Method for realizing one-tank type rapid production of caproic acid from kitchen waste by utilizing aerobic pretreatment

Abstract

The invention belongs to the technical field of solid waste resource utilization and biological fermentation, and particularly relates to a method for rapidly producing hexanoic acid from kitchen waste in a one-tank mode through aerobic pretreatment. The method comprises the following steps: firstly inoculating yeast for making hard liquor and red yeast rice into kitchen slurry for aerobic pretreatment, and then inoculating flora which is formed by mixing anaerobic sludge, vinasse and forest land soil and is subjected to enrichment treatment for anaerobic fermentation to generate caproic acid. According to the method, micro-aerobic pretreatment and anaerobic fermentation are combined to construct a fungus-bacterium synergistic fermentation system, so that the problems that electron donors depend on exogenous addition, the hydrolytic acidification efficiency is low, flora enrichment is difficult, the process is complicated and the like in the existing process of producing caproic acid from kitchen waste are solved, endogenous electron donors are efficiently generated, the caproic acid yield is optimized, and the method is suitable for industrial production. The production period is obviously shortened, and efficient resource utilization is realized.

Description

Technical Field

[0001] This invention belongs to the field of solid waste resource utilization and bio-fermentation technology, specifically involving a method for rapidly producing hexanoic acid from kitchen waste in a single batch using aerobic pretreatment. Background Technology

[0002] With the acceleration of urbanization, the amount of food waste generated is increasing year by year. According to statistics, the annual output of food waste is expected to reach a significant scale in the next few years. Improper treatment of food waste will not only lead to environmental pollution but also cause a huge waste of resources. Anaerobic fermentation, as a highly efficient resource recovery technology, has been widely used in the treatment of food waste. Its main products include methane, hydrogen, and volatile fatty acids (VFAs, such as acetic acid, propionic acid, butyric acid, and hexanoic acid). However, the storage and transportation conditions of methane and hydrogen are harsh and pose safety hazards; while short-chain fatty acids (such as acetic acid and propionic acid) have high solubility and high extraction costs, which limits their industrial application.

[0003] Hexanoic acid (C6H) 12 Hexanoic acid (O2), as a medium-chain fatty acid, has low water solubility (approximately 10.82 g / L), facilitating subsequent separation and extraction. It also has a low oxygen / carbon ratio (1:3) and high energy density (3452 kJ / mol). Hexanoic acid has wide applications in antibacterial agents, feed additives, flavorings, and biofuels, with high market value and promising economic prospects. Therefore, utilizing food waste for fermentation to produce hexanoic acid is considered an effective way to achieve its high-value utilization.

[0004] Currently, in the research on hexanoic acid production from anaerobic fermentation of food waste, the generation of hexanoic acid mainly depends on the chain elongation (CE) process, that is, the hexanoic acid-producing functional bacteria use ethanol or lactic acid as electron donors and acetic acid or butyric acid as electron acceptors to synthesize hexanoic acid through the "reverse β-oxidation" pathway. However, the existing technology has the following problems: (1) the source of electron donors mostly depends on external addition, such as ethanol or lactic acid, which leads to increased production costs; (2) the efficiency of the hydrolysis and acidification process of food waste is low and the substrate conversion rate is insufficient; (3) the fermentation system is prone to acidification inhibition (pH drops to 3-4), which is not conducive to the enrichment and activity of hexanoic acid-producing bacteria; (4) the traditional two-stage fermentation process is complex and has a long operation cycle, making it difficult to achieve rapid and efficient acid production.

[0005] Therefore, there is an urgent need to develop a fermentation method that can overcome acidification inhibition, achieve efficient generation of endogenous electron donors, and rapidly produce hexanoic acid, so as to simplify the process and improve the economic efficiency of food waste resource utilization. Summary of the Invention

[0006] To address the shortcomings of existing technologies, this invention provides a method for rapid one-tank production of hexanoic acid from kitchen waste using aerobic pretreatment. This method combines microaerobic pretreatment with anaerobic fermentation to construct a fungal-bacterial synergistic fermentation system. This solves the problems of electron donor dependence on external addition, low hydrolysis and acidification efficiency, difficulty in microbial enrichment, and complex processes in the existing process of producing hexanoic acid from kitchen waste. It optimizes the hexanoic acid yield, significantly shortens the production cycle, and achieves efficient resource utilization.

[0007] The technical solution of the present invention is: a method for rapidly producing hexanoic acid from kitchen waste in one batch using aerobic pretreatment, the steps of which include: first inoculating the kitchen waste slurry with Daqu or Daqu and red yeast rice for aerobic pretreatment, and then inoculating it with a microbial community composed of anaerobic sludge, distiller's grains and forest soil that has been enriched for anaerobic fermentation to produce hexanoic acid.

[0008] Furthermore, before aerobic pretreatment after inoculating the kitchen slurry with Daqu (a type of starter culture), or Daqu and red yeast rice, a mixing treatment is performed. During the mixing treatment, the temperature is controlled at 35℃, and the pH is maintained at 5.5±0.1. The mixing treatment method is physical stirring or mechanical stirring. The pH adjuster is an alkaline solution, including but not limited to calcium hydroxide solution and / or sodium hydroxide solution, preferably sodium hydroxide solution, with a concentration of 1.5-2.5M, preferably 2M.

[0009] The aerobic pretreatment involves discontinuous aeration. More specifically, aeration begins once at the start of the aerobic pretreatment, followed by discontinuous aeration at time intervals, ending with aeration ceasing. More specifically, after the start of the aerobic pretreatment, discontinuous aeration is performed at time intervals that divide the aerobic pretreatment time equally, with intervals ranging from 1 to 6 hours, from 4 to 6 hours, and even more specifically, from 6 hours. Each aeration session lasts 2 to 5 minutes, preferably 3 minutes. The aeration volume per session is 0.05-1 vvm, preferably 0.2-0.6 vvm. The volume ratio of air to CO2 is 1-5:1:1, preferably 2-4:1:1, and more preferably 3:1:1; the volume ratio of air to CO2 is 1-3:1, preferably 1.5-2:1.

[0010] The aerobic pretreatment time is 10-14 hours, preferably 12 hours.

[0011] As one implementation method, the aerobic pretreatment process includes: aerobic pretreatment for 12 hours, aeration once at 0 hours and 6 hours, aeration time of 3 minutes, aeration volume of 0.2 vvm, and the gas used for aeration is a mixture of air, CO2 and H2S, with a volume ratio of air, CO2 and H2S of 3:1:1.

[0012] In the aforementioned Daqu and Hongqu, VS 大曲 VS 红曲 The ratio is 0.5-2:1, preferably 0.8-1.5:1, and more preferably 1:1. The concentration of the kitchen slurry is 30-50 g VS / L, preferably 40 g VS / L. 餐厨浆液 :VS 大曲 =3-10:1, preferably 3-5:1, more preferably 4:1; VS 餐厨浆液 :VS 大曲和红曲 The ratio is 3-10:1, preferably 3-5:1, and more preferably 4:1. The kitchen slurry is preferably inoculated with Daqu (a type of yeast starter) and red yeast rice. Further, the Daqu and red yeast rice are first mixed thoroughly before being inoculated into the kitchen slurry.

[0013] The preparation of the kitchen waste slurry includes: removing impurities such as bones and plastics from kitchen waste, adding deionized water, and then crushing and homogenizing it at high speed to obtain the kitchen waste slurry. The kitchen waste slurry contains no obvious large particles and has a concentration of 30-50 g VS / L, preferably 40 g VS / L.

[0014] The kitchen slurry is inoculated with Daqu and red yeast rice for aerobic pretreatment to promote hydrolysis and acidification, generating small molecule substances such as ethanol and lactic acid.

[0015] The wet weight ratio of the anaerobic sludge, distiller's grains and forest soil is 1-2:1-3:1, preferably 1-1.5:1.5-2.5:1, and more preferably 1:2:1.

[0016] The steps of mixing and enriching the anaerobic sludge, distiller's grains, and forest soil include: (1) Mix the anaerobic sludge, distiller's grains and forest soil into a slurry; (2) Add acetic acid solution to the mud; (3) Culture of enriched microbial communities under anaerobic conditions.

[0017] Step (1): Add deionized water to the mixed anaerobic sludge, distiller's grains and forest soil, and mix well to form a slurry.

[0018] In step (1), the wet weight ratio of the anaerobic sludge, distiller's grains, and forest soil is 1-2:1-3:1, 1-1.5:1.5-2.5:1, and more preferably 1:2:1; the solid-liquid ratio of the mixture of anaerobic sludge, distiller's grains, and forest soil to deionized water is 1g:2-5mL, preferably 1g:3mL. The anaerobic sludge is selected from anaerobic sludge from a sewage treatment plant, the distiller's grains are selected from beer or liquor distiller's grains, and the forest soil is selected from the topsoil of broad-leaved forests.

[0019] Step (2): Add acetic acid solution to the mud until the initial concentration of acetic acid in the mud is 4-6 g / L, preferably 5 g / L.

[0020] Step (3): Nitrogen gas is introduced to remove oxygen and establish anaerobic conditions. The deoxygenation operation is carried out under ice bath conditions to reduce liquid evaporation.

