Method for synthesizing medium-chain hydroxy fatty acid from organic waste

Abstract

The invention provides a method for synthesizing medium-chain hydroxy fatty acid. The three-step cascade process comprises the following steps: (1) performing non-sterilization lactic acid fermentation on the organic wastes to obtain lactic acid-containing fermentation liquor; (2) adding a flora containing the genus Caproiciduces and / or the genus Anerococcus, and controlling the pH (Potential of Hydrogen) within the range of 5.5 to 7.5, in particular pH gt; 7.0, converting lactic acid into medium-chain fatty acid under a neutral condition; and (3) converting the medium-chain fatty acid into medium-chain hydroxy fatty acids such as hydroxyhexanoic acid, hydroxyheptanoic acid and hydroxycaprylic acid by utilizing the microorganisms for constructing the omega-oxidation path. The method breaks through the pH limit of the traditional chain extension process, can improve the ratio of octanoic acid to heptanoic acid, and provides diversified substrates for omega-oxidation. The product is a high-added-value fine chemical raw material, and can be used for biodegradable materials, special lubricants and medical intermediates. The technology realizes resourceful treatment of organic wastes, and provides a green path for biomanufacturing of medium-chain hydroxy fatty acids.

Description

Technical Field

[0001] The present invention relates to the technical field of organic waste treatment and resource utilization, and in particular to a method for synthesizing medium-chain hydroxy fatty acids from organic waste. Background Art

[0002] Medium-chain hydroxy fatty acids (MCHAs) are a class of fatty acids with carbon chains typically ranging from 6 to 12 carbon atoms and containing a hydroxyl functional group on the terminal methyl group. Due to their unique chemical structure and properties, they are widely used in biomedical materials, biodegradable polymers, cosmetics, specialty chemicals, and pharmaceutical synthesis. These compounds serve not only as monomers for high-performance materials but also as key intermediates for a variety of high-value-added products. With the deepening implementation of sustainable development strategies and the rapid expansion of the bio-based materials market, the development of green and efficient technologies for the preparation of MCHAs has important scientific and economic significance.

[0003] Take 6-hydroxyhexanoic acid as an example. This representative medium-chain hydroxy fatty acid is an important precursor for biodegradable materials such as polycaprolactone. The market price of industrial-grade hexanoic acid is approximately 10,000-15,000 yuan / ton, while the market price of industrial-grade 6-hydroxyhexanoic acid is as high as 15,000-25,000 yuan / ton, significantly increasing its value. Currently, the industrial methods for preparing medium-chain hydroxy fatty acids mainly include the following categories:

[0004] 1. Oxidation methods based on cyclic compounds: For example, the Baeyer-Villiger oxidation of cyclohexanone to produce 6-hydroxyhexanoic acid. While this method offers high selectivity and yield, it requires expensive catalysts such as Au-Pd bimetallic catalysts or TS-1 molecular sieves. Furthermore, the raw materials are completely dependent on petrochemical resources, making it unsuitable for green manufacturing.

[0005] 2. Chemical oxidation: For example, using the intermediate product of cyclohexane's uncatalyzed oxidation as the raw material, hydroxylated medium-chain fatty acids are obtained through multiple steps of separation and purification. This process is complex, has a high environmental impact, and the raw materials still rely on petrochemical resources.

[0006] 3. Biocatalysis: This involves using recombinant microorganisms to construct multi-step enzymatic cascade reactions. However, existing technologies primarily target petrochemical raw materials such as cyclohexane or cyclohexanol, requiring the addition of expensive cofactors such as NADH and ATP. This is costly and has limited environmental friendliness.

[0007] 4. Chemical conversion method: It is obtained by chemical conversion using diols or polyhydroxy acids as raw materials. Although the yield can reach 80-92%, the raw material cost is high, and the production process still requires the use of harmful chemicals such as precious metal catalysts or strong bases.

[0008] In recent years, the field of microbial metabolic engineering has focused on two strategies for the synthesis of hydroxylated fatty acids: direct oxidation of lipid substrates using the Pichia pastoris P450 enzyme system; and terminal hydroxylation of fatty acids using bacterial alkane hydroxylase systems. However, these approaches face fundamental challenges: they rely strictly on refined oils or pure fatty acids as substrates, which is costly. Furthermore, terminal oxidases rely on expensive cofactors such as NADH / NADPH, resulting in complex regeneration systems that are difficult to scale up, severely hindering industrialization and economic feasibility.

[0009] Unlike traditional strategies, our research innovatively proposes using medium-chain fatty acids as direct precursors to synthesize medium-chain hydroxy fatty acids, using lactic acid as the raw material for the synthesis of medium-chain fatty acids. This approach has the significant advantage of being able to obtain lactic acid from various waste products, significantly reducing raw material costs. However, the synthesis process from lactic acid to medium-chain fatty acids still faces significant technical bottlenecks that must be resolved to achieve the economic feasibility of the entire synthetic route.

[0010] The industry has long faced a technical bottleneck that has been widely recognized as insurmountable: the pH dilemma in traditional carbon chain extension processes. Specifically, lactic acid chain extension is typically performed at pH 6.5 or below. However, these acidic conditions cause the synthesized medium-chain fatty acids, such as caproic acid, to exist as free acids, resulting in strong product inhibition and severely limiting the final product concentration. While ethanol chain extension can initially proceed at a neutral pH (approximately 7.0), the pH naturally drops below 6.0 as caproic acid is synthesized and ethanol is utilized, similarly leading to free acid inhibition and hindering the continued accumulation of caproic acid. The paper "Long-Term Continuous Extraction of Medium-Chain Carboxylates by Pertraction With Submerged Hollow-Fiber Membranes and Advancements in Medium Chain Fatty Acids Production Through Chain Elongation: Key Mechanisms and Innovative Solutions for Overcoming Rate-Limiting Steps" reports that medium-chain fatty acid production generally remains limited to a bottleneck of 10-15 g / L unless costly online product extraction systems or immobilization techniques are employed. This fundamental contradiction seriously restricts the application scope and industrialization prospects of medium-chain fatty acid biosynthesis technology.

[0011] Through large-scale microbial resource screening and functional identification, the present invention has discovered and identified for the first time a class of Anaerococcus microorganisms that can efficiently catalyze the extension of lactic acid carbon chains under pH conditions higher than traditional processes (pH 6.5-7.5), and creatively constructed a composite bacterial community system with a wide pH adaptation range (pH 5.5-7.5). This breakthrough discovery not only effectively alleviates the above-mentioned free acid inhibition problem, but also brings two unexpected technical effects: First, when the pH is stably controlled within the range of 6.8-7.2, the yield of medium-chain fatty acids can exceed 20g / L, which is 80-100% higher than the traditional process; second, under pH>7.0 conditions, the product distribution changes significantly, and the yield of octanoic acid and heptanoic acid can be increased by 3-4 times, providing a more diverse substrate spectrum for subsequent hydroxylation reactions. This significant change in yield improvement and product distribution is unpredictable in the prior art and reflects the substantial technological breakthrough of the present invention. Based on this, the present invention successfully constructed a three-step cascade conversion system of "lactic acid fermentation + wide pH range carbon chain extension + ω-oxidation metabolism", realizing the full-process green conversion from organic waste to high-value-added hydroxylated medium-chain fatty acids.

[0012] Currently, conventional treatment methods for these organic wastes (such as anaerobic digestion to produce biogas, aerobic composting, etc.) have low product added value and cannot fully release the value of carbon resources in these wastes.

