Method for producing hexanoic acid and octanoic acid by using CO2 biorefinery of mixed flora

Abstract

The invention discloses a method for producing hexanoic acid and octanoic acid by using CO2 biorefinery through mixed flora, and relates to a method for producing hexanoic acid and octanoic acid by using CO2 biorefinery. The problems of low yield and low selectivity of caproic acid and octanoic acid produced by the existing CO2 biorefinery technology are solved. The method comprises the following steps: 1, enriching and domesticating mixed flora; and 2, fermenting the mixed flora. The method is used for producing the hexanoic acid and the octanoic acid through CO2 biorefinery of the mixed flora.

Description

Technical Field

[0001] This invention relates to a method for producing hexanoic acid and octanoic acid using CO2 biorefining. Background Technology

[0002] Hexanoic acid and octanoic acid are medium-chain fatty acids composed of 6 and 8 carbon atoms, respectively. They have high value and are commonly used in the food, pharmaceutical, and industrial sectors, primarily in the production of fragrances, preservatives, pharmaceuticals, lubricants, and plasticizers. Currently, their preparation methods mainly include physical extraction, chemical synthesis, and bio-fermentation. Physical extraction mainly involves the hydrolysis of coconut oil and palm oil rich in medium-chain fatty acid glycerides, as well as vacuum distillation of petroleum. Chemical synthesis mainly involves the oxidation of n-octanol and n-octanal, and carboxyl synthesis using olefins. Compared to physical extraction and chemical synthesis, bio-fermentation has the advantages of widely available raw materials and being environmentally friendly.

[0003] Since the Industrial Revolution, the extensive use of fossil fuels has propelled human modernization. However, their finite quantity and non-renewable nature have significantly limited the development of various industries and brought about severe environmental pollution. The combustion of fossil fuels remains the primary cause of global carbon emissions, and the ever-increasing concentrations of greenhouse gases such as CO2 are exacerbating the greenhouse effect. To address energy demands, environmental pollution, and global climate change, promoting the development of renewable energy technologies is urgently needed. CO2 is the most widely distributed carbon source, and its effective utilization can help mitigate the greenhouse effect. Therefore, using CO2 instead of conventional organic matter as a carbon source for biorefining to produce hexanoic acid and octanoic acid is a promising technology.

[0004] Traditional medium-chain fatty acid (MCFA) bio-fermentation primarily uses expensive raw materials such as glucose, sucrose, and grains, resulting in high production costs. Subsequent developments of fermentation technologies using waste materials like sludge, kitchen waste, and crop straw have reduced costs to some extent, but they suffer from low carbon utilization and low selectivity for MFAs in the products. Specifically, existing bio-fermentation methods for producing hexanoic acid and octanoic acid suffer from low carbon utilization, low yield, and low selectivity for MFAs. While third-generation biorefining technologies using CO2 as a substrate offer higher carbon utilization than traditional fermentation, the products are mainly low-value short-chain fatty acids, primarily acetic acid and butyric acid. Therefore, the current technologies still face the challenges of low MFA yield and low selectivity that urgently need to be addressed. Summary of the Invention

[0005] This invention aims to address the problems of low yield and low selectivity in the production of hexanoic acid and octanoic acid using existing CO2 biorefining technologies, and provides a method for producing hexanoic acid and octanoic acid using a mixed microbial community through CO2 biorefining.

[0006] A method for producing hexanoic acid and octanoic acid using CO2 biorefining with a mixed microbial community comprises the following steps:

[0007] I. Enrichment and domestication of mixed microbial communities:

[0008] Ethanol-containing enrichment medium was added to the enrichment and domestication reactor. Under a mixed atmosphere, the initial mixed bacterial population was inoculated and the pH was adjusted. Then, enrichment and domestication were carried out under a mixed atmosphere until the ethanol was completely consumed, and the enriched culture fermentation broth was obtained. A portion of the enriched culture fermentation broth was taken out and inoculated into a new enrichment and domestication reactor for the next cycle of enrichment and domestication. This process was repeated multiple times until the maximum content of hexanoic acid and octanoic acid obtained by enrichment and domestication in the cycle no longer increased compared with the previous cycle. The enrichment and domestication was then completed, and the fermentation broth after the enrichment and domestication was obtained.

[0009] II. Mixed microbial fermentation:

[0010] Ethanol-containing fermentation medium is added to the fermentation reactor. Under a mixed atmosphere, the fermentation broth after enrichment and domestication is inoculated and the pH is adjusted. Fermentation is then carried out under a mixed atmosphere until the ethanol is completely consumed. The fermentation ends, thus completing the method of producing hexanoic acid and octanoic acid by using CO2 biorefining with mixed microbial communities.

[0011] The beneficial effects of this invention are:

[0012] (1) This invention enriches the mixed microbial community that produces butyrate and butanol by fermenting straw, changes the community structure, strengthens the cooperation between strains, and changes the microbial community from straw as substrate to CO2 as the main substrate.

[0013] (2) In this invention, after obtaining acetic acid from CO2, the shorter carbon chain and lower value of acetic acid are converted into longer chain and higher value medium-chain fatty acids by using ethanol, thus realizing the production of hexanoic acid and octanoic acid from CO2.