[0021] Step (3): Cultivate the enriched microbial community under anaerobic conditions to obtain the precipitated sediment. The anaerobic cultivation conditions include: protection from light, temperature 35℃, pH maintained at 5.5-6.5, with the pH maintained by sodium hydroxide solution and / or calcium hydroxide solution at a concentration of 1.5-2.5M, preferably 2M, and external shaking or stirring. Further, the enriched microbial community is cultivated under anaerobic conditions for 5-6 days. Further, during the cultivation process, sodium hydroxide solution is used to adjust the pH to maintain 5.5-6.5, with a concentration of 1.5-2.5M, preferably 2M.

[0022] The anaerobic sludge, distiller's grains, and forest soil are mixed and enriched to enrich the hexanoic acid-producing carbon chain growth bacteria.

[0023] The volume of the microbial community, which is a mixture of anaerobic sludge, distiller's grains and forest soil and has been enriched, accounts for 3%-10% of the volume of the fermentation liquid in which the kitchen slurry is inoculated with Daqu or Daqu and red yeast rice are aerobically pretreated, preferably 5%.

[0024] The anaerobic fermentation time is 10-16 days, preferably 15.5 days.

[0025] The pH of the anaerobic fermentation is maintained at 5.5-6.5±0.1 throughout the entire process; or the pH is maintained at 5.5±0.1 for the first 0.095-0.1 of the total anaerobic fermentation time, and at 6.5±0.1 for the remaining time. As one implementation, the pH is maintained at 5.5±0.1 for 0-1.5 days and at 6.5±0.1 for 1.5-15.5 days; or the pH is maintained at 5.5±0.1 for the first 0.095-0.1 of the total anaerobic fermentation time, at 6.5±0.1 for the middle 0.25-0.28 of the total anaerobic fermentation time, and at 7.5±0.1 for the remaining time. As one implementation, the pH is maintained at 5.5±0.1 for 0-1.5 days, at 6.5±0.1 for 1.5-5.5 days, and at 7.5±0.1 for 5.5-15.5 days. As a preferred approach, the pH is maintained at 5.5±0.1 for the first 0.095-0.1 of the total anaerobic fermentation time, at 6.5±0.1 for the middle 0.25-0.28 of the total anaerobic fermentation time, and at 7.5±0.1 for the remaining time. Specifically, the pH is maintained at 5.5±0.1 for 0-1.5 days, at 6.5±0.1 for 1.5-5.5 days, and at 7.5±0.1 for 5.5-15.5 days.

[0026] The pH value is adjusted using an alkali agent, selected from any one or any combination of calcium hydroxide solution, sodium hydroxide solution, or zinc chloride solution. More specifically, it is selected from combinations of calcium hydroxide solution and / or sodium hydroxide solution, and / or zinc chloride solution with calcium hydroxide solution and / or sodium hydroxide solution. Even further, the pH value is adjusted using calcium hydroxide solution, and / or calcium hydroxide solution with sodium hydroxide solution, and / or calcium hydroxide solution, sodium hydroxide solution with zinc chloride solution. Still more specifically, the pH value is adjusted using calcium hydroxide solution throughout the entire process, or the pH value is adjusted using calcium hydroxide solution for the first 0.095-0.1% of the total anaerobic fermentation time, and the pH value is adjusted using calcium hydroxide solution and sodium hydroxide solution for the remaining time. As one implementation method, 0-1.5... The pH value is adjusted using calcium hydroxide solution for the first day, and then using a combination of calcium hydroxide and sodium hydroxide solution for days 1.5-15.5; or, for the first 0.095-0.1 of the total anaerobic fermentation time, calcium hydroxide solution is used to adjust the pH value; for the middle 0.25-0.28 of the total anaerobic fermentation time, calcium hydroxide and sodium hydroxide solution are used to adjust the pH value; and for the remaining time period, calcium hydroxide, sodium hydroxide, and zinc chloride solution are used to adjust the pH value. As one implementation method, calcium hydroxide solution is used to adjust the pH value for days 0-1.5, a combination of calcium hydroxide and sodium hydroxide solution for days 1.5-5.5, and a combination of calcium hydroxide, sodium hydroxide, and zinc chloride solution for days 5.5-15.5. As a preferred method, the pH value is adjusted using calcium hydroxide solution during the first 0.095-0.1% of the total anaerobic fermentation time, using calcium hydroxide and sodium hydroxide solution during the middle 0.25-0.28% of the total anaerobic fermentation time, and using calcium hydroxide, sodium hydroxide, and zinc chloride solution for the remaining time. As another implementation method, the pH value is adjusted using calcium hydroxide solution from 0 to 1.5 days, using calcium hydroxide and sodium hydroxide solution from 1.5 to 5.5 days, and using calcium hydroxide, sodium hydroxide, and zinc chloride solution from 5.5 to 15.5 days.

[0027] The concentration of the calcium hydroxide solution is 1.5-2.5M, preferably 2M; the concentration of the sodium hydroxide solution is 1.5-2.5M, preferably 2M; the concentration of the zinc chloride solution is 1.5-2.5M, preferably 2M; when adjusting the pH value using calcium hydroxide solution and sodium hydroxide solution, the molar ratio of calcium hydroxide in the calcium hydroxide solution to sodium hydroxide in the sodium hydroxide solution is 0.8-1.5:1, preferably 1:1; when adjusting the pH value using calcium hydroxide solution, sodium hydroxide solution and zinc chloride solution, the molar ratio of calcium hydroxide in the calcium hydroxide solution, sodium hydroxide in the sodium hydroxide solution and zinc chloride in the zinc chloride solution is 1-3:1-3:1, preferably 1-2:1-2:1, more preferably 2:2:1.

[0028] The temperature control for anaerobic fermentation includes: maintaining a temperature of 30-32℃ for the first 0.095-0.1% of the total anaerobic fermentation time, a temperature of 35-37℃ for the middle 0.25-0.28% of the total anaerobic fermentation time, and a temperature of 38℃ for the remaining time; furthermore, maintaining the temperature at 30-32℃ for the first 0.095-0.1% of the total anaerobic fermentation time and at 38℃ for the middle 0.25-0.28% of the total anaerobic fermentation time. The temperature is gradually increased and maintained at 35-37℃ during the first phase, and gradually increased and maintained at 38℃ during the remaining time period. As one implementation method, the temperature is 30-32℃ for 0-1.5 days, 35-37℃ for 1.5-5.5 days, and 38℃ for 5.5-15.5 days. Further, the temperature is maintained at 30-32℃ for 0-1.5 days, gradually increased and maintained at 35-37℃ for 1.5-5.5 days, and gradually increased and maintained at 38℃ for 5.5-15.5 days.

[0029] After the aerobic pretreatment, before inoculating the microbial community composed of a mixture of anaerobic sludge, distiller's grains, and forest soil that has undergone enrichment treatment, nitrogen gas is introduced to remove oxygen and establish an anaerobic environment. The microbial community composed of a mixture of anaerobic sludge, distiller's grains, and forest soil that has undergone enrichment treatment is then inoculated. Through gradient temperature control and pH regulation, a carbon chain elongation reaction is carried out to generate hexanoic acid.

[0030] In one implementation method, anaerobic fermentation lasts 15.5 days. From day 0-1.5, the temperature is maintained at 30-32℃; from day 1.5-5.5, the temperature is gradually increased and maintained at 35-37℃; from day 5.5-15.5, the temperature is gradually increased and maintained at 38℃. From day 0-1.5, the pH is adjusted to 5.5±0.1 using calcium hydroxide solution; from day 1.5-5.5, the pH is adjusted to 6.5±0.1 using a mixture of calcium hydroxide and sodium hydroxide solution, with a molar ratio of 1:1 for calcium hydroxide solution and sodium hydroxide solution; from day 5.5-15.5, the pH is adjusted to 7.5±0.1 using a mixture of calcium hydroxide, sodium hydroxide, and zinc chloride solution, with a molar ratio of 2:2:1 for calcium hydroxide, sodium hydroxide, and zinc chloride solution. The concentrations of the calcium hydroxide, sodium hydroxide, and zinc chloride solutions are all 2M.

[0031] Compared with the prior art, the beneficial effects of the present invention are: (1) Efficient generation of endogenous electron donors: Compared with no aerobic pretreatment (Comparative Example 1), microaerobic pretreatment (Example 4) utilizes fungi (such as yeast and mold in brewing koji) to rapidly decompose macromolecular organic matter in kitchen waste, generating high concentrations of ethanol (12.36~12.98 g COD / L) and lactic acid (17.91~28.57 g COD / L), without the need for external electron donors, thus reducing production costs.

[0032] (2) Significantly increased hexanoic acid production: Compared with the control group without aerobic pretreatment (hexanoic acid concentration of only 1.61 g COD / L), the present invention optimizes the process and the hexanoic acid production can reach 23.64 g COD / L, which is 13.68 times higher and close to its maximum solubility in water.

[0033] (3) One-tank rapid fermentation: Through the seamless connection between aerobic pretreatment and anaerobic fermentation, the traditional two-stage process is simplified, the fermentation cycle is shortened (completed within 12 to 16 days), and the production efficiency is improved.

