solution was prepared by dissolving nitrilotriacetic acid (2 g / L) in water and increasing the pH to 6.0 with 2 M KOH, followed by the addition of MnSO 4 ·H 2 O (1 g / L), Fe(SO 4 ) 2 (NH 4 ) 2 ·6H 2 O (0.8 g / L), CoCl 2 ·6H 2 O (0.2 g / L), ZnSO 4 ·7H2O (0.2 g / L), CuCl 2 ·2H 2 O (20 mg / L), NiCl 2 ·6H 2 O (20 mg / L), Na 2 MoO 4 ·2H 2 O (20 mg / L), Na 2 SeO 4 (20 mg / L), Na 2 WO 4 ·2H 2 O (20 mg / L).

[0116] Each of the eight components was screened at two levels (Table 1). The statistical program JMP was used to generate a Plackett-Burman matrix that described the final concentration of each factor in the 12 experiments conducted (Table 2). The experimental procedures were carried out in an anaerobic chamber to ensure an anaerobic environment. The anaerobic and sterile stock solutions were mixed together 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 achieve the final concentrations indicated in Table 2. 4.9 mL of each medium mixture was aliquoted into three wells of a 24-well deep-well plate, and 0.1 mL of inoculum (OD 600= 3.62) was added to all wells. The plate was placed in an anaerobic chamber and flushed with N 2 / CO 2 (80 / 20) and incubated at 60 °C without shaking at a final pressure of 0.8 bar. After 24 h, the plate was removed from the incubator and the culture from each well was transferred to a 96-well microtiter plate. Absorbance was read at 600 nm in a spectrophotometer (Table 3).

[0117] Table 1. Two levels selected for each factor tested using the Plackett-Burman design and fructose as the carbon source.

[0118]

[0119] Table 2. Plackett-Burman matrix used to examine which of the tested factors have a significant effect on the growth of Acetomicrobium thermoaceticum on fructose

[0120]

[0121] Table 3. Absorbance readings indicating biomass concentration after 24 h of fermentation in the Plackett-Burman experiment

[0122]

[0123] The results of the Plackett-Burman experiment were analyzed using JMP ( Figure 1 ). Yeast extract and MgCl 2 were the only components that had a significant effect on cell growth. MgCl 2 had a significant negative effect on cell growth, while yeast extract had a significant positive effect. Based on these results, response surface methodology (RSM) experiments were conducted to optimize the concentrations of these two components.

[0124] The response surface experiments were conducted using a central composite design with 2 center points and an axial point value of 1.414, and the corresponding matrix was generated by JMP (Table 4). High and low values were selected based on the Plackett-Burman experiment, and the center points and axial points were calculated by JMP. The axial points and center points were used to determine the curvature of the response surface. In the response matrix, -, +, 0, a, and A represent low value, high value, center point, lower axial point, and upper axial point, respectively. Yeast extract was shown to have a positive effect, so the low value of RSM was selected as the high value (2 g / L) in Plackett-Burman. The high value was arbitrarily selected as 10 g / L. Conversely, MgCl 2 ·6H 2O has a negative impact, so the low value in Placket - Burman was chosen as the high value (0.1 g / L) in the response surface experiment. The low value was chosen as 0.02 g / L.

[0125] Table 4. Response surface matrix for optimizing the concentrations of yeast extract and MgCl 2 for the growth of Moorella thermoacetica on fructose

[0126]

[0127] The entire procedure of the response surface experiment was carried out in an anaerobic chamber. Ten culture medium solutions were prepared according to the response surface matrix (Table 4), in which the concentrations of yeast extract and MgCl 2 ·6H 2 O were changed by adding stock solutions to the medium containing: MES monohydrate (final concentration 20 g / L; pH 6.5), NH 4 Cl (0.4 g / L), KH 2 PO 4 (0.5 g / L), trace elements (1% v / v), vitamins (1% v / v), CaCl 2 ·2H 2 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 was aliquoted into 3 wells of a 24 - well deep - well plate, and 0.1 mL of inoculum (OD 600 = 0.343) was added to all wells. The plate was placed in an anaerobic chamber and flushed with N 2 / CO 2 (80 / 20) with a final pressure of 0.8 bar. The chamber 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 5).

[0128] Table 5. Absorbance readings indicating the biomass concentration for the RSM experiment conducted on yeast extract and MgCl 2

[0129]

[0130] The results were analyzed by JMP to generate a second - order polynomial equation (Equation 1) and the corresponding response surface plot ( Figure 2 ). The analysis showed that when Moorella thermoacetica grows on fructose, the biomass concentration increases with the increase in the yeast extract concentration and the decrease in the MgCl 2 concentration.