[0014] (3) The method of producing hexanoic acid and octanoic acid by using CO2 biorefining with mixed microbial community of the present invention can effectively utilize greenhouse gases to produce high-value medium-chain fatty acids, and does not require sterilization operation. It has the advantages of low cost, simple operation and high carbon utilization rate. Attached Figure Description

[0015] Figure 1 The fatty acid content and proportion of each generation during the enrichment and domestication of the mixed microbial community in step one of Experiment Example 1;

[0016] Figure 2 This is a diagram showing the acid production from the fermentation of hexanoic acid and octanoic acid after the fermentation process in step two of Experiment Example 1.

[0017] Figure 3A comparison of the yield and selectivity of hexanoic acid and octanoic acid in the initial mixed microbial community in step one of Experiment Example 1, and the mixed microbial communities in Experiment Example 1 and Experiment 2 after the enrichment and domestication in step two.

[0018] Figure 4 This is a comparison diagram of the mixed bacterial community structure before and after enrichment and domestication in Experiment Example 1.

[0019] Figure 5 This is a diagram showing the acid production from the fermentation of hexanoic acid and octanoic acid after the fermentation process in step two of Experiment Example 2. Detailed Implementation

[0020] Specific Implementation Method 1: This implementation method describes a method for producing hexanoic acid and octanoic acid using CO2 biorefining with a mixed microbial community. It is carried out according to the following steps:

[0021] I. Enrichment and domestication of mixed microbial communities:

[0022] Ethanol-containing enrichment medium was added to the enrichment and domestication reactor. Under a mixed atmosphere, the initial mixed bacterial population was inoculated and the pH was adjusted. Then, enrichment and domestication were carried out under a mixed atmosphere until the ethanol was completely consumed, and the enriched culture fermentation broth was obtained. A portion of the enriched culture fermentation broth was taken out and inoculated into a new enrichment and domestication reactor for the next cycle of enrichment and domestication. This process was repeated multiple times until the maximum content of hexanoic acid and octanoic acid obtained by enrichment and domestication in the cycle no longer increased compared with the previous cycle. The enrichment and domestication was then completed, and the fermentation broth after the enrichment and domestication was obtained.

[0023] II. Mixed microbial fermentation:

[0024] Ethanol-containing fermentation medium is added to the fermentation reactor. Under a mixed atmosphere, the fermentation broth after enrichment and domestication is inoculated and the pH is adjusted. Fermentation is then carried out under a mixed atmosphere until the ethanol is completely consumed. The fermentation ends, thus completing the method of producing hexanoic acid and octanoic acid by using CO2 biorefining with mixed microbial communities.

[0025] In this specific implementation method, step one is carried out under a mixed atmosphere throughout the enrichment and domestication process and step two fermentation process, and the fermentation material of the previous cycle is inoculated in the next cycle.

[0026] The beneficial effects of this specific implementation method are:

[0027] (1) In this specific embodiment, the mixed microbial community for the fermentation of straw to produce butyrate and butanol is enriched, the community structure is changed, and the cooperation between strains is strengthened, so that the microbial community changes from straw as substrate to CO2 as the main substrate.

[0028] (2) In this specific embodiment, after obtaining acetic acid using CO2, ethanol is used to convert the acetic acid with a shorter carbon chain and lower value into medium-chain fatty acids with a longer chain and higher value, thereby realizing the production of hexanoic acid and octanoic acid from CO2.

[0029] (3) The method of producing hexanoic acid and octanoic acid by using CO2 biorefining with mixed microbial communities in this specific embodiment can effectively utilize greenhouse gases to produce high-value medium-chain fatty acids, and does not require sterilization operation. It has the advantages of low cost, simple operation and high carbon utilization rate.

[0030] Specific Implementation Method Two: This implementation method differs from Specific Implementation Method One in that the mixed atmosphere described in steps one and two is a mixture of hydrogen and carbon dioxide gases, and the volume ratio of hydrogen to carbon dioxide gases is (2~4):1. Everything else is the same as in Specific Implementation Method One.

[0031] Specific Implementation Method Three: This implementation method differs from Specific Implementation Method One or Two in that the initial mixed microbial community mentioned in Step One is enriched according to the following steps: A mixture of cow dung, pig manure compost, cornfield soil, and decaying wood is placed in a culture medium with straw as the carbon source and anaerobically fermented at a temperature of 35℃~38℃ and a stirring speed of 100rpm~120rpm for 3~5 months of continuous subculturing; the culture medium with straw as the carbon source is composed of pretreated straw, peptone, NaCl, CaCO3, yeast extract, and L-Cys, and the concentration of pretreated straw in the culture medium with straw as the carbon source is 5%. The concentrations of the pretreated straw are as follows: g / L~30 g / L, peptone concentration is 5 g / L~8 g / L, NaCl concentration is 5 g / L~8 g / L, CaCO3 concentration is 5 g / L~8 g / L, yeast extract concentration is 1 g / L~2 g / L, and L-Cys concentration is 0.3 g / L~1 g / L. The pretreated straw is prepared according to the following steps: the straw is cut into 1 cm~2 cm segments, and then soaked in a 1%~3% (w / w) NaOH solution at a temperature of 30℃~60℃ for 72 h~120 h. The soaked straw is then filtered and collected, and washed with water until the pH is neutral. Other steps are the same as in specific implementation method one or two.