[0013] Therefore, developing a method that can break through the limitations of existing technologies and achieve the direct and efficient conversion of organic waste into medium-chain hydroxy fatty acids through an innovative multi-step cascade biotransformation pathway not only requires solving a series of key technical bottlenecks, but also requires integrating multiple biocatalytic units into a collaborative system, which has major technical challenges and breakthrough significance. Summary of the Invention

[0014] The present invention addresses the technical problems in the existing technology such as low added value of resource utilization of organic waste and dependence of the preparation of hydroxy medium-chain fatty acids on petrochemical raw materials. It provides a method for synthesizing medium-chain hydroxy fatty acids from organic waste. Through a three-step cascade biotransformation pathway, the green conversion of organic waste into high-value-added hydroxy medium-chain fatty acids is achieved.

[0015] To achieve the above objectives, the present invention is implemented through the following technical solutions:

[0016] The present invention discloses a method for synthesizing medium-chain hydroxy fatty acids from organic waste, comprising the following steps:

[0017] (1) subjecting organic waste or simple carbohydrates to lactic acid fermentation under non-sterile conditions to obtain a fermentation liquid containing lactic acid;

[0018] (2) adding a composite bacterial consortium capable of synthesizing medium-chain fatty acids in a pH range of 5.5 to 7.5 to the fermentation broth, wherein the composite bacterial consortium comprises microorganisms that utilize a fatty acid synthesis pathway and a β-oxidation reverse cycle metabolic pathway; converting the lactic acid into medium-chain fatty acids under pH conditions of 5.5 to 7.5; wherein high pH fermentation conditions (especially pH>7.0) are beneficial for increasing the proportion of longer carbon-chain fatty acids such as octanoic acid and heptanoic acid.

[0019] (3) Using microorganisms that construct an ω-oxidation pathway, the medium-chain fatty acids are selectively converted into medium-chain hydroxy fatty acids.

[0020] The non-sterile conditions in the present invention refer to process conditions in which the raw materials and equipment are not strictly sterilized during the microbial fermentation process, and continuous fermentation is carried out in an open environment using naturally existing microbial communities or added functional bacteria. This method is more suitable for industrial large-scale production and reduces energy consumption and equipment requirements.

[0021] Preferably, in step (1), the pH value is controlled to be 5.8-7.0 during the fermentation process, and anaerobic fermentation is carried out at a temperature of 30-37° C. and a stirring speed of 80 r / min for 3-7 days;

[0022] When the raw material is a simple carbohydrate (such as glucose, sucrose, fructose, starch and other non-organic waste), lactic acid bacteria are added thereto for fermentation to produce lactic acid, and the amount of lactic acid bacteria added is 0.5-1.0%. Further preferably, when the raw material is a starchy material, gelatinization is first performed at 90-100°C, and α-amylase is added after cooling to 60-65°C for saccharification. After saccharification is completed, the material is cooled to 35°C, and lactic acid bacteria are added for lactic acid fermentation.

[0023] When the raw material is organic waste (such as kitchen waste, food processing waste, etc.), the pH is adjusted to 5.8-6.2, and lactic acid fermentation is carried out spontaneously by the lactic acid bacteria carried by the organic waste, without the need to add additional lactic acid bacteria. Of course, lactic acid bacteria can also be added to ferment and produce lactic acid.

[0024] Preferably, the lactic acid bacteria are added in the form of a bacterial solution or freeze-dried powder containing lactic acid bacteria. The freeze-dried powder is cultured with MRS medium for lactic acid bacteria at 30°C for 2 to 3 days, centrifuged, and the bacteria are taken to make 10 8 ~10 12 CFU / g of lactic acid bacteria freeze-dried powder.

[0025] MRS medium includes: 10 g casein peptone; 10 g beef extract; 5 g yeast extract; 5 g glucose; 5 g sodium acetate; 2 g diammonium citrate; 1 g Tween 80; 2 g dipotassium hydrogen phosphate; 0.2 g magnesium sulfate heptahydrate; 0.05 g manganese sulfate heptahydrate; 20 g calcium carbonate; 20 g agar; 1 L distilled water, pH 6.8.

[0026] Preferably, in step (2), the composite bacterial consortium comprises at least one of microorganisms of the genera Caproiciproducens and Anaerococcus; the microorganism constructing the ω-oxidation pathway is Escherichia coli, and the Escherichia coli is pre-cultured in M9 medium or LB medium containing 0.5% glucose, and then induced to express by any of the following methods:

[0027] (a) Add 0.1-1.0 mM IPTG to induce expression;

[0028] (b) Temperature induction from 30°C to 37-42°C;

[0029] (c) Add 0.01-0.2% L-arabinose or 0.1-1 μg / mL doxycycline to induce expression.

[0030] Preferably, in step (2), the fermentation is carried out at a temperature of 30 to 40° C., and the hydraulic retention time during the fermentation is 2 to 15 days.

[0031] Further preferably, when the carbon chain extension composite bacterial consortium is constructed with microorganisms of the genus Caproiciproducens as the core, the fermentation is carried out at a pH of 5.6 to 6.5 and a temperature of 30 to 37°C;

[0032] When the carbon chain extension complex is constructed with Anaerococcus microorganisms as the core, the fermentation is carried out at a pH of 6.0 to 7.5 and a temperature of 30 to 37°C;

[0033] When a carbon chain elongation mixed microbial flora in which microorganisms of the genera Caproiciproducens and Anaerococcus coexist is used, fermentation is carried out at a pH of 5.5 to 7.5 and a temperature of 30 to 37°C.

[0034] Preferably, in step (3), the reaction is carried out at a pH of 5.5 to 7.5, a temperature of 30 to 40° C., a rotation speed of 30 to 200 rpm, and a reaction time of 1 to 4 days.

[0035] Preferably, the medium-chain fatty acid is an organic carboxylic acid containing 6 to 12 carbon atoms, and the medium-chain hydroxy fatty acid is a fatty acid containing 6 to 12 carbon atoms and containing a hydroxyl group at the end.

[0036] Preferably, the ω-oxidation pathway is constructed by heterologously expressing the alkane hydroxylase system of Pseudomonas putida GPo1 to achieve selective hydroxylation of the terminal methyl group of medium-chain fatty acids.

[0037] Preferably, the alkane hydroxylation system includes alkane monooxygenase (alkB), rubredoxin (alkG) and rubredoxin reductase (alkT); a ribosome binding site is added to the gene of the hydrocarbon hydroxylation system, and the sequence is AAAGAGGAGAAA, that is, a specific ribosome binding site (AAAGAGGAGAAA) is added upstream of each gene to optimize protein expression level.

[0038] Preferably, the gene of the alkane hydroxylation system is connected to the plasmid vector by homologous recombination to form a corresponding recombinant plasmid, and the recombinant plasmid is transferred into a microorganism with a constructed ω-oxidation pathway;

[0039] The plasmid vector is selected from at least one of the following:

[0040] (a) pET-28a(+) vector for IPTG-induced expression;

[0041] (b) a λpR-pL vector containing the temperature-sensitive repressor protein cI857 for temperature-induced expression;

[0042] (c) pBAD series vectors for arabinose-induced expression;

[0043] (d) pTet series vectors for doxycycline-inducible expression.

[0044] For example, using E. coli BL21, select appropriate vectors based on different induction requirements to construct recombinant plasmids, and integrate three enzyme genes into the same expression vector through homologous recombination. For example, using the pET-28a(+) plasmid vector to construct the recombinant plasmid pET-28a(+)-alkB-alkG-alkT, and integrating three enzyme genes into the same expression vector through homologous recombination.