[0131] 0.738734065270186 +

[0132] + 0.119117490895689 * ((Yeast extract - 6) / 4) +

[0133] + - 0.00856188505625192 * ((MgCl 2 - 0.06) / 0.04) +

[0134] + ((Yeast extract - 6) / 4) * (((MgCl 2 - 0.06) / 0.04) * - 0.04625) +

[0135] + ((Yeast extract - 6) / 4) * (((Yeast extract - 6) / 4) * - 0.0595951861901537) +

[0136] + ((MgCl 2 - 0.06) / 0.04) * (((MgCl 2 - 0.06) / 0.04) * - 0.0161111425222695)

[0137] Equation 1. Quadratic polynomial equation obtained from the RSM experiment conducted on yeast extract and MgCl 2 Example 2

[0138] Example 2

[0139] This experiment describes the statistical optimization of selected trace elements for improving the growth of Thermoacetogenium phaeum on fructose. The Plackett - Burman design was used to identify which components have a significant effect on cell growth, and the response surface methodology (RSM) was used to determine the improved concentrations of these components. The factors tested in this experiment were the following 12 components: KAl(SO 4 ) 2 , CoCl 2 , CuCl 2 , H 3 BO 3 , FeSO 4 , MnSO 4 , NiCl 2 , nitrilotriacetic acid, Na 2 SeO 4 , Na 2 MoO 4 , Na 2 WO 4 and ZnSO 4 .

[0140] Prepare the following stock solutions and sterilize them: fructose (500 g / L), yeast extract (100 g / L), NH 4 Cl (40 g / L), MgCl 2 ·6H 2 O (20 g / L), CaCl 2 ·2H 2 O (10 g / L), KH 2 PO 4 (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; adjusted to pH 6 with KOH), MnSO 4 ·H 2 O (20 g / L), Fe(SO 4 )·7H 2 O (14 g / L), CoCl 2 ·6H 2 O (10 g / L), ZnSO 4 ·7H 2 O (4 g / L), KAl(SO 4 ) 2 ·12H 2 O (1 g / L), CuCl 2 ·2H 2 O (1 g / L), NiCl 2 ·6H 2 0 (1 g / L), Na 2 MoO 4 ·2H 2 O (1 g / L), Na 2 SeO 4 (1 g / L) and Na 2 WO 4 ·2H 2 O (1 g / L). Filter-sterilize the vitamin solution and it contains biotin (2 mg / L), folic acid (2 mg / L), pyridoxine hydrochloride (10 mg / L), thiamine-HCl (5 mg / L), riboflavin (5 mg / L), niacin (5 mg / L), D-(+)-calcium pantothenate (5 mg / L), vitamin B12 (0.5 mg / L), p-aminobenzoic acid (5 mg / L), lipoic acid (5 mg / L).

[0141] Initial screening was performed at two levels (Table 6). The statistical program JMP was used to generate a Plackett-Burman matrix that describes the final concentration of each factor in the 20 experiments conducted (Table 7). The experimental procedures were carried out in an anaerobic chamber to ensure an anaerobic environment. The anaerobic and sterile stock solutions were mixed together with MES monohydrate (final concentration 20 g / L; pH 6.5), fructose (5 g / L), yeast extract (0.5 g / L), NH 4 Cl (0.4 g / L), MgCl 2 ·6H 2 O (0.33 g / L), CaCl 2 ·2H 2 O (0.05 g / L), KH 2 PO 4 (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 indicated in Table 7. 490 μL of each medium mixture was aliquoted into 3 wells of a 96-well deep well plate, and 10 μL of inoculum (OD 600 = 0.6) was added to all wells. The plate was placed in an anaerobic chamber, flushed with N 2 / CO 2 (80 / 20), and incubated at 60 °C without shaking at a final pressure of 0.8 bar. After 24 hours, the plate was removed from the incubator, and the culture from each well was transferred to a 96-well microtiter plate. The absorbance was read at 630 nm in a spectrophotometer (Table 8).