[0032] Specific Implementation Method Four: This implementation method differs from Specific Implementation Methods One to Three in that: the inoculation amount of the initial mixed microbial community in step one is 5% to 10% of the volume of the ethanol-containing enrichment medium; the inoculation amount of the enrichment culture fermentation broth in each cycle in step one is 5% to 10% of the volume of the ethanol-containing enrichment medium. Everything else is the same as in Specific Implementation Methods One to Three.

[0033] Specific Implementation Method Five: This implementation method differs from Specific Implementation Methods One to Four in that: in step one, the initial mixed bacterial population is inoculated and the pH is adjusted to 5.5-7; in step one, enrichment and acclimatization are carried out for 32-52 days under mixed atmosphere, pH 5.5-7, temperature 35℃-37℃, and stirring speed 100rpm-120rpm. Everything else is the same as in Specific Implementation Methods One to Four.

[0034] Specific Implementation Method Six: This implementation method differs from Specific Implementation Methods One to Five in that: the ethanol-containing enrichment medium mentioned in step one is composed of ethanol, NaHCO3, NH4Cl, yeast extract, sodium 2-bromoethanesulfonate, L-cysteine, phosphate buffer, macro-element solution, acidic micro-element solution, alkaline micro-element solution, and vitamin solution; and the concentration of ethanol in the ethanol-containing enrichment medium is 7.7 g / L to 8.6 g / L, the concentration of NaHCO3 is 1 g / L to 2 g / L, the concentration of NH4Cl is 1 g / L to 2 g / L, the concentration of yeast extract is 1 g / L to 2 g / L, the concentration of sodium 2-bromoethanesulfonate is 2 g / L to 3 g / L, and the concentration of L-cysteine ​​is 0.5 g / L. The concentrations are as follows: ~1 g / L, phosphate buffer solution 20 mL / L~30 mL / L, macro-element solution 10 mL / L~15 mL / L, acidic trace element solution 1 mL / L~2 mL / L, alkaline trace element solution 1 mL / L~2 mL / L, and vitamin solution 0.2 mL / L~0.5 mL / L. Other aspects are the same as in specific embodiments one through five.

[0035] Specific Implementation Method Seven: This implementation method differs from Specific Implementation Methods One to Six in that the inoculation amount of the fermentation broth after the enrichment and domestication process in step two is 5% to 10% of the volume of the fermentation medium containing ethanol. Everything else is the same as in Specific Implementation Methods One to Six.

[0036] Specific Implementation Method Eight: This implementation method differs from Specific Implementation Methods One to Seven in that: in step two, the fermentation broth after enrichment and domestication is inoculated and the pH is adjusted to 5.5-7; in step two, fermentation is carried out for 21-22 days under mixed atmosphere, pH 5.5-7, temperature 35℃-37℃, and stirring speed 100-120 rpm. Everything else is the same as in Specific Implementation Methods One to Seven.

[0037] Specific Implementation Method Nine: This implementation method differs from Specific Implementation Methods One through Eight in that: the ethanol-containing fermentation medium described in step two is composed of ethanol, NaHCO3, NH4Cl, yeast extract, L-cysteine, phosphate buffer, macro-element solution, acidic micro-element solution, alkaline micro-element solution, and vitamin solution; and the concentration of ethanol in the ethanol-containing fermentation medium is 8.3 g / L to 11.3 g / L, the concentration of NaHCO3 is 1 g / L to 2 g / L, and the concentration of NH4Cl is... The concentrations of the following solutions are as follows: yeast extract (1 g / L to 2 g / L), L-cysteine ​​(0.5 g / L to 1 g / L), phosphate buffer (20 mL / L to 30 mL / L), macronutrient solution (10 mL / L to 15 mL / L), acidic micronutrient solution (1 mL / L to 2 mL / L), alkaline micronutrient solution (1 mL / L to 2 mL / L), and vitamin solution (0.2 mL / L to 0.5 mL / L). Other aspects are the same as in embodiments one through eight.

[0038] Specific Implementation Method 10: This implementation method differs from Specific Implementation Methods 1 to 9 in that: the phosphate buffer solution is composed of Na2HPO4·12H2O and KH2PO4; and the concentration of Na2HPO4·12H2O in the phosphate buffer solution is 50g / L~55g / L, and the concentration of KH2PO4 is 20g / L~25g / L.

[0039] The macro-element solution is composed of NaCl, CaCl2·2H2O and MgCl2·6H2O; and the concentration of NaCl in the macro-element solution is 30g / L~35g / L, the concentration of CaCl2·2H2O is 10g / L~15g / L, and the concentration of MgCl2·6H2O is 10g / L~15g / L.

