High-yield caproic acid bacterium and application of high-yield caproic acid bacterium in production of caproic acid by anaerobic fermentation of kitchen waste

Abstract

The invention discloses a high-yield caproic acid bacterium and application thereof in production of caproic acid by anaerobic fermentation of kitchen waste. According to the invention, a high-yield caproic acid bacterium-bacillus aryabhattai H1 (CCTCC NO: M 20241040) is obtained through separation and screening, and the caproic acid-producing complex microbial inoculant prepared by compatibility of the high-yield caproic acid bacterium, lactobacillus plantarum and saccharomyces cerevisiae can be used for realizing high-efficiency synthesis of caproic acid through one-step fermentation by taking kitchen waste enzymatic hydrolysate as a substrate. Under the operation condition that the water inlet load is 13.7 kg <-> COD / m < 3 > / d, the hexanoic acid yield reaches 10.96 g / L, the hexanoic acid yield reaches 0.35 g / g-COD / g-COD, and the hexanoic acid production intensity reaches 2.19 g / L / d.

Description

Technical Field

[0001] This invention belongs to the field of environmental microbiology technology, specifically relating to a hexanoic acid-producing bacterium and its application in the anaerobic fermentation of kitchen waste to produce hexanoic acid. Background Technology

[0002] With the significant increase in the amount of food waste separated from urban household waste, back-end reduction and resource recovery have become crucial aspects of ensuring the construction of "zero-waste cities." Food waste is rich in organic matter and has excellent bioavailability; it can be transformed into valuable products through biotechnology, thus "turning waste into treasure." Anaerobic digestion is one of the mainstream technologies for the resource recovery of food waste. To increase the added value of end products, fermentation-based acid production technology derived from anaerobic digestion has gradually gained attention. Among these technologies, using anaerobic microorganisms to convert organic waste into medium-chain fatty acids such as hexanoic acid has become a research hotspot in recent years. Compared to the short-chain fatty acids produced by traditional anaerobic fermentation, hexanoic acid has higher energy density and added value, and its low water solubility makes it easy to separate from the fermentation broth.

[0003] Food waste is rich in carbohydrates such as starch, but caproic acid-producing bacteria have a weak ability to directly utilize starch / sugar. Therefore, microorganisms such as lactic acid bacteria and yeast can first convert starch / sugar into lactic acid and ethanol, and then the caproic acid-producing bacteria can further convert lactic acid and ethanol into caproic acid. This patented method first hydrolyzes the starch in food waste into the liquid phase through enzyme pretreatment; and then screens out a high caproic acid-producing bacterium capable of co-metabolizing ethanol and lactic acid. When combined with *Lactobacillus plantarum* and *Saccharomyces cerevisiae*, it can efficiently produce caproic acid in a one-step fermentation process using the enzymatic hydrolysate of food waste as a substrate. Summary of the Invention

[0004] Purpose of the invention: In order to overcome the shortcomings of the prior art, this patent discloses a high-yield hexanoic acid bacteria and its application in the anaerobic fermentation of kitchen waste to produce hexanoic acid. By combining the high-yield hexanoic acid bacteria Bacillus aryabhattai H1 with Lactobacillus plantarum and Saccharomyces cerevisiae, a hexanoic acid-producing compound bacterial agent is prepared. It can use kitchen waste enzymatic hydrolysate as a substrate to achieve efficient one-step fermentation and synthesis of hexanoic acid, which significantly improves the efficiency of anaerobic fermentation to produce hexanoic acid using kitchen waste as raw material.

[0005] To achieve the above objectives, the technical solution of the present invention is as follows: The first objective of this invention is to provide a high-yield caproic acid-producing bacterium, which was deposited on May 23, 2024, at the China Center for Type Culture Collection (CCTCC), Wuhan, China, with accession number CCTCC NO: M20241040 and classification name: Bacillus aryabhattai H1.

[0006] In some embodiments of the present invention, the 16S rDNA sequence of the Bacillus argentea H1 is shown in SEQ ID NO.1.

[0007] A second objective of the present invention is to provide the use of the high caproic acid-producing bacterium, wherein the *Bacillus argentis* H1 is capable of producing caproic acid by fermentation using ethanol or lactic acid alone, or by co-metabolizing ethanol and lactic acid.