[0142] Table 6. Two levels selected for each factor tested using a Plackett-Burman design and fructose as the carbon source

[0143]

[0144] *NTA = nitrilotriacetic acid

[0145] Table 7. Plackett-Burman matrix used to examine which of the factors tested have a significant effect on the growth of Moorella thermoacetica on fructose

[0146]

[0147]

[0148] *NTA = nitrilotriacetic acid

[0149] Table 8. Absorbance readings indicating the biomass concentration after 24 hours of fermentation in the Plackett-Burman experiment

[0150]

[0151] Analyze the results of the Plackett - Burman experiment with JMP( Figure 3 ). The 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. Further test the five components with the strongest effects (Na 2 SeO 4 , CuCl 2 , NiCl 2 , KAl(SO 4 ) 2 and Na 2 MoO 4 ) using the response surface method in order to optimize their concentrations in the growth medium.

[0152] The response surface experiment is carried out with a central composite design having 3 center points, and the axial point value of 2 is selected, and the corresponding matrix is generated by JMP (Table 9). The high and low values are selected according to the Plackett - Burman experiment, and the center points and axial points are calculated by JMP. CuCl 2 ·2H 2 O and KAl(SO 4 ) 2 ·12H 2 O have a negative effect, so the high level in the RSM is selected as the low level in the Plackett - Burman, that is, for both, it is 0.05 mg / L. For both, the low level is arbitrarily selected as 0.01 mg / L. Na 2 MoO 4 ·2H 2 O, NiCl 2 ·6H 2 O and Na 2 SeO 4 have a positive effect, so the low concentration in the RSM is selected as the high level in the Plackett - Burman, which are 0.2, 0.5, and 0.2 mg / L respectively. The high levels are arbitrarily selected as 0.6, 1.5, and 1 mg / L respectively.

[0153] Table 9. For optimizing KAl(SO 4 ) 2 , CuCl 2 , Na 2 MoO 4 , NiCl 2 and Na 2 SeO 4Response surface matrix of the concentration

[0154]

[0155] The entire procedure for conducting the response surface experiment in the anaerobic chamber. Twenty-nine culture media solutions were prepared according to the response surface matrix (Table 9), where the concentration of Na was changed by adding the stock solution to the culture medium containing the following 2 SeO 4 , CuCl 2 ·2H 2 O, NiCl 2 ·6H 2 O, KAl(SO 4 ) 2 ·12H 2 O and Na 2 MoO 4 ·2H 2 O: MES monohydrate (final concentration 20 g / L; pH 6.5), NH 4 Cl (0.4 g / L), KH 2 PO 4 (0.5 g / L), CaCl 2 ·2H 2 O (0.05 g / L), MgCl 2 ·6H 2 O (0.33 g / L), NaCl (0.4 g / L), fructose (5 g / L), yeast extract (0.5 g / L), vitamins (1% v / v), CoCl 2 ·6H 2 O (2 mg / L), H 3 BO 3 (0.1 mg / L), FeSO 4 ·7H 2 O (2.24 mg / L), MnSO 4 ·H 2 O (10 mg / L), nitrilotriacetic acid (20 mg / L), Na 2 WO 4 ·2H 2 O (0.2 mg / L), ZnSO 4 ·7H 2 O (2 mg / L), resazurin (0.001 g / L) and cysteine-HCl (1 mM). 490 μL of each solution was aliquoted into 3 wells of a 96-well deep-well plate, and 10 μL of the inoculum (OD 600 = 0.948) was added to all wells. The plate was placed in an anaerobic chamber and flushed with N 2 / CO2 The plates were then rinsed with 4% paraformaldehyde (80 / 20) at a final pressure of 0.8 bar. The chamber was incubated at 60°C without shaking. After 24 hours, the plates were 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 10).

[0156] Table 10. Indications for KAl(SO 4 ) 2 , CuCl 2 、Na 2 MoO 4 、NiCl 2 and Na 2 SeO 4 Absorbance readout of biomass concentration for RSM experiments

[0157]

[0158] The results were analyzed using JMP to generate a quadratic polynomial equation (Equation 2) and the corresponding response surface plot ( Figure 4 ). The analysis showed that within the tested boundaries, biomass concentration increased with Na 2 SeO 4 concentration until a critical level, after which it begins to decrease. 2 MoO 4 The highest biomass concentration is reached at the concentration of 2 , the model showed that the highest biomass concentrations were achieved when using nickel concentrations close to the two boundaries tested.