[0040] The acidic trace element solution is composed of ZnCl2, CuCl2·H2O, CuSO4·5H2O, MnCl2·4H2O, CoCl2·6H2O, NiCl2·6H2O, H3BO4, and HCl solution; and the concentration of ZnCl2 in the acidic trace element solution is 0.05 g / L~0.1 g / L, the concentration of CuCl2·H2O is 0.02 g / L~0.05 g / L, and the concentration of CuSO4·5H2O is 0.01 g / L. The concentrations of the following components are specified: L~0.03 g / L, MnCl2·4H2O concentration 0.05 g / L~0.1 g / L, CoCl2·6H2O concentration 0.05 g / L~0.1 g / L, NiCl2·6H2O concentration 0.03 g / L~0.05 g / L, H3BO4 concentration 0.05 g / L~0.1 g / L, and HCl solution concentration 1 mL / L~2 mL / L; the HCl solution has a mass percentage of 95%~100%.

[0041] The alkaline trace element solution is composed of Na2SeO3·5H2O, Na2MoO4·2H2O, Na2WO4·2H2O, and NaOH; and the concentrations of Na2SeO3·5H2O, Na2MoO4·2H2O, Na2WO4·2H2O, and NaOH in the alkaline trace element solution are 0.03 g / L to 0.05 g / L, 0.01 g / L to 0.03 g / L, 0.03 g / L, 0.05 g / L, and 0.3 g / L to 0.5 g / L.

[0042] The vitamin solution comprises biotin, vitamin B3, pyridoxine hydrochloride, thiamine, riboflavin, cobalamin, calcium pantothenate, lipoic acid, para-aminobenzoic acid, and folic acid. The concentrations of biotin, vitamin B3, pyridoxine, pyridoxine, thiamine, riboflavin, cobalamin, calcium pantothenate, lipoic acid, para-aminobenzoic acid, and folic acid are all specified in the vitamin solution. The concentrations of biotin, pyridoxine, thiamine, riboflavin, cobalamin, calcium pantothenate, lipoic acid, and folic acid are all specified in the vitamin solution. Other aspects are the same as in embodiments one through nine.

[0043] The beneficial effects of the present invention are verified using the following embodiments:

[0044] Example 1:

[0045] A method for producing hexanoic acid and octanoic acid using CO2 biorefining with a mixed microbial community comprises the following steps:

[0046] I. Enrichment and domestication of mixed microbial communities:

[0047] Ethanol-containing enrichment medium was added to the enrichment and domestication reactor. Under a mixed atmosphere, the initial mixed bacterial population was inoculated and the pH was adjusted to 5.5-7. Then, enrichment and domestication were carried out under mixed atmosphere, pH 5.5-7, temperature 35℃ and stirring speed 110 rpm until the ethanol was completely consumed, and the enriched culture fermentation broth was obtained. A portion of the enriched culture fermentation broth was taken out and inoculated into a new enrichment and domestication reactor for the next cycle of enrichment and domestication. This process was repeated multiple times until the maximum content of hexanoic acid and octanoic acid obtained by enrichment and domestication in the cycle no longer increased compared with the previous cycle. The enrichment and domestication was then completed, and the fermentation broth after the enrichment and domestication was obtained, which is the mixed bacterial population after the enrichment and domestication was completed.

[0048] II. Mixed microbial fermentation:

[0049] Ethanol-containing fermentation medium is added to the fermentation reactor. Under a mixed atmosphere, the fermentation broth after enrichment and domestication is inoculated and the pH is adjusted to 5.5-7. Then, under the conditions of mixed atmosphere, pH 5.5-7, temperature 35℃ and stirring speed 110rpm, fermentation is carried out for 22 days until the ethanol is completely consumed and the fermentation ends. This completes the method of producing hexanoic acid and octanoic acid by using CO2 biorefining with mixed microbial communities.

[0050] The mixed atmosphere mentioned in Step 1 and Step 2 is a mixture of hydrogen and carbon dioxide gas, and the volume ratio of hydrogen to carbon dioxide gas is 4:1.

[0051] The initial mixed microbial community mentioned in step one is enriched according to the following steps: a mixture of cow dung, pig manure compost, cornfield soil, and decaying wood is placed in a culture medium with straw as the carbon source and anaerobically fermented at 35°C and a stirring speed of 120 rpm for 5 consecutive subcultures; the culture medium with straw as the carbon source consists of pretreated straw, peptone, NaCl, and CaCO3. The culture medium, composed of yeast extract and L-Cys, with straw as the carbon source, contains pretreated straw at concentrations of 10 g / L, peptone at 5 g / L, NaCl at 5 g / L, CaCO3 at 5 g / L, yeast extract at 1 g / L, and L-Cys at 0.5 g / L. The pretreated straw is prepared according to the following steps: the straw is cut into 1 cm to 2 cm segments, and then soaked in a 1% (w / w) NaOH solution at 50°C for 72 h. The soaked straw is then collected by filtration and washed with water until the pH is neutral.

[0052] The initial mixed microbial community in step one is inoculated at 10% of the volume of the ethanol-containing enrichment medium; the inoculation amount of the fermentation broth for each enrichment culture cycle in step one is 10% of the volume of the ethanol-containing enrichment medium.