[0045] More preferably, the E. coli is cultured to an OD 600 When the pH is about 0.6, induction is carried out by any of the following methods; then, medium-chain fatty acid fermentation broth is used as a substrate, and catalytic conversion is carried out for 1 to 4 days at a pH of 5.5 to 6.5, a temperature of 30 to 40° C., and a rotation speed of 150 to 200 rpm.

[0046] The induction method is:

[0047] (a) IPTG induction: 0.5 mM IPTG was added to induce alkBGT gene expression and cultured at 30°C for 12 h;

[0048] (b) Temperature induction: When the λpR-pL temperature-sensitive promoter system is used, the culture temperature is increased from 30°C to 37°C at a rate of 4°C per hour, maintained for 1 hour, and then increased to 42°C at a rate of 2.5°C per hour and cultured for another 8 hours.

[0049] (c) Chemical induction: When using the pBAD vector, 0.05% L-arabinose was added for induction; when using the pTet vector, 0.2 μg / mL doxycycline was added for induction, and cultured at 30°C for 12 hours.

[0050] The present invention has the following beneficial effects:

[0051] 1. Innovation in lactic acid fermentation strategy: Dedicated fermentation processes are designed for different types of carbohydrate substrates. In particular, for organic waste, a technical solution is proposed for the first time to use the lactic acid bacteria carried by the waste to carry out spontaneous lactic acid fermentation, which simplifies the process and reduces production costs.

[0052] 2. Expansion of the pH adaptation range for carbon chain extension: By exploring functionally complementary microbial populations centered around the genus Anaerococcus, the pH adaptation range for medium-chain fatty acid synthesis was significantly expanded from the traditional 5.5-6.5 to 5.5-7.5. In particular, efficient carbon chain extension was achieved under conditions of pH>7.0. This technological breakthrough significantly improved the robustness of the system and promoted the synthesis of longer carbon-chain fatty acids such as octanoic acid and heptanoic acid.

[0053] 3. Optimization of the ω-oxidation metabolic pathway: Through a carefully designed expression strategy for the Pseudomonas putida alkane hydroxylase system, highly selective hydroxylation of the terminal methyl groups of medium-chain fatty acids was achieved, with significantly improved catalytic efficiency and a substantial increase in the conversion rate of the target product.

[0054] 4. Synergistic Effects of the Three-Step Cascade Process: The three-step cascade process of "lactic acid fermentation + wide pH range carbon chain extension + ω-oxidation metabolism" in this invention is not a simple technical superposition, but rather achieves significant synergistic effects through careful design. First, the lactic acid fermentation stage is carried out under near-neutral conditions, creating an ideal starting environment for the subsequent wide pH range carbon chain extension, avoiding the inhibition of bacterial diversity by acidic conditions in traditional processes. Second, the high proportion of octanoic acid and heptanoic acid produced during wide pH range carbon chain extension, especially under conditions of pH > 7.0, provides a more diverse substrate spectrum for the ω-oxidation stage, significantly enriching the variety of final hydroxylated medium-chain fatty acids. Third, by appropriately adjusting the pH during the carbon chain extension stage, the proportion of fatty acids of different carbon chain lengths can be precisely controlled, thereby achieving targeted regulation of the final product variety. This full-process controllability is not achievable with simple combination technologies. It is particularly worth emphasizing that the efficient carbon chain extension reaction under neutral pH conditions discovered in this invention solves the bottleneck problem of "free acid inhibition" that has long plagued this field. This breakthrough not only increases the concentration of intermediate products, but also provides more favorable environmental conditions for the subsequent ω-oxidation reaction, significantly improving the overall process efficiency.

[0055] 5. Wide applicability of substrates: The technology of the present invention is not only applicable to simple sugar substrates, but can also efficiently utilize starchy materials and other types of organic waste (kitchen waste, food processing waste, livestock and poultry manure, straw, etc.), greatly expanding the source of raw materials and reducing production costs.

[0056] 6. Full-process low-energy green process: The entire technical route adopts biological transformation at room temperature and normal pressure, which has low energy consumption, is environmentally friendly, and complies with the concepts of green chemistry and circular economy. At the same time, the product has high added value and significant economic benefits.

[0057] 7. Substantial breakthrough of the present invention compared to the prior art: The present invention achieves a fundamental breakthrough in the pH range for the conversion of lactic acid into medium-chain fatty acids:

[0058] (a) Breaking down industry technical barriers: The industry has long held that the optimal pH range for converting lactic acid to medium-chain fatty acids, such as caproic acid, is strictly limited to 5.5-6.5. This has become an industry consensus and has been solidified by numerous publications and patents. This invention, for the first time, discovers and demonstrates that lactic acid can also be efficiently converted to medium-chain fatty acids under neutral to weakly alkaline conditions of pH > 7.0, completely overturning existing technical understanding.

[0059] (b) Overcoming fundamental microbiological barriers: Based on fundamental microbiological principles, a neutral environment (pH > 7.0) should theoretically be dominated by acetic acid bacteria, butyric acid bacteria, methanogenic bacteria, and propionic acid bacteria, making it difficult for hexanoic acid bacteria to dominate the community. The present invention successfully overcomes this fundamental microbiological barrier by discovering Anaerococcus microorganisms and their synergistic effects with traditional bacterial strains, which was not anticipated by those skilled in the art or discovered through conventional experimental design.

[0060] (c) Groundbreaking Process Window: This invention creatively expands the pH process window (5.5-7.5) for the conversion of lactic acid to medium-chain fatty acids, a global first. A search reveals no published technical literature reporting successful conversion of lactic acid to caproic acid at pH > 7.0, demonstrating the groundbreaking nature of this discovery rather than its obviousness.

[0061] (d) New mechanism for regulating product lineage: The neutral pH fermentation conditions of the present invention significantly alter the carbon chain length distribution of medium-chain fatty acids, increasing the proportion of longer carbon chain fatty acids such as octanoic acid and heptanoic acid. This product regulation capability, previously unattainable under weakly acidic conditions, provides strong support for the diversified synthesis of downstream hydroxy medium-chain fatty acids.

[0062] (e) Integration of innovative technology pathways: The present invention organically integrates spontaneous lactic acid fermentation of organic waste, carbon chain extension in a wide pH range, and ω-oxidation metabolic pathways to construct a new technology system from organic waste to high-value-added hydroxylated medium-chain fatty acids. Each link has substantial innovation, and the overall technology pathway is essentially different from previous patents.

[0063] In summary, the present invention provides a novel biosynthetic technology route for medium-chain hydroxy fatty acids. Through a three-step cascade transformation of "lactic acid fermentation + wide pH carbon chain elongation + ω-oxidation metabolism", it achieves the efficient and green conversion of organic waste into high-value-added medium-chain hydroxy fatty acids, providing a new approach for the resource utilization of organic waste and the manufacture of bio-based chemicals. DETAILED DESCRIPTION

[0064] The following is a clear and complete description of the technical solutions in the embodiments of the present invention. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of them. All other embodiments obtained by ordinary technicians in this field based on the embodiments of the present invention without making any creative efforts are within the scope of protection of the present invention.

[0065] Unless otherwise specified, the technical means used in the embodiments are conventional means well known to those skilled in the art.