[0159] 1.01783470999122+-0.000929736362079477*((KAl(SO 4 ) 2 -0.03) / 0.02)+

[0160] -0.0123591196301995*((CuCl 2 -0.03) / 0.02)+0.00973611111111112*((Na 2 MoO 4

[0161] -0.4) / 0.2)+0.00254166666666666*((NiCl 2 -1) / 0.5)+0.0833694065482988*((

[0162] Na 2 SeO 4-0.6) / 0.4)+(KAl(SO 4 ) 2 -0.03) / 0.02*(CuCl 2 -0.03) / 0.02*

[0163] -0.0106458333333333+(KAl(SO 4 ) 2 -0.03) / 0.02*(Na 2 MoO 4 -0.4) / 0.2*

[0164] -0.00589583333333332+(CuCl 2 -0.03) / 0.02*(Na 2 MoO 4 -0.4) / 0.2*

[0165] -0.00389583333333333+(KAl(SO 4 ) 2 -0.03) / 0.02*(NiCl 2 -1) / 0.5*

[0166] 0.00177083333333332+(CuCl 2 -0.03) / 0.02*(NiCl 2 -1) / 0.5*

[0167] 0.00135416666666668+(Na 2 MoO 4 -0.4) / 0.2*(NiCl 2 -1) / 0.5*

[0168] -0.0174791666666667+(KAl(SO 4 ) 2 -0.03) / 0.02*(Na 2 SeO 4 -0.6) / 0.4*

[0169] 0.00422916666666669+(CuCl 2 -0.03) / 0.02*(Na 2 SeO 4 -0.6) / 0.4*

[0170] 0.00706249999999999+(Na 2 MoO4 -0.4) / 0.2*(Na 2 SeO 4 -0.6) / 0.4*0.0138125+(

[0171] NiCl 2 -1) / 0.5*(Na 2 SeO 4 -0.6) / 0.4*-0.0046875+(KAl(SO 4 ) 2 -0.03) / 0.02

[0172] *(KAl(SO 4 ) 2 -0.03) / 0.02*0.071733898175278+(CuCl 2 -0.03) / 0.02*(CuCl 2

[0173] -0.03) / 0.02*0.0817094897534409+(Na 2 MoO 4 -0.4) / 0.2*(Na 2 MoO 4 -0.4) / 0.2

[0174] *0.0576734726195067+(NiCl 2 -1) / 0.5*(NiCl 2 -1) / 0.5*0.0594651392861734

[0175] +(Na 2 SeO 4 -0.6) / 0.4*(Na 2 SeO 4 -0.6) / 0.4*-0.048660176726902

[0176] Equation 2. The quadratic polynomial equation obtained from the RSM experiment conducted on KAl(SO 4 ) 2 , CuCl 2 , Na 2 MoO 4 , NiCl 2 and Na 2 SeO 4 and the quadratic polynomial equation obtained from the RSM experiment conducted on KAl(SO

[0177] Example 3.

[0178] This example describes the improved biomass yield of Thermoanaerobacter acetigenes in a growth medium containing fructose as a carbon source and new concentrations of certain nutrients.

[0179] The procedure was carried out in an anaerobic chamber to ensure an anaerobic environment. Sterile and anaerobic stock solutions of each component were prepared as in Example 1 and Example 2. Two media, designated "original" medium and "modified" medium, were prepared by mixing the stock solutions to achieve the concentrations shown in Table 11. Then, 49 mL of each medium was aliquoted into three 125 mL serum bottles and inoculated with a growth culture of Thermoanaerobacter acetigenes (OD 600 = 0.86) to achieve an initial OD 600 . The vials were incubated at 60 °C with stirring at 200 rpm using a magnetic stirrer. Biomass concentration was monitored using a real-time cell density monitoring device. The growth patterns in the two media are presented in Figure 5 , which shows that the modified medium supports faster growth and higher biomass density. Based on the endpoint manual OD 600 measurements, after 63 hours of fermentation, the biomass concentration in the modified medium was 63% higher compared to the original medium. At the same time, Figure 6 shows that the modified medium increased the acetate yield by an average of 60%.

[0180] Table 11. Concentrations of components in the "original" medium and "modified" medium using fructose as a carbon source for the fermentation of Thermoanaerobacter acetigenes

[0181]

[0182]

[0183] Example 4

[0184] This example describes the optimization of the medium composition for five macronutrients used particularly for growth with CO 2 and H 2 using the Plackett - Burman design and response surface methodology.