[0034] (4) Construction of synergistic microbial system: The efficiency of hydrolysis acidification and carbon chain elongation processes was enhanced by utilizing the synergistic effect of fungi and bacteria. At the same time, the functional bacterial community producing hexanoic acid was successfully enriched through gradient environmental regulation, which inhibited methane generation. Attached Figure Description

[0035] Figure 1 This is a schematic diagram of the process flow of the present invention.

[0036] Figure 2The following are comparative graphs showing the changes in the yield of major organic products during the fermentation process of Example 4 and Comparative Example 1: A: Graph showing the changes in the yield of major organic products in Comparative Example 1; B: Graph showing the changes in the yield of major organic products after 12 hours of aeration; C: Graph showing the changes in the yield of major organic products after 12 hours of aeration; D: Graph showing the changes in the yield of major organic products after 12 hours of aeration. Detailed Implementation

[0037] To make the technical means, creative features, objectives and effects of this invention easier to understand, the following embodiments are described in detail with reference to the accompanying drawings. It should be noted that the description of these embodiments is for the purpose of helping to understand this invention, but does not constitute a limitation of this invention.

[0038] In the following examples and comparative examples, the kitchen waste was collected from mixed kitchen waste from a university cafeteria in Shanghai, mainly consisting of rice, noodles, and vegetables. Anaerobic sludge was taken from the secondary sedimentation tank of a municipal wastewater treatment plant in Songjiang District, Shanghai, and distiller's grains came from fresh distiller's grains from breweries or distilleries. Forest soil was taken from the topsoil of uncontaminated broad-leaved forests. The Daqu and Hongqu fungal inoculants were commercially available products. These two types of distiller's grains were chosen because Daqu contains a rich enzyme system and diverse yeasts and molds, while Hongqu is known for its ability to produce amylase and saccharifying enzymes, and can also produce some beneficial secondary metabolites. In this text, VS stands for volatile solids.

[0039] Example 1 This embodiment utilizes different bacterial inoculums and inoculation ratios to enrich carbon chain growth bacteria to achieve rapid one-tank production of hexanoic acid through two-stage fermentation of kitchen waste. The specific steps are as follows: 1. Weigh the bacterial inoculum from each group using an electronic balance and place it separately into a large sterile beaker. Stir thoroughly with a sterile spatula to form a mixed inoculum source. The total amount of mixed inoculum source for each group can be scaled up as needed. The groupings are as follows: wet weight ratio of fresh distiller's grains and forest soil 1:1; wet weight ratio of anaerobic sludge and forest soil 1:1; wet weight ratio of fresh distiller's grains, forest soil, and anaerobic sludge 1:1:2; wet weight ratio of fresh distiller's grains, forest soil, and anaerobic sludge 2:1:1. Take 500 g of the mixture from each group and transfer it to a 2 L sterile serum bottle.

[0040] 2. Add approximately 1.5 L of sterile deionized water (solid-liquid ratio approximately 1:3, w / v) and stir with a glass rod to form a homogeneous slurry. Add sterile acetic acid solution to the slurry to bring the initial acetic acid concentration to approximately 5 g / L.

[0041] 3. Place the serum bottle in an ice bath (to reduce liquid evaporation), and through a long needle, purge high-purity N2 below the liquid surface at a flow rate of 1 L / min for 15-20 minutes, gently shaking the bottle during this time to thoroughly remove dissolved oxygen and oxygen from the headspace. Quickly remove the needle and seal the bottle with a sterile butyl rubber stopper and aluminum cap.

[0042] 4. Place the serum bottle in a 35°C constant-temperature shaker and incubate at 120 rpm in the dark. Inject an appropriate amount of 2M NaOH solution daily using a sterile syringe to adjust the pH to 5.5-6.5, at least three times a day. After 5-6 days of incubation, transfer the precipitated sediment to a sterile centrifuge bottle. Store at 4°C for later use; do not store for more than one week.

[0043] 5. Remove bones, plastics and other impurities from the kitchen waste, put it into a high-speed tissue homogenizer, add deionized water at a ratio of about 1:1 (w / w), and perform high-speed crushing and homogenization until a uniform slurry without obvious large particles is formed, and obtain a kitchen waste slurry sample with a concentration of 40 g VS / L.

[0044] 6. After grinding the yeast (Daqu and Hongqu) into a uniform powder using a mixer, weigh out the two powders in a 1:1 VS ratio according to their respective VS content, and mix them thoroughly in a sterile beaker. Add the mixed yeast to the kitchen waste slurry as an inoculum at a dosage of 10g VS / L. 餐厨垃圾 VS 酒曲 =4:1.

[0045] 7. The total volume is 1000 mL. Turn on the mechanical stirrer at 300 rpm to ensure thorough mixing of the materials. Simultaneously, start the water bath circulation to precisely control the temperature of all reactors at 35℃. During this stage, use 2M NaOH solution to stabilize the pH at 5.5.

[0046] 8. Prepare the air aeration pump and connect it to the gas flow controller (MFC) via corrosion-resistant piping (such as FEP or PFA tubing) to the air inlet of the fermenter. A 0.22μm hydrophobic sterile filter and a one-way valve must be installed before the air inlet (to prevent backflow of the fermentation broth). Aerate for 3 minutes at a total aeration rate of 0.2 vvm at hours 0, 4, and 8.

[0047] 9. At 12 hours, stop all aeration operations on time. Immediately introduce high-purity N2 into each reactor group for 5-6 minutes to thoroughly purge the oxygen from the top space of the reactor and the liquid phase, establishing a strictly anaerobic environment.

[0048] 10. Using a sterile peristaltic pump or in a sterile operating room, inoculate each reactor with 50 mL (5% v / v of the total fermentation broth volume) of the carbon chain growth microbial sludge enriched in Step 4. Reduce the stirring speed to 150 rpm to facilitate microbial floc formation.

[0049] XI. Temperature was controlled using a gradient over 16 days. From hour 12 to hour 48, the reactor temperature was set and maintained at a relatively low 30-32°C. From day 2 to day 6, the temperature was gradually increased and stabilized at 35-37°C. From day 6 until the end of the experiment (day 16), the temperature was further increased and stabilized at 38°C. The pH was maintained at 5.5 using a 2M NaOH solution during fermentation.

[0050] Samples were taken every two days during fermentation and placed in 10 mL centrifuge tubes. The fermentation broth was centrifuged at 8000 rpm for 10 min. The supernatant was then filtered to separate the microorganisms, and the contents of ethanol and hexanoic acid were detected. The results are shown in Table 1. Table 1. Investigation of different bacterial inoculums As can be seen from Table 1, when the wet weight ratio of fresh distiller's grains: forest soil: anaerobic sludge is 2:1:1, the maximum accumulation of ethanol in the supernatant reaches 10.89 g COD / L on the second day of fermentation, and the maximum accumulation of hexanoic acid in the supernatant reaches 11.98 g COD / L on the 14th day of fermentation.

[0051] Example 2 This embodiment utilizes different fungal inoculants and inoculation ratios to achieve rapid one-tank production of caproic acid through two-stage fermentation of kitchen waste. The specific steps are as follows: 1. Mix anaerobic sludge, fresh distiller's grains, and forest soil at a wet weight ratio of 2:1:1 to form a mixed inoculum. Transfer 500g of the mixture to a 2L sterile serum bottle.

[0052] Steps two, three, four, and five are the same as in Example 1.

[0053] VI. After grinding the Daqu and Hongqu into a uniform powder using a mixer, add the following groups of yeast to the kitchen waste slurry at a dosage of 10gVS / L: Daqu group, Hongqu group, and Daqu and Hongqu group (VS / L). 大曲 VS 红曲 =1:1), Daqu and Hongqu groups (VS 大曲 VS 红曲 =2:1). VS 餐厨垃圾 VS 酒曲 =4:1.

[0054] VII. The operation is the same as in Example 1, with a total volume of 1000 mL for each group.

[0055] Steps eight, nine, ten, and eleven are the same as in Example 1.

[0056] Samples were taken every two days during fermentation and placed in 10 mL centrifuge tubes. The fermentation broth was centrifuged at 8000 rpm for 10 min. The supernatant was then filtered to separate the microorganisms, and the contents of ethanol and hexanoic acid were detected. The results are shown in Table 2. Table 2. Investigation of different fungal inoculants As can be seen from Table 2, when VS 大曲 VS 红曲 = 1:1, on the second day of fermentation, the ethanol accumulation in the supernatant reached the maximum of 10.21 g COD / L, and on the 14th day of fermentation, the hexanoic acid accumulation in the supernatant reached the maximum of 13.74 g COD / L.

[0057] Example 3 This embodiment utilizes different aeration gases and mixing ratios to achieve rapid one-tank production of hexanoic acid through two-stage fermentation of kitchen waste. The specific steps are as follows: Steps one, two, three, four, and five are the same as in Example 2.

[0058] 6. After grinding the yeast (Daqu and Hongqu) into a uniform powder using a mixer, weigh out the two powders in a 1:1 VS ratio according to their respective VS content, and mix them thoroughly in a sterile beaker. Add the mixed yeast to the kitchen waste slurry as an inoculum at a dosage of 10g VS / L. 餐厨垃圾 VS 酒曲 =4:1.