[0053] The ethanol-containing enrichment medium described in step one is composed of ethanol, NaHCO3, NH4Cl, yeast extract, sodium 2-bromoethanesulfonate, L-cysteine, phosphate buffer, macro-element solution, acidic micro-element solution, alkaline micro-element solution, and vitamin solution. The concentrations of the ethanol-containing enrichment medium are as follows: ethanol 8.3 g / L (≈180 mmol / L), NaHCO3 1 g / L, NH4Cl 1 g / L, yeast extract 1 g / L, sodium 2-bromoethanesulfonate 2 g / L, L-cysteine ​​0.5 g / L, phosphate buffer 20 mL / L, macro-element solution 10 mL / L, acidic micro-element solution 2 mL / L, alkaline micro-element solution 2 mL / L, and vitamin solution 0.2 mL / L.

[0054] The inoculation amount of the fermentation broth after the enrichment and domestication process described in step two is 10% of the volume of the fermentation medium containing ethanol.

[0055] The ethanol-containing fermentation medium described in step two is composed of ethanol, NaHCO3, NH4Cl, yeast extract, L-cysteine, phosphate buffer, macro-element solution, acidic micro-element solution, alkaline micro-element solution, and vitamin solution. The concentrations of ethanol, NaHCO3, NH4Cl, yeast extract, L-cysteine, phosphate buffer, macro-element solution, acidic micro-element solution, alkaline micro-element solution, and vitamin solution in the ethanol-containing fermentation medium are: 8.3 g / L (≈180 mmol / L), 1 g / L, 1 g / L, 1 g / L, 0.5 g / L, 20 mL / L, 10 mL / L, 2 mL / L, 2 mL / L, and 0.2 mL / L.

[0056] The phosphate buffer solution is composed of Na2HPO4·12H2O and KH2PO4; and the concentration of Na2HPO4·12H2O in the phosphate buffer solution is 53 g / L, and the concentration of KH2PO4 is 20.5 g / L.

[0057] The macro-element solution is composed of NaCl, CaCl2·2H2O and MgCl2·6H2O; and the concentration of NaCl in the macro-element solution is 30 g / L, the concentration of CaCl2·2H2O is 11 g / L, and the concentration of MgCl2·6H2O is 10 g / L.

[0058] The acidic trace element solution is composed of ZnCl2, CuCl2·H2O, CuSO4·5H2O, MnCl2·4H2O, CoCl2·6H2O, NiCl2·6H2O, H3BO4, and HCl solution; and the concentrations of ZnCl2, CuCl2·H2O, CuSO4·5H2O, MnCl2·4H2O, CoCl2·6H2O, NiCl2·6H2O, and H3BO4 in the acidic trace element solution are 0.05 g / L, 0.038 g / L, 0.01 g / L, 0.05 g / L, 0.05 g / L, 0.03 g / L, 0.05 g / L, and 1 mL / L of HCl solution; the HCl solution has a mass percentage of 100%.

[0059] The alkaline trace element solution is composed of Na2SeO3·5H2O, Na2MoO4·2H2O, Na2WO4·2H2O, and NaOH; and the concentrations of Na2SeO3·5H2O, Na2MoO4·2H2O, Na2WO4·2H2O, and NaOH in the alkaline trace element solution are 0.03 g / L, 0.024 g / L, 0.033 g / L, and 0.4 g / L.

[0060] The vitamin solution comprises biotin, vitamin B3, pyridoxine hydrochloride, thiamine, riboflavin, cobalamin, calcium pantothenate, lipoic acid, para-aminobenzoic acid, and folic acid; and the concentrations of biotin, vitamin B3, pyridoxine hydrochloride, thiamine, riboflavin, cobalamin, calcium pantothenate, lipoic acid, para-aminobenzoic acid, and folic acid are 2 mg / L.

[0061] In step one of this embodiment, the ethanol consumption time for each generation is 52 days, 40 days, 33 days, 32 days, 40 days, and 35 days for the first to sixth generation, respectively.

[0062] In step one of this embodiment, an anaerobic bottle is used as an enrichment and acclimatization reactor. A mixed atmosphere is continuously introduced into the enrichment and acclimatization reactor to ensure a completely anaerobic state. Then, the anaerobic bottle is sealed with a rubber stopper and an anaerobic bottle cap. During the enrichment and acclimatization process, when the pH is lower than 5.5, it is adjusted to 6.5-7.0 using 1 mol / L NaOH. The headspace gas in the anaerobic bottle is measured every 1-2 days, and the mixed atmosphere is replenished to standard atmospheric pressure. The concentration of volatile acid in the fermentation broth is measured every 2-3 days.