[0185] The following stock solutions were prepared and sterilized: yeast extract (100 g / L), NH 4 Cl (40 g / L), MgCl 2 ·6H 2 O (20 g / L), CaCl 2 ·2H 2 O (10 g / L), KH 2 PO 4(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 contains nitrilotriacetic acid (1 g / L; adjusted to pH 6 with KOH), MnSO 4 ·H 2 O (20 g / L), Fe(SO 4 )·7H 2 O (14 g / L), CoCl 2 ·6H 2 O (10 g / L), ZnSO 4 ·7H2O (4 g / L), CuCl 2 ·2H 2 O (1 g / L), NiCl 2 ·6H 2 0 (1 g / L), Na 2 MoO 4 ·2H 2 O (1 g / L), Na 2 SeO 4 (1 g / L), and Na 2 WO 4 ·2H 2 O (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), niacin (5 mg / L), D-(+)-calcium pantothenate (5 mg / L), vitamin B12 (0.5 mg / L), p-aminobenzoic acid (5 mg / L), lipoic acid (5 mg / L).

[0186] Two levels are selected for each of the factors tested (Table 12). The statistical program JMP is used to generate a Plackett-Burman matrix that describes the final concentration of each factor in the 20 experiments conducted (Table 13). The experimental procedures are carried out in an anaerobic chamber to ensure an anaerobic environment. The anaerobic and sterile stock solutions are mixed together 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 indicated in Table 13. 490 μL of each medium mixture is aliquoted into 3 wells of a 96-well deep well plate, and 10 μL of inoculum is added to all wells. The plate is placed in an anaerobic chamber and flushed with H 2 / CO 2Rinse for 10 minutes with (80 / 20) and incubate at 60 °C without shaking at a final pressure of 0.6 bar. After 24 hours, remove the plates 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 14).

[0187] Table 12. Each factor tested using the Plackett-Burman design and CO 2 Two levels selected for each factor tested as a carbon source

[0188]

[0189] Table 13. Plackett-Burman matrix used to examine which of the factors tested have a significant effect on the growth of Moorella thermoacetica with CO 2 as a carbon source

[0190]

[0191]

[0192] Table 14. Absorbance readings indicating biomass concentration after 24 hours of fermentation in the Plackett-Burman experiment

[0193]

[0194] Analyze the results of the Plackett-Burman experiment using JMP ( Figure 7 ). The analysis shows that when using CO 2 as a carbon source, NH 4 Cl and KH 2 PO 4 have a significant negative impact on growth, while MgCl 2 and NaCl have a significant positive impact. CaCl 2 has no significant effect. Further test NH 4 Cl, KH 2 PO 4 , MgCl 2 and NaCl using the response surface method.

[0195] The response surface experiment was conducted using a central composite design with 3 center points, and the axial point value of 1 was selected, and the corresponding matrix was generated by JMP (Table 15). The high and low values were selected based on the Plackett-Burman experiment, and the center points and axial points were calculated by JMP. In Plackett-Burman, NH 4 Cl and KH 2 PO 4There is a negative impact, so the high concentration in RSM is the same as the low concentration in Plackett - Burman, which are 0.2 and 0.1 g / L respectively. The low values are selected as 0.02 and 0.01 g / L respectively. Since MgCl 2 and NaCl have a positive impact on growth, for both compounds, the low values in RSM are selected as 0.5 and 1 g / L respectively, and the high values are selected as 5 g / L.

[0196] Table 25. Response surface matrix for optimizing the concentrations of NH 2 Cl, MgCl 4 , KH 2 PO 2 and NaCl for the growth of Thermoacetogenium sp. with CO 4

[0197]

[0198] The entire procedure for the response surface experiment was carried out in an anaerobic chamber. Twenty - seven culture medium solutions were prepared according to the response surface matrix (Table 15), where the concentrations of NH 4 Cl, KH 2 PO 4 , MgCl 2 ·6H 2 O and NaCl were changed by adding stock solutions to the medium containing: MES monohydrate (final concentration 20 g / L; pH 6.5), CaCl 2 ·2H 2 O (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 aliquoted into 3 wells of a 96 - well deep - well plate, and 10 μL of inoculum was added to all wells. The plate was placed in an anaerobic chamber and flushed with H 2 / CO 2 (80 / 20) for 10 minutes, with a final pressure of 0.8 bar. The chamber 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 16).

[0199] Table 16. Absorbance readings indicating the biomass concentration for the RSM experiments conducted on NH 4 Cl, MgCl 2 , KH 2 PO 4 and NaCl

[0200] ​

[0201]

[0202] Through the JMP analysis results, a quadratic polynomial equation (Equation 3) is generated. MgCl is shown 2 The corresponding response surface plot of the trend is presented in Figure 7 When the carbon source is CO 2 , the biomass concentration increases with the MgCl 2 concentration until it reaches the critical point, after which the biomass concentration levels off even when the MgCl 2 concentration is further increased.