[0059] VII. The operation is the same as in Example 2, with the total volume for each group being 1000 mL.

[0060] 8. Prepare three independent gas cylinders: oil-free compressed air, high-purity CO2, and high-purity H2S. Connect the outlets of the three gas flow controllers (MFCs) via stainless steel tees or a small gas mixing chamber. The mixed gases (introduced at volume ratios of 1:0:0, 1:1:0, 1:1:1, 2:1:1, and 3:1:1) are connected to the fermenter's inlet via corrosion-resistant piping (such as FEP or PFA tubing). A 0.22μm hydrophobic sterile filter and a one-way valve must be installed before the inlet (to prevent backflow of the fermentation broth). Aerate for 3 minutes at a total aeration rate of 0.2 vvm at hours 0, 4, and 8.

[0061] Steps nine, ten, and eleven are the same as in Example 2.

[0062] Samples were taken every two days during fermentation and placed in 10 mL centrifuge tubes. The fermentation broth was centrifuged at 8000 rpm for 10 min. The supernatant was obtained, filtered, and the microorganisms were separated. The contents of ethanol and hexanoic acid were then detected. The results are shown in Table 3. Table 3. Investigation of different aeration gases As can be seen from Table 3, when the volume ratio of air:CO2:H2S in the aeration is 3:1:1, the ethanol accumulation in the supernatant reaches the maximum of 11.24 g COD / L on the second day of fermentation, and the hexanoic acid accumulation in the supernatant reaches the maximum of 14.85 g COD / L on the 12th day of fermentation.

[0063] Example 4 This embodiment utilizes different aeration intensities to achieve rapid one-tank production of hexanoic acid through two-stage fermentation of kitchen waste. The specific steps are as follows: Steps one through seven are the same as in Example 3.

[0064] 8. Prepare three independent gas cylinders: oil-free compressed air, high-purity CO2, and high-purity H2S. Connect the outlets of the three gas flow controllers (MFCs) via stainless steel tees or a small gas mixing chamber. The mixed gas (volume ratio 3:1:1) is connected to the fermenter's inlet via corrosion-resistant piping (such as FEP or PFA tubing). A 0.22μm hydrophobic sterile filter and a one-way valve must be installed before the inlet (to prevent backflow of the fermentation broth). Aerate at a rate of 0.2 vvm once (0h), twice (0h, 6h), and three times (0h, 4h, 8h) within the first 12 hours, each time for 3 minutes.

[0065] Steps nine, ten, and eleven are the same as in Example 3.

[0066] Samples were taken every two days during fermentation and placed in 10 mL centrifuge tubes. The fermentation broth was centrifuged at 8000 rpm for 10 min. The supernatant was then filtered to separate the microorganisms, and the contents of ethanol and hexanoic acid were detected. The results are shown in Table 4. Table 4. Investigation of different aeration intensities As can be seen from Table 4, when aeration was performed twice, the maximum accumulation of ethanol in the supernatant reached 12.98 g COD / L on the second day of fermentation, and the maximum accumulation of hexanoic acid in the supernatant reached 16.58 g COD / L on the 12th day of fermentation.

[0067] Example 5 This embodiment utilizes different pH control methods to achieve rapid one-tank production of hexanoic acid through two-stage fermentation of kitchen waste, specifically following these steps: Steps one through seven are the same as in Example 4.

[0068] 8. Prepare three independent gas cylinders: oil-free compressed air, high-purity CO2, and high-purity H2S. Connect the outlets of the three gas flow controllers (MFCs) via stainless steel tees or a small gas mixing chamber. Connect the mixed gases (volume ratio 3:1:1) to the fermenter inlet via corrosion-resistant piping (such as FEP or PFA tubing). A 0.22μm hydrophobic sterile filter and a one-way valve must be installed before the inlet (to prevent backflow of the fermentation broth). Aerate for 3 minutes at a total aeration rate of 0.2 vvm at hours 0 and 6.

[0069] Steps nine and ten are the same as in Example 4.

[0070] The temperature gradient control in step eleven is the same as in Example 4.

[0071] During fermentation, different pH control methods were investigated, and step twelve was added: pH control was performed using 2M NaOH solution during fermentation. Stable pH control throughout the entire process (5.5, 6.5, 7.5) and phased gradient control were implemented (stable at 5.5 for 12-48 hours, stable at 6.5 for 2-16 days, and stable at 5.5 for 12-48 hours, stable at 6.5 for 2-6 days, and stable at 7.5 for 6-16 days). Samples were taken every two days during fermentation, placed in 10mL centrifuge tubes, centrifuged at 8000 rpm for 10 min, and the supernatant was filtered to separate microorganisms. The ethanol and hexanoic acid contents were then measured, and the results are shown in Table 5. Table 5. Investigation of different pH control methods As can be seen from Table 5, when the pH is adjusted to increase in a stepwise manner (5.5+6.5+7.5), the ethanol accumulation in the supernatant reaches the maximum of 12.84 g COD / L on the second day of fermentation, and the hexanoic acid accumulation in the supernatant reaches the maximum of 18.36 g COD / L on the tenth day of fermentation.

[0072] Example 6 This embodiment utilizes different alkaline agents to adjust the pH and achieves rapid one-tank production of hexanoic acid through two-stage fermentation of kitchen waste. The specific steps are as follows: Steps one, two, and three are the same as in Example 5.

[0073] 4. Place the serum bottle in a 35°C constant-temperature shaker and incubate at 120 rpm in the dark. Inject an appropriate amount of 2M Ca(OH)₂ solution daily using a sterile syringe to adjust the pH to 5.5-6.5, at least three times a day. After 5-6 days of incubation, transfer the precipitated sediment to a sterile centrifuge bottle. Store at 4°C for later use; do not store for more than one week.

[0074] Steps five, six, seven, eight, nine, ten, and eleven are the same as in Example 5.

[0075] 12. In conjunction with temperature control, a phased pH gradient control strategy can also be implemented using different alkali agents (all at a concentration of 2M).

[0076] (1) During the first 12-48 hours, the pH value was strictly controlled within the acidic range of 5.5 ± 0.1 using Ca(OH)2 solution. From day 2 to day 6, the pH setting was increased to 6.5 ± 0.1. From day 6 to day 16, the pH was further increased to the weakly alkaline range of 7.5 ± 0.1.

[0077] (2) During the first 12-48 hours, the pH value was strictly controlled within the acidic range of 5.5 ± 0.1 using Ca(OH)₂ solution. From day 2 to day 6, the pH value was increased to 6.5 ± 0.1 using Ca(OH)₂ solution and NaOH solution (in a 1:1 ratio). From day 6 to day 16, the pH value was further increased to the weakly alkaline range of 7.5 ± 0.1 using Ca(OH)₂ solution and NaOH solution.

[0078] (3) During the first 12-48 hours, the pH value was strictly controlled within the acidic range of 5.5 ± 0.1 using Ca(OH)2 solution. From day 2 to day 6, the pH value was increased to 6.5 ± 0.1 using Ca(OH)2 solution and NaOH solution (in a 1:1 ratio). From day 6 to day 16, the pH value was further increased to the weakly alkaline range of 7.5 ± 0.1 using Ca(OH)2 solution, NaOH solution, and ZnCl2 solution (in a 1:1:1 ratio).

[0079] (4) During the first 12-48 hours, the pH value was strictly controlled within the acidic range of 5.5 ± 0.1 using Ca(OH)2 solution. From day 2 to day 6, the pH value was raised to 6.5 ± 0.1 using Ca(OH)2 solution and NaOH solution (in a ratio of 1:1). From day 6 to day 16, the pH value was further raised to the weakly alkaline range of 7.5 ± 0.1 using Ca(OH)2 solution, NaOH solution, and ZnCl2 solution (in a ratio of 2:2:1).

[0080] Samples were taken every two days during fermentation and placed in 10 mL centrifuge tubes. The fermentation broth was centrifuged at 8000 rpm for 10 min. The supernatant was then filtered to separate the microorganisms, and the contents of ethanol and hexanoic acid were detected. The results are shown in Table 6. Table 6. Investigation of different alkali agents As shown in Table 6, when the pH was adjusted in a stepwise manner, the pH was adjusted to 5.5 ± 0.1 using Ca(OH)₂ from 12 to 48 hours. From day 2 to 6, the pH was adjusted to 6.5 ± 0.1 using Ca(OH)₂ and NaOH (in a 1:1 ratio). From day 6 to 16, the pH was adjusted to 7.5 ± 0.1 using Ca(OH)₂, NaOH, and ZnCl₂ (in a 2:2:1 ratio). On day 2 of fermentation, the ethanol accumulation in the supernatant reached its maximum of 13.66 g COD / L, and on day 8 of fermentation, the hexanoic acid accumulation in the supernatant reached its maximum of 23.64 g COD / L.

[0081] Comparative Example 1 Using yeast as inoculum and kitchen waste as substrate, a fully anaerobic fermentation process is carried out, specifically following these steps: 1. Anaerobic sludge, fresh distiller's grains, and forest soil are mixed at a wet weight ratio of 2:1:1 to form a mixed inoculum. Take 500 g and transfer it to a 2 L sterile serum bottle.