[0008] In some embodiments of the present invention, when the *Bacillus argentea* H1 uses ethanol and lactic acid as a composite substrate, the hexanoic acid yield is 10 g / L or more.

[0009] The third objective of this invention is to provide a high-yield hexanoic acid compound microbial agent, the compound microbial agent comprising: Bacillus argentea H1, preservation number CCTCC NO: M 20241040; and further comprising a compatibility microbial agent, the compatibility microbial agent being Lactobacillus plantarum and / or Saccharomyces cerevisiae.

[0010] In some embodiments of the present invention, when the compound bacterial agent is Lactobacillus plantarum, the volume ratio of the activated bacterial liquid of Bacillus aureus H1 to Lactobacillus plantarum is (2-1):(1-2).

[0011] Furthermore, in some embodiments of the present invention, when the compound bacterial agent is Lactobacillus plantarum, the volume ratio of the activated bacterial liquid of Bacillus aureus H1 and Lactobacillus plantarum is 1:1.

[0012] In some embodiments of the present invention, when the compound microbial agent is Saccharomyces cerevisiae, the volume ratio of the activated bacterial liquid of Bacillus aureus H1 to Saccharomyces cerevisiae is (2-1):(1-2).

[0013] Furthermore, in some embodiments of the present invention, when the compound microbial agent is Saccharomyces cerevisiae, the volume ratio of the activated bacterial liquid of Bacillus aureus H1 and Saccharomyces cerevisiae is 1:1.

[0014] In some embodiments of the present invention, when the compound microbial agent is Lactobacillus plantarum and Saccharomyces cerevisiae, the volume ratio of the activated bacterial liquid of Bacillus aureus H1, Lactobacillus plantarum and Saccharomyces cerevisiae is (2-1):(1-2):(1-2).

[0015] Furthermore, in some embodiments of the present invention, when the compound microbial agent is Lactobacillus plantarum and Saccharomyces cerevisiae, the volume ratio of the activated bacterial liquid of Bacillus argentea H1, Lactobacillus plantarum and Saccharomyces cerevisiae is 1:1:1.

[0016] The fourth objective of this invention is to provide a method for preparing the compound bacterial agent as described above, comprising the following steps: activating and culturing the *Bacillus oryzae* H1 and the compatibility bacterial agent separately using LB liquid medium to obtain corresponding bacterial solutions, and when OD... 600 Once the bacterial solution reaches a concentration of 1.2-1.6, it is formulated into a compound bacterial agent.

[0017] The fifth objective of this invention is to provide the use of any of the above-described compound microbial agents in the anaerobic fermentation of kitchen waste to produce hexanoic acid.

[0018] In some embodiments of the present invention, in the enzymatic pretreatment reaction of kitchen waste, the enzymatic pretreatment conditions include: a material concentration of 100-250 g-TS / L; first, adding 1‰-3‰ of α-amylase and reacting for 3-4 h at a temperature of 70-90℃ and a pH of 5.0-6.5; then adding 0.5‰-1‰ of saccharifying enzyme and continuing the reaction for 3-4 h at a temperature of 50-60℃ and a pH of 5.0-6.0. Further, the material concentration is 150 g-TS / L.

[0019] In some embodiments of the present invention, in the anaerobic fermentation of kitchen waste to produce hexanoic acid, the inoculation amount of the compound microbial agent is 10%-20%.

[0020] In some embodiments of the present invention, the fermentation pH is 6.8-7.2 in the anaerobic fermentation reaction of kitchen waste to produce hexanoic acid.

[0021] In some embodiments of the present invention, the fermentation temperature in the anaerobic fermentation of kitchen waste to produce hexanoic acid is 32-38 ℃.

[0022] In some embodiments of the present invention, in the anaerobic fermentation of kitchen waste to produce hexanoic acid, the influent load is 10-15 kg-COD / m³. 3 / d. Furthermore, in some embodiments, the influent load is 13.7 kg-COD / m³. 3 / d.