[0203] 0.0969428571428572 + 0.00151851851851852 * ((NH 4 Cl - 0.11) / 0.09)

[0204] + 0.00124074074074074 * ((MgCl 2 - 2.75) / 2.25) + -0.00322222222222222 * ((KH 2 PO 4

[0205] - 0.055) / 0.045) + -0.00566666666666667 * ((NaCl - 3) / 2) + (NH 4 Cl - 0.11) / 0.09

[0206] *(MgCl 2 - 2.75) / 2.25 * 0.00239583333333333 + (NH 4 Cl - 0.11) / 0.09 * (KH 2 PO 4

[0207] - 0.055) / 0.045 * -0.00147916666666667 + (MgCl 2 - 2.75) / 2.25 * (KH 2 PO 4 - 0.055) /

[0208] 0.045 * -0.00185416666666667 + (NH 4 Cl - 0.11) / 0.09 * (NaCl - 3) / 2 *

[0209] 0.000770833333333332 + (MgCl 2-2.75) / 2.25*(NaCl - 3) / 2*

[0210] -0.000104166666666667+(KH 2 PO 4 -0.055) / 0.045*(NaCl - 3) / 2*

[0211] 0.000770833333333333+(NH 4 Cl - 0.11) / 0.09*(NH 4 Cl - 0.11) / 0.09*

[0212] -0.000257142857142861+(MgCl 2 -2.75) / 2.25*(MgCl 2 -2.75) / 2.25*

[0213] -0.00275714285714286+(KH 2 PO 4 -0.055) / 0.045*(KH 2 PO 4 -0.055) / 0.045*

[0214] 0.00307619047619048+(NaCl - 3) / 2*(NaCl - 3) / 2*-0.00259047619047619

[0215] Equation 3. From the RSM experiments on NH 4 Cl, MgCl 2 、KH 2 PO 4 and NaCl, the quadratic polynomial equation obtained

[0216] Example 5

[0217] This experiment describes the statistical optimization of selected trace elements and vitamins for improving the growth of Thermoacetogenium sp. with H 2 / CO 2 . The Plackett - Burman design was used to identify which components had a significant effect on cell growth, and the response surface methodology (RSM) was used to determine the optimized concentrations of these components. The factors tested in this experiment were the following 14 components: biotin, thiamine - HCl, D-(+)-calcium pantothenate, vitamin B12, niacin, lipoic acid, CoCl 2 、CuCl 2 、FeSO 4 、MnSO 4, NiCl 2 , Na 2 SeO 4 , Na 2 WO 4 , Na 2 MoO 4 .

[0218] Prepare the following stock solutions and sterilize them: yeast extract (100 g / L), NH 4 Cl (40 g / L), MgCl 2 ·6H 2 O (20 g / L), CaCl 2 ·2H 2 O (10 g / L), KH 2 PO 4 (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; adjusted to pH 6 with KOH), MnSO 4 ·H 2 O (20 g / L), Fe(SO 4 )·7H 2 O (14 g / L), CoCl 2 ·6H 2 O (10 g / L), ZnSO 4 ·7H2O (4 g / L), CuCl 2 ·2H 2 O (1 g / L), NiCl 2 ·6H 2 0 (1 g / L), Na 2 MoO 4 ·2H 2 O (1 g / L), Na 2 SeO 4 (1 g / L), Na 2 WO 4 ·2H 2 O (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), D-(+)-calcium pantothenate (1 g / L), vitamin B12 (1 g / L), p-aminobenzoic acid (1 g / L) and lipoic acid (1 g / L).

[0219] Initial screening was carried out at two levels (Table 17). The statistical program JMP was used to generate a Plackett - Burman matrix, which describes the final concentration of each factor in the 23 experiments conducted (Table 18). The experimental procedures were carried out in an anaerobic chamber to ensure an anaerobic environment. The anaerobic and sterile stock solutions were mixed together with MES monohydrate (final concentration 20 g / L; pH 6.5), yeast extract (0.5 g / L), NH 4 Cl (0.4 g / L), MgCl 2 ·6H 2 O (0.33 g / L), CaCl 2 ·2H 2 O (0.05 g / L), KH 2 PO 4 (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), ZnSO 4 ·7H 2 O (2 mg / L), nitrilotriacetic acid (20 mg / L), resazurin (0.001 g / L) and cysteine - HCl (1 mM) to achieve the final concentrations as indicated in Table 18. 490 μL of each medium mixture was aliquoted into 3 wells of a 96 - well deep - well plate, and 10 μL of inoculum was added to all wells. The plate was placed in an anaerobic chamber and flushed with H 2 / CO 2 (80 / 20) for 10 minutes and incubated at 60 °C without shaking at a final pressure of 0.8 bar. After 24 hours, the plate was removed from the incubator, and the culture from each well was transferred to a 96 - well microtiter plate. The absorbance was read at 630 nm in a spectrophotometer (Table 19).