[0063] In step two of this embodiment, a glass reagent bottle is used as the fermentation reactor. A two-way biofeeding bottle cap is used, with one end serving as the sampling port and the other end connected to a gas collection bag to provide the gaseous substrate (mixed atmosphere). The mixed atmosphere is continuously introduced into the fermentation reactor to ensure a completely anaerobic state and the reactor is sealed. Then, a gas collection bag containing 800 mL of the mixed atmosphere is connected to it. During fermentation, when the pH is below 5.5, it is adjusted to 6.5-7.0 using 4 mol / L NaOH. Whenever any gaseous substrate (hydrogen or carbon dioxide) in the gas collection bag is depleted, the gas ratio in the gas collection bag is measured and the gas collection bag is replaced. The concentration of volatile acid in the fermentation broth is measured every 2-3 days. This process is continued until all the ethanol in the culture medium is utilized, completing the fermentation.

[0064] Example 2: This example differs from Example 1 in that the concentration of ethanol in the ethanol-containing fermentation medium described in step 2 is 11.3 g / L (≈250 mmol / L). Everything else is the same as in Example 1.

[0065] Figure 1 The figure shows the fatty acid content and proportion of each generation during the enrichment and domestication of the mixed microbial community in step one of Experiment Example 1. As can be seen from the figure, the original microbial community has a strong ability to produce hexanoic acid, but no ability to produce octanoic acid. Starting from the second generation, the mixed microbial community begins to produce octanoic acid, and the total acid yield of fermentation increases, reaching its peak in the third generation. However, at this time, the butyric acid content in the product is relatively high, and the selectivity of hexanoic acid and octanoic acid is low. From the fourth to the sixth generation, the yield and selectivity of hexanoic acid and octanoic acid gradually stabilize.

[0066] The concentrations of hexanoic acid and octanoic acid were determined by gas chromatography. 1.5 mL of fermentation sample was placed in a 1.5 mL centrifuge tube and centrifuged at 12,000 rpm for 5 min. 1 mL of the supernatant was then placed in a 1.5 mL chromatographic vial, and 0.1 mL of 25% phosphoric acid was added and mixed thoroughly. Gas chromatography was performed using a flame ionization detector and a capillary column. The temperatures of the vaporization chamber, furnace column, and detector were set to 210 °C, 180 °C, and 210 °C, respectively. The hydrogen generator flow rate was set to 30 mL / min.

[0067] Figure 2The graph shows the acid production of hexanoic acid and octanoic acid after fermentation in step two of Experiment Example 1. As can be seen, ethanol was continuously consumed throughout the process, while acetic acid did not accumulate significantly. This may be due to the presence of many carbon-chain elongating bacteria in the mixed microbial community, which rapidly converted the produced acetic acid into longer-chain fatty acids. Butyric acid began to accumulate on day 7, peaked on day 16, and then slightly decreased. Hexanoic acid began to accumulate on day 4, surged on day 6, and gradually increased to 2.58 g / L on day 16, after which it slightly decreased to a final level of 2.17 g / L. The octanoic acid content remained almost unchanged until day 12, then surged on day 16, and with the conversion of butyric acid and hexanoic acid after day 16, the octanoic acid level ultimately increased to a final level of 2.49 g / L.

[0068] Figure 3 The figure shows a comparison of the yields and selectivity of hexanoic acid and octanoic acid in the initial mixed bacterial population of Experiment 1 (Step 1), Experiment 1, and the mixed bacterial populations after enrichment and acclimatization in Step 2 of Experiment 2. As can be seen from the figure, the original bacterial population without acclimatization had the highest hexanoic acid yield and selectivity (4.47 g / L and 78.93%, respectively), but this population did not possess the ability to produce octanoic acid. In contrast, the mixed bacterial population after acclimatization possessed the ability to produce octanoic acid, and the yields and selectivity of octanoic acid were almost identical to those of hexanoic acid: hexanoic acid yield was 2.17 g / L with a selectivity of 34.73%, and octanoic acid yield was 2.49 g / L with a selectivity of 39.85%. Increasing the ethanol content in the culture medium significantly improved the yield and selectivity of octanoic acid, reaching 4.17 g / L and 54.44%, respectively, while the yield of hexanoic acid was 1.77 g / L with a selectivity of 20.80%.

[0069] The community structure of the mixed microbial community after enrichment and domestication was examined, and the abundance of the main bacterial genera was compared: Figure 4 This is a comparison of the mixed bacterial community structure before and after enrichment and domestication in Experiment Example 1. As shown in the figure, the original bacterial community underwent significant changes after enrichment and domestication. The abundances of *Clostridium_sense_stricto_1* and *Paraclostridium*, which are related to the synthesis of short-chain fatty acids (especially butyric acid), decreased from 39.49% and 14.42% to 0.1% and 0.02%, respectively. The abundance of *Caproiciproducens*, which is related to the production of hexanoic acid, decreased from 20.88% to 0.68%. Meanwhile, the abundance of *Fonticella*, which can produce ethanol and lactic acid to provide precursors for subsequent reactions and maintain environmental adaptation, increased from 0% to 13.91%. The abundances of *Clostridium_sense_stricto_12* and *Rummeliibacillus*, which are related to the production of medium-chain fatty acids, increased from 0% and 0.005% to 6.31% and 12.96%, respectively.