[0023] In some embodiments of the present invention, the hexanoic acid yield in the anaerobic fermentation reaction of kitchen waste to produce hexanoic acid is not less than 10 g / L. Further, in some embodiments, the hexanoic acid yield reaches 10.96 g / L.

[0024] In some embodiments of the present invention, the hexanoic acid yield in the anaerobic fermentation reaction of kitchen waste to produce hexanoic acid is not less than 0.3 g-COD / g-COD. Further, in some embodiments, the hexanoic acid yield reaches 0.35 g-COD / g-COD.

[0025] In some embodiments of the present invention, the hexanoic acid production intensity in the anaerobic fermentation reaction of kitchen waste is not less than 2 g / L / d. Further, in some embodiments, the hexanoic acid production intensity reaches 2.19 g / L / d.

[0026] Beneficial effects: Food waste has a high organic matter content, making it ideal as a raw material for anaerobic fermentation to produce organic acids. However, the end products of conventional anaerobic fermentation are mainly short-chain fatty acids such as acetic acid and butyric acid. These short-chain carboxylic acids have low energy density, strong hydrophilicity, and are difficult to separate and purify, thus limiting their subsequent resource utilization. Compared with existing technologies, the method disclosed in this invention can effectively convert food waste into hexanoic acid, which has higher energy density and added value. Hexanoic acid is a medium-chain fatty acid with low water solubility, making it easy to separate and purify. Through enzymatic pretreatment of food waste, screening of high-hexanoic acid-producing bacteria (Bacillus aryabhattai) H1, and construction of a compound microbial agent, a one-step fermentation synthesis of hexanoic acid from food waste hydrolysate can be achieved. The hexanoic acid yield, production rate, and production intensity reached 10.96 g / L, 0.35 g-COD / g-COD, and 2.19 g / L / d, respectively. The technology described in this patent provides effective microbial resources for the biosynthesis of hexanoic acid from food waste, promoting the high-value utilization of food waste. Attached Figure Description

[0027] Figure 1 Changes in organic acid products during the enrichment culture of caproic acid-producing bacteria.

[0028] Figure 2 Colony morphology of Bacillus argentis H1.

[0029] Figure 3 Effect of the ratio of Bacillus aureus H1 to Lactobacillus plantarum on substrate transformation.

[0030] Figure 4 The effect of the ratio of Bacillus argentea H1 to Lactobacillus plantarum on the synthesis efficiency of hexanoic acid.

[0031] Figure 5 Effect of the ratio of Bacillus oryzae H1 to Saccharomyces cerevisiae on substrate transformation.

[0032] Figure 6 The effect of the ratio of Bacillus oryzae H1 to Saccharomyces cerevisiae on the synthesis efficiency of hexanoic acid.

[0033] Figure 7 The degradation effect of the main organic components of the enzymatic hydrolysate of kitchen waste under different influent loads.

[0034] Figure 8 Changes in organic acid products under different influent loads. Detailed Implementation

[0035] This invention discloses a high-yield hexanoic acid bacterium and its application in the anaerobic fermentation of food waste to produce hexanoic acid. Through targeted screening, a high-yield hexanoic acid bacterium—Bacillus aryabhattai H1—capable of metabolizing ethanol or lactic acid independently, and also co-metabolizing ethanol and lactic acid, was obtained. The hexanoic acid-producing compound bacterial agent prepared by combining Bacillus plantarum and Saccharomyces cerevisiae can achieve efficient one-step fermentation synthesis of hexanoic acid using food waste enzymatic hydrolysate as a substrate.

[0036] This invention screened out a high-producing caproic acid bacterium, Bacillus aryabhattai H1, capable of co-metabolizing ethanol and lactic acid. The accession number is CCTCC NO: M 20241040, the accession date is May 23, 2024, and the accession location is China Center for Type Culture Collection, Wuhan, China.

[0037] The *Bacillus aryabhattai* H1 strain possesses a strong hexanoic acid production capacity, capable of producing hexanoic acid through fermentation using ethanol or lactic acid alone, or through co-metabolism of ethanol and lactic acid. The screening process involved using anaerobic activated sludge pretreated at 121 °C for 15 minutes as the inoculum, and acclimation culture under substrate selection pressure using ethanol and lactic acid as a composite substrate. Hexanoic acid-producing bacteria were gradually enriched by increasing the influent substrate concentration, and then isolated and purified on a solid culture medium using ethanol and lactic acid as carbon sources. Further, fed-batch fermentation experiments were conducted to screen out the high-hexanoic acid-producing bacteria, identifying *Bacillus aryabhattai* H1, from the initial screening strains.