[0220]

[0221]

[0222]

[0223]

[0224]

[0225] Table 19. Absorbance readings indicating biomass concentration after 24 - hour fermentation in the Plackett - Burman experiment

[0226]

[0227] Analysis of the Results of Plackett-Burman Experiments with JMP( Figure 9 ). Among the concentrations tested, the components containing selenium, molybdenum, niacin, D-(+)-calcium pantothenate, and tungsten had a significant positive effect on growth, while the components containing copper, vitamin B12, cobalt, manganese, and thiamine had a significant negative effect. Based on these results, several response surface experiments were conducted, including the analysis of four components, Na 2 SeO 4 、Na 2 MoO 4 、niacin, and D-(+)-calcium pantothenate.

[0228] The response surface experiments were carried out using a central composite design with 3 center points, and the axial point values of 2 were selected, and the corresponding matrix was generated by JMP (Table 20). The high and low values were selected according to the Plackett-Burman experiment, and the center points and axial points were calculated by JMP. Since all factors had a positive effect in Plackett-Burman, the low level in RSM was selected as the high level in Plackett-Burman, that is, for Na 2 SeO 4 、Na 2 MoO 4 ·2H 2 O, niacin, and D-(+)-calcium pantothenate, they were 1, 0.6, 0.1, and 0.1 mg / L respectively. The high level of each factor was 10 times the low level, so that for Na 2 SeO 4 、Na 2 MoO 4 ·2H 2 O, niacin, and D-(+)-calcium pantothenate, they were 10, 6, 1, and 1 mg / L respectively.

[0229] Table 20. Response Surface Matrix for Optimizing the Concentrations of Na 2 for the Growth of Moorella thermoacetica with CO 2 SeO 4 、Na 2 MoO 4 、niacin, and D-calcium pantothenate

[0230]

[0231]

[0232] The entire procedure of the response surface experiment was carried out in an anaerobic chamber. According to the response surface matrix (Table 20), 27 culture medium solutions were prepared, in which the concentration of Na 2 SeO 4 、Na2 MoO 4 ·2H 2 O, concentrations of nicotinic acid and D-(+)-calcium pantothenate: MES monohydrate (final concentration 20 g / L; pH 6.5), NH 4 Cl (0.4 g / L), KH 2 PO 4 (0.5 g / L), CaCl 2 ·2H 2 O (0.05 g / L), MgCl 2 ·6H 2 O (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), pyridoxol-HCl (0.1 mg / L), thiamine-HCl (0.1 mg / L), riboflavin (0.05 mg / L), p-aminobenzoic acid (0.05 mg / L), lipoic acid (0.3 mg / L), vitamin B12 (0.0005 mg / L), KAl(SO 4 ) 2 ·12H 2 O (0.01 mg / L), CoCl 2 ·6H 2 O (2 mg / L), CuCl 2 ·2H 2 O (0.01 mg / L), MnSO 4 ·H 2 O (7.5 mg / L), FeSO 4 ·7H 2 O (5.6 mg / L), nitrilotriacetic acid (20 mg / L), Na 2 WO 4 ·2H 2 O (0.2 mg / L), ZnSO 4 ·7H 2 O (2 mg / L), resazurin (0.001 g / L) and cysteine-HCl (1 mM). 490 μL of each solution was aliquoted into 3 wells of a 96-well deep well plate, and 10 μL of inoculum was added to all wells. The plate was placed in an anaerobic chamber and flushed with H 2 / CO 2 (80 / 20) for 10 minutes, with a final pressure of 0.8 bar. The chamber 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. Absorbance was read at 630 nm in a spectrophotometer (Table 21).

[0233] Table 21. Indicating for Na 2SeO 4 、Na 2 MoO 4 Absorbance readings of biomass concentration for the RSM experiment with SeO, Na, MoO, niacin, and calcium D-pantothenate

[0234]

[0235] By analyzing the results with JMP, a second-order polynomial equation (Equation 4) and the corresponding response surface plot ( Figure 10 ) were generated. The analysis showed that within the tested concentrations and when using CO 2 as the carbon source, the biomass concentration increased with the decrease of Na 2 MoO 4 . At the same time, the biomass concentration increased with the increase of Na 2 SeO 4 concentration until a critical level was reached, after which it started to decline.