[0066] The technological innovation of the present invention in the construction of composite microbial communities is the core breakthrough in solving the problem of efficient conversion of organic waste into medium-chain hydroxy fatty acids. The construction of a composite microbial community of coexisting Caproiciproducens and Anaerococcus faces multiple technical challenges. First, microorganisms of the genus Anaerococcus have hardly been studied in the field of caproic acid production, and there has never been any report in the literature related to caproic acid synthesis in the past 10 years that this species has the ability to produce caproic acid. Secondly, there are significant differences in the environmental adaptability of these two types of microorganisms: microorganisms of the genus Caproiciproducens prefer a weakly acidic environment of pH 5.5-6.5, while microorganisms of the genus Anaerococcus can only remain active under neutral to weakly alkaline conditions of pH>6.5. In actual operation, since the system is usually maintained in a weakly acidic environment, Anaerococcus has almost no chance of growth, resulting in Caproiciproducens always being in an absolutely dominant position. This makes it an extremely challenging task to achieve true coexistence and synergy between the two types of microorganisms in the same system. In order to solve the above technical problems, the present invention adopts the following key strategies:

[0067] 1. Gradient acclimation: By setting a pH gradient of 5.8-7.5, long-term acclimation is used to gradually improve the tolerance of Caproiciproducens at high pH, ​​while enhancing the activity of Anaerococcus at lower pH;

[0068] 2. Co-culture optimization: Initial co-culture was performed at pH 6.5. This intermediate pH value allows both bacterial communities to maintain a certain level of activity, and after multiple rounds of acclimation, a stable symbiotic relationship is formed.

[0069] 3. Niche separation: By optimizing fermentation conditions to create spatial heterogeneity in the microbial community, the two types of bacteria can be allowed to perform their optimal activities in different areas of the system, thereby achieving functional complementarity rather than competition.

[0070] Therefore, the present invention successfully constructed a composite bacterial community system that can operate efficiently across a wide pH range of 5.5-7.5, completely breaking through the pH limitations of traditional medium-chain fatty acid fermentation and providing a new technical path for efficient medium-chain fatty acid biosynthesis. It is worth emphasizing that the stable operation of this composite bacterial community system requires not only precise control of process parameters but also a deep understanding of the metabolic interactions between microorganisms, reflecting the innovative breakthrough of the present invention in the field of microbial community engineering.

[0071] In order to make the objectives, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below with reference to specific embodiments.

[0072] Example 1: Lactic acid production by spontaneous lactic acid fermentation using kitchen waste

[0073] In this embodiment, food waste is used as raw material, and the lactic acid bacteria carried by the food waste are used to carry out spontaneous lactic acid fermentation. The specific steps are as follows:

[0074] (1) Raw material pretreatment: Fresh food waste (including rice, pasta, vegetables, etc.) was collected, and the bulk materials were crushed to a diameter of less than 5 mm. The total solid (TS) content was measured to be 17.8%, and the volatile solid (VS) content was 15.4%.

[0075] (2) Preparation of fermentation equipment: A 5 L glass fermentation tank was used, with a stirring paddle at the bottom and a pH electrode, a temperature sensor, and an inlet and outlet at the top.

[0076] (3) Fermentation process: 4 kg of pretreated food waste was added to the fermentation tank, the initial pH was adjusted to 7.0 (using 1 mol / L potassium hydroxide solution), the temperature was controlled at 35 °C, the stirring speed was set to 80 r / min, and fermentation was carried out under anaerobic conditions.

[0077] (4) Fermentation process monitoring: Samples were taken every 24 hours to measure pH, organic acid composition, and concentration. It was found that the pH dropped slightly (to approximately 6.7) during the initial fermentation period (0-24 hours), and then an automatic control system was used to maintain the pH within the range of 6.8-7.0.

[0078] (5) Fermentation results: After 5 days of fermentation, the concentration of lactic acid reached 23.6 g / L, accounting for 78.3% of the total organic acids. At the same time, a small amount of acetic acid (3.8 g / L) and butyric acid (2.1 g / L) were detected.

[0079] (6) Fermentation broth pretreatment: The fermentation broth was filtered through a 100-mesh sieve to remove solid residues and obtain a fermentation broth containing a high concentration of lactic acid for the next carbon chain extension reaction.

[0080] Example 2: Conversion of lactic acid into medium-chain fatty acids using Caproiciproducens microbial flora under acidic conditions

[0081] In this example, microorganisms of the genus Caproiciproducens were used as the core to construct a carbon chain extension bacterial community, and lactic acid was converted into medium-chain fatty acids mainly composed of caproic acid under weakly acidic conditions. The specific steps are as follows:

[0082] (1) Strain acquisition and activation: A composite bacterial community with Caproiciproducens as the dominant species was isolated from a wine cellar and activated by culturing in PYG medium (peptone 10 g / L, yeast extract 5 g / L, glucose 10 g / L, L-cysteine ​​hydrochloride 0.5 g / L) at 35°C under anaerobic conditions for 48 h.

[0083] (2) Inoculation and fermentation: 500 mL of the lactic acid fermentation broth obtained in Example 1 was taken, 50 mL of the activated bacterial solution (inoculation ratio 10%) was added, and the carbon chain extension reaction was carried out in a 2 L anaerobic fermentation tank.

[0084] (3) Fermentation condition control: adjust the initial pH to 6.0, use 2 mol / L potassium hydroxide solution to maintain the pH in the range of 5.8-6.2, control the temperature at 35 °C, and stir gently (60 r / min) to maintain uniformity without destroying the bacterial structure.

[0085] (4) Feeding strategy: The lactic acid concentration in the fermentation broth was measured every 24 hours. When the lactic acid concentration was lower than 5 g / L, an appropriate amount of lactic acid fermentation broth was added to restore it to 8-15 g / L.

[0086] (5) Fermentation process monitoring: Regular sampling to detect pH, lactic acid consumption and production of medium-chain fatty acids (C4-C8).

[0087] (6) Fermentation results: After 7 days of fermentation, the lactic acid conversion rate reached 92.3%, the hexanoic acid production was 11.8 g / L, the butyric acid production was 4.6 g / L, the octanoic acid production was 0.8 g / L, and the heptanoic acid production was 0.3 g / L.

[0088] Example 3: Conversion of lactic acid into medium-chain fatty acids using Anaerococcus microbial flora under neutral conditions

[0089] This example uses the Anaerococcus microorganism discovered for the first time by the present invention as the core to construct a carbon chain extension bacterial community, and converts lactic acid into medium-chain fatty acids containing a high proportion of octanoic acid under neutral conditions. The specific steps are as follows:

[0090] (1) Strain acquisition and screening: Functional bacteria that can convert lactic acid into medium-chain fatty acids under pH>7.0 were screened from wine cellars, and Anaerococcus microorganisms were confirmed as the dominant bacteria by 16S rDNA sequencing.

[0091] (2) Activation of bacterial strains: The screened bacterial colony was cultured in a modified PYG medium (10 g / L peptone, 5 g / L yeast extract, 5 g / L glucose, 10 g / L sodium lactate, 0.5 g / L L-cysteine ​​hydrochloride, pH adjusted to 7.2) at 37°C under anaerobic conditions for 72 h.

[0092] (3) Inoculation and fermentation: 500 mL of the lactic acid fermentation broth obtained in Example 1 was taken, 50 mL of the activated bacterial solution (inoculation ratio 10%) was added, and the carbon chain extension reaction was carried out in a 2 L anaerobic fermentation tank.

[0093] (4) Fermentation condition control: adjust the initial pH to 7.2, use 2 mol / L potassium hydroxide solution to maintain the pH in the range of 7.0-7.3, control the temperature at 37°C, and stir at 80 r / min.