[0038] The Bacillus aryabhattai H1 and the compatible inoculum (Lactobacillus plantarum and / or Saccharomyces cerevisiae) were activated and cultured separately in LB liquid medium. When OD 600 After reaching a concentration of 1.2-1.6, corresponding microbial agents were prepared in different proportions; and the optimal proportion was selected through batch fermentation experiments of kitchen waste enzymatic hydrolysate.

[0039] The optimal ratio of compound microbial agent was added to a continuous flow hexanoic acid production reactor of kitchen waste enzymatic hydrolysate at an inoculation rate of 10-20%, and the hexanoic acid production efficiency of the compound microbial agent under different influent loads was analyzed.

[0040] The enzymatic pretreatment step for kitchen waste includes: first, adding 1‰-3‰ of α-amylase and reacting at 70-90℃ and pH 5.0-6.5 for 3-4 hours; then adding 0.5‰-1‰ of saccharifying enzyme and continuing the reaction at 50-60℃ and pH 5.0-6.0 for another 3-4 hours. The material concentration is 100-250 g-TS / L.

[0041] The present invention will be further explained and illustrated below with specific embodiments. However, those skilled in the art will readily understand that the specific process conditions and results described in the embodiments are for illustrative purposes only and should not, and will not, limit the invention as described in detail in the claims.

[0042] Example 1: Enrichment culture of caproic acid-producing bacteria (1) Components of the enrichment medium for caproic acid-producing bacteria The medium contains 0.25 g / L ammonium chloride, 0.2 g / L magnesium sulfate heptahydrate, 0.23 g / L potassium dihydrogen phosphate, 0.31 g / L dipotassium hydrogen phosphate, 0.8 g / L sodium chloride, and 0.25 g / L hydrated cysteine ​​hydrochloride. Additionally, 1 g yeast extract, 1 mL vitamin solution, and 1 mL trace element solution are added per liter of medium. Ethanol and lactic acid are used as carbon sources in the medium.

[0043] (2) Enrichment culture of caproic acid-producing bacteria Anaerobic activated sludge pretreated at 121 °C for 15 minutes was used as the inoculum source for enrichment culture of caproic acid-producing bacteria in a mixed anaerobic bioreactor. The initial inoculum size was 20 g / L (based on VS). The molar ratio of ethanol to lactic acid in the influent culture medium was controlled at 1:1, and the initial influent concentration was 200 mmol / L (substrate concentration 400 mmol / L). Enrichment culture of caproic acid-producing bacteria was initiated after nitrogen stripping for 15 minutes. The reactor was operated in continuous fermentation mode, with the temperature and pH maintained at 36 °C and 7.0, respectively, and the stirring speed at 100 rpm. The entire enrichment process was divided into three stages: From 0 to 30 days, 4L of water was influent and effluent every two days, the hydraulic retention time was 5 days, and the concentrations of ethanol and lactic acid in the influent were both 200 mmol / L; From day 31 to 60, the influent concentrations of ethanol and lactic acid were both 250 mmol / L (substrate concentration 500 mmol / L), with 4L of water entering and leaving the tank per day and a hydraulic retention time of 2.5 days. From 61 to 120 days, the influent ethanol and lactic acid concentrations were both 300 mmol / L (substrate concentration 600 mmol / L), the influent and effluent were 4L per day, and the hydraulic retention time was 2.5 days.

[0044] like Figure 1 As shown, during the stable operation period (20-30 days) of the first enrichment stage, the concentrations of ethanol and lactic acid in the effluent were both below 0.5 g / L, with a conversion rate exceeding 95%. The average concentrations of acetic acid, butyric acid, and hexanoic acid in the effluent were 7.38 g / L, 7.08 g / L, and 5.57 g / L, respectively, while the concentrations of propionic acid and valeric acid were only 0.55 g / L and 0.89 g / L, respectively.