[0236] 0.118222222222222+0.00352777777777778*((Na 2 SeO 4 -5.5) / 4.5)+

[0237] -0.000611111111111111*((Na 2 MoO 4 -3.3) / 2.7)+-0.00252777777777778*((

[0238] niacin - 0.55) / 0.45)+-0.00138888888888889*((calcium D-pantothenate

[0239] -0.55) / 0.45)+(Na 2 SeO 4 -5.5) / 4.5*(Na 2 MoO 4 -3.3) / 2.7*0.00241666666666667

[0240] +(Na 2 SeO 4 -5.5) / 4.5*(niacin - 0.55) / 0.45*-0.000083333333333332

[0241] +(Na 2 MoO 4 -3.3) / 2.7*(niacin - 0.55) / 0.45*0.000583333333333334+(

[0242] Na 2 SeO 4 -5.5) / 4.5*(D - Calcium Pantothenate - 0.55) / 0.45*8.10795832051072e - 19

[0243] +(Na 2 MoO 4 -3.3) / 2.7*(D - Calcium Pantothenate - 0.55) / 0.45*

[0244] 0.000333333333333333+(Nicotinic Acid - 0.55) / 0.45*(D - Calcium Pantothenate - 0.55

[0245] ) / 0.45*0.0015+(Na 2 SeO 4 -5.5) / 4.5*(Na 2 SeO 4 -5.5) / 4.5*

[0246] -0.00401388888888889+(Na 2 MoO 4 -3.3) / 2.7*(Na 2 MoO 4 -3.3) / 2.7*

[0247] -0.000472222222222224+(Nicotinic Acid - 0.55) / 0.45*(Nicotinic Acid - 0.55) /

[0248] 0.45*0.00023611111111111+(D - Calcium Pantothenate - 0.55) / 0.45*(

[0249] D - Calcium Pantothenate - 0.55) / 0.45*-0.000805555555555556

[0250] Equation 4. From the RSM experiment on Na 2 SeO 4 、Na 2 MoO 4 、Nicotinic Acid and D - Calcium Pantothenate, the quadratic polynomial equation obtained

[0251] Example 6

[0252] This example describes the improved biomass yield of Moorella thermoacetica in a growth medium containing new concentrations of certain nutrients when using CO 2 as a carbon source.

[0253] The procedure is carried out in an anaerobic chamber to ensure an oxygen-free environment. According to Example 4 and Example 5, sterile and anaerobic stock solutions of each component are prepared. Two media, called "original" medium and "modified" medium, are prepared by mixing the stock solutions to achieve the concentrations shown in Table 22. 490 μL of each solution is aliquoted into 3 wells of a 96-well deep-well plate, and 10 μL of inoculum is added to all wells. The plate is placed in an anaerobic chamber and flushed with H 2 / CO 2 (80 / 20) for 10 minutes, with a final pressure of 0.8 bar. The chamber is incubated at 60 °C without shaking. After 24 hours, the plate is removed from the incubator and each solution is transferred to a 96-well microtiter plate. The absorbance ( Figure 11 ) is read at 630 nm in a spectrophotometer. Thus, the modified medium shows an average 27% increase in biomass concentration compared to the original medium.

[0254] Table 22. Concentrations of medium components in the "original" medium and "modified" medium used for the fermentation of Moorella thermoacetica with CO 2 as the carbon source

[0255]

[0256]

Claims

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

2. A method for increasing the growth efficiency of microorganisms that convert CO 2 , CO or a mixture thereof into at least one organic compound in anaerobic microbial fermentation, the method comprising growing the microorganisms in a culture medium containing nickel at a concentration in the range of 3.0 μM to 8.5 μM.

3. A method for improving the growth efficiency of a microorganism that converts CO 2 , CO or a mixture thereof into at least one organic compound in anaerobic microbial fermentation, the method comprising growing the microorganism in a culture medium containing molybdenum at a concentration of at least 1.5 μM.

4. The method according to any one of the preceding claims, 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. The method according to any one of the preceding claims, 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. The method according to any one of the preceding claims, 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. The method according to any one of the preceding claims, 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 method according to any one of the preceding claims, wherein the microorganism is capable of converting CO 2 into at least one organic compound.

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

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

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

13. The method according to any one of the preceding claims, wherein the anaerobic microbial fermentation produces the at least one organic compound from the culture medium and a gas comprising CO 2 , CO or a mixture thereof.

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

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

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