[0094] (5) Feeding strategy: A semi-continuous feeding method was used, replacing 30% of the fermentation broth every 48 h and supplementing with an equal amount of fresh lactic acid fermentation broth.

[0095] (6) Fermentation process tracking: Regular sampling was performed to detect lactic acid consumption and the production of medium-chain fatty acids (C4-C8), and the abundance changes of Anaerococcus strains were monitored by high-throughput sequencing.

[0096] (7) Fermentation Results: After 8 days of fermentation, the lactic acid conversion rate reached 85.7%, and the total yield of medium-chain fatty acids was 14.2 g / L. The product distribution was as follows: hexanoic acid 9.6 g / L, octanoic acid 1.4 g / L, heptanoic acid 1.8 g / L (12.7%), and butyric acid 1.4 g / L. Compared with Example 2, the proportions of octanoic acid and heptanoic acid were significantly increased.

[0097] (8) Microbial community analysis: At the end of fermentation, the relative abundance of Anaerococcus microorganisms in the bacterial community reached 43.7%, proving that they successfully occupied a dominant position under neutral conditions.

[0098] Example 4: Conversion of lactic acid into medium-chain fatty acids using a complex of Caproiciproducens and Anaerococcus bacteria over a wide pH range

[0099] This example uses the innovative composite bacterial system of the present invention to achieve efficient conversion of lactic acid to medium-chain fatty acids over a wide pH range. The specific implementation steps are as follows:

[0100] (1) Construction of composite bacterial consortium: The Caproiciproducens consortium in Example 2 and the Anaerococcus consortium in Example 3 were mixed in a volume ratio of 1:1 and co-cultured in a modified PYG medium at pH 6.5 for 48 hours to establish a stable functional complementary relationship between the two bacteria.

[0101] (2) Inoculation and fermentation: 1000 mL of the lactic acid fermentation broth obtained in Example 1 was taken, and 200 mL of the activated composite bacterial solution (inoculation ratio 20%) was added to carry out carbon chain extension reaction in a 3 L anaerobic fermentation tank.

[0102] (3) pH gradient experiment: Three parallel experiments were set up, and the pH was controlled at 5.8±0.2, 6.5±0.2, and 7.2±0.2, respectively. Other conditions remained the same, the temperature was 35°C, and the stirring speed was 70 r / min.

[0103] (4) Feeding strategy: The feeding method of replacing 25% of the fermentation liquid every 48 hours was adopted and the operation was continued for 15 days.

[0104] (5) Fermentation results: The product compositions under the three pH conditions are shown in Table 1.

[0105] Table 1 Production and composition of medium-chain fatty acids in each group

[0106] pH conditions Total yield (g / L) Hexanoic acid (g / L) Caprylic acid (g / L) Heptanoic acid (g / L) Lactic acid conversion rate (%) 5.8±0.2 14.4 12.5 1.5 1.4 95.3 6.5±0.2 20.8 15.0 4.8 3.2 84.3 7.2±0.2 23.6 16.0 3.2 2.3 94.8

[0107] (6) Dynamic monitoring of microbial communities: Metagenomic sequencing technology was used to monitor the abundance changes of the two core bacterial communities under different pH conditions. The results showed that at pH 5.8, Caproiciproducens dominated (relative abundance 58.3%); at pH 7.2, Anaerococcus dominated (relative abundance 47.5%); and at pH 6.5, the two bacterial communities formed a relatively balanced coexistence relationship.

[0108] (7) Long-term stability test: The system was operated continuously for 30 days at pH 6.8, and the medium-chain fatty acid production remained stable at 22.5±2.0 g / L, demonstrating that the composite bacterial community system has good long-term stability. At the same time, in a short-term test under high-load conditions (lactic acid concentration increased to 58 g / L), the medium-chain fatty acid production reached a maximum of 25.3 g / L, further confirming the processing capacity and potential of the system.

[0109] Example 5A: Construction of ω-oxidation pathway and production of hydroxylated medium-chain fatty acids

[0110] This example describes in detail the construction of the ω-oxidation metabolic pathway and the process of converting medium-chain fatty acids into hydroxylated medium-chain fatty acids. The specific steps are as follows:

[0111] (1) Gene acquisition and optimization: The alkB (alkane monooxygenase), alkG (rubredoxin), and alkT (rubredoxin reductase) genes were extracted from Pseudomonas putida GPo1 and synthesized after codon optimization. The ribosome binding site sequence AAAGAGGAGAAA was added upstream of each gene to optimize protein expression levels.

[0112] (2) Vector construction: Select one of the following vectors based on the induction requirements:

[0113] pET-28a(+) was used as an IPTG-inducible expression vector; pλ-pR-pL was used as a temperature-sensitive inducible expression vector; pBAD24 was used as an arabinose-inducible expression vector; and pTet-On was used as a doxycycline-inducible expression vector. The alkB, alkG, and alkT genes were integrated into the selected expression vector by homologous recombination to construct the corresponding recombinant plasmids.

[0114] (3) Strain transformation: The constructed recombinant plasmid was transformed into E. coli BL21 (DE3) by heat shock method, and positive clones were screened on LB plates (containing 50 μg / mL kanamycin).

[0115] (4) Strain culture and protein expression induction: The positive clones were inoculated into 250 mL of LB liquid medium containing 50 μg / mL kanamycin and cultured at 37°C and 200 rpm until the OD 600 About 0.6, using one of the following induction methods depending on the selected vector system:

[0116] Method A (IPTG induction): Add 0.5 mM IPTG to induce alkBGT gene expression and continue culturing at 30°C for 12 hours;

[0117] Method B (temperature induction): When using the pλ-pR-pL temperature-sensitive promoter system, increase the culture temperature in stages: first lower it to 30°C for 1 hour, then increase it to 37°C at a rate of 4°C per hour. After maintaining it for 1 hour, increase it to 42°C at a rate of 2.5°C per hour and continue incubating at this temperature for 8 hours. Before temperature induction, add 0.2% glycerol to the culture medium as a molecular chaperone stabilizer to reduce interference from heat shock proteins.

[0118] Method C (chemical induction): When using the pBAD system, L-arabinose was added at a final concentration of 0.05% to induce alkBGT gene expression; when using the pTet system, doxycycline was added at a final concentration of 0.2 μg / mL to induce alkBGT gene expression, and the cells were cultured at 30°C for 12 hours.

[0119] (5) Preparation of medium-chain fatty acid substrate: 50 mL of the medium-chain fatty acid mixture obtained in Example 4 was taken, the pH was adjusted to 5.0, and extracted three times with ethyl acetate (each time with equal volume). The organic phases were combined and the solvent was removed by rotary evaporation to obtain a medium-chain fatty acid mixture.

[0120] (6) Biotransformation reaction of hydroxylated medium-chain fatty acids: The induced engineered bacteria were collected and resuspended in 100 mL of M9 minimal medium (containing 0.4% glucose, 1 mM MgSO4, and 0.1 mM CaCl2), and 2 g / L of a medium-chain fatty acid mixture was added as a substrate. The biocatalytic reaction was carried out at pH 6.0, 35°C, and 180 rpm.

[0121] (7) Reaction process monitoring: Sampling was performed every 24 hours to measure the medium-chain fatty acid consumption and hydroxy medium-chain fatty acid production to monitor the reaction progress.