[0045] During the second phase of stable operation (45-60 days), the concentration of hexanoic acid in the effluent increased to 9.36 g / L, the concentration of acetic acid decreased significantly to 3.48 g / L, while the concentrations of butyric acid, propionic acid, and valeric acid did not change significantly.

[0046] When the influent substrate concentration was further increased to 600 mmol / L (stage 3), the concentrations of hexanoic acid, butyric acid, and acetic acid increased to 12.0 g / L, 8.81 g / L, and 4.84 g / L, respectively; while the production of propionic acid and valeric acid remained at a low level, at only 0.29 g / L and 0.58 g / L, respectively.

[0047] Table 1 shows the hexanoic acid fermentation parameters during the stable operation period under different influent substrate concentrations. The hexanoic acid yield (in electron equivalent) and production intensity in the first enrichment stage were 0.32 mol / mol and 1.11 g / L / d, respectively.

[0048] When the influent substrate concentration was increased to 500 mmol / L (stage 2), the yield and production intensity of hexanoic acid significantly increased to 0.43 mol / mol and 3.74 g / L / d, respectively, with the yield of hexanoic acid increasing by nearly 34.4%. When the influent substrate concentration was further increased to 600 mmol / L (stage 3), the hexanoic acid production intensity reached 4.80 g / L / d, and the hexanoic acid yield increased by approximately 7.0%.

[0049] The above operational data indicate that, under substrate selection pressure, as the concentration of influent substrate gradually increases, microorganisms with hexanoic acid-producing function are effectively enriched.

[0050] Table 1. Hexanoic acid synthesis efficiency during the enrichment culture of hexanoic acid-producing bacteria. Example 2 Screening of high caproic acid-producing bacteria (1) Components of the culture medium for isolating and purifying caproic acid-producing bacteria The medium contains 20 g / L sodium lactate, 10 g / L ethanol, 0.25 g / L ammonium chloride, 0.2 g / L magnesium sulfate heptahydrate, 0.23 g / L potassium dihydrogen phosphate, 0.31 g / L dipotassium hydrogen phosphate, 0.8 g / L sodium chloride, and 0.25 g / L cysteine ​​hydrochloride. Each liter of medium should also contain 1 g yeast extract, 1 mL vitamin solution, and 1 mL trace element solution, and the pH should be adjusted to 6.8. For solid medium, an additional 15 g / L agar should be added.

[0051] (2) Isolation and purification of high-yield caproic acid bacteria High-producing caproic acid bacteria were further screened from the enriched cultures of caproic acid-producing bacteria.

[0052] The isolation and purification steps are as follows: An appropriate amount of hexanoic acid-producing enriched culture was placed in an anaerobic tube, glass beads were added, and the mixture was shaken to disperse. Then, it was serially diluted using physiological saline. The diluted bacterial suspension was spread onto a solid culture medium for isolation and purification, and then incubated upside down in a 36℃ anaerobic incubator for 48 h. Single colonies on the plates were repeatedly streaked for isolation, resulting in the isolation and purification of five hexanoic acid-producing bacteria (H1-H5). After activation with LB medium, the initial screening strains were transferred at a 10% inoculum to anaerobic serum bottles containing 500 mL of liquid medium for fed-batch fermentation. 20 mL of fermentation broth was analyzed daily, and 20 mL of fresh medium was added daily until hexanoic acid production ceased to increase. Finally, strain H1, which produced the highest hexanoic acid, was isolated and screened. The colony morphology of hexanoic acid-producing strain H1 is shown in the figure below. Figure 2 As shown.

[0053] (3) Strain identification The 16S rDNA sequence of the hexanoic acid-producing strain H1, selected through screening, was amplified using universal bacterial primers (27F: 5'-AGAGTTTGATCATGGCTCAG-3'; 1492R: 5'-GGTTACCTTGTTACGACTT-3'). The sequence was then sent to Shanghai Sangon Biotech Co., Ltd. for sequencing, and the 16S rDNA sequence was compared with that in GenBank, as shown in SEQ ID NO. 01. The results showed that the 16S rDNA sequence of this strain was 1502 bp in length, with a 99.87% similarity to the 16S rDNA sequence of Bacillus abhattai.