[0070] Figure 5 The figure shows the acid production of hexanoic acid and octanoic acid after fermentation in step two of Experiment Example 2. As can be seen from the figure, after increasing the ethanol concentration in the culture medium, the trend of ethanol and acetic acid changes was almost unchanged compared with step two of Experiment Example 1. The butyric acid content first increased and then decreased. The hexanoic acid content showed an upward trend except for a slight decrease at 18 days, with a final yield of 1.77 g / L. The octanoic acid content increased slowly in the early stage, with two large increases at 15 days and 18 days, with a final yield of 4.17 g / L.

Claims

1. A method for producing hexanoic acid and octanoic acid by CO2 biorefining using a mixed microbial community, characterized in that... It is done in the following steps: I. Enrichment and domestication of mixed microbial communities: Ethanol-containing enrichment medium was added to the enrichment and domestication reactor. Under a mixed atmosphere, the initial mixed bacterial population was inoculated and the pH was adjusted. Then, enrichment and domestication were carried out under a mixed atmosphere until the ethanol was completely consumed, and the enriched culture fermentation broth was obtained. A portion of the enriched culture fermentation broth was taken out and inoculated into a new enrichment and domestication reactor for the next cycle of enrichment and domestication. This process was repeated multiple times until the maximum content of hexanoic acid and octanoic acid obtained by enrichment and domestication in the cycle no longer increased compared with the previous cycle. The enrichment and domestication was then completed, and the fermentation broth after the enrichment and domestication was obtained. II. Mixed microbial fermentation: Ethanol-containing fermentation medium is added to the fermentation reactor. Under a mixed atmosphere, the fermentation broth after enrichment and domestication is inoculated and the pH is adjusted. Fermentation is then carried out under a mixed atmosphere until the ethanol is completely consumed. The fermentation ends, thus completing the method of producing hexanoic acid and octanoic acid by using CO2 biorefining with mixed microbial communities.

2. The method for producing hexanoic acid and octanoic acid by CO2 biorefining using a mixed microbial community according to claim 1, characterized in that... The mixed atmosphere mentioned in Step 1 and Step 2 is a mixture of hydrogen and carbon dioxide gas, and the volume ratio of hydrogen to carbon dioxide gas is (2~4):

1.

3. The method for producing hexanoic acid and octanoic acid by CO2 biorefining using a mixed microbial community according to claim 1, characterized in that... The initial mixed microbial community mentioned in Step 1 is specifically enriched according to the following steps: A mixture of cow dung, pig manure compost, cornfield soil, and decaying wood is placed in a culture medium with straw as the carbon source and anaerobically fermented at a temperature of 35℃~38℃ and a stirring speed of 100rpm~120rpm for 3~5 months of continuous subculturing; the culture medium with straw as the carbon source is composed of pretreated straw, peptone, NaCl, CaCO3, yeast extract, and L-Cys, and the concentration of pretreated straw in the culture medium with straw as the carbon source is 5g / L~30g / L, and the concentration of peptone is... The concentrations of NaCl, CaCO3, yeast extract, and L-Cys are 5 g / L to 8 g / L, 5 g / L to 8 g / L, 1 g / L to 2 g / L, and 0.3 g / L to 1 g / L, respectively. The pretreated straw is prepared according to the following steps: the straw is cut into 1 cm to 2 cm segments, and then soaked in a 1% to 3% NaOH solution at a temperature of 30℃ to 60℃ for 72 h to 120 h. The soaked straw is then collected by filtration and washed with water until the pH is neutral.

4. The method for producing hexanoic acid and octanoic acid by CO2 biorefining using a mixed microbial community according to claim 1, characterized in that... The initial mixed microbial community in step one is inoculated at 5% to 10% of the volume of the ethanol-containing enrichment medium; the inoculation amount of the fermentation broth for each enrichment culture cycle in step one is 5% to 10% of the volume of the ethanol-containing enrichment medium.

5. The method for producing hexanoic acid and octanoic acid by CO2 biorefining using a mixed microbial community according to claim 1, characterized in that... In step one, the initial mixed microbial community was inoculated and the pH was adjusted to 5.5-7. In step one, the enrichment and acclimatization were carried out for 32-52 days under the conditions of mixed atmosphere, pH 5.5-7, temperature 35℃-37℃ and stirring speed 100rpm-120rpm.

6. The method for producing hexanoic acid and octanoic acid by CO2 biorefining using a mixed microbial community according to claim 1, characterized in that... The ethanol-containing enrichment medium described in step one consists of ethanol, NaHCO3, NH4Cl, yeast extract, sodium 2-bromoethanesulfonate, L-cysteine, phosphate buffer, macro-element solution, acidic micro-element solution, alkaline micro-element solution, and vitamin solution; and the concentrations of ethanol, NaHCO3, NH4Cl, yeast extract, sodium 2-bromoethanesulfonate, and L-cysteine ​​in the enrichment medium are 7.7 g / L to 8.6 g / L, 1 g / L to 2 g / L, 1 g / L to 2 g / L, 1 g / L to 2 g / L, 2 g / L to 3 g / L, and 0.5 g / L. The concentrations of the following solutions are as follows: ~1 g / L, phosphate buffer solution 20 mL / L~30 mL / L, macro-element solution 10 mL / L~15 mL / L, acidic micro-element solution 1 mL / L~2 mL / L, alkaline micro-element solution 1 mL / L~2 mL / L, and vitamin solution 0.2 mL / L~0.5 mL / L.