[0122] (8) Extraction and Analysis of Hydroxy Medium-Chain Fatty Acids: After 3 days of reaction, the reaction mixture was centrifuged to remove the bacterial cells. The supernatant was adjusted to pH 3.0 and extracted three times with ethyl acetate. The organic phases were combined and concentrated by rotary evaporation to obtain the crude product. The product was qualitatively and quantitatively analyzed by high-performance liquid chromatography (HPLC) and gas chromatography-mass spectrometry (GC-MS).

[0123] (9) Conversion Results: After 3 days of reaction, the medium-chain fatty acid conversion rate reached 72.3%, and the total yield of hydroxylated medium-chain fatty acids was 1.42 g / L, including 0.75 g / L of ω-hydroxyhexanoic acid, 0.48 g / L of ω-hydroxyoctanoic acid, and 0.19 g / L of ω-hydroxyheptanoic acid. The product selectivity (ω-hydroxylation vs. hydroxylation at other positions) was greater than 95%.

[0124] Example 5B: Comparison of performance of different induction systems

[0125] This example compares the performance of three systems: IPTG induction, temperature induction, and chemical induction in the production of hydroxylated medium-chain fatty acids. The specific steps are as follows:

[0126] (1) Construction of four expression systems: pET-28a(+)-alkB-alkG-alkT (IPTG-inducible system), pλ-alkB-alkG-alkT (temperature-inducible system), pBAD-alkB-alkG-alkT (arabinose-inducible system), and pTet-alkB-alkG-alkT (doxycycline-inducible system) recombinant plasmids were constructed and transformed into E. coli BL21 strain.

[0127] (2) Optimization of expression conditions: Optimize the conditions for each induction system:

[0128] IPTG induction: three IPTG concentrations of 0.1, 0.5, and 1.0 mM were tested;

[0129] Temperature induction: Comparison of three strategies: direct heating, two-stage heating, and slow linear heating;

[0130] Arabinose induction: three arabinose concentrations were tested: 0.01%, 0.05%, and 0.1%;

[0131] Doxycycline induction: three doxycycline concentrations of 0.1, 0.2, and 0.5 μg / mL were tested;

[0132] (3) Analysis of protein expression levels: The expression levels and soluble ratios of alkB, alkG, and alkT proteins under different induction systems were analyzed by SDS-PAGE and Western blot.

[0133] (4) Enzyme activity determination: The specific activity of the enzyme system under different induction systems was determined by the oxidation reaction of the standard substrate octane.

[0134] (5) Comparison of medium-chain hydroxy fatty acid production performance: Four induction systems were used to carry out the biotransformation reaction of medium-chain fatty acids, and the conversion rate, product distribution and selectivity were compared.

[0135] (6) Result analysis: The performance comparison results of the four induction systems are shown in Table 2 below.

[0136] Table 2 Conversion of medium-chain fatty acids by different induction systems

[0137]

[0138] (7) Conclusion: The four induction systems each have their own advantages and disadvantages: IPTG induction has the highest protein expression and conversion efficiency, but also the highest cost; temperature induction has the lowest cost but is difficult to control, resulting in lower protein expression and solubility; arabinose and doxycycline induction systems provide a good balance between expression and cost. Based on actual production needs and conditions, the most suitable induction system can be selected or multiple systems can be combined to achieve the best results.

[0139] Example 6: Directly using medium-chain fatty acid fermentation broth to produce medium-chain hydroxy fatty acids

[0140] This example describes in detail the efficient production process of hydroxylated medium-chain fatty acids by directly utilizing medium-chain fatty acid fermentation broth without extraction and purification steps. The specific steps are as follows:

[0141] (1) Preparation of engineered bacteria: According to steps (1) to (4) in Example 5, E. coli BL21 (DE3) engineered bacteria expressing the alkBGT gene were constructed and induced with IPTG, and the bacteria were collected by centrifugation.

[0142] (2) Preparation of fermentation broth raw materials: The fermentation broth obtained at pH 7.2 in Example 4 (containing 15.0 g / L hexanoic acid, 4.8 g / L octanoic acid, and 3.2 g / L heptanoic acid) was taken and the pH was adjusted to 6.5. The precipitate was removed by centrifugation (8000 × g, 10 min) to obtain a clarified fermentation supernatant.

[0143] (3) Construction of the biotransformation reaction system: The collected engineered bacteria were resuspended in 80 mL of M9 minimal medium (containing 0.5% glucose, 1 mM MgSO₄, and 0.1 mM CaCl₂), and then 20 mL of the fermentation broth supernatant (i.e., the fermentation broth ratio was 20%) was added to form a 100 mL reaction system. The final medium-chain fatty acid concentration in the reaction system was approximately 4.6 g / L, including 3.0 g / L hexanoic acid, 1.0 g / L octanoic acid, and 0.6 g / L heptanoic acid.

[0144] (4) Optimization of reaction conditions: Three reaction systems were set up, with the pH controlled at 6.0, 6.5, and 7.0, respectively. Other conditions were fixed at a temperature of 30 °C, a rotation speed of 160 rpm, and 2 g / L glucose was added as a co-substrate.

[0145] (6) Process monitoring: Sampling was performed every 12 hours to analyze substrate consumption and product formation, and quantitative and qualitative analysis was performed using HPLC and GC-MS.

[0146] (7) Conversion results: After 48 hours, the conversion effects under different pH conditions are shown in Table 3 below.

[0147] Table 3 Production results of hydroxylated medium-chain fatty acids using fermentation broth directly under different pH conditions

[0148]

[0149] (8) Comparison with Purified Substrate: Compared with Example 5, the total conversion rate of bioconversion using fermentation broth directly was slightly lower (68.2% vs. 72.3%), but due to the higher starting substrate concentration, the final hydroxylated medium-chain fatty acid yield increased by approximately 44.4% (2.05 g / L vs. 1.42 g / L). In addition, this process eliminates the fatty acid extraction and purification step, significantly reducing process costs and environmental impact.

[0150] (9) Inhibitory factor analysis: By adding different concentrations of lactic acid for control experiments, it was found that when the lactic acid concentration exceeded 5g / L, it would produce a 10-15% inhibitory effect on the alkBGT enzyme activity. This finding provides an important basis for subsequent process optimization.

[0151] (10) Large-scale verification: The above experiment was repeated in a 1 L reactor (pH 6.5 condition). After 48 hours of reaction, the total yield of hydroxylated medium-chain fatty acids reached 2.12 g / L, which was basically consistent with the results of the small-scale experiment, indicating that the process has good scalability potential.

[0152] Example 7: Optimization of lactic acid fermentation process for different organic wastes

[0153] This example optimizes the lactic acid fermentation process for various organic wastes and compares the effects of spontaneous lactic acid fermentation and the addition of lactic acid bacteria. The specific steps are as follows:

[0154] (1) Types of organic waste: Five common organic wastes were selected: a. kitchen waste; b. food processing waste; c. expired fruits and vegetables; d. residual activated sludge; e. livestock and poultry manure.

[0155] (2) Pretreatment method: Grind kitchen waste and food processing waste to <5mm; Mix the pomace and juice after juicing expired fruits and vegetables; Dehydrate the remaining activated sludge to a moisture content of 85%; Dilute livestock and poultry manure to a solid content of 15%.

[0156] (3) Experimental Design: Each organic waste was divided into two groups. Group A was subjected to spontaneous lactic acid fermentation (with the initial pH adjusted to 7.0); Group B was supplemented with 1% commercial lactic acid bacteria (Lactobacillus acidophilus). Other conditions remained the same: temperature 35°C, stirring speed 80 r / min, and fermentation time 6 days.