[0054] The strain was identified and named Bacillus aryabhattai H1 and deposited at the China Center for Type Culture Collection (CCTCC NO: M 20241040) in Wuhan, China, on May 23, 2024.

[0055] Example 3: One-step efficient synthesis of hexanoic acid from kitchen waste enzymatic hydrolysate using compound microbial agents. (1) Effect of compound Bacillus aureus H1 and Lactobacillus plantarum on hexanoic acid production After enzymatic pretreatment, food waste starch is effectively hydrolyzed into small sugar molecules such as glucose. However, caproic acid-producing bacteria have a weak ability to directly utilize glucose to produce caproic acid. *Lactobacillus plantarum* is a homofermentative lactic acid bacterium with a strong lactic acid production capacity, few byproducts, and strong environmental adaptability. Through the combination of *Bacillus aureus* H1 and *Lactobacillus plantarum*, *Lactobacillus plantarum* first efficiently converts small sugar molecules into lactic acid, and then *Bacillus aureus* H1 further utilizes the lactic acid to synthesize caproic acid.

[0056] The *Bacillus oryzae* H1 and *Lactobacillus plantarum* were activated and cultured in LB liquid medium, respectively, until OD... 600 After reaching a pH of 1.2-1.6, the mixture was compounded at different volume ratios (1:2, 1:1, 2:1). Then, 10% of the inoculum was transferred to a fermentation bottle containing 500 mL of kitchen waste enzymatic hydrolysate, and anaerobic batch fermentation to produce hexanoic acid was carried out at 36℃ and pH 7.0.

[0057] like Figure 3 As shown, reducing sugars and total sugars were effectively degraded in all experimental groups during the 12-day fermentation process. In the control group inoculated only with *Bacillus plantarum* H1, no significant accumulation of ethanol and lactic acid occurred during fermentation. When the ratio of *Lactobacillus plantarum* to *Bacillus plantarum* H1 activation solution was 1:2, the lactic acid accumulation reached its maximum of 12.78 g / L on day four. At ratios of 1:1 and 2:1, the maximum lactic acid accumulation significantly increased to 20.59 g / L and 22.87 g / L (4 days), respectively, before being gradually metabolized.

[0058] Liquid phase end-product components under different compounding ratios, such as Figure 4 As shown in the figure, the main products of each experimental group were acetic acid, butyric acid, hexanoic acid, and propionic acid. The yields of acetic acid, butyric acid, and hexanoic acid in the control group were 6.59 g / L, 11.13 g / L, and 4.74 g / L, respectively.

[0059] However, the introduction of *Lactobacillus plantarum* significantly increased the production of hexanoic acid. Under fermentation conditions with a mixture ratio of 1:2, 1:1, and 2:1, the hexanoic acid production reached 6.63 g / L, 8.41 g / L, and 7.29 g / L, respectively, representing increases of 39.9%, 77.4%, and 53.8% compared to the control group.

[0060] The above results indicate that Lactobacillus plantarum mediates the first directed conversion of hexoses into lactic acid, which is then further utilized by Bacillus argentis H1 to generate hexanoic acid.

[0061] (2) Effect of compound Bacillus argentisi H1 and Saccharomyces cerevisiae on hexanoic acid production Saccharomyces cerevisiae possesses highly efficient sugar conversion capabilities, rapidly converting small-molecule sugars such as glucose and maltose into ethanol, and exhibits good tolerance to environmental changes. By combining Bacillus aspergillus H1 with Saccharomyces cerevisiae, the Saccharomyces cerevisiae first efficiently converts small-molecule sugars in the enzymatic hydrolysate of kitchen waste into ethanol, and then Bacillus aspergillus H1 further utilizes the ethanol to synthesize hexanoic acid.

[0062] The *Bacillus oryzae* H1 and *Saccharomyces cerevisiae* were activated and cultured separately in LB liquid medium, when OD... 600After reaching a pH of 1.2-1.6, the mixture was compounded at different volume ratios (1:2, 1:1, 2:1). Then, 10% of the inoculum was transferred to a fermentation bottle containing 500 mL of kitchen waste enzymatic hydrolysate, and anaerobic batch fermentation to produce hexanoic acid was carried out at 36℃ and pH 7.0.