7. The method for producing hexanoic acid and octanoic acid by CO2 biorefining using a mixed microbial community according to claim 1, characterized in that... The inoculation amount of the fermentation broth after the enrichment and domestication process described in step two is 5% to 10% of the volume of the fermentation medium containing ethanol.

8. The method for producing hexanoic acid and octanoic acid by CO2 biorefining using a mixed microbial community according to claim 1, characterized in that... In step two, the fermentation broth after enrichment and domestication is inoculated and the pH is adjusted to 5.5-7. In step two, fermentation is carried out for 21-22 days under mixed atmosphere, pH 5.5-7, temperature 35℃-37℃ and stirring speed of 100rpm-120rpm.

9. The method for producing hexanoic acid and octanoic acid by CO2 biorefining using a mixed microbial community according to claim 1, characterized in that... The ethanol-containing fermentation medium described in step two is composed of ethanol, NaHCO3, NH4Cl, yeast extract, L-cysteine, phosphate buffer, macro-element solution, acidic micro-element solution, alkaline micro-element solution, and vitamin solution. The concentrations of ethanol, NaHCO3, NH4Cl, yeast extract, L-cysteine, phosphate buffer, macro-element solution, acidic micro-element solution, alkaline micro-element solution, and vitamin solution in the ethanol-containing fermentation medium are: 8.3 g / L to 11.3 g / L; 1 g / L to 2 g / L; 1 g / L to 2 g / L; 1 g / L to 2 g / L; 0.5 g / L to 1 g / L; 20 mL / L to 30 mL / L; 10 mL / L to 15 mL / L; 1 mL / L to 2 mL / L; 1 mL / L to 2 mL / L; and 0.2 mL / L to 0.5 mL / L.

10. A method for producing hexanoic acid and octanoic acid by CO2 biorefining using a mixed microbial community according to claim 6 or 9, characterized in that... The phosphate buffer solution is composed of Na2HPO4·12H2O and KH2PO4; and the concentration of Na2HPO4·12H2O in the phosphate buffer solution is 50 g / L~55 g / L, and the concentration of KH2PO4 is 20 g / L~25 g / L. The macro-element solution is composed of NaCl, CaCl2·2H2O and MgCl2·6H2O; and the concentration of NaCl in the macro-element solution is 30g / L~35g / L, the concentration of CaCl2·2H2O is 10g / L~15g / L, and the concentration of MgCl2·6H2O is 10g / L~15g / L. The acidic trace element solution is composed of ZnCl2, CuCl2·H2O, CuSO4·5H2O, MnCl2·4H2O, CoCl2·6H2O, NiCl2·6H2O, H3BO4, and HCl solution; and the concentration of ZnCl2 in the acidic trace element solution is 0.05 g / L~0.1 g / L, the concentration of CuCl2·H2O is 0.02 g / L~0.05 g / L, and the concentration of CuSO4·5H2O is 0.01 g / L. The concentrations of the following components are specified: L~0.03 g / L, MnCl2·4H2O concentration 0.05 g / L~0.1 g / L, CoCl2·6H2O concentration 0.05 g / L~0.1 g / L, NiCl2·6H2O concentration 0.03 g / L~0.05 g / L, H3BO4 concentration 0.05 g / L~0.1 g / L, and HCl solution concentration 1 mL / L~2 mL / L; the HCl solution has a mass percentage of 95%~100%. The alkaline trace element solution is composed of Na2SeO3·5H2O, Na2MoO4·2H2O, Na2WO4·2H2O, and NaOH; and the concentrations of Na2SeO3·5H2O, Na2MoO4·2H2O, Na2WO4·2H2O, and NaOH in the alkaline trace element solution are 0.03 g / L to 0.05 g / L, 0.01 g / L to 0.03 g / L, 0.03 g / L, 0.05 g / L, and 0.3 g / L to 0.5 g / L. The vitamin solution comprises biotin, vitamin B3, pyridoxine hydrochloride, thiamine, riboflavin, cobalamin, calcium pantothenate, lipoic acid, para-aminobenzoic acid, and folic acid. The concentrations of biotin, vitamin B3, pyridoxine, thiamine, riboflavin, cobalamin, calcium pantothenate, lipoic acid, and folic acid in the vitamin solution are as follows: biotin concentration 2 mg / L~4 mg / L; vitamin B3 concentration 5 mg / L~10 mg / L; pyridoxine hydrochloride concentration 10 mg / L~20 mg / L; thiamine concentration 5 mg / L~10 mg / L; riboflavin concentration 5 mg / L~10 mg / L; cobalamin concentration 0.1 mg / L~0.3 mg / L; calcium pantothenate concentration 5 mg / L~10 mg / L; lipoic acid concentration 5 mg / L~10 mg / L; para-aminobenzoic acid concentration 5 mg / L~10 mg / L; and folic acid concentration 2 mg / L~5 mg / L.

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