[0082] 2. Add approximately 1.5 L of sterile deionized water (solid-liquid ratio approximately 1:3, w / v) and stir with a glass rod to form a homogeneous slurry. Add sterile acetic acid solution to the slurry to bring the initial acetic acid concentration to approximately 5 g / L.

[0083] 3. Place the serum bottle in an ice bath and introduce high-purity N2 below the liquid surface through a long needle at a flow rate of 1 L / min for 15-20 minutes to thoroughly remove dissolved oxygen and oxygen from the headspace. Quickly remove the needle and seal the bottle with a sterile butyl rubber stopper and aluminum cap.

[0084] 4. Place the serum bottle in a 35°C constant temperature shaker and incubate at 120 rpm in the dark. Inject an appropriate amount of 2M NaOH solution daily using a sterile syringe to adjust the pH to 5.5-6.5, at least three times a day. After 5-6 days of incubation, transfer the precipitated sediment to a sterile centrifuge bottle for later use.

[0085] 5. Remove bones, plastics and other impurities from the kitchen waste, put it into a high-speed tissue homogenizer, add deionized water at a ratio of about 1:1 (w / w), and perform high-speed crushing and homogenization to obtain a kitchen waste slurry sample with a concentration of 40 gVS / L.

[0086] 6. After grinding the yeast (Daqu and Hongqu) into a uniform powder using a mixer, weigh out the two powders in a 1:1 VS ratio according to their respective VS content, and mix them thoroughly in a sterile beaker. Add the mixed yeast to the kitchen waste slurry as an inoculum at a dosage of 10g VS / L. 餐厨垃圾 VS 酒曲 =4:1.

[0087] 7. The total volume is 1000 mL. Turn on the mechanical stirrer at 300 rpm to ensure thorough mixing of the materials. Simultaneously, start the water bath circulation to precisely control the reactor temperature at 35℃. During this stage, use 2M NaOH to stabilize the pH at 5.5.

[0088] 8. Omit the aerobic pretreatment step. Immediately after completing step 7, introduce high-purity N2 into the reactor for 5-6 minutes to thoroughly purge the oxygen from the top space of the reactor and the liquid phase, establishing a strictly anaerobic environment.

[0089] 9. Using a sterile peristaltic pump, inoculate the reactor with 50 mL (5% v / v of the total fermentation broth volume) of the carbon chain growth bacteria substrate enriched in step four. Reduce the stirring speed to 150 rpm.

[0090] 10. Temperature was controlled using a gradient over 16 days: from hour 0 to hour 48, the temperature was maintained at 30-32℃; from day 2 to day 6, the temperature was increased and stabilized at 35-37℃; from day 6 until the end of the experiment, the temperature was stabilized at 38℃. Throughout the fermentation process, a 2M NaOH solution was used to maintain the pH at 5.5.

[0091] Test results: Without any aerobic pretreatment, the hydrolysis and acidification efficiency of the fermentation system was extremely low. On day 6 of fermentation, the highest accumulation of ethanol in the supernatant was only 2.7 g COD / L. By the end of fermentation (day 16), the highest yield of hexanoic acid in the supernatant was only 1.61 g COD / L.

[0092] Comparative Example 2 Using yeast as inoculum and kitchen waste as substrate, with oxygen as the aeration gas, the process is carried out according to the following steps: 1. Anaerobic sludge, fresh distiller's grains, and forest soil are mixed at a wet weight ratio of 2:1:1 to form a mixed inoculum. Take 500 g and transfer it to a 2 L sterile serum bottle.

[0093] 2. Add approximately 1.5 L of sterile deionized water (solid-liquid ratio approximately 1:3, w / v) and stir with a glass rod to form a homogeneous slurry. Add sterile acetic acid solution to the slurry to bring the initial acetic acid concentration to approximately 5 g / L.

[0094] 3. Place the serum bottle in an ice bath (to reduce liquid evaporation), and through a long needle, purge high-purity N2 below the liquid surface at a flow rate of 1 L / min for 15-20 minutes, gently shaking the bottle during this time to thoroughly remove dissolved oxygen and oxygen from the headspace. Quickly remove the needle and seal the bottle with a sterile butyl rubber stopper and aluminum cap.

[0095] 4. Place the serum bottle in a 35°C constant-temperature shaker and incubate at 120 rpm in the dark. Inject an appropriate amount of 2M NaOH solution daily using a sterile syringe to adjust the pH to 5.5-6.5, at least three times a day. After 5-6 days of incubation, transfer the precipitated sediment to a sterile centrifuge bottle. Store at 4°C for later use; do not store for more than one week.

[0096] 5. Remove bones, plastics and other impurities from the kitchen waste, put it into a high-speed tissue homogenizer, add deionized water at a ratio of about 1:1 (w / w), and perform high-speed crushing and homogenization until a uniform slurry without obvious large particles is formed, and obtain a kitchen waste slurry sample with a concentration of 40 g VS / L.

[0097] 6. After grinding the yeast (Daqu and Hongqu) into a uniform powder using a mixer, weigh out the two powders in a 1:1 VS ratio according to their respective VS content, and mix them thoroughly in a sterile beaker. Add the mixed yeast to the kitchen waste slurry as an inoculum at a dosage of 10g VS / L. 餐厨垃圾 VS 酒曲 =4:1.

[0098] 7. The total volume is 1000 mL. Turn on the mechanical stirrer at 300 rpm to ensure thorough mixing of the materials. Simultaneously, start the water bath circulation to precisely control the temperature of all reactors at 35℃. During this stage, use 2M NaOH solution to stabilize the pH at 5.5.

[0099] 8. Prepare a high-purity oxygen cylinder and connect it to the gas flow controller (MFC) via corrosion-resistant tubing (such as FEP or PFA tubing) to the fermenter's inlet. A 0.22μm hydrophobic sterile filter and a one-way valve must be installed before the inlet (to prevent backflow of the fermentation broth). Aerate for 3 minutes at a total aeration rate of 0.2 vvm at hours 0, 4, and 8.

[0100] 9. At 12 hours, immediately stop all aeration operations. Immediately introduce high-purity N2 into the reactor for 5-6 minutes to thoroughly purge the oxygen from the top space of the reactor and the liquid phase, establishing a strictly anaerobic environment.

[0101] 10. Using a sterile peristaltic pump or in a sterile operating room, inoculate the reactor with 50 mL (5% v / v of the total fermentation broth volume) of the carbon chain growth microbial sludge enriched in Step 4. Reduce the stirring speed to 150 rpm to facilitate microbial floc formation.

[0102] XI. Temperature was controlled using a gradient over 16 days. From hour 12 to hour 48, the reactor temperature was set and maintained at a relatively low 30-32°C. From day 2 to day 6, the temperature was gradually increased and stabilized at 35-37°C. From day 6 until the end of the experiment (day 16), the temperature was further increased and stabilized at 38°C. The pH was maintained at 5.5 using a 2M NaOH solution during fermentation.

[0103] Test results: Using yeast as inoculum and kitchen waste as substrate, under the condition of only oxygen being introduced during the aeration stage, the maximum ethanol accumulation in the supernatant reached 12.36 g COD / L on the 4th day of fermentation, and the maximum hexanoic acid concentration in the supernatant was only 7.22 g COD / L on the 12th day of fermentation.

[0104] Comparative Example 3 Using yeast as inoculum and kitchen waste as substrate, the aeration mode was changed to continuous aeration, which was accomplished by following these steps: 1. Anaerobic sludge, fresh distiller's grains, and forest soil are mixed at a wet weight ratio of 2:1:1 to form a mixed inoculum. Take 500 g and transfer it to a 2 L sterile serum bottle.

[0105] 2. Add approximately 1.5 L of sterile deionized water (solid-liquid ratio approximately 1:3, w / v) and stir with a glass rod to form a homogeneous slurry. Add sterile acetic acid solution to the slurry to bring the initial acetic acid concentration to approximately 5 g / L.

[0106] 3. Place the serum bottle in an ice bath (to reduce liquid evaporation), and through a long needle, purge high-purity N2 below the liquid surface at a flow rate of 1 L / min for 15-20 minutes, gently shaking the bottle during this time to thoroughly remove dissolved oxygen and oxygen from the headspace. Quickly remove the needle and seal the bottle with a sterile butyl rubber stopper and aluminum cap.

[0107] 4. Place the serum bottle in a 35°C constant-temperature shaker and incubate at 120 rpm in the dark. Inject an appropriate amount of 2M NaOH solution daily using a sterile syringe to adjust the pH to 5.5-6.5, at least three times a day. After 5-6 days of incubation, transfer the precipitated sediment to a sterile centrifuge bottle. Store at 4°C for later use; do not store for more than one week.

[0108] 5. Remove bones, plastics and other impurities from the kitchen waste, put it into a high-speed tissue homogenizer, add deionized water at a ratio of about 1:1 (w / w), and perform high-speed crushing and homogenization until a uniform slurry without obvious large particles is formed, and obtain a kitchen waste slurry sample with a concentration of 40 g VS / L.

[0109] 6. After grinding the yeast (Daqu and Hongqu) into a uniform powder using a mixer, weigh out the two powders in a 1:1 VS ratio according to their respective VS content, and mix them thoroughly in a sterile beaker. Add the mixed yeast to the kitchen waste slurry as an inoculum at a dosage of 10g VS / L. 餐厨垃圾 VS 酒曲 =4:1.