[0063] like Figure 5 As shown, under the combined action of *Bacillus oryzae* H1 and *Saccharomyces cerevisiae*, reducing sugars and total sugars in all experimental groups were effectively degraded within 12 days. When the ratio of *Saccharomyces cerevisiae* to *Bacillus oryzae* H1 was 1:2, 1:1, and 2:1, the maximum cumulative ethanol concentrations during fermentation were 7.09 g / L, 12.65 g / L, and 13.46 g / L (4 days), respectively, and were subsequently gradually converted.

[0064] Liquid phase end-product components under different compounding ratios, such as Figure 6 As shown in the figure. Compared with the control group (4.67 g / L hexanoic acid), under fermentation conditions with compound ratios of 1:2, 1:1 and 2:1, the hexanoic acid yields reached 6.85 g / L, 8.72 g / L and 7.61 g / L, respectively, which were 46.7%, 86.7% and 63.0% higher than the control group.

[0065] Similar to *Lactobacillus plantarum*, the combination of *Bacillus aureus* H1 and *Saccharomyces cerevisiae* significantly enhances the efficiency of the one-step synthesis of hexanoic acid from hexoses. *Saccharomyces cerevisiae* mediates the direct conversion of hexoses into ethanol, which is then further utilized by *Bacillus aureus* H1 to generate hexanoic acid. The hexanoic acid yield reaches its maximum when the ratio of *Bacillus aureus* H1 to *Saccharomyces cerevisiae* activation solution is 1:1.

[0066] (3) The continuous fermentation efficiency of hexanoic acid by a compound of Bacillus argentis H1, Lactobacillus plantarum and Saccharomyces cerevisiae The results of batch fermentation experiments showed that both Bacillus aureus H1 and Lactobacillus plantarum / Saccharomyces cerevisiae reached the highest hexanoic acid yield when the ratio was 1:1.

[0067] Therefore, the activated solutions of Bacillus oryzae H1, Lactobacillus plantarum, and Saccharomyces cerevisiae were further compounded at a volume ratio of 1:1:1. The compounded microbial agent (1L) was added to a continuous flow hexanoic acid reactor (5L) containing kitchen waste enzymatic hydrolysate. The analysis was performed under different influent COD loads (6.8 kg-COD / m³). 3 / d, 0-13d; 13.7 kg-COD / m 3 / d, 14-30d; 20.5kg-COD / m 3 The hexanoic acid production efficiency of compound bacterial agents ( / d, 31-45d).

[0068] like Figure 7As shown, the concentrations of reducing sugar, total sugar, protein, and COD in the enzymatic hydrolysate of influent kitchen waste were 37.3±1.9, 50.8±2.7 g / L, 10.2±0.3 g / L, and 68.3±4.4 g / L, respectively. Under low-load operating conditions (6.8 kg-COD / m³), 3 During the stable operation period of the reactor, the average concentrations of reducing sugar, total sugar and protein in the effluent were 1.78 g / L, 4.45 g / L and 4.72 g / L, respectively, with removal rates of 95.2%, 91.2% and 53.7%.

[0069] When the influent load increases to 13.7 kg-COD / m³ 3 At / d, the removal rates of reducing sugar, total sugar and protein were 94.3%, 86.3% and 44.4%, respectively.

[0070] When the influent load is further increased to 20.5 kg-COD / m³ 3 / d, the removal rates of the three were 92.3%, 83.2% and 32.6%, respectively.

[0071] Changes in organic acid products in the effluent of an anaerobic reactor, such as Figure 8 As shown, no significant accumulation of lactic acid and ethanol occurred during the entire fermentation process, and their effluent concentrations remained below 0.8 g / L, indicating that *Lactobacillus plantarum* and *Saccharomyces cerevisiae* could form a good "supply and demand balance" with *Bacillus aureus* H1. Hexanoic acid, butyric acid, and acetic acid were the main liquid-phase end products.