[0157] (4) The experimental results are shown in Table 4 below.

[0158] Table 4 Lactic acid fermentation process optimization results

[0159]

[0160] (5) Conclusion Analysis: The experimental results show that although the lactic acid production of spontaneous lactic acid fermentation is slightly lower than that of the solution with added lactic acid bacteria, it can still achieve a high lactic acid concentration and ratio for most organic waste (especially kitchen waste and expired fruits and vegetables), providing sufficient substrate for subsequent carbon chain extension. The pH fluctuation of spontaneous fermentation is small and remains in a range close to neutral, which is conducive to maintaining microbial diversity and laying the foundation for the subsequent construction of a carbon chain extension system with a wide pH range.

[0161] Example 8: Study on the effect of carbon chain extension on product distribution over a wide pH range

[0162] This example carefully controlled the pH gradient to study in detail the effect of pH on the distribution of medium-chain fatty acid products and explore the unique advantages of Anaerococcus microorganisms under pH>7.0 conditions. The specific steps are as follows:

[0163] (1) Experimental design: Seven different pH conditions (5.5, 6.0, 6.5, 7.0, 7.2, 7.4, 7.5) were set, the composite bacterial consortium (Caproiciproducens + Anaerococcus) in Example 4 was used as the bacterial species, the lactic acid fermentation broth obtained in Example 1 was used as the substrate, and other conditions remained consistent: temperature 35°C, stirring speed 70 r / min, and fermentation time 7 days.

[0164] (2) Precise pH control: A pH automatic control system is used to maintain the pH within the range of ±0.1 of the set value by adding 1 mol / L potassium hydroxide solution or 1 mol / L hydrochloric acid solution.

[0165] (3) Product analysis: The concentration of C2-C8 fatty acids was analyzed by gas chromatography, and the percentage of each component in the total amount of medium-chain fatty acids was calculated.

[0166] (4) Analysis of microbial community structure: High-throughput sequencing technology was used to analyze the changes in microbial community structure under different pH conditions, focusing on the relative abundance changes of the two genera Caproiciproducens and Anaerococcus.

[0167] (5) The experimental results are shown in Table 5 below.

[0168] Table 5 Percentage of each component in the total amount of medium-chain fatty acids at different pH

[0169]

[0170] (6) Result analysis: The data showed that as the pH increased, the proportion of octanoic acid and heptanoic acid showed a significant upward trend. At pH 7.5, the total proportion of octanoic acid and heptanoic acid reached 32.9%, which was nearly three times that of pH 5.5 (11.2%). This confirmed that high pH conditions are conducive to the synthesis of medium-chain fatty acids with longer carbon chains. Microbial community analysis showed that as the pH increased, the abundance of Anaerococcus microorganisms increased significantly, while the abundance of Caproiciproducens microorganisms decreased significantly, indicating that the two bacteria have complementary ecological niches under different pH conditions, which together ensure the stable operation of the system in a wide pH range.

[0171] (7) Organic acid utilization characteristics: During the experiment, the utilization of different organic acids by the composite bacterial community was monitored, and it was found that Anaerococcus microorganisms can effectively utilize acetic acid and butyric acid to carry out chain extension reactions with lactic acid. This characteristic is relatively rare in traditional carbon chain extension bacteria and is an important mechanism for their ability to function under neutral to weakly alkaline conditions.

[0172] Example 9: Demonstration of the entire process from organic waste to hydroxylated medium-chain fatty acids

[0173] This example demonstrates the complete conversion process from organic waste to hydroxylated medium-chain fatty acids, and conducts process integration and scale-up verification. The specific steps are as follows:

[0174] (1) Raw material preparation: 100 kg of mixed organic waste (70% kitchen waste and 30% food processing waste) was collected and preliminarily sorted and crushed.

[0175] (2) Lactic acid fermentation: The treated organic waste was added to a 500 L fermenter. The initial pH was adjusted to 7.0, the temperature was controlled at 35°C, and the stirring speed was 80 r / min. Spontaneous lactic acid fermentation was used for 6 days. The pH was monitored and maintained within the range of 6.8-7.0. At the end of fermentation, the lactic acid concentration reached 45.5 g / L.

[0176] (3) Solid-liquid separation: The lactic acid fermentation broth was subjected to solid-liquid separation using a plate and frame filter press to obtain approximately 400 L of liquid containing lactic acid.

[0177] (4) Carbon chain extension: 300 L of lactic acid fermentation broth was transferred to a 400 L anaerobic fermentation tank, inoculated with 30 L of the composite bacterial flora cultured in Example 4, and the pH was controlled at 6.8 and the temperature was 35°C to carry out the carbon chain extension reaction. A feeding strategy of replacing 25% of the fermentation broth every 48 hours was adopted, and the operation was continued for 10 days. After the reaction was completed, the total concentration of medium-chain fatty acids was 22.6 g / L, including 15.8 g / L (69.9%) of hexanoic acid, 4.2 g / L (18.6%) of octanoic acid, 2.6 g / L (11.5%) of heptanoic acid, and 2.5 g / L of butyric acid (not included in the total amount of medium-chain fatty acids).

[0178] (5) Option A: Medium-chain fatty acid extraction and conversion

[0179] a. Fatty Acid Extraction: The fermentation broth was adjusted to pH 4.0, and the cells were recovered using membrane separation technology. The supernatant was then fed into an extraction system. A three-stage countercurrent extraction process was performed using ethyl acetate (1:1 volume ratio), achieving an extraction efficiency of 92.7%. The organic phase was distilled under reduced pressure to recover the solvent, yielding 6.28 kg of a medium-chain fatty acid mixture.

[0180] b.ω-oxidation reaction: The engineered bacteria constructed in Example 5 were cultured in a 50 L fermenter until OD600 The cell culture medium was collected and resuspended in 35 L of M9 medium. 2.5 kg of medium-chain fatty acids were added as substrates (final concentration of about 7.1 g / L). The catalytic reaction was carried out at 35°C and 180 rpm for 3 days.

[0181] c. Extraction and Purification of Hydroxy Medium-Chain Fatty Acids: After the reaction, the cells were removed by centrifugation, and the supernatant was adjusted to pH 3.0 and extracted with ethyl acetate. After solvent recovery, 1.97 kg of crude hydroxy medium-chain fatty acids were obtained. The product was purified by molecular distillation to obtain 1.82 kg of refined hydroxy medium-chain fatty acids, with an overall yield of 72.8%.

[0182] d. Product analysis: GC-MS and nuclear magnetic resonance analysis confirmed the product composition: ω-hydroxyhexanoic acid 1.28 kg (70.3%), ω-hydroxyoctanoic acid 0.33 kg (18.1%), ω-hydroxyheptanoic acid 0.21 kg (11.6%), with an overall purity of >98%.

[0183] (6) Scheme B: Direct conversion of fermentation broth

[0184] a. Fermentation broth pretreatment: Take 100 L of the above medium-chain fatty acid fermentation broth, remove most of the bacteria and suspended matter by centrifugation (8000 × g, 15 minutes), adjust the pH to 6.5, and obtain a clarified fermentation supernatant.

[0185] b. Preparation of engineered bacteria: The engineered bacteria constructed in Example 5 were cultured in a 50 L fermenter to OD 600 About 0.6, add 0.5mM IPTG to induce for 12 hours, and collect the bacteria.