[0110] 7. The total volume is 1000 mL. Turn on the mechanical stirrer at 300 rpm to ensure thorough mixing of the materials. Simultaneously, start the water bath circulation to precisely control the temperature of all reactors at 35℃. During this stage, use 2M NaOH solution to stabilize the pH at 5.5.

[0111] 8. Prepare three independent gas cylinders: oil-free compressed air, high-purity CO2, and high-purity H2S. Connect the outlets of the three gas flow controllers (MFCs) via stainless steel tees or a small gas mixing chamber. Connect the mixed gases (volume ratio 3:1:1) to the fermenter inlet via corrosion-resistant piping (such as FEP or PFA tubing). A 0.22μm hydrophobic sterile filter and a one-way valve must be installed before the inlet (to prevent backflow of the fermentation broth). Aerate continuously for 12 hours at a rate of 0.2 vvm over 0-12 hours.

[0112] 9. At 12 hours, immediately stop all aeration operations. Immediately introduce high-purity N2 into the reactor for 5-6 minutes to thoroughly purge the oxygen from the top space of the reactor and the liquid phase, establishing a strictly anaerobic environment.

[0113] 10. Using a sterile peristaltic pump or in a sterile operating room, inoculate the reactor with 50 mL (5% v / v of the total fermentation broth volume) of the carbon chain growth microbial sludge enriched in Step 4. Reduce the stirring speed to 150 rpm to facilitate microbial floc formation.

[0114] XI. Temperature was controlled using a gradient over 16 days. From hour 12 to hour 48, the reactor temperature was set and maintained at a relatively low 30-32°C. From day 2 to day 6, the temperature was gradually increased and stabilized at 35-37°C. From day 6 until the end of the experiment (day 16), the temperature was further increased and stabilized at 38°C. The pH was maintained at 5.5 using a 2M NaOH solution during fermentation.

[0115] Samples were taken every two days during fermentation, placed in 10mL centrifuge tubes, and centrifuged at 8000r for 10 min. The supernatant was then filtered to separate the microorganisms, and the contents of ethanol and hexanoic acid were determined.

[0116] Test results: Under 12 hours of continuous aeration, the highest concentration of ethanol in the supernatant on day 6 was 12.8 gCOD / L. Ultimately, on day 16 of fermentation, continuous aeration caused oxygen pollution in the anaerobic fermentation, failing to successfully enrich the anaerobic caproic acid-producing bacteria, and no caproic acid was detected in the supernatant.

[0117] Comparative Example 4 Using kitchen waste as a substrate and enriched bacterial flora as an inoculum, without adding exogenous fungi, the process is carried out in the following steps: 1. Anaerobic sludge, fresh distiller's grains, and forest soil are mixed at a wet weight ratio of 2:1:1 to form a mixed inoculum. Take 500 g and transfer it to a 2 L sterile serum bottle.

[0118] 2. Add approximately 1.5 L of sterile deionized water (solid-liquid ratio approximately 1:3, w / v) and stir with a glass rod to form a homogeneous slurry. Add sterile acetic acid solution to the slurry to bring the initial acetic acid concentration to approximately 5 g / L.

[0119] 3. Place the serum bottle in an ice bath (to reduce liquid evaporation), and through a long needle, purge high-purity N2 below the liquid surface at a flow rate of 1 L / min for 15-20 minutes, gently shaking the bottle during this time to thoroughly remove dissolved oxygen and oxygen from the headspace. Quickly remove the needle and seal the bottle with a sterile butyl rubber stopper and aluminum cap.

[0120] 4. Place the serum bottle in a 35°C constant-temperature shaker and incubate at 120 rpm in the dark. Inject an appropriate amount of 2M NaOH solution daily using a sterile syringe to adjust the pH to 5.5-6.5, at least three times a day. After 5-6 days of incubation, transfer the precipitated sediment to a sterile centrifuge bottle. Store at 4°C for later use; do not store for more than one week.

[0121] 5. Remove bones, plastics and other impurities from the kitchen waste, put it into a high-speed tissue homogenizer, add deionized water at a ratio of about 1:1 (w / w), and perform high-speed crushing and homogenization until a uniform slurry without obvious large particles is formed, and obtain a kitchen waste slurry sample with a concentration of 40 g VS / L.

[0122] 6. The total volume is 1000 mL. Turn on the mechanical stirrer at 300 rpm to ensure thorough mixing of the materials. Simultaneously, start the water bath circulation to precisely control the temperature of all reactors at 35℃. During this stage, use 2M NaOH solution to stabilize the pH at 5.5.

[0123] 7. Prepare an air aeration pump and connect it to a gas flow controller (MFC) via corrosion-resistant piping (such as FEP or PFA tubing) to the fermenter's air inlet. A 0.22μm hydrophobic sterile filter and a one-way valve must be installed before the air inlet (to prevent backflow of the fermentation broth). Aerate for 3 minutes at a total aeration rate of 0.2 vvm at hours 0, 4, and 8.

[0124] 8. At 12 hours, immediately stop all aeration operations. Immediately introduce high-purity N2 into the reactor for 5-6 minutes to thoroughly purge the oxygen from the top space of the reactor and the liquid phase, establishing a strictly anaerobic environment.

[0125] 9. Using a sterile peristaltic pump or in a sterile operating room, inoculate the reactor with 50 mL (5% v / v of the total fermentation broth volume) of the carbon chain growth microbial sludge enriched in Step 4. Reduce the stirring speed to 150 rpm to facilitate microbial floc formation.

[0126] 10. Temperature was controlled using a gradient over 16 days. From hour 12 to hour 48, the reactor temperature was set and maintained at a relatively low 30-32°C. From day 2 to day 6, the temperature was gradually increased and stabilized at 35-37°C. From day 6 until the end of the experiment (day 16), the temperature was further increased and stabilized at 38°C. The pH was maintained at 5.5 using a 2M NaOH solution during fermentation.

[0127] Samples were taken every two days during fermentation, placed in 10mL centrifuge tubes, and centrifuged at 8000r for 10 min. The supernatant was then filtered to separate the microorganisms, and the contents of ethanol and hexanoic acid were determined.

[0128] Test results: In the absence of exogenous fungal inoculum, ethanol accumulation was slow and the concentration was low during the aerobic pretreatment stage, reaching a maximum of only 3.8 g COD / L on day 6. Finally, on day 16 of fermentation, no hexanoic acid was detected in the supernatant.

[0129] Comparative Example 5 Using kitchen waste as the substrate and the enriched bacterial flora as the inoculum, with only baker's yeast added as the fungal inoculum, the process is carried out in the following steps: 1. Anaerobic sludge, fresh distiller's grains, and forest soil are mixed at a wet weight ratio of 2:1:1 to form a mixed inoculum. Take 500 g and transfer it to a 2 L sterile serum bottle.

[0130] 2. Add approximately 1.5 L of sterile deionized water (solid-liquid ratio approximately 1:3, w / v) and stir with a glass rod to form a homogeneous slurry. Add sterile acetic acid solution to the slurry to bring the initial acetic acid concentration to approximately 5 g / L.

[0131] 3. Place the serum bottle in an ice bath (to reduce liquid evaporation), and through a long needle, purge high-purity N2 below the liquid surface at a flow rate of 1 L / min for 15-20 minutes, gently shaking the bottle during this time to thoroughly remove dissolved oxygen and oxygen from the headspace. Quickly remove the needle and seal the bottle with a sterile butyl rubber stopper and aluminum cap.

[0132] 4. Place the serum bottle in a 35°C constant-temperature shaker and incubate at 120 rpm in the dark. Inject an appropriate amount of 2M NaOH solution daily using a sterile syringe to adjust the pH to 5.5-6.5, at least three times a day. After 5-6 days of incubation, transfer the precipitated sediment to a sterile centrifuge bottle. Store at 4°C for later use; do not store for more than one week.

[0133] 5. Remove bones, plastics, and other impurities from the kitchen waste. Place it in a high-speed tissue homogenizer and add deionized water at approximately a 1:1 (w / w) ratio. Perform high-speed homogenization and slurrying until a homogeneous slurry without obvious large particles is formed, yielding a kitchen waste slurry sample with a concentration of 40 g VS / L. Add baker's yeast to the kitchen waste slurry as an inoculum at a dosage of 10 g VS / L. 餐厨垃圾 VS 酵母 =4:1.

[0134] 6. The total volume is 1000 mL. Turn on the mechanical stirrer at 300 rpm to ensure thorough mixing of the materials. Simultaneously, start the water bath circulation to precisely control the temperature of all reactors at 35℃. During this stage, use 2M NaOH solution to stabilize the pH at 5.5.

[0135] 7. Prepare an air aeration pump and connect it to a gas flow controller (MFC) via corrosion-resistant piping (such as FEP or PFA tubing) to the fermenter's air inlet. A 0.22μm hydrophobic sterile filter and a one-way valve must be installed before the air inlet (to prevent backflow of the fermentation broth). Aerate for 3 minutes at a total aeration rate of 0.2 vvm at hours 0, 4, and 8.