[0072] With an influent load of 6.8 kg-COD / m³ 3 Under operating conditions of 13.7 kg-COD / m³, the average concentrations of the three products during the steady-state period were 7.39 g / L, 8.42 g / L, and 7.31 g / L, respectively. When the influent load increased to 13.7 kg-COD / m³... 3 At / d, hexanoic acid production increased significantly, with the average concentration in the effluent reaching 10.96 g / L during the stable period, while the concentrations of acetic acid and butyric acid decreased to 5.68 g / L and 7.83 g / L, respectively.

[0073] However, when the influent load is further increased to 20.5 kg-COD / m³ 3 At / d, the yield of hexanoic acid decreased to 8.95 g / L.

[0074] Therefore, with an influent load of 13.7 kg-COD / m³ 3 The fermentation efficiency was optimal at 10.96 g / L, with hexanoic acid yield, production rate, and production intensity reaching 10.96 g / L, 0.35 g-COD / g-COD, and 2.19 g / L / d, respectively.

[0075] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A high-yield caproic acid bacterium, characterized in that, The high-yield caproic acid-producing bacterium was deposited on May 23, 2024, at the China Center for Type Culture Collection (CCTCC), Wuhan, China, with accession number CCTCC NO: M 20241040 and classification name: Bacillus aryabhattai H1.

2. The use of the high-yield caproic acid bacteria according to claim 1, characterized in that, The high-caproic acid-producing bacteria produce caproic acid by fermenting ethanol or lactic acid alone, or by co-metabolizing ethanol and lactic acid.

3. The use according to claim 1, characterized in that, When the high-yield hexanoic acid bacteria use ethanol and lactic acid as a composite substrate, the hexanoic acid yield is above 10 g / L.

4. A high-yield hexanoic acid-producing compound bacterial agent, characterized in that, The compound microbial agent includes: Bacillus abhattai H1, with accession number CCTCC NO: M 20241040; It also includes a compatibility microbial agent, which is Lactobacillus plantarum and / or Saccharomyces cerevisiae.

5. The compound microbial agent according to claim 4, characterized in that, In the compound microbial agent, when the compatibility microbial agent is *Lactobacillus plantarum*, the volume ratio of the activated bacterial solution of *Bacillus aureus* H1 to *Lactobacillus plantarum* is (2-1):(1-2), preferably 1:1; and / or When the inoculum is *Saccharomyces cerevisiae*, the volume ratio of the activated bacterial culture of *Bacillus aureus* H1 to *Saccharomyces cerevisiae* is (2-1):(1-2), preferably 1:1; and / or When the inoculant is Lactobacillus plantarum and Saccharomyces cerevisiae, the volume ratio of the activated bacterial liquid of Bacillus argentea H1, Lactobacillus plantarum and Saccharomyces cerevisiae is (2-1): (1-2): (1-2), preferably 1:1:

1.

6. A method for preparing the compound microbial agent as described in any one of claims 4-5, characterized in that, The steps include: activating and culturing *Bacillus oryzae* H1 and the corresponding bacterial agent separately using LB liquid medium to obtain the corresponding bacterial solutions; when OD... 600 Once the bacterial solution reaches a concentration of 1.2-1.6, it is formulated into a compound bacterial agent.

7. The use of the compound microbial agent as described in any one of claims 4-5 in the anaerobic fermentation of kitchen waste to produce hexanoic acid.

8. The use according to claim 7, characterized in that, Food waste requires enzymatic pretreatment with a material concentration of 100-250 g-TS / L. First, add 1‰-3‰ of α-amylase and react for 3-4 hours at a temperature of 70-90 ℃ and a pH of 5.0-6.

5. Then, add 0.5‰-1‰ of saccharifying enzyme and continue the reaction for 3-4 hours at a temperature of 50-60 ℃ and a pH of 5.0-6.

0.

9. The use according to claim 7, characterized in that, In the anaerobic fermentation of kitchen waste to produce hexanoic acid, the inoculum amount of the compound microbial agent is 10%-20%; and / or Fermentation pH is 6.8-7.2; and / or The temperature is 32-38 ℃.

10. The use according to any one of claims 7-9, characterized in that, Influent load is 10-15 kg-COD / m³ 3 / d.

Images