[0186] c. Direct Conversion: The harvested engineered bacteria were resuspended in 120 L of modified M9 medium (containing 0.5% glucose, 1 mM MgSO₄, 0.1 mM CaCl₂, and 0.2 U / mL glucose dehydrogenase). 80 L of pretreated fermentation supernatant (40% fermentation broth) was added and the reaction was continued at pH 6.5 and 30°C for 72 hours. The initial medium-chain fatty acid concentration in the reaction system was approximately 9.0 g / L.

[0187] d. Product Extraction and Analysis: After the reaction, the cells were removed by centrifugation, the supernatant was adjusted to pH 3.0, and extracted with ethyl acetate. After solvent recovery, 1.38 kg of crude hydroxylated medium-chain fatty acids were obtained. Purification by molecular distillation yielded 1.21 kg of refined hydroxylated medium-chain fatty acids, with a conversion rate of 53.5%. The product composition was: 0.83 kg (68.6%) of ω-hydroxyhexanoic acid, 0.23 kg (19.0%) of ω-hydroxyoctanoic acid, and 0.15 kg (12.4%) of ω-hydroxyheptanoic acid, with an overall purity of >95%.

[0188] (7) Comparison of the two solutions:

[0189] a. Yield Comparison: Alternative A (conversion after extraction) achieved a total conversion rate of 72.8% and a product purity of >98%. Alternative B (direct conversion) achieved a total conversion rate of 53.5% and a product purity of >95%. Although Alternative B had a lower conversion rate, it eliminated the extraction step, resulting in a higher overall process yield.

[0190] b. Cost Analysis: Option B eliminates the extraction and purification steps for medium-chain fatty acids, significantly reducing solvent consumption and process costs. Preliminary economic assessments indicate that the direct conversion process can reduce production costs by approximately 35-40%.

[0191] c. Process Challenges: Option B requires optimization of the engineered bacteria to improve their tolerance to the complex environment of the fermentation broth, especially their tolerance to residual lactic acid and various inhibitors.

[0192] (8) Full-process efficiency evaluation: Starting from 100 kg of organic waste, Scheme A can produce approximately 1.82 kg of hydroxylated medium-chain fatty acid product, while Scheme B can produce approximately 1.21 kg of product from 1 / 3 of the raw material amount. This translates to approximately 18-36 g of hydroxylated medium-chain fatty acid per kilogram of organic waste. By optimizing process parameters at each stage, the yield is expected to increase to 40-50 g / kg.

[0193] The above examples describe in detail the entire process of the present invention, from lactic acid fermentation and carbon chain elongation to ω-oxidation, and particularly demonstrate the application advantages of the new microbial system in a wide pH range. The full-process conversion efficiency from organic waste to hydroxylated medium-chain fatty acids is verified through actual cases.

[0194] Based on the understanding of the contents of the present invention, those skilled in the art can appropriately adjust the process parameters according to specific needs to obtain the best conversion effect, which should fall within the scope of protection determined by the claims of the present invention.

[0195] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art should fall within the scope of protection defined by the claims of the present invention.

Claims

1. A method for synthesizing medium-chain hydroxy fatty acids from organic waste, characterized by: The following steps are involved: (1) subjecting organic waste or simple carbohydrates to lactic acid fermentation under non-sterile conditions to obtain a fermentation liquid containing lactic acid; (2) adding a composite bacterial consortium capable of synthesizing medium-chain fatty acids in a pH range of 5.5 to 7.5 to the fermentation broth, wherein the composite bacterial consortium comprises microorganisms that utilize a fatty acid synthesis pathway and a β-oxidation reverse cycle metabolic pathway; converting the lactic acid into medium-chain fatty acids at a pH of 5.5 to 7.5; (3) Using microorganisms that construct an ω-oxidation pathway, the medium-chain fatty acids are selectively converted into medium-chain hydroxy fatty acids.

2. The method for synthesizing medium-chain hydroxy fatty acids from organic waste according to claim 1, wherein: In step (1), the pH value is controlled to be 5.8-7.0 during the fermentation process, and anaerobic fermentation is carried out at a temperature of 30-37° C. and a stirring speed of 80 r / min for 3-7 days; When the raw material is simple carbohydrates, lactic acid bacteria are added thereto for fermentation to produce lactic acid, and the amount of lactic acid bacteria added is 0.5-1.0%; when the raw material is organic waste, lactic acid bacteria are added thereto for fermentation to produce lactic acid or lactic acid fermentation is carried out spontaneously.

3. The method for synthesizing medium-chain hydroxy fatty acids from organic waste according to claim 2, wherein: The lactic acid bacteria are added in the form of a bacterial liquid or freeze-dried powder containing lactic acid bacteria. The freeze-dried powder is cultured with MRS medium for lactic acid bacteria at 30°C for 2-3 days, centrifuged, and the bacteria are taken to make 10 8 ~10 12 CFU / g of lactic acid bacteria freeze-dried powder.

4. The method for synthesizing medium-chain hydroxy fatty acids from organic waste according to claim 1, wherein: In step (2), the composite bacterial community comprises at least one of microorganisms of the genera Caproiciproducens and Anaerococcus; The microorganism constructing the ω-oxidation pathway is Escherichia coli. The Escherichia coli is pre-cultured in M9 medium or LB medium containing 0.5% glucose, and then induced to express by any of the following methods: (a) Add 0.1-1.0 mM IPTG to induce expression; (b) Temperature induction from 30°C to 37-42°C; (c) Add 0.01-0.2% L-arabinose or 0.1-1 μg / mL doxycycline to induce expression.

5. The method for synthesizing medium-chain hydroxy fatty acids from organic waste according to claim 1 or 4, characterized in that: In step (2), the fermentation is carried out at a temperature of 30 to 40° C., and the hydraulic retention time during the fermentation is 2 to 15 days.

6. The method for synthesizing medium-chain hydroxy fatty acids from organic waste according to claim 1, characterized in that: In step (3), the reaction is carried out at a temperature of 30 to 40° C. and a rotation speed of 30 to 200 rpm, and the reaction time is 1 to 4 days.

7. The method for synthesizing medium-chain hydroxy fatty acids from organic waste according to claim 1, characterized in that: The medium-chain fatty acid is an organic carboxylic acid containing 6 to 12 carbon atoms, and the medium-chain hydroxy fatty acid is a fatty acid containing 6 to 12 carbon atoms and containing a hydroxyl group at the end.

8. The method for synthesizing medium-chain hydroxy fatty acids from organic waste according to claim 1 or 4, characterized in that: The ω-oxidation pathway was constructed by heterologously expressing the alkane hydroxylase system of Pseudomonas putida GPo1.

9. The method for synthesizing medium-chain hydroxy fatty acids from organic waste according to claim 8, characterized in that: The alkane hydroxylation system includes alkane monooxygenase (alkB), rubredoxin (alkG) and rubredoxin reductase (alkT); a ribosome binding site is added to the gene of the hydrocarbon hydroxylation system, and the sequence is AAAGAGGAGAAA.

10. The method for synthesizing medium-chain hydroxy fatty acids from organic waste according to claim 9, characterized in that: The gene of the alkane hydroxylation system is connected to the plasmid vector through homologous recombination to form a corresponding recombinant plasmid, and the recombinant plasmid is transferred into a microorganism with a constructed ω-oxidation pathway; The plasmid vector is selected from at least one of the following: (a) pET-28a(+) vector for IPTG-induced expression; (b) a λpR-pL vector containing the temperature-sensitive repressor protein cI857 for temperature-induced expression; (c) pBAD series vectors for arabinose-induced expression; (d) pTet series vectors for doxycycline-inducible expression.

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