[0136] 8. At 12 hours, immediately stop all aeration operations. Immediately introduce high-purity N2 into the reactor for 5-6 minutes to thoroughly purge the oxygen from the top space of the reactor and the liquid phase, establishing a strictly anaerobic environment.

[0137] 9. Using a sterile peristaltic pump or in a sterile operating room, inoculate the reactor with 50 mL (5% v / v of the total fermentation broth volume) of the carbon chain growth microbial sludge enriched in Step 4. Reduce the stirring speed to 150 rpm to facilitate microbial floc formation.

[0138] 10. Temperature was controlled using a gradient over 16 days. From hour 12 to hour 48, the reactor temperature was set and maintained at a relatively low 30-32°C. From day 2 to day 6, the temperature was gradually increased and stabilized at 35-37°C. From day 6 until the end of the experiment (day 16), the temperature was further increased and stabilized at 38°C. The pH was maintained at 5.5 using a 2M NaOH solution during fermentation.

[0139] Samples were taken every two days during fermentation, placed in 10mL centrifuge tubes, and centrifuged at 8000r for 10 min. The supernatant was then filtered to separate the microorganisms, and the contents of ethanol and hexanoic acid were determined.

[0140] Test results: With baker's yeast as the fungal inoculum, ethanol accumulation was normal during the aerobic pretreatment stage, with a maximum concentration of 9.38 g COD / L on day 2. Finally, by the end of fermentation (day 16), the highest yield of hexanoic acid in the supernatant was only 6.97 g COD / L.

[0141] Comparative Example 6 Using yeast as inoculum and kitchen waste as substrate, without adding enriched carbon-chain growth bacteria, the process is carried out according to the following steps: 1. Remove bones, plastics and other impurities from kitchen waste, put it into a high-speed tissue homogenizer, add deionized water at a ratio of about 1:1 (w / w), and perform high-speed crushing and homogenization until a uniform slurry without obvious large particles is formed, and obtain a kitchen waste slurry sample with a concentration of 40 g VS / L.

[0142] 2. After grinding the yeast (Daqu and Hongqu) into a uniform powder using a mixer, weigh out the two powders in a 1:1 VS ratio according to their respective VS content, and mix them thoroughly in a sterile beaker. Add the mixed yeast to the kitchen waste slurry as an inoculum at a dosage of 10g VS / L. 餐厨垃圾 VS 酒曲 =4:1.

[0143] 3. The total volume is 1000 mL. Turn on the mechanical stirrer at 300 rpm to ensure thorough mixing of the materials. Simultaneously, start the water bath circulation to precisely control the temperature of all reactors at 35℃. During this stage, use 2M NaOH solution to stabilize the pH at 5.5.

[0144] 4. Prepare the air aeration pump and connect it to the gas flow controller (MFC) via corrosion-resistant piping (such as FEP or PFA tubing) to the air inlet of the fermenter. A 0.22μm hydrophobic sterile filter and a one-way valve must be installed before the air inlet (to prevent backflow of the fermentation broth). Aerate for 3 minutes at a total aeration rate of 0.2 vvm at hours 0, 4, and 8.

[0145] 5. At 12 hours, immediately stop all aeration operations. Immediately introduce high-purity N2 into the reactor for 5-6 minutes to thoroughly purge oxygen from the top space and liquid phase of the reactor, establishing a strictly anaerobic environment. Reduce the stirring speed to 150 rpm to facilitate the formation of microbial flocs.

[0146] VI. Temperature was controlled using a gradient over 16 days. From hour 12 to hour 48, the reactor temperature was set and maintained at a relatively low 30-32°C. From day 2 to day 6, the temperature was gradually increased and stabilized at 35-37°C. From day 6 until the end of the experiment (day 16), the temperature was further increased and stabilized at 38°C. The pH was maintained at 5.5 using 2M NaOH solution during fermentation.

[0147] Samples were taken every two days during fermentation, placed in 10mL centrifuge tubes, and centrifuged at 8000r for 10 min. The supernatant was then filtered to separate the microorganisms, and the contents of ethanol and hexanoic acid were determined.

[0148] Test results: In the absence of added bacterial inoculum, ethanol accumulation was normal during the aerobic pretreatment stage, with a peak concentration of 10.71 g COD / L on day 2. Ultimately, by the end of fermentation (day 16), the highest yield of hexanoic acid in the supernatant was only 2.97 g COD / L.

[0149] Comparative Example 7 Using yeast and enriched carbon-chain growth bacteria as inoculum, and kitchen waste as substrate, fermentation is carried out under constant conditions with no gradient temperature control and gradient pH. The specific steps are as follows: 1. Anaerobic sludge, fresh distiller's grains, and forest soil are mixed at a wet weight ratio of 2:1:1 to form a mixed inoculum. Take 500 g and transfer it to a 2 L sterile serum bottle.

[0150] 2. Add approximately 1.5 L of sterile deionized water (solid-liquid ratio approximately 1:3, w / v) and stir with a glass rod to form a homogeneous slurry. Add sterile acetic acid solution to the slurry to bring the initial acetic acid concentration to approximately 5 g / L.

[0151] 3. Place the serum bottle in an ice bath (to reduce liquid evaporation), and through a long needle, purge high-purity N2 below the liquid surface at a flow rate of 1 L / min for 15-20 minutes, gently shaking the bottle during this time to thoroughly remove dissolved oxygen and oxygen from the headspace. Quickly remove the needle and seal the bottle with a sterile butyl rubber stopper and aluminum cap.

[0152] 4. Place the serum bottle in a 35°C constant-temperature shaker and incubate at 120 rpm in the dark. Inject an appropriate amount of 2M NaOH solution daily using a sterile syringe to adjust the pH to 5.5-6.5, at least three times a day. After 5-6 days of incubation, transfer the precipitated sediment to a sterile centrifuge bottle. Store at 4°C for later use; do not store for more than one week.

[0153] 5. Remove bones, plastics and other impurities from the kitchen waste, put it into a high-speed tissue homogenizer, add deionized water at a ratio of about 1:1 (w / w), and perform high-speed crushing and homogenization until a uniform slurry without obvious large particles is formed, and obtain a kitchen waste slurry sample with a concentration of 40 g VS / L.

[0154] 6. After grinding the yeast (Daqu and Hongqu) into a uniform powder using a mixer, weigh out the two powders in a 1:1 VS ratio according to their respective VS content, and mix them thoroughly in a sterile beaker. Add the mixed yeast to the kitchen waste slurry as an inoculum at a dosage of 10g VS / L. 餐厨垃圾 VS 酒曲 =4:1.

[0155] 7. The total volume is 1000 mL. Turn on the mechanical stirrer at 300 rpm to ensure thorough mixing of the materials. Simultaneously, start the water bath circulation to precisely control the temperature of all reactors at 35℃. During this stage, use 2M NaOH solution to stabilize the pH at 5.5.

[0156] 8. Prepare three independent gas cylinders: oil-free compressed air, high-purity CO2, and high-purity H2S. Connect the outlets of the three gas flow controllers (MFCs) via stainless steel tees or a small gas mixing chamber. Connect the mixed gases (volume ratio 3:1:1) to the fermenter inlet via corrosion-resistant piping (such as FEP or PFA tubing). A 0.22μm hydrophobic sterile filter and a one-way valve must be installed before the inlet (to prevent backflow of the fermentation broth). Aerate for 3 minutes at a total aeration rate of 0.2 vvm at hours 0 and 6.

[0157] 9. At 12 hours, immediately stop all aeration operations. Immediately introduce high-purity N2 into the reactor for 5-6 minutes to thoroughly purge the oxygen from the top space of the reactor and the liquid phase, establishing a strictly anaerobic environment.

[0158] 10. Using a sterile peristaltic pump or in a sterile operating room, inoculate the reactor with 50 mL (5% v / v of the total fermentation broth volume) of the carbon chain growth microbial sludge enriched in Step 4. Reduce the stirring speed to 150 rpm to facilitate microbial floc formation.

[0159] 11. Within 16 days, the reactor temperature will be kept constant at 37°C, and the pH will be kept constant at 6.5 using 2M NaOH solution.

[0160] Samples were taken every two days during fermentation, placed in 10mL centrifuge tubes, and centrifuged at 8000r for 10 min. The supernatant was then filtered to separate the microorganisms, and the contents of ethanol and hexanoic acid were determined.

[0161] Test results: Under constant fermentation conditions, the maximum accumulation of ethanol in the supernatant reached 8.75 g COD / L on day 6 of fermentation, and the maximum accumulation of hexanoic acid in the supernatant reached 11.53 g COD / L on day 14 of fermentation.

[0162] Comparative Example 8 Using yeast and enriched carbon-chain growth bacteria as inoculum, and kitchen waste as substrate, the pH was controlled in stages using only a single alkali agent (NaOH) under optimal conditions. The process was carried out as follows: 1. Anaerobic sludge, fresh distiller's grains, and forest soil are mixed at a wet weight ratio of 2:1:1 to form a mixed inoculum. Take 500 g and transfer it to a 2 L sterile serum bottle.

[0163] 2. Add approximately 1.5 L of sterile deionized water and stir with a glass rod to form a homogeneous slurry. Add sterile acetic acid solution to the slurry to bring the initial concentration of acetic acid to approximately 5 g / L.

[0164] 3. Place the serum bottle in an ice bath, aerate it with high-purity N2 for 15-20 minutes, and then seal it.

[0165] 4. Place the serum bottle in a 35°C constant temperature shaker for incubation. Inject an appropriate amount of 2M Ca(OH)₂ solution daily using a sterile syringe to adjust the pH back to 5.5-6.5. Incubate for 5-6 days before use.

[0166] 5. Prepare a slurry sample with a concentration of 40 g VS / L from the kitchen waste.

[0167] 6. After grinding the yeast (Daqu and Hongqu) into a uniform powder using a mixer, weigh out the two powders in a 1:1 VS ratio according to their respective VS content, and mix them thoroughly in a sterile beaker. Add the mixed yeast to the kitchen waste slurry as an inoculum at a dosage of 10g VS / L. 餐厨垃圾 VS 酒曲 =4:1.

[0168] 7. The total volume is 1000 mL. Turn on the mechanical stirring (300 rpm) and water bath circulation (35℃).

[0169] 8. Prepare oil-free compressed air, high-purity CO2, and high-purity H2S gas cylinders, and mix them in a volume ratio of 3:1:1. Aerate for 3 minutes at a total aeration rate of 0.2 vvm at the 0th and 6th hours.

[0170] 9. After 12 hours, stop aeration and introduce high-purity N2 to establish an anaerobic environment.

[0171] 10. After inoculating the substrate with the enriched carbon chain growth bacteria from step four, reduce the stirring speed to 150 rpm.

[0172] 11. Within 16 days, the temperature will be controlled in a gradient: 30-32℃ for 12h-48h, 35-37℃ for 2-6 days, and 38℃ for 6-16 days.

[0173] 12. (Key difference from Example 6) In conjunction with temperature control, a phased pH gradient control is implemented, but only 2M NaOH solution is used as the regulator throughout the process: (1) During the 12h-48h period, the pH value was controlled at 5.5 ± 0.1.

[0174] (2) From day 2 to day 6, the pH setting was increased to 6.5 ± 0.1.

[0175] (3) From day 6 to day 16, the pH was further increased to 7.5 ± 0.1.

[0176] Samples were taken every two days during fermentation, placed in 10mL centrifuge tubes, and centrifuged at 8000r for 10 min. The supernatant was then filtered to separate the microorganisms, and the contents of ethanol and hexanoic acid were determined.

[0177] Test results: Under the step-by-step regulation of a single alkali agent during fermentation, the maximum accumulation of ethanol in the supernatant reached 9.19 g COD / L on day 4 of fermentation, and the maximum accumulation of hexanoic acid in the supernatant reached 14.53 g COD / L on day 12 of fermentation.

[0178] The experimental results of the above examples and comparative examples clearly show that the method proposed in this invention for rapid one-tank production of hexanoic acid from kitchen waste using aerobic pretreatment can significantly improve the production efficiency and final concentration of hexanoic acid. The core of this invention lies in the synergistic effect of "aerobic pretreatment" and "specific microbial system". The following situations cannot achieve efficient production of hexanoic acid: (1) No aerobic pretreatment, as shown in Comparative Example 1, the hexanoic acid yield is only 1.61 g COD / L; (2) Incomplete microbial system, as shown in Comparative Example 4 (no fungal inoculation) and Comparative Example 6 (no enriched bacteria inoculation), the hexanoic acid yields are 0 g COD / L and 2.97 g COD / L, respectively, which are far lower than the levels of the examples; (3) Improper pretreatment method, as shown in Comparative Example 3 (continuous aeration), which completely inhibits the hexanoic acid production process. This fully demonstrates that the two-stage process and specific microbial inoculation strategy described in this invention are necessary prerequisites for achieving efficient production.

[0179] Furthermore, the optimization of process details is also crucial in this invention. The composition of the aeration gas has a significant impact on acid production. As shown in Comparative Example 2, when only pure oxygen is used for aeration, the hexanoic acid yield is only 7.22 g COD / L, demonstrating the inventiveness of introducing CO2 and H2S in this invention. The type of fungi used for inoculation is equally critical. In Comparative Example 5, using a single baker's yeast instead of the compound yeast of this invention significantly reduced the hexanoic acid yield to 6.97 g COD / L, highlighting the superiority of the compound yeast strain in constructing an efficient metabolic network. Finally, the gradient control of environmental conditions is the finishing touch to further improve the yield. The hexanoic acid yield in Comparative Example 7 (constant condition fermentation) was only 11.53 g COD / L, while in Comparative Example 8, using a single alkali agent for gradient control, the yield was only 14.53 g COD / L, both significantly lower than the highest yield of 23.64 g COD / L achieved in Example 6 using a compound alkali agent for gradient control. In summary, this invention successfully overcomes the bottlenecks of existing technologies by integrating and optimizing microbial community construction and implementing a two-stage process and real-time air conditioning control strategy, thereby achieving efficient and high-value utilization of kitchen waste.

[0180] Figure 2 This is a graph showing the changes in the concentration of major metabolites under different aeration intensities in Example 4. Figure 2 A represents the results of the comparative example (without aeration). Figure 2 Examples B, 2C, and 2D show the results of 12 hours of aeration (1, 2, and 3 times, respectively). It can be seen that without aeration, ethanol and lactic acid accumulate slowly, and the yield of hexanoic acid is extremely low. In contrast, the example group, through aerobic pretreatment, rapidly accumulated high concentrations of ethanol and lactic acid as electron donors and carbon source precursors in the early fermentation stage (0-4 days), which were then efficiently converted to hexanoic acid in the anaerobic stage, demonstrating the effectiveness of the two-stage fermentation model of this invention.

[0181] In Example 4, the aerobic pretreatment stage (first 12 hours) promoted the growth of ethanol-producing yeasts such as *Pichia* and *Candida* (24.6%-35.8%), an increase of 12.4%-23.6% compared to Comparative Example 1. Furthermore, the aerobic pretreatment also promoted the enrichment of facultative anaerobic lactic acid bacteria such as *Leuconostoc* and *Streptococcus* (43.7%-56.8%), an increase of 21.1%-34.2% compared to Comparative Example 1. During the anaerobic stage (days 6-8), the relative abundance of the hexanoic acid-producing core functional bacterium *Clostridium sensu stricto* 12 significantly increased (9.5%-11.6%), while in Comparative Example 1, this species was almost not enriched (0.8%). This intuitively demonstrates that the method of the present invention can achieve orderly succession and efficient enrichment of functional microorganisms by regulating the environment.

Claims

1. A method for rapid one-tank production of hexanoic acid from kitchen waste using aerobic pretreatment, characterized by the following steps: include: The kitchen waste slurry is first inoculated with Daqu (a type of starter culture), or Daqu and red yeast rice for aerobic pretreatment, and then inoculated with a microbial community composed of anaerobic sludge, distiller's grains and forest soil that has been enriched for anaerobic fermentation to produce hexanoic acid.

2. The method according to claim 1, characterized in that, In the aforementioned Daqu and Hongqu, VS 大曲 VS 红曲 =0.5-2:

1.

3. The method according to claim 1, characterized in that, The concentration of the kitchen slurry is 30-50 g VS / L, VS 餐厨浆液 :VS 大曲 =3-10:1 or VS 餐厨浆液 :VS 大曲和红曲 =3-10:

1.

4. The method according to claim 1, characterized in that, The kitchen slurry is mixed before aerobic pretreatment after inoculation with Daqu or Daqu and red yeast rice.

5. The method according to claim 1, characterized in that, The aerobic pretreatment involves discontinuous aeration.

6. The method according to claim 1, characterized in that, The steps of mixing and enriching the anaerobic sludge, distiller's grains, and forest soil include: (1) Mix the anaerobic sludge, distiller's grains and forest soil into a slurry; (2) Add acetic acid solution to the mud; (3) Culture of enriched microbial communities under anaerobic conditions.

7. The method according to claim 6, characterized in that, The wet weight ratio of the anaerobic sludge, distiller's grains, and forest soil is 1-2:1-3:

1.

8. The method according to claim 1, characterized in that, The pH of the anaerobic fermentation is maintained at 5.5-6.5±0.1 throughout the entire process; or the pH is maintained at 5.5±0.1 for the first 0.095-0.1 of the total anaerobic fermentation time, and at 6.5±0.1 for the remaining time; or the pH is maintained at 5.5±0.1 for the first 0.095-0.1 of the total anaerobic fermentation time, at 6.5±0.1 for the middle 0.25-0.28 of the total anaerobic fermentation time, and at 7.5±0.1 for the remaining time.

9. The method according to claim 8, characterized in that, The pH value is adjusted using an alkaline agent, which is selected from any one or any combination of calcium hydroxide solution, sodium hydroxide solution, or zinc chloride solution.

10. The method according to claim 1, characterized in that, The temperature control for anaerobic fermentation is as follows: the temperature is 30-32℃ during the first period, which accounts for 0.095-0.1% of the total anaerobic fermentation time; the temperature is 35-37℃ during the middle period, which accounts for 0.25-0.28% of the total anaerobic fermentation time; and the temperature is 38℃ during the remaining period.

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